2,5-Disubstituted-1,3,4-oxadiazoles/thiadiazole as surface recognition moiety: design and synthesis of novel hydroxamic acid based histone deacetylase inhibitors

The enzymatic inhibition of histone deacetylase activity has come out as a novel and effectual means for the treatment of cancer. Two novel series of 2-[5-(4-substitutedphenyl)-[1,3,4]-oxadiazol/thiadiazol-2-ylamino]-pyrimidine-5-carboxylic acid (tetrahydro-pyran-2-yloxy)-amides were designed and synthesized as novel hydroxamic acid based histone deacetylase inhibitors. The antiproliferative activities of the compounds were investigated in vitro using histone deacetylase inhibitory assay and MTT assay. The synthesized compounds were also tested for antitumor activity against Ehrlich ascites carcinoma cells in Swiss albino mice. The efforts were also made to establish structure-activity relationships among synthesized compounds. The results of the present studying indicates 2,5-disubstituted 1,3,4-oxadiazole/thiadiazole as promising surface recognition moiety for development of newer hydroxamic acid based histone deacetylase inhibitor.     

Synthesis and antiproliferative activity of N-substituted 2-amino-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazoles

A number of N-substituted 2-amino-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazoles were synthesized and evaluated for their antiproliferative activities. The panel substitution included alkyl, aryl, and morpholinoalkyl derivatives. The structures of compounds were identified from elemental, IR, (1)H NMR, (13)C NMR and MS spectra analyses. The cytotoxicity in vitro against the four human cell lines: SW707 (rectal), HCV29T (bladder), A549 (lung), and T47D (breast) was determined. Alkyl and morpholinoalkyl derivatives exhibited significantly lower effect than phenyl ones. The highest antiproliferative activity was found for 2-(2,4-dichlorophenylamino)-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole, with ID(50) two times lower (SW707, T47D) than for cisplatin studied comparatively as the control compound.     

1,3,4-Oxadiazole-naphthalene hybrids as potential VEGFR-2 inhibitors: design,synthesis, antiproliferative activity,apoptotic effect,and in silico studies

In the current work, some 1,3,4-oxadiazole-naphthalene hybrids were designed and synthesised as VEGFR-2 inhibitors. The synthesised compounds were evaluated in vitro for their antiproliferative activity against two human cancer cell lines namely, HepG-2 and MCF-7. Compounds that exhibited promising cytotoxicity (5, 8, 15, 16, 17, and 18) were further evaluated for their VEGFR-2 inhibitory activities. Compound 5 showed good antiproliferative activity against both cell lines and inhibitory effect on VEGFR-2. Besides, it induced apoptosis by 22.86% compared to 0.51% in the control (HepG2) cells. This apoptotic effect was supported by a 5.61-fold increase in the level of caspase-3 compared to the control cells. Moreover, it arrested the HepG2 cell growth mostly at the Pre-G1 phase. Several in silico studies were performed including docking, ADMET, and toxicity studies to predict binding mode against VEGFR-2 and to anticipate pharmacokinetic, drug-likeness, and toxicity of the synthesised compounds.     

The activity of a new 2-amino-1,3,4-thiadiazole derivative 4ClABT in cancer and normal cells

