WO2015028094A1

WO2015028094A1 - Branimycin derivatives and their use for the treatment of bacterial infectious diseases

Info

Publication number: WO2015028094A1
Application number: PCT/EP2013/068055
Authority: WO
Inventors: Philip John Dudfield; John Lowther; Carole Annie Josette DELACHAUME; Renaud Henri Marcel LÉPINE; Amber Paula Marcella Thys; Julien Georges Pierre-Olivier Doyon; Mathieu Paul TOUMI; Friedrich Georg Hansske
Original assignee: Galapagos Nv
Priority date: 2013-09-02
Filing date: 2013-09-02
Publication date: 2015-03-05

Abstract

A compound is disclosed that has a formula represented by Formula (I). The novel compound of the invention may be prepared as a pharmaceutical composition, and may be useful in the treatment of infectious diseases, in particular bacterial infectious diseases. The compound may be active against a specific enzyme in the bacterial DNA replicative process, DNA polymerase IIIE.

Description

BRANIMYCIN DERIVATIVES AND THEIR USE FOR THE

TREATMENT OF BACTERIAL INFECTIOUS DISEASES

FIELD OF THE INVENTION

[0001] The present invention relates to a novel compound that is useful in the treatment of infectious diseases, in particular those causing significant morbidity in human medicine. In one aspect, the compound is active against a specific enzyme in the bacterial DNA replicative process, DNA polymerase HIE. The present invention also provides methods for the production of this novel compound, pharmaceutical compositions comprising the compound, and methods for the prevention and/or treatment of bacterial infectious diseases by administering the compound of the invention.

BACKGROUND OF THE INVENTION

[0002] The emergence of resistance to antibiotics in bacteria causing infections in the clinic presents a global and urgent medical threat. The Infectious Disease Society of America highlighted the most problematic species, the so called ESKAPE bacteria: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter (Clinical Infectious Diseases 2009; 48: 1-12) in which there is a danger that existing therapies will soon no longer be effective. Of particular concern is the methicillin resistant Staphylococcus aureus (MRSA) which was reported to be responsible for more deaths in US hospitals than HIV/AIDS and tuberculosis combined. Once firmly established in hospitals worldwide, MRSA has now emerged as a significant community-acquired pathogen. Community-acquired MRSA infections are increasing, and may now involve persons without risk factors predisposing for acquisition, including children.

[0003] The urgency is recognized by governments and health organizations alike, including the WHO which since 2004 has listed infections due to resistant bacteria at the top of their preliminary ranking of pharmaceutical gaps, (Kaplan W, Laing R. Priority Medicines for Europe and the World. Geneva: Department of Essential Drugs and Medicines Policy, World Health Organization, 2004).

[0004] The discovery and development of new agents with novel mechanisms of action is clearly imperative if delivery of therapies is to keep pace with the growing problem. Herein we describe a newly identified series of compounds, which are believed to show a novel mode of action designed to address the clinical needs of today and the future.

[0005] In the clinical setting, it is a clear advantage for a new antibiotic to have pharmacokinetic properties which give potential for oral administration as well allowing infrequent dosing, ideally once a day. Both properties increase patient convenience and compliance for treatment in the community, and also in terms of follow-on treatment at home after an initial hospital parenteral regimen. [0006] Whilst many naturally occurring compounds have potent anti-bacterial activity, this is often coupled with poor pharmacokinetic and related properties, resulting in compounds which do not exhibit therapeutically relevant in vivo activity, despite showing potent activity against whole bacterial cells when tested in vitro microbroth susceptibility.

[0007] One such family of natural polyketide antibiotics are the nargenicins and branimycin which have a tricyclic structure with either a 10- or a 9-membered lactone ring and which contain a unique ether bridge. In 1977, the nargenicin family of antibiotics was isolated by Pfizer and Upjohn scientists after aerobic fermentation of Nocardia argentinensis ATCC 31306. One of these compounds, nargenicin Al was subsequently patented and its structure elucidated (see W. D. Celmer, et al J. Am. Chem. Soc. 102 (1980) 4203-4209). Although in vitro antibacterial activity was shown, it was restricted to Gram-positive methicillin resistant bacteria Staphylococcus aureus (MRSA). It has also been shown by Kim SH, et al. {Biochemical Pharmacology 11 (2009) 1694-1701) that nargenicin Al induces cell differentiation and that it can be used as a possible treatment for neoplastic diseases. In 1998, branimycin was isolated from Actinomycete GW 60/1571. In vitro biological tests have shown it is active against Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Streptomyces viridochromogenes .

[0008] The present invention provides a novel compound which exhibits in vivo activity in animal models of infection, in particular when dosed orally. In a specific aspect, it also exhibits improved activity compared to the naturally occurring molecules. This compounds may also exhibit improved properties, including improved pharmacokinetic properties (e.g. solubility, bioavailability, stability and/or, exposure). In community settings it is desirable for drugs to be active via the oral route. The compound of the invention is efficacious in treating infections in vivo, particularly via the oral route and therefore potentially provides clinically effective treatment in mammals.

SUMMARY OF THE INVENTION

[0009] The present invention relates to a novel compound that may be useful for the treatment of bacterial infectious diseases. The present invention also provides methods for the preparation of the compound of the invention, intermediates for its preparation, pharmaceutical compositions comprising the compound of the invention and methods for treating bacterial infectious diseases by administering a compound of the invention.

[0010] In a first aspect the present invention provides 8-0-lH-pyrrole-2'-carbonylbranimycin as the compound of the invention.

[0011] Particularly, the invention relates to the compound of the invention according to Formula (I):

[0012] The present invention also relates to pharmaceutical compositions comprising the compound of the invention.

[0013] In a further aspect, the present invention provides pharmaceutical compositions comprising the compound of the invention, and a pharmaceutical carrier, excipient or diluent.

[0014] In another aspect the invention relates to the compound of the invention for use in therapy.

[0015] In another aspect, the invention relates to the use of the compound of the invention in the manufacture of a medicament for the treatment of bacterial infectious disease.

[0016] In a further aspect the invention relates to methods of treating a bacterial infectious disease selected from amongst those listed herein, and particularly, where said bacterial infectious disease is caused by Gram negative and/or Gram positive bacteria, which method comprises administering a therapeutically effective amount of the compound of the invention to a subject in need thereof.

[0017] In a further aspect, the present invention relates to the compound of the invention for use in the treatment of a bacterial infectious disease by inhibiting DNA polymerase HIE activity in the bacteria.

[0018] Accordingly, it is a principal object of this invention to provide the compound of the invention, which can treat or prevent bacterial infections. In a specific aspect, it is an object of this invention to inhibit DNA polymerase HIE activity in bacteria and thus prevent or treat bacterial infectious diseases.

[0019] A still further object of this invention is to provide pharmaceutical compositions that may be used in the treatment or prevention of bacterial infectious diseases, by inhibiting DNA polymerase HIE activity in bacteria.

[0020] In an additional aspect, this invention provides methods for preparation of the compound of the invention, with representative synthetic protocols and pathways disclosed herein.

[0021] Other objects and advantages will become apparent to those skilled in the art from a consideration of the ensuing detailed description. DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0022] It will be understood that the present invention covers all combinations of aspects, suitable, convenient and preferred groups described herein.

[0023] When describing the invention, which may include compounds, pharmaceutical compositions containing such compounds and methods of using such compounds and compositions, the following terms, if present, have the following meanings unless otherwise indicated. Unless otherwise stated, the term 'substituted' is to be defined as set out below. It should be further understood that the terms 'groups' and 'radicals' can be considered interchangeable when used herein.

[0024] When ranges are referred to herein, for example but without limitation, Ci_6 alkyl, the citation of a range should be considered a representation of each member of said range.

[0025] The articles 'a' and 'an' may be used herein to refer to one or to more than one (i.e. at least one) of the grammatical objects of the article. By way of example 'an analogue' means one analogue or more than one analogue.

[0026] The term 'comprise', and variations such as 'comprises' and 'comprising', throughout the specification and the claims which follow, unless the context requires otherwise, 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.

[0027] The term 'compound(s) of the invention' or 'compound(s) according to the invention', and equivalent expressions includes 8-0-lH-pyrrole-2'-carbonylbranimycin, compounds of Formula (I) (whether in solvated or unsolvated form), or its pharmaceutically acceptable salts (whether in solvated or unsolvated form). Suitably, said expression includes the pharmaceutically acceptable salts, and solvates (e.g. hydrates) thereof.

[0028] 'Pharmaceutically acceptable' means approved or approvable by a regulatory agency of the Federal or a state government or the corresponding agency in countries other than the United States, or that is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in mammals, and more particularly, in humans.

[0029] 'Pharmaceutically acceptable salt' refers to a salt of a compound that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic may be inorganic or organic acid addition salts and base addition salts. Specifically, such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethane-disulfonic acid, 2- hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2- naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2- ene-l-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine and the like. Salts further include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the compound contains a basic functionality, salts of non toxic organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like. The term 'pharmaceutically acceptable cation' refers to an acceptable cationic counter-ion of an acidic functional group. Such cations are exemplified by sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium cations, and the like.

[0030] 'Pharmaceutically acceptable vehicle' refers to a diluent, adjuvant, excipient or carrier with which a compound of the invention is administered.

[0031] The term 'prodrug' as used herein refers to compounds, including derivatives of the compounds of the invention, which have metabolically cleavable groups and are converted within the body e.g. by solvolysis or under physiological conditions into the compounds of the invention which are pharmaceutically active in vivo. Pharmaceutically acceptable prodrugs are described in: Bundgard, H. Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985, 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. Prodrugs include acid derivatives well known to practitioners of the art, such as, for example, esters prepared by reaction of the parent acid with a suitable alcohol, or amides prepared by reaction of the parent acid compound with a substituted or unsubstituted amine, or acid anhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters, amides and anhydrides derived from acidic groups pendant on the compounds of this invention are preferred prodrugs. In some cases it is desirable to prepare double ester type prodrugs such as (acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkylesters. Particularly useful are the CpCg alkyl, C2-Cg alkenyl, aryl, and C₇- Ci2 arylalkyl esters of the compounds of the invention.

[0032] 'Solvate' refers to forms of the compound that are associated with a solvent, usually by a solvolysis reaction. This physical association includes hydrogen bonding. Conventional solvents include water, ethanol, acetic acid and the like. The compounds of the invention may be prepared e.g. in crystalline form and may be solvated or hydrated. Suitable solvates include pharmaceutically acceptable solvates, such as hydrates, and further include both stoichiometric solvates and non- stoichiometric solvates. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. 'Solvate' encompasses both solution-phase and isolable solvates. Representative solvates include hydrates, ethanolates and methanolates.

[0033] The term 'isotopic variant' refers to a compound that contains unnatural proportions of isotopes at one or more of the atoms that constitute such compound For example, an 'isotopic variant' of a compound can contain one or more non-radioactive isotopes, such as for example, deuterium (²H or D), carbon-13 (¹³C), nitrogen-15 (¹⁵N), or the like. It will be understood that, in a compound where such isotopic substitution is made, the following atoms, where present, may vary, so that for example, any hydrogen may be ²H/D, any carbon may be ¹³C, or any nitrogen may be ¹⁵N, and that the presence and placement of such atoms may be determined within the skill of the art. Likewise, the invention may include the preparation of isotopic variants with radioisotopes, in the instance for example, where the resulting compounds may be used for drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e. ³H, and carbon-14, i.e. ¹⁴C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Further, compounds may be prepared that are substituted with positron emitting isotopes, such as ^UC, ¹⁸F, ¹⁵0 and ¹³N, and would be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. All isotopic variants of the compounds provided herein, radioactive or not, are intended to be encompassed within the scope of the invention.

[0034] The term 'isomer(s)' refers to compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed 'stereoisomers'.

[0035] 'Diastereomers' are stereoisomers that are not mirror images of one another and those that are non-superimposable mirror images of each other are termed 'enantiomers'. When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (-)-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a 'racemic mixture'.

[0036] 'Tautomers' refer to compounds that are interchangeable forms of a particular compound structure, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium through the movement of π electrons and an atom (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Another example of tautomerism is the aci- and nitro- forms of phenylnitromethane, that are likewise formed by treatment with acid or base. Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest.

[0037] 'Subject' refers to an animal, in particular a mammal and more particular to a human or a domestic animal serving as a model for a disease (for example guinea pigs, mice, rats, gerbils, cats, rabbits, dogs, monkeys, chimpanzees or like). Specifically the subject is a human. The terms 'patient' and 'subject' are used interchangeably herein.

[0038] 'Therapeutically effective amount' means the amount of a compound of the invention that, when administered to a subject for treating an infection, is sufficient to effect such treatment for the infection. For example, but without limitation, the treatment of an invention may involve decreasing the number of bacteria causing said infection in the patient. The "therapeutically effective amount" can vary depending on the compound, the infection and its severity, and the age, weight, physical condition, responsiveness etc., of the subject to be treated and will ultimately be at the discretion of the attendant physician.

[0039] 'Preventing' or 'prevention' refers to a reduction in risk of acquiring or developing an infection (i.e., causing at least one of the clinical symptoms of the infection not to develop in a subject that may be exposed to an infection-causing agent, or predisposed to the infection in advance of infection onset).

[0040] The term 'prophylaxis' is related to 'prevention', and refers to a measure or procedure the purpose of which is to prevent, rather than to treat or cure an infection. Non-limiting examples of prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization; and the administration of an anti-malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high.

