Monoamine oxidase inhibitors: Benzylidene-prop-2-ynyl-amines analogues

Article abstract of DOI:10.1248/bpb.33.725

A new series of benzylidene-prop-2-ynyl-amines analogues have been synthesized and evaluated for their monoamine oxidase A and B inhibitory activity by determination of IC₅₀ and selectivity index (SI). Among these inhibitors, benzhydrylidene-prop-2-ynyl-amine (2, IC₅₀ 32nM) and (3, 4-dimethoxy-benzylidene)-prop-2-ynylamine (10, IC₅₀ 14nM) provide the highest inhibitory potency toward monoamine oxidase (MAO) A and B respectively. (3,5-Dimethyl-1H-pyrrol-2-ylmethylene)-prop-2-ynyl-amine (1, SI=58.96) and compound (2, SI=0.34) were proved to be the superior selective inhibitors toward MAO-A and MAO-B respectively. Docking studies show that the imine moiety is located in hydrophobic pocket, bringing the propargyl group close to FAD which indicates that the different inhibitory potency toward MAO-A may be ascribable to both the distance between alkynyl group and N5 of FAD, and hydrogen bonding interactions between inhibitors and enzymes.

Full text of DOI:10.1248/bpb.33.725

April 2010
Notes
Biol. Pharm. Bull. 33(4) 725—728 (2010)
725
Monoamine Oxidase Inhibitors: Benzylidene-prop-2-ynyl-amines
Analogues
Zhao JIA,a Shen WEI,b and Qing ZHU*,a
a Institute of Bioengineering, Zhejiang University of Technology (Chaohui Campus); Hangzhou 310014, China: and
b Department of General Surgery, Jinhua Central Hospital; Jinhua 321000, China.
Received December 10, 2009; accepted January 6, 2010; published online January 15, 2010
A new series of benzylidene-prop-2-ynyl-amines analogues have been synthesized and evaluated for their
monoamine oxidase A and B inhibitory activity by determination of IC50 and selectivity index (SI). Among these
inhibitors, benzhydrylidene-prop-2-ynyl-amine (2, IC50ꢀ32 nM) and (3, 4-dimethoxy-benzylidene)-prop-2-ynyl-
amine (10, IC50ꢀ14 nM) provide the highest inhibitory potency toward monoamine oxidase (MAO) A and B re-
spectively. (3,5-Dimethyl-1H-pyrrol-2-ylmethylene)-prop-2-ynyl-amine (1, SIꢀ58.96) and compound (2, SIꢀ0.34)
were proved to be the superior selective inhibitors toward MAO-A and MAO-B respectively. Docking studies
show that the imine moiety is located in hydrophobic pocket, bringing the propargyl group close to FAD which
indicates that the different inhibitory potency toward MAO-A may be ascribable to both the distance between
alkynyl group and N5 of FAD, and hydrogen bonding interactions between inhibitors and enzymes.
Key words monoamine oxidase; inhibitor; benzylidene-prop-2-ynyl-amines analogue; docking study
Monoamine oxidase (EC 1.4.3.4; MAO), a FAD-contain- selectivity index (SI).
ing enzyme, is responsible for the degradation of various
neurotransmitters and xenobiotic amines. The following two MATERIALS AND METHODS
MAO isozymes: MAO-A and MAO-B, have been distin-
guished by substrates specificity, inhibitor selectivity and
Materials All commercially available solvents and
amino acid sequence. MAO-A is inhibited by low concentra- reagents were used without further purification. All moisture-
tions of clorgyline and preferentially catalyzes the oxidation sensitive reactions were carried out under a nitrogen atmo-
1
of 5-hydroxytryptamine (5-HT) and norephinephrine, sphere. H-NMR spectra were recorded at 300 MHz. MAO-
whereas MAO-B catalyzes the oxidation of benzylamine and A/B were prepared from beef liver and human placenta re-
is inhibited by (R)-deprenyl in nanomolar concentration.1) spectively according to the reported method.5)
Compounds that selectively inhibit MAO-A exhibit antide-
General Procedure for the Preparation of Benzylidene-
pressant activity, while MAO-B inhibitors are used clinically prop-2-ynyl-amine Derivatives. Procedure a6) To a so-
as adjuncts in the treatment of Parkinson’s disease.2) There- lution of aldehyde (2 mmol) in dry tetrahydrofuran (THF)
fore, the studies of MAO inhibitors (MAOI) have attracted (10 ml) was added propargylamine (0.205 ml) and MgSO4
significant attention, and a number of new MAOI including (0.6 g). The reaction mixture was stirred at room temperature
iproniazid, isatin and serotonin have been developed. Re- for 24 h. The progress of the reaction was monitored by TLC.
