Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7135—Compounds containing heavy metals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/545—Heterocyclic compounds
  • A61K47/546—Porphyrines; Porphyrine with an expanded ring system, e.g. texaphyrine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H1/00—Processes for the preparation of sugar derivatives
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
  • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H23/00—Compounds containing boron, silicon or a metal, e.g. chelates or vitamin B12

Definitions

• the present invention belongs to the field of the treatment of infectious diseases and relates to novel glycoclusters comprising a plurality of acetylated sialic acids for use in the treatment of infectious diseases, especially a SARS-CoV-2 infection.
• Coronaviruses are viruses of the subfamily Orthocoronavirinae, in the family Coronaviridae. They are enveloped viruses with a positive-sense single-stranded ribonucleic acid (RNA) genome and a helical nucleocapsid. Their name is due to their distinctive morphology, namely a series of club-shaped spikes projecting from the surface of their envelope that gives them a crown-like appearance. Coronaviruses are also characterized by an unusually large RNA genome and a specific replication strategy. RNA viruses and especially coronaviruses are responsible for a wide range of respiratory, systemic, gastrointestinal and neurologic diseases in mammals as well as birds.
• RNA viruses and especially coronaviruses are responsible for a wide range of respiratory, systemic, gastrointestinal and neurologic diseases in mammals as well as birds.
• Coronaviruses were first identified in humans about 50 years ago in the United Kingdom and the United States. They were since generally considered as causing only mild respiratory infectious diseases, e.g., the common cold. At the beginning of the 20 th century, two highly pathogenic coronaviruses were first identified: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). By contrast with previous coronavirus infections, SARS and MERS were severe respiratory diseases and accounted for hundreds of deaths. At the end of 2019, a new infectious respiratory illness outbroke in Wuhan (China), and the cause was identified in the end as a new human coronavirus SARS-CoV-2.
• SARS-CoV severe acute respiratory syndrome coronavirus
• MERS-CoV Middle East respiratory syndrome coronavirus
• SARS-CoV-2 shares about 80% identity with that of SARS-CoV and is about 96% identical to that of the bat coronavirus BatCoV RaTG13.
• the disease caused by SARS-COV-2 infection is named “coronavirus disease 2019” (“COVID-19”). CO VID- 19 quickly emerged as a severe world-scale pandemic in 2020.
• COVID-19 presents generally (about 80%) as a respiratory disease of mild severity whose symptoms may include fever, cough or other respiratory symptoms (such as mild breath shortness or chest tightness), headache, fatigue or muscle pain, and loss of smell and taste.
• COVID-19 pneumonia develop severe symptoms including dyspnea, hypoxia or pneumonia (“COVID-19 pneumonia”).
• COVID-19 pneumonia In a small number of cases (about 5%), critical symptoms such as respiratory failure, shock, or multiorgan failure are observed.
• ICU intensive care unit
• Permanent damage to organs has been observed in some cases, and some patients continue to experience a range of effects even months after recovery (“long COVID”).
• CO VID- 19 causes substantial suffering and death, and also endangers many health systems in the world.
• coronavirus infections in particular coronavirus respiratory infections causing diseases such as SARS, MERS or CO VID-19.
• coronavirus respiratory infections causing diseases such as SARS, MERS or CO VID-19.
• therapeutic agents highly efficient against coronavirus replication, with few or no significant adverse effects, having good chemical stability and/or low cost.
• SARS-CoV entry into host cells is mediated by its transmembrane spike (S) glycoprotein that forms homotrimers protruding from the viral surface.
• the S glycoprotein comprises two functional subunits responsible either for binding to the host cell receptor (S 1 subunit including the receptor-binding domain (RBD)) or for fusion of the viral and cellular membranes (S2 subunit).
• Angiotensin-converting enzyme 2 (ACE2) previously identified as the cellular receptor for SARS-CoV, also acts as a receptor of the new coronavirus (SARS-CoV-2).
• SARS-CoV-2 the S glycoprotein on the virion surface mediates receptor recognition and membrane fusion.
• cryo-electron microscopy structure obtained on the full-length human ACE2 in the presence of the RBD of the S glycoprotein of SARS-CoV-2 suggested simultaneous binding of two S-glycoprotein trimers to an ACE2 dimer.
• the S2 subunit is further cleaved by host proteases located immediately upstream of the fusion peptide, leading to the activation of the glycoprotein that undergoes extensive irreversible conformational changes, which facilitates the membrane fusion process.
• sialic acid derivatives could have a potential to act as competitive inhibitors to block the interactions between SARS-CoV-2 spike protein S and host cells, whose interactions usually mediate the first step of infection (Tortorici, M. A. et al., Nature Structural Molecular Biology, 2019, Vol. 26, pp. 481-489).
• sialic acid and its acetylated derivatives could likely bind, at least weakly, the spike protein of SARS-CoV-2, there is no direct quantitative evidence to define which one of these two carbohydrates would be the optimal partner (Yang, J. et al., Nature Communications, 2020, Vol. 11, Article number: 4541; Nguyen, K. et al., Viruses, May 2021, Vol. 13, No. 5, p. 927).
• individual glycans such as sialic acid or its derivatives generally have a relatively low affinity for their protein targets, typically moderate at best (Sauter, N. K. et al., Biochemistry 1989, Vol.
• the Applicants evidenced that, when at least two sialic acids or derivatives thereof are linked to specific macrocycles (such as porphyrins, pillararenes, calixarenes and fullerenes), the obtained glycoclusters are potent competitors of the SARS-CoV-2 early attachment to the host cells, despite the low affinity of the sialic acid.
• the glycoclusters of the invention are suitable for use in the treatment and/or prevention of an infectious disease such as COVID- 19.
• the present invention thus opens the way to new medicaments against viral infections such as COVID-19.
• This invention relates to a glycocluster comprising at least two acetylated- sialic acids covalently linked to a macrocycle, wherein said macrocycle is selected from porphyrins, pillararenes, calixarenes and fullerenes; and wherein each acetylated- sialic acid is independently selected from 4-O-acetylated-sialic acid, 7-O-acetylated-sialic acid, 8-O-acetylated-sialic acid and 9-O-acetylated-sialic acid.
• the glycocluster comprises at least four acetylated-sialic acids covalently linked to said macrocycle.
• each acetylated-sialic acid is independently selected from 7-O-acetylated- sialic acid and 9-O-acetylated-sialic acid.
• each acetylated-sialic acid is 9-O-acetylated-sialic acid.
• the macrocycle is selected from porphyrins, preferably selected from [Zn(tetraphenylporphyrin)] and tetraphenylporphyrin.
• the glycocluster further comprises at least one angiotensin-converting enzyme 2 (ACE2) binding inhibitor; preferably the angiotensinconverting enzyme 2 (ACE2) binding inhibitor is selected from ACE2 binding inhibitor peptides, ACE2 binding inhibitor proteins and anti-ACE2 antibodies or antigen-binding fragments thereof.
• ACE2 angiotensin-converting enzyme 2
• the glycocluster is a compound of formula (I), (la), (II), (III) or (IV) or a pharmaceutically acceptable salt and/or solvate thereof; wherein each L 1 is independently: a single bond or a linker selected from alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, arylalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl; wherein said alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, arylalkyl, heteroaryl, heteroarylalkyl or alkylheteroaryl optionally comprises at least one coupling product; each R 1 is independently selected from acetylated- sialic acids and ACE2 binding inhibitors; provided that at least two R 1 are acetylated-sialic acids; and M is a metal cation.
