ABSTRACT
A potent new lipopeptide antibiotic, A54145E(Asn(3)Asp(9)), was isolated from the fermentation broth of Streptomyces fradiae DA1489 engineered to delete genes encoding enzymes involved in hydroxylation of Asn(3) and methoxylation of Asp(9). The chemical structure predicted from the genetic changes in the biosynthetic pathway was determined by analyses of chemical transformations, D, L-amino acid quantitation by enantiomer labeling, tandem LC-MS/MS and 2D NMR techniques. These studies confirmed the primary amino acid sequence of A54145E(Asn(3)Asp(9)) predicted from the genetic engineering strategy, and also confirmed the structure and locations of three D-amino acids predicted from bioinformatic studies.
Subject(s)
Anti-Bacterial Agents/biosynthesis , Streptomyces/metabolism , Anti-Bacterial Agents/chemistry , Asparagine/analogs & derivatives , Asparagine/biosynthesis , Asparagine/chemistry , Asparagine/genetics , Lipoproteins/biosynthesis , Lipoproteins/chemistry , Lipoproteins/genetics , Magnetic Resonance Spectroscopy , Optical Rotation , Sarcosine/analogs & derivatives , Sarcosine/biosynthesis , Sarcosine/chemistry , Sarcosine/genetics , Spectrometry, Mass, Electrospray Ionization , Spectrophotometry, Infrared , Spectrophotometry, Ultraviolet , Streptomyces/geneticsABSTRACT
A54145 is a complex of lipopeptide antibiotics produced by Streptomyces fradiae. A54145 factors are structurally related to daptomycin, with four modified amino acids, only one of which is present in daptomycin. We generated three mutants defective in lptJ, lptK or lptL, whose gene products are involved in the formation of hydroxy-Asn(3) (hAsn(3)) and methoxy-Asp(9) (moAsp(9)). Each of the mutants produced novel lipopeptides related to A54145 and the profiles allowed assignment of functions for those genes. We constructed strains carrying different combinations of these genes coupled with a mutation in the lptI gene involved in the biosynthesis of 3-methyl-Glu(12) (3mGlu(12)), and all recombinants produced novel lipopeptides. One of the compounds displayed very good antibacterial activity in the presence of bovine surfactant, which interacts with daptomycin or A54145E to inhibit their antibacterial activities.
Subject(s)
Anti-Bacterial Agents/metabolism , Lipoproteins/biosynthesis , Streptomyces/genetics , Streptomyces/metabolism , DNA/chemistry , DNA/genetics , Fermentation , Genetic Complementation Test , Lipoproteins/genetics , Lipoproteins/metabolism , Microbial Sensitivity Tests , Mutagenesis, Insertional , Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
Daptomycin is a cyclic lipopeptide antibiotic approved for the treatment of skin and skin structure infections caused by Gram-positive pathogens and for that of bacteremia and right-sided endocarditis caused by Staphylococcus aureus. Daptomycin failed to meet noninferiority criteria for the treatment of community-acquired pneumonia, likely due to sequestration in pulmonary surfactant. Many analogues of daptomycin have been generated by combinatorial biosynthesis, but only two displayed improved activity in the presence of bovine surfactant, and neither was as active as daptomycin in vitro. In the present study, we generated hybrid molecules of the structurally related lipopeptide A54145 in Streptomyces fradiae and tested them for antibacterial activity in the presence of bovine surfactant. Hybrid A54145 nonribosomal peptide synthetase (NRPS) biosynthetic genes were constructed by genetic engineering and were expressed in combination with a deletion of the lptI methyltransferase gene, which is involved in the formation of the 3-methyl-glutamic acid (3mGlu) residue at position 12. Some of the compounds were very active against S. aureus and other Gram-positive pathogens; one compound was also highly active in the presence of bovine surfactant, had low acute toxicity, and showed some efficacy against Streptococcus pneumoniae in a mouse model of pulmonary infection.
