Design, synthesis, and enzyme kinetics of novel benzimidazole and quinoxaline derivatives as methionine synthase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.10.052

Methionine synthase catalyzes the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine, producing methionine and tetrahydrofolate. Benzimidazole and deazatetrahydrofolates derivatives have been shown to inhibit methionine synthase by competing with the substrate 5-methyltetrahydrofolate. In this study, a novel series of substituted benzimidazoles and quinoxalines were designed and assessed for inhibitory activity against purified rat liver methionine synthase using a radiometric enzyme assay. Compounds 3g, 3j, and 5c showed the highest activity against methionine synthase (IC₅₀: 20 μM, 18 μM, 9 μM, respectively). Kinetic analysis of these compounds using Lineweaver-Burk plots revealed characteristics of mixed inhibition for 3g and 5c; and uncompetitive inhibition for 3j. Docking study into a homology model of the rat methionine synthase gave insights into the molecular determinants of the activity of this class of compounds. The identification of these drug-like inhibitors could lead the design of the next generation modulators of methionine synthase.

Full text of DOI:10.1016/j.bmc.2013.10.052

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and enzyme kinetics of novel benzimidazole
and quinoxaline derivatives as methionine synthase inhibitors
Hosam Elshihawy a, Mohamed A. Helal b, Mohamed Said a, Mohamed A. Hammad c,
⇑
a Organic Chemistry Department, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt
c Eli & Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California,
1425 San Pablo Street, BCC 505, Los Angeles, CA 90033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Methionine synthase catalyzes the transfer of
a methyl group from 5-methyltetrahydrofolate to
Received 4 September 2013
Revised 19 October 2013
Accepted 29 October 2013
Available online xxxx
homocysteine, producing methionine and tetrahydrofolate. Benzimidazole and deazatetrahydrofolates
derivatives have been shown to inhibit methionine synthase by competing with the substrate 5-methyl-
tetrahydrofolate. In this study, a novel series of substituted benzimidazoles and quinoxalines were
designed and assessed for inhibitory activity against purified rat liver methionine synthase using a radio-
metric enzyme assay. Compounds 3g, 3j, and 5c showed the highest activity against methionine synthase
Keywords:
Benzimidazole
Quinoxaline
Methionine synthase
5-Methyltetrahydrofolate
Cancer
(IC50: 20 lM, 18 lM, 9 lM, respectively). Kinetic analysis of these compounds using Lineweaver–Burk
plots revealed characteristics of mixed inhibition for 3g and 5c; and uncompetitive inhibition for 3j.
Docking study into a homology model of the rat methionine synthase gave insights into the molecular
determinants of the activity of this class of compounds. The identification of these drug-like inhibitors
could lead the design of the next generation modulators of methionine synthase.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
methionine transmethylation. It is worth noting that MetS is the
only human enzyme that metabolizes MeTHF to regenerate the ac-
Cobalamin-dependant
methionine
synthase
(MetS)
tive form tetrahydrofolate. Therefore, it is essential in the recycling
of different biological forms of folates required for purine and
pyrimidine synthesis.7 In the methionine transmethylation cycle,
Met is transformed to the reactive s-adenosyl methionine
(AdoMet) which is a biologically iumportant methyl donor in-
volved in the synthesis of nucleic acids, epinephrine, and creatine.
The product of this methylation process is s-adenosyl homocyste-
ine which is readily hydrolysed to adenosine and homocysteine.
The latter is again methylated to methionine using MetS enzyme;
hence conserving the limited dietary supply of this essential amino
acid.8 Normal cells remain unaffected by Met restrictions if
supplemented with Hcy, as they are able to synthesize Met via
methylation of Hcy in sufficient quantities. Impaired function of
MetS could lead to megaloblastic anaemias and neurological
disorders.9 From a medicinal chemistry point of view, inhibition
of MetS would compromise DNA and RNA synthesis and provide
(EC.2.1.1.13) is one of the transmethylase enzymes that utilizes
cobalamin derivative methylcobalamin (Me-Cbl) as a cofactor.1
This enzyme plays a crucial role in folate cycle and methionine
(Met) metabolism. MetS catalyzes the transfer of the methyl group
from 5-methyltetrahydrofolate (MeTHF) to homocysteine (Hcy) via
the cobalamin cofactor (Cbl) which circulates between +1 and +3
oxidation states.2,3 The enzyme functions in two steps as a ping-
pong mechanism. First, it transfers methyl group from MeTHF to
enzyme-bound cobalamin (Cbl(I)) to generate methyl-cobala-
min(III) (Me-Cbl(III)) and tetrahydrofolate. Second, the methyl
group is transferred from the generated Me-Cbl(III) to Hcy produc-
ing Met with the regeneration of Cbl(I) (Fig. 1). Cobalamin-depen-
dent methionine synthase is found in mammalian tissues and
microorganisms, while the plant enzyme is cobalamin-indepen-
dent.4 Crystal structure of cobalamin-dependent MetS revealed
that it consists of four functional domains, namely, Hcy binding do-
main, MeTHF binding domain, cobalamin cofactor binding domain,
and the allosteric cofactor S-adenosyl-methionine (S-AdoMet)
binding domain.5,6 The reaction products, tetrahydrofolate and
Met are further metabolized via folate cycle and the one-carbon
a
valuable tool for chemotherapeutic intervention in some
cancers.10,11
Despite being an attractive target for rational drug design, MetS
is not yet exploited to produce clinically useful anticancer agents. A
number of studies have shown that frequent administration of eth-
anol resulted in the inhibition of methionine synthase via the
interference with the conversion of hydroxycobalamin to methyl-
cobalamin.12 It was also reported that nitrous oxide (N2O) inhibits
⇑
Corresponding author. Tel.: +1 323 442 1688; fax: +1 323 442 8067.
E-mail address: mhammad@usc.edu (M.A. Hammad).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.052
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2
H. Elshihawy et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
fore, we decided to keep these features fixed and introduce diver-
sity at position 2 of the benzimidazole or quinoxaline rings.
2.2. Chemistry
Phillips procedure was adapted for the synthesis of benzimid-
azole derivatives via a condensation reaction of o-phenylendiam-
ines 1a–b and the appropriate amino acids in the presence of
5.5 N HCl.18 Then, acetylation or benzoylatoion of the obtained
amines 2a–d was affected using acetic anhydruide or benzoyl chlo-
ride, respectively. This method has furnished benzimidazole deriv-
atives 3a–j in moderate yields (Scheme 1). Quinoxalines 5a–d have
been synthesized through a condensation reaction between the
appropriate aromatic aldehydes and 4-nitro-o-phenylendiamine
(1b) according to the reported procedure.19 The arylidene deriva-
tives produced (4a–d) have been refluxed with triethyl orthofor-
mate to give the required quinoxalines 5a–d.20 Finally, the target
quinoxalines 6a–b were obtained via the alkylation of 5b with
the appropriate alkyl halides, whilst the target compounds 7a–b
were obtained through the coupling of 5c with the appropriate ali-
phatic amines under the standard amide coupling conditions
(Scheme 2).
