1. Mol Divers
DOI 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 an
important role in a broad range of intracellular regulatory
mechanisms and in the pathophysiology of many human
diseases, including microbial and viral infections, cancer,
diabetes and neurodegenerative disorders. Recently, several
potent PKR inhibitors have been synthesized. However, the
enzyme’s multifunctional character and a multitude of PKR
downstream targets have prevented the successful transfor-
mation of such inhibitors into effective drugs. Thus, the
need for additional PKR inhibitors remains. With the help of
computer-aided drug-discovery tools, we designed and syn-
thesized potential PKR inhibitors. Indeed, two compounds
were found to inhibit recombinant PKR in pharmacologically
relevant concentrations. One compound, 6-amino-3-methyl-
2-oxo-N-phenyl-2,3-dihydro-1H-benzo[d]imidazole-1-car
boxamide, also showed anti-apoptotic properties. The novel
molecules diversify the existing pool of PKR inhibitors and
Electronic supplementary material The online version of this
article (doi:10.1007/s11030-016-9689-4) contains supplementary
material, which is available to authorized users.
B Arie Gruzman
gruzmaa@biu.ac.il
1 Division of Medicinal Chemistry, Department of Chemistry,
Faculty of Exact Sciences, Bar-Ilan University, 5290002
Ramat-Gan, Israel
2 Institute of Biochemistry, Food Science and Nutrition,
The Robert H. Smith Faculty of Agriculture, Food and
Environment, 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 Natural
Sciences and Center for Gene Manipulation in the Brain,
University of Haifa, 3498838 Haifa, Israel
provide a basis for the future development of compounds
based on PKR signal transduction mechanism.
Keywords PKR inhibitors · C16 · Benzoimidazole
derivatives · Computer modelling
Introduction
Protein kinase RNA-activated (PKR) is a member of the ser-
ine/threonine (Ser/Thr) kinase family that mediates a broad
spectrum 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 many
critical intracellular regulatory pathways, which are related
to 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 eukaryotic
initiation factor 2 alpha (eIF-2α) which plays an impor-
tant role in the regulation of protein synthesis in metabolic
stress, controls the translation initiation in various cells and
neurons and affects cognitive functions [10–13]. Phospho-
rylation of Ser51 in eIF-2 α by PKR inhibits total protein
synthesis, but selectively increases the production rates of
several proteins such as activating transcription factor 4
(ATF4) and beta-secretase 1 (BACE1) [14–17]. Several other
downstream PKR effector proteins were identified in the last
decade, including interferon regulatory factor 1, STATs, p53,
activating transcription factor 3, and IkK (which activates
NF-kappaB) [4,9,18–20].
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2. Mol Divers
In addition to the canonical protein synthesis PKR is also
involved in regulating CNS functions such as plasticity of
short-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 able
to inhibit the PKR RNA binding [26]. Two other known
PKR inhibitors were identified as ATP-recognizing domain
binders. In 2003, Jammi et al. discovered an imidazolo–
oxindole scaffold-based potent PKR inhibitor (C16) [2]. In
2011, 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) on
purified PKR [2] and exhibited biological effects in tissue
cultures [28]. However, the compound also affects PKR-
independent biochemical intracellular transduction mecha-
nisms. For example, in neurones, C16 modulates activity of
Jun N-terminal kinases (JNKs), the p38 MAP kinases, the
death-associated protein kinases (DAPKs), c-Raf, MEK1,
MKK6, and MKK7 pathways [28–30]. In addition, C16
also inhibits the activity of several cyclin-dependent kinases
(CDKs) including CDK2/CDK5 [30], and prevents Ab42-
induced apoptosis in C57BL/6J mouse embryo neuronal
cells. C16 also downregulated NF-kappaB in U937 human
monocytes following the reduction of IL-8 production. Fur-
thermore, C16 suppresses satratoxin G-induced apoptosis
in PC-12 neuronal cells, reduces HT-22 and HEK293T cell
cycle progression and blocks proliferation of MAC16 tumour
cells [5,28–31]. In addition, the compound showed impres-
sive biological activity in vivo. Tronel et al. reported that
C16 prevented neuronal loss and suppressed the inflam-
matory response in an acute excitotoxicity rat model [32].
