WO2020087122A1 - Novel antibiotic compounds - Google Patents

Novel antibiotic compounds Download PDF

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Publication number
WO2020087122A1
WO2020087122A1 PCT/AU2019/051196 AU2019051196W WO2020087122A1 WO 2020087122 A1 WO2020087122 A1 WO 2020087122A1 AU 2019051196 W AU2019051196 W AU 2019051196W WO 2020087122 A1 WO2020087122 A1 WO 2020087122A1
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WO
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group
alkyl
compound
mmol
radical
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PCT/AU2019/051196
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French (fr)
Inventor
Andrew Abell
Steven Polyak
William TIEU
Grant Booker
Kwang Lee JUN
Beatriz Blanco RODRIGEZ
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University Of Adelaide
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Priority claimed from AU2018904112A external-priority patent/AU2018904112A0/en
Application filed by University Of Adelaide filed Critical University Of Adelaide
Publication of WO2020087122A1 publication Critical patent/WO2020087122A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • This invention relates to new biotin derivatives having potent BPL inhibition activity.
  • Bacteraemia is a constant threat to modern society and represents a significant burden to the healthcare system.
  • Australia alone it is estimated that there are 200,000 episodes of hospital-acquired infections each year, accounting for up to 2 million bed days in hospital. Patients who develop infections remain in hospital three times longer than other patients, resulting in additional costs and a lack of available beds for alternative uses.
  • a significant number of these patients (estimated at 12,000 per year in healthcare) go onto develop blood stream infections. Of these 17 - 29% will die whilst in hospital, primarily due to ineffective treatment options.
  • Staphylococcus aureus is the most important of these pathogens with a 20% mortality rate by 30 days.
  • Biotin Protein Ligase is an essential enzyme found in all living organisms.
  • BPL attaches the prosthetic group biotin (Vitamin H) onto a class of enzymes known as the biotin-dependent carboxylases. These enzymes play essential roles in metabolic reactions such as membrane biogenesis; a key process in all living organisms.
  • inhibitors of the BPL enzyme from a pathogen would provide opportunities for either selective or broad-spectrum treatments for a range of non-viral infectious diseases.
  • Patent application WO 2009/062241 describes the crystal structure of the S. aureus biotin protein ligase and its use to identify molecules that interact with the biotin binding domain of the enzyme.
  • WO 2006/056007 describes methods for the identification of BPL inhibitors.
  • the biotin protein ligase in the various forms of the present invention may be any biotin protein ligase for which the identification of an inhibitor is desired.
  • the biotin protein ligase is a biotin protein ligase of a pathogenic organism of a human, animal or a plant.
  • Pathogenic organisms of humans or animals for which an inhibitor of a biotin protein ligase may be identified include bacteria, fungi or parasites.
  • Pathogenic organisms of plants for which an inhibitor of a biotin protein ligase may be identified include bacteria, fungi, insects or nematodes.
  • Pathogenic bacteria of humans include for example Acinetobacter calcoaceticus,
  • Acinetobacter Iwoffi Actinobacillus - all species, Actinomadura madurae, Actinomadura pelletieri, Actinomycetaceae - all members, Aeromonas hydrophila, Alcaligenes spp., Arachnia propionica, Arizona spp., Bacillus anthracis, Bacillus cereus, Bacteroides spp., Bartonella - all species, Bordetella - all species, Borrelia - all species, Brucella - all species, Campylobacter coli , Campylobacter fetus, Campylobacter jejuni,
  • Cardiobacterium hominis Chlamydia psittaci, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Clostridium botulinum, Clostridium chauvoei,
  • Clostridium difficile Clostridium haemolyticum, Clostridium histolyticum, Clostridium novyi, Clostridium perfringens, Clostridium septicum, Clostridium sordellii, Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium equi, Corynebacterium
  • Flavobacterium meningosepticum Francisella (Pasteurella) tularensis Type A, Francisella tularensis Type B, Francisella novocida, Haemophilus influenzae , Haemophilus ducreyi, Klebsiella - all species and all serotypes, Helicobacter - all species, Legionella - all species, Leptospira interrogans - all serovars, Listeria - all species, Mimae polymorpha, Moraxella - all species, Morganella morganii, Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium marinum, Mycobacterium paratuberculosis, Mycobacterium ajricanum, Mycobacterium avium/intracellulare, Mycobacterium bovis, Mycobacterium kansasii, Mycobacterium
  • Mycoplasma - all species Neisseria elongata, Neisseria gonorrhoeae, Neisseria meningitides, Nocar dia spp., Pasteurella multocida, Pasteurella. spp., Peptostreptococcus spp., Plesiomonas shigelloides, Porphyromonas spp., Prevotella spp., Proteus - all species, Providencia spp., Pseudomonas aeruginosa, Pseudomonas (Burkholderia) mallei,
  • Pseudomonas (Burkholderia) pseudomallei, Rickettsia - all species, Rhodococcus equi, Salmonella arizonae, Salmonella enteritidis, Salmonella typhimurium, Salmonella paratyphi, Salmonella typhi, Serpulina spp., Serratia liquefaciens, Serratia marcescens, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Shigella dysenteriae, Sphaerophorus necrophorus, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptobacillus moniliformis, Streptococcus spp., Treponema spp., Ureaplasma urealyticum, Vibrio spp., Yersinia (Pa
  • Pathogenic fungi of humans include for example Asperigillus fumigatus, Blastomyces dermatitidis, Candida spp., Cladophialophora bantiana, Coccidioides immitis,
  • Cryptococcus neoformans var. neoformans Cryptococcus neoformans var. gattii
  • Parasites of humans include for example Acanthamoeba spp., Ancylostoma duodenale, Angiostrongylus cantonensis, Angiostrongylus costaricensis, Anisakis simplex, Ascaris lumbricoides, Ascaris suum, Babesia divergens, Babesia microti, Balantidium coli, Blastocystis hominis, Brugia malayi, Brugia pahangi, Brugia timori, Capillaria spp., Contracaecum osculatum, Cryptosporidium spp., Cyclospora spp., Dicrocoelium dendriticum, Dientamoeba fragilis, Diphyllobothrium latum, Dracunculus medinensis, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Entamoeba histolytica
  • Mansonella (Dipetalonema) ozzardi, Mansonella (Dipetalonema) perstans, Mansonella
  • Pathogenic bacteria of plants include for example Erwinia species, including including Erwinia amylovora (Burrill) Winslowet al., Erwinia carotovora (Jones) Bergeyet al.
  • Pseudomonas species including Pseudomonas cichorii (Swingle) Stapp, Pseudomonas syringae pv.glycinea (Coerper) Younget al., Pseudomonas syringae pv.pisi (Sackett) Younget al., Pseudomonas syringae pv.
  • Dye Xanthomonas campestris pv.juglandis (Pierce) Dye, Xanthomonas cynarae Ride, Xanthomonas fragariae Kennedy et King, Agrobacterium species, and Corynebacterium species.
  • Pathogenic fungi of plants include for example Alternaria species, including Alternaria helianthi (Hansf.) Tub. et Nish., Alternaria brassicae (Berk.) Sacc. x Cabbage, Alternaria brassicae (Berk.) Sacc. x Rape, Alternaria brassicicola (Schwein.) Wiltshire, Alternaria dauci (Kiihn) Gr. et Sk. f. sp.solani (Ell.
  • Alternaria species including Alternaria helianthi (Hansf.) Tub. et Nish., Alternaria brassicae (Berk.) Sacc. x Cabbage, Alternaria brassicae (Berk.) Sacc. x Rape, Alternaria brassicicola (Schwein.) Wiltshire, Alternaria dauci (Kiihn) Gr. et Sk. f. sp.solani (Ell.
  • Botryotinia species including Botryotinia fuckeliana (de Bary) Whetzel x artichoke, Botryotinia fuckeliana (de Bary) Whetzel x blackcurrant, Botryotinia fuckeliana (de Bary) Whetzel x grapevine, Botryotinia fuckeliana (de Bary) Whetzel x hazel, Botryotinia fuckeliana (de Bary) Whetzel x raspberry, Botryotinia fuckeliana (de Bary) Whetzel x strawberry, Botryotinia fuckeliana (de Bary) Whetzel x strawberry, Botryotinia fuckeliana (de Bary) Whetzel x strawberry, Botryotinia fuckeliana (de Bary) Whetzel x strawberry, Botryotinia fuckeliana (de Bary) Whetzel x strawberry, Botryotinia fu
  • Glomerella species including Glomerella cingulata (Stoneman) Spaulding et Schrenk x almond, Glomerella cingulata (Stoneman) Spaulding et Schrenk x apple, Glomerella cingulata (Stoneman) Spaulding et Schrenk x olive, Glomerella glycines (Hori) Lehmann et Wolf; Gnomonia leptostyla (Fr.) Ces.
  • Glomerella species including Glomerella cingulata (Stoneman) Spaulding et Schrenk x almond, Glomerella cingulata (Stoneman) Spaulding et Schrenk x apple, Glomerella cingulata (Stoneman) Spaulding et Schrenk x olive, Glomerella glycines (Hori) Lehmann et Wolf; Gnomonia leptostyla (Fr.) Ces.
  • Leptosphaeria maculans Ces. et de Not. x rape, Leptosphaeria nodorum E.
  • Arx x cereals Mycocentrospora cladosporioides (Sacc.) P. Costaex Deighton; Mycoplasme de la Flavescence doree, Mycosphaerella pinodes (Berk et Blox.) Vestergren; Mycosphaerella sp.; Nectria galligena Bresad.; Peronospora species including Peronospora destructor (Berk.) Caspary x onion, Peronospora farinosa Fr. f. sp.beta Byford, Peronospora farinosa Fr. f.
  • Phytophthora species including Phytophthora cactorum (Lebert et Cohn) Schroter, Phytophthora capsici Leonian , Phytophthora fragariae Hickman, Phytophthora infestans (Mont.) de Bary x potato, Phytophthora infestans (Mont.) de Bary x tomato; Plasmopara species including Plasmopara helianthi Novot., Plasmopara viticola (Berk et Curtisex. de Bary) Berk et de Toni; Podosphaera leucotricha (Ell.
  • Pseudoperonospora humuli (Miyabe et Takah.) G. Wilson; Pseudopeziza species including Pseudopeziza medicaginis (Lib.) Sacc. f. sp.medicaginis-lupulinae Schmied., Pseudopeziza tracheiphila Muller-Thurgau; Puccinia species including Puccinia allii (DC.) Rudolph, Puccinia asparagi DC., Puccinia coronata Corda, Puccinia coronifera Kleb., Puccinia graminis Persoon f.
  • Sorosporium reiliana McAlp S phacelotheca species including Sphacelotheca reiliana (Kiihn) Clinton, Sphaerotheca fuliginea (Schlecht.) Pollacci, Sphaerotheca mors-uvae (Schwein.) Berk et Curtis; Spilocaea oleagina (Castagne) Hughes; Stereum hirsutum (Willd.) Pers.; Stigmina carpophila (Lev.) M.B. Ellis; Taphrina deformans (Berk.) Tul. var.persicae; Thanatephorus cucumeris (A.B.
  • Pathogenic insects of plants include insect pests of the orders of Lepidoptera, Coleoptera, Diptera, Homoptera, Hemiptera, Thysanoptera, and Orthoptera.
  • Pathogenic nematodes of plants include nematodes of the genera Heterodera, Globodera, Umbelliferae, Solanaceae, Pratylinchus, and Meloidogyne.
  • the biotin protein ligase is a biotin protein ligase of a pathogenic organism of a human.