cently, research into functions of MAO shows that MAOs After completion of the reaction, the solid was removed by
were flavoproteins which can be irreversibly inactivated by filtration and the solvent was distilled. Water (20 ml) was
pargyline and rasagiline derivates.3) The mechanism is illus- added, and the solution was extracted with dichloromethane.
trated in Fig. 1: propargylamine derivatives are oxidized by The organic layer was separated, washed with brine (20 ml)
MAO to the corresponding eyniminium species. These and saturation NaHCO3 (20 ml). The phases were separated,
highly electrophilic Michael acceptors bind the flavin group the organic phase dried over anhydrous MgSO4, filtered, and
with covalent bond and then lead to inactivation of the en- the solvent removed under vacuum. The residue was chro-
zyme by micromolar concentrations. Additionally, benzylim- matographed to afford the product.
1
ine derivatives were also reported to possess high inhibitory
Compound 1: Yꢀ79%, H-NMR (CDCl3) d: 8.15—8.14
activity toward MAO.4)
(t, 1H, Jꢀ2.5 Hz, CHꢀN), 5.72 (s, 1H, CH), 4.28—4.27 (t,
Encouraged by these results, in this paper, we designed 2H, Jꢀ2.5 Hz, CH2), 2.41—2.41 (s, 1H, CH), 2.177 (s, 3H,
and synthesized a series of benzylidene-prop-2-ynyl-amines CH3), 2.121 (s, 3H, CH3). ESI-MS m/z: 161.2 (Mꢁ1)ꢁ, IR
derivatives (1—21), and evaluated for their monoamine oxi- (KBr, cmꢂ1): nCꢀNꢀ1645.
1
dase A and B inhibitory activity by determination of IC50 and
Compound 3: Yꢀ85%, H-NMR (CDCl3) d: 8.64—8.63
(s, 1H, CHꢀN), 7.32—7.31 (d, 1H, Jꢀ2.5 Hz, CH), 7.28—
7.26 (m, 1H, CH2), 6.93—6.92 (d, 1H, Jꢀ2.5 Hz, CH),
4.57—4.56 (t, 2H, Jꢀ2.5 Hz, CH2), 2.61 (s, 1H, CH). ESI-
MS m/z: 194.2 (Mꢁ1)ꢁ, IR (KBr, cmꢂ1): nCꢀNꢀ1640.
Procedure b6) Ketone (2 mmol) was added to a solution
of propargylamine (0.4 ml) in dry CH2Cl2, and the reaction
flask was purged with N2 and cooled to ꢂ20 °C. TiCl4
(0.35 ml) was introduced by syringe, and the solution was
stirred at room temperature for 3 d. After completion of the
Fig. 1. Proposed Mechanism of Covalent Inactivation of MAO by Propar-
gylamine Inhibitors
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: zhuq@zjut.edu.cn

726
Vol. 33, No. 4
reaction, the mixture was filtered and the filtrate was washed
with brine (20 ml) and saturation NaHCO3 (20 ml). The
phases were separated, the organic phase dried over anhy-
drous MgSO4, filtered, and the solvent removed under vac-
uum. The residue was further purified by alumina column
chromatography to afford the product.
Reagents and reaction conditions: a: MgSO4, dry THF, rt, overnight; b: TiCl4, dry
THF, rt, 3 d; c: MgSO4, benzene, 80 °C, overnight, d: NaBH4, MeOH, 0 °C,
20 min.
