Patent Application
20050215490
This invention relates to methods of treating bacterial infections in a mammal using a compound of using a polyene polyketide of Formula I or its pharmaceutically acceptable salts.
Microbial natural products have been a reliable source of antimicrobial drugs. Streptomycin, erythromycin, tetracycline, pristinamycin, vancomycin and lincomycin are examples of antibiotics produced by microorganisms. However, the discovery of new antibiotics has dropped significantly over the past few decades. At the same time, problems related to resistance to current antibiotics by severe pathogens such as VRE and MRSA have been on the rise. According to the U.S. Center for Disease Control and Prevention, MRSA infections are common causes of hospital acquired infections, and a limited number of drugs remain effective against these infections. The number of hospital-acquired infections is growing as hospitals are increasingly treating patients suffering from compromised immune systems caused by such things as more cancer patients and the associated chemotherapy treatments, aging of the population, and increasing numbers of surgical operations such as organ transplants that require immunosuppressants. From the foregoing, there is a need for new antibiotic therapeutics that have higher efficacy, specificity or reduced side effects.
The invention encompasses a method of inhibiting bacterial infection, the method comprising contacting the bacterial cells with a therapeutically effective amount of a compound selected from Compounds 1, 2, 3, 4, 5, 6, 7 and pharmaceutically acceptable salts or prodrugs thereof:
such that growth of the bacterial infection is inhibited.
In another aspect the invention provides a method of inhibiting the growth of a bacterial infection, the method comprising contacting a bacterial cell with a therapeutically effective amount of a compound of Formula I:
such that growth of the bacterial infection is inhibited;
wherein,
In one embodiment of this aspect, A of the compound is —C(NH)NH2. Alternatively, in another embodiment, A of the compound is H. Alternatively, in still another embodiment, A of the compound is —C(NH)NHC(O)CH3. Alternatively, in yet another embodiment, A of the compound is
In another embodiment of the compound R4 is CH3. Alternatively, in yet another embodiment, R4 of the compound is H. The present invention further contemplates an embodiment of this aspect in which Z is
In a sub-class of this embodiment, R6, R7 and R8 are each H, and R5 is COOH or CO2CH3. Alternatively, the invention further contemplates an embodiment of this aspect in which Z is OH. The invention further contemplates an embodiment of this aspect in which D is
In a subclass of this embodiment R11 is H. The invention further contemplates an embodiment of this aspect in which D
In a subclass of this embodiment R11 is CH3. Alternative, in another embodiment of this aspect D is OH.
Specific embodiments of compounds of Formula I useful to inhibit the growth of bacterial infection are as follows:
or a pharmaceutically acceptable salt or prodrug of any one of Compounds 1-28.
In a further aspect, the invention relates to a method of inhibiting the growth of a bacterial infection, the method comprising contacting a bacterial cell with a pharmaceutical composition comprising of a compound of Formula I as defined above, or a pharmaceutically acceptable salt or prodrug thereof, with a pharmaceutically acceptable carrier, such as growth of the bacterial infection is inhibited. Specific embodiments of this aspect include a method of inhibiting the growth of bacterial infection, the method comprising contacting a bacterial cell with a phamaceutical composition comprising a compound of any one of Compounds 1-28 or a pharmaceutically acceptable salt or prodrug thereof, and a pharmaceutically acceptable carrier such that growth of the bacterial infection is inhibited.
The invention further encompasses a method of treating a bacterial infection in a mammal, comprising the step of administering to the mammal a therapeutically effective amount of a compound selected from the Compounds 1, 2, 3, 4, 5, 6, 7 and pharmaceutically acceptable salts or prodrugs thereof:
such that the bacterial infection is treated.
In another aspect, the invention provides a method of treating a bacteria infection in a mammal, comprising the step of administering to the mammal a therapeutically effective amount of a therapeutically effective amount of a compound of Formula I:
such that the bacterial infection is treated;
wherein,
Embodiment of Formula I useful in treating bacterial infection in a mammal are compounds of Formula I wherein A of the compound is —C(NH)NH2. Alternatively, in another embodiment, A of the compound is H. Alternatively, in still another embodiment, A of the compound is —C(NH)NHC(O)CH3. Alternatively, in yet another embodiment, A of the compound is
In another embodiment of the compound R4 is CH3. Alternatively, in yet another embodiment, R4 of the compound is H. The present invention further contemplates an embodiment of this aspect in which Z is
In a sub-class of this embodiment, R6, R7 and R8 are each H, and R5 is COOH or CO2CH3. Alternatively, the invention further contemplates an embodiment of this aspect in which Z is OH. The invention further contemplates an embodiment of this aspect in which D is
In a subclass of this embodiment R11 is H. The invention further contemplates an embodiment of this aspect in which D is
In a subclass of this embodiment R11 is CH3. Alternative, in another embodiment of this aspect D is OH. Specific compounds of Formula I useful to treat bacterial infection in a mammal are compound 1-28.
In a further aspect, the invention relates to a method of treating bacterial infection in a mammal, the method comprising contacting a bacterial cell with a pharmaceutical composition comprising of a compound of Formula I as defined above, or a pharmaceutically acceptable salt or prodrug thereof, with a pharmaceutically acceptable carrier, such that the bacterial infection is treated. Specific embodiments of this aspect include a method of treating bacterial infection in a mammal, the method comprising contacting a bacterial cell with a phamaceutical composition comprising a compound of any one of Compounds 1-28 or a pharmaceutically acceptable salt or prodrug thereof, and a pharmaceutically acceptable carrier such that the bacterial infection is treated.
In one embodiment, the bacterial infection is from a bacteria selected from the group consisting of Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Klebsiella pneumoniae, Enterobacter spp., Proteus spp., Pseudomonas aeruginosa, E. coli, Serratia marcesens, Staphylococcus aureus, Coagulase negative Staphylococcus, Haemophilus infuenzae, Bacillus anthracis, Mycoplasma pneumoniae, and Staphylococcus epidermidis. In another embodiment, the bacterial infection is caused by Gram-positive bacteria. In a further embodiment, the Gram-positive bacteria is a Staphylococcus or Enterococcus species.
According to one embodiment of this aspect, the compound is administered orally. In another embodiment, the compound is administered intravenously. In still another embodiment, the compound is administered intraperitoneally.
The present invention relates to methods of inhibiting the growth of bacterial cells. The method comprtises contacting bacterial cells with a polyene polyketide compound of Formula I, as exemplified herein as Compound 1, Compound 2 and Compound 7, which were isolated from strains of actinomycetes, Amycolatopsis sp. such as Amycolatopsis orientalis ATCC 43491, Amycolatopsis orientalis IDAC 220604-01, or a mutant or a variant thereof. Amycolatopsis orientalis ATCC 43491 may be obtained from the American Type Culture Collection (ATCC) P.O. Box 1549, Manassas, Va. 20108 USA; Amycolatopsis orientalis IDAC 220604-01 may be obtained from the International Depositary Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2.
The present invention further related to methods of treating a bacterial infection in a mammal, the method comprising administering a therapeutically effective amount of a polyene polyketide compound of the invention, such that the bacterial infection is treated. Compound 1, Compound 2 and Compound 7 are useful as a pharmaceutical, in particular for use as in inhibitor of bacterial cell growth. Alternatively a compound of Formula I can be used. Accordingly, certain aspects of the present invention relate to use of the polyene polyketide compounds of the present invention in to inhibit bacterial growth, and use of the polyene polyketides of the invention in the preparation of a medicament to treat bacterial infection.
The invention further related to pharmaceutically acceptable salts and derivatives of the compounds 1-7 and to methods for obtaining such compounds. One method of obtaining the compounds is by cultivating Amycolatopsis orientalis ATCC™ 43491, or a mutant or a variant thereof, under suitable Amycolatopsis sp. culture conditions preferably using the fermentation protocol described herein below.
