'Click' assembly of selective inhibitors for MAO-A

Article abstract of DOI:10.1016/j.bmcl.2010.08.104

In this Letter, an efficient strategy for the fast construction of 108 compounds library was developed using click chemistry. The fingerprint of inhibitory activity toward MAO-A/B against this library was obtained, and four hit compounds were identified as selective inhibitors toward MAO-A. Docking study was carried out to demonstrate the binding mode between a9 and MAO-A/B, and the result reveals that a9 localized in the 'aromatic cage' and oriented to establish π-π stacking interactions with Tyr407, Tyr444 and FAD in MAO-A rather than in MAO-B.

Full text of DOI:10.1016/j.bmcl.2010.08.104

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6222–6225
x
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
‘Click’ assembly of selective inhibitors for MAO-A
⇑
Zhao Jia, Qing Zhu
Institute of Bioengineering, Zhejiang University of Technology (chaohui campus), Hangzhou 310014, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this Letter, an efficient strategy for the fast construction of 108 compounds library was developed
using click chemistry. The fingerprint of inhibitory activity toward MAO-A/B against this library was
obtained, and four hit compounds were identified as selective inhibitors toward MAO-A. Docking study
was carried out to demonstrate the binding mode between a9 and MAO-A/B, and the result reveals that
Received 22 July 2010
Revised 18 August 2010
Accepted 20 August 2010
Available online 26 August 2010
a9 localized in the ‘aromatic cage’ and oriented to establish
p–p stacking interactions with Tyr407,
Tyr444 and FAD in MAO-A rather than in MAO-B.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Click chemistry
Monoamine oxidase
Docking study
Selective inhibitors
Monoamine oxidases (MAOs, EC 1.4.3.4) are flavin adenine dinu-
cleotide (FAD)-containing enzymes localized in the outer mitochon-
drial membranes of neuronal, glial and other cells. The following two
MAOisozymes:MAO-AandMAO-B, havebeendistinguishedbysub-
strate specificity, sensitivity to inhibitors, and amino acid sequences.
MAO-A, preferentially oxidizing norepinephrine and serotonin, is
selectively inhibited by nanomolar concentrations of clorgyline.
MAO-B preferentially deaminates b-phenylethylamine and benzyl-
amine, and is irreversibly inhibited by nanomolar concentrations
over, due to its modularity, good selectivity, and wide scope, the cop-
per(I)-catalyzed triazole formation has also been adopted widely for
high-throughput discovery of enzyme inhibitors. Ashraf Brik et al.
synthesized and screened HIV-1 PR inhibitors in the microtiter plate
format.11 Yao group developed small-molecule inhibitors targeting
caspases.12 On the other hand, we noticed that five-member ring
heterocycles with two or four nitrogen atoms have been found as
reversible and selective MAO-A/B inhibitors (Fig. 1).13 We infer that
1,2,3-triazole derivates containing 3 nitrogen atoms may be a new
type of inhibitor toward MAO-A/B. Herein, we report the ‘click’ syn-
thesis of 1,4-disubstituted-1H-1,2,3-triazoles, and high-throughput
screening of inhibitory activity toward MAO-A and B.
of L
-deprenyl.1 MAO-B inhibitor (MAO-B-I) is used as a coadjuvant
in the treatment of Parkinson’s disease and Alzheimer’s disease,2
while MAO-A inhibitor (MAO-A-I) is a new antidepressant and anti-
anxiety agent.3 Hence, selective inhibitors for MAO-A or MAO-B may
be useful therapeutic agents,4 and the amount of MAO inhibitors
(MAO-Is) was notably increased during the last decade.5 However,
a high rate of neuro degenerative diseases mutation still outpaces
conventional drug discovery efforts. Rapid synthesis of diverse ana-
logs of MAO-Is still remains a challenge.
