Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/06—Phosphorus compounds without P—C bonds
  • C07F9/08—Esters of oxyacids of phosphorus
  • C07F9/09—Esters of phosphoric acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/66—Phosphorus compounds
  • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • 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
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/06—Phosphorus compounds without P—C bonds
  • C07F9/08—Esters of oxyacids of phosphorus
  • C07F9/09—Esters of phosphoric acids
  • C07F9/091—Esters of phosphoric acids with hydroxyalkyl compounds with further substituents on alkyl
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/28—Phosphorus compounds with one or more P—C bonds
  • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the invention relates to new antibacterial compounds, their preparation and intermediates, their use as drugs and pharmaceutical compositions containing them.
• the invention relates to new compounds capable of inhibiting bacterial heptose biosynthesis and thereby lowering or suppressing bacterial virulence, as well as their antibacterial pharmaceutical applications in preventive or curative treatment or in combination therapy.
• the invention particularly relates to new compounds capable of inhibiting the GmhA enzyme of bacterial heptose synthesis, thereby lowering or suppressing bacterial virulence, as well as their antibacterial pharmaceutical applications .
• the lipopolysaccharide is a major component of the outer membrane of Gram-negative bacteria. It is composed of three regions: the lipid A, the core oligosaccharide and the 0 antigen.
• the core oligosaccharide is divided into the inner core and the outer core.
• the inner core consists in a motif of five sugars: two Kdo (Kdo: 3-deoxy-D-manno-octulosonic acid) and three successive heptoses.
• the first heptose transfer is catalysed by the Heptosyltransferase I (protein WaaC) and the second heptose transfer by the Heptosyltransferase II (protein WaaF) .
• ADP heptose The natural donor substrate of these transferases is ADP heptose, which is synthesized in bacteria from sedoheptulose-7-phosphate by the successive enzymatic steps catalyzed by the following enzymes: GmhA, HldE-K (former or other nomenclature: RfaE-K) , GmhB, HldE-AT (former or other nomenclature: RfaE-AT) and HldD (former or other nomenclature: RfaD, WaaD) (Journal of Bacteriology, 2002, 184, 363) . Heptose synthetic pathway is conserved among Gram negative bacterial species and is necessary for full LPS synthesis.
• inhibitors of bacterial GmhA By preventing full LPS development in Gram negative bacteria, inhibitors of bacterial GmhA would be expected to induce a high sensitivity to the host complement and therefore be able to prevent or inhibit bacterial infection. Such inhibitors would provide a novel way to treat or prevent bloodstream infections caused by pathogenic Gram negative bacteria, without affecting the commensal flora and with less selective pressure than conventional antibacterial agents.
• the invention relates to new compounds having the general formula (I)
• Ai and A2 identical or different, are H, (Ci-Ce) alkyl, (C1-C6) fluoroalkyl, (C2-C6) alkenyl, (C 2 -C 6 ) alkynyl, (Ci-C 6 ) alkyl-ORa, (Ci-C 6 ) alkyl-SR a ,
• - A3 is H, OH or form a carbonyl with A4;
• - A4 is H, OH or form a carbonyl with A3;
• - A5 is H, CR a RbOH, F , OH or forms a double bond with X in the case where X is CH;
• - A is H or F
• - X is CH 2 , CHF, CF 2 , CHOH, 0, S, NR a or a simple bond, or X is CH in the case where A5 forms with X a double bond;
• - Y is P(O) (ORa) (ORb) or P (0) (OR a ) (NR a R b ) ;
• - V is O or S
• a 7 is H, (Ci-C 6 ) alkyl, (Ci-Ce) fluoroalkyl ,
• R a and Rb are selected from the group consisting of H, (C1-C6) alkyl ,
• R a and R hereabove and hereafter in the processes, can be identical or different when they both appear in the definition of substituents Ai, A2, A5, A7, X and Y taken individually as well as when they appear in the definition of these substituents and of leaving groups, with respect to each other.
• the hydroxy substituent indicated in undetermined position can be in R, S or RS configuration and therefore the invention extends to the compounds of formula (I) in the form of pure diastereoisomers and mixtures of diastereoisomers .
• acid salts of the products of formula (I) there may be cited, among others, those formed with mineral acids, such as hydrochloric, hydrobromic, hydroiodic, sulfuric or phosphoric acid or with organic acids such as formic, acetic, trifluoroacetic, propionic, benzoic, maleic, fumaric, succinic, tartaric, citric, oxalic, glyoxylic, aspartic, alkanesulfonic acids, such as methanesulfonic and ethanesulfonic acids, arylsulfonic acids such as benzenesulfonic and para-toluenesulfonic acids.
• mineral acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric or phosphoric acid
• organic acids such as formic, acetic, trifluoroacetic, propionic, benzoic, maleic, fumaric, succinic, tartaric, citric, oxalic, gly
• alkaline salts of the products of formula (I) there may be cited, among others, those formed with mineral alkalis such as, for example, sodium, potassium, lithium, calcium, magnesium or ammonium or organic bases such as, for example, methylamine, ethylamine, propylamine, trimethylamine, diethylamine, triethylamine, N,N- dimethylethanolamine, tris (hydroxymethyl) aminomethane, ethanolamine, pyridine, piperidine, piperazine, picoline, dicyclohexylamine, morpholine, benzylamine, procaine, lysine, arginine, histidine, N-methylglucamine .
• mineral alkalis such as, for example, sodium, potassium, lithium, calcium, magnesium or ammonium or organic bases
• organic bases such as, for example, methylamine, ethylamine, propylamine, trimethylamine, diethylamine, triethylamine, N,N- dimethylethanol
• (C1-C6) alkyl means any linear, branched, mono or bicyclic hydrocarbon groups comprising 1 (or 3 for a cycle) to 6 carbon atoms, preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl, n-pentyl, isopentyl, neopentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [ 3.1.0 ] hexane, bicyclo [2.2.0 ] hexane, spiro [ 2.2 ] pentane or spiro [ 2.3 ] hexane, or means an alkyl chain including or substituted by a small cycloalkyl, itself possibly substituted by an alkyl, or means a small cycloalkyl substituted by alkyl;
• (C2-C6) alkenyl and “ (C2-C6) alkynyl as applied herein means any linear, branched or cyclic hydrocarbon groups of 2 to 6 carbon atoms, having at least one double bond or one triple bond and preferably ethenyl, propenyl, butenyl, cyclohexenyl , ethynyl, propargyl or butynyl .
• (C1-C6) fluoroalkyl as applied herein means any mono or polyfluoro linear, branched or cyclic alkyl and preferably mono, di or trifluoalkyl .
• cyclic hemiketal or cyclic hemiacetal forms is understood all possible lactol forms existing in equilibrium with the open forms, for compounds bearing carbonyl and hydroxyl groups susceptible of reacting intramolecularly as commonly observed in carbohydrate chemistry (see for example Monosaccharides: Their chemistry and their roles in natural products. P. Collins and R. Ferrier 1995 John Wiley & Sons or Carbohydrate Chemistry B.G. Davis and A.J. Fairbanks 2002 Oxford University Press) .
• Y is P (0) (OH) 2.
• X is C3 ⁇ 4, CHF, CF 2 , CHOH or 0.
• W is Wi as defined above.
• Ai and A2 are H or one of them is H and the other is (C1-C6) alkyl , or one of them is H and the other is fluoro (C1-C6) alkyl , or one of them is H and the other is (C1-C6) alkyl-OR a , or one of them is H and the other is (C1-C6) alkyl-SR a , or one of them is H and the other is COR a , a being as defined above, or one of them is H and the other is OH.
