Benzoxazoles as Selective Monoamine Oxidase B (MAO-B) Inhibitors

Full text of DOI:10.1002/bkcs.11704

Note
DOI: 10.1002/bkcs.11704
BULLETIN OF THE
V. S. Sawant et al.
KOREAN CHEMICAL SOCIETY
Benzoxazoles as Selective Monoamine Oxidase B (MAO-B) Inhibitors
Vikram S. Sawant,†,‡ Hyeri Park,† Soo Yeon Paek,† Jieon Lee,†,‡ Ji Won Choi,§ Ki Duk Park,§
†,‡,
Kyung Il Choi,† Jihye Seong,†,‡,§ Sanghee Lee,†, and Hyunah Choo
*
*
†Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology, Seoul
02792, Republic of Korea. *E-mail: slee19@kist.re.kr
‡Division of Bio-Medical Science and Technology, KIST School, Korea University of Science and
Technology, Seoul 02792, Republic of Korea. *E-mail: hchoo@kist.re.kr
§Convergence Research Center for Diagnosis Treatment Care of Dementia, Korea Institute of Science
and Technology, Seoul 02792, Republic of Korea
Received February 7, 2019, Accepted February 20, 2019
Keywords: Parkinson’s disease, MAO-B, Selective inhibitor, Benzoxazole
Parkinson’s disease (PD) is a central nervous system disor-
der accompanied by tremor, slow movement, motor impair-
ment, and dementia.1,2 These symptoms of PD mainly
result from the death of dopaminergic neurons in substantia
nigra (SN) and the deficiency of dopamine inside the
brain.3 Dopamine is a type of monoamine neurotransmitter
and acts as a chemical messenger to regulate neuronal sig-
naling in brain, thereby controls motor functions.4
caused an adverse effect because of undesirable action as a
sodium and calcium channel blocker.17 Therefore, the discov-
ery of novel and selective MAO-B inhibitor with different
molecular framework is still required.
Previously, we reported indole-substituted benzothiazoles
and benzoxazoles as MAO-B inhibitors and demonstrated
benzoxazole moiety working as a key pharmacophore for
selective and reversible MAO-B inhibitor.18 Herein, we
report piperidinyl- or pyrrolidinyl-substituted benzoxazole
derivatives as potent and selective MAO-B inhibitors. We
validated the reversible and competitive inhibitory effect of
the title compounds on human MAO-B. We further investi-
gated their binding modes against MAO-B via molecular
modeling to elucidate binding interaction.
To examine the substituent effect of the cyclic amine func-
tionality, we designed two classes of benzoxazoles with piperi-
dinyl or pyrrolidinyl moiety at 5 or 6 position of benzoxazole,
respectively. 5-Substituted benzoxazoles 4 were synthesized in
three steps from 2-amino-4-bromophenol 1 (Scheme 1).
2-Amino-4-bromophenol 1 underwent cyclization by reaction
with CH(OMe)3 at 120 ꢀC to produce 5-bromobenzoxazole
2 in 86% yield. 5-Bromobenzoxazole 2 was treated with NaO2-
SAr, Pd(OAc)2, and Cu(OAc)2 under acidic conditions to
afford 2-aryl-5-bromobenzoxazoles 3 by palladium-catalyzed
desulfitative C H arylation.19 Buchwald-Hartwig cross cou-
pling reactions of the compounds 3 with piperidine or pyrroli-
dine afforded the desired 5-substituted benzoxazoles 4 in
approximately 23–72% yields.