  • the biotin protein ligase is a biotin protein ligase from a bacterial pathogen of a human. More preferably, the biotin protein ligase is a biotin protein ligase from a bacterial pathogen of a human. More preferably, the biotin protein ligase is from a Gram-positive bacterium. Most preferably, the biotin protein ligase is from the Staphylococcus or Bacillus species. Most preferably the biotin protein ligase is from Staphylococcus aureus or
  • the present invention relates to a compound of formula (I)
  • Ri is selected from the group consisting of radical -(CH2) n - , where n is an integer between 1 and 10;
  • R 2 is selected from the group consisting of radical -(CH 2 )-, NH, NR 3 ,
  • M is selected from the group consisting of radical -(CH2) n -, NH, NR 3 ,
  • R 3 is H, or alkyl
  • R4 is selected from the group consisting of C3-C6 cyclo alkyl radical, radical -(CH2) n - , where n is an integer between 1 and 10;
  • R 5 is selected from the group consisting of:
  • R 6 , R 7 , R- 8 , R9 are selected from the group consisting of H, C 1 -C 5 alkyl, halogen, NH 2 , OH 2 , NR13R14, SH, SR14: where R 13 and R 14 are selected from the group consisting of H, Ci- C 5 alkyl;
  • Rio is selected from the group consisting of N, NH;
  • R 12 is selected from the group consisting of N, NH, O;
  • R 7 , R 8 , R 9 are selected from the group consisting of H, C 1 -C 5 alkyl, halogen, NH2, OH 2 , NR13R114, SH, SRI 4 :
  • R 9 and Rio are selected from the group consisting of H, C 1 -C 5 alkyl
  • Rio is selected from the group consisting of N, NH;
  • R 12 is selected from the group consisting of N, NH, O;
  • R 7 , R 8 are selected from the group consisting of H, C 1 -C 5 alkyl, halogen, NH 2 , OH 2 , NRi 3 Ri 4 , SH, SR I4 :
  • R 13 and R I4 are selected from the group consisting of H, Ci- C 5 alkyl
  • R 12 is selected from the group consisting of N, Mi, O;
  • X is selected from the group consisting of O, NR 13 R 14 , SR 14 ;
  • R12, R15, Ri6, Rn, Ri8, R19, R20 are selected from the group consisting of H, C1-C5 alkyl, halogen, MB, OH 2 , NR13R14, SH, SRI 4 :
  • R 13 and R 14 are selected from the group consisting of H, Ci- C 5 alkyl
  • n is an integer between 1 and 4;
  • X is a halogen selected from the group consisting of F, Br, Cl or I;
  • R 21 is selected from the group consisting of Ci-C 4 alkyl or C 3 -C 6 cyclo alkyl
  • R 22 is selected from the group consisting of Ci-C 4 alkyl
  • n is an integer between 1 and 4;
  • R- 23 is selected from the group consisting of H, Ci-C 4 alkyl
  • R- 24 is selected from the group consisting of H, Ci-C 4 alkyl
  • n is an integer between 1 and 4;
  • R-25 is selected from the group consisting of H, Ci-C 4 alkyl
  • R-26 is selected from the group consisting of H, Ci-C 4 alkyl
  • n is an integer between 1 and 4;
  • R-27 is selected from the group consisting of H, Ci-C 4 alkyl.
  • composition of general formula (I) including as an active ingredient at least one compound of formula (I) optionally together with one or more pharmacologically acceptable excipients.
  • composition of general formula (I) when used for the treatment of infection selected from the group consisting of bacterial, fungal, protozoan infections.
  • the pharmaceutical composition of general formula (I) when used for the treatment of infections caused by Staphylococcus aureus.
  • the pharmaceutical composition of general formula (I) when used for the treatment of infections caused by Staphylococcus aureus.
  • the bacterial infection is caused by a Gram negative bacterium or a Gram positive bacterium.
  • the bacterial infection is a Staphylococcus aureus infection.
  • Another aspect of this invention refers to a medicament or pharmaceutical composition
  • a medicament or pharmaceutical composition comprising at least one compound of formula (I) as defined above, or a pharmaceutically acceptable salt, isomer, prodrug or solvate thereof and a pharmaceutically acceptable carrier, adjuvant or vehicle.
  • alkyl refers to Ci -C 5 straight or branched, substituted or unsubstituted saturated chain radicals derived from saturated hydrocarbons by the removal of one hydrogen atom.
  • Representative examples of alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, and tert-butyl among others.
  • cycloalkyl refers to a substituted or unsubstituted aliphatic ring system having 3 to 6 carbon atoms, including, but not limited to cyclopropyl, cyclopentyl, cyclohexyl, norbornyl, and adamantyl among others.
  • Cycloalkyl groups can be unsubstituted or substituted with one, two or three substituents independently selected from lower alkyl, haloalkyl, alkoxy, thioalkoxy, amino, alkylamino, dialkylamino, hydroxy, halo, mercapto, nitro, carboxaldehyde, carboxy, alkoxycarbonyl and carboxamide. This term is meant to encompass cycloalkenyl and cycloalkynyl groups. "Cycloalkyl” includes cis or trans forms. Furthermore, the substituents may either be in endo or exo positions in the bridged bicyclic systems.
  • alkenyl and alkynyl carbon chains refers to carbon chains that may contain between 2-10 carbons, and between 1-5 double bonds.
  • the alkynyl carbon chain may contain between 1-5 triple bonds.
  • substituted refers to any group having (further substituted) one or more substituents.
  • the substituent is selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl, halogen, alkylene- COOH, ester, -OH, -SH, and -NH.
  • pharmaceutically acceptable salts in the context of this invention means any salt that is tolerated physiologically (normally meaning that it is not toxic, particularly, as a result of the counter-ion) when used in an appropriate manner for a treatment, applied or used, particularly, in humans and/or mammals.
  • physiologically acceptable salts may be formed with cations or bases and, in the context of this invention, are understood to be salts formed by at least one compound used in accordance with the invention -normally an acid (deprotonated)- such as an anion, particularly when used on humans and/or mammals.
  • physiologically acceptable salts may also be formed with anions or acids and, in the context of this invention, are understood as being salts formed by at least one compound used in accordance with the invention - normally protonated, for example in nitrogen - such as a cation and at least one physiologically tolerated anion, particularly when used on humans and/or mammals.
  • This definition specifically includes in the context of this invention a salt formed by a physiologically tolerated acid, i.e. salts of a specific active compound with physiologically tolerated organic or inorganic acids - particularly when used on humans and/or mammals.
  • salts are those formed with: hydrochloric acid, hydrobromic acid, sulphuric acid, methanesulfonic acid, formic acid, acetic acid, oxalic acid, succinic acid, malic acid, tartaric acid, mandelic acid, fumaric acid, lactic acid or citric acid.
  • Figure 1 shows a photograph of the anti -bacterial activity of compound 6 against S. aureus ATCC 49775;
  • Figure 2 compares the potency of compound 6 (solid squares) against a panel of antibiotics against S. aureus ATCC 49775;
  • Figure 3 shows a graph of the enzyme inhibition curve of compound 6 against S. aureus BPL
  • Figure 4 shows a graph of the activity of varying concentrations of compound 6 (BPL199, open circles) and compound 16 (BPL200, solid squares) against S. aureus BPL;
  • Figure 5 shows a graph of the stability of compounds 6 (BPL199, open circles) and compound 16 (BPL200, solid squares) in whole blood;
  • Figure 6A shows a graph of cytotoxicity of compound 6 (solid circles) against mammalian HEK 293 (kidney) cells;
  • Figure 6B shows a graph of cytotoxicity of compound 6 (solid circles) against mammalian HepG2 (liver) cells
  • Figure 7 shows a graph of the antibacterial activity of compound 16 (BPL200, red line) against & aureus ATCC49775
  • compositions of the present invention may be administered by different routes.
  • they may be administered orally in form of pharmaceutical preparations such as tablets, capsules, syrups and suspensions; also, parenterally in the form of solutions or emulsions, etc.
  • compositions may also be administered topically in the form of creams, pomades, balsams, etc. and transdermically for example through the use of patches or bandages. They may also be applied directly in the rectum as suppositories.
  • the preparations may comprise physiologically acceptable carriers, excipients, activators, chelating agents, stabilizers, etc. In case of injections, physiologically acceptable buffers, solubilising agents or isotonics may be added.
  • the antimicrobial activity of the compounds was determined by a microdilution broth method as recommended by the CLSI (Clinical and Laboratory Standards Institute, Document M07-A8, 2009, Wayne, Pa.) with cation-adjusted Mueller-Hinton broth (Trek Diagnostics Systems, U.K.).
  • Compounds were dissolved using DMSO.
  • Serial two-fold dilutions of each compound were made using DMSO as the diluent. Trays were inoculated with 5 x 10 4 CFU of S. aureus ATCC 49775 in a volume of 100 pL (final concentration of DMSO was 3.2 % (v/v)), and incubated at 35 °C for 16- 20 hours. Growth of the bacterium was quantitated by measuring the absorbance at 620 nm.
  • FIG. 1 shows the inhibitory effect of compound 6 against S. aureus ATCC 49775 with a wide zone of inhibition, similar to that shown for Erythromycin shows specific activity against S. aureus as shown in Figure 2 in which compound (6) compares favourably alongside other antibiotics such as erythromycin having a similar potency profile.
  • the reaction mixture contained: 50 mM Tris HCI pH 8.0, 3 mM ATP, 4.94 pM, 0.06 pM 3 H Biotin, 5.5 mM MgCL 100 mM KCI, 0.1 pM DTT and 10 pM biotin domain of S. aureus pyruvate carboxylase.
  • the reaction was initiated by the addition of BPL to a final concentration of 6.25 nM.
  • the inhibitory activity of compounds was determined by measuring S. aureus BPL activity in the presence of varying concentrations of compound.
  • a reaction mixture was prepared containing 50 mM Tris HC1, pH 8.0, 3 mM ATP, 5 mM biotin, 5.5 mM MgCl2, 100 mM KC1, 0.1 pM DTT, and 25 pM biotin domain of S. aureus pyruvate carboxylase (SaPC90) fused to GST. All compounds were dissolved in DMSO and then diluted into the reaction buffer to give a final concentration of 4% DMSO. The reaction was initiated by the addition of enzyme to give final concentrations of 4.5 nM of S. aureus BPL and final volume of 20 ul.
  • Enhancement solution (PerkinElmer, 50 pL) was added to each well and incubated for 10 min before reading the plate using a PerkinElmer Victor X5 multilabel reader (time-resolved fluorescence settings, 340 nm excitation, and 612 nm emission).
  • Figure 4 shows the activity of BPL from S. aureus measured in the presence of varying concentrations of the BPL inhibitors compound 6 (O) and compound 16 ( ⁇ ). Inhibition constants (K ⁇ ) were calculated using the Morrison equation. K ⁇ for compound 6 was 1.0 ⁇ 0.3 nM and Ki for compound 16 is 53 ⁇ 33 nM.
  • HEK 293 (kidney) and HepG2 (liver) cells were suspended in Dulbecco-modified Eagle’s medium containing 10% fetal bovine serum and then seeded in 96-well tissue culture plates at 10,000 cells per well. After 24 h, cells were treated with varying concentrations of Compound 6, such that the DMSO concentration was consistent at 2% (v/v) for HepG2 or 0.5% (v/v) for HEK293 in all wells. After treatment for 48 h, WST-l cell proliferation reagent (Roche Applied Science) was added to each well and incubated for 0.5 h at 37 °C.
  • the WST-l assay quantitatively monitors the metabolic activity of cells by measuring the hydrolysis of the WST-l reagent, the products of which are detectable at absorbance 450 nm.
  • Figure 6 shows no significant toxicity was observed at any concentration of Compound 6 for either HEK 293 (kidney, figure 6A) or HepG2 (liver, figure 6B) cells. Cells receiving no treatment are shown in red, and those with paracetamol (6.25 mM for HEK293, 25 mM for HepG2) are shown in blue.
  • Antimicrobial activity (minimal inhibitory concentration, MIC) of the BPL inhibitors and comparison antibiotics were determined by a microdilution broth method as recommended by the Clinical and Laboratory Standards Institute (Document M07-A8, 2009, Wayne, PA) with cation-adjusted Mueller-Hinton broth (Trek Diagnostics Systems). Compounds were dissolved using DMSO. Serial 2-fold dilutions of each compound were made using DMSO as the diluent. Trays were inoculated with 5 x 10 4 colony -forming units of S. aureus ATCC 49775 in a volume of 100 pl (final concentration of DMSO was 3.2% (v/v)) and incubated at 35 °C for 16 -20 h. Growth of the bacterium was quantitated by measuring the absorbance at 620 nm.
