Classifications

• • 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
• • 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

Definitions

• Novel oxoquinolizine compounds for the treatment and prophylaxis of bacterial infection
• the present invention relates to organic compounds useful for therapy and/or prophylaxis in a mammal, and in particular to inhibitors of DNA gyrase and/or topoisomerase IV useful for treatment and/or prophylaxis of bacterial infection.
• topoisomerase IV bacterial type IIA topoisomerases
• DNA Gyrase controls DNA supercoiling and relieves topological stress that occurs when the DNA strands are untwisted such as during replication.
• Topoisomerase IV primarily resolves linked chromosome dimers at the conclusion of DNA replication.
• Both enzymes can introduce double stranded DNA breaks; pass a second DNA strand through the break and rejoining the broken strands. The activity of both enzymes is driven by the binding and hydrolysis of ATP.
• Bacterial DNA gyrase consists of two A (GyrA) and two B (GyrB) subunits.
• Bacterial Topoisomerase IV is also a hetero-tetramer that consists of two C (ParC) and two E (ParE) subunits. The latter subunits bind ATP like GyrB in order to supply energy necessary for catalytic turnover of the enzymes.
• the present invention relates to novel compounds of formula (I),
• R 1 is Ci- 6 alkyl
• R 2 is halogen
• R 3 is H, halogen or cyano
• R 4 is (Ci-6alkyl)2amino or heterocyclyl
• R 5 is H, halogen or Ci-ealkyl
• R 6 is H, ((Ci- 6 alkyl) 2 amino)Ci- 6 alkyl, Ci-6alkyl, C 3- 7cycloalkyl, haloCi- 6 alkyl, halogen,
• R 7 is carboxy
• novel compounds of formula (I) are novel compounds of formula (I), their manufacture, medicaments based on a compound in accordance with the invention and their production as well as the use of compounds of formula (I) for the treatment or prophylaxis of bacterial infection.
• the use of compounds of formula (I) as DNA gyrase and/or topoisomerase IV inhibitors is also one of the objections of present invention.
• the compounds of formula (I) showed superior anti-bacterial activity, good solubility, good CC50 profiles, improved microsomal stability and/or improved PK profile.
• Ci-ealkyi denotes a saturated, linear or branched chain alkyl group containing 1 to 6, particularly 1 to 4 carbon atoms, for example methyl, ethyl, propyl, isopropyl, «-butyl, isobutyl, tert- butyl and the like.
• Particular“Ci-ealkyl” groups are methyl, ethyl and propyl.
• halogen and“halo” are used interchangeably herein and denote fluoro, chloro, bromo, or iodo.
• haloCi-ealkyl denotes an alkyl group wherein at least one of the hydrogen atoms of the alkyl group has been replaced by same or different halogen atoms, particularly fluoro atom.
• haloCi- 6 alkyl include monofluoro-, difluoro-or trifluoro-methyl, -ethyl or -propyl, for example 3,3,3 -trifluoropropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, fluoromethyl, difluoromethyl, trifluoromethyl and trifluoro ethyl.
• C3-7cycloalkyl denotes a saturated monocyclic or bicyclic carbon ring containing from 3 to 7 carbon atoms, particularly from 3 to 6 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[ 1.1.1 jpentanyl and the like.
• Particular“C 3-7 cycloalkyl” group is cyclopropyl.
• heterocyclyl denotes a monovalent saturated or partly unsaturated mono-, bicyclic or tricyclic ring system of 3 to 12 ring atoms, comprising 1, 2, or 3 ring heteroatoms selected from N, O and S, the remaining ring atoms being carbon.
• heterocyclyl is a monovalent saturated or partly unsaturated monocyclic or bicyclic ring system of 4 to 10 ring atoms, comprising 1, 2, or 3 ring heteroatoms selected from N, O and S, the remaining ring atoms being carbon.
• Examples for monocyclic saturated heterocyclyl are aziridinyl, oxiranyl, azetidinyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydro-thienyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl, morpholinyl, thiomorpholinyl, l,l-dioxo- thiomorpholin-4-yl, azepanyl, diazepanyl, homopiperazinyl, or oxazepanyl.
• heterocyclyl can be spiro ring, fused ring or bridged ring.