• A5 when A3 forms a carbonyl with A4, A5 is OH or CR a RbOH, R a and Rb being as defined above, or when A3 doesn't form a carbonyl with A4, A5 is H or F.
• A7 is H or (Ci-C 6 ) alkyl .
• the compounds of formula I may be prepared by any processes known to be applicable to the preparation of chemically related compounds (for non-limiting examples see in particular: Chem. Rev. 2006, 106, 3868; Tetrahedron 1997, 53, 16609; J. Med. Chem. 2010, 53, 5342; J. Med. Chem. 2010, 53, 7836) . Such processes may use known starting materials or intermediates which may be obtained by standard procedures of organic chemistry. The following processes provide a variety of non-limiting routes for the production of the compounds of formula (I) and their intermediates.
• Examples of processes to prepare compounds of formula (I) and salts thereof include in non-limiting manner the transformation into compounds of formula (I) of compounds of formula (II):
• PG is an appropriate protecting group
• PG include optionally substituted alkyl, aryl, aralkyl and silyl, in particular methyl, ethyl, phenyl, benzyl, para-methoxybenzyl , trimethylsilyl , triphenylsilyl , tertbutyldimethylsilyl or tertbutyldiphenylsilyl , acyl, in particular acetyl or benzoyl, benzyloxycarbonyl and supported polymer resin)
• Qi is H or OG;
• R a , b groups on Ai, A 2 , As , A 7 , X or Y groups are performed by known alkylation, acylation, alkoxylation, sulfenylation or amination reactions.
• LGi is an appropriate leaving group (non-limiting examples of LGi include hydroxyl, OP (0) (OR a ) 2, NR a Rb, OR a or halogen) and when appropriate X, Y, Ai, A2, A3, A4, A5, ⁇ , A 7 , V, Wi and W2 are optionally protected by one or several identical or different protecting groups PG defined as above.
• Non-limiting examples include phosphorylation with P(ORa) (ORb)LGi, P (ORa) (NR a Rb) LGi or (R a O) (R b O) P (0) -LGi, such as nucleophilic substitution in case LGi is halogen or diisopropylamine, or Mitsunobu reaction in case when LGi is hydroxy.
• phosphites synthesis with P (ORa) (ORb) LGi or P (OR a ) (NR a Rb) LGi subsequent oxidation to phosphates is performed (non-limiting example includes mCPBA or DDQ oxidation of phosphite to phosphate derivatives) .
• Ai, A 2 , A3, A 4 , A 5 , A 6 , A 7 , V, R a , Rb, Wi and W 2 , G and Qi are defined as above and when appropriate are optionally protected by one or several identical or different protecting groups PG, PG is as defined above, X is C3 ⁇ 4, CHF or CF2 and LG2 is an appropriate leaving group (non-limiting examples include 0R a , NR a Rb, halogen, alkyl/fluoroalkylsulfonyloxy arylsulfonyloxy, thioalkyl/aryl, diazonium, fluorosulfonyloxy, trialkylammonium, O-acyl and phosphonium) .
• Non-limiting examples of above reactions with compounds (IV) and (V) includes Arbuzov reaction of halides with trialkyl phosphites, base-catalyzed reactions between halides or aldehydes and H-phosphonate diesters with potassium hexamethylsilazane or sodium hydride (US4, 693, 742, DE2733658, Tet. Lett. 1993, 34, 8543); the addition of phosphites to aldehydes and aldimines (J. Am. Chem. Soc. 2008, 130, 10521) .
• Y, X, Ai, A 2 , A 3 , A 4 , A 6 , A 7 , V, R a , R b , Wi, W 2 , G and Qi are defined as above and when appropriate are optionally protected by one or several identical or different protecting groups PG, PG is as defined above and LG2 is an appropriate leaving group (non-limiting examples include OR a , NR a R b , halogen, alkyl/fluoroalkylsulfonyloxy arylsulfonyloxy, thioalkyl/aryl, diazonium, fluorosulfonyloxy, trialkylammonium, O-acyl and phosphonium) .
• Non-limiting examples of above reactions with compounds (VI) and (VII) include methylphosphonylation, fluoromethylphosphonylation or difluoromethylphosphonylation in the presence of bases such as BuLi or LDA, Arbuzov reaction of halides with trialkyl phosphites, base-catalyzed reactions between halides or aldehydes and H-phosphonate diesters with potassium hexamethylsilazane or sodium hydride (US 4,693,742, DE2733658, Tet. Lett. 1993, 34, 8543), addition of phosphites to aldehydes and aldimines (for example can be cited J. Am. Chem. Soc, 2008, 130, 10521) .
• Zi is defined as Y or G, and X, Y, Ai, A 2 , A3, A4, A5, ⁇ , A7, V, G and Qi are as above defined and when appropriate all of them are optionally protected by one or several identical or different protecting group PG, PG is as defined above, LG3 is an appropriate leaving group typically used to activate carbonyl group towards acylation (non-limiting examples include OR a , NR a Rb, halogen, O-acyl, uronium, phosphonium, imidazolium, succinimide-N-oxy, phtalimide-N-oxy, pentafluorophenyloxy) (Bioorg. Med. Chem. 1996, 6, 2077) .
• Zi is defined as Y or G, and Ai, A 2 , A3, A4, A5, Ae, X, Y, LG2, G and Qi are as above defined and when appropriate all of them are optionally protected by one or several identical or different protecting group PG, PG is as defined above.
• Non-limiting examples of above reactions with compounds (IX) and (X) include:
• Ai, A2, A3, A4, A5, ⁇ , A 7 , V, X, Zi and LG2, G and Qi are as defined above, when appropriate all of them are optionally protected by one or several identical or different protecting group PG, PG is as defined above.
• Non-limiting examples of above reactions with compounds (IX) include :
• substitution-type reactions of compound of formula (IX) with NH(CVA7)-OPG in the presence of base such as potassium carbonate (Tet. Lett. 2004, 45, 491) or solid-state synthesis with N-acyl Wang-O-hydroxylamine resin in the presence of a base such as 1,8- diazabicyclo [ 5.4.0 ] undec-7-ene (Org. Lett.
• HNA7-OG optionally in the presence of appropriate base
• suitable base include 4- dimethylaminopyridine, triethylamine, lithium hexamethyldisilazane
• reagents typically used to activate carbonyl group towards acylations include benzotriazol-l-ol (HOBT) , 1,1'- carbonyldiimidazole (CDI), dicyclohexylcarbodiimide (DCI), 2- (lH-7-azabenzotriazol-l-yl) --1, 1, 3, 3-tetramethyl uronium hexafluorophosphate methanaminium (HATU) , 1-ethyl- (3- (3- dimethylamino) propyl) -carbodiimide hydrochloride (EDAC) ) , followed as appropriate by one or more reactions including those listed above in the context of the transformation of compounds of formula (II) into compounds of formula (I) .
• HOBT 1,1'- carbonyldi
• Ai , A 2 , A3, A4, A5, ⁇ , A 7 , V, LG3, G and Qi , Z i and X are as above defined, and when appropriate all of them are optionally protected by one or several identical or different protecting group PG, PG is as defined above.
• LG3 can be an oxygen atom which belongs to Ai , A 2 , A3, A4 or A5, or the oxygen of either group OG of the compound of formula (XI), in which case G is no longer present on the group involved, so to form a lactone, which will be substituted in a similar way in the coupling with HNA7-OG.
• An illustration of such a lactone intermediate form is provided hereafter in the experimental part.