  • Figure 7 shows S. aureus ATCC49775 treated for 22 hours with varying concentrations of compound 16 (red curve). Erythromycin was included as a control (orange). Minimal inhibitory concentration of compound 16 is 4 pg/ml.

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Abstract

Novel antimicrobial inhibitors of Biotin Protein Ligase (BPL), which incorporate biotin, are described. The inhibitors have a structure that inhibit the essential metabolic enzyme BPL of pathogens and have been shown to be stable in whole blood and effective at reducing BPL activity in acceptable concentrations. The described compounds have shown to be effective against, for example, Staphylococcus aureus and compare favourably against erythromycin

Description

NOVEL ANTIBIOTIC COMPOUNDS
FIELD OF THE INVENTION
This invention relates to new biotin derivatives having potent BPL inhibition activity.
BACKGROUND
Bacteraemia is a constant threat to modern society and represents a significant burden to the healthcare system. In Australia alone it is estimated that there are 200,000 episodes of hospital-acquired infections each year, accounting for up to 2 million bed days in hospital. Patients who develop infections remain in hospital three times longer than other patients, resulting in additional costs and a lack of available beds for alternative uses. A significant number of these patients (estimated at 12,000 per year in healthcare) go onto develop blood stream infections. Of these 17 - 29% will die whilst in hospital, primarily due to ineffective treatment options. Staphylococcus aureus is the most important of these pathogens with a 20% mortality rate by 30 days. The ability to become pathogenic on entering the blood stream results in life-threatening bone and joint infections, endocarditis, pneumonia and septicaemia. Although the public profile of serious staphylococcal infections is that they are hospital-acquired, it is important to recognise that 60 % of serious S. aureus infections are now thought to begin in the community.
Conventional antibiotic treatment for S. aureus infections utilises penicillinase-resistant penicillin. However, resistance to this agent is now prevalent in Australian hospital- associated strains and community-associated strains. Multidrug resistant strains, such as the hospital strains of methicillin-resistant S. aureus (MRSA), are also now present in most Australian hospitals and vancomycin-intermediate S. aureus (visa) strains are starting to emerge.
A survey of Australian hospitals in 2005-06 revealed 24% of all S. aureus isolates were MRSA. Strains not susceptible to vancomycin (considered the last line of defence) pose a particular clinical problem, because there are very few agents available to treat them, and those that do exist (such as linezolid) have notable toxicity. Furthermore, the efficacy of vancomycin against vancomycin susceptible strains of MRSA is thought to be suboptimal. All this equates to a patient with MRSA infection costing a hospital five times more than one with methicillin-sensitive S. aureus infection.
There is a need to replenish the drug-discovery pipeline with new antibiotics to combat drug- resistant bacteria, including Staphylococcus aureus , which are responsible for a huge and growing health care problem
Biotin Protein Ligase (BPL) is an essential enzyme found in all living organisms. BPL attaches the prosthetic group biotin (Vitamin H) onto a class of enzymes known as the biotin- dependent carboxylases. These enzymes play essential roles in metabolic reactions such as membrane biogenesis; a key process in all living organisms. As the attachment of a biotin prosthetic group is absolutely required for normal enzyme function, inhibition of BPL activity can be used to inhibit cell growth.
Given the essential role of BPLs in all organisms, inhibitors of the BPL enzyme from a pathogen would provide opportunities for either selective or broad-spectrum treatments for a range of non-viral infectious diseases.
Patent application WO 2009/062241 describes the crystal structure of the S. aureus biotin protein ligase and its use to identify molecules that interact with the biotin binding domain of the enzyme. WO 2006/056007 describes methods for the identification of BPL inhibitors.
However, neither of these documents discloses inhibitors of BPL enzymes from pathogens.
It has now been surprisingly found that a certain class of compounds inhibit the essential metabolic enzyme BPL of pathogens, and their structure is described in this patent application. It is an object of the present invention to overcome, or at least substantially ameliorate, the disadvantages and shortcomings of the prior art.
Other objects and advantages of the present invention will become apparent from the following description, taking in connection with the accompanying drawings, wherein, by way of illustration and example, an embodiment of the present invention is disclosed.
The biotin protein ligase in the various forms of the present invention may be any biotin protein ligase for which the identification of an inhibitor is desired. Preferably, the biotin protein ligase is a biotin protein ligase of a pathogenic organism of a human, animal or a plant.
Pathogenic organisms of humans or animals for which an inhibitor of a biotin protein ligase may be identified include bacteria, fungi or parasites. Pathogenic organisms of plants for which an inhibitor of a biotin protein ligase may be identified include bacteria, fungi, insects or nematodes.
Pathogenic bacteria of humans include for example Acinetobacter calcoaceticus,
Acinetobacter Iwoffi, Actinobacillus - all species, Actinomadura madurae, Actinomadura pelletieri, Actinomycetaceae - all members, Aeromonas hydrophila, Alcaligenes spp., Arachnia propionica, Arizona spp., Bacillus anthracis, Bacillus cereus, Bacteroides spp., Bartonella - all species, Bordetella - all species, Borrelia - all species, Brucella - all species, Campylobacter coli , Campylobacter fetus, Campylobacter jejuni,
Cardiobacterium hominis, Chlamydia psittaci, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Clostridium botulinum, Clostridium chauvoei,
Clostridium difficile, Clostridium haemolyticum, Clostridium histolyticum, Clostridium novyi, Clostridium perfringens, Clostridium septicum, Clostridium sordellii, Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium equi, Corynebacterium
haemolyticum, Corynebacterium pseudotuberculosis, Corynebacterium pyogenes, Corynebacterium renale, Coxiella burnetii, Edwardsiella tarda, Eikenella corrodens, Enterobacter spp., Erysipelothrix rusiopathae (insidiosa), Escherichia coli
(enterotoxigenic/invasive/haemorrhagic strains), Flavobacterium meningosepticum, Francisella (Pasteurella) tularensis Type A, Francisella tularensis Type B, Francisella novocida, Haemophilus influenzae , Haemophilus ducreyi, Klebsiella - all species and all serotypes, Helicobacter - all species, Legionella - all species, Leptospira interrogans - all serovars, Listeria - all species, Mimae polymorpha, Moraxella - all species, Morganella morganii, Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium marinum, Mycobacterium paratuberculosis, Mycobacterium ajricanum, Mycobacterium avium/intracellulare, Mycobacterium bovis, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium malmoense, Mycobacterium microtic,
Mycobacterium scrofulaceum, Mycobacterium simiae, Mycobacterium szulga,
Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycobacterium xenopi,
Mycoplasma - all species, Neisseria elongata, Neisseria gonorrhoeae, Neisseria meningitides, Nocar dia spp., Pasteurella multocida, Pasteurella. spp., Peptostreptococcus spp., Plesiomonas shigelloides, Porphyromonas spp., Prevotella spp., Proteus - all species, Providencia spp., Pseudomonas aeruginosa, Pseudomonas (Burkholderia) mallei,
Pseudomonas (Burkholderia) pseudomallei, Rickettsia - all species, Rhodococcus equi, Salmonella arizonae, Salmonella enteritidis, Salmonella typhimurium, Salmonella paratyphi, Salmonella typhi, Serpulina spp., Serratia liquefaciens, Serratia marcescens, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Shigella dysenteriae, Sphaerophorus necrophorus, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptobacillus moniliformis, Streptococcus spp., Treponema spp., Ureaplasma urealyticum, Vibrio spp., Yersinia (Pasteurella) pestis, Yersinia spp..
Pathogenic fungi of humans include for example Asperigillus fumigatus, Blastomyces dermatitidis, Candida spp., Cladophialophora bantiana, Coccidioides immitis,
Cryptococcus neoformans var. neoformans, Cryptococcus neoformans var. gattii,
Emmonsia parva var. parva, Emmonsia parva var. crescens, Epidermophyton floccosum, Fonsecaea compacta, Fonsecaea pedrosoi, Histoplasma capsulatum var. capsulatum,, Histoplasma capsulatum var. duboisii, Histoplasma capsulatum var. farcinimosum, Madurella grisea, Madurella mycetomatis, Microsporum spp., Neotestudina rosatii, Paracoccidioides brasiliensis, Penicillium marneffei, Scedosporium apiospermum
(Pseudallescheria boydii), Scedosporium proliferans (inflatum), Sporothrix schenckii, Trichophyton spp.
Parasites of humans include for example Acanthamoeba spp., Ancylostoma duodenale, Angiostrongylus cantonensis, Angiostrongylus costaricensis, Anisakis simplex, Ascaris lumbricoides, Ascaris suum, Babesia divergens, Babesia microti, Balantidium coli, Blastocystis hominis, Brugia malayi, Brugia pahangi, Brugia timori, Capillaria spp., Contracaecum osculatum, Cryptosporidium spp., Cyclospora spp., Dicrocoelium dendriticum, Dientamoeba fragilis, Diphyllobothrium latum, Dracunculus medinensis, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Entamoeba histolytica, Enterobius vermicularis, Enterocytozoon bieneusi, Fasciola gigantica, Fasciola hepatica, Fasciolopsis buski, Giardia lamblia (Giardia intestinalis), Heterophyes spp., Hymenolepsis diminuta, Hymenolepsis nana (human origin), Isopora belli,
Leishmania brasiliensis, Leishmania donovani, Leishmania spp., Loa loa, Mansonella (Dipetalonema) ozzardi, Mansonella (Dipetalonema) perstans, Mansonella
(Dipetalonema) streptocerca, Metagonimus spp., Naegleria spp. (especially fowleri), Necator americanus, Onchocerca volvulus, Opisthorchis spp. (Clonorchis), Paragonimus spp., Plasmodium falciparum, Plasmodium spp., Pneumocystis carinii, Pseudoterranova decipiens, Sarcocystis suihominis, Schistosoma spp., Strongyloides spp., Taenia saginata, Taenia solium, Toxocara canis, Toxocara cati, Toxoplasma gondii, Trichinella nativa, Trichinella nelsoni, Trichinella pseudospiralis, Trichinella spiralis, Trichomonas vaginalis, Trichostrongylus spp., Trichuris trichiura, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Trypanosoma brucei brucei, Trypanosoma brucei gambiense, Trypanosoma rangeli, Wuchereria bancrofti.
Pathogenic bacteria of plants include for example Erwinia species, including including Erwinia amylovora (Burrill) Winslowet al., Erwinia carotovora (Jones) Bergeyet al.
subsp.carotovora;, Pseudomonas species including Pseudomonas cichorii (Swingle) Stapp, Pseudomonas syringae pv.glycinea (Coerper) Younget al., Pseudomonas syringae pv.pisi (Sackett) Younget al., Pseudomonas syringae pv. syringae van Hall x apple, Pseudomonas syringae pv.syringae van Hall x apricot, Pseudomonas syringae ssp.savastanoi pv.oleae;, Xanthomonas species including Xanthomonas campestris pv.campestris (Pammel) Dowson, Xanthomonas campestris pv.corylina Milleret ak, Xanthomonas campestris pv.graminis (Egliet al. ) Dye, Xanthomonas campestris pv.juglandis (Pierce) Dye, Xanthomonas cynarae Ride, Xanthomonas fragariae Kennedy et King, Agrobacterium species, and Corynebacterium species.
