Guanine riboswitch binding compounds and their use as antibiotics
09993491 · 2018-06-12
Assignee
Inventors
- Jerome Mulhbacher (Sherbrooke, CA)
- Daniel Lafontaine (Sherbrooke, CA)
- Francois Malouin (Eastman, CA)
- Marianne Allard (Sherbrooke, CA)
- Eric Marsault (Sherbrooke, CA)
Cpc classification
Y10T436/141111
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61K31/519
HUMAN NECESSITIES
A61K31/513
HUMAN NECESSITIES
A61K31/675
HUMAN NECESSITIES
A61K31/535
HUMAN NECESSITIES
A61K31/505
HUMAN NECESSITIES
A61K31/4412
HUMAN NECESSITIES
A61K31/54
HUMAN NECESSITIES
Y10T436/143333
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61K31/5383
HUMAN NECESSITIES
A61K31/542
HUMAN NECESSITIES
International classification
A61K31/505
HUMAN NECESSITIES
A61K31/535
HUMAN NECESSITIES
A61K31/54
HUMAN NECESSITIES
A61K31/513
HUMAN NECESSITIES
A61K31/542
HUMAN NECESSITIES
A61K31/519
HUMAN NECESSITIES
A61K31/5383
HUMAN NECESSITIES
C07D239/46
CHEMISTRY; METALLURGY
A61K31/675
HUMAN NECESSITIES
Abstract
The present invention includes novel compounds and pharmaceutically acceptable formulations of said compounds which exhibit antibiotic activity against microorganisms bearing a guanine riboswitch that controls the expression of the guaA gene, including organisms which are resistant to certain antibiotic families, and which are useful as antibacterial agents for treatment or prophylaxis of bacterial infections in animals or in humans, in particular but not limited to infections of the mammary gland, or their use as antiseptics, agents for sterilization or disinfection.
Claims
1. A method of treating a bacterial infection in a subject, said method comprising administering to said subject a therapeutically effective amount of a non ribosylable ligand of a guanine riboswitch, wherein said subject is infected by the bacterial infection prior to said administration, wherein said bacterial infection is caused by a pathogen bearing the guanine riboswitch, and wherein the guanine riboswitch controls the expression of guaA, wherein said pathogen: a) belongs to the genus Staphylococcus or Clostridium; or b) is Alkaliphilus metalliredigens; Alkaliphilus oremlandii; Bacillus anthracis; Bacillus cereus; Bacillus halodurans; Bacillus thuringiensis; Bacillus weihenstephanensis; Exiguobacterium sibiricum; Geobacillus kaustophilus; Geobacillus thermodenitrificans; Lysinibacillus sphaericus; Oceanobacillus iheyensis; Thermoanaerobacter pseudethanolicus; or Thermoanaerobacter X514, and wherein said non ribosylable ligand of guanine riboswitch is the compound 4-hydroxy-2,5,6-triaminopyrimidine (1.01) or a pharmaceutically acceptable salt, solvate or stereoisomer thereof.
2. The method of claim 1, wherein said subject is a cow or a human.
3. The method of claim 1, wherein said pathogen is Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Clostridium botulinum or Clostridium difficile.
4. The method of claim 1, wherein said infection is a mammary gland infection.
5. The method of claim 1, wherein said pathogen is a pathogen belonging to the genus Staphylococcus or Clostridium and wherein said pathogen is Staphylococcus aureus; Staphylococcus carnosus; Staphylococcus epidermidis; Staphylococcus haemolyticus, Staphylococcus saprophyticus, Clostridium acetobutylicum; Clostridium beijerinckii; Clostridium botulinum; Clostridium difficile; Clostridium novyi; Clostridium perfringens or Clostridium tetani.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) In the appended drawings:
(2)
(3)
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DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
(13) The gene guaA is essential for the survival or virulence of various microbial pathogens and encodes the enzyme GMP synthetase, which catalyses the synthesis of GMP (guanosine-5-monophosphate) (E.C. 6.3.5.2) from XMP (xanthosine 5-monophosphate). The present invention showed that, in some microbial pathogens, expression of the essential gene guaA is under control of the guanine riboswitch. As shown in the accompanying examples, the guanine riboswitch in S. aureus was found herein to control the expression of four genes present on the same mRNA transcript: xpt, pbuX, guaB and guaA. A bioinformatic analysis revealed that such guanine riboswitches are found in pathogen microorganisms such as Staphylococcus aureus and Clostridium difficile but not in microorganisms such as Bacillus subtilis and Streptococci in which a guanine riboswitch does not control the expression of guaA. See Table 3 below for examples of microorganisms which contain or not a guanine riboswitch that controls the expression of guaA.
(14) The present invention also relates to the unexpected discovery that some pathogen microorganisms, such as S. aureus, highly express guaA in specific environments during infection of an animal host such as a mammal. As shown in the accompanying examples, microarray and quantitative PCR gene expression analysis was performed on various S. aureus strains isolated from bovine intramammary infections. These studies reveal that guaA is highly expressed during infection in cows by prototypical S. aureus strains known to cause bovine mastitis as well as in strains known to cause persistent and chronic mastitis. Therefore, the present invention also relates to the surprising discovery that guanine riboswitches controlling the expression of the guaA gene are suitable targets for therapeutic antimicrobial intervention.
(15) The present invention also relates to the surprising discovery that guanine-like compounds modified to be unable to undergo ribosylation, are nevertheless capable of binding to or interacting with riboswitch aptamers and can affect expression of genes that are under riboswitch control. As shown in the accompanying examples, binding and competition assays measuring the ability of various compounds to displace a known guanine riboswitch ligand showed that the compounds of the present invention are capable of binding to the guanine riboswitch. Microarray gene expression analysis confirmed that the compounds of the present invention can not only bind to guanine riboswitches but also modulate expression of genes under control of the guanine riboswitch. Furthermore, the specificity and mechanism of this modulation was validated by studies combining a compound of the present invention with the metabolite GMP.
(16) The present invention also relates to the discovery that the compounds of the present invention can act as specific or selective antimicrobial agents to which microbial pathogens (that have the guaA gene under control of the guanine riboswitch) cannot develop resistance. The present invention also relates to the discovery that the compounds of the present invention have no substantial inhibitory activity against microorganisms in which the gene guaA is not controlled by a guanine riboswitch. As shown in the accompanying examples, the compounds of the present invention can demonstrate an anti-bacterial effect on S. aureus and C. difficile strains when grown in vitro (e.g., in culture media or milk) or in vivo (e.g., in mice mammary gland infections). The specificity of the compounds of the present invention was demonstrated by their failure to exert an antibacterial effect on strains (e.g., E. coli) lacking a guaA under control of the guanine riboswitch. The anti-bacterial effect of the compounds of the present invention were shown herein to be comparable to several well-known antibiotic compounds. Strikingly, unlike the well-known antibiotic compounds, ciprofloxacin and rifampicin, a S. aureus strain did not develop antibiotic resistance to a compound of the present invention. Therefore, it is also disclosed herein that the compounds of the present invention are able to inhibit the expression of the gene guaA, preventing rapid development of bacterial resistance in S. aureus.
