ANALOGS AND DERIVATIVES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. patent application serial number 61/474,798 filed on April 13, 2011, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates generally to the fields of organic chemistry, drug development, pharmacology, infectious diseases, and medicine. More particularly, the invention relates to the preparation of analogs and derivatives of two related classes of marine-derived terpenoid natural products, and their use as anti-bacterial agents.

BACKGROUND

[0003] Pseudopteroxazole (CMPD1) and homopseudopteroxazole (CMPD2) are two related natural products from Pseudopterogorgia elisabethae. Prior to the work described herein they were the only known members of this compound series. The pseudopterosins are a separate but structurally related class of terpenoids isolated from P. elisabethae and these include pseudopterosins G-J (represented by CMPD3). A variety of naturally occurring pseudopterosins are known which differ in the identity of the sugar moiety and in the stereochemistry of the aglycone backbone.

[0004] The development of pseudopteroxazoles as drugs has been hampered by their limited supply. Pseudopteroxazole and homopseudopteroxazole constitute respectively 0.0015% and 0.002% of the dry weight of P. elisabethae. Pseudopteroxazole has also been prepared by total synthesis; however, these lengthy multi-step syntheses are not economically practical for production purposes or analog synthesis. No syntheses of homopseudopteroxazole have previously been reported.

SUMMARY [0005] The invention is based on the development of new methods for converting pseudopterosins to pseudopteroxazoles. These methods have been used to make several new non-natural pseudopteroxazole analogs. These new non-natural pseudopteroxazole analogs, pseudopterosins and derivatives thereof, and various prenylated aromatic structural mimics of pseudopterosins/pseudopteroxazoles were shown to possess anti-bacterial activity, with some of these compounds exhibiting no or limited toxicity against mammalian cells.

[0006] Accordingly, the invention features several new compounds having the following general structures:

Wherein, for pseudopterosin series compounds: Xi can be (OCH ₂ ) ₄ CH ₃ and X ₂ can be OH; or Xi can be OMe (Me = methyl) and X ₂ can be OMe, OS0 ₂ CF ₃ , or OCONMe ₂ ; Y and R can be null (i.e. simple pseudopterosin derivatives); and Z can be null (i.e., a sp2 hybridized carbon). For oxazole and imidazole series compounds: Xi can be N, NH, N-alkyl (alkyl defined below), or O; X ₂ can be N, NH, N-alkyl, or O; Y can be C; R can be alkyl, aryl (aryl defined below); and Z can be null (i.e., sp2 hybridized carbon). For quinoxaline series: Xj and X ₂ can be N; Y can be R-C-C-R; R can be alkyl or aryl; and Z can be null or OMe.

[0007] The compounds of the invention include, e.g., CMPD16, CMPD17, CMPD18, CMPD19, CMPD20, CMPD22, CMPD23, CMPD24, CMPD25, CMPD26, CMPD27, CMPD28, CMPD29, CMPD30, CMPD31, CMPD32, CMPD33, CMPD34, CMPD35, CMPD36, CMPD37, CMPD38, CMPD39, CMPD40, CMPD41, CMPD42, CMPD43, CMPD44, CMPD45, CMPD46, CMPD47, CMPD48, and CMPD49 (described below).

[0008] Also within the invention is a method of producing a pseudopteroxazole, an isopseudopteroxazole, and derivatives thereof. This method includes the steps of: providing a solution of pseudopterosin aglycone; contacting the solution of pseudopterosin aglycone with an oxidant (e.g., air, oxygen gas, or a chemical oxidant) to yield a first reaction mixture comprising pseudopterosin aglycone and the oxidant; adding an aldehyde or an aldehyde equivalent (such as triethyl orthoformate) to the first reaction mixture to yield a second reaction mixture; adding an NH ₃ source to the second reaction mixture to yield a third reaction mixture; heating the third reaction mixture at least until the compound is formed; and purifying the compound from the heated third reaction mixture. During the step of heating the third reaction mixture at least until the compound is formed, additional reactants such as the aldehyde, the NH ₃ source, and/or the oxidant can be added to the third reaction mixture.

[0009] The invention additionally includes a method of producing a derivative of pseudopteroxazole or isopseudopteroxazole. This method includes the steps of: providing a solution of pseudopterosin aglycone; contacting the solution of pseudopterosin aglycone with an oxidant (e.g., silver salt) to yield a first reaction mixture comprising pseudopterosin aglycone and the oxidant; adding an amino acid to the first reaction mixture to yield a second reaction mixture; heating the second reaction mixture at least until the derivative is formed; and purifying the derivative from the heated second reaction mixture. During the step of heating the second reaction mixture at least until the derivative is formed, additional reactants such as the amino acid and the oxidant can be added to the second reaction mixture.

[0010] The invention further includes a method of producing a pseudopterosin quinoxaline derivative. This method includes the steps of: providing a solution of pseudopterosin aglycone; contacting the solution of pseudopterosin aglycone with an oxidant [e.g., a silver salt, Dess-Martin periodinane (DMP), and a chemical oxidant other than DMP] to yield a first reaction mixture comprising pseudopterosin aglycone and the oxidant; adding an ethylenediamine derivative to the first reaction mixture to yield a second reaction mixture; heating the second reaction mixture at least until the derivative is formed; and purifying the derivative from the heated second reaction mixture. During the step of heating the second reaction mixture at least until the derivative is formed, additional reactants including the ethylenediamine derivative and the oxidant can be added to the second reaction mixture.

[0011] In another aspect, the invention features a method of killing or retarding the growth of a bacterium by contacting a bacterial cell with a composition including at least one (e.g., 1, 2, 3, 4, or more) of the following compounds: CMPD1, CMPD4, CMPD15, CMPD16, CMPD17, CMPD19, CMPD20, CMPD25, CMPD27, CMPD28, CMPD29, CMPD30, CMPD31, CMPD32, CMPD33, CMPD34, CMPD35, CMPD36, CMPD37, CMPD38, CMPD39, CMPD41, CMPD42, and CMPD43. In one embodiment of this method, the bacterium is Mycobacterium tuberculosis and the composition includes at least one of the following compounds: CMPD1, CMPD15, CMPD20, CMPD25, CMPD28, CMPD29, CMPD30, CMPD31, CMPD32, CMPD33, CMPD34, CMPD35, CMPD36, CMPD38, or CMPD39. In another embodiment of this method, the bacterium is Mycobacterium tuberculosis in a non-replicating state and the composition includes at least one of the following compounds: CMPD1, CMPD15, CMPD20, CMPD25, CMPD28, CMPD29, CMPD30, CMPD31, CMPD32, CMPD33, CMPD34, CMPD35, CMPD36, or CMPD38.

[0012] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Commonly understood definitions of chemical terms can be found in Oxford Dictionary of Chemistry, John Daintith, ed., Oxford University Press, 2008 and R. T. Morrisson et al., Organic Chemistry, 6th edition, Addison-Wesley Publishing Co.: Boston, Mass., 1992.

[0013] As used herein with reference to the above general structures, "alkyl" refers to a carbon chain including methyl, ethyl, propyl, butyl, pentyl; the chain may be substituted internally or terminally with a functional group such as an hydroxyl, amine, thiol or secondary alkyl moiety.

[0014] As used herein with reference to the above general structures, "aryl" refers to an aromatic ring or a heteroaromatic ring including benzyl, pyridinyl, naphthyl etc.; and the ring may be substituted at one or more of the available positions (e.g. positions 2-6 for a benzene ring) with various groups including fluoro, methoxyl and hydroxyl moieties.

[0015] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions will control. In addition, the particular embodiments discussed below are illustrative only and not intended to be limiting. For example, although stereochemistry is shown in structures presented herein, these embodiments are only exemplary and all corresponding stereochemical analogs are intended to be included in the invention.

DETAILED DESCRIPTION

[0016] The invention provides methods of preparing pseudopterosin/pseudopteroxazole analogs and mimics, new compounds made using these methods, and methods of using such compounds as anti-bacterial agents. The analogs can be prepared semi-synthetically from naturally occurring, or synthetically derived, pseudopterosins or the corresponding aglycones (e.g., CMPD4 and CMPD5). The pseudopterosin/pseudopteroxazole mimics can be synthesized from commercially available materials, such as phenols.

[0017] The below described embodiments illustrate representative examples of these methods and compounds. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below.

General Methods

[0018] Methods involving conventional organic chemistry, medicinal chemistry, pharmaceutical sciences, microbiology, and drug development techniques are described herein. Such methods are described in: Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st edition (2005); Clinical Microbiology Procedures Handbook, Garcia and Isenberg (Editors), ASM Press (2010); Drug Discovery and Development, Mukund S. Chorghade (Editor) Wiley-Interscience; 1st edition (2007); The Practice of Medicinal Chemistry, 3rd Edition, Camille Georges Wermuth (Editor) Academic Press; 3rd edition (2008); and Clayden et al., Organic Chemistry, Oxford University Press, 1st edition (2000).