[0041] 'Treating' or 'treatment' of any infection refers, in one embodiment, to ameliorating the infection (i.e., arresting the infection or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof). In another embodiment 'treating' or 'treatment' refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, 'treating' or 'treatment' refers to modulating the infection, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In a further embodiment, 'treating' or 'treatment' relates to decreasing the bacterial load associated with the infection.

[0042] As used herein, the term 'bacterial infectious diseases' refers to diseases caused by bacterial infection and includes systemic infections (bacteremia and sepsis) and/or infections of any organ or tissue of the body. These organs or tissue include, without limitation, skeletal muscle, skin, bloodstream, kidneys, heart, lung and bone. These infections may be caused by Gram-positive or Gram-negative bacteria as described below. Specifically, said bacterial infectious disease is caused by Gram-positive bacteria. [0043] As used herein, the term 'Gram-negative bacteria' refers to bacteria which do not retain crystal violet dye in the Gram staining protocol and includes, but is not limited to, bacteria in the Genus Enterobacteriacae, including Escherichia spp. (including E. coli), Klebsiella spp., Enterobacter spp., Citrobacter spp., Serratia spp., Proteus spp., Providencia spp., Salmonella spp., Shigella spp., the genus Pseudomonas (including P. aeruginosa) and species such as Moraxella spp. (including M. catarrhalis), Haemophilus spp. and Neisseria spp.

[0044] As used herein, the term 'Gram-positive bacteria' refers to bacteria which are stained dark blue or violet by Gram staining and includes, but is not limited to, methiciiiin-susceptible and methicillin-resistant staphylococci (including Staphylococcus aureus, S. epidermidis, S. haemolyticus, S. hominis, S. saprophyticus, and coagulase-negative staphylococci), glycopeptideintermediary- susceptible S. aureus (GISA), penicillin-susceptible and penicillin-resistant streptococci (including Streptococcus pneumoniae, S. pyogenes, S. agalactiae, S. avium, S. bovis, S. lactis, S. sanguis and Streptococci Group C, Streptococci Group G and viridans streptococci), enter ococci (including vancom yci n-suscept iblc and vanco my c i n- res i si an I strains such as Enterococcus faecalis and E. faecium),. Clostridium difficile, Listeria monocytogenes, Corynebacterium jeikeium, Chlamydia spp (including C. pneumoniae) and Mycobacterium tuberculosis.

THE COMPOUND

[0045] The present invention is based on the identification that the compound of the invention may be useful for the treatment of bacterial infectious diseases, particularly in mammals. The present invention also provides methods for the preparation of the compounds of the invention, the intermediates for their preparation, pharmaceutical compositions comprising a compound of the invention and methods for the treatment of bacterial infectious diseases in mammals by administering the compound of the invention.

[0046] In a specific embodiment the compound of the invention is an inhibitor of DNA Polymerase HIE.

[0047] In a particular embodiment, as DNA polymerase HIE represents a novel target for antibacterial agents and the compound of the invention is an unexploited chemical class, the compound of the invention is active against bacterial strains which exhibit resistance to established classes of antibiotics. Therefore, in one embodiment the present invention provides the compound of the invention for use in the treatment of bacterial infectious diseases caused by strains resistant to established antibiotic classes. In a specific embodiment the present invention provides the compound of the invention for use in the treatment of bacterial infectious diseases caused by strains resistant to aminoglycosides, carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptide, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptides, quinolones, sulfonamides, fusidic acid, pseudomonic acids, rifamycins, lipoglycopeptides , novobiocin, and/or tetracyclines (e.g. glycylcyclines).

[0048] Accordingly, in one aspect the present invention provides 8-0-lH-pyrrole-2'- carbonylbranimycin as the compound of the invention.

[0049] The compound of the invention is a semi-synthetic derivative of the natural product branimycin, and the stereochemistry of the central core which corresponds to the parent compound is not affected by the semi-synthetic derivatisation to obtain the compound of the invention. Therefore, it is understood that only a single compound is generated via the synthetic methods described, the stereochemistry at each chiral centre is that found in the parent compound when produced via the fermentation of the producer strain as described in more detail in the examples herein.

[0050] In a further aspect the invention relates to a compound of the invention according to Formula (I):

[0051] In one embodiment the compound of the invention is not an isotopic variant.

[0052] In one aspect a compound of the invention according to any one of the embodiments herein described is a free base.

[0053] In one aspect a compound of the invention according to any one of the embodiments herein described is a salt, and specifically a salt of 8-0-lH-pyrrole-2'-carbonylbranimycin.

[0054] In one aspect a compound of the invention according to any one of the embodiments herein described is a pharmaceutically acceptable salt, and specifically a pharmaceutically acceptable salt of 8-0-lH-pyrrole-2'-carbonylbranimycin.

[0055] In one aspect a compound of the invention according to any one of the embodiments herein described is a solvate of the compound.

[0056] In one aspect a compound of the invention according to any one of the embodiments herein described is a solvate of a salt of a compound, in particular a solvate of a pharmaceutically acceptable salt.

[0057] Similarly, reference to intermediates, whether or not they themselves are claimed, is meant to embrace their salts, and solvates, where the context so permits. [0058] With regard to stereoisomers, the compounds of the invention have more than one asymmetric carbon atom. In the general formula(e) as drawn, the solid wedge shaped bond indicates that the bond is above the plane of the paper. The broken bond indicates that the bond is below the plane of the paper.

[0059] 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.

[0060] In a further aspect, the present invention provides a method for the synthesis of a compound according to Formula I, said method comprising:

(a) culturmg Saccharothrix xinjiangensis G60/1571 30xB2M21, (registered under accession number NCIMB 41952) or Actinomycete GW 60/1571,

(b) after harvest, generating a crude extract from the fermentation broth,

(c) isolating branimycin from the crude extract,

(d) reacting branimycin with a suitable hydroxyl protection agent R^pl-X to protect position C¹⁷ hydroxyl, wherein X is a leaving group and R^pl is a suitable protecting group,

(e) acylating the product of step (d) (Intermediate A) at the exposition with a reagent according to Formula A below, wherein R^p3 represents H or a protecting group selected from

alkyl, -CH₂-Ph, -Si(C alkyl)₃, -Si(C alkyl)(Ph)₂, tetrahydropyranyl, and allyl, wherein said alkyl, and phenyl groups may further be substituted with CM alkoxy,

Formula A

(f) removal of all protecting groups R^pl and R^p3 from the intermediate B obtained (e) above to yield the compound according to Formula I.

[0061] In one embodiment, in the method described above in step (a) branimycin is produced by culturing Saccharothrix xinjiangensis G60/1571 30xB2M21, (registered under accession number NCIMB 41952) ox Actinomycete GW 60/1571, in YM7.2 medium. Particularly, in step (a) branimycin is produced by culturing Saccharothrix xinjiangensis G60/1571 30xB2M21, (registered under accession number NCIMB 41952) or Actinomycete GW 60/1571, in YM7.2 medium at 28°C. In a specific embodiment, in step (a) branimycin is produced by culturing Saccharothrix xinjiangensis G60/1571 30xB2M21, (registered under accession number NCIMB 41952) or Actinomycete GW 60/1571 for between 90- 144L

[0062] In a further aspect, the present invention provides a method for the synthesis of a compound according to Formula I, said method comprising:

(a) reacting branimycin (Formula II) with a suitable hydroxyl protection agent R^pl-X to protect position C¹⁷ hydroxyl, wherein X is a leaving group and R^P1 is a suitable protecting group,

(b) acylating the product of step (a) (Intermediate A) at the exposition with a reagent according to Formula A below, wherein R^p3 represents H or a protecting group selected from

alkyl, -CH₂-Ph, -Si(C alkyl)₃, -Si(C alkyl)(Ph)₂, tetrahydropyranyl, and allyl, wherein said alkyl, and phenyl groups may further be substituted with C alkoxy,

Formula A

(c) removal of all protecting groups R^P1 and R^P3 from the intermediate B obtained in step

(b) above to yield the compound according to Formula I.

[0063] In one aspect, in the method described above in step (a) R^pl-X is a silyl ether group, particularly TES-C1, TBDPS-C1, TBDMS-C1, or TMS-C1.

[0064] In one further aspect, in the method described above in step (b) R^p3 is selected from - C(=0)OtBu, -CH₂-Ph, -C(=0)0(CH₂)₂Si(Me)₃, and 4-Me-Ph-S0₂-.

[0065] Unless indicated otherwise, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art.

[0066] While specified groups for each embodiment have generally been listed above separately, a compound of the invention may be one for which one or more variables (R groups and/or integers) is selected from one or more embodiments according to any of the Formula(e) listed above. Therefore, the present invention is intended to include all combinations of variables from any of the disclosed embodiments within its scope.

[0067] Alternatively, the exclusion of one or more of the specified variables from a group or an embodiment of said group, or combinations thereof is also contemplated by the present invention.

PHARMACEUTICAL COMPOSITIONS [0068] When employed as a pharmaceutical, the compound of the invention is typically administered in the form of a pharmaceutical composition. Such compositions can be prepared in a manner well known in the pharmaceutical art and comprise at least one active compound. Generally, a compound of the invention is administered in a therapeutically effective amount. The amount of the compound actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the infection to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

[0069] The pharmaceutical compositions of the invention can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intra-articular, intravenous, intramuscular, and intranasal. Depending on the intended route of delivery, the compound of this invention is preferably formulated as either injectable, including intravenous, or oral compositions or as salves, as lotions or as patches all for transdermal administration.

[0070] The compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. The term "unit dosage forms" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient, vehicle or carrier. Typical unit dosage forms include prefilled, premeasured ampoules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions. In such compositions, the compound of the invention is usually a minor component (from about 0.1 to about 50% by weight or preferably from about 1 to about 40% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.

[0071] Liquid forms suitable for oral administration may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors and the like. Solid forms may include, for example, any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0072] Injectable compositions are typically based upon injectable sterile saline or phosphate- buffered saline or other injectable carriers known in the art. As before, the active compound in such compositions is typically a minor component, often being from about 0.05 to 10%> by weight with the remainder being the injectable carrier and the like. [0073] Transdermal compositions are typically formulated as a topical ointment or cream containing the active ingredient(s), generally in an amount ranging from about 0.01 to about 20% by weight, preferably from about 0.1 to about 20% by weight, preferably from about 0.1 to about 10% by weight, and more preferably from about 0.5 to about 15% by weight. When formulated as an ointment, the active ingredients will typically be combined with either a paraffmic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with, for example an oil-in-water cream base. Such transdermal formulations are well-known in the art and generally include additional ingredients to enhance the dermal penetration of stability of the active ingredients or the formulation. All such known transdermal formulations and ingredients are included within the scope of this invention.

[0074] The compound of the invention can also be administered by a transdermal device. Accordingly, transdermal administration can be accomplished using a patch either of the reservoir or porous membrane type, or of a solid matrix variety.

[0075] The above-described components for orally administrable, injectable or topically administrable compositions are merely representative. Other materials as well as processing techniques and the like are set forth in Part 8 of Remington's Pharmaceutical Sciences, 17^th edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which is incorporated herein by reference.

[0076] A compound of the invention can also be administered in sustained release forms or from sustained release drug delivery systems. A description of representative sustained release materials can be found in Remington's Pharmaceutical Sciences.

[0077] The following formulation examples illustrate representative pharmaceutical compositions that may be prepared in accordance with this invention. The present invention, however, is not limited to the following pharmaceutical compositions.

[0078] Moreover, the compound of the present invention useful in the pharmaceutical compositions and treatment methods disclosed herein, is pharmaceutically acceptable as prepared and used. In this aspect of the invention, the pharmaceutical composition may additionally comprise further active ingredients suitable for use in combination with a compound of the invention.

Formulation 1 - Tablets

[0079] The compound of the invention may be admixed as a dry powder with a dry gelatin binder in an approximate 1 :2 weight ratio. A minor amount of magnesium stearate may be added as a lubricant. The mixture may be formed into 240-270 mg tablets (80-90 mg of active amide compound per tablet) in a tablet press.

Formulation 2 - Capsules

[0080] The compound of the invention may be admixed as a dry powder with a starch diluent in an approximate 1 : 1 weight ratio. The mixture may be filled into 250 mg capsules (125 mg of active amide compound per capsule). Formulation 3 - Liquid

[0081] The compound of the invention (125 mg), may be admixed with sucrose (1.75 g) and xanthan gum (4 mg) and the resultant mixture may be blended, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of microcrystalline cellulose and sodium carboxymethyl cellulose (11 :89, 50 mg) in water. Sodium benzoate (10 mg), flavor, and color may be diluted with water and added with stirring. Sufficient water may then be added with stirring. Sufficient water may be then added to produce a total volume of 5 mL.

Formulation 4 - Tablets

[0082] The compound of the invention may be admixed as a dry powder with a dry gelatin binder in an approximate 1 :2 weight ratio. A minor amount of magnesium stearate may be added as a lubricant. The mixture is formed into 450-900 mg tablets (150-300 mg of active amide compound) in a tablet press.

Formulation 5 - Injection

[0083] The compound of the invention may be dissolved or suspended in a buffered sterile saline injectable aqueous medium to a concentration of approximately 5 mg/mL.

Formulation 6 - Topical

[0084] Stearyl alcohol (250 g) and a white petrolatum (250 g) may be melted at about 75°C and then a mixture of the compound of the invention (50 g) methylparaben (0.25 g), propylparaben (0.15 g), sodium lauryl sulfate (10 g), and propylene glycol (120 g) dissolved in water (about 370 g) may be added and the resulting mixture may be stirred until it congeals.

METHODS OF TREATMENT

[0085] In one aspect, the present invention provides the compound of the invention for use as a medicament.

