Effect of lathyrane-type diterpenoids in neural stem cell physiology: Microbial transformations, molecular docking and dynamics studies.

Promoting endogenous neurogenesis for brain repair is emerging as a promising strategy to mitigate the functional impairments associated with various neurological disorders characterized by neuronal death. Diterpenes featuring tigliane, ingenane, jatrophane and lathyrane skeletons, frequently found in Euphorbia plant species, are known protein kinase C (PKC) activators and exhibit a wide variety of pharmacological properties, including the stimulation of neurogenesis. Microbial transformation of these diterpenes represents a green and sustainable methodology that offers a hitherto little explored approach to obtaining novel derivatives and exploring structure-activity relationships. In the present study, we report the biotransformation of euphoboetirane A (4) and epoxyboetirane A (5), two lathyrane diterpenoids isolated from Euphorbia boetica, by Mucor circinelloides MC NRRL3631. Our findings revealed the production of nine biotransformation products (6-14), including jatrophane derivatives originated through an unprecedented rearrangement from the parent lathyranes. The chemical structures and absolute configurations of the new compounds were elucidated through comprehensive analysis using NMR and ECD spectroscopy, as well as MS. The study evaluated how principal metabolites and their derivatives affect TGFα and NRG1 release, as well as their potential to promote proliferation or differentiation in cultures of NSC isolated from the SVZ of adult mice. In order to shed some light on the mechanisms underlying the ability of 12 as a neurogenic compound, the interactions of selected compounds with PKC δ-C1B were analyzed through molecular docking and molecular dynamics. Based on these, it clearly appears that the ability of compound 12 to form both acceptor and donor hydrogen bonds with certain amino acid residues in the enzyme pocket leads to a higher affinity compound-PKC complex, which correlates with the observed biological activity.

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.

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.

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.

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.

Efficient O-Acylation of Alcohols and Phenol Using Cp2TiCl as a Reaction Promoter.

A method has been developed for the conversion of primary, secondary, and tertiary alcohols, and phenol, into the corresponding esters at room temperature. The method uses a titanium(III) species generated from a substoichiometric amount of titanocene dichloride together with manganese(0) as a reductant, as well as methylene diiodide. It involves a transesterification from an ethyl ester, or a reaction with an acyl chloride. A radical mechanism is proposed for these transformations.

Biological activity of natural sesquiterpenoids containing a gem-dimethylcyclopropane unit.

The biological activities of aristolane, aromadendrane, ent-1,10-secoaromadendrane, 2,3-secoaromadendrane, ent-5,10-cycloaromadendrane, bicyclogermacrene, lepidozane, and maaliane terpenoids which contain the gem-dimethylcyclopropyl unit are described. Particular attention is given to their anti-viral, anti-microbial and cytotoxic activities. In the main text there are 119 references covering the literature from 1963-2014. The ESI contains tables listing 332 of these terpenoids, their occurrence and biological activity together with the related references.

12-Deoxyphorbols Promote Adult Neurogenesis by Inducing Neural Progenitor Cell Proliferation via PKC Activation.

BACKGROUND: Neuropsychiatric and neurological disorders frequently occur after brain insults associated with neuronal loss. Strategies aimed to facilitate neuronal renewal by promoting neurogenesis constitute a promising therapeutic option to treat neuronal death-associated disorders. In the adult brain, generation of new neurons occurs physiologically throughout the entire life controlled by extracellular molecules coupled to intracellular signaling cascades. Proteins participating in these cascades within neurogenic regions constitute potential pharmacological targets to promote neuronal regeneration of injured areas of the central nervous system. METHODOLOGY: We have performed in vitro and in vivo approaches to determine neural progenitor cell proliferation to understand whether activation of kinases of the protein kinase C family facilitates neurogenesis in the adult brain. RESULTS: We have demonstrated that protein kinase C activation by phorbol-12-myristate-13-acetate induces neural progenitor cell proliferation in vitro. We also show that the nontumorogenic protein kinase C activator prostratin exerts a proliferative effect on neural progenitor cells in vitro. This effect can be reverted by addition of the protein kinase C inhibitor G06850, demonstrating that the effect of prostratin is mediated by protein kinase C activation. Additionally, we show that prostratin treatment in vivo induces proliferation of neural progenitor cells within the dentate gyrus of the hippocampus and the subventricular zone. Finally, we describe a library of diterpenes with a 12-deoxyphorbol structure similar to that of prostratin that induces a stronger effect than prostratin on neural progenitor cell proliferation both in vitro and in vivo. CONCLUSIONS: This work suggests that protein kinase C activation is a promising strategy to expand the endogenous neural progenitor cell population to promote neurogenesis and highlights the potential of 12-deoxyphorbols as pharmaceutical agents to facilitate neuronal renewal.

nor-Mevaldic acid surrogates as selective antifungal agent leads against Botrytis cinerea. Enantioselective preparation of 4-hydroxy-6-(1-phenylethoxy)tetrahydro-2H-pyran-2-one.

