CN113354561B - Biguanide derivatives and their use and formulations - Google Patents
Biguanide derivatives and their use and formulations Download PDFInfo
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- C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
- C07C279/20—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups containing any of the groups, X being a hetero atom, Y being any atom, e.g. acylguanidines
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- C07C279/26—X and Y being nitrogen atoms, i.e. biguanides
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- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
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- A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a compound of formula I, a deuterio thereof or a pharmaceutically acceptable salt thereof,
Description
Technical Field
The invention belongs to the field of medicines, relates to a biguanide derivative and application and a preparation thereof, and particularly relates to a biguanide derivative used for treating intracellular bacterial infection, diseases caused by intracellular bacterial infection and glucose or lipid metabolism diseases.
Background
Autophagy is a cell-specific recognitionAnd degrading bacteria and viruses, and protecting cells from pathogen attack. Under normal conditions, the autophagosome wrapping the pathogenic bacteria in the cell can be fused with a normal lysosome to form an autophagososome, and various enzymes of the lysosome are utilized to degrade the pathogenic bacteria. However, in pathological situations, certain bacteria have evolved to escape host recognition, such as by inhibiting normal autophagy of cells by blocking lysosomal maturation, thereby promoting their survival. After infection of the host, the bacteria that reside primarily within the cell are called intracellular bacteria, and the persistence of intracellular bacteria is thought to lead to chronic infection. Common bacteria that survive intracellularly include Mycobacterium tuberculosis: (A), (B), (C)Mycobacterium tuberculosis, M. tuberculosis) Salmonella typhi (II)Salmonella typhi, S. typhi) Listeria (Listeria monocytogenes)Listeria) Golden yellow grape coccus (C)Staphylococcus aureus, S. aureus) And helicobacter pylori (H.pylori:Helicobacter pylori, H. pylori) And the like.
H. pyloriIs a gram-negative bacterium which is fixedly planted in the stomach and the duodenum and causes more than half of the population to be infected all over the world,H. pylorihas invasive properties and survives in host cells.H. pyloriVirulence factors such as secreted vacuolating toxin (VacA) damage the activity of a lysosome calcium ion channel TRPML1 of cells, the level of calcium in lysosomes is abnormally increased, hydrogen ions cannot be normally exchanged and ingested, the lysosome acidity is changed due to the environment, and lysosomes with damaged functions cannot normally degrade bacteria in autophagosomes. Living intracellularlyH. pyloriCan re-emerge under appropriate conditions to cause a new round of infection, so that treatment against intracellular bacteria can further improve the treatment ofH. pyloriClearance rate, and reduction of the occurrence of persistent recurrent infection.
Recent studies have shown that metformin, an agonist of adenylate activated protein kinase (AMPK), enhances lysosomal acidification, restores normal autophagy function in cells, and inhibits growth in vitro and in vivoH. pyloriAnd (4) growth. In addition, a plurality of clinical data show that metformin can assist tuberculosis by antagonizing typical intracellular infection of mycobacterium tuberculosisThe treatment of (1). Unfortunately, problems associated with the high water solubility and large amounts of metformin limit its use in the treatment of bacterial infections. Because the current problem of bacterial drug resistance is increasingly severe worldwide, the existence of intracellular bacteria is easy to cause intractable persistent infection, and a more efficient medicine for treating bacterial infection diseases is still needed clinically。
Disclosure of Invention
The biguanide derivatives of the present invention are capable of enhancing the agonistic activity against host cell AMPK as compared to metformin, and are safe, effective, low-toxic agents against intracellular bacteria. Meanwhile, the nanoparticles constructed by the biguanide derivatives of the invention are further improvedH. pyloriClearance rate, can be treatedH. pyloriAssociated persistent and recurrent infections.
The present invention provides in one aspect a compound of formula (I), a deutero-compound thereof, or a pharmaceutically acceptable salt thereof,
Wherein R is a substituted or unsubstituted saturated or unsaturated alkyl group having 10 to 20 carbon atoms.
In some embodiments, R is substituted or unsubstituted C 15-20 Alkyl, substituted or unsubstituted C 15-20 Alkenyl, or substituted or unsubstituted C 15-20 Alkynyl.
In some embodiments of the present invention, the substrate is, R is selected from the group consisting of n-decane, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl and n-eicosyl, n-decenyl, n-undecenyl, n-dodecenyl, n-tridecyl, n-tetradecyl, n-pentadecenyl, n-hexadecyl, n-heptadecenyl, n-octadecenyl, n-nonadecenyl, n-eicosenyl, 1,3-pentadecenyl, 2,5-pentadecenyl, 3,6-pentadecenyl, 4,7-pentadecenyl, 5,8-pentadecenyl, 6,9-pentadecenyl, 7,10-pentadecenyl, n-tetradecadienyl, 5852 zxft 3757-pentadecenyl, 4,7-pentadecenyl, 5,8-pentadecenyl, 3625-pentadecenyl 8,11-pentadecadienyl, 1,3-hexadecadienyl, 2,5-hexadecadienyl, 3,6-hexadecadienyl, 4,7-hexadecadienyl, 5,8-hexadecadienyl, 6,9-hexadecadienyl, 7,10-hexadecadienyl, 8,11-hexadecadienyl, 1,3-heptadecadienyl, 2,5-heptadecadienyl, 3,6-heptadecadienyl, 4,7-heptadecadienyl, 5,8-heptadecadienyl, 6,9-heptadecadienyl, 7,10-heptadecadienyl, 8,11-heptadecadienyl, 58 zxft 6258-heptadecadienyl; 1,3-octadecadienyl, 2,5-octadecadienyl, 3,6-octadecadienyl, 4,7-octadecadienyl, 5,8-octadecadienyl, 6,9-octadecadienyl, 7,10-octadecadienyl, 8,11-octadecadienyl, 9,12-octadecadienyl; 1,3-nonadienyl, 2,5-nonadienyl, 3,6-nonadienyl, 4,7-nonadienyl, 5,8-nonadienyl, 6,9-nonadienyl, 7,10-nonadienyl, 8,11-nonadienyl, 9,12-nonadienyl, 10,13-nonadienyl, 1,3-eicosadienyl, 2,5-eicosadienyl, 3,6-eicosadienyl, 9696 zxft 96-eicosadienyl, 329635 zxft 3235-eicosadienyl, etc 6,9-eicosadienyl, 7,10-eicosadienyl, 8,11-eicosadienyl, 9,12-eicosadienyl, 10,13-eicosadienyl, 1,4,7-heptadecyltrienyl, 2,5,8-heptadecyltrienyl, 3,6,9-heptadecyltrienyl, 4,7,10-heptadecyltrienyl, and 5,8,11-heptadecyltrienyl, n-decynyl, n-undecylenyl, n-dodecylnyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecylyl, n-heptadecynyl, n-octadecynyl, n-nonadecynyl, and n-eicosynyl.
