Mol DiversDOI 10.1007/s11030-016-9689-4

ORIGINAL ARTICLE

Design and synthesis of novel protein kinase R (PKR) inhibitors

Sagiv Weintraub1 · Tali Yarnitzky2,3 · Shirin Kahremany1 · Iliana Barrera4 ·Olga Viskind1 · Kobi Rosenblum4 · Masha Y. Niv2,3 · Arie Gruzman1

Received: 19 March 2016 / Accepted: 11 July 2016© Springer International Publishing Switzerland 2016

Abstract Protein kinase RNA-activated (PKR) plays animportant role in a broad range of intracellular regulatorymechanisms and in the pathophysiology of many humandiseases, including microbial and viral infections, cancer,diabetes and neurodegenerative disorders. Recently, severalpotent PKR inhibitors have been synthesized. However, theenzyme’s multifunctional character and a multitude of PKRdownstream targets have prevented the successful transfor-mation of such inhibitors into effective drugs. Thus, theneed for additional PKR inhibitors remains. With the help ofcomputer-aided drug-discovery tools, we designed and syn-thesized potential PKR inhibitors. Indeed, two compoundswere found to inhibit recombinant PKR in pharmacologicallyrelevant concentrations. One compound, 6-amino-3-methyl-2-oxo-N -phenyl-2,3-dihydro-1H-benzo[d]imidazole-1-carboxamide, also showed anti-apoptotic properties. The novelmolecules diversify the existing pool of PKR inhibitors and

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11030-016-9689-4) contains supplementarymaterial, which is available to authorized users.

B Arie Gruzmangruzmaa@biu.ac.il

1 Division of Medicinal Chemistry, Department of Chemistry,Faculty of Exact Sciences, Bar-Ilan University, 5290002Ramat-Gan, Israel

2 Institute of Biochemistry, Food Science and Nutrition,The Robert H. Smith Faculty of Agriculture, Food andEnvironment, 7610001 Rehovot, Israel

3 The Fritz Haber Research Center for Molecular Dynamics,The Hebrew University, 91904 Jerusalem, Israel

4 Sagol Department of Neurobiology, Faculty of NaturalSciences and Center for Gene Manipulation in the Brain,University of Haifa, 3498838 Haifa, Israel

provide a basis for the future development of compoundsbased on PKR signal transduction mechanism.

Keywords PKR inhibitors · C16 · Benzoimidazolederivatives · Computer modelling

Introduction

Protein kinase RNA-activated (PKR) is a member of the ser-ine/threonine (Ser/Thr) kinase family that mediates a broadspectrum of cellular transduction pathways [1,2]. Origi-nally, PKR was purified and characterized by Berry et al.as an important component of interferon-protective action[3]. Subsequently, the multifunctional role of PKR in manycritical intracellular regulatory pathways, which are relatedto severe human diseases, was revealed [4]. It was found,for example, that the enzyme plays a key role in the patho-physiology of cancer, inflammation, autoimmune diseases,diabetes, and chronic neurodegenerative disorders [5–9].

The main downstream target of PKR is the eukaryoticinitiation factor 2 alpha (eIF-2α) which plays an impor-tant role in the regulation of protein synthesis in metabolicstress, controls the translation initiation in various cells andneurons and affects cognitive functions [10–13]. Phospho-rylation of Ser51 in eIF-2 α by PKR inhibits total proteinsynthesis, but selectively increases the production rates ofseveral proteins such as activating transcription factor 4(ATF4) and beta-secretase 1 (BACE1) [14–17]. Several otherdownstream PKR effector proteins were identified in the lastdecade, including interferon regulatory factor 1, STATs, p53,activating transcription factor 3, and IkK (which activatesNF-kappaB) [4,9,18–20].

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In addition to the canonical protein synthesis PKR is alsoinvolved in regulating CNS functions such as plasticity ofshort-term and long-term memories [21–25].

Several PKR inhibitors have been reported so far. Carl-son et al. identified a peptide-based molecule named PAC(9-anilinoacridine-4-Hyp-Nap-Nap, where Hyp is trans-4-hydroxyproline and Nap is 1-napthylalanine), which is ableto inhibit the PKR RNA binding [26]. Two other knownPKR inhibitors were identified as ATP-recognizing domainbinders. In 2003, Jammi et al. discovered an imidazolo–oxindole scaffold-based potent PKR inhibitor (C16) [2]. In2011, Bryk et al. reported an additional compound, N -(2-(1H-indol-3-yl)ethyl)-4-(2-methyl-1H-indol-3-yl)pyrimidin-2-amine [27].

C16 demonstrated impressive inhibitory effects in phar-macologically relevant concentrations (in the nM range) onpurified PKR [2] and exhibited biological effects in tissuecultures [28]. However, the compound also affects PKR-independent biochemical intracellular transduction mecha-nisms. For example, in neurones, C16 modulates activity ofJun N-terminal kinases (JNKs), the p38 MAP kinases, thedeath-associated protein kinases (DAPKs), c-Raf, MEK1,MKK6, and MKK7 pathways [28–30]. In addition, C16also inhibits the activity of several cyclin-dependent kinases(CDKs) including CDK2/CDK5 [30], and prevents Ab42-induced apoptosis in C57BL/6J mouse embryo neuronalcells. C16 also downregulated NF-kappaB in U937 humanmonocytes following the reduction of IL-8 production. Fur-thermore, C16 suppresses satratoxin G-induced apoptosisin PC-12 neuronal cells, reduces HT-22 and HEK293T cellcycle progression and blocks proliferation ofMAC16 tumourcells [5,28–31]. In addition, the compound showed impres-sive biological activity in vivo. Tronel et al. reported thatC16 prevented neuronal loss and suppressed the inflam-matory response in an acute excitotoxicity rat model [32].This work confirmed the neuroprotective role of C16 whichwas described by Ingrand et al. in [33]. An interestingaspect of C16 activity in the CNS was reported by Sternet al. The authors showed that C16 improved long-termtaste memory in rodents [34]. In addition, C16 demon-strated strong antitumour activity in an adenocarcinomamurine model (MAC16). Moreover, in the same canceranimal models, C16 has been shown to attenuate mus-cle atrophy and slow the progression of cancer-relatedcachexia [5]. Finally, the imidazolo-oxindole derivative ofC16, imoxin, improved glucose homeostasis in obese dia-betic mice [35].

We have used a C16 scaffold to perform a structurallyinformed manual design of novel PKR inhibitors. The insilico part of the project included the identification ofthe putative binding pocket of PKR followed by a virtualdocking analysis of the designed compounds. Based onthese computer-modelling methods and synthetic consider-

ations, ten 1-methyl-1,3-dihydro-2H-benzo[d]imidazole-2-one derivatives were selected for synthesis. All compoundswere tested using a PKR activity assay (a recombinant pro-tein) in which the affinity of the potential inhibitors wasmeasured based on the competition between a test mole-cule and an immobilized PKR ligand-reporter. Two mole-cules, 6-amino-3-methyl-2-oxo-N -phenyl-2,3-dihydro-1H-benzo[d]imidazole-1-carboxamide (5) and 3-methyl-6-(met-hylsulphonamido)-2-oxo-N -phenyl-2,3-dihydro-1H-benzo[d]imidazole-1-carboxamide (6), inhibitedPKR in themicro-mole range. Compound 5 showed a cell-protective effectunder oxidative conditions similar to C16. These resultsprovide new chemotypes for the inhibition of the PKR path-way.