• bicyclic heterocyclyl are decahydronaphthyridine; azabicyclo[3 2.0]heptanyl; octahydrocyclopenta[b]pyrrolyl;
• octahydrocyclopenta[c]pyrrolyl l,3,3a,4,6,6a-hexahydrofuro[3,4-c]pyrrolyl; 1 ,3, 3a, 4, 6,6a- hexahydropyrrolo[3,4-c]pyrrolyl; l,7-diazaspiro[3.4]octanyl; 1 ,7-diazaspiro[4.4]nonanyl;
• Examples for partly unsaturated heterocyclyl are dihydrofuryl, imidazolinyl, dihydro-oxazolyl, tetrahydro-pyridinyl, or dihydropyranyl.
• Monocyclic or bicyclic heterocyclyl can be further substituted once, twice or three times by hydroxyCi-ealkyl, (Ci- 6 alkoxyC l-6 alkyl(C l-6 alkyl)amino)C i -6 alkyl, (C i-6alkyl) 2 amino, (C l-6 alkyl) 2 aminoC U6alkyl, (C3- 7cycloalkyl(Ci-6alkyl)amino)Ci-6alkyl, (C3-7cycloalkylamino)Ci-6alkyl, amino, aminoC l-6 alkyl, Ci- 6 alkoxy, Cu 6 alkoxyCi- 6 alkyl, Ci- 6 alkoxyCu 6 alkyl(Ci- 6 al
• enantiomer denotes two stereoisomers of a compound which are non- superimposable mirror images of one another.
• diastereomer denotes a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties, and reactivities.
• pharmaceutically acceptable salts denotes salts which are not biologically or otherwise undesirable.
• Pharmaceutically acceptable salts include both acid and base addition salts.
• pharmaceutically acceptable acid addition salt denotes those pharmaceutically acceptable salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and organic acids selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid
• pharmaceutically acceptable base addition salt denotes those pharmaceutically acceptable salts formed with an organic or inorganic base.
• acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts.
• Salts derived from pharmaceutically acceptable organic nontoxic bases includes salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine , lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine , theobromine, purines, piperizine, piperidine, /V-ethylpiperidine, and polyamine resins.
• substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine
• therapeutically effective amount denotes an amount of a compound or molecule of the present invention that, when administered to a subject, (i) treats or prevents the particular disease, condition or disorder, (ii) attenuates, ameliorates or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition or disorder described herein.
• the therapeutically effective amount will vary depending on the compound, the disease state being treated, the severity of the disease treated, the age and relative health of the subject, the route and form of administration, the judgement of the attending medical or veterinary practitioner, and other factors.
• pharmaceutical composition denotes a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient together with
• the present invention relates to a compound of formula (I),
• R 1 is Ci-ealkyl
• R 2 is halogen
• R 3 is H, halogen or cyano
• R 4 is (Ci-6alkyl)2amino or heterocyclyl
• R 5 is H, halogen or Ci- 6 alkyl
• R 6 is H, ((Ci-6alkyl)2amino)Ci-6alkyl, Ci- 6 alkyl, C 3-7 cycloalkyl, hak>Ci- 6 alkyl, halogen,
• R 7 is carboxy
• a further embodiment of present invention is (ii) a compound of formula (I) according to
• R 1 is Cnealkyl
• R 2 is halogen
• R 3 is H, halogen or cyano
• R 4 is (Ci- 6 alkyl)2amino
• morpholinyl said morpholinyl being unsubstituted or substituted by ((Ci-6alkyl)2amino)Ci_ ealkyl;
• R 5 is H, halogen or Ci- 6 alkyl
• R 6 is H, ((Ci-6alkyl)2amino)Ci-6alkyl, Ci- 6 alkyl, C3-7cycloalkyl, haloCi-ealkyl, halogen,
• R 7 is carboxy
• a further embodiment of present invention is (iii) a compound of formula (I) according to (ii), wherein
• R 1 is methyl
• R 2 is chloro or fluoro
• R 3 is H, chloro, fluoro or cyano
• R 4 is dimethylamino; dimethylamino-2,3,3a,4,6,6a-hexahydrofuro[2,3-c]pyrrolyl; methyl- 2, 3, 3a, 4,6, 6a-hexahydropyrrolo[2,3-c]pyrrolyl; methyl-2, 3, 3a, 4, 6, 6a- hexahydropyrrolo[3,4-b]pyrrolyi; methyl-2, 3, 4a, 5,7, 7a-hexahydropyrrolo[3, 4- b][l,4]oxazinyl; dimethylamino-3,3a,4,5,6,6a-hexahydro-lH-cyclopenta[c]pyrrolyl;
• R 5 is H, fluoro or methyl
• R 6 is H, (dimethylamino)methyl , methyl, cyclopropyl, trifluoromethyl, chloro,
• R 7 is carboxy
• a further embodiment of present invention is (iv) a compound of formula (I) according to (ii), wherein R 4 is 2,3,3a,4,6,6a-hexahydropyrrolo[2,3-c]pyrrolyl substituted by Ci- 6 alkyl;
• a further embodiment of present invention is (v) a compound of formula (I) according to (iv), wherein R 4 is methyl-2,3,3a,4,6,6a-hexahydropyrrolo[2,3-c]pyrrolyl; methyl-2, 3, 3a, 4, 6,6a- hexahydropyrrolo [3 ,4-b]pyrrolyl; or dimethyl amino-3 ,3a,4,5,6,6a-hexahydro- 1 H- cyclopenta[c]pyrrolyl.
