Glycomimetic ligands

Abstract

Glycan compounds and particles comprising glycan compounds are described herein. The compounds and particles described herein, or pharmaceutically acceptable salts thereof, are useful in methods of treating immune and inflammatory related diseases. # imgabs0 #

Description

Related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 295,698, filed December 31, 2021. The entire teachings of the above application are incorporated herein by reference. Background Art

[0002] Improved compositions and methods for modulating sialic acid binding to autoassociated pattern recognition receptors, known as sialic acid-binding immunoglobulin-type lectins (Siglecs), are needed to treat diseases caused by acute, chronic, or aberrant immune system activation. Summary of the Invention

[0003] In exemplary embodiments, the present invention is a compound represented by any one of the following structural formulas: or a pharmaceutically acceptable salt thereof.

[0004] In formulae (S-1), (S-2), (D-1) and (D-2): for each occurrence, R1 is independently -C(O)-A, wherein A is C1-C6 alkyl, C6-C 18 Aryl, (C6-C 18 )aryl(C1-C3)alkyl, 5-18 membered heteroaryl, (5-18 membered)heteroaryl(C1-C3)alkyl, C3-C8 cycloalkyl, (C3-C8)cycloalkyl(C1-C3)alkyl, 5-8 membered heterocycloalkyl, or (5-8 membered)heterocycloalkyl(C1-C3)alkyl, wherein one or two carbon atoms of the alkyl portion of A are each independently replaced by a heteroatom selected from N, O or S, and wherein A is optionally replaced by 1 to 3 R 11 Group substitution, each of the R 11 The groups are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, halogen, C 6- C 12 Aryl, 5-12 membered heteroaryl, cyano, or two R 11 The groups, together with the atoms to which they are attached, form a 5-7 membered heterocyclic group having 1 to 3 heteroatoms selected from N, O or S. Each R 11 is independently optionally substituted with 1 to 3 substituents selected from halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, or cyano; and further wherein: for each occurrence, R is independently -R L -R F , and where: for each occurrence, R L Independently: -O-(C1-C 12)alkylene-, -O-, -S-, -NR 100 -、-S-(C1-C 12 )alkylene-、-NR 101 -(C1-C 12 )alkylene-、-NR 101a -O-(C1-C 12 )alkylene-;-O-(CH2CH2O) m -、-O-(CH2CH2O) k -(CH2CH2)-、-NR 102 -X 100 -(C1-C 12 )alkylene-、-NR 102a -NR 102b -C(O)-(C1-C 12 ) alkylene, wherein R 100 、R 101 、R 101a 、R 102 、R 102a and R 102b are each independently H or C1-C3 alkyl, and X 100 is -O- or -NH-, and wherein m and k are each independently an integer from 1 to 12; for each occurrence, R F are independently: H, C1-C3 alkyl, -NH2, -NH-Fmoc, -NH-Boc, -NH-CBz, -NH-Troc, -NH-TFA, mono(C1-C3)alkylamino, di(C1-C3)alkylamino; -C(O)-R 103 , where R 103 is -H, -OH, or (C1-C3) alkyl; -SH, having the following structural formula Represents part; or click chemistry reagent.

[0005] In another exemplary embodiment, the present invention is a particle comprising a compound represented by the following structural formula (2). GLP (2).

[0006] In formula (2), P is a biocompatible polymer. For example, the biocompatible polymer includes at least one of polyglycolic acid, poly(lactic acid), poly(lactic-co-glycolic acid), polycaprolactone, poly(3-hydroxybutyric acid), poly(ethylene glycol), polyethylene oxide, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Pluronic F127), polyoxyethylene-polyoxypropylene block copolymer (Pluronic F68), poloxamer, poly(hydroxymethyl methacrylate), polyvinyl alcohol, poly(vinyl pyrrolidone), hyaluronic acid, heparin, heparin sulfate, polysialic acid, and chitosan; L is a covalent linker; and G is any one of the moieties represented by the following structural formulas: or a pharmaceutically acceptable salt thereof.The values ​​and exemplary values ​​of the variable R1 are as defined above for Formulas (S-1), (S-2), (D-1), and (D-2).

[0007] In another exemplary embodiment, the present invention is a method of preparing particles comprising a compound represented by the following structural formula (2): GLP (2), wherein P is a biocompatible polymer. For example, the biocompatible polymer comprises at least one of polyglycolic acid, poly(lactic acid), poly(lactic-co-glycolic acid), polycaprolactone, poly(3-hydroxybutyric acid), poly(ethylene glycol), polyethylene oxide, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Pluronic F127), polyoxyethylene-polyoxypropylene block copolymer (Pluronic F68), poloxamer, poly(hydroxymethyl methacrylate), polyvinyl alcohol, poly(vinyl pyrrolidone), hyaluronic acid, heparin, heparin sulfate, polysialic acid, and chitosan; L is a covalent linker; and G is any one of the moieties represented by structural formulas (S-1A), (S-2A), (D-1A), and (D-2A), or a pharmaceutically acceptable salt thereof, wherein the symbol represents the point of attachment to L, and the values ​​and exemplary values ​​of the variable R1 are as defined above with respect to formulas (S-1), (S-2), (D-1), and (D-2). The method comprises: F1 and R F2 Under conditions where the parts react with each other, the compound represented by structural formula (I) GR F1 (I) Reacting with a compound represented by structural formula (II) PR F2 (II), where R F1 and R F2Each is a reactive moiety, thereby generating the covalent -L- moiety.

[0008] In various exemplary embodiments, the compounds and particles described herein can be used in methods for treating diseases and conditions that are responsive to modulation (e.g., amplification, reduction, or elimination) of the activity of Siglec receptors. Such diseases and conditions include, but are not limited to, cancer, immune-related diseases and conditions, and inflammation-related diseases and conditions. For example, the compounds and particles described herein can be used in methods for treating a condition selected from cancer, ophthalmic diseases, fibrotic diseases, parasitic inflammation, fungal inflammation, viral inflammation, autoimmune inflammation, nervous system inflammation, neurodegeneration, skin inflammation, kidney inflammation, cardiovascular disease, gastrointestinal inflammation, or rheumatic disease.

[0009] In other embodiments, the compounds and particles described herein can be used to treat a condition selected from the group consisting of cancer (e.g., breast cancer, non-small cell lung cancer (NSCLC), prostate cancer, colorectal cancer, melanoma, pancreatic cancer, and myelofibrosis), diabetic retinopathy, idiopathic pulmonary fibrosis, liver fibrosis, sickle cell anemia, and acute respiratory distress syndrome (ARDS).

[0010] In another exemplary embodiment, the present invention is a compound represented by the following structural formula: or a pharmaceutically acceptable salt thereof. In formula (S000-C), for each occurrence, R is independently -R L -R F , and where: for each occurrence, R L are independently: -O-, -S-, -NR 100 -、-O-(C1-C 12 )alkylene-, -S-(C1-C 12 )alkylene-、-NR 101 -(C1-C 12 )alkylene-、-NR 101a -O-(C1-C 12 )alkylene-;-O-(CH2CH2O) m -、-O-(CH2CH2O) k -(CH2CH2)-、-NR 102 -X 100 -(C1-C 12 )alkylene-、-NR 102a -NR 102b -C(O)-(C1-C 12 ) alkylene, wherein R 100 、R 101 、R 101a、R 102 、R 102a and R 102b are each independently H or C1-C3 alkyl, and X 100 is -O- or -NH-, and wherein m and k are each independently an integer from 1 to 12; for each occurrence, R F are independently: H, C1-C3 alkyl, -NH2, -NH-Fmoc, -NH-Boc, -NH-CBz, -NH-Troc, -NH-TFA, mono(C1-C3)alkylamino, di(C1-C3)alkylamino; -C(O)-R 103 , where R 103 is -H, -OH, or (C1-C3) alkyl; -SH, having the following structural formula Represents the part; or click chemistry reagent, provided that –R L -R F Not –OH.

[0011] In another embodiment, the present invention is a particle comprising a compound represented by the following structural formula: GLP,

[0012] Wherein: P is a biocompatible polymer. For example, the biocompatible polymer comprises at least one of polyglycolic acid, poly(lactic acid), poly(lactic-co-glycolic acid), polycaprolactone, poly(3-hydroxybutyric acid), poly(ethylene glycol), polyethylene oxide, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Pluronic F127), polyoxyethylene-polyoxypropylene block copolymer (Pluronic F68), poloxamer, poly(hydroxymethyl methacrylate), polyvinyl alcohol, poly(vinyl pyrrolidone), hyaluronic acid, heparin, heparin sulfate, polysialic acid, and chitosan; L is a covalent linker; and G is represented by the following structural formula: or a pharmaceutically acceptable salt thereof, wherein: Represents the attachment point to L.

[0013] In another embodiment, the present invention is a method of treating a condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula (S000-C) or a pharmaceutically acceptable salt thereof, or a composition comprising particles comprising a molecule represented by the following structural formula: GLP, or a pharmaceutically acceptable salt thereof, wherein G is represented by the structural formula (S000-P), wherein the disease is influenza.

[0014] In another exemplary embodiment, the present invention is any one of the compounds listed in Table 4 or a pharmaceutically acceptable salt thereof.

[0015] In another exemplary embodiment, the present invention is Figures 6B to 6F A compound represented by any one of the structural formulae listed in , or a pharmaceutically acceptable salt thereof.

[0016] In another embodiment, the present invention is a compound or particle described herein for use in therapy. For example, for use in treating diseases and conditions that respond to modulation (e.g., amplification, reduction, or elimination) of Siglec receptor activity. Such diseases and conditions include, but are not limited to, cancer, immune-related diseases and conditions, and inflammation-related diseases and conditions, such as those described herein.

[0017] In another embodiment, the present invention is a compound or particle as described herein for use in the preparation of a medicament for use in treating diseases and conditions responsive to modulation (e.g., amplification, reduction, or elimination) of Siglec receptor activity. Such diseases and conditions include, but are not limited to, cancer, immune-related diseases and conditions, and inflammation-related diseases and conditions, such as those described herein.

[0018] The compounds, particles, compositions and methods described herein can be used to treat the conditions described herein, providing additional therapies for many difficult to treat diseases and conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The foregoing will be apparent from the following more particular description of various exemplary embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters represent like parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the invention.

[0020] Figure 1 A and Figure 1 B is 2D of compounds 22 and 23 of Scheme 1-6 (corresponding to compounds 240 and 250 of Scheme 10-10) 1 H-NMR spectrum.

[0021] Figure 2 Graphs showing the results of following the reactants and products of Reaction Schemes 1-7 and Reaction Schemes 10-11 using hydrophilic interaction liquid chromatography / electrospray ionization mass spectrometry (HILIC-LC / ESI-MS).

[0022] Figures 3A to 3M is a bar graph showing the results of microarray screening of compounds described herein for binding to the indicated Siglecs and viral particles (the vertical axis corresponds to fluorescence intensity measured in relative fluorescence units (RFU)).

[0023] Figures 4A to 4E ] are sensorgrams generated by measuring the binding constants of certain compounds of the invention by biolayer interferometry (BLI), as described in Example 6.

[0024] Figure 5 is the HSQC (600 MHz) 2D NMR spectrum of S106 azide.

[0025] Figures 6A to 6F The structural formula of the polymer-ligand conjugate is shown.

[0026] Figure 7 is a graph showing the viability of THP-1 monocyte-derived macrophages in an MTT assay as a function of nanoparticle concentration.

[0027] Figure 8 is a bar graph showing the inhibition of TNF-α production in LPS-challenged THP-1 cells after incubation with the nanoparticles described herein.

[0028] Figure 9 is a bar graph showing the inhibition of IL-6 production in LPS-challenged THP-1 cells after incubation with the nanoparticles described herein.

[0029] Figure 10 is a bar graph showing the inhibition of VEGF production in LPS-challenged THP-1 cells after incubation with the nanoparticles described herein.

[0030] Figures 11(A) and 11(B) are bar graphs showing the effects of tested nanoparticle formulations on fibroblast differentiation.

[0031] Figure 12 is a graph showing the changes in ROS generation in neutrophils as a function of nanoparticle dose after 1 hour of PMA treatment.

[0032] 13(A), 13(B), and 13(C) are graphs of MPO (myeloperoxidase) levels in neutrophil supernatants at different time points after PMA treatment of neutrophils in the presence of the indicated nanoparticles.

[0033] Figures 14(A) and 14(B) are bar graphs showing quantification of LysoBrite-labeled BV-2 microglia by spectrofluorometry after treatment with the indicated nanoparticle formulations.

[0034] Figure 15 Schematic diagram of the cell surface binding assay of recombinant Siglec-9 Fc.

[0035] Figure 16Shown is flow cytometric analysis of Siglec-9 Fc binding to PANC-1 cells + / - neuraminidase.

[0036] Figure 17 Shown is flow cytometric analysis of Siglec-9 Fc binding to PANC-1 cells + / - PLGA-based nanoparticles.

[0037] Figure 18 Shown are the results of flow cytometric analysis of Siglec-9 Fc binding to PANC-1 cells + / - PLGA-based nanoparticles.

[0038] Figure 19 Is with FluoSpheres TM NeutrAvidin TM Schematic diagram of exemplary S-series ligands coupled to labeled microspheres.

[0039] Figure 20 Siglec-9 Fc and PANC-1 cells + / - FluoSpheres are shown TM Results of flow cytometric analysis of microsphere binding.

[0040] Figure 21 A schematic diagram of the FLISA setup described herein is shown.

[0041] Figure 22 is a bar graph showing the FluoSpheres TM Results of FLISA analysis of microsphere binding to immobilized Siglec-9 Fc.

[0042] Figure 23 is a schematic diagram of the binding assay setup using HEK293T cells expressing Siglec-9.

[0043] Figure 24 is a histogram showing the results of flow cytometric analysis of the binding of Siglec-9-PE to HEK293T cells expressing Siglec-9 + / - PLGA-based nanoparticles. DETAILED DESCRIPTION

[0044] A description of exemplary embodiments of the present invention follows.

[0045] The present disclosure provides methods and compositions for modulating the activity of autoassociated pattern recognition receptors, such as Siglec (sialic acid-binding immunoglobulin-type lectins). The compositions provided include, for example, nanoparticles, microparticles, and other polymer structures decorated with modified glycans that can bind to, agonize, or antagonize autoassociated molecular pattern recognition receptors and infection-associated sialic acid binding moieties (which allow entry, proliferation, and evasion of immune surveillance in the host).

[0046] Binding to such autoassociated pattern recognition receptors, and / or agonism or antagonism of the activity of such receptors can address innate, adaptive, multimodal, inflammatory, or complement-mediated immune responses, thereby providing treatment for the following diseases: (1) acute inflammation, such as viral, bacterial, allergenic, transplant rejection, or autoimmune-induced inflammation; (2) chronic inflammation, such as chronic obstructive pulmonary disease or rheumatic disorders; and (3) innate and adaptive forms of chronic non-resolving inflammation, such as exudative or non-exudative macular degeneration or Alzheimer's disease.

[0047] Provided compositions can also be used to block or antagonize self-associated molecular pattern recognition receptors that allow cancer cells, infectious agents such as viral, bacterial, helminthic, parasitic, or damage-associated molecular patterns (DAMPs) to evade immune surveillance, detection, and clearance by the innate or adaptive immune systems.

[0048] Activation of Siglec 3, 5, 7, 8, 9, 10, 11, or 15 will dephosphorylate all activated (phosphorylated) tyrosine kinases within a given cell, resulting in an intracellular shutdown of activation of that particular cell. The modified oligosaccharide ligands and the presentation of these ligands to specific Siglec receptors determine their ability to agonize, antagonize, or block the receptor binding site.

[0049] Antagonizing Siglec 14 or 16, which activate inflammation through the immunoglobulin tyrosine kinase activation motif (ITAM), is another mechanism for suppressing inflammation. When Siglec 14 or 16 is activated, the ITAM is activated, and the tyrosine residues within the ITAM are phosphorylated by the SRC kinase family, resulting in a conformational change that makes the motif a docking site for proteins containing SH2 domains. Activating Siglec 14 and 16 can also be used to activate inflammation to treat infectious diseases or in the field of oncology.

[0050] Antagonizing or blocking the binding sites of Siglec 3, 5, 7, 8, 9, 10, 11 or 15 is a method for treating conditions that mimic surface sialic acid ligands to stimulate Siglec to escape immune surveillance or immune activation through self-associated molecular patterns (SAMPs). Conditions for which this method can be used include cancer and infection. Cancer has been shown to express sialic acid structures on its surface to escape immune activation by macrophages, natural killer (NK) cells and monocytes. Group B Streptococcus also expresses sialic acid ligands on its surface that bind to Siglec 7 to escape immune attack.

[0051] Siglecs 9 has been extensively studied in the context of fibrocytes and fibrosis. Siglec-9 is one of the major Siglecs in human blood monocytes and macrophages and regulates innate immunity. Siglec-9 expression is increased in alveolar and peripheral blood neutrophils in patients with chronic obstructive pulmonary disease (COPD). These fibrocytes are unique cells that have the pro-inflammatory properties of macrophages and the tissue remodeling properties of fibroblasts. In addition to fibrosis, neutrophils (including in models of diabetic retinopathy) have also shown a role for Siglec 9.

[0052] Glycophorin A is a sialoglycoprotein on the surface of erythrocytes that inhibits NET release / neutrophil activation through sialic acid binding to circulating Siglec-9. The interaction of lactoferrin with polySia enhances the inhibition of NET release. Glycophorin A, the most abundant sialoglycoprotein on erythrocytes, binds to neutrophil Siglec-9, a sialic acid recognition receptor known to inhibit innate immune cell activation in models of lung inflammation. The nanoparticles disclosed herein have been shown to effectively inhibit key pathways involved in the pathogenesis of Siglec 9-mediated diseases.

[0053] Sialic acid is also used as an entry point for several virus families, such as influenza A, influenza B, influenza C, SARS-CoV-1 or SARS-CoV-2. The binding receptors for these viruses can be hemagglutinin esterase (viral HE), neuraminidase (viral N) or parts of the viral capsid, such as the spike protein (viral SP), which binds to sialic acid ligands on the host cell surface and facilitates viral entry into the host cell, or CD147, a sialic acid-binding lectin used for infectious entry of SARS-CoV-2 and Plasmodium falciparum. Binding of these sialic acid receptors to decoy ligands prevents the virus from infecting the host cell and prevents the escape of viral particles from the infected cell.

[0054] There is a need for improved compositions and methods for stimulating sialic acid-binding autoassociated pattern recognition receptors to treat diseases caused by acute, chronic, or abnormal immune system activation. There is also a need to prevent these SAMP receptors from being occupied by cancers and infections to evade immune surveillance and avoid attack. The present invention provides nanoparticles capable of presenting ligands that will specifically and profoundly stimulate, block, or antagonize specific Siglec receptors.

[0055] Definitions of specific functional groups and chemical terms are described in more detail below.The chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th ed., John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd ed., Cambridge University Press, Cambridge, 1987.

[0056] The compounds described herein may contain one or more asymmetric centers and, therefore, may exist in various stereoisomeric forms (e.g., enantiomers and / or diastereomers). For example, the compounds described herein may be in the form of a single enantiomer, diastereomer, or geometric isomer, or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis. (See, e.g., Jacques et al., Enantiomers, Racemates and Resolutions, Wiley Interscience, New York, 1981; Wilen et al., Tetrahedron 33:2725 (1977); Eliel, EL Stereochemistry of Carbon Compounds, McGraw-Hill, NY, 1962; and Wilen, SH, Tables of Resolving Agents and Optical Resolutions, p. 268, EL Eliel, ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The invention additionally encompasses the compounds as individual isomers substantially free of other isomers, and alternatively as mixtures of various isomers.

[0057] In the formula, is a single bond, wherein the stereochemistry of the moiety directly attached thereto is not specified, --- is absent or is a single bond, and or Is a single bond or a double bond.

[0058] When a range of values ​​is listed, it is intended that every value and subrange within that range be included. For example, “C 1-6 "Alkyl" is intended to encompass C1, C2, C3, C4, C5, C6, C 1-6 、C 1-5 、C 1-4 、C 1-3 、C 1-2 、C 2-6 、C 2-5 、C 2-4 、C 2-3 、C3-6 、C 3-5 、C 3-4 、C 4-6 、C 4-5 and C 5-6 alkyl.

[0059] The term "alkyl" refers to a straight or branched chain saturated hydrocarbon radical having a specified range of carbon atoms (e.g., "C 1-16 An "alkyl" group can have from 1 to 16 carbon atoms. In some embodiments, an alkyl group has from 1 to 9 carbon atoms ("C 1-9 In some embodiments, an alkyl group has 1 to 8 carbon atoms ("C 1-8 In some embodiments, an alkyl group has 1 to 7 carbon atoms ("C 1-7 In some embodiments, an alkyl group has 1 to 6 carbon atoms ("C 1-6 In some embodiments, an alkyl group has 1 to 5 carbon atoms ("C 1-5 In some embodiments, an alkyl group has 1 to 4 carbon atoms ("C 1-4 In some embodiments, an alkyl group has 1 to 3 carbon atoms ("C 1-3 In some embodiments, an alkyl group has 1 to 2 carbon atoms ("C 1-2 In some embodiments, an alkyl group has 1 carbon atom ("C1 alkyl"). In some embodiments, an alkyl group has 2 to 6 carbon atoms ("C 2-6 C 1-6 Examples of alkyl groups include methyl (C1), ethyl (C2), propyl (C3) (e.g., n-propyl, isopropyl), butyl (C4) (e.g., n-butyl, tert-butyl, sec-butyl, isobutyl), pentyl (C5) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tert-pentyl alkyl), and hexyl (C6) (e.g., n-hexyl). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8), etc. Unless otherwise specified, each example of an alkyl group is independently unsubstituted ("unsubstituted alkyl") or substituted ("substituted alkyl") with one or more substituents (e.g., halogen, e.g., F). In certain embodiments, an alkyl group is an unsubstituted C 1-10 Alkyl (e.g. unsubstituted C 1-6Alkyl, for example -CH3(Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, for example, unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, for example, unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl (sec-Bu), unsubstituted isobutyl (i-Bu)). In certain embodiments, the alkyl group is substituted C 1-10 Alkyl (e.g. substituted C 1-6 Alkyl, such as -CF3, Bn).

[0060] The term "alkylene" refers to a divalent group of a linear, cyclic, or branched saturated hydrocarbon radical having a specified range of carbon atoms (e.g., "C 1-16 An "alkyl" group may have from 1 to 16 carbon atoms. An example of an alkylene group is a methylene group (-CH2-). An alkylene group may be substituted as described above for an alkyl group.

[0061] The term "haloalkyl" is a substituted alkyl group in which one or more hydrogen atoms are independently replaced by a halogen, such as fluorine, bromine, chlorine, or iodine. In some embodiments, the haloalkyl moiety has 1 to 8 carbon atoms ("C 1-8 In some embodiments, the haloalkyl moiety has 1 to 6 carbon atoms ("C 1-6 In some embodiments, the haloalkyl moiety has 1 to 4 carbon atoms ("C 1-4 In some embodiments, the haloalkyl moiety has 1 to 3 carbon atoms ("C 1-3 In some embodiments, the haloalkyl moiety has 1 to 2 carbon atoms ("C 1-2 Examples of haloalkyl groups include -CHF2, -CH2F, -CF3, -CH2CF3, -CF2CF3, -CF2CF2CF3, -CCl3, -CFCl2, -CF2Cl, and the like.

[0062] The term "hydroxyalkyl" is a substituted alkyl group in which one or more hydrogen atoms are independently replaced by a hydroxy group. In some embodiments, the hydroxyalkyl moiety has 1 to 8 carbon atoms ("C 1-8 In some embodiments, the hydroxyalkyl moiety has 1 to 6 carbon atoms ("C 1-6 In some embodiments, the hydroxyalkyl moiety has 1 to 4 carbon atoms ("C 1-4 In some embodiments, the hydroxyalkyl moiety has 1 to 3 carbon atoms ("C 1-3 In some embodiments, the hydroxyalkyl moiety has 1 to 2 carbon atoms ("C1-2 hydroxyalkyl").

[0063] The term "alkoxy" refers to an alkyl group, as defined herein, attached to the parent molecular moiety through an oxygen atom. In some embodiments, the alkoxy moiety has 1 to 8 carbon atoms ("C 1-8 In some embodiments, the alkoxy moiety has 1 to 6 carbon atoms ("C 1-6 In some embodiments, the alkoxy moiety has 1 to 4 carbon atoms ("C 1-4 In some embodiments, the alkoxy moiety has 1 to 3 carbon atoms ("C 1-3 Alkoxy”).

[0064] In some embodiments, the alkoxy moiety has 1 to 2 carbon atoms ("C 1-2 "alkoxy"). Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0065] The term "haloalkoxy" refers to a haloalkyl group, as defined herein, attached to the parent molecular moiety through an oxygen atom. In some embodiments, the alkoxy moiety has 1 to 8 carbon atoms ("C 1-8 In some embodiments, the alkoxy moiety has 1 to 6 carbon atoms ("C 1-6 In some embodiments, the alkoxy moiety has 1 to 4 carbon atoms ("C 1-4 In some embodiments, the alkoxy moiety has 1 to 3 carbon atoms ("C 1-3 In some embodiments, the alkoxy moiety has 1 to 2 carbon atoms ("C 1-2 "haloalkoxy"). Representative examples of haloalkoxy include, but are not limited to, difluoromethoxy, trifluoromethoxy, and 2,2,2-trifluoroethoxy.

[0066] The term "alkoxyalkyl" is a substituted alkyl group in which one or more hydrogen atoms are independently replaced by an alkoxy group as defined herein. In some embodiments, the alkoxyalkyl moiety has 1 to 8 carbon atoms ("C 1-8 In some embodiments, the alkoxyalkyl moiety has 1 to 6 carbon atoms ("C 1-6 In some embodiments, the alkoxyalkyl moiety has 1 to 4 carbon atoms ("C 1-4 In some embodiments, the alkoxyalkyl moiety has 1 to 3 carbon atoms ("C 1-3 In some embodiments, the alkoxyalkyl moiety has 1 to 2 carbon atoms ("C1-2 (alkoxyalkyl").

[0067] The term "heteroalkyl" refers to an alkyl group that further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within one or more terminal positions (i.e., inserted between adjacent carbon atoms) and / or located at one or more terminal positions of the parent chain. In certain embodiments, a heteroalkyl group refers to a saturated group ("heteroalkyl") having from 1 to 20 carbon atoms and 1 or more heteroatoms within the parent chain. 1-20 In some embodiments, a heteroalkyl group is a saturated group having 1 to 18 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC 1-18 In some embodiments, a heteroalkyl group is a saturated group having 1 to 16 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC 1-16 In some embodiments, a heteroalkyl group is a saturated group having 1 to 14 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC 1-14 In some embodiments, a heteroalkyl group is a saturated group having 1 to 12 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC 1-12 In some embodiments, a heteroalkyl group is a saturated group having 1 to 10 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroalkyl”). 1-10 In some embodiments, a heteroalkyl group is a saturated group having 1 to 8 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC 1-8 In some embodiments, a heteroalkyl group is a saturated group having 1 to 6 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC 1-6 In some embodiments, a heteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC 1-4 In some embodiments, a heteroalkyl group is a saturated group having 1 to 3 carbon atoms and 1 heteroatom within the parent chain (“heteroC 1-3 In some embodiments, a heteroalkyl group is a saturated group having 1 to 2 carbon atoms and 1 heteroatom within the parent chain (“heteroC 1-2In some embodiments, a heteroalkyl group is a saturated group having 1 carbon atom and 1 heteroatom (a "heteroC1 alkyl"). In some embodiments, a heteroalkyl group as defined herein is a partially unsaturated group having 1 or more heteroatoms and at least one unsaturated carbon (e.g., a carbonyl group) within the parent chain. For example, a heteroalkyl group may contain an amide or ester functional group within its parent chain such that one or more carbon atoms are unsaturated carbonyl groups. Unless otherwise specified, each instance of a heteroalkyl group is independently unsubstituted (an "unsubstituted heteroalkyl") or substituted (a "substituted heteroalkyl") with one or more substituents. In certain embodiments, a heteroalkyl group is an unsubstituted heteroC1 1-20 In certain embodiments, the heteroalkyl group is an unsubstituted heteroC 1-10 In certain embodiments, the heteroalkyl group is a substituted heteroC 1-20 In certain embodiments, the heteroalkyl group is an unsubstituted heteroC 1-10 alkyl.

[0068] The term "alkenyl" refers to a straight or branched chain hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In some embodiments, the alkenyl group has 2 to 9 carbon atoms ("C 2-9 In some embodiments, an alkenyl group has 2 to 8 carbon atoms ("C 2-8 In some embodiments, an alkenyl group has 2 to 7 carbon atoms ("C 2-7 In some embodiments, an alkenyl group has 2 to 6 carbon atoms ("C 2-6 In some embodiments, an alkenyl group has 2 to 5 carbon atoms ("C 2-5 In some embodiments, an alkenyl group has 2 to 4 carbon atoms ("C 2-4 In some embodiments, an alkenyl group has 2 to 3 carbon atoms ("C 2-3 In some embodiments, an alkenyl group has 2 carbon atoms.

[0069] ("C2 alkenyl"). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). C 2-4 Examples of alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. 2-6 Examples of alkenyl groups include the above-mentioned C 2-4Examples of alkenyl groups include pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl groups include heptenyl (C7), octenyl (C8), octatrienyl (C8), and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents. In certain embodiments, an alkenyl group is an unsubstituted C 2-10 In certain embodiments, an alkenyl group is a substituted C 2-10 In an alkenyl group, a C=C double bond with unspecified stereochemistry (e.g., -CH=CHCH3 or ) can be an (E)- or (Z)-double bond.

[0070] The term "heteroalkenyl" refers to an alkenyl group that further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within one or more terminal positions (i.e., inserted between adjacent carbon atoms) and / or located at one or more terminal positions of the parent chain. In certain embodiments, a heteroalkenyl group refers to a group having from 2 to 10 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain ("heteroalkenyl"). 2-10 In some embodiments, a heteroalkenyl group has 2 to 9 carbon atoms, at least one double bond, and 1 or more heteroatoms (“heteroC 2-9 (e.g., alkenyl)

[0071] In some embodiments, a heteroalkenyl group has 2 to 8 carbon atoms, at least one double bond, and 1 or more heteroatoms (“heteroC 2-8 In some embodiments, a heteroalkenyl group has 2 to 7 carbon atoms, at least one double bond, and 1 or more heteroatoms (“heteroC 2-7 In some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and 1 or more heteroatoms (“heteroC 2-6 In some embodiments, a heteroalkenyl group has 2 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms (“heteroC 2-5 In some embodiments, a heteroalkenyl group has 2 to 4 carbon atoms, at least one double bond, and 1 or 2 heteroatoms (“heteroC 2-4 In some embodiments, a heteroalkenyl group has 2 to 3 carbon atoms, at least one double bond, and 1 heteroatom (“heteroC 2-3 In some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatoms (“heteroC 2-6Unless otherwise specified, each instance of a heteroalkenyl group is independently unsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a “substituted heteroalkenyl”) with one or more substituents. In certain embodiments, a heteroalkenyl group is an unsubstituted heteroC 2-10 In certain embodiments, a heteroalkenyl group is a substituted heteroC 2-10 Alkenyl.

[0072] The term "alkynyl" refers to a straight or branched chain hydrocarbon group having from 2 to 10 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) ("C2-10 alkynyl"). In some embodiments, the alkynyl group has 2 to 9 carbon atoms ("C2-10 alkynyl"). 2-9 In some embodiments, an alkynyl group has 2 to 8 carbon atoms ("C 2-8 In some embodiments, an alkynyl group has 2 to 7 carbon atoms ("C 2-7 In some embodiments, an alkynyl group has 2 to 6 carbon atoms ("C 2-6 In some embodiments, an alkynyl group has 2 to 5 carbon atoms ("C 2-5 In some embodiments, an alkynyl group has 2 to 4 carbon atoms ("C 2-4 In some embodiments, an alkynyl group has 2 to 3 carbon atoms ("C 2-3 In some embodiments, an alkynyl group has 2 carbon atoms ("C2 alkynyl"). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). C 2_4 Examples of alkynyl groups include, but are not limited to, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. 2-6 Examples of alkenyl groups include the above-mentioned C 2-4 Examples of alkynyl groups include pentynyl (C5), hexynyl (C6), and the like. Additional examples of alkynyl groups include heptynyl (C7), octynyl (C8), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents. In certain embodiments, an alkynyl group is an unsubstituted C 2-10 In certain embodiments, the alkynyl group is a substituted C 2-10 Alkynyl.

[0073] The term "heteroalkynyl" refers to an alkynyl group that further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within one or more terminal positions (i.e., inserted between adjacent carbon atoms) and / or located at one or more terminal positions of the parent chain. In certain embodiments, a heteroalkynyl group refers to a group having from 2 to 10 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain ("heteroalkynyl"). 2-10 In some embodiments, a heteroalkynyl group has 2 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatoms (“heteroC 2-9 In some embodiments, a heteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond, and 1 or more heteroatoms (“heteroC 2-8 In some embodiments, a heteroalkynyl group has 2 to 7 carbon atoms, at least one triple bond, and 1 or more heteroatoms (“heteroC 2-7 In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or more heteroatoms (“heteroC 2-6 In some embodiments, a heteroalkynyl group has 2 to 5 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms (“heteroC 2-5 In some embodiments, a heteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms (“heteroC 2-4 In some embodiments, a heteroalkynyl group has 2 to 3 carbon atoms, at least one triple bond, and 1 heteroatom (“heteroC 2-3 In some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms (“heteroC 2-6 Unless otherwise specified, each instance of a heteroalkynyl group is independently unsubstituted (an “unsubstituted heteroalkynyl”) or substituted (a “substituted heteroalkynyl”) with one or more substituents. In certain embodiments, a heteroalkynyl group is an unsubstituted heteroalkynyl. 2-10 In certain embodiments, a heteroalkynyl group is a substituted heteroC 2-10 Alkynyl.

[0074] The term "carbocyclyl" or "carbocycle" refers to a non-aromatic cyclic hydrocarbon radical having from 3 to 14 ring carbon atoms ("C 3-14 In some embodiments, the carbocyclyl group has 3 to 10 ring carbon atoms ("C 3-10 In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms ("C3-8 In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms ("C 3-7 In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms ("C 3-6 In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms ("C 4-6 In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms ("C 5-6 In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms ("C 5-10 Carbocyclyl”). Exemplary C 3-6 Carbocyclyl groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like.

[0075] Example C 3–8 Carbocyclyl groups include but are not limited to the above C 3–6 Carbocyclyl groups and cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo [2.2.1] heptyl (C7), bicyclo [2.2.2] octyl (C8), etc. Exemplary C 3-10 Carbocyclyl groups include but are not limited to the above C 3-8 Carbocyclyl groups and cyclononyl (C9), cyclononenyl

[0076] (C9), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthyl (C 10 ), spiro[4.5]decyl (C 10 ) and the like. As shown in the foregoing examples, in certain embodiments, the carbocyclyl group is monocyclic ("monocyclic carbocyclyl") or polycyclic (e.g., containing a fused, bridged, or spirocyclic ring system such as a bicyclic system ("bicyclic carbocyclyl") or a tricyclic system ("tricyclic carbocyclyl")) and may be saturated or may contain one or more carbon-carbon double bonds or triple bonds. "Carbocyclyl" also includes a ring system in which the carbocyclyl ring as defined above is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the carbocyclyl ring, and in such a case, the number of carbons continues to indicate the number of carbons in the carbocyclyl system. Unless otherwise stated, each instance of a carbocyclyl group is independently unsubstituted ("unsubstituted carbocyclyl") or substituted ("substituted carbocyclyl") by one or more substituents. In certain embodiments, a carbocyclyl group is an unsubstituted C 3-14In certain embodiments, a carbocyclyl group is a substituted C 3-14 Carbocyclic group.

[0077] In some embodiments, "carbocyclyl" is a monocyclic saturated carbocyclyl group ("C 3-14 In some embodiments, a cycloalkyl group has 3 to 10 ring carbon atoms ("C 3-10 In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms ("C 3-8 In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms ("C 3-6 In some embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms ("C 4-6 In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms ("C 5-6 In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms ("C 5-10 Cycloalkyl”). C 5-6 Examples of cycloalkyl groups include cyclopentyl (C5) and cyclohexyl (C6). 3-6 Examples of the cycloalkyl group include the above-mentioned C 5-6 Cycloalkyl groups as well as cyclopropyl (C3) and cyclobutyl (C4). 3-8 Examples of the cycloalkyl group include the above-mentioned C 3-6 Cycloalkyl groups and cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an "unsubstituted cycloalkyl") or substituted (a "substituted cycloalkyl") with one or more substituents. In certain embodiments, a cycloalkyl group is an unsubstituted C 3-14 In certain embodiments, the cycloalkyl group is a substituted C 3-14 Cycloalkyl.

[0078] As used herein, the term "heterocyclyl" refers to an aromatic (also known as heteroaryl), unsaturated or saturated cyclic hydrocarbon group that includes at least one heteroatom in the ring.

[0079] For example, the term "heterocyclyl" or "heterocycle" refers to a radical of a 3- to 14-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("3-14 membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom when valence permits. Heterocyclyl groups can be monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., fused, bridged, or spiro ring systems such as bicyclic systems ("bicyclic heterocyclyl") or tricyclic systems ("tricyclic heterocyclyl")), and can be saturated or can contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings. "Heterocyclyl" also includes a ring system in which a heterocyclyl ring as defined above is fused to one or more carbocyclyl groups (wherein the point of attachment is on the carbocyclyl or heterocyclyl ring), or a ring system in which a heterocyclyl ring as defined above is fused to one or more aryl or heteroaryl groups (wherein the point of attachment is on the heterocyclyl ring), and in such cases, the number of ring members continues to indicate the number of ring members in the heterocyclyl ring system. Unless otherwise indicated, each instance of a heterocyclyl is independently unsubstituted ("unsubstituted heterocyclyl") or substituted ("substituted heterocyclyl") with one or more substituents. In certain embodiments, a heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, a heterocyclyl group is a substituted 3-14 membered heterocyclyl.

[0080] In some embodiments, the heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In some embodiments, the heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In some embodiments, the heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0081] Exemplary 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to, aziridinyl, oxiranyl, and thiirane. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thiirane. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrophenylthio, dihydrophenylthio, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxathiolanyl, and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing 3 heteroatoms include, but are not limited to, triazinyl. Exemplary 7-membered heterocyclyl groups containing 1 heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include, but are not limited to, azacyclooctanyl, oxecanyl, and thiocanyl. Exemplary bicyclic heterocyclyl groups include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothiophenyl, tetrahydrobenzothiophenyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, decahydroisoquinolyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthylimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepine yl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl, 1,2,3,4-tetrahydro-1,6-naphthyridinyl and the like.

[0082] The term "aryl" refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., sharing 6, 10, or 14 π electrons in the cyclic array) provided with 6-14 ring carbon atoms and zero heteroatoms ("C 6-14 In some embodiments, an aryl group has 6 ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C 10 "Aryl"; for example, naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms ("C 14 "Aryl"; e.g., anthracenyl). "Aryl" also includes ring systems in which the aryl ring, as defined above, is fused to one or more carbocyclyl or heterocyclyl groups, wherein the radical or point of attachment is on the aryl ring, and in such cases, the number of carbon atoms continues to indicate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an "unsubstituted aryl") or substituted (a "substituted aryl") with one or more substituents. In certain embodiments, an aryl group is an unsubstituted C 6-14 In certain embodiments, the aryl group is a substituted C 6-14 Aryl.

[0083] "Aralkyl" is a subset of "alkyl" and refers to an alkyl group substituted with an aryl group, where the point of attachment is on the alkyl portion.

