Patent Application
20080269146
The present invention relates to novel semi-synthetic macrolides having antimicrobial activity, in particular antibacterial activity. More particularly, the invention relates to 14-membered macrolides substituted at the 4″ position, processes for their preparation, compositions containing them and to their use in medicine.
Macrolide antibacterial agents are known to be useful in the treatment or prevention of bacterial infections. However, the emergence of macrolide-resistant bacterial strains has resulted in the need to develop new macrolide compounds. For example, EP 0 895 999, WO 03/042228, WO 04/101585, WO 04/101586, WO 04/101587, WO 04/101588, WO 04/101589, WO 04/101590, WO 04/039822, WO 05/108412, and WO 05/108413 describe derivatives modified at the 4″ position of the macrolide having antibacterial activity.
According to the present invention, we have now found novel 14-membered macrolides substituted at the 4″ position which also have antimicrobial activity.
Thus, the present invention provides the compound of formula (I)
and/or pharmaceutically acceptable derivatives thereof.
The term “pharmaceutically acceptable” as used herein means a compound which is suitable for pharmaceutical use. Salts and solvates of compounds of the invention which are suitable for use in medicine are those wherein the counterion or associated solvent is pharmaceutically acceptable. However, salts and solvates having non-pharmaceutically acceptable counterions or associated solvents are within the scope of the present invention, for example, for use as intermediates in the preparation of other compounds of the invention and their pharmaceutically acceptable salts and solvates.
The term “derivative” as used herein means any salt, solvate or prodrug, e.g. ester, of the compound of the invention, which upon administration to the recipient is capable of providing (directly or indirectly) a compound of the invention, or an active metabolite or residue thereof. Such derivatives are recognizable to those skilled in the art, without undue experimentation.
The term “pharmaceutically acceptable derivative” as used herein means any pharmaceutically acceptable salt, solvate or prodrug, e.g. ester, of the compound of the invention, which upon administration to the recipient is capable of providing (directly or indirectly) a compound of the invention, or an active metabolite or residue thereof. Such derivatives are recognizable to those skilled in the art, without undue experimentation. Nevertheless, reference is made to the teaching of Burger's Medicinal Chemistry and Drug Discovery, 5th Edition, Vol 1: Principles and Practice, which is incorporated herein by reference to the extent of teaching such derivatives. Examples of pharmaceutically acceptable derivatives are salts, solvates, esters, carbamates and phosphate esters. Additional examples of pharmaceutically acceptable derivatives are salts, solvates and esters. Further examples of pharmaceutically acceptable derivatives are salts and esters, such as salts.
The term “alkyl” as used herein as a group or a part of a group refers to a straight or branched hydrocarbon chain. Examples of “alkyl” as used herein include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, isobutyl, isopropyl, t-butyl, hexyl. In one aspect, “alkyl” refers to a C1-4alkyl group, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or t-butyl.
The compound of the present invention may be in the form of and/or may be administered as a pharmaceutically acceptable salt. For a review on suitable salts see Berge et al., J. Pharm. Sci., 1977, 66, 1-19.
Typically, a pharmaceutically acceptable salt may be readily prepared by using a desired acid or base as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. For example, an aqueous solution of an acid such as lactobionic acid may be added to a solution of a compound of formula (I) in a solvent such as acetonitrile, acetone or THF, and the resulting mixture evaporated to dryness, redissolved in water and lyophilised to obtain the acid addition salt as a solid. Alternatively, a compound of formula (I) may be dissolved in a suitable solvent, for example an alcohol such as isopropanol, and the acid may be added in the same solvent or another suitable solvent. The resulting acid addition salt may then be precipitated directly, or by addition of a less polar solvent such as diisopropyl ether or hexane, and isolated by filtration.
The skilled person will appreciate that the compound of formula (I) contains more than one basic group and therefore either mono (1:1 acid:compound of formula (I)) or bis salts (2:1) may be formed and are salts according to the present invention.
Suitable addition salts are formed from inorganic or organic acids which form non-toxic salts and examples are lactobionate, mandelate (including (S)-(+)-mandelate, (R)-(−)-mandelate and (R,S)-mandelate), hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate, hydrogen phosphate, acetate, trifluoroacetate, maleate, malate, fumarate, lactate, tartrate, citrate, formate, gluconate, succinate, ethyl succinate (4-ethoxy-4-oxo-butanoate), pyruvate, oxalate, oxaloacetate, saccharate, benzoate, sulphonates (e.g. methanesulphonate, ethanesulphonate, benzenesulphonate or p-toluenesulphonate) and isethionate. In one embodiment, suitable salts include phosphate, fumarate, and tartrate, for example bis-phosphate, D-tartrate and bis-fumarate.
Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium and salts with organic bases, including salts of primary, secondary and tertiary amines, such as isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexyl amine and N-methyl-D-glucamine.
The compound of the invention has both a basic and an acidic centre may therefore be in the form of a zwitterion.
Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as “solvates”. For example, a complex with water is known as a “hydrate”. Solvates of the compounds of the invention are within the scope of the invention. The salts of the compound of formula (I) may form solvates (e.g. hydrates) and the invention also includes all such solvates.
The term “prodrug” as used herein means a compound which is converted within the body, e.g. by hydrolysis in the blood, into its active form that has medical effects. Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, “Prodrugs as Novel Delivery Systems”, Vol. 14 of the A.C.S. Symposium Series; Edward B. Roche, ed., “Bioreversible Carriers in Drug Design”, American Pharmaceutical Association and Pergamon Press, 1987; and in D. Fleisher, S. Ramon and H. Barbra “Improved oral drug delivery: solubility limitations overcome by the use of prodrugs”, Advanced Drug Delivery Reviews (1996) 19(2) 115-130, each of which are incorporated herein by reference.
Prodrugs are any covalently bonded carriers that release a compound of formula (I) in vivo when such prodrug is administered to a patient. Prodrugs are generally prepared by modifying functional groups in a way such that the modification is cleaved, either by routine manipulation or in vivo, yielding the parent compound. Prodrugs include, for example, compounds of this invention wherein hydroxy, amine or sulfhydryl groups are bonded to any group that, when administered to a patient, cleaves to form the hydroxy, amine or sulfhydryl groups. Thus, representative examples of prodrugs include (but are not limited to) acetate, formate and benzoate derivatives of alcohol, sulfhydryl and amine functional groups of the compounds of formula (I). Further, in the case of a carboxylic acid (—COOH), esters may be employed, such as methyl esters, ethyl esters, and the like. Esters may be active in their own right and/or be hydrolysable under in vivo conditions in the human body. Suitable pharmaceutically acceptable in vivo hydrolysable ester groups include those which break down readily in the human body to leave the parent acid or its salt.
References hereinafter to a compound according to the invention include both compounds of formula (I) and their pharmaceutically acceptable derivatives.
With regard to stereoisomers, the compound of formula (I) has more than one asymmetric carbon atom. In the general formula (I) as drawn, the solid wedge shaped bond indicates that the bond is above the plane of the paper. The broken wedge shaped bond indicates that the bond is below the plane of the paper.
It will be appreciated that the substituents on the macrolide may also have one or more asymmetric carbon atoms, for example in a prodrug ester group. Thus, the compound of formula (I) may occur as individual enantiomers or diastereomers. All such isomeric forms are included within the present invention, including mixtures thereof.
Separation of diastereoisomers or cis and trans isomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or HPLC. A stereoisomeric mixture of the agent may also be prepared from a corresponding optically pure intermediate or by resolution, such as by HPLC, of the corresponding mixture using a suitable chiral support or by fractional crystallisation of the diastereoisomeric salts formed by reaction of the corresponding mixture with a suitable optically active acid or base, as appropriate.
The compounds of the invention may be in crystalline or amorphous form. Furthermore, some of the crystalline forms of the compounds of the invention may exist as polymorphs, which are included in the present invention.
It is to be understood that the present invention covers all combinations of the embodiments and representative examples described hereinabove.
Compounds of the invention include:
Compounds according to the invention may exhibit a broad spectrum of antimicrobial activity, in particular antibacterial activity, against a wide range of clinical pathogenic microorganisms. Using a standard microtiter broth serial dilution test, compounds of the invention have been found to exhibit useful levels of activity against one or more of a range of pathogenic microorganisims, for example gram positive bacteria. The compounds of the invention may be active against strains which include Staphylococcus aureus, Streptopococcus pneumoniae, Moraxella catarrhalis, Streptococcus pyogenes, Haemophilus influenzae, Enterococcus faecalis, Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila, such as Staphylococcus aureus, Streptopococcus pneumoniae, Enterococcus faecalis and Streptococcus pyogenes. The compounds of the invention may also be active against resistant strains, for example erythromycin resistant strains. Thus, for example, the compounds of the invention may be active against erythromycin resistant strains of Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus.
The compounds of the invention may therefore be useful for treating a variety of diseases caused by pathogenic microorganisms, in particular bacteria, in human beings and animals. It will be appreciated that reference to treatment includes acute treatment or prophylaxis as well as the alleviation of established symptoms.