The 2-amino-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole set are well known compounds with interesting in vitro and in vivo anti-cancer profiles. The aim of this study was an in vitro evaluation of the anti-cancer activity of a new synthesized aminothiadiazole derivative 2-(3-chlorophenyloamino)-5-(2,4-dihydroxyphenyl)- -1,3,4-thiadiazole 4ClABT. The effect on tumor cell proliferation, motility and morphology, DNA synthesis as well as the influence on normal cells was assessed. The antiproliferative activity of 4ClABT in tumor cells derived from peripheral cancers including breast carcinoma (T47D), colon carcinoma (HT-29), thyroid carcinoma (FTC-238), teratoma (P19), and T-cell leukemia (Jurkat E6.1), as well as cancers of the nervous system including rhabdomyosarcoma/medulloblastoma (TE671), brain astrocytoma (MOGGCCM) and glioma (C6) was studied by means of MTT assay. DNA synthesis level was determined in BrdU ELISA test. Wound assay model was applied for tumor cell motility assessment. Morphological changes induced by 4ClABT in cancer and normal cells were analyzed in HE staining specimens. Moreover, the influence of 4ClABT on normal cells including skin fibroblasts (HSF), hepatocytes (Fao), astroglia and neurons was studied by means of LDH assay. The tested compound inhibited the proliferation of tumor cells in dose-dependent fashion. The anti-cancer effect was attributed to decreased DNA synthesis, prominent changes in tumor cell morphology as well as reduced cell motility. In antiproliferative concentrations, 4ClABT was not toxic to normal cells. Our study showed prominent anti-cancer effects of the tested aminothiadiazole derivative in the absence of toxicity in normal cells. The obtained results confirmed the promising anti-cancer profile of previously tested 2-(monohalogenphenylamino)- -5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole derivatives (ClABT - chlorophenyl derivative, FABT and 3FABT - fluorophenyl derivatives and 4BrABT - bromophenyl derivative). The molecular mechanisms and the in vivo activity of aminothiadiazole derivatives will be the subject of further studies.     

2-Amino-1,3,4-thiadiazole derivative (FABT) inhibits the extracellular signal-regulated kinase pathway and induces cell cycle arrest in human non-small lung carcinoma cells

The anticancer potential of 2-amino-1,3,4-thiadiazole compounds has been documented by in vitro and in vivo studies. In our previous research, we described the synthesis as well as the antiproliferative and neuroprotective activities of 2-(4-fluorophenyloamino)-5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazole (FABT). The aim of the present study was to investigate the molecular mechanisms involved in FABT-induced growth inhibition in A549 lung carcinoma cells. Western blotting analysis revealed that FABT inhibited the activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, and Real-time PCR analysis showed no changes in the expression of P44ERK1 and CREB1 genes. Furthermore, FABT induced cell cycle arrest in the GO/G1 phase and enhanced p27/Kip1 expression. Our results suggest that FABT acts by inhibiting ERK1/2 pathway and cell cycle progression through G1 into S phase in A549 cells. Further studies are needed to completely explain the molecular mechanisms of anticancer action of this 2-aminothiadiazole derivative.     

Improved antiproliferative activity of 1,3,4-thiadiazole-containing histone deacetylase (HDAC) inhibitors by introduction of the heteroaromatic surface recognition motif

A series of 1,3,4-thiadiazole-containing hydroxamic acids, in accord with the common pharmacophore of histone deacetylase (HDAC) inhibitors (a Zn²⁺ binding moiety–a linker–a surface recognition motif), was identified as submicromolar HDAC inhibitors by our group. In this study, we continued our efforts to develop 1,3,4-thiadiazole bearing hydroxamate analogues by modifying the surface recognition motif. We found that 1,3,4-thiadiazoles having a heteroaromatic substituent showed better HDAC inhibitory activity in enzymatic assay and higher antiproliferative potency in cellular assay compared to SAHA.     

Synthesis and antitumor activities of novel hybrid molecules containing 1,3,4-oxadiazole and 1,3,4-thiadiazole bearing Schiff base moiety

A series of novel hybrid molecules containing 1,3,4-oxadiazole and 1,3,4-thiadiazole bearing Schiff base moiety were designed, synthesized and evaluated for their in vitro antitumor activities against SMMC-7721, MCF-7 and A549 human tumor cell lines by CCK-8 assay. The bioassay results demonstrated that most of the tested compounds showed potent antitumor activities, and some compounds exhibited stronger effects than positive control 5-fluorouracil (5-FU) against various cell lines. Among these compounds, compound 8d showed the best inhibitory effect against SMMC-7721 cells, with IC₅₀ value of 2.84 μM. Compounds 8k and 8n displayed highly effective antitumor activities against MCF-7 cells, with IC₅₀ values of 4.56 and 4.25 μM, respectively. Compounds 8a and 8n exhibited significant antiproliferative activity against A549 cells, with IC₅₀ values of 4.11 and 4.13 μM, respectively. The pharmacological results suggest that the substituents of phenyl ring on the 1,3,4-oxadiazole are vital for modulating antiproliferative activities against various tumor cell lines.     