• a further embodiment of present invention is (vi) a compound of formula (I) according to (iv), wherein R 5 is H.
• a further embodiment of present invention is (vii) a compound of formula (I) according to (vi), wherein R 6 is H.
• a further embodiment of present invention is (viii) a compound of formula (I) according to (ii), wherein
• R 1 is Ci- 6 alkyl
• R 2 is halogen
• R 3 is H, halogen or cyano
• R 4 is 2,3,3a,4,6,6a-hexahydropyrrolo[2,3-c]pyrrolyl substituted by Ci- 6 aikyl;
• R 5 is H
• R 6 is H
• R 7 is carboxy
• a further embodiment of present invention is (ix) a compound of formul a (I) according to (viii), wherein
• R 1 is methyl
• R 2 is chloro or fluoro
• R 3 is H, chloro, fluoro or cyano
• R 4 is methyi-2,3,3a,4,6,6a-hexahydropyrrolo[2,3-c]pyrrolyl;
• R 7 is carboxy
• the compounds of the present invention can be prepared by any conventional means. Suitable processes for synthesizing these compounds as well as their starting materials are provided in the schemes below and in the examples. All substituents, in particular, R 1 to R 7 are as defined above unless otherwise indicated. Furthermore, and unless explicitly otherwise stated, all reactions, reaction conditions, abbreviations and symbols have the meanings well known to a person of ordinary skill in organic chemistry.
• X 1 , X 2 , X 3 and X 4 are halogen.
• halogenation such as treatment of POCb or POBr 3 .
• Compound of formula (Ig) can also be obtained by additional halogenation of compound of formula (Ig) (when R 3 is H) with halogenating reagent, such as NCS, followed by di-/er/-butyl carbonate re -protection.
• X 1 , X 2 , X 3 and X 4 are halogen.
• compound of formula (Ig) can be prepared according to Scheme 2. Coupling of the compound of formula (Id) with di-halogenated pyridine can be achieved using palladium catalyst and phosphine ligands to give compound of formula (Ik). Cyclization of compound of formula (Ik) using palladium catalyst and phosphine ligands gives compound of formula (Im). Compound of formula (Im) can be subject to halogenation using halogenating reagent, such as NCS, NBS or NIS, to give compound of formula (If), which subsequently undergoes oxidation of the pyridine followed by halogenation, such as treatment of POCI3 or POBr 3 .
• halogenating reagent such as NCS, NBS or NIS
• compound of formula (If) can be obtained by treatment of compound of formula (If) (when R 3 is H) with halogenating reagent, such as NBS, to give compound of formula (If) (when R 3 is halogen, in particular bromo).
• halogenating reagent such as NBS
• compound of formula (If) can also be obtained by converting compound of formula (If) (when R 3 is halogen, in particular bromo) to compound of formula (If) (when R 3 is CN) via palladium mediated substitution or nucleophilic substitution.
• Compound of formula (Ig) can then be obtained through oxidation of the pyridine of compound of formula (If) followed by halogenation, such as treatment of POCI3 or POBrs.
• X 3 and X 4 are halogen, X 5 are halogen or OTf; Q 1 and Q 2 are boronic acids or esters.