Click chemistry, which is firstly developed by Sharpless, has en-
abled researchers to assemble two building blocks rapidly and effi-
ciently on both laboratory and production scales.6,7 The copper(I)-
catalyzed 1,3-cycloaddition is an ideal tool for achieving diversity
in just a few steps with satisfactory purity. Advantages of this reac-
tion in biological studies have recentlybeen demonstrated in several
applications: construction of fluorescent oligonucleotides for DNA
sequencing;8 chemically orthogonal high fidelity bioconjugation;9
and activity-based protein profiling in whole proteomes.10 More-
Firstly, R1 building block azide compounds (a–l) and R2 building
block (2, 3) were prepared according to the reported methods, while
the other compounds were commercial available (Scheme 1).14–16
Although Cu(I)-catalyzed 1,3-dipolar cycloaddition is a highly reli-
able process, which proceeds well in aqueous solvent and tolerates
virtually all functional groups. It is still essential to investigate the
minimum amount of catalysts used in the reaction owing to the sen-
sitivity of MAO-A/B toward Cu(I or II) ions. Therefore, to eliminate
interference arose from CuSO4 and sodium ascorbate in microplate
Ar
Ar
N
N
N
N
R
N
N
R'' N
N
N
CH3
R'
triazole
pyrazole
tetrazole
⇑
Corresponding author. Tel./fax: +86 571 88320781.
E-mail address: chmzhuq@hotmail.com (Q. Zhu).
Figure 1. Structures of five-member ring MAO-Is.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.104

Z. Jia, Q. Zhu / Bioorg. Med. Chem. Lett. 20 (2010) 6222–6225
6223
Cu(I)
N
N
N
R1 N3
R2
H
R2
+
hit compounds
R1
R1 building block
N3
R2 building block
OH
N
N3
N3
OH
N3
Br
HOOC
b
N
COOH
a
c
d
1
2
3
6
OEt
NO2
N
3
N3
N3
H
N
N3
Br
HO
Cl
N
OEt
e
f
g
h
4
5
N3
N3
O
N3
N3
H
O
N
H2N
NH2
Cl
N
i
H CO
H3C
3
O
7
8
9
j
k
l
Scheme 1. Click construction of library of 1,2,3-triazoles.
Table 1
screening system, the different amounts of catalysts were explored
using alkyne 1 and azide h as a model reaction. Finally, it was found
that, after incubation at room temperature for 24 h, 2% CuSO4 and
20% sodium ascorbate in t-BuOH/H2O (v/v 1/1) afforded full con-
sumption of the azide core and the approximately quantitative for-
mation of cycloaddition products. Next, the library was
constructed under the following condition: a solution of compound
a–l was dispensed into 1 mL vial containing a corresponding alkyne
and catalytic amount of CuSO4 and sodium ascorbate (Scheme1). LC/
MS was used for monitoring the reaction and yields ranged from 95%
to 99%.17
IC50 for four hit compounds against MAO-A/B
a
a
Compds
MAO-A IC50
,
(lM)
MAO-B IC50
,
(lM)
SIb
a1
a9
b9
c9
h9
na
na
0.97
19.62
50.72
0.83
510.84
56.88
62.07
115.92
526.23
2.89
0.95
139.66
a
The results are expressed as means. Data were analyzed by one-way analysis of
variance. The experiments were repeated three times.
b
SI = MAO-B IC50/MAO-A IC50
.
The inhibitory activity of 108 compounds against MAO-A/B was
determined using our reported fluorescence-based microplate as-
say,18 and four hit compounds (a9, b9, c9, d9) were identified
(Fig. 2). To study inhibitory efficacy, four compounds were resyn-
thesized in larger scale and values of IC50 against MAO-A/B were
further determined in Table 1.19
Our results reveal that compounds h9 and a9 are the most potent
inhibitors against MAO-A, and their IC50 values are 0.83
lM and
0.97 M, respectively. Meanwhile, compound a9 shows a remark-
l
able selectivity against MAO-A (SI = 526.63). Some further observa-
tions arose from the above screening results. First, compounds with
amino group are potent inhibitors toward MAO-A, indicating that
prop-2-yn-1-amine is a key building block for inhibitory activity.
