Discovery of highly selective and potent monoamine oxidase B inhibitors: Contribution of additional phenyl rings introduced into?2-aryl-1,3,4-oxadiazin-5(6H)-one

Article abstract of DOI:10.1016/j.ejmech.2017.02.059

Monoamine oxidase B (MAO-B) is a flavin adenine dinucleotide (FAD)-containing enzyme that plays a major role in the oxidative deamination of biogenic amines and neurotransmitters. Inhibiting MAO-B activity is a promising approach in the treatment of neuro

Full text of DOI:10.1016/j.ejmech.2017.02.059

Accepted Manuscript
Discovery of highly selective and potent monoamine oxidase B inhibitors: Contribution
of additional phenyl rings introduced into 2-aryl-1,3,4-oxadiazin-5(6H)-one
Jungeun Lee, Yeongcheol Lee, So Jung Park, Joohee Lee, Yeong Shik Kim, Young-
Ger Suh, Jeeyeon Lee
PII:
S0223-5234(17)30144-7
10.1016/j.ejmech.2017.02.059
EJMECH 9255
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 November 2016
Revised Date: 23 January 2017
Accepted Date: 27 February 2017
Please cite this article as: J. Lee, Y. Lee, S.J. Park, J. Lee, Y.S. Kim, Y.-G. Suh, J. Lee, Discovery of
highly selective and potent monoamine oxidase B inhibitors: Contribution of additional phenyl rings
introduced into 2-aryl-1,3,4-oxadiazin-5(6H)-one, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.02.059.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of highly selective and potent monoamine oxidase B
inhibitors: contribution of additional phenyl rings introduced
into 2-aryl-1,3,4-oxadiazin-5(6H)-one
Jungeun Lee, Yeongcheol Lee, So Jung Park, Joohee Lee, Yeong Shik Kim, Young-Ger Suh,
and Jeeyeon Lee*
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National
University, Seoul 08826, Korea
*Corresponding author.
E-mail address: jyleeut@snu.ac.kr (J. Lee).
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Abstract
Monoamine oxidase B (MAO-B) is a flavin adenine dinucleotide (FAD)-containing enzyme that
plays a major role in the oxidative deamination of biogenic amines and neurotransmitters. Inhibiting
MAO-B activity is a promising approach in the treatment of neurological disorders. Here, we report
a series of 2-aryl-1,3,4-oxadiazin-5(6H)-one derivatives as highly selective and potent MAO-B
inhibitors. Analysis of the binding sites of hMAO-A and hMAO-B led to design of linear analogs of
2-aryl-1,3,4-oxadiazin-5(6H)-one with an additional phenyl ring. Biological evaluation of the 26
new derivatives resulted in the identification of highly potent and selective inhibitors with optimal
physicochemical properties to potentially cross the blood-brain barrier (BBB). Compounds 18a, 18b,
18e and 25b potently inhibited MAO-B, with IC50 values of 4–25 nM and excellent SI over MAO-A
(18a > 25000, 18b > 8333 and 18e > 4000 and 25b > 4545). Docking results suggest that an optimal
linker between two aromatic rings on the 2-aryl-1,3,4-oxadiazin-5(6H)-one scaffold is a key element
in the binding and inhibition of MAO-B.
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1. Introduction
Monoamine oxidase (MAO), a flavin adenine dinucleotide (FAD)-containing enzyme, catalyzes the
oxidative deamination of a range of biogenic amines and monoamine neurotransmitters. Two
isoforms of MAO (MAO-A and MAO-B) are present in most mammalian tissues, with the two
distinguished by their tissue distribution and substrate/inhibitor preference [1-3]. Serotonin (5-HT)
and noradrenaline (NE) are the preferred substrates of MAO-A, whereas phenethylamine (PEA) and
benzylamine exhibit higher affinity toward MAO-B. Dopamine (DA) and tyramine are oxidized by
both isoforms [3-5]. Most mammalian peripheral tissues express both isoforms, but the proportions
of MAO vary among tissues. Human placenta expresses MAO-A, whereas platelets and brain have
high levels of MAO-B [6-8].
The ability of MAO to control the levels of several neurotransmitters, mainly in the central nervous
system (CNS), attracted the attention of many researchers, leading to the discovery of new drugs
targeting brain disorders [9-17]. MAO-A inhibitors are used in the treatment of depression [18, 19],
whereas selective MAO-B inhibitors, combined with levodopa, are used to treat early Parkinson’s
disease (PD), reducing the metabolic degradation of DA [20-23]. MAO-B activity in human brain
increases with age [24, 25], because of the predominant expression of MAO-B in glial cells [26].
Controlling the level of MAO-B activity may influence susceptibility to neurodegenerative diseases,
including Alzheimer’s disease (AD) and PD [27, 28]. The increased level of DA oxidation by MAO-
B in the elderly is related to the loss of dopaminergic neurons in the substantia nigra and has been
observed in patients with PD [29].
The earliest non-selective and irreversible inhibitors of MAOs showed severe side effects, including
tyramine-induced hypertensive crisis (Cheese reaction) [30, 31]. Therefore, research on additional
MAO inhibitors has focused on their selectivity for the two isoforms, MAO-A and MAO-B [32-42].
Clorgyline (1) in Fig. 1 is a potent and selective inhibitor of MAO-A [43-45], whereas selegiline (2)
and rasagiline (3) are selective irreversible inhibitors of MAO-B and are FDA-approved drugs for
PD [46-49]. Safinamide (4) is a selective and reversible inhibitor of MAO-B recently approved by
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the European Commission as an add-on therapy to levodopa or in combination with other
medications to treat patients with PD [22, 50]. Compounds 5–7 are previously reported inhibitors of
MAO-B, with IC50 values in the low nanomolar range [35, 38, 39].
NH2
Cl
N
HN
H
O
N
N
F
O
O
Cl
2
3
4
1
Selegiline (irreversible)
Clorgyline (irreversible)
Rasagiline (irreversible)
Safinamide (reversible)
h MAO-B IC50 98 nM
h MAO-B IC50 14 nM
h MAO-A IC50 710 nM
h MAO-B IC50 260 nM
h MAO-A IC50 0.42 nM
h MAO-B IC50 7 nM
h MAO-A IC50 580000 nM
h MAO-A IC50 1700 nM
H
N
Cl
Cl
O
O
H
N
N
H
Cl
O
O
O
O
O
Cl
HN
N
6
5
7
rat MAO-B IC50 13 nM
rat MAO-A IC50 5940 nM
h MAO-B IC50 8.0 nM
h MAO-B IC50 0.59 nM
h MAO-A IC50 > 10000 nM
h MAO-A IC50 > 10000 nM
Fig. 1. Structures of MAO inhibitors and potencies of previously reported MAO-B inhibitors.
