Agonist occupancy of a single monomeric element is sufficient to cause internalization of the dimeric beta2-adrenoceptor

A range of studies have indicated that many rhodopsin-like, family A G protein-coupled receptors, including the beta(2)-adrenoceptor, exist and probably function as dimers. It is less clear if receptors internalize as dimers and if agonist occupancy of only one element of a dimer is sufficient to cause internalization of a receptor dimer into the cell. We have used a chemogenomic approach to demonstrate that this is the case. Following expression of the wild type beta(2)-adrenoceptor, isoprenaline but not 1-(3'4'-dihydroxyphenyl)-3-methyl-1-butanone, which does not have significant affinity for the wild type receptor, caused receptor internalization. By contrast, 1-(3'4'-dihydroxyphenyl)-3-methyl-1-butanone, but not isoprenaline that does not have high affinity for the mutated receptor, caused internalization of Asp(113)Serbeta(2)-adrenoceptor. Following co-expression of wild type and Asp(113)Serbeta(2)-adrenoceptors each of isoprenaline and 1-(3'4'-dihydroxyphenyl)-3-methyl-1-butanone caused the co-internalization of both of these two forms of the receptor. Co-expressed wild type and Asp(113)Serbeta(2)-adrenoceptors were able to be co-immunoprecipitated and 1-(3'4'-dihydroxyphenyl)-3-methyl-1-butanone produced internalization of the wild type receptor that was not prevented by the beta-adrenoceptor antagonist propranolol that binds with high affinity only to the wild type receptor. These results demonstrate that agonist occupancy of either single binding site of the beta(2)-adrenoceptor dimer is sufficient to cause internalization of the dimer and that antagonist occupation of one of the two ligand binding sites is unable to prevent agonist-mediated internalization.     

Isolation of novel phenolic compounds with multidrug resistance (MDR) reversal properties from Onychium japonicum

We isolated seven novel compounds, namely, 3',4',6-trihydroxy-2,4-dimethoxy-3-(3″,4″-dihydroxybenzyl)chalcone (1), 3',6-dihydroxy-2,4,4'-trimethoxy-3-(3″,4″-dihydroxybenzyl)chalcone (2), α,β-dihydro-3',6-dihydroxy-2,4,6'-trimethoxy-3-(3″,4″-dihydroxybenzyl)chalcone (3), 3',4,4'-trihydroxy-2,6-dimethoxychalcone (4), 4',5,7-trihydroxy-6-(3″,4″-dihydroxybenzyl)flavone (5), 3-(3',4'-dihydroxybenzyl)-6,7-dihydroxycoumarin (6), 3-(3',4'-dihydroxyphenyl)-3,4-dihydroisocoumarin (7), as well as a known compound, 3',4',7-trihydroxy-5-methoxyflavanone (8) from the whole grass of Onychium japonicum, and elucidated their structures by spectroscopic methods. Compounds 1-3 exhibited significant multidrug resistance (MDR) reversal effects on MCF-7/ADR and Bel-7402/5-Fu cell lines.     

New bisabolane sesquiterpenes and coumarin from Leontopodium longifolium

From the roots of Leontopotium longifolium, three new bisabolane sesquiterpenes, rel-(1S,4R,5S,6R)-4,5-diacetoxy-6-[(R)-1,5-dimethylhexa-3,5-dienyl]-3-methylcyclohex-2-enyl (Z)-2-methylbut-2-enoate (1), rel-(1S,4R,5S,6R)-4,5-diacetoxy-6-[(R)-5-hydroxy-1,5-dimethylhex-3-enyl]-3-methylcyclohex-2-enyl (Z)-2-methylbut-2-enoate (2), rel-(1R,2S,4R,5S)-4-acetoxy-2-[(R)-5-hydroxy-1,5-dimethylhex-3-enyl]-5-methylcyclohexyl (Z)-2-methylbut-2-enoate (3), and a new coumarin, 2,3-dihydro-5-hydroxy-2-(1-methylethenyl)-7H-pyrano[2,3-g][1,4]benzodioxin-7-one (4) together with nine known compounds have been isolated. The structures of these compounds were established by spectroscopic methods. Compounds 1 and 2 exhibited moderate cytotoxic activities against human promyelocytic leukemia (HL-60) cells.     