Results

Computer-aided drug design has been used in this workfor developing potential PKR inhibitors. This methodol-ogy includes structure-based techniques, as done in previouswork by Levit et al. [36]. In this approach, we used kinasecomplexes Nek2: PDB code 2JAV, and Wee1A: PDB codewhich are structurally similar to PKR (PDB code 2A19).This enabled us to predict the ligand-binding site and sug-gest possible interactions with a ligand. Based on this data,C16 was docked into PKR (Fig. 1), and the putative interac-tions proposed by the best docked position (Fig. 2a, b) wereused as a template to evaluate the new proposed compounds.Specifically, the novel compounds were designed and drawn,and their 3D conformations were generated. These structureswere then virtually docked into the PKR-binding domain.Using the putative binding site and residues of PKR that mayinteract with C16, all of the compounds’ docked poses werescored based on binding energy and manually inspected.

The benzoimidazole ring was chosen as a central corescaffold in all ten compounds due to its ability to form a π/πstacking interaction with Phe 421 in the PKR active center.Phe 421 formed another important π/π contact with an imi-dazole ring. This interaction was mimicked by introducingdifferent aromatic residues into the structures of the synthe-sized compounds. In addition, the interaction between Lys296 and the electron-enriched thiazole ring in C16 was mim-icked by several electron-enriched functional groups. Finally,the hydrogen bonds of Cys 369 and Glu 367 with electrondonors and an acceptor in C16 were mimicked by nitrogen inan amide bond and a carbonyl group in the benzoimidazolering.

First, 3-methyl-6-nitro-2-oxo-N -phenyl-2,3-dihydro-1H-benzo[d]imidazole-1-carboxamide (4)was synthesized accord-ing to the literature [37], starting from commercially avail-able 2,4-dinitro-chlorobenzene as shown in Scheme 1. Thestarting molecule was converted to the corresponding sec-

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Scheme 1 a Methylamine,EtOH; b CH3CN, TEA, Pd-C,formic acid; c CDI, DMF; dPhNCO, TEA; e H2, 10% Pd-C

Scheme 2 aMethanesulphonylchloride,TEA; b Isobutyl chloroformate,TEA; c Succinic anhydride,acetic acid; d Glutaricanhydride, acetic acid

ondary amine: N -methyl-2,4-dinitroaniline (1). This wasdone by creating a Meisenheimer complex to evoke anaromatic nucleophilic substitution reaction [38] . The orange-colored product was obtained in high yield (93%). The nextstepwas the selective reduction of the ortho nitro group usingformic acid as a hydrogen donor in the presence of palladiumand triethylamine [39]. The reaction was extremely exother-mic, and the use of an ice bathwasnecessary.A red-tinted ani-line derivative, N 1-methyl-4-nitrobenzene-1,2-diamine (2),was obtained in a moderate yield (approx. 60 %). The com-pound underwent cyclization in dry DMF in presence of acarbonyldiimidazole. The intermediate bicyclic molecule: 1-methyl-5-nitro-1,3-dihydro-2H-benzo[d]imidazol-2-one (3)was conjugated with phenylisocyanate through the forma-tion of a urea bond. The structure of the correspondingbenzoimidazole derivative consisted of a novel molecule (4)which has not been reported before. Another benzoimidazolederivative (6-amino-3-methyl-2-oxo-N -phenyl-2,3-dihydro-1H-benzo[d]imidazole-1-carboxamide, 5) is included in theAuroraScreeningLaboratory chemical library, but its synthe-

sis has not been reported yet. The compoundwas synthesizedthrough the reduction of the nitro group using a Parrmachine.

In compounds 6-9, different substitutions to the amine inthe benzoimidazole of compound 5 were used (Scheme 2).In performing this manipulation, we investigated the rolethe positive aniline charge has on possible interactionswith the PKR active center. In addition, a negativelycharged carboxylic acid moiety was introduced using eitherethyl or propyl chain linkers. Compound 6 (3-methyl-6-(methylsulphonamido)-2-oxo-N -phenyl-2,3-dihydro-1H-benzo[d]imidazole-1-carboxamide) was synthesized usingmesylchloride which was coupled with the free amine toobtain amesitylate according to procedure described byMar-vel et al. [40].

The compound was obtained as a colorless solid inmoderate yield (approx. 30 %). The carbonate deriva-tive of 5 (isobutyl-1-methyl-2-oxo-3-(phenylcarbamoyl)-2,3-dihydro-1H-benzo[d]imidazol-5-yl-carbamate, 7) wasattained in good yield by coupling with isobutyl chlorofor-

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Fig. 1 In silico structure of the C16 PKR complex. C16 (cyan sticks)docked in PKR (grey ribbon), PDB code 2A19. (Color figure online)

mate in the presence of triethylamine. This compound wassynthetized to mimick the potential interactions (in additionto a hydrogen donor ability of an amide bond, as in com-pound 5) between an isopropyl moiety and a hydrophobicpocket in the active center which we predict to be formed byseveral lipophilic residues (Gly 278, Ile 273 and Val 281) inthe PKR-binding site (Supplementary Fig. 1).

Two different carboxy amide derivatives of 5 were pre-pared by the amidation of the free amine with either suc-cinic anhydride (4-((1-methyl-2-oxo-3-(phenylcarbamoyl)-2,3-dihydro-1H-benzo[d]imidazol-5-yl)amino)-4-oxobutan-oic acid, 8) or glutaric anhydride (5-((1-methyl-2-oxo-3-(phenylcarbamoyl)-2,3-dihydro-1H-benzo[d]imidazol-5-yl)-amino)-5-oxopentanoic acid, 9). Both compounds wereattained in relatively high yields (around 60%). In addition,two dimer molecules of 3 (a nitro and an aniline derivative)were synthesized as shown in Scheme 3.

The design of these two dimers was inspired by the workof Bryk et al., in which the authors showed that a moleculeconstructed from two indole rings conjugated to each otherby a pyrimidine linker exhibited significant PKR inhibitoryactivity [27]. Thus, twomolecules of 3were coupled throughan ethane linker bridging two nonmethylated nitrogen atomsto create a novel dimer: 3, 3′-(ethane-1,2-diyl)bis(1-methyl-5-nitro-1,3-dihydro-2H-benzo[d]imidazol-2-one) (10) (Sch-eme 3).

The crude green-colored solid product was purified bycolumn chromatography to yield pure product in 43 % yield.Both nitro groups in 10 were reduced to amines using ahigh-pressure hydrogenation in a Parr machine to obtaincompound 11 (3, 3′-(ethane-1,2-diyl)bis(5-amino-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one). We assumed thatthe introduction of the positively charged amino groupswould increase the binding affinity of the compound.

