Advocacy for the Medicinal Plant Artabotrys hexapetalus (Yingzhao) and Antimalarial Yingzhaosu Endoperoxides.

The medicinal plant Artabotrys hexapetalus (synonyms: A.uncinatus and A. odoratissimus) is known as yingzhao in Chinese. Extracts of the plant have long been used in Asian folk medicine to treat various symptoms and diseases, including fevers, microbial infections, ulcers, hepatic disorders and other health problems. In particular, extracts from the roots and fruits of the plant are used for treating malaria. Numerous bioactive natural products have been isolated from the plant, mainly aporphine (artabonatines, artacinatine) and benzylisoquinoline (hexapetalines) alkaloids, terpenoids (artaboterpenoids), flavonoids (artabotrysides), butanolides (uncinine, artapetalins) and a small series of endoperoxides known as yingzhaosu A-to-D. These natural products confer antioxidant, anti-inflammatory and antiproliferative properties to the plant extracts. The lead compound yingzhaosu A displays marked activities against the malaria parasites Plasmodium falciparum and P. berghei. Total syntheses have been developed to access yingzhaosu compounds and analogues, such as the potent compound C14-epi-yingzhaosu A and simpler molecules with a dioxane unit. The mechanism of action of yingzhaosu A points to an iron(II)-induced degradation leading to the formation of two alkylating species, an unsaturated ketone and a cyclohexyl radical, which can then react with vital parasitic proteins. A bioreductive activation of yingzhaosu A endoperoxide can also occur with the heme iron complex. The mechanism of action of yingzhaosu endoperoxides is discussed, to promote further chemical and pharmacological studies of these neglected, but highly interesting bioactive compounds. Yingzhaosu A/C represent useful templates for designing novel antimalarial drugs.

Eaton's reagent-mediated domino π-cationic arylations of aromatic carboxylic acids to Iasi-red polymethoxylated polycyclic aromatic hydrocarbons: products with unprecedented biological activities as tubulin polymerization inhibitors.

A rapid domino π-cationic arylation of aromatic carboxylic acids, mediated by Eaton's reagent, has been developed for the synthesis of Iasi-red polymethoxylated polycyclic aromatic hydrocarbons (PAHs). This route is currently the easiest method to obtain such popular PAH compounds, which bear in addition numerous methoxy groups. The domino process was generalized, the structure of the obtained red products and the mechanism of their formations were elucidated, and some of their photophysical properties were determined. Newly synthesized polymethoxylated-PAHs were tested for their interaction with tubulin polymerization as well as for their cytotoxicity on a panel of NCI-60 human cancer cell lines. Interestingly, one of these rubicene derivatives exhibited remarkable cytotoxicity in vitro, including inhibition of leukemia, colon, melanoma, CNS, and ovarian cancer cell lines with GI50 values in the low nanomolar range (GI50 < 10 nM).

Antioxidant activity of new benzo[de]quinolines and lactams: 2D-quantitative structure-activity relationships.

In order to predict the antioxidant activity of 7 polycyclic lactams, a two dimensional quantitative-structure activity relationships (2D-QSAR) study based on a 5-descriptor model was performed. The synthetic compounds built from a condensed lactam scaffold were screened for their abilities to inhibit the autoxidation of pyrogallol, a superoxide anion radical-dependent process. The ketone 2 (8,9-dihydro-7H-benzo[de]pyrrolo[1,2-a]quinoline-7,10(7aH)-dione) exhibited the most potent antioxidant activity in vitro. The oxidation mechanism was proved by the isolation and characterization of alcohol 5 formed in the reaction of ketone 2 with dissolved oxygen in methanol.

Synthesis and biological evaluation of phenstatin metabolites.

Previous investigations on the incubation of phenstatin with rat and human microsomal fractions revealed the formation of nine main metabolites. The structures of eight of these metabolites have been now confirmed by synthesis and their biological properties have been reported. Eaton's reagent was utilized as a convenient condensing agent, allowing, among others, a simple multigram scale preparation of phenstatin. Synthesized metabolites and related compounds were evaluated for their antiproliferative activity in the NCI-60 cancer cell line panel, and for their effect on microtubule assembly. Metabolite 23 (2'-methoxyphenstatin) exhibited the most potent in vitro cytotoxic activity: inhibition of the growth of K-562, NCI-H322M, NCI-H522, KM12, M14, MDA-MB-435, NCI/ADR-RES, and HS 578T cell lines with GI(50) values <10nM. It also showed more significant tubulin polymerization inhibitory activity than parent phenstatin (3) (IC(50)=3.2 µM vs 15.0 µM) and induced G2/M arrest in murine leukemia DA1-3b cells. The identification of this active metabolite led to the design and synthesis of analogs with potent in vitro cytotoxicity and inhibition of microtubule assembly.

