Discovery of antitumor diterpenoids from Casearia graveolens targeting VEGFR-2 to inhibit angiogenesis.

Eight novel clerodane diterpenoids (1-8) were isolated from the twigs of Casearia graveolens. Their structures were elucidated through comprehensive nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), and electronic circular dichroism (ECD) analyses. In addition to structural determination, surface plasmon resonance (SPR) assays were conducted to investigate molecular interactions, revealing that compound 8 exhibited high affinity for vascular endothelial growth factor receptor 2 (VEGFR2), a key regulator of tumor angiogenesis. Subsequent in vivo experiments demonstrated that compound 8 effectively inhibited angiogenesis and displayed significant antitumor activity by suppressing tumor proliferation and metastasis in zebrafish xenograft models. These findings suggest that compound 8 holds promise as an anticancer lead compound targeting VEGFR-2 to obstruct tumor angiogenesis.

Structurally diverse ent-clerodanoids from the aerial parts of Isodon scoparius.

Scoparodane C (1), a diterpenoid with a rare 3,4-seco-3-nor-2,11-epoxy-ent-clerodane scaffold, was obtained from the aerial parts of Isodon scoparius, along with isocopariusines A-E (2-6), five ent-clerodanoids featuring a 5/6-fused ring system, and isocopariusines F-H (7-9), three common ent-clerodanoids. The structures of these previously undescribed compounds were established by a combination of spectroscopic analysis, X-ray diffraction, chemical derivatization, and quantum chemical calculation. Remarkably, isocopariusine B (3) showed strong resistance reversal activity against fluconazole-resistant Candida albicans.

Neo-5,10-seco-clerodane diterpenoids from Schnabelia terniflora.

Three new neo-5,10-seco-clerodane diterpenoids (1-3), four previously undescribed ethoxy/methoxy acetal analogues (4-7), one new etherified labdane diterpenoid (8), and seven known diterpenoids (9-15) were isolated from the whole plant of Schnabelia terniflora. Their structures were established on the basis of extensive spectroscopic analysis, single-crystal X-ray diffraction data, calculated electronic circular dichroism (ECD), and Mo2(OAc)4-induced circular dichroism. Compounds 2 and 3 represent the first examples of neo-5,10-seco-clerodane diterpenoids containing a 1H-pyrrole-2,5-dione and a pyrrolidine-2,5-dione moiety, respectively. A plausible biosynthetic pathway for 1-3 is proposed. All diterpenoids were evaluated for their cytotoxic activity against non-small-cell lung cancer lines (A549 and H460) and gastric cancer lines (HGC27 and AGS). Among them, 2 and 14 showed moderate cytotoxicity against four cell lines.

Discovery of Novel Neo-Clerodane Derivatives as Potent Dual-Functional Antiosteoporosis Agents through Targeting Peroxisome Proliferator-Activated Receptor-γ.

A library of 31 natural neo-clerodanes isolated from Ajuga decumbens was assayed for antiosteoporosis. This results in 18 neo-clerodane osteoclastogenesis inhibitors, and compound 3 prevents bone loss in vivo. Further mechanistic studies demonstrated that these compounds inhibit osteoporosis by antagonizing peroxisome proliferator-activated receptor-γ (PPARγ). We designed and synthesized 17 compounds by chemically modifying the natural neo-clerodane 19 (highly potent and the major composition of A. decumbens extract) by means of structure-based drug design techniques. Among these neo-clerodane derivatives, compound 34 is the most potent osteoporosis inhibitor with a 46-fold improvement in inhibiting osteoclastogenesis (IC50 = 0.042 vs 1.92 µM), 11-fold increased activity in PPARγ antagonism (EC50 = 0.75 vs 8.35 µM), 66-fold enhancement in receptor affinity (KD = 0.27 vs 17.7 µM), and enhanced osteogenic promotion compared to 19. This underscores the potential of neo-clerodane diterpenoids as promising leads for osteoporosis treatment by targeting PPARγ.

