Concise asymmetric synthesis of a (1R,2S)-1-amino- 2-vinylcyclopropanecarboxylic acid-derived sulfonamide and ethyl ester.

The development and demonstration of short, robust and chromatography-free sequences for the preparation of a (1R,2S)-1-amino-2-vinylcyclopropane-carboxylic acid-derived sulfonamide and ethyl ester in ≥99% ee are described. Both compounds are common building blocks in multiple preparations of potent HCV NS3 protease inhibitors. The robustness of the asymmetric cyclopropanation of (E)-N-benzylideneglycine ethyl ester under phase transfer catalysis conditions is significantly improved based on a detailed mechanistic investigation that included an analysis of the catalyst decomposition pathway, a postulated model for the stereo-selectivity that was guided by calculations and rigorous quality control of the starting materials and reagents. Wet milling has been demonstrated to dramatically accelerate this phase transfer reaction. A bench stable benzylidene-protected primary 1-amino-2-vinylcyclopropane amide intermediate was isolated and its reliable enantiomeric enrichment was achieved by a controlled crystallization process. A chemical resolution procedure was identified using di-p-toluoyl-(D)-tartaric acid to access (1R,2S)-1-amino-2-vinyl-cyclopropanecarboxylic ester in high ee.

The novel plant-derived agent silvestrol has B-cell selective activity in chronic lymphocytic leukemia and acute lymphoblastic leukemia in vitro and in vivo.

Therapeutic options for advanced B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) are limited. Available treatments can also deplete T lymphocytes, leaving patients at risk of life-threatening infections. In the National Cancer Institute cell line screen, the structurally unique natural product silvestrol produces an unusual pattern of cytotoxicity that suggests activity in leukemia and selectivity for B cells. We investigated silvestrol efficacy using primary human B-leukemia cells, established B-leukemia cell lines, and animal models. In CLL cells, silvestrol LC(50) (concentration lethal to 50%) is 6.9 nM at 72 hours. At this concentration, there is no difference in sensitivity of cells from patients with or without the del(17p13.1) abnormality. In isolated cells and whole blood, silvestrol is more cytotoxic toward B cells than T cells. Silvestrol causes early reduction in Mcl-1 expression due to translational inhibition with subsequent mitochondrial damage, as evidenced by reactive oxygen species generation and membrane depolarization. In vivo, silvestrol causes significant B-cell reduction in Emu-Tcl-1 transgenic mice and significantly extends survival of 697 xenograft severe combined immunodeficient (SCID) mice without discernible toxicity. These data indicate silvestrol has efficacy against B cells in vitro and in vivo and identify translational inhibition as a potential therapeutic target in B-cell leukemias.

Forced Oxidative Degradation Pathways of the Imidazole Moiety of Daclatasvir.

Daclatasvir hydrochloride (DCV) is the active pharmaceutical ingredient of Daklinza, a marketed product for the treatment of hepatitis C viral infection. The intrinsic stability of daclatasvir was evaluated via a forced degradation study. DCV was found to be stable in the solid state. In solution, its carbamate moiety is susceptible to basic hydrolysis, whereas its imidazole is liable to base-mediated autoxidation to form degradants 1 and 3, 7-8, respectively. The imidazole moiety can also be oxidized to form degradants 6-7 in the presence of hydrogen peroxide or azobisisobutyronitrile. The chloro-adduct degradant 9 was also observed in hydrogen peroxide solution. Furthermore, the imidazole moiety is sensitive to photodegradation in solution. Degradants 2-8 were observed in a solution of DCV exposed to high intensity light/UV light; the formation of degradants 2 and 5-8 was postulated through 4 degradation pathways. The degradants 3 and 4 were deemed to be secondary degradants of 7 and 5, respectively.

Silvestrol, a potential anticancer rocaglate derivative from Aglaia foveolata, induces apoptosis in LNCaP cells through the mitochondrial/apoptosome pathway without activation of executioner caspase-3 or -7.