In some embodiments, the compound is:
In yet another aspect, the present invention provides a pharmaceutical composition comprising any of the above compounds, deuterated compounds thereof, or pharmaceutically acceptable salts thereof. In some embodiments, the pharmaceutical composition further comprises one or more of a urease inhibitor, an antibiotic, a proton pump inhibitor, a bismuth agent.
In a further aspect, the present invention provides a use of any of the above-mentioned compounds, deuterated compounds thereof, or pharmaceutically acceptable salts thereof, for the manufacture of a medicament for the treatment of intracellular bacterial infection, a disease caused by intracellular bacterial infection, or a disease of glucose or lipid metabolism. In some embodiments, wherein the intracellular bacteria is selected from the group consisting of mycobacterium tuberculosis, salmonella typhi, listeria, staphylococcus aureus, and helicobacter pylori. In some embodiments, the intracellular bacteria is preferably helicobacter pylori. In some embodiments, the disease caused by intracellular bacterial infection is selected from peptic ulcer, gastritis, gastroesophageal inflammation, non-esophagitis symptomatic gastroesophageal reflux disease, non-ulcer dyspepsia, acid-excess dyspepsia, upper gastrointestinal bleeding, gastric cancer and gastric MALT lymphoma. In some embodiments, the disorder of glucose or lipid metabolism is one or more of obesity, dyslipidemia, hyperglycemia, type I diabetes, or type II diabetes, and diabetic complications.
In another aspect, the present invention provides a self-assembled nanoparticle, comprising: (a) A compound, deuteride, or pharmaceutically acceptable salt thereof, of any of the above: (b) C 10-20 Fatty acid, wherein said C 10-20 The fatty acid and the compound, the deuteron thereof or the pharmaceutically acceptable salt thereof form a carrier structure of the self-assembled nanoparticle; and, (c) a urease inhibitor or antibiotic entrapped within the self-assembled nanoparticles. In some embodiments, the C is 10-20 The fatty acid is selected from capric acid, 9-decenoic acid, undecanoic acid, 10-undecenoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, or combinations thereof. In some embodiments, the C is 10-20 The fatty acid is preferably oleic acid, linoleic acid, linolenic acid. In some embodiments, the C is 10-20 The fatty acid is preferably linoleic acid. In some embodiments, the urease inhibitor is ebselen or acetohydroxamic acid. In some embodiments, the urease inhibitor is preferably ebselen. In some embodiments, the antibiotic is selected from amoxicillin, clarithromycin, metronidazole, tetracycline, levofloxacin, furazolidone, or a combination thereof.
In some embodiments, the self-assembling nanoparticle further comprises: (d) A polysaccharide coated on the exterior of the self-assembled nanoparticle. In some embodiments, the polysaccharide is selected from the group consisting of fucoidan, mannan, dextran, mannitol. In some embodiments, the polysaccharide is preferably Fucoidan (FU). In some embodiments, the self-assembled nanoparticle comprises: linoleic acid, which forms a carrier structure of the self-assembled nanoparticle with a biguanide derivative MU or ML; ebselen, which is entrapped and embedded in the self-assembled nanoparticles. In some embodiments, the self-assembled nanoparticle comprises: linoleic acid, which forms a carrier structure of the self-assembled nanoparticle with a biguanide derivative MU or ML; and ebselen, which is entrapped in the self-assembled nanoparticles; and FU which is coated outside the self-assembled nanoparticle. In some embodiments, the self-assembled nanoparticles have a particle size of 100 to 180 nm. In some embodiments, the particle size is preferably 125 nm or 150 nm. In some embodiments, the encapsulation efficiency of the self-assembled nanoparticle is greater than 75%. In some embodiments, the encapsulation efficiency of the self-assembled nano is preferably greater than 80%. In some embodiments, the self-assembled nano has a drug loading of greater than 55%.
The nanoparticles of the invention are taken up by endocytosis of host cells, thereby interfering with intracellularityH. pyloriCell membrane permeability of (2) to rupture bacteria and inhibitH. pyloriExerts resistance to internal urease activityH. pyloriThe effect is that, at the same time, the biguanide derivatives entering the cells can restore the degradation effect of the autophagosomes of the cells, and the advantages of the combined nanoparticles enhance the cellsH. pyloriThe cleaning effect is achieved, thereby eradicatingH. pyloriThe purpose of (1).
Drawings
FIG. 1: a. nuclear magnetic resonance hydrogen spectrum of intermediate 1a of biguanide derivative ML; b. mass spectrum of intermediate 1a of biguanide derivative ML.
FIG. 2: a. nuclear magnetic resonance hydrogen spectrum of intermediate 1b of biguanide derivative ML; b. mass spectrum of intermediate 1b of biguanide derivative ML.
FIG. 3: a. a nuclear magnetic resonance hydrogen spectrum of the biguanide derivative ML; b. mass spectrum of biguanide derivative ML.
FIG. 4: a. nuclear magnetic resonance hydrogen spectrum of intermediate 3a of biguanide derivative MU; b. mass spectrum of intermediate 3a of biguanide derivative MU.
FIG. 5: a. nuclear magnetic resonance hydrogen spectrum of intermediate 3b of biguanide derivative MU; b. mass spectrum of intermediate 3b of biguanide derivative MU.
FIG. 6: a. nuclear magnetic resonance hydrogen spectrum of biguanide derivative MU; b. mass spectrum of biguanide derivative MU.