Preparation of Pseudopteroxazole, Homopseudopteroxazole,

and Congeners Thereof

[0019] Pseudopteroxazole, homopseudopteroxazole and congeners thereof can be prepared from pseudopterosins or pseudopterosin aglycones following the general transformation shown below in Scheme 1. The disclosed transformations yield heterocyclic derivatives including oxazoles (CMPD6: XI =N, X2 =0; or X1=0, X2=N), imidazoles (CMPD6: X1=N, X2=NH & tautomer), and quinoxalines (CMPD7: X1=X2=N). Examples of specific procedures that can be utilized to impart heterocyclic functionality onto pseudopterosins and to generate the pseudopteroxazole congener are described below. Variations of the reaction conditions should be evident to those skilled in the art of organic synthesis, and these may be utilized to produce different heterocyclic congeners, including thiazoles, oxazines, oxadiazoles, etc. Variations in the reaction conditions may be utilized to affect the relative product ratios and yields.

[0020] Oxazole or imidazole functionality may be introduced into pseudopterosins semi- synthetically by a reaction between the aglycone, an ammonia source (NH ₃ or an ammonium salt NH ₄ X), and an aldehyde (Scheme 2; pseudopterosin denvatization; Method A). Following this methodology two different oxazole regioisomers (general structures CMPD9 & CMPDIO) or the imidazole derivative (represented by two tautomeric forms CMPD11 & CMPD12) may be produced in different ratios depending on the reaction conditions. The "R" group appending the oxazole or imidazole moiety is derived from the aldehyde and the "R" group can be varied by utilizing different aldehydes (or a different electrophile such as triethyl orthoformate) in the reaction. For example, the "R" group can be H, phenyl, naphthyl, 2-methoxy-phenyl, 2-fluoro-phenyl, 3-methoxy-phenyl, 3-fluoro-phenyl, 4- methoxy-phenyl, 4-fluoro-phenyl, 1,2-dimethoxy-phenyl, etc.

[0021] Alternatively, oxazole functionality may be introduced into a pseudopterosin semi- synthetically by reaction between the aglycone and an amino acid (Scheme 3; pseudopterosin derivatization; Method B). This process may be enhanced by utilizing an appropriate catalyst or oxidant [preferably a Ag(I) derivative]. Following this methodology, two different oxazole regioisomers (general structures CMPD9 & CMPDIO) may be produced in different ratios depending on the reaction conditions. The "R" group appending the oxazole moiety is derived from the amino acid and the "R" group can be varied by utilizing different amino acids in the reaction (e.g., alanine, asparagine, aspartic acid, arginine, cysteine, glutamine, glycine, glutamic acid, histidine, isoleucine, lysine, leucine, phenylalanine, methionine, serine, proline, tryptophan, threonine, tyrosine, valine, selenocysteine, pyrrolysine, taurine, carnitine, gaba, lanthionine, 2-aminoisobutyric acid, dehydroalanine, citrulline, or other amino acids having the structure (CH ₂ )nH, (CH ₂ )nOH, (CH ₂ )nC0 ₂ Me, or branched chain derivatives where n = 1-5).

[0022] Quinoxiline functionality may be introduced into pseudopterosins semi-synthetically by reaction between the aglycone, an appropriate catalyst or oxidant [such as a silver(I) salt], and ethylenediamine or a derivative of ethylenediamine (Scheme 4; pseudopterosin derivatization; Method C). Following this methodology, quinoxaline derivatives of general structures CMPD13 & CMPD14 may be produced in different ratios depending on the reaction conditions. The "Ri" and "R ₂ " groups appending the quinoxaline moiety are derived from the ethylenediamine derivative and these can be varied by utilizing different ethylenediamine derivatives in the reaction. . The "Ri" and "R ₂ " groups can for example be: H, (CH2)nH, (CH2)nOH, (CH2)nC02Me, or phenyl (where n = 1-5). The substituent "R ₃ " may be introduced by utilizing Dess-Martin periodinane (or alternative oxidants) and an alcohol in the reaction, or by other methods evident to those skilled in the art of chemistry. The "R ₃ " groups may include (CH2)nH or (CH2)nOH (where n = 1-5).

Anti-bacterial Activity

[0023] The anti-bacterial activity of the compounds described herein can be analyzed by methods well known in the art. In general, these methods involve adding the compound being tested to a culture of a particular species or strain of bacteria, and then assessing whether the compounds retard bacterial replication (i.e., exhibit bacteriostatic properties) and/or directly kill the bacteria (i.e., exhibit bacteriocidal properties). In the experiments described herein, the compounds were analyzed for activity against mycobacteria, Staphylococcus aureus, and enterococci. An example of a biological model to assay the ability of a compound to target non-replicating Mycobacterium tuberculosis (MTB) is the Low Oxygen Recovery Assay (LORA). Cho et al., Antimicrob. Agents Chemother. 2007, 51 : 1380-1385. This provides complimentary information to more traditional MTB assays, such as the microplate Alamar Blue assay (MABA) (Franzblau et al., J. Clin. Microbiol. 1998, 36:362-366) which may be used to measure the activity (MIC = Minimum Inhibitory Concentration) of compounds against the clinical MTB isolate H ₃ 7rv. The semi-synthetic pseudopteroxazole congeners described herein display anti-mycobacterial activity against different mycobacteria including activity against a non-replicating Mycobacterium tuberculosis (MTB) model in the LORA assay. These congeners can exhibit increased potency against mycobacteria compared to the natural pseudopteroxazoles (CMPD1 & CMPD2). A specific example of this is the semisynthetic compound CMPD29, which showed minimum inhibitory concentrations (MICs) of 13.2 μ& ^/ ηιί and 12.5 in the MABA and LORA assays, respectively, while displaying an IC ₅ 0 >128 μg/mL against Vero cells (see Falzari et al., Antimicrob. Agents Chemother. 2005, 49: 1447-1454). While pseudopteroxazole (CMPD1) has been previously shown to be active against MTB H ₃₇ rv, for the first time, activity against non-replicating MTB has been identified, as measured by the LORA assay (LORA MIC 49.8 μg/mL). Furthermore, pseudopteroxazole was found to be non-toxic against Vero cells, with 0% toxicity at 128 (Table 3, below). [0024] Pseudopterosin and certain pseudopterosin derivatives also display antibiotic activity, including activity against MTB and activity against non-replicating MTB in the LORA assay. Specific examples of this include the pseudopterosin mono methyl ether (CMPD15), which showed MICs of 30 μg/mL and 52.0 μg mL in the MABA and LORA assays, respectively, while displaying an IC50 >128 μg/mL against Vero cells. An additional aspect of this invention is the discovery that certain simplified prenylated aromatic structural mimics of pseudopterosins/pseudopteroxazoles (as represented by CMPD8) possess antimycobacterial activity, including LORA activity (Table 3, below). Compounds such as CMPD8 may be synthesized by prenylation reactions on various phenol or other aromatic derivatives.

[0025] Activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) was conducted using a microbroth dilution antibiotic susceptibility assay. Each compound was tested against each organism. Stock solutions of each compound were prepared and serially diluted into wells of a 96 well plate containing the bacteria in CAMH broth. The optical density of the wells in the plate was recorded at 600 nm at time zero and then again after incubation of the plates for 22 hours at 37°C. After subtracting the time zero OD ₆₀₀ from the final reading the percentages of microorganism survival relative to vehicle control wells were calculated and the IC50 was determined. Those compounds that displayed activity against MRSA and/or VRE are shown in Table 3 (below).

Reaction Conditions, Materials, and Purification

[0026] It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures. Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be used to prevent certain functional groups from undergoing undesired reactions. Suitable protecting groups for various functional groups as well as suitable conditions for protecting and deprotecting particular functional groups are well known in the art. See, for example, T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and references cited therein.

[0027] For compounds containing one or more chiral centers, if desired, such compounds can be prepared or isolated as pure stereoisomers, i.e., as individual enantiomers or diastereomers, or as stereoisomer-enriched mixtures. All such stereoisomers (and enriched mixtures) are included within the scope of this invention, unless otherwise indicated. Pure stereoisomers (or enriched mixtures) may be prepared using, for example, optically active starting materials or stereoselective reagents well-known in the art. Alternatively, racemic mixtures of such compounds can be separated using, for example, chiral column chromatography, chiral resolving agents and the like.

[0028] The starting materials for the reactions described herein are generally known compounds or can be obtained as described herein and/or by known procedures or modifications thereof which would be apparent to one of skill in the art. For example, many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wis., USA), Bachem (Torrance, Calif., USA), Emka-Chemce or Sigma (St. Louis, Mo., USA). Others may be prepared by procedures adapted from standard reference texts such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15 (John Wiley and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplemental (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley and Sons, 4.sup.th Edition), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

[00291 The compounds disclosed in this application may be purified by a variety of different techniques known to those skilled in the art of chemistry. These can include low, medium and high pressure chromatography utilizing a variety of different stationary phases, liquid-liquid extraction and counter current chromatography. Compounds may be utilized in a pure form, or as mixtures (e.g., a mixture of two regioisomers).

