Application of β-D-pyranose glucopyranosyl-lactic acid and its derivatives in the preparation of drugs for treating metabolic diseases and inflammation

Abstract

The present application discloses the use of β-D-pyranose-glucopyranosyl-lactic acid and its derivatives in the preparation of drugs for treating metabolic diseases and inflammation, and belongs to the technical field of drug use. The present application discloses the use of β-D-pyranose-glucopyranosyl-lactic acid and its derivatives in the preparation of drugs for treating metabolic diseases and inflammation. The metabolic diseases are one or more of hyperlipidemia, diabetes, fatty liver, obesity, hypertension and cardiovascular and cerebrovascular diseases; the inflammation is one or more of allergic encephalomyelitis, inflammatory bowel disease, rheumatoid arthritis, sepsis, acute pancreatitis, chronic pancreatitis, hyperuricemia and psoriasis. Please prove through relevant research that β-D-pyranose-glucopyranosyl-lactic acid and its derivatives have potential preventive and therapeutic effects on metabolic diseases and inflammation.

Description

Technical Field

[0001] The invention belongs to the technical field of drug application, and particularly relates to the application of β-D-pyranoglucopyranosyl-lactic acid and its derivatives in the preparation of drugs for treating metabolic diseases and inflammation. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic, idiopathic, nonspecific and relapsing gastrointestinal disease that is divided into two main types: Crohn's disease and ulcerative colitis (UC). [1] Ulcerative colitis is confined to the colon and is characterized by weight loss, diarrhea, and abdominal pain, affecting people of all ages. Although the etiology and pathogenesis remain unclear, it is generally believed to be the result of genetic and immunologic, microbial, and environmental factors. [2-4] Macrophages, as the mature form of monocytes, are involved in both innate and adaptive immunity. They are usually the first line of defense in innate immunity. [5] Once pathogens break through the epithelial barrier and invade the intestinal mucosa, they can be recognized by macrophages using Toll-like receptors, pattern recognition receptors (PRRs), etc., further inducing a series of pathway responses, such as NLRP3 inflammasome, which ultimately leads to cell membrane rupture, cytokines interleukin 1b (IL-1b) and IL-18, and the release of cell contents. This type of inflammatory cell death is called "heat death" [6-7] Like other innate immune responses, pyroptosis contributes to the host's self-defense against bacterial, fungal, and viral infections. [8] However, dysregulated thermoregulation closely contributes to the development of UC by disrupting the intestinal epithelial barrier and inducing adaptive immune dysregulation by promoting IL-17 production by Th17 cells and interferon-γ (IFN-γ) production by Th1 cells. [9-10] It has been demonstrated that inhibiting the thermogenic activity of macrophages may be a new strategy for treating experimental colitis [11-12] Immune cells can regulate their functions through metabolic reprogramming. Lactic acid, a metabolic substrate among hydroxy acids, has been re-recognized as an active signal in the function of regulatory immune cells. [13-14] Previous studies have revealed various mechanisms of how immune cells in diseased tissues respond to local accumulation of metabolites. Lactate is mostly present in the intestine in ionic form and is mainly absorbed and utilized by monocarboxylate transporter 1 (MCT1).

[15] In addition, lactate binds to the GPR81 receptor on the surface of intestinal macrophages, downregulating cyclic AMP (cAMP) and protein kinase A (PKA) signals, further inhibiting the expression of pro-inflammatory factors.

[16] Lactate protects the heart and ischemic neurons, promotes adult hippocampal neurogenesis, and inhibits inflammation after organ injury

[17] Lactate also promotes the emulsification of histone H3 lysine in macrophages and alters the polarization state of macrophages by increasing the expression of Arg-1 and other M2 macrophage genes.

[18] In cancer, under conditions of hypoxia and high extracellular lactate concentrations, lactate can be transported into cells for use as a metabolic substrate. Previous studies have shown that lactate can increase TH1 cell differentiation and interferon-γ (IFNγ) production.

[19] , and promotes M2-like polarization of tumor-associated macrophages (TAMs) and VEGF expression, partly mediated by activation of HIF1α

[20] The latter study raises the interesting idea that lactate has a homeostatic function in balancing the inflammatory effects of HIF1α and glycolytic metabolites. Liu et al.

[21] We expand our understanding of how high lactate concentrations in tumors promote the M2-type pro-tumorigenic TAM phenotype. Lactate uptake by macrophages activates mammalian target of rapamycin complex 1 (mTORC1)-mediated signaling, leading to reduced expression of the transcription factor TFEB and its downstream target genes, including Atp6v0d2, encoding the D2 subunit of the vacuolar proton pump, which acidifies lysosomes and promotes protein degradation. Attenuated expression of ATP6V0D2 leads to reduced lysosomal degradation of HIF2α, thereby inducing the expression of its target genes, including Veg f and the M2-like homologs Mrc1, Arg1, and Retnla. This ATP6V0D2-HIF2α pathway is pathologically relevant because Atp6v0d2 - / - Mice exhibited abnormal vascularization and increased tumor volume, which were reversed by inhibition of HIF2α. Thus, increased intracellular lactate concentrations in macrophages promote the M2 tumor growth phenotype through different mechanisms of activating HIF1α3 and HIF2α4. The inhibitory role of lactate is further supported by a recent study5 showing that tumor acidosis, in part due to the export of lactate and protons generated by aerobic glycolysis, induces a regulatory TAM phenotype that promotes tumor growth. A paper published in 2019 showed that increased intracellular lactate concentrations inhibited the cytoplasmic RIG-I-like receptor (RLR)

[22] induction of IFNβ. The inhibition of RLR signaling is independent of acidic pH and is mediated by direct binding of lactate molecules to the transmembrane domain of the RLR adaptor mitochondrial antiviral signaling protein (MAVS), which is anchored to the mitochondrial membrane. Lactate binding to MAVS attenuates its mitochondrial localization, interaction with RIG-I, aggregation and downstream signaling, as well as Ifnb activation. Inhibition of RLR signaling is biologically important because blocking lactate production by genetically or pharmacologically targeting the glycolytic enzyme lactate dehydrogenase leads to enhanced interferon production and viral clearance. This study identifies MAVS as a direct sensor of lactate and suggests that lactate may also indirectly promote polarization of cognate macrophages by inhibiting counter-balancing pro-inflammatory interferon-mediated pathways. Another surprising direct target of lactate was discovered in 2019 - lysine residues in histones

[23] Lactate is an important metabolic intermediate released by skeletal muscle and other organs, including adipose tissue, and is converted from glucose under the influence of insulin.

