Sterol profiling of Leishmania parasites using a new HPLC-tandem mass spectrometry-based method and antifungal azoles as chemical probes reveals a key intermediate sterol that supports a branched ergosterol biosynthetic pathway.

Human leishmaniasis is an infectious disease caused by Leishmania protozoan parasites. Current chemotherapeutic options against the deadly disease have significant limitations. The ergosterol biosynthetic pathway has been identified as a drug target in Leishmania. However, remarkable differences in the efficacy of antifungal azoles that inhibit ergosterol biosynthesis have been reported for the treatment of leishmaniasis. To better understand the sterol biosynthetic pathway in Leishmania and elucidate the mechanism underlying the differential efficacy of antifungal azoles, we developed a new LC-MS/MS method to study sterol profiles in promastigotes of three Leishmania species, including two L. donovani, one L. major and one L. tarentolae strains. A combination of distinct precursor ion masses and LC retention times allowed for specific detection of sixteen intermediate sterols between lanosterol and ergosterol using the newly developed LC-MS/MS method. Although both posaconazole and fluconazole are known inhibitors of fungal lanosterol 14α-demethylase (CYP51), only posaconazole led to a substantial accumulation of lanosterol in azole-treated L. donovani promastigotes. Furthermore, a key intermediate sterol accumulated by 40- and 7-fold when these parasites were treated with posaconazole and fluconazole, respectively, which was determined as 4α,14α-dimethylzymosterol by high resolution mass spectrometry and NMR spectroscopy. The identification of 4α,14α-dimethylzymosterol supports a branched ergosterol biosynthetic pathway in Leishmania, where lanosterol C4- and C14-demethylation reactions occur in parallel rather than sequentially. Our results suggest that selective inhibition of leishmanial CYP51 is insufficient to effectively prevent parasite growth and dual inhibitors of both CYP51 and the unknown sterol C4-demethylase may be required for optimal antiparasitic effect.

Exploiting Iminoquinones as Electrophilic at Nitrogen "N+" Synthons for C-N Bond Construction.

New methods for C-N bond construction exploiting the N-centered electrophilic character of iminoquinones are reported. Iminoquinones, generated in situ via the condensation of o-vinylanilines with benzoquinones, undergo acid-catalyzed cyclization to afford N-arylindoles in excellent yields. Under similar reaction conditions, homoallylic amines react analogously to afford N-arylpyrroles. Additionally, organometallic nucleophiles are shown to add to the nitrogen atom of N-alkyliminoquinones to provide amine products. Finally, iminoquinones are shown to be competent electrophiles for copper-catalyzed hydroamination.

Silylcyclopropanes by Selective [1,4]-Wittig Rearrangement of 4-Silyl-5,6-dihydropyrans.

4-Silyl-5,6-dihydropyrans undergo remarkably selective [1,4]-Wittig rearrangements to give silylcyclopropanes in good yields. The selectivity is independent of the silyl group, but it is influenced by the electronic character of the migrating center. Electron-rich and electron-neutral (hetero)aryl groups and aliphatic substituents at the migrating center lead to exclusive [1,4]-migration, whereas electron-deficient aryl groups predominantly afford [1,2]-Wittig products.

Formal α-Allylation of Primary Amines by a Dearomative, Palladium-Catalyzed Umpolung Allylation of N-(Aryloxy)imines.

N-(Aryloxy)imines, readily accessible by condensation/tautomerization of (pseudo)benzylic primary amines and 2,6-di-tert-butyl-1,4-benzoquinone, undergo efficient allylation to afford a wide range of homoallylic primary amines following hydrolytic workup. Deprotonation of N-(aryloxy)imines generates a delocalized 2-azaallyl anion-type nucleophile that engages in dearomative C-C bond-forming reactions with allylpalladium(II) electrophiles generated from allylic tert-butyl carbonates. This reactivity umpolung enables the formal α-allylation of (pseudo)benzylic primary amines. Mechanistic studies reveal that the apparent regioselectivity of the desired bond-forming event is a convergent process that is initiated by unselective allylation of N-(aryloxy)imines to give several regioisomeric species, which subsequently rearrange via stepwise [1,3]- or concerted [3,3]-sigmatropic shifts, ultimately converging to provide the desired regioisomer of the amine products.

