Novel tricyclic pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one: Design, synthesis, model and use as hMAO-B inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.02.045

Based on our recently reported selective hMAO-A inhibitors, on which, the intramolecular cyclization led to a very interesting change of isoform selectivity. A series of selective hMAO-B inhibitors (3a-3u) with novel scaffold of tricyclic pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one were designed and synthesized. Compound 3u (IC₅₀ = 221 nM) exhibited the best inhibitory activity and isoform selectivity against hMAO-B, superior to selegiline (IC₅₀ = 321 nM), which is a commercial selective hMAO-B inhibitor used to Parkinson's disease. Modeling study indicated that the selectivity of our compounds to hMAO-B is determined by at least two residues, i.e., Ile 199 and Cys 172 (or corresponded Phe 208 and Asn 181 of hMAO-A). These data support further studies to assess rational design of more efficiently selective hMAO-B inhibitors.

Full text of DOI:10.1016/j.bmc.2016.02.045

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel tricyclic pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one: Design,
synthesis, model and use as hMAO-B inhibitors
Rui Chen a,y, Jie Xiao c,y, Yong Ni b, Han-Fei Xu b, Min Zheng b, Xu Tong a, Tong-Tian Zhang a,
Chenzhong Liao b, , Wen-Jian Tang a,
⇑
⇑
a School of Pharmacy, Anhui Medical University, Hefei 230032, PR China
b School of Medical Engineering, Hefei University of Technology, Hefei 230009, PR China
c School of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on our recently reported selective hMAO-A inhibitors, on which, the intramolecular cyclization led
to a very interesting change of isoform selectivity. A series of selective hMAO-B inhibitors (3a–3u) with
novel scaffold of tricyclic pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one were designed and synthesized.
Compound 3u (IC50 = 221 nM) exhibited the best inhibitory activity and isoform selectivity against
hMAO-B, superior to selegiline (IC50 = 321 nM), which is a commercial selective hMAO-B inhibitor used
to Parkinson’s disease. Modeling study indicated that the selectivity of our compounds to hMAO-B is
determined by at least two residues, i.e., Ile 199 and Cys 172 (or corresponded Phe 208 and Asn 181 of
hMAO-A). These data support further studies to assess rational design of more efficiently selective
hMAO-B inhibitors.
Received 18 January 2016
Revised 26 February 2016
Accepted 29 February 2016
Available online xxxx
Keywords:
hMAO
Isoform selectivity
Benzoxazepinone
Selective hMAO-B inhibitor
Anti-Parkinson
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
MAO inhibitors were withdrawn from the clinic due to serious side
effects such as liver toxicity and hypertensive crisis.5
Monoamine oxidases (MAO, EC 1.4.3.4) are flavin-containing
amine oxidoreductases bound to the mitochondrial outer mem-
brane, which play essential roles in the catabolism of amine neuro-
transmitters in the peripheral and central nervous systems.1,2 The
human monoamine oxidases mainly have two isoforms, namely
hMAO-A and hMAO-B, with different three-dimensional struc-
tures, substrate specificity and inhibitor selectivity. hMAO-A has
more specific for serotonin and noradrenaline, while hMAO-B pref-
erentially deaminates phenylethylamine and benzylamine. Tyra-
mine, tryptamine and dopamine are common substrates for both
isoforms.3,4 Although both isoforms exhibit high sequence homol-
ogy, they differ in shapes and volumes of the catalytic sites allowed
to discovery selective inhibitors.
The reversible and selective hMAO inhibitors resulted in
improved safety profile and in renewed interests for the develop-
ment of novel selective MAO inhibitors. Actually, hMAO-A inhibi-
tors mainly act as antidepressant and anti-anxiety agents,
whereas hMAO-B inhibitors are used alone or in combination to
treat Alzheimer’s (AD) and Parkinson’s (PD) diseases.6–8 hMAO-B
elevation in PD patients may lead to an increased production of
hydrogen peroxide and other reactive oxygen species (ROS),
responsible for neuron degeneration. MAO-B-mediated ROS con-
tributes to neuropathology and antioxidant treatment can arrest
further progression of dopaminergic cell death.9,10 Thus, neurolog-
ical degeneration in the central nervous system could be associated
to the oxidative stress, and with an increased MAO-B activity.
Therefore, hMAO-B represents an attractive target for the treat-
ment of the neurodegenerative diseases, including PD.11 Two selec-
tive hMAO-B inhibitors, selegiline and rasagiline are marketed
drugs for the treatment of PD. But both are irreversible hMAO inhi-
bitors, which utilize the propargyl group to covalently bind to the
flavin ring of the cofactor, flavin adenine dinucleotide (FAD). In this
way, the bound enzymes could not work and thus enzyme activity
was blocked until the cell makes new enzymes.
MAO inhibitors have been showed to have potential uses in the
treatment of psychiatric and neurological diseases. The first
generation MAO inhibitors were introduced into clinical use as
antidepressants, however, many nonselective and irreversible
⇑
Corresponding authors. Tel./fax: +86 551 62901285 (C.L.), +86 551 65161115
(W.-J.T.).
E-mail addresses: czliao@hfut.edu.cn (C. Liao), ahmupharm@126.com
Both of AD and PD are related with elevated levels of hMAO-B in
the brain, therefore, novel, potent, selective and reversible hMAO-B
(W.-J. Tang).
y
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2016.02.045
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Chen, R.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.045

2
R. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
inhibitors have gained considerable attentions in the treatment of
these two diseases and other disorders.11 The development of syn-
thetic heterocyclic compounds as MAOIs progressed considerably
during the past decade. Different families of heterocycles contain-
ing nitrogen atoms have been used as scaffolds for the discovery of
selective and reversible hMAO inhibitors.12–19
Br
OH
O
N
O
N
O
N
N
R1
R1
R2
R2
hMAO-A inhibitors
hMAO-B inhibitors
Recently, we identified 3,5-diaryl-2-pyrazoline-1-ethanone
derivatives as potent and selective hMAO-A inhibitors.20 In this
work, we reported selective hMAO-B inhibitors (3a–3u) with a
novel scaffold of tricyclic pyrazolo[1,5-d][1,4]benzoxazepin-5
(6H)-one, which was derived from the selective hMAO-A inhibitors
mentioned above20 by the intramolecular cyclization of 1-etha-
none bromide with hydroxyl group. This cyclization led to a very
interesting change of isoform selectivity from hMAO-A to hMAO-
B. Amongst them, compound 3u (IC50 = 221 nM) exhibited the best
inhibitory activity and isoform selectivity against hMAO-B, supe-
rior to selegiline (IC50 = 321 nM), a selective hMAO-B inhibitor cur-
rently in the market for the treatment of Parkinson’s disease.