[24] showed that lactate activates the G protein-coupled receptor GPR81, which is expressed in adipocytes and mediates anti-lipolytic effects through Gi-dependent inhibition of adenylate cyclase. Using mice deficient in GPR81, Kashan Ahmed demonstrated that this receptor is not involved in the regulation of lipolysis during intensive exercise. However, insulin-induced inhibition of lipolysis and insulin-induced decrease in adipocyte cAMP levels were strongly reduced in mice lacking GPR81, although insulin-dependent lactate release from adipocytes was comparable in wild-type and GPR81-deficient mice. Thus, lactate and its receptor GPR81 unexpectedly act in autocrine and paracrine loops to mediate insulin-induced anti-lipolytic effects. These data suggest that lactate can directly regulate metabolic processes in a hormone-like manner, revealing a novel mechanism of lactate in the anti-lipid action of insulin.

[0003] Since the bioavailability of lactic acid in the body is extremely poor, lactic acid is used as a lead compound for structural modification. By connecting with glucose to form a β-glycosidic bond to form a prodrug, the bioavailability of lactic acid is increased, and the blood concentration of lactic acid is increased to treat inflammatory bowel disease and metabolic diseases.

[0004] References:

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[0007] [3].Piovani D,Danese S,Peyrin-Biroulet L,Nikolopoulos GK,Lytras T,Bonovas S.Environmental Risk Factors for Inflammatory Bowel Diseases:AnUmbrella Review of Meta-Analyses.Gastroenterol[Internet](2019)157(3):647–659.e4.

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[13] .Lee YS,Kim TY,Kim Y,et al.Microbiota-Derived Lactate AcceleratesIntestinal Stem-Cell-Mediated Epithelial Development.Cell Host Microbe[Internet](2018)24(6):833–846.e6.

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[16] .Ranganathan P,Shanmugam A,Swafford D,et al.GPR81,a Cell-SurfaceReceptor for Lactate,Regulates Intestinal Homeostasis and Protects Mice FromExperimental Colitis.J Immunol(2018)200(5):1781–9.

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[17] .Wang J,Cui Y,Yu Z,et al.Brain Endothelial Cells Maintain LactateHomeostasis and Control Adult Hippocampal Neurogenesis.Cell Stem Cell[Internet](2019)25(6):754–67.e9.

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[18] .Zhang D,Tang Z,Huang H,et al.Metabolic Regulation of GeneExpression by Histone Lactylation.Nat[Internet](2019)574(7779):575–80.

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[19] .Peng,M.et al.Aerobic glycolysis promotes T helper 1 celldifferentiation through an epigenetic mechanism.Science 354,481–484(2016).

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[20] .Colegio,O.R.et al.Functional polarization of tumour-associatedmacrophages by tumour-derived lactic acid.Nature 513,559–563(2014).

[0025]

[21] .Liu,N.et al.Lactate inhibits ATP6V0d2 expression in tumor-associated macrophages to promote HIF-2α-mediated tumorprogression.J.Clin.Invest.129,631–646(2019).

[0026]

[22] .Zhang,W.et al.Lactate is a natural suppressor of RLR signalingby targeting MAVS.Cell 178,176–189(2019).

[0027]

[23] . Zhang, D. et al. Metabolic regulation of gene expression by histonelactylation. Nature 574, 575–580 (2019).

[0028]

[24] .Kashan Ahmed(Ahmed K.et alAn autocrine lactate loop mediatesinsulin-dependent inhibition of lipolysis through GPR81.Cell Metab.2010 Apr7;11(4):311-9. Summary of the invention

[0029] Based on the important role of lactic acid in maintaining body homeostasis and normal physiological metabolism, the present invention provides, on one hand, the use of β-D-pyranoglucopyranosyl-lactic acid and its derivatives in the preparation of drugs for treating metabolic diseases.

[0030] The metabolic disease is one or more of hyperlipidemia, diabetes, fatty liver, obesity, hypertension and cardiovascular and cerebrovascular diseases;

[0031] On the other hand, the present application provides the use of β-D-pyranoglucopyranosyl-lactic acid and its derivatives in the preparation of drugs for treating inflammation.

[0032] The inflammation is one or more of allergic encephalomyelitis, inflammatory bowel disease, rheumatoid arthritis, sepsis, acute pancreatitis, chronic pancreatitis, hyperuricemia and psoriasis.

[0033] The medicine also includes other pharmaceutically acceptable adjuvants, carriers or diluents;

[0034] The pharmaceutical dosage form is selected from plain tablets, film-coated tablets, sugar-coated tablets, enteric-coated tablets, dispersible tablets, capsules, granules, oral solutions or oral suspensions.

[0035] The derivative of β-D-pyranoglucopyranosyl-lactic acid is a stereoisomer, a tautomer, a pharmaceutically acceptable salt, a solvate or a prodrug thereof of a β-D-pyranoglucopyranosyl-lactic acid compound.