• each L 1 is a linker selected from alkyl, heteroalkyl, alkylaryl, arylalkyl, heteroarylalkyl and alkylheteroaryl; wherein said alkyl, heteroalkyl, alkylaryl, arylalkyl, heteroarylalkyl or alkylheteroaryl comprises one or two coupling products; preferably at least one of said coupling products is triazolyl.
• each L 1 -R 1 is selected from -(CH2)m-R c -(CH2) n - R 1 , -O-(CH 2 ) m -R c -(CH 2 ) n -R 1 , -C(O)-(CH 2 ) m -R c -(CH 2 ) n -R 1 , -C(O)O-(CH 2 ) m -R c - (Cth/n-R 1 , -phenyl-(CH2)m-R c -(CH2)n-R 1 and -phenyl-O-(CH2)m-R c -(CH2) n -R 1 ; wherein R c is a coupling product, preferably triazolyl; m and n are independently an integer ranging from 1 to 8, preferably ranging from 1 to 4; and each R 1 is as defined hereinabove. According to one embodiment, each R 1 is a coupling product,
• the glycocluster is a compound of formula (011), (005), (008) or (014)
• each R 1 is of formula (9-AcSA) wherein the wavy line represents the point of attachment of R 1 to the glycocluster.
• This invention also relates to a pharmaceutical composition comprising a glycocluster according to the invention and at least one pharmaceutically acceptable carrier.
• This invention also relates to a glycocluster according to the invention or a pharmaceutical composition according to the invention, for use as a medicament.
• This invention also relates to a glycocluster according to the invention or a pharmaceutical composition according to the invention, for use in the treatment and/or prevention of an infectious disease, preferably a coronavirus or picomavirus infection, more preferably a SARS-CoV-2 infection.
• This invention also relates to a process for manufacturing a glycocluster according to the invention comprising a step of coupling of each of the acetylated- sialic acids with the macrocycle; preferably a coupling wherein the reaction between a terminal alkyne and an azide results in the formation of a triazolyl group.
• Alcohol or “ethanoyl”, represented by the symbol “Ac”, refers to the methyl acyl moiety of formula CH3-C(O)-.
• alkene or “alkenyl” refer to a linear or branched hydrocarbon chain comprising at least one double bond and typically from 2 to 12 carbon atoms, preferably 3 to 6 carbon atoms.
• alkenyl groups include ethenyl,
• Alkyl refers to a saturated linear or branched hydrocarbon chain, typically comprising from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms, more preferably from 1 to 3 carbon atoms.
• alkyl groups may be monovalent or divalent (z.e., “alkylene” groups are encompassed in “alkyl” definition).
• Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, z-propyl, n-butyl, z-butyl, 5-butyl and /-butyl, pentyl and its isomers (e.g., n-pentyl, zso-pentyl), and hexyl and its isomers (e.g., n-hexyl, z'.so-hcxyl).
• Preferred alkyl groups include methyl, ethyl, n-propyl, z-propyl, n-butyl, s-butyl and /-butyl.
• Alkylaryl refers to an aryl group substituted by an alkyl group: alkyl-aryl-.
• Alkylheteroaryl refers to a heteroaryl group substituted by an alkyl group: alky 1-hetero aryl- .
• Alkyne or “alkynyl” refer to a linear or branched hydrocarbon chain comprising at least one triple bond and typically from 2 to 12 carbon atoms, preferably 3 to 6 carbon atoms.
• alkynyl groups include ethynyl, 2-propynyl, 2-butynyl,
• “Amine” refers to the -Nth group and to secondary amines -NHR wherein R is different from hydrogen, preferably wherein R is an alkyl group.
• Amino refers to the group -Nth.
• aminooxy refers to a -O-Nth group.
• Aryl refers to a cyclic, polyunsaturated, aromatic hydrocarbyl group comprising at least one aromatic ring.
• Aryl groups may have a single ring (z.e., phenyl) or multiple aromatic rings fused together (e.g., naphthyl) or linked covalently.
• aryl groups have from 5 to 12 carbon atoms, preferably from 6 to 10 carbon atoms.
• the aromatic ring may optionally include one to two additional rings (either cycloalkyl, heterocycloalkyl or heteroaryl) fused thereto.
• Aryl is also intended to include the partially hydrogenated derivatives of the carbocyclic systems enumerated herein, as long as at least one ring is aromatic.
• Non-limiting examples of aryl groups include phenyl, biphenyl, biphenylenyl, 5- or 6-tetralinyl, naphthalen-1- or -2-yl, 4-, 5-, 6 or 7-indenyl, 1- 2-, 3-, 4- or 5-acenaphthylenyl, 3-, 4- or 5-acenaphthenyl, 1- or 2-pentalenyl, 4- or 5-indanyl, 5-, 6-, 7- or 8-tetrahydronaphthyl, 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl, 1-, 2-, 3-, 4- or 5-pyrenyl.
• a preferred aryl group is phenyl.
• Arylalkyl refers to an alkyl group substituted by an aryl group: aryl-alkyl-.
• (Cx-Cy) preceding a group means that the group comprises from x to y carbon atoms, in accordance to common terminology in the chemistry field.
• Carboxylic acid refers to the group of formula -COOH.
• Coupling function refers to a functional group capable to react with another functional group to form a covalent linkage, such as a bond or a linear group of atoms.
• a coupling function which is reactive under suitable reaction conditions is thus capable of chemically reacting with another coupling function on a different molecule to form a new covalent linkage.
• a coupling function generally represents a point of attachment for another molecule.
• Coupling functions generally include nucleophiles, electrophiles and/or photoactivatable groups.
• Coupling product refers to a residue of a coupling function that results from the reaction between two coupling functions in different molecules, for example a functionally related group of atoms (such as amide -C(O)-NH- group or a double bond) or a heterocycle (such as a divalent triazolyl group).
• a coupling product is what remains of one or two coupling function(s) after the coupling reaction between the two coupling functions.
• the coupling reaction between two coupling functions A and B can lead to the following coupling products as shown on Table 1 below, wherein X represents a halogen atom (e.g., Br or Cl).
• a coupling product may be comprised in a “linker” as defined herein.
• “Covalently linked’’ means that two moieties are covalently bound together either directly, i.e., by means of a single, double or triple covalent bond (typically a single bond), or indirectly, i.e., by means of a “linker” as described herein, which comprises a plurality of covalent bonds.
• “Glycocluster” refers to a cluster of glycans, i.e., a molecule or ensemble of molecules comprising a plurality of glycan units. Thus, a glycocluster comprises at least two polysaccharide, oligosaccharide and/or monosaccharide moieties, typically at least two monosaccharides.
• the glycan units are grouped by means of their bonding to a common scaffold (e.g., a macrocycle, a polymer or a metal nanoparticle) and are relatively close to each other.
• a common scaffold e.g., a macrocycle, a polymer or a metal nanoparticle
• Glycoclusters are often used for drug delivery, however in the present invention they may be used as inhibitors of cell binding and/or infectivity for use in the treatment of infectious diseases.
• Heteroalkyl refers to an alkyl group as defined hereinabove wherein one or more carbon atoms are replaced by a heteroatom selected from oxygen, nitrogen and sulfur. In heteroalkyl groups, the heteroatoms are bound along the alkyl chain only to carbon atoms, i.e., each heteroatom is separated from any other heteroatom by at least one carbon atom.
• the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized.
• a heteroalkyl is bond to another group or molecule only through a carbon atom, i.e., the bounding atom is not selected among the heteroatoms included in the heteroalkyl group.
• Non-limiting examples of heteroalkyl include alkoxy, ethers and polyethers, secondary amines, tertiary amines and thioethers.
