Design And Synthesis Of (+)-balasubramide Derivative Photoaffinity Molecules Probe

(+)-fluorobalasubramide（3C）is a derivative of eight membered lactam（+）-balasubramide extracted from Clausena in the central rainforest of Sri Lanka.Pharmacological studies have shown that（+）-fluorobalasubramide is able to inhibit the release of pro-inflammatory factors in BV-2 microglia induced by lipopolysaccharide in vitro At the same time,but it have also demonstrated that this compound possess excellent cytotoxicity.Further studies showed that compound 3C could regulate the activation of PGC-1a signaling pathway by promoting the phosphorylation of Ca MKKβand AMPK,and reduce the secretion of pro-inflammatory mediators in microglia and BV₂cells,thus promoting the expression of anti-inflammatory mediators.Because the target of the compound has been unclear,in order to clarify its mechanism,the team designed and synthesized 3C photoaffinity molecular probes of（+）-balasubramide derivative,which laid the foundation for the research of its target protein as follows:The preliminary structure-activity relationship studies on compound 3C have been completed by our research team,showing that the function group such as ether group,ester group and electron absorption group,introduced into the 5 position of（+）-fluorobalasubramide will not change its pharmacological action pattern,however,the type of function groups are able to affects the size of its pharmacological activity.Moreover,the group also introduces photoaffinity groups into 5-hydroxy groups to synthesize（+）-fluorobaractamide esters and ethers.Because of its weak efficacy,there is no way to fish out its target protein.In this study,we continue to design and synthesize other photoaffinity probes of（+）-balasubramide derivatives（3C）,that is,to introduce photoaffinity groups at other sites of 3C structure,to design and synthesize amide photoaffinity molecular probes of（+）-balasubramide derivatives,and to synthesize hydrocarbon photoaffinity molecular probes of（+）-balasubramide derivatives by introducing photoaffinity groups on indole nuclear nitrogen.Synthesis of key intermediates of carboxylic acid based photoaffinity probes containing diazirine group:Our team has achieved the synthesis of the key intermediate carboxylic acid photoaffinity probe 3-（3-（but-3-yn-1-yl）-3H-diazirin-3-yl）propanoic acid（T-13）.Based on this,the thesis improved the synthesis method of T-13,and increased the total yield of the target compound from 19.8%to36.2%.In this method,pentynic acid was used as starting material,EDCI/DMAP was used as condensing agent,and the intermediate methyl3-oxohept-6-ynoate was obtained by electrophilic reaction with Michaelis acid and refluxing in methanol;the intermediate reacted with methyl bromoacetate by S_N2 nucleophilic substitution reaction,then hydrolyzed and decarboxylated to obtain 4-carbonyl-6-alkynyl-1-octanoic acid,finally,the carboxyl photoaffinity molecular probe containing bisacridine group was obtained under the condition of ammonia methanol solution/HNOSO₃/I₂.Synthesis of amide based photoaffinity molecular probe（+）-balasubramide derivative:Because of the large steric hindrance of the amino group of the 3C indole ring,it is difficult to carry out the N-acylation of the 3C indole ring directly,so the intermediate（2S,3R）-N-（ethyl-2-（1H-indolyl））-3-phenyloxiranyl-2-amine was chosen for the N-acylation,at first,T-13was prepared into acyl chloride,and then（2S,3R）-N-（2-（1-（3-（3-（but-3-yn-1-yl）-3H-diazirin-3-yl）propanoyl）-1H-indol-3-yl）ethyl）-3-（4-（trifluor omethyl）phenyl）oxirane-2-carboxamide 2C+T₁was obtained by N-acylation with strong base Na H,then the target compound（+）-balasubramide derivative was obtained by cyclization in the presence of ytterbium trifluoromethanesulfonate,the yield of the three-step reaction was 15.8%.Due to the low yield of the above method,3C+T1 was synthesized from 3C in the presence of（Boc）₂O/2,6-dimethylpyridine/DMAP.The yield of 3C+T₁was 85.2%.Synthesis of the key intermediate iodinated photoaffinity probe containing diazirine group:The key intermediate 3-（butyl-3-ethynyl-1-yl）-3-（2-iodoethyl）-3H-diacrylpropane（T-10）was synthesized by the method reported by our lab,on this basis,the synthetic route was optimized,and the total yield was increased from 27.4%to 37.6%.Methyl 3-carbonyl-6-alkynylheptanoate was synthesized by electrophilic reaction of pentynoic acid with Michaelis acid and refluxing in methanol using EDCI/DMAP as condensation agent.Then ethylene glycol and carbonyl formed acetal to protect carbonyl group.Lithium aluminum hydride reduced ester group and removed protective group to obtain 1-hydroxy-6-heptanyl-3-one,the photoaffinity molecular probe containing bisacridine hydroxy group was formed in the condition of NH₃（in Me OH）/HNOSO₃/I₂,finally,the photoaffinity molecular probe containing diazirine group was obtained under the action of imidazole,triphenylphosphine and iodine.Synthesis of hydrocarbon photoaffinity molecular probes derived from（+）-balasubramide derivatives:According to the synthesis method of the amide photoaffinity probe of the（+）-balasolamide derivative（3C）,N-alkylation was carried out the step before the cyclization,and the anhydrous N,N-dimethylformamide as the solvent,the intermediate compound（2S,3R）-N-（2-（1-（2-（3-alkynylbutyl-3h-3-bisacridine）propionyl）-1H-indolyl）ethyl）-3-（4-（triflu oromethyl）phenyl）oxiranyl-2-formamide 2C+T₂was synthesized in the presence of potassium hydroxide,then the target compound（+）-balasubramide derivative（3C）hydrocarbons photoaffinity molecular probe 3C+T₂was synthesized by intramolecular cyclization catalyzed by ytterbium trifluoromethanesulfonate.That is,the original intramolecular cyclization is carried out first,then on indole ring nitrogen,instead of alkylation on indole ring nitrogen and then in the internal cyclization,which can effectively avoid the side reactions of partial product ring opening decomposition.In this thesis,a total of 23 intermediates and target compounds were synthesized,of which 6 were not reported.The structures of all the new compounds were confirmed by ¹H-NMR,¹³C-NMR and HRMS,and the research on the targets of anti-inflammatory effect by using（+）-balasubramide derivatives as photoaffinity molecular probes is in progress.