Subject(s)
Anti-Bacterial Agents/pharmacology , Daptomycin/analogs & derivatives , Animals , Anti-Bacterial Agents/biosynthesis , Anti-Bacterial Agents/chemistry , Cattle , Daptomycin/biosynthesis , Daptomycin/chemistry , Daptomycin/pharmacology , Female , Genes, Bacterial , Gram-Positive Bacteria/drug effects , Lipoproteins/biosynthesis , Lipoproteins/chemistry , Lipoproteins/genetics , Lipoproteins/pharmacology , Mice , Microbial Sensitivity Tests , Molecular Structure , Pneumonia, Pneumococcal/drug therapy , Protein Engineering , Pulmonary Surfactants/administration & dosage , Staphylococcus aureus/drug effects , Streptomyces/geneticsABSTRACT
Genetic engineering has been applied to reprogramme non-ribosomal peptide synthetases (NRPSs) to produce novel antibiotics, but little is known about what determines the efficiency of production. We explored module exchanges at nucleotide sequences encoding interpeptide linkers in dptD, a gene encoding a di-modular NRPS subunit that incorporates 3-methylglutamic acid (3mGlu(12)) and kynurenine (Kyn(13)) into daptomycin. Mutations causing amino acid substitutions, deletions or insertions in the inter-module linker had no negative effects on lipopeptide yields. Hybrid DptD subunits were generated by fusing the 3mGlu(12) module to terminal modules from calcium-dependent antibiotic (CDA) or A54145 NRPSs, and recombinants produced daptomycin analogues with Trp(13) or Ile(13) at high efficiencies. A recombinant expressing DptD with a hybrid Kyn(13) module containing a di-domain from a d-Asn module caused the production of a new daptomycin analogue containing Asn(13).
Subject(s)
Bacterial Proteins/metabolism , Daptomycin/biosynthesis , Peptide Synthases/metabolism , Recombinant Fusion Proteins/metabolism , Streptomyces/enzymology , Amino Acid Motifs , Amino Acid Sequence , Amino Acid Substitution , Anti-Bacterial Agents/biosynthesis , Bacterial Proteins/genetics , Base Sequence , Daptomycin/analogs & derivatives , Fermentation , Molecular Sequence Data , Peptide Synthases/genetics , Plasmids , Protein Engineering , Recombinant Fusion Proteins/genetics , Sequence Deletion , Streptomyces/genetics , Streptomyces/metabolismABSTRACT
Three daptomycin-related lipopeptides, A21978C1-3(d-Asn11) (2-4), were purified from the fermentation broth of a recombinant Streptomyces roseosporus strain. Their chemical structures were determined by analyses of the biosynthetic pathway, chemical transformations, d,l-amino acid quantitation by enantiomer labeling, tandem LC-MS/MS, and 2D-NMR techniques. Compounds 2-4 exhibited potent antibacterial activity against Staphylococcus aureus with MIC values of 0.6, 0.3, and 0.15 microM, respectively, well correlated to the acyl tail chain length.
Subject(s)
Anti-Bacterial Agents/isolation & purification , Daptomycin/analogs & derivatives , Daptomycin/isolation & purification , Streptomyces/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Asparagine/chemistry , Daptomycin/chemistry , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Organisms, Genetically Modified , Staphylococcus aureus/drug effects , Streptomyces/geneticsABSTRACT
Daptomycin, a cyclic lipopeptide produced by Streptomyces roseosporus, is the active ingredient of Cubicin (daptomycin-for-injection), a first-in-class antibiotic approved for treatment of skin and skin-structure infections caused by Gram-positive pathogens and bacteremia and endocarditis caused by Staphylococcus aureus, including methicillin-resistant strains. Genetic engineering of the nonribosomal peptide synthetase (NRPS) in the daptomycin biosynthetic pathway was exploited for the biosynthesis of novel active antibiotics. lambda-Red-mediated recombination was used to exchange single or multiple modules in the DptBC subunit of the NRPS to modify the daptomycin cyclic peptide core. We combined module exchanges, NRPS subunit exchanges, inactivation of the tailoring enzyme glutamic acid 3-methyltransferase, and natural variations of the lipid tail to generate a library of novel lipopeptides, some of which were as active as daptomycin against Gram-positive bacteria. One compound was more potent against an Escherichia coli imp mutant that has increased outer membrane permeability. This study established a robust combinatorial biosynthesis platform to produce novel peptide antibiotics in sufficient quantities for antimicrobial screening and drug development.
