Synthesis and biological evaluation of novel hydrazide based cytotoxic agents

Recently, we designed and synthesized a series of pyrroloquinoxaline compounds with hydrazine moiety linking a nitrogen-containing polycyclic group to a heteroaroyl system. Several derivatives, with attractive drug-like properties, were identified as promising cytotoxic agents, showing excellent potency in a panel of cancer cell lines. In the current study, we synthesized a further 19 new analogues to optimize their physicochemical properties and assess a coherent mechanism of action. Several chemical modifications were made to the reference compounds by varying the fused-ring system and/or the heteroacyl moiety. To evaluate their in vitro activity, we tested these compounds in six human cancer cell lines derived from different origins. Among them, two compounds ( and ) showed similar potency as the reference compounds with IC₅₀ values in the sub-micromolar range in all cell lines tested. Furthermore, compound showed excellent in vivo efficacy in our preliminary human ovarian cancer mouse xenograft studies. Flow cytometric studies indicated that both derivatives interrupted cell cycle progression in colorectal cancer HCT116 cell lines and ovarian cancer SKOV-3 cells. Further mechanistic studies revealed that and were able to induce reactive oxygen species in SKOV-3 cells with apparently different kinetic patterns. Considering their cytotoxicity profiles in a variety of in vitro and in vivo cancer models, these hydrazide based compounds seem to have considerable potentials as novel chemotherapeutics.     

Asiatic acid exerts anticancer potential in human ovarian cancer cells via suppression of PI3K/Akt/mTOR signalling

Context Asiatic acid, a triterpenoid compound extracted from the tropical medicinal plant Centella asiatica (Family: Apiaceae), has exhibited various biological activities.

Objective This study was performed to investigate the cytotoxic effects of asiatic acid on human ovarian cancer cells.

Materials and methods SKOV3 and OVCAR-3 ovarian cancer cells were exposed to different concentrations of asiatic acid (10–100?μg/mL) for 72 or 48?h. Cell viability, colony formation, cell cycle distribution, apoptotic response were examined. Involvement of the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway was tested.

Results At the concentration of 40?μg/mL, asiatic acid caused about 50% reduction in the viability of ovarian cancer cells, but had little effect on the viability of normal human ovarian epithelial cells. Asiatic acid at 10?μg/mL reduced colony formation of ovarian cancer cells by 25–30%. Asiatic acid-treated cells showed a cell cycle arrest at the G0/G1 phase and 7- to 10-fold increase in apoptosis. The phosphorylation levels of PI3K, Akt and mTOR were remarkably lower in asiatic acid-treated cells. Overexpression of constitutively active Akt partially reversed the cytotoxic effects of asiatic acid, as evidenced by increased cell viability and colony formation. Furthermore, knockdown of Akt mimicked the growth-suppressive activity of asiatic acid.

Discussion and conclusion These results provide first the evidence for the anticancer potential of asiatic acid in ovarian cancer cells, partially via inactivation of the PI3K/Akt/mTOR pathway. Asiatic acid may represent a potential therapeutic agent for ovarian cancer.     

Lack of effects of recombinant human interleukin-4 onin vitro colony formation of freshly explanted human tumor cells

Summary Interleukin-4 is a highly pleiotropic T-cell derived lymphokine that has been reported to stimulate a host cell-mediated antitumor response. Recombinant human interleukin-4 (rhuIL-4) is currently undergoing clinical phase I trials. We have studied the growth modulating effects of rhuIL-4 on a variety of freshly explanted human tumor specimens using anin vitro soft agar cloning system. Final concentrations of 0.1 to 10 ng/ml were used in continuous incubation experiments. Of 147 specimens, 73 (50%) were evaluable for the determination of tumor growth modulating activity. The most common tumor types recruited included breast, nonsmall cell lung, ovarian cancer and melanoma. Stimulation of tumor colony forming units (colony formation 1.5× controls) was observed in 0/73 tumors. Similarly, only 1/73 (1.3%) specimens (a non-small cell lung cancer) had a significant decrease in tumor colony forming units (colony formation 0.5× controls) at 1 ng/ml. We conclude that rhuIL-4 is not a direct modulator of tumor colony formationin vitro. However, antitumor effects could perhaps be achievedin vivo via the immune-modulating effects of Interleukin-4.     

