Synthesis and biological evaluation of potential acetylcholinesterase inhibitors based on a benzoxazine core

Article abstract of DOI:10.1002/ardp.201800024

With the purpose of expanding the structural variety of chemical compounds available as pharmacological tools for the treatment of Alzheimer's disease, we synthesized and evaluated a novel series of indole-benzoxazinones (Family I) and benzoxazine-arylpiperazine derivatives (Family II) for potential human acetylcholinesterase (hAChE) inhibitory properties. The most active compounds 7a and 7d demonstrated effective inhibitory profiles with K_i values of 20.3 ± 0.9 μM and 20.2 ± 0.9 μM, respectively. Kinetic inhibition assays showed non-competitive inhibition of AChE by the tested compounds. According to our docking studies, the most active compounds from both series (Families I and II) showed a binding mode similar to donepezil and interact with the same residues.

Full text of DOI:10.1002/ardp.201800024

Received: 30 January 2018
Revised: 8 March 2018
Accepted: 13 March 2018
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DOI: 10.1002/ardp.201800024
FULL PAPER
Synthesis and biological evaluation of potential
acetylcholinesterase inhibitors based on a benzoxazine core
Claudio Méndez-Rojas1
Carlos Gallardo-Garrido1
Gabriel Quiroz1
Mario Faúndez2
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C. David Pessoa-Mahana2
Hery Chung2
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Eduardo Gallardo-Toledo3
Claudio Saitz-Barría1
Ramiro Araya-Maturana4
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Marcelo J. Kogan3
María C. Zúñiga-López5
Patricio Iturriaga-Vásquez6
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Carla Valenzuela-Gutiérrez1
Hernán Pessoa-Mahana1
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1 Departamento de Química Orgánica y Fisicoquímica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, Chile
2 Departamento de Farmacia, Facultad de Química, Pontificia Universidad Católica de Chile, Santiago, Chile
3 Departamento de Química Farmacológica y Toxicológica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, Chile
4 Instituto de Química y Recursos Naturales, Universidad de Talca, Talca, Chile
5 Departamento de Química Inorgánica y Analítica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, Chile
6 Departamento de Ciencias Químicas y Recursos Naturales, Facultad de Ingeniería Ciencias, Universidad de la Frontera, Temuco, Chile
Correspondence
Abstract
Claudio Méndez-Rojas and Dr. Hernán Pessoa-
Mahana, Departamento de Química Orgánica y
Fisicoquímica, Facultad de Ciencias Químicas y
Farmacéuticas, Universidad de Chile, Casilla 233,
Santiago 1, Chile.
With the purpose of expanding the structural variety of chemical compounds
available as pharmacological tools for the treatment of Alzheimer's disease, we
synthesized and evaluated a novel series of indole-benzoxazinones (Family I) and
benzoxazine-arylpiperazine derivatives (Family II) for potential human acetylcholin-
esterase (hAChE) inhibitory properties. The most active compounds 7a and 7d
demonstrated effective inhibitory profiles with Ki values of 20.3 0.9 μM and
20.2 0.9 μM, respectively. Kinetic inhibition assays showed non-competitive
inhibition of AChE by the tested compounds. According to our docking studies,
the most active compounds from both series (Families I and II) showed a binding
mode similar to donepezil and interact with the same residues.
Email: claudiomendezr@gmail.com (CM-R);
hpessoa@ciq.uchile.cl (HP-M)
Funding information
FONDECYT, Grant numbers: 1130347,
1170269, 1150615
K E Y W O R D S
Alzheimer's disease, benzoxazinones, human acetylcholinesterase inhibition, indole
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1
INTRODUCTION
of the brain that mediate learning and memory functions. Based on
this hypothesis the cholinergic argumentation will improve the
Alzheimer's disease (AD) is
a
progressive neurodegenerative
cognition in AD.[3–5] Currently available treatment for patients
suffering from AD involve approved acetylcholinesterase (AChE)
inhibitors such as rivastigmine, donepezil, and galantamine which
avoid the hydrolysis of ACh thereby increasing its concentrations[6,7]
(Figure 1).
pathology with severe economic and social impact.[1,2] Nowadays,
drug research and development are based on the cholinergic
hypothesis which proposes that the selective loss of cholinergic
neurons results in a deficit of acetylcholine (ACh) in specific regions
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Arch Pharm Chem Life Sci. 2018;e1800024.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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FIGURE 1 AChE inhibitory drugs used in Alzheimer's disease
However, clinical efficiency is limited as available AChE inhibitors
can only ameliorate AD symptoms and, thus, the search for novel
compounds remains an emerging demand for the treatment of this
pathology. As an alternative strategy, the design of multi-targeted
drugs that can simultaneously act on ACh and other molecular targets
involved in the development of the disease[8–10] offer an interesting
approach to manage AD.
the present manuscript, we report the synthesis, docking, and
biological evaluation of Families I and II as potential AChE inhibitors
(Figure 3).
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2
RESULTS AND DISCUSSION
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2.1 Chemistry
It is well known that the indole and arylpiperazine rings
are recognized scaffolds endowed with serotonergic activity.[11,12]
Nevertheless, other studies[13,14] have also reported AChE inhibitory
activity in compounds containing these skeletons, as is shown in
Figure 2.
The indole benzoxazinone derivatives (Family I) were synthesized
by a three-step synthetic sequence as outlined in Scheme 1. The
3-(3-indolyl)-1-propanol analogues 2a,b were obtained in good yield
(85–90%) by means of a Fischer reaction between commercially
available substituted phenylhydrazines 1a,b, and 3,4-dihydro-2H-
pyran, under acidic reflux conditions.[18,19] The indolyl alcohols 2a,b
were subsequently converted to their tosylate derivatives 3a,b in 70%
yield. The corresponding 2-benzoxazinone intermediates 6a–f were
achieved in good yields (90–93%) by the reaction of substituted
2-aminophenols 5a–f with 2-chloroacetyl chloride.[20,21] The
2-aminophenol derivatives were obtained by iron metal reduction
of easily available substituted 2-nitrophenols 4a–f.
Our interest in the indole and arylpiperazine chemistry based on
the pharmacologic properties of both heterocyclic rings,[15,16]
encouraged us to carry out a synthetic work, connecting both scaffolds
to substituted 1,4-benzoxazine rings, a building block with potent
neuroprotecting properties,[17] and to explore their AChE inhibitory
properties. To the best of our knowledge, no studies have been carried
out with these molecular associations in AChE inhibition bioassays. In
Finally, compounds 3a,b reacted in a bimolecular nucleophilic
substitution with substituted 2H-benzo[b][1,4]oxazin-3(4H)-ones 6a–f
under basic conditions providing the indole benzoxazinone derivatives
7a–l in 48–59% yields.[22]
The 1H NMR spectra of intermediates 6a–f revealed, for all the
derivatives, downfield singlet signals for the amide N─H proton at δ:
10.66–10.87 ppm. In 13C NMR the existence of signals shifted
downfield between δ: 165.1 and 165.8 ppm confirmed the presence
of the amide carbonyl function. The 1H NMR spectra for the final
compounds, 7a–l, revealed signals at δ: 10.80–10.92 ppm range which
were assigned to the indolic N─H. The singlet signals between δ: 163.7
and 164.9 ppm in the 13C NMR spectra further confirmed the presence
of the amide carbonyl group.
FIGURE 2 The title compounds as examples of the indole and
arylpiperazine scaffold with AChE inhibitory activity

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FIGURE 3 Families I and II
On the other hand, the 1-(2,3-dihydro-4H-benzo[b][1,4]oxazin-4-
yl)-2-(4-phenylpiperazin-1-yl)ethan-1-one derivatives (Family II) were
obtained in 57–73% yields according to the reaction sequence
displayed in Scheme 2. The benzoxazinones 6a,b were reduced with
lithium aluminum hydride to the corresponding benzoxazines 8a,b
(87–92%) which were chromatographically purified and subsequently
reacted with 2-chloroacetyl chloride to provide 2-chloro-1-
(2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)ethan-1-one derivatives 9a,b
in 76 and 74% yields, respectively. Finally, the reaction of 9a,b
with commercially available arylpiperazine derivatives under basic
conditions, furnished the desired targets 10a–j in moderate to good
yields (57–73%).
In order to obtain information about the relevance of the
piperazine ring in the biological assays, four extra benzoxazinone
analogues 11a,b and 12a,b were synthesized by reaction of 9a,b
with 1-(2-fluorobenzyl)piperazine and 3-(4-fluorophenyl)-1H-pyrazole,
respectively, under the same conditions described for Family II (Scheme
2)andaddedasexploratory molecules (Figure 4). Inthe 11a,b analogues,
the piperazine ringlosestheresonance with thephenyl group, leading to
conformationally more flexible and basic structures, a condition that
SCHEME 1 Synthesis of indole benzoxazinone derivatives (Family I)