Salvinorin A ameliorates pilocarpine-induced seizures by regulating hippocampal microglia polarization.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia divinorum (Epling and Játiva) is a psychoactive plant traditionally used by the Latinos for various medicinal purposes. Salvinorin A (Sal A), the main bioactive constituent of S. divinorum, is a natural highly selective kappa opioid receptor (KOR) agonist. Considering the anti-inflammatory effect of S. divinorum and endogenous hippocampal dynorphin/kappa opioid receptor (KOR) system playing an anticonvulsant function, we hypothesis that Sal A can be a potential candidate to treat epilepsy. Here, we identified whether Sal A ameliorated epileptic seizures and neuronal damages in animal model and in vitro model and investigated its underlying mechanisms. MATERIALS AND METHODS: Mice epilepsy model was induced by pilocarpine following seizures assessed by Racine classification. Hippocampus tissues were obtained for genetic, protein, and histological investigation. Furthermore, lipopolysaccharide (LPS)-activated BV2 microglial cells were utilized to validate the anti-inflammatory and microglia polarization regulation effects of Sal A. RESULTS: Sal A treatment significantly prolonged the latency to status epileptics (SE) and shortened the duration of SE in the pilocarpine-induced model. It also alleviated neuronal damages via activation of the AMPK/JNK/p-38 MAPK pathway and inhibition of apoptosis-related protein in hippocampus tissues. Furthermore, Sal A dose-dependently reduced microglia-mediated expression of pro-inflammatory cytokines and increased anti-inflammatory factors levels in SE mice and LPS-activated BV2 microglial cells by regulating microglia polarization. In addition, the effect of Sal A in vitro was totally blocked by KOR antagonist nor-BNI. CONCLUSION: Sal A treatment protects against epileptic seizures and neuronal damages in pilocarpine-induced models by suppressing the inflammation response through regulating microglial M1/M2 polarization. This study might serve as a theoretical basis for clinical applications of Sal A and its analogs and provide a new insight into the development of anti-seizure drugs.

Targeted discovery of clerodane diterpenoids from Tinospora sinensis as immunomodulatory agents.

Under the guidance of MS/MS-based molecular networking, five new clerodane diterpenoid glucosides, tinosinesides R-V (1-5), along with 15 known diterpenoids (6-20), were isolated from the stems of Tinospora sinensis. Compound 1 represents the first example of diterpenoid bearing a thio sugar and compound 5 is the first 18,19-dinor-clerodane with cis-fused A/B ring. The structures of the new compounds were elucidated by spectroscopic means, and their absolute configurations were established on the basis of time-dependent density functional theory (TD-DFT) based electronic circular dichroism (ECD) calculation and chemical methods. Selected compounds were evaluated for their immunomodulatory effect and several compounds could enhance the proliferation of B lymphocytes. Preliminary mechanistic studies disclosed that 3 could promote B cell generation and inhibit B cell differentiation.

Discovery of Novel, Selective, and Nonbasic Agonists for the Kappa-Opioid Receptor Determined by Salvinorin A-Based Virtual Screening.

Modulating the kappa-opioid receptor (KOR) is a promising strategy for treating various human diseases. KOR agonists show potential for treating pain, pruritus, and epilepsy, while KOR antagonists show potential for treating depression, anxiety, and addiction. The diterpenoid Salvinorin A (SalA), a secondary metabolite of Salvia divinorum, is a potent and selective KOR agonist. Unlike typical opioids, SalA lacks a basic nitrogen, which encouraged us to search for nonbasic KOR ligands. Through structure-based virtual screening using 3D pharmacophore models based on the binding mode of SalA, we identified novel, nonbasic, potent, and selective KOR agonists. In vitro studies confirmed two virtual hits, SalA-VS-07 and SalA-VS-08, as highly selective for the KOR and showing G protein-biased KOR agonist activity. Both KOR ligands share a novel spiro-moiety and a nonbasic scaffold. Our findings provide novel starting points for developing therapeutics aimed at treating pain and other conditions in which KOR is a central player.