This work confirmed the neuroprotective role of C16 which
was described by Ingrand et al. in [33]. An interesting
aspect of C16 activity in the CNS was reported by Stern
et al. The authors showed that C16 improved long-term
taste memory in rodents [34]. In addition, C16 demon-
strated strong antitumour activity in an adenocarcinoma
murine model (MAC16). Moreover, in the same cancer
animal models, C16 has been shown to attenuate mus-
cle atrophy and slow the progression of cancer-related
cachexia [5]. Finally, the imidazolo-oxindole derivative of
C16, imoxin, improved glucose homeostasis in obese dia-
betic mice [35].
We have used a C16 scaffold to perform a structurally
informed manual design of novel PKR inhibitors. The in
silico part of the project included the identification of
the putative binding pocket of PKR followed by a virtual
docking analysis of the designed compounds. Based on
these 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 compounds
were tested using a PKR activity assay (a recombinant pro-
tein) in which the affinity of the potential inhibitors was
measured 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),inhibitedPKRinthemicro-
mole range. Compound 5 showed a cell-protective effect
under oxidative conditions similar to C16. These results
provide new chemotypes for the inhibition of the PKR path-
way.
Results
Computer-aided drug design has been used in this work
for developing potential PKR inhibitors. This methodol-
ogy includes structure-based techniques, as done in previous
work by Levit et al. [36]. In this approach, we used kinase
complexes Nek2: PDB code 2JAV, and Wee1A: PDB code
which 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) were
used as a template to evaluate the new proposed compounds.
Specifically, the novel compounds were designed and drawn,
and their 3D conformations were generated. These structures
were then virtually docked into the PKR-binding domain.
Using the putative binding site and residues of PKR that may
interact with C16, all of the compounds’ docked poses were
scored based on binding energy and manually inspected.
The benzoimidazole ring was chosen as a central core
scaffold in all ten compounds due to its ability to form a π/π
stacking 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 introducing
different aromatic residues into the structures of the synthe-
sized compounds. In addition, the interaction between Lys
296 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 electron
donors and an acceptor in C16 were mimicked by nitrogen in
an amide bond and a carbonyl group in the benzoimidazole
ring.
First, 3-methyl-6-nitro-2-oxo-N-phenyl-2,3-dihydro-1H-
benzo[d]imidazole-1-carboxamide(4)wassynthesizedaccord-
ing to the literature [37], starting from commercially avail-
able 2,4-dinitro-chlorobenzene as shown in Scheme 1. The
starting 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; d
PhNCO, TEA; e H2, 10% Pd-C
Scheme 2 a
Methanesulphonylchloride,
TEA; b Isobutyl chloroformate,
TEA; c Succinic anhydride,
acetic acid; d Glutaric
anhydride, acetic acid
ondary amine: N-methyl-2,4-dinitroaniline (1). This was
done by creating a Meisenheimer complex to evoke an
aromaticnucleophilicsubstitutionreaction[38].Theorange-
colored product was obtained in high yield (93%). The next
step was the selective reduction of the ortho nitro group using
formic acid as a hydrogen donor in the presence of palladium
and triethylamine [39]. The reaction was extremely exother-
mic,andtheuseofanicebathwasnecessary.Ared-tintedani-
line derivative, N1-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 a
carbonyldiimidazole. 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 corresponding
benzoimidazole derivative consisted of a novel molecule (4)
which has not been reported before. Another benzoimidazole
derivative(6-amino-3-methyl-2-oxo-N-phenyl-2,3-dihydro-
1H-benzo[d]imidazole-1-carboxamide, 5) is included in the
AuroraScreeningLaboratorychemicallibrary,butitssynthe-
sis has not been reported yet. The compound was synthesized
through the reduction of the nitro group using a Parr
machine.
In compounds 6-9, different substitutions to the amine in
the benzoimidazole of compound 5 were used (Scheme 2).
In performing this manipulation, we investigated the role
the positive aniline charge has on possible interactions
with the PKR active center. In addition, a negatively
charged carboxylic acid moiety was introduced using either
ethyl 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 using
mesylchloride which was coupled with the free amine to
obtain a mesitylate according to procedure described by Mar-
vel et al. [40].