(17) The present invention also relates to compounds of the following general formulas:
(18) Guanine
(19) ##STR00020##
(20) Formula 1.0:
(21) ##STR00021##
(22) Formula 1.01 (4-hydroxy-2,5,6-triaminopyrimidine here shown as the guanine riboswitch ligand 2,5,6-triaminopyrimidine-4-one):
(23) ##STR00022##
(24) Formula 1.13 (2,4-diamino-6-hydroxypyrimidine here shown as the guanine riboswitch ligand 2,6-diaminopyrimidine-4-one):
(25) ##STR00023##
(26) Formula 1.16 (4,5-diamino-6-hydroxy-2-mercaptopyrimidine here shown as the guanine riboswitch ligand 5,6-diamino-2-mercaptopyrimidine-4-one)
(27) ##STR00024##
(28) Formula 2.0:
(29) ##STR00025##
(30) Formula 2.02 (9-oxoguanine here shown as 5-Amino-6H-oxazolo[5,4-d]pyrimidin-7-one)
(31) ##STR00026##
(32) Formula 2.17 (5-amino-2-chloro-2,3-dihydrothiazolo[4,5]pyrimidine-7-(6H)-one)
(33) ##STR00027##
(34) Formula 3.0:
(35) ##STR00028##
(36) The compounds of the present invention bind to the guanine riboswitch binding site and yet are chemically unable to be ribosylated by the targeted pathogens. This property prevents incorporation of the compounds into cellular nucleosides, nucleotides or nucleic acids, and thus prevents broad, non-specific toxicity for bacterial species not targeted by the compound and for the mammalian or animal host in which the present compounds are used for therapeutic intervention.
(37) Definitions
(38) The use of the word a or an when used in conjunction with the term comprising in the claims and/or the specification may mean one but it is also consistent with the meaning of one or more, at least one, and one or more than one.
(39) As used in this specification and claim(s), the words comprising (and any form of comprising, such as comprise and comprises), having (and any form of having, such as have and has), including (and any form of including, such as includes and include) or containing (and any form of containing, such as contains and contain) are inclusive or open-ended and do not exclude additional, un-recited elements or method steps.
(40) As used herein, the terms molecule, compound, agent or ligand are used interchangeably and broadly to refer to natural, synthetic or semi-synthetic molecules or compounds. The term compound therefore denotes, for example, chemicals, macromolecules, cell or tissue extracts (from plants or animals) and the like. Non-limiting examples of compounds include peptides, antibodies, carbohydrates, nucleic acid molecules and pharmaceutical agents. The compound can be selected and screened by a variety of means including random screening, rational selection and by rational design using, for example, ligand modeling methods such as computer modeling. The terms rationally selected or rationally designed are meant to define compounds which have been chosen based on the configuration of interacting domains of the present invention (e.g., the guanine riboswitch). As will be understood by the person of ordinary skill, molecules having non-naturally occurring modifications are also within the scope of the term compound. For example, the compounds of the present invention can be modified to enhance their activity, stability, toxicity and/or bioavailability. The compounds or molecules identified in accordance with the teachings of the present invention have a therapeutic value in diseases or conditions related to microbial infections.
(41) The term subject or patient as used herein refers to an animal, preferably a mammal such as but not limited cattle such as cow; goat; ewe; ass; horse; pig; cat; dog; etc. or human who is the object of treatment, observation or experiment.
(42) As used herein, the terms pharmaceutically acceptable refer to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to animals (e.g., cows) or human. Preferably, as used herein, the term pharmaceutically acceptable means approved by regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
(43) The term carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compounds of the present invention may be administered. Sterile water or aqueous saline solutions and aqueous dextrose and glycerol solutions may be employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E. W. Martin.
(44) Compounds of the invention may be administered in a pharmaceutical composition. Pharmaceutical compositions may be administered in unit dosage form. Any appropriate route of administration may be employed, for example, parenteral, subcutaneous, intramuscular, intramammary, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraarticular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration. Examples of specific routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, intramammary; oral (e.g., inhalation); transdermal (topical); transmucosal, and rectal administration.
(45) Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer such compositions to patients. Methods well known in the art for making pharmaceutical compositions and formulations are found in, for example, Remington: The Science and Practice of Pharmacy, (20th ed.) ed. A. R. Gennaro A R., 2000, Lippincott: Philadelphia.
(46) Therapeutic formulations for oral administration, may be in the form of tablets or capsules; for transmucosal (e.g., rectal, intranasal) or transdermal/percutaneous administration may be in the form of ointments, powders, nasal drops, sprays/aerosols or suppositories; for topical administration, may be in the form of ointments, creams, gels or solutions; for parenteral administration (e.g., intravenously, intramuscularly, intradermal, intramammary, subcutaneously, intrathecally or transdermally), using for example injectable solutions. Furthermore, administration can be carried out sublingually or as ophthalmological preparations or as an aerosol, for example in the form of a spray. Intravenous, intramuscular or oral administration is a preferred form of use.
(47) Oral
(48) For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used for example in the form of tablets, troches, dragees, hard or soft gelatin capsules, solutions (e.g., syrups), emulsions or suspensions, or capsules. For the preparation of formulations for oral administration, the compounds of the present invention may be admixed with pharmaceutically inert, inorganic or organic excipients (e.g., pharmaceutically compatible binding agents, and/or adjuvant). The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. Examples of suitable excipients for tablets, dragees or hard gelatin capsules for example include lactose, maize starch or derivatives thereof, talc or stearic acid or salts thereof. Suitable excipients for use with soft gelatin capsules include for example vegetable oils, waxes, fats, semi-solid or liquid polyols etc.; according to the nature of the active ingredients it may however be the case that no excipient is needed at all for soft gelatin capsules.
(49) For the preparation of solutions and syrups, excipients which may be used include for example water, polyols, saccharose, invert sugar and glucose.
(50) Nasal
(51) For administration by inhalation, the compounds may be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Formulations for inhalation may contain excipients, or example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
(52) Transmucosal or Transdermal
(53) For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. For suppositories, and local or percutaneous application, excipients which may be used include for example natural or hardened oils, waxes, fats and semi-solid or liquid polyols.