[0036] The present application provides a preparation method of the β-D-pyranose glucopyranosyl-lactic acid, which comprises the following steps: using glucose as a raw material to prepare glycosyl trichloroacetimidate, then using D-lactic acid and benzyl bromide as raw materials to prepare benzyl lactate, finally reacting benzyl lactate with glycosyl trichloroacetimidate, and then removing the benzyl group to obtain the target compound.

[0037] The specific steps include:

[0038] (1) D-lactic acid was dissolved in N,N-dimethylformamide, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was added to the mixture with a syringe under stirring and stirred for 30 min. Benzyl bromide and potassium iodide were slowly added under stirring at 0°C. After stirring for 18 h at room temperature, the reaction solution was mixed with ethyl acetate and water. After separation, the organic phase was taken, and the aqueous phase was extracted with ethyl acetate for 4 times. The organic phases were combined, washed with 3 volumes of water, DMF in the organic phase was removed, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, and purified by column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain a white transparent liquid Bn-Lac;

[0039] (2) Acetic anhydride and sodium acetate were mixed and heated to reflux at 90°C. After complete dissolution, D-glucose was slowly added and reacted at 90°C for 4 hours. The mixture was cooled to room temperature, ice water was added by ultrasonication, and filtered to obtain a white solid. The white solid Glu1 was recrystallized from anhydrous ethanol to obtain a white solid.

[0040] (3) Dissolve the Glu1 obtained in step (2) in anhydrous dichloromethane, add BF3-Et2O and thiophenol at 0°C, react at room temperature for 5 hours, add saturated sodium bicarbonate to stop the reaction, extract with dichloromethane, wash with saturated sodium chloride aqueous solution, spin-dry the organic layer to obtain a yellow oily substance Glu2, and purify it by column chromatography (petroleum ether: ethyl acetate = 3:2) to obtain Glu2;

[0041] (4) Dissolve the Glu2 obtained in step (3) in anhydrous methanol, add 30% sodium methoxide / methanol solution, react for 15 h, purify by column chromatography (dichloromethane: methanol = 10: 1), add Amberlite IR 120 to adjust the pH to neutral, filter, spin dry the solvent, and vacuum dry to obtain Glu3;

[0042] (5) Dissolve the Glu3 obtained in step (4) in anhydrous N,N-dimethylformamide, add NaH to the reaction mixture in portions at 0°C. Stir at 0°C for 30 min, add benzyl bromide to the reaction mixture, and stir the mixture for 22 h. Slowly add cold distilled water to the solution; extract the aqueous phase with dichloromethane. Wash the collected organic phase with distilled water and saturated sodium chloride aqueous solution. 2 SO 4 The collected organic phase was dried on 400° C. The solvent was evaporated under reduced pressure and purified by column chromatography (petroleum ether:ethyl acetate=20:1) to obtain Glu4.

[0043] (6) Glu4 obtained in step (5) was dissolved in anhydrous dichloromethane, trichloroacetonitrile and 1,8-diazabicyclo[5.4.0]undec-7-ene were added at 0°C, and after the reaction was completed for 2 hours, Glu5 was obtained by column chromatography (petroleum ether: ethyl acetate = 10:1);

[0044] (7) Glu5 obtained in step (6) was placed in a round-bottom flask, 20 ml of anhydrous dichloromethane was added to dissolve, Bn-Lac obtained in step (1) was added under ice bath, TMSOTf was added under nitrogen protection, and after reacting at -20°C for 2 h, a saturated sodium chloride aqueous solution and a small amount of triethylamine were added to quench the reaction, the mixture was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate = 30:1) to obtain a colorless transparent liquid Glu6.

[0045] (8) Dissolve the Glu6 obtained in step (7) in 99% methanol, add 10% Pd-C and hydrogenate at room temperature under pressure for 24 hours, remove Pd-C by centrifugation, spin-dry the filtrate and recrystallize it from methanol-ethyl acetate to obtain Glu7, which is β-D-pyranose-lactic acid.

[0046] The specific reaction process is:

[0047]

[0048] (a)DMF,KI,DBU,rt.,overnight; (b)Ac 2 O / AcONa,90℃,reflux,4h; (c)BF 3 -Et 2 O,Ph-SH,DCM,rt.,5h;(d)MeOH / MeONa,rt.,15h;(e)0℃,NaH,DMF,30min,Bn-Br,rt.,22h;(f)CCl 3 CN,DBU,DCM,0℃rt.,2h; (g)Bn-Lac,TMSOTf,DCM,N 2 ,0℃–rt.,,2h;(h)Pd / C,H 2 ,MeOH,24h.

[0049] If not otherwise specified, the compounds of the present invention are understood to include free states and salts thereof. The term "salt" means acidic and / or basic salts formed with inorganic and / or organic acids and bases. In addition, the term "salt may include zwitterions (inner salts), such as when the compound of formula I contains a basic fragment such as an amine or a pyridine or imidazole ring, and an acidic fragment such as a carboxylic acid. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, such as acceptable metal and amine salts, in which the cation does not significantly contribute to the toxicity or biological activity of the salt. However, other salts may be useful, such as separation or purification steps used in the preparation process, and are therefore also included in the scope of the present invention. Salts of compounds of formula I can be formed as compounds of formula I with a certain amount of acid or base, such as an equal amount, in a medium such as in which the salt can be precipitated or in its aqueous medium, and then lyophilized.

[0050] Exemplary acid addition salts include acetates (e.g., formed with acetic acid or a trihaloacetic acid such as trifluoroacetic acid), adipates, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride (formed with hydrochloric acid), hydrobromide (formed with hydrochloric acid),

[0013] Examples of the invention include, but are not limited to, hydrobromic acid, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate (formed with maleic acid), methanesulfonate (formed with methanesulfonic acid), 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate (such as those formed with sulfuric acid), sulfonate, tartrate, thiocyanate, toluenesulfonate such as toluenesulfonate, undecanoate, and the like.

