Molecular modeling studies and synthesis of novel quinoxaline derivatives with potential anti-cancer activity as inhibitors of methionine synthase

Article abstract of DOI:10.1007/s00044-012-0307-4

Methionine synthase (MetS) catalyses the transfer of a methyl group from the methyltetrahydrofolate (MTHF) to homocysteine to produce methionine and tetrahydrofolate. MetS is over-expressed in the cytosol of certain breast and prostate tumor cells. In this article, we designed, synthesized, and evaluated the biological activity of a series of substituted quinoxaline derivatives that mimic the MTHF in the structure. The main aim was to develop inhibitors that could inhibit the enzyme reaction by blocking the binding of MTHF. These inhibitors were docked into the MTHF binding domain in such the same way as MTHF in its binding domain. Compound 4-({(6-nitro-quinoxalin-2-yl)methylamino} methyl)benzoic acid showed the lowest free energy of the binding (-152.62 kJ/mol) and showed the lowest IC₅₀ values of 45 ± 9 and 53 ± 9 μM against two types of cancer cell lines PC-3 and MCF-7, respectively.

Full text of DOI:10.1007/s00044-012-0307-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:3405–3415
DOI 10.1007/s00044-012-0307-4
ORIGINAL RESEARCH
Molecular modeling studies and synthesis of novel quinoxaline
derivatives with potential anti-cancer activity as inhibitors
of methionine synthase
•
Hosam Elshihawy Mohamed Hammad
Received: 2 April 2012 / Accepted: 24 October 2012 / Published online: 23 November 2012
ꢀ Springer Science+Business Media New York 2012
Abstract Methionine synthase (MetS) catalyses the
transfer of a methyl group from the methyltetrahydrofolate
(MTHF) to homocysteine to produce methionine and tetra-
hydrofolate. MetS is over-expressed in the cytosol of certain
breast and prostate tumor cells. In this article, we designed,
synthesized, and evaluated the biological activity of a series
of substituted quinoxaline derivatives that mimic the MTHF
in the structure. The main aim was to develop inhibitors
that zcould inhibit the enzyme reaction by blocking the
binding of MTHF. These inhibitors were docked into
the MTHF binding domain in such the same way as MTHF
in its binding domain. Compound 4-({(6-nitro-quinoxalin-
2-yl)methylamino}methyl)benzoic acid showed the lowest
free energy of the binding (-152.62 kJ/mol) and showed the
lowest IC50 values of 45 9 and 53 9 lM against two
types of cancer cell lines PC-3 and MCF-7, respectively.
cofactor (Halpern et al., 1974). MetS catalyses the transfer of
the methyl group from 5-methyltetrahydrofolate to homocys-
teine via the cobalamin cofactor (CH3-cobalamin), which cir-
culates between ?1 and ?3 oxidation states (Fig. 1). Crystal
structure of cobalamin-dependent MetS revealed that it consists
of four functional binding domains. They are homocysteine
binding domain, 5-methyltetrahydrofolate binding domain, the
third domain binds cobalamin cofactor, and the fourth domain
is an allosteric cofactor S-adenosy-L-methionine (S-AdoMet)
(Hoffman, 1982). The reaction products methionine and tetra-
hydrofolate (THF) are closely correlated to important bio-
chemical reactions of the methylation of DNA, lipids, proteins,
and polyamine (Kenyon et al., 2002; Pavillard et al., 2006).
Some recent studies have showed promising anti-tumor
and apoptosis activities in colon with S-adenosylmethionine
(SAMe) and its metabolite methylthioadenosine (MTA) (Tony
et al., 2011). Some investigations using quinoxaline derivatives
such as 3-aryl-2-quinoxaline-carbonitrile 1,4-di-N-oxide deriv-
atives showed hypoxic and cytotoxic activities against some of
human cell lines (Yunzhen et al., 2012).
Keywords Cobalamin ꢀ MCF-7 cells ꢀ Methionine ꢀ
Methionine synthase ꢀ PC-3 ꢀ
Quinoxaline derivatives and tumor
The present study showed a new approach for deter-
mining specific inhibitors of MetS. Quinoxaline derivatives
that resemble substructure of 5-MTHF (Fig. 2) have been
docked into the MTHF binding domain. The free energy of
binding of these ligand-receptor complexes have been
obtained and compared to the results obtained from cell
free assay and in vitro tumor cell cytotoxicity assay.
Introduction
Cobalamin-dependant methionine synthase (MetS) is one of
the transmethylase enzymes that utilizes cobalamin derivative
methylcobalamin [methylcobalamin (CH3-cobalamin I)] as a
H. Elshihawy (&)
Organic Chemistry Division, School of Pharmacy,
Suez Canal University, Ismailia 41522, Egypt
e-mail: h.elshihawy@uea.ac.uk; hosam_sh_27@hotmail.com
Materials and methods
Molecular modeling procedure
M. Hammad
Centre for Engineering in Medicine and Surgical Services,
Massachusetts General Hospital, Harvard Medical School,
Boston, MA, USA
Molecular modeling was carried out on Schrodinger com-
putational software workstation using Maestro 7.5 graphic
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Med Chem Res (2013) 22:3405–3415
Fig. 1 Different enzymes
involved in the folic acid cycle.
