Structural Modification Of Salidroside And Its Anti-hypoxia Activity

Oxygen is the basic substance for maintaining the normal metabolism of the body’s tissues and organs,and is of great importance for maintaining the body’s vital activities.Hypoxia refers to the pathological process of abnormal changes in tissue metabolism,function and morphological structure due to insufficient supply of oxygen to tissues or disorders of oxygen use.At present,domestic and foreign anti-hypoxia drugs are mainly divided into Chinese herbal medicine,Tibetan medicine,Western medicine and Chinese and Western medicine combination drugs.Among them,acetazolamide,nifedipine,non-steroidal anti-inflammatory drugs such as bendamycin hydrochloride,dexamethasone and other western drugs are mostly therapeutic drugs for other diseases,which have large adverse effects,while Tibetan drugs have the disadvantage of complex composition,so it is of great importance to develop new and efficient drugs for the prevention and treatment of hypoxic injury on the plateau and to formulate effective strategies for the prevention and treatment of hypoxic injury on the plateau.One of the current hot spots in the research and development of anti-hypoxic drugs is the discovery of anti-hypoxic active ingredients with precise efficacy and high safety based on traditional Chinese medicine and natural products.Studies have shown that salidroside has various pharmacological effects such as anti-cancer,anti-aging,anti-inflammatory,enhanced oxygen absorption,fatigue elimination,neuroprotection,hepatoprotection and antioxidant.In this paper,we designed and synthesized a series of new compounds using salidroside as the lead compound,in order to discover molecules with significant anti-hypoxic activity,reveal the conformational relationship pattern,and provide important experimental references for further discovery of efficient and safe new anti-hypoxic drugs.The following work was mainly accomplished.1.Combined with the study of anti-hypoxic conformational relationships of salidroside derivatives,25 structural analogues of salidroside were designed and synthesized with salidroside as the lead compound,including 10 compounds of R series,10 compounds of X series and 5 compounds of J series,the structures of which have not been reported in the literature and are new compounds.The structures of all compounds were corroborated by ¹H-NMR,¹³C-NMR and HRMS.2.The synthesis of 25 target compounds was achieved in good yields using p-hydroxyphenylethanol and 1,2,3,4,6-β-D-glucose pentaacetate as starting materials,after phenolic hydroxylation into ethers,oxyglucoside bond formation,deacetylation protection and Mannich reaction.In the synthesis of salidroside,a multi-step reaction and one-step purification strategy were used,which significantly shortened the synthesis time of the compounds and avoided the use of large amounts of organic solvents.In the synthesis of intermediate a and J series derivatives,it was found that the oxyglucosylation reaction could be carried out under argon-free protection after using1,3-dibromopropane and alkyl-protected phenolic hydroxyl groups,which simplified the experimental steps and had no significant effect on the yields.In the one-step synthesis of X-series derivatives from intermediate a,N,N-dimethylformamide was used as the reaction solvent,which allowed the reaction to proceed under milder conditions and in better yields.3.Using the anaerobic method of the Anaero Pack as a cellular hypoxia method,a hypoxia/reoxygenation（H/R）injury model was established for H9C2 cardiomyocytes,and a cellular hypoxia time of 8 h and a reoxygenation time of 24 h were determined.The optimal concentration of salidroside was determined to be 10μmol/L.The results of the initial screening showed that all 24 compounds significantly improved the cell viability of hypoxic H9C2 cells at a compound concentration of 25μmol/L.Eight of the compounds showed significantly better anti-hypoxic activity than the positive drug salidroside,namely R5,R7,X2,X3,X8,X10,J1 and J2.For the above eight compounds with better activity,five dosing concentrations:10,5,2.5,1 and 0.5μmol/L were set downwards for re-screening.The results showed that the cell viability values of compounds R7,X2,X8 and J1 were higher than those of salidroside at the dosing concentration of 10μmol/L,indicating that their anti-hypoxic activity was better than that of salidroside;the anti-hypoxic activity of compounds R7 and X8 was better than that of salidroside at the dosing concentrations of 5 and 2.5μmol/L;the anti-hypoxic activity of compound X8 was better than that of salidroside at the dosing concentration