[0051] Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, potassium, calcium and zinc salts; alkaline earth metal salts such as calcium and magnesium salts; barium, zinc and aluminum salts; salts formed with organic bases (such as organic amines) such as trialkylamines such as triethylamine, procaine, dibenzylamine, N-benzyl-β-phenylethylamine, 1-diphenylhydroxymethylamine, N,N'-bisbenzylethylenediamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, dicyclohexylamine or similar pharmaceutically acceptable amines and salts with amino acids such as arginine, lysine and the like. Basic nitrogen-containing groups can be quaternized with agents such as lower alkyl halides (such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides), dialkyl sulfates (such as dimethyl, diethyl, dibutyl and diamyl sulfates), long chain halides (such as decyl, dodecyl, tetradecyl and octadecyl chlorides, bromides and iodides), aralkyl halides (such as benzyl and phenethyl bromides) and others. Preferred salts include monohydrochloride, bisulfate, methanesulfonate, phosphate or nitrate salts.

[0052] The phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical evaluation, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0053] As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds, wherein the parent compound is modified with an acid or its basic salt. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic groups such as amines; and alkali or organic salts of acidic groups such as carboxylic acids. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound, such as from non-toxic inorganic or organic acids. For example, these conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid and nitric acid; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-ethoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid and isethionic acid and their analogs.

[0054] The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from the parent compound containing the basic or acidic fragment. Typically, these salts can be prepared from the free acid or basic form of these compounds with a suitable base or acid in a stoichiometric ratio in water or an organic solvent, or a mixture thereof; typically, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.

[0055] All stereoisomers of the compounds of the present invention are contemplated, both in mixtures and in pure or substantially pure form. Stereoisomers may include compounds of optical isomers through substitution of one or more chiral atoms, as well as compounds of optical isomers through restricted rotation of one or more bonds (atropisomers). The definition of the compounds of the present invention includes all possible stereoisomers and mixtures thereof. It particularly includes racemic forms and separated optical isomers with particular activity. The racemic forms are resolved by physical methods, such as distributed crystallization, separation or crystallization of stereoisomeric derivatives or separation by chiral column chromatography. Independent optical isomers such as salts formed with optically active acids are obtained from racemic salts by conventional methods and then crystallized.

[0056] Prodrugs and solvates of the compounds of the present invention are also contemplated. The term "prodrug" refers to a compound that undergoes a chemical reaction to generate a compound of formula I, and / or a salt and / or a solvate thereof, based on administration to a receptor, by metabolic or chemical methods. Any compound that is converted in vivo to provide a bioactive agent (i.e., a compound of formula I) is a prodrug within the scope and spirit of the present invention. Lactic acid and its derivatives have the disadvantages of high toxicity, low selectivity, and low water solubility, which limit the clinical application of lactic acid and its derivatives as anti-tumor drugs. Sugar is a polar compound with strong hydrophilicity. When sugar is introduced into a chemotherapeutic drug as an auxiliary group, it is often possible to change the physicochemical properties of the drug, improve water content, reduce the toxicity of the chemotherapeutic drug, and thus improve the selectivity of the chemotherapeutic drug.

[0057] "Pharmaceutically acceptable carrier" generally refers to a carrier that is generally accepted in the art for delivering biologically active agents to animals, especially mammals. The formulation of a pharmaceutically acceptable carrier is based on many factors well known to those of ordinary skill in the art. These include, without limitation, the type and identity of the active agent being formulated; the recipient to which the agent-containing composition is administered; the route of administration of the composition; and the targeted therapeutic indication. Pharmaceutically acceptable carriers include aqueous and non-aqueous liquid media, as well as a variety of solid and semi-solid dosage forms. These carriers include many different components and additives, and in addition to the active agent, these additional components are included in the formulation for a variety of reasons, such as stability of the active agent, adhesives, etc., which are well known to those of ordinary skill in the art.

[0058] The compounds of formula I of the present invention can be administered in any suitable manner for treating symptoms, depending on the site-specific treatment or the amount of drug delivered. Topical administration is generally preferred for systemic treatment of skin-related diseases, cancerous or precancerous symptoms, but other modes of delivery are also considered. For example, the compound is administered orally, such as in the form of tablets, capsules, granules, powders or liquid formulations including syrups; topically such as in the form of solutions, suspensions, gels or ointments; sublingual administration; buccal; parenteral administration such as by subcutaneous, intravenous, intramuscular or intrasternal injection or perfusion (such as sterile water or non-aqueous solutions or suspensions); nasal such as by inhalation spray; topically such as in the form of emulsions or ointments; rectally such as in the form of suppositories; or liposomes. A dosage unit formulation containing a non-toxic, pharmaceutically acceptable excipient or diluent can be administered. The compound can be administered in the form of immediate release or delayed release. Immediate release or delayed release can be obtained with a suitable pharmaceutical composition, and in the example of partial delayed release, a device such as a subcutaneous implant or an osmotic pump is used.

[0059] Exemplary compositions for oral administration include suspensions, which may contain, for example, microcrystalline cellulose for delivery, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners or flavorings known in the prior art; immediate release tablets may contain, for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and / or lactose and / or other excipients, binders, expanders, disintegrants, diluents and lubricants such as those known in the prior art. The compounds of the present invention may also be delivered orally by sublingual and / or buccal administration, such as compression molded, compressed or lyophilized tablets. Exemplary compositions may include rapidly dissolving diluents such as mannitol, lactose, sucrose and / or cyclodextrins. Included in these formulations may also be high molecular weight excipients such as cellulose. or polyethylene glycol (PEG); excipients that aid mucosal adhesion such as hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), sodium carboxymethyl cellulose (SCMC) and / or maleic anhydride copolymers (such as ); and controlled release agents such as polyacrylic acid copolymers (such as CARBOPOL ). Lubricants, glidants, flavors, colorants and stabilizers may also be added to facilitate preparation and use.