Further molecular modeling demonstrates that the selectivity of
our compounds to hMAO-B is determined by at least two residues,
i.e., Ile 199 and Cys 172 of hMAO-B (or corresponded Phe 208 and
Asn 181 of hMAO-A).
Figure 1. The general inhibitor design strategy in this study.
Data/restraints/parameters = 2891/0/199, Goodness of fit on
F2 = 1.053, Fine, R1 = 0.0506, wR(F2) = 0.1242. Compound 3m: Color-
less block crystals, yield, 47%; mp 224–225 °C; C17H12ClFN2O2,
M = 348.75, Monoclinic, space group I12/a1
b = 17.3453(4), c = 14.6200(3) (Å); = 90, b = 103.788(2),
V = 3136.37(12) Å3, T = 291(10) K, Z = 8, Dcalcd = 1.477 g/cm3,
;
a = 12.7349(3),
a
c
= 90,
F
(000) = 1440, Reflections collected/independent reflections =
2903/2565, Data/restraints/parameters = 2903/0/221, Goodness of
fit on F2 = 1.036, Fine, R1 = 0.0981, wR(F2) = 0.1018. Compound 3t:
Colorless plate crystals, yield, 50%; mp 173–175 °C; C18H15ClN2O2,
M = 326.77, Triclinic, space group Pꢀ1; a = 5.1296(2), b = 9.5222(5),
c = 16.1436(8) (Å);
a
= 79.376(4), b = 87.744(4),
c = 88.468(4),
V = 774.27(7) Å3,
T = 292(2) K, Z = 2,
Dcalcd = 1.402 g/cm3,
2. Results and discussion
2.1. Inhibitor design
F(000) = 340.0, Reflections collected/independent reflections =
2773/2582, Data/restraints/parameters = 2773/0/209, Goodness of
fit on F2 = 1.063, Fine, R1 = 0.0346, wR(F2) = 0.0985.
The molecular structures of compounds 3e, 3m and 3t are
shown in Figure 2. Crystallographic data (excluding structure
factors) for the structures have been deposited in the Cambridge
Crystallographic Data Center as supplementary publication Nos.
CCDC 1428287, 1040100 and 1040101.
Based on our recently reported 3,5-diaryl-2-pyrazoline-1-etha-
none derivatives as selective hMAO-A inhibitors,20 on which, the
intramolecular cyclization of ethanone bromide with hydroxyl
group produced a novel scaffold of tricyclic pyrazolo[1,5-d][1,4]
benzoxazepin-5(6H)-one derivatives (Fig. 1). The novel scaffold
could be nicely docked into the active site of hMAO-B, indicating
they have the potential to be developed as hMAO-B inhibitors.
Therefore, we synthesized a series of compounds (3a–3u) contain-
ing this scaffold. Interestingly, this intramolecular cyclization did
result in the change of isoform selectivity from hMAO-A to
hMAO-B, most of them showed reversible and selective hMAO
activity, for example, compound 3u did not show any activity
against hMAO-A, but had an IC50 value of 221 nM against hMAO-
B, superior to selegiline (IC50 = 321 nM).
2.4. Inhibition against hMAO-A and hMAO-B
The potential inhibitory activities of the synthesized com-
pounds 3a–3u against hMAO-A and hMAO-B were investigated
by measuring their effects on the production of hydrogen peroxide
(H2O2) from p-tyramine, using the Amplex Red MAO assay kit
(Molecular Probes, Inc., Eugene, Oregon, USA) and MAO isoforms
in microsomes prepared from insect cells infected with Recombi-
nant baculovirus containing cDNA inserts for hMAO-A or hMAO-
B. The inhibition of hMAO activity was evaluated using the general
method described by Santana et al.22 The test compounds did not
show any interference with the reagents used for a biochemical
assay. The results of hMAO-A and hMAO-B inhibition studies with
title compounds are showed in Table 1 together with the hMAO-B
selectivity index ([IC50(hMAO-A)]/[IC50(hMAO-B)]). An irreversible
and selective hMAO-A inhibitor, clorgyline and an irreversible and
selective hMAO-B inhibitor, selegiline were employed as the posi-
tive control compounds in this test.
Enzymatic assays revealed that most of tested compounds
exhibited inhibitory activities against hMAO, and showing selectiv-
ity toward hMAO-B. It is obvious from the data that compounds
3m and 3u exhibited the best activities against hMAO-B with
IC50 values of 242 and 221 nM, respectively, surpassing that of
the positive control selegiline (IC50 = 321 nM).23 Besides the most
potent inhibitory activity, compound 3u (IC50 = 221 nM) also
showed the highest selectivity (271.1-fold) with respect to the
hMAO-B isoform. Inspection of the chemical structures, it can be
concluded that among the synthesized compounds, the hMAO-B
inhibitory activity was affected by the substituent groups at C2,
C6 positions and the benzene ring of benzoxazepinone moiety
(Table 1). Further, the substituent on the benzene ring connected
to the pyrazole ring had impact on both of hMAO-A and hMAO-B
inhibition.
2.2. Chemistry
Pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one derivatives (3a–
3u, as shown in Table 1) were synthesized according to the proto-
col outlined in Scheme 1. After styrene ketones were prepared
through Claisen–Schmidt condensation of substituted salicylalde-
hyde with acetophenone, the key intermediate 2-pyrazoline
derivatives (1) were synthesized by the cyclization reaction of
excess hydrazine hydrate with the styrene ketones. The 2-pyrazo-
line-1-ethanone derivatives (2) were obtained through the acyla-
tion of compound
1 with a-bromoacyl chloride. The title
compounds 3a–3u were obtained from the cyclization reaction of
compound 2 in the present of NaHCO3 catalysis in ethanol.20,21
2.3. Crystal structure analysis
The structures of compounds 3e, 3m and 3t were determined
by X-ray crystallography. Crystal data of compound 3e: Colorless
plate crystals, yield, 87%; mp 168–169 °C;
C
17H13ClN2O2,
a = 13.3672(9),
a = 90, b = 97.002(7), c = 90,
Mr = 312.74, Monoclinic, space group P121/c1
;
b = 14.2568(10), c = 7.7793(6) (Å);
V = 1471.49(19) Å3, T = 293(2) K, Z = 4, Dx = 1.412 g/cm3, F(000) =
648.0, Reflections collected/independent reflections = 2891/1988,
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R. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Table 1
The chemical structures of compounds 3a–3u and their inhibitory activities and selectivities against hMAO-A and hMAO-Ba
R2
O
7
6
O
R3
5
4
N
8
N
3
9
R1
2
1
10
11
R4
Compd
R1
R2
R3
R4
MAO-A (nM)
MAO-B (nM)
SI MAO-Bc
b
b
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
Clorgyline
Selegiline
4-CH3O-Ph
Ph
4-i-Pr-Ph
4-CH3-Ph
4-Cl-Ph
4-F-Ph
2-Cl-Ph
2-CH3-Ph
4-CH3O-Ph
4-CH3-Ph
Ph
4-Cl-Ph
4-F-Ph
2-F-Ph
4-CH3O-Ph
4-CH3O-Ph
4-CH3-Ph
Ph
4-Cl-Ph
2-Cl-Ph
4-F-Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Br
H
H
H
H
H
H
—
0.8
17.0
—
0.9
33.7
42.1
—
22.0
2.4
23.4
9.3
19.2
102.7
10.2
—
2195 175
2878 213
b
3522 184
b
b
b
53260 2230
b
1782 126
55680 2540
1321 120
b
b
b
2722 189
1634 86
2568 98
504 56
242 27
584 95
3904 154
b
4712 172
4654 89
b
4756 215
467 22
b
b
CH3
CH3
CH3
CH3
CH3
CH3
b
b
b
45240 2680
34670 2030
452 26
1.3
1.7
132.7
11.7
271.1
0.0031
187.0
4055 122
348 104
b
221 28
b
189
7
b
321 11
a
b
c
Each IC50 value is the mean SEM from three experiments (n = 3).