Figure 1. The cobalamin-dependent methionine synthase catalyzed methyl group
transfer. Cbl(I): Cob(I)alamin, CH3-Cbl: methylcob(III)alamin, MeTHF: methyl
tetrahydrofolate.
the enzyme through oxidation of the cobalamin cofactor.13 Other
non-drug like inhibitors includes methylmercury, acetaldehyde,
and some cobalamin analogs.14,15 Therefore, we think that the de-
sign of drug-like small molecules as inhibitors of MetS has been
overlooked. In a recent study, Zhang et al. reported some N5-
substituted 8-deazatetrahydrofolates as folate analogs inhibiting
the enzyme by the formation of covalent bonds with the nucleo-
philic Cbl(I) cofactor. These inhibitors had shown some in vitro
inhibitory activity against HL-60 cells.16 In 2006, Banks et al. de-
signed simple benzimidazole and benzothiadiazole derivatives
and tested them in a cell free system for rat liver MetS inhibitory
activity.17 Despite being suitable drug leads, these compounds pos-
sessed limited diversity and only weak inhibitory activity against
2.3. Biological evaluation
The target compounds that were successfully synthesized were
tested for their inhibitory activity against highly purified rat liver
cobalamin-dependent methionine synthase (Table 1). Generally,
benzimidazoles with aliphatic side chains on carbon 2 showed sig-
nificantly lower activities compared to those with aromatic ones
(compare 3c and 3g). This could be attributed to the ability of
the bulkier aryl substituents to make favorable contacts with the
PABA binding pocket of MetS. We also noticed that the terminal
aryl ring should be isolated from the benzimidazole nucleus with
a polar three- to four-atom linker. Another feature that was found
to greatly enhance the inhibitory activity was the presence of nitro
group on carbon 5 of the benzimidazole ring (compare 3e and 3g).
This nitro group is the bioisoster of the amino group at carbon
atom 2 of the MeTHF pteridine ring. Phenylquinoxaline 5a and
its amino derivatives, 5b, 6a, and 6b showed only moderate activ-
ity, whilst substitution of the phenyl ring with a carboxylic acid
group increased the activity and gave the most active compound
the enzyme with IC50 values around 100 lM.
In the present study, we decided to explore the utility of this
benzimidazole scaffold to design MetS inhibitors that could com-
pete with MeTHF for its binding pocket. Benzimidazole and quin-
oxaline derivatives that resemble the chemical substructure of
MeTHF were designed by introducing alkyl and aryl side chains
on the nitrogen containing ring. The heterocyclic nucleus should
occupy the same pteridine-binding pocket while the diverse sub-
stituents could make contacts with other residues in the MeTHF
domain, originally binding PABA and glutamate part, to enhance
the activity and selectivity.
2. Results and discussion
2.1. Compounds design
in this study (compound 5c, IC50 = 9 lM). Further substitution on
the terminal carboxylic acid groups could not improve the inhibi-
tory activity (compounds 7a–b). The most active compound, 5c,
Scheme 1 possesses a similar shape to the natural ligand, tetrahy-
drofolate. In addition, it has less polar functional groups and more
rigid scaffold. Hence, it loses less entropy upon binding to the en-
zyme than tetrahydrofolate.
Methionine synthase is a bisubstrate enzyme binding both MeT-
FH as well as hmocysteine. It is possible that MetS inhibitors also
compete for the homocysteine binding domain in addition to the
MeTHF binding domain, or even bind at the site of the S-adenosyl
methionine reactivation cofactor.17 Therefore, the molecular mech-
anism of action of the most active compounds 5c, 3g, and 3j was fur-
ther investigated by studying the kinetic parameters of inhibition.
These compounds were tested at gradient concentrations of
Although the biochemical role of MetS in folate metabolism and
nucleotide biosynthesis is well established, no clinically useful
anticancer agents targeting this enzyme are available to date. As
mentioned above, there are also a limited number of drug-like
MetS inhibitors reported so far. We noticed that the weak benz-
imidazole and benzothiadiazole inhibitors designed by Banks
et al. possess a relatively small size compared to the large binding
site of MeTHF.17 These compounds were designed to mimic the
planar pteridine substructure of MeTHF and form polar contacts
with Asn458, Asp525, and Asn567 in the pteridine binding pocket.
On the other hand, the pocket created by Asn323, Arg516, and
Phe570, originally binding PABA, remains vacant. In order to en-
hance the activity and the drug-like properties of this series of
compounds, we envisioned a strategy in which acyl- and arylamino
side chains are introduced at carbon 2 of the benzimidazole nu-
cleus to occupy the PABA binding pocket and confer more binding
ability on the compounds. In addition, we decided to prepare a ser-
ies of quinoxaline derivatives to exploit their similar size and elec-
tronic characters to the pteridine nucleus of MeTHF. Also, Banks
et al. showed that the presence of free NH and 5-nitro group on
the benzimidazole ring is associated with stronger activity. There-
500 lM, 250 lM, 100 lM and 0 lM in a radiometric enzyme
assay.21 In this study we have only attempted the evaluation of the
inhibitory activity against the MeTHF binding site, keeping all other
parameters constant. The kinetic parameters of the enzyme inhibi-
tion were calculated and the Lineweaver-Burk plots were estab-
lished for the inhibited reactions as well as the uninhibited
reaction (Km = 28.6 lM, Vmax = 8.7 l
M minꢀ1) (Fig. 2 and S4).22 The
Lineweaver–Burk plots for the inhibited reactions showed that com-
pounds 5c and 3g exhibits the characteristics of mixed inhibition
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H. Elshihawy et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
Scheme 1. Reagents and conditions: (a) L-amino acid, 5.5 M HCl, reflux, 5 h; (b) RCOCl, TEA, THF, reflux, 3 h.
Scheme 2. Reagents and conditions: (a) ArCHO, EtOH, reflux, 12 h; (b) triethyl orthoformate, DMF, reflux, 24 h; (c) RBr, TEA, THF, reflux 24 h; (d) RCOOH, EDCI, HOBt, TEA,
DCM, rt, 12 h.
Table 1
steric cofactor (S-AdoMet) or to the surface of whole enzyme. Also,
due to the structure similarity, this nitrobenzimidazole 3j may dis-
Chemical structures and IC50 of the prepared benzimidazoles
X
place the dimethylbenzimidazole side chain of the cobalamin-cofac-
tor. The molecular determinants of the uncompetitive inhibition of
MetS will be the subject of future investigation in our laboratory.
N
(
)
n
O
R
N
H
NH
2.4. Homology modeling and docking studies
Compound
X
n
R
IC50 (lM)
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
H
NO2
NO2
H
1
2
1
2
1
2
1
2
1
2
CH3
CH3
CH3
CH3
Ph
Ph
Ph
Ph
NAa
NA
NA
NA
122
85
20
66
73
18
To examine the mode of binding of the designed inhibitors to
MetS and to investigate further their mechanism of action, com-
pounds 5c, 3g, and 3j were docked into the proposed MeTHF bind-
ing site in MetS. In the absence of a high-resolution crystal
structure of the mammalian enzyme, a model of the rat MetS
Hcy- and pterin-binding domains (residues: 1–620) was con-
structed, based on the X-ray crystal structure of MetS from Thermo-
toga maritima (PDB: 1Q8J).6 It has been reported that the MeTHF
binding domains of methyltransferases possess similar architec-
ture.23 The crystal structure of MetS from T. maritime showed that
four polar residues, Asn411, Asp473, Asn508, and Arg516, are crit-
ical for the interaction with the substrate MeTHF. Our final refined
model of rat MetS showed similar arrangement of the correspond-
ing polar residues within the binding site of MeTHF, namely,
Asn404, Asn458, Asp525, Asn567, and Arg579 (Fig. S3). Hence,
the developed model should be able to rationalize the binding of
the designed pterin isosteres into MetS.
