Identification and evolution of a diterpenoid phytoalexin oryzalactone biosynthetic gene in the genus Oryza.

The natural variation of plant-specialized metabolites represents the evolutionary adaptation of plants to their environments. However, the molecular mechanisms that account for the diversification of the metabolic pathways have not been fully clarified. Rice plants resist attacks from pathogens by accumulating diterpenoid phytoalexins. It has been confirmed that the composition of rice phytoalexins exhibits numerous natural variations. Major rice phytoalexins (momilactones and phytocassanes) are accumulated in most cultivars, although oryzalactone is a cultivar-specific compound. Here, we attempted to reveal the evolutionary trajectory of the diversification of phytoalexins by analyzing the oryzalactone biosynthetic gene in Oryza species. The candidate gene, KSLX-OL, which accounts for oryzalactone biosynthesis, was found around the single-nucleotide polymorphisms specific to the oryzalactone-accumulating cultivars in the long arm of chromosome 11. The metabolite analyses in Nicotiana benthamiana and rice plants overexpressing KSLX-OL indicated that KSLX-OL is responsible for the oryzalactone biosynthesis. KSLX-OL is an allele of KSL8 that is involved in the biosynthesis of another diterpenoid phytoalexin, oryzalexin S and is specifically distributed in the AA genome species. KSLX-NOL and KSLX-bar, which encode similar enzymes but are not involved in oryzalactone biosynthesis, were also found in AA genome species. The phylogenetic analyses of KSLXs, KSL8s, and related pseudogenes (KSL9s) indicated that KSLX-OL was generated from a common ancestor with KSL8 and KSL9 via gene duplication, functional differentiation, and gene fusion. The wide distributions of KSLX-OL and KSL8 in AA genome species demonstrate their long-term coexistence beyond species differentiation, suggesting a balancing selection between the genes.

OsWRKY10 extensively activates multiple rice diterpenoid phytoalexin biosynthesis genes to enhance rice blast resistance.

Phytoalexin is the main chemical weapon against pathogens in plants. Rice (Oryza sativa L.) produces a number of phytoalexins to defend against pathogens, most of which belong to the class of diterpenoid phytoalexins. Three biosynthetic gene clusters (BGCs) and a few non-BGC genes are responsible for rice diterpenoid phytoalexin biosynthesis. The corresponding regulatory mechanism of these phytoalexins in response to pathogen challenges still remains unclear. Here we identified a transcription factor, OsWRKY10, which positively regulates rice diterpenoid phytoalexin biosynthesis. Knockout mutants of OsWRKY10 obtained by CRISPR/Cas9 technology are more susceptible to Magnaporthe oryzae infection, while overexpression of OsWRKY10 enhances resistance to rice blast. Further analysis revealed that overexpression of OsWRKY10 increases accumulation of multiple rice diterpenoid phytoalexins and expression of genes in three BGCs and non-BGC genes in response to M. oryzae infection. Knockout of OsWRKY10 impairs upregulation of rice diterpenoid phytoalexin biosynthesis gene expression by blast pathogen and CuCl2 treatment. OsWRKY10 directly binds to the W-boxes or W-box-like elements (WLEs) of rice diterpenoid phytoalexin biosynthesis gene promoters to regulate gene expression. This study identified an extensive regulator (OsWRKY10) with broad transcriptional regulatory effects on rice diterpenoid phytoalexin biosynthesis genes, providing insight into the regulation of chemical defense to improve disease resistance in rice.

Chemical and transcriptomic analyses of leaf trichomes from Cistus creticus subsp. creticus reveal the biosynthetic pathways of certain labdane-type diterpenoids and their acetylated forms.

