Cytotoxic stress caused by azalamellarin D (AzaD) interferes with cellular protein translation by targeting the nutrient-sensing kinase mTOR.

Analogs of pyrrole alkaloid lamellarins exhibit anticancer activity by modulating multiple cellular events. Lethal doses of several lamellarins were found to enhance autophagy flux in HeLa cells, suggesting that lamellarins may modulate protein homeostasis through the interference of proteins or kinases controlling energy and nutrient metabolism. To further delineate molecular mechanisms and their targets, our results herein show that azalamellarin D (AzaD) cytotoxicity could cause translational attenuation, as indicated by a change in eIF2α phosphorylation. Intriguingly, acute AzaD treatment promoted the phosphorylation of GCN2, a kinase that transduces the integrated stress response (ISR), and prolonged exposure to AzaD could increase the levels of the phosphorylated forms of eIF2α and the other ISR kinase PKR. However, the effects of AzaD on ISR signaling were marginally abrogated in cells with genetic deletion of GCN2 and PKR, and evaluation of protein target engagement by CETSA revealed no significant interaction between AzaD and ISR kinases. Further investigation revealed that acute AzaD treatment negatively affected mTOR phosphorylation and signaling. The analyses by CETSA and computational modeling indicated that mTOR may be a possible protein target for AzaD. These findings indicate the potential for developing lamellarins as novel agents for cancer treatment.

Dihydro-10H-indeno[1,2-b]benzofurans and Tetrahydroindeno[1,2-c]isochromenes via Stereoselective Intramolecular Carbocation Cascade Cyclization.

Nazarov cyclization of the (E)-(2-stilbenyl)methanols under the catalysis of p-TsOH immobilized on silica (PTS-Si) proceeded to give the corresponding indanyl cation with the exclusive trans relationship at the two newly formed adjacent stereogenic centers. The ensuing intramolecular nucleophilic addition by the MOM-protected phenol (m = 0) or benzyl alcohol (m = 1) furnished the Indane-fused benzofuran [5/5] or isochroman [5/6] system, respectively, with the exclusive cis stereocontrol at the two-carbon ring junction. Thus, in a single step, from nonchiral starting materials, the intramolecular cascade carbocation cyclization (CCC) furnished the [5/5] or [5/6] oxygen-containing Indane fused-ring systems in moderate to good yields with excellent stereoselectivity on all three contiguous stereogenic centers.

Synthesis, Antiacetylcholinesterase Activity, and Molecular Dynamics Simulation of Aporphine-benzylpyridinium Conjugates.

A series of aporphines conjugated with an N-benzylpyridinium moiety through an amide-bond linkage were synthesized and evaluated for their acetylcholinesterase (AChE) inhibitory activity. The conjugation of the N-benzylpyridinium group significantly enhanced the AChE inhibitory activity of the core aporphine. The halogen substituents on the benzyl group affected the activity of the conjugates. Both (S)- and (R)-enantiomers of three conjugates with low IC50 values were synthesized and evaluated for their activities. All (S)-enantiomers exhibited higher activity than the corresponding (R)-enantiomers. The (S)-enantiomer of 2-chlorobenzylpyridinium-containing aporphine was the most potent inhibitor in this study with an IC50 value of 0.06 ± 0.003 µM. Molecular dynamics simulation analysis revealed that both enantiomers can interact with the AChE binding site, whereas the (S)-enantiomer possessed slightly stronger interaction than the (R)-enantiomer, presumably because of their different orientations, as evidenced by molecular docking. The N-benzylpyridinium dehydroaporphine conjugates were also synthesized but were less active than the corresponding aporphine conjugates.

Novel Potent Autophagy Inhibitor Ka-003 Inhibits Dengue Virus Replication.

Every year, dengue virus (DENV) affects millions of people. Currently, there are no approved drugs for the treatment of DENV infection. Autophagy is a conserved degradation process that was shown to be induced by DENV infection and required for optimal DENV replication. The modulation of autophagy is, therefore, considered an attractive target to treat DENV infection. This study carried out a high-content image screen analysis using Crispr-Cas9 GFP-LC3 knocked-in HeLa cells of a compound library synthesized from or inspired by natural products and their biocongener precursors to discover novel autophagy inhibitors. The screen identified Ka-003 as the most effective compound for decreasing the number of autophagic vacuoles inside cells upon autophagy induction. Ka-003 could inhibit autophagy in a dose-dependent manner at low micromolar concentrations. More importantly, Ka-003 demonstrated the concentration-dependent inhibition of DENV production in Crispr-Cas9 GFP-LC3 knocked-in THP-1 monocytes. The core structure of Ka-003, which is a methyl cyclohexene derivative, resembles those found in mulberry plants, and could be synthetically prepared in a bioinspired fashion. Taken together, data indicate that Ka-003 hampered autophagy and limited DENV replication. The low cytotoxicity of Ka-003 suggests its therapeutic potential, which warrants further studies for the lead optimization of the compound for dengue treatment.

