Combination of 1H and 13C NMR for quantitative analysis of the orange pigments produced by Monascus kaoliang KB9.

Two orange pigments, rubropunctatin (1) and monascorubrin (2), along with the yellow pigments, monascin (3) and ankaflavin (4), were isolated from M. kaoliang KB9-fermented rice, also known as red yeast rice. The orange pigments exhibit a broad spectrum of biological activities and appeared to be the major components of this fermented rice. In this work, quantitative 1H NMR (qHNMR) and 13C NMR experiments were used to determine the amounts of the two orange pigments in a crude extract in which most of the 1H NMR signals of the two compounds were indistinguishable. The quantitative values obtained by NMR techniques were found to be similar to those obtained by HPLC. Thus, the combined qHNMR with 13C experiment described in this work could be further developed to quantifying Monascus pigments or other invaluable natural products when qHNMR alone is insufficient for quantitative analysis.

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.

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.

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.

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.

Inhibitory effects of 2-substituted-1-naphthol derivatives on cyclooxygenase I and II.

Naphthol derivatives, 2-(3'-hydroxypropyl)-naphthalen-1-ol (2), 2-(3'-hydroxy-2'-methylpropyl)-naphthalen-1-ol (3) and 2-(3'-hydroxy-2',2'-dimethylpropyl)-naphthalen-1-ol (7) were synthesized and already reported by our group. Therefore in this paper we described further synthesis of their ether derivatives, 3-(1-methoxy-naphthalen-2-yl)-propan-1-ol (4), 3-(1-methoxy-naphthalen-2-yl)-2methyl-propan-1-ol (5), 3-(1-methoxy-naphthalen-2-yl)-2,2-dimethyl-propan-1-ol (8), 2-(3-methoxy-propyl)-naphthalen-1-ol (10) and 2-(3-methoxy-2,2-dimethyl-propyl)-naphthalen-1-ol (13). Compounds 4, 5 and 8 were prepared by methylation of compounds 2, 3 and 7, respectively while compounds 10 and 13 were prepared in good yield from naphthols 2 and 7, respectively. When tested for inhibitory activity, five compounds (2, 3, 7, 10 and 13) showed preferential inhibition of COX-2 over COX-1, while compounds 4, 5 and 8 lacked inhibitory effect on either the COX-1 or COX-2 isozyme. The structure-activity relationships of these naphthols analyzed by docking experiments, indicated that the presence of hydroxyl group at C-1 position on the naphthalene nucleus enhanced the anti-inflammatory activity towards COX-2 via hydrogen bonding to the COX-2 Val 523 side chain. When this hydroxyl group was replaced by methoxy group, there was no inhibition. C-2' Dimethyl substituents on the propyl chain also increased the inhibitory activity. All active compounds have the C-1 hydroxyl group aligned so as to form hydrogen bond with Val 523. The results provide a model for the binding of the naphthol derivatives to COX-2 and facilitate the design of more potent or selective analogs prior to synthesis.

Biologically active substances from amphibians: preliminary studies on anurans from twenty-one genera of Thailand.

Amphibian skin has been the source of a wide variety of biologically active substances, but less than one-third of the known genera of amphibians have been probed for such active substances. Skins of 21 genera of anurans from Thailand have now been investigated for noxious secretions, toxic substances, and alkaloids. Four genera of bufonid toads (Bufo, Ansonia, Leptophryne, Pedostipes) were toxic due to the presence of bufadienolides or bufadienolide-like compounds. Two species of ranid frogs (Rana raniceps, Rana signata) were toxic, perhaps due to the presence of toxic peptide(s). Two species of rhacophorid frogs (Polypedates) were slightly noxious/toxic. One species of microhylid frog (Kaloula pulchra) was noxious. Trace amounts of pumiliotoxin alkaloids were detected in a ranid frog (Limnonectes kuhli). A further 18 species did not exhibit noxious or toxic properties to a significant extent.

Synthesis of novel rhinacanthins and related anticancer naphthoquinone esters.

Rhinacanthin-M, -N and -Q, natural products isolated from the medicinal plant Rhinacanthus nasutus, and 39 novel naphthoquinone esters have been synthesized in excellent yield by esterification of naphthoquinone-3-(propan-3'-ols) with benzoic or naphthoic acids. Almost all the naphthoquinone esters that contain a C-3 hydroxy group showed significant cytotoxicities against KB, HeLa, and HepG2 cell lines. In contrast, ester derivatives lacking the C-3 hydroxy group were inactive to the cancer cell lines. Two methyl substituents on the C-2' of propyl chain conferred more potent cytotoxicity than when there is only one or no methyl group. Naphthoate esters exhibited greater cytotoxicity than benzoate esters. Computer modeling has been done to obtain a first look at the mode of action in connection with these observations.

Anti-HIV-1 limonoid: first isolation from Clausena excavata.

A limonoid, clausenolide-1-ethyl ether (1) and two coumarins, dentatin (2) and nor-dentatin (3), were isolated from Clausena excavata. Limonoid 1 was obtained from the crude ethanol extract of the rhizomes and the roots but had not previously been isolated from C. excavata and exhibited HIV-1 inhibitory activity. Coumarins 2 and 3, with their structures related to an anti-HIV-1 substance, (+)-calanolide A (4), were obtained from the crude chloroform extract of the rhizomes. Both induced toxicity to cells used in a syncytium assay for anti-HIV-1 activity. These compounds, 1-3, did not show any cytotoxic effect against KB and BC-1 cell lines (IC(50) value > 20 microgram/mL).