FIG. 7: effects of Metformin (MET) and biguanide derivatives (ML and MU) on the level of adenylate activated protein kinase (AMPK) activity in GES-1 human gastric epithelial cells. After MET, ML and MU are added to treat GES-1 cells, the steps of the enzyme-linked immunosorbent assay kit for human phosphorylated adenylate activated protein kinase are adopted for determination and comparison. As compared with control groupP<0.05, denotesP<0.001; in comparison with MET, #####indicatesP<0.001; compared with MU,. RepresentsP<0.01。
FIG. 8: metformin (MET) and biguanide derivatives (ML and MU) on RAW 264.7 macrophage intracellularH. pyloriEvaluation of the effect of removal of (1). In the constructed intracellular bacterial model, MET, ML and MU are added to treat GES-1 cells, and then the determination and comparison are carried out by adopting a coating plate method. As compared with control groupP<0.05; * DenotesP<0.001; in comparison with MET, # denotesP<0.05, #### # representsP<0.001; compared with MU,. RepresentsP<0.01。
FIG. 9: a. mean fluorescence intensity of biguanide derivative nanoparticles and C6 uptake at RAW 264.7 macrophages. Adding free coumarin-6 (C6), C6 labeled nanoparticles (ML-LA/C6 NPs and Fu/ML-LA/C6 NPs) andafter the cells were incubated with 4h, 1X 10 cells were collected 4 The fluorescence intensity of C6 was measured by flow cytometry for each cell and compared. b. Biguanide derivative nanoparticles and C6 mean fluorescence intensity taken up by GES-1 human gastric epithelial cells. Adding C6, C6 labeled nanoparticles (ML-LA/C6 NPs and Fu/ML-LA/C6 NPs) and incubating with cells for 4h, collecting 1 × 10 4 The fluorescence intensity of C6 was measured by flow cytometry for each cell and compared. Represents P compared with control group<0.001; in comparison with C6, # # denotesP<0.01, ##### # representsP<0.001; compared with ML-LA/C6,. RepresentsP<0.05,. RepresentsP<0.01,. RepresentsP<0.001。
FIG. 10: a. biguanide derivative nano particle in RAW 264.7 macrophageH. pyloriEvaluation of the effect of removal of (1). In the constructed intracellular bacteria model, the RAW 264.7 cells are added with the drug treatment, and then the determination and comparison are carried out by adopting a coating plate method. b. Biguanide derivative nano particle pair GES-1 human gastric epithelial cellH. pyloriEvaluation of the effect of removal of (1). In the constructed intracellular bacterial model, MET and ML are added to treat GES-1 cells, and then the determination and comparison are carried out by adopting a coating plate method. As compared with control groupP<0.01, denotesP<0.001; in comparison with MET, # denotesP<0.05, # # denotesP<0.01, ##### # representsP<0.001; compared with ML,. RepresentsP<0.01,. RepresentsP<0.001; in comparison with ML + LA + EB,. Diamond-solid showsP<0.05,. Diamond-solid. Sup. TheP<0.01; in contrast to ML-LA/EB,&to representP<0.05。
FIG. 11 biguanide derivative nanoparticles in RAW 264.7 macrophagesH. pyloriEvaluation of the cleaning effect of (4). In the constructed intracellular bacterial model, the results are observed by laser confocal after adding the drug treatment RAW 264.7 cells. Blue fluorescence labeling cell nucleus and green fluorescence labelingH. pyloriAnd marking lysosome by red fluorescence, wherein the overlay image is the overlay result of the three images.
FIG. 12 biguanide derivative nanoparticle pairs in GES-1 human gastric epithelial cellsH. pyloriEvaluation of the clearing effect. Adding drug treatment into the constructed intracellular bacterial modelAfter GES-1 cells, the results were observed by confocal laser observation. Blue fluorescence labeling of cell nuclei and green fluorescence labelingH. pyloriAnd marking lysosome by red fluorescence, wherein the overlay image is the overlay result of the three images.
Detailed Description
The term "alkyl" refers to a straight or branched chain hydrocarbon group having the indicated number of carbon atoms. E.g. C 10-20 Alkyl refers to a straight or branched chain hydrocarbon group having 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms, including but not limited to C 10 Alkyl radical, C 11-12 Alkyl radical, C 13-14 Alkyl radical, C 15-20 Alkyl radical, C 17 Alkyl radical, C 16-18 Alkyl and C 19-20 An alkyl group. Examples of the saturated alkyl group include n-decane, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl and n-eicosyl. In some embodiments, the alkyl group may be substituted with a halogen, a hydroxyl group, a mercapto group, or a heteroatom. "halogen" means fluorine, chlorine, bromine, or iodine. Heteroatoms are intended to include oxygen, nitrogen, sulfur. Examples of unsaturated alkyl groups include alkenyl, alkynyl, or combinations thereof.
The term "alkenyl" refers to a straight or branched chain hydrocarbyl group having the number of carbon atoms indicated by the prefix and containing at least one double bond. For example, (C) 2 -C 6 ) Alkenyl is intended to include ethenyl, propenyl, and the like. C 10-20 Alkenyl refers to straight or branched chain alkenyl of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms and includes, but is not limited to, C 10 Alkenyl radical, C 11-12 Alkenyl radical, C 13-14 Alkenyl radical, C 15-20 Alkenyl radical, C 17 Alkenyl radical, C 16-18 Alkenyl and C 19-20 An alkenyl group. Examples of alkenyl groups include n-decenyl, n-undecenyl, n-dodecenyl, n-tridecenyl, n-tetradecenyl, and C 15-20 An alkenyl group. C 15-20 Alkenyl comprises a straight or branched hydrocarbon group having 1,2 or 3 double bonds. C 15-20 The alkenyl group includes n-pentadecenyl, n-hexadecylene, n-heptadecenyl and n-octadecenylNonadecenyl and eicosenyl, 1,3-pentadecadienyl, 2,5-pentadecadienyl, 3,6-pentadecadienyl, 4,7-pentadecadienyl, 5,8-pentadecadienyl, 6,9-pentadecadienyl, 7,10-pentadecadienyl, 8,11-pentadecadienyl, 1,3-hexadecadienyl, 2,5-hexadecadienyl, 5678 zxf5678-hexadecadienyl, 4,7-hexadecadienyl, 5,8-hexadecadienyl, 9696 zxft 6296-hexadecadienyl, 329635 zxft 35-hexadecadienyl, 8,11-hexadecadienyl, 26 zxft 345274-heptadecadienyl, 34zxft 6296-hexadecadienyl, 35zjft 354258-heptadecadienyl, 35zzft 355258-heptadecadienyl, 35zxft 4258-heptadecadienyl, 35zjft 4258-heptadecadienyl, 35zjfet 4258-heptadecadienyl; 1,3-octadecadienyl, 2,5-octadecadienyl, 3,6-octadecadienyl, 4,7-octadecadienyl, 5,8-octadecadienyl, 6,9-octadecadienyl, 7,10-octadecadienyl, 8,11-octadecadienyl, 9,12-octadecadienyl; 1,3-nonadienyl, 2,5-nonadienyl, 3,6-nonadienyl, 4,7-nonadienyl, 5,8-nonadienyl, 6,9-nonadienyl, 7,10-nonadienyl, 8,11-nonadienyl, 9,12-nonadienyl, 10,13-nonadienyl, 5678 zxf5678-eicosadienyl, 2,5-eicosadienyl, 3,6-eicosadienyl, 9696 zxft 6296-eicosadienyl, 9635 zxft 3235-eicosadienyl, 6,9-eicosadienyl, heptadecadienyl, 3426 zxft 3474-heptadecadienyl, 34zxft 6296-heptadecadienyl, 35zzft 354258-heptadecadienyl, 35zzft 4258-heptadecadienyl, 35zjfet-4258-heptadecadienyl, 345258-heptadecadienyl, 34zxft 4235-heptadecadienyl, and 35zjfet-4258-heptadecadienyl. In some embodiments, the alkenyl group may be substituted with a halogen, a hydroxyl group, a mercapto group, or a heteroatom. "halogen" means fluorine, chlorine, bromine, or iodine. Heteroatoms are intended to include oxygen, nitrogen, sulfur.