Two additional compounds (14 and 15) were synthe-sized as shown in Scheme 4. We introduced an imidazolemoiety to the benzoimidazole ring in order to mimic theinteraction of C16 moeity with Phe 421. The important dif-

ference between our designed molecules and C16 is that inC16 the imidazole ring is connected to the main scaffold bya rigid double bond, while in compounds 14 and 15 the imi-dazole is conjugated through a flexible alkyl chain. As inthe synthesis of 1, the novel aminopropylimidazole precur-sor N -(3-(1H-imidazol-1-yl)propyl)-2,4-dinitroaniline (12)was prepared in good yield (93%). The compound wasconverted to its amine derivative: N 1-(3-(1H-imidazol-1-yl)propyl)-4-nitrobenzene-1,2-diamine (13) followed bycyclization to form 1-(3-(1H-imidazol-1-yl)propyl)-5-nitro-1,3-dihydro-2H-benzo[d]imidazol-2-one (14) and reductionof the nitro group to aniline to give 1-(3-(1H-imidazol-1-yl)propyl)-5-amino-1,3-dihydro-2H-benzo[d]imidazol-2-one(15) according to the procedures described above.

In total, 16 compounds were designed in silico based onour docking analysis and the synthetic feasibility of the com-pounds. Ten compounds were chosen for synthesis. Ninenovel synthesized C16 derivatives, namely 4, 6, 7, 8, 9, 10,11, 14, 15 and one known compound 5, were tested in vitro.A KINOMEscanTM assay (with recombinant human PKRas a targeted kinase) was used for the in vitro validation ofthe synthesized compounds. The KINOMEscanTM is a high-throughput system for screening compounds against largenumbers of human kinases. This is one of the most compre-hensive methods which were developed by DiscoveRx forindustrial use [41].

The assay performed by combining three components: aDNA-tagged kinase, an immobilized ligand and a test com-pound. The ability of a test compound to compete with theimmobilized ligand is measured by quantitative PCR of theDNA tag. All test compounds showed excellent solubility inDMSO. Thus, this solvent was used for the in vitro evaluationof our test compounds.

A summaryof theKdvalues obtainedbyKINOMEscanTM

is presented in Table 1. Two of the ten tested com-pounds showed significant affinity to PKR: compounds 5(Kd=27μM) and 6 (Kd=23 μM). Dose response curves forcompounds 5 and 6 are shown in the Supplemental Informa-tion (Supplementary Figs. 2 and 3, respectively).

An anti-apoptotic effect of C16 was reported in severalpublications [28,32]. Therefore, the possible anti-apoptoticeffect of 5 and 6 together with the parent molecule C16 wasevaluated in the human breast cancer cell line (MCF-7). Thiscell line was chosen for its high levels of PKR expressionand activity [42]. Apoptosis was induced using oxidativestress, created by the glucose oxidase/glucose system whichconstitutively generated hydrogen peroxide. Next, a standardMTT analysis was conducted. Only compound 5 and C16 at0.5 μM showed a significant cell-protective effect, as shownin Fig. 5a. However, compound 5 was more effective thanC16, which increased cell viability by approximately 15%compared with the 30% increase in cell protection shownby compound 5. In addition, the activity of caspase 3 (a

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Scheme 3 a K2CO3,dibromoethane; b H2, 10%Pd-C

Scheme 4 a 1-(3-Aminopropyl)imidazole, EtOH; b CH3CN,TEA, Pd-C, formic acid; c CDI,DMF; d CH3CN, TEA, Pd-C,formic acid

Table 1 In vitro-determined Kdand in silico-predicted active siteinteractions of test compounds

Entry Kd [μM] Val294 Lys296 Glu367 Cys369 Phe421 Asp432

4 Non active − + − − + +

5 27 + − − + + −6 23 − − + + + −7 Non active + − − − + +

8 Non active − − − − − +

9 Non active − − − + − +

10 Non active − + − + −11 Non active − − − + + −14 Non active − − − + + −15 Non active − − − + + −C16 0.21 − + + + + −

well-known apoptotic marker) was also measured in MCF-7 cells which were kept under induced oxidative stress inthe presence and absence of C16 and compounds 5 and 6[43]. In the same experiment, compound 5 greatly decreasedthe activity level of caspase 3 (Fig. 5b). Moreover, its effectwas significantly higher than that of C16 on caspase 3 activ-ity by approximately 18%. It is important to mention thatin both experiments Trolox (a known antioxidant and cyto-protective molecule) was used as a positive control agent[44]. Compound 6 was inactive in both oxidative stressassays.

Discussion

The potent PKR in vivo inhibitor C16was discovered in 2003[2]. However, because of its poor pharmacokinetic proper-ties, the compound did not become a useful drug. Thus, thestarting point of this research was to use the rigid polycyclicscaffold ofC16 that creates importantπ/π interactions for thedesign of active in vivo compounds suitable for use as parentmolecules with superior pharmacokinetic properties. Basedon our in silicowork, several new compoundswere designed,and the versatility of the synthetic approach presented hereenabled the production of several innovative compounds.

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The PKR inhibitory activity of ten novel compounds wastested in vitro. Recombinant PKR was used for this primaryscreening. Only two compounds were active (compounds5 and 6), and both showed affinity in the micromolar con-centration range. A molecular modelling approach was usedfor the analysis of the in vitro results. Compounds 4 and5 are predicted to make favourable interactions with thelipophilic moieties in the PKR active site via their benzoim-idazole domain (Supplementary Figs. 3 and 4, respectively).In addition to the benzoimidazole interaction with the criticalresidues in the active PKR center, compounds 4 and 5 canform a π/π stacking interaction by means of an additionalbenzene ring. It is also important to mention that the nitrogroups (in compound 4) and the amino groups (in compound5) are known as hydrogen-bond participants (depending onthe distance between corresponding donors/acceptors in theactive center) which might stabilize the binding capability ofthe molecule to the PKR active center. Interestingly, basedon the in silico analysis, compound 4 is able to form a σ/πstacking bond with Val 291, π/π stacking with Phe 421 anda hydrogen bond with Lys 296 and Asp 432 (SupplementaryFig. 4). In contrast, a hydrogen bond with an amine group incompound 5was not observed in silico. However, a carbonylgroup in the benzoimidazole moiety of 5 together with anamide group from a urea functional group might form twohydrogen bonds with Cys 369. The π/π stacking interac-tions were observed in silico for compound 5 as predicted.In addition, a noncovalent (σ −π) bonding between Val 294and the benzoimidazole moiety was also observed (Fig. 3).These differences explain why compound 5 was active andcompound 4 was not.

In compound 6, a positively charged amino group wasreplaced by a neutral sulphonylamide moiety, which couldparticipate in the formation of a hydrogen bond with Glu 367(with –NH– as a hydrogen donor). However, virtual dockingagainst PKR did not show such an interaction (Fig. 4).

Instead, Glu 367 interacted with the methylated amine inthe benzoimidazolemoiety by formation of a hydrogen bond.In addition, our docking simulations revealed that besidesπ/π interactions of the benzoimidazole core, another interac-tion was formed: A new hydrogen-bond interaction betweenCys 369 and the benzoimidazole’s carbonyl group in com-pound 6 (Fig. 4).