Cytotoxicity of sphingoid marine compound analogs in mono- and multilayered solid tumor cell cultures

Summary A subset of four synthetic sphingoid marine compound analogs was chosen from a preliminary in vitro cytotoxicity study for further analysis. The selected analogs were initially screened in monolayer cultures for their anticancer potential against a panel of eight human tumor cell lines, ovarian, colon and lung cancer, squamous cell carcinoma and leukemia producing IC₅₀ values ranging from 1.5 to 6.9 μM. In a secondary screening, the sphingoid analogs were evaluated against multilayered postconfluent cultures of A2780 ovarian cancer and WiDr colon cancer cells. In this model, compounds 5 and 8 were the most active derivatives showing EC₅₀ values in the range 25–32 μM. The performance of 5 and 8 against both cell lines was not dependent on the cell culture model as shown with resistance factor values in the range 8–12. Cell cycle studies in HL60 leukemia cells showed an arrest in G ₀/G ₁ at a low drug concentration (3 μM) but accumulation in S phase at a high drug concentration (9 μM). It can be concluded that the analogs showed a cell line independent activity, with an apparent selectivity against cells grown in more physiological three-dimensional condition compared to standard anticancer drugs.     

Ovarian cancer targeted hyaluronic acid-based nanoparticle system for paclitaxel delivery to overcome drug resistance

Abstract

Objective: Most primary human ovarian tumors and peritoneal implants, as well as tumor vascular endothelial cells, express the CD44 family of cell surface proteoglycans, the natural ligand for which is hyaluronic acid (HA). Paclitaxel (PTX) is an effective chemotherapeutic agent that is widely used for the treatment of several cancers, including ovarian cancer. This study aimed to develop a HA-based PTX-loaded nanoparticle system to improve the ovarian cancer therapeutic effects.

Methods: PTX-loaded cationic nanostructured lipid nanoparticles (PTX-NLCs) were prepared. HA-PE was then coated onto the PTX-NLCs by electrostatic adsorption to form HA-PTX-NLCs. In vitro tumor cell inhibition efficiency was analyzed on SKOV3 human ovarian cancer cells (SKOV3 cells) and PTX-resistant SKOV3 cells (SKOV3/PTX cells). In vivo anticancer ability was evaluated with mice bearing SKOV3 ovarian cancer cells xenografts.

Results: HA-PTX-NLCs had an average diameter of 163?nm, and PTX was incorporated with an efficiency of over 80%. The in vitro viability of SKOV3 cells and SKOV3/PTX cells was obviously inhibited by HA-PTX-NLCs. In the ovarian cancer cells model, significant reduction in tumor growth was observed, whereas the conventional PTX injection group did not achieve significance.

Conclusion: This study demonstrated that significantly improved results were obtained by the newly constructed HA-PTX-NLCs, in terms of in vitro and in vivo therapeutic efficacy. These findings strongly support the superiority of HA based nano-system for the PTX delivery, thus enhance the efficacy of ovarian cancer chemotherapy.     

Activity of gemcitabine in a human tumor cloning assay as a basis for clinical trials with gemcitabine

Summary 2′,2′-difluorodeoxycytidine (LY 188011, Gemcitabine, Gemzar^?) has recently been approved both in Europe for the treatment of patients with non-small cell lung cancer and in the United States for patients with pancreatic cancer. Since the initial discovery of the compound, we have been evaluating the in vitro activity of gemcitabine against human tumor colony-forming units taken directly from patients and growing in soft agar (in the human tumor cloning assay — HTCA). A total of 315 specimens have had gemcitabine tested against them with 44% giving evaluable results. Gemcitabine has been found to be active against colony-forming units from patients with non-small cell lung, breast, ovarian, and pancreatic cancers. A concentration-dependent in vitro response was noted with a higher in vitro response rate noted at 20 μg/ml than at 2.0 μg/ml. Based on subsequent clinical phase II data, the HTCA correctly predicted the wide spectrum of the clinical activity of gemcitabine.     

