Regiodivergent biosynthesis of bridged bicyclononanes.

Medicinal compounds from plants include bicyclo[3.3.1]nonane derivatives, the majority of which are polycyclic polyprenylated acylphloroglucinols (PPAPs). Prototype molecules are hyperforin, the antidepressant constituent of St. John's wort, and garcinol, a potential anticancer compound. Their complex structures have inspired innovative chemical syntheses, however, their biosynthesis in plants is still enigmatic. PPAPs are divided into two subclasses, named type A and B. Here we identify both types in Hypericum sampsonii plants and isolate two enzymes that regiodivergently convert a common precursor to pivotal type A and B products. Molecular modelling and substrate docking studies reveal inverted substrate binding modes in the two active site cavities. We identify amino acids that stabilize these alternative binding scenarios and use reciprocal mutagenesis to interconvert the enzymatic activities. Our studies elucidate the unique biochemistry that yields type A and B bicyclo[3.3.1]nonane cores in plants, thereby providing key building blocks for biotechnological efforts to sustainably produce these complex compounds for preclinical development.

Compound-Specific Isotope Analysis Provides Direct Evidence for Identifying the Source of Residual Pesticides Diazinon and Procymidone in the Soil-Plant System.

Compound-specific isotope analysis stands as a promising tool for unveiling the behavior of pesticides in agricultural environments. Using the commercial formulations of persistent fungicide procymidone (PRO) and less persistent insecticide diazinon (DIA), respectively, we analyzed the concentration and carbon isotope composition (δ13C) of the residual pesticides through soil incubation experiments in a greenhouse (for 150 days) and lab conditions (for 50-70 days). Our results showed that the magnitude of δ13C variation depends on pesticide specificity, in which PRO in the soil exhibited little variation in δ13C values over the entire incubation times, while DIA demonstrated an increased δ13C value, with the extent of δ13C variability affected by different spiking concentrations, plant presence, and light conditions. Moreover, the pesticides extracted from soils were isotopically overlapped with those from crop lettuce. Ultimately, the isotope composition of pesticides could infer the degradation and translocation processes and might contribute to identifying the source(s) of pesticide formulation in agricultural fields.

Efficacy and safety of add-on mirogabalin to conventional therapy for the treatment of peripheral neuropathic pain after thoracic surgery: the multicenter, randomized, open-label ADMIT-NeP study.

BACKGROUND: For chronic pain after thoracic surgery, optimal timing of its diagnosis and effective treatment remains unresolved, although several treatment options are currently available. We examined the efficacy and safety of mirogabalin, in combination with conventional pain therapy (nonsteroidal anti-inflammatory drugs and/or acetaminophen), for treating peripheral neuropathic pain (NeP) after thoracic surgery. METHODS: In this multicenter, randomized, open-label, parallel-group study, patients with peripheral NeP were randomly assigned 1:1 to mirogabalin as add-on to conventional therapy or conventional treatment alone. RESULTS: Of 131 patients of consent obtained, 128 were randomized (mirogabalin add-on group, 63 patients; conventional treatment group, 65 patients). The least squares mean changes (95% confidence interval [CI]) in Visual Analogue Scale (VAS) score for pain intensity at rest from baseline to Week 8 (primary endpoint) were - 51.3 (- 54.9, - 47.7) mm in the mirogabalin add-on group and - 47.7 (- 51.2, - 44.2) mm in the conventional group (between-group difference: - 3.6 [95% CI: - 8.7, 1.5], P = 0.161). However, in patients with Self-administered Leeds Assessment of Neuropathic Symptoms and Signs (S-LANSS) score (used for the screening of NeP) ≥ 12 at baseline, the greater the S-LANSS score at baseline, the greater the decrease in VAS score in the mirogabalin add-on group, while no such trend was observed in the conventional treatment group (post hoc analysis). This between-group difference in trends was statistically significant (interaction P value = 0.014). Chronic pain was recorded in 7.9% vs. 16.9% of patients (P = 0.171) at Week 12 in the mirogabalin add-on vs. conventional treatment groups, respectively. Regarding activities of daily living (ADL) and quality of life (QOL), changes in Pain Disability Assessment Scale score and the EQ-5D-5L index value from baseline to Week 8 showed significant improvement in the mirogabalin add-on group vs. conventional treatment group (P < 0.001). The most common adverse events (AEs) in the mirogabalin add-on group were dizziness (12.7%), somnolence (7.9%), and urticaria (3.2%). Most AEs were mild or moderate in severity. CONCLUSIONS: Addition of mirogabalin to conventional therapy did not result in significant improvement in pain intensity based on VAS scores, but did result in significant improvement in ADL and QOL in patients with peripheral NeP after thoracic surgery. TRIAL REGISTRATION: Japan Registry of Clinical Trials jRCTs071200053 (registered 17/11/2020).

