CN113354561B - Biguanide derivatives and their use and formulations - Google Patents

Abstract

The invention relates to a compound of formula I, a deuterio thereof or a pharmaceutically acceptable salt thereof,

Description

Biguanide derivatives and their applications and preparations

Technical Field

The present invention belongs to the field of medicine, and relates to biguanide derivatives and their application and preparation, and in particular to biguanide derivatives used for treating intracellular bacterial infection, diseases caused by intracellular bacterial infection, and glucose or lipid metabolism diseases.

Background Art

Autophagy is a process in which cells specifically recognize and degrade foreign pathogens such as bacteria and viruses, which can protect cells from invasion by pathogens. Under normal circumstances, autophagosomes encapsulating pathogens in cells can fuse with functioning lysosomes to form autophagolysosomes, which use a variety of lysosomal enzymes to degrade pathogens. However, under pathological conditions, some bacteria have evolved to evade host recognition, such as inhibiting normal cell autophagy by blocking lysosomal maturation and promoting their survival. After infecting the host, bacteria that mainly reside in cells are called intracellular bacteria, and the continued presence of intracellular bacteria is believed to lead to chronic infection. Common bacteria that survive in cells include Mycobacterium tuberculosis ( M. tuberculosis ), Salmonella typhi ( S. typhi ), Listeria ( Listeria ), Staphylococcus aureus ( S. aureus ) and Helicobacter pylori ( H. pylori ).

H. pylori is a Gram-negative bacterium that colonizes the stomach and duodenum, causing infection in more than half of the world's population. H. pylori has invasive properties and can survive in host cells. H. pylori damages the activity of the cell's lysosomal calcium channel TRPML1 by secreting vacuolating toxin (VacA) and other virulence factors. The abnormal increase in calcium levels in the lysosomes causes hydrogen ions to be unable to be exchanged and ingested normally, and the acidic environment of the lysosomes changes. The functionally impaired lysosomes cannot normally degrade bacteria in the autophagosomes. H. pylori that survives in the cell can re-exit the cell under appropriate conditions and cause a new round of infection. Therefore, treatment of intracellular bacteria can further improve the clearance rate of H. pylori and reduce the occurrence of persistent recurrent infections.

The latest studies have shown that metformin, an agonist of adenylate-activated protein kinase (AMPK), can enhance lysosomal acidification, restore normal cell autophagy, and inhibit the growth of H. pylori both in vitro and in vivo. Several other clinical data have found that metformin can assist in the treatment of tuberculosis by antagonizing typical intracellular infection of Mycobacterium tuberculosis. Unfortunately, metformin's high water solubility and large dosage limit its application in the treatment of bacterial infections. As the problem of bacterial resistance is becoming increasingly severe worldwide, the presence of intracellular bacteria can easily cause stubborn and persistent infections, and more effective drugs for the treatment of bacterial infections are still needed clinically .

Summary of the invention

Compared with metformin, the biguanide derivatives of the present invention can enhance the agonist activity of host cell AMPK and are safe, effective and low-toxic drugs for fighting intracellular bacteria. At the same time, the nanoparticles constructed from the biguanide derivatives of the present invention further improve the clearance rate of H. pylori and can treat persistent and recurrent infections associated with H. pylori .

In one aspect, the present invention provides a compound of formula (I), a deuterated substance thereof or a pharmaceutically acceptable salt thereof,

式（I）

Formula (I)

Here, 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, 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-tridecenyl, n-tetradecenyl, n-pentadecyl, n-hexadecenyl, n-heptadecenyl, n-octadecenyl, n-nonadecyl, and n-eicosyl, n-decenyl, n-undecenyl, n-dodecenyl, n-tridecenyl, n-tetradecenyl, n-pentadecyl, n-hexadecenyl, n-heptadecenyl, n-octadecenyl, n-nonadecyl, n-eicosyl, 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, 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, 9,12-heptadecadienyl; 1,3-octadecadienyl, 2,5-octadecadienyl, 1,3-nonadecadienyl, 2,5-nonadecadienyl, 3,6-nonadecadienyl, 4,7-nonadecadienyl, 5,8-nonadecadienyl, 6,9-nonadecadienyl, 7,10-nonadecadienyl, 8,11-nonadecadienyl, 9,12-nonadecadienyl, 1,3-nonadecadienyl, 2,5-nonadecadienyl, 3,6-nonadecadienyl, 4,7-nonadecadienyl, 5,8-nonadecadienyl, 6,9-nonadecadienyl, 7,10-nonadecadienyl, 8,11-nonadecadienyl, 9,12-nonadecadienyl, 10,13-nonadecadienyl, 1,3-eicosadienyl, 2,5-eicosadienyl, 3,6 -eicosadienyl, 4,7-eicosadienyl, 5,8-eicosadienyl, 6,9-eicosadienyl, 7,10-eicosadienyl, 8,11-eicosadienyl, 9,12-eicosadienyl, 10,13-eicosadienyl, 1,4,7-heptadecatrienyl, 2,5,8-heptadecatrienyl, 3,6,9-heptadecatrienyl, 4,7,10-heptadecatrienyl and 5,8,11-heptadecatrienyl, n-decynyl, n-undecynyl, n-dodecynyl, n-tridecynyl, n-tetradecynyl, n-pentadecaynyl, n-hexadecynyl, n-heptadecatrienyl, n-octadecynyl, n-nonadecatrienyl and n-eicosadienyl.

