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The current study was designed to identify new compounds as potential antiproliferative drug candidates. Synthesis of heteroaromatic bicyclic and monocyclic derivatives as purine bioisosters was employed. Their antiproliferative activity was studied against U937 cancer cells. The most effective compounds were evaluated for their selectivity against cancer cells, the possible mechanism of cell death, and their interference with DNA replication. Among the synthesized compounds, only three (4b, 4j and 4l) demonstrated a value of IC50 less than 20 μM. However, two of them (4b and 4l) were specific against cancer cells, with 4l presenting high selectivity. The presence of substituted pyrazolo[3,4- d]pyrimidine core is as essential for this activity as the presence of substituents at the thiol function in 6-position.

BackgroundThe first-pass complete recanalization by mechanical thrombectomy (MT) for the treatment of stroke remains limited due to the poor integration of the clot within current devices. Aspiration can help retrieval of the main clot but fails to prevent secondary embolism in the distal arterial territory. The dense meshes of extracellular DNA, recently described in stroke-related clots, might serve as an anchoring platform for MT devices.ObjectiveEvaluate the potential of DNA reacting surface toaid the retention of the main clot as well as of its small fragments within the thrombectomy deviceand improve the potential of MT procedures.MethodsDevice-suitable alloy experimental samples were coated with 15 different compounds and contacted with extracellular DNA or with human peripheral whole blood, to compare their binding to DNA versus flowing blood elements,in vitro. Clinical-grade MT devices were coated with two selected compounds and evaluated in functional bench tests aiming to studying clot retrieval and distal emboli release, concomitant with contact aspiration, using an M1 occlusion model.ResultsBinding properties of samples coated with all compounds were increased for DNA (≈ 3-fold) and decreased (≈ 5-fold) for blood elements, essentially platelet, as compared to the bare alloy samples,in vitro. Functional testing showed that surface modification with DNA-binding compounds improved clot retrieval and significantly reduced secondary embolism during experimental recanalization of occluded artery 3D model by thrombectomy procedures.ConclusionOur results suggest that device coating with DNA-binding compounds can considerably improve the outcome of MT procedures in stroke patients.What is already known on this topic –New mechanical thrombectomy device are being improved on the conformation and shape to increase the interaction clot on the physical point of view. However, none interact specifically with the structure or composition of the clot.What this study adds –The design of a chemical surface modification of the device opens the way for a specific targeting tool to increase the interaction with the clot on the molecular level.How this study might affect research, practice or policy –This new surface modification, which can be applied to all commercially available mechanical thrombectomy devices, leads to a decrease in secondary embolization which cannot and is not monitored during the procedure and responsible for new territory damage.

In pediatric acute myeloid leukemia (AML), intensive chemotherapy and allogeneic hematopoietic stem cell transplantation are the cornerstones of treatment in high-risk cases, with severe late effects and a still high risk of disease recurrence as the main drawbacks. The identification of targeted, more effective, safer drugs is thus desirable. We performed a high-throughput drug-screening assay of 1280 compounds and identified thioridazine (TDZ), a drug that was highly selective for the t(6;11)(q27;q23) MLL-AF6 (6;11)AML rearrangement, which mediates a dramatically poor (below 20%) survival rate. TDZ induced cell death and irreversible progress toward the loss of leukemia cell clonogenic capacity in vitro. Thus, we explored its mechanism of action and found a profound cytoskeletal remodeling of blast cells that led to Ca2+ influx, triggering apoptosis through mitochondrial depolarization, confirming that this latter phenomenon occurs selectively in t(6;11)AML, for which AF6 does not work as a cytoskeletal regulator, because it is sequestered into the nucleus by the fusion gene. We confirmed TDZ-mediated t(6;11)AML toxicity in vivo and enhanced the drug’s safety by developing novel TDZ analogues that exerted the same effect on leukemia reduction, but with lowered neuroleptic effects in vivo. Overall, these results refine the MLL-AF6 AML leukemogenic mechanism and suggest that the benefits of targeting it be corroborated in further clinical trials.

(1) Background: In the development of new and more effective anticancer approaches, combined treatments appear of great interest. Combination therapy could be of importance in the management of glioblastoma (GBM), a lethal malignancy that accounts for 42% of cancer of the central nervous system, with a median survival of 15 months. This study aimed to verify the activity on a glioblastoma cancer cell line of one of the most active compounds of a novel series of tubulin polymerization inhibitors based on the 1-(3′,4′,5′-trimethoxyphenyl)-2-aryl-1H-imidazole scaffold, used in combination with a miRNA inhibitor molecule targeting the oncomiRNA miR-10b-5p. This microRNA was selected in consideration of the role of miR-10b-5p on the onset and progression of glioblastoma. (2) Methods: Apoptosis was analyzed by Annexin-V and Caspase 3/7 assays, efficacy of the anti-miR-10b-5p was assessed by determining the miR-10b-5p content by RT-qPCR. (3) Results: The results obtained show that a “combination therapy” performed by combining the use of an anti-miR-10b-5p and a 1-(3′,4′,5′-trimethoxyphenyl)-2-aryl-1H-imidazole derivative is an encouraging strategy to boost the efficacy of anticancer therapies and at the same time to reduce side effects.

