The synthetic route to M
The synthetic route to M100907 developed by Rice was utilized, however, the chiral resolution was carried out at an earlier stage to provide the possibility of introducing different substituents onto the piperidinyl group. The conditions for this resolution were different from previously reported. The route to M100907-azide , began with the protection of commercially available guaiacol (). Guaiacol was reacted with TBDPSCl, imidazole and catalytic amount of DMAP at room temperature for 24 h to generate compound in 98% yield. The silyl protected was regioselectively -lithiated by -butyl lithium with TMEDA for 2 h at room temperature. Then Weinreb amide was added at -70 °C and the mixture was stirred at room temperature for 21 h to produce the ketone . Following reaction with the lithiated , the Boc group of was removed with TFA to give . Sodium borohydride reduction then provided the racemic alcohol (). Weinreb amide was synthesized from isonipecotic Pomalidomide (). BOC protection of isonipecotic acid by reaction of di-tertbutyldicarbonate, in a mixed solvent of 1,4-dioxane, acetonitrile and water in the presence of 1 N NaOH gave compound which was then reacted with -dimethylhydroxylamine hydrochloride, and the coupling reagent HBTU, in the presence of DIPEA to give the desired Weinreb amide in ∼80% yield over the two steps. For the chiral resolution of a later synthetic intermediate by the Rice group, methanol was used as the solvent to obtain the resolved salt with ()-mandelic acid. However, the solubility of the diastereomeric salt formed from compound and ()-mandelic acid was sufficiently high in methanol that the yield of recovered material was low. Using a 1:1 ratio of acetonitrile:methanol for the first recrystallization and 1:2 ratio for the second recrystallization, provided one diastereomeric salt in 36% overall yield as white crystals (). Aqueous workup of the diastereomeric salt with ammonium hydroxide afforded the enantiomer in a purity of >95% (). The optical purity was evaluated with ()-(−)-1,10- binaphthyl-2,2′-diyl hydrogen phosphate [(−)-BNP] as a H NMR shift reagent (). The benzylic proton adjacent to the hydroxyl group gave a doublet near 4.6 ppm that was resolved from all other aliphatic signals. This peak was followed on adding (−)-BNP. When one equivalent of (−)-BNP was added to the racemic in CDCl, the benzylic signal separated into 2 doublets, with -enantiomer at 4.4 ppm and the S-enantiomer at 4.5 ppm. Chemical shift changes of this signal were linear relative to the concentration of amine and (−)-BNP. Higher concentrations of (−)-BNP resulted in more significant proton shifting but with broadening of the proton signal. The resolved diastereomeric salt was partitioned between ammonium hydroxide and dichloromethane to obtain a single enantiomer amine (), which underwent -alkylation with (2-tosylethyl)-4-fluorobenzene to generate compound . Removal of the TBDPS group and reaction with linker provided azide-terminated M100907 () (). The alkyne needed to form the homobivalent was synthesized from intermediate through cleavage of the silyl group and alkylation with the tosylated PEG-alkyne (). The bivalent was then synthesized by formation a 1,2,3-triazole ring generated from the dipolar cycloaddition between the azide and the alkyne. This reaction was carried out by adding copper sulfate with sodium ascorbate in a mixture of and DMF and water at room temperature to form the triazole homodimer . Inhibition of 5-HTR-mediated signaling was determined by measuring the reduction of 5-HT (1 µM) stimulated intracellular calcium (Ca) release in CHO-K1 cells stably expressing the 5-HTR., Serotonin induces a concentration-dependent increase in Ca release with an EC of 4.2 nM (pEC = 8.38 ± 0.10) and 1 µM of 5-HT exhibited maximal intracellular Ca release (A). As expected, the active isomer (+)-M100907 displayed low nanomolar potency in inhibiting 5-HT (1 µM)-evoked Ca release (IC = 4.8 nM; pIC = 8.32 ± 0.40; B). None of the compounds displayed activity in the absence of 5-HT (data not shown) and all compounds retained sub-micromolar antagonist activity as shown in . Compound retained antagonist activity suggesting that tethering diethylene-azido linker on the catechol ring of M100907 does not disrupt antagonist properties. Although compounds and displayed slightly lower potency when compared to parent (+)-M100907, these compounds retained comparable potency to a previously published series of 5-HTR bivalent ligands which contain 14 and 17 ethylene glycol linkers., These results indicate that the 1,2,3-triazole ring formed in the click reaction does not significantly affect 5-HTR antagonist activity.