Pharmaceutical Formulations

[0030] The compounds described herein, derivatives and analogues thereof, and suitable salts of the foregoing can be included along with one or more pharmaceutically acceptable carriers or excipients to make pharmaceutical compositions which can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, insufflation, and intranasal administration. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985).

[0031] The active ingredient can be mixed with an excipient, diluted by an excipient, and/or enclosed within a carrier which can be in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. The compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile liquids for intranasal administration (e.g., a spraying device), or sterile packaged powders. In preparing a formulation, it may be necessary to mill the active compound to provide the appropriate particle size prior to combining with the other ingredients. Examples of excipients include: lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.

[0032] For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound. Tablets or pills may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate. [0033] Liquid forms of the compositions include aqueous solutions, aqueous or oil suspensions, and emulsions. To enhance serum half-life, the compounds may be encapsulated, introduced into the lumen of liposomes, prepared as a colloid. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al., U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028 each of which is incorporated herein by reference.

[0034] Other active ingredients might also be included in, or co-administered with, the pharmaceutical compositions. These might be other anti-bacterial drugs including penicillins and beta lactamase inhibitors such as ampicillin, bacampicillin, carbenicillin indanyl, mezlocillin, piperacillin, ticarcillin, amoxicillin-clavulanic acid, ampicillin-sulbactam, benzylpenicillin, cloxacillin, dicloxacillin, methicillin, oxacillin, penicillin g, penicillin v, piperacillin tazobactam, ticarcillin clavulanic acid, and nafcillin; cephalosporins such as cefadroxil, cefazolin, cephalexin, cephalothin, cephapirin, cephradine, cefaclor, cefamandol, cefonicid, cefotetan, cefoxitin, cefprozil, ceftmetazole, cefuroxime, loracarbef, cefdinir, ceftibuten, cefoperazone, cefixime, cefotaxime, cefpodoxime proxetil, ceftazidime, ceftizoxime, ceftriaxone, and cefepime; macrolides and lincosamines such as azithromycin, clarithromycin, clindamycin, dirithromycin, erythromycin, lincomycin, and troleandomycin; quinolones and fluoroquinolones such as cinoxacin, ciprofloxacin, enoxacin, gatifloxacin, grepafloxacin, levofloxacin, lomefioxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin, oxolinic acid, gemifloxacin, and perfloxacin; carbepenems such as imipenem-cilastatin and meropenem; monobactams such as aztreonam; aminoglycosides such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin and paromomycin; glycopeptides such as teicoplanin and vancomycin; tetracyclines such as demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, tetracycline, and chlortetracycline; sulfonamides such as mafenide, silver sulfadiazine, sulfacetamide, sulfadiazine, sulfamethoxazole, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole, and sulfamethizole; rifampins such as rifabutin, rifampin, and rifapentine; oxazolidonones such as linezolid, streptogramins, quinopristin, and dalfopristin; and others such as bacitracin, chloramphenicol, fosfomycin, isoniazid, methenamine, metronidazol, mupirocin, nitrofurantoin, nitrofurazone, novobiocin, polymyxin, spectinomycin, trimethoprim, colistin, cycloserine, capreomycin, ethionamide, pyrazinamide, para-aminosalicyclic acid, and erythromycin ethylsuccinate. For treating mycobaterial infections, these might include one or more of: ethambutol, isoniazid pyrazinamide, rifampicin, streptomycin aminoglycosides (e.g., amikacin and kanamycin), polypeptides (e.g., capreomycin, viomycin, and enviomycin), fluoroquinolones (e.g., ciprofloxacin, levofloxacin, and moxifloxacin), thioamides (e.g., ethionamide and prothionamide), cycloserine, p-aminosalicylic acid, rifabutin, macrolides such as clarithromycin, linezolid, thioacetazone, thioridazine, arginine, and R207910.

[0035] The compositions are preferably formulated in a unit dosage form, e.g., each dosage containing 5 to about 1200 mg (e.g., 5, 10, 15, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, and 1200 mg unit dosage forms) of the active ingredient. The amount administered to the patient will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the patient, the manner of administration, and the like all of which are within the skill of qualified physicians and pharmacists. In therapeutic applications, compositions are administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. Amounts effective for this use will depend on the disease condition being treated as well as by the judgment of the attending clinician depending upon factors such as the severity of the symptoms, the age, weight and general condition of the patient, and the like.

Methods of Use

[0036] The invention features methods for treating a patient (e.g., a human subject or an animal such as a dog or cat) having a bacterial infection (particularly a mycobacteria infection such as one caused by MTB, or a gram positive infection) by administering to the subject one or more of the compounds described herein in an amount and dosage schedule effective to cure, treat, reduce the number of infection-causing bacteria in a subject, and/or reduce a symptom of the infection.

EXAMPLES

[0037] In the following examples, the general experimental conditions and materials used were as follows:

[0038] NMR spectra were obtained on a Bruker 600 MHz NMR spectrometer operating at 600 and 150 MHz for Ή and ¹³ C, respectively. Chemical shifts are reported in ppm and were referenced to residual solvent signals: CDC1 ₃ (5H 7.26; 5c 77.0). Low resolution (nominal mass) mass spectra were obtained using a Finnigan LXQ ion trap mass spectrometer fitted with either an APCI or ESI source: samples were typically analyzed by LCMS using either an analytical HPLC or a UPLC column, with hyphenated MS-ELSD- UV detection. High resolution mass spectra were measured on a Bruker microTof Focus orthogonal ESI-TOF mass spectrometer. Optical rotations were measured on a Rudolp Autopol III Polarimeter. Infrared spectra were recorded using attenuated total reflectance, with samples deposited as a thin film on a Thermo Nicolet 6700 FT-IR spectrometer (Smart iTR). Flash chromatography was conducted utilizing a Teledyne Combiflash Rf, with UV detection triggered fraction collection using RediSep Columns.

[0039] Pseudopterosin starting materials (CMPD3) were isolated from samples of P. elisabethae obtained from Victory Reef, Bahamas. The pseudopterosins were purified by a combination of solvent extraction, liquid-liquid partitioning, and chromatography in a manner similar to that described in U.S. patent number 6,787,571. The major pseudopterosin in the sample possessed the pseudopterosin G-J aglycone skeleton (CMPD4). All other starting materials, reagents, and solvents were commercially available and were generally obtained from one of the following three suppliers: VWR, Sigma- Aldrich or Fisher Scientific.

Example 1 - Preparation of pseudopterosin G-J aglycone.

[0040] A mixture of pseudopterosins G-J (CMPD3) was refluxed, under an atmosphere of N ₂ , in MeOH H— I N HC1 for 1-3 hrs. After cooling, the sample was neutralized with aqueous NaHC0 ₃ , and then the aglycone was isolated by partitioning the sample between H ₂ 0 and an appropriate immiscible organic solvent such as EtOAc or CHC1 ₃ . Concentration of the organic phase yielded the aglycone (CMPD4), which may be used without further purification. The aglycone of pseudopterosin C is reportedly prone to decomposition on attempted purification, thus, the aglycone was stored dry at -20°C.

[0041] Aglycone (CMPD4): immobile brown oil; Ή NMR (CDC1 ₃ , 600 MHz), ¹³ C NMR (CDC1 ₃ , 150 MHz) δ 140.0, 139.9, 131.9, 131.4, 130.1 , 128.3, 125.3, 120.4, 44.4, 40.1, 36.9, 34.0, 32.0, 28.4, 27.8, 25.4, 23.1, 19.8, 17.5, 11.9; APCIMS m/z 301 [M+H] ⁺ .

Example 2 - Preparation of monomethyl ether of the aglycone of pseudopterosin G-J.

[0042] A solution of pseudopterosins G-J (CMPD4, 144 mg, 0.29 mmol), K ₂ C0 ₃ (1 g, excess), and Mel (caution, excess) in re-distilled dry acetone was refluxed under N ₂ for 6 hrs. The products were then partitioned between CHCI3 and H ₂ 0, and the organic phase was dried (MgS0 ₄ ), and then concentrated in vacuo to give an orange/brown oil (181 mg). This crude methyl ether was then dissolved in 1.5 N HCl in MeOH and refluxed under N ₂ for 3 hrs. The products were partitioned between CHCI3 and H ₂ 0 and the organic phase yielded the aglycone methyl ether (CMPD15, 83 mg, 0.26 mmol, 90% over 2 steps). CMPD15: [α] ²⁵ _D = +99 (c 0.5, CHCI3). IR v _max 3527, 2922, 2858, 1457, 1056 cm ^"1 ; APCIMS m/z 315 [M+H] ⁺ ; Ή NMR & ¹³ C NMR consistent with literature values. HRMS- ES miz [M + H] ⁺ 315.2304 (calcd for C ₂ iH ₃ i0 ₂ , 315.2319).