H
NO2
NO2
H
4-(NO2)Ph
4-(NO2)Ph
3j
NO2
Values were determined using highly purified rat liver methionine synthase.
a
NA: not active.
which is a pattern typically noticed in multisubstrate enzymes such
as MetS, whilst compound 3j shows the behavior of uncompetitive
inhibition. This uncompetitive inhibition indicates that there is no
direct interaction between the inhibitor and the MeTHF binding site.
Potential explanation includes the possibility that the inhibitor may
bind at other binding sites such as the binding site of the MetS allo-
Figure 2. Lineweaver–Burk plots for compounds 5c, 3g, and 3j, with respect to methyltetrahydrofolate (MeTHF), at inhibitor concentrations of 500
M.
lM, 250 lM, 100 lM, and
0
l
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H. Elshihawy et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
Compounds 5c, 3g, and 3j were docked into the MeTHF binding
site, defined by a sphere of 10 Å radius around the side chain nitro-
gen of Asn458, using CCDC Gold software. As expected, compound
5c showed more favorable interactions than 3g or 3j, taking into
consideration that 3j is an uncompetitive inhibitor. The docked
conformation of 5c shared a very similar binding mode to MeTHF
in the template crystal structure (PDB ID: 1Q8J) with the quinoxa-
line nucleus having the same position as the pterin ring of MeTHF.
Table 2 The nitro group, acting as a bioisotere of the pterin ring
amino group, forms a strong H bond with Ser461. On the other side
of the binding pocket, the carboxylic group shows another H bond
with Arg579 similar to that formed by the PABA fragment of
MeTHF in the template crystal structure (Fig. 3). Also, one of the
quinoxaline nitrogens forms a weak H bond with Asn484. This
extensive H bonding network, together with the relatively rigid
scaffold which is complementary to the MeTHF pocket, could ratio-
nalize the high potency of 5c (Fig. 3). Interestingly, mapping of the
electrostatic potential on the Connolly surface of 5c and tetrahy-
drofolate reveals an obvious similarity (Fig. 4). Also, the distances
between the groups responsible for the critical H bonding in both
compounds are similar, 13.2 Å and 13.5 Å for 5c and tetrahydrofo-
late, respectively (Fig. 4, black arrows).
Compound 3g shares a comparable binding pattern with 5c,
particularly its benzimidazole nucleus. However, it forms only
two H bonds with Asn458 and Asn 567. The unsubstituted benz-
amide moiety is not able to interact with Arg579 (Fig. 5). Finally,
we tried to dock the uncompetitive inhibitor 3j to understand its
inability to bind to the MeTHF pocket. A distance constraint be-
tween the benzimidazole NH and Asn458 was applied to force
the docking of this compound into the MeTHF binding site. This
Table 2
Chemical structures and IC50 of the quinoxaline series
Ar
O2N
N
N
Figure 3. Proposed binding mode of 5c in the homology model of rat MetS. (a)
Cartoon representation with amino acid residue colored cyan and 5c colored
yellow. (b) Surface representation of the binding site with 5c colored yellow. Shape
complementarity between 5c scaffold and the binding site is obvious.
Compound
Ar
IC50
90
(lM)
5a
NH2
5b
74
COOH
5c
5d
6a
9
70
101
NHEt
NHiPr
6b
7a
7b
NAa
82
CONHEt
CONHiPr
NA
Figure 4. Mapping of the electrostatic potential on the Connolly surface of 5c and
tetrahydrofolate. Black arrows indicate the functional groups responsible for the
critical H bonding with MetS.
Values were determined using highly purified rat liver methionine synthase.
a
NA: not active.
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H. Elshihawy et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
for the differential binding of the prepared compounds to MeTHF.
The ideal MetS competitive inhibitor should possess a benzo-fused
nitrogen heterocycle, as a pteridine isostere, connected via a polar
flexible linker to an aryl group which is able to occupy the PABA
binding pocket. It is worth noting that compounds reported in this
study represent the first class of non-folate MetS inhibitors target-
ing the PABA pocket. On the other hand, more detailed mechanistic
and computational studies on a large number of compounds are re-
quired to unveil the molecular determinants of uncompetitive
inhibition. This study revealed a new generation of lead com-
pounds that could help in the design of clinically useful MetS
inhibitors with potential anticancer activity.
4. Experimental
4.1. Chemistry
The structures of all tested compounds were confirmed by 1H
NMR, 13C NMR and mass spectrometry (ESI). The purities of the
tested compounds were determined by combustion analysis and
are 95% or higher. Commercial chemicals and solvents were re-
agent grade and used without further purification. 1H and 13C
NMR spectra were measured in DMSO-d6 on a Bruker ARX 400.
Chemical shifts are reported in parts per million (ppm) using tetra-
methylsilane (TMS) as an internal standard. Mass spectra were ob-
tained on a Fissous Instruments VG Platform 2 (ESI-MS system).
Combustion analysis was performed at the Microanalytical Center
of Cairo University, Egypt. The synthetic procedure for the com-
pounds is illustrated in Schemes 1 and 2.
4.1.1. General procedures for the synthesis of (1H-
benzimidazole-2-yl)alkylamines (2a–d)
General procedure for the synthesis of (1H-benzimidazole-2-
yl)alkylamines was adapted from the Phillips procedure.18
L-Amino
acid was added to a stirred solution of 4-nitro-o-phenylendiamine
and aqueous HCl (5.5 M). The mixture was heated under reflux for
5 h. The blue reaction mixture was cooled to room temperature.
The mixture was allowed to stand for overnight where upon the
desired (1H-benzimidazole-2-yl)alkylamine was crystallized in its
HCl salt form. The free base was obtained by neutralization of
the reaction mixture with 1 M K2CO3 solution followed by extrac-
tion with ethyl acetate. The extract was evaporated to dryness and
recrystallized from an ethanol.
Figure 5. Proposed binding mode of (a) 3g and (b) 3j in the homology model of rat
MetS. Cartoon representation is used with amino acid residues colored cyan and
compounds colored yellow.
4.1.2. 1-(1H-Benzimidazol-2-yl)methanamine (2a)
compound forms H bond with Asn458 only. In addition, the obvi-
ous internal strain in the docked conformation of 3j was found to
deteriorate its docking score. The bulky and relatively rigid nature
of this compound did not allow the proper extended orientation in
the MeTHF pocket adapted by 5c (Fig. 5). These factors could pro-
vide, at least, a partial explanation of the inability of 3j to compete
with MeTHF for MetS binding.