Discovery of a novel anti-osteoporotic agent from marine fungus-derived structurally diverse sirenins.

Thirteen new sirenin derivatives named eupenicisirenins C-O (1-13), along with a biosynthetically related known one (14), were isolated from the mangrove sediment-derived fungus Penicillium sp. SCSIO 41410. The structures, which possessed a rare cyclopropane moiety, were confirmed by extensive analyses of the spectroscopic data, quantum chemical calculations, and X-ray diffraction. Among them, eupenicisirenin C (1) exhibited the strongest NF-κB inhibitory activities, as well as suppressing effects on cGAS-STING pathway. Moreover, 1 showed the significant inhibitory effect on RANKL-induced osteoclast differentiation in bone marrow macrophages cells, and also displayed the therapeutic potential on prednisolone-induced zebrafish osteoporosis. Transcriptome analysis and the following verification tests suggested that its anti-osteoporotic mechanism is related to the extracellular matrix receptor interaction-related pathways. This study provided a promising marine-derived anti-osteoporotic agent for the treatment of skeletal disease.

Cytotoxic and Antioxidant Activity of Hypericum perforatum L. Extracts against Human Melanoma Cells from Different Stages of Cancer Progression, Cultured under Normoxia and Hypoxia.

Oxidative stress and the hypoxic microenvironment play a key role in the progression of human melanoma, one of the most aggressive skin cancers. The aim of our study was to evaluate the effect of Hypericum perforatum extracts of different origins (both commercially available (HpEx2) and laboratory-prepared from wild grown (HpEx12) and in vitro cultured (HpEx13) plants) and hyperforin salt on WM115 primary and WM266-4 lymph node metastatic human melanoma cells cultured under normoxic and hypoxic conditions. The polyphenol content, radical scavenging activity, and hyperforin concentration were determined in the extracts, while cell viability, apoptosis, ROS production, and expression of NRF2 and HO-1, important oxidative stress-related factors, were analyzed after 24 h of cell stimulation with HpExs and hyperforin salt. We found that cytotoxic, pro-apoptotic and antioxidant effects depend on the extract composition, the stage of melanoma progression, and the oxygen level. Hyperforin salt showed lower activity than H. perforatum extracts. Our study for the first time showed that the anticancer activity of H. perforatum extracts differs in normoxia and hypoxia. Importantly, the composition of extracts of various origins, including in vitro cultured, resulting in their unique properties, may be important in the selection of plants for therapeutic application.

Hyperforin: A natural lead compound with multiple pharmacological activities.

Hypericum perforatum L. (Clusiaceae), commonly known as St. John's wort, has a rich historical background as one of the oldest and most widely studied herbal medicines. Hyperforin is the main antidepressant active ingredient of St. John's wort. In recent years, hyperforin has attached increasing attention due to its multiple pharmacological activities. In this review, the information on hyperforin was systematically summarized. Hyperforin is considered to be a lead compound with diverse pharmacological activities including anti-depression, anti-tumor, anti-dementia, anti-diabetes and others. It can be obtained by extraction and synthesis. Further pharmacological studies and more precise detection methods will help develop a value for hyperforin. In addition, structural modification and pharmaceutical preparation technology will be beneficial to promoting the research progress of hyperforin based innovative drugs. Although these works are full of known and unknown challenges, researchers are still expected to make hyperforin play a greater value.

