MV1035 Overcomes Temozolomide Resistance in Patient-Derived Glioblastoma Stem Cell Lines.

Glioblastoma (GBM, grade IV glioma) represents the most aggressive brain tumor and patients with GBM have a poor prognosis. Until now surgical resection followed by radiotherapy and temozolomide (TMZ) treatment represents the standard strategy for GBM. We showed that the imidazobenzoxazin-5-thione MV1035 is able to significantly reduce GBM U87-MG cells migration and invasiveness through inhibition of the RNA demethylase ALKBH5. In this work, we focus on the DNA repair protein ALKBH2, a further MV1035 target resulting from SPILLO-PBSS proteome-wide scale in silico analysis. Our data demonstrate that MV1035 inhibits the activity of ALKBH2, known to be involved in GBM TMZ resistance. MV1035 was used on both U87-MG and two patient-derived (PD) glioma stem cells (GSCs): in combination with TMZ, it has a significant synergistic effect in reducing cell viability and sphere formation. Moreover, MV1035 induces a reduction in MGMT expression in PD-GSCs cell lines most likely through a mechanism that acts on MGMT promoter methylation. Taken together our data show that MV1035 could act as an inhibitor potentially helpful to overcome TMZ resistance and able to reduce GBM migration and invasiveness.

3D proteome-wide scale screening and activity evaluation of a new ALKBH5 inhibitor in U87 glioblastoma cell line.

The imidazobenzoxazin-5-thione MV1035, synthesized as a new sodium channel blocker, has been tested on tumoral cells that differ for origin and for expressed NaV pool (U87-MG, H460 and A549). In this paper we focus on the effect of MV1035 in reducing U87 glioblastoma cell line migration and invasiveness. Since the effect of this compound on U87-MG cells seemed not dependent on its sodium channel blocking capability, alternative off-target interaction for MV1035 have been identified using SPILLO-PBSS software. This software performs a structure-based in silico screening on a proteome-wide scale, that allows to identify off-target interactions. Among the top-ranked off-targets of MV1035, we focused on the RNA demethylase ALKBH5 enzyme, known for playing a key role in cancer. In order to prove the effect of MV1035 on ALKBH5 in vitro coincubation of MV1035 and ALKBH5 has been performed demonstrating a consequent increase of N6-methyladenosine (m6A) RNA. To further validate the pathway involving ALKBH5 inhibition by MV1035 in U87-MG reduced migration and invasiveness, we evaluated CD73 as possible downstream protein. CD73 is an extrinsic protein involved in the generation of adenosine and is overexpressed in several tumors including glioblastoma. We have demonstrated that treating U87-MG with MV1035, CD73 protein expression was reduced without altering CD73 transcription. Our results show that MV1035 is able to significantly reduce U87 cell line migration and invasiveness inhibiting ALKBH5, an RNA demethylase that can be considered an interesting target in fighting glioblastoma aggressiveness. Our data encourage to further investigate the MV1035 inhibitory effect on glioblastoma.

Drug repurposing approaches to fight Dengue virus infection and related diseases.

Dengue is a mosquito-borne viral disease caused by four antigenically distinct serotypes of Dengue Virus (DENV), namely DENV1-4 and is currently considered the most important arthropod-born viral disease in the world. An effective antiviral therapy to treat Dengue Virus infection is still missing and a number of replicative cycle inhibitors are currently under study. Considering the rapid spreading of DENV and the common timeframe required for bringing a new drug on the market, the repurposing of approved drugs used for different diseases to identify novel inhibitors of this pathogen represents an attractive approach for a rapid therapeutic intervention. Herein, we will describe the most recent drug repurposing approaches to fight DENV infection and their implications in antiviral drug-discovery.

Novel sodium channel antagonists in the treatment of neuropathic pain.