[0086] In a further aspect, the present invention provides the compound of the invention for use in the treatment of bacterial infectious diseases, particularly in mammals. In a particular embodiment, said bacterial infectious disease is caused by Gram-negative bacteria. In a specific embodiment, said bacterial infectious disease is caused by Gram-positive bacteria. In a further specific embodiment the present invention provides the compound of the invention for use in the treatment of bacterial infectious diseases caused by strains resistant to established antibiotic classes. In a further specific embodiment the present invention provides the compound of the invention for use in the treatment of bacterial infectious diseases caused by strains resistant to aminoglycosides, carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptide, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptides, quinolones, sulfonamides, fusidic acid, pseudomonic acids, rifamycins, lipoglycopeptides , novobiocin, and/or tetracyclines (e.g. glycylcyclines).

[0087] In a further aspect, the present invention provides the compound of the invention for use in manufacture of a medicament for the treatment of bacterial infectious diseases, particularly in mammals. In a particular embodiment, said bacterial infectious disease is caused by Gram-negative bacteria. In a specific embodiment, said bacterial infectious disease is caused by Gram-positive bacteria. In a further specific embodiment the present invention provides the compound of the invention for use in the manufacture of a medicament for the treatment of bacterial infectious diseases caused by strains resistant to established antibiotic classes. In a further specific embodiment, said bacterial infectious diseases are caused by strains resistant to aminoglycosides, carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptide, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptides, quinolones, sulfonamides, fusidic acid, pseudomonic acids, rifamycins, lipoglycopeptides , novobiocin, and/or tetracyclines (e.g. glycylcyclines).

[0088] In a further aspect, the present invention provides a method of treating bacterial infectious diseases, particularly in mammals, said method comprising administering a therapeutically effective amount of the compound of the invention, to a patient in need thereof. In a particular embodiment, said bacterial infectious disease is caused by Gram-negative bacteria. In a specific embodiment, said bacterial infectious disease is caused by Gram-positive bacteria. . In a further specific embodiment the bacterial infectious diseases are caused by strains resistant to established antibiotic classes. In a further specific embodiment, said bacterial infectious diseases are caused by strains resistant to aminoglycosides, carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptide, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptides, quinolones, sulfonamides, fusidic acid, pseudomonic acids, rifamycins, lipoglycopeptides , novobiocin, and/or tetracyclines (e.g. glycylcyclines).

[0089] In a specific embodiment, the present invention provides a compound of the invention for use in the treatment of bacterial infections caused by a Gram-positive bacteria selected from methicillin-susceptible and methicillin-resistant staphylococci (including Staphylococcus aureus, Staphylococcus epidermidis , Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, and coagulase-negative staphylococci), glycopeptides-intermediate susceptible Staphylococcus aureus (GISA), penicillin-susceptible and penicillin-resistant streptococci (including Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus avium, Streptococcus bovis, Streptococcus lactis, Streptococcus sanguis and Streptococci Group C (GCS), Streptococci Group G (GGS) and viridans streptococci), enterococci (including vancomycin- susceptible and vancomycin-resistant strains such as Enterococcus faecalis and Enterococcus faecium), Clostridium difficile, Listeria monocytogenes, Corynebacterium jeikeium, Chlamydia spp (including C. pneumoniae) and Mycobacterium tuberculosis. In a more specific embodiment the Gram-positive bacteria is Staphylococcus aureus, in particular methicillin-resistant S. aureus (MRSA).

[0090] In a specific embodiment, the present invention provides a compound of the invention for use in the manufacture of a medicament for the treatment of bacterial infections caused by a Gram- positive bacteria selected from methicillin-susceptible and methicillin-resistant staphylococci (including Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus , Staphylococcus hominis, Staphylococcus saprophyticus , and coagulase-negative staphylococci), glycopeptides-intermediate susceptible Staphylococcus aureus (GISA), penicillin-susceptible and penicillin-resistant streptococci (including Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus avium, Streptococcus bovis, Streptococcus lactis, Streptococcus sanguis and Streptococci Group C (GCS), Streptococci Group G (GGS) and viridans streptococci), enterococci (including vancomycin-susceptible and vancomycin-resistant strains such as Enterococcus faecalis and Enterococcus faecium), Clostridium difficile, Listeria monocytogenes, Corynebacterium jeikeium, Chlamydia spp (including C. pneumoniae) and Mycobacterium tuberculosis. In a more specific embodiment the Gram-positive bacteria is Staphylococcus aureus, in particular methicillin-resistant S. aureus (MRSA).

[0091] In a specific embodiment, the present invention provides a method of treating bacterial infections caused by a Gram-positive bacteria selected from methicillin-susceptible and methicillin- resistant staphylococci (including Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, and coagulase- negative staphylococci), glycopeptides-intermediate susceptible Staphylococcus aureus (GISA), penicillin-susceptible and penicillin-resistant streptococci (including Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus avium, Streptococcus bovis, Streptococcus lactis, Streptococcus sanguis and Streptococci Group C (GCS), Streptococci Group G (GGS) and viridans streptococci), enterococci (including vancomycin-susceptible and vancomycin- resistant strains such as Enterococcus faecalis and Enterococcus faecium), Clostridium difficile, Listeria monocytogenes, Corynebacterium jeikeium, Chlamydia spp (including C. pneumoniae) and Mycobacterium tuberculosis, said method comprising administering a therapeutically effective amount of the compound of the invention, to a patient in need thereof. In a more specific embodiment the Gram-positive bacteria is Staphylococcus aureus, in particular methicillin-resistant S. aureus (MRSA).

[0092] In a specific embodiment, the present invention provides a compound of the invention for use in the treatment of bacterial infections caused by a Gram-negative bacteria selected from bacteria in the Genus Enterobacteriacae, including Escherichia spp. (including Escherichia coli), Klebsiella spp., Enterobacter spp., Citrobacter spp., Serratia spp., Proteus spp., Providencia spp., Salmonella spp., Shigella spp., the genus Pseudomonas (including P. aeruginosa), Moraxella spp. (including M. catarrhalis), Haemophilus spp and Neisseria spp. In a more specific embodiment the Gram-negative bacteria is in the Genus Enterobacteriacae, including Escherichia spp. (including Escherichia coli), Klebsiella spp., Enterobacter spp., Citrobacter spp., Serratia spp., Proteus spp., Providencia spp., Salmonella spp., Shigella spp., or is P. aeruginosa.

[0093] In a specific embodiment, the present invention provides a compound of the invention for use in the manufacture of a medicament for the treatment of bacterial infections caused by a Gram- negative bacteria selected from bacteria in the Genus Enterobacteriacae, including Escherichia spp. (including Escherichia coli), Klebsiella spp., Enterobacter spp., Citrobacter spp., Serratia spp., Proteus spp., Providencia spp., Salmonella spp., Shigella spp., the genus Pseudomonas (including P. aeruginosa), Moraxella spp. (including M. catarrhalis), Haemophilus spp and Neisseria spp. In a more specific embodiment the Gram-negative bacteria is in the Genus Enterobacteriacae, including Escherichia spp. (including Escherichia coli), Klebsiella spp., Enterobacter spp., Citrobacter spp., Serratia spp., Proteus spp., Providencia spp., Salmonella spp., Shigella spp., or is P. aeruginosa.

[0094] In a specific embodiment, the present invention provides a method of treating bacterial infections caused by a Gram-negative bacteria selected from bacteria in the Genus Enterobacteriacae, including Escherichia spp. (including Escherichia coli), Klebsiella spp., Enterobacter spp., Citrobacter spp., Serratia spp., Proteus spp., Providencia spp., Salmonella spp., Shigella spp., the genus Pseudomonas (including P. aeruginosa), Moraxella spp. (including M. catarrhalis), Haemophilus spp and Neisseria spp, said method comprising administering a therapeutically effective amount of the compound of the invention, to a patient in need thereof. In a more specific embodiment the Gram-negative bacteria is in the Genus Enterobacteriacae, including Escherichia spp. (including Escherichia coli), Klebsiella spp., Enterobacter spp., Citrobacter spp., Serratia spp., Proteus spp., Providencia spp., Salmonella spp., Shigella spp., or is P. aeruginosa.

[0095] In a specific aspect, the present invention provides a compound of the invention for use in the treatment of bacterial infections caused by more than one strain of Gram-positive bacteria, or a bacterial infection caused by both Gram-positive and Gram-negative bacteria. These types of infections include intra-abdominal infections and obstetrical/gynecological infections.

[0096] In a specific aspect, the present invention provides a compound of the invention for use in the manufacture of a medicament for the treatment of bacterial infections caused by more than one strain of Gram-positive bacteria, or a bacterial infection caused by both Gram-positive and Gram- negative bacteria. These types of infections include intra-abdominal infections and obstetrical/gynecological infections.

[0097] In a specific aspect, the present invention provides a method of treating bacterial infections caused by more than one strain of Gram-positive bacteria, or a bacterial infection caused by both Gram-positive and Gram-negative bacteria, said method comprising administering a therapeutically effective amount of the compound of the invention, to a patient in need thereof. These types of infections include intra-abdominal infections and obstetrical/gynecological infections.

[0098] In a specific aspect, the present invention provides a compound of the invention for use in the treatment of endocarditis, nephritis, septic arthritis, intra-abdominal sepsis, bone and joint infections and / or osteomyelitis.

[0099] In a specific aspect, the present invention provides a compound of the invention for use in the manufacture of a medicament for the treatment of endocarditis, nephritis, septic arthritis, intraabdominal sepsis, bone and joint infections and / or osteomyelitis. [00100] In a specific aspect, the present invention provides a method of treating endocarditis, nephritis, septic arthritis, intra-abdominal sepsis, bone and joint infections and / or osteomyelitis, said method comprising administering a therapeutically effective amount of the compound of the invention, to a patient in need thereof.

[00101] In a further aspect, the present invention provides a compound of the invention for use in the treatment of bacterial infections in any organ or tissue in the body. In a specific embodiment, the present invention provides a compound of the invention for use in the treatment of skin and soft tissue infections, bacteremia, urinary tract infections and sexually transmitted bacterial infections. In a specific embodiment, the present invention provides a compound of the invention for use in the treatment of community acquired respiratory infections, including, without limitation, otitis media, sinusitis, chronic bronchitis and pneumonia. In a specific embodiment, the present invention provides a compound of the invention for use in the treatment of blood infections, in particular sepsis and/or septicemia.

[00102] In a further aspect, the present invention provides a compound of the invention for use in the manufacture of a medicament for the treatment of bacterial infections in any organ or tissue in the body. In a specific embodiment, the present invention provides a compound of the invention for use in the manufacture of a medicament for the treatment of skin and soft tissue infections, bacteremia, urinary tract infections and sexually transmitted bacterial infections. In a specific embodiment, the present invention provides a compound of the invention for use in the manufacture of a medicament for the treatment of community acquired respiratory infections, including, without limitation, otitis media, sinusitis, chronic bronchitis and pneumonia. In a specific embodiment, the present invention provides a compound of the invention for use in the manufacture of a medicament for the treatment of blood infections, in particular sepsis and/or septicemia.

[00103] In a further aspect, the present invention provides a method for the treatment of bacterial infections in any organ or tissue in the body, said method comprising administering a therapeutically effective amount of the compound of the invention, to a patient in need thereof. In a specific embodiment, the present invention provides a method for the treatment of skin and soft tissue infections, bacteremia, urinary tract infections and sexually transmitted bacterial infections, said method comprising administering a therapeutically effective amount of the compound of the invention, to a patient in need thereof. In a specific embodiment, the present invention provides a method for the treatment of community acquired respiratory infections, including, without limitation, otitis media, sinusitis, chronic bronchitis and pneumonia, said method comprising administering a therapeutically effective amount of the compound of the invention, to a patient in need thereof. In a specific embodiment, the present invention provides a method for the treatment or prophylaxis of blood infections, in particular sepsis and/or septicemia, said method comprising administering a therapeutically effective amount of the compound of the invention, to a patient in need thereof. [00104] The present invention provides the compound of the invention for use in the treatment or prevention of bacterial infections by inhibiting DNA polymerase HIE activity in the bacteria.

[00105] The present invention provides the compound of the invention for use in the manufacture of a medicament for use in the treatment or prevention of bacterial infections by inhibiting DNA polymerase HIE activity in the bacteria.

[00106] The present invention provides a method for the treatment or prevention of bacterial infections by inhibiting DNA polymerase HIE activity in the bacteria, said method comprising administering a therapeutically effective amount of a compound of the invention to a patient in need thereof.

[00107] A person of skill in the art will appreciate that the methods and uses described above, may also be applied to the use of pharmaceutical compositions comprising the compound of the invention.

[00108] A particular regimen of the present method comprises the administration to a subject suffering from a bacterial infectious disease, of a therapeutically effective amount of a compound of the invention for a period of time sufficient to reduce the level of infection in the subject, and preferably terminate said infection. A special embodiment of the method comprises administering of a therapeutically effective amount of the compound of the invention to a subject patient suffering from or susceptible to the development of a bacterial infectious disease, for a period of time sufficient to reduce or prevent, respectively, infection of said patient, and preferably terminate, said infection.

[00109] Injection dose levels range from about 0.1 mg/kg/h to at least 10 mg/kg/h, all for from about 1 to about 120 h and especially 24 to 96 h. A preloading bolus of from about 0.1 mg/kg to about 10 mg/kg or more may also be administered to achieve adequate steady state levels. The maximum total dose is not expected to exceed about 2 g/day for a 40 to 80 kg human patient.