• Heteroaryl refers to aromatic rings or aromatic ring systems comprising from 5 to 12 carbon atoms, preferably from 6 to 10 carbon atoms, having one or two rings which are fused together or linked covalently, wherein at least one ring is aromatic, and wherein one or more carbon atoms in one or more of these rings is replaced by oxygen, nitrogen and/or sulfur atoms.
• “Heteroaryl” may also be viewed as an “aryl” group as defined herein, wherein at least one carbon atom in the aryl group is replaced with a heteroatom and wherein the resulting molecule is chemically stable.
• the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized.
• heteroaryl groups include furanyl, thiophenyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, oxatriazolyl, thiatriazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl, dioxinyl, thiazinyl, triazinyl, imidazo[2,l-b][l,3] thiazolyl, thieno [3, 2-b] furanyl, thieno[3,2-b]thiophenyl, thieno[2,3-d][l,3]thiazolyl, thieno[2,3-d]imidazolyl, tetrazolo[l,
• Heteroarylalkyl refers to an alkyl group substituted by a heteroaryl group: heteroaryl-alkyl- .
• “Hydroxyl” refers to -OH group.
• Linker refers to a moiety that covalently binds two molecules to one another and comprises a series of multivalent atoms selected from C, N, O, S and P bound together by stable covalent bonds.
• the moiety typically incorporates 1 to 30 atoms, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30.
• a linker may be linear or non-linear, some linkers have pendant side chains or pendant functional groups or both.
• a linker is composed of any combination of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds and carbon-sulfur bonds.
• a linker consists of a combination of moieties selected from alkyl, -C(O)NH-, -C(O)O-, -NH-, -S-, -O-, -C(O)-, -S(O)-, -S(O)2 and 5- or 6-membered monocyclic aryls or heteroaryls.
• the linker comprises at least one “coupling product” as defined herein, typically one or two coupling products.
• “comprise” means that the linker can be interrupted by at least one coupling product (z.e., the coupling product is incorporated into the atomic chain of the linker) and/or that the linker can end with at least one coupling product (i.e., the coupling product terminates the linker).
• the coupling product is considered part of the linker, which means in particular that the atoms of the coupling product are counted among the total number of atoms of the linker.
• Microcycle refers to a molecule or ion containing a 12- or more membered ring such as pillararenes, calixarenes, porphyrins, fullerenes, crown ethers and cyclodextrins.
• “macrocycle” term and any specific genus thereof e.g., pillararenes, calixarenes, porphyrins and fullerenes
• macrocycle refer both to the macrocycle per se and to any macrocycle-based moiety resulting from the coupling of a macrocycle with another molecule, either directly by a single bound or through a “linker” as defined herein.
• “Peptide” refers to a linear polymer of amino acids of less than 50 amino acids linked together by peptide bonds.
• sialic acid or ‘ ‘SA” refers to a monosaccharide belonging to a class of alpha-keto acid sugars with a nine-carbon backbone, in accordance with the general knowledge in the art.
• sialic acid refers to acetylneuraminic acid (Neu5Ac) of the following formulae.
• Sialic acids may be “acetylated” in accordance with the general meaning in the art, i.e., a group in the sialic acid (typically a hydrogen atom) may be replaced by an acetyl group.
• Neu5Ac includes an acetylated amine (AcHN), in the present application, only sialic acids wherein the acetylation is on both a hydroxyl (OH) group and the amine are considered “acetylated” in the sense of the invention.
• an “acetylated-sialic acid” is a sialic acid wherein at least one hydroxyl (-OH) on positions 4, 7, 8 and 9 has been substituted by an acetyl group, thereby resulting in an acetate moiety (-OC(O)CH3).
• Synthetic methods for the acetylation of one or more positions in a sialic acid, including esterification reactions with acetic acid, are well-known in the art.
• an acetylated-sialic acid may refer to 9-O-acetyl-sialic acid (9-AcSA) of the following formulae (left: alpha- anomer, right: beta-anomer).
• a sialic acid or acetylated-sialic acid may be bound to another compound, be they natural or artificial, either directly by a single covalent bond or through a linker.
• sialic acids and/or acetylated-sialic acids are bound to a macrocycle in order to form a glycocluster.
• Non-acetylated hydroxyls may in particular be used as coupling functions to obtain a covalent linkage.
• sialic acid term and any specific genus thereof (e.g., acetylated-sialic acid) refer both to the sialic acid monosaccharide per se and to any sialic acid-based moiety resulting from the coupling of sialic acid with another molecule, either directly by a single bound or through a “linker” as defined herein. If necessary, the latter will be specifically referred to as a “residue of sialic acid” or “sialic acid residue”.
• a sialic acid is bound to another molecule by means of a non-acetylated hydroxyl (e.g., the hydroxyl on position 2), the remaining oxygen is considered as part of the sialic acid residue for definition and representation purposes.
• Silicon refers to the function -O-Si(R)3 wherein R represents for example alkyl or aryl.
• Triazolyl refers to a monovalent, divalent or trivalent derivative of the heteroaryl of general formula N " N (z.e., triazole), such as for example N ' N or groups.
• administering means providing a therapeutic agent (e.g., a compound of the invention) alone or as part of a pharmaceutically acceptable composition, to the patient in whom/which the condition, symptom, or disease is to be treated and/or prevented.
• a therapeutic agent e.g., a compound of the invention
• Human refers to a male or female subject at any stage of development, including neonate, infant, juvenile, adolescent and adult.
• Patient refers to an animal, typically a warm-blooded animal, preferably a human, who/which is awaiting the receipt of, or is receiving medical care, or is/will be the object of a medical procedure.
• a patient may also be the subject of preventive care or procedure.
• “Pharmaceutically acceptable” means that the ingredients of a composition are compatible with each other and not deleterious to the patient to which/whom it is administered.
• “Pharmaceutically acceptable carrier” refers to an excipient that does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory offices, such as, e.g., FDA Office or EMA.
• Prevent refers to delaying or precluding the onset of a condition and/or disease and/or any one of its attendant symptoms, barring a patient from acquiring a condition or disease, or reducing the risk for a patient of acquiring a condition and/or disease and/or any one of its attendant symptoms.
• Prodrug refers to a pharmacologically acceptable derivative of a therapeutic agent (e.g., a compound of the invention) whose in vivo biotransformation product is the therapeutic agent (active drug).
• Prodrugs are typically characterized by increased bioavailability and are readily metabolized in vivo into the active compounds.
• Non-limiting examples of prodrugs include amide prodrugs and carboxylic acid ester prodrugs, in particular alkyl esters, cycloalkyl esters and aryl esters.
• solvent refers to molecular complex comprising a compound along with stoichiometric or sub- stoichiometric amounts of one or more molecules of one or more solvents, typically the solvent is a pharmaceutically acceptable solvent such as, for example, ethanol.
• hydrate refers to when the solvent is water (H2O).
• “Therapeutic agent”, “active pharmaceutical ingredient” and “active ingredient” refer to a compound for therapeutic use and relating to health.
• a therapeutic agent e.g., a compound of the invention
• An active ingredient may also be indicated for improving the therapeutic activity of another therapeutic agent.
• “Therapeutically effective amount” refers to the amount of a therapeutic agent (e.g., a compound of the invention) that is sufficient to achieve the desired therapeutic or prophylactic effect in the patient to which/whom it is administered.
• Treat”, “treating” and “treatment” refer to alleviating, attenuating or abrogating a condition and/or disease and/or any one of its attendant symptoms, e.g., an infectious disease.
• This invention relates to a glycocluster comprising at least two acetylated- sialic acids covalently linked to a macrocycle.
