Synthetic Studies of 7‑Oxygenated Aporphine Alkaloids: Preparation of (−)-Oliveroline, (−)-Nornuciferidine, and Derivatives

7-Oxygenated aporphines <b>1</b>–<b>6</b> possessing anti-configurations have previously been reported. In order to explore their bioactivities, a synthesis was established by utilizing a diastereoselective reductive acid-mediated cyclization followed by palladium-catalyzed ortho-arylations. Moderate XPhos precatalyst loading (10 mol %) and short reaction times (30 min) were sufficient to mediate the arylations. Alkaloids <b>1</b>–<b>5</b> were successfully prepared, while (−)-artabonatine A was revised to syn-isomer <b>30</b>. Consequently, (−)-artabonatine E likely also has a syn-configuration (<b>31</b>)

Access to 6a-Alkyl Aporphines: Synthesis of (±)‑<i>N</i>‑Methylguattescidine

(−)-<i>N</i>-Methylguattescidine (<b>3</b>) is an alkaloid recently isolated from <i>Fissistigma latifolium</i> and assigned as a rare example of a 6a-alkyl aporphine. Herein, we report the synthesis of (±)-<b>3</b> and the des-hydroxyl derivative <b>4</b> using our previously reported <i>ortho</i>-phenol arylation methodology mediated by the XPhos precatalyst as a key synthetic step. In addition, substituents on the aryl halide portion of the <i>ortho</i>-phenol arylation substrates significantly influenced the formation of an oxidized side product

Synthesis of Tetracyclic Indoles via Intramolecular α-Arylation of Ketones

An intramolecular palladium(0)-mediated α-arylation of ketones applied to the synthesis of various substituted tetracyclic indoles is reported. Most significantly, the efficiency of the transformation was enhanced by the use of monoligated Pd(0) complexes. This methodology was extended to double α-arylation of ketones using one-pot reactions with either simultaneous addition or sequential addition of two aryl halides for producing aryl substituted tetracyclic indoles

Synthesis and Antiproliferative Activity Evaluation of the Disulfide-Containing Cyclic Peptide Thiochondrilline C and Derivatives

Thiochondrilline C (<b>4</b>) was previously isolated from <i>Verrucisispora</i> sp. and reported to have moderate cytotoxicity against human lung adenocarcinoma cells. Herein, we report the synthesis of thiochondrilline C by N-terminal peptide extension, oxidative disulfide bond formation, and heterocycle installation as key steps. Antiproliferative activities for the prepared natural product and several derivatives against the NCI 60 cancer cell line panel are also described. Derivative <b>22</b> was identified as a moderately potent antiproliferative agent (50% growth inhibition (GI₅₀) = 0.2–12.2 μM) with leukemia (average GI₅₀ = 1.8 ± 0.1 μM) and colon (average GI₅₀ = 2.4 ± 0.3 μM) cells being most sensitive

Divergent Approach for the Synthesis of Gombamide A and Derivatives

A synthesis of gombamide A (<b>1</b>) using N-terminal peptide extension, oxidative disulfide bond formation, and late-stage 4-hydroxystyrylamide installation has been achieved. This divergent method was also utilized to synthesize several gombamide A derivatives with modification to the 4-hydroxystyrylamide via cyclic peptide <b>2</b>. The natural product and four derivatives were found to be devoid of Na⁺/K⁺-ATPase activity at 10 μM. In addition, the compounds were not cytotoxic at 10 μM against a panel of cancer cells

Development of an ALK2-Biased BMP Type I Receptor Kinase Inhibitor

The bone morphogenetic protein (BMP) signaling pathway has essential functions in development, homeostasis, and the normal and pathophysiologic remodeling of tissues. Small molecule inhibitors of the BMP receptor kinase family have been useful for probing physiologic functions of BMP signaling <i>in vitro</i> and <i>in vivo</i> and may have roles in the treatment of BMP-mediated diseases. Here we describe the development of a selective and potent inhibitor of the BMP type I receptor kinases, LDN-212854, which in contrast to previously described BMP receptor kinase inhibitors exhibits nearly 4 orders of selectivity for BMP <i>versus</i> the closely related TGF-β and Activin type I receptors. <i>In vitro</i>, LDN-212854 exhibits some selectivity for ALK2 in preference to other BMP type I receptors, ALK1 and ALK3, which may permit the interrogation of ALK2-mediated signaling, transcriptional activity, and function. LDN-212854 potently inhibits heterotopic ossification in an inducible transgenic mutant ALK2 mouse model of fibrodysplasia ossificans progressiva. These findings represent a significant step toward developing selective inhibitors targeting individual members of the highly homologous BMP type I receptor family. Such inhibitors would provide greater resolution as probes of physiologic function and improved selectivity against therapeutic targets