INTRODUCTION: Effective and safe drugs for the treatment of neuropathic pain are still an unmet clinical need. Neuropathic pain, caused by a lesion or disease that affects the somatosensory system, is a debilitating and hampering condition that has a great economic cost and, above all, a tremendous impact on the quality of life. Sodium channels are one of the major players in generating and propagating action potentials. They represent an appealing target for researchers involved in the development of new and safer drugs useful in the treatment of neuropathic pain. The actual goal for researchers is to target sodium channels selectively to stop the abnormal signaling that characterizes neuropathic pain while leaving normal somatosensory functions intact. AREAS COVERED: This review covers the most recent publications regarding sodium channel blockers and their development as new treatments for neuropathic pain. The main areas discussed are the natural sources of new blockers, such as venom extracts and the recent efforts from many pharmaceutical companies in the field. EXPERT OPINION: There have been serious efforts by both the pharmaceutical industry and academia to develop new and safer therapeutic options for neuropathic pain. A number of different strategies have been undertaken; the main efforts directed towards the identification of selective blockers starting from both natural products or screening chemical libraries. At this time, researchers have identified and characterized selective compounds against NaV1.7 or NaV1.8 voltage-gated sodium channels but only time will tell if they reach the market.

Discovery of antitubercular 2,4-diphenyl-1H-imidazoles from chemical library repositioning and rational design.

TB, caused by Mycobacterium tuberculosis, is one of the deadliest infections worldwide. The co-infection with HIV and the emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains have further increased the burden for this disease. In the attempt to respond to the constant need of novel therapeutic options, we herein report the discovery of 2,4-diphenyl-1H-imidazoles as effective antitubercular agents, with MIC in the low micromolar range against actively replicating and persistent M. tuberculosis strains. The good activity, along with the lack of toxicity and the feasible synthesis, underscore their value as novel scaffolds for the development of new anti-TB drugs.

Sodium channel blockers: a patent review (2010 - 2014).

INTRODUCTION: Abnormal activity of voltage-gated sodium channels (VGSCs) is related to several pathological processes, including cardiac arrhythmias, epilepsy, cancer, neurodegenerative diseases, spasticity, chronic and neuropathic pain. As such VGSCs are considered important therapeutic targets. AREAS COVERED: This review summarized > 30 patents on sodium channel blockers, having beneficial effects on a number of diseases. Pubmed, http://www.sciencedirect.com/ , SciFinder Scholar, http://ep.espacenet.com/ were used as sources for this review and patents filed 2010 and July 2014 were examined. EXPERT OPINION: Over the past 4 years we assisted to a continuous effort in the discovery of new sodium channel blockers by a large number of pharmaceutical companies. All the different chemical classes presented, and here analyzed, could represent an important breakout but, the lack of precise structural information, with the incompleteness of the biological data hampered the possibility to understand the real 'state of the art' of any of these inventions. Upon analysis of a number of patents in this review, it remains clear that the major hurdle faced by the discovery teams is the ability to develop subtype selective compounds. The development of subtype selective blockers could, in theory, lead to more effective and better tolerated compounds.

Inhibition of NaV1.6 sodium channel currents by a novel series of 1,4-disubstituted-triazole derivatives obtained via copper-catalyzed click chemistry.

We have synthesized and evaluated a series of 1,4-disubstituted-triazole derivatives for inhibition of the rat Na(V)1.6 sodium channel isoform, an isoform thought to play an important role in controlling neuronal firing. Starting from a series of 2,4(1H)-diarylimidazoles previously published, we decided to extend the SAR study by replacing the imidazole with a different heterocyclic scaffold and by varying the aryl substituents on the central aromatic ring. The 1,4-disubstituted 1,2,3-triazoles were prepared employing the copper-catalyzed azide-alkyne cycloaddition (CuAAC). Many of the new molecules were able to block the rNa(v)1.6 currents at 10 µM by over 20%, displaying IC(50) values ranging in the low micromolar, thus indicating that triazole can efficiently replace the central heterocyclic core. Moreover, the introduction of a long chain at C4 of the central triazole seems beneficial for increased rNa(v)1.6 current block, whereas the length of N1 substituent seems less crucial for inhibition, as long as a phenyl ring is not direcly connected to the triazole. These results provide additional information on the structural features necessary for block of the voltage-gated sodium channels. These new data will be exploited in the preparation of new compounds and could result in potentially useful AEDs.