In vitro metabolism of phenstatin: potential pharmacological consequences.

Phenstatin and its derivatives are potential anticancer drug candidates according to their inhibitory properties on tubulin polymerization, cell growth and antivascular activity. However, at the present time, neither pharmacological nor metabolic studies have been conducted in order to strengthen the relevance of phenstatine as a drug discovery candidate. In the present work, the metabolic fate of phenstatin in rat and human microsomal preparations was studied to investigate the stability of this tubulin polymerization inhibitor and any effects of the metabolites on polymerization and on PC3 cancer cell proliferation. The metabolites were separated by high-performance liquid chromatography and, after their synthesis, characterized by simultaneous LC-DAD-UV and LC-ESI-MS analyses. Thus, eight metabolites were identified. The major biotransformation pathways are carbonyl reduction, O-methylation at C-3', O-methylation after aromatic hydroxylation at the position C-2' on phenyl B ring and O-demethylation on A ring. Four of the identified metabolites were as active or more active, than phenstatin in vitro. Moreover, the better stability of phenstatin versus CA-4 and the lack of quinone formation could justify the design of new analogues which could include various substituents on phenyl rings or linker group in order to modulate the metabolism of phenstatin toward even more active metabolites and so up-regulate the pharmacological activity.

Potent farnesyltransferase inhibitors with 1,4-diazepane scaffolds as novel destabilizing microtubule agents in hormone-resistant prostate cancer.

A new class of potent farnesyltransferase inhibitors based on a 1,4-diazepane scaffold was synthesized with protein farnesyltransferase inhibition potencies in the low nanomolar range. The compounds block the growth on two hormone-resistant tumor prostatic cell lines (DU145 and PC3). The advanced cellular evaluation of the more potent farnesyltransferase inhibitors was explored and revealed a disorganization of tubulin in PC3 cells.

Synthesis and biological activity of N-aroyl-tetrahydro-gamma-carbolines.

Research on dual inhibitors of both 5-LOX and COXs gained interest due to the overexpressions of these enzymes during the malignant state of the evolution of prostate cancer. In order to take part in this research, new N-aroyl-tetrahydro-gamma-carbolines issued from the modification of Indomethacin have been synthesised. As for the NSAIDs, the compounds have been tested for their activity against COX(1), COX(2) plus against 5-LOX and against the proliferation of malignant prostate cancer. Interesting cytotoxic activities and selectivities of some tetrahydro-gamma-carboline derivatives have been obtained.

Cytotoxic effects induced by docetaxel, gefitinib, and cyclopamine on side population and nonside population cell fractions from human invasive prostate cancer cells.

The present study has been undertaken to establish the therapeutic benefit of cotargeting epidermal growth factor receptor (EGFR) and sonic hedgehog pathways by using gefitinib and cyclopamine, respectively, for improving the efficacy of the current chemotherapeutic drug docetaxel to counteract the prostate cancer progression from locally invasive to metastatic and recurrent disease stages. The data from immuofluorescence analyses revealed that EGFR/Tyr(1173)-pEGFR, sonic hedgehog ligand, smoothened coreceptor, and GLI-1 were colocalized with the CD133(+) stem cell-like marker in a small subpopulation of prostate cancer cells. These signaling molecules were also present in the bulk tumor mass of CD133(-) prostate cancer cells with a luminal phenotype detected in patient's adenocarcinoma tissues. Importantly, the results revealed that the CD133(+)/CD44(high)/AR(-/low) side population (SP) cell fraction endowed with a high self-renewal potential isolated from tumorigenic and invasive WPE1-NB26 cells by the Hoechst dye technique was insensitive to the current chemotherapeutic drug, docetaxel. In contrast, the docetaxel treatment induced significant antiproliferative and apoptotic effects on the CD133(-)/CD44(low)/AR(+) non-SP cell fraction isolated from the WPE1-NB26 cell line. Of therapeutic interest, the results have also indicated that combined docetaxel, gefitinib, and cyclopamine induced greater antiproliferative and apoptotic effects on SP and non-SP cell fractions isolated from WPE1-NB26 cells than individual drugs or two-drug combinations. Altogether, these observations suggest that EGFR and sonic hedgehog cascades may represent the potential therapeutic targets of great clinical interest to eradicate the total prostate cancer cell mass and improve the current docetaxel-based therapies against locally advanced and invasive prostate cancers, and thereby prevent metastases and disease relapse.

