Design, synthesis and molecular docking study of new pyrimidine-based hydrazones with selective anti-proliferative activity against MCF-7 and MDA-MB-231 human breast cancer cell lines.

Efforts were directed on the design, synthesis and evaluation of the anticancer activity of some pyrimidine-based hydrazones against two breast cancer cell lines, MCF-7 and MDA-MB-231. Preliminary screening results revealed that some candidates scrutinized for their antiproliferative activities exhibited IC50 values of 0.87 µM-12.91 µM in MCF-7 and 1.75 µM-9.46 µM in MDA-MB-231 cells, indicating almost equal activities on both cell lines and better growth inhibition activities than those of the positive control 5-fluorouracil (5-FU) which displayed IC50 values of 17.02 µM and 11.73 µM respectively. Selectivity of the significantly active compounds was estimated against MCF-10A normal breast cells when compounds 7c, 8b, 9a and 10b exhibited superior activity for cancerous cells than for normal cells when compound 10b presented the best selectivity Index (SI) with respect to both MCF-7 and MDA-MB-231 cancer cells in comparison to the reference drug 5-FU. Mechanisms of their actions were explored by inspecting activation of caspase-9, annexin V staining and cell cycle analysis. It was noticed that compounds 7c, 8b, 8c 9a-c and 10b produced an increase in caspase-9 levels in MCF-7 treated cells with 10b inducing the highest elevation (27.13 ± 0.54 ng/mL) attaining 8.26-fold when compared to control MCF-7 which was higher than that of staurosporine (19.011 ± 0.40 ng/mL). The same compounds boosted caspase-9 levels in MDA-MB-231 treated cells when an increase in caspase-9 concentration reaching 20.40 ± 0.46 ng/mL (4.11-fold increase) was observed for compound 9a. We also investigated the role of these compounds for their increasing apoptosis ability against the 2 cell lines. Compounds 7c, 8b and 10b tested on MCF-7 cells displayed pre-G1 apoptosis and arrested cell cycle in particular at the S and G1 phases. Further clarification of their effects was made by modulating their related activities as inhibitors of ARO and EGFR enzymes when 8c and 9b showed 52.4% and 58.9% inhibition activity relative to letrozole respectively and 9b and 10b showed 36% and 39% inhibition activity of erlotinib. Also, the inhibition activity was verified by docking into the chosen enzymes.

Benzoxazole-appended piperidine derivatives as novel anticancer candidates against breast cancer.

Novel series of benzoxazole-appended piperidine derivatives were planned, synthesized and screened against two breast cancer cell lines. Considerable antiproliferative activity was observed for screened compounds (IC50 = 33.32 ± 0.2 µM to 7.31 ± 0.43 µM and 1.66 ± 0.08 µM to 12.10 ± 0.57 µM) against MCF-7 and MDA-MB-231 cell lines respectively being more potent than doxorubicin (IC50 = 8.20 ± 0.39 µM and 13.34 ± 0.63 µM respectively). Active compounds were submitted for enzyme inhibition assays when 4d and 7h demonstrated potent EGFR inhibition (0.08 ± 0.002 µM and 0.09 ± 0.002 µM respectively) compared to erlotinib (0.11 ± 0.003 µM). However, no one compound displayed effective ARO inhibition activity as tested compounds were less active than letrozole. Apoptosis inducing ability results implied that apoptosis was provoked by significant stimulation of caspase-9 protein levels (4.25-7.04-fold) upon treatment of MCF-7 cells with 4a, 7h, 9, 12e and 12f. Alternatively, MDA-MB-231 cells treated with 4d, 7a, 12b and 12c considerably increased caspase-9 levels (2.32-4.06-fold). Cell cycle arrest and annexin-V/Propidium iodide assays further confirmed apoptosis when tested compounds arrested cell cycle at various phases and demonstrated high annexin V binding affinity. Docking outcomes proved valuable binding affinities for compounds 4d and 7h to EGFR enzyme while compounds 4a and 12e, upon docking into the active site of ARO, failed to interact with heme, suggesting their inabilities to act as AIs. Therefore, these benzoxazoles can act as promising candidates exhibiting EGFR inhibition and apoptosis-promoting properties.

New functionalized 6-thienylpyrimidine-5-carbonitriles as antiproliferative agents against human breast cancer cells.

