In Vitro Cytotoxic Effects of Ferruginol Analogues in Sk-MEL28 Human Melanoma Cells.

Ferruginol is a promising abietane-type antitumor diterpene able to induce apoptosis in SK-Mel-28 human malignant melanoma. We aim to increase this activity by testing the effect of a small library of ferruginol analogues. After a screening of their antiproliferative activity (SRB staining, 48 h) on SK-Mel-28 cells the analogue 18-aminoferruginol (GI50 ≈ 10 µM) was further selected for mechanistic studies including induction of apoptosis (DAPI staining, p < 0.001), changes in cell morphology associated with the treatment (cell shrinkage and membrane blebbing), induction of caspase-3/7 activity (2.5 at 48 h, 6.5 at 72 h; p < 0.0001), changes in the mitochondrial membrane potential (not significant) and in vitro effects on cell migration and cell invasion (Transwell assays, not significant). The results were compared to those of the parent molecule (ferruginol, GI50 ≈ 50 µM, depolarisation of mitochondrial membrane p < 0.01 at 72 h; no caspases 3/7 activation) and paclitaxel (GI50 ≈ 10 nM; caspases 3/7 activation p < 0.0001) as a reference drug. Computational studies of the antiproliferative activity of 18-aminoferruginol show a consistent improvement in the activity over ferruginol across a vast majority of cancer cells in the NCI60 panel. In conclusion, we demonstrate here that the derivatisation of ferruginol into 18-aminoferruginol increases its antiproliferative activity five times in SK-MEL-28 cells and changes the apoptotic mechanism of its parent molecule, ferruginol.

Synergistic In Vitro Antiviral Effect of Combinations of Ivermectin, Essential Oils, and 18-(Phthalimid-2-yl)ferruginol against Arboviruses and Herpesvirus.

Combining antiviral drugs with different mechanisms of action can help prevent the development of resistance by attacking the infectious agent through multiple pathways. Additionally, by using faster and more economical screening methods, effective synergistic drug candidates can be rapidly identified, facilitating faster paths to clinical testing. In this work, a rapid method was standardized to identify possible synergisms from drug combinations. We analyzed the possible reduction in the antiviral effective concentration of drugs already approved by the FDA, such as ivermectin (IVM), ribavirin (RIBA), and acyclovir (ACV) against Zika virus (ZIKV), Chikungunya virus (CHIKV), and herpes virus type 2 (HHV-2). Essential oils (EOs) were also included in the study since they have been reported for more than a couple of decades to have broad-spectrum antiviral activity. We also continued studying the antiviral properties of one of our patented molecules with broad-spectrum antiviral activity, the ferruginol analog 18-(phthalimid-2-yl)ferruginol (phthFGL), which presented an IC99 of 25.6 µM for the three types of virus. In general, the combination of IVM, phthFGL, and oregano EO showed the greatest synergism potential against CHIKV, ZIKV, and HHV-2. For instance, this combination achieved reductions in the IC99 value of each component up to ~8-, ~27-, and ~12-fold for CHIKV, respectively. The ternary combination of RIBA, phthFGL, and oregano EO was slightly more efficient than the binary combination RIBA/phthFGL but much less efficient than IVM, phthFGL, and oregano EO, which indicates that IVM could contribute more to the differentiation of cell targets (for example via the inhibition of the host heterodimeric importin IMP α/ß1 complex) than ribavirin. Statistical analysis showed significant differences among the combination groups tested, especially in the HHV-2 and CHIKV models, with p = 0.0098. Additionally, phthFGL showed a good pharmacokinetic profile that should encourage future optimization studies.

Anticoronavirus Evaluation of Antimicrobial Diterpenoids: Application of New Ferruginol Analogues.

The abietane diterpene (+)-ferruginol (1), like other natural and semisynthetic abietanes, is distinguished for its interesting pharmacological properties such as antimicrobial activity, including antiviral. In this study, selected C18-functionalized semisynthetic abietanes prepared from the commercially available (+)-dehydroabietylamine or methyl dehydroabietate were tested in vitro against human coronavirus 229E (HCoV-229E). As a result, a new ferruginol analogue caused a relevant reduction in virus titer as well as the inhibition of a cytopathic effect. A toxicity prediction based on in silico analysis was also performed as well as an estimation of bioavailability. This work demonstrates the antimicrobial and specifically antiviral activity of two tested compounds, making these molecules interesting for the development of new antivirals.

