Glycosylated prodrugs

Abstract

Provided herein are compounds of Formula A-B wherein A is selected from a monosaccharide or disaccharide and B is a therapeutic moiety (e.g., selected from an anti-inflammatory moiety or a chemotherapeutic moiety); and pharmaceutically acceptable salts, stereoisomers, acids or solvates thereof. This compound can be a substrate for glucosylceramidase beta 2 (GBA2), and thus the compound is a prodrug that provides an active compound upon cleavage of the monosaccharide or disaccharide by GBA2. Also provided are pharmaceutical compositions comprising the compounds, and various medical and non-medical uses of the compounds and compositions.

Description

Technical Field

[0001] The present invention relates to compounds comprising a monosaccharide or a disaccharide and a therapeutic moiety (such as an anti-inflammatory moiety or a chemotherapeutic moiety), and pharmaceutically acceptable salts, stereoisomers, acids or solvates thereof. The compounds can be substrates for glucosylceramidase β2 (GBA2), and thus the compounds are prodrugs, which provide active compounds after cleavage of the monosaccharide or disaccharide by GBA2. Pharmaceutical compositions comprising the compounds are also provided, as well as various medical and non-medical uses of the compounds and compositions. Background Art

[0002] Corticosteroids (such as glucocorticoids) are a class of compounds commonly used in clinical practice because of their immunosuppressive effects, such as prednisolone and dexamethasone (Burns, 2016, The History of Cortisone Discovery and Development. Rheum Dis Clin North Am 42, 1-14, vii). They are used to treat various inflammatory and immune conditions (such as asthma, rheumatoid arthritis, inflammatory bowel disease, and psoriasis), hematological malignancies (such as leukemia), and to alleviate inflammatory complications of infectious diseases (such as tuberculosis and COVID-19) (Barnes, 2017, Glucocorticosteroids. Handb Exp Pharmacol 237, 93-115; Buttgereit, 2020, Views on glucocorticoid therapy in rheumatology: the age of convergence. Nat Rev Rheumatol 16, 239-246; Rhen and Cidlowski, 2005, Antiinflammatory action of glucocorticoids--new mechanisms for old drugs. N Engl J Med 353, 1711-1723).Although they are very effective in attenuating inflammation and other immune responses, their clinical use is limited by the severity of their side effects, which include increased risk of diabetes, endocrine system disorders, osteoporosis, delayed and interrupted wound healing, and inhibition of growth and fertility (Hoes et al., 2009, Adverse events of low-to medium-dose oral glucocorticoids in inflammatory diseases: a meta-analysis. Ann Rheum Dis 68, 1833-1838; McDonough et al., 2008, The epidemiology of glucocorticoid-associated adverse events. Curr Opin Rheumatol 20, 131-137; Schacke et al., 2002, Dissociation of transactivation from transrepression by a selective glucocorticoid receptor agonist leads to separation of therapeutic effects from side effects. Proc Natl Acad Sci US A 101, 227-232).

[0003] All actions of glucocorticoids are mediated by an intracellular receptor, the glucocorticoid receptor (GR), which is expressed in almost all cells of our body (Nicolaides et al., 2010). When activated by ligands, the cytoplasmic GR is activated and translocates to the nucleus, where it acts as a transcription factor, regulating the transcription of multiple target genes. GR regulates gene expression in multiple ways (reviewed in (Meijsing, 2015, Mechanisms of Glucocorticoid-Regulated Gene Transcription. Adv Exp Med Biol 872, 59-81; Miranda et al., 2013, Complex genomic interactions in the dynamic regulation of transcription by the glucocorticoid receptor. Mol Cell Endocrinol 380, 16-24; Ramamoorthy and Cidlowski, 2016, Corticosteroids: Mechanisms of Action in Health and Disease. Rheum Dis Clin North Am 42, 15-31, vii; Ratman et al., 2013, Beck, IM and De Bosscher, K. (2013). How glucocorticoid receptors modulate the activity of other transcription factors: a scope beyond tethering. Mol Cell Endocrinol 380, 41-54)). It can bind to specific sequences in the genome, so-called glucocorticoid response elements (GREs), as a dimer (although binding as a tetramer has also been described recently) and enhance or repress transcription of genes by recruiting specific transcriptional coregulators that modulate the local chromatin structure and the subsequent recruitment and activation of the transcriptional machinery. This process is generally referred to as transcriptional activation (transactivation), although binding to a specific class of GREs (so-called negative GREs) may lead to transcriptional repression. Alternatively, GR can regulate gene transcription without dimerization through interactions with other transcription factors. For example, it can physically interact with the p65 subunit of the NF-κB transcription factor complex, thereby inhibiting the activity of this transcription factor.Generally, this process is referred to as transcriptional repression, even though some interactions between GR and other transcription factors may result in enhanced transcriptional activity.

[0004] Initially, the transcriptional repression activity of GR was considered to be the main mechanism for the anti-inflammatory effect of glucocorticoids, while most of the side effects of glucocorticoids were considered to be caused by the transcriptional activation activity of GR (Schacke et al., 2002, Mechanisms involved in the side effects of glucocorticoids. Pharmacol Ther 96, 23-43; Schacke et al., 2004, Dissociation of transactivation from transrepression by a selective glucocorticoid receptor agonist leads to separation of therapeutic effects from side effects. Proc Natl Acad Sci USA 101, 227-232). Therefore, in order to improve the therapeutic rate of glucocorticoid drugs, a lot of efforts have been invested in developing new GR agonists that induce the transcriptional repression activity of GR rather than its transcriptional activation activity (Sundahl et al., 2015, Selective glucocorticoid receptor modulation: New directions with non-steroidal scaffolds. Pharmacol Ther 152, 28-41). However, although a variety of promising compounds have been developed (see, for example, Baiula et al., 2014, Mapracorat, a selective glucocorticoid receptor agonist, causes apoptosis of eosinophils infiltrating the conjunctiva in late-phase experimental ocular allergy. Drug Des Devel Ther 8, 745-757; Biggadike et al., 2007, Nonsteroidal glucocorticoid agonists: tetrahydronaphthalenes with alternative steroidal A-ring mimetics possessing dissociated (transrepression / transactivation) efficacy selectivity.J Med Chem 50, 6519-6534; Dewint et al., 2008, A plant-derived ligand favoring monomeric glucocorticoid receptorconformation with impaired transactivation potential attenuates collagen-induced arthritis. J Immunol 180, 2608-2615; Stock et al., 2017, Improved diseaseactivity with fosdagrocorat(PF-04171327), a partial agonist of the glucocorticoid receptor, in patients with rheumatoid arthritis: a Phase2randomized study. Int J Rheum Dis 20, 960-970; Zhang et al., 2020, Natural and synthetic compounds as dissociated agonists of glucocorticoid receptor. Pharmacol Res 156, 104802), but the success was limited, most likely because the immunosuppression of glucocorticoids may also depend on the transcriptional activation of anti-inflammatory genes and because the transcriptional repressive activity also produces some side effects (Clark, 2007, Anti-inflammatory functions of glucocorticoid-induced genes. Mol Cell Endocrinol 275, 79-97; Smoak and Cidlowski, 2004, Mechanisms of glucocorticoid receptor signaling during inflammation. Mech Ageing Dev 125, 697-706; Vandewalle et al., 2018, Therapeutic Mechanisms of Glucocorticoids.Trends Endocrinol Metab29,42-54). Therefore, it would be interesting to try to develop new methods for glucocorticoid drugs with reduced side effects, such as modifying the structure of existing GC. For example, anti-CD 163-dexamethasone conjugates designed to target activated macrophages showed improved therapeutic rates in rats (Thomsen et al., 2016, Anti-CD163-dexamethasone conjugate inhibits the acute phase response to lipopolysaccharide in rats. World J Hepatol 8,726-730), and conjugation of hydrolyzable polyethylene glycol (PEG) to prednisolone increased the retention time in rat lungs without causing any systemic side effects (Bayard et al., 2013, Polyethylene glycol-drug ester conjugates for prolonged retention of small inhaled drugs in the lung. J Control Release 171,234-240). In addition, the addition of γ-lactones and cyclic carbonates can make glucocorticoids more susceptible to inactivation by specific enzymes once they enter the bloodstream (Biggadike et al., 2000, Selective plasma hydrolysis of glucocorticoid gamma-lactones and cyclic carbonates by the enzyme paraoxonase: an ideal plasma inactivation mechanism. J Med Chem 43, 19-21).

[0005] Therefore, there is a need for improved corticosteroid-type compounds. In particular, it would be beneficial to provide useful prodrugs which have limited activity (eg as anti-inflammatory or anti-cancer agents) prior to conversion to an active form. Summary of the invention

[0006] The present invention provides a compound comprising a monosaccharide or disaccharide and an anti-inflammatory part or a chemotherapeutic part, and a pharmaceutically acceptable salt, stereoisomer, acid or solvate thereof. The compound can be a substrate of glucosylceramidase β2 (GBA2), and thus the compound is a prodrug, which provides an active compound after cleavage of the monosaccharide or disaccharide by GBA2.

[0007] We have noted that such glycosylated compounds may be relatively inactive until their sugar groups are removed by, for example, GBA2. The enzyme GBA2 is preferentially expressed in inflamed tissues (many cancer tissues also exhibit inflammation). This means that anti-inflammatory and chemotherapeutic compounds can be converted into prodrugs by the addition of a mono- or disaccharide moiety that can be cleaved by GBA2 at or near the site of inflammation, providing an active anti-inflammatory or chemotherapeutic compound in the inflamed tissue. This provides a new way to target therapeutic activity near the target tissue, thereby reducing off-target effects.

[0008] In a first aspect, the present invention provides a compound of formula I or a pharmaceutically acceptable salt, stereoisomer, acid or solvate thereof:

[0009] AB(I)

[0010] A is selected from monosaccharides and disaccharides. B is a therapeutic moiety and may suitably be selected from an anti-inflammatory moiety or a chemotherapeutic moiety.

[0011] The second aspect of the present invention provides a pharmaceutical composition comprising a compound of the first aspect. The composition can be formulated for non-oral administration. For example, the composition can be formulated for parenteral administration, such as the composition can be formulated for injection.

[0012] The third aspect provides a compound of the first aspect or a composition of the second aspect for use as a medicament. The medicament can be used to treat conditions associated with inflammation. The medicament can be used to treat conditions associated with inflammation by local conversion of the prodrug at the site of inflammation.

[0013] A fourth aspect provides a compound of the first aspect or a composition of the second aspect for use in treating inflammation.

[0014] A fifth aspect provides a compound of the first aspect or a composition of the second aspect for use in treating an inflammatory condition or other condition characterized by an overactive immune system.

[0015] A sixth aspect provides a compound of the first aspect or a composition of the second aspect for use in treating a condition characterised by hyperproliferation of cells belonging to the immune system.

[0016] The seventh aspect provides the compound of the first aspect or the composition of the second aspect for use in treating cancer.

[0017] The eighth aspect provides the use of the compound of the first aspect as an anti-inflammatory agent, comprising contacting the compound with cells expressing glucosylceramidase β2 (GBA2). The contact can be performed in vitro or in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Embodiments of the present invention will be further described below with reference to the accompanying drawings, in which:

[0019] Figure 1 The anti-inflammatory effects of ginsenosides via activation of GR are shown. A. Schematic representation of the experimental method used in the assay of the present invention. Zebrafish larvae were traumatized in the tail at 74 hpf. Chemical compound treatment was initiated 2 h before trauma and continued for 4 h after trauma. At this time point, the number of neutrophils that migrated to the injured area (indicated by a red box) was determined. B. Structures of the compounds used for treatment: beclomethasone (Bec), protopanaxadiol (PPD), F2, and Rb1. Glucose (Glc) groups conjugated to the steroid backbone are indicated in red. C. In wild type (gr + / + ) and Gr-deficient (gr - / - ) individuals after compound treatment. D. Relative mRNA levels of il1b, il6, and mmp9 determined by qPCR before and after trauma and compound treatment. EH. Relative mRNA levels of il1b, il6, and mmp9 determined by qPCR in the absence and presence of the nonspecific chemical GBA inhibitor miglustat (E), the absence and presence of the GBA1 inhibitor ME656 (F), the GBA2 inhibitor MZ31 (G), and the GBA2-deficient (gba2 - / - ) in individuals (H) of the Veh group. Data shown are the mean ± SEM of data pooled from three separate experiments (in C and EH), or the mean ± SEM of three separate experiments (in D). Statistical significance was determined by one-way or two-way ANOVA and Tukey post hoc test. Significant differences from the corresponding Veh group (grey bars, C, E, F, G, H) are indicated by *** (P < 0.001), and significant differences from the corresponding gr + / + (C) or the same compound / Veh (E, G) groups (same color bars, non-hatched) are indicated by ### (P<0.001), and the difference from the non-trauma / Veh group (D) is indicated by +++ (P<0.001).

[0020] Figure 2 The number of migrated neutrophils and macrophages in wild-type zebrafish larvae treated with Bec, PPD, F2, or Rb1 is shown. The data shown are the mean ± SEM of data pooled from three separate experiments. Statistical significance was determined by one-way or two-way ANOVA and Tukey post hoc test. Significant differences from the Veh group are indicated by *** (P < 0.001).

[0021] Figure 3A comparison of the side effects observed with beclomethasone relative to various ginsenosides is shown. A. Systemic glucose levels determined by ELISA in zebrafish larvae at 5 days post fertilization (dpf) treated with beclomethasone or ginsenosides PPD, F2, or Rb1 from 2hpf. B. Systemic glucose levels in 5dpf larvae with or without compound treatment with MZ31 after injury at 2dpf. C. Systemic cortisol levels determined by ELISA in zebrafish larvae at 5 days post fertilization (dpf) treated with beclomethasone or ginsenosides PPD, F2, or Rb1 from 2hpf. D. Systemic cortisol levels in 5dpf larvae with or without compound treatment with MZ31 after injury at 2dpf. E. Body length of zebrafish larvae at 5dpf after compound treatment from 2hpf. F. Length of regenerated caudal fin of 5dpf larvae injured at 2dpf after compound treatment with or without MZ31. G. Relative GFP fluorescence levels of 3dpf larvae of the Tg(9x GCRE-HSV.UI23:EGFP) line 24h after compound treatment, which is a reporter line for Gr transcriptional activation activity. H. Representative bright field microscopy images of caudal fins of 5dpf larvae from experimental groups shown in F. I. Representative fluorescence microscopy images of 3dpf larvae from experimental groups shown in G. Data shown are mean ± SEM of three separate experiments. Statistical significance was determined by one-way or two-way ANOVA and Tukey post hoc test. Significant differences from the corresponding Veh groups (AF, H, gray columns) are indicated by * (P < 0.05), ** (P < 0.01), *** (P < 0.001), differences from the same compound / Veh groups (E, G, same color columns, non-hatched lines) are indicated by # (P < 0.05), ## (P < 0.01), ### (P < 0.001), and differences from the corresponding F2 and Rb1-treated groups (C, D, H) are indicated by +++ (P < 0.001).

[0022] Figure 4 Further effects of beclomethasone treatment on regenerated tail fin length and mRNA levels in zebrafish larvae relative to various ginsenosides are shown. A. Length of regenerated tail fin of 5dpf larvae injured at 2dpf after treatment with different concentrations of Bec. B. Relative mRNA levels of fkbp5, pck1 and nfkbiaa determined by qPCR before and after trauma and after compound treatment. Data shown are mean ± SEM of three separate experiments. Statistical significance was determined by one-way or two-way ANOVA and Tukey post hoc test. Significant differences from the corresponding Veh group are indicated by *** (P<0.001), and differences from PPD, F2 and Rb1 treated groups are indicated by +++ (P<0.001).

[0023] Figure 5 The effects of trauma on the mRNA levels, protein levels and enzyme activities of Gba1 and Gba2 in the zebrafish larvae tested are shown. A. Relative mRNA levels of gba1 and gba2 determined by qPCR in control larvae and in 5dpf larvae 4h after trauma. B. Relative protein levels of Gba1 and Gba2 determined by band intensity in activity-based probe assays (as shown in Figure S3A) in control larvae and in 5dpf larvae 4h after trauma. C. Relative protein levels of Gba1 and Gba2 in the front and rear of the body of control and injured larvae. D. Enzyme activity of Gba2 measured in lysates of control and injured larvae of 5dpf (expressed as per mg protein). The data shown are the mean ± SEM of three separate experiments. Statistical significance was determined by one-way or two-way ANOVA and Tukey post hoc test. Significant differences from the corresponding control group are indicated by **(P<0.01), ***(P<0.001), and differences from the corresponding anterior group are indicated by ###(P<0.01).

[0024] Figure 6 Further evidence for the effects of trauma on enzyme activity, protein levels, and mRNA levels is shown, along with a gel generated from an activity-based probe assay. A. Representative image of a gel generated from an activity-based probe assay, showing Figure 3 Experimental groups shown in B. B. Representative images of gels generated from activity-based probe assays showing Figure 3 Experimental groups shown in C. C. Enzyme activity of Gba2 measured in lysates of control and injured larvae at 5 dpf (expressed per zebrafish larvae). D. Relative mRNA levels of gba1 and gba2 in HeLa cells determined by qPCR after 6 h of control and TNF-α treatment. E. Representative images of gels generated by activity-based probe assays showing the experimental groups shown in Figure S3F. F. Relative protein levels of Gba1 and Gba2 in HeLa cells determined by activity-based probe assays after 6 h of control and TNF-α treatment. Data shown are mean ± SEM of three separate experiments. Statistical significance was determined by one-way or two-way ANOVA and Tukey post hoc test. Significant differences from the corresponding control groups are indicated by **(P<0.01), ***(P<0.001).

[0025] Figure 7The anti-inflammatory activity of glucoronidated dexamethasone (GDex) and the side effects associated with it are shown in comparison with other compounds. A. The number of migrating neutrophils and macrophages in wild-type zebrafish larvae treated with Bec, dexamethasone (Dex), and glucuronidated dexamethasone (GDex) 4 h after trauma. B. The number of migrating neutrophils and macrophages in Gr-deficient (gr - / - ) individuals after compound treatment. C. Relative mRNA levels of il1b, il6il6, mmp9 and mmp13 determined by qPCR before and after wounding and after compound treatment. D. Systemic glucose levels determined by ELISA in zebrafish larvae at 5 days post fertilization (dpf) treated with Bec, Dex or G-Dex from 2hpf. E. Systemic cortisol levels determined by ELISA in zebrafish larvae at 5 days post fertilization (dpf) treated with Bec, Dex or G-Dex from 2hpf. F. Length of regenerated caudal fin of 5dpf larvae wounded at 2dpf after compound treatment. G. Relative GFP fluorescence levels of 3dpf larvae of the Tg(9xGCRE-HSV.UI23:EGFP) strain 24h after compound treatment. H. Relative mRNA levels of fkbp5, pck1, and nfkbiaa determined by qPCR before and after trauma and after compound treatment. The data shown are the mean ± SEM of the data pooled from three separate experiments (in AB), or the mean ± SEM of three separate experiments (in CH). Statistical significance was determined by one-way or two-way ANOVA and Tukey post hoc test. Significant differences from the corresponding Veh group are indicated by * (P < 0.05), ** (P < 0.01), *** (P < 0.001), and differences from Bec (if present) and Dex-treated groups are indicated by +++ (P < 0.001).