The novel cyclopenta[b]benzofuran, silvestrol, isolated from the fruits and twigs of Aglaia foveolata, has been found to exhibit very potent in vitro cytotoxic activity against several human cancer cell lines. Furthermore, it was active in the in vivo P388 murine leukemia model. In this study, the mechanism of cytotoxicity mediated by silvestrol in the LNCaP (hormone-dependent human prostate cancer) cell line was investigated. Silvestrol induced an apoptotic response, disrupted the mitochondrial trans-membrane potential and caused cytochrome c release into the cytoplasm. Immunoblot analysis indicated that, at the protein level, silvestrol produced an increase of Bcl-xl phosphorylation with a concomitant increase of bak. Furthermore, caspase-2, -9 and -10 appeared to be involved in silvestrol-mediated apoptosis. In contrast, the involvement of caspase-3 and -7 was not detected, either by immunoblot or caspase-3/-7-like activity analysis, indicating that these pathways do not play a crucial role in silvestrol-induced apoptosis. To investigate the relative contribution of the caspases, inhibition of apoptosis with four different cell-permeable inhibitors was studied (Boc-D-Fmk, Z-VDVAD-FMK Z-LEHD-FMK and Z-AEVD-FMK). Only the general caspase inhibitor, Boc-D-Fmk, completely inhibited the formation of apoptotic bodies. In contrast, caspase-2 and caspase-9 selective inhibitors induced about a 40% reduced apoptotic response, whereas the caspase-10 selective inhibitor caused about a 60% reduction in apoptosis compared to silvestrol only treated cells. Taken together, the studies described herein demonstrate the involvement of the apoptosome/mitochondrial pathway and suggest the possibility that silvestrol may also trigger the extrinsic pathway of programmed cell death signaling in tumor cells.

Anti-oxidant constituents of the roots and stolons of licorice (Glycyrrhiza glabra).

As part of a search for new cancer chemopreventive agents, a new chalcone derivative (1), a novel group of neolignan lipid esters (2), and seven known phenolic compounds (formononetin, glabridin, hemileiocarpin, hispaglabridin B, isoliquiritigenin, 4'-O-methylglabridin, and paratocarpin B) (3-9) were isolated from the roots and stolons of licorice (Glycyrrhiza glabra). The structures of compound 1 and the individual components of isolate 2 were elucidated using various spectroscopic and chemical methods. All isolates were tested in an authentic peroxynitrite anti-oxidant assay. Of these compounds, hispaglabridin B (6), isoliquiritigenin (7), and paratocarpin B (9) were found to be the most potent anti-oxidant agents. Furthermore, isoliquiritigenin (7) was demonstrated to prevent the incidence of 1,2-dimethylhydrazine-induced colon and lung tumors in mice when administered at a dose of 300 mg/kg.

Dioxadispiroketal Compounds and a Potential Acyclic Precursor from Amomum aculeatum.

Four new compounds having an unusual 1,7-dioxadispiro[5.1.5.2]-12-ene-11-one tricyclic ring system (1-4), their potential precursor, 5R-hydroxy-1-(4-hydroxyl-phenyl)-eicosan-3-one (5), and two known compounds, aculeatins A (6) and B (7), have been isolated from Amomum aculeatum. All compounds were characterized by spectroscopic methods and the configurations were established by 2D NOE correlations. Compounds 1-4, 6 and 7 showed cytotoxic activity against several human cancer cell lines.

Ixocarpalactone A isolated from the Mexican tomatillo shows potent antiproliferative and apoptotic activity in colon cancer cells.

