WO2024013349A1 - Benzamide adenine dinucleoside compounds and their uses - Google Patents
Benzamide adenine dinucleoside compounds and their uses Download PDFInfo
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- WO2024013349A1 WO2024013349A1 PCT/EP2023/069574 EP2023069574W WO2024013349A1 WO 2024013349 A1 WO2024013349 A1 WO 2024013349A1 EP 2023069574 W EP2023069574 W EP 2023069574W WO 2024013349 A1 WO2024013349 A1 WO 2024013349A1
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- pharmaceutically acceptable
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- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
- 125000000168 pyrrolyl group Chemical group 0.000 description 1
- 125000002294 quinazolinyl group Chemical group N1=C(N=CC2=CC=CC=C12)* 0.000 description 1
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- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 229940080313 sodium starch Drugs 0.000 description 1
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
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- 239000000600 sorbitol Substances 0.000 description 1
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- 239000003549 soybean oil Substances 0.000 description 1
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- 230000003019 stabilising effect Effects 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
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- 239000007929 subcutaneous injection Substances 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 229910021653 sulphate ion Inorganic materials 0.000 description 1
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- 229940095064 tartrate Drugs 0.000 description 1
- IOKGWQZQCNXXLD-UHFFFAOYSA-N tert-butyl n-(3-bromopropyl)carbamate Chemical compound CC(C)(C)OC(=O)NCCCBr IOKGWQZQCNXXLD-UHFFFAOYSA-N 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- FPGGTKZVZWFYPV-UHFFFAOYSA-M tetrabutylammonium fluoride Chemical compound [F-].CCCC[N+](CCCC)(CCCC)CCCC FPGGTKZVZWFYPV-UHFFFAOYSA-M 0.000 description 1
- MHXBHWLGRWOABW-UHFFFAOYSA-N tetradecyl octadecanoate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCCCCCCCCCCCCCC MHXBHWLGRWOABW-UHFFFAOYSA-N 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 125000001113 thiadiazolyl group Chemical group 0.000 description 1
- 125000001544 thienyl group Chemical group 0.000 description 1
- 229960000984 tocofersolan Drugs 0.000 description 1
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- 230000000699 topical effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- WBYWAXJHAXSJNI-VOTSOKGWSA-M trans-cinnamate Chemical compound [O-]C(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-M 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 125000004306 triazinyl group Chemical group 0.000 description 1
- 125000001425 triazolyl group Chemical group 0.000 description 1
- CYRMSUTZVYGINF-UHFFFAOYSA-N trichlorofluoromethane Chemical compound FC(Cl)(Cl)Cl CYRMSUTZVYGINF-UHFFFAOYSA-N 0.000 description 1
- 229940029284 trichlorofluoromethane Drugs 0.000 description 1
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
- C07H19/167—Purine radicals with ribosyl as the saccharide radical
-
- 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
- the present invention relates to benzamide adenine dinucleoside compounds which are useful as inhibitors of NAD kinases, to pharmaceutical composition comprising such compounds, and to uses of such compounds in the treatment or prevention of bacterial infections.
- the environmental bacterium and opportunistic human pathogen Pseudomonas aeruginosa is a Gram-negative bacterium responsible for a variety of acute infections and is a major cause of mortality in chronically infected cystic fibrosis patients. Due to increased resistance to antibiotics, P aeruginosa has been listed by the World Health Organization (WHO) among pathogenic bacteria for which new antibiotics or alternative therapeutic strategies are urgently needed (Tacconelli et al., Lancet Infect. Dis. 2018, 18, 318-327).
- WHO World Health Organization
- NADP + Bacterial nicotinamide adenine dinucleotide kinase
- NADK is therefore an essential enzyme for rapidly dividing cells, which need both high macromolecular turnover and an efficient response to oxidative stress, and therefore require high level of NADPH (Grose et al., Proc. Natl. Acad. Sci. USA 2006, 103, 7601- 7606). This is particularly important for bacteria in the context of antibiotic treatment. In fact, three main classes of antibiotics (quinolones, beta-lactams and aminoglycosides) stimulate the production of reactive oxygen species (ROS), which can participate in bacterial cell death (Van Acker et al., Trends Microbiol. 2017, 25, 456-466).
- ROS reactive oxygen species
- P aeruginosa NADK hereafter PaNADK
- LmNADK Listeria monocytogenes
- HsNADK human cytosolic NADK
- NAD kinases in particular P aeruginosa NADK (PaNADK) enzyme, Listeria monocytogenes (LmNADK) enzyme and/or human cytosolic NADK (HsNADK) enzyme.
- PaNADK P aeruginosa NADK
- LmNADK Listeria monocytogenes
- HsNADK human cytosolic NADK
- the invention therefore relates to compounds of general Formula I, their pharmaceutically acceptable salts or solvates thereof, as well as methods of use of such compounds or compositions comprising such compounds as inhibitors of NAD kinases.
- Y is CH or N; Z is O or S;
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising at least one compound according to the invention or a pharmaceutically acceptable salt or solvate thereof and at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.
- the invention further relates to compounds of Formula I or their pharmaceutically acceptable salts or solvates thereof for use in the prevention and/or treatment of bacterial infections.
- the invention relates to compounds of Formula I, as well as their tautomeric forms, stereoisomeric forms, mixture of stereoisomeric forms, pharmaceutically acceptable salts or solvates.
- Preferred compounds of Formula I or pharmaceutically acceptable salts or solvates thereof are those wherein one or more of X, R 1 , R 2 , R 3 and R 4 are defined as follows:
- Y is CH or N; in particular Y is CH;
- Z is O or S; in particular Z is O;
- the compound of Formula I includes its tautomeric forms, stereoisomeric forms, mixture of stereoisomeric forms.
- the compounds of Formula I are those wherein n is 1.
- the compounds of Formula I are those wherein m is 1. In one embodiment, the compounds of Formula I are those wherein n and m are 1.
- the compounds of Formula I are those wherein Y is CH.
- the compounds of Formula I are those wherein Z is O.
- the compounds of Formula I are those wherein R 1 is OH.
- the compounds of Formula I are those wherein R 1 is -NHSO 2 NR 2 R 3 wherein R 2 and R 3 are C1-C6-alkyl, in particular R 2 and R 3 are C1-C4-alkyl, more particularly R 2 and R 3 are C1-C2-alkyl, still more particularly R 2 and R 3 are methyl.
- the compounds of Formula I are those wherein R 1 is NH 2 .
- the compounds of Formula I are those wherein A is -O-.
- the compounds of Formula I are those of Formula II:
- R 1 and A are as defined above with respect to Formula I and any of its embodiments.
- Preferred compounds of Formula II or pharmaceutically acceptable salts or solvates thereof are those wherein one or more of R 1 and A are defined as follows:
- the compounds of Formula I are those of Formula III: III or pharmaceutically acceptable salts or solvates thereof, wherein
- R 1 , n and m are as defined above with respect to Formula I and any of its embodiments.
- Preferred compounds of Formula III or pharmaceutically acceptable salts or solvates thereof are those wherein one or more of R 1 , n and m are defined as follows:
- Preferred compounds of Formula IV or pharmaceutically acceptable salts or solvates thereof are those wherein one or more of R 1 , R 5 , n, m, and p are defined as follows:
- the compounds of Formula I are those of Formula V: or pharmaceutically acceptable salts or solvates thereof, wherein
- A, n and m are as defined above with respect to Formula I and any of its embodiments.
- Preferred compounds of Formula V or pharmaceutically acceptable salts or solvates thereof are those wherein one or more of A, n and m are defined as follows:
- the compounds of Formula I are those of Formula VI: or pharmaceutically acceptable salts or solvates thereof, wherein
- R 2 , A, n and m are as defined above with respect to Formula I and any of its embodiments.
- Preferred compounds of Formula VI or pharmaceutically acceptable salts or solvates thereof are those wherein one or more of R 2 , A, n and m are defined as follows:
- R 2 is C1-C6-alkyl, in particular R 2 is C1-C4-alkyl, more particularly R 2 is C1-C2-alkyl, still more particularly R 2 is ethyl;
- the compounds of Formula I are those of Formula VII: VII or pharmaceutically acceptable salts or solvates thereof, wherein
- R 2 , R 3 , A, n and m are as defined above with respect to Formula I and any of its embodiments.
- Preferred compounds of Formula VI or pharmaceutically acceptable salts or solvates thereof are those wherein one or more of R 2 , R 3 , A, n and m are defined as follows:
- R 2 and R 3 are independently C1-C6-alkyl, in particular R 2 and R 3 are independently C1- C4-alkyl, more particularly R 2 and R 3 are independently C1-C2-alkyl, still more particularly R 2 and R 3 are methyl;
- A is -O-;
- n is an integer from 1 to 4; in particular n is an integer from 1 to 3; more particularly n is an integer from 1 to 2; still more particularly n is 1; and m is an integer from 1 to 4; in particular m is an integer from 1 to 3; more particularly m is an integer from 1 to 2; still more particularly m is 1.
- the compounds of Formula I are those of Formula VIII: VIII or pharmaceutically acceptable salts or solvates thereof, wherein
- A, n and m are as defined above with respect to Formula I and any of its embodiments.
- Preferred compounds of Formula V or pharmaceutically acceptable salts or solvates thereof are those wherein one or more of A, n and m are defined as follows:
- A is -O-; n is an integer from 1 to 4; in particular n is an integer from 1 to 3; more particularly n is an integer from 1 to 2; still more particularly n is 1; and m is an integer from 1 to 4; in particular m is an integer from 1 to 3; more particularly m is an integer from 1 to 2; still more particularly m is 1.
- the compounds of the invention can be prepared by different ways with reactions known by the person skilled in the art. Reaction schemes as described in the Examples section illustrate by way of example different possible approaches.
- the compounds of the invention are indeed inhibitors, of NAD kinases, in particular PaNADK enzyme, LmNADK enzyme and/or HsNADK enzyme.
- the invention thus also provides the use of the compounds of the invention or pharmaceutically acceptable salts or solvates thereof as inhibitors of NAD kinases, in particular PaNADK enzyme, LmNADK enzyme and/or HsNADK enzyme.
- the invention relates to the use of compounds of Formula I or any of its subformulae, in particular those of Table 1 above, or pharmaceutically acceptable salts or solvates thereof, in the prevention and/or treatment of bacterial infections, in particular Gram-negative infections or Gram-positive infections, more particularly Gram-positive coccus infections.
- the inventors have demonstrated that the compounds of formula I or any of its subformulae, or pharmaceutically acceptable salts or solvates thereof, according to the present invention have the ability to inhibit NAD kinases, in particular PaNADK enzyme, LmNADK enzyme and HsNADK enzyme.
- the compounds of the invention or pharmaceutically acceptable salts or solvates thereof are therefore useful in the prevention and/or treatment of bacterial infections, in particular Gram-negative infections or Gram-positive infections, more particularly Gram-positive coccus infections.
- the invention thus also relates to a compound of the invention or a pharmaceutically acceptable salt or solvate thereof for use in preventing and/or treating bacterial infections, in particular Gram-negative infections or Gram-positive infections, more particularly Gram-positive coccus infections.
- the invention also relates to a method of treating or preventing bacterial infections, in particular Gram-negative infections or Gram-positive infections, more particularly Gram-positive coccus infections, comprising the administration of a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt or solvate thereof, to a patient in need thereof.
- bacterial infections in particular Gram-negative infections or Gram-positive infections, more particularly Gram-positive coccus infections
- the patient is a warm-blooded animal, more preferably a human.
- the invention further provides the use of a compound of the invention or a pharmaceutically acceptable salt or solvates thereof for the manufacture of a medicament for use in treating or preventing bacterial infections, in particular Gram-negative infections or Gram-positive infections, more particularly Gram-positive coccus infections.
- the patient is a warm-blooded animal, more preferably a human.
- a compound of the invention or a pharmaceutically acceptable salt or solvate thereof for use in inhibiting a NAD kinase, in particular PaNADK enzyme, LmNADK enzyme and/or HsNADK enzyme, in a patient in need of such treatment, comprising administering to said patient an effective amount of a compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof.
- a NAD kinase in particular PaNADK enzyme, LmNADK enzyme and/or HsNADK enzyme
- the invention also provides a method for inhibiting, a NAD kinase, in particular PaNADK enzyme, LmNADK enzyme and/or HsNADK enzyme, in a patient in need of such treatment, which comprises administering to said patient an effective amount of a compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof.
- a NAD kinase in particular PaNADK enzyme, LmNADK enzyme and/or HsNADK enzyme
- the patient is a warm-blooded animal, and even more preferably a human.
- the compound of the invention may be administered as a pharmaceutical formulation in a therapeutically effective amount by any of the accepted modes of administration, preferably by intravenous or oral route.
- Therapeutically effective amount ranges are typically from 0.1 to 50 000 pg/kg of body weight daily, preferably from 1 000 to 40 000 pg/kg of body weight daily, depending upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound, the route and the form of administration, the indication towards which the administration is directed, and the preferences and experience of the medical practitioner involved.
- One of ordinary skill in the art of treating such diseases will be able in reliance upon personal knowledge, to ascertain a therapeutically effective amount of the compound of the present invention for a given bacterial infection.
- the compounds of the invention may be administered as part of a combination therapy.
- a combination therapy comprising coadministration of, and compositions and medicaments which contain, in addition to a compound of the present invention, a pharmaceutically acceptable salt or solvate thereof as active ingredient, additional therapeutic agents and/or active ingredients.
- Such multiple drug regimens often referred to as combination therapy, may be used in the treatment or prevention of any bacterial infections, particularly those defined above.
- the methods of treatment and pharmaceutical compositions of the present invention may employ the compounds of the invention or their pharmaceutical acceptable salts or solvates thereof in the form of monotherapy, but said methods and compositions may also be used in the form of multiple therapy in which one or more compounds of Formula I or their pharmaceutically acceptable salts or solvates are co-administered in combination with one or more other therapeutic agents.
- the invention also provides pharmaceutical compositions comprising a compound of the invention or a pharmaceutically acceptable salt or solvate thereof and at least one pharmaceutically acceptable excipient.
- the invention also covers pharmaceutical compositions which contain, in addition to a compound of the present invention, a pharmaceutically acceptable salt or solvate thereof as active ingredient, additional therapeutic agents and/or active ingredients.
- the invention also provides a compound of the invention or a pharmaceutically acceptable salt or solvate thereof for use in a therapeutic treatment in humans or animals.
- Another object of this invention is a medicament comprising at least one compound of the invention, or a pharmaceutically acceptable salt or solvate thereof, as active ingredient.
- the compounds of the invention may be formulated as a pharmaceutical preparation comprising at least one compound of the invention and at least one pharmaceutically acceptable excipient, and optionally one or more further pharmaceutically active compounds.
- such a formulation may be in a form suitable for oral administration (e.g. as a tablet, capsule, or as an ingestible solution), for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration (including ocular), intracerebral administration, sublingual administration, aerosol administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, etc.
- oral administration e.g. as a tablet, capsule, or as an ingestible solution
- parenteral administration such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion
- topical administration including ocular
- intracerebral administration sublingual administration
- aerosol administration for administration by inhalation, by a skin patch, by an implant, by a suppository, etc.
- Such suitable administration forms which may be solid, semi-solid or liquid, depending on the manner of administration - as well as methods and carriers, diluents and excipients for use in the preparation thereof, will be clear to the skilled person; reference is made to the latest edition of Remington’s Pharmaceutical Sciences.
- the compound of the invention or a pharmaceutical composition comprising a compound of the invention can be administered orally in the form of tablets, coated tablets, pills, capsules, soft gelatin capsules, oral powders, granules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.
- the tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, a disintegrant such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, a binder such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia, a lubricant such as magnesium stearate, stearic acid, glyceryl behenate.
- solid compositions of a similar type may also be employed as fillers in hard gelatin capsules.
- Preferred excipients in this regard include lactose, saccharose, sorbitol, mannitol, potato starch, com starch, amylopectin, cellulose derivatives or gelatin.
- Hard gelatin capsules may contain granules of the compound of the invention.
- Soft gelatin capsules may be prepared with capsules containing the compound of the invention, vegetable oil, waxes, fat, or other suitable vehicle for soft gelatin capsules.
- the acceptable vehicle can be an oleaginous vehicle, such as a long chain triglyceride vegetable oil (e.g. com oil).
- Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water may contain the active ingredient in a mixture with dispersing agents, wetting agents, and suspending agents and one or more preservatives. Additional excipients, for example sweetening, flavouring and colouring agents, may also be present. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
- Liquid dosage forms for oral administration may include pharmaceutically acceptable, solutions, emulsions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water or an oleaginous vehicle.
- Liquid dosage form may be presented as a dry product for constitution with water or other suitable vehicle before use.
- compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, complexing agents such as 2-hydroxypropyl-beta-cyclodextrin, sulfobutylether-beta-cylodextrin, and sweetening, flavouring, perfuming agents, colouring matter or dyes with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
- adjuvants such as wetting agents, emulsifying and suspending agents, complexing agents such as 2-hydroxypropyl-beta-cyclodextrin, sulfobutylether-beta-cylodextrin, and sweetening, flavouring, perfuming agents, colouring matter or dyes with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
- an antioxidant such as butylated hydroxyanisol or al
- Finely divided powder of the compound of the invention may be prepared for example by micronisation or by processes known in the art.
- the compound of the invention may be milled using known milling procedures such as wet milling to obtain a particle size appropriate for tablet formation and for other formulation types.
- examples of such administration include one or more of: intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrastemally, intracranially, intramuscularly or subcutaneously administering the agent; and/or by using infusion techniques.
- the compound of the invention can be administered via the parenteral route with a ready available or a depot-type formulation.
- compositions for the parenteral administration of a ready available formulation may be in the form of a sterile injectable aqueous or oleagenous solution or suspension in a non-toxic parenterally- acceptable diluent or solvent and may contain formulatory agents such as suspending, stabilising dispersing, wetting and/or complexing agents such as cyclodextrin e.g. 2-hydroxypropyl-beta-cyclodextrin, sulfobutylether-beta- cylodextrin.
- formulatory agents such as suspending, stabilising dispersing, wetting and/or complexing agents such as cyclodextrin e.g. 2-hydroxypropyl-beta-cyclodextrin, sulfobutylether-beta- cylodextrin.
- the depot-type formulation for the parenteral administration may be prepared by conventional techniques with pharmaceutically acceptable excipient including without being limited to, biocompatible and biodegradable polymers (e.g. poly(P-caprolactone), poly(ethylene oxide), poly(glycolic acid), poly[(lactic acid)-co-(glycolic acid)...)], poly(lactic acid)...), non-biodegradable polymers (e.g. ethylene vinylacetate copolymer, polyurethane, polyester( amide), polyvinyl chloride%) aqueous and non-aqueous vehicles (e.g.
- biocompatible and biodegradable polymers e.g. poly(P-caprolactone), poly(ethylene oxide), poly(glycolic acid), poly[(lactic acid)-co-(glycolic acid)...)], poly(lactic acid)
- non-biodegradable polymers e.g. ethylene vinylacetate copolymer, polyurethane, polyester( amide), polyvinyl chloride
- the active ingredient may be in dry form such as a powder, crystalline or freeze-dried solid for constitution with a suitable vehicle.
- suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
- the compound of the present invention can be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, (for example from Ineos Fluor), carbon dioxide or other suitable gas.
- a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, (for example from Ineos Fluor), carbon dioxide or other suitable gas.
- a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, (for example from Ineos Fluor), carbon dioxide or other suitable gas
- Capsules and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound and a suitable powder base such as lactose or starch.
- a suitable powder base such as lactose or starch.
- the compound or salt of formula (I) is in a particle-size-reduced form, and more preferably the size-reduced form is obtained or obtainable by micronisation.
- the preferable particle size of the size-reduced (e.g. micronised) compound or salt or solvate is defined by a D50 value of about 0.5 to about 50 microns (for example as measured using laser diffraction).
- the compound of the present invention can be administered in the form of a suppository or pessary, or it may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder.
- the compound of the present invention may also be dermally or transdermally administered, for example, by the use of a skin patch. They may also be administered by the pulmonary or rectal routes. It may also be administered by the ocular route.
- the compound can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, it may be formulated in an ointment such as petrolatum.
- the agent of the present invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
- it can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
- any reference to compounds of the invention herein means the compounds as such as well as their pharmaceutically acceptable salts and solvates.
- halo refers to the atoms of the group 17 of the periodic table (halogens) and includes in particular fluorine (F), chlorine (C1), bromine (Br) and iodine (I) atom.
- Preferred halo groups are fluoro (F) and chloro (C1), fluoro being particularly preferred.
- alkyl by itself or as part of another substituent refers to a hydrocarbyl radical of Formula CnEhn+i wherein n is a number greater than or equal to 1.
- Alkyl groups may thus comprise 1 or more carbon atoms and generally, according to this invention comprise from 1 to 12, more preferably from 1 to 8 carbon atoms, and still more preferably from 1 to 6 carbon atoms.
- Alkyl groups within the meaning of the invention may be linear or branched.
- alkyl groups include but are not limited to methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, sec-pentyl, isopentyl, hexyl and isohexyl.
- alkenyl by itself or as part of another substituent refers to a hydrocarbyl radical at least one double carbon-carbon bond.
- Alkenyl groups may thus comprise 2 or more carbon atoms and generally, according to this invention comprise from 2 to 12, more preferably from 2 to 8 carbon atoms, and still more preferably from 2 to 6 carbon atoms.
- alkynyl by itself or as part of another substituent refers to a hydrocarbyl radical comprising at least one triple carbon-carbon bond.
- Alkynyl groups may thus comprise 2 or more carbon atoms and generally, according to this invention comprise from 2 to 12, more preferably from 2 to 8 carbon atoms, and still more preferably from 2 to 6 carbon atoms.
- alkoxy by itself or as part of another substituent refers to a -O-alkyl group, wherein alkyl is as defined above.
- alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, isoproxy, butoxy, sec-butoxy, isobutoxy, tert-butoxy, pentoxy, .scc-pcntoxy and isopentoxy.
- aminoalkyl by itself or as part of another substituent refers to a -alkyl-NHi group, wherein alkyl is as defined above.
- alkyloxycarbonyl by itself or as part of another substituent refers to a -C(O)- O-alkyl group, wherein alkyl is as defined above.
- haloalkyl or “halogenoalkyl” alone or in combination, refers to an alkyl radical having the meaning as defined above wherein one or more hydrogens are replaced with a halogen as defined above.
- Non-limiting examples of such haloalkyl radicals include chloromethyl, 1 -bromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1,1,1- trifluoroethyl and the like.
- cycloalkyl as used herein is a monovalent, saturated, or unsaturated monocyclic or bicyclic hydrocarbyl group.
- Cycloalkyl groups may comprise 3 or more carbon atoms in the ring and generally, according to this invention comprise from 3 to 10, more preferably from 3 to 8 carbon atoms, and still more preferably from 3 to 6 carbon atoms.