The compound was obtained as a colorless solid in
moderate 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) was
attained 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 was
synthetized to mimick the potential interactions (in addition
to a hydrogen donor ability of an amide bond, as in com-
pound 5) between an isopropyl moiety and a hydrophobic
pocket in the active center which we predict to be formed by
several lipophilic residues (Gly 278, Ile 273 and Val 281) in
the 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 were
attained 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 work
of Bryk et al., in which the authors showed that a molecule
constructed from two indole rings conjugated to each other
by a pyrimidine linker exhibited significant PKR inhibitory
activity [27]. Thus, two molecules of 3 were coupled through
an ethane linker bridging two nonmethylated nitrogen atoms
to 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 by
column chromatography to yield pure product in 43 % yield.
Both nitro groups in 10 were reduced to amines using a
high-pressure hydrogenation in a Parr machine to obtain
compound 11 (3, 3 -(ethane-1,2-diyl)bis(5-amino-1-methyl-
1,3-dihydro-2H-benzo[d]imidazol-2-one). We assumed that
the introduction of the positively charged amino groups
would increase the binding affinity of the compound.
Two additional compounds (14 and 15) were synthe-
sized as shown in Scheme 4. We introduced an imidazole
moiety to the benzoimidazole ring in order to mimic the
interaction of C16 moeity with Phe 421. The important dif-
ference between our designed molecules and C16 is that in
C16 the imidazole ring is connected to the main scaffold by
a rigid double bond, while in compounds 14 and 15 the imi-
dazole is conjugated through a flexible alkyl chain. As in
the 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 was
converted to its amine derivative: N1-(3-(1H-imidazol-
1-yl)propyl)-4-nitrobenzene-1,2-diamine (13) followed by
cyclization to form 1-(3-(1H-imidazol-1-yl)propyl)-5-nitro-
1,3-dihydro-2H-benzo[d]imidazol-2-one (14) and reduction
of 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 on
our docking analysis and the synthetic feasibility of the com-
pounds. Ten compounds were chosen for synthesis. Nine
novel 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 PKR
as a targeted kinase) was used for the in vitro validation of
the synthesized compounds. The KINOMEscanTM is a high-
throughput system for screening compounds against large
numbers of human kinases. This is one of the most compre-
hensive methods which were developed by DiscoveRx for
industrial use [41].
The assay performed by combining three components: a
DNA-tagged kinase, an immobilized ligand and a test com-
pound. The ability of a test compound to compete with the
immobilized ligand is measured by quantitative PCR of the
DNA tag. All test compounds showed excellent solubility in
DMSO. Thus, this solvent was used for the in vitro evaluation
of our test compounds.
AsummaryoftheKdvaluesobtainedbyKINOMEscanTM
is presented in Table 1. Two of the ten tested com-
pounds showed significant affinity to PKR: compounds 5
(Kd=27 μM) and 6 (Kd=23 μM). Dose response curves for
compounds 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 several
publications [28,32]. Therefore, the possible anti-apoptotic
effect of 5 and 6 together with the parent molecule C16 was
evaluated in the human breast cancer cell line (MCF-7). This
cell line was chosen for its high levels of PKR expression
and activity [42]. Apoptosis was induced using oxidative
stress, created by the glucose oxidase/glucose system which
constitutively generated hydrogen peroxide. Next, a standard
MTT analysis was conducted. Only compound 5 and C16 at
0.5 μM showed a significant cell-protective effect, as shown
in Fig. 5a. However, compound 5 was more effective than
C16, which increased cell viability by approximately 15%
compared with the 30% increase in cell protection shown
by compound 5. In addition, the activity of caspase 3 (a
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5. Mol Divers
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 Kd
and in silico-predicted active site
interactions 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 in
the presence and absence of C16 and compounds 5 and 6
[43]. In the same experiment, compound 5 greatly decreased
the activity level of caspase 3 (Fig. 5b). Moreover, its effect
was significantly higher than that of C16 on caspase 3 activ-
ity by approximately 18%. It is important to mention that
in 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 stress
assays.
Discussion
The potent PKR in vivo inhibitor C16 was discovered in 2003
[2]. However, because of its poor pharmacokinetic proper-
ties, the compound did not become a useful drug. Thus, the
starting point of this research was to use the rigid polycyclic
scaffold of C16 that creates important π/π interactions for the
design of active in vivo compounds suitable for use as parent
molecules with superior pharmacokinetic properties. Based
on our in silico work, several new compounds were designed,
and the versatility of the synthetic approach presented here
enabled the production of several innovative compounds.