1
Compound 2: Yꢀ87%, H-NMR (CDCl3) d: 7.75—7.72
(m, 4H, CH), 7.50—7.51 (t, 6H, Jꢀ2.5 Hz, CH), 4.11—4.12
(d, 2H, Jꢀ5 Hz, CH2), 2.22—2.21 (t, 1H, Jꢀ2.5 Hz, CH).
ESI-MS m/z: 220.2 (Mꢁ1)ꢁ, IR (KBr, cmꢂ1): nCꢀNꢀ1658.
Chart 1. Synthesis of Benzylidene-prop-2-ynyl-amines 1—23
Procedure c7) To a solution of aldehyde (2 mmol) in RESULTS AND DISCUSSION
benzene (8 ml) was added propargylamine (0.205 ml) and
MgSO4 (0.2 g). The reaction mixture was refluxed at 80 °C
Firstly, compounds 1—21 were synthesized from alde-
for 24 h. The progress of the reaction was monitored by TLC. hydes and propargylamine, and the experiments were sum-
After completion of the reaction, the reaction mixture was marized in Chart 1. Condensation of aldehydes (or ketone)
filtered and collected filtered. The solvent was distilled, water with propargylamine in the presence of dehydrating agents
(20 ml) was added, and the mixture was extracted with MgSO4 afforded benzylidene-prop-2-ynyl-amines in good
dichloromethane. The organic layer was separated, washed yields (70—99%). Then compound 3 and 21 were reduced to
with brine (20 ml) and saturation NaHCO3 (20 ml). The the corresponding secondary amines 22 and 23 with NaBH4.
phases were separated, the organic phase dried over anhy- All compounds were determined by MS and 1H-NMR.
drous MgSO4, filtered, and the solvent removed under vac-
uum. The residue was further purified by alumina column A/B, IC50 was determined with SPSS software using our re-
chromatography to afford the product.
ported fluorimetric method.11) All data including selectivity
To verify the ability of these compounds to inhibit MAO-
1
Compound 20: Yꢀ90%, H-NMR (CDCl3) d: 8.39—8.38 index (SI) toward MAO-A and B are presented in Table 1.
(t, 1H, Jꢀ2.5 Hz, CHꢀN), 6.93 (s, 2H, CH), 4.43—4.42 (t, Clorgyline and pargyline are commercial MAO inhibitors
2H, Jꢀ2.5 Hz, CH), 3.82 (s, 9H, CH3O), 1.16—1.15 (m, 1H, and their IC50 were included as references (24, 25 in
Jꢀ2.5 Hz, CH), ESI-MS m/z: 234.3 (Mꢁ1)ꢁ, IR (KBr, Table 1).12,13) As expected, nearly all compounds show good
cmꢂ1): nCꢀNꢀ1640.
inhibitory activity toward MAO-A/B. As can be seen from
Precedure d8) Benzylidene-prop-2-ynyl-amines (0.1 Table 1, benzhydrylidene-prop-2-ynyl-amine (2, IC50ꢀ
mmol) were dissolved in MeOH and cooled to 0 °C. The so- 32 nM) and (3,4-dimethoxy-benzylidene)-prop-2-ynyl-amine
lution was added NaBH4 (0.15 mmol). After 20 min, the sol- (10, IC50ꢀ14 nM) are the best inhibitors toward monoamine
vent was distilled. Water (20 ml) was added, and the mixture oxidase A and B respectively, while compound 1 (SIꢀ58.96)
was extracted with dichloromethane. The organic layer was is the most selective inhibitor toward MAO-A and compound
separated, washed with brine (20 ml) and saturation NaHCO3 2 (SIꢀ0.34) is the most selective inhibitor toward MAO-B.
(20 ml). The phases were separated, the organic phase dried
Structure–activity relationships (SARs) were examined
over anhydrous MgSO4, filtered, and the solvent removed based on IC50 and SI values (Table 1). Firstly, compound 16
under vacuum. The residue was further purified by alumina (nꢀ2) shows much better inhibitory activity than the others
column chromatography to afford the product.