In some embodiments, the compound is:

式（II）或

Formula (II) or

式（III）。

Formula (III).

In another aspect, the present invention provides a pharmaceutical composition comprising any of the above compounds, deuterated substances 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 or a bismuth agent.

In another aspect, the present invention provides the use of any of the above-mentioned compounds, their deuterated substances or pharmaceutically acceptable salts thereof in the preparation of drugs for treating intracellular bacterial infection, diseases caused by intracellular bacterial infection or glucose or lipid metabolism diseases. In some embodiments, the intracellular bacteria are selected from Mycobacterium tuberculosis, Salmonella typhi, Listeria, Staphylococcus aureus and Helicobacter pylori. In some embodiments, the intracellular bacteria are preferably Helicobacter pylori. In some embodiments, the disease caused by intracellular bacterial infection is selected from peptic ulcer, gastritis, peptic esophagitis, symptomatic gastroesophageal reflux disease without esophagitis, non-ulcer dyspepsia, hyperacid dyspepsia, upper gastrointestinal bleeding, gastric cancer and gastric MALT lymphoma. In some embodiments, the glucose or lipid metabolism disease 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, which comprises: (a) any of the above-mentioned compounds, deuterated substances thereof or pharmaceutically acceptable salts thereof; (b) C _10-20 fatty acids, wherein the C _10-20 fatty acids and the compounds, deuterated substances thereof or pharmaceutically acceptable salts thereof constitute the carrier structure of the self-assembled nanoparticles; and (c) urease inhibitors or antibiotics, wherein the urease inhibitors or antibiotics are encapsulated and embedded in the self-assembled nanoparticles. In some embodiments, the C _10-20 fatty acids are selected from decanoic acid, 9-decenoic acid, undecanoic acid, 10-undecenoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid or a combination thereof. In some embodiments, the C _10-20 fatty acids are preferably oleic acid, linoleic acid, linolenic acid. In some embodiments, the C _10-20 fatty acids are 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-assembled nanoparticles further comprise: (d) a polysaccharide, which is coated on the outside of the self-assembled nanoparticles. In some embodiments, the polysaccharide is selected from fucoidan, mannan, dextran, and mannitol. In some embodiments, the polysaccharide is preferably fucoidan (FU). In some embodiments, the self-assembled nanoparticles comprise: linoleic acid, which constitutes the carrier structure of the self-assembled nanoparticles with the biguanide derivatives MU or ML; ebselen, which is encapsulated and embedded in the self-assembled nanoparticles. In some embodiments, the self-assembled nanoparticles comprise: linoleic acid, which constitutes the carrier structure of the self-assembled nanoparticles with the biguanide derivatives MU or ML; and ebselen, which is encapsulated and embedded in the self-assembled nanoparticles; and FU, which is coated on the outside of the self-assembled nanoparticles. In some embodiments, the particle size of the self-assembled nanoparticles is 100-180 nm. In some embodiments, the particle size is preferably 125 nm or 150 nm. In some embodiments, the encapsulation rate of the self-assembled nanoparticles is greater than 75%. In some embodiments, the encapsulation efficiency of the self-assembled nanoparticles is preferably greater than 80%. In some embodiments, the drug loading of the self-assembled nanoparticles is greater than 55%.

The nanoparticles of the present invention are taken up by the endocytosis of the host cells, thereby interfering with the cell membrane permeability of intracellular H. pylori to rupture the bacteria, and inhibiting the internal urease activity of H. pylori to exert an anti- H. pylori effect. At the same time, the biguanide derivatives that enter the cells can restore the cell's autophagic lysosomal degradation effect, and the advantages of the combined nanoparticles enhance the intracellular H. pylori clearance effect, thereby achieving the purpose of eradicating H. pylori .

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: a. H NMR spectrum of intermediate 1a of biguanide derivative ML; b. Mass spectrum of intermediate 1a of biguanide derivative ML.

Figure 2: a. H NMR spectrum of the intermediate 1b of the biguanide derivative ML; b. Mass spectrum of the intermediate 1b of the biguanide derivative ML.

Figure 3: a. H NMR spectrum of the biguanide derivative ML; b. Mass spectrum of the biguanide derivative ML.

Figure 4: a. H NMR spectrum of the intermediate 3a of the biguanide derivative MU; b. Mass spectrum of the intermediate 3a of the biguanide derivative MU.

Figure 5: a. H NMR spectrum of the intermediate 3b of the biguanide derivative MU; b. Mass spectrum of the intermediate 3b of the biguanide derivative MU.

Figure 6: a. H NMR spectrum of the biguanide derivative MU; b. Mass spectrum of the biguanide derivative MU.

Figure 7: Effects of metformin (MET) and biguanide derivatives (ML and MU) on the activity level of adenylate-activated protein kinase (AMPK) in GES-1 human gastric epithelial cells. After adding MET, ML, and MU to treat GES-1 cells, the activity was determined and compared according to the steps of the human phosphorylated adenylate-activated protein kinase ELISA kit. Compared with the control group, * indicates P <0.05, *** indicates P <0.001; compared with MET, ### indicates P <0.001; compared with MU, ·· indicates P <0.01.