The compounds of the invention may also be more efficacious, show greater selectivity, have fewer side effects, have a longer duration of action, be more bioavailable by the preferred administration route, have more suitable pharmacodynamic or pharmacokinetic properties, or have other more desirable properties, for example, have better physical properties such as crystallinity than similar known compounds.
Thus, according to another aspect of the present invention we provide a compound of formula (I) or a pharmaceutically acceptable derivative thereof for use in therapy.
According to a further aspect of the invention we provide a compound of formula (I) or a pharmaceutically acceptable derivative thereof for use in the treatment or prophylaxis of systemic or topical microbial infections in a human or animal body.
According to a further aspect of the invention we provide the use of a compound of formula (I) or a pharmaceutically acceptable derivative thereof in the manufacture of a medicament for use in the treatment or prophylaxis of systemic or topical microbial infections in a human or animal body.
According to a yet further aspect of the invention we provide a method of treatment of the human or non-human animal body to combat microbial infections comprising administration to a body in need of such treatment of an effective amount of a compound of formula (I) or a pharmaceutically acceptable derivative thereof.
Examples of disease states in which the compounds of the invention may have utility include skin diseases such as eczema, psoriasis, allergic dermatitis, neurodermatitis, pruritis and hypersensitivity reactions; inflammatory conditions of the nose, throat or lungs such as asthma (including allergen-induced asthmatic reactions), rhinitis (including hayfever), nasal polyps, chronic obstructive pulmonary disease, interstitial lung disease, and fibrosis; inflammatory bowel conditions such as ulcerative colitis and Crohn's disease; atherosclerosis; diabetes after ischemia and reperfusion; myocardial infarction; stroke; cirrhosis; and auto-immune diseases such as rheumatoid arthritis.
Compounds of the invention may also have use in the treatment of conjunctiva and conjunctivitis.
While it is possible that, for use in therapy, a compound of the invention may be administered as the raw chemical it is preferable to present the active ingredient as a pharmaceutical formulation e.g. when the agent is in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
Accordingly, in one aspect, the present invention provides a pharmaceutical composition or formulation comprising a compound of the invention or a pharmaceutically acceptable derivative thereof in association with a pharmaceutically acceptable excipient, diluent and/or carrier. The excipient, diluent and/or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
In another aspect, the invention provides a pharmaceutical composition comprising, as active ingredient, a compound of the invention or a pharmaceutically acceptable derivative thereof in association with a pharmaceutically acceptable excipient, diluent and/or carrier for use in therapy, and in particular, in the treatment of human or animal subjects suffering from a condition susceptible to amelioration by an antimicrobial compound.
In another aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present invention and a pharmaceutically acceptable excipient, diluent and/or carrier (including combinations thereof).
There is further provided by the present invention a process of preparing a pharmaceutical composition, which process comprises mixing a compound of the invention or a pharmaceutically acceptable derivative thereof, together with a pharmaceutically acceptable excipient, diluent and/or carrier.
The compounds of the invention may be formulated for administration in any convenient way for use in human or veterinary medicine and the invention therefore includes within its scope pharmaceutical compositions comprising a compound of the invention adapted for use in human or veterinary medicine. Such compositions may be presented for use in a conventional manner with the aid of one or more suitable excipients, diluents and/or carriers. Acceptable excipients, diluents and carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical excipient, diluent and/or carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as—or in addition to—the excipient, diluent and/or carrier any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
Preservatives, stabilisers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
For some embodiments, the agents of the present invention may also be used in combination with a cyclodextrin. Cyclodextrins are known to form inclusion and non-inclusion complexes with drug molecules. Formation of a drug-cyclodextrin complex may modify the solubility, dissolution rate, bioavailability and/or stability property of a drug molecule. Drug-cyclodextrin complexes are generally useful for most dosage forms and administration routes. As an alternative to direct complexation with the drug the cyclodextrin may be used as an auxiliary additive, e.g. as a carrier, diluent or solubiliser. Alpha-, beta- and gamma-cyclodextrins are most commonly used and suitable examples are described in WO 91/11172, WO 94/02518 and WO 98/55148.
The compounds of the invention may be milled using known milling procedures such as wet milling to obtain a particle size appropriate for tablet formation and for other formulation types. Finely divided (nanoparticulate) preparations of the compounds of the invention may be prepared by processes known in the art, for example see International Patent Application No. WO 02/00196 (SmithKline Beecham).
The routes for administration (delivery) include, but are not limited to, one or more of: oral (e.g. as a tablet, capsule, or as an ingestable solution), topical, mucosal (e.g. as a nasal spray or aerosol for inhalation), nasal, parenteral (e.g. by an injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal, buccal, epidural and sublingual.
There may be different composition/formulation requirements depending on the different delivery systems. By way of example, the pharmaceutical composition of the present invention may be formulated to be delivered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be delivered by both routes.
Where the agent is to be delivered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.
Where appropriate, the pharmaceutical compositions can be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intravenously, intramuscularly or subcutaneously. For parenteral administration, the compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
It is to be understood that not all of the compounds need be administered by the same route. Likewise, if the composition comprises more than one active component, then those components may be administered by different routes.
The compositions of the invention include those in a form especially formulated for parenteral, oral, buccal, rectal, topical, implant, ophthalmic, nasal or genito-urinary use. For some applications, the agents of the present invention are delivered systemically (such as orally, buccally, sublingually), more preferably orally. Hence, preferably the agent is in a form that is suitable for oral delivery.
If the compound of the present invention is administered parenterally, then examples of such administration include one or more of: intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the agent, and/or by using infusion techniques.
For parenteral administration, the compound is best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
The compounds according to the invention may be formulated for use in human or veterinary medicine by injection (e.g. by intravenous bolus injection or infusion or via intramuscular, subcutaneous or intrathecal routes) and may be presented in unit dose form, in ampoules, or other unit-dose containers, or in multi-dose containers, if necessary with an added preservative. The compositions for injection may be in the form of suspensions, solutions, or emulsions, in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising, solubilising and/or dispersing agents. Alternatively the active ingredient may be in sterile powder form for reconstitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.
The compounds of the invention can be administered (e.g. orally or topically) in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.
The compounds of the invention may also be presented for human or veterinary use in a form suitable for oral or buccal administration, for example in the form of solutions, gels, syrups, mouth washes or suspensions, or a dry powder for constitution with water or other suitable vehicle before use, optionally with flavouring and colouring agents. Solid compositions such as tablets, capsules, lozenges, pastilles, pills, boluses, powder, pastes, granules, bullets or premix preparations may also be used. Solid and liquid compositions for oral use may be prepared according to methods well known in the art. Such compositions may also contain one or more pharmaceutically acceptable carriers and excipients which may be in solid or liquid form.
The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, calcium sulphate, dibasic calcium phosphate and glycine, manitol, pregelatinised starch, corn starch, potato starch, disintegrants such as sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia.
Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
Solid compositions of a similar type may also be employed as fillers in gelatin or HPMC (hydroxypropyl methylcellulose) capsules. Preferred excipients in this regard include microcrystalline cellulose, lactose, calcium carbonate, calcium sulphate, dibasic calcium phosphate and, manitol, pregelatinised starch, corn starch, potato starch or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
Capsules, may be filled with a powder (of medicament alone or as blend with selected filler(s)) or alternatively a liquid, each comprising one or more compounds of formula (I) and a carrier. Where the capsule is filled with a powder the compounds of formula (I) and/or the carrier may be milled or micronised to provide material with an appropriate particle size.
Compounds of the invention may be susceptible to acid degradation after ingestion and may therefore require a coating, such as an enteric coating, when administered orally as a tablet or capsule.
The tablet or capsule, as appropriate, may, for example be coated by a thin film such as a EUDRAGIT® film available from Röhm Pharma Polymers, which allows controlled dissolution in the gastrointestinal tract. The films are available as cationic polymers such as EUDRAGIT® E 100 (aminoalkyl methacylate copolymers) or as anionic acrylic polymers such as EUDRAGIT® L (methacrylic acid copolymers) and EUDRAGIT S. Permeable acrylic polymers such as EUDRAGIT® RL (ammonio methacrylate copolymer) and EUDRAGIT® RS are also available.
These coating formulations may be prepared as an aqueous dispersion including optional ingredients such as talc, silicone antifoam emulsion, polyethylene glycol. Alternatively the coating formulation may be prepared as an organic polymer solution.
Alternatively, tablets may be coated using OPADRY® (Surelease®) coating systems, available from Colorcon. Aqueous systems generally comprise up to 15% w/w of OPADRY®. Organic solvent systems generally comprise up to 5% w/w of OPADRY®.
The coatings may be prepared by known techniques, for example by;
Coatings can be applied by known techniques, using tablet coating machines.
The thickness of the coating applied is generally in the range 5 to 35 microns such as 10 to 30 microns, more specifically 10 or 20 microns, depending on the required effect.