Levodopa (L-dopa) is the major treatment for PD and
exerts its effect as a dopamine precursor.5 However, dopa-
resistance or serious complications are observed after an ini-
tial period of treatment.6 Besides L-dopa treatment, another
therapeutic way to restore dopamine function is the suppres-
sion of dopamine catabolism. Monoamine oxidase (MAO) is
involved in down-regulation of dopamine level which cata-
lyzes the deamination and degradation of dopamine.7 Inhibi-
tion of MAO blocks the breakdown of dopamine, thereby
prolongs the action of dopamine in the brain.8 There are two
isoforms of MAO-A and MAO-B. Although they share
about 70% of structure similarity, MAO-A and -B exhibit
different substrate specificity as well as diverse regional dis-
tribution in brain tissue.9 MAO-B has been regarded as a
therapeutic target for PD in clinic because the elevated activ-
ity of MAO-B was observed in the SN of PD patients.10
Currently, Selegiline and Rasagiline, both selective and irre-
versible MAO-B inhibitors, are used in clinics as a combina-
tion therapy with L-dopa (Figure 1).11 Both drugs increased
the amount of dopamine and efficiency of L-dopa.12 How-
ever, severe adverse effects such as psychosis, headache, diz-
ziness and nausea were reported due to neurotoxic
metabolites.13,14 Recently, more potent and selective MAO-B
inhibitor, Safinamide, was discovered as a reversible inhibitor
(Figure 1).8 Safinamide also prevented neuronal cell death in
animal models, improved motor function in clinical trials, and
eventually was approved by FDA for mid- to late-stage of PD
patients.15 All these evidences confirmed the importance of
non-covalent inhibitor for MAO-B because the irreversibility
induced short-lived actions of the inhibitors.16 Nevertheless, it
6-Substituted benzoxazoles 7 were prepared starting from
5-bromo-2-nitrophenol 5 (Scheme 2). After reduction of
5-bromo-2-nitrophenol
5
using SnCl2, the resulting
2-amino-5-bromophenol was directly converted into
6-bromobenzoxazole 6 by treatment with arylaldehyde fol-
lowed by DDQ oxidation. The final 6-subsitutued benzoxa-
zoles 7 were obtained in approximately 26–97% yields
through Buchwald–Hartwig cross coupling reaction.
For structure–activity relationship (SAR) study, the bio-
logical effect of synthesized benzoxazoles on human
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KOREAN CHEMICAL SOCIETY
Overall, compared to 5-substituted benzoxazole 4,
6-substituted benzoxazole 7 showed enhanced potency with
submicromolar IC50’s (from 103 to 820 nM) that demon-
strated an important role of 6-cyclic amine substitution in
the benzoxzole moiety as a pharmacophore. In para-
substitution on aryl moiety, replacement of X with fluorine,
hydrogen, and chlorine roughly decreased their potency
which implied that the size and electron withdrawing char-
acter on X were responsible for MAO-B inhibition. Particu-
larly, fluorine substitution on aryl ring dramatically
increased the selectivity index for MAO-A (7e and 7f,
175-fold and 169-fold). The effect on piperidinyl or pyrroli-
dinyl substitution was somewhat complicated. It seemed to
be associated with neighboring aryl group.
Based on all these SAR study, we identified the most
potent and selective MAO-B inhibitor 7e which proposed
comparable inhibitory profile with Safinamide.18 As a
result, 7e was tested for further biological evaluation.
Figure 1. Structures of known MAO-B inhibitors.
Inhibition of MAO-B by 7e was further validated by the
enzyme kinetic study, and the progress of reaction of MAO-B
was monitored in the absence or presence of 7e. By measuring
the rate, we observed the saturated plateau in the level of reac-
tion rate regardless of 7e treatment. Analysis of Michaelis–
Menten kinetics and Lineweaver-Burk plot revealed that Vmax
was unaffected with apparent shift in Km upon inhibitor treat-
ment. (Figure 2) These findings suggested that 7e should com-
pete with the substrate in the active site, which indicated the
competitive inhibition of 7e against MAO-B.
Scheme 1. Synthesis of 5-substituted benzoxazoles 4. Reagents
and conditions: a) CH(OMe)3, 120ꢀC; b) NaO2SAr, Pd(OAc)2,
Cu(OAc)2, TFA, DME, 120 ꢀC; c) amine, Pd2(dba)3, BINAP,
KOtBu, toluene, 120 ꢀC or amines, Pd(OAc)2, BINAP, NaOtBu,
toluene, 120 ꢀC.
Since we verified 7e as a competitive inhibitor working
at the active site, further confirmation for the reversibility
would be an important issue for therapeutic potential. With-
out any covalent warhead in chemical structure, we postu-
lated 7e as a reversible inhibitor. To confirm this
hypothesis, we investigated the recovery of enzyme func-
tion after inhibitor treatments. Selegiline and Safinamide
was used as irreversible and reversible control, respec-
tively.20,21 Each hMAO-B enzyme reaction in the absence
or presence of inhibitors was subjected to (1) MAO-B
activity assay or (2) washout followed by re-testing for
enzyme reaction.18 Compared to original enzyme activity
(Figure 2), wash-off of enzyme-binding of 7e or Safinamide
induced the recovery of MAO-B catalytic function, result-
ing in showing that %-inhibitions after washout of 7e and
Safinamide were 83.50% and 76.00%, respectively,
whereas Selegiline completely blocked the MAO-B even
after washing step (Table 2). These results indicated 7e as a
reversible inhibitor for hMAO-B along with Safinamide.