• references illustrating reactions as performed above with the compounds of formula (XI) include inter alia the following: Eur. J. Med. Chem. 2012, 51, 277; WO2011/045703; J. Med. Chem. 2011, 54, 6796; J. Med. Chem. 2012, 55, 6566; J. Org. Chem. 2010, 75, 3203.
• Ai, A2, A 4 , A5, ⁇ , A 7 , V, X, Zi, G and Qi are as above defined and when appropriate all of them are optionally protected by one or several identical or different protecting group PG, PG is as defined above.
• Non-limiting example includes reaction of epoxide opening with NH2-OG in the presence of base such as triethylamine (Tetrahedron: Asymmetry, 2004, 15, 3201) .
• X, Y, Ai, A 2 , A 3 , A 4 , A 5 , A 6 , A 7 , V, R a , Wi, W 2 , G and Qi are defined as above, and when appropriate all of them are optionally protected by one or several identical or different protecting groups PG, PG is as defined above.
• Non-limiting examples include methylphosphonylation, fluoromethylphosphonylation or difluoromethyl-phosphonylation with bases such as BuLi or LDA with optional presence of boron trifluoride diethyletherate (J. Med Chem. 2006, 49, 5309; J. Org. Chem. 1993, 58, 5779), phosphorylation with phosphoric acid with optional presence of Cul (US6949528) .
• Ai, A 2 , A3, A4, ⁇ , A 7 , V, R a , Rb, Wi, W 2 , G and Qi are defined as above, and when appropriate are optionally protected by one or several identical or different protecting groups PG, PG is as defined above, and X is C3 ⁇ 4, CHF, CF2.
• Non-limiting example includes base-catalyzed phosphonylation reactions between epoxydes and H-phosphonate diesters with BuLi as an example of base (Org. Lett. 2010, 12, 2302) .
• A3, A4, A5, ⁇ , A 7 , V, X, Zi, LG3, G and Qi are as above defined, when appropriate all of them are optionally protected by one or several identical or different protecting group PG, PG is as defined above.
• Non-limiting example includes reductive amination with protected hydroxylamine (Tet. Lett. 1998, 39, 2571) and reducing agents such as sodium cyanoborohydride or tris (acetoxy) borohydride) .
• reducing agents such as sodium cyanoborohydride or tris (acetoxy) borohydride
• Compounds of formula (I) are capable of inhibiting bacterial heptose synthesis which makes them useful as drugs for preventing or treating bacterial infections and another object of the invention is the use of the compounds of formula (I) as drugs, and in particular for the prevention and therapeutical treatment of severe infections due to Gram- negative bacteria able to disseminate in blood such as the non-limiting following species (spp.): Escherichia coli, Enterobacter r Salmonella , Shigella , Pseudomonas ,
• Burkholderia Acinetobacter, Neisseria , Klebsiella , Serratia , Citrobacter, Proteus , Yersinia , Haemophilus , Legionella , Moraxella and Helicobacter pylori.
• the invention also relates to pharmaceutical compositions comprising an effective amount of at least one compound of formula (I) such as above defined, in association with a pharmaceutically acceptable carrier.
• Said pharmaceutical compositions are advantageously formulated to be administered under topic, oral, parenteral, and preferably injectable routes, with individual doses appropriate for the patient to be treated.
• compositions according to the invention can be solid or liquid and be present in the pharmaceutical forms commonly used in human medicine, such as for example, plain or sugar- coated tablets, gelatin capsules, granules, suppositories, inhalation spray, injectable preparations, ointments, creams, gels; they are prepared according to the customary methods.
• the active ingredient ( s ) can be incorporated in same, using excipients which are customarily used in these pharmaceutical compositions, such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous vehicles, fatty substances of animal or vegetable origin, paraffin derivatives, glycols, various wetting agents, dispersants or emulsifiers, preservatives.
• excipients which are customarily used in these pharmaceutical compositions, such as talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous vehicles, fatty substances of animal or vegetable origin, paraffin derivatives, glycols, various wetting agents, dispersants or emulsifiers, preservatives.
• compositions can in particular be present in the form of a powder intended to be dissolved extemporaneously in an appropriate vehicle, for example, non-pyrogenic sterile water .
• the dose administered varies according to the condition treated, the patient in question, the administration route and the product envisaged. It can, for example, be comprised between 0.01 g and 10 g per day, by oral route in humans or by intramuscular or intravenous route.
• the drugs according to the invention can also be used, for the prevention as for the treatment of bacterial infections, in the form of associations with ant ibacterials or antivirulence agents or drugs reinforcing the host innate immunity.
• ant ibacterials or antivirulence agents or drugs reinforcing the host innate immunity are associated with lipophilic compounds that have difficulties crossing the full hydrophilic LPS membrane, such as macrolides, streptogramins , pleuromutilins , Fabl inhibitors, rifamycins and lipopeptides .
• GM-CSF Granulocyte macrophage colony- stimulating factor
• a further object of the invention is therefore the associations of the compounds of formula (I) with antibacterials and/or antivirulence agents and/or drugs reinforcing the host innate immunity and, in particular, with antibacterials such as macrolides, streptogramins , pleuromutilins , Fabl inhibitors, rifamycins, lipopeptides or with GM-CSF.
• a further object of the invention is the pharmaceutical compositions containing the above associations with a pharmaceutically acceptable carrier.
• compositions are advantageously formulated as described above.
• the doses administered vary according to the condition treated, the patient in question, the administration route and the associated active principles envisaged.
• the dose for the compound of formula (I) is the one indicated above and the dose for the associated active principle is the dose normally prescribed for such compound.
• the compound of formula (I) can be administered in association with Erythromycin at doses of 250 to 500mg every 6 hours in human (oral administration) or lg to 4g per day in human in divided doses every 6 hours (intravenous administration) or by continuous infusion.
• Figure 1 provides positive and negative controls obtained with a gel electrophoresis of (1) LPS of E. coli C7-AgmhA and (2) LPS of E. coli C7 wild type.
• CDCI3 is deuteriochloroform
• DMSO-d6 is hexadeuteriodimethylsulfoxide
• CD3OD is tetradeuteriomethanol .
• Mass spectra were obtained using electrospray ionization (ESI) techniques on an Agilent 1100 Series LCMS and 2795 Alliance Waters LCMS .
• Analtech Silica Gel GF and E. Merck Silica Gel 60 F-254 thin layer plates were used for thin layer chromatography. Flash chromatography was carried out on Flashsmart Pack cartridge irregular silica 40-60ym or spherical silica 20-40ym. Preparative thin layer chromatography was carried out on Analtech Silica Gel GF 1000 ym 20x20 cm.
• ESI electrospray ionization
• HPLC high pressure liquid chromatography
• LCMS liquid chromatography coupled with a mass spectrometer
• M in the context of mass spectrometry refers to the molecular weight
• MS mass spectrometry
• ESI or ES electrospray ionization
• APCI atmospheric-pressure chemical ionization
• HRMS high resolution mass spectrometry
• NMR nuclear magnetic resonance
• pH refers to potential of hydrogen
• TFA trifluoroacetic acid
• TEA triethylamine
• DIPEA triethylamine
• DIPEA triethylamine
• DIPEA triethylamine
• DIPEA triethylamine
• DIPEA triethylamine
• DIPEA triethylamine
• DIPEA triethylamine
• DIPEA triethylamine
• DIPEA triethylamine
• DIPEA triethyl
• Step 1 1 , 2 , 3 , 4 , 6-Penta-O-acetyl-D-altropyranose
• Step 2 Phenyl 2 , 3 , 4 , 6-tetra-O-acetyl-l-thio-a-D- altropyranoside (lb)
• the reaction mixture was allowed to warm at room temperature and it was stirred overnight, whereupon more thiophenol (8.8 mmol, 0.9 mL) and BF3.Et2 ⁇ 0 (0.04 mmol, 5.1 mL) were added. The mixture was stirred for more 4 h. Then it was diluted with dichloromethane, cooled to 0 °C and it was washed with a saturated solution of aqueous hydrogenocarbonate. The aqueous phase was extracted with dichloromethane. The combined organic phases were then washed with water and dried with magnesium sulfate.
