WO2023123008A1 - 一种双胍衍生物及其应用 - Google Patents

一种双胍衍生物及其应用 Download PDF

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WO2023123008A1
WO2023123008A1 PCT/CN2021/142309 CN2021142309W WO2023123008A1 WO 2023123008 A1 WO2023123008 A1 WO 2023123008A1 CN 2021142309 W CN2021142309 W CN 2021142309W WO 2023123008 A1 WO2023123008 A1 WO 2023123008A1
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mice
biguanide
cells
formula
metformin
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PCT/CN2021/142309
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English (en)
French (fr)
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全军民
许正双
李勤凯
付佳苗
刘思宇
王小权
徐炳琳
胡敏强
曹安琪
孙立峰
徐杰成
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北京大学深圳研究生院
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Priority to PCT/CN2021/142309 priority Critical patent/WO2023123008A1/zh
Publication of WO2023123008A1 publication Critical patent/WO2023123008A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/02—Guanidine; Salts, complexes or addition compounds thereof

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a biguanide derivative and its application.
  • Biguanide compounds such as metformin, phenformin, and buformin
  • metformin has always been the first-line drug for treating type II diabetes.
  • the hypoglycemic effect of metformin is mainly due to inhibition of hepatic gluconeogenesis and promotion of glucose absorption.
  • the main target of metformin in the cell is the mitochondria, because it is positively charged, making metformin more likely to accumulate in the mitochondrial matrix, and inhibit the mitochondrial respiratory chain complex I, thereby inhibiting the generation of ATP, leading to the accumulation of AMP, AMP/ATP An increase in the ratio activates AMPK.
  • AMPK is an important energy sensor in cells and a master regulator of metabolism and growth pathways.
  • AMPK phosphorylate insulin receptor and insulin receptor substrate-2 (IRS-2), thereby promoting the transport of glucose transporters; activated AMPK can also inhibit fatty acid synthesis and enhance ⁇ -oxidation, thereby promoting glucose Transport; increased AMP also inhibits adenylate cyclase from catalyzing the conversion of ATP to cAMP, thereby inhibiting gluconeogenesis.
  • IRS-2 insulin receptor and insulin receptor substrate-2
  • Diabetic patients are at increased risk of cancer, mainly due to long-term hyperinsulinemia, hyperglycemia, and fat-induced chronic inflammation.
  • my country conducted the largest study on the relationship between type 2 diabetes and cancer risk in mainland China so far. The results of the study showed that in the general population, men and women with diabetes had a higher risk of cancer than normal men. And women are 34% and 62% higher, and diabetic patients have a higher risk of liver cancer, pancreatic cancer, colon cancer, breast cancer and bladder cancer.
  • metformin use is associated with reduced cancer incidence and mortality in patients with diabetes.
  • Preclinical studies have also shown that metformin can directly kill tumor cells.
  • the anti-tumor mechanism of metformin is attributed to its indirect effect on the one hand, which is to reduce the levels of blood sugar and insulin in the body; on the other hand, it is attributed to its direct effect, which is to inhibit the mitochondrial respiratory chain complex I, activate AMPK and inhibit the mTORC1 pathway, etc. .
  • the results of in vivo and in vitro studies show that metformin needs to be much higher than its clinically accessible concentration to show significant antitumor activity.
  • the results of recent clinical trials of metformin in the treatment of pancreatic cancer also show that metformin does not significantly improve the patient's overall The survival rate shows that the antitumor effect of metformin still needs to be improved.
  • buformin and phenformin have stronger hypoglycemic effects and more significant anti-tumor effects, but these compounds can also activate the glycolysis pathway of tumor cells while efficiently inhibiting mitochondrial complex I , cause severe lactic acidosis, and activated glycolysis will also help tumor cells escape the killing effect of biguanide drugs. These properties limit the clinical application of existing biguanide compounds in tumor therapy.
  • metformin can also reduce the occurrence of cardiovascular diseases, improve symptoms related to polycystic ovary syndrome, and improve aging-related neurodegenerative diseases.
  • the risk of cardiovascular disease in diabetic patients is 5 times higher than that of normal people, and the risk of heart failure and death after myocardial infarction is 4 times higher than that of normal people.
  • Metformin can inhibit the early changes of atherosclerotic lesions, including reducing the adhesion of monocytes and leukocytes in the endothelium; inhibiting the differentiation of monocytes into macrophages, inhibiting the formation of foam cells, and reducing the proliferation of vascular smooth muscle cells.
  • Polycystic ovary syndrome is a disease caused by complex endocrine and metabolic abnormalities. Metabolic abnormalities are considered to be the core factor in the occurrence and development of polycystic ovary syndrome, and insulin sensitizers have been proposed as a feasible solution for the treatment of polycystic ovary syndrome. Metformin, as an insulin sensitizer, can improve multiple symptoms of polycystic ovary syndrome, such as promoting ovulation, improving follicle quality, and increasing pregnancy rate.
  • metformin can prolong the lifespan of diabetic patients or normal mice, and its mechanism of action can be attributed to the following points: 1) activate AMPK and inhibit mTOR; 2) regulate insulin/insulin-like growth factor -1 (IGF-1) signaling pathway; 3) reduce the production of reactive oxygen species (ROS); 4) regulate the expression of SIRTs.
  • IGF-1 insulin/insulin-like growth factor -1
  • ROS reactive oxygen species
  • metformin can also improve or benefit other inflammatory and metabolic-related diseases, such as non-alcoholic fatty liver disease, intestinal inflammation, arthritis, etc.
  • the object of the present invention is to provide a biguanide derivative.
  • Another object of the present invention is to provide a method for preparing the above-mentioned biguanide derivatives.
  • Another object of the present invention is to provide the application of the above-mentioned biguanide derivatives.
  • a biguanide derivative has a structure as shown in formula (I):
  • R is selected from H, or acyl
  • Acyl is selected from acetyl, propionyl, isopropionyl, butyryl and benzoyl and substituted benzoyl;
  • R 2 is selected from H, carboxyl, or ester group, and the ester group is selected from methyl ester or ethyl ester.
  • R 1 is H
  • R 2 is selected from H or carboxyl
  • R 2 is H
  • R 1 is selected from H, or an acyl group.
  • R 1 is H
  • R 2 is H
  • its specific structure is as follows:
  • the method comprises the following steps: reacting dicyandiamide with a compound represented by formula (II) in at least one organic solvent, and obtaining formula (I) after derivatization the indicated biguanide derivatives;
  • R is a mercapto protecting group, such as triphenylmethyl, diphenylmethyl or acetyl, etc.
  • R is selected from H or acyl; acyl is selected from acetyl, propionyl, isopropionyl, butyryl and benzoyl and substituted benzoyl;
  • R 2 is selected from H, carboxyl or ester group, and the ester group is selected from methyl ester or ethyl ester.
  • the organic solvent is a protic solvent, including solvents with suitable boiling points such as pentanol, butanol, propanol, and isobutanol.
  • the present invention also provides a medicine, which comprises a biguanide derivative represented by formula (I) or a non-toxic pharmaceutically acceptable salt thereof.
  • pharmaceutically acceptable salt refers to salts that retain the biological effectiveness and properties of the compounds of this invention and are not biologically or otherwise undesirable.
  • the compounds of the present invention may form acid and/or base salts by virtue of the presence of amino and/or carboxyl or similar groups.
  • Pharmaceutically acceptable acid addition salts can be prepared from inorganic or organic acids
  • pharmaceutically acceptable base addition salts can be prepared from inorganic or organic bases.
  • non-toxic pharmaceutically acceptable salt refers to a non-toxic salt formed with a non-toxic pharmaceutically acceptable inorganic or organic acid or inorganic or organic base.
  • such salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like, and from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, Malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, fumaric acid, methanesulfonic acid, and toluenesulfonic acid Acid etc.
  • inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like
  • organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, Malic acid, tartaric acid, citric acid, ascorbic acid, pamoi
  • the compounds of formula (I) according to the invention may be administered alone, preferably they are presented as pharmaceutical compositions.
  • the aforementioned pharmaceutical composition useful according to the present invention comprises at least one compound of formula (I) as defined above, and one or more pharmaceutically acceptable carriers and any other therapeutic ingredients of choice.
  • the active ingredients required in combination therapy may be combined into a single pharmaceutical composition for simultaneous administration.
  • compositions, carriers, diluents, and agents are used interchangeably when referring to compositions, carriers, diluents, and agents and mean that the substance can be administered to a mammal without adverse effects.
  • Physiological effects such as nausea, dizziness, heart noise, etc.
  • compositions in which the active ingredient is dissolved or dispersed are prepared as injectables, either as liquid solutions or suspensions; however, solid forms suitable for solution in, or suspension in, liquid prior to use can also be prepared.
  • the formulation may also be emulsified.
  • the above-mentioned pharmaceutical composition can be formulated into solid dosage forms, such as capsules, tablets, pills, powders, dragees or granules.
  • excipients and the content of active ingredients in the excipients are usually determined according to the solubility and chemical properties of the active compound, the specific mode of administration and the regulations observed in pharmaceutical practice.
  • excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate
  • disintegrants such as starch, alginic acid, and certain complex silicates
  • lubricants such as magnesium stearate, lauryl A combination of sodium alkyl sulfate and talc
  • aqueous suspensions When aqueous suspensions are used, they may contain emulsifying agents or suspension enhancers. Diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol and chloroform or mixtures thereof may also be used.
  • the above pharmaceutical composition can be administered to humans and animals in a suitable dosage form, by local or systemic administration, including oral administration, rectal administration, nasal cavity administration, buccal administration, eye administration, sublingual administration, transdermal administration, etc. Administration, rectal administration, topical administration, vaginal administration, parenteral administration (including subcutaneous injection, arterial injection, intramuscular injection, intravenous injection, intradermal injection, intrathecal injection, epidural injection), intracisternal injection Administration and intraperitoneal administration. It is to be understood that the preferred route may vary with, for example, the circumstances of the recipient.
  • the formulations may be prepared in unit doses by any methods well known in the art of pharmacy. Such methods include bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the above formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • the total daily dose of a compound of the invention is administered to a subject in single or multiple doses.
  • the present invention also provides the application of biguanide derivatives in the preparation of drugs for the prevention or treatment of metabolic and inflammation-related diseases, wherein the diseases include but not limited to polycystic ovary syndrome, cancer, neurodegenerative diseases, cardiovascular diseases, Obesity, diabetes, aging and non-alcoholic fatty liver disease, etc.
  • the present invention couples biguanide fragments with cysteamine through a disulfide bond to form a novel biguanide derivative.