The invention also relates to pharmaceutical compositions comprising a compound selected from 1-28 and its pharmaceutically acceptable salts and derivatives. Accordingly, certain aspects of the present invention relate to use to inhibit bacterial growth of a pharmaceutical compositions comprising a polyene polyketide compound of the present invention together with a pharmaceutically acceptable carrier, and use in the preparation of medicament to treat bacterial infection of a pharmaceutical compositions comprising a polyene polyketide compound of the present invention together with a pharmaceutically acceptable carrier.
Accordingly, certain aspects of the present invention relate to pharmaceutical compositions comprising the polyene polyketide compounds of the present invention together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit bacterial growth, and methods of using the pharmaceutical compositions to treat bacterial infections.
Molecular terms, when used in this application, have their common meaning unless otherwise specified. The meaning of certain terms and phrases used in the specification are provided below. Abreviations, as used herein, have the following meaning: Me refers to methyl (CH3), Et refers to ethyl (CH2CH3), Pr refers to n-propyl (CH2CH2CH3) and Ac refers to acetyl (C(O)CH3).
The term alkyl refers to a linear or branched hydrocarbon groups. Examples of alkyl groups include, without limitation, methyl, ethyl, n-propyl, isopropyl, n-butyl, pentyl, hexyl, heptyl, cyclopentyl, cyclohexyl, cyclohexymethyl, and the like. Alkyl may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro,.thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, oxo, guanidino and formyl.
The term alkenyl refers to linear, branched or cyclic hydrocarbon groups containing at least one carbon-carbon double bond. Examples of alkenyl groups include, without limitation, vinyl, 1-propene-2-yl, 1-butene-4-yl, 2-butene-4-yl, 1-pentene-5-yl and the like. Alkenyl may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, alkoxy, aryloxy, sulfinyl, sulfonyl, formyl, oxo and guanidino. The double bond portion(s) of the unsaturated hydrocarbon chain may be either in the cis or trans configuration.
The term cycloalkyl or cycloalkyl ring refers to a saturated or partially unsaturated carbocyclic ring in a single or fused carbocyclic ring system having from three to fifteen ring members. Examples of cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclohexyl, and cycloheptyl. Cycloalkyl may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.
The term heterocyclyl, heterocyclic or heterocyclyl ring refers to a saturated or partially unsaturated ring containing one to four hetero atoms or hetero groups selected from O, N, NH, NRx, PO2, S, SO or SO2 in a single or fused heterocyclic ring system having from three to fifteen ring members. Examples of a heterocyclyl, heterocyclic or heterocyclyl ring include, without limitation, morpholinyl, piperidinyl, and pyrrolidinyl. Heterocyclyl, heterocyclic or heterocyclyl ring may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, oxo, thiocarbonyl, imino, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.
The term amino acid refers to any natural amino acid, all natural amino acids are well known to a person skilled in the art.
The term halo is defined as a bromine, chlorine, fluorine or iodine.
The term aryl or aryl ring refers to common aromatic groups having “4n+2” electrons, wherein n is an integer from 1 to 3, in a monocyclic or conjugated polycyclic system and having from five to fifteen ring atoms. Aryl ring may include from 1 to 3 heteroatoms such as nitrogen, oxygen and sulphur atoms. Examples of aryl include, without limitation, phenyl, naphthyl, biphenyl, terphenyl, furyl, pyrrollyl, thienyl, pyridyl, oxazolyl, imidazolyl, pyrazolyl and indolyl groups. Aryl may optionally be substituted with one or more substituent group selected from acyl, amino, acylamino, acyloxy, azido, alkythio, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.
The compounds of the present invention can possess one or more asymetric carbon atoms and can exist as optical isomers forming mixtures of racemic or non-racemic compounds. The compounds of the present invention are useful as a single isomer or as a mixture of stereochemical isomeric forms. Diastereoisomers, i.e., nonsuperimposable stereochemical isomers, can be separated by conventional means such as chromatography, distillation, crystallization or sublimation. The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes.
The invention embraces isolated compounds. An isolated compound refers to a compound which represents at least 10%, 20%, 50% and 80% of the compound of the present invention present in a mixture, provided that the mixture comprising the compound of the invention-has demonstrable (i.e. statistically significant) biological activity including antimicrobial activity when tested in conventional biological assays known to a person skilled in the art.
As used herein, the term “treatment” refers to the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disorder, e.g., a disease or condition, a symptom of disease, or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of disease, or the predisposition toward disease.
As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a compound of the invention and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of polyene polyketide effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.
The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
The term “pharmaceutically acceptable salts” include acid addition salts and base addition salts. The nature of the salt is not critical, provided that it is pharmaceutically-acceptable. Without being limited, examples of acid addition salts include hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulphuric, phosphoric, formic, acetic, citric, tartaric, succinic, oxalic, malic, glutamic, propionic, glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic, algenic, β-hydroxybutyric, malonic, galactantic, galacturonic acid and the like. Suitable pharmaceutically-acceptable base addition salts of compounds of the invention include, but are not limited to, metallic salts made from aluminium, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine, procaine and the like. Additional examples of pharmaceutically acceptable salts are listed in Berge S M et al., Journal of Pharmaceutical Sciences, (1977) Vol. 66 no 1, pp. 1-19. All of these salts may be prepared by conventional means form the corresponding compounds of Formula I by treating with the appropriate acid or base.
Unless otherwise indicated, all numbers expressing quantities of ingredients and properties such as molecular weight, reaction conditions, MIC and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims are approximations. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of significant figures and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the examples, tables and figures are reported as precisely as possible. Any numerical values may inherently contain certain errors resulting from variations in experiments, testing measurements, statistical analysis and such.
In one aspect of this embodiment the invention relates to novel polyene polyketides, referred to herein as Compounds 1-7:
or, to a pharmaceutically acceptable salt or prodrug of any of Compounds 1 to 7. Compounds 1 to 7 may be characterized by any one or more of their physicochemical and spectral properties given below, such as mass, UV, and NMR spectroscopic data.
In another aspect the invention relates to a novel class of polyene polyketides represented by Formula I:
wherein,
In one embodiment the invention provides compounds of Formula I, wherein A is —C(NH)NH2; and all other groups are as previously defined; or a pharmaceutically acceptable salt or prodrug thereof.
In a further embodiment of the invention provides compounds of Formula I, wherein. A is H; and all other groups are as previously defined; or a pharmaceutically acceptable salt or prodrug thereof.
In a further embodiment the invention provides compounds of Formula I, wherein A is —C(NH)NHC(O)CH3; and all other groups are as previously defined; or a pharmaceutically acceptable salt or prodrug thereof.
In a further embodiment the invention provides compounds of Formula I, wherein A is
and all other groups are as previously defined; or a pharmaceutically acceptable salt or prodrug thereof.
In a further embodiment the invention provides compounds of Formula I, wherein R4 is CH3; and all other groups are as previously defined; or a pharmaceutically acceptable salt or prodrug thereof.
In a further embodiment the invention provides compounds of Formula I, wherein R4 is H; and all other groups are as previously defined; or a pharmaceutically acceptable salt or prodrug thereof.
In a further embodiment the invention provides compounds of Formula I, wherein Z is
and all other groups are as previously defined; or a pharmaceutically acceptable salt or prodrug thereof. In a subclass of this embodiment R6, R7 and R8 are each H, and R5 is COOH, all other groups are as previously defined; or a pharmaceutically acceptable salt or prodrug thereof.
In a further embodiment the invention provides compounds of Formula I, wherein Z is
and all other groups are as previously defined; or a pharmaceutically acceptable salt or prodrug thereof. In a subclass of this embodiment R6, R7 and Ra are each H, and R5 is CO2CH3, all other groups are as previously defined; or a pharmaceutically acceptable salt or prodrug thereof.