Our research group has explored nitrogen-containing heterocycle scaffolds, including 1,3,4-
oxadiazin-5(6H)-one scaffolds, to extend their activity profiles [51]. Although analogs of 1,3,4-
oxadiazin-5(6H)-one have been previously shown to act as MAO inhibitors [52-55], selective
inhibitors of human MAO-B have not yet been explored. Here, we describe a new class of MAO-B
inhibitors, constructed using a 2-aryl-4H-1,3,4-oxadiazin-5(6H)-one scaffold, with enhanced
potency and selectivity. Based on the structural features of MAO-A and MAO-B, we envisioned that
selectivity toward MAO-B could be achieved by introducing another phenyl ring and a linker unit
connecting two phenyl rings (Fig. 2). In this study, we describe the design and synthesis of a new
series of 2-aryl-4H-1,3,4-oxadiazin-5(6H)-one compounds with various linkers, along with their
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ability to inhibit the activity of human MAO enzymes. In addition, we describe molecular docking
studies to predict the mode of binding and interactions of selected compounds with MAO-B.
Fig. 2. Design strategy to obtain potent and selective inhibitors of MAO-B.
2. Results and discussion
2.1. Design and synthesis
To develop MAO-B selective inhibitors, we started by comparing the binding sites of hMAO-A
(PDB code 2BXR) [56] and hMAO-B (PDB code 2V5Z) [57], based on their reported protein
structures (Fig. 3). Although the overall structures of the two isoenzymes (MAO-A and MAO-B) are
similar, there are major differences in their active sites, which have different shapes and sizes [56].
The active site of hMAO-B consists of two cavities, an entrance cavity with a volume of 290 Å3 and
a larger substrate cavity with a volume of 420 Å3, resulting in a longer and narrower cavity than that
of hMAO-A. By contrast, hMAO-A has a single substrate cavity of ~550 Å3, which is less flat and
less elongated than the substrate cavity of hMAO-B [9, 56-58]. In designing selective MAO-B
inhibitors, we initially hypothesized that an additional phenyl ring on the 2-aryl-1,3,4-oxadiazin-
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5(6H)-one scaffold may contribute to selectivity for hMAO-B, owing to its longer and narrower
active site with two cavities, and may increase the potency of inhibitors by occupying both cavities
of MAO-B (Fig. 2).
Fig. 3. Active site of human MAO-A (A, PDB: 2BXR, co-crystallized with clorgyline) and MAO-B
(B, PDB: 2V5Z, co-crystallized with safinamide).
Most of compounds in the present study were synthesized as described in Schemes 1–3, with the
synthetic routes of 31, 39, 40 and 25f presented in the Supporting information. In general, the 1,3,4-
oxadiazine-5(6H)-one ring was synthesized by amide coupling of hydrazide with bromoacetic acid,
followed by a cyclization reaction in the presence of N,N-diisopropylethylamine (DIEA) (Scheme
1). The key intermediate 12 was obtained by nitro reduction of 10 or 11 in the presence of stannous
chloride as a reducing agent. After obtaining the common intermediate, a variety of linker units were
introduced between the two benzene rings to investigate the impact of the linker on inhibitory
activity (Scheme 2). These linker units included amide (14), sulfonamide (15), urea (16), alkyl
amine (17), azo (18), imine (19), olefin (25), inverse amide (31), inverse imine (39) and hydrazine
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(40) linkers. The final products 14–19 were prepared by reacting 12a with their corresponding
reagents. The intermediate 12a or 13 was reacted with differently substituted reagents at the R2
position to yield 18a–e and 19a–e. Nitrosobenzene 13 was obtained by oxidation of aniline 12a with
hydrogen peroxide, catalyzed by molybdenum oxide (MoO3), with 13 subsequently reacted with 3-
fluoroanline or m-toluidine to generate 18b or 18e with azo linkers. The derivatives of an olefin
linker were prepared as depicted in Scheme 3. Compounds 22a–e were prepared by Horner–
Wadsworth–Emmons reaction of aldehyde reagent 21 with commercially available phosphonates.
Reacting 22 with excess hydrazine hydrate in ethanol yielded compound 23. The final compounds
25a–e were synthesized by the general procedure described in Scheme 1 for the synthesis of the
1,3,4-oxadiazine-5(6H)-one scaffold. Reaction conditions and compound characterization are
described in the Experimental section. In NMR spectra, the imine peak for 19c was shown at 8.67
ppm, whereas trans olefin of stilbene moiety of 25d appeared at 7.36 and 7.31 ppm with the J value
of 16.4 Hz (Supporting). For 18a with the diazo linker, protons adjacent to the azo group were
merged to 8.00-7.91 ppm shown as multiplets
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Scheme 1. Reagents and conditions: a) Bromoacetic acid, EDCI, DMF, rt, 1 h; b) DIEA, DMF, 60
oC, 12 h, yield 68% in 2 steps; c) Methyl iodide, NaH, DMF, rt, 1.5 h, yield 67%; d) SnCl2·2H2O,
DMF, 70 oC, 1 h, yields 80% (12a) and 90% (12b).
Scheme 2. Reagents and conditions: a-1) 3-Chlorobenzoyl chloride, Et3N, DMF, rt, 1 h (for
compound 14), yield 35% in 2 steps; a-2) 3-Chlorobenzenesulfonyl chloride, pyridine, 0 oC, 1 h (for
compound 15), yield 60%; a-3) 3-Chlorophenyl isocyanate, DCM/DMF, rt, 12 h (for compound 16),
yield 53%; a-4) 3-Chlorobenzaldehyde, NaCNBH4, MeOH, 1% acetic acid, rt, 2 h (for compound
17), yield 75%; a-5) 3- or 4- Substituted nitrosobenzene, acetic acid, rt, 12 h (for compound 18),
yield 78–86%; a-6) 3- or 4- Substituted benzaldehyde, EtOH, reflux, 12 h (for compound 19), yield
45–80%; b) 3-Bromopropionitrile (for compound 20a) or 2-chloroacetamide (for compound 20b),
NaH, DMF, rt, 1 h, yield 95% (20a) and 72% (20b); c) MoO3, H2O2, H2O, MeOH, rt, 5 d, yield
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85%; d) 3-Fluoroaniline (for compound 18b) or m-toluidine (for compound 18e), acetic acid, rt, 12
h, yield 69% (18b) and 31% (18e).