[0060] Exemplary compositions for administration by nebulization or inhalation include solutions that may contain benzyl alcohol or other suitable preservatives, absorption enhancers to improve absorbability and / or biological activity, and / or other solubilizing or dispersing agents such as those known in the art.

[0061] Exemplary compositions for parenteral administration include injectable solutions or suspensions, which may contain, for example, suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution, isotonic sodium chloride solution, or other suitable dispersing or wetting and suspending agents, including synthetic mono- or diglycerides, and fatty acids including oleic acid.

[0062] Exemplary compositions for rectal administration include suppositories, which may contain, for example, suitable non-irritating excipients such as cocoa butter, synthetic glycerides or polyethylene glycols which are solid at ordinary temperatures but dissolve and / or melt in the stomach to release the drug.

[0063] Therapeutically effective amounts of the compounds of the invention may be determined by one of ordinary skill in the art and include exemplary doses for mammals ranging from about 0.05 to 1000 mg / kg; 1-1000 mg / kg; 1-50 mg / kg; 5-250 mg / kg; 250-1000 mg / kg, in terms of the amount of active compound per kilogram of body weight per day, which may be administered in a single dose or in separate divided doses, such as from 1 to 4 times per day. It will be appreciated that the specific dose level and dosage frequency for a particular recipient may vary depending on a variety of factors, including the activity of the specific compound used, the metabolic stability and length of action of the compound, age, body weight, general health, sex and diet of the recipient, mode and time of administration, rate of excretion, drug combination and severity of the particular disease. Preferred recipients for treatment include animals, most preferably humans, dogs, cats, horses and the like. DETAILED DESCRIPTION

[0064] The present invention is further described below by way of examples. It should be understood that the methods described in the examples of the present invention are only used to illustrate the present invention, rather than to limit the present invention, and simple improvements to the preparation methods of the present invention under the premise of the concept of the present invention belong to the scope of protection claimed in the present invention. All raw materials and solvents used in the examples were purchased from Sigma Biochemical and Organic Compounds for Research and Diagnostic Clinical Reagents.

[0065] Example 1 Preparation method of β-D-pyranose glucopyranosyl-lactic acid compound

[0066] The steps include:

[0067] (1) D-lactic acid (1 ml, 13.43 mmol) was dissolved in N,N-dimethylformamide (DMF) (10 ml), and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (2 ml, 13.42 mmol) was added to the mixture by syringe under stirring at 0°C and stirred for 30 min. Then, benzyl bromide (3.86 ml, 31.60 mmol) and potassium iodide (2.68 g, 16.12 mmol) were slowly added under stirring. After stirring at room temperature for 18 h, the reaction solution was mixed with ethyl acetate and water. After separation, the organic phase was collected and then extracted with ethyl acetate for 4 times. The organic phases were combined and washed with 3 volumes of water to remove DMF from the organic phase. The organic phase was washed with saturated sodium chloride solution and dried over anhydrous sodium sulfate. The mixture was purified by column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain a white transparent liquid Bn-Lac.

[0068] (2) Acetic anhydride (9.4 ml, 100 mmol) and sodium acetate (1.0 g, 13 mmol) were mixed and heated to reflux at 90°C. After complete dissolution, D-glucose (2.78 mmol) was slowly added and reacted at 90°C for 4 h. The mixture was cooled to room temperature, ice water was added by ultrasonication, and filtered to obtain a white solid. The white solid Glu1 was recrystallized from anhydrous ethanol.

[0069] (3) Dissolve 17 g of Glu1 obtained in step (2) in anhydrous dichloromethane and add BF 3 -Et 2 O (16.6 ml, 130.7 mmol), thiophenol (6.67 ml, 65.3 mmol), react at room temperature for 5 h, add saturated sodium bicarbonate to stop the reaction, extract with dichloromethane, wash with saturated sodium chloride aqueous solution, spin dry the organic layer to obtain yellow oily Glu2. Glu2 was purified by column chromatography (petroleum ether: ethyl acetate = 3:2).

[0070] (4) Dissolve the Glu2 obtained in step (3) in anhydrous methanol, add 30% sodium methoxide / methanol solution, react for 15 h, purify by column chromatography (dichloromethane: methanol = 10: 1), add Amberlite IR 120 to adjust the pH to neutral, filter, spin dry the solvent, and vacuum dry to obtain Glu3;

[0071] (5) Glu3 (3.06 g) obtained in step (4) was dissolved in anhydrous N, N-dimethylformamide (150 ml), and NaH (60%, 4.49 g, 112 mmol) was added to the reaction mixture in portions at 0°C. The mixture was stirred at 0°C for 30 min, and benzyl bromide (19.2 g, 112.26 mmol) was added to the reaction mixture, and the mixture was stirred for 22 h. Cold distilled water (100 ml) was slowly added to the solution. The aqueous phase was extracted with dichloromethane (3×50 mL). The collected organic phase was washed with distilled water (2×50 mL) and saturated sodium chloride aqueous solution (1×50 mL). 2 SO 4 The collected organic phase was dried on 400° C. The solvent was evaporated under reduced pressure and purified by column chromatography (petroleum ether:ethyl acetate=20:1) to obtain Glu4.

