A Biomimetic Approach to Premyrsinane-Type Diterpenoids: Exploring Microbial Transformation to Enhance Their Chemical Diversity.

Premyrsinane-type diterpenoids have been considered to originate from the cyclization of a suitable 5,6- or 6,17-epoxylathyrane precursor. Their biological activities have not been sufficiently explored to date, so the development of synthetic or microbial approaches for the preparation of new derivatives would be desirable. Epoxyboetirane A (4) is an 6,17-epoxylathyrane isolated from Euphorbia boetica in a large enough amount to be used in semi-synthesis. Transannular cyclization of 4 mediated by Cp2TiIIICl afforded premyrsinane 5 in good yield as an only diasteroisomer. To enhance the structural diversity of premyrsinanes so their potential use in neurodegenerative disorders could be explored, compound 5 was biotransformed by Mucor circinelloides NRRL3631 to give rise to hydroxylated derivatives at non-activated carbons (6-7), all of which were reported here for the first time. The structures and absolute configurations of all compounds were determined through extensive NMR and HRESIMS spectroscopic studies.

Enhancing Structural Diversity of Lathyrane Derivatives through Biotransformation by the Marine-Derived Actinomycete Streptomyces puniceus BC-5GB.11.

Lathyrane-type diterpenes have a wide range of biological activities. Among them, euphoboetirane A (1) exerts neurogenesis-promoting activity. In order to increase the structural diversity of this type of lathyrane and explore its potential use in neurodegenerative disorders, the biotransformation of 1 by Streptomyces puniceus BC-5GB.11 has been investigated. The strain BC-5GB.11, isolated from surface sediments collected from the intertidal zone of the inner Bay of Cadiz, was identified as Streptomyces puniceus, as determined by phylogenetic analysis using 16S rRNA gene sequence. Biotransformation of 1 by BC-5GB.11 afforded five products (3-7), all of which were reported here for the first time. The main biotransformation pathways involved regioselective oxidation at non-activated carbons (3-5) and isomerization of the ∆12,13 double bond (6). In addition, a cyclopropane-rearranged compound was found (7). The structures of all compounds were elucidated on the basis of extensive NMR and HRESIMS spectroscopic studies.

Isolation and Identification of 12-Deoxyphorbol Esters from Euphorbia resinifera Berg Latex: Targeted and Biased Non-Targeted Identification of 12-Deoxyphorbol Esters by UHPLC-HRMSE.

Diterpenes from the Euphorbia genus are known for their ability to regulate the protein kinase C (PKC) family, which mediates their ability to promote the proliferation of neural precursor cells (NPCs) or neuroblast differentiation into neurons. In this work, we describe the isolation from E. resinifera Berg latex of fifteen 12-deoxyphorbol esters (1-15). A triester of 12-deoxy-16-hydroxyphorbol (4) and a 12-deoxyphorbol 13,20-diester (13) are described here for the first time. Additionally, detailed structural elucidation is provided for compounds 3, 5, 6, 14 and 15. The absolute configuration for compounds 3, 4, 6, 13, 14 and 15 was established by the comparison of their theoretical and experimental electronic circular dichroism (ECD) spectra. Access to the above-described collection of 12-deoxyphorbol derivatives, with several substitution patterns and attached acyl moieties, allowed for the study of their fragmentation patterns in the collision-induced dissociation of multiple ions, without precursor ion isolation mass spectra experiments (HRMSE), which, in turn, revealed a correlation between specific substitution patterns and the fragmentation pathways in their HRMSE spectra. In turn, this allowed for a targeted UHPLC-HRMSE analysis and a biased non-targeted UHPLC-HRMSE analysis of 12-deoxyphorbols in E. resinifera latex which yielded the detection and identification of four additional 12-deoxyphorbols not previously isolated in the initial column fractionation work. One of them, identified as 12-deoxy-16-hydroxyphorbol 20-acetate 13-phenylacetate 16-propionate (20), has not been described before.

Rescue of neurogenesis and age-associated cognitive decline in SAMP8 mouse: Role of transforming growth factor-alpha.