Alternatively, the tablet or a capsule, as appropriate, may be filled into another capsule (preferably a HPMC capsule such as Capsugel®) to provide either a tablet in capsule or capsule in capsule configuration, which when administered to a patient yields controlled dissolution in the gastrointestinal tract thereby providing a similar effect to an enteric coating.
Thus in one aspect the invention provides a solid dose formulation of a compound of formula (I) wherein said formulation has an enteric coating.
In another aspect the invention provides a solid dose formulation comprising a protective capsules as outer layer, for example as a tablet in a capsule or as a capsule in a capsule.
The compounds of the invention may also be administered orally in veterinary medicine in the form of a liquid drench such as a solution, suspension or dispersion of the active ingredient together with a pharmaceutically acceptable carrier or excipient.
The compounds of the invention may also, for example, be formulated as suppositories e.g. containing conventional suppository bases for use in human or veterinary medicine or as pessaries e.g. containing conventional pessary bases.
The compounds according to the invention may be formulated for topical administration, for use in human and veterinary medicine, in the form of ointments, creams, gels, hydrogels, lotions, solutions, shampoos, powders (including spray or dusting powders), pessaries, tampons, sprays, dips, aerosols, drops (e.g. eye ear or nose drops) or pour-ons.
For application topically to the skin, the agent of the present invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
Alternatively, it can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
The compounds may also be dermally or transdermally administered, for example, by use of a skin patch.
For ophthalmic use, the compounds can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.
As indicated, the compound of the present invention can be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134AT″″) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate.
Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound and a suitable powder base such as lactose or starch.
For topical administration by inhalation the compounds according to the invention may be delivered for use in human or veterinary medicine via a nebuliser.
The compounds of the invention may also be used in combination with other therapeutic agents. The invention thus provides, in a further aspect, a combination comprising a compound of the invention or a pharmaceutically acceptable derivative thereof together with a further therapeutic agent.
When a compound of the invention or a pharmaceutically acceptable derivative thereof is used in combination with a second therapeutic agent active against the same disease state the dose of each compound may differ from that when the compound is used alone. Appropriate doses will be readily appreciated by those skilled in the art. It will be appreciated that the amount of a compound of the invention required for use in treatment will vary with the nature of the condition being treated and the age and the condition of the patient and will be ultimately at the discretion of the attendant physician or veterinarian. The compounds of the present invention may for example be used for topical administration with other active ingredients such as corticosteroids or antifungals as appropriate.
The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising a combination as defined above together with a pharmaceutically acceptable carrier or excipient comprise a further aspect of the invention. The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations by any convenient route.
When administration is sequential, either the compound of the invention or the second therapeutic agent may be administered first. When administration is simultaneous, the combination may be administered either in the same or different pharmaceutical composition.
When combined in the same formulation it will be appreciated that the two compounds must be stable and compatible with each other and the other components of the formulation. When formulated separately they may be provided in any convenient formulation, conveniently in such manner as are known for such compounds in the art.
The compositions may contain from 0.01-99% of the active material. For topical administration, for example, the composition will generally contain from 0.01-10%, more preferably 0.01-1% of the active material.
Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular individual may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.
For oral and parenteral administration to humans, the daily dosage level of the agent may be in single or divided doses.
For systemic administration the daily dose as employed for adult human treatment it will range from 2-100 mg/kg body weight, preferably 5-60 mg/kg body weight, which may be administered in 1 to 4 daily doses, for example, depending on the route of administration and the condition of the patient. When the composition comprises dosage units, each unit will preferably contain 200 mg to 1 g of active ingredient. The duration of treatment will be dictated by the rate of response rather than by arbitrary numbers of days.
The compound of formula (I) and pharmaceutically acceptable derivatives thereof may be prepared by the general methods outlined hereinafter, said methods constituting a further aspect of the invention.
A comprehensive discussion of the ways in which sensitive groups may be protected and methods for cleaving the resulting protected derivatives is given by for example T. W. Greene and P. G. M Wuts in Protective Groups in Organic Synthesis 2nd ed., John Wiley & Son, Inc 1991 and by P. J. Kocienski in Protecting Groups, Georg Thieme Verlag 1994 which are incorporated herein by reference. Examples of suitable amino protecting groups include acyl type protecting groups (e.g. formyl, trifluoroacetyl and acetyl), aromatic urethane type protecting groups (e.g. benzyloxycarbonyl (Cbz) and substituted Cbz, and 9-fluorenylmethoxycarbonyl (Fmoc)), aliphatic urethane protecting groups (e.g. t-butyloxycarbonyl (Boc), isopropyloxycarbonyl and cyclohexyloxycarbonyl) and benzyl type protecting groups (e.g. benzyl, trityl and chlorotrityl). Examples of suitable oxygen protecting groups may include for example silyl groups, such as trimethylsilyl, triethylsilyl or tert-butyldimethylsilyl; ethers such as tetrahydropyranyl or tert-butyl; or esters such as acetate or benzoate. Hydroxy groups may be protected by reaction of for example acetic anhydride, benzoic anhydride or a trialkylsilyl chloride in an aprotic solvent. Examples of aprotic solvents are dichloromethane, N,N-dimethylformamide, dimethylsulfoxide, tetrahydrofuran and the like. A carboxylate group may be protected as an ester for example an ethyl or benzyl ester.
The compounds of general formula (I) and derivatives thereof may be purified by conventional methods known in the art. For example, the compounds may be purified by HPLC using an aqueous solution of an acid such as formic acid or trifluoroacetic acid with an organic co-solvent such as acetonitrile or methanol. Alternatively, said compounds may be purified by crystallisation, chromatography and/or differential partition between aqueous and organic solvents.
Compounds of formula (I) may be prepared by reaction of a 4″ aldehyde compound of formula (II)
or a derivative thereof wherein:
one or more functional groups may be protected (for example with benzoyl, triethyl silyl, benzyloxycarbonyl or by formation of a bicyclic ketal by interaction of the 9-ketone with the 12-OH and an alcohol, for example methanol). Alternatively or in addition, the 3′-dimethylamino group may be in the form of an N-Oxide or as part of a 2′-O-3′-N-bis-benzyloxycarbonyl moiety as described by Flynn et al (J. Amer. Chem. Soc. 1955, 77, 3104-3106),
with the amine of formula (III), wherein R1 is hydrogen or a carboxylic acid protecting group, and R* represents hydrogen or methyl, in a reductive amination reaction,
which may be carried out in a suitable solvent, such as DCM, methanol, dioxan or DMF, under neutral to mildly acidic conditions. Suitable reducing agents include, for example, sodium cyanoborohydride, sodium triacetoxyborohydride, tetrabutylammonium triacetoxyborohydride or a polymer bound borohydride. A borohydride in a solvent containing acetic acid wherein the triacetoxyborohydride is formed in situ may also be employed. Alternatively, palladium on charcoal and hydrogen may be employed to effect the reduction. Suitable reagents for adjusting acidity include acetic acid and sodium acetate.
Appropriate chemical transformations are then carried out to achieve, where necessary and in any order, methylation of the nitrogen in the linker, re-methylation of the 3′ amine and removal of one or more protecting groups as described above.
Methylation of the nitrogen in the linker and/or re-methylation of the 3′ position may be effected preferably by a reductive alkylation reaction with formaldehyde. This may be carried out in a solvent, such as DCM, methanol or DMF, under neutral to mildly acidic conditions. Suitable reducing agents include, for example, sodium cyanoborohydride, sodium triacetoxyborohydride, tetrabutylammonium triacetoxyborohydride, or a polymer bound borohydride. A borohydride in a solvent containing acetic acid wherein triacetoxyborohydride is formed in situ may also be employed. Alternatively, palladium on charcoal and hydrogen may be employed to effect the reduction. Suitable reagents for adjusting acidity include acetic acid and sodium acetate. Suitable temperatures are in the region 0-50° C. typically 20° C. Alternatively reductive methylation may be achieved using aqueous formaldehyde and formic acid at elevated temperatures for example in refluxing chloroform.
Methylation may also be effected by treatment with an alkylating agent, for example methyl iodide in the presence of a base, for example N(CH2CH3)(CH[CH3]2)2 or potassium carbonate.
Compound (II) or a derivative thereof may be prepared from compound (IIa)
or a derivative thereof wherein:
one or more functional groups may be protected (for example with benzoyl, triethyl silyl, benzyloxycarbonyl or by formation of a bicyclic ketal by interaction of the 9-ketone with the 12-OH and an alcohol, for example methanol). Alternatively or in addition, the 3′-dimethylamino group may be in the form of an N-Oxide or as part of a 2′-O-3′-N-bis-benzyloxycarbonyl moiety as described by Flynn et al (J. Amer. Chem. Soc. 1955, 77, 3104-3106),
by oxidative cleavage, for example, using osmium tetroxide and sodium periodate. Alternatively ozone at a low temperature, 0° C. or below, typically −50 to −78° C. in the presence of an acid, for example TFA (trifluoroacetic acid) may be employed. In the absence of acid, oxidation of —N(CH3)2 on the moiety below, may occur
The N-oxide may be reduced at various stages of the process, as required, for example, by treatment with a suitable reducing agent, such as triphenyl phosphine under appropriate conditions as described in J. of Antib. 41 (1988) 1029-1047 or with Zn powder as described in EP 0 985 679.
