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 R ¹ is selected from the group consisting of -OH, -NHC(=O)NR ² R ³ and -NHSO2NR ² R ³ , F, NR ² R ³ , OR ⁴ , N ₃ and NH ₂ wherein R ² and R ³ are independently selected from H, C1-C6-alkyl and C1-C12- heteroalkyl comprising one or more oxygen atom, and R ⁴ 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(CH ₂ ) _P R ⁵ wherein p is an integer from 1 to 6; and R ⁵ is selected from the group consisting of -NH ₂ , -C=CH, -COOH, -COOR ⁶ and -OR ⁶ wherein R ⁶ is C1-C6-alkyl; or

R ¹ and A form together -NHC(=O)(CH ₂ ) _q NHC(=O)(CH ₂ )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, R ¹ , R ² , R ³ and R ⁴ are defined as follows:

R ¹ is selected from the group consisting of -OH, -NHC(=O)NR ² R ³ and -NHSO ₂ NR ² R ³ wherein R ² and R ³ are independently H or C1-C6-alkyl, in particular R ² and R ³ are independently H or C1-C4-alkyl, more particularly R ² and R ³ are independently H or C1- C2-alkyl, still more particularly R ² and R ³ are H or methyl; for example, R ¹ is selected from the group consisting of -OH, -NHC(=O)NHR ² and -NHSO ₂ NR ² R ³ wherein 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; in a particular example R ¹ is selected from the group consisting of -OH, - NH2, -NHC(=O)NR ² R ³ and -NHSO2NR ² R ³ wherein R ² and R ³ are independently H or C1- C6-alkyl, in particular R ² and R ³ are independently H or C1-C4-alkyl, more particularly R ² and R ³ are independently H or C1-C2-alkyl, still more particularly R ² and R ³ are H or methyl; in a more particular example, R ¹ is selected from the group consisting of -OH, - NH2, -NHC(=O)NHR ² and -NHSO2NR ² R ³ wherein 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; 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) _P R ⁵ 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 R ⁵ is -NH2 or -C=CH; or

R ¹ and A form together -NHC(=O)(CH2) _q NHC(=O)(CH2) _r N 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 R ¹ is OH.

In one embodiment, the compounds of Formula I are those wherein R ¹ is -NHC(=O)NHR ² wherein 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.

In one embodiment, 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.

In one embodiment, the compounds of Formula I are those wherein R ¹ is NH ₂ .

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(CH ₂ ) _P R ⁵ 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 R ⁵ is -NH ₂ or -C=CH.

In one embodiment, the compounds of Formula I are those wherein R ¹ and A form together -NHC(=O)(CH ₂ ) _q NHC(=O)(CH ₂ ) _r N 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 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:

R ¹ is selected from the group consisting of -OH, -NHC(=O)NHR ² and -NHSO2NR ² R ³ wherein 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 or ethyl; for example R ¹ is selected from the group consisting of -OH, -NH2, -NHC(=O)NHR ² and -NHSO2NR ² R ³ wherein 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 or ethyl;

A is -O- or N(CH2) _P R ⁵ 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 R ⁵ is -NH2 or -C=CH; or

R ¹ and A form together -NHC(=O)(CH2) _q NHC(=O)(CH2) _r N 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

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:

R ¹ is selected from the group consisting of -OH, -NHC(=O)NHR ² and -NHSO2NR ² R ³ wherein 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 or ethyl; for example R ¹ is selected from the group consisting of -OH, -NH2, -NHC(=O)NHR ² and -NHSO2NR ² R ³ wherein 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 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 R ¹ , R ⁵ , 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 R ¹ , R ⁵ , n, m, and p are defined as follows:

R ¹ is -OH or -NHC(=O)NHR ² wherein 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; 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 R ⁵ is -NH ₂ 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) _P R ⁵ 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 R ⁵ is -NH ₂ or -C=CH, in particular R ⁵ is -NH ₂ ; 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

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;

A is -O- or N(CH2) _P R ⁵ 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 R ⁵ is -NH2 or -C=CH, in particular R ⁵ 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

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.

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.

Particularly preferred compounds of the invention are those listed in Table 1 hereafter: Table 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, 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.