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SCHEME 2 Synthesis of arylpiperazine benzoxazine derivatives (Family II)
could favor interactions in the active site of the enzyme. On the other
hand, the piperazine ring moiety was replaced by a pyrazole ring in the
12a,b analogues providing a conformationally more rigid structure with
weaker basic properties.
this purpose, the rate of enzyme activity was measured at six different
concentrations of substrate ATC. The obtained Michaelis–Menten
parameters along with the Lineweaver–Burk plots (Figure 5) allows us
to conclude that both compounds would display a non-competitive
inhibition mechanism for AChE, with Ki values of 20.3 0.9 μM and
20.2 0.9 μM for 7a and 7d, respectively (Table 2). An inhibition curve
dose dependent for compounds 7a, 7d, 11a, and 11b is shown in
Supporting Information Figure S1.
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2.2 Biological activity
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2.2.1 AChE inhibitory activity
The AChE inhibition assays for all the synthesized compounds were
performed according to Ellman's method[23] in comparison to standard
drug donepezil.[24] In a first approach, an inhibitory screening was
carried out for all compounds with a final concentration of 100 μM.
The compounds selected to construct cholinesterase inhibition curves
to obtain IC50 data were only those with inhibition values over than
50% on cholinesterase inhibition activity.
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2.3 Docking study
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2.3.1 Molecular docking studies on Family I
In order to gain a better insight on the binding mode and possible
interactions of the targets compounds (Family I) with AChE enzyme,
docking studies were performed by AutoDock 4.0. Based on the
obtained docking poses, all compounds were analyzed using the
known inhibitor donepezil as reference structure. Donepezil binding
mode has been well characterized and three main interactions have
been described,[25–27] namely: 1) the benzyl moiety π-stacking against
Trp86 in the active site; 2) the piperidine nitrogen involved in a water-
mediated hydrogen bond with Tyr337 and Tyr341; and 3) the
indanone moiety π-stacking against Trp286 in the peripheral anionic
site (PAS). The PAS consists of five residues (Tyr72, Asp74, Tyr124,
Trp286, and Tyr341) clustered around the entrance to the active site
gorge.[25–28]
In this first assay, compounds 7a, 7d, 11a, and 11b showed the
best inhibitory effect against AChE activity (Table 1). Subsequent
cholinesterase inhibition curves were performed to obtain the IC50
values for each compound (Table 2).
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2.2.2 Kinetic study of AChE inhibition
Based on the previous obtained results, and with the aim to assess the
kinetic mode of AChE inhibition of the target compounds, the most
active compounds 7a and 7d were subjected to kinetics studies. For
FIGURE 4 Benzoxazinone analogues as exploratory molecules

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TABLE 1 Acetylcholinesterase inhibition assay of the synthesized compounds on plasma at 100 μM concentration expressed as percentage
Compound
% AChE inhibition SD
95.96 0.45
79.94 2.72
47.49 2.30
48.67 0.62
80.03 1.00
49.27 4.83
48.84 0.94
41.29 0.89
45.66 0.36
46.67 1.68
36.43 1.47
45.03 1.52
15.73 6.82
Compound
10a
% AChE inhibition SD
14.96 1.71
13.68 2.88
12.16 6.59
27.81 2.53
34.10 1.84
11.09 2.47
6.15 1.52
Donepezil
7a
7b
7c
7d
7e
7f
7g
7h
7i
10b
10c
10d
10e
10f
10g
10h
10i
15.75 8.31
20.53 1.17
28.36 9.86
72.04 1.91
65.68 1.94
17.18 4.69
28.69 0.14
10j
7j
11a
7k
7l
11b
12a
12b
Data represent the mean of percentage SD of three experiments, each with triplicate samples. Donepezil was used as positive control.
The best values are in bold.
Docking results show two main binding modes in which the
analyzed compounds bind along narrow deep gorge, lined
predominantly with aromatic residues[27] and interact with residues
in the PAS (Tyr72, Tyr124, Trp286, and Trp341). Both binding modes
can be overlapped with the binding pose of donepezil and some of the
key interactions appear to be conserved.
and 7d displayed the most potent activity. Figure 6 shows the
superimposed structures of representative compounds 7a and 7b along
with the co-crystallized donepezil in the active site of AChE showing the
two main binding poses observed in the docking studies.
a
Compounds 7a, 7c, 7d, 7g, and 7j share a similar binding pose
where 1) the indole nitrogen can participate in a water-mediated
hydrogen bond with Tyr337 and Tyr341; 2) the indole ring stacks
against Tyr337; 3) the benzoxazinone ring stacks against Trp286 while
its carbonyl oxygen hydrogen bonds with the amine group of Phe295.
On the other hand, compounds 7b, 7e, 7f, 7h, 7i, and 7k share a second
binding mode in which 1) the indole ring stacks against Tyr341 leaving
the indole nitrogen too far to hydrogen-bond with Tyr337; 2) the
benzoxazinone carbonyl oxygen hydrogen-bonds with Arg296; and 3)
the indanone ring lays at an angle that makes aromatic stacking with
Trp286 less likely although side-chain flexibility could permit some
degree of staking. Among the synthesized compounds, compounds 7a
TABLE 2 Ki values of compounds 7a and 7d and inhibitory
potency assays of donepezil and synthesized compounds 7a, 7d,
11a, and 11b on plasma cholinesterase activity measured as IC50
value
Compound
Donepezil
7a
(IC50 SD)a (μM)
Ki (μM)
0.0238[24]
2.17 0.15
21.87 2.61
25.80 3.63
33.37 3.52
34.33 2.88
20.3 0.9
FIGURE 5 Lineweaver–Burk plot of acetylthiocholine (ATC)
degradation by plasma cholinesterase with or without the synthesized
compound 7a (A) and 7d (B). The plot shows the dose-dependent
inhibition of acetylthiocholine degradation by plasma cholinesterase
in the presence of the synthesized compounds 7a and 7d at their
IC10, IC25, IC50 concentrations. Each point represents the mean
percentage SD of three experiments, each with triplicate samples
7d
20.2 0.9
11a
Not measured
Not measured
11b
aData represent the mean SD of three experiments, each with triplicate
samples. The most potent activities are shown in italics.

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FIGURE 6 Superimposed structures of representative compounds 7a (cyan) in (A) and 7b (orange) in (B) with co-crystallized ligand
donepezil (blue) showing the two main binding modes observed. Black-dashed lines represent hydrogen bonds and aromatic stacking is shown
with green lines. Water 931 is shown as a green sphere
Comparison with the well-characterized binding mode of done-
pezil shows that most of the aromatic stacking interactions and the
water-mediated hydrogen bonds are conserved in the first binding
mode (compounds 7a, 7c, 7d, 7g, and 7j), while only one of donepezil
aromatic interactions is properly maintained in the second binding
mode (compounds 7b, 7e, 7f, 7h, 7i, and 7k). This is in line with the
highest activity values observed for compounds 7a and 7d whereas
lower activity was observed in compounds 7b and 7k.
Compound 7l (15.73 6.82%) showed a flipped orientation with
the benzoxazinone ring facing toward the active site gorge and the
indole ring extending to the surface of the enzyme. This different
docking pose could be related to the lowest inhibition observed for this
compound.
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2.3.2 Molecular docking studies on Family II
The lower activity values together with the similar binding poses
obtained for compounds 7b, 7e, 7f, 7h, 7i, and 7k suggest that
relatively larger substituents at position 6 of the benzoxazinone ring
are unfavorable for activity. This is in agreement with the reported
crystal structure of rhAChE in complex with donepezil, where the
narrow gorge created by Tyr337 and Tyr341 produces tight packing in
this region and can accommodate only small substituents (e.g., H, F).[25]
Docking results for series II show a common binding mode for the two
most active compounds of Family II (11a,b). Similar to series I, the two
compounds can be overlapped with the co-crystallized ligand
donepezil and seem to share some of the key interactions (Figure 7).
Compounds 11a and 11b share a common orientation with the
dihydrobenzoxazine ring laying toward the PAS and the phenyl ring
extending toward the active site gorge. This orientation was not
FIGURE 7 Superimposed structures of compounds 11a (yellow) and 11b (light green) with co-crystallized ligand donepezil (blue) are shown
in (A) and (B), respectively. Black-dashed lines represent hydrogen bonds and aromatic stacking is shown with green lines. Water 931 is
shown as a green sphere. Overlapping docking poses of each compound and co-crystallized ligand donepezil is shown in the inset

