Discovery and synthesis of sulfur-containing 6-substituted 5,8-dimethoxy-1,4-naphthoquinone oxime derivatives as new and potential anti-MDR cancer agents.

Multi-drug resistance (MDR) to anticancer drugs is the primary impediment to successful treatment of cancer. Hunting for new compounds with potent anti-MDR activity is an effectual approach to conquer cancer drug resistance. In this work, 33 new sulfur-containing 1,4-naphthoquinone oxime derivatives were prepared and investigated for their cytotoxicity against a panel of tumor cell lines and fibroblast normal cell line. Cell-based assay showed that most of target compounds displayed more potent cytotoxic potency than positive controls. Meanwhile, all of compounds were non-toxic to normal cells. More importantly, the cytotoxic activity of these oxime derivatives toward drug-resistant cancer cell lines was found to be much stronger than that toward drug-susceptible cell lines (anti-drug resistance coefficient (ADRC) > 1). Of these, compound 12 m was identified as the most effective molecule with IC50 values in the range of 0.29 ±â€¯0.01 to 1.33 ±â€¯0.05 µM toward MDR sublines. Further mechanism studies demonstrated that 12 m could inhibit colony formation, cause G1 phase arrest and promote cell apoptosis mediated by augmenting Bax/Bcl-2 ratio of Bel7402/5-FU cells. Our findings provide promising start points for development of sulfur-containing 1,4-naphthoquinone oxime derivatives as potential anti-MDR agents.

Advance in Anti-tumor Mechanisms of Shikonin, Alkannin and their Derivatives.

Shikonin, alkannin and their derivatives, the main ingredient of Lithospermum erythrorhizon and Arnebia euchroma (Royle) Johnst native to Inner Mongolian and Northwest of China respectively, hold promising potentials for antitumor effects via multiple-target mechanisms. This review will emphasize the importance of their antitumor activity in apoptosis, necroptosis and immunogenic cell death, and expound the relationship of their antitumor activity and naphthoquinone scaffold that could generate ROS and alkylating agent. Meanwhile, the antitumor mechanisms of naturally-occurring shikonin, alkannin and their derivatives, which were divided into the direct interaction involved in alkylating agent, covalently binding the DNA and protein, as well as the indirect interaction mediated by ROS, nonspecifically influencing the mitochondria or multiple signal pathways, will be systematically summarized and discussed.

Synthesis and biological evaluation of sulfur-containing shikonin oxime derivatives as potential antineoplastic agents.

As a continuation of our research on developing potent and potentially safe antineoplastic agents, a set of forty five sulfur-containing shikonin oxime derivatives were synthesized and evaluated for their in vitro cytotoxic activity against human colon cancer (HCT-15), gastric carcinoma (MGC-803), liver (Bel7402), breast (MCF-7) cancer cells and human skin fibroblast (HSF) cells. All the synthesized compounds exhibited potent cytotoxic activity selectively towards HCT-15 cells and did not display apparent toxicity to the normal HSF cells, some of which were more or comparatively effective to the parent compound against HCT-15, MGC-803 and Bel7402 cells. The most active agent 9m displayed high potency against human cancer cells with IC50 ranging from 0.27 ± 0.02 to 9.23 ± 0.12 µM. The structure-activity relationships (SARs) studies suggested that the nature of substituent group in the side chain is important for antitumor potency in vitro. Additionally, nitric oxide release studies revealed that the amount of nitric oxide generated from these oxime derivatives was relatively low. Furthermore, cellular mechanism investigations indicated that compound 9m could arrest cell cycle at G1 phase and induce a strong apoptotic response in HCT-15 cells. Moreover, western blot studies revealed that compound 9m induced apoptosis through the down-regulation of Bcl-2 and up-regulation of Bax, caspase 3 and 9. For all these reasons, compound 9m hold promising potential as antineoplastic agent.

Shikonin Derivative DMAKO-05 Inhibits Akt Signal Activation and Melanoma Proliferation.

DMAKO-05((S)-1-((5E,8E)-5,8-bis(hydroxyimino)-1,4-dimethoxy-5,8-dihydronaphthalen-2-yl)-4-methylpent-3-enyl 3-methylbutanoate) is a novel oxime derivative of shikonin, the major component extracted from Chinese herb Lithospermun erythrorhizon. Here, we report that DMAKO-05 had an antitumor activity against mouse melanoma cell line B16F0. Our studies indicated that DMAKO-05 not only inhibited B16F0 proliferation and migration but also led to cell cycle arrest at G1 phase and cell apoptosis, in which DMAKO-05 triggered mitochondrial-mediated apoptosis signal including caspase-9/3 and PARP. In response to DMAKO-05 treatment, the Akt-mediated survival signals were remarkably attenuated in B16F0 cells. Collectively, DMAKO-05 has a strong cytotoxicity in B16F0 cells via inhibiting Akt activation, inducing G1 arrest, and promoting B16F0 cell apoptosis. DMAKO-05 might serve as a potential candidate lead compound for melanoma.

