Leonardus B S Kardono

Natural Product Libraries to Accelerate the High Throughput Discovery of Therapeutic Leads

A high-throughput (HT) paradigm generating LC-MS-UV-ELSD-based natural product libraries to discover compounds with new bioactivities and or molecular structures is presented. To validate this methodology, an extract of the Indo-Pacific marine sponge Cacospongia mycofijiensis was evaluated using assays involving cytoskeletal profiling, tumor cell lines, and parasites. Twelve known compounds were identified including latrunculins (1-4, 10), fijianolides (5, 8, 9), mycothiazole (11), aignopsanes (6, 7), and sacrotride A (13). Compounds 1-5 and 8-11 exhibited bioactivity not previously reported against the parasite T. brucei, while 11 showed selectivity for lymphoma (U937) tumor cell lines. Four new compounds were also discovered including aignopsanoic acid B (13), apo-latrunculin T (14), 20-methoxy-fijianolide A (15), and aignopsane ketal (16). Compounds 13 and 16 represent important derivatives of the aignopsane class, 14 exhibited inhibition of T. brucei without disrupting microfilament assembly, and 15 demonstrated modest microtubule-stabilizing effects. The use of removable well plate libraries to avoid false positives from extracts enriched with only one or two major metabolites is also discussed. Overall, these results highlight the advantages of applying modern methods in natural products-based research to accelerate the HT discovery of therapeutic leads and/or new molecular structures using LC-MS-UV-ELSD-based libraries.

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.

Phenylacetic acid derivatives and a thioamide glycoside fromEntada phaseoloides

Abstract Three new compounds, 2-hydroxy-5-butoxyphenylacetic acid, 2-β- d -glucopyranosyloxy-5-butoxyphenylacetic acid, and entadamide A-β- d -glucopyranoside, in addition to the new natural product 2,5-dihydroxyphenylacetic acid methyl ester, have been isolated and characterized from seeds ofEntada phaseoloides collected in Indonesia. None of these compounds was found to demonstrate significant cytotoxicity for cultured human cancer cells, but 2-hydroxy-5-butoxyphenylacetic acid and 2,5-dihydroxyphenylacetic acid methyl ester gave ED50 values of 1.0 and 1.7 μg ml−1, respectively, with cultured P-388 cells.

Cytotoxic triterpenes from the twigs of Celtis philippinensis.

Two triterpene esters, 3beta-trans-sinapoyloxylup-20(29)-en-28-ol (1) and 3beta-trans-feruloyloxy-16 beta-hydroxylup-20(29)-ene (2), were isolated as cytotoxic constituents from the chloroform-soluble extract of the twigs of Celtis philippinensis, along with five known triterpenes, 3beta-O-(E)-feruloylbetulin (3), 3beta-O-(E)-coumaroylbetulin (4), betulin (5), 20-epibryonolic acid (6), and ursolic acid (7). The structures of 1 and 2 were assigned from their 1D and 2D NMR spectroscopic data. All isolates were evaluated for cytotoxicity against several human cancer cell lines.

Flavonoids and a proanthocyanidin from rhizomes of Selliguea feei

Abstract Three novel compounds, as well as a bitter-tasting flavonoid glycoside of known structure, kaempferol-3-O-β- d -glucopyranoside-7-O-α- l -rhamnopyranoside, were isolated from the rhizomes of Selliguea feei. The structures of the new compounds were determined as (−)-4β-carboxymethyl epiafzelechin (3′-deoxydryopteric acid), epiafzelechin-(4β→8, 2β→O→7)-epiafzelechin-(4β→8)-3′-deoxydryopteric acid methyl ester (selligueain B), and (+)-afzelechin-O-β- 4′- d -glucopyranoside.

Dereplication of Saccharide and Polyol Constituents of Candidate Sweet‐tasting Plants: Isolation of the Sesquiterpene Glycoside Mukurozioside IIb as a Sweet Principle of Sapindus rarak

In the search for new potently sweet compounds from plants, the rapid identification and quantification of free sugars and polyols in a crude plant extract is important for dereplication purposes, wherein compounds of known structure or biological activity are removed from further consideration. Accordingly, plants found to have high levels of free sugars and polyols are regarded as lower priority leads when screening for novel natural sweeteners. In the present study, gas chromatography–mass spectrometry was used to examine the sugar/polyol content of six sweet-tasting species, comprised of the pericarp of Dialium indum L. (Leguminosae), the stem of Drypetes floribunda Hutchinson (Euphorbiaceae), the fruit of Hymenaea oblongifolia Huber var. palustris (Ducke) Lee and Langenheim (Leguminosae), the rhizomes of Imperata cylindrica (L.) Beauvois (Gramineae), the fruit of Manilkara zapota (L.) van Royen (Sapotaceae), and the pericarp of Sapindus rarak DC. (Sapindaceae). The total yields of sugars/polyols in these plant parts were 1.9, 6.1, 7.8, 4.5, 10.8 and 2.9% w/w, respectively. Several uncommon polyols were identified, including bornesitol in D. floribunda and quebrachitol in S. rarak. It is likely that the sweet taste of the plants containing more than 5% of sugars/polyols is imparted as a result of the high free sugar and/or polyol content. Owing to its low level of free sugars, S. rarak pericarp was chosen for further study, and the known sesquiterpene glycoside mukurozioside IIb (1) was isolated in high yield (6.3% w/w) as a sweet-tasting constituent. Preliminary evaluations, comprised of mouse acute toxicity and bacterial mutagenesis determinations, indicated the safety of 1. The compound was subsequently rated by a human taste panel as having about the same sweetness potency as sucrose.