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6. Mol Divers
The PKR inhibitory activity of ten novel compounds was
tested in vitro. Recombinant PKR was used for this primary
screening. Only two compounds were active (compounds
5 and 6), and both showed affinity in the micromolar con-
centration range. A molecular modelling approach was used
for the analysis of the in vitro results. Compounds 4 and
5 are predicted to make favourable interactions with the
lipophilic 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 critical
residues in the active PKR center, compounds 4 and 5 can
form a π/π stacking interaction by means of an additional
benzene ring. It is also important to mention that the nitro
groups (in compound 4) and the amino groups (in compound
5) are known as hydrogen-bond participants (depending on
the distance between corresponding donors/acceptors in the
active center) which might stabilize the binding capability of
the molecule to the PKR active center. Interestingly, based
on the in silico analysis, compound 4 is able to form a σ/π
stacking bond with Val 291, π/π stacking with Phe 421 and
a hydrogen bond with Lys 296 and Asp 432 (Supplementary
Fig. 4). In contrast, a hydrogen bond with an amine group in
compound 5 was not observed in silico. However, a carbonyl
group in the benzoimidazole moiety of 5 together with an
amide group from a urea functional group might form two
hydrogen 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 294
and the benzoimidazole moiety was also observed (Fig. 3).
These differences explain why compound 5 was active and
compound 4 was not.
In compound 6, a positively charged amino group was
replaced by a neutral sulphonylamide moiety, which could
participate in the formation of a hydrogen bond with Glu 367
(with –NH– as a hydrogen donor). However, virtual docking
against PKR did not show such an interaction (Fig. 4).
Instead, Glu 367 interacted with the methylated amine in
the benzoimidazole moiety by formation of a hydrogen bond.
In addition, our docking simulations revealed that besides
π/π interactions of the benzoimidazole core, another interac-
tion was formed: A new hydrogen-bond interaction between
Cys 369 and the benzoimidazole’s carbonyl group in com-
pound 6 (Fig. 4).
For compounds 8 and 9, modelling revealed that an
electrostatic interaction between the negatively charged car-
boxylic acid group and a positively charged primary amine of
lysine 296 was not likely to be formed. However, a carboxyl
group of compound 9 was predicted to create a hydrogen
bond with the amide hydrogen of Phe 278. The short linker
between a carboxy group and the amide in compound 8 did
not allow similar interactions to form (Supplementary Fig.
5a,b). According to the docking simulations, compound 10
formsπ/πstackinginteractionsbetweenthecoreofthemole-
cule and Phe 421. (In practice, this interaction was doubled.)
Also, Lys 296 interacted through a cation-π bond with one
of the benzoimidazole moieties (Supplementary Fig. 6a, b).
In compound 11, an analysis of the docked pose revealed
that together with the obvious hydrophobic interactions with
the core which we described above, the amine group in one
of the benzoimidazole domains interacted with a carbonyl of
Ile 273 (Supplementary Fig. 6a, b). Interestingly, the in silico
model predicted that two additional noncovalent bonds may
be possible, both σ − π interactions. The first one was created
between Val 281 and one of the benzoimidazole moieties,
and a second between Gly 372 and another benzoimidazole
moiety. Moreover, a hydrogen bond between Cys 369 and
a carbonyl in one of the benzoimidazole domains was also
detected as a possible option.
Finally, in silico analysis of the mode of interaction of
compounds 14 and 15 showed that when they are at the PKR
binding site, they adopt stable conformations in which the
benzoimidazole scaffold interacted with Phe 421. However,
thenewlyintroducedimidazoleringdidnotformasignificant
interaction with the PKR active center (Supplementary Fig.
7a, b). In addition, Cys 369 may interact with the nitro group
of compound 14 and with a carbonyl in the benzoimidazole
moiety of compound 15. Also, a hydrogen bond could be
formed between the amine group (compound 15) and the
carbonyl 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 the
PKR 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 key
factor that contributes to the success of this reaction is the
introduction of a strong electron withdrawing group, such as
a nitro group, into the aromatic system [38]. Therefore, we
also used a nitro moiety in our synthesis in the first step, as
shown in Scheme 1. The second step was the reduction of
the nitro group by a mix of TEA, formic acid and 10% Pd/C.
