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Since the discovery of compound BIX01294 over 10 years ago, only a very limited number of nonquinazoline inhibitors of H3K9-specific methyltransferases G9a and G9a-like protein (GLP) have been reported. Herein, we report the identification of a novel chemotype for G9a/GLP inhibitors, based on the underinvestigated 2-alkyl-5-amino- and 2-aryl-5-amino-substituted 3 H-benzo[ e][1,4]diazepine scaffold. Our research efforts resulted in the identification 12a (EML741), which not only maintained the high in vitro and cellular potency of its quinazoline counterpart, but also displayed improved inhibitory potency against DNA methyltransferase 1, improved selectivity against other methyltransferases, low cell toxicity, and improved apparent permeability values in both parallel artificial membrane permeability assay (PAMPA) and blood-brain barrier-specific PAMPA, and therefore might potentially be a better candidate for animal studies. Finally, the co-crystal structure of GLP in complex with 12a provides the basis for the further development of benzodiazepine-based G9a/GLP inhibitors. Since the discovery of compound BIX01294 over 10 years ago, only a very limited number of nonquinazoline inhibitors of H3K9-specific methyltransferases G9a and G9a-like protein (GLP) have been reported. Herein, we report the identification of a novel chemotype for G9a/GLP inhibitors, based on the underinvestigated 2-alkyl-5-amino- and 2-aryl-5-amino-substituted 3H-benzo[e][1,4]diazepine scaffold. Our research efforts resulted in the identification 12a (EML741), which not only maintained the high in vitro and cellular potency of its quinazoline counterpart, but also displayed improved inhibitory potency against DNA methyltransferase 1, improved selectivity against other methyltransferases, low cell toxicity, and improved apparent permeability values in both parallel artificial membrane permeability assay (PAMPA) and blood-brain barrier-specific PAMPA, and therefore might potentially be a better candidate for animal studies. Finally, the co-crystal structure of GLP in complex with 12a provides the basis for the further development of benzodiazepine-based G9a/GLP inhibitors.

Chemical manipulations performed on 2-methyl-3-carbethoxyquinoline (1), a histone acetyltransferase inhibitor previously identified by our research group and active at the sub-millimolar/millimolar level, led to compounds bearing higher alkyl groups at the C2-quinoline or additional side chains at the C6-quinoline positions. Such compounds displayed at least threefold improved inhibitory potency toward p300 protein lysine acetyltransferase activity; some of them decreased histone H3 and H4 acetylation levels in U937 cells and induced high degrees of apoptosis (three compounds >10-fold higher than compound 1) after treatment of U937 cells.

Structure-activity relationships (SARs) within the 4-phenylquinazoline-2- carboxamide series of translocator protein (TSPO) ligands have been explored further by the synthesis and TSPO binding affinity evaluation of N-benzyl-N-ethyl/methyl derivatives variously decorated at the 6-, 2′-, 4′-, and 4″-positions. Most of the compounds showed high affinity with Ki values in the nanomolar/subnanomolar range. A pharmacophore model was developed and employed to better address SAR data presented by the new TSPO ligands. A subset of the new compounds (5, 8, 12, and 19) were tested for their ability to inhibit the viability of human glioblastoma cell line U343. The observed antiproliferative effect was demonstrated to be specific for compound 19, endowed with the best combination of binding affinity and efficacy. Furthermore, the ability of 19 to induce mitochondrial membrane dissipation (Δψm) substantiated the intracellular pro-apoptotic mechanism activated by the binding of this class of ligands to TSPO. © 2014 American Chemical Society.

Chemical manipulations undertaken on some bis(bromo- and dibromo-phenol) compounds previously reported by us as wide-spectrum epigenetic inhibitors let us to identify bis (bromo- and dibromo-methoxyphenyl) derivatives highly selective for PR-SET7 and EZH2 (compounds 4, 5, 9, and 10). Western blot analyses were carried out in U937 cells to determine the effects of such compounds on the methyl marks related to the tested enzymes (H3K4me1, H3K9me2, H4H20me1, and H3K27me3). The 1,5-bis(3-bromo-4-methoxyphenyl)penta-1,4-dien-3- one 4 (EC50 vs EZH2 = 74.9 μM), tested in U937 cells at 50 μM, induced massive cell death and 28% of granulocytic differentiation, highlighting the potential use of EZH2 inhibitors in cancer. © 2012 Elsevier Masson SAS. All rights reserved.

A series of novel 4-phenylquinazoline-2-carboxamides (1-58) were designed as aza-isosters of PK11195, the well-known 18 kDa translocator protein (TSPO) reference ligand, and synthesized by means of a very simple and efficient procedure. A number of these derivatives bind to the TSPO with K i values in the nanomolar/subnanomolar range, show selectivity toward the central benzodiazepine receptor (BzR) and exhibit structure-affinity relationships consistent with a previously published pharmacophore/topological model of ligand-TSPO interaction. © 2012 American Chemical Society.

