Synthesis of 3-benzyl-2-substituted quinoxalines as novel monoamine oxidase A inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.11.088

A new series of 3-benzyl-2-substituted quinoxalines have been synthesized by means of microwave enhancement of nucleophilic substitution reaction involving the corresponding 2-chloroquinoxaline analogs and substituted amines or hydrazine. The synthesized

Full text of DOI:10.1016/j.bmcl.2005.11.088

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1753–1756
Synthesis of 3-benzyl-2-substituted quinoxalines
as novel monoamine oxidase A inhibitors
Seham Y. Hassan,a Sherine N. Khattab,a Adnan A. Bekhitb,* and Adel Amera,*,
aDepartment of Chemistry, Faculty of Science, University of Alexandria, Alexandria 21521, Egypt
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria 21521, Egypt
Received 26 February 2005; revised 26 November 2005; accepted 29 November 2005
Available online 13 December 2005
Abstract—A new series of 3-benzyl-2-substituted quinoxalines have been synthesized by means of microwave enhancement of nucle-
ophilic substitution reaction involving the corresponding 2-chloroquinoxaline analogs and substituted amines or hydrazine. The
synthesized compounds were evaluated for their monoamine oxidase A and B inhibitory activity by determination of their IC50.
All the newly synthesized compounds showed more selective inhibitory activity toward MAO-A than MAO-B. In addition, the acute
toxicity of the synthesized compounds was determined. This work may be a fruitful matrix of the synthesis of a new series of novel
MAO-A inhibitors with good safety margins.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Human monoamine oxidases A and B (MAO-A and B)
are the most intensively investigated flavin-dependent
amine oxidases. This is due to their roles in the metabo-
lism of neurotransmitters such as serotonin and dopa-
mine.2,3 MAO-A and MAO-B are separate gene
products of ꢀ70% sequence identity with both isoforms
containing 8 a-S-cysteinyl-FAD coenzymes as the sole
redox cofactors and retaining different but partly over-
lapping substrate and inhibitor specificities. Due to the
key role played by monoamine oxidases (MAOs) in
the metabolism of neurotransmitters, MAO inhibitors
(MAOIs) represent a useful tool for the treatment of
several neurological diseases.4 MAOs are implicated in
a large number of neurological disorders such as Parkin-
son’s disease and depression, and have been important
targets for drug, therapy over the past 40 years.2 Among
selective MAOIs, MAO-A inhibitors are used as antide-
pressant and antianxiety drugs and are claimed to pro-
tect neuronal cells against apoptosis.5 There are many
different structures of MAOIs due to the fact that the ac-
tive sites of the MAO-A are still unknown today which
limits the design of potent selective MAOIs.6,7 The
three-dimensional structures of both MAO-A and
MAO-B are therefore of interest. However, both en-
zymes are bound to the outer mitochondrial membrane
through a C-terminal polypeptide segment and this fea-
ture has made structural investigation by X-ray crystal-
lography more difficult. With the recent high resolution
crystal structure of human MAO-B8 and a lower resolu-
tion structure of rat MAO-A,9 new insights into the
molecular basis of MAO inhibition are now available.
MAOIs can be classed into three categories: (1) irrevers-
ible inhibitors, (2) Ôquasi-irreversible’ inhibitors, and (3)
reversible inhibitors. Those belonging to the irreversible
class include the acetylenic inhibitors and the arylalkyl
hydrazines.8 These mechanism-based inhibitors form
covalent adducts with the flavin. Stable N (5) flavocya-
nine adducts (acetylenic inhibitors) and C (4a) adducts
(proposed) are formed on hydrazine inhibition. Inhibi-
tion by acetylenic inhibitors occurs in a single turnover
event, while phenethylhydrazine inhibition requires
ꢀ15 turnovers/inhibition event. Attempts to determine
the structure of hydrazine-inactivated MAO-B have
been unsuccessful due to the lability of the adduct in
the X-ray beam. The Ôquasi-irreversible’ inhibitors are
so classified since denaturation of the inhibited enzyme
results in their dissociation and include phenylcyclopro-
pylamine (Ôtranylcypromine’) and the N-(2-aminoeth-
yl)benzamides (which belong to the Ôlazabemide’ class
of MAO-B inhibitors). It was worthy to design a hybride
structure of reversible and irreversible inhibitors to
study the effect of such molecular variation on MAO
inhibitor activity.
Keywords: MAO-A inhibitors; 3-Benzyl-2-substituted quinoxalines.
