Synthesis and Selective Anticancer Activity Evaluation of 2-phenylacrylonitrile Derivatives as Tubulin Inhibitors.

OBJECTIVE: This study aimed at synthesizing 13 series of novel derivatives with 2-phenylacrylonitrile, evaluating antitumor activity both in vivo and in vitro, and obtaining novel tubulin inhibitors. METHOD: The 13 series of 2-phenylacrylonitrile derivatives were synthesized by Knoevenagel condensation and the anti-proliferative activities were determined by MTT assay. The cell cycle and apoptosis were analyzed by flow cytometer. Quantitative cell migration was performed using 24-well Boyden chambers. The proteins were detected by western blotting. in vitro kinetics of microtubule assembly was measured using ELISA kit for Human ß-tubulin (TUBB). Molecular docking was done by Discovery Studio (DS) 2017 Client online tool. RESULTS: Among the derivatives, compound 1g2a possessed strong inhibitory activity against HCT116 (IC50 = 5.9 nM) and BEL-7402 (IC50 = 7.8 nM) cells. Compound 1g2a exhibited better selective antiproliferative activities and specificities than all the positive control drugs, including taxol. Compound 1g2a inhibited proliferation of HCT116 and BEL-7402 cells by arresting them in the G2/M phase of the cell cycle, inhibited the migration of HCT116 and BEL-7402 cells and the formation of cell colonies. Compound 1g2a showed excellent tubulin polymerization inhibitory activity on HCT116 and BEL-7402 cells. The results of molecular docking analyses showed that 1g2a may inhibit tubulin to exert anticancer effects. CONCLUSION: Compound 1g2a shows outstanding antitumor activity both in vivo and in vitro and has the potential to be further developed into a highly effective antitumor agent with little toxicity to normal tissues.

Development of Combretastatin A-4 Analogues as Potential Anticancer Agents with Improved Aqueous Solubility.

Combretastatin A-4 (CA-4) is a potent tubulin polymerisation inhibitor. However, the clinical application of CA-4 is limited owing to its low aqueous solubility and the easy conversion of the olefin double bond from the more active cis- to the less active trans-configuration. Several structural modifications were investigated to improve the solubility of CA-4 derivatives. Among the compounds we synthesized, the kinetic solubility assay revealed that the solubility of compounds containing a piperazine ring increased the most, and the solubility of compounds 12a1, 12a2, 15 and 18 was increased 230-2494 times compared with that of the control compound (Z)-3-(4-aminophenyl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile (9a). In addition, these synthesised stilbene nitriles had high anticancer cell (AGS, BEL-7402, MCF-7, and HCT-116) selectivity over L-02 and MCF-10A normal cells while maintaining micromolar activity against cancer cells. The most cytotoxic compound is 9a, and the IC50 value is 20 nM against HCT-116 cancer cells. Preliminary studies indicated that compound 12a1 had excellent plasma stability and moderate binding to rat plasma proteins, suggesting it is a promising lead compound for the development of an anticancer agent.

Synthesis, and evaluation of in vitro and in vivo anticancer activity of 14-substituted oridonin analogs: A novel and potent cell cycle arrest and apoptosis inducer through the p53-MDM2 pathway.

A series of novel oridonin derivatives bearing various substituents on the 14-OH position were designed and synthesised. Their antitumour activity was evaluated in vitro against three human cancer cell lines (HCT116, BEL7402, and MCF7). Most tested derivatives showed improved anti-proliferative activity compared to the lead compound oridonin and the positive control drug 5-fluorouracil (5-Fu). Among them, compound C7 (IC50 = 0.16 µM) exhibited the most potent anti-proliferative activity against HCT116 cells; it was about 43- and 155-fold more efficacious than that of oridonin (IC50 = 6.84 µM) and 5-Fu (IC50 = 24.80 µM) in HCT116 cancer cells. Interestingly, the IC50 value of compound C7 in L02 normal cells was 23.6-fold higher than that in HCT116 cells; it exhibited better selective anti-proliferative activity and specificity than oridonin and 5-Fu. Furthermore, compound C7 possibly induced cell cycle arrest and apoptosis by regulating the p53-MDM2 signalling pathway. Notably, C7 displayed more significant suppression of tumour growth than oridonin in colon tumour xenograft models where the tumour growth inhibition rate was 85.82%. Therefore, compound C7 could be a potential lead compound for the development of a novel antitumour agent.