[0072] (6) Glu4 (1.0 g, 1.9 mmol) obtained in step (5) was dissolved in anhydrous dichloromethane (10 ml), and trichloroacetonitrile (1.71 ml, 18.99 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (0.17 ml, 1.14 mmol) were added at 0°C. After the reaction was completed for 2 h, Glu5 was obtained by column chromatography (petroleum ether: ethyl acetate = 10:1);

[0073] (7) Glu5 (1 g, 1.49 mmol) obtained in step (5) was placed in a round-bottom flask, 20 ml of anhydrous dichloromethane was added to dissolve, Bn-Lac (0.24 ml, 2.22 mmol) obtained in step (1) was added under ice bath, TMSOTf (0.183 ml, 2.22 mmol) was added under nitrogen protection, and after reacting at -20°C for 2 h, a saturated sodium chloride aqueous solution and a small amount of triethylamine were added to quench the reaction, and the mixture was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate = 30:1) to obtain a colorless transparent liquid Glu6.

[0074] (8) Glu6 (1.0 g, 1.45 mmol) obtained in step (7) was dissolved in 99% methanol, 10% Pd-C (200 mg) was added and hydrogenated at room temperature for 24 h under pressure. Pd-C was removed by centrifugation, the filtrate was dried and recrystallized from methanol-ethyl acetate to obtain Glu7, i.e. β-D-pyranose-lactic acid.

[0075] The structural formula of the β-D-glucopyranosyl-lactic acid is:

[0076]

[0077] The nuclear magnetic hydrogen spectrum data of the β-D-pyranose glucopyranosyl-lactic acid is:

[0078] 1 H NMR(500MHz,Deuterium Oxide)δ4.92(d,J=3.8Hz,1H),4.35(q,J=7.0Hz,1H),3.76(dd,J=12.1,2.2Hz,1H),3. 68–3.58(m,4H),3.45(dd,J=9.9,3.8Hz,1H),3.33–3.29(m,1H),1.38(d,J=7.0Hz,3H).

[0079] Test Example 1 β-D-pyranose-glucopyranosyl-lactic acid has the effect of reducing inflammation in vitro

[0080] 1. Experimental Methods

[0081] Mouse macrophage RAW264.7 cell line was stimulated with 1μg / mL lipopolysaccharide (LPS) to induce inflammatory response for 3h and 24h, and the same concentration of lactic acid (Lac) and β-D-pyranosyl-lactic acid compound Gly-Lac (20mM) were used for intervention. After the stimulation, the cells were collected and the expression levels of inflammatory factors interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and monocyte chemoattractant 1 (MCP-1) were determined by RT-qPCR.

[0082] 2. Experimental results

[0083] As shown in Table 1, the same concentration of Lac and Gly-Lac (20mM) were applied to the LPS-induced in vitro inflammatory model, and the expression levels of inflammatory factors IL-6, TNF-α and MCP-1 were determined by RT-qPCR. The results showed that the expression levels of TNF-α in RAW264.7 cells stimulated by LPS for 3h and MCP-1 and IL-6 in RAW264.7 cells stimulated for 24h were significantly upregulated compared with the normal group (P<0.01); compared with the model group, the high and low concentration groups of Gly-Lac could significantly reduce the expression of IL-6, TNF-α and MCP-1, and the difference was statistically significant (p<0.01); indicating that Gly-Lac has an anti-inflammatory effect.

[0084] Table 1 Effects of high and low concentrations of Lac and Gly-Lac on LPS-induced inflammation in vitro (±s, n=6)

[0085]

[0086] Note: Compared with the blank control group, ## P<0.01, ### P<0.001; compared with the model group, * P<0.05, ** P<0.01, *** P<0.01.

[0087] In summary, the Gly-Lac prepared in the present invention has a significant effect of inhibiting inflammation induced by LPS and is better than lactic acid.

[0088] Experimental Example 2 β-D-pyranose-glucopyranosyl-lactic acid has the effect of treating colitis in mice induced by dextran sulfate sodium salt

[0089] 1. Experimental Methods

[0090] 1.1 Animal Model:

[0091] Forty C57BL / 6 mice were taken and adapted for 1 week. The mice were randomly assigned to four groups, divided into a blank group (PBS), a sodium dextran sulfate group (DSS), a sodium lactate group (DSS+Lac), and a β-D-pyranosyl-lactic acid group (DSS+Gly-Lac). Ten mice per cage were housed at room temperature (standard light cycle, 12 / 12-h light / dark) with free access to food and water. The treatment method was maintained for 7 days. Mice in the DSS+PBS, DSS+Lac, and DSS+Gly-Lac groups received 2% (w / v) DSS in drinking water, while mice in the PBS group received normal drinking water, and the drug-treated groups were gavaged with 15mM Lac or Gly-Lac every day. At the same time, the weight and feces of the mice were recorded every day. The disease activity index (DAI) was scored according to Table 2. The feces of the mice were collected in sterile Eppendorf tubes and stored at -80°C. On the last day, mice were cervically dislocated under anesthesia, blood was collected, colon was removed and its length was measured, and tissues were fixed in 4% paraformaldehyde solution or stored at -80 °C until further analysis.

[0092] 1.2 Measurement indicators

[0093] Disease index determination: The body weight and feces of mice were recorded daily, and their disease activity index (DAI) was scored according to Table 2.

[0094] Table 2 Disease Activity Index (DAI) scoring system.

[0095] score Percentage of weight loss Stool consistency Blood in stool 0 0 normal Negative 1 1-5％ Soft stools Light blue 2 5-10％ Mucoid stool blue 3 10-20％ Diluted stool Dark Blue 4 >20％ —— Naked bloody stool

[0096] Cytokine determination: To quantify cytokines in serum, three relevant cytokines were selected, interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α). They were determined by ELISA and expressed in nanograms per milliliter.

