Cytotoxic and Nitric Oxide Production‐Inhibitory Activities of Limonoids and Other Compounds from the Leaves and Bark of Melia azedarach

Nine limonoids, 1 – 9 , one apocarotenoid, 11 , one alkaloid, 12 , and one steroid, 13 , from the leaf extract; and one triterpenoid, 10 , five steroids, 14 – 18 , and two flavonoids, 19 and 20 , from the bark extract of Melia azedarach L. (Chinaberry tree; Meliaceae) were isolated. Among these compounds, three compounds, 4 – 6 , were new, and their structures were established as 3‐deacetyl‐28‐oxosalannolactone, 3‐deacetyl‐28‐oxosalanninolide, and 3‐deacetyl‐17‐defurano‐17,28‐dioxosalannin, respectively, on the basis of extensive spectroscopic analyses and comparison with literature data. All of the isolated compounds were evaluated for their cytotoxic activities against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK‐BR‐3) cancer cell lines. 3‐Deacetyl‐4′‐demethyl‐28‐oxosalannin ( 3 ) against HL60 and AZ521 cells, and methyl kulonate ( 10 ) against HL60 cells exhibited potent cytotoxicities with IC₅₀ values in the range of 2.8–5.8 μM . In addition, upon evaluation of compounds 1 – 13 against production of nitric oxide (NO) in mouse macrophage RAW 264.7 cells induced by lipopolysaccharide (LPS), seven, i.e., trichilinin B ( 1 ), 4 , ohchinin ( 7 ), 23‐hydroxyohchininolide ( 8 ), 21‐hydroxyisoohchininolide ( 9 ), 10 , and methyl indole 3‐carboxylate ( 12 ), inhibited production of NO with IC₅₀ values in the range of 4.6–87.3 μM with no, or almost no, toxicity to the cells (IC₅₀ 93.2–100 μM ). Western blot analysis revealed that compound 7 reduced the expression levels of the inducible NO synthase (iNOS) and COX‐2 proteins in a concentration‐dependent manner. Furthermore, compounds 5, 6, 13 , and 18 – 20 exhibited potent inhibitory effects (IC₅₀ 299–381 molar ratio/32 pmol TPA) against Epstein? Barr virus early antigen (EBV‐EA) activation induced by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in Raji cell line.     

Cytotoxic and Apoptosis‐Inducing Activities of Steviol and Isosteviol Derivatives against Human Cancer Cell Lines

Seventeen steviol derivatives, i.e., 2 – 18 , and 19 isosteviol derivatives, i.e., 19 – 37 , were prepared from a diterpenoid glycoside, stevioside ( 1 ). Upon evaluation of the cytotoxic activities of these compounds against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK‐BR‐3) cancer cell lines, nine steviol derivatives, i.e., 5 – 9 and 11 – 14 , and five isosteviol derivatives, i.e., 28 – 32 , exhibited activities with single‐digit micromolar IC₅₀ values against one or more cell lines. All of these active compounds possess C(19)‐O‐acyl group, and among which, ent‐kaur‐16‐ene‐13,19‐diol 19‐O‐4′,4′,4′‐trifluorocrotonate ( 14 ) exhibited potent cytotoxicities against four cell lines with IC₅₀ values in the range of 1.2–4.1 μM . Compound 14 induced typical apoptotic cell death in HL60 cells upon evaluation of the apoptosis‐inducing activity by flow‐cytometric analysis. These results suggested that acylation of the 19‐OH group of kaurane‐ and beyerane‐type diterpenoids might be useful for enhancement of their cytotoxicities with apoptosis‐inducing activity.     