Similarly, the term "alkynyl" refers to a linear or branched hydrocarbyl group containing at least one triple bond and having the number of carbon atoms indicated by the prefix. Examples of alkynyl groups include ethynyl, 1-and 3-propynyl, 3-butynyl, and the higher homologs and isomers. C 10-20 Alkynyl means 10Straight or branched alkynyl of 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms and includes, but is not limited to, C 10 Alkynyl, C 11-12 Alkynyl, C 13-14 Alkynyl, C 15-20 Alkynyl, C 17 Alkynyl, and C 16-18 Alkynyl and C 19-20 Alkynyl. Examples of alkynyl groups include n-decynyl, n-undecylenyl, n-dodecylenyl, n-tridecynyl, n-tetradecynyl, n-pentadecynyl, n-hexadecylenyl, n-heptadecynyl, n-octadecynyl, n-nonadecynyl, and n-eicosynyl. In some embodiments, the alkynyl group can be substituted with a halogen, a hydroxyl group, a thiol group, or a heteroatom. "halogen" means fluorine, chlorine, bromine, or iodine. Heteroatoms are intended to include oxygen, nitrogen, sulfur.
Some of the compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. "hydrate" refers to a complex formed by the association of molecules or ions of water molecules and solutes. "solvate" refers to a complex of a solvent molecule and a solute bound by a molecular or ionic combination. The solvent may be an organic compound, an inorganic compound, or a mixture thereof. Solvates are intended to include hydrates. Some examples of solvents include, but are not limited to, methanol (MT), N-Dimethylformamide (DMF), tetrahydrofuran (THF), dichloromethane (DCM), trifluoroacetic acid (TFA), dioxane (Diox), dimethyl sulfoxide (DMSO), and water. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Some compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
Formulations and administration
The present invention provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier or excipient and a compound described herein, or a pharmaceutically acceptable salt or solvate thereof. In exemplary embodiments, the present invention provides pharmaceutical formulations comprising a compound described herein. In one embodiment, the pharmaceutical formulation or composition comprises a compound of formula (I):
Wherein the R group is as defined above.
The invention also contemplates the use of pharmaceutically acceptable deuterated or other non-radioactively substituted compounds of the compounds. Deuterium substitution is to replace one or more or all hydrogen in the active molecular group of the medicine with isotope deuterium, and because the deuterium is non-toxic and non-radioactive, and is stabilized about 6~9 times compared with carbon-hydrogen bond, the deuterium substitution can seal the metabolic site and prolong the half-life period of the medicine, thereby reducing the therapeutic dose, and simultaneously does not influence the pharmacological activity of the medicine, and the deuterium substitution is considered as an excellent modification method.
These compounds will generally be useful in the treatment of human diseases. However, they may also be used to treat similar or identical indications in other animals. The compounds described herein can be administered by different routes, including injection (intravenous, intraperitoneal, subcutaneous, and intramuscular), oral, transdermal, rectal, by approach, or by inhalation. These dosage forms should allow the compound to reach the target cells. Other factors are well known in the art and include such considerations as toxicity and dosage forms which prevent the compound or composition from exerting its effect.
In some embodiments, the compositions will comprise pharmaceutically acceptable carriers or excipients, such as fillers, binders, disintegrants, glidants, lubricants, complexing agents, solubilizing agents, and surfactants, which may be selected to facilitate administration by a particular route. Examples of carriers include calcium carbonate, calcium phosphate, various sugars (e.g., lactose, glucose, or sucrose), various types of starch, cellulose derivatives, gelatin, lipids, liposomes, nanoparticles, and the like. The carrier also includes physiologically compatible liquids as solvents or for suspension, including, for example, sterile aqueous solutions for injection (WFI), physiological saline solutions, dextrose solutions, hank's solution, ringer's solution, vegetable oils, mineral oils, animal oils, polyethylene glycols, liquid paraffin, and the like. Excipients may also include, for example, gumsSilicon dioxide, silica gel, talc, magnesium silicate, calcium silicate, sodium aluminosilicate, magnesium trisilicate, powdered cellulose, macrocrystalline cellulose, carboxymethylcellulose, croscarmellose sodium, sodium benzoate, calcium carbonate, magnesium carbonate, stearic acid, aluminum stearate, calcium stearate, magnesium stearate, zinc stearate, sodium stearyl fumarate, matting powder (syloid), stearowet C, magnesium oxide, starch, sodium carboxymethyl starch, glycerol monostearate, glycerol dibehenate, glycerol palmitostearate, hydrogenated vegetable oil, hydrogenated cottonseed oil, castor seed oil mineral oil, polyethylene glycol (such as PEG 4000-8000), polyoxyethylene glycol, poloxamer, povidone, crospovidone, croscarmellose sodium, alginic acid, casein, divinylbenzene copolymer of methacrylic acid, docusate sodium, cyclodextrin (such as 2-hydroxypropyl-delta-cyclodextrin), polysorbate (such as polysorbate 80), cetrimide, d-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS), magnesium sulfate, sodium lauryl sulfate, polyethylene glycol ether, fatty acid esters of polyethylene glycol, or polyoxyethylene sorbitan fatty acid esters (such as Tween) ® ) Polyoxyethylene sorbitan fatty acid esters, sorbitan fatty acid esters (such as sorbitan fatty acid esters from fatty acids like oleic, stearic or palmitic acid), mannitol, xylitol, sorbitol, maltose, lactose monohydrate or spray dried lactose, sucrose, fructose, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, dextrates, dextrans, dextrins, dextrose, cellulose acetate, maltodextrin, dimethicone, polydextrose, chitosan, gelatin, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), hydroxyethylcellulose, and the like.
Pharmaceutical formulations may be presented in unit dosage form containing a predetermined amount of active ingredient per unit dose. Such unit doses may contain, for example, 0.5 mg to 1 g (preferably 1 mg to 700 mg, more preferably 5 mg to 100 mg) of a compound of the present invention (in free form, in any form of solvate (including hydrate), or salt), in amounts that depend on the disorder, route of administration, age, weight, and condition of the patient being treated. Preferred unit dose formulations are those containing a daily dose, a weekly dose, a monthly dose, sub-doses thereof or appropriate proportions of the active ingredients. Moreover, such pharmaceutical formulations may be prepared by any of the methods well known in the art of pharmacy.
The pharmaceutical formulations may be presented in a form suitable for administration by any suitable route, for example by the oral (including capsules, tablets, liquid filled capsules, disintegrating tablets, immediate release tablets, sustained release tablets, controlled release tablets, oral strips, solutions, syrups, buccal tablets and sublingual tablets), nasal, pulmonary, rectal, vaginal, topical (including transdermal) or injection (including subcutaneous, intramuscular, intravenous or intradermal) route. These formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier, excipient or diluent. Generally, carriers, excipients, or diluents used in pharmaceutical formulations are "nontoxic" in the sense that they are safe for administration in amounts found in pharmaceutical compositions, and "inert" in the sense that they do not appreciably react with or cause an undesirable effect on the therapeutic activity of the active ingredient.