  • the biguanide derivative can simultaneously inhibit two energy metabolism pathways of glycolysis and oxidative phosphorylation, reduce the production of lactic acid, and more effectively inhibit tumor growth. grow. It has shown good therapeutic effects in the treatment of other diseases, such as lowering blood sugar, treating polycystic ovary syndrome, cardiovascular protection, delaying aging, and treating non-alcoholic fatty liver.
  • Figure 1 shows the characterization data of MC001, where A is the hydrogen spectrum of MC001, and B is the carbon spectrum of MC001;
  • Figure 2 shows the characterization of the reduced form of MC001 in cells
  • Figure 3 shows that MC001 inhibits mitochondrial complex I, wherein, A is the comparison of the effects of different compounds on inhibiting mitochondrial complex I, and B is the curve fitting of MC001 inhibiting mitochondrial complex I;
  • Figure 4 shows the verification results of MC001 simultaneously inhibiting oxidative phosphorylation and glycolysis; A, C are inhibition of respiration; B, D are inhibition of glycolysis;
  • Figure 5 shows that MC001 does not produce too much lactic acid;
  • A is the effect of the drug on the production of lactic acid at the cellular level, and B is the detection of serum lactic acid levels in mice;
  • Figure 6 shows the effect of the compound on the viability of tumor cells
  • A is the cell survival curve obtained by fitting
  • B is the calculated half inhibitory concentration of MC001 and metformin on different cells
  • Figure 7 shows the anti-tumor activity of MC001 in vivo;
  • A is the HCT116 tumor growth curve in nude mice
  • B is the CT26 tumor growth curve in BALB/c nude mice
  • C is the CT26 tumor growth curve in BALB/c wild-type mice;
  • Figure 8 shows that MC001 promotes the infiltration and function of CD8+ T cells in the tumor microenvironment; A is the proportion of CD8-positive T cells in the tumor microenvironment, and B is the content of granzyme B in CD8-positive T cells;
  • Figure 9 shows the inhibition of MC001 on TGF ⁇ secretion and downstream signaling pathways;
  • A is the level of TGF ⁇ in mouse serum,
  • B and C are the cell level, the TGF ⁇ content in the medium supernatant and the expression of TGF ⁇ downstream target proteins in cells level;
  • Figure 10 shows that MC001 inhibits tumor metastasis
  • Figure 11 shows that MC001 promotes the activity of STING agonists.
  • A is MC001 combined with ADU-S100, B is MC001 combined with cGAMP;
  • Figure 12 shows that MC001 reduces the fasting blood glucose of the T2DM mouse model and improves its glucose tolerance;
  • A is the content of fasting blood glucose 16 hours after administration in high-fat fed (HFD) mice;
  • B shows that the mice were injected with 2g/kg The change of blood glucose in mice after glucose over time, that is, glucose tolerance;
  • Figure 13 shows that MC001 reduces the secretion of insulin in HFD mice;
  • Figure A shows the changes in insulin secretion into the blood of mice stimulated by glucose over time;
  • Figure B shows the fasting insulin levels of mice in each experimental group;
  • Figure 14 shows that MC001 improves insulin sensitivity in HFD mice
  • Figure 15 shows that MC001 achieves the effect of sugar control without producing lactic acid toxicity and liver and kidney toxicity;
  • A is the detection of lactic acid level, and
  • B, C, D, E are the detection of liver and kidney toxicity;
  • Figure 16 shows that MC001 relieves dihydrotestosterone (DHT)-induced granulosa cell luteinization disorder in polycystic ovary syndrome (PCOS) mice;
  • a and B are mouse ovary morphology and follicle classification and counting respectively, and
  • C is mouse ovary mRNA Expression,
  • D is mouse ovary immunohistochemical staining;
  • Figure 17 shows that MC001 alleviates the inhibitory effect of luteinization caused by DHT at the cellular level
  • a and B are the Cyp11a1 immunofluorescence staining morphology and fluorescence intensity quantification of granulosa cells
  • C is the concentration of progesterone in the supernatant of granulosa cells
  • D is the granule Cellular mRNA expression
  • Figure 18 shows the effect of MC001 on the degree of aortic lesion in mice with atherosclerosis induced by high-fat diet;
  • A is a representative map of aortic arch lesion, and B is a statistical map of lesion;
  • Figure 19 shows the effect of MC001 on the CRP content in serum of atherosclerotic mice induced by high-fat diet
  • Figure 20 shows the effect of MC001 on conventional blood lipid indexes in the serum of atherosclerotic mice induced by high-fat diet;
  • the content of protein cholesterol Figure E shows the ratio of total cholesterol and high-density lipoprotein cholesterol in mouse serum.
  • Figure 21 shows that MC001 improves the exercise capacity of aging mice;
  • A is the duration in the rotarod test after the aging mice are given drug treatment;
  • B is the grip test result of the aging mice;
  • Figure 22 shows that MC001 can increase the number of lymphocytes in the blood of aging mice and improve thymus function;
  • A is the ratio of neutrophils, lymphocytes, and monocytes in the blood, and
  • B is the HE staining map of thymus;
  • Figure 23 shows that MC001 reduces the metabolic toxicity of aging mice;
  • Figure A is the lactic acid content level of aging mice;
  • Figure B is the activity of alanine aminotransferase in aging mice;
  • Figure C is the creatinine level of aging mice;
  • Figure 24 shows that MC001 relieves liver damage in non-alcoholic fatty liver mice;
  • a and B are toxicity indicators, and
  • C and D are triglyceride and total cholesterol content respectively;
  • Figure 25 shows that MC001 alleviates the phenomenon of fat vacuoles in the liver of non-alcoholic fatty liver model mice; A is HE staining of liver slices, and B is the statistical result of the number of liver vacuoles;
  • Figure 26 shows the effect of MC001 on ferroptosis; A is that MC001 inhibits the increase of ROS caused by the ferroptosis inducer erastin, and B is that MC001 inhibits the increase of lipidROS caused by the ferroptosis inducer erastin;
  • Figure 27 shows the effect of MC001 on the accumulation of lipid droplets in cells
  • A is oil red staining, reflecting the accumulation of lipid droplets in HEPG2 cells caused by oleic acid and the changes of lipid droplets after drug treatment
  • B is a statistical graph
  • Figure 28 shows the effect of MC001 on cellular lipid peroxidation; A is Bodipy staining, and B is a statistical graph;
  • Figure 29 shows the effect of MC001 on cellular inflammatory response and ROS level;
  • A is the expression of NOS2
  • B is the expression of TNF- ⁇
  • C is the expression of IL-1 ⁇ ;
  • Figure 30 shows the effect of flow cytometry detection of MC001 on the apoptosis of SH-SY5Y cells induced by MPP+;
  • Figure 31 shows the effect of TH staining on the loss of dopaminergic neurons caused by MPTP to observe MC001;
  • Figure 32 shows the effect of MC001 on oxidative stress in the brain of MPTP model mice.
  • the image on the left is the imaging result, and the image on the right is the optical density statistical result;
  • Figure 33 shows the effect of MC001 on GSH levels in mice;
  • Figure A shows the effect of the time gradient of a single dose of MC001 on the ratio of reduced glutathione GSH to total glutathione, and
  • Figure B shows the effect of long-term treatment of MC001 on MPTP mice The effect of GSH on the total glutathione level in the brain.
  • Figure C shows the effect of different doses of MC001 on the level of GSH.
  • the present invention connects 2-mercaptoethyl biguanide and cysteamine through a disulfide bond to produce a novel biguanide derivative MC001 (1-(2-((2-aminoethyl) perthio) ethyl) biguanide) .
  • MC001 (1-(2-((2-aminoethyl) perthio) ethyl) biguanide)
  • MC001 enters the cells, it is converted into 2-mercaptoethylbiguanide and cysteamine in a reducing environment.
  • Cysteamine inhibits glycolysis
  • 2-mercaptoethylbiguanide inhibits oxidative phosphorylation, thereby achieving The purpose of dual inhibition of two energy metabolic pathways, glycolysis and oxidative phosphorylation.
  • the target compound of the following examples was prepared by a method similar to that described in Example 1, the difference from Example 1 was that the amine compound corresponding to the structure of the target compound was used to react with dicyandiamide.
  • Example 4 confirms the reduced form of MC001 in cells
  • MC001 is reduced to 2-mercaptoethyl biguanide (162.0809) and cysteamine in the cell. Since cysteamine itself exists in the cell, the present invention uses 2-mercaptoethyl biguanide to characterize MC001 reduction in cells. Thus, the present invention confirms that MC001 is reduced to 2-mercaptoethylbiguanide and cysteamine in cells.
  • This example uses Complex I Activity Assay kit, in vitro detection of mitochondrial complex I activity. 5mM MC001, phenformin and metformin were premixed with the mitochondrial solution for 4 hours, and the substrate was added to detect the absorbance at 340nm immediately.
  • the concentration gradient of MC001 (20mM, 10mM, 5mM, 2.5mM, 1.25mM, 0.625mM, 0.3125mM, 0.156mM, 0.078mM, 0mM) was further set to detect the activity of mitochondrial complex I.
  • the IC 50 value of MC001 inhibition of mitochondrial complex I in vitro is about 155 ⁇ M calculated by fitting.
  • Example 6 Verifies that MC001 simultaneously inhibits oxidative phosphorylation and glycolysis
  • seahorse instruments are used to detect the oxygen consumption rate and glycolysis rate of cells.
  • HCT116 cells were inoculated in 24-well seahorse culture plates 36 hours in advance, with 2 ⁇ 104 cells per well. After 24 hours of culture, they were treated with drugs and treated for 12 hours. Then, the cells were washed according to the kit instructions, and the mitochondrial stress kit was diluted. Drugs, and finally on the computer to detect the oxygen consumption of each stage of the cell.
  • Example 7 verifies that MC001 does not produce excessive lactic acid
  • this example uses 200mM MC001, metformin, phenformin and buformin to treat HCT116 cells, and after 24 hours, detect the content of lactic acid in the supernatant.
  • the administration concentrations of MC001 and metformin were 20mg/kg, 100mg/kg and 200mg/kg, and the administration concentration of phenformin was 200mg /kg, the lactic acid content in the blood was analyzed one week after administration, the results are shown in Figure 5B, the lactic acid level of the phenformin treatment group at a dose of 200 mg/kg was significantly higher than that of MC001 at the same concentration, and different concentrations of MC001 caused the accumulation of lactic acid in the body
  • the average level of metformin is comparable to that of metformin and does not cause excessive lactic acid accumulation.
  • the present embodiment In order to verify the anti-tumor activity of the novel biguanide compound MC001, the present embodiment first used the MTT method in various tumor cell lines (human colon cancer cells: HCT116, Caco2, SW480; mouse colon cancer cells: CT26; human liver cancer cells: HepG2 ; Human lung cancer cells: NCI-H460). After the cells were treated with drugs for 48 hours, MTT was added, cultured for 2 hours, the supernatant was removed, and the absorbance was measured after adding DMSO for dissolution.
