Synthesis And Anticancer Activity Of New Hybrid 3-Methylidene-2,3-Dihydro-1,8-Naphthyridinones.

Synthesis of molecular hybrids, obtained by combination of two or more pharmacophoric groups of different bioactive substances in order to produce more efficient drugs, is now a frequently used approach in medicinal chemistry. Following this strategy, we synthetized a library of 3-methylidene-1-tosyl-2,3-dihydro-1,8-naphthyridin-4(1H)-ones, combining a 1,8-naphthyridin-4-one motif with an exo-methylidene bond conjugated with a carbonyl group, pharmacophoric units that are present in many natural, biologically active compounds with anticancer potential. We reasoned that such bifunctional conjugates may have enhanced cytotoxic activity. The title compounds were synthesized in a four step reaction sequence. ß-Ketophosphonate, obtained from methyl N-tosylnicotinate and diethyl methylphosphonate, was reacted with various aldehydes giving 3-diethoxyphosphoryl-2,3-dihydro-1,8-naphthyridin-4(1H)-ones as keto-enol tautomers. Later, these compounds were transformed into 3-methylidene-1-tosyl-2,3-dihydro-1,8-naphthyridin-4(1H)-ones applying the Horner-Wadsworth-Emmons methodology. Then, the cytotoxicity of the new compounds was assessed on two cancer cell lines, promyelocytic leukemia HL-60 and breast cancer adenocarcinoma MCF-7, and for comparison, on human umbilical vein endothelial cells HUVEC. The most active and selective analog, 2-ethyl-3-methylidene-1-tosyl-2,3-dihydro-1,8-naphthyridin-4(1H)-one 4 a was chosen for more detailed studies on HL-60 cell line, to determine molecular mechanisms of its anticancer activity. It was shown that 4 a strongly inhibited proliferation and induced apoptosis which could be attributed to its ability to cause DNA damage.

New Uracil Analog with Exocyclic Methylidene Group Can Reverse Resistance to Taxol in MCF-7 Cancer Cells.

Introduction: Taxol (Tx), a microtubule-stabilizing drug, has been widely used as a chemotherapeutic in several types of cancer. However, the development of resistance limited its application. One of the strategies used to prevent the emergence of drug resistance is combination treatment, involving at least two drugs. The aim of the current study was to assess if a new uracil analog, 3-p-bromophenyl-1-ethyl-5-methylidenedihydrouracil (U-359) can prevent the development of Tx resistance in breast cancer cells. Methods: The cytotoxicity of the new drug was tested in MCF-7 (hormone receptor (ER, PR) positive cell-line) and MCF-10A cell lines using MTT method. For the detection of apoptosis and necrosis, the Wright and Giemsa staining was used. Gene expression was measured by real-time PCR, and changes in the protein levels were evaluated by ELISA and bioluminescent method. Results: We investigated the effect of Tx and U-359 on cancer MCF-7 and normal MCF-10A cells alone and in combination. Tx co-administered with U-359 inhibited proliferation of MCF-7 cells to 7% while the level of ATPase drastically decreased to 14%, compared with effects produced by Tx alone. The apoptosis process was induced through the mitochondrial pathway. These effects were not seen in MCF-10A cells, showing the wide safety margin. The obtained results have shown that U-359 produced a synergistic effect with Tx probably by reducing Tx resistance in MCF-7 cells. To elucidate the possible mechanism of resistance, expression of tubulin III (TUBIII), responsible for microtubule stabilization and tau and Nlp proteins, responsible for microtubule dynamics, was assessed. Conclusion: Combination of Tx with U-359 reduced overexpression of TUBIII and Nlp. Thus, U-359 may stand for a potential reversal agent for the treatment of MDR in cancer cells.

Anticancer Properties of 3-Dietoxyphosphorylfuroquinoline-4,9-dione.