[0026] Figure 8The anti-inflammatory activity of glucosidated prednisolone (GPdn) and the side effects associated with it are shown compared to other compounds. A. Number of migrated neutrophils after treatment with Bec, Dex, GDex, prednisolone (Pdn), and glucosidated prednisolone (GPdn) in the absence and presence of the Gba2 inhibitor MZ31. B. Systemic glucose levels determined by ELISA in zebrafish larvae at 5 days post fertilization (dpf) treated with Bec, Dex, G-Dex, Pdn, or G-Pdn from 2 hpf. C. Systemic glucose levels in 5 dpf larvae with or without compound treatment with MZ31 after injury at 2 dpf. D. Systemic cortisol levels determined by ELISA in zebrafish larvae at 5 days post fertilization (dpf) treated with Bec, Dex, G-Dex, Pdn or G-Pdn from 2hpf. E. Systemic cortisol levels in 5dpf larvae with or without compound treatment with MZ31 after injury at 2dpf. Data shown are the mean ± SEM of data pooled from three separate experiments (in A) or the mean of three separate experiments (performed in triplicate) in BE. Statistical significance was determined by one-way or two-way ANOVA and Tukey post hoc test. Significant differences from the corresponding Veh groups (grey columns) are indicated by * (P<0.05) and *** (P<0.001), differences from the corresponding groups treated with non-glycoidlated forms of the compounds (Dex or Pdn) are indicated by +++ (P<0.001), and differences from the Veh groups of the MZ31-treated groups (same color columns, non-hatched columns) are indicated by # (P<0.05) and ### (P<0.001).

[0027] Fig. 9Anti-inflammatory activity and side effects associated with gentibiosidated prednisolone (GbPdn) compared to glucoside prednisolone and prednisolone are shown. A. Structures of compounds used for treatment: prednisolone (Pdn), glucoside prednisolone (GPdn), and gentiobiosidated prednisolone (GdPdn). Glucose (Glc) and gentiobiose (Gb) groups conjugated to the steroid backbone of Pdn are indicated in red. B. Number of migrated neutrophils after treatment with Pdn, GPdn, and gentiobiosidated Pdn (GbPdn) in the absence and presence of the Gba2 inhibitor MZ31. C. Relative mRNA levels of il1b, il6, and il8 determined by qPCR before and after trauma and after compound treatment. D. Systemic glucose levels determined by ELISA in control and injured zebrafish larvae treated with Pdn, GPdn, or GbPdn from 2hpf to 5dpf. E. Systemic glucose levels determined by ELISA in zebrafish larvae treated with Pdn, GPdn, or GbPdn from 2hpf to 5dpf after control treatment or gba2 overexpression using CMV:gba2 plasmid. F. Systemic cortisol levels determined by ELISA in control and injured zebrafish larvae treated with Pdn, GPdn, or GbPdn from 2hpf to 5dpf. G. Systemic cortisol levels determined by ELISA in zebrafish larvae treated with Pdn, GPdn, or GbPdn from 2hpf to 5dpf after control treatment or gba2 overexpression using CMV:gba2 plasmid. Data shown are mean ± SEM of data pooled from three separate experiments (in B) or mean ± SEM of three separate experiments (in CG). Statistical significance was determined by one-way or two-way ANOVA and Tukey post hoc test. Significant differences from the corresponding Veh group (grey columns) are indicated by * (P < 0.05), ** (P < 0.01), *** (P < 0.001), differences from the corresponding Pdn-treated group (pink columns) are indicated by +++ (P < 0.001), differences from the Veh group of the MZ31-treated group (B) and differences from the wounded control group (D, F) or CMV:gba2 (E, G) group (same color, non-hatched columns) are indicated by # (P < 0.05), ## (P < 0.01), ### (P < 0.001).

[0028] Fig.10Further effects of treatment of zebrafish larvae with gentiobiosylated prednisolone (GbPdn), glucosylated prednisolone (GPdn) and prednisolone (Pdn) are shown. A. Length of regenerated caudal fin of 5dpf larvae injured at 2dpf after treatment with different concentrations of Pdn. B. Relative mRNA levels of gba2 determined by qPCR in control larvae and after overexpression of gba2 using CMV:gba2 plasmid. C. Body length of zebrafish larvae at 5dpf after compound treatment from 2hpf. D. Length of regenerated caudal fin of 5dpf larvae injured at 2dpf after compound treatment with or without MZ31. E. Relative GFP fluorescence levels of 3dpf larvae of the Tg(9xGCRE-HSV.UI23:EGFP) strain 24h after compound treatment. F. Relative GFP fluorescence levels of 3dpf juveniles of the Tg(9x GCRE-HSV.UI23:EGFP) strain after overexpression of gba2 using CMV:gba2 plasmid 24h after compound treatment. G. Representative fluorescence microscopy images of 3dpf juveniles of selected experimental groups shown in EF. H. Relative mRNA levels of fkbp5, pck1 and nfkbiaa determined by qPCR before and after wounding and after compound treatment. I. Relative mRNA levels of fkbp5, pck1 and nfkbiaa with and without gba2 overexpression (using CMV:gba2 plasmid) and compound treatment. Data shown are mean ± SEM of three separate experiments. Statistical significance was determined by one-way or two-way ANOVA and Tukey post hoc test. Significant differences from the corresponding Veh group (grey column) are indicated by *** (P < 0.001), differences from the Pdn-treated group (pink column) are indicated by +++ (P < 0.001), and differences from the corresponding MZ31-treated group (D, same color, non-hatched column) are indicated by ### (P < 0.001).

[0029] Fig.11Comparative effects of in vitro treatment of human cells with gentiobiosylated prednisolone (GbPdn), glucosylated prednisolone (GPdn), and prednisolone (Pdn) are shown. A. Relative binding affinity was determined in vitro using the PolarScreen glucocorticoid receptor (GR) competitor assay. Fluorescence polarization levels are plotted, reflecting competition for binding of the fluorescent ligand to the receptor by increasing concentrations of compounds Dex, Pdn, G-Pdn, and Gb-Pdn. The IC50 level for each compound is shown. B. Levels of nuclear translocation of the GR in HeLa cells determined using immunocytochemistry and confocal microscopy following treatment with Pdn, GPdn, and GbPdn in the absence and presence of TNF-α and / or MZ31. C-F. Relative mRNA levels of IL1B (C), IL8 (D), FKBP5 (E), and NFKBIA (F) in HeLa cells determined by qPCR after 6 h of treatment with Pdn, G-Pdn, or Gb-Pdn in the absence and presence of TNF-α and / or MZ31. G. Relative mRNA levels of FKBP5 in HeLa cells after 6 h of treatment with Pdn, G-Pdn, or Gb-Pdn in the absence and presence of TNF-α, and after lentiviral-mediated shRNA knockdown of GBA1 or GBA2. H. Relative mRNA levels of FKBP5 and NFKBIA in HeLa cells after 6 h of treatment with Pdn, G-Pdn, or Gb-Pdn after overexpression of GBA2 by transfection of CMV:GBA2 plasmid. Data shown are mean ± SEM of three separate experiments. Statistical significance was determined by one-way or two-way ANOVA with Tukey post hoc test. Significant differences from the corresponding Veh groups (grey columns) are indicated by *(P<0.05), **(P<0.01), ***(P<0.001), differences from the Pdn-treated group (pink columns) are indicated by +(P<0.05), ++(P<0.01), +++(P<0.001), and differences from the MZ31-treated Veh group (B, same color, non-hatched columns) are indicated by ##(P<0.01).

[0030] Fig.12 Further effects of in vitro treatment of human cells with gentiobiosylated prednisolone (GbPdn), glucosylated prednisolone (GPdn) and prednisolone (Pdn) are shown. Fig.11Representative confocal microscopy images of the experimental groups shown in B show nuclear translocation of GR (green fluorescence) in HeLa cells. Nuclear DAPI staining is shown in blue. BD. Relative mRNA levels of MMP13 (B), GILZ (C), and SGK1 (D) in HeLa cells determined by qPCR after 6 h of treatment with Pdn, GPdn, or GbPdn in the absence and presence of TNF-α and / or MZ31. E. Relative mRNA levels of NFKBIA in HeLa cells after 6 h of treatment with Pdn, GPdn, or GbPdn in the absence and presence of TNF-α, and after lentiviral-mediated shRNA knockdown of GBA1 or GBA2. The data shown are the mean ± SEM of three separate experiments. Statistical significance was determined by one-way or two-way ANOVA and Tukey post hoc test. Significant differences from the corresponding Veh groups (grey columns) are indicated by ** (P < 0.01), *** (P < 0.001), and differences from the Pdn-treated groups (pink columns) are indicated by ++ (P < 0.01), +++ (P < 0.01).

[0031] Fig.13 A schematic diagram of the steps followed in the collagen antibody-induced arthritis (CAIA) mouse model experiment is provided. Mice received CAIA-cocktail on day 1 and then LPS treatment on day 4. Prednisolone (Pdn) or gentiobiosylated prednisolone (GbPdn) were treated daily from day 7 to day 15.

[0032] Fig.14 The therapeutic effects and side effects of prednisolone (Pdn) and gentiobiosylated prednisolone (GbPdn) obtained in the CAIA model are illustrated. A. Arthritis scores of mice measured daily from day 5 to day 15. B. Serum IL-6 levels determined by ELISA on days 10 and 15. C. Serum insulin levels determined by ELISA on day 14. D. Serum corticosterone levels determined by enzyme immunoassay on day 15. E. Liver tissue mRNA levels of Fkbp5, Pck1, and Gilz determined by qPCR on day 15. E. Muscle tissue mRNA levels of Redde1, Foxo3, and Klf15 determined on day 15. G. Gilz and Fkbp5 mRNA levels in hind paw ankle joint tissue determined on day 15. Statistically significant differences from the Veh group are indicated by *** (P<0.001).

[0033] Fig.15Gba2 expressed near sites of inflammation was shown to remove the entire gentiobiose group from gentiobiosylated prednisolone (GbPdn). Concentrations of GbPdn, glucosylated prednisolone (GPdn), and prednisolone (Pdn) were determined by LC-MS analysis in extracts of zebrafish larvae following tail wounding and treatment with 25 μM GbPdn (2 h before and 4 h after wounding). A. Concentrations determined in extracts of control (uninjured) and injured wild-type larvae treated with vehicle or the Gba2 inhibitor MZ31. B. Concentrations determined in extracts of control (uninjured) and injured gba2 - / - Statistically significant differences from the control / Veh group are indicated by *** (P < 0.001). DETAILED DESCRIPTION

[0034] Throughout the description and claims of this specification, the words "comprise" and "comprising" and their variations mean "including but not limited to", and they are not intended to (and do not) exclude other parts, additives, components, integers or steps. Throughout the description and claims of this specification, unless the context requires otherwise, the singular includes the plural. In particular, where the indefinite article is used, the specification should be understood as considering plural as well as singular, unless the context requires otherwise.

[0035] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All features disclosed in this specification (including any accompanying claims, abstract and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any foregoing embodiments. The invention extends to any novel one or any novel combination of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one or any novel combination of the steps of any method or process so disclosed.

[0036] The reader should pay attention to all papers and documents related to this application that are filed concurrently with or prior to this specification, which are open to public inspection together with this specification, and the contents of all these papers and documents are incorporated herein by reference.

[0037] definition

[0038] The following explanations of terms and methods are provided to better describe the present disclosure and guide those of ordinary skill in the art to practice the present disclosure.

[0039] The present invention relates in particular to the treatment of diseases. The term "treatment" and the therapies included in the present invention include the following and combinations thereof: (1) hindering, for example, delaying the onset and / or progression of an event, state, disorder or condition, or at least one clinical or subclinical symptom thereof, for example, preventing, reducing or delaying the development of an event, state, disorder or condition, or its recurrence (in the case of maintenance treatment or secondary prevention); (2) preventing or delaying the appearance of clinical symptoms of an event, state, disorder or condition developed in an animal (e.g., a human), the animal may suffer from or be susceptible to the state, disorder or condition, but has not yet experienced or shown clinical or subclinical symptoms of the state, disorder or condition; and / or (3) alleviating and / or curing an event, state, disorder or condition (e.g., causing regression of at least one of an event, state, disorder or condition or its clinical or subclinical symptoms, curing the patient or causing the patient to enter remission). The benefit to the patient to be treated may be statistically significant or at least perceptible to the patient or physician. It should be understood that a drug may not necessarily produce a clinical effect in every patient to whom it is administered; therefore, in any individual patient or even in a particular patient population, treatment may fail or be only partially successful, and the meaning of the terms "treatment" and "prevention" and cognate terms should be understood accordingly. The compositions and methods described herein can be used to treat and / or prevent the above-mentioned conditions.

[0040] The term "prevention" includes reference to therapeutic treatments for the purpose of maintaining health or inhibiting or delaying the onset and / or progression of an event, state, disorder or condition, e.g., to reduce the chance of an event, state, disorder or condition occurring. The result of prevention can be, for example, maintaining health or delaying the onset and / or progression of an event, state, disorder or condition. It should be remembered that treatment may fail in any individual patient or even in a particular patient population, and this paragraph should be understood accordingly.

[0041] As used herein, the term "alkyl" includes reference to straight or branched chain alkyl moieties having up to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms. The term includes reference to, for example, methyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, sec-butyl, or tert-butyl), pentyl, hexyl, and the like. In particular, the alkyl group may be a "C1-C8 alkyl group," i.e., an alkyl group having 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms; a "C1-C6 alkyl group," i.e., an alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms; a "C1-C4 alkyl group," i.e., an alkyl group having 1, 2, 3, or 4 carbon atoms; or a "C1-C3 alkyl group," i.e., an alkyl group having 1, 2, or 3 carbon atoms. The term "lower alkyl" includes reference to alkyl groups having 1, 2, 3 or 4 carbon atoms.

[0042] As used herein, the term "alkenyl" includes reference to straight or branched alkenyl moieties having up to 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) carbon atoms. The term includes reference to, for example, ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like. In particular, the alkenyl group may be a "C3-C8 alkenyl," i.e., an alkenyl group having 3, 4, 5, 6, 7, or 8 carbon atoms; a "C3-C6 alkyl," i.e., an alkenyl group having 3, 4, 5, or 6 carbon atoms; a "C3-C4 alkyl," i.e., an alkenyl group having 3 or 4 carbon atoms. The term "lower alkenyl" includes reference to alkyl groups having 2, 3, or 4 carbon atoms. The alkenyl group can be monounsaturated (i.e., contain a single carbon-carbon double bond) or polyunsaturated (i.e., contain two or more carbon-carbon double bonds, such as 2, 3, or 4 carbon-carbon double bonds). For example, the alkenyl group can be alkadienyl, alkatrienyl, etc.

[0043] As used herein, the term "cycloalkyl" includes reference to alicyclic moieties having 3, 4, 5, or 6 carbon atoms. The group can be a bridged ring system or a polycyclic system. The more common cycloalkyl groups are monocyclic. The term includes reference to groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0044] The term "heteroalkyl" by itself or in combination with another term, unless otherwise stated, refers to a stable straight or branched chain or cyclic hydrocarbon radical, or combinations thereof, consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si and S, wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom O, N, P, S and Si may be placed at any interior position of the heteroalkyl radical or at the position at which the alkyl radical is attached to the rest of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, –CH=CH-N(CH3)-CH3, O-CH3, -O-CH2-CH3, and –CN. Up to two heteroatoms may be consecutive, for example, -CH2-NH-OCH3 and –CH2-O-Si(CH3)3. Similarly, the term "heteroalkylene" by itself or as part of another substituent refers to a divalent radical derived from a heteroalkyl group, for example, but not limited to, -CH2-CH2-S-CH2-CH2- and –CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms may also occupy either or both chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). In addition, for alkylene and heteroalkylene linking groups, the direction of the linking group is not implied by the direction in which the molecular formula of the linking group is written. For example, the formula -C(O)2R'- represents -C(O)2R'- and -R'C(O)2-. As described above, heteroalkyl groups as used herein include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)R', -C(O)NR', ​​-NR'R", -OR', -SR', and / or -SO2R'. When "heteroalkyl" is recited, followed by a specific heteroalkyl group, such as -NR'R", etc., it should be understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive. Instead, the specific heteroalkyl groups are recited for clarity. Therefore, the term "heteroalkyl" should not be interpreted herein to exclude specific heteroalkyl groups, such as -NR'R", etc.

[0045] The term "heterocycloalkyl" as used herein includes reference to a saturated heterocyclic moiety having 3, 4, 5, 6 or 7 ring carbon atoms and 1, 2, 3, 4 or 5 ring heteroatoms selected from nitrogen, oxygen, phosphorus and sulfur. For example, heterocycloalkyl can include 3, 4 or 5 ring carbon atoms and 1 or 2 ring heteroatoms selected from nitrogen and oxygen. The group can be a polycyclic system, but more commonly monocyclic. The term includes reference to groups such as azetidinyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, oxiranyl, pyrazolidinyl, imidazolyl, indolizidinyl, piperazinyl, thiazolidinyl, morpholinyl, thiomorpholinyl, quinolizidinyl etc.

[0046] As used herein, the term "halogen" includes reference to F, Cl, Br or I, such as F, Cl or Br. In a particular class of embodiments, halogen is F or Cl, with F being more common.

[0047] Unless otherwise indicated, the term "halogen" by itself or as part of another substituent refers to a fluorine, chlorine, bromine or iodine atom; for example, the halogen can be fluorine, chlorine or bromine. In addition, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by a corresponding number of halogens. For example, the term "halo (C1-C4) alkyl" refers to, but is not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0048] As used herein, the term "alkoxy" includes reference to -O-alkyl, wherein the alkyl is straight or branched, and contains 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In one class of embodiments, the alkoxy has 1, 2, 3, or 4 carbon atoms, such as 1, 2, or 3 carbon atoms. The term includes reference to, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like. The term "lower alkoxy" includes reference to alkoxy having 1, 2, 3, or 4 carbon atoms.

[0049] The term "haloalkoxy" as used herein refers to an alkoxy group in which one or more hydrogen atoms are replaced by a corresponding number of halogens.

[0050] Unless otherwise indicated, each of the above terms (e.g., "alkyl," "cycloalkyl," "heteroalkyl," and "alkoxy") is meant to include substituted and unsubstituted forms of the groups shown. When a substituent is R-substituted (e.g., Rx-substituted alkyl, where "x" is an integer), the substituent may be substituted with one or more R groups as permitted by the rules of valence, where each R group is optionally different (e.g., Rx-substituted alkyl may include multiple Rx groups, where each Rx group is optionally different). Some examples of substituents for each type of group are provided below.

[0051] The term "substituted" used herein when referring to a moiety means that one or more, especially 1 to 5, more especially 1, 2 or 3 hydrogen atoms in the moiety are replaced independently of each other by the corresponding number of described substituents. Unless otherwise indicated, exemplary substituents include -OH, -CN, -NH2, -NH (C1-C6 alkyl), -N (C1-C4 alkyl) 2, =O, -halogen, -C1-C6 alkyl, -C2-C6 alkenyl, -C1-C6 haloalkyl, -C1-C6 haloalkoxy and -C2-C6 haloalkenyl, -C1-C6 alkylcarboxylic acid (e.g. -CH3COOH or -COOH). When the substituent is -C1-C6 alkyl or -C1-C6 haloalkyl, the C1-C6 chain is optionally interrupted by an ether bond (-O-) or an ester bond (-C(O)O-). Exemplary substituents for substituted alkyl groups may include -OH, -CN, -NH2, =O, -halogen, -CO2H, -C1-C6 haloalkyl, -C1-C6 haloalkoxy and -C2-C6 haloalkenyl, -C1-C6 alkylcarboxylic acid (e.g., -CH3COOH or -COOH). For example, exemplary substituents for alkyl groups may include -OH, -CN, -NH2, =O, -halogen.