[0084] The term "heteroaryl" refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., sharing 6, 10, or 14 π electrons in a cyclic array) provided with ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-14 membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom when valence permits. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. "Heteroaryl" includes ring systems in which the heteroaryl ring as defined above is fused to one or more carbocyclyl or heterocyclyl groups, wherein the point of attachment is on the heteroaryl ring, and in such cases, the number of ring members continues to indicate the number of ring members in the heteroaryl ring system. "Heteroaryl" also includes ring systems in which the heteroaryl ring as defined above is fused to one or more aryl groups, wherein the point of attachment is on the aryl or heteroaryl ring, and in such cases, the number of ring members indicates the number of ring members in the fused polycyclic (aryl / heteroaryl) ring system. For polycyclic heteroaryl groups in which one ring contains no heteroatoms (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be on either ring, i.e., the ring bearing the heteroatom (e.g., 2-indolyl) or the ring containing no heteroatoms (e.g., 5-indolyl).

[0085] In some embodiments, the heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In some embodiments, the heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, the heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl group has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl group has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, a 5-6 membered heteroaryl group has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted (an "unsubstituted heteroaryl") or substituted (a "substituted heteroaryl") with one or more substituents. In certain embodiments, a heteroaryl group is an unsubstituted 5-14 membered heteroaryl group. In certain embodiments, a heteroaryl group is a substituted 5-14 membered heteroaryl group.

[0086] Exemplary 5-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, aza Oxalic acid thia Examples of 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indolizinyl, and purinyl. Examples of 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Examples of tricyclic heteroaryl groups include, but are not limited to, phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.

[0087] "Heteroaralkyl" is a subset of "alkyl" and refers to an alkyl group substituted with a heteroaryl group, where the point of attachment is on the alkyl portion.

[0088] The prefix "ene" is attached to a group to indicate that the group is a divalent moiety, for example, alkylene is a divalent moiety of alkyl, alkenylene is a divalent moiety of alkenyl, alkynylene is a divalent moiety of alkynyl, heteroalkylene is a divalent moiety of heteroalkyl, heteroalkenylene is a divalent moiety of heteroalkenyl, heteroalkynylene is a divalent moiety of heteroalkynyl, carbocyclylene is a divalent moiety of carbocyclyl, heterocyclylene is a divalent moiety of heterocyclyl, arylene is a divalent moiety of aryl, and heteroarylene is a divalent moiety of heteroaryl.

[0089] Unless otherwise expressly provided, group is optionally substituted.Term " optionally substituted " refers to substituted or unsubstituted.In certain embodiments, alkyl, alkenyl, alkynyl, assorted alkyl, assorted alkenyl, assorted alkynyl, carbocyclic radical, heterocyclic radical, aryl and heteroaryl group are optionally substituted. " optionally substituted " refers to can be substituted or unsubstituted group (for example, " substituted " or " unsubstituted " alkyl, " substituted " or " unsubstituted " alkenyl, " substituted " or " unsubstituted " alkynyl, " substituted " or " unsubstituted " assorted alkyl, " substituted " or " unsubstituted " assorted alkenyl, " substituted " or " unsubstituted " alkynyl, " substituted " or " unsubstituted " assorted alkenyl, " substituted " or " unsubstituted " assorted alkynyl, " substituted " or " unsubstituted " carbocyclic radical, " substituted " or " unsubstituted " heterocyclic radical, " substituted " or " unsubstituted " aryl or " substituted " or " unsubstituted " heteroaryl group). In general, the term "substituted" means that at least one hydrogen present on a group is replaced by a permitted substituent, for example, a substituent that produces a stable compound when substituted, such as a compound that does not spontaneously undergo transformation (e.g., by rearrangement, cyclization, elimination or other reactions). Unless otherwise stated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is the same or different at each position. The term "substituted" is intended to include substitution by all permissible substituents of an organic compound, including any substituent described herein that causes the formation of a stable compound. The present invention encompasses any and all such combinations to achieve a stable compound. For the purposes of the present invention, a heteroatom such as nitrogen may have a hydrogen substituent and / or any suitable substituent as described herein that satisfies the valence of the heteroatom and results in the formation of a stable moiety. The present invention is not intended to be limited in any way by the exemplary substituents described herein.

[0090] Exemplary carbon atom substituents include, but are not limited to, halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR aa 、-ON(R bb )2、-N(R bb )2、-N(R bb )3 + X - 、-N(OR cc )R bb 、-SH、-SR aa 、-SSR cc 、-C(=O)R aa 、-CO2H、-CHO、-C(OR cc )3. -CO2R aa 、-OC(=O)R aa 、-OCO2R aa 、-C(=O)N(Rbb )2、-OC(=O)N(R bb )2、-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-OC(=NR bb )N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-C(=O)NR bb SO2R aa 、-NR bb SO2R aa 、-SO2N(R bb )2、-SO2R aa 、-SO2OR aa 、-OSO2R aa 、-S(=O)R aa 、-OS(=O)R aa 、-Si(R aa )3、-OSi(R aa )3、-C(=S)N(R bb )2、-C(=O)SR aa 、-C(=S)SR aa 、-SC(=S)SR aa 、-SC(=O)SR aa 、-OC(=O)SR aa 、-SC(=O)OR aa 、-SC(=O)R aa 、-P(=O)(R aa )2、-P(=O)(OR cc )2、-OP(=O)(R aa )2、-OP(=O)(OR cc )2、-P(=O)(N(R bb )2)2、-OP(=O)(N(R bb)2)2、-NR bb P(=O)(R aa )2、-NR bb P(=O)(OR cc )2、-NR bb P(=O)(N(R bb )2)2、-P(R cc )2、-P(OR cc )2、-P(R cc )3 + X – 、-P(OR cc )3 + X – 、-P(R cc )4、-P(OR cc )2、-OP(R cc )2、-OP(R cc )3 + X – 、-OP(OR cc )2、-OP(OR cc )3 + X – 、-OP(R cc )4、-OP(OR cc )4、-B(R aa )2、-B(OR cc )2, -BR aa (OR cc ), C 1-10 Alkyl, C 1-10 Perhalogenated alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, hetero C 1-10 Alkyl, hetero C 2-10 Alkenyl, hetero C 2-10 Alkynyl, C 3-10 Carbocyclic group, 3-14 membered heterocyclic group, C 6-14 aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently replaced by 0, 1, 2, 3, 4, or 5 R dd Group substitution; wherein X – is a counterion; or the two geminal hydrogens on the carbon atom are substituted by groups =O, =S, =NN(R bb )2, =NNR bb C(=O)R aa 、=NNR bb C(=O)OR aa 、=NNR bb S(=O)2R aa 、=NRbb or = NOR cc Replace; R aa Each instance of is independently selected from C 1-10 Alkyl, C 1-10 Halogenated alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, hetero C 1-10 Alkyl, hetero C 2-10 Alkenyl, hetero C 2-10 Alkynyl, C 3-10 Carbocyclic group, 3-14 membered heterocyclic group, C 6-14 Aryl, and 5-14 membered heteroaryl, or two R aa The groups are connected to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4, or 5 R dd Group substitution; R bb Each instance of is independently selected from hydrogen, -OH, -OR aa 、-N(R cc )2, -CN, -C(=O)R aa 、-C(=O)N(R cc )2, -CO2R aa 、-SO2R aa 、-C(=NR cc )OR aa 、-C(=NR cc )N(R cc )2、-SO2N(R cc )2, -SO2R cc 、-SO2OR cc 、-SOR aa 、-C(=S)N(R cc )2, -C(=O)SR cc 、-C(=S)SR cc 、-P(=O)(R aa )2、-P(=O)(OR cc )2、-P(=O)(N(R cc )2)2、C 1-10 Alkyl, C 1-10 Perhalogenated alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, hetero C 1-10 Alkyl, hetero C 2-10 Alkenyl, hetero C 2-10 Alkynyl, C 3-10 Carbocyclic group, 3-14 membered heterocyclic group, C 6-14 aryl, and 5-14 membered heteroaryl, or two Rbb The groups are connected to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4, or 5 R dd Group substitution; wherein X – is the counterion; R cc Each instance of is independently selected from hydrogen, C 1-10 Alkyl, C 1-10 Perhalogenated alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, hetero C 1-10 Alkyl, hetero C 2-10 Alkenyl, hetero C 2-10 Alkynyl, C 3-10 Carbocyclic group, 3-14 membered heterocyclic group, C 6-14 aryl, and 5-14 membered heteroaryl, or two R cc The groups are connected to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4, or 5 R dd Group substitution; R dd Each instance of is independently selected from halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OR ee 、-ON(R ff )2、-N(R ff )2、-N(R ff )3 + X – 、-N(OR ee )R ff 、-SH、-SR ee 、-SSR ee 、-C(=O)R ee 、-CO2H、-CO2R ee 、-OC(=O)R ee 、-OCO2R ee 、-C(=O)N(R ff )2、-OC(=O)N(R ff )2、-NR ff C(=O)R ee 、-NR ff CO2R ee 、-NR ff C(=O)N(R ff )2、-C(=NR ff )OR ee 、-OC(=NR ff )Ree 、-OC(=NR ff )OR ee 、-C(=NR ff )N(R ff )2、-OC(=NR ff )N(R ff )2、-NR ff C(=NR ff )N(R ff )2、-NR ff SO2R ee 、-SO2N(R ff )2, -SO2R ee 、-SO2OR ee 、-OSO2R ee 、-S(=O)R ee 、-Si(R ee )3、-OSi(R ee )3、-C(=S)N(R ff )2, -C(=O)SR ee 、-C(=S)SR ee 、-SC(=S)SR ee 、-P(=O)(OR ee )2、-P(=O)(R ee )2、-OP(=O)(R ee )2、-OP(=O)(OR ee )2、C 1-6 Alkyl, C 1-6 Perhalogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, hetero C 1-6 Alkyl, hetero C 2-6 Alkenyl, hetero C 2-6 Alkynyl, C 3-10 Carbocyclic group, 3-10 membered heterocyclic group, C 6-10 Aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl are independently replaced by 0, 1, 2, 3, 4, or 5 R gg Group substitution, or two geminal R dd Substituents may be linked to form =O or =S; where X – is the counterion; R ee Each instance of is independently selected from C 1-6 Alkyl, C 1-6 Perhalogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, hetero C 1-6 Alkyl, hetero C 2-6 Alkenyl, hetero C2-6 Alkynyl, C 3-10 Carbocyclic group, C 6-10 aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently replaced by 0, 1, 2, 3, 4, or 5 R gg Group substitution; R ff Each instance of is independently selected from hydrogen, C 1-6 Alkyl, C 1-6 Perhalogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, hetero C 1-6 Alkyl, hetero C 2-6 Alkenyl, hetero C 2-6 Alkynyl, C 3-10 Carbocyclic group, 3-10 membered heterocyclic group, C 6-10 Aryl and 5-10 membered heteroaryl, or two R ff The groups are connected to form a 3-10 membered heterocyclyl or 5-10 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4, or 5 R gg and R gg Each instance of is independently halogen, -CN, -NO2, -N3, -SO2H, -SO3H, -OH, -OC 1-6 Alkyl, -ON(C 1-6 Alkyl)2, -N(C l-6 Alkyl)2, -N(C l-6 Alkyl)3 + X – 、-NH(C l-6 Alkyl)2 + X – 、-NH2(C 1-6 alkyl) + X – 、-NH3 + X – 、-N(OC 1-6 Alkyl)(C l-6 Alkyl), -N(OH)(C l-6 Alkyl), -NH(OH), -SH, -SC 1-6 Alkyl, -SS(C l-6 alkyl), -C(=O)(C l-6 alkyl), -CO2H, -CO2(C 1-6 alkyl), -OC(=O)(C l-6 Alkyl), -OCO2(C 1-6alkyl), -C(=O)NH2, -C(=O)N(C 1-6 alkyl)2, -OC(=O)NH(C 1-6 alkyl), -NHC(=O)(C l-6 Alkyl), -N(C l-6 alkyl)C(=O)(C 1-6 Alkyl), -NHCO2(C 1-6 alkyl), -NHC(=O)N(C l-6 alkyl)2, -NHC(=O)NH(C l-6 alkyl), -NHC(=O)NH2, -C(=NH)O(C l-6 alkyl), -OC(=NH)(C l-6 alkyl), -OC(=NH)OC l-6 Alkyl, -C(=NH)N(C l-6 Alkyl)2, -C(=NH)NH(C l-6 alkyl), -C(=NH)NH2, -OC(=NH)N(C 1-6 Alkyl)2, -OC(=NH)NH(C 1-6 alkyl), -OC(=NH)NH2, -NHC(=NH)N(C 1-6 Alkyl)2, -NHC(=NH)NH2, -NHSO2(C 1-6 Alkyl), -SO2N(C 1-6 Alkyl)2, -SO2NH(C 1-6 alkyl), -SO2NH2, -SO2(C 1-6 Alkyl), -SO2O(C 1-6 Alkyl), -OSO2(C 1-6 alkyl), -SO(C 1-6 Alkyl), -Si(C l-6 alkyl)3, -OSi(C l-6 alkyl)3, -C(=S)N(C l-6 Alkyl)2, -C(=S)NH(C l-6 alkyl), -C(=S)NH2, -C(=O)S(C l-6 alkyl), -C(=S)SC 1-6 Alkyl, -SC(=S)SC 1-6 Alkyl, -P(=O)(OC 1-6 alkyl)2, -P(=O)(C 1-6 alkyl)2, -OP(=O)(C l-6 alkyl)2, -OP(=O)(OC l-6 Alkyl)2, C 1-6 Alkyl, C 1-6Perhalogenated alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, hetero C 1-6 Alkyl, hetero C 2-6 Alkenyl, hetero C 2-6 Alkynyl, C 3-10 Carbocyclic group, C 6-10 Aryl, 3-10 membered heterocyclic group, 5-10 membered heteroaryl; or two geminal R gg Substituents may be linked to form =O or =S; where X – It is a counter ion.

[0091] The term "halo" or "halogen" refers to fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br), or iodine (iodo, -I).

[0092] The term "hydroxyl" or "hydroxy" refers to the group -OH. By extension, the term "substituted hydroxyl" or "substituted hydroxy" refers to a hydroxy group in which the oxygen atom directly attached to the parent molecule is replaced by a group other than hydrogen, and includes groups selected from: -OR aa 、-ON(R bb )2, -OC(=O)SR aa 、-OC(=O)R aa 、-OCO2R aa 、-OC(=O)N(R bb )2、-OC(=NR bb )R aa 、-OC(=NR bb )OR aa 、-OC(=NR bb )N(R bb )2, -OS(=O)R aa 、-OSO2R aa 、-OSi(R aa )3、-OP(R cc )2、-OP(R cc )3 + X – 、-OP(OR cc )2、-OP(OR cc )3 + X – 、-OP(=O)(R aa )2、-OP(=O)(OR cc )2 and -OP(=O)(N(R bb )2)2, where X – 、R aa 、Rbb and R cc is as defined herein.

[0093] The term "amino" refers to the group -NH2. By extension, the term "substituted amino" refers to monosubstituted amino, disubstituted amino, or trisubstituted amino. In certain embodiments, the "substituted amino" is a monosubstituted amino or disubstituted amino group.

[0094] The term "monosubstituted amino" refers to an amino group in which the nitrogen atom directly attached to the parent molecule is replaced by one hydrogen and one group other than hydrogen, and includes groups selected from: -NH(R bb ),-NHC(=O)R aa 、-NHCO2R aa 、-NHC(=O)N(R bb )2, -NHC(=NR bb )N(R bb )2, -NHSO2R aa 、-NHP(=O)(OR cc )2 and -NHP(=O)(N(R bb )2)2, where R aa 、R bb and R cc is as defined herein, and wherein the group -NH(R bb ) of R bb Not hydrogen.

[0095] The term "disubstituted amino" refers to an amino group in which the nitrogen atom directly attached to the parent molecule is replaced by two groups other than hydrogen, and includes groups selected from: -N(R bb ) 2 、-NR bb C(=O)R aa 、-NR bb CO2R aa 、-NR bb C(=O)N(R bb )2、-NR bb C(=NR bb )N(R bb )2、-NR bb SO2R aa 、-NR bb P(=O)(OR cc )2 and -NR bb P(=O)(N(R bb )2)2, where R aa 、R bb and R ccis as defined herein, with the proviso that the nitrogen atom directly attached to the parent molecule is not replaced by hydrogen.

[0096] The term "trisubstituted amino" refers to an amino group wherein the nitrogen atom directly attached to the parent molecule is substituted with three groups selected from -N(R bb )2 and -N(R bb )3 + X – A group in which R bb and X – is as defined herein.

[0097] The term "sulfonyl" refers to a group selected from -SO2N(R bb )2, -SO2R aa and SO2OR aa A group in which R aa and R bb is as defined herein.

[0098] The term "sulfinyl" refers to the group -S(=O)R aa , where R aa is as defined herein.

[0099] The term "acyl" refers to a group having the following general formula: -C(=O)R X1 、-C(=O)OR X1 、-C(=O)-OC(=O)R X1 、-C(=O)SR X1 、-C(=O)N(R X1 )2、-C(=S)R X1 、-C(=S)N(R X1 )2、-C(=S)O(R X1 )、-C(=S)S(R X1 ),-C(=NR X1 )R X1 、-C(=NR X1 )OR X1 、-C(=NR X1 )SR X1 , and -C(=NR X1 )N(R X1 )2, where R X1is hydrogen; halogen; substituted or unsubstituted hydroxy; substituted or unsubstituted thiol; substituted or unsubstituted amino; substituted or unsubstituted acyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkenyl; substituted or unsubstituted alkynyl; substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthiooxy, heteroaliphaticthiooxy, alkylthiooxy, heteroalkylthiooxy, arylthiooxy, heteroarylthiooxy, mono- or di-aliphatic amino, mono- or di-heteroaliphatic amino, mono- or di-alkylamino, mono- or di-heteroalkylamino, mono- or di-arylamino, or mono- or di-heteroarylamino; or two R X1 The groups taken together form a 5- to 6-membered heterocyclyl ring.

[0100] Exemplary acyl groups include aldehydes (-CHO), carboxylic acids (-C0H), ketones, acyl halides, esters, amides, imines, carbonates, carbamates, and ureas. Acyl substituents include, but are not limited to, any of the substituents described herein that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocycle, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphatic amino, heteroaliphatic amino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphatic oxy, heteroaliphatic oxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphatic thiooxy, heteroaliphatic thiooxy, alkylthiooxy, heteroalkylthiooxy, arylthiooxy, heteroarylthiooxy, acyloxy, etc., each of which may or may not be further substituted).

[0101] The term "carbonyl" refers to a group wherein the carbon directly attached to the parent molecule is sp 2 hybridized and substituted with oxygen, nitrogen or sulfur atoms, such as a group selected from the group consisting of: ketone (e.g., -C(=O)R aa ), carboxylic acids (e.g., -CO2H), aldehydes (CHO), esters (e.g., -CO2R aa 、-C(=O)SR aa 、-C(=S)SR aa ), amides (e.g., -C(=O)N(R bb )2、C(=O)NR bb SO2R aa 、-C(=S)N(Rbb )2) and imine (e.g., -C(=NR bb )R aa 、-C(=NR bb )OR aa ), C(=NR bb )N(R bb )2), where R aa and R bb is as defined herein.

[0102] The term "oxo" refers to the group =0, and the term "thiooxo" refers to the group =S.

[0103] The term "cyano" refers to the group -CN.

[0104] The term "azide" refers to the group -N3.

[0105] Nitrogen atoms may be substituted or unsubstituted when valence permits, and include primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen atom substituents include, but are not limited to, hydrogen, -OH, -OR aa 、-N(R cc )2, -CN, -C(=O)R aa 、-C(=O)N(R cc )2, -CO2R aa 、-SO2R aa 、-C(=NR bb )R aa 、-C(=NR cc )OR aa 、-C(=NR cc )N(R cc )2、-SO2N(R cc )2, -SO2R cc 、-SO2OR cc 、-SOR aa 、-C(=S)N(R cc )2, -C(=O)SR cc 、-C(=S)SR cc 、-P(=O)(OR cc )2、-P(=O)(R aa )2、-P(=O)(N(R cc )2)2、C 1-10 Alkyl, C 1-10 Perhalogenated alkyl, C 2-10 Alkenyl, C 2-10 Alkynyl, hetero C 1-10 Alkyl, hetero C 2-10 Alkenyl, hetero C 2-10 Alkynyl, C 3-10Carbocyclic group, 3-14 membered heterocyclic group, C 6-14 aryl, and 5-14 membered heteroaryl, or two R attached to the N atom cc The groups are connected to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl group is independently replaced by 0, 1, 2, 3, 4, or 5 R dd group substituted, and wherein R aa 、R bb 、R cc and R dd is as defined herein.

[0106] In certain embodiments, the substituent present on the nitrogen atom is a nitrogen protecting group (also referred to herein as an "amino protecting group"). Nitrogen protecting groups include, but are not limited to, -OH, -OR aa 、-N(R cc h, -C(=O)R aa 、-C(=O)N(R cc )2.

[0107] -CO2R aa 、-SO2R aa 、-C(=NR cc )R aa 、-C(=NR cc )OR aa 、-C(=NR cc )N(R cc )2、-SO2N(R cc )2, -SO2R cc 、-SO2OR cc 、-SOR aa 、-C(=S)N(R cc )2, -C(=O)SR cc 、-C(=S)SR cc 、C 1-10 Alkyl (e.g., aralkyl, heteroaralkyl), C 2-10 Alkenyl, C 2-10 Alkynyl, hetero C 1-10 Alkyl, hetero C 2-10 Alkenyl, hetero C 2-10 Alkynyl, C 3-10 Carbocyclic group, 3-14 membered heterocyclic group, C 6-14 aryl, and 5-14 membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl are independently replaced by 0, 1, 2, 3, 4, or 5 R dd group substituted, and wherein R aa 、Rbb 、R cc and R dd Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd ed., John Wiley & Sons, 1999, which is incorporated herein by reference.

[0108] For example, a nitrogen protecting group such as an amide group (e.g., -C(=O)R aa ) include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropionamide, picolinamide, 3-pyridylformamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-phenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxyacylamino)acetamide, 3-(p-hydroxyphenyl)propionamide, 3-(o-nitrophenyl)propionamide, 2-methyl-2-(o-nitrophenoxy)propionamide, 2-methyl-2-(o-phenylazophenoxy)propionamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamamide, N-acetylmethionine derivatives, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

[0109] Nitrogen protecting groups such as carbamate groups (e.g., -C(=O)OR aa) include but are not limited to methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfonylamino)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 2,7-di-tert-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthenyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacylcarbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethyl carbamate (Ad carbamic acid 1-(4-biphenylyl)ethyl ester (Bpoc), 1-(3,5-di-tert-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarbamyl)ethyl carbamate, tert-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc) , allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolinyl carbamate, N-hydroxypiperidinyl carbamate, alkyl dithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate esters, 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphoniumethyl carbamate (Peoc), 2-triphenylphosphoniumisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl) methyl carbamate, tert-amyl carbamate, S-benzylthiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropyl methyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylformamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylformamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furylmethyl carbamate, 2- Iodoethyl ester, isobornyl carbamate, isobutyl carbamate, isonicotinoyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-tert-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzyl carbamate.

[0110] Nitrogen protecting groups such as sulfonamide groups (e.g., -S(=O)2R aa ) include, but are not limited to, p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), -trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylmethylsulfonamide.

[0111] Other nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)acyl derivatives, N'-toluenesulfonylaminoacyl derivatives, N'-phenylaminothioacyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiosuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilazide adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5- -substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N-2, 7-Dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fern), N-2-pyridylmethylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, N-p-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylamine, N-(5,5-dimethyl-3-oxo-1-cyclohexyl)amine

[0014] Examples include benzophenone, ...In certain embodiments, the nitrogen protecting group is benzyl (Bn), tert-butyloxycarbonyl (BOC), benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), trifluoroacetyl, triphenylmethyl, acetyl (Ac), benzoyl (Bz), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), 2,2,2-trichloroethyloxycarbonyl (Troc), triphenylmethyl (Tr), tosyl (Ts), p-bromophenylsulfonyl (Bs), nitrobenzenesulfonyl (Ns), methanesulfonyl (Ms), trifluoromethanesulfonyl (Tf), or dansyl (Ds).

[0112] In certain embodiments, the substituent present on the oxygen atom is an oxygen protecting group (also referred to herein as a "hydroxy protecting group"). Oxygen protecting groups include, but are not limited to, -R aa 、-N(R bb )2, -C(=O)SR aa 、-C(=O)R aa 、-CO2R aa 、-C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-S(=O)R aa 、-SO2R aa 、-Si(R aa )3、-P(R cc )2、-P(R cc )3 + X – 、-P(OR cc )2、-P(OR cc )3 + X – 、-P(=O)(R aa )2、-P(=O)(OR cc )2 and -P(=O)(N(R bb )2)2, where X – 、R aa 、R bb and R ccOxygen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd ed., John Wiley & Sons, 1999, which is incorporated herein by reference.

[0113] Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxymethyl (MOM), methylthiomethyl (MTM), tert-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), tert-butoxymethyl, 4-pentenyloxymethyl (POM), silyloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7aoctahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl , 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylhydrogen selenyl)ethyl, tert-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p'-dinitrodiphenylmethyl, 5-dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl 1-Benzodithiolan-2-yl, 1-Benzoisothiazolyl S, 1-Benzodithiolan-2-yl, 1-Benzodithiolan-3 ...S dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethyltert-hexylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), tert-butylmethoxyphenylsilyl (TEMPS), formates, benzoylformates, acetates, chloroacetates, dichloroacetates, trichloroacetates, trifluoroacetates Acid esters, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinic acid ester), 4,4-(ethylenedithio)pentanoate (levulinic acid dithioacetal), pivalate, adamantane ester, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (trimethylbenzoate / mesitoate), methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate ( TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonium)ethyl carbonate (Peoc), isobutyl carbonate, vinyl carbonate, allyl carbonate, tert-butyl carbonate (BOC or Boc), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzyl carbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, S-benzylthiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(Methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenyl acetate, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethyl diaminophosphoryl ester, alkyl N-phenyl carbamate, borate, dimethylphosphinothioate, alkyl 2,In certain embodiments, the oxygen protecting group is tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), triphenylsilyl (TPS), triethylsilyl (TES), trimethylsilyl (TMS), triisopropylsiloxymethyl (TOM), acetyl (Ac), benzoyl (Bz), allyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-trimethylsilylethyl carbonate, methoxymethyl (MOM), 1-ethoxyethyl yl (EE), 2-methoxy-2-propyl (MOP), 2,2,2-trichloroethoxyethyl, 2-methoxyethoxymethyl (MEM), 2-trimethylsilylethoxymethyl (SEM), methylthiomethyl (MTM), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), p-methoxyphenyl (PMP), triphenylmethyl (Tr), methoxytrityl (MMT), dimethoxytrityl (DMT), allyl, p-methoxybenzyl (PMB), tert-butyl, benzyl (Bn), allyl or pivaloyl (Piv).

[0114] In certain embodiments, the substituent present on the oxygen atom is an oxygen protecting group (also referred to herein as a "hydroxy protecting group"). Oxygen protecting groups include, but are not limited to, -R aa 、-N(R bb )2, -C(=O)SR aa 、-C(=O)R aa 、-CO2R aa 、-C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-S(=O)R aa 、-SO2R aa 、-Si(R aa )3、-P(R cc )2、-P(R cc )3 + X – 、-P(OR cc )2、-P(OR cc )3 + X – 、-P(=O)(R aa )2、-P(=O)(ORcc )2 and -P(=O)(N(R bb )2)2, where X – 、R aa 、R bb and R cc Oxygen protecting groups are well known in the art and include those described in detail in: Protecting Groups in Organic Chemistry

[0115] Protective Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd ed., John Wiley & Sons, 1999, which is incorporated herein by reference.

[0116] Exemplary oxygen protecting groups include, but are not limited to, methyl, methoxymethyl (MOM), methylthiomethyl (MTM), tert-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), tert-butoxymethyl, 4-pentenyloxymethyl (POM), silyloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7aoctahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl , 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylhydrogen selenyl)ethyl, tert-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p'-dinitrodiphenylmethyl, 5-dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl 1-Benzodithiolan-2-yl, 1-Benzoisothiazolyl S, 1-Benzodithiolan-2-yl, 1-Benzodithiolan-3 ...S dioxide, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethyltert-hexylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), tert-butylmethoxyphenylsilyl (TEMPS), formates, benzoylformates, acetates, chloroacetates, dichloroacetates, trichloroacetates, trifluoroacetates Acid esters, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinic acid ester), 4,4-(ethylenedithio)pentanoate (levulinic acid dithioacetal), pivalate, adamantane ester, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (trimethylbenzoate / mesitoate), methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate ( TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonium)ethyl carbonate (Peoc), isobutyl carbonate, vinyl carbonate, allyl carbonate, tert-butyl carbonate (BOC or Boc), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzyl carbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, S-benzylthiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylvalerate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(Methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenyl acetate, isobutyrate, monosuccinate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethyl diaminophosphoryl ester, alkyl N-phenyl carbamate, borate, dimethylphosphinothioate, alkyl 2,In certain embodiments, the oxygen protecting group is tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), triphenylsilyl (TPS), triethylsilyl (TES), trimethylsilyl (TMS), triisopropylsiloxymethyl (TOM), acetyl (Ac), benzoyl (Bz), allyl carbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-trimethylsilylethyl carbonate, methoxymethyl (MOM), 1-ethoxyethyl yl (EE), 2-methoxy-2-propyl (MOP), 2,2,2-trichloroethoxyethyl, 2-methoxyethoxymethyl (MEM), 2-trimethylsilylethoxymethyl (SEM), methylthiomethyl (MTM), tetrahydropyranyl (THP), tetrahydrofuranyl (THF), p-methoxyphenyl (PMP), triphenylmethyl (Tr), methoxytrityl (MMT), dimethoxytrityl (DMT), allyl, p-methoxybenzyl (PMB), tert-butyl, benzyl (Bn), allyl or pivaloyl (Piv).

[0117] In certain embodiments, the substituent present on the sulfur atom is a sulfur protecting group (also known as a "thiol protecting group"). Sulfur protecting groups include but are not limited to -R aa 、-N(R bb )2, -C(=O)SR aa 、-C(=O)R aa 、-CO2R aa 、-C(=O)N(R bb )2、-C(=NR bb )R aa 、-C(=NR bb )OR aa 、-C(=NR bb )N(R bb )2、-S(=O)R aa 、-SO2R aa 、-Si(R aa )3、-P(R cc )2、-P(R cc )3 + X – 、-P(OR cc )2、-P(OR cc )3 + X – 、-P(=O)(R aa )2、-P(=O)(ORcc )2 and -P(=O)(N(R bb )2)2, where R aa 、R bb and R cc is as defined herein. Sulfur protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3rd ed., John Wiley & Sons, 1999, which is incorporated herein by reference. In certain embodiments, the sulfur protecting group is acetamidomethyl, t-Bu, 3-nitro-2-pyridinesulfenyl, 2-pyridine-sulfenyl, or triphenylmethyl.

[0118] Additionally, the following terms are used herein: "Ac" refers to an acyl group; "Fmoc" refers to the fluorenylmethoxycarbonyl protecting group; "tBoc" refers to the tert-butyloxycarbonyl protecting group; "Cbz" refers to a benzyl chlorocarbonate protecting group; "Troc" refers to the trichloroethyl chloroformate protecting group; "TFA" refers to the trifluoroacetamide protecting group; · "[1,3]dioxolyl group" means a moiety having the following structural formula

[0119] As used herein, the term "click chemistry reagent" (interchangeably used with "click reagent" or "click moiety") refers to any chemical moiety that reacts with its corresponding click moiety (the two reactive moieties are referred to as a "click reagent pair") to produce a "click reaction product" via an electrocycloaddition reaction (a "click chemistry reaction" or "click reaction"). Typically, click reactions occur in aqueous solvents under mild conditions and may include any of the following: neutral pH, ambient temperature, and low reactant concentrations. Exemplary click reagent pairs include, but are not limited to, Cu(I)-catalyzed azide-alkyne click chemistry (CuAAC); strain-promoted azide-alkyne click chemistry (SPAAC), and tetrazine and olefin (trans-cyclooctene).

[0120] Any suitable click reagent pair can be used in the present invention. Examples of click reagents include:

[0121] - Reagents including a bicyclo[6.1.0]nonyne (BCN) group, for example:

[0122] – Reagents including alkynes, such as:

[0123] – reagents including azides, such as N3-CH2-;

[0124] – Reagents including the dibenzocyclooctyne (DBCO) group, such as:

[0125] – Reagents including trans-cyclooctene, such as:

[0126] – Reagents including tetrazines, such as:

[0127] "Sialic acid" refers to naturally occurring or synthetically derived neuraminic acid or any chemical modification of neuraminic acid. The structural formula of neuraminic acid is as follows:

[0128] As used herein, the terms "poly(ethylene glycol)", "polyethylene oxide" and "polyoxyethylene" are used interchangeably and refer to a polyethylene glycol having the structural formula H-(O-CH2CH2) m -OH polymers, where m is the number of repeating units. The polymer molecular weight can vary from 300 g / mol to 10,000,000 g / mol.

[0129] As used herein, the term "particle" includes microparticles and nanoparticles as defined herein.

[0130] As used herein, the term "particle" includes microparticles and nanoparticles as defined herein.

[0131] As used herein, "subject" refers to a subject who is treated according to the provided treatment methods. The subject can be a human, primate, dog, cat, cow, horse, mouse, etc. Subjects also refer to those animals used in laboratory experiments.

[0132] As used herein, "nanoparticle" refers to a particle composed of one or more polymers, the size of which is nanometer (nm) level, including a linear dimension range between 10 nanometers and 2000 nanometers. As used herein, "linear dimension" refers to the distance between any two points on the surface of the nanoparticle measured in a straight line. The nanoparticles disclosed herein can be irregular, elliptical, spindle-shaped, rod-shaped, cylindrical, pancake-shaped, disc-shaped, spherical, biconcave or red blood cell-shaped. Linear dimension can be measured using a variety of methods, including but not limited to transmission electron microscopy or adjustable resistive pulse sensing, which are some standard methods for determining the size of nanoparticles. One of the widely used techniques for measuring the size of nanoparticles is dynamic light scattering (DLS), which can provide the diameter and polydispersity of nanoparticles. DLS assumes that the nanoparticles are spherical in nature, and the size of the nanoparticles is the average diameter (or radius) of this assumed sphere. In such measurements, nanoparticles can be described as having a size range of 10nm to 1000nm or 1nm to 500nm.

[0133] As used herein, "microparticles" refer to microscopic particles composed of one or more polymers, having a size on the micrometer (μm) scale, including a maximum cross-sectional width of less than 1000 μm and greater than or equal to 1 μm.

[0134] As used herein, "polymer" refers to one or more molecules composed of multiple repeating structural units linked by covalent bonds. As used herein, "polymer particle" refers to a solid or porous particle, in contrast to the shell-like structure of liposomes and polymersomes and the relatively open structure of hydrogel particles. As used herein, "hydrogel particle" refers to a cross-linked network of polymer chains that is hydrophilic but stable in aqueous environments.

[0135] As used herein, the term "biocompatible polymer" refers to a polymer that does not undesirably interfere with the biological function of a tissue. In some cases, a biocompatible polymer can be biodegradable, bioabsorbable, and bioerodible. Biodegradable, bioabsorbable, and bioerodible, as well as degradable, eroded, and absorbed, are used interchangeably (unless the context indicates otherwise) and refer to polymers and materials that are capable of being degraded or absorbed when exposed to body fluids (e.g., blood) and their components (e.g., enzymes), and that can be gradually reabsorbed, absorbed, and / or eliminated by the body.

[0136] As used herein, the term "treatment" or "treating" characterizes a method or process whose purpose is to: (1) delay or prevent the onset of a disease, disorder, or condition; (2) slow or stop the progression, aggravation, or worsening of one or more symptoms of a disease, disorder, or condition; (3) ameliorate the symptoms of a disease, disorder, or condition; (4) reduce the severity or incidence of a disease, disorder, or condition; or (5) cure a disease, disorder, or condition. Treatment can be administered before the onset of a disease, disorder, or condition to serve a prophylactic or preventive effect. Alternatively, or additionally, treatment can be administered after the onset of a disease, disorder, or condition to serve a therapeutic effect.

[0137] As used herein, "effective amount" means the amount of an active compound agent that elicits a desired biological response in a subject, e.g., an amount that results in treatment as defined herein above. In one embodiment, the effective amount of a compound of the invention is from about 0.01 mg / kg / day to about 1000 mg / kg / day, from about 0.1 mg / kg / day to about 100 mg / kg / day, or from about 0.5 mg / kg / day to about 50 mg / kg / day.

[0138] particles

[0139] In one aspect, the present disclosure provides nanoparticles comprising polymers that are tethered to the surface of the nanoparticles by covalent chemical conjugation of ligands (e.g., compounds represented by structural formulas S-1, S-2, D-1, D-2, S-1-1, S-2-1, D-1-1, D-2-1, or moieties represented by S-1A, S-2A, D-1A, D-2A, also referred to herein as sialic acid-containing ligands). The nanoparticles can be used to contact immune cells expressing sialic acid-binding immunoglobulin-type lectins (Siglecs) to modulate inflammatory processes. It has been determined that providing ligands capable of targeting and binding to immune cells expressing sialic acid-binding immunoglobulin-type lectins (Siglecs) can be used to modulate inflammatory responses in target cells and the associated environment.

[0140] Presentation of ligands on the surface of nanoparticles means decorating the nanoparticles with ligands so that they can bind to Siglec receptors on target cells or organisms. These ligands are appropriately provided to bind to, activate, or block the receptors.

[0141] A single nanoparticle can be decorated with a multivalent ligand, which will allow the single nanoparticle to bind multivalently to different Siglec receptors, thereby regulating the inflammatory response. Nanoparticles decorated with unique ligands can also be mixed with nanoparticles decorated with other ligands that may target different Siglec receptors, again achieving the desired regulation of the inflammatory response. In some embodiments, the presentation of the ligand on the surface of the nanoparticle or microparticle can increase the cellular uptake of the particle by at least about two times, at least about three times, at least about four times, at least about five times, at least about six times, or at least about 10 times. In some embodiments, the presentation of the ligand on the surface of the nanoparticle or microparticle can reduce the inflammatory response. In non-limiting embodiments, the reduction in inflammatory response is more than about two times, more than about three times, more than about four times, more than about five times, more than about 10 times, more than about 20 times, more than about 50 times, more than about 100 times, more than about 500 times, or more than about 1000 times.

[0142] Nanoparticles or microparticles can be used for systemic delivery or local delivery to target diseased tissues in subjects in need of treatment, thereby regulating the inflammatory response in the subject to resolve innate and adaptive inflammation, activate innate and / or adaptive immunity when it is desired to enhance immune surveillance, or reduce the infectivity of infectious organisms. The targeted immune cells or viruses should have Siglec receptors or viral sialic acid ligand binding regions, respectively. The activities of the innate immune system include, for example, cellular responses of the innate immune system; non-cellular / humoral responses of the innate immune system, the complement system, the alternative complement pathway, the amplification loop of the alternative complement pathway, and / or the amplification loop of the alternative complement pathway activated by complement factor H. The activities of the adaptive immune system involve dendritic cell maturation and presentation to T cells, T cell activation, T cell regulation, T cell checkpoint inhibition or activation, neutrophil NETosis, and B cell activation. Reduced infectivity includes reduced viral entry into host cells, reduced viral particle replication, or reduced inflammatory response to viral infection.