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 8.10 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%. 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 (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. Coupling constants (J values) are reported in Hz. The complete assignment of ' H and ¹³ C signals was performed by analysis of the correlated homonuclear { ' HJ H I-COSY and heteronuclear { ¹ H, ¹³ C}- HMBC, { ' H, ^{I 3} C}-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

General synthetic scheme 1. Building block 10, A = O

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,

-0 °C; (g) H2O2, K ₂ CO ₃ , 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. NaHCO ₃ (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, BF ₃ ·Et ₂ O (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%). ¹ H 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, CH ₂ 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). ¹³ C NMR (100 MHz, DMSO-d ₆ ): δ 70.64 (C-5'), 71.55, 71.71 and 72.95 (CH ₂ 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 [C ₃₃ H ₃₁ NO ₄ +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%). ¹ H 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, CONH ₂ ). ¹³ C 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 [C ₁₅ H ₁₉ NO ₅ +H] ⁺ 254.1023, found 254.1025. 3-(β-D-ribofuranosyl)benzamide (5) A 1M solution of BBr ₃ 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%). ¹ H NMR (400 MHz, DMSO-d ₆ ): 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, CONH ₂ ), 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, CONH ₂ ). ¹³ C 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 Na ₂ SO ₄ , 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%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 1.30 (s, 3H, CH ₃ isop), 1.55 (s, 3H, CH ₃ 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, CONH ₂ ). ¹³ C 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 [C ₁₅ H ₁₉ NO ₅ +Na] ⁺ 316.1155, found 316.1151. Route B 3-(β-D-ribofuranosyl)benzonitrile (7) A 1 M solution of BBr ₃ 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%). ¹ H 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). ¹³ C 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 [C ₁₂ H ₁₃ NO ₄ -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 Na ₂ SO ₄ , 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%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 1.30 (s, 3H, CH ₃ isop), 1.55 (s, 3H, CH ₃ 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). ¹³ C 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 Na ₂ SO ₄ , 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%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 1.30 (s, 3H, CH ₃ isop), 1.55 (s, 3H, CH ₃ 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). ¹³ C 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 [C ₁₈ H ₁₉ NO ₄ +H] ⁺ 314.1387, found 314.1385. 3-(2,3-O-isopropylidene-5-O-propargyl-β-D-ribofuranosyl)ben zamide (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 H ₂ O, 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 K ₂ CO ₃ (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%). ¹ H 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, CH ₂ -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, CONH ₂ ), 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, CONH ₂ ). ¹³ C 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%). ¹ H 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). ¹³ C 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 [C ₁₅ H ₁₈ N ₄ O ₄ +H] ⁺ 319.1401, found 319.1381. 5-Azido-2,3-O-isopropylidene-1β-(3-(carbamoyl)phenyl)-1,5-d i-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%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 1.36 (s, 3H, CH ₃ isop), 1.63 (s, 3H, CH ₃ 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, CONH ₂ ), 7.44 (t, 1H, H Ph), 7.55 (d, 1H, H Ph), 7.76 (d, 1H, H Ph), 7.89 (s, 1H, H Ph). ¹³ C 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%). ¹ H 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 [C ₂₁ H ₂₃ N ₃ O ₈ S+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 NEt ₃ (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 Na ₂ SO ₄ , 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. ¹ H NMR (400 MHz, DMSO-d6): δ 1.38 (s, 9H, CH3 Boc), 1.91 (quint. J = 6.6 Hz, 2H, CH ₂ ), 3.03 (td, J = 6.6 Hz, J = 5.8 Hz, 2H, NCH ₂ ), 3.51 (t, J = 6.6 Hz, 2H, CH ₂ ), 6.88 (t, J = 5.8 Hz, 1H, NH). ¹³ C NMR (100 MHz, DMSO-d6): δ 28.7 (3C, CH3 Boc), 32.7 (CH2), 33.2 (CH2), 39.0 (CH ₂ ), 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 K ₂ CO ₃ (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%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 1.30 (s, 3H, CH ₃ isop), 1.37 (s, 9H, CH ₃ 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, CONH ₂ ), 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, CONH ₂ ). ¹³ C NMR (100 MHz, DMSO-d6): δ 25.91 (CH3 isop), 27.84 (CH3 isop), 28.72 (3C, CH3 Boc), 29.64 (CH ₂ ), 38.41 (CH ₂ ), 47.21 (CH ₂ ), 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 [C ₂₃ H ₃₅ N ₃ O ₆ +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%). ¹ H 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, CH ₂ NBoc), 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, CONH ₂ ), 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, CONH ₂ ). ¹³ C 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 (CH ₂ -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 [C ₂₆ H ₃₇ N ₃ O ₆ +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 K ₂ CO ₃ (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%). ¹ H NMR (400 MHz, DMSO-d6): δ 0.10 (s, 9H, CH3 TMS), 1.30 (s, 3H, CH3 isop), 1.55 (s, 3H, CH ₃ isop), 1.59 (quint, 2H, CH ₂ ), 2.26 (t, 2H, CH ₂ ), 2.64 (t, 2H, CH ₂ ), 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, CONH ₂ ). ¹³ C 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%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 0.11 (s, 9H, CH ₃ TMS), 1.30 (s, 3H, CH ₃ isop), 1.55 (s, 3H, CH ₃ isop), 1.58 (quint., 2H, CH ₂ ), 2.25 (t, 2H, CH ₂ -C≡), 2.59 (t, 2H, CH ₂ ), 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, CONH ₂ ), 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). ¹³ C NMR (100 MHz, DMSO-d6): δ 25.91 (CH3 isop), 27.84 (CH3 isop), 28.72 (3C, CH3 Boc), 29.64 (CH ₂ ), 38.41 (CH ₂ ), 47.21 (CH ₂ -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 (CONH ₂ ). 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 SiO ₂ , 0 to 5% MeOH in DCM) affording 19 (145 mg, 50%). ¹ H NMR (400 MHz, DMSO-d6): δ 1.30 (s, 3H, CH3 isop), 1.55 (s, 3H, CH3 isop), 1.94 (s, 3H, CH ₃ OAc), 2.05 (s, 3H, CH ₃ OAc), 2.10 (s, 3H, CH ₃ 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, CONH ₂ ), 8.22 (s, 1H, H- 2a). ¹³ C 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 (CONH ₂ ), 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%). ¹ H NMR (400 MHz, DMSO-d ₆ ): 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, NH ₂ ), 7.77 (d, 1H, H Ph), 7.86 (s, 1H, H Ph), 7.95 (br, 1H, CONH2), 8.17 (s, 1H, H-2a). ¹³ C 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 (CONH ₂ ). HRMS (ESI ⁺ -TOF): m/z calcd for [C ₂₅ H ₂₈ N ₆ O ₉ +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 SiO ₂ , 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. ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 0.95 (t, 3H, CH ₃ Et), 1.29 and 1.30 (each s, 6H, CH ₃ 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, CH ₂ - 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, CONH ₂ ), 7.43 (t, J = 7.7 Hz, 1H, H Ph), 7.52 (d, 1H, H Ph), 7.61 (br, 2H, NH ₂ ), 7.81 (d, 1H, H Ph), 7.85 (s, 1H, H Ph), 8.00 (br, 1H, CONH2), 8.22 (s, 1H, H-2a). ¹³ C NMR (100 MHz, DMSO-d6): δ 16.03 (CH3 Et), 25.72, 25.87, 27.56 and 27.81 (CH3 isop), 34.54 (CH ₂ Et), 41.91 (C-5'a), 58.83 (CH ₂ -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%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 0.96 (t, 3H, CH ₃ Et), 2.95-3.03 (m, 2H, CH ₂ 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 CH ₂ -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, NH ₂ ), 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). ¹³ C 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 (CONH ₂ ). HRMS (ESI ⁺ -TOF): m/z calcd for [C ₂₈ H ₃₄ N ₈ O ₉ +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 SiO ₂ , 3 to 6% MeOH in DCM; 20 g SiO ₂ , 2 to 3% MeOH in ethylacetate) affording 25 (0.14 g 25%). ¹ H 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, CH ₂ -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, CONH ₂ ), 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, CONH ₂ ), 8.20 (s, 1H, H-2a). ¹³ C 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 (CONH ₂ ). HRMS (ESI ⁺ -TOF): m/z calcd for [C ₃₃ H ₄₂ N ₈ O ₁₀ S+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%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 0.96 (t, 3H, CH ₃ Et), 2.95-3.03 (m, 2H, CH ₂ 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 CH ₂ -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 NH ₂ ), 7.80 (d, J = 7.8 Hz, 1H, H Ph), 7.85 (s, 1H, H Ph), 7.98 (br, 1H, CONH ₂ ), 8.20 (s, 1H, H-2a). ¹³ C 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 [C28H34N ₈ O ₉ +H] ⁺ 627.2506, found 627.2522.