6-Thienylpyrimidine-5-carbonitrile derivatives were synthesized and screened for their in vitro antiproliferative activities against two human breast cancer cell lines in comparison to 5-fluorouracil as a reference. Compounds 2, 3a-c, and 6b evolved as the most active congeners against both cell lines, while others showed selectivity for only one cell line. Compound 2 exerted its effect through inhibition of the epidermal growth factor receptor (EGFR), while 6b showed less aromatase inhibitory activity than letrozole. The rest of the tested compounds did not show significant inhibition, and it can be assumed that they exert their antiproliferative activity through different target mechanisms. In addition, caspase-9 protein activation assays, cell cycle analysis using flow cytometry, and annexin V-fluorescein isothiocyanate-propidium iodide (FITC/PI) dual staining assays were performed for the most active compounds. All the tested compounds were found to be potent pyrimidine derivatives able to initiate apoptosis in MCF-7 and MDA-MB-231 cells.

New thieno[3,2-d]pyrimidine-based derivatives: Design, synthesis and biological evaluation as antiproliferative agents, EGFR and ARO inhibitors inducing apoptosis in breast cancer cells.

An array of newly synthesized thieno[3,2-d]pyrimidine-based derivatives and thienotriazolopyrimidines hybridized with some pharmacophoric anticancer fragments were designed, synthesized and assessed for their in vitro antiproliferative activity against MCF-7 and MDA-MB-231 breast cancer cell lines using erlotinib and pictilisib as reference standards in the MTT assay. In general, many compounds were endowed with considerable antiproliferative activity (IC50 = 0.43-1.31 µM). Some of the tested compounds, namely 3c, 5b, 5c, 9d, 10, 11b and 13 displayed remarkable antiproliferative activity against both cell lines. Meanwhile, compounds 2c-e, 3b, 4a, 5a, 9c and 15b showed noticeable selectivity against MCF-7 cells while compounds 2b, 3a, 4b, 6a-c, 7, 8, 9b and 12 exhibited considerable selectivity against MDA-MB-231 cells. Further mechanistic evidences for their anticancer activities were provided by screening the most potent compounds against MCF-7 and/or MDA-MB-231 cells for EGFR and ARO inhibitory activities using erlotinib and letrozole as reference standards respectively. Results proved that, in general, tested compounds were better EGFRIs than ARIs. In addition, significant overexpression in caspase-9 level in treated MCF-7 breast cell line samples was observed for all tested compounds with the 4-fluorophenylhydrazone derivative 2d exhibiting the highest activation. In treated MDA-MB-231 breast cell line samples, 11b was found to highly induce caspase-9 level thereby inducing apoptosis. Cell cycle analysis and Annexin V-FITC/PI assay were also assessed for active compounds where results indicated that all tested compounds induced preG1 apoptosis and cell cycle arrest at G2/M phase. Compound 9d, as an inhibitor of ARO, was observed to downregulate the downstream signaling proteins HSP27 and p-ERK in MCF-7 cells. Furthermore, compound 11b downregulated EGFR expression as well as the downstream signaling protein p-AKT. Docking experiments on EGFR and ARO enzymes supported their in vitro results. Thus, the thienotriazolopyrimidines 11b and 12 showing good EGFR inhibition and the thieno[3,2-d]-pyrimidine derivatives 3b and 9d, eliciting the best ARO inhibition activity, can be considered as new candidates as anti-breast cancer agents that necessitate further development.

Antiproliferative activity, enzymatic inhibition and apoptosis-promoting effects of benzoxazole-based hybrids on human breast cancer cells.

New benzoxazole derivatives containing 1,3,4-oxadiazole, 1,2,4-triazole or triazolothiadiazine rings were synthesized and screened for their in vitro antiproliferative activities against MCF-7 and MDA-MB-231 breast cancer cell lines using MTT assay. Doxorubicin, cisplatin and 2-(4-aminophenyl)benzothiazole (CJM 126) were used as references. The most active compounds 7a, 8d, 8e and 10c were screened for their antiproliferative activities against MCF-10A normal breast cells where compounds 8e and 7a were the most selective towards MCF-7 and MDA-MB-231 cell lines, respectively compared to CJM 126. In vitro enzymatic inhibition assays of epidermal growth factor receptor (EGFR) and aromatase (ARO) enzymes were performed. Compound 7a showed inhibition of EGFR comparable to that of erlotinib while compound 8e exhibited nearly half the inhibitory activity of erlotinib towards EGFR and was more potent inhibitor of ARO than letrozole. Caspase-9 activation assay, cell cycle analysis and Annexin-V/ Propidium iodide assay performed for compounds 7a, 8d, 8e and 10c demonstrated over expression of caspase-9 protein level, pre G1 apoptosis and high annexin V binding affinity. Therefore, these compounds are considered as potent apoptosis-promoting agents. The predicted docking studies and in silico chemo-informatic properties of compounds 7a and 8e were appropriate. Compounds 7a and 8e are promising anti-breast cancer agents exhibiting potent apoptosis-promoting properties.