Scheme 3. Reagents and conditions: a) 3- or 4- Substituted diethyl benzylphophonate, NaH, THF, rt,
1 h, yield 50–85% ; b) Hydrazine monohydrate, EtOH, reflux, 12 h, yield 44–77%; c) Bromoacetic
acid, EDCI, DMF, rt, 1 h; d) DIEA, DMF, 70 oC, 1 d, yield 24–26% in 2 steps.
2.2. In vitro inhibition of MAO
All the synthesized analogs of 1,3,4-oxadiazin-5(6H)-one were tested for inhibition of human MAO-
A and MAO-B activity. The corresponding IC50 values and selectivity indices of these compounds
relative to MAO-B are shown in Tables 1–3. The ability of these compounds to inhibit the enzymatic
activities of hMAO-A and hMAO-B was tested by measuring the production of hydrogen peroxide
(H2O2) from p-tyramine using a fluorescence-based assay kit (see Experimental section) [59]. The
biological evaluation of 26 new derivatives resulted in the identification of highly potent and
selective inhibitors of MAO-B.
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First, we examined 10 derivatives that included various linkers between two phenyl rings, including
amide (14), sulfonamide (15), urea (16), alkyl amine (17), azo (18c), imine (19c), olefin (25c),
inverse amide (31), inverse imine (39) and hydrazine (40) linkers (see Table 1). In general, linkers
containing a π bond between two atoms (Ar–X = X–Ar, X = C or N, 18c, 19c, 25c and 39) showed
good inhibitory activities against MAO-B, with IC50 values of 287 nM, 257 nM, 173 nM, and 551
nM, respectively. Introduction of one more atom between the two phenyl groups (16 and 40) or
bulky linker units such as sulfonamide (15) and urea (16) reduced the inhibitory activity against
MAO-B compared with the smaller linkers. Introduction of a flexible linker (17, IC50 = 2690 nM)
dramatically reduced the inhibitory effect against MAO-B when compared with rigid linkers (18c,
19c and 25c). These results indicate that proper linker units with optimal distance between two
aromatic units are essential for inhibiting MAO-B. Introduction of an alkyl substituent at the R1
position of the 1,3,4-oxadiazin-5(6H)-one ring significantly reduced inhibitory activities when
compared with unsubstituted compounds (20a, 20b vs 14; 19f vs 19c; 25f vs 25c).
Table 1
In vitro activities on hMAO of 1,3,4-oxadiazinone derivatives: Variation of linker or R1 position.
O
Cl
O
N
N
R1
L
L=linker
hMAO-B
(IC50, nM)a
hMAO-A
(IC50, nM)a
Compd. Linker
R1
SIb
H
H
H
284 ± 14
>100000
>100000
>100000
> 357
nd
14
15
16
>10000
2891 ± 234
> 34
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H
2690 ± 144
287 ± 200
257 ± 49
>100000
>100000
>100000
>100000
>100000
>100000
>10000
>37
17
H
> 348
> 385
> 578
> 344
> 182
>18
18c
19c
25c
31
H
H
173 ± 17
H
288 ± 21
H
551 ± 133
554 ± 45
39
H
40
CH2CH2CN
CH2CONH2
CH3
4228 ± 199
3692 ± 538
2033 ± 420
>10000
>100000
>100000
>100000
>100000
> 24
> 27
> 49
nd
20a
20b
19f
25f
CH2CH3
aEach IC50 value is the mean ± standard error of the mean (SEM) of more than three independent
experiments. bSelectivity index. It was calculated as IC50(hMAO-A)/ IC50(hMAO-B).
Notably, all of the compounds listed in Table 1 exhibited higher inhibitory activities against MAO-B
than MAO-A. Most compounds did not show any inhibitory effects on MAO-A at 100 µM
concentrations, except for 40. Based on the first structure-activity relationship (SAR) analysis, we
selected three compounds with relatively small linkers having a π bond with good inhibitory
activities against MAO-B (18c, IC50 = 287 nM; 19c, IC50 = 257 nM; 25c, IC50 = 173 nM) for further
optimization.
To further explore the effect of substitutions at the R2 position of the first phenyl ring, we prepared
R2 substituted analogs with azo, olefin and imine linkers (Tables 2 and 3). We compared the effects
of 4-Cl and 3-Cl substituted series on MAO-B activity to evaluate the contribution of meta or para
substitution (Table 2). Because the compounds with 3-Cl substitution (18c, 25c and 19c) at the R2
position had slightly more potent inhibitory activities against MAO-B than those with 4-Cl
substitution (18d, 25d and 19d), we fixed the substituent at the meta position of the phenyl ring.
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Table 2
In vitro activities on hMAO of 1,3,4-oxadiazinone derivatives: meta or para substitution at R2
position.
hMAO-B
(IC50, nM)a
hMAO-A
(IC50, nM)a
SIb
Compd.
R2
X1=X2
N=N
C=C
C=N
N=N
C=C
C=N
287 ± 200
173 ± 17
257 ± 49
392 ± 47
542 ± 111
433 ± 35
>100000
>100000
>100000
>100000
>100000
>100000
> 348
> 578
> 385
> 255
> 185
> 231
18c
25c
19c
18d
25d
19d
3-Cl
4-Cl
aEach IC50 value is the mean ± SEM of more than three independent experiments. bSelectivity index.
It was calculated as IC50(hMAO-A)/ IC50(hMAO-B).
Interestingly, H, 3-F and 3-CH3 analogs significantly differed in inhibitory activities against MAO-B
among the three linkers, azo, olefin and imine (Table 3). In general, the potency of 3-H, 3-F and 3-
CH3 derivatives increased in the order of imine < olefin < azo. Compounds with azo linkers (18a,
18b and 18e) showed greater inhibition of MAO-B than did compounds with olefin or imine linkers.
