Design And Synthesis Of (+)-flubalasubramide Photoaffinity Molecules Probe

(+)-Flubalasubramide was discovered as a lead compound with anti-neuroinflammation role in our previous studies on the structure-activity relationships of natural product（+）-balasubramide present in clausena Indica.Our previous pharmacologic study showed that this lead compound could inhibit neuroinflammation by promoting the M₂ phenotype polarization of microglia through the AMPK/JNK and AMPK/PGC-Iαpathways,but its specific target protein for anti-neuroinflammation is not clear.In order to clarify the pharmacological mechanism of（+）-flubalasubramide,photo-affinity molecular probes for（+）-flubalasubramide was designed and synthesized.These studies are the important basis of specific target protein for anti-neuroinflammation.And the work is as follows:Further structure-activity relationship studies to determine the positions of（+）-flubalasubramide where the probes can be connected:In the previous study,our group had carried out preliminary research on the structure-activity relationship of 5-hydroxy and 6-phenyl Flubalasubramide.The results showed that pharmacological activity of5-ester-containing,5-ether-containing,6-benzene ring with electron-withdrawing substituents flubalasubramide would not decrease.And the different sites of substituents would not change the type of pharmacological action,but only changes the magnitude of pharmacological activity,indicating that these sites on（+）-flubalasubramide can substitute photophiles.Then we investigated the effect of introduction of substituents in N atom positions of the indole ring of（+）-flubalasubramide,so as to determine whether the photoaffinity probe molecule can be introduced at this position.Therefore,some substituents in N atom positions of the indole ring of（+）-flubalasubramide were designed and synthesized,for evaluating the anti-inflammatory activity lay the foundation.But 5-OH of the eight-membered lactam ring of（+）-flubalasubramide would affect the substitution reaction of the nitrogen atom of the indole ring.Thus,according to the retrosynthetic analysis,it was determined that tryptophan was used as the starting material,and nitrogen-substituted tryptophan hydrochloride was obtained after the protection of primary amine,through alkylation and deprotection of primary amines.Afterwards,it was subjected to asymmetric epoxidation to obtain 2S,3R-p-trifluoromethyl methyl cinnamate and then through ester amine condensation reaction under alkaline condition;finally,N-substituted derivatives of（+）-flubalasubramide on the indole ring of（+）-Flubalasubramide was obtained by a five-step asymmetric synthesis route such as Lewis acid-catalyzed cyclization.Synthesis of photoaffinity labeled molecular probe of（+）-Flubalasubramide ether T-14:According to the previous reports,Meldrum’s acid,the starting material,was carried on reactions such as condensation,protection,reduction,and deprotection to give T-8.Then T-8 underwent ammoniation in a methanol solution of ammonia and then underwent condensation with hydroxylamine-O-sulfonic acid to give dipropidine.Then dipropidine underwent oxidation reaction with iodine（oxidant）and triethylamine（catalyst）to give T-9.After iodine reaction of T-9 and iodine under the catalysis of triphenylphosphine and imidazole were carried on to give T-10.At last,T-10 and（+）-flubalasubramide underwent ether reaction to give the target compound T-14.There are seven steps in the whole route with a total yield of 23%（based on Meldrum’s acid）.The route improves the key step of the route which was reported,such as the synthesis of 3-carbonyl-6-alkynylheptanoate and2-（3-But-3-ynyl-3H-diazirin-3-yl）-ethanol,it avoids using of highly toxic reagents,ultra-low temperature（-78℃）anhydrous and anaerobic reaction conditions,and simplifying operations.Synthesis of photoaffinity labeled molecular probe of（+）-flubalasubramide ester T-15:Synthesis of the part of T-15,T-11,have reported before which required eight step reactions with harsh reaction conditions and the reagents（such as potassium cyanide）that pollute to the environment.Thus,a new synthetic route was redesigned,methyl3-oxohept-6-ynoateT-5a was subjected to S_N2 nucleophilic substitution with methyl bromoacetate to give T-12,then underwent decarboxylation of T-12 to obtain 4-carbonyl-6-alkynyl-1-octanoic acid T-13.After three steps of reaction of T13,such as ammoniation,condensation,and oxidation to give the key intermediate photophile molecular T-11 that containing a carboxyl group.At last,T-11 and（+）-flubalasubramide underwent esterification reaction to give the target compound T-14.There are five steps in the whole route with a total yield of 23%（based on Meldrum’s acid）.In particular,the final three-step reaction of ammoniation,condensation,and oxidation achieved"one-pot cooking",which greatly simplified the operation.The designed synthetic route of the key intermediate T-11 was shortened from the eight-step reaction reported to the four-step reaction,which simplified the process and improved the yield.In this paper,a total of 36 compounds including all intermediates and target compounds were synthesized,20 of them were not reported.All new compounds have been identified by ¹H-NMR,¹³C-NMR and HRMS,The biological evaluation of（+）-flubalasubramide indole ring N-substituted derivatives,ester and ether probes are under studying.