Antiestrogenic Activity and Possible Mode of Action of Certain New Nonsteroidal Coumarin-4-acetamides.

The preparation of certain 2-(2-oxo-2H-chromen-4-yl)-N-substituted acetamides IIIa-h was planned as a step in the development of new modified nonsteroidal antiestrogens. The purity of target compounds IIIa-h was checked by thin-layer chromatography (TLC), and their structures were confirmed using various spectroscopic tools including IR, 1H-NMR, 13C-NMR, and MS spectroscopy. Viability tests were applied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay to evaluate the cytotoxic effect of the synthesized compounds against two breast cancer cell lines, MCF-7 and MDA-MB-231. Compound IIIb proved the most active against MCF-7 cells, with an IC50 value of 0.32 µM. The results of an analysis of in vitro antiestrogenic activity indicated that only compound IIIb exhibited antiestrogenic activity; its IC50 value of 29.49 µM was about twice as potent as that of the reference compound, MIBP. The aromatase activity was evaluated for the synthesized target compounds IIIa-g and the intermediates Ib and IIa. A significant aromatase inhibition was observed for the intermediate Ib and compound IIIe, with IC50 values of 14.5 and 17.4 µM, respectively. Compound IIIb, namely 7-methoxy-4-(2-oxo-2-(piperidin-1-yl)ethyl)-2H-chromen-2-one, could be used as an antiestrogen and/or cytotoxic agent with selective activity against tumor cells.

2-Anilinopyrimidine derivatives: Design, synthesis, in vitro anti-proliferative activity, EGFR and ARO inhibitory activity, cell cycle analysis and molecular docking study.

Novel anti-proliferative agents possessing pyrimidine scaffolds were designed, synthesized and evaluated for their IC50 values using MTT assay. Most compounds displayed good to excellent activity against the two tested breast cancer lines (MCF-7 and MDA-MB-231) as compared to 5-FU. The observed IC50 values for active compounds ranged from 0.27 to 10.57 µM in MCF-7 compared to the reference drug 5-FU (IC50 = 10.80 µM) and from 0.73 to 29.07 µM in MDA-MB-231 (IC50 for 5-FU = 11.40 µM). SAR analysis indicated that compounds 2c, 3b with hydrazone functionalities and compound 12 possessing pyrazolone ring exhibited superior activities. The most promising compounds were evaluated for their inhibitory activity against epidermal growth factor receptor (EGFR) and aromatase (ARO) enzymes and were further tested for caspase-9 activation, apoptosis and Annexin V/PI staining. Results of enzyme-based experiments indicated that the tested compounds 2c and 12 exert their activities through EGFR inhibition while compound 3b exhibited remarkable ARO inhibition activity. Furthermore, they remarkably induce caspase-9 activation and showed pre G1 apoptosis and cell cycle arrest at G2/M phase. In addition, docked compounds displayed good binding affinities to the target enzymes. Binding interaction details for the most promising inhibitors with the active site of the target enzymes EGFR and ARO utilizing MOE-dock method are also reported.

Benzoxazole derivatives as new generation of anti-breast cancer agents.

New 2-substituted benzoxazole derivatives were synthesized and screened for their in vitro anti-proliferative activities against MCF-7 and MDA-MB-231 cell lines. Compounds 4b, 4d and 11c eliciting the highest activity against MCF-7 cells were further assayed for their cytotoxic activities against A431 and HCC827 cancer cells in addition to their in vitro inhibition of wild and mutated epidermal growth factor receptor (EGFR) enzymes. Compound 11c was the most active against A431 cells and it displayed a potent inhibition of EGFRWT while compounds 4b and 4d elicited higher potencies than erlotinib against mutated EGFRL858R. Compounds 4a, 6c and 8a showed the most potent cytotoxic activity against MDA-MB-231 cancer cells where compounds 4a and 6c were slightly less potent aromatase (ARO) inhibitors than letrozole. MCF-7 cells treated with compounds 4b, 4d, 11c and MDA-MB-231 cells treated with compounds 4a, 6c and 8a showed remarkable over-expression of caspase-9 protein level and elicited pre G1 apoptosis and cell cycle arrest at G2/M phase in addition to high annexin V binding affinity indicating significant apoptosis. Chemo-informatic and docking properties were also predicted. Docking results revealed that docked compounds displayed binding modes with EGFR and ARO enzymes comparable to that of the reference ligands. The benzoxazole derivatives 11c and 6c possessing amide and dithiocarbamate moieties respectively were found to be potent apoptosis-inducing anti-breast cancer agents with acceptable physicochemical properties. They exert their activity via inhibition of EGFR and ARO enzymes respectively.