In addition, compounds with a small (F) or no substituent at the meta position (18a, 18b and 25b)
were more potent inhibitors in the presence of azo or olefin linkers, with IC50 values of 4, 12, and 22
nM, respectively. These results indicate that the steric effect of the R2 group in the presence of an
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azo or olefin linker plays a key role in the modulation of MAO-B inhibitory activity. Interestingly,
the azo derivative with 3-CH3 (18e) was 10-fold more active than the derivative with 3-Cl (18c). The
hydrophobic property of 3-CH3 in the azo series may be responsible for its increased potency
because of hydrophobic environments of the active site of human MAO-B [57]. None of the
compounds in Table 3 inhibited MAO-A at 100 µM concentrations. Compounds with azo linkers
and 3-H, 3-F and 3-CH3 substituents at the R2 position (18a, 18b and 18e) and an olefin derivative
with a 3-F substituent at R2 (25b) showed increased potency and selectivity, with IC50 values of 4–25
nM and selectivity indices (SI) greater than 4000. Compared with the current MAO-B inhibitors
(selegiline, IC50 = 7 nM; rasagiline, IC50 = 14 nM; safinamide, IC50 = 98 nM) [47, 50], these
compounds have similar or more potent activities, along with excellent SIs.
To determine the binding mode of studied compounds, a time-dependency of enzyme inhibition was
measured. If the compounds form covalent adduct with the enzyme, a time-dependent reduction of
enzyme activity would be expected. 18a, 25b, 19c, and 19e were preincubated with MAO-B for 0,
15, 30, and 60 mins prior to starting the enzyme reaction. As shown in Figure 4, enzyme activity was
not reduced with increased preincubation time, indicating that selected compounds were reversible
inhibitors of MAO-B. Based on the Lineweaver-Burk plots (Figure S1), the selected compounds are
likely to be competitive MAO-B inhibitors.
Table 3
In vitro activities on hMAO of 1,3,4-oxadiazinone derivatives: R2 variation with selected linkers.
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hMAO-B
(IC50, nM)a
hMAO-A
(IC50, nM)a
Compd.
X1=X2
N=N
R2
SIb
H
4 ± 3
>100000
> 25000
> 8333
> 4000
> 606
> 4545
> 296
ndc
18a
18b
18e
25a
25b
25e
19a
19b
19e
3-F
12 ± 10
25 ± 5
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
3-CH3
H
165 ± 95
22 ± 1
C=C
C=N
3-F
3-CH3
H
338 ± 47
>10000
1526 ± 91
1270 ± 229
3-F
> 65
3-CH3
> 78
Clorgylined (1)
61350e
4e
Selegiline (2)
Rasagiline (3)
7f
1700f
710f
242
50
14f
6 ± 3
98g
Safinamided (4)
580000g
5918
b
aEach IC50 value is the mean ± SEM of three independent experiments. Selectivity index. It was
c
d
calculated as IC50(hMAO-A)/ IC50(hMAO-B). Not determined. Positive controls; safinamide:
e
reversible MAO-B inhibitor, clorgyline: irreversible MAO-A inhibitor. Values reported in ref 44.
fValues reported in ref 47. gValues reported in ref 50.
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18000
16000
14000
12000
10000
8000
6000
4000
2000
0
0 min.
15 min.
30 min.
60 min.
Compounds
Fig. 4. Time dependency of MAO-B inhibition in the presence of 18a, 25b, 19c, and 19e. Data are
expressed as the mean ± SEM of three independent experiments
2.3. Molecular docking
.
To understand the possible binding mode of the synthesized compounds, we performed flexible
docking simulations of azo linker compounds (18a and 18b) and imine derivatives (19a and 19b),
using the X-ray cocrystal structure of human MAO-B with safinamide (4, PDB code 2V5Z, see
Experimental section) [57]. All the tested ligands (18a, 18b, 19a and 19b) occupied both the
substrate and entrance cavities in the active site of MAO-B. The 4H-1,3,4-oxadiazin-5(6H)-one ring,
the relatively polar part of these molecules, was oriented in the substrate cavity toward FAD,
whereas the two phenyl rings were directed towards the hydrophobic pocket in the entrance cavity.
Potential interactions with the gate residues (Leu171 and Ile199) may have resulted in compounds
residing in both cavities [9]. Docking results in Fig. 5 showed that compounds containing an azo
group (18a and 18b) appeared to properly occupy the hydrophobic entrance pocket by interacting
with Leu164, Leu171, Ile199, Ile316 and Tyr326. In particular, Tyr326, a critical residue in the
recognition of inhibitors [60-62], exhibits a π-π interaction with the phenyl group in the middle. The
compounds 18a and 18b were also stabilized by hydrogen bonding interactions between the N-H of
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the 4H-1,3,4-oxadiazin-5(6H)-one ring and the carbonyl group of Cys172 (Fig. 5A and D). In
addition, the π-sulfur interaction with Cys172 appears to play an important role. In contrast,
compounds with imine linkers (19a and 19b) do not exhibit hydrogen-bonding interactions with
Cys172, shifting them towards the FAD binding side (Fig. 5B and E). Consequently, these
compounds may not well fit into the buried hydrophobic pocket in the entrance cavity. These
docking results suggest that optimizing the linker between the two aromatic rings is a key element in
the binding to and inhibition of MAO-B.
Fig. 5. Predicted binding mode of 18a (A), 19a (B), 18b (D) and 19b (E) in the human MAO-B
(PDB code: 2V5Z) active site. (C) Overlaid structure of 18a (magenta) and 19a (green). (D)
Overlaid structure of 18b (magenta) and 19b (green). Only the relevant residue side chains are
shown.
