Effect of natural and synthetic benzyl benzoates on calmodulin

The present investigation describes the effect of the spasmolytic benzylbenzoates 1-9 from Brickellia veronicifolia on CaM using a functional in vitro enzymatic assay. Bovine brain PDE1 was used as a monitoring enzyme. The most active natural inhibitors of the system CaM-PDE1 were benzyl benzoates 3-5, which inhibited the activity of PDE1 in a concentration-dependent manner. In addition, three series of analogs of compound 4, compounds 10a-32a, were prepared and assayed. The benzyl benzoates from the first series, namely 10a-24a, possess no substituents on ring B but different number and position of hydroxyl or methoxy groups in ring A. The second group (25-32a), on the other hand, possesses an A ring identical to that on compound 4, but different substituents in Ring B. The most active compounds were 14a, 15a and 30a. These compounds were two to six times more potent than chlorpromazine, a well known CaM inhibitor. Benzyl benzoates 14a and 15a have methoxyl groups at C-2/C-4 and C-3/C-4 in ring A, respectively; while 30a, in addition to the methoxyl groups at C-2/C-6 of ring A, hold a benzoyloxy moiety at C-3' of ring B. Kinetic studies revealed that compounds 3, 4, 14a, 15a and 30a behave as competitive CaM antagonists.     

Cholestane and spirostane glycosides from the rhizomes of Dioscorea septemloba

Cholestane glycosides, dioseptemlosides A (1) and B (2), together with six spirostane glycosides, dioseptemlosides C-H (3-8), were isolated from the rhizomes of Dioscorea septemloba. Their structures were established on the basis of physical data, spectroscopic analysis (HRESIMS, 1D and 2D NMR), and chemical methods. Spirostane aglcones containing hydroxyl group at C-7, as found in compounds 4-7, were reported in the family Dioscoreaceae for the first time. These compounds did not show considerable inhibitory anti-tumor activities at a concentration of 10 microM.     

Homoisoflavanones from Disporopsis aspera

From cytotoxic extracts of the roots of Disporopsis aspera Engl. (Liliaceae) a homoisoflavanone, disporopsin (3-(2',4'-dihydroxy-benzyl)-5,7-dihydroxy-chroman-4-one) (1) and three rare methyl-homoisoflavanones, 3-(4'-hydroxy-benzyl)-5,7-dihydroxy-6-methyl-chroman-4-one (2), 3-(4'-hydroxy-benzyl)-5,7-dihydroxy-6,8-dimethyl-chroman-4-one (3) and 3-(4'-hydroxy-benzyl)-5,7-dihydroxy-6-methyl-8-methoxy-chroman-4- one (4) along with five other known compounds, N-trans-feruloyl tyramine (5), adenine (6), 5-(hydroxymethyl)-2-furfural (7), beta-sitosterol (8) and beta-sitosteryl glucopyranoside (9) were isolated. The structures of compounds 1-2 were elucidated by spectral data (1, 2-D NMR and EIMS). The four homoisoflavanones (1-4) were found to be cytotoxic against a series of human cancer cell lines (HCT15, T24S, MCF7, Bowes, A549 and K562) with IC(50) ranging from 15 to 200 microM. Possible biosynthesis routes for homoisoflavonoids (1-4) are discussed.     

Stemodane skeletal rearrangement: chemistry and microbial transformation

Solvolytic rearrangement of the C/D ring system of the tetracyclic diterpenoid stemodinone (2) afforded the compounds 15(13-->12)abeo-13beta-hydroxystemaran-2-one (5) and 15(8-->9)abeo-8beta(H)-12beta-hydroxystachan-2-one (10). Terpene 5 possesses a novel diterpene skeleton. Oxidation of these compounds yielded their respective diketones. Bioconversion of 5 by Rhizopus oryzae yielded 15(13-->12)abeo-7beta,13beta-dihydroxystemaran-2-one (18) while microbial transformation of 10 provided 15(8-->9)abeo-8beta(H)-6alpha,12beta-dihydroxystachan-2-one (19), 15(8-->9)abeo-8beta(H)-7beta,12beta-dihydroxystachan-2-one (20) and 15(8-->9)abeo-8beta(H)-6alpha,12beta,14beta-trihydroxystachan-2-one (21). A rationale for the formation of the rearranged compounds is proposed.     