[0097] 2. Measurement results

[0098] 2.1β-D-pyranosyl-glucopyranosyl-lactic acid can alleviate the clinical symptoms of DSS-induced colitis

[0099] The effect of oral administration of β-D-pyranoglucopyranosyl-lactic acid compounds was evaluated in mice with DSS-induced acute colitis model. The body weight of mice treated with DSS decreased over time compared with the water+PBS group. In addition, DSS-induced weight loss was effectively alleviated in the DSS+LAC and DSS+Gly-Lac groups, indicating that lactic acid and β-D-pyranoglucopyranosyl-lactic acid compound treatment significantly prevented DSS-induced weight loss (Table 3). In addition, the results of DAI were consistent with the results of body weight changes (Table 3). DSS treatment also shortened the colon length on the last day (Table 3). Colon shortening was positively correlated with DSS-induced colon inflammation and edema. In addition, the spleen index (spleen / body weight) reflected the severity of colitis. Compared with the control group, treatment with lactic acid and β-D-pyranoglucopyranosyl-lactic acid compounds significantly prolonged the length of the colon and reduced the spleen index (Table 3). Therefore, β-D-pyranoglucopyranosyl-lactic acid treatment significantly improved the disease symptoms of mice with DSS colitis.

[0100] Table 3 Effects of lactic acid and glucose lactic acid on colitis induced by dextran sulfate sodium (DSS) in mice (±s, n=6)

[0101] Group Weight loss ratio Disease Index Spleen index Colon length CON 0.47％±0.012％ —— 0.0042±0.0004 8.22±0.51 DSS <![CDATA[24.09%±2.803% ## ]]> <![CDATA[11.14±0.11 ## ]]> <![CDATA[0.0052±0.001 ### ]]> <![CDATA[5.23±0.73 ## ]]> DSS+Lac <![CDATA[16.42%±3.219% ** ]]> <![CDATA[7.64±0.16 ** ]]> <![CDATA[0.0046±0.00069 ** ]]> 6.91±0.39 DSS+Gly-Lac <![CDATA[17.19%±0.469% ** ]]> <![CDATA[5.96±0.09 ** ]]> <![CDATA[0.0047±0.00045 ** ]]> <![CDATA[7.07±0.43 * ]]>

[0102] Note: Compared with the blank control group, ## P<0.01, ## P<0.001; compared with the model group, * P<0.05, ** P<0.01.

[0103] β-D-glucopyranosyl-lactic acid compounds modulate the immune response in DSS-induced colitis

[0104] To further confirm the anti-inflammatory properties of Gly-Lac, the levels of pro-inflammatory and anti-inflammatory mediators in serum were analyzed. The levels of TNF-α and IL-1β in the DSS group were higher than those in the control group. β-D-pyranoglucopyranosyl-lactic acid treatment also reduced the levels of pro-inflammatory TNF-α, IL-6, and IL-1β compared with the DSS group (P<0.05) (Table 4). These results suggest that β-D-pyranoglucopyranosyl-lactic acid compounds show intestinal anti-inflammatory activity in DSS-induced colitis.

[0105] Table 4 Effects of lactate and glucose lactate on inflammatory cytokines in mouse serum (±s, n=6)

[0106]

[0107] Note: Compared with the blank control group, ## P<0.01, ####P<0.0001; compared with the model group, ** P<0.01, **** P<0.0001.

[0108] Taken together, these results suggest that β-D-glucopyranosyl-lactic acid displays intestinal anti-inflammatory activity in DSS-induced colitis.

[0109] Experimental Example 3 β-D-pyranose-glucopyranosyl-lactic acid has the effect of reducing fatty degeneration of hepatocytes in vitro

[0110] 1. Experimental Methods

[0111] The model of fatty degeneration of normal human liver cells LO2 cells induced by free fatty acids (FFA) was established. When LO2 cells covered the bottom of the dish, 2×10 5 The cells were seeded in 6-well plates at a density of 10 cells / mL and incubated at 37°C and 5% CO 2 After culturing for 24 hours under the conditions, the experimental groups were divided into normal control group, model group, low concentration group of 1.5mM and high concentration group of 15mM lactic acid (Lac) and low concentration group of 1.5mM and high concentration group of 15mM β-D-pyranose glucopyranosyl-lactic acid (Gly-Lac). Normal control group: cultured in DMEM medium containing 10% fetal bovine serum; model group: cultured in medium containing FFA; drug administration group: cultured in medium containing FFA and various high and low concentration compounds. Cultured for 24 hours under each experimental condition.

[0112] Determination of triglyceride (TG) / protein content: (1) discard the remaining culture medium in the well, wash with PBS 1-2 times, add trypsin solution to digest the cells and prepare cell suspension, divide the cell suspension in each well into two parts (cell ① and cell ②), and transfer them into 1.5mL EP tubes respectively; (2) wash the cells twice with 4℃ PBS, centrifuge at 4℃, 1000rpm for 5min, and discard the supernatant; (3) absorb the residual liquid in the EP tube on absorbent paper, add 100μL isopropanol to cell tube ①, shake well and place in an ice box; add 100μL PBS to cell tube ②, shake well and place in an ice box; (4) lyse the cells with an ultrasonic cell crusher at 4℃, centrifuge at 4℃, 13500rpm for 10min, collect the supernatant, use a triglyceride kit (strictly follow the instructions for operation and measurement) to measure the TG content in the supernatant of cell ①, and use a BCA protein quantification kit (strictly follow the instructions for operation and measurement) to measure the protein content of cell ②, and convert it into the ratio of TG / protein content.