• the glycocluster and “the compound of the invention” and similar wordings are synonyms.
• the glycocluster comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15 or 16 acetylated sialic acids covalently linked to the macrocycle. In one embodiment, the glycocluster comprises at least four acetylated sialic acids covalently linked to the macrocycle. According to one embodiment, the glycocluster comprises four acetylated sialic acids covalently linked to the macrocycle (“tetramer”). According to one embodiment, the glycocluster comprises ten acetylated sialic acids covalently linked to the macrocycle (“decamer”). According to one embodiment, the glycocluster comprises twelve acetylated sialic acids covalently linked to the macrocycle (“dodecamer”).
• each acetylated- sialic acid not fully acetylated, i.e., wherein at least one OH group on positions 4, 7, 8 and 9 is not substituted by an acetyl.
• each acetylated- sialic acid is acetylated at 1, 2, 3 or 4 positions among positions 4, 7, 8 and 9.
• each acetylated- sialic acid is acetylated at 1, 2 or 3 positions.
• each acetylated- sialic acid is acetylated at 1 or 2 positions.
• each acetylated- sialic acid is acetylated at positions 7 and 9.
• each acetylated-sialic acid comprises at least one OH group. In one embodiment, each acetylated-sialic acid comprises one OH group. In one embodiment, each acetylated-sialic acid comprises two OH groups. In one embodiment, each acetylated-sialic acid comprises three OH groups. In one embodiment, each acetylated-sialic acid comprises four OH groups.
• each acetylated-sialic acid is acetylated at one position only, i.e., each acetylated-sialic acid is independently selected from, 4-O- acetylated-sialic acid, 7-O-acetylated-sialic acid, 8-O-acetylated-sialic acid and 9-O- acetylated-sialic acid.
• each acetylated-sialic acid is independently selected from 7-O-acetylated-sialic acid, 8-O-acetylated-sialic acid and 9- O-acetylated-sialic acid.
• each acetylated-sialic acid is independently selected from 7-O-acetylated-sialic acid and 9-O-acetylated-sialic acid.
• each acetylated-sialic acid is 4-O-acetylated-sialic acid.
• each acetylated-sialic acid is 7-O-acetylated-sialic acid.
• each acetylated-sialic acid is 8-O-acetylated-sialic acid. In one embodiment, each acetylated-sialic acid is 9-O-acetylated-sialic acid.
• sialic acids that are not fully acetylated, in particular sialic acids wherein only one OH is acetylated, are advantageous in terms of virus affinity or inhibition, in particular over SARS-CoV-2.
• the macrocycle is selected from porphyrins, pillararenes, calixarenes and fullerenes.
• the macrocycle is selected from porphyrins.
• the porphyrin is selected from porphin or tetraphenylporphyrin.
• the porphyrin has a metal cation coordinated by the four nitrogen atoms and no hydrogen attached to the nitrogen atoms (“porphyrin chelate”).
• Non-limiting examples include [Zn(porphin)] and [Zn(tetraphenylporphyrin)].
• the porphyrin does not have any cation coordinated by the four nitrogen atoms and two hydrogens are attached to the nitrogen atoms (“porphyrin free base”).
• the macrocycle is selected from pillararenes.
• the pillararene is selected from pillar[5]arenes.
• the macrocycle is selected from calixarenes.
• the calixarene is selected from calix[4]arenes.
• the macrocycle is selected from fullerenes.
• the macrocycle is selected from Ceo-fullerenes, also called buckminsterfullerenes.
• the macrocycle may be non-substituted (unsubstituted) apart from the acetylated-sialic acid substituents.
• the macrocycle may comprise further substituents, e.g., water- solubilizing substituents or substituents susceptible to interact with a virus.
• the glycocluster comprises at least one angiotensin-converting enzyme 2 (ACE2) binding inhibitor.
• ACE2 binding inhibitor is an ACE2 binding inhibitor peptide or protein.
• the ACE2 binding inhibitor is an ACE2 binding inhibitor peptide.
• the ACE2 binding inhibitor is an ACE2 binding inhibitor protein.
• the ACE2 binding inhibitor is an anti-ACE2 antibody or antigen-binding fragments thereof, such as an anti-ACE2 monoclonal antibody.
• the ACE2 binding inhibitor peptide is designed according to the sequence of the ACE2 receptor in complex with the RBD domain of the SI glycoprotein.
• the ACE2 binding inhibitor peptide is selected from the peptides [22-44] consisting of the amino acid sequence EEQAKTFLDKFNHEAEDLFYQSS (SEQ ID NO: 1), [351-357] consisting of the amino acid sequence LGKGDFR (SEQ ID NO: 2), [22-57] consisting of the amino acid sequence EEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEE (SEQ ID NO: 3) and [22-44-g-351-357] consisting of the amino acid sequence EEQAKTFLDKFNHEAEDLFYQSSGLGKGDFR (SEQ ID NO: 4); as described in reference document: Yang, J. et al., Nature Communications, 2020, Vol. 11, Article number: 4541.
• the glycocluster comprises at least one other substituent susceptible to bind with S protein such as antibody, antibody fragments, nanobody or lectin.
• the glycocluster is a compound of formula (I)
• M is a metal cation such as zinc, iron, copper, manganese, silver, gold, cobalt, nickel, tin, cadmium, lead or vanadium cations.
• the metal cation has a positive charge of two or three, preferably a positive charge of two.
• M is a metal cation selected from zinc (II), iron (II), iron (III), copper (II), copper (III), manganese (II), manganese (III), silver (II), silver (III), gold (III), cobalt (II), cobalt (III), nickel (II), nickel (III), tin (II), cadmium (II), lead (II), vanadium (II) and vanadium (III).
• the glycocluster is a compound of formula (la)
• each L 1 is independently a single bond or a linker selected from alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, arylalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl; wherein the alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, arylalkyl, heteroaryl, heteroarylalkyl or alkylheteroaryl optionally comprises at least one coupling product.
• each L 1 is a linker selected from alkyl, heteroalkyl, alkylaryl, arylalkyl, heteroarylalkyl and alkylheteroaryl; wherein the alkyl, heteroalkyl, alkylaryl, arylalkyl, heteroarylalkyl or alkylhetero aryl comprises at least one coupling product.
• the linker comprises one or two coupling product(s).
• the linker comprises one coupling product (i.e., only one coupling product).
• at least one of the coupling products is triazolyl.
• each R 1 is independently selected from acetylated- sialic acids and ACE2 binding inhibitors; provided that at least two R 1 are acetylated- sialic acids.
• each lA-R 1 is selected from -(CH 2 ) m -R c - (CH 2 ) n -R 1 , -O-(CH 2 ) m -R c -(CH 2 ) n -R 1 , -C(O)-(CH 2 ) m -R c -(CH 2 ) n -R 1 , -C(O)O-(CH 2 ) m - R c -(CH 2 ) n -R 1 , -phenyl-(CH 2 ) m -R c -(CH 2 ) n -R 1 and -phenyl-O-(CH 2 ) m -R c -(CH 2 ) n -R 1 ; wherein R c is a coupling product, and m and n are independently an integer ranging from 1 to 8.
• R c is triazolyl.
• m is an integer ranging from 1 to 6, preferably ranging from 1 to 4, more preferably ranging from 2 to 4, more preferably 2 or 3.
• n is an integer ranging from 1 to 4, preferably ranging from 1 to 3, more preferably 1 or 2, more preferably 1.
• each l R 1 is -phenyl-O-(CH2)m-R c -(CH2) n -R 1 ; wherein R c is a coupling product, and m and n are independently an integer ranging from 1 to 8.