of 1μmol/L;when the administered concentration was reduced to 0.5μmol/L,the anti-hypoxic activity of all eight compounds was comparable to that of salidroside,with no significant difference.4.Human renal epithelial cells 293T,human lung epithelial cells BEAS-2B,rat cardiomyocytes H9C2,and human normal hepatocytes LO2 were selected for toxicity testing.The results showed that the compounds R7,X8 and J1 were not significantly toxic to the four types of cells.The compound X2 showed some toxicity to all four cells with increasing concentrations:at concentrations≥1μmol/L,X2 showed greater cytotoxicity to LO2 cells;at concentrations≥2.5μmol/L,X2 showed greater cytotoxicity to BEAS-2B cells;at concentrations≥25μmol/L,X2 gradually showed less cytotoxicity to 293T and H9C2 cells.5.The software Discovery Studio 20.0 and the web tool Swiss ADME were used to predict the physicochemical and pharmacokinetic properties of salidroside and 25derivatives,focusing on the relevant properties of compounds R7,X2,X8 and J1 with good cellular anti-hypoxic activity.The predicted results showed that salidroside and its four derivatives have good water solubility;the ability to cross the blood-brain barrier into the CNS after oral administration of salidroside,R7,X2 and X8 cannot be accurately predicted,suggesting that they may have difficulty crossing the blood-brain barrier,while compound J1 may have a relatively low blood-brain barrier transmission rate;salidroside and its four derivatives do not inhibit the expression of Cytochrome P4502D6 enzymes and do not have significant hepatotoxicity;compounds R7,X2 and J1 have good intestinal absorption,while salidroside and X8 have reduced absorption and are moderately well absorbed;also none of the five compounds are highly bound to plasma proteins,thus affecting the pharmacological action of the compounds.Swiss ADME predictions showed an increase in CLog P for all four compounds,indicating an increase in lipid solubility compared to salidroside.The values of molecular weight,hydrogen bond acceptor,hydrogen bond donor,number of rotatable bonds and polar surface area were in accordance with Lipinski’s rules and others.6.The results of atmospheric hypoxia experiments showed that compared with saline,salidroside prolonged the survival time of hypoxic mice by 12.03%,and compounds R7,X8 and J1 prolonged the survival time of hypoxic mice by 21.39%,12.72%and 24.21%,respectively;compared with salidroside group,R7 and J1significantly prolonged the survival time of hypoxic mice,and the results were significantly different（P<0.05）.Hypobaric hypoxia experiments showed that after 8 h of hypoxic stimulation at a simulated altitude of 8000 m,the number of white blood cells（WBC）,lymphocytes（Lym）and red blood cells（RBC）in the blood of mice decreased,and the concentrations of hemoglobin（HGB）,red blood cell volume（HCT）and platelets（PLT）decreased.Salidroside and the three derivatives were able to slow down this decline to some extent.At the same time,after being stimulated by hypoxia,the malondialdehyde content in the serum of mice would be significantly increased,while salidroside,compound R7,X8 and J1 could significantly reduce the malondialdehyde content in the serum of hypoxic mice（P<0.01）,and compound R7 and J1 could further reduce the MDA content in the serum compared with the salidroside group,and the results were significantly different（P<0.05 or P<0.01）.In conclusion,25 novel salidroside derivatives were designed and synthesized with salidroside as the lead compound,all of which have not been reported in the literature.In vitro and in vivo anti-hypoxic activity evaluation and cytotoxicity experiments showed that compounds R7,X8 and J1 have significant anti-hypoxic activity.The preliminary conformational relationship pattern of the above compounds was summarized and analyzed.When the amine methyl group was introduced in the neighboring position of phenolic hydroxyl group of salidroside,the chain amine was more favorable to the activity than the cyclic amine,and when the amine propyl group was introduced in the phenolic hydroxyl group,the activity was related to the carbon number of the amine,and the compound activity was better than that of salidroside when the carbon number of the chain amine was 4 or 6 and the carbon number of the cyclic amine was 4;when the alkyl group was introduced in the phenolic hydroxyl group,the compound activity gradually decreased with the increase of the carbon number.The results provide an important experimental reference for the design and synthesis of such compounds,the evaluation of anti-hypoxic activity and thus the discovery of effective anti-hypoxic molecules.