In a further embodiment the invention provides compounds of Formula I, wherein Z is OH and all other groups are as previously defined; or a pharmaceutically acceptable salt or prodrug thereof.
In a further embodiment the invention provides compounds of Formula I, wherein D is
and all other groups are as previously defined or a pharmaceutically acceptable salt or prodrug thereof. In a subclass of this embodiment R11 is H and all other groups are as previously defined; or a pharmaceutically acceptable salt or prodrug thereof.
In a further embodiment the invention provides compounds of Formula I, wherein D is
and all other groups are as previously defined; or a pharmaceutically acceptable salt or prodrug thereof. In a subclass of this embodiment R11 is CH3 and all other groups are as previously defined; or a pharmaceutically acceptable salt or prodrug thereof.
In another embodiment the invention provides compounds of Formula I, wherein D is OH; and all other groups are as previously defined; or a pharmaceutically acceptable salt or prodrug thereof.
The following are exemplary compounds of the invention:
or a pharmaceutically acceptable salt or prodrug of any one of Compound 1-28. Certain embodiments may exclude one or more of the compounds of Formula I. The compounds of the invention may be formulated into pharmaceutical compositions comprised compounds of Formula I in combination with a pharmaceutical acceptable carrier as discussed in Section IV below.
In one embodiment, compounds 1-7 may be obtained by cultivating a strain of actinomycetes, Amycolatopsis sp. such as Amycolatopsis orientalis ATCC 43491, Amycolatopsis orientalis IDAC 220604-01, or a mutant or a variant thereof. Amycolatopsis orientalis ATCC 43491 may be obtained from the American Type Culture Collection (ATCC) P.O. Box 1549, Manassas, Va. 20108 USA; Amycolatopsis orientalis IDAC 220604-01 may be obtained from the International Depositary Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2. The deposit of Amycolatopsis orientalis IDAC 220604-01 was made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Purposes of Patent Procedure. The deposited strain will be irrevocably and without restriction or conditions released to the public upon the issuance of a patent. The deposited strains are provided merely as a convenience to those skilled in the art and are not an admission that a deposit is required for enablement, such as that required under 35 U.S.C.§112. Indeed it is to be understood that the present invention is not limited to use of a particular strain of Amycolatopsis orientalis. Rather, the invention contemplates the use of other Formula I producing organisms, such as mutant or variants of Amycolatopsis orientalis ATCC 43491 or Amycolatopsis orientalis IDAC 220604-01 that can be derived by well known means such as X-ray irradiation, ultraviolet irradiation, treatment with nitrogen mustard, phage exposure, antibiotic selection and the like, or through the use of recombinant genetic engineering as described in co-pending parent application U.S. Ser. No. 10/937,379. In addition, the polyene polyketides of the invention may be biosynthesized by various microorganisms. Microorganisms that may synthesize the compounds of the present invention include but are not limited to bacteria of the order Actinomycetales, also referred to as actinomycetes. Non-limiting examples of members belonging to the genera of Actinomycetes include Nocardia, Geodermatophilus, Actinoplanes, Micromonospora, Nocardioides, Saccharothrix, Amycolatopsis, Kutzneria, Saccharomonospora, Saccharopolyspora, Kitasatospora, Streptomyces, Microbispora, Streptosporangium, Actinomadura. The taxonomy of actinomycetes is complex and reference is made to Goodfellow (1989) Suprageneric classification of actinomycetes, Bergey's Manual of Systematic Bacteriology, Vol. 4, Williams and Wilkins, Baltimore, pp 2322-2339, and to Embley and Stackebrandt, (1994), and The molecular phylogeny and systematics of the actinomycetes, Annu. Rev. Microbiol. 48, 257-289 (1994), for genera that may synthesize the compounds of the invention, incorporated herein in its entirety by reference.
Microorganisms producing polyene polyketides of the invention are cultivated in culture media containing known nutritional sources for actinomycetes, having assimilable sources of carbon, nitrogen plus optional inorganic salts and other known growth factors, at a pH of about 6 to about 9. Suitable media include, without limitation, growth media provided in Table 1. Microorganisms are cultivated at incubation temperatures of about 20° C. to about 40° C. for about 3 to about 40 days. The culture media inoculated with the polyene polyketide-producing microorganisms, may be aerated by incubating the inoculated culture media with agitation, for example shaking on a rotary shaker, or a shaking water bath. Aeration may also be achieved by the injection of air, oxygen or an appropriate gaseous mixture to the inoculated culture media during incubation. Following cultivation the polyene polyketide compounds can be extracted and isolated from the cultivated culture media by techniques known to a skilled person in the art and/or disclosed herein, including for example centrifugation, chromatography, adsorption, filtration. For example, the cultivated culture media can be mixed with a suitable organic solvent such as n-butanol, n-butyl acetate and 4-methyl-2-pentanone, the organic layer can be separated for example, by centrifugation followed by the removal of the solvent, by evaporation to dryness or by evaporation to dryness under vacuum. The resulting residue can optionally be reconstituted with for example water, ethyl ether, ethanol acetate, methanol or a mixture thereof, and re-extracted in a two-phase system with a suitable organic solvent such as hexane, carbon tetrachloride, methylene chloride or a mixture thereof. After removal of the solvent, the compound can be further purified by the use of standard techniques such as chromatography.
The polyene polyketides-biosynthesized by microorganism may optionally be subjected to random and/or directed chemical modifications to form compounds that are derivatives or structural analogs. Such derivative or structural analogs having similar functional activity are within the scope of the present invention. Polyene polyketides may optionally be modified using methods known in the art and described in co-pending application U.S. Ser. No. 10/937,379.
Unless otherwise indicated, all the components are in gm/L
To a liter of media GA add: 10 ml KH2PO4 (0.5% solution); 80 ml CaCl2.2H2O (3.68% solution); 15 ml L-proline (20% solution); 100 ml TES buffer (5.73% solution, pH 7.2); 5 ml NaOH (1N solution).
The pH is adjusted as marked prior to the addition of CaCO3.
Solution of trace elements contains: ZnCl2 40 mg; FeCl3.6H2O (200 mg); CuCl2.2H2O (10 mg); (NH4)6MO7O24.4H2O (10 mg) per litre.
The invention relates to a pharmaceutical composition comprising a polyene polyketide, as described in Section II and a pharmaceutically acceptable carrier as described below. The pharmaceutical composition comprising the polyene polyketide is useful for treating a variety of bacterial infections.
The compounds of the present invention, or pharmaceutically acceptable salts or prodrugs thereof, can be formulated for oral, intravenous, intramuscular, subcutaneous, topical or parenteral administration for the therapeutic or prophylactic treatment of diseases, particularly bacterial infections. For oral or parental administration, compounds of the present invention can be mixed with conventional pharmaceutical carriers and excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, wafers and the like. The compositions comprising a compound of this present invention will contain from about 0.1% to about 99.9%, about 1% to about 98%, about 5% to about 95%, about 10% to about 80% or about 15% to about 60% by weight of the active compound.
The pharmaceutical preparations disclosed herein are prepared in accordance with standard procedures and are administered at dosages that are selected to reduce, prevent, or eliminate bacterial infection (See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. and Goodman and Gilman's the Pharmaceutical Basis of Therapeutics, Pergamon Press, New York, N.Y. for a general description of the methods for administering various antimicrobial agents for human therapy, the contents of which are incorporated herein by reference). The compositions of the present invention can be delivered using controlled (e.g., capsules) or sustained release delivery systems (e.g., bioerodable matrices). Exemplary delayed release delivery systems for drug delivery that are suitable for administration of the compositions of the invention (preferably of Formula I) are described in U.S. Pat. No. 4,452,775 (issued to Kent), U.S. Pat. No. 5,039,660 (issued to Leonard), U.S. Pat. No. 3,854,480 (issued to Zaffaroni).