Discovery of Novel Bioactive Tanshinones and Carnosol Analogues against Breast Cancer.

The abietane diterpenoids ferruginol (1), tanshinone IIA (3), and carnosol (4) are well-known for their interesting pharmacological properties, including antitumor, similar to other natural and semisynthetic abietanes. In this study, a pair of semisynthetic C18-functionalized analogues of 3 and 4 were prepared from the commercially available (+)-dehydroabietylamine or readily obtained methyl dehydroabietate. Semisynthetic ferruginol (1) and some selected analogues, together with the synthesized analogues, were tested in vitro for the inhibition of proliferation in four breast cancer cell lines, SUM149, MDA-MB231, T47D, and MCF07. As a result, several tested abietane analogues decreased cell proliferation and enhanced cell death, with IC50 in the range 1.3-18.7 µM. This work demonstrates the antitumor activities of two tested compounds, making these molecules interesting for the development of new anticancer agents.

Semisynthetic Abietic and Dehydroabietic Acid Derivatives and Triptoquinone Epimers Interfere with LPS-Triggered Activation of Dendritic Cells.

Abietic acid (AA), dehydroabietic acid (DHA) and triptoquinones (TQs) are bioactive abietane-type diterpenoids, which are present in many edible vegetables and medicinal herbs with health-promoting properties. Evidence suggests that beneficial effects of diterpenes operate, at least in part, through effects on cells in the immune system. Dendritic cells (DCs) are a key type of leukocyte involved in the initiation and regulation of the immune/inflammatory response and natural or synthetic compounds that modulate DC functions could be potential anti-inflammatory/immunomodulatory agents. Herein, we report the screening of 23 known semisynthetic AA and DHA derivatives, and TQs, synthesized previously by us, in a multi-analyte DC-based assay that detects inhibition of pro-inflammatory cytokine production. Based on the magnitude of the inhibitory effect observed and the number of cytokines inhibited, a variety of activities among compounds were observed, ranging from inactive/weak to very potent inhibitors. Structurally, either alcohol or methyl ester substituents on ring A along with the introduction of aromaticity and oxidation in ring C in the abietane skeleton were found in compounds with higher inhibitory properties. Two DHA derivatives and two TQs exhibited a significant inhibition in all pro-inflammatory cytokines tested and were further investigated. The results confirmed their ability to inhibit, dose dependently, LPS-stimulated expression of the co-stimulatory molecules CD40 and/or CD86 and the production of the pro-inflammatory cytokines IL-1ß, IL-6, IL-12 and TNFα. Our results demonstrate that DC maturation process can be targeted by semisynthetic DHA derivatives and TQ epimers and indicate the potential of these compounds as optimizable anti-inflammatory/immunomodulatory agents.

Antifungal Activity of Amphiphilic Perylene Bisimides.

Perylene-based compounds, either naturally occurring or synthetic, have shown interesting biological activities. In this study, we report on the broad-spectrum antifungal properties of two lead amphiphilic perylene bisimides, compounds 4 and 5, which were synthesized from perylene-3,4,9,10-tetracarboxylic dianhydride by condensation with spermine and an ammonium salt formation. The antifungal activity was evaluated using a collection of fungal strains and clinical isolates from patients with onychomycosis or sporotrichosis. Both molecules displayed an interesting antifungal profile with MIC values in the range of 2-25 µM, being as active as several reference drugs, even more potent in some particular strains. The ammonium trifluoroacetate salt 5 showed the highest activity with a MIC value of 2.1 µM for all tested Candida spp., two Cryptococcus spp., two Fusarium spp., and one Neoscytalidium spp. strain. Therefore, these amphiphilic molecules with the perylene moiety and cationic ammonium side chains represent important structural features for the development of novel antifungals.

Antiviral Profiling of C-18- or C-19-Functionalized Semisynthetic Abietane Diterpenoids.