Inactive at 60 M, IC50 values obtained under the assumption that the corresponding IC50 against either hMAO-A or hMAO-B is the highest concentration tested.
SI MAO-B = [IC50(MAO-A)]/[IC50(MAO-B)].
l
R2
H
C
R3
O
.
O
N2H4 H2O
HO
40% NaOH / H2O
EtOH, 60°C
R1
CH3
+
R1
HO
R3
EtOH, reflux
R2
O
R4
R2
R2
O
HO
O
HO
NH
O
R2
R1
R4 CHBrCCl
NaHCO3, EtOH
30 - 70°C
N
R1
N
R3
N
N
Br
R3
R1
DMAP, CH2Cl2
N
R3
R4
O
1
2
3a - 3u
Scheme 1. Synthesis of pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one derivatives 3a–3u.
Figure 2. An ORTEP view of the molecular structures of compounds 3e, 3m and 3t.
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4
R. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Compounds with halogen substituents at the benzene ring in
the benzoxazepinone moiety have great influence on the hMAO-
B inhibition, for example, compounds 3i–3n with 10-chloro substi-
tution at the benzene ring were found better in terms of potency
than the corresponding compounds 3a–3h without the sub-
stituents. Compound 3o with 8,10-dibromo substituents showed
more potent hMAO-B inhibitory activity (IC50 = 467 nM) than the
monochloro substituted compound 3i (IC50 = 2722 nM) and the
compound without substitution 3a (IC50 > 60 lM). This trend can
also be observed among compounds 3b, 3k, compounds 3d, 3j,
compounds 3f, 3m, and compounds 3e, 3l, etc.
From Table 1, it is intuitive that the volumes of the para-substi-
tuted groups at the benzene ring in the C2 position have influences
on the activity. For hMAO-B, the smaller of this substituent, the
better of the inhibitory activity. The methyl group at the R2 posi-
tion also has great impact on the inhibitory activity against hMAO:
it can increase the potency against B-isoform but retard the
potency against A-isoform. For example, compound 3f has an
IC50 value of 1782 nM against hMAO-B; when the R2 position is
methyl (compound 3u), the IC50 value was changed to 221 nM,
having an 8-fold increment.
2.5. Molecular docking study
Structure-based docking methods have been successfully
employed to detect the binding poses and energies of small ligands
to their receptors.24 To explore the binding models of the com-
pounds discussed herein to hMAO-B, explain the SAR and isoform
selectivity between hMAO-A and hMAO-B and further guide the
design of more potent and specific hMAO-B inhibitors, these com-
pounds were docked into the active site of hMAO-B using Glide
5.9 of the Schrödinger suite. The standard precision (SP) of Glide25
was employed. The PDB code used of the hMAO-B complex was
2V5Z which has an X-ray resolution of 1.6 Å and a R-factor of
0.227.13 In this complex, a selective non-covalent inhibitor resides
in the binding pocket. During the docking process, the thiol group
of Cys 172 was allowed to adopt different orientations since in the
crystal structure of 2V5Z, an intramolecular hydrogen bond
between this thiol group and the protein can be observed, however,
in the crystal structure of 2XFQ,26 this hydrogen bond does not exist.
Among the many docking poses produced by Glide SP, a pose
having a score of ꢀ9.896 kJ/mol of compound 3u can explain the
SAR and selectivity of our compounds to both hMAO-A and
hMAO-B very well. From the docking pose of compound 3u to
hMAO-B, it can be seen that this compound fills the binding pocket
of hMAO-B very well, mainly forming hydrophobic interactions
with surrounding hydrophobic residues (Fig. 3A and B). Further-
more, a hydrogen bond can be observed in the docking complex
of compound 3u to hMAO-B.
Figure 3. Putative binding pose of compound 3u to hMAO-B. (A) Ribbon diagram of
the docked complex of 3u (yellow) to hMAO-B. The FAD is represented as a sphere
model. (B) The residues of hMAO-B around the docked compound 3u. A hydrogen
bond between 3u and Cys 172 is indicated by a magenta dashed line. (C) The
different residues between hMAO-A (orange) and hMAO-B (green) around the
docking pose of compound 3u.
The benzene ring in the benzoxazepinone moiety is in the
hydrophobic sub-pocket composed of Leu 171, Tyr 326, Leu 328,
Tyr 398 and Phe 343, so increasing both of the hydrophobicity
and volume may enhance the inhibition against hMAO-B. It is well
known that halogen atoms can increase the hydrophobicity and
volume and therefore they have wide applications in medicinal
chemistry.27 Indeed, when one or two hydrogen atoms in this ben-
zene ring were substituted by halogens, such as chlorine or bro-
mine, the binding affinities were enhanced a lot. For hMAO-B,
inhibitions against hMAO-A were much lower or even lost. In the
docking pose of compound 3u to hMAO-B, another benzene ring
connected to the pyrazole moiety inserts into a hydrophobic sub-
pocket and has hydrophobic interactions with the surrounding
residues such as Phe 168, Leu 171, Ile 199, Ile 316 and Tyr 326.