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observed for the least active compound from series II. All three main
interactions of donepezil can be established by the benzylpiperazine
compounds 11a and 11b (aromatic stacking with Trp286, water-
mediated hydrogen bond with Tyr337 and Tyr341, and aromatic
stacking with Trp86). Similar docking interactions have also been
described for a series of benzylpiperazinyl indanone derivatives.[29] On
the other hand, lacking the piperazine nitrogen, compounds 12a and
12b do not show a hydrogen bond interaction and only share the
aromatic stacking interaction with Trp286 while its benzyl moiety
could interact with Trp337. This is in line with the lower inhibitory
activity observed by compounds 12a,b as compared to 11a,b. A
supplementary docking file has been added for compounds 11a, 11b,
and 12a. Docking score and main molecular interactions for both series
are shown in Table 3.
pose but only the aromatic stacking interaction with Trp286 was
shared possibly explaining the lower inhibition observed. With respect
to Family II these compounds showed poor inhibitory activity, which is
in line with the different orientations observed in this series with
respect to donepezil (docking figures not shown).
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4
EXPERIMENTAL
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4.1 Chemistry
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4.1.1 General
Melting points were determined on a hot-stage apparatus and are
uncorrected. The IR spectra were recorded in KBr discs on an FT-IR
Bruker IFS 55 spectrophotometer and wavenumbers are reported in
cm−1. The 1H and 13C NMR spectra were obtained on a Bruker DRX-
300 spectrometer (300 and 75 MHz, respectively) in CDCl3 or DMSO-
d6. Chemical shifts were recorded in ppm (δ) relative to TMS as an
internal standard. J values are given in Hz. Micro-analyses were carried
out on a Fisons EA 1108 analyzer. High resolution mass spectra were
recorded on a DSA-TOF AxION 2 TOF MS (Perkin Elmer, Shelton, CT,
USA), positive mode. Silica gel Merck 60 (70–230 mesh) and aluminum
sheets coated with silica gel 60 F254 were used for column and TLC
chromatography, respectively.
In summary, the most active compounds from each series of
Family I show a binding mode similar to the known inhibitor donepezil
and interact with the same residues. In both series, the compounds
extend near the entrance of the active site. Here the benzoxazine
portion faces the PAS overlapping with donepezil indanone ring, while
the indole or phenyl portions extend toward the enzymes active site.
The similar binding poses with respect to donepezil suggest a non-
competitive inhibition mechanism that can be further supported by the
kinetics study for the analyzed compounds 7a and 7d.
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
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3
CONCLUSIONS
3-(1H-3-Indolyl)-1-propanol (2a)[18,19]
The purpose of this study was to examine the effect of incorporating
bioactive heterocyclic rings such as arylpiperazine and indolylpropyl
moieties to benzoxazine ring derivatives in the search of novel ligands
with potential acetylcholinesterase inhibitory properties. Twenty-six
compounds were synthesized classified in two families (I and II).
Compounds 7a and 7d belonging to family I were the most active
compounds against AChE with IC50 values of 21.87 and 25.80 μM,
respectively. The substitution pattern of compound 7a (R; R1 = H) and
7d (R = H, R1 = 7F) together with a SAR analysis of Family I allows us to
draw several conclusions: i) A C-6-substitution on the benzoxazine ring
is unfavorable for the inhibitory activity for a variety of substituents; ii)
a C-7 substitution pattern on the benzoxazine ring seems to be
beneficial for inhibitory activity; iii) the presence of a fluorine atom at
C-5 (R = F) in the indole ring for compounds 7g–l resulted in lower
AChE inhibitory activity, even compound 7j bearing a C-7 substitution
showed poor activity. A possible explanation could involve the
inductive electron-withdrawing effect of the fluorine atom disrupting
an aromatic interaction between the indole ring and Trp86.
To a stirred solution of commercial phenylhydrazine hydrochloride
(1000 mg, 7 mmol) in N,N-DMA (10 mL), H2SO4 4% w/w (10 mL,
0.04 mmol), and 3,4-dihydro-2H-pyran (0.63 mL, 6.9 mmol) were
added, and the mixture was heated at 100°C and then kept at reflux
temperature for 2 h. After this time, the reaction was quenched with
water (50 mL) and extracted with AcOEt (3 × 50 mL). The organic layer
was dried over anhydrous Na2SO4 and evaporated in vacuo. The crude
product was purified by column chromatography on silica gel (AcOEt)
to furnish 1083 mg, (90%) of pure 2a as a brown oil. IR: cm−1: 3417
(N─H), 3329 (O─H), 3055 (C─H arom.), 2926 (C─H aliph.). 1H NMR
(CDCl3): δ 1.78 (s, 1H, OH), 1.96 (q, 2H, In─CH2─CH2─CH2─OH,
J = 7.6 Hz), 2.83 (t, 2H, In─CH2─CH2─CH2─OH, J = 7.5 Hz), 3.69
(t, 2H, ─CH2OH, J = 6.5 Hz), 6.93 (d, 1H, 2-H, J = 2.1 Hz), 7.10 (td, 1H,
5-H or 6-H, Jo = 7.3 Hz, Jm = 1.2 Hz), 7.18 (td, 1H, 6-H or 5-H,
Jo = 7.1 Hz, Jm = 1.2 Hz), 7.32 (d, 1H, 4-H, J = 8.0 Hz), 7.60 (d, 1H, 7-H,
J = 7.8 Hz) and 8.03 (s, 1H, N─H). 13C NMR (CDCl3): δ 21.4, 32.9, 62.6,
111.2, 115.9, 118.9, 119.2, 121.4, 122.0, 127.5, and 136.4 ppm.
On the other hand, the presence of relatively larger substituents at
the C-6 position of the benzoxazinone ring in compounds 7b, 7e, 7f,
7h, 7i, and 7k resulted unfavorable for the inhibitory activity, with the
lowest inhibition displayed by compound 7l probably as a result of a
different fitting pose on the active site of the AChE enzyme. In relation
to the exploratory molecules, compounds 11a and 11b showed higher
inhibition values of 72.04 and 65.68%, respectively. Due to their close
structural relation to Family I, docking results show a similar binding
3-(5-Fluoro-1H-3-indolyl)-1-propanol (2b)[18,19]
Prepared from 4-fluor-phenylhydrazine hydrochloride (1000 mg,
6 mmol), H2SO4 4% w/w (10 mL, 0.04 mmol), and 3,4-dihydro-2H-pyran
(0.54 mL, 6 mmol) to afford pure 2b as an oily product. IR (cm−1):
3629–3250 (O─H and N─H), 3060 (C─H arom), 2939 (C─H aliph),
1581 (C C). 1H NMR (CDCl3): δ 1.79 (s, 1H, OH), 1.95 (q, 2H,

In─CH2─CH2─CH2─OH, J = 7.6 Hz), 2.79(t, 2H, In─CH2─CH2─CH2─OH,

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TABLE 3 AutoDock binding energies and types of interactions obtained by the docking calculations for donepezil, Series I derivatives and the
most active molecules from Series II
Binding energy
Compound
(kcal/mol)
Main types of interactions (residues <5 Å)
H bonds (Phe295, wat-Tyr341-Tyr337)
Donepezil
−9.61
Aromatic (Trp86, Trp286)
Hydrophobic (His447, Gly448, Gly120, Gly121, Phe338, Tyr124, Leu289, Phe297, Val294, Ser333, Tyr72)
H bonds (Phe295, wat-Tyr337-Tyr341)
7a
7b
7c
7d
7e
7f
−9.89
−10.24
−9.75
−9.81
−9.76
−10.36
−9.56
−10.05
−9.48
−9.48
−9.74
Aromatic (Trp86, Trp286, Tyr337)
Hydrophobic (His447, Tyr124, Tyr72, Phe297, Gly448, Val294)
H bonds (Arg296)
Aromatic (Trp286, Tyr341)
Hydrophobic (Phe338, Tyr337, Phe295, Phe297, Asp74, Tyr72, Gly448, Val294, Tyr124)
H bonds (Phe295, wat-Tyr337-Tyr341)
Aromatic (Trp86, Trp286, Tyr337)
Hydrophobic (His447, Tyr124, Tyr72, Phe297, Gly448, Val294)
H bonds (Phe295, wat-Tyr337-Tyr341)
Aromatic (Trp86, Trp286, Tyr337)
Hydrophobic (His447, Tyr124, Tyr72, Phe297, Gly448, Val294)
H bonds (Arg296)
Aromatic (Trp286, Tyr341)
Hydrophobic (Phe338, Tyr337, Phe295, Phe297, Asp74, Tyr72, Gly448, Val294, Tyr124)
H bonds (Arg296)
Aromatic (Trp286, Tyr337)
Hydrophobic (Trp86, Tyr341, Leu289, His447, Phe338, Phe295, Phe297, Val294, Tyr72, Tyr124)
H bonds (Phe295, wat-Tyr337-Tyr341)
7g
7h
7i
Aromatic (Trp86, Trp286, Tyr337)
Hydrophobic (His447, Tyr124, Tyr72, Phe297, Gly448, Val294)
H bonds (Arg296)
Aromatic (Trp286, Tyr341)
Hydrophobic (Phe338, Tyr337, Phe295, Phe297, Asp74, Tyr72, Gly448, Val294, Tyr124)
H bonds (Arg296)
Aromatic (Trp286, Tyr341)
Hydrophobic (Phe338, Tyr337, Phe295, Phe297, Asp74, Tyr72, Gly448, Val294, Tyr124)
H bonds (Phe295)
7j
Aromatic (Trp86, Trp286, Tyr337)
Hydrophobic (Tyr72, Val294, Phe297, Tyr124, His447, Gly448, Phe338, Tyr341, Tyr124)
H bonds (Arg296)
7k
Aromatic (Trp286, Tyr341)
Hydrophobic (Phe338, Tyr337, Phe295, Phe297, Asp74, Tyr72, Gly448, Val294, Tyr124)
H bonds (Phe297)
7l
−10.24
−10.69
Hydrophobic (Thr83, Trp86, His447, Tyr337, Phe338, Phe295, Phe297, Tyr124, Val294, Tyr341, Gly342,
Leu289, Trp286)
11a
H bonds (wat-Tyr341-Tyr337)
Aromatic (Trp286, Trp86)
Hydrophobic (Gly448, His447, Phe338, Gly120, Gly121, Phe295, Tyr72, Tyr124, Val294, Phe297)
12a
−9.32
Aromatic (Trp286, Trp337)
(Continues)