For compounds 8 and 9, modelling revealed that anelectrostatic interaction between the negatively charged car-boxylic acid group and a positively charged primary amine oflysine 296 was not likely to be formed. However, a carboxylgroup of compound 9 was predicted to create a hydrogenbond with the amide hydrogen of Phe 278. The short linkerbetween a carboxy group and the amide in compound 8 didnot allow similar interactions to form (Supplementary Fig.5a,b). According to the docking simulations, compound 10formsπ/π stacking interactions between the core of themole-

cule and Phe 421. (In practice, this interaction was doubled.)Also, Lys 296 interacted through a cation-π bond with oneof the benzoimidazole moieties (Supplementary Fig. 6a, b).

In compound 11, an analysis of the docked pose revealedthat together with the obvious hydrophobic interactions withthe core which we described above, the amine group in oneof the benzoimidazole domains interacted with a carbonyl ofIle 273 (Supplementary Fig. 6a, b). Interestingly, the in silicomodel predicted that two additional noncovalent bonds maybe possible, both σ −π interactions. The first onewas createdbetween Val 281 and one of the benzoimidazole moieties,and a second between Gly 372 and another benzoimidazolemoiety. Moreover, a hydrogen bond between Cys 369 anda carbonyl in one of the benzoimidazole domains was alsodetected as a possible option.

Finally, in silico analysis of the mode of interaction ofcompounds 14 and 15 showed that when they are at the PKRbinding site, they adopt stable conformations in which thebenzoimidazole scaffold interacted with Phe 421. However,the newly introduced imidazole ring did not forma significantinteraction with the PKR active center (Supplementary Fig.7a, b). In addition, Cys 369 may interact with the nitro groupof compound 14 and with a carbonyl in the benzoimidazolemoiety of compound 15. Also, a hydrogen bond could beformed between the amine group (compound 15) and thecarbonyl of the amide moiety of Gly 431.

The section of synthetic chemistry includes the synthe-sis of the main scaffold (the substituted benzoimidazole),which was chosen according to the in silico model of thePKR active center. The synthesis includes the use of a well-known nucleophilic aromatic substitution reaction (SNAr)[38] with high yielding outcome. It is also known that a keyfactor that contributes to the success of this reaction is theintroduction of a strong electron withdrawing group, such asa nitro group, into the aromatic system [38]. Therefore, wealso used a nitro moiety in our synthesis in the first step, asshown in Scheme 1. The second step was the reduction ofthe nitro group by a mix of TEA, formic acid and 10% Pd/C.The final compoundswere then successfully purified and iso-lated by column chromatography. The last step, a cyclizationtoward the creation of substituted benzoimidazoles, was alsorapid and very efficient. All ten compounds presented in thiswork were synthesized using this synthetic strategy.

Taken together, the in vitro (recombinant PKR) and in sil-ico results reveal that besides the π/π stacking interactionsbetween the core of the active molecules and the PKR activecenter, Cys 369 is able to form two hydrogen bonds withcompound 5. Moreover, the same residue interacts with acarbonyl in the benzoimidazole domain of compound 6. It isimportant to mention that several inactive compounds have asimilarmode of interactionwith the PKRactive center, whichcan be seen in compounds 11, 14 and 15. However, com-pounds 11 and 15 do not fit precisely in the PKRactive center,

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Fig. 2 Predicted interactionswith C16. a 2D representationof the interactions between C16(cyan lines) and PKR (residuescolored according to descriptionin the figure itself). b Putativeinteractions between C16 (cyansticks) and PKR (grey sticks).Hydrogen-bond interactions areshown as blue dotted lines. Inaddition, pi-interactions can beformed between Phe 421 and thearomatic rings of the ligand.(Color figure online)

which explains the lack of activity of these compounds. Incompound 14, the bond between Cys 369 and the carbonylin the benzoimidazole moiety is replaced by the interactionbetween Cys 369 and the nitro group. This changemight alsobe the reason for the lack of activity in compound 14. Inter-

estingly, compound 6, which has the lowest Kd (23μM),showed the most structural similarity to the binding modeof C16: both compounds shared a binding to Glu 367, Cys369 and Phe 421. In accordance with these in silico results,compound 5 was not able to form an interaction with Glu

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Fig. 3 Schematicrepresentation of the putativeinteractions between PKR andcompound 5

Fig. 4 Schematic representation of the putative interactions between PKR and compound 6

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Fig. 5 Anti-apoptotic effect of compound 5 in MCF-7 cells. a TheMCF-7 cells were grown as described in “Materials and methods” andtreated with C16 (0.5 μM), compounds 5 and 6 (0.5 μM), Trolox (T,50 μM), and DMSO (0.1%) for 24h. Afterward, glucose oxidase (GO,50mU/ml) and glucose (23.5mM, final concentration) were introducedinto the medium for 1.15 min. Standard MTT (described in Methods)was used for evaluating the effect of the test compounds on cell via-bility. b The MCF-7 cells were grown as described in “Materials and

methods” and treatedwith C16 (0.5μM), compounds 5 and 6 (0.5μM),Trolox (T, 50 μM), and DMSO (0.1%), for 24h. Afterward, the cellswere washed and lysated using the lysis buffer described in “Materialsand methods.” The obtained lysates were diluted by a factor of five andused for the detection of caspase 3 activity levels with a commerciallyavailable kit, according to the protocol provided in the kit. *p < 0.05,n=3. mean ± SE. &, the significant difference between cells whichwere treated by C16 and compound 5

367. It would be beneficial to further investigate the criticalrole of Cys 369 in the inhibition of the PKR activity. Suchan investigation could be done using classical mutagenesisapproaches.

Although both compounds 5 and 6 have shown significantaffinity to recombinant PKR in relatively high concentra-tions and were 100-fold less potent than C16, we decided totest their anti-apoptotic activity in a cellular model. We wereencouraged by the data published by Atkinson et al., Grayet al. and Islam et al. that showed C16 biological effects incell cultures in concentrations higher than 0.21 μM, all theway to 5μM [29,31,45] . This range of the active concentra-tions of C16 for cellular assays was also reported by others[28,46].

Couturier et al. showed that C16 has an anti-apoptoticeffect in primary murine mixed co-cultures [28]. The pos-sible cytoprotective effects of both active compounds andC16 itself were investigated in MCF-7 cells. We decided touse C16 and our new compounds in three different concen-trations: 5, 1, and 0.5 μM [28]. An oxidative stress modelwas chosen for the induction of apoptosis as described inthe Methods section. Compound 6 was inactive in all threeconcentrations. In contrast, C16 and compound 5 showed asignificant cytoprotective effect under oxidative stress condi-tions when the cells were already pretreated with the lowestconcentrations of both compounds: 0.5 μM (Fig. 5a). Inter-estingly, compound 5wasmore effective than C16 (by nearly17%). Moreover, compound 5 showed a similar cytoprotec-tive effect compared to the well-known antioxidant Trolox.

