A Novel Cell-Permeable, Selective, and Noncompetitive Inhibitor of KAT3 Histone Acetyltransferases...

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A Novel Cell-Permeable, Selective, and Noncompetitive Inhibitor ofKAT3 Histone Acetyltransferases from a Combined MolecularPruning/Classical Isosterism ApproachCiro Milite,† Alessandra Feoli,† Kazuki Sasaki,‡,§ Valeria La Pietra,∥ Amodio Luca Balzano,†

Luciana Marinelli,∥ Antonello Mai,⊥ Ettore Novellino,∥ Sabrina Castellano,*,†,# Alessandra Tosco,*,†

and Gianluca Sbardella*,†

†Epigenetic Med Chem Lab, Dipartimento di Farmacia, Universita degli Studi di Salerno, Via Giovanni Paolo II 132, I-84084 Fisciano,Salerno, Italy‡Chemical Genetics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan§Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 4-1-8Honcho, Kawaguchi, Saitama 332-0012, Japan∥Dipartimento di Farmacia, Universita di Napoli “Federico II” Via D. Montesano 49, I-80131 Naples, Italy⊥Dipartimento di Chimica e Tecnologie del Farmaco, “Sapienza” Universita di Roma, P.le A. Moro 5, I-00185 Rome, Italy#Dipartimento di Medicina e Chirurgia, Universita degli Studi di Salerno, Via Salvador Allende, I-84081 Baronissi, Salerno, Italy

*S Supporting Information

ABSTRACT: Selective inhibitors of the two paralogue KAT3 acetyltransferases (CBP and p300) may serve not only as preciouschemical tools to investigate the role of these enzymes in physiopathological mechanisms but also as lead structures for thedevelopment of further antitumor agents. After the application of a molecular pruning approach to the hardly optimizable andnot very cell-permeable garcinol core structure, we prepared many analogues that were screened for their inhibitory effects usingbiochemical and biophysical (SPR) assays. Further optimization led to the discovery of the benzylidenebarbituric acid derivative7h (EML425) as a potent and selective reversible inhibitor of CBP/p300, noncompetitive versus both acetyl-CoA and a histoneH3 peptide, and endowed with good cell permeability. Furthermore, in human leukemia U937 cells, it induced a marked andtime-dependent reduction in the acetylation of lysine H4K5 and H3K9, a marked arrest in the G0/G1 phase and a significantincrease in the hypodiploid nuclei percentage.

■ INTRODUCTION

After 50 years from its discovery,1 lysine acetylation is far frombeing thoroughly understood and remains an intriguing topicfor study. Among the other histone posttranslationalmodifications, this chemical modification neutralizes thepositive charge of lysine side chains, weakening the interactionsbetween histones and DNA and, consequently, relaxing thechromatin structure and making chromosomal DNA moreaccessible.2,3 The enzymes responsible for this transformation,lysine acetyltransferases (KATs),4 are generally classified intotwo major categories, type-A and type-B. The type-B KATs are

predominantly cytoplasmic and acetylate free histones but notthose already deposited into chromatin. Even if they are a morediverse family of enzymes than the type-Bs, nuclear (type-A)KATs can be classified into different groups by structuralhomology and biochemical mechanisms of actions: GCN5(general control nonderepressible 5)-related N-acetyltrans-ferases (GNATs) (including GCN5, PCAF, ELP3, HAT1,and HPA2), the MYST (MOZ, YBF2/SAS3, SAS2 and TIP60)

Received: December 19, 2014

Article

pubs.acs.org/jmc

family, p300 (adenoviral E1A-associated protein of 300 kDa)/CBP (CREB, cyclic-AMP response element binding protein),general transcription factor KATs, and nuclear hormonereceptor-related KATs.5,6

The two paralogues p300 and CBP (KAT3A and KAT3B,respectively; also called CBP/p300) were first described asbinding partners of the adenovirus early region 1A (E1A) andthe cAMP-regulated enhancer (CRE) binding protein,respectively,7,8 but it was later demonstrated that these twoproteins contribute to transcriptional regulation through theirhistone acetyltransferase activity.9,10 The structure of theirHAT domain suggests a “hit-and-run” (Theorell−Chance)catalytic mechanism in which, after binding of acetyl-CoA, thelysyl residue of the substrate peptide snakes through the p300tunnel and reacts with the acetyl group.11

Interacting with a large number of transcription factors, CBPand p300 are involved in different cellular processes; thisimplies that the dysregulation of their activity leads to manyhuman diseases, including cancer. Mutations in the CBP (rarelyp300) gene causes Rubinstein−Taybi syndrome, characterizedby a short stature, moderate to severe intellectual disability,

distinctive facial features, and broad thumbs and first toes.12

Moreover, CBP and p300 were demonstrated to be involved inhematopoietic homeostasis, such that mutations in the CBP/p300 interaction domain of different transcription factors werefound in hematologic malignancies13,14 and chromosomaltranslocations involving CBP or p300 genes are associatedwith leukemia and lymphomas.15−17

CBP and p300 promote prostate cancer progression byactivating androgen receptor-regulated transcription18 andcolon cancer progression by microsatellite instability19 andare involved in the development of drug resistance.20 Apartfrom cancer, CBP/p300 interactions with transcription factorsare involved in the development of a variety of diseases,21

comprising viral diseases,22,23 cognitive disorders, and Alz-heimer’s disease,24−27 diabetes,28−30 and cardiovascular dis-eases.31−34

For all the above considerations, there is an urgent need formodulators of CBP/p300 activity not only as useful tools todissect the role of these KATs in physiological and pathologicalmechanisms but also as potential leads for the development ofdrug candidates for specific diseases. The inhibitors of CBP/

Figure 1. Indirect (top) and direct (bottom) inhibitors of p300. Selective inhibitors are indicated in blue.

Journal of Medicinal Chemistry Article

DOI: 10.1021/jm5019687J. Med. Chem. XXXX, XXX, XXX−XXX

Figure 2. Flowchart of our molecular pruning approach.

Scheme 1a

aReagents and conditions: (a) malonic acid, Ac2O, MW (60 °C, 10 min); (b) Bz2O, MW (160 °C, 30 min), neat; (c) appropriate benzoic acid,DCC, DMAP, TEA, THF, room temperature, overnight; (d) aqueous 6 N HCl, methanol, 1 h; (e) appropriate benzaldehyde, ethanol, reflux, 1 h; (f)Zn, AcOH, room temp, 1 h.

p300 described so far are of two types: the compounds thatinhibit the binding of other proteins targeting the interactingdomains and the derivatives that directly affect the

acetyltransferase activity. To the first class belong thebromodomain-interacting molecules, such as the 5-isoxazolyl-benzimidazoles recently reported as potent and selective

Table 1. Effects of Compounds 1−9 on the Activity of p300 and PCAF

% inhibition (at 100 μM, mean ± SD)a,b,c IC50 (μM)e,f

compd R1 R2, R3 p300/KAT3Bd PCAF/KAT2Bd p300/KAT3Bd

1a Bn H 84.8 ± 0.2 −14.5 ± 7.81b Bn 4-OH 54.4 ± 0.21c Bn 3-OH, 4-OH 48.2 ± 2.22a i-Bu H 34.1 ± 1.2 19.3 ± 2. 42b i-Bu 4-OH 39.7 ± 0.9 3.4 ± 8.22c i-Bu 3-OH, 4-OH 8.4 ± 2.1 −13.2 ± 11.63a allyl H 28.1 ± 3.8 −2.7 ± 0.13b allyl 4-OH 19.1 ± 1.8 3.6 ± 1.83c allyl 3-OH, 4-OH 4.9 ± 1.9 5.0 ± 1.74a Bn H 83.9 ± 6.74b Bn 4-OH 60.3 ± 1.14c Bn 3-OH, 4-OH 59.9 ± 0.65a i-Bu H 23.1 ± 4.05b i-Bu 4-OH 64.6 ± 0.5 −10.5 ± 9.65c i-Bu 3-OH, 4-OH 24.3 ± 2.4 −7.5 ± 7.46a allyl H 28.9 ± 10.36b allyl 4-OH 23.7 ± 0.6 −10.5 ± 9.66c allyl 3-OH, 4-OH 1.1 ± 2.3 −0.6 ± 1.67a Bn H NTg

7b Bn 4-OH 99.2 ± 0.6 −2.3 ± 2.8 2.1 ± 0.27c Bn 3-OH, 4-OH 96.15 ± 0.15 −162.4 ± 8.2 1.6 ± 0.17d Bn 3-OH, 4-OMe 97.28 ± 0.22 −55.6 ± 9.1 5.9 ± 0.37e Bn 3-OMe, 4-OH 97.06 ± 0.18 −25.8 ± 16.4 1.5 ± 0.27f Bn 3-OMe, 4-OMe 88.25 ± 0.85 −8.2 ± 3.7 11.4 ± 0.67g Bn 4-OMe 93.59 ± 1.39 −22.2 ± 1.8 8.0 ± 0.48a i-Bu H NTg

8b i-Bu 4-OH 94.03 ± 0.17 −34.1 ± 2.4 8.5 ± 0.38c i-Bu 3-OH, 4-OH 91.85 ± 0.22 10.6 ± 2.0 5.4 ± 0.28d i-Bu 3-OH, 4-OMe 84.68 ± 0.35 9.3 ± 1.0 31.4 ± 0.98e i-Bu 3-OMe, 4-OH 96.43 ± 0.67 9.2 ± 0.4 4.2 ± 0.38f i-Bu 3-OMe, 4-OMe 45.82 ± 2.91 3.6 ± 3.28g i-Bu 4-OMe 53.78 ± 5.01 3.1 ± 1.49a allyl H NTg

9b allyl 4-OH 64.71 ± 0.71 3.3 ± 0.79c allyl 3-OH, 4-OH 61.65 ± 2.46 3.0 ± 1.29d allyl 3-OH, 4-OMe 49.48 ± 2.10 5.1 ± 1.69e allyl 3-OMe, 4-OH 67.33 ± 0.01 6.6 ± 0.5 26.4 ± 0.79f allyl 3-OMe, 4-OMe 31.15 ± 0.48 11.7 ± 0.29g allyl 4-OMe 59.85 ± 1.96 3.6 ± 3.2curcumin 93.45 ± 0.81 6.5 ± 0.2AA 102.9 ± 2.3 (IC50 33.9 ± 0.7)e,f

C646 86% @10 μMh <10% @10 μMh 1.6 ± 0.2h

aCompounds were tested at a 100 μM fixed concentration. bEnzyme inhibition percentage calculated with respect to DMSO. cValues are the means± SD determined for at least two separate experiments, and they are indicated as percentages. dHistone H3 was used as substrate (5 μM), and[acetyl-3H]-acetyl coenzyme A (3.08 μM) was used as acetyl donor. eCompounds were tested in 10-concentration IC50 mode with 3-fold serialdilutions starting at 100 μM. fData were analyzed with GraphPad Prism software (version 6.0) for IC50 curve fitting.

gCompounds 7a, 8a, and 9awere revealed to be quite sensitive to hydrolysis and any attempt to obtain a pure sample of such derivatives was unsuccessful. hLit.: see refs 48 and49.

ligands,35 those targeting the TAZ1 domain, such aschetomin,36 and those (namely sekikaic acid and lobaric acid)targeting the GACKIX domain37 (Figure 1). The second classinhibit the enzymatic activity of the HAT domain, and inaddition to many nonselective inhibitors (e.g., the naturalproducts anacardic acid,38 garcinol,39 curcumin,40 plumbagin,41

and their analogues42−45 or semisynthetic derivatives;22,46

Figure 1), the only selective p300 HAT inhibitors describedto date are the bisubstrate inhibitor Lys−CoA conjugate,47 therecently described pyrazolone C646,48,49 and the isogarcinolderivative LTK-14.22 The mechanisms for p300 HAT inhibitionby the latter have recently been elucidated, and it has beendemonstrated that, different from the nonspecific HATinhibitors garcinol and isogarcinol, it behaves as a non-competitive inhibitor of both acetyl-CoA and histones.50

From these results, the molecular scaffold of LTK-14 couldbe considered potentially promising for further elaboration toobtain potent and specific p300 inhibitors. Yet it still retains thesynthetic complexity of garcinol and isogarcinol that limitsstudies on structure−activity relationships.Structural simplification represents a drug design strategy to

simplify the structure and shorten synthetic routes whilekeeping or enhancing the biological activity of a natural

bioactive compound.51 Recently, the application of thisapproach to anacardic acid as a lead compound allowed us todisclose diethyl pentadecylidenemalonate (SPV106, Figure 1)as the first mixed activator/inhibitor of protein acetyltrans-ferases.52,53 Derivative SPV106 exhibits inhibitory propertiesagainst CBP/p300, with a potency comparable to anacardicacid, and at the same time, it enhances the acetylating activity ofPCAF. As a result of its peculiar activity profile, SPV106 wassuccessfully used as a chemical probe to correlate Duchennecardiomyopathy to PCAF-mediated lysine acetylation levels ofconnexin 43,54 to investigate the role of KAT enzymes in theregulation of the extinction of conditioned fear and neuronalplasticity,55 to study the role of PCAF acetyltransferase activityin the regulation of nitroglycerin-dependent arterial relaxa-tion,56 and to investigate their role in wound healing.57

Prompted by these successful results, we decided to apply amolecular pruning approach to the molecular scaffold ofgarcinol in which the lead structure was simplified step-by-stepto identify the minimal structural elements required for KATinhibitory activity. Our intention was to rapidly gain access tothe compounds and promptly achieve a comprehensive SARframework. Therefore, we prioritized the straightforwardness ofthe synthesis and applied a classical nitrogen-for-carbon

Figure 3. Inhibitory activities of compounds 1−9: the heat maps depict the percentage of inhibition of p300 activity at a 100 μM concentration ofcompounds 1−9 (top panel) and the IC50 values for selected compounds 7−9 (bottom).

isosteric substitution to replace the polyisoprenylated benzo-phenone core with the more accessible N-substitutedbarbiturate ring. Alternative chains for the pendant prenylmoieties were also considered (Figure 2).The three series of derivatives resulting from this approach,

namely benzoylbarbiturates A, benzylbarbiturates B, andbenzylidenebarbiturates C (Figure 2), were screened using invitro biochemical assays to study their ability to inhibit CBP/p300 and PCAF. The biological effects of the selectedcompounds on the human leukemia U937 cell line were alsoevaluated. Herein, we report the discovery of the benzyliden-barbituric acid derivative 7h (EML425) as a potent andselective inhibitor of CBP/p300.