[0052] Of course, it is understood that substituents are only located where they are chemically possible, and one skilled in the art will have no difficulty determining (experimentally or theoretically) whether a particular substitution is possible. For example, an amino or hydroxyl group with a free hydrogen may be unstable if attached to a carbon atom by an unsaturated (e.g., olefinic) bond. Furthermore, it is of course understood that the substituents described herein may themselves be substituted with any substituent, provided that the above-mentioned limitations on appropriate substitutions recognized by one skilled in the art are met.

[0053] When steric considerations dictate the position of substituents on a group, the isomer with the lowest conformational energy may be preferred.

[0054] When a compound, part, process or product is described as "optionally" having a feature, the disclosure includes such compounds, parts, processes or products having that feature as well as such compounds, parts, processes or products not having that feature. Thus, when a moiety is described as "optionally substituted," the disclosure includes both unsubstituted and substituted moieties.

[0055] When two or more moieties are described as being "independently" or "each independently" selected from a series of atoms or groups, this means that the moieties may be the same or different. The identity of each moiety is therefore independent of the identity of one or more other moieties.

[0056] The term "molecular radical" by itself or as part of another substituent refers to a monovalent group derived from a specified molecular species. For example, a molecular radical of a given molecular species may correspond to the group remaining after abstracting a hydrogen atom or a hydroxyl group from the given molecular species.

[0057] The term "pharmaceutically acceptable" as used herein includes reference to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human or animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications within the scope of reasonable medical judgment, commensurate with a reasonable benefit / risk ratio. The term includes acceptability for both human and veterinary purposes.

[0058] The term "pharmaceutically acceptable salt" refers to salts of active compounds that are prepared with relatively nontoxic acids or bases, depending on the specific substituents found on the compounds described herein. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, which can be either pure or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, which can be either pure or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid, and salts derived from relatively nontoxic organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are amino acid salts such as arginine salts, and organic acids such as glucuronic acid or galacturonic acid salts, and the like (see, e.g., Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain basic and acidic functional groups, which allow these compounds to be converted into base or acid addition salts.

[0059] The term "acid" in connection with sugar-containing compounds disclosed herein is intended to include forms of compounds in which the sugar (and / or at least one monosaccharide residue thereof) contains a carboxylic acid. Such a carboxylic acid may form part of an aldonic acid or uronic acid. When the terminal aldehyde group in an aldose is oxidized, an aldonic acid is obtained, for example, oxidation of D-glucose at C1 produces D-gluconic acid. When the hydroxyl group furthest from the carbonyl group of the sugar is oxidized to a carboxylic acid, a uronic acid is obtained, for example, oxidation of D-glucose produces the corresponding uronic acid D-glucuronic acid (D-glucoronate). Exemplary sugar acids include gluconic acid, glucuronic acid, galactonic acid, galacturonic acid, idonic acid, iduronic acid, altronic acid, and altruronic acid; and D- and L-enantiomers thereof (e.g., D-gluconic acid, D-glucuronic acid, D-galactonic acid, D-galacturonic acid, L-idonic acid and L-iduronic acid, L-altronic acid and L-altruronic acid).

[0060] The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.

[0061] Some compounds of the present invention can exist in non-solvated form and solvated form, including hydrated form. Usually, solvated form is equivalent to non-solvated form, and is included in the scope of the present invention. Some compounds of the present invention can exist in multiple crystallization or amorphous form. In general, all physical forms are equal for the intended use of the present invention, and are within the scope of the present invention.

[0062] Certain compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, tautomers, geometric isomers and individual isomers are all included within the scope of the present invention. The compounds of the present invention do not include those compounds known in the art that are too unstable to be synthesized and / or separated.

[0063] As used herein, the term "prodrug" represents a compound that is converted into a parent compound or other active compound in vivo, for example, by hydrolysis in the blood. Examples of such prodrugs are pharmaceutically acceptable carboxylates. A detailed discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14, ACS, Symposium Series, Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987; H. Bundgaard, ed., Design of Prodrugs, Elsevier, 1985; and Judkins et al., Synthetic Communications, 26 (23), 4351-4367 (1996); and Richard B Silverman, The organic chemistry of drug design and drug action, especially pages 497 to 546; each of which is incorporated herein by reference. The compounds of the invention may represent prodrugs (eg comprising a potential anti-inflammatory agent or a potential anti-cancer agent) wherein hydrolysis of the mono- or disaccharide moiety, eg by the action of GBA2, provides an active anti-inflammatory agent or an active anti-cancer agent.

[0064] The term "pharmaceutical composition" as used herein includes reference to a composition comprising at least one active compound (or prodrug) and optionally one or more additional pharmaceutically acceptable ingredients (e.g., a pharmaceutically acceptable carrier). Unless the context indicates otherwise, all references to "compositions" herein refer to pharmaceutical preparations.

[0065] As used herein, the term "product" or "product of the invention" includes reference to any product containing a compound of the invention. In particular, the term "product" relates to a composition or formulation, such as a pharmaceutical composition, containing a compound of the invention.

[0066] As used herein, the term "therapeutically effective amount" refers to the amount of a drug or pharmaceutical agent that is calculated to provide (or will provide) the desired therapeutic response in a mammal (animal or human), within the scope of sound pharmacological judgment. The therapeutic response can be, for example, to cure a disease, disorder or condition, delay the progression of a disease, disorder or condition, or prevent a disease, disorder or condition.

[0067] The term "GBA2" as used herein refers to glucosylceramidase β2 as designated by the HUGO Gene Nomenclature Committee. This enzyme is a microsomal β-glucosidase known to catalyze the hydrolysis of bile acid 3-O-glucosides. We have determined that the enzyme GBA2 is selectively expressed at sites of inflammation, such as at sites of inflammation induction in zebrafish. The compounds provided herein (such as compounds of Formula I or Formula II) comprising a therapeutic moiety (such as an anti-inflammatory moiety or a chemotherapeutic moiety) covalently linked to a monosaccharide or disaccharide may have a monosaccharide or disaccharide that is actively hydrolyzed by GBA2. Therefore, GBA2 can selectively release an active therapeutic moiety, such as an anti-inflammatory agent or an active chemotherapeutic moiety, at the site of inflammation. Therefore, the compounds of the present invention are suitable for targeted delivery of active therapeutic moieties (including but not limited to active anti-inflammatory agents or active chemotherapeutic moieties) to sites of inflammation. It is readily understood that such targeted delivery may be beneficial for enabling a wide range of therapeutic moieties to reach sites of inflammation. Inflammation is an integral part of many pathological processes and conditions, and by generating active drugs in situ from the prodrug compounds of the present invention, the method can be used to deliver therapeutic moieties suitable for treating these conditions (not just alleviating inflammatory responses).

[0068] Although little is known about which sugars mammalian GBA2 can cleave from lipophilic compounds such as cholesterol and steroid hormones, in addition to the results provided in the present disclosure, some findings suggest that there is a level of specificity that can be exploited. GBA2 has been shown to cleave β-D-glucosylated compounds (i.e., compounds with D-glucose groups attached by β-glycosidic bonds) (Marques et al., (2016). "Glucosylated cholesterol in mammalian cells and tissues: formation and degradation by multiple cellular beta-glucosidases." J Lipid Res 57(3):451-463). GBA2 also cleaves β-D-galactosylated compounds (Akiyama et al., (2020). "Glucocerebrosidases catalyze a transgalactosylation reaction that yields a newly-identified brain sterol metabolite, galactosylated cholesterol." J Biol Chem 295(16):5257-5277). In contrast, this is not the case for β-xylosylated compounds (Boer et al., (2021). "Human glucocerebrosidase mediates formation of xylosyl-cholesterol by beta-xylosidase and transxylosidase reactions." J Lipid Res62:100018), therefore GBA2 may be considered specific for aldohexose. In the aldohexose group, there may be additional specificity, as observed in studies testing iminosugar deoxynojirimycin derivatives as GBA2 inhibitors (Wennekes et al., (2010). "Dual-action lipophilic iminosugar improves glycemic control in obese rodents by reduction of visceral glycosphingolipids and buffering of carbohydrate assimilation." J Med Chem 53(2):689-698; Ghisaidoobe et al., (2014). "Identification and development of biphenyl substitutediminosugars as improved dual glucosylceramide synthase / neutralglucosylceramidase inhibitors." J Med Chem 57(21):9096-9104; Lahav et al., (2017). "AFluorescence Polarization Activity-Based Protein Profiling Assay in the Discovery of Potent, Selective Inhibitors for Human NonlysosomalGlucosylceramidase." J Am Chem Soc 139(40):14192-14197). For example, based on these studies, it was shown that L-idose can also form an efficient substrate for GBA2 cleavage.

[0069] Previous studies have suggested that GBA2 is an exoglycosidase, i.e. an enzyme that can only cleave monosaccharides from lipophilic compounds. However, in this application we have demonstrated for the first time that GBA2 can actually remove disaccharides from steroid molecules, thus indicating that it is in fact an endoglycosidase, capable of cleaving disaccharide moieties from lipophilic compounds such as corticosteroids.

[0070] The term "corticosteroid" as used herein refers to a class of steroid hormones, including natural corticosteroids produced by vertebrates and synthetic analogs of such compounds. Similar to other steroid molecules, the carbon atoms of corticosteroids can be numbered according to the following hypothetical parent skeleton structure:

[0071]

[0072] One group of preferred corticosteroids are glucocorticoids, which generally have anti-inflammatory activity. The anti-inflammatory effect may be mediated by reducing the levels of pro-inflammatory mediators (e.g., by transcriptional inhibition of gene expression) and / or increasing the levels of anti-inflammatory mediators (e.g., by transcriptional activation of gene expression).

[0073] Compound

[0074] In one aspect, the present invention provides a compound of formula I as described above or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof. These compounds may represent prodrugs, wherein the glycosylated compound has reduced biological activity compared to the corresponding unglycosylated (i.e., non-glycosylated) compound.

[0075] In the compound of formula I, A can be selected from glucose, galactose, mannose, fructose and any acid form thereof, and any disaccharide formed by one or two sugars selected from glucose, galactose, mannose, fructose and any acid form thereof. The acid form of glucose, galactose, mannose and fructose can be an aldonic acid or uronic acid; for example, the acid form of glucose, galactose, mannose and fructose can be an uronic acid, such as glucuronic acid. When the terminal aldehyde group in the aldose is oxidized, an aldonic acid is obtained, for example, the oxidation of D-glucose at C1 produces D-gluconic acid. When the hydroxyl group farthest from the carbonyl group of the sugar is oxidized to a carboxylic acid, a uronic acid is obtained, for example, the oxidation of D-glucose produces the corresponding uronic acid D-glucuronic acid.

[0076] In the compound of formula I, A can be an aldohexose. A can be selected from glucose, galactose, idose, altrose and any acid form thereof, and any disaccharide formed by one or two sugars selected from glucose, galactose, idose, altrose and any acid form thereof. The acid form of glucose, galactose, idose, altrose can be an aldonic acid or a uronic acid; for example, the acid form of glucose, galactose, idose, altrose can be a uronic acid, such as glucuronic acid. When the terminal aldehyde group in the aldose is oxidized, an aldonic acid is obtained, for example, the oxidation of D-glucose at C1 produces D-gluconic acid. When the hydroxyl group farthest from the carbonyl group of the sugar is oxidized to a carboxylic acid, a uronic acid is obtained, for example, the oxidation of D-glucose produces the corresponding uronic acid D-glucuronic acid. Hydroxyl groups at C2 and / or C3 positions may have an effect on GBA2 cleavage (van den Berg et al., (2011). "Assessment of partially deoxygenated deoxynojirimycin derivatives as glucoseylceramide synthase inhibitors." ACS Med Chem Lett 2(7):519-522). In view of this, A can be selected from D-glucose, D-galactose, L-idose, L-altrose and any acid form thereof, and any disaccharide formed by one or two sugars selected from D-glucose, D-galactose, L-idose, L-altrose and any acid form thereof. The acid form of D-glucose, D-galactose, L-idose, L-altrose can be aldonic acid or uronic acid; for example, the acid form of D-glucose, D-galactose, L-idose, L-altrose can be uronic acid, such as glucuronic acid.

[0077] In the compound of formula I, A can be a disaccharide formed by one or two of glucose, galactose, mannose, fructose and any acid form thereof (such as aldonic acid or uronic acid). A can be a disaccharide formed by one or two of glucose, galactose, mannose, fructose and its uronic acid form. A can be a disaccharide formed by one or two of glucose, galactose, mannose and fructose. For example, A can be gentiobiose. A can be a monosaccharide selected from glucose, galactose, mannose, fructose and any acid form thereof (such as aldonic acid or uronic acid). A can be a monosaccharide selected from glucose, galactose, mannose, fructose and its uronic acid form. For example, A can be glucose or glucuronic acid. A can be a monosaccharide selected from glucose, galactose, mannose and fructose. For example, A can be glucose.

[0078] In the compound of formula I, A can be a disaccharide formed by two aldohexoses. For example, A can be a disaccharide formed by one or two of glucose, galactose, idose, altrose and any acid form thereof (such as aldonic acid or uronic acid). A can be a disaccharide formed by one or two of glucose, galactose, idose, altrose and any uronic acid form thereof. A can be a disaccharide formed by one or two of glucose, galactose, idose and altrose. For example, A can be gentiobiose. Hydroxyl groups at the C2 and / or C3 positions may have an effect on GBA2 cleavage (van den Berg et al., (2011), supra). In view of this, A can be a disaccharide formed by one or two of D-glucose, D-galactose, L-idose, L-altrose and any acid form thereof (such as aldonic acid or uronic acid). A can be a disaccharide formed by one or two of D-glucose, D-galactose, L-idose, L-altrose and any uronic acid form thereof. A may be a disaccharide formed from one or two of D-glucose, D-galactose, L-idose and L-altrose. For example, A may be gentiobiose.

[0079] In some embodiments, disaccharides are preferred because disaccharide prodrugs may have reduced activity compared to the corresponding monosaccharide prodrugs, while both compounds will provide an unglycosylated compound with the same activity. Therefore, compounds containing disaccharides and monosaccharides should provide similar levels of targeted activity (after the glycans are removed), while disaccharides may provide less off-target activity side effects.

[0080] In the compounds of formula I, B can be any therapeutic moiety that provides lower biological activity by adding a monosaccharide or disaccharide as described above. Exemplary classes of therapeutic moieties include anti-inflammatory moieties, chemotherapeutic moieties, anti-infective moieties, analgesic moieties, anti-angiogenic moieties, and anti-fibrotic moieties.

[0081] Suitably, B can be an anti-inflammatory moiety. The anti-inflammatory moiety can be or include a corticosteroid (e.g., a glucocorticoid), a nonsteroidal glucocorticoid receptor agonist, a nonsteroidal anti-inflammatory drug, a disease-modifying antirheumatic drug, or rapamycin. The anti-inflammatory moiety can be or include a corticosteroid (e.g., a glucocorticoid). The anti-inflammatory moiety can be or include a nonsteroidal glucocorticoid receptor agonist. The anti-inflammatory moiety can be or include a nonsteroidal anti-inflammatory drug. The anti-inflammatory moiety can be or include a disease-modifying antirheumatic drug. The anti-inflammatory moiety can be or include rapamycin.

[0082] Corticosteroids can be selected from hydrocortisone type corticosteroids, triamcinoloneacetonide type corticosteroids, metasone type corticosteroids, betamethasone dipropionate type corticosteroids and methylprednisoloneaceponate type corticosteroids. Corticosteroids can be hydrocortisone type corticosteroids. Corticosteroids can be triamcinoloneacetonide type corticosteroids. Corticosteroids can be metasone type corticosteroids. Corticosteroids can be betamethasone dipropionate type corticosteroids. Corticosteroids can be methylprednisoloneaceponate type corticosteroids. The corticosteroids of these categories will be further defined below.

[0083] Based on the cross-sensitivity of corticosteroid-induced allergic contact dermatitis, four structural classes of corticosteroid drugs have been defined (Coopman and Dooms-Goossens, Cross-reactions in topical corticosteroid contact dermatitis. Contact Dermatitis. 1988 Aug; 19(2): 145-6; Coopman et al., Identification of cross-reaction patterns in allergic contact dermatitis from topical corticosteroids. Br J Dermatol. 1989 Jul; 121(1): 27-34; Isaksson, Corticosteroids. Dermatol Ther. 2004; 17(4): 314-20; Jacob and Steele, Corticosteroid classes: a quick reference guide including patch test substances and cross-reactivity. J Am Acad Dermatol. 2006 Apr; 54(4): 723-7). The following paragraphs give a description and general structure of each class of corticosteroids. It should be noted that in these paragraphs on the classes of corticosteroids, reference is made to "Cnn," where "nn" is a number referring to the designated numbered carbon atom of the corticosteroid.

[0084] Category A: Hydrocortisone-type corticosteroids are corticosteroids with no substitutions on the corticosteroid D ring or C11 carbon chain, but include C11 and / or C21 acetates and the C21 pivalate thioester hydrocortisone.

[0085]

[0086] Category B: Triamcinolone-type corticosteroids are corticosteroids with a C16, C17-cis, -diol or -ketal chain structure.

[0087]

[0088] Class C: Betamethasone-type corticosteroids are corticosteroids with C16 alkyl substitution.

[0089]

[0090] Category D: Hydrocortisone-17-butyrate type corticosteroids are corticosteroids with long chain esters at C17 and / or C21.

[0091]

[0092] The D category can be further divided into two subcategories (Matura and Goossens, Reactions to corticosteroids: some new aspects regarding cross-sensitivity. Cutis. 2000 Jan; 65(1): 43-5; Goossens et al., Contact allergy to corticosteroids. Allergy. 2000 Aug; 55(8): 698-704), the D1 category and the D2 category. The D1 category, betamethasone dipropionate type corticosteroids, includes corticosteroids with methyl substitution on C16, halogenation on the B ring, and long side chain esters on C17 and / or C21. The D2 category, methylprednisolone acetonide type corticosteroids, includes corticosteroids without methyl substitution on C16, without halogenation, with long side chain esters on C17, and possibly with side chains on C21.

[0093] In the compound of formula I, B can be or include the following molecular groups: alclomethasone, amcinonide, beclomethasone, betamethasone, budesonide, cloprednisone, ciclometasone, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol, cortifen, hydrocortisone, cortisone, cortivazol, cortodoxone, deflazacort, desonide, desoxim etasone, dexamethasone, dichlorisone, diflucortolone, diflorasone, difluprednate, formocortal, fluazacort, flucloronide, fludrocortisone, flucortin, flucortolone, flumetasone, flunisolide, fluocinolone, flucinolone acetateacetonide, fluocinonide, fluocortin, fluorometholone, fluperolone, fluprednidene, fluprednisolone, flurandrenolide, fluticasone, halcinonide, halobetasol, halometasone, hydrocortamate, icometasone, isoflurane, ethacrynic acid The invention may be selected from the group consisting of lotoprednol, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone, nicocortonide, paramethasone, prednicarbate, prednisolone, prednimustine, prednisone, procinonide, tixocortol, triamcinolone or triamcinolone acetonide or any ester thereof. The ester may be or include acetate, adamantate, benzoate, benzofuran carboxylate, benzoyl-β-aminoisobutyrate, butyrate, butylate, hexanoate, carboxylate, chlorambucil, chlorobenzoyl, cipecilate, cyclopentanepropionate, cyclopentylpropionate, cyclopropylcarboxylate, cypionate, dicibate, dichloroacetate, diethylaminoacetate, enanthate, enbutate, cyclopentylpropionate, cyclopropylcarboxylate, cyclopentylpropionate ... The invention also includes but is not limited to esters of the compounds of the present invention, such as etabonate (ethyl carbonate), fuorate, hemisuccinate, caproate, isobutyrate, isonicotinate, linoleate, meta-sulfobenzoate, metembonate, palmitate, phosphate, piperidine acetate, pivalate, propionate, salicylate, stearyl glycolate, succinate, sulfoheptyl, sulfate, tebutate (tert-butyl acetate), tetrahydrophthalate, troxundate, valerate or xanthate.