An additional step in the investigation of the action mech-anism of compound 5 was a measurement of the possibleeffect of the compound on the level of caspase-3, a knownapoptotic marker [47,48]. C16 and compound 5 were activein the lowest concentrationof the three chosen for the test (5, 1and 0.5 μM). Compound 5was also more effective than C16(by 19%) and surprisingly, also more effective than Trolox(by 25%). Compound 6 did not show any inhibitory activityon caspase 3 levels in MCF-7 cells (Fig. 5b). These resultspositively correlated with the results obtained in the viabil-ity assay which was conducted in identical conditions to thecaspase 3 experiment.

It is clear that the affinity of PKR inhibitors to itsactive center and the level of inhibition of the enzyme ina pure protein-based assay do not always correlate withthe inhibitory activity of such molecules in cellular assays.Many factors, such as solubility in a medium, intracellularmetabolic activation or inactivation, the rate of the cellularmembrane penetration, intracellular accumulation, off-targetbinding and intervention in other cellular signal transductionmechanisms can dramatically influence the biological effectof an inhibitor. Although in the pure-protein affinity assayC16 was 100-fold more potent than compound 5, in the cel-lular assay, both compounds induced the anti-apoptotic effectat identical concentrations.Moreover, compound 5wasmoreeffective than the parent molecule.

Therefore, we believe that compound 5 inhibits PKR inhigh concentrations in the free cell system (KINOMEscanTM),but may be in nanomolar concentrations in the cells. This

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compound might inhibit some other kinases and lead to theobserved anti-apoptotic effects. In addition, an intracellu-lar transformation of compound 5 is possible (but unlikelyin the recombinant PKR assay), and the possibly obtainingmetabolite might be the cause of the determined cellulareffect. More research is needed to determine the exact cel-lular targets of compound 5. It is important to mention thatdespite the fact that several PKB inhibitors have been devel-oped so far, there is still a great need for effective, selectiveand nontoxic compounds, due to their possible anticancerand anti-inflammatory therapeutic potential.

Conclusions

With the use of molecular modelling methods, 16 molecules,15 of them novel, were designed based on the known PKRinhibitor C16 and its predicted interactions with PKR. Com-pound 5 (a known molecule) was synthesized by a novelsynthetic pathway. Nine other new molecules were synthe-sized in our laboratory. Two molecules, 5 and 6, showedsignificant PKR binding in cell-free assay. Although the Kdvalues of both compounds were higher (27 and 23 μM) thanthe Kd value of C16 (0.21μM), both compounds were testedin cellular assays.

Compound 5 showed a significant cell-protective effectunder oxidative stress conditions, in similar concentration toC16 (0.5 μM). Moreover, compound 5 was more effectivethan C16. The molecule we report here may be used as astarting point for the development of potent PKR transduc-tion mechanism inhibitors and as a novel biochemical toolfor the exploration of the PKR signal transduction pathway.

There is a dire need for new therapeutic agents againstdevastating human diseases in which PKR is involved, suchas Alzheimer’s, cancer and others. These newly identifiedmolecules can be used as a basis for the future developmentof such drugs.

Materials and methods

The organic solvents (HPLC grade) were obtained fromFrutarom Ltd. (Haifa, Israel). The melting points weredetermined with a Fisher-Johns melting point apparatus(Palmerton, PA). The 1H NMR and 13C NMR spectra wererecorded at room temperature on a Bruker Advance NMRspectrometer (Vernon Hills, IL) operating at 300 and 400MHz, andwere in accordwith the assigned structures. Chem-ical shift values were reported relative to the TMS that wasused as an internal standard. The samples were prepared bydissolving the synthesized compounds in either DMSO-d6(δH = 2.50 ppm, δC = 39.52 ppm) or CDCl3 (δH = 7.26ppm, δC = 77.16 ppm). Chemical shifts were expressed

in δ (ppm) and coupling constants (J) in hertz units. Thesplitting pattern abbreviations are as follows: s, singlet; d,doublet; t, triplet; q, quartet; quint, quintet; m, unresolvedmultiplet due to the field strength of the instrument; dd, dou-blet of doublet. A QTof micro spectrometer (Micromass,Milford, MA) in the positive ion mode was used for massspectrometry. Data were processed using massLynX v.4.1calculation and deconvolution software (Waters Corporation,Milford, MA). Column chromatography was performed onMerck Silica gel 60 (230–400mesh; Merck, Darmstadt, Ger-many).Analytical thin-layer chromatographywas carried outon pre-coated Merck Silica gel 60F254 (Merck) sheets usingUV absorption for visualization. The purity of the final com-pounds was confirmed using high-field NMR analysis. Allanalytical data (including the NMR images) are shown inthe Supplemental data. Elemental analysis was conductedby Perkin-Elmer 2400 series II Analyzer (Waltham, MA,USA), and the results for all synthesized compounds areshown in the Supplemental material (Supplementary Table1). The purity of all compounds was above 95%. BSA,D-glucose, MT reagent, and the protease inhibitor cocktailwere purchased from Sigma-Aldrich Chemicals (Rehovot,Israel). Glycerol and sodium fluoride were obtained fromMerck (Whitehouse Station, NJ). Mercaptoethanol, phenyl-methanesulphonylfluoride (PMSF), sodium orthovanadate,sodium-β-glycerophosphate, sodiumpyrophosphate andSDSwere purchased from Alfa Aesar (Ward Hill, MA). Fetalcalf serum (FCS), l-glutamine, EMEM and antibiotics werepurchased fromBiological Industries (Beth-Haemek, Israel).TheCaspase 3 assay colorimetric apoptotic kitwas purchasedfrom Abcam (Cambridge, MA, USA).

Cell culture

The human breast cancer cell line (MCF-7) obtained bycourtesy of Dr. E. Alpert (Quiet Therapeutics, (Ness Ziona,Israel)was used for experiments. Cellswere grown inEagle’sminimum essential medium (EMEM) containing 10 % fetalbovine serum (FBS), 1mM glutamine, 100μg/mL penicillinand 100 μg/mL streptomycin at 37◦ C in a 5% CO2 humid-ified atmosphere. Cells were seeded (100,000cell/mL) in a6-well plate.

MTT assay

We described this test in a previous publication [49]. Inbrief, cells were incubated with MTT (2mg/mL) in a growthmedium for 30min at 37 ◦C. Themediumwas then aspirated,andDMSOwas added to solubilize the cells and colored crys-tals. Absorbance at 570nm was measured in a SpectraMaxM5 spectrophotometer (Sunnyvale, CA, USA). The obtained

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results were normalized by total protein content in culturecells, which was measured using the Bradford reagent.

Apoptosis assay

TheCaspase 3 assay colorimetric apoptotic kitwas purchasedfrom Abcam (Cambridge, MA, USA) and used as per themanufacturer’s instructions. Absorbance at 405nmwasmea-sured in a SpectraMax M5 spectrophotometer (Sunnyvale,CA, USA).