*
Corresponding authors. Tel.: +2 012 2219463; fax: +1 781 8465563
(A.A); e-mail: ShKh2@link.net
See Ref. 1.
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.088

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S. Y. Hassan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1753–1756
As part of our medicinal chemistry program aimed at
the search for novel quinoxaline-based bioactive mole-
cules,10,11 we report herein our attempt to design a
new lead compound by combining the different possible
active sites of two previously known MAO-A inhibitors
(namely, moclobemide and pargyline) in a quinoxaline
model as shown in Figure 1.
For the synthesis of desired compounds, Scheme 1 was
adopted.
The final compounds 4a–4h were evaluated for their
MAO-A inhibitory activity in vitro by the method de-
scribed by Undenfriend et al.15 using serotonin (5HT)
as substrate. The method depends on the determination
of MAO-A activity of rat liver mitochondria.16 All com-
pounds under test were used at a final concentration of
1 · 10ꢁ4 M. The results were expressed as percentage
inhibition of the activity of MAO-A (Table 1).
Attempt to synthesize the 3-benzyl-2-(2-morpholin-4-
yl-ethyl)amino-quinoxaline 4a from the reaction of
3-benzyl-2-chloroquinoxaline with the 2-morpholin-4-
ylethylamine in n-butanol for 48 h reflux gave a mixture
of products, while the separation of the target molecule
was troublesome. The reaction was repeated in the pres-
ence of ammonium chloride,12 affording also a mixture
of undesired products. However, we were able to obtain
our aimed product in very high yield by effecting the
reaction using microwave radiation of the reactants in
isopropanol in a Pyrex-glass open vessel. The reaction
mixture was irradiated in a domestic microwave oven
for 15 min. Similarly, compounds 4b–4h were pre-
Furthermore, the synthesized compounds 4a–4h were
tested to determine their activity toward MAO-A and
MAO-B selectivity in the presence of the specific sub-
strate, serotonin or benzylamine, respectively. Bovine
brain mitochondria were isolated according to Bas-
ford.17 The activity of MAO-A and MAO-B was deter-
mined by the fluorimetric method, according to
Matsumoto et al.18 The mitochondrial fractions were
preincubated at 38 ꢁC for 30 min before adding the spe-
cific inhibitor, L-depreny (l0.5 lM), to determine the
MAO-A activity and clorgyline (10.5 lM) to determine
the MAO-B activity. The incubation mixture contained
(0.1 ml, 0.25 M) phosphate buffer, pH 7.4, mitochondri-
al suspension (6 mg/1 ml), the specific substrate for
MAO-A or MAO-B (0.1 mM), and test compounds at
four different concentrations ranging from 5 lM to
0.1 mM dissolved in propylene glycol. The mixture
was incubated in a shaking water bath at 37 ꢁC for
60 min. The reaction was quenched by adding perchloric
acid. The samples were centrifuged at 10000g for 5 min
and the supernatant was completed to 2.7 ml of 1 N
NaOH and measured on a Perkin-Elmer Lf 45 Spectro-
fluorimeter. Protein concentration was determined
according to the reported method.19 The MAO-A and
MAO-B results were expressed as IC50 (Table 1). The
selectivity index is also given in (Table 1).
1
pared.13,14 Their structures were established by IR, H
NMR, and elemental analyses.14
Cl
H
N
N
N
O
O
Pargyline
Moclobemide
H
N
N
N
N
The results revealed that all the test compounds 4a–4h
showed MAO-A inhibitory activity higher than that of
MAO-B. Compounds 4a, 4b, and 4g are the most selec-
tive compounds as MAO-A inhibitors.
O
Figure 1. Planned modification and newly designed MAO-A inhibitor.
R1
N
R1
R2
N
N
POCl3
R2
R3
N
H
O
Cl
R3
2
1
4
1
2
3
Compound
R
R
R
R
R1
R2
N
4a
4b
4c
4d
4e
4f
-CH -CH -N(CH -CH ) O
2 2 2 2 2
H
H
H
H
4
R NH (3) ,isopropanol
2
-CH -CH -N(CH -CH ) O
H
Cl
H
H
H
H
Cl
H
2
2
2
2 2
15 min M.W., 600W
-CH -CH -N(CH -CH ) O
2 2 2 2 2
Cl Cl
N
R3
N
-CH -CH -OH
H
H
H
H
H
H
H
H
2
2
R4
-CH -CH -NH-CH
3
2
2
H
-NH
2
2
2
4
4g
4h
-NH
-NH
Cl Cl
Scheme 1.