- Examples of cycloalkyl groups include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
- heteroatom refers to any atom that is not carbon or hydrogen.
- Non-limiting examples of such heteroatoms include nitrogen, oxygen, sulfur, and phosphorus.
- Preferred heteroatoms according to the invention are nitrogen, oxygen and sulfur.
- heteroalkyl refers to an alkyl radical having the meaning as defined above wherein one or more carbons are replaced with a heteroatom as defined above. Preferred heteroatoms are nitrogen and oxygen.
- heterocyclyl refers to non-aromatic, fully saturated or partially unsaturated cyclic groups (for example, 3 to 7 member monocyclic, 7 to 11 member bicyclic, or containing a total of 3 to 10 ring atoms) which have at least one heteroatom in at least one carbon atom-containing ring.
- Each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3 or 4 heteroatoms selected from nitrogen, oxygen and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quatemized.
- heterocyclic group may be attached at any heteroatom or carbon atom of the ring or ring system, where valence allows.
- heterocyclyl groups include but are not limited to aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, azepanyl, piperazinyl, morpholinyl.
- Preferred heterocyclyl groups according to the invention are azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl and morpholinyl.
- aryl refers to a polyunsaturated, aromatic hydrocarbyl group having a single ring (i.e. phenyl) or multiple aromatic rings fused together (e.g. naphthyl), typically containing 5 to 12 atoms; preferably 6 to 10, wherein at least one ring is aromatic.
- aryl groups include but are not limited to phenyl, biphenyl, 1 -naphthyl (or naphthalene- 1-yl), 2-naphthyl (or naphthalene-2-yl), anthracenyl, indanyl, indenyl, 1 ,2,3,4- tetrahydronaphthyl.
- Preferred aryl group according to the invention is phenyl.
- heteroaryl refers but is not limited to 5 to 12 carbon-atom aromatic rings or ring systems containing 1 to 2 rings which are fused together, typically containing 5 to 6 atoms; at least one of which is aromatic, in which one or more carbon atoms in one or more of these rings is replaced by oxygen, nitrogen and/or sulfur atoms where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized.
- heteroaryl groups include but are not limited to pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, furanyl, benzofuranyl, pyrrolyl, indolyl, thiophenyl, benzothiophenyl, imidazolyl, benzimidazolyl, pyrazolyl, indazolyl, oxazolyl, benzoxazolyl, isoxazolyl, benzisoxazolyl, thiazolyl, and benzothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, dioxazolyl, dithiazolyl and tetrazolyl.
- Preferred heteroaryl group according to the invention is thiazolyl.
- haloaryl or “halogenoaryl” alone or in combination, refers to an aryl radical having the meaning as defined above wherein one or more hydrogens are replaced with a halogen as defined above.
- the compounds of the invention containing a basic functional group may be in the form of pharmaceutically acceptable salts.
- Pharmaceutically acceptable salts of the compounds of the invention containing one or more basic functional groups include in particular the acid addition salts thereof. Suitable acid addition salts are formed from acids which form nontoxic salts.
- Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, cinnamate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate,
- the salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.
- the degree of ionization in the salt may vary from completely ionized to almost nonionized.
- solvate is used herein to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol.
- solvent molecules for example, ethanol.
- hydrate is employed when said solvent is water.
- the compounds of the invention include compounds of the invention as hereinbefore defined, including all polymorphs and crystal habits thereof, prodrugs and isomers thereof (including optical, geometric and tautomeric isomers) and isotopically-labeled compounds of the invention.
- salts of the compounds of the invention are preferred, it should be noted that the invention in its broadest sense also includes non-pharmaceutically acceptable salts, which may for example be used in the isolation and/or purification of the compounds of the invention.
- non-pharmaceutically acceptable salts which may for example be used in the isolation and/or purification of the compounds of the invention.
- salts formed with optically active acids or bases may be used to form diastereoisomeric salts that can facilitate the separation of optically active isomers of the compounds of the invention.
- patient refers to a warm-blooded animal, more preferably a human, who/which is awaiting or receiving medical care or is or will be the object of a medical procedure.
- human refers to subjects of both genders and at any stage of development (i.e. neonate, infant, juvenile, adolescent, adult). In one embodiment, the human is an adolescent or adult, preferably an adult.
- treat are meant to include alleviating or abrogating a condition or disease and/or its attendant symptoms.
- therapeutically effective amount means the amount of active agent or active ingredient which is sufficient to achieve the desired therapeutic or prophylactic effect in the individual to which it is administered.
- administration means providing the active agent or active ingredient, alone or as part of a pharmaceutically acceptable composition, to the patient in whom/which the condition, symptom, or disease is to be treated.
- pharmaceutically acceptable is meant that the ingredients of a pharmaceutical composition are compatible with each other and not deleterious to the patient thereof.
- excipient means a substance formulated alongside the active agent or active ingredient in a pharmaceutical composition or medicament. Acceptable excipients for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington’s Pharmaceutical Sciences, 21 st Edition 2011. The choice of excipient can be selected with regard to the intended route of administration and standard pharmaceutical practice. The excipient must be acceptable in the sense of being not deleterious to the recipient thereof.
- the at least one pharmaceutically acceptable excipient may be for example, a binder, a diluent, a carrier, a lubricant, a disintegrator, a wetting agent, a dispersing agent, a suspending agent, and the like.
- Figure 1 Inhibition of the enzymatic activity of PaNADK by compound 1.
- A The steady state rate constants of PaNADK activity were determined at 25 nM PaNADK, 4 mM MgATP, 0.05-1 mM NAD + and at different concentrations of compound 1, as indicated in the figure. Global competitive inhibition fittings are shown as lines.
- B Lineweaver-Burk representation of the data showing a significant competitive inhibition of PaNADK by compound 1.
- FIG. 1 Evaluation of compound 1 as a novel anti-Pseudomonas molecule.
- A Effect of compound 1 on P aeruginosa growth in liquid LB cultures. P aeruginosa growth curves in presence of compound 1 at 20 pM (in 0.1% DMSO) or in presence of 0.1% DMSO as control. Graphs represent the mean of three independent experiments.
- B The efficacy of compound 1 against P aeruginosa was tested with embryos injured in the tail fin and bath infected with PAO1 suspension at approximately 8.10 7 CFU/mL in presence of compound 1 at 20 pM (in 0.1% DMSO). Injured embryos in the control group were treated with PA01 suspension in presence of DMSO at 0.1%.
- APTS p-toluenesulfonic acid
- DNA deoxyribonucleic acid
- EDTA ethylenediaminetetraacetic acid
- ESI electrospray ionization
- g gram(s)
- h hour(s)
- HsNADK human cytosolic NADK
- LmNADK NADK from Listeria monocytogenes
- NAD + nicotinamide adenine dinucleotide
- PaNADK NADK from Pseudomonas aeruginosa
- PBS Phosphate Buffered Saline PCR: Polymerase Chain Reaction
- TEAA triethylammonium acetate
- UV ultra-violet wt: weight
- HPLC purification was carried out on an Agilent system (1100 Series) equipped with a diode array detector using a C18 reverse phase column (Kromasil, 5 pm, 100 A, 250x10 mm) and a linear gradient of acetonitrile in 10 mM triethylammonium acetate (TEAA) buffer over 15 or 20 min at a flow rate of 4 mL/min. Retention time (R t ) and elution conditions are specified. The purity of tested compounds was at least 96% pure as determined by HPLC. NMR spectra were recorded on Bruker Avance 400 ( ⁇ H at 400.13 MHz and 13 C at 100.62 MHz). Chemical shifts ( ⁇ 5) are reported in ppm relative to the solvent signals.
- Reagents and conditions (a) 5 -bromobenzonitrile, nBuLi, THF, -78 °C; MeOH -78 °C to rt; (b) BF 3 -OEt 2 , Et 3 SiH, -0 °C to rt; (c) TMSOK, THF, reflux; (d) 1 M BBr 3 in DCM, - 78 °C to rt; (e) APTS, acetone, 2,2-dimethoxypropane; (f) Propargyl bromide, NaH, DMF,
- reaction mixture was heated at 60°C for 1 h under argon atmosphere, concentrated under reduced pressure and the residue was purified by flash column chromatography (40 g SiO 2 , 0 to 5% MeOH in DCM) affording 19 (145 mg, 50%).
- reaction mixture was concentrated to dryness and the residue was purified by flash column chromatography (30 g SiO 2 , 3 to 6% MeOH in DCM; 20 g SiO 2 , 2 to 3% MeOH in ethylacetate) affording 25 (0.14 g 25%).
- the reaction mixture was concentrated to dryness and the residue was purified by flash column chromatography (20 g SiO 2 , 2 to 4% MeOH in DCM) affording a mixture of coupling and homocoupling products (4/1 ratio, 0.55 g), which was used in the next step without further purification.
- the mixture of coupling and homocoupling products (0.54 g) was first treated with aq. ammonia (1 mL) in MeOH (4 mL) at room temperature overnight. After removal of the volatiles, the residue was purified by flash column chromatography (20 g SiO2, 3 to 10% MeOH in DCM) affording deacetylated coupling product 27b (0.35 g, 78% in two steps).
- reaction mixture was heated at 60°C for 1 h under argon atmosphere.
- the reaction mixture was concentrated to dryness and the residue was purified by flash column chromatography (40 g SiO 2 , 0 to 8% MeOH in DCM) affording 35 (0.20 g containing 0.11 eq triethylammonium salt), which was used in the next step without further purification.
- Bromide 38 Compound 38 was synthesized as described previously in Clement et al., Eur. J. Med. Chem.2023, 246, 114941. Bromide 39 To a solution of bromide 38 (0.58 g, 1.5 mmol) in DMF (15 mL) was added di-tert-butyl dicarbonate (1.10 g, 2.25mmol). After 18 hours at room temperature, volatiles were removed under reduced pressure and the residue was purified by flash column chromatography (35 g SiO2, 2 to 5% MeOH in DCM) to give 39 (0.53 g, 73%).
- EGD-e monocytogenes strain EGD-e by using the Vent DNA polymerase, dNTPs, and the following primers: 5′-GGAATTCCATATGAAATATATGATTACTTCCAAAGGA- 3′, and 5′-CGGCGCTCGAGTTAATCTTCAATAAACGAATCGTGTAC-3′.
- the amplified DNA was cloned into the expression vector pET22b (Novagen) at the NdeI and XhoI restriction sites, and the corresponding plasmid was used for transforming the E. coli strain BL21(DE3)/pDIA17 for protein expression.
- Transformants were grown at 37 °C in 2YT medium (Difco) in the presence of chloramphenicol and ampicillin at 30 ⁇ g/mL. When the absorbance reached 1.5 at 600 nm, the expression of the recombinant protein was induced by the addition of 1 mM isopropyl- ⁇ -D-1-thiogalactopyranoside, and growth was continued for three more hours at 37 °C. The cells were then pelleted by centrifugation and served as source for protein purification. Soluble protein was purified using cobalt-agarose affinity chromatography followed by size exclusion chromatography.
- the protein was concentrated to 6–7 mg/mL in 50 mM KH2PO4 pH 7.5 and 100 mM NaCl, aliquoted, flash frozen in liquid nitrogen and stored at –20 °C.
- the sequence of the recombinant protein corresponds to the sequence of L. monocytogenes NADK 1 (UniProtKB Q8Y8D7) with a LEHHHHHH tag at the C-terminus.
- the expression, solubility and purity of LmNADK were tested on SDS-PAGE gel (InvitrogenTM NuPAGETM 4-12 % gel).
- the molar absorption coefficient and the absorbance value for 0.1% of the 6His-tagged protein were estimated with the ABIM software (http://bioserv.cbs.cnrs.fr/ABIM/w3bb/d_abim/) at 26,740 M -1 cm -1 and 0.863 (g/L) -1 cm -1 , respectively.
- ABIM software http://bioserv.cbs.cnrs.fr/ABIM/w3bb/d_abim/
- PaNADK protein was overexpressed in E. coli BL21 (DE3) cells in 2-YT broth with 100 pg/mL ampicillin. The protein expression was induced at ODeoo of 0.7-0.8 with 1 mM isopropyl P-D-l -thiogalactopyranoside (IPTG) overnight at 25 °C.
- the harvested cells were re-suspended in buffer A (50 mM sodium phosphate pH 8.3, 150 mM NaC1, 10 mM imidazole, 2 mM DTT) supplemented with 1 tablet of cOmpleteTM EDTA-free protease inhibitor cocktail (Roche), 5 pg/mL of lysozyme (Euromedex), 5 pg/mL of DNase (Sigma Aldrich).
- Bacterial cell disruption was achieved by sonication at 40% amplitude during 5 minutes (5 sec on, 5 sec off) with 6 mm probe on ice. Cellular debris were removed by centrifugation at 20,000 g for 30 minutes and followed by filtration through 0.45 pm syringe filter.
- the bacteria lysate was loaded onto a HisTrap FF column equilibrated with buffer A using Akta Pure system (GE Healthcare) at 4 °C.
- the column was washed with buffer A during 15 column volumes to remove unspecific binding proteins, followed by elution using buffer B (50 mM sodium phosphate pH 8.3, 150 mM NaC1, 500 mM imidazole, 2 mM DTT) with a linear gradient of imidazole from 10 mM to 500 mM in 20 column volumes.
- the fractions containing the protein were pooled and concentrated using Amicon Ultra 15 centrifugal filters (3 kDa, Merck Millipore) at 4 °C.
- the concentrated protein was loaded onto a 16/60 Superdex 200 pre-equilibrated with buffer C (50 mM NaH2PO4/Na2HPO4 pH 8.3, 100 mM NaC1, 5 mM DTT). Finally, the protein was concentrated till 30 mg/mL, aliquoted, flash frozen in liquid nitrogen and stored at -80 °C.
- the sequence of the recombinant protein corresponds to the sequence of P aeruginosa PAG NADK (UniProtKB Q9HZC0) with a LEHHHHHH tag at the C-terminus.
- HsNADK Molecular cloning, expression and purification of the HsNADK gene
- LmNADK the coding region of cytoplasmic HsNADK was amplified using standard PCR cloning (see above) and cloned into the same over-expression plasmid pET22b (Novagen) at the Ndel and Xhol restriction sites.
- the corresponding plasmid was sequenced and then used for transforming the E. coli strain BL21(DE3)/pDIA17 for protein over-expression.
- HsNADK protein was overexpressed in E. coli BL21 (DE3) cells in auto inducible ZYM broth with 100 pg/mL ampicillin. The bacteria were incubated 5 h at 37 °C and overnight at 25 °C. The harvested cells were re-suspended in buffer A (50 mM Tris pH 8.0, 150 mM NaC1, 2 mM DTT) supplemented with 1 tablet of cOmpleteTM EDTA-free protease inhibitor cocktail (Roche) and 5 pg/mL of lysozyme (Euromedex). Bacterial cell disruption was achieved by sonication at 40% amplitude during 5 minutes (5 sec on, 5 sec off) with 6 mm probe on ice.
- the fractions containing the protein were pooled and concentrated using Amicon Ultra 15 centrifugal filters (10 kDa, Merck Millipore) at 4 °C.
- the concentrated protein was loaded onto a 26/60 Superdex 200 preequilibrated with buffer A.
- the protein was dialyzed 3 h and overnight against buffer C (50 mM Tris pH 7.5, 2 mM DTT), concentrated till 10 mg/mL, aliquoted, flash frozen in liquid nitrogen and stored at -80 °C.
- the sequence of the recombinant protein corresponds to the sequence of H. sapiens cytosolic NADK (UniProtKB 095544) with a LEHHHHHH tag at the C-terminus.
- HsNADK 6His-tagged protein
- the enzymatic reaction was followed at 30 °C in a CLARIOstar plate reader (BMG LABTECH) by measuring the absorbance at 340 nm (OD340) using an enzymatic coupled system involving glucose-6-phosphate dehydrogenase (G6PDH).
- the reaction mixture was made in a half area 96-well microplate (Greiner Bio-One C1ear UV-Star).
- Buffers used were 50 mM Bis-Tris pH 7.0, 100 mM NaC1, 1 mM MgCh with HsNADK, 50 mM BisTris pH 7.0, 2 mM NaCitrate Tribasic Dihydrate, 1 mM MgCh with LmNADK and 50 mM Tris pH 7.5, 1 mM MgCh, 5 mM DTT with PaNADK.
- Reaction mixtures contained 25 nM NADK, 0.1-2 mM NAD (with HsNADK or LmNADK) or 0.05-1 mM NAD (with PaNADK), 4 mM MgATP, 5 mM G6P, 1 U/mL G6PDH and different concentrations of inhibitors.
- Buffer, NAD and inhibitor solutions (total volume 25 pL, concentration 4x) were dispensed into the microplate using an OT-2 lab robot (Opentrons).
- the 25 pL of mixture containing G6PDH, MgATP and glucose-6-phosphate at a 4x concentration were dispensed using a manual repeating pipette.
- the reaction was started at time 0 by adding and mixing 50 pL of NADK at a 2x concentration using a multichannel pipette.
- OD340 were measured every 32 s during 32 min.
- the values of the inhibition constants were determined from the raw values of k ss at different concentrations of NAD and inhibitor using a competitive inhibition equation (Eq. 1).
- OD340 values were converted to concentrations of produced NADH (i.e. NAD consumed) using a molar absorption coefficient for NADH of 6,220 M' 1 cm' 1 .
- the value of the steady state rate constant (k ss ) given in the text corresponds to the initial slope of NAD consumed per second divided by the concentration of NADK.
- GraFit software version 7.0.3, Erithacus Software Ltd.
- the synthesized compounds were assayed for cytotoxicity on MRC-5 (human fetal lung fibroblasts) cells.
- the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 25 mM glucose, 10% (v/v) serum, 1% penicillin/streptomycin and kept under 5% CO2 at 37°C.
- DMEM Dulbecco’s modified Eagle’s medium
- the cell viability was assessed using the CellTiter Gio kit from Promega (G7572) after 72 h incubation time using four concentrations of compounds (from 1 to 50 pM) in triplicate. No cytotoxicity was observed up to concentrations of 50 ⁇ M
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Abstract
The inventors have now succeeded in developing compounds of Formula (I), described below, having the advantage of inhibiting NAD kinases, in particular P. aeruginosa NADK (PaNADK) enzyme, Listeria monocytogenes (LmNADK) enzyme and/or human cytosolic NADK (HsNADK) enzyme. The present invention relates to benzamide adenine dinucleoside compounds which are useful as inhibitors of NAD kinases, to pharmaceutical composition comprising such compounds, and to uses of such compounds in the treatment or prevention of bacterial infections.
Description
BENZAMIDE ADENINE DINUCLEOSIDE COMPOUNDS AND THEIR USES
The present invention relates to benzamide adenine dinucleoside compounds which are useful as inhibitors of NAD kinases, to pharmaceutical composition comprising such compounds, and to uses of such compounds in the treatment or prevention of bacterial infections.
BACKGROUND OF THE INVENTION
The environmental bacterium and opportunistic human pathogen Pseudomonas aeruginosa is a Gram-negative bacterium responsible for a variety of acute infections and is a major cause of mortality in chronically infected cystic fibrosis patients. Due to increased resistance to antibiotics, P aeruginosa has been listed by the World Health Organization (WHO) among pathogenic bacteria for which new antibiotics or alternative therapeutic strategies are urgently needed (Tacconelli et al., Lancet Infect. Dis. 2018, 18, 318-327).
Bacterial nicotinamide adenine dinucleotide kinase (NADK, EC 2.7.1.23) has been identified as a promising target for the development of new antibiotics (Gelin et al., Structure 2012, 20, 1107-1117), NADK being the sole enzyme that phosphorylates nicotinamide adenine dinucleotide (NAD+) to NADP+. NADP+ is an essential coenzyme converted by different redundant enzymes into NADPH, to provide reducing power in many processes such as oxidative stress response, fatty acid and nucleotide biosynthesis (Agledal et al., Redox Rep. 2010, 15, 2-10; Pollak et al., Biochem. J. 2007, 402, 205-218). NADK is therefore an essential enzyme for rapidly dividing cells, which need both high macromolecular turnover and an efficient response to oxidative stress, and therefore require high level of NADPH (Grose et al., Proc. Natl. Acad. Sci. USA 2006, 103, 7601- 7606). This is particularly important for bacteria in the context of antibiotic treatment. In fact, three main classes of antibiotics (quinolones, beta-lactams and aminoglycosides) stimulate the production of reactive oxygen species (ROS), which can participate in bacterial cell death (Van Acker et al., Trends Microbiol. 2017, 25, 456-466). In addition, inactivation of the gene encoding NADK is fatal for all bacteria tested so far (Gerdes et al., J. Bacteriol. 2002, 184, 4555-4572; Thanassi et al., Nucleic Acids Res. 2002, 30, 3152-
3162; Zalacain et al., J. Mol. Microbiol. Biotechnol. 2003, 6, 109-126; Kobayashi et al., Proc. Natl. Acad. Sci. USA 2003, 100, 4678-4683; Sassetti et al., Mol. Microbiol. 2003, 48, 77-84; Spaans et al., Front Microbiol. 2015, 6, 742).
The originality of the enzymatic mechanism of NADK and of its NAD+ binding mode has been shown by a structural approach (Poncet-Montange et al., J. Biol. Chem. 2007, 282, 33925-33934). Based on the analysis of the structure of the NADK 1 from Listeria monocytogenes (LmNADK), a class of antibacterial compounds that specifically mimics the particular conformation that NAD+ adopts in the NADK binding site was developed. Selective inhibition of nicotinamide adenine dinucleotide kinases by dinucleoside disulfide mimics of nicotinamide adenine dinucleotide analogues (Petrelli et al., Bioorg Med Chem. 2009, 17, 5656-5664). Compounds targeting bacterial NADKs were synthesized (Paoletti et al., Eur. J. Med. Chem. 2016, 124, 1041-1056). A propargyl-linked di-adenosine compound active against the Gram-positive bacterium Staphylococcus aureus both in vitro and in vivo in a mouse animal model of infection was also developed (Gelin et al., ACS Infect. Dis. 2020, 6, 422-435).
There is however still a need for compounds active as inhibitors of NAD kinases, in particular on P aeruginosa NADK (hereafter PaNADK) enzyme, Listeria monocytogenes (hereafter LmNADK) enzyme and/or human cytosolic NADK (hereafter HsNADK) enzyme.
SUMMARY OF THE INVENTION
The inventors have now succeeded in developing compounds of Formula I, described below, having the advantage of inhibiting NAD kinases, in particular P aeruginosa NADK (PaNADK) enzyme, Listeria monocytogenes (LmNADK) enzyme and/or human cytosolic NADK (HsNADK) enzyme.
These compounds are useful in the prevention and/or treatment of bacterial infections, in particular Gram-negative infections (against P aeruginosa for example) and Gram-positive infections, more particularly Gram-positive coccus infections (especially S. aureus).
The invention therefore relates to compounds of general Formula I, their pharmaceutically acceptable salts or solvates thereof, as well as methods of use of such compounds or compositions comprising such compounds as inhibitors of NAD kinases.