To elucidate the binding mode between benzoxazole
inhibitors and MAO-B, computational study was performed
using co-crystal structure of hMAO-B and safinamide
(PDB: 2V5Z).22,23 The most potent two benzoxazoles 7e
and 7f were docked smoothly to the active site of hMAO-
B. The orientation of the piperidine/pyrrolidine ring was
positioned inside of the active site near to flavin adenine
dinucleotide (FAD) and the fluoro-substituted phenyl group
was positioned to the entrance of the binding pocket
MAO-B (hMAO-B) was evaluated with Safinamide as a
positive control. Since all the compounds, except 4c, exhib-
ited more than 50% of MAO-B inhibition at 10 μM from
initial test, they were subjected to determine IC50 values
(Table 1). To confirm selectivity against MAO-A, we also
measured their inhibitory effect against human MAO-A
(hMAO-A) with clorgyline as a positive control and esti-
mated the selectivity index. Similar to the potency, most of
compounds also showed selectivity against MAO-A over 4-
to 175-folds.
Scheme 2. Synthesis of 6-substituted benzoxazoles 7. Reagents
and conditions: a) i) SnCl2, EtOH, 70 ꢀC; ii) ArCHO, MeOH,
ꢀ
50 C; iii) DDQ, DCM, rt.; b) amine, Pd2(dba)2, BINAP, KOtBu,
toluene, 120 ꢀC.
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Table 1. IC50 value for hMAO-B and selectivity index against
hMAO-A of benzoxazoles 4 and 7.
and 7f. Additional interaction with Ile199, Ile316, Gln206,
and Leu171 provided extra stability to the complex by
π-sigma, π-alkyl, and π-donor hydrogen bond interactions.
According to the molecular docking study, various hydro-
phobic interactions described the molecular evidence for
binding mode between 7e and MAO-B.
The benzoxazoles with piperidine and pyrrolidine were
synthesized and biologically evaluated as selective MAO-B
inhibitors. Based on SAR analysis, we verified the impor-
tance of cyclic amine on 6-position and fluorine moiety on
para-position of the aryl ring. Finally, we identified the
most potent and selective compound 7e among benzoxazole
analogues with competitive inhibition and reversibility.
Modeling study elucidated the structural understanding for
binding mode between MAO-B and 7e through various
hydrophobic interaction. In conclusion, the novel selective
and reversible MAO-B inhibitor 7e was discovered, of
which further study such as pharmacokinetic profiling or
in vivo efficacy in animal model are in due course.
MAO-B
Selectivity index
(SI)b
Compd
X
n
IC50 (nM)a
4a
4b
4c
4d
4e
4f
7a
7b
7c
7d
7e
7f
H
H
Cl
Cl
F
1
0
1
0
1
0
1
0
1
0
1
0
501 Æ 45
2724 Æ 251
--c
>19
>4
--d
4324 Æ 800
700 Æ 86
466 Æ 85
656 Æ 123
743 Æ 260
820 Æ 373
332 Æ 89
103 Æ 18
286 Æ 53
51 Æ 1
>18
>25
>41
>31
>36
>44
>88
>175
>169
>196
F
H
H
Cl
Cl
F
F
Safinamide
a
All of values were obtained by three independent experiments at
least.
b
Selectivity index (SI) = IC50 (hMAO-A)/IC50 (hMAO-B). All of
IC50 values about hMAO-A postulated >10 000 nM due to low
values of %-inhibition (under 28.6% at 10 μM).
Not determined.
Experimental
c
Synthesis.
6-Bromo-2-(4-fluorophenyl)benzo[d]oxazole
d
Not calculated.