New Clerodane Diterpenoids from Tinospora Capillipes with Antibacterial and Anti-Inflammatory Properties.

Four new clerodane diterpenoids, namely tinocapills A-D (1-4), and one known analogue (5) were isolated from the roots of Tinospora capillipes in the present study. The structures of these new compounds, including their absolute configurations, were determined through a combination of detailed spectroscopic analysis and theoretical statistical approaches, including electronic circular dichroism (ECD) analyses and quantum mechanical (QM)-NMR methods. Additionally, the stereostructure of 5 was confirmed via X-ray diffraction analysis. Furthermore, all these isolates were evaluated for their antibacterial and anti-inflammatory activities. Compounds 1, 2 and 5 demonstrated antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) with MICs ranging from 4-64 µg/mL, and compounds 3 and 4 exhibited potential anti-inflammatory effects by suppressing LPS-induced TNF-α and NO releases in RAW264.7 cells.

Acute Effects of Hallucinogens on Functional Connectivity: Psilocybin and Salvinorin-A.

The extent of changes in functional connectivity (FC) within functional networks as a common feature across hallucinogenic drug classes is under-explored. This work utilized fMRI to assess the dissociative hallucinogens Psilocybin, a classical serotonergic psychedelic, and Salvinorin-A, a kappa-opioid receptor (KOR) agonist, on resting-state FC in nonhuman primates. We highlight overlapping and differing influence of these substances on FC relative to the thalamus, claustrum, prefrontal cortex (PFC), default mode network (DMN), and DMN subcomponents. Analysis was conducted on a within-subject basis. Findings support the cortico-claustro-cortical network model for probing functional effects of hallucinogens regardless of serotonergic potential, with a potential key paradigm centered around the claustrum, PFC, anterior cingulate cortices (ACC), and angular gyrus relationship. Thalamo-cortical networks are implicated but appear dependent on 5-HT2AR activation. Acute desynchronization relative to the DMN for both drugs was also shown. Our findings provide a framework to understand broader mechanisms at which hallucinogens in differing classes may impact subjects regardless of the target receptor.

Three neo-Clerodane Diterpenoids from Tinospora cordifolia Stems and Their Arginase Inhibitory Activities.

Three neo-clerodane diterpenoids, including two new tinocordifoliols A (1) and B (2) and one known tinopanoid R (3), were isolated from the ethyl acetate-soluble fraction of the 70% ethanol extract of Tinospora cordifolia stems. The structures were elucidated by various spectroscopic methods, including one dimensional (1D) and 2D-NMR, high resolution-electrospray ionization (HR-ESI)-MS, and electronic circular dichroism (ECD) data. The T. cordifolia extract and all isolated compounds 1-3 possessed arginase I inhibitory activities. Among them, 3 exhibited moderate competitive inhibition of human arginase I (IC50 = 61.9 µM). Furthermore, docking studies revealed that the presence of a ß-substituted furan in 3 may play a key role in the arginase I inhibitory activities.

Identification of Scutebarbatine B metabolites in rats using UHPLC-Q-Orbitrap-MS/MS and exploration of its mechanism of reversal multidrug resistance in breast cancer by network pharmacology and molecular docking studies.

Scutebarbatine B (SBT-B) is a neo-clerodane diterpenic compound isolated from Scutellaria barbata D. Don (S. barbata), which has been reported to exhibit inhibitory P-glycoprotein (P-gp) property in MCF-7/ADR cells. However, its metabolism and molecular mechanism of reversal multidrug resistance (MDR) in breast cancer remains unclear. This study investigated the metabolite profile of SBT-B in rats by UHPLC-Q-Orbitrap-MS/MS, and explored its mechanism of reversal MDR through network pharmacology and molecular docking studies. A total of 16 Phase I metabolites and 2 Phase II metabolites were identified, and 18 metabolites were all newly discovered metabolites as novel compounds. The metabolic pathway of SBT-B mainly includes oxidization, reduction, hydrolysis, acetylation and glycination. Meanwhile, network pharmacology analyses showed that SBT-B mainly regulated p27 phosphorylation during cell cycle progression, p53 signaling pathway, influence of Ras and Rho proteins on G1 to S Transition. Molecular docking studies revealed that SBT-B exhibits the potential to inhibit P-gp expression by selectively binding to GLN721 and ALA981 residue sites at the interface of P-gp. In addition, SBT-B exhibits moderate binding affinity with CDK2 and E2F1. This study illustrated the major metabolic pathways of SBT-B in vivo, clarified detailed information on SBT-B metabolites in rats, and uncovered the potential mechanism of SBT-B reversal MDR in breast cancer, providing new insights for the development of P-gp inhibitors.