(54) Parenteral
(55) Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Solutions or suspensions used for parenteral application can include the following components: a sterile diluent such as water for injection (where water soluble), saline solution, fixed oils (e.g., paraffin oil), polyalkylene glycols such as polyethylene glycols, glycerine, propylene glycol or other synthetic solvents, oils of vegetable origin, or hydrogenated napthalenes; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; reducing agents such as dithiothreitol, buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for compounds of the invention include ethylenevinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The parenteral preparation can also be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
(56) For intravenous or intramammary administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
(57) Liposomal suspensions (including liposomes targeted to specific cell types) can also be used as pharmaceutically acceptable carriers. A variety of liposomal formulations suitable for delivering a compound to an animal have been described and demonstrated to be effective in delivering a variety of compound, including, e.g., small molecules, nucleic acids, and polypeptides.
(58) As mentioned earlier, medicaments containing the compounds of the present invention are also an object of the present invention, as is a process for the manufacture of such medicaments, which process comprises bringing one or more of the compounds of the present invention to, if desired, one or more other therapeutically valuable substances into a galenical administration form.
(59) Salts, Esters, Hydrates and Solvates
(60) The compounds of the present invention include pharmacologically acceptable salts and ester derivatives thereof as well as hydrates or solvates thereof and all stereoisomeric forms of the referenced compounds. The compounds and pharmacologically acceptable esters thereof of the present invention can form pharmacologically acceptable salts if necessary.
(61) Salts
(62) The terms pharmacologically acceptable salt thereof refer to a salt to which the compounds of the present invention can be converted. Preferred examples of such a salt include alkali metal salts such as a sodium salt, a potassium salt, a lithium salt, magnesium or calcium salts; alkaline earth metal salts such as a calcium salt and a magnesium salt; metal salts such as an aluminium salt, an iron salt, a zinc salt, a copper salt, a nickel salt and a cobalt salt; amine salts such as inorganic salts including an ammonium salt; organic salts or ammonium salts such as a t-octylamine salt, a dibenzylamine salt, a morpholine salt, a glucosamine salt, a phenylglycine alkyl ester salt, an ethylenediamine salt, an N-methylglucamine salt, a guanidine salt, a diethylamine salt, a triethylamine salt, a dicyclohexylamine salt, an N,N-dibenzylethylenediamine salt, a chloroprocaine salt, a procaine salt, a diethanolamine salt, an N-benzyl-phenethylamine salt, a piperazine salt, a tetramethylammonium salt and a tris(hydroxymethyl)aminomethane salt; inorganic acid salts such as hydrohalic acid salts such as a hydrofluoride, a hydrochloride, a hydrobromide or a hydroiodide, a nitrate, a perchlorate, a sulfate or a phosphate; lower alkanesulfonates such as a methanesulfonate, trifluoromethanesulfonate or an ethanesulfonate; arylsulfonates such as a benzenesulfonate or a p-toluenesulfonate and the like, which are non toxic to living organisms; organic acid salts such as an acetate, a malate, adipate, a fumarate, a succinate, a citrate, alginate, ascorbate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, cinnamate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, itaconate, lactate, maleate, mandelate, sulfonate, methanesulfonate, trifluoromethanesulfonates, ethanesulfonates 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, tartrate, thiocyanate, tosylate, and undecanoate, a tartrate, an oxalate or a maleate; and amino acid salts such as a glycine salt, a lysine salt, an arginine salt, an omithine salt, histidine, a glutamate or an aspartate salt. Additionally, basic nitrogen containing groups may be quaternized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates including dimethyl, diethyl, and dibutyl sulfate; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and strearyl chlorides, bromides and iodides, aralkyl halides including benzyl and phenethyl bromides, and others. For further example, see S. M. Berge, et al. Pharmaceutical Salts, J. Pharm. Sci. 1977, 66, 1-19. Such salts can be formed quite readily by those skilled in the art using standard techniques.
(63) Preferred example of the salts formed with an acidic group present in the compounds of the present invention include metal salts such as alkali metal salts (e.g., sodium salts, potassium salts and lithium salts), alkali earth metal salts (e.g., calcium salts and magnesium salts), aluminum salts and iron salts; amine salts such as inorganic amine salts (e.g., ammonium salts) and organic amine salts (e.g., t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycinealkyl ester salts, ethylenediamine salts, N-methylglucamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts. N-benzylphenethylamine salts, piperazine salts, tetramethylammonium salts and tris(hydroxymethyl)aminomethane salts; and amino acid salts such as glycine salts, lysine salts, arginine salts, ornithine salts, glutamates and aspartates.
(64) All salts are intended to be pharmaceutically acceptable salts within the scope of the invention and all salts are considered equivalent to the free forms of the corresponding compounds for purposes of the invention.
(65) Esters
(66) Physiologically/pharmaceutically acceptable esters are also useful as active medicaments. The term pharmaceutically acceptable esters embraces esters of the compounds of the present invention, in which hydroxy groups (e.g., in carboxylic acid) have been converted to the corresponding esters and may act as a prodrug which, when absorbed into the bloodstream of a warm-blooded animal, may cleave in such a manner as to release the drug form and permit the drug to afford improved therapeutic efficacy. Such esters can be formed with inorganic or organic acids such as nitric acid, sulphuric acid, phosphoric acid, citric acid, formic acid, maleic acid, acetic acid, succinic acid, tartaric acid, methanesulphonic acid, p-toluenesulphonic acid and the like, which are non toxic to living organisms. Further examples are the esters with aliphatic or aromatic acids such as acetic acid or with aliphatic alcohol (e.g., alkyl esters, including methyl, ethyl, propyl, isopropyl, butyl, isobutyl or pentyl esters, and the like) or aromatic alcohols (e.g., benzyl ester)
(67) Esters can be prepared from their corresponding acids or salts by a variety of methods known to those skilled in the art, such as, for example, by first transforming the acid to the acid chloride and then reacting the acid chloride with a suitable alcohol. Other suitable methods for making esters are described in Kemp and Vellaccio, 1980.
(68) Where esters of the invention have a basic group, such as an amino group, the compound can be converted to a salt by reacting it with an acid, and in the case where the esters have an acidic group, such as a sulfonamide group, the compound can be converted to a salt by reacting it with a base. The compounds of the present invention encompass such salts.
(69) Salts and esters of the compounds of the present invention may be prepared by known method by employing appropriate starting materials or intermediate compounds that are readily available and/or are described herein.
(70) Generally, a desired salt of a compound of this invention can be prepared in situ during the final isolation and purification of a compound by means well known in the art. For example, a desired salt can be prepared by separately reacting the purified compound in its free base or free acid form with a suitable organic or inorganic acid, or suitable organic or inorganic base, respectively, and isolating the salt thus formed. In the case of basic compounds, for example, the free base is treated with anhydrous HCl in a suitable solvent such as THF, and the salt isolated as a hydrochloride salt. In the case of acidic compounds, the salts may be obtained, for example, by treatment of the free acid with anhydrous ammonia in a suitable solvent such as ether and subsequent isolation of the ammonium salt. These methods are conventional and would be readily apparent to one skilled in the art.
