In vitro and in silico studies of naphthoquinones and peptidomimetics toward Plasmodium falciparum plasmepsin V.

Plasmodium proteases play both regulatory and effector roles in essential biological processes in this important pathogen and have long been investigated as drug targets. Plasmepsin V from P. falciparum (PfPMV) is an essential protease that processes proteins for export into the host erythrocyte and is a focus of ongoing drug development efforts. In the present study, recombinant protein production, inhibition assays, binding studies as well as molecular docking and molecular dynamics simulation studies were used to investigate the mode of binding of a PEXEL-based peptidomimetic and naphthoquinone compounds to PfPMV. Consistent with our previous study, refolded PfPMVs were produced with functional characteristics similar to the soluble counterpart. Naphthoquinone compounds inhibited PfPMV activity by 50% at 50 µM but did not affect pepsin activity. The IC50 values of compounds 31 and 37 against PfPMV were 22.25 and 68.94 µM, respectively. Molecular dynamics simulations revealed that PEXEL peptide interacted with PfPMV active site residues via electrostatic interactions while naphthoquinone binding preferred van der Waal interactions. P1'-Ser of the PfEMP2 substrate formed an additional H-bond with Asp365 promoting the catalytic efficiency. Additionally, the effect of metal ions on the secondary structure of PfPMV was examined. Our results confirmed that Hg2+ ions reversibly induced the changes in secondary structure of the protein whereas Fe3+ ions induced irreversibly. No change was observed in the presence of Ca2+ ions. Overall, the results here suggested that naphthoquinone derivatives may represent another source of antimalarial inhibitors targeting aspartic proteases but further chemical modifications are required.

A facile palladium catalysed 3-component cascade route to functionalised isoquinolinones and isoquinolines.

Palladium catalysed three component cascade process, involving coupling of 2-iodobenzoates, -benzaldehydes, or acetophenones with substituted allenes and ammonium tartrate as an ammonium surrogate, provides a novel and facile route to substituted functionalised isoquinolinones and isoquinolines in good yields.

Biosynthetic origins of menisporopsin A.

Menisporopsin A, produced by Menisporopsis theobromae, shows antimalarial, antimycobacterial, and cytotoxic activities. Here, we report the first (13)C incorporations at individual carbons of menisporopsin A using sodium [1-(13)C] and [2-(13)C] acetate. This result indicates that each of the subunits of the pentalactone menisporopsin A is assembled by a polyketide synthase.

Stereoselective Pd(0) catalysed five component cascade synthesis of complex Z,Z-bisallylamines.

Catalytic 5-component cascade chemistry provides an effective stereo- and regioselective route to novel multi-functional Z,Z-bisallylamines. The process, which is capable of considerable further extension, utilises ammonium tartrate as a novel ammonia source which avoids the use of ammonia gas or aqueous ammonia.

Synthesis of novel naphthoquinone aliphatic amides and esters and their anticancer evaluation.

Fourteen new naphthoquinone aliphatic amides and seventeen naphthoquinone aliphatic esters were synthesized in nine to ten steps from 1-hydroxy-2-naphthoic acid with 9-25% overall yield for the amides, and 16-21% overall yield for the esters. The key step of the amide synthesis is a coupling reaction between amine and various aliphatic acids using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as a coupling agent while for the ester synthesis, DCC/DMAP or CDI was used as the coupling reagent between aliphatic acids and naphthoquinone alcohol. Both naphthoquinone amides and esters were evaluated for their anticancer activity against KB cells. It was found that naphthoquinone aliphatic amides showed stronger anticancer activity than those of the esters when the chains are longer than 7-carbon atoms. The optimum chain of amides is expected to be 16-carbon atoms. In addition, naphthoquinone aliphatic esters with α-methyl on the ester moiety possessed much stronger anticancer activity than the straight chains. Decatenation assay revealed that naphthoquinone amide with 16-carbon atoms chain at 15 µM and 20 µM can completely inhibit hTopoIIα activity while at 10 µM the enzyme activity was moderately inhibited. Molecular docking result also showed the same trend as the cytotoxicity and decatenation assay.

Biophysical and molecular docking studies of naphthoquinone derivatives on the ATPase domain of human topoisomerase II.

Numerous naphthoquinone derivatives, such as rhinacanthins function as anticancer drugs, which target hTopoII. The structure of hTopoII contains both an ATPase domain and a DNA binding domain. Several drugs bind to either one or both of these domains, thus modifying the activity of hTopoII. The naphthoquinone esters and amides used in this study showed that their hTopoIIα inhibitory activity was inversely proportional to ATP concentration. In order to better characterize the inhibitory action of these compounds, sufficient quantities of soluble functional hTopoII-ATPase domain were required. Therefore, both the alpha and beta isoforms of the hTopoII-ATPase domain were over-expressed in Escherichia coli. The hTopoIIα-ATPase activity was reduced in the presence of naphthoquinone derivatives. Additionally, a molecular docking study revealed that the selected naphthoquinone ester and amide bind to the ATP-binding domain of hTopoIIα. Collectively, the results here provide for the first time a novel insight into the interaction between naphthoquinone esters and amides, and the ATP-binding domain of hTopoIIα. The further elucidation of the mechanism of action of the naphthoquinone esters and amides inhibitory activity is essential.