海洋真菌Arthrinium sp. UJNMF0008代谢产物的研究

为丰富海洋真菌的化学多样性,发现海洋真菌活性代谢产物,对海洋沉积物来源真菌Arthriniumsp.UJNMF0008的化学成分及其生物活性进行研究,采用硅胶柱色谱、凝胶柱色谱、反向柱色谱和高效液相色谱等方法从海洋沉积物来源真菌Arthriniumsp.UJNMF0008的发酵提取物中分离到5个化合物,通过核磁共振、质谱等方法,结合文献对照,鉴定了化合物的结构分别为arthoneF(1)、arthoneG(2)、sydoxanthoneC(3)、(3R,4R)-cis-4-hydroxymellein(4)和2-(2′S-hydroxypropyl)-5-methyl-7-hydroxychromone(5),其中化合物1和2是新化合物,化合物3首次从该属真菌中分离到。活性测试显示,化合物1~5在50μmoL/L的测试浓度下均没有表现出明显的抗氧自由基活性、抗菌活性以及NO释放抑制活性。     

Synthetic studies of proanthocyanidins. Highly stereoselective synthesis of the catechin dimer,procyanidin-B3

A stereoselective synthesis of benzylated procyanidin-B3, a condensed catechin dimer, is described. Condensation of 5,7,3',4'-tetrabenzylcatechin with (2R,3S,4S)-5,7,3',4'-tetrabenzyloxy-3-acetoxy-4-methoxyflavan as an electrophile in the presence of TiCl4 led to octabenzylated procyanidin-B3 stereoselectively.     

羊角拗茎的化学成分及其抗炎活性研究

探讨羊角拗茎的化学成分及其抗炎活性,采用硅胶、Sephadex LH-20等色谱分离技术分离纯化羊角拗茎乙醇提取物的乙酸乙酯萃取物中的化合物,通过质谱和核磁共振等波谱技术对其结构进行鉴定,确定出了13个化合物,包括常春藤皂苷元(1)、(-)-loliolide(2)、(3S,5R,6S,7E)-3,5,6-trihydroxy-7-megastigmen-9-one(3)、(3R,6R,7E)-3-hydroxy-4,7-megastigmadien-9-one(4)、松脂素(5)、表松脂素(6)、4,4′-dihydroxy-3,3′-dimethoxybenzophenone(7)、6-羟基柚皮素(8)、东莨菪素(9)、6-羟基-7,8-二甲氧基香豆素(10)、吲唑(11)、香草酸(12)、对羟基苯甲酸(13)。其中化合物1～4和6～13为首次从羊角拗中分离得到。采用LPS诱导小鼠单核巨噬细胞RAW 264.7体外细胞炎症模型测试结果表明,化合物5能显著抑制NO的产生,IC₅₀值为18.09±1.09μM。     

Substrate specificity of catechol oxidase from Lycopus europaeus and characterization of the bioproducts of enzymic caffeic acid oxidation

The substrate specificity of catechol oxidase from Lycopus europaeus towards phenols is examined. The enzyme catalyzes the oxidation of o-diphenols to o-quinones without hydroxylating monophenols, the additional activity of tyrosinase. Substrates containing a -COOH group are inhibitors for catechol oxidase. The products of enzymic oxidation of caffeic acid were analyzed and isolated by HPLC with diode array detection. The neolignans of the 2,3-dihydro-1,4-benzodioxin type (3, 6-8), 6,7-dihydroxy-1-(3,4-dihydroxyphenyl)-2,3-dicarboxy-1,2-dihydro naphthaline (1) 6,7-dihydroxy-1-(3,4-dihydroxyphenyl)-3-carboxynaphthaline (5) and 2,6-bis-(3',4'-dihydroxyphenyl)-1-carboxy-3-oxacyclo-(3,0)-pent an-2-on-1-ene (4) were formed. A reaction mechanism for the formation of (1, 4 and 5) is discussed.     