Mulberry anthracnose antagonists (iturins) produced by Bacillus amyloliquefaciens RC-2

Bacillus amyloliquefaciens strain RC-2 produced seven antifungal compounds (1-7) secreted into the culture filtrate. These compounds inhibited the development of mulberry anthracnose caused by the fungus, Colletotrichum dematium. Chemical structural analyses by NMR and FAB-MS revealed that all these compounds were iturins (cyclic peptides with the following sequence: L-Asn --> D-Tyr --> D-Asn --> L-Gln --> L-Pro --> D-Asn --> L-Ser --> D-beta-amino acid -->) and compounds 1-6 are identical to iturins A-2-A-7, respectively. Compound 7 (iturin A-8) is a new iturin, which has a -(CH(2))(10)CH(CH(3))CH(2)CH(3) group as a side chain in the beta-amino acid in the molecule.     

Three types of sesquiterpenes from rhizomes of Atractylodes lancea

Eight sesquiterpenes, including four guaiane-types containing an interesting epoxy unit (1-4), a rare tricyclic carbon skeleton-type (5) and three eudesmane-types (6-8), along with five known compounds, were isolated from rhizomes of Atractylodes lancea. The structures and relative configurations of 1-8 were determined by analysis of spectroscopic data, and the absolute configuration of 8 was assigned by application of the CD technique. Compounds 1, 2 and 4 were evaluated for their cytotoxic effects against P388 and A549 cells, but all were inactive. Possible biosynthetic pathways for sesquiterpenes (1-8) were discussed.     

Synthesis of 3-alkyl(aryl)-4-alkylidenamino-4,5-dihydro-1H-1,2,4-triazol-5-ones and 3-alkyl-4-alkylamino-4,5-dihydro-1H-1,2,4-triazol-5-ones as antitumor agents

A series of 3-alkyl-4-phenylethylidenamino- (8) and 3-alkyl-4-(3-phenylallylidenamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones (9) was synthesized from the reaction of the corresponding 3-alkyl(aryl)-4-amino-4,5-dihydro-1H-1,2,4-triazol-5-ones (1), with phenylacetaldehyde and cinnamaldehyde. 3-Alkyl-4-(2-phenylethylamino)- (10) and 3-alkyl-4-(3-phenylpropylamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones (11) were obtained from the selective reduction of compounds (8) and (9) with NaBH₄. The in vitro antitumor activity of the novel compounds was screened and the highest inhibition of tree tumor cell lines was observed for the compounds containing phenylethylenamino and phenylethylamino groups at position 4 of 1,2,4-triazol ring.     

Design and synthesis of methyl 2-methyl-7,7-dihalo-5-phenyl-2-azabicyclo[4.1.0]hept-3-ene-4-carboxylates with calcium channel antagonist activity

A group of methyl 2-methyl-7,7-dihalo-5-(substituted-phenyl)-2-azabicyclo[4.1.0]hept-3-ene-4-carboxylates were prepared by reaction of dihalocarbenes (:CX2, X = Br, Cl) with methyl 1-methyl-4-(substituted-phenyl)-1,4-dihydropyridine-3-carboxylates. In vitro calcium channel antagonist activities were determined using a guinea pig ileum longitudinal smooth muscle assay. The title compounds exhibited weaker CC antagonist activity (10(-5)-10(-6)M range) than the reference drug nifedipine (1.4 x 10(-8)M). Structure-activity relationship studies showed that the position of a nitro substituent on the C-5 phenyl ring where the relative potency order was ortho > meta > para, and the size and/or electronegativity of the C-7 geminal-dihalo substituents (Br > Cl), were determinants of calcium channel antagonist activity. This class of compounds did not exhibit any inotropic effect on guinea pig left atria. A dihalocyclopropyl moiety is a potential bioisostere for the 2-methyl-3-methoxycarbonylvinyl moiety present in the calcium channel antagonist nifedipine.     