  • tumor cell lines human colon cancer cells: HCT116, Caco2, SW480; mouse colon cancer cells: CT26; human liver cancer cells: HepG2 ; Human lung cancer cells: NCI-H460.
  • a subcutaneous xenograft model of HCT116 cells was established in nude mice in this example. Metformin and MC001 were administered with water respectively. Tumor volumes were measured every other day for a total of 12 days of dosing. Results As shown in Figure 7A, the tumor growth of the MC001 group was significantly slower than that of the control group and the metformin group, showing significant anti-tumor activity, while metformin did not show significant anti-tumor activity at the same dose.
  • the CT26-Luc xenograft tumor model was established in BALB/c nude mice and BALB/c wild mice at the same time, and the tumor volume was observed and recorded every other day.
  • the antitumor activity of MC001 and metformin in BALB/c wild-type mice was significantly better than that in BALB/c nude mice, as shown in Figure 7B and C, indicating that MC001 and metformin
  • the anti-tumor activity of the drug comes partly from its immunomodulatory function.
  • Example 10 MC001 promotes the infiltration and function of CD8+ T cells in the tumor microenvironment
  • the spleen T cells of the BALB/c wild-type CT26 tumor model mouse in Example 9 were further analyzed by flow cytometry in this example.
  • Example 11 MC001 inhibits TGF ⁇ secretion and downstream signaling pathways
  • MC001 dose-dependently significantly inhibited the secretion level of TGF ⁇ in murine CT26 colon cancer cells, and MC001 could also effectively inhibit the phosphorylation of Smad 3 downstream of TGF ⁇ and the expression of Snail.
  • a CT26 xenograft tumor model was established in BALB/c mice, and when the tumor grew to 50 mm 3 , MC001 was administered intragastrically (the concentration of MC001 was 0.1 mg/mL, 0.5 mg /mL, 2mg/mL), one week later, the TGF ⁇ in mouse serum was quantitatively analyzed by ELISA kit.
  • Example 12 MC001 inhibits tumor metastasis
  • this example established a tumor metastasis model by injecting CT26-luc cells into the tail vein, administered MC001 (0.5 mg/mL) with water, and performed imaging of mice at 4 weeks to observe tumor metastasis and planting conditions.
  • Example 13 MC001 promotes the activity of STING agonists
  • TGF ⁇ inhibits the phosphorylation of IRF3 through a series of indirect mechanisms, preventing the activation of the STING pathway, thereby affecting the function of STING agonists in regulating the tumor microenvironment. Therefore, this example verifies whether MC001 can enhance the activity of STING agonists.
  • THP1 cells were treated with 2 ⁇ g/mL ADU-S100 or cGAMP in combination with MC001, samples were collected 8 hours later, and RNA was extracted for RT-PCR analysis. Results As shown in Figure 11, MC001 can enhance the activation of type I interferon induced by ADU-S100 and cGAMP.
  • Example 14 MC001 reduces fasting blood sugar in T2DM mouse model and improves its glucose tolerance
  • this example selects an obese mouse model (HFD mouse) induced by a high-fat diet as the research object of the type 2 diabetes mouse model.
  • the control group Ctrl, MC001 0.1mg/mL group, MC001 1mg/mL group and metformin Met 1mg/ml group were respectively set up, and the administration was carried out by drinking water.
  • mice After 5 weeks of administration, the mice were fasted for 16 hours, and then the blood glucose of the mice was detected using a Roche handheld blood glucose meter. Subsequently, the mice were intraperitoneally injected with glucose (a dose of 2 g/kg), and blood was collected at different time points to detect blood sugar levels. The blood samples corresponding to the time points were also saved for the insulin detection of the subsequent glucose stimulation.
  • MC001 can improve fasting blood sugar in HFD mice at a dose of 0.1 mg/ml, and its ability to control blood sugar is close to that of metformin 1 mg/mL. In the GTT glucose challenge experiment (B in Figure 12), MC001 also showed better blood sugar control ability than metformin.
  • Example 15 MC001 reduces the secretion of insulin in HFD mice
  • Example 14 Taking the HFD mice in Example 14 as the object, take blood samples at the corresponding time points after glucose injection, and use ELISA to detect the content of insulin in them, and obtain the results of glucose-stimulated insulin secretion and the basis of HFD mice after administration. insulin levels.
  • both MC001 and Metformin can significantly reduce the level of glucose-stimulated insulin secretion, and at the same time the basal insulin is also at a lower level after administration, as shown in Figure 13 B shows that the drug can improve insulin response, and then achieve Better control of sugar levels while reducing insulin secretion.
  • Embodiment 16 MC001 improves the insulin sensitivity of HFD mice
  • the insulin sensitivity test (insulin tolerance test, ITT) was performed on the HFD mice in Example 14, and the insulin signal response was further measured. After the mice were fasted for 6 hours, insulin was injected intraperitoneally (the dose for HFD mice was 1 U/kg), and then the blood glucose levels at different time points were detected.
  • Example 17 MC001 achieves sugar control effect without producing lactic acid toxicity
  • the lactic acid content of the HFD mice in Example 14 was detected using a lactic acid detection kit to detect the lactic acid toxicity produced by the drug.
  • MC001 did not significantly increase lactic acid in mice while reducing blood sugar.
  • Metformin with the same efficacy significantly increased lactic acid levels in mice, which was consistent with the clinical results Consistent, thus further illustrating the superiority of MC001, showing greater clinical value.
  • Nanjing Jiancheng Biochemical Kit was used to detect the contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST) (hepatotoxicity indicators) and nephrotoxicity indicators (creatinine and urea) in serum of HFD mice.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • nephrotoxicity indicators creatinine and urea
  • Example 18 MC001 relieves dihydrotestosterone (DHT)-induced granulosa cell luteinization disorder in polycystic ovary syndrome (PCOS) mice
  • This example is to verify the role of MC001 in the treatment of polycystic ovary syndrome (PCOS).
  • PCOS polycystic ovary syndrome
  • Long-term exogenous DHT stimulation can induce mice and other symptoms similar to human PCOS, such as ovulation disorders, polycystic ovaries, and hyperandrogenism.
  • DHT slow-release tubes were implanted subcutaneously in mice to maintain high androgen stimulation for 56 days, and 0.1mg/ml Metformin and MC001 were continuously given to drink water on the 28th day. At the end of the experiment, the mouse ovaries were taken for section observation.
  • MC001 alleviated the reduction of mouse follicles caused by DHT, corrected the abnormal expression of granulosa cell luteinization gene caused by DHT, and relieved the DHT-induced luteinization disorder of granulosa cells.
  • Example 19 MC001 relieves the inhibition of luteinization caused by DHT at the cellular level
  • Granulosa cells obtained from prepubescent mouse ovaries were treated with 10 ⁇ M forskolin and 20 nM phorbol alcohol (PMA) for 6 hours to luteinize them. At the same time, the cells were treated with 1nM DHT to establish the PCOS cell model.
  • PMA phorbol alcohol
  • DHT inhibition attenuated the positive signal of granulosa cells
  • MC001 reversed this effect of DHT at both protein and mRNA levels.
  • the process of luteinization of granulosa cells produces a large amount of progesterone, and MC001 can alleviate the decrease of progesterone caused by DHT.
  • Example 20 Effect of MC001 on the degree of aortic lesion in mice with atherosclerosis induced by high-fat diet
  • the 5-week-old ApoE gene-deficient C57BL/6J strain mice were randomly divided into groups, and high-fat induction was carried out with Western diet. From the 8th week, the MC001 group was administered by drinking water at a dose of 1 mg/ mL; starting from the 12th week, the MC001 delayed group began to be administered orally at a dose of 1 mg/mL; the control group received normal water until the end of the 16th week.
  • the experimental animals were euthanized. After dissection, 10 mL of PBS was perfused from the right atrial appendage, and then 10 mL of tissue fixative (4% paraformaldehyde). The aorta was completely separated under a stereoscope, and the blood vessels were peeled off with fat forceps. Adhesive adipose tissue. The isolated aorta was stained grossly by Oil Red O staining, and photographed and recorded. The lesion area of the aortic arch in the experimental group and the control group was counted using ImageJ, and the difference was statistically significant.
  • the MC001 group and the MC001 delayed group were administered with drinking water from the 8th week and the 12th week, respectively, at a dose of 1 mg/mL, and continuously administered
  • the risk of cardiovascular disease in the body was evaluated by detecting the level of C-reactive protein (CRP) in the serum of mice, so as to evaluate the impact of the compound on the occurrence of cardiovascular disease in the body.
  • CRP C-reactive protein
  • both the MC001 group and the MC001 delayed administration group can significantly reduce the CRP content in the serum of mice in the experimental group, indicating that MC001 has an excellent predictive and improving effect on the occurrence of cardiovascular diseases in the body .
  • Example 22 Effect of MC001 on routine blood lipid indexes in serum of mice with atherosclerosis induced by high-fat diet
  • Example 20 The method described in Example 20 was used to induce the mouse atherosclerosis model with a high-fat diet, and the administration was carried out in groups.
  • the MC001 group and the MC001 delay group were administered with water from the 8th week and the 12th week, respectively.
  • the dose was 1 mg/mL, and the administration continued until the end of the 16th week.
  • the mice were euthanized, and the serum was taken for routine blood lipid detection.
  • MC001 can significantly reduce the content of low-density lipoprotein (LDL-C) in the body's serum and increase the content of high-density lipoprotein (HDL-C), but it will also increase to a certain extent Triglyceride content, but had no significant effect on total cholesterol; while delayed administration of MC001 had no significant effect on the four blood lipid items. Nevertheless, administration of MC001 at the early stage of atherosclerosis process or delayed administration at the late stage can significantly reduce the ratio of total cholesterol to high-density lipoprotein in the body, showing that it has a positive effect on cardiovascular diseases such as atherosclerosis and coronary heart disease. Significant predictive improvement.
  • LDL-C low-density lipoprotein
  • HDL-C high-density lipoprotein
  • Example 23 MC001 improves the exercise capacity of aging mice
  • Example 24 MC001 can increase the number of lymphocytes in the blood of aging mice and improve thymus function
  • the whole blood of the mice in Example 23 was taken, and the blood cells of the aged mice were counted by WBC classification using an automatic blood cell counter.
  • senescent cells had increased neutrophils, decreased lymphocytes, and increased monocytes
  • mice treated with MC001 had increased lymphocytes, neutrophils, and neutrophils.