Synthesis, structure, and bioactivity of N'-substituted benzylidene-3,4,5-trimethoxybenzohydrazide and 3-acetyl-2-substituted phenyl-5-(3,4,5-trimethoxyphenyl)-2,3-dihydro-1,3,4-oxadiazole derivatives

Some 3-acetyl-2-substituted phenyl-5-(3,4,5-trimethoxyphenyl)-2,3-dihydro-1,3,4-oxadiazole derivatives were synthesized by cyclization reaction of N'-substituted benzylidene-3,4,5-trimethoxybenzohydrazide in acetic anhydride. Their structures were verified by elemental analysis, IR, (1)H NMR, and (13)C NMR. Compound 3i was provided with X-ray crystallographic data. The compounds were evaluated for their antiproliferative activities against some cancer cells in vitro by MTT method. Among them, 2a, 2b, 2c, 2f, 3l, and 3m were highly effective against PC3 cells and 2a, 2c, and 2f showed moderate activities against Bcap37 and BGC823 cells. The IC(50) values of high active compounds 2a, 2b, 2c, 2f, 3l, and 3m against PC3 cells were 0.2, 1.8, 0.2, 1.2, 1.7, and 0.3muM, respectively.     

Synthesis and antiproliferative activity of imidazo[2,1-b][1,3,4]thiadiazole derivatives

A series of 2,5,6-substituted imidazo[2,1-b][1,3,4]thiadiazole derivatives have been prepared and were tested for antiproliferative activity on cancer cells at the National Cancer Institute. Results showed that molecules with a benzyl group at position 2, exhibited an increase in activity for the introduction of a formyl group at the 5 position. The compound 2-benzyl-5-formyl-6-(4-bromophenyl)imidazo[2,1-b][1,3,4]thiadiazole 22 has been chosen for understanding the mechanism of action by various molecular and cellular biology studies. Results obtained from cell cycle evaluation analysis, analysis of mitochondrial membrane potential and Annexin V-FITC by flow cytometric analysis, ROS production and expression of apoptotic and DNA-repair proteins suggested that compound 22 induced cytotoxicity by activating extrinsic pathway of apoptosis, however, without affecting cell cycle progression.     

Synthesis,antioxidant and antiproliferative activities of 1,3,4-thiadiazoles derived from phenolic acids

Two 2-amino-1,3,4-thiadiazoles containing phenolic hydroxyl groups were combined with different carboxylic acid chlorides giving sixteen amide derivatives with good antioxidant and antiproliferative potential. The compound 3′c with an adamantane ring displayed excellent DPPH radical scavenging activity and good cytotoxic activity against human acute promyelocytic leukemia HL-60 cells, while 1,3,4-thiadiazole 3′h with 4-chlorophenyl moiety was found to be the most effective in inhibition of survival of lung carcinoma A549 cells. All examined thiadiazoles except 3a and 3′a exerted higher cytotoxic activities on A549 and HL-60 cancer cells when compared with normal fibroblasts MRC-5, pointing to selectivity in their antiproliferative action. Some of the most active novel compounds 3c, 3′c, 3′g and 3′h induced significant increase in the percentage of HL-60 cells in the subG1 cell cycle phase in comparison with the control cells. The induction of cell death in HL-60 cells by these compounds was at least partially dependent on activation of caspase-3 and caspase-8. The compounds 3c and 3′c exerted strong antiangiogenic activity. Furthermore, compounds 3c, 3′c, 3′g and 3′h showed the ability to down-regulate the MMP2 and VEGFA expression levels in the treated HL-60 cells when compared with the control cell samples.     