Constituents of the stems of Macrococculus pomiferus and their inhibitory activities against cyclooxygenases-1 and -2

As part of our program directed towards the discovery of new cancer chemopreventive agents from plants, the EtOAc-soluble extract of the stems of M. pomiferus was found to inhibit the enzyme cyclooxygenase-2 (COX-2). Bioassay-directed fractionation of this extract led to the isolation of two dibenzylbutyrolactone lignans, (8R,8'R)-3'-O-demethyl-5-hydroxymatairesinol (1) and (8R,8'R)-3'-O-demethyl-5-methoxymatairesinol (2), as well as seven known compounds, (-)-5'-methoxyyatein (3), blumenol A, (-)-deoxypodophyllotoxin (anthricin), (-)-deoxypodorhizone, 2,6-dimethoxyhydroquinone, 4-hydroxybenzaldehyde, and beta-sitosterol glucoside. The structures of compounds 1 and 2 were determined using spectroscopic data (1D and 2D NMR, and HREIMS), and the 8R and 8'R absolute stereochemistry was established for both 1 and 2 on the basis of their CD spectra. All isolates obtained in the present study were evaluated for their inhibitory effects with both COX-1 and -2. Of these, only 5'-methoxyyatein (3) showed weak activity against COX-2, while all other compounds isolated were inactive. The COX-2 inhibitory activity of the EtOAc extract was also traced to the presence of several common fatty acids by LC-MS.     

Indole alkoloids from Nauclea officinalis with weak antimalarial activity

Five indole alkaloids (naucleofficines A-E) were isolated from the stems (with bark) of Nauclea officinalis: (E)-2-(1-beta-d-glucopyranosyloxybut-2-en-2-yl)-3-(hydroxymethyl)-6,7-dihydro-indolo[2,3-a]quinolizin-4(12H)-one (1), (E)-1-propenyl-12-beta-d-glucopyranosyloxy-2,7,8-trihydro-indolo[2,3-a]pyran[3,4-g]quinolizin-4,5(13H)-dione (2), (E)-2-(1-hydroxybut-2-en-2-yl)-11-beta-d-glucopyranosyloxy-6,7-dihydro-indolo[2,3-a]quinolizin-4(12H)-one (3), (E)-1-propenyl-4-hydroxy-2,4a,7,8,13b,14,14a-hepthydro-(4alpha,4abeta,13balpha,14abeta)indolo[2,3-a]pyran[3,4-g]quinolizin-5(13H)-one (4) and 1-(1-hydroxyethyl)-10-hydroxy-7,8-dihydro-indolo[2,3-a]pirydine[3,4-g]quinolizin-5(13H)-one (10-hydroxyangustoline) (5), together with two known compounds, naucleidinal (6) and angustoline (7). All of the structures of the seven compounds above were elucidated by spectroscopic methods including use of 1D- and 2D-NMR spectroscopic analyses. Compounds 2 and 3 are rare examples of monoterpene indole alkaloids with a glucopyranosyloxy group attached to position C-12. In vitro activity screening of the above seven compounds showed weak to moderate inhibitory activity against Plasmodium falciparum.     

Chemosystematic investigations of irregular diterpenes in Anisotome and related New Zealand Apiaceae

A chemosystematic HPLC-UV and HPLC-MS investigation of New Zealand members of the Apiaceae was performed. Diterpenes were identified and quantified in methanolic extracts from subaerial parts of 28 taxa and 54 samples of Aciphylla, Anisotome, Apium, Gingidia, Lignocarpa, Oreomyrrhis, and Scandia. Six diterpenes (1-2, 4-7) and four polyacetylenes (8-11) were identified. The known compounds were the diterpenes anisotomenoic acid 1, anisotomene-1-ol 2, 16-acetoxyanisotomenoic acid 4 and anisotomene-1,12-diol 5; and the polyacetylenes falcarinol 8, falcarindiol 9, (+)-9(Z),17-octadecadiene-12,14-diyne-1,11,16-triol 10, and (+)-9(Z),17-octadecadiene-12,14-diyne-1,11,16-triol 1-acetate 11. New irregular diterpenes 13,14-dihydroanisotom-12E-ene-1,14-diol 6 and 14-methoxy-13,14-dihydroanisotom-12E-ene-1-ol 7 were isolated from A. haastii. Isomers of the new semi-synthetic diterpene 16-hydroxyanisotomenoic acid 3 were detected in extracts of Anisitone flexuosa. Structure elucidation was performed by HR mass spectrometry and 1D and 2D NMR spectroscopy. In crude extracts, compounds were identified by their HPLC retention times and their on-line HPLC-UV and MS spectra. Anisotomene diterpenes occurred in eight out of 16 species of the genus Anisotome, but were not detected in any of the other genera. In contrast, polyacetylenes were present in all the genera investigated.     