Herein, the antitumor activity of a novel synthetic analog with 5,8-quinolinedione scaffold, diethyl (2-(2-chlorophenyl)-4,9-dioxo-4,9-dihydrofuro [3,2-g]quinolin-3-yl)phosphonate (AJ-418) was investigated on two breast cancer cell lines. This analog was selected from a small library of synthetic quinolinediones on the basis of its strong antiproliferative activity against MCF-7 and MDA-MB-231 cells and 4-5-fold lower cytotoxicity towards healthy MCF-10A cells. The morphology of MCF-7 and MDA-MB-231 cancer cells treated with AJ-418 changed drastically, while non-tumorigenic MCF-10A cells remained unaffected. In MCF-7 cells, after 24 h incubation, the increased number of apoptotic cells coincided with a decrease in proliferation and cell viability. The 24 h treatment of MDA-MB-231 cells with the tested compound reduced their cell viability and proliferation rate; however, a significant pro-apoptotic effect was visible only after longer incubation times (48 h and 72 h). Then, the maximum tolerated dose (MTD) of compound AJ-418 in C3H mice after subcutaneous administration was determined to be 160 mg/kg, showing that this analog was well tolerated and can be further evaluated to assess its potential therapeutic effect in tumor-bearing mice.

Synthesis and Anticancer Properties of New 3-Methylidene-1-sulfonyl-2,3-dihydroquinolin-4(1H)-ones.

Quinolinones have been known for a long time as broad-spectrum synthetic antibiotics. More recently, the anticancer potential of this group of compounds has been investigated. Following this direction, we obtained a small library of 3-methylidene-1-sulfonyl-2,3-dihydroquinolin-4(1H)-ones with various substituents at positions 1, 2, 6, and 7 of the quinolinone ring system. The cytotoxic activity of the synthesized analogs was tested in the MTT assay on two cancer cell lines in order to determine the structure-activity relationship. All compounds produced high cytotoxic effects in MCF-7, and even higher in HL-60 cells. 2-Ethyl-3-methylidene-1-phenylsulfonyl-2,3-dihydroquinolin-4(1H)-one, which was over 5-fold more cytotoxic for HL-60 than for normal HUVEC cells, was selected for further tests. This analog was shown to inhibit proliferation and induce DNA damage and apoptosis in HL-60 cells.

Stereoselective Synthesis and Anticancer Activity of 2,6-Disubstituted trans-3-Methylidenetetrahydropyran-4-ones.

In this report, we present efficient and stereoselective syntheses of 2,6-disubstituted trans-3-methylidenetetrahydropyran-4-ones and 2-(4-methoxyphenyl)-5-methylidenetetrahydropyran-4-one that significantly broaden the spectrum of the available methylidenetetrahydropyran-4-ones with various substitution patterns. Target compounds were obtained using Horner-Wadsworth-Emmons methodology for the introduction of methylidene group onto the pyranone ring. 3-Diethoxyphosphoryltetrahydropyran-4-ones, which were key intermediates in this synthesis, were prepared by fully or highly stereoselective addition of Gilman or Grignard reagents to 3-diethoxyphosphoryldihydropyran-4-ones. Addition occurred preferentially by axial attack of the Michael donors on the dihydropyranone ring. Relative configurations and conformations of the obtained adducts were assigned using a detailed analysis of the NMR spectra. The obtained methylidenepyran-4-ones were evaluated for cytotoxic activity against two cancer cell lines (HL-60 and MCF-7). 2,6-Disubstituted 3-methylidenetetrahydropyran-4-ones with isopropyl and phenyl substituents in position 2 were more cytotoxic than analogs with n-butyl substituent. Two of the most cytotoxic analogs were then selected for further investigation on the HL-60 cell line. Both analogs induced morphological changes characteristic of apoptosis in cancer cells, significantly inhibited proliferation and induced apoptotic cell death. Both compounds also generated DNA damage, and one of the analogs arrested the cell cycle of HL-60 cells in the G2/M phase. In addition, both analogs were able to inhibit the activity of topoisomerase IIα. Based on these findings, the investigated analogs may be further optimized for the development of new and effective topoisomerase II inhibitors.

Synthesis and structure-activity relationship study of novel 3-diethoxyphosphorylfuroquinoline-4,9-diones with potent antitumor efficacy.