Synthesis and antiproliferative studies of 5-aryl-2-(3-thienylamino)-1,3,4-thiadiazoles

A series of 5-aryl-2-(3-thienylamino)-1,3,4-thiadiazoles 3a–m were synthesized in good yields in two steps starting from thiophen-3-isothiocyanates. Those compounds as well as the thiosemicarbazide intermediates 2a–m were screened for their antiproliferative activity against a panel of six cancer cell lines. Among them, two 5-aryl-2-(3-thienylamino)-1,3,4-thiadiazoles (3f and 3i) have shown very interesting results with IC₅₀ <10 μM on three cell lines.     

Synthesis, biological evaluation and molecular docking studies of 1,3,4-thiadiazole derivatives containing 1,4-benzodioxan as potential antitumor agents

A series of 1,3,4-thiadiazole derivatives containing 1,4-benzodioxan (2a-2s) have been synthesized to screen for FAK inhibitory activity. Compound 2p showed the most potent biological activity against HEPG2 cancer cell line (EC(50)=10.28 μg/mL for HEPG2 and EC(50)=10.79 μM for FAK), which was comparable to the positive control. Docking simulation was performed to position compound 2p into the FAK structure active site to determine the probable binding model. The results of antiproliferative and Western-blot assay demonstrated that compound 2p possessed good antiproliferative activity against HEPG2 cancer cell line. Therefore, compound 2p with potent FAK inhibitory activity may be a potential anticancer agent against HEPG2 cancer cell.     

Novel steroidal 1,3,4-thiadiazines: Synthesis and biological evaluation in androgen receptor-positive prostate cancer 22Rv1 cells

A flexible approach to previously unknown spirofused and linked 1,3,4-thiadiazine derivatives of steroids with selective control of heterocyclization patterns is disclosed. (N-Arylcarbamoyl)spiroandrostene-17,6′ [1,3,4]thiadiazines and (N-arylcarbamoyl)17-[1′,3′,4′]thiadiazine-substituted androstenes, novel types of heterosteroids, were prepared from 16β,17β-epoxypregnenolone and 21-bromopregna-5,16-dien-20-one in good to high yields by the treatment with oxamic acid thiohydrazides. The synthesized compounds were screened for antiproliferative activity against the human androgen receptor-positive prostate cancer cell line 22Rv1. Most of (N-arylcarbamoyl)17-[1′,3′,4′]thiadiazine-substituted androstenes exhibit better antiproliferative potency (IC₅₀ = 2.1–6.6 µM) than the antiandrogen bicalutamide. Compounds 7d with IC₅₀ = 3.0 μM and 7j with IC₅₀ = 2.1 μM proved to be the most active in the series under study. Lead synthesized compound 7j downregulates AR expression and activity in 22Rv1 cells. NF-κB activity is also blocked in 7j-treated 22Rv1 cells. Apoptosis is considered as a possible mechanism of 7j-induced cell death.     

Synthesis, biological evaluation, and molecular docking studies of 1,3,4-thiadiazol-2-amide derivatives as novel anticancer agents

A series of 1,3,4-thiadiazol-2-amide derivatives (5a-5y) have been designed and synthesized, and their biological activities were also evaluated as potential antiproliferation and FAK inhibitors. Among all the compounds, 5h showed the most potent activity in vitro, which inhibited the growth of MCF-7 and B16-F10 cell lines with IC(50) values of 0.45 and 0.31 μM, respectively. Compound 5h also exhibited significant FAK inhibitory activity (IC(50)=5.32 μM). Docking simulation was performed to position compound 5h into the FAK structure active site to determine the probable binding model. The results of antiproliferative and Western-blot assay demonstrated that compound 5h possessed good antiproliferative activity. Therefore, compound 5h with potent FAK inhibitory activity may be a potential anticancer agent.     