• each R 1 is selected from 7-O-acetylated-sialic acid and 9-O-acetylated-sialic acid. In one embodiment, each R 1 is 7-O-acetylated-sialic acid. In one embodiment, each R 1 is 9-O-acetylated-sialic acid.
• the glycocluster is a compound of formula (II) wherein L 1 and R 1 are as defined hereinabove under formulae (I) and (la).
• each L 1 -R 1 is -O-(CH2)m-R c -(CH2) n -R 1 ; wherein R c is a coupling product, and m and n are independently an integer ranging from 1 to 8.
• R c is triazolyl.
• m is an integer ranging from 1 to 6, preferably ranging from 1 to 4, more preferably ranging from 2 to 4, more preferably 2 or 3.
• n is an integer ranging from 1 to 4, preferably ranging from 1 to 3, more preferably 1 or 2, more preferably 1.
• the glycocluster is a compound of formula (III) wherein L 1 and R 1 are as defined hereinabove under formulae (I) and (la).
• each l R 1 is -O-(CH2)m-R c -(CH2) n -R 1 ; wherein R c is a coupling product, and m and n are independently an integer ranging from 1 to 8.
• R c is triazolyl.
• m is an integer ranging from 1 to 6, preferably ranging from 1 to 4, more preferably ranging from 2 to 4, more preferably 2 or 3.
• n is an integer ranging from 1 to 4, preferably ranging from 1 to 3, more preferably 1 or 2, more preferably 1.
• the glycocluster is a compound of formula (IV) wherein L 1 and R 1 are as defined hereinabove under formulae (I) and (la).
• each lA-R 1 is -C(O)O-(CH2)m-R c -(CH2) n -R 1 ; wherein R c is a coupling product, and m and n are independently an integer ranging from 1 to 8.
• R c is triazolyl.
• m is an integer ranging from 1 to 6, preferably ranging from 1 to 4, more preferably ranging from 2 to 4, more preferably 2 or 3.
• n is an integer ranging from 1 to 4, preferably ranging from 1 to 3, more preferably 1 or 2, more preferably 1.
• the glycocluster is a compound of formula (Oi l) or a pharmaceutically acceptable salt or solvate thereof.
• the glycocluster is a compound of formula (005) or a pharmaceutically acceptable salt or solvate thereof.
• the glycocluster is a compound of formula (008) or a pharmaceutically acceptable salt or solvate thereof.
• the glycocluster is a compound of formula (014) or a pharmaceutically acceptable salt or solvate thereof.
• each R 1 is as described under formulae (I) and (la).
• each R 1 is of formula (9AcSA) wherein the wavy line represents the point of attachment of R 1 to the glycocluster.
• the glycocluster is selected from the compound of formula (Oi l), (005), (008) and (014) hereinabove, wherein each R 1 is of formula (9AcSA), or a pharmaceutically acceptable salt or solvate thereof, i.e., compounds Oil, 005, 008 and 014 as represented in Example 1 herein, or a pharmaceutically acceptable salt or solvate thereof.
• All references herein to a compound of the invention include references to salts, preferably pharmaceutically acceptable salts, solvates, multi component complexes and/or liquid crystals thereof.
• All references herein to a compound of the invention include references to polymorphs and/or crystal habits thereof.
• All references to a compound of the invention include references to pharmaceutically acceptable prodrugs thereof.
• All references to a compound of the invention include references to isotopically-labelled compounds, including deuterated compounds.
• a compound of the invention e.g., “glycocluster” or “formula (I)” and subformulae thereof contains at least one asymmetric centre(s) and thus may exist as different stereoisomeric forms.
• all references to a compound of the invention include references to all possible stereoisomers and includes not only the racemic compounds but the individual enantiomers and their non-racemic mixtures as well.
• a compound is desired as a single enantiomer, such single enantiomer may be obtained by stereospecific synthesis, by resolution of the final product or any convenient intermediate, or by chiral chromatographic methods as each are known in the art. Resolution of the final product, an intermediate, or a starting material may be carried out by any suitable method known in the art.
• the compounds of the invention may be in the form of pharmaceutically acceptable salts.
• Pharmaceutically acceptable salts include the acid addition and base salts thereof. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, 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, pa
• Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, 2-(diethylamino)ethanol, diolamine, ethanolamine, glycine, 4-(2-hydroxyethyl)- morpholine, lysine, magnesium, meglumine, morpholine, olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts. When a compound contains an acidic group as well as a basic group the compound may also form internal salts, and such compounds are within the scope of the invention. When a compound contains a hydrogen-donating heteroatom (e.g., NH), the invention also covers salts and/or isomers formed by transfer of said hydrogen atom to a basic group or atom within the molecule.
• a hydrogen-donating heteroatom e.g
• salts of compounds of the invention may be prepared by one or more of these methods: (i) by reacting the compound with the desired acid; (ii) by reacting the compound with the desired base; (iii) by removing an acid- or base-labile protecting group from a suitable precursor of the compound or by ringopening a suitable cyclic precursor, e.g., a lactone or lactam, using the desired acid; and/or (iv) by converting one salt of the compound 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 non-ionized.
• This invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising a compound of the invention as described herein and at least one pharmaceutically acceptable carrier.
• the pharmaceutical composition further comprises at least another therapeutic agent.
• the therapeutic agent is an antiviral agent.
• the at least another therapeutic agent is an angiotensin-converting enzyme 2 (ACE2) binding inhibitor, as described herein.
• ACE2 binding inhibitor is vectorized by the glycocluster.
• the compound of the invention may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration.
• This invention also relates to a compound of the invention as described herein, or a pharmaceutical composition of the invention as described herein, for use as a medicament.
• This invention also relates to a compound of the invention as described herein, or a pharmaceutical composition of the invention as described herein, for use in the treatment and/or prevention of an infectious disease.
• the infectious disease is a coronavirus infection or a picomavirus infection. In one embodiment, the infectious disease is a coronavirus infection.
• the coronavirus is an alpha coronavirus or a beta coronavirus, preferably a beta coronavirus.
• alpha coronaviruses include human coronavirus 229E (HCoV-229E) and human coronavirus NL63 (HcoV- NL63) also sometimes known as HcoV-NH or New Haven human coronavirus.
• Non-limiting examples of beta coronaviruses include human coronavirus OC43 (HcoV- OC43), human coronavirus HKU1 (HcoV-HKUl), Middle East respiratory syndrome- related coronavirus (MERS-CoV) previously known as novel coronavirus 2012 or HcoV- EMC, severe acute respiratory syndrome coronavirus (SARS-CoV) also known as SARS- CoV- 1 or SARS-classic, and severe acute respiratory syndrome coronavirus (SARS- CoV-2) also known as 2019-nCoV or novel coronavirus 2019.
• HcoV- OC43 human coronavirus OC43
• HKU1 HcoV-HKUl
• MERS-CoV Middle East respiratory syndrome- related coronavirus
• SARS-CoV severe acute respiratory syndrome coronavirus
• SARS-CoV- 1 severe acute respiratory syndrome coronavirus
• SARS- CoV-2 severe acute respiratory syndrome coronavirus
• 2019-nCoV 2019-nCoV or novel
• the coronavirus is selected from HcoV-229E, HcoV-NL63, HcoV-OC43, HcoV-HKUl, MERS-CoV, SARS-CoV- 1 and SARS-CoV-2. In one embodiment, the coronavirus is selected from MERS-CoV, SARS-CoV- 1 and SARS-CoV-2.
• the coronavirus is a SARS coronavirus.
• the coronavirus is SARS-CoV- 1 or SARS-CoV-2.