■ RESULTS AND DISCUSSIONChemistry. All the target compounds were prepared

starting from pyrimidine-2,4,6-triones 13−15. As shown inScheme 1, urea intermediates 10−12, promptly synthesized

from commercially available amines and triphosgene, weretreated with malonic acid in acetic anhydride under microwaveirradiation to provide pyrimidine-2,4,6-triones 13−1558−60 inhigh yields (95−98%). Acylation of pyrimidine-2,4,6-trioneswith benzoic anhydride under microwave irradiation straight-forwardly gave compounds 1a, 2a, and 3a. Unfortunately,substituted benzoic anhydrides only gave poor yields (<25%)under the same conditions. Conversely, running the acylationreaction with the appropriate benzoic acid (4-acetoxybenzoicacid or 2-ethoxybenzo[d][1,3]dioxole-5-carboxylic acid61) inthe presence of DCC and DMAP in dry THF resulted inincreased yields, furnishing the protected phenol and catecholderivatives that, after deprotection with HCl in methanol, gavethe corresponding compounds 1b,c, 2b,c, and 3b,c. Knoeve-nagel reaction between pyrimidine-2,4,6-triones 13−15 and theappropriate benzaldehyde provided benzylidenepyrimidine-2,4,6-triones 7a−h, 8a−g, and 9a−g in excellent yields.Unfortunately, compounds 7a, 8a, and 9a were revealed to

Figure 4. Live-cell studies of p300 inhibition by compound 7b. (A) Absorbance spectrum shows that compound 7b lacks intrinsic fluorescence andis devoid of interference in the emission wavelengths recorded for Histac. (B) Changes in emission ratios and emission ratio time courses (C) ofCOS-7 cells expressing Histac treated with derivative 7b at 30 μM. (D) Representative nucleus of a COS-7 cell transfected with the Histac FRETreporter and treated with 30 μM 7b is shown. Emission ratio (480 nm/535 nm) was calculated for each pixel and pseudocolored according to a“rainbow” color map, with lower values at the blue end of the spectrum and higher values in red. Corresponding phase-contrast images of COS7 cellsare shown in gray scale. Derivative 7b at a final concentration of 30 μM was added to the culture at 0 min.

be quite sensitive to hydrolysis, and any attempt to obtain apure sample of such derivatives was unsuccessful. Derivatives7a−c, 8a−c, and 9a−c were finally reduced with zinc powder inacetic acid to furnish the corresponding saturated compounds4a−c, 5a−c, and 6a−c.Library Design and KAT Inhibitory Assays. Our

molecular simplification strategy applied to the garcinolstructure is schematically depicted in Figure 2. First, thepruning of the condensed cyclohexanone ring turned thebicycle core into the simple cyclohexanetrione. Then theisosteric replacement of nitrogen for carbon provided the easilysynthesizable barbituric acid moiety, providing the first series ofanalogues (compounds of type A in Figure 2). The removal ofthe benzoyl carbonyl group and the introduction ofunsaturation yielded the second (B) and third (C) series ofderivatives, respectively. Subsequently, we decided to explorethe importance of the nature of the carbon chain on the twonitrogen atoms and the significance of the substitution patternon the phenyl ring. Therefore, we introduced an aliphaticsaturated (i-butyl), unsaturated (allyl), or aromatic (benzyl)chain in place of the prenyl substituent on the nitrogens,whereas the phenyl ring was decorated with one or twohydroxyl groups.The first 24 molecules (compounds 1a−c to 6a−c, 7b,c,

8b,c, and 9b,c62) of the initial set of compounds weresynthesized, and their effect on the catalytic activity of thehuman recombinant acetyltransferase enzymes p300 and PCAFwas determined at a fixed 100 μM concentration in HotSpotHAT activity assays, which were performed by ReactionBiology Corporation (Malvern, PA, USA) according to thecompany’s standard operating procedure, using curcumin,40

anacardic acid (AA),38 and C64648 as reference compounds.The results are reported in Table 1 and summarized as heatmaps in Figure 3. We noticed that derivatives of the generalstructure C (Figure 3) were endowed with the highestinhibitory activity against p300; therefore, we decided to alsosynthesize a second set of 12 5-benzylidenebarbituricderivatives (compounds 7d−g, 8d−g, and 9d−g), with theaim of further evaluating the role of the substitution pattern onthe phenyl ring. Indeed, most of the 5-benzylidenebarbiturates(in particular, derivatives 7b−g and 8b, 8c, and 8e) displayed agreater than 90% inhibition of p300 activity, and therefore IC50values were determined (Table 1).As shown in Table 1 and in Figure 3, the activity of the

compounds was markedly affected by the nature of thesubstituent on the nitrogen atoms. In fact, derivatives withbenzyl substituents were always more active than thecorresponding i-butyl-substituted analogues and even moreactive than the allyl-substituted ones (compare, for example, theactivity of compounds 7b and 7c with those of derivatives 8band 8c and 9b and 9c, respectively). With regard to thesubstitution pattern on the phenyl ring, the presence of a 4-hydroxyl substituent was crucial for the inhibitory potency ofthe compounds, which was further increased by the presence ofa second oxygen atom in position 3. In fact, derivative 7bresulted in more potency than curcumin and derivatives 7c and7e were more potent than both curcumin and C646 (Table 1).On the contrary, replacement of the 4-hydroxyl with a methoxygroup reduced the activity of the resulting derivatives (comparethe activities of compounds 7b, 7c, and 7e with those ofcompounds 7g, 7d, and 7f, respectively). It is noteworthy thatthe inhibition activity was selective, as all the compounds werescarcely active against PCAF (Table 1).

Live-Cell Studies of p300 Inhibition. Then we resolvedto investigate whether the identified inhibitors were able totarget acetyltransferase enzymes in cells. To this aim, wedecided to use an assay recently reported by Cole et al.49 basedon the application of a FRET-based reporter, Histac, in live-cellstudies of p300/CBP HAT inhibition. Histac is a FRET-basedreporter system recently developed to allow the visualization ofprotein acetylation in living cells.63 Briefly, it is a FRET-basedindicator, consisting of a fusion protein of a BRDTbromodomain and histone H4 flanked by eCFP and Venusfluorescent proteins, designed to show a change in FRET uponacetylation of lysine residues K5 and K8 in the H4 tail andupon a conformational change that involves the association ofthe bromodomain and the acetylated lysines.Compound 7b, one of the most active compounds in vitro

(Table 1), did not show intrinsic fluorescence and did notinterfere in the emission wavelengths recorded for Histac(Figure 4A), enabling us to examine its ability to influence theHistac FRET signal in COS-7 cells. Therefore, COS-7 cellsexpressing Histac were challenged with 7b at a finalconcentration of 30 μM and then imaged. As shown in Figure4, we observed a strong reduction in the fluorescence ratio (480nm/535 nm) when incubating COS-7 cells transfected withHistac with derivative 7b at a concentration of 30 μM,comparable in magnitude but opposite in direction to thechange in emission ratio induced by the HDAC inhibitor TSA(Figure 4B). It is worth noting that the effect of compound 7bon the emission ratio began immediately after exposure of cellsto the compound and reached a minimum after approximately40 min of incubation, with a clear tendency to return back tothe initial level after a longer time.This assay confirmed a strong inhibition of p300 by 7b in a

live-cell system, although it appears not to be persistent forprolonged incubation times.

SPR-Based Binding Assays. Benzylidenbarbituric acidderivatives 7−9, together with compounds 1a and 4a, chosenas the best inhibitors among the benzoylbarbiturates A and thebenzylbarbiturates B, respectively (Figure 3), were tested bysurface plasmon resonance (SPR) assays to establish whethertheir inhibitor effects were due to direct binding to p300.Toward this aim, the human recombinant KAT3B (aa 1284−

1673) catalytic domain was immobilized (up to approximately10000 RU, response units) on a sensor chip, and tested ligandswere injected at different concentrations over the proteinsurface. To reduce false positives from detergent-sensitive,nonspecific aggregation-based binding, 0.005% NP20 wasadded to the running buffer in all experiments. In addition,to evaluate potential nonspecific binding, all compounds wereinjected on immobilized myoglobin. Equilibrium dissociationconstant (KD) values were derived from the ratio between thekinetic dissociation (kd) and association (ka) constants obtainedby fitting data from all the injections at different concentrationsof each compound using the simple 1:1 Langmuir binding fitmodel in the BIAevaluation software. All compounds wereshown to interact with the immobilized recombinant p300/KAT3B HAT domain, even if a KD measurement was possibleonly for derivatives 4a (KD = (1.1 ± 0.7) × 10−6) and 1a (KD =(2.4 ± 1.3) × 10−7). For the benzylidenebarbituric inhibitors oftype C, while setting up the injection conditions, we observedan unexpected SPR response for subsequent injections at thesame concentration. Specifically, all derivatives gave a high SPRsignal for the first injection immediately after dilution from thestock solution in DMSO (the time passed from the dilution to

the real contact of the molecule with the sensor chip wasestimated to be 2 min), while subsequent injections of the samedilution (after 10 and 15 min) gave a strongly reduced signal.The SPR response of the binding of compound 7b, taken asrepresentative of the series, to immobilized hKAT3B/p300 isshown in Figure 5, whereas all the other sensorgrams aredisplayed in Figure S1 (Supporting Information). On the otherhand, a fresh dilution from stock produced, again, a high signalsuperimposable on the first injection (data not shown).Chemical Stability Assays. The time-dependent outcomes

of the SPR and live-cell assays, together with the instability of

compounds 7a, 8a, and 9a with respect to hydrolysis, promptedus to evaluate the stability of the barbituric acid derivatives inaqueous solution. Therefore, compounds 7b−g, as well asderivatives 1a and 4a, were incubated with phosphate-bufferedsaline (PBS) solution at 25 °C, and the presence of hydrolysisproducts in the samples was evaluated by HPLC analysis after2, 10, 30, 60 min and 24 h (Figure S2, SupportingInformation). The experimental results indicated that the 5-benzoyl- and 5-benzylbarbituric acid derivatives 1a and 4a,respectively, were stable under the conditions routinely used inthe biological assays (Figure S2, bottom panels, Supporting

Figure 5. Sensorgrams obtained from subsequent injections (after 2, 10, and 15 min) of a 10 μM solution of compound 7b on immobilizedhKAT3B/p300 (catalytic domain, aa 1284−1673).

Figure 6. (A) Chemical structure and apparent permeability (Papp, calculated using PAMPA) of derivative 7h. (B) Sensorgrams obtained from theSPR interaction analysis of compound 7h to immobilized hKAT3B/p300 (catalytic domain, aa 1284−1673, top row). Compound was injected at 0.5,1, 5, and 10 μM, giving the concentration-dependent signals shown. (C−D) Concentration−response inhibition of p300 (left panel) and CBP (rightpanel) by compound 7h. The compound was tested in 10-concentration IC50 mode with 3-fold serial dilutions starting at 100 μM. Data wereanalyzed using GraphPad Prism software (version 6.0) for curve fitting, using a sigmoidal concentration−response with a variable slope equation.