[00110] For the prevention and/or treatment of bacterial infectious diseases, the regimen for treatment will typically last from 1 to 30 days. For the treatment of such infections oral dosing is preferred for patient convenience and tolerance. With oral dosing, one to five and especially two to four and typically three oral doses per day are representative regimens. Alternatively, once a day dosing is preferred for patient convenience. Using these dosing patterns, each dose provides from about 0.01 to about 20 mg/kg of the compound of the invention, with particular doses each providing from about 0.1 to about 10 mg/kg and especially about 1 to about 5 mg/kg. Alternatively, the bacterial infectious disease may be treated via the parenteral route in a hospital based setting.

[00111] Transdermal doses are generally selected to provide similar or lower blood levels than are achieved using injection doses.

[00112] In an alternative aspect, the compound of the invention may be used to prevent the onset of a condition. In this aspect, the compound of the invention will be administered to a patient at risk for developing the condition, typically on the advice and under the supervision of a physician, at the dosage levels described above. Patients at risk for developing a particular condition generally include those that have been exposed to a particular bacterial infectious agent, who have a suppressed immune system or those who have been identified by screening to be particularly susceptible to developing the condition, for example, but without limitation, patients diagnosed with cystic fibrosis or patients undergoing invasive surgery.

[00113] The compound of the invention can be administered as the sole active agent or it can be administered in combination with other therapeutic agents, including other compounds that demonstrate the same or a similar therapeutic activity, and that are determined to be safe and efficacious for such combined administration. In a specific embodiment, co-administration of two (or more) agents allows for significantly lower doses of each to be used, thereby reducing the side effects seen.

[00114] In one embodiment, the compound of the invention is co-administered with another therapeutic agent for the treatment and/or prevention of bacterial infectious diseases; particular agents include but are not limited to antibiotics. In a particular embodiment, the compound of the invention is co-administered with another therapeutic agent for the treatment and/or prevention of infections of any organ of the human body; particular agents include but are not limited to: aminoglycosides, carbacephem, carbapenems, cephalosporins, glycopeptides, lincosamides, macrolides, monobactams, nitrofurans, penicillins, polypeptides, quinolones, sulfonamides, tetracyclins, anti-mycobacterial agents, as well as chloramphenicol, fosfomycin, linezolid, metronidazole, mupirocin, rifamycin, thiamphenicol and tinidazole.

[00115] In one embodiment, the compound of the invention is co-administered with an additional therapeutic agent for the treatment and/or prevention of bacterial infectious diseases caused by Gram- negative bacteria, wherein said additional therapeutic agent is an efflux pump inhibitor or a membrane permeabilising agent.

[00116] By co-administration is included any means of delivering two or more therapeutic- agents to the patient as part of the same treatment regime, as will be apparent to the skilled person. Whilst the two or more agents may be administered simultaneously in a single formulation this is not essential. The agents may be administered in different formulations and at different times. Therefore, in one aspect the present invention provides the co-administration of a compound of the invention with one or more additional therapeutic agents where the active agents are present in the same pharmaceutical composition. Alternatively, the present invention provides the co-administration of a compound of the invention with one or more additional therapeutic agents, where each active agent is administered via a separate pharmaceutical composition.

[00117] In a specific embodiment, the compound of the invention may be used in combination with a companion diagnostic test to confirm the presence of one or more of the bacterial strains as described herein. GENERAL SYNTHETIC PROCEDURES

General

[00118] The compound of the invention can be prepared from readily available starting materials using the following general methods and procedures. 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.

[00119] Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and P. G. M. Wuts, Protecting Groups in Organic Synthesis, Wiley-Blackwell; 4th Revised edition edition (2006), and references cited therein.

[00120] The compound of the invention may be prepared from known or commercially available starting materials and reagents by one skilled in the art of organic synthesis.

[00121] All reagents were of commercial grade and were used as received without further purification, unless otherwise stated. Commercially available anhydrous solvents were used for reactions conducted under inert atmosphere. Reagent grade solvents were used in all other cases, unless otherwise specified. Column chromatography was performed on silica standard (35-70 μιη). Thin layer chromatography was carried out using pre-coated silica gel 60 F-254 plates (thickness 0.25 mm). ^lH NMR spectra were recorded on a Broker Advance 400 NMR spectrometer (400 MHz) or a Broker Advance 300 NMR spectrometer (300 MHz). Chemical shifts (δ) for ^lH NMR spectra are reported in parts per million (ppm) relative to tetramethylsilane (δ 0.00) or the appropriate residual solvent peak as internal reference. Multiplicities are given as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m) and broad (br). Electrospray MS spectra were obtained on a Waters platform LC/MS spectrometer. Analytic LCMS: Columns used, Waters Acquity UPLC BEH CI 8 1.7μηι, 2.1mm ID x 50mm L or Waters Acquity UPLC BEH C18 1.7μιη, 2.1mm ID x 30mm L. All the methods are using MeCN/H₂0 gradients. MeCN and H₂0 contain either 0.1% Formic Acid or NH₃ (lOmM). Preparative LCMS: Column used, Waters XBridge Prep C18 5μιη ODB 30mm ID x 100mm L. All the methods are using MeOH/H₂0 gradients. MeOH and H₂0 contain either 0.1% Formic Acid or 0.1%) Diethylamine. Microwave heating was performed with a Biotage Initiator. Hydrogenation reaction was performed using H-Cube ®, HC-2.SS (SS reaction line version)

[00122] The following is a list of abbreviations used in the experimental section: ACN min

Acetonitrile minute

mL milliliter

AcOH Acetic acid μΕ microliter

Molecular weight calculated

Ac₂0 Acetic anhydride MW

MS Mes'd Measured mass

BAIB [bis(acetoxy)iodo]benzene

MPLC Medium Pressure Liquid

Boc tert-Butyloxy-carbonyl Chromatography br s broad singlet NMR Nuclear Magnetic Resonance cat. Pd/C Palladium on Carbon 10 %

Catalytic amount

ppm part-per-million d doublet

DCM Dichloromethane q quadruplet

Rt

DCC NN'-Dicyclohexylcarbodiimide Room temperature

DCE 1 ,2-Dichloroethene, s singlet

DIPEA Chloromethyl 2-

N,N-Diisopropylethylamine SEMC1

trimethylsilylethyl

DMAP 4-Dimethylaminopyridine

SM Starting Material

DMSO Dimethyl sulfoxide

t triplet

EtOAc Ethyl acetate

TCDI Γ, 1 '-thiocarbonyl diimidazole

Et₂0 Diethyl ether

TEOC 2-Trimethylsilylethyl carbamate

EtOH Ethanol

TFA Trifluoroacetic acid eq. equivalents

THF Tetrahydrofuran g gram

h hour TEMPO 2,2,6,6-teramethyl- 1 - piperidinyloxyl

High Pressure Liquid TESC1 Triethylchlorosilane

HPLC- Chromatography-Mass

MS/CAD spectrometry/ Charged Aerosol TBAF Tetra-nbutylammonium fluoride

Detector TLC Thin layer chromatography

LCMS Liquid Chromatography- Mass

Spectrometry TsCl p-Toluenesulfonylchloride m multiplet Tris(dimethylamino)sulphonium

TASF

MeCN Acetonitrile difluoro(trimethyl)silicate

MeOH Methanol TBDPS-C1 tert-Butylchlorodiphenylsilane

MtBE Methyl-Tert-Butyl-Ether TBDMS-C1 tert-Butyldimethylsilyl chloride mg milligram TMS-C1 Trimethylsilyl chloride

[00123] The naturally occurring parent molecule branimycin may be prepared by the fermentation procedure described in M Speitling PhD thesis in 2000 or by total synthesis using the methods as described in by S. Marchart et. al. in Angew. Chem. Int. Ed (2010) 49 (11): 2050-2053 "Total synthesis of the Antibiotic Branimycin". Synthetic Preparation of The Compounds of the Invention, and the comparison Compounds

[00124] Scheme 1 below shows the general procedure for the synthesis of the compound of the invention. The conversion of branimycin to compound 1 may be achieved by a sequence involving suitable conditions for the protection of the 17-hydroxyl, acylation of the 8-position with a protected or unprotected pyrrole-2-carboxylic acid derivative and removal of the protecting groups. Exemplary methods are described below, protecting groups suitable are typically silyl ether derivatives such as, but not restricted to, TES, TBDPS, TBDMS, TMS and related, but a person of skill in the art is aware of suitable alternatives.

Scheme 1

(II) Intermediate A

Intermediate B Compound 1

Method A: General method for C-l 7-hydrox l group protection

(I I) Intermediate A wherein R^pl is a suitable protecting group, for example SiEt₃.

Intermediate A: general procedure

[00125] A solution of starting compound (II) (1.0 eq.) in a solvent such as a halogenated solvent at 0°C is treated with imidazole (1.7 - 2.8 eq.) in presence of a catalytic base (e.g. DMAP (cat.)) and a chlorotrialkylsilane (e.g. TESC1 (1.1 - 1.3 eq.)) and stirred at 0°C or from 0°C to room temperature for lh to 16h. A saturated solution of NH₄C1 is added and the mixture diluted with DCM. The aqueous phase is extracted with DCM. The combined organic layers are dried over Na₂S0₄, filtered and concentrated under vacuum to give the desired product, which is purified by silica gel chromatography or may be used in the next step without further purification.

Representative Intermediates A

Intermediate Al:

[00126] A solution of branimycin (2.0 g, 4.15 mmol, 1.0 eq.) in DCM (33 mL) at 0°C was treated with imidazole (706 mg, 10.4 mmol, 2.5 eq.), DMAP (cat.) and TESC1 (871 μΕ, 5.19 mmol, 1.25 eq.) and stirred at 0°C for lh 15 min. Further amounts of TESC1 (8x70 μΕ) and imidazole (1x140 mg) were added until complete conversion is reached. Then saturated solution of NH₄C1 was added and the mixture diluted with DCM. The aqueous phase was extracted with DCM. The combined organic layers were dried over Na₂S0₄, filtered and concentrated under vacuum to give the desired product, which was used in the next step without further purification.

[00127] An alternative route to Intermediate Al : To a solution of branimycin (2.12 g, 4.4 mmol, 1 eq.) in dry THF (44 mL), imidazole (750 mg, 11.0 mmol, 2.5 eq.) was added at rt followed by DMAP (cat.). At 0°C chlorotriethylsilane (965 μΕ, 5.7 mmol, 1.30 eq.) was added over 15 min and the resulting white suspension was vigorously stirred for 120 min keeping the temperature at ~ 0°C. The reaction mixture was diluted with water and EtOAc, the aqueous layer was extracted with EtOAc (250mL). The combined organic layers were washed with brine, dried over Na₂S0₄ and concentrated. The residue was purified on Si0₂ with (Et₂0/Petroleum ether: 95/5): to give the 17-TES protected Branimycin as a white solid.

Method B: General method for the synthesis of acylation reagents

[00128] To a solution of pyrrole-2-carboxylic acid (2.0 g, 18 mmol, 1.0 eq.) in a (1 :1) mixture of dry DMF/THF (60 mL) was added Et₃N (12.6 mL, 90 mmol, 5.0 eq.) followed at rt by benzyl bromide (90 mmol, 10 mL, 5.0 eq.), the resulting white suspension was vigorously stirred at rt for one day. The reaction was poured into a mixture of Et₂0 and a saturated aqueous solution of NaHCOs (50mL), the aqueous layer was extracted with Et₂0 (2xl00mL) and EtOAc (lOOmL). The combined organic layers were washed with brine dried over Na₂S0₄, filtered and concentrated. Chromatography on Si0₂ with gradient from pure petroleum ether to EtO Ac/petroleum ether: 10/90: gave the protected pyrrole as a white solid.

[00129] To a solution of the O-benzyl protected pyrrole (3.53 g, 17.5 mmol, 1.0 eq.) in dry THF (40 mL) was added at rt Et₃N (2.7 mL, 19.2 mmol, 1.1 eq.) followed by di-tert-butyl dicarbonate (4.2 g, 19.2 mmol, 1.1 eq.) and DMAP (cat.), the resulting yellow solution was stirred at rt for 60 min and concentrated in vacuo. Chromatography on Si0₂ with (EtOAc/Petroleum ether: 5/95): gave the fully protected pyrrole as a yellow oil.

[00130] To a solution of the fully protected pyrrole (1.38 g, 4.6 mmol) in absolute EtOH (25 mL), purged with N₂ was added Pd/C (10% wt, 450 mg) followed by 1 ,4-cyclohexadiene (2.2 mL, 25 mmol, 5.5 eq.), the resulting black solution was vigorously stirred at rt for 120 min. The reaction mixture was filtered through a pall Seitz filter (washed with MeOH -200 mL). The clear solution was concentrated in vacuo and a white solid of N-Boc pyrrole carboxylic acid was formed upon standing at rt.