Subject(s)
Anti-Bacterial Agents/biosynthesis , Daptomycin/analogs & derivatives , Daptomycin/biosynthesis , Amino Acid Sequence , Anti-Bacterial Agents/chemistry , Daptomycin/chemistry , Gene Deletion , Molecular Sequence Data , Molecular Structure , Mutation/genetics , Sequence Alignment , Staphylococcus aureus/chemistry , Staphylococcus aureus/metabolism , Streptomyces/chemistry , Streptomyces/metabolismABSTRACT
In many peptide antibiotics, modified amino acids are important for biological activity. The amino acid 3-methyl-glutamic acid (3mGlu) has been found only in three cyclic lipopeptide antibiotics: daptomycin and the A21978C family produced by Streptomyces roseosporus, calcium-dependent antibiotic produced by Streptomyces coelicolor and A54145 produced by Streptomyces fradiae. We studied the non-ribosomal peptide synthetase genes involved in A21978C biosynthesis and the downstream genes, dptG, dptH, dptI and dptJ predicted to encode a conserved protein of unknown function, a thioesterase, a methyltransferase (MTase) and a tryptophan 2,3-dioxygenase respectively. Deletion of dptGHIJ reduced overall lipopeptide yield and led to production of a series of novel A21978C analogues containing Glu12 instead of 3mGlu12. Complementation by only dptI, or its S. coelicolor homologue, glmT, restored the biosynthesis of the 3mGlu-containing compounds in the mutant. Compared with A21978C, the Glu12-containing derivatives were less active against Staphylococcus aureus. Further genetic analyses showed that members of the dptGHIJ locus cooperatively contributed to optimal A21978C production; deletion of dptH, dptI or dptJ genes reduced the yield significantly, while expression of dptIJ or dptGHIJ from the strong ermEp* promoter substantially increased lipopeptide production. The results indicate that these genes play important roles in the biosynthesis of daptomycin, and that dptI encodes a Glu MTase.
Subject(s)
Daptomycin/biosynthesis , Glutamic Acid/metabolism , Methyltransferases/genetics , Streptomyces/genetics , Amino Acid Motifs/genetics , Amino Acid Sequence , Anti-Bacterial Agents/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chromatography, High Pressure Liquid/methods , Chromatography, Liquid/methods , Glutamic Acid/analogs & derivatives , Mass Spectrometry/methods , Methyltransferases/metabolism , Molecular Sequence Data , Molecular Structure , Mutation/genetics , Sequence Homology, Amino Acid , Streptomyces/chemistry , Streptomyces/metabolismABSTRACT
Three common plant-derived pentacyclic triterpenoids, oleanolic acid (1), betulinic acid (2) and ursolic acid (3), have been found to exhibit moderate anti-tubercular activity in a microplate alamar blue assay. In order to facilitate the discovery of novel anti-tubercular leads with diverse chemical structures, a new and rapid GC-EI/MS method was developed simultaneously and unambiguously to dereplicate 1-3 as their methyl esters with limits of detection of 25.6, 26.9 and 26.8 ng, respectively.