Information). On the other hand, as hypothesized, the 5-benzylidenebarbituric acid derivatives, while stable in organicsolvents, were revealed to be unstable in aqueous solutions. Foralmost all the compounds, degradation became clearly evidentafter 10 min of incubation in an aqueous medium, and after 24h, only the presence of hydrolysis products was detected. Theeffect was especially marked for derivatives bearing a hydroxylgroup in the phenyl ring, for which degradation was almostcomplete after only 30 min of incubation in the aqueousmedium (7b−e, Figure S2, Supporting Information). Thesefindings, which are consistent with the SPR results and live-cellassays, suggested that the IC50 values provided by thebiochemical assays might have underestimated the potency ofthe 5-benzylidenebarbituric acid derivatives.Optimization of Chemical Stability. Regardless of their

high in vitro inhibitory activity against p300, the highsusceptibility of benzylidenebarbituric derivatives of type C tohydrolysis appeared to us as a major issue to be addressedbefore considering any further development of the compounds.Therefore, we resolved to modify their structure to overcomethe aqueous instability problem while preserving the activity ofthe parent compounds. We supposed that the insertion of twomethyl groups in the ortho positions of the benzylidene moietyof derivative 7b, one of the most potent compounds designed,could protect the double bond from hydrolysis. As the crucial4-hydroxyl group was not involved, this modification wasexpected to not significantly affect the inhibitory potency.Hence, we synthesized derivative 7h (EML425, Figure 6), andwe first evaluated its chemical stability. HPLC analysis revealedthat the compound did not decompose after exposure to PBSbuffer solution over a period of 24 h (Figure S3, SupportingInformation). Additionally, we evaluated the cell permeabilityof 7h with the well-validated parallel artificial membranepermeability assay (PAMPA) technique,64,65 using the highlypermeable drug propranolol and the poorly permeable drugfurosemide as references. The compound showed an apparentpermeability value (Papp) of 1.9 × 10−6 cm/s, similar to that ofpropranolol (Papp = 4.1 × 10−6 cm/s), and very different fromfurosemide (Papp = 0.09 × 10−6 cm/s).

Once both chemical stability and cell permeability wereestablished, derivative 7h was tested for inhibition of therecombinant acetyltransferase enzymes p300/CBP andKAT2A/2B (Figure 6). We were pleased to find that theintroduction of two methyl groups in the structure of 7bresulted in an analogue (derivative 7h) endowed with potencyand selectivity comparable to those of the parent compound. Infact, 7h efficiently inhibited both p300 and CBP (IC50 values of2.9 and 1.1 μM, respectively; Figure 6C,D) while beingpractically inactive against the enzymes GCN5 and PCAF.It is important to note that due to the time-dependent

instability of aqueous solutions of 5-benzylidenebarbiturates7b−g, a direct comparison of the KAT inhibitory potency ofcompound 7h with those of derivatives 7b−g might not be verymeaningful.

Mechanism of p300 Inhibition. The conjugated doublebond in compound 7h is potentially electrophilic and couldserve as a Michael acceptor and covalently modify the targetenzyme. Therefore, even if the results of the SPR experimentswere not consistent with this hypothesis (Figure 6B), wedecided to investigate whether 7h could generate adducts withthe nucleophile β-mercaptoethanol. HPLC chromatographyanalysis revealed no evidence of reaction under the bufferconditions used in the enzymatic assay (Figure S4, SupportingInformation), thus suggesting that 7h acts as a reversible p300inhibitor.Subsequently, we resolved to evaluate the kinetic mechanism

of p300 inhibition by compound 7h using an AlphaLISAhomogeneous proximity immunoassay. This type of assayallows one to measure enzyme activity by detecting theacetylation of a biotinylated histone H3 peptide usingstreptavidin-coated donor beads and AlphaLISA acceptorbeads conjugated to an antibody directed against the modifiedsubstrate (PerkinElmer). In the presence of the acetyltransfer-ase enzyme, streptavidin donor beads captured by thebiotinylated histone peptide bearing the mark of interest andthe antimark acceptor beads come into proximity. Upon laserirradiation of the donor beads at 680 nm, short-lived singletoxygen molecules produced by the donor beads can come into

Figure 7. (A) Plot of 1/v vs 1/[H3] at a fixed acetyl-CoA concentration (3 μM) and three different concentrations of 7h shows noncompetitiveinhibition. 7h Ki = 9.4 μM, α Ki = 6.8 μM, H3 apparent Km = 30 nM. (B) Plot of 1/v vs 1/[acetyl-CoA] at a fixed H3 concentration (0.05 μM) andthree different concentrations of 7h shows noncompetitive inhibition. 7h Ki = 2 μM, α Ki = 5.7 μM, acetyl-CoA apparent Km = 194 nM.

proximity with the acceptor beads to generate an amplifiedchemiluminescent signal at 615 nm.We explored the inhibitory effect of 7h by performing two

sets of experiments: in the first one, we measured enzymeactivity by varying the histone H3 peptide substrateconcentration while keeping the acetyl-CoA concentrationconstant, while in the second set, we varied the acetyl-CoAconcentration while keeping the H3 peptide concentrationconstant. Figure 7 shows the double-reciprocal plots at threedifferent inhibitor concentrations (0, 5, and 10 μM). Fromthese data, derivative 7h was shown to be a noncompetitiveinhibitor versus both acetyl-CoA and a histone H3 peptide, ableto bind both the free enzyme and the enzyme−substratecomplex, even with unequal affinity constants determined bythe two secondary plots reported in Figure S5 (SupportingInformation).66

Molecular Modeling Studies. As mentioned above,Kundu et al. previously described a noncompetitive (versusboth acetyl-CoA and a histone H3 peptide) mechanism ofinhibition of p300 for isogarcinol derivative LTK-14, for whichan alternative binding site was hypothesized with the aid of ablind docking procedure and then confirmed by site-directedmutagenesis studies.50 This finding was in agreement with aprevious report by Marmorstein and Cole demonstrating theexistence of such a second pocket near the substrate lysinebinding groove and close to the acetylation site (Figure 8A)and showing its importance for peptide substrate binding.11

Herein, to give an insight into the mechanism of action of 7hat an atomic level, a blind docking of this compoundcomprising the entire enzyme (Protein Data Bank (PDB) IDcode 3BIY) was performed. 7h was mainly found, in addition tothe canonical acetyl-CoA and histone pockets, in the same siteas described for LTK-14. On the basis of the structuralsimilarities between 7h and the isogarcinol derivative LTK-14,it appeared reasonable that 7h could occupy the same bindingpocket. A second docking run focused only on the LTK-14pocket, furnishing a clear picture of the 7h binding mode. Asshown in Figure 8B, the barbituric core of 7h is sandwichedbetween W1436 and Y1397 and the C6 carbonyl oxygen H-bonds to the Y1394 hydroxyl group. One of the two benzylmoieties protrudes toward the substrate groove and forms π-interactions with Y1397 and Y1446, while the other benzylgroup makes a T-shaped contact with Y1397 and hydrophobiccontacts with the E1505 carbon chain. The substitutedbenzylidene moiety stretches along the external wall of the

pocket and establishes many hydrophobic interactions with theside chains of the surrounding residues W1436, C1438, andY1446. Finally, the hydroxyl substituent of 7h H-bonds withthe S1441 side chain and the formation of a second H-bondcould be favored by reorientation of the nearby hydroxyl groupof Y1446.Even if a direct comparison of the inhibitory potency of

compound 7h with those of derivatives 7b−g should be takencautiously, this binding mode hypothesis is consistent with theextensive SAR studies discussed above. In fact, bothbenzoylbarbituric (A) and benzylbarbituric (B) scaffolds areendowed with different geometry and rigidity with respect tothe benzylidenebarbituric counterpart (C), therefore, theirbinding poses are not totally superimposable. This is clearlyevidenced by the superimposition of 7h and the N-benzyl-substituted benzoyl derivative 1a. As shown in Figure S6(Supporting Information), only one phenyl ring could beaccommodated inside the pocket together with the barbituriccore, while the other two aromatic moieties are mainly solventexposed. Among the benzylidenebarbiturates, both compounds8 and 9 are weaker binders than the corresponding 7derivatives. In fact, replacement of the two benzyl substituentson the nitrogen atoms with i-Bu or allyl groups causes the lossof the copious π-interactions described above and theestablishment of minimal hydrophobic contacts. With regardto the most potent compounds 7b−g, the binding pose of 7bresulting from the docking calculations is mostly super-imposable with that of 7h, although the lack of the two methylgroups allows for a better accommodation of the benzylidenemoiety, so that the 4-OH substituent forms H-bonds with theside chain of D1444 rather than with S1441 (Figure S7A,Supporting Information). This difference may account for theslightly better activity of 7b with respect to 7h (IC50s of 2.1 and2.9 μM, respectively). The binding affinity is further improvedwhen two hydroxyl substituents are present on C3 and C4 ofthe benzylidene moiety, thus enabling the formation of two H-bonds with both D1444 and S1441 residues (e.g., 7c, IC50 = 1.6μM, Figure S7B, Supporting Information). Replacement of thehydroxyl substituent in the aromatic 3-position with a methoxygroup on the one hand induces the loss of the H-bond toD1444, and on the other hand, allows for hydrophobicinteractions with the Y1446 side chain. Accordingly, derivatives7e and 7c were found to be practically equipotent (IC50s of 1.5and 1.6 μM, respectively). In contrast, the methoxy substituentis not well tolerated in the aromatic 4-position because of the

Figure 8. (A) Magenta spheres indicating the position of the alternative binding site near the substrate tunnel. (B) Putative binding pose of 7h(violet sticks) in the alternative site of p300, shown as a white cartoon. Interacting residues are shown as sticks and colored by atom type, while theH-bonds are represented by black dashed lines.

steric hindrance of the residues forming the pocket wall (S1441and C1438). This could explain the relatively lower activities ofderivatives 7g, 7d, and 7f (IC50s of 8.0, 5.9, and 11.4 μM,respectively).Effects on Cell Cycle, Cell Viability, and Acetylation

Levels. With the stable, potent and selective p300/CBPinhibitor 7h in our hands, we turned our attention toinvestigating its cellular effects. Unfortunately, live-cell studiesof p300 inhibition were not feasible because the compound hasan absorbance maximum at 440 nm, the same wavelength forCFP absorbance (Figure S8, Supporting Information). There-fore, we could not use Histac to gauge the intracellular HATinhibitory activity of 7h. Consequently, we resolved to screenthe effect of 7h on the cell cycle and cell viability in the humanleukemia U937 cell line. To this aim, we incubated U937 cellswith different concentrations of the compound (from 10 to 100μM) for 24 h and assessed cell viability using the MTT assay.We observed that after 24 h, the tested concentrations of thecompound did not significantly reduce the number ofmetabolically active cells in comparison with vehicle (FigureS9, Supporting Information). Then, we analyzed the cell cycleprogression and the percentage of sub-G1 hypodiploid nucleiand observed that, after 72 h of incubation, the treatment with100 μM 7h (Figure 9A−B) induced a marked arrest in the G0/G1 phase and a significant increase in hypodiploid nucleipercentage, as detected by the Nicoletti method.67

We next investigated the effects of compound 7h on cellularhistone acetylation, using modification-specific antibodiesagainst H3K9ac and H4K5ac (Figure 9C). Cells were incubatedfor the indicated times (24, 48, or 72 h) with 7h (100 μM) orthe reference compound garcinol (5 μM),39 and the histoneextracts were then immunoblotted with antibodies to specific

histone acetylation sites. As represented in Figure 9C, weobserved that compound 7h induced a marked and time-dependent reduction in the acetylation of lysine H4K5 andH3K9 in U937 cells.Taken together, all of these findings corroborate the

inhibitory effect of 7h on the intracellular histone acetylationmediated by p300/KAT3B and are consistent with theimportance of this enzyme for control of the G1/S transition.In fact, p300 is required for the orderly G1/S transition inhuman cancer cells, and selective inhibitors, such as C646,induce cell cycle arrest in the G1 phase and apoptosis.48,49,68−71