Pathogenic fungi of plants include for example Alternaria species, including Alternaria helianthi (Hansf.) Tub. et Nish., Alternaria brassicae (Berk.) Sacc. x Cabbage, Alternaria brassicae (Berk.) Sacc. x Rape, Alternaria brassicicola (Schwein.) Wiltshire, Alternaria dauci (Kiihn) Gr. et Sk. f. sp.solani (Ell. et Mart.) Neerg., Alternaria radicina (Meier) Drechsler et Eddy; Armilaria species including Armilaria mellea (Vahl) Kummer x grapevine, Armilaria mellea (Vahl) P. Kummer x fig; Ascochyta species including Ascochyta fabae Speg., Ascochyta hortorum (Speg.) Smith, Ascochyta pisi Lib.; Athelia species including Athelia rolfsii (Curzi) Tu et Kimbrough; Botryotinia species including Botryotinia fuckeliana (de Bary) Whetzel x artichoke, Botryotinia fuckeliana (de Bary) Whetzel x blackcurrant, Botryotinia fuckeliana (de Bary) Whetzel x grapevine, Botryotinia fuckeliana (de Bary) Whetzel x hazel, Botryotinia fuckeliana (de Bary) Whetzel x raspberry, Botryotinia fuckeliana (de Bary) Whetzel x strawberry, Botryotinia fuckeliana (de Bary) Whetzel x sunflower, Botrytis fabae Sardina; Bremia species including Bremia lactucae Regel x artichoke, Bremia lactucae Regel x lettuce; Ceratobasidium cereale Murray et Burpee; Cercospora beticola Sacc.; Chondrostereum species including
Chondrostereum purpureum (Pers.) Pouzar x apple, Chondrostereum purpureum (Pers.) Pouzar xPrunus sp.; Cladosporium cucumerinum Ell. et Arthur; Claviceps purpurea (Fr.) Tub; Colletotrichum species including Colletotrichum lagenarium (Pass.) Ellis et Halsted, Colletotrichum trifolii Bain et Essary, Colletotrichum truncatum (Schwein); Coniothyrium diplodiella (Speg.) Sacc.; Corticium solani (Prill et Delacr.) Bourdot et Galzin, Corticium solani (Prill et Delacr.) Bourdot et Galzin; Coryneum beijerinckii Oudemans; Cronartium ribicola J.C. Fischer; Cryphonectria parasitica (Murrill) Barr; Cryptosporella viticola (Reddick) Shear; Diaporthe species including Diaporthe cinerescens (Sacc.), Diaporthe eres Nitschke, Diaporthe helianthi Muntanola-Cvetkovic Mihaljcevic et Petrov, Diaporthe phaseolorum var.sojae (Lehman) Wehmeyer; Didymella species including Didymella applanata (Niessl.) Sacc., Didymella lycopersici Klebahn; Diplocarpon earliana (Ell. et Ev.) Wolf; Drepanopeziza ribis (Kleb.) Hohnel; Elsinoe ampelina Shear; Erysiphe species including Erysiphe betae (Vanha) Weltzien, Erysiphe cichoracearum DC.; Erysiphe graminis DC.avenae, Erysiphe polyfaga Hammarlund; Eutypa armeniacae Hansford et Carter; Fulvia fulva (Cooke) Ciferri; Fusarium species including Fusarium oxysporum f sp.asparagi Cohen, Fusarium oxysporum Schlecht. f. sp.lini (Bolley) Snyd. et Hansen, Fusarium oxysporum Schlecht. f. sp.melonis (L. et C.) Snyd. et Hansen x Cucumber, Fusarium oxysporum Schlecht. f. sp.melonis (L. et C.) Snyd. et Hansen x melon, Fusarium oxysporum Schlecht. f. sp.melonis (L. et C.) Snyd. et Hansen x watermelon, Fusarium oxysporum Schlecht. f. sp.pisi (Linford) Snyd. et Hansen, Fusarium oxysporum Schlecht. f. sp.radicis-lycopersici Jarvis et Shoem., Fusarium roseum (Link) Snyd. et Hansen x asparagus, Fusarium roseum (Link) Snyd. et Hansen x gramineae; Fusicoccum amygdali Delacr.; Glomerella species including Glomerella cingulata (Stoneman) Spaulding et Schrenk x almond, Glomerella cingulata (Stoneman) Spaulding et Schrenk x apple, Glomerella cingulata (Stoneman) Spaulding et Schrenk x olive, Glomerella glycines (Hori) Lehmann et Wolf; Gnomonia leptostyla (Fr.) Ces. et de Not; Guignardia baccae (Cavara) Jacz.; Helicobasidium brebissonii (Desm.) Donk x asparagus, Helicobasidium brebissonii (Desm.) Donk x beet; Kabatiella zeae Narita et Hiratsuka, Leptosphaeria maculans (Desm.) Ces. et de Not. x cabbage; Leptosphaeria species including
Leptosphaeria maculans (Desm.) Ces. et de Not. x rape, Leptosphaeria nodorum E.
Miiller; Leucostoma cinctum (Fr.) Hohnel, Leveillula taurica (Lev.) Arn.; Marssonina panattoniana (Berlese) Magnus; Mastigosporium rubricosum (Dearn. et Barth.) Sprague; Monilinia species including Monilinia fructigena (Aderhold et Ruhl.) Honey ex Whetzel, Monilinia laxa (Aderhold et Ruhland) Honey xPrunus spp.; Monographella nivalis (Schaffnit) E. Miiller et v. Arx x cereals; Mycocentrospora cladosporioides (Sacc.) P. Costaex Deighton; Mycoplasme de la Flavescence doree, Mycosphaerella pinodes (Berk et Blox.) Vestergren; Mycosphaerella sp.; Nectria galligena Bresad.; Peronospora species including Peronospora destructor (Berk.) Caspary x onion, Peronospora farinosa Fr. f. sp.beta Byford, Peronospora farinosa Fr. f. sp.spinaciae Byford, Peronospora viciae (Berk.) Caspary f. sp.pisi Sydow; Pestalotiopsis menezesiana (Bres. et Torr) Bres. et Torr.; Phakopsora species including Phakopsora euvitis , Phakopsora ampelopsidis , and
Phakopsora vitis, Phoma apiicola Klebhan, Phoma exigua Desm., Phoma medicaginis Mal. et Roum v.pinodella (L.K. Jones) Boer x lucerne; Phoma medicaginis Mal. et Roum v.pinodella (L.K. Jones) Boer x peas; Phragmidium rubi-idaei (DC.) P. Karsten;
Phytophthora species including Phytophthora cactorum (Lebert et Cohn) Schroter, Phytophthora capsici Leonian , Phytophthora fragariae Hickman, Phytophthora infestans (Mont.) de Bary x potato, Phytophthora infestans (Mont.) de Bary x tomato; Plasmopara species including Plasmopara helianthi Novot., Plasmopara viticola (Berk et Curtisex. de Bary) Berk et de Toni; Podosphaera leucotricha (Ell. et Ev.) Salmon; Polymyxa betae Keskin; Pseudocercosporella herpotrichoides (Fron) Deighton; Pseudoperonospora species including Pseudoperonospora cubensis (Berk et Curtis) Rostovtsev,
Pseudoperonospora humuli (Miyabe et Takah.) G. Wilson; Pseudopeziza species including Pseudopeziza medicaginis (Lib.) Sacc. f. sp.medicaginis-lupulinae Schmied., Pseudopeziza tracheiphila Muller-Thurgau; Puccinia species including Puccinia allii (DC.) Rudolph, Puccinia asparagi DC., Puccinia coronata Corda, Puccinia coronifera Kleb., Puccinia graminis Persoon f. sp.avenae, Puccinia hordei Otth, Puccinia anomala Rostrup, Puccinia recondita Roberge f. sp.recondita, Puccinia rubigo-vera Winter, Puccinia striiformis Westendorp f. sp.tritici; Pyrenochaeta lycopersici R. Schneider et Gerlach; Pyrenopeziza brassicae B. Sutton et Rawlinson; Pyrenophora species including Pyrenophora graminea Ito et Kuribay, Pyrenophora teres Drechsler; Rhynchosporium secalis (Oudem.) J. Davis; Rosellinia necatrix Prill; Sclerotinia species including Sclerotinia laxa Aderhold et Ruhland, Sclerotinia minor Jagger, Sclerotinia sclerotiorum (Lib.) de Bary x artichoke, Sclerotinia libertiana Fuckel, Sclerotinia sclerotiorum (Lib.) de Bary x carott, Sclerotinia libertiana Fuckel, Sclerotinia sclerotiorum (Lib.) de Bary x rape, Sclerotinia libertiana Fuckel, Sclerotinia sclerotiorum (Lib.) de Bary x rsunflower, Sclerotinia libertiana Fuckel, Sclerotinia sclerotiorum (Lib.) de Bary x soybean, Sclerotinia libertiana Fuckel,
Sclerotinia trifoliorum J. Eriksson; Sclerotium bataticola Taub., Sclerotium cepivorum Berk; Septoria apiicola Speg.; Setosphaeria turcica (Luttrell) Leonard et Suggs;
Sorosporium reiliana McAlp; S phacelotheca species including Sphacelotheca reiliana (Kiihn) Clinton, Sphaerotheca fuliginea (Schlecht.) Pollacci, Sphaerotheca mors-uvae (Schwein.) Berk et Curtis; Spilocaea oleagina (Castagne) Hughes; Stereum hirsutum (Willd.) Pers.; Stigmina carpophila (Lev.) M.B. Ellis; Taphrina deformans (Berk.) Tul. var.persicae; Thanatephorus cucumeris (A.B. Frank) Donk x artichoke, Thanatephorus cucumeris (A.B. Frank) Donk x chicory, Uncinula necator (Schw.) Burr.; Urocystis cepulae Frost; Uromyces betae Kickx, Ustilago maydis (DC.) Corda; Venturia inaequalis (Cooke) Winter, Venturia pirina Aderhold; Verticillium species including Verticillium albo-atrum Reinke et Berthold x lucerne, Verticillium albo-atrum Reinke et Berthold x sunflower, Verticillium dahliae Klebahn x artichoke, Verticillium dahliae Klebahn x strawberry, Verticillium dahliae Klebahn x sunflower, Verticillium dahliae Klebahn x tomato.
Pathogenic insects of plants include insect pests of the orders of Lepidoptera, Coleoptera, Diptera, Homoptera, Hemiptera, Thysanoptera, and Orthoptera.
Pathogenic nematodes of plants include nematodes of the genera Heterodera, Globodera, Umbelliferae, Solanaceae, Pratylinchus, and Meloidogyne.
In one embodiment, the biotin protein ligase is a biotin protein ligase of a pathogenic organism of a human.
Preferably, the biotin protein ligase is a biotin protein ligase from a bacterial pathogen of a human. More preferably, the biotin protein ligase is a biotin protein ligase from a bacterial pathogen of a human. More preferably, the biotin protein ligase is from a Gram-positive bacterium. Most preferably, the biotin protein ligase is from the Staphylococcus or Bacillus species. Most preferably the biotin protein ligase is from Staphylococcus aureus or
Bacillus athracis.
SUMMARY OF THE INVENTION
The present invention relates to a compound of formula (I)
Figure imgf000011_0001
wherein
Ri is selected from the group consisting of radical -(CH2)n- , where n is an integer between 1 and 10;
R2 is selected from the group consisting of radical -(CH2)-, NH, NR3,
M is selected from the group consisting of radical -(CH2)n-, NH, NR3,
R3 is H, or alkyl;
R4 is selected from the group consisting of C3-C6 cyclo alkyl radical, radical -(CH2)n- , where n is an integer between 1 and 10;
R5 is selected from the group consisting of:
adenine;
benzoxazolone, benzoxazole, benzofuran, benzimidazole,
Figure imgf000011_0002
, where:
R6, R7, R-8, R9 are selected from the group consisting of H, C1-C5 alkyl, halogen, NH2, OH2, NR13R14, SH, SR14: where R13 and R14 are selected from the group consisting of H, Ci- C5 alkyl;
Rio is selected from the group consisting of N, NH;
R11 is selected from the group consisting of CFh, C=0;
R12 is selected from the group consisting of N, NH, O;
Figure imgf000012_0001
, where:
Rs, R7, R8, R9 are selected from the group consisting of H, C1-C5 alkyl, halogen, NH2, OH2, NR13R114, SH, SRI4:
where R9 and Rio are selected from the group consisting of H, C1-C5 alkyl;
Rio is selected from the group consisting of N, NH;
R11 is selected from the group consisting of CH2, C=0;
R12 is selected from the group consisting of N, NH, O;
Figure imgf000012_0002
, where:
Rs, R7, R8, are selected from the group consisting of H, C1-C5 alkyl, halogen, NH2, OH2, NRi3Ri4, SH, SRI4:
where R13 and RI4 are selected from the group consisting of H, Ci- C5 alkyl;
Rio is selected from the group consisting of N, NH; Rn is selected from the group consisting of CFh, C=0;
R12 is selected from the group consisting of N, Mi, O;
R R
napthyl,
Figure imgf000013_0002
Figure imgf000013_0001
where:
X is selected from the group consisting of O, NR13R14, SR14;
R12, R15, Ri6, Rn, Ri8, R19, R20 are selected from the group consisting of H, C1-C5 alkyl, halogen, MB, OH2, NR13R14, SH, SRI4:
where R13 and R14 are selected from the group consisting of H, Ci- C5 alkyl;
Figure imgf000013_0003
n is an integer between 1 and 4;
X is a halogen selected from the group consisting of F, Br, Cl or I;
R21 is selected from the group consisting of Ci-C4 alkyl or C3-C6 cyclo alkyl; R22 is selected from the group consisting of Ci-C4 alkyl;
Figure imgf000013_0004
n is an integer between 1 and 4;
R-23 is selected from the group consisting of H, Ci-C4 alkyl;
R-24 is selected from the group consisting of H, Ci-C4 alkyl;
Figure imgf000014_0001
n is an integer between 1 and 4;
R-25 is selected from the group consisting of H, Ci-C4 alkyl;
R-26 is selected from the group consisting of H, Ci-C4 alkyl;
Figure imgf000014_0002
n is an integer between 1 and 4;
R-27 is selected from the group consisting of H, Ci-C4 alkyl.