The pharmaceutically-acceptable compositions of the present invention comprise one or more compounds of the present invention in association with one or more non-toxic, pharmaceutically-acceptable carriers and/or diluents and/or adjuvants and/or excipients, collectively referred to herein as “carrier” materials, and if desired other active ingredients. The compositions may contain common carriers and excipients, such as corn starch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid. The compositions may contain crosarmellose sodium, microcrystalline cellulose, sodium starch glycolate and alginic acid.
Tablet binders that can be included are acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (Providone), hydroxypropyl methylcellulose, sucrose, starch and ethylcellulose.
Lubricants that can be used include magnesium stearate or other metallic stearates, stearic acid, silicon fluid, talc, waxes, oils and colloical silica. Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring or the like can also be used. It may also be desirable to add a coloring agent to make the dosage form more esthetic in appearance or to help identify the product comprising a compound of the present invention.
For oral use, solid formulations such as tablets and capsules are particularly useful. Sustained released or enterally coated preparations may also be devised. For pediatric and geriatric applications, suspension, syrups and chewable tablets are especially suitable. For oral administration, the pharmaceutical compositions are in the form of, for example, a tablet, capsule, suspension or liquid. The pharmaceutical composition is preferably made in the form of a dosage unit containing a therapeutically-effective amount of the active ingredient. Examples of such dosage units are tablets and capsules. For therapeutic purposes, the tablets and capsules which can contain, in addition to the active ingredient, conventional carriers such as binding agents, for example, acacia gum, gelatin, polyvinylpyrrolidone, sorbitol, or tragacanth; fillers, for example, calcium phosphate, glycine, lactose, maize-starch, sorbitol, or sucrose; lubricants, for example, magnesium stearate, polyethylene glycol, silica or talc; disintegrants, for example, potato starch, flavoring or coloring agents, or acceptable wetting agents. Oral liquid preparations generally are in the form of an aqueous or oily solution, suspensions, emulsions, syrups or elixirs may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous agents, preservatives, coloring agents and flavoring agents. Examples of additives for liquid preparations include acacia, almond oil, ethyl alcohol, fractionated coconut oil, gelatin, glucose syrup, glycerin, hydrogenated edible fats, lecithin, methyl cellulose, methyl or propyl para-hydroxybenzoate, propylene glycol, sorbitol, or sorbic acid.
For intravenous (IV) use, compounds of the present invention can be dissolved or suspended in any of the commonly used intravenous fluids and administered by infusion. Intravenous fluids include, without limitation, physiological saline or Ringer's solution.
Formulations for parental administration can be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions. These solutions or suspensions can be prepared from sterile powders or granules having one or more of the carriers mentioned for use in the formulations for oral administration. The compounds can be dissolved in polyethylene glycol, propylene glycol, ethanol, corn oil, benzyl alcohol, sodium chloride, and/or various buffers.
For intramuscular preparations, a sterile formulation of compounds of the present invention or-suitable soluble salts forming the compound, can be dissolved and administered in a pharmaceutical diluent such as Water-for-Injection (WFI), physiological saline or 5% glucose. A suitable insoluble form of the compound may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g. an ester of a long chain fatty acid such as ethyl oleate.
For topical use the compounds of present invention can also be prepared in suitable forms to be applied to the skin, or mucus membranes of the nose and throat, and can take the form of creams, ointments, liquid sprays or inhalants, lozenges, or throat paints. Such topical formulations further can include chemical compounds such as dimethylsulfoxide (DMSO) to facilitate surface penetration of the active ingredient.
For application to the eyes or ears, the compounds of the present invention can be presented in liquid or semi-liquid form formulated in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints or powders.
For rectal administration the compounds of the present invention can be administered in the form of suppositories admixed with conventional carriers such as cocoa butter, wax or other glyceride.
Alternatively, the compounds of the present invention can be in powder form for reconstitution in the appropriate pharmaceutically acceptable carrier at the time of delivery. In another embodiment, the unit dosage form of the compound can be a solution of the compound or a salt thereof in a suitable diluent in sterile, hermetically sealed ampoules.
The amount of the compound of the present invention in a unit dosage comprises a therapeutically-effective amount of at least one active compound of the present invention which may vary depending on the recipient subject, route and frequency of administration. A recipient subject refers to a plant, a cell culture or an animal such as an ovine or a mammal including a human.
According to this aspect of the present invention, the novel compositions disclosed herein are placed in a pharmaceutically acceptable carrier and are delivered to a recipient subject (including a human subject) in accordance with known methods of drug delivery. In general, the methods of the invention for delivering the compositions of the invention in vivo utilize art-recognized protocols for delivering the agent with the only substantial procedural modification being the substitution of the compounds of the present invention for the drugs in the art-recognized protocols.
Likewise, the methods for using the claimed composition for treating cells in culture, for example, to eliminate or reduce the level of bacterial contamination of a cell culture, utilize art-recognized protocols for treating cell cultures with antibacterial agents with the only substantial procedural modification being the substitution of the compounds of the present invention for the agents used in the art-recognized protocols.
The compounds of the present invention provide a method for treating microbial infections. As used herein the term “unit dosage” refers to a quantity of a therapeutically-effective amount of a compound of the present invention that elicits a desired therapeutic response. As used herein the phrase “therapeutically-effective amount” means an amount of a compound of the present invention that prevents the onset, alleviates the symptoms, or stops the progression of a bacterial infection. The term “treating” is defined as administering, to a subject, a therapeutically-effective amount of at least one compound of the present invention, both to prevent the occurrence of a bacterial infection, or to control or eliminate a bacterial infection. The term “desired therapeutic response” refers to treating a recipient subject with a compound of the present invention such that a bacterial infection is reversed, arrested or prevented in a recipient subject.
The compounds of the present invention can be administered as a single daily dose or in multiple doses per day. The treatment regime may require administration over extended periods of time, e.g., for several days or for from two to four weeks. The amount per administered dose or the total amount administered will depend on such factors as the nature and severity of the infection, the age and general health of the recipient subject, the tolerance of the recipient subject to the compound and the type of the bacterial infection.
A compound according to this invention may also be administered in the diet or feed of a patient or animal. The diet for animals can be normal foodstuffs to which the compound can be added or it can be added to a premix.
The compounds of the present invention may be taken in combination, together or separately with any known clinically approved antibiotic to treat a recipient subject in need of such treatment.
In one embodiment, the present invention relates to a method of inhibiting bacterial growth. Compounds described herein can possess antbacterial activity. The compounds are effective in inhibiting bacterial infections in vivo. Examples of bacterial organisms that may be controlled by the compositions and methods of this invention include, but are not limited to the following organisms: Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium, Klebsiella pneumoniae, Enterobacter spp., Proteus spp., Pseudomonas aeruginosa, Escherichia coli, Serratia marcesens, Staphylococcus aureus, Haemophilus infuenzae, Bacillus anthracis, Mycoplasma pneumoniae, and Coagulase negative Staphylococcus including Staphylococcus epidermidis. In a preferred embodiment, pharmaceutical compositions and compounds of the invention will be useful in controlling or treating infections caused by Gram-positive bacteria. None limiting examples of Gram-positive bacterial growth that may be inhibited by the compounds and pharmaceutical compositions of the invention include Staphylococci and Enterococci. Compositions and methods will be useful for controlling, or reducing the advancement, severity or effects of nosocomial or non-nosocomial infections. Examples of nosocomial uses include, but are not limited to, urinary tract infections, pneumonia, surgical wound infections, bacteremia and therapy for febrile neutropenic patients. Examples of non-nosocomial uses include but are not limited to urinary tract infections, pneumonia, prostatitis, skin and soft tissue infections and intra-abdominal infections.