Four new immunomodulating steroidal glycosides from the stems of Stephanotis mucronata

Four new C-21 steroidal glycosides, mucronatosides E (1), F (2), G (3), and H (4), were isolated from the stems of Stephanotis mucronata. Two of them had the rare aglycone with a double bond between C-6 and C-7. Their structures were elucidated on the basis of spectroscopic data and chemical evidence. These isolated compounds were assayed for their immunological activities in vitro against concanavalin A (Con A)- and lipopolysaccharide (LPS)-induced proliferation of mice splenocytes. Compounds 2 and 4 showed immunosuppressive activities in a dose-dependent manner, while compounds 1 and 3 possessed immunoenhancing activities.     

E,E-2-Benzylidene-6-(nitrobenzylidene)cyclohexanones: syntheses, cytotoxicity and an examination of some of their electronic, steric, and hydrophobic properties

Three series of structurally isomeric 2-benzylidene-6-(nitrobenzylidene) cyclohexanones 1-3 were prepared and evaluated against human Molt/C8 and CEM T-lymphocytes as well as murine L1210 cells. The IC(50) values of the majority of compounds are less than 10microM and in some assays, the figures for 1d and 1e are submicromolar. Correlations were discerned between cytotoxic potencies and the atomic charges on one of the olefinic carbon atoms, the torsion angles between an aryl ring, and the adjacent unsaturated group as well as logP values. Three representative compounds were examined for their effect on respiration in rat liver mitochondria.     

Novel 1-(azacyclyl)-3-arylsulfonyl-1H-pyrrolo[2,3-b]pyridines as 5-HT6 agonists and antagonists

1-Aminoethyl-3-arylsulfonyl-1H-indoles 1 are 5-HT(6) receptor ligands with modest activity in a 5-HT(6) cyclase assay. Introduction of an additional nitrogen in the indole ring provides 1-aminoethyl-3-arylsulfonyl-1H-pyrrolo[2,3-b]pyridines 2 with both enhanced 5-HT(6) affinity and cyclase activity, many acting as 5-HT(6) agonists. We constrained the basic side chain as part of a ring to make 1-(azacyclyl)-3-arylsulfonyl-1H-pyrrolo[2,3-b]pyridines incorporating a pyrrolidinyl 3 or piperidinyl 4 ring system. Preparation of compounds 3 and 4 required synthesis of the key intermediates, 1-(pyrrolidin-3-yl)-1H-pyrrolo[2,3-b]pyridines 7 and 1-(piperidin-3-yl)-1H-pyrrolo[2,3-b]pyridines 8, respectively. Intermediates 7 were prepared through alkylation of 7-azaindole while the intermediates 8 required an alternate synthesis. The compounds of both series 3 and 4 were shown to have high binding affinities for the 5-HT(6) receptor. The in vitro functional activity at the 5-HT(6) receptor varied depending on various functionalities including the selection of the arylsulfonyl, the substitution on the arylsulfonyl group, the ring size, and the substitution on the basic amine moiety producing either 5-HT(6) receptor agonists or antagonists.     

Antimicrobial constituents of Scrophularia deserti

A study of the chemistry and antibacterial activity of Scrophularia deserti led to the isolation of eight compounds, including the metabolite 3(zeta)-hydroxy-octadeca-4(E),6(Z)-dienoic acid (1). The known compounds ajugoside (2), scropolioside B (3), 6-O-alpha-L-rhamnopyranosylcatalpol (4), buddlejoside A(8) (5), scrospioside A (6), laterioside (7) and 3R-1-octan-3-yl-3-O-beta-D-glucopyranoside (8) were also isolated. Compounds 1-3 exhibited moderate antibacterial activity against strains of multidrug and methicillin-resistant Staphylococcus aureus (MRSA) and a panel of rapidly growing mycobacteria with minimum inhibitory concentration (MIC) values ranging from 32 to 128 microg/ml.     