Example 3 - Preparation of a dimethyl ether (CMPD16).

[0043] A stirred solution of CMPD15 (25.5 mg, 0.083 mmol) in dry THF (5 mL), under N ₂ , was allowed to react with an excess of NaH for 2 hr. Mel was then added (200 μί, excess) and the solution was left stirring at room temperature overnight. The reaction was then carefully quenched with MeOH (1 mL), and excess Mel was removed under a stream of N ₂ . The products were then partitioned between EtOAc and H ₂ 0. The EtOAc fraction was concentrated in vacuo to give the crude product which was purified by flash chromatography (4 g silica column, hexane/MTBE gradient). The desired dimethyl ether (CMPD16) was obtained as a colorless amorphous semi-solid (18.2 mg, 0.055 mmol, 67 %). CMPD16: amorphous solid; [a] ²⁵ _D +69.5 (c 0.573, CHCI3); IR v _max 2924, 2861, 1460, 1069 cm ^"1 ; Ή NMR (CDCI3, 600 MHz) δ 4.99 (d, 1H, J = 9.2 Hz), 3.88 (s, 3H), 3.81 (s, 3H), 3.73 (q, 1H, J = 8.8Hz), 3.26 (m, 1H), 2.15-2.05 (m, 3H), 2.11 (s, 3H), 2.00 (m, 1H), 1.76 (br s, 3H), 1.71 (br s, 3H), 1.39 (m, 1H), 1.31-1.23 (m, 2H), 1.28 (d, 3H, J = 6.9Hz), 1.07 (d, 3H, J = 6.0Hz), 0.98 (m, 1H); ¹³ C NMR (CDC1 ₃ , 150 MHz) δ 149.4, 149.0, 135.3, 134.0, 133.0, 130.9, 128.5, 128.4, 60.1, 59.8, 44.0, 40.1, 37.3, 34.0, 31.3, 28.3, 27.5, 25.4, 24.3, 20.0, 17.5, 12.1; APCIMS m/z 329 [M+H] ⁺ ; HRMS-ES miz [M + H] ⁺ 329.2459 (calcd for C ₂₂ H ₃₃ 0 ₂ , 329.2475).

Example 4 - Preparation of a monopentyl ether (CMPD17).

[0044] A solution of pseudopterosins G-J (CMPD3, 102 mg, 0.2 mmol), K ₂ C0 ₃ (1 g, excess) and iodopentane (2 mL, excess) in re-distilled dry acetone was refluxed under N ₂ for 18 hrs. The products were then partitioned between EtOAc and H ₂ 0 and the organic phase was dried (MgS0 ₄ ) and then concentrated in vacuo to give an orange/brown oil (127 mg). This crude product was then dissolved in 1.5 M HCl in MeOH (10 mL) and refluxed under N ₂ for 2 hrs. The products were partitioned between EtOAc and H ₂ 0 and the organic phase dried in vacuo to yield the crude product, which was then purified by flash chromatography (43 g C18 column, MeOH/H ₂ 0 gradient) to yield the pentyl ether (CMPD17) as an immobile oil (40.3 mg, 0.11 mmol, 52% over 2 steps). CMPD17: immobile oil; [ ] ²⁵ _D +71.6 (c 0.787, CHC1 ₃ ); IR v _max 3523, 2924, 2857, 1456, 1056 cm ^"1 ; Ή NMR (CDC1 ₃ , 600 MHz) δ 5.70 (s, 1H), 4.97 (d, 1H, J = 9.2 Hz), 3.84 (m, 1H), 3.75 (m, 1H), 3.67 (m, 1H), 3.17 (m, 1H), 2.17 (m, 1H), 2.07 (s, 3H), 2.06-2.02 (m, 2H), 1.97 (m, 1H), 1.80 (m, 2H), 1.73 (s, 3H), 1.68 (s, 3H), 1.46 (m, 2H), 1.40 (m, 2H), 1.34-1.31 (m, 1H), 1.30 (d, 3H, J = 6.8 Hz), 1.23-1.21 (m, 2H), 1.03 (d, 3H, J = 6.0Hz), 0.95 (m, 1H), 0.95 (t, 3H, 7.2 Hz); ¹³ C NMR (CDC1 ₃ , 150 MHz) δ 144.8, 142.8, 135.6, 131.3, 129.8, 128.3, 126.7, 125.5, 73.6, 44.7, 40.2, 37.0, 34.0, 32.1, 30.1, 28.8, 28.2, 27.8, 25.4, 23.0, 22.6, 20.0, 17.5, 14.0, 12.7; APCIMS m/z 371 [M+H] ⁺ ; HRMS-ES m/z [M + H] ⁺ 371.2942 (calcd for C ₂₅ H ₃₉ 0 ₂ , 371.2945).

Example 5 - Preparation of a triflate (CMPD18). [0045] To a stirred, ice-cooled solution of CMPD15 (178 mg, 0.56 mmol) and Hunig's base (2 mL) in dry toluene (8 mL) was added triflic anhydride (6 mmol) in DCM. The reaction was stirred under N ₂ , and allowed to warm to room temperature overnight before being partitioned between DCM and aqueous HCl (1 N). Concentration of the organic phase in vacuo yielded the crude inflate; purification was achieved by flash chromatography (40 g silica column, hexane/EtOAc gradient) to yield the triflate (CMPD18, 177 mg, 0.4 mmol, 70%, -90% pure by ELSD LCMS). A portion of the product was further purified by RP-HPLC (Phenomenex, phenylhexyl, 5 μηι, 250 x 10 mm, 4.0 mL/min) using a gradient of MeOH/H ₂ 0 (9:1 for 1 mins, then to 10:0 over 1-4 mins; eluted across 9.9 to 10.25 min) to yield a white amorphous solid. CMPD18: amorphous solid; [ot] ²⁵ D +62.8 (c 0.205, CHC1 ₃ ); IR v _max 2928, 2869, 1416, 1206, 1139 cm ^-1 ; Ή NMR (CDC1 ₃ , 600 MHz) δ 4.93 (d, 1H, J= 9.3 Hz), 3.74 (s, 3H), 3.72 (m, 1H,), 3.23 (dd, 1H, J = 7.2, 14.6 Hz), 2.18-2.06 (m, 3H), 2.12 (s, 3H), 2.01 (m, 1H), 1.74 (br s, 3H), 1.70 (br s, 3H), 1.37 (m, 1H), 1.26-1.21 (m, 2H), 1.25 (d, 3H, J = 6.9 Hz), 1.04 (d, 3H, J = 5.9 Hz), 0.97 (m, 1H); ¹³ C NMR (CDC1 ₃ , 150 MHz) δ 147.9, 140.1 , 139.7, 136.7, 132.6, 129.7 (2xQ, 129.5, 118.7 (q, J = 320 Hz), 117.6, 60.8, 44.0, 39.8, 37.4, 33.9, 30.9, 28.6, 27.0, 25.4, 23.3, 19.8, 17.6, 12.5; APCIMS m/z 447 [M+H] ⁺ ; HRMS-ES mlz [M + H] ⁺ 447.1804 (calcd for C ₂₂ H ₃ oF ₃ 0 ₄ S, 447.1811).

Example 6 - Preparation of a carbamate (CMPD19).

[0046] A stirred solution of CMPD15 (25.5 mg, 0.083 mmol) in dry THF (5 mL), under N ₂ , was allowed to react with an excess of NaH for 2 hrs. Dimethyl carbamoyl chloride was then added (200 μΐ., excess) and the solution was left stirring overnight. The reaction was then carefully quenched with MeOH (1 mL) and the products were portioned between EtOAc and H ₂ 0. The EtOAc fraction was concentrated in vacuo to give the crude product which was purified by flash chromatography (4 g silica column, hexane/MTBE gradient). The carbamate was obtained as an amorphous solid (CMPD19, 13.7 mg, 0.035 mmol, 43%). Carbamate (CMPD19): amorphous solid; [a] D +110.2 (c 0.07, CHC1 ₃ ); IR v _max 2924, 2861, 1723 (CO), 1451, 1386, 1164 cm ^-1 ; Ή NMR (CDC1 ₃ , 600 MHz) δ 4.97 (d, 1H, J= 9.2 Hz), 3.70 (s, 3H), 3.71-3.67 (m, 1H,), 3.16 (s, 3H), 3.04 (s, 3H), 3.03 (m, 1H), 2.14- 2.10 (m, 2H), 2.07 (s, 3H), 2.02-2.07 (m, 2H), 1.94-1.98 (m, 1H) 1.73 (br s, 3H), 1.68 (br s, 3H), 1.34-1.16 (m, 6H), 1.01 (d, 3H, J = 6.1 Hz), 0.99 (m, 1H); ¹³ C NMR (CDC1 ₃ , 150 MHz) δ 154.6, 148.6, 140.9, 136.4, 135.3, 130.6, 128.7, 128.3, 60.5, 44.2, 40.0, 37.3, 36.8, 36.4, 33.7, 31.5, 28.6, 27.3, 25.4, 23.8, 19.9, 17.5, 12.3. (two quaternary carbon resonances overlap); APCIMS m/z 386 [M+H] ⁺ ; HRMS-ES m/z [M + Na] ⁺ 408.2505 (calcd for C ₂₄ H ₃₅ N0 ₃ Na, 408.2509).