• the coronavirus is SARS-CoV (also referred to as SARS-CoV- 1) causing severe acute respiratory syndrome (SARS).
• the coronavirus is SARS-CoV-2 causing COVID- 19.
• any reference to a “coronavirus” or to “SARS-CoV-2” encompass any variant thereof currently identified.
• the coronavirus is an original haplotype of the SARS-CoV-2 pandemic (lineage A or B), or a variant thereof.
• the coronavirus is SARS-CoV-2 Alpha (lineage B.1.1.7, and sub-lineages thereof).
• the coronavirus is SARS-CoV-2 Beta (lineage B.1.351, and sub-lineages thereof).
• the coronavirus is SARS-CoV-2 Gamma (lineage P.l, and sub-lineages thereof).
• the coronavirus is SARS-CoV-2 Delta (lineages B.1.617.2, XD, XF, XS, and sub-lineages thereof). In one embodiment, the coronavirus is SARS-CoV-2 Epsilon (lineages B.1.427, B 1.429, and sub-lineages thereof). In one embodiment, the coronavirus is SARS-CoV-2 Zeta (lineage P.2, and sub-lineages thereof). In one embodiment, the coronavirus is SARS-CoV-2 Eta (lineage B.1.525, and sub-lineages thereof). In one embodiment, the coronavirus is SARS-CoV-2 Theta (lineage P.3, and sub-lineages thereof).
• the coronavirus is SARS-CoV-2 Iota (lineage B.1.526, and sub-lineages thereof). In one embodiment, the coronavirus is SARS-CoV-2 Kappa (lineage B.1.617.1, and sub-lineages thereof). In one embodiment, the coronavirus is SARS-CoV-2 Lambda (lineage C.37, and sub-lineages thereof). In one embodiment, the coronavirus is SARS- CoV-2 Mu (lineage B.1.621, and sub-lineages thereof). In one embodiment, the coronavirus is SARS-CoV-2 Omicron (lineages B.1.1.529, BA.l, BA.2, BA.3, BA.4, BA.5, XE, and sub-lineages thereof). A list of all SARS-CoV-2 variants can be found on the cov-lineages website, at https://cov-lineages.org/lineage list.html.
• the COVID-19 is moderate COVID- 19. In one embodiment, the COVID-19 is mild-to-moderate COVID- 19. In one embodiment, the COVID-19 is mild COVID-19. In one embodiment, the COVID-19 is mild-to- severe COVID-19. In one embodiment, the COVID-19 is severe COVID-19.
• the subject suffering from COVID- 19 may or may not be hospitalized.
• COVID- 19 severity is assessed according to the World Health Organization (WHO) criteria of severity, which are as follows:
• WHO World Health Organization
• CO VID-19 severity and/or progression is assessed with the WHO 10-point progression scale as indicated in Table 2 below.
• Table 2 WHO 10-point progression scale of COVID-19
• the subject to be treated according to the present invention is suffering from CO VID- 19 and has a score on the WHO 10-point progression scale of COVID- 19 (as described in Table 2) ranging from 1 to 5, preferably ranging from 2 to 4; ranging from 3 to 6, preferably ranging from 4 to 5; or ranging from 5 to 9, preferably ranging from 6 to 8.
• the compound or pharmaceutical composition of the invention may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant), by inhalation spray, nasal, vaginal, rectal, sublingual, or topical routes of administration.
• parenteral e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant
• inhalation spray nasal, vaginal, rectal, sublingual, or topical routes of administration.
• an appropriate dosage level may be from about 0.01 to 500 mg per kg patient body weight per day (mg/kg/day), which can be administered in single or multiple doses.
• the dosage level will be from about 0.1 to about 250 mg/kg/day, preferably from about 0.5 to about 100 mg/kg/day, more preferably from about 2.5 to about 20 mg/kg/day.
• the compounds may be administered on a regimen of 1 to 4 times per day, preferably once or twice per day. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular diseases and the host undergoing therapy.
• This invention also relates to the use of a compound of the invention as described herein, or a pharmaceutical composition of the invention as described herein, in the manufacture of a medicament for the treatment and/or prevention of an infectious disease.
• This invention also relates to a method for the treatment and/or prevention of an infectious disease in a subject in need thereof, comprising a step of administering to the subject a therapeutically effective amount of a compound of the invention as described herein, or of a pharmaceutical composition of the invention as described herein.
• This invention also relates to the use of a compound of the invention as described herein, or a pharmaceutical composition of the invention as described herein, in the treatment and/or prevention of an infectious disease. Glycocluster synthesis
• the glycocluster may be prepared by any synthetic method known in the art.
• appropriate synthetic methods for creating a covalent linkage between the acetylated-sialic acid and macrocycle are part of the general knowledge of a person skilled in organic chemistry.
• the acetylated-sialic acid may be linked to the macrocycle by means of at least one non-acetylated hydroxyl group on position 2, 4, 7, 8 or 9.
• the acetylated-sialic acid is linked to the macrocycle by means of the hydroxyl on position 2 or 4.
• the acetylated-sialic acid is linked to the macrocycle by means of the hydroxyl on position 2.
• the hydroxyl on position 2 is especially advantageous for linking the acetylated-sialic acid to the macrocycle without impairing the biological activity associated with the other side of the acetylated-sialic acid.
• a non-acetylated hydroxyl group is used as a coupling function and reacted with a corresponding coupling function of the macrocycle (e.g., halocarbon, carboxylic acid or amine) to form a linkage between the acetylated-sialic acid and the macrocycle (e.g., an ether, ester or amide bond).
• a corresponding coupling function of the macrocycle e.g., halocarbon, carboxylic acid or amine
• the hydroxyl group is first substituted by a group comprising a coupling function other than hydroxyl and/or converted into a coupling function other than hydroxyl, then the other coupling function is reacted with a corresponding coupling function of the macrocycle to form a linkage between the acetylated-sialic acid and the macrocycle.
• the glycocluster is prepared by Copper(I)-catalysed Azide-Alkyne Cycloaddition (CuAAC).
• CuAAC Copper(I)-catalysed Azide-Alkyne Cycloaddition
• the glycocluster is prepared in presence of a copper (I) catalyst such as a copper (I) salt (e.g., copper bromide or copper iodide), or a mixture of a copper (II) salt (e.g., copper sulfate) and a reducing agent (e.g., sodium ascorbate).
• a copper (I) catalyst such as a copper (I) salt (e.g., copper bromide or copper iodide), or a mixture of a copper (II) salt (e.g., copper sulfate) and a reducing agent (e.g., sodium ascorbate).
• the acetylated-sialic acid comprises a -O-(CH2) n -N3 group wherein n is an integer ranging from 1 to 8.
• the macrocycle comprises at least two -O-(CH2)m-N3 groups wherein m is an integer ranging from 1 to 8.
• Figure 1 is a scheme showing the method of preparation of the SA- or 9-AcSA- derived glycoclusters 004-014 from a-propargyl sialic acid (a-p-SA) 001 or a-propargyl acetylated sialic acid (a-p-9-AcSA) 002 and azide compounds 003, 006, 009 and 012 (which are shown in Scheme 3 in Example 1-d).
• a-p-SA a-propargyl sialic acid
• a-p-9-AcSA a-propargyl acetylated sialic acid
• Figure 2 is a box plot showing the specific binding probabilities (BP) measured between the S 1 functionalized tip and the surface coated with 9- AcS A, SA or streptavidin, before and after blocking with free 1 mM 9- AcS A, as described in Example 2.1.
• One data point represents the binding frequency (BF) obtained for 1024 FD curves.
• the square in the box indicates the mean, the coloured box the 25 th and 75 th percentiles, and the whiskers the highest and lowest values.