Antitumor activity of DMAKO-05, a novel shikonin derivative, and its metabolism in rat liver microsome.

The antitumor activity of shikonin derivatives is largely dependent on the generation of superoxide radicals and the alkylation activity of their naphthoquinone moiety. However, our recent study showed that 1,4-dioxime-5,8-dimethoxynaphthalene (DMAKO-05), a novel shikonin derivative, displayed more potential antitumor activity and less toxicity compared to fluorouracil (5-FU) both in vitro and in vivo, even though the hydroxyl and carbonyl groups of its naphthoquinone structure were shielded. To understand the underlying mechanisms, we investigated the metabolism of DMAKO-05 in rat liver microsomes. The kinetic analysis indicated that DMAKO-05 underwent a biphasic metabolism in rat liver microsomes. The inhibition experiments showed that CYP1A and CYP3A were the major enzymes in the metabolism of DMAKO-05, along with partial contribution from CYP2A. In addition, the structures of eight DMAKO-05 metabolites, which were characterized by accurate mass and MS/MS fragmentograms, implied that DMAKO-05 was mainly metabolized through the oxygenation of its naphthoquinone nucleus and the hydrolysis of its side chain, instead of the oxidation of hydroxyimine to ketone. Therefore, DMAKO-05 should not be considered as a traditional naphthoquinone prodrug.

Design, synthesis, and biological evaluation of shikonin and alkannin derivatives as potential anticancer agents via a prodrug approach.

To minimize the cytotoxicity of shikonin and alkannin that arises through the generation of reactive oxygen species (ROS) and alkylation of the naphthazarin ring, two series of novel core-scaffold-modified shikonin and alkannin derivatives were designed. These derivatives, which differ in their configurational and positional isomerism (R-, S-, and 2- and 6-isomers) were synthesized in high enantiomeric excess (>99 % ee). The selectivity of the dimethylated derivatives was significantly higher than the parent shikonin in vitro, but some side effects were still observed in vivo. Surprisingly, the dimethylated diacetyl derivatives with poor anticancer activity in vitro showed tumor-inhibiting effects similar to paclitaxel without any toxicity in vivo. The anticancer activity of these derivatives is in agreement with their low ROS generation and alkylating capacity, emphasizing their potential as prodrugs. This strategy provides means to address the nonspecific cytotoxicity of naphthazarin analogues toward normal cells.

[Antitumor effect research progress of shikonin and its derivatives].

Shikonin, the main active ingredient of Lithospermum, and its derivatives have been proved to have antitumor effects, and the anti-tumor mechanisms involve multiple targets. Based on recent literatures, this review focuses on the antitumor effects and its mechanisms. More emphases are given on the aspects of induction of apoptosis, induction of necrosis, acting on matrix metalloproteinase, acting on the protein tyrosine kinase and antiangiogenesis. The current status and problems of shikonin derivatives in antitumor effects are simply summarized and lookout for the development of antitumor drugs with shikonin as leading compounds.

Synthesis and antitumour activity of ß-hydroxyisovalerylshikonin analogues.

A series of novel ß-hydroxyisovalerylshikonin analogues bearing oxygen-containing substituents at the side-chain hydroxyl of shikonin were designed and synthesized. The cytotoxicities of these compounds were evaluated in vitro against multi-drug resistant (MDR) cell lines DU-145 and HeLa. Most compounds exhibited significant inhibitory activity on both cell lines. The structure-activity relationship showed the analogues with ether substituents displayed the most potent antitumour activity and selective cytotoxicity towards DU-145. Among the compounds with ether substituents, increasing the steric hindrance in the carbon bearing ß-hydroxyl or replace the ß-hydroxyl with acetoxy or methoxy would lead to the decline of cytotoxicity.

Semi-synthesis and antitumor activity of 6-isomers of 5, 8-O-dimethyl acylshikonin derivatives.