Second, unsubstituted aromatic rings in R1 building block (a9, h9)
have more potent inhibitory activity toward MAO-A than substi-
tuted derivatives (b9, c9). In order to shed light on the binding mode
of MAO-A/B with these inhibitors, compound a9 was computation-
ally docked into MAO-A/B using AutoDock.20 Crystal structures of
MAO-A and B used for molecular docking were retrieved from the
Protein Data Bank (PDB) as targets.21,22
After docking into the active site of MAO-A/B, Figure 3 illustrates
the most favorable binding mode for a9. Firstly, the pose of a9 shows
that the phenyl ring fits into the ‘aromatic cage’ and sandwiched be-
tween Tyr407 and Tyr444 to establish
p–p stacking interactions.
Moreover, the phenyl moiety is able to establish a T-shaped
p–p
interaction with the FAD aromatic ring. Secondly, a hydrogen bond
Figure 2. The fingerprint of inhibitory activity against MAO-A (left)/B (right).
between the protonated amino group of compound a9 and the car-
Strongest inhibitory activity is visualized in red according to the scale shown.

6224
Z. Jia, Q. Zhu / Bioorg. Med. Chem. Lett. 20 (2010) 6222–6225
References and notes
1. Regina, G. L.; Silvestri, R.; Artico, M.; Lavecchia, A.; Novellino, E.; Befani, O.;
Turini, P.; Agostinelli, E. J. Med. Chem. 2007, 50, 922.
2. Marcaida, J. A.; Schwid, R. S.; White, W. B. Mov. Disord. 2006, 21, 1716.
3. Youdim, B. H. M.; Edmondson, D.; Tipton, K. F. Nat. Rev. Neurosci. 2006, 7, 295.
4. Youdim, M. B.; Bakhle, H. Y. S. Br. J. Pharmacol. 2006, 147, 287.
5. Zhao, J.; Shen, W.; Zhu, Q. Biol. Pharm. Bull. 2010, 33, 725.
6. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004.
7. Lewis, W. G.; Green, L. G.; Grynspan, F.; Radic, Z.; Carlier, P. R.; Taylor, P.; Finn,
M. G.; Sharpless, K. B. Angew. Chem. Weinheim Bergstr. Ger. 2002, 114, 1095.
8. Seo, T. S.; Li, Z.; Ruparel, H.; Ju, J. J. Org. Chem. 2003, 68, 609.
9. Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.; Finn, M. G. J. Am.
Chem. Soc. 2003, 125, 3192.
10. Speers, A. E.; Adam, G. C.; Cravatt, B. F. J. Am. Chem. Soc. 2003, 125, 4686.
11. Brik, A.; Muldoon, J.; Elder, J. H.; Goodsell, D. S.; Olson, A. J.; Fokin, V. V.;
Sharpless, K. B.; Wong, C. H. ChemBioChem 2003, 4, 1246.
12. Ng, S.; Yang, P.; Kitty, C. Y. T.; Rajavel, S.; Yao, S. Q. Org. Biomol. Chem. 2008, 6, 844.
13. Wouters, J.; Ooms, F.; Jegham, S.; Koenig, J. J., et al Eur. J. Med. Chem. 1997, 32, 721.
14. Experimental procedure for alkyne 2 and 3: the pyridine (1 mmol) was dissolved
in dry dichloromethane and 3-bromopropyne (1.5 mmol) was added. The
mixture was stirred at room temperature over night to afford the desired
products as the resultant precipitate.
4-Hydroxy-1-(prop-2-ynyl) pyridinium (2): yield 75%. 1H NMR (D2O): d 7.41–
7.39 (d, 2H, J = 5 Hz, CH), 6.35–6.34 (d, 2H, J = 5 Hz, CH), 4.56 (s, 2H, CH2), 2.59–
2.58 (d, 1H, J = 5 Hz, C„CH). MS (ESI) m/z 134.1 (M+).
3-Hydroxy-1-(prop-2-ynyl) pyridinium (3): yield 67%. 1H NMR (D2O): d 8.32–
8.29 (m, 2H, CH), 7.80–7.78 (m, 1H, CH),7.74–7.71 (m, 1H, CH),5.27 (s, 2H, CH2),
3.11–3.10 (d, 1H, J = 5 Hz, C„CH). MS (ESI) m/z 134.1 (M+).