Induction of oxidative stress

Oxidative stress conditions were induced using glucose oxi-dase (GO, 50mU/mL). Glucose oxidase with high levels ofglucose (23.5mM instead of the usual 5.5mM) was addedto the growing medium of MCF-7 cells [50]. This resultedin an elevated H2O2 concentration in the medium (reaching29.0 ± 9.6μM in 4h of incubation). The concentration ofH2O2 generated by the glucose oxidase/glucose system wasdetermined as described [51].

Computational modelling

Before docking, all ligands were prepared in the DiscoveryStudio (DS3.5, Accelrys) using the “Prepare Ligands” mod-ule [52]. A set (not exceeding 255) of the most effectivelow-energy conformations was generated for each molecule.All conformers within 20kcal/mol of the global energy min-imum were included in the set.

Molecular docking of C16 and the designed small-molecule compounds was performed using CDocker asimplemented in DS3.5. CDocker is a CHARMm-baseddocking method which uses a molecular dynamics (MD)simulated annealing-based algorithm for ligand conforma-tion generation and docking. Default algorithm settings wereused for docking. The final ligand poses were selected basedon their docking score and manual inspection.

The crystal structure of PKR (PDB code 2A19)was down-loaded from the PDB (http://www.rcsb.org/pdb/home/home.do) and used for docking.

PKR affinity assay

ThePKRaffinity of the synthesized compoundswas obtainedusingLeadHunterTM DiscoveryServices (DiscoveRxCorpo-ration, Fremont, CA, USA).

For the assay (KinomeScan analysis), PKR-tagged T7phage strains were prepared in an E. coli host derived from

the BL21 strain. E. coli were grown to the log-phase andinfected with the T7 phage, then incubated and shaken at32 ◦C until lysis. The lysates were centrifuged and filteredto remove cell debris. The remaining kinases were producedin HEK-293 cells and subsequently tagged with DNA forqPCR detection. Streptavidin-coated magnetic beads weretreated with biotinylated small molecule ligands for 30minat room temperature to generate affinity resins for the kinaseassays. The ligated beads were blocked with excess biotinand washed with a blocking buffer (SeaBlock (Pierce), 1%BSA, 0.05% Tween 20 and 1mM DTT) to remove unboundligands and to reduce nonspecific binding. Binding reactionswere assembled by combining kinases, ligand affinity beadsand test compounds in a 1× binding buffer (20% SeaBlock,0.17× PBS, 0.05% Tween 20 and 6mMDTT). All reactionswere performed in polystyrene 96-well plates in a total vol-ume of 0.135mL. The assay plates were incubated at roomtemperature and shaken for 1h, and the affinity beads werewashed with wash buffer (1× PBS and 0.05% Tween 20).The beads were then resuspended in an elution buffer (1×PBS, 0.05% Tween 20 and 0.5 μM nonbiotinylated affinityligand), then incubated at room temperature and shaken for30min. The kinase concentration in the eluateswasmeasuredby qPCR.

Statistical analysis

Statistical significance (p < 0.05) was calculated amongexperimental groups using the two-tailed Student’s t-test.The Graphpad program was used [53].

Synthetic procedures

Synthesis of (2,4-dinitrophenyl)-methylamine (1)

Methylamine (40% solution) (8.22mL, 0.237mol) wasadded to a solution of 1-chloro-2,4-dinitrobenzene (3g,0.0148mol) in ethanol (30mL) at 0 ◦C and stirred at roomtemperature (RT) for 15h. The reaction was monitored byTLC (EtOAc:Hexane, 1:4). The reaction solution was con-centrated, and hotwaterwas added to the final crudematerial.The precipitatewas filtered andwashedwith hexane to obtaincompound 1 (2.7g, 93%) as an orange solid. m.p.: 170 ◦C.

1H NMR (300 MHz, DMSO- d6) : δ 8.88 (s, 1H), 8.81(s,1H), 8.24 (d, J = 9.3 Hz, 1H), 7.10 (d, J = 9.3 Hz, 1H),3.04 (s, 3H) ppm. 13C NMR (300 MHz, CDCl3): δ 148.7,134.5, 129.9, 129.5, 123.4, 115.1, 30.2 ppm. MS (CI): m/z(C7H7N3O4, MH+) 198.

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Synthesis of N-methyl-4-nitrobenzene-1,2-diamine (2)

TEA (5.64g, 0.0404mol) and 10% Pd/C (0.108g) wereadded to a solution of (1) (2g, 0.0101mol) in CH3CN.The flask was chilled to −15 ◦C, after which formic acid(2.07mL, 0.0505mol) was added while maintaining the tem-perature at −15 ◦C. The solution was stirred at RT for 4.5hfollowed by heating at 80◦C for 10min. The reaction wasmonitored by TLC (EtOAc:Hexane, 1:1). The resulting mix-ture was filtered, the solid residue was washed with MeOH,and thefiltratewas concentrated andpurifiedby columnchro-matography (EtOAc:Hexane, 3:7) to obtained compound 2(1 g, 59%) as a red solid. m.p.: 172 ◦C.

1H NMR (300 MHz, DMSO- d6) : δ 7.55 (d, J = 8.7,2.7 Hz, 1H), 7.40 (s, 1H), 6.41 (d, J = 8.7 Hz, 1H), 6.13 (s,1H), 5.08 (s, 2H), 2.83 (s, 3H) ppm. 13C NMR (400 MHz,DMSO- d6): δ 143.6, 136.5, 134.4, 115.9, 106.9, 106.4, 29.6ppm. MS (CI): m/z (C7H9N3O2, MH+) 168.

Synthesis of 1-methyl-5-nitro-1H-benzo[d]imidazol-2(3H)-one (3)

Di-imidazol-1-yl-methanone (2.91g, 0.0179mol) was addedto a solution of (2) (1g, 0.00598mol) in DMF (7mL) at 0◦C. After 10min, the temperature was allowed to reach RT,and the reaction mix was stirred for 2 h. The progress of thereaction was monitored by TLC (EtOAc:Hexane, 1:1). Theresulting reaction mixture was quenched with ice. A brownsolid precipitated, and was then filtered and analysed. Theobtained material was compound 3 (1g, 66%). m.p.: 234 ◦C

1H NMR (300 MHz, DMSO- d6) : δ 8.02 (dd, J = 8.7,2.1 Hz, 1H), 7.76 (d, J = 2.4 Hz, 1H), 7.29 (d, J = 8.7 Hz,1H), 3.35 (s, 3H) ppm. 13C NMR (400 MHz, DMSO- d6):δ 154.7, 141.4, 136.5, 128.2, 117.6, 107.2, 103.7, 26.8 ppm.MS (ESI): m/z (C8H7N3O3,MH+) 194.

Synthesis of 3-methyl-6-nitro-2-oxo-N -phenyl-2,3-dihydro-1H-benzoimidazole-1-carboxamide (4)

TEA (0.0366g, 0.000362mol) was mixed with a solution of(3) (0.07g, 0.000362mol) in toluene (20mL) at 0 ◦C andphenylisocyanate (0.043g, 0.000362mol) was added undera nitrogen atmosphere. The mixture was then refluxed for2 h. The progress of the reaction was monitored by TLC(DCM: Hexane, 1:1). The mixture was concentrated underreduced pressure. The crude material was purified using col-umn chromatography (eluent: DCM) to obtain compound 4(0.05g, 45%) as a white solid. m.p.: 260 ◦C.