[0143] Several types and configurations of nanoparticles are encompassed by the present disclosure. For example, nanoparticles can be composed of a variety of materials, including but not limited to biodegradable polymers, biocompatible polymers, bioabsorbable polymers, or combinations thereof.

[0144] The polymer backbone of the nanoparticles connecting the sialic acid-containing part can be composed of naturally occurring polymers such as carbohydrates or proteins, or can be composed of synthetic polymers. The polymer backbone will have unique terminal functional groups to achieve the sialic acid-containing part being bound to the nanoparticle surface. The polymer backbone can first be combined with multiple sialic acid ligands by chemical conjugation methods and then form nanoparticles, or the polymer backbone can first form nanoparticles and then the functional groups exposed on the nanoparticle surface can be combined with the sialic acid-containing part by chemical conjugation methods. Suitable nanoparticles include polymer particles and hydrogel particles.

[0145] Suitable polymers for preparing nanoparticles include, but are not limited to, poly(lactide-co-glycolide)-poly(ethylene glycol) or poly(lactide-co-glycolide)-block-poly(ethylene glycol) or poly(lactide-co-glycolide) containing click chemistry functional groups (e.g., azide or alkyne, such as DBCO, etc.) or amine reactive esters such as succinimide esters. Such polymers include, but are not limited to, PLGA-PEG-DBCO and PLGA-PEG-NHS. Other polymers include poly(N-acetylglucosamine) (chitin), chitosan, poly(3-hydroxyvalerate), poly(lactide-co-glycolide (e.g., poly(D,L-lactide-co-glycolide), poly(L-lactide-co-glycolide)), poly(3-hydroxybutyrate), poly(4-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyorthoesters, polyanhydrides, poly(glycolic acid), poly(glycolide), poly(lactic acid) (e.g., poly(L-lactic acid), poly(D,L-lactic acid)), poly(lactide) (e.g., poly(D,L-lactide)), poly((D,L)-lactide)-b-poly(ethylene glycol)-azide, poly(DL-lactide)-b-poly(ethylene glycol)-methyltetrazine, poly(L-lactide-co-D,L-lactide), poly(D,L-lactide) -b-poly(ethylene glycol)-carboxylic acid, poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide), poly(lactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(lactide-co-glycolide), poly(lactide-co-glycolide)-b-poly(ethylene glycol)-azide, poly(lactide-co-glycolide)-b-poly(ethylene glycol)-alkyne, poly((D,L)lactic acid)-b-poly (Ethylene glycol)-azide, poly((D,L)lactic acid)-b-poly(ethylene glycol)-alkyne, poly(caprolactone), poly(caprolactone)-b-poly(ethylene glycol), polycaprolactone-b-poly(ethylene glycol), poly(lactide-co-caprolactone)-b-poly(ethylene glycol)-b-poly(lactide-co-caprolactone), poly(L-lactide-co-caprolactone), poly(L-lactide-co-caprolactone), poly(D,

[0014] Examples of the present invention include polyols, e.g., oligosaccharides ... and copolymers other than polyacrylates, vinyl halide polymers and copolymers (e.g., polyvinyl chloride), polyvinyl ethers (e.g., polyvinyl methyl ether), polyvinylidene halides (e.g., polyvinylidene chloride), poly(vinylidene fluoride), poly(vinylidene fluoride-co-hexafluoropropylene), polyacrylonitrile, polyvinyl ketone, polyvinyl aromatics (e.g., polystyrene), polyvinyl esters (e.g., polyvinyl acetate), acrylonitrile-styrene polymers, ABS resins, polyamides (e.g., nylon 66 and polycaprolactam), polycarbonates (including tyrosine-based polycarbonate), polyoxymethylene, polyimides, polyethers, polyurethanes, rayon, triacetate rayon, cellulose, cellulose acetate, cellulose butyrate, cellulose acetate butyrate, cellophane, cellulose nitrate, cellulose propionate, cellulose ethers, carboxymethyl cellulose, and fullerenes.

[0146] In one aspect, the nanoparticles are formed from polycaprolactone, and in other embodiments, from polymers comprising polyglycolic acid, poly(lactic acid), poly(lactic-co-glycolic acid), polycaprolactone, poly(3-hydroxybutyric acid). In some embodiments, the nanoparticles can be formed from poly(lactide) (PLA), poly(glycolide) (PGA), polylactic-glycolic acid (PLGA), poly(butyl cyanoacrylate) (PBCA), or N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers. In another aspect, the nanoparticles are formed from polymers such as poly(ethylene glycol), polyethylene oxide, Pluronic F127, Pluronic F68, poloxamers, poly(hydroxymethyl methacrylate), polyvinyl alcohol, and poly(vinyl pyrrolidone).

[0147] In certain embodiments, nanoparticle is formed by the mixture of biodegradable and non-biodegradable polymer, as block copolymer (BCP), comprises the preferred embodiment of PLGA-block-PEG.Block copolymer comprises the polymer with two or more different polymer subunits connected by covalent bond.In certain embodiments, nanoparticle is formed by the mixture of biodegradable and non-biodegradable polymer, as block copolymer, comprises the preferred embodiment of PLGA-block-PEG.On the other hand, nanoparticle is formed with the form of hydrogel nanoparticle by naturally derived polymer, comprises and is formed by collagen, hyaluronic acid, heparin, heparin sulfate, chitosan and alginate.

[0148] Methods for synthesizing nanoparticles are well known to those skilled in the art. (e.g., see Spence et al., Science Translational Medicine, 2015, 7:303 303ra140 and references cited therein), for example, methods for synthesizing nanoparticles with known degradation rates are well known to those skilled in the art, as described in U.S. Patent No. 6,451,338 to Gregoriadis et al., U.S. Patent No. 6,168,804 to Samuel et al., and U.S. Patent No. 6,258,378 to Schneider et al., which are hereby incorporated by reference in their entirety.

[0149] The chemical connection of the ligand described herein to the nanoparticle surface can be achieved by using click chemistry. In this type of reaction, a chemical reaction occurs between the terminal functional groups of the nanoparticle polymer and the terminal functional groups of the ligand (referred to herein as "terminal functional group conjugated pairs") so that the polymer is connected to the ligand containing sialic acid. The type of terminal functional group found on the polymer surface and its binding partner ligand will determine the type of click chemistry reaction for chemically connecting the ligand to the nanoparticle surface. In addition, the selection of polymers with specific terminal functional groups can be used to control the type, density and spatial arrangement of the ligand conjugate partners presented on the nanoparticle surface. In an embodiment, the polymer has a specific functional group that provides a chemical conjugation site on the nanoparticle surface formed, including azide, alkyne, aryl ester, amide, amine, aryl amide, aldehyde, acetyl, substituted aryl ester, alkyl ester, alkyl ketone, aryl ketone, substituted aryl ketone, ketone, alkyl halide, aminooxy (amnioxy), alcohol, aza-inium salt, carboxylic acid, ester, amide, bicyclononyne, dihydrazide, halocarbonyl, halogen In certain embodiments, the polymer is a poly(lactide-co-glycolide)-poly(ethylene glycol) or poly(lactide-co-glycolide)-block-poly(ethylene glycol) or poly(lactide-co-glycolide)-block-poly(ethylene glycol) having at least one of the functional groups listed directly above.

[0150] Such conjugation sites provide locations for the connection of ligands to the surface of nanoparticles by performing click chemistry reactions. In one example, nanoparticles are formed from PLGA-PEG polymers having azide or alkyne terminal functional groups. In non-limiting embodiments, blends of different polymers with different terminal functional groups can be used. Such polymers include, for example, PLGA-PEG-alkyne, PLGA-PEG-ester, and PLGA-PEG-DBCO. In a particular aspect, blends of PLGA-PEG-alkyne and PLGA-PEG-carboxylic acid can be prepared into nanoparticles. In another particular aspect, blends of PLGA-PEG-alkyne and PLGA-PEG-ester can be prepared into nanoparticles. In another particular embodiment, blends of PLGA-PEG-DBCO and PLGA-PEG-carboxylic acid can be prepared into nanoparticles. In another particular aspect, blends of PLGA-PEG-DBCO and PLGA-PEG-ester can be prepared into nanoparticles.

[0151] Although PLGA polymers can have free terminal alkyne groups, many of these will be buried in the particle matrix and unavailable for binding on the particle surface. In some embodiments, further alkyne groups can be introduced into the particles by providing a second polymer or copolymer surfactant or coating in addition to the first PGLA polymer or copolymer of the particles. Suitably, the second polymer or copolymer can be branched or linear and can have multiple terminal alkyl groups, wherein the alkyl groups contain only carbon and hydrogen and form a homologous series having the general formula CnH2n+1. In other embodiments, sialic acid-containing ligands can be attached to the particles, such as polymeric nanoparticles, via covalent bonds.

[0152] In other embodiments, the ligand comprises a terminal functional group (i.e., a conjugation site) for attachment to the surface of the nanoparticle. Such terminal functional groups include azide, alkyne, aryl ester, amide, amine, arylamide, aldehyde, acetyl, substituted aryl ester, alkyl ester, alkyl ketone, aryl ketone, substituted aryl ketone, ketone, alkyl halide, aminooxy, alcohol, aza-ylide, carboxylic acid, ester, amide, bicyclononyne, dihydrazide, halocarbonyl, halosulfonyl, hydrazide, N-hydroxysuccinimide, norbornene, oxa-norbornadiene, succinyl, hydroxybenzoic acid, benzophenone ... Imidoesters, isothiocyanates, iodoacetamides, mono- and difluorocyclooctynes, maleimides, methylcyclopropenes, isocyanopropionates, hydrazines, nitriles, nitro groups, phosphines, phosphine azides, tetrazines, methyltetrazines, trans-cyclooctenes, strained alkynes, dibenzocyclooctynes, diarylazacyclooctanones, propargyl groups, isonitriles, azadibenzylcyclooctynes, vinyl groups, sulfonyl esters, thioesters, thiocarboxylates, thioesters, sulfonyl halides, thiols, and thioalkenes. Such conjugation sites provide locations for the attachment of ligands to the nanoparticle surface by performing click chemistry reactions.

[0153] In one embodiment, a click chemistry reaction is used to connect the ligand to the nanoparticle surface. Such click chemistry reactions are characterized by a class of biocompatible small molecule reactions commonly used for bioconjugation, which is used for chemical connection to modify other molecules, biomolecules, nanoparticles and other surfaces. In general, click chemistry reactions have the following characteristics: modularity, insensitivity to solvent parameters, high chemical yield, insensitivity to oxygen and water, regiospecificity and stereospecificity, and large thermodynamic driving force (>20kcal / mol), which is conducive to reacting with a single reaction product. Click chemistry reactions provide high reaction specificity and can control regiospecificity and stereospecificity. Reaction specificity is particularly useful for achieving the desired presentation of sialic acid ligands on the nanoparticle surface, thereby allowing optimal binding of nanoparticles to immune cell Siglec receptors. The bonds formed by the click reaction during the conjugation process provide accessibility to highly stable covalent bonds between the sialic acid ligands and the nanoparticles, which do not rearrange or react, nor do they cause degradation or hydrolysis under biological conditions.

[0154] A variety of different click chemistry reactions can be used to attach ligands to the surface of the nanoparticles. The use of such click chemistry reactions provides a controlled reaction medium for generating nanoparticles with a desired density and spatial arrangement of sialic acid ligands. The density and spatial arrangement of sialic acid ligands on the nanoparticle surface can be controlled, for example, by controlling the amount of polymer with functional groups used to form the nanoparticles, the polymer molecular weight, the polymer density, the number of functional groups per polymer, the solvent, the type of functional group, the ligand concentration, the type of click chemistry employed, and the type of click chemistry conjugate pair.

[0155] In various embodiments, the average molecular weight of a polymer (eg, PEG, PLGA, PEG-PLGA block copolymer) can be determined by any method known in the art, such as anion exchange chromatography, gel permeation chromatography, viscosity measurement, and the like.

[0156] Click chemistry reactions for tethering ligands to polymers are well known to those skilled in the art and include, for example, Huisgen 1,3-dipolar cycloadditions, copper(I)-catalyzed azide-alkyne cycloadditions (CuAAC) to produce 1,3-substituted products, ruthenium-catalyzed alkyne-azide cycloadditions (RuAAC) to produce 1,5-substituted triazoles, strain-promoted alkyne-azide cycloadditions (SPAAC) to produce 1,4-substituted products, strain-promoted alkyne-nitrone cycloadditions, Diels-Alder reactions of olefins and tetrazines with reversed demand, tetrazine trans-cyclooctene ligations, thiol-ene reactions, thiol-yne reactions, Staudinger reactions, [4+1] cycloadditions, tetracycloheptane ligations [2+2+2] cycloadditions, norbornene cycloadditions, and olefin tetrazole photoclick reactions.

[0157] Binding the ligand to the nanoparticle surface is performed in such a way that the ligand is presented with maximum binding affinity to the Siglec receptor expressed on the surface of the immune cell or the sialic acid ligand receptor expressed on the surface of the viral particle. The ligand density can be controlled to provide the desired multivalent (multivalent / polyvalent) ligand to Siglec receptor interaction upon contact with the immune cell, as such interaction is associated with a desired cellular immune response. The multivalent ligand-receptor interaction can be controlled based on the ligand density provided on the nanoparticle surface, and this density can affect the response elicited by the immune cell upon contact.

[0158] As disclosed, the use of click chemistry requires that both the nanoparticle and the polymer of the sialic acid ligand be functionalized to allow the desired conjugation of the ligand to the nanoparticle surface. In one embodiment, a terminal alkyne is presented on the nanoparticle surface for covalent chemical conjugation to the sialic acid ligand presenting an azide by performing the following reactions: copper (I) catalyzed azide-alkyne (CuAAC) reaction; copper-free reaction; strain-promoted azide-alkyne (SPAAC) reaction; tetrazine-olefin ligation reaction, or trans-cyclooctene (TCO)-tetrazine reaction. The azide functional group can be added to the ligand using, for example, the sialyltransferase ST8SIA4.

[0159] In one aspect, ligand / nanoparticle conjugate pairs can be prepared by copper(I) azide-alkyne cycloaddition of ligands with azide and nanoparticles with alkyne functionality, or ligands with alkynes and nanoparticles with azide functionality. Ligands with dibenzylcyclooctyne, difluorooctyne, or diarylazacyclooctanone can be reacted with azides via SPACC. Ligands with trans-cyclooctene can be reacted with nanoparticles with tetrazine functionality.

[0160] Nanoparticles were prepared by mixing poly(D,L-lactide-co-glycolide-COOH)-PEG-COOH (PLGA 10,000 Da-PEG-COOH 5000 Da) and poly(lactide-co-glycolide)-b-poly(ethylene glycol)-alkyne (PLGA-PEG-alkyne; 10,000 Da PLGA:1,000 PEG Da) at a 75:25 (w / w) ratio of PLGA-PEG-COOH:PLGA-alkyne (DBCO). This 75:25 ratio is an example of the density of alkyne functional groups on the nanoparticle surface. Other ratios of PLGA-PEG-COOH to PLGA-PEG-alkyne (DBCO) used to prepare nanoparticles were 95:5, 90:10, 85:15, 80:20, 70:30, 65:35, 60:40, 55:45, and 50:50. The ratio of PLGA-PEG-COOH to PLGA-PEG-alkyne (DBCO) was designed to provide sufficient space between the functional groups to allow efficient conjugation of polymeric ligands and to achieve the desired ligand density.

[0161] Nanoparticles can be prepared that contain one or more polymers with different click chemistry functional groups paired with their sialic acid-containing ligand conjugation partners, thereby allowing one or more types of ligands to be presented at different densities and / or spatial arrangements on the nanoparticle surface. By employing one or more nanoparticle polymer / ligand pairs, the use of different conjugated pairs can be designed into the nanoparticle.

[0162] By providing different click chemistry functional groups on the nanoparticle surface, different types of ligands can be conjugated to the nanoparticle surface. The density of different functional groups can be controlled by the ratio between different polymers, the concentration of polymer, the type of click chemistry conjugate, the type of click chemistry reaction and the size and shape of the ligand containing sialic acid. The quantity of the different ligands that can be presented on the surface can be determined by those skilled in the art. In a non-limiting embodiment, the quantity of the different ligands present on the nanoparticle surface is in the range of 1 to 20. The quantity of different ligands includes, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In another embodiment, the quantity of the different ligands present on the nanoparticle surface is in the range of 2 to 20. The quantity of different ligands includes, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In another embodiment, the nanoparticles will comprise at least two different ligands. In another embodiment, the nanoparticles will comprise at least three different ligands. In another embodiment, the nanoparticles will comprise at least four different ligands. In another embodiment, the nanoparticles will comprise at least five different ligands.

[0163] In general, the functional group density on the nanoparticle surface determines the maximum ligand density that can be covalently chemically bound to the nanoparticle surface via click chemistry. The ligand density can be controlled and quantified based on the number of functional groups per square nanometer surface area.

[0164] The ligand density on the surface of the nanoparticles can be controlled by a variety of methods, including chemical conjugation techniques, ligand density on the polymer, ligand type, solvent, pH, and ionic strength. The ligand density on the nanoparticle surface can be adjusted so that immune cells in contact with such nanoparticles will produce an immunomodulatory response, including an anti-inflammatory biological response. Control of the ligand density can also be used to adjust the intensity of the desired anti-inflammatory response.

[0165] In some embodiments, the sialic acid-containing ligands can be present on the nanoparticles in groups of at least 2, at least 5, at least 10, at least 15, at least 20, or at least 25, at least 50, at least 100, at least 200, or at least 400. In some embodiments, the sialic acid-containing ligands can be spaced apart on the surface of the nanoparticles so that they or the nanoparticles can bind to more than one Siglec receptor. In some embodiments, the sialic acid-containing ligands can be spaced apart on the surface of the nanoparticles so that they or the nanoparticles can bind to multiple Siglec receptors present on a single cell type, and the number of Siglec receptors present on the plasma membrane may vary.

[0166] In some embodiments, the nanoparticles may comprise a polymer comprising a sialic acid-containing ligand at a concentration in the range of 0.05 nmol / mg (sialic acid-containing ligand to nanoparticle) to 250 nmol / mg (sialic acid-containing ligand to nanoparticle), preferably 0.5 nmol / mg to 25 nmol / mg, and most preferably 0.5 to 15 nmol sialic acid-containing ligand / mg nanoparticle. In embodiments, a device may be coated with such nanoparticles. In alternative embodiments, the device may be formed from a polymer, for example, where the device is a microparticle or nanoparticle, wherein the sialic acid-containing ligand is provided in the polymer at a concentration in the range of 0.05 nmol / mg (sialic acid-containing ligand to nanoparticle) to 250 nmol / mg (sialic acid-containing ligand to nanoparticle), preferably 1 nmol / mg to 25 nmol / mg, and most preferably 2 to 15 nmol sialic acid-containing ligand / mg nanoparticle.

[0167] In some embodiments, the nanoparticles may have a maximum cross-sectional width or diameter of less than about 1000 nm, less than about 500 nm, less than about 250 nm, or less than about 200 nm. In embodiments, the nanoparticles may have a width greater than about 1 nm, greater than about 10 nm, greater than about 50 nm, or greater than about 100 nm. In embodiments, nanoparticles coated with sialic acid or sialic acid analogs may have a maximum cross-sectional width or diameter ranging from about 130 nm to about 170 nm, more preferably a width of about 150 nm. In embodiments, these size ranges may be the average width of the nanoparticles. In embodiments, at least 80% of the nanoparticles are within the disclosed ranges.

[0168] Suitably, in some embodiments, about at least 80%, more preferably at least 90%, of the particles have a maximum cross-sectional width between 130 nm and 170 nm. In embodiments, the average maximum cross-sectional width of the particles may be 150 nm, and the width of the particles may be no greater than or less than a value that is not within one standard deviation of 150 nm. In some embodiments, the volume of the nanoparticles may be equal to the volume of a sphere having a diameter between 10 nm and 500 nm, suitably between 50 nm and 250 nm, or between 100 nm and 200 nm, or between 130 nm and 170 nm.

[0169] In one example, the volume of the nanoparticle can be equal to the volume of a sphere with a diameter of approximately 100 nm.

[0170] In another aspect of the present invention, the connection of nanoparticles to ligands provides a means for the nanoparticles to evade the immune system, i.e., through opsonization and phagocytosis by the reticuloendothelial system (RES). PEGylation of known nanoparticles (i.e., coating the nanoparticles with polyethylene glycol) can provide a barrier from immune cell detection. However, PEG has the disadvantages of toxicity, immunogenicity, reduced cellular uptake, reduced binding, and non-biodegradable or bioabsorbable properties. Coating nanoparticles with sialic acid-containing ligands overcomes the disadvantages of PEG and provides a natural, non-immunogenic nanoparticle coating that can evade RES and immune detection. Therefore, the nanoparticles disclosed herein have the ability to evade immune detection and mitigate immunogenic responses.

[0171] Nanoparticles or microparticles disclosed herein may further comprise a bioactive agent encapsulated in the nanoparticle, adhered to its surface, or integrated into its structure. For example, the nanoparticles may further comprise at least one of an antibiotic, an antiviral agent, an anti-inflammatory agent, a cytokine, a cytokine inhibitor, an immunomodulator, an immunotoxin, an anti-angiogenic agent, an antihypertensive agent, an anti-edema agent, a radiosensitizer, an oligonucleotide comprising DNA or RNA, a peptide, an anticancer agent, or any combination thereof. Methods for preparing nanoparticles comprising a bioactive agent encapsulated in the nanoparticle, adhered to its surface, or integrated into its structure are well known to those skilled in the art.

[0172] Modified oligosaccharides

[0173] Disclosed herein are modified oligosaccharides, which are generally referred to as S-series (or S-1XX and S-2XX) compounds and D-series (or D-1XX and D-2XX) compounds, and are also referred to herein as sialic acid-containing ligands. The S-series compounds are compounds represented by the following general structural formulas, or pharmaceutically acceptable salts thereof: (S-1) (or S-1XX compound), or (S-2) (or S-2XX compound). The D-series compounds are compounds represented by the following general structural formulas, or pharmaceutically acceptable salts thereof: (D-1) (or D-1XX compound), or (D-2) (or D-2XX compound).

[0174]

[0175] Values ​​and exemplary values ​​for the variables R and R1 in formulas (S-1), (S-2), (D-1), and (D-2) are defined below.

[0176] Example compounds of the S and D series and their syntheses are described in Examples 1A, 1B, and 2 below.

[0177] Conjugation of oligosaccharides to polymers and nanoparticle production

[0178] Also described herein are nanoparticles comprising a polymer conjugated to the above oligosaccharide having the general structure GLP, wherein P is a biocompatible polymer and G is any one of the moieties represented by the following structural moieties (also referred to herein as sialic acid-containing ligands): or a pharmaceutically acceptable salt thereof. Values ​​and exemplary values ​​for the variables R, R1, and L in Formulas (S-1A), (S-2A), (D-1A), and (D-2A) are described below.

[0179] Example compounds of conjugated oligosaccharides of formula (S-1A), (S-2A), (D-1A), (D-2A) and their synthesis are described in Example 3 below.

[0180] Indications

[0181] In various exemplary embodiments, the compounds and particles described herein can be used in methods for treating diseases and conditions that respond to modulation (e.g., amplification, reduction, or elimination) of the activity of Siglec receptors. Such diseases and conditions include, but are not limited to, cancer, immune-related diseases and conditions, and inflammation-related diseases and conditions. Such diseases and conditions include, but are not limited to, breast cancer, non-small cell lung cancer (NSCLC), prostate cancer, colorectal cancer, melanoma, pancreatic cancer, and myelofibrosis, diabetic retinopathy, idiopathic pulmonary fibrosis, liver fibrosis, sickle cell anemia, and acute respiratory distress syndrome (ARDS).

[0182] In further embodiments, the present disclosure provides methods of treating immune and inflammatory related diseases including, but not limited to, dry and wet macular degeneration, retinal vascular disease, diabetic retinopathy, diabetic macular edema, cystoid macular edema, proliferative diabetic retinopathy, proliferative vitreoretinopathy, dry eye, allergic conjunctivitis, rheumatoid arthritis, inflammatory arthritis, lupus, nephritis, immune complex nephropathy, allergic esophagitis, allergic gastritis, hepatitis, fibrotic liver disease, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, sepsis, bacterial and viral infections, influenza, SARS-CoV-1 and SARS-CoV-2, HIV / AIDS, group B streptococcal infection, Neisserial infection, cancer involving solid organs, non-small cell lung cancer, colorectal cancer, prostate cancer, uveal melanoma, cutaneous malignant melanoma, myelofibrosis, or hematopoietic cancer, in each case by administering such a pharmaceutical composition to a subject suffering from the disease. The present disclosure provides a method for regulating an inflammatory response in a cell, the method comprising: providing a sialic acid-containing ligand to the cell, wherein the sialic acid-containing ligand is present on a nanoparticle, thereby suppressing the proinflammatory response in the cell or increasing the anti-inflammatory response in the cell. In certain embodiments, the method suppresses the proinflammatory response. In alternative embodiments, the method increases the anti-inflammatory response. In some embodiments, the method enhances the proinflammatory response in situations such as infection or cancer.

[0183] Thus, provided are methods of treating an inflammatory disease in a subject in need thereof, comprising administering sialic acid or an analog thereof to the subject, wherein the sialic acid-containing ligand is present on a nanoparticle such that a proinflammatory immune response is suppressed or an anti-inflammatory immune response is increased in the subject.

[0184] The method may include: identifying a subject having a proinflammatory immune response and / or having a condition associated with or caused by a proinflammatory immune response, or being at risk for developing a proinflammatory immune response or a condition associated with or caused by a proinflammatory immune response; and administering a sialic acid-containing ligand to the subject, wherein the sialic acid-containing ligand is present on a nanoparticle.

[0185] In specific embodiments, the method can be used to treat a subject with a lung disease, including pulmonary and non-inflammatory conditions, but not limited to tuberculosis, chronic obstructive pulmonary disease (COPD), asthma, acute lung injury, acute respiratory distress syndrome, cystic fibrosis, bronchiectasis, pulmonary fibrosis, interstitial lung disease, pulmonary vascular disease, influenza, viral pneumonia, bacterial pneumonia, allergic bronchitis, non-allergic bronchitis, rhinitis, and fibrosing alveolitis.

[0186] In some embodiments, the method can be used to treat rheumatic diseases, including but not limited to rheumatoid arthritis, fibromyalgia, systemic lupus erythematosus, systemic sclerosis (scleroderma), psoriatic arthritis, ankylosing spondylitis, Sjögren's syndrome, polymyalgia rheumatica, gout, osteoarthritis, infectious arthritis, and juvenile idiopathic arthritis.

[0187] In some embodiments, the method can be used to treat gastrointestinal inflammatory diseases, including but not limited to Crohn's disease, ulcerative colitis, irritable bowel syndrome, celiac disease, diverticulitis, gastroesophageal reflux, lactose intolerance, peptic ulcer, cholecystitis, gastritis, colitis, pancreatitis, autoimmune hepatitis, hepatitis, infectious hepatitis, and pancreatitis.

[0188] In some embodiments, the method can be used to treat cardiovascular disease, including but not limited to septic shock, atherosclerosis, diastolic dysfunction, heart failure, cardiac fibrosis, coxsackie myocarditis, congenital heart block, autoimmune myocarditis, giant cell myocarditis, and inflammation.

[0189] In some embodiments, the method can be used to treat renal inflammation, including but not limited to renal transplant rejection, glomerulonephritis, acute nephritis, cystitis, prostatitis, diabetic nephritis, diabetic nephropathy, and urinary tract infection.

[0190] In some embodiments, the method can be used to treat inflammatory conditions of the skin, including but not limited to dermatitis, eczema, inflammatory rashes, scleroderma, keloids, acne, sarcoidosis, tinea cruris, tinea corporis, tinea pedis, tinea capitis, onychomycosis, rosacea, vitiligo, lichen sclerosus, autoimmune urticaria, dermatomyositis, and hidradenitis suppurativa.

[0191] In some embodiments, the method can be used to treat nervous system inflammation and degeneration, including but not limited to neuromyelitis, multiple sclerosis, encephalitis, neurosarcoids, Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease.

[0192] In some embodiments, the method can be used to treat autoimmune inflammatory diseases, including but not limited to diabetes, SLE, multiple sclerosis, Sjögren's syndrome, Addison's disease, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, celiac disease, pernicious anemia, alopecia areata, autoimmune hepatitis, autoimmune angioedema, autoimmune encephalomyelitis, autoimmune inner ear disease, Guillain-Barré syndrome, Kawasaki disease, Lambert-Eaton syndrome, Vogt-Koyanagi-Harada syndrome, systemic vasculitis, giant cell arteritis, sarcoidosis, and polyarteritis nodosa.

[0193] In some embodiments, the method can be used to treat viral inflammation, including but not limited to influenza A, influenza B, influenza C, SARS-CoV1, SARS-CoV2, Newcastle disease, Sendai virus, polyomavirus, HIV, flavivirus, calicivirus, herpes virus, picoronavirus, and coronavirus.

[0194] In some embodiments, the method can be used to treat fungal inflammatory conditions, including but not limited to fungemia, fungal abscesses, fungal keratitis, candidiasis, tinea pedis, and tinea cruris.

[0195] In some embodiments, the method can be used to treat parasitic inflammatory diseases, including but not limited to amebiasis, giardiasis, toxoplasmosis, and toxocariasis.

[0196] In some embodiments, the method can be used to treat fibrotic diseases, including but not limited to idiopathic pulmonary fibrosis, myelofibrosis, liver fibrosis, cardiac fibrosis with diastolic dysfunction and CHF, renal fibrosis, retinal fibrosis, skin fibrosis, and scarring.

[0197] In some embodiments, the method can be used to treat life-threatening acute inflammation, including but not limited to sepsis and cytokine storm. In specific embodiments, methods for treating a variety of ocular inflammatory diseases are provided, such as macular degeneration, uveitis, optic neuritis, neuromyelitis and inflammation caused by eye infection, eye exposure to drugs and toxins, and general immune disorders including autoimmune disorders. In non-limiting embodiments, useful methods for preventing, treating or improving macular degeneration in patients are provided, such as dry macular degeneration, wet macular degeneration, geographic atrophy, intermediate macular degeneration and age-related macular degeneration. Methods for treating, preventing or improving ocular inflammation including macular degeneration include administering a composition of sialic acid ligand nanoparticles to a patient suffering from or at risk of developing ocular inflammation such as macular degeneration.

[0198] In some embodiments, the ophthalmic formulation is provided in the form of eye drops, eye ointments, or eye injections. For eye injections, intravitreal or subconjunctival injections can be used to administer the nanoparticles.

[0199] The additional compound administered in combination for use in the method for treating, preventing or ameliorating macular degeneration can be co-administered with a pharmaceutical composition comprising nanoparticles for treating macular degeneration. For example, anti-angiogenic drugs for treating wet age-related macular degeneration, such as pegaptanib sodium, ranibizumab, bevacizumab, aflibercept, brolucizumab, etc., can be used in combination. Although specific embodiments of the present disclosure have been shown and described, it will be apparent to those skilled in the art that changes and modifications can be made without departing from the broader aspects of the present disclosure. Therefore, the appended claims will encompass within their scope all such changes and modifications that fall within the true spirit and scope of the present disclosure.

[0200] In exemplary embodiments, the compounds described herein may be used in methods of treating the following disorders and conditions:

[0201] Ophthalmic diseases such as age-related macular degeneration, dry eye, diabetic retinopathy, allergic eye conditions, and ocular fibrosis.

[0202] Neurological diseases such as Alzheimer's "neuroinflammation / degeneration, ALS, multiple sclerosis, neuropsychiatry "schizophrenia".

[0203] Cancers such as solid tumors (breast, lung, colon, prostate, brain "glioblastoma"), lymphomas, and leukemias.

[0204] Infections, such as viral infections including influenza, avian influenza and SARS-COV-2, bacterial infections such as meningococcal infection, streptococcal pneumonia and group B streptococcus.

[0205] Allergic conditions such as anaphylaxis, food allergies, and contact allergies.

[0206] Fibrosis, such as pulmonary fibrosis, hepatitis-induced fibrosis, and myelofibrosis.

[0207] Autoimmune conditions such as uveitis, arthritis, systemic lupus erythematosus, sepsis.

[0208] Kidney disease, such as AHUS or glomerulonephritis.

[0209] GI conditions, such as Crohn's disease or ulcerative colitis.

[0210] Heart diseases, such as atherosclerosis, "macrophage-driven" cardiac fibrosis, ischemic cardiomyopathy, and heart failure.

[0211] Inflammatory conditions.

[0212] In other embodiments, the compositions disclosed herein can inhibit pro-inflammatory responses and increase anti-inflammatory responses.

[0213] In one aspect, the pro-inflammatory response can be inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%. In alternative embodiments, the anti-inflammatory response can be increased by at least 10%, at least 20%, at least 30%, at least 40%, and at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99%.

[0214] Proinflammatory cytokines may be measured to determine the efficacy of the nanoparticle drug treatment. Such measurements may be made during actual treatment of the subject, or alternatively, during animal testing of the nanoparticles disclosed herein. In embodiments, proinflammatory cytokines may include, for example, TNF-α and IL-6. Anti-inflammatory cytokines, such as IL-10, may also be measured as appropriate. The skilled artisan will be aware of suitable assays for measuring such cytokines. For example, a Bio-Plex TM Cytokine Assay (Bio-Rad). To determine whether cells produce more or less pro-inflammatory cytokines, a suitable method can be used to resuspend the cells and culture them at 2×10 5 Cells are plated in 96-well plates at 100 cells / ml and 200 μl / well. The cells are then allowed to adhere to the plates overnight and treated with a range of LPS and ligand concentrations for 24 hours. The supernatant is then removed and stored at -70°C. Cytokine levels can then be assessed by ELISA (R&D Systems). It will be appreciated that similar methods can be used to determine anti-inflammatory cytokines.

[0215] In an embodiment, TNF-α levels can be appropriately measured by coating a 96-well plate with a TNF-α capture antibody diluted in 1× phosphate buffered saline (PBS) overnight. All steps can be performed at room temperature. The wells can be washed three times with 1× PBS / 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) and then blocked for one hour with 1% BSA (BDH) dissolved in 1× PBS. The washing step can be repeated, and 50 μl of treated cell supernatant or a standard ranging from 2000 pg / ml to 0 pg / ml can be added to the wells and left for 2 hours. The supernatant is then aspirated, the wells are washed three times, and 50 μl of TNF-α detection antibody diluted in 1% BSA / 1× PBS is added for 2 hours. Similarly, the wells can be washed three times, and a horseradish peroxidase (HRP)-conjugated antibody diluted in 1% BSA / 1× PBS at a dilution of 1 / 200 can be added for 20 minutes. At this stage, the plate can be covered with aluminum foil. After washing the wells, 3,3',5,5'-tetramethylbenzidine (TMB) can be added for 20 minutes and then protected from light again. The reaction can be stopped by adding 1M hydrochloric acid, and the absorbance at 450 nM can be read on a microplate reader. The TNF-α concentration can then be extrapolated based on the standard curve. It will be appreciated that similar methods can be applied to determine levels of other cytokines, substituting a TNF-α detection antibody for the detection antibody or other agents specific for the applicable cytokine.

[0216] In an embodiment, IL-10 levels can be appropriately measured by coating a 96-well plate with an IL-10 capture antibody diluted in 1× phosphate-buffered saline (PBS) overnight. All steps can be performed at room temperature. The wells can be washed three times with 1× PBS / 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) and then blocked for one hour with 1% BSA (BDH) dissolved in 1× PBS. The wash step can be repeated, and 50 μl of treated cell supernatant or standards ranging from 2000 pg / ml to 0 pg / ml can be added to the wells and left for 2 hours. The supernatant is then aspirated, the wells are washed three times, and 50 μl of IL-10 detection antibody diluted in 1% BSA / 1× PBS is added for 2 hours. Similarly, the wells can be washed three times, and a horseradish peroxidase (HRP)-conjugated antibody diluted in 1% BSA / 1× PBS at a dilution of 1 / 200 can be added for 20 minutes. At this stage, the plate can be covered with aluminum foil. After washing the wells, 3,3',5,5'-tetramethylbenzidine (TMB) can be added for 20 minutes and then protected from light again. The reaction can be stopped by adding 1M hydrochloric acid, and the absorbance at 450 nM can be read on a microplate reader. The IL-10 concentration can then be extrapolated based on the standard curve. It will be appreciated that similar methods can be applied to determine levels of other cytokines, substituting an IL-10 detection antibody for the detection antibody or other reagents specific for the applicable cytokine.

[0217] In order to determine whether the subject or test animal for treatment produces a greater or lesser pro-inflammatory response, a method that can be used is to analyze serum cytokine levels. For example, this can be achieved by collecting 50 μl of blood from the subject for treatment using a capillary tube. This blood is allowed to coagulate for 30 minutes at room temperature before centrifugation at 1300 rpm to precipitate red blood cells. The serum is poured into a clean microcentrifuge tube and analyzed by ELISA. For a more comprehensive analysis, a larger volume of blood (approximately 600 μl-1 ml) can be collected by direct puncture of the heart, thereby allowing a larger volume of serum to be collected and analyzed by ELISA or other such techniques. Other suitable techniques for determining whether the subject or test animal for treatment produces a greater or lesser pro-inflammatory response will be known in the art, particularly techniques for detecting and measuring cytokines.

[0218] Pharmaceutical composition

[0219] As used herein, "pharmaceutically acceptable carriers or excipients" may include any and all medically suitable inactive ingredients and / or solvents, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonicity agents, thickeners or emulsifiers, preservatives, solid binders, fillers, bulking agents, lubricants, etc., to suit the particular dosage form desired. Remington's Pharmaceutical Sciences, 18th ed., A.R. Geunaro (Mack Publishing Co., Easton, Pa., 1990) discloses various carriers used in formulating pharmaceutical compositions and known techniques for preparing them.

[0220] The present disclosure further provides the medicine or veterinary composition of the nanoparticle that comprises the ligand connection containing sialic acid disclosed herein.Such pharmaceutical composition is mixed with and is compatible with its expected route of administration.The example of route of administration includes parenteral and non-parenteral administration method simultaneously, including for example intravenous, intravitreal, oral, intraocular, subretinal, under the Tenon's fascia (subtenons), intrascleral, periocular, intravenous, nasal cavity and oral inhalation, intramuscular, intraarterial, intraspinal, intrathecal, intracranial, intradermal, percutaneous (topical), through mucosal, subcutaneous, lung lavage, gastric lavage, intrahepatic, subcutaneous and rectal administration.

[0221] Suitably, in some embodiments, nanoparticles can be administered parenterally. After parenteral administration, nanoparticles can selectively accumulate in specific tissues or body parts. In certain embodiments, nanoparticles can deliver therapeutic payload to cells or tissues. In certain embodiments, nanoparticles can enter diseased tissues through enhanced permeability and retention.

[0222] In general, the pharmaceutical composition provided comprises an effective amount of nanoparticles and a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant and / or carrier. Such compositions include: diluents with a variety of buffer contents (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizers (e.g., tween 80, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal (thimersol), benzyl alcohol) and fillers (e.g., lactose, mannitol). Such compositions may affect the physical state, stability, in vivo release rate and in vivo clearance rate of the nanoparticles. See, for example, Remington's Pharmaceutical Sciences [Lemington's Pharmaceutical Sciences], 18th edition (1990, Mack Publishing Co. [Mark Publishing Company], Easton, Pennsylvania (Easton, Pa.) 18042) pp. 1435-1712, which is incorporated herein by reference. The composition may be prepared in liquid form, or may be formulated as a dry powder, for example in lyophilized form.

[0223] The term "pharmaceutically acceptable salts" also refers to salts prepared from the compounds disclosed herein, or any other compound described herein, that have an acidic functional group, such as a carboxylic acid functional group, and a pharmaceutically acceptable inorganic or organic base.