Cytotoxic Activities and Anti‐Tumor‐Promoting Effects of Microbial Transformation Products of Prenylated Chalcones from Angelica keiskei

Three prenylated chalcones, 4‐hydroxyderricin ( 1 ), xanthoangelol ( 2 ), and xanthoangelol F ( 3 ), isolated from Angelica keiskei, were transformed by the fungus Aspergillus saitoi. These chalcones were converted to flavanones (i.e., 4, 8 , and 12 ), and prenyl‐chain‐hydrated (i.e., 5, 7, 9 – 11 , and 13 ) and ring‐B‐hydroxylated (i.e., 6 ) chalcones. The structures of three new metabolites, 7, 9 , and 13 , were established as 2″,3″‐dihydro‐4,3″‐dihydroxyderricin, 6″,7″‐dihydro‐7″‐hydroxyxanthoangelol, and 6″,7″‐dihydro‐7″‐hydroxyxanthoangelol F, respectively. Upon evaluation of cytotoxic activities of compounds 1 – 13 , the metabolite 7 exhibited potent cytotoxicity against HL60 cells, and this cell death was revealed to be mostly due to apoptosis. In addition, compounds 1 – 4, 7 – 10, 12 , and 13 were examined for their inhibitory effects on the induction of Epstein? Barr virus early antigen (EBV‐EA) by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in Raji cells. All compounds tested showed inhibitory effects against EBV‐EA activation with potencies higher than that of β‐carotene. Furthermore, the metabolite 13 exhibited inhibitory effect on skin tumor promotion in an in vivo two‐stage mouse skin carcinogenesis test based on 7,12‐dimethylbenz[a]anthracene (DMBA) as initiator, and with TPA as promoter.     

Cytotoxic and Melanogenesis‐Inhibitory Activities of Limonoids from the Leaves of Azadirachta indica (Neem)

Seventeen limonoids (tetranortriterpenoids), 1 – 17 , including three new compounds, i.e., 17‐defurano‐17‐(2,5‐dihydro‐2‐oxofuran‐3‐yl)‐28‐deoxonimbolide ( 14 ), 17‐defurano‐17‐(2ξ‐2,5‐dihydro‐2‐hydroxy‐5‐oxofuran‐3‐yl)‐28‐deoxonimbolide ( 15 ), and 17‐defurano‐17‐(5ξ‐2,5‐dihydro‐5‐hydroxy‐2‐oxofuran‐3‐yl)‐2′,3′‐dehydrosalannol ( 17 ), were isolated from an EtOH extract of the leaf of neem (Azadirachta indica). The structures of the new compounds were elucidated on the basis of extensive spectroscopic analyses and comparison with literature. Upon evaluation of the cytotoxic activities of these compounds against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK‐BR‐3) cancer cell lines, seven compounds, i.e., 1 – 3, 12, 13, 15 , and 16 , exhibited potent cytotoxicities with IC₅₀ values in the range of 0.1–9.9 μM against one or more cell lines. Among these compounds, cytotoxicity of nimonol ( 1 ; IC₅₀ 2.8 μM ) against HL60 cells was demonstrated to be mainly due to the induction of apoptosis by flow cytometry. Western blot analysis suggested that compound 1 induced apoptosis via both the mitochondrial and death receptor‐mediated pathways in HL60 cells. In addition, when compounds 1 – 17 were evaluated for their inhibitory activities against melanogenesis in B16 melanoma cells, induced with α‐melanocyte‐stimulating hormone (α‐MSH), seven compounds, 1, 2, 4 – 6, 15 , and 16 , exhibited inhibitory activities with 31–94% reduction of melanin content at 10 μM concentration with no or low toxicity to the cells (82–112% of cell viability at 10 μM ). All 17 compounds were further evaluated for their inhibitory effects against the Epstein? Barr virus early antigen (EBV‐EA) activation induced by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in Raji cells.     

Cytotoxic and Apoptosis‐inducing Activities of Taraxastane‐type Triterpenoid Derivatives in Human Cancer Cell Lines

Twenty‐eight taraxastane‐type triterpenoid derivatives 4  –  31 were prepared from the naturally occurring triterpenoids faradiol ( 1 ) and heliantriol C ( 3 ). The cytotoxic activities of these compounds and arnidiol ( 2 ) were evaluated in leukemia (HL60), lung (A549), duodenal (AZ521), and breast (SK‐BR‐3) cancer cell lines. 21‐Oxoarnidiol ( 18 ) and faradiol 3,16‐di‐O‐l ‐alaninate ( 31 ) exhibited potent cytotoxicity, with 50% inhibitory concentrations of 0.5 – 2.7 μm . In particular, flow cytometric analysis indicated that compound 31 induced typical apoptotic cell death in HL60 cells. These results suggested that taraxastane‐type triterpenoid derivatives might provide useful antitumor agents with apoptosis‐inducing activity.     