Increasing the volume of this benzene ring may attenuate the inhi-
bition against hMAO-B, for example, the IC50 of compound 3b was
2878 nM, however, when the hydrogen atom at the para-position
was replaced by bulky groups (see compounds 3a, 3d, 3e), the
binding affinities dropped. Since fluorine atom has a similar
VDW radius as hydrogen atom and it can increase the hydropho-
bicity of benzene, it is not surprised to see that compound 3f had
better IC50 against hMAO-B than compound 3b.
compound 3a had no inhibition against hMAO-B even at 60 lM,
however, when R4 was chlorine (compound 3i, see Table 1), the
IC50 was 2722 nM; when both R3 and R4 were bromine (compound
3o), the IC50 was dramatically changed to 467 nM. This trend also
can be observed among compounds 3b, 3k, compounds 3d, 3j,
compounds 3f, 3m, and compounds 3e, 3l, etc. For the correspond-
ing sub-pocket in hMAO-A, because the residues are same as the
ones in hMAO-B, this trend also can be perceived, although the
Almost of all compounds discussed herein showed no activity
against hMAO-A, which is also explainable from the docking pose
of compound 3u: when superimposing the crystal structure of
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R. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
hMAO-A (PDB code: 2Z5X28) with our docking complex of hMAO-B,
it can be seen that in hMAO-A, the corresponding residue of Ile 199
of hMAO-B is Phe 208, which would have conflicts with our com-
pounds (see Fig. 3C) and therefore dramatically reduce the inhibi-
tion against hMAO-A or do not inhibit hMAO-A at all. It is
interesting to notice that the carbonyl group of compound 3u can
form a hydrogen bond with the residue of Cys 172 of hMAO-B. Nev-
ertheless, in this corresponding position in hMAO-A, the residue is
Asn 181, which could not form a hydrogen bond with the carbonyl
group. We can see that in compounds 3p–3u, there is an extra
methyl group in the R2 position, which is close to the carbonyl
group. This methyl group may have extra hydrophobic interactions
with the residue of Tyr 398 and Cys 172 in hMAO-B, as indicated by
the docking study, which can explain why compounds 3p–3u had
better inhibitions than the corresponding compounds without the
methyl group. However, in hMAO-A, the corresponding residue of
Cys 172 of hMAO-B is Asn 181, which would have conflict with
the methyl group and cause worse inhibition. Residue of Tyr 326
of hMAO-B is close to the docked pose of compound 3u, and has
hydrophobic interaction with it. In hMAO-A, the equivalent residue
is Ile 335, which is smaller than Tyr. Another different residue
around the docked compound is Leu 171 of hMAO-B and corre-
sponding Ile 180 of hMAO-A. Both of these two residues may also
have impacts on the isoform selectivity.
Since all compounds discussed herein did not show inhibitions
or showed very weak activities against hMAO-A, it is not meaning-
ful to try to identify the docking pose of these weak inhibitors to
hMAO-A using docking methods.
All in all, we can see that the selectivity of our compounds to
hMAO-B is determined by at least two residues, i.e., Ile 199 and
Cys 172 of hMAO-B (or corresponded Phe 208 and Asn 181 of
hMAO-A). A- and B-isoforms of h-MAO share about 70% sequence
identities; in the binding pockets, only few residues between them
are different. Employing the difference of the residues in the bind-
ing pockets of hMAO-A and hMAO-B, it is feasible to design and
identify isoform selective hMAO inhibitors.
were recorded in CDCl3 or DMSO-d6 on a Bruker AV300 spectrom-
eter at frequencies of 300 MHz and 75 MHz, respectively. Chemical
shifts are reported in parts per million (d) downfield from the sig-
nal of TMS as internal standards. Spin multiplicities are given as s
(singlet), d (doublet), t (triplet), q (quartet) or m (multiplet). High
resolution mass spectra (HRMS) were obtained on an Agilent
1260-6221 TOF mass spectrometry.
4.2. Procedures for the synthesis of title compounds 3a–3u
To a solution of aromatic methyl ketones (10 mmol) and salicy-
laldehyde (11 mmol) in ethanol (10 mL) was added 40% NaOH
aqueous solution (2 mL) dropwise and the reaction was carried
out at 60 °C for 4–10 h until the disappearance of starting material
(monitored by TLC). The mixture was poured into cold water and
neutralized with 2 M HCl to a pH in the range of 2–3. The resulting
precipitate was collected, washed with water and dried to give
chalcones. The chalcone was treated with 5 times excess of hydra-
zine hydrate in dry ethanol and refluxed for 3–6 h. The reaction
mixture was then poured into ice-cold water. The solid was fil-
tered, washed and recrystallized from ethanol to afford respective
pyrazoline (1). A mixture of compound 1 and a-bromoacyl chloride
in CH2Cl2 was added 4-dimethylaminopyridine (DMAP) and the
reaction was stirred overnight. The reaction mixture was washed
with water and brine, dried with anhydrous Na2SO4, filtrated and
concentrated in vacuo. The resulting residue was then purified by
column chromatography to give the N-acylated product 2. NaHCO3
(3.0 mmol) was added to an ethanol (20 mL) solution of compound
2 (2.0 mmol), then the reaction mixture was stirred at 30 °C until
the disappearance of starting material (monitored by TLC). EtOAc
(100 mL) was added to the reaction mixture, then washed with
water and brine, dried with anhydrous Na2SO4, filtrated and con-
centrated in vacuo. The residue was purified by chromatography
on a silica gel column (petroleum/EtOAc, 1:1 ? 1:2) to give title
compounds 3a–3u.
4.2.1. 2-(4-Methoxy-phenyl)-1,11b-dihydro-pyrazolo[1,5-d][1,4]
benzoxazepin-5(6H)-one (3a)
3. Conclusion
The title compound was prepared from 4-methoxy-phenyl
methyl ketone and salicylaldehyde in a yield of 19%: white pow-
der; mp 231–233 °C; 1H NMR (300 MHz, DMSO-d6) d (ppm):
3.73–3.88 (m, 4H, CH3O, 1-Ha), 3.97 (dd, 1H, J1 = 9.1 Hz,
J2 = 17.9 Hz, pyrazole 1-Hb), 4.36 (d, 1H, J = 16.0 Hz, 6-Ha), 5.01
(d, 1H, J = 16.0 Hz, 6-Hb), 6.02 (dd, 1H, J1 = 8.8 Hz, J2 = 11.3 Hz,
11b-H), 7.03–7.85 (8H, Ar-H). TOF-HRMS: m/z [M+H]+ calcd for
In this work, based on our recently reported selective hMAO-A
inhibitors,19 a series of selective hMAO-B inhibitors (3a–3u) with a
novel scaffold of tricyclic pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-
one were designed and synthesized. Compared with our early
reported of selective hMAO-A inhibitors, the intramolecular
cyclization led to this interesting change of isoform selectivity.
Compound 3u (IC50 = 221 nM) exhibited best inhibitory activity
and isoform selectivity against hMAO-B, better than selegiline
(IC50 = 321 nM). Further docking study shows that the selectivity
of our compounds to hMAO-B is determined by at least two resi-
dues, i.e., Ile 199 and Cys 172 (or corresponded Phe 208 and Asn
181 of hMAO-A). Two other residues, i.e., Tyr 326 and Leu 171 of
hMAO-B, may also have impacts on the selectivity. This striking
selectivity profile of compounds reported herein imply that we
can further develop selective hMAO-B and/or hMAO-A inhibitors
based on this novel scaffold using a diversity evolution strategy.29
C18H17N2O3: 309.1234; found: 309.1233.