[0224] Also included are pharmaceutically acceptable salts of the compounds of the invention. For example, acidic salts of compounds containing amines or other basic groups can be obtained by reacting the compounds of the invention with a suitable organic or inorganic acid to form pharmaceutically acceptable anionic salt forms. Examples of anionic salts include acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camphorsulfonate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, propionate lauryl sulfate, ethanesulfonate, fumarate, glyceptate, gluconate, glutamate, glycolylaminophenylarsinate, hexylresorcinate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, methanesulfonate, methylsulfate, mucate, naphthenate, nitrate, pamoate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theoclate, tosylate, and triethyl iodide.

[0225] Salts of the compounds of the invention containing carboxylic acid or other acidic functional groups can be prepared by reaction with a suitable base. Pharmaceutically acceptable salts can be made with bases that provide pharmaceutically acceptable cations, including alkali metal salts (particularly sodium and potassium), alkaline earth metal salts (particularly calcium and magnesium), aluminum salts, and ammonium salts, as well as salts made from physiologically acceptable organic bases such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine, tris-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, dehydroabietinamine, N,N'-didehydroabietinamine, glucosamine, N-methylglucosamine, collidine, quinine, quinoline, and basic amino acids such as lysine and arginine.

[0226] Therefore, in a first exemplary embodiment, the present invention is a compound represented by any one of the following structural formulas: or a pharmaceutically acceptable salt thereof.

[0227] In a first aspect of the first exemplary embodiment, for each occurrence, R1 is independently -C(O)-A, wherein A is C1-C6 alkyl, C6-C 18 Aryl, (C6-C 18 )aryl(C1-C3)alkyl, 5-18 membered heteroaryl, (5-18 membered)heteroaryl(C1-C3)alkyl, C3-C8 cycloalkyl, (C3-C8)cycloalkyl(C1-C3)alkyl, 5-8 membered heterocycloalkyl, or (5-8 membered)heterocycloalkyl(C1-C3)alkyl; wherein one or two carbon atoms of the alkyl portion of A are each independently replaced by a heteroatom selected from N, O, or S. (Examples of the alkyl portion of A including heteroatoms are CH3-CH2-O-CH2-CH2-, CH3-NH-CH2-, CH3-N(CH3)-CH2-, CH3-S-CH2-CH2-, etc.). Further, A is optionally replaced by 1 to 3 R 11 Group substitution, each of the R 11 The groups are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, halogen, C 6- C 12 Aryl, 5-12 membered heteroaryl, cyano, or two R 11 The groups, together with the atoms to which they are attached, form a 5-7 membered heterocyclic group having 1 to 3 heteroatoms selected from N, O or S; wherein each R 11is independently optionally substituted with 1 to 3 substituents selected from halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, or cyano; and further wherein: for each occurrence, R is independently -R L -R F , and where: for each occurrence, R L Independently: -O-(C1-C 12 )alkylene-, -O-, -S-, -NR 100 -、-S-(C1-C 12 )alkylene-、-NR 101 -(C1-C 12 )alkylene-、-NR 101a -O-(C1-C 12 )alkylene-;-O-(CH2CH2O) m -、-O-(CH2CH2O) k -(CH2CH2)-、-NR 102 -X 100 -(C1-C 12 )alkylene-、-NR 102a -NR 102b -C(O)-(C1-C 12 ) alkylene, wherein R 100 、R 101 、R 101a 、R 102 、R 102a and R 102b are each independently H or C1-C3 alkyl, and X 100 is -O- or -NH-, and wherein m and k are each independently an integer from 1 to 12; for each occurrence, R F are independently: H, C1-C3 alkyl, -NH2, -NH-Fmoc, -NH-Boc, -NH-CBz, -NH-Troc, -NH-TFA, mono(C1-C3)alkylamino, di(C1-C3alkyl)amino; -(O)-R 103 , where R 103 is -H, -OH, or (C1-C3)alkyl; -SH, by the following structural formula Represents part; or click chemistry reagent.

[0228] In a second aspect of the first exemplary embodiment, the compound is represented by any one of the following structural formulas: or a pharmaceutically acceptable salt thereof.The values ​​and exemplary values ​​of the variables in formulas (S-1-1), (S-2-1), (D-1-1), and (D-2-1) are defined above for the first aspect.

[0229] In a third aspect of the first exemplary embodiment, the click chemistry reagent comprises an azide, a C2-C3 alkyne, a tetrazine, a trans-cyclooctene, or a cyclooctyne. The remaining values ​​and exemplary values ​​are as defined above for the first and second aspects.

[0230] In a fourth aspect of the first exemplary embodiment, the click chemistry reagent is an azide, a C2-C3 alkyne, or a moiety represented by any one of the following structural formulas: Where: R co is hydrogen or halogen; X is absent or is O; and in each case, R 0 independently selected from hydrogen, halogen, C 1-8 Alkyl, C 1-8 Alkoxy, C6-C 12 Aryl, 5-8 membered heteroaryl, C 3-8 Cycloalkyl, or 3-8 membered heterocyclic group; any two or more R 0 The groups may optionally form, together with the atoms to which they are attached, an unsaturated, saturated, or aromatic 5-8 membered ring; and R td It is hydrogen, C 1-8 Alkyl, C 1-8 Alkoxy, C6-C 12 Aryl, 5-8 membered heteroaryl, C 3-8 The remaining values ​​and exemplary values ​​are as defined above for the first to third aspects.

[0231] In a fifth aspect of the first exemplary embodiment, the click reagent is any one of an azide, a C2-C3 alkyne, or a moiety represented by the following structural formula: Where R* is H or methyl, for each occurrence, R # are independently H or C1-C3 alkyl, and R X is -NH-C(O)O-. The remaining values ​​and exemplary values ​​are as defined above for the first to fourth aspects.

[0232] In a sixth aspect of the first exemplary embodiment, for each occurrence, R is independently -O-(CH2) x -NH2 or -O-(CH2) x-NH-Fmoc, wherein for each occurrence, x is independently an integer from 1 to 10, for example, x is 5. The remaining values ​​and exemplary values ​​are as defined above for the first to fifth aspects.

[0233] In another aspect, for each occurrence, R is independently -O-(CH2) x -N3, wherein for each occurrence, x is independently an integer from 1 to 10, for example, x is 5.

[0234] In a seventh aspect of the first exemplary embodiment, the cycloalkyl portion of portion A is selected from C6-C7 cycloalkyl; the heterocycloalkyl portion of portion A is selected from 5-6 membered heterocycloalkyl having 1 or 2 heteroatoms selected from N, O or S; the aryl portion of portion A is selected from phenyl or naphthyl; and the heteroaryl portion of portion A is selected from a moiety represented by any one of the following structural formulas: where X 1 、X 2 and X 3 Each independently selected from NR H , O or S, where R H is H or C1-C3 alkyl; and wherein each R, if present, 11 are independently selected from cyano, halogen, phenyl, halophenyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C3 alkoxy, or two R 11 The groups, together with the atoms to which they are attached, form a [1,3]dioxolone group optionally substituted with one or two methyl or ethyl groups.The remaining values ​​and exemplary values ​​are as defined above for the first to sixth aspects.

[0235] In an eighth aspect of the first exemplary embodiment, for each occurrence, R1 is independently selected from a moiety represented by the following structural formula, wherein the wavy line represents the point of attachment of R1 to the nitrogen atom:

[0236] The remaining values ​​and exemplary values ​​are as defined above for the first to seventh aspects.

[0237] In a ninth aspect of the first exemplary embodiment, the compound is any one of compounds S-101 to S-131 or D-101 to D-131, wherein the moiety R is -O-(CH2)5-NH-Fmoc.

[0238] In a further aspect, the compound is any one of compounds S-101 to S-131 or D-101 to D-131, wherein the moiety R is -O-(CH2)5-NH2 or -O-(CH2)5-N3.

[0239] In a second exemplary embodiment, the present invention is a particle comprising a compound represented by the following structural formula: GLP.

[0240] In a first aspect of the second exemplary embodiment, P is a biocompatible polymer; L is a covalent linker; and G is any one of the moieties represented by the following structural formulas: or a pharmaceutically acceptable salt thereof.

[0241] In formulae (S-1A), (S-2A), (D-1A) and (D-2A): Symbol Represents the point of attachment to L; for each occurrence, R1 is independently -C(O)-A, wherein A is C1-C6 alkyl, C6-C 18 Aryl, (C6-C 18 )aryl(C1-C3)alkyl, 5-18 membered heteroaryl, (5-18 membered)heteroaryl(C1-C3)alkyl, C3-C8 cycloalkyl, (C3-C8)cycloalkyl(C1-C3)alkyl, 5-8 membered heterocycloalkyl, or (5-8 membered)heterocycloalkyl(C1-C3)alkyl; wherein one or two carbon atoms of the alkyl portion of A are each independently replaced by a heteroatom selected from N, O, or S. (Examples of alkyl portions of A including heteroatoms are CH3-CH2-O-CH2-CH2-, CH3-NH-CH2-, CH3-N(CH3)-CH2-, CH3-S-CH2-CH2-, etc.) A is optionally replaced by 1 to 3 R 11 Group substitution, each of the R 11 The groups are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, halogen, C 6- C 12 Aryl, 5-12 membered heteroaryl, cyano, or two R 11 The groups, together with the atoms to which they are attached, form a 5-7 membered heterocyclic group having 1 to 3 heteroatoms selected from N, O or S; wherein each R 11 Independently optionally substituted with 1 to 3 substituents selected from halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, or cyano.

[0242] In a second aspect of the second exemplary embodiment, the portion G is represented by any one of the following structural formulae: or a pharmaceutically acceptable salt thereof. The remaining values ​​and exemplary values ​​are as defined above for the first aspect.

[0243] In some aspects, the biocompatible polymer can comprise at least one biocompatible polymer selected from the group consisting of polyglycolic acid, poly(lactic acid), poly(lactic-co-glycolic acid), polycaprolactone, poly(3-hydroxybutyric acid), poly(ethylene glycol), polyethylene oxide, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (Pluronic F127), polyoxyethylene-polyoxypropylene block copolymer (Pluronic F68), poloxamer, poly(hydroxymethyl methacrylate), polyvinyl alcohol, poly(vinyl pyrrolidone), hyaluronic acid, heparin, heparin sulfate, polysialic acid, and chitosan. For example, the polymer can comprise the copolymer PLGA-PEG. The remaining values ​​and exemplary values ​​are as defined above for the first and second aspects.

[0244] In a third aspect, the biocompatible polymer comprises at least one of polyglycolic acid, poly(lactic acid), poly(lactic-co-glycolic acid), polycaprolactone, poly(3-hydroxybutyric acid), polyethylene oxide, polyoxyethylene-polyoxypropylene block copolymer, poly(hydroxymethyl methacrylate), polyvinyl alcohol, poly(vinyl pyrrolidone), hyaluronic acid, heparin, heparin sulfate, polysialic acid, and chitosan. The remaining values ​​and exemplary values ​​are as defined above for the first and second aspects.

[0245] In a fourth aspect, the biocompatible polymer comprises poly(L-lactic acid), poly(D-lactic acid), poly(D / L-lactic acid), copolymers thereof, or combinations thereof. The remaining values ​​and exemplary values ​​are as defined above for the first and second aspects.

[0246] In a fifth aspect, the biocompatible polymer comprises polyethylene oxide, a polyoxyethylene-polyoxypropylene block copolymer, a copolymer thereof, or a combination thereof.The remaining values ​​and exemplary values ​​are as defined above for the first and second aspects.

[0247] In a sixth aspect of the second exemplary embodiment, the polymer P is represented by the following structural formula:

[0248] The symbols represents the point of attachment of the polymer to the linker L, and further wherein: y is an integer from 0 to 1000, x is an integer from 0 to 1000, and m is an integer from 0 to 450, with the proviso that x and y are not both 0. The remaining values ​​and exemplary values ​​are as defined above for the first to fifth aspects.

[0249] In some examples of the sixth aspect, y is an integer from 0 to 500, x is an integer from 0 to 500, and m is an integer from 0 to 250. For example, in various fields, m is 50-200, or m is 75-175, or m is 90-150, or m is 90-140, or m is 100-130. In various fields, x is 50-200, or x is 75-175, or x is 90-150, or x is 90-140, or x is 100-130. In other aspects, y is 5-100, or y is 10-75, or y is 15-50. Any combination of the value of x, y and m based on the above various fields is also contemplated. For example, m can be 100-130, x can be 100-130 and y can be 15-50.

[0250] In another example of the sixth aspect, x is an integer from 90 to 140; y is an integer from 10 to 75; and m is an integer from 90 to 140.

[0251] The remaining values ​​and exemplary values ​​are as defined above for the first to fifth aspects.

[0252] In a seventh aspect, the particle further comprises one or more of: acid-terminated PLGA (PLGA-COOH); acid-terminated PLGA-PEG copolymer, wherein the acid moiety terminates a PEG block (PLGA-PEG-COOH); PLGA-PEG copolymer, wherein the PEG block is terminated with a moiety represented by the following structural formula: or PLGA-PEG copolymers, wherein the PEG block is terminated by a moiety represented by the following structural formula: The dotted line indicates the point of attachment of the capping moiety to the PEG block.

[0253] In an example of the seventh aspect, the particle comprises at least one of: a blend of 75% by weight PLGA-COOH and 25% by weight PLGA-PEG-DBCO; a blend of 75% by weight PLGA-COOH and 25% by weight PLGA-PEG-NHS; a blend of 90% by weight PLGA-PEG-COOH and 10% by weight PLGA-PEG-DBCO; and PLGA-PEG-NHS.

[0254] The remaining values ​​and exemplary values ​​are as defined above for the first to sixth aspects.

[0255] In an eighth aspect of the second exemplary embodiment, the linker L comprises a moiety that is a product of a click chemistry reaction. The remaining values ​​and exemplary values ​​are as defined above for the first to seventh aspects.

[0256] In a ninth aspect of the second exemplary embodiment, the linker L comprises a moiety represented by any one of the following structural formulae, wherein the symbol and Each independently represents the point of attachment to the other part of the linker L, to P, or to G: Where R* is H or methyl, for each occurrence, R # are independently H or C1-C3 alkyl, and R X is -NH-C(O)O-, and R 200 is -H or C1-C3 alkyl. Examples of additional moieties of the linker are R as defined above for the first exemplary embodiment or any aspect thereof. L An example of an additional part of a linker is -O-(C1-C 12 )alkylene, such as a -O-(CH2)5 moiety. The remaining values ​​and exemplary values ​​are as defined above for the first to eighth aspects.

[0257] In a tenth aspect of the second exemplary embodiment, polymer P is PLGA(10k)-PEG(5k).The remaining values ​​and exemplary values ​​are as defined above for the first to ninth aspects.

[0258] In an eleventh aspect of the second exemplary embodiment, the weight of G per unit weight P (ligand density) is from 10 to 75 μg / mg. The remaining values ​​and exemplary values ​​are as defined above for the first to tenth aspects.

[0259] In a twelfth aspect of the second exemplary embodiment, the weight of G (also referred to herein as sialic acid-containing ligand) per weight of total solids is from about 1 μg / mg to about 1000 μg / mg.

[0260] In a third exemplary embodiment, the invention is a method of treating a disorder in a subject in need thereof.

[0261] In a first aspect of the third exemplary embodiment, the method comprises administering to a subject an effective amount of a compound or a composition comprising the particles described herein or a pharmaceutically acceptable salt thereof, wherein the condition is selected from cancer, ophthalmic disease, fibrotic disease, parasitic inflammation, fungal inflammation, viral inflammation, autoimmune inflammation, nervous system inflammation, neurodegeneration, skin inflammation, kidney inflammation, cardiovascular disease, gastrointestinal inflammation, or rheumatic disease.

[0262] In a fourth exemplary embodiment, the invention is a method of treating a condition in a subject in need thereof.

[0263] In a first aspect of the fourth exemplary embodiment, the method comprises administering to a subject an effective amount of a compound or a composition comprising the particles described herein or a pharmaceutically acceptable salt thereof, wherein the condition is selected from breast cancer, non-small cell lung cancer (NSCLC), prostate cancer, colorectal cancer, melanoma, pancreatic cancer, myelofibrosis, diabetic retinopathy, idiopathic pulmonary fibrosis, liver fibrosis, sickle cell anemia, and acute respiratory distress syndrome (ARDS).

[0264] In a fifth exemplary embodiment, the invention is a pharmaceutical composition comprising a compound or particle as described herein for the first and second exemplary embodiments and various aspects and exemplary aspects thereof, or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier.

[0265] In a sixth exemplary embodiment, the present invention is a method of preparing particles comprising a molecule represented by the following structural formula: GLP.

[0266] The values ​​and exemplary values ​​of the variables G, L, and P of the sixth exemplary embodiment are defined above for the first and second exemplary embodiments and various aspects thereof.

[0267] In a first aspect of the sixth exemplary embodiment, the method comprises: F1 and R F2 Under conditions where the parts react with each other, the compound represented by structural formula (I) GR F1 (I) Reacting with a compound represented by structural formula (II) PR F2 (II), where R F1 and R F2 Each is a reactive moiety, thereby generating the covalent -L- moiety.

[0268] In the second aspect of the sixth exemplary embodiment, R F1 and R F2 is a pair of click reagents, and wherein the conditions are suitable for causing R F1 and R F2 Click reaction between, wherein the linker L includes R F1 and R F2 The remaining values ​​and exemplary values ​​of the variables are as defined above for the first aspect.

[0269] In a third aspect, the biocompatible polymer comprises at least one biocompatible polymer selected from the group consisting of polyglycolic acid, poly(L-lactic acid), poly(lactic-co-glycolic acid), polycaprolactone, poly(3-hydroxybutyric acid), polyethylene oxide, polyoxyethylene-polyoxypropylene block copolymer, poly(hydroxymethyl methacrylate), polyvinyl alcohol, poly(vinyl pyrrolidone), hyaluronic acid, heparin, heparin sulfate, polysialic acid, and chitosan. The remaining values ​​and exemplary values ​​are as defined above for the first and second aspects.

[0270] In a seventh exemplary embodiment, the present invention is a compound represented by the following structural formula:

[0271] or a pharmaceutically acceptable salt thereof. In a first aspect of the seventh exemplary embodiment, for each occurrence, R is independently -R L -R F , and where: for each occurrence, R L are independently: -O-, -S-, -NR 100 -、-O-(C1-C 12 )alkylene-, -S-(C1-C 12 )alkylene-、-NR 101 -(C1-C 12 )alkylene-、-NR 101a -O-(C1-C 12 )alkylene-;-O-(CH2CH2O) m -、-O-(CH2CH2O) k -(CH2CH2)-、-NR 102 -X 100 -(C1-C 12 )alkylene-、-NR 102a -NR 102b -C(O)-(C1-C 12 ) alkylene, wherein R 100 、R 101 、R 101a 、R 102 、R 102a and R 102b are each independently H or C1-C3 alkyl, and X 100 is -O- or -NH-, and wherein m and k are each independently an integer from 1 to 12; for each occurrence, R F Independently it is: H, C1-C3 alkyl, -NH2, -NH-Fmoc, -NH-Boc, -NH-CBz, -NH-Troc, -NH-TFA, mono(C1-C3)alkylamino, di(C1-C3)alkylamino; -C(O)-R 103 , where R 103 is -H, -OH, or (C1-C3)alkyl; -SH, by the following structural formula the part represented; Or click Chemical Reagents.

[0272] In one aspect of the seventh exemplary embodiment, –R L -R F Not –OH.

[0273] In various further aspects, the values ​​and exemplary values ​​of the variable R are described above with respect to the first to sixth exemplary embodiments and various aspects thereof.

[0274] In an eighth exemplary embodiment, the present invention provides a particle comprising a molecule represented by the following structural formula: GLP.

[0275] In a first aspect of the eighth exemplary embodiment, P is a biocompatible polymer, L is a covalent linker; and G is represented by the following structural formula: or a pharmaceutically acceptable salt thereof, wherein the symbol Represents the attachment point to L.

[0276] In other respects, the values ​​and exemplary values ​​for the variables L and P are described above with respect to the second exemplary embodiment and various aspects thereof.

[0277] In a second aspect, the biocompatible polymer comprises at least one of polyglycolic acid, poly(lactic acid), poly(lactic-co-glycolic acid), polycaprolactone, poly(3-hydroxybutyric acid), polyethylene oxide, polyoxyethylene-polyoxypropylene block copolymer, poly(hydroxymethyl methacrylate), polyvinyl alcohol, poly(vinyl pyrrolidone), hyaluronic acid, heparin, heparin sulfate, polysialic acid, and chitosan. The remaining values ​​and exemplary values ​​are as defined above for the first and further aspects of the eighth embodiment.

[0278] In a second aspect, the biocompatible polymer comprises poly(L-lactic acid), poly(D-lactic acid), poly(D / L-lactic acid), copolymers thereof, or combinations thereof. The remaining values ​​and exemplary values ​​are as defined above for the first and further aspects of the eighth embodiment.

[0279] In a third aspect, the biocompatible polymer comprises polyethylene oxide, a polyoxyethylene-polyoxypropylene block copolymer, copolymers thereof, or combinations thereof.The remaining values ​​and exemplary values ​​are as defined above for the first and further aspects of the eighth embodiment.

[0280] In a fourth aspect, the polymeric scaffold comprises the block copolymer PLGA-PEG.The remaining values ​​and exemplary values ​​are as defined above for the first and further aspects of the eighth embodiment.

[0281] In a fifth aspect, the polymer P is represented by the following structural formula: The symbols represents the point of attachment of the polymer to the linker L, and further wherein: y is an integer from 0 to 1000, x is an integer from 0 to 1000, and m is an integer from 0 to 450, with the proviso that x and y are not both 0. The remaining values ​​and exemplary values ​​are as defined above for the first to fourth and further aspects of the eighth embodiment.

[0282] In certain instances of the fifth aspect, y is an integer from 0 to 500, x is an integer from 0 to 500, and m is an integer from 0 to 250. For example, in various aspects, m is 50-200, or m is 75-175, or m is 90-150, or m is 90-140, or m is 100-130. In various aspects, x is 50-200, or x is 75-175, or x is 90-150, or x is 90-140, or x is 100-130. In further aspects, y is 5-100, or y is 10-75, or y is 15-50. In embodiments of the fifth aspect, x is an integer from 90 to 140; y is an integer from 10 to 75; and m is an integer from 90 to 140. The remaining values ​​and exemplary values ​​are as defined above for the first to fourth and further aspects of the eighth embodiment.

[0283] In a sixth aspect, the particle further comprises one or more of: acid-terminated PLGA (PLGA-COOH); acid-terminated PLGA-PEG copolymer, wherein the acid moiety terminates a PEG block (PLGA-PEG-COOH); PLGA-PEG copolymer, wherein the PEG block is terminated with a moiety represented by the following structural formula: or PLGA-PEG copolymers, wherein the PEG block is terminated by a moiety represented by the following structural formula: The dotted line indicates the point of attachment of the capping moiety to the PEG block.

[0284] The remaining values ​​and exemplary values ​​are as defined above for the first to fifth and further aspects of the eighth embodiment.

[0285] In a seventh aspect, the particles comprise at least one of: a blend of 75% by weight PLGA-COOH and 25% by weight PLGA-PEG-DBCO; a blend of 75% by weight PLGA-COOH and 25% by weight PLGA-PEG-NHS; a blend of 90% by weight PLGA-PEG-COOH and 10% by weight PLGA-PEG-DBCO; and PLGA-PEG-NHS. The remaining values ​​and exemplary values ​​are as defined above for the first to fourth and further aspects of the eighth embodiment.

[0286] In a ninth exemplary embodiment, the invention is a pharmaceutical composition comprising a compound as described for the seventh exemplary embodiment, or any aspect thereof, or a particle as described for the eighth exemplary embodiment, or any aspect thereof, or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier.

[0287] In a tenth exemplary embodiment, the present invention is a method for treating a condition in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of a compound as described above for the seventh exemplary embodiment and various aspects thereof, or a pharmaceutically acceptable salt thereof, or a composition comprising particles as described above for the eighth exemplary embodiment and various aspects thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the ninth exemplary embodiment. In one aspect, the condition is influenza. The influenza can be human influenza or avian influenza.

[0288] In an eleventh exemplary embodiment, the present invention is any one of the compounds represented by the structural formula listed in Table 4, or a pharmaceutically acceptable salt thereof, wherein the variable R is -O-(CH2)5-NH2, -O-(CH2)5-NH-Fmoc, -O-(CH2)5-N3 or -O-(CH2)5-biotin.

[0289] In a twelfth exemplary embodiment, the present invention is a compound represented by any one of the following structural formulae or a pharmaceutically acceptable salt thereof:

[0290]

[0291]

[0292]

[0293]

[0294]

[0295] (S106-NP01);

[0296]

[0297]

[0298]

[0299]

[0300] wherein, for each occurrence, x is independently an integer from 90 to 140; y is independently an integer from 10 to 75; and m is independently an integer from 90 to 140. Examples

[0301] Example 1A: Synthesis of S-Compounds - Procedure A

[0302] The following reaction scheme is used to synthesize 6-sulfo-sialyl Lewis X derivatives (S series compounds).

[0303] Scheme 1: Attachment of terminally modified fragments

[0304] In Scheme 1, the notation S1XX represents a compound in which the substituent at position 5 of the terminal sialic acid is one of the fragments described below in Table 1, and the notation S2XX represents a compound in which position 9 of the terminal sialic acid is substituted with one of the fragments described below in Table 1.

[0305] I. Synthesis of Precursors 30 and 35

[0306] Precursor compounds 30 and 35 of Scheme 1 were synthesized as follows.

[0307] 1. Synthesis of galactose structural units.

[0308] Plan 1-1

[0309]

[0310] The following conditions were used in the reaction shown in Scheme 1-1: a) BzCl, pyridine; b) AcOH, Ac2O, HBr; c) Ag2CO3, acetone:H2O; d) CNCCl3, DBU.

[0311] More specifically, in a 1 L round-bottom flask in an ice bath, galactose (30 g, 166 mmol) was dissolved in 400 mL of pyridine. During 1 h, benzoyl chloride (154 mL, 1.33 mol) was added dropwise to the cooled solution. After adding all the benzyl chlorides, the reaction was taken out of the ice bath and mixed at room temperature for 2 hours, which resulted in the formation of a large amount of salts. The elution was performed by TLC (30% EtOAC / Hex, R f =0.8) monitor reaction and when completing, remove salt by vacuum filtration.Excess pyridine is evaporated under reduced pressure, and crude material is dissolved among the EtOAc (1L), uses 5%H continuously SO (5x 100mL), saturated CuSO (2x 100mL), 1MHCl (2x 100mL), salt solution (2x 100mL) washing, through MgSO Drying, and evaporated under reduced pressure solvent.The oily matter of gained is transferred in the 2L round-bottomed flask equipped with condenser and magnetic bar and is placed in oil bath.Under vigorous stirring, round-bottomed flask is heated to reflux, with adding MeOH (about 1L in total).This process causes forming desired full benzoylation galactose 1, is collected and in next step without further purification by vacuum filtration and promptly uses.In ice bath, under vigorous stirring, 1 is suspended in diacetyl anhydride (50mL) and acetic acid (80mL). To this suspension was added dropwise hydrogen bromide in acetic acid (204 mL, 833 mmol) over 1 h; the mixture was allowed to reach room temperature. After approximately 2 h, the reaction mixture became translucent orange. The precipitate was analyzed by TLC (20% EtOAc / Hex, R f =0.50) was observed for complete conversion, the reaction was diluted with EtOAc (1 L), extracted with DI water (5 x 200 mL), quenched with sodium bicarbonate (100 mL of DI water, then solid sodium bicarbonate), brine (2 x 100 mL), dried over MgSO4 and the volume was reduced to yield 2 (104 g, 95% over two steps) as a white solid. To a solution of 2 (104 g, 158 mmol) in acetone (300 mL) and water (14.2 mL) was added freshly prepared Ag2CO3 (22 g, 79 mmol). The reaction was kept in the dark at room temperature and analyzed by TLC (30% EtOAc / Hex, R f=0.45). When all the starting material was consumed, the reaction was filtered through celite, reduced in vacuo, dissolved in EtOAc (500 mL), washed with saturated sodium bicarbonate (3x100 mL), brine (2x 100 mL), dried over MgSO4, and reduced in volume to yield the desired product 3. The fully benzoylated galactol 3 was dissolved in DCM (316 mL) containing trichloroacetonitrile (48 mL, 470 mmol). To this solution was added DBU dropwise until the reaction pH reached approximately 10. The product was analyzed by TLC (15% EtOAc / Hex, R f =0.2) to monitor the progress of the reaction and reduce the reaction volume when all the starting material has been consumed. The desired product 4 was purified by flash chromatography (15% EtOAc / Hex) to give the desired α-imidate (79.05 g, 68% over two steps) as a white solid. 1 The H data are consistent with those reported in the literature.

[0312] 2. Synthesis of N-trifluoroacetyl-glucosamine structural units.

[0313] Plan 1-2

[0314]

[0315] The following conditions were used in the reaction shown in Scheme 1-2: a) ethyl trifluoroacetate, sodium methoxide; b) Ac2O; c) hydrazine acetate; d) CNCCl, DBU; e) 5-azidopentan-1-ol, TMSOTf; f) sodium methoxide; g) benzaldehyde dimethyl acetal; CSA; h) Ac2O; i) TFA, triethylsilane.

[0316] More specifically, glucosamine hydrochloride (GlcNH , 10 g, 46.3 mmol) was added to a 500 mL round-bottom flask containing MeOH (130 mL) and sodium methoxide (51 mL, 1 M stock solution). The solution was stirred at room temperature until the GlcNAH dissolved, at which time ethyl trifluoroacetate (6.2 mL, 51 mmol) was added in one go. The mixture was stirred at room temperature and the color was analyzed by TLC (20% MeOH / DCM, R f =0.5) monitoring. After completion, the solvent was reduced in vacuo to provide crude 5. In an ice bath, 5 was dissolved in pyridine (70 mL). During 30 min, acetic anhydride (22 mL, 231.5 mmol) was added dropwise to the solution, and the reaction was then warmed to room temperature. The product was analyzed by TLC (40% EtOAC / hex, R f=0.48) assessment shows that the reaction was complete after 2 hours. Crude reaction EtOAc (500mL) is diluted and water-soluble impurities are extracted with HCl (5x 100mL) and saline (3x 100mL). The organic layer is dried over MgSO4 and dried under reduced pressure to produce tetraacetate intermediate 6. Crude intermediate 6 is dissolved in THF (156mL) and in the solution is added a methanol solution of freshly prepared 2M acetic acid hydrazine (27.8mL, 55.5mmol). By TLC (1: 1EtOAc / Hex, R f =0.47) to monitor the deprotection of the epimeric acetates and when all the starting material was consumed, the reaction volume was reduced and the crude 7 was dissolved in EtOAc (500 mL), washed with 1 M HCl (3 x 100 mL), brine (2 x 100 mL), dried over MgSO4 and dried under reduced pressure. Intermediate 7 was dissolved in DCM (176 mL) and to this solution was added trichloroacetonitrile (9.2 mL, 96.6 mmol). The reaction was mixed at room temperature and the pH was adjusted to 9 by the addition of DBU. After 1 hour the crude product was analyzed by TLC (40% EtOAc / Hex, R f =0.51) product formation was observed. The reaction volume was reduced in vacuo and the product was purified by flash chromatography (20%→40% EtOAc / Hex) to afford the desired imidate 8 (15.2 g, 52% over 4 steps) as a white foam. To a 500 mL round-bottom flask was added 7 (12.7 g, 23.2 mmol), 5-azidopentan-1-ol (3.6 g, 28 mmol) and Molecular sieves (5 g). The components were dissolved in DCM (232 mL) and stirred at room temperature for 30 min, after which the reaction was placed in an ice bath for 30 min. TMSOTf (840 μL, 4.64 mmol) was added to the cooled solution and the product was analyzed by TLC (40% EtOAc / Hex, R f =0.4) to monitor the reaction. After the starting material was converted to the glycosylated product, the reaction was quenched by adding triethylamine (970 μL, 6.96 mmol) and the product 9 was purified by flash chromatography (35% EtOAc / Hex) to produce a white solid (10 g, 85%). The protected advanced intermediate 9 (10 g, 19.5 mmol) was suspended in MeOH (195 mL) and the pH was adjusted to 8 by adding sodium methoxide. The reaction was analyzed by TLC (10% MeOH / DCM, R f =0.45) the desired triol was observed and the reaction was separated using Dowex 50WX8H +The resin was neutralized. The reaction was filtered and MeOH was removed under reduced pressure to yield 10 as a light yellow wax, which was carried on to the next step without further purification. 10 (7.5 g, 19.4 mmol) was dissolved in ACN (190 mL) and benzaldehyde dimethyl acetal (3.2 mL, 21.3 mmol) and camphorsulfonic acid were added to this solution until pH reached 3. The reaction mixture was moved to a rotary evaporator, which was distilled at 50 ° C under reduced pressure on a rotary evaporator. The product was analyzed by TLC (40% acetone / Hex, R f =0.6) to monitor the reaction and after all the starting material has been consumed, the pH of the solution is adjusted to 7 with triethylamine. The solvent is removed to give the benzylidene intermediate 11 as a brown solid, which is dissolved in DCM (87 mL). Pyridine (6 mL, 77.6 mmol) and acetic anhydride (3.6 mL, 38.8 mmol) are added to the reaction and it is stirred at room temperature. The results are shown in Table 1 using TLC (40% acetone / Hex, R f =0.65) to monitor the reaction progress. After consuming all the starting materials, the reaction was decanted, resuspended in EtOAc (300 mL), washed with 1M HCl (3x50 mL), brine (2x 50 ml), dried over MgSO4, and dried in vacuo. The resulting solid was recrystallized using EtOAc and Hex to produce the 3-O-Ac product 12 (6.2 g, 67% over 3 steps). Selective benzyl ring opening was achieved by dissolving 12 (2 g, 3.88 mmol) and DCM (33 mL). The reaction was placed in an ice bath, and triethylsilane (3.2 mL, 19.4 mmol) and trifluoroacetic acid (1.48 mL, 19.4 mmol) were added to the cooled solution. The product was analyzed by TLC (40% acetone / Hex, R f =0.62) and when all 12 was consumed, the reaction was diluted with EtOAc (200 mL) and extracted with sodium bicarbonate (3 x 50 mL), brine (2 x 50 mL), and dried over MgSO 4. The crude reaction mixture was purified by flash chromatography (30% acetone / Hex) to provide 13 (1.52 g, 76%) as a clear oil, which turned to a white solid upon standing.

[0317] 3. Disaccharide glycosylation

[0318] Options 1-3

[0319]

[0320] The following conditions were used in the reactions shown in Schemes 1-3: TMSOTf, 4AMS.

[0321] More specifically, acceptor 13 (1.52 g, 3 mmol) and donor 4 (2.8 g, 3.75 mmol) were dissolved in DCM (30 mL) and to this solution was added MS (2 g). The solution was stirred at room temperature for 30 min and then placed in an ice bath for 30 min. TMSOTf (135 μL, 0.75 mmol) was added to the cooled solution in one portion. The reaction was kept on ice under vigorous stirring. After 30 min, TLC (30% EtOAc / Hex, R f = 0.35) indicated complete disappearance of 4 and 13 and production of the desired disaccharide 14. The reaction was quenched with triethylamine (167 μL, 1.2 mmol) and purified by flash chromatography (30% EtOAc / Hex) to yield pure 14 (1.3 g, 40%) as a white foam.

[0322] 4.6-O-Benzyl Deprotection

[0323] Options 1-4

[0324]

[0325] The following conditions were used in the reactions shown in Schemes 1-4: sodium dithionite / sodium bromate.

[0326] More specifically, fully protected disaccharide 14 (1.1 g, 1 mmol) was dissolved in EtOAc (20 mL) and H O (20 mL) with vigorous mixing. Sodium dithionite (870 mg, 5 mmol) was added to the two-phase solution. The reaction was placed in an ice bath and sodium bromate (150 mg, 1 mmol) was added, resulting in the formation of bromine radicals in situ (the solution turned yellow / orange). The bromine radicals were analyzed by TLC (40% acetone / Hex, R f =0.56) to monitor the reaction. Additional sodium bromate (50 mg, 0.3 mol) was added as needed to convert all 14 → 15. The reaction was quenched by adding saturated sodium thiosulfate and purified by flash chromatography (30% acetone / hex) to give the desired 3-OH product (0.62 g, 62%) as a white solid foam.

[0327] 5.6-O-Sulfation, global deprotection, and amine acetylation.

[0328] Options 1-5

[0329]

[0330] The following conditions were used in the reactions shown in Schemes 1-5: a) chlorosulfonic acid, TEA; b) NaOH; c) Ac2O.

[0331] More specifically, 15 (620 mg, 0.61 mmol) was dissolved in THF (5 mL) containing triethylamine (1.3 mL, 9.2 mmol) in an ice bath, to which was added chlorosulfonic acid (405 μL, 6.1 mmol). The reaction was mixed on ice for 1 hour, then allowed to reach room temperature and mixed overnight. The results were analyzed by TLC (10% MeOH / DCM, R f =0.58) and desalted by LH-20 size exclusion chromatography (eluting with 1:1 MeOH / DCM). The recovered 3-O-sulfated 16 was suspended in MeOH (8 mL) and H2O (4 mL). NaOH (1.1 mL, 5.49 mmol, 5 M stock solution) was added to the reaction mixture and the solution was vigorously stirred at room temperature, resulting in global deprotection to provide 17, which was determined by TLC (EtOH:NH4OH:H2O-7:3:2, R f =0.82) was observed. The reaction pH was adjusted with concentrated AcOH and the solvent was reduced by rotary evaporation. The deprotected crude 17 was redissolved in MeOH (10 mL) and acetic anhydride (115 μL, 1.22 mmol) and triethylamine (170 μL, 1.22 mmol) were added to the solution. Amine acetylation was achieved by mixing at room temperature. 6-sulfo-N-acetylaminolactoside (18) was purified by Bio-Gel P-2 size chromatography (eluting with ammonium bicarbonate (100 mM)). The fractions containing the product were freeze-dried to give the desired product (203 mg, 55% over 3 steps) as a white fluffy solid.

[0332] 6. Chemical Synthesis of 6-Sulfo-N-acetylglucosamine

[0333] Plans 1-6

[0334]

[0335] The following conditions were used in the reactions shown in Schemes 1-6: a) Ac2O, sodium acetate; b) TMSOTf; c) 5-azidopentan-1-ol, TMSOTf; d) sodium methoxide; e) sulfur trioxide-pyridine.