Method B2: N-2-(trimethylsilyl)ethyl-pyrrole-2-carboxylic acid

B2.a: Introduction of 2-(trimethylsilyl)ethyl protecting group on 1H pyrrole-2-carbaldehyde

[00131] To a suspension of sodium hydride (164.0 mg, 4.10 mmol, 1.3 eq, 60% in mineral oil) in DMF (2.5 mL), lH-2-pyrrole-carbaldehyde (300.0 mg, 3.15 mmol, 1.0 eq) was added at 0°C, and the mixture was stirred at the same temperature for 20 minutes. A solution of 4-nitrophenyl-2- trimethylsilyl- ethyl carbonate (1.16 g, 4.10 mmol, 1.3 eq) in DMF (2.5 mL) was added, and the reaction temperature was left stirring at at 0°C for 1 hour. Water (80 mL) was added to reaction mixture and extracted with EtOAc (80 mL). Organic layer was washed with H₂0 (2 x 80 mL), and then with brine (80 mL). Organic layer was separated, dried over Na₂SO i, filtered, and concentrated under vacuum. Crude product (944.8 mg) was purified using Biotage SP purification system (10 g column, fraction size: 6 mL, weak eluant: n-hexane, strong eluant: «-hexane:EtOAc=6: l, elution of product at 5% of strong eluant) to afford N-2-(trimethylsilyl)ethyl-pyrrole-2-carbaldehyde was obtained. ¾ NMR (DMSO-d₆) δ: 10.18 (s, 1H), 7.59 (m, 1H), 7.14 (m, 1H), 6.43 (m, 1H), 4.18-4.62 (m, 2H), 0.92-1.32 (m, 2H), 0.06 (s, 9H).

B2.b: Oxidation of N-2-(trimethylsilyl)ethyl-pyrrole-carbaldehyde

Mr =239.35 Mr =255.35

[00132] To a solution of N-2-(trimethylsilyl)ethyl-pyrrole-2-carbaldehyde (420.0 mg, 1.76 mmol, 1.0 eq) in THF (15 mL) and i-BuOH (15 mL), 2,3-dimethyl-2-butene (1.67 mL, 14.1 mmol, 8.0 eq) was added at 0°C. Subsequently, a solution of sodium chlorite (478.4 mg, 5.29 mmol, 3.0 eq) and sodium dihydrogenphosphate dihydrate (825.3 mg, 5.29 mmol, 3.0 eq) in H₂0 (5 mL) was added dropwise to the solution and the mixture was stirred vigorously at 0°C for 1 hour, and then the temperature was raised to room temperature. Reaction was stirred for 5 hours at room temperature. The mixture was then diluted with a saturated solution of NH₄C1 (100 mL) and extracted with EtOAc (100 mL). Organic layer was washed with brine (100 mL), dried over Na₂S0₄, filtered, and concentrated under vacuum. Crude product (549.2 mg) was purified using Biotage SP purification system (10 g column, fraction size: 6 mL, weak eluant: DCM, strong eluant: 5% DCM/MeOH, elution of product at 0-20% of strong eluant) to afford N-2-(trimethylsilyl)ethyl-pyrrole-2-carboxylic acid ^lH NMR (DMSO-d₆) δ: 12.75 (s, 1H), 7.38 (m, 1H), 6.83 (m, 1H), 6.14-6.39 (m, 1H), 4.21-4.53 (m, 2H), 1.26 (d, 1H), 0.96-1.15 (m, 1H), 0.04 (s, 9H). Method B3: l-tosyl-pyrrole-2-carboxylic acid

B3.a: synthesis of Methyl l-tosyl-pyrrole-2-carboxylate

[00133] To a suspension of sodium hydride (115.2 mg, 2.88 mmol, 1.2 eq, 60% in mineral oil) in DMF (2.5 mL) methyl 2-pyrrole-carboxylate (300.0 mg, 2.40 mmol, 1.0 eq) was added at 0°C, and the mixture was stirred at the same temperature for 20 minutes. A solution of 4-toluenesulfonyl chloride (549.1 mg, 2.88 mmol, 1.2 eq) in DMF (2.5 mL) was added, and the reaction temperature was left stirring at at 0°C for 3 hours. Water (80 mL) was added to reaction mixture and extracted with EtOAc (80 mL). Organic layer was washed with brine (80 mL), dried over Na₂S0₄, filtered, and concentrated under vacuum. Crude product (757.2 mg, purity: >90%) was triturated with n-hexane (10 mL). White precipitate was filtered to afford methyl 1 -tosyl-pyrrole-2-carboxylate

[00134] ^lH NMR (DMSO-dg) δ: 7.94-7.99 (m, 2H), 7.93 (m, IH), 7.55 (m, 2H), 7.18 (m, IH), 6.50-6.60 (m, IH), 3.75 (s, 3H), 2.49 (s, 3H).

B3.b: Hydrolysis of Methyl l-tosyl-pyrrole-2-carboxylate.

[00135] To a solution of methyl l-tosyl-pyrrole-2-carboxylate (490.0 mg, 1.75 mmol, 1.0 eq) in THF (7.5 mL) a solution of LiOH (84.0 mg, 3.51 mmol, 2.0 eq) in H₂0 (2.5 mL) was added at room temperature. After 40 hours of stirring, reaction mixture was poured into H₂0 (100 mL), and pH was adjusted with 2M HC1 to 3. Acidic aqeous layer was extracted with EtOAc (2 x 80 mL). Organic layers were combined, dried over Na₂SO i, filtered, and concentrated under vacuum. Crude product (0.45 g) was triturated with n-hexane (15 mL). Light yellow precipitate was filtered, and product 1- tosyl-pyrrole-2-carboxylic acid was obtained. Method B4: l-tosyl-pyrrole-2-carbox lic acid

Synthesis of l-Tosyl-pyrrole-2-carbaldehyde

Mr =249.29

Mr =190.65

[00136] To a suspension of sodium hydride (151.6 mg, 3.15 mmol, 1.2 eq, 60% in mineral oil) in DMF (2.5 mL) lH-pyrrole-2-carbaldehyde (300.0 mg, 3.15 mmol, 1.0 eq) was added at 0°C, and the mixture was stirred at the same temperature for 20 minutes. A solution of 4-toluenesulfonyl chloride (722.6 mg, 3.79 mmol, 1.2 eq) in DMF (2.5 mL) was added, and the reaction temperature was left stirring at at 0°C for 1 hour. Water (80 mL) was added to reaction mixture and extracted with EtOAc (80 mL). Organic layer was washed with brine (80 mL), dried over Na₂S0₄, filtered, and concentrated under vacuum. Crude product (887.0 mg, purity: 90%) was triturated with n-hexane (10 mL). Dark violet precipitate occurred, which was filtered, and product 1 -tosyl-pyrrole-2-carbaldehyde was obtained. ^lH NMR (DMSO-d₆) δ: 9.97 (s, 1Η), 8.03 (d, 2Η), 7.98 (m, 1Η), 7.57 (m, 2Η), 7.38 (m, 1Η), 6.67 (m, 1Η), 2.49 (s, 3Η).

B4.b: Oxidation of l-tosyl-pyrrole-2-carbaldehyde

[00137] To a solution of 1 -tosyl-pyrrole-2-carbaldehyde (0.61 g, 2.45 mmol, 1.0 eq) in THF (25 mL) and i-BuOH (25 mL) 2,3-dimethyl-2-butene (2.33 mL, 19.6 mmol, 8.0 eq) was added at 0°C. Subsequently, a solution of sodium chlorite (0.66 g, 7.34 mmol, 3.0 eq) and sodium dihydrogenphosphate dihydrate (1.15 g, 7.34 mmol, 3.0 eq) in H₂0 (8 mL) was added dropwise to the solution and the mixture was stirred vigorously at 0°C for 1 hour, and then was left to reach RT. After 16 hours of stirring, mixture was diluted with a saturated solution of NH₄C1 (100 mL) and extracted with EtOAc (100 mL). Organic layer was washed with brine (100 mL), dried over Na₂SO i, filtered, and concentrated under vacuum. Crude product (971.5 mg) was triturated with n-hexane (15 mL). Light yellow precipitate was formed, which was filtered, and product l-tosyl-pyrrole-2-carboxylic acid was obtained. ^lH NMR (DMSO-d₆) δ: 12.50-12.90 (m, 1H), 7.93 (d, 2H), 7.87 (m, 1H), 7.45- 7.58 (m, 2H), 7.11 (m, 1H), 6.46-6.54 (m, 1H), 2.49 (s, 3H). Method C: general methods for the synthesis of Intermediates B: - acylation of C8-hydroxyl

Intermediate A Intermediate B

Method C.a: General method for C/8-O-acylation using trichloroacetyl reagent R -C(=0)CC

[00138] To a solution of Intermediate A (1.0 eq.) in DMF at 0°C under N₂ is added NaH (4.0 eq.). The reaction mixture is stirred for 10 to 30 min before adding the appropriate trichloroacetyl reagent R -C(=0)CCh, where R⁴ is a protected or unprotected pyrrole group (1.2 - 1.25 eq.). The reaction mixture is stirred from 0°C to room temperature overnight. A saturated solution of NH₄C1 is added and the mixture extracted with EtOAc. The combined organic layers are dried over Na₂S0₄, filtered and concentrated under vacuum. The desired product is obtained after purification by flash chromatography or preparative TLC to afford the desired product.

[00139] Trichloroacetyl reagent R⁴-C(=0)CC is either commercially available or can be readily prepared by methods known in the art.

Method C.b: General method for C/8-O-acylation using carboxylic acid R -C (=0)011 or R^4p-C(=0)OH

[00140] A solution of Intermediate A (1.0 eq.) in DCM is treated with the appropriate DCC supported (1.5 - 3.0 eq.), carboxylic acid R -C(=0)OH where R⁴ is a protected or unprotected pyrrole group (for example those intermediates prepared according to Method B above) (1.1 - 3.0 eq.) and DMAP (0.1 - 3.0 eq.), and then stirred at room temperature or at 35°C for 16h to 5 days. If necessary more reagents are added until the reaction reaches complete conversion. The reaction mixture is filtered and the solid washed with DCM and THF. The filtrate is concentrated under vacuum. The residue can be taken up in DCM or Et₂0 and washed with saturated NaHC0₃ and saturated NH₄C1 solutions, dried over Na₂S0₄, filtered and concentrated under vacuum prior to purification; or directly purified by flash chromatography or preparative TLC to afford the desired product. [00141] To a solution of N-Boc pyrrole carboxylic acid (970 mg, 4.6 mmol, 1.15 eq.) in dry DCM (12 mL) was added at rt Et₃N (1.68 mL, 12 mmol, 3.0 eq.) followed by 2-Methyl-6-nitrobenzoic anhydride (2.04 g, 5.8 mmol, 1.45 eq.) and DMAP (cat.), the resulting pale yellow solution was stirred at rt for 60 min, then was added at rt a solution of both solids 17-TES branimycin (2.38 g, 4.0 mmol, 1.0 eq.) and DMAP (538 mg, 4.4 mmol, 1.1 eq.) in dry DCM (22 mL, via the syringe pump, the syringe was rinsed with 3 mL of dry DCM after complete addition) over 60 min, the resulting yellow solution was stirred at rt overnight. The reaction was quenched with an aqueous solution of NaHC0₃ and diluted with DCM, the aqueous layer was extracted with DCM (150 mL). The combined organics layers were washed with brine, filtered through a phase separator and concentrated. The residue was purified on S1O₂ with (EtO Ac/Petroleum ether: 22/78): to give as the main fraction the 8- (Boc-pyrrole-2-yl)-17-TES-branimycin as a white foam.

Representative Intermediates B

Intermediate Bl: wherein R^pl is

Intermediate B

[00142] To a solution of Intermediate Al (250 mg, 0.414 mmol, 1.0 eq.) in DMF (4.2 mL) at 0°C under N₂, NaH (66 mg, 1.66 mmol, 4.0 eq.) was added. The reaction mixture was stirred for 10-30 min before adding trichloroacetyl-(lH-pyrrol-2-yl) (110 mg, 0.52 mmol, 1.25 eq.). The reaction mixture was stirred from 0°C to room temperature overnight. A saturated solution of NH₄C1 was added and the mixture extracted with EtO Ac. The combined organic layers were dried over Na₂S0₄, filtered and concentrated under vacuum. Purification of the residue by flash chromatography using heptane/EtOAc (75/25 to 60/40) afforded the desired product. Intermediate B2: wherein R^pl is SiEt₃, and R^F2 = Boc

Intermediate B

[00143] This intermediate was prepared by Method C.b starting from Intermediate Al and pyrrole-l,2-dicarboxylic acid 1-tert-butyl ester (obtained as per Tetrahedron Letters (1987) 28, 48, p. 6025).

Intermediate B3: 8-N-2-(trimethylsilyl)ethyl-pyrrole-l 7-TES-branimycin

[00144] N-2-(Trimethylsilyl)ethyl-pyrrole-2-carboxylic acid (70.0 mg, 0.21 mmol, 1.3 eq), 2- methyl-6-nitrobenzoic anhydride (116.4 mg, 0.34 mmol, 1.6 eq) and triethylamine (88.5 μL, 0.63 mmol, 3.0 eq) were stirred at RT in DCM (5.0 mL) for 2 hours. Then a solution of 17-TES- branimycin (126 mg, 0.21 mmol, 1.0 eq) and 4-dimethylaminopyridine (28.4 mg, 0.23 mmol, 1.1 eq) in DCM (2.5 mL) were added dropwise over 2.5 hours, and reaction mixture was left stirring at RT. After 16 hours of stirring, reaction mixture was poured into a saturated solution of NaHCOs (50 mL), and extracted with DCM (2 x 50 mL). Organic layers were combined, and washed with brine (75 mL). Organic layer was dried over Na₂SO i, filtered, and concentrated under vacuum. Crude product (235.5 mg) was purified on Biotage SP purification System (10 g column, fraction size: 6 mL, weak eluant: n-hexane, strong eluant: «-hexane:EtOAc=4: l, elution of product at 50% of strong eluant) to afford 8-N-2-(trimethylsilyl)ethyl-pyrrole- 17-TES-branimycin.