Figure 8: Evaluation of the clearance effect of metformin (MET) and biguanide derivatives (ML and MU) on H. pylori in RAW 264.7 macrophages. In the constructed intracellular bacteria model, GES-1 cells were treated with MET, ML and MU, and then the results were compared by the spread plate method. Compared with the control group, * indicates P <0.05; *** indicates P <0.001; compared with MET, # indicates P < 0.05, ### indicates P <0.001; compared with MU, ·· indicates P < 0.01.

Figure 9: a. Average fluorescence intensity of biguanide derivative nanoparticles and C6 uptake in RAW 264.7 macrophages. After adding free coumarin-6 (C6) and C6-labeled nanoparticles (ML-LA/C6 NPs and Fu/ML-LA/C6 NPs) and incubating the cells for 4 hours, 1×10 ⁴ cells were collected, and the fluorescence intensity of C6 was measured by flow cytometry and compared. b. Average fluorescence intensity of biguanide derivative nanoparticles and C6 uptake in GES-1 human gastric epithelial cells. After adding C6 and C6-labeled nanoparticles (ML-LA/C6 NPs and Fu/ML-LA/C6 NPs) and incubating the cells for 4 hours, 1×10 ⁴ cells were collected, and the fluorescence intensity of C6 was measured by flow cytometry and compared. Compared with the control group, *** indicates P <0.001; compared with C6, ## indicates P < 0.01, ### indicates P <0.001; compared with ML-LA/C6, · indicates P < 0.05, ·· indicates P < 0.01, and ··· indicates P < 0.001.

Figure 10: a. Evaluation of the clearance effect of biguanide derivative nanoparticles on H. pylori in RAW 264.7 macrophages. In the constructed intracellular bacterial model, RAW 264.7 cells were treated with drugs, and then the results were compared using the plate spreading method. b. Evaluation of the clearance effect of biguanide derivative nanoparticles on H. pylori in GES-1 human gastric epithelial cells. In the constructed intracellular bacterial model, GES-1 cells were treated with MET and ML, and then the results were compared using the plate spreading method. Compared with the control group, ** indicates P < 0.01, *** indicates P <0.001; compared with MET, # indicates P < 0.05, ## indicates P < 0.01, ### indicates P <0.001; compared with ML, ·· indicates P < 0.01, ··· indicates P <0.001; compared with ML+LA+EB, ◆ indicates P < 0.05, ◆◆ indicates P <0.01; compared with ML-LA/EB, & indicates P < 0.05.

Figure 11. Evaluation of the effect of biguanide derivative nanoparticles on the clearance of H. pylori in RAW 264.7 macrophages. In the constructed intracellular bacterial model, RAW 264.7 cells were treated with drugs and the results were observed by laser confocal microscopy. Blue fluorescence marks the nucleus, green fluorescence marks H. pylori , and red fluorescence marks lysosomes. The overlay image is the overlay result of the above three images.

Figure 12. Evaluation of the effect of biguanide derivative nanoparticles on the clearance of H. pylori in GES-1 human gastric epithelial cells. In the constructed intracellular bacterial model, GES-1 cells were treated with drugs and the results were observed by laser confocal microscopy. Blue fluorescence marks the cell nucleus, green fluorescence marks H. pylori , and red fluorescence marks lysosomes. The overlay image is the overlay result of the above three images.

DETAILED DESCRIPTION

The term "alkyl" refers to a straight or branched hydrocarbon group with a specified number of carbon atoms. For example, a _C10-20 alkyl group refers to a straight or branched hydrocarbon group with 10, 11, 12, 13, 14, ₁₅ , ₁₆ , _{17, 18} , 19 or 20 carbon atoms, including but not limited to _C10 alkyl, _C11-12 alkyl, _C13-14 alkyl, C15-20 alkyl, C17 alkyl, C16-18 alkyl and _C19-20 alkyl. Examples of saturated alkyl groups 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 halogen, hydroxyl, sulfhydryl or heteroatom. "Halogen" refers to fluorine, chlorine, bromine or iodine. Heteroatoms are intended to include oxygen, nitrogen, sulfur. Examples of unsaturated alkyl groups include alkenyl, alkynyl or a combination thereof.