The term “inhibition” when used in conjunction the antibacterial methods refers to a suppression, killing, stasis or destruction of bacterial cells. The antibacterial methods preferably result in prevention, reduction or elimination of invasive activity and related growth of bacterial cells. The term “effective amount” when used in conjunction with antibacterial methods refers to the amount of the compound sufficient to result in the inhibition of growth bacterial cells. The inhibition of bacterial growth according to this method can be monitored in several ways. First bacterial cells grown in vitro can be treated with the compound and monitored for growth or death relative to the same cells cultured in the absence of the compound. A cessation of growth or a slowing of the growth rate, e.g. by 10% of more, is indicative of bacterial cell inhibition. Alternatively, bacterial cell inhibition can be monitored by administering the compound to an animal model having a bacterial infection of interest. Examples of experimental aminal bacterial models are known in the art and described in the examples herein. A convenient means to measure antibacterial activity is by Minimal Inhibitory Concentration (MIC), which is the minimal concentration of an agent necessary to inhibit a standard (control) dose of bacteria. Another convenient means to measure antibacterial activity is Minimal Bactericidal Concentration (MBC), which is the minimal concentration of an agent necessary to kill a standard (control) dose of bacteria.
In a related embodiment, the invention provides a method of decreasing bacterial quantity in a biological sample. This method comprises the step of contacting the biological sample with a polyene polyketide of Formula I, a compound as described herein, or a pharmaceutically acceptable derivative or prodrug thereof. This method is effective if the number of bacteria decreases by at least 10%, and preferably more, e.g., 25%, 50%, 75% or even 100% after contacting the biological sample with a polyene polyketide of Formula I, a compound as described herein, or a pharmaceutically acceptable derivative or prodrug thereof.
In another embodiment, the present invention relates to a method for treating bacterial infection in a mammalian subject in need thereof, comprising the step of administering to the mammal a therapeutically effective amount of a polyene poketide of Formula I, a compound described herein or a pharmaceutically acceptable derivative or prodrug thereof in combination with a physiologically acceptable carrier. In one embodiment, the compound is represented by Formula I. In another embodiment, the compound is selected from Compounds 1-27.
Pharmaceutical compositions effective to treat or prevent a bacterial infection comprise any one of Compounds 1 to 7, a compound of Formula I, or a pharmaceutically acceptable derivative or prodrug thereof, in an amount sufficient to measurably decrease bacterial quantity, and a pharmaceutically acceptable carrier. The term “measurably decrease bacterial quantity”, as used herein means a measurable change in the number of bacteria between a sample containing the inhibitor and a sample not containing the inhibitor.
Agents which increase the susceptibility of bacterial organisms to antibiotics are known. For example, U.S. Pat. No. 5,523,288, U.S. Pat. No. 5,783,561 and U.S. Pat. No. 6,140,306 describe methods of using bactericidal/permeability-increasing protein (BPI) for increasing antibiotic susceptibility of gram-positive and gram-negative bacteria. Agents that increase the permeability of the outer membrane of bacterial organisms have been described by Vaara, M. in Microbiological Reviews (1992) pp. 395-411, and the sensitization of gram-negative bacteria has been described by Tsubery, H., et al, in J. Med. Chem. (2000) pp. 3085-3092.
For the method of the invention related to treatment of subjects with a bacterial infection, a typical effective unit dose of any one of Compounds 1 to 7, a compound of Formula I as described herein or a pharmaceutically acceptable derivative or prodrug thereof given orally or parenterally would be from about 5 to about 100 mg/kg of body weight of the subject with a daily dose ranging from about 15 to about 300 mg/kg of body weight of the subject.
Another preferred embodiment of this invention relates to a method, as described above, of treating a bacterial infection in a mammal in need thereof, but further comprising the step of administering to the mammal an agent which increases the susceptibility of bacterial organisms to antibiotics.
According to another preferred embodiment, the invention provides a method, as described above, of decreasing bacterial quantity in a biological sample, but further comprising the step of contacting the biological sample with an agent which increases the susceptibility of bacterial organisms to antibiotics.
Methods of decreasing bacterial quantity are effective if the number of bacteria decreases at least 10%, and preferably more, e.g., 25%, 50%, 75% or even 100% after contacting the biological sample with any one of Compounds 1 to 7, a compound of Formula I as described herein, or a pharmaceutically acceptable derivative or prodrug thereof.
In addition to the compounds of this invention, pharmaceutically acceptable derivatives or prodrugs of the compounds of this invention may also be employed in compositions to treat or prevent the above-identified disorders. A “pharmaceutically acceptable derivative or prodrug” means any pharmaceutically acceptable salt, ester, salt of an ester or other derivative of a compound of this invention which, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention or an inhibitorily active metabolite or residue thereof. Particularly favored derivatives or prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. Pharmaceutically acceptable prodrugs of the compounds of this invention include, without limitation, esters, amino acid esters, phosphate esters, metal salts and sulfonate esters.
Amycolatopsis orientalis ATCC™ 43491 was cultivated under aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbon, assimilable sources of nitrogen, inorganic salts and vitamins. Preferred carbon sources are glucose, glycerol and the like. Preferred nitrogen sources are beef extract, malt extract, yeast extract, and the like. Representative media are provided in Table 1.
Compounds 1, 2, 7, 8 and 15 were produced by the following procedure: Amycolatopsis orientalis ATCC™ 43491 was maintained and sporulated on agar plates of ISP2 medium (Difco™). The innoculum for the production phase was prepared by adding two loopfull of the spores obtained from the surface of the ISP2 agar plate to a 125-ml flask containing 25 ml of ITSB medium (Zahn et al. (2001). Applied and Environmental Microbiology 76, 377-386) composed of 30 g trypticase soy broth (Bacto™), 3 g yeast extract, 2 g MgSO4, 5 g glucose, 4 g maltose made up to one liter with distilled water. The flasks are shaken (250 rpm) for about 60 hours at 28° C. and then 10 ml of the culture is used to, inoculate each 2-L flasks containing ten glass beads and 500 ml of sterile production medium OA consisting of glucose 10 g, glycerol 5 g, corn steep liquor 3 g, beef extract 3 g, malt extract 3 g, yeast extract 3 g, calcium carbonate 2 g, thiamine 0.1 g made up to one liter with distilled water (Kanzaki et al. (1998). Biosci Biotechnol Biochem 62; 438-442). The medium was adjusted at pH 7.0, and then 1 ml of silicon defoamer-oil (Chem Service) was added to each flask before sterilization. The fermentation batches are incubated aerobically under stirring (200 rpm) at 28° C. for a period of 4 days. A fermentation period of 7 days without defoamer-oil was also used to produce Compound 1.
Compounds 1, 2, 7, 8 and 15 could also be produced in other media including JA, GA, RM, NA, CA, and CB (Table 1). Compounds 1 and 2 were further produced as described above using a preferred strain, namely Amycolatopsis orientalis IDAC 220604-01.
Procedure 1: (for 12×500 mL of fermentation)
a) The whole fermentation broth at harvest was centrifuged at 3500 rpm for 20 minutes and the supernatant liquid was decanted and discarded. The residual mycelial pellet was treated with methanol (200 mL/L of original fermentation broth volume), stirred and centrifuged. The methanolic supernatant liquid was removed and the mycelial solid was extracted with acetone, the same manner as the methanol extraction. The combined methanol and acetone extracts are evaporated to dryness to a crude residue.
b) The crude residue of a) was partitioned between 100 mL(per litre of fermentation) of chloroform (CHCl3) and 100 mL(per litre of fermentation) of methanol (MeOH) in water (3:2) buffered to pH 10 with ammonium hydroxide (NH4OH) and at 10 mM ammonium bicarbonate (NH4HCO3) salt concentration. The two layers were separated and the methanol:water layer evaporated to dryness. The residue from the upper phase was partitioned between n-butanol (100 ml/L of fermentation) and water (100 ml/L of fermentation), buffered as above. The butanol layer was concentrated to a orange-brown residue.