(nꢀ0), and this phenomenon indicates that the distance be-
1
Compound 23: Yꢀ73%, H-NMR (CDCl3) d: 7.33—7.31 tween imine group and aromatic ring affects the anti-MAO
(d, 2H, Jꢀ10 Hz, CH), 7.30—7.28 (t, 2H, Jꢀ5 Hz, CH), activity. Additionally, most compounds with electron donat-
7.24—7.26 (d, 1H, Jꢀ10 Hz, CH), 3.88 (s, 2H, CH2), 3.40— ing group such as 4, 7—10, and 18, display better inhibitory
3.39 (d, 2H, Jꢀ5 Hz, CH2), 2.25—2.24 (t, 1H, Jꢀ2.5 Hz, activity than comounds 3, 5, 15, 17, 19 bearing electron
NH), 2.04 (s, 1H, CH). ESI-MS m/z: 146.3 (Mꢁ1)ꢁ, IR withdrawing groups. We also noticed that compounds 2 and
(KBr, cmꢂ1): nN–Hꢀ1495.
12 showed higher inhibitory activity toward MAO-B than all
Biochemistry Briefly, newly synthesized amines (ranged the other compounds, which implied imines with bulky
from 1—1000 mM), mitochondria (6 mg/ml) and probe (0.04 group were favorable inhibitors against MAO-B. In addition,
mM) were added to borate buffer (0.05 M, pH 8.4). The assays in order to study the function of imines, compounds 3 and 21
were carried out at 37 °C for 3 h, and the fluorescent signals were reduced to the corresponding amines 22 and 23 by
were recorded at lex 360 nm and lem 460 nm on a Spectrum sodium borohydride. Although no general rules could be
M2 spectrofluorometer.
found whether double bond exerts positive effect on the in-
Molecular Modeling Molecular modeling and graphics hibitory activity toward MAO-A/B, comparison of com-
manipulations were performed by using the Discover studio pounds 3 with 22, 21 with 23 indicates that reduced species
2.1 software. Protein Data Bank crystallographic models of increase the inhibitor selectivity toward MAO-B: the values
human MAO-A (2BXR)9) and MAO-B (1GOS)10) were con- of SI decrease from 40.6 and 39.83 to 2.63 and 27.13 respec-
sidered for the docking studies. Both structures were ob- tively.
tained as adducts with two similar compounds: clorgyline for
To better understand MAO-inhibitor interactions and the
2BXR and pargyline for 1GOS, covalently linked to the FAD selectivity toward different MAO isoforms, docking studies
N5 nitrogen. Starting geometries of 1 and 2 complexes with were performed in the following discussions. Crystal struc-
MAO-A and MAO-B were generated by the Flexible docking tures of MAO-A and B were retrieved from the Protein Data
method.
Bank (PDB) as targets, and selective inhibitors 1 and 2 were
considered for docking studies by using the Discover studio

April 2010
727
Table 1. Structures and Monoamine Oxidase Inhibitory Activity of 1—23a)
IC50 (mM)
Preparation
method
Item
R1
R2
SIb)
MAO-A
MAO-B
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
3,5-Dimethyl-2-pyrrolyl
Phenyl
H
Phenyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
a
b
a
a
c
a
a
c
c
c
c
c
c
a
a
c
a
c
c
c
a
d
d
0.048
0.11
0.90
0.26
1.37
0.88
0.14
0.10
0.19
0.014
0.16
5.36
2.57
3.88
2.98
16.65
1.04
0.65
1.71
0.55
0.18
0.89
4.01
0.0022
(0.0014)8)
—
2.83
0.032
36.54
0.84
1.61
1.37
2.43
0.48
0.63
0.051
1.14
2.34
3.48
9.91
8.28
137.39
10.62
1.90
2.19
4.39
58.96
0.34
40.60
3.23
1.18
1.56
17.36
4.80
3.32
3.64
7.13
0.44
1.35
2.55
2.78
8.25
10.21
2.92
1.28
7.98
39.83
2.63
27.13
—
2-Hydroxyl-4-chloro-phenyl
2-Hydroxyl-1-naphthyl
2-Chloro-4-fluoro-phenyl
2-Hydroxyl-3-ethoxy-phenyl
2,5-Dihydroxy-phenyl
