DNA Methylation Targeting: The DNMT/HMT Crosstalk Challenge.

Chromatin can adopt a decondensed state linked to gene transcription (euchromatin) and a condensed state linked to transcriptional repression (heterochromatin). These states are controlled by epigenetic modulators that are active on either the DNA or the histones and are tightly associated to each other. Methylation of both DNA and histones is involved in either the activation or silencing of genes and their crosstalk. Since DNA/histone methylation patterns are altered in cancers, molecules that target these modifications are interesting therapeutic tools. We present herein a vast panel of DNA methyltransferase inhibitors classified according to their mechanism, as well as selected histone methyltransferase inhibitors sharing a common mode of action.

Amine coupling versus biotin capture for the assessment of sulfonamide as ligands of hCA isoforms.

This work was dedicated to the development of a reliable SPR method allowing the simultaneous and quick determination of the affinity and selectivity of designed sulfonamide derivatives for hCAIX and hCAXII versus hCAII, in order to provide an efficient tool to discover drugs for anticancer therapy of solid tumors. We performed for the first time a comparison of two immobilization approaches of hCA isoforms. First one relies on the use of an amine coupling strategy, using a CM7 chip to obtain higher immobilization levels than with a CM5 chip and consequently the affinity with an higher precision (CV% < 10%). The second corresponds to a capture of proteins on a streptavidin chip, named CAP chip, after optimization of biotinylation conditions (amine versus carboxyl coupling, biotin to protein ratio). Thanks to the amine coupling approach, only hCAII and hCAXII isoforms were efficiently biotinylated to reach relevant immobilization (3000 RU and 2700 RU, respectively) to perform affinity studies. For hCAIX, despite a successful biotinylation, capture on the CAP chip was a failure. Finally, concordance between affinities obtained for the three derivatives to CAs isozymes on both chips has allowed to valid the approaches for a further screening of new derivatives.

Synthesis and biological evaluation of di-aryl urea derivatives as c-Kit inhibitors.

Inhibition of receptor tyrosine kinases (RTKs) continued to be a successful approach for the treatment of many types of human cancers and many potent small molecules kinase inhibitors have been discovered the last decade. In the present study, we describe the synthesis of thienopyrimidine derivatives and their pharmacological evaluation against nine kinases (EGFR, PDGFR-ß, c-Kit, c-Met, Src, Raf, VEGFR-1, -2 and -3). Most of the synthesized compounds showed from moderate to potent activities against c-Kit with IC50 values in the nanomolar range. Among them, 4-anilino(urea)thienopyrimidine analogs showed selectivity and potent c-Kit inhibition with IC50 values less than 6 nM. Docking simulation was performed for the most promising compound 9 into the c-Kit active site to determine the potential binding mode. This study reveal that the 4-anilino(urea)thienopyrimidine is an interesting scaffold to design novel potent and selective c-Kit inhibitors which may make promising candidates for cancers where c-Kit receptors are overexpressed.

Quinazoline derivatives as anticancer drugs: a patent review (2011 - present).

INTRODUCTION: Quinazoline is one of the most studied moieties in medicinal chemistry due to the wide range of biological properties such as the anticancer, antibacterial, anti-inflammatory, antimalarial and antihypertensive activities. During the past decades, several patents and articles have been published in international peer-reviewed literature regarding the discovery and development of original and promising quinazoline derivatives for cancer treatment. Although quinazolines are well known to inhibit EGFR, there is also a large panel of other therapeutic protein targets. AREAS COVERED: This review summarized the new patents and articles published about quinazoline derivatives as anticancer drugs since 2011. EXPERT OPINION: Since 2011, a lot of quinazoline compounds have shown EGFR inhibition. Unlike the first-generation EGFR inhibitors, they inhibit both wild-type and mutated EGFR. In recent years, a number of studies on quinazoline synthesis have been reported and used by several medicinal chemistry groups for better and easier development of new derivatives. Therefore, several patents have been approved for the use of quinazoline compounds as inhibitors of other kinases, histone deacetylase, Nox and some metabolic pathways. Because of the large number of proteins targeted, some high structural diversity is observed in patented quinazoline compounds. Due to the vast applications of quinazoline derivatives, development of novel quinazoline compounds as anticancer drugs remains a promising field.