2,6-Diphenylthiazolo[3,2-b][1,2,4]triazoles as telomeric G-quadruplex stabilizers.

The design and synthesis of 2,6-diphenylthiazolo[3,2-b][1,2,4]triazoles characterized by a large aromatic building block bearing cationic side chains are reported. These molecules are evaluated as telomeric G-quadruplex stabilizers and for their selectivity towards duplex DNA by competition experiments. Two compounds (14a, 19) were found active with high selectivity for telomeric G-quadruplex over duplex DNA.

Synthesis and biological evaluation of a new family of anti-benzylanilinosulfonamides as CA IX inhibitors.

We report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors prepared regio- and stereoselectively by reacting sulfanilamide with ethyl trans-phenylglycidate in the presence of cobalt(II) chloride. Various derivatizations of the ester moiety in the parent compound led to a small library of derivatives (2R,3R and 2S,3S) which displayed interesting inhibitory activities towards the human tumor-associated isoform CA IX. One of the new compounds shows high selectivity in inhibiting hCA IX compared to the two physiologically relevant, cytosolic isozymes hCA I and hCA II. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active sites of hCA IX and hCA II.

New NSAIDs-NO hybrid molecules with antiproliferative properties on human prostatic cancer cell lines.

The design of profen hybrids containing a NO donor moiety connected to an aliphatic spacer led to compounds with a similar cyclooxygenase inhibition compared to their parent profen and with significant antiproliferative activities on PC3 cells. However, inhibition of COX-2 pathway alone did not seem sufficient to inhibit cancer cell proliferation, and NO-release in a time-dependent manner strongly contributes to this activity.

Synthesis, cytotoxicity, DNA interaction, and topoisomerase II inhibition properties of novel indeno[2,1-c]quinolin-7-one and indeno[1,2-c]isoquinolin-5,11-dione derivatives.

Indeno[2,1- c]quinolin-7-ones and 6 H-indeno[1,2- c]isoquinolin-5,11-diones, bearing two cationic aminoalkyl side chains, were synthesized and evaluated for DNA interaction, topoisomerases inhibition, and cytotoxicity against human cancer cell lines. They displayed strong interaction with DNA and one indeno[1,2- c]isoquinolin-5,11-dione bearing side chains at N-6 and C-8 positions ( 6a) was a potent human topoisomerase II inhibitor with high cytotoxicity toward HL60 cells. An increased topoisomerase II inhibition is found with (a) a cationic aminoalkyl side chain at the C-8 rather than at the C-9 position, (b) a dimethylaminoethoxy side chain at the C-8 position introduced on the N-6 monosubstituted derivative, going with suppression of topoisomerase I poisoning, and (c) a dimethylaminoethyl rather than a dimethylaminopropyl side chain at the N-6 position. The cytotoxicity was only partially reduced when using the topoisomerase II-mutated mitoxantrone-resistant HL60/MX2 cell line, suggesting that additional targets are involved in their mechanism of action. These indeno[1,2- c]isoquinolin-5,11-dione derivatives represent new DNA-topoisomerase II interfering anticancer molecules.

Synthesis and biological activities of a series of 4,5-diaryl-3-hydroxy-2(5H)-furanones.

A series of thirteen 4,5-diaryl-3-hydroxy-2(5H)-furanones were synthesized. They were evaluated for their antioxidant potencies and inhibitory properties of 5-lipoxygenase, cyclooxygenases, HIV-1 integrase and PC3 cell proliferation. New hits were discovered either in the anti-proliferation test or in the HIV anti-integrase test.