Viral infections affect several million patients annually. Although hundreds of viruses are known to be pathogenic, only a few can be treated in the clinic with available antiviral drugs. Naturally based pharmacotherapy may be a proper alternative for treating viral diseases. Several natural and semisynthetic abietane-type diterpenoids have shown important antiviral activities. In this study, a biological evaluation of a number of either C-18- or C-19-functionalized known semisynthetic abietanes against Zika virus, Dengue virus, Herpes virus simplex type 1, and Chikungunya virus are reported. Semisynthetic abietane ferruginol and its analogue 18-(phthalimid-2-yl)ferruginol displayed broad-spectrum antiviral properties. The scale-up synthesis of this analogue has been optimized for further studies and development. This molecule displayed an EC50 between 5.0 and 10.0 µM against Colombian Zika virus strains and EC50 = 9.8 µM against Chikungunya virus. Knowing that this ferruginol analogue is also active against Dengue virus type 2 (EC50 = 1.4 µM, DENV-2), we can conclude that this compound is a promising broad-spectrum antiviral agent paving the way for the development of novel antivirals.

Biological Profiling of Semisynthetic C19-Functionalized Ferruginol and Sugiol Analogues.

The abietane-type diterpenoids are significant bioactive compounds exhibiting a varied range of pharmacological properties. In this study, the first synthesis and biological investigation of the new abietane-diterpenoid (+)-4-epi-liquiditerpenoid acid (8a) together with several of its analogs are reported. The compounds were generated from the readily available methyl callitrisate (7), which was obtained from callitrisic acid present in Moroccan Sandarac resin. A biological evaluation was conducted to determine the effects of the different functional groups present in these molecules, providing basic structure-activity relationship (SAR) elements. In particular, the ferruginol and sugiol analogs compounds 10-16 were characterized by the presence of a phenol moiety, higher oxidization states at C-7 (ketone), and the hydroxyl, methyl ester or free carboxylic acid at C19. The biological profiling of these compounds was investigated against a panel of six human solid tumor cell lines (HBL-100, A549, HeLa, T-47D, SW1573 and WiDr), four parasitic Leishmania species (L. donovani, L. infantum, L. guyanensis and L. amazonensis) and two malaria strains (3D7 and K1). Furthermore, the capacity of the compounds to modulate gamma-aminobutyric acid type A (GABAA) receptors (α1ß2γ2s) is also described. A comparison of the biological results with those previously reported of the corresponding C18-functionalized analogs was conducted.

Synthesis of bodinieric acids A and B, both C-18 and C-19-functionalized abietane diterpenoids: DFT study of the key aldol reaction.

The first synthesis of C-18- and C-19-bifunctionalized abietane diterpenoids, bodinieric (or callicapoic) acids, via an aldol reaction has been developed. This key aldol reaction was very sensitive to steric hindrance. This fact has been studied by deuterium exchange experiments and DFT methods. Optimization of this reaction led to the synthesis of anti-inflammatory bodinieric acids A and B, starting from abietic acid.

Synthesis and Biological Studies of (+)-Liquiditerpenoic Acid A (Abietopinoic Acid) and Representative Analogues: SAR Studies.

The first semisynthesis and biological profiling of the new abietane diterpenoid (+)-liquiditerpenoic acid A (abietopinoic acid) (7) along with several analogues are reported. The compounds were obtained from readily available methyl dehydroabietate (8), which was derived from (-)-abietic acid (1). Biological comparison was conducted according to the different functional groups, leading to some basic structure-activity relationships (SAR). In particular, the ferruginol and sugiol analogues 7 and 10-16 were characterized by the presence of an acetylated phenolic moiety, an oxidized C-7 as a carbonyl, and a different functional group at C-18 (methoxycarbonyl, carboxylic acid, and hydroxymethyl). The biological properties of these compounds were investigated against a panel of six representative human tumor solid cells (A549, HBL-100, HeLa, SW1573, T-47D, and WiDr), five leukemia cellular models (NALM-06, KOPN-8, SUP-B15, UoCB1, and BCR-ABL), and four Leishmania species ( L. infantum, L. donovani, L. amazonensis, and L. guyanensis). A molecular docking study pointed out some targets in these Leishmania species. In addition, the ability of the compounds to modulate GABAA receptors (α1ß2γ2s) is also reported. The combined findings indicate that these abietane diterpenoids offer a source of novel bioactive molecules with promising pharmacological properties from cheap chiral-pool building blocks.

