Assignment of Absolute Configuration to Enantiomers of Anti-Alzheimer Drug Candidate Blarcamesine

Blarcamesine is a promising investigational drug for the treatment of Alzheimer's disease. The international nonproprietary name blarcamesine refers to a racemic compound, although it seems likely that it will be marketed in an enantiopure form. A resolution process has been described in the literature, but the absolute configurations of the enantiomers have not yet been disclosed. In the present study, crystals of (R)-(-)- and (S)-(+)-mandelate salts of (+)- and (-)-blarcamesine and also that of (R)-(+)-blarcamesine itself, suitable for single-crystal X-ray diffraction measurement were prepared and the absolute configurations of (+)- and (-)-blarcamesine have been determined

Position-Dependent Sequence Motif Preferences of SpCas9 are Largely Determined by Scaffold-Complementary Spacer Motifs

Streptococcus pyogenes Cas9 (SpCas9) nuclease exhibits considerable position-dependent sequence preferences. The reason behind these preferences is not well understood and is difficult to rationalise, since the protein establishes interactions with the target-spacer duplex in a sequence-independent manner. We revealed here that intramolecular interactions within the single guide RNA (sgRNA), between the spacer and the scaffold, cause most of these preferences. By using in cellulo and in vitro SpCas9 activity assays with systematically designed spacer and scaffold sequences and by analysing activity data from a large SpCas9 sequence library, we show that some long (>8 nucleotides) spacer motifs, that are complementary to the RAR unit of the scaffold, interfere with sgRNA loading, and that some motifs of more than 4 nucleotides, that are complementary to the SL1 unit, inhibit DNA binding and cleavage. Furthermore, we show that intramolecular interactions are present in the majority of the inactive sgRNA sequences of the library, suggesting that they are the most important intrinsic determinants of the activity of the SpCas9 ribonucleoprotein complex. We also found that in pegRNAs, sequences at the 3′ extension of the sgRNA that are complementary to the SL2 unit are also inhibitory to prime editing, but not to the nuclease activity of SpCas9

Study on the Alkylation Reactions of N(7)-Unsubstituted 1,3-Diazaoxindoles

The chemistry of the 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one (1,3-diazaoxindole) compound family, possessing a drug-like scaffold, is unexplored. In this study, the alkylation reactions of N(7)-unsubstituted 5-isopropyl-1,3-diazaoxindoles bearing various substituents at the C(2) position have been investigated. The starting compounds were synthesized from the C(5)-unsubstituted parent compounds by condensation with acetone and subsequent catalytic reduction of the 5-isopropylidene moiety. Alkylation of the thus obtained 5-isopropyl derivatives with methyl iodide or benzyl bromide in the presence of a large excess of sodium hydroxide led to 5,7-disubstituted derivatives. Use of butyllithium as the base rendered alkylation in the C(5) position possible with reasonable selectivity, without affecting the N(7) atom. During the study on the alkylation reactions, some interesting by-products were also isolated and characterized

Thermal Ring Contraction Reactions of 9‐Aryl‐5H,7H‐[1,2,5]thiadiazolo[3,4‐h][2,3,4]benzothiadiazepine 6,6‐Dioxides. Experimental and Computational Studies for Understanding the Course of the Transformations

When refluxing with sodium hydrogen carbonate in acetonitrile, 7-chloro-5-(4-fluorophenyl)-1,3-dihydro-2,3,4-benzothiadiazepine 2,2-dioxide afforded, after loss of dinitrogen and subsequent ring contraction, the corresponding sulfone in 83% yield. Similar treatment of the related thiadiazolo-fused tricycles, i.e. 9-aryl-5<i>H</i>,7<i>H</i>-[1,2,5]­thiadiazolo­[3,4-<i>h</i>]­[2,3,4]­benzothiadiazepine 6,6-dioxides, resulted in a substantially different product mixture: formation of sultines and benzocyclobutenes was observed, while only small amounts of the sulfones were formed, if any. Density functional theory calculations support the mechanism proposed for the transformations involving a zwitterionic intermediate formed by the tautomerization of the thiadiazepine ring followed by dinitrogen extrusion. When starting from 7-chloro-substituted 2,3,4-benzothiadiazepine 2,2-dioxide, the formation of sulfone via <i>o</i>-quinodimethane is the preferred pathway from the zwitterion. However, in the case of thiadiazolobenzothiadiazepine 6,6-dioxides it has been found that the ring closure of the zwitterion leading to the formation of sultines was kinetically preferred over the loss of sulfur dioxide leading to <i>o</i>-quinodimethane, which is the key intermediate to benzocyclobutene-type products. The calculations explain the differences observed between the product distributions of the chloro-substituted and the thiadiazolo-fused derivatives

A novel tool for structure assignment of hydroxylated metabolites of(arylpiperazinylbutyl)oxindole derivatives based on relative HPLC retention times

tIncubation of oxindole derivatives containing an arylpiperazine pharmacophore in rat liver microsomesin vitro formed several metabolites hydroxylated at various positions of the aromatic rings of the oxin-dole carbocycle or the arylpiperazine moiety. In order to substitute the sites of metabolic attack onthese positional isomers, the exact structure of the molecules had to be identified. As polarities of thecompounds depend on the site of hydroxylation, we measured retention times of the metabolites usingreversed-phase HPLC. It was noted that the relative retention times (RRT, the ratio of the retention timeof the metabolite and the parent compound) fell into distinct narrow ranges for metabolites identifiedby MS spectra as positional isomers. These RRT ranges correlated with the positions of hydroxylation.The hypothesis was validated by synthesis of hydroxy compounds of known structure and by deter-mination of their RRT values. Change in the chromatographic parameters such as column type, eluent,gradient time and temperature did not impede the identification of the sites of hydroxylation as the RRTpattern remained similar to the original one. The new empirical method proposed in our study can beused for tentative identification of hydroxy metabolites and orient the direction of efforts to synthesizemetabolically stable compounds