The cycle starts with THF and
ends up with
methyltetrahydrofolate (MTHF)
which is in turn used by
methionine synthase as a
substrate and metabolizes it to
produce essential amino acid
methionine
acids and acidic amino acids such as glutamic acid, aspartic
acid, asparagine, and glutamine were deprotonated. Prime
1.5 was used to check the energy content of the protein
segments and loops. The docking process involved the
standard precision docking (SP) in which ligand poses that
were anticipated to have poor energies would be rejected
from the conditioned set up of the hierarchal filter. SP
docking is appropriate for screening ligands of unknown
quality in large numbers. SP is a soft docking program that
was adept at identifying ligands that have a reasonable
propensity to bind and 20 % of the final poses produced
from the SP docking were subjected to the Extra Precision
mode of Glide docking (XP) to perform the more expen-
sive docking simulation on worthwhile poses. XP docking
mode is harder than SP docking mode in that it penalizes
the poses that violate established charges. Flexible docking
was selected to generate conformations of all possible
ligand poses, which is more realistic as this occurs in
reality because the protein undergoes side chain and back
bone movement or both, upon ligand binding. Five- and
six-membered rings were allowed to flip and amide bonds
which were not cis or trans configuration were penalized.
5,000 poses per ligand for the initial phase of docking with
a scoring window for keeping poses of 100 kJ/mol were set
up. The best poses which fulfill those conditions were
subjected to energy minimization on the OPLS-AA non-
bonded-interaction grid with a distance dielectric constant
of 2 and maximum number of conjugate gradient steps of
500 iterations. The ligands of the poses selected by the
initial screening were subsequently minimized in the field
of the receptor using a standard molecular mechanics
energy function (OPLS-AA force field) in conjunction with
a distance-dependant dielectric model. Finally, the lowest
Fig. 2 The interception of MTHF with its binding domain of
methionine synthase forming hydrogen bonding with the key amino
acids of the receptor. The image showed that MTHF is docked
horizontally deep inside the receptor where the pteridine nucleus is
positioned at right hand side and the aromatic and c glutamate side
chain at the orifice of the receptor at left hand side
user interface (GUI) and Red Hat Linux nash Enterprise
version V 4.1.18 on Batchmin V 9.1 modeling engine. The
atomic coordinates for the polypeptide segments of MTHF
and Hcy binding domains of MetS enzyme extracted from
human liver were obtained from the protein bank database
[PDB (Brookhaven protein database)]. Explicit calcium ion
counter ions were included instead of cadmium and posi-
˚
tioned at a 4 A distance from the homocysteine binding
site. The basic and acidic amino acids were neutralized at
pH 7 2 by protonation of the terminal amino groups and
basic amino acids such as histidine, lysine, and arginine.
Moreover, the terminal carboxylic acid groups of amino
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Med Chem Res (2013) 22:3405–3415
3407
energy poses obtained in this fashion were subjected to a
Monte Carlo procedure that examines nearby torsion
minima. The complex was minimized using the conjugate
gradients algorithm until an energy convergence criterion
of 0.1 kJ/mol was reached with iteration cycle of 10,000.
Molecular dynamics (MD) at 300 K were then performed
on the solvated system for a 10 ps equilibration and 100 ps
of production employing a 1 fs time step using OPLS 2005
force field, from which 100 structures were sampled at 1 ps
intervals and averaged. The final averaged structure was
then finally minimized (Cairns et al., 2001).
C, 48.42; H, 2.73; N; 18.27. C9H6ClN3O2 requires:
C, 48.34; H, 2.70; N, 18.79 %.
N-[(6-Nitroquinoxalin-2-yl)methyl]benzenamine [2]
Aniline (2.21 mL, 0.238 mmol) dissolved in Tetrahydro-
furan (25 mL) and triethylamine (2 mL) was added to the
stirred solution. 2-Chloromethyl-6-nitroquinoxaline (3.33 g,
0.149 mmol) was added dropwise to the stirred solution,
which was then heated under reflux for 100 h. The reaction
mixture was evaporated under reduced pressure. The crude
product was dissolved in dichloromethane and washed with
water (3 9 10 mL). The organic layer was dried over
MgSO4, filtered, and concentrated under reduced pressure
to yield a brown precipitate, which recrystallized from
acetone to give the product as bright yellow crystals
(2.12 g, 63.6 %); m.p. 235–237 ꢁC (Woo-Jin and Chao-
Jun, 2006).
Experimental procedure
4-Nitro-phenylen-1,2-diamine and 2-iodobenzoic acid
(IBX) were obtained from Aldrich (Poole, UK). Aromatic
amine compounds were obtained from Avocado (Hew-
sham, UK). 3-Chloro-2-oxopropanal was gifted from a
colleague. Deuterated solvents were from Goss (Glossop,
UK). Melting points were determined using an Electro-
thermal IA9200 digital melting point apparatus. IR spectra
were recorded on a Perkin Elmer (Paragon 1000) FT-IR
Spectrophotometer (Perkin Elmer, Seer Green, UK). 1H
and 13C NMR spectra were acquired at 270.05 and
67.80 MHz, respectively, on a JEOL GX270 spectrometer
(JEOL UK, Welwyn, UK); 13C assignments were made
using the DEPT135 experiment, and coupling constant
(J) was measured in hertz. Mass spectra were obtained
from IPI, University of Bradford, West Yorkshire, UK.