S. Y. Hassan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1753–1756
1755
Table 1. Effect of some quinoxaline derivatives on the activity of MAO-A and MAO-B of rat liver mitochondria
Compound
% MAO-A inhibition
MAO-A IC50
MAO-B IC50
Selectivity index (SI)a
4a
4b
4c
4d
4e
4f
46.13 1.28
50.52 1.46
39.99 1.67
45.97 1.54
40.92 2.74
54.09 + 1.36
49.48 1.84
41.99 3.12
1.3 · 10ꢁ9 0.03
1.7 · 10ꢁ9 0.04
7.3 · 10ꢁ8 0.12
3.7 · 10ꢁ9 0.05
9.2 · 10ꢁ8 0.04
8.8 · 10ꢁ9 0.06
2.1 · 10ꢁ9 0.08
7.6 · 10ꢁ9 0.06
8.4 · 10ꢁ4 0.08
3.6 · 10ꢁ4 0.16
9.6 · 10ꢁ4 0.12
8.6 · 10ꢁ5 0.24
7.9 · 10ꢁ4 0.24
8.4 · 10ꢁ4 0.32
5.7 · 10ꢁ4 0.42
7.4 · 10ꢁ5 0.36
646153
211764
13150
23243
8977
95454
271428
9736
4g
4h
The results are expressed as means SEM. Data were analyzed by one-way analysis of variance. Student’s t test for unpaired observations was used.
P value = <0.001 and was significant. The number of experiments was 6.
a SI = MAO-B IC50/MAO-A IC50
.
ate, filtered, and washed with water. The crude product
was recrystallized from ethanol. Compound 2a: This
compound was obtained as pink crystals, 2.35 g (92.27%)
yield, mp 85 ꢁC (Lit 84–85 ꢁC).23 Compound 2b: This
compound was obtained as pale pink crystals, 1.96 g
(67.7%) yield, mp 120 ꢁC, 1H NMR (CDCl3): d 4.46 (s,
2H, CH2), 7.27 (m, 4H, H-20, H-30, H-50, H-60Ph), 7.76
(dt, 2H, H-6Q, H-7Q), 7.99, 8.07 (2m, 2H, H-5Q, H-8Q).
Anal. Calcd for C15H10Cl2N2: C, 62.31; H, 3.49; N, 9.69.
Found: C, 62.57; H, 3.36; N, 9.51. Compound 2c: This
compound was obtained as pale pink crystals, 2.25 g
(69.5%) yield, mp 122 ꢁC; 1H NMR (CDCl3): d 4.47 (s,
2H, CH2), 7.26 (br s, 5H, Ph), 8.09, 8.21 (2s, 2H, H-5Q, H-
8Q). Anal. Calcd for C15H9Cl3N2: C, 55.67; H, 2.80; N,
8.66. Found: C, 55.83; H, 2.67; N, 8.53.
The test compounds 4a–4h were further evaluated for
their oral acute toxicity in male mice using a literature
method.20,21 The results indicated that test compounds
proved to be non-toxic and well tolerated by the exper-
imental animals up to 250 mg/kg, although no mortality
was recorded at 500 mg/kg. Moreover, these compounds
were tested for their toxicity through the parenteral
route.22 The results revealed that all the test compounds
were non-toxic up to 125 mg/kg. We could conclude that
the synthesis and biochemical evaluation of the new ser-
ies of compound 4 led to the design of a novel class of
MAO-A inhibitors with a good safety margin.
14. General procedure for the preparation of 3-benzyl-2-
substituted aminoquinoxalines 4: (1 mmol) of 2-benzyl-3-
chloroquinoxaline derivatives 2 was dissolved in 5 ml
isopropyl alcohol in a Pyrex-glass open vessel. (2 mmol) of
the primary amine 3 was added to the reaction mixture.
The reaction mixture was irradiated in a domestic micro-
wave oven for 15 min. After removal of the solvent, the
product was separated by gradiant column chromatogra-
phy using hexane and ethyl acetate as eluents.
References and notes
1. Part 18 of the series: synthetic reactions and structural
studies of heterocycles containing nitrogen, for part 17 see:
Khattab, Sh. N.; El Massry, A. M.; El-Faham, A.; Bekhit,
A. A.; Amer, A. J. Heterocyclic Chem. 2004, 41, 387.
2. Cesura, A. M.; Pletscher, A. Prog. Drug Res. 1992, 38,
171.