Labdane-related diterpenoids (LRDs), a subgroup of terpenoids, exhibit structural diversity and significant commercial and pharmacological potential. LRDs share the characteristic decalin-labdanic core structure that derives from the cycloisomerization of geranylgeranyl diphosphate (GGPP). Labdanes derive their name from the oleoresin known as 'Labdanum', 'Ladano', or 'Aladano', used since ancient Greek times. Acetylated labdanes, rarely identified in plants, are associated with enhanced biological activities. Chemical analysis of Cistus creticus subsp. creticus revealed labda-7,13(E)-dien-15-yl acetate and labda-7,13(E)-dien-15-ol as major constituents. In addition, novel labdanes such as cis-abienol, neoabienol, ent-copalol, and one as yet unidentified labdane-type diterpenoid were detected for the first time. These compounds exhibit developmental regulation, with higher accumulation observed in young leaves. Using RNA-sequencing (RNA-seq) analysis of young leaf trichomes, it was possible to identify, clone, and eventually functionally characterize labdane-type diterpenoid synthase (diTPS) genes, encoding proteins responsible for the production of labda-7,13(E)-dien-15-yl diphosphate (endo-7,13-CPP), labda-7,13(E)-dien-15-yl acetate, and labda-13(E)-ene-8α-ol-15-yl acetate. Moreover, the reconstitution of labda-7,13(E)-dien-15-yl acetate and labda-13(E)-ene-8α-ol-15-yl acetate production in yeast is presented. Finally, the accumulation of LRDs in different plant tissues showed a correlation with the expression profiles of the corresponding genes.

Identification and structural modification of ent-rosane diterpenoids from Euphorbia milii inhibiting RANKL-induced osteoclastogenesis.

Phytochemical study on Euphorbia milii, a common ornamental plant, resulted in the identification of thirteen new ent-rosane diterpenoids (1-13), three new ent-atisane diterpenoids (14-16), and a known ent-rosane (17). Their structures were delineated using spectroscopic data, quantum chemical calculations, and X-ray diffraction experiments. Euphomilone F (1) represented a rare ent-rosane-type diterpenoid with a 5/7/6 skeleton. Euphoainoid G (8) was a rare rosane diterpenic acid. Compounds 9 and 10 carried infrequent tetrahydrofuran rings, and compounds 11-13 was 18-nor-ent-rosane diterpenoids. All isolates were evaluated for their inhibitory effects on RANKL-induced osteoclasts. Notably, compounds with aromatic ester groups (2-7) showed promising activities (IC50 < 10 µM), underscoring the significance of acylated A-ring moieties in the ent-rosane skeleton for anti-osteoclastogenesis. Thirteen synthetic derivatives were obtained through esterification of 17. Of these, compound 27 exhibited remarkable improvement, with an IC50 of 0.8 µM, more than a 12-fold increase in potency compared to the parent compound 17 (IC50 > 10 µM). This work presents a series of new ent-rosane diterpenoids with potential antiosteoporosis agents.

Brunonianines D-F, three new C19-diterpenoid alkaloids from the Delphinium brunonianum, with therapeutic effect on ovarian cancer in vitro and in vivo.

The current standard treatment for ovarian cancer consists of surgery to reduce the size of the tumor, followed by treatment with chemotherapeutic drugs, which have major side effects. Therefore, finding a new natural product drug with fewer side effects is a strategy. Delphinium brunonianum (D. brunonianum) is a traditional Tibetan medicine, mainly from southern Tibet, China, whereas the chemical constituents in this plant remain elusive. The major metabolites in the dichloromethane fraction of D. brunonianum were analyzed and purified by HPLC and various column chromatography techniques. Nine diterpenoid alkaloids (1-9) and one amide alkaloid (10) were isolated from D. brunonianum, including three novel C19-type diterpenoid alkaloids (Brunonianines D-F) (1-3). Their structures were elucidated by 1D/2D NMR, HR-ESI-MS and single-crystal X-ray diffraction analyses. All compounds were evaluated for toxicity in four tumor cell lines. Most of the compounds exhibited potent inhibitory effects on Skov-3 cell lines, with IC50 values ranging from 2.57 to 8.05 µM. The western blotting experiment was used to further analyze the expression levels of molecules in the Bax/Bcl-2/Caspase-3 signaling pathway for compound 1. Molecular docking was performed to predict the binding modes of Brunonianine D with target proteins. In vivo experiments were also performed and evaluated in real time by monitoring the size of the Skov-3 tumor. Additionally, tumor H&E staining and the TUNEL assay used to evaluate anti-tumor effects.

Identification of diterpenoids from Salvia castanea Diels f. tomentosa Stib and their antitumor activities.