Solvent-free desymmetrisation of meso-dialdehyde 1 with chiral 1-phenylethan-1-ol, led to preparation of 4-silyloxy-6-alkyloxytetrahydro-2H-pyran-2-one (+)-3a with a 96:4 dr Deprotected lactone (+)-19a and the related racemic lactones 16a-18a present a lactone moiety resembling the natural substrate of HMG-CoA reductase and their antifungal properties have been evaluated against the phytopathogenic fungi Botrytis cinerea and Colletotrichum gloeosporioides. These compounds were selectively active against B. cinerea, while inactive against C. gloeosporioides.

Biologically active diterpenes containing a gem-dimethylcyclopropane subunit: an intriguing source of PKC modulators.

Covering: 1973 to 2013. The biological activities of tigliane, lathyrane, ingenane, casbane, jatropholane and premyrsinane diterpenoids which contain the gem-dimethylcyclopropyl unit are described. Particular attention is given to their anti-viral, anti-microbial and cytotoxic activities. In the main text there are 132 references. The electronic supplementary information contains tables listing 424 of these diterpenoids, their occurrence and biological activity together with the references.

The asymmetric total synthesis of cinbotolide: a revision of the original structure.

The structure 3,4-dihydroxy-2,4,6,8-tetramethyldec-8-enolide (1) was assigned to a metabolite of Botrytis cinerea, but the spectra of several synthetic analogues had significant differences from that of 1. Examination of the constituents of a B. cinerea mutant that overproduces polyketides gave sufficient quantities of 1, now named cinbotolide, for chemical transformations. These led to a revised γ-butyrolactone structure for the metabolite. This structure has been confirmed by an asymmetric total synthesis, which also established its absolute configuration.

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.

A shared biosynthetic pathway for botcinins and botrylactones revealed through gene deletions.

Isotopic labelling experiments and the study of mutants with disrupted genes encoding botcinic acid have revealed a common link in the biosynthesis of the polyketide toxins excreted by Botrytis cinerea: botcinins and botrylactones. Furthermore, the results reported here shed light on the origin of the starter unit, thereby solving a long-standing mystery in the biosynthesis of botcinins.

Phytotoxic activity and metabolism of Botrytis cinerea and structure-activity relationships of isocaryolane derivatives.

Research has been conducted on the biotransformation of (8S,9R)-isocaryolan-9-ol (4a) and (1S,2S,5R,8S)-8-methylene-1,4,4-trimethyltricyclo[6.2.1.0(2,5)]undecan-12-ol (5a) by the fungal phytopathogen Botrytis cinerea. The biotransformation of compound 4a yielded compounds 6-9, while the biotransformation of compound 5a yielded compounds 10-13. The activity of compounds 4a and 5a against B. cinerea has been evaluated. (8R,9R)-Isocaryolane-8,9-diol (6), a major metabolite of compound 4a, shows activity compared to its parent compound 4a, which is inactive. The effect of isocaryolanes 3, 4a, and 5a, together with their biotransformation products 6-8, 10, and 14-17, on the germination and radicle and shoot growth of Lactuca sativa (lettuce) has also been determined. Compounds 7-13 are described for the first time.

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.

Biotransformation of clovane derivatives. Whole cell fungi mediated domino synthesis of rumphellclovane A.

Here we describe the biotransformation of clovane derivatives by filamentary fungi Pestalotiopsis palustris and Penicillium minioluteum, and the application of the latter to the synthesis and determination of the absolute configuration of rumphellclovane A (2). Methoxyclovanol (1), a growth inhibitor of the phytopathogen Botrytis cinerea, is metabolised by P. palustris to yield rumphellclovane A (2), a natural compound recently isolated from the gorgonian coral Rumphella antipathies, two new compounds, (1R,2S,5S,8R,9S,10R)-2-methoxyclovane-9,10-diol (5) and (1S,2S,5S,7R,8R,9R)-2-methoxyclovane-7,9-diol (6), hydroxylated in positions not easily accessed by classic synthetic chemistry, and clovanodiols 3 and 4. P. minioluteum is able to selectively transform methoxyclovanol (1) into clovanodiols 3 and 4 and, in turn, lactone 8, the putative intermediate in the above mentioned synthesis of rumphellclovane A (2), into compound 2 via a domino process. The ability of P. minioluteum to carry out the cleavage of ethers on clovane derivatives is also evaluated.

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.

Biotransformation of bioactive isocaryolanes by Botrytis cinerea.

The metabolism of the fungistatic agent (8R,9R)-8-methoxyisocaryolan-9-ol (4) by the fungus Botrytis cinerea has been investigated. Biotransformation of compound 4 yielded compounds 5 and 6-9. No dihydrobotrydial is observed after 4 days of incubation of compound 4. Separate biotransformation of (8R,9R)-isocaryolane-8,9-diol (5) yielded compounds 7-11. The evaluation of the fungistatic activity against B. cinerea of compounds 4, 5, and 6 is reported. (4R,8R,9R)-8-Methoxyisocaryolane-9,15-diol (6), a major metabolite of (8R,9R)-8-methoxyisocaryolan-9-ol (4), shows a much reduced biological activity when compared with the parent compound. Isocaryolane derivatives 6-11 are described for the first time.