D-ring-opened phragmalin-type limonoids from Chukrasia tabularis var. velutina

Five new D-ring-opened phragmalin-type limonoids, tabulalins A-E (1-5, resp.), were isolated from the stem bark of Chukrasia tabularis var. velutina. In the structures of these new isolates, the D-ring (C(16)/C(17) δ-lactone ring) of phragmalins was cleaved, and rare C(16)/C(30) δ-lactone ring in 1-3 or C(16)/C(8) γ-lactone ring in 4 and 5 were formed. The structures of these new compounds were elucidated based on extensive 1D- and 2D-spectroscopic analyses (HSQC, HMBC, and ROESY) and HR-ESI-MS. The major compounds, 2, 3, and 5, were evaluated for their inhibitory activities against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in a macrophage (RAW264.7) cell line with IC(50) values of 15.3±0.6, 13.0±0.5, and 17.1±0.7 μM, respectively.     

Efficacy of a DNA vaccine delivered in attenuated Salmonella typhimurium against Eimeria tenella infection in chickens

The efficacy of an oral DNA vaccine carrying the Eimeria tenella 5401 antigen gene delivered by attenuated Salmonella typhimurium was examined in an experimental challenge study. The DNA vaccine preparation was made by transforming the recombinant plasmid pcDNA3-5401 into the attenuated S. typhimurium strain (Dam(-) and PhoP(-)) (designated hereafter as ZJ111/pcDNA3-5401). The chickens were randomly divided into six groups, 50 per group. Group A were given PBS as control. Chickens in group B were fed with 10(8) colony forming units (CFU) of attenuated S. typhimurium carrying pcDNA3. Group C were immunised with 100 microg of the recombinant 5401 protein via intramuscular injection. Groups D to F orally received ZJ111/pcDNA3-5401 at doses of 10(7), 10(8) and 10(9)CFU per chicken, respectively. All immunisations were boosted 2 weeks later. The immunised chickens were challenged with 6x10(4) homologous sporulated oocysts 14 days after the second immunisation. No significant differences in body weight were detected between the groups before immunisation and at week 4 after the booster immunisation. The ZJ111/pcDNA3-5401 was eventually eliminated from the spleen and liver on week 6 post-immunisation. The plasmid pcDNA3-5401 was stably maintained in over 80% of the attenuated S. typhimurium population after 100 generations of growth in antibiotic-free media. Oral immunisation of chickens with ZJ111/pcDNA3-5401 elicited specific humoral responses and stimulated proliferation of peripheral blood lymphocytes. The lymphocyte proliferation response was significantly higher in all vaccinated groups than in the control chickens. Antibody response was significantly lower in group C than in groups immunised with strain ZJ111/pcDNA3-5401. Vaccination with the strain ZJ111/pcDNA3-5401 at 10(8) (group E) and 10(9) (group F) CFU per chicken provided 55.0 and 57.5% protection against E. tenella challenge, respectively. These results have important implications for the development of DNA vaccines against avian coccidiosis by bacteria-vectored oral delivery system.     

Commercially processed dry ginger (Zingiber officinale): composition and effects on LPS-stimulated PGE2 production