MÉNDEZ-ROJAS ET AL.
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|
TABLE 3 (Continued)
Binding energy
(kcal/mol)
Compound
12b
Main types of interactions (residues <5 Å)
Hydrophobic (Trp86, Gly448, His448, Phe338, Tyr124, Phe297, Val294, Tyr341)
Aromatic (Trp286, Trp337)
−9.23
Hydrophobic (Trp86, Gly448, His448, Phe338, Tyr124, Phe297, Val294, Tyr341, Tyr72)
H bonds (wat-Tyr341-Tyr337)
11b
−10.87
Aromatic (Trp286, Trp86)
Hydrophobic (Gly448, His447, Phe338, Gly120, Gly121, Gly122, Phe295, Tyr72, Tyr124, Val294, Phe297)
J = 7.6 Hz), 3.70 (t, 2H, ─CH2─OH, J = 6.5 Hz), 6.92 (td, 1H, 6-H, Jo = 9.2 Hz,
m = 2.4 Hz), 7.02 (d, 1H, 2-H, J = 2.0 Hz), 7.23 (m, 2H, 4-H and 7-H) and 8.11
114.5 (d, 4JC-F = 4.4 Hz), 123.6, 127.5 (d, 3JC-F = 9.4 Hz), (2x)127.8, (2x)
1
J
129.9, 133.9, 133.1, 144.8, and 157.7 (d, JC-F = 234.4 Hz) ppm.
(s, 1H, NH). 13C NMR (CDCl3): δ 21.3, 32.8, 62.5, 103.8 (d, 2JC-F = 23.2 Hz),
110.3 (d, 2′JC-F = 26 Hz), 111.7 (d, 3′JC-F = 9.4 Hz), 116.1 (d, 4JC-F = 4.4 Hz),
123.2, 127.9 (d, 3JC-F = 10 Hz), 132.9 and 157.7 (d, 1JC-F = 234 Hz) ppm.
|
4.1.2 General procedure for the synthesis of
2H-benzo[b][1,4]oxazin-3(4H)-one derivatives 6a–f
with 2H-benzo[b][1,4]oxazin-3(4H)-one (6a) as a
model[21,22]
3-(1H-Indol-3-yl)-propyl-4-methylbenzenesulfonate (3a)[18,19]
To a solution of 3-(1H-indolyl)-3-propanol 2a (1100 mg, 6.2 mmol) in
dry dichloromethane (30 mL), anhydrous Et3N (0.85 mL, 6.2 mmol),
tosyl chloride (1.175 mg, 6.2 mmol) and DMAP as a catalyst (12.0 mg,
0.098 mmol) were added. The mixture was stirred for 20 h at room
temperature under nitrogen atmosphere subsequently concentrated
under vacuum and purified by column chromatography AcOEt/
n-hexane (1:2), to give pure 3a as a brown solid (1421 mg; 70%).
Mp: 84–86°C. IR: cm−1: 3386 (N─H), 3038 (C─H aromatic), 2929–2903
(C─Haliphatic).1HNMR(CDCl3): δ2.02 (q, 2H, In─CH2─CH2─CH2─OTs),
2.43 (s, 3H, CH3─Ts), 2.80 (t, 2H, In─CH2─CH2─CH2─OTs, J = 7.3 Hz),
4.07 (t, 2H, ─CH2─OTs, J = 6.2 Hz), 6.89 (s, 1H, 2-H), 7.08 (td, 1H, 5-H or
6-H Ind, Jo = 7.8 Hz, Jm = 1.1 Hz), 7.17 (d, 1H, 6-H or 5-H Ind, Jo = 7.0 Hz,
To a stirred solution of 2-aminophenol 5a (2000 mg, 18.33 mmol) in
dry CH3CN (70 mL) was added dropwise chloroacetyl chloride
(1.48 mL; 18.33 mmol) and K2CO3 (2533 mg; 18.33 mmol) at room
temperature. The suspension was subsequently heated under reflux
conditions for 20 h. After completion the reaction mixture was cooled
to room temperature quenched with water (100 mL) and the resulting
mixture extracted with CH2Cl2 (5 × 50 mL). The combined organic
layers were washed with acidified water, dried over anhydrous
Na2SO4, and concentrated in vacuo. The organic crude was purified by
silica gel column chromatography (AcOEt/n-hexane) (1:1, v/v) to give
6a (2536 mg, 92.7%) as a white solid. Mp: 170–171°C. 1H NMR
(DMSO-d6): δ 4.51 (s, 2H, 2-H), 6.82–6.92 (m, 4H) and 10.66 (s, 1H,
NH). 13C NMR (DMSO-d6): δ 67.2, 116.3, 116.6, 122.8, 123.5, 127.7,
143.7, and 165.4 ppm.
J
m = 1.1 Hz), 7.32 (t, 3H, 4-H Ind, 3-H and 5-H Ts, J = 9.1 Hz), 7.49
(d, 1H, 4-H Ind, J = 7.8 Hz), 7.77 (d, 2H, 2-H and 6-H Ts, J = 8.3 Hz)
and 7.98 (s, 1H, N─H). 13C NMR (CDCl3): δ 20.9, 21.7, 29.1, 70.1,
111.2, 114.4, 118.7, 119.3, 121.8, 122.0, 127.2, (2x)127.9, (2x)
129.9, 133.2, 136.4, and 144.7.
6-Chloro-4H-benzo[1,4]oxazin-3-one (6b)[30–32]
Prepared from 2-amino-4-chlorophenol 5b (1000 mg; 6.96 mmol),
chloroacetyl chloride (0.56 mL; 6.96 mmol) and anhydrous K2CO3
(962 mg; 6.96 mmol) to give 6b (1163 mg; 91%) of a light brown solid.
Mp: 214–215°C. 1H NMR (DMSO-d6): δ 4.64 (s, 2H, 2-H), 6.95–7.01
(m, 3H) and 10.87 (s, 1H, NH). 13C NMR (DMSO-d6): δ 67.1, 115.7,
118.1, 122.9, 126.2, 129.2, 142.6, and 165.1.
3-(5-Fluoro-1H-indol-3-yl)-propyl-4-methylbenzenesulfonate
(3b)[18,19]
To a solution of 3-(5-fluoro-1H-3-indolyl)-1-propanol 2b (1000 mg,
5.0 mmol) in dry dichloromethane (30 mL), anhydrous Et3N (0.72 mL,
5.0 mmol), tosyl chloride (953 mg, 5.0 mmol), and DMAP as a catalyst
(12.0 mg, 0.098 mmol) were added. The organic crude was column
chromatographed to afford pure 3b (1260 mg, 70%) as a light brown
solid. Mp: 75–77°C. IR (cm−1): 3391 (N─H), 1396 (SO2), 1356 (SO2).
1H NMR (CDCl3): δ 1.99 (q, 2H, In─CH2─CH2─CH2─OTs, J = 7.3 Hz),
2.43 (s, 3H, CH3─Ts), 2.74 (t, 2H, In─CH2─CH2─CH2─OTs,
J = 7.3 Hz), 4.07 (t, 2H, In─CH2─CH2─CH2─OTs, J = 6.2 Hz), 6.90
(m, 2H, 2-H and 6-H Ind), 7.09 (dd, 1H, 4-H Ind, Jo = 9.6 Hz,
6-Fluoro-4H-benzo[1,4]oxazin-3-one (6c)[30,31]
Prepared from 2-amino-4-fluorophenol 5c (1500 mg; 11.8 mmol),
chloroacetyl chloride (0.95 mL; 11.8 mmoles) and anhydrous
K2CO3 (1631 mg; 11.8 mmol) to give 6c (1785 mg, 93%) of a white
solid. Mp: 205–206°C. 1H NMR (DMSO-d6): δ 4.61 (s, 2H, 2-H),
6.73–6.82 (m, 2H, 7-H and 8-H), 7.02 (dd, 1H, 5-H, Jo = 8.7 Hz,
J
m = 2.4 Hz), 7.24 (dd, 1H, 7-H Ind, Jo = 9.3 and Jm = 3.4 Hz), 7.31 (d,
J
m = 5.1 Hz), 10.85 (s, 1H, NH). 13C NMR (DMSO-d6): δ 67.2,
103.2 (d, 2JC-F = 27 Hz), 109.2 (d, 2′JC-F = 24 Hz), 117.5 (d, 3JC-F = 9 Hz),
2H, 3-H and 5-H Ts, J = 8.0 Hz), 7.77 (d, 2H, 2-H and 6-H Ts, J = 8.3 Hz)
and 8.03 (s, 1H, NH). 13C NMR (CDCl3): δ 20.8, 21.7, 29.3, 69.9, 103.5
(d, 2JC-F = 23.2 Hz), 110.4 (d, 2′JC-F = 26.5 Hz), 111.8 (d, 3′JC-F = 9.4 Hz),
3′
4
1
128.9 (d,
J
C-F
= 12 Hz), 140.1 (d, JC-F = 2.3 Hz), 157.7 (d, JC-F =
236 Hz), 165.4.