(71) The compounds of this invention may be esterified by a variety of conventional procedures including reacting the appropriate anhydride, carboxylic acid or acid chloride with the alcohol group of a compound of this invention. The appropriate anhydride is reacted with the alcohol in the presence of a base to facilitate acylation such as 1,8-bis[dimethylamino]naphthalene or N,N-dimethylaminopyridine. Or, an appropriate carboxylic acid can be reacted with the alcohol in the presence of a dehydrating agent such as dicyclohexylcarbodiimide, 1-[3-dimethylaminopropyl]-3-ethylcarbodiimide or other water soluble dehydrating agents which are used to drive the reaction by the removal of water, and, optionally, an acylation catalyst. Esterification can also be effected using the appropriate carboxylic acid in the presence of trifluoroacetic anhydride and, optionally, pyridine, or in the presence of N,N-carbonyldiimidazole with pyridine. Reaction of an acid chloride with the alcohol can be carried out with an acylation catalyst such as 4-DMAP or pyridine.
(72) One skilled in the art would readily know how to successfully carry out these as well as other known methods of esterification of alcohols.
(73) Hydrates
(74) As used herein the terms, pharmaceutically acceptable hydrate refer to the compounds of the instant invention crystallized with one or more molecules of water to form a hydrated form.
(75) Prodrugs and Solvates
(76) Prodrugs and solvates of the compounds of the invention are also contemplated herein. A discussion of prodrugs is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems (1987) 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, (1987) Edward B. Roche, ed., American Pharmaceutical Association and Pergamon Press. The term prodrug means a compound (e.g., a drug precursor) that is transformed in vivo to yield a compound of the present invention or a pharmaceutically acceptable salt, hydrate or solvate of the compound. The transformation may occur by various mechanisms (e.g., by metabolic or chemical processes), such as, for example, through hydrolysis in blood. A discussion of the use of prodrugs is provided by T. Higuchi and W. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.
(77) For example, if a compound of the present invention or a pharmaceutically acceptable salt, hydrate or solvate of the compound contains a carboxylic acid functional group, a prodrug can comprise an ester formed by the replacement of the hydrogen atom of the acid group with a group such as, for example, (C1-C8)alkyl, (C2-C12)alkanoyloxymethyl, 1-(alkanoyloxy)ethyl having from 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, 1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl, di-N,N(C1-C2)alkylamino(C2-C3)alkyl (such as -dimethylaminoethyl), carbamoyl-(C1-C2)alkyl, N,N-di(C1-C2)alkylcarbamoyl-(C1-C2)alkyl and piperidino-, pyrrolidino- or morpholino(C2-C3)alkyl, and the like.
(78) Similarly, if a compound of the present invention contains an alcohol functional group, a prodrug can be formed by the replacement of the hydrogen atom of the alcohol group with a group such as, for example, (C1-C6)alkanoyloxymethyl, 1-((C1-C6)alkanoyloxy)ethyl, 1-methyl-1-((C1-C6)alkanoyloxy)ethyl, (C1-C6)alkoxycarbonyloxymethyl, N(C1-C6)alkoxycarbonylaminomethyl, succinoyl, (C1-C6)alkanoyl, -amino(C1-C4)alkanyl, arylacyl and -aminoacyl, or -aminoacyl--aminoacyl, where each -aminoacyl group is independently selected from the naturally occurring L-amino acids, P(O)(OH)2, P(O)(O(C1-C6)alkyl)2 or glycosyl (the radical resulting from the removal of a hydroxyl group of the hemiacetal form of a carbohydrate), and the like.
(79) If a compound of the present invention incorporates an amine functional group, a prodrug can be formed by the replacement of a hydrogen atom in the amine group with a group such as, for example, R-carbonyl, RO-carbonyl, NRR-carbonyl where R and R are each independently (C1-C10)alkyl, (C3-C7)cycloalkyl, benzyl, or R-carbonyl is a natural -aminoacyl or natural -aminoacyl, C(OH)C(O)OY1 wherein Y1 is H, (C1-C6)alkyl or benzyl, C(OY2)Y3 wherein Y2 is (C1-C4) alkyl and Y3 is (C1-C6)alkyl, carboxy (C1-C6)alkyl, amino(C1-C4)alkyl or mono-N or di-N,N(C1-C6)alkylaminoalkyl, C(Y4)Y5 wherein Y4 is H or methyl and Y5 is mono-N or di-N,N(C1-C6)alkylamino morpholino, piperidin-1-yl or pyrrolidin-1-yl, and the like.
(80) One or more compounds of the invention may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the invention embrace both solvated and unsolvated forms. Solvate means a physical association of a compound of this invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. Solvate encompasses both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like. Hydrate is a solvate wherein the solvent molecule is H.sub.2O.
(81) One or more compounds of the invention may optionally be converted to a solvate. Preparation of solvates is generally known. Thus, for example, M. Caira et al, J. Pharmaceutical Sci., 93(3), 601-611 (2004) describe the preparation of the solvates of the antifungal fluconazole in ethyl acetate as well as from water. Similar preparations of solvates, hemisolvate, hydrates and the like are described by E. C. van Tonder et al, AAPS Pharm Sci Tech., 5(1), article 12 (2004); and A. L. Bingham et al, Chem. Commun., 603-604 (2001). A typical, non-limiting, process involves dissolving the inventive compound in desired amounts of the desired solvent (organic or water or mixtures thereof) at a higher than ambient temperature, and cooling the solution at a rate sufficient to form crystals which are then isolated by standard methods. Analytical techniques such as, for example I. R. spectroscopy, show the presence of the solvent (or water) in the crystals as a solvate (or hydrate).
(82) Stereoisomers, Enantiomers, Racemates, Tautomers
(83) The compounds of the present invention have asymmetric carbon atoms and can exist in the form of optically pure enantiomers or as racemates. The invention embraces all of these forms.
(84) For purposes of this Specification, pharmaceutically acceptable tautomer means any tautomeric form of any compound of the present invention.
(85) The purification of enantiomers and the separation of isomeric mixtures of a compound of the present invention may be accomplished by standard techniques known in the art.
(86) The pharmaceutical compositions may also contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts for the variation of osmotic pressure, buffers, coating agents or antioxidants. As mentioned earlier, they may also contain other therapeutically valuable agents.
(87) Dosages
(88) The dosages in which the compounds of the present invention are administered in effective amounts depend on the nature of the specific active ingredient, the age and the requirements of the patient and the mode of application. In general, daily dosages of about 1 mg-1000 mg, preferably 5 mg-500 mg, per day come into consideration.
(89) The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a compound of the present invention can include a series of treatments.