Subject(s)
Antitubercular Agents/chemistry , Lavandula/chemistry , Oleanolic Acid/chemistry , Tanacetum parthenium/chemistry , Triterpenes/chemistry , Valerian/chemistry , Chromatography, Gas , Molecular Structure , Pentacyclic Triterpenes , Spectrometry, Mass, Electrospray Ionization , Betulinic Acid , Ursolic AcidABSTRACT
Nematicidal bioassay-guided fractionation of the n-hexane extract of the seeds of Jubaea chilensis led to the identification of eight known fatty acids and a mixture of triglycerides, reported for the first time for this species. In addition, their corresponding methyl esters were identified to be artifacts generated during the extraction and isolation procedures by using GC-EI-MS and chemical transformation methods. The fatty acid composition of the triglycerides was analyzed by GC-EI-MS and chemical transformation techniques. Among the 17 compounds, only lauric acid and myristic acid exhibited significant inhibitory effects on the movement of Caenorhabditis elegans with minimum inhibitory concentrations (MIC) of 75 microg/ml.
Subject(s)
Antinematodal Agents/isolation & purification , Arecaceae/chemistry , Fatty Acids/isolation & purification , Seeds/chemistry , Triglycerides/isolation & purification , Animals , Antinematodal Agents/toxicity , Fatty Acids/toxicity , Gas Chromatography-Mass Spectrometry , Nematoda/drug effects , Triglycerides/toxicityABSTRACT
The structure of an iridolactone isolated from Valeriana laxiflora was established as (4R,4aR,6S,7S,7aS)-6-hydroxy-7-hydroxymethyl-4-methylperhydrocyclopenta[c]pyran-1-one chloroform solvate, C(10)H(16)O(4).CHCl(3). The two rings are cis-fused. The delta-lactone ring adopts a slightly twisted half-chair conformation with approximate planarity of the lactone group and the cyclopentane ring adopts an envelope conformation. The hydroxy group, the hydroxymethyl group and the methyl group all have beta orientations. The absolute configuration was determined using anomalous dispersion data enhanced by the adventitious inclusion of a chloroform solvent molecule. Hydrogen bonding, crystal packing and ring conformations are discussed in detail.
Subject(s)
Cyclopentanes/chemistry , Pyrans/chemistry , Valerian/chemistry , Chloroform , Crystallography, X-Ray , Hydrogen Bonding , Models, Molecular , Plant Leaves/chemistry , Plant Roots/chemistry , Plant Stems/chemistry , SolventsABSTRACT
Two new sesquiterpenoids, (1S,4S,5R,10R)-1-hydroxy-6-isobutyryloxy-10H-9-oxofuranoeremophilane (1) and 1alpha-hydroxy-6beta-(2xi-methylbutyryloxy)-10alphaH-9-oxofuranoeremophilane (2), along with 21 known constituents, were isolated from the n-hexane and dichloromethane extracts of the above-ground biomass and roots of Senecio chionophilus. The structures of 1 and 2 were elucidated on the basis of spectroscopic evidence and chemical transformation methods. The absolute configuration of 1 was determined by Mosher ester methodology. All of the isolates were evaluated for their antitubercular potential against Mycobacterium tuberculosis in a microplate Alamar Blue assay. Compound 2, 6beta-angeloyloxy-1alpha-hydroxy-10alphaH-9-oxofuranoeremophilane (3), and 4'-hydroxyacetophenone (6) exhibited mild antitubercular activity at minimum inhibitory concentrations of 119, 114, and 121 microg/mL, respectively.
