Two Cytochrome P450 Enzymes Form the Tricyclic Nested Skeleton of Meroterpenoids by Sequential Oxidative Reactions.

Meroterpenoid clavilactones feature a unique benzo-fused ten-membered carbocyclic ring unit with an α,ß-epoxy-γ-lactone moiety, forming an intriguing 10/5/3 tricyclic nested skeleton. These compounds are good inhibitors of the tyrosine kinase, attracting a lot of chemical synthesis studies. However, the natural enzymes involved in the formation of the 10/5/3 tricyclic nested skeleton remain unexplored. Here, we identified a gene cluster responsible for the biosynthesis of clavilactone A in the basidiomycetous fungus Clitocybe clavipes. We showed that a key cytochrome P450 monooxygenase ClaR catalyzes the diradical coupling reaction between the intramolecular hydroquinone and allyl moieties to form the benzo-fused ten-membered carbocyclic ring unit, followed by the P450 ClaT that exquisitely and stereoselectively assembles the α,ß-epoxy-γ-lactone moiety in clavilactone biosynthesis. ClaR unprecedentedly acts as a macrocyclase to catalyze the oxidative cyclization of the isopentenyl to the nonterpenoid moieties to form the benzo-fused macrocycle, and a multifunctional P450 ClaT catalyzes a ten-electron oxidation to accomplish the biosynthesis of the 10/5/3 tricyclic nested skeleton in clavilactones. Our findings establish the foundation for the efficient production of clavilactones using synthetic biology approaches and provide the mechanistic insights into the macrocycle formation in the biosynthesis of fungal meroterpenoids.

Two novel diterpenes from the stems and leaves of tropical seagrass Enhalus acoroides in the South China sea.

Two novel diterpenes Enhoidin A (1) and Enhoidin B (2) featuring an unusual gibberellane skeleton were isolated from the stems and leaves of Enhalus acoroides. Their structures were elucidated on the basis of spectroscopic analysis including 1D and 2D NMR techniques and HR-ESI-MS. This is the first time that this type of lactone ring between C-18 and C-20 has been found among gibberellanes from the tropical seagrasses. Evaluation of the all compounds for cytotoxicity against four human cancer cell lines (MCF-7, HCT-116, HepG-2 and HeLa), and showed moderate cytotoxic activities.

Blocking Effect of Demethylzeylasteral on the Interaction between Human ACE2 Protein and SARS-CoV-2 RBD Protein Discovered Using SPR Technology.

The novel coronavirus disease (2019-nCoV) has been affecting global health since the end of 2019, and there is no sign that the epidemic is abating. Targeting the interaction between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and the human angiotensin-converting enzyme 2 (ACE2) receptor is a promising therapeutic strategy. In this study, surface plasmon resonance (SPR) was used as the primary method to screen a library of 960 compounds. A compound 02B05 (demethylzeylasteral, CAS number: 107316-88-1) that had high affinities for S-RBD and ACE2 was discovered, and binding affinities (KD, µM) of 02B05-ACE2 and 02B05-S-RBD were 1.736 and 1.039 µM, respectively. The results of a competition experiment showed that 02B05 could effectively block the binding of S-RBD to ACE2 protein. Furthermore, pseudovirus infection assay revealed that 02B05 could inhibit entry of SARS-CoV-2 pseudovirus into 293T cells to a certain extent at nontoxic concentration. The compoundobtained in this study serve as references for the design of drugs which have potential in the treatment of COVID-19 and can thus accelerate the process of developing effective drugs to treat SARS-CoV-2 infections.

Triterpenoid saponins from Clinopodium chinense (Benth.) O. Kuntze and their biological activity.

Four new ursane-type triterpenoid saponins, clinopoursaponins A-D (1-4), six new oleanane-type triterpenoid saponins, clinopodiside VII-XII (5-10), as well as eight known triterpene analogues (11-18), were isolated from the aerial parts of Clinopodium chinense (Benth.) O. Kuntze. The structures of the new compounds were determined based on extensive spectral analyses, including 1D (1H and 13C) and 2D NMR experiments (COSY, NOESY, HSQC, 2D TOCSY, HSQC-TOCSY and HMBC), HR-ESI-MS and chemical methods. Compounds 1-18 were evaluated for their protective effects against anoxia/reoxygenation-induced apoptosis in H9c2 cells and cytotoxicities against murine mammary carcinoma cell line 4T1. Compounds 8, 9 and 18 exhibited significant protective effects, while compound 1 exhibited cytotoxic activity with IC50 value of 7.4 µm compared to 7.6 µm for the positive control 10-hydroxycamptothecin.

Calenduloside E Analogues Protecting H9c2 Cardiomyocytes Against H2O2-Induced Apoptosis: Design, Synthesis and Biological Evaluation.

Modulation of apoptosis is therapeutically effective in cardiomyocytes damage. Calenduloside E (CE), a naturally occurring triterpenoid saponin, is a potent anti-apoptotic agent. However, little is known about its synthetic analogues on the protective effects in apoptosis of cardiomyocytes. The present research was performed to investigate the potential protective effect of CE analogues against H2O2-induced apoptosis in H9c2 cardiomyocytes and the underlying mechanisms. Sixteen novel CE anologues have been designed, synthesized and biological evaluation. Among the 16 CE anologues, as well as the positive control CE tested, compound 5d was the most effective in improving cardiomyocytes viability. Pretreatment with anologue 5d inhibited ROS generation, maintained the mitochondrial membrane potential and reduced apoptotic cardiomyocytes. Moreover, exposure to H2O2 significantly increased the levels of Bax, cleaved caspase-3, and cleaved PARP, and decreased the level of Bcl-2, resulting in cell apoptosis. Pretreatment with anologue 5d (0.02-0.5 µg/mL) dose-dependently upregulated antiapoptotic proteins and downregulated proapoptotic proteins mentioned above during H2O2-induced apoptosis. These results suggested that CE analogues provide protection to H9c2 cardiomyocytes against H2O2-induced oxidative stress and apoptosis, most likely via anti-apoptotic mechanism, and provided the basis for the further optimization of the CE analogues.