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2.4. Physicochemical properties
To understand the overall properties of the described derivatives, we calculated several
physicochemical parameters, including lipophilicity (ClogP) and topological polar surface area
(tPSA). The ClogP value of all compounds was lower than 5 and the molecular weight of all
compounds was lower than 500, in agreement with Lipinski’s rule-of-5 for drug-likeness [63]. The
tPSA value of all analogs were within the OK (140 > PSA > 61 Å2) or good range (PSA ≤ 61 Å2) for
intestinal absorption [64]. In particular, compounds with an olefin linker (black dot) exhibited
optimal tPSA value for good intestinal absorption (PSA ≤ 61 Å2) [64]. To further evaluate their
ability to cross the blood-brain barrier (BBB), we selected four inhibitors (18a, 18b, 18e and 25b)
with high potency toward MAO-B, as shown by IC50 values < 100 nM (Table 4). Properties used to
evaluate the ability of compounds to cross the BBB included ClogP, MW, tPSA and numbers of
hydrogen bond donors (HBD). The physicochemical properties of selected compounds (18a, 18b,
18e and 25b) were within the suggested limits (clogP = 2–5, PSA < 90 Å2, MW < 500, HSD < 3)
and compound 25b were in the preferred range for BBB penetration (clogP = 2–4, PSA < 70 Å2,
MW < 450, HSD = 0–1) (Fig. 6.) [65]. Their tPSA values ranged between 50.4 and 73.0 and their
MW was lower than that of the reference drug, safinamide. The tPSA values of the compounds in
Fig. 6 were mainly determined by the linkers of these molecules. These results indicate that the
linker moiety of the structure may be a critical element affecting the physicochemical properties as
well as the potency of MAO-B inhibitors.
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6
5
4
3
2
1
Azo linker
Olefin linker
Imine linker
Safinamide
Æ
18e
18b
18a
25b
Æ
25
50
75
100
125
40
61
70
90
140
PSA_2D
Fig. 6. Calculated physicochemical properties of the all tested compounds. Preferred range for
crossing BBB is shown in gray color. Suggested limits for crossing BBB are shown in the dashed
box.
Table 4
Calculated physicochemical properties of selected compounds and reference molecule
hMAO-B inhibition
(IC50, nM)
Molecular
weight
H-bond
donors
Compd.
ClogP
tPSA
4
280.30
298.28
294.31
18
3.17
3.38
3.66
73.0
73.0
73.0
1
1
1
18a
18b
18e
12
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22
6
296.30
302.34
2.99
2.48
50.4
65.6
1
2
25b
Safinamide
2.5. Cytotoxicity assay
Cytotoxicity of 18a, 25b, 19c, and 19e toward PC-12, PC-12 Adh, and SH-SY5Y cells were
measured at 10 and 50 µM using the WST-1 assay. PC-12 cells which are neuroblastoma cells were
used because they are similar to dopaminergic neurons. Cell viability was not significantly reduced
with the tested compounds except with 25b. More than 90% of cells were viable after treatment of
18a, 25b, 19c, and 19e, indicating low toxicity of studied compounds at higher concentration (50
µM). We also tested neuroprotective effect of the selected compounds (Figure S2). 5 µM rotenone or
150 µM H2O2 were treated to induce neurotoxicity, but no significant neuroprotective effect was
observed. Further optimization for screening condition along with additional synthesis of derivatives
based on oxadiazin-5(6H)-one scaffold will be pursued soon to discover neuroprotective agents.
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10 µM
50 µM
120
100
80
60
40
20
0
A
Compounds
120
100
80
60
40
20
0
B
10 µM
50 µM
Compounds
120
100
80
60
40
20
0
C
10 µM
50 µM
Compounds
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Fig. 7. Cytotoxicity of the representative compounds against PC-12 (A), PC-12 Adh (B), and SH-
SY5Y (C) cells. Data are expressed as a percentage of control (± S.E.M, n = 3 in triplicate).
Untreated cells were used as control.
3. Conclusion
We have identified new derivatives of 2-aryl-4H-1,3,4-oxadiazin-5(6H)-one as selective MAO-B
inhibitors. Structural modification and analysis of SAR led to the identification of inhibitors with
low nanomolar potency and excellent SI. The compounds with 3-H, 3-F and CH3 substituents at the
R2 in azo compounds (18a, 18b and 18e) and a 3-F substituent at R2 in an olefin derivative (25b)
potently inhibited MAO-B with IC50 values of 4–25 nM and excellent SIs (18a > 25000, 18b > 8333
and 18e > 4000 and 25b > 4545). Molecular docking with the compounds containing azo (18a and
18b) and imine (19a and 19b) linkers provided insights into the main interactions and the
importance of linker units for enzyme-inhibitor binding. Furthermore, the molecules with high
potency, as shown by lower IC50 values, had optimal calculated physicochemical properties allowing
oral administration and BBB penetration. Notably, the tPSA values of 4H-1,3,4-oxadiazin-5(6H)-
one derivatives were mainly affected by the linkers. Together with the SAR findings, the docking
simulations and physicochemical properties suggested that the linker modification of the 2-aryl-4H-
1,3,4-oxadiazin-5(6H)-one scaffold is a major strategy to optimize the potency and properties of a
new class of MAO-B inhibitors.
4. Experimental procedures
4.1. Chemistry
Starting materials, reagents and solvents were purchased from Alfa aesar (Ward Hill, MA, USA),
TCI (Tokyo, Japan) and Sigma-Aldrich (St. Louis, MO, USA). Melting points were determined on
1
B-540 melting point analyzer (Buchi, Postfach, Flawil, Switzerland). H NMR spectra (300, 400
MHz) and 13C NMR (75, 100, 125, 200 MHz) were recorded on GEMINI 2000 (VARIAN, Palo
21

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Alto, CA, USA), JNM-LA300 (JEOL, Tokyo, Japan) or AVANCE 400 (Bruker, Billerica, MA,
USA). Chemical shifts were expressed in ppm (δ) and were referenced to the residual solvent peak.
Analytical thin layer chromatography (TLC) was carried out using precoated silica gel 60F254 (layer
thickness, 0.25 mm; Merck, Darmstadt, Germany), and chromatography was performed using
ZEOprep 60 silica gel (40–63 µm; Zeochem, Lake Zurich, Switzerland). Mass spectra were recorded
on a 6130 Single Quadrupole LC/MS (Agilent Technologies, Santa Clara, CA, USA) and High-
resolution mass spectra (HRMS) were acquired under fast atom bombardments (FAB) condition on a
JMS-700 MStation (JEOL, Tokyo, Japan). Preparative HPLC was performed on an YL9100
reversed-phase HPLC (Younglin, Anyang, South Korea).