Disparate Effects of Two Clerodane Diterpenes of Giant Goldenrod (Solidago gigantea Ait.) on Bacillus spizizenii.

New substances with antimicrobial properties are needed to successfully treat emerging human, animal, or plant pathogens. Seven clerodane diterpenes, previously isolated from giant goldenrod (Solidago gigantea) root, were tested against Gram-positive Bacillus subtilis, Bacillus spizizenii and Rhodococcus fascians by measuring minimal bactericidal concentration (MBC), minimal inhibitory concentration (MIC) and half-maximal inhibitory concentration (IC50). Two of them, Sg3a (a dialdehyde) and Sg6 (solidagoic acid B), were proved to be the most effective and were selected for further study. Bacillus spizizenii was incubated with the two diterpenes for shorter (1 h) or longer (5 h) periods and then subjected to genome-wide transcriptional analyses. Only a limited number of common genes (28 genes) were differentially regulated after each treatment, and these were mainly related to the restoration of cell membrane integrity and to membrane-related transports. Changes in gene activity indicated that, among other things, K+ and Na+ homeostasis, pH and membrane electron transport processes may have been affected. Activated export systems can be involved in the removal of harmful molecules from the bacterial cells. Inhibition of bacterial chemotaxis and flagellar assembly, as well as activation of genes for the biosynthesis of secondary metabolites, were observed as a general response. Depending on the diterpenes and the duration of the treatments, down-regulation of the protein synthesis-related, oxidative phosphorylation, signal transduction and transcription factor genes was found. In other cases, up-regulation of the genes of oxidation-reduction processes, sporulation and cell wall modification could be detected. Comparison of the effect of diterpenes with the changes induced by different environmental and nutritional conditions revealed several overlapping processes with stress responses. For example, the Sg6 treatment seems to have caused a starvation-like condition. In summary, there were both common and diterpene-specific changes in the transcriptome, and these changes were also dependent on the length of treatments. The results also indicated that Sg6 exerted its effect more slowly than Sg3a, but ultimately its effect was greater.

New clerodane diterpenoids from Callicarpa pseudorubella and their antitumor proliferative activity.

Six previously undescribed clerodane diterpenes, cardorubellas A-F (1-6), along with seven known ones (7-13), were isolated from the aerial parts of Callicarpa pseudorubella. Their chemical structures were established by analysis of 1D and 2D NMR, HR-ESI-MS, X-ray diffraction, and electronic circular dichroism (ECD) data. Notably, cardorubella B (2) represented the first examples of naturally occurring succinic anhydride-containing clerodane diterpenes derivatives. The anti-proliferative activities of these compounds were assessed. Remarkably, compound 2 exhibited comparable inhibitory activity against HEL cell lines, surpassing the positive control with an IC50 value of 14.01 ± 0.77 µM, compared to 17.02 ± 4.70 µM for 5-fluorouracil.

Clerodane diterpenoids with in-vitro anti-neuroinflammatory activity from the tuberous root of Tinospora sagittata (Menispermaceae).