Identification of bicyclic compounds that act as dual inhibitors of Bcl-2 and Mcl-1.

Elevated expression of anti-apoptotic proteins, such as Bcl-2 and Mcl-1 contributes to poor prognosis and resistance to current treatment modalities in multiple cancers. Here, we report the design, synthesis and characterization of benzimidazole chalcone and flavonoid scaffold-derived bicyclic compounds targeting both Bcl-2 and Mcl-1 by optimizing the structural differences in the binding sites of both these proteins. Initial docking screen of Bcl-2 and Mcl-1 with pro-apoptotic protein Bim revealed possible hits with optimal binding energies. All the optimized bicyclic compounds were screened for their in vitro cytotoxic activity against two oral cancer cell lines (AW8507 and AW13516) which express high levels of Bcl-2 and Mcl-1. Compound 4d from the benzimidazole chalcone series and compound 6d from the flavonoid series exhibited significant cytotoxic activity (IC50 7.12 µM and 17.18 µM, respectively) against AW13516 cell line. Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) analysis further demonstrated that compound 4d and compound 6d could effectively inhibit the Bcl-2 and Mcl-1 proteins by displacing their BH3 binding partners. Both compounds exhibited potent activation of canonical pathway of apoptosis evident from appearance of cleaved Caspase-3 and PARP. Further, treatment of oral cancer cells with the inhibitors induced dissociation of the BH3 only protein Bim from Mcl-1 and Bak from Bcl-2 but failed to release Bax from Bcl-xL thereby confirming the nature of compounds as BH3-mimetics selectively targeting Bcl-2 and Mcl-1. Our study thus identifies bicyclic compounds as promising candidates for anti-apoptotic Bcl-2/Mcl-1 dual inhibitors with a potential for further development.

Cascade synthetic strategies opening access to medicinal-relevant aliphatic 3- and 4-membered N-heterocyclic scaffolds.

Cascade reactions are often 'employed' by nature to construct structurally diverse nitrogen-containing heterocycles in a highly stereoselective fashion, i.e., secondary metabolites important for pharmacy. Nitrogen-containing heterocycles of three- and four-membered rings, as standalone and bicyclic compounds, inhibit different enzymes and are pharmacophores of approved drugs or drug candidates considered in many therapies, e.g. anticancer, antibacterial or antiviral. Domino transformations are in most cases in line with modern green chemistry concepts due to atom economy, one-pot procedures often without use the protective groups, time-saving and at markedly lower costs than multistep transformations. The tandem approaches can help to obtain novel N-heterocyclic scaffolds, functionalized according to structural requirements of the target in cells, taking into account the nature of functional group and stereochemistry. On the other hand cascade strategies allow to modify small N-heterocyclic rings in a systematic way, which is beneficial for structure-activity relationship (SAR) analyses. This review is focused on the biological relevance of the N-heterocyclic scaffolds with smaller 3- and 4-membered rings among approved drugs and leading structures of drug candidates. The cascade synthetic strategies offering N-heterocyclic scaffolds, at relatively good yields and high stereoselectivity, are discussed here. The review covers mainly years from 2015 to 2021.

Direct Detection of Glutathione Biosynthesis, Conjugation, Depletion and Recovery in Intact Hepatoma Cells.