Design,synthesis, in silico and antiproliferative evaluation of novel pyrazole derivatives as VEGFR‐2 inhibitors

As the blockade of the VEGFR‐2 signaling pathway is a viable approach in cancer therapy, the present study focuses on a series of pyrazole based VEGFR‐2 inhibitors that were designed on the basis of the hybridization approach, supported by docking and in silico computational studies. The designed compounds were synthesized through facile synthetic methods and the structures were confirmed by ¹H NMR, ¹³C NMR, MS and elemental analysis. The compounds were screened for in vitro antiproliferative activity against the HT‐29 (human colon cancer) and MCF‐7 (human breast cancer) cell lines by MTT assay. The compounds were also studied for in vitro inhibitory activity against VEGFR‐2 kinase. Among all the tested compounds, compound 6h emerged as a potent agent in the antiproliferative study against HT‐29 and MCF‐7 cells, with IC₅₀ values of 2.36 and 6.59 μM, respectively. Moreover, the same compound exhibited the highest VEGFR‐2 inhibitory activity with an IC₅₀ value of 1.89 μM. In docking studies, the designed compounds showed similar and essential key interactions as those of known VEGFR‐2 inhibitors. The present study may lead to new molecules in the development of anticancer agents targeting VEGFR‐2.     

Synthesis and Antitumor Activity of 5′-deoxy-4′-thio-l-nucleosides

A series of novel 5′-deoxy-4′-thio-l -nucleosides was designed and synthesized. The absolute configuration of the target compound 23α was confirmed by X-ray crystallography. The antitumor activities of the target compounds were tested against the growth of human carcinoma of colon (LOVO), human leukemia cell line (CEM) and human breast cancer cell line (MDA-MB-435) cells in vitro. 6-cyclopentylamino and 6-cyclohexylamino purine compounds 26 and 27, both in α-configuration and in β-form, exhibited strong inhibition to CEM.     

In vitro antitumor effect of recombinant human tumor necrotizing factor on cultured human cancer cell lines and freshly isolated lung cancer cells by the human tumor clonogenic assay

Summary In vitro antitumor effects of human recombinant tumor necrotizing factor (rH-TNF) were examined against nine lung cancer cell lines including six non small and three small cell lung cancer, four stomach cancer cell lines and 30 freshly isolated lung cancer cell samples by the human tumor clonogenic assay. rH-TNF did not show any inhibitory effect on the colony formations of lung and stomach cancer cell lines, except for PC10 established from squamous cell carcinoma even at the high concentration. The overall response rate of fresh material was 11.5%. The colony formations of only two materials from 20 patients without prior chemotherapy were significantly suppressed by rH-TNF in vitro. Three specimens of adenocarcinoma exhibited more than 70% decrease in colony number by treating with 100 and 1000 u/ml of rH-TNF resulting in the response rate of 15.8% (3/19). From these results, it can be concluded that rH-TNF has modest direct cytotoxic effect on lung cancer, and additional study against adenocarcinoma of the lung might be warranted.     

Synthesis and anticancer activity of Indolin-2-one derivatives bearing the 4-thiazolidinone moiety

A novel series of indolin‐2‐one derivatives containing the 4‐thiazolidinone moiety ( 5a—5p ) was synthesized and the cytotoxicity of these derivatives was evaluated in vitro against three human cancer cell lines (HT‐29, H460 and MDA‐MB‐231) by standard MTT assay. Some prepared compounds exhibited significant cytotoxicity against different human cancer cell lines. Several potent compounds were further evaluated against one normal cell line (WI‐38). In particular, the promising compound 5h showed remarkable cytotoxicity and selectivity against the HT‐29 and H460 cancer cell lines (IC₅₀ = 0.016 µmol/L, 0.0037 µmol/L, respectively).     