Herein we report an efficient synthesis of a series of regioisomeric N,O-syn and N,O-anti 3-diethoxyphosphorylfuroquinoline-4,9-diones combining furoquinoline-5,8-dione skeleton, present in several highly cytotoxic compounds, with diethoxyphosphoryl moiety. The cytotoxic activity of the obtained analogs was tested against two human cancer cell lines: promyelocytic leukemia HL-60 and breast cancer adenocarcinoma MCF-7 and for comparison on human umbilical vein endothelial cells HUVEC and mammary gland/breast MCF-10 A cells. Several diethoxyphosphorylfuroquinoline-4,9-diones proved to be highly cytotoxic for cancer cells with IC50 values even below 0.1 µM. Interestingly, N,O-syn 3-diethoxyphosphorylfuroquinoline-4,9-diones were 3- to 7-fold more active against HL-60 cells than the respective N,O-anti regioisomers. The most promising analogs 9c and 9i, with the highest cancer/healthy cells cytotoxicity ratio, were further evaluated to establish their mode of action. In HL-60 cells these analogs enhanced intracellular ROS generation and NAD(P)H:quinone oxidoreductase 1 (NQO1) depletion which led to the cell cycle arrest in the S-phase, reduced cell proliferation, DNA damage and apoptosis.

The search for opioid analgesics with limited tolerance liability.

Reducing the well-known side effects of opioids prescribed to treat chronic pain remains unresolved, despite extensive research in this field. Among several options to tackle this problem the synthesis of multifunctional compounds containing hybridized structures gained a lot of interest. Recently, extensively investigated are combinations of opioid agonist and antagonist pharmacophores embodied in a single molecule. To this end, agonism at the µ opioid receptor (MOR) with simultaneous antagonism at the δ opioid receptor (DOR) emerged as a promising avenue to obtaining novel analogs devoid of serious adverse effects associated with morphine-based analgesics. In this review we covered up-to-date research on the synthesis of peptide-based ligands with MOR agonist/DOR antagonist profile.

A New Hybrid δ-Lactone Induces Apoptosis and Potentiates Anticancer Activity of Taxol in HL-60 Human Leukemia Cells.

In the search for new drug candidates, researchers turn to natural substances isolated from plants which may be either used directly or may serve as a source for chemical modifications. An interesting strategy in the design of novel anticancer agents is based on the conjugation of two or more biologically active structural motifs into one hybrid compound. In this study, we investigated the anticancer potential of 4-benzyl-5,7-dimethoxy-4-methyl-3-methylidene-3,4-dihydro-2H-chroman-2-one (DL-247), a new hybrid molecule combining a chroman-2-one skeleton with an exo-methylidene bond conjugated with a carbonyl group, in human myeloid leukemia HL-60 cell line. The cytotoxicity of the new compound was tested using MTT assay. The effect of DL-247 on cell proliferation and apoptosis induction were studied by flow cytometry, fluorometric assay and ELISA analysis. DL-247 displayed high cytotoxic activity (IC50 = 1.15 µM, after 24 h incubation), significantly inhibited cell proliferation and induced apoptosis by both, the intrinsic and extrinsic pathways. A combination of DL-247 with taxol exhibited a strong synergistic effect on DNA damage generation, apoptosis induction and inhibition of cell growth.

New uracil analog U-332 is an inhibitor of NF-κB in 5-fluorouracil-resistant human leukemia HL-60 cell line.

BACKGROUND: 5-Fluorouracil (5-FU) is an antimetabolite that interferes with DNA synthesis and has been widely used as a chemotherapeutic drug in various types of cancers. However, the development of drug resistance greatly limits its application. Overexpression of ATP-binding cassette (ABC) transporters in many types of cancer is responsible for the reduction of the cellular uptake of various anticancer drugs causing multidrug resistance (MDR), the major obstacle in cancer chemotherapy. Recently, we have obtained a novel synthetic 5-FU analog, U-332 [(R)-3-(4-bromophenyl)-1-ethyl-5-methylidene-6-phenyldihydrouracil], combining a uracil skeleton with an exo-cyclic methylidene group. U-332 was highly cytotoxic for HL-60 cells and showed similar cytotoxicity in the 5-FU resistant subclone (HL-60/5FU), in which this analog almost completely abolished expression of the ATP-binding cassette (ABC) transporter, multidrug resistance associate protein 1 (ABCC1). The expression of ABC transporters is usually correlated with NF-κB activation. The aim of this study was to determine the level of NF-κB subunits in the resistant HL-60/5-FU cells and to evaluate the potential of U-332 to inhibit activation of NF-κB family members in this cell line. METHODS: Anti-proliferative activity of compound U-332 was assessed by the MTT assay. In order to disclose the mechanism of U-332 cytotoxicity, quantitative real-time PCR analysis of the NF-κB family genes, c-Rel, RelA, RelB, NF-κB1, and NF-κB2, was investigated. The ability of U-332 to reduce the activity of NF-κB members was studied by ELISA test. RESULTS: In this report it was demonstrated, using RT-PCR and ELISA assay, that members of the NF-κB family c-Rel, RelA, RelB, NF-κB1, and NF-κB2 were all overexpressed in the 5-FU-resistant HL-60/5FU cells and that U-332 potently reduced the activity of c-Rel, RelA and NF-κB1 subunits in this cell line. CONCLUSIONS: This finding indicates that c-Rel, RelA and NF-κB1 subunits are responsible for the resistance of HL-60/5FU cells to 5-FU and that U-332 is able to reverse this resistance. U-332 can be viewed as an important lead compound in the search for novel drug candidates that would not cause multidrug resistance in cancer cells.