Nuclear magnetic resonance (NMR) spectroscopy was used to monitor glutathione metabolism in alginate-encapsulated JM-1 hepatoma cells perfused with growth media containing [3,3'-13C2]-cystine. After 20 h of perfusion with labeled medium, the 13C NMR spectrum is dominated by the signal from the 13C-labeled glutathione. Once 13C-labeled, the high intensity of the glutathione resonance allows the acquisition of subsequent spectra in 1.2 min intervals. At this temporal resolution, the detailed kinetics of glutathione metabolism can be monitored as the thiol alkylating agent monobromobimane (mBBr) is added to the perfusate. The addition of a bolus dose of mBBr results in rapid diminution of the resonance for 13C-labeled glutathione due to a loss of this metabolite through alkylation by mBBr. As the glutathione resonance decreases, a new resonance due to the production of intracellular glutathione-bimane conjugate is detectable. After clearance of the mBBr dose from the cells, intracellular glutathione repletion is then observed by a restoration of the 13C-glutathione signal along with wash-out of the conjugate. These data demonstrate that standard NMR techniques can directly monitor intracellular processes such as glutathione depletion with a time resolution of approximately < 2 min.

The Evidence for Effective Inhibition of INa Produced by Mirogabalin ((1R,5S,6S)-6-(aminomethyl)-3-ethyl-bicyclo [3.2.0] hept-3-ene-6-acetic acid), a Known Blocker of CaV Channels.

Mirogabalin (MGB, Tarlige®), an inhibitor of the α2δ-1 subunit of voltage-gated Ca2+ (CaV) channels, is used as a way to alleviate peripheral neuropathic pain and diabetic neuropathy. However, to what extent MGB modifies the magnitude, gating, and/or hysteresis of various types of plasmalemmal ionic currents remains largely unexplored. In pituitary tumor (GH3) cells, we found that MGB was effective at suppressing the peak (transient, INa(T)) and sustained (late, INa(L)) components of the voltage-gated Na+ current (INa) in a concentration-dependent manner, with an effective IC50 of 19.5 and 7.3 µM, respectively, while the KD value calculated on the basis of minimum reaction scheme was 8.2 µM. The recovery of INa(T) inactivation slowed in the presence of MGB, although the overall current-voltage relation of INa(T) was unaltered; however, there was a leftward shift in the inactivation curve of the current. The magnitude of the window (INa(W)) or resurgent INa (INa(R)) evoked by the respective ascending or descending ramp pulse (Vramp) was reduced during cell exposure to MGB. MGB-induced attenuation in INa(W) or INa(R) was reversed by the further addition of tefluthrin, a pyrethroid insecticide known to stimulate INa. MGB also effectively lessened the strength of voltage-dependent hysteresis of persistent INa in response to the isosceles triangular Vramp. The cumulative inhibition of INa(T), evoked by pulse train stimulation, was enhanced in its presence. Taken together, in addition to the inhibition of CaV channels, the NaV channel attenuation produced by MGB might have an impact in its analgesic effects occurring in vivo.

Inflammation Regulation via an Agonist and Antagonists of α7 Nicotinic Acetylcholine Receptors in RAW264.7 Macrophages.

The α7 nicotinic acetylcholine receptor (nAChR) is widely distributed in the central and peripheral nervous systems and is closely related to a variety of nervous system diseases and inflammatory responses. The α7 nAChR subtype plays a vital role in the cholinergic anti-inflammatory pathway. In vivo, ACh released from nerve endings stimulates α7 nAChR on macrophages to regulate the NF-κB and JAK2/STAT3 signaling pathways, thereby inhibiting the production and release of downstream proinflammatory cytokines and chemokines. Despite a considerable level of recent research on α7 nAChR-mediated immune responses, much is still unknown. In this study, we used an agonist (PNU282987) and antagonists (MLA and α-conotoxin [A10L]PnIA) of α7 nAChR as pharmacological tools to identify the molecular mechanism of the α7 nAChR-mediated cholinergic anti-inflammatory pathway in RAW264.7 mouse macrophages. The results of quantitative PCR, ELISAs, and transcriptome analysis were combined to clarify the function of α7 nAChR regulation in the inflammatory response. Our findings indicate that the agonist PNU282987 significantly reduced the expression of the IL-6 gene and protein in inflammatory macrophages to attenuate the inflammatory response, but the antagonists MLA and α-conotoxin [A10L]PnIA had the opposite effects. Neither the agonist nor antagonists of α7 nAChR changed the expression level of the α7 nAChR subunit gene; they only regulated receptor function. This study provides a reference and scientific basis for the discovery of novel α7 nAChR agonists and their anti-inflammatory applications in the future.