The residue was further purified by HPLC (Waters Autopurification System with ACD), using a Waters Xterra MS C18 column (5μ, 19×150 mm), and a gradient of 10 mM aqueous NH4HCO3, pH 10/acetonitrile 85:15 to 25:75 over 30 min at 19 mL/min, UV detector set at 261 nm. The sample was loaded as a suspension in DMSO/MeOH (3:1). The pooling of eluate gave samples of Compound 7 (<1 mg, RT: 16-17 min) and a mixture of Compounds 1 and 2 (1.04 g, non-freezedried, RT: 11.8-12.1 min).
Alternatively, the first purification (step 2) was also accomplished using a Phenomenex Max-RP C12 column (4μ, 21.2×250 mm) using the gradient buffer (as above) and acetonitrile 89.5:10.5 to 20:80 over 25 minutes, with the same flow and UV detection. Fractions were collected at a retention time of 16.5-17 minutes (mixture of Compounds 1 and 2) and at RT: 21-22 minutes (Compound 7).
The mixture of Compounds 1 and 2 is further purified by HPLC (Waters Autopurification System with ACD), using a RCM Column (Novapak C-18, 6μ, 40×200 mm), and a gradient of 10 mM aqueous ammonium acetate (NH4OAc) to pH 5 with glacial acetic acid/acetonitrile 80:20 to 20:80 over 25 min at 35 mL/min. Fractions were collected and freeze-dried to give pure Compound 1 (RT: 18.5-18.8 minutes, 224.5 mg) and pure Compound 2 (RT: 17.3-17.6 minutes, 34.6 mg).
Procedure 2:
The crude residue of 1 a) (see Procedure 1) was partitioned between 100 mL(per litre of fermentation) of hexanes and 100 mL(per litre of fermentation) of methanol (MeOH) in water (3:2) buffered to pH 10 with ammonium hydroxide (NH4OH) and at 10 mM ammonium bicarbonate (NH4HCO3) salt concentration. The two layers were separated and the methanol:water layer evaporated to dryness. The procedure was repeated and concentrated to dryness.
The crude residue was purified by solid phase extraction. Methanol washed Diaion® HP-20 resin (30 mL) was added to the crude residue. The mixture was added to a column made of 75 mL of methanol washed HP-20 resin and eluted with a mixture of ethanol and pH 10 buffered aqueous ammonium carbonate following the following gradient:
Fractions 4, 5 and 6 were combined and concentrated to give a mixture of Compounds 1 and 2 and Compound 7 was found in the concentrated fraction 7.
The mixture of Compounds 1 and 2 was further purified by HPLC (Waters Autopurification System with ACD), using a Symmetry C18 column (5μ, 30×100 mm), and a gradient of 10 mM aqueous NH4OAc, made to pH 5 with glacial acetic acid/acetonitrile 74:26 to 50:50 over 20 min at 39 mL/min. The collection was triggered by UV 261 nm (PDA). The sample was loaded as a suspension in DMSO:MeOH (3:1). The pooling of eluates gave pure Compound 1 (RT: 14.9-15.2 min) and pure Compound 2 (RT: 14.1-14.2 min), generally with a ratio of 5:1. Compound 7 had a retention time of 13-14 minutes using the same conditions.
Compounds 1, 2 and 7 were produced by fermentation as described in Example 1 and isolated as described in Example 2.
Compound 1 is named 3,4,5-trihydroxy-6-[1-[11-(2-hydroxy-5-oxocyclopent-1-enylcarbamoyl)-1-methyldodeca-2,4,6,8,10-pentaenyl]-2,4,10-trimethyl-16-(N-methyl-guanidino)-hexadeca-2,8,12-trienyloxy]-tetrahydropyran-2-carboxylic acid.
Compound 1 structure determination was based on mass, UV and NMR data. The NMR data detailed in Table 2 was collected at 500 MHz in d4-MeOH including 1H-NMR spectrum of
Mass spectra (
The UV spectrum for Compound 1 exhibited UV λmax at 258.77 and 362.77 nm in methanol in accordance with the methylpentaene amide of the 2-aminocyclopenta-1,3-dione tautomer.
Compound 2 is named 6-{16-guanidino-1-[11-(2-hydroxy-5-oxocyclopent-1-enylcarbamoyl)-1-methyldodeca-2,4,6,8,10-pentaenyl]-2,4,10-trimethylhexadeca-2,8,12-trienyloxy}-3,4,5-trihydroxy-tetrahydro-pyran-2-carboxylic acid.
Structure of Compound 2 was confirmed by 1H (
Compound 7 is named 6-[1-(11-carboxy-1-methyldodeca-2,4,5,8,10-pentaenyl)-2,4,10-trimethyl-16-(N-methylguanidino)-hexadeca-2,8,12-trienyloxy]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid.
Structure analysis of Compound 7 was accomplished by spectral data analysis including 1H NMR (
Analysis of the 1H NMR (
Compounds 3, 4 and 5 were prepared according to the following procedure. A 0.1M solution of sodium hydroxide in methanol (334 μL, Fisher Chemicals) was added to Compound 1 (20 mg) in methanol (2 mL). Dimethyl sulfate (5.68 μL, Sigma) was added and the reaction stirred at room temperature for 24 hours. Additional sodium hydroxide in methanol (334 μL) and dimethyl sulfate (10 μL) were added and the reaction stirred for an additional 24 hours. A third portion of sodium hydroxide in methanol (400 μL) and dimethyl sulfate (15 μL) were added and the reaction mixture stirred for an additional 24 hours. The reaction was monitored by TLC (Merck Silica gel 60 F254, eluting with 7% methanol in chloroform) visualized under UV. The reaction was concentrated in vacuo.
The crude residue was purified by multiple injection on an HPLC Waters Auto-Purification System using a Symmetry (C-18, 5μ, 30×100 mm) column and the following eluent: A (10 mM ammonium acetate in water (10 mM NH4OAc)) and B acetonitrile (MeCN), 74:26 to 50:50 A:B gradient in 20 minutes, 40 mL/min. The fractions having retention times 9.4, 11.5 and 15.5 minutes were collected to give respectively Compound 4 (0.53 mg), Compound 3 (5.36 mg) and Compound 5 (4.04 mg).
Structures of Compounds 3, 4 and 5:
Compound 3 is named 3,4,5-trihydroxy-6-[1-[11-(2-hydroxy-5-oxocyclopent-1-enylcarbamoyl)-1-methyldodeca-2,4,6,8,10-pentaenyl]-2,4,10-trimethyl-16-(N-methyl-guanidino)-hexadeca-2,8,12-trienyloxy]-tetrahydropyran-2-carboxylic acid methyl ester.
Structure determination of Compound 3 was accomplished by spectral data analysis including 1H NMR (
Compound 4 is named 3,4,5-trihydroxy-6-[1-[11 -(2-methoxy-5-oxo-cyclopent-1-enylcarbamoyl)-1-methyldodeca-2,4,6,8,10-pentaenyl]-2,4,10-trimethyl-16-(N-methyl-guanidino)-hexadeca-2,8,12-trienyloxy]-tetrahydropyran-2-carboxylic acid.
Structure determination of Compound 4 was accomplished by spectral data analysis including 1H NMR (
Compound 5 is named 3,4,5-trihydroxy-6-[1-[11 -(2-methoxy-5-oxo-cyclopent-1-enylcarbamoyl)-1-methyldodeca-2,4,6,8,10-pentaenyl]-2,4,10-trimethyl-16-(N-methyl-guanidino)-hexadeca-2,8,12-trienyloxy]-tetrahydropyran-2-carboxylic acid methyl ester.