[0094] In the compound of formula I, B can be a chemotherapeutic moiety. The chemotherapeutic moiety can be or include an alkylating agent (such as cisplatin), a nitrosourea (such as carmustine), an antimetabolite (such as methotrexate and pentostatin), an antitumor antibiotic (such as doxorubicin), a topoisomerase inhibitor (such as irinotecan) and a mitotic inhibitor (such as paclitaxel) or other molecular groups of chemotherapeutic drugs that do not fit neatly into any of these categories.

[0095] In the compounds of formula I, B may be an anti-infective agent. In the compounds of formula I, B may be an analgesic agent. In the compounds of formula I, B may be an anti-angiogenic agent. In the compounds of formula I, B may be an anti-fibrotic agent.

[0096] In one embodiment, the present invention provides a compound of formula II or a pharmaceutically acceptable salt, stereoisomer, acid or solvate thereof:

[0097]

[0098] R 1 Selected from monosaccharides or disaccharides. 2 R is selected from H, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C3-C8 alkenyl. 3 and R 4 Each independently selected from H, OH, C1-C4 substituted or unsubstituted alkyl, OR 9 、OC(O)R 9 ; or R 3 and R 4 Together with the carbon atoms to which they are attached, they form a substituted or unsubstituted 5- or 6-membered heterocycloalkyl group. 5 , R 6 , R 7 and R 8 Each is independently selected from H, OH, halogen, substituted or unsubstituted -C1-C4 alkyl or OR 10 . R 9 is selected from H or -C1-C8 substituted or unsubstituted alkyl. X is selected from =O or -OH.

[0099] X may be =0. X may be -OH.

[0100] The compound may be a compound of formula IIIa or IIIb or a pharmaceutically acceptable salt, stereoisomer, acid or solvate thereof:

[0101]

[0102] The compound may be a compound of formula IVa or IVb or a pharmaceutically acceptable salt, stereoisomer or solvate thereof:

[0103]

[0104] The compound may be a compound of formula Va, Vb, Vc or Vd or a pharmaceutically acceptable salt, stereoisomer, acid or solvate thereof:

[0105]

[0106] In each of Formulae IIIa, IIIb, IVa, IVb, Va, Vb, Vc and Vd, the substituents are defined as follows. 1 Selected from monosaccharides or disaccharides. 2 R is selected from H, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C3-C8 alkenyl. 3 and R 4 Each independently selected from H, OH, C1-C4 substituted or unsubstituted alkyl, OR 9 、OC(O)R 9 ; or R 3 and R 4 Together with the carbon atoms to which they are attached, they form a substituted or unsubstituted 5- or 6-membered heterocycloalkyl group. 5 , R 6 , R 7 and R 8 Each is independently selected from H, OH, halogen, substituted or unsubstituted C1-C4 alkyl or OR 10 . R 9 Selected from H or -C1-C8 substituted or unsubstituted alkyl.

[0107] In any of Formulae II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc and Vd, the substituents may be further defined as follows:

[0108] R 1 It can be selected from glucose, galactose, mannose, fructose and any acid form thereof, and any disaccharide formed by one or two sugars selected from glucose, galactose, mannose, fructose and any acid form thereof. The acid form of glucose, galactose, mannose and fructose can be aldonic acid or uronic acid; for example, the acid form of glucose, galactose, mannose and fructose can be uronic acid, such as glucuronic acid.

[0109] R 1 It can be selected from aldohexose and any disaccharide formed from two aldohexoses (which may be the same or different). 1It can be selected from glucose, galactose, idose, altrose and any acid form thereof, and any disaccharide formed by one or two sugars selected from glucose, galactose, idose, altrose and any acid form thereof. The acid form of glucose, galactose, idose and altrose can be an aldonic acid or a uronic acid; for example, the acid form of glucose, galactose, idose and altrose can be a uronic acid, such as glucuronic acid. The hydroxyl group at the C2 and / or C3 position may have an effect on GBA2 cleavage (van den Berg et al., (2011), supra). In view of this, R 1 It can be selected from D-glucose, D-galactose, L-idose, L-altrose and any acid form thereof, and any disaccharide formed by one or two sugars selected from D-glucose, D-galactose, L-idose, L-altrose and any acid form thereof. The acid form of D-glucose, D-galactose, L-idose and L-altrose can be aldonic acid or uronic acid; for example, the acid form of D-glucose, D-galactose, L-idose and L-altrose can be uronic acid, such as glucuronic acid.

[0110] R 1 It can be a disaccharide formed from one or two of glucose, galactose, mannose, fructose and any acid form thereof (such as aldonic acid or uronic acid). 1 It can be a disaccharide formed from one or two of glucose, galactose, mannose, fructose and their uronic acid forms. 1 It can be a disaccharide formed from one or two of glucose, galactose, mannose and fructose. 1 It may be gentiobiose. 1 It may be a monosaccharide selected from glucose, galactose, mannose, fructose and any acid form thereof (such as aldonic acid or uronic acid). 1 It can be a monosaccharide selected from glucose, galactose, mannose, fructose and their uronic acid forms. 1 It can be glucose or glucuronic acid. 1 It can be a monosaccharide selected from glucose, galactose, mannose and fructose. 1 It may be glucose.

[0111] R 1 It can be a disaccharide formed from two aldohexoses (which may be the same or different). 1 It can be a disaccharide formed from one or two of glucose, galactose, idose, altrose and any acid form thereof (such as aldonic acid or uronic acid). 1 It is a disaccharide formed by one or two of glucose, galactose, idose, altrose and their uronic acid forms. 1 It can be a disaccharide formed from one or two of glucose, galactose, idose and altrose.1 It may be gentiobiose. Hydroxyl groups at the C2 and / or C3 positions may have an effect on GBA2 cleavage (van den Berg et al., (2011), supra). In view of this, R 1 It may be a disaccharide formed from one or two of D-glucose, D-galactose, L-idose, L-altrose and any acid form thereof (such as aldonic acid or uronic acid). 1 It may be a disaccharide formed from one or two of D-glucose, D-galactose, L-idose, L-altrose and their uronic acid forms. 1 It can be a disaccharide formed from one or two of D-glucose, D-galactose, L-idose and L-altrose. 1 It may be gentiobiose.

[0112] Where R 1 Compounds that are disaccharides are sometimes preferred because such compounds may represent disaccharide prodrugs that have reduced activity compared to the corresponding monosaccharide prodrugs, whereas both compounds would provide an unglycosylated compound with the same activity. Thus, compounds containing disaccharides and monosaccharides should provide similar levels of targeted activity (after the monosaccharide or disaccharide moiety is removed), whereas compounds containing R 1 Compounds that are disaccharides may provide fewer off-target activity side effects.

[0113] R 2 R may be selected from H and substituted or unsubstituted C1-C8 alkyl. 2 It can be H. R 2 It may be a substituted or unsubstituted C1-C8 alkyl group. 2 It may be a substituted C1-C8 alkyl group. 2 It may be an unsubstituted C1-C8 alkyl group.

[0114] R 3 Can be selected from H, OH and OC(O)R 9 . R 3 R may be selected from H and OH. 3 Can be selected from H and OC(O)R 9 . R 3 Can be selected from OH and OC(O)R 9 . R 3 It can be H. R 3 It can be OH. 3 Can be OC(O)R 9 .

[0115] R 9 It may be a substituted or unsubstituted -C1-C8 alkyl group. 9 It may be a substituted -C1-C8 alkyl group. 9It may be an unsubstituted -C1-C8 alkyl group.

[0116] R 4 R may be selected from H, OH or C1-C4 alkyl. 4 R may be selected from H, OH or methyl. 4 It can be H. R 4 It can be OH. 4 It may be a C1-C4 alkyl group (eg, methyl group).

[0117] R 3 and R 4 Together with the carbon atoms to which they are attached, they can form a substituted or unsubstituted 5- or 6-membered heterocycloalkyl group. 3 and R 4 Together with the carbon atom to which they are attached, they may form a substituted or unsubstituted 1,3-dioxolane ring. 3 and R 4 Together with the carbon atoms to which they are attached, they can form a substituted 1,3-dioxolane. 1,3-dioxolane can be substituted at the 2-position by 1 or 2 groups selected from -C1-C4 alkyl (such as methyl), 5- or 6-membered cycloalkyl or 5- or 6-membered heterocycle (such as furan). 1,3-dioxolane can be substituted at the 2-position by 1 or 2 groups selected from -C1-C4 alkyl (such as 1 or 2 methyl). 1,3-dioxolane can be substituted at the 2-position by a 5- or 6-membered cycloalkyl. 1,3-dioxolane can be substituted at the 2-position by a 5- or 6-membered heterocycle (such as furan).

[0118] In some compounds, R 2 Yes H, R 3 Yes OH, R 4 It's H.

[0119] In some compounds, R 2 Yes H, R 3 and R 4 Together with the carbon atom to which they are attached, they form a substituted or unsubstituted 1,3-dioxolane ring.

[0120] In some compounds, R 2 Yes H, R 4 It is a C1-C4 alkyl group (such as methyl).

[0121] In some compounds, R 2 Yes H, R 3 It is OC(O)R 9 , R 4 is H; optionally, wherein R 9 It is a substituted or unsubstituted -C1-C8 alkyl group.

[0122] R 5 R may be selected from H and halogen.5 R may be selected from H, H and Cl. 5 It can be H. R 5 It can be F. R 5 It can be Cl.

[0123] R 6 and R 7 They can each be H.

[0124] R 8 R may be selected from H and halogen. 8 R may be selected from H, F and Cl. 8 It can be H. R 8 It can be F. R 8 It can be Cl.

[0125] R 10 R may be selected from H or -C1-C8 (e.g. -C1-C4) substituted or unsubstituted alkyl. 10 It can be H. R 10 The alkyl group may be -C1-C6 (eg -C1-C4) substituted or unsubstituted.

[0126] The compound may be selected from:

[0127] or a pharmaceutically acceptable salt, stereoisomer, acid or solvate thereof.

[0128] The compound may be a substrate for glucosylceramidase β2 (GBA2).

[0129] The sugar groups of compounds that are substrates for GBA2 will be hydrolyzed by the activity of the enzyme GBA2. Two approaches can be used to determine GBA2 activity, genetic and pharmacological, to 1) determine if a particular effect of the compound is dependent on the presence and / or activity of the enzyme, and 2) determine in vitro if the compound is converted by GBA2.

[0130] For 1), to determine whether a particular effect of a compound is dependent on the presence and / or activity of GBA2, it can be done in a) cultured cells or animal models that are genetically modified to be deficient in GBA2 as a result of such modification (e.g., mice or zebrafish (Yildiz et al., Mutation of beta-glucosidase 2 causes glycolipid storage disease and impaired male fertility. J Clin Invest. 2006 Nov; 116(11):2985-94; Lelieveld et al., Role of β-glucosidase 2 in aberrant glycosphingolipid metabolism: model of glucocerebrosidase deficiency in zebrafish. J Lipid Res. 2019 Nov; 60(11):1851-1867)), or b) cultured cells or animal models in the presence of a chemical inhibitor of GBA2 (Wennekes et al., Dual-action lipophilic iminosugar improves glycemic control in obese rodents by reduction of This effect was studied in (Visceral glycosphingolipids and buffering of carbohydrate assimilation. J Med Chem. 2010 Jan 28; 53(2): 689-98; Marques et al., Reducing GBA2Activity Ameliorates Neuropathology in Niemann-Pick Type CMice. PLoS One. 2015 Aug 14; 10(8): e0135889).

[0131] For 2), in order to determine whether the compound is converted by GBA2 in vitro, the protocol previously described by Lelieveld et al. (2019) (supra); and Marques et al., Glucosylated cholesterol in mammalian cells and tissues: formation and degradation by multiple cellular beta-glucosidases. J Lipid Res. 2016 Mar; 57 (3): 451-63 was used. In brief, human cell cultures expressing GBA2 (eg, HeLa or HEK293T) were cultured. For this experiment, cells were harvested in phosphate-buffered saline (PBS), centrifuged and resuspended in 25 mM potassium phosphate buffer (25 mM Kpi pH 6.5, 0.1% Triton-X100 and 25 U / mL benzonase) supplemented with 0.1% (v / v) Triton-X100 and benzonase, and lysed using ultrasound. The cell homogenate was added to a mixture of the compound in McIlvaine buffer (100 μL, including 3.75 mM 4MU-β-Glc, 0.1% (w / v) BSA and 25 μM cholesterol in 150 mM McIlvaine pH 4 or pH 5.2, and 1% ethanol, supplemented with 0.1% (v / v) Triton-X100 and / or 0.2% (w / v) sodium taurocholate) with appropriate pH and supplemented with additives. After incubation at 37° C. with shaking for 1 h, 5 μL of the sample was transferred to 200 μL of stop buffer (1 M glycine-NaOH, pH 10.3). Proteins were precipitated by adding methanol and chloroform (2:1 (v / v)), and after centrifugation, lipids in the supernatant were extracted using the Bligh-Dyer extraction method (MeOH:CHCl3:H2O, 1:1:0.9) and measured using liquid chromatography / mass spectrometry (LC / MS).

[0132] Composition and administration

[0133] According to another aspect of the present invention, a pharmaceutical composition comprising the compounds of the present invention is provided. For example, the pharmaceutical composition may include a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc or Vd. The composition may provide a compound mixed with at least one pharmaceutically acceptable adjuvant, carrier or diluent. The composition may be formulated for non-oral administration, particularly for non-oral systemic administration. The composition may be formulated for parenteral administration. The composition may be formulated for injection (e.g., for intravenous or intramuscular injection).

[0134] The compounds or compositions of the present invention can be administered orally, topically, intravenously, subcutaneously, orally, rectally, dermally, nasally, tracheally, bronchially, by any other parenteral route, as an oral or nasal spray or by inhalation. Preferably, the compounds or compositions are formulated for parenteral administration. The compounds can be administered in the form of pharmaceutical preparations comprising compounds in the form of free compounds or compounds in the form of, for example, pharmaceutically acceptable nontoxic organic or inorganic acid or base addition salts, in pharmaceutically acceptable dosage forms. Depending on the condition to be treated and the patient and the route of administration, the compositions can be administered in different doses.

[0135] Non-oral administration forms may be preferred. The reason for this is that the glycan portion (e.g., monosaccharide or disaccharide) of the compounds of the invention may mask the activity of the rest of the disclosed compounds. The glycan portion is removed to provide an active unglycosylated portion. In non-oral administration, this glycan removal can be performed at or near the site of inflammation (e.g., by GBA2-catalyzed hydrolysis). This provides active compounds at or near the site of inflammation, and activity away from the site of inflammation is generally reduced, which can reduce side effects, especially when the compound is administered systemically. If the compound is administered orally, this advantage may be reduced because the glycan portion may be hydrolyzed by the intestinal microbiota.

[0136] Therefore, the pharmaceutical compounds of the present invention can be administered parenterally ("parenteral" as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion) or orally to a host to obtain a therapeutic effect (such as an anti-inflammatory or chemotherapeutic effect). For example, the pharmaceutical compounds of the present invention can be administered by intravenous injection or infusion. For larger animals (such as humans), the compounds can be administered alone or as a composition in combination with a pharmaceutically acceptable diluent, excipient or carrier.

[0137] The actual dosage level of the active ingredient in the pharmaceutical preparation and pharmaceutical composition of the present invention can be varied so as to obtain the amount of the active compound that effectively achieves the desired therapeutic response for a specific patient, composition and mode of administration. The selected dosage level will depend on the activity of the specific compound, the route of administration, the severity of the condition being treated, and the condition and previous medical history of the treated patient. However, those skilled in the art can start the dosage of the compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. Suitable dosages are generally in the range of 0.01-100 mg / kg / day, for example, in the range of 0.1-50 mg / kg / day.

[0138] The pharmaceutical composition of the present invention for parenteral (e.g., intravenous) injection can include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injection solutions or dispersions before use. The example of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles includes water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (e.g., olive oil) and injectable organic esters, such as ethyl oleate. Suitable mobility can be maintained, for example, by using coating materials, such as lecithin, by maintaining required particle size in the case of dispersions, and by using surfactants. The composition for parenteral injection can represent a preferred composition of the present invention.

[0139] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. By including various antibacterial and antifungal agents, such as parabens, chlorobutanol or phenol sorbic acid, it is possible to ensure that the action of microorganisms is inhibited. It may also be necessary to include isotonic agents, such as sugars or sodium chloride. The absorption of injectable drug forms may be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin).

[0140] Compositions for rectal or vaginal administration may be in the form of suppositories which may be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

[0141] Topical dosage forms of the compounds of the invention include powders, sprays, creams, foams, gels, ointments and inhalants. The active compound is mixed with a pharmaceutically acceptable carrier and any required preservative, buffer or propellant (which may be required) under aseptic conditions. Ophthalmic preparations, eye ointments, powders and solutions are also considered to be within the scope of the present invention.

[0142] Compositions according to the present subject matter may also include inactive components. Suitable inactive components are well known in the art and are described in standard textbooks, such as Goodman and Gillman: The Pharmacological Bases of Therapeutics, 13th edition, Brunton et al., ed., McGraw-Hill Education (2017) and Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Co., Easton, Pa. (1990), which are incorporated herein by reference in their entirety.

[0143] Compositions can be used in combination with additional pharmaceutical dosage forms to enhance their effectiveness in treating any of the conditions described herein. In this regard, the formulations of the invention can be administered as part of a regimen that also includes any other drugs and / or pharmaceutical dosage forms known in the art to be effective in treating any of these conditions.

[0144] use

[0145] The compounds of the present invention represent novel monosaccharide- and disaccharide-containing derivatives of therapeutic moieties such as anti-inflammatory and chemotherapeutic agents. Thus, these compounds are useful in the treatment of inflammation and / or cancer.

[0146] We have determined that therapeutic agents (such as anti-inflammatory or anti-cancer agents) can be converted into prodrugs by the addition of mono- or disaccharides to provide compounds comprising a glycan (monosaccharide or disaccharide) portion and a therapeutic (such as anti-inflammatory or chemotherapeutic) portion.

[0147] GBA2, such as GBA2 present at or near a site of inflammation, is believed to hydrolyze the glycan moiety, thereby providing the active aglyconic form of the therapeutic (eg, anti-inflammatory or chemotherapeutic) moiety.