A facile access to novel steroidal 17-2′-(1′,3′,4′)-oxadiazoles,and an evaluation of their cytotoxic activities in vitro

Novel types of 17-exo-heterocycles in the Δ⁵ androstene series carrying an 1,3,4-oxadiazole moiety were efficiently synthesized via aldehyde N-acylhydrazone intermediates, obtained from the microwave-assisted condensation of 3β-hydroxy- or 3β-acetoxyandrost-5-ene-17β-carbaldehyde with different acylhydrazides. The subsequent phenyl iodonium diacetate-induced oxidative cyclization proceeded under mild conditions. The synthesized compounds were subjected to in vitro pharmacological studies to investigate their antiproliferative activities on four malignant adherent cell lines (HeLa, MCF7, A2780 and A431), and exhibited the highest potency against HeLa cells, some of them revealing action comparable to that of the reference agent cisplatin.     

Differential antiproliferative activities of IFNs alpha, beta and gamma: kinetics of establishment of their antiproliferative effects and the rapid development of resistance to IFNs alpha and beta

Interferons have been recognized to have potent in vitro antiproliferative activities in mouse and human systems. To further investigate the kinetics of development of interferons' antiproliferative activities, mouse B-16 melanoma cells were treated with MuIFN-alpha, MuIFN-beta or MuIFN-gamma for various initial periods of time during an 8 day cloning assay. With MuIFN-alpha and MuIFN-beta treatments, maximal expression of antiproliferative activity was attained with 2 to 4 days of interferon treatment. In contrast, with MuIFN-gamma treatment, expression of antiproliferative activity increased with progressively longer periods of time of MuIFN-gamma treatment. These results suggested that B-16 melanoma cells were initially sensitive to all three of the interferons but rapidly became resistant to MuIFN-alpha and MuIFN-beta after 2 to 4 days of treatment. This suggestion was confirmed by cell growth kinetics experiments. The cells which were resistant to the antiproliferative activity of the MuIFN-alpha remained sensitive to the antiviral activity of MuIFN-alpha, suggesting that MuIFN-alpha and MuIFN-beta regulate their antiviral and antiproliferative responses via different mechanisms. The cells which were resistant to the antiproliferative activities of MuIFN-alpha and MuIFN-beta remained sensitive to MuIFN-gamma, suggesting that they were not generally resistant to antiproliferative effects. The cells which were resistant to the antiproliferative activities of the interferons gradually lost their resistance with a half-life of 11 days when they were cultured in the absence of interferons. The differential antiproliferative actions of alpha, beta and gamma interferons observed with murine B-16 melanoma were confirmed in the human system with G-361 melanoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural determinants of heparin's growth inhibitory activity. Interdependence of oligosaccharide size and charge

The glycosaminoglycan heparin inhibits the growth of several cell types in vitro including smooth muscle cells and rat cervical epithelial cells. The commercially available heparin which has antiproliferative activity is a structurally heterogeneous polymer that undergoes extensive modifications during maturation. In this report we have performed structure-function studies on heparin's antiproliferative activity using three different cell types: both rat and calf vascular aortic smooth muscle cells and rat cervical epithelial cells. The minimal oligosaccharide size requirements for antiproliferative activity were determined for the three cell types by using oligosaccharide fragments of defined length prepared by nitrous acid cleavage and gel filtration and a synthetic pentasaccharide. The size requirements are similar but not identical for the different cell types. Hexasaccharide fragments are antiproliferative for all three cell types but the synthetic pentasaccharide inhibits the growth of only the rat and calf vascular aortic smooth muscle cells. The interdependence between size and charge for antiproliferative activity was investigated using chemically modified oligosaccharides as well as oligosaccharides prepared from heparin and separated into fractions of differing charge by ion-exchange chromatography. There is a strong interdependence between size and charge for antiproliferative activity. For example, increasing the charge of inactive tetrasaccharide fragments by O-oversulfation makes them antiproliferative whereas reducing the charge of active larger fragments causes them to loose their antiproliferative activity. Finally the importance of 2-O-sulfate glucuronic acid moieties for antiproliferative activity was investigated using heparin preparations that lack 2-O-sulfate glucuronic acid. These compounds possess antiproliferative activity indicating that 2-O-sulfate glucuronic acid is not required for antiproliferative activity.     

Inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate formation in Ca2+-mobilizing-hormone-activated cells.

The inositol trisphosphate liberated on stimulation of guinea-pig hepatocytes, pancreatic acinar cells and dimethyl sulphoxide-differentiated human myelomonocytic HL-60 leukaemia cells is composed of two isomers, the 1,4,5-trisphosphate and the 1,3,4-trisphosphate. Inositol 1,4,5-trisphosphate was released rapidly, with no measurable latency on hormone stimulation, and, consistent with its proposed role as an intracellular messenger for Ca2+ mobilization, there was good temporal correlation between its formation and Ca2+-mediated events in these tissues. There was a definite latency before an increase in the formation of inositol 1,3,4-trisphosphate could be detected. In all of these tissues, however, it formed a substantial proportion of the total inositol trisphosphate by 1 min of stimulation. In guinea-pig hepatocytes, where inositol trisphosphate increases for at least 30 min after hormone application, inositol 1,3,4-trisphosphate made up about 90% of the total inositol trisphosphate by 5-10 min. In pancreatic acinar cells, pretreatment with 20 mM-Li+ caused an increase in hormone-induced inositol trisphosphate accumulation. This increase was accounted for by a rise in inositol 1,3,4-trisphosphate; inositol 1,4,5-trisphosphate was unaffected. This finding is consistent with the observation that Li+ has no effect on Ca2+-mediated responses in these cells. The role, if any, of inositol 1,3,4-trisphosphate in cellular function is unknown.     

Design, synthesis, biological evaluation and molecular modeling of 1,3,4-oxadiazoline analogs of combretastatin-A4 as novel antitubulin agents

A total of 20 novel 1,3,4-oxadiazoline analogs (6a-6t) of combretastatin A-4 with naphthalene ring were designed, synthesized, and evaluated for biological activities as potential tubulin polymerization inhibitors. Among these compounds, 6n showed the most potent antiproliferative activities against multiple cancer cell lines and retained the microtubule disrupting effects. Docking simulation was performed to insert compound 6n into the crystal structure of tubulin to determine the probable binding model. These results indicated oxadiazoline compounds bearing the naphthyl moiety are promising tubulin inhibitors.     

Metabolism of inositol 1,3,4-trisphosphate to a new tetrakisphosphate isomer in angiotensin-stimulated adrenal glomerulosa cells

Angiotensin stimulates rapid and prominent increases in inositol polyphosphates and their metabolites in bovine glomerulosa cells labeled with [3H]inositol. In addition to the early formation of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and inositol 1,3,4-trisphosphate (Ins-1,3,4-P3), as well as their intermediate product, inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4), delayed increases in two new InsP4 isomers were consistently observed by high resolution high performance liquid chromatography. Studies on the metabolism of purified Ins-1,3,4,5-P4 preparations, labeled with [3H]inositol and 32P to monitor sites of dephosphorylation, were performed in permeabilized glomerulosa cells. In addition to rapid degradation of Ins-1,3,4,5-P3 to Ins-1,3,4-P3 and then to Ins-3,4-P2, there was delayed formation of one of the putative InsP4 isomers observed during AII stimulation in intact cells. The kinetics of formation of the new InsP4 isomer, and the lack of phosphate in its 5 position based on isotope ratios, were consistent with its origin from Ins-1,3,4-P3. This was confirmed by the conversion of [3H]Ins-1,3,4-P3 to the new InsP4 isomer in permeabilized cells by a kinase distinct from that which phosphorylates Ins-1,4,5-P3. These results have demonstrated that the dephosphorylation sequence of Ins-1,4,5-P3 metabolism is accompanied by a complex cycle of higher phosphorylations with formation of new intermediates of potential significance in cellular regulation.