Antitumor‐Promoting Effects and Cytotoxic Activities of Dammar Resin Triterpenoids and Their Derivatives

Nineteen known triterpenoids, 1 – 19 , and one known sesquiterpenoid, 20 , were isolated from dammar resin obtained from Shorea javanica K. & V. (Dipterocarpaceae). One of the acidic triterpenoids, dammarenolic acid ( 1 ), was converted to fourteen derivatives, namely, an alcohol, 21 , an aldehyde, 22 , and twelve L ‐amino acid conjugates, 23 – 34 . Compounds 1 – 34 were examined for their inhibitory effects on the induction of Epstein–Barr virus early antigen (EBV‐EA) by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) in Raji cells, a known primary screening test for antitumor promoters. All of the compounds tested, except for compounds 4, 5, 12 – 14, 16 , and 17 , showed inhibitory effects against EBV‐EA activation with potencies either comparable with or stronger than that of β‐carotene, a known natural antitumor promoter. In addition, (20S)‐20‐hydroxy‐3,4‐secodammara‐4(28),24‐dien‐3‐al ( 22 ) exhibited inhibitory effects on skin tumor promotion in an in vivo two‐stage mouse skin carcinogenesis test based on 7,12‐dimethylbenz[a]anthracene (DMBA) as initiator, and with TPA as promoter. Furthermore, evaluation of the cytotoxic activities of compounds 1 – 34 against human cancer cell lines showed that reduction (i.e., 21 and 22 ) or conjugation with L ‐amino acids (i.e., 23 – 34 ) of compound 1 enhanced the cytotoxicity against human melanoma cell line CRL1579.     

Discovery and Pharmacophore Studies of Novel Pyrazole‐Based Anti‐Melanoma Agents

Due to the rising incidence and lack of effective treatments, malignant melanoma is the most dangerous form of skin cancer, so that new treatment strategies are urgently needed. Several recent developments indicate that the V600E mutant BRAF (BRAF^V600E) is a validated target for antimelanoma‐drug development. Based on in silico screening results, a series of novel pyrazole derivatives has been designed, synthesized, and evaluated in vitro for their inhibitory activities against BRAF^V600E melanoma cells. Compound 3d exhibited the most potent inhibitory activity with an IC₅₀ value of 0.63 μM for BRAF^V600E and a GI₅₀ value of 0.61 μM for mutant BRAF‐dependent cells. Furthermore, the QSAR modeling and the docking simulation of inhibitor analogs provide important pharmacophore clues for further structural optimization.     

Cytostatic/Cytotoxic Effects of 5‐Fluorouridine Nucleolipids on Colon,Hepatocellular, and Renal Carcinoma Cells: in vitro Identification of a Potential Cytotoxic Multi‐Anticancer Drug

The insufficient penetration through the cell membranes is one of the major drawbacks of chemotherapeutics such as 5‐fluorouracil (5‐FU; 1 ). To improve the penetration, a useful strategy is the attachment of lipophilic moieties. Thus, we have synthesized a series of nucleolipid derivatives of 5‐fluorouridine (5‐FUrd; 2a ), carrying lipophilic moieties at N(3) and/or at the 2′,3′‐O position, i.e., 3a, 3b, 4 – 7 , and tested their cytostatic/cytotoxic activities towards three carcinoma cell lines (colon (HT‐29), hepatocellular (HepG2), and renal (RENCA)) in comparison with 5‐FU ( 1 ) and 5‐FUrd ( 2a ). After 48 h of incubation, four derivatives, 3a, 3b, 5 , and 7 , showed inhibitory effects on the survival of HT‐29, HepG2, and RENCA cells. Additionally, to differentiate between anticancer and side‐effects, we tested the cytotoxicity of the derivatives in human macrophages. Interestingly, the derivatives 4, 5 , and 6 did not exhibit any effects on survival of THP‐1 macrophages. Furthermore, we investigated the apoptosis induction of compound 1 and 2a , and the above‐mentioned derivatives in HT‐29 cells. Derivative 5 showed the highest significant (p<0.05; p<0.01) increase of the apoptosis at 80 μM after 2‐h or 4‐h treatment, as well as after 6‐h incubation at 40 μM (p<0.05). Real‐time PCR revealed that 40‐μM derivative 5 showed a 1.8‐fold increase of the pro‐apoptotic caspase‐3 gene and a twofold significant increase (p<0.01 and p<0.05 vs. control and 1 , resp.) of the tumor suppressor TP53 gene, whereas the other compounds did not show any effect. We demonstrated that some 5‐FUrd derivatives such as compound 5 are more effective than 5‐FU or 5‐FUrd concerning a cytotoxic (vs. cytostatic (5‐FU, 5‐FUrd)) effect on different cancer cell lines, but without cytotoxic side‐effects on differentiated macrophages. Thus, compound 5 is suggested as a novel potent cytotoxic multi‐anti‐cancer drug.     