Functionalized Chromans from ortho-Quinone Methides and Arylallenes.

ortho-Quinone methides (o-QMs) underwent formal [4 + 2]-cycloaddition reactions with arylallenes regioselectively at the styrenyl olefin to furnish the corresponding 3-methylene-2-arylchromans in moderate to good yields (up to 88%). When R ≠ H, the reactions also proceeded with moderate stereoselectivity (up to 5:1) which was governed by the nature of the R group. The 3-methylene-2-arylchromans could serve as common intermediates for further functionalization including epoxidation, oxidative cleavage/Baeyer-Villiger oxidation, Riley oxidation, acid-catalyzed rearrangement, and Pd-catalyzed cross-coupling reactions to furnish the corresponding derivatives in moderate to good yields.

Dulcisenes C-E, polyoxygenated cyclohexenes, from Uvaria dulcis dunal and their cytotoxic activity.

Dulcisenes C-E, undescribed polyoxygenated cyclohexenes and twenty-one known compounds were isolated from the dichloromethane extract of the leaves of Uvaria dulcis Dunal. The structures of these undescribed compounds were determined by spectroscopic data analyses, including 1D and 2D NMR, IR, and MS techniques; their absolute configurations were analyzed by NOESY and ECD spectra. Cytotoxicity of sixteen more abundant isolates was evaluated. Cherrevenone and 2',3'-dihydroxy-4',6'-dimethoxychalcone exhibited cytotoxic activity against some cancer cell lines with IC50 values in the range of 3.3-11.8 µM.

Syntheses of 3-Aryl Tetrahydroisoquinolines via an Intermolecular [4 + 2] Cycloaddition of Sultines with Imines.

The development of an intermolecular aza-Diels-Alder (DA) cycloaddition of sultines and imines is reported. By exploiting sultines as o-quinodimethane precursors and aryl imines as dienophiles in the presence of Cu(OTf)2, an aza-DA reaction proceeds to provide a wide variety of 3-aryl tetrahydroisoquionlines in moderate to excellent yield (up to 89%). The synthetic utility of these products was demonstrated in the preparation of tetracyclic N-heterocycles, including a tetrahydroprotoberberine skeleton.

Total Synthesis of Palodesangrens A and C.

Palodesangrens A and C along with the common tetracyclic core are prepared from simple benzaldehyde and acetophenone derivatives in a 10-step longest linear sequence which featured the Diels-Alder reaction forming the cyclohexene moiety, LiAlH4 isomerization, stereoselective acid-catalyzed cyclization forming the chroman moiety, regioselective iodination/vinyl Suzuki cross-coupling reaction, and ring-closing metathesis (RCM) forming the 2H-pyran-2-one. Overall, the desired palodesangrens A and C are obtained in 6.1% and 6.4% yields, respectively.

Roles of autophagy in relation to mitochondrial stress responses of HeLa cells to lamellarin cytotoxicity.

As a promising class of bioactive marine pyrrole alkaloids, lamellarins reportedly act on multiple targets to suppress the vitality of various cancer cell lines. Nevertheless, an in-depth understanding of the molecular mechanisms governing their cytotoxicity is still in demand. Here we report that while activating intrinsic apoptosis, up to 5 µM of lamellarins and their lactam-containing analogs, azalamellarins, also induced mitochondrial stress responses and autophagy in HeLa cervical cancer cells. Detailed characterization of the mitochondria in the treated cells revealed shifted abundance of the two optic atrophy protein 1 (Opa1) isoforms, disturbed morphology, and dissipated membrane potential, leading to PTEN-induced kinase-1 (PINK1) and microtubule-associated protein 1 light chain 3-II (LC3-II) accumulation as a molecular signature of mitophagy. Furthermore, an acute treatment with lamellarins also modulated cellular autophagy flux as evidenced by elevated LC3-II levels, LC3 puncta formation, and p62 degradation. Surprisingly, clustered regularly interspaced short palindromic repeats (CRISPR)-based suppression of autophagy transiently affected the number of apoptotic cells induced by these compounds. Our findings illustrate the potential of these alkaloids for further development into prospective anti-cancer agents.