4.2.2. 2-Phenyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxa-
zepin-5(6H)-one (3b)
The title compound was prepared from phenyl methyl ketone
and salicylaldehyde in a yield of 22%: white powder; mp 191–
193 °C; 1H NMR (300 MHz, CDCl3)
d (ppm): 3.69 (dd, 1H,
J1 = 11.5 Hz, J2 = 17.4 Hz, 1-Ha), 3.92 (dd, 1H, J1 = 9.3 Hz,
J2 = 17.4 Hz, 1-Hb), 4.48 (d, 1H, J = 16.7 Hz, 6-Ha), 5.05 (d, 1H,
J = 16.7 Hz, 6-Hb), 6.01 (dd, 1H, J1 = 8.8 Hz, J2 = 11.3 Hz, 11b-H),
7.17–8.03 (9H, Ar-H); 13C NMR (75 MHz, CDCl3) d (ppm): 35.4 (1-
C), 56.3 (11b-C), 72.9 (6-C), 121.4, 124.6, 125.2, 127.3 (2C), 128.7
(2C), 130.6, 130.7, 130.8, 131.0, 156.1 (2-C), 158.0, 164.7. TOF-
HRMS: m/z [M+H]+ calcd for C17H15N2O2: 279.1128; found:
279.1127.
4. Experimental section
4.1. Chemicals and instrumentation
All starting materials, unless noted elsewhere, were obtained
from commercial companies and were used without purification.
The reactions were monitored by thin layer chromatography
(TLC) on pre-coated silica GF254 plates. Melting points (mp) were
determined on a XT4MP apparatus (Taike Corp., Beijing, China)
and are uncorrected. Proton (1H) and carbon (13C) NMR spectra
4.2.3. 2-(4-Isopropyl-phenyl)-1,11b-dihydro-pyrazolo[1,5-d]
[1,4]benzoxazepin-5(6H)-one (3c)
The title compound was prepared from 4-isopropyl-phenyl
methyl ketone and salicylaldehyde in a yield of 18%: white pow-
der; mp 180–181 °C; 1H NMR (300 MHz, CDCl3) d (ppm): 1.28 (d,
Please cite this article in press as: Chen, R.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.045

6
R. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
6H, J = 6.9 Hz), 2.97 (dt, 1H, J1 = 6.9 Hz, J2 = 13.8 Hz, CH), 3.67 (dd,
1H, J1 = 11.5 Hz, J2 = 17.3 Hz, 1-Ha), 3.90 (dd, 1H, J1 = 9.2 Hz,
J2 = 17.3 Hz, 1-Hb), 4.47 (d, 1H, J = 16.7 Hz, 6-Ha), 5.04 (d, 1H,
J = 16.7 Hz, 6-Hb), 6.00 (dd, 1H, J1 = 9.5 Hz, J2 = 11.1 Hz, 11b-H),
7.18–7.93 (8H, Ar-H); 13C NMR (75 MHz, CDCl3) d (ppm): 23.8
(2C), 34.2 (CH), 35.4 (1-C), 56.1 (11b-C), 72.9 (6-C), 121.4, 124.6,
125.2, 126.8 (2C), 127.5 (2C), 128.1, 130.6, 130.9, 152.3, 156.1
(2-C), 158.0, 164.5. TOF-HRMS: m/z [M+H]+ calcd for C20H21N2O2:
321.1598; found: 321.1598.
4.2.9. 10-Chloro-2-(4-methoxy-phenyl)-1,11b-dihydro-pyrazolo
[1,5-d][1,4]benzoxazepin-5(6H)-one (3i)
The title compound was prepared from 4-methoxy-phenyl
methyl ketone and 5-chloro-salicylaldehyde in a yield of 27%:
white powder; mp 235–236 °C; 1H NMR (300 MHz, CDCl3) d
(ppm): 3.67 (dd, 1H, J1 = 11.4 Hz, J2 = 17.3 Hz, 1-Ha), 3.81 (dd, 1H,
J1 = 9.2 Hz, J2 = 17.3 Hz, 1-Hb), 3.87 (s, 3H, CH3O), 4.45 (d, 1H,
J = 16.6 Hz, 6-Ha), 5.02 (d, 1H, J = 16.6 Hz, 6-Hb), 5.93 (dd, 1H,
J1 = 9.3 Hz, J2 = 11.2 Hz, 11b-H), 6.92–7.95 (7H, Ar-H); 13C NMR
(75 MHz, CDCl3) d (ppm): 35.6 (1-C), 55.5 (CH3O), 55.9 (11b-C),
72.8 (6-C), 114.2 (2C), 122.8, 122.9, 125.2, 129.1 (2C), 130.4,
130.5, 132.6, 155.7 (2-C), 156.5, 162.0, 164.0. TOF-HRMS: m/z
[M+H]+ calcd for C18H16ClN2O3: 343.0844; found: 343.0845.
4.2.4. 2-p-Tolyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]benzoxa-
zepin-5(6H)-one (3d)
The title compound was prepared from p-methyl-phenyl
methyl ketone and salicylaldehyde in a yield of 23%: white
powder; mp 252–253 °C; 1H NMR (300 MHz, CDCl3) d (ppm):
2.41 (s, 3H, CH3), 3.66 (dd, 1H, J1 = 11.4 Hz, J2 = 17.3 Hz, 1-Ha),
3.90 (dd, 1H, J1 = 9.3 Hz, J2 = 17.3 Hz, 1-Hb), 4.47 (d, 1H,
J = 16.7 Hz, 6-Ha), 5.04 (d, 1H, J = 16.7 Hz, 6-Hb), 5.99 (dd, 1H,
J1 = 9.6 Hz, J2 = 11.1 Hz, 11b-H), 7.19–7.90 (8H, Ar-H). TOF-
HRMS: m/z [M+H]+ calcd for C18H17N2O2: 293.1285; found:
293.1294.
4.2.10. 10-Chloro-2-p-tolyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]
benzoxazepin-5(6H)-one (3j)
The title compound was prepared from p-methyl-phenyl
methyl ketone and 5-chloro-salicylaldehyde in a yield of 20%:
white powder; mp 214–215 °C; 1H NMR (300 MHz, CDCl3) d
(ppm): 2.42 (s, 3H, CH3), 3.68 (dd, 1H, J1 = 11.4 Hz, J2 = 17.4 Hz, 1-
Ha), 3.82 (dd, 1H, J1 = 9.2 Hz, J2 = 17.4 Hz, 1-Hb), 4.46 (d, 1H,
J = 16.6 Hz, 6-Ha), 5.02 (d, 1H, J = 16.6 Hz, 6-Hb), 5.94 (dd, 1H,
J1 = 9.6 Hz, J2 = 11.3 Hz, 11b-H), 7.12–7.86 (7H, Ar-H); 13C NMR
(75 MHz, CDCl3) d (ppm): 21.6 (CH3), 35.6 (1-C), 55.9 (11b-C),
72.8 (6-C), 122.8, 125.2, 127.3 (2C), 127.5, 129.5 (2C), 130.4,
130.5, 132.5, 141.7, 156.0 (2-C), 156.5, 164.1. TOF-HRMS: m/z
[M+H]+ calcd for C18H16ClN2O2: 327.0895; found: 327.0897.