Neuropathological aging is associated with memory impairment and cognitive decline, affecting several brain areas including the neurogenic niche of the dentate gyrus of the hippocampus (DG). In the healthy brain, homeostatic mechanisms regulate neurogenesis within the DG to facilitate the continuous generation of neurons from neural stem cells (NSC). Nevertheless, aging reduces the number of activated neural stem cells and diminishes the number of newly generated neurons. Strategies that promote neurogenesis in the DG may improve cognitive performance in the elderly resulting in the development of treatments to prevent the progression of neurological disorders in the aged population. Our work is aimed at discovering targeting molecules to be used in the design of pharmacological agents that prevent the neurological effects of brain aging. We study the effect of age on hippocampal neurogenesis using the SAMP8 mouse as a model of neuropathological aging. We show that in 6-month-old SAMP8 mice, episodic and spatial memory are impaired; concomitantly, the generation of neuroblasts and neurons is reduced and the generation of astrocytes is increased in this model. The novelty of our work resides in the fact that treatment of SAMP8 mice with a transforming growth factor-alpha (TGFα) targeting molecule prevents the observed defects, positively regulating neurogenesis and improving cognitive performance. This compound facilitates the release of TGFα in vitro and in vivo and activates signaling pathways initiated by this growth factor. We conclude that compounds of this kind that stimulate neurogenesis may be useful to counteract the neurological effects of pathological aging.

Pharmacological Potential of Lathyrane-Type Diterpenoids from Phytochemical Sources.

Lathyrane diterpenoids are one of the primary types of secondary metabolites present in the genus Euphorbia and one of the largest groups of diterpenes. They are characterized by having a highly oxygenated tricyclic system of 5, 11 and 3 members. These natural products and some synthetic derivatives have shown numerous interesting biological activities with clinical potential against various diseases, such as cytotoxic activity against cancer cell lines, multi-drug resistance reversal, antiviral properties, anti-inflammatory activity and their capability to induce proliferation or differentiation into neurons of neural progenitor cells. The structure of the lathyrane skeleton could be considered privileged because its framework is able to direct functional groups in a well-defined space. The favorable arrangement of these makes interaction possible with more than one target. This review aims to highlight the evidence of lathyranes as privileged structures in medicinal chemistry. Chemical structures of bioactive compounds, the evaluation of biological properties of natural and semisynthetic derivatives, and the exploration of the mechanisms of action as well as target identification and some aspects of their targeted delivery are discussed.

The complemented mutant complΔBcstc7niaD, in the STC7 of Botrytis cinerea led to the characterization of 11,12,13-tri-nor-eremophilenols derivatives.

Botrytis cinerea has high potential for the production of specialized metabolites. The recent resequencing of the genome of the B05.10 strain using PacBio technology and the resulting update of the Ensembl Fungi (2017) database in the genome sequence have been instrumental in identifying new genes that could be involved in secondary metabolism. Thus, a new sesquiterpene cyclase (STC) coding gene (Bcstc7) has been included in the gene list from this phytopathogenic fungus. We recently constructed the null and complement transformants in STC7 which enabled us to functionally characterize this STC. Deletion of the Bcstc7 gene abolished (+)-4-epi-eremophilenol biosynthesis, and could then be re-established by complementing the null mutant with the Bcstc7 gene. Chemical analysis of the complemented transformant suggests that STC7 is the principal enzyme responsible for the key cyclization step of farnesyl diphosphate (FDP) to (+)-4-epi-eremophil-9-en-11-ols. A thorough analysis of the metabolites produced by two wild-type strains, B05.10 and UCA992, and the complemented mutant complΔBcstc7niaD, revealed the isolation and structural characterization of six 11,12,13-tri-nor-eremophilene derivatives, in addition to a large number of known eremophilen-11-ol derivatives. The structural characterization was carried out by extensive spectroscopic techniques. The biosynthesis of these compounds is explained by a retroaldol reaction or by dehydration and oxidative cleavage of C11-C13 carbons. This is the first time that this interesting family of degraded eremophilenols has been isolated from the phytopathogenous fungus B. cinerea.

Phorbol Diesters and 12-Deoxy-16-hydroxyphorbol 13,16-Diesters Induce TGFα Release and Adult Mouse Neurogenesis.

A small library of phorbol 12,13-diesters bearing low lipophilicity ester chains was prepared as potential neurogenic agents in the adult brain. They were also used in a targeted UHPLC-HRMS screening of the latex of Euphorbia resinifera. Two new 12-deoxy-16-hydroxyphorbol 13,16-diesters were isolated, and their structures were deduced using two-dimensional NMR spectroscopy and NOE experiments. The ability of natural and synthetic compounds to stimulate transforming growth factor alpha (TFGα) release, to increase neural progenitor cell proliferation, and to stimulate neurogenesis was evaluated. All compounds that facilitated TGFα release promoted neural progenitor cell proliferation. The presence of two acyloxy moieties on the tigliane skeleton led to higher levels of activity, which decreased when a free hydroxyl group was at C-12. Remarkably, the compound bearing isobutyryloxy groups was the most potent on the TGFα assay and at inducing neural progenitor cell proliferation in vitro, also leading to enhanced neurogenesis in vivo when administered intranasally to mice.