Hantzsch 1,4-dihydropyridines containing a diazen-1-ium-1,2-diolate nitric oxide donor moiety to study calcium channel antagonist structure-activity relationships and nitric oxide release

A group of racemic 3-isopropyl 5-[(2-piperazin-1-yl)ethyl] 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates (12a-c), 3-isopropyl 5-{2-[4-nitrosopiperazinyl]ethyl} 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates (14a-c) and 3-isopropyl 5-{2-[(O(2)-acetoxymethyldiazen-1-ium-1,2-diolate)(N,N-dialkylamino or 4-piperazin-1-yl)]ethyl} 1,4-dihydro-2,6-dimethyl-4-(pyridyl)-3,5-pyridinedicarboxylates (22-30) were prepared using modified Hantzsch reactions. This group of compounds (12a-c, 14a-c, and 22-30) exhibited less potent calcium channel antagonist activity (IC(50)=0.11 to 3.35muM range) than the reference drug nifedipine (IC(50)=0.01 microM). The point of attachment of the isomeric C-4 substituent was a determinant of calcium channel antagonist activity providing the potency profile 2-pyridyl3-pyridyl4-pyridyl. The N-nitrosopiperazinyl compounds (14a-c) did not release nitric oxide. The prodrugs 22-30 that have a C-5 2-[(O(2)-acetoxymethyldiazen-1-ium-1,2-diolate)(N,N-dialkylamino or 4-piperazin-1-yl)]ethyl ester substituent, upon incubation with guinea pig serum, undergo consecutive cleavage of the O(2)-acetoxymethyl moiety to give a nitric oxide donor diazenium-1-ium-1,2-diolate species that subsequently releases nitric oxide. The extent of nitric oxide released from the diazen-1-ium-1,2-diolate group is dependent upon the nature of the amino functionality attached directly to the diazen-1-ium N-1 position where the nitric oxide release profile is 1,4-piperazinyl>N-Et>N-(n-Bu)>N-Me upon exposure to guinea pig serum esterase(s). The results from this study suggest this class of hybrid calcium channel antagonist/nitric oxide donor prodrugs should release the vasodilator nitric oxide in vivo, preferentially in the vascular endothelium, to enhance the smooth muscle calcium channel antagonist effect to produce a combined synergistic antihypertensive effect.     

Role of nitric oxide in radiation-induced initiation of mammary tumorigenesis in rats.

Nitric oxide (NO) and its reaction products have been shown to cause DNA damage and to be mutagenic. To elucidate whether NO produced by irradiation participates in the initiation of mammary tumorigenesis, we performed experiments using the nitric oxide-specific scavenger Fe(2+)-diethyldithiocarbamate complex (Fe(DETC)(2)) or a selective inhibitor for inducible nitric oxide synthase (iNOS), S,S(')-(4-phenylene-bis(1,2-ethanedinyl))bis-isothiourea (1,4-PB-ITU). Mother rats at day 21 of lactation were injected simultaneously with diethyldithiocarbamate intraperitoneally and Fe(2+)-citrate subcutaneously to form Fe(DETC)(2), in vivo, and then irradiated with 1.5Gy gamma-rays immediately after the injection. An additional injection of chemicals followed twice at 8 and 24h after the irradiation in the same manner. Both control and treated rats were then implanted with diethylstilbestrol pellets as a tumor promoter. The mammary tumor incidence in the experimental group was significantly reduced to one-fourth of that in the irradiated-alone group as the control. On the other hand, when mother rats took drinking water containing 0.005% 1,4-PB-ITU for 6 days from 3 days prior to irradiation at day 21 of lactation, a low tumor incidence in the iNOS inhibitor-treated groups was observed in the 1-year period. This report is the first to show that the NO derived from iNOS is an important radical for radiation-induced initiation of tumorigenesis of mammary glands in rats.     

Constituents of Asarum sieboldii with inhibitory activity on lipopolysaccharide (LPS)-induced NO production in BV-2 microglial cells

Bioassay-guided fractionation of the root extract of Asarum sieboldii led to the isolation of the four active compounds (-)-sesamin (1), (2E,4E,8Z,10E)-N-(2-methylpropyl)dodeca-2,4,8,10-tetraenamide (2), kakuol (3), and '3,4,5-trimethoxytoluene' (=1,2,3-trimethoxy-5-methylbenzene; 4), in terms of inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production. Compounds 1-4 showed potent inhibition of NO production, with IC(50) values in the low nanomolar-to-micromolar range. Also isolated were the known compounds methylkakuol (5), '3,5-dimethoxytoluene', safrole, asaricin, methyleugenol, and (-)-asarinin, which were found to be inactive in the above assay. Among the ten known isolates, compounds 1, 2, and 5 were found for the first time in this plant.     