4.1.1. Synthesis of the intermediates (12a–b)
4.1.1.1. 2-(4-Aminophenyl)-4H-1,3,4-oxadiazin-5(6H)-one (12a)
A solution of the hydrazide 8 (2.0 g, 11.04 mmol), bromoacetic acid (1.7 g, 12.14 mmol) and EDCI
(3.2 g, 16.56 mmol) in DMF (28 mL) was stirred at room temperature for 1 h. The resulting mixture
was diluted with EtOAc, washed with H2O, and dried over Na2SO4. The crude solid was used for the
next reaction without purification. DIEA (9.6 mL, 55.20 mmol) was added to a solution of the crude
product 9 in DMF and the solution was stirred overnight at 70 ºC. After cooling, the residue was
taken up in H2O and extracted with EtOAc. The combined extracts were dried over Na2SO4 and
concentrated. The crude product was sonicated with MeOH for 5 minutes. The resulting precipitate
was filtered to afford the corresponding 1,3,4-oxadiazin-5(6H)-one 10 (1.7 g, 68% in 2 steps). To the
nitro compound 10 (0.8 g, 3.62 mmol) in DMF (9 mL) was added SnCl2·2H2O (3.3 g, 14.47 mmol).
After stirring at 70 ºC for 1 h, the reaction mixture was cooled down to room temperature and
diluted with EtOAc, washed with saturated NaHCO3 solution, and dried over Na2SO4. Intermediate
1
12a was obtained by filtration with MeOH. Light brown solid (0.55 g, 80%). H NMR (300 MHz,
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DMSO-d6) δ 10.78 (s, 1H), 7.42 (d, J = 8.6 Hz, 2H), 6.53 (d, J = 8.6 Hz, 2H), 5.66 (s, 2H), 4.64 (s,
2H).
4.1.1.2. 2-(4-Aminophenyl)-4-methyl-4H-1,3,4-oxadiazin-5(6H)-one (12b)
To a solution of NaH (4 mg, 0.09 mmol) in DMF (0.4 mL) was added compound 10 (20 mg, 0.09
mmol) in DMF (0.4 mL) at 0 ºC and then the mixture was stirred at 0 ºC for 0.5 h. After adding
methyl iodide (6 µL, 0.68 mmol) in DMF (0.2 mL), the mixture was stirred at room temperature for
1 h and was then quenched with H2O. The reaction mixture was extracted with EtOAc, and dried
over Na2SO4. The crude product was sonicated with DCM/Hexane to provide 11 (14 mg, 67%). To
the nitro compound 11 (14 mg, 0.06 mmol) was added DMF (0.4 mL) and SnCl2·2H2O (4 mg, 0.09
mmol). After 1 h of stirring at 70 ºC, the reaction mixture was cooled down to room temperature and
diluted with saturated NaHCO3 solution. The product was extracted with EtOAc, washed with brine,
and dried over Na2SO4. Purification by silica gel column chromatography (DCM/MeOH = 80/1, Rf =
0.2) afforded 12b as a light yellow solid (11 mg, 90%). 1H NMR (300 MHz, DMSO-d6) δ 7.45 (d, J
= 8.4 Hz, 2H), 6.54 (d, J = 8.6 Hz, 2H), 5.72 (s, 2H), 4.69 (s, 2H), 3.20 (s, 3H).
4.1.2. Procedure for synthesis of 14–17
4.1.2.1. 3-Chloro-N-[4-(5-oxo-5,6-dihydro-4H-1,3,4-oxadiazin-2-yl)phenyl]benzamide (14)
A solution of the 3-chlorobenzoic acid (100 mg, 0.64 mmol) in THF (3.2 mL) was treated at room
temperature with few drops of DMF followed by oxalyl chloride (60 µL, 0.71 mmol). The reaction
mixture was stirred at 0 ºC for 1 h and the volatiles were removed under reduced pressure. The crude
mixture was dissolved in DMF (1.2 mL), and 12a (50 mg, 0.26 mmol) was added followed by Et3N
(70 µL, 0.52 mmol). The mixture was stirred at room temperature for 0.5 h, diluted with H2O,
extracted with EtOAc, and dried over Na2SO4. After adding MeOH, the precipitate was filtered and
washed to afford the amide compound 14 as a light yellow solid (30 mg, 35% in 2 steps). Mp 265–
266 °C; 1H NMR (300 MHz, DMSO-d6) δ 11.02 (s, 1H), 10.55 (s, 1H), 8.00 (s, 1H), 7.91 (d, J = 7.7
23

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Hz, 1H), 7.87 (d, J = 9.0 Hz, 2H), 7.76 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 8.6 Hz, 1H), 7.57 (t, J = 7.9
Hz, 1H), 4.76 (s, 2H); 13C NMR (75 MHz, DMSO-d6) δ 164.24, 161.09, 147.02, 140.96, 136.62,
133.19, 131.52, 130.39, 127.42, 126.60 (2C), 126.51, 124.98, 119.85 (2C), 64.54; LCMS (ESI) m/z
330 [M + H]+; HRMS (FAB) m/z calcd for C16H13ClN3O3 [M + H]+ 330.0645, found 330.0651.
4.1.2.2.
3-Chloro-N-(4-(5-oxo-5,6-dihydro-4H-1,3,4-oxadiazin-2-yl)phenyl)benzenesulfonamide
(15)
To a solution of 12a (20 mg, 0.11 mmol) in pyridine (0.5 mL) at 0 ºC was added 3-
chlorobenzenesulfonyl chloride (15 µL, 0.11 mmol). The reaction mixture was stirred at 0 ºC for 1 h,
acidified with 1 N HCl solution. The product was extracted with EtOAc, washed with brine, and
dried over Na2SO4. After adding DCM, the precipitate was filtered to afford the sulfonamide
1
compound 15 as a light pink solid (22 mg, 60%). Mp 276 °C; H NMR (300 MHz, DMSO-d6) δ
10.99 (s, 1H), 10.75 (s, 1H), 7.78 (s, 1H), 7.71 (d, J = 8.10 Hz, 1H), 7.64 (d, J = 8.8 Hz, 2H), 7.61–
7.56 (m, 2H), 7.17 (d, J = 8.8 Hz, 2H), 4.70 (s, 2H); 13C NMR (75 MHz, DMSO-d6) δ 161.04,
146.67, 141.00, 139.30, 133.87, 133.13, 131.44, 127.22 (2C), 126.14, 125.54, 125.35, 119.35 (2C),
64.52; LCMS (ESI) m/z 366 [M + H]+; HRMS (FAB) m/z calcd for C15H13ClN3O4S [M + H] +
366.0315, found 366.0324.