Impairment of botrydial production in Botrytis cinerea allows the isolation of undescribed polyketides and reveals new insights into the botcinins biosynthetic pathway.

Botrytis cinerea is a necrotrophic fungal pathogen that affects a total of 586 genera representing approximately 1400 plant species. This pathogen produces two families of phytotoxins involved in its infection process i.e. botrydial and its relatives, and botcinic and botcineric acids and their relatives, botcinins. The botrydial biosynthetic cluster consists of seven genes, where the gene BcBOT4 encodes a cytochrome P450 monooxygenase that was shown to catalyse regio- and stereospecific hydroxylation at position C-4 of the presilphiperfolan-8-ß-ol skeleton. The null mutant bcbot4Δ halted the production of botrydial and its derivatives, and instead accumulated tricyclic presilphiperfolane alcohol and overproduced a significant number of polyketides. A detailed study of the bcbot4Δ mutant led us to the isolation and characterization of five undescribed polyketides, three derived from botcinic and botcineric acids (botcinins H, I, J), one derived from the initial pentaketide (botcinin K), and one cinbotolide derivative (cinbotolide D). Botcinins are tetra-methylated tetraketides biosynthesized by the sequential assembly of a pentaketide (C10) based on an acetate primer unit which is lost through a retro-Claisen type C-C bond cleavage. The structural characterization of botcinin K showed a basic chemical structure corresponding to a botcinin (C14) derivative obtained directly from the original per-methylated pentaketide leading to the biosynthesis of botrylactone and other botcinins, confirming the previously proposed biosynthetic route.

Effects of classical PKC activation on hippocampal neurogenesis and cognitive performance: mechanism of action.

Hippocampal neurogenesis has widely been linked to memory and learning performance. New neurons generated from neural stem cells (NSC) within the dentate gyrus of the hippocampus (DG) integrate in hippocampal circuitry participating in memory tasks. Several neurological and neuropsychiatric disorders show cognitive impairment together with a reduction in DG neurogenesis. Growth factors secreted within the DG promote neurogenesis. Protein kinases of the protein kinase C (PKC) family facilitate the release of several of these growth factors, highlighting the role of PKC isozymes as key target molecules for the development of drugs that induce hippocampal neurogenesis. PKC activating diterpenes have been shown to facilitate NSC proliferation in neurogenic niches when injected intracerebroventricularly. We show in here that long-term administration of diterpene ER272 promotes neurogenesis in the subventricular zone and in the DG of mice, affecting neuroblasts differentiation and neuronal maturation. A concomitant improvement in learning and spatial memory tasks performance can be observed. Insights into the mechanism of action reveal that this compound facilitates classical PKCα activation and promotes transforming growth factor alpha (TGFα) and, to a lesser extent, neuregulin release. Our results highlight the role of this molecule in the development of pharmacological drugs to treat neurological and neuropsychiatric disorders associated with memory loss and a deficient neurogenesis.

Bond reactivity indices approach analysis of the [2+2] cycloaddition of jatrophane skeleton diterpenoids from Euphorbia gaditana Coss to tetracyclic gaditanone.

The reaction mechanism of the intramolecular [2 + 2] cycloaddition from a jatrophane precursor to the gaditanane skeleton, an unprecedented 5/6/4/6-fused tetracyclic ring framework recently isolated from Euphorbia spp., was studied using the bond reactivity indices approach. Furthermore, six diterpenoids, including three undescribed jatrophanes isolated from E. gaditana Coss, were described. The structures of these compounds were deduced by a combination of 2D NMR spectroscopy and ECD data analysis.

Synthesis of Degraded Limonoid Analogs as New Antibacterial Scaffolds against Staphylococcus aureus.

Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) have become serious infections in humans and ruminants. S. aureus strains are showing rapid changes to develop resistance in traditional antibiotic-containing systems. In the continuous fierce fight against the emergent multi-drug resistant bacterial strains, straightforward and scalable synthetic procedures to produce new active molecules are in demand. Analysis of molecular properties points to degraded limonoids as promising candidates. In this article, we report a simple synthetic approach to obtain degraded limonoid analogs as scaffolds for new antibacterial molecules. The minimum inhibitory concentrations against S. aureus were evaluated for the stereoisomer mixtures by the broth microdilution method. Analysis of results showed that the acetylated derivatives were the most active of them all.

A novel PKC activating molecule promotes neuroblast differentiation and delivery of newborn neurons in brain injuries.

Neural stem cells are activated within neurogenic niches in response to brain injuries. This results in the production of neuroblasts, which unsuccessfully attempt to migrate toward the damaged tissue. Injuries constitute a gliogenic/non-neurogenic niche generated by the presence of anti-neurogenic signals, which impair neuronal differentiation and migration. Kinases of the protein kinase C (PKC) family mediate the release of growth factors that participate in different steps of the neurogenic process, particularly, novel PKC isozymes facilitate the release of the neurogenic growth factor neuregulin. We have demonstrated herein that a plant derived diterpene, (EOF2; CAS number 2230806-06-9), with the capacity to activate PKC facilitates the release of neuregulin 1, and promotes neuroblasts differentiation and survival in cultures of subventricular zone (SVZ) isolated cells in a novel PKC dependent manner. Local infusion of this compound in mechanical cortical injuries induces neuroblast enrichment within the perilesional area, and noninvasive intranasal administration of EOF2 promotes migration of neuroblasts from the SVZ towards the injury, allowing their survival and differentiation into mature neurons, being some of them cholinergic and GABAergic. Our results elucidate the mechanism of EOF2 promoting neurogenesis in injuries and highlight the role of novel PKC isozymes as targets in brain injury regeneration.

Lathyrane, Premyrsinane, and Related Diterpenes from Euphorbia boetica: Effect on in Vitro Neural Progenitor Cell Proliferation.

Lathyrane-type diterpenes previously have been proven to promote proliferation of neural precursor cells (NPCs) by targeting and activating one or more protein kinase C (PKC) isozymes. Aiming to find new drug candidates with a lathyrane skeleton to modulate adult neurogenesis through PKC activation, a phytochemical study of a methanol extract of the aerial parts of Euphorbia boetica was carried out. Seven new diterpenes, representing the premyrsinane (1-3), myrsinane (4, 5), and cyclomyrsinane types (6, 7), along with three known diterpenes, belonging to the cyclomyrsinane (8) and lathyrane types (9, 10), were isolated. The chemical structures and relative configurations of the new compounds were determined by extensive NMR spectroscopic studies and comparison with known compounds. The absolute configurations for compounds 2, 3, 6, and 7 were proposed, based on a comparison of the experimental ECD spectra of compounds 2 and 7 with those of known related compounds. The activity of lathyrane compounds 9 and 10 as promoters of NPC proliferation was evaluated using a neurosphere assay. Both compounds increased the size of neurospheres in a dose-dependent manner when proliferation was stimulated by the epidermal growth factor and the basic fibroblast growth factor.

Structural and biosynthetic studies on eremophilenols related to the phytoalexin capsidiol, produced by Botrytis cinerea.

A thorough study of the fermentation broth of three strains of Botrytis cinerea which were grown on a modified Czapek-Dox medium supplemented with 5 ppm copper sulphate, yielded five undescribed metabolites. These metabolites possessed a sesquiterpenoid (+)-4-epi-eremophil-9-ene carbon skeleton which was enantiomeric to that of the phytoalexin, capsidiol. The isolation of these metabolites when the fungus was stressed, suggests that they may be potential effectors used by B. cinerea to circumvent plant chemical defences against phytopathogenic fungi. The biosynthesis of these compounds has been studied using 2H and 13C labelled acetate.

Gaditanone, a Diterpenoid Based on an Unprecedented Carbon Skeleton Isolated from Euphorbia gaditana.

A novel diterpenoid, gaditanone (2), which possesses an unprecedented 5/6/4/6-fused gaditanane tetracyclic ring skeleton, and a new jatrophane (1) were isolated from the aerial parts of Euphorbia gaditana. The chemical structures and absolute configurations were determined by extensive spectroscopic NMR studies and ECD data analysis. A proposed biosynthetic pathway is presented for compound 2.

The formation of sesquiterpenoid presilphiperfolane and cameroonane metabolites in the Bcbot4 null mutant of Botrytis cinerea.