10 of 17
MÉNDEZ-ROJAS ET AL.
|
7-Fluoro-4H-benzo[1,4]oxazin-3-one (6d)[22,30]
3.77 (s, 1H, NH), 4.17 (t, 2H, 2-H, J = 4.3 Hz), 6.24–6.34 (m, 2H, 7-H
and 8-H Ar), 6.67 (dd, 1H, 5-H, Jo = 5.4 Hz, Jm = 4.3 Hz). 13C NMR
(CDCl3): δ 40.7, 67.2, 101.8 (d, 2JC-F = 26 Hz), 104.2 (d, 2′JC-F = 23 Hz),
Prepared from 2-amino-5-fluorophenol 5d (1500 mg; 11.8 mmol),
chloroacetyl chloride (0.95 mL; 11.8 mmoles) and anhydrous K2CO3
(1631 mg; 11.8 mmol) to give 6d (1726 mg, 90%) of a white solid. Mp:
201–202°C. 1H NMR (DMSO-d6): δ 4.64 (s, 2H, 2-H), 6.85 (td, 1H, 6-H,
Jo = 8.6 Hz, Jm = 2.6 Hz), 6.90–6.96 (m, 2H, 5-H and 8-H), 10.78 (s, 1H,
3
3′
4
117.0 (d, JC-F = 10 Hz), 134.5 (d,
J
C-F
= 11 Hz), 139.9 (d, JC-F =
1
2.2 Hz) and 157.7 (d, JC-F = 237 Hz).
2
NH). 13C NMR (DMSO-d6) δ: 67.1, 104.5 (d, JC-F = 26 Hz), 109.1 (d,
2-Chloro-1-(2,3-dihydro-benzo[1,4]oxazin-4-yl)-ethanamide
(9a)
2′
J
= 23 Hz), 116.7 (d, 3JC-F = 10 Hz), 124.3 (d, 4JC-F = 3.0 Hz), 144.5
C-F
1
(d, 3′JC-F = 12 Hz), 158.2 (d, JC-F = 237 Hz), 164.7.
To a stirred solution of 3,4-dihydro-2H-benzo[1,4]oxazine 8a
(2684 mg; 19.86 mmol) in dry THF (60 mL) was added dry triethyl-
amine (2.38 mL; 19.86 mmol) and chloroacetyl chloride (1.61 mL;
19.86 mmol), the mixture was stirred for 2 h under nitrogen atmo-
sphere at room temperature. After completion water was added
(100 mL) and the mixture extracted with AcOEt (3 × 100 mL). The
combined organic layers were dried over anhydrous Na2SO4 and
concentrated under reduced pressure, the organic crude was purified
by silica gel column chromatography AcOEt/n-hexane (1:1) to yield
compound 9a (3180 mg; 76%) as a colorless oil which gradually
solidified under vacuum. Mp: 104.2–105.5°C. 1H NMR (CDCl3) δ: 3.87
(t, 2H, 2-H, J = 4.8 Hz), 4.30 (t, 2H, 3-H, J = 4.4 Hz), 4.64 (s, 2H,
N─CO─CH2─Cl), 6.85–6.93 (m, 2H, 6-H and 8-H), 7.08 (t, 1H, 7-H,
J = 7.5 Hz), 7.85 (br.s, 1H, 5-H). 13C NMR (CDCl3) δ: 40.2, 43.7, 66.1,
115.4, 116.8, 118.9, 122.2, 135.6, 143.1, 165.2. HRMS: (EI) calcd. for
6-Methoxy-4H-benzo[1,4]oxazin-3-one (6e)[30]
Prepared from 2-amino-4-methoxyphenol 5e (1000 mg; 7.18 mmol),
chloroacetyl chloride (0.58 mL; 7.18 mmol) and anhydrous K2CO3
(992 mg; 5.32 mmol) to give 6e (1146 mg, 89%) of a light violet solid.
Mp: 164–165°C. 1H NMR (DMSO-d6) δ: 3.73 (s, 3H, OCH3), 4.53
(s, 2H, 2-H), 6.50–6.58 (m, 2H, 5-H, 8-H), 6.92 (m, 1H, 7-H), 10.68
(s, 1H, NH). 13C NMR (DMSO-d6) δ: 55.8, 67.3, 102.3, 108.0, 117.0,
128.5, 137.6, 155.0, 165.8 ppm.
6-Bromo-4H-benzo[1,4]oxazin-3-one (6f)[31,33]
Prepared from 2-amino-4-bromophenol 5f (1000 mg; 5.32 mmol),
chloroacetyl chloride (0.43 mL; 5.32 mmol) and anhydrous K2CO3
(735 mg; 5.32 mmol) to give 6f (1116 mg, 92%) of a light brown solid.
Mp: 184–185°C. 1H NMR (DMSO-d6): δ 4.64 (s, 2H, 2-H), 6.97 (d, 1H,
8-H, J = 8.5 Hz), 7.09 (d, 1H, 5-H, J = 2.3 Hz), 7.12 (dd, 1H, 7-H,
Jo = 8.5 Hz, Jm = 2.3 Hz) and 10.87 (s, 1H, NH). 13C NMR (DMSO-d6) δ:
67.1, 113.7, 118.4, 118.5, 125.9, 129.5, 143.1, 165.1 ppm.
C10H10ClNO2 (M+1): 212.0478. Found: 212.0512.
2-Chloro-1-(6-fluoro-2,3-dihydro-benzo[1,4]oxazin-4-yl)-
ethanamide (9b)
Prepared from 6-fluoro-3,4-dihydro-2H-benzo[1,4]oxazine 8b
(1485 mg; 9.69 mmol) and chloroacetyl chloride (0.79 mL;
9.69 mmol) to provide 9b (1645 mg; 74%) as a white solid. Mp:
99.1–100.8°C. 1H NMR (CDCl3) δ: 3.87 (t, 2H, 3-H J = 4.8 Hz), 4.28 (t,
2H, 2-H J = 4.7 Hz), 4.70 (s, 2H, N─CO─CH2─Cl), 6.91–6.96 (m, 2H,
7-H and 8-H), 7.87 (br.s, 1H, 5-H). 13C NMR (CDCl3) δ: 40.1, 44.0, 65.6,
|
4.1.3 General procedure for the synthesis of
3,4-dihydro-2H-benzo[1,4]oxazine derivatives 8a,b
with 3,4-dihydro-2H-benzo[1,4]oxazine (8a) as a
model[22,34,35]
2
2′
3
To a suspension of LiAlH4 (1069 mg; 28.16 mmol) in dry THF (60 mL) at
0°C was slowly added a solution of 4H-benzo[1,4]oxazin-3-one 6a
(2000 mg; 13.41 mmol) in dry THF (50 mL). The mixture was stirred
under nitrogen atmosphere for 2 h at room temperature, after
completion the reaction the mixture was cautiously quenched with a
mixture of AcOEt/H2O (1:1 v/v) and the salts formed were filtered
over celite. The precipitate was washed with AcOEt and the mother
liquors dried with anhydrous Na2SO4 and concentrated in vacuo. The
crude product was purified by silica gel column chromatography with
AcOEt/n-hexane (1:1 v/v) to give 8a as a brown oil (1667 mg; 92%).
1H NMR (CDCl3): δ 3.39 (s, 2H, 2-H, J = 4.5 Hz), 3.50 (s, 1H, NH), 4.23
(s, 2H, 3-H, J = 4.4 Hz), 6.77 (dd, 1H, 5-H, Jo = 7.6 Hz, Jm = 1.8 Hz), 6.65
(td, 1H, 6-H or 7-H, Jo = 7.7 Hz, Jm = 1.6 Hz), 6.71–6.80 (m, 2H, 7-H or
6-H and 8-H). 13C NMR (CDCl3): δ 40.9, 65.1, 115.6, 116.7, 118.7,
121.2, 133.6, 144.1 ppm.
110.2 (d, JC-F = 26 Hz), 112.6 (d,
J
C-F
= 23 Hz), 118.2 (d, JC-F
=
3′
4
10 Hz), 126.4 (d,
J
= 11 Hz), 143.2 (d, JC-F = 2.2 Hz), 155.6
C-F
1
(d, JC-F = 237 Hz), 165.6 ppm. HRMS: (EI) calcd. for C10H9ClFNO2
(M+1): 230.0384. Found: 230.0394.
|
4.1.4 General procedure for the synthesis of 4-(3-
(1H-indol-3-yl)propyl)-2H-benzo[b][1,4]oxazin-3(4H)-
one derivatives 7a–l, with 4-(3-(1H-indol-3-yl)propyl)-
2H-benzo[b][1,4]oxazin-3(4H)-one (7a) as a model
To a solution of 2H-benzo[b][1,4]oxazin-3(4H)-one 6a (0.5 g 3.4 mmol)
in dry DMF (5 mL) was added NaH 60% dispersion in mineral oil (0.16 g,
6.8 mmol) the mixture was heated at 100°C for 30 min and then 3-(1H-
indol-3-yl)propyl-4-methylbenzenesulfonate 3a (1.1 g, 3.4 mmol)
was incorporated, the mixture remained at 100°C for 48 h. After
completion, the reaction mixture was quenched with water (50 mL)
and extracted with AcOEt (2 × 50 mL). The organic layers were dried
over anhydrous Na2SO4 and concentrated under vacuum to provide a
crude solid which was purified by silica gel column chromatography
(AcOEt/n-hexane 2:1) to give 7a as a pure white solid (0.51 g, 50%).
6-Fluoro-3,4-dihydro-2H-benzo[1,4]oxazine (8b)[36]
Prepared from 6-fluoro-4H-benzo[1,4]oxazin-3-one 6c (1500 mg;
9 mmol) and LiAlH4 (717 mg; 18.9 mmol) to give 8b (1200 mg; 87%)
as a brown light oil. 1H NMR (CDCl3) δ: 3.36 (t, 2H, 3-H, J = 4.5 Hz),