The term "alkenyl" refers to a straight or branched chain hydrocarbon 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 vinyl, propenyl, etc. C _10-20 alkenyl refers to a 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, C _11-12 alkenyl, C 13-14 alkenyl, C _15-20 alkenyl, C ₁₇ alkenyl, C _16-18 alkenyl and C _19-20 alkenyl. Examples of alkenyl groups include n-decenyl, n-undecenyl, n-dodecenyl, n-tridecenyl, n-tetradecenyl and C _15-20 alkenyl. C _15-20 alkenyl contains a straight or branched chain hydrocarbon group of 1, 2 or 3 double bonds. C _15-20 alkenyl includes n-pentadecenyl, n-hexadecene, n-heptadecenyl, n-octadecene, n-nonadecenyl and n-eicosenyl, 1,3-pentadecanadienyl, 2,5-pentadecanadienyl, 3,6-pentadecanadienyl, 4,7-pentadecanadienyl, 5,8-pentadecanadienyl, 6,9-pentadecanadienyl, 7,10-pentadecanadienyl, 8,11-pentadecanadienyl, 1,3-hexadecene, 2,5-hexadecene, 3,6-hexadecene, 4,7-hexadecene, 5,8-pentadecanadienyl, 6,9-pentadecanadienyl, 7,10-pentadecanadienyl, 8,11-pentadecanadienyl, ,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, 9,12-heptadecadienyl; 1,3-octadecadienyl, 2,5-octadecadienyl, 3,6-octadecadienyl, 4,7-heptadecadienyl, 5,8-heptadecadienyl, 6,9-heptadecadienyl, 7,10-heptadecadienyl, 8,11-heptadecadienyl, 9,12-heptadecadienyl; 7-octadecadienyl, 5,8-octadecadienyl, 6,9-octadecadienyl, 7,10-octadecadienyl, 8,11-octadecadienyl, 9,12-octadecadienyl; 1,3-nonadecadienyl, 2,5-nonadecadienyl, 3,6-nonadecadienyl, 4,7-nonadecadienyl, 5,8-nonadecadienyl, 6,9-nonadecadienyl, 7,10-nonadecadienyl, 8,11-nonadecadienyl, 9,12-nonadecadienyl, 10,13-nonadecadienyl, 1,3-eicosadienyl, 2,5-eicosadienyl, 3,6-eicosadienyl, 4,7-eicosadienyl, 5,8-eicosadienyl, 6,9-eicosadienyl, 7,10-eicosadienyl, 8,11-eicosadienyl, 9,12-eicosadienyl, 10,13-eicosadienyl, 1,4,7-heptadecatrienyl, 2,5,8-heptadecatrienyl, 3,6,9-heptadecatrienyl, 4,7,10-heptadecatrienyl, 5,8,11-heptadecatrienyl. In some embodiments, the alkenyl group may be substituted with halogen, hydroxyl, sulfhydryl, or heteroatoms. "Halogen" refers to fluorine, chlorine, bromine, or iodine. Heteroatoms are intended to include oxygen, nitrogen, and sulfur.

Similarly, the term "alkynyl" refers to a straight or branched chain hydrocarbon group containing at least one triple bond and having the number of carbon atoms shown in the prefix. Examples of alkynyl include ethynyl, 1- and 3-propynyl, 3-butynyl, and higher order homologues and isomers. _C10-20 alkynyl refers to a straight or branched chain alkynyl of 10, 11, 12, 13, 14, 15, 16, ₁₇ , 18, 19 or 20 carbon atoms, and includes but is not limited to _C10 alkynyl, _C11-12 alkynyl, _C13-14 alkynyl, _C15-20 alkynyl, C17 alkynyl, and _C16-18 alkynyl and _C19-20 alkynyl. Examples of alkynyl groups include n-decynyl, n-undecynyl, n-dodecynyl, n-tridecynyl, n-tetradecynyl, n-pentadecinyl, n-hexadecynyl, n-heptadecynyl, n-octadecynyl, n-nonadecinyl, and n-eicosynyl. In some embodiments, the alkynyl may be substituted with halogen, hydroxyl, sulfhydryl, or heteroatoms. "Halogen" refers to fluorine, chlorine, bromine, or iodine. Heteroatoms are intended to include oxygen, nitrogen, sulfur.

Some compounds of the present invention may exist in non-solvated forms as well as solvated forms (including hydrated forms). "Hydrate" refers to a complex formed by the combination of water molecules and molecules or ions of a solute. "Solvate" refers to a complex in which a combination of solvent molecules and molecules or ions of a solute is combined. 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,N-dimethylformamide (DMF), tetrahydrofuran (THF), dichloromethane (DCM), trifluoroacetic acid (TFA), dioxane (Diox), dimethyl sulfoxide (DMSO), and water. Generally, solvated forms are equivalent to non-solvated forms and are included within the scope of the present invention. Some compounds of the present invention may exist in multiple crystalline or amorphous forms. Generally, all physical forms are equivalent for the intended applications of the present invention, and they are intended to be included within the scope of the present invention.

Preparation and administration

The present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a compound described herein or a pharmaceutically acceptable salt or solvate thereof. In an exemplary embodiment, the present invention provides a pharmaceutical formulation comprising a compound described herein. In one embodiment, the pharmaceutical formulation or composition comprises a compound of formula (I):

式（I）

Formula (I)

wherein the R group is as defined above.

The present invention also contemplates the use of pharmaceutically acceptable deuterated compounds or other non-radioactive substituted compounds of the compound. Deuteration is the replacement of one or more or all hydrogens in the active molecular group of the drug with the isotope deuterium, which is considered to be an excellent modification method because it is non-toxic and non-radioactive, and is about 6 to 9 times more stable than carbon-hydrogen bonds. It can block metabolic sites and prolong the half-life of the drug, thereby reducing the therapeutic dose without affecting the pharmacological activity of the drug.

These compounds are usually used to treat human diseases. However, they can 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, intubation or inhalation. These dosage forms should allow the compound to reach the target cells. Other factors are well known in the art, including considerations such as toxicity and dosage forms that prevent the compound or composition from exerting its effect.