The amount of each compound administered can be determined by standard procedures and takes into account, for example, the following factors: compound activity (in vitro activity, e.g. Compound IC) 50 Target, or in vivo activity in an animal pharmacodynamic model), pharmacokinetic results in an animal model (e.g., biological half-life or bioavailability), age, size, and weight of the subject, and subject-related diseases. The importance of these factors, as well as others, is well known to those skilled in the art. Generally, the dosage will range from about 0.01 to 50 mg/kg, or about 0.1 to 20 mg/kg. Multiple dose administration may be used.
The compounds described herein may also be used in combination with other therapies for the treatment of the same diseases. Such combination use includes administering the compound and one or more other therapeutic agents at different times, or administering the compound and one or more other therapeutic agents in combination. In some embodiments, the dosage of one or more compounds of the invention or other therapeutic agents used in combination can be modified by methods well known to those skilled in the art, for example, by reducing the amount of the compound or therapeutic agent used relative to that used alone.
It is to be understood that combination includes use with other therapies, drugs, medical procedures, and the like, wherein the other therapies or procedures can be administered at a time different from the compounds described herein (e.g., within a short time, such as within several hours (e.g., within 1,2, 3, 4-24 hours), or within a longer time (e.g., within 1-2 days, 2-4 days, 4-7 days, 1-4 weeks)), or at the same time as a compound of the invention. Combinations also include use with a therapy or medical procedure (e.g., surgery) that is administered once or infrequently, and the compounds of the invention are administered shortly or longer before or after the other therapy or procedure. In some embodiments, the invention provides for the delivery of a compound of the invention and one or more additional pharmacotherapeutic agents administered by different routes, or administered by the same route. Co-administration by any route of administration includes delivery of a compound of the invention and one or more other pharmacotherapeutic agents delivered together in any formulation by the same mode of administration, including formulations in which the two compounds are chemically linked together so that they maintain their therapeutic activity upon administration. In one aspect, the other drug therapy can be co-administered with a compound described herein. Co-administration of the combination includes the use of a common formulation or formulations of the compounds that are chemically combined together, or the administration of two or more compounds in different formulations by the same or different routes within a short period of time of each other (e.g., within 1 hour, 2 hours, 3 hours, up to 24 hours). Co-administration of the separate formulations includes co-administration by delivery from one device, e.g., the same inhalation device, the same syringe, etc., or administration from separate devices within a short time of each other. Co-formulation of the compounds described herein with one or more additional pharmacotherapeutic agents administered by the same route includes materials prepared together so that they can be administered by one device, including different compounds combined in one formulation, or compounds modified to be chemically linked but still maintain their biological activity. Such chemically linked compounds may have a linker which is substantially retained in vivo, or which breaks down in vivo to separate the two active components. In some embodiments, the compounds of the present invention may be used with urease inhibitors, antibiotics, proton pump inhibitors, bismuth agents, or combinations thereof. The antibiotic is selected from amoxicillin, clarithromycin, metronidazole and tetracycline. In some embodiments, the proton pump inhibitor is selected from lansoprazole, omeprazole, pantoprazole, rabeprazole, and esomeprazole. In some embodiments, the bismuth agent is selected from the group consisting of bismuth salicylate, bismuth potassium citrate, and bismuth pectin. In some embodiments, the urease inhibitor is ebselen or acetohydroxamic acid.
Adenylate activated protein kinase (AMPK)
AMPK is a cellular energy receptor and regulator, and plays an important role in maintaining cellular metabolic balance. The activated AMPK can enhance the catabolism of the body to promote the generation of ATP and inhibit the anabolism to reduce the consumption of ATP by acute regulation of key enzyme activities in metabolic links and chronic regulation of the expression of key transcription factors. Evidence for the regulatory role of AMPK on glucose and lipid metabolism makes it a potential drug target for the treatment of diabetes and metabolic syndrome. AMPK inhibits hepatic gluconeogenesis and lipid production, while reducing hepatic lipid deposition by increasing lipid oxidation, thereby improving glucose and lipid profiles. Studies have shown that leptin and adiponectin (adipokines) regulate glucose and lipid metabolism by activating AMPK, thereby exerting their anti-diabetic effects. Among them, leptin stimulates muscle fatty acid oxidation by direct activation of AMPK and via hypothalamic adrenergic pathways, adiponectin stimulates glucose uptake and fatty acid oxidation in vitro by activation of AMPK. In addition, AMPK exerts its hypoglycemic effect by inhibiting the expression of gluconeogenesis critical genes such as genes encoding phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6 Pase). And at the pharmacological level, the concept of AMPK as a target for the treatment of metabolic syndrome has been supported by a large number of studies: two major classes of existing antidiabetic drugs, the thiazolidinediones (rosiglitazone, troglitazone and pioglitazone) and biguanides (metformin and phenformin) activate AMPK in both in vitro cell experiments as well as in vivo. There is evidence that AMPK mediates the antidiabetic effects of rosiglitazone. The research proves that the metformin can activate the AMPK in vitro and in vivo by inhibiting the compound I, and if the upstream kinase LKBl of the AMPK is knocked out, the blood sugar reducing effect of the metformin is completely prevented, so that the key effect of the AMPK in mediating the antidiabetic effect of the metformin is proved. Many AMPK activators (e.g., a-769662 and PT 1) are capable of exerting an anti-diabetic effect in vivo. The compounds of the present invention are potent AMPK activators.
AMPK has been used as a promising target for the treatment of several renal diseases. AMPK is reported to be a modulator of several ion channels, transporters and pumps in the kidney, and treatment with AMPK activators is beneficial for preventing kidney damage in various disease settings. Furthermore, AMPK activation induces autolysis in cells, and this has been demonstrated to have a protective effect on the kidney in several animal models.
The term "diseases of glucose or lipid metabolism" refers to the metabolic syndrome caused by abnormalities in glucose metabolism or lipid metabolism. In some embodiments of the invention, a "disorder of glucose or lipid metabolism" refers to a disorder that can be treated by modulating the activity of AMPK (e.g., AMPK activation). "diseases of glucose or lipid metabolism" which may be treated by the compounds of the present invention include, but are not limited to, obesity, nephropathy, dyslipidemia, hyperglycemia, type I diabetes, type II diabetes and diabetic complications.
The term "C 10-20 Fatty acids "generally refer to straight or branched chain fatty acids of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms and include, but are not limited to, C 10 Fatty acid, fatty acid,C 11-12 Fatty acid, C 13-14 Fatty acid, C 15-20 Fatty acid, C 18 Fatty acid, C 16-18 Fatty acids, and C 19-20 A fatty acid. C 10-20 Examples of fatty acids include, but are not limited to: capric acid, 9-decenoic acid, undecanoic acid, 10-undecenoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, or linolenic acid. In a preferred embodiment, C 10-20 The fatty acid is C 18 Mono-and/or di-unsaturated fatty acid.
The present invention is further explained below by way of specific examples, but the present invention is not limited to the examples.