■ CONCLUSIONSThe two paralogue proteins p300 and CBP are transcriptionalcoactivators with intrinsic histone acetyltransferase activity. Inparticular, p300 plays a crucial role in cell cycle progression,differentiation, and apoptosis, with a distinct associationbetween aberrant p300 activity and malignancies. The activityof this enzyme is required for G1/S transition in cancer cells,and its inhibition induces apoptosis in prostate cancer cells andsuppresses cellular growth in melanoma cells. Therefore, p300has been indicated as a tumor promoter and selective inhibitorsmay serve not only as invaluable chemical tools to study therole of this enzyme in physiopathological mechanisms but alsoas a prospective approach for antitumor therapy.In addition to many nonselective inhibitors (both naturally

occurring and of synthetic origin), the only selective p300/CBPHAT inhibitors described prior to this study were thebisubstrate inhibitor Lys−CoA conjugate,47 which is not cellpermeable, the pyrazolone C646,48 described as competitiveversus acetyl-CoA and noncompetitive versus a histonesubstrate, and the isogarcinol derivative LTK14,22,50 again not

Figure 9. Cell cycle analysis (A) and percentages of cells with hypodiploid nuclei (B) in U937 cells by fluorescence-activated cell sorting (FACS).The U937 cells were treated with the compounds 7h and garcinol at the indicated concentrations for 72 h, stained with propidium iodide, andsubjected to flow cytometric analysis to determine the distribution of cells in each phase of the cell cycle. Data are reported as the mean ± SD of atleast three independent experiments. (C) Western blot analyses performed with compound 7h at 100 μM for 24, 48, and 72 h on the acetylation ofthe specific lysine residues H4K5 (upper lane) and H3K9 (middle lane) in histone extracts from U937 leukemic monocyte lymphoma cells.Acetylation was detected by immunoblotting with antibodies specific for histone acetylation sites as indicated. Total histone H3 was used to checkfor equal loading. Garcinol (5 μM) was used as a reference compound.

very cell permeable and hardly optimizable due to its structuralcomplexity.After the application of a molecular pruning approach to the

garcinol core structure, followed by the isosteric replacement ofnitrogen for carbon, we were able to identify the barbituric acidmoiety as a useful scaffold to easily prepare numerousanalogues and achieve an inclusive SAR framework. Theresulting three series of derivatives (benzoylbarbiturates A,benzylbarbiturates B, and benzylidenebarbiturates C) werescreened for their inhibitory effects against CBP/p300 andPCAF using biochemical and biophysical (SPR) assays. Live-cell studies of p300/CBP HAT inhibition with selectedderivatives were also performed using a FRET-based reportersystem, Histac. Further optimization of the chemical stability ofthe lead compound led to the identification of a relativelypotent and selective small molecule p300 HAT inhibitor, thebenzylidenebarbituric acid derivative 7h (EML425).72 Thecompound was shown to be a reversible inhibitor, non-competitive versus both acetyl-CoA and a histone H3 peptide,and able to bind both the free enzyme and the enzyme−substrate complex, even with unequal affinity constants. Thebest scoring docking poses suggested that the binding site for7h is an alternative pocket lying near the substrate lysinebinding groove and close to the acetylation site. Additionally,the proposed binding mode is consistent with the exper-imentally determined SARs. Additional experiments (site-directed mutagenesis and X-ray studies) are ongoing todefinitively validate these assumptions.Derivative 7h was found to be more potent and selective

than curcumin and anacardic acid, with a potency comparableto that of C646. It is noteworthy that it is endowed with goodcell permeability (Papp = 1.9 × 10−6 cm/s), as assessed by thePAMPA technique. Furthermore, in human leukemia U937cells, it induced a marked and time-dependent reduction in theacetylation of lysine H4K5 and H3K9, a marked arrest in theG0/G1 phase and a significant increase in hypodiploid nucleipercentage.Therefore, 7h may be an invaluable chemical probe not only

for mechanistic studies of p300-mediated lysine acetylation butalso to further investigate the biological role of this KATenzyme and its implications in physiological and/or patho-logical processes.

■ EXPERIMENTAL SECTIONChemistry. General Directions. All chemicals were purchased from

Aldrich Chimica (Milan, Italy) and were of the highest purity. Allsolvents were reagent grade and, when necessary, were purified anddried by standard methods. All reactions requiring anhydrousconditions were conducted under a positive atmosphere of nitrogenin oven-dried glassware. Standard syringe techniques were used foranhydrous addition of liquids. Reactions were routinely monitored byTLC performed on aluminum-backed silica gel plates (Merck DC,Alufolien Kieselgel 60 F254) with spots visualized by UV light (λ =254, 365 nm) or using a KMnO4 alkaline solution. Solvents wereremoved using a rotary evaporator operating at a reduced pressure of∼10 Torr. Organic solutions were dried over anhydrous Na2SO4.Chromatographic purification was done on an automated flash-chromatography system (IsoleraOne, Biotage) using cartridges packedwith KP-SIL, 60 Å (40−63 μm particle size). High performance liquidchromatography (HPLC) was performed on a Shimadzu SPD 20AUV/vis detector (λ = 220 nm) using C-18 column PhenomenexSynergi Fusion RP 80A (75mm × 4.60 mm; 4 μm) at 25 °C using amobile phase A (water + 0.1% TFA) and B (MeCN + 0.1% TFA) at aflow rate of 1 mL/min. Melting points were determined on a StuartSMP30 melting point apparatus in open capillary tubes and are

uncorrected. 1H NMR spectra were recorded at 300 MHz on a BrukerAvance 300 spectrometer. Chemical shifts are reported in δ (ppm)relative to the internal reference tetramethylsilane (TMS). Massspectra were recorded on a Finnigan LCQ DECA TermoQuest (SanJose, USA) mass spectrometer in electrospray positive and negativeionization modes (ESI-MS). Purity of tested compounds wasestablished by combustion analysis, confirming a purity ≥95%.Elemental analyses (C, H, N) were performed on a PerkinElmer2400 CHN elemental analyzer; the analytical results were within±0.4% of the theoretical value.

1,3-Dibenzylpyrimidine-2,4,6(1H,3H,5H)-trione (13).60 A 10mL CEM pressure vessel equipped with a stirrer bar was charged withmalonic acid (300 mg, 2.88 mmol), acetic anhydride (4 mL), and 1,3-dibenzylurea 10 (691 mg, 2.88 mmol). The microwave vial was sealedand heated in a CEM Discover microwave synthesizer to 60 °C(measured by the vertically focused IR temperature sensor) for 10min. After cooling to room temperature, the reaction mixture wasdiluted with cold water (20 mL) and extracted with EtOAc (3 × 10mL). The combined organic layers were washed with brine, dried,filtered, and concentrated in vacuo to give the title compound (870mg) in 98% yield as a white solid, mp 136−138 °C. 1H NMR (300MHz, DMSO-d6): δ 7.38−7.22 (m, 10H), 4.92 (s, 4H), 3.92 (s, 2H).ESI m/z: 307 [M − H]+.

1,3-Diisobutylpyrimidine-2,4,6(1H,3H,5H)-trione (14).58 Syn-thesized in 95% yield starting from 1,3-diisobutylurea 11 following theprocedure described for 13. White solid, mp 68−69 °C. 1H NMR (300MHz, DMSO-d6): δ 3.78 (s, 2H), 3.57 (d, J = 7.3 Hz, 4H), 2.01−1.87(m, 2H), 0.85 (d, J = 6.8 Hz, 12H). ESI m/z: 239 [M − H]+.

1,3-Diallylpyrimidine-2,4,6(1H,3H,5H)-trione (15).59 Synthe-sized in 95% yield starting from 1,3-diallylurea 12 following theprocedure described for 13. White solid, mp 74−76 °C. 1H NMR (300MHz, DMSO-d6): δ 5.87−5.71 (m, 2H), 5.23−5.06 (m, 4H), 4.36−4.29 (m, 4H), 3.81 (s, 2H). ESI m/z: 207 [M − H]+.

5-Benzoyl-1,3-dibenzylpyrimidine-2,4,6(1H,3H,5H)-trione(1a). In a 10 mL CEM pressure vessel equipped with a stirrer bar, 1,3-dibenzylpyrimidine-2,4,6(1H,3H,5H)-trione 13 (123 mg, 0.40 mmol)and benzoic anhydride (181 mg, 0.80 mmol) were melted at 80 °Ctogether. The microwave vial was sealed and heated in a CEMDiscover microwave synthesizer to 160 °C (measured by the verticallyfocused IR temperature sensor) for 30 min. After cooling to roomtemperature, the reaction mixture was taken up with CH2Cl2 (20 mL),washed with brine (20 mL), dried, filtered, and concentrated in vacuo.The crude was purified by silica gel chromatography (CH2Cl2/EtOAc)to give the title compound (155 mg) in 94% yield as a white solid, mp193−194 °C (decomp). 1H NMR (300 MHz, DMSO-d6): δ 7.59−7.51 (m, 3H), 7.46−7.42 (m, 2H), 7.32−7.26 (m, 10H), 5.01 (s, 4H).13C NMR (75 MHz, DMSO-d6): δ 189.98, 164.28, 150.18, 136.72,135.43, 131.26, 128.30, 128.21, 127.48, 127.27, 127.06, 109.04, 95.47,44.00. ESI m/z: 411 [M − H]+. Anal. (C25H20N2O4) C, H, N.

5-Benzoyl-1,3-diisobutylpyrimidine-2,4,6(1H,3H,5H)-trione(2a). Synthesized in 94% yield starting from 14 following theprocedure described for 1a. Yellow solid, mp 170−175 °C (decomp).1H NMR (300 MHz, DMSO-d6): δ 7.41−7.23 (m, 5H), 3.57 (d, J =7.5 Hz, 4H), 2.01−1.94 (m, 2H), 0.79 (d, J = 6.8 Hz, 12H). 13C NMR(75 MHz, DMSO-d6): δ 195.31, 162.80, 143.59, 129.10, 127.70,127.01, 109.04, 93.13, 46.17, 26.71, 19.88, 19.75. ESI m/z: 343 [M −H]+. Anal. (C19H24N2O4) C, H, N.

5-Benzoyl-1,3-diallylpyrimidine-2,4,6(1H,3H,5H)-trione (3a).Synthesized in 96% yield starting from 15 following the proceduredescribed for 1a. Yellow solid, mp 154−158 °C (decomp). 1H NMR(300 MHz, DMSO-d6): δ 7.43−7.27 (m, 5H), 5.85−5.74 (m, 2H),5.06−5.01 (m, 4H), 4.35−4.33 (m, 4H). 13C NMR (75 MHz, DMSO-d6): δ 193.61, 162.61, 150.91, 142.92, 134.00, 129.30, 127.69, 127.02,115.41, 93.58, 41.69. ESI m/z: 311 [M − H]+. Anal. (C17H16N2O4) C,H, N.

5-(4-Acetoxybenzoyl)-1,3-dibenzylpyrimidine-2,4,6-(1H,3H,5H)-trione (1i). To a solution of 4-acetoxybenzoic acid (126mg, 0.70 mmol), DCC (157 mg, 0.76 mmol), and DMAP (8 mg, 0.07mmol) in dry THF (20 mL) at 0 °C, TEA (0.21 mL, 1.6 mmol) wasadded. The temperature was allowed to rise to 20 °C, and a solution of

1,3-dibenzylpyrimidine-2,4,6(1H,3H,5H)-trione 13 (246 mg, 0.80mmol) in dry THF (5 mL) was added dropwise. The resultingmixture was stirred at room temperature for 24 h, and then the solventwas evaporated. The residue was taken up with DCM (30 mL),washed with 1 N HCl (2 × 30 mL) and brine (30 mL), dried, filtered,and concentrated in vacuo. The crude was purified by silica gelchromatography (CH2Cl2/EtOAc) to give the title compound (178mg) in 54% yield as a white solid, mp 146−149 °C (decomp). 1HNMR (300 MHz, DMSO-d6): δ 7.57 (d, J = 8.2 Hz, 2H), 7.51−7.03(m, 10H), 7.07 (d, J = 8.2 Hz, 2H), 4.95 (s, 4H), 2.30 (s, 3H). ESI m/z: 469 [M − H]+.5-(4-Acetoxybenzoyl)-1,3-diisobutylpyrimidine-2,4,6-

(1H,3H,5H)-trione (2i). Synthesized in 51% yield starting from 14following the procedure described for 1i. White solid, mp 122−125 °C(decomp). 1H NMR (300 MHz, DMSO-d6): δ 7.49 (d, J = 7.8 Hz,2H), 7.07 (d, J = 7.8 Hz, 2H), 3.59 (d, J = 8.2 Hz, 4H), 2.28 (s, 3H),2.03−1.89 (m, 2H), 0.81 (d, J = 6.9 Hz, 12H). ESI m/z: 401 [M −H]+.5-(4-Acetoxybenzoyl)-1,3-diallyl-pyrimidine-2,4,6-