dH (DLSO-d6): 9.22 (1 H, s, H-6), 8.93 (1 H, s, H-3),
8.75 (1 H, d, J3 1.4, H-8), 8.01 (1 H, d, J3 1.4, H-9),
7.25–6.34 (5 H, m, H-14–18), 3.62 (2 H, s, H-11), NH
signal was not observed; dC (DMSO-d6): 160 (C-2), 152
(C-7), 150 (C-3), 148 (C-13), 147 (C-10), 146 (C-5), 137
(C-9), 136 (C-15, C-17), 130 (C-6), 128 (C-8), 120 (C-16),
118 (C-14, C-18) 35 (C-11); DEPT 135 (DLSO-d6): 137
(C-9), 136 (C-15, C-17), 130 (C-6), 128 (C-8), 120 (C-16),
118 (C-14, C-18) 35 (C-11); tmax/cm-1 (KBr): 3120 (N–
H), 3122 (C–H, sp2), 2722 (C–H, sp3), 1378 (C–H, bend);
MS (ES): m/z 281 (M?•, 100 %), 210 (30), 187 (40), 166
(30); Elemental analysis: Found: C, 64.44; H, 4.33; N;
19.72. C15H12N4O2 requires: C, 64.28; H, 4.32; N,
19.99 %.
2-Chloromethyl-6-nitroquinoxaline [1]
A
solution of 4-nitro-phenylen-1,2-diamine (0.223 g,
1 mmol) and 3-chloro-2-oxopropanal (0.104 g, 1 mmol) in
dry glacial acetic acid (5 mL) was stirred at rt in the with
catalytic amount of IBX (0.028 g, 0.01 mmol). The reac-
tion mixture was filtered. NaHCO3 (5 mL, 5 % g/v) was
added to the filtrate and extracted with diethyl ether
(2 9 5 mL). The combined organic layers were washed
with brine (2 9 5 mL) and dried over MgSO4. The solvent
was evaporated under reduced pressure and the crude
product was crystallized with ethanol to give the product as
bright yellow crystals (0.13 g, 58 %); m.p. 213–216 ꢁC
(lit. 215–216 ꢁC) (Majid and Khadijeh, 2006).
4-Methyl-N-[(6-nitroquinoxalin-2-yl)methyl]
benzenamine [3]
Method 2.2 was followed using P-toluidine (0.25 g,
0.238 mmol) dissolved in absolute ethanol (25 mL), tri-
ethylamine (2 mL), and 2-chloromethyl-6-nitroquinoxaline
(3.33 g, 0.149 mmol). The reaction mixture was heated
under reflux for 79 h and then evaporated under reduced
pressure. The crude product was dissolved in ethyl acetate
and washed with water (3 9 10 mL). The organic layer
was dried over MgSO4, filtered, and concentrated under
reduced pressure to yield a yellow precipitate, which
recrystallized from ethanol to give the product as bright
yellow crystals (0.14 g, 56 %); m.p. 270–272 ꢁC (Woo-Jin
and Chao-Jun, 2006).
dH (DLSO-d6): 8.48 (1 H, d, J4 1.2, H-6), 8.16 (1 H, s,
H-3), 7.75 (1H, dd, J3 1.2, J4 2.3, H-8), 7.58 (1 H, d, J3 2,3,
H-9), 4.62 (2 H, s, CH2), NH signal was not observed; dC
(DMSO-d6): 158 (C-2), 148 (C-7), 147 (C-3), 144 (C-10),
140 (C-5), 130 (C-9), 125 (C-6), 122 (C-8), 45 (CH2);
DEPT 135 (DLSO-d6): 147 (C-3), 130 (C-9), 125 (C-6),
122 (C-8), 45 (CH2); tmax/cm-1 (KBr): 3098 (C–H, sp2),
2891 (C–H, sp3), 1300 (C–H, bend); MS (ES): m/z 224
(M?•, 95 %), 131 (11), 119 (8); Elemental analysis: Found:
dH (DLSO-d6): 8.76–6.32 (8 H, m, H-3, H-6, H-8, H-9,
H-14, H-15, H-17, H-18), 3.81 (2 H, s, H-11), NH signal
was not observed; dC (DMSO-d6): 158 (C-2), 156 (C-7),
152 (C-3), 144 (C-10, C-13), 138 (C-5), 137 (C-9), 129 (C-
15, C-17), 126 (C-16), 126 (C-6), 123 (C-8), 116 (C-14,
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Med Chem Res (2013) 22:3405–3415
C-18), 43 (C-11), 25 (C-19); DEPT 135 (DLSO-d6): 152
(C-3), 129 (C-15, C-17), 137 (C-9), 126 (C-6), 123 (C-8),
116 (C-14, C-18) 43 (C-11), 25 (C-19); MS (ES): m/z 295
(M?•, 50 %), 230 (20), Elemental analysis: Found: C,
65.00; H, 4.34; N; 19.01. C16H14N4O2 requires: C, 65.30;
H, 4.79; N, 19.04 %.