First synthesis and anticancer activity of novel naphthoquinone amides.

Sixteen novel naphthoquinone aromatic amides were synthesized by a new route starting from 1-hydroxy-2-naphthoic acid in nine or ten steps with good to excellent yield. Amide formation reaction was carried out by using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as an efficient condensing agent leading to carboxamides in high yield. The key step for converting naphthol to 3-hydroxynaphthoquinone was the Fremy's salt oxidation followed by hydroxylation with tert-butyl hydroperoxide and triton B. Anticancer activity of these new naphthoquinone amides were evaluated and benzamide 22 showed potent inhibition against NCI-H187 cell lines while naphthamides 23 and 43 were the most potent inhibition against KB cells. The decatenation assay revealed that compounds 24 and 43 at 20 µM can inhibit hTopoIIα activity while three other compounds, namely compounds 22, 23, and 45, exhibited hTopoIIα inhibitory activity at final concentration of 50 µM. Docking experiment revealed the same trend as the cytotoxicity and decatenation assay. Therefore, naphthamides 24 and 43 can be promising target molecules for anticancer drug development.

Regioselective ring opening of exo- and endo-3,4-benzylidene acetals of arabinopyranoside derivatives with Lewis acids and reducing agents.

Dioxolane type 3,4-benzylidene acetals of benzyl ß-l-arabinose either as a mixture or pure exo- and endo-isomers cleavaged with BF(3)·OEt(2)/Et(3)SiH in dichloromethane or acetonitrile regioselectively, provided the 4-O-benzyl-3-hydroxy derivative. The reaction with TiCl(4)/Et(3)SiH or Cu(OTf)(2)/Et(3)SiH provided a mixture of 3- and 4-O-benzyl derivatives whereas with Cu(OTf)(2)/BH(3)·THF gave only hydrolyzed product. The regioselectivity of the reaction was proved to be directed by the acetyl substitution at C-2. Benzyl substitution provided a mixture of 3- and 4-O-benzyl derivatives in 1:1 ratio whereas non-substitution yielded the same mixture in 2:1 ratio.

Determination of a pyranocoumarin and three carbazole compounds in Clausena excavata by RP-HPLC.

Clausenidin, O-methylmukonal, 3-formyl-2,7-dimethoxycarbazole, and clauszoline-J, isolated from the rhizomes and roots of Clausena excavata, exhibit anti-HIV-1 activity in a syncytial assay with EC(50) values of 5.3, 12.0, 29.1, and 34.2 microM, respectively. Due to the highly active anti-HIV-1 property, quantitative analysis of four compounds are investigated. The direct analysis of these four compounds in the crude extracts of the combined rhizomes and roots of Clausena excavata from ten various sources in Thailand by high-performance liquid chromatography is accomplished. Chromatographic separation is achieved on a C(18) column, and the mobile phase is a mixture of methanol and distilled water in a mode of isocratic or gradient elution detected at 254 nm at a flow rate of 0.6 mL/min for clausenidin, at 274 nm at a flow rate of 0.6 mL/min for O-methylmukonal, at 298 nm at a flow rate of 0.4 mL/min for 3-formyl-2,7-dimethoxycarbazole, and at 242 nm at a flow rate of 0.4 mL/min for clauszoline-J. This is the first quantitative analysis of these four anti-HIV-1 compounds from the crude extract without prior isolation and purification steps.

Synthesis and anticancer evaluation of naphthoquinone esters with 2'-cyclopentyl and 2'-cyclohexyl substituents.

Twelve novel naphthoquinone esters containing cyclopentyl and cyclohexyl substituents at C-2' of the propyl chain were synthesized by starting from 1-hydroxy-2-naphthoic acid via alkylation with cyclopentyl ester and cyclohexyl ester. They were evaluated for cytotoxicity against three cancer cell lines (human epidermoid carcinoma (KB), human cervical carcinoma (HeLa), and human hepatocellular carcinoma (HepG(2))). In comparison to naphthoquinone esters with the 2',2'-dimethyl group, the naphthoquinones with a 2'-cyclopentyl substituent showed stronger activity than those with a 2'-cyclohexyl substituent, but less than that with the 2',2'-dimethyl group. This work provides new information about the effect of 2'-position substituents on the cytotoxicity of naphthoquinone ester analogues.