Example 1 Synthesis and characterization of biguanide derivatives ML
An exemplary synthetic route for the biguanide derivatives ML of the present invention is as follows:
5.6 g of linoleic acid, 4.8 g of N-tert-butyl-1,2-ethylenediamine, 4.6. 4.6 g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and g of 1-hydroxybenzotriazole were taken, anhydrous dichloromethane was added, the reaction was performed overnight at room temperature, and after drying and concentration, purification was performed by column chromatography to obtain intermediate 1a 7.4g with a yield of 88%. The structure verification is shown in figure 1:
1 H NMR (400 MHz, DMSO) δ 7.75 (d, J = 5.0 Hz, 1H), 6.76 (d, J = 5.4 Hz, 1H), 5.39 – 5.29 (m, 4H), 3.05 (dd, J = 12.2, 6.1 Hz, 2H), 2.97 – 2.89 (m, 2H), 2.74 (t, J = 6.3 Hz, 2H), 2.02 (dd, J = 13.6, 6.7 Hz, 6H), 1.48 (dd, J = 14.3, 7.3 Hz, 2H), 1.38 – 1.19 (m, 14H), 0.85 (t, J = 7.0 Hz, 3H)；ESI-MS m/z: 445.40 [M+Na] + 。
7.4g of the above compound 1a was dissolved in methylene chloride, and trifluoroacetic acid was added thereto to react at room temperature, followed by sufficient concentration and drying to obtain intermediate 1b 4.8 g in 85% yield. The structural verification is shown in fig. 2:
1 H NMR (400 MHz, DMSO) δ 8.01 (t, J = 5.5 Hz, 1H), 7.90 (s, 3H), 5.48 – 5.26 (m, 4H), 3.28 (q, J = 6.3 Hz, 2H), 2.91 – 2.80 (m, 2H), 2.74 (t, J = 6.2 Hz, 2H), 2.08 (dd, J = 13.6, 5.9 Hz, 2H), 2.02 (dd, J = 13.4, 6.7 Hz, 4H), 1.50 (dd, J = 14.1, 7.1 Hz, 2H), 1.32 – 1.09 (m, 14H), 0.90 – 0.78 (m, 3H)；ESI-MS m/z: 323.30 [M] + 。
adding 4.8 g of the intermediate 1b, 2.21 g dicyandiamide and 4.26 g ferric chloride into dioxane, reacting at 100 ℃ overnight, drying, concentrating and purifying by column chromatography to obtain the product 1.9 g with 31% yield. The structure verification is shown in fig. 3:
1 H NMR (400 MHz, DMSO) δ 8.06 (s, 2H), 7.86 (s, 1H), 7.47 (s, 1H), 5.42 – 5.22 (m, 4H), 3.23 – 3.07 (m, 4H), 2.73 (t, J = 6.1 Hz, 2H), 2.10 – 1.93 (m, 6H), 1.47 (s, 2H), 1.30 (dd, J = 36.0, 16.5 Hz, 18H), 0.86 (t, J = 6.6 Hz, 3H)；ESI-MS m/z:408.35 [M+2H] + 。
example 2 Synthesis and characterization of biguanide derivatives MU
An exemplary synthetic route for the biguanide derivatives MU of the present invention is as follows:
3.7 g of undecanoic acid, 4.8 g of N-tert-butyl-1,2-ethylenediamine, 4.6 g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 3.2 g of 1-hydroxybenzotriazole were taken, anhydrous dichloromethane was added, reacted overnight at room temperature, dried and concentrated, and then purified by column chromatography to obtain 5.9 g of intermediate 3a, yield 90%, structural verification is shown in FIG. 4:
1 H NMR (400 MHz, DMSO) δ 6.16 (s, 1H), 5.91 (s, 1H), 3.70 (s, 1H), 3.59 – 3.58 (d, J = 4.0 Hz, 1H), 3.53 – 3.51 (t, J = 4.0 Hz, 1H), 3.44 – 3.42 (t, J = 4.0 Hz, 1H), 2.32 – 2.29 (t, J = 4.0 Hz, 2H), 1.56 (dd, J = 14.0, 7.0 Hz, 2H), 1.33 (s, 9H), 1.29 (m, 14H), 0.96 – 0.94 (t, J = 4.0 Hz, 3H)；ESI-MS m/z: 351.32 [M+Na] + 。
5.8 g of the compound 3a was dissolved in dichloromethane, and trifluoroacetic acid was added thereto to react at room temperature, followed by sufficient concentration and drying to obtain 3.4 g of intermediate 3b in 85% yield. The structural verification is shown in fig. 5:
1 H NMR (400 MHz, DMSO) δ 7.02 (s, 1H), 3.69 – 3.58 (dt, J = 35.2, 8.0 Hz, 2H), 2.83 – 2.80 (t, J = 8.0 Hz, 2H), 2.18 – 2.16 (d, J = 8.0 Hz, 4H), 1.81 (s, 2H), 1.50 (dd, J = 14.2, 7.0 Hz, 2H), 1.30 (m, 14H), 0.96 – 0.94 (m, 3H)；ESI-MS m/z: 229.31 [M] + 。
3.2 g of the intermediate 3b, 2.2 g dicyandiamide and 4.2 g ferric chloride are added into dioxane to react at 100 ℃ overnight, and after drying and concentration, the product is purified by column chromatography, so that 1.5 g is obtained with the yield of 35%. The structural verification is shown in fig. 6:
1 H NMR (400 MHz, DMSO) δ 6.66 (s, 1H), 5.05 (s, 1H), 4.65 (s, 1H), 3.84 (s, 1H), 3.80 (s, 1H), 3.68 (s, 1H), 3.59 – 3.57 (d, J = 8.0 Hz, 1H),2.30 – 2.27 (t, J = 6.1 Hz, 2H), 1.50 (dd, J = 14.0, 7.0 Hz, 2H), 1.37 – 1.00 (m, 14H), 1.00 (s, 1H), 0.95 – 0.94 (t, J = 4.0 Hz, 3H), 0.54 (s, 2H)；ESI-MS m/z:314.29 [M+2H] + 。
example 3 activation of adenylate-activated protein kinase by biguanide derivatives
The activation of adenylate activated protein kinase (AMPK) by the biguanide derivatives was determined using the Elisa kit method. GES-1 human gastric epithelial cells were seeded in 12-well plates and cultured for 24h, the medium was aspirated off and the drugs (i.e., MET at a concentration of 100. Mu.g/mL and biguanide derivatives ML and MU at a concentration of 100. Mu.g/mL) were added and incubation continued for 24h. Centrifuging to collect cells, adding PBS, blowing, beating, mixing uniformly, repeatedly freezing and thawing at-20 ℃ for 5 times, and detecting the AMPK phosphorylation level of GES-1 cells in each group according to a method shown in a use instruction of a human phosphorylation adenylate activated protein kinase enzyme-linked immunosorbent assay kit. The degree of AMPK activation by the drug group was expressed as the degree of phosphorylated AMPK in the drug group cells relative to the level of phosphorylated AMPK in the blank group cells, and the results are shown in fig. 7. After the treatment with the biguanide derivatives MU and ML of the present invention, the phosphorylation level of AMPK of GES-1 cells was significantly enhanced and was more active than the metformin hydrochloride positive control group. In addition, compared with MU, ML has more significant activation effect on AMPK, and ML with AMPK enhancing activity can better assist antibacterial drugs in clearing intracellular bacteria.