Substituted 9-Anthraldehydes from Dibenzocycloheptanol Epoxides via Acid-Catalyzed Epoxide Opening/Semipinacol Rearrangement.

Starting from benzaldehyde derivatives, the corresponding dibenzocycloheptenol could be prepared in five steps. Under both substrate (secondary vs tertiary alcohol and the substituents on the aromatic ring(s)) and condition control, the subsequent epoxidation and acid-catalyzed epoxide opening/semipinacol rearrangement/aromatization afforded the corresponding 9-anthraldehydes in good yields, up to 88% over two steps. The presence of the electron-withdrawing group(s) on the aromatic ring(s) suppressed the rate of the epoxidation while the subsequent semipinacol rearrangement step required heating; the presence of the electron-donating group(s), on the other hand, frequently led to the decomposition during the epoxidation. From the mechanistic studies, the semipinacol rearrangement of the epoxide could precede the ionization at the bisbenzylic position, yielding the aldehyde intermediate. The ensuing dehydrative aromatization led to the formation of 9-anthraldehyde. Conversely, nucleophilic addition to the aldehyde and dehydrative aromatization with concomitant loss of formic acid led to anthracene.

An Expeditious Modular Hybrid Strategy for the Diversity-Oriented Synthesis of Lamellarins/Azalamellarins with Anticancer Cytotoxicity.

A modular hybrid strategy has been developed for the diversity-oriented synthesis of lamellarins/azalamellarins. The common pentacyclic pyrrolodihydroisoquinoline lactone/lactam core was formed via the Michael addition/ring closure (Mi-RC) and the copper(I) thiophene-2-carboxylate (CuTC)-catalyzed C-O/C-N Ullmann coupling. Subsequent direct functionalization at C1, DDQ-mediated C5═C6 oxidation, and global deprotection of all benzyl-type O- and N-protecting groups furnished the desired lamellarins/azalamellarins. The late-stage functionalization at C1 provided a handle to accommodate a wider scope of functional groups as they need to tolerate only the DDQ oxidation and global deprotection. Moreover, with the C1-H pyrrole as the late-stage common intermediate, it was also possible to divergently exploit not only its nucleophilic nature to react with some electrophilic species but also some transition-metal-catalyzed cross-coupling reactions (via the intermediacy of the C1-iodopyrrole) to incorporate diversity at this position. Overall, this strategy simplifies the preparation of lamellarins/azalamellarins; including the Mi-RC, these C1-structurally diverse analogues could be prepared efficiently in 6-7 steps from the easily accessed 1-acetoxymethyldihydroisoquinoline and ß-nitrocinnamate. Some selected azalamellarins were evaluated for their inhibitory effect against HeLa cervical cancer cells. An acute induction of intrinsic apoptosis was detected and may lead to growth suppression of or cytotoxicity against cancer cells.

Chemoselective acid-catalyzed [4 + 2]-cycloaddition reactions of ortho-quinone methides and styrenes/stilbenes/cinnamates.

ortho-Quinone methides (o-QMs) generated from the corresponding benzyl acetate precursors chemoselectively underwent the formal [4 + 2]-cycloadditions with the olefin of styrene, stilbene, or cinnamate derivatives by using different transition metal salts or Brønsted acids. Such selectivity was obtained when these olefins either separately acted as the dienophiles or were simultaneously present on the same dienophiles. Complete selectivity was also achieved between the stilbene olefin and acetylene to furnish the key chroman intermediate for the subsequent ring-closing metathesis (RCM), affording the corresponding tetracyclic 5H-dihydronaphtho[1,2-c]chromene.

Atalantiaphyllines A-G, prenylated acridones from Atalantia monophylla DC. and their aromatase inhibition and cytotoxic activities.

Seven previously undescribed acridones, named atalantiaphyllines A-G, along with twenty-six known compounds were isolated from the dichloromethane extracts of roots and stems of Atalantia monophylla DC. Their structures were elucidated by analysis of extensive NMR and HRMS data. Aromatase inhibition, cytotoxicity against MOLT-3, HepG2, A549 and HuCCA-1 cell lines and DPPH radical scavenging activity of these compounds were evaluated. Most of the tested acridones exhibited higher potency in inhibiting aromatase than the positive control, ketoconazole, with IC50 values in the range of 0.08-2.0 µM. In the cytotoxicity assay, cycloataphylline A, N-methylbuxifoliadine E and atalantiaphylline G were selectively cytotoxic against MOLT-3 cell line with IC50 values of 8.0, 5.4 and 9.8 µM, respectively, while only atalaphyllidine exhibited highest antioxidant activity as evaluated by DPPH free radical scavenging assay with an IC50 value of 22.4 µM.