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 IC

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.

Many clinically used agents active in cancer chemotherapy exert their activity through the induction of cell death (apoptosis) by targeting microtubules, altering protein function or inhibiting DNA synthesis. The benzo[b]thiophene scaffold holds a pivotal place as a pharmacophore for the development of anticancer agents, and, in addition, this scaffold has many pharmacological activities. We have developed a flexible method for the construction of a new series of 2-aryl-3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophenes as potent antiproliferative agents, giving access to a wide range of substitution patterns at the 2-position of the 6-methoxybenzo[b]thiophene common intermediate. In the present study, all the synthesized compounds retained the 3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophene moiety, and the structure-activity relationship was examined by modification of the aryl group at its 2-position with electron-withdrawing (F) or electron-releasing (alkyl and alkoxy) groups. We found that small substituents, such as fluorine or methyl, could be placed in the para-position of the 2-phenyl ring, and these modifications only slightly reduced antiproliferative activity relative to the unsubstituted 2-phenyl analogue. Compounds 3a and 3b, bearing the phenyl and para-fluorophenyl at the 2-position of the 6-methoxybenzo[b]thiophene nucleus, respectively, exhibited the greatest antiproliferative activity among the tested compounds. The treatment of both Caco2 (not metastatic) and HCT-116 (metastatic) colon carcinoma cells with 3a or 3b triggered a significant induction of apoptosis as demonstrated by the increased expression of cleaved-poly(ADP-ribose) polymerase (PARP), receptor-interacting protein (RIP) and caspase-3 proteins. The same effect was not observed with non-transformed colon 841 CoN cells. A potential additional effect during mitosis for 3a in metastatic cells and for 3b in non-metastatic cells was also observed.

Three different series of cis-restricted analogues of combretastatin A-4 (CA-4), corresponding to thirty-nine molecules that contained a pyrrole nucleus interposed between the two aryl rings, were prepared by a palladium-mediated coupling approach and evaluated for their antiproliferative activity against six human cancer cell lines. In the two series of 1,2-diaryl pyrrole derivatives, results suggested that the presence of the 3′,4′,5′-trimethoxyphenyl moiety at the N-1 position of the pyrrole ring was more favorable for antiproliferative activity. In the series of 3,4-diarylpyrrole analogues, three compounds (11i-k) exhibited maximal antiproliferative activity, showing excellent antiproliferative activity against the CA-4 resistant HT-29 cells. Inhibition of tubulin polymerization of selected 1,2 pyrrole derivatives (9a, 9c, 9o and 10a) was similar to that observed with CA-4, while the isomeric 3,4-pyrrole analogues 11i-k were generally from 1.5- to 2-fold more active than CA-4. Compounds 11j and 11k were the only compounds that showed activity as inhibitors of colchicine binding comparable to that CA-4. Compound 11j had biological properties consistent with its intracellular target being tubulin. This compound was able to block the cell cycle in metaphase and to induce significant apoptosis at a concentration of 25 nM, following the mitochondrial pathway, with low toxicity for normal cells. More importantly, compound 11j exerted activity in vivo superior to that of CA-4P, being able to significantly reduce tumor growth in a syngeneic murine tumor model even at the lower dose tested (5.0 mg/kg).

Glutamate cytotoxicity is implicated in neuronal death in different neurological disorders including stroke, traumatic brain injury, and neurodegenerative diseases. Adenosine is a nucleoside that plays an important role in modulating neuronal activity and its receptors have been identified as promising therapeutic targets for glutamate cytotoxicity. The purpose of this study is to elucidate the role of adenosine and its receptors on glutamate-induced injury in PC12 cells and to verify the protective effect of the novel A1 adenosine receptor positive allosteric modulator, TRR469. Flow cytometry experiments to detect apoptosis revealed that adenosine has a dual role in glutamate cytotoxicity, with A2A and A2B adenosine receptor (AR) activation exacerbating and A1 AR activation improving glutamate-induced cell injury. The overall effect of endogenous adenosine in PC12 cells resulted in a facilitating action on glutamate cytotoxicity, as demonstrated by the use of adenosine deaminase and selective antagonists. However, enhancing the action of endogenous adenosine on A1ARs by TRR469 completely abrogated glutamate-mediated cell death, caspase 3/7 activation, ROS production, and mitochondrial membrane potential loss. Our results indicate a novel potential therapeutic strategy against glutamate cytotoxicity based on the positive allosteric modulation of A1ARs.