Pharmacomodulations around the 4-oxo-1,4-dihydroquinoline-3-carboxamides, a class of potent CB2-selective cannabinoid receptor ligands: consequences in receptor affinity and functionality.

CB2 receptor selective ligands are becoming increasingly attractive drugs due to the potential role of this receptor in several physiopathological processes. Thus, the development of our previously described series of 4-oxo-1,4-dihydroquinoline-3-carboxamides was pursued with the aim to further characterize the structure-affinity and structure-functionality relationships of these derivatives. The influence of the side chain was investigated by synthesizing compounds bearing various carboxamido and keto substituents. On the other hand, the role of the quinoline central scaffold was studied by synthesizing several 6-, 7-, or 8-chloro-4-oxo-1,4-dihydroquinolines, as well as 4-oxo-1,4-dihydronaphthyridine and 4-oxo-1,4-dihydrocinnoline derivatives. The effect of these modifications on the affinity and functionality at the CB2 receptor was studied and allowed for the characterization of new selective CB2 receptor ligands.

Design, synthesis and biological evaluation of substituted dioxodibenzothiazepines and dibenzocycloheptanes as farnesyltransferase inhibitors.

A new series of FTase inhibitors containing a tricyclic moiety--dioxodibenzothiazepine or dibenzocycloheptane--has been designed and synthesized. Among them, dioxodibenzothiazepine 18d displayed significant inhibitory FTase activity (IC(50)=17.3 nM) and antiproliferative properties.

Combined targeting of epidermal growth factor receptor and hedgehog signaling by gefitinib and cyclopamine cooperatively improves the cytotoxic effects of docetaxel on metastatic prostate cancer cells.

The epidermal growth factor receptor (EGFR) and hedgehog cascades provide a critical role in prostate cancer progression and contribute to the resistance to clinical therapies and disease relapse. Therefore, we evaluated, for the first time, the antiproliferative and cytotoxic effects induced by a combination of selective inhibitors of EGFR tyrosine kinase and smoothened hedgehog signaling element, gefitinib and cyclopamine, with a current chemotherapeutic drug used in the clinics, docetaxel, on some metastatic prostate cancer cell lines. Immunohistochemical analyses revealed that sonic hedgehog (SHH) expression was enhanced in 39% of primary prostatic adenocarcinomas (Gleason scores 4-10) compared with the corresponding normal tissues of the same prostate gland from 32 prostate cancer patients. The confocal microscopy and Western blot analyses have also indicated the high expression levels of SHH and EGFR in metastatic LNCaP, DU145, and PC3 cells. Moreover, the results revealed that the drugs, alone or in combination, at lower concentrations inhibited the growth of EGF plus SHH-stimulated and serum-stimulated androgen-responsive LNCaP-C33 and androgen-independent LNCaP-C81, DU145, and PC3 cells. Importantly, the combined docetaxel, gefitinib, and cyclopamine also caused a higher rate of apoptotic death of prostate cancer cells compared with individual agents. The cytotoxic effects induced by these drugs in PC3 cells seem to be mediated in part through the cellular ceramide production and activation of caspase cascades via a mitochondrial pathway and the release of cytochrome c into the cytosol. Additionally, the combined agents were more effective at suppressing the invasiveness of PC3 cells through Matrigel in vitro than the single drugs. These findings indicate that the combined use of inhibitors of EGF-EGFR and hedgehog signaling with docetaxel could represent a more promising strategy for treatment in patients with metastatic and androgen-independent prostate cancer.

COX-2/5-LOX dual acting anti-inflammatory drugs in cancer chemotherapy.

Emerging reports now indicate alterations of arachidonic acid metabolism with carcinogenesis and many COX and LOX inhibitors (used for the treatment of inflammatory diseases) are being investigated as potential anticancer drugs. Results from clinical trials seem to be encouraging but a better knowledge of the dynamic balance that shifts toward lipoxygenases (and different isoforms of LOXs) and cyclooxygenase-2 are essential to progress in the design of new drugs more specially directed on chemoprevention or chemotherapy of human cancers. So, on the basis of these results, it seemed useful to study the advantages of combination of COX inhibitor with LOX inhibitor and a next step will be the conception of dual inhibitors able to induce the anticarcinogenic and/or to inhibit the procarcinogenic enzymes responsible for polyunsaturated fatty acid metabolism. After a rapid summary of some recent reviews published on the involvement of different COX and LOX isoforms present in human cells, we will discuss on cross-talk reported between the downstream pathways which contribute to the development and progression of human cancers. This will lead us to evoke and to justify alternative strategies to develop agents that modulate multiple targets simultaneously with the aim of enhancing efficacy or improving safety relative to drugs that address only a single enzyme.