1H NMR (400 MHz, CDCl3) : δ 10.62 (s, 1H), 9.20 (d,J = 2 Hz, 1H), 8.26 (dd, J = 8.8, 2.4 Hz, 1H), 7.62 (m,2H), 7.40 (m, 2H), 7.18 (m, 1H), 7.13 (d, J = 8.8 Hz, 1H),3.55 (s, 3H) ppm. 13C NMR (400 MHz, CDCl3) : δ 147.7,

136.4, 129.0, 124.7, 120.5, 120.2, 111.5, 107.0, 27.5 ppm.MS (ESI): m/z (C15H12N4O4, MH+) 313.

Synthesis of 6-amino-3-methyl-2-oxo-N -phenyl-2,3-dihydro-1H-benzoimidazole-1-carboxamide (5)

Pd/C (0.4g) was added to solution (4) (1.84g, 0.00589mol)in EtOH (50mL), and hydrogenation was carried out in aParr shaker for 3 h. The resulting mixture was filtered andconcentrated under reduced pressure. Recrystallization fromDCM and EtOH gave rise to compound 5 (0.91g, 55%) as acream-colored solid. m.p.: 160 ◦C.

1H NMR (400 MHz, CDCl3) : δ 10.98 (s, 1H), 7.74 (d,J = 2.4 Hz, 1H), 7.61 (m, 2H), 7.36 (m, 2H), 7.13 (m, 1H),6.8 (d, J = 8.4 Hz, 1H), 6.60 (dd, J = 8.4, 2.4 Hz, 1H),3.41 (s, 3H) ppm. 13C NMR (600 MHz, CDCl3) : δ 154.8,148.6, 141.4, 136.6, 128.7, 128.3, 117.7, 115.6, 113.8, 107.3,103.7, 26.9 ppm. MS (ESI): m/z (C15H14N4O2,MH+) 283.

Synthesis of 3-methyl-6-(methylsulphonamido)-2-oxo-N-phenyl-2,3-dihydro-1H-benzoimidazole-1-carboxamide(6)

TEA (0.0538g, 0.000531mol) was mixed with a solu-tion of (5) (0.1g, 0.000354mol) in DCM (20mL) at 0◦Candmethanesulphonylchloride (0.0609g, 0.000531mol)wasadded under a nitrogen atmosphere. The reactionmixwas leftfor 12 h at RT. The white solid residue that formed was fil-tered and washed with DCM and EtOH to give compound 6(0.04g, 31%). m.p.: 170 ◦C.

1H NMR (400 MHz, DMSO- d6) : δ 10.84 (s, 1H), 8.07(s, 1H), 7.61 (d, J = 8 Hz, 2H), 7.39 (m, 3H), 7.19 (m, 2H),3.43 (s, 3H), 2.08 (s, 3H) ppm. 13CNMR (600MHz, DMSO-d6): δ 152.9, 148.2, 136.8, 129.1, 136.8, 129.1, 128.6, 127.2,124.3, 119.8, 118.1, 109.3, 109.1, 27.3 ppm. MS (ESI): m/z(C16H16N4O4S,MH+) 361.

Synthesis of isobutyl (1-methyl-2-oxo-3- (phenylcarbamoyl)-2,3-dihydro-1H-benzo[d]imidazol-5-yl)carbamate(7)

Compound 7, a white solid, was synthesized according tothe procedure described above for compound (6) (0.062g,46%). m.p.: 178 ◦C.

1H NMR (300 MHz, CDCl3) : δ 10.87 (s, 1H), 8.15 (d,J = 2.1 Hz, 1H), 7.62 (m, 3H), 7.37 (m, 2H), 7.14 (m, 1H),6.9 (d, J = 8.4 Hz, 1H), 6.63 (s, 1H), 3.96 (d, J = 6.9 Hz,2H), 3.46 (s, 3H), 1.98 (m, 1H), 0.97 (d, J = 6.9 Hz, 6H)ppm. 13C NMR (400 MHz, DMSO- d6) : δ 154.5, 153.7,148.5, 133.2, 128.7, 128.3, 126.4, 115.6, 113.8, 111.0, 99.8,69.9, 27.5, 26.3, 18.9 ppm. MS (ESI): m/z (C20H22N4O4,MH+) 383.

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Synthesis of 4-((1-methyl-2-oxo-3-(phenylcarbamoyl)-2,3-dihydro-1H-benzo[d]imidazol-5-yl)amino)-4-oxobutanoic acid (8)

Succinic anhydride (0.0532g, 0.000531mol) was added to asolution of (5) (0.1g, 0.000354mol) in acetic acid (10mL),at RT, under a nitrogen atmosphere. The reaction was left atRT for 12 h. The gray solid residue that formed was filteredand washed with H2O and Et2O, resulting in compound 8(0.082g, 61%). m.p.: 221 ◦C.

1H NMR (400 MHz, DMSO- d6) : δ 10.91 (s, 1H), 10.04(s, 1H), 8.45 (d, J = 2 Hz, 1H), 7.61 (m, 2H), 7.52 (dd,J = 8.8, 2 Hz, 1H), 7.4 (m, 2H), 7.23 (d, J = 8.4, 1H),7.17 (m, 1H), 3.39 (s, 3H), 2.53 (m, 4H) ppm. 13C NMR(600 MHz, DMSO- d6): δ 173.8, 169.8, 152.9, 148.3, 137.1,134.6, 129.1, 125.8, 125.1, 124.1, 119.7, 114.9, 108.3, 106.2,30.9, 28.8, 27.1 ppm. MS (ESI): m/z (C19H18N4O5, MH+)383.

Synthesis of 5-((1-methyl-2-oxo-3-(phenylcarbamoyl)-2,3-dihydro-1H-benzo[d]imidazol-5-yl)amino)-5-oxopentanoic acid (9)

Compound 9, a white solid, was prepared as described above.Glutaric anhydride (0.0606g, 0.000531mol)was used for thesynthesis (0.077g, 55%) instead of succinic anhydride. m.p.:186 ◦C.

1H NMR (400 MHz, CDCl3) : δ 10.81 (s, 1H), 8.14 (s,1H), 7.59 (m, 2H), 7.37 (m, 2H), 7.16 (m, 1H), 7.06 (s,2H), 3.39 (s, 3H), 2.32 (m, 2H), 2.12 (m, 2H), 1.86 (m, 2H)ppm. 13C NMR (600 MHz, DMSO- d6) : δ 173.8, 169.8,152.9, 148.3, 137.1, 134.6, 129.1, 125.8, 125.1, 124.1, 119.7,114.9, 108.3, 106.2, 30.9, 28.8, 27.1 ppm. MS (ESI): m/z(C20H22N4O4, MH+) 397.