Inhibition of tumor cell growth and angiogenesis by 7-aminoalkoxy-4-aryloxy-quinazoline ureas, a novel series of multi-tyrosine kinase inhibitors.

Several regulatory and signaling molecules governing angiogenesis are targets of interest for the development of drugs in the cancer, including growth factors such as Vascular Endothelial Growth Factor (VEGF) and Platelet-Derived Growth Factor (PDGF). A series of 4-aryloxy-6,7-dimethoxyquinazolines, previously synthesized in our laboratory, has shown a nanomolar inhibition of kinase enzymatic activity of VEGFR, PDGFR and c-Kit. We have therefore studied the impact of the variation in the 7-position substitution of the quinazoline core. Substitution by aminoalkoxy chains led to new highly potent ATP-competitive inhibitors of VEGFR, PDGFR and c-Kit enzyme with IC50 values in the nanomolar range and this substitution has increased greatly antiproliferative activity on cancer cell lines (PC3, MCF7, HT29) and HUVEC (human umbilical vein endothelial cells). One of the most promising compounds (36) was assessed for its ability to limit the induction of web like network of capillary tubes by the human umbilical vascular endothelial cells (HUVEC) and for its ability to inhibit invasion.

New selective carbonic anhydrase IX inhibitors: synthesis and pharmacological evaluation of diarylpyrazole-benzenesulfonamides.

Carbonic anhydrase (CA) IX expression is increased upon hypoxia and has been proposed as a therapeutic target since it has been associated with poor prognosis, tumor progression and pH regulation. We report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors, 4-(5-aryl-2-hydroxymethyl-pyrazol-1-yl)-benzenesulfonamides. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active site of hCA IX. Pharmacological studies revealed high hCA IX inhibitory potency in the parameters nanomolar range. This study showed that the position of sulfonamide group in meta of the 1-phenylpyrazole increase a selectivity hCA IX versus hCA II of our compounds. An in vitro antiproliferative screening has been performed on the breast cancer MDA-MB-231 cell using doxorubicin as cytotoxic agent and in presence of selected CA IX inhibitor. The results shown that the cytotoxic efficiency of doxorubicin in an hypoxic environment, expressed in IC50 value, is restored at 20% level with 1µM CA IX inhibitor.

Synthesis and structure-activity relationships of (aryloxy)quinazoline ureas as novel, potent, and selective vascular endothelial growth factor receptor-2 inhibitors.

In our continuing search for medicinal agents to treat proliferative diseases, quinazoline derivatives were synthesized and evaluated pharmacologically as epithelial growth factor receptor and vascular endothelial growth factor receptor 2 (VEGFR-2) tyrosine kinase inhibitors. A quantitative structure-activity relationship analysis was conducted to rationalize the structure-activity relationship and to predict how similar the inhibitor-binding profiles of two protein kinases are likely to be on the basis of the docking of lead coumpounds into the ATP-binding site. This model was used to direct the synthesis of new compounds. A series of N-(aromatic)-N'-{4-[(6,7-dimethoxyquinazolin-4-yl)oxy]phenyl}urea were identified as potent and selective inhibitors of the tyrosine kinase activity of VEGFR-2 (fetal liver kinase 1, kinase insert domain-containing receptor). An efficient route was developed that enabled the synthesis of a wide variety of analogues with substitution on several positions of the template. Substitution of diarylurea, competitive with ATP, afforded several analogues with low nanomolar inhibition of enzymatic activity of VEGFR-2. In this paper, we describe the synthesis, structure-activity relationships, and pharmacological characterization of the series.