Synthesis and characterisation of (Z)-styrylbenzene derivatives as potential selective anticancer agents.

To identify anticancer agents with high potency and low toxicity, a series of (Z)-styrylbenzene derivatives were synthesised and evaluated for anticancer activities using a panel of nine cancer cell lines and two noncancerous cell lines. Most derivatives exhibited significant anti-proliferative activities against five cancer cell lines, including MGC-803 and BEL-7402. (Z)-3-(p-Tolyl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile (6h) showed a strong inhibitory effect on MGC-803 cells (IC50 < 0.01 µM) and exhibited stronger anti-proliferative activity than taxol (IC50 < 0.06 ± 0.01 µM). The IC50 value of 6h in L-02 cells was 10,000-fold higher than in MGC-803 cells. Compound 6h inhibited proliferation of BEL-7402 cells by arresting at the G2/M phase through up-regulation of cyclin B1 expression, down-regulation of cyclin A and D1 expression, and induction of apoptosis. In addition, 6h inhibited the migration of BEL-7402 cells and the formation of cell colonies.

Design and Synthesis of Novel Dehydroepiandrosterone Analogues as Potent Antiproliferative Agents.

The aim of the present study was to determine the cytotoxic effects of a series of novel dehydroepiandrosterone derivatives containing triazole at the C16 position on human cancer cells. The cancer cells used in the present study were A549, Hela, HepG-2, BEL7402, MCF-7, and HCT116. Several of the synthesised compounds exhibited potent antiproliferative effects. The most promising compound was (E)-3-hydroxy-16-((1-(4-iodophenyl)-1H-1,2,3-triazole-4-yl)methylene)-10,13-dimet-hyl-1,3,4,7,8,9,10,11,12,13,15,16-dodecahydro-2H-cyclopenta[a]phenanthren-17(14)-one (compound 2n), which showed considerably high antiproliferative activity in the HepG-2 cell line, with an IC50 value of 9.10 µM, and considerably high activity against the MCF-7 cell line, with an IC50 value of 9.18 µM. Flow cytometry assays demonstrated that compound 2n exerted antiproliferative effects by arresting cells in the G2 phase of the cell cycle and inducing apoptosis.

Synthesis and pharmacological evaluation of 2,3-diphenyl acrylonitriles-bearing halogen as selective anticancer agents.

Eighteen novel 2,3-diphenyl acrylonitrile derivatives bearing halogens were designed, synthesized, and evaluated for biological activity. Preliminary in vitro results indicated that the majority of the compounds with a para-substituted halogen had considerable antiproliferative activity against five human cancer cell lines, including MGC-803, AGS, and BEL-7402, with IC50 values in the range of 0.46-100 µm. No significant toxic effects on the non-cancerous human liver cell line L-02 were observed. The selective inhibitory activities against cancer cells were significantly better than that of the control lead compound CA-4 and CA-4P. Particularly, potent activities were found for the derivatives of 3-(4-halogen phenyl)-2-(3,4,5-trimethoxyphenyl)acrylonitrile, such as 5c (4-fluoro), 5f (4-bromo), 5h (4-chloro), and 5k (4-trifluoro- methyl), for AGS with IC50 values of 0.75 ± 0.24, 0.68 ± 0.21, 0.41 ± 0.05, and 1.49 ± 0.92 µm, respectively. The antiproliferative effects of 5f were attributed to cell-cycle arrest in the G2 /M phase, induction of cellular apoptosis, suppression of cell migration, and inhibition of cell colony formation in AGS cells.

Design and Synthesis of C-19 Isosteviol Derivatives as Potent and Highly Selective Antiproliferative Agents.

Six series of novel isosteviol derivatives; modified in the C-19 position; were synthesized; and their antiproliferative activity was evaluated against three human cancer cell lines (HCT-116; BEL-7402; HepG2) and the human L02 normal cell line in vitro. Most of the derivatives tested here exhibited improved antiproliferative activity with high selectivity when compared with the parent compound isosteviol and the positive control drug 5-fluorouracil. Among these derivatives; compound 5d exhibited the most potent antiproliferative activity and commendable selectivity between cancer and normal cells. In addition; compound 5d inhibited the colony formation of HCT-116 cells in a concentration-dependent manner. Further studies revealed that compound 5d arrested the HCT-116 cell cycle in the S phase; and western blot analysis demonstrated the mechanism may be correlated with a change in the expression of cyclin A; cyclin B1; and cyclin E1. Furthermore; the results of a docking study that involved placing compound 5d into the CDK2/cyclin A binding site revealed that its mode of action was possibly as a CDK2/cyclin A inhibitor.