15. Typical experimental procedure for aryl azide: 4-bromide aniline (0.15 mol) was
dissolved in (40 ml) 50% H2SO4 at 0–5 °C, followed by addition of sodium
nitrite (0.20 mol). After 1 h, sodium azide (0.3 mol) dissolved in 15 ml of water
was added drop wise, and the reaction is allowed to continue over night. The
resultant precipitate was extracted with chloroform and washed successively
with water. The organic layer was dried over anhydrous sodium sulfate, and
the solvent stripped out in rotary evaporator to get crude product. The residue
was chromatographed on silica gel using 10% EtOAc in hexane to give 1-Azido-
4-bromobenzene (d): yield 84%. 1H NMR (CDCl3): d 7.48–7.46 (d, 2H, J = 5 Hz,
CH), 6.92–6.91 (d, 2H, J = 5 Hz, CH),. MS (EI) m/z 197.1 (M+, 100%), 199.1 (M++2,
96%). IR (KBr, cmÀ1): 2137 (–N3).
16. Preparation of compound
h and i: To a solution of 4-bromo-pyridine
hydrochloride (20 mmol) in ethanol/water = 1/1 (40 ml) was added NaOH
(0.4 g) and NaN3 (3 g). The reaction mixture was refluxed at 110 °C for 2 h. The
progress of the reaction was monitored by TLC. After completion of the
reaction, the solvent was distilled, and the mixture was extracted with
dichloromethane. The organic layer was separated, washed with brine (20 ml)
and saturation NaHCO3 (20 ml), anhydrous MgSO4 dried. The solvent was
evaporated to afford product h. Compound
h
was dissolved dry
Figure 3. The interacting mode and orientations of compound 9 (in sticks at center)
in the active sites of MAO-A (up), and MAO-B (down). FAD cofactor (yellow), and
interacting key residues (gray) are shown in stick models. Hydrogen bonds are
displayed as dashed green line.
dichloromethane and MeI (2.5 ml) was added. The mixture was stirred at
room temperature over night. The resultant precipitate was filtered to afford
product i.
4-Azidopyridine (h): yield 86%. 1H NMR (CDCl3): d 8.50–8.48 (m, 2H, CH), 7.65–
7.63 (m, 2H, CH). MS (ESI) m/z 121.2 (M+H)+. IR (KBr, cmÀ1): 2121 (–N3).
4-Azido-1-methylpyridinium (i): yield 93%. 1H NMR(D2O): d 8.49–8.47 (m, 2H,
CH), 7.50–7.48 (m, 2H, CH), 4.14 (s, 3H, CH3). MS(ESI) m/z 135.2 (M). IR (KBr,
cmÀ1): 2119 (–N3).
bonyl oxygen of Ser209 side chain is observed. Thirdly, docking re-
sults demonstrate that a9 is embedded in a large hydrophobic pock-
et formed by Tyr 69, Phe208, Val210, Gly216 and Met350.
Unexpectedly, in the active site of MAO-B, the phenyl ring is posi-
tioned underneath the enzymatic ‘aromatic cage’ formed by
17. Microplate-based screening of MAO inhibitors: 20
corresponding alkyne (1.0 equiv) were dispensed. Ten microliter of CuSO4
(400 M), and 10 L of sodium ascorbate (4 mM) were added, and t-BuOH/H2O
lL of azide (10 mM) and a
l
l
(v/v 1/1) was added to keep the mixture at 200
by LC–MS and was completed after 24 h.
lL. The reaction was monitored
Tyr398, Tyr435and FAD, thus a9 fails toform any p–pstacking inter-
action with MAO-B. In addition, docking study also displays that no
hydrogen bond is observed between the enzyme and the inhibitor.