Structure determination of Compound 5 was accomplished by spectral data analysis including 1H NMR (
Diazomethane was generated by standard methods in an Aldrich diazomethane-generator [Z41, 173-6]. Approx. 2 mL 5N sodium hydroxide was added dropwise to 450 mg 1-methyl-3-nitro-1-nitrosoguanidine and the generated gaseous diazomethane was condensed in 4 mL of diethyl ether at 0° C. The diazomethane solution was added dropwise to a solution of Compound 1 (10 mg) in 6 mL of methanol. The reaction was carried out at room temperature with magnetic stirring maintained for 15 minutes until completion. The reaction product was evaporated at 30° C. under a gentle stream of N2. The crude material was purified by multiple injections on HPLC using a YMC ODS-A 10×250 mm column and a gradient of 5 mM NH4OAc/acetonitrile at 5 mL/min (90:10 for 1 min, 90:10-30:70 o. 20 min). The combined yield of pure Compound 3 derived from two separate 10 mg batches was 0.99 mg.
Compound 6 is named 6-{16-(N′-acetyl-N-methylguanidino)-1-[11-(2-hydroxy-5-oxocyclopent-1-enylcarbamoyl)-1-methyldodeca-2,4,6,8,10-pentaenyl]-2,4,10-trimethylhexadeca-2,8,12-trienyloxy}-3,4,5-trihydroxytetrahydropyran-2-carboxylic acid.
Compound 6 was prepared by acetylation of Compound 1 using the following procedure. Acetic anhydride (3.38 μL, Sigma) was added to a solution of Compound 1 (20 mg) in methanol (2 mL) and the reaction stirred at room temperature for 72 hours. Additional portions of acetic anhydride (20 μL) were added after 24 and 48 hours. The reaction was monitored by TLC (Merck Silica gel 60 F254, eluting with 7% methanol in chloroform) visualized under UV. The reaction was concentrated in vacuo.
The crude residue was purified by multiple injection on an HPLC Waters Auto-Purification System using a Symmetry (C-18, 5μ, 30×100 mm) column and the following eluent: A (10 mM ammonium acetate in water (10 mM NH4OAc)) adjusted to pH 5 with glacial acetic acid and B acetonitrile (MeCN), 74:26 to 50:50 A:B gradient in 20 minutes, 40 mL/min. The fractions having retention times 9.4, 11.5 and 15.5 minutes were collected to give Compound 6 (6.43 mg).
Structure determination of Compound 6 was accomplished by spectral data analysis including 1H NMR (
In biological Examples 7, 8 and 9, culture media (ex: MH, BHI and Agar) were supplied by Quelab Laboratories Inc, Montreal, Canada. Indicator strains were supplied by American Type Culture Collection (ATCC™, Manassas, Va., USA).
Antibacterial activity of Compounds 1 and 2 (Table 4) was measured by determining the minimal inhibitory concentration (MIC) necessary to obtain a complete inhibition of bacteria growth in eight indicator strains, namely Staphylococcus aureus (ATCC™ 6538P), Staphylococcus aureus MRS3 (ATCC™ 700699), Staphylococcus epidermidis (ATCC™ 12228), Bacillus subtilis (ATCC™ 23857), Bacillus megaterium (ATCC™ 14581), Enterococcus faecalis VRE-1 (ATCC™ 29212), Enterococcus faecalis VRE-2 (ATCC™ 51299) and Micrococcus luteus (ATCC™ 9341). Indicator strains preparation and MIC determination were performed according to the National Committee for Clinical Laboratory Standards (NCCLS) guideline M7-A5 Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-Fifth Edition. (NCCLS document M7-A5, ISBN 1-56238-394-9; NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pa. 19087-1898 USA), the content of which is incorporated herein by reference.
Compounds 1 and 2 were prepared as 100× stock solution in DMSO, with concentrations ranging from 3.2 mg/ml to 0.00625 mg/ml (a two-fold dilution series over 12 points). An aliquot of each 100× stock solution was diluted 50-fold in test medium described below to give a set of ten (10) 2× solutions ranging from 64 to 0.125 μg/mL. 50 μl of each of the ten 2× solutions was aliquoted into the corresponding well of a 12-well row, with the final well reserved for medium alone control. Each test was made in duplicate.
Vancomycin (Sigma™) used as positive control compounds, was prepared as 2× stock solutions in sterile, double-distilled water (ddH2O) ranging from 64 μg/ml to 0.00125 μg/ml (a two-fold dilution series over 10 points). An aliquot of 50 μl corresponding to each concentration (at 2×) was then transferred to 96-well microplates to obtain a series of eleven two-fold dilutions.
An isolated colony of each of the eight indicator strains was used to inoculate tubes containing 3 ml of test medium. Mueller-Hinton test medium was used for Staphylococcus aureus (ATCC™ 6538P), Staphylococcus aureus MRS3 (ATCC™ 700699), Staphylococcus epidermidis (ATCC™ 12228), Bacillus subtilis (ATCC™ 23857), Bacillus megaterium (ATCC™ 14581) and Micrococcus luteus (ATCC™ 9341) indicator strains, and BHI (Brain-Heart Infusion broth) test medium was used for Enterococcus faecalis VRE-1 (ATCC™ 29212) and Enterococcus faecalis VRE-2 (ATCC™ 51299) indicator strains. Cells were grown overnight at 28° C. with shaking. Inoculum density for each indicator strain was adjusted to OD600=0.1 in 5 ml 0.85% saline, then further diluted 1/100 in appropriate medium. 50 μl of the final dilution (in test medium) of each indicator strain was added to each well of a 12-well row. This brings the final dilution of the test article or control compound in solution to 1×. The final inoculum is approximately 5×105 CFU/ml.
The indicator strains were incubated with 11 concentrations of each of Compounds 1 and 2, Vancomycin (Sigma™) control compound and one media alone control. For MIC determination, assay plates were incubated at 35° C. for 18 hours. The MIC for each indicator was assessed as the lowest concentration of compound resulting in total absence of growth and is shown below in Table 4.
Antibacterial activitiy of Compounds.3, 4, 5, 6 and 7 are accomplished using the same method in a panel of bacterial strains and Vancomycin as positive control.
Antibacterial efficacy of Compounds 1, 2, 3, 4, 5 and 6 on bacterial strain Staphylococcus aureus NRRL B-313 (ATCC™ 6538P) was determined at different pH concentrations. These results are shown in Table 5 together with the antibacterial activity of Compound 7 on the same strain.
*ND: not determined
Minimal bactericidal concentration (MBC) was also determined for Compounds 1 and 2. MBC value was measured as an indicator of cidal activity of the compound, i.e. to show that the compound actually kills bacteria instead of only inhibiting their growth. MBC were measured against three Staphylococcus strains, namely sp. aureus MSSA (ATCC™ 6538P), sp. aureus MRSA-hVISA (ATCC™ 700699) and sp. epidermidis (ATCC™ 12228). The results, shown in Table 6 below, were consistent with bactericidal activity (MBC/MIC ratio were of 1 to 2).
MBC determination was performed according to the NCCLS guideline M26-A: Methods for Determining Bactericidal Activity of Antimicrobial Agents (NCCLS document M7-A5, ISBN 1-56238-394-9; NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pa. 19087-1898 USA), the content of which is incorporated herein by reference.
Microdilutions broth assays were performed using the same procedure as for MIC determination, Example 6. 24 hours post-incubation, cells were collected in wells corresponding to the MIC, 2×MIC and 4×MIC. Collected cells were plated on agar media. MBC were determined as the drug concentration at which more than 99.9% growth inhibition was observed. As a reference for 100% growth, cells grown in media only were also plated.