A compound of formula (IIa) or a derivative thereof, can be formed by palladium-catalysed allylation of a suitably protected 4″ hydroxy compound, for example by 2′,11-bis-silylation and conversion of the 9-ketone to a bicyclic ketal by interaction with the 12-OH and an alcohol, for example methanol. Further guidance to O-allylation can be gained from Stoner et al J. Org Chem 68, 8847-8852, references therein (palladium catalysed allylation of 6-OH in erythromycin derivatives) and Freiberg et al in U.S. Pat. No. 5,288,709 (4″-O allylation using NaN(TMS)2/allyl bromide on a highly modified and protected erythromycin derivative).
In another embodiment of the invention compounds of formula (I), may be prepared by reaction of a compound of formula (IIb) wherein L is a suitable leaving group such as halogen, mesylate or tosylate,
or a derivative thereof wherein:
one or more functional groups may be protected (for example with benzoyl, triethyl silyl, benzyloxycarbonyl or by formation of a bicyclic ketal by interaction of the 9-ketone with the 12-OH and an alcohol, for example methanol). Alternatively or in addition, the 3′-dimethylamino group may be in the form of an N-Oxide or as part of a 2′-O-3′-N-bis-benzyloxycarbonyl moiety as described by Flynn et al (J. Amer. Chem. Soc. 1955, 77, 3104-3106),
with a compound of formula (III) wherein:
R1 is hydrogen or an carboxylic acid protecting group, and R* represents hydrogen, methyl or a nitrogen protecting group which facilitates deprotonation of the NH group, such as benzyloxycarbonyl,
in an appropriate aprotic solvent, such as DCM, DMF or THF, at a temperature of −78 to 100° C. such as 0 to 50° C., in the presence of a base, for example LiN(TMS)2, DBU, potassium carbonate or a sterically hindered tertiary amine, for example N(CH2CH3)(CH[CH3]2)2,
Appropriate chemical transformations are then carried out to achieve, where necessary and in any order, methylation of the nitrogen in the linker, re-methylation of the 3′ amine and removal of one or more protecting groups as described above.
In another embodiment of the invention, compounds of formula (I) may be prepared by reaction of a compound of formula (IV):
or a derivative thereof; wherein:
R* represents methyl, hydrogen or a nitrogen protecting group which facilitates deprotonation of the NH group, such as benzyloxycarbonyl; and one or more functional groups may be protected (for example with benzoyl, triethyl silyl, benzyloxycarbonyl or by formation of a bicyclic ketal by interaction of the 9-ketone with the 12-OH and an alcohol, for example methanol). Alternatively or in addition, the 3′-dimethylamino group may be in the form of an N-Oxide or as part of a 2′-O-3′-N-bis-benzyloxycarbonyl moiety as described by Flynn et al (J. Amer. Chem. Soc. 1955, 77, 3104-3106),
with the alkylating agent (V), wherein R1 is hydrogen or a carboxylic acid protecting group and L is a suitable leaving group such as halogen, mesylate or tosylate,
in an appropriate aprotic solvent such as DCM, DMF or THF at a temperature of −78 to 100° C. such as 0 to 50° C., in the presence of a base for example LiN(TMS)2, DBU, potassium carbonate or a sterically hindered tertiary amine, for example N(CH2CH3)(CH[CH3]2)2.
Appropriate chemical transformations are then carried out to achieve, where necessary and in any order, methylation of the nitrogen in the linker, re-methylation of the 3′ amine and removal of one or more protecting groups as described above.
In another embodiment of the invention, compounds of formula (I) may be prepared by reacting a compound of formula (IV) or a derivative thereof; wherein R* represents methyl or hydrogen, wherein one or more functional groups may be protected (for example with benzoyl, triethyl silyl, benzyloxycarbonyl or by formation of a bicyclic ketal by interaction of the 9-ketone with the 12-OH and an alcohol, for example methanol). Alternatively or in addition, the 3′-dimethylamino group may be in the form of an N-Oxide or as part of a 2′-0-3′-N-bis-benzyloxycarbonyl moiety as described by Flynn et al (J. Amer. Chem. Soc. 1955, 77, 3104-3106),
with a compound of formula (Va)
wherein R1 is hydrogen or a carboxylic acid protecting group,
in a reductive alkylation reaction which may be carried out in a suitable solvent, such as DCM, methanol or DMF, under neutral to mildly acidic conditions. Suitable reducing agents include, for example, sodium cyanoborohydride, sodium triacetoxyborohydride, tetrabutylammonium triacetoxyborohydride or a similar polymer bound borohydride. Sodium borohydride in a solvent, such as acetic acid wherein the triacetoxyborohydride is formed in situ may also be employed. Alternatively, palladium on charcoal and hydrogen may be employed to effect the reduction. Suitable reagents for adjusting acidity include acetic acid and sodium acetate.
Appropriate chemical transformations are then carried out to achieve, where necessary and in any order, methylation of the nitrogen in the linker, re-methylation of the 3′ amine and removal of one or more protecting groups as described above.
Compound (IIb) or a derivative thereof can be prepared by converting the primary alcohol in compound (IVa) or a derivative of it:
into a suitable leaving group such as tosyl, mesyl or halogen by reactions well known to the skilled person.
Compound (IVa) or a derivative thereof may be prepared by treatment of compound (II) or a derivative thereof with a reducing agent such as sodium borohydride.
All the chemistry above may also be carried out with analogues of (III), (V) or (Va) containing a double or triple bond as shown below:
If intermediates (III′), (III″), (V′), (V″), (Va′) or (Va″) are utilised then an additional hydrogenation step is carried out to reduce the unsaturated bond before the compound of formula (I) is obtained.
Compounds of formula (III), (III′) and (III″), may be prepared as shown in scheme 1 below.
In the above scheme:
P1 represents an amine protecting group, such as Boc or benzyloxycarbonyl, and R* is hydrogen or methyl, and the dotted bond is a single, double or triple bond.
In step 1) the acid is converted to an acid chloride using a reagent such as phosgene, oxallyl chloride or thionyl chloride with DMF as catalyst at a temperature of 0-40° C., typically 20° C. for about 3 h. Alternative starting materials may have, for example, chlorine, bromine or a sulfonyloxy group, for example triflate, in place of the iodine atom, and chlorine or a sulfonyloxy group for example triflate in place of the fluorine atom.
In step 2) reaction with ethyl 3-(dimethylamino)acrylate may be effected in DMF or another suitable solvent at a temperature of 60-120° C. typically 90° C. for about 2.5 h.
In step 3) reaction with N,N-dimethyl hydrazine occurs at temperatures between 0-40° C., typically 20° C. for about 1.5 h.
In step 4) cyclisation takes place in DMF or another suitable solvent in the presence of a base such as potassium carbonate at a temperature of 50-120° C. typically 70° C. for about 2 h.
In step 5) a Sonogashira reaction of a suitable acetylene occurs in the presence of a suitable catalyst for example copper (I) iodide and dichlorobis(triphenylphosphine)palladium (IIb) in a suitable solvent for example acetonitrile and base for example triethylamine at temperatures between 0-80° C., typically 20° C. for about 1.5 h.
Where P1=benzyloxycarbonyl and R*=H, a compound of type (III″) where R*=benzyloxycarbonyl is thereby obtained.
In step 6) hydrogenation of the unsaturated bond takes place in a suitable solvent for example DCM, ethanol or a mixture of the two, using a catalyst, for example palladium on carbon, typically at 20° C. with a typical reaction time of 24 h. In the case where P1 is benzyloxycarbonyl this group will also be removed to give an intermediate (III).
In step 7) where P1 is Boc, treatment with a strong acid, for example HCl in dioxan or TFA in DCM, removes the Boc protection.
In step 8) treatment with a base such as potassium carbonate in a two phase system such as water/DCM or chloroform at temperatures between 0-30° C., typically 20° C. gives the amino ester free base.
In step 9) a Heck reaction using a suitable olefin may be carried out to give an intermediate where the dotted bond is a double bond. Alternatively, a Suzuki reaction with a borane, derived for example by hydroboration of a suitable olefin, may be carried out to give a product where the dotted bond is a single bond. In these cases, where P1=benzyloxycarbonyl and R*=H, a compound of type (III) or (III′) where R*=benzyloxycarbonyl will be obtained.
Such an intermediate where the dotted line shown represents either a single or double bond and where P1 is Boc may be subjected to the conditions of step 7 and step 8 to give a compound of type (III) or (III′).