(90) Toxicity and Therapeutic Efficacy
(91) Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
(92) Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
(93) Kits
(94) The present invention also encompasses kits comprising the compounds of the present invention. For example, the kit can comprise one or more compounds or agents binding to a guanine riboswitch and inhibit or reduce the expression of the gene guaA. The kit may optionally include one or more control samples. The compounds or agents can be packaged in a suitable container. The kit can further comprise instructions for using the kit.
(95) The present invention also relates to methods for preparing the above-mentioned compounds. In an embodiment, the above-mentioned method is that defined in Examples 13 and 16 below.
(96) The present invention is illustrated in further details by the following non-limiting examples.
Example 1
guaA Expression in S. Aureus Isolates from Infected Cows and from Milk
(97) The present invention relates to the discovery that some pathogen microorganisms, such as S. aureus, sustain a significant level of expression of the gene guaA (comparable to some known essential genes) in specific environments during infection of a mammalian or animal host. The present invention also discloses the sustained expression of guaA in several S. aureus isolates, including those causing persistent and chronic bovine mastitis, during infection in a cow. The present invention also discloses that the expression of guaA in several S. aureus isolates, including those causing persistent and chronic bovine mastitis, was relatively higher during infection in cows compared to the expression of guaA in the corresponding S. aureus isolates grown in vitro in freshly collected milk obtained from a healthy cow.
(98) DNA microarray gene expression analysis was used to identify genes that were expressed (at different time points) by various S. aureus strains in vivo during bovine mastitis. Mastitis infection in two cows were examined: Cow #310 and cow #5325. The results of this analysis with respect to all genes are summarized in the Venn diagram presented in
(99) TABLE-US-00001 TABLE 1 Mastitic milk samples in which the expression of guaA was shown to be greater than the average expression of all genes on microarrays for 4 different S. aureus strains at 2 different time points in two animals. S. aureus strains Cow Day of infection SHY97-3609 3 557 1290 310 8 .circle-solid. .circle-solid. .circle-solid. .circle-solid. 310 10 .circle-solid. .circle-solid. .circle-solid. .circle-solid. 5325 10 .circle-solid.
Symbols: .circle-solid., expression of guaA greater than the average expression of all genes on arrays;
, expression of guaA was detected but was below the average expression of all genes on arrays;
, not tested by DNA arrays.
(100) Depicted in
(101) Briefly, four different Staphylococcus aureus strains (clinical isolate SHY97-3906 and chronic isolates 3, 557 and 1290) were inoculated in 8 multiparous Holstein cows in mid lactation at the Dairy and Swine Research and Development Centre of Agriculture and Agri-Food Canada in Sherbrooke, QC, Canada. Each quarter of the mammary gland of the cows was infused with 50 CFU of each S. aureus strain. After the inoculation, milk was collected from each quarter of each cow every 2-4 days in the morning for a total of 18 days. S. aureus was then isolated from the mastitic milk and RNA was extracted from the in vivo grown S. aureus. With the RNA, fluorescent probes for hybridization to DNA arrays were generated through an aminoallyl cDNA labelling procedure. The fluorescent probes (labeled with Cy5) were hybridized to a DNA array that contained a selection of 530 known or putative genes. Only genes with a Cy5 signal intensity of 100%, i.e., greater or equal to the mean Cy5 intensity of the entire array were analyzed. The results were confirmed by quantitative PCR and the level of expression of genes found to be strongly expressed during mastitis by S. aureus was compared to that of in vitro grown S. aureus.
Example 2
Polycistronic RNA Encoding xpt/pbuX/guaB/guaA
(102) The present invention relates to the discovery that the guanine riboswitch of S. aureus controls the expression of a polycistronic mRNA encoding xpt/pbuX/guaB/guaA and not only the non-essential genes xpt and pbuX, contradicting the teachings of the prior art that the S. aureus guanine riboswitch controls genes that are not essential for survival or virulence of the pathogen. Example 1 showed that there was a sustained expression of guaA in S. aureus during infection in cows.
(103) Shown in
Example 3
Ability of Compounds of the Invention to Bind Guanine Riboswitches
(104) The compounds of the present invention are able to compete with guanine-like ligands for binding to the guanine riboswitch. Structural modifications of these and other related compounds influence this property.
(105) Shown in
(106) TABLE-US-00002 TABLE 2 Description of the properties of various known guanine riboswitch ligands Appropriate Compound Structure Relative binding Ribosylation properties guanine (1 M)
Example 4
Growth Inhibition of Bacterial Species Possessing a Guanine Riboswitch that Controls the Expression of the guaA Gene by Compounds of the Present Invention
(107) As disclosed herein, compounds of the present invention (such as 4-hydroxy-2,5,6-triaminopyrimidine (compound 1.01)) are able to inhibit the growth of bacteria if such bacteria possess a guanine riboswitch that controls the expression of the guaA gene. Also, the compounds of the present invention (e.g., compound 1.01) do not substantially inhibit the growth of bacteria that have the guaA gene but not the guanine riboswitch that controls its expression.
(108) Shown in Table 3 below are examples of the genes and operons controlled by guanine riboswitches in several bacterial species following a bioinformatic analysis involving a sequence homology algorithm as described in Mulhbacher and Lafontaine, 2007. Briefly, the identification of riboswitches controlling guaA was realized using the Rfam database or a published article (Barrick J E and Breaker R R, Genome Biol. 2007) which repertories guanine riboswitches. The NCBI database was then blasted to find the associate genes. The identification of new guanine riboswitches in newly sequenced genomes could also be realized using the RNAmotif solftware. Genes corresponding to guaA are highlighted. Accession numbers for the genome of the bacterium and positions corresponding to the beginning of the riboswitch and of the gene or the operon containing guaA are also presented. Consistent with the teachings of the present invention, bacterial strains (e.g., S. aureus and C. difficile) expressing the guaA gene under the control of a guanine riboswitch are expected to be susceptible to the antimicrobial effects of the compounds of the present invention. Also consistent with the teachings of the present invention, bacterial strains expressing the guaA gene independently from the guanine riboswitch (e.g., E. coli and Bacillus subtilis) are expected to be not significantly affected by the antimicrobial effects of the compounds of the present invention.
