7-Amino-[1,2,4]triazolo[1,5-a][1,3,5]triazines as CK1δ inhibitors: Exploring substitutions at the 2 and 5-positions.

CK1δ is a serine-threonine kinase involved in several pathological conditions including neuroinflammation and neurodegenerative disorders like Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis. Specifically, it seems that an inhibition of CK1δ could have a neuroprotective effect in these conditions. Here, a series of [1,2,4]triazolo[1,5-a][1,3,5]triazines were developed as ATP-competitive CK1δ inhibitors. Both positions 2 and 5 have been explored leading to a total of ten compounds exhibiting IC50s comprised between 29.1 µM and 2.08 µM. Three of the four most potent compounds (IC50 < 3 µM) bear a thiophene ring at the 2 position. All compounds have been submitted to computational studies that identified the chain composed of at least 2 atoms (e.g., nitrogen and carbon atoms) at the 5 position as crucial to determine a key bidentate hydrogen bond with Leu85 of CK1δ. Most potent compounds have been tested in vitro, resulting passively permeable to the blood-brain barrier and, safe and slight neuroprotective on a neuronal cell model. These results encourage to further structural optimize the series to obtain more potent CK1δ inhibitors as possible neuroprotective agents to be tested on models of the above-mentioned neurodegenerative diseases.

Disclosing the Preferential Mercury Chelation by SeCys Containing Peptides over Their Cys Analogues.

Methylmercury, mercury (II), and mercury (I) chlorides were found to react with vasopressin, a nonapeptide hormone cyclized by two cysteine residues, and its mono- and diselenium analogues to form several mercury-peptide adducts. The replacement of Cys by SeCys in vasopressin increased the reactivity toward methylmercury, with the predominant formation of -Se/S-Hg-Se-bridged structures and the consequent demethylation of methylmercury. In competitive experiments, CH3HgCl reacted preferentially with the diselenium analogue rather than with vasopressin. The diselenium peptide also showed the capability to displace the CH3Hg moiety bound to S in vasopressin. These results open a promising perspective for the use of selenopeptides for methylmercury chelation and detoxification strategies.

Apoptosis Pathways Triggered by a Potent Antiproliferative Hybrid Chalcone on Human Melanoma Cells.

The World Health Organization reported that approximately 324,000 new cases of melanoma skin cancer were diagnosed worldwide in 2020. The incidence of melanoma has been increasing over the past decades. Targeting apoptotic pathways is a potential therapeutic strategy in the transition to preclinical models and clinical trials. Some naturally occurring products and synthetic derivatives are apoptosis inducers and may represent a realistic option in the fight against the disease. Thus, chalcones have received considerable attention due to their potential cytotoxicity against cancer cells. We have previously reported a chalcone containing an indole and a pyridine heterocyclic rings and an α-bromoacryloylamido radical which displays potent antiproliferative activity against several tumor cell lines. In this study, we report that this chalcone is a potent apoptotic inducer for human melanoma cell lines SK-MEL-1 and MEL-HO. Cell death was associated with mitochondrial cytochrome c release and poly(ADP-ribose) polymerase cleavage and was prevented by a non-specific caspase inhibitor. Using SK-MEL-1 as a model, we found that the mechanism of cell death involves (i) the generation of reactive oxygen species, (ii) activation of the extrinsic and intrinsic apoptotic and mitogen-activated protein kinase pathways, (iii) upregulation of TRAIL, DR4 and DR5, (iv) downregulation of p21Cip1/WAF1 and, inhibition of the NF-κB pathway.

Silver (I) N-Heterocyclic Carbene Complexes: A Winning and Broad Spectrum of Antimicrobial Properties.