Synthesis of 5‐Fluorouridine Nucleolipid Derivatives and Their Cytostatic/Cytotoxic Activities on Human HT‐29 Colon Carcinoma Cells

One of the major drawbacks of chemotherapeutics is their insufficient penetration through cell membranes due to a high hydrophobicity. Thus, we have synthesized a series of selected nucleolipid derivatives of 5‐fluorouridine (5‐FUrd; 2a ), carrying lipophilic moieties at N(3) and/or in the 2′,3′‐O‐position (i.e., 3a – 7a and 3c ), and tested their cytostatic/cytotoxic activities using HT‐29 human colon carcinoma cells, in comparison with, e.g., 5‐FU ( 1 ) and 5‐FUrd ( 2a ). Incorporation and intracellular localization of the substances under test were performed after conjugation with the fluorochrome Atto 425. We showed that all 5′‐O‐labelled Atto 425 derivatives were incorporated by the human HT‐29 cells and accumulated in their cytoplasm. Moreover, after 24‐h treatment of HT‐29 human colon carcinoma cells, 1 or 2a (10, 20, 40, or 80 μM ) revealed a significant (14–23 or 33–45%, resp.) decrease of the viability in comparison with the (negative) control. Interestingly, derivatives 3a and 3c (40 and 80 μM ) led to a significant (77–95 or 89–96%, resp.) inhibition of survival of human HT29 cells, i.e., these two substances were ca. 63–72% or ca. 75%, respectively more effective than 5‐FU ( 1 ; positive control). Furthermore, derivative 5a showed a significant, i.e., 30 and 86%, inhibition of the survival at 40 and 80 μM , respectively in comparison with the (negative) control. Some synthesized 5‐FUrd derivatives turned out to be more effective than 5‐FU ( 1 ) or 5‐FUrd ( 2a ).     

Limonoids and Flavonoids from the Flowers of Azadirachta indica var. siamensis,and Their Melanogenesis‐Inhibitory and Cytotoxic Activities

A new limonoid, 7‐O‐acetyl‐7‐O‐debenzoyl‐22‐hydroxy‐21‐methoxylimocinin ( 2 ), and two new flavonoids, 3′‐(3‐hydroxy‐3‐methylbutyl)naringenin ( 7 ) and 4′‐O‐methyllespedezaflavanone C ( 9 ), along with nine known compounds, including two limonoids, 1 and 3 , and seven flavonoids, 4 – 6, 8 , and 10 – 12 , were isolated from a MeOH extract of the flowers of Azadirachta indica A.Juss. var. siamensis Valeton (Siamese neem tree; Meliaceae). The structures of new compounds were elucidated on the basis of extensive spectroscopic analysis and comparison with literature data. All of these compounds were evaluated for their melanogenesis‐inhibitory activities in B16 melanoma cells induced with α‐melanocyte‐stimulating hormone (α‐MSH). Compound 2 (16.9% melanin content at 30 μM ), 6‐deacetylnimbin ( 3 ; 49.6% melanin content at 100 μM ), and kaempferide ( 10 ; 41.7% melanin content at 10 μM ) exhibited inhibitory effects with no, or almost no, toxicity to the cells (81.0–111.7% cell viability). In addition, evaluation of their cytotoxic activities against HL60, A549, AZ521, and SK‐BR‐3 human cancer cell lines, isoazadironolide ( 1 ), 4′‐O‐methyl‐8‐prenylnaringenin ( 5 ), euchrestaflavanone A ( 8 ), 9 , and 3‐methoxy‐3′‐prenylnaringenin ( 12 ) revealed potent cytotoxicities against one or more cell lines with IC₅₀ values in the range of 4.5–9.9 μM .     