Molecular mechanisms of apoptosis induced by a novel synthetic quinolinone derivative in HL-60 human leukemia cells.

The mortality rates for acute myeloid leukemia are very high, necessitating the search for novel chemotherapeutic candidates. Herein, we investigated the anticancer potential of a new synthetic compound, 2-ethyl-3-methyliden-1-tosyl-2,3-dihydroquinolin-4-(1H)-one (AJ-374) against myeloid leukemia HL-60 cell line. This analog was selected from the small library of synthetic dihydroquinolinones on the basis of its strong antiproliferative activity against HL-60 cells and 30-fold lower cytotoxicity towards healthy HUVEC cells. AJ-374 promoted the arrest of the cells in the subG0/G1 phase of the cell cycle in the first 24 h. Treatment of HL-60 cells with AJ-374 caused an increase in annexin-V positive cells, activation of caspase-8, -9 and -3, dissipation of the mitochondrial membrane potential and enhancement of FAS protein level. Apoptosis induction triggered by this quinolinone was blocked by the pre-treatment of the cells with caspase-8, -9 and -3 inhibitors. The obtained results indicated that AJ-374-induced apoptosis was executed by both, the extrinsic and intrinsic pathways. The cytotoxic activity of AJ-374 was also associated with down-regulation of the mitogen-activated protein kinase (MAPK) pathway and was independent of reactive oxygen species generation. Taken together, these results suggest that AJ-374 exerts a potent anticancer effect on leukemia cells, with a wide safety margin, which makes this analog an attractive drug candidate for further testing.

Synthesis of 2,2,6-Trisubstituted 5-Methylidene-tetrahydropyran-4-ones with Anticancer Activity.

In our continuous search for new, relatively simple 2-alkylidene-1-oxoheterocycles as promising anticancer drug candidates, herein we report an efficient synthesis of 2,2,6-trisubstituted 5-methylidenetetrahydropyran-4-ones. These compounds were obtained in a four step reaction sequence, in which starting diethyl 2-oxopropylphosphonate was transformed into 2,2-disubstituted 5-diethoxyphosphoryldihydropyran-4-ones, followed by Michael addition of various Grignard reagents and Horner-Wadsworth-Emmons reaction of the obtained adducts with formaldehyde. Stereochemistry of the intermediate Michael adducts is also discussed. Final 5-methylidenetetrahydropyran-4-ones were tested for their possible antiproliferative effect against three cancer cell lines and 6-isopropyl-2,2-dimethyl-5-methylidenetetrahydropyran-4-one (11c), which showed very high cytotoxic activity against HL-60 human leukemia cells and was three times more active than known anticancer drug carboplatin, was selected for further biological evaluation, in order to disclose its mechanism of action. The obtained results indicated that 11c induced apoptosis in HL-60 cells and caused the arrest of the cell cycle in the G2/M phase, which may suggest its cytotoxic and cytostatic activity.

New Uracil Analogs with Exocyclic Methylidene Group as Potential Anticancer Agents.