  • Granulocytes and monocytes decreased, indicating that MC001 may improve thymus function in aging mice, thereby increasing the proportion of blood lymphocytes and reducing the number of inflammatory cells.
  • Example 26 After dissecting the aged mouse in Example 26, the thymus was taken for paraffin embedding, sectioned, fixed, and HE stained.
  • Example 25 MC001 reduces metabolic toxicity in aging mice
  • mice serum in Example 23 was taken, and the lactic acid content, alanine aminotransferase activity and creatinine content of the mice were measured using the mouse metabolic index detection kit built in Nanjing.
  • MC001 can significantly reduce the levels of the three, and improve the metabolic toxicity in the aging process of mice.
  • Example 26 MC001 relieves liver damage in mice with non-alcoholic fatty liver
  • This example uses a high-fat diet and carbon tetrachloride injection to induce obesity C57BL6/J mice (HFD mice) as a non-alcoholic fatty liver model, using water feeding, with 0.05mg/ml or 0.1mg/ml dose of MC001 Drugs were administered to C57 mice at the 4th week, 10th week, and 12th week of high-fat diet respectively, and 30 mg/kg obeticholic acid was intragastrically administered at the 10th week as a control. After 14 weeks of hyperlipidemia, the mouse serum was collected, and the contents of LT, AST, TG, and TC were detected using A commercial kit.
  • Example 27 Medication of MC001 can alleviate the fat cavitation phenomenon in the liver of non-alcoholic fatty liver model mice
  • the NAFLD model was dissected to obtain mouse livers, and paraffin sections were prepared, and liver vacuoles were observed by HE staining.
  • Embodiment 28 Effect of MC001 on ferroptosis
  • the occurrence and development of non-alcoholic fatty liver are related to ferroptosis.
  • the present invention firstly verifies the effect of MC001 on the changes of ROS and LipidROS content caused by erastin. 10 ⁇ M erastin and different concentrations of MC001 were simultaneously added to HepG2 cells to stimulate for 24 hours, and after incubation with DCFH-DA or BODIPY dye, the fluorescence luminescence rate was detected by flow cytometry.
  • HEPG2 cells were stimulated with oleic acid in this example, and changes in lipid droplet content were detected. After being stimulated by 300 ⁇ M OA for 24 hours, the culture medium was replaced with different concentrations of MC001 for 24 hours, oil red staining solution was added for staining, and the accumulation of red lipid droplets was observed under a microscope.
  • this example chooses to stimulate HEPG2 cells with oleic acid, and detect the change of Lipid ROS content. After being stimulated by 300 ⁇ M OA for 24 hours, the culture medium was replaced with different concentrations of MC001 for 24 hours, and BODIPY staining solution was added to incubate and stain, and the fluorescence luminescence was detected by flow cytometry.
  • Example 31 MC001 inhibits glial cell activation
  • BV2 cells were used as a model to observe the effect of MC001 on the expression of inflammatory factors.
  • LPS was used to stimulate mouse glioma Bv2 cells, and the changes in the levels of inflammation-related factors were detected. After being stimulated by 1 ⁇ g/ml LPS for 2 hours, MC001, metformin and cysteamine were added for 24 hours, and then quantitative PCR was used to detect the transcription of related inflammatory factors.
  • MC001 can significantly inhibit the activation of NF-kB signaling caused by LPS, leading to the downregulation of a series of pro-inflammatory cytokines, such as iNOS, TNF- ⁇ , IL-1 ⁇ .
  • Example 32 MC001 inhibits nerve cell apoptosis caused by MPP +
  • MPP + is a neurotoxin that interferes with the mitochondrial oxidative phosphorylation process, leading to blockage of cellular ATP production and ultimately cell death. For this reason, this example uses flow cytometry to detect whether MC001 can protect the apoptosis caused by MPP+ in the human SH-SY5Y nerve cell line.
  • Example 33 MC001 inhibits the loss of dopaminergic neurons caused by MPTP
  • MPTP can be oxidized to MPP + by monoamine oxidase in vivo, and then cause Parkinson's similar disease symptoms, which can be used as a typical drug-derived animal model of PD disease.
  • MC001 can inhibit neural validation and inhibit apoptosis, we carried out in vivo intervention studies using the MPTP model, and observed tyrosine hydroxylase (TH) by immunofluorescence staining. The results are shown in Figure 31, compared with metformin, MC001 can significantly inhibit the loss of TH positive cells induced by MPTP.
  • Example 34 MC001 inhibits oxidative stress in the brain caused by MPTP