Antiproliferative Activity and Phototoxicity of some Methyl Derivatives of 5–Methoxypsoralen and 5–Methoxyangelicin

Abstract: The in vitro antiproliferative activity and in vivo phototoxicity of some methyl derivatives of 5–methoxypsoralen and 5–methoxyangelicin, i.e. 4,4′–dimethyl–5–methoxyangelicin (compound I), 3,4′–dimethyl–5–methoxyangelicin (compound II), 4,4′–dimethyl–5–methoxypsoralen (compound III); and 3.4′–dimethyl–5–methoxypsoralen (compound IV), have been investigated. The effects of the compounds were evaluated in vitro on HL60 and A431 cells, using 5–methoxypsoralen as the reference compound. In both cell lines compound I, II and III showed better antiproliferative activity than compound IV and 5–methoxypsoralen. Scanning electron microscopy revealed that all the compounds induced the formation of blebs and blisters on a A431 cell surface. Significant variations in the nuclear area strictly related to the toxicity of the compounds have been shown in both cell lines. Skin irritancy in vivo was evaluated by mean of histopathological responses on guinea–pig skin. For each compound a damage index was determined by morphometrical analysis of empty spaces in the epidermis. Histopathology revealed skin phototoxicity of compounds which lacked erythemogenic activity by visual scoring. By coupling cytotoxicity data in vitro to skin sensitization ones in vivo, compound I proved a promising candidate for use in clinical trials since due to a high inhibitory effect on the growth of human cell lines coupled to low skin phototoxicity.     

The rationale for mTOR inhibition in epithelial ovarian cancer

The AKT/mTOR signaling pathway is frequently overexpressed in human epithelial ovarian cancer and an attractive target for therapy. In vivo mouse models were confirmative for in vitro findings, where the administration of mTOR inhibitors in ovarian cancer xenografts showed antitumoral as well as antiangiogenic effects. Phase I – II trials are now ongoing with mTOR inhibitors in ovarian cancer patients, some in combination with conventional cytotoxic agents. If further development of mTOR inhibition in ovarian cancer is pursued, studying combinations of mTOR inhibitors with other new targeted therapies would be of interest. mTOR inhibitors in the adjuvant setting could have potential, since, for the moment, there is no standard maintenance therapy in ovarian cancer. A crucial challenge will be to identify strong predictive biomarkers. This review highlights the rationale for the use of mTOR inhibitors in ovarian cancer and summarizes the available preclinical findings.     

Bufadienolides from the Bufo viridis toad venom exert cytotoxic effects on cancer cells by inducing cell apoptosis and cell cycle arrest

A series of bufadienolides were isolated from the Bufo viridis toad venom, and their cytotoxic activities against three human cancer cell lines (HeLa, HT-29, MCF7) and a non-cancer cell line (L-O2) were explored using the MTT assay in vitro. All of nine compounds exhibited cytotoxic activities against the three cancer cell lines, with compound D4 exhibiting potent cytotoxic activity against HeLa cells and was better than positive control. Herein, we further evaluated the effect of compound D4 on HeLa cells. The results revealed that compound D4 has excellent cytotoxic effect on HeLa cells by inhibiting cell colony formation and migration, promoting cell apoptosis, increasing reactive oxygen species (ROS) levels and arresting of HeLa cells in S and G2/M phases. These findings encourage further work on the chemistry and bioactivity of the Bufo viridis toad venom.     

Synthesis and <Emphasis Type="Italic">in Vitro</Emphasis> cytotoxic activities of 2-alkyl-2,3-dihydro-1<Emphasis Type="Italic">H</Emphasis>-2,6-diazacyclopenta[<Emphasis Type="Italic">b</Emphasis>]anthracene-5,10-diones

A series of 2-alkyl-2,3-dihydro-1H-2,6-diazacyclopenta[b]anthracene-5,10-diones (4a–f) was synthesized and their in vitro cytotoxic activities were evaluated against six human cancer cell lines (HCT15, SK-OV-3, SNB19, A549, MCF7 and MCF7/ADR). They all appeared to be less potent than doxorubicin against all doxorubicin sensitive human cancer cell lines tested. However, these compounds retained considerable cytotoxic activity against the doxorubicin-resistant cell line MCF7/ADR, implying their therapeutic potential to treat doxorubicin-resistant tumors. The most active compound 4c was equipotent with doxorubicin against HCT15 cell line.     