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&prime, 4&prime, 5&prime, 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&prime, 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 &mu, M 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.

A new class of inhibitors of tubulin polymerization based on the 2-amino-3-(3′,4′,5′-trimethoxybenzoyl)benzo[b]furan molecular scaffold was synthesized and evaluated for in vivo and in vitro biological activity. These derivatives were synthesized with different electron-releasing or electron-withdrawing substituents at one of the C-4 through C-7 positions. Methoxy substitution and location on the benzene part of the benzo[b]furan ring played an important role in affecting antiproliferative activity, with the greatest activity occurring with the methoxy group at the C-6 position, the least with the substituent at C-4. The same effect was also observed with ethoxy, methyl or bromine at the C-6 position of the benzo[b]furan skeleton, with the 6-ethoxy-2-amino-3-(3′,4′,5′-trimethoxybenzoyl)benzo[b]furan derivative 4f as the most promising compound of the series. This compound showed remarkable antiproliferative activity (IC50: 5 pM) against the Daoy medulloblastoma cell line, and 4f was nearly devoid of toxicity on healthy human lymphocytes and astrocytes. The potent antiproliferative activity of 4f was derived from its inhibition of tubulin polymerization by binding to the colchicine site. The compound was also examined for in vivo activity, showing higher potency at 15 mg/kg compared with the reference compound combretastatin A-4 phosphate at 30 mg/kg against a syngeneic murine mammary tumor.

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. ispartof: BIOORGANIC CHEMISTRY vol:97 ispartof: location:United States status: published

Induction of apoptosis is a common chemotherapeutic mechanism to kill cancer cells The thiazole system has been reported over the past decades as a building block for the preparation of anticancer agents. A novel series of 2-arylalkylamino-4-amino-5-(3&prime, 4&prime, 5&prime, trimethoxybenzoyl)-thiazole derivatives designed as dual inhibitors of tubulin and cyclin-dependent kinases (CDKs) were synthesized and evaluated for their antiproliferative activity in vitro against two cancer cell lines and, for selected highly active compounds, for interactions with tubulin and cyclin-dependent kinases and for cell cycle and apoptosis effects. Structure-activity relationships were elucidated for various substituents at the 2-position of the thiazole skeleton. Among the synthesized compounds, the most active analogues were found to be the p-chlorobenzylamino derivative 8e as well as the p-chloro and p-methoxyphenethylamino analogues 8f and 8k, respectively, which inhibited the growth of U-937 and SK-MEL-1 cancer cell lines with IC50 values ranging from 5.7 to 12.2 &mu, M. On U-937 cells, the tested compounds 8f and 8k induced apoptosis in a time and concentration dependent manner. These two latter molecules did not affect tubulin polymerization (IC50 &gt, 20 &mu, M) nor CDK activity at a single concentration of 10 &mu, M, suggesting alternative targets than tubulin and CDK for the compounds.

STAT5 is a transcription factor, a member of the STAT family of signaling proteins. STAT5 is involved in many types of cancer, including chronic myelogenous leukemia (CML), in which this protein is found constitutively activated as a consequence of BCR-ABL expression. The neuroleptic drug pimozide was recently reported to act as an inhibitor of STAT5 phosphorylation and is capable of inducing apoptosis in CML cells in vitro. Our research group has synthesized simple derivatives of pimozide with cytotoxic activity and that are able to decrease the levels of phosphorylated STAT5. In this work we continued the search for novel STAT5 inhibitors, synthesizing compounds in which the benzoimidazolinone ring of pimozide is either maintained or modified, in order to obtain further structure–activity relationship information for this class of STAT5 inhibitors. Two compounds of the series showed potent cytotoxic activity against BCR-ABL-positive and pSTAT5-overexpressing K562 cells and were able to markedly decrease the levels of phosphorylated STAT5.