Synthesis of 3, 3′-(ethane-1,2-diyl)bis(1-methyl-5-nitro-1,3-dihydro-2H-benzo[d]imidazol-2-one) (10)

K2CO3 (0.1431g, 0.00103mol) was added to a solution of(3) (0.1g, 0.000517mol) in DMF (10mL), after which asupply of 1,2-dibromoethane (0.0486g, 0.000258mol) wasadded to the reaction mix. The reaction was carried outunder a nitrogen atmosphere. The mixture was refluxed for4h. The progress of the reaction was monitored by TLC(EtOAc:CHCl3, 2:8). The resulting reaction mixture wasquenched with ice, and the obtained green solid was filteredand purified using column chromatography (EtOAc:CHCl3,2:8) to obtain compound 10 (0.0458g, 43%). m.p.: 284 ◦C.

1H NMR (300 MHz, DMSO- d6) : δ 7.97 (d, J = 9Hz, 2H), 7.77 (s, 2H), 7.23 (d, J = 8.7 Hz, 2H), 4.29 (s,4H), 3.2 (s, 6H) ppm. 13C NMR (600 MHz, DMSO- d6) :δ 154.1, 141.4, 135.1, 128.7, 118.0, 107.4, 102.9, 27.2 ppm.MS (ESI): m/z (C18H16N6O6, MH+) 413.

Synthesis of 3, 3′-(ethane-1,2-diyl)bis(5-amino-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one) (11)

Compound 11 was synthesized according to the proceduredescribed for preparing compound (5). Recrystallization,however, was conducted under different conditions. A mixof EtOH and Et2O was used to obtain a pure compound 11(0.082g, 24%) as a pale brown solid. m.p.: 280 ◦C.

1HNMR (300MHz, DMSO- d6) : δ 6.73 (d, J = 8.1 Hz,2H), 6.26 (m, 4H), 4.71 (s, 4H), 3.92 (s, 4H), 3.16 (s, 6H)ppm. 13C NMR (600 MHz, DMSO- d6) : δ 153.7, 143.7,129.7, 120.9, 108.1, 107.0, 93.9, 26.7 ppm. MS (ESI): m/z(C18H20N6O6, MH+) 353.

Synthesis of N-(3-(1H-imidazol-1-yl)propyl)-2,4-dinitroaniline (12)

1-(3-Aminopropyl) imidazole (17.72mL, 0.1485mol) wasadded to a solution of 1-chloro-2,4-dinitrobenzene (3g,0.0148mol) in ethanol (30mL) at 0 ◦C and stirred at RTfor 15h. The progress of the reaction was monitored by TLC(EtOAc:EtOH, 1:1). The reaction solution was concentrated,and hot water was added. The obtained yellow solid precipi-tate was filtered andwashedwith hexane to obtain compound12 (4.0454g, 93%). m.p.: 141 ◦C.

1H NMR (600 MHz, DMSO- d6) : δ 8.84 (d, J = 2.4Hz, 1H), 8.83 (bs, 1H), 8.23 (dd, J = 9, 2.4 Hz, 1H), 7.63(s,1H), 7.19 (s, 1H), 7.15 (d, J = 9.6 Hz, 1H), 6.88 (s,1H), 4.07 (m, 2H), 3.47 (m, 2H), 2.08 (m, 2H) ppm. 13CNMR (600 MHz, DMSO- d6): δ 148.0, 137.2, 134.7, 129.9,128.4, 123.6, 119.2, 115.1, 43.6, 40.3, 29.4 ppm. MS (CI):m/z (C12H13N5O4, MH+) 292.

Synthesis N1-(3-(1H-imidazol-1-yl)propyl)-4-nitrobenzene-1,2-diamine (13)

Compound 13 was synthesized using the same proceduredescribed for the synthesis of compound (2). Recrystalliza-tion fromDCM and Et2O gave a dark red-colored compound13 (0.841g, 94%). m.p.: 170 ◦C.

1H NMR (400 MHz, DMSO- d6):): δ 8.85 (m, 1H), 8.84(bs, 1H), 8.23 (dd, J = 9.6, 3 Hz, 1H), 8.14 (s,1H), 7.64(s, 1H),7.2 (s, 1H), 7.16 (d, J = 9.6 Hz, 1H), 6.89 (s, 1H),4.06 (m, 2H), 3.50 (m, 2H), 2.08 (m, 2H) ppm. 13C NMR(600MHz,DMSO- d6) : δ 163.0, 147.9, 137.2, 134.7, 129.9,129.8, 128.3, 123.6, 119.2, 115.1, 43.6, 40.2, 29.4 ppm. MS(CI): m/z (C12H15N5O2,MH+) 262.

Synthesis of 1-(3-(1H-imidazol-1-yl)propyl)-5-nitro-1,3-dihydro-2H-benzo[d]imidazol-2-one (14)

Di-imidazol-1-yl-methanone (1.86g, 0.0114mol) was addedto a solution of (13) (1g, 0.00382mol) inDMF (7mL) at 0 ◦C.

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After 10min, the solution was allowed to reach RT, and thereaction mix was stirred for 2 h. The progress of the reactionwasmonitored byTLC (EtOAc:DCM, 1:1). The reactionwasquenched with ice and the obtained brown precipitate wasfiltered to yield compound 14 (0.614g, 56%). m.p.: 227 ◦C.

1H NMR (400 MHz, DMSO- d6):): δ 11.47 (s, 1H), 8.01(m, 1H), 7.76 (d, J = 2.4 Hz, 1H), 7.64 (s, 1H), 7.30 (d,J = 8.8Hz, 1H), 7.21 (s, 1H), 6.88 (s, 1H), 4.02 (m, 2H), 3.86(m, 2H), 2.10 (m, 2H) ppm. 13C NMR (600 MHz, DMSO-d6): δ 154.5, 141.5, 137.2, 135.6, 128.4, 128.2, 119.3, 119.1,117.7, 107.3, 103.9, 43.6, 37.9, 29.4 ppm. MS (CI): m/z(C13H13N5O3, MH+) 288.

Synthesis of 1-(3-(1H-imidazol-1-yl)propyl)-5-amino-1,3-dihydro-2H-benzo[d]imidazol-2-one (15)

Compound 15 was prepared as described in the procedurefor the synthesis of compound (2). Compound 15 (0.143g,32%) was obtained as a colorless oil.

1H NMR (400 MHz, DMSO- d6) : δ 10.46 (s, 1H), 7.72(s, 1H), 7.25 (s, 1H), 6.91 (s, 1H), 6.70 (d, J = 8.4 Hz,1H), 6.30 (d, J = 2 Hz, 1H), 6.24 (d, J = 8.4, 2 Hz, 1H),3.98 (m, 2H), 3.64 (m, 2H), 2.01 (m, 2H) ppm. 13C NMR(600 MHz, DMSO- d6): δ 154.3, 143.6, 137.3, 129.2, 129.8,128.1, 121.1, 119.4, 107.9, 106.7, 95.7, 43.6, 37.1, 29.6 ppm.MS (CI): m/z (C13H15N5O, MH+) 258.

Acknowledgments This study was partly supported by a Bar-Ilan-University new faculty Grant for A.G. This study was also supportedby a KAMIN program grant (Israel Ministry of Industry, Trade andLabour) for M.Y.N. and K.R. We would like to thank Nechama-SaraCohen for the English editing of the manuscript.

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