Subject(s)
Antitubercular Agents/isolation & purification , Mycobacterium tuberculosis/drug effects , Plants, Toxic/chemistry , Senecio/chemistry , Sesquiterpenes/isolation & purification , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Chile , Microbial Sensitivity Tests , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Plant Roots/chemistry , Sesquiterpenes/chemistry , Sesquiterpenes/pharmacology , StereoisomerismABSTRACT
Previous collaborative work by our group has led to the discovery of several plant isolates and derivatives with activities in in vivo models of cancer chemoprevention, including deguelin, resveratrol, bruceantin, brassinin, 4'-bromoflavone, and oxomate. Using a panel of in vitro bioassays to monitor chromatographic fractionation, a diverse group of plant secondary metabolites has been identified as potential cancer chemopreventive agents from mainly edible plants. Nearly 50 new compounds have been isolated as bioactive principles in one or more in vitro bioassays in work performed over the last five years. Included among these new active compounds are alkaloids, flavonoids, stilbenoids, and withanolides, as well as a novel stilbenolignan and the first representatives of the norwithanolides, which have a 27-carbon atom skeleton. In addition, over 100 active compounds of previously known structure have been obtained. Based on this large pool of potential cancer chemopreventive compounds, structure-activity relationships are discussed in terms of the quinone reductase induction ability of flavonoids and withanolides and the cyclooxygenase-1 and -2 inhibitory activities of flavanones, flavones and stilbenoids. Several of the bioactive compounds were found to be active when evaluated in a mouse mammary organ culture assay, when used as a secondary discriminator in our work. The compounds (2 S)-abyssinone II, (2 S)-2',4'-dihydroxy-2"-(1-hydroxy-1-methylethyl)dihydrofuro[2,3- h]-flavanone, 3'-[gamma-hydroxymethyl-( E)-gamma-methylallyl]-2,4,2',4'-tetrahydroxychalcone 11'- O-coumarate, isolicoflavonol, isoliquiritigenin, and ixocarpalactone A are regarded as promising leads as potential cancer chemopreventive agents.
Subject(s)
Anticarcinogenic Agents/therapeutic use , Neoplasms/drug therapy , Phytotherapy , Plant Extracts/therapeutic use , Plants, Medicinal , Animals , Anticarcinogenic Agents/chemistry , Humans , Mice , Plant Extracts/chemistry , Structure-Activity RelationshipABSTRACT
Antitubercular bioassay-guided fractionation of the n-hexane- and CH (2)Cl (2)-soluble extracts of the above-ground biomass and roots of Valeriana laxiflora led to the isolation of a new iridolactone, (4R,5R,7S,8S,9S)-7-hydroxy-8-hydroxymethyl-4-methyl-perhydrocyclopenta[ c]pyran-1-one ( 1), and a new lignan, (+)-1-hydroxy-2,6-bis- epi-pinoresinol ( 2), along with eleven known compounds, betulin ( 3), betulinic acid ( 4), 5,7-dihydroxy-3,6,4'-trimethoxyflavone ( 5), 23-hydroxyursolic acid ( 6), oleanolic acid ( 7), tricin ( 8), ursolic acid ( 9), ferulic acid, (+)-1-hydroxypinoresinol, prinsepiol, and 5,7,3'-trihydroxy-4'-methoxyflavone. The structures of compounds 1 and 2 were elucidated on the basis of spectroscopic evidence. The absolute stereochemistry of 1 was determined by chemical transformations and Mosher ester procedures. In a microplate alamar blue assay against Mycobacterium tuberculosis, compounds 2 - 9 exhibited minimum inhibitory concentrations (MIC) of 15.5 - 127 microg/mL, while the other isolates were regarded as inactive (MIC > 128 microg/mL). In addition, all the isolates were tested for cytotoxicity against African green monkey Vero cells in order to evaluate their selectivity potential.
Subject(s)
Antitubercular Agents/pharmacology , Mycobacterium tuberculosis/drug effects , Phytotherapy , Plant Extracts/pharmacology , Valerianaceae , Animals , Antitubercular Agents/administration & dosage , Antitubercular Agents/therapeutic use , Chlorocebus aethiops , Humans , Microbial Sensitivity Tests , Plant Extracts/administration & dosage , Plant Extracts/therapeutic use , Vero Cells/drug effectsABSTRACT
Bioactivity-directed fractionation of extracts of two Diospyros maritima bark samples from Indonesia,one collected at sea level in a beach forest in Java and the other collected at a slight elevation away from the sea shore on the island of Lombok, yielded a diverse set of secondary metabolites. The naphthoquinone plumbagin (1), although found in extracts of both specimens, constituted a much larger percentage of the former sample, which also yielded a series of plumbagin dimers, maritinone (2), chitranone (3), and zeylanone (4). The latter sample yielded a new naphthoquinone derivative, (4S)-shinanolone (5), and a new natural product coumarin, 7,8-dimethoxy-6-hydroxycoumarin (6), along with three other analogues of plumbagin, 2-methoxy-7-methyljuglone (7), 3-methoxy-7-methyljuglone (8), and 7-methyljuglone (9). The structures of compounds 5 and 6 were elaborated by physical, spectral, and chemical methods. All of the isolates were evaluated in both cytotoxicity and antimicrobial assays, and structure-activity relationships of these naphthoquinones are proposed. Plumbagin (1) and maritinone (2) were evaluated also for in vivo antitumor activity in the hollow fiber assay, but both were found to be inactive.