MÉNDEZ-ROJAS ET AL.
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|
Mp: 149.1–150.1°C. IR: ν (cm−1): 3286 (N─H), 3057 (C─H arom.),
aliph.), 1678 (C ON), 1509 (C C). 1H NMR (DMSO-d6): δ: 1.93 (m,
2H, 2′-H), 2.75 (t, 2H, 3′-H, J = 7.5 Hz), 3.98 (t, 2H, 1′-H, J = 7.5 Hz),
4.68 (s, 2H, 3″-H), 6.84 (td, 1H, 6-H, Jo = 8.7 Hz and Jm = 2.8 Hz), 6.94–
6.98 (m, 2H, 5″-H, 8″-H), 7.07 (t, 1H, 5-H, J = 7.3 Hz), 7.12–7.16 (m,
2H, 2-H, 7″-H), 7.35 (d, 1H, 4-H, J = 8.0 Hz), 7.46 (d, 1H, 7-H,
J = 7.8 Hz), 10.80 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 22.3, 27.6, 40.8,


1655 (C ON), 1502 (CC). 1H NMR (DMSO-d6) δ: 1.94 (m, 2H, 2′-H),

2.76 (t, 2H, 3′-H, J = 7.6 Hz), 3.98 (t, 2H, 1′-H, J = 7.5 Hz), 4.64 (s, 2H,
3″-H), 6.92–7.14 (m, 6H, 5-H, 6-H and 5″-H, 6″-H, 7″-H, 8″-H), 7.18
(d, 1H, 2-H, J = 1.8 Hz), 7.35 (d, 1H, 4-H, J = 8.1 Hz), 7.46 (d, 1H, 7-H,
J = 7.7 Hz), 10.83 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 21.8, 27.1, 40.7,
67.0, 111.3, 113.5, 115.3, 116.6, 118.1, 118.2, 120.9, 122.3, 122.6,
123.4, 127.0, 128.3, 136.3, 145.0, 163.9. Anal. calcd. for C19H18N2O2:
C, 74.49; H, 5.92; N, 9.14. Found: C, 73.85; H, 6.13; N, 9.21.
2
2′
67.6, 104.9 (d, JC-F = 26 Hz), 109.3 (d
J
= 22.7 Hz), 111.8,
C-F
3
114.0, 116.7 (d, JC-F = 10.0 Hz), 118.6, 118.7, 121.4, 122.8, 125.5
4
3′
(d, JC-F = 2.8 Hz), 127.5, 136.8, 146.4 (d,
J
C-F
= 12.2 Hz), 158.4
1
(d, JC-F = 240 Hz), 163.8. Anal. calcd. for C19H17FN2O2: C, 70.36; H,
4-(3-(1H-Indol-3-yl)propyl)-6-chloro-2H-benzo[b][1,4]oxazin-
3(4H)-one (7b)
5.28; N, 8.64. Found: C, 69.97; H, 5.12; N, 8.62.
To a solution of 6-chloro-2H-benzo[b][1,4]oxazin-3(4H)-one 6b (0.5 g
2.7 mmol) in dry DMF (5 mL) was added NaH 60% (0.13 g, 5.4 mmol),
and 3-(1H-indol-3-yl)propyl-4-methylbenzenesulfonate 3a (0.89 g,
2.7 mmol) to give 7b (0,49 g, 53%) as a yellow solid. Mp: 169.2–
170.1°C. IR: ν (cm−1): 3412 (N─H), 3057 (C─H arom.), 2956 (C─H
4-(3-(1H-Indol-3-yl)propyl)-6-methoxy-2H-benzo[b][1,4]oxazin-
3(4H)-one (7e)
To a solution of 7-methoxy-2H-benzo[b][1,4]oxazin-3(4H)-one 6e
(0.5 g, 2.8 mmol) in dry DMF (5 mL) was added NaH 60% (0.13 g,
5.6 mmol) and 3-(1H-indol-3-yl)propyl-4-methylbenzenesulfonate 3a
(0.92 g, 2.8 mmol) to give 7e (0.52 g, 55.4%) of a gray solid. Mp: 165.6–
166.4°C. IR: ν (cm−1): 3425 (N─H), 3052 (C─H arom.), 2987 (C─H
aliph.), 1697 (C ON), 1494 (C C). 1H NMR (DMSO-d6) δ: 1.92 (m,
2H, 2′-H), 2.75 (t, 2H, 3′-H, J = 7.3 Hz), 3.98 (t, 2H,1′-H, J = 7.3 Hz),
4.66 (s, 2H, 3″-H), 6.96 (t, 1H, 6-H, J = 7.4), 7.0–7.09 (m, 3H, 5-H, 5″-H,
8″-H), 7.17 (br.s, 2H, 2-H, 6″-H), 7.34 (d, 1H, 4-H, J = 8.1 Hz), 7.47 (d,
1H, 7-H, J = 7.8 Hz), 10.80 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 22.2,
27.4, 40.6, 67.4, 111.9, 113.9, 115.6, 118.4, 118.6, 118.7, 121.4,
122.8, 123.3, 127.0, 127.5, 130.2, 136.8, 144.4, 164.2. Anal. calcd. for


aliph.), 1713 (C ON), 1610 (C C). 1H NMR (DMSO-d6) δ: 1.93 (m,
2H, 2′-H), 2.76 (t, 2H, 3′-H, J = 7.3 Hz), 3.57 (s, 3H, OMe), 3.97 (t, 2H,
1′-H, J = 7.5 Hz), 4.56 (s, 2H, 3″-H), 6.56 (dd, 1H, 6″-H, Jo = 8.7,


Jm = 2.7), 6.60 (d, 1H, 8″-H, Jm = 2.5), 6.93 (d, 1H, 5″-H, J = 8.7 Hz),
6.96 (t, 1H, 5-H, J = 7.2 Hz), 7.06 (t, 1H, 6-H, J = 7.1 Hz), 7.17 (d, 1H,
2-H, J = 1.8 Hz), 7.34 (d, 1H, 4-H, J = 8 Hz), 7.47 (d, 1H, 7-H, J = 7.8 Hz),
10.81 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 22.2, 27.7, 40.7, 55.8, 67.7,
102.2, 108.3, 111.8, 114.0, 117.5, (2x)118.6, 121.4, 122.9, 127.5,
129.6, 136.8, 139.4, 155.4, 164.8. Anal. calcd. for C20H20N2O3: C,
71.41; H, 5.99; N, 8.33. Found: C, 71.20; H, 5.82; N, 8.14.
C19H17ClN2O2: C, 66.96; H, 5.03; N, 8.22. Found: C, 66.48; H, 5.01; N,
8.14.
4-(3-(1H-Indol-3-yl)propyl)-6-fluoro-2H-benzo[b][1,4]oxazin-
3(4H)-one (7c)
To a solution of 6-fluoro-2H-benzo[b][1,4]oxazin-3(4H)-one 6c (0.5 g,
3.0 mmol) in dry DMF (5 mL) was added NaH 60% (0.14 g, 6.0 mmol)
and 3-(1H-indol-3-yl)propyl-4-methylbenzenesulfonate 3a (0.99 g,
3.0 mmol) to give 7c (0.52 g, 54%) as a light brown solid. Mp:
4-(3-(1H-Indol-3-yl)propyl)-6-bromo-2H-benzo[b][1,4]oxazin-
3(4H)-one (7f)
To a solution of 6-bromo-2H-benzo[b][1,4]oxazin-3(4H)-one 6f (0.5 g,
2.2 mmol) in dry DMF (5.0 mL) was added NaH 60% (0.11 g, 4.4 mmol) and
3-(1H-indol-3-yl)propyl-4-methylbenzenesulfonate 3a (0.72 g, 2.2 mmol)
to give 7f (0.43 g, 51%) of a light red solid. Mp: 191.5–192.5°C. IR: ν(cm−1):
172.1–172.7°C. IR: ν (cm−1): 3410 (N─H), 3056 (C─H arom.), 2980
1