Yield 61%; mp 269–270 °C. 1H NMR (400 MHz, DMSO-d6): d
7.61 (2H, dd, 3J 9.40, 4J 3.20, 4-H, 7-H), 7.27 (2H, m, 3J 9.40, 4J
3.20, 5-H, 6-H), 4.43 (2H, s, CH2), 5.02 (3H, br, s, NH, NH2,); 13C
NMR (400 MHz, DMSO-d6): d 147.56 (C-2), 132.51 (C-3a, C-7a),
126.16 (C-5, C-6), 114.99 (C-4, C-7), 35.17 (C-10); DEPT (135)
(DMSO-d6); CH2: 35.17, CH: 126.16, 114.99; IR (KBr): 3050 (C–H,
sp2), 2850 (C–H, sp3), 1450 and 1350 (C–H, bend) cmꢀ1; MS (EI):
m/z 147.19 (M+). Anal. Calcd for C8H9N3: C, 65.29; H, 6.16; N,
28.55. Found; C, 64.99; H, 6.36; N, 28.43.
3. Conclusion
MetS inhibitors represent a promising class of chemotherapeu-
tic agents. Based on the scaffold of MeTHF and the previously re-
ported inhibitors of the benzimidazole class, a novel series of
benzimidazoles and quinoxalines were designed and tested in a
cell-free assay for MetS inhibitory activity. Some of the prepared
derivatives showed significant inhibition of the MetS reaction,
especially relatively small quinoxalines and nitrobenzimidazoles.
The most three active compounds, 5c, 3g, and 3j were evaluated
in a kinetic enzyme assay and, interestingly, the results indicated
that there is more than one mode of interaction with the enzyme.
Homology modeling and docking studies gave partial explanation
4.1.3. 2-(1H-Benzimidazol-2-yl)ethanamine (2b)
Yield 66%; mp 269–270 °C. 1H NMR (400 MHz, DMSO-d6): d
8.57 (2H, br, s, NH2), 7.79 (2H, dd, 3J 9.40, 4J 3.21, 4-H, 7-H), 7.53
(2H, dd, 3J 9.40, 4J 3.21, 5-H, 6-H), 3.56 (4H, m, CH2CH2); 13C
NMR (400 MHz, DMSO-d6): d 150.8 (C-2), 131.6 (C-3a, C-7a),
126.1 (C-5, C-6), 114.3 (C-4, C-7), 36.3 (C-20), 25.2 (C-10); IR
(KBr): 3500 (N–H), 3050 (C–H, sp2), 2850 (C–H, sp3), 1450 and
1350 (C–H, bend) cmꢀ1; MS (EI): m/z 161.17 (M+). Anal. Calcd for
C9H11N3: C, 67.06; H, 6.88; N, 26.07. Found; C, 67.35; H, 6.80;
N, 26.77.
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H. Elshihawy et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
4.1.4. 1-(5-Nitro-1H-benzimidazol-2-yl)methanamine (2c)
Yield 40%; mp 248–251 °C. 1H NMR (400 MHz, DMSO-d6): d
10.90 (1H, br, s, NH), 8.90 (2H, br, s, NH2), 8.51 (1H, d, 4J 1.97,
4-H), 8.15 (1H, dd, 3J 8.90, 4J 2.23, 6-H), 7.78 (1H, d, 3J 8.90, 7-H),
4.41 (2H, s, CH2); 13C NMR (400 MHz, DMSO-d6): d 153.7 (C-2),
143.2 (C-7a), 142.5 (C-5), 138.0 (C-3a), 118.7 (C-6), 115.3 (C-7),
112.9 (C-4), 36.92 (CH2NH2); DEPT 135 (DMSO-d6) CH2: 36.92,
CH: 118.7, 115.3, 112.9; IR (KBr): 3400 (N–H), 3050 (C–H, sp2),
2850 (C–H, sp3), 1500 and 1350 (N–O); MS (EI): m/z 192.08 (M+);
Anal. Calcd for C8H8N4O2ꢁ2HClꢁ1/4H2O: C, 35.60; H, 3.89; N,
20.77. Found; C, 35.92; H, 3.80; N, 20.43.
2850 (C–H, sp3), 1580 (C@O),1550 and 1350 (N–O) cmꢀ1; MS
(EI): m/z 234.11 (M+); Anal. Calcd for C10H10N4O3: C, 51.28; H,
4.30; N, 23.92. Found; C, 51.41; H, 4.10; N, 23.70.
4.1.10. N-(2(5-Nitro-1H-benzimidazole-2-yl)ethyl)acetamide
(3d)
Pale yellow powder; yield 42%; mp 208–209 °C. 1H NMR
(400 MHz, DMSO-d6): d 8.43 (1H, d, 4J 2.20, 4-H), 8.10 (2H, dd, 3J
8.90, 4J 2.20, 6-H, NH), 7.76 (1H, d, 3J 8.90, 7-H), 3.56 (2H, t, 3J
3
6.68, 20-H), 3.08 (2H, t, J 6.68, 10-H), 1.82 (3H, s, CH3); 13C NMR
(400 MHz, DMSO-d6): d 170.7 (C@O0), 157.4 (C-2), 145.3 (C-7a),
133.2 (C-5), 131.3 (C-3a), 121.3 (C-6), 115.3 (C-7), 110.9 (C-4),
36.9 (C-20), 28.2 (C-10), 23.0 (CH3); IR (KBr): 3222 (N–H), 3061
(C–H, sp2), 2931 (C–H, sp3), 1665 (C@O), 1500 and 1344 (N@O)
cmꢀ1; MS (EI): m/z 248.19 (M+); Anal. Calcd for C11H12N4O3: C,
53.22; H, 4.87; N, 22.57. Found; C, 53.33; H, 4.66; N, 22.26.
4.1.5. 2-(5-Nitro-1H-benzimidazol-2-yl)ethanamine (2d)
Yield 58%; mp >300 °C (decomp.). 1H NMR (400 MHz, DMSO-
d6): d 11.74 (1H, br, s, NH), 8.53 (1H, d, 4-H), 8.29 (1H, dd, 3J 6.2,
6-H), 7.89 (1H, d, 3J 6.2, 7-H), 3.52 (4H, m, CH2CH2); NH2 not
observed; 13C NMR (400 MHz, DMSO-d6): d 155.9 (C-2), 144.6
(C-7a), 137.7 (C-5), 133.5 (C-3a), 120.5 (C-6), 115.2 (C-7), 111.2
(C-4), 36.4 (C-20), 26.1 (C-10); IR (KBr): 3450 (N–H), 3050 (C–H,
sp2), 2850 (C–H, sp3), 1500 and 1350 (N–O); MS (EI): m/z 206.22
(M+); Anal. Calcd for C9H12N4O2ꢁ2HClꢁH2O: C, 36.53; H, 4.32; N,
18.77. Found: C, 36.38; H, 4.73; N, 18.86.
4.1.11. N-(1H-Benzimidazole-2-yl-methyl)benzamide (3e)
Long white needles; Yield 95%; mp 234–235 °C. 1H NMR
(400 MHz, DMSO-d6): d 12.35 (1H, br, s, NH), 9.19 (1H, t, 3J 5.7, CH2-
NH), 7.95 (2H, d, 3J 6.9, 50-H, 90-H), 7.56–7.47 (5H, m, 4-H, 7-H, 60-H,
70-H, 80-H), 7.13 (2H, dd, 3J 7.4, 5-H, 6-H), 4.69 (2H, d, 3J 5.7, CH2);
13C NMR (400 MHz, DMSO-d6): d 167.06 (C@O), 152.94 (C-2),
142.94 (C-3a, C-7a), 134.50 (C-70), 131.99 (C-40), 128.86 (C-60, C-
70), 128.03 (C-50, C-90), 122.32 (C-5, C-6), 118.89 (C-4, C-7), 38.36
(C-10); IR (KBr): 3050 (C–H, sp2), 2850 (C–H, sp2), 1700 (C@O),
1450 and 1350 (C–H, bend) cmꢀ1; MS (EI) m/z 251.11 (M+); Anal
Calcd for C15H13N3O: C, 71.70; H, 5.21; N, 16.72. Found: C, 71.02;
H, 5.38; N, 16.57.