Four new diterpenoid tropolones, salvirrddones A-D (1-4), and four new icetexanes, salvirrddices A-D (9-12), along with thirteen new 11,12-seco-norabietane diterpenoids, salvirrddnor A-M (14-24, 31, 32) and sixteen known compounds (5-8, 13, 25-30, 33-37), were isolated from the roots and rhizomes of Salvia castanea Diels f. tomentosa Stib. Their structures were elucidated by comprehensive spectroscopic analyses, quantum chemical calculations, and X-ray crystallography. Structurally, compounds 1-8 represent a class of rare natural products featuring a unique cyclohepta-2,4,6-trienone moiety with diterpenoid skeletons. Bioassays showed that only diterpenoid tropolones 3, 5, 6, and 7 exhibited significant activity against several human cancer cell lines with IC50 values ranging from 3.01 to 11.63 µM. Additionally, 3 was shown to inhibit Hep3B cell proliferation, block the G0/G1 phase of the cell cycle, induce mitochondrial dysfunction and oxidative stress, promote apoptosis, as well as inhibit migration and invasion in vitro. Meanwhile, 3 demonstrated anti-proliferative, pro-apoptotic, and migration-inhibitory effects in the Hep3B xenograft zebrafish model in vivo. Network pharmacological analysis and molecular docking results suggested that 3 may treat hepatocellular carcinoma (HCC) through the PI3K-Akt signaling pathway, as well as by binding PARP1 and CDK2 targets. Overall, the present results extremely expand the repertoire of diterpenoids from natural products and may provide a novel chemical scaffold for the discovery of new antitumor drugs.

Anti-necroptosis and anti-ferroptosis compounds from the Deep-Sea-Derived fungus Aspergillus sp. MCCC 3A00392.

Eight undescribed (1-8) and 46 known compounds (9-54) were isolated from the deep-sea-derived Aspergillus sp. MCCC 3A00392. Compounds 1-3 were three novel oxoindolo diterpenoids, 4-6 were three bisabolane sesquiterpenoids, while 7 and 8 were two monocyclic cyclopropanes. Their structures were established by exhaustive analyses of the HRESIMS, NMR, and theoretical calculations of the NMR data and ECD spectra. Compounds 10, 33, 38, and 39 were able to inhibit tumor necrosis factor (TNF)-induced necroptosis in murine L929 cell lines. Functional experiments verified that compounds 10 and 39 inhibited necroptosis by downregulating the phosphorylation of RIPK3 and MLKL. Moreover, compound 39 also reduced the phosphorylation of RIPK1. Compounds 10, 33, and 34 displayed potent inhibitory activities against RSL-3 induced ferroptosis with the EC50 value of 3.0 µM, 0.4 µM, and 0.1 µM, respectively. Compound 10 inhibited ferroptosis by the downregulation of HMOX1, while compounds 33 and 34 inhibited ferroptosis through regulation of NRF2/SLC7A11/GCLM axis. However, these compounds only showed weak effect in either the necroptosis or ferroptosis relative mouse disease models. Further studies of pharmacokinetics and pharmacodynamics might improve their in vivo bioactivities.

Structure-activity relationships of natural and synthetic lathyrane-based diterpenoids for suppression of NLRP3-driven inflammation and gouty arthritis.

Lathyrane-based diterpenoid is one of the critical bioactive elements of Euphorbia lathyris L., a widely used traditional Chinese medicine for the treatment of inflammation and infection. In this study, we introduced and evaluated seven synthetic or natural lathyrane-based diterpenoids with the same core structure but notable structural variations at specific positions, for their anti-inflammatory and gout-alleviating properties. There was no significant cytotoxicity below 10 µM among the initial test of the cell counting kit 8 of the seven candidate derivatives (compounds 13 to 19) in this work. Furthermore, maintaining the acyloxy group at 15-C position and the strongly hydrophobic aryl structure at 3-C and 5-C positions, compounds 15 (Euphorbia factor L3, EFL3) and 17 strikingly inhibited the production of IL-1ß related to the actuation of the inflammasome in our study. The ELISA assay indicated that the anti-inflammatory effects of EFL3 were better associated with MSU stimulation than other second-line pathways triggered by inflammasome. Further examinations on the acute paw gout model in C57BL/6 mice showed that EFL3 had a significantly inflammatory retarding effect by intraperitoneal injection. It decreased swelling volume as well as the cleavage and activation of local IL-1ß and casepas-1 in the paw. To conclude, our findings reveal several potential key structure-activity relationships that govern the anti-inflammatory effects of lathyrane-type diterpenoids, the dispensable acyl group at the 15-C position, the importance of maintaining the spatial structure of the B-ring, and the potential importance of hydrophobic substituents at the 3-C position. These insights may provide guidance for the structural design of lathyrane-type agents in the future; furthermore, we found that the lathyrane-based diterpenoid EFL3 is a potential agent for gout that is expected to provide a novel therapeutic strategy for inflammation intervention.