Kinetic, Mechanistic, and Structural Modeling Studies of Truncated Wild-Type Leucine-Rich Repeat Kinase 2 and the G2019S Mutant

Leucine-rich repeat kinase 2 (LRRK2), a large and complex protein that possesses two enzymatic properties, kinase and GTPase, is one of the major genetic factors in Parkinson’s disease (PD). Here, we characterize the kinetic and catalytic mechanisms of truncated wild-type (t-wt) LRRK2 and its most common mutant, G2019S (t-G2019S), with a structural interpretation of the kinase domain. First, the substitution of threonine with serine in the LRRKtide peptide results in a much less efficient substrate as demonstrated by a 26-fold decrease in <i>k</i>_cat and a 6-fold decrease in binding affinity. The significant decrease in <i>k</i>_cat is attributed to a slow chemical transfer step as evidenced by the inverse solvent kinetic isotope effect in the proton inventory and pL (pH or pD)-dependent studies. The shape of the proton inventory and pL profile clearly signals the involvement of a general base (p<i>K</i>_a = 7.5) in the catalysis with a low fractionation factor in the ground state. We report for the first time that the increased kinase activity of the G2019S mutant is substrate-dependent. Homology modeling of the kinase domain (open and closed forms) and structural analysis of the docked peptide substrates suggest that electrostatic interactions play an important role in substrate recognition, which is affected by G2019S and may directly influence the kinetic properties of the enzyme. Finally, the GTPase activity of the t-G2019S mutant was characterized, and the mutation modestly decreases GTPase activity without significantly affecting GTP binding affinity

Selective and Potent Urea Inhibitors of Cryptosporidium parvum Inosine 5′-Monophosphate Dehydrogenase

Cryptosporidium parvum and related species are zoonotic intracellular parasites of the intestine. Cryptosporidium is a leading cause of diarrhea in small children around the world. Infection can cause severe pathology in children and immunocompromised patients. This waterborne parasite is resistant to common methods of water treatment and therefore a prominent threat to drinking and recreation water even in countries with strong water safety systems. The drugs currently used to combat these organisms are ineffective. Genomic analysis revealed that the parasite relies solely on inosine-5′-monophosphate dehydrogenase (IMPDH) for the biosynthesis of guanine nucleotides. Herein, we report a selective urea-based inhibitor of C. parvum IMPDH (<i>Cp</i>IMPDH) identified by high-throughput screening. We performed a SAR study of these inhibitors with some analogues exhibiting high potency (IC₅₀ < 2 nM) against <i>Cp</i>IMPDH, excellent selectivity >1000-fold versus human IMPDH type 2 and good stability in mouse liver microsomes. A subset of inhibitors also displayed potent antiparasitic activity in a Toxoplasma gondii model

Akt signaling contributes to autocrine TNFα production in multiple cell types.

<p>FADD deficient Jurkat cells were treated with TNFα followed by measurement of (A) human TNFα mRNA levels by qRT-PCR and normalized using human 18S RNA or (B) western blot at 9 hr. RAW 264.7 or J774A.1 cells were treated with zVAD.fmk (100 uM or 50 uM respectively) followed by (C,E) measurement of TNFα mRNA levels by qRT-PCR or (D,F) western blot at 9 hr. (G) Akt null mouse lung fibroblasts expressing Myr-Akt or K179M were treated with zVAD.fmk and TNFα followed by measurement of TNFα mRNA levels by qRT-PCR at 9 hr. (H) Mouse lung fibroblasts expressing only endogenous Akt1 or Akt2 were treated with zVAD.fmk and TNFα followed by measurement of TNFα mRNA levels by qRT-PCR at 9 hr.</p

mTORC1 contributes to the regulation of necroptosis.

<p>(A) L929 cells were treated with zVAD.fmk or TNFα for 9 hr and harvested for western blot. (B) Cell under serum free condition were treated with bFGF or bFGF/zVAD.fmk for the indicated amounts of time, followed by western blotting using the indicated antibodies. (C) Necroptosis was induced by zVAD.fmk or TNFα in L929 cell in the presence of inhibitors of Akt(Akt inh. VIII) and mTOR (rapamycin, Torin-1 and PI-103). (D) L929 cells with mTOR siRNA knockdown were harvested for western blot or treated with zVAD.fmk or TNFα for 24 hrs. Cell viability was determined 24 hr after activation of necroptosis. In all graphs, average±SD was plotted.</p