4.1.6. General procedures for the acylation of (1H-
benzimidazole-2-yl)alkylamines
Starting amino compound (0.238 mmol) was dissolved in THF
(25 mL) and triethylamine (10 drops) was added to the stirred
solution. Acyl halide (0.149 mmol) was added dropwise to the stir-
red solution, which was then heated under reflux for 3 h. The reac-
tion mixture was concentrated under reduced pressure. The
residue was dissolved in EtOAc and washed with aq. K2CO3 solu-
tion followed by brine. The organic layer was then dried over Na2-
SO4, filtered, and concentrated in vacuo. The residue was purified
by silica gel column chromatography eluting with 1–5 methanol/
DCM v/v.
4.1.12. N-(2-(1H-Benzimidazole-2-yl)ethyl)benzamide (3f)
Fine white needles; Yield 76%; mp 308–310 °C. 1H NMR
(400 MHz, DMSO-d6): d 12.40 (1H, s, NH), 8.70 (1H, t, 3J 5.20,
4
NH), 7.83 (2H, dd, 3J 8.40, J. 60-H, 100-H), 7.52–7.42 (5H, m, 4-H,
7-H, 80-H, 70H, 90-H), 7.11 (2H, dd, 3J 9.10, 4J 2.80, 5-H, 6-H), 3.73
3
3
(2H, q, J, 7.15, 20-H), 3.12 (2H, t, J 7.15, 10-H); 13C NMR
(400 MHz, DMSO-d6): d 167.3 (C@O), 152.7 (C-2), 134.4 (C-3a, C-
7a), 131.9 (C-50), 131.3 (C-80), 128.8 (C-70, C-90), 127.8 (C-60, C-
100) 126.1 (C-5, C-6), 114.2 (C-4, C-7), 37.6 (C-20), 27.6 (C-10); DEPT
135 (600 MHz; DMSO-d6) CH2: 38.6, 29.4; CH: 131.7, 128.8, 127.7,
121.8, 121.7; IR (KBr): 3300 (N–H), 3025 (C–H, sp2), 2850 (C–H,
sp3), 1635 (C@O); 1550 (N–H bend) cmꢀ1; MS (EI) m/z 265.18
(M+). Anal. Calcd for C16H15N3O: C, 72.43; H, 5.70; N, 15.84. Found;
C, 72.88; H, 5.55; N, 15.66.
4.1.7. N-((1H-Benzimidazole-2-yl)methyl)acetamide (3a)
Pale brown powder; yield 85%; mp 196–197 °C. 1H NMR
(400 MHz, DMSO-d6): d 9.26 (1H, d, 3J 5.44, NH), 7.45 (2H, dd, 3J
9.40, 4J 3.21, 4-H, 7-H), 7.11 (2H, dd, 3J 9.15, 4J 3.21, 5-H, 6-H),
3
4.47 (2H, d, J 5.69, 10-H), 1.89 (3H, s, CH3); 13C NMR (400 MHz,
DMSO-d6): d 170.1 (C@O), 153.2 (C-2), 131.2 (C-3a, C-7a), 126.3
(C-5, C-6), 114.4 (C-4, C-7), 37.6 (C-10), 23.1 (C–CH3); MS (EI): m/
z 189.15 (M+). Anal. Calcd for C10H11N3O: C, 63.48; H, 5.86; N,
22.21. Found; C, 63.55; H, 5.79; N, 21.70.
4.1.13. N-((5-Nitro-1H-benzimidazole-2-yl)methyl)benzamide
(3g)
4.1.8. N-(2-(1H-Benzimidazole-2-yl)ethyl)acetamide (3b)
White powder; yield 80%; mp 186–187 °C. 1H NMR (400 MHz,
DMSO-d6): d 8.05 (1H, t, 3J 5.40, NH), 7.47 (2H, dd, 4-H, 7-H),
Pale yellow crystals; yield 68%; mp 244–246 °C. 1H NMR
(400 MHz, DMSO-d6): d 9.29 (1H, t, 3J 5.93, NH), 8.41 (1H, d, 4J
2.22, 4-H), 8.10 (1H, dd, 3J 8.90, 4J 2.22, 6-H), 7.94 (2H, dd, 3J
3
7.11 (2H, dd, 3J 9.10, 4J 3.10, 5-H, 6-H), 3.36 (2H, t, J 7.17, 20-H),
3
2.49 (2H, m, 10-H), 1.78 (3H, s, CH3); 13C NMR (400 MHz, DMSO-
d6): d 169.85 (C-40), 153.4 (C-2), 133.6 (C-3a, C-7a), 121.74 (C-5,
C-6), 121.7 (C-4, C-7), 37.8 (C-20), 29.5 (C-10), 23.2 (CH3). MS (EI):
m/z 203.16 (M+). Anal Calcd for: C11H13N3O: C, 65.01; H, 6.45; N,
20.68. Found: C, 65.09; H, 6.65; N, 20.38.
8.16, 50-H, 90-H), 7.67 (1H, d, J 8.90, 7-H), 7.66–7.47 (3H, m, 60-
H, 70-H, 80-H), 4.77 (2H, d, 3J 5.93, CH2); 13C NMR (400 MHz,
DMSO-d6): d 167.9 (C@O), 157.3 (C-2), 145.3 (C-7a), 135.8 (C-5),
133.3 (C-3a), 132.5 (C-70), 131.4 (C-40), 128.9 (C-60, C-80), 128.3
(C-50, C-90), 121.4 (C-6), 115.5 (C-7), 111.2 (C-4), 37.0 (C-10); DEPT
(135) (600 MHz; DMSO-d6) CH2: 37.0, CH: 132.5, 128.9, 128.3,
121.4, 115.5, 111.2; IR (KBr): 3500 (N–H), 3050 (C–H, sp2), 2850
(C–H, sp3), 1625 (C@O, conj.) 1550 and 1350 (N@O); 1300 (C–N)
cmꢀ1; MS (EI) m/z 296.20 (M+); Anal Calcd for: C15H12N4O3: C,
60.81; H, 4.08; N, 18.91. Found: C, 61.32; H, 3.99; N, 18.88.