Mirogabalin alleviates nociceptive hypersensitivity without causing sedation in a mouse model of post-traumatic trigeminal neuropathy.

Post-traumatic trigeminal neuropathy (PTTN) is a chronic sensory disorder that afflicts patients with nerve injury caused by orofacial and dental surgery or cervicofacial trauma. Currently, effective treatment strategies for PTTN are lacking, and patients treated with conventional drugs for PTTN experience adverse effects such as drowsiness and drug addiction. In the present study, we investigated whether mirogabalin, a novel gabapentinoid, could be an effective treatment for PTTN induced by distal infraorbital nerve chronic constriction injury (dIoN-CCI) in the mouse. Increased facial grooming time and hyper-responsiveness to acetone were observed in dIoN-CCI mice. These pain-related behaviors were attenuated by intraperitoneal injection of mirogabalin. In particular, mirogabalin significantly diminished the increase in facial grooming time. The analgesic effect of mirogabalin injection started 45 min after the injection and persisted for 6 h. Additionally, 10 mg/kg mirogabalin did not affect locomotor activity in the open field test, suggesting that it does not cause sedation. Together, the current findings suggest that mirogabalin could be a valuable therapeutic drug for PTTN following orofacial surgeries without sedative side effects.

Assessment of Ferula hermonis Boiss fertility effects in immature female rats supported by quantification of ferutinin via HPLC and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: Ferula hermonis is a small shrub renowned for its aphrodisiac abilities. Middle East herbalists have utilized Ferula hermonis seed and root as an aphrodisiac folk medicine to treat women's frigidity and male erectile and sexual dysfunction. AIM OF THE STUDY: Assessment of follicle-stimulating hormone-like (FSH), luteinizing hormone-like (LH), and estrogenic activities of the methanolic extract (ME) of the roots of Ferula hermonis on female reproductive function. MATERIALS AND METHODS: The methanolic extract was prepared from the root of F. hermonis and studied at dose level 6 mg/kg in immature female rats for FSH-like, LH-like, and estrogenic activities. These activities were determined by analyzing gross anatomical features, relative organ weight, and serum level of FSH, LH, progesterone and estrogen hormones, and histopathological characteristics. Quantification of the main phytoestrogenic component ferutinin carried out by HPLC. In addition, molecular docking for the binding affinity of ferutinin inside active sites of both estrogen receptor alpha (ERα) and FSH receptor (FSHR) was performed to predict the potential role of ferutinin in regulating the female reproductive process. RESULTS: Ferula hermonis (ME) showed potent FSH-like, LH-like activities and moderate estrogenic effect at the dose of 6 mg/kg. The content of ferutinin in F. hermonis was estimated to be 92 ± 1.33 mg/g of the methanolic extract. Molecular docking of ferutinin with ERα and FSHR displayed strong interaction with target proteins. CONCLUSIONS: Based on results, it can be concluded that Ferula hermonis can be considered as a suitable female fertility improving agent.

LY395756 promotes NR2B expression via activation of AKT/CREB signaling in the juvenile methylazoxymethanol mice model of schizophrenia.