In some embodiments, the composition may include a pharmaceutically acceptable carrier or excipient, such as a filler, a binder, a disintegrant, a glidant, a lubricant, a complexing agent, a solubilizer, and a surfactant, which may be selected to facilitate administration by a specific route. Examples of carriers include calcium carbonate, calcium phosphate, various sugars (such as lactose, glucose, or sucrose), various types of starch, cellulose derivatives, gelatin, lipids, liposomes, nanoparticles, and the like. Carriers also include physiologically compatible liquids as solvents or for suspensions, including, for example, sterile aqueous solutions for injection (WFI), physiological saline solutions, glucose solutions, Hank's solutions, Ringer's solutions, vegetable oils, mineral oils, animal oils, polyethylene glycols, liquid paraffin, and the like. Excipients may also include, for example, colloidal silicon dioxide, silica gel, talc, magnesium silicate, calcium silicate, sodium aluminosilicate, magnesium trisilicate, powdered cellulose, macrocrystalline cellulose, carboxymethyl cellulose, croscarmellose sodium, sodium benzoate, calcium carbonate, magnesium carbonate, stearic acid, aluminum stearate, calcium stearate, magnesium stearate, zinc stearate, sodium stearyl fumarate, syloid, stearowet C, magnesium oxide, starch, sodium starch glycolate, glyceryl monostearate, glyceryl dibehenate, glyceryl palmitostearate, hydrogenated vegetable oil, hydrogenated cottonseed oil, castor seed oil, mineral oil, polyethylene glycols (e.g., PEG 4000-8000), polyoxyethylene glycol, poloxamer, povidone, crospovidone, croscarmellose sodium, alginic acid, casein, divinylbenzene methacrylate copolymer, docusate sodium, cyclodextrin (e.g., 2-hydroxypropyl-delta-cyclodextrin), polysorbate (e.g., polysorbate 80), cetrimide, d-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS), magnesium lauryl sulfate, sodium lauryl sulfate, polyethylene glycol ethers, di-fatty acid esters of polyethylene glycol, or polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan ester ^Tween® ), polyoxyethylene sorbitan fatty acid esters, sorbitan fatty acid esters (such as sorbitan fatty acid esters from fatty acids such as oleic acid, stearic acid or palmitic acid), mannitol, xylitol, sorbitol, maltose, lactose, lactose monohydrate or spray-dried lactose, sucrose, fructose, calcium phosphate, calcium hydrogen phosphate, calcium phosphate, calcium sulfate, dextrates, dextran, dextrin, dextrose, cellulose acetate, maltodextrin, dimethicone, polydextrose, chitosan, gelatin, hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxyethyl cellulose, etc.

Pharmaceutical preparations can be presented in unit dose form, each unit dose containing a predetermined amount of active ingredient. 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 the compound of the present invention (free form, any form of solvate (including hydrate) or salt), depending on the condition to be treated, the route of administration, the age, weight and condition of the patient. Preferred unit dose formulations are those containing daily doses, weekly doses, monthly doses, their sub-doses or suitable proportions of active ingredients. Moreover, such pharmaceutical preparations can be prepared by any method well known in the pharmaceutical field.

Pharmaceutical preparations may be prepared in a form suitable for administration by any appropriate route, such as 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) routes. These preparations may be prepared by any method known in the pharmaceutical art, such as by combining the active ingredient with a carrier, excipient or diluent. Generally, the carrier, excipient or diluent used in the pharmaceutical preparation is "non-toxic" and "inert", the former meaning that it is safe to take in the amount in the pharmaceutical composition, and the latter meaning that it does not significantly react with the active ingredient or cause an undesirable effect on the therapeutic activity of the active ingredient.

The dosage of each compound can be determined by standard procedures and takes into account factors such as compound activity (in vitro activity, such as compound IC ₅₀ vs. target, or in vivo activity in animal efficacy models), pharmacokinetic results in animal models (such as biological half-life or bioavailability), age, size and weight of the subject, and diseases associated with the subject. The importance of these factors and other factors is well known to those skilled in the art. In general, the dosage range will be about 0.01 to 50 mg/kg, or about 0.1 to 20 mg/kg. Multiple doses can be used.

The compounds described herein may also be used in combination with other therapies for treating the same disease. Such combination 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 present invention or other therapeutic agents used in combination may be modified by methods well known to those skilled in the art, such as reducing the dosage relative to the use of the compound or therapeutic agent alone.