• the crude was purified by column chromatography on silica gel (hexane/EtOAc, from 7:3 to 3:2) to afford the phenyl thioglycoside as a colorless oil (4, 82%, ratio - pyr/ ⁇ -pyr . /fur . , 1:0.1:0.15), along with recovered starting material (244 mg, 6%) .
• Step 4 Phenyl l-thio- 6-O-triisopropylsilyl-a-D- altropyranoside (Id)
• Step 5 Phenyl 2 , 3 , 4-tri-0-benzyl-l-thio-6-0- triisopropylsilyl-a-D-altropyranoside (le)
• Step 6 Phenyl 2 , 3 , 4-tri-O-benzyl-l-thio-a-D-altropyranoside (If)
• Step 8 2 , 3 , 4-Tri-0-benzyl-6-0- [bis (phenoxy) phosphoryl] -D- ltropyranose (lh)
• Step 9 2 , 3 , 4-Tri-0-benzyl-6-0- [bis (phenoxy) phosphoryl] -D- altrono-5-lactone (li)
• the catalyst was filtered off and the eluent was concentrated in vaccum. The residue was dissolved in water and washed with diethyl ether. The aqueous phase was lyophilized to give a white hygroscopic solid which was then dissolved in water (HPLC grade) and subjected to gel filtration using a PD-10 Sephadex G 25 column (water as eluent) . The eluent was lyophilized to afford the title compound (32.6 mg, 90%) .
• Step 11 D-altronohydroxamic acid 6- (dihydrogen phosphate) (lk)
• Example 2 iV-hydroxy-iV-formylamino-l-deoxy-D-ribitol-5- phosphate (monosodium salt) Step 1: Triisopropyl- ( (2R,3R,4R) -2 , 3 , 4-tris-benzyloxy-5 , 5- bis-eth lsulfanyl-pentyloxy) -silane (2a)
• ethylsulfanyl-pentyloxy) -silane (356 mg, 0.52 mmol) in a mixture of 5:1 acetone : water (12 mL) and the reaction mixture was stirred for 30 min at 0°C.
• a saturated aqueous solution of sodium hydrogenocarbonate (5 mL) was added, then a saturated aqueous solution of sodium thiosulfate (5 mL) was added and the mixture was stirred for 15 min.
• the organic solvent was evaporated off and the remaining aqueous phase was extracted with diethyl ether (2 x 10 mL) .
• Step 3 O-Benzyl-N- ( (2S , 3S , 4R) -2 , 3 , 4-tris-benzyloxy-5- triisopropylsilanyloxy-pentyl) -hydroxylamine (2c)
• Step 4 N-Benzyloxy-N- ( (2S , 3S , 4R) -2 , 3 , 4-tris-benzyloxy-5- triisopropylsilanyloxy-pentyl) -formamide (2d)
• Step 5 N-Benzyloxy-N- ( (2S , 3S , 4R) -2 , 3 , 4-tris-benzyloxy-5- hydroxy-pentyl) -formamide (2e)
• N-Benzyloxy-N- ( (2S, 3S, 4R) -2, 3, 4-tris-benzyloxy-5-hydroxy- pentyl ) -formamide (100 mg, 0.18 mmol) was evaporated with toluene two times, dried at high vacuum for 12 h and taken up in anhydrous dichloromethane (1.5 mL, 1.1 ppm) .
• Dibenzyl diisopropylphosphoramidite reagent (91 yL, 0.27 mmol) was added followed by slow addition of lH-tetrazole (0.45 M in acetonitrile, 600 yL, 0.27 mmol) and the solution was stirred at room temperature for 40 minutes until TLC indicated complete conversion of the starting material.
• reaction mixture was then cooled to -78°C, meta-chloroperoxybenzoic acid (70%, 71 mg, 0.28 mmol) was taken up in dichloromethane (1 mL) and added slowly to the reaction mixture which was stirred for 20 minutes at the given temperature.
• TEA 40 yL, 0.28 mmol was added and the reaction mixture was allowed to warm to room temperature.
• the reaction mixture was diluted with dichloromethane (10 mL) , washed with a saturated aqueous solution of sodium hydrogenocarbonate (2 x, 5 mL) , dried over magnesium sulfate and reduced to dryness.
• Step 7 iV-hydroxy-iV-formylamino-l-deoxy-D-ribitol-5-phosphate (monosodium salt)
• Step 1 2 , 3 , 4-Tri-O-benzyl-l- (N-benzyloxy-N-formylamino) -1- deoxy-5-oxo-D-ribitol (3a)
• Step 2 Dibenzyl [2 , 3 , 4-tri-O-benzyl-l- (N-benzyloxy-N- formylamino) -l-deoxy-D-ribo-5 (E) -hex-enitol) ] 6-phosphonate (3b)
• Step 3 [6- (Formyl-hydroxy-amino) - (3R, 4J?, 5S) -3, , 5- trihydroxy-hexyl] -phosphonic acid (Exam le 3)
• Step 1 Methyl (li , 2R, 3R, 4J?) -2 , 3-O-isopropylidene- trifluoromethanesulfonyl-beta-D-ribofuranoside (3-II-a)
• Step 3 [ (2R, 3R, 4J?) -2- (3 , 4 , 5-Trihydroxy-tetrahydro-furan-2- yl) -ethyl] -phosphonic acid dimethyl ester (3-II-c)
• Step 5 (6-Benzyloxyamino- (3i , 4i , 5S) -3 , 4 , 5-trihydroxy-hexyl) - phosphonic acid dimethyl ester (3-II-e)
• Step 6 [6-(Benzyloxy-formyl-amino)-(3R,4R,5S)-3,4,5- trihydroxy-hexyl] -phosphonic acid dimethyl ester (3-II-f)
• Step 7 [6- (Formyl-hydroxy-amino) - (3J?, 4J?, 5S) -3, 4, 5- trihydroxy-hexyl] -phosphonic acid dimethyl ester (3-II-g)
• compound 3-II-f (93mg, 0.23mmol) was dissolved in MeOH (0.9mL) . Pd/C 10% Degussa type E101 (19mg, 20%w/w) was added. The autoclave was purged three times with nitrogen (2bar) and then three times with 3 ⁇ 4 (2bar) . The pressure was set to lbar of 3 ⁇ 4 and the reaction mixture was stirred at 22°C for 1 hour. The autoclave was depressurized and purged with nitrogen.
• Step 8 [6- (Formyl-hydroxy-amino) - (3R, 4J?, 5S) -3, , 5- trihydroxy-hexyl] -phosphonic acid (monosodium salt)
• Step 1 [l-Benzyloxy-2- (3J?, 4J?, 5J?, 6J?) - (6-methoxy-2 , 2-dimethyl- tetrahydro-furo [3 , 4-d] [1 , 3] dioxol-4-yl) -ethyl] -phosphonic acid diethyl ester (4a)
• Step 2 [1-Hydroxy-2- (3J?, 4J?, 5J?, 6J?) - (6-methoxy-2 , 2-dimethyl- tetrahydro-furo [3 , 4-d] [1 , 3] dioxol-4-yl) -ethyl] -phosphonic acid diethyl ester (4b)
• compound 4a (1.2g, 2.56mmol) was dissolved in EtOH (6mL) . Pd/C 10% Degussa type E101 (360mg, 30%w/w) was added. The autoclave was purged three times with nitrogen (5bar) and then three times with 3 ⁇ 4 (5bar) . The pressure was set to lObar of 3 ⁇ 4 and the reaction mixture was stirred at 50°C for 6 hours. The autoclave was depressurized and purged with nitrogen.