(1H,3H,5H)-trione (3i). Synthesized in 58% yield starting from 15following the procedure described for 1a. White solid, mp 118−121°C (decomp). 1H NMR (300 MHz, DMSO-d6): δ 7.51 (d, J = 8.2 Hz,2H), 7.06 (d, J = 8.2 Hz, 2H), 6.01−5.60 (m, 2H), 5.16−4.88 (m,4H), 4.39−4.28 (m, 4H), 2.28 (s, 3H). ESI m/z: 369 [M − H]+.1,3-Dibenzyl-5-(2-ethoxybenzo[d][1,3]dioxole-5-carbonyl)-

pyrimidine-2,4,6(1H,3H,5H)-trione (1j). Synthesized in 58% yieldstarting from 15 and 2-ethoxybenzo[d][1,3]dioxole-5-carboxylic acid61

following the procedure described for 1i. White solid, mp 214−215 °C(decomp). 1H NMR (300 MHz, DMSO-d6): δ 7.38−7.07 (m, 13H),6.91 (d, J = 8.0 Hz, 1H), 4.94 (s, 4H), 3.71 (q, J = 7.1 Hz, 2H), 1.19 (t,J = 7.1 Hz, 3H). ESI m/z: 499 [M − H]+.1,3-Diisobutyl-5-(2-ethoxybenzo[d][1,3]dioxole-5-carbonyl)-

pyrimidine-2,4,6(1H,3H,5H)-trione (2j). Synthesized in 56% yieldstarting from 14 and 2-ethoxybenzo[d][1,3]dioxole-5-carboxylic acidfollowing the procedure described for 1i. White solid, mp 169−170 °C(decomp). 1H NMR (300 MHz, DMSO-d6): δ 7.13−6.90 (m, 4H),3.72 (q, J = 7.2 Hz, 2H), 3.58 (d, J = 7.3 Hz, 4H), 2.03−1.89 (m, 2H),1.06 (t, J = 7.2 Hz, 3H), 0.81 (d, J = 6.6 Hz, 12H). ESI m/z: 431 [M −H]+.1,3-Diallyl-5-(2-ethoxybenzo[d][1,3]dioxole-5-carbonyl)-

pyrimidine-2,4,6(1H,3H,5H)-trione (3j). Synthesized in 52% yieldstarting from 15 and 2-ethoxybenzo[d][1,3]dioxole-5-carboxylic acidfollowing the procedure described for 1i. White solid, mp 190−191 °C(decomp). 1H NMR (300 MHz, DMSO-d6): δ 7.13−6.89 (m, 4H),5.94−5.61 (m, 2H), 5.44−4.72 (m, 4H), 4.55−4.16 (m, 4H), 3.70 (q, J= 7.1 Hz, 2H), 1.18 (t, J = 7.1 Hz, 3H). ESI m/z: 399 [M − H]+.1,3-Dibenzyl-5-(4-hydroxybenzoyl)pyrimidine-2,4,6-

(1H,3H,5H)-trione (1b). To a solution of 1i (235 mg, 0.50 mmol) inmethanol (2 mL), aqueous 6 M HCl solution (5 mL) was added. Theresulting mixture was stirred at room temperature for 1 h and thenconcentrated in vacuo and extracted with EtOAc (3 × 10 mL). Thecombined organic phases were washed with brine (10 mL), dried,filtered, and concentrated in vacuo. The crude was purified by silica gelchromatography (CH2Cl2/EtOAc) to give the title compound (193mg) in 90% yield as a white solid, mp 270−273 °C (decomp). 1HNMR (300 MHz, DMSO-d6): δ 9.75 (brs, 1H), 7.44 (d, J = 7.5 Hz,2H), 7.29−7.19 (m, 10H), 6.65 (d, J = 7.5 Hz, 2H), 4.92 (s, 4H). 13CNMR (75 MHz, DMSO-d6) δ 193.11, 162.12, 159.65, 151.98, 139.00,133.17, 130.72, 127.92, 127.20, 126.42, 113.80, 92.90, 42.62. ESI m/z:427 [M − H]+. Anal. (C25H20N2O5) C, H, N.1,3-Diisobutyl-5-(4-hydroxybenzoyl)pyrimidine-2,4,6-

(1H,3H,5H)-trione (2b). Synthesized in 87% yield starting from 2ifollowing the procedure described for 1b. White solid, mp 235−236°C (decomp). 1H NMR (300 MHz, DMSO-d6) δ 10.32 (brs, 1H),7.52 (d, J = 8.4 Hz, 2H), 6.80 (d, J = 8.4 Hz, 2H), 3.66 (d, J = 7.3 Hz,4H), 2.06−1.93 (m, 2H), 0.86 (d, J = 6.7 Hz, 12H). 13C NMR (75MHz, DMSO-d6) δ 189.24, 161.50, 150.24, 131.78, 124.97, 114.19,94.19, 47.59, 26.71, 19.82. ESI m/z: 359 [M − H]+. Anal.(C19H24N2O5) C, H, N.1,3-Dial lyl-5-(4-hydroxybenzoyl)pyrimidine-2,4,6-

(1H,3H,5H)-trione (3b). Synthesized in 92% yield starting from 3i

following the procedure described for 1b. White solid, mp 245−247°C (decomp). 1H NMR (300 MHz, DMSO-d6) δ 9.80 (brs, 1H), 7.29(d, J = 8.2 Hz, 2H), 6.66 (d, J = 8.2 Hz, 2H), 5.86−5.72 (m, 2H),5.06−4.99 (m, 4H), 4.36−4.30 (m, 4H). 13C NMR (75 MHz, DMSO-d6) δ 193.20, 162.61, 159.65, 150.97, 132.94, 130.62, 115.45, 113.73,93.97, 41.82, 40.35. ESI m/z: 327 [M − H]+. Anal. (C17H16N2O5) C,H, N.

1,3-Dibenzyl-5-(3,4-dihydroxybenzoyl)pyrimidine-2,4,6-(1H,3H,5H)-trione (1c). Synthesized in 85% yield starting from 1jfollowing the procedure described for 1b. White solid, mp 250−252°C (decomp). 1H NMR (300 MHz, DMSO-d6): δ 9.04 (brs, 2H),7.31−7.06 (m, 10H), 9.95−6.90 (m, 1H), 6.62 (d, J = 7.3 Hz, 1H),4.94 (s, 4H). 13C NMR (75 MHz, DMSO-d6) δ 194.70, 193.77,162.21, 151.88, 151.05, 148.17, 143.93, 138.91, 137.86, 133.56, 127.94,127.25, 126.52, 121.09, 116.35, 114.09, 94.13, 92.66, 43.13, 42.70. ESIm/z: 443 [M − H]+. Anal. (C25H20N2O6) C, H, N.

5-(3,4-Dihydroxybenzoyl)-1,3-diisobutylpyrimidine-2,4,6-(1H,3H,5H)-trione (2c). Synthesized in 87% yield starting from 2jfollowing the procedure described for 1b. White solid, mp 227−229°C (decomp). 1H NMR (300 MHz, DMSO-d6): δ 9.36, 9.26 (2brs,1H), 8.93, 8.84 (2brs, 1H), 7.00, 6.86 (2s, 1H), 6.93, 6.76 (2d, J = 7.2Hz, 1H) 6.68−6.57 (m, 1H), 3.60, 3.54 (2d, J = 7.2 Hz, 4H), 2.02−1.91 (m, 2H), 0.81 (d, J = 6.7 Hz, 12H). 13C NMR (75 MHz, DMSO-d6) δ 194.58, 192.35, 163.43, 151.60, 148.47, 147.72, 143.88, 133.87,121.36, 120.57, 116.57, 116.19, 114.12, 94.08, 93.33, 46.70, 26.73,19.90. ESI m/z: 375 [M − H]+. Anal. (C19H24N2O6) C, H, N.

1,3-Diallyl-5-(3,4-dihydroxybenzoyl)pyrimidine-2,4,6-(1H,3H,5H)-trione (3c). Synthesized in 85% yield starting from 3jfollowing the procedure described for 1b. White solid, mp 203−205°C (decomp). 1H NMR (300 MHz, DMSO-d6): δ 9.33, 9.31 (2brs,1H), 8.92, 8.90 (2brs, 1H), 7.04, 6.89 (2s, 1H), 9.96, 6.77 (2d, J = 8.1Hz, 1H), 6.70−6.62 (m, 1H), 5.86−5.74 (m, 2H), 5.08−5.01 (m, 4H),4.36−4.32 (m, 4H). 13C NMR (75 MHz, DMSO-d6) δ 194.57, 192.63,162.31, 160.02, 155.83, 150.84, 148.30, 143.84, 133.97, 133.38, 121.32,116.49, 115.48, 114.13, 109.02, 95.21, 92.67, 41.80. ESI m/z: 343 [M− H]+. Anal. (C17H16N2O6) C, H, N.

5-Benzylidene-1,3-dibenzylpyrimidine-2,4,6(1H,3H,5H)-tri-one (7a). A solution of 13 (148 mg, 0.48 mmol) and benzaldehyde(51 mg, 0.42 mmol) in absolute ethanol (5 mL) was heated underreflux for 1 h. After cooling to room temperature, the solvent wasevaporated to give TLC pure derivative 7a. Any attempt to obtain apure sample of the title compound failed (small amounts ofbenzaldehyde were always detected). Therefore, compound 7a wasused in the next step without further purification.

5-Benzylidene-1,3-diisobutylpyrimidine-2,4,6(1H,3H,5H)-tri-one (8a). Synthesized starting from 14 and benzaldehyde followingthe procedure described for 7a and used in the next step withoutfurther purification.

5-Benzylidene-1,3-diallylpyrimidine-2,4,6(1H,3H,5H)-trione(9a). Synthesized starting from 15 and benzaldehyde following theprocedure described for 7a and used in the next step without furtherpurification.

1,3-Dibenzyl-5-(4-hydroxybenzylidene)pyrimidine-2,4,6-(1H,3H,5H)-trione (7b). Synthesized in 94% yield starting from 13and 4-hydroxybenzaldehyde following the procedure described for 7aand purified by recrystallization from ethanol. Yellow solid, mp 223−225 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.90 (brs, 1H), 8.38 (s,1H), 8.33 (d, J = 8.4 Hz, 2H), 7.35−7.23 (m, 10H), 6.89 (d, J = 8.4Hz, 2H), 5.06 (s, 4H). 13C NMR (75 MHz, DMSO-d6) δ 163.39,162.50, 160.51, 157.55, 150.91, 138.47, 137.04, 136.97, 128.23, 127.32,127.18, 127.04, 126.97, 123.77, 115.54, 113.54, 44.90, 44.28. ESI m/z:411 [M − H]+. Anal. (C25H20N2O4) C, H, N.

1,3-Diisobutyl-5-(4-hydroxybenzylidene)pyrimidine-2,4,6-(1H,3H,5H)-trione (8b). Synthesized in 94% yield starting from 14and 4-hydroxybenzaldehyde following the procedure described for 7aand purified by recrystallization from ethanol. Yellow solid, mp 216−217 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.82 (brs, 1H), 8.31−8.28 (m, 3H), 6.89 (d, J = 8.5 Hz, 2H), 3.70 (d, J = 7.2 Hz, 4H), 2.06−1.96 (m, 2H), 0.84 (d, J = 6.6 Hz, 12H. 13C NMR (75 MHz, DMSO-d6): δ 163.04, 162.64, 160.74, 156.79, 151.11, 138.15, 123.79, 115.44,

113.81, 48.34, 47.68, 26.79, 19.89. ESI m/z: 343 [M − H]+. Anal.(C19H24N2O4) C, H, N.1,3-Diallyl-(4-hydroxybenzylidene)pyrimidine-2,4,6-

(1H,3H,5H)-trione (9b). Synthesized in 94% yield starting from 15and 4-hydroxybenzaldehyde following the procedure described for 7aand purified by recrystallization from ethanol. Yellow solid, mp 166−167 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.87 (brs, 1H), 8.33−8.30 (m, 3H), 6.89 (d, J = 8.5 Hz, 2H), 5.91−5.79 (m, 2H), 5.19−5.09(m, 4H), 4.46−4.43 (m, 4H). 13C NMR (75 MHz, DMSO-d6): δ163.21, 162.10, 160.16, 157.05, 150.05, 138.29, 132.65, 132.54, 123.74,116.34, 116.28, 115.48, 113.65, 43.61, 43.02. ESI m/z: 311 [M − H]+.Anal. (C17H16N2O4) C, H, N.1,3-Dibenzyl-5-(3,4-dihydroxybenzylidene)pyrimidine-