4.2.5. 2-(4-Chloro-phenyl)-1,11b-dihydro-pyrazolo[1,5-d][1,4]
benzoxazepin-5(6H)-one (3e)
The title compound was prepared from 4-chloro-phenyl methyl
ketone and salicylaldehyde in a yield of 18%: white powder; mp
250–251 °C; 1H NMR (300 MHz, CDCl3) d (ppm): 3.65 (dd, 1H,
J1 = 11.5 Hz, J2 = 17.4 Hz, 1-Ha), 3.90 (dd, 1H, J1 = 9.4 Hz,
J2 = 17.4 Hz, 1-Hb), 4.47 (d, 1H, J = 16.7 Hz, 6-Ha), 5.04 (d, 1H,
J = 16.7 Hz, 6-Hb), 6.02 (dd, 1H, J1 = 9.6 Hz, J2 = 11.1 Hz, 11b-H),
7.19–7.94 (8H, Ar-H). TOF-HRMS: m/z [M+H]+ calcd for
4.2.11. 10-Chloro-2-phenyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]
benzoxazepin-5(6H)-one (3k)
The title compound was prepared from phenyl methyl ketone
and 5-chloro-salicylaldehyde in a yield of 24%: white powder;
mp 220–221 °C; 1H NMR (300 MHz, CDCl3) d (ppm): 3.70 (dd, 1H,
J1 = 11.4 Hz, J2 = 17.4 Hz, 1-Ha), 3.84 (dd, 1H, J1 = 9.2 Hz,
J2 = 17.4 Hz, 1-Hb), 4.46 (d, 1H, J = 16.6 Hz, 6-Ha), 5.02 (d, 1H,
J = 16.6 Hz, 6-Hb), 5.96 (dd, 1H, J1 = 9.5 Hz, J2 = 11.2 Hz, 11b-H),
7.12–7.96 (8H, Ar-H). TOF-HRMS: m/z [M+H]+ calcd for
C17H14ClN2O2: 313.0738; found: 313.0735.
4.2.6. 2-(4-Fluoro-phenyl)-1,11b-dihydro-pyrazolo[1,5-d][1,4]
benzoxazepin-5(6H)-one (3f)
The title compound was prepared from 4-fluoro-phenyl methyl
ketone and salicylaldehyde in a yield of 20%:, white powder; mp
229–230 °C; 1H NMR (300 MHz, CDCl3) d (ppm): 3.66 (dd, 1H,
J1 = 11.5 Hz, J2 = 17.3 Hz, 1-Ha), 3.90 (dd, 1H, J1 = 9.4 Hz,
J2 = 17.3 Hz, 1-Hb), 4.48 (d, 1H, J = 16.7 Hz, 6-Ha), 5.05 (d, 1H,
J = 16.7 Hz, 6-Hb), 6.00 (dd, 1H, J1 = 9.6 Hz, J2 = 11.1 Hz, 11b-H),
7.19–7.99 (8H, Ar-H). TOF-HRMS: m/z [M+H]+ calcd for C17H14FN2O2:
297.1034; found: 297.1033.
C17H14ClN2O2: 313.0644; found: 313.0737.
4.2.12. 10-Chloro-2-(4-chloro-phenyl)-1,11b-dihydro-pyrazolo
[1,5-d][1,4]benzoxazepin-5(6H)-one (3l)
The title compound was prepared from 4-chloro-phenyl methyl
ketone and 5-chloro-salicylaldehyde in a yield of 21%: yellow pow-
der; mp 206–207 °C; 1H NMR (300 MHz, CDCl3) d (ppm): 3.67 (dd,
1H, J1 = 11.5 Hz, J2 = 17.4 Hz, 1-Ha), 3.82 (dd, 1H, J1 = 9.3 Hz,
J2 = 17.4 Hz, 1-Hb), 4.46 (d, 1H, J = 16.7 Hz, 6-Ha), 5.03 (d, 1H,
J = 16.7 Hz, 6-Hb), 5.98 (dd, 1H, J1 = 9.4 Hz, J2 = 11.2 Hz, 11b-H),
7.14–7.92 (7H, Ar-H). TOF-HRMS: m/z [M+H]+ calcd for
4.2.7. 2-(2-Chloro-phenyl)-1,11b-dihydro-pyrazolo[1,5-d][1,4]
benzoxazepin-5(6H)-one (3g)
The title compound was prepared from 2-chloro-phenyl methyl
ketone and salicylaldehyde in a yield of 26%: white powder; mp
160–162 °C; 1H NMR (300 MHz, CDCl3) d (ppm): 3.86 (dd, 1H,
J1 = 11.3 Hz, J2 = 17.9 Hz, 1-Ha), 4.15 (dd, 1H, J1 = 9.5 Hz,
J2 = 17.9 Hz, 1-Hb), 4.49 (d, 1H, J = 16.7 Hz, 6-Ha), 5.06 (d, 1H,
J = 16.7 Hz, 6-Hb), 6.03 (dd, 1H, J1 = 9.5 Hz, J2 = 11.4 Hz, 11b-H),
7.20–7.97 (8H, Ar-H). TOF-HRMS: m/z [M+H]+ calcd for
C17H13Cl2N2O2: 347.0349; found: 347.0349.
4.2.13. 10-Chloro-2-(4-fluoro-phenyl)-1,11b-dihydro-pyrazolo
[1,5-d][1,4]benzoxazepin-5(6H)-one (3m)
The title compound was prepared from 4-fluoro-phenyl methyl
ketone and 5-chloro-salicylaldehyde in a yield of 25%: colorless
crystals; mp 224–225 °C; 1H NMR (300 MHz, CDCl3) d (ppm):
3.68 (dd, 1H, J1 = 11.5 Hz, J2 = 17.4 Hz, 1-Ha), 3.82 (dd, 1H,
J1 = 9.3 Hz, J2 = 17.4 Hz, 1-Hb), 4.46 (d, 1H, J = 16.7 Hz, 6-Ha), 5.03
(d, 1H, J = 16.7 Hz, 6-Hb), 5.97 (dd, 1H, J1 = 9.6 Hz, J2 = 11.2 Hz,
11b-H), 7.10–7.97 (7H, Ar-H). TOF-HRMS: m/z [M+H]+ calcd for
C17H13ClFN2O2: 331.0644; found: 331.0645.