A novel class of KAT modulators (long chain alkylidenemalonates, LoCAMs) has been identified. Variations of the alkyl chain length can change the activity profile from inhibition of both KAT3A/KAT2B (as derivative 2a) to the peculiar profile of pentadecylidenemalonate 1b, the first activator/inhibitor of histone acetyltransferases. Together with the powerful apoptotic effect (particularly notable if considering that anacardic acid and other KAT inhibitors are not cell permeable) appoint them as valuable biological tools to understand the mechanisms of lysine acetyltransferases. (C) 2011 Elsevier Ltd. All rights reserved.

An inexpensive and regioselective approach to dihydrothieno[3,2-g]quinoline-4,9-dione is reported. A combination of a mild version of Skraup reaction with a sequential substitution/Michael addition allowed the selective preparation in acceptable yield of a pharmacologically important quinone derivative, previously obtained only in trace and together with the other regioisomer.

We identified a series of 4-hydroxyquinolines bearing a C1 to C15 alkyl chain at the C2 position and a carbethoxy/carboxy group at the C3 position of the quinoline nucleus (MC compounds), endowed with selective inhibitory activity against the p300/CBP HAT enzymes. Enzyme inhibition was investigated using in vitro HAT assays and by western blot analysis of cellular lysates to examine the acetylation levels of histone H3 and alpha-tubulin. When tested in U937 cells, some compounds displayed pro-apoptotic or cytodifferentiating properties.

Pentadecylidenemalonate 1b, a simplified analogue of anacardic acid, was identified as the first mixed activator/inhibitor of histone acetyltransferases (HATs). It potentiates PCAF HAT activity while inhibiting those of p300/CBP and recombinant CBP. The remarkable apoptotic effect together with the ability to selectively acetylate histone versus non-histone substrates appoint 1b as a lead for the development of anticancer drugs.

A number of new compounds bearing two ortho-bromo- and ortho, ortho-dibromophenol moieties linked through a saturated/unsaturated, linear/(poly)cyclic spacer (compounds 1- 9) were prepared as simplified analogues of AMI-5 (eosin), a recently reported inhibitor of both protein arginine and histone lysine methyltransferases (PRMTs and HKMTs). Such compounds were tested against a panel of PRMTs (RmtA, PRMT1, and CARM1) and against human SET7 (a HKMT), using histone and nonhistone proteins as a substrate. They were also screened against HAT and SIRTs, because they are structurally related to some HAT and/or SIRT modulators. From the inhibitory data, some of tested compounds ( 1b, 1c, 4b, 4f, 4j, 4l, 7b, and 7f) were able to inhibit PRMTs, HKMT, HAT, and SIRTs with similar potency, thus behaving as multiple ligands for these epigenetic targets (epi-MLs). When tested on the human leukemia U937 cell line, the epi-MLs induced high apoptosis levels [i.e., 40.7% ( 4l) and 42.6% ( 7b)] and/or massive, dose-dependent cytodifferentiation [i.e., 95.2% ( 1c) and 96.1% ( 4j)], whereas the single-target inhibitors eosin, curcumin, and sirtinol were ineffective or showed a weak effect.

A convenient method for double Michael additions to quinone systems catalyzed by Al 2 O 3 , is reported. The advantages of this method include the use of a cheap and environment-friendly catalyst, a straightforward isolation of the pure product by filtration, high yields, and excellent selectivity, thus providing rapid access to useful building blocks for the preparation of biologically active quinones.

