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The chemical modification of natural compounds is a promising strategy to improve their frequently poor bioavailability and low potency. This study aimed at synthesizing chemical derivatives of carvone, a natural monoterpene with anti-inflammatory properties, which we recently identified, and evaluating their potential anti-inflammatory activity. Fourteen chemical derivatives of carvone were synthesized, purified and their chemical structures confirmed. Noncytotoxic concentrations of the test compounds were selected based on the resazurin reduction assay. Among the tested compounds, four significantly reduced the lipopolysaccharides-induced protein levels of the inducible isoform of the nitric oxide synthase and nitric oxide production and showed a dual effect on pro-IL-1 protein levels in the Raw 264.7 cell line. The Ligand Express drug discovery platform was used to predict the targets of the test compounds, and an enrichment analysis was performed to group the different biological processes and molecular and cellular functions of the tested compounds. Moreover, Ligand Express also predicted that all chemicals evaluated have intestinal and blood-brain barrier permeability, do not inhibit P-gp and do not interact with major receptors. Although presenting anti-inflammatory and some advantageous ADME properties, the tested compounds still have low potency and specificity but may provide novel structures the further chemical modification of which may yield more promising drugs.

To explore the molecular mechanisms underlying the anti-inflammatory activity of (R)-(-)-carvone, we evaluated its ability to inhibit the signaling pathways involving the mitogen-activated protein kinases (MAPKs) and the transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). (R)-(-)-carvone significantly decreased c-Jun N-terminal kinase (JNK) 1phosphorylation, but not that of the other MAPKs, induced by bacterial lipopolysaccharides (LPS) in the RAW 264.7 macrophage cell line. Although (R)-(-)-carvone significantly inhibited resynthesis of the inhibitor of NF-κB (IκB)-α induced by LPS, it did not interfere with the canonical NF-κB activation pathway, suggesting that it may interfere with its transcriptional activity. (R)-(-)-carvone also showed a tendency to decrease the levels of acetylated NF-κB/p65 in the nucleus, without affecting the activity and protein levels of Sirtuin-1, the major NF-κB/p65 deacetylating enzyme. Interestingly, the nuclear protein levels of the transcription factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and the expression of its target,, heme oxygenase-1 (HO-1), an antioxidant enzyme, also showed a tendency to increase in the presence of (R)-(-)-carvone. Taken together, these results suggest that the ability of (R)-(-)-carvone to inhibit JNK1 and to activate Nrf2 can underlie its capacity to inhibit the transcriptional activity of NF-κB and the expression of its target genes. This study highlights the diversity of molecular mechanisms that can be involved in the anti-inflammatory activity of monoterpenes.

The signaling pathways involved in age-related inflammation are increasingly recognized as targets for the development of preventive and therapeutic strategies. Our previous study elucidated the structure-activity relationship of monoterpene compounds derived from p -menthane as potential anti-inflammatory drugs and identified (S)-(+)-carvone as the most potent among the compounds tested. This study aims at identifying the molecular mechanism underlying the anti-inflammatory properties of (S)-(+)-carvone. The murine macrophage cell line, Raw 264.7, was stimulated with bacterial lipopolysaccharide (LPS) to simulate inflammation. Western blot was used to assess protein levels and post-translational modifications. The subcellular localization of NF-κB/p65 was visualized by immunocytochemistry. An in vitro fluorometric assay was used to measure Sirtuin-1 (SIRT1) activity. (S)-(+)-carvone inhibited LPS-induced JNK1 phosphorylation, but not that of p38 and ERK1/2 and also did not affect the phosphorylation and degradation of the NF-κB inhibitor, IκB-α. Accordingly, (S)-(+)-carvone did not affect LPS-induced phosphorylation of NF-κB/p65 on Ser536 and its nuclear translocation, but it significantly decreased LPS-induced IκB-α resynthesis, a NF-κB-dependent process, and NF-κB/p65 acetylation on lysine (Lys) 310. Deacetylation of that Lys residue is dependent on the activity of SIRT1, which was found to be increased by (S)-(+)-carvone, while its protein levels were unaffected. Taken together, these results show that (S)-(+)-carvone is a new SIRT1 activator with the potential to counteract the chronic low-grade inflammation characteristic of age-related diseases.

Mint species are widely used in traditional and conventional medicine as topical analgesics for osteoarthritic pain and for disorders of the gastrointestinal and respiratory tracts which are all associated with chronic inflammation. To identify the structural determinants of anti-inflammatory activity and potency which are required for chemical optimization towards development of new anti-inflammatory drugs, a selected group of monoterpenes especially abundant in mint species was screened by measuring bacterial lipopolysacharide (LPS)-induced nitric oxide (NO) production in murine macrophages. Nine compounds significantly decreased LPS-induced NO production by more than 30%. IC 50 values were calculated showing that the order of potency is: (S)-(+)-carvone > (R)-(-)-carvone > (+)-dihydrocarveol > (S)-8-hydroxycarvotanacetone > (R)-8-hydroxycarvotanacetone > (+)-dihydrocarvone > (-)-carveol > (-)-dihydrocarveol > (S)-(-)-pulegone. Considering the carbon numbering relative to the common precursor, limonene, the presence of an oxygenated group at C6 conjugated to a double bond at C1 and an isopropenyl group and S configuration at C4 are the major chemical features relevant for activity and potency. The most potent compound, (S)-(+)-carvone, significantly decreased the expression of NOS2 and IL-1β in macrophages and in a cell model of osteoarthritis using primary human chondrocytes. (S)-(+)-carvone may be efficient in halting inflammation-related diseases, like osteoarthritis.