The evolution of antibacterial resistance has arisen as the main downside in fighting bacterial infections pushing researchers to develop novel, more potent and multimodal alternative drugs.Silver and its complexes have long been used as antimicrobial agents in medicine due to the lack of silver resistance and the effectiveness at low concentration as well as to their low toxicities compared to the most commonly used antibiotics. N-Heterocyclic Carbenes (NHCs) have been extensively employed to coordinate transition metals mainly for catalytic chemistry. However, more recently, NHC ligands have been applied as carrier molecules for metals in anticancer applications. In the present study we selected from literature two NHC-carbene based on acridinescaffoldand detailed nonclassicalpyrazole derived mono NHC-Ag neutral and bis NHC-Ag cationic complexes. Their inhibitor effect on bacterial strains Gram-negative and positivewas evaluated. Imidazolium NHC silver complex containing the acridine chromophore showed effectiveness at extremely low MIC values. Although pyrazole NHC silver complexes are less active than the acridine NHC-silver, they represent the first example of this class of compounds with antimicrobial properties. Moreover all complexesare not toxic and they show not significant activity againstmammalian cells (Hek lines) after 4 and 24 h. Based on our experimental evidence, we are confident that this promising class of complexes could represent a valuable starting point for developing candidates for the treatment of bacterial infections, delivering great effectiveness and avoiding the development of resistance mechanisms.

Forward Precision Medicine: Micelles for Active Targeting Driven by Peptides.

Precision medicine is based on innovative administration methods of active principles. Drug delivery on tissue of interest allows improving the therapeutic index and reducing the side effects. Active targeting by means of drug-encapsulated micelles decorated with targeting bioactive moieties represents a new frontier. Between the bioactive moieties, peptides, for their versatility, easy synthesis and immunogenicity, can be selected to direct a drug toward a considerable number of molecular targets overexpressed on both cancer vasculature and cancer cells. Moreover, short peptide sequences can facilitate cellular intake. This review focuses on micelles achieved by self-assembling or mixing peptide-grafted surfactants or peptide-decorated amphiphilic copolymers. Nanovectors loaded with hydrophobic or hydrophilic cytotoxic drugs or with gene silence sequences and externally functionalized with natural or synthetic peptides are described based on their formulation and in vitro and in vivo behaviors.

Design, synthesis and biological evaluation of 2-alkoxycarbonyl-3-anilinoindoles as a new class of potent inhibitors of tubulin polymerization.

A new class of inhibitors of tubulin polymerization based on the 2-alkoxycarbonyl-3-(3',4',5'-trimethoxyanilino)indole molecular skeleton was synthesized and evaluated for antiproliferative activity, inhibition of tubulin polymerization and cell cycle effects. The results presented show that the methoxy substitution and location on the indole nucleus plays an important role in inhibition of cell growth, and the most favorable position for the substituent was at C-6. In addition, a small-size ester function (methoxy/ethoxycarbonyl) at the 2-position of the indole core was desirable. Also, analogues that were alkylated with methyl, ethyl or n-propyl groups or had a benzyl moiety on the N-1 indolic nitrogen retained activity equivalent to those observed in the parent N-1H analogues. The most promising compounds of the series were 2-methoxycarbonyl-3-(3',4'.5'-trimethoxyanilino)-5-methoxyindole 3f and 1-methyl-2-methoxycarbonyl-3-(3',4'.5'-trimethoxyanilino)-6-methoxy-indole 3w, both of which target tubulin at the colchicine site with antitubulin activities comparable to that of the reference compound combretastatin A-4.

Synthesis and Biological Evaluation of New Antitubulin Agents Containing 2-(3',4',5'-trimethoxyanilino)-3,6-disubstituted-4,5,6,7-tetrahydrothieno[2,3-c]pyridine Scaffold.