• Compound of formula (I) can be prepared according to Scheme 3. Introducing R 4 to compound of formula (Ig) can be achieved either through nucleophilic substitution with amine and a base for certain C-N bond formation (with R 4 bearing a nucleophilic N), or a Buchwald- Hartwig Cross Coupling Reaction for certain C-N bond formation (with R 4 bearing a basic N), to give compound of formula (Ih). Further coupling of compound of formula (Ih) with compound of formula (Io) to give compound of formula (Ii) can be achieved using a palladium catalyzed Suzuki coupling. Chiral separation can be achieved on compound of formula (Ih) or compound of formula (Ii).
• Some special compound of formula (Ii) need to reverse the Suzuki coupling for C-C bond formation by converting compound of formula (Ih) to compound of formula (Ij) as a boronic ester or boronic acid then coupling with compound of formula (Ip). Ester hydrolysis such as NaOH in ethanol followed by deprotection of compound of formula (Ii) in the presence of an acid, such as trifluoroacetic acid, then affords compound of formula (I).
• This invention also relates to a process for the preparation of a compound of formula (I) comprising the reaction of compound of formula (Ii),
• an acid which can be for example trifluoroacetic acid
• R 1 to R 7 are defined above.
• a compound of formula (I) when manufactured according to the above process is also an object of the invention.
• compositions or medicaments containing the compounds of the invention and a therapeutically inert carrier, diluent or excipient, as well as methods of using the compounds of the invention to prepare such compositions and medicaments.
• compounds of formula (I) may be formulated by mixing at ambient temperature at the appropriate pH, and at the desired degree of purity, with physiologically acceptable carriers, i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed into a galenical administration form.
• the pH of the formulation depends mainly on the particular use and the concentration of compound, but preferably ranges anywhere from about 3 to about 8.
• a compound of formula (I) is formulated in an acetate buffer, at pH 5.
• the compounds of formula (I) are sterile.
• the compound may be stored, for example, as a solid or amorphous composition, as a lyophilized formulation or as an aqueous solution.
• compositions are formulated, dosed, and administered in a fashion consistent with good medical practice.
• Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
• The“effective amount” of the compound to be administered will be governed by such considerations, and is the minimum amount necessary to reduced bacterial load or improve host survival through the inhibition of bacterial DNA gyrase and/or Topoisomerase IV. For example, such amount may be below the amount that is toxic to normal cells, or the mammal as a whole.
• the pharmaceutically effective amount of the compound of the invention administered parenterally per dose will be in the range of about 0.1 to 1000 mg/kg, alternatively about 1 to 100 mg/kg of patient body weight per day.
• oral unit dosage forms such as tablets and capsules, preferably contain from about 5 to about 5000 mg of the compound of the invention.
• the compounds of the invention may be administered by any suitable means, including oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal and epidural and intranasal, and, if desired for local treatment, intralesional administration.
• Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
• the compounds of the present invention may be administered in any convenient administrative form, e.g., tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc.
• Such compositions may contain components conventional in pharmaceutical preparations, e.g., diluents, carriers, pH modifiers, sweeteners, bulking agents, and further active agents.
• a typical formulation is prepared by mixing a compound of the present invention and a carrier or excipient.
• Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, e.g., Ansel, Howard C., et al., Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004;
• the formulations may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament).
• buffers stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or aid in the manufacturing
• An example of a suitable oral dosage form is a tablet containing about 10 to 500 mg of the compound of the invention compounded with about 40 to 400mg anhydrous lactose, about 5 to 50 mg sodium croscarmellose, about 5 to 50 mg polyvinylpyrrolidone (PVP) K30, and about 1 to 10 mg magnesium stearate.
• the powdered ingredients are first mixed together and then mixed with a solution of the PVP.
• the resulting composition can be dried, granulated, mixed with the magnesium stearate and compressed to tablet form using conventional equipment.
• An example of an aerosol formulation can be prepared by dissolving the compound, for example 5 to 1000 mg) of the invention in a suitable buffer solution, e.g. a phosphate buffer, adding a tonicifier, e.g. a salt such sodium chloride, if desired.
• the solution may be filtered, e.g., using a 0.2 micron filter, to remove impurities and contaminants.
• An embodiment therefore, includes a pharmaceutical composition comprising a compound of formula (I), or a stereoisomer or pharmaceutically acceptable salt thereof.
• a pharmaceutical composition comprising a compound of formula (I), or a stereoisomer or pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier or excipient.
• Another embodiment includes a pharmaceutical composition comprising a compound of formula (I) for use in the treatment and/or prophylaxis of bacterial infections.
• the compounds of this invention may be administered, as part of a single or multiple dosage regimen, orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir.