(109) TABLE-US-00003 TABLE 3 Genes and operons controlled by guanine riboswitches in various bacterial strains guaA or operon containing guanine guaA or operon gene or operon controlled guaA controlled by riboswitch containing Bacteria Gram by guanine riboswitch guanine riboswitch genome starting guaA ending Acholeplasma_laidlawii + uraA Alkaliphilus_metalliredigens + xpt; purE guaA NC_009633 944301 947544 Alkaliphilus_oremlandii + pur operon guaA NC_009922 582562 585305 Bacillus_amyloliquefaciens + xpt, pbuX operon; pbuG; pur operon Bacillus_anthracis + uraA; GntR; xpt, pbuX guaA NC_003997 260657 262446 operon; pur operon; NC_007530 260657 262446 nupC NC_005945 260670 262459 Bacillus_cereus + pbuG; pur operon; xpt, guaA NC_012472 272003 273792 pbuX operon; GntR; NC_011658 269262 271050 nupC NC_011773 261264 263053 NC_004722 259617 261405 NC_003909 294525 296313 NC_011725 259207 260995 NC_011772 251188 252976 NC_011969 266283 268071 NC_006274 265891 267680 NC_009674 271090 272876 Bacillus_clausii + pbuG; pur operon; xpt, pbuX operon Bacillus_halodurans + xpt, pbuX operon; pur guaA NC_002570 648458 650224 Operon; uraA Bacillus_licheniformis + pur operon; xpt, pbuX operon; pbuG; nupG Bacillus_pumilus + pbuG; xpt, pbuX operon; uraA; pur operon Bacillus_subtilis + pbuG; pur operon; yxjA; xpt, pbuX operon Bacillus_thuringiensis + pbuG; pur operon; xpt, guaA NC_008600 274659 276448 pbuX operon; GntR; NC_005957 266593 268382 nupC Bacillus_weihenstephanensis + uraA; pur operon; guaA NC_010184 262234 264023 xpt, pbuX operon; GntR; nupC Bdellovibrio_bacteriovorus putative secreted nuclease; hypothetical protein Clostridium_acetobutylicum + uraA; xptpbuX operon guaB, guaA NC_003030 2824936 2821591 operon Clostridium_beijerinckii + uraA; xptpbuX operon guaB, guaA NC_009617 398946 402351 operon Clostridium_botulinum + uraA; pur operon; pbuX, guaB, guaA NC_009495 3506948 3503782 xpt operon; uraA, apt operon NC_010520 3584820 3581653 operon; uraA NC_009697 3482936 3479770 NC_009698 3380044 3376878 NC_010516 3567858 3564691 NC_010674 396433 399735 NC_010723 391849 395150 NC_009699 3600582 3597415 Clostridium_difficile + uraA; xpt; pbuX guaA NC_009089 256232 258074 Clostridium_kluyveri + uraA Clostridium_novyi + xpt, pbuG guaB, guaA NC_008593 2143257 2139825 operon Clostridium_perfringens + xpt; uraA guaB, guaA NC_003366 2618404 2615047 operon NC_008261 2822240 2818882 NC_008262 2482608 2479246 Clostridium_tetani + guaB, guaA NC_004557 2551375 2549717 operon Desulfitobacterium_hafniense + purL Desulfotomaculum_reducens + add; uraA, purE operon; uraA; adenylosuccinate lyase Enterococcus_faecalis + xpt, pbuX, pur operon Exiguobacterium_sibiricum + xpt, pbuX operon; uraA; guaA NC_010556 459675 461411 pur operon Fusobacterium_nucleatum pur operon Geobacillus_kaustophilus + pbuG; pur operon guaA NC_006510 272489 274168 Geobacillus_thermodenitrificans + pbuG; pur operon; xpt, guaA NC_009328 252216 253897 pbuX operon Lactobacillus_acidophilus + uraA, pbuG operon; xpt, pbuX operon Lactobacillus_brevis + uraA Lactobacillus_delbrueckii_bulgaricus + xpt, pbuX operon Lactobacillus_fermentum + uraA Lactobacillus_gasseri + uraA Lactobacillus_helveticus + uraA Lactobacillus_johnsonii + uraA Lactobacillus_plantarum + uraA; add Lactobacillus_reuteri + uraA Lactobacillus_salivarius + xpt, pbuX Lactococcus_lactis + xpt, pbuX Leuconostoc_mesenteroides + uraA Listeria_innocua + xpt, pbuX operon; uraA Listeria_monocytogenes + xpt, pbuX operon; uraA Listeria_welshimeri + xpt, pbuX operon; uraA Lysinibacillus_sphaericus + uraA, pbuG operon; pur guaA NC_010382 217326 219120 operon; xpt, pbuX operon; pbuE Oceanobacillus_iheyensis + xpt, pbuX operon guaA NC_004193 760489 762329 Oenococcus_oeni + guaB; xpt, pbuX operon Pediococcus_pentosaceus + uraA Shewanella_halifaxensis uraA Shewanella_pealeana uraA Staphylococcus_aureus + xpt, pbuX, guaB, NC_002951 460076 465308 guaA operon NC_009632 466145 471377 NC_009487 466075 471307 NC_003923 410563 415795 NC_009782 430774 436006 NC_002758 430771 436003 NC_002745 430814 436046 NC_007795 378013 383245 NC_009641 425751 430983 NC_007622 383514 388746 NC_007793 436156 441388 NC_010079 436051 441283 NC_002952 441067 446299 NC_002953 409208 414440 Staphylococcus_carnosus + xpt, pbuX, guaB, NC_012121 46344 51725 guaA operon Staphylococcus_epidermidis + xpt, pbuX, guaB, NC_004461 2433030 2427604 guaA operon NC_002976 54833 60259 Staphylococcus_haemoliticus + xpt, pbuX, guaB, NC_007168 2598923 2593514 guaA operon Staphylococcus_saprophyticus + xpt, pbuX, guaB, NC_007350 2407693 2403978 guaA operon Streptococcus_agalactiae + xpt, pbuX operon Streptococcus_pneumoniae + xpt, pbuX operon Streptococcus_pyogenes + xpt, pbuX operon Streptococcus_thermophilus + xpt, pbuX operon Thermoanaerobacter_pseudethanolicus + uraA guaA NC_010321 1760593 1758904 Thermoanaerobacter_X514 + uraA guaA NC_010320 528317 531544 Thermoanaerobacter_tengcongensis + uraA/purE Vibrio_sp uraA Vibrio_splendidus uraA
(110)
Example 5
Preclusion from Ribosylation of the Compounds of the Present Invention
(111) Shown schematically in
(112) The effects of several antimicrobial compounds on the growth and survival of various strains of E. coli are summarized in Table 4 and presented in the form of minimum inhibitory concentrations (MIC, in g/mL). Briefly, minimal inhibitory concentration of compounds 1.01 and 1.13 was determined using the microdilution method in 96-well microplates. Bacteria were inoculated at 10.sup.5 CFU/mL and incubated at 37 C. for 24 h in Muller-Hinton cation adjusted media. Then OD.sub.595 nm was read on microplate reader.
(113) TABLE-US-00004 TABLE 4 Minimum inhibitory concentrations (MIC) of various antibiotic compounds against E. coli strains E. coli E. coli E. coli Compounds ATCC35695 AcrAB/ Imp Compound 1.01 >5000 >5000 >5000 Compound 1.13 >5000 >5000 >5000 Vancomycin >128 >128 0.25 Erythromicin 128 1.0-4.0 0.125-1.0 All concentrations are in g/mL.