(6). 5-Bromo-2-nitrophenol (500 mg, 2.29 mmol) was dis-
solved in ethanol (25 mL) and then tin(II) chloride dihy-
drate (2.59 g, 11.46 mmol) was added to this flask. The
Michaelis-Menten Plot
Lineweaver-Burk Plot
0.8
0.6
0.4
0.2
0.0
15
10
5
Control
1 μM
ꢀ
resulting mixture was stirred for 2 h at 70 C. After the
500 nM
100 nM
reaction was completed, the mixture was poured onto ice,
neutralized to pH 7 with NaHCO3, and extracted with
EtOAc. The combined organic layers were dried over
MgSO4, filtered and evaporated. This compound was used
for the next reaction without further purification. This com-
pound (300 mg, 1.59 mmol) was dissolved in methanol
(15 mL) and 4-fluorobenzaldehyde (0.156 mL, 1.59 mmol)
was added to this solution. The reaction mixture was stirred
Control
1 μM
500 nM
100 nM
0
1000
2000
[Substrate](μM)
3000
4000
5000
-0.010 -0.005 0.000 0.005 0.010 0.015 0.020
1/[S]
Figure 2. Validation of mechanism for MAO-B inhibition by 7e.
The reaction rates were measured at different concentrations of
MAO-B substrate, benzylamine (0.0625, 0.125, 0.25, 0.5, 1, 2, and
4 mM) in absence or presence of 7e with indicated concentrations.
ꢀ
at 50 C for 12 h. After concentration, CH2Cl2 (10 mL)
was added to the residue and the resulting solution was
treated with DDQ (383 mg, 1.69 mmol). After stirring at
room temperature for 30 min, the mixture was diluted with
additional CH2Cl2 and washed with saturated Na2CO3. The
organic layer was dried over MgSO4. After evaporation,
the crude compound was purified by slica gel column chro-
matography (ethyl acetate: hexane = 20:80) to obtain the
(Figure 3). Major interactions of benzoxazole based on
π-sulfur interactions with Cys172 and π–π interactions with
Tyr326 revealed the importance of core structure. π-Donor
hydrogen bonding between Pro104 and fluoro-substituted
aryl group explained high potency of 7e and 7f by fluorine
incorporation. On the other hand, carbon-hydrogen interac-
tion between piperidine and Tyr435/Leu171 was detected
only in 7e, which distinguished the potency between 7e
1
desired product 6 (280 mg, 0.96 mmol) in 62.5% yield: H
NMR (400 MHz, CDCl3) δ 8.24–8.22 (m, 2H), 7.75 (dd,
J 1.6, 0.4 Hz, 1H), 7.62 (dd, J = 8.4. 0.4 Hz, 1H), 7.49
(dd, J = 8.4, 2.0 Hz, 1H), 7.24–7.20 (m, 2H).
Table 2. Reversibility test of MAO-B inhibitor 7e.
2-(4-Fluorophenyl)-6-(piperidin-1-yl)benzo[d]oxazole (7e).
Piperidine (0.95 ml, 9.6 mmol), Pd2(dba)3 (5 mg, 0.0048
mmol), BINAP (9 mg, 0.014 mmol) and KOtBu (161 mg,
1.44 mmol) were added to a solution of 6-bromo-2-phenyl-
benzo[d]oxazole (140 mg, 0.48 mmol) in tolueneꢀ(10 ml).
The reaction mixture was stirred for 4 h at 120 C. After
cooling to room temperature, H2O was added and then
extracted with CH2Cl2. The organic solvent layers were dried
over MgSO4, filtered and evaporated. The residue was
%-Inhibition
%-Inhibition
Inhibitor
before washouta
after washouta
Reversibility
7e
82.78
100
95.69
83.50
0
76.00
Reversible
Irreversible
Reversible
Selegiline
Safinamide
a
%-inhibition was normalized by control in absence of inhibitor. All
enzyme reactions were performed with 1 μM of inhibitors and
100 mM of substrates.
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control experiments, appropriate dilutions of the vehicles
were carried out. To exclude the signal noise from reagents
or compounds, background absorbance was measured from
the sample only without enzyme, then subtracted from orig-
inal raw data. For analysis of kinetic experiment, GraphPad
Prism was used to characterize enzyme reaction constants.
Acknowledgments. This research is supported by the Origi-
nal
Technology
Research
Program
(NRF-
2016M3C7A1904344) funded by the National Research
Foundation of Korea (NRF). And this work is additionally
funded by the Korea Institute of Science and Technology
(KIST) Institutional Program (2E29190 and 2E29222).
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