In preference, the pharmaceutical composition of general formula (I) including as an active ingredient at least one compound of formula (I) optionally together with one or more pharmacologically acceptable excipients.
In preference, the pharmaceutical composition of general formula (I) when used for the treatment of infection selected from the group consisting of bacterial, fungal, protozoan infections.
In preference, the pharmaceutical composition of general formula (I) when used for the treatment of infections caused by Staphylococcus aureus. In preference, when
Figure imgf000015_0001
cyclo alkyl and R22 = C2 alkyl.
In preference, when
Figure imgf000015_0002
In preference, when
Figure imgf000015_0003
CH3.
In preference, when
Figure imgf000015_0004
In preference, the compound of formula (I) where Ri is -(0¼)h and n=3.
In preference, the compound of formula (I) where R2 is radical NH. In preference, the compound of formula (I) where R4 is -(CH2)n and n=4.
In preference, the compound of formula (I) where R5 is radical adenine.
In preference, the compound of formula (
Figure imgf000016_0001
Use of at least one compound according to formula (I) as an active ingredient in the preparation of a medicament for the treatment and/or prevention of bacterial infections, fungal infections, and protozoan infections to a subject in need thereof.
In preference, the bacterial infection is caused by a Gram negative bacterium or a Gram positive bacterium.
In preference, the bacterial infection is a Staphylococcus aureus infection.
Another aspect of this invention refers to a medicament or pharmaceutical composition comprising at least one compound of formula (I) as defined above, or a pharmaceutically acceptable salt, isomer, prodrug or solvate thereof and a pharmaceutically acceptable carrier, adjuvant or vehicle.
The term“alkyl” as used herein alone or in combination refers to Ci -C5 straight or branched, substituted or unsubstituted saturated chain radicals derived from saturated hydrocarbons by the removal of one hydrogen atom. Representative examples of alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, and tert-butyl among others. The term "cycloalkyl" as used herein alone or in combination refers to a substituted or unsubstituted aliphatic ring system having 3 to 6 carbon atoms, including, but not limited to cyclopropyl, cyclopentyl, cyclohexyl, norbornyl, and adamantyl among others. Cycloalkyl groups can be unsubstituted or substituted with one, two or three substituents independently selected from lower alkyl, haloalkyl, alkoxy, thioalkoxy, amino, alkylamino, dialkylamino, hydroxy, halo, mercapto, nitro, carboxaldehyde, carboxy, alkoxycarbonyl and carboxamide. This term is meant to encompass cycloalkenyl and cycloalkynyl groups. "Cycloalkyl" includes cis or trans forms. Furthermore, the substituents may either be in endo or exo positions in the bridged bicyclic systems.
The term "alkenyl" and "alkynyl" carbon chains, if not specified, refers to carbon chains that may contain between 2-10 carbons, and between 1-5 double bonds. The alkynyl carbon chain may contain between 1-5 triple bonds.
The term "substituted" refers to any group having (further substituted) one or more substituents. In some embodiments, the substituent is selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocyclyl, halogen, alkylene- COOH, ester, -OH, -SH, and -NH.
The term“pharmaceutically acceptable salts” in the context of this invention means any salt that is tolerated physiologically (normally meaning that it is not toxic, particularly, as a result of the counter-ion) when used in an appropriate manner for a treatment, applied or used, particularly, in humans and/or mammals. These physiologically acceptable salts may be formed with cations or bases and, in the context of this invention, are understood to be salts formed by at least one compound used in accordance with the invention -normally an acid (deprotonated)- such as an anion, particularly when used on humans and/or mammals. These physiologically acceptable salts may also be formed with anions or acids and, in the context of this invention, are understood as being salts formed by at least one compound used in accordance with the invention - normally protonated, for example in nitrogen - such as a cation and at least one physiologically tolerated anion, particularly when used on humans and/or mammals. This definition specifically includes in the context of this invention a salt formed by a physiologically tolerated acid, i.e. salts of a specific active compound with physiologically tolerated organic or inorganic acids - particularly when used on humans and/or mammals. Examples of this type of salts are those formed with: hydrochloric acid, hydrobromic acid, sulphuric acid, methanesulfonic acid, formic acid, acetic acid, oxalic acid, succinic acid, malic acid, tartaric acid, mandelic acid, fumaric acid, lactic acid or citric acid.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows a photograph of the anti -bacterial activity of compound 6 against S. aureus ATCC 49775;
Figure 2 compares the potency of compound 6 (solid squares) against a panel of antibiotics against S. aureus ATCC 49775;
Figure 3 shows a graph of the enzyme inhibition curve of compound 6 against S. aureus BPL;
Figure 4 shows a graph of the activity of varying concentrations of compound 6 (BPL199, open circles) and compound 16 (BPL200, solid squares) against S. aureus BPL;
Figure 5 shows a graph of the stability of compounds 6 (BPL199, open circles) and compound 16 (BPL200, solid squares) in whole blood;
Figure 6A shows a graph of cytotoxicity of compound 6 (solid circles) against mammalian HEK 293 (kidney) cells;
Figure 6B shows a graph of cytotoxicity of compound 6 (solid circles) against mammalian HepG2 (liver) cells; Figure 7 shows a graph of the antibacterial activity of compound 16 (BPL200, red line) against & aureus ATCC49775
DETAILED DESCRIPTION OF THE INVENTION
The pharmaceutical compositions of the present invention may be administered by different routes. For example, they may be administered orally in form of pharmaceutical preparations such as tablets, capsules, syrups and suspensions; also, parenterally in the form of solutions or emulsions, etc.
They may also be administered topically in the form of creams, pomades, balsams, etc. and transdermically for example through the use of patches or bandages. They may also be applied directly in the rectum as suppositories. The preparations may comprise physiologically acceptable carriers, excipients, activators, chelating agents, stabilizers, etc. In case of injections, physiologically acceptable buffers, solubilising agents or isotonics may be added.
The working examples included in the present specification describe in detail suitable processes to obtain several of the compounds according to general formula (I). In the light of these examples, it is within the general knowledge of the expert in the field to obtain the compounds not explicitly exemplified by suitable modifications of the working examples. It is also obvious for the expert in the field that these examples are only illustrative and should not be taken as a limitation of the scope of the invention.
EXAMPLES
Figure imgf000020_0001
6-Aminopurine
Figure imgf000020_0002
Figure imgf000020_0003
Synthesis of 2-(4-(6-amino-9H-purin-9-v0butv0isoindoline-l.3-dione (13
To a suspension of 6-aminopurine (1.1 g, 8.14 mmol) in DMF (11 mL) was added K2C03 (1.8 g 13.23 mmol) and N-(4-bromobutyl)phthalamide (2.75 G, 9.75 mmol) and the mixture was stirred at 70 °C overnight. The mixture was allowed to cool down to room temperature and partitioned between ethyl acetate and water. The aqueous phase was extracted with ethyl acetate. The organics combined were washed with brine (x2) and water (xl), dried over sodium sulphate anh., filtrated and evaporated under reduced pressure. The solid obtained was triturated from diethyl ether affording the title compound as a pale yellow solid (2.5 g, 84%).
3H NMR (500 MHz, DMSO- d): d 8.12 (s, 1H, ArH), 8.07 (s, 1H, ArH), 7.84 (m, 4H, ArH), 7.16 (s, 2H, NH2), 4.17 (t, J= 6.7 Hz, 2H, CH2), 3.61 (t, J= 6.7 Hz, 2H, CH2), 1.83 (dd, J = 14.7, 6.9 Hz, 2H, CH2), 1.56 (dt, J= 13.8, 6.9 Hz, 2H, CH2).
13C NMR (125 MHz, DMSO- d): d 167.9 (2xCO), 155.9 (C), 152.3 (CH), 149.5 (C), 140.8 (CH), 134.3 (2xC), 131.6 (2xCH), 123.0 (2xCH), 118.7 (C), 42.4 (CH2), 36.8 (CH2), 26.7 (CH2), 25.1 (CH2) ppm.
Figure imgf000021_0001
1 2
Synthesis of 9-(4-aminobutvO-9H-purin-6-amine (2)
To a suspension of 2-(4-(6-amino-9H-purin-9-yl)butyl)isoindoline-l,3-dione 1 (2.5 g, 7.43 mmol) in ethanol (125 mL) was added hydrazine hydrate (3.5 mL, 114.45 mmol, 15 eq) and the mixture was stirred at reflux overnight. The mixture was allowed to cooled down to room temperature and concentrated under reduced pressure. A solid was formed which was filtrated and washed with dichloromethane. Trituration from methanol afforded the title compound (l .Og, 67%) as a colourless solid.
3H NMR (500 MHz, DMSO-d6): d 8.12 (s, 1H, ArH), 8.11 (s, 1H, ArH), 7.14 (s, 2H, NH2), 4.11 (t, J= 7.1 Hz, 2H, CH2), 3.06 (br s, 2H, NH2), 2.51 (t, J = 6.9 Hz, 2H, CH2), 1.79 (dd, J= 14.9, 7.3 Hz, 2H, CH2) and 1.28 (dd, J= 14.9, 7.3 Hz, 2H, CH2).
13C NMR (125 MHz, DMSO-d6): d 155.9 (C) 152.3 (CH), 149.5 (C), 140.8 (CH), 118.7 (C), 42.8 (CH2), 41.0 (CH2), 30.0 (CH2) and 26.9 (CH2) ppm.
MS HRMS (ESI) calcd for CgHisNe (M + H+): 207.1358, found 207.1365.
Figure imgf000022_0001
CSI Sulfamoylating Agent A
Synthesis of f/rvV-butoxycarbonyl )ff4-fdimethyliminio)pyridin- l (4H)- isulfonyl iamide
Figure imgf000022_0002
(A)
To a solution of /er/-butyl alcohol (1.7 mL, 18.63 mmol) in dry CDM (15 ml) at 0 °C was added dropwise chlorosulfonyl isocyanate (CSI) (1.6 mL, 18.63 mmol), followed by addition of DMAP (4.55 g, 37.25 mmol). The reaction mixture was stirred at room temperature for 1 h, and then washed with water (3 x 15 mL). The organic layers were combined and dried over Na2S04, filtered, concentrated in vacuo. The crude compound was then crystallized from MeCN to afford a colourless powder (4.50 g, 80 %).