Example 4 Effect of biguanide derivatives on the eradication of intracellular bacteria
Quantitative evaluation of biguanide derivatives in cells by the spread plate methodH. pyloriThe cleaning effect of (1). Inoculating GES-1 human gastric epithelial cells into a cell pore plate for culturing for 24h, and collectingH. pyloriAdding the bacterial suspension into a pore plate to be incubated with cells, removing the culture medium by suction, and adding a dual-resistant-free culture medium containing gentamicin to kill cells which do not enter the cellsH. pyloriThe medium was aspirated and washed with sterile PBS, and the culture was continued for 24h by the addition of the drugs (i.e., metformin hydrochloride MET at a concentration of 100. Mu.g/mL and biguanide derivatives ML and MU at a concentration of 100. Mu.g/mL). Adding 0.1% tea saponin solution to lyse cells, and collecting cell lysate for use. Coating a part of cell lysate on a plate, culturing at 37 ℃ in a microaerobic environment for 72 h, and quantifying intracellularly by using a plate counting methodH. pyloriThe results are shown in FIG. 8. In cells after ML and MU treatmentH. pyloriThe levels were all significantly reduced and were lower than the bacterial levels after MET treatment. ML decreased intracellular bacterial levels more significantly than the MU group, and ML was more potent in decreasing intracellular bacterial load.
Example 5 preparation and characterization of self-assembled nanoparticles of biguanide derivatives.
4 kinds of self-assembled nano particles (ML-LA NPs, ML-LA/EB NPs, FU/ML-LA NPs and FU/ML-LA/EB NPs) of biguanide derivatives are prepared by a selective nano precipitation method. The preparation method comprises the following steps: taking DMF solutions of ML, LA and EB with different volumes, mixing, fixing the concentration of ML and LA to be 25 mM, adding or not adding EB solution (6.2 mM) to ensure that the mass ratio of ML to EB is 6:1, dripping into 900 mu L pure water or FU water solution (1 mg/mL) under stirring, and continuing stirring for 2 min to obtain 4 biguanide derivative self-assembled nanoparticles. The particle size, PDI and zeta potential of the 4 kinds of nanoparticles were measured, and as shown in Table 1, the 4 kinds of nanoparticles all had smaller particle size and PDI, wherein ML-LA NPs and ML-LA/EB NPs have positive charges, and the particle size was about 125 nm. FU/ML-LA NPs and FU/ML-LA/EB NPs have negative charges, and the particle sizes of the two are obviously about 25 nm compared with ML-LA NPs and ML-LA/EB NPs, which proves the successful coating of FU.
TABLE 1 particle size, PDI and potential of the nanoparticles
A quantitative analysis method of 4 types of nanoparticles is established by adopting a high performance liquid chromatography, and the encapsulation efficiency and the drug loading rate of each component are measured, and the results are shown in Table 2, wherein the encapsulation efficiency of 4 types of nanoparticles is higher than 80%, and the drug loading rate is higher than 55%.
TABLE 2 encapsulation efficiency and drug loading of nanoparticles
Example 6 cellular uptake Effect of biguanide derivative nanoparticles
The uptake of nanoparticles (prepared according to example 5) by RAW 264.7 macrophages and GES-1 human gastric epithelial cells was quantified using flow cytometry. Culturing RAW 264.7 cells and GES-1 cells in 12-well plate for 24h, adding 2 mL free C6, C6 labeled nanoparticles (ML-LA/C6 NPs and FU/ML-LA/C6 NPs), incubating with cells for 4h, washing with PBS 2 times, and collecting 1 × 10 4 The fluorescence intensity of each cell was measured by flow cytometry, and the results are shown in FIG. 9. In RAW 264.7 cells, the mean fluorescence intensity of free C6 was 1.52X 10 5 ML-LA/C6 NPs of 2.01X 10 5 FU/ML-LA/C6 NPs of 2.11X 10 5 . Compared to free C6, ML-LA/C6 NPs and FU/ML-LA/C6 NPs significantly enhanced the ability to be taken up by both cells. FU/ML-LA/C6 NPs showed higher cellular uptake rates than ML-LA/C6 NPs. The same tendency of the two nanoparticles to be taken up by cells is detected in GES-1 cells, and the ML-LA/C6 NPs and FU/ML-LA/C6 NPs can be further proved to improve the capability of being taken up by cells.
Example 7 Effect of biguanide derivative nanoparticles on the removal of intracellular bacteria
Quantitative evaluation of biguanide derivative nanoparticles (prepared according to example 5) to intracellular by the coating plate methodH. pyloriThe cleaning effect of (1). Culturing GES-1 human gastric epithelial cells and RAW 264.7 macrophages in a cell-well plate for 24h, and collectingH. pyloriAdding the bacterial suspension into a pore plate to be incubated with cells, removing the culture medium by suction, and adding a dual-resistant-free culture medium containing gentamicin to kill cells which do not enter the cellsH. pyloriThe medium was aspirated and washed with sterile PBS. The following dosing groups were set: free MET, free ML + LA + EB, ML-LA NPs, ML-LA/EB NPs and FU/ML-LA/EB NPs, the same drug concentrations were consistent across the groups (i.e., MET 100. Mu.g/mL, ML 100. Mu.g/mL, LA 70. Mu.g/mL, EB 17. Mu.g/mL, FU 90. Mu.g/mL). The above-mentioned medicine 2 mL is added into each well, and the culture is continued for 24h. Adding 0.1% tea saponin solution to lyse cells, and collecting cell lysate for use. Coating a part of cell lysate on a plate, culturing at 37 ℃ in a microaerobic environment for 72 h, and quantifying intracellularly by using a plate counting methodH. pyloriThe results are shown in FIG. 10. In both cells after treatment with the biguanide derivative MLH. pyloriThe levels were all significantly reduced and were lower than the intracellular bacterial levels after metformin hydrochloride treatment. In the ML + LA + EB mixed drug group, the number of intracellular bacteria is further reduced, so the addition of LA and EB can play a role in killing the intracellular bacteria in cooperation with ML. In RAW 264.7 cells, the intracellular bacterial numbers of ML-LA/EB NPs and FU/ML-LA/EB NPs were reduced by 78% and 87%, respectively, showing higher intracellular bacterial killing ability than the free drug group, and the GES-1 cells had the same tendency of reducing intracellular bacterial load. Therefore, the FU/ML-LA/EB NPs can obviously enhance the medicine intake and improve the intracellular level of the medicine so as to play a role in killing intracellular bacteria.