• the line in the box indicates the median.
• N 9 maps examined over 3 independent experiments. P-values were determined by two-sample t test in Origin.
• Figure 3 is a graph showing the binding frequency (BF) plotted between SI and 9-AcSA as a function of the hold time, as described in Example 2.2. Least-squares fits of the data to a mono-exponential decay curve (line) provides average kinetic on-rates (k on ) of the probed interaction. Further calculation (k o ff/k on ) leads to KD. One data point represent the BF obtained for 1024 FD curves.
• Figures 4 to 9 show the results of a screening of the anti-binding properties of SA- or 9-AcSA-derived glycoclusters, as described in Example 2.5.
• Figures 4-8 are histograms showing the inhibiting efficiency of the tested molecules, which is evaluated by measuring the binding probability (BP) of the interaction between 9-AcSA and SARS-CoV-2 before and after incubation with the tested molecules at increasing concentration (1-100 pM).
• BP binding probability
• Figure 9 is a graph showing the reduction of the binding probability (BP) after incubation with the acetylated tested molecules 9-AcSA-a-p, 005, 008, 011 and 014 at increasing concentration (1-100 pM) as described in Example 2.5.
• Figures 10 and 11 show the results of a probing of 9-AcSA-porphyrin glycocluster 011 efficiency to inhibit SARS-CoV-2 binding to acetylated SA on model surfaces (9-O-acetylated SA) and living cells (CHO-cells) at low concentrations, as described in Example 2.6.
• Figure 10 is a box plot showing the relative binding values of the interaction between SARS-Cov-2 and 9-O-acetylated SA model surfaces before and after incubation with 9-AcSA-porphyrin glycocluster Oil at increasing concentration (0.001-100 pM).
• Figure 11 is a box plot showing the relative binding values of the interaction between SARS-Cov-2 and CHO-cells before and after incubation with 9-AcSA-porphyrin glycocluster Oil at increasing concentration (0.1-10 pM).
• Figure 12 is a histogram showing the results of an infectivity assay, namely, the infectivity measured in the presence of free SA, free 9-AcSA, SA-porphyrin 010 and 9-AcSA porphyrin 011, as described in Example 2.7. Each dot shows the infectivity from a well.
• the colored box indicates the mean and the whiskers the s.d. of the mean value.
• the line in the box indicates the median. P values were determined by two-sample t test in Origin.
• Figures 13A-B are two box plots of the binding probability (BP, in %) between SI and Lec2 or CHO cells ( Figure 13 A) or between SARS-CoV-2 and Lec2 or CHO cells ( Figure 13B).
• n 10 ( Figure 13A) or 12 ( Figure 13B) maps examined over three independent experiments. P values were determined by two-sample t-test in Origin.
• Example 1 Materials, methods and results for synthesis and characterization
• Example 1-a General materials and methods
• Example 1-b Synthesis and characterization of biotinylated sialic acids
• Compound (B4) (“biot-SA”): A solution of compound B3 (345 mg, 0.371 mmol, 1 equiv.) and NaOMe (40 mg, 0.742 mmol, 2 equiv.) in dry MeOH (20 mL) was stirred at 0°C for 30 min, then warmed up to room temperature and stirred for another 1.5 h. Afterwards, Amberlyst®15 ion-exchange resin was added to neutralize the base. The resin was filtered and washed with water (2 x 5 mL). The filtrate was evaporated under reduced pressure to afford a white solid.
• the white solid was dissolved in water (15 mL) and LiOH.H2 ⁇ D (35 mg, 0.831 mmol, 3 equiv.) was added. The solution was stirred at room temperature for 1 h before adding Amberlyst®15 ion- exchange. Afterwards, the reaction mixture was filtered, the resin was washed with water (2 x 5 mL) and the filtrate was lyophilized to obtain white solid (186 mg, 0.249 mmol, 90% yield).
• Compound (B5) (“biot-9- AcS A”): To a solution of compound B4 (200 mg, 0.267 mmol, 1 equiv.) and trimethyl orthoacetate (0.34 mL, 2.67 mmol, 10 equiv.) in dry dimethylsulfoxide (DMSO) (1.2 mL) was added -tolucncsul Ionic acid monohydrate (5.0 mg, 0.027 mmol, 0.1 equiv.). The solution was stirred at room temperature for 12 h. Then, DCM (50 mL) was added to precipitate the crude product.
• DMSO dry dimethylsulfoxide
• the crude was purified by Cis silica gel flash chromatography using H 2 O/MeOH (0-1/3, gradient) as eluent.
• the fractions containing compound B5 were combined and concentrated under reduced pressure.
• the concentrated solution was lyophilized to afford the desired compound as a white solid (25 mg, 0.0316 mmol, 12%).
• Example 1-c Synthesis and characterization of sialic acid derivatives
• Intermediate compound 003 was prepared according to known procedures (Nierengarten, I. et al., Chemical Communications, 2012, Vol. 48, pp. 8072-8074).
• Intermediate compound 006 was prepared according to known procedures (Tikad, A. et al., Chemistry: A European Journal, 2016, Vol. 22, pp. 13147-13155).
• Intermediate compound 009 was prepared according to known procedures (Tikad, A. et al., Chemistry: A European Journal, 2016, Vol. 22, pp. 13147-13155; and Liu, Y. et al., Angewandte Chemie (International Edition English), 2016, Vol. 55, pp. 7952-7957).
• Intermediate compound 012 was prepared according to known procedures (Nierengarten, J. F. et al., Chemical Communications, 2010, Vol. 46, pp. 3860-3862).
• SA-glycoclusters Compounds 004, 007, 010 and 013 (“SA-glycoclusters”) and 005, 008, 011 and 014 (“9-As-SA-glycoclusters”) were prepared by grating the clickable a-propargyl sialic acids 001 and 002 to multimeric azides 003, 006, 009 and 0012 using either a combination of copper(II) sulfate and sodium L-ascorbate, or copper(I)bromide dimethyl sulphide, as shown in Figure 1.
• a specific method was optimized for the porphyrin tetramers 010 and 011, to avoid ion exchange between copper and zinc and to cope with the solubility properties of 009.
• a lower catalyst amount and a ternary solvent system (THF/DMSO/H2O, 3:3:1) were employed.
• the tetravalent porphyrin conjugates 010 and 011 were obtained in 61% and 87% yields, respectively.
• the calix[4] arenes 007 and 008 and fullerenes 013 and 014 were also obtained in high yields using similar coupling and purification protocols. All the multimeric species were characterized by NMR, 13 C NMR and mass spectrometry to confirm the completion of all reactions.
• SARS-CoV-2 (BavPatl strain, European Virology Archives) was grown in Vero E6 and used at passage 3. 500 pL of passage 3 stock (supernatants of infected cells) was added to a 6-well dish. The 6-well dish was placed in a UV Stratalinker 1800 (Stratagene) without a lid and virus-containing supernatant was exposed to 5000 J of UV irradiation. Virus inactivation was confirmed by adding 10 pL of the UV treated supernatant to a 48-well plate containing 50000 naive Vero E6 cells and monitoring infection 48 hours later by indirect immunofluorescence assay.
• Gold-coated silicon substrates were first washed with ethanol and cleaned by UV-0 treatment (Jetlight) for 15 minutes. The surfaces were then incubated overnight at 4°C in a biotinylated bovine serum albumin (BBSA) solution (25 pg.mL' 1 in PBS, Sigma). After rinsing with PBS, a drop of streptavidin (10 pg.mL' 1 in PBS, Sigma) was pipetted onto the BBSA surface for 1 h at 4°C, followed by rinsing with PBS.