New Cassane Diterpenoids from Caesalpinia sappan and their Antiplasmodial Activity.

One new cassane diterpene possessing an unusual N bridge between C-19 and C-20 named caesalsappanin R (1), as well as another new diterpene caesalsappanin S (2), were isolated from the seeds of Caesalpinia sappanwith methanol extract. Their structures were determined by spectroscopic analysis and examined alongside existing data from prior studies. Their biological activities were profiled by their antiplasmodial activity.

[Studies on chemical constituents of Clinopodium chinense].

Twenty-eight compounds were isolated and purified from Clinopodium chinense by Sephedax LH-20, ODS, MCI and preparative HPLC. Their structures were identified as apigenin (1), apigenin-7-O-ß-D-glucopyranoside (2), apigenin-7-O-ß-D-glucuronopyranoside (3), thellungianol (4), apigenin-7-O-ß-D-rutinoside (5), luteolin (6), luteolin-4'-O-ß-D-glucopyranoside (7), apigenin-7-O-ß-D-pyranglycuronate butyl ester (8), luteolin-7-O-ß-D-rutinoside (9), luteolin-7-O-ß-D-noehesperidoside (10), acacetin (11), acacetin-7-O-ß-D-glucuronopyranoside (12), buddleoside (13), naringenin (14), pruning (15), nairutin (16), isosakuranetin (17), isosakuranin (18), didymin (19), hesperidin (20), kaempferol (21), quercetin (22), kaempferol-3-O-α-L-rahmnoside (23), p-hydroxycinnamic acid (24), caffeic acid (25), cis-3-[2-[1-(3,4-dihydroxy-phenyl)-1 -hydroxymethyl]-1,3-ben-zodioxol-5-yl]-(E)-2-propenoic acid (26), mesaconic acid (27), gentisic acid 5-O-ß-D-(6'-salicylyl)-glucopyranoside (28). Among them, compounds 7, 9-10, 12, 23, 26-28 were isolated from the Clinopodium for the first time. The protective effects of compounds 1-6, 8-17 and 19 against H2O2-induced H9c2 cardiomyocyte injury were tested, compounds 15 exhibited significantly protective effects. Compared with the cell viability of (62.12±6.18)% in the model, pruning exhibited viabilities of (84.25±7.36)% at 25.0 mg•L⁻¹, respectively, using quercetin as a positive control [cell viability of (84.55±8.26)%, 20 mg•L⁻¹].

Four new phenolic acid with unusual bicycle [2.2.2] octane moiety from Clerodendranthus spicatus and their anti-inflammatory activity.

Four new phenolic acids, clerodens A-D (1-4) possessing an unusual bicycle [2.2.2] octane moiety were isolated from the whole plants of Clerodendranthus spicatus. Their structures were elucidated by extensive spectroscopic methods, including NMR, MS, and ECD data. All isolates were evaluated for their anti-inflammatory activities on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7, and compound 4 showed significant inhibitory activities with IC50 value of 6.8 µM.

[Cassae-type diterpenes from seeds of Caesalpinia minax].

Fifteen cassaen-type diterpenes were isolated from the 95% ethanolic extract of the seeds of C. minax through various chromatographic techniques. Their structures were identified on the basis of spectroscopic data as pulcherralpin (1), caesalpinin ML (2), chamaetexane C (3), chamaetexane D (4), 6ß, 18-diacetoxycassan-13, 15-diene (5), neocaesalpin K (6), neocaesalpin MP (7), neocaesalpin M (8), neocaesalpin Q (9), neocaesalpin P (10), neocaesalpin R (11), caesaldekarin D (12), caesaldekarin A (13), caesaldekarin b (14), 3ß,6α-diacetoxyvouacapane (15). Among them, compounds 14, 9-11 were isolated from this plant for the first time.

Norcassane- and cassane-type furanoditerpenoids from the seeds of Caesalpinia sappan.

Three novel furanoditerpenoids, norcaesalpinin J (1) featuring an unusual 20-norcassane hydroperoxide and phangininoxys B (2) and C (3) possessing cassane hemiketal skeletons, were isolated from the seeds of Caesalpinia sappan. Their structures were elucidated by extensive spectroscopic methods. All isolates were evaluated for the cytotoxic activities on three human cancer cell lines.

Novel dinorcassane- and cassane-type diterpenes from the seeds of Caesalpinia minax.

Two novel diterpenes, norcaesalpinin I (1) featuring an unusual ring C-contracted dinorcassane and caesalpinin U (2) possessing a highly oxygenated furanocassane skeleton were isolated from the seeds of Caesalpinia minax. Their structures were determined by different spectroscopic methods. A plausible biosynthetic pathway of 1 was proposed. The cytotoxic activity of compounds 1 and 2 against HepG2 and HeLa human tumor cell lines was evaluated.

Two new degradative cassane-type diterpenes isolated from Caesalpinia minax.

Cassane-type diterpenes are main bioactive constituents of Caesalpinia minax HANCE. As a part of our ongoing chemical investigation of C. minax, two new degradative cassane-type diterpenes, named caesalpins I (1) and J (2), were isolated from the EtOAc extract of the seeds of C. minax. The structures were elucidated by means of spectroscopic analysis.