A. Toxicity: Acute toxicities were determined using CD-1 female mice (supplied by Charles River Laboratories, Wilmington, Mass.) having a bodyweight of 18-25 g, and about 6 weeks of age. Compounds 1 and 2 at doses ranging from 1 to 70 mg/kg (formulated in 20% propylene glycol/water for a total volume of 10 μL/g of weight) were administered intraperitoneal (i.p.) and intravenous (i.v.) to groups of 5 mice (5 mice per concentration, per mode of administration). Two groups of 3 mice were injected with vehicle only. Mice were observed immediately after injection, within 4 hours and once a day for the subsequent days. Acute toxicity studies of Compound 1 in mice indicated no observable effect and high dose tolerability: 70 mg/kg intraperitoneal and 50 mg/kg intravenous (an MTD of 250 mg/kg was also determined by oral administration). Compound 2 showed no observable effect at 25 mg/kg when administered i.v. (in 20% propylene glycol). In vitro Haemolytic activity results are also shown in Table 7, along with the in vivo toxicity and efficacy of Compound 1. No Haemolytic activity was observed for Compounds 1 and 2 at a concentration of 128 μg/ml compared to Amphotericin (EC50: 3 μg/mL).
B. Efficacy: In vivo efficacy studies were performed on ICR mice (supplied by Charles River Laboratories, Wilmington, Mass.) having a bodyweight of 20±2 g. Mice (38) were inoculated i.p. with an LD90-100 dose (1×106 CFU/mouse) of Staphylococcus aureus (Smith strain, ATCC™ 13709) suspended in 0.5 mL of BHI containing 5% mucin. Compound 1 at doses 20, 30, 40, 50, 60 and 70 mg/kg (formulated in 20% propylene glycol/water for a total volume of 10 μL/g of weigth) was administered i.p. one hour post infection (5 mice for each concentration). Another group of 5 mice was treated i.v. with a 10 mg/kg dose of Vancomycin. A last group of 3 mice was administered vehicle only. Mortality was recorded for 7 days. In vivo efficacy studies showed 100% protection at the lowest dose tested (20 mg/kg i.p.) against an acute lethal infection with S. aureus.
*Concentration to achieve 50% hemolysis of SRBC, control: Amphotericin EC50: 3 μg/mL
**Lowest dose tested i.p. gave 100% protection against S. aureus (Smith) (ATCC ™ 13709)
To a methylene choride solution of Dess-Martin periodinane (1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)one, Sigma-Aldrich Co.) is added a solution of Compound 8 in methylene chloride and the reaction stirred at room temperature for 1 hour. The mixture is diluted with diethyl ether and a saturated aqueous sodium bicarbonate solution containing sodium thiosulfate. Organic layer is separated and washed with saturated aqueous sodium bicarbonate, water and brine, dried over magnesium sulfate, filtered and concentrated in vacuo. The crude residue is purified by HPLC according to the procedure described in Example 4. Pure Compound 10 is obtained by pooling and concentrating the appropriate eluate fractions.
LAH (lithium aluminum hydride) is added to a 0° C. solution of Compound 1 in THF (tetrahydrofuran). After hydrogen gas has stopped evolving, the reaction is allowed to warm to room temperature and stirred overnight. Water is slowly added and 1M hydrochloric solution is used to acidify the solution carefully. The mixture is extracted three times with ethyl acetate. Organic layers are combined and washed with saturated aqueous sodium bicarbonate, water and brine, dried over magnesium sulfate, filtered and concentrated in vacuo. Pure Compound 11 is obtained by pooling and concentrating the appropriate fractions of HPLC purification according to Example 4.
Compound 23 is prepared by modification of the guanidino group of Compound 1 according to the procedure described in Argoudelis et al., J. Antibiotics, Vol. XL, No. 6, June 1987, pp 750-760. A mixture of 200 mg of Compound 1 in 1.2 ml water, 1.0 ml of absolute ethanol,1.0 ml of 2,4-pentadione and 120 mg of sodium bicarbonate is stirred at 90° C. for 3 hours. The mixture is allowed to cool to room temperature, concentrated to dryness, dissolved in 5 ml of 2N acetic acid, and purified by HPLC as described in Example 4. Pure Compound 23 is obtained by pooling and concentrating the appropriate eluate fractions.
Compound 24 is prepared as follows. See Bartzatt et al., Biotechnol. Appl. Biochem. (2002) 36, 89-93. A Wheaton-type double-chamber device (Wheaton Co., Millville, N.J., U.S.A.) is utilized to generate diazopropane (CH3CH2CHN2). Approx. 5.0 mg of Compound 1 is placed into an organic solvent (ethyl acetate/diethyl ether, 1:1, v/v) and the diazopropane gas formed is allowed to dissolve in the mixture. Diazopropane is generated by mixing 0.15 ml of 5 M NaOH with 0.15 g of 3-nitro-1-nitroso-1-propylguanidine. Excess diazoalkane and the solvent are removed by nitrogen gas flow or under vacuum. The remaining residue is dissolved in methanol, and the methanol solution is purified by HPLC as described in Example 4. Pure Compound 24 is obtained by pooling and concentrating the appropriate eluate fractions.
Compound 25 is prepared by acylation of Compound 1 as follows. Acetic anhydride (4.5 equivalents) is added dropwise to a solution of 50 mg/ml of Compound 1 and in acetonitrile and pyridine (9:1). The mixture is stirred under reflux and monitored by TLC (see example 5). The solvent is removed under vacuum and the residue is dissolved in methanol. The methanol solution is purified by HPLC as described in Example 4. Pure Compound 25 is obtained by pooling and concentratin the appropriate eluate fractions.
To a solution of Compound 25 in diethyl ether/ethyl acetate (1/1) is added 1 equivalent of diazomethane in diethyl ether. The reaction mixture is allowed to stand at room temperature overnight. Excess diazomethane and the solvent are removed by nitrogen gas flow or under vacuum. The remaining residue is dissolved in methanol, and the methanol solution is purified by HPLC as described in Example 5. Pure Compound 26 is obtained by pooling and concentrating the appropriate eluate fractions.
Compound 27 is prepared by acylation of Compound 1 as follows. Acetic anhydride (3.2 equivalents) is added dropwise to a solution of 50 mg/ml of Compound 1 in acetonitrile. The mixture is stirred under reflux and monitored by TLC (see Example 5). The solvent is removed under vacuum and the residue is dissolved in methanol. The methanol solution is purified by HPLC as described in Example 4. Pure Compound 27 is obtained by pooling and concentrating the appropriate eluate fractions.
To a solution of Compound 27 in diethyl ether is added 1 equivalent of diazomethane in diethyl ether. The reaction mixture is allowed to stand at room temperature overnight. Excess diazomethane and the solvent is removed by nitrogen gas flow or under vacuum. The remaining residue is dissolved in methanol, and the methanol solution is purified by HPLC as described in Example 4. Pure Compound 28 is obtained by pooling and concentrating the appropriate eluate fractions.
All patents, patent applications, and published references cited herein are hereby incorporated by reference in their entirety. While this invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
This application is a continuation-in-part application of U.S. application Ser. No. 10/937,379 filed Sep. 10, 2004, which claims priority from U.S. provisional application 60/501,821 filed Sep. 11, 2003, U.S. provisional application 60/574,922 filed May 28, 2004 and U.S. provisional application 60/581,707 filed Jun. 23, 2004. The entire teachings of the above application are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60501821 | Sep 2003 | US | |
| 60574922 | May 2004 | US | |
| 60581707 | Jun 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10937379 | Sep 2004 | US |
| Child | 10975545 | Oct 2004 | US |