INTRODUCTION: Synaptic N-methyl-d-aspartate receptor subtype 2B(NR2B) is significantly reduced in prefrontal cortex (PFC) in the neurodevelopmental methylazoxymethanol (MAM) model of schizophrenia (SCZ). Recent research has shown that LY395756 can effectively restore NR2B levels and improve cognitive performance in juvenile MAM mice model. However, the underlying mechanisms of these beneficial effects remain unclear. MATERIALS AND METHODS: Juvenile MAM mice model of SCZ is used in our study. Synaptic membrane protein levels were examined by western blotting under different treatment conditions. Interaction of cAMP-response element binding protein (CREB) and the promoter of NR2B was detected by the chromatin immunoprecipitation (ChIP) assay. Further examination of signaling pathway that mediates NR2B expression was also investigated by western blotting. RESULTS: In the PFC of the juvenile MAM mice schizophrenia model, CREB was found to directly bind with the promoter of NR2B. LY395756 activated the phosphorylation of AKT. Phosphorylated AKT subsequently induced the phosphorylation of CREB, and the activated CREB promoted the expression of NR2B. Subsequent experiments showed that the dephosphorylation of CREB induced by protein phosphatase 1 (PP1) can inhibit NR2B levels. Taken together, these findings support that the AKT/CREB signaling pathway is essential for the promoting effect of LY395756 on synaptic NR2B in PFC in juvenile MAM mice SCZ model. CONCLUSIONS: Our investigation has identified a novel mechanism by which LY395756 increases NR2B expression in juvenile MAM mice SCZ model. The AKT/CREB signaling pathway warrants further research as a potential direction for clinical treatment of SCZ.

Investigating Bicyclobutane-Triazolinedione Cycloadditions as a Tool for Peptide Modification.

Acyl bicyclobutanes are shown to engage in strain-promoted cycloaddition reactions with a diverse array of triazolinedione reagents. The synthesis of an orthogonally protected urazole building block enabled the facile preparation of amino acid- and peptide-derived triazolinediones that undergo cycloaddition reactions to afford novel peptide conjugates. The additive-free and fully atom-economical nature of the transformation is a promising starting point for the generalization of this cycloaddition reaction for the functionalization of biomolecules.

Bicyclic Picomolar OGA Inhibitors Enable Chemoproteomic Mapping of Its Endogenous Post-translational Modifications.

Owing to its roles in human health and disease, the modification of nuclear, cytoplasmic, and mitochondrial proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) has emerged as a topic of great interest. Despite the presence of O-GlcNAc on hundreds of proteins within cells, only two enzymes regulate this modification. One of these enzymes is O-GlcNAcase (OGA), a dimeric glycoside hydrolase that has a deep active site cleft in which diverse substrates are accommodated. Chemical tools to control OGA are emerging as essential resources for helping to decode the biochemical and cellular functions of the O-GlcNAc pathway. Here we describe rationally designed bicyclic thiazolidine inhibitors that exhibit superb selectivity and picomolar inhibition of human OGA. Structures of these inhibitors in complex with human OGA reveal the basis for their exceptional potency and show that they extend out of the enzyme active site cleft. Leveraging this structure, we create a high affinity chemoproteomic probe that enables simple one-step purification of endogenous OGA from brain and targeted proteomic mapping of its post-translational modifications. These data uncover a range of new modifications, including some that are less-known, such as O-ubiquitination and N-formylation. We expect that these inhibitors and chemoproteomics probes will prove useful as fundamental tools to decipher the mechanisms by which OGA is regulated and directed to its diverse cellular substrates. Moreover, the inhibitors and structures described here lay out a blueprint that will enable the creation of chemical probes and tools to interrogate OGA and other carbohydrate active enzymes.

Measurement of S-glutathionylated proteins by HPLC.