Figure imgf000022_0003
2 3
Synthesis of tert-butyl N-(4-(6-amino-9H-purin-9-v0butv0sulfamoylcarbamate f3)
A solution of 9-(4-aminobutyl)-9H-purin-6-amine 2 (1.6 g, 7.75 mmol, 1 eq) trietylamine (2.1 mL, 15.5 mmol, 2 eq) and A (2.6 g, 7.75 mmol 1 eq) in dichloromethane (15 mL) was stirred at room temperature overnight. The solvent was removed under reduced pressure. Purification by flash chromatography (5% methanol: dichloromethane) afforded the title compound (664 mg, 23%) as a colourless solid. 3H NMR (500 MHz, DMSO- d): d 10.77 (s, 1H, NH), 8.12 (s, 1H, Ar), 8.11 (s, 1H, ArH), 7.55 (br s, 1H, NH), 7.16 (s, 2H, NH2), 4.12 (t, J= 7.0 Hz, 2H, CH2), 2.90 (dd, J= 13.0, 6.7 Hz, 2H, CH2), 1.82 (m, 2H, CH2), 1.41 (m, 2H, CH2) and 1.38 (s, 9H, /Bu) ppm.
13C NMR (125 MHz, DMSO- d): d 155.9 (CO), 152.3 (C), 150.6 (CH), 149.5 (C), 140.7 (CH), 118.7 (C), 81.0 (C), 42.4 (CH2), 42.2 (CH2), 27.7 (3xC¾), 26.7 (CH2), and 25.8 (CH2) ppm.
MS HRMS (ESI) calcd for Ci4H24N704S (M + H+): 386.1676, found 386.1605.
Figure imgf000023_0001
Synthesis of the sulphonamide precursor (43
A solution of tert-butyl N-(4-(6-amino-9H-purin-9-yl)butyl)sulfamoylcarbamate 3 (664 mg, 1.72 mmol, 1 eq) in dichloromethane (12 mL) cooled down to 0 °C was treated with trifluoroacetic acid (1.2 mL, 15.7 mmol, 9 eq) and stirred at room temperature. After 2 h trifluoroacetic acid (1.2 mL, 15.7 mmol, 9 eq) was added. After being stirred at room temperature overnight the solvent was removed under reduced pressure. The title compound was obtained as a colourless solid (641 mg, 59%) and was used without further purification.
MS HRMS (ESI) calcd for C9Hi5N7Na02S (M + Na+): 308.0906, found 308.0883.
Figure imgf000024_0001
Synthesis of 2.5-dioxopyrrolidin-l-yl 5-(Y3aS.4S.6aRV2-oxohexahvdro-lH-thieno[3.4- dlimidazol-4-vOpentanoate (53 d-Biotin (5.0 g, 20.5 mmol) and N-hydroxysuccinimide (2.3 g, 20.5 mmol, 1.0 eq) were dissolved in hot DMF (150 mL). 1 -Ethyl-3 -(3 -dimethylaminopropyl)carbodiimide hydrochloric salt (4.7 g, 2.4.6 mmol, 1.2 eq) was added, and the solution was stirred overnight at room temperature. The mixture was partitioned between ethyl acetate and water and the aqueous phase was extracted with ethyl acetate (x3). The organics combined were dried over sodium sulphate anh., filtrated and evaporated under reduced pressure. The solid obtained was triturated from diethyl ether affording the title compound (4g, 57%) as a colourless solid.
3H NMR (500 MHz, DMSO-76) d 6.41 (s, 1H, NH), 6.35 (s, 1H, NH), 4.51-4.20 (m, 1H, CHH), 4.20-4.07 (m, 1H, CH H), 3.11 (dd, J= 12.3, 6.5 Hz, 1H, CHH), 2.87-2.78 (m, 5H, CH2+CH2+CH H), 2.67 (t, j= 7.4 Hz, 2H, CH2), 2.58 (d, j= 12.2 Hz, 1H, CHH), 1.65 (dt, J = 15.2, 7.7 Hz, 3H, CH2+CH H), 1.55-1.46 (m, 1H, GftH) and 1.43 (dd, 7 = 12.4, 6.4 Hz, 2H, CH2) ppm.
13C NMR (125 MHz, DMSO-d6): 170.2 (2xCO), 168.9 (CO), 162.6 (CO), 61.0 (CH), 59.2 (CH), 55.2 (CH), 40.0 (CH2), 30.0 (CH2), 27.8 (CH2), 27.6 (CH2), 25.43 (2xCH2), 24.30 and (CH2) ppm.
Figure imgf000025_0001
Synthesis of N-(N-(4-(6-amino-9H-purin-9-v0butv0sulfamov0-5- oxohexahvdro-
Figure imgf000025_0002
lH-thieno[3.4-dlimidazol-4-vOpentanamide (61
A suspension of the sulfonamide precursor 4 (641 mg, 2.225 mmol, 1 eq), CsC03 (520 mg, 2.7 mmol, 1.2 eq) and 5 (844 mg, 2.4 mmol, 1.1 eq) in DMF (2.8 mL) was stirred at room temperature overnight. The solvent was removed under reduced pressure and the crude was purified by flash chromatography (10-20% MeOH-DCM) affording the title compound (500 mg, 45%) as a colourless solid.
3H NMR (500 MHz, DMSO- d): d 11.26 (s, 1H, NH), 8.13 (s, 1H, ArH), 8.11 (s, 1H, ArH), 7.59 (t, J = 5.7 Hz, 1H, NH), 7.20 (s, 2H, NH2), 6.50 (s, 1H, NH), 6.38 (s, 1H, NH), 4.30 (dd, J = 7.6, 5.2 Hz, 1H, CH), 4.20-4.07 (m, 3H, CH2+C#H), 3.12-3.04 (m, 1H, CH H), 2.88 (dd, j= 12.9, 6.7 Hz, 2H, CH2), 2.80 (dd, j= 12.4, 5.1 Hz, 1H, CHR), 2.57 (d, j= 12.4 Hz, 1H, CH H), 2.18 (t, J = 7.4 Hz, 2H, CH2), 1.91-1.73 (m, 2H, CH2), 1.67-1.55 (m, 1H, CHR ), 1.55-1.36 (m, 5H, CH2+CH2+CHtf) and 1.35-1.21 (m, 2H, CH2) ppm.
13C NMR (125 MHz, DMSO- d): 171.5 (CO), 162.8 (CO), 155.9 (C), 152.4 (CH), 149.5 (C), 140.8 (CH), 118.7 (C), 61.1 (CH), 59.2 (CH), 55.4 (CH), 48.6 (CH2), 42.4 (CH2), 42.2 (CH2), 35.0 (CH2), 28.0 (2xCH2), 26.8 (CH2), 25.8 (CH2) and 24.3 (CH2) ppm.
MS HRMS (ESI) calcd for Ci9H3oN904S2 (M + H+): 512.1857, found 512.1834.
Figure imgf000026_0003
d-biotin 7
Synthesis of fer/-butyl f4-ff3aVAS'.6a/s>)-2-oxohexahvdro- l //-thieno[3.4- /]imidazol-4- yl ibutyl icarbamate (73
To a suspension of d-biotin (300 mg, 1.23 mmol) were added diphenylphosphoryl azide (320 mg, 1.35 mmol) and TEA (136 mg, 1.35 mmol) in /er/-butanol (10 mL) and heated at reflux for 18 h. Solvents were evaporated; the residue was dissolved in DCM (50 mL) and washed with water and brine. The organic layer was dried over Na2S04 and concentrated under reduced pressure. The residue was purified by flash chromatography on silica (DCM:MeOH = 25: 1) to yield a white solid (197 mg, 51 %).
¾ NMR (300 MHz, 2% CD3OD, CDCh) d 4.48-4.58 (m, 1H), 4.30-4.40 (m, 1H), 3.27-3.38 (m, 1H), 3.05-3.18 (m, 2H), 2.93 (dd, J= 4.5, 8.7 Hz, 1H), 1.30-1.80 (m, 6H), 1.44 (s, 9H).
Figure imgf000026_0001
Synthesis of 4-aminobutvOtetrahvdro-liT-thieno[3.4-<71imidazol-2(3i7V
Figure imgf000026_0002
one hydrochloride salt (81
To a solution of compound 7 (150 mg, 0.47 mmol) in MeOH (5 mL) was added 6N HC1 (5 mL) and stirred overnight. The mixture was concentrated under reduced pressure, the precipitate was filtered, washed with ice water (2 x 10 mL) and dried with vacuum filtration to yield a white solid (89 mg, 87 %).
NMR (300 MHz, DMSO-^) d 6.48 (br s, 1H), 6.37 (br s, 1H), 3.08-3.14 (m, 1H), 2.85 (dd, J= 5.1, 12.6 Hz, 1H), 2.52-2.59 (m, 3H), 1.25-1.64 (m, 6H); 13C NMR (75 MHz, DMSO-r¾) d 162.7, 61.6, 59.2, 55.5, 40.1, 32.1, 28.4, 28.3, 26.3.
diphenyl carbonate
Figure imgf000027_0001
TEA, THF/water(1/9)
Figure imgf000027_0002
Figure imgf000027_0003
Synthesis of phenyl f4-ff3aVAV.6ari>)-2-oxohexahvdro- l //-thieno[3.4- /]imidazol-4- vObutvOcarbamate t9)
To a mixture of compound 8 (300 mg, 1.19 mmol) in water/THF (9: 1, 3.33 mL) were added diphenyl carbonate (255 mg, 1.19 mmol) and TEA (0.33 mL, 2.38 mmol) and the mixture was stirred for 6 h. The mixture was poured into water and extracted with DCM. The organic layer was washed with cold aqueous 10 % NaOH solution and brine. The organic layer was dried over Na2S04 and concentrated. The residue was purified by flash chromatography on silica (DCMMeOH = 10: 1) to yield a white solid (223 mg, 56 %).
3H NMR (500 MHz, DMSO- e) d 7.71 (t, J= 5.7 Hz, 1H), 7.40 - 7.33 (m, 2H), 7.22 - 7.15 (m, 1H), 7.11 - 7.06 (m, 2H), 6.43 (s, 1H), 6.35 (s, 1H), 4.31 (ddt, j= 7.6, 5.0, 1.1 Hz, 1H), 4.14 (ddd, j= 7.8, 4.5, 1.9 Hz, 1H), 3.12 (ddd, j= 8.2, 6.4, 4.4 Hz, 1H), 3.05 (q, j= 6.6 Hz, 2H), 2.83 (dd, J = 12.4, 5.1 Hz, 1H), 2.58 (d, J= 12.4 Hz, 1H), 1.69 - 1.58 (m, 1H), 1.57 - 1.27 (m, 5H);
13C NMR (126 MHz, DMSO- e) d 162.68, 154.27, 151.11, 129.18, 124.77, 121.69, 60.93, 59.19, 55.43, 40.22, 39.82, 29.20, 27.98, 25.80.
Figure imgf000027_0004
80% 2-steps
10
Synthesis of fer/-butyl (4-bromobutvOcarbamate (103
To a solution of 4-amino- 1 -butanol (1.50 g, 16.8 mmol) in MeCN (150 mL) were added TEA (4.68 mL) and Boc20 (4.03 g, 18.5 mL) at 0 °C. The mixture was stirred for 6 h and quenched with water. The mixture was poured onto water and extracted with EtOAc three times. The combined organic layers were dried over Na2S04 and concentrated under reduced pressure. The crude product 6 (2.68 g) was used without further purification. The crude product 6 was dissolve in THF (130 mL) followed by addition of Ph3P (7.06 g, 26.9 mmol). Then CBr4 (8.92 g, 26.9 mmol) was slowly added to the mixture. After 3 h, the solution was filtered through Celite® pad and washed with Et20. The solvents were removed under reduced pressure. The residue was purified by flash chromatography on silica (hexane:EtOAc = 3 : 1) to yield a colourless oil (4.38 g, 80 %). 7 Hz,
Figure imgf000028_0001
To a suspension of adenine (1.10 g, 8.14 mmol) in DMF (12 mL) was added Cs2C03 (3.98 g, 12.2 mmol) and bromide 10 (2.45 g, 9.77 mmol). The mixture was stirred overnight, poured onto water and extracted with EtOAc twice. The combined organic layers were washed with brine and water. The organic layer was dried over Na2S04 and concentrated under reduced pressure. The residue was purified by flash chromatography on silica (DCM:MeOH = 15: 1 10: 1) to yield a white solid (1.70 g, 68 %).