Example 7 - Aldehyde Route to Pseudopteroxazole Derivatives.

[0047] In a general procedure for the synthesis of pseudopteroxazole congeners from the pseudopterosin aglycone (CMPD4) with an ammonium source and an aldehyde, a mixture of the pseudopterosin aglycone (CMPD4), and NH ₄ OAc (> 1 equiv) in AcOH or another solvent is stirred at room temperature for approximately 10 mins. During this time air may be bubbled into the flask, or an oxidant may be added. The aldehyde is then added and the reaction refluxed until all the starting aglycone has been consumed (monitored by TLC, LCMS etc). The products are typically isolated by partitioning the products between H ₂ 0 and an appropriate organic solvent (e.g. EtOAc) and then the organic soluble material is purified chromatographically. Where necessary or desired, individual regioisomers may be separated by HPLC. Typically this procedure yields a -3:1 ratio of the pseudopteroxazole- like regioisomer (CMPD9) versus the isopseudopteroxazole-like regioisomer (CMPD10).

Example 8 - Preparation of pseudopteroxazole (CMPD1) and isopseudopteroxazole (CMPD20).

[0048] A sample of the aglycone (CMPD4, 179 mg, 0.6 mmol) was dissolved in AcOH (5 mL) and air was bubbled into the solution for 10 min. Paraformaldehyde (100 mg) and NH ₄ OAc (1 g) were then added and the reaction was then refluxed for 7 hr. The reaction products were then partitioned between EtOAc and H ₂ 0, and the EtOAc soluble material was then subjected to reversed phase flash chromatography (43 g C-18 column) using a MeOH/H ₂ 0 gradient (40:60 to 100:0). After analysis by LCMS, the fractions were recombined and concentrated in vacuo. One fraction was a mixture of pseudopteroxazole and isopseudopteroxazole (95.5 mg, 0.31 mmol, 52%; 3:1 ratio CMPD1:CMPD20 by NMR). A more polar fraction contained hydrated pseudopteroxazoles (38 mg, 12 mmol, 19%), that inter-converted to the pseudopteroxazoles upon standing in CDC1 ₃ over a few days (total yield pseudopteroxazole derivatives = 71%). The pseudopteroxazole regioisomers were separated by normal phase flash chromatography (40 g Silica column) using a MTBE/Hexane gradient (0: 100 to 25:75) to give pure pseudopteroxazole (CMPD1, 52 mg, 0.17 mmol, 29%) and isopseudopteroxazole (CMPD20, 12.5 mg, 0.04 mmol, 7%).

[0049] Pseudopteroxazole (CMPD1): oil; [α] ²⁵ _D +100 (c 0.2, CHC1 ₃ ); ¹ H NMR (see Fig. 1) consistent with literature values. APCIMS m/z 310 [M+H] ⁺ ; HRMS-ES m/z [M + H] ⁺ 310.2154 (calcd for C ₂₁ H ₂₈ NO, 310.2165).

[0050] CMPD20: oil; [α] ²⁵ _D +114 {c 1.29, CHC1 ₃ ); IR v _max 2946, 2921, 2855, 1445, 1088 cm ^-1 ; ¹ H NMR see Fig. 1; APCIMS m/z 310 [M+H] ⁺ ; HRMS-ES m/z [M + H] ⁺ 310.2150 (calcd for C ₂₁ H ₂₈ NO, 310.2165).

Example 9 - Preparation of o-Anisaldehyde derivatives (CMPD22 & CMPD23).

[0051] A sample of pseudopterosins G-J (CMPD3, 38 mg, 0.078 mmol) was refluxed in methanolic HC1 (1.5 N, 10 mL) under N ₂ for 2.5 h. The solvent was removed under a stream of nitrogen and then NH ₄ C0 ₂ H (1 g, excess) and AcOH (4 mL) were added to the residue. After the subsequent effervescence ceased, air was bubbled into the vessel for 10 mins, and then ortAo-anisaldehyde (45 mg, 0.3 mmol) was added. The reaction was then heated at 110°C overnight. The cooled reaction contents was then partitioned between CHCI3 and H ₂ 0, and the CHCI ₃ layer was concentrated in vacuo to give a residue that was purified by flash chromatography (silica, hexane/ethylacetate) to give the title compounds (16.7 mg, 0.040 mmol, 52%; 2.6: 1 ratio CMPD22:CMPD23). A portion of this material was further purified by RP-HPLC (Phenomenex, phenylhexyl, 5 μχα, 250 x 10 mm, 4.0 mL/min) eluted with MeOH/H ₂ 0 (isocratic 95:1) to give CMPD23 (1.3 mg, eluted 14.9 to 15.6 min) and CMPD22 (4.8 mg, eluted 15.6 to 16.8 min).

[0052] CMPD22: amorphous solid; [α] ²⁵ D +76 (c 0.25, CHCI ₃ ); IR v _max 2920, 2853, 1464, 1257, 1025 cm ^-1 ; ¹ H NMR see Fig. 1; APCIMS m/z 416 [M+H] ⁺ ; HRMS-ES m/z [M + H] ⁺ 416.2568 (calc'd for C ₂₈ H ₃₄ NO ₂ , 416.2584). [00531 CMPD23: amorphous solid; [α] ²⁵ _D +72 (c 0.07, CHC1 ₃ ); IR v _max 2923, 2854, 1484, 1257, 1025 cm ^-1 ; Ή see Fig. 1 ; APCIMS m/z 416 [M+H] ⁺ ; HRMS-ES m/z [M + H] ⁺ 416.2569 (calc'd for C ₂₈ H ₃₄ NO ₂ , 416.2584).

Example 10 - Preparation of a /?-Fluoroaldehyde derivative (CMPD24).

A solution of the aglycone (CMPD4, 41 mg, 0.14 mmol), NH ₄ OAc (1 g, excess) and 4- fluorobenzaldehyde (800 μΙ , excess) was refluxed in AcOH (4 mL) for 16 h. The cooled reaction mixture was subsequently partitioned between EtOAc and aqueous HCl (1 N). The organic soluble material was purified by flash chromatography over silica (hexane/ethyl acetate 10:0 to 9: 1 gradient) to give a mixture of the mixed oxazole regioisomers (27.6 mg, 0.068 mmol, 49 %; 3.5: 1 ratio normal:iso regioisomer). A portion of the major regioisomer (CMPD24) was further purified by RP-HPLC (Phenomenex, phenylhexyl, 5 /an, 250 x 10 mm, 4.0 mL/min) using isocratic MeOH (eluted across 10.0 to 10.4 min).

[0054] CMPD24: immobile oil; [α] ²⁵ _D +99 (c 0.14, CHC1 ₃ ); IR v _max 2922, 2855, 1501 , 841 cm ^-1 ; ¹ H NMR (CDC1 ₃ , 600 MHz) δ 8.23 (m, 2H, H-23/H-27), 7.19 (app. t, 2H, J = 8.6 Hz, H-24/H-26), 5.00 (d, 1H, J = 9.4 Hz, H-14), 3.96 (m, 1H, H-l), 3.31 (m, 1H, H-7), 2.48 (s, 3H, H-20), 2.31-2.10 (m, 4H), 1.79 (s, 3H, H-17), 1.69 (s, 3H, H-16), 1.54 (d, 3H, J = 6.8 Hz, H-19), 1.48-1.42 (m, 1H, H-6b), 1.32-1.26 (m, 2H), 1.11 (m, 1H, H-5b), 1.06 (d, 3H, J = 5.7 Hz, H-18); ¹³ C NMR (CDC1 ₃ , 150 MHz) δ 164.4 (d, J = 251.4 Hz), 160.6, 147.9, 140.1 , 136.3, 134.7, 130.8, 129.54 (d, J = 8.6 Hz), 128.7, 126.4, 124.3, 121.9, 115.9 (d, J =22.1 Hz), 44.8, 40.1 , 36.5, 34.5, 32.3, 30.4, 28.1, 25.4, 22.3, 19.8, 17.6, 13.5. APCIMS m/z 404 [M+H] ⁺ ; HRMS-ES m/z [M + H] ⁺ 404.2384 (calc'd for C ₂₇ H ₃₁ FNO, 404.2384).