122 (C-16), 120 (C-14, C-18) 42 (C-11); DEPT 135
(DLSO-d6): 152 (C-3), 137 (C-9), 136 (C-15, C-17), 132
(C-6), 130 (C-8), 122 (C-16), 120 (C-14, C-18) 42 (C-11);
MS (ES): m/z 316 (M?11, 66 %), 300 (100), 122 (40), 166
(30); Elemental analysis: Found: C, 57.22; H, 3.90; N;
17.72. C15H11ClN4O2 requires: C, 57.24; H, 3.53; N,
17.80 %.
3-Methyl-N-[(6-nitroquinoxalin-2-yl)methyl]
benzenamine [4]
4-[(6-Nitroquinoxalin-2-yl)methylamino)benzoic
acid [6]
Method 2.2 was followed using m-toluidine (0.25 g,
0.238 mmol) dissolved in absolute ethanol (25 mL), tri-
ethylamine (2 mL), and 2-chloromethyl-6-nitroquinoxaline
(3.33 g, 0.149 mmol). The reaction mixture was heated
under reflux for 74 h and then concentrated under reduced
pressure. The crude product was dissolved in ethyl acetate
and washed with water (3 9 10 mL). The organic layer
was evaporated to give a yellow precipitate, which recrys-
tallized from ethanol to give the product as bright yellow
crystals (0.06 g, 24 %); m.p. 266–267 ꢁC (Woo-Jin and
Chao-Jun, 2006).
Method 2.2 was followed using p-amino-benzoic acid
(0.137 g, 0.01 mmol) dissolved in THF (25 mL), tri-
ethylamine (2 mL), and 2-chloromethyl-6-nitroquinoxaline
(0.223 g, 0.01 mmol). The reaction mixture was heated under
reflux for 200 h and then concentrated under reduced pres-
sure. Thecrude product wasdissolvedindichloromethaneand
washed with water (3 9 10 mL). The organic layer was
evaporated to give a brown precipitate, which recrystallized
from methanol to give the product as brown crystals (0.2 g,
40 %); m.p. 215–216 ꢁC (Woo-Jin and Chao-Jun, 2006).
dH (DLSO-d6): 9.51 (1 H, brs, COOH), 9.01 (1 H, s,
H-6), 8.63 (1 H, s, H-3), 8.75 (1 H, d, J3 1.4, H-8), 8.12 (1
H, d, J3 1.6, H-9), 7.54–6.38 (4 H, m, H-14, H-15, H-17,
H-18), 4.62 (2 H, s, H-11), 2.1 (1 H, brs, H-12); dC
(DMSO-d6): 168 (C-19), 162 (C-2), 153 (C-13), 151 (C-7),
148 (C-3), 145 (C-10), 143 (C-5), 140 (C-15, C-17), 138
(C-9), 135 (C-6), 131 (C-8), 128 (C-16), 121 (C-14, C-18),
34 (C-11); DEPT 135 (DLSO-d6): 148 (C-3), 140 (C-15,
C-17), 138 (C-9), 135 (C-6), 131 (C-8), 121 (C-14, C-18),
34 (C-11); tmax/cm-1 (KBr): 3510 (O–H), 3155 (N–H),
3210 (C–H, sp2), 2756 (C–H, sp3), 1421 (C–H, bend); MS
(ES): m/z 325 (M?•, 66 %), 300 (45), 210 (40), 150 (30);
Elemental analysis: Found: C, 59.00; H, 3.33; N; 17.12.
C16H12N4O4 requires: C, 59.26; H, 3.73; N, 17.28 %.
dH (DLSO-d6): 8.46–6.32 (8 H, m, H-3, H-6, H-8, H-9,
H-14, H-15, H-17, H-18), 4.01 (2 H, s, H-11), NH signal
was not observed; dC (DMSO-d6): 161 (C-2), 156 (C-7),
155 (C-3), 147 (C-13), 143 (C-5, C-10), 140 (C-9), 139
(C-15), 134 (C-17), 130 (C-16), 124 (C-6), 123 (C-8), 117
(C-14), 116 (C-18), 40 (C-11), 23 (C-19); DEPT 135
(DLSO-d6): 155 (C-3), 137 (C-9), 134 (C-17), 130 (C-16),
124 (C-6), 123 (C-8), 117 (C-14), 116 (C-18), 40 (C-11), 23
(C-19); MS (ES): m/z 295 (M?•, 60 %), 220 (33), 100 (20);
Elemental analysis: Found: C, 65.40; H, 4.81; N; 19.05.
C16H14N4O2 requires: C, 65.30; H, 4.79; N, 19.04 %.
4-Chloro-N-[(6-nitroquinoxalin-2-yl)methyl]
benzenamine [5]
Method 2.2 was followed using p-chloro-aniline (0.303 g,
0.238 mmol) dissolved in THF (30 mL), triethylamine
(2 mL), and 2-chloromethyl-6-nitroquinoxaline (3.33 g,
0.149 mmol). The reaction mixture was heated under reflux
for 100 h and then concentrated under reduced pressure.
The crude product was dissolved in ethyl acetate and
washed with water (3 9 10 mL). The organic layer was
evaporated to give a brown precipitate, which recrystallized
from acetone to give the product as bright yellow crystals
(0.06 g, 19 %); m.p. 242–244 ꢁC (Woo-Jin and Chao-Jun,
2006).