Sodium channel blockers as therapeutic target for treating epilepsy: recent updates.

The voltage-gated sodium channels (VGSCs) are a family of membrane proteins forming a pore, through which they selectively conduct sodium ions inward and outward cell's plasma membranes in response to variations of membrane potentials, playing a fundamental role in controlling cellular excitability. Growing evidences suggest that abnormal VGSCs are involved in the pathophysiology of both acquired and inherited epilepsy. Approximately two dozen drugs are currently marketed for the treatment of epilepsy and most of them act as sodium channel blockers, preventing the return of the channels to the active state by stabilizing the inactive form. Despite the many drugs on the market, 30% of patients continue to experience seizures even in the presence of optimal doses of AEDs, while others continue to suffer from medication induced side effects. Thus, there is a great need to continue the search for new AEDs that are not only more effective, but also have a better side effects profile. For this reason, many efforts have been made in the recent years to identify new sodium channel blockers for the treatment of epilepsy. These studies have led to different classes of compounds, characterized by a great structural diversity. The aim of this review is to provide an introduction on the structure and function of the sodium channels, followed by a brief historical perspective on the sodium channel blockers in use as anticonvulsant drugs. Moreover, it will focus on the medicinal chemistry of the sodium channel blockers recently published (2008-2011) and the drug design/molecular modeling studies related to the receptor.

Anticonvulsant activity of 2,4(1H)-diarylimidazoles in mice and rats acute seizure models.

2,4(1H)-Diarylimidazoles have been previously shown to inhibit hNa(V)1.2 sodium (Na) channel currents. Since many of the clinically used anticonvulsants are known to inhibit Na channels as an important mechanism of their action, these compounds were tested in two acute rodent seizure models for anticonvulsant activity (MES and scMet) and for sedative and ataxic side effects. Compounds exhibiting antiepileptic activity were further tested to establish a dose response curve (ED(50)). The experimental data identified four compounds with anticonvulsant activity in the MES acute seizure rodent model (compound 10, ED(50)=61.7mg/kg; compound 13, ED(50)=46.8mg/kg, compound 17, ED(50)=129.5mg/kg and compound 20, ED(50)=136.7mg/kg). Protective indexes (PI=TD(50)/ED(50)) ranged from 2.1 (compound 10) to greater than 3.6 (compounds 13, 17 and 20). All four compounds were shown to inhibit hNa(V)1.2 in a dose dependant manner. Even if a correlation between sodium channel inhibition and anticonvulsant activity was unclear, these studies identify four Na channel antagonists with anticonvulsant activity, providing evidence that these derivatives could be potential drug candidates for development as safe, new and effective antiepileptic drugs (AEDs).

NMR analysis of a series of imidazobenzoxazines.

The complete (1)H and (13)C NMR assignment of a series of imidazobenzoxazines by a combination of one- and two-dimensional experiments (COSY, HSQC and HMBC) is studied. Moreover, 2D NOESY and 1D selective NOESY are reported. This procedure allows the identification of the regioisomers obtained.

Sodium channel blockers for neuropathic pain.

IMPORTANCE OF THE FIELD: The voltage-gated sodium channels (VGSCs) play a fundamental role in controlling cellular excitability and their abnormal activity is related to several pathological processes, including cardiac arrhythmias, epilepsy, neurodegenerative diseases, spasticity, chronic and neuropathic pain. In particular, neuropathic pain (e.g., postherpetic and trigeminal neuralgia, diabetic neuropathy and spinal cord injury) is a serious clinical problem that affects a high percentage of the world population. Because an altered sodium channel isoform expression profile has been considered one reason for the changes in neuronal excitability, there is a continuous quest for new selective molecules targeting sodium channels for the treatment of chronic pain. AREAS COVERED IN THIS REVIEW: PubMed, http://www.sciencedirect.com/ , SciFinder Scholar and http://ep.espacenet.com/ were used as sources for this review and patents between 2007 and September 2009 were taken into account for the sodium channel blockers molecular classes reviewed and discussed herein. WHAT THE READER WILL GAIN: The sodium channel blockers reported in this review have been categorized into different molecular classes on the basis of their wide structural diversity. This classification, somewhat arbitrary, does not necessarily reflect the presence of pharmacophoric elements but offers a useful way to discuss and comment on structurally homogenous classes of chemotypes recently patented. TAKE HOME MESSAGE: The continuous discoveries in the field of sodium channel blockers, highlighted by the increasing numbers of patent applications published in the last few years and by the numbers of compounds currently in clinical development, underline the importance of this target for the treatment of neuropathic pain. The great difficulty in the design of new selective and active structures, not obtained from old VGSC blockers that are often associated with high risk of adverse effects, is a strong challenge for medicinal chemistry research.