[0336] More specifically, N-acetylglucosamine (100 g, 450 mmol) was added portionwise to a refluxing solution of acetic anhydride (340 mL, 3375 mmol) and sodium acetate (37.2 g, 450 mmol). The mixture was stirred at reflux until all the material was dissolved. TLC (100% EtOAc, R f=0.47) illustrate that the reaction is complete. Excess acetic anhydride is removed under reduced pressure and the crude material is dissolved in EtOAc (1L) and washed with saturated sodium bicarbonate (10x 150mL), saline (3x 100), through MgSO4 drying, and deduction in a vacuum to provide the fully acetylated N-acetylglucosamine (19, 129g, 74%) that is a white solid. 19 (5.4g, 13.9mmol) is dissolved in DCE (69mL) and TMSOTf (2.5mL, 13.87mmol) is added to this solution. Solution is warmed to 60 ℃ to promote oxazoline formation, which is determined by TLC (40% acetone / hex, R f =0.3). The reaction was quenched with TEA (2.2 mL, 15.18 mmol), the solvent was reduced, and the product was purified by flash chromatography (40% acetone / hex) to give 20 (3.9 g, 85%) as a clear oil. Triacetylated oxazoline 20 (4.36 g, 13.24 mmol) was dissolved in DCE (66 mL). To this solution were added 5-azidopentan-1-ol (3.4 g, 26.48 mmol) and MS (3 g). The combination was mixed at room temperature before TMSOTf (2.4 mL, 13.24 mmol) was added and the reaction was warmed to 60°C to promote glycosylation. The results were analyzed by TLC (90% EtOAc / Hex, R f =0.55) monitor the reaction and when all starting materials are converted to product, the reaction is quenched with TEA (1.84mL, 13.24mmol) and cooled to room temperature. The solvent is removed under reduced pressure and glycosylated N-acetylglucosamine is recovered by flash chromatography (80% → 90% EtOAc / Hex) to provide 21 (5g, 80%) as a white solid. 21 (14.3g, 31.37mmol) is dissolved in MeOH (156mL) and the solution is adjusted to pH 9 with sodium methoxide. The product is purified by TLC (20% MeOH / DCM, R r =0.76) and upon completion, the reaction was monitored with Dowex 50WX8 H +The resin is neutralized with pH. The resin is removed by filtration and MeOH is removed by decompression to produce triol 22 (9.3g, 90%) as a white solid. 6-O-sulfation is added by dissolving 22 (600mg, 1.8mmol) in DMF (36mL) and placing the mixture in an ice bath. Sulfur trioxide-pyridine complex (287mg, 1.8mmol) is added to the cooled solution. The reaction is stirred overnight at 4°C for three days, and other sulfur trioxide-pyridine (287mg, 1.8mmol) is added every day. The reaction is monitored by TLC (20% MeOH / DCM, Rf=0.23) and quenched with an aliquot of NaOH (5M, pH→8). The solvent is removed, and the reaction is purified by Bio-Gel size exclusion chromatography. The fraction containing the product is freeze-dried to produce 23 (514mg, 69%) as a white powder.

[0337] Figure 1 A and 1B show 2D of compounds 22 and 23 of Schemes 1-6. 1 H-NMR spectrum.

[0338] 7. Enzymatic Installation of β1,4-Gal to Form 6-Sulfo-N-acetyllactosamine

[0339] Schemes 1-7

[0340]

[0341] β1,4-Gal was installed by dissolving 5 mg 6S-GlcNAc (23, 5 mg, 11.7 μmol) and UDP-galactose (10 mg, 17.6 μmol) in Tris buffer (585 μL, pH 7.3, 0.1 M) containing MnCl2 (10 mM). To this solution was added B4GALT1 (50 μg, 1% wt / wt) and calf intestinal alkaline phosphatase (CIAP, 5.85 μL, 1 kU stock solution). The reaction was incubated at 37 ° C for three hours. The HPLC was performed by LC-ESI-MS (equipped with Waters XBridge BEH, amide column, 2.5 μm, The reaction progress was monitored at 2.1 x 150 mm (flow rate: 0.25 mL / min, A = 10 mM ammonium formate, B = ACN. Linear gradient: 80% → 60% B over 18 min).

[0342] The reactions were monitored by following the reactants and products using hydrophilic interaction liquid chromatography / electrospray ionization mass spectrometry (HILIC-LC / ESI-MS). Figure 2 The results of such tracking are shown.

[0343] The residual starting material was converted to the product by adding excess UDP-Gal (5 mg, 5.85 μmol) and B4GALT1 (25 μg). The reaction mixture was passed through PALL Filtration was performed through centrifugal spin filters (3k MWCO) and the filtrate was purified by Bio-Gel P2 size exclusion chromatography. Product containing fractions were freeze dried to afford the desired disaccharide 24 (4.8 mg, 70%).

[0344] 8.6S-LacNAc Azide Reduction and Fmoc Installation

[0345] Schemes 1-8

[0346]

[0347] The following conditions were used in the reactions shown in Schemes 1-8: a) Pd / C, H2, b) Fmoc-OSu.

[0348] More specifically, 24 (100 mg, 0.17 mmol) was dissolved in a 1:1 mixture of H2O / t-BuOH (8 mL) (containing 5% wt / wt Pd / C). The reaction vessel was evacuated three times and kept under an H2 atmosphere to promote the reduction of the azide to a primary amine. After the azide was completely reduced, the reaction was filtered through a 0.22 μm syringe filter and the filtrate was freeze-dried to provide 25, which was subsequently dissolved in H2O (8 mL) containing NaHCO3 (0.51 mmol). In a separate container, FmocOSu (114 mg, 0.34 mmol) was dissolved in ACN (8 mL). An aqueous solution containing 25 was added dropwise to the solution ACN. The mixed solution was vigorously vortexed to produce 26, which was analyzed by C18 TLC (30% ACN / water; R f =0.3) The mixture was reduced in vacuo and the product was purified by Bio-Gel P2 size exclusion chromatography to yield 26 (103 mg, 77% over 2 steps).

[0349] 11H NMR (600 MHz, D2O) δ 7.23 - 7.21 (m, 4H, Fmoc H1, H1’, H4, H4’), 6.95 (m, 4H, Fmoc H2, H2’, H3, H3’), 4.40 - 4.39 (d, J = Hz, 1H, Gal H1), 4.21 - 4.16 (m, 3H, GlcNAc H1, H6), 4.06 - 4.02 (m, 2H, Fmoc H8), 3.79 - 3.78 (m, 1H, Gal H4), 3.73 - 3.40 (m, 11H, Gal, H2, H3, H5, H6, GlcNAc H2, H3, H4, H5, Fmoc H7, lipid chain H5a), 3.11 - 3.10 (m, 1H, H5b), 2.67 - 2.65 (m, 1.5H, H1), 2.32 (m, 0.5H, H1), 1.79 (s, 3H, GlcNAc, Ac, CH3), 1.16 - 1.12 (m, 1.5H, H2), 1.00 - 0.98 (m, 2H, H4), 0.87 - 0.83 (m, 1.5H, H3), 0.69 (m, 1H, H2, H3). 13 13C NMR (151 MHz, D2O) δ 173.90 (GlcNAc, -CO-), 157.49 (Fmoc, -CO-), 143.62 (Fmoc C6, C6’), 140.76 (Fmoc C5, C5’), 127.60 (Fmoc C3, C3’), 127.09 (Fmoc C2, C2’), 124.86 (Fmoc C1, C1’), 119.75 (Fmoc C4, C4’), 102.37 (Gal, C1), 101.03 (GlcNAc, C1), 77.19 (Gal, C3), 75.22 (GlcNAc, C4), 72.47 (GlcNAc, C5), 72.42 (Gal, C5), 72.15 (GlcNAc, C3), 70.95 (Gal, C2), 70.01 (C5), 68.58 (Gal, C4), 66.11 (GlcNAc, C6), 65.88 (Fmoc, C8), 60.94 (Gal, C6), 54.99 (GlcNAc, C2), 46.77 (Fmoc, C7), 40.27 (C1), 28.50 (C4), 28.20 (C2), 22.21 (C3, GlcNAc, CH3).

[0350] 9. Enzymatic Installation of α2,3 Neu5Az

[0351] Scheme 1 - 9

[0352] Amine-protected 6-sulfo-LacNAc (26, 50 mg, 63 μmol) and ManNAz (27, 33 mg, 126 μmol) were dissolved in Tris buffer (0.1 M, pH 8.5, 6.3 mL) containing MgCl2 (20 mM), sodium pyruvate (630 μmol) and cytidine-5'-triphosphate (CTP, 252 μmol). Sialyl aldolase was added to this mixture. 1 (PmNANA, 660 μg), CMP-sialic acid synthetase 2 (NmCSS, 660 μg) and sialyltransferase 1 3-5 (PmST1, 500 μg). The reaction was incubated at 37 ° C with gentle agitation for 2 hours. The reaction was quenched by adding EtOH (8 mL) and placed under refrigeration. The protein precipitate was removed by centrifugation and the supernatant was reduced by rotary evaporation. The crude reaction mixture was purified by preparative HPLC (Agilent 1200, Eclipse XDB-C18, 21.2x 250mm, 7 μm. A solvent = 50mM ammonium bicarbonate; B = ACN, λ = 262m) (linear gradient: 10% → 60% B, over 30min, flow rate: 20mL / min). The fraction containing the compound was freeze-dried to trisaccharide 28 (59 mg, 83%).

[0353] 1H NMR(600MHz,D2O)δ7.57-7.52(m,2H,Fmoc H4,H4’),7.41-7.35(m,2H,Fmoc H1,H1’),7.22-7.14(m,4H,Fmoc H2,H2’,H3,H3’),4.46-4.45(d,1H,Gal H1),4.27-4.15(m,5H,Fmoc H8,GlcNAc H1,H6),4.00-3.99(m,1H,GlcNAc H4),3.93(m,3H,Fmoc H7,Neu5Az,Az,-CH2),3.83-3.75(m,4H,Gal H4,Neu5Az H5,H6,H9a),3.62-3.46(m,12H,H5a,Gal H3,H5,H6,GlcNAc H2,H3,H5,Neu5Az H4,H7,H8,H9b),3.44-3.41(t,1H,Gal H2),3.26-3.14(m,1H,H5b),2.74(m,1.5H,H1),2.64-2.62(dd,1H,Neu5Az H3a),2.29(m,0.5H,H1),1.81(s,3H,GlcNAc Ac,-CH3),1.71-1.67(t,1H,Neu5Az H3b),1.24(m,1.5H,H2),1.08(m,2H,H4),0.94(m,1.5H,H3),0.67(m,1H,H2,H3)。 13C NMR(151MHz,D2O)δ174.12(Neu5Az,C1),173.72(GlcNAc,Ac,-CO-),170.98(Neu5Az,Az,-CO-) ,157.94(Fmoc,C9,-CO-),143.67(Fmoc,C6),140.86(Fmoc,C5),127.85(Fmoc,C3),127.29(Fmoc c,C2),124.87(Fmoc,C1),119.96(Fmoc,C4),102.07(Gal,C1),101.01(GlcNAc,C1),99.56(Ne u5Az,C2),77.18(Gal,C3),75.27(GlcNAc,C4),74.95(Neu5Az,C7),72.46(GlcNAc,C5),72.43( GlcNAz,C3),72.18(Gal,C5),71.39(Gal,C4),70.14(C5),69.38(Gal,C2),68.15(Neu5Az,C4),67.96(Neu5Az,C8),67.38(Neu5Az,C6),66.17(GlcNAz,C6),65.74(Fmoc,C8),62.46(Neu5Az, C9),60.93(Gal,C6),54.96(GlcNAz,C2),51.83(Neu5Az,Az,CH2),51.74(Neu5Az,C5),46.98(Fmoc,C7),40.13(C1),39.53(Neu5Az,C3),28.26(C4),28.10(C2),22.11(GlcNAc,Ac,CH3 and C3).

[0354] 10. Enzymatic Installation of α1,3-fucose

[0355] program 1-10

[0356] The trisaccharide 28 (59 mg, 52 μmol) was dissolved in Tris buffer (5.2 mL, 0.1 M, pH 7.5) containing MgCl2 (10 mM) and GDP-1-fucose (78 μmol). To this solution, fucosyltransferase 6 (FUT6, 1.7 mg) and CIAP (52 μL, 1 kU stock solution) were added. The mixture was incubated at 37 ° C overnight. EtOH (5.2 mL) was added, the reaction was quenched and the solution was kept at 0 ° C for 1 hour. Insoluble proteins were removed by centrifugation and the supernatant was reduced in vacuo and purified by preparative HPLC (as described for 28). The fractions containing the compound were freeze-dried to produce tetrasaccharide 29 (52 mg, 78%) as a white powder.

[0357] 1H NMR(600MHz,D2O)δ7.75-7.74(d,2H,Fmoc H4,H4’),7.54-7.52(d,2H,Fmoc H1,H1’),7.35-7.33(t,2H,Fmoc H3,H3’),7.28-7.25(t,2H,Fmoc H2,H2’),4.95-4.94(d,1H,Fuc H1),4.67-4.61(m,1H,Fuc H5),4.58(m,0.5H,Fmoc H8),4.48-4.44(m,2.5H,Gal H1,Fmoc H8),4.36(m,1H,GlcNAc H1),4.24-4.19(m,2H,GlcNAc H6),4.12(m,1H,Fmoc H7),3.98-3.96(m,1H,Gal H3),3.93(s,2H,Neu5Az,Az,-CH2),3.87-3.80(m,4H,Fuc H4,GalH4,Neu5Az H5,H6),3.76-3.61(m,9H,H5a,Fuc H3,Gal H5,GlcNAc H2,H3,H5,Neu5Ac H4,H7,H9a),3.54-3.51(m,4H,Fuc H2,Gal H6,Neu5Ac H9b),3.47-3.44(m,2H,GlcNAc H4,Neu5Ac H8),3.39-3.36(m,2H,H5b,Gal H2),2.80(m,1.5H,H1),2.64-2.61(dd,1H,Neu5AzH3a),2.33(m,0.5H,H1),1.81(s,3H,GlcNAc Ac,-CH3),1.71-1.67(t,1H,Neu5Ac H3b),1.32(m,1.5H,H2),1.15(m,2H,H4),1.04-0.99(m,4.5H,H3,Fuc H6),0.71(m,1H,H2,H3)。 13CNMR (151 MHz, d2o) δ 173.98 (Neu5Az, C1), 173.40 (GlcNAc, Ac, -CO-), 171.01 (Neu5Az, Az, -CO-), 158.18 (Fmoc, C9, -CO-), 143.77 (Fmoc, C6), 140.94 (Fmoc, C5), 127.91 (Fmoc, C3), 127.36 (Fmoc, C2), 124.88 (Fmoc, C1), 120.03 (Fmoc, C4), 101.09 (Gal, C1), 100.75 (GlcNAc, C1), 99.15 (Neu5Az, C2), 98.47 (Fuc, C1), 75.35 (Gal, C3), 74.69 (GlcNAc, C4), 74.63 (Neu5Az, C7), 72.81 (GlcNAc, C5), 72.62 (GlcNAc, C3), 72.53 (Fuc, C4), 71.82 (Gal, C5), 71.11 (Gal, C4), 70.21 (C5), 69.30 (Gal, C2), 69.10 (Fuc, C3), 67.97 (Neu5Az, C4), 67.94 (Neu5Az, C8), 67.70 (Fuc, C2), 67.10 (Neu5Az, C6), 66.62 (Fuc, C5), 65.80 (GlcNAc, C6), 65.61 (Fmoc, C8), 62.54 (Neu5Az, C9), 61.31 (Gal, C6), 55.60 (GlcNAc, C2), 51.82 (Neu5Az, Az, CH2), 51.73 (Neu5Az, C5), 47.19 (Fmoc, C7), 40.04 (C1), 39.58 (Neu5Az, C3), 28.18 (C4), 28.06 (C2), 22.11 (GlcNAc, Ac, CH3), 22.07 (C3), 15.22 (Fuc, C6).

[0358] 11. Azide reduction; preparation of handles for library generation.

[0359] Scheme 1-11

[0360]

[0361] Tetrasaccharide 29 (52 mg, 41 μmol) was dissolved in H2O (2 mL) and HCl-washed Zn (266.5 mg, 4.1 mmol) and AcOH (820 μmol) were added to the solution. The reaction was placed in an ice bath and vigorously mixed for 1 hour. The reaction was monitored by ESI-MS and, when complete conversion of 30 was achieved, the reaction was filtered through a celite pad and dried under reduced pressure. The crude reaction mixture was purified by Bio-Gel P2 size exclusion chromatography. The fraction containing the compound was freeze-dried to produce reduced tetrasaccharide 30 (42 mg, 82%).

[0362] 1 H NMR(600MHz,D2O)δ7.77-7.76(d,2H,Fmoc H4,H4'),7.55-7.54(d,2H,Fmoc H1,H1'),7.36-7.34(t,2H,Fmoc H3,H3'),7.29-7.27(t,2H,Fmoc H2,H2'),4.95(d,1H,FucH1),4.67-4.61(m,1.5H,Fuc H5,Fmoc H8),4.48-4.46(m,2.5H,Gal H1,Fmoc H8),4.37(m,1H,GlcNAc H1),4.22-4.15(m,3H,GlcNAc H6,Fmoc H7),3.97-3.96(m,1H,Gal H3),3.87-3.36(m,23H,H5,Fuc H2,H3,H4,Gal H2,H4,H5,H6,GlcNAc H2,H3,H4,H5,Neu5Az,H4,H5,H6,H7,H8,H9,Az,-CH2),2.81(m,1.5H,H1),2.64-2.61(dd,1H,Neu5Az H3a),2.34(m,0.5H,H1),1.82(s,3H,GlcNAc Ac,-CH3),1.70-1.66(t,1H,Neu5Ac H3b),1.33(m,1.5H,H2),1.15(m,2H,H4),1.04-1.00(m,4.5H,H3,Fuc H6),0.72(m,1H,H2,H3). 13C NMR(151MHz,D2O)δ174.00(Neu5Ac,C1),173.91(GlcNAc,Ac,-CO-),167.38(Neu5Az,Az,-CO-),158.16(Fmo c,C9,-CO-),143.79(Fmoc,C6),140.94(Fmoc,C5),127.92(Fmoc,C3),127.37(Fmoc,C2),124.89(Fmoc,C1), 120.03(Fmoc,C4),101.18(Gal,C1),100.75(GlcNAc,C1),99.43(Neu5Ac,C2),98.50(Fuc,C1),75.24(Gal,C 3),74.71(GlcNAc,C4),74.65(Neu5Ac,C7),72.84(GlcNAc,C5),72.75(GlcNAc,C3),72.53(Fuc,C4),71.82( Gal,C5),71.14(Gal,C4),70.22(C5),69.29(Gal,C2),69.10(Fuc,C3),68.36(Neu5Ac,C4),67.91(Neu5Ac,C 8),67.70(Fuc,C2),67.11(Neu5Ac,C6),66.62(Fuc,C5),65.82(GlcNAc,C6),65.59(Fmoc,C8),62.32(Neu5A c,C9),61.35(Gal,C6),55.62(GlcNAc,C2),51.74(Neu5Ac,C5),47.22(Fmoc,C7),40.44(Neu5Az,Az,CH2),4 0.04(C1),39.69(Neu5Ac,C3),28.17(C4),28.06(C2),22.12(GlcNAc,Ac,CH3),22.06(C3),15.24(Fuc,C6).

[0363] The synthesis of 6-sulfo-9-amino-sialyl Lewis X was performed using the same protocol as described above for Schemes 1-8 to 1-11.

[0364] 12. Construction of 6-sulfo-9-amino-sialyl Lewis X

[0365] Plan 1-12

[0366]

[0367] Compounds 33 to 35 were obtained. 1 H-NMR spectrum:

[0368] Compound 33

[0369] 1 H NMR (600 MHz, D2O) δ 7.48 - 7.43 (m, 2H, Fmoc H4, H4’), 7.36 - 7.28 (m, 2H, Fmoc H1, H1’), 7.15 - 7.10 (m, 4H, Fmoc H2, H2’, H3, H3’), 4.46 - 4.45 (d, 1H, Gal H1), 4.24 - 4.19 (m, 5H, Fmoc H8, GlcNAc H1, H6), 3.98 - 3.96 (m, 2H, Gal H4, GlcNAc H4), 3.87 - 3.82 (m, 2H, Fmoc H7, Neu5Az H6), 3.74 - 3.71 (t, 1H, Neu5Az H5), 3.62 - 3.46 (m, 12H, H5a, Gal H3, H5, H6, GlcNAc H2, H3, H5, Neu5Az H4, H7, H8, H9a), 3.43 - 3.38 (m, 2H, Gal H2, Neu5Az H9b), 3.21 - 3.09 (m, 1H, H5b), 2.72 (m, 1.5H, H1), 2.63 - 2.60 (dd, 1H, Neu5Az H3a), 2.29 (m, 0.5H, H1), 1.90 (s, Neu5Az, Az, -CH3), 1.80 (s, 3H, GlcNAc Ac, -CH3), 1.69 - 1.65 (t, 1H, Neu5Az H3b), 1.21 (m, 1.5H, H2), 1.06 (m, 2H, H4), 0.91 (m, 1.5H, H3), 0.67 (m, 1H, H2, H3). 1313C NMR (151 MHz, D2O) δ 174.77 (Neu5Ac, Ac, -CO-), 174.07 (Neu5Ac, C1), 173.69 (GlcNAc, Ac, -CO-), 157.84 (Fmoc, C9, -CO-), 143.65 (Fmoc, C6), 140.83 (Fmoc, C5), 127.79 (Fmoc, C3), 127.23 (Fmoc, C2), 124.86 (Fmoc, C1), 119.92 (Fmoc, C4), 102.06 (Gal, C1), 101.03 (GlcNAc, C1), 99.50 (Neu5Ac, C2), 77.12 (Gal, C3), 75.34 (GlcNAc, C4), 74.96 (Neu5Ac, C7), 72.59 (GlcNAc, C5), 72.44 (GlcNAc, C3), 72.14 (Gal, C5), 70.10 (C5), 69.84 (Gal, C4), 69.37 (Gal, C2), 68.58 (Neu5Ac, C4), 68.31 (Neu5Ac, C8), 67.32 (Neu5Ac, C6), 66.05 (GlcNAc, C6), 65.79 (Fmoc, C8), 60.93 (Gal, C6), 54.96 (GlcNAc, C2), 52.97 (Neu5Ac, C9), 51.65 (Neu5Ac, C5), 46.90 (Fmoc, C7), 40.15 (C1), 39.64 (Neu5Ac, C3), 28.32 (C4), 28.12 (C2), 22.13 (GlcNAc, Ac, CH3 and C3), 22.01 (Neu5Ac, Ac, CH3).

[0370] Compound 34:

[0371] 1H NMR(600MHz,D2O)δ7.75-7.74(d,2H,Fmoc H4,H4’),7.53-7.52(d,2H,Fmoc H1,H1’),7.35-7.33(t,2H,Fmoc H3,H3’),7.28-7.25(t,2H,Fmoc H2,H2’),4.95-4.94(d,1H,Fuc H1),4.67-4.61(m,1H,Fuc H5),4.58(m,0.5H,Fmoc H8),4.50-4.48(d,1H,Gal H1),4.37(m,1.5H,Fmoc H8),4.36(m,1H,GlcNAc H1),4.25-4.18(m,2H,GlcNAc H6),4.12(m,1H,Fmoc H7),3.97-3.95(m,2H,Gal H4,GlcNAc H4),3.87-3.80(m,2H,Fuc H4,Neu5AzH6),3.76-3.52(m,13H,H5a,Fuc H2,H3,Gal H5,H6,GlcNAc H2,H3,H5,Neu5Ac H4,H5,H7,H9a),3.47-3.36(m,5H,H5b,Gal H2,GlcNAc H4,Neu5Ac H8,H9b),2.80(m,1.5H,H1),2.63-2.60(dd,1H,Neu5Az H3a),2.33(m,0.5H,H1),1.90(s,Neu5Az,Az,-CH3),1.81(s,3H,GlcNAc Ac,-CH3),1.69-1.65(t,1H,Neu5Ac H3b),1.32(m,1.5H,H2),1.15(m,2H,H4),1.14-0.99(m,4.5H,H3,Fuc H6),0.71(m,1H,H2,H3)。 1313C NMR (151 MHz, D2O) δ 174.78 (Neu5Ac, Ac, -CO-), 173.99 (Neu5Ac, C1), 173.36 (GlcNAc, Ac, -CO-), 158.16 (Fmoc, C9, -CO-), 143.77 (Fmoc, C6), 140.94 (Fmoc, C5), 127.91 (Fmoc, C3), 127.36 (Fmoc, C2), 124.89 (Fmoc, C1), 120.03 (Fmoc, C4), 101.06 (Gal, C1), 100.77 (GlcNAc, C1), 99.05 (Neu5Ac, C2), 98.48 (Fuc, C1), 75.37 (Gal, C3), 74.70 (GlcNAc, C4), 74.65 (Neu5Ac, C7), 72.83 (GlcNAc, C5), 72.65 (GlcNAc, C3), 72.62 (Fuc, C4), 71.83 (Gal, C5), 70.21 (C5), 69.56 (Gal, C4), 69.29 (Gal, C2), 69.11 (Fuc, C3), 68.48 (Neu5Ac, C4), 68.12 (Neu5Ac, C8), 67.71 (Fuc, C2), 67.04 (Neu5Ac, C6), 66.62 (Fuc, C5), 65.70 (GlcNAc, C6), 65.62 (Fmoc, C8), 61.31 (Gal, C6), 55.60 (GlcNAc, C2), 53.05 (Neu5Ac, C9), 51.65 (Neu5Ac, C5), 47.19 (Fmoc, C7), 40.05 (C1), 39.68 (Neu5Ac, C3), 28.18 (C4), 28.06 (C2), 22.12 (GlcNAc, Ac, CH3, C3), 21.99 (Neu5Ac, Ac, CH3), 15.22 (Fuc, C6).

[0372] Compound 35:

[0373] 1H NMR(600MHz,D2O)δ7.76-7.75(d,2H,Fmoc H4,H4’),7.55-7.54(d,2H,Fmoc H1,H1’),7.36-7.33(t,2H,Fmoc H3,H3’),7.29-7.26(t,2H,Fmoc H2,H2’),4.95(d,1H,FucH1),4.67-4.61(m,1.5H,Fuc H5,Fmoc H8),4.47-4.46(m,2.5H,Gal H1,Fmoc H8),4.37(m,1H,GlcNAc H1),4.29-4.15(m,3H,GlcNAc H6,Fmoc H7),3.99-3.93(m,2H,Gal H3,Neu5AzH8),3.88-3.85(m,1H,GlcNAc H3),3.76-3.30(m,19H,H5,Fuc H2,H3,H4,Gal H2,H4,H5,H6,GlcNAc H2,H4,H5,Neu5Az,H4,H5,H6,H7,H8,H9a),2.90-2.87(t,1H,Neu5Ac H9b),2.80(m,1.5H,H1),2.67-2.64(dd,1H,Neu5Az H3a),2.34(m,0.5H,H1),1.89(s,Neu5Az,Az,-CH3),1.82(s,3H,GlcNAc Ac,-CH3),1.64-1.60(t,1H,Neu5Ac H3b),1.32(m,1.5H,H2),1.15-0.98(m,4.5H,H3,Fuc H6),0.71(m,1H,H2,H3)。 13C NMR(151MHz,D2O)δ174.89(Neu5Ac,Ac,-CO-),174.03(Neu5Ac,C1),173.56(GlcNAc,Ac,-CO-),158.19(Fmo c,C9,-CO-),143.80(Fmoc,C6),140.96(Fmoc,C5),127.92(Fmoc,C3),127.37(Fmoc,C2),124.89(Fmoc,C1) ,120.03(Fmoc,C4),101.10(Gal,C1),100.77(GlcNAc,C1),98.99(Neu5Ac,C2),98.52(Fuc,C1),75.47(Gal ,C3),74.80(GlcNAc,C4),74.58(Neu5Ac,C7),72.70(GlcNAc,C5),72.47(GlcNAc,C3),72.40(Fuc,C4),71. 82(Gal,C5),70.23(C5),70.08(Gal,C4),69.28(Gal,C2),69.11(Fuc,C3),68.05(Neu5Ac,C4),67.83(Neu5 Ac,C8),67.68(Fuc,C2),66.73(Neu5Ac,C6),66.61(Fuc,C5),65.78(GlcNAc,C6),65.59(Fmoc,C8),61.34( Gal,C6),55.67(GlcNAc,C2),51.55(Neu5Ac,C5),47.23(Fmoc,C7),42.24(Neu5Ac,C9),40.24(C1),40.04( Neu5Ac,C3), 28.18(C4), 28.07(C2), 22.12(GlcNAc,Ac,CH3,C3), 21.96(Neu5Ac,Ac,CH3), 15.23(Fuc,C6).

[0374] 13. General protocol for Fmoc deprotection

[0375] Deprotection of the S series compounds is generally performed according to Schemes 1 to 13. After deprotection, compounds S1XX and S2XX shown in Scheme 13 are also referred to herein as S1XX-amine and S2XX-amine (see, e.g., Example 8, where S212-amine and S217-amine were conjugated to nanoparticles).

[0376] Plan 1-13

[0377]

[0378] The following conditions were used in the reactions shown in Schemes 1-13: H2O / triethylamine.

[0379] In particular, compound S1XX or S2XX (0.15 μmol) was dissolved in 4: 1 (v / v) H2O / triethylamine solution (150 μL). During the reaction time of 3 hours, the solution was periodically mixed using a vortexer. The progress was monitored by ESI-MS, and when the starting material was converted into product, the organic material was extracted with EtOAc (500 μL). The aqueous layer was separated and freeze-dried to produce the desired deprotected amine target in the form of a white amorphous solid. The material was used for microarray analysis without further purification.

[0380] II. Library of S-compounds according to Example 1A - Procedure A

[0381] Tetrasaccharide (30 or 35, 1 mg, 0.8 μmol) is dissolved in DMF (100 μL) containing diisopropylethylamine (DIPEA, 4 μmol). To this solution, acyl chloride (1.6 μmol) is added and the reaction is mixed for 30 min using a vortexer. The reaction is monitored by ESI, and when the complete conversion of the product is achieved, the product is purified by HPLC chromatography (under the following conditions: Agilent 1200HPLC, Eclipse XDB-C8, 4.6 x 250 mm, 5 μm analytical column, A solvent = 50 mM ammonium bicarbonate; B = ACN, λ = 262 m), linear gradient: 10% → 60% B, over 30 min, flow rate 1 mL / min). The fraction containing the desired product is freeze-dried to produce the desired product as a white fluffy solid. Table 1 shows 31 R1-acyl chloride fragments of the library for synthesizing S series compounds.

[0382] Table 1

[0383]

[0384] The following compounds were synthesized and their identities were confirmed by 1 H-NMR confirmed. For the compounds below, the naming convention is such that R has the following formula:

[0385] The general procedure referenced below in the SXX-Fmoc Examples is General Procedure A.

[0386] Compound S101-Fmoc

[0387]

[0388] Prepared by using the general procedure (0.15 μmol). 1H NMR(600MHz,D2O)δ8.29(m,1H),7.94-7.87(m,3H),7.78-7.73(m,3H),7.56-7.50(m,4H),7.37-7.35(t ,2H),7.30-7.27(t,2H),4.95-4.95(d,1H),4.67-4.61(m,1.5H),4.48-4.47(m,2.5H),4.39-4.38(m,1H ),4.22-4.17(m,3H),3.97-3.95(m,1H),3.88-3.36(m,23H),2.81(m,1.5H),2.64-2.61(dd,1H),2.34(m ,0.5H),1.82(s,3H),1.71-1.67(t,1H),1.34(m,1.5H),1.17(m,2H),1.04-1.00(m,4.5H),0.73(m,1H).

[0389] Compound S102-Fmoc

[0390]

[0391] Prepared by using the general procedure (0.15 μmol). 1 H NMR(600MHz,dmso)δ8.74-8.72(t,1H),8.30-8.29(dd,1H),8.14(m,1H),8.00-7.99(m,1H),7.96-7.94(dd,1H),7.89-7.86(m ,3H),7.68-7.63(m,3H),7.55-7.52(m,3H),7.41-7.38(t,2H),7.32-7.30(t,2H),7.25-7.24(t,1H),4.82(d,1H),4.44-4.36( m,4H),4.26-4.17(m,4H),4.12-3.96(m,6H),3.90-3.88(m,1H),3.75(m,1H),3.69-3.46(m,12H),3.38(m,1H),3.25-3.19(m, 3H),2.94(m,1H),2.60-2.59(m,1H),1.77(s,3H),1.44-1.38(m,2H),1.37-1.36(m,2H),1.24-1.21(m,2H),0.99-0.97(d,3H).

[0392] Compound S103-Fmoc

[0393]

[0394] Prepared using the general procedure (0.15 μmol). 1 H NMR(600MHz,dmso)δ8.75-8.73(t,1H),8.06-8.05(m,1H),7.87-7.86(m,3H),7.79-7.78(d,1H),7.75-7.74(d,1H ),7.68-7.67(d,2H),7.41-7.38(t,2H),7.32-7.30(t,2H),7.25-7.23(t,1H),7.15-7.13(t,1H),4.82-4.81(d,1H ),4.58-4.35(m,4H),4.26-4.17(m,4H),4.11-3.82(m,7H),3.73(m,1H),3.69-3.46(m,12H),3.38(m,1H),3.25-3 .18(m,3H),2.93(m,1H),2.71-2.60(m,1H),1.76(s,3H),1.44-1.36(m,4H),1.24-1.21(m,2H),0.98-0.96(d,3H).

[0395] Compound S104-Fmoc

[0396] Prepared using the general procedure (0.15 μmol). 1 H NMR(600MHz,dmso)δ8.82-8.80(t,1H),8.07-8.06(m,1H),7.87-7.86(m,3H),7.77-7.76(d,1H),7.68-7.66( m,3H),7.56(s,1H),7.47-7.44(t,1H),7.41-7.38(t,2H),7.33-7.30(m,3H),7.25-7.23(t,1H),4.82-4.81(d ,1H),4.55-4.35(m,3H),4.26-4.10(m,5H),4.07-3.87(m,5H),3.73(m,1H),3.68-3.37(m,13H),3.24-3.18( m,3H),2.93(m,1H),2.70-2.60(m,1H),1.76(s,3H),1.44-1.35(m,4H),1.24-1.21(m,2H),0.98-0.97(d,3H).

[0397] Compound S105-Fmoc

[0398]

[0399] Prepared using the general procedure (0.15 μmol). 1 H NMR(600MHz,dmso)δ9.13-9.11(t,1H),8.23-8.22(d,1H),8.16-8.15(d,1H),8.10-8.08(m,1H),7.89-7.86(m, 3H),7.68-7.67(d,2H),7.64-7.56(m,2H),7.40-7.38(t,2H),7.32-7.30(t,2H),7.25-7.23(t,1H),4.82-4.81 (d,1H),4.58-4.35(m,3H),4.26-4.03(m,10H),3.94-3.90(m,2H),3.73(m,1H),3.68-3.37(m,13H),3.25-3.18 (m,3H),2.93(m,3H),2.71-2.60(m,1H),1.76(s,3H),1.44-1.34(m,4H),1.25-1.21(m,2H),0.98-0.97(d,3H).

[0400] Compound S106-Fmoc

[0401]

[0402] Prepared using the general procedure (0.15 μmol). 1 H NMR(600MHz,dmso)δ9.02-9.00(t,1H),8.58-8.56(d,1H),8.17-8.14(m,2H),8.10-8.07(m,2H),7.88-7.8 6(m,4H),7.73-7.67(m,3H),7.40-7.38(t,2H),7.32-7.30(t,2H),7.25-7.23(t,1H),4.82-4.81(d,1H),4 .57-4.35(m,3H),4.26-3.98(m,11H),3.89-3.87(m,1H),3.74(m,1H),3.68-3.34(m,13H),3.25-3.18(m,3 H),2.93(m,3H),2.71-2.60(m,1H),1.76(s,3H),1.45-1.35(m,4H),1.24-1.22(m,2H),0.98-0.97(d,3H).

[0403] Compound S107-Fmoc

[0404]

[0405] Prepared using the general procedure (0.15 μmol). 1 H NMR(600MHz,dmso)δ8.78-8.76(t,1H),8.05-8.04(m,2H),7.97-7.96(d,2H),7.87-7.86(m,3H),7.78-7.76(d,2H), 7.73-7.72(d,2H),7.68-7.67(d,2H),7.49-7.46(t,2H),7.40-7.38(t,3H),7.32-7.30(t,2H),7.25-7.23(t,1H),4 .82-4.81(d,1H),4.58-4.36(m,3H),4.26-3.98(m,10H),3.90-3.87(m,2H),3.74(m,1H),3.69-3.37(m,13H),3.25- 3.18(m,3H),2.93(m,3H),2.71-2.60(m,1H),1.77(s,3H),1.44-1.34(m,4H),1.24-1.21(m,2H),0.98-0.97(d,3H).

[0406] Compound S108-Fmoc

[0407]

[0408] Prepared using the general procedure (0.15 μmol). 1 H NMR(600MHz,dmso)δ8.82-8.80(t,1H),8.04-8.03(m,1H),7.89-7.86(m,5H),7.68-7.67(d,2H),7.54 -7.53(d,2H),7.41-7.38(t,2H),7.32-7.30(t,2H),7.25-7.23(t,1H),4.82-4.81(d,1H),4.57-4.35 (m,3H),4.26-3.95(m,10H),3.89-3.84(m,2H),3.73(m,1H),3.68-3.37(m,13H),3.25-3.18(m,3H),2 .93(m,3H),2.70-2.60(m,1H),1.76(s,3H),1.45-1.34(m,4H),1.24-1.21(m,2H),0.98-0.96(d,3H).

[0409] Compound S109-Fmoc

[0410]

[0411] Prepared using the general procedure (0.135 μmol). 1 H NMR(600MHz,dmso)δ8.76-8.74(t,1H),8.04-8.03(m,1H),7.95-7.92(dd,1H),7.89-7.86(m,3H),7.6 8-7.67(d,2H),7.41-7.38(t,2H),7.32-7.28(m,4H),7.25-7.23(t,1H),4.82-4.81(d,1H),4.58-4.3 5(m,3H),4.26-3.95(m,10H),3.88-3.84(m,2H),3.73(m,1H),3.69-3.36(m,13H),3.24-3.17(m,3H), 2.93(m,3H),2.70-2.60(m,1H),1.76(s,3H),1.45-1.35(m,4H),1.24-1.22(m,2H),0.98-0.96(d,3H).

[0412] Compound S110-Fmoc

[0413]

[0414] Prepared using the general procedure (0.135 μmol). 1 H NMR(600MHz,dmso)δ9.01-8.99(t,1H),8.12,-8.08(m,2H),8.02-8.00(d,1H),7.95-7.94(d,1H),7.89 -7.86(m,3H),7.68-7.67(d,2H),7.46-7.38(m,4H),7.32-7.30(t,2H),7.25-7.23(t,1H),4.82-4.81(d ,1H),4.58-4.35(m,3H),4.26-3.98(m,10H),3.91-3.87(m,2H),3.74(m,1H),3.68-3.36(m,13H),3.24 -3.19(m,3H),2.94-2.60(m,4H),1.76(s,3H),1.45-1.34(m,4H),1.25-1.21(m,2H),0.98-0.96(d,3H).

[0415] Compound S111-Fmoc

[0416]

[0417] Prepared using the general procedure (0.15 μmol). 1 H NMR(600MHz,dmso)δ8.46-8.44(t,1H),8.11-8.05(m,2H),7.94-7.92(dd,1H),7.89-7.86(m,3H),7.68 -7.67(d,2H),7.48-7.44(td,1H),7.41-7.38(t,2H),7.32-7.30(t,2H),7.25-7.23(t,1H),4.82-4.81( d,1H),4.57-4.36(m,3H),4.26-4.01(m,10H),3.97-3.87(m,2H),3.74(m,1H),3.69-3.36(m,13H),3.25 -3.18(m,3H),2.93-2.60(m,4H),1.77(s,3H),1.44-1.36(m,4H),1.24-1.21(m,2H),0.98-0.97(d,3H).

[0418] Compound S112-Fmoc

[0419]

[0420] Prepared using the general procedure (0.15 μmol). 1 H NMR(600MHz,dmso)δ8.48-8.46(t,1H),8.12-8.10(m,2H),7.91-7.86(m,4H),7.68-7.57 (m,4H),7.41-7.38(t,2H),7.32-7.30(t,2H),7.25-7.23(t,1H),4.82-4.81d,1H),4.58 -4.36(m,3H),4.26-3.87(m,12H),3.74(m,1H),3.68-3.37(m,13H),3.24-3.18(m,3H),2 .96-2.59(m,4H),1.77(s,3H),1.45-1.36(m,4H),1.24-1.21(m,2H),0.98-0.97(d,3H).