Two novel series of compounds based on the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine and 4,5,6,7-tetrahydrobenzo[b]thiophene molecular skeleton, characterized by the presence of a 3',4',5'-trimethoxyanilino moiety and a cyano or an alkoxycarbonyl group at its 2- or 3-position, respectively, were designed, synthesized, and evaluated for antiproliferative activity on a panel of cancer cell lines and for selected highly active compounds, inhibition of tubulin polymerization, and cell cycle effects. We have identified the 2-(3',4',5'-trimethoxyanilino)-3-cyano-6-methoxycarbonyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine derivative 3a and its 6-ethoxycarbonyl homologue 3b as new antiproliferative agents that inhibit cancer cell growth with IC50 values ranging from 1.1 to 4.7 µM against a panel of three cancer cell lines. Their interaction with tubulin at micromolar levels leads to the accumulation of cells in the G2/M phase of the cell cycle and to an apoptotic cell death. The cell apoptosis study found that compounds 3a and 3b were very effective in the induction of apoptosis in a dose-dependent manner. These two derivatives did not induce cell death in normal human peripheral blood mononuclear cells, suggesting that they may be selective against cancer cells. Molecular docking studies confirmed that the inhibitory activity of these molecules on tubulin polymerization derived from binding to the colchicine site.

3-Aryl/Heteroaryl-5-amino-1-(3',4',5'-trimethoxybenzoyl)-1,2,4-triazoles as antimicrotubule agents. Design, synthesis, antiproliferative activity and inhibition of tubulin polymerization.

Many natural and synthetic substances are known to interfere with the dynamic assembly of tubulin, preventing the formation of microtubules. In our search for potent and selective antitumor agents, a novel series of 1-(3',4',5'-trimethoxybenzoyl)-5-amino-1,2,4-triazoles were synthesized. The compounds had different heterocycles, including thiophene, furan or the three isomeric pyridines, and they possessed a phenyl ring bearing electron-releasing or electron-withdrawing substituents at the 3-position of the 5-amino-1,2,4-triazole system. Most of the twenty-two tested compounds showed moderate to potent antiproliferative activities against a panel of solid tumor and leukemic cell lines, with four (5j, 5k, 5o and 5p) showing strong antiproliferative activity (IC50 < 1 µM) against selected cancer cells. Among them, several molecules preferentially inhibited the proliferation of leukemic cell lines, showing IC50 values 2-100-fold lower for Jurkat and RS4;11 cells than those for the three lines derived from solid tumors (HeLa, HT-29 and MCF-7 cells). Compound 5k strongly inhibited tubulin assembly, with an IC50 value of 0.66 µM, half that obtained in simultaneous experiments with CA-4 (IC50 = 1.3 µM).

Synthesis and biological evaluation of alpha-bromoacryloylamido indolyl pyridinyl propenones as potent apoptotic inducers in human leukaemia cells.

The combination of two pharmacophores into a single molecule represents one of the methods that can be adopted for the synthesis of new anticancer molecules. To investigate the influence of the position of the pyridine nitrogen on biological activity, two different series of α-bromoacryloylamido indolyl pyridinyl propenones 3a-h and 4a-d were designed and synthesized by a pharmacophore hybridization approach and evaluated for their antiproliferative activity against a panel of six human cancer cell lines. These hybrid molecules were prepared to combine the α-bromoacryloyl moiety with two series of indole-inspired chalcone analogues, possessing an indole derivative and a 3- or 4-pyridine ring, respectively, linked on either side of 2-propen-1-one system. The structure-activity relationship was also investigated by the insertion of alkyl or benzyl moieties at the N-1 position of the indole nucleus. We found that most of the newly synthesized displayed high antiproliferative activity against U-937, MOLT-3, K-562, and NALM-6 leukaemia cell lines, with one-digit to double-digit nanomolar IC50 values. The antiproliferative activities of 3-pyridinyl derivatives 3f-h revealed that N-benzyl indole analogues generally exhibited lower activity compared to N-H or N-alkyl derivatives 3a-b and 3c-e, respectively. Moreover, cellular mechanism studies elucidated that compound 4a induced apoptosis along with a decrease of mitochondrial membrane potential and activated caspase-3 in a concentration-dependent manner.

Gaucher Disease: A Glance from a Medicinal Chemistry Perspective.