• the compound was obtained as a mixture of diastereomers in a ratio 3:1.
• Step 4 (6-Benzyloxyimino- (3J?, 4J?, 5J?) -1 , 3 , 4 , 5-tetrahydroxy- hexyl) -phosphonic acid diethyl ester (4d)
• the compound was obtained as a mixture of diastereomers in a ratio 3:1 and a mixture of oxime E and Z: 9:1.
• Step 5 (6-Benzyloxyamino- (3J?, 4J?, 5S) -1 , 3 , 4 , 5-tetrahydroxy- hexyl) -phosphonic acid diethyl ester (4e)
• Step 7 [6- (Benzyloxy-formyl-amino) - (3J?, 4J?, 5S) -1 , 3 , 4 , 5- tetrahydroxy-hexyl] -phosphonic acid diethyl ester (4f)
• Step 8 [6- (Formyl-hydroxy-amino) - (3J?, 4J?, 5S) -1 , 3 , 4 , 5- tetrahydroxy-hexyl] -phosphonic acid diethyl ester (4g)
• compound 4f (185mg, 0.40mmol) was dissolved in MeOH (4.5mL). Pd/C 10% Degussa type E101 (37mg, 20%w/w) was added. The autoclave was purged three times with nitrogen (2bar) and then three times with 3 ⁇ 4 (2bar) . The pressure was set to lbar of 3 ⁇ 4 and the reaction mixture was stirred at 22 °C for 1 hour. The autoclave was depressurized and purged with nitrogen.
• the compound was obtained as a mixture of diastereomers in a ratio 3:1.
• the compound was obtained as a mixture of diastereomers in a ratio 3:1.
• Example 5 [ (2S , 3S , 4S) -5- (Formyl-hydroxy-amino) -2 , 3 , 4 trihydroxy-pentyl] -phosphonic acid sodium salt (Sodium 1-(N benzyloxy-N-formylamino) -1 , 5-dideoxy-D-ribitol] 5 phosphonate)
• Step 1 2, 3, 4-Tri-O-benzyl-l- (W-benzyloxy-W-formylamino) -1, 5- dideoxy-5-iodo-D-ribitol
• Step 2 Diethyl [2,3, 4-tri-O-benzyl-l- (iV-benzyloxy-iV- formylamino) -1 , 5-dideoxy-D-ribitol] 5-phosphonate (5b)
• Step 3 Sodium 1- (N-benzyloxy-N-formylamino) -1 , 5-dideoxy-D- ribitol] 5-phosphonate (Example 5)
• reaction mixture was filtered through a syringe filter and the filter was successively washed with MeOH/H20 1:1 (15 mL) .
• the filtrate was concentrated, and the residue was taken up in H2O and neutralized with NaHC03 (10 mg, 0.12 mmol) and concentrated.
• the residue was flashed (MeCN/H20 1: 1) through a short plug of ZIC-HILIC silica gel (500 mg) .
• Step 1 Dibenzyl [2, 3, 4-tri-O-benzyl-l- (N-benzyloxy-N- formylamino) -l-deoxy-5, 6-dihydroxy-D-ribo-hexitol) ] 6- phosphonate (6a)
• Hexenitol 3b (50 mg, 0.06 mmol, obtained as described in Example 3, Step 2) was dissolved in 2:1 acetone/water (0.75 mL) and NMO (10 mg, 0.1 mmol) was added. Then potassium osmate (2.25 mg, 6.2 ymol) was added and the reaction was stirred overnight at laboratory temperature. Since TLC (EtOAc/Hex 1:2) showed only minor conversion, another 0.1 eq. of potassium osmate and 1.6 eq. of NMO were added. This was repeated 3 times until an acceptable conversion was reached (after 5 days) . The reaction was quenched by adding satd. aq. a2S03 and extraction into EtOAc.
• Step 2 Sodium 1- (N-benzyloxy-N-formylamino) -l-deoxy-5 , 6- dihydroxy-D-ribo-hexitol) ] 6-phosphonate (Example 6)
• Example 6 Compound 6a (24 mg, 0.03 mmol) was dissolved in 1:2 THF/water mixture and Pd(OH)2 catalyst (4 mg, 20% w/w) was added. The reaction was stirred overnight under hydrogen atmosphere (1 bar) and checked by TLC (Chloroform/Metanol/Water 10:10:3). After completeness, solid sodium bicarbonate (4 mg, 0.09 mmol) was added, and all volatiles were removed. Then the crude was purified by repeated ZIC-HILIC chromatography (Column: Sequant ZIC HILIC 10x250 mm, Merck, Gradient MeCN/H 2 0 85 to 40 %, flow rate 3 mL, fraction size 2 mL) .
• Example 7 [ (3R, 4R, 5S) -6- (Formyl-hydroxy-amino) -3 , 4 , 5 , 7- tetrahydroxy-heptyl] -phosphonic acid (Diastereoisomer 1)
• Step 1 Triisopropyl- ( (2R,3S,4S) -2 , 3 , 4-tris-benzyloxy-hex-5- enylox -silane (7a)
• Step 2 (3S , 4R, 5R) -3 , 4 , 5-Tris-benzyloxy-6- triisopropylsilanyloxy-hexane-1 , 2-diol (7b)
• Step 3 (3S , 4R, 5R) -3 , 4 , 5-Trisbenzyloxy-6- triisopropylsilanyloxy-l-trityloxyhexan-2-ol (7c) .
• Step 5 O-Benzyl-N- ( (2S , 3S , 4R) -2 , 3 , 4-tris-benzyloxy-5- triisopropylsilanyloxy-l-trityloxymethyl-pentyl) - hydroxylamine (7e)
• Step 6 N-Benzyloxy-N- ( (2S , 3S , 4R) -2 , 3 , 4-tris-benzyloxy-5- triisopropylsilanyloxy-l-trityloxymethyl-pentyl) -formamide
• reaction mixture was concentrated under reduced pressure, obtained oily residue was purified by chromatography on silica gel (eluent cyclohexane/EtOAc 95/5) to afford compound 7f (3.36 g, 68%) as yellow oil .
• Step 7 N-Benzyloxy-N- ( (2S , 3S , 4R) -2 , 3 , 4-tris-benzyloxy-5- hydroxy-l-trityloxymethyl-pentyl) -formamide (7h, diastereoisomers 1 and 2 .
• Step 8 N-Benzyloxy-N- ( (2S , 3S , 4S) -2 , 3 , 4-tris-benzyloxy-5-oxo- 1-trityloxymethyl-pentyl) -formamide (7i-DIAl)
• Step 9 [ (E) - (3R,4R,5S) -3,4,5-Tris-benzyloxy-6- (benzyloxy- formyl-amino) -7-trityloxy-hept-l-enyl] -phosphonic acid dibenzyl ester (7j-DIAl)
• reaction mixture was stirred at -78°C for 30min, then a solution of the compound 7i-DIAl (2.20 g, 2.65 mmol, leq.) in THF (48 mL) was added dropwise. After stirring for lh at RT the reaction mixture was quenched with NH4CI saturated aqueous solution, and then extracted with EtOAc. Combined organic phases were washed with brine, dried over a2S04 and concentrated under reduced pressure. Obtained oily residue was purified by chromatography on silica gel (eluent cyclohexane/EtOAc 80/20 to 70/30) to afford compound 7j-DIAl (2.64g, 92%) as white foam.