Synthesis of cytotoxic novel 9,11-secosterol analogs: Structure/activity studies.

In an effort to determine the pharmaceutical utility and the structural requirements for activity against tumor cell lines, 30 novel 9,11-secosterol analogues with different side chains and degrees of oxidation at C-9 were synthesized starting from hecogenin. Evaluation of the synthesized compounds for cytotoxicity against KB, HeLa and MCF-7 cell lines revealed that some important structural features are required for activity. The presence of a cholesterol-type side chain, which appears to play a major role in determining the biological activity, the existence of a ketone functional at C-9 is also crucial for anticancer activity whereas hydroxyl/ketone function at C-22 on the side chain did not increase cytotoxicity.

Further study on synthesis and evaluation of 3,16,20-polyoxygenated steroids of marine origin and their analogs as potent cytotoxic agents.

A series of new polyoxygenated steroid derivatives with various steroid skeleton moieties were synthesized. Antitumor activity of the compounds against three tumor cell lines (Breast cancer MCF7, lung cancer NCI and oral cancer KB) were evaluated. Compounds with aromatic A ring of this series exhibited the most potent cytotoxicities in all tested cells. The absence of OH at C-16 or lack of cholesterol like side chain at C-20 in the steroid skeleton apparently result in decreased cytotoxicity. The compound became inactive when the side chain contains double bond at C-24-C-25. When hydroxyl group at C-3 was protected no cytotoxicities against MCF7 and NCI and considerable low cytotoxicity against KB cell lines were observed.

Transforming rhinacanthin analogues from potent anticancer agents into potent antimalarial agents.

Twenty-six novel naphthoquinone aliphatic esters were synthesized by esterification of 1,4-naphthoquinone alcohols with various aliphatic acids. The 1,4-naphthoquinone alcohols were prepared from 1-hydroxy-2-naphthoic acid in nine steps with excellent yields. Twenty-four of the novel synthetic naphthoquinone esters showed significant antimalarial activity with IC(50) values in the range of 0.03-16.63 microM. The length of the aliphatic chain and the presence of C-2' substituents on the propyl chain affected the activity. Interestingly, compounds 31 and 37 showed very good antimalarial activity and were not toxic to normal Vero cells, and the PTI values of 31 (>1990.38) and 37 (1825.94) are excellent. Both 31 and 37 showed potent inhibition against P. falciparum 3D7 cyt bc(1) and no inhibition on rat cyt bc(1). They showed IC(50) values in the nanomolar range, providing full inhibition of cyt bc(1) with one molecule inhibitor bound per cyt bc(1) monomer at the Q(o) site.

Induction of apoptosis by rhinacanthone isolated from Rhinacanthus nasutus roots in human cervical carcinoma cells.

Rhinacanthone, a main bioactive naphthoquinone, isolated from roots of Rhinacanthus nasutus KURZ, (family Acanthaceae), a Thai traditional medicine, has been reported to possess anticancer effects, although the anticancer mechanism is still unclear. Therefore, we investigated the effects of rhinacanthone on cell proliferation, cell cycle progression and apoptosis induction in human cervical carcinoma (HeLa) cells. beta-Lapachone, an anticancer drug having a chemical structure related to rhinacanthone, was used as a positive control. The results demonstrated that rhinacanthone inhibited proliferation of HeLa cells in a dose-dependent manner and had greater efficacy than that of beta-lapachone: IC(50) values of the compound ranged from 1.2+/-0.1 to 5.5+/-0.86 muM for 2-24 h time periods. Rhinacanthone-treated HeLa cells displayed several apoptotic features as evidenced by the appearance of chromatin condensation, internucleosomal DNA fragmentation, increase in the proportion of sub G(1) apoptotic cells, and externalization of annexin-V. The apoptotic processes by the treatment with rhinacanthone involved in a marked increase in the level of pro-apoptotic protein Bax and decrease in the levels of anti-apoptotic proteins Bcl-2 and survivin as well as subsequent activation of caspase-9 and caspase-3. Moreover, rhinacanthone increased the expression of apoptosis-inducing factor (AIF) which would translocate from mitochondria to nucleus through cytosol, and induce apoptosis through caspase independent signaling pathway. Taken together, our findings for the first time demonstrate that rhinacanthone-induced apoptosis in HeLa cells is mediated primarily through the mitochondria-dependent signaling pathway, suggesting that it may be a promising agent for the treatment of human cervical cancer.

Pinostrobin from Boesenbergia pandurata is an inhibitor of Ca2+-signal-mediated cell-cycle regulation in the yeast Saccharomyces cerevisiae.