The final compounds were then successfully purified and iso-
lated by column chromatography. The last step, a cyclization
toward the creation of substituted benzoimidazoles, was also
rapid and very efficient. All ten compounds presented in this
work 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 interactions
between the core of the active molecules and the PKR active
center, Cys 369 is able to form two hydrogen bonds with
compound 5. Moreover, the same residue interacts with a
carbonyl in the benzoimidazole domain of compound 6. It is
important to mention that several inactive compounds have a
similar mode of interaction with the PKR active center, which
can be seen in compounds 11, 14 and 15. However, com-
pounds 11 and 15 do not fit precisely in the PKR active center,
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7. Mol Divers
Fig. 2 Predicted interactions
with C16. a 2D representation
of the interactions between C16
(cyan lines) and PKR (residues
colored according to description
in the figure itself). b Putative
interactions between C16 (cyan
sticks) and PKR (grey sticks).
Hydrogen-bond interactions are
shown as blue dotted lines. In
addition, pi-interactions can be
formed between Phe 421 and the
aromatic rings of the ligand.
(Color figure online)
which explains the lack of activity of these compounds. In
compound 14, the bond between Cys 369 and the carbonyl
in the benzoimidazole moiety is replaced by the interaction
between Cys 369 and the nitro group. This change might also
be the reason for the lack of activity in compound 14. Inter-
estingly, compound 6, which has the lowest Kd (23 μM),
showed the most structural similarity to the binding mode
of C16: both compounds shared a binding to Glu 367, Cys
369 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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8. Mol Divers
Fig. 3 Schematic
representation of the putative
interactions between PKR and
compound 5
Fig. 4 Schematic representation of the putative interactions between PKR and compound 6
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9. Mol Divers
Fig. 5 Anti-apoptotic effect of compound 5 in MCF-7 cells. a The
MCF-7 cells were grown as described in “Materials and methods” and
treated 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 introduced
into 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 treated with C16 (0.5 μM), compounds 5 and 6 (0.5 μM),
Trolox (T, 50 μM), and DMSO (0.1%), for 24h. Afterward, the cells
were washed and lysated using the lysis buffer described in “Materials
and methods.” The obtained lysates were diluted by a factor of five and
used for the detection of caspase 3 activity levels with a commercially
available kit, according to the protocol provided in the kit. *p < 0.05,
n=3. mean ± SE. &, the significant difference between cells which
were treated by C16 and compound 5
367. It would be beneficial to further investigate the critical
role of Cys 369 in the inhibition of the PKR activity. Such
an investigation could be done using classical mutagenesis
approaches.
Although both compounds 5 and 6 have shown significant
affinity to recombinant PKR in relatively high concentra-
tions and were 100-fold less potent than C16, we decided to
test their anti-apoptotic activity in a cellular model. We were
encouraged by the data published by Atkinson et al., Gray
et al. and Islam et al. that showed C16 biological effects in
cell cultures in concentrations higher than 0.21 μM, all the
way to 5 μM [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-apoptotic
effect in primary murine mixed co-cultures [28]. The pos-
sible cytoprotective effects of both active compounds and
C16 itself were investigated in MCF-7 cells. We decided to
use C16 and our new compounds in three different concen-
trations: 5, 1, and 0.5 μM [28]. An oxidative stress model
was chosen for the induction of apoptosis as described in
the Methods section. Compound 6 was inactive in all three
concentrations. In contrast, C16 and compound 5 showed a
significant cytoprotective effect under oxidative stress condi-
tions when the cells were already pretreated with the lowest
concentrations of both compounds: 0.5 μM (Fig. 5a). Inter-
estingly, compound 5 was more effective than C16 (by nearly
17%). 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 possible
effect of the compound on the level of caspase-3, a known
apoptotic marker [47,48]. C16 and compound 5 were active
inthelowestconcentrationofthethreechosenforthetest(5,1
and 0.5 μM). Compound 5 was also more effective than C16
(by 19%) and surprisingly, also more effective than Trolox
(by 25%). Compound 6 did not show any inhibitory activity
on caspase 3 levels in MCF-7 cells (Fig. 5b). These results
positively correlated with the results obtained in the viabil-
ity assay which was conducted in identical conditions to the
caspase 3 experiment.