Design and synthesis of new triazoles linked to xanthotoxin for potent and highly selective anti-gastric cancer agents.

Two series of xanthotoxin-triazole derivatives were designed, synthesized, and studied for their antiproliferative properties. The in vitro cytotoxicity of the compounds in the AGS cancer cell line and the L02 normal cell line was evaluated via MTT assay. Among the synthesized compounds, 9-((1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)methoxy)-7H-furo[3,2-g]chromen-7-one (6p) was found to have the greatest antiproliferative activity against AGS cells (IC50=7.5µM) and showed better activity than the lead compound (xanthotoxin, IC50>100µM) and the reference drug (5-fluorouracil, IC50=29.6µM) did. The IC50 value of 6p in L02 cells was 13.3 times higher than that in the AGS cells. Therefore, the compound exhibited better therapeutic activity and specificity compared with the positive control 5-fluorouracil. Cell cycle analysis revealed that compound 6p inhibited cell growth via the induction of S/G2 phase arrest in AGS cells. Compound 6p was identified as a promising lead compound for the further development and identification of 1,2,3-triazole-based anticancer agents.

Synthesis and biological evaluation of novel 7-hydroxy-4-phenylchromen-2-one-linked to triazole moieties as potent cytotoxic agents.

A new series of novel 7-hydroxy-4-phenylchromen-2-one (1a)-linked 1,2,4-triazoles were synthesised using a click chemistry approach. All derivatives were subjected to 3-(4,5-dimethylthiazol-yl)-diphenyl tetrazolium bromide (MTT) cytotoxicity screening against a panel of six different human cancer cell lines (AGS, MGC-803, HCT-116, A-549, HepG2, and HeLa) to assess their cytotoxic potential. Among the tested molecules, some of the analogues showed better cytotoxic activity than that shown by the 7-hydroxy-4-phenylchromen-2-one (1a). Of the synthesised 1,2,4-triazoles,the 7-((4-(4-Chlorophenyl)-4H-1,2,4-triazol-3-yl)methoxy)-4-phenyl-2H-chromen-2-one (4d) showed the best activity, with an IC50 of 2.63 ± 0.17 µM against AGS cells. Further flow cytometry assays demonstrated that compound 4d exerts its antiproliferative effects by arresting cells in the G2/M phase of the cell cycle and by inducing apoptosis. Collectively, our results indicate that the 1,2,4-triazole derivatives have a significantly stronger antitumour activity than 1,2,3-triazole derivatives. Most of the compounds exhibited better antitumour activity than the positive control drug 5-fluorouracil.

Pharmacological evaluation of 9,10-dihydrochromeno[8,7-e][1,3]oxazin-2(8H)-one derivatives as potent anti-inflammatory agent.

BACKGROUND: Non-steroidal anti-inflammatory drugs (NSAIDs) are the most widely administered drugs for the treatment of inflammation. However, they usually cause some unexpected side effects. Coumarins and their derivatives exhibit broad-spectrum biological activities. In order to develop new anti-inflammatory drugs with high anti-inflammatory activity and less side effects, a series of 9-substituted-9,10-dihydrochromeno[8,7-e][1,3]oxazin-2(8H)-one derivatives were designed, synthesized, and screened for their anti-inflammatory activities. METHODS: We investigated the effect of compound 9-(2-chlorophenyl)-9,10-dihydrochromeno[8,7-e][1,3]oxazin-2(8H)-one (B3) on lipopolysaccharide (LPS)-induced cytokine levels in RAW 264.7 cells at concentrations between 6.25µg/ml and 25µg/ml. Concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured by enzyme-linked immunosorbent assay (ELISA). Moreover, mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) activation was investigated by western blot assay. RESULTS: Compound B3 could inhibit inflammatory responses via suppression of the NF-κB and MAPK signaling pathways. Docking study of the prepared compounds was performed for the study of interaction of molecules with the active site of TNF-α. CONCLUSION: 9,10-Dihydrochromeno[8,7-e][1,3]oxazin-2(8H)-one derivatives showed anti-inflammatory activity. Compound B3 was the most potent. The results of this study are encouraging further investigations to develop compound B3 as a novel therapeutic agent for inflammatory disorders.