Such an intermediate where the dotted line shown represents a double bond and where P1 is Boc may be subjected to the conditions of steps 6, 7 and 8 to give a compound of type (III).
Compound (IV) or a derivative thereof may be prepared by reductive amination of compound (II) or a derivative thereof, for example using the general conditions described above for reductive aminations.
Compounds of formula (V) and (Va), defined above, can be prepared by employing the method shown in scheme 2 below.
In step 1) a Sonogashira reaction with propargyl alcohol can be effected in the presence of a suitable catalyst for example copper (I) iodide and dichlorobis(triphenylphosphine)palladium (II) in a suitable solvent, for example acetonitrile, and base, for example triethylamine, at temperatures between 0-80° C., typically 20° C. for about 1 h. Alternatively, a Heck reaction using allyl alcohol may be carried out to give an intermediate where the dotted bond is a double bond. Alternatively, a Suzuki reaction with a borane, derived for example by hydroboration of allyl alcohol, may be carried out to give a product where the dotted bond is a single bond. Alternative starting materials may have chlorine, bromine or a sulfonyloxy group, for example triflate, in place of the iodine atom.
Where required, step 2) may be carried out in which hydrogenation of a multiple bond takes place in a suitable solvent for example DCM, ethanol or a mixture of the two, using a catalyst, for example palladium on carbon, typically at 20° C.
In step 3) oxidation of the alcohol to an aldehyde is carried out, for example, using Swern conditions (DMSO and oxalyl chloride in the presence of triethylamine) or manganese dioxide to give aldehydes of type (Va), (Va′) or (Va″) where the dotted line represents a single, double or triple bond respectively.
In step 4) a leaving group, such as halogen, mesylate or tosylate is introduced by chemistry well known to those skilled in the art to give compounds of formula (V), (V′) or (V″) where the dotted line represents a single, double or triple bond respectively.
A compound of formula (I) may also be prepared by coupling a compound of formula (X) or a derivative thereof; wherein:
the dotted line represents a double or triple bond, R* represents methyl, hydrogen or a nitrogen protecting group such as benzyloxycarbonyl; and one or more functional groups may be protected (for example with benzoyl, triethyl silyl, benzyloxycarbonyl or by formation of a bicyclic ketal by interaction of the 9-ketone with the 12-OH and an alcohol, for example methanol). Alternatively or in addition, the 3′-dimethylamino group may be in the form of an N-Oxide or as part of a 2′-O-3′-N-bis-benzyloxycarbonyl moiety as described by Flynn et al (J. Amer. Chem. Soc. 1955, 77, 3104-3106) and where the dotted line represents a double or triple bond,
with a suitable 6-halo quinolone in a Sonogashira, Heck or Suzuki reaction.
Appropriate chemical transformations are then carried out to achieve, where necessary and in any order, methylation of the nitrogen in the linker, re-methylation of the 3′ amine, reduction of an unsaturated bond in the linker and removal of one or more protecting groups as described above.
A compound of formula (X), or a derivative thereof may be prepared by reacting a compound of type (II) or (lib) or a derivative of either with a suitable reagent for example N-methylpropargylamine in an alkylation or reductive alkylation reaction. An intermediate of formula (X) may also be prepared by reacting a compound of type (IV) or a derivative thereof with a suitable reagent for example propargyl bromide or propenal in an alkylation or reductive alkylation reaction.
In another embodiment of the invention, compounds of formula (I) may be prepared by reacting a compound (VI)
or a derivative thereof wherein:
one or more functional groups other than the 4″-hydroxyl may be protected (for example with benzoyl, triethyl silyl, benzyloxycarbonyl or by formation of a bicyclic ketal by interaction of the 9-ketone with the 12-OH and an alcohol, for example methanol). The ketone could also be protected, as for example, an oxime. Alternatively or in addition, the 3′-dimethylamino group may be in the form of an N-Oxide or as part of a 2′-O-3′-N-bis-benzyloxycarbonyl moiety as described by Flynn et al (J. Amer. Chem. Soc. 1955, 77, 3104-3106), or the 9-ketone may be protected as an oxime derivative,
with a compound of formula (VII)
wherein R1 is hydrogen or a carboxylic acid protecting group, and R* represents hydrogen methyl or a nitrogen protecting group such as benzyloxycarbonyl, and L is a suitable leaving group such as halogen, mesylate or tosylate, and X is oxygen or the C═X moiety is CH2;
in an appropriate aprotic solvent, such as DCM, DMF, DMSO or THF, at a temperature of −78 to 100° C., such as 0 to 50° C., in the presence of a base, for example LiN(TMS)2, DBU, potassium tert-butoxide, potassium carbonate or a sterically hindered tertiary amine, for example N(CH2CH3)(CH[CH3]2)2.
Appropriate chemical transformations are then carried out to achieve, where necessary and in any order, methylation of the nitrogen in the linker, re-methylation of the 3′ amine, and removal of one or more protecting groups as described above. Where a compound of formula (VII) in which X is oxygen is utilised, reduction of the amide to an amine is also required.
A compound of formula (VII), as defined above, where the C═X moiety is CH2, can be prepared by reacting a compound of formula (III) with ethylene oxide or a haloethanol in the presence of a suitable base where appropriate in a suitable solvent for example ethanol, DCM or DMF followed by conversion of the resulting hydroxyl group to a leaving group by reaction with for example thionyl chloride or a sulfonic anhydride in the presence of a suitable base where appropriate in a suitable solvent for example DCM or THF.
A compound of formula (VII), as defined above, where X is oxygen, can be prepared by reacting a compound of formula (III) with a halo acetyl derivative for example chloroacetyl chloride in the presence of a suitable base for example pyridine in a suitable solvent for example dichloromethane.
All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.
In order that the invention may be more fully understood the following examples are given by way of illustration only.
The following abbreviations may appear in the text: 9-BBN for 9-borabicyclo[3.3.1]nonane, BOC for t-butoxycarbonyl, DBU for 1,8-diazabicyclo[5.4.0]undec-7-ene, DCM for dichloromethane, DMAP for 4-dimethylaminopyridine, DMF for N,N-dimethylformamide, DMSO for dimethyl sulfoxide, EtOAc for ethyl acetate, EtOH for ethanol, MeCN for acetonitrile, MeOH for methanol, TFA for trifluoroacetic acid, THF for tetrahydrofuran, MgSO4 for anhydrous magnesium sulphate and Na2SO4 for anhydrous sodium sulphate.
In the procedures that follow, reference to an Intermediate by number is typically provided. This is provided merely for assistance to the skilled chemist to identify the starting material used. The starting material may not necessarily have been prepared from the batch referred to. In addition, the preparation of an Example compound is typically presented as a series of individual reaction steps, for example (a), (b), (c), etc. This is also provided merely for assistance to the skilled chemist to identify a suitable sequence of reaction steps to prepare the Example. Although each of the reaction steps indicated will have been carried out as described, the steps (a), (b), (c), etc. may not have been performed in one continuous sequence from the same batch of starting materials.
Where Example compounds are isolated as salts these are typically characterised and the stoichiometry determined using proton NMR, for example by considering the chemical shift values, the integrated number of protons, and by assignment of one or more equivalent peak(s) from the acid and from the parent base.
6-O-Methyl-erythromcyin A (108 g) in THF (500 mL) under an atmosphere of argon was treated portionwise with carbonyldiimidazole (43.2 g) with ice bath cooling, the cooling bath was then removed. After 24 h additional THF (300 mL) and water (20 mL) were added followed by the dropwise addition of water (500 mL). After stirring for 3 h a thick white precipitate had formed. The solid was filtered off, washed with cold water and dried under vacuum to yield the title compound (107.58 g) as a white solid. The mother liquors yielded further title compound (9.0 g); ESMS m/z 842.7 [M+H]+.
4″-O-(1-Imidazol-1-yl-carbonyl)-6-O-methyl-erythromycin (50 g) in DCM (200 mL) was cooled to 0° C. under argon and treated with allyl alcohol (23.8 mL) and DBU (9.1 mL). The reaction was stirred at 0° C. for 2 h and at 20° C. for 2 h. The reaction mixture was quenched with 3% aqueous citric acid (200 mL), the phases separated, and the organic phase washed with saturated sodium hydrogen carbonate. After drying and evaporation to dryness, the residue was triturated with petroleum ether (bp 40-60° C.) with stirring then filtered and dried to give the title compound as a white solid (41 g); ESMS m/z 832.6 [M+H]+.
4″-O-(Allyloxycarbonyl)-6-O-methyl-erythromycin A (10 g) dissolved in dry pyridine (30 mL) was treated with chlorotrimethylsilane (12.0 mL). After stirring for 1 h the pyridine was evaporated and the residue partitioned between toluene/diethyl ether and saturated aqueous sodium hydrogen carbonate. The organic layer was separated and the aqueous phase extracted with a fresh portion of diethyl ether. The combined organic layers were washed with brine, dried and evaporated to yield the title compound as a foam (10.5 g); ESMS m/z 990.8 [M+H]+.