Bioactivity-guided isolation of cytotoxic sesquiterpenes of Rolandra fruticosa

Cytotoxicity-guided fractionation of a methanol extract of the leaves and twigs of Rolandra fruticosa using the HT-29 human colon cancer cell line led to the isolation of seven sesquiterpene lactones, including the hitherto unknown isorolandrolide, 13-methoxyisorolandrolide (1), and bourbonenolide, 2alpha,13-diacetoxy-4alpha-hydroxy-8alpha-isobutyroyloxybourbonen-12,6alpha-olide (2), as well as five known compounds, 13-acetoxyrolandrolide (3), 8-desacyl-13-acetoxyrolandrolide-8-O-tiglate (4), 2-epi-glaucolide E (5), 2alpha,13-diacetoxy-4alpha-hydroxy-8alpha-methacryloyloxybourbonen-12,6alpha-olide (6), and 2alpha,13-diacetoxy-4alpha-hydroxy-8alpha-tigloyloxybourbonen-12,6alpha-olide (7). The structures of the two sesquiterpenes were elucidated on the basis of spectroscopic methods. All isolates were evaluated for their cytotoxicity using the HT-29 cell line, and only 13-acetoxyrolandrolide (3) was found to possess a potent inhibitory effect against this cell line. Compounds 3, 5 and 6 were also tested in a NF-kappaB (p65) inhibition assay, and 3 was assessed in an in vivo hollow fiber assay.

Bioactive prenylated flavonoids from the stem bark of Artocarpus kemando.

Four known prenylated flavonoids, artonins E (1) and O (2), artobiloxanthone (3), and cycloartobiloxanthone (4), were isolated from the stem bark ofArtocarpus kemando by bioassayguided fractionation using the DNA strand-scission and the KB cytotoxicity assays as monitors. Compounds1 and3 exhibited strong DNA strand-scission activity, and all four compounds were found to be cytotoxic.

(+)-Altholactone exhibits broad spectrum immune modulating activity by inhibiting the activation of pro-inflammatory cytokines in RAW 264.7 cell lines.

An evaluation of Indonesian plants to identify compounds with immune modulating activity revealed that the methanolic extract of an Alphonsea javanica Scheff specimen possessed selective anti-inflammatory activity in a nuclear factor-kappa B (NF-κB) luciferase and MTT assay using transfected macrophage immune (Raw264.7) cells. A high-throughput LC/MS-ELSD based library approach of the extract in combination with the NF-κB and MTT assays revealed the styryl lactone (+)-altholactone (2) was responsible for the activity. Compound 2, its acetylated derivate (+)-3-O-acetylaltholactone (3), and the major compound of this class, (+)-goniothalmin (1), were further evaluated to determine their anti-inflammatory potential in the NF-κB assay. Concentration-response studies of 1-3 indicated that only 2 possessed NF-κB based anti-inflammatory activity. Compound 2 reduced the LPS-induced NO production, phosphorylation of IκBα, and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) using Western blot analysis. Further studies using qPCR indicated 2 reduced the expression of eight pro-inflammatory cytokines/enzymes (0.8-5.0μM) which included: COX-2, iNOS, IP-10, IL-1β, MCP-1, GCS-F, IL-6 and IFN-β. These results indicated that 2 displays broad spectrum immune modulating activity by functioning as an anti-inflammatory agent against LPS-induced NF-κB signaling. Conversely the selective cytotoxicity and in vivo anti-tumor and anti-inflammatory activity previously reported for 1 do not appear to arise from a mechanism that is linked to the NF-κB immune mediated pathway.

A flavan-3-ol glycoside from bark of Plumeria rubra

Abstract A novel flavan-3-ol glycoside, plumerubroside, was isolated from a water-soluble extract of the stem bark of Plumeria rubra and its structure and conformation elucidated by spectroscopic means as (2R,3S)-3,4′-dihydroxy-7,3′,5′-trimethoxyflavan-5-O-β- d -glucopyranoside.