Natural Product-Inspired Molecules for Covalent Inhibition of SHP2 Tyrosine Phosphatase.

Natural products have been playing indispensable roles in the development of lifesaving drug molecules. They are also valuable sources for covalent protein modifiers. However, they often are scarce in nature and have complex chemical structures, which are limiting their further biomedical development. Thus, natural product-inspired small molecules which still contain the essence of the parent natural products but are readily available and amenable for structural modification, are important and desirable in searching for lead compounds for various disease treatment. Inspired by the complex and diverse ent-kaurene diterpenoids with significant biological activities, we have created a synthetically accessible and focused covalent library by incorporating the bicyclo[3.2.1]octane α-methylene ketone, which is considered as the pharmacophore of ent-kaurene diterpenoids, as half of the structure, and replacing the other half with much less complex but more druglike scaffolds. From this library, we have identified and characterized selective covalent inhibitors of protein tyrosine phosphatase SHP2, an important anti-cancer therapeutic target. The success of this study demonstrated the importance of creating and evaluating natural product-inspired library as well as their application in targeting challenging disease targets.

Isolation, structure modification, and anti-rheumatoid arthritis activity of isopimarane-type diterpenoids from Orthosiphon aristatus.

Orthosiphon aristatus is a well-known folkloric medicine and herb for Guangdong soup for the treatment of rheumatism in China. Eight isopimarane-type and migrated pimarane-type diterpenoids (1-8), including a new one with a rarely occurring α,ß-unsaturated diketone C-ring, were isolated from O. aristatus. Their structures were determined by spectroscopic methods and quantum chemical calculations. Furthermore, the most abundant compound, orthosiphol K, was structurally modified by modern synthetic techniques to give seven new derivatives (9-15). The anti-rheumatoid arthritis activity of these diterpenoids were evaluated on a TNF-α induced MH7A human rheumatoid fibroblast-like synoviocyte model. Compound 10 showed the most potent activity among these compounds. Based on their inhibitory effects on the release levels of IL-1ß, the preliminary structure-activity relationships were concluded. Furthermore, western blot analysis revealed that 10 could increase the expression of IκBα and decrease the expression of NF-κB p65, and the expression levels of COX-2 and NLRP3 proteins were consequently down-regulated.

Identifying chemical markers in wine-processed Salvia miltiorrhiza using ultrahigh-performance liquid chromatography-quadrupole-time-of-flight-tandem mass spectrometry.

To find the chemical markers of wine-processed Salvia miltiorrhiza (WSM), 76 constituents, including diterpenoid quinones and phenolic acids in Salvia miltiorrhiza (SM) and WSM, were profiled using ultrahigh-performance liquid chromatography-quadrupole-time-of-flight-tandem mass spectrometry (UPLC-Q-TOF-MS/MS) in positive- and the negative-ion modes. Thirty compounds were screened out as candidate differential components using chemometrics analysis, and the concentration of most compounds increased after processing with wine. Seven compounds, namely tanshinone IIA, magnesium lithospermate B, salvianolic acid G, cryptotanshinone, isocryptotanshinone, salvianolic acid B, and rosmarinic acid, were selected as chemical markers of WSM using variable importance of the project. This study revealed the chemical markers of WSM and confirmed that WSM can improve the extraction and solubility effect of chemical constituents.

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.

Two Novel Diterpenoid Alkaloids from Aconitum Pendulum.

Two previously uncharacterized compounds, an aconitine-type C19-diterpenoid alkaloid (1) and a napelline-type diterpenoid alkaloid C20-diterpenoid alkaloid (2), as well as ten known compounds (3-12), were isolated from Aconitum pendulum. Their structures were elucidated based on spectroscopic data, including 1D and 2D NMR, IR, HR-ESI-MS, and single-crystal X-ray diffraction analysis. The anti-insecticidal activities of these compounds were evaluated by contact toxicity tests against two-spotted spider mites, and compounds 1, 2, and 9 showed moderate contact toxicity, with LC50 values of 0.86±0.09, 0.95±0.23, and 0.89±0.19 mg/mL, respectively. This study highlights the potential use of diterpenoid alkaloids as natural plant-derived pesticides for the management of plant pests.