Upon searching plant extracts for inhibitors of the Ca(2+) signaling pathway using the zds1Delta-yeast proliferation based assay, a crude rhizome extract of Boesenbergia pandurata was found to be strongly positive, and from this extract pinostrobin, alpinetin, and pinocembrin chalcone were isolated as active components. Further biochemical experiments confirmed that pinostrobin possesses inhibitory activity on the Ca(2+) signals involved in the control of G2/M phase cell cycle progression in Saccharomyces cerevisiae.

First synthesis of 3,16,20-polyoxygenated cholestanes, new cytotoxic steroids from the gorgonian Leptogorgia sarmentosa.

Using tigogenin as starting material, (20S)-20-hydroxycholestane-3,6-dione (1), (16S, 20S)-16,20-dihydroxycholestan-3-one (2), (20S)-20-hydroxycholest-1-ene-3,16-dione (3) and (20S)-20-hydroxycholest-4-ene-3,16-dione (4), natural polyoxygenated steroids from the gorgonian, Leptogorgia sarmentosa, were synthesized in four steps. Antitumor activity against three tumor cell lines (breast cancer, MCF7, lung cancer NCI and oral cancer KB) was evaluated. Two compounds (3 and 4) showed strong activity against NCI (IC(50) 6.16 and 10.51 microM) and moderate activity against MCF7 and KB, the IC(50) being in the range 30.65-47.22 microM. Compound 2 showed moderate activity against NCI (IC(50) 42.68 microM) but was inactive against MCF7 and KB whereas compound 1 showed no activity against all tested cells.

Identification of Saccharomyces cerevisiae Tub1 alpha-tubulin as a potential target for NKH-7, a cytotoxic 1-naphthol derivative compound.

In a screening for small-molecule compounds that alleviate the deleterious effects of external CaCl(2) on zds1 Delta strain yeast, we found 2-((1-(hydroxymethyl) cyclohexyl) methyl) naphthalen-1-ol (NKH-7) to be an active compound. NKH-7 also inhibited cell growth at higher concentrations. To identify its target in growth inhibition, we isolated NKH-7-resistant mutants and selected those mutants that exhibited dominant or semi-dominant resistance specifically to NKH-7. By gene cloning, a TUB1 mutant gene encoding alpha-tubulin with a Ser248Pro mutation was identified. Deletion of the TUB3 gene, a minor gene encoding alpha-tubulin, led to supersensitivity to NKH-7. Cellular tubulin-containing arrays as visualized by green fluorescent protein (GFP)-labeled alpha-tubulin diminished rapidly on exposure to the inhibitor. The mutation was situated proximal to the alpha-beta interface of alpha-tubulin in microtubule protofilaments, suggesting the possibility that NKH-7 affects the hydrolysis of GTP bound to beta-tubulin. A functional connection perhaps exists between the tubulin inhibition and Ca(2+)-dependent cell-cycle regulation.

Synthesis of novel 2-(2'-cyclopentyl)- and 2-(2'-cyclohexyl) substituted 1-naphthol derivatives with anticyclooxygenase activity.

Eight novel 2-(2'-cyclopentyl)- and 2-(2'-cyclohexyl) substituted 1-naphthol derivatives were synthesized in good yield starting from 1-hydroxy-2-naphthoic acid. Two of them, 2-((1-(hydroxymethyl)cyclopentyl)methyl)naphthalene-1-ol (8) and 2-((1-(hydroxymethyl)cyclohexyl)methyl)-naphthalene-1-ol (9) showed anticyclooxygenase activity on COX-2 with IC(50) values of 19.90 microM and 7.77 microM, respectively and 9 also inhibited COX-1 (5.55 microM), while the other six were inactive on both isozymes. Molecular docking experiments indicated that the orientation of the active naphthols is different from that of the inactive ones. Two evidences playing important roles for the inhibition by the active compounds, are 1) C-1 and C-3' hydroxyl groups formed hydrogen bonds with COX-2/COX-1 Val523/Ile523 and Arg120, respectively, 2) hydrogen at C-5 of the naphthalene nucleus was attracted rather close to the phenolic group of Tyr385 due to van der Waals interaction.

Anti-HIV-1 constituents from Clausena excavata: Part II. Carbazoles and a pyranocoumarin.

Three carbazole derivatives, O-methylmukonal (1), 3-formyl-2,7-dimethoxycarbazole (2) and clauszoline J (3), and a pyranocoumarin, clausenidin (4), were isolated from the rhizomes and roots of Clausena excavata. Compound 1, isolated from this plant for the first time, has not been reported previously as having anti-HIV-1 activity. Compounds 1-4 displayed anti-HIV-1 activity in a syncytial assay with EC(50) values of 12, 29.1, 34.2 and 5.3 microm, respectively, and thus exhibited potential therapeutic index (PTI) values of 56.7, 8.0, 1.6 and 7.0, respectively. All isolated compounds demonstrated a lack of cytotoxicity against the KB and BC-1 cancer cell lines.