Twenty-six clerodane diterpenoids have been isolated from T. sagittata, a plant species of traditional Chinese medicine Radix Tinosporae, also named as "Jin Guo Lan". Among them, there are eight previously undescribed clerodane diterpenoids (tinotanoids A-H: 1-8), and 18 known diterpenoids (9-26). The absolute configurations of compounds 1, 2, 5, 8, 13, 17 and 20 were determined by single-crystal X-ray diffraction. Compound 1 is the first example of rotameric clerodane diterpenoid with a γ-lactone ring which is constructed between C-11 and C-17; meanwhile, compounds 3 and 4 are two pairs of inseparable epimers. Compounds 2, 12 and 17 demonstrated excellent inhibitory activity on NO production against LPS-stimulated BV-2 cells with IC50 values of 9.56 ± 0.69, 9.11 ± 0.53 and 11.12 ± 0.70 µM, respectively. These activities were significantly higher than that of the positive control minocycline (IC50 = 23.57 ± 0.92 µM). Moreover, compounds 2, 12 and 17 dramatically reduced the LPS-induced upregulation of iNOS and COX-2 expression. Compounds 2 and 12 significantly inhibited the levels of pro-inflammatory cytokines TNF-α, IL-1ß and IL-6 that were increased by LPS stimulation.

Three New Clerodane Diterpenoids and Their NLRP3 Inflammasome Activation Inhibitory Activity from Callicarpa arborea Roxb.

Three new compounds callicarpenoids A-C (1-3), were isolated from the stems of Callicarpa arborea Roxb together with fifteen known compounds (4-18). The structures of these compounds were elucidated using advanced spectroscopic techniques, including 1D and 2D NMR, UV, IR, HR-ESI-MS, ECD, ORD, and quantum chemical calculations. Compound 3, a rare rearranged diterpenoid with a fused 5/6-ring system demonstrated strong potential as an inhibitor of the NLRP3 inflammasome activation with an IC50 value of 3.153 µM. It effectively reduced GSDMD-NT production, inhibited caspase-1 activation, and suppressed IL-1ß secretion, thereby mitigating NLRP3 inflammasome-induced pyroptosis in J774A.1 cells. These findings suggest that compound 3 warrants further research and development as a promising NLRP3 inflammasome inhibitor.

A New Clerodane from the Leaves of Croton krabas and Its Cholinesterase Inhibitory Activities.

Chromatographic separation of the leaves of Croton krabas resulted in the isolation of one new clerodane, crotoeurin D (1), along with two known compounds, 6S-crotoeurin C (2) and blumenol A (3). Their structures were determined based on extensive nuclear magnetic resonance spectroscopic data analysis and mass spectrometry. The absolute configuration of the new clerodane was assigned by nuclear overhauser effect spectroscopy correlations and electronic circular dichroism calculations. Compound 1 exhibited significant acetylcholinesterase and butyrylcholinesterase inhibitory activities. Moreover, the binding modes of 1 revealed that its structure formed strong hydrogen bonds and hydrophobic interactions with the active sites of both enzymes.

Stereochemical assignment of clerodane-type diterpenes from the fruits of Casearia grewiifolia and their ability to inhibit PCSK9 expression.

More than 20 natural products have been reported to modulate PCSK9-mediated cholesterol regulation, and small-molecule-derived proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors continue to be developed and identified. Here, twelve undescribed clerodane-type diterpenes (1-9 and 12-14) and two known compounds were isolated from the chloroform-soluble extract of the dried fruits of Casearia grewiifolia Vent. using a PCSK9 mRNA expression monitoring assay. Among the undescribed compounds, the stereochemistry of two diastereomeric grewiifolins A and B (1 and 2) were extensively elucidated using 2D Nuclear Overhauser Effect Spectroscopy (NOESY) experiments, excitation-sculptured indirect detection experiments (EXSIDE), interproton distance analyses, and computational calculations that included quantum chemical shift calculations combined with DP4+ analysis. All isolates were assessed for their inhibitory activity against PCSK9 and IDOL mRNA expression. Among the compounds tested, compound 3 inhibited PCSK9 and IDOL mRNA expression.

Tinopanoids K-T, clerodane diterpenoids with anti-inflammatory activity from Tinospora crispa.