The A3 adenosine receptor (A3 AR) subtype is a novel, promising therapeutic target for inflammatory diseases, such as rheumatoid arthritis (RA) and psoriasis, as well as liver cancer. A3 AR is coupled to inhibition of adenylyl cyclase and regulation of mitogen-activated protein kinase (MAPK) pathways, leading to modulation of transcription. Furthermore, A3 AR affects functions of almost all immune cells and the proliferation of cancer cells. Numerous A3 AR agonists, partial agonists, antagonists, and allosteric modulators have been reported, and their structure-activity relationships (SARs) have been studied culminating in the development of potent and selective molecules with drug-like characteristics. The efficacy of nucleoside agonists may be suppressed to produce antagonists, by structural modification of the ribose moiety. Diverse classes of heterocycles have been discovered as selective A3 AR blockers, although with large species differences. Thus, as a result of intense basic research efforts, the outlook for development of A3 AR modulators for human therapeutics is encouraging. Two prototypical selective agonists, N6-(3-Iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA; CF101) and 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA; CF102), have progressed to advanced clinical trials. They were found safe and well tolerated in all preclinical and human clinical studies and showed promising results, particularly in psoriasis and RA, where the A3 AR is both a promising therapeutic target and a biologically predictive marker, suggesting a personalized medicine approach. Targeting the A3 AR may pave the way for safe and efficacious treatments for patient populations affected by inflammatory diseases, cancer, and other conditions.

A series of 3-(3′,4′,5′-trimethoxyphenyl)-4-substituted 1H-pyrazole and their related 3-aryl-4-(3′,4′,5′-trimethoxyphenyl)-1-H-pyrazole regioisomeric derivatives, prepared as cis-rigidified combretastatin A-4 (CA-4) analogues, were synthesized and evaluated for their in vitro antiproliferative against six different cancer cell lines and, for selected highly active compounds, inhibitory effects on tubulin polymerization, cell cycle effects and in vivo potency. We retained the 3′,4′,5′-trimethoxyphenyl moiety as ring A throughout the present investigation, and a structure-activity relationship (SAR) information was obtained by adding electron-withdrawing (OCF3, CF3) or electron-releasing (alkyl and alkoxy) groups on the second aryl ring, corresponding to the B-ring of CA-4, either at the 3- or 4-position of the pyrazole nucleus. In addition, the B-ring was replaced with a benzo[b]thien-2-yl moiety. For many of the compounds, their activity was greater than, or comparable with, that of CA-4. Maximal activity was observed with the two regioisomeric derivatives characterized by the presence of a 4-ethoxyphenyl and a 3′,4′,5′-trimethoxyphenyl group at the C-3 and C-4 positions, and vice versa, of the 1H-pyrazole ring. The data showed that the 3′,4′,5′-trimethoxyphenyl moiety can be moved from the 3- to the 4-position of the 1H-pyrazole ring without significantly affecting antiproliferative activity. The most active derivatives bound to the colchicine site of tubulin and inhibited tubulin polymerization at submicromolar concentrations. In vivo experiments, on an orthotopic murine mammary tumor, revealed that 4c inhibited tumor growth even at low concentrations (5 mg/kg) compared to CA-4P (30 mg/kg).

Numerous structure-activity relationship (SAR) studies of ligands of the A3 adenosine receptor (AR) have generated selective agonists, antagonists, partial agonists, and allosteric modulators. The efficacy of nucleoside agonists may be reduced, while retaining affinity, by successive structural changes. Subnanomolar affinity and selectivity of >10,000-fold have been achieved for various compound classes, but often with a pronounced species dependence, especially for diverse heterocyclic antagonists. Two prototypical A3AR agonists, IB-MECA and Cl-IB-MECA, are being evaluated clinically for treating autoimmune inflammatory disorders and liver diseases. The design of A3AR orthosteric ligands is now largely guided by computational approaches, in which the receptor is modeled by homology to X-ray structures of the A2AAR and other G protein-coupled receptors (GPCRs). Thus, we have amassed a large body of data concerning the relationship of receptor structure and function and have supported many of the hypotheses generated with experimental data.

Inhibition of microtubule function using tubulin targeting agents has received growing attention in the last several decades. The indole scaffold has been recognized as an important scaffold in the design of novel compounds acting as antimitotic agents. Indole-based chalcones, in which one of the aryl rings was replaced by an indole, have been explored in the last few years for their anticancer potential in different cancer cell lines. Eighteen novel (3',4',5'-trimethoxyphenyl)-indolyl-propenone derivatives with general structure 9 were synthesized and evaluated for their antiproliferative activity against a panel of four different human cancer cell lines. The highest IC50 values were obtained against the human promyelocytic leukemia HL-60 cell line. This series of chalcone derivatives was characterized by the presence of a 2-alkoxycarbonyl indole ring as the second aryl system attached at the carbonyl of the 3-position of the 1-(3',4',5'-trimethoxyphenyl)-2-propen-1-one framework. The structure-activity relationship (SAR) of the indole-based chalcone derivatives was investigated by varying the position of the methoxy group, by the introduction of different substituents (hydrogen, methyl, ethyl or benzyl) at the N-1 position and by the activity differences between methoxycarbonyl and ethoxycarbonyl moieties at the 2-position of the indole nucleus. The antiproliferative activity data of the novel synthesized compounds revealed that generally N-substituted indole analogues exhibited considerably reduced potency as compared with their parent N-unsubstituted counterparts, demonstrating that the presence of a hydrogen on the indole nitrogen plays a decisive role in increasing antiproliferative activity. The results also revealed that the position of the methoxy group on the indole ring is a critical determinant of biological activity. Among the synthesized derivatives, compound 9e, containing the 2-methoxycarbonyl-6-methoxy-N-1H-indole moiety exhibited the highest antiproliferative activity, with IC50 values of 0.37, 0.16 and 0.17 μM against HeLa, HT29 and MCF-7 cancer cell lines, respectively, and with considerably lower activity against HL-60 cells (IC50: 18 μM). This derivative also displayed cytotoxic properties (IC50 values ∼1 μM) in the human myeloid leukemia U-937 cell line overexpressing human Bcl-2 (U-937/Bcl-2) via cell cycle progression arrest at the G2-M phase and induction of apoptosis. The results obtained also demonstrated that the antiproliferative activity of this molecule is related to inhibition of tubulin polymerisation. The presence of a methoxy group at the C5- or C6-position of the indole nucleus, as well as the absence of substituents at the N-1-indole position, contributed to the optimal activity of the indole-propenone-3',4',5'-trimethoxyphenyl scaffold. ispartof: JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY vol:33 issue:1 pages:1225-1238 ispartof: location:England status: published