Derivatives of Iressa, a specific epidermal growth factor receptor inhibitor, are powerful apoptosis inducers in PC3 prostatic cancer cells.

The tyrosine kinase activity of the epidermal growth factor receptor (EGFR) is widely involved in signaling pathways and often deregulated in cancer. Its role in the development of prostate cancer is well established, and therapeutic strategies such as blockade of the intracellular tyrosine kinase domain with small-molecule tyrosine kinase inhibitors have been proposed. Herein we describe the synthesis and in vitro pharmacological properties of C6- and C7-substituted 4-anilinoquinazolines, analogues of Iressa and powerful proapoptotic inducers in hormone-independent prostate cancer PC3 cell lines.

Novel combination therapy against metastatic and androgen-independent prostate cancer by using gefitinib, tamoxifen and etoposide.

In this study, we evaluated, for the first time, the antiproliferative and cytotoxic effects induced by a combination of a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, gefitinib, with other chemotherapeutic drugs including estrogen receptor-beta (ER-beta) antagonist (tamoxifen) and topoisomerase II inhibitor (etoposide) on some metastatic prostate cancer (PC) cell lines. Immunohistochemial analyses revealed that EGFR expression was enhanced in 38% of primary prostatic adenocarcinomas (Gleason scores 4-10) as compared to the corresponding normal tissues of the same prostate gland from 32 PC patients. The RT-PCR and Western blot data have also indicated the higher expression levels of EGFR and ER-beta transcripts and proteins in metastatic LNCaP, DU145 and PC3 cells relative to nonmalignant normal prostate cells. Moreover, the results from MTT and FACS analyses revealed that the drugs, alone or in combination at lower concentrations, inhibited the growth of 17beta-estradiol (E2) plus EGF and serum-stimulated androgen-responsive LNCaP-C33 and androgen-independent LNCaP-C81, DU145 and PC3 cells. Importantly, the combined gefitinib, tamoxifen and etoposide also caused a higher rate of apoptotic death of PC cells as compared to single agents. The cytotoxic effects induced by these drugs in PC3 cells appear to be mediated through the accumulation of cellular ceramide and activation of caspase cascades via a mitochondrial pathway. These findings indicate that the combined use of inhibitors of EGF-EGFR and E2-ER-beta signaling with etoposide, which act by increasing the cellular ceramide levels and caspase activity, represents a promising strategy for a more effective treatment of metastatic PC forms.

Improvement of cytotoxic effects induced by mitoxantrone on hormone-refractory metastatic prostate cancer cells by co-targeting epidermal growth factor receptor and hedgehog signaling cascades.

The results of the present study revealed for the first time the possibility to use a combination of mitoxantrone with gefitinib and cyclopamine for inhibiting the growth of epidermal growth factor (EGF), sonic hedgehog- (SHHNp), and serum-stimulated androgen-sensitive LNCaP-C33 and androgen-independent (AI) LNCaP-C81, DU145 and PC3 prostate cancer (PC) cells. The supra-additive anti-proliferative effects of drugs were mediated via a blockade of the PC3 cells in the G(1) and G(2)M phases of the cell cycle. Importantly, the combination of mitoxantrone plus gefitinib and/or cyclopamine also caused a higher rate of apoptotic death of PC cells including enriched fraction of CD44(high) PC3 cell subpopulation as compared to the individual agents or bi-combination of drugs. The cytotoxic effects induced by mitoxantrone, gefitinib and cyclopamine on PC3 cells appear to be at least partly mediated through the depolarization of the mitochondrial membrane, release of cytochrome c into the cytosol, hydrogen peroxide production and activation of caspase cascades. These findings indicate that the simultaneous blockade of EGF-EGFR and sonic hedgehog tumorigenic signaling cascades may represent a promising strategy for improving the efficacy of current mitoxantrone-based therapies against incurable AI and metastatic PCs in the clinics.