Compound 4a: This compound was obtained as yellow
crystals, 0.259 g (74.3%) yield, mp 86 ꢁC; IR (KBr): 3393
3. Shih, J. C.; Chen, K.; Ridd, M. J. Annu. Rev. Neurosci.
1999, 22, 197.
4. Rang, H. P., Dale, M. M., Eds.; Pharmacology, ELBS:
Churcill Livingstone, 1998; p 513.
5. Youdin, M. B. H.; Finberg, J. P. M.; Tipton, K. F.
Catecholamines I. In Trendelenburg, U., Weiner N., Eds.;
Springer-Verlag: Berlin, 1998; p 119.;
6. Mai, A.; Artico, M.; Esposito, M.; Ragno, R.; Sbardella,
G.; Massa, S. IL Farmaco 2003, 58, 231.
1
(NH) cmꢁ1; H NMR (CDCl3): d 2.34 (t, 4H, 2 CH2-N)
2.49 (t, 2H, CH2-N), 3.53 (q, 2H, CH2), 3.59 (t, 4H, CH2-
O), 4.27 (s, 2H, CH2), 5.49 (br s, 1H, NH), 7.27 (m, 5H,
Ph), 7.38, 7.54 (2dt, 2H, H-6Q, H-7Q), 7.69, 7.90 (2d, 2H,
H-5Q, H-8Q). Anal. Calcd for C21H24N4O: C, 72.39; H,
6.94; N, 16.08. Found: C, 72.61; H, 6.74; N, 15.89.
Compound 4b: This compound was obtained as yellow
crystals, 0.266 g (69.47%) yield, mp 77 ꢁC; IR (KBr): 3400
7. Chimenti, F.; Secci, D.; Bolasco, A.; Chimenti, P.;
Granese, A.; Befani, O.; Turini, P.; Alcaro, S.; Ortuso,
F. Bioorg. Med. Chem. Lett. 2004, 14, 3697.
1
(NH) cmꢁ1; H NMR (CDCl3): d 2.37 (br s, 4H, 2 CH2-
N), 2.51 (t, 2H, CH2-N), 3.53 (q, 2H, CH2), 3.62 (br s, 4H,
CH2-O), 4.22 (s, 2H, CH2), 5.43 (br s, 1H, NH), 7.20, 7.26
(2d, 4H, Ph), 7.39, 7.55 (2t, 2H, H-6Q, H-7Q), 7.69, 7.88
(2d, 2H, H-5Q, H-8Q). Anal. Calcd for C21H23ClN4O: C,
65.87; H, 6.05; N, 14.63. Found: C, 66.01; H, 5.87; N,
14.39.
´
8. Binda, C.; Li, M.; Hubalek, F.; Restelli, N.; Edmondson,
D. E.; Mattevi, A. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
9750.
9. Ma, J.; Yoshimura, M.; Yamashita, E.; Nakagawa1, A.;
Ito, A.; Tsukihara, T. J. Mol. Biol. 2004, 338, 103.
10. El-Faham, A.; El Massry, A. M.; Amer, A. Lett. Pept. Sci.
2002, 9, 49.
11. Abdul-Ghani, M.; Khattab, Sh. N.; El Massry, A. M.; El-
Faham, A.; Amer, A. Org. Prep. Proced. Int. 2004, 36,
121.
12. Contreras, J. M.; Parrot, I.; Sippl, W.; Rival, Y. M.;
Wermuth, C. G. J. Med. Chem. 2001, 44, 2707.
13. General procedure for the preparation of 2-benzyl-3-
chloroquinoxaline derivatives 2: a mixture of 3-benzyl-1H-
quinoxalin-2-one derivatives 1 (10 mmol) and phosphoryl
chloride (30 ml) was refluxed for 2 h, cooled, poured over
crashed ice, neutralized with saturated sodium bicarbon-
Compound 4c: This compound was obtained as yellow
crystals, 0.325 g (77.88%) yield, mp 140 ꢁC; IR (KBr):
1
3386 (NH) cmꢁ1; H NMR (CDCl3): d 2.36 (br s, 4H, 2
CH2-N) 2.50 (t, 2H, CH2-N), 3.47 (q, 2H, CH2), 3.61 (br s,
4H, CH2-O), 4.22 (s, 2H, CH2), 5.68 (br s, 1H, NH), 7.27
(m, 5H, Ph), 7.77, 7.97 (2s, 2H, H-5Q, H-8Q). Anal. Calcd
for C21H22Cl2N4O: C, 60.44; H, 5.31; N, 13.43. Found: C,
60.22; H, 5.57; N, 13.68.