Synthesis,Characterization, and Biological Activities of Pendant Arm‐Pyridyltetrazole Copper(II) Complexes: DNA Binding/Cleavage Activity and Cytotoxic Studies

2‐(1H‐Tetrazol‐5‐yl)pyridine ( L ) has been reacted separately with Me₂NCH₂CH₂Cl?HCl and ClCH₂CH₂OH to yield two regioisomers in each case, N,N‐dimethyl‐2‐[5‐(pyridin‐2‐yl)‐1H‐tetrazol‐1‐yl]ethanamine ( L1 )/N,N‐dimethyl‐2‐[5‐(pyridin‐2‐yl)‐2H‐tetrazol‐2‐yl]ethanamine ( L2 ) and 2‐[5‐(pyridin‐2‐yl)‐1H‐tetrazol‐1‐yl]ethanol ( L3 )/2‐[5‐(pyridin‐2‐yl)‐2H‐tetrazol‐2‐yl]ethanol ( L4 ), respectively. These ligands, L1 – L4 , have been coordinated with CuCl₂?H₂O in 1 : 1 composition to furnish the corresponding complexes 1 – 4 . EPR Spectra of Cu complexes 1 and 3 were characteristic of square planar geometry, with nuclear hyperfine spin 3/2. Single X‐ray crystallographic studies of 3 revealed that the Cu center has a square planar structure. DNA binding studies were carried out by UV/VIS absorption; viscosity and thermal denaturation studies revealed that each of these complexes are avid binders of calf thymus DNA. Investigation of nucleolytic cleavage activities of the complexes was carried out on double‐stranded pBR322 circular plasmid DNA by using a gel electrophoresis experiment under various conditions, where cleavage of DNA takes place by oxidative free‐radical mechanism (OH ^? ). In vitro anticancer activities of the complexes against MCF‐7 (human breast adenocarcinoma) cells revealed that the complexes inhibit the growth of cancer cells. The IC₅₀ values of the complexes showed that Cu complexes exhibit comparable cytotoxic activities compared to the standard drug cisplatin.     

Phenolic Composition,Anti‐Inflammatory,Antioxidant, and Antimicrobial Activities of Alchemilla mollis (Buser) Rothm.

The current study was designed to evaluate the antioxidant, anti‐inflammatory and antimicrobial activities of Alchemilla mollis (Buser ) Rothm . (Rosaceae) aerial parts extracts. Chemical composition was analyzed by spectrophotometric and chromatographic (HPLC) techniques. The antioxidant properties assessed included DPPH^· and ABTS^·+ radical scavenging, β‐carotene‐linoleic acid co‐oxidation assay. Antimicrobial activity was evaluated with disc diffusion and micro dilution method. In order to evaluate toxicity of the extracts, with the sulforhodamine B colorimetric assay L929 cell line (mouse fibroblast) was used. The anti‐inflammatory activities of the potent antioxidant extracts (methanol, 70% methanol, and water extracts) were determined by measuring the inhibitory effects on NO production and pro‐inflammatory cytokine TNF‐α levels in lipopolysaccharide stimulated RAW 264.7 cells. 70% methanol and water extracts which were found to be rich in phenolic compounds (184.79 and 172.60 mg GAE/g extract) showed higher antioxidant activity. Luteolin‐7‐O‐glucoside was the main compound in the extracts. Ethyl acetate and 70% methanol extracts showed higher antibacterial activity against Staphylococcus aureus and Salmonella enteritidis with MIC value of 125 μg/ml. 70% methanol extract potentially inhibited the NO and TNF‐α production (18.43 μm and 1556.22 pg/ml, respectively, 6 h).     