  • the ROS detection reagent was orthotopically injected into the mouse ventricle 15 minutes after the mouse brain fluorescence signal was observed and the fluorescence value was quantitatively analyzed.
  • Example 35 MC001 improves GSH level in mice

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Abstract

本发明属于生物医药领域，具体涉及一种双胍衍生物及其应用。本发明的双胍衍生物的结构式如式(I)所示。本发明通过同时抑制糖酵解和氧化磷酸化两条能量代谢通路，减少乳酸的生成，增强化合物的抗肿瘤效果，并从多个疾病出发评价了该化合物的治疗潜力和应用价值。

Description

一种双胍衍生物及其应用 技术领域
发明属于生物医药领域，具体涉及一种双胍衍生物及其应用。
背景技术
双胍类化合物，例如二甲双胍、苯乙双胍、丁双胍，是较早发现具有降糖作用的化合物，其中，二甲双胍一直是治疗II型糖尿病的一线药物。二甲双胍的降糖作用主要是由于抑制肝脏糖异生和促进葡萄糖吸收。二甲双胍在细胞内的主要靶标是线粒体，因为其带正电荷，使二甲双胍更容易在线粒体基质中积累，并抑制线粒体呼吸链复合物I，从而抑制ATP的生成，导致AMP的积累，AMP/ATP的比值提高会激活AMPK。AMPK是细胞内重要的能量感受器，是新陈代谢和生长通路的主要调控者。AMPK激活后会打开ATP的分解代谢途径，关闭消耗ATP的合成代谢途径，以维持细胞能量代谢平衡。激活的AMPK还可以磷酸化胰岛素受体和胰岛素受体底物-2(IRS-2)，从而促进葡萄糖转运蛋白的转运；活化的AMPK也可以抑制脂肪酸合成和增强β-氧化，进而促进葡萄糖的转运；AMP的增加还会抑制腺苷酸环化酶催化ATP转化为cAMP，从而抑制了糖异生。
糖尿病患者患癌症的风险增加，这主要是由于长期的高胰岛素血症、高血糖和脂肪诱导的慢性炎症。2018年，我国进行了迄今为止规模最大的一项有关中国大陆II型糖尿病与癌症风险之间关系的研究，研究结果表明在普通人群中，患糖尿病的男性和女性患癌症的风险分别比正常男性和女性高34％和62％，糖尿病患者患肝癌，胰腺癌，结肠癌，乳腺癌和膀胱癌等的风险较高。一系列回溯性分析研究表明二甲双胍的使用与糖尿病患者的癌症发病率和死亡率降低相关。临床前研究也表明二甲双胍能够直接杀死肿瘤细胞。
二甲双胍的抗肿瘤机制一方面归因于其间接作用，即降低机体血糖和胰岛素等的水平；另一方面，归因于其直接作用即抑制线粒体呼吸链复合物I、激活AMPK以及抑制mTORC1通路等。体内体外的研究结果表明，二甲双胍需要远 高于其临床可及浓度才显示出明显的抗肿瘤活性，最近二甲双胍治疗胰腺癌的临床试验结果也表明，在治疗剂量下二甲双胍并不能显著提高患者的整体生存率，显示二甲双胍的抗肿瘤效果还有待提高。相对于二甲双胍，丁双胍和苯乙双胍具有更强的降糖作用，同时也具备更显著的抗肿瘤效果，但这些化合物在高效抑制线粒体复合物I的同时也会激活肿瘤细胞的糖酵解途径，引起严重的乳酸酸中毒，激活的糖酵解也会帮助肿瘤细胞逃逸双胍药物的杀伤作用，这些性质限制了现有双胍化合物在肿瘤治疗中的临床应用。
二甲双胍除了降低血糖和抗肿瘤的作用，还可以降低心血管疾病的发生，改善多囊卵巢综合征相关症状，改善衰老相关的神经退行性疾病等。糖尿病患者心血管疾病的发生风险比正常人高5倍，且心肌梗死后心力衰竭和死亡的风险是正常人的4倍。二甲双胍可抑制动脉粥样硬化病变的早期改变，包括减少内皮中单核细胞和白细胞的粘附；抑制单核细胞向巨噬细胞的分化，抑制泡沫细胞的形成，减少血管平滑肌细胞的增殖等。多囊卵巢综合征是复杂的内分泌及代谢异常所致的疾病。代谢异常被认为是多囊卵巢综合征发生和发展的核心因素，胰岛素致敏剂已被提出是治疗多囊卵巢综合征的可行性方案。二甲双胍作为胰岛素增敏剂可以改善多囊卵巢综合征的多个症状，例如促进排卵、提高卵泡质量、提高妊娠率等。回溯型研究和临床前的实验结果表明，二甲双胍可以延长糖尿病患者或者正常小鼠的寿命，其作用机制可以归结为以下几点：1)激活AMPK,抑制mTOR；2)调节胰岛素/胰岛素样生长因子-1(IGF-1)信号通路；3)减少活性氧(ROS)的产生；4)调节SIRTs表达。除了这里列举的具体适应症之外，二甲双胍也可以改善或有益于其他炎症和代谢相关的疾病，如非酒精性脂肪肝、肠道炎症、关节炎等。
由于双胍类化合物在癌症治疗中的局限性，即肿瘤杀伤活性差或容易造成酸中毒、引起肿瘤代谢补偿而逃逸杀伤作用。因此需提供一种全新的双胍衍生物，以突破双胍类化合物局限性，同时扩大其适用疾病范围。
发明内容
本发明的目的在于提供一种双胍衍生物。
本发明的再一目的在于提供上述双胍衍生物的制备方法。
本发明的再一目的在于提供上述双胍衍生物的应用。
根据本发明具体实施方式的一种双胍衍生物，其结构如式(I)所示：
Figure PCTCN2021142309-appb-000001
其中，R 1选自H，或酰基；酰基选自乙酰基、丙酰基、异丙酰基、丁酰基和苯甲酰基及取代的苯甲酰基；
R 2选自H、羧基，或酯基，酯基选自甲酯或乙酯。
根据本发明具体实施方式的双胍衍生物，R 1为H，R 2选自H或羧基。
根据本发明具体实施方式的双胍衍生物，R 2为H，R 1选自H，或酰基。
根据本发明具体实施方式的双胍衍生物，在式(I)基础上，R 1为H，R 2为H，其具体结构如下：
Figure PCTCN2021142309-appb-000002
根据本发明具体实施方式的双胍衍生物的制备方法，所述方法包括以下步骤：将双氰胺与式(Ⅱ)所示化合物在至少一种有机溶剂中反应，经衍生后得到式(I)所示的双胍衍生物；
或者，双氰胺与式(III)所示化合物在至少一种有机溶剂中反应，经衍生后得到式(I)所示的双胍衍生物。
Figure PCTCN2021142309-appb-000003
R为巯基保护基，例如三苯基甲基、二苯基甲基或乙酰基等；
R 1选自H或酰基；酰基选自乙酰基、丙酰基、异丙酰基、丁酰基和苯甲酰基及取代的苯甲酰基；
R 2选自H、羧基或酯基，酯基选自甲酯或乙酯。
本发明技术方案中，有机溶剂选择质子性溶剂，其中包括戊醇、丁醇、丙醇、异丁醇等沸点适合的溶剂。
本发明还提供一种药物，所述药物包含式(I)所示的双胍衍生物或其非毒性药学上可接受的盐。
术语“药学上可接受的盐”指保留了本发明的化合物的生物有效性和特性的盐，且不在生物方面或其他方面是不理想的。在许多实例中，本发明的化合物利用氨基和/或羧基或类似的基团的存在可形成酸式和/或碱盐。药学上可接受的酸式加成盐可由无机或有机酸制备，而药学上可接受的碱式加成盐可由无机或有机碱制备。药学上可接受的盐的综述见Berge((1977)J.Pharm.Sd，vol.66，151)。“非毒性药学上可接受的盐”指与非毒性的药学上可接受的无机或有机酸或无机或有机碱形成的非毒性的盐。例如，上述盐包括那些衍生自无机酸例如盐酸、氢溴酸、硫酸、氨基磺酸、磷酸、硝酸等，以及由有机酸例如乙酸、丙酸、琥珀酸、乙醇酸、硬脂酸、乳酸、苹果酸、酒石酸、枸橼酸、抗坏血酸、扑酸、马来酸、羟基马来酸、苯乙酸、谷氨酸、苯甲酸、水杨酸、磺胺酸、富马酸、甲磺酸和甲苯磺酸等。
本发明的式(I)的化合物可单独施用，优选将它们作为药物组合物提供。根据本发明有用的上述药物组合物，不管是用于兽药还是用于人类用途，包含至少一种具有上述定义的式(I)的化合物、和一种或多种药学上可接受的载体和任选的其它治疗成分。
在某些优选实施方案中，联合治疗中所需的活性成分可合并到单个药物组合物中以同时给药。
当述及组合物、载体、稀释剂和试剂时，本文使用的术语“药学上可接受的”和它们语法上的变化可相互交换使用且表示该物质可施用于哺乳动物而不 会产生不良的生理效果例如恶心、头晕、心嘈等。
其中溶解或分散有活性成分的药物组合物的制备方法是现有技术中已知的，且不需要基于配方对其进行限定。通常这种组合物以液体溶液或混悬液制备成注射剂；然而，也可制备适于在使用前溶解或混悬在液体中的固体形式。制剂也可为乳化的。特别地，上述药物组合物可配置成固体剂型，例如胶囊、片剂、丸剂、粉剂、糖丸剂或颗粒剂。
通常根据活性化合物的溶解性和化学性质、给药的具体模式和药学实践中遵守的规定来确定赋形剂的选择和赋形剂中活性成分的含量。例如，赋形剂(如乳糖、柠檬酸钠、碳酸钙、磷酸二钙)和崩解剂(如淀粉、褐藻酸和某些复合硅酸盐)与润滑剂(如硬脂酸镁、十二烷基硫酸钠和滑石粉)结合可用于制备片剂。为了制备胶囊，使用乳糖和高分子量聚乙二醇是有利的。当采用水混悬液时，其可包含乳化剂或混悬促进剂。也可以使用稀释剂如蔗糖、乙醇、聚乙二醇、丙二醇、甘油和氯仿或其混合物。
上述药物组合物可以适合的剂型施用于人和动物，通过局部或全身给药，包括口服给药、直肠给药、鼻腔给药、口腔给药、眼部给药、舌下给药、透皮给药、直肠给药、局部给药、阴道给药、肠胃外给药(包括皮下记注射、动脉注射、肌肉注射、静脉注射、皮内注射、鞘内注射、硬膜外注射)、脑池内给药和腹腔给药。应当理解的是优选的途径可随着例如接受者的情况而变化。
配方可按照药剂领域公知的任何方法制备成单位剂量。这种方法包括使活性成分和由一种或多种助剂组成的载体相结合。通常上述配方通过将活性成分与液体载体或极细固体载体或两者均匀且密切地结合而制备，然后如果需要对产品成形。以单次或多次剂量施用于对象的本发明的化合物的每日总剂。
同时，本发明还提供双胍衍生物在制备预防或治疗代谢和炎症相关疾病的药物方面的应用，其中，所述疾病包括但不限于多囊卵巢综合征、癌症、神经变性疾病、心血管疾病、肥胖症、糖尿病、衰老和非酒精性脂肪肝等。
本发明的有益效果：
本发明通过二硫键将双胍片段与半胱胺偶联形成新型双胍衍生物，双胍衍生物能够同时抑制糖酵解和氧化磷酸化两条能量代谢通路，减少乳酸产生，更有效的抑制肿瘤的生长。在其他疾病治疗，如降低血糖，治疗多囊卵巢综合征、心血管保护、延缓衰老，治疗非酒精性脂肪肝等方面展示出良好的治疗效果。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1显示MC001表征数据，其中A为MC001的氢谱，B为MC001的碳谱；
图2显示MC001细胞内还原形式的表征情况；
图3显示MC001抑制线粒体复合物I，其中，A为不同化合物抑制线粒体复合物I的效果对比，B为MC001抑制线粒体复合物I的曲线拟合；
图4显示MC001同时抑制氧化磷酸化和糖酵解的验证结果；A、C为呼吸作用的抑制；B、D为糖酵解的抑制作用；
图5显示MC001不过多产生乳酸；A为细胞水平上药物对乳酸产生的影响，B为检测小鼠血清乳酸的水平；
图6显示化合物对肿瘤细胞活力的影响；A为拟合得到的细胞存活曲线，B为计算得到的MC001和二甲双胍对不同细胞的半数抑制浓度；
图7显示MC001体内抗肿瘤活性；A为裸鼠中HCT116肿瘤生长曲线，B为BALB/c裸鼠中CT26肿瘤生长曲线，C为BALB/c野生型小鼠中CT26肿瘤生长曲线；