4.1.9. N-((5-Nitro-1H-benzimidazole-2-yl)methyl)acetamide
(3c)
Yellow powder; yield 72%; mp 214–216 °C. 1H NMR (400 MHz,
DMSO-d6): d 8.63 (1H, t, 3J 5.93, CH2NH), 8.40 (1H, d, 4J 2.22, 4-H),
8.10 (1H, dd, 3J 8.90, 4J 2.22, 6-H), 7.67 (1H, d, 3J 8.90, 7-H), 4.52
(2H, d, 3J 5.69, CH2), 3.34 (1H, br, s, NH), 1.93 (3H, s, CH3); 13C
NMR (400 MHz, DMSO-d6): d 171.3 (C@O), 157.8 (C-2), 144.6 (C-
5), 138.0 (C-7a), 133.9 (C-3a), 120.3 (C-6), 115.3 (C-7), 111.5 (C-
4), 36.8 (C-10), 22.9 (CH3); DEPT (135) (DMSO-d6); CH3: 22.9;
CH2: 36.8; CH: 120.3; 115.3, 111.5; IR (KBr): 3050 (C–H, sp2),
4.1.14. N-(2-(5-Nitro-1H-benzimidazole-2-yl)ethyl)benzamide
(3h)
Yellow powder; yield 98%, mp 197–200 °C. 1H NMR (400 MHz,
DMSO-d6): d 8.69 (1H, s, NH), 8.40 (1H, d, 4J 2.22, 4-H), 8.09 (1H,
3
dd 3J 8.90, 4J 2.22, 6-H), 7.82 (2H, d, J 7.90, 60-H, 100-H), 7.67
Please cite this article in press as: Elshihawy, H.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.10.052

H. Elshihawy et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
(1H, d, 3J 8.90, 7-H), 7.55–7.42 (3H, m 3J 7.70, 70-H, 80-H, 90-H), 3.37
4.1.20. 4-(((2-Amino-5-nitrophenyl)imino)methyl)benzoic acid
(4c)
3
3
(2H, q, J 6.70, 20-H), 3.17 (2H, t, J 6.93, 10-H), 1-H not observed;
13C NMR (400 MHz, DMSO-d6): d 167.4 (C@O), 157.5 (C-2), 145.3
(C-7a), 135.7 (C-5), 134.4 (C-3a), 131.9 (C-70), 131.3 (C-50), 128.8
(C-70, C-90), 127.8 (C-60, C-100), 121.3 (C-1), 115.3 (C-7), 110.8 (C-
4), 37.6 (C-20), 28.2 (C-10); DEPT (135) (600 MHz; DMSO-d6) CH2:
37.6, 28,2; CH: 131.9, 128.8, 127.8, 121.3, 115.3, 110.8; IR (KBr):
3308 (N–H), 3098 (C–H, sp2), 2850 (C–H, sp3), 1641.17 (C@O),
1505 and 1338 (NO) cmꢀ1; MS (EI): m/z 310.21 (M+); Anal. Calcd
for C16H14N4O3: C, 61.93; H, 4.55; N, 18.06. Found; C, 62.30; H,
4.49; N, 17.80.
Brown crystalline solid; 77%; mp 270 °C. 1H NMR (400 MHz,
DMSO-d6): d 9.12 (1H, br s, OH), 8.31 (1H, d, 3J 1.20, 5-H), 7.87
(1H, d, 4J 3.20, 3-H), 7.77 (1H, d, 3J 9.40, 6-H), 6.65 (4H, m, ArH),
5.69 (1H, s, 7-H), 5.40 (2H, br s, NH2); IR (KBr): 3400 (OH), 3170
(NH2), 3050 (C–H, sp2), 1450 and 1350 (C–H, bend), 1660 (C@O),
1390 (NO2) cmꢀ1; MS (EI): m/z 285.09 (M+). Anal. Calcd for:
C14H11N3O4: C, 58.95; H, 3.89; N, 14.73. Found: C, 59.33; H, 4.01;
N, 14.50.
4.1.21. 4-Nitro-N-(phenylethylidene)-1,2-phenylenediamine
(4d)
4.1.15. N-((1H-Benzimidazole-2-yl)methyl)4-nitrobenzamide
(3i)
Yellow solid; yield 41%; mp 199 °C. 1H NMR (400 MHz, DMSO-
d6): d 8.55 (2H, br s, NH2), 8.40 (1H, d, 3J 3.12, 5-H), 8.07 (1H, s, 4J
1.20, 3-H), 8.02 (1H, d, 3J 3.12, 6-H), 7.88 (5 H, m, ArH), 6.01 (1H, t,
3J 2.22, 7-H), 2.20 (2H, d, 3J 2.12, CH2); MS (EI): m/z 255.19 (M+).
Anal. Calcd for: C14H13N3O2: C, 65.87; H, 5.13; N, 16.46. Found: C,
65.58; H, 4.74; N, 16.21.
Pale yellow powder; yield 44%; mp 128–130 °C. 1H NMR
4
(400 MHz, DMSO-d6): d 8.20 (2H, dd, 3J 8.16, J 2.22, 60-H, 80H),
7.67 (2H, dd, 3J 8.41, 4-H, 7-H), 7.50 (2H, dd, 3J 8.41, 5-H, 6-H),
7.14 (5H, m, ArH), 3.92 (4H, s, 2CH2), NHs not observed; 13C NMR
(400 MHz, DMSO-d6): d 154.01 (C-70), 148.81 (C-40), 147.32 (C-2),
147.02 (C-3a, C-7a), 130.31 (C-50, C-90), 129.71 (C-5, C-6), 123.85
(C-60, C-80), 121.92 (C-4, C-7), 52.02 (C-30), 46.65 (C-10); IR (KBr):
3400 (C–N), 3067 (C–H, sp2), 2832 (C–H, sp3), 1517 and 1344 (N–
O) cmꢀ1; MS (EI): m/z 296.17 (M+). Anal. Calcd for: C15H12N4O3:
C, 60.81; H, 4.08; N, 18.91. Found: C, 80.18; H, 4.14; N, 19.20.
4.1.22. General procedures for the synthesis of 7-nitro-2-
arylquinoxaline (5a–d)
4-Nitro-N-arylidene-1,2-phenylenediamine 4a–d (8.5 mmol)
was dissolved in 15 ml DMF at room temperature and triethyl
orthoformate (6.36 mL, 43 mmol) was added. The mixture was
heated under reflux for 24 h and subsequently allowed to cool to
room temperature and evaporated to dryness. The residue was
purified using column chromatography eluting with toluene/ethyl
acetate 2:1.
4.1.16. 4-Nitro-N-(2-(5-nitro-1H-benzimidazol-2-
yl)ethyl)benzamide (3j)
Yellow powder; yield 36%; mp 198–200 °C. 1H NMR (400 MHz,
DMSO-d6): d 8.43 (1H, d, 4J 2.22, 4-H), 8.35 (2H, d, 3J 8.65, 70-H, 90-
H), 8.10 (1H, dd, 3J 8.90, 4J 2.22, 6-H), 7.90 (2H, d, 3J 8.65, 60-H, 100-
H), 7.70 (1H, d, 3J 8.90, 7-H), 4.42 (2H, s, 40-H, CH2), 3.44 (4H, m, 3J
5.93, 10-H, 20-H). 13C NMR (400 MHz, DMSO-d6): d 155.4 (C-2),
148.2 (C-80), 145.6 (C-7a), 138.8 (C-5), 138.2 (C-50), 136.2 (C-3a),
132.0 (C-60, C-100), 124.1 (C-70, C-90), 120.9 (C-6), 115.3 (C-7),
111.1 (C-4), 49.5 (C-40), 43.7 (C-20), 38.9 (C-10); DEPT (135)
(DMSO-d6) CH2: 49.5, 43.7, 38.9; CH: 132.0, 124.1, 120.9 115.3;
111.1; MS (EI): m/z 355.11 (M+). Anal. Calcd for C16H13N5O5: C,
54.09; H, 3.69; N, 19.71. Found; C, 53.59; H, 3.77; N, 19.66.