It is clear that the affinity of PKR inhibitors to its
active center and the level of inhibition of the enzyme in
a pure protein-based assay do not always correlate with
the inhibitory activity of such molecules in cellular assays.
Many factors, such as solubility in a medium, intracellular
metabolic activation or inactivation, the rate of the cellular
membrane penetration, intracellular accumulation, off-target
binding and intervention in other cellular signal transduction
mechanisms can dramatically influence the biological effect
of an inhibitor. Although in the pure-protein affinity assay
C16 was 100-fold more potent than compound 5, in the cel-
lular assay, both compounds induced the anti-apoptotic effect
at identical concentrations. Moreover, compound 5 was more
effective than the parent molecule.
Therefore, we believe that compound 5 inhibits PKR in
highconcentrationsinthefreecellsystem(KINOMEscanTM),
but may be in nanomolar concentrations in the cells. This
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10. Mol Divers
compound might inhibit some other kinases and lead to the
observed anti-apoptotic effects. In addition, an intracellu-
lar transformation of compound 5 is possible (but unlikely
in the recombinant PKR assay), and the possibly obtaining
metabolite might be the cause of the determined cellular
effect. More research is needed to determine the exact cel-
lular targets of compound 5. It is important to mention that
despite the fact that several PKB inhibitors have been devel-
oped so far, there is still a great need for effective, selective
and nontoxic compounds, due to their possible anticancer
and anti-inflammatory therapeutic potential.
Conclusions
With the use of molecular modelling methods, 16 molecules,
15 of them novel, were designed based on the known PKR
inhibitor C16 and its predicted interactions with PKR. Com-
pound 5 (a known molecule) was synthesized by a novel
synthetic pathway. Nine other new molecules were synthe-
sized in our laboratory. Two molecules, 5 and 6, showed
significant PKR binding in cell-free assay. Although the Kd
values of both compounds were higher (27 and 23 μM) than
the Kd value of C16 (0.21 μM), both compounds were tested
in cellular assays.
Compound 5 showed a significant cell-protective effect
under oxidative stress conditions, in similar concentration to
C16 (0.5 μM). Moreover, compound 5 was more effective
than C16. The molecule we report here may be used as a
starting point for the development of potent PKR transduc-
tion mechanism inhibitors and as a novel biochemical tool
for the exploration of the PKR signal transduction pathway.
There is a dire need for new therapeutic agents against
devastating human diseases in which PKR is involved, such
as Alzheimer’s, cancer and others. These newly identified
molecules can be used as a basis for the future development
of such drugs.
Materials and methods
The organic solvents (HPLC grade) were obtained from
Frutarom Ltd. (Haifa, Israel). The melting points were
determined with a Fisher-Johns melting point apparatus
(Palmerton, PA). The 1H NMR and 13C NMR spectra were
recorded at room temperature on a Bruker Advance NMR
spectrometer (Vernon Hills, IL) operating at 300 and 400
MHz, and were in accord with the assigned structures. Chem-
ical shift values were reported relative to the TMS that was
used as an internal standard. The samples were prepared by
dissolving the synthesized compounds in either DMSO-d6
(δ H = 2.50 ppm, δ C = 39.52 ppm) or CDCl3 (δ H = 7.26
ppm, δ C = 77.16 ppm). Chemical shifts were expressed
in δ (ppm) and coupling constants (J) in hertz units. The
splitting pattern abbreviations are as follows: s, singlet; d,
doublet; t, triplet; q, quartet; quint, quintet; m, unresolved
multiplet 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 mass
spectrometry. Data were processed using massLynX v.4.1
calculation and deconvolution software (Waters Corporation,
Milford, MA). Column chromatography was performed on
Merck Silica gel 60 (230–400mesh; Merck, Darmstadt, Ger-
many). Analytical thin-layer chromatography was carried out
on pre-coated Merck Silica gel 60F254 (Merck) sheets using
UV absorption for visualization. The purity of the final com-
pounds was confirmed using high-field NMR analysis. All
analytical data (including the NMR images) are shown in
the Supplemental data. Elemental analysis was conducted
by Perkin-Elmer 2400 series II Analyzer (Waltham, MA,
USA), and the results for all synthesized compounds are
shown in the Supplemental material (Supplementary Table
1). The purity of all compounds was above 95%. BSA,
D-glucose, MT reagent, and the protease inhibitor cocktail
were purchased from Sigma-Aldrich Chemicals (Rehovot,
Israel). Glycerol and sodium fluoride were obtained from
Merck (Whitehouse Station, NJ). Mercaptoethanol, phenyl-
methanesulphonylfluoride (PMSF), sodium orthovanadate,
sodium-β-glycerophosphate,sodiumpyrophosphateandSDS
were purchased from Alfa Aesar (Ward Hill, MA). Fetal
calf serum (FCS), l-glutamine, EMEM and antibiotics were
purchased from Biological Industries (Beth-Haemek, Israel).