In conclusion, an efficient strategy for the fast construction 108
compounds library was developed using click chemistry. The fin-
gerprint of inhibitory activity toward MAO-A/B against this library
was obtained, and four hit compounds, especially a9 exhibited
excellent inhibitory activity and selectivity toward MAO-A. Dock-
ing study was carried out to demonstrate the binding mode be-
tween a9 and MAO-A/B, and the result reveals that compound a9
18. Lu, Y.; Dai, B.; Dai, Y.; Zhu, Q. Chin. Chem. Lett. 2008, 19, 947. The reaction
mixture was diluted into 96-well microplate from 0 to 100 M. The inhibitory
activity against MAO-A/B was determined according the reported method, and
l
the fluorescent signals were recorded at kex360 nm and kem460 nm on
a
SpectrumM2 spectrofluorometer..
19. General procedure for the Cu+-catalyzed [3+2] cycloaddition of azides and alkynes:
Azide (1.6 mmol, 1.2 equiv) and then alkyne (1.3 mmol, 1.0 equiv) were
suspended in a 1:1 mixture of water and t-BuOH (1.5 mL each) in a 10-mL
one-neck round bottle equipped with a small magnetic stirring bar. To this was
added copper sulfate solution (1 M, 20
lL) and sodium ascorbate solution (1 M,
200 L). The mixture was stirred for 24 h at room temperature, after which
l
time TLC analysis indicated complete consumption of starting materials. The
solvent was distilled, water (20 mL) was added, and the mixture was extracted
with dichloromethane. The organic layer was separated, washed with brine
(20 mL) and saturation NaHCO3 (20 ml), anhydrous MgSO4 dried. The solvent
was evaporated to afford crude product, which was further purified by alumina
column chromatography to offer product as a yellow solid.
can establish
p–p stacking interactions with Tyr407, Tyr444 and
FAD in MAO-A rather than in MAO-B. We expect this strategy will
contribute greatly to develop more selective and potent inhibitors
against MAO-A as well as MAO-B.
(1-(Pyridin-4-yl)-1H-1,2,3-triazol-4-yl) methanamine (h9): 91%. 1H NMR(D2O): d
8.55–8.54 (m, 2H, CH), 8.35 (s, 1H, –CH@C), 7.71–7.70 (m, 2H, CH), 3.85 (s, 2H,
CH2). MS (ESI) m/z 176.1 (M+H)+.
Acknowledgments
(1-Phenyl-1H-1,2,3-triazol-4-yl)methanamine (a9): 95%. 1H NMR(D2O): d 8.02 (s,
1H, –CH@C), 7.44–7.43 (m, 2H, CH), 7.37–7.31 (m, 3H, CH), 3.75 (s, 2H, CH2).
MS (ESI) m/z 175.1 (M+H)+.
This work was supported by Qianjiang Scholars Fund, Zhejiang
Province (No. 2006R10016), and Zhejiang Natural Science Fund
(No. Y2080303).
4-(4-(Aminomethyl)-1H-1,2,3-triazol-1-yl)benzoic
acid
(b9):
93%.
1H

Z. Jia, Q. Zhu / Bioorg. Med. Chem. Lett. 20 (2010) 6222–6225
6225
NMR(DMSO): d 8.95 (s, 1H, –CH@C), 8.18–8.16 (m, 2H, CH), 8.06–8.04 (m, 2H,
CH),4.25 (s, 2H, CH2). MS (ESI) m/z 219.1 (M+H)+.
21. De Colibus, L.; Min, L.; Binda, C.; Lustig, A.; Edmondson, D. E.; Mattevi, A. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 12684. Data deposition: www.pdb.org (PDB ID
code 2BXR, 2BXS, and 2BYB)..
22. Binda, C.; Newton-Vinson, P.; Hubalek, F.; Edmondson, D. E.; Mattevi, A. Nat.
Struct. Biol. 2002, 9, 22. Data deposition: www.pdb.org (PDB ID code 1GOS)..
3-(4-Aminomethyl-[1,2,3]triazol-1-yl)-benzoic acid (c9): 93%. 1H NMR(DMSO): d
8.87 (s, 1H, –CH@C), 8.33 (s, 1H, CH), 8.02–8.02 (m, 2H, CH),7.69 (s, 1H, CH),
4.21 (s, 2H, CH2). MS (ESI m/z 219.1 (M+H)+.
20. Goodsell, D. S.; Morris, G. M.; Olson, A. J. J. Mol. Recognit. 1996, 9, 1.