4.1.23. 7-Nitro-2-phenylquinoxaline (5a)
Yellow crystalline solid; yield 74%; mp 210 °C. 1H NMR
(400 MHz, DMSO-d6): d 8.90–6.46 (9H, m, ArH); IR (KBr): 3020
(C–H, sp2), 1630 (C@C, sp2), 1440 (C@N), 1330 (NO2) cmꢀ1; MS
(EI): m/z 251.15 (M+); Anal Calcd for C14H9N3O2: C, 66.93; H,
3.61; N, 16.73. Found: C, 67.04; H, 4.01; N, 16.22.
4.1.24. 4-(7-Nitroquinoxalin-2-yl)aniline (5b)
Brown crystalline solid; 47%; mp 222 °C. 1H NMR (400 MHz,
DMSO-d6): d 9.22 (2H, br s, NH2), 8.90–6.05 (8H, m, ArH); IR
(KBr): 3400 (forked NH2), 3100 (C–H, sp2), 1699 (C@C, sp2), 1546
(C@N), 1300 (NO2) cmꢀ1; MS (EI): m/z 266.17 (M+); Anal. Calcd
for C14H10N4O2: C, 63.15; H, 3.79; N, 21.04. Found: C, 63.07; H,
4.11; N, 21.40.
4.1.17. General procedure for the synthesis of 4-nitro-N-
arylidene-1,2-phenylenediamine
4-Nitro-o-phenylendiamine 1 (2.69, 17.6 mmol) was suspended
in 10 ml dry ethanol. The appropriate aldehyde (20.6 mmol) was
dissolved in another 10 ml of dry ethanol and added to the above
suspension. The mixture was heated under reflux for 12 h. The
reaction was cooled on an ice bath and the precipitate was col-
lected by filtration and washed with ethanol.
4.1.25. 4-(7-Nitroquinoxalin-2-yl)benzoic acid (5c)
Pale yellow solid, yield 25; mp 310 °C. 1H NMR (400 MHz,
DMSO-d6): d 9.52 (1H, br s, OH), 7.99–6.25 (8H, m, ArH); IR
(KBr): 3550 (OH), 3206 (C–H, sp2), 1600 (C@C, sp2), 1396 (NO2)
cmꢀ1; MS (EI): m/z 295.10 (M+); Anal. Calcd for C15H9N3O4: C,
61.02; H, 3.07; N, 14.23. Found: C, 61.37; H, 3.51; N; 14.06.
4.1.18. 4-Nitro-N-benzylidene-1,2-phenylenediamine (4a)
Brown crystalline solid; yield 84%; mp 120 °C. 1H NMR
(400 MHz, DMSO-d6): d 7.91 (1H, dd, 3J 4.40, 4J 3.20, 5-H), 7.87
(1H, s, 3-H), 7.77 (1H, d, 3J 9.40, 6-H), 6.65 (5H, m, ArH), 5.69
(1H, s, 7-H), 5.40 (2H, brs, NH2); IR (KBr): 3200 (NH2), 3050 (C–
H, sp2), 1450 and 1350 (C–H, bend) cmꢀ1; MS (EI): m/z 241.09
(M+). Anal. Calcd for: C13H11N3O2: C, 64.72; H, 4.60; N, 17.42.
Found: C, 64.28; H, 4.19; N, 17.20.
4.1.26. 2-Benzyl-7-nitroquinoxaline (5d)
Brown crystalline solid; yield; 47%; mp 280 °C. 1H NMR
(400 MHz, DMSO-d6): d 9.50–6.05 (9 H, m, ArH), 2.99 (2H, s,
CH2); IR (KBr): 1590 (C@C, sp2), 1309 (NO2) cmꢀ1; MS (EI): m/z
266.17 (M+); Anal. Calcd for C15H11N3O2: C, 67.92; H, 4.18; N,
15.84. Found: C, 67.07; H, 4.51; N; 15.26.
4.1.19. 4-Nitro-N-(4-aminobenzylidene)-1,2-phenylenediamine
(4b)
Green crystalline solid; 64%; mp 210 °C. 1H NMR (400 MHz,
DMSO-d6): d 8.32 (2H, br s, NH2), 8.01 (1H, d, 3J 2.10, 5-H), 7.97
(1H, s, 3-H), 7.72 (1H, d, 3J 2.10, 6-H), 6.65–6.22 (5H, m, ArH),
5.99 (1H, s, 7-H), IR (KBr): 3200 (NH2), 3050 (C–H, sp2), 1450 and
1350 (NO2, asymmetric bending) cmꢀ1; MS (EI): m/z 256.20 (M+).
Anal. Calcd for: C13H12N4O2: C, 60.93; H, 4.72; N, 21.86. Found: C,
60.83; H, 4.70; N, 22.08.
4.1.27. General procedure for the synthesis of N-alkyl-4-(7-
nitroquinoxalin-2-yl)aniline (6a–b)
4-(7-Nitroquinoxalin-2-yl)aniline (5b) (0.12 g, 0.476 mmol)
was dissolved in 25 ml THF and, then, triethylamine (10 drops)
was added to the stirred solution. Alkyl bromide (0.149 mmol)
was added dropwise to the mixture, which was then heated under
reflux for 24 h. The reaction mixture was concentrated under
Please cite this article in press as: Elshihawy, H.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.10.052

8
H. Elshihawy et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
reduced pressure. The crude product was dissolved in dichloro-
methane and washed with water (3 ꢂ 10 mL). The organic layer
was dried over MgSO4, filtered and concentrated under reduced
pressure to yield a yellow precipitate, which was purified with col-
umn chromatography eluting with DCM/ethylacetate 2:5.
collection commenced at the beginning of each peak until it tailed
off. The active fractions were pooled, desalted, and concentrated
using an Amicon ultrafiltrator fitted with a 30 kDa membrane. Fur-
ther purification of the enzyme was performed using a hydroxyap-
atite column eluting with potassium phosphate buffer (gradient:
20–500 mM). The active fractions of MetS were collected, deion-
ized and concentrated by ultrafiltration. The final enzyme prepara-
tion was stored at 20 °C, out of direct light.
4.1.28. N-Ethyl-4-(7-nitroquinoxalin-2-yl)aniline (6a)
Bright yellow crystals; yield 75%; mp 213–216 °C. 1H NMR
(400 MHz, DMSO-d6): d 8.94 (1H, s, NH), 8.50–6.50 (13H, m,
ArH), 3.99 (2H, q, 3J 2.1, CH2), 1.33 (3H, t, 3J 2.1, CH3); MS (EI) m/
z 294.21 (M+); Anal. Calcd for C16H14N4O2: C, 65.30; H, 4.79; N,
19.04. Found: C, 65.00; H, 5.09; N; 18.86.
4.2.2. Methionine synthase assay
MetS activity was determined according to the procedure re-
ported by Kenyon et al.26 Reactions contained 50 mM phosphate
buffer (pH 7.4), 227
dithiothreitol, 40 M S-AdoMet, 60
zyme source, and DMSO solution of the inhibitor (5
lM
14C-5-methyltetrahydrofolate, 23 mM
4.1.29. N-Isopropyl-4-(7-nitroquinoxalin-2-yl)aniline (6b)
Yellow solid; yield 25%; mp 262–265 °C. 1H NMR (400 MHz,
DMSO-d6): d 8.99–6.50 (8H, m, ArH), 4.94 (1H, s, NH), 2.99–1.01
(11H, m, CHaliphatic); MS (EI) m/z 308.15 (M+); Anal. Calcd for
l
l
M hydroxycobalamin, the en-
lM). The reac-
tion mixture was incubated in light-excluding sealed serum vials
under N2 gas. After pre-incubation for 5 min, the reaction was ini-
C17H16N4O2: C, 66.22; H, 5.23; N, 18.17. Found: C, 65.90; H, 5.29;
tiated by the addition of 500
lM homocysteine and incubated at
N; 17.79.