¾ NMR (500 MHz, DMSO- e) d 8.12 (s, 1H), 8.11 (s, 1H), 7.16 (s, 2H), 6.80 (t, J = 5.8 Hz, 1H), 4.12 (t, J= 7.0 Hz, 2H), 2.92 (q, J= 6.6 Hz, 2H), 1.76 (m, 2H), 1.35 (s, 11H);
13C NMR (126 MHz, DMSO- e) d 155.93, 155.58, 152.31, 149.52, 140.78, 118.74, 77.38, 42.57, 28.23, 26.83, 26.65.
Figure imgf000029_0001
Synthesis of fer/-butyl (4-(6-benzamido- purin-9-vObutvOcarbamate (123
Figure imgf000029_0002
To a mixture of compound 11 (1.03 g, 3.36 mmol) in pyridine (5 mL) was slowly added BzCl (0.39 mL, 3.36 mmol) and the mixture was stirred at 100 °C for 1 h. The mixture was concentrated under reduced pressure and the residue was purified by flash chromatography on silica (DCM:MeOH = 20: 1) to yield a white solid (0.83 g, 60 %).
3H NMR (600 MHz, CDCh) d 9.30 (s, 1H), 8.74 (s, 1H), 8.06 - 7.93 (m, 3H), 7.59 - 7.52 (m, 1H), 7.47 (t, J= 7.8 Hz, 2H), 4.71 (t, J= 6.2 Hz, 1H), 4.28 (t, J = 7.3 Hz, 2H), 3.16 (q, J= 6.7 Hz, 2H), 2.40 (s, 1H), 1.92 (m, 2H), 1.50 (m, 2H), 1.39 (s, 9H);
13C NMR (151 MHz, CDCh) d 164.93, 156.21, 152.56, 152.23, 149.60, 143.12, 133.74, 132.81, 128.88, 128.02, 123.14, 79.46, 53.55, 43.69, 39.55, 28.48, 27.36, 27.22.
Figure imgf000029_0003
34% 2-steps
12 13
Synthesis of fer/-butyl fN-f4-f6-benzamido-9//-purin-9-yl )butyl isulfamoyl icarbamate (131
To a mixture of compound 12 (820 mg, 2.00 mmol) in DCM (9 mL) was added TFA (0.83 mL, 10.8 mmol) and the mixture was stirred for 3 hours. TEA (1.5 mL) was added dropwise to the reaction mixture to form a white solid. The solid was collected by filtration and washed with DCM. To a mixture of the collected solid in THF (20mL) were added TEA (0.45 mL, 4.00 mmol) and sulfamoylating agent A (602 mg 2.00 mmol) and the mixture was stirred at 60°C overnight. The mixture was concentrated under reduced pressure and the residue was purified by flash chromatography on silica (DCM:acetone:MeOH = 50:25: 1) to yield a white sold (320 mg, 34 %). 3H NMR (500 MHz, CDCh) d 9.26 (br s, 1H), 8.79 (s, 1H), 8.10 - 7.99 (m, 3H), 7.66 - 7.57 (m, 1H), 7.52 (t, j = 7.6 Hz, 2H), 5.81 (br s, 1H), 4.33 (t, j = 7.1 Hz, 2H), 3.14 (q, j = 5.9 Hz, 2H), 2.03 (m, 2H), 1.69 - 1.55 (m, 2H), 1.46 (s, 9H);
13C NMR (126 MHz, CDCh) d 152.55, 152.43, 150.60, 149.74, 143.23, 133.69, 132.92, 128.92, 128.24, 123.26, 83.93, 68.10, 43.56, 43.14, 28.14, 27.34, 26.15.
Figure imgf000030_0001
13 14
Synthesis of N-(9-(4-(sulfamoylamino)butylV9i7-purin-6-vnbenzamide (143
To a mixture of compound 13 (140 mg, 0.286 mmol) in DCM (5 mL) was added TFA (0.5 mL) and the mixture was stirred for 2 h. The mixture was concentrated under reduced pressure and the residue was purified by flash chromatography on silica (DCM:MeOH = 6: 1) to yield a white solid (105 mg, 94 %).
3H NMR (500 MHz, DMSO- e) d 11.11 (br s, 1H), 8.73 (s, 1H), 8.50 (s, 1H), 8.09 - 8.00 (m, 2H), 7.69 - 7.60 (m, 1H), 7.55 (t, J= 7.7 Hz, 2H), 6.47 (m, 3H), 4.28 (t, J= 7.1 Hz, 2H), 2.91 (q, J= 6.5 Hz, 2H), 1.91 (m, 2H), 1.52 - 1.40 (m, 2H);
13C NMR (126 MHz, DMSO- e) d 152.42, 151.31, 150.01, 144.66, 133.46, 132.34, 128.42, 128.40, 125.28, 42.96, 41.87, 26.73, 26.11.
Figure imgf000030_0002
yl ibutyl icarbamoyl isulfamoyl )amino)butyl purin-6-yl ibenzamide (153 To a mixture of compound 9 (77 mg, 0.23 mmol) and compound 14 (74 mg, 0.19 mmol) in MeCN (10 mL) was added DBU (43 pL, 0.29 mmol) and the resulting mixture was refluxed for 17 h. The solvent was evaporated under reduced pressure. The residue was dissolved in EtOAc and the organic layer was washed with 0.1N HC1, brine, and water. The organic layer was dried over Na2S04 and concentrated under reduced pressure. The residue was purified by flash chromatography on silica (DCM:MeOH = 9: 1) to yield a white solid (58 mg, 48%).
3H NMR (500 MHz, DMSO- e) d 11.11 (br s, 1H), 9.74 (br s, 1H), 8.72 (s, 1H), 8.47 (s, 1H), 8.09 - 8.00 (m, 2H), 7.70 - 7.60 (m, 1H), 7.55 (t, J= 7.7 Hz, 2H), 7.35 (br s, 1H), 6.41 (s, 1H), 6.34 (s, 1H), 6.26 (m, 1H), 4.27 (m, 3H), 4.11 (ddd, J= 7.5, 4.5, 1.7 Hz, 1H), 3.07 (ddd, j= 8.4, 6.3, 4.4 Hz, 1H), 3.01 (q, j= 6.6 Hz, 2H), 2.93 (m, 2H), 2.79 (dd, j= 12.5, 5.1 Hz, 1H), 2.56 (d, J= 12.5 Hz, 1H), 1.89 (m,2H), 1.62 (m, 1H), 1.54 - 1.24 (m, 7H);
13C NMR (126 MHz, DMSO- e) d 162.68, 152.43, 152.28, 151.32, 150.02, 144.62, 132.33, 128.44, 128.42, 128.40, 125.30, 60.97, 59.17, 55.43, 54.87, 42.81, 42.11, 29.37, 27.94, 26.59, 25.88, 25.86, 25.79.
Figure imgf000031_0001
Synthesis of (3aS,.4.S,.6ai?V4-(4-(Y(Y4-(6-amino-9i/-purin-9- yl ibutyl isulfamoyl )carbamoyl )amino)butyl )tetrahvdro- l //-thieno[3.4- /]imidazol-2f3//)- one (161
To a solution of compound 15 (50 mg, 0.079 mmol) in MeOH (1.6 mL) was added 30 % NH4OH (2.4 mL) and the mixture was stirred overnight. The mixture was concentrated under reduced pressure and the residue was purified by flash chromatography on silica (DCM:MeOH = 4: 1) to yield a white solid (22 mg, 53 %).
*H NMR (500 MHz, DMSO- 6) d 9.74 (br s, 1H), 8.13 (s, 1H), 8.11 (s, 1H), 7.33 (br s, 1H), 7.17 (s, 2H), 6.47 (s, 1H), 6.35 (s, 1H), 6.21 (br s, 1H), 4.29 (dd, j = 7.7, 5.0 Hz, 1H), 4.17 - 4.09 (m, 3H), 3.08 (ddd, J= 8.6, 6.2, 4.4 Hz, 1H), 3.01 (q, J= 6.6 Hz, 2H), 2.90 (q, J= 6.6 Hz, 2H), 2.80 (dd, J= 12.4, 5.1 Hz, 1H), 2.57 (d, j= 12.4 Hz, 1H), 1.87 - 1.77 (m, 2H), 1.67 - 1.56 (m, 1H), 1.51 - 1.19 (m, 7H);
13C NMR (126 MHz, DMSO- e) d 162.72, 155.91, 152.33, 149.51, 140.71, 118.67, 61.01, 59.17, 55.45, 54.87, 48.57, 42.42, 42.14, 38.81, 29.38, 27.97, 26.75, 25.82, 25.78.
MS HRMS (ESI) calcd for C19H30N10O4S2 (M + H+): 527.1971, found 527.1966.
Biological data
Antimicrobial susceptibility assays
The antimicrobial activity of the compounds was determined by a microdilution broth method as recommended by the CLSI (Clinical and Laboratory Standards Institute, Document M07-A8, 2009, Wayne, Pa.) with cation-adjusted Mueller-Hinton broth (Trek Diagnostics Systems, U.K.). Compounds were dissolved using DMSO. Serial two-fold dilutions of each compound were made using DMSO as the diluent. Trays were inoculated with 5 x 104 CFU of S. aureus ATCC 49775 in a volume of 100 pL (final concentration of DMSO was 3.2 % (v/v)), and incubated at 35 °C for 16- 20 hours. Growth of the bacterium was quantitated by measuring the absorbance at 620 nm.
It has been surprisingly found that the present invention being the compound of general formula (I), in particular when n=4, m=8 (-C4H8) and Ri = adenine; N-(N-(4-(6-amino-9H- purin-9-yl)butyl)sulfamoyl)-5-((4S)-2-oxohexahydro-lH-thieno[3,4-d]imidazol-4- yl)pentanamide (compound 6), has specific antibacterial activity against Staphylococcus aureus.
The antibacterial activity of compound (6) unexpected properties of inhibition against biotin protein ligase. Figure 1 shows the inhibitory effect of compound 6 against S. aureus ATCC 49775 with a wide zone of inhibition, similar to that shown for Erythromycin shows specific activity against S. aureus as shown in Figure 2 in which compound (6) compares favourably alongside other antibiotics such as erythromycin having a similar potency profile. Enzyme inhibition assays for Compound 6
Quantitation of BPL catalyzed 3H-biotin incorporation into the biotin domain substrate was performed. The reaction mixture contained: 50 mM Tris HCI pH 8.0, 3 mM ATP, 4.94 pM, 0.06 pM 3H Biotin, 5.5 mM MgCL 100 mM KCI, 0.1 pM DTT and 10 pM biotin domain of S. aureus pyruvate carboxylase. The reaction was initiated by the addition of BPL to a final concentration of 6.25 nM. After 10 minutes at 37°C, 90 pL of stopping buffer (50 mM Tris HCI pH 8.0, 110 mM EDTA) was added to each reaction and 100 pL of each reaction was added to the wells of 96-well HTS multiscreen plate with added immobilon-P (Merck Millipore) that had been pre-treated with 50 pL of 70% ethanol and 400 pL of MQ H20. The plate wells were washed by the addition of Tris buffered saline. Quantitation of protein- bound radiolabelled biotin was determined by liquid scintillation. The ICso value of each compound was determined from a dose-response curve by varying the concentration of inhibitor under the same enzyme concentration. The inhibition constant (Ki) was analysed with GraphPad prism (version 6) by fitting the data to the Morrison equation, where [S] is the substrate concentration ([biotin] = 5 pM) and Km is the affinity of the enzyme for biotin (S. aureus BPL = 1 pM).
Figure 3 shows the enzyme inhibition curve of compound 6 against S. aureus with IC50 = 4.3 nM, and Ki = 0.72 nM.