TheCaspase3assaycolorimetricapoptotickitwaspurchased
from Abcam (Cambridge, MA, USA).
Cell culture
The human breast cancer cell line (MCF-7) obtained by
courtesy of Dr. E. Alpert (Quiet Therapeutics, (Ness Ziona,
Israel) was used for experiments. Cells were grown in Eagle’s
minimum essential medium (EMEM) containing 10 % fetal
bovine serum (FBS), 1mM glutamine, 100 μg/mL penicillin
and 100 μg/mL streptomycin at 37◦ C in a 5% CO2 humid-
ified atmosphere. Cells were seeded (100,000cell/mL) in a
6-well plate.
MTT assay
We described this test in a previous publication [49]. In
brief, cells were incubated with MTT (2mg/mL) in a growth
medium for 30 min at 37 ◦C. The medium was then aspirated,
andDMSOwasaddedtosolubilizethecellsandcoloredcrys-
tals. Absorbance at 570nm was measured in a SpectraMax
M5 spectrophotometer (Sunnyvale, CA, USA). The obtained
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11. Mol Divers
results were normalized by total protein content in culture
cells, which was measured using the Bradford reagent.
Apoptosis assay
TheCaspase3assaycolorimetricapoptotickitwaspurchased
from Abcam (Cambridge, MA, USA) and used as per the
manufacturer’s instructions. Absorbance at 405nm was mea-
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 of
glucose (23.5mM instead of the usual 5.5mM) was added
to the growing medium of MCF-7 cells [50]. This resulted
in an elevated H2O2 concentration in the medium (reaching
29.0 ± 9.6 μM in 4h of incubation). The concentration of
H2O2 generated by the glucose oxidase/glucose system was
determined as described [51].
Computational modelling
Before docking, all ligands were prepared in the Discovery
Studio (DS3.5, Accelrys) using the “Prepare Ligands” mod-
ule [52]. A set (not exceeding 255) of the most effective
low-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 as
implemented in DS3.5. CDocker is a CHARMm-based
docking method which uses a molecular dynamics (MD)
simulated annealing-based algorithm for ligand conforma-
tion generation and docking. Default algorithm settings were
used for docking. The final ligand poses were selected based
on 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
The PKR affinity of the synthesized compounds was obtained
usingLeadHunterTM DiscoveryServices(DiscoveRxCorpo-
ration, Fremont, CA, USA).
For the assay (KinomeScan analysis), PKR-tagged T7
phage strains were prepared in an E. coli host derived from
the BL21 strain. E. coli were grown to the log-phase and
infected with the T7 phage, then incubated and shaken at
32 ◦C until lysis. The lysates were centrifuged and filtered
to remove cell debris. The remaining kinases were produced
in HEK-293 cells and subsequently tagged with DNA for
qPCR detection. Streptavidin-coated magnetic beads were
treated with biotinylated small molecule ligands for 30min
at room temperature to generate affinity resins for the kinase
assays. The ligated beads were blocked with excess biotin
and washed with a blocking buffer (SeaBlock (Pierce), 1%
BSA, 0.05% Tween 20 and 1mM DTT) to remove unbound
ligands and to reduce nonspecific binding. Binding reactions
were assembled by combining kinases, ligand affinity beads
and test compounds in a 1× binding buffer (20% SeaBlock,
0.17× PBS, 0.05% Tween 20 and 6mM DTT). All reactions
were performed in polystyrene 96-well plates in a total vol-
ume of 0.135mL. The assay plates were incubated at room
temperature and shaken for 1h, and the affinity beads were
washed 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 affinity
ligand), then incubated at room temperature and shaken for
30min. The kinase concentration in the eluates was measured
by qPCR.