37 °C for 30 min. The reaction was terminated by the addition of
ice-cold water (400 lM). The reaction mixture was passed through
4.1.30. General procedure for the synthesis of N-alkyl-4-(7-
nitroquinoxalin-2-yl)benzamide (7a–b)
a 0.5 ꢂ 5 cm AG1-X8 resin column. [14C] Methionine was eluted
with 2 mL of water and quantified using a liquid scintillation coun-
ter (Packard Tricarb 1900CA; Perkin Elmer).
To a solution of amine HCl (0.3 mmol) in CH2Cl2 (5 ml) were
added 4-(7-nitroquinoxalin-2-yl)benzoic acid (5c) (106 mg,
0.36 mmol), HOBt (42 mg, 0.36 mmol), EDCI (90 mg, 0.45 mmol)
and Et3N (0.21 mL, 1.5 mmol), and this mixture was stirred at room
temperature for 12 h. The mixture was then partitioned between
CH2Cl2 and saturated NaHCO3 aqueous solution and the organic
layer was washed with brine, dried over Na2SO4, filtered, and con-
centrated in vacuo. The crude residue was purified by silica gel col-
umn chromatography CHCl3/MeOH (96:4) to yield the required
amide.
4.2.3. Determination of Km and Vmax for
5-methyltetrahydrofolate
In all the three assays, a constant concentration of Hcy (500
was used. Assays were incubated for 10 min at varying concentra-
tions of 5-methyltetrahydrofolate (0, 50, 100, 500 and 1000 M).
lM)
l
Initially, the incubation time and the enzyme concentration were
altered in order to establish conditions for linear kinetics. Then,
the protein concentration was adjusted to ensure that the initial
velocities were correctly estimated.
4.1.31. N-Ethyl-4-(7-nitroquinoxalin-2-yl)benzamide (7a)
Bright yellow needles; yield 27%; mp 274–276 °C. 1H NMR
(400 MHz, DMSO-d6): d 9.44 (1H, s, NH), 9.22 (1H, s, OH), 8.93–
6.91 (8H, m, ArH), 3.01 (2H, q, 3J 4.1, CH2), 2.63 (3H, t, 3J 4.66,
CH3); IR (KBr): 3600 (amide N–H stretching), 3166 (C–H, sp2),
4.3. Homology modeling
Multiple sequences of three MetS enzymes were used in the
alignment process. Sequences of human, rat, Thermotoga maritima
MetS were aligned using the web interface ClustalW (Fig. S2).27
The multiple sequence alignment obtained in the previous step
was used together with the crystal structure of Thermotoga mariti-
ma MetS (PDB: 1Q8J) as an input for the building of the homology
model of the rat MetS using MODELLER 9v3 software.28 After all
the hydrogens were added, the crude model was minimized in a
stepwise manner using OPLS-2005 force field29 in Macromodel
v8.0 (Schrodinger Inc., Portland, OR, USA) in order to relieve any
steric clashes or distorted geometries. Five steps of minimization
were carried out. In the first step, all heavy atoms were kept fixed
(force constant: 500 kcal/mol/Å2) and only water and hydrogens
were allowed to minimize and relax their positions. During the
second step protein side chains were kept flexible while keeping
the protein backbone restrained with the same force constant.
The last three steps involved minimization of the whole protein
with gradually reducing force constraints of 50, 25, and 10 kcal/
1690 (amide C@O stretching), 1600 (C@C, sp2), 1396 (NO2) cmꢀ1
;
MS (EI) m/z 322.19 (M+); Anal. Calcd for C17H14N4O3: C, 63.35; H,
4.38; N, 17.38. Found: C, 63.90; H, 4.99; N; 17.06.
4.1.32. N-Isopropyl-4-(7-nitroquinoxalin-2-yl)benzamide (7b)
Bright yellow solid; yield 24%; mp 200–202 °C. 1H NMR
(400 MHz, DMSO-d6): d 9.20 (1H, s, NH), 8.81–7.01 (8H, m, ArH),
4.01 (1H, m, CH), 3.99 (4H, m, CH2), 1.33 (6H, m, CH3); MS (EI)
m/z 336.21 (M+); Anal. Calcd for C18H16N4O3: C, 64.28; H, 4.79; N,
16.66. Found: C, 64.90; H, 5.00; N; 16.06.
4.2. Biological evaluation
4.2.1. Enzyme purification
Methionine synthase was isolated and purified from rat liver
according to the reported procedure.24,25 Rat liver homogenate
was prepared in the presence of a cocktail of protease inhibitors.
The cytosolic fraction was obtained following a series of centrifu-
gation steps. This preparation was initially purified using batch
chromatography on DEAE cellulose. The speedy removal of un-
wanted proteins in the unbound fraction enriched the enzyme
preparation and facilitated the next step of the purification which
was performed using column chromatography. Anion exchange
chromatography using DEAE and Q-sepharose resins was used to
purify MetS through electrostatic attractions. Bound proteins were
eluted by increasing the ionic strength of the buffer to 500 mm
NaCl. The bound proteins were eluted at 2 mL/min and fractions
were collected at selected intervals throughout the run. Fraction
mol/Å2 on the C atoms of the backbone. The five minimization
a
steps consisted of 6000 steps each, in which the first 1000 steps
were steepest descents, and the last 5000 were conjugate
gradient.30
4.4. Docking
Compounds 5c, 3g, and 3j were built and energy-minimized
using MMFF charges and the MMFF force field as implemented in
Sybyl X.1 with 2000 steps of the conjugate gradient method to a
gradient of 0.001 kcal/Å.31 CCDC GOLD 4.12 software was used
for docking of the minimized compounds into the putative binding
Please cite this article in press as: Elshihawy, H.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.10.052

H. Elshihawy et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
9
pocket of the developed homology model of rat MetS.32 This soft-
ware uses a genetic algorithm (GA) to explore possible ligand bind-
ing modes by changing dihedrals of ligand rotatable bonds, ligand
ring geometries, and dihedrals of protein OH and NH2 groups. The
binding site was defined to include all amino acid residues within
10 Å of the side chain nitrogen of Asn458 with no distance re-
straints unless otherwise indicated. Docking was carried out using
the standard mode settings and GoldScore, a molecular mechanics-
like function depending on protein–ligand hydrogen bonding, pro-
tein–ligand van der Waals score, ligand intramolecular hydrogen
bonding and ligand intramolecular strain.33 A total of 10 genetic
algorithm runs were performed for each ligand with early termina-
tion criteria set to 1.5 Å root-mean-square deviation (RMSD) value.
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