A total of 17 structurally diverse clerodane diterpenoids, including ten undescribed clerodane diterpenoids (tinopanoids K-T, 1-10) and seven known compounds (11-17), were isolated from the vines and leaves of Tinospora crispa. Compound 3 has not only bear the dominant substituents of γ-hydroxy-α, ß-unsaturated-γ-lactone with anti-inflammatory activity, but also a ternary epoxy structure at C-3/C-4. The planar structures and relative configurations of the clerodane diterpenoids were elucidated by spectroscopic data interpretation. The absolute configurations of compounds 1, 4, 8 and 13 were determined by single-crystal X-ray crystallographic, while that of compound 3 was determined using computed ECD data and single crystal X-ray diffraction of related p-bromobenzoate ester (3a). Subsequently, all compounds were evaluated for their inhibitory effect on nitric oxide (NO) production of LPS-activated BV-2 cells, and compounds 3 and 8 exhibited better NO inhibitory potency, with IC50 values of 5.6 and 13.8 µM than the positive control minocycline (Mino, IC50 = 22.9 µM). The corresponding results of western blot analysis and qRT-PCR revealed that compound 3 can significantly inhibit the inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) protein expressions, mRNA levels of pro-inflammatory cytokins of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and interleukin 1ß (IL-1ß). The underlying mechanism by which compound 3 exerted anti-neuroinflammatory effects was investigated by western blot and immunofluorescence assay, which suggested compound 3 inhibited LPS induced neuroinflammation via the suppression of toll-like receptor 4 (TLR4) dependent Signal Transducer and Activator of Transcription 3 (Stat3) and mitogen-activated protein kinase (MAPK) signaling pathways, and the activation of Heme Oxygenase-1 (HO-1) mediated signals.

Discovery of neo-Clerodane Diterpenoids from Ajuga campylantha as Neuroprotective Agents against Ferroptosis and Neuroinflammation.

Twelve new neo-clerodane diterpenoids, eight undescribed methoxy/ethoxy acetal analogues, and one new nor-iridane monoterpenoid were isolated from Ajuga campylantha. Their structures were elucidated using a combination of spectroscopic data, quantum chemical calculations, and X-ray crystallography. This research reveals the distinctive structural features of A. campylantha diterpenes, including distinct C rings and 4,18-double bonds, distinguishing them from diterpenes of other plants in the Ajuga genus. Compound 2 represents the first example of a 19(5→6)-abeo-clerodane formed through a Wagner-Meerwein rearrangement. The isolated compounds were assessed for their neuroprotective effects against RSL3-induced ferroptosis in HT22 cells and LPS-induced neuroinflammation in BV-2 cells. Notably, compound 7 inhibits ferroptosis (EC50 = 10 µM) with a potentially new mechanism of action. The preliminary structure-activity relationship studies revealed that the furan-clerodane diterpenoids possess potential ferroptosis inhibitory activity, while the lactone-clerodanes do not. This study represents the first report of furan-containing clerodanes within the Ajuga genus, providing fresh insights into the phytochemistry and pharmacological potential of A. campylantha.

Clerodane diterpenoids from the Uygur medicine Salvia deserta with immunosuppressive activity.

Six undescribed clerodane diterpenoids along with five known ones were isolated from the aerial parts of Salvia deserta, a traditional Uygur medicine. Their chemical structures including absolute configurations were elucidated by extensive spectroscopic analysis (including 1D and 2D NMR, HRESIMS, and IR), combined with calculated ECD method and single-crystal X-ray diffraction analysis. All the compounds possessed a terminal α,ß-unsaturated-γ-lactone moiety, and were assayed for their immunosuppressive activity via inhibiting the secretion of cytokines TNF-α and IL-6 in macrophages RAW264.7. Among them, (5R,8R,9S,10R)-18-nor-cleroda-2,13-dien-16,15-olide-4-one obviously suppressed the secretion of TNF-α and IL-6 with IC50 values of 8.55 and 13.65 µM, respectively.