Impact of aryloxy-linked quinazolines: a novel series of selective VEGFR-2 receptor tyrosine kinase inhibitors.

Three series of 6,7-dimethoxyquinazoline derivatives substituted in the 4-position by aniline, N-methylaniline and aryloxy entities, targeting EGFR and VEGFR-2 tyrosine kinases, were designed and synthesized. Pharmacological activities of these compounds have been evaluated for their enzymatic inhibition of VEGFR-2 and EGFR and for their antiproliferative activities on various cancer cell lines. We have studied the impact of the variation in the 4-position substitution of the quinazoline core. Substitution by aryloxy groups led to new compounds which are selective inhibitors of VEGFR-2 enzyme with IC(50) values in the nanomolar range in vitro.

Design, synthesis, and DNA-binding of N-alkyl(anilino)quinazoline derivatives.

New N-alkylanilinoquinazoline derivatives 5, 12, 20, and 22 have been prepared from 4-chloro-6,7-dimethoxyquinazoline 3, 4-chloro-6,7-methylenedioxyquinazoline 19, and commercially available anilines. Differents classes of compounds substituted by an aryloxygroup (6a-c, 16a,b, and 17a,b), (aminophenyl)ureas (12a,b and 13a-f), anilines (4a-m, 20a,b), N-alkyl(aniline) (5a-m, 21a,b, 22a,d), and N-aminoalkyl(aniline) (22e-g) have been synthesized. These molecules were evaluated for their cytotoxic activities and as potential DNA intercalating agents. We studied the strength and mode of binding to DNA of these molecules by DNA melting temperature measurements, fluorescence emission, and circular dichroism. The results of various spectral and gel electrophoresis techniques obtained with the different compounds, in particular compounds 5g and 22f, revealed significant DNA interaction. These experiments confirm that the N-aminoalkyl(anilino)-6,7-dimethoxyquinazoline nucleus is an efficient pharmacophore to trigger binding to DNA, via an intercalative binding process.

Design, solid-phase synthesis, and biological evaluation of novel 1,5-diarylpyrrole-3-carboxamides as carbonic anhydrase IX inhibitors.

Following previous studies we herein report the synthesis and the pharmacological evaluation of a new class of human carbonic anhydrase (hCA) inhibitors, 1,5-diarylpyrrole-3-carboxamides prepared by a solid-phase strategy involving a PS(HOBt) resin. A molecular modeling study was conducted in order to simulate the binding mode of this new family of enzyme inhibitors within the active site of hCA IX. This study revealed that the 3-position of the pyrrole was opened to the solvent, so we introduced an amino side-chain, protonated at physiological pH both to enhance the aqueous solubility and to decrease the cell membrane penetration. This strategy consisted of preparing membrane-impermeant inhibitors that may selectively target the tumor-associated hCA IX. Physico-chemical characterizations including aqueous solubility and lipophilic parameters are described. Pharmacological studies revealed high hCA IX inhibitory potency in the nanomolar range. Some compounds are selective for hCA IX displaying hCA I/hCA IX and hCA II/hCA IX ratios higher than 20 and 5, respectively.

Quinazoline-urea, new protein kinase inhibitors in treatment of prostate cancer.

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor-2 (VEGFR-2), two protein tyrosine kinases, are involved in pathological disorders and the progression of different types of carcinomas. Concomitant inhibition of both tyrosine kinase activities appears to be an attractive target for cancer chemotherapy. A series of new quinazoline derivatives substituted by amide, urea, or carbamic acid ester groups have been synthesized. The biological activities of these new compounds have been evaluated for their enzyme inhibition and antiproliferative activities.

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.

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.

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.

Cytotoxic effects induced by a combination of cyclopamine and gefitinib, the selective hedgehog and epidermal growth factor receptor signaling inhibitors, in prostate cancer cells.