Synthesis and biological evaluation of 1,4-dihydropyridine calcium channel modulators having a diazen-1-ium-1,2-diolate nitric oxide donor moiety for the potential treatment of congestive heart failure

A group of racemic 4-aryl(heteroaryl)-1,4-dihydro-2,6-dimethyl-3-nitropyridines possessing nitric oxide donor O(2)-acetoxymethyl-1-(N-ethyl-N-methylamino, or 4-ethylpiperazin-1-yl)diazen-1-ium-1,2-diolate, C-5 ester substituents were synthesized by coupling the respective 4-aryl(heteroaryl)-1,4-dihydro-2,6-dimethyl-3-nitropyridine-5-carboxylic acids with either O(2)-acetoxymethyl-1-[N-(2-methylsulfonyloxyethyl)-N-methylamino]diazen-1-ium-1,2-diolate, or O(2)-acetoxymethyl-1-[4-(2-methylsulfonyloxyethyl)piperazin-1-yl]diazen-1-ium-1,2-diolate. Compounds having a C-4 2-pyridyl, 4-pyridyl, 2-trifluoromethylphenyl, or benzofurazan-4-yl substituent exhibited more potent smooth muscle calcium channel antagonist activity (IC(50)'s in the 0.37-1.09 microM range) than related analogs having a C-4 3-pyridyl substituent (IC(50)'s=3.03-9.14 microM range) relative to the reference drug nifedipine (IC(50)=9.13 nM). The point of attachment of C-4 isomeric pyridyl substituents was a determinant of smooth muscle calcium channel antagonist activity where the relative potency profile was 4-pyridyl>2-pyridyl>3-pyridyl. Replacement of the C-5 methyl ester substituent of methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate (Bay K 8644) by an O(2)-acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate, or O(2)-acetoxymethyl-1-(4-ethylpiperazin-1-yl)diazen-1-ium-1,2-diolate, C-5 ester substituent provided compounds, which exhibited a lower, yet respectable, cardiac positive inotropic effect (IC(50)'s=4.82 and 4.05 microM, respectively) relative to the reference drug Bay K 8644 (IC(50)=0.30 microM). All compounds released nitric oxide upon incubation with either phosphate buffer at pH7, or porcine liver esterase. However, the percentage nitric oxide released was up to 3-fold higher (76%) when these O(2)-acetoxymethyl-1-(alkylamino)diazen-1-ium-1,2-diolates were incubated with guinea pig serum. These results suggest that *NO would be released in vivo, upon cleavage by nonspecific serum esterases, preferentially in the vascular endothelium where it may enhance smooth muscle calcium channel antagonist activity.     

Synthesis of fused bicyclic thioglycosides of N-acylated glucosamine as analogues of mycothiol

The synthesis of a fused bicyclic thioglycoside analogue of mycothiol, (3R)-3-acetylamino-4-one-6,7-dihydro-(1',2'-dideoxy-beta-D-glucopyranoso)[2',1'-f]-1,5-thiazepane (5), is reported. Treatment of phthalimido-protected peracetylated glucosamine with N-acetyl-cysteine and boron trifluoride-etherate gave the beta-linked thioglycoside, which was deprotected and cyclized, using HOBt and EDCl to form the lactam and giving the target structure. This mycothiol mimic and its tri-O-acetate will be investigated as potential inhibitors of enzymes involved in the biosynthesis of mycothiol. The protected derivative also has the potential to be an alpha-selective N-cysteinyl glucosamine donor; however, initial glycosylation attempts failed due to the apparent stability of the fused bicyclic system.     

1,5-Disubstituted indole derivatives as selective human neuronal nitric oxide synthase inhibitors

A series of 1,5-disubstituted indole derivatives was designed, synthesized and evaluated as inhibitors of human nitric oxide synthase. A variety of flexible and restricted basic amine side chain substitutions was explored at the 1-position of the indole ring, while keeping the amidine group fixed at the 5-position. Compounds having N-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)- (12, (R)-12, (S)-12 and 13) and N-(1-(1-methylazepan-4-yl)- side chains (14, 15, (-)-15 and (+)-15) showed increased inhibitory activity for the human nNOS isoform and selectivity over eNOS and iNOS isoforms. The most potent compound of the series for human nNOS (IC(50)=0.02 μM) (S)-12 showed very good selectivity over the eNOS (eNOS/nNOS=96-fold) and iNOS (iNOS/nNOS=850-fold) isoforms.     

Calmodulin-independent nitric oxide synthase from rat polymorphonuclear neutrophils.