Intermediate B4: 8-N-Ts-pyrrole-17-TES-branimycin

Esteriflcation of 17-TES Branimycin with N-tosyl-pyrrole-2-carboxylic acid

[00145] N-Ts-pyrrole-2-carboxylic acid (350.2 mg, 1.32 mmol, 1.3 eq), 2-methyl-6-nitrobenzoic anhydride (557.7 mg, 1.62 mmol, 1.6 eq) and triethylamine (426.5 \L, 3.05 mmol, 3.0 eq) were stirred at room temperature in DCM (22.5 mL) for 2 hours. Then a solution of 17-TES-Branimycin (606.0 mg, 1.02 mmol, 1.0 eq) and 4-dimethylaminopyridine (136.8 mg, 1.12 mmol, 1.1 eq) in DCM (7.5 mL) were added dropwise over 3 hours, and reaction mixture was left stirring at room temperature. After 2 hours of stirring, reaction mixture was poured into a saturated solution of NaHC0₃ (70 mL), and extracted with DCM (2 x 50 mL). Organic layers were combined, and washed with brine (80 mL). Organic layer was dried over Na₂SO i, filtered, and concentrated under vacuum. Crude product (1.053 g, 87% wanted product, 13%> bis-acylated product) was purified on Biotage SP purification system (25 g column, fraction size: 9 mL, weak eluant: n-hexane, strong eluant: n- hexane:EtOAc=2: l, elution of product at 75%> of strong eluant) to afford 8-N-Ts-pyrrole-17-TES- branimycin.

Method D: General method for protecting group removal Method Dl: Protecting group removal

[00146] To a cooled solution of the 8-(Boc-pyrrole-2-carbonyl)-17-TES-branimycin (2.2 g, 2.79 mmol) in dry DCM (50 mL) at 0°C was added TFA (11 mL, 144 mmol, 50 eq.) over 15 min and the resulting solution was stirred at 0 °C for 75 min. The reaction was quenched at 0°C by slow addition of K₂CO₃ solid (-12 g) until complete neutralization, the mixture was stirred at 0°C for 5 min and at rt for 20 min, diluted with DCM and filtrated. The K₂CO₃ solid was washed with DCM until no product could be detected and the filtrate was concentrated. The residue was purified on S1O₂ with (Et₂0/ Petroleum ether/Acetone: 80/10/10): to give the desired compound as a white foam.

[00147] A solution of starting compound (1.0 eq.) in THF at 0°C or room temperature is treated with a 1M solution of TBAF in THF (1.2 - 3.2 eq.), and allowed to stir at 0°C or at room temperature for 10 min to 4h. The reaction mixture is then concentrated to dryness, or diluted with DCM, washed with brine or with saturated solution of NH₄C1, dried over Na₂S0₄, filtered and concentrated under vacuum. The residue is purified by flash chromatography to afford the desired product.

Method E: General method for simultaneous deprotection of -C/8and, if present, of -C/17 (removal of protective groups)

Scheme 5.

Intermediate B Compound 1

[00148] A solution of starting compound (1.0 eq.) in DCM at 0°C is treated with TFA (50 eq.) and stirred at 0°C for 30 min to lh. The reaction mixture is concentrated under vacuum and the residue is purified by flash chromatography or preparative TLC to afford the desired product.

Method El: De rotection of 8-N-2-(trimethylsilyl)ethyl-pyrrole-17-TES-Branimycin with TFA

[00149] 8-N-2-(Trimethylsilyl)ethyl-pyrrole-17-TES-branimycin (150.0 mg, 0.18 mmol, 1.0 eq) was dissolved in DCM (5 mL) at 0°C, and trifluoroacetic acid (668.0 L, 9.0 mmol, 50.0 eq) was added. After 30 minutes reaction was stopped by adding K₂CO₃ (s, 746.3 mg, 5.4 mmol, 30.0 eq) to the reaction mixture at 0°C. Reaction was left stirring at 0°C for 5 minutes, and then 10 minutes at room temperature. Reaction mixture was diluted with DCM (25 mL), and filtered. Solid was washed with DCM, until no product could be detected (checked by UV). The filtrate was concentrated under vacuum to give white foam (111.2 mg), which was purified on a Biotage SP purification system (10 g column, fraction size: 6 mL, strong eluant: 5% MeOH/DCM, weak eluant: DCM, elution of product at 50% of strong eluant) to afford desired product.

Method E2: Deprotection of 8-N-2-(trimethylsilyl)ethyl-pyrrole-17-TES-Branimycin with TASF

[00150] 8-N-2-(Trimethylsilyl)ethyl-pyrrole-17-TES-branimycin (1.60 mg, 1.32 μηιοΐ, 1.0 eq) was dissolved in DMF (0.2 mL) and tris(dimethylamino)sulfonium difluorotrimethylsilicate (2.64 mg, 6.59 μmol, 5.0 eq) was added. Reaction mixture was monitored by OAUPLC-MS and TLC (eluant: 5%MeOH/DCM). After 2 hours of stirring at room temperature, the reaction proceeded quantitatively.

Method E3: Deprotection ofN-Tosyl-pyrrole-17-TES-Branimycin with TASF

[00151] 8-N-Ts-pyrrole-17-TES-branimycin (1.0 mg, 1.18 μιηοΐ, 1.0 eq) was dissolved in DMF (0.2 mL) and tris(dimethylamino)sulfonium difluorotrimethylsilicate (1.63 mg, 5.92 μιηοΐ, 5.0 eq) was added. Reaction mixture was monitored by UPLC-MS and TLC (eluant: 5%MeOH/DCM). After 7 hours of stirring at room temperature, the deprotection was completed.

Branimycin

[00152] This compound was prepared by following fermentation process.

[00153] For the preparation of the first seed stage, one mycelial cell bank working copy was quickly thawed at 37°C in a water bath and the glycerol-culture of Saccharothrix xinjiangensis G60/1571 30xB2M21, registered under accession number NCIMB 41952 (12.5 mL) transferred to 1L baffled Erlenmeyer flasks containing YM7.2 medium (200 mL). It should be recognized that other media may be used to optimize various aspects of production). This first seed stage was cultivated at 28°C for 48 h on a rotary shaker, analyzed microscopically and pooled prior to transfer to the second seed stage. To prepare the second seed stage, a fermenter containing 20 L YM7.2 medium (200 mL) was inoculated from the first seed stage using 1% first seed stage inoculum. The second seed stage was grown for 48 h at 28°C with an air flow rate of 14 L per min and an initial stirrer speed of 400 rpm and pooled prior to inoculation of MC production medium (3000 L). [00154] Fermentation was carried out in a 4000 L stirred tank fermenter at 28°C with an air flow rate of 300 L per min and an initial stirrer speed of 400 rpm. Dissolved oxygen was controlled online during fermentation. During fermentation, foaming was controlled by automatic addition of antifoaming agent Silfoam Se2, Struktol J647 and water (1 : 1 :1). It would be appreciated by a person of skill in the art that other types of antifoaming agents can be used, such as other type of polypropylene glycols, silicones, esters, fatty acids, fats, and sulfonates. Compound production was monitored on a daily basis by use of LC-MS/UV/ELSD analysis. Fermentations were carried-out for 90 to 144h, typically for 120h. In the fermentations described above, any stirring system known to a person of skill in the art may be used, for example a conventional paddle stirrer, a turbine-stirrer system or the Ekato InterMIG impeller.

[00155] After harvest, around 2% Celite 512 were added to the fermentation broth. The fermentation broth was filtered through 24 chambers (17.28 m²) with a filtration performance of 526.24 L per hour. 125 L resin (LEWATIT VP OC 1064 MD PH) were loaded to a 800 L column (cross section 770 mm; height 27 cm). The supernatant was pumped through the column with a flow rate of 3500 L per hour for three hours. Afterwards the culture broth was extracted with 350 L per hour. The loaded resin was eluted two times with double volume acetone in portions. The acetone was evaporated until a residual water phase was visible. The water phase was extracted three times with equal amounts of ethyl acetate. The ethyl acetate extracts were combined and the solvent was removed under reduced pressure yielding around 750 g crude extract.

[00156] The crude extract was then subject to normal phase MPLC using a Biotage Isolera Flash purification system with silica cartridge (800 g) as the solid phase and gradient solvent mixture DCM:MeOH from 1 :0 to 96:4 as eluent to obtain a branimycin- enriched fraction that was further purified on reversed phase MPLC using a Biotage Isolera Flash purification system with CI 8 cartridge (800 g), gradient ACN:H₂0 0% to 100% to give branimycin as a pure product.

YM7.2 medium

Dissolve in 1000 mL of distilled H₂0

Adjust pH to 7.2 before sterilization

Autoclave MC medium

Dissolve in 1000 mL of tap H₂0

Adjust pH to 7.2 before sterilization

Autoclave

Baleomycin

[00157] This compound was prepared by following fermentation process.

[00158] For the preparation of the first seed stage, one mycelial cell bank working copy was quickly thawed at 37°C in a water bath and the glycerol-culture of Saccharothrix xinjiangensis G60/1571 30xB2M21, registered under accession number NCIMB 41952 (12.5 mL) transferred to 1L baffled Erlenmeyer flasks containing Celmer-79a medium (200 mL). This first seed stage was cultivated at 28°C for 48 h on a rotary shaker, analyzed microscopically and pooled prior to transfer to the second seed stage. To prepare the second seed stage, 18 x 1 L baffled Erlenmeyer flasks containing Celmer-79a medium (200 mL) were inoculated from the first seed stage using 5% first seed stage inoculum. The second seed stage was grown for 48 h at 28°C on a rotary shaker (120 rpm, 10 cm stroke) and pooled prior to inoculation of SGG production medium (90 L).

[00159] Fermentation was carried out in a 140 L stirred tank fermenter at 28°C with an air flow rate of 63 L per min and an initial stirrer speed of 200 rpm. Dissolved oxygen was cascade-controlled at 30% via agitation. Pure oxygen was added at a flow rate of 20 L per min by using the on/off control when the agitation speed reaches the maximum value. To prevent foaming during fermenter sterilization, Antifoam A (Sigma Aldrich, #10794, 30% aqueous emulsion of silicon polymer) was added to the medium at 0.1 % (v/v). During fermentation, foaming was controlled by automatic addition of antifoaming agent Desmophen® 2061 BD (Bayer, solvent- free linear polypropylene ether polyol). It would be appreciated by the skill in the art that other types of antifoaming agents can be used, such as other type of polypropylene glycols, silicones, esters, fatty acids, fats, and sulfonates. Compound production was monitored on a daily basis by use of HPLC-MS/CAD analysis. Fermentations were typically carried-out for 120 h. [00160] After harvest, the broth was then extracted 3 times for 12 h on a rotary shaker with EtOAc (1 : 1). Decantation of EtOAc phase and evaporation of the solvent was performed to obtain the EtO Ac extract. This later extract was further solubilized with pentane (1.5 L). After decantation, the pentane phase was slowly removed to give the defatted extract as the insoluble residue.

[00161] The defatted extract was then solubilized twice with MeOH (1 L) per ~100g extract. After filtration, the MeOH was separated from the insoluble residue and dried to obtain the final extract.

[00162] The equivalent of lOOg of final extract was then treated with MtBE (1L) and filtrated. The residue was then treated with DCM (500 mL) and NH₄OH (500 mL). The DCM phase was collected and dried down to provide crude extract.

[00163] The crude extract was then subject to normal phase MPLC using a Biotage Isolera Fash purification system with silica cartridge (800 g) as the solid phase and gradient solvent mixture DCM:MeOH from 1 :0 to 96:4 as eluent to obtain a baleomycin-enriched fraction that was further purified on preparative HPLC using a Varian preparative HPLC system, a Nucleodur Sphinx CI 8 250 x 40 mm column, gradient ACN:H₂0 10% to 100% to give compound 1 (baleomycin) as a pure product.

[00164] Celmer-79a medium

Dissolve in 1000 mL of distilled H₂0

Adjust pH to 7.2 before sterilization

Autoclave

* added after sterilization from sterile stock solution (c=0.2g/mL)

[00165] SGG medium

Ingredients g/L

Glucose* 10.0

Glycerol 10.0

Starch 10.0

Cornsteep powder 2.5

Yeast extract 2.0

Tryptone 5.0

NaCl 1.0

CaC0₃ 3.0

Dissolve in 1000 mL of tap H₂0

Adjust pH to 7.2 before sterilization

Sterilize

* added after sterilization from sterile stock solution (c=0.3g/mL)

Synthesis of the compound of the invention: Compound 1: 8-0-lH-pyrrole-2'- carbonylbranimycin

[00166] This compound was prepared by Method F starting from Intermediate B 1.

[00167] A solution of Intermediate B. 1 (740 mg, 0.937 mmol, 1.0 eq.) in THF (37 mL) at 0°C was treated with a 1M solution of TBAF in THF (1.41 mL, 1.41 mmol, 1.5 eq.). The reaction mixture was stirred at 0°C for 2.5h, then diluted with DCM, washed with brine, dried over Na₂S0₄, filtered and concentrated at room temperature under vacuum. The residue was purified by flash chromatography (using DCM/EtOAc 1 :0 to 0: 1) to afford the desired product.

[00168] The reference compounds are listed in Table 1A, and Compound 1 is listed in Table IB below. The NMR spectral data of Compound 1 is given in Table 2 below.