Compound 4d: This compound was obtained as yellow
crystals, 0.196 g (70.2%) yield, mp 73–74 ꢁC; IR (KBr):
3393 (NH), 3280 (br s, OH) cmꢁ1 1H NMR (CDCl3):
;
d 2.98 (br s, 1H, OH), 3.58 (q, 2H, CH2), 3.72 (t, 2H,

1756
S. Y. Hassan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1753–1756
CH2-O), 4.27 (s, 2H, CH2), 5.17 (br s, 1H, NH), 7.28 (m,
Compound 4h: This compound was obtained as yellow
crystals, 0. 256 g (80.23%) yield, mp 225 ꢁC; IR (KBr):
5H, Ph), 7.40, 7.55 (2dt, 2H, H-6Q, H-7Q), 7.65, 7.91 (2d,
2H, H-5Q, H-8Q). Anal. Calcd for C17H17N3O: C, 73.10;
H, 6.13; N, 15.04. Found: C, 73.25; H, 5.93; N, 14.86.
Compound 4e: This compound was obtained as yellow
crystals, 0.252 g (86.19%) yield, mp 65 ꢁC; IR (KBr): 3400,
3382 (NH) cmꢁ1; 1H NMR (CDCl3): d 1.95 (br s, 1H, NH,
D2O exchangable), 2.26 (s, 3H, CH3), 2.68 (t, 2H, CH2),
3.51 (q, 2H, CH2), 4.22 (s, 2H, CH2), 5.39 (br s, H, NH,
D2O exchangable), 7.27 (m, 5H, Ph), 7.36, 7.51 (2dt, 2H,
H-6Q, H-7Q), 7.67, 7.88 (2d, 2H, H-5Q, H-8Q). Anal.
Calcd for C18H20N4: C, 73.94; H, 6.89; N, 19.16. Found:
C, 74.13; H, 6.59; N, 18.94.
3410 (NH) cmꢁ1 1H NMR (CDCl3): d 4.26 (s, 2H,
;
CH2), 4.86 (br s, 2H, NH2), 7.28 (m, 5H, Ph), 7.73, 8.03
(2s, 2H, H-5Q, H-8Q), 8.69 (br s, 1H, NH). Anal. Calcd
for C15H12Cl2N4: C, 56.44; H, 3.79; N, 17.55. Found: C,
56,69; H, 3.57; N, 17.28.
15. Undenfriend, S.; Weissbach, H.; Clark, C. T. J. Biol.
Chem. 1955, 215, 337.
16. Schneider, W. C. J. Biol. Chem. 1948, 176, 259.
17. Basford, R. E. Methods Enzymol. 1967, 10, 96.
18. Matsumoto, T.; Suzuki, O.; Furuta, T.; Asai, M.;
Kurokawa, Y.; Rimura, Y.; Katsumata, Y.; Takahashi,
I. Clin. Biochem. 1985, 18, 126.
19. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
20. Verma, M.; Tripathi, M.; Saxena, A. K.; Shanker, K. Eur.
J. Med. Chem. 1994, 29, 941.
21. Litchfield, J. T.; Wilcoxon, F. J. Pharmacol. Exp. Ther.
1949, 96, 99.
22. Bekhit, A. A.; Fahmy, H. T. Y. Arch. Pharm. Pharm.
Med. Chem. 2003, 336, 111.
23. El Massry, A. M. Heterocyclic Commun. 1999, 5, 555.
Compound 4f: This compound was obtained as red crystals,
0.23 g (92.0%) yield, mp 154 ꢁC (Lit 155-157 ꢁC).23
Compound 4g: This compound was obtained as red
crystals, 0.202 g (71.1%) yield, mp 135 ꢁC; IR (KBr): 3400
(NH) cmꢁ1; 1H NMR (CDCl3): 4.18 (s, 2H, CH2), 6.84 (m,
2H, NH2), 7.15–7.36 (m, 4H, Ph), 7.55–7.63 (m, 2H, H-6Q,
H-7Q), 7.74, 7.84 (2d, 2H, H-5Q, H-8Q), 8.61 (br s, 1H,
NH). Anal. Calcd for C15H13ClN4: C, 63.27; H, 4.60; N,
19.68. Found: C, 63.01; H, 4.82; N, 19.94.