Microtubules are recognized as crucial components of the mitotic spindle during cell division, and, for this reason, the microtubule system is an attractive target for the development of anticancer agents. Continuing our search strategy for novel tubulin targeting-compounds, a new series of 2-alkoxycarbonyl-3-(3′,4′,5′-trimethoxyanilino)-5-aryl/heteroarylthiophene derivatives was designed, synthesized and demonstrated to act as tubulin polymerization inhibitors at the colchicine site. A structure-activity relationship study on the phenyl at the 5-position of the thiophene ring was performed by introducing a variety of substituents containing electron-releasing and electron-withdrawing groups, with the 2-alkoxycarbonyl-3-(3′,4′,5′-trimethoxyanilino)thiophene scaffold being the minimum structural requirement for activity. Of the tested compounds, derivatives 4a, 4c, 4i and 4k possessed the highest overall potency and displayed high antiproliferative activities at submicromolar concentrations, with IC50 values ranging from 0.13 to 0.84 μM against four different cancer cell lines. Three agents (4a, 4c and 4i) in the present series had similar effects, and these were comparable to those of the reference compound combretastatin A-4 (CA-4) as inhibitors of tubulin assembly. The antitubulin effects correlated with the cytostatic activities and indicate that these compounds inhibit cell growth through inhibition of tubulin polymerization by binding at the colchicine site. Compound 4c, containing the 2′-thienyl ring at the 5-position of the 2-methoxycarbonyl-3-(3′,4′,5′-trimethoxyanilino)thiophene scaffold, exhibited substantial antiproliferative activity with a mean IC50 value of 140 nM, inhibited tubulin polymerization with an IC50 value of 1.2 μM, similar to that of CA-4 (IC50: 1.1 μM), and induced apoptosis in HeLa cells.

The 2-amino-3-(p-chlorobenzoyl)thiophene scaffold has been widely employed as a pharmacophore for the identification of small molecules acting as allosteric modulators at the adenosine A1 receptor. A new series of 2-amino-3-(p-chlorobenzoyl)-4-benzyl-5-arylthiophene derivatives, characterized by the absence as well as the presence of electron-releasing or electron-withdrawing groups on the phenyl ring at the 4- and 5-positions of the thiophene ring, were identified as positive allosteric enhancers at the adenosine A1 receptor in binding (saturation, competition and dissociation kinetics) and functional assays. To better understand the positional requirements of substituents on the 2-amino-3-(p-chlorobenzoyl)thiophene core, the corresponding regioisomeric 4-aryl-5-benzylthiophene analogues were synthesized and found to possess reduced allosteric enhancer activity.

Adenosine is an ubiquitous nucleoside involved in various physiological and pathological functions by stimulating A1, A2A, A2B and A3 adenosine receptors (ARs). Allosteric enhancers to A1ARs may represent novel therapeutic agents because they increase the activity of these receptors by mediating a shift to their active form in the A1AR-G protein ternary complex. In this manner, they are able to amplify the action of endogenous adenosine, which is produced in high concentrations under conditions of metabolic stress. A1AR allosteric enhancers could be used as a justifiable alternative to the exogenous agonists that are characterized by receptor desensitization and downregulation. In this review, an analysis of some of the most interesting allosteric modulators of A1ARs has been reported.