• parenteral as used includes subcutaneous, intracutaneous, intravenous, intramuscular, intra- articular, intrasynovial, intrastemal, intrathecal, intralesional and intracranial injection or infusion techniques.
• the pharmaceutical compositions of the invention will be administered from about 1 to 5 times per day or alternatively, as a continuous infusion upon improvement of a patient’s condition.
• the compounds of the invention are useful for treatment and/or prophylaxis of bacterial infection in humans or other animals by administering to the subject in need of a therapeutically effective amount of compound of formula (I), or a pharmaceutically acceptable salt, or enantiomer or diastereomer thereof.
• the compounds and methods of the invention are particularly well suited for human patients infected by pathogens that include Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa.
• bacterial organisms that may also be controlled by the compounds of the invention include, but not limited to, the following Gram-Positive and Gram-Negative organisms: Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalis,
• Enterococcus faecium Enterobacter spp. species, Proteus spp. species, Serratia marcescens, Staphylococcus aureus, Coag. Neg. Staphylococci, Haemophilus influenzae, Bacillus anthraces, Mycoplasma pneumoniae, Moraxella catarrhalis, Chlamydophila pneumoniae, Chlamydia trachomatis, Legionella pneumophila, Mycobacterium tuberculosis, Helicobacter pylori, Staphylococcus saprophyticus, Staphylococcus epidermidis, Francisella tularensis, Yersinia pestis, Clostridium difficile, Bacteroides spp. species Neisseria gonorrhoeae, Neisseria meningitidis, Burkholderia pseudomallei, Burkholderia mallei, Borrelia burgdorferi,
• Mycobacterium avium complex Mycobacterium abscessus, Mycobacterium kansasii, E. coli and Mycobacterium ulcerans.
• bacterial infections may include, but not limited to, upper respiratory infections, lower respiratory infections, ear infections, pleuropulmonary and bronchial infections, complicated urinary tract infections, uncomplicated urinary tract infections, intra-abdominal infections, cardiovascular infections, a blood stream infection, sepsis, bacteremia, CNS infections, skin and soft tissue infections, GI infections, bone and joint infections, genital infections, eye infections, or granulomatous infections.
• specific bacterial infections include, but not limited to, uncomplicated skin and skin structure infections (uSSSI),
• cSSSI complex skin and skin structure infections
• catheter infections pharyngitis, sinusitis, otitis externa, otitis media, bronchitis, empyema, pneumonia, community-acquired bacterial pneumoniae (CABP), hospital-acquired pneumonia (HAP), hospital-acquired bacterial pneumonia, ventilator-associated pneumonia (VAP), diabetic foot infections, vancomycin resistant enterococci infections, cystitis and pyelonephritis, renal calculi, prostatitis, peritonitis, complicated intra-abdominal infections (cIAI) and other inter-abdominal infections, dialysis- associated peritonitis, visceral abscesses, endocarditis, myocarditis, pericarditis, transfusion- associated sepsis, meningitis, encephalitis, brain abscess, osteomyelitis, arthritis, genital ulcers, urethritis, vaginitis, cer
• the invention relates to the use of a compound of formula (I) for the treatment and/or prophylaxis of bacterial infection.
• the invention relates to the use of a compound of formula (I) for the preparation of a medicament for the treatment and/or prophylaxis of bacterial infection.
• Another embodiment includes a method for the treatment or prophylaxis of bacterial infection which method comprises administering an effective amount of a compound of formula (I), or pharmaceutically acceptable salt, or enantiomer or diastereomer thereof.
• CCso concentration results in the death of 50 percent of the cells
• HATXJ 1 - [Bis(dimethylamino)methylene] - 1 H- 1 ,2,3-triazolo[4,5-b]pyridinium 3- oxid hexafluorophosphate
• HPLC-UV high performance liquid chromatography with ultraviolet detector
• Acidic condition A: 0.1% formic acid and 1% acetonitrile in H 2 0; B: 0.1% formic acid in acetonitrile;
• Mass spectra generally only ions which indicate the parent mass are reported, and unless otherwise stated the mass ion quoted is the positive mass ion (M+H) + .