Evaluation of biguanide derivative nanoparticles (prepared according to example 5) for intracellular targeting using fluorescence stainingH. pyloriThe cleaning effect of (1). CollectingH. pyloriAfter the bacterial suspension is incubated with CFDA-SE fluorescent dye for 30 min, the precipitate is collected by centrifugation and washed three times by sterile PBS, and then the precipitate is added into a cell well plate inoculated with GES-1 human gastric epithelial cells and RAW 264.7 macrophages after being resuspended by a double-antibody-free culture medium for co-incubation. Suction deviceAbandoning the culture medium and adding a double-resistance-free culture medium containing gentamicin to kill the cells which do not enter the cellsH. pyloriThe medium was aspirated and washed with sterile PBS, and the incubation continued with 24h by adding the above drug solution. Removing culture medium containing medicinal liquid, washing with sterile PBS, staining cell nucleus with DAPI dye and lysosome with DAD 99 dye, and observing inside and outside of cell with laser scanning confocal microscopeH. pyloriThe levels were evaluated to evaluate the effect of the biguanide derivatives on their clearance, and the results are shown in fig. 11 and 12. Green fluorescent representationH. pyloriRed fluorescence indicates lysosomes in the cells, and blue fluorescence indicates nuclei. In the model group, the green and red fluorescence are highly overlapping, indicating that the bacteria are mostly aggregated in lysosomes after being taken up by the cells. In both cells after treatment with the biguanide derivative MLH. pyloriThe levels were all significantly reduced and the green fluorescence intensity was significantly weaker than after metformin hydrochloride treatment. In the ML + LA + EB mixed drug group, the green fluorescence intensity was further reduced. The ML-LA/EB NPs and FU/ML-LA/EB NPs groups showed lower green fluorescence intensity than the free drug group, indicating that the two groups have higher intracellular bacteria killing capability, wherein the FU/ML-LA/EB NPs group has more obvious effect and is consistent with the quantitative result of the coating plate method.
Claims (12)
1. A compound of formula I or a pharmaceutically acceptable salt thereof,
Wherein R is an alkyl or alkenyl group having 10 to 20 carbon atoms.
2. The compound of claim 1, wherein R is C 15-20 Alkyl or C 15-20 An alkenyl group.
3. A compound according to claim 1, which is a pharmaceutically acceptable salt thereof, wherein R is selected from the group consisting of n-decane, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl and n-eicosyl, n-decenyl, n-undecenyl, n-dodecenyl, n-tridecyl, n-tetradecyl, n-pentadecenyl, n-hexadecyl, n-heptadecenyl, n-octadecenyl, n-nonadecenyl, n-eicosenyl, 1,3-pentadecenyl, 2,5-pentadecenyl, 3,6-pentadecenyl, 4,7-pentadecenyl, 5,8-pentadecenyl, 6,9-pentadecenyl, 7,10-pentadecenyl, ft 3525-pentadecenyl 8,11-pentadecadienyl, 1,3-hexadecadienyl, 2,5-hexadecadienyl, 3,6-hexadecadienyl, 4,7-hexadecadienyl, 5,8-hexadecadienyl, 6,9-hexadecadienyl, 7,10-hexadecadienyl, 8,11-hexadecadienyl, 1,3-heptadecadienyl, 2,5-heptadecadienyl, 3,6-heptadecadienyl, 4,7-heptadecadienyl, 5,8-heptadecadienyl, 6,9-heptadecadienyl, 7,10-heptadecadienyl, 8,11-heptadecadienyl, 58 zxft 6258-heptadecadienyl; 1,3-octadecadienyl, 2,5-octadecadienyl, 3,6-octadecadienyl, 4,7-octadecadienyl, 5,8-octadecadienyl, 6,9-octadecadienyl, 7,10-octadecadienyl, 8,11-octadecadienyl, 9,12-octadecadienyl; 1,3-nonadienyl, 2,5-nonadienyl, 3,6-nonadienyl, 4,7-nonadienyl, 5,8-nonadienyl, 6,9-nonadienyl, 7,10-nonadienyl, 8,11-nonadienyl, 9,12-nonadienyl, 10,13-nonadienyl, 5678 zxf5678-eicosadienyl, 2,5-eicosadienyl, 3,6-eicosadienyl, 9696 zxft 6296-eicosadienyl, 9635 zxft 3235-eicosadienyl, 6,9-eicosadienyl, heptadecadienyl, 3426 zxft 3474-heptadecadienyl, 34zxft 6296-heptadecadienyl, 354258-heptadecadienyl, 35zzft 4258-heptadecadienyl, 35zxft 4258-heptadecadienyl, 35z5258-heptadecadienyl, 35zxft 4258-heptadecadienyl, 345258-heptadecadienyl, and 35zxft 4258-heptadecadienyl.
4. The compound of claim 3, wherein the compound has the structure of formula (II) or formula (III):
5. A pharmaceutical composition comprising a compound of any one of claims 1-4, or a pharmaceutically acceptable salt thereof.
6. Use of a compound of any one of claims 1-4, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating an intracellular bacterial infection, a disease caused by an intracellular bacterial infection;
wherein the intracellular bacteria is helicobacter pylori.
7. The use according to claim 6, wherein the disease caused by intracellular bacterial infection is selected from peptic ulcer, gastritis, peptic esophagitis, symptomatic gastroesophageal reflux disease without esophagitis, non-ulcer dyspepsia, gastrorrhagia, upper gastrointestinal bleeding, gastric cancer and gastric MALT lymphoma.
8. A self-assembling nanoparticle comprising:
(a) A compound of any one of claims 1-4 or a pharmaceutically acceptable salt thereof;
（b）C 10-20 fatty acid, wherein said C 10-20 The fatty acid and the compound or the pharmaceutically acceptable salt thereof form a carrier structure of the self-assembled nanoparticle, wherein C is 10-20 The fatty acid is selected from capric acid, 9-decenoic acid, undecanoic acid, 10-undecenoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, or combinations thereof; and (c) a second step of,
(c) A urease inhibitor or antibiotic entrapped within the self-assembled nanoparticle, wherein the urease inhibitor is ebselen or acetohydroxamic acid, wherein the antibiotic is selected from amoxicillin, clarithromycin, metronidazole, tetracycline, levofloxacin, furazolidone, or a combination thereof.
9. The self-assembling nanoparticle of claim 8, further comprising: (d) A polysaccharide coated on the exterior of the self-assembling nanoparticle, wherein the polysaccharide is selected from the group consisting of fucoidan, mannan, dextran, and mannitol.
10. The self-assembling nanoparticle of claim 8, wherein the C 10-20 The fatty acid is linoleic acid.
11. The self-assembling nanoparticle of claim 8, wherein the urease inhibitor is ebselen.
12. The self-assembling nanoparticle of claim 9, wherein the polysaccharide is fucoidan.
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