Cefoperazone induces esterase B expression by EstR and esterase B enhances cefoperazone activity at the periplasm.

The occurrence of antibiotic resistance bacteria has become a major threat to public health. We have recently discovered a transcriptional activator that belongs to MarR family, EstR, and an esterase B (EstB) with a newly proposed de-arenethiolase activity from Sphingobium sp. SM42. De-arenethiolase activity involves the removal of the small aromatic side chain of cephalosporin antibiotics as an excellent leaving group by the enzymatic CS bond cleavage. Here, we report the regulation of estB through EstR as an activator in response to a third generation cephalosporin, cefoperazone, antibiotic. Cefoperazone induced the expression of estB in wild type Sphingobium sp., but not in the estR knockout strain, and the induction was restored in the complemented strain. Moreover, we revealed the importance of EstB localization in periplasm. Since EsB has the ability to inactivate selected ß-lactam antibiotics in vitro, it is possible that the enzyme works at the periplasmic space of Gram negative bacteria similar to ß-lactamases. EstB was genetically engineered by incorporating NlpA binding motif, or OmpA signal sequence, or SpyTag-SpyCatcher to the estB gene to mobilize it to different compartments of periplasm; inner membrane, outer membrane, and periplasmic space, respectively. Surprisingly, we found that Sphingobium sp. SM42 and E. coli expressing EstB at the periplasm were more sensitive to cefoperazone. The possible drug enhancement mechanism by enzyme was proposed. This work might lead to a novel strategy to tackle antibiotic resistance problem.

Molecular docking study of lamellarin analogues and identification of potential inhibitors of HIV-1 integrase strand transfer complex by virtual screening.

Molecular docking has been applied to elucidate the binding of lamellarin analogues with HIV-1 integrase strand transfer complex (PDB ID: 5U1C). The results suggest hydrogen bond interaction with residue Glu92 is key, and stabilisation by π-π stacking interactions with DNA base is chiefly influential to strand transfer activity. Other residues involved in hydrogen bonding are Cys65, His67, Asp64, Asp116 and chelation with Mg2+ ion was seen for certain analogues. Furthermore, hydrophobic interactions can be accounted for several amino acids including Asp64, Cys65, Asp116, His67, Glu92, Tyr143, Phe121, Gly118, Pro142 and Val72, as well as the DNA base. The molecular docking results are in line with the reported literatures of other inhibitors and strand transfer activity observed previously by Faulkner. We further employed molecular docking simulation to virtually screen and identified 4 novel potential inhibitors of HIV-1 integrase strand transfer complex from a Chembridge diversity collection of 25,132 small molecule compounds; Chembridge ID compound codes: 22850303, 27553460, 24578440 and 27591056. The candidates clearly formed hydrogen bonding interactions with important residues: His67 and Glu92. In addition, hydrophobic interactions were seen with residues similar to interactions with lamellarin analogues. The calculated drug-like scores are suggestive of these compounds to have clinical potential and ADMET predictions implied of their acceptable pharmacokinetic and toxicity profiles.

Total Synthesis of Palodesangren B Trimethyl Ether and D Dimethyl Ether via a Late-Stage Formation of 2H-Pyran-2-one of the Tetrahydrobenzo[c]pyranochromenone Core.

In four steps from the tricyclic core, palodesangren B trimethyl ether and palodesangren D dimethyl ether could be synthesized in 29 and 18% overall yields, respectively. A reaction sequence comprising the regioselective MgCl2-mediated Casnati-Skattebøl ortho-formylation of phenol, Wittig methylenation, acryloylation, and Ru(II)-catalyzed ring-closing metathesis (RCM) led to the formation of the final 2H-pyran-2-one ring of the desired tetracyclic core.

Stereoselective Convergent Synthesis of Tetrahydro-5 H-benzo[ c]fluorene via Nine-Membered Ring-Closing Metathesis and Transannular Acid-Mediated Cyclization/Nucleophilic Addition.