In a general aspect, the invention provides compounds of general Formula I:
its tautomeric forms, stereoisomeric forms, mixture of stereoisomeric forms, pharmaceutically acceptable salts or solvates thereof, wherein R1 is selected from the group consisting of -OH, -NHC(=O)NR2R3 and -NHSO2NR2R3, F, NR2R3, OR4, N3 and NH2 wherein R2 and R3 are independently selected from H, C1-C6-alkyl and C1-C12- heteroalkyl comprising one or more oxygen atom, and R4 is C1-C6-alkyl or C1- C12-heteroalkyl comprising one or more oxygen atom; n is an integer from 1 to 4; m is an integer from 1 to 4;
Y is CH or N;
Z is O or S; and
A is selected from the group consisting of -O-, -S-, -Se- and N(CH2)PR5 wherein p is an integer from 1 to 6; and R5 is selected from the group consisting of -NH2, -C=CH, -COOH, -COOR6 and -OR6 wherein R6 is C1-C6-alkyl; or
R1 and A form together -NHC(=O)(CH2)qNHC(=O)(CH2)rN wherein q and r are independently integers from 1 to 6.
In another aspect, the present invention provides a pharmaceutical composition comprising at least one compound according to the invention or a pharmaceutically acceptable salt or solvate thereof and at least one pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.
The invention further relates to compounds of Formula I or their pharmaceutically acceptable salts or solvates thereof for use in the prevention and/or treatment of bacterial infections.
DETAILED DESCRIPTION OF THE INVENTION
As detailed above, the invention relates to compounds of Formula I, as well as their tautomeric forms, stereoisomeric forms, mixture of stereoisomeric forms, pharmaceutically acceptable salts or solvates.
Preferred compounds of Formula I or pharmaceutically acceptable salts or solvates thereof are those wherein one or more of X, R1, R2, R3 and R4 are defined as follows:
R1 is selected from the group consisting of -OH, -NHC(=O)NR2R3 and -NHSO2NR2R3 wherein R2 and R3 are independently H or C1-C6-alkyl, in particular R2 and R3 are independently H or C1-C4-alkyl, more particularly R2 and R3 are independently H or C1- C2-alkyl, still more particularly R2 and R3 are H or methyl; for example, R1 is selected from the group consisting of -OH, -NHC(=O)NHR2 and -NHSO2NR2R3 wherein R2 and R3 are independently C1-C6-alkyl, in particular R2 and R3 are independently C1-C4-alkyl,
more particularly R2 and R3 are independently C1-C2-alkyl, still more particularly R2 and R3 are methyl; in a particular example R1 is selected from the group consisting of -OH, - NH2, -NHC(=O)NR2R3 and -NHSO2NR2R3 wherein R2 and R3 are independently H or C1- C6-alkyl, in particular R2 and R3 are independently H or C1-C4-alkyl, more particularly R2 and R3 are independently H or C1-C2-alkyl, still more particularly R2 and R3 are H or methyl; in a more particular example, R1 is selected from the group consisting of -OH, - NH2, -NHC(=O)NHR2 and -NHSO2NR2R3 wherein R2 and R3 are independently C1-C6- alkyl, in particular R2 and R3 are independently C1-C4-alkyl, more particularly R2 and R3 are independently C1-C2-alkyl, still more particularly R2 and R3 are methyl; n is an integer from 1 to 4; in particular n is an integer from 1 to 3; more particularly n is an integer from 1 to 2; still more particularly n is 1; m is an integer from 1 to 4; in particular m is an integer from 1 to 3; more particularly m is an integer from 1 to 2; still more particularly m is 1;
Y is CH or N; in particular Y is CH;
Z is O or S; in particular Z is O; and
A is -O- or N(CH2)PR5 wherein p is an integer from 1 to 6, in particular p is an integer from 1 to 4, more particularly p is 3, and R5 is -NH2 or -C=CH; or
R1 and A form together -NHC(=O)(CH2)qNHC(=O)(CH2)rN wherein q and r are independently integers from 1 to 6, in particular q and r are independently integers from 1 to 4, more particularly q and r are independently integers from 1 to 2, still more particularly q and r are 1.
In one embodiment, the compound of Formula I includes its tautomeric forms, stereoisomeric forms, mixture of stereoisomeric forms.
In one embodiment, the compounds of Formula I are those wherein n is 1.
In one embodiment, the compounds of Formula I are those wherein m is 1.
In one embodiment, the compounds of Formula I are those wherein n and m are 1.
In one embodiment, the compounds of Formula I are those wherein Y is CH.
In one embodiment, the compounds of Formula I are those wherein Z is O.
In one embodiment, the compounds of Formula I are those wherein R1 is OH.
In one embodiment, the compounds of Formula I are those wherein R1 is -NHC(=O)NHR2 wherein R2 is C1-C6-alkyl, in particular R2 is C1-C4-alkyl, more particularly R2 is C1-C2- alkyl, still more particularly R2 is ethyl.
In one embodiment, the compounds of Formula I are those wherein R1 is -NHSO2NR2R3 wherein R2 and R3 are C1-C6-alkyl, in particular R2 and R3 are C1-C4-alkyl, more particularly R2 and R3 are C1-C2-alkyl, still more particularly R2 and R3 are methyl.
In one embodiment, the compounds of Formula I are those wherein R1 is NH2.
In one embodiment, the compounds of Formula I are those wherein A is -O-.
In one embodiment, the compounds of Formula I are those wherein A is N(CH2)PR5 wherein p is an integer from 1 to 6, in particular p is an integer from 1 to 4, more particularly p is 3, and R5 is -NH2 or -C=CH.
In one embodiment, the compounds of Formula I are those wherein R1 and A form together -NHC(=O)(CH2)qNHC(=O)(CH2)rN wherein q and r are integers from 1 to 6, in particular q and r are integers from 1 to 4, more particularly q and r are integers from 1 to 2, still more particularly q and r are 1.
In one embodiment, the compounds of Formula I are those of Formula II:
II or pharmaceutically acceptable salts or solvates thereof, wherein R1 and A are as defined above with respect to Formula I and any of its embodiments.
Preferred compounds of Formula II or pharmaceutically acceptable salts or solvates thereof are those wherein one or more of R1 and A are defined as follows:
R1 is selected from the group consisting of -OH, -NHC(=O)NHR2 and -NHSO2NR2R3 wherein R2 and R3 are independently C1-C6-alkyl, in particular R2 and R3 are independently C1-C4-alkyl, more particularly R2 and R3 are independently C1-C2-alkyl, still more particularly R2 and R3 are methyl or ethyl; for example R1 is selected from the group consisting of -OH, -NH2, -NHC(=O)NHR2 and -NHSO2NR2R3 wherein R2 and R3 are independently C1-C6-alkyl, in particular R2 and R3 are independently C1-C4-alkyl, more particularly R2 and R3 are independently C1-C2-alkyl, still more particularly R2 and R3 are methyl or ethyl;
A is -O- or N(CH2)PR5 wherein p is an integer from 1 to 6, in particular p is an integer from 1 to 4, more particularly p is 3, and R5 is -NH2 or -C=CH; or
R1 and A form together -NHC(=O)(CH2)qNHC(=O)(CH2)rN wherein q and r are independently integers from 1 to 6, in particular q and r are independently integers from 1
to 4, more particularly q and r are independently integers from 1 to 2, still more particularly q and r are 1.
In one embodiment, the compounds of Formula I are those of Formula III:
III or pharmaceutically acceptable salts or solvates thereof, wherein
R1, n and m are as defined above with respect to Formula I and any of its embodiments.
Preferred compounds of Formula III or pharmaceutically acceptable salts or solvates thereof are those wherein one or more of R1, n and m are defined as follows:
R1 is selected from the group consisting of -OH, -NHC(=O)NHR2 and -NHSO2NR2R3 wherein R2 and R3 are independently C1-C6-alkyl, in particular R2 and R3 are independently C1-C4-alkyl, more particularly R2 and R3 are independently C1-C2-alkyl, still more particularly R2 and R3 are methyl or ethyl; for example R1 is selected from the group consisting of -OH, -NH2, -NHC(=O)NHR2 and -NHSO2NR2R3 wherein R2 and R3 are independently C1-C6-alkyl, in particular R2 and R3 are independently C1-C4-alkyl, more particularly R2 and R3 are independently C1-C2-alkyl, still more particularly R2 and R3 are methyl or ethyl;
n is an integer from 1 to 4; in particular n is an integer from 1 to 3; more particularly n is an integer from 1 to 2; still more particularly n is 1; and m is an integer from 1 to 4; in particular m is an integer from 1 to 3; more particularly m is an integer from 1 to 2; still more particularly m is 1. In one embodiment, the compounds of Formula I are those of Formula IV:
IV or pharmaceutically acceptable salts or solvates thereof, wherein R1, R5, n, m, and p are as defined above with respect to Formula I and any of its embodiments.
Preferred compounds of Formula IV or pharmaceutically acceptable salts or solvates thereof are those wherein one or more of R1, R5, n, m, and p are defined as follows:
R1 is -OH or -NHC(=O)NHR2 wherein R2 is C1-C6-alkyl, in particular R2 is C1-C4-alkyl, more particularly R2 is C1-C2-alkyl, still more particularly R2 is ethyl; n is an integer from 1 to 4; in particular n is an integer from 1 to 3; more particularly n is an integer from 1 to 2; still more particularly n is 1; and
m is an integer from 1 to 4; in particular m is an integer from 1 to 3; more particularly m is an integer from 1 to 2; still more particularly m is 1. p is an integer from 1 to 6; in particular p is an integer from 1 to 4; more particularly p is 3; and R5 is -NH2 or -C=CH.
In one embodiment, the compounds of Formula I are those of Formula V:
or pharmaceutically acceptable salts or solvates thereof, wherein
A, n and m are as defined above with respect to Formula I and any of its embodiments.
Preferred compounds of Formula V or pharmaceutically acceptable salts or solvates thereof are those wherein one or more of A, n and m are defined as follows:
A is -O- or N(CH2)PR5 wherein p is an integer from 1 to 6, in particular p is an integer from 1 to 4, more particularly p is 3, and R5 is -NH2 or -C=CH, in particular R5 is -NH2; n is an integer from 1 to 4; in particular n is an integer from 1 to 3; more particularly n is an integer from 1 to 2; still more particularly n is 1; and
m is an integer from 1 to 4; in particular m is an integer from 1 to 3; more particularly m is an integer from 1 to 2; still more particularly m is 1.
In one embodiment, the compounds of Formula I are those of Formula VI:
or pharmaceutically acceptable salts or solvates thereof, wherein
R2, A, n and m are as defined above with respect to Formula I and any of its embodiments.
Preferred compounds of Formula VI or pharmaceutically acceptable salts or solvates thereof are those wherein one or more of R2, A, n and m are defined as follows:
R2 is C1-C6-alkyl, in particular R2 is C1-C4-alkyl, more particularly R2 is C1-C2-alkyl, still more particularly R2 is ethyl;
A is -O- or N(CH2)PR5 wherein p is an integer from 1 to 6, in particular p is an integer from 1 to 4, more particularly p is 3, and R5 is -NH2 or -C=CH, in particular R5 is -C=CH; n is an integer from 1 to 4; in particular n is an integer from 1 to 3; more particularly n is an integer from 1 to 2; still more particularly n is 1; and
m is an integer from 1 to 4; in particular m is an integer from 1 to 3; more particularly m is an integer from 1 to 2; still more particularly m is 1.
In one embodiment, the compounds of Formula I are those of Formula VII:
VII or pharmaceutically acceptable salts or solvates thereof, wherein
R2, R3, A, n and m are as defined above with respect to Formula I and any of its embodiments. Preferred compounds of Formula VI or pharmaceutically acceptable salts or solvates thereof are those wherein one or more of R2, R3, A, n and m are defined as follows:
R2 and R3 are independently C1-C6-alkyl, in particular R2 and R3 are independently C1- C4-alkyl, more particularly R2 and R3 are independently C1-C2-alkyl, still more particularly R2 and R3 are methyl; A is -O-; n is an integer from 1 to 4; in particular n is an integer from 1 to 3; more particularly n is an integer from 1 to 2; still more particularly n is 1; and
m is an integer from 1 to 4; in particular m is an integer from 1 to 3; more particularly m is an integer from 1 to 2; still more particularly m is 1.
In one embodiment, the compounds of Formula I are those of Formula VIII:
VIII or pharmaceutically acceptable salts or solvates thereof, wherein
A, n and m are as defined above with respect to Formula I and any of its embodiments.
Preferred compounds of Formula V or pharmaceutically acceptable salts or solvates thereof are those wherein one or more of A, n and m are defined as follows:
A is -O-; n is an integer from 1 to 4; in particular n is an integer from 1 to 3; more particularly n is an integer from 1 to 2; still more particularly n is 1; and m is an integer from 1 to 4; in particular m is an integer from 1 to 3; more particularly m is an integer from 1 to 2; still more particularly m is 1.
The compounds of the invention can be prepared by different ways with reactions known by the person skilled in the art. Reaction schemes as described in the Examples section illustrate by way of example different possible approaches.
The compounds of the invention are indeed inhibitors, of NAD kinases, in particular PaNADK enzyme, LmNADK enzyme and/or HsNADK enzyme. The invention thus also provides the use of the compounds of the invention or pharmaceutically acceptable salts or solvates thereof as inhibitors of NAD kinases, in particular PaNADK enzyme, LmNADK enzyme and/or HsNADK enzyme.
Accordingly, in a particularly preferred embodiment, the invention relates to the use of compounds of Formula I or any of its subformulae, in particular those of Table 1 above, or pharmaceutically acceptable salts or solvates thereof, in the prevention and/or treatment of bacterial infections, in particular Gram-negative infections or Gram-positive infections, more particularly Gram-positive coccus infections.
APPLICATIONS
The inventors have demonstrated that the compounds of formula I or any of its subformulae, or pharmaceutically acceptable salts or solvates thereof, according to the present invention have the ability to inhibit NAD kinases, in particular PaNADK enzyme, LmNADK enzyme and HsNADK enzyme.
The compounds of the invention or pharmaceutically acceptable salts or solvates thereof are therefore useful in the prevention and/or treatment of bacterial infections, in particular Gram-negative infections or Gram-positive infections, more particularly Gram-positive coccus infections.
The invention thus also relates to a compound of the invention or a pharmaceutically acceptable salt or solvate thereof for use in preventing and/or treating bacterial infections, in particular Gram-negative infections or Gram-positive infections, more particularly Gram-positive coccus infections.
In other terms, the invention also relates to a method of treating or preventing bacterial infections, in particular Gram-negative infections or Gram-positive infections, more particularly Gram-positive coccus infections, comprising the administration of a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt or solvate thereof, to a patient in need thereof. Preferably the patient is a warm-blooded animal, more preferably a human.
The invention further provides the use of a compound of the invention or a pharmaceutically acceptable salt or solvates thereof for the manufacture of a medicament for use in treating or preventing bacterial infections, in particular Gram-negative infections
or Gram-positive infections, more particularly Gram-positive coccus infections. Preferably the patient is a warm-blooded animal, more preferably a human.
According to a further feature of the present invention, there is provided a compound of the invention or a pharmaceutically acceptable salt or solvate thereof for use in inhibiting a NAD kinase, in particular PaNADK enzyme, LmNADK enzyme and/or HsNADK enzyme, in a patient in need of such treatment, comprising administering to said patient an effective amount of a compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof. In other terms, the invention also provides a method for inhibiting, a NAD kinase, in particular PaNADK enzyme, LmNADK enzyme and/or HsNADK enzyme, in a patient in need of such treatment, which comprises administering to said patient an effective amount of a compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof. Preferably, the patient is a warm-blooded animal, and even more preferably a human.
According to the present invention, the compound of the invention may be administered as a pharmaceutical formulation in a therapeutically effective amount by any of the accepted modes of administration, preferably by intravenous or oral route.
Therapeutically effective amount ranges are typically from 0.1 to 50 000 pg/kg of body weight daily, preferably from 1 000 to 40 000 pg/kg of body weight daily, depending upon numerous factors such as the severity of the disease to be treated, the age and relative health of the subject, the potency of the compound, the route and the form of administration, the indication towards which the administration is directed, and the preferences and experience of the medical practitioner involved. One of ordinary skill in the art of treating such diseases will be able in reliance upon personal knowledge, to ascertain a therapeutically effective amount of the compound of the present invention for a given bacterial infection.
According to one embodiment, the compounds of the invention, their pharmaceutical acceptable salts or solvates may be administered as part of a combination therapy. Thus, are included within the scope of the present invention embodiments comprising coadministration of, and compositions and medicaments which contain, in addition to a
compound of the present invention, a pharmaceutically acceptable salt or solvate thereof as active ingredient, additional therapeutic agents and/or active ingredients. Such multiple drug regimens, often referred to as combination therapy, may be used in the treatment or prevention of any bacterial infections, particularly those defined above.
Thus, the methods of treatment and pharmaceutical compositions of the present invention may employ the compounds of the invention or their pharmaceutical acceptable salts or solvates thereof in the form of monotherapy, but said methods and compositions may also be used in the form of multiple therapy in which one or more compounds of Formula I or their pharmaceutically acceptable salts or solvates are co-administered in combination with one or more other therapeutic agents.
The invention also provides pharmaceutical compositions comprising a compound of the invention or a pharmaceutically acceptable salt or solvate thereof and at least one pharmaceutically acceptable excipient. As indicated above, the invention also covers pharmaceutical compositions which contain, in addition to a compound of the present invention, a pharmaceutically acceptable salt or solvate thereof as active ingredient, additional therapeutic agents and/or active ingredients.
The invention also provides a compound of the invention or a pharmaceutically acceptable salt or solvate thereof for use in a therapeutic treatment in humans or animals.
Another object of this invention is a medicament comprising at least one compound of the invention, or a pharmaceutically acceptable salt or solvate thereof, as active ingredient.
Generally, for pharmaceutical use, the compounds of the invention may be formulated as a pharmaceutical preparation comprising at least one compound of the invention and at least one pharmaceutically acceptable excipient, and optionally one or more further pharmaceutically active compounds.
By means of non- limiting examples, such a formulation may be in a form suitable for oral administration (e.g. as a tablet, capsule, or as an ingestible solution), for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or
intravenous infusion), for topical administration (including ocular), intracerebral administration, sublingual administration, aerosol administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, etc. Such suitable administration forms - which may be solid, semi-solid or liquid, depending on the manner of administration - as well as methods and carriers, diluents and excipients for use in the preparation thereof, will be clear to the skilled person; reference is made to the latest edition of Remington’s Pharmaceutical Sciences.
For example, the compound of the invention or a pharmaceutical composition comprising a compound of the invention can be administered orally in the form of tablets, coated tablets, pills, capsules, soft gelatin capsules, oral powders, granules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.
The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, a disintegrant such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, a binder such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia, a lubricant such as magnesium stearate, stearic acid, glyceryl behenate. Solid compositions of a similar type may also be employed as fillers in hard gelatin capsules. Preferred excipients in this regard include lactose, saccharose, sorbitol, mannitol, potato starch, com starch, amylopectin, cellulose derivatives or gelatin. Hard gelatin capsules may contain granules of the compound of the invention.
Soft gelatin capsules may be prepared with capsules containing the compound of the invention, vegetable oil, waxes, fat, or other suitable vehicle for soft gelatin capsules. As an example, the acceptable vehicle can be an oleaginous vehicle, such as a long chain triglyceride vegetable oil (e.g. com oil).
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water may contain the active ingredient in a mixture with dispersing agents, wetting agents, and suspending agents and one or more preservatives. Additional
excipients, for example sweetening, flavouring and colouring agents, may also be present. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
Liquid dosage forms for oral administration may include pharmaceutically acceptable, solutions, emulsions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water or an oleaginous vehicle. Liquid dosage form may be presented as a dry product for constitution with water or other suitable vehicle before use. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, complexing agents such as 2-hydroxypropyl-beta-cyclodextrin, sulfobutylether-beta-cylodextrin, and sweetening, flavouring, perfuming agents, colouring matter or dyes with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof. These compositions may be preserved by the addition of an antioxidant such as butylated hydroxyanisol or alpha-tocopherol.
Finely divided powder of the compound of the invention may be prepared for example by micronisation or by processes known in the art. The compound of the invention may be milled using known milling procedures such as wet milling to obtain a particle size appropriate for tablet formation and for other formulation types.
If the compound of the present invention is administered parenterally, then examples of such administration include one or more of: intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrastemally, intracranially, intramuscularly or subcutaneously administering the agent; and/or by using infusion techniques.
The compound of the invention can be administered via the parenteral route with a ready available or a depot-type formulation.
The pharmaceutical compositions for the parenteral administration of a ready available formulation may be in the form of a sterile injectable aqueous or oleagenous solution or suspension in a non-toxic parenterally- acceptable diluent or solvent and may contain formulatory agents such as suspending, stabilising dispersing, wetting and/or complexing
agents such as cyclodextrin e.g. 2-hydroxypropyl-beta-cyclodextrin, sulfobutylether-beta- cylodextrin.
The depot-type formulation for the parenteral administration may be prepared by conventional techniques with pharmaceutically acceptable excipient including without being limited to, biocompatible and biodegradable polymers (e.g. poly(P-caprolactone), poly(ethylene oxide), poly(glycolic acid), poly[(lactic acid)-co-(glycolic acid)...)], poly(lactic acid)...), non-biodegradable polymers (e.g. ethylene vinylacetate copolymer, polyurethane, polyester( amide), polyvinyl chloride...) aqueous and non-aqueous vehicles (e.g. water, sesame oil, cottonseed oil, soybean oil, castor oil, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils, propylene glycol, DMSO, THF, 2-pyrrolidone, N- methylpyrrolidinone, N-vinylpyrrolidinone... ).
Alternatively, the active ingredient may be in dry form such as a powder, crystalline or freeze-dried solid for constitution with a suitable vehicle. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
As indicated, the compound of the present invention can be administered intranasally or by inhalation and is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, (for example from Ineos Fluor), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound and a suitable powder base such as lactose or starch. For compositions suitable and/or adapted for inhaled administration, it is preferred that the compound or salt of formula (I) is in a particle-size-reduced form, and more preferably the size-reduced form is obtained or obtainable by micronisation. The preferable particle size of the size-reduced (e.g. micronised) compound or salt or solvate is defined by
a D50 value of about 0.5 to about 50 microns (for example as measured using laser diffraction).
Alternatively, the compound of the present invention can be administered in the form of a suppository or pessary, or it may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder. The compound of the present invention may also be dermally or transdermally administered, for example, by the use of a skin patch. They may also be administered by the pulmonary or rectal routes. It may also be administered by the ocular route. For ophthalmic use, the compound can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, it may be formulated in an ointment such as petrolatum.
For topical application to the skin, the agent of the present invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, it can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
DEFINITIONS
The definitions and explanations below are for the terms as used throughout the entire application, including both the specification and the claims.