(114) The results depicted in Table 4 above show that the guanine switch-less bacterium Escherichia coli is not inhibited by compounds 1.01 and 1.13 while the E. coli strain is highly permeable (strain E. coli Imp) to large antibiotic molecules like erythromycin and vancomycin or lacks the efflux pump AcrAB (strain E. coli AcrAB/) that is able to pump toxic molecules out of the cell. These results demonstrate the antimicrobial specificity of the compounds of the present invention (e.g., compounds 1.01 and 1.13) for bacteria that possess a guanine riboswitch controlling the expression of guaA. These results also demonstrate that the compounds of the present invention (e.g., compounds 1.01 and 1.13) are not broadly toxic to other bacteria lacking a guanine riboswitch controlling the expression of guaA. Thus, the compounds of the present invention allow for selective treatment of microbial pathogens affecting animals and humans. For example, compound 1.01 is sufficiently distinct from the natural guanine riboswitch ligand (guanine), to prevent incorporation of compounds into cellular nucleosides, nucleotides or nucleic acids, thus preventing toxicity for the mammalian or animal host in which such a compound is used for therapeutic intervention.
Example 6
Minimal Inhibitory Concentrations and Bactericidal Activities of Compounds of the Present Invention
(115) As disclosed herein, the compounds of the present invention (e.g., compounds 1.01 and 1.13) are able to specifically and selectively inhibit the growth and/or kill bacterial species that possess the guanine riboswitch controlling the expression of guaA such as S. aureus and C. difficile. The compounds of the present invention demonstrate an anti-bacterial effect on prototypical bacterial strains or bacterial stains causing persistent and chronic bovine mastitis in vitro (e.g., in culture media or milk) and in vivo (e.g., in mice mammary gland infections). Furthermore, the anti-bacterial effect of the compounds of the present invention (e.g., compound 1.01) is as rapid as that of the well-known antibiotic ciprofloxacin that is used in human and veterinary medicine.
(116) The effect of various antibiotic compounds, including compounds 1.13 and 1.01, on the growth of S. aureus strain ATCC 29213 in vitro in Muller-Hinton cation adjusted media was tested and is shown in
Example 7
Bactericidal Activity of Compound 1.01 on Various S. Aureus Strains
(117) The bactericidal activity of compound 1.01 on various strains of S. aureus (including 3 chronic strains and 3 prototypical strains) was determined. These strains include prototypical S. aureus strains ATCC 29213, Newbould 305, SHY97-3906 as well as S. aureus strains isolated from persistent and chronic bovine mastitis cases (isolates #3, #557, #1290). C. difficile strain Cd6 was also examined. The bacteria were inoculated at 10.sup.5 CFU/mL in Muller-Hinton cation adjusted media in absence or presence of 600 g/mL compound 1.01 and grown for 4 hours. The results are summarized in Table 5 below and are expressed as the mean fold reduction of CFU/mL (i.e., the number of CFU/mL of control bacteria divided by the number of CFU/mL of treated bacteria). These results indicate that compound 1.01 has a significant anti-bacterial effect against all the strains tested, with the highest effect being against S. aureus strain ATCC 29213.
(118) TABLE-US-00005 TABLE 5 Bactericidal activity of compound 1.01 on S. aureus and C. difficile strains Fold reduction of CFU/mL (control/treated) Strains Mean (log10) SD S. aureus ATCC 29213 6.67 0.58 S. aureus Newbould 305 4.86 1.42 S. aureus Chronic #3 5.11 1.25 S. aureus Chronic #1290 5.42 1.56 S. aureus Chronic #557 4.38 1.86 S. aureus SHY97-3906 6.35 1.26 C. difficile (Cd6) 5.42 1.02
Example 8
Mode of Action of Compounds of the Present Invention
(119) To verify the specificity and explore the mechanism of action of compound 1.01, a transcriptomic microarray analysis was used to follow the expression of more then 500 S. aureus genes. Compound 1.01 was used alone or in combination with GMP and the results are shown in
(120)
Example 9
Inhibitory Effect of Compounds of the Present Invention During In Vivo Infection in Mice and Cows
(121) The compounds of the present invention (e.g., compound 1.01) are able to inhibit the growth and/or kill microbial pathogens possessing the guanine riboswitch controlling the expression of guaA during infection of the mammary glands in the mouse.
(122) The efficacy of compound 1.01 in treating a S. aureus infection of the mouse mammary glands was examined. Briefly, mouse mammary glands were infected with 100 CFU of S. aureus. Four hours after the inoculation, the glands were treated with vehicle (PBS), 10, 50 or 100 g/gland of compound 1.01. Six hours later, glands were excised, homogenized and plated on Mueller-Hinton agar. CFU were then counted after 18 hours at 35 C. The results of these studies are shown in
Example 10
Compounds of the Present Invention Prevent Rapid Development of Bacterial Resistance in S. Aureus
(123) The present invention relates to the discovery that the compounds of the present invention are not only able to inhibit the expression of the gene guaA when it is under riboswitch control, but can also prevent development of bacterial resistance. Specifically,
Example 11
Illustrative Compounds of the Present Invention
(124) The present invention relates to a number of small molecule compounds having a selective antimicrobial activity on microbial pathogens containing guaA under control of the guanine riboswitch but having no substantial antimicrobial activity against microorganisms lacking a guaA-controlled by the guanine riboswitch. The compounds of the present invention fit the structural requirements for binding to the guanine riboswitch binding site and yet are chemically unable to be ribosylated by the targeted pathogens. The latter is meant at preventing incorporation of the compounds into cellular nucleosides, nucleotides or nucleic acids, and thus preventing broad, non-specific toxicity for the mammalian or animal host in which the present compounds are used for therapeutic intervention.
(125) Representative structures of the compounds of the present invention derived from formulas 1.0, 2.0 and 3.0 are shown in
Example 12
Effect of Compounds of the Present Invention on the Treatment of Infections
(126) In addition to intramammary infections, S. aureus is a pathogen also found in skin and skin structure infections, respiratory tract infections, blood and urine (Garau et al., 2009; Araki et al, 2002; Corey, 2009). Clostridium infections in humans such as those caused by C. difficile, C. botulinum, C. tetani and C. perfringens (Leffler and Lamont, 2009; Khanna, 2008), in poultry by C. perfringens (Van Immerseel et al., 2004) and in pigs are also of importance and the colonization of pigs by MRSA is also a problem linked to human health (Songer and Uzal, 2005; Khanna et al., 2008). The invention can therefore also be used for treatment of a variety of clostridial or staphylococcal infections occurring at body sites other than the mammary glands in animals and in humans.
Example 13
Aspects Relating to Synthesis of Compounds of the Present Invention
(127) ##STR00037##
(128) Analogue A can be synthesized from chloronitropyrimidine (A1) (Patel et al., 2007). Synthesis of A1 as described by Sircar et al., 1986 to give the precursor aminomercaptopyrimidine (A2). The latter can then be derivatized with ethyl oxoacetate to give the desired product A (RH). This scheme will allow additional functionalization as well.