Synthesis and bioactivity evaluation of 2,3-diaryl acrylonitrile derivatives as potential anticancer agents.

Thirty novel derivatives of 2,3-diaryl acrylonitrile were synthesized and evaluated for biological activity. Preliminary investigations of antitumor activity in vitro showed that most of the synthesized compounds have significant antiproliferative effects on human cancer cell lines, such as BEL-7402, HeLa, and HCT116 with IC50 values in the range of 0.13-60.23µM without significant toxic effects on the non-cancerous human liver cell line L-02. In particular, compounds 4d and 4p were found to be the most potent against HeLa (4.20µM) and HCT116 cells (0.13µM), respectively, with superior or similar in vitro efficacy to that of the broad-spectrum anticancer drug taxol.

Antiphotoaging effects of light-emitting diode irradiation on narrow-band ultraviolet B-exposed cultured human skin cells.

BACKGROUND: Antiaging effects of light-emitting diodes (LEDs) have been clinically demonstrated using one or two wavelengths. The mechanism is unclear. OBJECTIVE: To examine direct and indirect photobiomodulation effects of LEDs on narrow-band ultraviolet B (NB-UVB)-induced photoaging using seven different wavelengths alone or in combination. MATERIALS AND METHODS: Four LED wavelengths were chosen based on type I collagen and metalloproteinase (MMP)-1 expression. NB-UVB-irradiated fibroblasts or keratinocytes were irradiated using these four wavelengths. The expression of collagen and MMP-1 in fibroblasts with or without conditioned medium from LED-irradiated keratinocytes and the expression of proinflammatory cytokines in the LED-irradiated keratinocytes were examined. RESULTS: Irradiation with four wavelengths (630, 660, 830, and 850 nm) significantly increased the number of viable fibroblasts. These four wavelengths also increased type I collagen expression, particularly four combinations (630/830, 660/850, 630/850, and 660/830 nm). The fibroblasts cultured with the keratinocyte conditioned medium, particularly with a combination of 630/850 or 660/830 nm, increased collagen levels. Low tumor necrosis factor alpha (TNF-α) and high transforming growth factor beta 1 (TGF-ß1) expression was detected in the LED-irradiated keratinocytes. CONCLUSION: The combination of 630/850- or 660/830-nm irradiation led to better direct and indirect antiphotoaging outcomes than single LED wavelengths in NB-UVB-irradiated cultured normal human skin cells.

Light-emitting diodes at 830 and 850 nm inhibit melanin synthesis in vitro.

Treatment of hyperpigmentation remains a challenge. Because of the positive effects of low-energy Nd:YAG lasers on the treatment of melasma, it is suggested that laser-like light-emitting diodes (LEDs) can potentially ameliorate hyperpigmentation. We evaluated the effect of seven different LED wavelengths on melanogenesis. LED irradiation at 830 nm (dose-dependent, from 1 to 20 J/cm2) and 850 nm (1 J/cm2) significantly reduced melanin production and tyrosinase expression, not only in a normal human melanocyte monoculture both with and without forskolin stimulation but also in a three-dimensional multiple cell type culture. It reduced melanin content via inactivation of the apoptosis signal-regulating kinase and extracellular signal-regulated kinase 1/2 pathways. The level of phosphorylated cyclic AMP response element-binding protein was also decreased by LED irradiation. Moreover, LED irradiation reduced melanogenesis through decreased expression of tyrosinase family genes (tyrosinase-related protein-1 and 2, and microphthalmia-associated transcription factor). These results indicate that LEDs could potentially be used to treat melanin-overproducing skin conditions.

A two-photon turn-on probe for glucose uptake.

We report a two-photon turn-on probe (AS1) that can be excited by 780 nm femto-second pulses and visualize glucose uptake and the changes in the intracellular glucose concentration in live cells and tissue by two-photon microscopy.

Two-photon probes for Zn2+ ions with various dissociation constants. Detection of Zn2+ ions in live cells and tissues by two-photon microscopy.