In this paper we describe an extension SAR study of pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine nucleus as A3AR antagonist. Our initial aim was to replace the phenylcarbamoyl moiety at the 5 position of PTP nucleus with a thiourea functionality to evaluate the contribution of new structural modification against the A3AR. The synthesized 12-25 were not characterized by the predicted side chain but by a 1,3-disubstituted guanidine and are shown to be interesting A3AR antagonists.

A series of 1,3,6-triphenylpyrazolo[3,4- b ]pyridin-4-one derivatives was designed, synthesized and evaluated for cytotoxic activity in A375 human melanoma and human erythroleukemia (HEL) cells. The new pyrazolopyridones displayed comparable activities to the antitumor compound etoposide. The inhibitory effect of compounds 17, 18, 27 and 32 against topoisomerase II-mediated cleavage activities was measured finding good correlation with the results obtained from MTS assay. Docking studies into bacterial topoisomerase II (DNA Gyrase), topoisomerase IIα and topoisomerase IIβ binding sites in the DNA binding interface were performed.

5-(2-(4-Methoxyphenyl)ethyl)-2-amino-3-methylcarboxylate thiophene (TR560) is the prototype drug of a recently discovered novel class of tumor-selective compounds that preferentially inhibit the proliferation of specific tumor cell types (e.g. leukemia/lymphoma). Here, we further increased tumor selectivity by simplification of the molecule through replacing the 4-methoxyphenyl moiety by an alkyl chain. Several 2-amino-3-methylcarboxylate thiophene derivatives containing at C-5 an alkyl group consisting of at least 6 (hexyl) to 9 (nonyl) carbon units showed pronounced anti-proliferative activity in the mid-nanomolar range with 500- to 1000-fold tumor cell selectivity. The compounds preferentially inhibited the proliferation of T-lymphoma CEM and Molt/4, prostate PC-3, kidney Caki-1 and hepatoma Huh-7 tumor cells, but were virtually inactive against other tumor cell lines including B-lymphoma Raji and cervix carcinoma HeLa cells. The novel prototype drug 3j (containing a 5-heptyl chain) elicited a cytotoxic, rather than cytostatic activity, already after 4 h of exposure. The unusual tumor selectivity could not be explained by a differential uptake (or efflux) of the drug by sensitive versus resistant tumor cells. Exposure of a fluorescent derivative of 3j revealed pronounced uptake of the drug in the cytoplasm, no visible appearance in the nucleus, and a predominant localization in the endoplasmic reticulum. These observations may be helpful to narrow down the intracellular localization and identification of the molecular target of the 5-substituted thiophene derivatives. publisher: Elsevier articletitle: Pronounced anti-proliferative activity and tumor cell selectivity of 5-alkyl-2-amino-3-methylcarboxylate thiophenes journaltitle: European Journal of Medicinal Chemistry articlelink: http://dx.doi.org/10.1016/j.ejmech.2017.03.044 content_type: article copyright: © 2017 The Authors. Published by Elsevier Masson SAS. ispartof: European Journal of Medicinal Chemistry vol:132 pages:219-235 ispartof: location:France status: published

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.

The A(3) adenosine receptor (A(3)AR) subtype is a novel, promising therapeutic target for inflammatory diseases, such as rheumatoid arthritis (RA) and psoriasis, as well as liver cancer. A(3)AR is coupled to inhibition of adenylyl cyclase and regulation of mitogen-activated protein kinase (MAPK) pathways, leading to modulation of transcription. Furthermore, A(3)AR affects functions of almost all immune cells and the proliferation of cancer cells. Numerous A(3)AR agonists, partial agonists, antagonists, and allosteric modulators have been reported, and their structure–activity relationships (SARs) have been studied culminating in the development of potent and selective molecules with drug-like characteristics. The efficacy of nucleoside agonists may be suppressed to produce antagonists, by structural modification of the ribose moiety. Diverse classes of heterocycles have been discovered as selective A(3)AR blockers, although with large species differences. Thus, as a result of intense basic research efforts, the outlook for development of A(3)AR modulators for human therapeutics is encouraging. Two prototypical selective agonists, N6-(3-Iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA; CF101) and 2-chloro-N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA; CF102), have progressed to advanced clinical trials. They were found safe and well tolerated in all preclinical and human clinical studies and showed promising results, particularly in psoriasis and RA, where the A(3)AR is both a promising therapeutic target and a biologically predictive marker, suggesting a personalized medicine approach. Targeting the A(3)AR may pave the way for safe and efficacious treatments for patient populations affected by inflammatory diseases, cancer, and other conditions.

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.