It is to be understood that the combined use includes use with other therapies, drugs, medical procedures, etc., wherein the other therapies or procedures may be administered at a different time from the compounds described herein (e.g., within a short period of time, such as within a few hours (e.g., 1, 2, 3, 4-24 hours), or over a longer period of time (e.g., 1-2 days, 2-4 days, 4-7 days, 1-4 weeks)), or at the same time as the compounds of the present invention. The combined use also includes use with a therapy or medical procedure (e.g., surgery) that is administered once or infrequently, and administering the compounds described herein within a short period of time or within a longer period of time before or after the other therapy or procedure. In some embodiments, the present invention provides delivery of the compounds described herein and one or more other drug therapeutic agents, which are administered by different routes, or by the same route. The combined administration of any route of administration includes delivery of the compounds described herein and one or more other drug therapeutic agents, which are delivered together in any formulation form by the same administration method, including two compounds chemically linked together so that they maintain their therapeutic activity when administered. In one aspect, the other drug therapy can be co-administered with the compounds described herein. The combined use of co-administration includes the use of a co-formulation or a formulation of chemically bound compounds, or the administration of two or more compounds in different formulations in the same or different routes within a short time relative to each other (such as within 1 hour, 2 hours, 3 hours, up to 24 hours). The co-administration of a separate formulation includes co-administration through the delivery of a device, such as the same inhalation device, the same syringe, etc., or by a separate device within a short time relative to each other. The co-formulation of the compounds described herein and one or more additional drug therapeutic agents administered by the same route includes preparing materials together so that they can be administered by a device, including different compounds combined in a formulation, or modified compounds that can be chemically connected but still maintain their biological activity. This chemically connected compound may have a linker that is substantially maintained in vivo, or the linker decomposes in vivo to separate the two active components. In some embodiments, the compounds of the present invention may be used together with urease inhibitors, antibiotics, proton pump inhibitors, bismuth agents, or combinations thereof. The antibiotic may be 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 bismuth salicylate, potassium bismuth citrate and bismuth pectin. In some embodiments, the urease inhibitor is ebselen or acetohydroxamic acid.

Adenylate-activated protein kinase (AMPK)

AMPK is a cell energy sensor and regulator, and plays an important role in maintaining cellular metabolic balance. Activated AMPK can enhance the body's catabolism and promote ATP production through acute regulation of key enzyme activities in metabolic links and chronic regulation of key transcription factor expression. On the one hand, it can inhibit anabolism and reduce ATP consumption. Evidence of AMPK's regulatory effects on glucose and lipid metabolism makes it a potential drug target for the treatment of diabetes and metabolic syndrome. AMPK can inhibit hepatic gluconeogenesis and lipid production, while reducing hepatic lipid deposition by increasing lipid oxidation, thereby improving glucose and lipid distribution. Studies have shown that leptin and adiponectin (adipokine) regulate glucose and lipid metabolism by activating AMPK, thereby exerting their anti-diabetic effects. Among them, leptin directly activates AMPK and stimulates muscle fatty acid oxidation via the hypothalamic adrenergic pathway, and adiponectin stimulates glucose uptake and fatty acid oxidation in vitro by activating AMPK. In addition, AMPK exerts its hypoglycemic effect by inhibiting the expression of key genes for gluconeogenesis, such as genes encoding phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). 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: the two main types of existing antidiabetic drugs, namely thiazolidinediones (rosiglitazone, troglitazone and pioglitazone) and biguanides (metformin and phenformin), activate AMPK in in vitro cell experiments and in vivo. There is evidence that AMPK mediates the antidiabetic effect of rosiglitazone. Studies have shown that metformin can activate AMPK both in vitro and in vivo by inhibiting complex I. If the upstream kinase LKB1 of AMPK is knocked out, the hypoglycemic effect of metformin is completely blocked, proving the key role of AMPK in mediating the antidiabetic effect of metformin. Many AMPK activators (e.g., A-769662 and PT1) are able to exert anti-diabetic effects in vivo. The compounds of the present invention are effective AMPK activators.

AMPK has been proposed as a promising target for the treatment of several kidney diseases. AMPK is reported to be a regulator of several ion channels, transporters and pumps in the kidney, and treatment with AMPK activators is beneficial in preventing kidney damage in various disease settings. In addition, AMPK activation induces autolysis in cells, which has been shown to protect the kidneys in several animal models.

The term "glucose or lipid metabolism disease" refers to metabolic syndrome caused by abnormal glucose metabolism or lipid metabolism. In some embodiments of the present invention, "glucose or lipid metabolism disease" refers to a disease that can be treated by regulating the activity of AMPK (e.g., AMPK activation). "Glucose or lipid metabolism diseases" that can 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 acid" generally refers to a straight or branched fatty acid of 10, _{11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms, and includes but is not limited to C 10 fatty} acids, C _11-12 fatty acids, C _13-14 fatty acids, C 15-20 fatty acids, C ₁₈ fatty acids, C _16-18 fatty acids, and C _19-20 fatty acids. Examples of C _10-20 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, the C _10-20 fatty acid is a C ₁₈ mono- and/or di-unsaturated fatty acid acid.

The present invention is further explained below by means of specific examples, but the present invention is not limited to the examples.

Example 1. Synthesis and identification of biguanide derivative ML

An exemplary synthesis route of the biguanide derivative ML of the present invention is as follows:

Take 5.6 g of linoleic acid, 4.8 g of N-tert-butyl-1,2-ethylenediamine, 4.6 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 3.24 g of 1-hydroxybenzotriazole, add anhydrous dichloromethane, react at room temperature overnight, dry and concentrate, and purify by column chromatography to obtain 7.4 g of intermediate 1a 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 = 5.4Hz, 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] ⁺ .

Take 7.4 g of the above compound 1a, dissolve it in dichloromethane, then add trifluoroacetic acid, react at room temperature, fully concentrate and dry, and obtain 4.8 g of intermediate 1b with a yield of 85%. The structural verification is shown in Figure 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] ⁺ .