Recently, the purification of nitric oxide synthase (EC 1.14.23) from rat cerebellum has been reported, and the enzyme is a calmodulin-requiring enzyme (Bredt, D. S., and Snyder, S. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685). In this paper, nitric oxide synthase has been purified to near homogeneity from the cytosol fraction of rat polymorphonuclear neutrophils. The purification procedure involves affinity chromatography with adenosine 2',5'-diphosphate-agarose and an anion exchange column, DEAE-Bio-Gel A. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrated as a single protein band with Mr = 150,000. The molecular weight was estimated to be 150,000 by gel filtration on a Superose 12 HR 10/30. The purified enzyme was unstable with a half-life of 3 h at pH 7.4 and 4 degrees C. The enzyme activity required the presence of Ca2+, NADPH, FAD, and (6R)-5,6,7,8-tetrahydro-L-biopterin. Calmodulin antagonists (W5, W7, W13, and trifluoperazine dihydrochloride) did not inhibit the enzyme activity, and the addition of calmodulin was also ineffective for the increase in the enzyme activity. The neutrophil enzyme appears to be a calmodulin-independent type of nitric oxide synthase.     

3-Hydroxy-4-methyl-5-pentyl-2-iminopyrrolidine: a potent and highly selective inducible nitric oxide synthase inhibitor

(3S,4S,5R)-2-Imino-4-methyl-5-pentyl-3-pyrrolidinol hydrochloride (1) is a potent inducible nitric oxide synthase (i-NOS) inhibitor that has three times the selectivity of its parent, (+)-cis-4-methyl-5-pentylpyrrolidin-2-imine hydrochloride (2).     

Very long-chain phenylpropyl and phenylbutyl esters from Taxus baccata needle cuticular waxes

The cuticular wax of Taxus baccata L. needles was found to contain four different classes of long-chain esters that were identified by various chemical transformations with product assignment employing GC-MS. Homologous series of (1) 3-(4'-hydroxyphenyl)-propyl esters of C(20)-C(36) fatty acids, (2) 4-(4'-hydroxyphenyl)-2-butyl esters of C(18)-C(28) fatty acids, (3) 3-(3',4'-dihydroxyphenyl)-propyl esters of C(20)-C(32) fatty acids, and (4) 4-(3',4'-dihydroxyphenyl)-2-butyl esters of C(18)-C(28) fatty acids were identified. The four compound classes amounted to 0.1-3.6 micro g/cm(2) of needle surface area, corresponding to 0.2-7.6% of the wax mixture, respectively. While both phenylpropyl ester series had a maximum for the homolog containing tetracosanoic acid, in the phenylbutyl esters homologs containing eicosanoic and docosanoic acids predominated.     

(3Z)-2-Acetylamino-3-octadecen-1-ol as a potent apoptotic agent against HL-60 cells

(2R,3Z)-, (2R,3E)-, (2S,3Z) and (2S,3E)-2-Acetylamino-3-octadecen-1-ol, and (2R)- and (2S)-2-acetylamino-octadecan-1-ol were prepared using the Wittig olefination of Garner's aldehyde (N-Boc-N,O-isopropylidene-L- or D-serinal) from L- or D-serine. The apoptotic activities of these saturated and unsaturated 2-acetylaminoalcohols were examined in human leukemia HL-60 cells using MTT assay. Among the newly synthesized compounds, the cis-isomers were the most potent. Despite their simple structures, (2R,3Z)- and (2S,3Z)-2-acetylamino-3-octadecen-1-ol showed high and comparable apoptotic activities compared with N-acetyl-D-erythro-sphingosine (D-e-C2-Cer, a well-known inducer of apoptosis). Their apoptotic activities were in the order D-e-C2-Cer approximately L-e-C2-Cer approximately (2R,3Z)- approximately (2S,3Z)->(2R,3E)- approximately (2S,3E)- approximately (2R)- approximately (2S)-derivative. Qualitative analysis of DNA fragmentation caused by these compounds was conducted using agarose gel electrophoresis, and typical DNA fragmentation was found in the cases of (2R,3Z)- and (2S,3Z)-isomers such as C2-Cer, but not trans and saturated isomers. The morphological features of the cells, the proteolytic processing of pro-caspase-3, and the cleavage of PARP as a result of exogenous treatment with (2R,3Z)- and (2S,3Z)-isomers indicated that cell death induced by these compounds was apoptosis. These observations suggest that these newly synthesized compounds, (3Z)-2-Acetylamino-3-octadecen-1-ol, have similar characteristics and apoptosis-inducing activities against HL-60 cells with C2-Cer.     