Antibacterial Indole-diterpenoid Alkaloids from the Marine Fungus Penicillium sp. ZYX-Z-718.

Two new indole-diterpenoids, penpaxilloids F and G (1 and 2), along with 11 known analogues (3-13), were isolated from the marine fungus Penicillium sp. ZYX-Z-718. The structures of the new compounds were identified by extensive spectroscopic analyses including HR-ESI-MS, UV, and NMR, as well as theoretical NMR chemical shifts and ECD calculations. Compounds 6 and 10 showed antibacterial activity against Gram-positive bacteria including Staphylococcus aureus, Bacillus subtilis, and MRSA with MIC values ranging from 16.0 to 32.0 µg/mL.

Diterpenoid Alkaloids from Delphinium ajacis and Their Anti-inflammatory Activity.

Three novel diterpenoid alkaloids, comprising two C19 -diterpenoid alkaloids (1 and 2) and one C20 -diterpenoid alkaloid (3), were isolated from Delphinium ajacis, alongside the six known compounds (4-9). Their structures were elucidated by spectroscopic methods (MS, UV, IR, 1D and 2D NMR) and chemical properties. Simultaneously, the anti-inflammatory properties of all compounds (1-9) was conducted, focusing on nitric oxide (NO) production in LPS-induced BV-2 cells. The results indicated compounds 1-3, 7, and 8 have potential anti-inflammatory activity.

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.

Diterpenoid Alkaloids from Delphinium liangshanense.

Two new C19-diterpenoid alkaloids of the lycoctonine-type (liangshanine A and liangshanine B) and nineteen known compounds (3-21) were isolated from the whole plant of Delphinium liangshanense W. T. Wang, and all the compounds were identified by different spectroscopic analyses, such as IR, HR-ESI-MS and NMR. All the compounds were isolated from this plant for the first time and tested for the anti-proliferation effects on MH7 A and SF9 cells to figure their anti-rheumatoid arthritis and anti-insect activity, but none of them showed remarkable activity.

Pseudoamaolide P, a 1,2:3,4:9,10:9,19-tetraseco-cycloartane triterpene spiroketal lactone from seeds of Pseudolarix amabilis.

A 1,2:3,4:9,10:9,19-tetraseco-cycloartane triterpene spiroketal lactone, pseudoamaolide P (1), two new labdane-type diterpenoids, pseudoamains A and B (2-3), and four known cembrane-type diterpenoids (4-7) were isolated from the seeds of Pseudolarix amabilis. The structures of these compounds were elucidated by spectroscopic analyses, including HRESIMS, 1D-, and 2D-NMR. The anti-inflammatory activities of the compounds were evaluated by suppressing the transcription of the NF-κB-dependent reporter gene in LPS-induced 293 T/NF-κB-luc cells. All compounds do not show potent activity.

A new cassane diterpenoid from the seed of Caesalpinia sappan.

In this study, a previously undescribed cassane diterpenoid, named caesalpinin JF (1), along with two known cassane diterpenoids caesanine C (2) and tomocinol B (3), was isolated from 95% EtOH extract of the seeds of Caesalpinia sappan Linn. Additionally, three known compounds including pulcherrin R (4), syringaresinol-4'-O-ß-D-glucopyranoside (5) and kaempferol (6) were also identified. The structures of the isolated compounds were elucidated by comprehensive 1D and 2D NMR spectroscopic analyses. Additionally, electronic circular dichroism (ECD) calculation was used to identify the absolute structure of compound 1. Among the isolated compounds, compound 1 displayed a potent anti-neuroinflammation with an IC50 value of 9.87 ± 1.71 µM.

Two new diterpenoid alkaloids from the roots of Aconitum nagarum and their cytotoxic activity.

A chemical investigation on the roots of Aconitum nagarum afforded two undescribed C19-diterpenoid alkaloids nagarumines D and E (1 and 2). The structures of the new compounds were elucidated by spectral methods such as 1D and 2D (1H-1H COSY, HMQC, and HMBC) NMR spectroscopy, as well as HR-ESI-MS. The two isolated alkaloids were tested in vitro for cytotoxic activity against five gastric tumor cell lines. Consequently, compound 2 exhibited some cytotoxicities against several human cancer cell lines with IC50 value less than 20.0 µM.