The intermediate 1b 4.8 g, dicyandiamide 2.21 g and ferric chloride 4.26 g were added to dioxane, reacted at 100°C overnight, dried and concentrated, and purified by column chromatography to obtain 1.9 g of the product with a yield of 31%. The structural verification is shown in Figure 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 identification of biguanide derivative MU

An exemplary synthesis route of the biguanide derivative MU of the present invention is as follows:

Take 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, add anhydrous dichloromethane, react at room temperature overnight, dry and concentrate, and purify by column chromatography to obtain 5.9 g of intermediate 3a with a yield of 90%. The structure verification is shown in Figure 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, 7.0Hz, 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] ⁺ .

Take 5.8 g of the above compound 3a, dissolve it in dichloromethane, then add trifluoroacetic acid, react at room temperature, fully concentrate and dry, and obtain 3.4 g of intermediate 3b with a yield of 85%. The structural verification is shown in Figure 5:

¹ H NMR (400 MHz, DMSO) δ 7.02 (s, 1H), 3.69 – 3.58 (dt, J = 35.2, 8.0Hz, 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 of dicyandiamide and 4.2 g of ferric chloride were added to dioxane, reacted at 100 °C overnight, dried and concentrated, and purified by column chromatography to obtain 1.5 g of the product with a yield of 35%. The structural verification is shown in Figure 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 AMP-activated protein kinase by biguanide derivatives

Elisa kit method was used to determine the activation effect of biguanide derivatives on adenylate-activated protein kinase (AMPK). GES-1 human gastric epithelial cells were inoculated in 12-well plates and cultured for 24 h. The culture medium was aspirated and drugs (i.e., MET at a concentration of 100 μg/mL and biguanide derivatives ML and MU at a concentration of 100 μg/mL) were added and incubated for another 24 h. The cells were collected by centrifugation and mixed by blowing PBS, placed at -20 °C and repeatedly frozen and thawed 5 times. The phosphorylation level of AMPK in GES-1 cells in each group was detected according to the method indicated in the instruction manual of the human phosphorylated adenylate-activated protein kinase enzyme-linked immunosorbent assay kit. The degree of activation of AMPK in the drug-added group was expressed as the phosphorylated AMPK level of the cells in the drug group relative to the level of phosphorylated AMPK in the blank group, and the results are shown in Figure 7. After treatment with the biguanide derivatives MU and ML of the present invention, the phosphorylation level of AMPK in GES-1 cells was significantly enhanced, and the activation ability was stronger than that of the metformin hydrochloride positive control group. In addition, compared with MU, ML has a more significant activation effect on AMPK, and ML with AMPK enhancing activity can better assist antimicrobial drugs in eliminating intracellular bacteria.

Example 4. Effect of biguanide derivatives on the elimination of intracellular bacteria

The plate-spreading method was used to quantitatively evaluate the clearance effect of biguanide derivatives on intracellular H. pylori . GES-1 human gastric epithelial cells were inoculated in cell well plates and cultured for 24 h. The H. pylori suspension was collected and added to the well plates for co-incubation with the cells. The culture medium was aspirated and a medium without biguanide containing gentamicin was added to kill H. pylori that had not entered the cells. The culture medium was aspirated and washed with sterile PBS. Drugs (i.e., metformin hydrochloride MET at a concentration of 100 μg/mL and biguanide derivatives ML and MU at a concentration of 100 μg/mL) were added and cultured for another 24 h. 0.1% tea saponin solution was added to lyse the cells, and the cell lysate was collected and set aside. Part of the cell lysate was spread on the plate, and after culturing at 37 °C for 72 h under a microaerobic environment, the plate count method was used to quantify the intracellular H. pylori level. The results are shown in Figure 8. After treatment with ML and MU, the H. pylori levels in the cells were significantly reduced and were lower than the bacterial levels after treatment with MET. ML reduced the level of intracellular bacteria more significantly than the MU group, and ML had a stronger ability to reduce the intracellular bacterial load.

Example 5. Preparation and characterization of self-assembled nanoparticles of biguanide derivatives.

Nanoprecipitation method was selected to prepare four kinds of biguanide derivative self-assembled nanoparticles (ML-LA NPs, ML-LA/EB NPs, FU/ML-LA NPs and FU/ML-LA/EB NPs). The preparation method is as follows: different volumes of ML, LA and EB DMF solutions were mixed, the concentrations of ML and LA were fixed at 25 mM, EB solution (6.2 mM) was added or not, and the mass ratio of ML to EB was 6:1. Then, 900 μL of pure water or FU aqueous solution (1 mg/mL) was added dropwise under stirring, and stirring was continued for 2 min to obtain four kinds of biguanide derivative self-assembled nanoparticles. The particle size, PDI and zeta potential of the four kinds of nanoparticles were measured. The results are shown in Table 1. All four kinds of nanoparticles have small particle size and PDI. Among them, ML-LA NPs and ML-LA/EB NPs have positive charges and particle sizes of about 125 nm. FU/ML-LANPs and FU/ML-LA/EB NPs were negatively charged, and the particle sizes of both were significantly larger than those of ML-LA NPs and ML-LA/EB NPs by about 25 nm, demonstrating the successful encapsulation of FU.

Table 1 Particle size, PDI and potential of nanoparticles

High performance liquid chromatography was used to establish a quantitative analysis method for the four nanoparticles, and the encapsulation efficiency and drug loading of each component were determined. The results are shown in Table 2. The encapsulation efficiency of the four nanoparticles was higher than 80%, and the drug loading was greater than 55%.