We recently discovered that 5, 8-O-dimethyl acylshikonin derivatives displayed the selectivity towards MCF-7 and no toxicity to normal cells. Herein, a series of the corresponding 6-isomers of 5, 8-O-dimethyl acylshikonin derivatives were synthesized starting from shikonin. In vitro evidence of the cytotoxicities indicated that most of thecompounds were more active than or comparative to shikonin and retained the selectivity against MCF-7, MDA-MB-231 besides no toxicity in the normal cells. Also, in vivo anticancer activity of the positional isomers 5p, 6c further showed that 6-isomers of 5, 8-O-dimethyl acylshikonin derivatives were more active than their corresponding 2-isomers. Thus, we may conclude that the position of the side chain of shikonin attached to 5,8-dimethoxy -1,4-naphthoquinone is associated with the antitumor activity.

Comparative study on enantiomeric excess of main akannin/shikonin derivatives isolated from the roots of three endemic Boraginaceae plants in China.

This work systematically investigated the enantiomeric excess (e.e.) of main components isolated from the roots of three endemic Boraginaceae plants distributed extensively in China, named Arnebia euchroma (Royle) Johnst (A.e.), Lithospermum erythrorhizon Sieb. et Zucc. (L.e.) and Onosma confertum W. W. Smith (O.c.), and the optical purity of their hydrolysis products separately, by means of three different approaches. The influence of HCl on the e.e. values of the major constituents was also studied. Analysis of the absolute configurations and e.e. values of all the derivatives acquired was performed by CD and chiral-HPLC respectively. The results of the main constituents demonstrated that A.e. mainly yields S-form naphthoquinone derivatives, while the R-form is predominant in the derivatives of L.e. and O.c. The optical purity of alkannin and shikonin and their derivatives was not influenced by acid treatment in the course of separation and hydrolysis. Additionally, it was found that 100% e.e. of shikinon could be acquired from a specific shikinon ester derivative, ß,ß-dimethylacrylshikonin occurring in the roots of O.c., as did 100% e.e. of alkannin from ß,ß-dimethylacrylalkannin contained in the roots of A.e.

Semi-synthesis and anti-tumor activity of 5,8-O-dimethyl acylshikonin derivatives.

A set of twenty-two 5,8-O-dimethyl acylshikonin derivatives were designed and synthesized starting from shikonin. The cell-based investigation demonstrated that these dimethylated derivatives were less active than or equally effective to shikonin. However, the selective cytotoxicities toward MCF-7 were found among these derivatives, together with no toxicity in the normal cell. Furthermore, compounds 3f, 3p, 3r were subjected to KM mice suffering from S-180 carcinoma subcutaneously, which possessed more potent than Fluorouracil, a typical anticancer drug used clinically. So we may conclude that the modification to the mother nucleus of shikonin via the methylation is an available approach to acquiring anti-tumor agents with higher selectivity and lower toxicity.

[Progress in the study of drug delivery system based on nanoparticles to overcome multi-drug resistance].

Multi-drug resistance (MDR) of cancer cells is a major cause of failure in chemotherapy. To the majority of anti-cancer drugs, tumor cells are able to generate a multi-drug resistance; but there is no common views on the mechanism of MDR. This review summarizes the use of drug delivery system based on nanoparticles to overcome MDR in recent years. Three kinds including non-modified, ligand-modified and multifunctional drug delivery systems are described. Especially, the mechanism of reversing MDR based on nanoparticles is covered. Through efficiently offsetting and antagonizing the action of pumping drugs out of the tumor cells, drug delivery system based on nanoparticles can increase the concentration of the drug in tumors, while reduce the side effects on normal cells and overcome multi-drug resistance. The use of drug-loaded nanoparticles, which combines nanotechnology with the strategy of active and passive targeting administration, has shown significant prospect improving cancer therapy.

Synthesis and antitumor activity of 6- and 2-(1-substituted-thio-4-methylpent-3-enyl)-5,8-dimethoxynaphthalene-1,4-diones.

In an attempt to develop potent and selective antitumor agents, a series of 6- and 2-(1-substituted-thio-4-methylpent-3-enyl)-5,8-dimethoxynaphthalene-1,4-diones were designed and synthesized. The cytotoxicities of these compounds were evaluated in vitro against BEL-7402, HT-29 and SPC-A1 cell lines. The pharmacological results showed that most of the prepared compounds displayed the excellent selective cytotoxicity toward HT-29 cells. From the structure-activity relationships we may conclude that the introduction of a thioether functional group at the 1'-position in the side chain of shikonin is associated with an increase in cytotoxicity.