2,4,6(1H,3H,5H)-trione (7c). Synthesized in 86% yield starting from13 and 3,4-dihydroxybenzaldehyde following the procedure describedfor 7a and purified by recrystallization from EtOAc/hexane. Yellowsolid, mp 165−167 °C. 1H NMR (300 MHz, DMSO-d6); δ 10.53 (brs,1H), 9.52 (brs, 1H), 8.29 (s, 1H), 8.21 (d, J = 2.0 Hz, 1H), 7.66 (dd, J= 8.1, 2.0 Hz, 2H), 7.36−7.24 (m, 10H), 6.87 (d, J = 8.1 Hz, 1H), 5.06(s, 4H). 13C NMR (75 MHz, DMSO-d6): δ 158.07, 152.90, 150.91,144.85, 137.04, 131.78, 128.23, 127.19, 126.99, 124.27, 121.42, 115.41,112.93, 44.88, 44.32. ESI m/z: 427 [M − H]+. Anal. (C25H20N2O5) C,H, N.5-(3,4-Dihydroxybenzylidene)-1,3-diisobutylpyrimidine-

2,4,6(1H,3H,5H)-trione (8c). Synthesized in 86% yield starting from14 and 3,4-dihydroxybenzaldehyde following the procedure describedfor 7a and purified by recrystallization from EtOAc/hexane. Yellowsolid, mp 184−186 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.43 (brs,1H), 9.47 (brs, 1H), 8.22 (s, 1H), 8.16 (d, J = 2.0 Hz, 1H), 7.62 (dd, J= 8.2, 2.0 Hz, 2H), 6.86 (d, J = 8.2 Hz, 1H), 3.70 (d, J = 7.2 Hz, 4H),2.06−1.96 (m, 2H), 0.87 (d, J = 6.7 Hz, 12H). 13C NMR (75 MHz,DMSO-d6): δ 158.52, 153.76, 152.38, 146.03, 132.60, 125.50, 122.46,116.58, 114.51, 49.44, 49.04, 28.02, 21.14. ESI m/z: 359 [M − H]+.Anal. (C19H24N2O5) C, H, N.1,3-Diallyl-5-(3,4-dihydroxybenzylidene)pyrimidine-2,4,6-

(1H,3H,5H)-trione (9c). Synthesized in 96% yield starting from 15and 3,4-dihydroxybenzaldehyde following the procedure described for7a and purified by recrystallization from EtOAc/hexane. Yellow solid,mp 132−133 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.43 (brs, 1H),9.50 (brs, 1H), 8.24 (s, 1H), 8.20 (d, J = 2.1 Hz, 1H), 7.65 (dd, J = 8.4,2.1 Hz, 2H), 6.87 (d, J = 8.4 Hz, 1H), 5.92−5.79 (m, 2H), 5.19−5.09(m, 4H), 4.46−4.43 (m, 4H). 13C NMR (75 MHz, DMSO-d6): δ157.55, 152.67, 150.05, 144.81, 132.63, 131.51, 124.23, 121.31, 116.25,115.35, 113.04, 43.55, 43.04. ESI m/z: 327 [M − H]+. Anal.(C17H16N2O5) C, H, N.1,3-Dibenzyl-5-(3-hydroxy-4-methoxybenzylidene)-

pyrimidine-2,4,6(1H,3H,5H)-trione (7d). Synthesized in 98% yieldstarting from 13 and 3-hydroxy-4-methoxybenzaldehyde following theprocedure described for 7a and purified by recrystallization fromethanol. Yellow solid, mp 192−193 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.46 (brs, 1H), 8.32 (s, 1H), 8.11 (d, J = 2.2 Hz, 1H), 7.73 (dd, J= 8.7, 2.2 Hz, 1H), 7.35−7.22 (m, 10H), 7.08 (d, J = 8.7 Hz, 1H), 5.05(s, 4H), 3.89 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 162.41,160.36, 157.57, 153.22, 150.87, 145.77, 136.99, 136.92, 130.58, 128.20,127.31, 127.15, 127.01, 126.95, 125.38, 120.42, 114.44, 111.32, 55.78,44.92, 44.29. ESI m/z: 441 [M − H]+. Anal. (C26H22N2O5) C, H, N.1,3-Diisobutyl-5-(3-hydroxy-4-methoxybenzylidene)-

pyrimidine-2,4,6(1H,3H,5H)-trione (8d). Synthesized in 98% yieldstarting from 14 and 3-hydroxy-4-methoxybenzaldehyde following theprocedure described for 7a and purified by recrystallization fromethanol. Yellow solid, mp 154−155 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.42 (brs, 1H), 8.25 (s, 1H), 8.06 (d, J = 2.2 Hz, 1H), 7.69 (dd, J= 8.6, 2.2 Hz, 1H), 7.07 (d, J = 8.6 Hz, 1H), 3.89 (s, 3H), 3.70 (d, J =7.1 Hz, 4H), 2.14−1.92 (m, 2H), 0.87 (d, J = 6.7 Hz, 12H). 13C NMR(75 MHz, DMSO-d6): δ 162.55, 160.59, 156.78, 152.91, 151.10,145.73, 130.17, 125.42, 120.26, 114.71, 111.27, 55.74, 48.38, 47.70,26.78, 19.90. ESI m/z: 373 [M − H]+. Anal. (C20H26N2O5) C, H, N.1,3-Diallyl-5-(3-hydroxy-4-methoxybenzylidene)pyrimidine-

2,4,6(1H,3H,5H)-trione (9d). Synthesized in 96% yield starting from15 and 3-hydroxy-4-methoxybenzaldehyde following the procedure

described for 7a and purified by recrystallization from ethanol. Yellowsolid, mp 153−154 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.44 (s,1H), 8.27 (s, 1H), 8.10 (d, J = 2.2 Hz, 1H), 7.72 (dd, J = 8.6, 2.2 Hz,1H), 7.07 (d, J = 8.6 Hz, 1H), 5.91−5.79 (m, 2H), 5.21−5.09 (m,4H), 4.47−4.43 (m, 4H), 3.89 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 163.29, 161.28, 158.34, 154.33, 151.28, 147.02, 133.88, 133.77,131.66, 126.62, 121.58, 117.61, 117.54, 115.79, 112.54, 57.02, 44.90,44.29. ESI m/z: 341 [M − H]+ Anal. (C18H18N2O5) C, H, N.

1,3-Dibenzyl-5-(4-hydroxy-3-methoxybenzylidene)-pyrimidine-2,4,6(1H,3H,5H)-trione (7e). Synthesized in 98% yieldstarting from 13 and 4-hydroxy-3-methoxybenzaldehyde following theprocedure described for 7a and purified by recrystallization fromethanol. Yellow solid, mp 169−170 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.63 (brs, 1H), 8.38 (s, 1H), 8.23 (d, J = 2.1 Hz, 1H), 7.90 (dd,J = 8.6, 2.1 Hz, 1H), 7.36−7.22 (m, 10H), 6.91 (d, J = 8.6 Hz, 1H),5.06 (s, 4H), 3.80 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 162.52,160.63, 157.71, 153.43, 146.92, 137.00, 132.26, 128.21, 127.28, 127.00,124.12, 118.37, 115.34, 113.51, 55.59, 44.85, 44.31. ESI m/z: 441 [M− H]+. Anal. (C26H22N2O5) C, H, N.

1,3-Diisobutyl-5-(4-hydroxy-3-methoxybenzylidene)-pyrimidine-2,4,6(1H,3H,5H)-trione (8e). Synthesized in 98% yieldstarting from 14 and 4-hydroxy-3-methoxybenzaldehyde following theprocedure described for 7a and purified by recrystallization fromethanol. Yellow solid, mp 108−109 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.56 (brs, 1H), 8.33 (s, 1H), 8.21 (d, J = 2.0 Hz, 1H), 7.89 (dd,J = 8.4, 2.0 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 3.84 (s, 3H), 3.73 (d, J =7.3 Hz, 4H), 2.10−1.98 (m, 2H), 0.89 (d, J = 6.7 Hz, 12H). 13C NMR(75 MHz, DMSO-d6): δ 162.72, 160.90, 157.00, 153.08, 151.11,146.89, 131.83, 124.15, 118.33, 115.30, 113.78, 55.63, 48.32, 47.67,26.81, 19.90. ESI m/z: 373 [M − H]+. Anal. (C20H26N2O5) C, H, N.

1,3-Diallyl-5-(4-hydroxy-3-methoxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (9e). Synthesized in 93% yield starting from15 and 4-hydroxy-3-methoxybenzaldehyde following the proceduredescribed for 7a and purified by recrystallization from ethanol. Yellowsolid, mp 130−132 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.60 (brs,1H), 8.34 (s, 1H), 8.28 (d, J = 2.0 Hz, 1H), 7.89 (dd, J = 8.6, 2.0 Hz,1H), 6.92 (d, J = 8.6 Hz, 1H), 5.93−5.80 (m, 2H), 5.20−5.09 (m,4H), 4.47−4.44 (m, 4H), 3.83 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 162.13, 160.28, 157.29, 153.28, 150.03, 146.93, 132.60, 132.15,124.12, 118.39, 116.26, 115.32, 113.58, 55.64, 43.59, 43.02. ESI m/z:341 [M − H]+. Anal. (C18H18N2O5) C, H, N.

1,3-Dibenzyl-5-(3,4-dimethoxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (7f). Synthesized in 91% yield starting from13 and 3,4-dimethoxybenzaldehyde following the procedure describedfor 7a and purified by recrystallization from ethanol. Yellow solid, mp173−174 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.40 (s, 1H), 8.15(d, J = 2.1 Hz, 1H), 7.95 (dd, J = 8.6, 2.1 Hz, 1H), 7.36−7.24 (m, 8H),7.12 (d, J = 8.6 Hz, 1H), 5.06 (s, 4H), 3.88 (s, 3H), 3.77 (s, 3H). 13CNMR (75 MHz, DMSO-d6): δ 162.35, 157.21, 153.86, 150.85, 147.77,136.96, 136.90, 131.28, 128.20, 127.33, 127.29, 127.03, 126.99, 125.21,116.95, 114.95, 111.11, 55.83, 55.47, 44.91, 44.31. ESI m/z: 455 [M −H]+. Anal. (C27H24N2O5) C, H, N.

1,3-Diisobutyl-5-(3,4-dimethoxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione (8f). Synthesized in 91% yield starting from14 and 3,4-dimethoxybenzaldehyde following the procedure describedfor 7a and purified by recrystallization from ethanol. Yellow solid, mp174−175 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.35 (s, 1H), 8.12(d, J = 2.1 Hz, 1H), 7.93 (dd, J = 8.7, 2.1 Hz, 1H), 7.12 (d, J = 8.7 Hz,1H), 3.89 (s, 3H), 3.80 (s, 3H), 3.71 (d, J = 7.2 Hz, 4H), 2.08−1.97(m, 2H), 0.87 (d, J = 6.7 Hz, 12H). 13C NMR (75 MHz, DMSO-d6): δ162.53, 160.75, 156.53, 153.59, 151.09, 147.76, 130.86, 125.28, 116.94,115.21, 111.09, 55.82, 55.52, 48.39, 47.69, 26.86, 26.81, 19.91. ESI m/z: 387 [M − H]+. Anal. (C21H28N2O5) C, H, N.