Unless otherwise stated, any reference to compounds of the invention herein, means the compounds as such as well as their pharmaceutically acceptable salts and solvates.
When describing the compounds of the invention, the terms used are to be construed in accordance with the following definitions, unless indicated otherwise.
The term “unsubstituted” as used herein means that a radical, a group or a residue carries no substituent. The term “substituted” means that a radical, a group or a residue carries one or more substituents. The term “N-substituted” means that the one or more substituents are carried on a N atom of the radical, group or residue.
The term “halo” or “halogen” refers to the atoms of the group 17 of the periodic table (halogens) and includes in particular fluorine (F), chlorine (C1), bromine (Br) and iodine (I) atom. Preferred halo groups are fluoro (F) and chloro (C1), fluoro being particularly preferred.
The term “alkyl” by itself or as part of another substituent refers to a hydrocarbyl radical of Formula CnEhn+i wherein n is a number greater than or equal to 1. Alkyl groups may thus comprise 1 or more carbon atoms and generally, according to this invention comprise from 1 to 12, more preferably from 1 to 8 carbon atoms, and still more preferably from 1 to 6 carbon atoms. Alkyl groups within the meaning of the invention may be linear or branched. Examples of alkyl groups include but are not limited to methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, sec-pentyl, isopentyl, hexyl and isohexyl.
The term “alkenyl” by itself or as part of another substituent refers to a hydrocarbyl radical at least one double carbon-carbon bond. Alkenyl groups may thus comprise 2 or more carbon atoms and generally, according to this invention comprise from 2 to 12, more preferably from 2 to 8 carbon atoms, and still more preferably from 2 to 6 carbon atoms.
The term “alkynyl” by itself or as part of another substituent refers to a hydrocarbyl radical comprising at least one triple carbon-carbon bond. Alkynyl groups may thus comprise 2 or more carbon atoms and generally, according to this invention comprise from 2 to 12, more preferably from 2 to 8 carbon atoms, and still more preferably from 2 to 6 carbon atoms.
The term “alkoxy” by itself or as part of another substituent refers to a -O-alkyl group, wherein alkyl is as defined above. Examples of alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, isoproxy, butoxy, sec-butoxy, isobutoxy, tert-butoxy, pentoxy, .scc-pcntoxy and isopentoxy.
The term “aminoalkyl” by itself or as part of another substituent refers to a -alkyl-NHi group, wherein alkyl is as defined above.
The term “alkyloxycarbonyl” by itself or as part of another substituent refers to a -C(O)- O-alkyl group, wherein alkyl is as defined above.
The term “haloalkyl” or “halogenoalkyl” alone or in combination, refers to an alkyl radical having the meaning as defined above wherein one or more hydrogens are replaced with a halogen as defined above. Non-limiting examples of such haloalkyl radicals include chloromethyl, 1 -bromoethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1,1,1- trifluoroethyl and the like.
The term “cycloalkyl” as used herein is a monovalent, saturated, or unsaturated monocyclic or bicyclic hydrocarbyl group. Cycloalkyl groups may comprise 3 or more carbon atoms in the ring and generally, according to this invention comprise from 3 to 10, more preferably from 3 to 8 carbon atoms, and still more preferably from 3 to 6 carbon atoms. Examples of cycloalkyl groups include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
The term “heteroatom” as used herein refers to any atom that is not carbon or hydrogen. Non-limiting examples of such heteroatoms include nitrogen, oxygen, sulfur, and phosphorus. Preferred heteroatoms according to the invention are nitrogen, oxygen and sulfur.
The term “heteroalkyl” as used herein by itself or as part of another group, refers to an alkyl radical having the meaning as defined above wherein one or more carbons are replaced with a heteroatom as defined above. Preferred heteroatoms are nitrogen and oxygen.
The terms “heterocyclyl”, “heterocycloalkyl” or “heterocyclo” as used herein by itself or as part of another group refer to non-aromatic, fully saturated or partially unsaturated cyclic groups (for example, 3 to 7 member monocyclic, 7 to 11 member bicyclic, or containing a total of 3 to 10 ring atoms) which have at least one heteroatom in at least one
carbon atom-containing ring. Each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3 or 4 heteroatoms selected from nitrogen, oxygen and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quatemized. The heterocyclic group may be attached at any heteroatom or carbon atom of the ring or ring system, where valence allows. Examples of heterocyclyl groups include but are not limited to aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, azepanyl, piperazinyl, morpholinyl. Preferred heterocyclyl groups according to the invention are azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl and morpholinyl.
The term “aryl” as used herein refers to a polyunsaturated, aromatic hydrocarbyl group having a single ring (i.e. phenyl) or multiple aromatic rings fused together (e.g. naphthyl), typically containing 5 to 12 atoms; preferably 6 to 10, wherein at least one ring is aromatic. Examples of aryl groups include but are not limited to phenyl, biphenyl, 1 -naphthyl (or naphthalene- 1-yl), 2-naphthyl (or naphthalene-2-yl), anthracenyl, indanyl, indenyl, 1 ,2,3,4- tetrahydronaphthyl. Preferred aryl group according to the invention is phenyl.
The term “heteroaryl” as used herein by itself or as part of another group refers but is not limited to 5 to 12 carbon-atom aromatic rings or ring systems containing 1 to 2 rings which are fused together, typically containing 5 to 6 atoms; at least one of which is aromatic, in which one or more carbon atoms in one or more of these rings is replaced by oxygen, nitrogen and/or sulfur atoms where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. Examples of heteroaryl groups include but are not limited to pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, furanyl, benzofuranyl, pyrrolyl, indolyl, thiophenyl, benzothiophenyl, imidazolyl, benzimidazolyl, pyrazolyl, indazolyl, oxazolyl, benzoxazolyl, isoxazolyl, benzisoxazolyl, thiazolyl, and benzothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, dioxazolyl, dithiazolyl and tetrazolyl. Preferred heteroaryl group according to the invention is thiazolyl.
The term “haloaryl” or “halogenoaryl” alone or in combination, refers to an aryl radical having the meaning as defined above wherein one or more hydrogens are replaced with a halogen as defined above.
The compounds of the invention containing a basic functional group may be in the form of pharmaceutically acceptable salts. Pharmaceutically acceptable salts of the compounds of the invention containing one or more basic functional groups include in particular the acid addition salts thereof. Suitable acid addition salts are formed from acids which form nontoxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, cinnamate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts.
Pharmaceutically acceptable salts of compounds of Formula I and subformulae may for example be prepared as follows:
(i) reacting the compound of Formula I or any of its subformulae with the desired acid; or
(ii) converting one salt of the compound of Formula I or any of its subformulae to another by reaction with an appropriate acid or by means of a suitable ion exchange column.
All these reactions are typically carried out in solution. The salt, may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionization in the salt may vary from completely ionized to almost nonionized.
The term “solvate” is used herein to describe a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term “hydrate” is employed when said solvent is water.
The compounds of the invention include compounds of the invention as hereinbefore defined, including all polymorphs and crystal habits thereof, prodrugs and isomers thereof (including optical, geometric and tautomeric isomers) and isotopically-labeled compounds of the invention.
In addition, although generally, with respect to the salts of the compounds of the invention, pharmaceutically acceptable salts are preferred, it should be noted that the invention in its broadest sense also includes non-pharmaceutically acceptable salts, which may for example be used in the isolation and/or purification of the compounds of the invention. For example, salts formed with optically active acids or bases may be used to form diastereoisomeric salts that can facilitate the separation of optically active isomers of the compounds of the invention.
The term “patient” refers to a warm-blooded animal, more preferably a human, who/which is awaiting or receiving medical care or is or will be the object of a medical procedure.
The term “human” refers to subjects of both genders and at any stage of development (i.e. neonate, infant, juvenile, adolescent, adult). In one embodiment, the human is an adolescent or adult, preferably an adult.
The terms “treat”, “treating” and “treatment”, as used herein, are meant to include alleviating or abrogating a condition or disease and/or its attendant symptoms.
The term “therapeutically effective amount” (or more simply an “effective amount” or “suitable dose”) as used herein means the amount of active agent or active ingredient which is sufficient to achieve the desired therapeutic or prophylactic effect in the individual to which it is administered.
The term “administration”, or a variant thereof (e.g. “administering”), means providing the active agent or active ingredient, alone or as part of a pharmaceutically acceptable composition, to the patient in whom/which the condition, symptom, or disease is to be treated.
By “pharmaceutically acceptable” is meant that the ingredients of a pharmaceutical composition are compatible with each other and not deleterious to the patient thereof.
The term “excipient” as used herein means a substance formulated alongside the active agent or active ingredient in a pharmaceutical composition or medicament. Acceptable excipients for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington’s Pharmaceutical Sciences, 21st Edition 2011. The choice of excipient can be selected with regard to the intended route of administration and standard pharmaceutical practice. The excipient must be acceptable in the sense of being not deleterious to the recipient thereof. The at least one pharmaceutically acceptable excipient may be for example, a binder, a diluent, a carrier, a lubricant, a disintegrator, a wetting agent, a dispersing agent, a suspending agent, and the like.
The present invention will be better understood with reference to the following figures and examples. These examples are intended to representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.
FIGURES
Figure 1. Inhibition of the enzymatic activity of PaNADK by compound 1. (A) The steady state rate constants of PaNADK activity were determined at 25 nM PaNADK, 4 mM MgATP, 0.05-1 mM NAD+ and at different concentrations of compound 1, as indicated in the figure. Global competitive inhibition fittings are shown as lines. (B) Lineweaver-Burk representation of the data showing a significant competitive inhibition of PaNADK by compound 1.
Figure 2. Evaluation of compound 1 as a novel anti-Pseudomonas molecule. (A) Effect of compound 1 on P aeruginosa growth in liquid LB cultures. P aeruginosa growth curves in presence of compound 1 at 20 pM (in 0.1% DMSO) or in presence of 0.1% DMSO as control. Graphs represent the mean of three independent experiments. (B) The efficacy of compound 1 against P aeruginosa was tested with embryos injured in the tail fin and bath infected with PAO1 suspension at approximately 8.107 CFU/mL in presence of compound 1 at 20 pM (in 0.1% DMSO). Injured embryos in the control group were treated with
PA01 suspension in presence of DMSO at 0.1%. A significant difference (P<0.01) is found in the survival curve of treated versus non-treated embryos. (C) The toxicity of compound 1 at 20 pM was monitored after immersion of embryos injured in the tail fin. For all experiments, the embryo survival was monitored for 45 h and survival curves were represented with a Kaplan-Meier representation. Graphs represent the pool of three minimum independent experiments (n = 120 larvae for compound 1 test or 60 larvae for toxicity test, in total per condition). ** means P value <0.01, ns means no significant difference.
EXAMPLES
ABBREVIATIONS
In the context of the present invention, the following abbreviations and empirical formulae are used:
APTS: p-toluenesulfonic acid
°C: degree Celsius
DBU: l,8-diazabicyclo[5.4.0]undec-7-ene
DCM: dichloromethane
DIEA: A,A-diisopropylethylamine
DMSO: dimethylsulfoxide
DNA: deoxyribonucleic acid
DPPA: diphenylphosphoryl azide
DTT: dithio threitol
EDTA: ethylenediaminetetraacetic acid
ESI: electrospray ionization g: gram(s) h: hour(s)
HRMS: High-resolution mass spectra HPLC: high performance liquid chromatography
HsNADK: human cytosolic NADK
LC-MS: liquid chromatography /mass spectrometry
LmNADK: NADK from Listeria monocytogenes
M: mole(s) per liter mg: milligram(s)
MHz: megahertz pL: microliter(s) mL: milliliter(s) mmol: millimole(s) mol: mole(s)
NAD+: nicotinamide adenine dinucleotide
NMR: nuclear Magnetic Resonance
PaNADK: NADK from Pseudomonas aeruginosa
PBS: Phosphate Buffered Saline
PCR: Polymerase Chain Reaction
PyBOP: (Benzo triazol- l-yloxy)tripyrrolidinophosphonium hexafluorophosphate
TBAI: tetra-n-butylammonium iodide
TEAA: triethylammonium acetate
TFA: trifluoroacetic acid
THF: tetrahydrofuran
TLC: thin-layer chromatography
TMSOK: potassium trimethylsilanolate
UV: ultra-violet wt: weight
Other features, properties and advantages of the invention will emerge more clearly from the description and examples that follow.
SYNTHESIS
1. Material and instrumentation
All commercially available reagents and solvents, unless otherwise stated, were used without purification. Anhydrous reactions were carried out under an argon atmosphere. Analytical thin-layer chromatography (TLC) was performed on TLC plates pre-coated with silica gel 60 F254. Compounds were visualized with UV light (254 nm) and by spraying with a mixture of ethanol/anisaldehyde/sulfuric acid/acetic acid (90/5/4/1), followed by heating. Reactions were also monitored using an HPLC system (Agilent 1100 equipped with a C18 reverse phase column) coupled to a mass spectrometer (ESI source). Flash chromatography was performed with silica gel 60 (230-400 mesh). HPLC purification was carried out on an Agilent system (1100 Series) equipped with a diode array detector using a
C18 reverse phase column (Kromasil, 5 pm, 100 A, 250x10 mm) and a linear gradient of acetonitrile in 10 mM triethylammonium acetate (TEAA) buffer over 15 or 20 min at a flow rate of 4 mL/min. Retention time (Rt) and elution conditions are specified. The purity of tested compounds was at least 96% pure as determined by HPLC. NMR spectra were recorded on Bruker Avance 400 (^H at 400.13 MHz and 13C at 100.62 MHz). Chemical shifts (<5) are reported in ppm relative to the solvent signals. Coupling constants (J values) are reported in Hz. The complete assignment of ' H and 13C signals was performed by analysis of the correlated homonuclear { ' HJ H I-COSY and heteronuclear {1H,13C}- HMBC, { ' H,I 3C}-HSQC spectra. High-resolution mass spectra (HRMS) were recorded with a Q-Tof Micro mass spectrometer under electrospray ionization in positive ionization mode using a mobile phase of acetonitrile/water with 0.1% formic acid.
2. Synthesis procedures
Reagents and conditions: (a) 5 -bromobenzonitrile, nBuLi, THF, -78 °C; MeOH -78 °C to rt; (b) BF3-OEt2, Et3SiH, -0 °C to rt; (c) TMSOK, THF, reflux; (d) 1 M BBr3 in DCM, - 78 °C to rt; (e) APTS, acetone, 2,2-dimethoxypropane; (f) Propargyl bromide, NaH, DMF,
-0 °C; (g) H2O2, K2CO3, DMSO.
3-(2,3,5-tri-O-benzyl-p-D-ribofuranosyl)benzonitrile (3)
The title compound was prepared following reported procedures with slight modifications. To a solution of 3 -bromobenzonitrile (3.81 g, 21 mmol) in dry THF (72 mL) containing activated 4A molecular sieves (5 g) was slowly added n-BuLi (2.5 M in hexane, 9.2 mL) at -78°C under an argon atmosphere. Stirring was maintained for 20 min after addition was completed, then a solution of ribonolactone 1 (2.51 g, 6 mmol) in THF (24 mL) was slowly added to the reaction mixture at -78°C. After 30 min, anhydrous MeOH (7.3 mL) was added at the temperature was allowed to warm to room temperature. The reaction mixture was neutralized by adding 2N HC1 (12 mL) and extracted with sat. NaHCO3 (150 mL). The organic phase was dried, concentrated under reduced pressure and the residue was purified by flash column chromatography (180 g silica gel, 5 to 15% ethyl acetate in cyclohexane) affording hemiacetal 2 (2.77 g, 88.5%).
To a solution of 2 (2.75 g, 5.3 mmol) in dry CH2Cl2 (26.5 mL) at 0°C was added Et3SiH (3.37 mL, 21.1 mmol). After stirring for 5 min, BF3·Et2O (1.31 mL, 10.6 mmol) was slowly added and the resulting mixture was warmed to room temperature over 10 min. Then Et3SiH (7.39 mL, 53 mmol) was added, and the reaction mixture was concentrated under reduced pressure. The residue was purified by flash column chromatography (5 to 10% ethyl acetate in cyclohexane) to give 3 (2.23 g, 83.2%). 1H NMR (400 MHz, DMSO-d6): δ 3.64 (dd, J = 3.9 Hz, J = 10.6 Hz, 1H, H-5'), 3.71 (dd, J = 3.7 Hz, J = 10.6 Hz, 1H, H-5''), 3.93 (dd, J = 5.2 Hz, J = 6.4 Hz, 1H, H-2'), 4.11 (t, J = 4.4 Hz, 1H, H-3'), 4.29 (q, J = 3.6 Hz, 1H, H-4'), 4.44-4.61 (m, 6 H, CH2 Bn), 4.95 (d, J = 6.6 Hz, 1H, H-1'), 7.18-7.40 (m, 15 H, H Bn), 7.50 (t, 1H, H Ph), 7.71 (d, 1H, H Ph), 7.75 (d, 1H, H Ph), 7.78 (s, 1H, H Ph). 13C NMR (100 MHz, DMSO-d6): δ 70.64 (C-5'), 71.55, 71.71 and 72.95 (CH2 Bn), 77.72 (C-3'), 81.28 (C-1'), 82.03 (C-4'), 83.85 (C-2'), 119.18 and 111.74 (Cq), 127.87, 127.93, 128.02, 128.09, 128.29, 128.59, 128.67, 128.75, 129.86, 130.10, 131.63 and 131.89 (CH Ph), 138.37, 138.66 and 142.78 (Cq Ph). HRMS (ESI+-TOF): m/z calcd for [C33H31NO4+H]+ 506.2326, found 506.2320. Route A 3-(2,3,5-tri-O-benzyl-β-D-ribofuranosyl)benzamide (4) To compound 3 (2.22 g, 4.4 mmol) in anhydrous THF (11 mL) was added TMSOK (0.845 g, 6.6 mmol) in a sealed tube. After stirring at 70°C for 24 h, ethanol was added to the reaction mixture, then volatiles were removed under reduced pressure and the residue was purified by flash column chromatography (50 g silica gel, 0 to 2% MeOH in DCM) affording 4 (1.92 g, 83.5%). 1H NMR (400 MHz, DMSO-d6): δ 3.54 (dd, J = 4.8 Hz, J = 11.6 Hz, 1H, H-5'), 3.59 (dd, J = 4.4 Hz, J = 11.6 Hz, 1H, H-5''), 3.71 (dd, J = 5.4 Hz, J = 7.1 Hz, 1H, H-2'), 3.83 (t, J = 4.6 Hz, J = 8.3 Hz, 1H, H-4'), 3.90 (dd, J = 3.6 Hz, J = 5.4 Hz, 1H, H-3'), 4.60 (d, J = 7.1