4.1.2.3. 1-(3-Chlorophenyl)-3-(4-(5-oxo-5,6-dihydro-4H-1,3,4-oxadiazin-2-yl)phenyl)urea (16)
The compound 12a (20 mg, 0.11 mmol) was suspended in DCM/DMF (10/1, 1 mL) and 3-
chlorophenyl isocyanate (20 µL, 0.16 mmol) was added. The mixture was stirred at room
temperature for 5 h. The volatiles were removed under reduced pressure. MeOH was added and the
1
solid was filtered to yield desired product as yellow solid (29 mg, 53%). Mp 308–309 °C; H NMR
(400 MHz, DMSO-d6) δ 10.96 (s, 1H), 9.00 (s, 1H), 8.94 (s, 1H), 7.71–7.69 (m, 3H), 7.54 (d, J = 8.8
Hz, 2H), 7.33–7.28 (m, 2H), 7.02 (d, J = 7.0 Hz, 1H), 4.74 (s, 2H); 13C NMR (100 MHz, DMSO-d6)
24

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δ 161.16, 152.13, 147.30, 141.67, 140.99, 133.20, 130.39, 126.91 (2C), 123.18, 121.68, 117.77 (2C),
117.71, 116.78, 64.54; LCMS (ESI) m/z 345 [M + H]+; HRMS (FAB) m/z calcd for C16H14ClN4O3
[M + H] + 345.0754, found 345.0756.
4.1.2.4. 2-(4-((3-Chlorobenzyl)amino)phenyl)-4H-1,3,4-oxadiazin-5(6H)-one (17)
To a solution of 12a (20 mg, 0.11 mmol) in 1 mL of MeOH was added 0.1 mL of acetic acid,
followed by 3-chlorobenzaldehyde (20 µL, 0.16 mmol) and NaCNBH3 (13 mg, 0.21 mmol). The
reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with
NaHCO3 in H2O, extracted with EtOAc, and dried over Na2SO4. After adding DCM, the residue was
filtered to afford 17 as yellow solid (25 mg, 75%). Mp 218–219 °C; 1H NMR (300 MHz, DMSO-d6)
δ 10.79 (s, 1H), 7.46 (d, J = 8.8 Hz, 2H), 7.37 (s, 1H), 7.35–7.26 (m, 3H), 6.89 (t, J = 6.2 Hz, 1H),
13
6.58 (d, J = 8.8 Hz, 2H), 4.63 (s, 2H), 4.33 (d, J = 6.2 Hz, 2H); C NMR (75 MHz, DMSO-d6) δ
161.30, 150.38, 148.36, 142.50, 133.05, 130.21, 127.44 (2C), 126.80, 126.68, 125.77, 116.85,
111.67 (2C), 64.44, 45.31; LCMS (ESI) m/z 316 [M + H]+; HRMS (FAB) m/z calcd for
C16H15ClN3O2 [M + H] + 316.0853, found 316.0854.
4.1.3. General procedure for the synthesis of diazo linker 18
To a solution of 12a (300 mg, 1.57 mmol) in MeOH (0.3 mL) were added H2O2 (0.8 mL), H2O (0.6
mL) and MoO3 (23 mg, 0.16 mmol). The resulting solution was stirred at room temperature for 5 d.
H2O was added, and the precipitate was filtered to yield desired product 13 as a yellow solid (270
mg, yield 85%). To a solution of 12a or 3-substituted aniline (1 equiv.) in glacial acetic acid (0.4
mL) was added 3- or 4- substituted nitrosobenzene (1 equiv.) or 13 and the resulting mixture was
stirred overnight at room temperature. The crude mixture was diluted with EtOAc, washed with
saturated NaHCO3 solution, and dried over Na2SO4. The precipitate in MeOH was filtered to afford
diazo compounds 18a–e.
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4.1.3.1. (E)-2-(4-(Phenyldiazenyl)phenyl)-4H-1,3,4-oxadiazin-5(6H)-one (18a)
1
Light brown solid (yield 78%). Mp 257–259 °C; H NMR (400 MHz, DMSO-d6) δ 11.18 (s, 1H),
8.00–7.91 (m, 6H), 7.61 (m, 3H), 4.84 (s, 2H); 13C NMR (200 MHz, DMSO-d6) δ 161.08, 152.72,
151.93, 146.38, 132.28, 131.94, 129.54 (2C), 127.10 (2C), 122.76 (2C), 122.70 (2C), 64.68; LCMS
(ESI) m/z 281 [M + H]+; HRMS (FAB) m/z calcd for C15H12N4O2 [M + H]+ 281.1039, found
281.1045.
4.1.3.2. (E)-2-(4-((3-Fluorophenyl)diazenyl)phenyl)-4H-1,3,4-oxadiazin-5(6H)-one (18b)
1
Yellow solid (yield 69%). Mp 257–258 °C; H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 8.02–
8.00 (m, 4H), 7.82 (d, J = 8.0 Hz, 1H), 7.71–7.66 (m, 2H), 7.46 (td, J = 8.8, 2.0 Hz, 1H), 4.84 (s,
2H); 13C NMR (200 MHz, DMSO-d6) δ 162.66 (d, J = 243.2 Hz), 161.06, 153.42 (d, J = 6.4 Hz),
152.43, 146.28, 132.73, 131.34 (d, J = 7.8 Hz), 127.12 (2C), 122.98 (2C), 120.56, 118.55 (d, J =
22.0 Hz), 107.73 (d, J = 22.2 Hz), 64.68; LCMS (ESI) m/z 299 [M + H]+; HRMS (FAB) m/z calcd
for C15H11FN4O2 [M + H]+ 299.0944, found 299.0948.
4.1.3.3. 2-(4-((3-Chlorophenyl)diazenyl)phenyl)-4H-1,3,4-oxadiazin-5(6H)-one (18c)
Yellow solid (yield 83%). Mp 276–278 °C; 1H NMR (300 MHz, DMSO-d6) δ 11.19 (s, 1H), 7.98 (s,
4H), 7.93–7.90 (m, 2H), 7.67–7.65 (m, 2H), 4.83 (s, 2H); 13C NMR (75 MHz, DMSO-d6) δ 161.01,
152.86, 152.44, 146.26, 134.19, 132.75, 131.32, 131.28, 127.09 (2C), 122.97 (2C), 122.62, 121.04,
64.66; LCMS (ESI) m/z 315 [M + H]+; HRMS (FAB) m/z calcd for C15H12ClN4O2 [M + H] +
315.0649, found 315.0661.