• the titled compound was synthesized according to the following scheme:
• the reaction mixture was stirred at 160 °C for 6 h before it was cooled to room temperature and poured into water (100 mL). Then the mixture was extracted with EtOAc (200 mL) and the organic layer was collected and washed with water (50 mL) two times and brine (30 mL) two times, dried over anhy. sodium sulfate. The separated organic layer was concentrated in vacuo and the residue was purified by flash chromatography (silica gel,
• the titled compound was synthesized according to the following scheme:
• the titled compound was synthesized according to the following scheme:
• the titled compound was synthesized according to the following scheme:
• the titled compound was synthesized according to the following scheme:
• the titled compound was synthesized according to the following scheme:
• Step (c) Preparation of ethyl 4-oxo-7-(trifluoromethylsulfonyloxy)quinolizine-3-carboxylate (compound BE 8)
• ethyl 7 -hydroxy-4-oxo-quinolizine-3 -carboxylate_(4.0 g, 17.15 mmol, compound B1.7) in DCM (64 mL) was added triethylamine (3.47 g, 34.3 mmol) and NN- bis(trifluoromethylsulfonyl)aniline (12.25 g, 34.3 mmol) slowly at 0 °C.
• the titled compound was synthesized according to the following scheme:
• the titled compound was synthesized according to the following scheme:
• the titled compound was synthesized according to the following scheme:
• the titled compound was synthesized according to the following scheme:
• the titled compound was synthesized according to the following scheme:
• Step fa) Preparation of ethyl -7-bromo-l-iodo-4-oxo-quinolizine-3 -carboxyl ate (compound B 6.2)
• ethyl 7-bromo-4-oxo-4H-quinolizine-3-carboxylate (1.00 g, 3.38 mmol, compound B 6.1)
• DMF 15 mL
• N -iodosuccinimide (1.90 g, 8.44 mmol.
• the reaction mixture was stirred at 40 °C for 2 h before it was poured into 10% aq. Na 2 S0 3 solution (150 mL) and stirred for additional 30 min..
• the titled compound was synthesized according to the following scheme:
• the titled compound was synthesized according to the following scheme:
• Step (b) Preparation of - cA-N,N-dimethyl-l,2.3,3a,4.5,6.6a-octahvdrocvclopenta[c1pyrrol-
• the titled compound was synthesized according to the following scheme:
• the titled compound was synthesized according to the following scheme:
• the titled compound was synthesized according to the following scheme:
• the titled compound was synthesized according to the following scheme:
• the titled compound was synthesized according to the following scheme:
• Step (b) Preparation of ethyl 7-(8-((ter/-butoxycarbonyl fmethyl ammo -4-(dimethylamino -5.6- difluoro-9H-pyrido[2.3-blmdol-3-yl -4-oxo-4H-qumolizine-3-carboxylate
• Step (a) Preparation of fer/-butvh3-bromo-5.6-difluoro-4-(cA-5-methyl-2.3,3aA6,6a- hexahvdropyrrolo[2.3-clpyrrol-l-yl)-9H-pYridoi2.3-blindol-8-yl)(methyl)carbamate
• reaction mixture was stirred at 25 °C for 3 hr before 2-isopropoxy-4,4,5 ,5 -tetramethyl- 1 ,3 ,2- dioxaborolane (162.3 mg, 0.872 mmol) was added and the reaction mixture was stirred at 25 °C for another 1 hr.
• the mixture was then poured into aq. NH 4 Cl solution (50 mL) and extracted by EtOAc (100 mL) two times.
• the combined organic layer was washed by aq. NH 4 CI solution (50 mL), brine (50 ml) two times, dried over anhy.
• the reaction mixture was then poured into water (50 mL), adjusted to pH ⁇ 7 by aq. HC1 solution (0.1 M), and extracted with EtOAc (50 mL). The organic layer was washed with aq. CaCl 2 solution (100 mL, 1 N) two times and brine (100 mL) two times. The organic layer was then dried over anhy.