Synthesis,Structure, and Biological Activities of a New μ‐Oxamido‐Bridged Dicopper(II) Complex: The Influence of Hydrophobicity of Bridging Ligand on DNA Binding and Cytotoxic Activities

A new μ‐oxamido‐bridged dicopper(II) complex, [Cu₂(papo)(H₂O)‐ (phen)]Cl·CH₃OH·H₂O, where H₃papo and phen represent N‐(2‐hydroxyphenyl)‐N'‐(3‐aminopropyl)oxamide and 1,10‐phenanthroline, respectively, has been synthesized and characterized by elemental analysis, molar conductivity measurement, infrared and electronic spectra studies, and single‐crystal X‐ray diffraction. The complex crystallizes in the triclinic space group P‐1. Each copper(II) ion is located in a slightly distorted square‐pyramidal environment. The Cu···Cu distance through the oxamide bridge is 5.1848(7) Å. The three‐dimensional supramolecular structure is built‐up by hydrogen bonds and π–π stacking interactions. The dicopper(II) complex exhibits cytotoxic activity against the SMMC‐7721 and A549 cell lines. The reactivity toward herring sperm DNA and protein bovine serum albumin (BSA) reveals that the dicopper(II) complex can interact with the DNA by the intercalation mode, and effectively quench the intrinsic fluorescence of BSA via a static mechanism. The influence of hydrophobicity of the bridging ligand on DNA‐binding properties and in vitro cytotoxic activities of this kind of dicopper(II) complexes was investigated.     

Essential Oil of Azorella cryptantha Collected in Two Different Locations from San Juan Province,Argentina: Chemical Variability and Anti‐Insect and Antimicrobial Activities

The essential oils (EOs) of two populations of Azorella cryptantha (Clos) Reiche , a native species from San Juan Province, were obtained by hydrodistillation in a Clevenger‐type apparatus and characterized by GC‐FID and GC/MS analyses. The compounds identified amounted to 92.3 and 88.7% of the total oil composition for A. cryptantha from Bauchaceta (Ac‐BAU) and Agua Negra (Ac‐AN), respectively. The EO composition for the two populations was similar, although with differences in the identity and content of the main compounds and also in the identity of minor components. The main compounds of the Ac‐BAU EO were α‐pinene, α‐thujene, sabinene, δ‐cadinene, δ‐cadinol, trans‐β‐guaiene, and τ‐muurolol, while α‐pinene, α‐thujene, β‐pinene, γ‐cadinene, τ‐cadinol, δ‐cadinene, τ‐muurolol, and a not identified compound were the main constituents of the Ac‐AN EO, which also contained 3.0% of oxygenated monoterpenes. The repellent activity on Triatoma infestans nymphs was 100 and 92% for the Ac‐AN and Ac‐BAU EOs, respectively. Regarding the toxic effects on Ceratitis capitata, the EOs were very active with LD₅₀ values lower than 11 μg/fly. The dermatophytes Microsporum gypseum, Trichophyton rubrum, and T. mentagrophytes and the bacterial strains Escherichia coli LM₁, E. coli LM₂, and Yersinia enterocolitica PI were more sensitive toward the Ac‐AN EO (MIC 125 μg/ml) than toward the Ac‐BAU EO. This is the first report on the composition of A. cryptantha EO and its anti‐insect and antimicrobial properties.     

Synthesis of a Natural Chalcone and Its Prenyl Analogs – Evaluation of Tumor Cell Growth‐Inhibitory Activities,and Effects on Cell Cycle and Apoptosis

Six prenyl (=3‐methylbut‐2‐en‐1‐yl) chalcones (=1,3‐diphenylprop‐2‐en‐1‐ones), 2 – 7 , and one natural non‐prenylated chalcone, 1 , have been synthesized and evaluated for their in vitro growth‐inhibitory activity against three human tumor cell lines. A pronounced dose‐dependent growth‐inhibitory effect was observed for all prenylated derivatives, except for 7 . The chalcone possessing one prenyloxy group at C(2′), i.e., 2 , was the most active derivative against the three human tumor cell lines (5.9<GI₅₀<7.7 μM ). The majority of compounds caused an increase in percentage of apoptotic cells and/or they interfered with cell cycle distribution in the MCF‐7 cell line.