4. Sonogashira cross-coupling reactions with alkyne 15

EXAMPLE 4

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 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). HRMS (ESI ⁺ -TOF): m/z calcd for [C ₃₅ H ₅₀ N ₈ O ₁₀ +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%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 1.68-1.73 (m, 2H, CH ₂ ), 1.74 (s, 3H, CH ₃ Ac), 2.68- 2.75 (m, 2H, CH ₂ ), 2.74-2.88 (m, 4H, CH ₂ , 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, CH ₂ -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, NH ₂ ), 7.77 (d, 1H, H Ph), 7.86 (s, 1H, H Ph), 8.02 (br, 1H, CONH ₂ ), 8.16 (s, 1H, H-2a). ¹³ C 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 (CONH ₂ ), 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, NCH ₂ ), 2.71 (t, 2H, NCH ₂ -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, CONH ₂ ), 8.21 (s, 1H, H-2a). ¹³ C 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 (NCH ₂ ), 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 (CONH ₂ ). 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 (CH ₃ Et), 1.63-1.72 (m, 2H, CH ₂ -C≡), 2.23 (dt, 2H, NCH ₂ ), 2.70 (t, 2H, CH ₂ ), 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, CONH ₂ ), 7.41 (t, J = 7.5 Hz, 1H, H Ph), 7.50-7.56 (br, 3H, H Ph and NH ₂ ), 7.77 (d, 1H, H Ph), 7.84 (s, 1H, H Ph), 7.96 (br, 1H, CONH2), 8.22 (s, 1H, H-2a). ¹³ C NMR (100 MHz, DMSO-d ₆ ): δ 15.99 (CH ₂ ), 16.07 (CH ₃ Et), 26.76 (CH ₂ ), 34.58 (CH ₂ Et), 42.22 (C-5'a), 43.93 (CH ₂ -C≡), 53.48 (NCH ₂ ), 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 (PPh ₃ , pyridine, NH ₄ OH) 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-isopro pylidene-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 SiO ₂ , 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. ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 1.32 (s, 3H, CH ₃ isop), 1.34 (s, 9H, CH ₃ 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, NH ₂ ), 8.18 (t, 1H, NHCO), 8.19 (s, 1H, H-2), 8.32 (s, 1H, H-8). ¹³ C NMR (100 MHz, DMSO-d ₆ ): δ 25.76 (CH ₃ isop), 26.32 and 26.39 (CH ₂ residual phosphine), 27.54 (CH ₃ isop), 28.63 (3C, CH ₃ Boc), 36.21 (CH ₂ ), 37.18 (CH ₂ NBoc), 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-isopropylide ne-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 Na ₂ S ₂ O ₃ (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 SiO ₂ , 2 to 5% MeOH in DCM) to give 34 (0.38 g, 54%). ¹ H NMR (400 MHz, DMSO-d6): δ 1.32 (s, 3H, CH3 isop), 1.34 (s, 9H, 3 CH3 Boc), 1.54 (s, 3H, CH ₃ isop), 3.32-3.44 (m, 2H, H-5' and H-5"), 3.52 (d, 2H, CH ₂ ), 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). ¹³ C NMR (100 MHz, DMSO-d ₆ ): δ 25.71 (CH ₃ isop), 27.59 (CH ₃ isop), 28.60 (3C, CH ₃ 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 [C ₂₀ H ₂₈ BrN ₇ O ₆ +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 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. ¹ H NMR (400 MHz, DMSO-d6): δ 1.17 (t, 3H, CH3 of OEt), 1.29 (s, 3H, CH3 isop), 1.30 (s, 3H, CH ₃ isop), 1.33 (s, 9H, 3 CH ₃ Boc), 1.51 (s, 3H, CH ₃ isop), 1.55 (s, 3H, CH ₃ 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, CONH ₂ ), 7.44 (t, 1H, H Ph), 