4.1.3.4. (E)-2-(4-((4-Chlorophenyl)diazenyl)phenyl)-4H-1,3,4-oxadiazin-5(6H)-one (18d)
26

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1
Light brown solid (yield 86%). Mp 275–277 °C; H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H),
13
8.00–7.96 (m, 4H), 7.94 (d, J = 8.8 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H), 4.84 (s, 2H); C NMR (125
MHz, DMSO-d6) δ 161.06, 152.56, 150.48, 146.31, 136.43, 132.54, 129.68 (2C), 127.11 (2C),
124.37 (2C), 122.88 (2C), 64.68; LCMS (ESI) m/z 315 [M + H]+; HRMS (FAB) m/z calcd for
C15H11ClN4O2 [M + H]+ 315.0649, found 315.0638.
4.1.3.5. (E)-2-(4-(m-Tolyldiazenyl)phenyl)-4H-1,3,4-oxadiazin-5(6H)-one (18e)
Brown solid (yield 37%). Mp 243–244 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.18 (s, 1H), 7.96 (m,
13
4H), 7.73 (m, 2H), 7.51 (t, J = 7.6 Hz, 1H), 7.41 (d, J = 7.6 Hz, 1H), 4.83 (s, 2H), 2.44 (s, 3H); C
NMR (125 MHz, DMSO-d6) δ 161.07, 152.75, 152.01, 146.39, 139.06, 132.57, 132.19, 129.32,
127.09 (2C), 122.69 (2C), 122.65, 120.41, 64.67, 20.85; LCMS (ESI) m/z 295 [M + H]+; HRMS
(FAB) m/z calcd for C16H14N4O2 [M + H]+ 295.1195, found 295.1196.
4.1.4. General procedure for the preparation of imine 19a–f
A mixture of amine compound 12a (1 equiv.) and appropriate aldehyde (1 equiv.) in EtOH (1 mL)
was refluxed overnight. After cooling, the precipitate was filtered to yield imine derivatives 19a–f.
4.1.4.1. (E)-2-(4-(Benzylideneamino)phenyl)-4H-1,3,4-oxadiazin-5(6H)-one (19a)
1
Light brown solid (yield 45%). Mp 206–207 °C; H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H),
8.66 (s, 1H), 7.97–7.94 (m, 2H), 7.82 (d, J = 8.4 Hz, 2H), 7.57–7.53 (m, 3H), 7.32 (d, J = 8.8 Hz,
2H), 4.78 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 161.65, 161.12, 153.23, 147.02, 135.78,
131.76, 128.83 (4C), 127.25, 127.04 (2C), 121.19 (2C), 64.59; LCMS (ESI) m/z 280 [M + H]+;
HRMS (FAB) m/z calcd for C16H14N3O2 [M + H] + 280.1086, found 280.1098.
27

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4.1.4.2. (E)-2-(4-((3-Fluorobenzylidene)amino)phenyl)-4H-1,3,4-oxadiazin-5(6H)-one (19b)
1
Light brown solid (yield 71%). Mp 200–202 °C; H NMR (400 MHz, DMSO-d6) δ 11.05 (s, 1H),
8.68 (s, 1H), 7.82 (d, J = 8.8 Hz, 2H), 7.79 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 9.2 Hz, 1H), 7.58 (dd, J
= 13.8, 8.0 Hz, 1H), 7.40 (td, J = 8.4, 2.8 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H), 4.78 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) δ 162.37 (d, J = 243.1 Hz), 161.13, 160.48 (d, J = 2.8 Hz), 152.71, 146.97,
138.25 (d, J = 7.5 Hz), 130.99 (d, J = 7.9 Hz), 127.61, 127.08 (2C), 125.30 (d, J = 2.6 Hz), 121.28
(2C), 118.57 (d, J = 21.3 Hz), 114.50 (d, J = 21.9 Hz), 64.61; LCMS (ESI) m/z 298 [M + H]+;
HRMS (FAB) m/z calcd for C16H13FN3O2 [M + H] + 298.0992, found 298.1000.
4.1.4.3. (E)-2-(4-((3-Chlorobenzylidene)amino)phenyl)-4H-1,3,4-oxadiazin-5(6H)-one (19c)
Yellow solid (yield 61%). Mp 194–195 °C; 1H NMR (300 MHz, DMSO-d6) δ 11.07 (s, 1H), 8.67 (s,
1H), 7.98 (s, 1H), 7.91 (d, J = 7.3 Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.64–7.54 (m, 2H), 7.34 (d, J =
8.4 Hz, 2H), 4.78 (s, 2H); 13C NMR (75 MHz, DMSO-d6) δ 161.09, 160.29, 152.66, 146.93, 137.83,
133.67, 131.34, 130.77, 128.07, 127.62, 127.41, 127.05 (2C), 121.27 (2C), 64.59; LCMS (ESI) m/z
314 [M + H]+; HRMS (FAB) m/z calcd for C16H13ClN3O2 [M + H]+ 314.0696, found 314.0688.
4.1.4.4. (E)-2-(4-((4-Chlorobenzylidene)amino)phenyl)-4H-1,3,4-oxadiazin-5(6H)-one (19d)
1
Light brown solid (yield 80%). Mp 251–253 °C; H NMR (300 MHz, DMSO-d6) δ 11.06 (s, 1H),
8.68 (s, 1H), 7.96 (d, J = 8.6 Hz, 2H), 7.81 (d, J = 8.6 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.33 (d, J =
8.4 Hz, 2H), 4.78 (s, 2H); 13C NMR (75 MHz, DMSO-d6) δ 161.39, 160.72, 153.14, 147.24, 136.63,
134.91, 130.71 (2C), 129.28 (2C), 127.74, 127.33 (2C), 121.55 (2C), 64.89; LCMS (ESI) m/z 314
[M + H]+; HRMS (FAB) m/z calcd for C16H13ClN3O2 [M + H]+ 314.0696, found 314.0689.
4.1.4.5. (E)-2-(4-((3-Methylbenzylidene)amino)phenyl)-4H-1,3,4-oxadiazin-5(6H)-one (19e)
28