Physalis philadelphica Lam, commonly known as a tomatillo, is a staple of the Mesoamerican cuisine. In our laboratory, an ethyl acetate-soluble extract and four withanolides [ixocarpalactone A (IxoA), ixocarpalactone B, philadelphicalactone B, and withaphysacarpin] were isolated. Studies conducted on Hepa-1c1c7 hepatoma cells revealed that withanolides were potent inducers of quinone reductase, suggesting possible cancer chemoprotective activity. Here we evaluated the antiproliferative properties of the withanolides in SW480 human colon cancer cells. IxoA, which is present in the edible part of the tomatillo, was selected for further evaluation. SW480 cells treated with IxoA showed cell cycle arrest in the G2/M phase, up-regulation of hyper-phosphorylated retinoblastoma, and down-regulation of E2F-1 and DP-1. On the basis of flow cytometry analysis, ethidium bromide/acridine orange, and 4',6-diamidino-2-phenylindole staining, it was found that IxoA induces apoptosis in SW480 cells. Moreover, increased concentrations of the pro-apoptotic protein, BIM/BOD, were found by western blot analysis and immunocytochemistry. Morphological examination revealed vacuole formation in cells treated with IxoA, and Oil Red O staining showed that the vacuole content was nonlipid. Furthermore, immunocytochemistry demonstrated increased concentrations of mucin 3 in IxoA-treated SW480 cells. These findings suggest that chemicals present in tomatillos (e.g. IxoA) may have cancer chemopreventive properties.

Beccaridiol, an unusual 28-nortriterpenoid from the leaves of Diplectria beccariana.

A C29-triterpene, beccaridiol (1), a dihydrochalcone natural product, 2',4'-dihydroxy-3-(4-methoxyphenyl)-propiophenone (2), as well as three known compounds, 4'-hydroxy-1',2'-dihydro-beta-ionone, 4'-O-methyldavidigenin (3), and ursolic acid, have been isolated from an EtOAc-soluble extract of the leaves of Diplectria beccariana. Beccaridiol (1) was characterized as an ursane-type 28-nortriterpene possessing an unusual aromatic E-ring by spectroscopic data interpretation. The relative configuration of this unusual isolate was established by analyzing the observed NOESY NMR correlations, and the absolute stereochemistry of 1 was then determined based on the circular dichroism (CD) spectrum of its 2,3-di-p-bromobenzoate (1b) derivative. All isolates were evaluated for their potential cancer chemopreventive properties utilizing a cell culture assay to determine quinone reductase induction.

Silvestrol regulates G2/M checkpoint genes independent of p53 activity.

As previously reported, silvestrol, a rocaglate derivative isolated from Aglaia foveolata, has similar potency to paclitaxel and camptothecin against cultured human cancer cells. Furthermore, silvestrol can inhibit cancer cell growth in mice without noticeable toxicity when administered up to 5 mg/kg body weight (the highest dose tested). The purpose of the current study was to evaluate the mechanism of silvestrol's cytotoxicity in human prostate cancer cells (LNCaP). The molecular signature induced in LNCaP cells by silvestrol was evaluated using microarray analysis. The results revealed that 20 apoptosis and cell cycle related genes were significantly altered in LNCaP cells exposed to silvestrol. These included UBL-3, p21 and p300, which were up-regulated, and p53, which was down-regulated. Since p53 expression is governed primarily at the level of translation, p53 was also evaluated by Western blot. Silvestrol caused a dose-dependent decrease in p53 protein within 30 min of exposure with no p53 detectable after 6 h. Down-regulation of p53 by silvestrol was associated with down-regulation of MDM2 and not prevented by lactacystin suggesting that silvestrol-induced degradation of p53 is not mediated by the proteasome. A slight decrease in cyclin B was observed within 6 h of silvestrol exposure and phosphatase Cdc25C protein, which activates Cdc2, was also decreased. These data demonstrate that cytotoxicity induced by silvestrol in LNCaP cells is associated with a block in the cell cycle at the G2/M checkpoint and alterations in the expression of genes regulating apoptosis and cell cycle in a manner independent of p53.

Rocaglaol induces apoptosis and cell cycle arrest in LNCaP cells.