[0421] Compound S113-Fmoc

[0422]

[0423] Prepared using the general procedure (0.15 μmol). 1H NMR(600MHz,dmso)δ8.62-8.60(t,1H),8.03-8.02(m,1H),7.89-7.86(m,3H),7.77-7.76(d,2H) ,7.68-7.67(d,2H),7.41-7.38(t,2H),7.32-.30(t,2H),7.27-7.23(m,3H),4.82-4.81(d,1H),4 .57-4.35(m,3H),4.26-3.83(m,12H),3.73(m,1H),3.69-3.38(m,13H),3.25-3.18(m,3H),2.96- 2.60(m,4H),2.34(s,3H),1.76(s,3H),1.44-1.35(m,4H),1.24-1.21(m,2H),0.98-0.96(d,3H).

[0424] Compound S114-Fmoc

[0425]

[0426] Prepared using the general procedure (0.15 μmol). 1 H NMR(600MHz,dmso)δ7.91-7.86(m,5H),7.68-7.67(d,2H),7.41-7.38(t,2H),7.33-7.30(t,2H),7. 25-7.23(t,1H),4.82-4.81(d,1H),4.58-4.36(m,3H),4.26-3.98(m,10H),3.88-3.87(m,1H),3.74- 3.71(m,2H),3.68-3.38(m,13H),3.23-3.15(m,3H),2.96-2.59(m,4H),2.17-2.13(m,1H),1.77(s, 3H),1.69-1.67(m,4H),1.59-1.57(m,1H),1.45-1.36(m,4H),1.32-1.11(m,7H),0.98-0.97(d,3H).

[0427] Compound S115-Fmoc (0.15 μmol) was prepared using the general procedure.

[0428]

[0429] Compound S116-Fmoc

[0430]

[0431] Prepared using the general procedure (0.15 μmol). 1 H NMR(600MHz,dmso)δ9.48(s,1H),9.11-9.09(t,1H),8.24-8.19(m,2H),8.07(m,1H),8.00-7.97(m,2H),7.89-7.8 6(m,3H),7.68-7.67(d,2H),7.40-7.38(t,2H),7.32-7.30(t,2H),7.25-7.23(t,1H),4.82-4.81(d,1H),4.58-4.3 5(m,3H),4.26-4.07(m,10H),4.05-3.98(m,2H),3.89-3.87(m,1H),3.73(m,1H),3.69-3.42(m,12H),3.26-3.18(m ,3H),2.96-2.92(m,3H),2.71-2.59(m,1H),1.76(s,3H),1.44-1.35(m,4H),1.24-1.21(m,2H),0.98-0.97(d,3H).

[0432] Compound S117-Fmoc

[0433]

[0434] Prepared using the general procedure (0.525 μmol). 1H NMR(600MHz,dmso)δ9.16-9.14(t,1H),8.36(s,1H),8.33-8.32(d,1H), 8.27-8.25(td,1H),8.22-8.20(m,2H),8.14-8.13(d,1H),7.92-7.90(d, 1H),7.87-7.86(d,2H),7.85-7.82(t,1H),7.68-7.58(m,5H),7.41-7.3 8(t,2H),7.32-7.30(t,2H),7.25-7.23(t,1H),4.82-4.81(d,1H),4.63- 4.62(d,1H),4.47(m,1H),4.37-4.36(d,2H),4.27-4.23(m,4H),4.19-4 .17(m,3H),4.08-4.01(m,3H),3.98-3.97(m,1H),3.90-3.88(m,1H),3.7 5-3.37(m,16H),3.21-3.19(m,1H),2.95-2.93(m,3H),2.71-2.59(m,1H ),1.77(s,3H),1.45-1.36(m,4H),1.24-1.21(m,2H),0.98-0.97(d,3H).

[0435] Compound S118-Fmoc

[0436]

[0437] Prepared using the general procedure (0.15 μmol).

[0438] 1H NMR(600MHz,D2O)δ7.80(dt,J=14.0,6.4Hz,5H),7.71(s,1H),7.56(d,J=7.5Hz,2H),7.43(p,J=6.9Hz,2H),7 .39–7.31(m,3H),7.29(t,J=7.5Hz,2H),4.95(d,J=4.1Hz,1H),4.46(d,J=7.8Hz,1H),4.38(d,J=7.5Hz,1H),4 .20(d,J=23.9Hz,3H),3.94(dd,J=9.6,3.2Hz,1H),3.90–3.33(m,28H),2.81(s,1H),2.59(dd,J=12.6,4.9Hz ,1H),1.81(s,3H),1.65(t,J=12.1Hz,1H),1.34(s,1H),1.21–1.14(m,0H),1.02(d,J=6.6Hz,5H),0.73(s,1H)

[0439] Compound S119-Fmoc (0.15 μmol) was prepared using the general procedure.

[0440]

[0441] Compound S120-Fmoc

[0443]

[0444] Prepared using the general procedure (0.15 μmol)

[0445] 1H NMR(600MHz,D2O)δ7.78(d,J=7.6Hz,2H),7.56(d,J=7.6Hz,2H),7.36(t,J=7.5Hz,2H),7.29(t,J=7.5Hz,2H) ,4.95(d,J=3.4Hz,1H),4.46(d,J=7.8Hz,1H),4.38(d,J=7.7Hz,1H),4.25–4.16(m,3H),3.95(d,J=10.0Hz,1H ),3.86(t,J=8.8Hz,1H),3.82–3.35(m,26H),2.81(s,2H),2.61(dd,J=12.4,4.6Hz,1H),2.05(s,2H),1.81(s, 1H), 1.66 (t, J = 12.1Hz, 1H), 1.33 (s, 1H), 1.21–1.18 (m, 2H), 1.02 (d, J = 6.6Hz, 5H), 0.85 (s, 8H), 0.72 (s, 1H).

[0446] Compound S121-Fmoc

[0447]

[0448] Prepared using the general procedure (0.15 μmol).

[0449] 1H NMR(600MHz,D2O)δ7.77(d,J=7.6Hz,2H),7.65(dd,J=8.6,1.8Hz,2H),7.56(d,J=7.5Hz,2H),7.48(d,J=8.7Hz,2 H),7.36(t,J=7.5Hz,2H),7.28(t,J=7.5Hz,2H),4.94(d,J=3.9Hz,1H),4.46(d,J=7.9Hz,1H),4.38(d,J=7.7Hz,1 H),4.28–4.12(m,3H),4.03–3.92(m,3H),3.86(t,J=8.8Hz,1H),3.84–3.34(m,23H),2.81(s,2H),2.61(dd,J=12. 4,4.5Hz,1H),1.81(s,1H),1.67(t,J=12.2Hz,1H),1.33(s,1H),1.19(s,9H),1.02(d,J=6.6Hz,5H),0.72(s,1H).

[0450] Compound S122-Fmoc

[0451]

[0452] Prepared using the general procedure (0.15 μmol).

[0453] 1H NMR(600MHz,D2O)δ7.77(d,J=7.6Hz,2H),7.61–7.51(m,6H),7.36(t,J=7.5Hz,2H),7.28(t,J=7.5H z,2H),4.94(d,J=4.0Hz,1H),4.46(d,J=7.8Hz,1H),4.37(s,1H),4.27–4.14(m,3H),4.03–3.91(m,3 H),3.86(t,J=9.3Hz,1H),3.83–3.31(m,23H),2.81(s,2H),2.61(dd,J=12.4,4.5Hz,1H),1.81(s,2 H), 1.67 (t, J = 12.2Hz, 1H), 1.33 (s, 1H), 1.17 (d, J = 20.2Hz, 2H), 1.02 (d, J = 6.5Hz, 5H), 0.72 (s, 1H).

[0454] Compound S123-Fmoc

[0455]

[0456] Prepared using the general procedure (0.15 μmol).

[0457] 1H NMR(600MHz,D2O)δ7.88–7.83(m,1H),7.80–7.77(m,4H),7.72–7.68(m,1H),7.56(d,J=7.6Hz,2H),7.36(t,J=7.5Hz,2H), 7.28(t,J=7.5Hz,2H),4.94(d,J=4.0Hz,1H),4.46(d,J=8.1Hz,1H),4.38(d,J=7.5Hz,1H),4.28–4.12(m,3H),4.01(s,2H), 3.96(d,J=10.0Hz,1H),3.86(t,J=9.1Hz,1H),3.84–3.32(m,22H),3.06(q,J=8.0,7.4Hz,1H),2.81(s,2H),2.61(dd,J=12. 3,4.7Hz,1H),1.81(s,3H),1.67(t,J=12.2Hz,1H),1.33(s,1H),1.19(d,J=6.4Hz,2H),1.02(d,J=6.6Hz,5H),0.72(s,1H).

[0458] Compound S124-Fmoc

[0459]

[0460] Prepared using the general procedure (0.15 μmol).

[0461] 1H NMR (600MHz, D2O) δ7.77(d,J=7.6Hz,2H),7.68(d,J=9.0Hz,2H),7.56(d,J=7.5Hz,2H),7.36(t,J=7.5Hz,2H),7. 28(t,J=7.5Hz,2H),6.94(d,J=9.0Hz,2H),4.94(d,J=4.0Hz,1H),4.46(d,J=8.1Hz,1H),4.38(d,J=7.4Hz,1H),4. 26–4.13(m,3H),4.01–3.92(m,3H),3.85(t,J=8.8Hz,1H),3.83–3.30(m,25H),2.81(s,2H),2.61(dd,J=12.6,4.7 Hz,1H),1.81(s,3H),1.67(t,J=12.2Hz,1H),1.33(s,1H),1.23–1.10(m,6H),1.02(d,J=6.6Hz,5H),0.72(s,1H).

[0462] Compound S125-Fmoc

[0463]

[0464] Prepared using the general procedure (0.15 μmol).

[0465] 1H NMR(600MHz,D2O)δ7.77(d,J=7.6Hz,2H),7.56(d,J=7.5Hz,2H),7.52(s,1H),7.47(d,J=8.0Hz,1H),7.39–7.32( m,3H),7.31–7.25(m,3H),4.94(d,J=4.0Hz,1H),4.46(d,J=7.9Hz,1H),4.38(d,J=7.7Hz,1H),4.26–4.16(m,3H) ,4.03–3.91(m,3H),3.86(t,J=8.9Hz,1H),3.83–3.22(m,22H),2.81(s,2H),2.61(dd,J=12.5,4.7Hz,1H),2.26( s,3H),1.81(s,1H),1.67(t,J=12.2Hz,1H),1.33(s,1H),1.21–1.11(m,4H),1.02(d,J=6.6Hz,5H),0.72(s,1H).

[0466] Compound S126-Fmoc

[0467]

[0468] Prepared using the general procedure (0.15 μmol).

[0469] 1H NMR(600MHz,D2O)δ7.78(d,J=7.6Hz,2H),7.56(d,J=7.5Hz,2H),7.49(s,0H),7.41–7.25(m,6H),4 .95(d,J=3.9Hz,1H),4.47(d,J=8.1Hz,1H),4.38(d,J=4.4Hz,1H),4.27–4.14(m,3H),4.03–3.92(m ,6H),3.86(t,J=8.9Hz,1H),3.84–3.33(m,23H),2.81(s,2H),2.62(dd,J=12.5,4.6Hz,1H),1.81( s,3H),1.68(t,J=12.3Hz,1H),1.33(s,1H),1.20–1.15(m,2H),1.03(d,J=6.6Hz,5H),0.72(s,1H).

[0470] Compound S127-Fmoc

[0471]

[0472] Prepared using the general procedure (0.075 μmol).

[0473] 1H NMR(600MHz,D2O)δ7.78(d,J=7.6Hz,2H),7.56(d,J=7.6Hz,2H),7.36(t,J=7.5Hz,2H),7.33–7.23(m,5H),7.20(t, J=7.2Hz,1H),4.94(d,J=4.0Hz,1H),4.46(d,J=7.9Hz,1H),4.38(d,J=7.8Hz,1H),4.25–4.13(m,3H),3.94(d,J=9.8 Hz,1H),3.85(t,J=9.3Hz,1H),3.81(d,J=3.2Hz,1H),3.78–3.35(m,50H),2.81(t,J=6.0Hz,2H),2.59(dd,J=12.4,4 .6Hz,1H),1.81(s,3H),1.64(t,J=12.2Hz,1H),1.33(s,1H),1.22–1.17(m,2H),1.02(d,J=6.6Hz,5H),0.72(s,1H).

[0474] Compound S128-Fmoc (0.15 μmol) was prepared using the general procedure.

[0475]

[0476] Compound S129-Fmoc

[0477]

[0478] Prepared using the general procedure (0.15 μmol).

[0479] 1H NMR(600MHz,D2O)δ7.78(d,J=7.6Hz,2H),7.57(dd,J=17.6,7.3Hz,3H),7.36(t,J=7.5Hz,2H),7.28(t,J=7.5Hz,2H ),7.04(t,J=9.1Hz,1H),4.94(d,J=4.0Hz,1H),4.46(d,J=8.1Hz,1H),4.38(d,J=5.7Hz,2H),4.25–4.14(m,7H),4. 01–3.92(m,6H),3.86(t,J=8.7Hz,1H),3.83–3.32(m,23H),2.81(t,J=5.4Hz,3H),2.61(dd,J=12.1,4.4Hz,1H),2. 17(s,2H),1.81(s,2H),1.67(t,J=12.2Hz,1H),1.33(s,1H),1.20–1.13(m,6H),1.02(d,J=6.6Hz,5H),0.72(s,1H).

[0480] Compound S130-Fmoc

[0481]

[0482] Prepared using the general procedure (0.105 μmol).

[0483] 1H NMR(600MHz,D2O)δ7.78(d,J=7.6Hz,2H),7.56(d,J=7.6Hz,2H),7.36(t,J=7.5Hz,2H),7.29(t,J=7.5Hz ,2H),6.84(s,1H),4.95(d,J=2.9Hz,2H),4.47(d,J=8.0Hz,1H),4.38(d,J=5.6Hz,2H),4.27–4.14(m,4H ),4.02–3.91(m,1H),3.91–3.21(m,24H),2.81(t,J=4.9Hz,3H),2.66–2.59(m,1H),2.16–2.07(m,7H),1 .81(s,2H),1.68(t,J=12.6Hz,1H),1.33(s,1H),1.22–1.12(m,1H),1.03(d,J=6.5Hz,5H),0.72(s,1H).

[0484] Compound S131-Fmoc

[0485]

[0486] Prepared using the general procedure (0.15 μmol).

[0487] 1H NMR(600MHz,D2O)δ8.06(s,1H),7.99–7.93(m,1H),7.86–7.81(m,1H),7.77(d,J=7.6Hz,2H),7.59–7.53(m,3H),7.36(t, J=7.5Hz,2H),7.28(t,J=7.5Hz,2H),4.94(d,J=4.0Hz,1H),4.46(d,J=7.9Hz,1H),4.38(d,J=6.3Hz,1H),4.27–4.13(m,6 H),4.01(s,2H),3.96(d,J=10.1Hz,1H),3.86(t,J=8.9Hz,1H),3.85–3.25(m,23H),2.81(t,J=6.3Hz,2H),2.61(dd,J=12 .6,4.7Hz,1H),1.81(s,1H),1.67(t,J=12.2Hz,1H),1.33(s,1H),1.23–1.16(m,2H),1.02(d,J=6.6Hz,5H),0.72(s,1H).

[0488] The following compounds were synthesized according to the general procedure:

[0489] Compound S201-Fmoc (0.15 μmol) was prepared using the general procedure.

[0491]

[0492] Compound S202-Fmoc (0.135 μmol) was prepared using the general procedure.

[0493]

[0494] Compound S203-Fmoc (0.135 μmol) was prepared using the general procedure.

[0495]

[0496] Compound S204-Fmoc (0.15 μmol) was prepared using the general procedure.

[0497]

[0498] Compound S205-Fmoc (0.15 μmol) was prepared using the general procedure.

[0499]

[0500] Compound S206-Fmoc (0.15 μmol) was prepared using the general procedure.

[0501]

[0502] Compound S207-Fmoc (0.15 μmol) was prepared using the general procedure.

[0503]

[0504] Compound S208-Fmoc (0.075 μmol) was prepared using the general procedure.

[0505]

[0506] Compound S209-Fmoc (0.15 μmol) was prepared using the general procedure.

[0507]

[0508] Compound S210-Fmoc (0.15 μmol) was prepared using the general procedure.

[0509]

[0510] Compound S211-Fmoc (0.15 μmol) was prepared using the general procedure.

[0511]

[0512] Compound S212-Fmoc (0.15 μmol) was prepared using the general procedure.

[0513]

[0514] Compound S213-Fmoc

[0515]

[0516] Prepared using the general procedure (0.075 μmol).

[0517] 1H NMR(600MHz,D2O)δ7.74(d,J=7.5Hz,2H),7.53(d,J=8.4Hz,5H),7.34(t,J=7.5Hz,2H),7.30–7. 24(m,2H),7.13(d,J=6.4Hz,3H),4.93(s,1H),4.43(t,J=7.2Hz,4H),4.34(s,1H),4.24–4.04(m, 2H),3.95–3.36(m,26H),2.90–2.75(m,4H),2.62(d,J=10.4Hz,1H),2.18(s,3H),1.89–1.78(m,1 0H), 1.63 (t, J = 12.3Hz, 1H), 1.35 (s, 1H), 1.20–1.19 (m, 2H), 1.02 (d, J = 6.6Hz, 5H), 0.73 (s, 1H).

[0518] The following compounds were synthesized according to the general procedure:

[0519] Compound S214-Fmoc (0.075 μmol) was prepared using the general procedure.

[0520]

[0521] Compound S215-Fmoc (0.15 μmol) was prepared using the general procedure.

[0522]

[0523] Compound S216-Fmoc (0.15 μmol) was prepared using the general procedure.

[0524]

[0525] Compound S217-Fmoc (0.15 μmol) was prepared using the general procedure.

[0526]

[0527] Compound-Fmoc (0.15 μmol) was prepared using the general procedure.

[0528] S218

[0529]

[0530] Compound S219-Fmoc (0.15 μmol) was prepared using the general procedure.

[0531]

[0532] Compound S220-Fmoc

[0533]

[0534] Prepared using the general procedure (0.15 μmol).

[0535] 1H NMR(600MHz,D2O)δ7.78(d,J=7.6Hz,2H),7.56(d,J=7.5Hz,2H),7.36(t,J=7.5Hz,2H),7.28(t,J=7.5Hz,2H),4.94(d,J=3.9 Hz,1H),4.47(d,J=8.1Hz,1H),4.39(d,J=5.6Hz,1H),4.27(d,J=10.5Hz,1H),4.19(t,J=13.0Hz,2H),3.92(dd,J=9.6,2.9Hz ,2H),3.90–3.82(m,2H),3.81–3.20(m,22H),2.81(s,2H),2.61(dd,J=12.5,4.8Hz,1H),2.12–1.95(m,2H),1.90–1.85(m,3H ),1.82(s,2H),1.62(t,J=12.2Hz,1H),1.34(s,1H),1.19(d,J=6.6Hz,2H),1.03(d,J=6.5Hz,4H),0.84(s,5H),0.73(s,2H).

[0536] Compound S221-Fmoc

[0537]

[0538] Prepared using the general procedure (0.15 μmol).

[0539] 1H NMR(600MHz,D2O)δ7.81–7.67(m,2H),7.63–7.50(m,4H),7.43–7.30(m,4H),7.31–7.22(m,2H),4.9 3(d,J=4.0Hz,1H),4.42(d,J=8.2Hz,1H),4.38–4.30(m,1H),4.08(d,J=9.6Hz,2H),4.05–3.13(m,26 H),2.84(q,J=9.0,6.9Hz,2H),2.62(dd,J=12.4,4.6Hz,1H),1.86(s,3H),1.82(s,3H),1.63(t,J=1 2.2Hz, 1H), 1.36–1.33 (m, 1H), 1.19–1.18 (m, 2H), 1.12 (s, 11H), 1.02 (d, J = 6.7Hz, 5H), 0.73 (s, 1H).

[0540] Compound-Fmoc

[0541]

[0542] Prepared using the general procedure (0.15 μmol).

[0543] 1H NMR(600MHz,D2O)δ7.79–7.68(m,2H),7.59–7.40(m,6H),7.33(t,J=7.6Hz,2H),7.26(t,J=7 .5Hz,2H),4.93(d,J=4.0Hz,1H),4.47–4.38(m,1H),4.35(d,J=5.6Hz,1H),4.22–4.05(m,2H) ,3.97–3.31(m,26H),2.84(s,2H),2.62(dd,J=12.4,4.5Hz,1H),1.87(s,3H),1.82(s,3H),1. 62(t,J=12.2Hz,1H),1.35(s,1H),1.19(d,J=7.0Hz,2H),1.02(d,J=6.5Hz,5H),0.74(s,1H).

[0544] Compound S223-Fmoc (0.15 μmol) was prepared using the general procedure.

[0545]

[0546] 1H NMR(600MHz,D2O)δ7.93–7.42(m,8H),7.37–7.29(m,2H),7.26(t,J=7.5Hz,2H),4.93(d ,J=4.0Hz,1H),4.48–4.40(m,1H),4.37(d,J=6.0Hz,1H),4.21–4.06(m,2H),4.00–3.32( m,26H),2.91–2.74(m,2H),2.63(dd,J=12.5,4.6Hz,1H),1.87(s,3H),1.82(s,3H),1.6 3(t,J=12.2Hz,1H),1.34(s,1H),1.20–1.19(m,2H),1.02(d,J=6.6Hz,5H),0.73(s,1H).

[0547] Compound S224-Fmoc

[0548]

[0549] Prepared using the general procedure (0.15 μmol).

[0550] 1H NMR(600MHz,D2O)δ7.71(d,J=7.7Hz,2H),7.67–7.47(m,4H),7.32(q,J=8.6,8.1Hz,2H),7.26(q,J=9 .2,7.5Hz,2H),6.97–6.75(m,2H),4.93(d,J=3.9Hz,1H),4.50–4.38(m,1H),4.37–4.28(m,1H),4.18 –4.04(m,2H),4.03–3.21(m,28H),2.90–2.77(m,2H),2.62(dd,J=12.4,4.5Hz,1H),1.86(s,3H),1.8 2(s,3H),1.63(t,J=12.2Hz,1H),1.35(s,1H),1.20–1.17(m,2H),1.02(d,J=6.6Hz,5H),0.74(s,1H).

[0551] Compound S225-Fmoc

[0552]

[0553] Prepared using the general procedure (0.15 μmol).

[0554] 1H NMR(600MHz,D2O)δ7.79–7.70(m,2H),7.56–7.49(m,2H),7.45–7.20(m,8H),4.93(d,J=4.1Hz,1H), 4.42(d,J=9.3Hz,1H),4.32(d,J=6.3Hz,1H),4.23–4.04(m,2H),3.98–3.47(m,23H),3.36(d,J=9.3 Hz,3H),2.88–2.76(m,2H),2.62(dd,J=12.5,4.5Hz,1H),2.19(s,3H),1.86(s,3H),1.81(s,3H),1. 63(t,J=12.2Hz,1H),1.36–1.33(m,1H),1.19(d,J=6.5Hz,2H),1.02(d,J=6.6Hz,5H),0.73(s,1H).

[0555] Compound S226-Fmoc

[0556]

[0557] Prepared using the general procedure (0.15 μmol).

[0558] 1H NMR(600MHz,D2O)δ7.76(d,J=7.9Hz,2H),7.55(d,J=7.5Hz,2H),7.40–7.32(m,3H),7. 32–7.18(m,4H),4.93(d,J=4.1Hz,1H),4.45–4.33(m,2H),4.24–4.13(m,2H),4.05–3. 31(m,26H),2.82(s,2H),2.63(dd,J=12.4,4.6Hz,1H),1.89(s,3H),1.81(s,3H),1.62 (t,J=12.2Hz,1H),1.34(s,1H),1.20–1.19(m,2H),1.02(d,J=6.6Hz,5H),0.73(s,1H).

[0559] Compound S227-Fmoc

[0560]

[0561] Prepared using the general procedure (0.15 μmol).

[0562] 1H NMR(600MHz,D2O)δ7.76(d,J=7.8Hz,2H),7.54(d,J=7.8Hz,2H),7.35(t,J=7.1Hz,2H),7.31–7.09(m,7H),4 .94(d,J=4.1Hz,1H),4.49–4.34(m,2H),4.25–4.08(m,3H),3.90(dd,J=9.8,3.2Hz,1H),3.86–3.51(m,19H), 3.45–3.23(m,6H),3.19(dd,J=14.1,8.8Hz,1H),2.87–2.74(m,2H),2.62(dd,J=12.5,4.6Hz,1H),1.84(d,J= 20.1Hz, 6H), 1.61 (t, J = 12.2Hz, 1H), 1.33 (s, 1H), 1.19 (d, J = 6.6Hz, 2H), 1.03 (d, J = 6.5Hz, 5H), 0.72 (s, 1H).

[0563] Compound S228-Fmoc (0.15 μmol) was prepared using the general procedure.

[0564]

[0565] Compound S229-Fmoc

[0566]

[0567] Prepared using the general procedure (0.15 μmol).

[0568] 1H NMR(600MHz,D2O)δ7.75(dd,J=32.6,7.7Hz,1H),7.62–7.39(m,4H),7.30(dq,J=43.3,9.1,7.4Hz ,4H),7.08–6.87(m,1H),4.93(d,J=4.0Hz,1H),4.51–4.29(m,2H),4.26–4.06(m,2H),4.02–3.24( m,26H),2.91–2.76(m,1H),2.62(dd,J=12.4,4.5Hz,1H),2.08(s,3H),1.84(d,J=28.4Hz,6H),1.6 3(t,J=12.2Hz,1H),1.38–1.32(m,1H),1.20(d,J=6.5Hz,2H),1.02(d,J=6.6Hz,5H),0.73(s,1H).

[0569] Compound S230-Fmoc (0.15 μmol) was prepared using the general procedure.

[0570]

[0571] Compound S231-Fmoc

[0572]

[0573] Prepared using the general procedure (0.15 μmol).

[0574] 1H NMR(600MHz,D2O)δ8.00–7.79(m,2H),7.69(dd,J=31.0,7.9Hz,3H),7.59–7.39(m,3H),7.37–7 .20(m,4H),4.93(d,J=4.0Hz,1H),4.47–4.40(m,1H),4.39–4.33(m,1H),4.22–4.06(m,2H),4. 03–3.15(m,26H),2.90–2.75(m,1H),2.63(dd,J=12.4,4.6Hz,1H),1.88(s,3H),1.82(s,3H),1 .63(t,J=12.2Hz,1H),1.34(s,1H),1.19(d,J=6.6Hz,2H),1.02(d,J=6.7Hz,5H),0.74(s,1H).

[0575] Example 1B-S-Synthesis of Compounds - Procedure B

[0576] I. Synthesis

[0577] The following reaction scheme is used to synthesize 6-sulfo-sialyl Lewis X derivatives (S series compounds).

[0578] Scheme 10: Attachment of terminally modified fragments

[0579] In Scheme 10, the designation S1XX represents a compound wherein the substituent at position 5 of the terminal sialic acid is one of the fragments described herein in Table 1, and the designation S2XX represents a compound wherein position 9 of the terminal sialic acid is substituted with one of the fragments described in Table 1. The compounds prepared according to Scheme 10 all have And referred to herein as S1XX-azide and the like.

[0580] I. Synthesis of Precursors 300 and 350

[0581] Precursor compounds 300 and 350 of Scheme 10 were synthesized as follows.

[0582] 1. Synthesis of galactose structural units.

[0583] Plan 10-1

[0584] The following conditions were used in the reaction shown in Scheme 10-1: a) BzCl, pyridine; b) AcOH, Ac2O, HBr; c) Ag2CO3, acetone:H2O; d) CNCCl3, DBU. Note that Scheme 10-1 shows the same transformation as Scheme 1-1 of Procedure A. The compound numbers in Scheme 10-1 are different from those in Scheme 1-1, but the compounds are the same.

[0585] More specifically, in a 1L round-bottom flask in an ice bath, galactose (30 g, 166 mmol) was dissolved in 400 mL of pyridine. During 1 h, benzoyl chloride (154 mL, 1.33 mol) was added dropwise to the cooling solution. After adding all the benzoyl chlorides, the reaction was taken out of the ice bath and mixed at room temperature for 2 hours, which resulted in the formation of a large amount of salts. The elution was performed by TLC (30% EtOAC / Hex, R f =0.8) monitor reaction and when completing, remove salt by vacuum filtration.Excess pyridine is evaporated under reduced pressure, and crude material is dissolved among the EtOAc (1L), uses 5%H continuously SO (5x 100mL), saturated CuSO (2x 100mL), 1MHCl (2x 100mL), salt solution (2x 100mL) washing, through MgSO Drying, and evaporated under reduced pressure solvent.The oily matter of gained is transferred in the 2L round-bottomed flask equipped with condenser and magnetic bar and is placed in oil bath.Under vigorous stirring, round-bottomed flask is heated to reflux, with adding MeOH (about 1L in total).This process causes forming desired full benzoylation galactose 10, is collected and in next step without further purification by vacuum filtration and promptly uses.In ice bath, under vigorous stirring, 10 is suspended in diacetyl anhydride (50mL) and acetic acid (80mL). To this suspension was added dropwise hydrogen bromide in acetic acid (204 mL, 833 mmol) over 1 h; the mixture was allowed to reach room temperature. After approximately 2 h, the reaction mixture became translucent orange. The precipitate was analyzed by TLC (20% EtOAc / Hex, R f=0.50) was observed for complete conversion, the reaction was diluted with EtOAc (1 L), extracted with DI water (5 x 200 mL), quenched with sodium bicarbonate (100 mL of DI water, then solid sodium bicarbonate), brine (2 x 100 mL), dried over MgSO4 and the volume was reduced to give 20 (104 g, 95% over two steps) as a white solid. To a solution of 20 (104 g, 158 mmol) in acetone (300 mL) and water (14.2 mL) was added freshly prepared Ag2CO3 (22 g, 79 mmol). The reaction was kept in the dark at room temperature and analyzed by TLC (30% EtOAc / Hex, R f =0.45). When all the starting material was consumed, the reaction was filtered through celite, reduced in vacuo, dissolved in EtOAc (500 mL), washed with saturated sodium bicarbonate (3x100 mL), brine (2x 100 mL), dried over MgSO4, and reduced in volume to yield the desired product 3. The fully benzoylated galactol 30 was dissolved in DCM (316 mL) containing trichloroacetonitrile (48 mL, 470 mmol). To this solution was added DBU dropwise until the reaction pH reached about 10. The product was analyzed by TLC (15% EtOAc / Hex, R f =0.2) to monitor the progress of the reaction and reduce the reaction volume when all the starting material has been consumed. The desired product 40 was purified by flash chromatography (15% EtOAc / Hex) to give the desired α-imidate (79.05 g, 68% over two steps) as a white solid. 1 The H data are consistent with those reported in the literature.

[0586] 2. Synthesis of N-phthalimido-glucosamine structural unit.

[0587] Plan 10-2

[0588] The following conditions are used in the reaction shown in Scheme 10-2: a) phthalic anhydride, sodium methoxide; b) acetic anhydride, pyridine; c) ethylenediamine / acetic acid; d) trichloroacetonitrile, DBU; e) 5-amino-Cbz-pentan-1-ol, TMSOTf; f) sodium methoxide; g) TBDPS-Cl, imidazole.

[0589] Glucosamine hydrochloride (20g, 93mmol) is dissolved in MeOH (100mL) containing sodium methoxide (1M, 110mL) and mixed at room temperature for 30min. The reaction mixture is filtered to remove NaCl and the filtrate is added to a 500mL round-bottom flask. Phthalic anhydride (15.1g, 102mmol) is added to this solution and the mixture is vigorously mixed under reflux for 2 hours. During this period, a product in the form of a white insoluble material is formed. After 2 hours, the reaction is cooled in an ice bath and the insoluble material (50) is filtered and washed with ice-cold MeOH and dried under reduced pressure. At 4°C, solid material 50 is suspended in pyridine (130mL) and acetic anhydride (52mL, 560mmol) and catalytic DMAP are added dropwise to this solution. The mixture is kept on ice until all acetic anhydride have been added and afterwards, the reaction is warmed to room temperature and mixed overnight. Monitor reaction process with TLC (1:1EtOAc / Hex) and after completing, under reduced pressure remove excessive pyridine and diacetyl oxide.The roughage material gained is dissolved among the EtOAc (1L), and thoroughly washed with 1M HCl (10x 150mL), sodium bicarbonate (5x 100mL), salt solution (2x 100mL), and through MgSO Dry.Under reduced pressure remove solvent to produce white solid 60 (35g), it is promptly proceeded to next step without other purification.At room temperature, ethylenediamine (5.88mL, 88mmol) and acetic acid (5mL, 88mmol) are dissolved among the THF (366mL) and mix 30min.In this solution, add 60 (35g) and allow solution to stir and spend the night. The reaction was monitored by TLC (1: 1 EtOAc / Hex) and after completion, THF was removed by decompression, the crude material was redissolved in EtOAc (500 mL), washed with 1M HCl (5 x 75 mL), saturated sodium bicarbonate (3 x 50 mL), brine (2 x 50 mL), dried over MgSO4, and reduced in vacuo to provide 70 as a white solid. 70 (27.1 g) was dissolved in DCM (311 mL) containing trichloroacetonitrile (7.5 mL, 75 mmol). DBU was added dropwise to the mixture until the reaction pH → 10. The reaction progress was monitored by TLC (1: 1 EtOAc / Hex) and when complete, the solvent was removed by decompression and the product was purified by flash chromatography (1: 1 EtOAc / Hex (containing 0.1% TEA)) to produce 80 (25.2 g, 46% yield over 4 steps) as a clear oil. 1 H NMR and mass spectral data were consistent with those reported in the literature. 80 (12.6 g, 21.7 mmol) and 5-amino-Cbz-pentan-1-ol (5.15 g, 21.7 mmol) were dissolved in a mixture containing flame-dried To the 4-nitro-2-oxo-1-yl ... 1 H NMR and mass spectrometry data were consistent with those reported in the literature. 90 (8 g, 12.2 mmol) was dissolved in MeOH (120 mL) and to this solution was added sodium methoxide in methanol (1 M stock solution) dropwise until the reaction pH stabilized at approximately 9. It is important to note that Keep the pH above 9 as there is a risk of phthalimide ring opening . Monitor the reaction by TLC (10% MeOH / DCM) and confirm by MALDI-MS. When removing all acetates, use Amberchrom 50WX8 hydrogen form, 200-400 mesh (Sigma#217514) to neutralize the reaction pH. Separate the resin by vacuum filtration and remove the reaction solvent under reduced pressure to produce the desired triol 100 in a sticky white foam. In 100, add DMF (120mL) and imidazoles (1.66g, 24.4mmol). This solution is placed in an ice bath and during 1 hour, TBDPS-Cl (4.7mL, 18.3mmol) is dropwise added into the mixture. Allow the reaction to be warming up to room temperature overnight and next day, monitor the reaction by TLC (100% EtOAc). Remove DMF via rotary evaporation and the crude product is redissolved in EtOAc (500mL). The organic layer was washed three times with 1M HCl (3×50 mL), brine (2×50 mL), dried over MgSO 4 , reduced in volume and purified by flash chromatography (40% EtOAc / Hex) to provide 110 (6.53 g, 70% over two steps) as a white foam. 1 H NMR and mass spectrometric data were consistent with those details reported in the literature.

[0590] 3. Disaccharide glycosylation

[0591] Plan 10-3

[0592] The following conditions were used in the reaction shown in Scheme 10-3: TMSOTf, 4AMS.

[0593] More specifically, acceptor 110 (6.53 g, 8.5 mmol) and donor 40 (6.3 g, 8.5 mmol) were dissolved in a 1% ethanol solution containing MS in DCM (85mL) and mixed at room temperature for 30min. In a dry ice bath, the reaction mixture is cooled to -40 DEG C and TMSOTf (308 μ L, 1.7 mmol) is added to the mixture. During 1 hour, the reaction is slowly warmed to -20 DEG C. During this period, the reaction is monitored by TLC (40% EtOAc / Hex) and after completion, quenched with TEA (474 ​​μ L, 3.4 mmol). Note: this reaction is regioselective glycosylation. If the reaction temperature increases too much, or an excess of donor (40) is used, there is the possibility of overglycosylation at the 3-position. The reaction is filtered through diatomite and purified by flash chromatography (40% EtOAc / Hex) to produce the product 120 (5.1 g, 44% yield) in white foam.

[0594] 1 H (600MHz, CDCl3), δ (ppm): GlcNPhth: 5.13 (H1), 4.18 (dd, J=10.8, 8.6Hz, H2), 4.60 (H3), 4.09 (H4), 3.46 (H5), 3.79 (H6a, H6b); Gal: 5.11 (H1), 5.85 ( dd,J=10.5,8.1Hz,H2),5.57(dd,J=10.5,3.5Hz,H3),5.95(d,J=3.3Hz,H4 ), 4.29 (H5), 4.69 (ddd, J=11.7, 4.3, H6a), 4.37 (ddd, J=11.6, 8.3Hz, H6b).

[0595] From HSQC 13 C (75MHz, CDCl3), δ (ppm): GlcNPhth: 97.8 (C1), 56.1 (C2), 69.8 (C3) 80.2 (C4), 75.3 ( C5), 61.7 (C6); Gal: 101.5 (C1), 69.6 (C2), 71.6 (C3), 67.9 (C4), 72.5 (C5), 62.6 (C6).

[0596] MALDI-TOF MS m / z C 77 H 76 N2O 18 SiNa(M+Na) + Calculated value = 1367.4755, Found value = 1367.6501

[0597] 4. Debenzoylation, phthalimide deprotection, and overall acetylation

[0598] Plan 10-4

[0599] The following conditions were used in the reaction shown in Scheme 10-4: a) sodium methoxide in methanol; b) hydrazine hydrate; c) acetic anhydride and pyridine.

[0600] More specifically, disaccharide 120 (4.7 g, 3.5 mmol) was dissolved in MeOH (175 mL) and an aliquot of sodium methoxide (1 M stock solution) was added to the reaction until the reaction pH was approximately 9. Special care was taken not to raise the pH above 9, as increased alkalinity could result in undesirable opening of the imide ring. The progress of the reaction was monitored by MALDI-MS, and upon removal of all benzoyl groups, the reaction pH was neutralized with Amberchrom 50WX8 Hydrogen Form, the resin was removed by vacuum filtration, and the MeOH was reduced under reduced pressure to produce 130, which was carried to the next step without purification. 130 was dissolved in EtOH (175 mL) and hydrazine hydrate (50%-60%, 1.6 mL, 17.5 mmol) was added to this solution. The reaction was stirred at reflux until deprotection of the phthalimido group was achieved (monitored by MALDI-MS). In 40% phthalimide, 1-dimethyl-1-oxo-2-oxo-1-propene was added 4-nitro-2-oxo-1-propene.After successful phthalimide deprotection, the reaction was cooled, any insoluble material was filtered through diatomite, and the filtrate was deducted to produce crude product 140, which was carried out downwards without additional purification. 140 was dissolved in pyridine (65mL) and catalytic DMAP was added to the reaction. The reaction was placed in an ice bath and acetic anhydride (6.6mL, 70mmol) was added dropwise. After adding all anhydrides, the reaction was warming up to room temperature. The reaction was monitored by TLC (40% EtOAc / Hex). After conversion to the product, the reaction was quenched with MeOH (20 mL), the solvent was reduced in vacuo, and the crude material was redissolved in EtOAc (500 mL), washed with 1 M HCl (10 x 50 mL), saturated copper sulfate (2 x 50 mL), saturated sodium bicarbonate (5 x 50), brine (2 x 50), dried over MgSO4, reduced, and purified by flash chromatography (40% EtOAc / Hex) to give 150 (2.03 g, 55% over three steps) as a white foam.