A series of Zn(2+)-selective two-photon fluorescent probes (AZnM1-AZnN) that had a wide range of dissociation constants (K(d) (TP) =8 nm-12 µM) were synthesized. These probes showed appreciable water solubility (>3 µM), cell permeability, high photostability, pH insensitivity at pH>7, significant two-photon action cross-sections (86-110 GM) upon complexation with Zn(2+), and can detect the Zn(2+) ions in HeLa cells and in living tissue slices of rat hippocampal at a depth of >80 µm without mistargeting and photobleaching problems. These probes can potentially find application in the detection of various amounts of Zn(2+) ions in live cells and intact tissues.

Mechanisms by which the inhibition of specific intracellular signaling pathways increase osteoblast proliferation on apatite surfaces.

Osteoblasts proliferate slowly on the surface of calcium phosphate apatite which is widely used as a substrate biomaterial in bone regeneration. Owing to poor adhesion signaling in the cells grown on the calcium phosphate surface, inadequate growth factor signaling is generated to trigger cell cycle progression. The present study investigated an intracellular signal transduction pathway involved in the slow cell proliferation in osteoblasts grown on the calcium phosphate surface. Small GTPase RhoA and phosphatase and tensin homolog (PTEN) were more activated in cells grown on the surface of calcium phosphate apatite than on tissue culture plate. Specific inhibition of RhoA and PTEN induced the cells on calcium phosphate apatite surface to proliferate at a similar rate as cells on tissue culture plate surface. Specific inhibition of ROCK, which is a downstream effector of RhoA and an upstream activator of PTEN also increased proliferation of these osteoblasts. Present results indicate that physical property of calcium phosphate crystals that impede cell proliferation may be surmounted by the inhibition of the RhoA/ROCK/PTEN pathway to rescue delayed proliferation of osteoblasts on the calcium phosphate apatite surface. In addition, specific inhibition of ROCK promoted cell migration and osteoblast differentiation. Inhibition of the RhoA/ROCK/PTEN intracellular signaling pathway is expected to enhance cell activity to promote and accelerate bone regeneration on the calcium phosphate apatite surface.

Hyperosmotic Stimulus Down-regulates 1alpha, 25-dihydroxyvitamin D(3)-induced Osteoclastogenesis by Suppressing the RANKL Expression in a Co-culture System.

The hyperosmotic stimulus is regarded as a mechanical factor for bone remodeling. However, whether the hyperosmotic stimulus affects 1alpha, 25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3))-induced osteoclastogenesis is not clear. In the present study, the effect of the hyperosmotic stimulus on 1alpha,25(OH)(2)D(3)-induced osteoclastogenesis was investigated in an osteoblast-preosteoclast co-culture system. Serial doses of sucrose were applied as a mechanical force. These hyperosmotic stimuli significantly evoked a reduced number of 1alpha,25(OH)(2)D(3)-induced tartrate-resistant acid phosphatase-positive multinucleated cells and 1alpha,25(OH)(2)D(3)-induced bone-resorbing pit area in a co-culture system. In osteoblastic cells, receptor activator of nuclear factor kappaB ligand (RANKL) and Runx2 expressions were down-regulated in response to 1alpha,25(OH)(2)D(3). Knockdown of Runx2 inhibited 1alpha,25(OH)(2)D(3)-induced RANKL expression in osteoblastic cells. Finally, the hyperosmotic stimulus induced the overexpression of TonEBP in osteoblastic cells. These results suggest that hyperosmolarity leads to the down-regulation of 1alpha,25(OH)(2)D(3)-induced osteoclastogenesis, suppressing Runx2 and RANKL expression due to the TonEBP overexpression in osteoblastic cells.

Detection of mercury in fish organs with a two-photon fluorescent probe.

We report a two-photon fluorescent probe (AHg1) that can be excited by 780 nm femto-second pulses, shows high photostability and negligible toxicity, and can visualize the site of Hg(2+) accumulation, but can also estimate trace amounts of mercury ions in fresh fish organs by two-photon microscopy.

A two-photon fluorescent probe for thiols in live cells and tissues.

We report a two-photon fluorescent probe (ASS) that can be excited by 780 nm femtosecond pulses and detect thiols in live cells and living tissues at a 90-180 microm depth without interference from other biologically relevant species by two-photon microscopy.