Microwave assisted efficient synthesis of imidazole-based privileged structures.

A simple and efficient microwave assisted synthesis of imidazobenzoxazines, imidazobenzoxazin-5-ones, and imidazobenzoxazin-5-thiones with broad chemistry scope is described. The molecules were prepared both under conventional as well as microwave heating conditions, to provide in high yields with clean and scalable reactions a small library of imidazole-based privileged structures for drug discovery.

Recent advances in the medicinal chemistry of sodium channel blockers and their therapeutic potential.

The voltage-gated sodium channels (VGSCs) play a fundamental role in controlling cellular excitability. Abnormal activity of sodium channels is related to several pathological processes, including cardiac arrhythmias, epilepsy, chronic pain, neurodegenerative diseases and spasticity. In view of this, VGSCs are considered important therapeutic targets for the treatment of these disorders. To date, nine VGSC isoforms have been identified and have a distinct pattern of expression within the human body. In addition, VGSCs also have distinct electrophysiological profiles with differing activation and inactivation states. As such, there is a concerted effort to develop not only isoform selective antagonists, but also antagonists that exhibit state selectivity, particularly to the inactivated state of the channel. This review will provide a brief historical prospective and will primarily focus on recent advances in the development of isoform specific and state selective sodium channel antagonists and the medicinal chemistry involved, surveying the emerging therapeutic fields.

2,4(5)-diarylimidazoles as inhibitors of hNaV1.2 sodium channels: pharmacological evaluation and structure-property relationships.

Sodium (Na) channels continue to represent an important target for the development of novel anticonvulsants. We have synthesized and evaluated a series of 2,4(5)-diarylimidazoles for inhibition of the human neuronal Na(V)1.2 Na channel isoform. Starting with the unsubstituted lead compound previously published 3, SAR studies were performed introducing substituents with different physico-chemical properties. Lipophilicity (log D(7.4)) and basicity (pK(a)) of the compounds were measured and submitted for QSPR investigations. Some of the active compounds described had IC(50) values that were considerably lower than our lead compound. In particular, the m-CF(3) disubstituted 22 was the most active compound, inhibiting hNa(V)1.2 currents within the nanomolar concentration range (IC(50)=200 nM). In comparison, lamotrigine and phenytoin, two clinically used anticonvulsant drugs known to inhibit Na channels, had IC(50)'s values that were greater than 100 microM.

Chiral NMR discrimination of the diastereoisomeric salts of the H3-antagonist 2-[3-(1H-imidazol-4-ylmethyl)piperidin-1-yl]-1H-benzimidazole.

Diastereomeric salts with optically pure (S)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetic acid (MTPA) were used to discriminate the enantiomers of the chiral H(3)-antagonist 2-[3-(1H-imidazol-4-ylmethyl)piperidin-1-yl]-1H-benzimidazole. Chemical-shift differences (Delta delta) in NMR spectra strongly depend on solvent and stoichiometric ratio. The better observable differentiation occurred for the proton at the 2-position of the imidazole ring.

5-Benzylidene-hydantoins: synthesis and antiproliferative activity on A549 lung cancer cell line.