BACKGROUND: Hybrid molecules combining uracil skeleton with methylidene exo-cyclic group were designed in the search for novel anticancer drug candidates. OBJECTIVE: Two series of racemic 5-methylidenedihydrouracils, either 1,3-disubstituted or 1,3,6-trisubstituted were synthesized and tested for their possible cytotoxic activity against two cancer cell lines (HL-60 and MCF-7) and two healthy cell lines (HUVEC and MCF-10A). The most cytotoxic analogs were re-synthesized as pure enantiomers. The analog designated as U-332 [(R)-3-(4-bromophenyl)-1-ethyl-5-methylidene-6-phenyldihydrouracil], which had a very low IC50 value in HL-60 cell line (0.77µM) and was the most selective towards cancer cells was chosen for further experiments on HL-60 cell line, in order to determine the possible mechanism involved in its antineoplastic action. METHODS: Cytotoxic activities of compound was assessed by the MTT assay. In order to explore the mechanism of U-332 activity, we performed quantitative real-time PCR analysis of p53 and p21 genes. Apoptosis, cell proliferation and DNA damage in HL-60 cells were determined using the flow cytometry. The ability of U-332 to determine GADD45ɑ protein level in HL-60 cells incubated with U-332 was analyzed by ELISA test. RESULTS: U-332 was shown to generate excessive DNA damage (70% of the cell population), leading to p53 activation, resulting in p21 down-regulation and a significant increase of GADD45α protein, responsible for the cell cycle arrest in G2/M phase. CONCLUSION: U-332 can be used as a potential lead compound in the further development of novel uracil analogs as anticancer agents.

New uracil analogs as downregulators of ABC transporters in 5-fluorouracil-resistant human leukemia HL-60 cell line.

Overexpression of ATP-binding cassette (ABC) transporters causing multidrug resistance (MDR) in cancer cells is one of the major obstacles in cancer chemotherapy. The 5-FU resistant subclone (HL-60/5FU) of the human HL-60 promyelocytic leukemia cell line was selected by the conventional method of continuous exposure of the cells to the drug up to 0.08 mmol/L concentration. HL-60/5FU cells exhibited six-fold enhanced resistance to 5-FU than HL-60 cells. RT-PCR and ELISA assay showed significant overexpression of MDR-related ABC transporters, ABCB1, ABCG2 but especially ABCC1 in the HL-60/5FU as compared with the parental cell line. Three novel synthetic 5-methylidenedihydrouracil analogs, U-236, U-332 and U-359, selected as highly cytotoxic for HL-60 cells in MTT test, showed similar cytotoxicity in the resistant cell line. When co-incubated with 5-FU, these analogs were found to down-regulate the expression of all three transporters. However, the most pronounced effect was caused by U-332 which almost completely abolished ABCC1 expression in the resistant HL-60/5FU cells. Additionally, U-332 inhibited the activity of ATPase, an enzyme which catalyzes hydrolysis of ATP, providing energy to efflux drugs from the cells through the cellular membranes. Taken together, the obtained data suggest that acquired 5-FU resistance in HL-60/5FU cells results from overexpression of ABCC1 and that targeting ABCC1 expression could be a potential approach to re-sensitize resistant leukemia cells to 5-FU. The synthetic uracil analog U-332, which can potently down-regulate ABC transporter expression and therefore disturb drug efflux, can be considered an efficient ABCC1 regulator in cancer cells.

Synthesis and Cytotoxic Evaluation of 3-Methylidenechroman-4-ones.

In the search for new anticancer agents, a library of variously substituted 3-methylidenechroman-4-ones was synthesized using Horner-Wadsworth-Emmons methodology. Acylation of diethyl methylphosphonate with selected ethyl salicylates furnished 3-diethoxyphosphorylchromen-4-ones which were next used as Michael acceptors in the reaction with various Grignard reagents. The adducts were obtained as the mixtures of trans and cis diastereoisomers along with a small amount of enol forms. Their relative configuration and preferred conformation were established by NMR analysis. The adducts turned up to be effective Horner-Wadsworth-Emmons reagents giving 2-substituted 3-methylidenechroman-4-ones, which were then tested for their possible cytotoxic activity against two leukemia cell lines, HL-60 and NALM-6, and against MCF-7 breast cancer cell line. All new compounds (14a-o) were highly cytotoxic for the leukemic cells and showed a moderate or weak effect on MCF-7 cells. Analog 14d exhibited the highest growth inhibitory activity and was more potent than carboplatin against HL-60 (IC50 = 1.46 ± 0.16 µM) and NALM-6 (IC50 = 0.50 ± 0.05 µM) cells. Further tests showed that 14d induced apoptosis in NALM-6 cells, which was mediated mostly through the extrinsic pathway.

Involvement of a coumarin analog AD-013 in the DNA damage response pathways in MCF-7 cells.