A Unified Strategy for the Syntheses of the Isoquinolinium Alkaloids Berberine, Coptisine, and Jatrorrhizine.

Total syntheses of the antibacterial alkaloids berberine, coptisine, and jatrorrhizine have been achieved in four steps through a unified route. The key step of this strategy is an efficient intramolecular Friedel-Crafts alkoxyalkylation which, following oxidation, establishes the isoquinolinium core of these natural products. Herein, the design and development of this synthetic strategy, which has enabled the shortest and most efficient syntheses of these alkaloids reported to date, is described.

Exploiting Alkylquinone Tautomerization for the Total Synthesis of Calothrixin A and B.

The pentacyclic alkaloid calothrixin B (1) has been synthesized in 5 steps from murrayaquinone A (9). The key step involved the union of boryl aniline 31 with brominated murrayaquinone A (26). In this transformation, alkylquinone 26 undergoes tautomerization to a quinone methide, which is intercepted by boryl aniline 31 to forge a new C-N bond. An intramolecular Suzuki coupling, followed by dehydrogenative aromatization, completed the synthesis of calothrixin B. Subsequent N-oxidation of calothrixin B delivered calothrixin A. The successful synthesis of these alkaloids and the challenges that led to the development of the final synthesis plan are reported herein.

Copper-Catalyzed Amidation of Primary and Secondary Alkyl Boronic Esters.

The oxidative copper-catalyzed cross-coupling of functionalized alkyl boronic esters with primary amides is reported. Through the identification of appropriate diketimine ligands, conditions for efficient coupling of both primary and secondary alkyl boronic esters with diverse primary amides, including acetamide, have been developed.

Exploiting Alkylquinone Tautomerization: Amine Benzylation.

A general protocol for the synthesis of benzylic amines via side-chain amination of alkylquinones is reported. The reactions are initiated by the tautomerization of an alkylquinone to the corresponding quinone methide, which is subsequently trapped in situ by an amine nucleophile. This process is promoted by tertiary amines in protic solvents under mild conditions and is compatible with many functional groups. 1,2- and 1,4-benzoquinones, as well as naphthoquinones, participate in this reaction using a wide range of primary and secondary amines/anilines. The synthetic utility of this transformation is also explored.

Stereoconvergent [1,2]- and [1,4]-Wittig rearrangements of 2-silyl-6-aryl-5,6-dihydropyrans: a tale of steric vs electronic regiocontrol of divergent pathways.

The regiodivergent ring contraction of diastereomeric 2-silyl-5,6-dihydro-6-aryl-(2H)-pyrans via [1,2]- and [1,4]-Wittig rearrangements to the corresponding α-silylcyclopentenols or (α-cyclopropyl)acylsilanes favor the [1,4]-pathway by ortho and para directing groups in the aromatic appendage and/or by sterically demanding silyl groups. The [1,2]-pathway is dominant with meta directing or electron-poor aromatic moieties. Exclusive [1,2]-Wittig rearrangements are observed when olefin substituents proximal to the silyl are present. cis and trans diastereomers exhibit different reactivities, but converge to a single [1,2]- or [1,4]-Wittig product with high diastereoselectivity and yield.

Selective formation of secondary amides via the copper-catalyzed cross-coupling of alkylboronic acids with primary amides.

For the first time, a general catalytic procedure for the cross-coupling of primary amides and alkylboronic acids is demonstrated. The key to the success of this reaction was the identification of a mild base (NaOSiMe3) and oxidant (di-tert-butyl peroxide) to promote the copper-catalyzed reaction in high yield. This transformation provides a facile, high-yielding method for the monoalkylation of amides.

[1,2]- and [1,4]-Wittig Rearrangements of α-Alkoxysilanes: Effect of Substitutions at both the Migrating Benzylic Carbon and the Terminal sp2 Carbon of the Allyl Moiety.

Substituted α-alkoxysilanes can be deprotonated by alkyllithium bases and made to undergo Wittig rearrangements to afford the #x0005B;1,4]- and [1,2]-rearranged products in varying ratios. Substitution at the benzylic migrating carbon and/or at the allylic carbon of the allyl moiety impacts the rearrangement reaction, influencing the reactivity as well as the [1,4]-/[1,2]-selectivity. Diastereomeric α-alkoxysilanes show different reactivities with the syn diastereomer being the more reactive isomer.