Rare diseases are particular pathological conditions affecting a limited number of people and few drugs are known to be effective as therapeutic treatment. Gaucher disease, caused by a deficiency of the lysosomal enzyme glucocerebrosidase, belongs to this class of disorders, and it is considered the most common among the Lysosomal Storage Diseases. The two main therapeutic approaches are the Enzyme Replacement Therapy (ERT) and the Substrate Reduction Therapy (SRT). ERT, consisting in replacing the defective enzyme by administering a recombinant enzyme, is effective in alleviating the visceral symptoms, hallmarks of the most common subtype of the disease whereas it has no effects when symptoms involve CNS, since the recombinant protein is unable to significantly cross the Blood Brain Barrier. The SRT strategy involves inhibiting glucosylceramide synthase (GCS), the enzyme responsible for the production of the associated storage molecule. The rational design of new inhibitors of GCS has been hampered by the lack of either the crystal structure of the enzyme or an in-silico model of the active site which could provide important information regarding the interactions of potential inhibitors with the target, but, despite this, interesting results have been obtained and are herein reviewed.

New Insights into the Behavior of NHC-Gold Complexes in Cancer Cells.

Among the non-platinum antitumor agents, gold complexes have received increased attention owing to their strong antiproliferative effects, which generally occur through non-cisplatin-like mechanisms of action. Several studies have revealed that many cytotoxic gold compounds, such as N-heterocyclic carbene (NHC)-gold(I) complexes, are potent thioredoxin reductase (TrxR) inhibitors. Many other pathways have been supposed to be altered by gold coordination to protein targets. Within this frame, we have selected two gold(I) complexes based on aromatic ligands to be tested on cancer cells. Differently from bis [1,3-diethyl-4,5-bis(4-methoxyphenyl)imidazol-2-ylidene]gold(I) bromide (Au4BC), bis [1-methyl-3-acridineimidazolin-2-ylidene]gold(I) tetrafluoroborate (Au3BC) inhibited TrxR1 activity in vitro. Treatment of Huh7 hepatocellular carcinoma (HCC) cells, and MDA-MB-231 triple-negative breast cancer (TNBC) cells, with Au4BC inhibited cell viability, increased reactive oxygen species (ROS) levels, caused DNA damage, and induced autophagy and apoptosis. Notably, we found that, although Au3BC inhibited TrxR1 activity, no effect on the cell viabilities of HCC and BC cells was observed. At the molecular level, Au3BC induced a protective response mechanism in Huh7 and MDA-MB-231 cells, by inducing up-regulation of RAD51 and p62 protein expression, two proteins involved in DNA damage repair and autophagy, respectively. RAD51 gene knock-down in HCC cells increased cell sensitivity to Au3BC by significant reduction of cell viability, induction of DNA damage, and induction of apoptosis and autophagy. All together, these results suggest that the tested NHC-Gold complexes, Au3BC and Au4BC, showed different mechanisms of action, either dependent or independent of TrxR1 inhibition. As a result, Au3BC and Au4BC were found to be promising candidates as anticancer drugs for the treatment of HCC and BC.

[1,2,4]Triazolo[1,5-c]pyrimidines as Tools to Investigate A3 Adenosine Receptors in Cancer Cell Lines.

The A3 adenosine receptor is an interesting target whose role in cancer is controversial. In this work, a structural investigation at the 2-position of the [1,2,4]triazolo[1,5-c]pyrimidine nucleus was performed, finding new potent and selective A3 adenosine receptor antagonists such as the ethyl 2-(4-methoxyphenyl)-5-(methylamino)-[1,2,4]triazolo[1,5-c]pyrimidine-8-carboxylate (20, DZ123) that showed a Ki value of 0.47 nM and an exceptional selectivity profile over the other adenosine receptor subtypes. Computational studies were performed to rationalize the affinity and the selectivity profile of the tested compounds at the A3 adenosine receptor and the A1 and A2A adenosine receptors. Compound 20 was tested on both A3 adenosine receptor positive cell lines (CHO-A3 AR transfected, THP1 and HCT16) and on A3 negative cancer cell lines, showing no effect in the latter and a pro-proliferative effect at a low concentration in the former. These interesting results pave the way to further investigation on both the mechanism involved and potential therapeutic applications.