Mass spectrometric analysis of catechol-histidine adducts from insect cuticle

Adducts of catechols and histidine, which are produced by reactions of 1,2-quinones and p-quinone methides with histidyl residues in proteins incorporated into the insect exoskeleton, were characterized using electrospray ionization mass spectrometry (ESMS), tandem electrospray mass spectrometry (ESMS-MS, collision-induced dissociation), and ion trap mass spectrometry (ITMS). Compounds examined included adducts obtained from acid hydrolysates of Manduca sexta (tobacco hornworm) pupal cuticle exuviae and products obtained from model reactions under defined conditions. The ESMS and ITMS spectra of 6-(N-3')-histidyldopamine [6-(N-3')-His-DA, pi isomer] isolated from M. sexta cuticle were dominated by a [M + H]+ ion at m/z 308, rather than the expected m/z 307. High-resolution fast atom bombardment MS yielded an empirical formula of C14H18N3O5, which was consistent with this compound being 6-(N-1')-histidyl-2-(3, 4-dihydroxyphenyl)ethanol [6-(N-1')-His-DOPET] instead of a DA adduct. Similar results were obtained when histidyl-catechol compounds linked at C-7 of the catechol were examined; the (N-1') isomer was confirmed as a DA adduct, and the (N-3') isomer identified as an (N-1')-DOPET derivative. Direct MS analysis of unfractionated cuticle hydrolysate revealed intense parent and product ions characteristic of 6- and 7-linked adducts of histidine and DOPET. Mass spectrometric analysis of model adducts synthesized by electrochemical oxidative coupling of N-acetyldopamine (NADA) quinone and N-acetylhistidine (NAcH) identified the point of attachment in the two isomers. A prominent product ion corresponding to loss of CO2 from [M + H]+ of 2-NAcH-NADA confirmed this as being the (N-3') isomer. Loss of (H2O + CO) from 6-NAcH-NADA suggested that this adduct was the (N-1') isomer. The results support the hypothesis that insect cuticle sclerotization involves the formation of C-N cross-links between histidine residues in cuticular proteins, and both ring and side-chain carbons of three catechols: NADA, N-beta-alanyldopamine, and DOPET.     

Design and synthesis of orally bioavailable inhibitors of inducible nitric oxide synthase. Part 1: synthesis and biological evaluation of dihydropyridin-2-imines

Dihydropyridin-2-imines were synthesized and biologically evaluated both in vitro and in vivo using a nitric oxide inhibition assay. Compounds 1, 4, 5 and 7-11 exhibited potent activity in the inducible nitric oxide (iNOS) inhibition assay. Of these 5, 6, 9 and 10 showed 5- to 11-fold increases in isoform selectivity. Compounds 1, 5, 9 and 10 showed potent inhibitory activity in the NOx accumulation assay in mice. Compounds 1 and 5 also showed good bioavailability (BA) when given orally.     

Bi-bicyclic and bi-tricyclic compounds from Dendrobium thyrsiflorum

One bi-bicyclic and two bi-tricyclic derivatives of coumarin-benzofuran, phenanthrene-phenanthrene and phenanthrene-phenanthraquinone, along with seven known compounds, were isolated from stems of Dendrobium thyrsiflorum Rchb.f. (Orchidaceae). On the basis of chemical, NMR (1H, 13C, HMQC, HMBC and NOESY) and mass spectrometry data, their structures were elucidated as denthyrsin [3-(5',6'-dimethoxybenzofuran-2'-yl)-6,7-dimethoxy-2H-chromen-2-one; 1], denthyrsinol (4,5'-dimethoxy-[1,1']biphenanthrenyl-2,5,4',7'-tetraol; 2), and denthyrsinone (7,4',7'-trihydroxy-2,2',8'-trimethoxy-[5,1']biphenanthrenyl-1,4-dione; 3). Compounds 1-3 and denthyrsinin (1,5,7-trimethoxyphenanthrene-2,6-diol; 4) showed significant cytotoxic activities against Hela (13.5, 9.3, 9.9 and 2.7 microM, respectively), K-562 (0.45, 1.6, 6.0 and 2.3 microM, respectively) and MCF-7 (18.1, not tested, 3.5 and 4.8 microM, respectively) cell lines.