This foam was dissolved in acetonitrile (˜100 mL) and the resultant solution cooled to about 5° C. A seed crystal (prepared by purifying a previous sample of the title compound by chromatography on silica gel eluting with 33-70% diethyl ether in hexane followed by maintaining a 100 mg/mL solution in acetonitrile at approx 5° C. for two weeks) was added and immediate crystallisation commenced. After standing at about 5° C. overnight the mixture was filtered to yield the title compound mono acetonitrile solvate as a crystalline solid (5.2 g).
(9S)-4″-O-(Allyloxycarbonyl)-9-dihydro-9-methoxy-2′,11-bis-O-trimethylsilyl-9,12-anhydro-6-O-methyl-erythromycin A mono acetonitrile solvate (15.0 g) in dry THF (100 mL) under argon was treated with tetrakis(triphenylphosphine)palladium (0.36 g) and the resultant mixture heated at reflux for 1.5 h. Allyl t-butyl carbonate (5 mL) (F. Houlihan et al, Can. J. Chem. 1985, 63, 153) was added and heating continued for a further 3.75 h. After cooling and standing overnight at 20° C. the THF was evaporated and the dark brown residue taken up in 40/60 petroleum ether (100 mL). The solution was treated with charcoal, filtered and evaporated. The solid was then taken up in acetonitrile, re-evaporated and dried under vacuum overnight to yield 15.89 g. The product (12.8 g) was dissolved in acetonitrile (25 mL) and 10% aqueous acetic acid (130 mL). After stirring at 20° C. for 6 h diethyl ether (50 mL) was added and the layers separated, the organic layer was extracted with water and the combined aqueous extracts made basic by the addition of potassium carbonate. The organic product was extracted with EtOAc (2×100 mL), dried and evaporated to give the title compound as a solid (10.0 g); ESMS m/z 788.7 [M+H]+.
4″-O-(Allyloxycarbonyl)-6-O-methyl-erythromycin A (272.02 g) was dissolved in DCM (1360 mL) and DBU (208 mL) was added. The solution was cooled to 0-5° C. and triethylsilyl chloride (220 mL.) was added dropwise over 50 minutes. The reaction mixture was allowed to warm up to room temperature and stirred for 24 h.
The reaction mixture was washed successively with water (0.98 L), saturated NaHCO3 (0.98 L) and the aqueous phase extracted with DCM. The combined organic phases were washed with brine (0.98 L). The organic phase was concentrated under reduced pressure. The residue was dissolved in methanol (1 L) and pyridine hydrochloride (37.79 g) in methanol (360 mL) added. The reaction mixture was stirred for 18 h. DCM (2.72 L) was added and the solution was washed with water (1.632 L), and saturated NaHCO3 (1.632 L). The organic phase was evaporated under reduced pressure. The residue was dissolved in THF (1.65 L) and evaporated under reduced pressure. This was repeated. The residue was dissolved in THF (3300 mL), degassed and flushed with nitrogen. Pd(PPh3)4 (14.82 g) was added and the solution heated to reflux and stirred under nitrogen for 6 hours.
More Pd(PPh3)4 (1.50 g) in THF (120 mL) was added over 10 min followed by dropwise addition of tertbutylallylcarbonate (162 mL) over 30 min. The reaction mixture was stirred at 65° C. for 16 hours. The solution was cooled to 60° C., activated charcoal (27.5 g) added and the mixture heated to reflux for 40 min, then cooled to room temperature, filtered through celite and the filtered solid washed with THF (2 L).
The filtrate was concentrated under vacuum, the residue was dissolved in acetonitrile (1 L) and evaporated under reduced pressure. This was repeated. The residue was dissolved in acetonitrile (1 L) and acetic acid (3 L) and water (5 L) added. The reaction mixture was stirred at room temperature for 70 hours.
The reaction mixture was basified to pH 9 with solid K2CO3. The resulting suspension was extracted twice with EtOAc (2×2.5 L). The combined organic phases were washed with water (2 L) and concentrated by 80% under vacuum. Methylcyclohexane (1 L) was added and the mixture concentrated by 80% under vacuum. This was repeated, then a third portion of methylcyclohexane (1 L) was added and the suspension filtered off. The solid was washed with methylcyclohexane (3×350 mL) and dried under vacuum at 42° C. to give the title compound as a white powder (115.4 g). δc (100 MHz; CDCl3) 9.0, 10.5, 12.3, 15.9, 16.0, 18.0, 19.0, 19.7, 20.9, 21.5, 21.6, 28.4, 35.5, 37.1, 39.0, 39.2, 40.1, 44.9, 45.3, 49.6, 50.6, 64.7, 64.8, 68.0, 69.0, 71.0, 73.4, 74.2, 75.0, 76.5, 78.2, 78.4, 80.2, 86.5, 96.2, 102.3, 117.1, 134.8, 175.9, 221.1.
4″-O-Allyl-6-O-methyl-erythromycin A (95.8 g) in DCM (1 L) and methanol (100 mL) was cooled to −70° C. under argon and TFA (18.8 mL) added. Ozonized oxygen was bubbled through until a blue colour developed (1.25 h). Argon was bubbled through the mixture to flush out the ozone, then dimethyl sulfide (35.7 mL) and triethylamine (50.5 mL) were added. The reaction was stirred at −70° C. for 30 min then removed from the cooling bath. After 0.5 h the reaction (now at −30° C.) was warmed to 0° C. in a water bath and stirred for a further 0.5 h.
The reaction mixture was washed with water (500 mL), dried (Na2SO4) and evaporated to dryness. The residue was dissolved in toluene and evaporated under reduced pressure (three times each 300 mL) to give the title compound (103.7 g) which was used without purification; ESMS m/z 822.7 [M+MeOH+H]+, 834.6 [M+HCO2]−.
A stirred suspension of 2-fluoro-5-iodobenzoic acid (99.7 g) in DCM (1 L) at 20° C. was treated with oxalyl chloride (49.8 mL) and DMF (0.3 mL). After 3 h further DMF (0.1 mL) was added. After a further 2 h the clear solution was evaporated and re-evaporated from toluene (3×200 mL). The acid chloride was re-dissolved in toluene (1.5 L) and treated with triethylamine (79.2 mL) and ethyl 3-(dimethylamino)acrylate (65.3 g). After stirring for 2.5 h at 90° C. the mixture was filtered and evaporated. The residue was redissolved in EtOAc, washed with saturated sodium hydrogen carbonate solution (2×), water, saturated brine, dried (MgSO4) and treated with decolourising charcoal for 0.5 h. The mixture was filtered, evaporated, redissolved in diethyl ether and allowed to crystallise. The solid was filtered off, washed with diethyl ether and dried to give the title compound (91.2 g). On concentration and seeding a second crop was obtained (9.0 g); ESMS m/z 391.9 [M+H]+.
A stirred suspension of Intermediate 3a (50 g) in EtOH (500 mL) was treated with 1,1-dimethylhydrazine (10.7 mL). After stirring for 2.5 h the clear solution was evaporated. The residue was dissolved in DMF (500 mL), treated with potassium carbonate (26.5 g) and the mixture stirred at 70° C. for 2 h. After cooling to 20° C. the mixture was poured into water containing excess citric acid, the solid filtered off, washed with water and dried to give the title compound (48.07 g); ESMS m/z 386.9 [M+H]+.
A stirred mixture of Intermediate 3b (48.07 g) and copper (I) iodide (2.36 g) in triethylamine (510 mL) and MeCN (1 L) was stirred under argon for 15 min. N-t-butoxycarbonyl-N-methylpropargylamine (31.72 g) and dichlorobis(triphenylphosphine)palladium (II) (2.8 g) were added. After 1.5 h the mixture was evaporated and redissolved in DCM. The solution was washed with saturated sodium hydrogen carbonate solution (2×), water, saturated brine, dried (Na2SO4) and evaporated. The residue was triturated with diethyl ether. The solid was filtered off washed with diethyl ether and dried, then boiled in EtOAc, filtered, the solution diluted with diethyl ether and allowed to crystallise to give the title compound (13.88 g). The solid insoluble in hot EtOAc was dissolved in hot ethanol and treated with decolourising charcoal. The hot mixture was filtered and evaporated to give more of the title compound (32.57 g); δH (250 MHz; CDCl3) 1.43 (3H, t, J=7.1 Hz), 1.50 (9H, s), 2.92 (6H, s), 2.99 (3H, s), 4.29 (2H, br s), 4.42 (2H, q, J=7.1 Hz), 7.68 (1H, dd, J=1.9 & 8.8 Hz), 8.02 (1H, d, J=8.8 Hz), 8.50 (1H, d, J=1.9 Hz), 8.73 (1H, s).