Modulation of orphan nuclear receptor Nur77-mediated apoptotic pathway by acetylshikonin and analogues.

Shikonin derivatives, which are the active components of the medicinal plant Lithospermum erythrorhizon, exhibit many biological effects including apoptosis induction through undefined mechanisms. We recently discovered that orphan nuclear receptor Nur77 migrates from the nucleus to the mitochondria, where it binds to Bcl-2 to induce apoptosis. Here, we report that certain shikonin derivatives could modulate the Nur77/Bcl-2 apoptotic pathway by increasing levels of Nur77 protein and promoting its mitochondrial targeting in cancer cells. Structural modification of acetylshikonin resulted in the identification of a derivative 5,8-diacetoxyl-6-(1'-acetoxyl-4'-methyl-3'-pentenyl)-1,4-naphthaquinones (SK07) that exhibited improved efficacy and specificity in activating the pathway. Unlike other Nur77 modulators, shikonins increased the levels of Nur77 protein through their posttranscriptional regulation. The apoptotic effect of SK07 was impaired in Nur77 knockout cells and suppressed by cotreatment with leptomycin B that inhibited Nur77 cytoplasmic localization. Furthermore, SK07 induced apoptosis in cells expressing the COOH-terminal half of Nur77 protein but not its NH(2)-terminal region. Our data also showed that SK07-induced apoptosis was associated with a Bcl-2 conformational change and Bax activation. Together, our results show that certain shikonin derivatives act as modulators of the Nur77-mediated apoptotic pathway and identify a new shikonin-based lead that targets Nur77 for apoptosis induction.

A novel bieremophilanolide from Ligularia lapathifolia.

A new bieremophilanolide was isolated from the roots and rhizomes of Ligularia lapathifolia. Its structure was established as 8,8'-bi-3beta-angeloyloxy-eremophil-7(11)-en-12,8alpha(14beta,6alpha)-diolide (1) by IR, MS, 1D, and 2D NMR experiments.

Sensitized photooxidation of furanoeremophilane with singlet oxygen and their biogenetic relationship.

In an attempt to explore the biogenetic relationship of furanoeremophilane derivatives and eremophilan-8alpha,12-olides, produced in Ligularia and their structure-activity relationship, we studied the photosensitized oxidation of furanoeremophilane-type sesquiterpenes. Under the condition of several solvents solution Irradiation with a 200 W incandescent lamp of furanoeremophilan-14beta,6alpha-olide isolated from Ligularia vellerea, in various solutions with methylene blue, rose bengal, toluidine blue and safranine T gave several products. The products were isolated by chromatographic procedure and their structures were elucidated as eremophilan-14beta,6alpha,8alpha,12-diolide derivatives by NMR, IR and MS methods. A reaction mechanism has been proposed.

[Studies on dissolution rate in vitro of silymarin dropping pill].

OBJECTIVE: To test the dissolution rate of silymarin dropping pill as well as to be compared with other three commercial products of the silymarin. METHOD: By UV spectrophotometry, we studied the dissolution conditions of silymarin dropping pill and compared its dissolution rate with Yiganling tablets (film-coating, sugar-coating) and Legalon capsule which are available in the market. RESULT: The dissolution parameters T50 and Td of silymarin dropping pill, Yiganling tablet (film-coating), Yiganling tablet (sugar-coating) and Legalon capsule are 6.78, 9.85 min, 51.01, 73.78 min, 74.35, 86.97 min and 53.10, 72.65 min. CONCLUSION: The dissolution rate of silymarin dropping pill is superior to that of two kinds of Yiganling tablets and Legalon capsule.

A new silymarin preparation based on solid dispersion technique.

Silymarin is a hepatoprotective agent that is poorly soluble in water. The present study describes a new preparation of solid dispersions in the form of "dripping pills" designed to enhance solubility. Dripping pills of silymarin were prepared at a 1:4 ratio by the traditional fusion method with the use of a mixture of silymarin and polyethylene glycol 6000 (PEG 6000). The prepared dripping pills were spherical and 3 to 4 mm in diameter, with an average weight of 30 mg per pill and with each pill containing 5 mg of silymarin. The dissolution rates of silymarin in dripping pill and of 3 other silymarin preparations, including Yiganling Film-Coating Tablet, Yiganling Sugar-Coating Tablet, and Legalon Capsule, were determined in pH 1.2 medium. The dissolution rate (T50) of the silymarin dripping pill was found to be significantly higher (by a factor of 7.5-11) than those of the other 3 preparations.