Cinnamic acid derivatives linked to arylpiperazines as novel potent inhibitors of tyrosinase activity and melanin synthesis.

A novel series of twenty-seven cinnamides constituted by cinnamic acid derivatives liked to 1-aryl piperazines were synthesized and evaluated for their potential inhibitory diphenolase activity of mushroom tyrosinase. Among them, the presence of a 3-chloro-4-fluorophenyl moiety at the N-1 position of piperazine ring was essential for a potent tyrosinase inhibitory effect, with the 3-nitrocinnamoyl (19p) and 2-chloro-3-methoxycinnamoyl (19t) derivatives as the most potent compounds of the series, with IC50 of 0.16 and 0.12 µM, respectively, resulting much active than kojic acid, whose IC50 value was 17.76 µM. In general, all compounds characterized by the presence of a 1-(3-chloro-4-fluorophenyl)piperazine moiety showed an excellent potency, and the nature, position and number of the substituents on the aryl of the cinnamic acid did not affect significantly the anti-tyrosinase activity. The molecular docking to the active site of the enzyme has been also performed to investigate the nature of enzyme-inhibitor interactions. Furthermore, for selected highly active compounds, their ability to inhibit melanogenesis in the A375 human melanoma cells and in vivo zebrafish model was also evaluated. One of the most potent compounds of series (19t) significantly reduced the pigmentation of zebrafish at 50 µM, unfortunately showing 100% mortality in the Fish Embryo Acute Toxicity (FET) test at the same concentration, Moreover, the zebrafish assay reveals that also compound 19r (IC50:0.51 µM against mushroom tyrosinase) effectively reduces melanogenesis with no acute toxicity effects and it could be proposed as potential candidate to treat tyrosinase-mediated hyperpigmentation.

Design, Synthesis and Biological Investigation of 2-Anilino Triazolopyrimidines as Tubulin Polymerization Inhibitors with Anticancer Activities.

A further investigation aiming to generate new potential antitumor agents led us to synthesize a new series of twenty-two compounds characterized by the presence of the 7-(3',4',5'-trimethoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidine pharmacophore modified at its 2-position. Among the synthesized compounds, three were significantly more active than the others. These bore the substituents p-toluidino (3d), p-ethylanilino (3h) and 3',4'-dimethylanilino (3f), and these compounds had IC50 values of 30-43, 160-240 and 67-160 nM, respectively, on HeLa, A549 and HT-29 cancer cells. The p-toluidino derivative 3d was the most potent inhibitor of tubulin polymerization (IC50: 0.45 µM) and strongly inhibited the binding of colchicine to tubulin (72% inhibition), with antiproliferative activity superior to CA-4 against A549 and HeLa cancer cell lines. In vitro investigation showed that compound 3d was able to block treated cells in the G2/M phase of the cell cycle and to induce apoptosis following the intrinsic pathway, further confirmed by mitochondrial depolarization and caspase-9 activation. In vivo experiments conducted on the zebrafish model showed good activity of 3d in reducing the mass of a HeLa cell xenograft. These effects occurred at nontoxic concentrations to the animal, indicating that 3d merits further developmental studies.

Allantodapsone is a Pan-Inhibitor of Staphylococcus aureus Adhesion to Fibrinogen, Loricrin, and Cytokeratin 10.