Using techniques previously employed to identify ginger constituents in fresh organically grown Hawaiian white and yellow ginger varieties, partially purified fractions derived from the silica gel column chromatography and HPLC of a methylene chloride extract of commercially processed dry ginger, Zingiber officinale Roscoe, Zingiberaceae, which demonstrated remarkable anti-inflammatory activity, were investigated by gas chromatography-mass spectrometry. In all, 115 compounds were identified, 88 with retention times (R(t)) >21 min and 27 with <21 min. Of those 88 compounds, 45 were previously reported by us from fresh ginger, 12 are cited elsewhere in the literature and the rest (31) are new: methyl [8]-paradol, methyl [6]-isogingerol, methyl [4]-shogaol, [6]-isoshogaol, two 6-hydroxy-[n]-shogaols (n=8 and 10), 6-dehydro-[6]-gingerol, three 5-methoxy-[n]-gingerols (n=4, 8 and 10), 3-acetoxy-[4]-gingerdiol, 5-acetoxy-[6]-gingerdiol (stereoisomer), diacetoxy-[8]-gingerdiol, methyl diacetoxy-[8]-gingerdiol, 6-(4'-hydroxy-3'-methoxyphenyl)-2-nonyl-2-hydroxytetrahydropyran, 3-acetoxydihydro-[6]-paradol methyl ether, 1-(4'-hydroxy-3'-methoxyphenyl)-2-nonadecen-1-one and its methyl ether derivative, 1,7-bis-(4'-hydroxy-3'-methoxyphenyl)-5-methoxyheptan-3-one, 1,7-bis-(4'-hydroxy-3'-methoxyphenyl)-3-hydroxy-5-acetoxyheptane, acetoxy-3-dihydrodemethoxy-[6]-shogaol, 5-acetoxy-3-deoxy-[6]-gingerol, 1-hydroxy-[6]-paradol, (2E)-geranial acetals of [4]- and [6]-gingerdiols, (2Z)-neral acetal of [6]-gingerdiol, acetaldehyde acetal of [6]-gingerdiol, 1-(4-hydroxy-3-methoxyphenyl)-2,4-dehydro-6-decanone and the cyclic methyl orthoesters of [6]- and [10]-gingerdiols. Of the 27 R(t)<21 min compounds, we had found 5 from fresh ginger, 20 others were found elsewhere in the literature, and two are new: 5-(4'-hydroxy-3'-methoxyphenyl)-pent-2-en-1-al and 5-(4'-hydroxy-3'-methoxyphenyl)-3-hydroxy-1-pentanal. Most of the short R(t) compounds are probably formed by thermal degradation during GC (which mimics cooking) and/or commercial drying. The concentrations of gingerols, the major constituents of fresh ginger, were reduced slightly in dry ginger, while the concentrations of shogaols, the major gingerol dehydration products, increased.     

Design and synthesis of alkyl 7,7-dihalo-3-methyl-5-(nitrophenyl)-2-azabicyclo[4.1.0]hept-3-ene-4-carboxylates with calcium channel antagonist activity

A group of alkyl 7,7-dihalo-3-methyl-5-(2- or 3-nitrophenyl)-2-azabicyclo[4.1.0]hept-3-ene-4-carboxylates were prepared by reaction of dihalocarbenes (:CX(2), X=Br, Cl) with alkyl 2-methyl-4-(2- or 3-nitrophenyl)-1,4-dihydropyridine-3-carboxylates. In vitro calcium channel antagonist activities were determined using a guinea pig ileum longitudinal smooth muscle assay. The title compounds exhibited weaker CC antagonist activity (10(-5) to 10(-7)M range) than the reference drug nifedipine (1.4 x 10(-8)M). Structure-activity relationships showed that the position (ortho or meta) of the nitro-substituent on the C-5 phenyl ring, the size (van der Waal's radius for Br and Cl are 1.95 and 1.80A, respectively) and/or electronegativity (Cl>Br) of the C-7 geminal halogen atoms do not appear to have a significant effect on CC antagonist activity. In contrast, the effect of the alkyl ester substituent was more pronounced where compounds having a Me or Et alkyl ester group showed superior potency (IC(50) in the 10(-7)M range) relative to the reference drug nifedipine (IC(50)=1.40 x 10(-8)M). Replacement of a 2-methyl-3-methoxycarbonylvinyl moiety present in nifedipine by a bioisosteric geminal-dihalocyclopropyl moiety provided a novel class of calcium channel antagonists that do not exhibit any inotropic effect on guinea pig atria.     

Analogues of 1-(3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo-[5,6]-cyclohepta [1,2-b]pyridin-11-yl)piperidine as inhibitors of farnesyl protein transferase.

The synthesis of several 4-pyridylacetyl N-oxide derivatives of 4-(3-bromo-6,11-dihydro-5H-benzo[5,6]-cyclohepta[1,2-b]-pyridin-11-yl)pi perazine/piperidine 3 is described. This study was aimed at identifying fomesyl protein transferase (FPT) inhibitors in these two series of tricycles containing different phenyl ring substituents. The in vitro activity profile of the initial group of compounds 7a-7g led to the synthesis of the 8-methyl-10-methoxy and 8-methyl-10-bromo analogues 7i, 13i, and 13j. The 11R(-) enantiomers of these compounds were found to exhibit potent in vitro FPT inhibition activity.