• Step fa) Preparation of cis-tert- butyl N-G4-G1 -methyl-23.3a,4.6.6a-hexahydropYrrolo [3,4- blp Y rrol-5-yl1-3-bromo-6-fluoro-9H-pyridoi2.3-b1indol-8-yll-N -methyl- carbamate
• Step (b) Preparation of c/.s-i4-il-methyl-2,3,3a,4,6.6a-hexahydropyrroloi3,4-blpyrrol-5-yll-8- iter/-butoxycarbonylfmethyl)aminol-6-fluoro-9H-pYrido i2.3-blindol-3-yliboronic acid
• Step (c) Preparation of cA-ethyl 7-i8-iter/-butoxycarbonyl-fmethyl)amino]-6-fluoro-4-fl- methyl-2, 3,3a,4.6.6a-hexahvdropyrrolo[3,4-b]pyrrol-5-yl)-9H-pyridoi2,3-blindol-3-yl1-4-oxo- quinolizine-3 -carboxylate
• Step (e) Preparation of 7-!4-[(3ai?,6ai? -l-methyl-2,3,3a,4,6,6a-hexahvdropyrroloi3,4-blpyrrol-5- yll-8-rter/-butoxycarbonyl(methyl amino1-6-fluoro-9H-pyridoi2.3-b1mdol-3-vH-4-oxo- quinolizine-3 -carboxylic acid
• Step (f) Preparation of 7-i4-i(3ai?.6ai? -l-methyl-2.3,3a,4,6,6a-hexahvdropyrrolor3,4-b1pyrrol-5- vH-6-fluoro-8-(methylamino -9H-pyridoi2,3-b1mdol-3-vH-4-oxo-quinolizine-3-carboxylic acid (Example 3.01 A) To a mixture of 7-[4-[(3ai?,6ai?)-l-methyl-2,3,3a,4,6,6a-hexahydropyrrolo[3,4-b]pyrrol-5- yl]-8-[/er/-butoxycarbonyl(methyl)amino]-6-fluoro-9H-pyrido[2,3-b]indol-3-yl]-4-oxo- quinolizine-3 -carboxylic acid (55 mg, 0.09 mmol) in DCM (5
• Step (b) Preparation of /ert-butyl (4-(Yl£,3i?)-l-(Ytert-butoxycarbonyl)aminoV 5- azaspiroi2.41heptan-5-yl)-3-chloro-5.6-difluoro-9H-pyridor2.3-b1indol-8-yl) carbamate
• the resulting reaction mixture was degassed with N 2 five times and then stirred at 70 °C for 2 hr under N 2 . After it was cooled down to r.l, the mixture was diluted with EtOAc (100 mL), poured into water (100 mL), and extracted with EtOAc (100 mL) two times.
• Step (d) Preparation of 7-(8-((fert-butoxycarbonyl)(methyl)amino)-4-((l l S.5i?)-l- ((tert- butoxycarbonyl)amino)-5-azaspiro[2.41heptan-5-yl)-5,6-difluoro-9H-pyridoi2,3-b1indol-3-yl)-4- oxo-4H-quinolizine-3 -carboxylic acid
• Step (e) Preparation of 7-(4-(fl t S , ,3i? -l-amino-5-azaspiro[2.41heptan-5-yl - 5 uncomfortable6-difluoro-8- (methylaminoV9H-pyrido[2.3-b]indol-3-ylV4-oxo-4H-quinolizine-3-carboxylic acid
• Step (a) Preparation of cis-tert- butyl (3 -bromo-4-( 1 -((tert-butoxycarbonvOamino - 5 - azaspiro[2.4]heptan-5-vD -5-chloro-6-fluoro-9H-pyridol2.3-blindol-8-yl fmethyl carbamate and trans-tert- butyl 3 -bromo-4-( 1 -(Ytert-butoxycarbonyl)aminoV5 -azaspiro G 2.41heptan-5-vD-5-
• Step (b) Preparation of fer/-butyl (3-bromo-4-((l A37? - 1 -(Yfert-butoxycarbonyl amino) -5- azaspiroi2.4iheptan-5-yl -5-chloro-6-fluoro-9H-pyridoi2,3-b1indol-8-yl) carbamate and fe/7-butyl (3-bromo-4-((li?.3 > y)-l-((ter/-butoxycarbonyl amino -5 -azaspiro G 2 41heptan-5 -vD-5 - chloro-6-fluoro-9H-pyridoi2.3-b1mdol-8-yl carbamate and fer -butyl (3-bromo-4-
• Step id) Preparation of 7-(8-((ter/-butoxycarbonyl (methyl amino -4-((l5,3i? -l-((ter/- butoxycarbonyl amino -5-azaspiror2.41heptan-5-yl -5-chloro-6-fluoro-9H-pyrido[2.3-b1indol-3- vD-4-oxo-4H-quinolizine-3-carboxylic acid
• the assay used a 10-points Iso-Sensitest broth medium to measure quantitatively the in vitro activity of the compounds against S. aureus ATCC29213, K. pneumoniae ATCC 10031, and A. baumannii ATCC17978.