Benzylidene hydantoins have been recently reported as a new class of EGFR inhibitors. We describe here a simple and efficient methodology for the parallel solution-phase synthesis of a library of 5-benzylidene hydantoins, which were evaluated for antiproliferative activity on the human lung adenocarcinoma A549 cell line. Various substituents at positions 1, 3 and 5 on the hydantoin nucleus were examined. In the presence of a 5-benzylidene group and of a lipophilic substituent at position 1, most of the tested compounds inhibited cell proliferation at a concentration of 20 microM. Compound 7 (UPR1024), bearing 1-phenethyl and (E)-5-p-OH-benzylidene substituents, was found to be the most active derivative of the series. It inhibited EGFR autophosphorylation and induced DNA damage in A549 cells. Compound 7 and other synthesized 5-benzylidene hydantoin derivatives increased p53 levels, suggesting that the dual mechanism of action was a common feature shared by compound 7 and other member of the series.

2,4(5)-Diarylimidazoles: synthesis and biological evaluation of a new class of sodium channel blockers against hNa(v)1.2.

A small family of novel 2,4(5)-diarylimidazoles were prepared through a simple and efficient synthesis and evaluated as potential inhibitors of hNa(v)1.2 sodium channel currents. One member of this series (4) exhibited profound inhibition of Na(v)1.2 currents, emerging as a promising lead compound for further structure-activity relationship studies for the development of novel sodium channel blockers.

Dual mechanisms of action of the 5-benzylidene-hydantoin UPR1024 on lung cancer cell lines.

In this study, we examined the mechanism of action of the novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor 5-benzylidene-hydantoin UPR1024, whose structure was designed to interact at the ATP-binding site of EGFR. The compound had antiproliferative and proapoptotic effects when tested on the non-small cell lung cancer cell line A549. The growth inhibitory effect was associated with an accumulation of the cells in the S phase of the cell cycle. Moreover, UPR1024 induced significant level of DNA strand breaks associated with increased expression of p53 and p21(WAF1) proteins, suggesting an additive mechanism of action. The presence of wild-type p53 improved the drug efficacy, although the effect was also detectable in p53 null cells. We also noted apoptotic cell death after treatment with UPR1024 at concentrations above 10 mumol/L for >24 h, with involvement of both the extrinsic and intrinsic pathways. The present data show that UPR1024 may be considered a combi-molecule capable of both blocking EGFR tyrosine kinase activity and inducing genomic DNA damage. UPR1024 or its derivatives might serve as a basis for development of drugs for the treatment of lung cancer in patients resistant to classic tyrosine kinase inhibitors.

Synthesis and stability in biological media of 1H-imidazole-1-carboxylates of ROS203, an antagonist of the histamine H3 receptor.

A series of carbamate derivatives of the H(3) antagonist ROS203 (1) were prepared, and their lipophilicity and steric hindrance were modulated by introducing linear or branched alkyl chains of various lengths. In vitro stability studies were conducted to evaluate how structural modulations affect the intrinsic reactivity of the carbamoyl moiety and its recognition by metabolic enzymes. Linear alkyl carbamates were the most susceptible to enzymatic hydrolysis, with bioconversion rates being higher in rat liver and plasma. Chain ramification significantly enhanced the enzymatic stability of the set, with two derivatives (1g and 1h) being more stable by a factor of 8-40 than the ethyl carbamate 1a. Incubation with bovine serum albumin (BSA) showed a protective role of proteins on chemical and porcine-liver esterase (PLE)-catalyzed hydrolysis. Ex vivo binding data after i.v. administration of 1h revealed prolonged displacement of the labeled ligand [(3)H]-(R)-alpha-methylhistamine ([(3)H]RAMHA) from rat-brain cortical membranes, when compared to 1. However, the high rates of bioconversion in liver, as well as the chemical instability of 1h, suggest that further work is needed to optimize the enzymatic and chemical stability of these compounds.

A practical synthesis of 2,4(5)-diarylimidazoles from simple building blocks.

A simple and efficient approach to selectively obtain 2,4(5)-diarylimidazoles suppressing formation of 2-aroyl-4(5)-arylimidazoles is described. The yield of each of the two products strongly depends on the reaction conditions employed. This reaction provides a simple method to prepare small libraries of biologically active compounds by parallel synthesis.