Rocaglaol is a cytotoxic cyclopenta[b]benzofuran isolated from the bark of Aglaia crassinervia. It exhibited in vitro cytotoxic activity against Lu1, LNCaP and MCF-7 cells with ED50 values of 13.8, 23.0 and 9.2 nM, respectively. DAPI staining indicated that LNCaP cells treated with rocaglaol underwent apoptosis. In order to determine whether rocaglaol-induced apoptosis is mediated by the mitochondrial pathway, apoptosis-related mitochondrial-associated proteins were studied. Rocaglaol treatment induced Bax expression through 12 to 72 h of exposure, while Bcl-xl expression was slightly decreased through 48 h, and decreased more significantly by 72 h. Cleaved caspase-9 expression was detected at 72 h, and cleaved caspase-7 was increased through 48 to 72 h. Consequently, the large fragment (89 kDa) of PARP resulting from caspase cleavage was detected at 12, 24 and 48 h, and especially at 72 h. Cleaved PARP expression was also detected at 72 h. Since rocaglaol caused dose-dependent G2/M phase arrest of LNCaP cells as indicated by flow cytometric analysis, the protein levels of cell cycle-related genes were measured. Rocaglaol treatment (230 nM) did not change cyclin B after 24- to 60-h treatment. The expression of cdc2 was not changed and phospho-cdc2 (Tyr 15) increased after 36-, 48- and 60-h treatment. In addition, protein phosphatase Cdc25C, which functions as a mitotic activator by dephosphorylation of Cdc2, decreased in a time-dependent manner after rocaglaol treatment. Taken together, these results suggest that rocaglaol is a potent anticancer drug that induces apoptosis of LNCaP cells through the mitochondrial pathway and its G2/M-phase cell cycle arrest is associated with the down-regulation of Cdc25C and the dephosphorylation of Cdc2.

Antioxidant xanthones from the pericarp of Garcinia mangostana (Mangosteen).

As part of ongoing research on cancer chemopreventive agents from botanical dietary supplements, Garcinia mangostana L. (commonly known as mangosteen) was selected for detailed study. Repeated chromatography of a CH2Cl2-soluble extract of the pericarp led to the isolation of two new highly oxygenated prenylated xanthones, 8-hydroxycudraxanthone G (1) and mangostingone [7-methoxy-2-(3-methyl-2-butenyl)-8-(3-methyl-2-oxo-3-butenyl)-1,3,6-trihydroxyxanthone, 2], together with 12 known xanthones, cudraxanthone G (3), 8-deoxygartanin (4), garcimangosone B (5), garcinone D (6), garcinone E (7), gartanin (8), 1-isomangostin (9), alpha-mangostin (10), gamma-mangostin (11), mangostinone (12), smeathxanthone A (13), and tovophyllin A (14). The structures of compounds 1 and 2 were elucidated by spectroscopic data analysis. Except for compound 2, which was isolated as a minor component, the antioxidant activities of all isolates were determined using authentic and morpholinosydnonimine-derived peroxynitrite methods, and compounds 1, 8, 10, 11, and 13 were the most active. Alpha-mangostin (10) inhibited 7,12-dimethylbenz[alpha]anthracene-induced preneoplastic lesions in a mouse mammary organ culture assay with an IC50 of 1.0 microg/mL (2.44 microM).

Potential cancer chemopreventive agents from Arbutus unedo.

A phytochemical study of the petroleum ether and ethyl acetate extracts of the entire plant of Arbutus unedo led to the isolation of a new sterol, 7beta-hydroxystigmast-4-en-3-one (1), and nine known compounds of the flavan, steroid, and terpenoid types. The structure of 1 was determined by spectroscopic data interpretation in combination with molecular modeling calculations. The absolute stereochemistry of C-7 was assigned as S for compound 1 based on the obtained CD spectral data. Activity in the JB6 cell transformation assay was found for pomolic acid 3-acetate (4). All isolates obtained were evaluated in a cyclooxygenase-2 (COX-2) inhibition assay.

Activity-guided isolation of the chemical constituents of Muntingia calabura using a quinone reductase induction assay.