4-[(6-Nitroquinoxalin-2-yl)methylamino)benzoyl
chloride [7]
4-[(6-Nitroquinoxalin-2-yl)methylamino)benzoic acid (0.402 g,
1.24 mmol) was dissolved in dry dichloromethane
(25 mL). Oxalyl chloride (0.15 mL, 1.24 mmol) was added
dropwise to the stirred solution and the mixture was left
stirring for 6 h. The solvent was removed under reduced
pressure to afford a light yellow precipitate, which was
recrystallized from dry dichloromethane to give a prod-
uct with white powder (0.23 g, 76 %); m.p. 90–92 ꢁC
(Kitamura and Yoshida, 2003).
dH (DLSO-d6): 9.01 (1 H, s, H-6), 8.95 (1 H, s, H-3),
8.75 (1 H, dd, J3 1.5, J4 2.5, H-8), 8.50 (1 H, d, J3 1.5, H-9),
8.00–6.30 (5 H, m, H-14, H-15, H-17, H-18), 4.02 (2 H, s,
H-11), NH signal was not observed; dC (DMSO-d6): 162
(C-2), 155 (C-7), 152 (C-3), 150 (C-13), 142 (C-10), 140
(C-5), 137 (C-9), 136 (C-15, C-17), 132 (C-6), 130 (C-8),
dH (DLSO-d6): 8.85 (1 H, s, H-6), 8.60 (1 H, s, H-3),
8.77 (1 H, d, J3 1.4, H-8), 8.12 (1 H, d, J3 1.6, H-9),
7.53–6.66 (4 H, m, H-14, H-15, H-17, H-18), 4.62 (2 H, s,
H-11), 1.7 (1 H, brs, H-12).
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Table 1 The calculated energy terms for the interaction of a series of compounds with MTHF binding domain and their RMSD values
˚
RMSD (A)
Ligand
Energy term (kJ/mol)
IC50 (lM)
DEBind
DEComplex
DEReceptor
DELigand
PC-3
MCF-7
1
2
3
4
5
6
8
9
-105.51
-130.93
-132.91
-138.91
-122.93
-152.62
-150.19
-145.89
-202.72
-267.08
-275.87
-290.85
-341.15
-390.03
-331.05
-341.55
-186.09
-190.24
-185.05
-218.42
-285.62
-326.11
-280.94
-299.94
88.88
54.09
42.09
66.48
67.40
88.70
100.08
104.28
70 19
66 19
5.23
2.51
3.12
4.67
4.42
1.6
56
8
61
6
57 14
77 10
70 13
68 14
79 14
75
53
47
49
4
9
6
9
45
49
66
9
6
6
1.9
2.81
The table represents a collection of information about the binding algorithms of the different docked ligands into the receptor
DEBind The free binding energy algorithm results from Eq. 1
DEComplex The free binding energy of the ligand-receptor complex
DEReceptor The free binding energy of the receptor
DELigand The free binding energy of the ligand
Fig. 4 Image of compound 8 docked into the MTHF binding domain
of MetS. The left hand side represents the outermost part of the
enzyme and the orifice of the receptor and the right hand side
represents the innermost part of the enzyme. Compound 8 makes
hydrogen bonds with arginine 516 and asparagine 508
Fig. 3 Image of compound 6 docked into the MTHF binding domain
of MetS. The left hand side represents the outermost part of the
enzyme and the orifice of the receptor and the right hand side
represents the innermost part of the enzyme. Compound 6 makes
hydrogen bond with asparagine 411
reduced pressure. The crude product was dissolved in
dichloromethane and washed with water (3 9 10 mL). The
organic layer was evaporated to give a brown precipitate,
which recrystallized from methanol to give the product as
brown crystals (0.11 g, 32 %); m.p. 260–261 ꢁC (Woo-Jin
and Chao-Jun, 2006).
4-[(6-Nitroquinoxalin-2-yl)methylamino)-N-
methylbenzamide [8]
Method 2.2 was followed using methylamine (0.031 mL,
0.01 mmol) dissolved in THF (25 mL), triethylamine
(2 mL), and 4-[(6-nitroquinoxalin-2-yl)methylamino)ben-
zoyl chloride (0.342 g, 0.01 mmol). The reaction mixture
was heated under reflux for 12 h and concentrated under
dH (DLSO-d6): 9.06 (1 H, s, H-6), 8.68 (1 H, s, H-3),
8.60 (1 H, dd, J3 2.6, J4 1.2, H-8), 8.07 (1 H, d, J3 2.6, H-9),
8.00 (1 H, br s, H-20), 7.34–6.78 (4 H, m, H-14, H-15,
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Fig. 5 Image of compound 1
docked into the MTHF binding
domain of MetS. The left hand
side represents the outermost
part of the enzyme and the
orifice of the receptor, and the
right hand side represents the
innermost part of the enzyme.
Compound 1 makes hydrogen
bond with arginine 516
Elemental analysis: Found: C, 60.02; H, 4.33; N; 20.52.
C17H15N5O3 requires: C, 60.53; H, 4.48; N, 20.76 %.