Botrytis cinerea is a polyphagous fungal parasite which causes serious damage to more than 200 plant species and consequent economic losses for commercial crops. This pathogen produces two families of phytotoxins, the botryanes and botcinins, which are involved in the infection mechanism. The B. cinerea genome has provided a complete picture of the genes involved in the biosynthesis of its secondary metabolites. The botrydial biosynthetic gene cluster has been identified. This cluster consists of seven genes, where the genes BcBOT1, BcBOT3 and BcBOT4 encode three mono-oxygenases. A study of the Bcbot4Δ null mutant revealed that this mono-oxygenase was involved in the hydroxylation at C-4 of the probotryane skeleton (C-11 of the presilphiperfolane skeleton). A detailed study of the Bcbot4Δ null mutant has been undertaken in order to study the metabolic fate of the presilphiperfolan-8-ol intermediate biosynthesized by this organism and in particular by this strain. As a result three new presilphiperfolanes and three new cameroonanes have been identified. The results suggest that the absence of the oxygen function at C-11 of the presilphiperfolane skeleton permits rearrangement to a cameroonane whilst hydroxylation at C-11 precludes this rearrangement. It is possible that the interactions of the C-11 hydroxylated derivatives perturb the stereo-electronic requirements for the migration of the C-11:C-7 sigma bond to C-8.

ELAC (3,12-di-O-acetyl-8-O-tigloilingol), a plant-derived lathyrane diterpene, induces subventricular zone neural progenitor cell proliferation through PKCß activation.

BACKGROUND AND PURPOSE: Pharmacological strategies aimed to facilitate neuronal renewal in the adult brain, by promoting endogenous neurogenesis, constitute promising therapeutic options for pathological or traumatic brain lesions. We have previously shown that non-tumour-promoting PKC-activating compounds (12-deoxyphorbols) promote adult neural progenitor cell (NPC) proliferation in vitro and in vivo, enhancing the endogenous neurogenic response of the brain to a traumatic injury. Here, we show for the first time that a diterpene with a lathyrane skeleton can also activate PKC and promote NPC proliferation. EXPERIMENTAL APPROACH: We isolated four lathyranes from the latex of Euphorbia plants and tested their effect on postnatal NPC proliferation, using neurosphere cultures. The bioactive lathyrane ELAC (3,12-di-O-acetyl-8-O-tigloilingol) was also injected into the ventricles of adult mice to analyse its effect on adult NPC proliferation in vivo. KEY RESULTS: The lathyrane ELAC activated PKC and significantly increased postnatal NPC proliferation in vitro, particularly in synergy with FGF2. In addition ELAC stimulated proliferation of NPC, specifically affecting undifferentiated transit amplifying cells. The proliferative effect of ELAC was reversed by either the classical/novel PKC inhibitor Gö6850 or the classical PKC inhibitor Gö6976, suggesting that NPC proliferation is promoted in response to activation of classical PKCs, particularly PKCß. ELAC slightly increased the proportion of NPC expressing Sox2. The effects of ELAC disappeared upon acetylation of its C7-hydroxyl group. CONCLUSIONS AND IMPLICATIONS: We propose lathyranes like ELAC as new drug candidates to modulate adult neurogenesis through PKC activation. Functional and structural comparisons between ELAC and phorboids are included.

Mild Epoxidation of Allylic Alcohols Catalyzed by Titanium(III) Complexes: Selectivity and Mechanism.

A novel methodology for the epoxidation of a broad range of primary, secondary, and tertiary allylic alcohols is described using tert-butyl hydroperoxide as oxidant and Ti(III) species generated by reduction of Ti(IV) complexes, with manganese (0) in 1,4-dioxane under mild reaction conditions. The reaction proceeded with wide substrate scope and high chemo- and diastereoselectivity. A mechanistic pathway for the reaction is also discussed.

Lathyrane Diterpenes from the Latex of Euphorbia laurifolia.

Two new macrocyclic diterpenes, 2-epi-latazienone (4) and 15ß-acetoxy-7ß-nicotinoyloxy-3ß,8α-di-(2-methylpropanoyloxy)-4αH,9αH,l1αH-lathyra- 5E,12E-dien-14-one (5), and three known lathyrane-type diterpenes (1-3) were isolated from Euphorbia laurifolia latex. Their structures were determined on the basis of a detailed analysis of their 1D and 2D NMR spectroscopic and mass spectral data.