Synthesis and Biological Evaluation of Novel Pyrazoles and Pyrazolo[3,4‐d]pyrimidines Incorporating a Benzenesulfonamide Moiety

Synthesis and biological evaluation of novel pyrazoles and pyrazolo[3,4‐d]pyrimidines are reported. Fourteen compounds were selected by the NCI and tested for their preliminary in‐vitro anticancer activity, whereas all the synthesized compounds were evaluated for their in‐vitro antimicrobial activity. Compound 12a was proven to possess the highest anticancer activity with a broad spectrum profile. It showed particular effectiveness towards leukemia HL‐60 (TB), K‐562, non‐small cell lung cancer NCI‐H23, and colon cancer HT 29, KM 12 cell lines (GI₅₀ = 6.59, 4.44, 1.37, 3.33, and 9.63 μM, respectively). Out of the synthesized compounds, thirteen derivatives were found to display pronounced antimicrobial activity especially against P. aeruginosa. Compounds 2c , 5b , 10 , 11b , 17b , 18b , and 19 were proven to be the most active with a broad spectrum of activity. Compound 19 was found to be equipotent to ampicillin against B. subtilis, whereas compounds 11b and 19 were four times superior to ampicillin against P. aeruginosa, while compounds 5b and 18b were equipotent to ampicillin against the same organism. Moreover, compounds 2c , 10 , and 11b were nearly equipotent to ampicillin against E. coli. On the other hand, compounds 2c , 5b , 10 , 11a , 17b , and 18b exerted nearly half the activity of clotrimazole against C. albicans.     

Synthesis,Cytotoxicity, and Pro‐Apoptosis Activity of Etodolac Hydrazide Derivatives as Anticancer Agents

Etodolac hydrazide and a novel series of etodolac hydrazide‐hydrazones 3 – 15 and etodolac 4‐thiazolidinones 16 – 26 were synthesized in this study. The structures of the new compounds were determined by spectral (FT‐IR, ¹H NMR, ¹³C NMR, HREI‐MS) methods. Some selected compounds were determined at one dose toward the full panel of 60 human cancer cell lines by the National Cancer Institute (NCI, Bethesda, USA). 2‐(1,8‐Diethyl‐1,3,4,9‐tetrahydropyrano[3,4‐b]indole‐1‐yl)acetic acid[(4‐chlorophenyl)methylene]hydrazide 9 demonstrated the most marked effect on the prostate cancer cell line PC‐3, with 58.24% growth inhibition at 10^?5 M (10 µM). Using the MTT colorimetric method, compound 9 was evaluated in vitro against the prostate cell line PC‐3 and the rat fibroblast cell line L‐929, for cell viability and growth inhibition at different doses. Compound 9 exhibited anticancer activity with an IC₅₀ value of 54 µM (22.842 µg/mL) against the PC‐3 cells and did not display any cytotoxicity toward the L‐929 rat fibroblasts, compared to etodolac. In addition, this compound was evaluated for caspase‐3 and Bcl‐2 activation in the apoptosis pathway, which plays a key role in the treatment of cancer.     

Effect of a Vitamin D<Subscript>3</Subscript> derivative (B3CD) with postulated anti-cancer activity in an ovarian cancer animal model