Table 1A - reference compounds

Table IB - Compound 1

9] Table 2: NMR Data of The Compound of the Invention

Cpd # NMR Data

lH NMR (300 MHz, CDC1₃) δ ppm 9.11 (1 H, br. s.), 6.99 - 7.05 (1 H, m), 6.87 - 6.92 (1 H, m), 6.28 - 6.33 (1 H, m), 5.94 - 6.03 (1 H, m), 5.72 (1 H, d), 5.41 (1 H,

1 dd), 5.25 (1 H, t), 5.06 (1 H, dd), 4.27 (1 H, d), 4.05 - 4.15 (2 H, m), 3.39 - 3.63 (10 H, m), 3.33 (3 H, s), 3.30 (3 H, s), 3.01 - 3.13 (2 H, m), 2.85 - 2.99 (1 H, m), 2.68 - 2.79 (1 H, m), 2.54 - 2.61 (2 H, m), 2.51 (1 H, d), 1.73 (3 H, s), 1.24 - 1.35 (3 H, m). Biological Examples

Biological Example 1 : Antibacterial activity assay

[00170] Compounds were tested for antimicrobial activity against a panel of organisms according to standard procedures described by the National Committee for Clinical Laboratory Standards (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that grow Aerobically 7^th edition Approved Standard M7-A7 Wayne PA:CLSI 2006) except that all testing was performed at 37° C. Compounds were dissolved in 100% DMSO and were diluted to the final reaction concentration (0.06 μg/mL-64 μg/mL) in microbial growth media. In all cases the final concentration of DMSO incubated with cells was less than or equal to 2.5%. For minimum inhibitory concentration (MIC) calculations, 2-fold dilutions of compounds were added to wells of a microtiter plate containing 5x 10⁴ bacterial cells in a final volume of 200 μΐ. of media (Mueller-Hinton Broth). The MIC value is defined as the lowest compound concentration inhibiting visible growth of the test organism. The MIC (in μg/mL) values of representative compounds of the present invention together with comparisons with the previously reported naturally occurring molecules are listed in Tables 3A-3C below. Whilst data against specific strains is reported herein, the specific strains selected are not critical for identifying active compounds. A person of skill in the art can readily select alternative or additional strains for MIC calculations.

[00171] Strains used:

S. aureus strains:

• Sal ATCC 13709 Sensitive strain

• Sa26 ATCC 25923 Sensitive strain

• Sa4 NCTC 6571 Sensitive strain

• Sa2 Clinical strain registered under accession number NCIMB 41953

MRSA - Erythromycin Resistant - Fluoroquinolone resistant

H. influenza strains

Hi3 ATCC 31517 Sensitive strain

Hi4 Efflux pump KO Sensitive strain

Hi 106 ATCC 51907 Sensitive strain

Hi47 Clinical strain Ampicillin Resistant

HilO Clinical strain Sensitive strain

E. coli strains:

• Eel ATCC 25922 Sensitive strain

• Ec50 Efflux KO Sensitive strain

• EC113 Efflux KO Sensitive strain [00172] Table 3A - S. aureus

[00173] Table 3B - H. influenza strains

Table 3C - E. coli

Biological Example 2 - in vitro activity against DNA III Polymerase E.

[00174] The replicative DNA polymerase III, a subunit, from Staphylococcus aureus (Biocat73824) was purified from a recombinant strain, containing a pBluePet-DnaE(AAl-1022) construct.

[00175] The E.coli strain BL21(DE3) was transformed with the pBluePet-Z¾∞£'(AAl -1022) construct and was grown in Terrific Broth (TB) medium under 220 rpm shaking at 37°C. Terrific Broth was prepared by following procedure: tryptone peptone ((12 g, DIFCO, #211705), Bacto™ yeast extract (24 g, BD, # 212750) and glycerol (4 mL) were added to water (900 mL final volume), sterilized and the volume was adjusted to 1000 mL by addition of 100 mL of KH₂P0₄ (170 mM) and K₂HP0₄ (720 mM) stock solution. When OD₆oo reached 0.5, bacteria were induced with 1 mM isopropyl β-D-l - thiogalactopyranoside and left at 25°C for 14-16 hours under 220 rpm shaking. Cells were harvested by centrifugation at 6,000 x g and frozen at -20°C before use. Expression yield was determined after lysis using B-PER^® Bacterial Protein Extraction Reagent (Thermo Scientific, #78248) as described by the manufacturer, giving 100 mg/ L of soluble protein.

[00176] A total of 16 g (wet weight) of E.coli BL21(DE3) paste was suspended in 11 volumes of lysis buffer (50 mM Tris pH 8, 50 mM NaCl, 10% glycerol 100 mM lysozyme and protease cocktail inhibitor (Roche Diagnostics, # 11873580001)). The pellet was homogenized at 4°C by magnetic stirring for 15 min. Cells were broken by sonication (150 pulses of 4 sec (8 sec off) using a 13 mm diameter probe, in icy bath). To hydrolyze DNA and RNA, 250 U/ mL of Benzonase^® nuclease (Novagen, #70746-3) was added before ultracentrifugation at 142,400 x g for one hour at 4°C. Supernatant was recovered and loaded on a 5 mL Histrap™ column HP (GE Healthcare, 17-5248-02) preequilibrated in buffer A (50 mM Tris pH 8, 20 mM NaCl, 10% glycerol, lOmM β- Mercaptoethanol). The column was first washed with 10 column- volumes of buffer A + 1.3 M NaCl then with 10 column- volumes of buffer A + 30 mM Imidazole to remove unspecific binding. Bound proteins were eluted with a 10-column- volume linear gradient of buffer B (50 mM Tris pH 8, 20 mM NaCl, 10%) glycerol, 10 mM β-Mercaptoethanol, 500 mM imidazole). Fractions containing DnaE from Staphylococcus aureus, as determined by SDS-PAGE analysis, were pooled giving 70 mg of 95%) pure target protein (final yield: 87.5 mg/ L).

[00177] A radioactive filterplate assay was used to assess inhibitory activity of compounds on DNA Polymerase Ilia. Five μΕ of a dilution series of compound, starting from 100 μΜ highest concentration, 1/5 dilution, was added to the wells of a 96 well plate. Recombinant enzyme was diluted to 0.47μg/mL in a buffer containing 20mM Tris pH7.5, 8mM DTT, lOmM MgOAc, 0.05% CHAPS and 10μΕ thereof was added to the compound dilutions. The reaction was started with the addition of 10μΕ substrate in the same buffer, containing activated calf thymus DNA (Sigma, D4522), dATP, dGTP, dCTP, dTTP (Invitrogen) and [a-33P]-dTTP (Perkin Elmer, NEG605H001) at a concentration of 62^g/mL, 50nM and 7.5μΟ/ηιΕ respectively. The mixture was incubated at 30°C for 120 minutes and terminated by addition of phosphoric acid. Samples were transferred to filter plates and incorporated radioactivity was measured by the Topcount. Data were converted to percent inhibition with respect to positive and negative controls. IC₅o values were calculated using Graph Prism™ software, values are shown in Table 4 below

Semiquantitative scoring:

S. aureus E. coli

0.001-1 μg/mL *** ###

λ .ΟΛΟ μ^π^ ** ##

>10 μg/mL * #

Table 4 S. aureus E. coli

Nargenicin Al *** ###

Nargenicin Bl *** #

Nodusmicin *** ##

Branimycin *** ##

Baleomycin ** ##

Compound 1 *** ##

Biological Example 3 - broad panel S. aureus in vitro assay

[00178] In this example, the activity of Compound 1 against panels of methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), methicillin-susceptible S. epidermidis (MSSE) and methicillin-resistant S. epidermidis (MRSA) was investigated at IHMA Europe Sari. The MRSA panel also contained a variety of phenotypes including CMRSA 2, 3/6, 7, 8, 9, USA 300, 700 & 1000, and isolates resistant to daptomycin, linezolid or vancomycin. The data clearly show that Compound 1 exhibited good activity against all isolates tested in the study. Globally, the minimum and maximum MICs were 0.12 and 4 μg/mL. Specifically, ranges were often narrower than this depending on the phenotypes of MSSA, S. epidermidis and especially MRSA tested. It is noteworthy that resistance to all other drugs had no effect on the activity of Compound 1. 3.1 Clinical Isolates

[00179] The following clinical isolates were tested in this example.

3.1.1 Staphylococcus aureus

• MSSA (n = 100) and MRSA (n = 100)

• USA300: n = 10

• European ST398: n = 10

• 2 isolates each of the following lineages: CMRSA2; CMRSA10; CMRSA7; CMRSA 3/6, CMRSA8; CMRSA9; USA1000; USA700, ST88,

• VRSA (n = 7)

• VISA (10 isolates)

• Linezolid-resistant (3 isolates)

• Daptomycin-resistant (5 isolates)

3.1.2 Staphylococcus epiderm idis

• MSSE (n = 25) and MRSE (n = 25)

3.2 Compounds [00180] Compound 1 was dissolved in DMSO to make a stock solution of 6400 μ^ιηΐ.. This solution was then diluted 1 :10 in water, then diluted into cation-adjusted Mueller-Hinton broth (CAMHB) for the sequential dilutions used in the broth microdilution panels. Daptomycin (Cubist, lot# MCB2007), linezolid ( BMS, lot# K0200228)), levofloxacin (Sigma, lo# WA20608), trimethoprim/sulfamethoxazole (Sigma lot# 103K1266/Sigma lot# 103K1237), vancomycin (Sigma, lot# 015K0825), ceftriaxone (Sigma, lot# 083K0521), and tigecycline (Pfizer, Inc, lot#FOA0044901) were dissolved to 640 μg/mL according CLSI specifications (CLSI, Ml 00-21, 2012). The stock solutions were further diluted into CAMHB for the sequential dilutions used in the broth microdilution panels. mecA mediated oxacillin resistance was confirmed by either the CLSI cefoxitin disc method (CLSI, Ml 00-21, 2012) or previous testing for the presence of the mecA gene.

3.3 Antimicrobial Susceptibility Testing

[00181] Minimum inhibitory concentration (MIC) endpoints were determined by broth microdilution according to CLSI guidelines (CLSI, Ml 00-21, 2012). Panels were prepared at IHMA using cation-adjusted Mueller-Hinton broth. Colonies were taken directly from a second-pass culture plate and prepared to a suspension equivalent of the 0.5 McFarland standard using normal saline. Inoculation of the MIC plates took place within 15 minutes after adjustment of the inoculum suspension turbidity. The panels were incubated at 35 °C for 16 to 20 hours before reading the MIC endpoints. Quality control testing was performed each day of testing as specified by the CLSI using S. aureus ATCC 29213 and E. faecalis ATCC 29212.

3.4 Results

[00182] Compound 1 exhibited MIC ranges of 0.12 - 4 μg/mL against all S. aureus and and 0.5 - 4 μg/mL against all S. epidermidis. MIC90's for all S. aureus were 1 - 2 μg/mL and for all S. epidermidis were 4 μg/mL. See Table 5 for results from Compound 1 and the control antibiotics used.

[00183] Compound 1 exhibited consistent activity against all phenotypes tested in the study, including MSSA, MSSA VISA, MRSA, CMRSA 2, 3/6, 7, 8 & 9, daptomycin and linezolid-resistant isolates, clonal MRSA ST398 and ST88 and the USA types 300, 700 & 1000.

[00184] Furthermore, activity of Compound 1 was good also against MRSA, VISA & VRSA. The activity against S. epidermidis was slightly lower as compared with MSSA and MRSA though maximum MICs were 4 μg/mL against MSSE and 2/250 strains of S. aureus exhibited the highest MIC 4μg/ml, compared to 18/50 strains of S. epidermidis.

[00185] The overall MIC distributions for Compound 1 were relatively narrow with a maximum range of six doubling dilutions. Furthermore, activity of Compound 1 against the CLSI quality control isolate S. aureus ATCC 29213 was 1 μg/mL on each day of quality control testing. Table 5

3.4 Conclusions

[00186] This study demonstrates that Compound 1 has activity against all resistance phenotypes including MSSA, MRSA, multidrug resistance strains including fluoroquinolone, as well as Linezolid resistance. Moreover CAM-1 has not shown cross resistance with existing antibiotics, in particular four daptomycin resistant strains, 2 linezolid resistant strains and 15 VISA/VRSA strains were inhibited by <lug/mL of Compound 1.

Biological Example 4 - in vivo efficacy

[00187] The compound of the invention and comparative examples from the parent molecules were tested in several in vivo models of infection. In particular data from infection with S. aureus in thigh, lung and groin is presented herein.

3.1 Thigh Model of Infection

[00188] The neutropenic mouse thigh infection model is well known and has been used extensively for determination of pharmacokinetic/pharmacodynamic (PK/PD) index determination and prediction of antibiotic efficacy in patients since its description by W. A. Craig, J. Redington, and S. C. Ebert, J. Antimicrob. Chemother. 27[Suppl. C] :29-40, 1991.

[00189] The in vivo antibacterial activity was established by infecting both thighs of male CD-I mice (Charles River Lab, Lyon, France) weighing 19-23 g with Methicillin Resistant S. aureus (MRSA) inoculum. Before infection, mice were rendered neutropenic (neutrophil^ 100/mm³) by injecting them with cyclophosphamide (SIGMA, St Louis) intraperitoneally 4 days (150mg/kg of body weight) and 1 day (lOOmg/kg) before thigh infection. The inoculum was prepared from Methicillin Resistant clinical isolate of S. aureus. The optical density of a broth culture of freshly plated bacteria was adjusted to 0.1 at an absorbance at 590nm, then put at 37°C in shaken culture for about lh30 until a OD= 0.3 at 590nm. After a 1/10,000 dilution into physiological saline, O. lmL of this inoculum suspension was injected into each thigh.