1,3-Diallyl-5-(3,4-dimethoxybenzylidene)pyrimidine-2,4,6-(1H,3H,5H)-trione (9f). Synthesized in 97% yield starting from 15and 3,4-dimethoxybenzaldehyde following the procedure described for7a and purified by recrystallization from ethanol. Yellow solid, mp169−170 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.36 (s, 1H), 8.20(d, J = 2.1 Hz, 1H), 7.94 (dd, J = 8.7, 2.1 Hz, 1H), 7.12 (d, J = 8.7 Hz,1H), 5.87−5.81 (m, 2H), 5.21−5.09 (m, 4H), 4.47−4.44 (m, 4H),

3.89 (s, 3H), 3.81 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 153.24,151.43, 148.08, 145.04, 141.27, 139.07, 123.74, 122.47, 116.49, 108.29,107.62, 107.56, 106.26, 102.38, 47.09, 46.81, 34.90, 34.31. ESI m/z:355 [M − H]+. Anal. (C19H20N2O5) C, H, N.1,3-Dibenzyl-5-(4-methoxybenzylidene)pyrimidine-2,4,6-

(1H,3H,5H)-trione (7g). Synthesized in 97% yield starting from 13and 4-methoxybenzaldehyde following the procedure described for 7aand purified by recrystallization from ethanol. Yellow solid, mp 156−157 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.42 (s, 1H), 8.35 (d, J =8.8 Hz, 2H), 7.36−7.24 (m, 10H), 7.08 (d, J = 8.8 Hz, 2H), 5.06 (s,4H), 3.87 (s, 3H). 13C NMR (75 MHz, DMSO-d6): δ 163.66, 162.31,160.41, 156.96, 150.88, 137.49, 136.96, 136.89, 128.20, 127.34, 127.23,127.04, 126.97, 125.13, 115.01, 113.93, 55.65, 44.92, 44.33. ESI m/z:425 [M − H]+. Anal. (C26H22N2O4) C, H, N.1,3-Diisobutyl-5-(4-methoxybenzylidene)pyrimidine-2,4,6-

(1H,3H,5H)-trione (8g). Synthesized in 91% yield starting from 14and 4-methoxybenzaldehyde following the procedure described for 7aand purified by recrystallization from ethanol. Yellow solid, mp 136−138 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.35 (s, 1H), 8.32 (d, J =8.8 Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H), 3.88 (s, 3H), 3.70 (d, J = 7.0 Hz,4H), 2.08−1.94 (m, 2H), 0.87 (d, J = 6.7 Hz, 12H). 13C NMR (75MHz, DMSO-d6): δ 163.41, 162.46, 160.64, 156.18, 151.09, 137.21,125.17, 115.24, 113.86, 55.62, 48.38, 47.73, 26.80, 19.90. ESI m/z: 357[M − H]+. Anal. (C20H26N2O4) C, H, N.1,3-Diallyl-5-(4-methoxybenzylidene)pyrimidine-2,4,6-

(1H,3H,5H)-trione (9g). Synthesized in 95% yield starting from 15and 4-methoxybenzaldehyde following the procedure described for 7aand purified by recrystallization from ethanol. Yellow solid, mp 116−118 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.37 (s, 1H), 8.34 (d, J =8.9 Hz, 2H), 7.08 (d, J = 8.9 Hz, 2H), 5.91−5.79 (m, 2H), 5.21−5.09(m, 4H), 4.47−4.43 (m, 4H), 3.88 (s, 3H). 13C NMR (75 MHz,DMSO-d6): δ 163.55, 161.95, 160.07, 156.48, 150.03, 137.35, 132.59,132.48, 125.12, 116.39, 116.35, 115.10, 113.90, 55.65, 43.65, 43.07.ESI m/z: 325 [M − H]+. Anal. (C18H18N2O4) C, H, N.1,3-Dibenzyl-5-(2,6-dimethyl-4-hydroxybenzylidene)-

pyrimidine-2,4,6(1H,3H,5H)-trione (7h). Synthesized in 72% yieldstarting from 13 and 4-methoxy-2,6-dimethylbenzaldehyde followingthe procedure described for 7a and purified by recrystallization fromethanol. Yellow solid, mp 180−181 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.60 (s, 1H), 8.47 (s, 1H), 7.39−7.22 (m, 10H), 6.47 (s, 2H),5.04 (s, 2H), 4.94 (s, 2H), 2.05 (s, 6H). 13C NMR (75 MHz, DMSO-d6): δ 161.35, 159.30, 158.15, 156.66, 138.10, 136.86, 128.23, 128.15,127.57, 127.26, 127.11, 127.01, 120.40, 114.15, 44.88, 44.12, 20.12.ESI m/z: 439 [M − H]+. Anal. (C27H24N2O4) C, H, N.1,3,5-Tribenzylpyrimidine-2,4,6(1H,3H,5H)-trione (4a). To an

ice cooled solution of 7a (119 mg, 0.30 mmol) in AcOH (10 mL), zincpowder (196 mg, 3.00 mmol) was added. The resulting mixture wasstirred at room temperature for 1 h, the solid was filtered off, and thesolvent was evaporated. The residue was taken up with EtOAc (20mL) and washed with brine. The organic phase was dried, filtered, andconcentrated in vacuo. The residue was purified by silica gelchromatography (CH2Cl2/EtOAc) to give the title compound (81mg) in 68% yield as a white solid, mp 110−112 °C.1H NMR (300MHz, DMSO-d6): δ 7.27−7.07 (m, 13H), 6.93−6.91 (m, 2H), 4.92−4.80 (m, 4H), 4.31 (t, J = 4.9 Hz, 1H).73 13C NMR (75 MHz, DMSO-d6): δ 168.09, 150.88, 136.57, 136.25, 136.02, 129.60, 128.83, 128.25,128.11, 127.79, 127.65, 127.45, 127.20, 127.15, 127.02, 126.77, 50.06,44.26, 34.91. ESI m/z: 397 [M − H]+. Anal. (C25H22N2O3) C, H, N.5-Benzyl-1,3-diisobutylpyrimidine-2,4,6(1H,3H,5H)-trione

(5a). Synthesized in 65% yield starting from 8a following theprocedure described for 4a. White solid, mp 104−105 °C. 1H NMR(300 MHz, DMSO-d6): δ 7.22−7.20 (m, 3H), 7.05−7.02 (m, 2H),4.14 (t, J = 4.8 Hz, 1H), 3.53−3.50 (m, 4H), 3.35 (d, J = 2.4, 1H),1.88−1.79 (m, 2H), 0.76 (d, J = 6.7 Hz, 6H), 0.65 (d, J = 6.7 Hz, 6H).13C NMR (75 MHz, DMSO-d6): δ 169.41, 152.58, 137.91, 130.10,129.47, 128.04, 51.07, 49.15, 36.06, 27.84, 21.05, 20.86. ESI m/z: 329[M − H]+. Anal. (C19H26N2O3) C, H, N.5-Benzyl-1,3-diallyl-pyrimidine-2,4,6(1H,3H,5H)-trione (6a).

Synthesized in 69% yield starting from 9a following the proceduredescribed for 4a. White solid, mp 94−95 °C. 1H NMR (300 MHz,

DMSO-d6): δ 7.26−7.16 (m, 3H), 7.04−6.88 (m, 2H), 5.69−5.58 (m,2H), 5.12−4.91 (m, 4H), 4.32−4.09 (m, 5H), 3.32 (d, J = 5.2 Hz,2H). 13C NMR (75 MHz, DMSO-d6): δ 170.61, 151.05, 132.64,130.66, 129.15, 118.12, 117.44, 50.96, 43.95, 35.99. ESI m/z: 297 [M− H]+. Anal. (C17H18N2O3) C, H, N.

1,3-Dibenzyl-5-(4-hydroxybenzyl)pyrimidine-2,4,6-(1H,3H,5H)-trione (4b). Synthesized in 71% yield starting from 7bfollowing the procedure described for 4a. White solid, mp 148−150°C. 1H NMR (300 MHz, DMSO-d6): δ 9.32 (brs, 1H), 7.27−7.22 (m,6H), 7.05−7.02 (m, 4H), 6.67 (d, J = 8.4 Hz, 2H), 6.49 (d, J = 8.4 Hz,2H), 4.93−4.78 (m, 4H), 4.17 (t, J = 4.7 Hz, 1H), 3.26 (d, J = 4.7 Hz,2H). 13C NMR (75 MHz, DMSO-d6): δ 168.25, 156.26, 150.85,136.20, 129.91, 128.06, 127.11, 126.97, 126.17, 115.13, 50.35, 44.18,34.85. ESI m/z: 413 [M − H]+. Anal. (C25H22N2O4) C, H, N.

1,3-Diisobutyl-5-(4-hydroxybenzyl)pyrimidine-2,4,6-(1H,3H,5H)-trione (5b). Synthesized in 66% yield starting from 8bfollowing the procedure described for 4a. White solid, mp 112−114°C. 1H NMR (300 MHz, 300 MHz, DMSO-d6): δ 9.35 (s, 1H), 6.74(d, J = 8.2 Hz, 2H), 6.61 (d, J = 8.2 Hz, 2H), 4.19(t, J = 4.7 Hz, 1H,3.55−3.44 (m, 4H), 1.90−1.83 (m, 2H), 0.80 (d, J = 6.7 Hz, 6H), 0.72(d, J = 6.7 Hz, 6H). 13C NMR (75 MHz, DMSO-d6): δ 170.30, 156.70,150.22, 130.47, 122.44, 115.03, 51.17, 48.27, 36.14, 26.62, 19.81,19.76. ESI m/z: 345 [M − H]+. Anal. (C19H26N2O4) C, H, N.

1,3-Diallyl-5-(4-hydroxybenzyl)pyrimidine-2,4,6(1H,3H,5H)-trione (6b). Synthesized in 61% yield starting from 9b following theprocedure described for 4a. White solid, mp 100−102 °C. 1H NMR(300 MHz, 300 MHz, DMSO-d6): δ 9.30 (s, 1H), 6.78 (d, J = 8.4 Hz,2H), 6.60 (d, J = 8.4 Hz, 2H), 5.69−5.60 (m, 2H), 5.07−4.92 (m,4H), 4.26−4.15 (m, 4H) 4.16 (t, J = 4.7 Hz, 1H), 3.21 (d, J = 6.7 Hz,2H). 13C NMR (75 MHz, DMSO-d6): δ 168.84, 157.19, 151.07,130.89, 123.25, 116.69, 115.94, 51.31, 43.90, 36.00. ESI m/z: 313 [M− H]+. Anal. (C17H18N2O4) C, H, N

1,3-Dibenzyl-5-(3,4-dihydroxybenzyl)pyrimidine-2,4,6-(1H,3H,5H)-trione (4c). Synthesized in 63% yield starting from 7cfollowing the procedure described for 4a. White solid, mp 140−142°C. 1H NMR (300 MHz, DMSO-d6): δ 8.82 (brs, 2H), 7.28−7.21 (m,6H), 7.05−7.02 (m, 4H), 6.49−6.45 (m, 2H), 6.13 (dd, J = 8.1, 2.1Hz, 1H), 4.99−4.76 (m, 4H), 4.17 (t, J = 4.6 Hz, 1H), 3.22 (d, J = 4.7Hz, 2H). 13C NMR (75 MHz, DMSO-d6): δ 169.54, 152.03, 146.34,145.49, 137.49, 129.35, 128.25, 128.14, 120.68, 117.87, 116.73, 51.60,45.44, 36.15. ESI m/z: 429 [M − H]+. Anal. (C25H22N2O5) C, H, N

5-(3,4-Dihydroxybenzyl)-1,3-diisobutylpyrimidine-2,4,6-(1H,3H,5H)-trione (5c). Synthesized in 66% yield starting from 8cfollowing the procedure described for 4a. White solid, mp 130−132°C. 1H NMR (300 MHz, DMSO-d6): δ 8.73 (s, 1H), 8.66 (s, 1H),6.51 (d, J = 8.0 Hz, 1H), 6.41 (d, J = 2.1 Hz, 1H), 6.23 (dd, J = 8.0, 2.1Hz, 1H), 3.93 (t, J = 4.6 Hz, 1H), 3.55−3.44 (m, 4H), 3.15 (d, J = 4.7Hz, 2H), 1.92−1.78 (m, 2H), 0.78 (d, J = 6.7 Hz, 6H), 0.64 (d, J = 6.7Hz, 6H). 13C NMR (75 MHz, DMSO-d6): δ 169.65, 152.66, 146.23,145.41, 128.03, 120.69, 117.63, 116.51, 51.35, 49.11, 36.44, 27.81,21.14, 20.81. ESI m/z: 361 [M − H]+. Anal. (C19H26N2O5) C, H, N.