Hz, 1H, H-1'), 7.32 (br, 1H, CONH2), 7.40 (t, 1H, H Ph), 7.55 (d, 1H, H Ph), 7.77 (d, 1H, H Ph), 7.86 (s, 1H, H Ph), 7.92 (br, 1H, CONH2). 13C NMR (100 MHz, DMSO-d6): δ 62.50 (C-5'), 71.85 (C-3'), 77.94 (C-2'), 83.33 (C-1'), 85.71 (C-4'), 125.91, 126.84, 128.36 and 129.56 (CH Ph), 134.60 and 141.93 (Cq Ph), 168.46 (CO). HRMS (ESI+-TOF): m/z calcd for [C15H19NO5+H]+ 254.1023, found 254.1025. 3-(β-D-ribofuranosyl)benzamide (5) A 1M solution of BBr3 in DCM (14.6 mL) was added dropwise to a stirred and cooled to – 78°C solution of 4 (1.91 g, 3.6 mmol) in anhydrous DCM (72 mL) containing molecular sieves. After a 1 h at –78°C, the mixture was allowed to warm to room temperature and stirred overnight. The reaction was quenched by adding diethyl ether/water (4/1, 180 mL) at 4°C for 20 min, then the volatiles were removed under reduced pressure. Purification by flash column chromatography (115 g silica gel, 10 to 12% MeOH in DCM) afforded 5 (0.76 g, 83%). 1H NMR (400 MHz, DMSO-d6): δ 3.54 (dd, J = 4.8 Hz, J = 11.6 Hz, 1H, H-5'), 3.59 (dd, J = 4.4 Hz, J = 11.6 Hz, 1H, H-5''), 3.71 (dd, J = 5.4 Hz, J = 7.1 Hz, 1H, H-2'), 3.83 (t, J = 4.6 Hz, J = 8.3 Hz, 1H, H-4'), 3.90 (dd, J = 3.6 Hz, J = 5.4 Hz, 1H, H-3'), 4.60 (d, J = 7.1 Hz, 1H, H-1'), 7.32 (br, 1H, CONH2), 7.40 (t, 1H, H Ph), 7.55 (d, 1H, H Ph), 7.77 (d, 1H, H Ph), 7.86 (s, 1H, H Ph), 7.92 (br, 1H, CONH2). 13C NMR (100 MHz, DMSO-d6): δ 62.51 (C-5'), 71.85 (C-3'), 77.94 (C-2'), 83.33 (C-1'), 85.71 (C-4'), 125.91, 126.84, 128.36 and 129.56 (CH Ph), 134.59 and 141.93 (Cq Ph), 168.46 (CO). HRMS (ESI+-TOF): m/z calcd for [C15H19NO5+H]+ 254.1023, found 254.1025. 3-(2,3-O-isopropylidene-β-D-ribofuranosyl)benzamide (6)
To a suspension of 5 (0.76 g, 3.0 mmol) in acetone (30 mL) were added 2,2- dimethoxypropane (1.11 mL, 9.0 mmol) and APTS (0.57 g, 3.0 mmol). After stirring overnight at room temperature, the reaction was quenched by addition of 1M Na2CO3 (15 mL), concentrated under reduced pressure, then extracted twice with ethyl acetate. The organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure. Purification by flash column chromatography (40 g silica gel, 5% MeOH in DCM) afforded 6 (0.58 g, 66%). 1H NMR (400 MHz, DMSO-d6): δ 1.30 (s, 3H, CH3 isop), 1.55 (s, 3H, CH3 isop), 3.56 (dd, 1H, H-5'), 3.61 (dd, 1H, H-5''), 4.01 (q, J = 4.7 Hz, J = 8.8 Hz, 1H, H-4'), 4.53 (dd, J = 5.4 Hz, J = 6.7 Hz, 1H, H-2'), 4.69 (dd, J = 3.8 Hz, J = 6.7 Hz, 1H, H-3'), 4.79 (d, J = 5.4 Hz, 1H, H-1'), 4.92 (t, 1H, OH), 7.36 (br, 1H, CONH2), 7.44 (t, 1H, H Ph), 7.53 (d, 1H, H Ph), 7.80 (d, 1H, H Ph), 7.85 (s, 1H, H Ph), 7.96 (br, 1H, CONH2). 13C NMR (100 MHz, DMSO-d6): δ 25.86 (CH3 isop), 27.86 (CH3 isop), 61.99 (C-5'), 82.22 (C-3'), 84.99 (C-1'), 85.23 (C-4'), 86.54 (C-2'), 114.34 (Cq isop), 125.70, 127.24, 128.69 and 129.34 (CH Ph), 134.85 and 140.55 (Cq Ph), 168.23 (CO). HRMS (ESI+-TOF): m/z calcd for [C15H19NO5+Na]+ 316.1155, found 316.1151. Route B 3-(β-D-ribofuranosyl)benzonitrile (7) A 1 M solution of BBr3 in DCM (0.8 mL) was added dropwise to a stirred and cooled to – 78°C solution of 3 (100 mg, 0.2 mmol) in anhydrous DCM (4 mL) containing molecular sieves. After a 1 h at –78°C, the mixture was allowed to warm to room temperature and stirred overnight. The reaction was quenched by adding diethyl ether/water (4/1, 10 mL) at 4°C for 20 min, then the volatiles were removed under reduced pressure. Purification by flash column chromatography (82 g silica gel, 5 to 8% MeOH in DCM) afforded 7 (37 mg, 79%). 1H NMR (400 MHz, DMSO-d6): δ 3.54 (dd, J = 4.3 Hz, J = 11.7 Hz, 1H, H-5'), 3.60 (dd, J = 4.0 Hz, J = 11.7 Hz, 1H, H-5''), 3.68 (dd, J = 5.3 Hz, J = 7.4 Hz, 1H, H-2'), 3.84-3.88 (m,
1H, H-4'), 3.93 (dd, J = 3.2 Hz, J = 5.2 Hz, 1H, H-3'), 4.63 (d, J = 7.4 Hz, 1H, H-1'), 7.55 (t, 1H, H Ph), 7.72 (d, 1H, H Ph), 7.74 (d, 1H, H Ph), 7.85 (br, 1H, H Ph). 13C NMR (100 MHz, DMSO-d6): δ 62.30 (C-5'), 71.88 (C-3'), 78.27 (C-2'), 82.23 (C-1'), 86.06 (C-4'), 111.56 (CN), 119.40 (Cq), 129.73, 129.96, 131.51 and 131.53 (CH Ph), 143.71 (Cq Ph). HRMS (ESI+-TOF): m/z calcd for [C12H13NO4-H]+ 234.0772, found 234.0767. 3-(2,3-O-isopropylidene-β-D-ribofuranosyl)benzonitrile (8) To a suspension of 7 (0.30 g, 1.28 mmol) in acetone (13 mL) were added 2,2- dimethoxypropane (0.47 mL, 3.84 mmol) and APTS (0.24 g, 1.28 mmol). After stirring overnight at room temperature, the reaction was quenched by addition of 1 M Na2CO3 (6.5 mL), volatiles were removed under reduced pressure, then extracted twice with DCM (3x50 mL). The organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure. Purification by flash column chromatography (25 g silica gel, 0 to1.5% MeOH in DCM) afforded 8 (0.30 g, 86%). 1H NMR (400 MHz, DMSO-d6): δ 1.30 (s, 3H, CH3 isop), 1.55 (s, 3H, CH3 isop), 3.56 (dd, J = 4.7 Hz, J = 5.4 Hz, 2H, H-5' and H-5''), 4.04 (q, J = 4.4 Hz, 1H, H-4'), 4.50 (dd, J = 5.7 Hz, J = 6.4 Hz, 1H, H-2'), 4.70 (dd, J = 3.6 Hz, J = 6.6 Hz, 1H, H-3'), 4.83 (d, J = 5.4 Hz, 1H, H-1'), 4.96 (t, 1H, OH), 7.58 (t, 1H, H Ph), 7.71 (d, 1H, H Ph), 7.78 (dt, 1H, H Ph), 7.85 (s, 1H, H Ph). 13C NMR (100 MHz, DMSO-d6): δ 25.87 (CH3 isop), 27.85 (CH3 isop), 61.87 (C-5'), 82.12 (C-3'), 84.51 (C-1'), 85.08 (C-4'), 86.51 (C-2'), 111.89 (CN), 114.25 (Cq isop), 119.18 (Cq), 129.97, 130.05, 131.45 and 131.99 (CH Ph), 142.26 (Cq Ph). HRMS (ESI+-TOF): m/z calcd for [C15H17NO4+H]+ 276.1230, found 276.1231. 3-(2,3-di-O-isopropylidene-5-O-propargyl-β-D-ribofuranosyl)benzonitrile (9)
Sodium hydride (60% in oil, 32 mg, 0.8 mmol) was added to an ice-cooled solution of 8 (55 mg, 0.2 mmol) in DMF (2 mL) containing molecular sieves. After stirring at 0°C for 2 h, a solution of propargyl bromide (80% in toluene, 45 µL, 0.4 mmol) was added dropwise. After stirring at 0°C for 1 h, acetic acid (60 µL) was added and the reaction was stirred for an additional 1 h. Water (3 mL) was added, the mixture was extracted with DCM (3x30 mL). The organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure. Purification by flash column chromatography (10 g silica gel, 0 to 1% MeOH in DCM) afforded 9 (54 mg, 86%). 1H NMR (400 MHz, DMSO-d6): δ 1.30 (s, 3H, CH3 isop), 1.55 (s, 3H, CH3 isop), 3.45 (t, J = 2.4 Hz, 1H, C≡CH), 3.62-3.70 (m, 2H, H-5' and H-5''), 4.17 (q, J = 4.5 Hz, 1H, H-4'), 4.21 (d, J = 2.4 Hz, 2H, CH2-C≡), 4.56 (dd, J = 5.2 Hz, J = 6.5 Hz, 1H, H-2'), 4.68 (dd, J = 3.8 Hz, J = 6.6 Hz, 1H, H-3'), 4.87 (d, J = 5.0 Hz, 1H, H-1'), 7.60 (t, 1H, H Ph), 7.51 (d, 1H, H Ph), 7.70 (d, 1H, H Ph), 7.78 (s, 1H, H Ph), 7.79 (d, 1H, H Ph). 13C NMR (100 MHz, DMSO-d6): δ 25.85 (CH3 isop), 27.78 (CH3 isop), 58.41 (CH2-C≡), 69.90 (C-5'), 77.91 (C≡CH), 80.42 (C≡CH), 82.23 (C-3'), 83.07 (C-4'), 84.68 (C-1'), 86.37 (C-2'), 111.93 (CN), 114.49 (Cq isop), 119.10 (Cq Ph), 129.92, 130.15, 131.39 and 132.08 (CH Ph), 140.28 (Cq Ph), 141.99 (Cq Ph). HRMS (ESI+-TOF): m/z calcd for [C18H19NO4+H]+ 314.1387, found 314.1385. 3-(2,3-O-isopropylidene-5-O-propargyl-β-D-ribofuranosyl)benzamide (10) From 7 (route A): Sodium hydride (60% in oil, 84 mg, 2.1 mmol) was added at –10°C to a solution of 9 (0.21 g, 0.7 mmol) in DMF (21 mL) containing molecular sieves. After stirring at –10°C for 2 h, a solution of propargyl bromide (80% in toluene, 78 µL, 0.7 mmol) in DMF (1 mL) was added dropwise. After 2 h at –10°C, acetic acid (210 µL) was added and the reaction was stirred for an additional 1 h. Water was added, the mixture was extracted with DCM (3 times). The organic layers were dried over Na2SO4, filtered and concentrated under
reduced pressure. Purification by flash column chromatography (25 g silica gel, 0 to 3% MeOH in DCM) afforded 10 (94 mg, 41%). From 9 (route B, conditions 1): To an ice-cooled solution of 9 (94 mg, 0.3 mmol) in DMSO (5 mL) in the presence of K2CO3 (180 mg, 1.3 mmol) was added H2O2 (35% wt in H2O, 0.166 mL, 1.9 mmol). After stirring for 2 h at room temperature, volatiles were removed by lypohilization. Purification by flash column chromatography (12 g silica gel, 3% MeOH in DCM) afforded 10 (69 mg, 70%). From 9 (route B, conditions 2b): To a solution of 9 (0.36 g, 1.16 mmol) in MeOH (23 mL) was added K2CO3 (0.48 g, 3.5 mmol) and urea-hydrogen peroxide adduct (0.54 g, 5.8 mmol). After stirring for 8 h at room temperature, the reaction was judged incomplete and urea-hydrogen peroxide adduct (0.46 g, 5.2 mmol) was added portionwise. After 2 days, volatiles were removed by lyophilization. Purification by flash column chromatography (30 g silica gel, 2 to 3% MeOH in DCM) afforded 10 (0.28 g, 73%). 1H NMR (400 MHz, DMSO-d6): δ 1.31 (s, 3H, CH3 isop), 1.55 (s, 3H, CH3 isop), 3.45 (t, J = 2.4 Hz, 1H, C≡CH), 3.66 (d, J = 5.0 Hz, 2H, H-5' and H-5''), 4.14 (dd, J = 5.0 Hz, J = 9.2 Hz, 1H, H-4'), 4.22 (d, J = 2.4 Hz, 2H, CH2-C≡), 4.57 (dd, J = 5.2 Hz, J = 6.7 Hz, 1H, H- 2'), 4.67 (dd, J = 4.0 Hz, J = 6.8 Hz, 1H, H-3'), 4.82 (d, J = 5.2 Hz, 1H, H-1'), 7.36 (br, 1H, CONH2), 7.45 (t, 1H, H Ph), 7.51 (d, 1H, H Ph), 7.83 (d, 1H, H Ph), 7.85 (s, 1H, H Ph), 7.99 (br, 1H, CONH2). 13C NMR (100 MHz, DMSO-d6): δ 25.85 (CH3 isop), 27.80 (CH3 isop), 58.42 (CH2-C≡), 69.93 (C-5'), 77.90 (C≡CH), 80.50 (C≡CH), 82.25 (C-3'), 85.01 (C-4'), 85.40 (C-1'), 86.46 (C-2'), 114.64 (Cq isop), 125.74, 127.28, 128.76 and 129.24 (CH Ph), 134.90 and 140. 28 (Cq Ph), 168.24 (CO). HRMS (ESI+-TOF): m/z calcd for [C18H21NO5+Na]+ 354.1312, found 354.1299.
2. Building blocks 15 & 17, A = NR
5-Azido-2,3-O-isopropylidene-ip-(3-(carbamoyl)phenyl)-l,5-di-deoxy-D-ribofuranose
(11) To a solution of 6 (0.73 g, 2.5 mmol) in anhydrous dioxane (25 mL) were added dropwise DBU (1.12 mL, 7.5 mmol) and DPPA (1.08 mL, 5.0 mmol). After stirring for 1.5 h at room temperature, TBAI (92 mg, 0.25 mmol), 15-crown-15 ether (50 pL, 0.25 mmol) and NaNs (0.81 g, 12.5 mmol) were added to the mixture. The reaction was completed after heating the solution at 100°C for 1.5 h. Water was added, the mixture was extracted with ethyl acetate (twice), the organic layers were dried over NaiSCL, filtered and concentrated under reduced pressure. Purification by flash column chromatography (55 g silica gel, 2 to 4% MeOH in DCM) afforded 11 (0.79 g, 99%).
1H NMR (400 MHz, CDCl3): δ 1.36 (s, 3H, CH3 isop), 1.63 (s, 3H, CH3 isop), 3.45 (dd, J = 4.4 Hz, J = 13.2 Hz, 1H, H-5'), 3.72 (dd, J = 3.5 Hz, J = 13.2 Hz, 1H, H-5''), 4.26 (dd, J = 4.3 Hz, J = 8.0 Hz, 1H, H-4'), 4.54 (dd, J = 5.5 Hz, J = 7.0 Hz, 1H, H-2'), 4.69 (dd, J = 4.4 Hz, J = 7.0 Hz, 1H, H-3'), 4.93 (d, J = 5.5 Hz, 1H, H-1'), 6.36 (br, 2H, CONH2), 7.45 (t, 1H, H Ph), 7.56 (d, 1H, H Ph), 7.82 (dt, 1H, H Ph), 7.85 (s, 1H, H Ph). 13C NMR (100 MHz, CDCl3): δ 25.46 (CH3 isop), 27.44 (CH3 isop), 52.22 (C-5'), 81.86 (C-3'), 82.93 (C-4'), 85.43 (C-14), 86.81 (C-2'), 115.63 (Cq isop), 124.08, 127.25, 128.63 and 128.89 (CH Ph), 133.80 and 139.67 (Cq Ph), 169.30 (CO). HRMS (ESI+-TOF): m/z calcd for [C15H18N4O4+H]+ 319.1401, found 319.1381. 5-Azido-2,3-O-isopropylidene-1β-(3-(carbamoyl)phenyl)-1,5-di-deoxy-D-ribofuranose (12) To a stirred solution of 11 (0.79 g, 2.5 mmol) in pyridine (25 mL) was added triphenylphosphine (0.98 g, 3.75 mmol). After 2 h at room temperature, aq. ammonia (32%, 25 mL) was added and the mixture was stirred overnight at room temperature. Volatiles were removed under reduced pressure and the residue was purified by flash column chromatography (60 g 7734 Merck silica gel, 10 to 15% MeOH in DCM) affording 12 (0.66 g, 90%). 1H NMR (400 MHz, CDCl3): δ 1.36 (s, 3H, CH3 isop), 1.63 (s, 3H, CH3 isop), 2.99 (dd, J = 6.2 Hz, J = 13.2 Hz, 1H, H-5'), 3.12 (dd, J = 4.2 Hz, J = 13.2 Hz, 1H, H-5''), 4.08-4.12 (m, 1H, H-4'), 4.53 (dd, J = 5.4 Hz, J = 6.9 Hz, 1H, H-2'), 4.63 (dd, J = 4.5 Hz, J = 7.0 Hz, 1H, H-3'), 4.89 (d, J = 5.4 Hz, 1H, H-1'), 6.05 and 6.45 (br, 2H, CONH2), 7.44 (t, 1H, H Ph), 7.55 (d, 1H, H Ph), 7.76 (d, 1H, H Ph), 7.89 (s, 1H, H Ph). 13C NMR (100 MHz, CDCl3): δ 25.46 (CH3 isop), 27.44 (CH3 isop), 52.22 (C-5'), 81.86 (C-3'), 82.93 (C-4'), 85.43 (C-14), 86.81 (C-2'), 115.63 (Cq isop), 124.61, 126.90, 129.33 and 129.56 (CH Ph), 133.72 and 140.32 (Cq Ph), 169.20 (CO). HRMS (ESI+-TOF): m/z calcd for [C15H20N2O4+H]+ 293.1493, found 293.1487.
1β-(3-(Carbamoyl)phenyl)-2,3-O-isopropylidene-1,5-di-deoxy-5-(2- nitrophenylsulfonyl)amino-D-ribofuranose (13) To a stirred solution of 12 (0.655 g, 2.2 mmol) in pyridine (22 mL) was added 2- nitrobenzenesulfonyl chloride (1.13 g, 5.1 mmol). After 2 h at room temperature, volatiles were removed and the residue was purified by flash column chromatography (100 g silica gel, 3% MeOH in DCM) affording 13 (0.77 g, 74%). 1H NMR (400 MHz, CDCl3): δ 1.36 (s, 3H, CH3 isop), 1.62 (s, 3H, CH3 isop), 3.42-3.55 (m, 2H, H-5' and H-5"), 4.21 (q, J = 4.1 Hz, J = 6.3 Hz, 1H, H-4'), 4.55 (dd, J = 5.6 Hz, J= 6.7 Hz, 1H, H-2'), 4.75 (dd, J = 4.5 Hz, J = 7.0 Hz, 1H, H-3'), 4.88 (d, J = 5.5 Hz, 1H, H- 1'), 5.73 (br, 1H, CONH2), 5.99 (t, 1H, NH), 6.54 (br, 1H, CONH2), 7.45-7.53 (m, 2H, H Ph), 7.75-7.82 (m, 3H, H Ph), 7.85-7.95 (s, 2H, H Ph), 8.16-8.22 (m, 1H, H Ph). HRMS (ESI+-TOF): m/z calcd for [C21H23N3O8S+H]+ 478.1279, found 478.1275. 3-N-Boc-bromopropylamine To a suspension of 3-bromopropylamine hydrobromide (2.19 g, 10.0 mmol) in DCM (50 mL) was added NEt3 (1.67 mL, 12.0 mmol) followed by di-tert-butyl dicarbonate (2.18 g, 10.0 mmol). After 18 hours at room temperature, the crude was washed twice with a 5% aqueous citric acid solution (2x 50 mL) and once with brine (50 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure to yield 3-N-Boc- bromopropylamine (2.23 g, 94%) as a white solid, which was used in the next step without further purification. 1H NMR (400 MHz, DMSO-d6): δ 1.38 (s, 9H, CH3 Boc), 1.91 (quint. J = 6.6 Hz, 2H, CH2), 3.03 (td, J = 6.6 Hz, J = 5.8 Hz, 2H, NCH2), 3.51 (t, J = 6.6 Hz, 2H, CH2), 6.88 (t, J = 5.8 Hz, 1H, NH). 13C NMR (100 MHz, DMSO-d6): δ 28.7 (3C, CH3 Boc), 32.7 (CH2), 33.2 (CH2), 39.0 (CH2), 78.1 (Cq tBu), 156.1 (Cq Boc).
HRMS (ESI-TOF): m/z calcd for [C8H16BrNO2+Na]+ 260.0262 and 262.0242, found 260.0265 and 262.0249. 1β-(3-(carbamoyl)phenyl)-1,5-di-deoxy-2,3-O-isopropylidene-5-(3-N-Boc- propylamino)-D-ribofuranose (14) To a stirred solution of nosylated 13 (0.76 g, 1.6 mmol) in anhydrous DMF (16 mL) were added K2CO3 (0.66 g, 4.8 mmol) and N-Boc-bromo-propylamine (0.57 mL, 2.4 mmol). After 30 h at room temperature, the reaction was nearly complete (LC-MS monitoring). Thiophenol (0.33 mL, 3.2 mmol) was added and the reaction mixture was stirred overnight. The volatiles were removed under reduced pressure and the residue was purified by flash column chromatography (80 g SiO2, 5 to 10% MeOH in DCM) to give 14 (0.54, 76%). 1H NMR (400 MHz, DMSO-d6): δ 1.30 (s, 3H, CH3 isop), 1.37 (s, 9H, CH3 Boc), 1.55 (s, 3H, CH3 isop), 2.57-2.67 (m, 2H, CH2), 2.77-2.89 (m, 2H, H-5' and H-5"), 2.92-3.01 (m, 2H, CH2NBoc), 4.03-4.10 (m, 1H, H-4'), 4.52-4.60 (m, 1H, H-2'), 4.64-4.71 (m, 1H, H-3'), 4.80 (d, J = 5.0 Hz, 1H, H-1'), 6.79 (br, 1H, NHBoc), 7.38 (br, 1H, CONH2), 7.45 (t, 1H, H Ph), 7.53 (d, 1H, H Ph), 7.82 (d, 1H, H Ph), 7.85 (s, 1H, H Ph), 8.00 (br, 1H, CONH2). 13C NMR (100 MHz, DMSO-d6): δ 25.91 (CH3 isop), 27.84 (CH3 isop), 28.72 (3C, CH3 Boc), 29.64 (CH2), 38.41 (CH2), 47.21 (CH2), 51.29 (C-5'), 77.86 (Cq Boc), 83.20 and 83.33 (C-3' and C-4'), 85.24 (C-1'), 86.52 (C-2'), 114.62 (Cq isop), 125.73, 127.31, 128.74 and 129.31 (CH Ph), 134.93 and 140.26 (Cq Ph), 156.11 (CO Boc), 169.19 (CONH2). HRMS (ESI+-TOF): m/z calcd for [C23H35N3O6+H]+ 450.2599, found 450.2584. 1β-(3-(carbamoyl)phenyl)-1,5-di-deoxy-2,3-O-isopropylidene-5-(N-propargyl)-5-(3-N- Boc-propylamino)-D-ribofuranose (15) To a stirred solution of 14 (0.54 g, 1.2 mmol) in anhydrous DMF (12 mL) were added DIEA (0.63 mL, 3.6 mmol) and propargyl bromide (80% in toluene, 0.20 mL, 1.8 mmol). After 2.5 h at room temperature, volatiles were removed under reduced pressure and the
residue was purified by flash column chromatography (80 g SiO2, 5 to 10% MeOH in DCM) affording 15 (0.50, 85%). 1H NMR (400 MHz, DMSO-d6): δ 1.30 (s, 3H, CH3 isop), 1.37 (s, 9H, CH3 Boc), 1.47- 1.50 (m, 2H, CH2), 1.55 (s, 3H, CH3 isop), 2.47-2.52 (m, 2H, CH2), 2.62-2.74 (m, 2H, H-5' and H-5"), 2.90-3.00 (m, 2H, CH2NBoc), 3.10 (t, J = 2.2 Hz, 1H, C≡CH), 3.45 (br, 2H, CH2-C≡), 4.02-4.08 (m, 1H, H-4'), 4.55 (dd, J = 5.3 Hz, J = 6.8 Hz, 1H, H-2'), 4.63 (dd, J = 4.2 Hz, J = 6.8 Hz, 1H, H-3'), 4.78 (d, J = 5.3 Hz, 1H, H-1'), 6.73 (br, 1H, NHBoc), 7.37 (br, 1H, CONH2), 7.45 (t, 1H, H Ph), 7.50 (td, 1H, H Ph), 7.81 (td, 1H, H Ph), 7.83 (s, 1H, H Ph), 8.00 (br, 1H, CONH2). 13C NMR (100 MHz, DMSO-d6): δ 25.91 (CH3 isop), 27.84 (CH3 isop), 28.72 (3C, CH3 Boc), 29.64 (CH2), 38.41 (CH2), 47.21 (CH2), 51.29 (C-5'), 76.04 (C≡CH), 77.83 (Cq Boc), 79.47 (CH2-C≡CH), 83.20 (C-4'), 83.79 (C-3'), 85.14 (C-1'), 86.36 (C-2'), 114.74 (Cq isop), 125.71, 127.23, 128.75, 129.13 (CH Ph), 134.96 and 140.30 (Cq Ph), 156.05 (CO Boc), 169.21 (CONH2). HRMS (ESI+-TOF): m/z calcd for [C26H37N3O6+H]+ 488.2755, found 488.2740. 1β-(3-(carbamoyl)phenyl)-1,5-di-deoxy-2,3-O-isopropylidene-5-(5-trimethylsilyl-pent- 4-ynyl)amino-D-ribofuranose (16) To a stirred solution of 13 (0.185 g, 0.4 mmol) in anhydrous DMF (4 mL) were added K2CO3 (0.166 g, 1.2 mmol) and iodo-alkyne-TMS (0.15 mg, 0.6 mmol). After 2.5 h at room temperature, the reaction was complete (LC-MS monitoring). Thiophenol (82 µL, 0.8 mmol) was then added and the mixture was stirred at room temperature overnight. After removal of the volatiles under reduced pressure, the residue was purified by flash column chromatography (30 g SiO2, 5 to 6% MeOH in DCM) affording 16 (0.11, 63%). 1H NMR (400 MHz, DMSO-d6): δ 0.10 (s, 9H, CH3 TMS), 1.30 (s, 3H, CH3 isop), 1.55 (s, 3H, CH3 isop), 1.59 (quint, 2H, CH2), 2.26 (t, 2H, CH2), 2.64 (t, 2H, CH2), 2.72-2.83 (m, 2H, H-5' and H-5"), 4.01-4.07 (m, 1H, H-4'), 4.55 (dd, J = 5.4 Hz, J = 6.7 Hz, 1H, H-2'), 4.66 (dd, J = 4.0 Hz, J = 6.7 Hz, 1H, H-3'), 4.77 (d, J = 5.4 Hz, 1H, H-1'), 7.36 (br, 1H,
CONH2), 7.45 (t, 1H, H Ph), 7.52 (d, 1H, H Ph), 7.84 (d, 1H, H Ph), 7.84 (s, 1H, H Ph), 8.00 (br, 1H, CONH2). 13C NMR (100 MHz, DMSO-d6): δ 0.63 (CH3 TMS), 17.40 (CH2), 25.92 (CH3 isop), 27.85 (CH3 isop), 28.62 (CH2), 48.68 (CH2), 51.55 (C-5'), 83.20 (C-3'), 83.79 (C-4'), 84.66 (≡C- TMS), 85.19 (C-1'), 86.54 (C-2'), 108.43 (C≡C-TMS), 114.53 (Cq isop), 125.69, 127.28, 128.73 and 129.27 (CH Ph), 134.93 and 140.38 (Cq Ph), 169.18 (CONH2). HRMS (ESI+-TOF): m/z calcd for [C23H34N2O4Si+H]+ 431.2361, found 431.2356. 1β-(3-(carbamoyl)phenyl)-1,5-di-deoxy-2,3-O-isopropylidene-5-(N-propargyl)-(5- trimethylsilyl-pent-4-ynyl)amino-D-ribofuranose (17) To a stirred solution of 16 (0.10 g, 0.23 mmol) in anhydrous DMF (2.5 mL) were added DIEA (0.12 mL, 0.69 mmol) and propargyl bromide (80% in toluene, 38 µL, 0.35 mmol). After 2 h at room temperature, volatiles were removed under reduced pressure and the residue was purified by flash column chromatography (25 g SiO2, 0 to 3% MeOH in DCM) affording 17 (91 mg, 84%). 1H NMR (400 MHz, DMSO-d6): δ 0.11 (s, 9H, CH3 TMS), 1.30 (s, 3H, CH3 isop), 1.55 (s, 3H, CH3 isop), 1.58 (quint., 2H, CH2), 2.25 (t, 2H, CH2-C≡), 2.59 (t, 2H, CH2), 2.64-2.76 (m, 2H, H-5' and H-5"), 3.10 (br, 1H, C≡CH), 3.45 (br, 2H, CH2-C≡), 4.02-4.08 (m, 1H, H- 4'), 4.55 (dd, J = 5.2 Hz, J = 6.9 Hz, 1H, H-2'), 4.61 (dd, J = 4.2 Hz, J = 6.9 Hz, 1H, H-3'), 4.77 (d, J = 5.2 Hz, 1H, H-1'), 7.36 (br, 1H, CONH2), 7.45 (t, 1H, H Ph), 7.50 (d, 1H, H Ph), 7.81 (td, 1H, H Ph), 7.83 (s, 1H, H Ph), 7.99 (br, 1H, CONH2). 13C NMR (100 MHz, DMSO-d6): δ 25.91 (CH3 isop), 27.84 (CH3 isop), 28.72 (3C, CH3 Boc), 29.64 (CH2), 38.41 (CH2), 47.21 (CH2-C≡), 51.29 (C-5'), 76.04 (C≡CH), 77.83 (Cq Boc), 79.47 (CH2-C≡CH), 83.20 (C-4'), 83.79 (C-3'), 85.14 (C-1'), 86.36 (C-2'), 114.74 (Cq isop), 125.71, 127.23, 128.75, 129.13 (CH Ph), 134.96 and 140.30 (Cq Ph), 169.21 (CONH2). HRMS (ESI+-TOF): m/z calcd for [C26H36N2O4Si+H]+ 469.2517, found 469.2512.