[0148] Inflammatory conditions are characterized by excessive and / or uncontrolled and / or chronic inflammation (collectively referred to herein as "overactivity" of the immune system). Inflammation is the body's response to harmful stimuli (such as pathogens, damaged cells, or irritants). It is a general, nonspecific response involving the immune system. The five main symptoms are fever, pain, redness, swelling, and loss of function. The function of inflammation is to eradicate pathogens or substances and damaged cells and tissues from the body and to initiate tissue repair. However, excessive (usually chronic) inflammation is associated with many clinical problems. It may become uncomfortable, it may prevent or delay the normal progression of the healing response, it may prolong the injury, and it may cause a variety of diseases. These diseases include autoimmune diseases caused by abnormal immune responses to functional body parts, such as rheumatoid arthritis, allergies caused by excessive responses to foreign substances (usually harmless), such as hay fever, atopic dermatitis, and allergic asthma, and inflammation associated with infectious diseases, such as COVID-19 and tuberculosis.

[0149] Conditions characterized by excessive proliferation of cells belonging to the immune system include leukemias (acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML). ALL is a cancer of the lymphoid blood cells characterized by the production of large numbers of immature lymphocytes, while AML is a cancer of the myeloid blood cells characterized by the rapid growth of abnormal cells that accumulate in the bone marrow and blood and interfere with normal blood cell production.

[0150] In addition, it should be noted that cancer was found to be associated with inflammation as early as the 19th century (Balkwill et al., Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell. 2005 Mar; 7 (3): 211-7; Mantovani et al., Cancer-related inflammation. Nature. 2008 Jul 24; 454 (7203): 436-44; Multhoff et al., Chronic inflammation in cancer development. Front Immunol. 2012 Jan 12; 2: 98; Danforth, The Role of Chronic Inflammation in the Development of Breast Cancer. Cancers (Basel). 2021 Aug 3; 13 (15): 3918). Inflammatory components are present in the microenvironment of most tumor tissues, including those that are not causally related to obvious inflammatory processes. The main features of cancer-related inflammation (CRI) include the infiltration of leukocytes, especially tumor-associated macrophages (TAMs) (Kimm et al., Tumor-Associated Macrophages-Implications for Molecular Oncology and Imaging. Biomedicines. 2021Apr 2; 9(4): 374), which may express GBA2. Without wishing to be bound by any theory, it is believed that glycosylated prodrugs of chemotherapeutic agents that are GBA2 substrates (such as compounds disclosed herein, such as compounds of Formulas I and II) will induce targeted activity to tumors and their microenvironment, where they are locally converted to a more active (non-glycosylated) form. This can result in reduced side effects compared to direct administration of the corresponding non-glycosylated chemotherapeutic agent.

[0151] One aspect of the present invention provides a compound of the present invention or a pharmaceutical composition of the present invention for use as a medicine. For example, the compound can be a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc or Vd. The composition may include a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc or Vd. This application can utilize a compound of the present invention, which incorporates any suitable therapeutic portion that is desired to be provided to the inflammation site. Suitably, this application can use a compound of the present invention incorporating an anti-inflammatory portion or a chemotherapeutic portion.

[0152] Inflammation and / or inflammatory conditions that result in upregulation of GBA2 are associated with a variety of conditions. The compounds of the invention and disclosure (e.g., compounds of any of Formulas I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc, or Vd) or pharmaceutical compositions comprising such compounds can be used to treat any such condition or condition.

[0153] While the compounds of the present invention are well suited for the delivery of anti-inflammatory agents, they can also be used to target other therapeutic moieties to sites of inflammation.Since inflammation is found in many pathological processes and conditions, a wide range of therapeutic moieties can be effectively targeted in this manner.

[0154] Since inflammation is known to be associated with infection, the compounds of the present invention may contain an anti-infective agent as a therapeutic moiety. Such compounds of the present invention are useful as medicaments for treating infection and / or treating inflammation-related complications of infection.

[0155] Since pain is known to be associated with a number of processes involving inflammation, the compounds of the present invention may contain an analgesic as a therapeutic moiety. Such compounds of the present invention are useful as medicaments for relieving pain, particularly pain associated with inflammation.

[0156] Inflammation is also known to be associated with many processes where excessive or pathological neovascularization occurs. Therefore, the compounds of the present invention may include an anti-angiogenic agent as a therapeutic moiety. Such compounds of the present invention may be used as medicaments for treating conditions associated with excessive vascularization. By way of example only, these conditions may include cancer, psoriasis, vascular retinopathy (such as proliferative diabetic retinopathy or wet macular degeneration), and wound healing.

[0157] The role of inflammation in wound healing provides for the medical use of compounds of the invention comprising an anti-fibrotic agent as a therapeutic moiety. Such compounds of the invention are useful as drugs to inhibit scar formation.

[0158] Thus, suitable therapeutic moieties that may be incorporated into the compounds of the invention are not limited to anti-inflammatory or chemotherapeutic moieties, but may also include, by way of example only, anti-fibrotic, analgesic, anti-angiogenic, or anti-infective moieties.

[0159] The use in any of the above applications may include non-oral administration of the compound or composition to a patient. Non-oral administration may include parenteral administration. For example, non-oral administration may include injection.

[0160] One aspect of the present invention provides a compound of the present invention or a pharmaceutical composition of the present invention for treating inflammation. This aspect also provides a compound of the present invention or a pharmaceutical composition of the present invention for relieving inflammation. For example, the compound can be a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc or Vd. The composition may include a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc or Vd. This application may use a compound of the present invention incorporating an anti-inflammatory portion.

[0161] The use may include non-oral administration of the compound or composition to the patient. Non-oral administration may include parenteral administration. For example, non-oral administration may include injection.

[0162] Another aspect of the present invention provides a method for treating inflammation, comprising administering (e.g., non-oral administration) an effective amount of a compound of the present invention or a pharmaceutical composition of the present invention to a patient. For example, the compound can be a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc, or Vd. The composition may include a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc, or Vd. This application can use a compound of the present invention incorporating an anti-inflammatory portion.

[0163] A related aspect provides the use of a compound of the invention in the preparation of a medicament for treating inflammation.

[0164] One aspect of the present invention provides compounds of the present invention or pharmaceutical compositions of the present invention for treating inflammatory conditions or other conditions characterized by an overactive immune system. For example, the compound can be a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc or Vd. The composition may include a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc or Vd. This application may use a compound of the present invention incorporating an anti-inflammatory portion.

[0165] The inflammatory condition or other condition may be selected from arthritis, osteoarthritis, gout, rheumatoid arthritis, other autoimmune disorders such as systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD, including Crohn's disease, ulcerative colitis), multiple sclerosis, chronic inflammatory demyelinating polyneuropathy (CIPD)), infectious diseases such as COVID-19 and tuberculosis, asthma, chronic obstructive pulmonary disease (COPD), allergic and non-allergic rhinitis and sinusitis, tendinitis, nasal polyps, skin diseases such as rash, dermatitis, pruritus, eczema, fungal skin diseases, lichen (sclerosis or erythema) and psoriasis, eye diseases such as uveitis, conjunctivitis, macular edema, otitis, thyroiditis, sarcoidosis, myositis, vasculitis, hemorrhoids, transplant recipient organ rejection and graft-versus-host disease.

[0166] Inflammatory conditions or other conditions may be selected from rheumatoid arthritis, other autoimmune disorders such as systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), thyroiditis, asthma, chronic obstructive pulmonary disease (COPD), allergic and non-allergic rhinitis, respiratory tract infections such as COVID-19, nasal polyps, psoriasis, eczema, dermatitis, eye diseases, and graft-versus-host disease. Inflammatory conditions or other conditions may be selected from rheumatoid arthritis, other autoimmune disorders such as systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), and thyroiditis.

[0167] The use may include non-oral administration of the compound or composition to the patient. Non-oral administration may include parenteral administration. For example, non-oral administration may include injection.

[0168] Another aspect of the present invention provides a method for treating an inflammatory condition or other conditions characterized by an overactive immune system, comprising administering to the patient (e.g., non-oral administration) an effective amount of a compound of the present invention or a pharmaceutical composition of the present invention. For example, the compound can be a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc, or Vd. The composition may include a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc, or Vd.

[0169] The inflammatory condition or other condition may be selected from arthritis, osteoarthritis, gout, rheumatoid arthritis, other autoimmune disorders such as systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD, including Crohn's disease, ulcerative colitis), multiple sclerosis, chronic inflammatory demyelinating polyneuropathy (CIPD)), infectious diseases such as COVID-19 and tuberculosis, asthma, chronic obstructive pulmonary disease (COPD), allergic and non-allergic rhinitis and sinusitis, tendinitis, nasal polyps, skin diseases such as rash, dermatitis, pruritus, eczema, fungal skin diseases, lichen (sclerosis or erythema) and psoriasis, eye diseases such as uveitis, conjunctivitis, macular edema, otitis, thyroiditis, sarcoidosis, myositis, vasculitis, hemorrhoids, transplant recipient organ rejection and graft-versus-host disease.

[0170] Inflammatory conditions or other conditions may be selected from rheumatoid arthritis, other autoimmune disorders such as systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), thyroiditis, asthma, chronic obstructive pulmonary disease (COPD), allergic and non-allergic rhinitis, respiratory tract infections such as COVID-19, nasal polyps, psoriasis, eczema, dermatitis, eye diseases, and graft-versus-host disease. Inflammatory conditions or other conditions may be selected from rheumatoid arthritis, other autoimmune disorders such as systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), and thyroiditis.

[0171] A related aspect provides the use of a compound of the invention in the preparation of a medicament for treating an inflammatory condition or other condition characterized by an overactive immune system. The inflammatory condition or other condition characterized by an overactive immune system may be further defined as above.

[0172] One aspect of the invention provides a compound of the invention or a pharmaceutical composition of the invention for use in treating a condition characterized by excessive proliferation of cells belonging to the immune system. For example, the compound can be a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc, or Vd. The composition can include a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc, or Vd. This application can use a compound of the invention incorporated with a chemotherapeutic moiety.

[0173] The condition may be selected from acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML).

[0174] The use may include non-oral administration of the compound or composition to the patient. Non-oral administration may include parenteral administration. For example, non-oral administration may include injection.

[0175] Another aspect of the present invention provides a method for treating a condition characterized by excessive proliferation of cells belonging to the immune system, comprising administering (e.g., non-oral administration) an effective amount of the compounds of the present invention or the pharmaceutical composition of the present invention to the patient. For example, the compound can be a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc or Vd. The composition may include a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc or Vd. This application can use the compounds of the present invention incorporated into a chemotherapy portion.

[0176] The condition may be selected from acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML).

[0177] A related aspect provides the use of a compound of the invention in the preparation of a medicament for treating a condition characterised by hyperproliferation of cells belonging to the immune system. A condition characterised by hyperproliferation of cells belonging to the immune system may be further defined as above.

[0178] One aspect of the present invention provides a compound of the present invention or a pharmaceutical composition of the present invention for treating cancer. Cancer can be selected from acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML). For example, the compound can be a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc or Vd. The composition may include a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc or Vd. This application can use a compound of the present invention incorporated with a chemotherapy moiety.

[0179] The use may include non-oral administration of the compound or composition to the patient. Non-oral administration may include parenteral administration. For example, non-oral administration includes injection.

[0180] Another aspect of the present invention provides a method for treating cancer, comprising administering (e.g., non-oral administration) an effective amount of the compounds of the present invention or the pharmaceutical composition of the present invention to a patient. Cancer can be selected from acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML). For example, the compound can be a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc or Vd. The composition may include a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc or Vd. This application can use the compounds of the present invention incorporated into a chemotherapy portion.

[0181] A related aspect provides the use of the compounds of the invention in the preparation of a medicament for treating cancer. The cancer may be selected from acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML). For example, the compound may be a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc or Vd. The composition may include a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc or Vd. This application may use the compounds of the invention incorporated with a chemotherapy moiety.

[0182] One aspect of the present invention provides the use of a compound of the present invention as an anti-inflammatory agent. The use comprises contacting the compound with a cell expressing glucosylceramidase β2 (GBA2). This use may use a compound of the present invention incorporating an anti-inflammatory moiety.

[0183] The use may be an in vitro use. The cell may be a tissue or an organ. The cell may be in cell culture. The compound may be a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc or Vd. The composition may include a compound of any formula I, II, IIIa, IIIb, IVa, IVb, Va, Vb, Vc or Vd.

[0184] Determination

[0185] The biological activity of the compounds of the invention may be assessed using any suitable assay known to those skilled in the art. Exemplary assays for assessing the compounds of the invention are provided in the following paragraphs.

[0186] Materials and Methods

[0187] Zebrafish (Danio rerio) were housed and handled according to the guidelines of the Zebrafish Model Organism Database (https: / / zfin.org / ) and in accordance with the instructions of the local Animal Welfare Committee of Leiden University. To maintain circadian rhythmicity, adult fish were exposed to a 14 h light and 10 h dark cycle. Fertilization was performed by natural spawning at the beginning of the light period. Fertilized eggs were collected and cultured in egg water (60 μg / ml instant marine sea salt and 0.0025% methylene blue) at 28 °C. The following zebrafish strains were used in this study: AB / TL wild type, transgenic line Tg(mpx:GFP i114 / mpeg1:mCherry umsF001)(Bernut et al., (2014). Mycobacterium abscessus cording prevents phagocytosis and promotes abscess formation. Proc Natl Acad Sci U S A 111, E943-952; Renshaw et al., (2006). A transgenic zebrafish model of neutrophilic inflammation. Blood 108, 3976-3978) and Tg(9xGCRE-HSV.UI23:EGFP) ia20 (Benato et al., (2014). A living biosensor model to dynamically trace glucocorticoid transcriptional activity during development and adult life in zebrafish. Mol Cell Endocrinol 392, 60-72) and the mutant strain gba2 - / - (Lelieveld et al., (2019). Role of beta-glucosidase 2 in aberrant glycosphingolipid metabolism: model of glucocerebrosidase deficiency in zebrafish. J Lipid Res 60, 1851-1867) and gr s357 (Ziv et al., (2013). An affective disorder in zebrafish with mutation of the glucocorticoid receptor. Mol Psychiatry 18, 681-691).

[0188] Compounds beclomethasone, prednisolone, dexamethasone, dexamethasone, PPD, Rb1, F2 and miglustat were purchased from Sigma-Aldrich (Burlington, MA, USA), dexamethasone-glucuronide (referred to as glucuronidated dexamethasone, GDex) was purchased from Santa Cruz (Dallas, TX, USA), and ME656 and MZ31 were provided by Dr. Hans Aerts (Leiden University, The Netherlands). Compounds 21-O-Glc-prednisolone (glucuronidated prednisolone, GPdn) and 21-O-Gb-prednisolone (gentiobiosylated prednisolone, GbPdn) were synthesized as described in the Examples herein.

[0189] Tail fin cut-off assay

[0190] For tail fin amputation experiments, cells from Tg(mpx:GFP i114 / mpeg1:mCherry umsF001 ) strains at 3 days post fertilization (dpf) were anesthetized in 0.02% buffered aminobenzoic acid ethyl ester (tricaine) in egg water and placed on 2% agarose-coated culture dishes. The tail was partially cut off with a 1 mm sapphire blade (World Precision Instruments) under a Leica M165C stereomicroscope (Leica Microsystems, Wetzlar, Germany). Chemical treatment (or 0.1% DMSO as vehicle treatment) started 2 h before cutting (pretreatment) and continued for 4 h after tail fin cutting (unless otherwise stated, 20 larvae per group), at which time the larvae were fixed in 4% paraformaldehyde (PFA) overnight at 4°C and stored at 4°C until further examination. In some experiments, treatment with megludec, ME656 or MZ31 was started at 2 dpf for 24 h, and these treatments were continued during pretreatment and treatment with other chemicals. In order to i114 / mpeg1:mCherry umsF001 ) strain, using a Leica MZ16FA fluorescence stereomicroscope with LAS 3.7 software. Neutrophils were detected by GFP fluorescence and macrophages by mCherry fluorescence. The number of neutrophils was determined by blinded manual counting in predefined areas. - / - or gr s357When larvae of mutant strains were grown, neutrophils were labeled using the TSA fluorescein detection kit (PerkinElmer) according to the manufacturer's instructions to specifically stain myeloperoxidase (Mpx)-positive cells.

[0191] Measurement of larval body length and caudal fin regeneration

[0192] When regeneration of the caudal fin after trauma or larval body length was used as a readout, chemical treatment of embryos began at 2 hours post fertilization (hpf) and continued until 5 dpf. During this period, the solution was refreshed every day. At 5 dpf, larvae were imaged using a Leica MZ16FA fluorescence stereomicroscope supported by LAS 3.7 software, and larval length was measured using ImageJ software (15 larvae per treatment group). For regeneration experiments, the caudal fin was removed at 2 dpf and larvae were imaged at 5 dpf (15 larvae per treatment group). Newly grown tissue could be distinguished from old tissue, allowing accurate measurement of the size of the newly grown tissue using ImageJ software.

[0193] Whole-body glucose measurement using a colorimetric assay

[0194] Zebrafish embryos were treated with chemicals at 2 hpf as described, with solutions refreshed daily until 5 dpf. At 5 dpf, larvae were collected in Eppendorf tubes and 100 μL of ice-cold glucose buffer was added to each sample. The larvae were homogenized at 8,000 rpm for 3 min. The homogenate was then centrifuged at 11,000 rpm for 8 min at 4°C, and the supernatant was stored at -20°C. Systemic glucose concentration was determined using a glucose colorimetric assay kit (Cayman Chemical, USA) according to the manufacturer's instructions. In each experiment, three biological replicates were used for each treatment group, and colorimetric assays were performed using technical duplicates.

[0195] Systemic cortisol measurement using ELISA

[0196] Zebrafish embryos were chemically treated at 2 hpf, as described, with solutions renewed daily until 96 hpf. Then, treatments were stopped and embryos were incubated in egg water until 5 dpf to avoid cross-reaction with the cortisol antibody compounds used in the ELISA. Then, treatments were stopped and replaced with egg water. At 5 dpf, larvae were collected in Eppendorf tubes and 100 μL of ice-cold phosphate-buffered saline (PBS) was added. The larvae were homogenized at 8,000 rpm for 3 min. Ethyl acetate was added to the homogenate, and the supernatant was collected and evaporated. 150 μl volume of 0.2% bovine serum albumin (Sigma Aldrich) dissolved in phosphate buffered saline (PBS) was added to the sample and frozen. Systemic cortisol in zebrafish larvae was determined by ELISA (Demeditec Diagnostics GmbH, Kiel-Wellsee, Germany) according to the manufacturer's instructions. In each experiment, three biological replicates were used for each treatment group, and colorimetric determinations were performed using technical replicates.

[0197] Transcriptional activation activity of Gr in a zebrafish reporter line

[0198] Tg(9xGCRE-HSV.UI23:EGFP) ia20 The zebrafish reporter line expresses enhanced green fluorescent protein (EGFP) under the control of a promoter containing an array of 9 GREs. To investigate the effects of chemical treatments on Gr transcriptional activation activity, 15 zebrafish embryos (2 dpf) per group were treated with the indicated compounds for 24 h. Treatment-induced Gr transcriptional activation activity was assessed using a Leica MZ16FA fluorescence stereomicroscope supported by LAS 3.7 software. The integrated intensity of the EGFP signal in the larvae was determined using ImageJ software.

[0199] Overexpression of gba2 in zebrafish

[0200] To overexpress gba2 in zebrafish, a plasmid containing the cDNA encoding the zebrafish gba2 gene fused to the CMV promoter was used (pDEST-zeo-zGBA2) (Lelieveld et al., 2020). The plasmid was injected into zebrafish embryos at the one-cell stage using an automated microinjection system version 3AMS-03 (Life Science Methods Bv), which was diluted in nuclease-free water (1 nl / egg, final concentration of 80 pg / egg). After injection, chemical treatments were performed as indicated.