[00190] The test compound was dissolved in methyl cellulose at 0.5% (for oral treatment) or polyethyleneglycol 200 at 20% (for parenteral treatments) to give a solution of 2 mg/mL (pH=7.0). This solution was diluted with vehicle to give 0.6 mg/mL solutions. One hour post infection (PI), animals were treated orally (po) or via parenteral routes (ip, sc or iv) as indicated in the experimental tables. A group of untreated mice received only the corresponding vehicle.

[00191] Administration was repeated seven hours post-infection (PI) for the twice a day (BID) model or at 24h PI for the once a day (QD) model. Twenty four hours PI for the BID model or 48h PI for the QD model, all mice were euthanized, each thigh was removed, and the bacterial burden in the thigh muscles was enumerated after tissue homogenization and plating.

[00192] The results of the in vivo efficacy test are summarized in Table 6A, which provides a representative example of the results obtained for Compound 1.

[00193] In this model, Ciprofloxacin is used as the negative control, and Vancomycin (parenteral) and/or Linezolid (parenteral or oral) is used as the positive control. A test compound is considered active when it shows a log reduction equivalent to the positive control in the same study.

Table 6A - Thigh Sa - (Nd of treatment

Study Compound Dose Route Frequency Sample Log reduction Active mg/kg time, PI vs. untreated

1 Vancomycin 50 SC BID 7h -4.01 Y

Ciprofloxacin 50 SC -1.27 N

Branimycin 100 IP -0.3 N

2 Vancomycin 50 SC BID 7h -2.57 Y

Ciprofloxacin SC -1.07 N

Compound 1 IP -3.55 Y

3 Vancomycin 50 SC BID 7h -1.52 Y

Ciprofloxacin SC 0.22 N

Nargenicin Al SC -4.52 Y

4 Vancomycin 15 SC BID 24h -3.96 Y

Ciprofloxacin 50 SC -0.05 N

Compound 1 5 IP -1.16 N

15 -3.32 Y

50 -4.74 Y Study Compound Dose Route Frequency Sample Log reduction Active mg/kg time, PI vs. untreated

5 Ciprofloxacin 50 SC BID 24h -0.27 N

Vancomycin 50 sc -4.27 Y

Linezolid 50 PO -4.07 Y

50 sc -4.64 Y

Nargenicin Al 5 IP 0.28 N

15 IP -0.55 N

50 IP -5.40 Y

200 PO -4.47 Y

50 sc -3.28 Y

Compound 1 5 IV -0.13 N

6 Ciprofloxacin 50 sc BID 24h -0.26 N

Linezolid 25 PO -4.54 Y

Linezolid 50 PO -3.49 Y

Nargenicin Al 50 PO -2.02 Y

100 PO -4.06 Y

Compound 1 25 IV -3.92 Y

7 Ciprofloxacin 50 sc BID 24h -0.08 N

Linezolid 15 PO -2.98 Y

Linezolid 50 PO -5.13 Y

Compound 1 15 PO -4.13 Y

50 PO -6.72 Y

8 Ciprofloxacin 50 sc QD 48h -0.39 N

Vancomycin 50 sc -3.2 Y

Linezolid 50 PO -2.78 Y

Compound 1 50 PO -4.63 Y

3.2 Lung Model of Infection

[00194] To see if compounds are potentially efficacious to treat pneumonia, they were tested in a lung infection model in mice induced by a methicillin resistant S. aureus clinical isolate Sa2.

[00195] The in vivo antibacterial activity was established by infecting lungs of male CBAJ mice (Charles River Lab, Lyon, France) weighing 19-23 g with Methicillin Resistant S. aureus Sa2 (MRS A) inoculum. Before infection, mice were rendered neutropenic (neutrophil^ 100/mm³) by injecting them with cyclophosphamide (SIGMA, St Louis) intraperitoneally 4 days (150mg/kg of body weight) and 1 day (lOOmg/kg) before thigh infection. The inoculum was prepared from Methicillin Resistant clinical isolate of S. aureus Sa2. The optical density of a broth culture of freshly plated bacteria was adjusted to 0.1 at an absorbance at 590nm, then put at 37°C in shaken culture for about lh30 until a OD= 0.3 at 590nm. After a 1/50 dilution into physiological saline, 0.05mL of this inoculum suspension was instilled intranasally.

[00196] The test compound was dissolved in methyl cellulose at 0.5% to give a solution of 2 mg/ml (pH=7.0). This solution was diluted with vehicle to give 1, 0.6, and 0.2 mg/mL solutions. Two hour post infection (PI), animals were treated orally, compounds were diluted in methyl cellulose at 0.5%), a group of untreated mice was administered only the vehicle. Administration was repeated twenty hours post-infection (PI) for the twice a day (BID) model or 24h and 48h PI for the once a day (QD) model. Twenty four hours PI for the BID model, or 72h PI for the QD all mice were euthanized, each lung was removed and the bacterial burden in the lungs enumerated after tissue homogenization and plating.

[00197] The results of the in vivo efficacy test are summarized in Table 6B, which provides a representative example of the results obtained for Compound 1.

[00198] In this model, Levofloxacin or Ciprofloxacin is used as the negative control, and Vancomycin (parenteral or oral) and/or Linezolid (oral) is used as the positive control. A test compound is considered active when it shows a log reduction equivalent to the positive control in the same study.

Table 6B - Lung Sa (Nd of Treatment

Study Compound Tested Route Frequency Sample Log Active dose time, reduction

mg/kg post vs.

infection untreated

1 Vancomycin 50 SC BID 24h -2.00 Y

Levofloxacin PO -0.32 N

Linezolid PO -2.92 Y

Compound 1 PO -3.36 Y

2 Levofloxacin 50 PO BID 24h -0.55 N

Linezolid 15 -3.29 Y

Linezolid 50 -4.64 Y

Compound 1 15 -3.91 Y

50 -2.99 Y

Linezolid 25 -3.10 Y

Cipro 50 -0.82 N

12.5 -0.39 N

Nargenicin Al

25 -2.17 Y

3 PO BID 24h

50 -2.57 Y

6.25 -0.04 N

Compound 1

12.5 -1.9 Y

25 -3.48 Y

5 -0.37 N

Linezolid

15 -1.49 Y

4 PO QD 72h

Ciprofloxacin 50 -0.91 N

Compound 1 5 -1.57 Y 15 -3.62 Y

Linezolid -2.37 Y

Ciprofloxacin -0.44 N

5 25 PO QD 72h

Compound 1 Y

-3.65

3.3 Skin Model of Infection (groin)

[00199] To see if compounds are potentially efficacious to treat skin infections, they were tested in an abscess model in mice induced by a methicillin resistant S. aureus clinical isolate Sa2.

[00200] The in vivo antibacterial activity was established by infecting the groin of male CD1 mice (Charles River Lab, Lyon, France) weighing 19-23 g with Methicillin Resistant S. aureus Sa2 (MRSA) inoculum. The inoculum was prepared from Methicillin Resistant clinical isolate of S. aureus Sa2. An overnight culture of the strain was diluted 1/10,000 in physiological water and then 0.5mL was injected subcutaneously into the groin.

[00201] The test compound was dissolved in methyl cellulose at 0.5% or polyethylene glycol 200 at 20% (depending on administration route) to give a solution of 2 mg/mL (pH=7.0). Two hour post infection (PI), animals were treated orally or by parenteral routes (ip or sc) depending on experiment, a group of untreated mice was administrated only with the corresponding vehicle. Administration was repeated seven hours post-infection (PI) and 24h PI. Thirty one hours PI, all mice were euthanized, each groin was removed and the bacterial burden in the lungs enumerated after tissue homogenization and plating.

[00202] The results of the in vivo efficacy test are summarized in Table 5C, which provides a representative example of the results obtained for Compound 1.

[00203] In this model, Ciprofloxacin or Levofloxacin is used as the negative control, and Vancomycin (parenteral) and/or Linezolid (oral) is used as the positive control. A test compound is considered active when it shows a log reduction equivalent to the positive control in the same study.

Table 7C - Groin Sa (Nd of Treatment

Study Sample Log Active

Tested

reduction

Compound dose Route Frequency time, post

mg/kg infection vs.

untreated

1 Ciprofloxacin SC -1.57 N

Vancomycin SC -3.59 Y

50 BID 31h

Nargenicin Al PO -1.11 N

IP -3.13 Y

2 Levofloxacin -1.00 N

Linezolid -2.44 Y

50 PO BID 31h

Compound 1 Y Print Out (Original in Electronic Form)

(This sheet is not part of and does not count as a sheet of the international application)

The indications made below relate to

the deposited microorganism(s) or

other biological material referred to in

the description on:

-1 Paragraph number 0060, 0061, 00153, 00158

-3 Identification of deposit

-3-1 Name of depositary institution NCIMB NCIMB Ltd.

-3-2 Address of depositary institution Ferguson Building, Craibstone Estate,

Bucksburn, Aberdeen AB21 9YA, United Kingdom

-3-3 Date of deposit 05 April 2012 (05.04.2012)

-3-4 Accession Number NCIMB 41952

-4 Additional Indications saccharothrix xinjiangensis G60/1571

30xB2M21; receipt of deposit enclosed; name/address of depositor: Galapagos SASU, 102 Avenue Gaston, 93230

Romainville, France; Statement by depostior authorizing applicant enclosed -5 Designated States for Which

Indications are Made All designations

The indications made below relate to

the deposited microorganism(s) or

other biological material referred to in

the description on:

-1 Paragraph number 00171, 00172, 00194, 00199

-3 Identification of deposit

-3-1 Name of depositary institution NCIMB NCIMB Ltd.

-3-2 Address of depositary institution Ferguson Building, Craibstone Estate,

Bucksburn, Aberdeen AB21 9YA, United Kingdom

-3-3 Date of deposit 05 April 2012 (05.04.2012)

-3-4 Accession Number NCIMB 41953

-4 Additional Indications staphylococcus aureus Sa 2; receipt of deposit enclosed; name/address of depositor: Galapagos SASU, 102 Avenue Gaston, 93230 Romainville, France;

Statement by depostior authorizing applicant enclosed

-5 Designated States for Which

Indications are Made All designations Print Out (Original in Electronic Form)

FOR RECEIVING OFFICE USE ONLY -4 This form was received with the

international application:

YES

(yes or no)

-4-1 Authorized officer

Peschier-Van den Berg, Y.

FOR INTERNATIONAL BUREAU USE ONLY -5 This form was received by the

international Bureau on:

-5-1 Authorized officer

Claims

WE CLAIM

1. A compound which is 8-0-lH-pyrrole-2'-carbonylbranimycin or a pharmaceutically

acceptable salt, or a solvate, or a solvate of the pharmaceutically acceptable salt.

2. A compound according to Formula (I):

or a pharmaceutically acceptable salt, or a solvate, or a solvate of the pharmaceutically acceptable salt.

3. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmaceutically effective amount of the compound according to any one of claims 1 to 2.

4. The pharmaceutical composition according to claim 3 comprising a further therapeutic agent.

5. The compound according claim 1 or 2, or a pharmaceutical composition according to claim 3 or 4, for use in medicine.

6. The compound according to claim 1 or 2, or a pharmaceutical composition according to claim 3 or 4, for use in the treatment of bacterial infectious diseases.

7. A method for the treatment, or prevention of bacterial infectious diseases comprising administering an amount of a compound according to any one of claims 1 to 3, or a pharmaceutical composition according to claim 4 or 5, said method comprising administering a therapeutically effective amount of said compound or said pharmaceutical composition, to a patient in need thereof. The method according to claim 7, wherein the compound according to claim 1 or 2, or a pharmaceutical composition according to claim 3 or 4, is administered in combination with a further therapeutic agent.

9. A method for the synthesis of a compound according to claim 1 or 2, said method comprising:

(a) reacting branimycin (Formula II) with a suitable hydroxyl protection agent R^pl-X to protect position C¹⁷ hydroxyl, wherein X is leaving group and R^pl is a suitable protecting group,

alkyl, -C(=0)Ci_-6 alkyl, -CH₂-Ph, -Si(C alkyl)₃, -Si(C alkyl)(Ph)₂, tetrahydropyranyl, and allyl, wherein said alkyl, and phenyl groups may further be substituted with C alkoxy, NO₂, or halo and

Formula A

(c) removal of all protecting groups R^pl and R^p3 from the intermediate B obtained in step (b) above to yield the compound according to Formula I.

10. A method for the synthesis of a compound according to claim 1 or 2 said method comprising:

(a) culturing Saccharothrix xinjiangensis G60/1571 30xB2M21, (registered under accession number NCIMB 41952), or Actinomycete GW 60/1571 ;

(b) after harvest, generating a crude extract from the fermentation broth

(c) isolating branimycin from the crude extract,

(d) reacting branimycin with a suitable hydroxyl protection agent R^pl-X to protect position C¹⁷ hydroxyl, wherein X is leaving group and R^pl is a suitable protecting group,

alkyl, -CH₂-Ph, -Si(C alkyl)₃, -Si(C alkyl)(Ph)₂, tetrahydropyranyl, and allyl, wherein said alkyl, and phenyl groups may further be substituted with C₁.₄ alkoxy, N0₂, or halo and

Formula A

(f) removal of all protecting groups R^P1 and R^P3 from the intermediate B obtained (e) above to yield the compound according to Formula I.

The method according to claim 9 or 10, wherein in step (a) R^pl-X is a silyl ether group, particularly TES-Cl, TBDPS-Cl, TBDMS-Cl, or TMS-Cl.

The method according to any one of claims 9 to 11, wherein in step (b) R^P3 is selected from - C(=0)OtBu, -CH₂-Ph, -C(=0)0(CH₂)₂Si(Me)₃, and 4-Me-Ph-S0₂-.

13. A compound produced by the method of any one of claims 9 to 12.