Statistical analysis
Statistical significance (p < 0.05) was calculated among
experimental 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) was
added to a solution of 1-chloro-2,4-dinitrobenzene (3g,
0.0148mol) in ethanol (30mL) at 0 ◦C and stirred at room
temperature (RT) for 15h. The reaction was monitored by
TLC (EtOAc:Hexane, 1:4). The reaction solution was con-
centrated, and hot water was added to the final crude material.
The precipitate was filtered and washed with hexane to obtain
compound 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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12. Mol Divers
Synthesis of N-methyl-4-nitrobenzene-1,2-diamine (2)
TEA (5.64g, 0.0404mol) and 10% Pd/C (0.108g) were
added 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 4.5h
followed by heating at 80◦C for 10min. The reaction was
monitored by TLC (EtOAc:Hexane, 1:1). The resulting mix-
ture was filtered, the solid residue was washed with MeOH,
andthefiltratewasconcentratedandpurifiedbycolumnchro-
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, 29.6
ppm. 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 added
to 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 the
reaction was monitored by TLC (EtOAc:Hexane, 1:1). The
resulting reaction mixture was quenched with ice. A brown
solid precipitated, and was then filtered and analysed. The
obtained 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 and
phenylisocyanate (0.043g, 0.000362mol) was added under
a nitrogen atmosphere. The mixture was then refluxed for
2 h. The progress of the reaction was monitored by TLC
(DCM: Hexane, 1:1). The mixture was concentrated under
reduced 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 a
Parr shaker for 3 h. The resulting mixture was filtered and
concentrated under reduced pressure. Recrystallization from
DCM and EtOH gave rise to compound 5 (0.91g, 55%) as a
cream-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◦C
andmethanesulphonylchloride(0.0609g,0.000531mol)was
added under a nitrogen atmosphere. The reaction mix was left
for 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. 13C NMR (600 MHz, 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- (phenylcarba
moyl)-2,3-dihydro-1H-benzo[d]imidazol-5-yl)carbamate
(7)
Compound 7, a white solid, was synthesized according to
the 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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13. Mol Divers
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 a
solution of (5) (0.1g, 0.000354mol) in acetic acid (10mL),
at RT, under a nitrogen atmosphere. The reaction was left at
RT for 12 h. The gray solid residue that formed was filtered
and 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 the
synthesis (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 a
supply of 1,2-dibromoethane (0.0486g, 0.000258mol) was
added to the reaction mix. The reaction was carried out
under a nitrogen atmosphere. The mixture was refluxed for
4h. The progress of the reaction was monitored by TLC
(EtOAc:CHCl3, 2:8). The resulting reaction mixture was
quenched with ice, and the obtained green solid was filtered
and 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 = 9
Hz, 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 procedure
described for preparing compound (5). Recrystallization,
however, was conducted under different conditions. A mix
of EtOH and Et2O was used to obtain a pure compound 11
(0.082g, 24%) as a pale brown solid. m.p.: 280 ◦C.
1H NMR (300 MHz, 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) was
added to a solution of 1-chloro-2,4-dinitrobenzene (3g,
0.0148mol) in ethanol (30mL) at 0 ◦C and stirred at RT
for 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 and washed with hexane to obtain compound
12 (4.0454g, 93%). m.p.: 141 ◦C.
1H NMR (600 MHz, DMSO- d6) : δ 8.84 (d, J = 2.4
Hz, 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. 13C
NMR (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 procedure
described for the synthesis of compound (2). Recrystalliza-
tion from DCM and Et2O gave a dark red-colored compound
13 (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
(600 MHz, 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 added
toasolutionof(13)(1g,0.00382mol)inDMF(7mL)at0 ◦C.
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14. Mol Divers
After 10min, the solution was allowed to reach RT, and the
reaction mix was stirred for 2 h. The progress of the reaction
was monitored by TLC (EtOAc:DCM, 1:1). The reaction was
quenched with ice and the obtained brown precipitate was
filtered 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 procedure
for 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 supported
by a KAMIN program grant (Israel Ministry of Industry, Trade and
Labour) for M.Y.N. and K.R. We would like to thank Nechama-Sara
Cohen for the English editing of the manuscript.
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