Example 11 - Amino Acid Route to Pseudopteroxazole Derivatives

[0055] The NMR data for the compounds prepared in this section are tabulated for easy comparison in Fig. 1. In a general procedure for the synthesis of pseudopteroxazole congeners from the pseudopterosin aglycone and amino acids, a mixture of the pseudopterosin aglycone (CMPD4) and an oxidant (typically Ag ₂ 0, 0.7 equiv) is refluxed in MeOH or EtOH for a short period (<1 hr). A given amino acid (>1 equiv) is then added and the reaction further refluxed until the starting aglycone has been consumed (monitored by TLC, LCMS etc). Additional batches of the amino acid or the oxidant may be added periodically (typically hourly) to drive the reaction to completion. The crude products are typically isolated by filtering the cooled crude reaction products through celite. Purification is achieved chromatographically. Where necessary or desired, individual regioisomers may be separated by HPLC. Typically this procedure yields an -9: 1 ratio of the pseudopteroxazole-like regioisomer versus the isopseudopteroxazole-like regioisomer.

Example 12 - Synthesis of pseudopteroxazole and isopseudopteroxazole via the pseudopterosin G-J aglycone and glycine.

[0056] A sample of CMPD4 (33.8 mg, 0.11 mmol) and Ag ₂ 0 (18.7 mg, 0.08 mmol) was refluxed in MeOH (30 mL) for one hr. The solution was then cooled to room temperature, and glycine (85 mg, 1.3 mmol) was added. The reaction mixture was refluxed for a further 19 hrs, with additional Ag ₂ 0 (0.08 mmol) being added two hrs into this timeframe. The cooled crude reaction mixture was then filtered through celite and dried in vacuo. The crude product (35 mg) was subjected to flash chromatography on a 13 g CI 8 column eluted with a gradient of MeOH/H ₂ 0 (from 4:6 to 1 :0) to yield a mixture of pseudopteroxazole and isopseudopteroxazole (21.4 mg, 0.069 mmol, 61.5%, 10: 1 ratio CMPDl: CMPD20 by NMR). Subsequently, the individual regioisomers were purified by flash chromatography on a 4 g silica column eluted with a gradient of hexane/ MTBE (from 100% hexane to 95% hexane over 15 min) to yield CMPDl and CMPD20. Example 13 - Preparation of homopseudopteroxazole (CMPD2).

[0057] Homopseudopteroxazole was synthesized from the pseudopterosin G-J aglycone (14.2 mg, 0.047 mmol), Ag ₂ O (2.3 equiv) and 2-amino heptanoic acid (3.1 equiv) following the general procedure. After purification by MPLC on silica, homopseudopteroxazole (CMPD2) was obtained in 34% isolated yield (6.1 mg).

[0058] CMPD2: ¹ H NMR see Fig. 1; APCIMS m/z 380 [M+H] ⁺ ; HRMS-ES mlz [M + H] ⁺ 380.2944 (calcd for C ₂₆ H ₃₈ NO, 380.2948).

Example 14 - Preparation of an alanine product (CMPD25).

[0059] The pseudopteroxazole C-21 methyl derivative (CMPD25) was synthesized from the pseudopterosin aglycone (CMPD4, 9.1 mg, 0.03 mmol), Ag ₂ 0 (2.4 equiv) and alanine (2.9 equiv) following the general procedure. After purification by MPLC on CI 8 the product was obtained in 33% isolated yield (3.2 mg). CMPD25: Thin film; [α] ²⁵ D +93 (c 0.38, CHC1 ₃ ); IR v _max 2922, 2855, 1609, 1583, 1445, 1376, 1063 cm ^-1 ; ¹ H see Fig. 1; APCIMS m/z 324 [M+H] ⁺ ; HRMS-ES mlz [M + H] ⁺ 324.2318 (calcd for C ₂₂ H ₃₀ NO, 324.2322).

Example 15 - Preparation of an isoleucine product (CMPD26).

[0060] The pseudopteroxazole C-21 sec-butyl derivative (CMPD26) was synthesized from the pseudopterosin aglycone (CMPD4, 10.5 mg, 0.035 mmol), Ag ₂ 0 (2.2 equiv) and isoleucine (4.3 equiv) following the general procedure. After purification by MPLC on C18 the product was obtained in 32 % isolated yield (4.1 mg). CMPD26: immobile oil; [α] ²⁵ D +104 (c 0.21, CHC1 ₃ ); IR v _max 2956, 2924, 2871, 1571, 1446 cm ^"1 ; Ή NMR see Fig. 1 ; APCIMS m/z 366 [M+H] ⁺ ; HRMS-ES mlz [M + H] ⁺ 366.2783 (calcd for C ₂₅ H ₃₆ NO, 366.2791).

Example 16 - Preparation of a phenylalanine product (CMPD27).

[0061] The pseudopteroxazole C-21 benzyl derivative (CMPD27) was synthesized from the pseudopterosin aglycone (CMPD4, 10.5 mg, 0.035 mmol), Ag ₂ 0 (2.0 equiv) and phenylalanine (4.5 equiv) following the general procedure. After purification by MPLC on C18 the product was obtained in 19 % isolated yield (2.6 mg). CMPD27: immobile oil; [α] ²⁵ _D +58.6 (c .130, CHC1 ₃ ); IR v _max 2922, 2855, 1603, 1574, 1453, 1374, 1062 cm ^-1 ; ¹ H NMR see Fig. 1; APCIMS m/z 400 [M+H] ⁺ ; HRMS-ES mlz [M + H] ⁺ 400.2615 (calcd for C ₂₈ H ₃₄ NO, 400.2635).

Example 17 - Preparation of a threonine product (CMPD28).

[0062] The pseudopteroxazole C-21 (1 -hydroxy ethyl) derivative (CMPD28) was synthesized from the pseudopterosin aglycone (CMPD4, 51 mg, 0.17 mmol), Ag ₂ 0 (2.0 equiv) and threonine (8.2 equiv) following the general procedure. After purification by MPLC on C18 the product was obtained in 29% isolated yield (17.2 mg). CMPD28: orange immobile oil; [α] ²⁵ _D +86 (c 0.57, CHC1 ₃ ); IR v _max 3359, 2924, 2856, 1446, 1374, 1073 cm ^-1 ; ¹ H NMR see Fig. 1 ; APCIMS m/z 354 [M+H] ⁺ ; HRMS-ES mlz [M + H] ⁺ 354.2422 (calcd for C ₂₃ H ₃₂ NO ₂ , 354.2428).

Example 18 - Preparation of a histidine product (CMPD29).

[0063] The pseudopteroxazole C-21 (lH-imidazol-4-yl) methyl derivative (CMPD29) was synthesized from the pseudopterosin aglycone (CMPD4, 193 mg, 0.64 mmol), Ag ₂ C0 ₃ (1.4 equiv) and histidine (6.7 equiv) following the general procedure. After purification by MPLC on diol-silica the product was obtained in 23% isolated yield (57.5 mg). In the MPLC purified product the ratio of normal to inverse regioisomer was 2.4:1. A portion of this material was subjected to RP-HPLC (Phenomenex, phenylhexyl, 5 μαι, 250 x 10 mm, 2.9 mL/min) eluted with MeOH:H ₂ 0:HC0 ₂ H (70:30:0.1). While the regioisomers eluted as one asymmetric peak (19.8 to 20.7 min), peak shaving lead to the isolation of an enriched fraction (3: 1 ratio of normal to inverse regioisomer), which was the material used for biological evaluation. CMPD29: orange immobile oil; [a] ²⁵ _D +129.4 (c 0.09, CHC1 ₃ ); IR v _max 2948, 2921, 2856, 1446, 1085 cm ^"1 ; Ή NMR see Fig. 1 ; APCIMS m/z 390 [M+H] ⁺ ; HRMS-ES m/z [M + H] ⁺ 390.25 390.2540).

Example 19 - Preparation of a methionine product (CMPD30).

[0064] The pseudopteroxazole C-21 (2-(methylthio)ethyl) derivative (CMPD30) was synthesized from the pseudopterosin aglycone (CMPD4, 20.5 mg, 0.068 mmol), Ag ₂ 0 (2.0 equiv) and methionine (10 equiv) following the general procedure. After purification by MPLC on CI 8 the product was obtained in 34% isolated yield (8.8 mg).

[0065] CMPD30: orange immobile oil; [α] ²⁵ _D +82 (c 0.44, CHC1 ₃ ); IR v _max 2921, 2854, 1605, 1576, 1445, 1375, 1063 cm ^"1 ; ¹ H NMR (CDC1 ₃ , 600 MHz) Ή NMR see Fig. 1 ; APCIMS m/z 384 [M+H] ⁺ ; HRMS-ES m/z [M + H] ⁺ 384.2344 (calcd for C ₂₄ H ₃₄ NOS, 384.2356).

Example 20 - Preparation of an asparagine product (CMPD31).