(C─H aliph.), 1696 (C ON), 1499 (C C). H NMR (DMSO-d6): δ: 1.92
(m, 2H, 2′-H), 2.75 (t, 2H, 3′-H, J = 7.5 Hz), 3.98 (t, 2H, 1′-H, J = 7.6 Hz),
4.63 (s, 2H, 3″-H), 6.83 (td, 1H, 6-H, Jo = 8.6 Hz and Jm = 2.8 Hz), 6.96
(t, 1H, 5-H, J = 7.1 Hz), 7.0–7.1 (m, 3H, 5″-H, 6″-H, 8″-H), 7.17 (d, 1H,
2-H, J = 2.0 Hz), 7.34 (d, 1H, 4-H, J = 8.0 Hz), 7.47 (d, 1H, 7-H,
J = 7.8 Hz), 10.80 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 22.3, 27.6, 40.8,

3411 (N─H), 3078 (C─H arom.), 2958 (C─H aliph.), 1682 (C ON), 1599
(C C). 1H NMR (DMSO-d6) δ: 1.91 (m, 2H, 2′-H), 2.75 (t, 2H, 3′-H,

J = 7.3 Hz), 3.98 (t, 2H, 1′-H, J = 7.4 Hz), 4.66 (s, 2H, 3″-H), 6.94–6.98 (m,
2H,6-H,5″-H), 7.06 (t, 1H, 5-H, J = 7.2 Hz),7.13–7.17 (m, 2H, 6″-H, 8″-H),
7.29 (d, 1H, 2-H, J = 1.4 Hz), 7.34 (d, 1H, 4-H, J = 8 Hz), 7.47 (d, 1H, 7-H,
J = 7.8 Hz), 10.81 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 22.2, 27.4, 40.7,
67.4, 111.9, 113.9, 114.6, 118.3, 118.6, 118.7, 118.9, 121.4, 122.8, 126.3,
127.5, 130.5, 136.8, 144.8, 164.2. Anal. calcd. for C19H17BrN2O2:
C, 59.23; H, 4.45; N, 7.27. Found: C, 59.11; H, 4.38; N, 7.23.
2
2′
67.6, 105.0 (d, JC-F = 26 Hz), 109.3 (d
J
C-F
= 23 Hz), 111.8, 114.0,
3
4
116.7 (d JC-F = 10.0 Hz), 118.6, 118.7, 121.4, 122.8, 125.5 (d, JC-
3′
1
F = 2.8 Hz), 127.50, 136.8, 146.4 (d,
J
C-F
= 12 Hz), 158.2 (d, JC-
F = 237 Hz), 163.8. Anal. calcd. for C19H17FN2O2: C, 70.36; H, 5.28; N,
8.64. Found: C, 70.09; H, 5.32; N, 8.56.
4-(3-(1H-Indol-3-yl)propyl)-7-fluoro-2H-benzo[b][1,4]oxazin-
3(4H)-one (7d)
4-(3-(5-Fluoro-1H-indol-3-yl)propyl)-2H-benzo[b][1,4]oxazin-
3(4H)-one (7g)
To a solution of 7-fluoro-2H-benzo[b][1,4]oxazin-3(4H)-one 6d (0.5 g,
3.0 mmol) in dry DMF (5 mL) was added NaH 60% (0.14 g, 6.0 mmol)
and 3-(1H-indol-3-yl)propyl-4-methylbenzenesulfonate 3a (0.99 g,
3.0 mmol) to give 7d (0.48 g, 50%) as a brown solid. Mp: 185.2–
186.1°C. IR: ν (cm−1): 3372 (N─H), 3080 (C─H Arom.), 2982 (C─H
To a solution of 2H-benzo[b][1,4]oxazin-3(4H)-one 6a (0.5 g, 3.4 mmol)
in dry DMF (5 mL) was added NaH 60% (0.16 g, 6.8 mmol) and 3-(5-
fluoro-1H-indol-3-yl)propyl-4-methylbenzenesulfonate 3b (1.16 g,
3.4 mmol) to give 7g (0.56 g, 52%) as a white solid. Mp: 133.4–
134.3°C. IR: ν (cm−1): 3426 (N─H), 3080 (C─H arom.), 2982 (C─H

12 of 17
MÉNDEZ-ROJAS ET AL.
|
aliph.), 1697 (C ON), 1607 (C C). 1H NMR (DMSO-d6) δ: 1.90 (m,
2H, 2′-H), 2.72 (t, 2H, 3′-H, J = 7.5 Hz), 3.97 (t, 2H, 1′-H, J = 7.6 Hz),
4.63 (s, 2H, 3″-H), 6.90 (td, 1H, 6-H, Jo = 9.2 Hz and Jm = 2.5 Hz),
6.97–7.03 (m, 3H, 5″-H, 6″-H, 8″-H), 7.10–7.14 (m, 1H, 7″-H), 7.21
(dd, 1H, 4-H, Jo = 10.0 Hz and Jm = 2.4 Hz), 7.25 (d, 1H, 2-H, J = 2.1 Hz),
7.33 (dd, 1H, 7-H, Jo = 8.8 Hz and Jm = 4.6 Hz), 10.90 (s, 1H,
NH). 13C NMR (DMSO-d6) δ: 22.2, 27.6, 40.0, 67.5, 103.4
7-Fluoro-4-(3-(5-fluoro-1H-indol-3-yl)propyl)-2H-benzo[b][1,4]-
oxazin-3(4H)-one (7j)


To a solution of 7-fluoro-2H-benzo[b][1,4]oxazin-3(4H)-one 6d (0.5 g,
3.0 mmol) in dry DMF (5 mL) was added NaH 60% (0.14 g, 6.0 mmol)
and 3-(5-fluoro-1H-indol-3-yl)propyl-4-methylbenzenesulfonate 3b
(1.04 g, 3.0 mmol) to give 7j (0.51 g, 50%) as a white solid. Mp:
184.3–185.2°C. IR: ν (cm−1): 3473 (N─H), 3080 (C─H arom.), 2981
2
2′
3
1


(d, JC-F = 23.2 Hz), 109.4 (d,
J
C-F
= 26.0 Hz), 112.7 (d, JC-F
=
(C─H aliph.), 1678 (C ON), 1608 (C C). H NMR (DMSO-d6) δ: 1.90
(q, 2H, 2′-H, J = 7.5 Hz), 2.71 (t, 2H, 1′-H, J = 7.5 Hz), 3.97 (t, 2H, 3-H,
J = 7.4 Hz), 4.67 (s, 2H, 3″-H), 6.84–6.98 (m, 3H, 6-H, 5″-H, 7″-H),
7.10–7.23 (m, 2H, 4-H, 8″-H), 7.25 (d, 1H, 2-H, J = 2.1 Hz), 7.33 (dd,
1H, 7-H, Jo = 8.9 and Jm = 4.6 Hz), 10.90 (s, 1H, NH). 13C NMR (DMSO-
4
9.4 Hz), 114.3 (d, JC-F = 5.0 Hz), 115.8, 117.1, 123.1, 123.9,
3′
125.0, 127.7 (d,
J
= 10.0 Hz), 128.8, 133.4, 145.9, 157.2
C-F
1
(d, JC-F = 210 Hz), 164.4. Anal. calcd. for C19H17FN2O2: C, 70.36; H,
5.28; N, 8.64. Found: C, 70.14; H, 5.21; N, 8.56.
d6) δ: 22.2, 27.5, 40.7, 67.5, 103.3 (d, 2JC-F = 22.7 Hz), 105.0 (d, 2′JC-F
=
3
2″
6-Chloro-4-(3-(5-fluoro-1H-indol-3-yl)propyl)-2H-benzo[b][1,4]-
oxazin-3(4H)-one (7h)
26 Hz), 109.2 (d, JC-F = 11.6 Hz), 109.5 (d,
J
C-F
= 15.5 Hz), 112.7
3′
4
3″
(d,
J
C-F
= 9.4 Hz), 114.3 (d, JC-F = 4.4 Hz), 116.7 (d,
J
= 9.4 Hz),
C-F
4′
3″′
To a solution of 6-chloro-2H-benzo[b][1,4]oxazin-3(4H)-one 6b (0.5 g,
2.7 mmol) in dry DMF (5 mL) was added NaH 60% (0.13 g, 5.4 mmol)
and 3-(5-fluoro-1H-indol-3-yl)propyl-4-methylbenzenesulfonate 3b
(0.95 g, 2.7 mmol) to give 7h (0.48 g, 49%) as a light brown solid.
125.0, 125.4 (d,
J
= 3.3 Hz), 127.7 (d,
J
C-F
= 10.0 Hz), 133.4,
C-F
2″′
1
146.4 (d,
J
= 12.2 Hz), 157.0 (d, JC-F = 230.5 Hz), 158.4 (d,
C-F
1′
J
= 240.5 Hz), 163.8. Anal. calcd. for C19H16F2N2O2: C, 66.66; H,
C-F
4.71; N, 8.18. Found: C, 66.57; H, 4.65; N, 8.01.
Mp: 130.8–131.5°C. IR: ν (cm−1): 3365 (N─H), 3081 (C─H arom.),
1