C17H14ClN2O2: 313.0738; found: 313.0738.
4.2.8. 2-o-Tolyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]
benzoxazepin-5(6H)-one (3h)
The title compound was prepared from o-methyl-phenyl
methyl ketone and salicylaldehyde in a yield of 22%: yellow pow-
der; mp 180–182 °C; 1H NMR (300 MHz, CDCl3) d (ppm): 2.67 (s,
3H, CH3), 3.71 (dd, 1H, J1 = 11.4 Hz, J2 = 17.3 Hz, 1-Ha), 3.95 (dd,
1H, J1 = 9.3 Hz, J2 = 17.3 Hz, 1-Hb), 4.48 (d, 1H, J = 16.7 Hz, 6-Ha),
5.05 (d, 1H, J = 16.7 Hz, 6-Hb), 5.96 (dd, 1H, J1 = 9.5 Hz,
J2 = 11.1 Hz, 11b-H), 7.18–7.58 (8H, Ar-H). TOF-HRMS: m/z
[M+H]+ calcd for C18H17N2O2: 293.1285; found: 293.1291.
4.2.14. 10-Chloro-2-(2-fluoro-phenyl)-1,11b-dihydro-pyrazolo
[1,5-d][1,4]benzoxazepin-5(6H)-one (3n)
The title compound was prepared from 2-fluoro-phenyl methyl
ketone and 5-chloro-salicylaldehyde in a yield of 21%: yellow
Please cite this article in press as: Chen, R.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.045

R. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
powder; mp 219–221 °C; 1H NMR (300 MHz, CDCl3) d (ppm): 3.81
(ddd, 1H, J1 = 3.0 Hz, J2 = 11.5 Hz, J3 = 18.3 Hz, 1-Ha), 3.95 (ddd, 1H,
J1 = 2.6 Hz, J2 = 9.3 Hz, J3 = 18.3 Hz, 1-Hb), 4.47 (d, 1H, J = 16.7 Hz,
6-Ha), 5.04 (d, 1H, J = 16.7 Hz, 6-Hb), 5.96 (m, 1H), 7.11–8.22
(7H, Ar-H). TOF-HRMS: m/z [M+H]+ calcd for C17H13ClFN2O2:
331.0644; found: 331.0643.
122.4, 123.9, 126.1, 128.3, 128.4 (2C), 129.0 (2C), 129.2, 129.5,
136.9, 155.1 (2-C), 155.2, 167.4. TOF-HRMS: m/z [M+H]+ calcd for
C18H16ClN2O2: 327.0895; found: 327.0890.
4.2.20. 2-(2-Chloro-phenyl)-6-methyl-1,11b-dihydro-pyrazolo
[1,5-d][1,4]benzoxazepin-5(6H)-one (3t)
The title compound was prepared from 2-chloro-phenyl methyl
ketone and salicylaldehyde in a yield of 28%: white crystals; mp
173–175 °C; 1H NMR (300 MHz, CDCl3) d (ppm): 1.56 (d, 3H,
J = 6.8 Hz, CH3), 3.89–4.10 (m, 2H, 1-Ha, 1-Hb), 5.05 (q, 1H,
J = 6.8 Hz, 6-H), 5.97 (t, 1H, J = 10.8 Hz, 11b-H), 7.05–7.93 (8H,
Ar-H). TOF-HRMS: m/z [M+H]+ calcd for C18H16ClN2O2: 327.0895;
found: 327.0894.
4.2.15. 8,10-Dibromo-2-(4-methoxy-phenyl)-1,11b-dihydro-
pyrazolo[1,5-d][1,4]benzoxazepin-5(6H)-one (3o)
The title compound was prepared from 4-methoxy-phenyl
methyl ketone and 3,5-dibromo-salicylaldehyde in a yield of 20%:
white powder; mp 269–270 °C; 1H NMR (300 MHz, DMSO-d6) d
(ppm): 3.75 (dd, 1H, J1 = 11.6 Hz, J2 = 17.8 Hz, 1-Ha), 3.87 (s, 1H),
3.99 (dd, 1H, J1 = 8.6 Hz, J2 = 17.8 Hz, 1-Hb), 4.44 (d, 1H,
J = 16.5 Hz, 6-Ha), 5.02 (d, 1H, J = 16.5 Hz, 6-Hb), 6.02 (dd, 1H,
J1 = 8.8 Hz, J2 = 11.3 Hz, 11b-H), 6.92–7.91 (6H, Ar-H); 13C NMR
(75 MHz, DMSO-d6) d (ppm): 40.5 (1-C), 60.3 (CH3O), 60.9 (11b-
C), 76.2 (6-C), 118.9 (2C), 121.7, 122.9, 127.7, 132.6, 134.0 (2C),
139.8, 140.4, 158.3 (2-C), 161.1, 166.6, 167.8. TOF-HRMS: m/z [M
+H]+ calcd for C18H15Br2N2O3: 466.9425; found: 466.9426.
4.2.21. 2-(4-Fluoro-phenyl)-6-methyl-1,11b-dihydro-pyrazolo
[1,5-d][1,4]benzoxazepin-5(6H)-one (3u)
The title compound was prepared from 4-fluoro-phenyl methyl
ketone and salicylaldehyde in a yield of 21%: white powder; mp
189–190 °C; 1H NMR (300 MHz, CDCl3) d (ppm): 1.55 (d, 3H,
J = 6.8 Hz, CH3), 3.76 (d, 1H, J = 2.1 Hz, pyrazole 4-Ha), 3.79 (d,
1H, J = 2.7 Hz, pyrazole 4-Hb), 5.05 (q, 1H, J = 6.8 Hz, oxazepane
2-H), 5.96 (t, 1H, J = 10.7 Hz, pyrazole 5-H), 7.04–7.94 (8H, Ar-H).
TOF-HRMS: m/z [M+H]+ calcd for C18H16FN2O2: 311.1190; found:
311.1186.
4.2.16. 2-(4-Methoxy-phenyl)-6-methyl-1,11b-dihydro-pyrazolo
[1,5-d][1,4]benzoxazepin-5(6H)-one (3p)
The title compound was prepared from 4-methoxy-phenyl
methyl ketone and salicylaldehyde in a yield of 26%: white pow-
der; mp 240–241 °C; 1H NMR (300 MHz, CDCl3) d (ppm): 1.55 (d,
3H, J = 6.8 Hz, CH3), 3.75 (d, 1H, J = 2.2 Hz, 1-Ha), 3.78 (d, 1H,
J = 2.7 Hz, 1-Hb), 3.86 (s, 3H, CH3O), 5.05 (q, 1H, J = 6.8 Hz, 6-H),
5.92 (t, 1H, J = 10.6 Hz, 11b-H), 6.90–7.87 (8H, Ar-H); 13C NMR
(75 MHz, CDCl3) d (ppm): 17.4 (CH3), 38.4 (1-C), 55.4 (CH3O),
58.0 (11b-C), 76.1 (6-C), 114.1 (2C), 122.4, 123.3, 123.8, 126.1,
128.7, 128.9 (2C), 129.4, 155.3 (2-C), 155.8, 161.7, 167.1. TOF-
HRMS: m/z [M+H]+ calcd for C19H19N2O3: 323.1390; found:
323.1391.