[0066] The pseudopteroxazole C-21 acetamide derivative (CMPD31) was synthesized from the pseudopterosin aglycone (CMPD4, 174 mg, 0.58 mmol), Ag ₂ 0 (1.3 equiv) and asparagine (7.8 equiv) following the general procedure. After purification by MPLC on diol-silica the product was obtained in 20% isolated yield (42.1 mg). CMPD31: light orange immobile oil; [oc] ²⁵ _D +107.3 (c 0.32, CHCI3); IR v _max 3342 (br), 3191, 2948, 2922, 2855, 1679 (CO), 1388, 1062 cm ^"1 ; 'H NMR see Fig. 1 ; APCIMS m/z 367 [M+H] ⁺ ; HRMS- ES m/z [M + Na] ⁺ 389.2190 (cal 389.2199).

Example 21 - Preparation of a glutamine product (CMPD32).

[0067] The pseudopteroxazole propanamide derivative (CMPD32) was synthesized from the pseudopterosin aglycone (CMPD4, 51 mg, 0.17 mmol), Ag ₂ 0 (1.4 equiv) and glutamine (8 equiv) following the general procedure. After purification by MPLC on CI 8 the product was obtained in 16% isolated yield (10.3 mg). CMPD32: orange immobile oil; [ ] ²⁵ _D +73 (c 0.29, CHC1 ₃ ); IR v _max 3346, 3198, 2923, 2855, 1672 (CO), 1444, 1374, 1064 cm ^"1 ; Ή NMR see Fig. 1 ; APCIMS m/z 381 [M+H] ⁺ ; HRMS-ES m/z [M + H] ⁺ 381.2525 (calcd for C ₂₄ H ₃₃ N ₂ 0 ₂ , 381.2537).

Example 22 - Preparation of a methyl glutamate product (CMPD33)

[0068] The pseudopteroxazole methyl propanate derivative (CMPD33) was synthesized from the pseudopterosin aglycone (CMPD4, 51 mg, 0.17 mmol), Ag ₂ 0 (1.4 equiv) and 2- amino-5-methoxy-5-oxopentanoic acid (7.9 equiv) following the general procedure. After purification by MPLC on CI 8 (43% yield at this stage) and then on silica the product was obtained in 25% isolated yield (16.6 mg). CMPD33: pale yellow oil; [α] ²⁵ _D + 82 (c 0.35, CHC1 ₃ ); IR v _max 2950, 2922, 2855, 1743 (CO), 1606, 1578, 1438, 1 166 cm ^'1 ; Ή NMR see Fig. 1; APCIMS m/z 396 [M+H] ⁺ ; HRMS-ES mlz [M + Na] ⁺ 418.2336 (calcd for C ₂₅ H ₃₃ NO ₃ Na, 418.2353).

Example 23 - Preparation of a free acid from methyl glutamate product (CMPD34)

[0069] To a portion of the above ester (CMPD33, 10 mg, 0.25 mmol) in THF (3 mL) was added LiOH solution (12 mL, 1 N, aqueous). The mixture was stirred for 21 hrs at 35°C. Dilute HC1 (0.5 N) was then added to adjust the pH to ~1 and the mixture was then partitioned between EtOAc and H ₂ 0. The combined EtOAc extracts were concentrated in vacuo to yield the carboxylic acid CMPD34 (7.2 mg, 0.19 mmol, 75%).

[0070] CMPD34: amorphous light yellow solid; [α] ²⁵ _D + 73.0 (c 0.36, CHC1 ₃ ); IR v _max 3000 (broad), 2921, 2856, 1714 (CO), 1576, 1443, 1 167 cnT ¹ ; Ή NMR see Fig. 1; APCIMS m/z 382 [M+H] ⁺ ; HRMS-ES mlz [M + H] ⁺ 382.2360 (calcd for C ₂₄ H ₃₂ NO ₃ , 382.2377).

Example 24 - Preparation of a quinoxaline derivative (CMPD35). [0071] A sample of pseudopterosins G-J (CMPD3, 0.041 mmol) was refluxed in methanolic HC1 (1.5 N, 10 mL) under N ₂ for 2.5 hrs. The crude mixture was then partitioned between EtOAc and H ₂ 0, and the EtOAc phase was concentrated in vacuo to give the crude aglycone. This material was dissolved in EtOH (15 mL) and air was bubbled through the sample for 10 mins. Ethylenediamine (50 iL, excess) and Ag ₂ 0 (14 mg, 0.065 mmol) were then added and the reaction was refluxed for 1.5 h. The reaction mixture was then filtered through Celite and partitioned between EtOAc and H ₂ 0. The EtOAc phase was concentrated and then purified by flash chromatography (silica, hexane/MTBE) to give CMPD35 (2 mg, 0.0063 mmol, 15% over two steps) as a yellow oil. CMPD35: yellow oil; [a] ²⁵ _D +103 (c 0.03, CHC1 ₃ ); IR v _max 2921, 2858, 1470 cm ^"1 ; Ή NMR (CDC1 ₃ , 600 MHz) δ 8.74 (m, 2H, H-20, H-21), 5.04 (d, 1H, J = 9.1Hz, H-14), 4.11 (app. q, 1H, J = 8.5 Hz, H- 1), 4.06 (app. q, 1H, J = 7.3 Hz, H-7), 2.64 (s, 3H, H-20), 2.33-2.39 (m, 1H), 2.23-2.28 (m, 1H), 2.18-2.23 (m, 1H), 2.11-2.17 (m, 1H), 1.82 (s, 3H, H-17), 1.70 (s, 3H, H-16), 1.54 (m, 1H), 1.40-1.42 (m, 2H), 1.39 (d, 3H, J = 6.9 Hz, H-19), 1.32-1.37 (m, 1H), 1.24-1.26 (m, 1H), 1.11 (d, 3H, J = 6.2 Hz, H-18) 1.09-1.14 (m, 1H); ¹³ C NMR (CDC1 ₃ , 150 MHz) δ 142.1, 142.0, 141.9, 141.3, 141.0, 140.1, 137.0, 132.2, 129.7, 129.7, 44.3, 39.5, 37.4, 33.8, 30.7, 28.5, 26.8, 25.5, 24.7, 20.4, 17.7, 12.8; APCIMS m/z 321 [M+H] ⁺ ; HRMS-ES m/z [M + H] ⁺ 321.2313 (calcd for C ₂₂ H ₂₉ N

Example 25 - Preparation of a quinoxaline (CMPD35) (alternate method) and the methyl ether derivatives (CMPD36).

[0072] To a solution of the pseudopterosin aglycone (CMPD4, 26.3 mg, 0.088 mmol) in DCM (10 mL + two drops H ₂ 0) was added Dess-Martin periodinane (68 mg, 0.16 mmol). After stirring for 15 min, MeOH (2 mL) and ethylenediamine (20 drops) were added. After another 45 min the solvent was removed in vacuo, and then isopropyl alcohol was added (20 mL). The solution was then refluxed overnight. After LCMS analysis additional ethylenediamine (6 drops) was added, and the solution refluxed for a further 24 hr. The reaction products were partitioned between EtOAc and H ₂ 0 and the organic phase was concentrated in vacuo to give an orange brown gum (31.8 mg). Purification by flash chromatography on diol modified silica (hexane - MTBE gradient) yielded the quinoxaline (CMPD35, 4.6 mg, 16 %) and the methyl ether (CMPD36, 1.6 mg, 5%). CMPD36: amorphous semi solid; [ ] ²⁵ _D -5.0 (c 0.08, CHC1 ₃ );IR v _max 2925, 2866, 1470, 1079 cm ^"1 ; Ή NMR (CDC1 ₃ , 600 MHz) δ 8.76 (s, 2H, H-20, H-21), 3.89 (app. q, 1H, J = 6.9 Hz), 3.71 (m, 1H), 3.20 (s, 3H, OMe), 2.79 (s, 3H, H-20), 2.49 (m, 1H), 2.21-2.15 (m, 3H), 1.89 (dd, 1H, J = 9.5 & 14.5 Hz, H-14-a), 1.68-1.62 (m, 2H), 1.54-1.50 (m, 1H), 1.41 (d, 3H, J = 6.9 Hz, H-19), 1.27 (s, 6H), 1.26 (m, 2H), 1.13 (d, 3H, J = 6.5 Hz); ¹³ C NMR (CDC1 ₃ , 150 MHz) 5144.7, 142.3, 141.8, 141.6, 141.3, 140.4, 136.5, 129.8, 75.3 (C-15), 49.2 (C-23), 48.6 (C-14), 42.3, 38.2, 34.7, 32.2, 30.3, 29.4, 25.8, 25.7, 25.4, 24.0, 21.0. 12.6; APCIMS m/z 353 [M+H] ⁺ ; HRMS-ES m/z [M + H] ⁺ 353.2582 (calcd for C ₂₃ H ₃₃ N ₂ 0, 353.2587).