The diene methyl ethers or acetates, constructed from the Li-Br exchange/addition reactions of 2-vinylbenzaldehydes and 2-(but-3-en-1-yl)bromoarenes followed by etherification or acetylation of the corresponding alcohols, smoothly underwent the ring-closing metathesis (RCM) by using Hoveyda-Grubbs II as a catalyst to provide the corresponding benzannulated ( Z)-cyclononenes as single products in good yields (up to 75%). The ensuing one-pot acid-mediated transannular cyclization/nucleophilic addition at C7 furnished the corresponding tetrahydro-5 H-benzo[ c]fluorenes as single stereoisomers with the exclusive cis stereochemistry at the ring junction (C5-C6) and trans at the site of nucleophilic attack (C6-C7) on the three contiguous stereogenic centers in good to excellent yield (up to 94%). The developed strategy was general; the reaction conditions were compatible with hydride, azide, and electron-rich aromatics as nucleophiles. In addition, various methoxylated benzannulated cyclononene acetates could be employed as substrates. Thus, tetrahydro-5 H-benzo[ c]fluorenes could be prepared in four steps from appropriately substituted bromoarenes and benzaldehydes in good yields (up to 56%) with excellent stereo- and regio-control.

The esterase B from Sphingobium sp. SM42 has the new de-arenethiolase activity against cephalosporin antibiotics.

The esterase B (EstB) from Sphingobium sp. SM42, which was previously reported to be active towards dibutyl phthalate, can cleave some small aromatic ring side chains from cephalosporin derivatives. A new name, de-arenethiolase, has been proposed to represent this activity. We present the in vitro characterization of the activity of purified EstB toward cephalosporin substrates. Interestingly, EstB was highly active against cefoperazone and cefazolin resulting in 83 and 67% decreases in killing zone diameter, respectively. EstB also demonstrated a moderate activity towards ceftriaxone (18%) and cefotaxime (16%) while exhibiting no activity against cephalosporin C and cefixime. HPLC analysis indicated that EstB catalyzed the cleavage of the C-S bond found in cephalosporin derivatives to release the corresponding free aromatic ring side chains.

Selective Divergent Synthesis of Indanols, Indanones, and Indenes via Acid-Mediated Cyclization of ( Z)- and ( E)-(2-Stilbenyl)methanols and Its Application for the Synthesis of Paucifloral F Derivatives.

Starting from bromo/iodobenzaldehyde derivatives, the corresponding ( Z)- and ( E)-(2-stilbenyl)methanols could be prepared in 2-5 steps via Pd-catalyzed cross-coupling reactions (Sonogashira and Heck reactions) followed by aryllithium/aryl Grignard addition. For the ( E)-stilbenes, subsequent acid-mediated cyclization using p-TsOH immobilized on silica (PTS-Si) at low temperatures furnished the 2,3- trans-1-indanols with complete stereocontrol at the C2-C3. Further oxidization of the alcohol provided the indanones, which are structurally related to the natural product paucifloral F. At higher temperatures, 1,2- and 2,3-disubstituted indenes could be selectively prepared in good to excellent yields. On the other hand, the ( Z)-stilbenes, under similar conditions (PTS-Si), did not give the indanols; only the 1,2-disubstituted indenes could be obtained. To gain further insights into the stereochemistry at C2-C3 for the ( Z)-stilbenes, hydride or azide was employed as a nucleophile; the corresponding indane products were obtained with the cis stereochemistry at the C2-C3. Thus, the ( Z)- or ( E)-olefin geometry of the substrate directed the stereoselective indanyl cyclization to furnish the cis or trans at the C2-C3 ring junction, respectively, while reaction conditions controlled the selectivity of the product types.

Bioinspired Diastereoconvergent Synthesis of the Tricyclic Core of Palodesangrens via Diels-Alder Reaction, LiAlH4-Mediated Isomerization, and Acid-Mediated Cyclization.

The cyclohexene moiety of the tricyclic 6,7-diaryl-tetrahydro-6 H-benzo[ c]chromene core of palodesangrens could be assembled in a biomimetic and step-economical fashion by the Diels-Alder reaction between the electron-rich ( E)-1,3-butadienylarenes as the diene and the electron-deficient chalcones as the dienophile. During the reduction of ketone to the corresponding alcohol by LiAlH4, the mixture of endo and exo isomers underwent a novel diastereoconvergent LiAlH4-mediated isomerization to install the desired stereochemistry at C10a. Subsequent pyran ring closure under acidic conditions installed the stereochemistry at the remaining C6. Overall, the tricyclic core of palodesangrens could be prepared in three steps and up to 38% yield.