In eukaryotic cells, genes are complexed with core histones and other chromosomal proteins to form the chromatin. The basic unit of chromatin is the nucleosome, a nucleoprotein particle that consists of 147 base pairs of DNA wrapped around a core of histones (H2A, H2B, H3, and H4). The histone lysineand arginine-rich N-terminal tails protrude out of the histone core and are the sites of many types of post-translational modifications such as acetylation, methylation, and phosphorylation. The post-translational modification of histone tails regulates the level of chromatin condensation, and is in turn important for gene transcription. Histone acetylation is one of the best understood histone modifications. The highly regulated activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs) are responsible for the control of specific acetylation levels. Indeed, actively transcribed regions of chromatin (euchromatin) are hyperacetylated in comparison with condensed regions (heterochromatin), which are not accessible to transcription factors. In this scenario, small molecule inhibitors of HDACs can affect the heritable changes in gene expression of specific genes, and are used as drugs for cancer therapy. Histone methylation has also been shown to be important in establishing stable gene-expression patterns. Histone methylation does not alter the overall charge of the histone tails, but has an influence on basicity, hydrophobicity, and on the affinity for anionic molecules such as DNA. Histone tails can be mono-, di-, and trimethylated on the e-amino group of lysine residues, and either monoor dimethylated on arginine residues. Depending on the context, lysine methylation provides either activating or repressing modification. Thus, trimethylation of Lys9 in histone H3 is associated primarily with transcriptional silencing, whereas Lys4 methylation correlates with transcriptional activation. Moreover, aberrant histone methylation has been linked to a number of human diseases such as cancer. Protein arginine methyltransferases (PRMTs) are grouped into two major classes, type I enzymes catalyzing the formation of asymmetric w-N,N-dimethylarginine tails, and type II enzymes catalyzing the formation of symmetric w-N,N-dimethylarginine tails. To date, no mutations have been identified in PRMTs in tumour cells. However, the coactivator-associated arginine methyltransferase (CARM1/PRMT4) is over-expressed in both grade-3 breast tumours and in hormone-dependent prostate tumours. In addition to their role in histone modification, PRMTs target several proteins involved in cell proliferation, signal transduction, mRNA splicing, RNA transport, and protein–protein interactions. PRMT1 regulates the nuclear cytoplasmic shuttling of the heterogeneous nuclear ribonuleoprotein (hnRNP) Npl3p, and methylates Arg3 in H4 facilitating acetylation of H4 by the HAT p300, which leads to transcriptional activation. CARM1 binds the p160 family of nuclear hormone receptor coactivators, and enhances the nuclear receptor-mediated transcription activation through methylation of H3. Whereas studies on PRMTs are in their infancy, it is likely that they hold crucial roles in chromatin remodelling with regulation of gene expression and cellular processes. As such, PRMTs are likely to provide useful targets in the design of new anticancer agents. In 2004, a series of dyes and dye-like compounds were evaluated as small molecule modulators of PRMT and histone lysine methyltransferase (HKMT) activity. In this screen, AMI-1 was described as the first specific PRMT inhibitor, and AMI-5 was one of the most potent, though less selective, compounds (Figure 1). Recently, the fungal metabolite chaetocin was identified and characterized as the first specific inhibitor of the HKMT SU ACHTUNGTRENNUNG(VAR)3-9 (Figure 1). As a part of our medicinal chemistry project aimed at discovering new entities as small molecule modulators of epigenetic targets, we chose the AMI-5 chemical structure as a template and designed a new series of simplified analogues starting from a pharmacophore hypothesis. In this hypothesis, we identified the presence of two o-bromoor o,o-dibromophenol moieties as crucial for having antimethyltransferase activity, and inserted a hydrophobic spacer between the above fragments. In particular, we prepared a series of substituted 1,5-diphenyl-1,4-pentadien-3-ones 1–12 (Figure 2), in which some of them share two or more bro[a] Prof. A. Mai, Dr. S. Valente, Dr. A. Perrone, Dr. R. Ragno, Dr. S. Simeoni Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici Universit$ degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Roma (Italy) Fax: (+39)06-491491 E-mail : antonello.mai@uniroma1.it [b] Dr. D. Cheng, Prof. M. T. Bedford University of Texas M.D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957 (USA) Fax: (+1)512-237-2475 E-mail : mtbedford@mdanderson.org [c] Prof. G. Sbardella Dipartimento di Scienze Farmaceutiche, Universit$ degli Studi di Salerno, via Ponte Don Melillo, 84084 Fisciano (SA) (Italy) [d] Prof. G. Brosch Division of Molecular Biology, Biocenter, Innsbruck Medical University, FritzPreglstrasse 3, 6020 Innsbruck (Austria) [e] Dr. A. Nebbioso, Dr. M. Conte, Prof. L. Altucci Dipartimento di Patologia Generale, Seconda Universit$ degli Studi di Napoli, vico L. De Crecchio 7, 80138 Napoli (Italy) Fax: (+39)081-450-169 E-mail : lucia.altucci@unina2.it Supporting information for this article is available on the WWW under http://www.chemmedchem.org or from the author. Supporting information includes experimental procedures, characterization data for compounds 1–14, molecular modelling investigation, and further biological data on U937 cell line.

Molecular modeling analysis of compounds belonging to the recently published series of dihydro-alkoxy-benzyl-oxopyrimidines (DABOs), such as S-DABOs and DATNOs, gave support to the design of new 2, 6-disubstituted benzyl-DABO derivatives as highly potent and specific inhibitors of the HIV-1 reverse transcriptase (RT). To follow up on the novel DABO derivatives, we decided to investigate the effect of electron-withdrawing substituents in the benzyl unit of the S-DABO skeleton versus their anti-HIV-1 activity. Such chemical modifications impacted the inhibitory activity, especially when two halogen units were introduced at positions 2 and 6 in the phenyl portion of the benzyl group bound to C-6 of the pyrimidine ring. Various 5-alkyl-2-(alkyl(or cycloalkyl)thio)-6-(2, 6-dichloro(or 2,6-difluoro)phenylmethyl)-3, 4-dihydropyrimidin-4(3H)-ones were then synthesized and tested as anti-HIV-1 agents in both cell-based and enzyme (recombinant reverse transcriptase, rRT) assays. Among the various mono- and disubstituted phenyl derivatives, the most potent were those containing a 6-(2,6-difluorophenylmethyl) substituent (F-DABOs), which showed EC50's ranging between 40 and 90 nM and selectivity indexes up to >/=5000. An excellent correlation was found between EC50 and IC50 values which confirmed that these compounds act as inhibitors of the HIV-1 RT. The structure-activity relationships of the newly synthesized pyrimidinones are presented herein.