Staphylococcus aureus infections have become a major challenge in health care due to increasing antibiotic resistance. We aimed to design small molecule inhibitors of S. aureus surface proteins to be developed as colonization inhibitors. We identified allantodapsone in an initial screen searching for inhibitors of clumping factors A and B (ClfA and ClfB). We used microbial adhesion assays to investigate the effect of allantodapsone on extracellular matrix protein interactions. Allantodapsone inhibited S. aureus Newman adhesion to fibrinogen with an IC50 of 21.3 µM (95% CI 4.5-102 µM), minimum adhesion inhibitory concentration (MAIC) of 100 µM (40.2 µg/mL). Additionally, allantodapsone inhibited adhesion of Lactococcus lactis strains exogenously expressing the clumping factors to fibrinogen (L. lactis ClfA, IC50 of 3.8 µM [95% CI 1.0-14.3 µM], MAIC 10 µM, 4.0 µg/mL; and L. lactis ClfB, IC50 of 11.0 µM [95% CI 0.9-13.6 µM], MAIC 33 µM, 13.3 µg/mL), indicating specific inhibition. Furthermore, the dapsone and alloxan fragments of allantodapsone did not have any inhibitory effect. Adhesion of S. aureus Newman to L2v loricrin is dependent on the expression of ClfB. Allantodapsone caused a dose dependent inhibition of S. aureus adhesion to the L2v loricrin fragment, with full inhibition at 40 µM (OD600 0.11 ± 0.01). Furthermore, recombinant ClfB protein binding to L2v loricrin was inhibited by allantodapsone (P < 0.0001). Allantodapsone also demonstrated dose dependent inhibition of S. aureus Newman adhesion to cytokeratin 10 (CK10). Allantodapsone is the first small molecule inhibitor of the S. aureus clumping factors with potential for development as a colonization inhibitor. IMPORTANCE S. aureus colonization of the nares and the skin provide a reservoir of bacteria that can be transferred to wounds that can ultimately result in systemic infections. Antibiotic resistance can make these infections difficult to treat with significant associated morbidity and mortality. We have identified and characterized a first-in-class small molecule inhibitor of the S. aureus clumping factors A and B, which has the potential to be developed further as a colonization inhibitor.

Design, Synthesis, and Biological Evaluation of 6-Substituted Thieno[3,2- d]pyrimidine Analogues as Dual Epidermal Growth Factor Receptor Kinase and Microtubule Inhibitors.

The clinical evidence for the success of tyrosine kinase inhibitors in combination with microtubule-targeting agents prompted us to design and develop single agents that possess both epidermal growth factor receptor (EGFR) kinase and tubulin polymerization inhibitory properties. A series of 6-aryl/heteroaryl-4-(3',4',5'-trimethoxyanilino)thieno[3,2- d]pyrimidine derivatives were discovered as novel dual tubulin polymerization and EGFR kinase inhibitors. The 4-(3',4',5'-trimethoxyanilino)-6-( p-tolyl)thieno[3,2- d]pyrimidine derivative 6g was the most potent compound of the series as an antiproliferative agent, with half-maximal inhibitory concentration (IC50) values in the single- or double-digit nanomolar range. Compound 6g bound to tubulin in the colchicine site and inhibited tubulin assembly with an IC50 value of 0.71 µM, and 6g inhibited EGFR activity with an IC50 value of 30 nM. Our data suggested that the excellent in vitro and in vivo profile of 6g may be derived from its dual inhibition of tubulin polymerization and EGFR kinase.

The discovery of new and more potent chloropyramine (C4) analogues for the potential treatment of invasive breast cancer.

Breast cancer is the second most common cancer worldwide, accounting for 25% of all female cancers. Although the survival rate has increased significantly in the past few decades, patients who develop secondary site metastasis as well as those diagnosed with triple negative breast cancer still represent a real unmet medical challenge. Previous studies have shown that chloropyramine (C4) inhibits FAK-VEGFR3 signalling. More recently, C4 is reported to have SASH1 inducing properties. However, C4 exerts its antitumour and antiangiogenic effects at high micromolar concentrations (>100 µm) that would not be compatible with further drug development against invasive breast cancer driven by FAK signalling. In this study, molecular modelling guided structural modifications have been introduced to the chloropyramine C4 scaffold to improve its activity in breast cancer cell lines. Seventeen compounds were designed and synthesized, and their antiproliferative activity was evaluated against three human breast cancer lines (MDA-MB-231, BT474 and T47D). Compound 5c was identified to display an average activity of IC50  = 23.5-31.3 µm, which represents a significant improvement of C4 activity in the same assay model. Molecular modelling and pharmacokinetic studies provided more promising insights into the mechanistic features of this new series.