The objective of this study was to encapsulate a synthetic compound, the 4-[(2E)-N'-(2,2'-bithienyl-5-methylene)hydra-zinecarbonyl]-6,7-dihydro-1-phenyl-1H-pyrazolo[3,4-d]pyridazin-7-one (T6) in glucan-rich particles mainly composed by the cell wall of Saccharomyces cerevisiae (GPs) and to study their individual and combined activity on Leishmania infantum. The possible mechanism of action of T6 was also investigated. Our results showed the activity of T6 compound in both promastigote (IC 50  = 2.5 μg/mL) and intracellular amastigote (IC 50  = 1.23 μg/mL) forms. We also found activity against intracellular amastigote forms (IC 50  = 8.20 μg/mL) when the T6 compound was encapsulated in GPs. Another interesting finding was the fact that T6 encapsulated in GPs showed a significant decrease in J774A1 macrophage toxicity (CC 50  ≥ 18.53 μg/mL) compared to the T6 compound alone (IC 50  = 2.27 μg/mL). Through electron microscopy and biochemical methodologies, we verified that the activity of T6 in promastigote forms of L. infantum was characterized by events of cell death by apoptosis like increased ROS production, cell shrinkage, phosphatidylserine exposure and DNA fragmentation. We conclude that T6 can be considered a promising anti-Leishmania compound, and that the use of GPs for drug encapsulation is an interesting approach to the development of new effective and less toxic formulations., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Context/objective: Cell lines used to study the role of the G protein-coupled receptor 30 (GPR30) or G protein-coupled estrogen receptor (GPER) as a mediator of estrogen responses have yielded conflicting results. This work identified a simple assay to predict cell line competence for pharmacological studies of GPR30., Materials and Methods: The phosphorylation or expression levels of ERK1/2, Akt, c-Fos and eNOS were evaluated to assess GPR30 activation in response to known agonists (17β-estradiol and G-1) in MCF-7 and T-47D breast cancer cell lines and in bovine aortic endothelial cells. GPR30 expression was analyzed by qRT-PCR and Western blot with two distinct antibodies directed at its carboxy and amino terminals., Results: None of the agonists, at any of the concentrations tested, activated any of those target proteins. Additional experiments excluded the disruption of the signaling pathway, interference of phenol red in the culture medium and constitutive proteasome degradation of GPR30 as possible causes for the lack of response of the three cell lines. Analysis of receptor expression showed the absence of clearly detectable GPR30 species of 44 and 50-55 kDa previously identified in cell lines that respond to 17β-estradiol and G-1., Discussion and Conclusion: Cells that do not express the 44 and 50-55 kDa species do not respond to GPR30 agonists. Thus, the presence or absence of these GPR30 species is a simple and rapid manner to determine whether a given cell line is suitable for pharmacological or molecular studies of GPR30 modulation.

Seven steroid epoxides were prepared from 5α-pregn-2-en-20-one and 5α-pregn-3-en-20-one and their side-chain derivatives. All compounds were tested in vitro for binding to γ-aminobutyric acid (GABAA) receptor, some of them also in vivo for anticonvulsant action. 2α,3α-Epoxy-5α-pregnan-20-one inhibited the TBPS binding to the GABAA receptor and showed a moderate anticonvulsant action in immature rats. In contrast, its 3α,4α-isomer was inactive. More polar epoxide derivatives, modified at the side chain were less active or inactive. Noteworthy, diol 20, the product of trans-diaxial opening of the 2α,3α-epoxide 4, was not able to inhibit the TBPS binding, showing that the activity of the epoxide is due to the compound itself and not to its hydrolytic product. The 3α-hydroxyl group is known to be essential for the GABAA receptor binding. Despite the shortness of in vivo effects which are probably due to metabolic inactivation of the products prepared, our results show that the 2α,3α-epoxy ring is another structural pattern with ability to bind the GABAAR., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Steroids, a widespread class of natural organic compounds occurring in animals, plants and fungi, have shown great therapeutic value for a broad array of pathologies. The present overview is focused on the anticancer activity of steroids, which is very representative of a rich structural molecular diversity and ability to interact with various biological targets and pathways. This review encompasses the most relevant discoveries on steroid anticancer drugs and leads through the last decade and comprises 668 references.