Enzyme inhibition assays of compound 6 and 16
The inhibitory activity of compounds was determined by measuring S. aureus BPL activity in the presence of varying concentrations of compound. A reaction mixture was prepared containing 50 mM Tris HC1, pH 8.0, 3 mM ATP, 5 mM biotin, 5.5 mM MgCl2, 100 mM KC1, 0.1 pM DTT, and 25 pM biotin domain of S. aureus pyruvate carboxylase (SaPC90) fused to GST. All compounds were dissolved in DMSO and then diluted into the reaction buffer to give a final concentration of 4% DMSO. The reaction was initiated by the addition of enzyme to give final concentrations of 4.5 nM of S. aureus BPL and final volume of 20 ul. After 20 mins at 37 °C the reaction was terminated with 180 pL of stop buffer (50 mM Tris, 5.5 mM EDTA, pH 8), and three 50 pL aliquots were added to each well of a white Lumitrac-600 96-well plate (Greiner) that had been precoated with a polyclonal anti-GST antibody (Sigma-Aldrich, 50 pL per well, 1 :40 000 dilution) at 4 °C overnight, followed by blocking for 2 h (200 pL per well) with 1% BSA in TBS at 37 °C. After incubation at 37 °C for 1 h the plate was then washed 5 times (200 pL per well) with TBS containing 0.1% Tween-20. Europium labelled streptavidin (PerkinElmer) was diluted to 0.1 pg/mL in TBS containing 0.1% Tween-20. The streptavidin probe (50 pL per well) was incubated for 30 min at 37°C followed by washing 5 times with TBS containing 0.1% Tween-20 followed by another 5 washes in sterile water. Enhancement solution (PerkinElmer, 50 pL) was added to each well and incubated for 10 min before reading the plate using a PerkinElmer Victor X5 multilabel reader (time-resolved fluorescence settings, 340 nm excitation, and 612 nm emission). The inhibition constant ( ) was analysed with GraphPad prism (version 6) by fitting the data to the Morrison equation, where [S] is the substrate concentration ([biotin] = 5 pM) and Km is the affinity of the enzyme for biotin (S. aureus BPL = 1 pM) and Et is the enzyme concentration (4.5 nM).
Figure 4 shows the activity of BPL from S. aureus measured in the presence of varying concentrations of the BPL inhibitors compound 6 (O) and compound 16 (■). Inhibition constants (K\) were calculated using the Morrison equation. K\ for compound 6 was 1.0 ± 0.3 nM and Ki for compound 16 is 53 ± 33 nM.
Stability of BPL inhibitors in whole blood
Fresh whole rat blood (490pL) spiked with lOpL 0.5 mg/mL of either compound 6 or compound 16 in separate tubes (dissolved in methanol/water 50/50). Samples were mixed well and incubated at 22 °C for up to 24 h. Blood was then extracted for LCMS by protein precipitation (10 pL blood + lOOpL 1.4% ZnS04 + 50pL methanol) The concentration of compound remaining at various time points was measured by LCMS, as shown in figure 5.
Cytotoxicity of compound 6 against mammalian cell culture lines
HEK 293 (kidney) and HepG2 (liver) cells were suspended in Dulbecco-modified Eagle’s medium containing 10% fetal bovine serum and then seeded in 96-well tissue culture plates at 10,000 cells per well. After 24 h, cells were treated with varying concentrations of Compound 6, such that the DMSO concentration was consistent at 2% (v/v) for HepG2 or 0.5% (v/v) for HEK293 in all wells. After treatment for 48 h, WST-l cell proliferation reagent (Roche Applied Science) was added to each well and incubated for 0.5 h at 37 °C. The WST-l assay quantitatively monitors the metabolic activity of cells by measuring the hydrolysis of the WST-l reagent, the products of which are detectable at absorbance 450 nm. Figure 6 shows no significant toxicity was observed at any concentration of Compound 6 for either HEK 293 (kidney, figure 6A) or HepG2 (liver, figure 6B) cells. Cells receiving no treatment are shown in red, and those with paracetamol (6.25 mM for HEK293, 25 mM for HepG2) are shown in blue.
Antibacterial susceptibility assays
Antimicrobial activity (minimal inhibitory concentration, MIC) of the BPL inhibitors and comparison antibiotics were determined by a microdilution broth method as recommended by the Clinical and Laboratory Standards Institute (Document M07-A8, 2009, Wayne, PA) with cation-adjusted Mueller-Hinton broth (Trek Diagnostics Systems). Compounds were dissolved using DMSO. Serial 2-fold dilutions of each compound were made using DMSO as the diluent. Trays were inoculated with 5 x 104 colony -forming units of S. aureus ATCC 49775 in a volume of 100 pl (final concentration of DMSO was 3.2% (v/v)) and incubated at 35 °C for 16 -20 h. Growth of the bacterium was quantitated by measuring the absorbance at 620 nm.
Anti-staphylococcus activity of compound 16
Figure 7 shows S. aureus ATCC49775 treated for 22 hours with varying concentrations of compound 16 (red curve). Erythromycin was included as a control (orange). Minimal inhibitory concentration of compound 16 is 4 pg/ml.
Although the invention has been herein shown and described in what is conceived to be the most practical and preferred embodiments, it is recognized that departures can be made within the scope of the invention, which is not to be limited to the details described herein but it is to be accorded the full scope of the appended claims so as to embrace any and all equivalent structures and applications.

Claims

Claims
1. A compound of formula (I)
Figure imgf000037_0001
wherein
Ri is selected from the group consisting of radical -(CH2)n- , where n is an integer between 1 and 10;
R2 is selected from the group consisting of radical -(CH2)-, NH, NR3,
M is selected from the group consisting of radical -(0¾) -, NH, NR3,
R3 is H, or alkyl;
R4 is selected from the group consisting of C3-C6 cyclo alkyl radical, radical -(0¾) - , where n is an integer between 1 and 10;
R5 is selected from the group consisting of:
adenine;
benzoxazolone, benzoxazole, benzofuran, benzimidazole,
Figure imgf000037_0002
, where:
R6, R7, R8, R9 are selected from the group consisting of H, C1-C5 alkyl, halogen, NH2, OH2, NR13R14, SH, SR14: where R13 and R14 are selected from the group consisting of H, Ci- C5 alkyl;
Rio is selected from the group consisting of N, NH;
R11 is selected from the group consisting of CFh, C=0;
R12 is selected from the group consisting of N, NH, O;
Figure imgf000038_0001
, where:
Rs, R7, R8, R9 are selected from the group consisting of H, C1-C5 alkyl, halogen, NH2, OH2, NR13R114, SH, SRI4:
where R9 and Rio are selected from the group consisting of H, C1-C5 alkyl;
Rio is selected from the group consisting of N, NH;
R11 is selected from the group consisting of CH2, C=0;
R12 is selected from the group consisting of N, NH, O;
Figure imgf000038_0002
, where:
Rs, R7, R8, are selected from the group consisting of H, C1-C5 alkyl, halogen, NH2, OH2, NRi3Ri4, SH, SRI4:
where R13 and RI4 are selected from the group consisting of H, Ci- C5 alkyl;
Rio is selected from the group consisting of N, NH; Rn is selected from the group consisting of CFh, C=0;
R12 is selected from the group consisting of N, NH, O;
R R
napthyl,
Figure imgf000039_0002
Figure imgf000039_0001
where:
X is selected from the group consisting of O, NR13R14, SR14;
R12, R15, Ri6, Rn, Ri8, R19, R20 are selected from the group consisting of H, C1-C5 alkyl, halogen, MB, OH2, NR13R14, SH, SRI4: where R13 and R14 are selected from the group consisting of H, Ci- C5 alkyl;
Figure imgf000039_0003
n is an integer between 1 and 4;
X is a halogen selected from the group consisting of F, Br, Cl or I;
R21 is selected from the group consisting of Ci-C4 alkyl or C3-C6 cyclo alkyl; R22 is selected from the group consisting of Ci-C4 alkyl;
Figure imgf000039_0004
n is an integer between 1 and 4;
R-23 is selected from the group consisting of H, Ci-C4 alkyl; R-24 is selected from the group consisting of H, Ci-C4 alkyl;
Figure imgf000040_0001
n is an integer between 1 and 4;
R-25 is selected from the group consisting of H, Ci-C4 alkyl;
R-26 is selected from the group consisting of H, Ci-C4 alkyl;
Figure imgf000040_0002
n is an integer between 1 and 4;
R-27 is selected from the group consisting of H, Ci-C4 alkyl.
2. A pharmaceutical composition of claim 1 including as an active ingredient at least one compound of formula (I) optionally together with one or more pharmacologically acceptable excipients.
3. The pharmaceutical composition of claim 2, when used for the treatment of infection selected from the group consisting of bacterial, fungal, protozoan infections.
4. The pharmaceutical composition of claim 1 , when used for the treatment of infections caused by Staphylococcus aureus.
5. The compound of any one of the above claims wherein when R5 =
Figure imgf000041_0001
, n=3, X =F, R21 = C3 cyclo alkyl and R22 =
C2 alkyl.
6. The compound of any one of the above claims wherein when R5
Figure imgf000041_0002
7. The compound of any one of the above claims wherein when R5
Figure imgf000041_0003
8. The compound of any one of the above claims wherein, when
Figure imgf000041_0004
, n=3 and R27 = H.
9. The compound of any one of the above claims wherein the compound of formula (I) where Ri is -(CH2)n and n=3.
10. The compound of any one of the above claims where R2 is radical NH.
11. The compound of any one of the above claims where R4 is -(CH2)n and n=4.
12. The compound of any one of the above claims where R5 is radical adenine.
13. The compound of any one of the above claims wherein where R5 is
Figure imgf000042_0001
14. Use of at least one compound according to formula (I) of any of the above claims as an active ingredient in the preparation of a medicament for the treatment and/or prevention of bacterial infections, fungal infections, and protozoan infections to a subject in need thereof.
15. The use of claim 14, wherein the bacterial infection is caused by a Gram negative bacterium or a Gram positive bacterium.
16. The use of claim 14 or 15, wherein the bacterial infection is a Staphylococcus aureus infection
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140296177A1 (en) * 2011-09-23 2014-10-02 Monash University Novel antimicrobial compounds

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140296177A1 (en) * 2011-09-23 2014-10-02 Monash University Novel antimicrobial compounds

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BOCKMAN, M. R. ET AL.: "Avoiding Antibiotic Inactivation in Mycobacterium tuberculosis by Rv3406 through Strategic Nucleoside Modification", ACS INFECTIOUS DISEASES, vol. 4, no. 7, 17 April 2018 (2018-04-17), pages 1102 - 1113, XP055668249, DOI: 10.1021/acsinfecdis.8b00038 *
BOCKMAN, M. R. ET AL.: "Targeting Mycobacterium tuberculosis Biotin Protein Ligase (MtBPL) with Nucleoside-Based Bi-substrate Adenylation Inhibitors", JOURNAL OF MEDICINAL CHEMISTRY, vol. 58, no. 18, 24 August 2015 (2015-08-24), pages 7349 - 7369, XP055291760, DOI: 10.1021/acs.jmedchem.5b00719 *
DATABASE CAS 25 September 2015 (2015-09-25), "1H- Thieno[3,4-d]imidazole-4-pentanamide, N-[[[[(1R,3S)-3-(6-amino-9H-purin-9- yl)cyclopentyl]methyl]amino]sulfonyl]hexahydro-2-oxo-, (3 aS,4S ,6aR)- Chemical formula as referenced in its entirety", retrieved from STN Database accession no. 1808182-63-9 *
DATABASE CAS 25 September 2015 (2015-09-25), "1H- Thieno[3,4-d]imidazole-4-pentanamide, N-[[[[(1S,4R)-4-(6-amino-9H-purin-9-y1)-2- cyclopenten-l-yl]methyl ] amino ]sulfonyl]hexahydro-2-oxo-, (3aS,4S,6aR)- Chemical formula as referenced in its entirety", retrieved from STN Database accession no. 1808182-61-7 *
DATABASE CAS 25 September 2015 (2015-09-25), "1H- Thieno[3,4-d]imidazole-4-pentanamide,N-[[[[(1R,2R,3S,4R)-4-(6-amino-9H-purin-9- yl)-2,3-dihydroxycyclopentyl]methyl]amino]sulfonyl]hexahydro-2-oxo-, (3aS,4S,6aR)- Chemical formula as referenced in its entirety", retrieved from STN Database accession no. 1808182-55-9 *
LEE, K. J. ET AL.: "Sulfonamide-Based Inhibitors of Biotin Protein Ligase as New Antibiotic Leads", ACS CHEMICAL BIOLOGY, vol. 14, no. 9, 13 August 2019 (2019-08-13), pages 1990 - 1997, XP055705509 *

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