[0601] 1H (600MHz, CDCl3), δ (ppm): GlcNAc: 4.35 (d, J = 7.8Hz, H1), 4.07 (H2), 5.01 (H3), 4.11 (H4), 3.31 (H5), 3.94 (H6a), 3.89 (H6b); Gal: 4.76(d,J=8.0Hz,H1),5.06(H2),4.92(dd,J=3.5Hz,H3),5.31(dd,J=3.2Hz,H4),3.76(dd,J=6.6Hz,H5),4.11(H6a,H6b)

[0602] From HSQC 13 C (75MHz, CDCl3): δ (ppm): GlcNAc: 101.1 (C1), 53.6 (C2), 72.3 (C3), 73.9 (C4), 75.4 ( C5), 61.3 (C6); Gal: 100.5 (C1), 69.3 (C2), 71.0 (C3), 67.2 (C4), 70.7 (C5), 61.3 (C6).

[0603] MALDI-TOF MS m / z C 53 H 70 N2NaO 18 SiNa(M+Na) + Calculated value = 1073.4285, Found value = 1073.5575

[0604] 5.6-O-TBDPS deprotection.

[0605] Plan 10-5

[0606] The following conditions were used in the reaction shown in Scheme 10-5: a) HF-pyridine

[0607] More particularly, 150 (2.03 g, 1.93 mmol) is dissolved in pyridine (15 mL) and placed in an ice bath. To this solution, HF-pyridine (70%, 2.3 mL, 19.3 mmol) is added. After adding all HF-pyridines, the reaction is maintained on ice for 30 min and then warmed to room temperature. The reaction is monitored with TLC (40% acetone / hex) and after completion, content is transferred to EtOAc (300 mL) and extracted with 1 M HCl, salt water, and dried over MgSO. The crude reaction is purified by flash chromatography (using 1: 1 acetone / hex) to produce 160 (1.12 g, 72%) in the form of white foam.

[0608] 1H (600MHz, CDCl3), δ (ppm): GlcNAc: 4.40 (d, J = 8.2Hz, H1), 3.96 (H2), 5.06 (H3), 3.91 (H4), 3.35 (H5), 3.86 (H6a), 3.72 (H6b) ;

[0609] From HSQC 13 C (75MHz, CDCl3), δ (ppm): GlcNAc: 101.3 (C1), 54.1 (C2), 73.0 (C3), 74.9 (C4), 75.0 ( C5), 60.7 (C6); Gal: 100.9 (C1), 69.8 (C2), 70.9 (C3), 68.0 (C4), 70.4 (C5), 61.2 (C6).

[0610] MALDI-TOF MS m / z C 37 H 52 N2O 18 Na(M+Na) + Calculated value = 835.3113, Found value = 835.4063

[0611] 6.6-O Sulfate Installation

[0612] Plan 10-6

[0613] The following conditions were used in the reaction shown in Scheme 10-6: a) Sulfur trioxide pyridine complex in DMF.

[0614] More specifically, 160 (1.34 g, 1.65 mmol) was dissolved in 33 mL of DMF at room temperature. Sulfur trioxide pyridine complex (787 mg, 4.9 mmol) was added to the mixture and the reaction was stirred overnight. Product formation was monitored by TLC (10% MeOH / DCM). When all starting materials were consumed, the reaction was quenched by adding 3 mL of a 1:1 mixture of MeOH / TEA. The solvent was removed under reduced pressure and the product was desalted using size exclusion chromatography (2.5 x 100 cm, eluted with 1:1 MeOH / DCM, 4 min / fraction, 4-5 mL / fraction) on an LH-20. The product was found to elute in fractions 66–76 and to produce a white foam when dried. The product 170 was moved to the next step without additional purification.

[0615] 7. Overall deacetylation

[0616] Plan 10-7

[0617] The following conditions were used in the reaction shown in Scheme 10-7: a) Sodium methoxide in methanol.

[0618] More specifically, 170 (1.25 g, 12.8 mmol) was dissolved in MeOH (50 mL) and the pH was adjusted to about 9 with sodium methoxide in methanol (1 M stock solution). The progress of the reaction was monitored by TLC (7:3:2 EtOH:NH 4 OH:H 2 O) and ESI-MS. When all acetate groups were removed, the reaction was concentrated using Amberchrom H as described above. + The resin neutralization reaction pH is then removed by vacuum filtration. The filtrate is dried under reduced pressure and the corresponding solid is redissolved in the ammonium bicarbonate of minimum volume 100mM. Material is loaded onto a Bio-Rad P2 size exclusion column (60x 5cm) and eluted with 100mM ammonium bicarbonate, with every kind of fraction representing an elution of 15min (approximately 40mL / fraction). The fraction is stained with 5% sulfuric acid in ethanol and confirmed by ESI. The fraction containing product is collected, concentrated under reduced pressure, and placed on a freeze dryer to produce 180 (726mg, 80% productive rate) that is a white fluffy solid.

[0619] 1 H (600MHz, CDCl3), δ (ppm): GlcNAc: 4.38 (H1), 3.58 (H2), 3.63 (H5), 4.26 (H6a), 4.17 (H6b), 1.85 (NHAc); Gal: 4.38 (H1), 3.38 (H2), 3.55 (H3), 3.79 (H4), 3.62 (H6a and H6b).

[0620] From HSQC 13 C (75MHz, CDCl3), δ (ppm): GlcNAc: 102.1 (C1), 55.2 (C2), 72.6 (C5), 66.0 (C6), 22.1 (NHAc); Gal: 102.1 (C1), 71.2 (C2), 72.3 (C3), 68.8 (C4), 61.0 (C6)

[0621] ESI-TOF MS m / z C 27 H 41 N2O 16 S(MH) -Calculated value = 681.2182, Found value = 681.0994

[0622] 8. Cbz deprotection

[0623] Plan 10-8

[0624] The following conditions were used in the reaction shown in Scheme 10-8: a) Pd / C, H2, 1:1 t-BuOH / H2O.

[0625] More specifically, 180 (100 mg, 0.14 mmol) was dissolved in a 1:1 mixture of t-BuOH / H2O (1.5 mL) and to this solution was added catalytic approximately 5% (wt / wt) Pd / C. The reaction was mixed overnight under a hydrogen atmosphere. The progress of the reaction was monitored by TLC (7:3:2 EtOH:NH4OH:H2O) and upon completion, the reaction was filtered through a 0.2 μm Whatman syringe filter and freeze-dried to yield 190 (73 mg, 91%) as a white solid.

[0626] 9. Azide Installation

[0627] Plan 10-9

[0628] The following conditions were used in the reaction shown in Scheme 10-9: a) 1-(azidosulfonyl)-1H-imidazol-3-ium chloride, copper(II) sulfate, potassium carbonate.

[0629] More specifically, 190 (72 mg, 0.13 mmol), 1-(azidosulfonyl)-1H-imidazol-3-ium chloride (80 mg, 0.38 mmol), copper sulfate (10 μmol) and potassium carbonate (79 mg, 0.57 mmol, 579 μL of a 1 M stock solution) were dissolved in water (1.96 mL). The reaction was mixed at room temperature and monitored by TLC (7:3:2 EtOH:NH4OH:H2O). When all the starting materials were consumed, Cuprisorb was added. TM Add to the reaction, which was incubated at room temperature for 2 hours to remove the copper from the solution. The resin was removed by filtration, the reaction volume was reduced in vacuo, and the product was purified by P2 size exclusion (70 x 2.5 cm column, 100 mM ammonium bicarbonate, 8 min / fraction, approximately 5 mL / fraction). Fractions containing product (26-32) were combined and dried under reduced pressure to yield 200 (67 mg, 89% yield) as a white solid.

[0630] 1H (600MHz, CDCl3), δ (ppm): GlcNAc: 4.39 (H1), 3.59 (H2), 3.64 (H5), 4.26 (ddd, J = 10.9Hz, H6a), 4.17 (ddd, J=11.0, 3.7Hz, H6b); Gal: 4.41 (H1), 3.38 (H2), 3.53 (H3), 3.77 (H4), 3.60 (H6a and H6b)

[0631] From HSQC 13 C (75MHz, CDCl3), δ (ppm): GlcNAc: 102.5 (C1), 55.1 (C2), 65.9 (C6), 21.9 (NHAc); Gal: 101.3 (C1), 71.1 (C2), 72.1 (C3), 68.7 (C4), 61.0 (C6)

[0632] ESI-TOF MS m / z C 19 H 33 N4O 14 S(MH) - Calculated value = 573.1719, Found value = 573.0749

[0633] 10. Chemical Synthesis of 6-Sulfo-N-acetylglucosamine Plan 10-10

[0634] The following conditions were used in the reaction shown in Scheme 10-10: a) AcO, sodium acetate; b) TMSOTf; c) 5-azidopentan-1-ol, TMSOTf; d) sodium methoxide; e) sulfur trioxide-pyridine. Note that Scheme 10-10 shows the same transformation as Scheme 1-6 of Procedure A. The compound numbers in Scheme 10-10 are different from those in Scheme 1-6, but the compounds are the same.

[0635] More specifically, N-acetylglucosamine (100 g, 450 mmol) was added portionwise to a refluxing solution of acetic anhydride (340 mL, 3375 mmol) and sodium acetate (37.2 g, 450 mmol). The mixture was stirred at reflux until all the material was dissolved and TLC analysis (100% EtOAc, R f=0.47) illustrates that the reaction is complete. Excess acetic anhydride is removed under reduced pressure and the crude material is dissolved in EtOAc (1L) and washed with saturated sodium bicarbonate (10x 150mL), brine (3x 100), dried over MgSO4, and decremented in a vacuum to provide the fully acetylated N-acetylglucosamine (210, 129g, 74%) as a white solid. 210 (5.4g, 13.9mmol) is dissolved in DCE (69mL) and TMSOTf (2.5mL, 13.87mmol) is added to this solution. The solution is warmed to 60°C to promote oxazolidinone formation, which is determined by TLC (40% acetone / hex, R f =0.3). The reaction was quenched with TEA (2.2 mL, 15.18 mmol), the solvent was reduced, and the product was purified by flash chromatography (40% acetone / hex) to give 220 (3.9 g, 85%) as a clear oil. Triacetylated oxazolidinone 220 (4.36 g, 13.24 mmol) was dissolved in DCE (66 mL). To this solution were added 5-azidopentan-1-ol (3.4 g, 26.48 mmol) and MS (3 g). The combination was mixed at room temperature before the addition of TMSOTf (2.4 mL, 13.24 mmol). The reaction was heated to 60°C to promote glycosylation, and its progress was determined by TLC (90% EtOAc / Hex, R f =0.55) monitoring. When all starting materials were converted into products, the reaction was quenched with TEA (1.84 mL, 13.24 mmol) and cooled to room temperature. The solvent was removed under reduced pressure and glycosylated N-acetylglucosamine was recovered by flash chromatography (80 → 90% EtOAc / Hex) to provide 230 (5 g, 80%) as a white solid. 230 (14.38 g, 31.37 mmol) was dissolved in MeOH (156 mL) and the solution was adjusted to pH 9 with sodium methoxide in methanol (1 M stock solution). The product was purified by TLC (20% MeOH / DCM, R r =0.76) and upon completion, the reaction was monitored with Dowex 50WX8 H +The resin is neutralized with pH. The resin is removed by filtration and MeOH is removed by decompression to produce triol 240 (9.3 g, 90%) as a white solid. 6-O-sulfation is achieved by dissolving 240 (600 mg, 1.8 mmol) in DMF (36 mL) and placing the mixture in an ice bath. Sulfur trioxide-pyridine complex (287 mg, 1.8 mmol) is added to the cooled solution. The reaction is stirred overnight at 4 ° C for three days, and other sulfur trioxide-pyridine (287 mg, 1.8 mmol) is added every day. The reaction is monitored by TLC (20% MeOH / DCM, Rf=0.23) and quenched with an aliquot of NaOH (5 M, pH → 8). The solvent is removed, and the reaction is purified by Bio-Gel size exclusion chromatography. The fraction containing the product is freeze-dried to produce 250 (514 mg, 69%) as a white powder.

[0636] Figure 1 A and 1B show 2D of compounds 240 and 250 of Scheme 10-10. 1 H-NMR spectrum.

[0637] 11. Enzymatic installation of β1,4-Gal to form 6-sulfo-N-acetyllactosamine

[0638] Plan 10-11 (Same transformations as Schemes 1-7 of Procedure A - numbered differently)

[0639] The installation of β1,4-Gal was achieved by dissolving 250 (5 mg, 11.7 μmol) and UDP-galactose (10 mg, 17.6 μmol) in Tris buffer (585 μL, pH 7.3, 0.1 M) containing MnCl2 (10 mM). To this solution was added B4GALT1 (50 μg, 1% wt / wt) and calf intestinal alkaline phosphatase (CIAP, 5.85 μL, 1 kU stock solution). The reaction was incubated at 37 ° C for three hours. The HPLC was performed by LC-ESI-MS (equipped with Waters XBridge BEH, amide column, 2.5 μm, The reaction progress was monitored at 2.1 x 150 mm (flow rate: 0.25 mL / min, A = 10 mM ammonium formate, B = ACN. Linear gradient: 80% → 60% B over 18 min).

[0640] The reaction was monitored by following the crude reaction using hydrophilic interaction liquid chromatography / electrospray ionization mass spectrometry (HILIC-LC / ESI-MS). Figure 2The results of such tracking are shown. If the reaction was not complete, excess UDP-Gal (5 mg, 5.85 μmol) and B4GALT1 (25 μg) were added to the reaction. The reaction mixture was passed through PALL Filtration was performed through centrifugal spin filters (3k MWCO) and the filtrate was purified by Bio-Gel P2 size exclusion chromatography. Product containing fractions were freeze dried to afford the desired disaccharide 200 (4.8 mg, 70%).

[0641] 12. Chemoenzymatic Synthesis of CMP-Neu5-Glycine

[0642] Plan 10-12

[0643] The following conditions were used in the reactions shown in Schemes 10-12: a) sodium pyruvate, cytidine triphosphate disodium salt; PmNanA; NmCSS, PmPPA; b) Lindlar catalyst, H2.

[0644] More specifically, ManAz (260, 100 mg, 0.38 mmol), disodium cytidine triphosphate (219 mg, 0.42 mmol) and sodium pyruvate (210 mg, 1.91 mmol) were dissolved in Tris buffer (100 mM, pH 8.5, 19 mL) containing MgCl2 (10 mM).To the reaction were added sialic acid aldolase (PmNanA) from Pasteurella multocida (Li, Y.; Yu, H.; Cao, H.; Lau, K.; Muthana, S.; Tiwari, VK; Son, B.; Chen, X., Pasteurella multocida sialic acid aldolase: a promising biocatalyst. Appl Microbiol Biot. 2008, 79(6), 963.), CMP-sialic acid synthetase (NmCSS) from Neisseria meningitidis group B (Yu, H.; Yu, H.; Karpel, R.; Chen, X., Chemoenzymatic synthesis of CMP–sialic acid derivatives by a one-pot two-enzyme system: comparison of substrate flexibility of three microbial CMP–sialic acid synthetases [Chemoezymatic synthesis of CMP-sialic acid derivatives by a one-pot two-enzyme system: comparison of substrate flexibility of three microbial CMP–sialic acid synthetases]. Bioorg Med Chem [Bioorganic and Medicinal Chemistry] 2004, 12(24), 6427-6435.) and inorganic pyrophosphatase (PmPPA) (1 mg) from Pasteurella multocida (see: Lau, K.; Thon, V.; Yu, H.; Ding, L.; Chen, Y.; Muthana, MM; Wong, D.; Huang, R.; Chen, X., Highly efficient chemoenzymatic synthesis of [small beta]1-4-linked galactosides with promiscuous bacterial [small beta]1-4-galactosyltransferases [Efficient chemoenzymatic synthesis of [β]1-4-linked galactosides using promiscuous bacterial [β]1-4-galactosyltransferases]. Chem Commun [Chemical Communications] 2010, 46(33), 6066-6068.) and incubate the reaction at 37°C overnight.When all 260 was consumed (as determined by TLC), calf intestinal alkaline phosphatase (CIAP, 1000 units) was added to the reaction and the mixture was incubated again at 37°C overnight. On the third day, ice-cold ethanol (17 mL) was added to the mixture and all insoluble material was removed by centrifugation. The supernatant was concentrated and CMP-Neu5Az (270) was purified by P2 size exclusion chromatography (5 x 60 cm, eluted with 100 mM ammonium bicarbonate, 15 min / fraction or approximately 35 mL). Product-positive fractions were combined to produce a white fluffy solid (185 mg, 74%), the analytical details of which were consistent with those found in the literature. 270 (185 mg, 0.28 mmol) was dissolved in 20 mM ammonium hydroxide (28 mL) and to this mixture was added Lindlar's catalyst (9 mg). The mixture was stirred under a hydrogen atmosphere until no starting material could be observed by TLC (7:3:2 EtOH:NH4OH:H2O).The catalyst was removed using a 0.2 μm syringe filter and the filtrate was dried by lyophilization to give 280 as a white solid (219 mg, 92%), which was used without further purification.

[0645] Compound 280 was analyzed by LC-MS (using Waters XBridge BEH, amide column, 2.5 μm, 2.1x150mm, using ESI as the detector, flow rate: 0.25mL / min; 80%→60% A over 18 min (A=10mM ammonium formate, pH 3.4, B=acetonitrile)) for analysis. The peak of compound 280 eluted at 19.0 minutes. The molecular mass of compound 280 was determined using electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS); m / z C 20 H 31 N5O 16 P(MH) - Calculated = 628.1509, Found = 628.0397.

[0646] 13. Enzymatic Installation of α2,3-Neu5-glycine

[0647] Plan 10-13

[0648] The following conditions were used in the reactions shown in Schemes 10-13: a) PmST1, CIAP.

[0649] More specifically, 200 (70 mg, 0.12 mmol) and 280 (115 mg, 183 mmol) were dissolved in Tris buffer (100 mM, pH 8.5, 12 mL) containing sialyltransferase-I (PmSt1) from Pasteurella multocida (700 μg) (Yu H, Chokhawala H, Karpel R, Yu H, Wu B, Zhang J, Zhang Y, Jia Q, Chen X. A multifunctional Pasteurella multocida sialyltransferase: A powerful tool for the synthesis of sialoside libraries. J Am Chem Soc. 2005; 127: 17618–17619.) and CIAP (120 U). The reaction was incubated at 30 ° C until all 200 was consumed (as determined by TLC (7: 3: 2 EtOH: NH 4 OH: HO). After all the starting disaccharides were consumed, ice-cold EtOH was added to the mixture (12 mL) and insoluble material was removed by centrifugation. The supernatant was concentrated and purified by P2 size exclusion chromatography (2.5 x 70 cm, eluted with 100 mM ammonium bicarbonate, 8 min / fraction). The fractions containing the desired product were combined and concentrated by lyophilization to produce 290 (82 mg, 77% yield) as a white solid.

[0650] Compound 290 was purified by LC-MS HPLC (using Waters XBridge BEH, amide column, 2.5 μm, 2.1 x 150 mm, using ESI as the detector, flow rate: 0.25 mL / min; 80% → 60% A over 18 min (A = 10 mM ammonium formate, pH 3.4, B = acetonitrile). The peak of compound 290 eluted at 11.2 minutes. The molecular mass of compound 290 was determined using ESI-TOF MS: m / z C 30 H 51 N6O 22 S(MH) - Calculated value = 879.2783, found value = 879.1189.

[0651] 14. Enzymatic Installation of α1,3-fucose and FUT6

[0652] Plan 10-14

[0653] The following conditions were used in the reactions shown in Schemes 10-14: a) FUT6, GDP-fucose.

[0654] More generally, 290 (71 mg, 80.8 μmol) and GDP-fucose (102 mg, 161 μmol) were dissolved in sodium cacodylate buffer (100 mM, pH 6.5, 4 mL) containing MnCl2 (10 mM). To this solution, FUT6 (50 μg) and CIAP (40 units) were added. The reaction was incubated at 37°C overnight and the next day, the reaction was analyzed by Shimadzu LC-ESI-IT-TOF (Waters XBridge BEH, amide column, 2.5 μm, 2.1 x 150 mm, using ESI as the detector, flow rate: 0.25 mL / min; 20% → 40% A, progress monitored over 18 min (A = 10 mM ammonium formate, pH 3.4, B = acetonitrile). If the reaction was incomplete, additional aliquots of FUT6 were added until the reaction was complete. Upon completion, ice-cold EtOH (4 mL) was added and insoluble material was removed by centrifugation. The product was purified by P2 chromatography (70 x 2.5 cm, 100 mM ammonium bicarbonate, 8 min / fraction). Fractions containing product were combined to yield 300 (65 mg, 79%) as a white, fluffy solid.

[0655] 1 H (600MHz, CDCl3), δ (ppm): GlcNAc: 4.42 (d, J = 7.9Hz, H1), 3.75 (H2), 3.73 (H3), 3.86 (H4), 3.64 (H5), 4. 23 (H6a and H6b); Gal: 4.47 (d, J=7.8Hz, H1), 3.36 (H2), 3.97 (dd, J=9.9, 3.1Hz, H2), 3.82 (H3), 3.46 (H4), 3. 55 (H6a and H6b); Fuc: 4.96 (d, J = 3.9 Hz, H1), 3.52 (H2), 3.75 (H3), 3.64 (H4), 4.68 (H5), 1.04 (d, J = 6.6 Hz, CH3); Neu5 glycine: 2.61 (dd, J = 12.4, 6.0 Hz, H3-equatorial), 1.68 (dd, J = 12.2 Hz, H3-axial), 3.57 (H4), 3.75 (H9a and H9b).

[0656] From HSQC 13C (75MHz, CDCl3), δ (ppm): GlcNAc: 100.9 (C1), 55.8 (C2), 74.7 (C3), 73.0 (C4), 72.9 (C5), 65.9 (C6); Gal: 101.3 (C1), 69.6 (C2), 75.4 (C3), 67.2 (C 4),67.9(C5),61.3(C6);Fuc:98.8(C1),67.0(C2),69.2(C3),71.8(C4), 66.9(C5)15.9(CH3); Neu5 glycine:39.5(C3),72.6(C4),51.7(C5),62.7(C9)

[0657] Compound 300 was analyzed by HPLC LC-MS (using Waters XBridge BEH, amide column, 2.5 μm, 2.1 x 150 mm, using ESI as the detector, flow rate: 0.25 mL / min; 80% → 60% A over 18 min (A = 10 mM ammonium formate, pH 3.4, B = acetonitrile). The peak of compound 300 eluted at 13 minutes. The molecular mass of compound 300 was determined using ESI-TOF MS: m / z C 36 H 61 N6O 26 S(MH) - Calculated value = 1025.3362, found value = 1025.1684.

[0658] 15. Enzymatic Synthesis of 9-AminoCMP-Neu5Ac

[0659] Plan 10-15

[0660] The following conditions can be used in the reactions shown in Schemes 10-15: a) sodium pyruvate, cytidine triphosphate disodium salt; PmNanA; NmCSS, PmPPA; b) Lindella's catalyst, H2.

[0661] More specifically, synthesis 330 may be performed according to the same scheme as shown for synthesis 280 .

[0662] 16. Enzymatic Installation of α2,3-Neu5Ac-9-amino

[0663] Plan 10-16

[0664] The following conditions can be used in the reactions shown in Schemes 1-16: a) PmST1, CIAP.

[0665] More specifically, synthesis 340 may be performed according to the same scheme as shown for synthesis 290 .

[0666] 17. Enzymatic Installation of α1,3-fucose and FUT6

[0667] Plan 10-17

[0668] The following conditions can be used in the reactions shown in Schemes 10-17: a) FUT6, GDP-fucose.

[0669] More specifically, 350 can be synthesized according to the same scheme as that of 300 .

[0670] II. Library of S-compounds according to Procedure B

[0671] Exemplary S-compounds were prepared according to Procedure B as exemplified below.

[0672] S106-Synthesis of Azides

[0673] Plan 10-18

[0674] The following conditions were used in the reactions shown in Schemes 10-18: a) Triethylamine.

[0675] More specifically, 300 (14 mg, 13.66 μmol) and quinoline-2-carbonyl-NHS ester (11 mg, 54.6 μmol) were dissolved in a 1:1 mixture of H O / DMF (683 μL) and triethylamine (11 μL, 81.9 μmol) was added to this solution. The reaction mixture was incubated at 37° C. overnight and the next day, the progress was monitored by LC-ESI-MS (equipped with a Shimadzu C18 5 μm, 50×4.6 mm column, flow rate: 0.25 mL / min, gradient: 0→100% acetonitrile over 10 min (solvent A=10 mM ammonium formate, pH 3.4)). When all starting material was consumed, the product was purified by P2 chromatography (1.5 x 80 cm, 100 mM ammonium bicarbonate, 15 drops / fraction) and the product-containing fractions were combined and lyophilized to yield S106-azide (11 mg, 68%) as a white fluffy solid.

[0676] LC-MS of trace amounts of S106-azide was performed using the same conditions as described above for monitoring the progress of the reaction. The product S106-azide eluted at 6 minutes. The molecular mass of compound S106-azide was determined using ESI-TOF MS: m / z C 46 H 66 N7O27 S(MH - )Calculated value = 1180.3733Found value = 1180.1682.

[0677] Figure 5 The HSQC (600 MHz) 2D NMR spectrum of S106 azide is shown.

[0678] The following representative compounds were prepared generally according to Scheme 10, and in particular according to Schemes 10-18. All compounds were analyzed by LC-ESI-MS (equipped with a Shimadzu C18 5 μm, 50 x 4.6 mm column, flow rate: 0.25 mL / min, gradient: 0→100% acetonitrile over 10 min (solvent A=10 mM ammonium formate, pH 3.4)).

[0679] S101-Azide:

[0680] S101-Azide: The product eluted at 6.75 min. The molecular mass of S101-azide was determined using ESI-TOF. MS: m / z C 47 H 67 N6O 27 S(MH - )Calculated value = 1179.3780, Found value = 1179.1732.

[0681] S103-Azide:

[0682] S103-Azide: The product eluted at 5.6 min. The molecular mass of S103-azide was determined using ESI-TOF. MS: m / z C 41 H 63 N6O 27 S2(MH - )Calculated value = 1135.3188, Found value = 1135.1208.

[0683] S105-Azide:

[0684] S105-Azide: The product eluted at 6.5 min. The molecular mass of S105-azide was determined using ESI-TOF. MS: m / z C 44 H 64 N7O 27 S2(MH -)Calculated value = 1186.3297Found value = 1186.0807.

[0685] S107-Azide

[0686] S107-azide: The product eluted at 7 min. The molecular mass of S107-azide was determined using ESI-TOF. MS: m / z C 49 H 69 N6O 27 S(MH - )Calculated value = 1205.3937, Found value = 1205.1807.

[0687] S110-Azide:

[0688] The product eluted at 6.6 min. The molecular mass of S110-azide was determined using ESI-TOF. MS: m / z C 45 H 65 N6O 27 S2(MH - )Calculated value = 1185.3345, Found value = 1185.1246.

[0689] S112-Azide: The molecular mass of S112-azide was determined by ESI-TOF. MS: m / zC 45 H 64 ClN6O 27 S2(MH - )Calculated value = 1219.2955, Found value = 1219.1099.

[0690] Example 2: Synthesis of D-compound library

[0691] The following reaction scheme is used to synthesize D series compounds.

[0692] Scheme 2: Attachment of terminally modified fragments

[0693] In Scheme 2, the notation D1XX represents a compound in which the N-acetyl group at position 5 of the terminal sialic acid is substituted with one of the fragments described below, and the notation D2XX represents a compound in which position 9 of the terminal sialic acid is substituted with one of the fragments described below.

[0694] I. Synthesis of Precursors 45 and 46

[0695] Precursor compounds 45 and 46 of Scheme 2 were synthesized as follows.

[0696] 1. Chemical Synthesis of Lactose Connected to an End-Dimeric Linker

[0697] Plan 1-2

[0698]

[0699] The following conditions were used in the reaction shown in Scheme 1-2: a) BzCl, pyridine; b) HBr-AcOH / Ac2O; c) 5-azidopentan-1-ol, AgOTf; d) sodium methoxide.

[0700] More specifically, lactose (25 g, 73 mmol) was suspended in pyridine (300 mL) and the mixture was placed in an ice bath. Benzoyl chloride (100 mL, 876 mmol) was added dropwise to the cooled solution over 30 min. After all the benzoyl chloride had been added, the reaction mixture was allowed to reach room temperature and vigorously distilled until the lactose was fully benzoylated, as determined by TLC (30% EtOAc / Hex, R f =0.30). The contents of the reaction mixture were added to an ice / water mixture, which resulted in the formation of an oily layer upon standing. The aqueous phase was decanted, retaining the oily material at the bottom, which was dissolved in boiling EtOH and placed at -20°C overnight to produce the product 36 as a white / yellow solid, which was carried downward without further purification. In an ice bath, the fully benzylated lactose was suspended in acetic acid (142 mL) and acetic anhydride (47 mL). Over a 1 hour period, HBr-AcOH (176 mL, 10 equivalents) was added dropwise to the suspension. After all the HBr-AcOH was added, the reaction was allowed to reach room temperature and stirred vigorously. The product was analyzed by TLC (30% EtOAc / Hex, R f =0.44) to monitor the progress of the reaction. The reaction was worked up by diluting in EtOAc (2 L), the acid was extracted with water (5 x 250 mL), quenched with sodium bicarbonate (the reaction was added to a beaker and solid sodium bicarbonate was added to the stirred solution), brine (2 x 200 mL), dried over MgSO4, and reduced to yield the anomer-isomeric bromide product (3, 74.9 g, 91%, 2 steps) as a white solid. 37 (75 g, 66 mmol) was dissolved in 20 g of MS and 5-azidopentan-1-ol (9.3 g, 72 mmol) in toluene (660 mL). The mixture was stirred at room temperature for 30 min and then cooled to -80 ° C in a dry ice bath. Silver trifluoromethanesulfonate (21 g, 82.5 mmol) was added to the cooled reaction. The reaction mixture was analyzed by TLC (30% EtOAc / Hex, R f=0.34) and after complete conversion, the reaction mixture was filtered through a pad of celite, reduced in volume, and purified by flash chromatography (30% EtOAc / Hex) to give 38 (53.8 g, 69%) as a white solid. 38 (53.8 g, 45.5 mmol) was dissolved in MeOH (455 mL) and the pH was adjusted to 9 by adding 1 M sodium methoxide solution. The product was analyzed by TLC (20% MeOH / DCM, R f =0.57) to monitor the reaction, and Dowex 50WX8 H + The resin (pH adjusted to neutral) was quenched and the solvent was reduced to produce a brown solid, which was washed with acetone (acetone removes the benzyl methyl ester) to provide a light brown solid (39, 17.3 g, 84%).

[0701] 2. Chemoenzymatic Synthesis of Fmoc-Protected α2,3-Sialyl Lactose

[0702] Plan 2-2

[0703]

[0704] The following conditions were used in the reaction shown in Scheme 2-2: a) PmST1, NmCSS, PmPPA, Neu5Ac, CTP; b) Pd / C, H2, c) Fmoc-OSu.

[0705] More specifically, lactose 39 (100 mg, 0.22 mmol) containing an end epimeric linker was dissolved in Tris buffer (11 mL, 100 mM, pH-8.5) containing MgCl (10 mM), N-acetylneuraminic acid (102 mg, 0.33 mmol) and cytidine-5'-triphosphate (159 mg, 0.33 mmol). The solution was adjusted to pH 8.5 using 5 M NaOH. PmST1 (1 mg), NmCSS (2 mg) and PmPPA (2 mg) were added to the solution. The reaction mixture was incubated at 37° C. with periodic inversion of the reaction vessel. Progress was monitored by TLC (7:3:2 EtOH, NH OH, H O) and upon observation of complete conversion, the enzyme was removed by precipitation (facilitated by the addition of EtOH (11 mL)). The mixture was placed at −20° C. for 2 hours and insoluble material was removed by centrifugation. Bio-Gel P2 size exclusion chromatography is used to purify the crude product. The fraction containing the product is concentrated and freeze-dried to produce trisaccharide 40 (142 mg, 87%) as a white fluffy solid. 40 (142 mg, 0.19 mmol) is dissolved in t-BuOH / H2O (1.9 mL, 1:1 v / v) containing Pd / C (7 mg). The reaction is mixed at room temperature under a hydrogen atmosphere and the reaction process is monitored by ESI-MS. A 0.2 μm PALL Acrodisc syringe filter is used to remove the catalyst and the solution is freeze-dried to produce a white fluffy solid (41). 41 is dissolved in DI water (9.5 mL) containing sodium bicarbonate (4.5 mmol). In a separate vial, Fmoc-OSu (128 mg, 0.38 mmol) is dissolved in ACN (9.5 mL), and under vigorous mixing, 41 is added dropwise to this vial. The progress of the reaction was monitored by ESI-MS and when all the starting material was converted to the desired product, the reaction was reduced in vacuo and purified by preparative HPLC (Agilent 1200, Eclipse XDB-C18, 21.2 x 250 mm, 7 μm. A solvent = 50 mM ammonium bicarbonate; B = ACN, λ = 262 m) (linear gradient: 10 → 60% B over 30 min, flow rate: 20 mL / min). The fractions containing the compound were freeze-dried to give the Fmoc-protected trisaccharide 42 (100 mg, 56% over 2 steps) as a white fluffy solid.

[0706] 3. Enzymatic Synthesis of GD3-Azide Derivatives

[0707] Plan 2-3

[0708]

[0709] The following conditions were used in the reactions shown in Schemes 2-3: a) CSTII, CMP-Neu5Ac-9-N3; b) CSTII, CMP-Neu5Az.

[0710] More specifically, 42 (50 mg, 53 μmol) was dissolved in Tris buffer (5.3 mL, 100 mM, pH 8.5) containing MgCl2 (10 mM), CMP-Neu5Ac-9-N3 or CMP-Neu5Az (106 μmol), CSTIIΔ32 I53S (1 mg), and CIAP (53 μL, 1 kU stock solution). 6 The reaction was incubated at 37°C and monitored by ESI-MS. When no additional product conversion was observed, the reaction mixture was passed through PALL Filtration was performed through centrifugal spin filters (3k MWCO) and the filtrate was purified by Bio-Gel P2 size exclusion chromatography. Product-containing fractions were freeze-dried to afford the desired tetrasaccharides 43 (40 mg, 59%) and 44 (44 mg, 65%) as white fluffy solids.

[0711] 4. General Protocol for Reduction of α2,8-Azido-Sialic Acid

[0712] Plan 2-4

[0713]

[0714] The following conditions were used in the reactions shown in Schemes 2-4: Zn, AcOH, H2O.

[0715] More specifically, 44 (30 mg, 23.5 μmol) was dissolved in H o (3 mL) and acid-treated Zn (60 mg) was added to this solution. The mixture was placed in an ice bath and acetic acid (100 μL) was added to the cooled solution. The reaction was monitored by ESI-MS and after all starting materials were converted into products, the reaction was quenched with saturated ammonium bicarbonate (pH 7). The reaction was filtered with a 0.2 μm PALL Acrodisc syringe filter and purified by preparative HPLC (Agilent 1200, Eclipse XDB-C18, 21.2 x 250 mm, 7 μm. A solvent = 50 mM ammonium bicarbonate; B = ACN, λ = 262 m) (linear gradient: 10 → 60% B, over 30 min, flow rate: 20 mL / min). The fraction containing the compound was freeze-dried to produce the Fmoc-protected trisaccharide 45 (20 mg, 69%) as a white fluffy solid.

[0716] 5. General protocol for Fmoc deprotection

[0717] Deprotection of compounds of the D series is generally carried out according to Schemes 1-13 above.

[0718] II.D-Libraries of Compounds

[0719] Tetrasaccharide (45 or 46, 1 mg, about 0.8 μmol) is dissolved in DMF (100 μL) containing diisopropylethylamine (DIPEA, 4 μmol). To this solution, acyl chloride (1.6 μmol) is added and the reaction is mixed for 30 min using a vortexer. The reaction is monitored by ESI, and when the complete conversion of the product is achieved, the product is passed through HPLC chromatography (under the following conditions: Agilent 1200 HPLC, Eclipse XDB-C8, 4.6 x 250 mm, 5 μm analytical column, A solvent = 50 mM ammonium bicarbonate; B = ACN, λ = 262 m), linear gradient: 10% → 60% B, over 30 min, flow rate 1 mL / min) purification. The fraction containing the desired product is freeze-dried to produce the desired product as a white fluffy solid.

[0720] The following D series compounds were prepared using the terminally modified fragments listed in Table 1 above. 1 The identity of the compound was confirmed by H-NMR mass spectrometry.

[0721] Compound D101 (0.15 μmol) was prepared using the general procedure.

[0722]

[0723] Compound D102 (0.15 μmol) was prepared using the general procedure.

[0725]

[0726] Compound D103 (0.15 μmol) was prepared using the general procedure.

[0727]

[0728] Compound D104 (0.15 μmol) was prepared using the general procedure.

[0729]

[0730] Compound D105 (0.15 μmol) was prepared using the general procedure

[0731]

[0732] Compound D106 (0.15 μmol) was prepared using the general procedure

[0733]

[0734] Compound D107 (0.15 μmol) was prepared using the general procedure

[0735]

[0736] Compound D108 (0.15 μmol) was prepared using the general procedure

[0737]

[0738] Compound D109 (0.15 μmol) was prepared using the general procedure

[0739]

[0740] Compound D110 (0.15 μmol) was prepared using the general procedure

[0741]

[0742] Compound D111 (0.15 μmol) was prepared using the general procedure

[0743]

[0744] Compound D112 (0.15 μmol) was prepared using the general procedure

[0745]

[0746] Compound D113 (0.15 μmol) was prepared using the general procedure

[0747]

[0748] Compound D114 (0.15 μmol) was prepared using the general procedure

[0749]

[0750] Compound D115 (0.15 μmol) was prepared using the general procedure

[0751]

[0752] Compound D116 (0.15 μmol) was prepared using the general procedure

[0753]

[0754] Compound D117 (0.15 μmol) was prepared using the general procedure

[0755]

[0756] Compound D118 (0.15 μmol) was prepared using the general procedure

[0757]

[0758] Compound D119 (0.15 μmol) was prepared using the general procedure

[0759]

[0760] Compound D120 (0.15 μmol) was prepared using the general procedure

[0761]

[0762] Compound D121 (0.15 μmol) was prepared using the general procedure

[0763]

[0764] Compound D122 (0.15 μmol) was prepared using the general procedure

[0765]

[0766] Compound D123 (0.15 μmol) was prepared using the general procedure

[0767]

[0768] Compound D124 (0.15 μmol) was prepared using the general procedure

[0769]

[0770] Compound D125 (0.15 μmol) was prepared using the general procedure

[0771]

[0772] Compound D126 (0.15 μmol) was prepared using the general procedure

[0773]

[0774] Compound D127 (0.15 μmol) was prepared using the general procedure

[0775]

[0776] Compound D128 (0.15 μmol) was prepared using the general procedure

[0777]

[0778] Compound D129 (0.15 μmol) was prepared using the general procedure

[0779]

[0780] Compound D130 (0.15 μmol) was prepared using the general procedure

[0781]

[0782] Compound D131 (0.15 μmol) was prepared using the general procedure

[0783]

[0784] Compound D201 (0.15 μmol) was prepared using the general procedure

[0785]

[0786] Compound D202 (0.15 μmol) was prepared using the general procedure

[0787]

[0788] Compound D203 (0.15 μmol) was prepared using the general procedure

[0789]

[0790] Compound D204 (0.15 μmol) was prepared using the general procedure

[0791]

[0792] Compound D205 (0.15 μmol) was prepared using the general procedure

[0793]

[0794] Compound D206 (0.15 μmol) was prepared using the general procedure

[0795]

[0796] Compound D207 (0.15 μmol) was prepared using the general procedure

[0797]

[0798] Compound D208 (0.15 μmol) was prepared using the general procedure

[0799]

[0800] Compound D209 (0.15 μmol) was prepared using the general procedure

[0801]

[0802] Compound D210 (0.15 μmol) was prepared using the general procedure

[0803]

[0804] Compound D211 (0.15 μmol) was prepared using the general procedure

[0805]

[0806] Compound D212 (0.15 μmol) was prepared using the general procedure

[0807]

[0808] Compound D213 (0.15 μmol) was prepared using the general procedure

[0809]

[0810] Compound D214 (0.15 μmol) was prepared using the general procedure

[0811]

[0812] Compound D215 (0.15 μmol) was prepared using the general procedure

[0813]

[0814] Compound D216 (0.15 μmol) was prepared using the general procedure

[0815]

[0816] Compound D217 (0.15 μmol) was prepared using the general procedure

[0817]

[0818] Compound D218 (0.15 μmol) was prepared using the general procedure

[0819]

[0820] Compound D219 (0.15 μmol) was prepared using the general procedure

[0821]

[0822] Compound D220 (0.15 μmol) was prepared using the general procedure

[0823]

[0824] Compound D221 (0.15 μmol) was prepared using the general procedure

[0825]

[0826] Compound D222 (0.15 μmol) was prepared using the general procedure

[0827]

[0828] Compound D223 (0.15 μmol) was prepared using the general procedure

[0829]

[0830] Compound D224 (0.15 μmol) was prepared using the general procedure

[0831]

[0832] Compound D225 (0.15 μmol) was prepared using the general procedure

[0833]

[0834] Compound D226 (0.15 μmol) was prepared using the general procedure

[0835]

[0836] Compound D227 (0.15 μmol) was prepared using the general procedure

[0837]

[0838] Compound D228 (0.15 μmol) was prepared using the general procedure

[0839]

[0840] Compound D229 (0.15 μmol) was prepared using the general procedure

[0841]

[0842] Compound D230 (0.15 μmol) was prepared using the general procedure

[0843]

[0844] Compound D231 (0.15 μmol) was prepared using the general procedure

[0845]

[0846] Example 3: Conjugation of polymers and polysaccharides

[0847] The following synthetic schemes are used to conjugate polysaccharides to polymers. In these schemes, only the carbohydrate units directly bonded to the linker groups are used to partially represent the polysaccharides. Additional preparation schemes are provided in Example 8.