4-[(6-Nitroquinoxalin-2-yl)methylamino)-N-
ethylbenzamide [9]
Method 2.2 was followed using ethylamine (0.045 mL,
0.01 mmol) dissolved in THF (25 mL), triethylamine
(2 mL), and 4-[(6-nitroquinoxalin-2-yl)methylamino)ben-
zoyl chloride (0.342 g, 1 mmol). The reaction mixture was
heated under reflux for 22 h and concentrated under
reduced pressure. The crude product was dissolved in
dichloromethane and washed with water (3 9 10 mL). The
organic layer was evaporated to give a brown precipitate,
which recrystallized from methanol to give the product as
brown crystals (0.10 g, 29 %); m.p. 255–256 ꢁC (Woo-Jin
and Chao-Jun, 2006).
dH (DLSO-d6): 8.66 (1 H, s, H-6), 8.63 (1 H, s, H-3),
8.60 (1 H, d, J3 2.9, H-8), 8.12 (1 H, d, J3 2.9, H-9),
7.57–6.38 (4 H, m, H-14, H-15, H-17, H-18), 4.62 (2 H, s,
H-11), 2.2 (1 H, br s, OH), 2.1 (1 H, brs, H-12), 3.2 (2 H, q,
J3 3.2, H-21), 3.00 (1 H, br s, H-20), 1.2 (3 H, t, J3 3.2,
H-22); dC (DMSO-d6): 170 (C-19), 163 (C-2), 160 (C-13),
158 (C-7), 153 (C-3), 145 (C-10, C-5), 145 (C-15, C-17),
144 (C-9), 130 (C-6), 129 (C-8), 128 (C-16), 127 (C-14,
C-18), 37 (C-11), 26 (C-21), 20 (C-22); DEPT 135
(DLSO-d6): 153 (C-3), 145 (C-15, C-17), 144 (C-9), 130
(C-6), 129 (C-8), 127 (C-14, C-18), 37 (C-11), 26 (C-21), 20
(C-22); MS (ES): m/z 352 (M?•, 90 %), 320 (50), 200 (30),
150 (30); Elemental analysis: Found: C, 61.34; H, 4.45; N;
20.02. C18H17N5O3 requires: C, 61.53; H, 4.88; N, 19.93 %.
Fig. 6 Image of compound 2 docked into the MTHF binding domain
of MetS. The left hand side represents the outermost part of the
enzyme and the orifice of the receptor, and the right hand side
represents the innermost part of the enzyme. Compound 2 makes
hydrogen bond with asparagine 411
H-17, H-18), 4.32 (2 H, s, H-11), 4.1 (1 H, br s, H-12), 2.9
(3 H, s, H-21); dC (DMSO-d6): 169 (C-19), 164 (C-2), 155
(C-13), 153 (C-7), 150 (C-3), 146 (C-10, C-5), 133 (C-15,
C-17), 132 (C-9), 131 (C-6), 130 (C-8), 128 (C-16), 127
(C-14, C-18), 35 (C-11), 26 (C-21); DEPT 135 (DLSO-
d6): 150 (C-3), 133 (C-15, C-17), 132 (C-9), 131 (C-6), 130
(C-8), 127 (C-14, C-18), 34 (C-11), 26 (C-21); MS (ES):
m/z 338 (M?•, 71 %), 305 (40), 200 (30), 150 (30);
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Med Chem Res (2013) 22:3405–3415
3411
Fig. 7 Image of compound 3
docked into the MTHF binding
domain of MetS. The left hand
side represents the outermost
part of the enzyme and the
orifice of the receptor, and the
right hand side represents the
innermost part of the enzyme.
Compound 3 makes hydrogen
bonds with asparagine 411 and
asparagine 508
Fig. 8 Image of compound 4
docked into the MTHF binding
domain of MetS. The left hand
side represents the outermost
part of the enzyme and the
orifice of the receptor, and the
right hand side represents the
innermost part of the enzyme.
Compound 4 makes hydrogen
bond with asparagine 508
Biological evaluation
containing 100 lM of l-homocysteine thiolactone (abbre-
viated M–H?). Media was supplemented with 10 % 1 kDa
cut-off dialyzed fetal calf serum (HL60, 20 %), sodium
pyruvate (1 mM), L-glutamine (2 lM), hepes (24 lM), and
sodium bicarbonate (2 lM). Cells in exponential growth
were plated (500–1,000 9 10-3 cells per well) in complete
M ? H-, medium, containing L-methionine (100 lM).
After an initial incubation of 24 h at 37 ꢁC, the medium
was changed to M–H- and cells were treated with
Cell lines were supplied by the European Collection of Cell
Cultures (ECACC); human prostate adenocarcinoma cells
(PC-3) and breast carcinoma (MCF-7) were investigated.
Cells were preserved in vitro at 37 ꢁC in a 5 % carbon
dioxide, 95 % air dampened incubator. Cells were grown
in RPMI media containing 100 lM of l-methionine
(abbreviated M ? H) or methionine-free RPMI media
123

3412
Med Chem Res (2013) 22:3405–3415
Fig. 9 Image of compound 5 docked into the MTHF binding domain of MetS. The left hand side represents the outermost part of the enzyme
and the orifice of the receptor, and the right hand side represents the innermost part of the enzyme
transferred to fresh M ? H- medium (recovery). Cells
were then incubated for a further 5 days at 37 ꢁC before
performing the MTT assay. Cell culture media was col-
lected at appropriate times during recovery and stored at
-80 ꢁC. Appropriate control experiments were performed
without the inhibitors.
MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazo-
lium bromide) was used to assess the cytotoxicity of the
tested compounds in cell culture. Following the above
described treatments, media were recovered, and the cells
were treated with fresh medium containing MTT (0.6
mg/mL). The cells were incubated for 4 h at 37 ꢁC, the
medium was removed and replaced with DMSO (150 lL)
to dissolve the resulting crystals. The absorbance of this
solution was recorded at 550 nm in a plate reader. The final
DMSO concentration was 0.1 % and each plate contained
the blank group (complete medium 200 lL/well and no
cells) and the control group (complete medium 200
lL/well and untreated cells). After 96 h of exposure time,
Fig. 10 Image of compound 9 docked into the MTHF binding
20 lL of MTT solution (5 mg/mL) was added and incu-
domain of MetS. Compound 9 made a hydrogen bond with asparagine
bated in the dark at 37 ꢁC for 4 h. The supernatant was
removed and formazan crystals were dissolved in DMSO
411
(150 lL) (Lawrence et al., 2006).
The results of the IC50 values were listed down in
Table 1.
compound concentrations between 100 and 0.1 lM. The
tested compounds were dissolved in DMSO. After 24 h,
the cells were washed twice with Hanks solution and
123

Med Chem Res (2013) 22:3405–3415
3413
O
OH
OH
O
O
H
O
H2N
H2N
NO2
I
I
O
O
O
O
+
Cl
+
H
Cl
O
O
HOAC
OH
O
H2O
O
H
N
OH
O
N
NO2
O
I
NO2
I
+
N
N
H
O
Cl
O
Cl
Fig. 11 Mechanism of the synthesis of quinoxaline derivatives. The
mechanism involves the activation of the 4-chlorobut-2-enal for the
nucleophilic substitution reactions with IBX. The nucelophilic
substitution started with the attack of the free lone pair nitrogen
atoms of the 4-nitro-phenylen-1,2-diamine at the carbonyl carbon and
b carbon atoms of 4-chlorobut-2-enal
Results
enzyme. The root mean square deviations (RMSD) for the
˚
both compounds were 1.6 and 1.9 A, respectively (Figs. 3,
Compound
4-[{(6-nitro-quinoxalin-2-yl)methylamino}
4) (Danishpajooh et al., 2001). This means that both
compounds most likely will produce competitive inhibition
and high concentration of the two compounds will displace
MTHF from its receptor and, therefore, inhibit the enzyme
reaction. The percentage inhibition, IC50 values, and the
free energy of the binding of the synthesized compounds
were listed in Table 1.
methyl]benzoic acid showed the lowest free energy of
binding and the highest percentage of inhibition to the
enzyme reaction free cell assay technique (89 %) and has got
IC50 values of 45 9 and 53 9 lM when screened
against PC-3 and MCF-7 cells. Compound 2-chloromethyl-
6-nitroquinoxaline showed the lowest percentage of inhibi-
tion of the enzyme reaction (55 %), high IC50 values
(70 19 lM with PC-3 and 66 19 lM with MCF-7), and
the highest free binding energy -105.51 kJ/mol among
the synthesized compounds in this article. Compounds
N-[(6-nitroquinoxalin-2-yl)methyl]benzenamine, 4-methyl-
N-[(6-nitroquinoxalin-2-yl)methyl]benzenamine, 3-methyl-
N-[(6-nitroquinoxalin-2-yl)methyl]benzenamine, and 4-
chloro-N-[(6-nitroquinoxalin-2-yl)methyl]benzenamine showed
moderate percentage of inhibition of the enzyme reaction
(65.9 %, 65 %, 45 %, and 45 %), IC50 values (56 8,
57 14, 77 10, and 70 13 lM with PC-3 and 61 6,
68 14, 79 14, and 75 14 lM with MCF-7), and
moderate free binding energies -105.51, -130.93, -132.91,
-138.91, and -122.93 kJ/mol (Table 1).
Compound1 made a hydrogen bondwith arginine 516, and
the quinoxaline nucleus docked vertically inside the pocket
to avoid the van der Waals repulsive interactions (Fig. 5).
The compounds achieved low free energy of binding of
-105.51 kJ/mol and % inhibition of 55 %. This could be
attributed to the steric hindrance caused bythe rotamer methyl
chloride moiety withthe asparagine 411 and asparticacid390.
The nitro group of the compound 2 made a hydrogen bond
with amino group of asparagine 411 whereas the phenyl ring
tended to point away from the polar argentine 516 positioned
at the surface of the receptor and started to bend over to
avoid the high energy barrier among the atoms of the ligand
and the key residues of the enzyme cavity (Fig. 6). Com-
pound 2 showed a moderate free energy of binding
-130.93 kJ/mol and % inhibition of 65.9. Compounds 3, 4,
and 5 tended to move away from the key amino acids of the
receptor to avoid the polar-nonpolar repulsive interaction
between the phenyl moiety and arginine 508 and 516
(Figs. 7, 8, 9) to avoid the steric clashes between the ligand
and the amino acids of the receptor. These ligands had
moderate free energy of binding -132.91, -138.91, and
-122.93 kJ/mol, respectively, and % inhibition of 65, 45,
and 45, respectively. CH3CH2COPh moiety of compound 9
started to unfold and stretch out along the binding domain.