A solution of Intermediate 3c (32.57 g) in DCM (300 mL) was treated with 10% Pd/C (2 g) and stirred for 1 min. The catalyst was filtered off and replaced with fresh (2 g). The mixture was hydrogenated at ambient temperature and pressures overnight, filtered and evaporated. The residue was triturated with diethyl ether. The solid was filtered off washed with diethyl ether and dried to give the title compound (26.33 g). The ether solution was evaporated, redissolved in EtOH and treated with decolourising charcoal. Filtration, evaporation and trituration with diethyl ether gave a second crop (3.96 g); ESMS m/z 432.2 [M+H]+.
A stirred solution of Intermediate 3d (30.29 g) in DCM (120 mL) was treated with 4M HCl in 1,4-dioxan (120 mL). After 1 h the mixture was evaporated and the residue partitioned between DCM and 10% K2CO3 solution. The DCM solution was collected, washed with saturated brine, dried (Na2SO4) and evaporated. The gummy solid was triturated with diethyl ether then light petroleum 40-60 was added portionwise. The solid was filtered off, washed with light petroleum 40-60 and dried to yield the title compound (19 g); ESMS m/z 332.1 [M+H]+.
To a stirred solution of ethyl 6-(3-t-butoxycarbonylmethylaminopropyl)-1-(dimethylamino)-4-oxo-1,4-dihydro-3-quinolinecarboxylate (518 g) in chloroform (2070 mL) was added a mixture of 4M HCl in dioxane (1070 mL, containing 20-30% water) over 1 hour. The reaction was stirred for a further 1 h. The phases were separated, the organic layer was concentrated in vacuo and combined with the aqueous layer. To this was added chloroform (3 L). Potassium carbonate (475 g) in water (4 L) was added, the organics were separated and the aqueous re-extracted with chloroform (3 L and 1.5 L). The combined organics were washed with brine (3 L) before being dried over magnesium sulphate, filtered and concentrated in vacuo to give a solid. The material was triturated with diethyl ether (1.5 L) and stirred for 20 mins before the addition of hexane (750 L). The suspension was stirred for a further 10 mins before being filtered and dried to give 328 g, of the title product as an off white solid.
DIMS m/z 332.2 [M+H]+.
a) 4″-O-{2-[(3-[3-Carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycin A ethyl ester Intermediate 2 (91.5 g) in DCM (1 L) was stirred under argon with ethyl 6-(3-methylaminopropyl)-1-(dimethylamino)-4-oxo-1,4-dihydro-3-quinolinecarboxylate (33.6 g) and 3 A molecular sieves (45 g) for 30 min, then sodium triacetoxy borohydride (45.1 g) was added. After 40 min the solution was decanted from the molecular sieves, which were extracted with more DCM. The combined DCM solutions were washed twice with water and once with 5% potassium carbonate, dried (Na2SO4) and evaporated under reduced pressure to give the crude product (110.6 g). This was purified using 2 Biotage 75 800 g columns eluting with 0-6.4% (10:1 MeOH/0.880 NH3) in DCM to give the title compound (65.1 g); ESMS m/z 1106.0 [M+H]+.
b) 4″-O-{2-[(3-[3-Carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycin A D-tartrate salt Example 1a (65.1 g) in THF (550 mL) was treated with 0.5M LiOH (283 mL) at room temperature under argon. After 2.5 h 1 M sodium dihydrogen phosphate was added to give a pH of approximately 11, and the mixture extracted with diethyl ether (500 mL). The aqueous layer was treated with a small amount of phosphoric acid, giving a pH of 10.4 when a solid rapidly separated. This was collected by filtration and washed with diethyl ether and water to give 125 g of wet solid. A portion of this (109 g) was slurried in water and ethyl acetate and the pH adjusted to 7.2 with 1 M sodium dihydrogen phosphate. The ethyl acetate was evaporated under reduced pressure and the aqueous extracted with 2×400 mL DCM, the combined extracts were dried (Na2SO4) and evaporated under reduced pressure to give the product free base (47.8 g). This material was dissolved in acetone (3.2 L) at 45° C. and treated with D-tartaric acid (6.66 g in 240 mL water) and seeded with authentic material. After stirring and allowing to cool for 2 h followed by ice bath cooling to 15° C., the solid was filtered off, washed with acetone and dried under reduced pressure to give the title compound as a white powder (43.9 g); ESMS m/z 1077.9 [M+H]+. δH (500 MHz; CD3OD+1 drop D2O), inter alia 4.32 (2H, s, tartrate) 2.81 (3H, s, 22″-CH3), 2.84 (6H, 7′-, 8′-CH3).
δc (125 MHz, HSQC/HMBC, CD3OD+1 drop D2O) 8.6, 9.7, 11.2, 15.0, 15.7, 17.1, 18.0, 19.4, 20.5, 20.5, 20.8, 25.3, 30.3, 31.7, 34.5, 37.9 38.4, 38.7, 38.8, 39.6, 44.8, 45.0, 45.0, 48.9, 49.9, 55.6, 55.8, 64.4, 64.8, 66.6, 67.3, 69.1, 69.4, 73.5, 74.3, 74.7, 76.8, 78.4, 78.4, 80.6, 87.0, 96.2, 101.7, 108.7, 117.6, 124.4, 125.6, 135.5, 139.4, 140.0, 143.3, 168.0, 177.2, 176.6, 177.7, 221.5.
Example 1 (18 g) in DCM (150 mL) and water (10 mL) was shaken with saturated aqueous sodium hydrogen carbonate solution (30 mL). The aqueous layer was extracted with DCM (3×50 mL), the four organic extracts combined, dried (Na2SO4) and evaporated under reduced pressure to a foam. This material was taken up in acetone and evaporated to dryness to give a white solid foam, (14.2 g). Similar material from another preparation (0.6 g) was added, and the material was crystallised twice from acetone/water (1:1, 11 mug) to give the title compound, after drying under vacuum over phosphorus pentoxide (10.748 g); mp 144-145° C., ES m/z 1077.6 [M+H]+. δH (400 MHz, CDCl3) inter alia 2.25 (6H, 7′-, 8′-CH3), 2.26 (3H, s, 22″-CH3).
Ethanol (3 mL) was added to 4″-O-{2-[(3-[3-Carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycin A (809.0 mg). To the solution, seeds of crystalline free-base (from an ethanol crystallisation which had been seeded with crystals produced by crystallisation from acetone/supercritical fluid carbon dioxide) were added. The solution was stirred overnight at room temperature which led to the formation of crystalline white solid. The solid was filtered, washed with ethanol and analysed using FT-IR and DSC. Obtained about 503.5 mg of crystalline free-base.
DSC showed a single endotherm maximum at 192.38° C.
4″-O-{2-[(3-[3-Carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycin A (9.1 g) in acetonitrile (450 mL) was treated with a solution of phosphoric acid (1.656 g) in water (40 mL) and acetonitrile (90 mL). The hazy solution was filtered through kieselguhr and allowed to crystallize overnight at 20° C. and 3 h at 4° C. The white solid was filtered off, washed with acetonitrile and dried to give the title compound (8.238 g); ESMS m/z 1077.9 [M+H]+, OH (400 MHz, CD3OD) inter alia 2.77 (3H, s, 22″-CH3), 2.84 (6H, 7′-, 8′-CH3)
4″-O-Allyl-6-O-methyl-erythromycin A (5.0 g) in dry DCM (50 mL) under argon was treated with triethylamine (1.41 mL). Benzoyl chloride (0.95 mL) was then added dropwise. The resultant mixture was stirred for 2 h, then additional benzoyl chloride (0.14 mL) was introduced. After stirring for a further 1 h, the mixture was quenched by the addition of 1 N NaOH. The layers were separated and the organic phase was washed with brine, then dried (Na2SO4) and concentrated in vacuo. Chromatography on silica eluting with 0-10% (2M NH3 in MeOH) in DCM gave the title compound as a white foam (5.6 g); ES m/z 892.5 [M+H]+.
Example 5a (862 mg) in DCM (20 mL) and methanol (2 mL) was cooled to −70° C. and TFA (143 μL) added. Ozonized oxygen was bubbled through until a blue colour developed. Oxygen and then argon were bubbled through the mixture to flush out the ozone, then dimethyl sulfide (283 μL) and triethylamine (404 μL) were added. The reaction was then removed from the cooling bath and allowed to warm to room temperature. The reaction mixture was then diluted with water, the organic layer removed and then washed three times with water, dried (Na2SO4) and evaporated to dryness to afford the title compound which was used without purification; ES m/z 912.5 [M+H2O+H]+.
Example 5b in DCM (12 mL) was stirred under argon with ethyl 6-(3-methylaminopropyl)-1-(dimethylamino)-4-oxo-1,4-dihydro-3-quinolinecarboxylate (301 mg) and 3 A molecular sieves (300 mg), then sodium triacetoxy borohydride (411 mg) was added. After 20 min the solution was decanted from the molecular sieves. The DCM solution was washed with water and NaHCO3 solution. The combined aqueous phases were re-extracted with DCM, the combined DCM solutions were then washed with brine and then dried (Na2SO4) and concentrated in vacuo. Chromatography on silica eluting with 0-17% (2M NH3 in MeOH) in DCM gave the title compound as a colourless foam (648 mg); ES m/z 1209.6 [M+H]+.