1,3-Diallyl-5-(3,4-dihydroxybenzyl)pyrimidine-2,4,6-(1H,3H,5H)-trione (6c). Synthesized in 62% yield starting from 9cfollowing the procedure described for 4a. White solid, mp 135−136°C. 1H NMR (300 MHz, DMSO-d6): δ 8.76−8.71 (m, 2H), 6.54 (d, J= 8.0 Hz, 1H), 6.41 (d, J = 2.1 Hz, 1H), 6.23 (dd, J = 8.0, 2.1 Hz, 1H),5.70−5.59 (m, 2H), 5.09−4.94 (m, 4H), 4.29−4.17 (m, 4H), 3.98 (t, J= 4.7 Hz, 1H), 3.14 (d, J = 4.7 Hz, 2H). 13C NMR (75 MHz, DMSO-d6): δ 167.96, 150.15, 144.97, 144.17, 131.95, 126.76, 119.59, 116.51,116.39, 115.31, 50.32, 42.98, 35.39. ESI m/z: 329 [M − H]+. Anal.(C17H18N2O5) C, H, N

Live Cell Studies. COS7 cells was cultured in DMEMsupplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in 5% CO2. Plasmid encoding Histac wastransfected into COS7 cells with FuGENE HD transfection reagent(Roche) and cultured for 24 h at 37 °C in 5% CO2. Before imaging,the culture medium was replaced with phenol red-free growthmedium. The transfected cells were observed at 37 °C in 5% CO2using Olympus IX81 microscope with a UIC-QE cooled charged-coupled device camera (Molecular Devices). Fluorescent images were

collected using MetaFluor (Universal Imaging) with a 440AF21excitation filter, a 455DRLP dichroic mirror, and two emission filters(480AF30 for CFP and 535AF26 for Venus).Surface Plasmon Resonance. SPR analyses were performed on a

Biacore 3000 optical biosensor equipped with research-grade CM5sensor chips (Biacore AB). Recombinant p300/KAT3B (Enzo LifeSciences, catalogue no. BML-SE451; GenBank accession no.NM_001429) HAT domain was immobilized (10 μg/mL in 100mM sodium acetate, pH 4.5) at a flow rate of 10 μL/min by usingstandard amine-coupling protocols to obtain densities of 8−9 kRU.Myoglobin was used as negative control, and one flow cell was leftempty for background subtractions. All compounds were dissolved inDMSO (100%) to obtain 50 mM solutions and diluted in HBS-P (10mM HEPES pH 7.4, 0.15 M NaCl, 0.005% surfactant P20), alwaysmaintaining a final 0.2% DMSO concentration. Binding experimentswere performed at 25 °C by using a flow rate of 30 μL/min, with 60 smonitoring of association and 200 s monitoring of dissociation.Regeneration of the surfaces was performed, when necessary, by a 10 sinjection of 5 mM NaOH.The simple 1:1 Langmuir binding fit model of the BIAevaluation

software was used for determining equilibrium dissociation constants(KD) and kinetic dissociation (kd) and association (ka) constants byusing eqs 1 and 2:

= × × − − ×Rt

k C R R k Rdd

( )a max d (1)

where R represents the response unit, C is the concentration of theanalyte, and

=K k k/D d a (2)

Cell Viability Assay. U937 cell line (derived from a humanhistiocytic lymphoma) was cultured in RPMI-1640 medium (Sigma)supplemented with 10% (v/v) fetal bovine serum (Sigma), 100 U/mLpenicillin, and 100 μg/mL streptomycin (Sigma) at 37 °C in a 5% CO2atmosphere. Cell viability of U937 was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.Then 200 μL (2.5 × 105 cells/mL) of cells, seeded in 96-wellmicrotiter plates, were exposed for 24 h to different concentrations ofselected compounds ranging from 10 to 100 μM in media containing0.2% DMSO. The mitochondrial-dependent reduction of MTT toformazan was used to assess cell viability. The experiments werecarried out in quadruplicate, and all the values were expressed aspercentage of the control containing 0.2% DMSO.Parallel Artificial Membrane Permeability Assay (PAMPA).

Donor solution (0.5 mM) was prepared by diluting 1 mM dimethylsulfoxide (DMSO) compound stock solution using phosphate buffer(pH 7.4, 0.01 M). Filters were coated with 5 μL of a 1% (w/v)dodecane solution of phosphatidylcholine. Donor solution (150 μL)was added to each well of the filter plate. To each well of the acceptorplate were added 300 μL of solution (50% DMSO in phosphatebuffer). Compound 7h, propanolol, and furosemide were tested intriplicate. The sandwich was incubated for 24 h at room temperatureunder gentle shaking. After the incubation time, the sandwich plateswere separated and 250 μL of the acceptor plate were transferred to aUV quartz microtiter plate and measured by UV spectroscopy, using aMultiskan GO microplate spectrophotometer (Thermo Scientific) at250−500 nm at step of 5 nm. Reference solutions (250 μL) wereprepared diluting the sample stock solutions to the same concentrationas that with no membrane barrier. The apparent permeability valuePapp is determined from the ratio r of the absorbance of compoundfound in the acceptor chamber divided by the theoretical equilibriumabsorbance (determined independently) applying the Faller74

modification of Sugano75 equation:

= −+

× −PV V

V V Atr

( )ln(1 )app

D R (3)

In this equation, VR is the volume of the acceptor compartment (0.3cm3), VD is the donor volume (0.15 cm3), A is the accessible filter area(0.24 cm2), and t is the incubation time in seconds.

Kinetic Characterization of 7h Inhibitor. To explore themechanisms of p300 inhibition by 7h, we took advantage of anAlphaLisa homogeneous proximity immunoassay using the Perki-nElmer assay kit no. AL114. Reactions were performed as suggested bymanufacturer, briefly each assay containing 5 nM p300 (EnzoLifeScience no. BML-SE451), 3 μM Acetyl CoA, and 50 nMbiotinylated H3 (1−21) peptide (Anaspec no. 61702) in 10 μL ofassay buffer (50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 1 mM DTT,0.01% Tween-20, 0.01% BSA, 330 nM TSA) was incubated at roomtemperature for 15 min in a White opaque OptiPlate-384(PerkinElmer no. 6007299). Reactions were stopped by addinggarcinol (final concentration 50 μM) and antiacetyl histone H3 lysine9 (H3K9Ac) acceptor beads (PerkinElmer no. AL114, finalconcentration 20 μg/mL). After 60 min of incubation at roomtemperature, 20 μg/mL final concentration of Alpha StreptavidinDonor beads (PerkinElmer no. 6760002) were added in subdued lightand incubated in the dark for 30 min at room temperature. Signalswere read in Alpha mode with a PerkinElmer Enspire plate reader.

To determine the pattern of inhibition of 7h, different set of assayswere performed maintaining one substrate constant and varying theother in the presence of three different concentrations of inhibitor (0,5, and 10 μM). In the first set of experiments, H3 peptide was variedfrom 1.5 to 50 nM while holding AcCoA constant at 3 μM, and in thesecond set AcCoA was varied from 15.6 nM to 1 μM while holding H3peptide constant at 50 nM. Reactions were performed in duplicate anddata were fit to equations to determine the kind of inhibition.

Cell Cycle Analysis. For cell cycle analysis, 500 μL of U937 cells(2.5 × 105 cells/mL) were seeded in 24-well plastic plates andincubated with 100 μM 7h for 72 h. After this period of treatment, 500μL of hypotonic buffer (33 mM sodium citrate, 0.1% Triton X-100, 50μg/mL propidium iodide) was added to cell suspensions. Cells wereanalyzed with a FACScan flow cytometer (Becton Dickinson, CA) andquantitative analysis of cell cycle distribution and hypodiploid nucleiwas performed using ModFit LT Macintosh software (Verity SoftwareHouse, Inc., ME). All the experiments were performed at least intriplicate.

Western Blot Analysis of Acetyl-lysines. First, 10 mL of U937cells (2.5 × 105 cells/mL) were seeded and incubated with 100 μM 7hfor 24, 48 and 72 h. After treatments cells were then harvested andwashed three times with PBS 1× (Sigma) then resuspended in lysisbuffer (10 mM Tris pH 8, 1 mM KCl, 1.5 mM MgCl2, and 1 mMDTT) supplemented with protease inhibitor cocktail (Sigma). Allsubsequent manipulations were performed at 4 °C. After incubationfor 1 h on a rotator, nuclei were collected by centrifugation for 10 minat 10000g. The nuclear pellets were suspended in 0.4 N H2SO4 and,after incubation for 2 h, were centrifuged for 15 min at 12000g.Histones contained in the supernatant were precipitated adding TCAat a final concentration of 33% and incubating overnight at 4 °C. Aftercentrifugation at 10000g, for 30 min, histone pellets were washed twicewith acetone, air-dried, and finally redissolved in 50 μL of water.Protein concentration was determined using the Bradford assay, and 1μg of histones from each sample was loaded on 15% SDS-PAGE andtransferred onto a nitrocellulose membrane. Primary antibodies usedwere: antihistone H4 (acetyl K5) (Abcam catalogue no. ab61236),antihistone H3 (acetyl K9) (Abcam catalogue no. ab10812), andantihistone H3 (Abcam catalogue no. ab1791).

Molecular Modeling. Docking Simulations. The Autodockprogram (version 4.2)76 was used just for the blind docking. Thestructures of the ligands were first generated through the DundeePRODRG2 Server (http://davapc1.bioch.dundee.ac.uk/prodrg).77 Forthe protein setup, to create initial coordinates for docking studies, allwater molecules of the crystal structure (Protein Data Bank (PDB) IDcode 3BIY) were removed and excluded from calculations. Thedocking area has been defined by a box large enough to comprise theentire protein and centered on the macromolecule. Grid points of 126× 126 × 126 with 0.550 Å spacing were calculated around the dockingarea for all the ligand atom types using the Autodock Tool AutoGrid4.One hundred separate docking calculations were performed, each oneconsisting of 25 × 106 energy evaluations using the Lamarckian geneticalgorithm local search (GALS) method. A low-frequency local search

according to the method of Solis and Wets78 was applied to dockingtrials to ensure that the final solution represents a local minimum.Each docking run was performed with a population size of 150, and300 rounds of Solis and Wets local search were applied with aprobability of 0.06. A mutation rate of 0.02 and a crossover rate of 0.8were used to generate new docking trials for subsequent generations.The docking results from each of the 100 calculations were clusteredon the basis of root-mean square deviation (rmsd = 3.0 Å) betweenthe Cartesian coordinates of the ligand atoms and were ranked on thebasis of the free energy of binding. The pocket detected as alternativebinding site was further investigated with a subsequent “focused”docking step in order to characterize the 7h and its derivatives bindingmodes. The “focused” docking was performed with the aid of Glidesoftware (version 5.5; Schrodinger, LLC, New York).79 The latter waspreferred over Autodock because it was able to retain, during thedocking simulation, a correct geometry of the hydroxy- and methoxy-benzylidene moieties of derivatives 7b−7g. The structures of theinhibitors were first generated through the Dundee PRODRG2 server.Then geometry optimized ligands were prepared using Lig-Prep(version 2.3, Schrodinger, LLC, New York), as implemented inMaestro. The target protein was prepared through the ProteinPreparation Wizard of the graphical user interface Maestro (version9.3, Schrodinger, LLC, New York, NY) and the OPLS-200180,81 forcefield. Water molecules were removed. Hydrogen atoms were added,and minimization was performed until the rmsd of all heavy atoms waswithin 0.3 Å of the crystallographically determined positions. Thebinding pocket was identified by placing a 15 Å cube centered on theY1397 residue. Molecular docking calculations were performed withthe aid of Glide 5.5 in standard precision (SP) mode, using Glidescorefor ligand ranking. For multiple ligand docking experiments, an outputmaximum of 5000 ligand poses per docking run with a limit of 100poses for each ligand was adopted.

■ ASSOCIATED CONTENT*S Supporting InformationElemental analysis and copies of 1H NMR and 13C NMRspectra of all final compounds, SPR analysis of compounds 7c−g, 8b−e, and 9e, stability assays, additional kinetic assays,additional molecular modeling studies, UV absorbancespectrum of 7h, and U937 cells viability (MTT) assay. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Authors*For S.C.: phone, +39-089-96-9244; fax, +39-089-96-9602; E-mail, scastellano@unisa.it.*For A.T.: phone, +39-089-96-9797; fax, +39-089-96-9602; E-mail, tosco@unisa.it.*For G.S.: phone, +39-089-96-9770; fax, +39-089-96-9602; E-mail, gsbardella@unisa.it.Author ContributionsA.F. and C.M. contributed equally to this work. The manuscriptwas written through contributions of all authors. All authorshave given approval to the final version of the manuscript.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis study was supported by grants from the Italian Ministerodell’Istruzione, dell’Universita e della Ricerca (MIUR), Progettidi Ricerca di Interesse Nazionale (PRIN 2009PX2T2E, PRIN2012ZHN9YH), the Italian Ministero dell’Istruzione, dell’Uni-versita e della Ricerca (MIUR), Futuro in Ricerca(RBFR10ZJQT), the European Union’s Seventh Framework

Programme (FP7 COST/TD0905), and the Universita diSalerno (Italy). C.M. was supported by Universita di Salernowith postdoctoral research fellowships and A.F. with apredoctoral fellowship. We thank Prof. Minoru Yoshida forserving as advisor in live-cell studies of p300 inhibition.

■ ABBREVIATIONS USED

ALPHA, amplified luminescent proximity homogeneous assay;CBP, CREB binding protein; CREB, c-AMP response elementbinding protein; DMEM, Dulbecco’s Modified Eagle’sMedium; DTT, dithiothreitol; E1A, adenovirus early region1A; GALS, genetic algorithm local search; GCN5, generalcontrol non derepressible-5; GNAT, GCN5-related N-acetyl-transferase; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesul-fonic acid; MOZ, monocytic leukemia zinc finger protein;MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; MYST, MOZ, YBF2/SAS3, SAS2, TIP60 N-acetyltransferase; p300, E1A-associated protein of 300 kDa;PCAF, p300/CBP-associated factor; RPMI medium, RoswellPark Memorial Institute medium; SPR, surface plasmonresonance

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A Novel Cell-Permeable, Selective, and Noncompetitive Inhibitor of KAT3 Histone Acetyltransferases...

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