Example 26 - Prenylation of 2,6-dimethoxyphenol: synthesis of CMPD37, CMPD38 & CMPD39.

[0073] A solution of 2-methyl-3-buten-2-ol (640 mg, 7.4 mmol) in DCM (3 mL) was added dropwise to a stirred mixture of 2,6-dimethoxyphenol (612 mg, 3.97 mmol) and TsOH (19 mg, cat) in DCM/MeOH (3: 1, 30 mL). After stirring for 96 hrs the solution was refluxed for 20 hrs and then partitioned between H ₂ 0 and EtOAc. The organic phase was concentrated to yield a crude oil (793 mg) which was subjected to flash chromatography on CI 8 (H ₂ O^MeOH gradient) to yield the mono-prenylated product (CMPD37, 239 mg, 1.08 mmol, 27%), the di-prenylated product (CMPD38, 96 mg, 0.33 mmol, 8 %), and the triprenylated product (CMPD39, 20 mg, 0.055 mmol, 1.4%) along with recovered starting material (321 mg, 52%).

[0074] CMPD37: oil; IR v _max 3456, 2931, 2835, 1493, 1288, 1090 cm ^"1 ; Ή NMR (CDC1 ₃ , 600 MHz) δ 6.63 (d, 1H, J = 8.5 Hz), 6.60 (d, 1H, J = 8.4 Hz), 5.53 (s, 1H, OH), 5.27 (m, 1H, H-8), 3.863 (s, 3H, OMe), 3.861 (s, 3H, OMe), 3.30 (d, 2H, J = 7.3 Hz); 1.73 (s, 6H, H-10 & H-l l); ¹³ C NMR (CDCI3, 150 MHz) δ 145.9, 145.2, 138.5, 132.0, 127.8, 123.1, 1 19.1, 106.4, 60.4, 56.2, 28.0, 25.7, 17.7; APCIMS m/z 223 [M+H] ⁺ ; HRMS-ES m/z [M + Naf 245.1 142 (calcd for Ci ₃ H, ₈ 0 ₃ Na, 245.1148).

[0075] CMPD38: pale yellow oil; IR v _max 3440, 2964, 2912, 1854, 1497, 1309, 11 16 cm ^"1 ; 1H NMR (CDC1 ₃ , 600 MHz) δ 6.50 (s, IH, H-5), 5.23 (m, IH, olefinic), 5.07 (m, IH, olefinic), 3.85 (s, 3H, OMe), 3.83 (s, 3H, OMe), 3.32 (d, 2H, J = 6.5 Hz), 3.25 (d, 2H, J = 6.5 Hz), 1.77 (s, 3H), 1.75 (s, 3H), 1.71 (s, 3H), 1.68 (s, 3H). ¹³ C NMR (CDC1 ₃ , 150 MHz) δ 145.5, 145.4, 136.7, 132.1, 131.0 (2*C), 126.0, 123.7, 123.4, 107.6, 60.6, 56.1, 31.4, 25.7, 25.6, 25.1, 17.9, 17.8; APCIMS m/z 291 [M+H] ⁺ ; HRMS-ES m/z [M + Naf 313.1763 (calcd for C _]8 H ₂₆ 0 ₃ Na, 313.1774).

[0076] CMPD39: colorless oil; IR v _max 3400, 2964, 2912, 2855, 1456, 1087 cm ^"1 ; Ή NMR (CDCI ₃ , 600 MHz) δ 5.49 (s, IH, OH), 5.09 (m, 2H, olefinic), 4.97 (m, IH, olefinic), 3.80 (s, 6H, OMe), 3.32 (d, 4H, J = 6.5 Hz), 3.24 (d, 4H, J = 6.5 Hz), 3.23 (d, 2H, J = 6.0 Hz), 1.74 (s, 6H), 1.68 (broad overlapping singlets, 12H). ¹³ C NMR (CDC1 ₃ , 150 MHz) δ 144.2, 140.1, 131.2, 131.0, 130.7, 129.5, 123.9, 123.8, 60.9, 27.8, 25.6, 25.55, 25.53, 17.90, 17.91; APCIMS m/z 359 [M+H] ⁺ ; HRMS-ES m/z [M + Naf 381.2409 (calcd for C ₂₃ H ₃₄ 0 ₃ Na, 381.2400).

Example 27 - Preparation of 4-(3-hydroxy-3-methylbutyl)-2,3,6-trimethylphenol (CMPD40).

[0077] A sample of 2-methyl-3-buten-2-ol (730 mg, 8.4 mmol) was added dropwise to a stirred mixture of 2,3,6-trimethylphenol (1.05 g, 7.7 mmol) in formic acid (10 mL) at 55°C over about five mins (exothermic). After stirring for 2.5 hrs at 50-55°C the solution was partitioned between aqueous NaHC0 ₃ solution (saturated) and EtOAc. The organic phase was concentrated to yield a crude oil (1.85 g) which was subjected to flash chromatography on CI 8 (H ₂ 0->MeOH gradient) to yield several fractions. One of these fractions (F 19-22) contained 4-(3-hydroxy-3-methylbutyl)-2,3,6-trimethylphenol (CMPD40, 313 mg, 1.4 mmol, 18%). CMPD40: amorphous white solid; I v _max 3400, 2969, 2942, 1480, 1203, 1087 cm ^"1 ; ^l H NMR (CDC1 ₃ , 600 MHz) δ 6.79 (s,lH), 4.54 (s, 1H), 2.63 (m, 2H), 2.21 (s, 3H), 2.20 (s, 3H), 2.19 (s, 3H), 1.67 (m, 2H), 1.31 (s, 6H). ¹³ C NMR (CDC1 ₃ , 150 MHz) δ 150.1, 130.1, 132.3, 128.5, 122.3, 119.9, 71.0, 46.3, 29.2, 28.4, 15.8, 15.3, 12.1; APCIMS m/z 221 [M-H] ^" ; HRMS-ES m/z [M +Na] ⁺ 245.1508 (calcd for Ci ₄ H ₂₂ 0 ₂ Na, 245.1512).

Example 28 - Biological Assays

[0078] The MABA, LORA and Vero cell assays were all conducted as previously described. Cho et al., Antimicrob. Agents Chemother. 2007, 51, 1380-1385; Franzblau, et al., J. Clin. Microbiol. 1998, 36, 362-366; and Falzari et al., Antimicrob. Agents Chemother. 2005, 49, 1447-1454. Activity against Mycobacterium smegmatis ATCC 12051 and Mycobacterium diernhoferi ATCC 19340 was conducted using a microbroth dilution antibiotic susceptibility assay. Testing was conducted in accordance with Clinical Laboratory Standards Institute testing standards (Susceptibility testing of Mycobacteria, Nocardiae and other aerobic actinomycetes; Approved Standard. M24-A Volume 23, number 18. Microbroth dilution method for antimicrobial testing of fast growing mycobacteria). Compounds were tested against each organism in triplicate. Compound were prepared in sterile 20% DMSO and serially diluted to generate a range of 8 concentrations. Replicates were performed on separate plates. Each plate contained four un- inoculated contamination controls (media + 20% DMSO), four untreated controls (media + 20% DMSO + organism) and one column containing a concentration range of a control antibiotic. Control antibiotics tested included rifampicin, ciprofloxacin and doxycycline. Growth was assessed by visual inspection after 5 days of incubation at 30°C. Growth was assessed relative to untreated control wells. Results are shown in Table 3.

[0079] Activity against methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA) and vancomycin-resistant Enterococcus faecium Ef 379 (VRE) was conducted using the microbroth dilution antibiotic susceptibility assay. Testing was conducted in accordance with Clinical Laboratory Standards Institute testing standards (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard, Sixth Ed, M7-A6 Volume 23, number 2). Compounds were tested against each organism in triplicate. Compounds were prepared in sterile 20% DMSO and serially diluted to generate a range of 8 concentrations. Each plate contained eight un-inoculated contamination controls (media + 20% DMSO), eight untreated controls (media + 20% DMSO + organism), and one column containing a concentration range of a control antibiotic (vancomycin or rifampicin, for MRSA and VRE, respectively). The optical density of the plate was recorded at 600 nm at time zero and then again after incubation of the plates for 22 hours at 37°C. After subtracting the time zero OD ₆ oo from the final reading the percentages of microorganism survival relative to vehicle control wells were calculated and the IC50 was determined. Results are shown in Table 3.

Table 3

Example 29 - Semi-synthetic Pseudopterosins.

[0080] Using the methods described above additional semi-synthetic pseudopterosins were prepared. These include those listed in Table 4 below.

[0081] Pseudopteroxazole was tested against drug resistant TB strains. The results shown in Table 5 below indicate that pseudopteroxazole has similar activity to second line anti- tubercular drugs and suggest that this compound exerts its activity by a novel mechanism of action.

Other Embodiments

[0082] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0083] What is claimed is: