Sulforaphane glycoconjugate compounds, synthesis and uses thereof

Abstract

The present invention relates to sulforaphane glycoconjugate compounds, as well as to the synthesis and therapeutic uses thereof for treating inflammatory diseases.

Description

[0001] DESCRIPTION

[0002] Sulforaphane glycoconjugates, synthesis and uses

[0003] The present invention relates to sulforaphane glycoconjugates, their synthesis, and their therapeutic uses for the treatment of inflammatory diseases. The present invention falls within the field of organic chemistry and biotechnology, specifically in the pharmaceutical sector.

[0004] BACKGROUND OF THE INVENTION

[0005] Sulforaphane (SFN) is widely known for its antioxidant and anti-inflammatory properties. Its chemical structure allows it to act as a potent inducer of phase II detoxification enzymes and a modulator of transcription factors, such as nuclear factor (erythroid factor 2)-related 2 (Nrf2). However, the low bioavailability and stability of SFN have been significant barriers to its direct therapeutic use.

[0006] Regarding the extraction and stability of SFN, numerous studies discuss methods for extracting SFN from cruciferous vegetables and its instability under physiological conditions. In fact, in recent years, since 2020, many studies have discussed in detail different strategies to inhibit SFN degradation and increase its yield. Separation, purification, and determination methods for the compound have been described, but there is still an urgent need to develop new strategies to optimize these processes, both in terms of purification and chemical synthesis, while maintaining its stability. Furthermore, there is also a need to understand its mechanism of action (Men X., et al. Food Sci Biotechnol.).

[0007] 2023 Oct 16;33(3):539-556). Therefore, these data suggest that SFN research is an active and developing area, with a focus on improving extraction and stabilization techniques to maximize the benefits of this compound.

[0008] In addition, other studies address the degradation of SFN and its rapid conversion to inactive forms in the human body. The short half-life of SFN in the body is a topic of great interest to scientific research, and several studies have addressed the rapid degradation of SFN and its conversion to inactive forms, which limits its therapeutic potential. Among the mechanisms involved in this degradation are: myrosinase, an enzyme present in plant cells, catalyzes the hydrolysis of the SFN precursor, glucoraffinin, releasing active SFN. However, myrosinase is inactivated by heat and stomach acid, limiting the availability of SFN after ingestion (Sun J, et al. Food Chem. 2021 Oct 30; 360:130007). Also, SFN is rapidly metabolized in the liver and small intestine through conjugation with sulfate and glucuronide groups, forming inactive metabolites that are excreted in the urine and feces (Yang G, Drug Metab Rev.2017 May;49(2): 105-138), and intestinal bacteria metabolize SFN to compounds such as p-hydroxyphenylpropionic acid (p-HPA) and diindolylmethane acid (DIM), with less biological activity than the original SFN (Santana-Gálvez, J. et al Int. J. Mol. Sci. 2020, 21, 3108).

[0009] Related to the above, several patents describe methods for stabilizing SFN. These include contacting SFN with an analogue and a cyclodextrin to form a stable complex (WG / 2008 / 091608, July 31, 2008, PHARMAGRA LABS, INC.). Other patents also describe combining SFN with a phytosterol and / or phytostanol or its ester, providing different combinations to stabilize the bioactive compound (WO / 2014 / 008341, March 15, 2014, NUTRAMAX LABORATORIES, INC.). Patent EP2919775, November 19, 2013, ROSENGREN ANDERS & AXELSSON ANNIKA, describes SFN as a therapeutic agent for treating or reducing hepatic insulin resistance. Specifically, it provides that SFN reduces the liver's insulin resistance index relative to the index of other metabolic tissues.Related to the biological effect of SFN, the method for preparing magnolol-SFN conjugates and their biological activity have also been described, showing a significantly superior antitumor effect compared to magnolol and sulforaphane separately. These magnolol-SFN conjugates have a broad spectrum of antitumor activity and strong pharmacological potential. Therefore, they are considered antitumor compounds with development potential, and this also provides new ideas and approaches for the development of novel antitumor drugs (EP4083019, 02.11.2022, ZENG YONGCHANG).

[0010] Therefore, the state of the art to date has identified the limitations of SFN in terms of stability and bioavailability, and has proposed various partial solutions. However, it is necessary to provide a more complete and effective solution to significantly improve the therapeutic properties of SFN, especially in the treatment of inflammatory diseases. DESCRIPTION OF THE INVENTION

[0011] The present invention provides sulforaphane glycoconjugates (SFN) with applications in medicine, providing a more effective and safer alternative for the management of chronic inflammatory conditions such as, for example, rheumatoid arthritis and inflammatory bowel disease.

[0012] Sulforaphane (SFN) has the following formula:

[0013]

[0014] Its conjugation with sugars, particularly specific carbohydrates such as mannose and fucose, suitably functionalized, has improved its stability and solubility, reducing cytotoxic effects at high concentrations. Furthermore, it achieves an enhanced anti-inflammatory effect compared to free SFN. Therefore, this conjugation not only improves the physicochemical properties of SFN but also enhances its biological efficacy, offering a complete and effective solution for therapeutic use.

[0015] A first aspect of the present invention relates to a glycoconjugate compound of sulforaphane (SFN), including any of its pharmaceutically acceptable salts, of general formula (I) or (II):

[0016] (YO)

[0017]

[0018] where:

[0019] R 1 is a glycosyl radical. In a preferred embodiment, R 1is fucosyl or mannosyl, more preferably D-mannosyl or L-fucosyl. The glycosyl group may be attached to the rest of the molecule by the carbon 1 or anomeric carbon of the glycosyl group or by a carbon other than the anomeric carbon, preferably attached to the rest of the molecule by the anomeric carbon of the glycosyl group;

[0020] And it can be O or S; and

[0021] n can be 0 or 1.

[0022] In a more preferred embodiment of the compound of the invention (I) n is 0 and more preferably the compound is selected from the following:

[0023]

[0024] In another preferred embodiment, n is 1 in the compound of formula (I) and Y can be O or S. In a more preferred embodiment, the compound is selected from the following:

[0025]

[0026] In another preferred embodiment of the present invention, the compound of formula (II) is O, more preferably the compound of formula (II) has the following structure:

[0027]

[0028] Another aspect of the present invention relates to the procedure for obtaining the compounds of the invention.

[0029] In particular, the synthesis of the compounds of the invention of formula (I), when n= O, is carried out by coupling between a glycosylisothiocyanate (R 1 -NCS), suitably protected, and 4-(methylsulfinyl)butan-1-amine, as shown in the following scheme:

[0030] R 1 -NCS

[0031]

[0032] where: R 1It is a glycosyl as previously defined with all -OH groups protected and the -NCS group may be attached to the carbon 1 or anomeric position of the glycosyl group or to a carbon other than the anomeric carbon, preferably attached to the anomeric carbon of the glycosyl group.

[0033] Following coupling, the -OH groups of the glycosyl are deprotected, preferably with NaOMe in MeOH.

[0034] In a preferred embodiment, the glycosylisothiocyanate can be selected from the following compounds:

[0035] OAc ^.

[0036] i OAc

[0037] AcO

[0038] AcO

[0039]

[0040] NCS AcO In another preferred embodiment, the compound 4-(methylsulfinyl)butan-1-amine is obtained by reaction of tert-butyl (4-(methylsulfinyl)butyl) carbamate with trifluoroacetic acid in dichloromethane.

[0041] When n= 1 the synthesis of the compounds of formula (I) of the invention is carried out by coupling the glycosyl compounds functionalized with an amine group of structure R 1 -Y-(CH2)2-NH2 with SFN in a solvent, preferably dichloromethane, followed by deprotection, preferably with NaOMe in MeOH; where: Y has been previously defined, and R 1 It is a glycosyl as previously defined with all -OH groups protected and the glycosyl can be attached by the carbon 1 or anomeric position of the glycosyl to group Y or by a carbon other than the anomeric carbon to group Y, preferably attached by the anomeric carbon of the glycosyl to group Y.

[0042] In a preferred embodiment, the glycosyl compounds of R structure 1 -Y-(CH2)2-NH2 are selected from the following compounds:

[0043] _ NH2

[0044] AND

[0045]

[0046] where: And has been previously defined.

[0047] The compounds of the invention of formula (II) can be synthesized by the following steps: the reaction of the SFN in a solvent, preferably dichloromethane, with the multivalent platform (1,3-bis(prop-2-yn-1-yloxy)-2-((prop-2-yn-1-yloxy)methyl)propan-2-amine) to yield 1-(1,3-bis(prop-2-yn-1-yloxy)-2-((prop-2-yn-1-yloxy)methyl)propan-2-yl)-3-(4-(methylsulfinyl)butyl)thiourea. The subsequent copper-catalyzed (CuAAC) 1,3-dipolar cycloaddition reaction with the corresponding glycosyls functionalized with an azide group of structure R 1 -Y-(CH2)2-Ns, followed by deprotection, preferably with NaOMe in MeOH as shown in the following scheme: 1) R 1 -Y-(CH2)2-N3

[0048] 2) lack of protection

[0049]

[0050] where: R 1e and Y have been previously defined in glycosyls functionalized with an amine group.

[0051] This conjugation process significantly improves the stability and solubility of the SFN, crucial aspects for its efficacy in therapeutic applications. The resulting glycoconjugates, i.e., the compounds of formula (I) or (II) of the present invention, exhibit increased solubility in aqueous solutions, which has been validated in in vitro studies, and show no toxic effects at high concentrations, allowing the concentration used to be doubled without cytotoxic effects.

[0052] From a biological standpoint, the SFN glycoconjugates of formula (I) or (II) of the present invention exhibit an enhanced anti-inflammatory effect compared to free SFN, where these sugars act as adjuvants, thus improving the solubility and effects of SFN. These glycoconjugates are able to inhibit the nuclear factor kappa light chain enhancer (NF-κB) signaling pathway and modulate dendritic cells (DCs) more effectively. Inhibition of the NF-κB pathway results in more efficient DC maturation, suggesting a specific and novel mechanism of action. Furthermore, the SFN glycoconjugates induce a more potent and specific immune response, increasing IL-10 production and the activation of regulatory T cells (Tregs).This effect is achieved thanks to functionalization with saccharides that interact with C-type lectin receptors (CLRs), such as the CDs-specific non-integrin intercellular adhesion receptor-3 (DC-SIGN), on CDs, facilitating their cellular entry through specific recognition.

[0053] Interaction with these receptors allows the glycoconjugates of the invention to induce a mature and fully activated state in the CDs, a finding that represents a significant advance.

[0054] Therefore, another aspect of the present invention relates to a compound of formula (I) or (II) as described in the first aspect of the present invention, for use as a medicament.

[0055] The SFN glycoconjugates of the present invention have applications in the treatment of various inflammatory diseases, including autoimmune diseases, chronic inflammatory diseases, and cancer. Their improved stability and solubility, along with their enhanced safety profile and greater biological efficacy, make them ideal candidates for the development of advanced therapies. Furthermore, their ability to modulate the immune response makes them suitable for applications in immunotherapy and vaccine design.

[0056] Another aspect of the invention relates to a compound of formula (I) or (II) as described in the first aspect of the present invention, for use in the treatment and / or prevention of inflammatory diseases, autoimmune diseases, or cancer, preferably selected from breast cancer, prostate cancer, colon cancer, and lung cancer. Autoimmune diseases are characterized by a dysregulated immune response that attacks the body's own tissues, causing chronic inflammation. Because SFN glycoconjugates possess anti-inflammatory and immune-modulating properties, they are particularly useful in the treatment of various autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, and type 1 diabetes. Furthermore, they are effective in the management of inflammatory bowel diseases, including Crohn's disease and ulcerative colitis.

[0057] Another aspect of the present invention relates to a pharmaceutical composition comprising a compound of formula (I) or (II) as described in the first aspect of the present invention, together with a pharmaceutically acceptable excipient and / or vehicle. Furthermore, the invention relates to a method of treating an inflammatory, autoimmune, or cancerous disease as previously described, comprising administering to a subject a therapeutically effective amount of a compound of formula (I) or (II) as described in the first aspect of the present invention.

[0058] A final aspect of the present invention relates to the use of a compound of formula (I) or (II) as described in the first aspect of the present invention for the preparation of a medicament for the treatment and / or prevention of inflammatory, autoimmune diseases or cancer as previously described.

[0059] The compounds of the invention of formula (I) or (II) may be in the form of free compounds or as solvates, and it is intended that both forms are within the scope of the present invention. In this sense, the term “solvate,” as used herein, includes pharmaceutically acceptable solvates, i.e., solvates of the compound that can be used in the preparation of a medicament. In one particular embodiment, the solvate is a hydrate. The solvates can be obtained by conventional solvation methods well known to those skilled in the art.

[0060] The compounds of formula (I) or (II) of the present invention for therapeutic use are prepared in solid form or aqueous suspension in a pharmaceutically acceptable diluent. These preparations may be administered by any appropriate route of administration, for which purpose the preparation shall be formulated in the pharmaceutical form suitable for the chosen route of administration. In one particular embodiment, the administration of the compound of formula (I) or (II) is effected orally, topically, rectally, or parenterally (including subcutaneous, intraperitoneal, intradermal, intramuscular, intravenous, etc.).

[0061] The compounds described in the present invention, their pharmaceutically acceptable salts, solvates, and pharmaceutical compositions containing them can be used in conjunction with other drugs to provide combination therapy. These additional drugs may form part of the same pharmaceutical composition or, alternatively, may be provided as a separate composition for administration concurrently or separately with the pharmaceutical composition comprising a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof. The present invention covers all isomers of the compounds of formula (I) or (II), including all possible stereoisomers. The different isomeric forms can be separated or resolved from each other by conventional methods, or any given isomer can be obtained by conventional synthetic methods or by stereospecific, stereoselective, or asymmetric synthesis.

[0062] Definitions according to the present invention:

[0063] The term "glycoconjugate" refers to any carbohydrate-derived compound covalently bonded to another molecule, which can be a protein, lipid, or other compound.

[0064] The term “glycosyl” or “glycosyl” in the present invention refers to a radical resulting from the removal of a hydroxyl group (OH) from a natural or synthetic saccharide or sugar, and which is attached to the rest of the molecule through the carbon that has lost the hydroxyl group. In a preferred embodiment of the invention, the glycosyl radical is attached to the rest of the molecule through the anomeric carbon (C-1).

[0065] The term “treatment or prevention” as used herein, unless otherwise indicated, means to reverse, alleviate, inhibit the progression of, or prevent the disorder or condition to which it applies in such terms, or one or more symptoms of such disorder or condition.

[0066] The term “excipients, adjuvants, and / or vehicles” refers to molecular entities or substances with which the active ingredient is administered. Such excipients, adjuvants, or pharmaceutical vehicles may be sterile liquids, such as water and oils, including petroleum-based or those of animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and similar products; excipients; disintegrants; wetting agents; or diluents. Suitable pharmaceutical excipients and vehicles are generally known to a person skilled in the art.

[0067] The term “therapeutically effective amount” means the amount of a compound necessary for the treatment or prevention of the disease, disorder, or condition to be effective. Throughout the description and claims, the word “comprises” and its variants are not intended to exclude other technical features, additives, components, or steps. For those skilled in the art, other objects, advantages, and features of the invention will become apparent partly from the description and partly from the practice of the invention. The following examples and figures are provided for illustrative purposes and are not intended to limit the scope of the present invention.

[0068] BRIEF DESCRIPTION OF THE FIGURES

[0069] Fig. 1.- Shows a significant improvement in the cytotoxicity of the compounds of the invention with respect to free SFN.

[0070] Fig. 2.- Shows the maturation (CD80, CD83, CD86, HLA-DR and PD-L1) of the CDs treated with free SFN against the compounds of the invention.

[0071] Fig. 3.- Shows the inhibition of the p65 NF-KB pathway in the presence of the compounds of the invention.

[0072] EXAMPLES

[0073] The invention will then be illustrated by means of tests carried out by the inventors, which demonstrate the effectiveness of the product of the invention.

[0074] 1. Synthesis of compounds of formula (I) and (II):

[0075] Parameters and characteristics: The spectra of 1 H- and 13¹³C NMR spectra were recorded using Bruker AVIII300 and NE0300 spectrometers for solutions in CDCh and CD3OD. Chemical shift values ​​(5) are given in ppm, and coupling constant values ​​( ) are given in Hz. Spectra were calibrated using the residual solvent signal (CDCI3: 5(H) = 7.26, 5(C) = 77.16; CD3OD: 5(H) = 3.31, 5(C) = 49.00). Signal assignment was confirmed by 2D NMR spectra (COSY and HSQC). High-resolution mass spectra (HRESIMS) were recorded on a Q-Exactive spectrometer. Analytical thin-layer chromatography (TLC) was performed for qualitative purposes using silica gel 60 F254 (Merck). The chromatographic plates were developed by exposure to UV light followed by development with ninhydrin, [(NH4)eMoO4, Ce(SÜ4)2, H2SO4, H2O] reagent, or p-anisaldehyde. Silica gel 60 (Merck, 40-60 and 63-200 pm) was used for the purification of compounds by column chromatography.

[0076] Concentrations of reagents: For the synthesis of glycoconjugates, the reagents and solvents were used directly from the commercial source without prior purification or distillation, in the quantities indicated at each stage of the experimental development.

[0077] SFNMan and SFNFuc synthesis scheme:

[0078] Chen, X. et al, Synthesis, 2011, 24, 3991-3996 HO^^ NH2 x- s x^^ NH BQc 1) MCPBA, CH2CI2, -10 ° C>

[0079] 2) TFA, CH2CI2, ta

[0080] 1

[0081] 1 ) AC2O, l2t.a. OH 2) HBr / AcOH, 0 °C 1)(2), CH2CI 2i Et3N, ta

[0082] 3) NBU4I, KSCN, MeCN, 4A MS, reflux 2) NaOMe, MeOH, ta

[0083]

[0084] L-Fucose

[0085] Synthesis of derivative 2

[0086] A solution of meta-chloroperbenzoic acid (MCPBA) (1.35 g, 5.47 mmol) in CH2Cl2 (10 mL) was added dropwise to a solution of compound 1 (Chen, X. et al., Synthesis, 2011, 24, 3991–3996) (1.20 g, 5.47 mmol) in CH2Cl2 (10 mL) previously cooled to -10 °C, and the resulting mixture was stirred at -10 °C for 30 min. After this time, the reaction crude was washed twice with a saturated aqueous solution of NaHCO3 and water. The aqueous phase was extracted three times with CH2Cl2, and the collected organic phases were washed with a saturated aqueous solution of NaCl, dried over Na2SO4, filtered, and concentrated to dryness. The residue obtained was purified by silica gel column chromatography (CH2CI2 -* Ch C^MeOH 20:1) obtaining the corresponding sulfoxide (1.26 g, 5.35 mmol, 98%) in the form of oil.To a solution of this compound (250 mg, 1.06 mmol) in CH2CI2 (5 mL), thinfluoroacetic acid (TFA) (1.7 mL) was added and the reaction mixture was stirred at room temperature (ta) for 2.5 h. Subsequently, the crude was concentrated to dryness to obtain compound 2 (450 mg, 1.06 mmol, quantitative) in the form of a colorless oil.

[0087] 1 H-NMR (CD3OD, 300 MHz, 5 ppm) 53.01-2.97 (m, 2H), 2.94-2.76 (m, 2H), 2.66 (s, 3H), 1.92-1.76 (m, 4H). HRESIMS m / z obsd. 136.0789, calc, for C5H14NOS [M] + : 136.0791.

[0088] Synthesis of SFNMan: A solution of 2 (166 mg, 0.393 mmol) in anhydrous CH2CI2 (1.5 mL) and EtsN (183 pL) was added to a solution of 3 (Avvakumova, S. et al Chem. Commun., 2014, 50, 11029-11032) (102 mg, 0.262 mmol) in anhydrous CH2CI2 (1.5 mL), and the reaction mixture was stirred at room temperature for 2 h. After this time, the crude was concentrated to dryness and the resulting residue was purified by silica gel column chromatography (CH2Cl2:MeOH 20:1 10:1) yielding peracetylated SFNMan (128 mg, 0.244 mmol, 93%) in the form of a colorless oil. To a solution of this compound (110 mg, 0.210 mmol) in anhydrous MeOH (2 mL), NaOMe (6 mg, 0.1 mmol) was added and after stirring the reaction mixture for 3 h, Amberlite IR 120 (H + ) up to pH = 5. Subsequent filtration of the resin and evaporation of the solvent allowed obtaining SFNMan (72.9 mg, 0.205 mmol, 98%) in the form of a colorless oil. 1H-NMR (CD3OD, 300 MHz, 5 ppm, diastereoisomer mixture) 5 5.61-5.50 (m, 1H, H-1), 3.91-3.35 (m, 8H, H-2, H-3, H-4, H-5, H-6, H-19, H-4, H-5, H-6, H-19, H-4, 2.2. H-4′), 2.66 (s, 3H, -CW3), 1.80 (sa, 4H, H-2′, H-3′). 13 C-NMR (CD3OD, 75.4 MHz, 5 ppm, diastereoisomer mixture) 5 184.6, 184.2 (C=S), 83.5, 83.0 (C-1), 79.4, 75.9, 75.7, 72.4, 71.1, 68.3, 68.3. 62.9, 62.6 (C-2, C-3, C-4, C-5, C-6), 54.3, 45.1, 44.9 (CT, C-4′), 38.1 (-CH3), 29.1, 29.0, 21.0, 20.9 (C-2′, C-3′). HRESIMS m / z obsd. 379.0974, cale, for Ci2H24N20eNaS2 [M+Na] + : 379.0968.

[0089] Synthesis of SFNFuc: To a solution of I₂ (8 mg, 0.03 mmol) in AC₂O (2.9 mL) at 0 °C, L-fucose (500 mg, 3.05 mmol) was added in two portions, and the resulting mixture was stirred overnight. After this time, the crude reaction was added to a saturated aqueous solution of Na₂S₂U₃ at 0 °C, followed by the addition of a saturated aqueous solution of NaHCO₃. The aqueous phase was extracted four times with CH₂Cl₂, and the pooled organic phases were dried over Na₂S₄, filtered, and concentrated to dryness, yielding peracetylated L-fucose, which was used directly in the next reaction step without further purification. To a solution of this compound in AcOH (2 mL) at 0 °C, HBr (33% in AcOH, 2 mL) was added and the reaction mixture was stirred at this temperature for 5 min. Subsequently, the crude reaction was added to a saturated aqueous solution of NaHCOs at 0 °C, and extracted three times with Et2U.The collected organic phases were washed with a saturated aqueous solution of NaCl, dried over Na₂S₄, filtered, and concentrated to dryness to obtain the corresponding peracetylated fucopyranosyl bromide. This compound was dissolved in anhydrous MeCN (5 mL) and added to a solution of KSCN (592 mg, 6.09 mmol) and NBU₄I (1.13 g, 3.06 mmol) in anhydrous MeCN (20 mL) on 4A molecular sieves, which had been previously stirred at room temperature for 2 h. After stirring the resulting mixture for 2 h, the crude reaction was diluted with CH₂Cl₂, filtered over Celita®, and the filtrate concentrated to dryness. The resulting residue was dissolved in CH₂Cl₂ and washed with water. The aqueous phase was extracted four times with CH2CI2 and the collected organic phases were washed with a saturated aqueous solution of NaCI, dried over Na2SÜ4, filtered and concentrated to dryness.The residue obtained was purified by silica gel column chromatography (Et2O:Cyclohexane, 15% E12U) yielding 4 (80.0 mg, 0.241 mmol, 8%, 3 stages) as a colorless oil. A solution of 4 (76.3 mg, 0.230 mmol) in anhydrous CH2Cl2 (1.3 mL) and Et3N (160 pL) was added to a solution of 2 (148 mg, 0.345 mmol) in anhydrous CH2Cl2 (1.3 mL), and the reaction mixture was stirred at room temperature for 2 h. After this time, the crude was concentrated to dryness and the resulting residue was purified by silica gel column chromatography (CH2Cl2:MeOH 20:1 10:1) yielding peracetylated SFNFuc (99.3 mg, 0.213 mmol, 93%) in the form of a colorless oil. To a solution of this compound (80.9 mg, 0.173 mmol) in anhydrous MeOH (1.7 mL), NaOMe (5 mg, 0.1 mmol) was added and after stirring the reaction mixture for 3 h, Amberlite IR 120 (H) was added. +) up to pH = 5. Subsequent filtration of the resin and evaporation of the solvent allowed obtaining SFNFuc (54.7 mg, 0.161 mmol, 93%) in the form of a light yellow solid. 1 H-NMR (CD3OD, 300 MHz, 5 ppm, J Hz, mixture of diastereoisomers) 5 5.12 (sa, 1H, H-1), 3.78-3.53 (m, 6H, H-5, H-2, H-3, H-4, HT), 2.97-2.79 (m, 2H, H-4'), 2.66 (s, 3H, -CH3), 1.85-1.74 (m, 4H, H-2', H-3'), 1.25 (d, 3H, J = 6.5, -CW3-fuc). 13 C-NMR (CD3OD, 75.4 MHz, 5 ppm, mixture of diastereoisomers) 5 185.0 (C=S), 85.2 (C-1), 75.8, 73.2, 73.1, 71.1 (C-2, C-3, C-4, C-5), 54.3, 45.1, 44.9 (C-1', C-4'), 38.1 (-CH3), 29.2, 29.1, 20.9 (C-2', C-3'), 16.9 (-CH3-fuc). HRESIMS m / z obsd. 363.1026, cal, for Ci2H24N2O5NaS2 [M+Na] + : 363.1019.

[0090] Synthesis scheme of compound 6

[0091] NHBoc 1)TFA, CH2CI2t.a.

[0092] 2) SFN, CH2CI2' Et3N, ta

[0093] 3) NaOMe, MeOH, ta

[0094]

[0095] 5 6 To a solution of compound 5 (Moreno-Vargas, AJ et al. Eur. J. Org. Chem. 2008, 2973-2982) (101 mg, 0.225 mmol) in CH2CI2(1.5 mL), TFA (1.7 mL) was added and the reaction mixture was stirred at high temperature for 20 min. Subsequently, the crude was concentrated to dryness to obtain the corresponding ammonium ethylthiofucopyranoside thlfluoroacetate (126 mg, 0.225 mmol, quantitative) in the form of a yellowish oil. A solution of this compound (112 mg, 0.204 mmol) in anhydrous CH2CI2 was added to SFN (LD-Sulforaphane) (29.3 mg, 0.157 mmol), followed by Et3N (110 pL, 0.785 mmol) and the reaction mixture was allowed to evolve under stirring at room temperature for 2 h. After this time, the crude was concentrated to dryness and the resulting residue was purified by silica gel column chromatography (CH2CI2:MeOH 20:1 -> 10:1) obtaining the peracetylated compound 6 (72.9 mg, 0.138 mmol, 88%) in the form of a white foam.To a solution of this compound (58.6 mg, 0.111 mmol) in anhydrous MeOH (1.5 mL), NaOMe (3 mg, 0.05 mmol) was added and after stirring the reaction mixture for 3 h, Amberlite IR 120 (H) was added. + ) up to pH = 5. Subsequent filtration of the resin and evaporation of the solvent allowed obtaining 6 (41.8 mg, 0.104 mmol, 94%) in the form of a white solid.

[0096] 1 H-NMR (CD3OD, 300 MHz, 5 ppm, J Hz, mixture of diastereoisomers) 5 5.39 (d, 1H, i,2= 5.6, H-1), 4.31-4.24 (m, 1H, H-5), 4.05 (dd, 1H,2,3= 10.1, H-2), 3.72-3.67 and 3.52-3.45 (2 m, 5H, H-4, H-1” and H-1' or H-2'), 3.60 (dd, 1H, J 3A = 3.3, H-3), 2.96-2.69 (m, 4H, H-4” and H-2' or H-1'), 2.65 (s, 3H, -CW3), 1.86-1.70 (m, 4H, H-2”, H-3”), 1.24 (d, 3H, J = 6.5, -C / 73-fuc). 13C-NMR (CD3OD, 75.4 MHz, 5 ppm, diastereoisomer mixture) 5 183.5 (C=S), 87.8 (C-1), 73.4 (C-4), 72.3 (C-3), 69.4 (C-2), 68.3 (C-5), 54.4, 45.3, 45.3. 30.5 (CT, C-2', C-1”, C-4”), 38.1 (-CH3), 29.2, 21.0 (C-2”, C-3”), 16.7 (-CH3-fuc). HRESIMS m / z obsd. 423.1060, cale, for Ci4H 28 N2O5NaS3[M+Na] + : 423.1053.

[0097] Scheme of compound 8 synthesis

[0098] 1) H2Pd / C, MeOH, ta

[0099] 2) SFN, CH2CI2, Et3N, ta

[0100] 3) NaOMe, MeOH, ta

[0101]

[0102] 1. To a solution of 7 (Sardzík, R. et al. Beilstein J. Org. Chem. 2010, 6, 699-703) (51 mg, 0.097 mmol) in MeOH (3 mL) Pd / C was added and the reaction mixture was stirred at room temperature under a H2 atmosphere for 3.5 h. After this time, the crude was filtered over celita® and concentrated to dryness. The residue obtained was purified by silica gel column chromatography (CH2CI2:MeOH 10:1 -> 5:1) yielding the corresponding peracetylated aminoethylmannopyranoside (27 mg, 0.069 mmol, 71%) as a colorless oil. A solution of SFN (LD-Sulforaphane) (15 mg, 0.080 mmol) in anhydrous CH2CI2 (1 mL) was added to this compound (27 mg, 0.069 mmol), followed by EtsN (48 pL, 0.34 mmol) and the reaction mixture was allowed to evolve under stirring at ta for 6 h.After this time, the crude oil was concentrated to dryness and the resulting residue was purified by silica gel column chromatography (CH2Cl2:MeOH 20:1 -> 10:1), yielding peracetylated compound 8 (29 mg, 0.051 mmol, 74%) as a colorless oil. To a solution of this compound (29 mg, 0.051 mmol) in anhydrous MeOH (1 mL), NaOMe (2 mg, 0.04 mmol) was added, and after stirring the reaction mixture for 3 h, Amberlite IR 120 (H) was added. + ) up to pH = 5. Subsequent filtration of the resin and evaporation of the solvent yielded 8 (19 mg, 0.047 mmol, 92%) in the form of a white solid.

[0103] 1 H-NMR (CD3OD, 300 MHz, 5 ppm, mixture of diastereoisomers) 54.78-4.76 (m, 1H, H-1), 3.87-3.50 (m, 12H, H-2, H-3, H-4, H-5, H-6, H-1', H-2', H-1”), 2.95-2.78 (m, 2H, H-4”), 2.65 (s, 3H, -CH3), 1.86-1.72 (m, 4H, H-2”, H-3”). 13C-NMR (CD3OD, 75.4 MHz, 5 ppm, diastereoisomer mixture) 5 182.9 (C=S), 101.8 (C-1), 74.8, 72.5, 72.1, 68.6, 67.4, 62.9, 54.4, 4.4,4.9 (C-4.4.4). C-3, C-4, C-5, C-6, C-1', C-2', C-1”, C-4”), 38.1 (-CH3), 29.3, 21.0 (C-2”, C-3”). HRESIMS m / z obsd. 423.1218, cale, for Ci4H28N2O7NaS2 [M+Na] + : 423.1230.

[0104] Scheme of synthesis of compounds 10 and 11

[0105] 1) the CuSO4, NaAsc THF:H2O, MW 80 °C

[0106] 2) NaOMe, MeOH, ta

[0107]

[0108] Synthesis of 10 To a solution of SFN (LD-Sulforaphane) (72.7 mg, 0.410 mmol) in CH2CI2 (4 mL) 9 (Chabre, YM et al. J. Org. Chem. 2008, 73, 5602-5605) (103 mg, 0.438 mmol) was added. The reaction mixture was allowed to proceed under stirring at 40 °C for 24 h. Then, Et3N (103 pL, 0.734 mmol) was added and allowed to proceed for 7 h at 40 °C. It was then concentrated to dryness and the crude obtained was purified by silica gel column chromatography (AcOEtMeOH 15:1) obtaining 10 (87.3 mg, 0.212 mmol, 52%) as a yellowish liquid.

[0109] 1 H-NMR (300 MHz, CDCI3, 5 ppm, J Hz, mixture of diastereoisomers) 5 8.20 (s, 1 H, NH), 6.35 (s, 1 H, NH), 4.20 (d, 6H, J= 2.4, -CH2-O-), 3.65 (s, 8H, -CH2-O-, H-1), 2.75 (t, 2H, J = 7.3, H-4), 2.55 (s, 3H, -CW3), 2.45 (t, 3H, J = 2.4, C QH), 1.90-1.70 (m, 4H, H-3, H-2). 13C-RMN (75.4 MHz, CDC , 5 ppm, diastereoisomeric mixture) 5 182.0 (C=S), 78.5 (-C=CH), 75.6 (-C CH), 69.0 (-CH2-O-), 61.1 (-CH2-O-), 58.8 (C-4), 54.0 (C-1), 45.6 (Cq), 38.6 (-CH3), 28.0, 20.0 (C-2, C-3). HRESIMS m / z obsd. 412.1372, cale, para Ci9H26O4N2NaS2 [M+Na] + : 435.1372.

[0110] Synthesis of 11

[0111] A dissolution of the compound there (Combemale, S. et al. Molecules 2014, 19, 1120-1149) (68 mg, 0.16 mmol):

[0112]

[0113] there'

[0114] In THF:H2O 3:1 (2 mL), CuSCU (3 mg, 0.02 mmol), sodium ascorbate (AscNa) (6 mg, 0.03 mmol) and 10 (20 mg, 0.048 mmol) were added. The reaction mixture was heated in a microwave reactor (80 °C, 45 min, 2 cycles). The crude reaction was concentrated to dryness and purified by silica gel column chromatography (AcOEtMeOH 5:1) yielding 11 peracetylated (45 mg, 0.027 mmol, 56%) as a colorless oil. To a solution of this compound (28 mg, 0.017 mmol) in anhydrous MeOH (1 mL), NaOMe (2 mg, 0.04 mmol) was added and after stirring the reaction mixture for 3 h, Amberlite IR 120 (H + ) up to pH= 5. Subsequent filtration of the resin and evaporation of the solvent allowed obtaining 11 (19 mg, 0.016 mmol, 94%) in the form of colorless oil.

[0115] 1H-RMN (CD3OD, 300 MHz, 5 ppm, mixed diastereoisomers) 5 8.07 (s, 3H, H-triazol), 4.75-4.74 (m, 3H, Man), 4.68-4.65 (m, 12H), 4.17-4.10 (m, 3H, Man), 3.92-3.83 (m, 3H, Man), 3.80-3.56 (m, 21 H), 3.53-3.48 (m, 2H, H-1), 3.25-3.19 (m, 3H, Man), 2.94-2.75 (m, 2H, H-4), 2.65 (s, 3H, -CW3), 1.77-1.63 (m, 4H, H-2, H-3). 13 C-RMN (CD3OD, 75.4 MHz, 5 ppm, mixed diastereoisomers) 5182.7 (C=S), 145.3 (Cq triazol), 126.2 (C triazol), 101.6 (C Man), 74.9 (C Man), 72.5, 71.9, 70.2, 68.3; 66.7 (C Man), 65.2, 63.0, 62.8; 54.3 (C-4), 51.4; 45.3 (C-1), 38.2 (-CH3), 29.1, 21.0 (C-2, C-3). HRESIMS m / z obsd.

[0116] 1182.4263, calc, para C43H73NiiO22NaS2[M+Na] + : 1182.4265.

[0117] 2. Validation of the efficacy and cytotoxicity of the compounds

[0118] The efficacy and cytotoxicity of the SFN glycoconjugates (SFNMan and SFNFuc) were validated as described in the continuation.

[0119] The cytotoxicity of SFN glycoconjugates dissolved in saline and DMSO was evaluated by comparing them to free SFN in dendritic cells (DCs). DCs were cultured in 96-well plates at 37°C and 5% CO2 for 48 hours. Before the addition of LPS (lipopolysaccharide, an inducer of chronic inflammation at 100 ng / mL), the DCs were pre-incubated with the SFN glycoconjugates for 1 hour. Cell viability was determined using the LIVE / DEAD viability kit, and the results were expressed as percentages of SFN expression in DCs by flow cytometry.

[0120] No toxic effects were observed at high concentrations (above 25 pM), allowing the concentration used to be doubled without cytotoxic effects compared to free SFN. Above 10 pM, SFN is cytotoxic (Fig. 1).

[0121] 3. Biological effects of the compounds of the invention

[0122] Biological efficacy was evaluated by in vitro assays that showed inhibition of the NF-KB pathway, maturation and efficient secretion of cytokines by CDs (increase in IL-10 production and reduction of IL-5 and TNF-α) and proliferation of regulatory T cells (Tregs).

[0123] Evaluation of the anti-inflammatory activity of SFN glycoconjugates using dendritic cells (DCs): DCs are cultured in 96-well plates at 37°C and 5% CO2 for 10 minutes in the presence of SFNMan and SFNFuc (25 pM), both in the absence and presence of LPS (an inducer of chronic inflammation at 100 ng / mL). For effective intracellular staining of p65 NF-κB, DCs are fixed with 1% paraformaldehyde and permeabilized using BD Phosflow Perm Buffer III. Specific monoclonal antibodies against p65 NF-κB-PE are used. Flow cytometry is employed to evaluate changes in this signaling pathway. The results are analyzed as percentages of p65 NF-κB-PE expression, thus measuring the inhibition of the NF-κB pathway. The results showed that LPS activates p65 NF-KB, a process that was significantly attenuated by SFN glycoconjugates reaching levels similar to the specific p65 NF-KB blocker, MG132.

[0124] Analysis of DC maturation and activation by flow cytometry. DCs were cultured in 96-well plates at 37°C and 5% CO2 for 48 hours in the presence of SFN glycoconjugates (SFNMan and SFNFuc), both in the absence and presence of LPS. To assess the effects of the SFN glycoconjugates, DCs were pre-incubated with the glycoconjugates for 1 hour before the addition of LPS. Cells were stained with fluorochrome-labeled monoclonal antibodies (see Table 1) to assess maturation. Flow cytometry was used to analyze the expression levels of maturation and activation markers, including CD80, CD83, CD86, HLA-DR, and PD-L1. Results are expressed as percentage expression of each surface marker on DCs.The study demonstrates that dendritic cells (DCs) pretreated with glycoconjugates for one hour, followed by LPS stimulation, significantly reduce the expression of all surface markers compared to those treated with LPS alone. Both the mannose-conjugated (SFNMan+LPS) and fucose-conjugated (SFNFuc+LPS) DCs induce a significant increase in markers compared to SFN-only treatment (SFN+LPS), particularly CD83 and PD-L1. Furthermore, we observed an increase in CD86 with SFNFuc+LPS compared to SFNFuc alone without an inflammatory environment.

[0125] Table 1: Monoclonal antibodies for the CDs phenotype.

[0126] Monoclonal Fluorochrome Clone Company CD86 Pacific Blue IT2.2 BioLegend HLA-DR Pacific Orange L243 EXBIO Praha, CD80 Isothiocyanate BB1 ​​BioLegend defFIuorescein

[0127] PD-L1 Phycoerythrin 29E.2A3 BioLegend CD83 Aiophycocyanin HB15e BioLegend

[0128] Cytokine Production Assessment: Cytokine production (IFN-γ, IL-17, IL-5, TNF-α, and IL-10) is quantified using the ProcartaPlex Multiplex Human Kit. After 48 hours of incubation in the presence of SFN glycoconjugates, both in the absence and presence of LPS, the supernatants from the CD cultures are collected. Samples (80 pL) and standards are incubated overnight at 4°C with a magnetic bead mixture. Biotinylated antibodies are then added, and the plates are incubated for 30 minutes at room temperature (ta). Streptavidin is then added, and the plates are incubated for another 30 minutes at room temperature (ta). After incubation, the plates are washed and prepared for detection using Bio-Plex 200, and the results are expressed as the concentration (pg / mL) of each cytokine.The production of the proinflammatory cytokines IL-5 and TNF-α tended to decrease with treatment with all glycoconjugates, including mannose and fucose, compared to LPS. This highlights the importance of SFN glycoconjugates in enhancing the protective effect of SFN against inflammatory stimuli such as LPS. Regarding the regulatory cytokine IL-10, a significant increase in its production was observed in dendritic cells pretreated with SFN glycoconjugates before LPS restimulation.

[0129] Activation of regulatory T cells (Treqs) in mixed immune cell (DC) cultures: As previously mentioned, autologous DCs are prestimulated in 96-well plates with SFN glycoconjugates (25 pM) and free SFN in the presence or absence of LPS for 48 hours. Proliferation analysis is performed using 5,6-carboxyfluorescein diacetate N-succinimidyl ester (CFSE), which labels CD14- cells. These cells are exposed to the prestimulated DCs in a 10:1 ratio in a total volume of 250 pL of medium for 6 days at 37°C and 5% CO2. CFSE is used to assess the proliferative responses of T cells, B cells, and Treg cells. Furthermore, the proliferative response of T reg cells is confirmed by the ability to produce anti-inflammatory cytokines such as IL-10 and pro-inflammatory cytokines such as IFN-γ.To this end, Brefeldin A was added at a concentration of 1 / 1000 for 3 hours, followed by cell fixation using the Cytofix / CytoPerm fixation / permeabilization solution kit and FOXP3 labeling. The results are expressed as percentages of cells weakly expressing CFSE, as well as the percentages of IL-10 produced by Treg cells. Regarding the results, the SFNMan glycoconjugate significantly increased T cell proliferation (CD3+CD4+FOXP3+ Tregs) under LPS restimulation, compared to unstimulated cells. Furthermore, this glycoconjugate, after LPS restimulation, significantly increased B cell proliferation (CD3-CD19+) compared to SFN and SFNMan. On the other hand, SFNFuc did not show a proliferative immune response, further underscoring the importance of the nature of SFN conjugates in the cellular response. Results:.

[0130] • Improved solubility and stability of glycosylated SFN compared to free SFN. In solubility studies, glycoconjugates showed a significant increase in aqueous solubility compared to free SFN. When analyzing the soluble concentrations of both compounds, glycosylated SFN was found to dissolve more effectively in aqueous media, thus promoting its absorption and bioavailability. This increase in solubility is related to the addition of hydrophilic carbohydrate groups (mannose or fucose), which increase the compound's affinity for water. By increasing its water solubility, the concentration of organic solvents, which can affect cell viability, decreases (Fig. 1).

[0131] • Increased DC maturation and activation, as well as IL-10 production. First, the expression levels of maturation markers such as CD83, CD86, HLA-DR, and PD-L1 were evaluated using flow cytometry (Fig. 2). The results indicated that DCs treated with SFN glycoconjugates (SFNMan and SFNFuc) showed a significant increase in the expression of these markers compared to DCs treated only with LPS or free SFN. This indicates greater DC maturation, especially in the presence of the glycoconjugates. In fact, compared to controls, the SFN conjugates, particularly SFNMan, induced higher levels of CD83 and CD86 than SFN alone, demonstrating that conjugated carbohydrates enhance SFN's ability to activate DCs.Similarly, dendritic cells (DCs) pretreated with SFN glycoconjugates and then stimulated with LPS showed a significant increase in the production of IL-10, a cytokine with key immunoregulatory functions. This increase was especially notable in DCs treated with SFNMan+L and SFNFuc+L, surpassing even the results of treatment with SFN alone.

[0132] • Effective inhibition of the NF-KB pathway in CDs treated with SFN glycoconjugates.

[0133] Effective inhibition of the NF-κB pathway in dendritic cells (DCs) treated with sulfonylureas (SFN) has been demonstrated in previous studies (Ana M Múnera-Rodríguez et al., Biomed Pharmacother. 2024 Aug: 177:117056). SFN, known for its anti-inflammatory properties, and its carbohydrate conjugates (such as mannose and fucose) have shown an increased capacity to inhibit the activation of the NF-κB pathway, which is crucial in the inflammatory response (Fig. 3). In NF-κB activation studies, the p65 subunit, a key marker of the NF-κB pathway, is used to measure the activation of this pathway in DCs. The results showed that DCs treated with SFN glycoconjugates exhibited a significant decrease in activated p65 levels compared to DCs treated only with lupus phosphate (LPS), which activates NF-κB. This was evidenced by flow cytometry.

[0134] Furthermore, free SFN also showed inhibition of NF-κB activation, but SFN glycoconjugates, especially those containing mannose (SFNMan), proved more effective in this inhibition. Under conditions with SFN and LPS glycoconjugates, a much greater reduction in NF-κB activation was observed compared to treatment with free SFN or LPS alone, suggesting that glycoconjugates enhance SFN's ability to inhibit this inflammatory pathway.

[0135] SFN glycoconjugates also showed significant inhibition of the production of NF-κB-regulated proinflammatory cytokines, such as TNF-α and IL-6, compared to LPS treatment alone. This reinforces the conclusion that SFN glycoconjugates not only inhibit NF-κB signaling but also reduce the inflammatory response mediated by this pathway.

[0136] • Increased proliferation of IL-10 producing regulatory T cells.

[0137] Treg cells showed increased proliferation when dendritic cells (DCs) were treated with SFN glycoconjugates. Treg proliferation was significantly higher with the SFNMan+L treatment than with SFN+L alone, indicating that glycoconjugates enhance the ability of DCs to activate IL-10-producing Tregs. This activation is crucial for their role in suppressing excessive immune responses and promoting tolerance. In addition to Treg activation, the SFNMan conjugate also showed increased proliferation of CD19+ B cells compared to SFN alone, suggesting that the nature of the glycoconjugate influences the activation of different cell lineages. This reinforces the idea that conjugates enhance the immune response more broadly.

Claims

CLAIMS 1. A compound of general formula (I) or (II): (YO) (II) where: R 1 is a glycosyl radical, And you select between O and S, and n is 0 or 1.

2. Compound according to claim 1, wherein R 1 The choice is between mannosyl and fucosyl.

3. Compound according to any of claims 1 or 2, wherein R 1 The selection is between D-mannosyl and L-fucosyl.

4. Compound according to claims 1 to 3, wherein n is 0.

5. Compound according to any of claims 1 to 4, wherein the compound of formula (I) is selected from:

6. Compound according to claims 1 to 3, wherein n is 1 and Y is selected from O and S.

7. Compound according to any of claims 1 to 3 or 6, wherein the compound of formula (I) is selected from:

8. Compound according to any of claims 1 to 3, wherein the compound of formula (II) is:

9. Compound as described in any of claims 1 to 8 for use as a medicament.

10. Compound as described in any of claims 1 to 8 for use in the treatment and / or prevention of inflammatory, autoimmune diseases or cancer.

11. Compound for use according to claim 10, wherein the diseases are selected from rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, type 1 diabetes, breast cancer, prostate cancer, colon cancer, lung cancer and inflammatory bowel disease.

12. Compound for use according to claim 11, wherein the diseases are selected from rheumatoid arthritis and inflammatory bowel disease.

13. Compound for use according to any of claims 11 or 12, wherein the inflammatory bowel diseases are selected from Crohn's disease and ulcerative colitis.

14. Pharmaceutical composition comprising a compound described in any of claims 1 to 8, together with a pharmaceutically acceptable excipient and / or vehicle.

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