(129) ##STR00038##
(130) Pyridopyrimidine B can be synthesized following a procedure reported by Urleb et al., 1990 for a similar analogue. Pterin (XN) is commercially available.
(131) ##STR00039##
(132) Oxazole derivative D was synthesized similarly to the synthesis of a methylated analogue (Miller et al, 2002).
(133) ##STR00040##
(134) Pyrrole derivative E can be synthesized as described by Taylor and Young, 1995.
Example 14
Bio-Informatics Search of Guanine Riboswitches
(135) Natural riboswitches contain aptamer domains which are typically very conserved in their sequence and structure given that they must bind cellular metabolites that are conserved through evolution. These aptamer domains exhibit a conserved scaffold of base-paired helices that organize the overall fold of each aptamer. The identities of each base in these helices most often reflect their need to be base-paired but in contrast, the identities of bases that are in direct contact with the bound metabolite are very highly conserved given precise role in the formation of the ligand binding site. Thus, it is possible to define a consensus sequence, representing all possible aptamer sequences, which can be used to perform computational searches in biological sequence databases. For instance, by translating the consensus sequence into an algorithm search, the software RNAmotif (Macke et al., 2001) can screen for all aptamer occurrences in the RefSeq microbial database (Pruitt et al., 2005). The output of the software gives genomic locations of all aptamer found. There is already a subgroup of aptamer sequences for various riboswitch families that have been retrieved by the scientific community that can be found at the Rfam database (Griffiths-Jones et al., 2005), but because it was observed that it is only partially complete, it was found preferable to build a database using RNAmotif.
(136) Below is an example of a consensus sequence used for the bioinformatic search. The meaningfulness of the characters and commands described in the consensus are very well described in the original RNAmotif article (Macke et al., 2001). This corresponds to possible guanine riboswitches that could adopt a similar structures than the known guanine riboswitch.
(137) TABLE-US-00006 parms wc += gu; descr h5( minlen=4, maxlen=10, mispair=0, ends=mm) ss ( seq={circumflex over ()}UA.\{0,1\}$) h5( minlen=5, maxlen=8, mispair=3, ends=mm) ss ( seq={circumflex over ()}.\{0,1\}AU.\{0,3\}GG$ ) h3 ss ( seq={circumflex over ()}GNNNCUAC$ ) h5( minlen=5, maxlen=8, mispair=2, ends=mm ) ss ( seq={circumflex over ()}CC.\{0,3\}A.\{0,1\}$) h3 ss ( seq={circumflex over ()}N\{0,1\}C.\{0,1\}$) h3 H, Hoogsteen face; MI, mutual information; nt, nucleotides;
Example 15
Ability of Compounds of the Invention to Inhibit S. Aureus Growth
(138) As disclosed herein, the compounds of the present invention (e.g., compounds 1.16, 2.02 and 2.17) (1.16 and 2.17 were purchased from Sigma-Aldrich and Toronto Research Chemical respectively) are able to specifically and selectively inhibit the growth and/or kill bacterial species that possess the guanine riboswitch controlling the expression of guaA such as S. aureus. As reported in Table 6 (below), compounds 1.16, 2.02 and 2.17 are able to bind guanine riboswitch and to inhibit S. aureus growth with a MIC of 0.128, 1.024 and 0.128 mg/mL, respectively.
(139) The effect of the antibiotic compounds of formula 2.0, including compounds 2.02 and 2.17, on the growth of S. aureus strain ATCC 29213 in vitro in Muller-Hinton cation adjusted media allow to confirm the efficiency of compounds of general formula 2.0 to achieve antibiotic activity. For each compound, antibiotic activity was also tested on E. coli strain ATCC 35695 and no antimicrobial activity of the compounds (1.16, 2.02 and 2.17) was observed. The MICs were determined using the microdilution method in 96-well microplates and the bacteria were incubated at 37 C. for 24 h following inoculation.
(140) As required, the compounds of Table 6 present no ribosylation site, so this will prevent their incorporation in DNA and their mutagenic potential.
(141) TABLE-US-00007 TABLE 6 Description of the properties of various guanine riboswitch ligands Guanine S. aureus riboswitch growth Appropriate Compound Structure binding inhibition Ribosylation properties 1.16
Example 16
Route to Afford Compound 2.02: 5-Amino-6H-oxazolo[5,4-d]pyrimidin-7-one
(142) ##STR00044##
5-Formamino-2-amino-4,6-dihydroxypyrimidine
(143) ##STR00045##
(144) Sodium pieces (242 mg, 10.5 mmol) were added to MeOH (20 mL). After all the sodium had reacted, guanidine hydrochloride (1.00 g, 10.5 mmol) was added and the reaction mixture was stirred for 15 min. The reaction mixture was then heated to reflux and during this time, diethylacetamido malonate (2.13 g, 10.5 mmol) was added. After 2 h, more MeOH (20 mL) was added and the reaction mixture was refluxed overnight. The reaction mixture was then cooled to rt, filtered, washed with MeOH and chloroform and dried under vacuum at 50 C. The resulting solid was dissolved in water (25 mL) and was precipitated by the addition of 50% hydrochloric acid. The solution was filtered and the solid was washed with water and acetone and was finally dried under vacuum at 50 C. to afford compound 7 as a white solid (900 mg, 50%). mp >300 C. .sup.1H NMR (300 MHz, D.sub.2O) (ppm) 8.19 (s, 1H). .sup.13C NMR (75.5 MHz, DMSO-d.sub.6) (ppm) 174.9, 168.2, 154.5, 92.6, 21.9.
5-Amino-6H-oxazolo[5,4-d]pyrimidin-7-one
(145) ##STR00046##
(146) Compound 7 (125 mg, 0.73 mmol) was dissolved in 12 M H.sub.2SO.sub.4 (1.50 mL) using an ultrasonic bath. The dark yellow solution was stirred at rt overnight. THF (5 mL) was then added at 0 C. to form a yellow precipitate. The mixture was stored at 4 C. for 2 h and was centrifuged to obtain compound 8 as a yellow solid. .sup.1H NMR (300 MHz, DMSO-d.sub.6) (ppm) 8.71 (br s, 1H), 7.45 (br s, 1H). .sup.13C NMR (75.5 MHz, DMSO-d.sub.6) (ppm) 158.6, 152.3, 84.2. LRMS (m/z, relative intensity) 137 (MH.sup.+NH.sub.3, 100), 153 (MH.sup.+, 50), 159 (MNa.sup.+NH3, 90). HRMS calculated for C.sub.5H.sub.6N.sub.4O.sub.3: 153.0413, found: 152.9617.
(147) Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.
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