[0201] Quantitative PCR (qPCR) analysis

[0202] For experiments in zebrafish, 3 dpf embryos were used to measure gene expression. Groups of 15 larvae were collected in TRIzol reagent (Invitrogen) and mRNA was isolated using the miRNeasy mini kit (Qiagen) according to the manufacturer's instructions. For experiments in HeLa cells, samples were collected from individual wells of 6-well plates (80% confluence) in TRIzol reagent and mRNA was isolated using the RNeasy mini kit. DNA-free TM Zebrafish and cell samples were treated with DNAse using the iScript cDNA Synthesis Kit (Ambion). cDNA synthesis was performed using the iScript cDNA Synthesis Kit (Bio-Rad), using 1 μg RNA per sample. For qPCR, 10 μM forward primer and 10 μM reverse primer, 12.5 μL LiQ SYBRGreen Supermix (Bio-Rad), and 2 μL cDNA were added to the qPCR reaction mixture. The total volume of each mixture was 25 μL, which was divided into two aliquots with a volume of 12.5 μL. qPCR reactions were performed on the MyiQ-Single Color Real-Time PCR Detection System (Bio-Rad), with initial denaturation at 95°C for 3 min and 40 cycles of 95.5°C for 15 s, 60°C for 15 s, and 72°C for 30 s. The cycle threshold (Ct value, i.e., the number of cycles to reach the fluorescence intensity threshold) was determined for each sample. The expression of ppial (peptidylprolyl isomerase Ab (cyclophilin A)) of zebrafish samples and 18S rRNA of human cells was used to standardize the gene expression level of each sample. The ΔΔCt method was used to calculate the fold change of each sample (compared with the respective control group). In each experiment, three biological replicates were used for each treatment group, and the reactions were repeated.

[0203] Cell culture, transfection, and shRNA gene knockdown

[0204] HeLa cells (purchased from ATCC) were cultured in Dulbecco's modified Eagle (DMEM) high glucose (HG) medium without phenol red and supplemented with 10% fetal calf serum (FCS) and 10% Glutamax (Sigma-Aldrich). Cells were maintained at 37°C and 5% CO2. 24-48h before treatment, cells were seeded in 6-well plates and allowed to adhere. After adhesion and reaching 80% confluence, cells were treated with the specified chemicals (or 0.01% DMSO as a vehicle) for 6h.

[0205] To overexpress GBA2 in HeLa cells, a plasmid containing the cDNA encoding the human GBA2 gene fused to the CMV promoter (pDEST-zeo-hGBA2 (Lelieveld et al., 2020)) was transfected into HeLa cells at about 70% confluence. The plasmid was mixed with FuGENE HD transfection reagent (Promega, Madison, WI, USA) and 500 μl of serum-free DMEM. The mixture was incubated at room temperature for 20 min and added to HeLa cells cultured in supplemented DMEM. After 2 days, the medium was changed and chemical treatments were performed for 6 h as indicated.

[0206] To knock down GBA1 and GBA2, lentiviral particles containing shRNA for GBA1 and GBA2 were generated. For this purpose, we used plasmids developed by the RNAi Consortium containing shRNA for GBA1 (TRCN0000029294) and GBA2 (TRCN0000049585). These plasmids were mixed with packaging plasmids (psPAX2) and lentiviral envelope vectors (pMD2.G) in 30 μl of transfection reagent (Lipo293D). The mixture was incubated at room temperature for 20 min and added to HEK293T Lenti-X cells cultured in complete DMEM medium. After 6 h, the medium was replaced with complete DMEM containing 25 mM HEPES. After 24 h, the cells were washed twice with PBS, the medium was replaced with fresh DMEM containing HEPES, and the lentiviral particles were harvested at 48 and 72 h. The medium in the flask was collected into a 50 ml sterile falcon tube and centrifuged at 3,000 rpm for 7 min. The supernatant was filtered through a 0.45 μm filter. Titration was performed to find the optimal amount of virus required for effective knockdown. Based on these experiments, an aliquot (2 ml) of lentivirus was added to 3 ml of serum-free DMEM containing 10 μg / ml polycondensate. The mixture was added to Hela cells cultured in DMEM supplemented with fresh serum at 70-80% confluence in a T75 flask. The medium was replaced after 12-16 h, and after 48 h, the cells were seeded into 6 well plates. Then, as described above, chemical treatment was added for 6 h, and cells were collected for qPCR analysis, which was also used to confirm GBA1 and GBA2 knockdown.

[0207] Activity-based probe assays

[0208] Activity-based probe (ABP) assays were performed as previously described by Lelieveld et al. (2019). For experiments in zebrafish, 5 dpf larvae (10 per sample) were cut and euthanized with 0.02% tricaine on ice 4 h after cutting. Excess water was removed, 50 μL of homogenization buffer (25 mM KPI, pH 6.5, 0.1% v / v Triton-X) was added, and the samples were homogenized by sonication on ice and stored at -20 °C. In addition, to determine the enzyme activity at the site of inflammation, larvae were cross-sectioned into two parts 4 h after cutting: the part in front of the yolk extension and the part behind the yolk extension, and they were collected separately (30 per sample). For experiments in HeLa cells, cells were grown to 80-90% confluence and treated with vehicle (0.001% DMSO) or TNF-α for 6 h. Homogenization buffer (25 mM KPI, pH 6.5, 0.1% v / v Triton-X) was added and samples were sonicated on ice and stored at -20°C.

[0209] The β-glucosidase probe (JJB367-alkylCy5) in DMSO was diluted in McIlvaine buffer (citric acid / sodium hydrogen phosphate: 100mM / 200mM mixture, pH 5.8), and 10μL was added to 10μL protein sample (final probe concentration of 10μM in 1% DMSO) and incubated at 37°C for 30min. Laemmli sample buffer (15L) was added and the samples were incubated at 95°C for 5min. Afterwards, they were immediately loaded onto a 12% SDS-PAGE gel (Bio-Rad Laboratories). The gel was run at 90V for about 2h and analyzed using a Typhoon FLA 9000 gel imager (GE Healthcare; Cy5 (λEX: 635nm, λEM: 665nm), 750V, pixel size 100μm). The intensity of the protein bands was quantified using ImageJ software. In each experiment, four biological replicates were used, except for the experiment where the larvae were divided into anterior and posterior parts. In the latter experiments, two biological replicates were used. The total protein concentration of the samples was quantified using a Bradford assay kit (Bio-Rad) to standardize the total protein concentration.

[0210] Enzyme activity assay

[0211] Zebrafish larvae were cut off at 5 dpf and euthanized with 0.02% tricaine on ice 4 hours after cutting and collected in Eppendorf tubes (10 larvae per sample). All excess water was removed, 50 μL of homogenization buffer (25 mM KPI, pH 6.5, 0.1% Triton-X) was added, and the samples were homogenized by ultrasonic treatment on ice and stored at -20°C. 12.5 μL of the homogenate (equivalent to about 1 larvae) was added to a black flat-bottom 96-well plate on ice with 12.5 μL of Milli-Q water containing 5% DMSO (with and without GBA1 or GBA2 inhibitors). GBA1 inhibitor (ME656) solution was prepared at a final concentration of 1 μM. GBA2 inhibitor (MZ31) was used at a final concentration of 100 nM. The plate was incubated at 37°C for 30 min to allow the inhibitor to bind to the enzyme. A volume of 100 μL of 4MU (4-methylumbelliferyl-β-D-glucopyranoside) in 150 mM McIlvaine buffer (pH 5.8) was added. The blank contained 12.5 μL of homogenate, 12.5 μL of Milli-Q containing 5% DMSO, and 100 μL of 150 mM McIlvaine buffer (pH 5.8). After a brief shaking, the plate was incubated at 37°C for 2 h. After the incubation period, the plate was placed on ice and 200 μL of stop buffer (1 M NaOH-glycine, pH 10.3) was added to each well. After stopping the reaction, a 4MU standard (10 μL of a known 4MU standard (1 nmol), 15 μL of KPI buffer, 100 μL of 150 mM McIlvaine (pH 5.8), and 200 μL of stop buffer) was prepared and added to individual wells. Subsequently, a blank of 4MU standard (10 μL Milli-Q, 15 μL KPI buffer, 100 μL 150 mM McIlvaine (pH 5.8), and 200 μL stop buffer) was prepared and added to a separate well. The fluorescence intensity in all wells was then measured using a fluorescence spectrometer LS-55 (PerkinElmer). Three biological replicates were used. The specific activity under each condition was calculated using the following formula (Kallemeijn et al., (2017). Investigations on therapeutic glucocerebrosidases through paired detection with fluorescent activity-based probes. PLoS One 12, e0170268):

[0212]

[0213] Where Arbs X is the absorbance of the sample, Arbs blank is the absorbance of the blank, and Arbs 4MU std is the absorbance of the 4MU standard. To standardize the enzyme activity obtained per mg of protein, Pierce TM Total protein concentration was measured by BCA protein assay kit (Fisher Scientific).

[0214] Competitor binding assay

[0215] To determine the relative GR binding affinities of different compounds, PolarScreen was used according to the manufacturer's instructions. TM Glucocorticoid receptor competitor assay kit (ThermoFisher). Briefly, test compounds were first dissolved in DMSO to a 10 mM (100x) stock concentration and further diluted in GR buffer (100 mM potassium phosphate (pH 7.4), 200 mM Na2MoO4, 1 mM EDTA, and 20% DMSO). Serial dilutions of compounds were transferred to Black 384-well plate, add Fluormone GS red TM , then add full-length GR (partially purified receptor in storage buffer). The plate was incubated for 4 h in the dark at room temperature and fluorescence polarization was measured with a CLARIOStar microplate reader (BMG Labtech). The values ​​obtained were normalized to the maximum measured value (no ligand) and the minimum control value (10 μM dexamethasone), and the relative percentage of polarization was determined. The dose-response curve was fitted so that the IC50 value of each compound could be calculated. The experiment was performed in duplicate.

[0216] Induced arthritis assay

[0217] The mouse collagen antibody induced arthritis (CAIA) model can be used to study the effects of compounds in treating inflammatory diseases.Although the method has been described below for compound GbPdn, the skilled person will readily understand how to generalize it to any related compound.

[0218] Male BALB / cAnNCrl mice (±8 weeks old) were purchased from Charles River. Four or five mice were housed per cage in individually ventilated cages under specific pathogen-free (SPF) conditions. The experiments were reviewed by the LUMC Animal Welfare Unit and performed under a license granted by the Central Authority for Animal Scientific Procedures and study number AVD11600202115391 / PE.15391.01.002, in accordance with the Dutch Animal Experimentation Act and EU Directive 2010 / 63 / EU.

[0219] All mice received 1.5 mg CAIA mixture (Chondrex) intravenously on day 1, followed by an intraperitoneal (ip) injection of 50 μg LPS (Chondrex) on day 4. The mice were randomly divided into six groups. There were 4 mice in each group, except for the 25 mg / kg Pdn and GbPdn groups (each group had 5 mice). Pdn and GbPdn were diluted in 10% EtOH (vehicle) and administered daily by intraperitoneal injection from day 7 to day 15, with doses of 12.5 and 25 mg / kg for Pdn and 12.5, 25, 50 mg / kg for GbPdn. Arthritis was scored daily from day 5 to day 15 as described by Jansen et al. (Jansen DT et al., Abatacept decreases disease activity in aabsence of CD4(+)Tcells in a collagen-induced arthritis model. Arthritis Res Ther 2015; 17:220). Scoring was blinded. Blood was collected by tail vein sampling on day 0 (75 μl), day 10 (40 μl), day 14 (40 μl) and day 15 (75 μl). Blood collection on day 14 was performed after a 6 h fast. On day 15, mice were sacrificed with carbon dioxide, and tissue samples were collected and immediately frozen in liquid nitrogen. The humane endpoints applied were a total body weight loss of >20% compared to the day of LPS treatment or an arthritis score of 45.

[0220] IL-6 concentrations in serum collected on days 10 and 15 were determined by ELISA using an IL-6 mouse ELISA kit (Invitrogen) according to the manufacturer's protocol. Insulin concentrations in serum collected on day 14 were determined using an ultrasensitive mouse insulin ELISA kit (CrystalChem) according to the manufacturer's protocol. Corticosterone concentrations in serum collected on day 15 were determined using a corticosterone HS (high sensitivity) enzyme immunoassay kit (Immunodiagnostic Systems) according to the manufacturer's protocol. Expression levels of various GR target genes in tissue samples collected on day 15 (homogenized in TRIzol reagent (ThermoFisher)) were determined by qPCR.

[0221] LC / MS cleavage confirmation assay

[0222] The ability of GBA2 to cleave sugars from compounds can be assessed by the following assay performed in zebrafish, using liquid chromatography / mass spectrometry to evaluate the resulting products. In the following method, the compound GPdn was used, along with appropriate standards of Pdn and GPdn. This method can be adapted to other glycosylated compounds by selecting appropriate standards to prepare LC / MS calibration curves.

[0223] 100 wild-type zebrafish larvae (in the presence or absence of the Gba2 inhibitor MZ31) or gba2- / - larvae were traumatized in the tail and treated with GbPdn for 6 h at 5 dpf (2 h before and 4 h after trauma). For the group treated with MZ31, the compound was added 24 h before trauma. The samples were collected in a cell strainer and quickly washed three times with MilliQ water to remove residual treatment solution. The washed larvae were transferred to a 2 ml Eppendorf tube and immediately immersed in liquid nitrogen. The larvae were freeze-dried overnight and then homogenized with glass beads (5 mm) at 30 Hz for 2 min. The fish powder was ultrasonically extracted with 1 ml 80% methanol for 20 min. The mixture was filtered through a 0.22 μm RC membrane.

[0224] To perform LC / MS, calibration curves were prepared by adding 2.5, 5, 25, 100, 500, 1000 and 5000 pg / ml of prednisolone, 21-glucose prednisolone and 21-gentiobiose prednisolone to blank samples (from untreated larvae). These compounds were analyzed by using an Acquity BEH C18 column (50×2.1 mm, 1.7 μm (Waters, USA)) on a Shimadzu LC-30AD (Japan) coupled to a SCIEX Q-Trap 6500+ (Framingham, MA, USA). Separation was performed using three mobile phases: (A) water containing 0.1% acetic acid; (B) ACN: methanol containing 0.1% acetic acid (9:1, v / v); (C) isopropanol containing 0.1% acetic acid, 25% B and 1% C at a flow rate of 0.5 ml / min in 3 min at 40°C. Multiple reaction monitoring (MRM) was used in MS / MS acquisition, using electrospray positive ion mode, spray voltage 4.5 kV, capillary temperature 600 °C, sheath gas 50, auxiliary gas 50, curtain gas 40, collision energy 20 V. Data were acquired using Sciex Analyst software (version 1.7, Framingham, MA, USA), and peak integration was performed using Sciex OS (version 1.4.0, Framingham, MA, USA).

[0225] Example

[0226] In the synthetic examples (Examples 1 and 2), unless otherwise stated, all reagents used were of higher grade of the American Chemical Society (ACS) and were used without further purification. NMR characterization was obtained using a Bruker 500 (500 MHz) using DMSO-d6. HRMS analysis was performed on a Shimadzu Nexera X2 UHPLC system using a Waters Acquity HSS C18 column (2.1×100 mm, 1.8 μm) equipped with a diode array detector at 30° C. The following solvent system was used with a flow rate of 0.5 mL / min: solvent A, 0.1% formic acid in water; solvent B, 0.1% formic acid in acetonitrile. The gradient elution was as follows: 95:5 (A / B) for 1 min, 95:5 to 15:85 (A / B) for 6 min, 15:85 to 0:100 (A / B) for 1 min, 0:100 (A / B) for 3 min, and then back to 95:5 (A / B) for 3 min. The system was connected to a Shimadzu 9030 QTOF mass spectrometer (ESI ionization) which was internally calibrated with an API-TOF reference mass solution kit from Agilent (5.0 mM purine, 100.0 mM ammonium trifluoroacetate, and 2.5 mM hexa(1H,1H,3H-tetrafluoropropoxy)phosphazene), diluted to achieve a mass count of 10,000. Preparative HPLC was run on a BESTA-Technik system equipped with a Dr.Maisch Reprosil Gold 120C18 column (25×250 mm, 10 μm) and an ECOM flash UV detector with a monitoring wavelength of X nm. The following solvent system with a flow rate of 12 mL / min was used: solvent A, 95 / 5 solution of 0.1% TFA in water / acetonitrile; solvent B, 5 / 95 solution of 0.1% TFA in water / acetonitrile. Gradient elution was as follows: 95:5 (A / B) for 2 min, 95:5 to 0:100 (A / B) for 55 min, 0:100 (A / B) for 2 min, then returned to 95:5 (A / B) for 1 min, and 95:5 (A / B) for 2 min.

[0227] Although the examples and reaction schemes provided illustrate the synthesis of specific glycosylated corticosteroids, as the skilled artisan will appreciate, these methods can be generalized to synthesize other glycosylated corticosteroids, such as those with different sugars and / or with different corticosteroids.

[0228] Example 1: Synthesis of 21-O-glucose-prednisolone

[0229]

[0230] Solution 1

[0231] 21-O-Glucose-prednisolone was prepared according to Reaction Scheme 1. Under argon atmosphere, prednisolone (0.50 g, 1.39 mmol), silver trifluoromethanesulfonate (1.1 equivalents, 0.39 g, 1.53 mmol), ground Molecular sieves (1.2 g) were stirred at room temperature for 1 h in 10 mL of DCM. The reaction mixture was cooled to 0 ° C, and bromine-α-D-glucose tetraacetate (1.1 equivalents, 0.63 g, 1.53 mmol) in 10 mL of DCM was added dropwise within 1 h. After stirring the reaction mixture at 0 ° C for 1 h, the ice-water bath was removed and the mixture was stirred at room temperature overnight. The next day, the reaction mixture was filtered through diatomaceous earth, and a saturated sodium bicarbonate solution was added to the filtrate. After 2.5 h, the organic layer and the aqueous layer were separated. The organic layer was then washed with brine, dried over sodium sulfate and concentrated in vacuo. The mixture was then applied to a silica gel column and eluted with a gradient of 100% DCM to 70:30 DCM / acetone to obtain a partially purified intermediate product, which was considered suitable for subsequent deprotection steps. The fully acetylated glucose-prednisolone conjugate was then dissolved in 3 ml of anhydrous methanol, and 1.5 ml of anhydrous chloroform and 0.32 ml of a 25% wt solution of sodium methoxide in methanol were added dropwise under an argon atmosphere. After stirring for 1 h, Dowex 50WX8 (H+) resin was added to neutralize the mixture. The resin was then removed by filtration and the filtrate was concentrated in vacuo. The residue was purified using RP-HPLC and lyophilized to obtain 125 mg of 21-O-Glc-prednisolone in 2 steps with a yield of 17% as a fluffy white powder. C27H38O10[M+H]+ calculated value 523,2538, found value (HRMS) 523,2538.

[0232] 1H NMR (500MHz, DMSO) δ7.32(d,J=10.1Hz,1H),6.16(dd,J=10.1,1.9Hz,1H),5.91(t,J=1.6Hz,1H),5.28(s,1H),4.78(d,J=18.2Hz, 1H),4.59(s,1H),4.34(d,J=18.2Hz,1H),4.30–4.23(m,1H),4.17(d,J=7.8Hz,1H),3.69(dd,J=11.8,2.0Hz,2H),3.49–3.37(m,1 H),3.16–3.07(m,2H),3.06–2.97(m,2H),2.58–2.51(m,2H),2.33–2.22(m,1H),2.11–1.94(m,3H),1.87(dd,J=13.7,3.6Hz,1H), 1.70–1.57(m,4H),1.44–1.35(m,5H),1.34–1.22(m,1H),1.01(qd,J=13.2,4.6Hz,1H),0.89(dd,J=11.0,3.5Hz,1H),0.78(s,3H).

[0233] 13 C NMR (126MHz, DMSO) δ208.53,185.70,171.05,157.26,127.61,121.72,102.41,89.25,77.53,77.19,73.85,71.69,70.53,69.03,61.61, 55.85,51.58,47.19,44.31,40.56,40.39,40.23,40.06,39.89,39.73,39.56,39.43,34.50,33.24,31.86,31.51,24.08,21.37,17.45.

[0234] Example 2: Synthesis of 21-O-gentiobiose-prednisolone

[0235]

[0236] Solution 2

[0237] 21-O-gentiobiose-prednisolone was prepared according to Reaction Scheme 2. Under argon atmosphere, prednisolone (0.50 g, 1.39 mmol), silver triflate (1.1 equivalents, 0.39 g, 1.53 mmol), ground Molecular sieves (1.2 g) were stirred at room temperature for 1 h in 10 mL of DCM. The reaction mixture was cooled to 0 ° C and bromo-α-D-gentiobiose octaacetate (1.1 equivalents, 1.10 g, 1.53 mmol) in 10 mL of DCM was added dropwise over 1 h. After stirring the reaction mixture at 0 ° C for 1 h, the ice-water bath was removed and the mixture was stirred at room temperature overnight. The next day, the reaction mixture was filtered through diatomaceous earth and a saturated sodium bicarbonate solution was added to the filtrate. After 2.5 h, the organic layer and the aqueous layer were separated. The organic layer was then washed with brine, dried over sodium sulfate and concentrated in vacuo. The mixture was then applied to a silica gel column and eluted with a gradient of 100% DCM to 70:30 DCM / acetone to obtain a partially purified intermediate product, which was considered suitable for subsequent deprotection steps. The fully acetylated gentiobiose-prednisolone conjugate was then dissolved in 3 ml of anhydrous methanol, and 1.5 ml of anhydrous chloroform and 0.32 ml of a 25% wt solution of sodium methoxide in methanol were added dropwise under an argon atmosphere. After stirring for 1 h, Dowex 50WX8 (H+) resin was added to neutralize the mixture. The resin was then removed by filtration and the filtrate was concentrated in vacuo. The residue was purified using RP-HPLC and lyophilized to obtain 221 mg of 21-O-gentiobiose-prednisolone as a fluffy white powder in 2 steps with a yield of 23%. 33 H 48 O 15 [M+H]+ calcd. 685,3066, found (HRMS) 685,3067.

[0238] 1H NMR (500MHz, DMSO) δ7.33(d,J=10.1Hz,1H),6.15(dd,J=10.0,1.9Hz,1H),5.91(t,1H),5.24(s,1H),4.83(d,J=18.3Hz,1H),4.61(s,1H),4.33(s,1H) ,4.29(s,1H),4.19(dd,J=10.1,7.8Hz,2H),3.96(dd,J=11.5,1.9Hz,1H),3 .66(dd,J=11.8,1.7Hz,1H),3.58(dd,J=11.4,5.9Hz,1H),3.44(dd,J=11.6 ,5.2Hz,1H),3.34–3.27(m,1H),3.18–3.10(m,3H),3.08–3.01(m,3H),2.99 –2.94(m,1H),2.56–2.51(m,2H),2.32–2.25(m,1H),2.07–1.97(m,2H),1.8 6(dd,J=13.8,3.7Hz,1H),1.73–1.52(m,2H),1.45–1.33(m,4H),1.28(dd,J =11.4, 6.3Hz, 1H), 0.99 (qd, J = 13.1, 4.6Hz, 1H), 0.88 (m, 1H), 0.78 (s, 3H).

[0239] 13 C NMR (126MHz, DMSO) δ208.54,185.75,171.12,157.37,127.58,122.09,103.86, 102.78,89.19,77.39,77.22,77.07,76.08,74.00,73.74,72.27,70.56,70.12 ,68.97,68.74,61.53,55.85,47.28,47.25,44.32,40.51,40.34,40.17,40.00,39.84,39.67,39.50,39.45,34.50,33.36,31.87,31.48,24.05,21.38,17.45.

[0240] Example 3: Anti-inflammatory effects of ginsenosides

[0241] It has been previously reported that ginsenosides have anti-inflammatory effects by activating GR (He et al., 2020, GinsenosideRg1 Acts as a Selective Glucocorticoid Receptor Agonist with Anti-Inflammatory Action without Affecting Tissue Regeneration in ZebrafishLarvae.Cells 9). To study this effect in more detail, local inflammatory responses were induced in zebrafish larvae by damaging the caudal fin ( Figure 1 A), which causes leukocytes to migrate to the injured area and increases the expression of pro-inflammatory genes (Renshaw et al., 2006, Blood 108, 3976-3978; Xie et al., 2019, Dis Model Mech12). Using this model, it was first investigated whether the anti-inflammatory effects of ginsenosides depend on their glycosylation pattern. To this end, the ginsenoside proginaphanadiol (PPD), F2 and Rb1 were used. PPD is not glycosylated, and F2 and Rb1 are composed of the aglycone PPD with two glycones at the C-3 and C-20 positions of the steroid skeleton. In F2, these glycosyl groups are formed by the monosaccharide glucose, and in Rb1 by the disaccharide gentiobiose, which is composed of two glucose molecules connected by a β(1->6) bond. As a positive control, a synthetic version of the anti-inflammatory glucocorticoid drug beclomethasone (Bec) was used. The structures of these compounds are as shown in Figure 1 As shown in B.

[0242] The results showed that, as previously reported, Bec reduced the migration of neutrophils into the injured area, whereas the migration of macrophages was unaffected. Ginsenosides PPD, F2, and Rb1 all showed similar inhibitory effects on neutrophil migration ( Figure 1 C and Figure 2 As shown previously for ginsenoside Rg1, this ginsenoside effect is mediated by Gr, since the inhibition of neutrophil migration was abolished in zebrafish larvae from a mutant strain deficient in Gr function ( Figure 1 C). Similar results were obtained when we used the expression of pro-inflammatory genes as a readout. Bec, PPD, F2, and Rb1 strongly suppressed the wound-induced increase in the expression of il1b, il6, and mmp9 ( Figure 1D). As the effects of ginsenosides appear to be independent of the glycosylation pattern, we hypothesized that glycosylated ginsenosides are metabolized to the aglycone form. One class of enzymes capable of catalyzing the hydrolysis of the β-glycosidic bond between PPD and the glycosyl group is the β-glucosidase, of which there are two isoforms in vertebrates. Lysosomal acid β-glucosidases, also known as glucocerebrosidase (GBA1), have been well studied because deficiency of this enzyme causes the most common lysosomal storage disorder in humans, Gaucher disease, which is characterized by the accumulation of tissue macrophages filled with glucosylceramide. In addition, there exists a non-lysosomal glucosylceramidase (GBA2) that is structurally unrelated to GBA1, is located near the cell surface, and differs in enzyme characteristics such as specificity for artificial substrates and inhibitors.

[0243] The role of GBA1 and GBA2 enzymes in the anti-inflammatory effects of ginsenosides was tested in zebrafish using the nonspecific chemical GBA inhibitor miglustat and the specific GbA1 and GBA2 inhibitors ME656 and MZ31, respectively. The reduction of neutrophil migration by F2 and Rb1 was abolished in the presence of miglustat and MZ31, whereas these inhibitors did not affect the effects of PPD ( Figure 1 E, G). However, treatment with ME656 did not alter the effects of any ginsenosides ( Figure 1 F), indicating that this effect is Gba2-specific. To confirm the dependence of the action of glycosylated ginsenosides on Gba2, a Gba2-deficient mutant strain was used. In juveniles of this strain, F2 and Rb1 did not affect neutrophil migration, while PPD did ( Figure 1 H), similar to the results obtained with GBA2 inhibitors. Taken together, these data suggest that the effects of glycosylated ginsenosides are dependent on GBA2, rather than GBA1, to hydrolyze these compounds to their aglycone forms.

[0244] Example 4: Effects of glycosylated ginsenosides on side effects compared with classic glucocorticoid drugs

[0245] Other ginsenosides were investigated to determine whether similar low-level adverse effects and possible side effects depend on the glycosylation pattern of the ginsenosides. To this end, zebrafish embryos were treated with Bec, PPD, F2, and Rb1, and the effects on glucose and cortisol levels, larval length, and tissue regeneration after injury were monitored, which can be considered as surrogates for several well-known glucocorticoid side effects observed in the clinic. The anti-inflammatory glucocorticoid drug Bec was used as a positive control and indeed induced an increase in glucose levels ( Figure 3 A), and cortisol levels ( Figure 3 C) larval length ( Figure 3 E) and tissue regeneration ( Figure 3F, G). Ginsenoside PPD did not affect glucose levels ( Figure 3 A), but did reduce cortisol concentrations ( Figure 3 C) larval length ( Figure 3 E) and regeneration ( Figure 3 Interestingly, glycosylated ginsenosides F2 and Rb1 also did not show any effect on glucose ( Figure 3 A), but they also did not affect cortisol concentrations ( Figure 3 C) larval length ( Figure 3 E) or tissue regeneration ( Figure 3 F, G). After injury, glycosylated ginsenosides Rb1 and F2 did show minor effects on cortisol and tissue regeneration, which were GBA2-dependent, as demonstrated using the chemical GBA2 inhibitor MZ31 ( Figure 3 D, F, H), which may indicate that trauma increases the activity of Gba2, which then hydrolyzes Rb1 and F2.

[0246] The remaining question was why PPD did not affect glucose levels, while it showed similar (although sometimes smaller) effects as Bec on all other readouts. As it is known that the increase in glucose concentration induced by glucocorticoids is solely dependent on the transcriptional activation activity of the GR, we hypothesized that PPD binding to the Gr would not induce this transcriptional activation activity. To test this hypothesis, we used a reporter strain of fish in which the GFP gene is fused to a promoter containing a GRE. Treatment of larvae from this strain with Bec resulted in a massive increase in the GFP signal throughout the body of the larvae, whereas PPD (as well as F2 and Rb1) did not cause an increase in fluorescence intensity ( Figure 3 G, I). In addition, PPD did not increase the transcriptional activation activity of Gr, which is known to target genes fkbp5, pck1 (which plays an important role in the glucocorticoid-induced increase in cortisol concentrations), and nfkbiaa ( Figure 4 B) expression, confirming that PPD does not induce the transcriptional activation activity of GR.

[0247] This suggests that the side effects of PPD are relatively minor, as there is no induction of Gr transcriptional activation activity, and that these side effects disappear after glycosylation of the aglycones ginsenosides. Taken together with the observed effects of ginsenosides on the inflammatory response, these results suggest that glycosylation of ginsenosides inactivates these compounds and that they can be converted to their active aglycone form by hydrolysis by the enzyme GBA2, the activity of which is specifically increased in inflamed tissues.

[0248] Example 5: GBA2 abundance and activity are locally increased at sites of inflammation

[0249] To test whether GBA2 activity is increased in inflamed tissues, we injured the caudal fin of zebrafish larvae and measured gba1 and gba2 mRNA concentrations, GBA1 and GBA2 protein levels, and GBA2 activity levels. The results showed that trauma increased gba2 mRNA levels in zebrafish larvae ( Figure 5 A) Gba2 protein level ( Figure 5 B and 6A) and Gba2 enzyme activity ( Figure 5 D and 6C). To investigate whether this increase was confined to the inflamed tissue at the wound site, the larvae were divided into anterior and posterior parts (the latter containing the wound area). The wound-induced increase in GBA2 protein levels was observed only in the posterior part of the body but not in the anterior part ( Figure 5 C and 6B). No effects of trauma on gba1 mRNA were observed ( Figure 5 A) and GBA1 protein levels ( Figure 5 B and 6A). These data suggest that the concentration and activity of GBA2 are specifically increased at sites of inflammation.

[0250] Example 6: Effect of dexamethasone glucuronidation on anti-inflammatory activity and side effects

[0251] Since glycosylation of ginsenoside PPD eliminates its side effects, we hypothesized that novel glucocorticoid drugs with reduced side effects could be developed by conjugating sugar groups to synthetic glucocorticoid drugs such as dexamethasone. A glucuronidated form of dexamethasone (i.e., dexamethasone with glucuronic acid conjugated to the C21 position) is commercially available (GDex), in which the glucose group is conjugated at the C-21 position via a β-glycosidic bond. This compound is being developed as a drug for inflammatory bowel disease on the basis that it is hydrolyzed by β-glucosidases in the gut microbiota. First, we determined the anti-inflammatory effects of GDex using our zebrafish caudal fin trauma model. The results showed that GDex, like Bec and Dex, inhibited neutrophil migration without having any effect on macrophage migration ( Figure 7 A). This inhibitory effect of Bec, Dex, and GDex on neutrophil migration was absent in larvae of the gr mutant strain, indicating that the effects of these compounds are mediated by Gr. Furthermore, GDex strongly reduced the wound-induced increase in the expression of the proinflammatory genes il1b, il6, mmp9, and mmp13, an effect similar to the reduction observed after Dex treatment ( Figure 7 C). To investigate possible side effects of GDex, we measured glucose and cortisol levels and larval length after treatment with Bec, Dex, and GDex. Like Bec, Dex increased glucose concentrations and decreased cortisol levels and larval length, whereas GDex showed only a very slight decrease in cortisol levels ( Figure 7 D, E, F). Moreover, like glycosylated ginsenosides, GDex did not induce the transcriptional activation activity of Gr, whereas Bec and Dex did, as shown using the GRE:GFP reporter line of fish ( Figure 7 G) and the expression levels of Gr genes fkbp5, pck1 and nfkbiaa ( Figure 7 These data suggest that GDex acts similarly to glycosylated ginsenosides Rb1 and F2, meaning that it is inactive outside of inflamed tissues but is active at these sites of inflammation.

[0252] Example 7: Effect of glycosylation of prednisolone on anti-inflammatory activity and side effects

[0253] To investigate whether similar modifications to the glucocorticoid drug prednisolone (Pdn) would result in the same reduction in side effects, we synthesized a glycosylated form of prednisolone (GPdn) in which a glucose group was conjugated at the C-21 position of Pdn. Using our zebrafish caudal fin trauma model, we showed that GPdn inhibited neutrophil migration, similar to Bec, Dex, GDex, and Pdn ( Figure 8 A). Like GDex, GPdn did not increase glucose levels (while Bec, Dex, and Pdn did, Figure 8 B), and only slightly reduced Cortisol concentrations (whereas Bec, Dex, and Pdn caused significantly greater reductions, Figure 8 D) Under trauma conditions, GPdn caused a slight increase in glucose concentration (although this effect was still significantly smaller than that of Pdn, Figure 8 C), and the decrease in cortisol concentration was greater than that in uninjured larvae (and this effect was still smaller than that of Pdn, Figure 8 E) These additional side effects observed after wounding were reduced after treatment with MZ31, indicating that these effects are dependent on GBA2-mediated conversion of GPdn to Pdn at the wound site.

[0254] Interestingly, the inhibitory effects of GDex and GPdn on neutrophil migration were independent of GBA2 activity, unlike the effects of glycosylated ginsenosides, as demonstrated using the GBA2 inhibitor MZ31 ( Figure 8 A). This suggests that, like PPD, the uncleaved form is still able to bind and activate Gr, sufficient to reduce neutrophil migration. It is therefore reasonable to assume that conjugation of a single monosaccharide is insufficient to inactivate glucocorticoids and that larger, more bulky modifications are required. We therefore synthesized a second glycosylated form of prednisolone in which the sugar group conjugated to the C-21 position is formed by the disaccharide gentiobiose. The structures of Pdn, GPdn, and this gentiobiose-glycosylated prednisolone (GbPdn) are shown in Fig. 9A. Treatment of zebrafish larvae with GbPdn results in a reduction in neutrophil migration after caudal fin trauma ( Fig. 9 B) and inhibition of the expression of pro-inflammatory genes il1b, il6, and il8 ( Fig. 9 C), and these effects were similar to those observed with Pdn and GPdn treatments ( Fig. 9 B, C). MZ31 abolished the effect of GbPdn, but not GPdn, on neutrophil migration, indicating that GbPdn is an inactive molecule that requires hydrolysis by GBA2 to remove the bulky gentiobiose group.

[0255] Furthermore, GbPdn did not affect glucose or cortisol concentrations ( Fig. 9 B, D), nor does it affect the length of the larvae ( Fig.10 A). A small effect on caudal fin regeneration was observed. This effect was significantly smaller than that of Pdn and was dependent on Gba2 activity ( Fig.10 D). Under traumatic conditions, both GPdn and GbPdn induce an increase in glucose levels ( Fig. 9 B) and a decrease in cortisol concentrations ( Fig. 9 D), which may be the result of increased GBA2 activity. Using the zebrafish GRE:GFP reporter line, we showed that neither GPdn nor GbPdn induced the transcriptional activation activity of Gr ( Fig.10 E, G), and the lack of this effect was confirmed when we investigated the expression of the Gr target genes fkbp5, pck1, and nfkbiaa ( Fig.10 H). Apparently, conjugation of monosaccharides is sufficient to abolish the ability of Pdn to induce Gr transcriptional activation activity and eliminate nearly all glucocorticoid side effects in this model.

[0256] To demonstrate whether the side effects of glycosylated glucocorticoids are caused by a lack of GBA2 activity outside of inflamed tissues, we overexpressed GBA2 in whole zebrafish larvae by injecting a plasmid containing the gba2 cDNA driven by a constitutively active CMV promoter. qPCR analysis confirmed the overexpression, showing a significant increase in gba2 mRNA concentration ( Fig.10 B). When Gba2 was overexpressed systemically, Pdn, GPdn, and GbPdn showed similar adverse effects on glucose and cortisol concentrations and transcriptional activation. Using the GRE:GFP reporter line ( Fig.10 F, G and by studying the expression of Gr target genes fkbp5, pck1 and nfkbiaa ( Fig.10I) demonstrated the latter effect. These data suggest that the lack of GBAa2 activity in most of the body ensures that glycosylated glucocorticoids remain in their inactive glycosylated state, in which they cannot cause harm to the body.

[0257] Example 8: In vitro studies of GR activation in human cells

[0258] The in vitro binding of Dex, Pdn, GPdn, and GbPdn to human GR was studied using a competitive ligand binding assay. The results showed that Bec and Pdn had similar binding affinities (IC50 of about 4.8 and about 12 nM, respectively). Pdn had a slightly lower affinity (IC50 of about 350 nM), but GbPdn had a significantly lower affinity (IC50 of about 17 mM) ( Fig.11 A). Apparently, glucosylation of Pdn resulted in an approximately 30-fold decrease in the binding affinity of the ligand, whereas gentiobiosylation reduced the affinity by approximately 1400-fold. Using HeLa cell cultures, the translocation of GR to the nucleus was determined following administration of Pdn, GPdn, and GbPdn. The greatest nuclear translocation was observed following treatment with Pdn and GPdn, whereas GbPdn only slightly increased the level of translocation compared to vehicle treatment ( Fig.11 B, 12A). These data suggest that gentiobiosylation may be advantageous over glucosylation when designing prodrugs, as conjugation of the disaccharide results in a greater degree of inactivation of the steroid. Interestingly, upon co-treatment with the proinflammatory cytokine TNF-α GbPdn, similar to Pdn and GPdn, induced complete translocation, and this effect was dependent on GBA2 activity, as it was abolished upon MZ31 treatment ( Fig.11 B).