• Stock compounds in DMSO were serially two-fold diluted (range from 50 to 0.097 mM final concentration) in 384 wells microtiter plates and inoculated with 49 pL the bacterial suspension in Iso-Sensitest broth medium to have a final cell concentration of ⁇ 5 x 10 5 CFU/mL in a final volume/well of 50 pL/well.
• Microtiter plates were incubated at 35 ⁇ 2 °C.
• GP-6 (Example 4.131 disclosed in WO 2012/125746 Al) was used as the reference compound in Table 6-8.
• Table 6 IC 50 values of the compounds of this invention against S. aureus, K. pneumoniae and baumannii
• HepG2 assay The cytotoxicity of compounds of present invention was assessed by HepG2 assay.
• HepG2 cells ATCC HB-8065TM
• 20pL of Cell titer blue buffer were added to each well of the cells, which were incubated for 3 hours at 37°C, and the fluorescence value was measured by a plate reader (Molecular Device SpectraMax M2).
• the CCso value of each compound is plotted with GraphPad Prism using four parameter logistic equation. Results of cytotoxicity are given in Table 8.
• SDPK Single dose PK study
• the single dose PK in female CD-l mouse was performed to assess compound’s pharmacokinetic properties.
• One group of animals were dosed via bolus intravenous (IV) of the respective compound.
• Blood samples (approximately 30 mT) were collected via saphenous vein at 5 mins, 15 min, 30 min, lhr, 2hr, 4hr and 7hr. For last time point (24hr), samples were collected via cardiac puncture while the mouse was under anesthesia. Blood samples were placed into tubes containing EDTA-K2 anticoagulant (2 pL of 0.5M per tube) and centrifuged at 3000 rpm for 10 minutes at 4°C within half an hour to separate plasma from the samples.
• EDTA-K2 anticoagulant 2 pL of 0.5M per tube
• MIC Minimal Inhibitory Concentration: The MIC of antibacterial compounds against multiple species and strains was determined using the broth microdilution method according to M07-A10 Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI (2015) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved Standard, Tenth Edition. CLSI Document M7-A10 (ISBN 1-56238-987-4). Wayne, PA: Clinical and Laboratory Standards Institute, 19087, USA.) . In short, bacterial strain preparation was performed on Dl and D2.
• the bacterial strains were revived from storage frozen two days before the MIC screening, and streaked onto surface of agar plates, incubated in an ambient-air incubator with humidity for 20-24 hr at 35 ⁇ 2°C. 5-10 well-isolated colonies of similar morphology were selected and re-streaked onto fresh agar plates using sterile loops and incubated for 20-24 hr at 35 ⁇ 2°C.. Bacterial strains were subcultured two times to ensure fresh inoculums for MIC screening.
• Bacterial inoculum was diluted by 1 :280 for Gram-positive strains and 1 :400 for Gram-negative strains into corresponding medium broth (CAMHB, CAMHB+3% lysed horse blood, HTM) (e.g. 35.6 pL of inoculum into 10 mL of CAMHB or 25 pL of inoculum into 10 mL of CAMHB).
• HTM medium broth
• Lor compound plate the compound stock solutions were prepared in 100% DMSO on the day of MIC screening and used immediately.
• Compound stock concentration [(highest testing concentration) x 100 pL / 2 pL] (e.g.
• the compound plates were prepared by dispensing lOOpL of each stock solution in the first well of a 96 well microtiter plate (MTP) at a concentration lOO-fold higher than the final concentration desired in broth. Eleven serial 2-fold dilutions of the highest concentration were made in DMSO for new compounds and for reference compounds (GP-6 and ciprofloxacin). lpL of each well was transferred in a new MTP, which served as the test plate by subsequent inoculation. The bacterial suspension prepared above is dispensed at 98pL/well within 15 minutes after preparation.
• Negative controls (lack of bacterial cells) and growth control wells (lack of compound) were included in all plates. MTPs were incubated for 18-24 hours at 35 ⁇ 2°C in ambient air. The MIC was recorded and CPUs were calculated on Day 4. The assay plate was placed on the top of MIC reader, and the magnification mirror was adjusted to read each wells, recording growth status as raw data. Photo images of each assay plates were recorded using Q Count 530(Spiral Biotech). . The MIC value of each compound, expressed as pg/mL, was determined as the lowest concentration required for complete growth inhibition (no visible growth). Results of MIC were summarized in Table 10.
• Cip Ciprofloxacin

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