Pyrazoles containing thiophene, thienopyrimidine and thienotriazolopyrimidine as COX-2 selective inhibitors: Design, synthesis, in vivo anti-inflammatory activity, docking and in silico chemo-informatic studies.

New thiophene and annulated thiophene pyrazole hybrids were synthesized and screened for their in vitro COX-1/COX-2 enzymatic inhibition and in vivo anti-inflammatory activities. All compounds were more COX-2 selective inhibitors than COX-1 with compound 13 exhibiting the highest COX-2 selectivity index. Compounds 3, 6a, 9 and 11 were the most promising in the acute anti-inflammatory assay while compounds 3, 5, 6a, 6c, 9, 10, 11 and 13 exerted promising anti-inflammatory activity in the sub-acute anti-inflammatory assay. Compounds 3, 6a, 6c, 9, 10 and 11 were evaluated for their ED50 values and were more potent than diclofenac sodium while compounds 6a, 6c and 9 were of greater potency than celecoxib with compound 6a being the most potent showing ED50 = 0.033 mmol/kg. These compounds were non-toxic and proved to be gastrointestinal safe compared to indomethacin, diclofenac sodium and celecoxib. Docking studies into COX-2 active site (PDB code 3LN1) revealed that compounds 3, 6a, 6c, 9, 10, 11 and 13 had binding modes and energies comparable to that of celecoxib. Compounds 3, 9, 10 and 11 complied with Lipinski's RO5 while compounds 6a and 6c showed one violation whereas compound 13 deviated by 2 violations. Compounds 6a, 6c and 13 showed 100% plasma protein binding (PPB) and showed no aqueous solubility while compounds 3, 10 and 11 demonstrated the best drug likeness model scores. Therefore, the thiophene analog 3 and the thienopyrimidine derivatives 10 and 11 are promising anti-inflammatory candidates that exert moderate selective COX-2 inhibition with acceptable physicochemical properties.

Design and synthesis of some ß-carboline derivatives as multi-target anticancer agents.

AIM: Some anticancer ß-carbolines exhibited dual inhibition of topo-I and KSP. METHODOLOGY/RESULTS: Novel ß-carbolines were synthesized and screened for their anticancer activity according to the NCI protocol. Five dose assays results indicated that compounds 9, 10, 12, 17 and 20 were potent and non selective anticancer agents; the sulfanyltriazole 12 was the most potent. Compounds 10, 12 and 20 showed dual topo-I and KSP inhibition with compound 12 being the most potent. Active compounds elicited Pre-G1 apoptosis and cell cycle arrest at G2/M phase of melanoma MDA-MB-435 cells. Docking results, in silico physicochemical and absorption, distribution, metabolism, excretion (ADME) properties were appropriate. CONCLUSION: Compounds 10, 12 and 20 are potent apoptosis-inducing multitarget anticancer agents that act via dual inhibition of topo-I and KSP-ATPase.

Design, synthesis, anticancer screening, docking studies and in silico ADME prediction of some ß-carboline derivatives.

BACKGROUND: Medicinal interest has focused on ß-carbolines as anticancer agents. METHODOLOGY/RESULTS: Several ß-carbolines were designed, synthesized and evaluated for their cytotoxic activity against MCF-7 and A-549 cancer cell lines using MTT assay. Compounds 13a, 13c, 13d and 20a were the most promising showing high selectivity indices. Compounds 13c and 20a showed potent inhibition of topoisomerase (topo-I) and kinesin spindle protein (KSP/Eg5 ATPase) which was confirmed by their docking results into the active site of both enzymes. In silico physicochemical calculations predicted that compounds 13a, 13d and 20a obeyed Lipinski's rule of five. CONCLUSION: Compounds 13c and 20a are multitarget anticancer leads that act as potent inhibitors for both topo-I and/or KSP ATPase.

Synthesis and biological evaluation of novel N3-substituted dihydropyrimidine derivatives as T-type calcium channel blockers and their efficacy as analgesics in mouse models of inflammatory pain.

