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  • The co crystal structure of blebbistatin bound to

    2024-02-10

    The co-crystal structure of ()-blebbistatin ()- bound to myosin II (PDB: ) was used to scout for possible favorable interactions with the residues lining the binding pocket. Looking at A, we hypothesized that -oriented hydrophilic moieties (e.g. hydroxymethyl) on C of the blebbistatin scaffold might engage in hydrogen bonding with the carboxylate of the neighboring Arg238 residue. This additional bonding possibility would be absent in the -oriented diastereoisomers. Further, both - and -oriented larger functionalities (e.g. allyloxymethyl) might optimize filling of the binding pocket (B). Given these observations and the ready availability of racemic pyroglutamic WAY 208466 dihydrochloride synthesis (), the synthesis of both diastereoisomers of analogs (±)- and (±)- was envisioned. Due to the exploratory nature of this investigation, the syntheses in this letter start from racemic substrates and do not focus on obtaining enantiomerically pure compounds. The precursor (±)-pyroglutamic acid (±)- was chosen as an ideal starting point for the synthesis of the proposed blebbistatin analogs (±)-- (). This strategy required the preparation of quinolinone (±)- as a key intermediate. The pathway () commenced with the synthesis of amide (±)- which could be accessed two separate routes. In a first method, allyl protection of the carboxyl group of (±)-pyroglutamic acid (±)- (step , 97%) preceded Goldberg-type -arylation of compound (±)- with iodobenzene (). The rather low conversion of the latter step (step , 25%) prompted us to use an alternative method for the preparation of amide (±)-. In this approach, Chan-Lam-type -arylation of the unprotected (±)-pyroglutamic acid (±)- was performed with phenylboronic acid () (step , 85%)., Subsequent allyl protection of the carboxyl group of compound (±)- afforded amide (±)- (step , 93%) in a much higher yield than the first method. Amide (±)- was reacted with POCl and amine to furnish amidine (±)- (step , 21%) which was subsequently treated with LiHMDS to induce intramolecular cyclization. However, this resulted in a complex mixture, containing only trace amounts of quinolinone (±)- (step ). These side reactions presumably occurred due to the presence of an acidic hydrogen in the -position of the allyl ester (±)-. We thus opted to reduce the ester functionality in (±)- (). Sakai et al. had reported on the direct and selective reduction of esters to the corresponding ethers in the presence of secondary amides with EtSiH and catalytic amounts of InBr. We applied these conditions in an attempt to selectively convert the allyl ester in amide (±)- to an allyl ether in amide (±)- (, step ). A clean conversion to a sole product was obtained. Unfortunately, reduction of the tertiary amide, rather than the ester, occurred affording allyl ester (±)-. No trace of amide (±)- was observed. Therefore, a detour was made by first completely reducing the allyl ester in amide (±)- to primary alcohol (±)- (step , 93%), after which the latter was allyl protected to yield amide (±)- (step , 92%). Preparation of amidine (±)- was achieved by the action of POCl and amine (step , 52%). Next, sequential treatment with LiHMDS and an equimolar mixture of oxaziridines and resulted in a clean one-pot ring closure-α-hydroxylation sequence towards (±)-2-(allyloxymethyl)blebbistatin (±)-. (step , diastereoisomeric ratio 84:16, 90%). This mixture of diastereoisomers was allyl deprotected with Pd(PPh) and KCO, but only a 50% conversion toward (±)-2-(hydroxymethyl)blebbistatin (±)- was obtained for each diastereoisomer (step , 30–36%). Likely, a higher conversion could have been achieved by adding multiple portions of Pd(PPh). By chance, we observed that a yellow precipitate persisted when trying to dissolve the crude reaction mixture in THF. This precipitate was isolated and identified as the minor diastereoisomer of compound (±)- in pure form. The remaining major diastereoisomer was subsequently purified automated flash chromatography.