The objective of the present study was to test the hypothesis that Calcidiol derivative B3CD qualifies as a potential anti-cancer drug in vivo employing an ovarian cancer xenograft model in mice. In addition, the selectivity of B3CD on viability and proliferation of platinum-resistant human ovarian cancer cell lines in comparison to control cell lines was analyzed in vitro. B3CD displayed cell line-specific cytotoxicity screened against a panel of ovarian and other carcinoma cell lines, endothelial and control cells. B3CD, at sub-cytotoxic concentrations, revealed stronger effects on the proliferation of SKOV-3 ovarian cancer cells vs. primary fibroblasts as determined by BrdU incorporation analysis. Treatment with B3CD at 0.5 μM resulted in highly condensed chromatin and fragmented nuclei in SKOV-3 cells but not in primary fibroblasts. B3CD induced cell death at low drug concentrations (≤0.5 μM) in SKOV-3 ovarian cancer cells is mediated by the p38 MAPK signaling pathway: B3CD induced p38 MAPK expression and activation in SKOV-3 cells and inhibition of p38 signaling counteracted B3CD induced cell death in vitro. An ovarian cancer cell animal model (human SKOV-3 cell derived xenografts in nude mice) revealed that tumor growth in few B3CD treated mice accelerated while the majority of B3CD treated mice displayed delayed tumor growth or full tumor regression. B3CD possesses anti-ovarian cancer properties in vitro and in vivo. We propose the further development of non-calcemic bromoacetoxy derivatives of vitamin D₃ as potential anti-cancer therapeutics.     

Genetic toxicology and preliminary in vivo studies of nitric oxide donor tocopherol analogs as potential new class of antiatherogenic agents

Nitric oxide donor tocopherol analogs were found to be incorporated in low-density lipoprotein to release nitric oxide into the hydrophobic core of the lipoprotein, thus inhibiting lipid oxidation processes associated with atheroma plaque formation. Previously, we studied their cytotoxicity against human and murine macrophages as first selection for in vivo studies. Herein, we examined both the in vitro mutagenic and DNA-damage effects of selected compounds to further evaluate drug potential. While the compounds of interest were nongenotoxics in both experimental tests (Ames and alkaline comet), one of the potential blood metabolites exhibited genotoxicity (alkaline comet test), and the furazan derivative was mutagenic (Ames test). Two selected (nitrooxy and furoxan) compounds were studied in long- and short-term in vivo treatment, and in these conditions, animal toxicity was not evidenced, suggesting the possibility of these compounds as potential antiatherogenic drugs.     

Folic Acid Targeting of Protein Conjugates into Ascites Tumour Cells from Ovarian Cancer Patients

Abstract

Despite a wealth of in vitro data describing the use of folic acid for drug and DNA delivery into ovarian cancer cell lines, there have been no reports describing the targeting of such compounds to freshly isolated tumour cells. We have carried out a study to determine the usefulness of folic acid as a targeting ligand for ovarian cancer by measuring the uptake of folic acid—BSA-FITC in tumour cells isolated from the ascitic fluid of ovarian cancer patients. In 7 out of 7 patients we have found folic acid mediated uptake of the fluorescently labelled albumin, with the accumulation (average cell fluorescence) and differential uptake (ratio between receptor mediated and fluid phase uptake) varying between patients. Accumulation of folic acid—albumin-FITC occurs in ascites tumour cells expressing the epithelial cell marker EMA, with a significant proportion of EMA negative cells also accumulating the conjugate. There is no correlation between cell cycle and uptake of folic acid-BSA-FITC. These results suggest that folic acid-targeting of therapeutics is a promising approach for the treatment of ovarian cancer.     

Inhibitors of Tubulin Assembly Identified through Screening a Compound Library

Tubulin is the proposed target for drugs against cancer and helminths and is also a validated target in kinetoplastid parasites. With the aim of identifying new lead compounds against Leishmania sp., tubulin isolated from L. tarentolae was used to screen a 10 000 compound library. One compound, Chembridge No. 7992831 ( 5 ), displayed an IC₅₀ of 13 μm against Leishmania tubulin in an in vitro assembly assay and showed a greater than threefold selectivity over mammalian tubulin. Another compound, Chembridge No. 9067250 ( 8 ), exhibited good activity against mammalian tubulin (IC₅₀ = 5.0 μm ). This compound was also toxic to several cancer cell lines with IC₅₀ values in the region of 1 μm . Subsequent testing of analogues of 8 contained within the library identified two compounds with greater potency against mammalian tubulin (IC₅₀ values of 1.1 and 2.8 μm ). The more potent antitubulin agent also showed promising activity against cancer cell lines in vitro, with IC₅₀ values ranging from 0.18 to 0.73 μm .