• BBSA biotinylated bovine serum albumin
• the BBSA-streptavidin surfaces were immersed for Ih in a biotinylated biot-SA (B4) or biot-9-AcSA (B5) solution (10 pg.mL' 1 in PBS), followed by a final PBS rinsing.
• the surfaces showed a homogeneous and stable morphology under repeated scanning and exhibited a thickness of 1.1 ⁇ 0.1 nm.
• the thickness of the deposited layer was estimated by scanning a small area (1 pm x 1 pm) of the surface at high forces to remove the attached biomolecules, followed by imaging larger squares of the same region (5 pm x 5 pm) at a lower force.
• AFM tip functionalization NHS-PEG24-Ph- aldehyde linkers (Broadpharm) were used.
• AFM tips MSCT-D probes, Bruker were immersed in chloroform for 10 min, rinsed with ethanol, dried in a gentle stream of filtered nitrogen, cleaned for 15 min in an ultraviolet radiation and ozone cleaner (JetLight), and immersed overnight in an ethanolamine solution [3.3 g of ethanolamine hydrochloride in 6.6 niL of dimethyl sulfoxide (DMSO)]. The cantilevers were then washed three times with DMSO and three times with ethanol, and dried with nitrogen.
• DMSO dimethyl sulfoxide
• a Nanoscope Multimode 8 (Bruker) was operated in force-volume (contact) mode to conduct the force spectroscopy experiments on model surfaces (Nanoscope software v9.1). MSCT-D probes (nominal spring constant of 0.03 N m’ 1 ) were used to record 5 pm x 5 pm maps, with a ramp size of 200 nm, a maximum force of 500 pN, and no surface delay. The sample was scanned using a line frequency of 1 Hz, and 32 pixels per line (32 lines). Both approach and retraction speed were kept constant at 1 pm s’ 1 .
• FD-curve based AFM was used to compare SARS-CoV-2 binding to SA (Neu5Ac) and 9-AcSA and characterize the binding free-energy landscape of the interaction to 9-AcSA.
• biotinylated-SA either biot-9-AcSA (B5) or biot-SA (B4) were immobilized onto streptavidin-coated surfaces and validated by AFM imaging and scratching experiments, revealing a 1.1 ⁇ 0.1 nm thick deposited layer. The interaction between the spike SI subunit and the SA-coated surfaces was monitored by FD-based AFM.
• binding probabilities (BP) (fraction of curves showing binding events) were measured before and after incubation with different concentrations (0, 1, 10 and 100 pM respectively) of SA-glycoclusters (004, 007, 010 and/or 013) and four 9-AsSA- glycoclusters (005, 008, 011 and/or 014).
• BP binding probabilities
• the BP was determined at a certain contact time (t), which is the time the tip is in contact with the surface.
• V e ff 47tr e ff 3 ) represents the volume in which the interaction can take place. This results in a half-sphere, since only half of the S 1 molecules can interact with its corresponding receptor on the substrate.
• CHO cells were cultured in Ham’ s F12 medium (Sigma) supplemented with 10% FBS (Fetal Bovine Serum), penicillin (100 U mL-1), streptomycin (100 pg mL' 1 ) (Invitrogen) and 2mM L- glutamine (Sigma). Cells were incubated at 37 °C with 5% of CO2 and in an environment saturated in humidity. Transduction of Lec2 cells
• Lec2 cells were transduced to express nuclear eGFP as well as cytoplasmic mCherry using H2BeGFP and actin-mCherry-expressing lentiviruses.
• the sample was scanned using a frequency of 0.125 Hz and 128 or 256 pixels per line.
• AFM images and FD curves were analysed using the Nanoscope analysis software (vl.9, Bruker), Origin, and ImageJ (vl.52e).
• Individual FD curves detecting unbinding events between the cell surface and SI or SARS-CoV-2 were analysed using the Nanoscope analysis and Origin software.
• the baseline of the retraction curve was corrected using a linear fit on the last 30% of the retraction curve.
• the loading rate (slope) of each rupture event was determined.
• Optical images were analysed using Zen Blue software (Zeiss).
• the live cell experiments were conducted in the same manner as described above by scanning a suitable area of confluent layers of cells, followed by adding either 10 nM, 100 nM, 1 pM or 10 pM of 9-AcSA porphyrin 011 to the culture medium. The same area was then scanned again to monitor potential changes after addition of the 9-AcSA oligomer.
• pCGl SARS-CoV-2 spike protein with a C-terminal truncation of 18 amino acid residues plasmid was transfected in HEK-293 cells.
• VSV-deltaG virions were transduced in cells with MOI 5 FFU per cell.
• MOI 5 FFU per cell.
• the transduced cells were cultured in DMEM supplemented with 5% FBS, 1% penicillin, 1% streptomycin, 2 mM L-Glutamine, 1 mM Na-Pyruvate and NEAA, as well as anti-VSV-G antibody (1:1000).
• the produced viruses were collected from the media the day after the transduction. Cell debris were cleared by centrifugation (1250 x g, 10 min) and with a 0.22
• A549 or A549 ACE2 stable cells (1 x 10 4 ) were seeded in a 96-well plate.
• the mixture of the pseudotyped virus at MOI 5 and either SA (Neu5Ac), 9-AcSA, SA- porphyrin glycocluster 010 or 9-AcSA-porphyrin glycocluster 011 (as shown on Example 1-d) at increasing concentration (0.001 pM, 0.01 pM, O.lpM, 1 pM, 10 pM) were incubated for 15 min at room temperature.
• the mixture was added in the media and the cells were incubated for 1 hour.
• the cells were washed with PBS and incubated in fresh cell culture media for 24 hours.
• the infectivity was monitored via fluorescence and the images were taken with the bioimager device (Amersham Typhoon). The number of infected cells were counted by Fiji.
• Binding free-energy landscape of the interaction between SI and 9-AcSA was characterized using single-molecule dynamic force spectroscopy (DFS).
• DFS single-molecule dynamic force spectroscopy
• Non-replicating SARS-CoV-2 particles i.e., native SARS-CoV-2 virions inactivated through UV radiation
• 9-AcSA The binding of these non-replicating SARS-CoV-2 particles to 9-AcSA was evaluated, by grafting the whole virions onto the AFM tip. The interaction was probed at moderate (1 pm.s' 1 ) and fast (20 pm.s' 1 ) pulling speed, and the DFS plot were reconstructed and overlaid with the data obtained with the purified SI domain.
• the glycoclusters of the invention may be used in the treatment and/or prevention of an infectious disease such as COVID- 19. 6. Characterization of the inhibition of an AcSA-derived porphyrin
• the Applicants unexpectedly evidenced that a plurality of AcSA covalently linked to porphyrins lead to especially significant inhibition of the SARS-CoV-2 even in low concentrations, despite the low affinity of the free AcSA.
• the AcSA-porphyrin-based glycoclusters of the invention may be used in the treatment and/or prevention of an infectious disease such as COVID- 19.
• VSV G-deleted vesicular stomatitis virus
• VSV-SARS-CoV-2 GFP reporter protein
• A549-ACE2 were infected with a MOI of 5 of the VSV-SARS-CoV-2. Infectivity was monitored by measuring the GFP fluorescence in the cells 24h post-infection. While A549 cells are not infected by the VSV-SARS-CoV-2, overexpression of ACE2 strongly enhanced infection.
• This cell-based assay confirms the previous results on the effect of sialic acids on SARS-CoV-2 binding and further evidences that effective inhibition of the virus binding to its receptor by a glycocluster of the invention leads to a significant drop in infectivity.

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