Activity-guided fractionation of an EtOAc-soluble extract of the leaves of Muntingia calabura collected in Peru, using an in vitro quinone reductase induction assay with cultured Hepa 1c1c7 (mouse hepatoma) cells, resulted in the isolation of a flavanone with an unsubstituted B-ring, (2R,3R)-7-methoxy-3,5,8-trihydroxyflavanone (5), as well as 24 known compounds, which were mainly flavanones and flavones. The structure including absolute stereochemistry of compound 5 was determined by spectroscopic (HRMS, 1D and 2D NMR, and CD spectra) methods. Of the isolates obtained, in addition to 5, (2S)-5-hydroxy-7-methoxyflavanone, 2',4'-dihydroxychalcone, 4,2',4'-trihydroxychalcone, 7-hydroxyisoflavone and 7,3',4'-trimethoxyisoflavone were found to induce quinone reductase activity.

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.

Isolation and absolute stereochemistry of coussaric acid, a new bioactive triterpenoid from the stems of Coussarea brevicaulis.

Coussaric acid (1), a triterpenoid based on an ursane skeleton, and an oleanane-type triterpene acid, 3-epi-spathodic acid (2), as well as four known compounds, barbinervic acid, scutellaric acid, stigmasterol and stigmasterol glucoside, have been isolated from an EtOAc-soluble extract of the stems of Coussarea brevicaulis. The structures of compounds 1 and 2 were elucidated on the basis of spectroscopic investigation, and single-crystal X-ray crystallography was used to confirm the structure of 1. The absolute stereochemistry of 1 was established by chemical transformations and by the Mosher ester procedure. The potential of the isolates and chemical transformation products to induce quinone reductase was evaluated in mouse Hepa lclc7 hepatoma cells.

Compounds obtained from sida acuta with the potential to induce quinone reductase and to inhibit 7,12-dimethylbenz[a]anthracene-induced preneoplastic lesions in a mouse mammary organ culture model.

Activity-guided fractionation of the EtOAc-soluble extract of the whole plants of Sida acuta using a bioassay based on the induction of quinone reductase (QR) in cultured Hepa 1c1c7 mouse hepatoma cells, led to the isolation of ten active compounds of previously known structure, quindolinone (1), cryptolepinone (2), 11-methoxyquindoline (3), N-trans-feruloyltyramine (4), vomifoliol (5), loliolide (6), 4-ketopinoresinol (7), scopoletin (8), evofolin-A (9), and evofolin-B (10), along with five inactive compounds of known structure, ferulic acid, sinapic acid, syringic acid, (+/-)-syringaresinol, and vanillic acid. These isolates were identified by physical and spectral data measurement. A new derivative of quindolinone, 5,10-dimethylquindolin-11-one (1a) was synthesized and characterized spectroscopically. Of the active substances, compounds 1-3 and 1a exhibited the most potent QR activity, with observed CD (concentration required to double induction) values ranging from 0.01 to 0.12 microg/mL. Six compounds were then evaluated in a mouse mammary organ culture assay, with cryptolepinone (2), N-trans-feruloyltyramine (4), and 5,10-dimethylquindolin-11-one (1a) found to exhibit 83.3, 75.0, and 66.7% inhibition of 7,12-dimethylbenz[a]anthracene-induced preneoplastic lesions, respectively, at a dose of 10 microg/mL.

Improving the efficiency of quantitative (1)H NMR: an innovative external standard-internal reference approach.

The classical internal standard quantitative NMR (qNMR) method determines the purity of an analyte by the determination of a solution containing the analyte and a standard. Therefore, the standard must meet the requirements of chemical compatibility and lack of resonance interference with the analyte as well as a known purity. The identification of such a standard can be time consuming and must be repeated for each analyte. In contrast, the external standard qNMR method utilizes a standard with a known purity to calibrate the NMR instrument. The external standard and the analyte are measured separately, thereby eliminating the matter of chemical compatibility and resonance interference between the standard and the analyte. However, the instrumental factors, including the quality of NMR tubes, must be kept the same. Any deviations will compromise the accuracy of the results. An innovative qNMR method reported herein utilizes an internal reference substance along with an external standard to assume the role of the standard used in the traditional internal standard qNMR method. In this new method, the internal reference substance must only be chemically compatible and be free of resonance-interference with the analyte or external standard whereas the external standard must only be of a known purity. The exact purity or concentration of the internal reference substance is not required as long as the same quantity is added to the external standard and the analyte. The new method reduces the burden of searching for an appropriate standard for each analyte significantly. Therefore the efficiency of the qNMR purity assay increases while the precision of the internal standard method is retained.