[0848] Synthesis Example 3-1: Preparation of polysaccharides carrying click reagents

[0849] Synthesis Scheme 3-1

[0850]

[0851] Synthesis Scheme 3-2

[0852]

[0853] Synthesis Scheme 3-3

[0854]

[0855] Synthesis Scheme 3-4

[0856]

[0857] Synthesis Scheme 3-5

[0858]

[0859] Synthesis Scheme 3-6

[0860]

[0861] For each of the synthetic schemes 3-1 to 3-6, the terminal epimeric linker was modified by the corresponding click reagent using the following general scheme. Amine-terminated polysaccharide (1 equivalent) and click reagent (5 equivalents) were dissolved in DMF to a final polysaccharide concentration of 10 mM. Diisopropylethylamine (DIPEA, 5 equivalents) was added to this solution and the reaction was incubated at 37 ° C until no additional starting material was observed by ESI-MS. The reaction was purified by P-2 size exclusion chromatography (eluting with 0.1 M ammonium bicarbonate). The fractions containing the product were combined and the solvent was removed by lyophilization.

[0862] The skilled artisan will appreciate that many other click chemistry reagents are commercially available and can be used depending on the needs, availability, and desirability of the final product. For example, for the synthesis of Scheme 3-1, any of the following commercially available click reagents (from suppliers such as Broadpharma, Sigma-Millipore, or Fischer Scientific) can be used:

[0863]

[0864]

[0865]

[0866]

[0867]

[0868] Synthesis Example 3-2: Production of Exemplary Nanoparticles Using a Polymer with DBCO.

[0869] Nanoparticles were prepared using an emulsion method. A representative example of nanoparticle production includes the following. PLGA(10k)-PEG(5k)-COOH and PLGA(10k)-PEG(5k)-DBCO were weighed at a weight ratio of 3:1 and dissolved in an organic solvent mixture. The polymer concentration in the organic solvent (59.5% ethyl acetate and 40.5% benzyl alcohol) was 37 mg / mL. 10 mL of the polymer solution was added to 40 mL of an aqueous phase consisting of either 0 or 0.1% Tween 80 in water and homogenized using a rotating stator to form a coarse emulsion. The coarse emulsion was further homogenized into a fine nanoemulsion using a microfluidizer with three channels. The nanoemulsion was then quenched by adding 450 mL of cold water to harden the nanoparticles. The quenched nanoparticle solution was then concentrated and washed by tangential flow filtration to remove the organic solvent. The concentration of the polymerized nanoparticles was determined by evaporating water from a known volume of the nanoparticle solution. The nanoparticle solution was then prepared for conjugation with an oligosaccharide ligand.

[0870] Synthesis Example 3-3: Ligand-Azide Conjugation to the Core DBCO Nanoparticle Surface via SPAAC Chemistry

[0871] The purified N3-ligand prepared as described above was conjugated to the surface of purified PLGA-DBCO nanoparticles via a SPAAC copper-free click chemistry protocol. Briefly, the nanoparticles with DBCO were incubated with ligand-azide at 4°C, room temperature, or 37°C overnight. After the conjugation reaction was complete, the nanoparticles were washed using TFF to remove unreacted components and the buffer was replaced with an appropriate isotonic solution (such as 10% sucrose). The nanoparticle solution was then sterile filtered through a 0.2 μm filter.

[0872] Synthesis Examples 3-4: Exemplary Nanoparticles Prepared by Pre-conjugation of Ligand-Azide with DBCO-Polymer Additional production of pellets

[0873] Nanoparticles were prepared using an emulsion method. A representative example of nanoparticle production is described below. 10 mg of ligand-azide was dissolved in 0.6 mL of DMSO or DMF or acetonitrile or some appropriate solvent. The dissolved ligand solution was added to 15 mg of PLGA (10k) -PEG (5k) -DBCO (dissolved in an organic solvent mixture of ethyl acetate: benzyl alcohol). The mixture was stirred and mixed overnight to allow the ligand-azide to be conjugated to the PLGA-PEG-DBCO via azide-DBCO click chemistry coupling. After mixing overnight, 285 mg of PLGA (10k) -PEG (5k) -COOH was added to the reaction mixture and the solution was mixed / stirred until it was visually clear.

[0874] The final volume of the organic solvent was 3.03 mL, containing 2.43 mL of ethyl acetate:benzyl alcohol in a 60:40 volume ratio and 0.6 mL of DMSO. To introduce the fluorescent moiety into the nanoparticles, the polymer fluorescein-PLGA can be used. The total fraction of fluorescein polymer in the total polymer used to prepare the nanoparticles was 1% (by weight), i.e., 3 mg of PLGA-fluorescein was also added to the polymer solution.

[0875] This polymer solution was added to 27 mL of cold water (saturated with ethyl acetate) and homogenized using an IKA T-18 rotating stator to form a coarse emulsion. The coarse emulsion was further homogenized into a fine nanoemulsion using a microfluidizer with 3 channels (Microfluidics LM10). The nanoemulsion was then quenched by adding 270 mL of cold water to harden the nanoparticles. The quenched nanoparticle solution was then concentrated and washed with cold water by tangential flow filtration (KR2i TFF, Repligen) to remove the organic solvent and unreacted ligand-azide. The concentration of the polymerized nanoparticles was determined by evaporating water from a known volume of the nanoparticle solution. Sucrose was added to the nanoparticle solution at 10% wt / wt and filtered using a 0.2 μm Millipore syringe filter. The nanoparticle solution was then frozen at -20°C. The size of the nanoparticles was measured using dynamic light scattering using a Malvern Zetasizer. Dynamic light scattering (DLS) uses the constant random thermal motion of particles and molecules, known as Brownian motion, to measure size. Particles diffuse at a rate related to their size, with smaller particles diffusing faster than larger particles. The diffusion rate is measured by illuminating the particles with a laser to create a speckle pattern. Fluctuations in scattered intensity at specific angles are detected using a sensitive photodiode detector. The intensity changes are analyzed using a digital autocorrelator to generate a correlation function. This curve is analyzed to determine the size and size distribution of the particles. Nanoparticles are diluted with clean water to a concentration of approximately 0.1-1.0 mg / mL and measured in a Zetasizer. Each value generated is the average of three readings.

[0876] Synthesis Examples 3-5: Conjugation of polysaccharides to polymers via amide coupling

[0877] Synthesis Scheme 3-7

[0878] Carboxylic acid-terminated PLGA or PLGA-PEG are dissolved in DCM to a final concentration of 5mM. The solution is mixed at room temperature, and EDC and NHS or sulfo-NHS are added to this sample to provide a final concentration of 25mM for each reagent. The solution is stirred at room temperature for one hour to produce the activated ester of desired NHS or sulfo-NHS. By adding MeOH (10x reaction volume), the activated ester is precipitated and collected by centrifugation (2,000g, continuing 10min). The activated PLGA-PEG-NHS precipitation is dissolved in DCM again and precipitated a total of three times in MeOH to ensure that residual EDC and NHS or sulfo-NHS are removed completely. By dissolving the polysaccharide containing amine and the activated PLGA in the mixture of an organic solvent to promote the reaction of the NHS group on the amine component of polysaccharide-amine and the PLGA-PEG-NHS, promote amide formation. The selection of organic solvent can be optimized for each polysaccharide-amine and PLGA-PEG-NHS system. For example, typically, the polysaccharide is dissolved in DMSO or DMF and, if necessary, a small amount of water is added. The PLGA-PEG-NHS block copolymer is dissolved in DMSO or DMF. The polysaccharide and PLGA-PEG-NHS solutions should be clear and then mixed to initiate the reaction. N,N-diisopropylethylamine (DIPEA) is added to this reaction mixture to couple the PLGA-PEG and polysaccharide. The reaction is carried out for at least 4 hours or more. After the reaction is completed, the organic solvent mixture is emulsified into the aqueous phase to produce a nanoemulsion. It is expected that unreacted components in the reaction will be removed during the manufacture of the nanoparticles. The aqueous phase used in the nanoemulsion formation process can be weakly alkaline (pH>7.2) to facilitate the hydrolysis of unreacted NHS groups to COOH.

[0879] Synthetic Examples 3-6: Conjugation of polysaccharides to polymers via azide / alkyne click coupling

[0880] Synthesis Scheme 3-8

[0881] The azide-containing polysaccharide (>1.2 equivalents) and the alkyne-containing polymer (1 equivalent) are mixed in a solvent system containing DMSO or DMF, benzyl alcohol, and ethyl acetate. The reaction is allowed to proceed for at least 4 hours until completion. Unreacted alkyne groups in the polymer can be capped by adding an excess of the azide-containing molecule. After the reaction is completed, the organic solvent mixture is emulsified into the aqueous phase to produce a nanoemulsion. It is expected that unreacted components of the reaction will be removed during the manufacture of the nanoparticles.

[0882] Synthesis Example 3-7: Conjugation of polysaccharides to polymers via copper-free click coupling

[0883] Synthesis Scheme 3-9A

[0884] Synthesis Scheme 3-9B

[0885] Synthesis Scheme 3-9C

[0886] The azide-containing polysaccharide (1 equivalent) and the cyclooctyne-containing polymer (1 equivalent) are mixed in a solvent system containing DMSO or DMF, benzyl alcohol, and ethyl acetate. The reaction is allowed to proceed for at least 4 hours until completion. Unreacted alkyne groups in the polymer can be capped by adding an excess of azide-containing molecules. After the reaction is completed, the organic solvent mixture is emulsified into the aqueous phase to produce a nanoemulsion. It is expected that unreacted components of the reaction will be removed during the manufacture of the nanoparticles.

[0887] Synthesis Scheme 3-9D

[0888] The thiol-containing polysaccharide (1 equivalent) and the maleimide-containing polymer (1 equivalent) are mixed in a solvent system containing DMSO or DMF, benzyl alcohol and ethyl acetate. The reaction is carried out for at least 4 hours until completion. Unreacted maleimide groups in the polymer can be capped by adding an excess of thiol-containing molecules such as cysteine. After the reaction is carried out to completion, the organic solvent mixture is emulsified into the aqueous phase to produce a nanoemulsion. It is expected that unreacted components in the reaction will be removed during the manufacture of the nanoparticles.

[0889] The above-mentioned method for fabricating nanoparticles can be applied to various coupling chemistries, including maleimide-thiol or cysteine, NHS and amine, click chemistry, etc.

[0890] Example 4: Microarray Printing and Screening

[0891] The compounds described herein were initially tested for binding to Siglec targets using microarray printing technology as described below.

[0892] The Scienion sciFLEXARRAYER S3 non-contact microarrayer (equipped with a Scienion PDC80 nozzle (Scienion)) was used to obtain the NHS-ester activated glass slides ( All compounds are printed on slide H (Schott company). Individual samples are dissolved in sodium phosphate buffer (50 μ L, 0.225 M, pH 8.5), concentration 100 μ M and with 10 repetitions, about 400 pL dot volume, at 20 ℃ and 50% humidity, print. Each slide has 24 subarrays, adopts 3x 8 layout. After printing, slide is hatched 8h in humidity chamber, then at 40 ℃, with 5mM ethanolamine in Tris buffer (pH 9.0, 50mM) blocking 30min. Rinse the sealed slide with DI water, spin dry, and store in desiccator at room temperature for future use.

[0893] Screening is performed by incubating the slides with the protein solution for a specified period of time, followed by washing and drying. The buffers used in the screening are TSM buffer (TSM, 20 mM Tris·Cl, pH 7.4, 150 mM NaCl, 2 mM CaCl2, and 2 mM MgCl2), TSM binding buffer (TSMBB, TSM buffer (containing 0.05% Tween-20 and 1% BSA)), and TSM wash buffer (TSMWB, TSM buffer (containing 0.05% Tween-20)). A typical washing procedure involves sequentially immersing the glass slides in TSM wash buffer (2 min, containing 0.05% Tween 20), TSM buffer (2 min), and water (2 x 2 min), followed by spin drying.

[0894] For screening of Fc-tagged Siglec-2, -3, -5, -6, -7, and -9, slides were incubated with Siglec-2 (Fc-tagged, R&D Systems Catalog No. 1968-SL-050, 1 μg / mL in TSMBB), Siglec-3 (Fc-tagged, R&D Systems Catalog No. 1137-SL-050, 1 μg / mL in TSMBB), Siglec-5 (Fc-tagged, R&D Systems Catalog No. 1072-SL-050, 20 μg / mL in TSMBB), Siglec-6 (Fc-tagged, R&D Systems Catalog No. 2859-SL-050, 1 μg / mL in TSMBB), Siglec-7 (Fc-tagged, R&D Systems Catalog No. 3961-SL-050, 1 μg / mL in TSMBB), Siglec-8 (Fc-tagged, R&D Systems Catalog No. 4061-SL-050, 1 μg / mL in TSMBB), Siglec-9 (Fc-tagged, R&D Systems Catalog No. 5961-SL-050, 1 μg / mL in TSMBB), Siglec-10 (Fc-tagged, R&D Systems Catalog No. 5961-SL-050, 1 μg / mL in TSMBB), Siglec-11 (Fc-tagged, R&D Systems Catalog No. 5961-SL-050, 1 μg / mL in TSMBB), Siglec-12 (Fc-tagged, R&D Systems Catalog No. 5961-SL-050, 1 μg / mL in TSMBB), Siglec-13 (Fc-tagged, R&D Systems Catalog No. 5961-SL-050, 1 μg / mL in The cells were incubated with either Siglec7 (Fc-labeled, R&D Systems catalog number 1138-SL-050, 20, 10, and 51 μg / mL in TSMBB) or Siglec9 (Fc-labeled, R&D Systems, 10, 3, and 1 μg / mL in TSMBB) for 1 h, followed by washing and incubation with goat anti-human IgG antibody (Alexa Fluor 647-conjugated, Jackson ImmunoResearch, 10 μg / mL) for 30 min. For screening of streptavidin-labeled enterovirus proteins B3 / 1013, A2 / 2018, B2 / 039, A4 / P4 and hemagglutinins of H5VN and H3N8, slides were incubated with protein (50 μg / mL in TSMBB) for 1 h, followed by washing and incubation with anti-streptavidin labeled antibody solution (StrepMAB-Classic Oyster 645, IBA Life Sciences 2-1555-050, 10 μg / mL) for 30 min. For screening of MERS spike protein, slides were incubated with a premixed mixture of MERS spike protein (Fc-labeled, 50 μg / mL) and pAb-LS nanoparticles (50 μg / mL) for 1 h, followed by washing and incubation with goat anti-human IgG antibody solution (Alexa Fluor 647-conjugated, Jackson ImmunoResearch, 10 μg / mL) for 30 min. After washing and drying, slides were scanned using a GenePix 4000B microarray scanner (Molecular Devices) at a suitable excitation wavelength with a resolution of 5 μm. Various gain and PMT values ​​were used to ensure that all signals were within the linear range of the scanner detector and that there was no signal saturation. Images were analyzed using GenePix Pro 7 software (version 7.2.29.2, Molecular Devices). Data were analyzed using Excel macros (http: / / zenodo.org / record / 5146251).The highest and lowest values ​​of total fluorescence intensity of the replicates were removed, and the remaining values ​​were used to calculate the mean and standard deviation.

[0895] The results are shown in Figures 3A to 3M (the vertical axis corresponds to the fluorescence intensity measured in relative fluorescence units (RFU)).

[0896] Example 5: Surface plasmon resonance analysis of Siglec binding

[0897] The binding K of the compounds of the present invention was determined using surface plasmon resonance (SPR) technology. d .

[0898] Surface / sample conjugation:

[0899] All analyses were performed on a GE Biacore T100 machine. Standard EDC / NHS amide conjugation chemistry (flow cells 1, 2, 3, and 4) was used to coat streptavidin (2,000 reaction units, Thermo Fisher Scientific No. 434301) on an S-series CM5 chip (Cytiva No. 29149603). All flow cells were subsequently blocked with ethanolamine and the chip surface was stabilized overnight with a degassed solution of PBS-P (10 mM NaHPO, 1.8 mM KHPO, 137 mM NaCl, 2.7 mM KCl, and 0.5% surfactant P20 (Cytiva No. BR100054), pH 7.4) flowing (10 μL / min).

[0900] To stabilize the baseline, a biotinylated compound was introduced, dissolved in DI water to a concentration of 50 μg / mL (total volume = 200 μL). Using the T100 software in manual mode, a 50 μg / mL biotinylated disialoside solution was introduced into flow cell 2 at a flow rate of 30 μL / min for 90 seconds, followed by introduction of PBS-P at a flow rate of 30 μL / min for 90 seconds. This process was repeated three times, after which no additional biotin conjugation was observed.

[0901] Siglec-3 screening

[0902] Lyophilized hFc-Siglec-3 (R&D Systems, No. 1137-SL-050) was reconstituted in PBS-P buffer to a concentration of 200 μg / mL and equilibrated on ice for 30 min before analysis. A binding assay was set up to measure the interaction between siglec-3 and biotinylated sialoside by subtracting the response units measured from flow cells 2 and 1. The analysis was performed by introducing three different concentrations of siglec-3: 200 μg / mL, 100 μg / mL, and 50 μg / mL, using the following parameters:

[0903] a. Flow rate = 30 μL / min; b. Contact time = 60 s; c. Dissociation time = 90 s; d. Stabilization time = 30 s.

[0904] Siglec-5 screening

[0905] Lyophilized hFc-Siglec-5 (R&D Systems, No. 1072-SL-050) was reconstituted in PBS-P buffer to a concentration of 200 μg / mL and equilibrated on ice for 30 min before analysis. A binding assay was set up to measure the interaction between siglec-3 and biotinylated sialoside by subtracting the response units measured from flow cells 2 and 1. The assay was performed by introducing three different concentrations of siglec-5: 200 μg / mL, 100 μg / mL, and 50 μg / mL, using the following parameters:

[0906] a. Flow rate = 30 μL / min; b. Contact time = 60 s; c. Dissociation time = 90 s; d. Stabilization time = 30 s.

[0907] Siglec-7 screening

[0908] Lyophilized hFc-Siglec-7 (R&D Systems, No. 1138-SL-050) was reconstituted in PBS-P buffer to a concentration of 200 μg / mL and equilibrated on ice for 30 min before analysis. A binding assay was set up to measure the interaction between siglec-7 and biotinylated sialoside by subtracting the response units measured from flow cells 2 and 1. The assay was performed by introducing three different concentrations of siglec-7: 200 μg / mL, 100 μg / mL, and 50 μg / mL, using the following parameters:

[0909] a. Flow rate = 30 μL / min; b. Contact time = 60 s; c. Dissociation time = 90 s; d. Stabilization time = 30 s.

[0910] Siglec-9 screening

[0911] Lyophilized hFc-Siglec-9 (R&D Systems, No. 1139-SL-050) was reconstituted in PBS-P buffer to a concentration of 200 μg / mL and equilibrated on ice for 30 min before analysis. A binding assay was set up to measure the interaction between siglec-9 and biotinylated sialoside by subtracting the response units measured from flow cells 2 and 1. The analysis was performed by introducing siglec-9 at concentrations of 100 μg / mL, 50 μg / mL, and 25 μg / mL using the following parameters:

[0912] a. Flow rate = 30 μL / min; b. Contact time = 60 s; c. Dissociation time = 90 s; d. Stabilization time = 30 s.

[0913] Siglec-E screening

[0914] Lyophilized mFc-Siglec-E (R&D Systems, No. 5806-SL-050) was reconstituted in PBS-P buffer to a concentration of 200 μg / mL and equilibrated on ice for 30 min before analysis. A parallel binding assay was set up to measure the interaction between Siglec-E and biotinylated sialoside by subtracting the respo...

Claims

1. A compound represented by any one of the following structural formulas: or a pharmaceutically acceptable salt thereof, in: For each occurrence, R1 is independently -C(O)-A, wherein A is C1-C6 alkyl, C6-C 18 Aryl, (C6-C 18 )aryl(C1-C3)alkyl, 5-18 membered heteroaryl, (5-18 membered)heteroaryl(C1-C3)alkyl, C3-C8 cycloalkyl, (C3-C8)cycloalkyl(C1-C3)alkyl, 5-8 membered heterocycloalkyl, or (5-8 membered)heterocycloalkyl(C1-C3)alkyl; wherein one or two carbon atoms in the alkyl portion of A are optionally each independently replaced by a heteroatom selected from N, O or S; and wherein A is optionally replaced by 1 to 3 R 11 group substituted, each of the R 11 The groups are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, halogen, C 6- C 12 Aryl, 5-12 membered heteroaryl and cyano, or two R 11 The groups, together with the atoms to which they are attached, form a 5-7 membered heterocyclyl having 1 to 3 heteroatoms selected from N, O or S; Each R 11 is independently optionally substituted with 1 to 3 substituents selected from halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, or cyano; and And further wherein: For each occurrence, R is independently -R L -R F , and where: For each occurrence, R L Independently are: -O-(C1-C 12 )alkylene-, -O-, -S-, -NR 100 -、-S-(C1-C 12 )alkylene-, -NR 101 -(C1-C 12 )alkylene-, -NR 101a -O-(C1-C 12 )alkylene-; -O-(CH2CH2O) m -、-O-(CH2CH2O) k -(CH2CH2)-、-NR 102 -X 100 -(C1-C 12 )alkylene-, -NR 102a -NR 102b -C(O)-(C1-C 12 ) alkylene, wherein R 100 , R 101 , R 101a , R 102 , R 102a and R 102b are each independently H or C1-C3 alkyl, and X 100 is -O- or -NH-, and wherein m and k are each independently an integer from 1 to 12; For each occurrence, R F Independently are: H, C1-C3 alkyl, -NH2, -NH-Fmoc, -NH-Boc, -NH-CBz, -NH-Troc, -NH-TFA, mono(C1-C3)alkylamino, di(C1-C3)alkylamino; -C(O)-R 103 , where R 103 is -H, -OH, or (C1-C3)alkyl; -SH, with the following structural formula Represents part; or click chemistry reagent.

2. The compound of claim 1, wherein the compound is represented by any one of the following structural formulas: or a pharmaceutically acceptable salt thereof.

3. The compound of claim 1 or 2, wherein the click chemistry reagent comprises an azide, a C2-C3 alkyne, a tetrazine, trans-cyclooctene, or a cyclooctyne.

4. The compound of claim 3, wherein the click chemistry reagent is an azide, a C2-C3 alkyne, or a moiety represented by any one of the following structural formulas: in: R co is hydrogen or halogen; X is absent or is O; and in each case, R 0 are independently selected from hydrogen, halogen, C 1-8 Alkyl, C 1-8 Alkoxy, C6-C 12 Aryl, 5-8 membered heteroaryl, C 3-8 Cycloalkyl, or 3-8 membered heterocyclic group; any two or more R 0 The groups, together with the atoms to which they are attached, optionally form an unsaturated, saturated, or aromatic 5-8 membered ring; and R td It is hydrogen, C 1-8 Alkyl, C 1-8 Alkoxy, C6-C 12 Aryl, 5-8 membered heteroaryl, C 3-8 Cycloalkyl or 3-8 membered heterocyclic group.

5. The compound of claim 3, wherein the click reagent is any one of an azide, a C2-C3 alkyne, or a moiety represented by the following structural formula: Where R* is H or methyl, for each occurrence, R # are independently H or C1-C3 alkyl, and R X It is -NH-C(O)O-.

6. The compound of claim 1, wherein for each occurrence, R is independently -O-(CH2) x -NH2 or -O-(CH2) x -NH-Fmoc, wherein x is independently an integer from 1 to 10 for each occurrence.

7. The compound of claim 6, wherein x is 5.

8. The compound according to any one of claims 1 to 7, wherein: The cycloalkyl portion of part A is selected from C6-C7 cycloalkyl; The heterocycloalkyl portion of moiety A is selected from 5-6 membered heterocycloalkyl having 1 or 2 heteroatoms selected from N, O or S; The aryl portion of moiety A is selected from phenyl or naphthyl; and The heteroaryl moiety of moiety A is selected from moieties represented by any one of the following structural formulae: Where X 1 , X 2 and X 3 Each independently selected from NR H , O or S, where R H is H or C1-C3 alkyl; and further If present, each R 11 independently selected from cyano, halogen, phenyl, halophenyl, C 1- C6 alkyl, C1-C6 haloalkyl, C1-C3 alkoxy, or two R 11 The groups, together with the atoms to which they are attached, form a [1,3]dioxolyl group optionally substituted with one or two methyl or ethyl groups.

9. The compound of any one of claims 1-8, wherein for each occurrence, R1 is independently selected from a moiety represented by the following structural formula, wherein the wavy line represents the point of attachment of R1 to the nitrogen atom:

10. The compound of any one of claims 1-9, wherein the compound is any one of compounds S-101 to S-131 or D-101 to D-131, wherein the moiety R is -O-(CH2)5-NH-Fmoc.

11. A particle comprising a compound represented by the following structural formula: GLP, in: P is a biocompatible polymer; L is a covalent linker; and G is any one of the moieties represented by the following structural formulas: or a pharmaceutically acceptable salt thereof, in: symbol represents the point of attachment to L; For each occurrence, R1 is independently -C(O)-A, wherein A is C1-C6 alkyl, C6-C 18 Aryl, (C6-C 18 )aryl(C1-C3)alkyl, 5-18 membered heteroaryl, (5-18 membered)heteroaryl(C1-C3)alkyl, C3-C8 cycloalkyl, (C3-C8)cycloalkyl(C1-C3)alkyl, 5-8 membered heterocycloalkyl, or (5-8 membered)heterocycloalkyl(C1-C3)alkyl; wherein one or two carbon atoms in the alkyl portion of A are optionally each independently replaced by a heteroatom selected from N, O or S; and wherein A is optionally replaced by 1 to 3 R 11 group substituted, each of the R 11 The groups are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, halogen, C 6- C 12 Aryl, 5-12 membered heteroaryl and cyano, or two R 11 The groups, together with the atoms to which they are attached, form a 5-7 membered heterocyclyl having 1 to 3 heteroatoms selected from N, O or S; Each R 11 Independently optionally substituted with 1 to 3 substituents selected from halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, or cyano.

12. The particle of claim 11, wherein the portion G is represented by any one of the following structural formulas: or a pharmaceutically acceptable salt thereof.

13. The particle of claim 11 or 12, wherein the biocompatible polymer comprises the copolymer PLGA-PEG.

14. The particle of any one of claims 11-12, wherein P is represented by the following structural formula: The symbol represents the point of attachment of the polymer to the linker L, and further wherein: y is an integer from 0 to 1000, x is an integer from 0 to 1000, and m is an integer from 0 to 450, with the proviso that x and y are not 0 at the same time.

15. The particle of claim 14, wherein y is an integer from 0 to 500, x is an integer from 0 to 500, and m is an integer from 0 to 250.

16. The particle of any one of claims 11-15, wherein the linker L comprises a moiety that is a product of a click chemistry reaction.

17. A particle as described in any one of claims 11 to 16, wherein the linker L comprises a portion represented by any one of the following structural formulas, wherein the symbols ----- and Each independently represents the point of attachment to the other part of the linker L, to P, or to G: Where R* is H or methyl, for each occurrence, R # are independently H or C1-C3 alkyl, and R X is -NH-C(O)O-, and R 200 It is –H or C1-C3 alkyl.

18. The particle of any one of claims 11-17, wherein the P is PLGA(10k)-PEG(5k).

19. The particle according to any one of claims 11 to 18, wherein the weight of G per unit weight of P (ligand density) is 10 to 75 μg / mg.

20. A method of treating a condition in a subject in need thereof, the method comprising: An effective amount of a compound as described in any one of claims 1-10 or 31-33, or a particle as described in any one of claims 11-19, 34-37, 39, 42, 43 and 45, or a pharmaceutically acceptable salt thereof, is administered to a subject, wherein the condition is selected from cancer, ophthalmic disease, fibrotic disease, parasitic inflammation, fungal inflammation, viral inflammation, autoimmune inflammation, nervous system inflammation, neurodegeneration, skin inflammation, kidney inflammation, cardiovascular disease, gastrointestinal inflammation, or rheumatic disease.

21. A method of treating a condition in a subject in need thereof, the method comprising: An effective amount of a compound as described in any one of claims 1 to 10 or 31-33, or a particle as described in any one of claims 11-19, 34-37, 39, 42, 43 and 45, or a pharmaceutically acceptable salt thereof, is administered to a subject, wherein the condition is selected from breast cancer, non-small cell lung cancer (NSCLC), prostate cancer, colorectal cancer, melanoma, pancreatic cancer, myelofibrosis, diabetic retinopathy, idiopathic pulmonary fibrosis, liver fibrosis, sickle cell anemia, and acute respiratory distress syndrome (ARDS).

22. A pharmaceutical composition comprising the compound of any one of claims 1 to 10 or the particles of any one of claims 11 to 19 or a pharmaceutically acceptable salt thereof in a pharmaceutically acceptable carrier.

23. A method of preparing particles comprising a molecule represented by the following structural formula: GLP, in: P is a biocompatible polymer; L is a covalent linker; and G is any one of the moieties represented by the following structural formulas: or a pharmaceutically acceptable salt thereof, in: symbol represents the point of attachment to L; For each occurrence, R1 is independently -C(O)-A, wherein A is C1-C6 alkyl, C6-C 18 Aryl, (C6-C 18 )aryl(C1-C3)alkyl, 5-18 membered heteroaryl, (5-18 membered)heteroaryl(C1-C3)alkyl, C3-C8 cycloalkyl, (C3-C8)cycloalkyl(C1-C3)alkyl, 5-8 membered heterocycloalkyl, or (5-8 membered)heterocycloalkyl(C1-C3)alkyl; wherein one or two carbon atoms in the alkyl portion of A are optionally each independently replaced by a heteroatom selected from N, O or S; and wherein A is optionally replaced by 1 to 3 R 11 group substituted, each of the R 11 The groups are independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, halogen, C 6- C 12 Aryl, 5-12 membered heteroaryl, cyano, or two R 11 The groups, together with the atoms to which they are attached, form a 5-7 membered heterocyclyl having 1 to 3 heteroatoms selected from N, O or S; Each R 11 is independently optionally substituted by 1 to 3 substituents selected from halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, or cyano, The method includes: In the case of F1 and R F2 Under conditions where the parts react with each other, the compound represented by structural formula (I) G-R F1 (I) Reaction with a compound represented by structural formula (II) PR F2 (II), Where R F1 and R F2 Each is a reactive part, This creates the -L- portion of the covalent linker.

24. The method of claim 23, wherein R F1 and R F2 is a click reagent pair, and wherein the conditions are suitable for causing R F1 and R F2 A click reaction between the two, wherein the linker L comprises R F1 and R F2 The parts of the products of the click chemistry reaction between them.

25. A compound represented by the following structural formula: or a pharmaceutically acceptable salt thereof, in: For each occurrence, R is independently -R L -R F , and where: For each occurrence, R L Independently are: -O-, -S-, -NR 100 -、-O-(C1-C 12 )alkylene-, -S-(C1-C 12 )alkylene-, -NR 101 -(C1-C 12 )alkylene-, -NR 101a -O-(C1-C 12 )alkylene-; -O-(CH2CH2O) m -, -O-(CH2CH2O)k-(CH2CH2)-, -NR 102 -X 100 -(C1-C 12 )alkylene-, -NR 102a -NR 102b -C(O)-(C1-C 12 ) alkylene, wherein R 100 , R 101 , R 101a , R 102 , R 102a and R 102b are each independently H or C1-C3 alkyl, and X 100 is -O- or -NH-, and wherein m and k are each independently an integer from 1 to 12; For each occurrence, R F Independently are: H, C1-C3 alkyl, -NH2, -NH-Fmoc, -NH-Boc, -NH-CBz, -NH-Troc, -NH-TFA, mono(C1-C3)alkylamino, di(C1-C3)alkylamino; -C(O)-R 103 , where R 103 is -H, -OH, or (C1-C3)alkyl; -SH, with the following structural formula Represents part; or click chemistry reagents, The premise is – R L -R F Not –OH.

26. A particle comprising a compound represented by the following structural formula: GLP, in: P is a biocompatible polymer; L is a covalent linker; and G is represented by the following structural formula: or a pharmaceutically acceptable salt thereof, in: symbol Represents the point of attachment to L.

27. The particle of claim 26, wherein the polymer comprises the block copolymer PLGA-PEG.

28. A method of treating a condition in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of the compound of claim 25 or a pharmaceutically acceptable salt thereof, or a composition comprising the particles of any one of claims 26-27 or a pharmaceutically acceptable salt thereof, The disease is influenza.

29. Any one of the compounds represented by the structural formula listed in Table 4, or a pharmaceutically acceptable salt thereof, wherein the variable R is -O-(CH2)5-NH2, -O-(CH2)5-NH-Fmoc, -O-(CH2)5-N3 or -O-(CH2)5-biotin.

30. A compound represented by any one of the following structural formulae or a pharmaceutically acceptable salt thereof: in, For each occurrence, x is independently an integer from 90 to 140; y is independently an integer from 10 to 75; and m is independently an integer from 90 to 140.

31. The compound of claim 1, wherein for each occurrence, R is independently -O-(CH2) x -N3, wherein for each occurrence, x is independently an integer from 1 to 10.

32. The compound of claim 31, wherein x is 5.

33. A compound as described in any one of claims 1-9, wherein the compound is any one of compounds S-101 to S-131 or D-101 to D-131, wherein the moiety R is -O-(CH2)5-NH2 or -O-(CH2)5-N3.

34. The particle of claim 17, wherein the additional portion of the linker is -O-(C1-C 12 )alkylene-.

35. The particle of any of claims 11, 12 or 26, wherein the biocompatible polymer comprises at least one of polyglycolic acid, poly(lactic acid), poly(lactic-co-glycolic acid), polycaprolactone, poly(3-hydroxybutyric acid), polyethylene oxide, polyoxyethylene-polyoxypropylene block copolymers, poly(hydroxymethyl methacrylate), polyvinyl alcohol, poly(vinyl pyrrolidone), hyaluronic acid, heparin, heparin sulfate, polysialic acid, and chitosan.

36. The particle of any of claims 11, 12 or 26, wherein the biocompatible polymer comprises poly (L-lactic acid), poly (D-lactic acid), poly (D / L-lactic acid), copolymers thereof, or combinations thereof.

37. The particle of any one of claims 11, 12 or 26, wherein the biocompatible polymer comprises polyethylene oxide, a polyoxyethylene-polyoxypropylene block copolymer, a copolymer thereof, or a combination thereof.

38. The method of claim 23, wherein the biocompatible polymer comprises at least one biocompatible polymer selected from the group consisting of polyglycolic acid, poly(L-lactic acid), poly(lactic-co-glycolic acid), polycaprolactone, poly(3-hydroxybutyric acid), polyethylene oxide, polyoxyethylene-polyoxypropylene block copolymers, poly(hydroxymethyl methacrylate), polyvinyl alcohol, poly(vinyl pyrrolidone), hyaluronic acid, heparin, heparin sulfate, polysialic acid, and chitosan.

39. The particle of claim 14, wherein: x is an integer from 90 to 140; y is an integer from 10 to 75; and m is an integer from 90 to 140.

40. The particle of claim 26, wherein P is represented by the following structural formula: The symbol represents the point of attachment of the polymer to the linker L, and further wherein: y is an integer from 0 to 1000, x is an integer from 0 to 1000, and m is an integer from 0 to 450, with the proviso that x and y are not 0 at the same time.

41. The particle of claim 40, wherein: x is an integer from 90 to 140; y is an integer from 10 to 75; and m is an integer from 90 to 140.

42. The particle of any one of claims 11, 12 or 26, wherein the particle further comprises one or more of the following: Acid-terminated PLGA (PLGA-COOH); Acid-terminated PLGA-PEG copolymers, wherein the acid moiety terminates the PEG block (PLGA-PEG-COOH); PLGA-PEG copolymers, wherein the PEG block is terminated with a moiety represented by the following structural formula: or PLGA-PEG copolymers, wherein the PEG block is terminated by a moiety represented by the following structural formula: The dotted line indicates the point of attachment of the capping moiety to the PEG block.

43. The particle of claim 42, wherein the particle comprises at least one of: a blend of 75% by weight PLGA-COOH and 25% by weight PLGA-PEG-DBCO; a blend of 75% by weight PLGA-COOH and 25% by weight PLGA-PEG-NHS; a blend of 90% by weight PLGA-PEG-COOH and 10% by weight PLGA-PEG-DBCO; and PLGA-PEG-NHS.

44. A pharmaceutical composition comprising the compound of claim 25 or the particles of claim 26 or a pharmaceutically acceptable salt thereof in a pharmaceutically acceptable carrier.

45. The particle of any one of claims 11-18, 34-37, 39, 42, 43 and 45, wherein the amount of G per unit weight of the particle is from 1 μg / mg to 1000 μg / mg.

46. ​​A method of treating a condition responsive to modulation of Siglec receptor activity in a subject in need thereof, the method comprising administering to the subject a compound as described in any one of claims 1-10 or 31-33 or a particle as described in any one of claims 11-19, 34-37, 39, 42, 43 or 45.

47. The method of claim 46, wherein the activity of at least one of Siglec 3, 5, 7, 8, 9, 10, 11, 14, 15, or 16 is modulated.

48. The method of claim 46, wherein the disorder is selected from cancer, an immune-related disorder, and an inflammation-related disorder.

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