4-Benzyloxy-phenyl
4-Methoxy-5,6-dimethyl-phenyl
3,4-Dimethoxy-phenyl
4-(Pyridin-2-yl)-phenyl
4-Carbomethoxyl-biphenyl
2-Methyl-5,6-dimethoxy-phenyl
4-[(2-Hydroxy-ethyl)-methyl-amino]-phenyl
2-Chloro-quinol-3-yl
trans-2-(Furan-2-yl)-ethenyl
4-Chloro-3-hydroxy-phenyl
2,3-Dimethyl-phenyl
5-Bromo-2-methoxy-phenyl
3,4,5-Trimethoxy-phenyl
Phenyl
H
H
H
H
H
H
7.17
2.34
108.78
—
2-Hydroxyl-4-chloro-phenyl
Phenyl
Clorgyline
25
Pargyline
—
0.038
—
—
(0.038)9)
a) Results are expressed as means IC50. Data were analyzed by one-way analysis of variance. All experiments were repeated three times. b) SIꢀIC50 of MAO-B/IC50 of
MAO-A.
Fig. 2. Binding Modes of Compound 1 and 2 into the Binding Cavity of (a1) Compound 1 with MAO-A; (a2) Compound 1 with MAO-B; (b1) Compound
2 with MAO-A; and (b2) Compound 2 with MAO-B
For clarity, only interacting residues are displayed. Ligands (gray) are represented as stick models. FAD cofactor (light gray), and interacting key residues (gray) are represented
as line models. H-bonds are shown as dashed white lines.
2.1 software. In the active center, Ile199 was previously iden- Phe343, and aromatic cage contains FAD, Tyr398 and
tified as the “gate” residue, and two hydrophobic areas of the Tyr435. Docking studies display that the imine moiety is lo-
enzyme are implicated in the most stable binding mode. Hy- cated in hydrophobic pocket bringing the propargyl group
drophobic pocket is delimited by Ile198, Ile199, Gln206, close to FAD. Figure 2 illustrates the most favorable binding

728
Vol. 33, No. 4
mode of 1 and 2 with MAO-A/B, which shows that propargyl
Acknowledgements This work was supported by Qian-
group in compound 1 stay closer to N5 of FAD in MAO-A jiang Outstanding Researcher Program (No. 2006R10016)
than in MAO-B. Moreover, hydrogen bonding interactions and Zhejiang Natural Science Fund (No. Y2080303).
are discovered between carboxyl hydrogen of Phe208 and hy-
droxyl oxygen of compound 1 (Fig. 2a1). Interactions of REFERENCES
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In conclusion, a new series of benzylidene-prop-2-ynyl-
amines analogues have been synthesized and evaluated for
their monoamine oxidase A and B inhibitory activity by de-
termination of IC50 and SI. Benzhydrylidene-prop-2-ynyl-
amine (2) and (3,4-dimethoxy-benzylidene)-prop-2-ynyl-
amine (10) show the best inhibitory activity against MAO-A
and B respectively. Additionally, (3,5-dimethyl-1H-pyrrol-2-
yl-methylene)-prop-2-ynyl-amine (1, SIꢀ58.96) was proved
to be the superior inhibitor against MAO-A and compound
2 (SIꢀ0.34) possesses the best selective inhibitory activity
toward MAO-B compared to those of other compounds. To
some extent, SAR analysis clarifies structural requirements
for this new class of anti-MAO agents, including: (i) bulky
structure of the inhibitor is favorable for MAO-B inhibitory
activity; (ii) the structure of imine seems to play a significant
role in the inhibitory activity. (iii) the electron density of
aromatic ring has effect inhibitory activity and selectivity.
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inhibitory potency of compound 1 toward MAO-A may be
ascribable to both the distance between alkynyl group and
N5 of FAD, and hydrogen bonding interactions between 1
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