This was attributed to the electrostatic interactions occurred
between the amide group of the ligand and arginine 516
Discussion
Molecular modeling investigations were done on the
MTHF receptor and the designed inhibitors were docked
into the receptor on the same docking pose of the MTHF
substrate (Fig. 2). Compounds 4-[(6-nitroquinoxalin-2-yl)
methylamino)benzoic acid and 4-[(6-nitroquinoxalin-2-yl)
methylamino)-N-methylbenzamide have been docked on
the same pose orientation of the natural substrate of the
123

3414
Med Chem Res (2013) 22:3405–3415
4
6
9
5
O2N
N
3
O
H
O2N
NH2
NH2
IBX
7
8
+
2
Cl
N
HOAC
10
1
Cl
1
NH2
CH3
NH2
NH2
NH2
NH2
TEA
TEA
TEA
TEA
TEA
CH3
Cl
COOH
4
6
4
N
4
6
6
9
5
5
O2N
N
O2N
12
5
4
12
3
3
2
12
O2N
N
6
9
3
2
4
7
8
7
5
H
6
9
7
8
H
O2N
N
14
17
H
12
14
17
3
14
13
O2N 7
5 N
2
13
7
8
8
N
12
H
N
13
3
2
15
N
19
14
17
15
15
10
9
2
10
N
13
N
10 N
H
N
9
11
N
14
17
11
1
15
1
1
11
10
13
19
N
8
CH3
11
16
1
18
15
18
18
10
N
16
16
COOH
Cl
18
11
1
17
16
CH3
6
19
18
2
16
5
3
4
Cl
O
O
Cl
4
N
6
9
5
O2N
12
3
2
7
H
14
13
8
N
15
10
N
11
1
19
18
16
COCl
17
7
CH3NH2
CH3CH2NH2
4
6
5
4
6
O2N
N
12
3
2
5
7
H
O2N
N
12
14
3
2
13
8
7
N
H
15
14
10
N
13
9
8
11
N
1
15
19 O
NH
10
N
9
18
11
1
16
19
17
21
22
18
O
16
20
17
20
NH
8
9
CH3
H3C
21
Scheme 1 Mechanism of the synthesis of quinoxaline derivatives. The
mechanism involves the activation of the 4-chlorobut-2-enal for the
nucleophilic substitution reactions with IBX. The nucelophilic substitution
started with the attack of the free lone pair nitrogen atoms of the 4-nitro-
phenylen-1,2-diamine at the carbonyl carbon and beta carbon atoms of
4-chlorobut-2-enal. Subesequently nucleophilic substitution reaction
occurred by the attack of various aromatic amines on the produced
2-Chloromethyl-6-nitroquinoxaline in the presence of triethyl amine to
form the corresponding compounds 2–6. Oxylyl chloride will react with
compound 6toproduce thecorresponding arylchloride compound7 which
in turn will react with aliphatic amine compounds methyl and theyl amines
to produce final compounds 8 and 9
(Fig. 10). Therefore, the free energy of binding slightly
increased and scored -145.89 kJ/mol and % inhibition
increased to 70 %.
Conclusions
The results obtained from this study showed new genera-
tion of compounds that could probably be used as drugs
that could be used in the treatment of breast and prostate
carcinoma. Quinoxaline derivatives showed a meaningful
inhibition of the methionine synthase reaction for both the
somatic and cancer cells. Meanwhile, the somatic cells
have the ability to use homocysteine as a precursor of
biosynthase of methionine. The synthesized ligands
showed from moderate to high inhibitory activity on the
enzyme reaction with low concentration requirement to
inhibit the growth of the cancer cells.
In this article we synthesized quinoxaline derivatives in
the presence of IBX. Compound 1 was obtained by the
condensation of 4-nitro-phenylen-1,2-diamine derivatives
with 3-chloro-2-oxopropanal in the presence of IBX in
acetic acid (Scheme 1). The reaction took longer time in
the absence of IBX with many side products; therefore,
IBX was used to reduce the side products and decrease the
required reaction time, and facilitate the reaction condi-
tions. We suggest that IBX can activate the carbonyl
compounds as shown in Fig. 11.
123

Med Chem Res (2013) 22:3405–3415
3415
Acknowledgments My gratitude to Andrew Healy (University of
Bradford, IPI) for measuring the mass spectra and to Dennis Farewell
(University of Bradford, IPI) who had trained me on using NMR.
Kenyon SH, Waterfield CJ, Timbrell JA (2002) Methionine synthase
activity and sulphur amino acid levels in the rat liver tumour
cells HTC and Phi-1. Biochem Pharmacol 63:381–391
Kitamura M, Yoshida M (2003) Synthesis of quinolines and 2H-
dihydropyrroles by nucleophilic substitution at the nitrogen atom
of oxime derivatives. Synthesis 15:2415–2426
Lawrence T, Scott A, Mark A (2006) A short new azulene synthesis.
J Am Chem Soc 102:6311–6314
Appendix: The free energy of binding
Majid M, Khadijeh B (2006) Facile synthesis of quinoxaline
derivatives using o-iodoxybenzo acid (IBX) at room tempera-
ture. ARKIVOC xvi:16–22
DEBind ¼ DEComplex ꢁ ðDEReceptor þ DELigand
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