Table 2 Encapsulation efficiency and drug loading of nanoparticles

Example 6. Cellular uptake effect of biguanide derivative nanoparticles

Flow cytometry was used to quantitatively analyze the uptake of nanoparticles (prepared according to Example 5) by RAW 264.7 macrophages and GES-1 human gastric epithelial cells. RAW 264.7 cells and GES-1 cells were cultured in 12-well plates for 24 h, 2 mL of free C6 and C6-labeled nanoparticles (ML-LA/C6 NPs and FU/ML-LA/C6 NPs) were added and incubated with the cells for 4 h, washed twice with PBS, 1×10 ⁴ cells were collected, and the fluorescence intensity of C6 was measured by flow cytometry. The results are shown in Figure 9. In RAW 264.7 cells, the average fluorescence intensity of free C6 was 1.52×10 ⁵ , ML-LA/C6 NPs was 2.01×10 ⁵ , and FU/ML-LA/C6 NPs was 2.11×10 ⁵ . Compared with 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 a higher cellular uptake rate than ML-LA/C6 NPs. The same trend of cellular uptake of the two nanoparticles was detected in GES-1 cells, further proving that ML-LA/C6 NPs and FU/ML-LA/C6 NPs can improve their ability to be taken up by cells.

Example 7. Effect of biguanide derivative nanoparticles on the removal of intracellular bacteria

The coating plate method was used to quantitatively evaluate the clearance effect of biguanide derivative nanoparticles (prepared according to Example 5) on intracellular H. pylori . GES-1 human gastric epithelial cells and RAW 264.7 macrophages were cultured in cell well plates for 24 h, H. pylori bacterial suspension was collected and added to the well plates for co-incubation with cells, the culture medium was aspirated and a medium without double antibiotics containing gentamicin was added to kill H. pylori that did not enter the cells, the culture medium was aspirated and washed with sterile PBS. The following dosing groups were set: free MET, free ML, free ML+LA+EB, ML-LA NPs, ML-LA/EB NPs and FU/ML-LA/EB NPs, and the concentration of the same drug was kept consistent in each group (i.e., MET was 100 μg/mL, ML was 100 μg/mL, LA was 70 μg/mL, EB was 17 μg/mL, and FU was 90 μg/mL). 2 mL of the above drugs were added to each well and cultured for 24 h. 0.1% tea saponin solution was added to lyse the cells, and the cell lysate was collected for standby use. Part of the cell lysate was coated on a plate, and after culturing at 37 °C for 72 h under a microaerobic environment, the plate counting method was used to quantify the intracellular H. pylori level, and the results are shown in Figure 10. After treatment with the biguanide derivative ML, the H. pylori levels in both cells were significantly reduced and lower than the intracellular bacterial levels after metformin hydrochloride treatment. In the ML+LA+EB mixed drug group, the number of intracellular bacteria was further reduced, so the addition of LA and EB can synergize with ML to kill intracellular bacteria. In RAW 264.7 cells, the number of intracellular bacteria of ML-LA/EB NPs and FU/ML-LA/EB NPs decreased by 78% and 87%, respectively, showing a higher intracellular bacterial killing ability than the free drug group, and the same trend of reducing intracellular bacterial load was found in GES-1 cells. It can be seen that the FU/ML-LA/EB NPs of the present invention can significantly enhance the uptake of drugs, increase the level of drugs reaching the intracellular level, and play a role in killing intracellular bacteria.

Fluorescence staining was used to evaluate the effect of biguanide derivative nanoparticles (prepared according to Example 5) on the removal of intracellular H. pylori . After collecting the H. pylori bacterial suspension and incubating it with CFDA-SE fluorescent dye for 30 min, the precipitate was collected by centrifugation and washed three times with sterile PBS, and then resuspended with a medium without double antibodies and added to a cell plate with GES-1 human gastric epithelial cells and RAW264.7 macrophages for co-incubation. The culture medium was discarded and a medium without double antibodies containing gentamicin was added to kill H. pylori that did not enter the cell. The culture medium was discarded and washed with sterile PBS, and the above-mentioned drug solution was added to continue incubation for 24 h. The culture medium containing the drug solution was removed and washed with sterile PBS, and the cell nucleus was stained with DAPI dye and the lysosome was stained with DAD 99 dye, respectively. The intracellular and extracellular H. pylori levels were observed using a laser scanning confocal microscope to evaluate the removal effect of biguanide derivatives on it, and the results are shown in Figures 11 and 12. Green fluorescence indicates H. pylori , red fluorescence indicates lysosomes in cells, and blue fluorescence indicates cell nuclei. In the model group, green fluorescence and red fluorescence highly overlapped, indicating that most of the bacteria accumulated in lysosomes after being taken up by cells. After treatment with the biguanide derivative ML, the H. pylori levels in both cells were significantly reduced, and the green fluorescence intensity was significantly weaker than that 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 both have higher intracellular bacteria killing ability, among which the FU/ML-LA/EB NPs group has a more significant effect, which is consistent with the quantitative results of the above-mentioned coating plate method.

Claims (12)

1. A compound of formula I or a pharmaceutically acceptable salt thereof,

formula (I)

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):

formula (II) and

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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