Potential anticancer activity of naturally occurring and semisynthetic derivatives of aculeatins A and B from Amomum aculeatum.

Activity-guided fractionation of hexanes- and CHCl 3-soluble extracts of Amomum aculeatum leaves, collected in Indonesia, led to the isolation of three new dioxadispiroketal-type ( 3- 5) and two new oxaspiroketal-type ( 6 and 7) derivatives. Nine semisynthetic derivatives ( 1a- 1h and 2a) of the parent compounds, aculeatins A ( 1) and B ( 2), were prepared. All isolates and semisynthetic compounds were tested against a small panel of human cell lines. Of these, aculeatin A ( 1; ED 50 0.2-1.0 microM) was found to be among the most cytotoxic of the compounds tested and was further evaluated in an in vivo hollow fiber assay; it was found to be active against MCF-7 (human breast cancer) cells implanted intraperitoneally at doses of 6.25, 12.5, 25, and 50 mg/kg. However, when 1 was tested using P388 lymphocytic leukemia and human A2780 ovarian carcinoma in vivo models, it was deemed to be inactive at the doses used.

Activity-guided isolation of cytotoxic constituents from the bark of Aglaia crassinervia collected in Indonesia.

Activity-guided fractionation of a CHCl(3)-soluble partition of the MeOH extract of the bark of Aglaia crassinervia collected in Indonesia led to the isolation of three new glabretal-type triterpenoids, aglaiaglabretols A-C (1-3), as well as nine known compounds, 3-epi-cabraleahydroxylactone (4), cabraleahydroxylactone (5), rocaglaol (6), 2beta,3beta-dihydroxy-5alpha-pregn-17(20)-(E)-16-one, scopoletin, and mixtures of cabraleadiol and epicotillol and of beta-sitosterol and stigmasterol. The structures of compounds 1-3 were determined on the basis of spectroscopic and chemical methods. The structure of aglaiaglabretol A (1) was confirmed by single-crystal X-ray analysis, and the absolute stereochemistry of this isolate was established by the Mosher ester method. The cytotoxic activity of all isolates and several chemical transformation products obtained in the present study was evaluated. The known cyclopenta[b]benzofuran, rocaglaol (6), was found to be significantly active and comparable in potency to the positive controls, paclitaxel and camptothecin. Aglaiaglabretol B (2) was further tested in an in vivo hollow fiber model.

Cytotoxic flavaglines and bisamides from Aglaia edulis.

Two new cyclopenta[b]benzofurans, aglaroxin A 1-O-acetate (2) and 3'-methoxyaglaroxin A 1-O-acetate (3), a new benzo[b]oxepine, 19,20-dehydroedulisone A (4), and five new cyclopenta[bc]benzopyrans, edulirin A (5), edulirin A 10-O-acetate (6), 19,20-dehydroedulirin A (7), isoedulirin A (8), and edulirin B (9), were isolated from the bark of Aglaia edulis, along with one known cyclopenta[b]benzofuran, aglaroxin A (1). Additionally, four new amides, aglamides A-D (10-13), as well as three known compounds, aglalactone, scopoletin, and 5-hydroxy-3,6,7,4'-tetramethoxyflavone, were isolated from the leaves and/or twigs of this species. The structures of the new compounds (2-13) were elucidated by interpretation of their spectroscopic data. All isolates obtained in this study were evaluated for cytotoxicity against both several human cancer cell lines (Lu1, LNCaP, and MCF-7) and a nontumorigenic (HUVEC) cell line. Among these isolates, the cyclopenta[b]benzofurans (1-3) exhibited potent in vitro cytotoxic activity (ED50 range 0.001 to 0.8 microg/mL). Aglaroxin A 1-O-acetate (2) was further evaluated in the in vivo P388 lymphocytic leukemia model, by intraperitoneal injection, but found to be inactive in this model.