[0259] The anti-inflammatory effects of GPdn and GbPdn in human cells were investigated by treating HeLa cells with TNF-α. This treatment induced the expression of the pro-inflammatory gene IL1B ( Fig.11 C) IL8( Fig.11 D) and MMP13 ( Fig.12 B), all three genes were strongly repressed by Pdn. GPdn and GbPdn similarly inhibited the TNF-α-induced increase in the expression of these genes. For GbPdn, the inhibition of IL1B and IL8 was dependent on the activity of GBA2, as its effect was reduced after MZ31 treatment. These results are consistent with the GBA2 dependence of the anti-inflammatory effects of GbPdn that we observed in zebrafish.

[0260] The expression levels of GR target genes FKBP5 and NFKBIA, GILZ, and SGK1 were measured to investigate the transcriptional activation activity of GR in HeLa cells. Pdn strongly increased the expression of these genes, whereas GPdn and GbPdn did not increase or only moderately increased their expression levels ( Fig.11 TNF-α treatment barely altered the expression of these genes in GPdn-treated cells but did increase the expression of these genes in GbPdn-treated cells, an effect that was Gba2-dependent as it was strongly reduced by MZ31 ( Fig.11 E, F, 12C, D). This effect of GBA2 was confirmed in our experiments using lentivirus-mediated shRNA knockdown of GBA1 or GBA2. GBA2 knockdown, but not GBA1 knockdown, resulted in the inhibition of TNF-α-induced increase in FKBP5 and NFKBIA expression ( Fig.11 G, 12E). Finally, we overexpressed GBA2 by transfecting HeLa cells with a plasmid containing a CMV:GBA2 DNA construct. In these GBA2-overexpressing cells, Pdn, GPdn, and GbPdn similarly increased the expression levels of FKBP5 and NFKBIA. These data suggest that TNF-α increases the activity of GBA2, which increases transcriptional activation of genes after treatment with GbPdn, but has very limited effects after treatment with GPdn.

[0261] Example 9: Collagen antibody-induced arthritis (CAIA) mouse experiment

[0262] The induced arthritis assay described in the "Assays" section above was performed to investigate the effects of GbPdn in a mammalian model of inflammatory disease. This used the collagen antibody-induced arthritis (CAIA) model in mice, where arthritis was induced by injection of an anti-collagen antibody cocktail on day 1 and LPS on day 4 ( Fig.13 Subsequently, we treated mice daily with vehicle, Pdn (12.5 or 25 mg / kg), or GbPdn (12.5, 25, or 50 mg / kg). Arthritis scores were determined daily from day 5 to day 15. Vehicle-treated mice reached high arthritis scores (approximately 15), and these levels remained stable until day 14 ( Fig.14 A). In contrast, the scores of the Pdn and GbPdn treated groups of mice decreased rapidly after the start of treatment. The group treated with Pdn showed the greatest reduction (scores of approximately 0) on day 11 of the experiment, while the groups treated with 12.5, 25 and 50 mg / kg of GbPdn induced the greatest effects on days 15, 14 and 13, respectively ( Fig.14 A).

[0263] As a second readout of the inflammatory response in these animals, we measured serum IL-6 levels on days 10 and 15. The results of this experiment showed that IL-6 levels were significantly reduced in all treatment groups compared to the vehicle group, with the 12.5 mg / kg GbPdn group showing slightly higher IL-6 concentrations than the other groups treated with Pdn or GbPdn ( Fig.14 B). On day 15, IL-6 levels were undetectable in the serum of all groups treated with Pdn or GbPdn, except in the 12.5 mg / kg GbPdn group, where very low levels were detected in two (out of four) mice in the 12.5 mg / kg GbPdn group. Taken together, these data suggest that GbPdn is a highly effective anti-inflammatory drug that exhibits only delayed activity compared to Pdn.

[0264] To investigate whether GbPdn induces side effects in arthritic mice, we investigated the effects of our treatment on serum insulin and corticosterone levels (on days 14 and 15, respectively), which are biomarkers of glucocorticoid side effects. Our data showed that Pdn treatment significantly increased insulin levels, but GbPdn treatment did not change insulin concentrations ( Fig.14 C). In addition, Pdn strongly suppressed corticosterone levels, whereas GbPdn had no effect on corticosterone levels ( Fig.14 D). These data suggest that GbPdn does not induce any adverse effects in this animal model, consistent with our hypothesis that this compound is activated only at sites of inflammation.

[0265] Finally, we investigated (using qPCR) the mRNA levels of several GR target genes in liver and muscle tissues as well as in the ankle joint tissues of the hind paw. Our data showed that after Pdn treatment, the levels of Fkbp5, Pck1, and Gilz mRNA in liver tissues ( Fig.14 E) The levels of Redde1, Foxo3 and Klf15 mRNA in muscle tissue ( Fig.14 F) and the levels of Gilz and Fkbp5 mRNA in joints ( Fig.14 G) were increased in the joints. Interestingly, GbPdn treatment did not affect the expression of the GR target genes studied in the liver or muscle tissue. However, in the GbPdn-treated group, the mRNA levels of Gilz and Fkbp5 increased in the joints, indicating that inflammation in this tissue leads to the local conversion of GbPdn to Pdn and subsequent activation of GR.

[0266] Example 10: Mass spectrometric confirmation of gentiobiose cleavage

[0267] The liquid chromatography / mass spectrometry cleavage confirmation assay described in the "Assay" section above was performed to investigate whether GbPdn is converted to Pdn by Gba2 in one step, or whether the two glucose groups are cleaved separately. Concentration levels of GbPdn, Pdn, and the possible intermediate metabolite glucose-prednisolone (GPdn) were determined in injured and uninjured zebrafish larvae after treatment with 25 μM GbPdn for 6 h (2 h before and 4 h after trauma). The experiment was performed in wild-type zebrafish in the presence or absence of MZ31, and in larvae from the gba2- / - strain. The results of this experiment showed that GbPdn was primarily converted to Pdn, and this was only observed in injured larvae ( Fig.15 A). Interestingly, the high rate of conversion of GBPdn to Pdn in injured larvae was almost completely abolished in the presence of MZ31 ( Fig.15 A) and was not observed in injured gba2- / - larvae ( Fig.15 B). These results suggest that this transformation is dependent on inflammation-induced Gba2 activity. Notably, very low concentrations of GPdn ( Fig.15 A, B), indicating that GbPdn is barely converted to GPdn but that Gba2 removes the entire gentiobiose group from GbPdn in a single step.

Claims

1. Compounds of formula I: AB (I) in: A is selected from monosaccharides and disaccharides; and B is selected from an anti-inflammatory moiety or a chemotherapeutic moiety; or a pharmaceutically acceptable salt, stereoisomer, acid or solvate thereof.

2. The compound according to claim 1, wherein A is selected from glucose, galactose, mannose, fructose and any acid form thereof, and any disaccharide formed by one or two of glucose, galactose, mannose, fructose and any acid form thereof.

3. The compound according to claim 1 or 2, wherein A is a disaccharide formed from one or two of glucose, galactose, mannose, fructose and any acid form thereof; optionally, wherein the disaccharide is gentiobiose.

4. The compound according to claim 1 or 2, wherein A is a disaccharide formed by two aldohexoses; optionally, wherein A is a disaccharide formed by one or two of glucose, galactose, idose, altrose and any acid form thereof.

5. The compound according to claim 1 or 2, wherein A is a monosaccharide selected from glucose, galactose, mannose, fructose and any acid form thereof; optionally, wherein the monosaccharide is glucose or glucuronic acid.

6. A compound according to any one of the preceding claims, wherein B is an anti-inflammatory moiety.

7. The compound of claim 6, wherein the anti-inflammatory moiety is or comprises a corticosteroid (such as a glucocorticoid), a nonsteroidal glucocorticoid receptor agonist, a nonsteroidal anti-inflammatory drug, a disease-modifying antirheumatic drug, or rapamycin.

8. A compound according to any one of the preceding claims, wherein B is or comprises a corticosteroid (such as a glucocorticoid) selected from the group consisting of hydrocortisone-type corticosteroids, triamcinolone-acetonide-type corticosteroids, methasone-type corticosteroids, betamethasone dipropionate-type corticosteroids and methylprednisolone acetonide-type corticosteroids.

9. The compound according to any one of the preceding claims, wherein B is or comprises a molecular group consisting of: alclomethasone, amcinonide, beclomethasone, betamethasone, budesonide, cloprednisone, cyclomethasone, ciclesonide, clobetasol, clobetasone, clocortolone, clopredniol, cortifen, cortisol (hydrocortisone), cortisone, cortivazole, cortodoxone, deflazacort, desonide, desoximetasone, dexamethasone, diclofenac, diflucortolone, diflorasone, difluprednate, formocort, flurazepam, flucloroxolone, fludrocortolone fluocortolone, fluocortidine, fluocortolone, flumethasone, flunisolide, fluocinolone, fluocinolone acetate, fluocinolone, fluorometholone, fluperone, fluprednidine, fluprednisolone, flurandrenolide, fluticasone, halcinonide, halobetasol, halometasone, hydrocortamide, icomethasone, isofluprednisolone, ethanoprednol, maprednione, medrysone, methylprednisone, methylprednisolone, mometasone, nicronide, paramethasone, prednicarbate, prednisolone, prednimustine, prednisone, prednisolone, tixocortolone, triamcinolone or triamcinolone acetonide or any ester thereof.

10. The compound according to claim 9, wherein the compound is an ester thereof, optionally wherein the ester comprises a group selected from acetate, adamantanoate, benzoate, benzofurancarboxylate, benzoyl-β-aminoisobutyrate, butyrate, butyl, hexanoate, carboxylate, chlorambucil, chlorobenzoyl, pyroacetate, cyclopentanepropionate, cyclopentylpropionate, cyclopropylcarboxylate, cyclopentylpropionate, diazepam, dichloroacetate, diethylaminoacetate, heptanoate, butyl acetate, ethyl carbonate (ethyl carbonate), furoate, hemisuccinate, hexanoate, isobutyrate, isonicotinate, linoleate, m-sulfobenzoate, pamoate, palmitate, phosphate, piperidine acetate, pivalate, propionate, salicylate, stearoylglycolate, succinate, sulfoheptyl, sulfate, tert-butyl acetate (tert-butyl acetate), tetrahydrophthalate, kojic acid ester, valerate or xanthate.

11. A compound according to any one of the preceding claims, wherein B is a chemotherapeutic moiety.

12. Compounds of formula II: in: R 1 Selected from monosaccharides or disaccharides; R 2 is selected from H, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C3-C8 alkenyl, R 3 and R 4 Each independently selected from H, OH, C1-C4 substituted or unsubstituted alkyl, OR 9 、OC(O)R 9 ; or R 3 and R 4 Together with the carbon atoms to which they are attached, they form a substituted or unsubstituted 5- or 6-membered heterocycloalkyl group; R 5 , R 6 , R 7 and R 8 Each is independently selected from H, OH, halogen, substituted or unsubstituted -C1-C4 alkyl or OR 10 ; R 9 is selected from H or -C1-C8 substituted or unsubstituted alkyl; and R 10 Selected from H or -C1-C8 substituted or unsubstituted alkyl; X is selected from =O or -OH; or a pharmaceutically acceptable salt, stereoisomer, acid or solvate thereof.

13. The compound according to claim 12, wherein R 1 The sugar is selected from glucose, galactose, mannose, fructose and any acid form thereof, and any disaccharide formed from one or two of glucose, galactose, mannose, fructose and any acid form thereof.

14. The compound according to claim 12 or 13, wherein R 1 It is a disaccharide formed by one or two of glucose, galactose, mannose, fructose and any acid form thereof.

15. The compound according to claim 12 or 13, wherein R 1 is a disaccharide formed by two aldohexoses and any of their acid forms, optionally wherein R 1 It is a disaccharide formed by one or two of glucose, galactose, idose and altrose.

16. A compound according to any one of claims 12 to 15, wherein R 1 It's gentiobiose.

17. The compound according to claim 12 or 13, wherein R 1 is a monosaccharide selected from glucose, galactose, mannose, fructose and any acid form thereof; optionally, wherein the monosaccharide is glucose or glucuronic acid.

18. A compound according to any one of claims 12 to 17, wherein R 2 is selected from H and substituted or unsubstituted C1-C8 alkyl; optionally, wherein R 2 It's H.

19. A compound according to any one of claims 12 to 18, wherein R 3 Selected from H, OH and OC(O)R 9 .

20. A compound according to any one of claims 12 to 19, wherein R 9 is a substituted or unsubstituted -C1-C8 alkyl group; optionally, wherein R 8 It is an unsubstituted -C1-C8 alkyl group.

21. A compound according to any one of claims 12 to 20, wherein R 4 is selected from H, OH or C1-C4 alkyl; optionally, wherein R 4 Selected from H, OH or methyl.

22. A compound according to any one of claims 12 to 18, wherein R 3 and R 4 Together with the carbon atoms to which they are attached, they form a substituted or unsubstituted 5- or 6-membered heterocycloalkyl group.

23. A compound according to any one of claims 11 to 17 or 21, wherein R 3 and R 4 Together with the carbon atom to which they are attached, they form a substituted or unsubstituted 1,3-dioxolane ring.

24. The compound according to claim 23, wherein the 1,3-dioxolane is substituted at position 2 by 1 or 2 groups selected from -C1-C4 alkyl (such as methyl), 5- or 6-membered cycloalkyl or 5- or 6-membered heterocycle (such as furan).

25. A compound according to any one of claims 12 to 17, wherein R 2 Yes H, R 3 Yes OH, R 4 It's H.

26. A compound according to any one of claims 12 to 17, wherein R 2 Yes H, R 3 and R 4 Together with the carbon atom to which they are attached, they form a substituted or unsubstituted 1,3-dioxolane ring.

27. A compound according to any one of claims 12 to 17, wherein R 2 Yes H, R 4 is C1-C4 alkyl; optionally, wherein R 2 Yes H, R 4 It's methyl.

28. A compound according to any one of claims 12 to 17, wherein R 2 Yes H, R 3 It is OC(O)R 9 , R 4 is H; optionally, wherein R 9 It is a substituted or unsubstituted -C1-C8 alkyl group.

29. A compound according to any one of claims 12 to 28, wherein R 10 It's H.

30. A compound according to any one of claims 12 to 29, wherein R 5 is selected from H and halogen; optionally, wherein R 5 Selected from H, F and Cl.

31. A compound according to any one of claims 12 to 30, wherein R 8 is selected from H and halogen; optionally, wherein R 8 Selected from H, F and Cl.

32. A compound according to any one of claims 12 to 31, wherein R 6 and R 7 Each is H.

33. A compound according to any one of claims 12 to 32, wherein X is OH.

34. A compound according to any one of claims 12 to 32, wherein X is O.

35. Compounds of formula (Vb): in: R 1 Selected from monosaccharides or disaccharides and any acid form thereof; R 2 Selected from H, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C1-C8 alkoxy, substituted or unsubstituted C3-C8 alkenyl; R 3 and R 4 Each independently selected from H, OH, C1-C4 substituted or unsubstituted alkyl, OR 9 、OC(O)R 9 ; or R 3 and R 4 Together with the carbon atoms to which they are attached, they form a substituted or unsubstituted 5- or 6-membered heterocycloalkyl group; R 5 , R 6 , R 7 and R 8 Each is independently selected from H, OH, halogen, substituted or unsubstituted -C1-C4 alkyl or OR 10 ; R 9 is selected from H or -C1-C8 substituted or unsubstituted alkyl; and R 10 Selected from H or -C1-C8 substituted or unsubstituted alkyl; or a pharmaceutically acceptable salt, stereoisomer, acid or solvate thereof.

36. The compound according to claim 35, wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 Any one or more of as further defined in any one of claims 13 to 32.

37. The compound according to claim 35, wherein R 1 It is a disaccharide formed by two aldohexose and any acid form. 2 Yes H, R 3 Yes OH, R 4 is H; optionally, wherein R 6 Yes H, R 7 Yes H, R 8 Yes H, R 9 It's H.

38. The compound according to claim 35 or 37, wherein R 1 is a disaccharide formed from one or two of D-glucose, D-galactose, L-idose, L-altrose and any acid form thereof; optionally, wherein R 1 It's gentiobiose.

39. The compound according to any one of the preceding claims, wherein the compound is a substrate for glucosylceramidase β2 (GBA2).

40. A compound selected from: or a pharmaceutically acceptable salt, stereoisomer, acid or solvate thereof.

41. Compounds: or a pharmaceutically acceptable salt, stereoisomer, acid or solvate thereof.

42. A pharmaceutical composition comprising a compound according to any one of the preceding claims.

43. The composition of claim 42, wherein the composition is formulated for non-oral administration, optionally wherein the composition is formulated for parenteral administration, further optionally wherein the composition is formulated for injection.

44. A compound according to any one of claims 1 to 41 or a pharmaceutical composition according to claim 42 or 43 for use as a medicament.

45. A compound according to any one of claims 1 to 42 or a pharmaceutical composition according to claim 42 or 43 for use in treating inflammation.

46. ​​A compound according to any one of claims 1 to 41 or a pharmaceutical composition according to claim 42 or 43 for use in treating an inflammatory condition or other condition characterized by an overactive immune system.

47. A compound or composition according to claim 46, wherein the inflammatory condition or other condition is selected from arthritis, osteoarthritis, gout, rheumatoid arthritis, other autoimmune disorders (such as systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD, including Crohn's disease, ulcerative colitis), multiple sclerosis, chronic inflammatory demyelinating polyneuropathy (CIPD)), infectious diseases (such as COVID-19 and tuberculosis), asthma, chronic obstructive pulmonary disease (COPD), allergic and non-allergic rhinitis and sinusitis, tendonitis, nasal polyps, skin diseases (such as rash, dermatitis, pruritus, eczema, dermatophytosis, lichen (sclerosis or erythema) and psoriasis), eye diseases (such as uveitis, conjunctivitis, macular edema), otitis, thyroiditis, sarcoidosis, myositis, vasculitis, hemorrhoids, transplant recipient organ rejection and graft-versus-host disease.

48. A compound or composition according to claim 46 or 47, wherein the inflammatory or other condition is selected from rheumatoid arthritis, other autoimmune disorders (such as systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD)) and thyroiditis.

49. A compound according to any one of claims 1 to 41 or a pharmaceutical composition according to claim 42 or 43 for use in treating a condition characterized by excessive proliferation of cells belonging to the immune system, optionally wherein the condition is selected from acute lymphocytic leukemia (ALL) or acute myeloid leukemia (AML).

50. A compound according to any one of claims 1 to 41 or a pharmaceutical composition according to claim 42 or 43 for use in the treatment of cancer.

51. according to any one of claims 44 to 50, the compound or composition, wherein the use comprises non-oral administration of the compound or composition to a patient, optionally, wherein the administration comprises parenteral administration, further optionally, wherein the administration comprises injection.

52. Use of a compound according to any one of claims 1 to 41 as an anti-inflammatory agent, comprising contacting the compound with a cell expressing glucosylceramidase β2 (GBA2) (optionally in vitro).

53. The use according to claim 52, wherein the cell is located in a tissue or an organ.

54. The use according to claim 53, wherein the cell is in cell culture.

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