Example 5c (200 mg) in THF (12 mL) was treated with 0.5M LiOH (6 mL) at room temperature under argon. After 3 h 40 drops of AcOH were added and the mixture was concentrated in vacuo. Chromatography on silica eluting with 0-20% (9:1 MeOH/0.880 NH3) in DCM gave the title compound as a colourless foam (200 mg); ES m/z 1181.6 [M+H]+.
Example 5d (200 mg) in MeOH (10 mL) was refluxed for 48 h under argon. The crude product was triturated with hexanes. Chromatography on silica eluting with 5-16% (9:1 MeOH/0.880 NH3) in DCM gave the title compound as a colourless foam (130 mg); ES m/z 1077.6 [M+H]+.
Isopropanol (2 mL) was added to 4″-O-{2-[(3-[3-Carboxy-1-dimethylamino-4-oxo-1,4-dihydro-6-quinolinyl]propyl)methylamino]ethyl}-6-O-methyl-erythromycin A, (231.7 mg). The solution was stirred for 15 minutes at room temperature and then fumaric acid (52.42 mg) was added to the solution. The slurry was heated to 50° C. and left stirring overnight. The slurry was filtered, washed with i-propanol and dried in a vacuum oven at 50° C. with a slow flow of nitrogen, to give the title compound (112.2 mg). δH (400 MHz, (CD3)2SO) inter alia 2.26 (3H, s, 22″-CH3), 2.44 (6H, 7′-, 8′-CH3), 6.55 (4H, s, fumarate) DSC showed a single endotherm maximum at 215.43° C.
Whole-cell antimicrobial activity was determined by broth microdilution using the Clinical and Laboratory Standards (CLSI) recommended procedure (Document M7-A6A7, Methods for Dilution Susceptibility Tests for Bacteria that Grow Aerobically). Compounds were dissolved in DMSO and diluted into water to produce a 640 μg/mL stock solution. The stock solution was further diluted 1:10 into Haemophilus Test Media to produce a 64 μg/mL working stock solution. A Microlab AT Plus 2 (Hamilton Co., Reno, Nev.) was used to prepare serial two-fold dilutions (50 μL aliquots) of the working stock in a 96 well microtitre plate. After the compounds were diluted, a 50 μL aliquot of the test isolate (˜1×106 cfu/ml) was added to each well of the microtitre plate. The final test concentrations ranged from 0.016-16 μg/mL. Inoculated plates were incubated at 35° C. in ambient air for 18 to 24 hours. The minimum inhibitory concentration (MIC) was determined as the lowest concentration of compound that inhibited visible growth. The compounds in the above examples generally gave MICs less than 1 μg/mL against erythromycin-sensitive and erythromycin-resistant strains of Streptococcus pneumoniae and/or Streptococcus pyogenes.
However, it will appreciated by a person skilled in the art that compounds of the invention may have different levels of activity against different strains of the same bacteria.
The MICs of compounds can be determined against 42 strains of Haemophilus influenzae. The 90th percentile of these values is termed the MIC90, while the 50th percentile is termed the MIC50. The geometric mean of the 42 values may also be calculated.
For these studies, CD-1 mice are acclimated to a restricted diet (8 grams/day) for 4 days prior to infection. Once on study, mice receive their daily ration each morning. An inoculum is obtained by placing 40 μl of Haemophilus influenzae H-128 onto Chocolate II agar plates and putting them into the CO2 incubator for approximately 18 hours. 20 colonies are taken from the plates and put into 50 mls Mueller-Hinton broth with 5% Fildes and placed into CO2 incubator for approximately 24 hours. The conical tube is centrifuged at 4000 rpms for 20 minutes at 37° C. The pellet is resuspended in 3 mls of Mueller Hinton broth with 5% Fildes. That tube is serially diluted 1:10 into saline to 10-7. 10-5-10-7 dilutions (200 μl each) are plated in triplicate for enumeration of the inoculum (done by scoring colony-forming units after growing overnight in CO2 incubator). From the original conical tube, a 1:5 dilution of bacteria:melted nutrient agar is made and placed directly into 38-41° C. water bath.
For the infection, mice are anesthetized with isoflurane using an anesthesia machine. The anesthetized mice are then infected with 20 μl/mouse of bacterial suspension in molton nutrient agar by direct intra-bronchial instillation via intratrachial intubation. Mice are dosed at 1, 7, 24, 31, 48, 55, 72 and 79 hours post-infection with the corresponding compounds. Mice are sacrificed at 96 hours by CO2 overdose and the lungs excised for enumeration of viable bacteria numbers after growing overnight in CO2 incubator.
The bioavailability/oral exposure of compounds of the present invention may be tested using the following protocols in rat or mouse.
Rat Animal Model—Rats are surgically implanted with a Tygon catheter and are allowed a 4 day post-surgical recovery period prior to study initiation. Food (PMI 5002 certified diet) and water are provided ad-libitum until placed into a commercial auto sampling system (Culex, Bioanalytical Systems, West Lafayette, Ind.). Animals are acclimated to the caging and tethering system on the automated system for a minimum of 24 hours prior to study initiation. During acclimation and throughout the duration of the study, animals are given 5 food pellets/day (≈20 g) and free access to water.
Dose Preparation—Oral target doses in the rat are 50 mg/kg at 16 mL/kg and intravenous target doses are 5 mg/kg at 4 mL/kg. The intravenous dose is infused over a 1 hour period. The final dose Solution compositions are: 1.0% DMSO; 20% Encapsin (w/v) in water (po), or saline (iv) pH 3.5-4.0. Dose solution aliquots are taken (N=3, 50-μL) by weight and frozen at −80° C. until subsequent LC/MS/MS analysis along with blood/water samples.
Study regimen and sample collection—During the PO dose session, animals are orally dosed with a stainless steel gavage needle with a standard dose volume of 16 mL/kg. Rats are then allowed a 2 day washout period and crossed over for a second IV dose session in which animals are infused for 1 hour (4 mL/kg) into the femoral vein while blood samples are removed via the femoral artery catheter. When blood samples are removed from the femoral artery by the Culex instrument, samples are retained in a fraction collector at 33° F. in heparanized glass tubes. The standard timepoints at which blood samples are collected are as follows:
po: 0, 15, 30, 45, 60, 90, 120, 180, 240, 360, 480, 720, 960 and 1440 min. (75 uL per sample)
iv: 0, 20, 40, 60, 65, 75, 90, 120, 180, 240, 360, 480, 960, 1440 and 1800 min. (75 uL per sample)
Duplicate blood samples are aliquoted (25 uL) and 25 uL of water is added by a Tecan® robotic pipetting instrument. The blood/water lysates are then stored at −80° C. until subsequent quantitation by LC/MS/MS.
PK analysis—Non-compartmental pharmacokinetic analysis is performed using WinNonlin version 4.1; NCA and an internal DMPK software program, MPKR 1.74.
Mouse Animal Model—Mice are acclimated to a 2 pellets (≈8 g)/day (PMI 5002 certified diet) for a minimum of 3 days and water is provided ad-libitum. Mice are individually housed in polycarbonate shoeboxes.
Dose Preparation—Oral target doses in the mice are 300 mg/kg at 16 mL/kg. The final dose Solution compositions are: 1.0% DMSO; 20% Encapsin (w/v) in water (po), pH 3.5-5.5. Dose solution aliquots are taken (N=2-3, 50-μL) by weight and frozen at −80° C. until subsequent LC/MS/MS analysis along with blood/water samples.
Study regimen and sample collection—During the PO dose session, animals are orally dosed with a stainless steel gavage needle with a standard dose volume of 16 mL/kg. Blood samples are removed from the femoral artery via the lateral tail veins into lithium heparinized microfuge tubes. The standard timepoints at which blood samples are collected are as follows: po: 20, 40, 60, 90, 120, 180, 240, 360, 480, and 1440 (25 uL per sample). The blood is aliquoted (25 uL) and 25 uL of water is added immediately. The blood/water lysates are then stored at −80° C. until subsequent quantitation by LC/MS/MS.
PK analysis—Non-compartmental pharmacokinetic analysis is performed using WinNonlin version 4.1; NCA and an internal DMPK software program, MPKR 1.74.
Throughout the specification and the claims which follow, unless the context requires otherwise, the word ‘comprise’, and variations such as ‘comprises’ and ‘comprising’, will be understood to imply the inclusion of a stated integer or step or group of integers but not to the exclusion of any other integer or step or group of integers or steps.
The application of which this description and claims forms part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the following claims:
| Number | Date | Country | Kind |
|---|---|---|---|
| 2005/012038 | Nov 2005 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2006/010731 | 11/7/2006 | WO | 00 | 5/9/2008 |