Although the blockade of the hedgehog cascade by using cyclopamine has been reported to inhibit the growth of some cancer cell types, few studies on the mechanism by which this drug alone or in combination with other cytotoxic agents induces its cytotoxic effect have been reported. In our study, we evaluate, for the first time, the antiproliferative and cytotoxic effects induced by a combination of selective SMO inhibitor, cyclopamine and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, gefitinib on metastatic prostate cancer (PC) cells. The results revealed that cyclopamine, alone or at a lower concentration in combination with gefitinib, inhibited the growth of sonic hedgehog- (SHH), epidermal growth factor- (EGF) and serum-stimulated androgen-sensitive LNCaP-C33 and LNCaP-LN3 and androgen-independent LNCaP-C81, DU145 and PC3 cells. The antiproliferative effect of cyclopamine and gefitinib, alone or in combination, was mediated via a blockade of the PC3 cells in the G1 phase of the cell cycle. Importantly, the combined cyclopamine and gefitinib also caused a higher rate of apoptotic death of PC cells compared to single agents. The cytotoxic effect induced by these drugs in PC3 cells appears to be mediated at least, in part, via the mitochondrial pathway through the depolarization of the mitochondrial membrane and the release of cytochrome c and reactive oxygen species into the cytosol. This was also accompanied by the activation of caspase cascades, PARP cleavage and DNA fragmentation. Additionally, the combined cyclopamine and gefitinib were more effective at suppressing the invasiveness of PC3 cells through matrigel in vitro as the drugs alone. These findings indicate that the simultaneous blockade of SHH-GLI-1 and EGF-EGFR signaling, which results in the growth arrest and massive rate of apoptotic cell death, represents a promising strategy for a more effective treatment of metastatic PC forms.

New antiproliferative benzoindolinothiazepines derivatives.

New benzoindolinothiazepines containing a piperazine moiety are described as potent antiproliferative agents against PC3 human prostatic cell lines. This activity could be explained by an accumulation of cells in G1 phase.

Synergistic antiproliferative and apoptotic effects induced by mixed epidermal growth factor receptor inhibitor ZD1839 and nitric oxide donor in human prostatic cancer cell lines.

BACKGROUND: The specific inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, ZD1839 induces potent antitumoral effects on several advanced cancer types. The present study was undertaken to determine whether the combination of ZD1839 with an agent donating nitric oxide (NO(*)), sodium nitroprusside (SNP) results in a synergy of anticarcinogenic responses on metastatic prostate cancer (PC) cells. METHODS: The antiproliferative and apoptotic/necrotic effects of ZD1839 and SNP alone or in combination were estimated on EGF- and serum-stimulated LNCaP, DU145, and PC3 cells by MTT growth tests, trypan blue dye exclusion method, and flow cytometric analyses. Moreover, the cellular ceramide levels were evaluated by the diacylglycerol kinase enzymatic method and the amounts of cytosolic cytochrome c by ELISA assays. RESULTS: ZD1839 and SNP alone or in combination at lower concentrations induced an inhibition of EGF- and serum-stimulated growth of LNCaP, DU145, and PC3 concomitant with an arrest in the G1 phase of cellular cycle. Interestingly, the mixed ZD1839 and SNP also caused a more substantial apoptotic/necrotic death of these PC cells as compared to drugs alone. Moreover, we have observed that an inhibition of acidic sphingomyelinase, hydrogen peroxide (H(2)O(2)) accumulation and caspase cascades results in a significant reduction of apoptotic/necrotic death induced by mixed ZD1839 and SNP in EGF-stimulated PC3 cells. In addition, the combined ZD1839 plus SNP also induced a higher cellular ceramide and reactive oxygen species (ROS) production, mitochondrial transmembrane potential decrease, and cytochrome c amount released into cytosol as compared to drugs alone. CONCLUSIONS: The simultaneous use of EGFR inhibitor and compound releasing NO(*) might lead to a synergy in the ceramide and ROS production which might cause cellular membrane damages resulting in a massive apoptotic/necrotic death of metastatic PC cells.