图8显示MC001促进CD8+T细胞在肿瘤微环境的浸润和功能；A为肿瘤微环境中CD8阳性T细胞比例，B为CD8阳性T细胞中颗粒酶B含量；
图9显示MC001对TGFβ分泌及下游信号通路的抑制；A为小鼠血清中TGFβ的水平，B和C分别是细胞水平上，培养基上清中的TGFβ含量和细胞内TGFβ下游靶蛋白的表达水平；
图10显示MC001抑制肿瘤转移；
图11显示MC001促进STING激动剂的活性。A为MC001联用ADU-S100,B为MC001联用cGAMP；
图12显示MC001减少T2DM小鼠模型的禁食血糖，改善其葡萄糖耐受；A为高脂喂养(HFD)小鼠给药后16小时禁食血糖的含量；B图为小鼠注射2g/kg葡萄糖后小鼠血糖随时间的变化，即葡萄糖耐量；
图13显示MC001降低HFD小鼠的胰岛素的分泌；A图为葡萄糖刺激的小鼠分泌到血液中胰岛素随时间的变化；B图为各个实验组小鼠的禁食胰岛素水平；
图14显示MC001改善HFD小鼠的胰岛素敏感性；
图15显示MC001实现控糖效果的同时不产生乳酸毒性和肝肾毒性；A为乳酸水平检测，B、C、D、E为肝肾毒性检测；
图16显示MC001缓解双氢睾酮(DHT)诱导的多囊卵巢综合征治(PCOS)小鼠的颗粒细胞黄体化障碍；A和B分别是小鼠卵巢形态、卵泡分类计数,C是小鼠卵巢mRNA表达，D是小鼠卵巢免疫组织化学染色；
图17显示MC001在细胞水平上缓解由DHT引起的黄体化抑制作用；A和B分别是颗粒细胞的Cyp11a1免疫荧光染色形态、荧光强度定量，C是颗粒细胞上清中孕酮浓度，D是颗粒细胞mRNA表达；
图18显示MC001对高脂饮食诱导的动脉粥样硬化小鼠主动脉病变程度的影响；A为主动脉弓病变代表图，B为病变统计图；
图19显示MC001对高脂饮食诱导的动脉粥样硬化小鼠血清中CRP含量的影响；
图20显示MC001对高脂饮食诱导的动脉粥样硬化小鼠血清中常规血脂指标的影响；图20A-D分别表示小鼠血清中总胆固醇、甘油三酯、低密度脂蛋白胆固醇和高密度脂蛋白胆固醇的含量，图E表示小鼠血清总胆固醇和高密度脂蛋白胆固醇含量的比值。
图21显示MC001改善衰老小鼠的运动能力；A为衰老小鼠给与药物处理后转棒实验中的持续时间；B为衰老小鼠的抓力测试结果；
图22显示MC001能够增加衰老小鼠血液中淋巴细胞数量，改善胸腺功能；A为血液中嗜中性粒细胞、淋巴细胞、单核细胞的比例，B为胸腺HE染色图；
图23显示MC001降低衰老小鼠的代谢毒性；A图为衰老小鼠的乳酸含量水平；B图为衰老小鼠的谷丙转氨酶活性；C图为衰老小鼠的肌酐水平；
图24显示MC001缓解非酒精性脂肪肝小鼠的肝损伤；A和B为毒性指标，C和D分别是甘油三酯和总胆固醇含量；
图25显示MC001缓解非酒精性脂肪肝模型小鼠肝脏的脂肪空泡现象；A为肝脏切片HE染色，B为肝脏空泡数量统计结果；
图26显示MC001对铁死亡的影响；A为MC001抑制由铁死亡诱导剂erastin引起的ROS增加，B为MC001抑制由铁死亡诱导剂erastin引起的lipidROS增加；
图27显示MC001对细胞脂滴积聚的影响；A为油红染色，体现油酸引起的HEPG2细胞中脂滴的积累和药物处理后脂滴的变化，B为统计图；
图28显示MC001对细胞脂质过氧化的影响；A为Bodipy染色，B为统计图；
图29显示MC001对细胞炎症反应及ROS水平的影响；A为NOS2表达，B为TNF-α表达，C为IL-1β表达；
图30显示流式细胞检测MC001对MPP+引起SH-SY5Y细胞凋亡的影响；
图31显示TH染色观察MC001对MPTP引起多巴胺能神经元丢失的影响；
图32显示MC001对MPTP模型小鼠脑部氧化应激的影响。左图为成像结果，右图为光密度统计结果；
图33显示MC001对小鼠体内GSH水平的影响；A图为MC001单剂量的时间梯度对还原型谷胱甘肽GSH占总谷胱甘肽比例的影响，B图为MC001长期处理对MPTP小鼠脑部GSH占总谷胱甘肽水平的影响，C图为不同剂量的MC001对GSH水平的影响。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将对本发明的技术方 案进行详细的描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式，都属于本发明所保护的范围。
本发明通过二硫键连接2-巯基乙基双胍和半胱胺产生了一种新型的双胍衍生物MC001(1-(2-((2-氨基乙基)过硫基)乙基)双胍)。MC001进入细胞后，在还原环境中转化为2-巯基乙基双胍和半胱胺，半胱胺发挥抑制糖酵解的功能，2-巯基乙基双胍发挥抑制氧化磷酸化的功能，从而达到对糖酵解和氧化磷酸化两条能量代谢通路双重抑制的目的。
MC001进细胞后还原过程如下：
Figure PCTCN2021142309-appb-000004
实施例1合成MC001
胱胺二盐酸盐与双氰胺在正丁醇中回流过夜,初步获得含杂质产物MC001，反应结束冷却至35℃左右有固体析出，取出正丁醇，再次加入正丁醇加热回流溶解、冷却，取出正丁醇，加入甲醇超声溶解，旋蒸，反复三次，再除去剩余的溶剂，得到的白色泡沫状产物即MC001。合成路线如下：
Figure PCTCN2021142309-appb-000005
MC001表征: 1H NMR(500MHz,CD 3CN)δ7.83(s,4H),6.60(s,5H),3.51(t,J＝6.2Hz,2H),3.33–3.23(m,2H),3.10–2.99(m,2H), 2.94(t,J＝6.2Hz,2H)，见图1-A。
13C NMR(100MHz,CD 3CN)δ160.39(s),158.91(s),39.95(s),38.97(s),38.34(s),34.63(s)，见图1-B。
通过与实施例1所述相似的方法制备以下实施例的目标化合物，与实施例1的不同之处在于使用了对应于目标化合物结构中的胺化合物与双氰胺反应。
实施例2合成MC-003
Figure PCTCN2021142309-appb-000006
称取2克化合物2与1.0当量三苯基甲醇(TrtOH)于圆底烧瓶中，抽气换气，加入三氟乙酸(TFA)反应1小时。反应结束后，旋干TFA，加入乙酸乙酯，用2摩尔/升NaOH溶液调pH至碱性，乙酸乙酯层用无水Na 2SO 4干燥，旋干即得化合物2。
称取2克化合物3与1.0当量化合物1于圆底烧瓶中，抽气换气，加入浓盐酸(HCl)，正丁醇(0.2摩尔/升)，升温回流反应过夜。反应结束后，旋干正丁醇，用少量甲醇溶解，柱层析分离(洗脱剂为二氯甲烷:甲醇＝10:1)纯化得到化合物4。
称取80毫克化合物4于25毫升圆底烧瓶中，抽气换气，加入干燥二氯甲烷2毫升，TFA 2毫升(反应体系变黄色)，再加入2.0当量三乙基硅烷(反应体系变无色)。反应完成后，用干燥二氯甲烷彻底旋干溶剂，粘在瓶子上的 白色固体用少量乙醚，石油醚洗涤除去杂质，即得产物MC-002(高分辨质谱：m/z 162.08083)。
称取1克化合物5与2当量化合物6于圆底烧瓶中，抽气换气，加入甲醇和水(0.2摩尔/升)，室温反应15小时。反应结束后，二氯甲烷萃取，水洗，二氯甲烷层用无水Na 2SO 4干燥，旋干，用少量二氯甲烷溶解，柱层析分离(洗脱剂为二氯甲烷:甲醇＝8:1)纯化得到化合物7(高分辨质谱：m/z 271.02167)。
称取20毫克化合物7与1当量MC-002于圆底烧瓶中，抽气换气，加入2mL甲醇，室温反应15小时。反应结束后，刮板(洗脱剂为二氯甲烷:甲醇＝8:1)纯化得到化合物MC-003(高分辨质谱：C 9H 18N 6O 3S 2m/z[M+H] +323.09537)。
实施例3合成MC-004
Figure PCTCN2021142309-appb-000007
称取20毫克化合物MC-003于圆底烧瓶中加入2mL甲醇进行溶解，冰水浴冷却后加入1当量三甲基硅基重氮甲烷(TMSCHN 2)，室温反应15小时。反应结束后，刮板(洗脱剂为二氯甲烷:甲醇＝9:1)纯化得到化合物MC-004(高分辨质谱：C 10H 20N 6O 3S 2m/z[M+H] +337.11121)。
实施例4确认MC001在细胞内的还原形式
使用200μM的MC001与细胞孵育2小时，再用甲醇重悬细胞并反复冻融，离心后取上清进行质谱鉴定。结果见图2。
结果如图3所示，MC001在细胞内被还原为2-巯基乙基双胍(162.0809)和半胱胺，由于半胱胺本身在细胞内存在，本发明用2-巯基乙基双胍来表征MC001在细胞内的还原。从而，本发明确认了MC001在细胞内还原为2-巯基乙基双胍和半胱胺。
实施例5验证MC001抑制线粒体复合物I
本实施例使用
Figure PCTCN2021142309-appb-000008
Complex I Activity Assay试剂盒，在体外检测线粒体复合物I活性。将5mM的MC001、phenformin和metformin与线粒体溶液预混4小时，最后加底物立即检测340nm吸光值。
结果图3中A所示，当药物浓度为5mM时，MC001和苯乙双胍(Phenformin)都表现出显著的线粒体复合物I抑制活性，而二甲双胍(Metformin)并未观察到抑制作用。
进一步设置MC001的浓度梯度(20mM、10mM、5mM、2.5mM、1.25mM、0.625mM、0.3125mM、0.156mM、0.078mM、0mM)，检测线粒体复合物I活性。如图4中B所示，通过拟合计算出MC001在体外对线粒体复合物I抑制的IC 50值约为155μM。
实施例6验证MC001同时抑制氧化磷酸化和糖酵解
本实施例利用seahorse仪器检测细胞氧气消耗速率和糖酵解速率。HCT116细胞提前36小时接种于24孔seahorse培养板，每孔2×10 4个细胞，培养24小时后，加药处理，处理时间12小时，再按照试剂盒说明书清洗细胞，稀释线粒体压力试剂盒配套药物，最后上机检测细胞各阶段的耗氧量。
结果如图4所示，加入寡霉素抑制ATP合酶(复合物Ⅴ)，该药可以影响或降低通过ETC的电子流，引起线粒体呼吸或OCR减少，再加入解偶联剂，该药会破坏质子梯度和线粒体膜电位，引起电子在ETC不受限制地传递，同时复合物Ⅳ的耗氧达到最大，最后加入鱼藤酮抑制复合物Ⅰ，抑制线粒体呼吸。加入寡霉素之前的耗氧量为基础呼吸值，寡霉素之前的细胞外酸化值为基础糖酵解。从图4中可以看出MC001和苯乙双胍几乎完全抑制了细胞基础氧气消耗，也能够明显抑制糖酵解，而苯乙双胍在一定程度上会增加基础糖酵解。二甲双胍对氧化磷酸化和糖酵解影响不大。
实施例7验证MC001不过多产生乳酸
为了在体内外验证MC001对乳酸产生的影响，本实施例使用200mM MC001、 二甲双胍、苯乙双胍和丁双胍处理HCT116细胞，24小时后，检测上清中乳酸含量。
图5中A，可以看出MC001和二甲双胍(Metformin)不会造成明显的乳酸积累，而苯乙双胍(Phenformin)和丁双胍(Buformin)在相同浓度下会显著增加乳酸积累。体内实验用不同浓度的MC001、二甲双胍和苯乙双胍分别给小鼠灌胃，其中MC001和二甲双胍的给药浓度为20mg/kg，100mg/kg和200mg/kg，苯乙双胍的给药浓度为200mg/kg，给药一周后分析血液中乳酸的含量，结果如图5B所示，200mg/kg剂量下苯乙双胍处理组的乳酸水平要显著高于相同浓度下MC001，不同浓度MC001造成体内乳酸积累的平均水平与二甲双胍的水平相当，不造成过渡的乳酸积累。