Low-voltage-activated calcium channels are important regulators of neurotransmission and membrane ion conductance. A plethora of intracellular events rely on their modulation. Accordingly, they are implicated in many disorders including epilepsy, Parkinson's disease, pain and other neurological diseases. Among different subfamilies, T-type calcium channels, and in particular the CaV3.2 isoform, were shown to be involved in nociceptive neurotransmission. The role of CaV3.2 in pain modulation was supported by demonstrating selective antisense oligonucleotide-mediated CaV3.2 knockdown, in vivo antinociceptive effects of T-type blockers, and pain attenuation in CaV3.2 knockout formalin-induced pain model. These Emerging investigations have provided new insights into targeting T-type calcium channels for pain management. Within this scope, various T-type calcium channel blockers have been developed such as mibefradil and ethosuximide. Although being active, most of these molecules interact with other receptors as well. This addresses the need for T-selectivity. Few selective T-type channel blockers of diverse chemical classes were developed such as ABT-639 and TTA-P2. Interestingly, R(-) efonidipine which is a dihydropyridine (DHP) showed T-channel selectivity. Systematic modification of 1,4-dihydropyridine scaffold introduced novel derivatives with 40-fold T-type selectivity over L-type calcium channels. Along these lines, substitution of the DHP core with various analogues favored T-selectivity and may serve as novel pharmacophores. Several dihydropyrimidine (DHPM) mimics were introduced by Squibb as potential candidates. As a continuation of this approach, the current study describes the synthesis of Novel N3 substituted DHPMs with structure similarities to the active DHPs. Different functional groups were introduced to the N3 position through a spacer to gain more information about activity and selectivity. Furthermore, the spacer aims at improving the metabolic stability of the molecules. Initial screening data by whole patch clamp technique showed a robust inhibition of Cav3.2 T-type channels by eleven compounds. Interestingly, four compounds of these were efficient selective T-type blockers. Based on selectivity and efficiency, two compounds were selected for in vivo evaluation in mouse models of inflammatory pain. Results showed effective attenuation of nociception and mechanical hypersensitivity.

Design, facile synthesis and anthelmintic activity of new O-substituted 6-methoxybenzothiazole-2-carbamates. Part II.

In the framework of pursuing the design and synthesis of a new series of substituted 6-methoxybenzothiazole-2-carbamates as potential anthelmintics, and as a continuation of the expended efforts in part I, we have set out to develop novel compounds with enhanced anthelmintic activity by blocking the 6-position of benzothiazole with side chains of different polarities. Guided by the findings in part I, and reporting the paramphistomicidal activity of oxadiazoline derivatives V and VI, we aimed to synthesize target benzothiazoles designed to comprise some planar heterocyclic ring systems, namely, 1,3,4-oxadiazoles and 1,2,4-triazoles, bearing a variety of hydrophobic and hydrophilic components. The synthesis of the desired compounds was primarily achieved by cyclization of 6-acetohydrazide, 1. The in vitro paramphistomicidal activity of all synthesized carbamates was evaluated. Four synthesized carbamates exhibited notable activity. Compound 24, methyl 6-[(5-(4-bromophenacylsulfanyl)-[1,3,4]-oxadiazol-2-yl)methoxy]benzothiazole-2-carbamate, displayed an equipotent effect to the reference drug oxyclozanide at a concentration of 80 µg mL-1; compounds 9, 10 and 23 showed high orders of anthelmintic effect. A structural computational study on the polar nature and hydrophilic-lipophilic properties of the synthesized carbamates was undertaken to discuss their structure-activity relationship (SAR). Besides, pharmacophore mapping was performed using eight active compounds as a training set. The generated pharmacophore model revealed five common features and was validated using fenbendazole, triclabendazole and triclabendazole sulfoxide.

Ethyl 5-methyl-7-phenyl-1,2,4-triazolo[4,3-a]pyrimidine-6-carboxyl-ate.

In the title compound, C15H14N4O2, the triazolo-pyrimidine ring system is almost planar (r.m.s. deviation = 0.02 Å) and the phenyl ring is inclined to its mean plane by 42.45 (9)°. The carboxyl group is inclined to the triazolo-pyrimidine ring mean plane by 57.8 (3)°. In the mol-ecule, there is a short C-H⋯O contact involving the carbonyl O atom and an H atom of the adjacent methyl substituent. In the crystal, neighbouring mol-ecules are linked by C-H⋯O hydrogen bonds, forming chains propagating along [010]. There are also weak π-π inter-actions present involving the pyridine and phenyl rings of neighbouring chains [inter-centroid distance = 3.8580 (16) Å].