Virtual screening, SAR, and discovery of 5-(indole-3-yl)-2-[(2-nitrophenyl)amino] [1,3,4]-oxadiazole as a novel Bcl-2 inhibitor.

A new series of oxadiazoles were designed to act as inhibitors of the anti-apoptotic Bcl-2 protein. Virtual screening led to the discovery of new hits that interact with Bcl-2 at the BH3 binding pocket. Further study of the structure-activity relationship of the most active compound of the first series, compound 1, led to the discovery of a novel oxadiazole analogue, compound 16j, that was a more potent small-molecule inhibitor of Bcl-2. 16j had good in vitro inhibitory activity with submicromolar IC50 values in a metastatic human breast cancer cell line (MDA-MB-231) and a human cervical cancer cell line (HeLa). The antitumour effect of 16j is concomitant with its ability to bind to Bcl-2 protein as shown by an enzyme-linked immunosorbent assay (IC50  = 4.27 µm). Compound 16j has a great potential to develop into highly active anticancer agent.

Design, synthesis and biological evaluation of 3-substituted-2-oxindole hybrid derivatives as novel anticancer agents.

The 2-oxindole nucleus is the central core to develop new anticancer agents and its substitution at the 3-position can effect antitumor activity. Utilizing a pharmacophore hybridization approach, a novel series of antiproliferative agents was obtained by the modification of the structure of 3-substituted-2-oxindole pharmacophore by the attachment of the α-bromoacryloyl moiety, acting as a Michael acceptor, at the 5-position of 2-oxindole framework. The impact of the substituent at the 3-position of 2-oxindole core on the potency and selectivity against a panel of seven different cancer cell lines was examined. We found that these hybrid molecules displayed potent antiproliferative activity against a panel of four cancer cell lines, with one-to double digit nanomolar 50% inhibitory concentrations (IC50). A distinctive selective antiproliferative activity was obtained towards CCRF-CEM and RS4; 11 leukemic cell lines. In order to study the possible mechanism of action, we observed that the two most active compounds namely 3(E) and 6(Z) strongly induce apoptosis that follow the mitochondrial pathway. Interestingly a decrease of intracellular reduced glutathione content (GSH) and reactive oxygen species (ROS) production was detected in treated cells compared with controls suggesting that these effects may be involved in their mechanism of action.

Synthesis and Biological Evaluation of 2-Methyl-4,5-Disubstituted Oxazoles as a Novel Class of Highly Potent Antitubulin Agents.

Antimitotic agents that interfere with microtubule formation are one of the major classes of cytotoxic drugs for cancer treatment. Multiple 2-methyl-4-(3',4',5'-trimethoxyphenyl)-5-substituted oxazoles and their related 4-substituted-5-(3',4',5'-trimethoxyphenyl) regioisomeric derivatives designed as cis-constrained combretastatin A-4 (CA-4) analogues were synthesized and evaluated for their antiproliferative activity in vitro against a panel of cancer cell lines and, for selected highly active compounds, interaction with tubulin, cell cycle effects and in vivo potency. Both these series of compounds were characterized by the presence of a common 3',4',5'-trimethoxyphenyl ring at either the C-4 or C-5 position of the 2-methyloxazole ring. Compounds 4g and 4i, bearing a m-fluoro-p-methoxyphenyl or p-ethoxyphenyl moiety at the 5-position of 2-methyloxazole nucleus, respectively, exhibited the greatest antiproliferative activity, with IC50 values of 0.35-4.6 nM (4g) and 0.5-20.2 nM (4i), which are similar to those obtained with CA-4. These compounds bound to the colchicine site of tubulin and inhibited tubulin polymerization at submicromolar concentrations. Furthermore, 4i strongly induced apoptosis that follows the mitochondrial pathway. In vivo, 4i in a mouse syngeneic model demonstrated high antitumor activity which significantly reduced the tumor mass at doses ten times lower than that required for CA-4P, suggesting that 4i warrants further evaluation as a potential anticancer drug.