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

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

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

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

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

The pentacyclic alkaloid calothrixin B (<b>1</b>) has been synthesized in 5 steps from murrayaquinone A (<b>9</b>). The key step involved the union of boryl aniline <b>31</b> with brominated murrayaquinone A (<b>26</b>). In this transformation, alkylquinone <b>26</b> undergoes tautomerization to a quinone methide, which is intercepted by boryl aniline <b>31</b> 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

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

The pentacyclic alkaloid calothrixin B (<b>1</b>) has been synthesized in 5 steps from murrayaquinone A (<b>9</b>). The key step involved the union of boryl aniline <b>31</b> with brominated murrayaquinone A (<b>26</b>). In this transformation, alkylquinone <b>26</b> undergoes tautomerization to a quinone methide, which is intercepted by boryl aniline <b>31</b> 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

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-<i>tert</i>-butyl peroxide) to promote the copper-catalyzed reaction in high yield. This transformation provides a facile, high-yielding method for the monoalkylation of amides