• Step 10 [ (E) - (3R, 4R, 5S , 6R) -3,4, 5-Tris-benzyloxy-6- (benzyloxy-formyl-amino) -7-hydroxy-hept-l-enyl] -phosphonic acid dibenzyl ester (7k-DIAl)
• Step 11 [ (3R, 4R, 5S) -6- (Formyl-hydroxy-amino) -3 , 4 , 5 , 7- tetrahydroxy-heptyl] -phosphonic acid (71-DIA1,
• the crude residue was purified via semi-preparative HPLC using HILIC-type column (XBridge PrepAmide 5ym, OBD 19x50mm) (sample dissolved in 4 mL of the mixture H20/acetonitrile 1/2; gradient H20/acetonitrile 15/85 to 60/40 in 12 minutes then 60/40) .
• the fractions containing the product were lyophilized and purified again in the same chromatography conditions to afford compound 71-DIA1 (Example 7) (15 mg, 20%) as white foam.
• Example 8 [ (3R, 4R, 5S) -6- (Formyl-hydroxy-amino) -3 , 4 , 5 , 7- tetrahydroxy-heptyl] -phosphonic acid (81-DIA1, Diastereoisomer 2)
• Step 1 N-Benzyloxy-N- ( (2S,3S,4S) -2 , 3 , 4-tris-benzyloxy-5-oxo- 1-trityloxymethyl-pentyl) -formamide (8i-DIA2)
• Compound 8i-DIA2 was prepared according to procedure analogous to that described in Example 7, Step 8 using solution of 7h-DIA2 (2.85 g, 3.44 mmol, 1 eq.) in DCM (80 mL) , W-methyl-morpholine oxide (805 mg, 6.88 mmol ; 2 eq.) and tetrapropylammonium peruthenate (121 mg ; 0.344 mmol, 0,leq.) . Purification of the crude oily product by chromatography on silica gel (eluent cyclohexane/EtOAc 80/20) afforded compound 8i-DIA2 (1.85g, 65%) as white solid.
• Step 2 [ (E) - (3R, 4R, 5S) -3,4, 5-Tris-benzyloxy-6- (benzyloxy- formyl-amino) -7-trityloxy-hept-l-enyl] -phosphonic acid dibenzyl ester (8j-DIA2)
• Step 3 [ (E) - (3R, 4R, 5S , 6R) -3,4, 5-Tris-benzyloxy-6- (benzyloxy- formyl-amino) -7-hydroxy-hept-l-enyl] -phosphonic acid dibenzyl ester (8k-DIA2)
• Step 4 [ (3R, 4R, 5S) -6- (Formyl-hydroxy-amino) -3 , 4 , 5 , 7- tetrahydroxy-heptyl] -phosphonic acid (81-DIA2,
• Compound 81-DIA2 was prepared according to procedure analogous to that described in Example 7, Step 11 using a solution of the compound 8k-DIA2 (200 mg, 0.24mmol, leq.) in the mixture of THF (15 mL) and i-PrOH (15 mL) , water (0.22 mL) and two portions of Pd/C 10% (20 mg) .
• Example 9 [l-Fluoro-6- (formyl-hydroxy-amino) - (3J?, 4J?, 5S) - 3 , 4 , 5-trihydroxy-hexyl] -phosphonic acid, monosodium salt
• Step 1 Trifluoro-methanesulfonic acid 1- (diethoxy- phosphoryl) - (3J?, 4J?, 5J?, 6J?) -2- (6-methoxy-2-methyl-tetrahydro- furo[3,4-d] [l,3]dioxol-4-yl) -ethyl ester (9a)
• Step 2 [1-Fluoro- (3J?, 4J?, 5J?, 6R) -2- (6-methoxy-2 , 2-dimethyl- tetrahydro-furo [3 , 4-d] [1,3] dioxol-4-yl) -ethyl] -phosphonic acid diethyl ester (9b)
• Step 4 (6-Benzyloxyimino-l-fluoro- (3J?, 4J?, 5J? ⁇ -3 , 4 , 5- trihydroxy-hexyl) -phosphonic acid diethyl ester (9d) + Q ° .HCI -HCO , (1. eq.) 3 ⁇ 4 f
• Step 5 (6-Benzyloxyamino-l-fluoro- (3J?, 4J?, 5S) -3 , 4 , 5- trihydroxy-hexyl) -phosphonic acid diethyl ester (9e)
• Step 7 [l-Fluoro-6- (formyl-hydroxy-amino) - (3J?, 4J?, 5S)
• the compound was obtained as a mixture of diastereomers in a ratio 55:45.
• Step 1 Synthesis of (2R, 3S , 4S) -5 , 5-bis (ethylthio) pentane- 1,2,3,4-tetraol (10a) .
• Step 2 Synthesis of (2S , 3S , 4R) -1 , 1-bis (ethylthio) -5- ( (triisopropylsilyl) oxy) entane-2 , 3 , 4-triol (10b)
• the crude reaction mixture was diluted with water, extracted with ethylacetate, dried over anhydrous sodium sulfate, filter and concentrated under reduced pressure to obtain crude product which was purified by column chromatography on silica gel (100-200 mesh) and 0-50% EtOAc : n-hexane as eluent to afford the title product 10b (36.0 g, 78.0%) as light yellow liquid.
• Step 3 Synthesis of triisopropyl ( ( (2R, 3S , 4S) -2 , 3 , 4- tris (benzyloxy) -5 , 5-bis (ethylthio) pentyl) oxy) silane (10c).
• Step 4 Synthesis of (2S , 3S , 4R) -2 , 3 , 4-tris (benzyloxy) -5- ( (triisopropylsilyl) oxy) entanal (lOd) .
• Step 5 Synthesis of O-benzyl-N- ( (2R, 3R, 4R) -2 , 3 , 4-tris (benzyloxy) -5- ( (triisopropylsilyl) oxy) pentyl) hydroxylamine (lOe) .
• the intermediate oxime (25g) was dissolved in acetic acid (130 mL) and sodium cyanoborohydride (12.9g, 208.33 mmol) was added in portions to the reaction mixture.
• the reaction mixture was stirred for 20 min at RT .
• the reaction mixture was diluted with water, extracted with DCM, washed with saturated NaHC03 solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
• the crude reaction mixture was purified by column chromatography on silica gel (100-200 mesh) and 0-10% EtOAc : n-hexane as eluent to afford title product lOe (18.0 g ,53%) as colorless oil, ESMS (M+H) 684.47.
• Step 6 Synthesis of N- (benzyloxy) -N- ( (2R, 3R, 4R) -2,3, 4-tris (benzyloxy) -5- ( (triisopropylsilyl) oxy) pentyl) formamide (lOf) .
• Step 7 Synthesis of N- (benzyloxy) -N- ( (2R, 3R, 4R) -2 , 3 , 4-tris (benzyloxy) -5-hydroxypentyl) formamide (lOg) .
• Step 8 Synthesis of N- (benzyloxy) -N- ( (2R, 3R, 4S) -2,3, 4-tris (benzyloxy) -5-oxopentyl) form mide (lOh) .
• Step 9 Synthesis of dimethyl ( (3R, 4S , 5R,E) -3 , 4 , 5- tris (benzyloxy) -6- (N- (benzyloxy) formamido) hex-l-en-l-yl) phosphonate (lOi) .
• Step 10 Synthesis of dimethyl ( (3R, 4S , 5R) -3 , 4 , 5- tris (benzyloxy) -6- (N- (benzyloxy) formamido) hexyl) phosphonate (10j).