A Sonogashira coupling strategy was employed to synthesize a new series of allosteric modulators for the A1 adenosine receptor based on the 2-amino-3-(p-chlorobenzoyl)-4-substituted thiophene skeleton, with a two-carbon (rigid or flexible) linker between the 5-position of the thiophene ring and a (hetero)aryl or alkyl moiety. Among the compounds characterized by the presence of a common phenylacetylene moiety at the 5-position of the thiophene ring, the neopentyl substitution at the 4-position supported a strong activity. In the series of 4-neopentyl derivatives, the presence of an acetylene spacer at the 5-position of the thiophene is optimal for activity, whereas reduction of the acetylene to an ethyl moiety decreased activity, both in functional and binding assays. Derivatives 4e, 4g-h, 4j, 4l, and 4m were the most promising compounds in binding (saturation and competition) and functional cAMP studies, being able to potentiate agonist [(3)H]CCPA binding to the A1 receptor, with 4e as the best compound of the series. The latter compound also retarded the dissociation of another radiolabeled agonist, [(3)H]NECA, from the receptor.

2-Amino-3-benzoyl thiophenes have been widely reported to act as allosteric enhancers at the A1 adenosine receptor. Their activity can be increased considerably by appropriate substitutions at the 4- and 5-positions of the thiophene ring. Substituent size at the thiophene C-4 position seemed to be a factor closely related to activity, with the 4-neopentyl (2,2-dimethylpropyl) substitution showing the greatest enhanced activity. A wide series of 2-amino-3-aroyl-4-neopentylthiophene derivatives with general structure 3, characterized by the presence of different substituents (bromine, aryl and heteroaryl) at the 5-position of the thiophene ring, have been identified as potent AEs at the A1AR. With only one exception, all of the synthesized compounds proved to be superior to the reference compound PD 81,723 in a functional assay. Derivatives 3p, 3u, 3am, 3ap and 3ar were the most active compounds in binding (saturation and competition) and functional cAMP studies, being able to potentiate agonist [(3)H]CCPA binding to the A1 receptor.

Signal Transducer and Activator of Transcription 5 (STAT5) protein, a component of the STAT family of signaling proteins, is considered to be an attractive therapeutic target because of its involvement in the progression of acute myeloid leukemia. In an effort to discover potent molecules able to inhibit the phosphorylation-activation of STAT5, twenty-two compounds were synthesized and evaluated on the basis of our knowledge of the activity of 2-(3’,4’,5’-trimethoxybenzoyl)-3-iodoacetamido-6-methoxy benzo[b]furan derivative 1 as a potent STAT5 inhibitor. Most of these molecules, structurally related to compound 1, were characterized by the presence of a common 3’,4’,5’-trimethoxybenzoyl moiety at the 2-position of different benzoheterocycles such as benzo[b]furan, benzo[b]thiophene, indole and N-methylindole. Effects on biological activity of the iodoacetamido group and of different moieties (methyl and methoxy) at the C-3 to C-7 positions were examined. In the series of benzo[b]furan derivatives, moving the iodoacetylamino group from the C-4 to the C-5 or C-6 positions did not significantly affect antiproliferative activity. Compounds 4, 15, 20 and 23 blocked STAT5 signals and induced apoptosis of K562 BCR-ABL positive cells. For compound 23, the trimethoxybenzoyl moiety at the 2-position of the benzo[b]furan core was not essential for potent inhibition of STAT5 activation.

Cytostatic agents often do not discriminate in their cytostatic potential between different tumor cell types in vitro. In this study, several 2-aminothiophene-3-carboxylic acid ester derivatives were discovered that show an unusual cytostatic selectivity for several T-cell (but not B-cell) lymphoma, prostate cancer, kidney carcinoma and hepatoma cell lines. Their 50 % cytostatic concentrations were generally in the higher nanomolar range and were approximately 20- to 50-fold lower for these tumor cell types than for any other tumor cell line or non-tumorigenic cells. The tumor-selective compounds caused a more preferential suppression of protein synthesis than DNA or RNA synthesis and the prototype compound 3 resulted in an accumulation of prostate cancer cells in the G1 phase of their cell cycle. Compound 3 was also shown to induce apoptosis in prostate cancer cells. The 2-aminothiophene-3-carboxylic acid ester derivatives represent novel candidate cytostatic agents to be further explored for their tumor-selective potential.

Two new series of inhibitors of tubulin polymerization based on the 2-(alkoxycarbonyl)-3-(3',4',5'-trimethoxyanilino)benzo[b]thiophene and thieno[2,3-b]pyridine molecular skeletons were synthesized and evaluated for antiproliferative activity on a panel of cancer cell lines, inhibition of tubulin polymerization, cell cycle effects, and in vivo potency. Antiproliferative activity was strongly dependent on the position of the methyl group on the benzene portion of the benzo[b]thiophene nucleus, with the greatest activity observed when the methyl was located at the C-6 position. Also, in the smaller thieno[2,3-b]pyridine series, the introduction of the methyl group at the C-6 position resulted in improvement of antiproliferative activity to the nanomolar level. The most active compounds (4i and 4n) did not induce cell death in normal human lymphocytes, suggesting that the compounds may be selective against cancer cells. Compound 4i significantly inhibited in vivo the growth of a syngeneic hepatocellular carcinoma in Balb/c mice.