3. Sonogashira cross-coupling reactions with alkyne 10 EXAMPLE 1
Coupling product 19 To an argon-purged flask containing bromide 18 (284 mg, 0.6 mmol) and alkyne 10 (133 mg, 0.4 mmol) in THF (4 mL) were added under argon atmosphere triethylamine (167 µL, 1.2 mmol), CuI (8 mg, 0.04 mmol) and tetrakis(triphenylphosphine)palladium (23 mg, 0.02 mmol). The reaction mixture was heated at 60°C for 1 h under argon atmosphere, concentrated under reduced pressure and the residue was purified by flash column chromatography (40 g SiO2, 0 to 5% MeOH in DCM) affording 19 (145 mg, 50%).
1H NMR (400 MHz, DMSO-d6): δ 1.30 (s, 3H, CH3 isop), 1.55 (s, 3H, CH3 isop), 1.94 (s, 3H, CH3 OAc), 2.05 (s, 3H, CH3 OAc), 2.10 (s, 3H, CH3 OAc), 3.80 (d, J = 5.3 Hz, 2H, H- 5'b and H-5''b), 4.13-4.22 (m, 2H, H-4'b and H-5'a), 4.32-4.38 (m, 1H, H-4'a), 4.42 (dd, J = 3.5 Hz, J = 12.0 Hz, 1H, H-5"a), 4.59 (dd, J = 5.2 Hz, J = 6.6 Hz, 1H, H-2'b), 4.72 (dd, J = 4.1Hz, J = 6.8 Hz, 1H, H-3'b), 4.82 (d, J = 5.2 Hz, 1H, H-1'b), 5.73-5.78 (m, 1H, H-3'a), 6.16 (d, J = 4.3 Hz, 1H, H-2'a), 6.21 (dd, J = 4.3 Hz, J = 6.0 Hz, 1H, H-1'a), 7.40 (br, 1H, CONH2), 7.44 (t, J = 7.5 Hz, 1H, H Ph), 7.53 (d, 1H, H Ph), 7.66 (br, 2H, NH2), 7.82 (dd, J = 6.3 Hz, J = 6.3 Hz, 1H, H Ph), 7.86 (s, 1H, H Ph), 8.02 (br, 1H, CONH2), 8.22 (s, 1H, H- 2a). 13C NMR (100 MHz, DMSO-d6): δ 20.65 (CH3 OAc), 20.76 (CH3 OAc), 20.82 (CH3 OAc), 25.84 (CH3 isop), 27.79 (CH3 isop), 58.83 (CH2C≡), 62.87 (C-5'a), 70.17 (C-3'a), 70.43 (C-5'b), 71.88 (C-2'a), 75.30 (C≡CH), 79.64 (C-4'a), 82.19 (C-3'b), 82.89 (C-4'b), 85.42 (C-1'b), 86.45 (C-2'b), 87.29 (C-1'a), 93.12 (C≡CH), 114.75 (Cq isop), 119.36 (C-5), 125.80, 127.29, 128.78 and 129.22 (CH Ph), 132.73 (C-8), 134.92 and 140.19 (Cq Ph), 149.14 (C-4), 154.56 (C-2), 156.53 (C-6), 168.17 (CONH2), 169.86 (2C, CO Ac), 170.42 (CO Ac). HRMS (ESI+-TOF): m/z calcd for [C33H39N6O12+H]+ 723.2620, found 723.2618. Compound 20 Compound 19 (133 mg, 0.18 mmol) was treated with 28% aq. ammonia (1 mL) in MeOH (2 mL) at room temperature for 5 h. After removal of the volatiles, the residue was then treated with an ice-cooled solution of 70% aqueous TFA (5 mL) for 4 h, then the volatiles were removed by lyophilization and the residue was purified by reverse phase HPLC (linear gradient of 5 to 25% acetonitrile in 10 mM TEAA over 15 min) affording 20 as a white foam (40 mg, 40%). 1H NMR (400 MHz, DMSO-d6): δ 3.49-3.60 (m, 1H, H-5'a), 3.66-3.84 (m, 4H, H-5''a, H- 2'b, H-5'b and H-5"b), 3.87-3.94 (m, 1H, H-3'b), 3.97-4.04 (m, J = 6.3 Hz, 2H, H-4'a and H-4'b), 4.18-4.24 (m, 1H, H-3'a), 4.64 (s, 2H, CH2-C≡), 4.65 (d, 1H, H-1'b), 4.95-5.15 (m, 3H, H-2'a, OH-2'b and OH-3'b), 5.20 (br, 1H, OH-3'a), 5.45 (br, 1H, OH-2'a), 5.52 (dd, 1H,
OH-5'), 5.97 (d, J = 6.8 Hz, 1H, H-1'a), 7.32 (br, 1H, CONH2), 7.42 (t, J = 7.8 Hz, 1H, H Ph), 7.55 (d, 1H, H Ph), 7.64 (br, 2H, NH2), 7.77 (d, 1H, H Ph), 7.86 (s, 1H, H Ph), 7.95 (br, 1H, CONH2), 8.17 (s, 1H, H-2a). 13C NMR (100 MHz, DMSO-d6): δ 58.79 (CH2-C≡), 62.64 (C-5'a), 71.29 (C-5'b), 71.45 (C-3'a), 72.07 (C-2'a and C-3'b), 75.58 (C≡CH), 77.61 (C-2'b), 83.27 (C-4'b), 83.91 (C- 1'b), 87.17 (C-4'a), 89.90 (C-1'a), 92.68 (C≡CH), 119.83 (C-5), 126.03, 126.88, 128.53 and 129.37 (CH Ph), 133.48 (C-8), 134.67 and 141.58 (Cq Ph), 148.89 (C-4), 153.89 (C-2), 156.64 (C-6), 168.45 (CONH2). HRMS (ESI+-TOF): m/z calcd for [C25H28N6O9+H]+ 557.1991, found 557.1987. EXAMPLE 2
Compound 21
Compound 21 was prepared as described previously (Paoletti, J. et al., Eur. J. Med. Chem. 2016, 124, 1041-1056). Coupling product 22 To a degassed solution (3 times) of bromide 21 (155 mg, 0.34 mmol) and alkyne 10 (82 mg, 0.17 mmol) in THF (3 mL) were added under argon atmosphere triethylamine (117 µL, 0.84 mmol), CuI (6 mg, 0.03 mmol) and tetrakis(triphenylphosphine)palladium (17 mg, 0.015 mmol). The reaction mixture was heated at 60°C for 2.5 h under argon atmosphere. The reaction mixture was concentrated to dryness and the residue was purified by flash column chromatography (20 g SiO2, 0 to 8% MeOH in DCM) affording 22 (77 mg containing 0.2 eq. triethylammonium salt), which was used in the next step without further purification. 1H NMR (400 MHz, DMSO-d6): δ 0.95 (t, 3H, CH3 Et), 1.29 and 1.30 (each s, 6H, CH3 isop), 1.51 and 1.55 (each s, 3H, CH3 isop), 2.93-3.02 (m, 2H, CH2 Et), 3.09-3.19 (m, 1H, H-5'a), 3.33-3.40 (m, 1H, H-5"a), 3.79 (d, 2H, H-5'b and H-5"b), 4.08-4.15 (m, 1H, H-4'a), 4.16-4.21 (m, 1H, H-4'b), 4.59 (dd, J = 5.4 Hz, J = 6.6 Hz, 1H, H-2'b), 4.67 (s, 2H, CH2- C≡), 4.71 (dd, J = 4.2 Hz, J = 6.8 Hz, 1H, H-3'b), 4.83 (d, J = 5.2 Hz, 1H, H-1'b), 5.00 (dd J = 3.5 Hz, J = 6.3 Hz, 1H, H-3'a), 5.57 (dd, J = 2.6 Hz, J = 6.3 Hz, 1H, H-2'a), 5.78 (t, 1H, NHEt), 6.01 (t, 1H, NH-5'), 6.14 (d, J = 2.5 Hz, 1H, H-1'a), 7.37 (br, 1H, CONH2), 7.43 (t, J = 7.7 Hz, 1H, H Ph), 7.52 (d, 1H, H Ph), 7.61 (br, 2H, NH2), 7.81 (d, 1H, H Ph), 7.85 (s, 1H, H Ph), 8.00 (br, 1H, CONH2), 8.22 (s, 1H, H-2a). 13C NMR (100 MHz, DMSO-d6): δ 16.03 (CH3 Et), 25.72, 25.87, 27.56 and 27.81 (CH3 isop), 34.54 (CH2 Et), 41.91 (C-5'a), 58.83 (CH2-C≡), 70.56 (C-5'b), 75.62 (C≡CH), 82.26 (C-3'b), 82.38 (C-3'a), 82.90 (C-4'b), 82.99 (C-2'a), 85.44 (C-1'b), 86.12 (C-4'a), 86.45 (C- 2'b), 89.71 (C-1'a), 92.88 (C≡CH), 114.75 (Cq isop), 119.36 (C-5), 125.78, 127.29, 128.76 and 129.18 (CH Ph), 132.73 (C-8), 134.92 and 140.19 (Cq Ph), 149.14 (C-4), 154.50 (C- 2), 156.53 (C-6), 158.27 (CO), 168.19 (CONH2). HRMS (ESI+-TOF): m/z calcd for [C33H43N8O9+H]+ 707.3148, found 707.3119.
Compound 23 Compound 22 (65 mg, 0.09 mmol) was treated with an ice-cooled solution of 70% aqueous TFA (1 mL) for 2.5 h, then the volatiles were removed by lyophilization and the residue was purified by reverse phase HPLC (linear gradient of 10 to 25% acetonitrile in 10 mM TEAA over 15 min) affording 23 (12 mg, 22%). 1H NMR (400 MHz, DMSO-d6): δ 0.96 (t, 3H, CH3 Et), 2.95-3.03 (m, 2H, CH2 Et), 3.18- 3.27 (m, 1H, H-5'a), 3.40-3.49 (m,1H, H-5''a), 3.71-3.82 (m, 3H, H-2'b, H-5'b and H-5"b), 3.87-3.94 (m, 2H, H-4'a and H-3'b), 3.98-4.03 (m, 1H, H-4'b), 4.16-4.21 (m, 1H, H-3'a), 4.63-4.66 (m, 3H, H-1'b and CH2-C≡), 5.07 (d, 1H, OH-3'b), 5.09-5.15 (m, 2H, H-2'a, OH- 2'b), 5.23 (d, 1H, OH-3'a), 5.45 (br, 1H, OH-2'a), 5.81 (t, 1H, NHEt), 5.96 (d, J = 6.3 Hz, 1H, H-1'a), 6.08 (t, 1H, NH-5'a), 7.34 (br, 1H, CONH2), 7.42 (t, J = 7.5 Hz, 1H, H Ph), 7.54 (d, J = 6.3 Hz, 1H, H Ph), 7.57 (br, 2H, NH2), 7.77 (d, J = 7.8 Hz, 1H, H Ph), 7.85 (s, 1H, H Ph), 7.97 (br, 1H, CONH2), 8.23 (s, 1H, H-2a). 13C NMR (100 MHz, DMSO-d6): δ 16.08 (CH3), 34.58 (CH2), 42.19 (C-5'a), 58.81 (CH2- C≡), 71.31 (C-5'b), 71.55 (C-3'b), 71.68 (C-3'a), 72.06 (C-4'b), 75.59 (C≡CH), 77.60 (C- 2'a), 83.27 (C-1'b), 83.95 (C-4'a), 85.02 (C-2'b), 89.47 (C-1'a), 92.60 (C≡CH), 119.74 (C- 5), 126.03, 126.88, 128.53 and 129.35 (CH Ph), 133.38 (C-8), 134.69 and 141.57 (Cq Ph), 149.37 (C-4), 154.33 (C-2), 156.53 (C-6), 158.38 (CO), 168.45 (CONH2). HRMS (ESI+-TOF): m/z calcd for [C28H34N8O9+H]+ 627.2506, found 627.2522. EXAMPLE 3
Reaction scheme
Compound 24 Compound 24 was prepared as described previously (Paoletti, J. et al., Eur. J. Med. Chem. 2016, 124, 1041-1056). Coupling product 25 To a degassed solution (3 times) of bromide 24 (0.37 g, 0.75 mmol) and alkyne 10 (0.275 g, 0.83 mmol) in THF (7.5 mL) were added under argon atmosphere triethylamine (314 µL, 2.25mmol), CuI (14 mg, 0.075 mmol) and tetrakis(triphenylphosphine)palladium (44 mg, 0.038mmol). The reaction mixture was heated at 60°C for 1.5 h under argon atmosphere. The reaction mixture was concentrated to dryness and the residue was purified by flash column chromatography (30 g SiO2, 3 to 6% MeOH in DCM; 20 g SiO2, 2 to 3% MeOH in ethylacetate) affording 25 (0.14 g 25%). 1H NMR (400 MHz, DMSO-d6): δ 1.31 and 1.32 (each s, 6H, CH3 isop), 1.53 and 1.55 (each s, 6H, CH3 isop), 2.56 (s, 6H, CH3), 3.08-3.17 (m, 1H, H-5'a), 3.20-3.28 (m, 1H, H- 5"a), 3.79 (d, 2H, H-5'b and H-5"b), 4.19 (q, 1H, H-4'b), 4.23-4.29 (m, 1H, H-4'a), 4.59 (dd, J = 5.4 Hz, J = 6.6 Hz, 1H, H-2'b), 4.65 (s, 2H, CH2-C≡), 4.71 (dd, J = 4.2 Hz, J = 6.8 Hz, 1H, H-3'b), 4.84 (d, J = 5.2 Hz, 1H, H-1'b), 5.08 (dd J = 2.8 Hz, J = 6.2 Hz, 1H, H- 3'a), 5.54 (dd, J = 2.5 Hz, J = 6.3 Hz, 1H, H-2'a), 6.16 (d, J = 2.5 Hz, 1H, H-1'a), 7.35 (br, 1H, CONH2), 7.43 (t, J = 7.5 Hz, 1H, H Ph), 7.53 (d, J = 6.3 Hz, 1H, H Ph), 7.60-7.70 (m,
3H, NH-5' and NH2), 7.80 (d, J = 7.8 Hz, 1H, H Ph), 7.85 (s, 1H, H Ph), 7.98 (br, 1H, CONH2), 8.20 (s, 1H, H-2a). 13C NMR (100 MHz, DMSO-d6): δ 25.62, 25.87, 27.47 and 27.80 (CH3 isop), 37.92 (2C, NCH3), 45.15 (C-5'a), 58.79 (CH2-C≡), 70.52 (C-5'b), 75.40 (C≡CH), 82.23 (C-3'b), 82.43 (C-3'a), 82.87 (C-4'b), 83.03 (C-2'a), 85.42 (C-1'b), 85.53 (C-4'a), 86.44 (C-2'b), 90.50 (C- 1'a), 92.97 (C≡CH), 113.85 (Cq isop), 114.75 (Cq isop), 119.44 (C-5), 125.78, 127.28, 128.76 and 129.19 (CH Ph), 132.73 (C-8), 134.90 and 140.19 (Cq Ph), 148.58 (C-4), 154.29 (C-2), 156.60 (C-6), 168.18 (CONH2). HRMS (ESI+-TOF): m/z calcd for [C33H42N8O10S+H]+ 743.2817, found 743.2795. Compound 26 Compound 25 (130 mg, 0.18 mmol) was treated with an ice-cooled solution of 70% aqueous TFA (2 mL) for 2 h, then water was added to the mixture and the volatiles were removed by lyophilization. Purification by reverse phase HPLC (linear gradient of 10 to 30% acetonitrile in 10 mM TEAA over 15 min) affording 26 (12 mg, 22%). 1H NMR (400 MHz, DMSO-d6): δ 0.96 (t, 3H, CH3 Et), 2.95-3.03 (m, 2H, CH2 Et), 3.18- 3.27 (m, 1H, H-5'a), 3.40-3.49 (m,1H, H-5''a), 3.71-3.82 (m, 3H, H-2'b, H-5'b and H-5"b), 3.87-3.94 (m, 2H, H-4'a and H-3'b), 3.98-4.03 (m, 1H, H-4'b), 4.16-4.21 (m, 1H, H-3'a), 4.63-4.66 (m, 3H, H-1'b and CH2-C≡), 5.07 (d, 1H, OH-3'b), 5.09-5.15 (m, 2H, H-2'a, OH- 2'b), 5.23 (d, 1H, OH-3'a), 5.45 (br, 1H, OH-2'a), 5.81 (t, 1H, NHEt), 6.16 (d, J = 2.5 Hz, 1H, H-1'a), 7.35 (br, 1H, CONH2), 7.43 (t, J = 7.5 Hz, 1H, H Ph), 7.53 (d, J = 6.3 Hz, 1H, H Ph), 7.60-7.70 (m, 3H, NH-5' and NH2), 7.80 (d, J = 7.8 Hz, 1H, H Ph), 7.85 (s, 1H, H Ph), 7.98 (br, 1H, CONH2), 8.20 (s, 1H, H-2a). 13C NMR (100 MHz, DMSO-d6): δ 16.08 (CH3), 34.58 (CH2), 42.19 (C-5'a), 58.81 (CH2- C≡), 71.31 (C-5'b), 71.55 (C-3'b), 71.68 (C-3'a), 72.06 (C-4'b), 75.59 (C≡CH), 77.60 (C- 2'a), 83.27 (C-1'b), 83.95 (C-4'a), 85.02 (C-2'b), 89.47 (C-1'a), 92.60 (C≡CH), 119.74 (C- 5), 126.03, 126.88, 128.53 and 129.35 (CH Ph), 133.38 (C-8), 134.69 and 141.57 (Cq Ph), 149.37 (C-4), 154.33 (C-2), 156.53 (C-6), 158.38 (CO), 168.45 (CONH2).
HRMS (ESF-TOF): m/z calcd for [C28H34N8O9+H]+ 627.2506, found 627.2522.