• Step 11 Synthesis of ( (3R, 4S , 5R) -3 , 4 , 5-tris (benzyloxy) (N- (benzyloxy) formamido) hexyl) phosphonic acid (10k).
• Step 12 Synthesis of ( (3R, 4S , 5R) -3,4, 5-trihydroxy-6- (N- hydroxyformamido) hexyl) hosphonic acid
• reaction mass was filtered by a micro filter, concentrated and crude compound was further purified by prep HPLC with Zic Hillic ( 19x250mm, 5 ⁇ ) column with 20:80 5mM ammonium acetate :ACN, flow rate 25 mL and desired fraction was lyophilized to obtain title compound, ( (3R, 4S, 5R) -3, 4, 5-trihydroxy- 6- (N-hydroxyformamido) hexyl ) phosphonic acid (0.024 g, 28%) as off white solid.
• Example 11 (3R, 4R, 5R) -3 , 4 , 5-trihydroxy-6- (N- hydroxyformamido) hexylphosphonic acid.
• Step 1 (2R,3R,4S) -5, 5-bis (ethylthio) pentane-1, 2, 3, 4-tetraol (Ha) .
• Step 4 (2S , 3R, 4R) -2 , 3 , 4-tris (benzyloxy) -5-
• Step 5 O-benzyl-N- ( (2R, 3S , 4R) -2 , 3 , 4-tris (benzyloxy) (triisopropylsilyloxy) pentyl) hydroxylamine (lie) . 9 Bn
• the intermediate oxime (14.9 g) obtained was dissolved in acetic acid (150 ml) and sodium cyanoborohydride (7.7g, 122.5 mmol) was added in portions to the reaction mixture.
• the reaction mixture was stirred for 30 min at RT, then diluted with water, extracted with DCM, washed with saturated NaHC03 solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
• the crude reaction mixture was purified by column chromatography on silica gel (100-200 mesh) and 0-10% ethyl acetate in hexane as eluent to afford title product lie (12. Og, 60%) as colorless oil.
• Step 6 N- (benzyloxy) -N- ( (2R, 3S , 4R) -2,3, 4-tris (benzyloxy) -5- (triis ropylsilyloxy) entyl) formamide (llf) .
• Step 8 N- (benzyloxy) -N- ( (2R, 3S , 4S) -2 , 3 , 4-tris (benzyloxy) oxopentyl) formamide (llh)
• Step 9 Dimethyl (3R, 4R, 5R,E) -3 , 4 , 5-tris (benzyloxy) -6- (N- (benzyloxy) formamido) hex-l-enylphosphonate (Hi)
• the crude reaction mixture was purified by flash chromatography using silica gel column (40g) and 0-80% ethyl acetate in hexane as eluent to afford pure title product Hi (2.1 g, 55.40%) as a colorless oil.
• Step 10 Dimethyl (3R, 4R, 5R) -3 , 4 , 5-tris (benzyloxy) -6- (N- (benzyloxy) formamido) hexylphosphonate (11j )
• Step 11 Ethyl ( (3R, 4R, 5R) -3 , 4 , 5-tris (benzyloxy) h roxyformamido) hexyl) hosphinic aci (Ilk)
• reaction mass was filtered by a micro filter, concentrated and crude compound was further purified by prep HPLC with Zic-Hilic column (19x250mm, 5ym, gradient MeCN /5M ammonium acetate, 80 to 30%, flow rate 25 mL/min) and desired fraction was lyophilized to obtain title compound (3R,4R,5R)- 3,4, 5-trihydroxy- 6- (N-hydroxylformamido) hexylphosphonic acid
• Step 1 [1 , 1-Difluoro-2- (3J?, 4J?, 5J?, 6J?) - (6-methoxy-2 , 2- dimethyl-tetrahydro-furo [3 , 4-d] [1,3] dioxol-4-yl) -ethyl] hosphonic acid diethyl ester (12a)
• Step 2 [1 , 1-Difluoro- (2J?) -2- (3J?, 4J?) - (3 , 4 , 5-trihydroxy- tetrahydro-furan-2-yl) -ethyl] -phosphonic acid diethyl ester (12b)
• Step 3 (6-Benzyloxyimino-l , 1-difluoro- (3J?, 4J?, 5J?) -3 , 4 , 5- trih droxy-hexyl) -phosphonic acid diethyl ester (12c)
• Step 4 (6-Benzyloxyamino-l , 1-difluoro- (3J?, 4J?, 5S) trihydroxy-hexyl) -phosphonic acid diethyl ester (12d)
• Step 5 (6-Benzyloxyamino-l , 1-difluoro- (3J?, 4J?, 5S) -3 , 4 , 5- trihydroxy-hexyl) -phosphonic acid mono-ethyl ester sodium salt (12e)
• compound 12f 60 mg, 0.13 mmol was dissolved in MeOH (0.6 mL) .
• Pd/C 10% Degussa type E101 (12 mg, 20%w/w) was added.
• the autoclave was purged three times with nitrogen (2 bar) and then three times with 3 ⁇ 4 (2 bar) .
• the pressure was set to 1 bar of 3 ⁇ 4 and the reaction mixture was stirred at 22°C for 1 hour.
• the autoclave was depressurized and purged with nitrogen.
• Step 8 [1 , 1-Difluoro-6- (formyl-hydroxy-amino) - (3i , 4i , 5S) - 3 , 4 , 5-trihydroxy-hexyl] -phosphonic acid (monosodium salt) (Example 12)
• the reaction mixture was heated to 30 °C and stirred during 5 hours until LC-MS indicated complete conversion.
• the reaction mixture was then lyophilized.
• Step 1 Dibenzyl [2 , 3 , 4-tri-O-benzyl-l- (iV-benzyloxy-iV- formylamino) -1 , 6-dideoxy-D-allo/L-talo-hexitol) ] 6- phosphonate (13a)
• Step 2 Sodium [1- (IV-hydroxy-iV-formylamino) -1 , 6-dideoxy-D- allo/L-talo-hexitol) ] 6-phosphonate (Example 13) .
• Example 13a Compound 13a (20 mg, 0.024 mmol) was dissolved in 1:2 THF/water and 20% Pd(OH) 2 -C catalyst (4 mg) was added. The reaction was stirred overnight (18 h) under hydrogen atmosphere (1 bar) and checked by TLC (Chloroform/Methanol/Water 10:10:3) . The catalyst was then filtered off and the filtrate was washed with 1:1 water/methanol mixture (1 :1) and purified by HILIC separation (column: Sequant ZIC HILIC 10x250 mm, Merck, Gradient MeCN/H 2 0 85 to 40 %, flow rate 3 mL, fraction size 2 mL) .
• Example 14 Sodium 1- (N-benzyloxy-N-formylamino) -1-deoxy-D- ribo-5- (E) -hexenitol 6-phosphonate
• the filtrate was concentrated and purified by ZIC-HILIC chromatography (column: Sequant ZIC HILIC 10x250 mm, Merck, Gradient MeCN/H 2 0 85 to 40 %, flow rate 3 mL, fraction size 2 mL) . Purity of fractions was checked by 1 R NMR and product containing fractions were pooled and lyophilized to give title compound (3.56 mg, 54 %) as white solid. To remove residual sodium bicarbonate, the material was passed over DOWEX-50 (H+) and the pH of the filtrate was adjusted to 7.0 by addition of 0.01 M NaOH.