Recent developments indicate that CB(2) receptor ligands have the potential to become therapeutically important. To explore this potential, it is necessary to develop compounds with high affinity for the CB(2) receptor. Very recently, we have identified the oxazinoquinoline carboxamides as a novel class of CB(2) receptor full agonists. In this paper we describe the medicinal chemistry of a new series of heteroaryl-4-oxopyridine/7-oxopyrimidine derivatives. Some of the reported compounds showed high affinity and potency at the CB(2) receptor while showing only modest affinity for the centrally expressed CB(1) cannabinoid receptor. Moreover, we found that the functionality of these ligands is controlled by the nature of the heteroaryl function condensed with the pyridine ring. In 3,5-cyclic adenosine monophosphate (cAMP) assays, the novel series show dose-dependent effects on the modulation of forskolin-induced cAMP production, revealing different behaviors as full agonists, partial agonists, and inverse agonists.

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Tubulin binding agents (TBAs) are drugs commonly used in cancer therapy as antimitotics. In the last years it has been described that TBAs, like combretastatin A-4 (CA-4), present also vascular disrupting activity and among its derivatives we identified three analogues endowed with potent microtubule depolymerizing activity, higher than that of the lead compound. In this paper we have investigated the anti-vascular activity of these derivatives. We tested the anti-angiogenic effects in human umbilical endothelial cells (HUVEC) and in vivo in chick chorioallantoic membrane assay (CAM), and in a syngeneic tumor mouse model. The three molecules, compound 1 : 1-(3,4,5-trimethoxyphenyl)-5-(4-ethoxyphenyl)-1H-1,2,4-triazole; compound 2 : (1-(3,4,5-trimethoxyphenyl)-5-(4-ethoxyphenyl)-1H-tetrazole, compound- 3 (4-amino-2-p-tolylaminothiazol-5-yl)-(3,4,5-trimethoxyphenyl)-methanone) showed a moderate effect on the growth of HUVEC cells at concentrations below 200 nM. At lower concentrations (5–20 nM), in particular compound 2 , they induced inhibition of capillary tube formation, inhibition of endothelial cell migration and affected endothelial cell morphology as demonstrated by the alteration of the microfilaments network. Moreover, they also increased permeability of HUVEC cells in a time dependent manner. In addition, compounds 1 and 3 , as well as the reference compound CA-4, inhibited VEGF-induced phosphorylation of VE-cadherin and in addition compound 3 prevented the VEGF-induced phosphorylation of FAK. In CAM assay, both compounds 2 and 3 efficiently counteracted the strong angiogenic response induced by bFGF, even at the lowest concentration used (1 pmol/egg). Moreover in a syngenic mouse model, compounds 1 – 3 after a single i.p. injection (30 mg/kg), showed a stronger reduction of microvascular density. Altogether our results identified these derivatives as potential new vascular disrupting agents candidates.

Activation of A1 adenosine receptors (ARs) has been associated with anxiolytic-like effects in different behavioral tests, but development of A1AR agonists for therapeutic use has been hampered, most likely due to the presence of side effects. With the aim to identify a safer approach for the treatment of anxiety, we investigated, in mice, the anxiolytic-like properties of a novel A1AR positive allosteric modulator, TRR469. Acute administration of TRR469 (0.3–3 mg/kg) resulted in robust anxiolytic-like effects in the elevated plus maze, the dark/light box, the open field and the marble burying tests. The magnitude of the anxiolytic action of TRR469 was comparable to that obtained with benzodiazepine diazepam (1 mg/kg). The use of the A1AR antagonist DPCPX (3 mg/kg) suggested that the effects of TRR469 were mediated by this receptor subtype. In contrast to diazepam, the novel positive allosteric modulator did not potentiate the sedative effect of ethanol (3.5 g/kg) evaluated by the loss of righting reflex. While diazepam produced motor coordination impairment in the rotarod test, this effect being enhanced by the presence of ethanol (1.5 g/kg), TRR469 did not elicit locomotor disturbances either when administered alone or in the presence of ethanol. In vitro, TRR469 was able to increase the number of A1AR recognizable by the agonist radioligand [3H]-CCPA in mouse brain regions involved in emotional processes. TRR469 markedly increased the affinity of the agonist CCPA, suggesting the capability, in vivo, to increase the affinity of endogenous adenosine. Taken together, these findings indicate that the positive allosteric modulation of A1AR may represent a promising approach for the treatment of anxiety-related disorders.