A theoretical study on NHC-catalysed enantioselective cycloaddition of ketenes and 3-aroylcoumarins: mechanism and enantioselectivity.

NHC-catalysed enantioselective cycloaddition of ketenes to 3-aroylcoumarins to yield dihydrocoumarin-fused dihydropyranones has been investigated using DFT methods at the B3LYP/6-31G* and MPWB1K/6-311G** computational levels. Two plausible mechanisms have been studied: the "ketene-first" mechanism A and the "coumarin-first" mechanism B. An analysis of the activation Gibbs free energies involved in the two competitive pathways makes it possible to rule out the pathway associated with the "coumarin-first" mechanism B. The first step of the "ketene-first" mechanism A is the formation of zwitterionic intermediate IN1-Zvia a nucleophilic attack of NHC 1 on ketene 2. A [4 + 2] cycloaddition through the nucleophilic attack of enolate IN1-Z on the conjugated double bond of the benzoyl group of coumarin 3, viaTS3-SS-a2 or TS3-RR-a2, yields IN3. Finally, the extrusion of the catalyst through TS5 leads to the final products, either 4-SS or 4-RR. Enantioselectivity observed in the experimental results is determined in the transition states TS3-SS-a2/TS3-RR-a2. In this pathway, the intramolecular hydrogen-bonding between the hydroxyl group of the IN1-Z adduct and the carbonyl oxygen of the original ketene group directs the final stereochemistry throughout the entire process.

A new mechanism for internal nucleophilic substitution reactions.

A new mechanism for the classic internal nucleophilic substitution reactions SNi by means of computational studies in the gas-phase, DCM and acetonitrile is reported. Despite the importance of the SNi mechanism, since the mid-1990s this mechanism has remained unexplored. This study focused mainly on the comparison between the mechanisms postulated to date for the SNi reactions and a new mechanism suggested by us that fits better the experimental observations. This comparative study has been applied to the conversion of ethyl, neopentyl, isopropyl and tert-butyl chlorosulfites into the corresponding alkyl chlorides. This new mechanism occurs through two transition structures. For primary and secondary substrates, the first transition structure is a 6-center syn-rearrangement of the alkanesulfonyl chloride that produces the corresponding olefin by simultaneous expulsion of HCl and SO2. The olefin, HCl and SO2 form a molecular complex. The final syn-addition of HCl to the olefin leads to alkyl chloride with the retention of configuration. For tertiary substrates, a variation of the previous mechanism is postulated with the intervention of contact ion pairs. It is of great importance to emphasize that this new mechanism is able to explain some experimental observations such as the presence of olefins in these types of reactions and the low reactivity of some systems such as neopentyl chlorosulfite. Our results pave the way to a new mechanistic perspective in similar reactions which will need further studies and validation.

Synthesis and antileishmanial activity of C7- and C12-functionalized dehydroabietylamine derivatives.

Abietane-type diterpenoids, either naturally occurring or synthetic, have shown a wide range of pharmacological actions, including antiprotozoal properties. In this study, we report on the antileishmanial evaluation of a series of (+)-dehydroabietylamine derivatives functionalized at C7 and/or C12. Thus, the activity in vitro against Leishmania infantum, Leishmania donovani, Leishmania amazonensis and Leishmania guyanensis, was studied. Most of the benzamide derivatives showed activities at low micromolar concentration against cultured promastigotes of Leishmania spp. (IC50 = 2.2-46.8 µM), without cytotoxicity on J774 macrophage cells. Compound 15, an acetamide, was found to be the most active leishmanicidal agent, though it presented some cytotoxicity on J774 cells. Among the benzamide derivatives, compounds 8 and 10, were also active against L. infantum intracellular amastigotes, being 18- and 23-fold more potent than the reference compound miltefosine, respectively. Some structure-activity relationships have been identified for the antileishmanial activity in these dehydroabietylamine derivatives.