4.3. Crystal structure determination for compounds 3e, 3m and
3t
X-ray single-crystal diffraction data for compounds 3e, 3m and
3t was collected on a Bruker SMART APEX CCD diffractometer at
293(2) K using MoKa radiation (k = 0.71073 Å) by the x scan
mode. The program SAINT was used for integration of the diffrac-
tion profiles. The structure was solved by direct methods using
the SHELXS program of the SHELXTL package and refined by full-
matrix least-squares methods with SHELXL.30 The corrections for
LP factors were applied. Multi-scan symmetry-related measure-
ment was used as experimental absorption correction type. The
non-hydrogen atoms of compounds 3e, 3m and 3t were refined
with anisotropic thermal parameters whereas all hydrogen atoms
were generated theoretically onto the parent atoms and refined
isotropically with fixed thermal factors.
4.2.17. 6-Methyl-2-p-tolyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]
benzoxazepin-5(6H)-one (3q)
The title compound was prepared from p-methyl-phenyl
methyl ketone and salicylaldehyde in a yield of 28%: white pow-
der; mp 195–196 °C; 1H NMR (300 MHz, CDCl3) d (ppm): 1.55 (d,
3H, J = 6.8 Hz, CH3), 2.40 (s, 3H, CH3), 3.76 (d, 1H, J = 2.8 Hz, 1-
Ha), 3.79 (d, 1H, J = 3.5 Hz, 1-Hb), 5.05 (q, 1H, J = 6.8 Hz, 6-H),
5.93 (t, 1H, J = 10.6 Hz, 11b-H), 7.03–7.83 (8H, Ar-H). TOF-HRMS:
m/z [M+H]+ calcd for C19H19N2O2: 307.1441; found: 307.1441.
4.4. Enzymatic assay
Enzymatic hMAO-A and hMAO-B activity of compounds was
determined by a fluorimetric method.22 Briefly, 0.1 mL of sodium
phosphate buffer (0.05 M, pH 7.4) containing various concentra-
tions of the test drugs (new compounds or reference inhibitors).
The appropriate amounts of recombinant hMAO-A or hMAO-B
was adjusted to the same reaction velocity in the presence of both
isoforms the same concentration of substrate: 165 pmol of p-tyra-
4.2.18. 6-Methyl-2-phenyl-1,11b-dihydro-pyrazolo[1,5-d][1,4]
benzoxazepin-5(6H)-one (3r)
The title compound was prepared from phenyl methyl ketone
and salicylaldehyde in a yield of 25%: white powder; mp 258–
259 °C; 1H NMR (300 MHz, CDCl3)
d (ppm): 1.55 (d, 3H,
J = 6.8 Hz, CH3), 3.78 (d, 1H, J = 3.2 Hz, 1-Ha), 3.81 (d, 1H,
J = 3.8 Hz, 1-Hb), 5.05 (q, 1H, J = 6.8 Hz, 6-H), 5.96 (t, 1H,
J = 10.6 Hz, 11b-H), 7.03–7.93 (9H, Ar-H). TOF-HRMS: m/z [M+H]+
calcd for C18H17N2O2: 293.1285; found: 293.1297.
mine/min (hMAO-A: 1.1
p-tyramine oxidized top hydroxyphenylacetaldehyde/min/mg pro-
tein; hMAO-B: 7.5 g protein; specific activity: 22 nmol of p-tyra-
lg protein; specific activity: 150 nmol of
l
mine transformed/min/mg protein). The mixture was incubated for
4.2.19. 2-(4-Chloro-phenyl)-6-methyl-1,11b-dihydro-pyrazolo
[1,5-d][1,4]benzoxazepin-5(6H)-one (3s)
15 min at 37 °C, placed in the dark fluorimeter chamber. After this
incubation period, the reaction was started by adding 200 lM of
The title compound was prepared from 4-chloro-phenyl methyl
ketone and salicylaldehyde in a yield of 20%: yellow powder; mp
182–184 °C; 1H NMR (300 MHz, CDCl3) d (ppm): 1.55 (d, 3H,
J = 6.8 Hz, CH3), 3.75 (d, 1H, J = 3.2 Hz, 1-Ha), 3.79 (d, 1H,
J = 3.8 Hz, 1-Hb), 5.05 (q, 1H, J = 6.8 Hz, 6-H), 5.96 (t, 1H,
J = 10.7 Hz, 11b-H), 7.05–7.88 (8H, Ar-H); 13C NMR (75 MHz,
CDCl3) d (ppm): 17.3 (CH3), 38.4 (1-C), 58.3 (11b-C), 76.1 (6-C),
10-acetyl-3,7-dihydroxyphenoxazine reagent (Amplex Red assay
kit, Molecular Probes, Inc., Eugene, OR), 1 U/mL horseradish perox-
idase, and 1 mM p-tyramine. The production of H2O2 and, conse-
quently, of resorufin, was quantified at 37 °C in a multidetection
microplate fluorescence reader based on the fluorescence gener-
ated (excitation, 545 nm, emission, 590 nm) over a 15 min period,
during which the fluorescence increased linearly.
Please cite this article in press as: Chen, R.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.045

8
R. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
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LigPrep 2.6 in the Schrödinger suite, and then docked into the
active site of the hMAO-B (PDB code: 2V5Z) by employing the pro-
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Acknowledgments
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This work was supported by the National Natural Science Foun-
dation of China (21572003), Natural Science Foundation of Anhui
Higher Education Institutions (KJ2016A339) and National Training
Programs of Innovation and Entrepreneurship for Undergraduates
(201410366041).
Supplementary data
Crystallographic data (excluding structure factors) for the struc-
ture had been deposited with the Cambridge Crystallographic Data
Center as supplementary publication Nos. CCDC 1428287 (3e),
1040100 (3m) and 1040101 (3t). These data can be obtained free
of charge via the URL http://www.ccdc.cam.ac.uk (or from the
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223
336033; e-mail: deposit@ccdc.cam.ac.uk). Supplementary data
(representative 1H and 13C NMR spectra of compounds 3a–3u)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.2016.02.045.
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Please cite this article in press as: Chen, R.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.02.045