Subject(s)
Diospyros/chemistry , Naphthoquinones/isolation & purification , Plants, Medicinal/chemistry , Aspergillus niger/drug effects , Bacteria/drug effects , Candida albicans/drug effects , Coumarins/chemistry , Coumarins/isolation & purification , Coumarins/pharmacology , Drug Screening Assays, Antitumor , Humans , Indonesia , KB Cells/drug effects , Microbial Sensitivity Tests , Naphthoquinones/chemistry , Naphthoquinones/pharmacology , Plant Bark/chemistry , Saccharomyces cerevisiae/drug effectsABSTRACT
As a result of a bioactivity-guided search for novel, plant-derived cancer chemopreventive agents, ixocarpalactone A (5) was isolated previously as a potent quinone reductase inducer from the leaves and stems of Physalis philadelphica. In the present study, this promising lead compound was reisolated in gram quantities for in vivo biological testing. During the course of this work, four additional minor new withanolides were also obtained and characterized, namely, 2,3-dihydro-3beta-methoxyixocarpalactone A (1), 2,3-dihydro-3beta-methoxyixocarpalactone B (2), 2,3-dihydroixocarpalactone B (3), and 4beta,7beta,20R-trihydroxy-1-oxowitha-2,5-dien-22,26-olide (4). However, compounds 1 and 2 were determined using liquid chromatography (LC)-MS-MS to be artifacts generated during the extraction and isolation procedure. Ixocarpalactone A was detected in the fresh fruits (tomatillos) of P. philadelphica by LC-MS-MS analysis at a concentration of 143+/-4.53 ppb.
Subject(s)
Ergosterol/analogs & derivatives , Ergosterol/chemistry , Ergosterol/pharmacology , Lactones/chemistry , Lactones/pharmacology , NAD(P)H Dehydrogenase (Quinone)/biosynthesis , Physalis/chemistry , Animals , Artifacts , Cell Line, Tumor , Chromatography, Liquid , Crystallography, X-Ray , Enzyme Induction/drug effects , Fruit/chemistry , Lactones/isolation & purification , Liver Neoplasms, Experimental/enzymology , Magnetic Resonance Spectroscopy , Mass Spectrometry , Mice , Plant Leaves/chemistry , Plant Stems/chemistry , Structure-Activity RelationshipABSTRACT
Three new prenylated dihydrochalcones, (+/-)-nicolaioidesins A, B, and C (1-3), as well as a new natural product, 5-styrylfuran-2-carboxylic acid methyl ester (4), along with four known compounds, 2'-hydroxy-4',6'-dimethoxychalcone (5), (+/-)-5-hydroxy-7-methoxyflavanone (6), (+/-)-5-hydroxy-7,4'-dimethoxyflavanone, and panduratin A, were isolated from the roots of Renealmia nicolaioides, using a bioassay to determine the induction of quinone reductase (QR) activity with cultured Hepa lclc7 mouse hepatoma cells. Among these isolates, 5 and 6 induced QR activity, with observed concentrations to double activity (CD) values of 1.7 and 0.9 microg/mL, respectively, while the other constituents were not regarded as being active (CD >10 microg/mL). The chemical structures of 1-4 were elucidated by spectroscopic methods. A biogenetic pathway for the formation of (+/-)-nicolaioidins A-C (1-3) is proposed.