7.50 (d, 1H, H Ph), 7.59 (br, 2H, NH ₂ ), 7.81 (d, 1H, H Ph), 7.83 (s, 1H, H Ph), 7.98 (br, 1H, CONH ₂ ), 8.18 (br, 1H, 5'-NHCO), 8.26 (s, 1H, H-2a). ¹³ C NMR (100 MHz, DMSO-d6): δ 9.14 (CH3 of OEt), 25.67 and 25.92 (CH3 isop), 27.57 and 27.81 (CH ₃ isop), 28.58 (3C, CH ₃ Boc), 41.19 (C-5’a), 43.70 (CH ₂ NHBoc), 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-CH ₂ ), 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 [C ₄₂ H ₅₅ N ₉ O ₁₂ +H] 878.4043, found 878.4035. 3-(5'-Deoxy-5'-(N-(3-(5'-deoxy-5'-(2-aminoethylamido)-adenos in-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); t _R = 8.8 min. The crude product (9/10) was used in the cyclisation step without further purification. ¹ H 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, COCH ₂ ), 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, CONH ₂ ), 7.42 (t, 1H, H Ph), 7.52 (s, 2H, NH ₂ ), 7.53 (d, 1H, H Ph), 7.76 (d, 1H, H Ph), 7.85 (s, 1H, H Ph), 7.98 (br, 1H, CONH ₂ ), 8.22 (s, 1H, H-2a), 8.60 (br, 1H, 5'-NHCO). ¹³ C 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 (NCH ₂ CO), 71.87 (C-3’a), 72.25 (C-2'a), 73.38 (C-2'b), 74.21 (C≡C- CH ₂ ), 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. ¹ H 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 CH ₂ -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, CONH ₂ ), 8.21(s, 1H, H-2a), 8.47 (t, 1H, 5'-NHCO). ¹³ C 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- CH ₂ ), 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 (CONH ₂ ), 170.00 (CONH), 170.37 (CONH). HRMS (ESI-TOF) m/z calcd for [C ₂₉ H ₃₃ N ₉ O ₉ +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%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 1.32 (s, 3H, CH ₃ isop), 1.36 (s, 9H, CH ₃ Boc), 1.54 (s, 3H, CH ₃ 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, NH ₂ ), 8.15 (s, 1H, H-2). ¹³ C NMR (100 MHz, DMSO-d ₆ ): δ 25.71 (CH ₃ isop), 27.59 (CH ₃ isop), 28.60 (3C, CH ₃ 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(PPh ₃ ) 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. ¹ H 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, CH ₂ Et ₃ N), 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, CONH ₂ ), 7.43 (t, J = 7.5 Hz, 1H, H Ph), 7.52 (d, 1H, H Ph), 7.63 (br, 2H, NH ₂ ), 7.80 (d, 1H, H Ph), 7.84 (br, 1H, H Ph), 8.00 (br, 1H, CONH2), 8.21 (s, 1H, H-2a). ¹³ C NMR (100 MHz, DMSO-d ₆ ): δ 25.63 and 25.84 (CH ₃ isop), 27.51 and 27.77 (CH ₃ isop), 28.61 (CH ₃ Boc), 42.40 (C-5'a), 58.77 (CH ₂ -C≡), 70.43 (C-5'b), 75.47 (CH ₂ -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%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 1.86 (s, 3H, CH ₃ ), 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, CONH ₂ ), 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). ¹³ C NMR (100 MHz, DMSO-d ₆ ): δ 43.59 (C-5'a), 58.79 (CH ₂ -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 (CONH ₂ ). 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 k _ss 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 (k _ss ) 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).

Table 2: Inhibition Constant (Ki) nd: not determined

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