4. Sonogashira cross-coupling reactions with alkyne 15
Coupling product 27b
To a degassed solution (3 times) of bromide 18 (0.28 g, 0.6 mmol) and alkyne 15 (0.48 g, 1 mmol) in THF (5 mL) were added under argon atmosphere triethylamine (0.21 mL, 1.5 mmol), Cui (10 mg, 0.05 mmol) and tetrakis(triphenylphosphine)palladium (29 mg, 0.025 mmol). The reaction mixture was heated at 60°C for 3 h under argon atmosphere. The
reaction mixture was concentrated to dryness and the residue was purified by flash column chromatography (20 g SiO2, 2 to 4% MeOH in DCM) affording a mixture of coupling and homocoupling products (4/1 ratio, 0.55 g), which was used in the next step without further purification. The mixture of coupling and homocoupling products (0.54 g) was first treated with aq. ammonia (1 mL) in MeOH (4 mL) at room temperature overnight. After removal of the volatiles, the residue was purified by flash column chromatography (20 g SiO2, 3 to 10% MeOH in DCM) affording deacetylated coupling product 27b (0.35 g, 78% in two steps). HRMS (ESI+-TOF): m/z calcd for [C35H50N8O10+H]+ 753.3566, found 753.3564. Compound 28 The deacetylated product 27b (0.34 g, 0.45 mmol) was treated with an ice-cooled solution of 70% aqueous TFA (5 mL) for 3 h, then the volatiles were removed by lyophilization and the residue was purified by reverse phase HPLC (linear gradient of 5 to 25% acetonitrile in 10 mM TEAA over 15 min) affording 28 as acetate salt (0.24 g, 83%). 1H NMR (400 MHz, DMSO-d6): δ 1.68-1.73 (m, 2H, CH2), 1.74 (s, 3H, CH3 Ac), 2.68- 2.75 (m, 2H, CH2), 2.74-2.88 (m, 4H, CH2, H-5'b and H-5"b), 3.54 (dd, J = 4.0 Hz, J = 12.2 Hz, 1H, H-5'a), 3.70 (dd, J = 3.6 Hz, J = 12.2 Hz, 1H, H-5"a), 3.74 (t, 1H, H-2'b), 3.83 (t, 1H, H-3'b), 3.89 (br, 2H, CH2-C≡), 3.94-3.98 (m, 1H, H-4'b), 3.98-4.03 (m, 1H, H- 4'a), 4.21 (dd, J = 2.3Hz, J = 5.2 Hz, 1H, H-3'a), 4.64 (d, J = 6.1 Hz, 1H, H-1'b), 5.00 (d, J = 5.5 Hz, J = 6.8 Hz, 1H, H-2'a), 6.02 (d, J = 6.8 Hz, 1H, H-1'a), 7.35 (br, 1H, CONH2), 7.42 (t, J = 7.7 Hz, 1H, H Ph), 7.53 (d, 1H, H Ph), 7.62 (br, 2H, NH2), 7.77 (d, 1H, H Ph), 7.86 (s, 1H, H Ph), 8.02 (br, 1H, CONH2), 8.16 (s, 1H, H-2a). 13C NMR (100 MHz, DMSO-d6): δ 23.50 (CH3 Ac), 27.52 (CH2), 38.59 (CH2), 43.76 (CH2C≡), 51.89 (CH2), 56.58 (C-5'b), 62.72 (C-5'a), 71.51 (C-3'a), 72.20 (C-2'a), 73.31 (C- 3'b), 74.83 (C≡CH), 77.38 (C-2'b), 83.04 (C-4'b), 84.31 (C-1'b), 87.13 (C-4'a), 90.07 (C- 1'a), 93.08 (C≡CH), 119.70 (C-5), 125.86, 126.82, 128.55 and 129.30 (CH Ph), 134.00 (C-
8), 134.72 and 141.69 (Cq Ph), 148.89 (C-4), 153.66 (C-2), 156.57 (C-6), 168.52 (CONH2), 174.15 (CO Ac). HRMS (ESI+-TOF): m/z calcd for [C28H36N8O8+H]+ 613.2729, found 613.2709. 5. Sonogashira cross-coupling reactions with alkyne 17 EXAMPLE 5
Coupling product 29a To a degassed solution (3 times) of bromide 21 (96 mg, 0.21 mmol) and alkyne 17 (82 mg, 0.17 mmol) in THF (2 mL) were added under argon atmosphere triethylamine (71 µL, 0.51
mmol), CuI (8 mg, 0.04 mmol) and tetrakis(triphenylphosphine)palladium (24 mg, 0.02 mmol). The reaction mixture was heated at 60°C for 1 h under argon atmosphere. The reaction mixture was concentrated to dryness and the residue was purified by flash column chromatography (20 g SiO2, 2 to 4% MeOH in DCM) affording 29a (83 mg containing 0.18 eq. triethylammonium salt), which was used in the next step without further purification. HRMS (ESI+-TOF): m/z calcd for [C40H58N9O8Si+H]+ 844.4172, found 844.4158. Compound 30 To the coupling product 29a (75 mg) in THF (1) was added TBAF (1M in THF, 96 µL). After 1.5 h at room temperature, the volatiles were removed under reduced pressure and the residue was purified by flash column chromatography (20 g SiO2, 5 to 8% MeOH in DCM) affording free alkyne 29b (65 mg, 50% in two steps).
0.95 (CH3 Et), 1.63-1.72 (m, 2H, CH2), 1.29 (s, 3H, CH3 isop), 1.31 (s, 3H, CH3 isop), 1.50 (s, 3H, CH3 isop), 1.56 (s, 3H, CH3 isop), 2.23 (dt, 2H, NCH2), 2.71 (t, 2H, NCH2-C≡), 2.75 (t, 2H, ≡CH), 2.80-2.86 (m, 2H, H-5'b and H- 5"b), 2.94-3.02 (m, 2H, CH2 Et), 3.10-3.20 (m, 1H, H-5'a), 3.32-3.40 (m, 1H, H-5"a), 3.91 (s, 2H, CH2-C≡), 4.09-4.14 (m, 2H, H-4'a and H-4'b), 4.58 (dd, J = 5.3 Hz, J = 6.8 Hz, 1H, H-2'b), 4.65 (dd, J = 4.2 Hz, J = 6.8 Hz, 1H, H-3'b), 4.79 (d, J = 5.3 Hz, 1H, H-1'b), 4.98 (dd, J = 3.3 Hz, J = 6.4 Hz, 1H, H-3'a), 5.61 (dd, J = 2.7 Hz, J = 6.4 Hz, 1H, H-2'a), 5.77 (t, 1H, NHEt), 6.00 (t, 1H, NH-5'), 6.17 (d, J = 2.7 Hz, 1H, H-1'a), 7.37 (br, 1H, CONH2), 7.44 (t, J = 7.5 Hz, 1H, H Ph), 7.51 (d, 1H, H Ph), 7.57 (br, 1H, NH2), 7.81 (d, 1H, H Ph), 7.84 (s, 1H, H Ph), 8.00 (br, 1H, CONH2), 8.21 (s, 1H, H-2a). 13C NMR (100 MHz, DMSO-d6): δ 15.91 (CH2), 16.03 (CH3 Et), 25.72 and 25.92 (CH3 isop), 26.62 (CH2), 27.57 and 27.82 (CH3 isop), 34.58 (CH2 Et), 40.70 (C-5'a), 41.92 (CH2- C≡), 53.39 (NCH2), 55.96 (C-5'b), 71.63 (Cq isop), 74.65 (C≡CH), 82.45 (C-3'a), 82.79 (C-2'a), 83.73 (C-3'b), 85.15 (C-1'b), 86.06 (C-4'a), 86.31 (C-2'b), 90.01 (C-1'a), 93.24 (C≡CH), 113.95 and 114.88 (Cq isop), 119.28 (C-5), 125.76, 127.25, 128.77 and 129.13
(CH Ph), 133.15 (C-8), 134.95 and 140.17 (Cq Ph), 148.86 (C-4), 154.30 (C-2), 156.45 (C- 6), 158.27 (CO), 168.18 (CONH2). HRMS (ESI+-TOF): m/z calcd for [C37H50N9O8+H]+ 772.3777, found 772.3762. Compound 29b (52 mg, 0.067 mmol) was treated with an ice-cooled solution of 70% aqueous TFA (1 mL) for 4 h, then the volatiles were removed by lyophilization and the residue was purified by reverse phase HPLC (linear gradient of 0 to 50% acetonitrile in 10 mM TEAA over 15 min) affording 30 (36 mg, 78%).
0.96 (CH3 Et), 1.63-1.72 (m, 2H, CH2-C≡), 2.23 (dt, 2H, NCH2), 2.70 (t, 2H, CH2), 2.74 (t, 2H, ≡CH), 2.75-2.86 (m, 2H, H-5'b and H-5"b), 2.94-3.04 (m, 2H, CH2 Et), 3.17-3.28 (m, 1H, H-5'a), 3.39-3.50 (m, 1H, H-5"a), 3.74 (dd, 1H, H-2'b), 3.82 (t, 1H, H-3'b), 3.86-3.90 (m, 3H, CH2-C≡ and H-4'b), 3.91-3.98 (m, 1H, H-4'a), 4.18 (dd, J = 4.7 Hz, J = 7.8 Hz, 1H, H-3'a), 4.63 (d, J = 6.4 Hz, 1H, H-1'b), 5.00 (d, J = 5.3 Hz, 1H, OH-3'b), 5.09 (d, J = 6.5 Hz, 1H, OH-2'b), 5.13 (q, J = 6.0 Hz, 1H, H- 2'a), 5.19 (d, J = 4.7 Hz, 1H, OH-3'a), 5.41 (d, J = 6.1 Hz, 1H, OH-2'a), 5.80 (t, 1H, NHEt), 5.99 (d, J = 6.2 Hz, 1H, H-1'a), 6.08 (t, 1H, NH-5'), 7.34 (br, 1H, CONH2), 7.41 (t, J = 7.5 Hz, 1H, H Ph), 7.50-7.56 (br, 3H, H Ph and NH2), 7.77 (d, 1H, H Ph), 7.84 (s, 1H, H Ph), 7.96 (br, 1H, CONH2), 8.22 (s, 1H, H-2a). 13C NMR (100 MHz, DMSO-d6): δ 15.99 (CH2), 16.07 (CH3 Et), 26.76 (CH2), 34.58 (CH2 Et), 42.22 (C-5'a), 43.93 (CH2-C≡), 53.48 (NCH2), 56.57 (C-5'b), 71.48 (C-2'a), 71.61 (C- 3'a), 73.29 (C-3'b), 74.89 (C≡CH), 77.28 (C-2'b), 83.22 (C-4'b), 84.17 (C-1'b), 84.87 (C- 4'a), 89.58 (C-1'a), 93.02 (C≡CH), 119.60 (C-5), 125.94, 126.82, 128.55 and 129.25 (CH Ph), 133.97 (C-8), 134.72 and 141.62 (Cq Ph), 148.35 (C-4), 154.10 (C-2), 156.44 (C-6), 158.39 (CO), 168.52 (CONH2). HRMS (ESI+-TOF): m/z calcd for [C33H41N9O8+H]+ 692.3151, found 692.3130. 6. Macrocycle EXAMPLE 6
5 ’ - Amino-5 ’ -Deoxy-2' ,3 ’ -O-isopropylidene-adenosine (33b) The title compound was obtained in two steps from commercially available 2',3'-O- isopropylidene- adenosine following reported procedures. First, adenosine derivative was reacted with of DPPA (2 eq.) and DBU (3 eq.) in dioxane (10 mL/mmol) for 3 h, followed
by NaN3 (5 eq.), TBAI (0.1 eq.) and 15-crown-5 (0.1 eq.) yielding 5’-azido derivative 33a in 85% yield. Staudinger reduction of 33a (PPh3, pyridine, NH4OH) gave 33b in 92% yield (Paoletti et al., Eur. J. Med. Chem.2016, 124, 1041-1056; Clément et al., Molecules 2020, 25, 4893). NMR spectra was identical to that reported in the literature. 5’-Deoxy-5’-(3-(N-Boc-amino)ethylamido)-2',3’-O-isopropylidene-adenosine (33c) To a solution of 33b (0.46 g, 1.5 mmol) in anhydrous DMF (15 mL) were added N-Boc- glycine (0.315 g, 1.8 mmol), DIEA (0.78 mL, 4.5 mmol) and PyBOP (0.78 g, 1.5 mmol). After stirring for 2 h at room temperature, the volatiles were removed under reduced pressure and the residue purified by flash column chromatography (60 g SiO2, 2 to 5% MeOH in DCM) to give 33c (0.63 g, 91%) containing residual phosphine reagent (<10%), which was used in the next step without further purification. 1H NMR (400 MHz, DMSO-d6): δ 1.32 (s, 3H, CH3 isop), 1.34 (s, 9H, CH3 Boc), 1.54 (s, 3H, CH3 isop), 1.73 (m, 1.3H, CH2 phosphine), 2.28 (m, 2H, CH2NBoc), 3.00-3.05 (m, 1.3H, CH2 phosphine), 3.13 (q, 2H, CH2CO), 3.35-3.43 (m, 2H, H-5' and H-5"), 4.17-4.23 (m, 1H, H-4'), 4.90 (dd, J = 2.3 Hz, J = 5.7 Hz, 1H, H-3'), 5.38-5.43 (m, 1H, H-2'), 6.11 (d, J = 2.4 Hz, 1H, H-1’), 6.72 (br, 1H, NHBoc), 7.35 (br, 2H, NH2), 8.18 (t, 1H, NHCO), 8.19 (s, 1H, H-2), 8.32 (s, 1H, H-8). 13C NMR (100 MHz, DMSO-d6): δ 25.76 (CH3 isop), 26.32 and 26.39 (CH2 residual phosphine), 27.54 (CH3 isop), 28.63 (3C, CH3 Boc), 36.21 (CH2), 37.18 (CH2NBoc), 41.15 (C-5’), 46.03 and 46.34 (CH2 phosphine), 78.02 (Cq Boc), 82.13 (C-2’), 83.18 (C-3'), 84.45 (C-4’), 89.73 (C-1’), 114.00 (Cq isop), 119.86 (C-5), 140.58 (C-8), 149.20 (C-4), 153.20 (C-2), 155.92 (CO Boc), 156.69 (C-6), 171.24 (CONH). HRMS (ESI-TOF) m/z calcd for [C21H31N7O6+H] 478.2409, found 478.2395. 5'-Deoxy-5'-(2-(N-Boc-amino)ethylamido)-2',3'-O-isopropylidene-8-bromo-adenosine (34) To a strirred solution of 33c (0.62 g, 1.3 mmol) in a mixture of dioxane/acetate buffer pH 5.3 (17 mL/10 mL) was added dropwise bromine (0.14 mL, 2.7 mmol). After stirring for 2
h at room temperature, the reaction was quenched by addition of saturated aqueous solution Na2S2O3 (15 mL) and the mixture was extracted with ethyl acetate (2 x 50 mL). The combined organic phases were washed with aqueous NaCl (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography (30 g SiO2, 2 to 5% MeOH in DCM) to give 34 (0.38 g, 54%). 1H NMR (400 MHz, DMSO-d6): δ 1.32 (s, 3H, CH3 isop), 1.34 (s, 9H, 3 CH3 Boc), 1.54 (s, 3H, CH3 isop), 3.32-3.44 (m, 2H, H-5' and H-5"), 3.52 (d, 2H, CH2), 4.19-4.24 (m, 1H, H- 4'), 4.96 (dd, J = 3.0 Hz, J = 6.0 Hz, 1H, H-3'), 5.62 (dd, J = 2.6 Hz, J = 6.0 Hz, 1H, H-2'), 6.02 (d, J = 2.7 Hz, 1H, H-1’), 6.91 (t, 1H, NHBoc), 7.53 (br, 2H, NH2), 8.08 (t, 1H, 5'- NHCO), 8.21 (s, 1H, H-2). 13C NMR (100 MHz, DMSO-d6): δ 25.71 (CH3 isop), 27.59 (CH3 isop), 28.60 (3C, CH3 Boc), 41.06 (C-5’), 43.67 (NCH2), 78.44 (Cq Boc), 82.23 (C-3'), 82.57 (C-2'), 85.22 (C- 4’), 91.08 (C-1’), 113.90 (Cq isop), 119.81 (C-5), 126.63 (C-8), 150.30 (C-4), 153.47 (C- 2), 155.52 (C-6), 156.26 (CO Boc), 170.15 (CONH). HRMS (ESI-TOF) m/z 156.27 (CO Boc), calcd for [C20H28BrN7O6+H] 542.1357 and 544.1338, found 542.1355 and 544.31. 3-(5'-Deoxy-5'-(N-[3-(5'-deoxy-5'-(2-(N-Boc-amino)ethylamido)-2',3'-O- isopropylidene-adenosin-8-yl)prop-2-yn-1-yl]-2-(ethyl ethanoate)amino)-2',3'-O- isopropylidene-β-D-ribofuranosyl)benzamide (35) To a degassed solution (3 times) of bromide 34 (0.20 g, 0.37 mmol) and alkyne 32 (0.18 g, 0.44 mmol) in THF (4 mL) were added under argon atmosphere triethylamine (71 µL, 0.51 mmol), CuI (8 mg, 0.04 mmol) and tetrakis(triphenylphosphine)palladium (24 mg, 0.02 mmol). The reaction mixture was heated at 60°C for 1 h under argon atmosphere. The reaction mixture was concentrated to dryness and the residue was purified by flash column chromatography (40 g SiO2, 0 to 8% MeOH in DCM) affording 35 (0.20 g containing 0.11 eq triethylammonium salt), which was used in the next step without further purification.
1H NMR (400 MHz, DMSO-d6): δ 1.17 (t, 3H, CH3 of OEt), 1.29 (s, 3H, CH3 isop), 1.30 (s, 3H, CH3 isop), 1.33 (s, 9H, 3 CH3 Boc), 1.51 (s, 3H, CH3 isop), 1.55 (s, 3H, CH3 isop), 2.85 (dd, 1H, 1H, H-5'b), 2.97 (dd, 1H, H-5"b), 3.32-3.49 (m, 2H, H-5'a and H-5"a), 3.53 (d, 2H, CH2NHBoc), 3.58 (br, 2H, NCH2COOEt), 4.02 (br, 2H, CH2-C≡), 4.09 (q, 2H, CH2 of OEt), 4.12-4.18 (m, 1H, H-4'b), 4.20-4.25 (m, 1H, H-4'a), 4.57 (dd, J = 5.5 Hz, J = 6.6 Hz, 1H, H-2'b), 4.72 (dd, J = 4.5 Hz, J = 6.9 Hz, 1H, H-3'b),4.79 (d, J = 5.3 Hz, H-1'b), 4.89-4.94 (m, 1H, H-3'a), 5.50-5.55 (m, 1H, H-2'a), 6.14 (d, J = 3.0 Hz, H-1'a), 6.92 (t, 1H, NHBoc), 7.36 (br, 1H, CONH2), 7.44 (t, 1H, H Ph), 7.50 (d, 1H, H Ph), 7.59 (br, 2H, NH2), 7.81 (d, 1H, H Ph), 7.83 (s, 1H, H Ph), 7.98 (br, 1H, CONH2), 8.18 (br, 1H, 5'-NHCO), 8.26 (s, 1H, H-2a). 13C NMR (100 MHz, DMSO-d6): δ 9.14 (CH3 of OEt), 25.67 and 25.92 (CH3 isop), 27.57 and 27.81 (CH3 isop), 28.58 (3C, CH3 Boc), 41.19 (C-5’a), 43.70 (CH2NHBoc), 44.39 (CH2-C≡), 55.36 and 55.47 (NCH2CO and C-5’b), 60.57 (CH2), 74.40 (C≡C-CH2), 78.44 (Cq Boc), 82.29 (C-3'a), 82.68 (C-2'a), 83.10 (C-4’b), 83.33 (C-3'b), 84.90 (C-4'a), 85.20 (C-1’b), 86.19 (C-2'b), 90.38 (C-1’a), 93.05 (C≡C-CH2), 113.92 and 114.91 (2C, Cq isop), 119.41 (C-5a), 125.75, 127.29, 128.77, 129.11 (CH Ph), 132.98 (C-8a), 134.95 (Cq Ph), 140.05 (Cq Ph), 148.72 (C-4), 154.38 (C-2a), 156.27 (CO Boc), 156.51 (C-6), 168.14 (COOEt), 170.19 (CONH), 170.63 (CONH). HRMS (ESI-TOF) m/z calcd for [C42H55N9O12+H] 878.4043, found 878.4035. 3-(5'-Deoxy-5'-(N-(3-(5'-deoxy-5'-(2-aminoethylamido)-adenosin-8-yl)prop-2-yn-1-yl)- 2-(acetic acid)amino)-β-D-ribofuranosyl)benzamide (36) To a solution of compound 35 (0.18 g) in DCM (1 mL) was added dioxane/water (1 mL/2 mL) and 2N NaOH (85 µL, 0.34 mmol). After stirring for 2 h at room temperature (LC-MS monitoring), volatiles were removed by lyophilization. The residue was then treated by an ice-cooled solution of 60% aqueous TFA (4 mL) for 4 h, volatiles were removed by lyophilization. An aliquot (1/10) of the crude product was purified by reverse phase HPLC (linear gradient of 5 to 30% acetonitrile in 10 mM TEAA over 15 min) affording 36 (7 mg); tR = 8.8 min. The crude product (9/10) was used in the cyclisation step without further purification.