实施例8检测MC001对肿瘤细胞活力的影响
为了验证新型双胍类化合物MC001的抗肿瘤活性，本实施例首先用MTT法在多种肿瘤细胞系(人结肠癌细胞：HCT116，Caco2，SW480；小鼠结肠癌细胞：CT26；人肝癌细胞：HepG2；人肺癌细胞：NCI-H460)中进行检测。细胞加药处理48小时后，加入MTT，培养2小时，去上清，加入DMSO溶解后检测吸光度。
结果如图6所示，MC001的抗肿瘤活性相对于二甲双胍有显著的提高，对多种肿瘤具有明显的抑制，尤其针对Caco2和HCT116细胞抑制活性提高近100倍。
实施例9检验MC001体内抗肿瘤活性
为了进一步在机体水平验证MC001的抗肿瘤活性，本实施例在裸鼠中建立了HCT116细胞皮下异种移植模型。分别用二甲双胍和MC001喂水给药。隔天测量肿瘤的体积，总共给药12天。结果如图7A所示，MC001组的肿瘤生长明显较对照组和二甲双胍组缓慢，展示出显著的抗肿瘤活性，而二甲双胍在同样剂量下没有体现出明显的抗肿瘤活性。
在BALB/c裸鼠和BALB/c野生鼠中同时建立CT26-Luc移植瘤模型，隔天观察记录肿瘤的体积。相同的药物处理条件下，MC001和二甲双胍在BALB/c野生 型鼠中的抗肿瘤活性要明显优于在BALB/c裸鼠中的抗肿瘤活性，如图7B和C所示，说明MC001和二甲双胍的抗肿瘤活性部分来自于其免疫调节功能。
实施例10 MC001促进CD8+T细胞在肿瘤微环境的浸润和功能
为了验证MC001对肿瘤微环境免疫细胞的影响，本实施例用流式细胞仪对实施例9中的BALB/c野生型CT26肿瘤模型鼠的脾脏T细胞进行进一步分析。
结果如图8中A所示，MC001显著增加CD8 +T细胞的数量，图8B展示MC001可以明显提高CD8 +T细胞主要的杀伤性分子granzyme B的含量。
实施例11 MC001抑制TGFβ分泌及下游信号通路
本实施例在细胞及机体水平检测了MC001对TGFβ分泌水平和下游信号通路的影响。使用MC001处理CT26细胞，48小时后使用ELISA试剂盒检测培养基上清中TGFβ的含量。
如图9B和C所示，MC001剂量依赖地显著抑制鼠源CT26结肠癌细胞TGFβ的分泌水平，同时MC001也可以有效地抑制TGFβ下游Smad 3的磷酸化和Snail的表达。
进一步在机体水平上对此效应进行验证，在BALB/c鼠中建立CT26移植瘤模型，当肿瘤长到50mm 3时开始给灌胃给药MC001(MC001给药浓度为0.1mg/mL,0.5mg/mL,2mg/mL)，一周之后使用ELISA试剂盒对小鼠血清中的TGFβ进行定量分析。
结果如图9A所示，荷瘤小鼠TGFβ水平相对于健康小鼠有显著升高，MC001在不同的给药剂量均显著降低荷瘤小鼠血清中TGFβ的水平。
实施例12 MC001抑制肿瘤转移
为了验证MC001对肿瘤转移的抑制活性，本实施例通过尾静脉注射CT26-luc细胞建立肿瘤转移模型，喂水给药MC001(0.5mg/mL)，4周时进行小鼠成像，观测肿瘤的转移和定植情况。
结果如图10所示，对照组中肿瘤主要定植在颈部、肺部、后肢内侧和生殖器，一半以上的小鼠已出现2个以上的转移灶，且多数已经形成非常明显的肿瘤组织(成像时一只小鼠已死亡)。而MC001组相比于对照组肿瘤细胞在体内的定植要少许多，且已形成的肿瘤组织要明显小于对照组。继续饲养小鼠统计小鼠的存活率，MC001给药组能够显著提高小鼠的存活率。
实施例13 MC001促进STING激动剂的活性
TGFβ通过一系列间接的机制抑制IRF3的磷酸化，阻止STING通路的激活，从而影响STING激动剂调节肿瘤微环境发挥功能。于是本实施例验证MC001是否能够增强STING激动剂活性。使用2μg/mL ADU-S100或cGAMP与MC001联用处理THP1细胞，8小时后收样，提取RNA进行RT-PCR分析。结果图11所示，MC001能够增强由ADU-S100和cGAMP引起的一型干扰素的激活。
实施例14 MC001减少T2DM小鼠模型的禁食血糖，改善其葡萄糖耐受
为有效评估MC001药物在二型糖尿病(T2DM)中的作用效果，本实施例选择高脂饮食诱导的肥胖小鼠模型(HFD鼠)为II型糖尿病小鼠模型研究对象。分别设置对照组Ctrl，MC001 0.1mg/mL组，MC001 1mg/mL组和二甲双胍Met 1mg/ml组，采用饮水的方式进行给药。
给药5周后，对小鼠禁食16小时，随后使用罗氏手持式血糖计检测小鼠血糖。随后给小鼠以腹腔注射的方式注射葡萄糖(剂量为2g/kg)，在不同的时间点采血，检测血糖含量。对应时间点的血样同时保存用于后续葡糖糖刺激的胰岛素检测。如图12中A所示，MC001能够以0.1mg/ml的剂量改善HFD小鼠的禁食血糖，其控糖能力和二甲双胍1mg/mL接近。而在GTT葡萄糖挑战实验中(图12中B)，MC001同样表现出比二甲双胍更佳的血糖控制能力。
实施例15 MC001降低HFD小鼠的胰岛素的分泌
以实施例14中的HFD小鼠为对象，取葡糖糖注射后的对应时间点的血样， 使用ELISA的方式检测其中胰岛素的含量，得到葡萄糖刺激的胰岛素分泌结果和用药后HFD小鼠的基础胰岛素水平。
如图13中A所示，MC001和Metformin都能够显著降低葡萄糖刺激的胰岛素分泌水平，同时给药后基础胰岛素也处于较低水平，见图13中B说明用药能够改善胰岛素的响应，进而在实现更好的控糖水平的同时降低胰岛素的分泌。
实施例16 MC001改善HFD小鼠的胰岛素敏感性
对实施例14中HFD小鼠进行胰岛素敏感性实验(胰岛素耐受实验，ITT)，进一步测定胰岛素信号响应。小鼠禁食6小时后，采用腹腔注射的方式注射胰岛素(HFD小鼠剂量为1U/kg)，随后检测不同时间点的血糖含量。
结果如图14所示，MC001和Metformin都能明显改善DB胰岛素敏感性，而MC001在0.1mg/mL的剂量下和Metformin作用相当。进一步说明了药物能够改善胰岛素的信号响应，从而改善胰岛素敏感性，实现更好的控糖效果。
实施例17 MC001实现控糖效果的同时不产生乳酸毒性
使用乳酸检测试剂盒检测实施例14中HFD小鼠的乳酸含量，以检测药物产生的乳酸毒性。
如图15中A所示，0.1mg/mL的MC001在实现降糖的同时，并不显著增加小鼠乳酸，相比之下，同等疗效的Metformin显著提高了小鼠的乳酸水平，与临床结果一致，从而进一步说明MC001的优越性，展现出较大的临床价值。
随后使用南京建成生化试剂盒，检测HFD小鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)的含量(肝脏毒性指标)和肾毒指标(肌酐和尿素)。
结果如图15中B、C、D、E所示，MC001和Metformin在HFD小鼠中都没有产生明显的肝肾毒性。而MC001以低剂量(0.1mg/mL)长周期给药的方式，不仅能够降低禁食血糖，改善葡萄糖耐受，提高胰岛素敏感性，同时不影响胰岛素分泌和其他肝肾毒性的改变，尤其是乳酸毒性。在这些指标中，MC001均表现出作为降糖药物的更大潜力。
实施例18 MC001缓解双氢睾酮(DHT)诱导的多囊卵巢综合征治(PCOS)小鼠的颗粒细胞黄体化障碍
本实施例在于验证MC001在多囊卵巢综合征治(PCOS)疗上的作用。长期外源DHT刺激可以诱导小鼠等表现类似人PCOS样症状，如排卵障碍、多囊性卵巢和高雄激素血症等。在小鼠皮下埋置DHT缓释管维持高雄激素刺激，持续56天，第28天开始连续给于0.1mg/ml Metformin、MC001饮水。实验结束时取小鼠卵巢做切片观察。
结果如图16所示，MC001缓解了由DHT引起的小鼠卵泡的减少，纠正了DHT导致的颗粒细胞黄体化基因表达异常，缓解DHT诱导的颗粒细胞黄体化障碍。
实施例19 MC001在细胞水平上缓解由DHT引起的黄体化抑制作用
使用10μM毛猴菌素(forskolin)和20nM佛波醇(PMA)处理青春前小鼠卵巢获得的颗粒细胞6小时，使其发生黄体化。同时用1nM DHT处理细胞，建立PCOS细胞模型。
如图17所示，DHT抑制减弱了颗粒细胞阳性信号，MC001在蛋白水平和mRNA水平均逆转了DHT这种作用。颗粒细胞黄体化过程产生大量孕酮，而MC001可以缓解由DHT造成的孕酮的降低。
实施例20 MC001对高脂饮食诱导的动脉粥样硬化小鼠主动脉病变程度的影响
选取5周龄的ApoE基因缺陷的C57BL/6J品系小鼠，随机分组后采用西方饮食进行高脂诱导，从第8周开始，采取饮水给药的方式对MC001组进行给药，剂量为1mg/mL；从第12周开始，对MC001延迟组开始口服给药，剂量为1mg/mL；对照组正常给水，持续到第16周结束。
实验结束后，将实验动物安乐死，解剖后，从右心耳先灌注10mLPBS，再 灌注10mL组织固定液(4％多聚甲醛)，在体视镜下将主动脉完整分离出来，并用脂肪镊剥离血管外黏附脂肪组织。采用油红O染色的方法对分离出的主动脉进行大体染色，拍照记录，使用ImageJ统计实验组和对照组动物主动脉弓病变面积，并统计差异显著性。
如图18所示，在对建模成功的动脉粥样硬化小鼠进行周期性间歇给药后，相对于对照组，MC001组小鼠主动脉弓的病变面积显著减少，而MC001延迟给药组仅微弱减少斑块面积，说明MC001在动脉粥样硬化早期能够直接改善血管斑块病变的发生，晚期给药则不能逆转。
实施例21 MC001对高脂饮食诱导的动脉粥样硬化小鼠血清中CRP含量的影响
在动脉粥样硬化小鼠建模成功后，采用如实施例20中描述方法，对MC001组和MC001延迟组分别从第8周和第12周开始饮水给药，剂量为1mg/mL，连续给药至第16周结束，通过检测小鼠血清中C-反应蛋白(CRP)水平，评估机体心血管疾病的发生风险，从而评价化合物对机体心血管疾病发生的影响。
如图19所示，与对照组相比，MC001组和MC001延迟给药组均能够极显著降低实验组小鼠血清中CRP的含量，说明MC001对机体心血管疾病的发生具有优良的预测改善作用。
实施例22 MC001对高脂饮食诱导的动脉粥样硬化小鼠血清中常规血脂指标的影响
采用如实施例20所述方法进行高脂饮食诱导小鼠动脉粥样硬化模型，并按照分组进行给药，对MC001组和MC001延迟组分别从第8周和第12周开始饮水给药，剂量为1mg/mL，连续给药至第16周结束，对小鼠进行安乐死，并取血清，进行常规血脂含量的检测。
结果如图20所示，与对照组相比，MC001可以显著降低机体血清中低密度脂蛋白(LDL-C)含量，增加高密度脂蛋白(HDL-C)的含量，但也会一定程度 增加甘油三酯含量，而对总胆固醇无显著影响；而延迟给MC001，对血脂四项均无显著影响。尽管如此，在动脉粥样硬化进程早期即给药和延迟到晚期给药MC001，均可以显著降低机体总胆固醇与高密度脂蛋白比值，显示出对动脉粥样硬化、冠心病等心血管疾病具有显著的预测改善作用。