Coumarin is a plant-derived compound but as such has no medical uses. Several synthetic coumarin analogs have been shown to possess anti-proliferative activity and to induce apoptosis in cancer cells. Here, we explored DNA damage responses in MCF-7 cells treated with our novel synthetic hybrid compound AD-013, which integrates a coumarin moiety and an α-methylene-δ-lactone motif. The mRNA expression of several genes engaged in DNA-damage-induced responses was assessed by quantitative real-time PCR. The protein levels of a few members of phosphoinositide-3-kinases family (ATM, ATR and DNA-PK) and BRCA1 were assessed by ELISA, while p53 was evaluated by western blot method. AD-013 down-regulated DNA-PK gene expression but increased the level of ATM/ATR and p53. The new analog completely inhibited BRCA1 and greatly decreased the activity of BRCA1 protein, engaged in DNA damage repair. Exposure of MCF-7 cells to a coumarin analog AD-013 led to DNA damage and decreased expression of several repair-associated genes.

Supramolecular architecture in azaheterocyclic phosphonates. III. Structures of an ethyl phosphonamidate and an ethyl phosphonate.

The novel crystal structures of ethyl (S)-P-(4-oxo-4H-benzo[4,5]thiazolo[3,2-a]pyrimidin-3-yl)-N-[(R)-1-phenylethyl]phosphonamidate, C20H20N3O3PS, I, and diethyl (4-isopropyl-2-oxo-3,4-dihydro-2H-benzo[4,5]thiazolo[3,2-a]pyrimidin-3-yl)phosphonate, C18H25N2O4PS, II, were characterized by X-ray diffraction analysis. The crystal packing of I is dominated by two infinite stacks composed of symmetry-independent molecules linked by distinctively different hydrogen-bond systems. The structure of II shows a ladder packing topology similar to those observed in related phosphorylated azaheterocycles. Structural studies are supplemented by calculations on the interactions stabilizing the molecular assemblies using the PIXEL method. Additionally, fingerprint plots derived from the Hirshfeld surfaces were generated for each structure to characterize the crystal packing arrangements in detail. The aromaticities of the heterocyclic moieties have been investigated using HOMA and HOMHED parametrization and compared with structures reported previously.

Synthesis of 3-Methylidene-1-tosyl-2,3-dihydroquinolin-4(1H)-ones as Potent Cytotoxic Agents.

An efficient synthetic strategy to 3-methylidene-2,3-dihydroquinolin-4(1H)-ones variously substituted in position 2 has been developed. The title compounds were synthesized in the reaction sequence involving reaction of diethyl methylphosphonate with methyl 2-(tosylamino)benzoate, condensation of thus formed diethyl 2-oxo-2-(2-N-tosylphenyl)ethylphosphonate with various aldehydes followed by successful application of the obtained 3-(diethoxyphosphoryl)-1,2-dihydroquinolin-4-ols as Horner-Wadsworth-Emmons reagents for the olefination of formaldehyde. Also, enantioselective approach to the target compounds has been evaluated using 3-dimenthoxyphosphoryl group as a chiral auxiliary. Single X-ray crystal analysis of (2S)-3-(dimenthoxyphosphoryl)-2-phenyl-1-tosyldihydroquinolin-4-ol revealed the presence of strong resonance-assisted hydrogen bond (RAHB). The obtained 3-methylidene-2,3-dihydroquinolin-4(1H)-ones were then tested for their cytotoxic activity against two leukemia cell lines NALM-6 and HL-60 and a breast cancer MCF-7 cell line. All compounds showed very high cytotoxic activity with the IC50 values mostly below 1 µm in all three cancer cell lines. The selected analogs were also tested on human umbilical vein endothelial cells (HUVEC) and on human mammary gland/breast cells (MCF-10A) to evaluate their influence on normal cells. Since one of the most serious problems in cancer chemotherapy is the development of drug resistance, the mRNA levels and activity of ABCB1 transporter considered to be the most important factor engaged in drug resistance, were evaluated in MCF-7 cells treated with two selected analogs. Both compounds were strong ABCB1 transporter inhibitors that could prevent efflux of anticancer drugs from cancer cells.

Involvement of α-methylene-γ- and δ-lactones in the suppression of multidrug resistance in MCF-7 cells.