1H NMR (400 MHz, DMSO-d6): δ 2.97-3.05 (m, 2H, H-5'b and H-5"b), 3.27 (q, 2H, CH2), 3.37-3.45 (m, 1H, H-5'a), 3.47 (q, 2H, COCH2), 3.53-3.62 (m, 1H, H-5"a), 3.74 (t, 1H, H- 3'b), 3.86 (t, 1H, H-2'b), 3.88-4.06 (m, 5H, CH2, H-4'b, H-3'a and H-4'a), 4.63 (d, J = 6.1 Hz, 1H, H-1'b), 4.93 (pt, J = 6.4 Hz, 1H, H-2'a), 6.19 (d, J = 6.9 Hz, 1H, H-1’a), 7.29 (br, 1H, CONH2), 7.42 (t, 1H, H Ph), 7.52 (s, 2H, NH2), 7.53 (d, 1H, H Ph), 7.76 (d, 1H, H Ph), 7.85 (s, 1H, H Ph), 7.98 (br, 1H, CONH2), 8.22 (s, 1H, H-2a), 8.60 (br, 1H, 5'-NHCO). 13C NMR (100 MHz, DMSO-d6): δ 41.63 (C-5'a), 42.25 (COCH2NH2), 44.59 (CH2-C≡), 56.51 (C-5'b), 58.59 (NCH2CO), 71.87 (C-3’a), 72.25 (C-2'a), 73.38 (C-2'b), 74.21 (C≡C- CH2), 77.45 (C-2'b), 77.44 (C-3’b), 84.26 (C-1’b), 84.92 (C-4’a), 89.26 (C-1’a), 94.08 (C≡C-CH2), 119.69 (C-5a), 125.85, 126.87, 128.59 and 129.28 (CH Ph), 134.27 (C-8a), 134.68 (Cq Ph), 141.73 (Cq Ph), 149.02 (C-4a), 153.89 (C-2a), 156.50 (C-6a), 168.44 (CONH), 168.97 (CONH), 174.56 (COOH). HRMS (ESI-TOF) m/z calcd for [C29H35N9O10+H] 670.2580, found 670.2581. Macrocycle 37 A solution of crude 36 (100 mg, 0.15 mmol) and DIEA (155 µL, 0.9 mmol) in DMF (37mL) was added dropwise over 2 h to a stirred solution of PyBOP (117 mg, 0.2 mmol) in DMF (2 mL). After stirring for 4 h at room temperature, PyBOP (78 mg, 0.1 mmol) in DMF (1mL) and DIEA (52 µL, 0.3 mmol) were added to the mixture. After overnight stirring at room temperature, the reaction mixture was concentrated to dryness, solubilized in H2O/acetonitrile (10 mL, 9/1) and purified by reverse phase HPLC (linear gradient of 10-30% acetonitrile in 10 mM TEAA buffer over 15 min) affording 37 (20 mg, 21%). tR = 7.3 min. 1H NMR (400 MHz, DMSO-d6): δ 2.91-2.96 (m, 2H, H-5'b and H-5"b), 3.42-3.52 (m, 5H, CH2, NCH2CO, H-5"a and H-5'a), 3.75-3.90 (m, 5H, H-2'b, H-3'b, H-4'b, and CH2CO), 3.75-3.90 (m, 4H, H-3'a, H-4'a and CH2-C≡), 4.67 (d, J = 5.7 Hz, 1H, H-1'b), 4.83-4.90 (d, 1H, H-2'a), 5.05 (d, 1H, OH-3'b), 5.15 (d, 1H, OH-2'b), 5.23 (d, 1H, OH-3'a), 5.47 (d, 1H, OH-2'a), 5.91 (d, J = 4.1 Hz, 1H, H-1'a), 6.89 (t, 1H, NHCO), 7.33 (br, 1H, CONH2), 7.41
(t, 1H, H Ph), 7.55 (d, 1H, H Ph), 7.59 (br, 2H, NH2), 7.77 (d, 1H, H Ph), 7.96 (br, 1H, CONH2), 8.21(s, 1H, H-2a), 8.47 (t, 1H, 5'-NHCO). 13C NMR (100 MHz, DMSO-d6): δ 39.37 (NHCH2), 43.10 and 43.17 (C-5'a and CH2-C≡), 56.63 (NCH2CO), 57.77 (C-5'b), 68.94 (C-3’a), 71.24 (C-2'a), 73.36 (C-3'b), 76.09 (C≡C- CH2), 77.23 (C-2'b), 79.97 (C-4’b), 82.92 (C-4’a), 84.60 (C-1’b), 88.67 (C-1’a), 92.22 (C≡C-CH2), 118.45 (C-5a), 125.89, 126.91, 128.60 and 129.28 (CH Ph), 131.86 (C-8a), 134.73 (Cq Ph), 141.52 (Cq Ph), 150.12 (C-4a), 154.60 (C-2a), 156.31 (C-6a), 168.38 (CONH2), 170.00 (CONH), 170.37 (CONH). HRMS (ESI-TOF) m/z calcd for [C29H33N9O9+H] 652.2474, found 652.2474. EXAMPLE 7
Reaction scheme
Bromide 38 Compound 38 was synthesized as described previously in Clement et al., Eur. J. Med. Chem.2023, 246, 114941. Bromide 39 To a solution of bromide 38 (0.58 g, 1.5 mmol) in DMF (15 mL) was added di-tert-butyl dicarbonate (1.10 g, 2.25mmol). After 18 hours at room temperature, volatiles were removed under reduced pressure and the residue was purified by flash column chromatography (35 g SiO2, 2 to 5% MeOH in DCM) to give 39 (0.53 g, 73%). 1H NMR (400 MHz, DMSO-d6): δ 1.32 (s, 3H, CH3 isop), 1.36 (s, 9H, CH3 Boc), 1.54 (s, 3H, CH3 isop), 3.11-3.22 (m, 2H, H-5' and H-5"), 4.16-4.22 (m, 1H, H-4'), 5.02 (dd, J = 3.0 Hz, J = 6.0 Hz, 1H, H-3'), 5.64 (dd, J = 2.2 Hz, J = 6.2 Hz, 1H, H-2'), 6.01 (d, J = 2.2 Hz, 1H, H-1’), 7.15 (br t, 1H, NHBoc), 7.54 (br, 2H, NH2), 8.15 (s, 1H, H-2). 13C NMR (100 MHz, DMSO-d6): δ 25.71 (CH3 isop), 27.59 (CH3 isop), 28.60 (3C, CH3 Boc), 42.30 (C-5’), 78.44 (Cq Boc), 82.23 (C-3'), 82.70 (C-2'), 85.62(C-4’), 91.18 (C-1’),
113.90 (Cq isop), 119.81 (C-5), 126.63 (C-8), 150.30 (C-4), 153.37 (C-2), 155.52 (C-6), 156.06 (CO Boc). HRMS (ESI-TOF): m/z calcd for [C18H25BrN6O5+H] 485.1148 and 487.1130, found 485.1144 and 487.1121. Coupling product 40 To a solution of alkyne 10 (0.42 g, 1.27 mmol) and bromide 39 (0.51 g, 1.06 mmol) in THF (10 mL) was added triethylamine (0.45 mL, 3.18 mmol). The reaction mixture was degassed with argon (3 times) before adding CuI (20 mg, 10 mol%) and Pd(PPh3)4 (61 mg, 5 mol%) followed by argon degassing (3 times). After stirring for 2 h at 60 °C, volatiles were removed and the residue was purified by two successive flash column chromatography (40 g SiO2, 2 to 5% MeOH in DCM) to give 40 (0.71 g, 85%) still containing 0.5 eq. triethylammonium salt. 1H NMR (400 MHz, D2O): δ 1.18 (t, 4.5H, CH3 Et3N), 1.29 (s, 3H, CH3 isop), 1.30 (s, 3H, CH3 isop), 1.36 (s, 9H, CH3 Boc), 1.48 (s, 3H, CH3 isop), 1.54 (s, 3H, CH3 isop), 3.10 (q, 3H, CH2 Et3N), 3.15-3.27 (m, 2H, H-5'a and H-5''a), 3.75-3.84 (m, 2H, H-5'b and H-5"b), 4.15-4.24 (m, 2H, H-4'a and H-4'b), 4.59 (dd, 1H, H-2'b), 4.66 (s, 2H, CH2-C≡), 4.72 (dd, 1H, H-3'b), 4.83 (d, 1H, H-1'b), 4.99 (dd, 1H, H-3'a), 5.52 (dd, 1H, H-2'a), 6.12 (d, J = 4.3 Hz, 1H, H-1'a), 7.21 (t, 1H NH), 7.38 (br, 1H, CONH2), 7.43 (t, J = 7.5 Hz, 1H, H Ph), 7.52 (d, 1H, H Ph), 7.63 (br, 2H, NH2), 7.80 (d, 1H, H Ph), 7.84 (br, 1H, H Ph), 8.00 (br, 1H, CONH2), 8.21 (s, 1H, H-2a). 13C NMR (100 MHz, DMSO-d6): δ 25.63 and 25.84 (CH3 isop), 27.51 and 27.77 (CH3 isop), 28.61 (CH3 Boc), 42.40 (C-5'a), 58.77 (CH2-C≡), 70.43 (C-5'b), 75.47 (CH2-C≡C), 78.29 (Cq Boc), 82.14 and 82.19 (C-3'a and C-3'b), 82.82 and 82.85 (C-2'a and C-4'b), 85.13 (C-4'a), 85.38 (C-1'b), 86.41 (C-2'b), 89.97 (C-1'a), 93.12 (CH2-C≡C), 113.97 (Cq), 114.73 (Cq), 119.39 (C-5), 125.78, 127.26, 128.75 and 129.18 (CH Ph), 132.73 (C-8), 134.85 and 140.16 (Cq Ph), 148.73 (C-4), 154.33 (C-2), 156.16 (CO), 156.54 (C-6), 168.14 (CONH2).
HRMS (ESI-TOF) m/z calcd for [C36H45N7O10+H] 736.3301, found 736.3267. Compound 41 To compound 40 (0.69 g, 0.94 mmol) was added an ice-cold solution of TFA (70% in water, 10 mL). The mixture was stirred at 4°C for 1 h and allowed to slowly warm to room temperature. After 4 h, water was added and volatiles were removed by lyophilization. An aliquot (1/10) of the crude product was purified by reverse phase HPLC (0-50% acetonitrile in 10 mM TEAA buffer, linear gradient over 15 min, tR = 8.7 min) to yield 41 (22 mg, 40%). 1H NMR (400 MHz, DMSO-d6): δ 1.86 (s, 3H, CH3), 2.84 (dd, 1H, H-5'a), 2.90 (dd, 1H, H-5"a), 3.72-3.83 (m, 3H, H-2'b, H-5'b and H-5"b), 3.88-3.95 (m, 2H, H-3'b and H-4'a), 3.99-4.04 (m, 1H, H-4'b), 4.26 (dd, 1H, H-3'a), 4.64 (s, 2H, CH2-C≡), 4.65 (d, 1H, H-1'b), 5.08 (t, 2H, H-2'a), 5.96 (d, J = 6.3 Hz, 1H, H-1'a), 7.32 (br, 1H, CONH2), 7.41 (t, 1H, H Ph), 7.54 (d, 1H, H Ph), 7.57 (br, 2H, NH2), 7.77 (d, 1H, H Ph), 7.85 (s, 1H, H Ph), 7.96 (br, 1H, CONH2), 8.18 (s, 1H, H-2a). 13C NMR (100 MHz, DMSO-d6): δ 43.59 (C-5'a), 58.79 (CH2-C≡), 71.28 (C-5'b), 71.41 (C-3'a), 71.72 (C-2'a), 70.05 (C-3'b), 75.58 (CH2-C≡C), 77.64 (C-2'b), 83.24 (C-4'b), 83.87 (C-1'b), 85.68 (C-4'a), 89.49 (C-1'a), 92.58 (CH2-C≡C), 119.67 (C-5), 126.01, 126.87, 128.52 and 129.38 (CH Ph), 133.32 (C-8), 134.61 and 141.59 (Cq Ph), 149.33 (C-4), 154.19 (C-2), 156.50 (C-6), 168.44 (CONH2). HRMS (ESI-TOF) m/z calcd for [C25H29N7O8+H] 556.2150, found 556.2135. BIOLOGICAL EVALUATION LmNADK expression and purification As in Poncet-Montange et al. (J. Biol. Chem.2007, 282, 33925–33934). In brief, the NAD kinase 1 (EC 2.7.1.23) coding sequence was amplified by PCR from the genomic DNA isolated from L. monocytogenes strain EGD-e by using the Vent DNA polymerase, dNTPs, and the following primers: 5′-GGAATTCCATATGAAATATATGATTACTTCCAAAGGA-
3′, and 5′-CGGCGCTCGAGTTAATCTTCAATAAACGAATCGTGTAC-3′. The amplified DNA was cloned into the expression vector pET22b (Novagen) at the NdeI and XhoI restriction sites, and the corresponding plasmid was used for transforming the E. coli strain BL21(DE3)/pDIA17 for protein expression. Transformants were grown at 37 °C in 2YT medium (Difco) in the presence of chloramphenicol and ampicillin at 30 μg/mL. When the absorbance reached 1.5 at 600 nm, the expression of the recombinant protein was induced by the addition of 1 mM isopropyl-β-D-1-thiogalactopyranoside, and growth was continued for three more hours at 37 °C. The cells were then pelleted by centrifugation and served as source for protein purification. Soluble protein was purified using cobalt-agarose affinity chromatography followed by size exclusion chromatography. The protein was concentrated to 6–7 mg/mL in 50 mM KH2PO4 pH 7.5 and 100 mM NaCl, aliquoted, flash frozen in liquid nitrogen and stored at –20 °C. The sequence of the recombinant protein corresponds to the sequence of L. monocytogenes NADK 1 (UniProtKB Q8Y8D7) with a LEHHHHHH tag at the C-terminus. The expression, solubility and purity of LmNADK were tested on SDS-PAGE gel (Invitrogen™ NuPAGE™ 4-12 % gel). The molar absorption coefficient and the absorbance value for 0.1% of the 6His-tagged protein were estimated with the ABIM software (http://bioserv.cbs.cnrs.fr/ABIM/w3bb/d_abim/) at 26,740 M-1 cm-1 and 0.863 (g/L)-1 cm-1, respectively. Molecular cloning of the PaNADK gene and purification A synthetic gene encoding PaNADK was optimized for expression in E. coli and ordered from Integrated DNA Technologies, BVBA (Leuven). It was amplified with forward primer 5’-CCCTTTCATATGATGGAGCCCTTCCGCAAT-3’ and reverse primer 5’- CCCAAACTCGAGGTCACCCCCTCCTAAACG-3’ including cleavage sites of restriction enzymes for cloning into pET-22b(+) vector (Q5 Hot Start High-Fidelity 2× Master Mix). The PCR product was then digested using the NdeI and Xho1 restriction enzymes, purified (NucleoSpin Gel and PCR clean-up kit, Macherey-Nagel), and ligated (T4 ligase) into linearized pET-22b(+) vector at the same restriction sites. At the C-
terminus, the construct codes for two additional amino acids (LE) followed by a 6His-tag. The gene sequence of PaNADK was checked by sequencing (Genewiz).
PaNADK protein was overexpressed in E. coli BL21 (DE3) cells in 2-YT broth with 100 pg/mL ampicillin. The protein expression was induced at ODeoo of 0.7-0.8 with 1 mM isopropyl P-D-l -thiogalactopyranoside (IPTG) overnight at 25 °C. The harvested cells were re-suspended in buffer A (50 mM sodium phosphate pH 8.3, 150 mM NaC1, 10 mM imidazole, 2 mM DTT) supplemented with 1 tablet of cOmplete™ EDTA-free protease inhibitor cocktail (Roche), 5 pg/mL of lysozyme (Euromedex), 5 pg/mL of DNase (Sigma Aldrich). Bacterial cell disruption was achieved by sonication at 40% amplitude during 5 minutes (5 sec on, 5 sec off) with 6 mm probe on ice. Cellular debris were removed by centrifugation at 20,000 g for 30 minutes and followed by filtration through 0.45 pm syringe filter. The bacteria lysate was loaded onto a HisTrap FF column equilibrated with buffer A using Akta Pure system (GE Healthcare) at 4 °C. The column was washed with buffer A during 15 column volumes to remove unspecific binding proteins, followed by elution using buffer B (50 mM sodium phosphate pH 8.3, 150 mM NaC1, 500 mM imidazole, 2 mM DTT) with a linear gradient of imidazole from 10 mM to 500 mM in 20 column volumes. The fractions containing the protein were pooled and concentrated using Amicon Ultra 15 centrifugal filters (3 kDa, Merck Millipore) at 4 °C. The concentrated protein was loaded onto a 16/60 Superdex 200 pre-equilibrated with buffer C (50 mM NaH2PO4/Na2HPO4 pH 8.3, 100 mM NaC1, 5 mM DTT). Finally, the protein was concentrated till 30 mg/mL, aliquoted, flash frozen in liquid nitrogen and stored at -80 °C.
The sequence of the recombinant protein corresponds to the sequence of P aeruginosa PAG NADK (UniProtKB Q9HZC0) with a LEHHHHHH tag at the C-terminus.
The expression, solubility and purity of PaNADK were tested on SDS-PAGE gel (Invitrogen™ NuPAGE™ 4-12 % gel). The molar absorption coefficient and the absorbance value for 0.1% of the 6His-tagged protein were estimated with the AB IM software (http://bioserv.cbs.cnrs.fr/ABIM/w3bb/d_abim/) at 14,650 M'1 cm'1 and 0.441 (g/L)'1 cm'1, respectively.
Molecular cloning, expression and purification of the HsNADK gene
As for LmNADK, the coding region of cytoplasmic HsNADK was amplified using standard PCR cloning (see above) and cloned into the same over-expression plasmid pET22b (Novagen) at the Ndel and Xhol restriction sites. The corresponding plasmid was sequenced and then used for transforming the E. coli strain BL21(DE3)/pDIA17 for protein over-expression.
HsNADK protein was overexpressed in E. coli BL21 (DE3) cells in auto inducible ZYM broth with 100 pg/mL ampicillin. The bacteria were incubated 5 h at 37 °C and overnight at 25 °C. The harvested cells were re-suspended in buffer A (50 mM Tris pH 8.0, 150 mM NaC1, 2 mM DTT) supplemented with 1 tablet of cOmplete™ EDTA-free protease inhibitor cocktail (Roche) and 5 pg/mL of lysozyme (Euromedex). Bacterial cell disruption was achieved by sonication at 40% amplitude during 5 minutes (5 sec on, 5 sec off) with 6 mm probe on ice. Cellular debris were removed by centrifugation at 20,000 g for 30 minutes and followed by filtration through 0.45 pm syringe filter. The bacteria lysate was loaded onto a HisTrap FF column equilibrated with buffer A using Akta Pure system (GE Healthcare) at 4 °C. The column was washed with buffer A during 15 column volumes to remove unspecific binding proteins, followed by elution using buffer B (50 mM Tris pH 8.0, 150 mM NaC1, 300 mM imidazole, 2 mM DTT) with a linear gradient of imidazole from 10 mM to 300 mM in 15 column volumes. The fractions containing the protein were pooled and concentrated using Amicon Ultra 15 centrifugal filters (10 kDa, Merck Millipore) at 4 °C. The concentrated protein was loaded onto a 26/60 Superdex 200 preequilibrated with buffer A. Finally, the protein was dialyzed 3 h and overnight against buffer C (50 mM Tris pH 7.5, 2 mM DTT), concentrated till 10 mg/mL, aliquoted, flash frozen in liquid nitrogen and stored at -80 °C.
The sequence of the recombinant protein corresponds to the sequence of H. sapiens cytosolic NADK (UniProtKB 095544) with a LEHHHHHH tag at the C-terminus.
The expression, solubility and purity of HsNADK were tested on SDS-PAGE gel (Invitrogen™ NuPAGE™ 4-12% gel). The molar absorption coefficient and the absorbance value for 0.1% of the 6His-tagged protein, hereafter named HsNADK, were
estimated with the ABIM software (http://bioserv.cbs.cnrs.fr/ABIM/w3bb/d_abim/) at 41,250 M'1 cm'1 and 0.820 (g/L)'1 cm'1, respectively.
In vitro enzymatic activity measurements and inhibition assays
The enzymatic reaction was followed at 30 °C in a CLARIOstar plate reader (BMG LABTECH) by measuring the absorbance at 340 nm (OD340) using an enzymatic coupled system involving glucose-6-phosphate dehydrogenase (G6PDH). The reaction mixture was made in a half area 96-well microplate (Greiner Bio-One C1ear UV-Star). Buffers used were 50 mM Bis-Tris pH 7.0, 100 mM NaC1, 1 mM MgCh with HsNADK, 50 mM BisTris pH 7.0, 2 mM NaCitrate Tribasic Dihydrate, 1 mM MgCh with LmNADK and 50 mM Tris pH 7.5, 1 mM MgCh, 5 mM DTT with PaNADK. Reaction mixtures contained 25 nM NADK, 0.1-2 mM NAD (with HsNADK or LmNADK) or 0.05-1 mM NAD (with PaNADK), 4 mM MgATP, 5 mM G6P, 1 U/mL G6PDH and different concentrations of inhibitors. Buffer, NAD and inhibitor solutions (total volume 25 pL, concentration 4x) were dispensed into the microplate using an OT-2 lab robot (Opentrons). The 25 pL of mixture containing G6PDH, MgATP and glucose-6-phosphate at a 4x concentration were dispensed using a manual repeating pipette. Finally, the reaction was started at time 0 by adding and mixing 50 pL of NADK at a 2x concentration using a multichannel pipette. OD340 were measured every 32 s during 32 min.
The values of the inhibition constants were determined from the raw values of kss at different concentrations of NAD and inhibitor using a competitive inhibition equation (Eq. 1).
A Lineweaver Burk transformation of the raw values was used to produce an illustrative double-reciprocal plot of the data. OD340 values were converted to concentrations of produced NADH (i.e. NAD consumed) using a molar absorption coefficient for NADH of 6,220 M'1 cm'1. The value of the steady state rate constant (kss) given in the text corresponds to the initial slope of NAD consumed per second divided by the concentration
of NADK. Global fittings and graphical representations of kinetic parameters were made using GraFit software (version 7.0.3, Erithacus Software Ltd).
Cytotoxicity (PF-CCB, IP)
The synthesized compounds were assayed for cytotoxicity on MRC-5 (human fetal lung fibroblasts) cells. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 25 mM glucose, 10% (v/v) serum, 1% penicillin/streptomycin and kept under 5% CO2 at 37°C. The cell viability was assessed using the CellTiter Gio kit from Promega (G7572) after 72 h incubation time using four concentrations of compounds (from 1 to 50 pM) in triplicate. No cytotoxicity was observed up to concentrations of 50 μM
Claims
I, its tautomeric forms, stereoisomeric forms, mixture of stereoisomeric forms, pharmaceutically acceptable salts or solvates thereof, wherein
R1 is selected from the group consisting of -OH, -NHC(=O)NR2R3 and -NHSO2NR2R3, F, NR2R3, OR4, N3 and NH2 wherein R2 and R3 are independently selected from H, C1-C6-alkyl and C1-C12- heteroalkyl comprising one or more oxygen atom, and R4 is C1-C6-alkyl or C1- C12-heteroalkyl comprising one or more oxygen atom; n is an integer from 1 to 4; m is an integer from 1 to 4; Y is CH or N;
Z is O or S; and
A is selected from the group consisting of -O-, -S-, -Se- and N(CH2)PR5 wherein p is an integer from 1 to 6; and R5 is selected from the group consisting of -NH2, -C=CH, -COOH, -COOR6 and -OR6 wherein R6 is C1-C6-alkyl; or
R1 and A form together -NHC(=O)(CH2)qNHC(=O)(CH2)rN wherein q and r are independently integers from 1 to 6.
2. The compound according to claim 1, wherein n is 1.
3. The compound according to claim 1 or 2, wherein m is 1.
4. The compound according to any one of claims 1 to 3, wherein Y is CH.
5. The compound according to any one of claims 1 to 4, wherein Z is O.
II, a pharmaceutically acceptable salt or a solvate thereof, wherein R1 and A are as defined in claim 1.
7. The compound according to any one of claims 1 to 5, having the Formula III:
a pharmaceutically acceptable salt or a solvate thereof, wherein
R1, n and m are as defined in claim 1.
IV, a pharmaceutically acceptable salt or a solvate thereof, wherein
R1, R5, n, m, and p are as defined in claim 1.
9. The compound according to any one of claims 1 to 5, having the Formula V:
a pharmaceutically acceptable salt or a solvate thereof, wherein
A, n and m are as defined in claim 1.
VI,
a pharmaceutically acceptable salt or a solvate thereof, wherein
R2, A, n and m are as defined in claim 1.
VII, a pharmaceutically acceptable salt or a solvate thereof, wherein
R3, A, n and m are as defined in claim 1.
12. The compound according to any one of claims 1 to 5, having the Formula VIII:
VIII, a pharmaceutically acceptable salt or a solvate thereof, wherein A, n and m are as defined in claim 1. The compound according to claim 1, selected from the group consisting of:
A pharmaceutical composition comprising a compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt or solvate thereof and at least one pharmaceutically acceptable excipient. A compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt or solvate thereof for use in the prevention and/or treatment of bacterial infections.
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