S-glutathionylated proteins (GSSP), i.e., protein-mixed disulfides with glutathione (GSH), are considered a suitable biomarker of oxidative stress. In fact, they occur within cells at low level and their concentration increases markedly under pro-oxidant conditions. Plasma is something different, since it is physiologically rich in S-thiolated proteins (RSSP), i.e., protein-mixed disulfides with various types of low molecular mass thiols (LMM-SH). However, albumin, which is largely the most abundant plasma protein, possesses a cysteine residue at position 34 that is mostly reduced (about 60%) under physiological conditions, but easily involved in the formation of additional RSSP in the presence of oxidants. The quantification of GSSP requires special attention to sample handling, since their level can be overestimated as a result of artefactual oxidation of GSH. We have developed the present protocol to avoid this methodological problem. Samples should be treated as soon as possible after their collection with the alkylating agent N-ethylmaleimide that masks -SH groups and prevents their oxidation. The GSH released from mixed disulfides by reduction with dithiothreitol is then labeled with the fluorescent probe monobromobimane and quantified by HPLC. The method can be applied to many different biological samples, comprising blood components, red blood cell plasma membrane, cultured cells, and solid organs from animal models.

Terpestacin, a toxin produced by Phoma exigua var. heteromorpha, the causal agent of a severe foliar disease of oleander (Nerium oleander L.).

Since 1987, several cytochalasins were isolated from Phoma exigua var. heteromorpha, the causal agent of foliar blight disease of oleander (Nerium oleander L.), and chemically and biologically characterised. During the purification process of a large-scale production of cytochalasins A and B, necessary to continue the study on their anticancer activity, a metabolite having a different carbon skeleton compared to that of cytochalasans, was isolated. It was identified as terpestacin, a well-known toxic fungal stestertepenoid, isolated for the first time from P. exigua var. heteromorpha, by spectroscopic investigation (essentially 1D and 2D 1H and 13C-NMR and ESI MS) and optical methods in comparison with the literature data. Terpestacin and some its derivatives (including a natural one, fusaproliferin) were prepared and tested for their biological activity. Terpestacin and fusaproliferin had some inhibitory effects on seed germination of Phelipanche ramosa, whereas none of the compounds caused phytotoxic effects on weed leaves.[Formula: see text].

Reassignment of crispatene, isolation and chemical characterization of stachydrine, isolated from the marine mollusk Elysia crispata.

The molluscan genus Elysia Risso, 1818 (Sacoglossa) is composed of shell-less herbivore sea slugs. From these marine organisms, polyketides such as polypropynates have been isolated and showed cytotoxic, antibiotic, and antifungal, and antiviral properties. In this work, we reported the isolation, and structure elucidation of two compounds isolated from marine mollusk E. crispata. Both compounds isolated, crispatene (1) and stachydrine (2), were purified by HPLC. The chemical structure of compound (1) was reassigned through 1D and 2D NMR experiments and high-resolution electrospray ionization mass spectrometry (HRESIMS). On the other hand, this is the first time that compound (2) has been found in this species of mollusk or the marine environment, previously, (2) has only been found in terrestrial plants or bacteria in symbiosis with plants.

Synthesis of sp3-rich chiral bicyclo[3.3.1]nonanes for chemical space expansion and study of biological activities.

Chiral sp3-rich bicyclo[3.3.1]nonane scaffolds 10-12 were synthesized as single diastereomers from aldehyde 9, which was prepared from 4,4-dimethoxycyclohexa-2,5-dienone through a copper-catalyzed enantioselective reduction. Three different types of intramolecular addition reactions were studied: SmI2-mediated reductive cyclization, base-promoted aldol reaction, and one-pot Mannich reaction. We succeeded in introducing three side-chains to scaffold 11 and construct an sp3-rich compound library in both enantiomeric variants by simply changing the chirality of the ligands. The biological evaluation revealed that all synthesized compounds exhibited a concentration-dependent inhibition of hypoxia-inducible factor-1 (HIF-1) transcriptional activity, with IC50 values in the range of 17.2-31.7 µM, whereas their effects on cell viability were varied (IC50 = 3.5 to > 100 µM). The most active compound 16f inhibits the accumulation of HIF-1α protein and mRNA in hypoxia, indicating that it has a mechanism of action distinctly different from other known compounds bearing the common bicyclo[3.3.1]nonane skeleton.