Subject(s)
Antineoplastic Agents, Phytogenic/isolation & purification , Chalcone/isolation & purification , Plants, Medicinal/chemistry , Zingiberaceae/chemistry , Animals , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Carcinoma, Hepatocellular , Chalcone/analogs & derivatives , Chalcone/chemistry , Chalcone/pharmacology , Chalcones , Chromatography, High Pressure Liquid , Drug Screening Assays, Antitumor , Inhibitory Concentration 50 , Mice , Molecular Conformation , Molecular Structure , NAD(P)H Dehydrogenase (Quinone)/metabolism , Peru , Plant Roots/chemistry , Structure-Activity Relationship , Tumor Cells, Cultured/drug effectsABSTRACT
Activity-guided fractionation of an ethyl acetate extract of the twigs of Hernandia ovigera, using a soft agar assay with JB6 murine epidermal cells, led to the isolation of two new naturally occurring aryltetralin lignans, (7R,8S,7'R,8'R)-(+)-7'-acetyl-5'-methoxypicropodophyllin (1) and (7R,8S,7'R,8'R)-(+)-7'-acetylpicropodophyllin (2), of which 2 was previously identified as a synthetic product, along with eight known compounds, epiyangambin (3), caruilignan C, diasesartemin, (+)-epimagnolin A, N-methylcorydaldine, (-)-5'-methoxyyatein, (+)-syringaresinol dimethyl ether, and (-)-yatein. The structures and stereochemistry of 1 and 2 were determined using spectroscopic methods. Compounds 2 and 3 exhibited significant inhibition of the transformation of murine epidermal JB6 cells, with IC50 values of 0.15 and 0.4 microg/mL, respectively, while the other isolates were regarded as inactive (IC50 >4 microg/mL).
Subject(s)
Anticarcinogenic Agents/isolation & purification , Lignans/isolation & purification , Plants, Medicinal/chemistry , Animals , Anticarcinogenic Agents/chemistry , Anticarcinogenic Agents/pharmacology , Cells, Cultured/drug effects , Epidermis/drug effects , Indonesia , Inhibitory Concentration 50 , Lignans/chemistry , Lignans/pharmacology , Mice , Molecular Conformation , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Activity-guided fractionation of an ethyl acetate extract of the aerial parts of Tithonia diversifolia, using an antiproliferation bioassay performed with human colon cancer (Col2) cells, led to the isolation of three new sesquiterpenoids, 2alpha-hydroxytirotundin (1), tithofolinolide (2), and 3alpha-acetoxydiversifolol (3), along with eight known sesquiterpene lactones, 3beta-acetoxy-8beta-isobutyryloxyreynosin (4), tagitinin C (5), 1beta,2alpha-epoxytagitinin C (6), 4alpha,10alpha-dihydroxy-3-oxo-8beta-isobutyryloxyguaia-11(13)-en-12,6alpha-olide (7), 3alpha-acetoxy-4alpha-hydroxy-11(13)-eudesmen-12-oic acid methyl ester, 17,20-dihydroxygeranylnerol, tagitinin A, and tirotundin. These isolates were evaluated for their potential as cancer chemopreventive agents, by measuring antiproliferative activity in Col2 cells and induction of cellular differentiation in human promyelocytic leukemia (HL-60) cells. Selected compounds were then investigated for their ability to inhibit 7,12-dimethylbenz[a]anthracene-induced preneoplastic lesions in a mouse mammary organ culture assay. Among these isolates, 5 and 6 showed significant antiproliferative activity, 2, 4, and 7 induced HL-60 cellular differentiation, and 4 significantly inhibited (63.0% at 10 microg/mL) lesion formation in the mouse mammary organ culture assay. The chemical structures of 1-3 were elucidated by spectroscopic analysis. The absolute configurations of 1 and 2 were determined by Mosher ester methodology.