• Example 15 ( (3S , 5R) -3 , 5-dihydroxy-6- (N-hydroxyformamido) - hexyl) hosphonic acid
• Step 1 Dithiocarbonic acid O- [ (5R, 6S , 6aR) -5- (2 , 2-dimethyl- [1,3] dioxolan-4-yl) -2 , 2-dimethyl-tetrahydro-furo [2 , 3- d] [1 , 3] dioxol-6-yl] ester S-methyl ester (15a)
• Step 2 (5S , 6aR) -5- (2 , 2-Dimethyl- [1 , 3] dioxolan-4-yl) -2 , 2- dimethyl-tetrahydro-furo [2 , 3-d] [1,3] dioxole
• Step 3 1- ( (5S , 6aR) -2 , 2-Dimethyl-tetrahydro-furo [2 , 3- d] [1,3] dioxol-5-yl) -ethane-1 , 2-diol
• Step 4 (5S , 6aR) -2 , 2-Dimethyl-tetrahydro-furo [2,3- d [1 , 3] dioxole-5-carbaldehyde (15d)
• Step 5 [ (E) -2- ( (5S , 6aR) -2 , 2-Dimethyl-tetrahydro-furo [2 , 3- d] [1 , 3] dioxol-5-yl) -vinyl] -phosphonic acid dimethyl ester (15e)
• Step 6 [2- ( (5S , 6aR) -2 , 2-Dimethyl-tetrahydro-furo [2 , 3- d] [1 , 3] dioxol-5-yl) -ethyl] -phosphonic acid dimethyl ester (15f)
• Step 7 [2- ( (2S , 4R) -4 , 5-Dihydroxy-tetrahydro-furan-2-yl) - ethyl] -phosphonic acid dimethyl ester (15g)
• Step 8 ( (3S , 5R) -6-Benzyloxyimino-3 , 5-dihydroxy-hexyl) - phosphonic acid dimethyl ester (15i)
• Step 9 ( (3S , 5R) -6-Benzyloxyamino-3 , 5-dihydroxy-hexyl) - phosphonic acid dimethyl ester (15i)
• Step 10 [ (3S , 5R) -6- (Benzyloxy-formyl-amino) -3 , 5-dihydroxy- hexyl] -phosphonic acid dimethyl ester (15j)
• Solutions for IV administration have been prepared by diluting the compound of example 2 at 1-20 mg/mL in physiological serum (0.9% sodium chloride in non-pyrogenic water) or in glucose solution (1-5% glucose in non-pyrogenic water), with and without addition of erythromycin (1-5 mg/mL) .
• the assay buffer "AB” contained 50 mM Hepes pH7.5, 1 mM MnCl 2 , 25 mM KC1, 0.012% Triton-X100, 1 mM dithiothreitol (DTT) and 0.1 ⁇ Myelin basic protein (MBP) .
• the following components were added in a white polystyrene Costar plate up to a final volume of 30 yL : 10 yL inhibitor dissolved in DMSO/water 50/50, and 20 yL GmhA of E. coli in AB . After 30min of pre-incubation at room temperature, 30 yL of Substrates mix in AB were added in each well to a final volume of 60 yL .
• This reaction mixture was then composed of 2 nM GmhA, 3 ⁇ sedoheptulose-7-phosphate (Sigma), 3 ⁇ ATP (Sigma) and 50nM HldE of E. coli in assay buffer. After 30 minutes of incubation at room temperature, 100 yL of the revelation mix were added to a final volume of 160 yL, including the following constituents at the respective final concentrations: 10000 light units/mL luciferase (Sigma), 20 ⁇ D-luciferin (Sigma) , 100 ⁇ N-acetylcysteamine (Aldrich) . Luminescence intensity was immediately measured on Luminoskan (Thermofisher) and converted into inhibition percentages.
• E. coli C7 (018:K1:H7) is a Newborm Meningitidis E. coli (NMEC) strain which displays a typical LPS made of Lipid A successively branched with the inner and outer core oligosaccharides, and finally with the O-antigen repeats.
• the inner core contains several heptose residues.
• An inhibitor of the LPS heptosylation pathway should therefore reduce dramatically the size of LPS from full-length to the so- called x Re-LPS' limited to lipid A branched with 2 Kdo residues.
• a simple way of monitoring LPS size and composition consists in running LPS gel electrophoresis (Figure 1) : a wild type E. coli strain displays several bands including those for full and core LPS but none for Re-LPS. On the contrary, a delta-hldE mutant defective for LPS-heptosylation biosynthesis displays only the Re-LPS band.
• Bacterial culture The effect of heptosylation inhibitors on E. coli LPS was assessed as described below.
• the compounds to be tested were prepared in deionised water/DMSO (50/50) solutions and added (25 yL) in sterile culture microtubes.
• the strain used in this study was E. coli C7 (018:K1:H7) .
• the bacteria were isolated on tryptic soy agar (TSA) overnight. Isolated colonies were cultured in 10 mL of Luria-Bertani medium (LB) at 37°C up to an optical density (OD) of typically 0.15.
• LB Luria-Bertani medium
• LPS extraction Bacterial cultures were normalized via OD determination, pelleted and washed with 1 mL Phosphate- Buffer-Saline (PBS) . The pellets were then denatured for 10 min at 95-100°C in 50 yL of Sodium dodecyl sulphate 0.2% (SDS), beta-mercaptoethanol 1%, Glycerol 36%, Tris pH 7.4 30 mM and bromophenol blue 0.001%. Samples were cooled down to room temperature, supplemented with 1.5 ⁇ of proteinase K at 20 mg/mL, incubated for 1 hour at 55°C and centrifuged for 30 min at 13,000 rpm at 25°C. The resulting supernatant, containing LPS, was finally analysed by SDS-PAGE electrophoresis .
• PBS Phosphate- Buffer-Saline
• LPS SDS-PAGE electrophoresis Polyacrylamide gels (16%/4% acrylamide for separation and concentration respectively) were prepared, loaded with 8 yL of LPS extracts and migrated.
• Silver staining Gels were incubated overnight in 5% acetic acid/40% ethanol/deionised water, treated by 1% periodic acid/5% acetic acid for 15 min, washed 4 times for 10 min in deionised water and finally incubated for 18 min in the dark in a silver nitrate solution composed of 56 mL NaOH 0. IN, 4 mL ammoniac 33%, 45 mL AgN03 5% (Tsai and Frasch) and 195 mL deionised water.
• the compound to be tested was diluted in 50 mM HEPES buffer pH 7.4 from 10 mM stock solution. 5 yL of compound dilution or buffer were distributed in a sterile clear round bottom 96-well polystyrene microplate (Corning) . 5 yL of serial 2- fold dilutions of reference antibiotics (Erythromycin (Fluka) , Rifampicin (Fluka) , or Synercid® (Monarch Pharmaceuticals) ) in DMSO were added. An exponentially growing preculture of E.
• coli C7 (018:K1:H7) in LB was diluted to le4 cfu/mL and supplemented with 100 ⁇ G6P, and 90 yL of this suspension were added to the microplates. After overnight incubation at 37°C, the minimal inhibitory concentration (MIC) of the antibiotics were determined for each test compound concentration, as the lowest antibiotic concentration for which no bacterial pellet is visible without magnification.
• MIC minimal inhibitory concentration
• Compound described in example 2 displays in the presence of 100 ⁇ G6P a 32-fold potentiation of erythromycin (MIC from 32 to 1 yg/mL) , and a 16-fold potentiation of rifampicin (MIC from 4 to 0.25 yg/mL) and Synercid® (MIC from 128 to 8 yg/mL) at concentrations ⁇ 1 mM.
• Such inhibitors provide a novel way to treat or prevent bloodstream infections caused by pathogenic Gram negative bacteria, without affecting the commensal flora and with less selective pressure than conventional antibacterial agents.

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