实施例23 MC001改善衰老小鼠的运动能力
本实施例购买了24周龄C57小鼠作为衰老鼠模型，评估了MC001在抗衰老中的应用。
对衰老小鼠以饮水的方式，给与0.1mg/mL MC001用药4周后，使用转棒实验和前爪抓力测试实验检测其运动能力的改善。
结果如图21所示，MC001给药后明显改善了衰老小鼠的转棒上停留时间，其抓力显著增加，说明MC001能够改善衰老小鼠的大脑控制运动的协调能力、肌肉运动耐力和力量。
实施例24 MC001能够增加衰老小鼠血液中淋巴细胞数量，改善胸腺功能
取实施例23中小鼠的全血，使用全自动血球计数仪，对衰老小鼠的血液细胞进行WBC分类计数。
如图22中A所示，衰老细胞相比于年轻成年小鼠的嗜中性粒细胞增加，淋巴细胞减少，单核细胞增多，而给与MC001处理后的小鼠淋巴细胞增多，嗜中性粒细胞和单核细胞减少，表明MC001可能改善衰老小鼠的胸腺功能，进而增加了血液淋巴细胞的比例，降低炎症细胞的数量。
进一步将实施例26中衰老小鼠解剖后，取胸腺部分进行石蜡包埋，切片，固定，HE染色。
结果如图22B所示，MC001给药后，小鼠胸腺的脂肪化明显减少，CMJ边界更加规整，胸腺实质部分增加，胸腺组织再生，功能得到改善。这也与小鼠血液的淋巴细胞增加一致，胸腺功能的改善被认为是伴随着抗衰老的一项重要特点。
实施例25 MC001降低衰老小鼠的代谢毒性
取实施例23中的小鼠血清，使用南京建成的小鼠代谢指标检测试剂盒，对小鼠的乳酸含量，谷丙转氨酶活性和肌酐含量进行了测定，
结果如图23所示，MC001能够显著降低三者的水平，改善小鼠衰老过程中的代谢毒性。
实施例26 MC001缓解非酒精性脂肪肝小鼠的肝损伤
本实施例使用高脂饮食及四氯化碳注射诱导肥胖C57BL6/J小鼠(HFD鼠)作为非酒精性脂肪肝模型，采用喂水方式，以0.05mg/ml或0.1mg/ml剂量的MC001分别在高脂第4周、第10周、第12周开始对C57小鼠用药，在第10周灌胃30mg/kg的奥贝胆酸作为对照。高脂14周后，采集小鼠血清，使用A商用试剂盒检测LT、AST、TG、TC含量。
结果如图24所示，非酒精性脂肪肝模型小鼠在用药后血液ALT、AST、TG、TC含量均降低，表明MC001能缓解小鼠肝脏毒性，同时减少高脂引起的血脂含量增加。
实施例27 MC001用药能够缓解非酒精性脂肪肝模型小鼠肝脏的脂肪空泡现象
本实施例解剖NAFLD模型取得小鼠肝脏，制得石蜡切片，使用HE染色观察肝脏空泡现象。
如图25所示，非酒精性脂肪肝模型小鼠在使用MC001后肝脏脂肪空泡明显减少，表明MC001能缓解NASH小鼠肝脏脂肪化。
实施例28 MC001对铁死亡的影响
非酒精脂肪肝的发生发展和铁死亡相关，本发明首先验证MC001对由erastin引起的ROS与LipidROS含量变化的影响。将10μM erastin与不同浓 度的MC001同时加入到HepG2细胞中刺激24小时，用DCFH-DA或BODIPY染料孵育后，流式细胞仪检测荧光发光率。
结果如图26所示，erastin刺激显著提高了细胞内ROS与LipidROS含量，MC001可浓度依赖性的抑制由erastin诱导的ROS的增加，表明MC001可以抑制细胞铁死亡。
实施例29 MC001对细胞脂滴积聚的影响
为在体外评估MC001对细胞脂质积累的影响，本实施例选择用油酸刺激HEPG2细胞，检测脂滴含量变化。300μM OA刺激24小时后，再换新培养基加入不同浓度MC001处理24小时，加入油红染色液染色，显微镜下观察红色脂滴积累情况。
结果如图27所示，MC001用药后，油酸引起的脂滴积累被明显抑制。表明MC001对脂滴积聚有一定的抑制作用。
实施例30 MC001对细胞脂质过氧化的影响
为在体外评估MC001对细胞脂质过氧化的影响，本实施例选择用油酸刺激HEPG2细胞，检测Lipid ROS含量变化。300μM OA刺激24小时后，再换新培养基加入不同浓度MC001处理24小时，加入BODIPY染色液孵育染色，流式细胞仪检测荧光发光情况。
结果如图28所示，MC001用药后，油酸引起的脂质过氧化被明显抑制。表明MC001对脂质过氧化有一定的抑制作用。
实施例31 MC001抑制胶质细胞活化
神经胶质细胞过度活化引起的炎症反应是包括帕金森病在内的多数神经退行性疾病的重要的致病机制之一。为此，本实施例以BV2细胞为模型，观察MC001对其炎症因子表达的影响。
本实施例选择用LPS刺激小鼠胶质瘤Bv2细胞，检测其炎症相关因子的含 量变化。经1μg/ml LPS刺激2小时后，再加入MC001、二甲双胍以及半胱胺处理24小时后使用定量PCR仪检测相关炎症因子的转录情况。
结果如图29所示，MC001可显著抑制LPS引起的NF-kB信号激活，导致一系列促炎性细胞因子的下调，如iNOS、TNF-α、IL-1β。
实施例32 MC001抑制MPP +引起的神经细胞凋亡
MPP +是一种神经毒素，可干扰线粒体氧化磷酸化过程，导致细胞ATP产生受阻，最终引起细胞死亡。为此，本实施例利用流式细胞术在人SH-SY5Y神经细胞系种检测了MC001能否保护MPP+引起的细胞凋亡。
SH-SY5Y细胞使用MPP +处理2小时后在培养基中加入MC001或二甲双胍继续共孵育24小时后使用annexin V/PI染色检测细胞凋亡/坏死情况。
结果如图30所示，与二甲双胍相比，MC001可明显地抑制MPP +引起的细胞凋亡。
实施例33 MC001抑制MPTP引起的多巴胺能神经元丢失
MPTP作为一种神经毒素前体，可在体内被单胺氧化酶氧化为MPP +，进而引起帕金森类似的疾病症状，可作为典型的PD疾病药源性动物模型使用。
基于上述实验结果，即MC001可抑制神经验证、抑制细胞凋亡，我们利用MPTP模型开展了体内干预研究，并利用免疫荧光染色观察了酪氨酸羟化酶(TH)。结果如图31所示，与二甲双胍相比，MC001可明显地抑制MPTP引起的TH阳性细胞丢失。
实施例34 MC001抑制MPTP引起的脑部的氧化应激
由于上述结果发现MC001可抑制MPTP引起的TH阳性细胞丢失，以及体外研究发现其抑制氧化应激水平，本实施例进一步利用染色观察小鼠脑部ROS水平。
使用MPTP建模，在使用MC001或二甲双胍处理后，将ROS检测试剂原位 注射入小鼠脑室后15分钟观察小鼠脑部荧光信号并进行了荧光值的定量分析。
实验结果如图32所示，与二甲双胍相比，MC001可明显地抑制MPTP引起的小鼠脑部氧化应激水平。
实施例35 MC001提高小鼠体内GSH水平
由于胞内还原型谷胱甘肽GSH水平在抗氧化及抗炎症过程中的发挥了至关重要的作用，接下去探讨了MC001是否具备提升GSH的能力。
首先，使用单剂量注射MC001后1小时，3小时进行鼠尾静脉采血，使用试剂盒检测其GSH以及总谷胱甘肽水平。另外，使用MPTP模型鼠检测了其在MC001或二甲双胍处理后小鼠纹状体以及血清中的GSH以及总谷胱甘肽水平。
实验结果如图33所示，单剂量处理小鼠仅1-3小时即可显著提高其血清内GSH占总谷胱甘肽的比例。另外在MPTP模型中同样亦发现MC001能够补偿MPTP处理所引起的GSH水平的降低。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (10)

  1. 一种双胍衍生物，其特征在于，其结构如式(I)所示：
    Figure PCTCN2021142309-appb-100001
    其中，R 1选自H或酰基；
    R 2选自H、羧基或酯基。
  2. 根据权利要求1所述的双胍衍生物，其特征在于，R 1为H，R 2选自H或羧基。
  3. 根据权利要求1所述的双胍衍生物，其特征在于，R 2为H，R 1选自H或酰基。
  4. 根据权利要求1所述的双胍衍生物，其特征在于，R 1为H，R 2为H。
  5. 权利要求1所述的双胍衍生物的制备方法，其特征在于，所述方法包括以下步骤：将双氰胺与式(Ⅱ)或式(III)所示化合物在至少一种有机溶剂中反应，经衍生后得到式(I)所示的双胍衍生物，
    Figure PCTCN2021142309-appb-100002
    式(Ⅱ)中，R为巯基保护基；
    式(III)中，R 1选自H或酰基；
    R 2选自H、羧基或酯基。
  6. 一种药物，其特征在于，所述药物以式(I)所示的双胍衍生物或其非毒性药学上可接受的盐作为活性成分。
  7. 根据权利要求6所述的药物，其特征在于，所述药物被配制成片剂、胶囊剂、注射剂、丸剂、颗粒剂、粉剂或液体剂。
  8. 权利要求1-4任一项所述的双胍衍生物在制备预防或治疗代谢和炎症 相关疾病的药物方面的应用。
  9. 根据权利要求8所述的双胍衍生物的用途，其特征在于，所述疾病包括多囊卵巢综合征、癌症、神经变性疾病、心血管疾病、肥胖症、糖尿病、衰老和/或非酒精性脂肪肝。
  10. 据权利要求8所述的双胍衍生物的用途，其特征在于，所述疾病包括糖尿病、多囊卵巢综合征、癌症和非酒精性脂肪肝。
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* Cited by examiner, † Cited by third party
Title
KHATRI, DILIP; RAJORA, SONAL; BANU, TAHIRA; TALESARA, G. L.: "Synthesis of bis-1, 2-aryl-biguanidino-oxy, 3-biguanidinopropane as potential antimalarials", ASIAN JOURNAL OF CHEMISTRY, CHEMIC PUBLISHING, SAHIBADAD, IN, vol. 11, no. 4, 13 October 1999 (1999-10-13), IN , pages 1438 - 1444, XP009547375, ISSN: 0970-7077 *
WAN TAO, CHEN YUXUAN, PAN QI, XU XIAOJIE, KANG YU, GAO XUE, HUANG FEIHE, WU CHUANBIN, PING YUAN: "Genome editing of mutant KRAS through supramolecular polymer-mediated delivery of Cas9 ribonucleoprotein for colorectal cancer therapy", JOURNAL OF CONTROLLED RELEASE, ELSEVIER, AMSTERDAM, NL, vol. 322, 1 June 2020 (2020-06-01), AMSTERDAM, NL , pages 236 - 247, XP093074581, ISSN: 0168-3659, DOI: 10.1016/j.jconrel.2020.03.015 *

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