BACKGROUND: The development of multidrug resistance to chemotherapy remains a challenge in the treatment of cancer and is a major factor causing failure of many forms of chemotherapy. The ATP binding cassette (ABC) family of proteins are efflux pumps that transport various potentially dangerous substances out of the cells. Several of the ABC transporters are related to chemoresistance, as the rapidly dividing malignant cells use them to protect themselves from medical interventions. Inhibitors of ABC transporters have the potential to enhance the efficacy of anticancer drugs. Two new synthetic compounds, AD-06 and AD-013, were tested as possible multidrug resistance inhibitors in MCF-7 cells. METHODS: The cytotoxicity of new compounds was tested in MCF-7 and MCF-10A cell lines using the MTT method. Gene expression was measured by real-time PCR and changes in the protein levels were evaluated by flow cytometry and ELISA. A method based on the use of a fluorescent dye, being a marker of the ABC transporter activity, was used for screening the tested compounds as potential multidrug resistance inhibitors. RESULTS: AD-06 and AD-013 down-regulated NF-κB mRNA levels and decreased the population of cells with activated NF-κB. Both compounds were found to be strong ABCB1 and ABCG2 transporter inhibitors. They showed synergistic effects when incubated with taxol or oxaliplatin. CONCLUSIONS: α-Methylene-γ- and -δ-lactones AD-06 and AD-013 are promising lead structures for further development as multidrug resistance inhibitors.

Anticancer Properties of a New Hybrid Analog AD-013 Combining a Coumarin Scaffold with an α-methylene-δ-lactone Motif.

BACKGROUND: Coumarin is a natural phytochemical but as such has no medical uses. However, various natural and synthetic coumarin analogs attract attention due to their interesting biological properties. OBJECTIVE: Here, we evaluated and compared anticancer properties of a new synthetic hybrid compound AD- 013, which integrates a coumarin moiety and an α-methylene-δ-lactone motif, with novobiocin, a natural antibiotic bearing a coumarin scaffold. METHODS: Cytotoxic activities of compound AD-013 and novobiocin were assessed by the MTT assay. In order to explore the mechanism of anticancer activity of analog AD-013, we performed quantitative real-time PCR analysis of apoptosis- and cell cycle-related genes. The ability of AD-013 and novobiocin to induce apoptosis and DNA damage was studied by flow cytometry. RESULTS: The cytotoxic activity of this new compound was compared with the activity of a coumarin-based antibiotic novobiocin against two cancer cell lines, MCF-7 and HL-60 and also against normal human cells, MCF- 10A and HUVEC. AD-013 was much more cytotoxic than novobiocin in both cancer cell lines and showed some selectivity against MCF-7 cancer cells as compared with MCF-10A healthy cells. Expression levels of the pro-apoptotic genes significantly increased while the anti-apoptotic genes, were down-regulated for both compounds in both cancer cell lines. AD-013 was able to inhibit cell proliferation, generate DNA damage and induce apoptosis. The obtained data showed that this compound caused the cell cycle arrest in subG0/G1 in both cancer cell lines. CONCLUSION: The new hybrid analog was a much stronger apoptosis inducer than novobiocin and activated the intrinsic pathway of apoptosis.

Synthesis of 4,4-Disubstituted 3-Methylidenechroman-2-ones as Potent Anticancer Agents.

The synthesis of a new library of 4,4-disubstituted 3-methylidene-3,4-dihydro-2H-chroman-2-ones applying Horner-Wadsworth-Emmons methodology for the construction of an exo-methylidene moiety is reported. Corresponding 3-diethoxyphosphorylchroman-2-ones were synthesized in a three-step reaction sequence consisting of O-methylation of ethyl 2-diethoxyphosphoryl-3-oxoalkanoates, followed by reaction of the obtained 2-diethoxyphosphoryl-3-methoxy-2-alkenoates with phenols or 1-naphthol. The resulting 3-diethoxyphosphorylochromen-2-ones proved to be effective Michael acceptors in reactions with various Grignard reagents. Preliminary biological evaluations showed that many of the synthesized 3-methylidenechroman-2-ones possess very high cytotoxic activity against NALM-6 and HL-60 cancer cell lines (IC50 <1.0 µm) as well as high activity against the MCF-7 cancer cell line (IC50 <10 µm). Furthermore, two of the highly active 3-methylidenechroman-2-ones with geminal methyl and ethyl substituents at position 4 showed promising therapeutic indexes of 10 and 13 in tests against human umbilical vein endothelial cells (HUVECs).