[0113] 2. Experimental results

[0114] As shown in Table 5, high and low concentrations of Lac and Gly-Lac (1 mM, 10 mM) were applied to LO 2After 24 hours, the triglyceride content in the cells was determined using a triglyceride kit. The results showed that compared with the blank group, the intracellular triglyceride content in the model group was significantly higher than that in the blank group; compared with the model group, the high and low concentrations of Lac and the high and low concentrations of Gly-Lac groups could significantly reduce the intracellular triglyceride content, and the difference was statistically significant (p<0.01); the reduction effect of the high-dose Gly-Lac group was greater than that of the low-dose group, indicating that β-D-pyranose-glucopyranosyl-lactic acid compounds have the effect of reducing fatty degeneration of hepatocytes in vitro and can inhibit FFA-induced intracellular lipid deposition in a dose-dependent manner.

[0115] Table 5 Effects of high and low concentrations of lactic acid and glucose-lactate on triglyceride accumulation in hepatocytes (±s, n=6)

[0116]

[0117]

[0118] Note: Compared with the blank control group, #### P<0.0001; compared with the model group, ** P<0.01, **** P<0.0001.

[0119] In summary, the compounds prepared in the present invention have β-D-pyranoglucopyranosyl-lactic acid (Gly-Lac), which can improve the pathological changes of the colon of mice with colitis, downregulate proinflammatory cytokines, improve spleen index and have the effect of reducing fatty degeneration of hepatocytes in vitro.

[0120] The above is a further description of the present invention in conjunction with specific embodiments, but these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

Claims

1. Application of β-D-pyranoglucopyranosyl-lactic acid in the preparation of drugs for treating metabolic diseases caused by triglycerides.

2. The use according to claim 1, characterized in that: The medicine also includes other pharmaceutically acceptable adjuvants, carriers or diluents.

3. The use according to claim 1, characterized in that: The drug dosage form is selected from plain tablets, film-coated tablets, sugar-coated tablets, enteric-coated tablets, dispersible tablets, capsules, granules, oral solutions or oral suspensions.

4. The use according to claim 1, characterized in that: The preparation method of β-D-pyranose glucopyranosyl-lactic acid comprises the following steps: using glucose as a raw material to prepare glycosyl trichloroacetimidate, then using D-lactic acid and benzyl bromide as raw materials to prepare benzyl lactate, finally reacting benzyl lactate with glycosyl trichloroacetimidate, and then removing the benzyl group to obtain the target compound.

5. The use according to claim 1, characterized in that: The preparation method of β-D-pyranose glucopyranosyl-lactic acid specifically comprises the following steps: (1) D-lactic acid was dissolved in N,N-dimethylformamide, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was added to the mixture with a syringe under stirring and stirred for 30 min. Benzyl bromide and potassium iodide were slowly added under stirring. After stirring for 18 h at room temperature, the reaction solution was mixed with ethyl acetate and water. After separation, the organic phase was taken, and the aqueous phase was extracted with ethyl acetate for 4 times. The organic phases were combined, washed with 3 volumes of water, and DMF in the organic phase was removed. The organic phase was washed with a saturated sodium chloride solution and dried with anhydrous sodium sulfate. The organic phase was purified by column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain a white transparent liquid Bn-Lac; (2) Mix acetic anhydride and sodium acetate and heat under reflux at 90°C. After they are completely dissolved, slowly add D-glucose and react at 90°C for 4 hours. Cool to room temperature, add ice water by ultrasonication, and filter to obtain a white solid. Recrystallize from anhydrous ethanol to obtain a white solid Glu1. (3) Dissolve the Glu1 obtained in step (2) in anhydrous dichloromethane, add BF3-Et2O and thiophenol at 0°C, react at room temperature for 5 hours, add saturated sodium bicarbonate to stop the reaction, extract with dichloromethane, wash with saturated sodium chloride aqueous solution, spin-dry the organic layer to obtain a yellow oily substance Glu2, and purify it by column chromatography (petroleum ether: ethyl acetate = 3:2) to obtain Glu2; (4) Dissolve the Glu2 obtained in step (3) in anhydrous methanol, add 30% sodium methoxide / methanol solution, react for 15 h, purify by column chromatography (dichloromethane: methanol = 10: 1), add Amberlite IR 120 to adjust the pH to neutral, filter, spin dry the solvent, and vacuum dry to obtain Glu3; (5) Dissolve the Glu3 obtained in step (4) in anhydrous N,N-dimethylformamide, add NaH to the reaction mixture in portions at 0°C, stir at 0°C for 30 min, add benzyl bromide to the reaction mixture, stir the mixture for 22 h, and slowly add cold distilled water to the solution; extract the aqueous phase with dichloromethane, wash the collected organic phase with distilled water and saturated sodium chloride aqueous solution, dry the collected organic phase over Na2SO4, and evaporate the solvent under reduced pressure; purify by column chromatography (petroleum ether:ethyl acetate=20:1) to obtain Glu4; (6) Glu4 obtained in step (5) was dissolved in anhydrous dichloromethane, trichloroacetonitrile and 1,8-diazabicyclo[5.4.0]undec-7-ene were added at 0°C, and after the reaction was completed for 2 hours, Glu5 was obtained by column chromatography (petroleum ether: ethyl acetate = 10:1); (7) Glu5 obtained in step (6) was placed in a round-bottom flask, 20 ml of anhydrous dichloromethane was added to dissolve, Bn-Lac obtained in step (1) was added under ice bath, TMSOTf was added under nitrogen protection, and after reacting at -20°C for 2 h, a saturated sodium chloride aqueous solution and a small amount of triethylamine were added to quench the reaction, the mixture was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether: ethyl acetate = 30:1) to obtain a colorless transparent liquid Glu6; (8) Dissolve the Glu6 obtained in step (7) in 99% methanol, add 10% Pd-C and hydrogenate at room temperature under pressure for 24 hours, remove Pd-C by centrifugation, spin-dry the filtrate and recrystallize it from methanol-ethyl acetate to obtain Glu7, which is β-D-pyranose-lactic acid.

6. The use according to claim 5, characterized in that: The reaction equation of the preparation method is:

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