2952 (C─H aliph.), 1695 (C ON), 1603 (C C). H NMR (DMSO-d6) δ:
1.90 (q, 2H, 2′-H, J = 7.4 Hz), 2.73 (t, 2H, 1′-H, J = 7.3 Hz), 3.96 (t, 2H,
3-H, J = 7.5 Hz), 4.65 (s, 2H, 3″-H), 6.91 (td, 1H, 6-H, Jo = 9.2 Hz and
4-(3-(5-Fluoro-1H-indol-3-yl)propyl)-6-methoxy-2H-benzo[b]-
[1,4]oxazin-3(4H)-one (7k)
To a solution of 6-methoxy-2H-benzo[b][1,4]oxazin-3(4H)-one 6e
(0.5 g, 2.8 mmol) in dry DMF (5 mL) was added NaH 60% (0.13 g,
5.6 mmol) and 3-(5-fluoro-1H-indol-3-yl)propyl-4-methylbenzenesul-
fonate 3b (0.97 g, 2.8 mmol) to give 7k (0.47 g, 48 %) as a light brown
solid. Mp: 161.5–162.3°C. IR: ν (cm-1): 3434 (N─H), 3052 (C─H
J
m = 2.5 Hz), 7.01 (m, 2H, 5″-H and 8″-H), 7.13 (m, 1H, 6″-H), 7.20–
7.28 (m, 2H, 2-H, 4-H), 7.34 (dd, 1H, 7-H, Jo = 8.8 and Jm = 4.5 Hz),
10.92 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 22.0, 27.2, 40.0, 67.3, 103.2
2
2′
3
(d, JC-F = 23 Hz), 109.4 (d,
J
C-F
= 26 Hz), 112.7 (d, JC-F = 9,8 Hz),
4
arom.), 2987 (C─H aliph.), 1712 (C ON), 1518 (C C). 1H NMR
(DMSO-d6) δ: 1.90 (q, 2H, 2′-H, J = 7.4 Hz), 2.72 (t, 2H, 1′-H,
J = 7.3 Hz), 3.60 (s, 3H, Ar-OMe), 3.95 (t, 2H, 3-H, J = 7.5 Hz), 4.44
(s, 2H, 3″-H), 6.55 (dd, 1H, 6″-H, Jo = 8.7 and Jm = 2.7 Hz), 6.61 (d, 1H,
8″-H, Jm = 2.6 Hz), 6.90 (td, 1H, 6-H, Jo = 9.2 and Jm = 2.4 Hz), 6.93 (d,
1H, 5″-H, Jo = 8.7 Hz), 7.22 (dd, 1H, 4-H, Jo = 10.1 and Jm = 2.3 Hz),
7.26 (d, 1H, 2-H, J = 1.9 Hz), 7.32 (dd, 1H, 7-H, Jo = 8.8 and Jm = 4.6 Hz),
10.91 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 22.1, 27.6, 40.1, 55.9, 67.7,


114.1 (d, JC-F = 4.9 Hz), 115.5, 118.3, 123.2, 124.9, 126.9, 127.6 (d,
3′
J
= 9.3 Hz), 130.1, 133.40, 144.2, 157.0 (d, 1JC-F = 231 Hz), 164.1.
C-F
Anal. calcd. for C19H16ClFN2O2: C, 63.60; H, 4.50; N, 7.81. Found:
C, 62.97; H, 4.46; N, 7.75.
6-Fluoro-4-(3-(5-fluoro-1H-indol-3-yl)propyl)-2H-benzo[b][1,4]-
oxazin-3(4H)-one (7i)
To a solution of 6-fluoro-2H-benzo[b][1,4]oxazin-3(4H)-one 6c (0.5 g,
3.0 mmol) in dry DMF (5 mL) was added NaH 60% (0.14 g, 6.0 mmol)
and 3-(5-fluoro-1H-indol-3-yl)propyl-4-methylbenzenesulfonate 3b
(1.04 g, 3.0 mmol) to give 7i (0.55 g, 54%) as a light purple solid. Mp:
2
2′
102.3, 103.3 (d, JC-F = 22.7 Hz), 108.2, 109.4 (d,
112.7 (d, 3JC-F = 10 Hz), 114.3 (d, 4JC-F = 4.4 Hz), 117.4, 125.1, 127.7 (d,
J
C-F
= 26.5 Hz),
3′
1
J
= 9.4 Hz), 129.6, 133.4, 139.4, 155.4, 157.1 (d, JC-F = 231 Hz),
C-F
176.2–177.1°C. IR: ν (cm−1): 3279 (N─H), 3060 (C─H arom.), 2979
164.8. Anal. calcd. for C20H19F2N2O3: C, 67.79; H, 5.40; N, 7.91.
Found: C, 67.36; H, 5.32; N, 7.87.
1


(C─H aliph.), 1697 (C ON), 1604 (C C). H NMR (DMSO-d6) δ: 1.91
(q, 2H, 2′-H, J = 7.5 Hz), 2.73 (t, 2H, 1′-H, J = 7.5 Hz), 3.96 (t, 2H, 3-H,
J = 7.5 Hz), 4.63 (s, 2H, 3″-H), 6.82 (td, 1H, 6″-H, Jo = 8.5 Hz and
6-Bromo-4-(3-(5-fluoro-1H-indol-3-yl)propyl)-2H-benzo[b][1,4]-
oxazin-3(4H)-one (7l)
J
m = 2.8 Hz), 6.91 (td, 1H, 6-H, Jo = 9.2 Hz and Jm = 2.5 Hz), 7.02
(dd, 2H, 5″-H, 8″-H, Jo = 8.9 Hz and Jm = 5.4 Hz), 7.20–7.28 (m, 2H,
2-H, 4-H), 7.34 (dd, 1H, 7-H, Jo = 8.8 Hz and Jm = 4.7 Hz), 10.91
(s, 1H, NH). 13C NMR (DMSO-d6) δ: 21.6, 26.9, 40.2, 67.0, 102.8
(d, 2JC-F = 23.1 Hz), 103.0 (d, 2′JC-F = 28.5 Hz), 108.9 (d, 2″JC-F = 25.8 Hz),
To a solution of 6-bromo-2H-benzo[b][1,4]oxazin-3(4H)-one 6f (0.5 g,
2.2 mmol) in dry DMF (5 mL) was added NaH 60% (0.11 g, 4.4 mmol)
and 3-(5-fluoro-1H-indol-3-yl)propyl-4-methylbenzenesulfonate 3b
(0.76 g, 2.2 mmol) to give 7l (0.52 g, 59 %) as a gray solid. Mp:
2″′
3
4
109.0 (d,
J
C-F
= 23.1 Hz), 112.2 (d, JC-F = 9.9 Hz), 113.8 (d, JC-F
=
195.3–196.1°C. IR: ν (cm−1): 3423 (N─H), 3080 (C─H arom.), 2960
1
4.4 Hz), 117.3 (d, 3′JC-F = 9.9 Hz), 124.5, 127.2, (d, 3″JC-F = 9.9 Hz), 129.5
(C─H aliph.), 1682 (C ON), 1599 (C C). H NMR (DMSO-d6) δ: 1.89
(q, 2H, 2′-H, J = 7.5 Hz), 2.73 (t, 2H, 1′-H, J = 7.34 Hz), 3.96 (t, 2H, 3-H,
J = 7.5 Hz), 4.66 (s, 2H, 3″-H), 6.91 (td, 1H, 6-H, Jo = 9.1 and


3″′
4′
1
(d,
J
C-F
= 11.0 Hz), 132.9, 141.2 (d,
J
C-F
= 2.2 Hz), 156.6 (d, JC-F =
1′
231 Hz), 157.7 (d,
J
C-F
= 237 Hz), 163.9. Anal. calcd. for
C19H16F2N2O2: C, 66.66; H, 4.71; N, 8.18. Found: C, 66.26; H, 4.68;
Jm = 2.5 Hz), 6.96 (d, 1H, 5″-H, Jo = 8.6 Hz), 7.14 (dd, 1H, 6″-H,
N, 8.10.
Jo = 8.5 and Jm = 1.9 Hz), 7.20–7.27 (m, 3H, 2-H, 4-H, 8″-H), 7.34 (dd,

MÉNDEZ-ROJAS ET AL.
13 of 17
|
1H, 7-H, Jo = 8.8 and Jm = 4.5 Hz), 10.92 (s, 1H, NH). 13C NMR (DMSO-
(0.43 g, 2.4 mmol) and K2CO3 (0.33 g, 2.4 mmol) to give pure 10c
d6) δ: 21.6, 26.8, 39.5, 66.9, 102.8 (d, 2JC-F = 23.1 Hz), 109.0 (d, 2′JC-F
=
(0.53 g, 63%) as a light red solid. Mp: 119.1–119.4°C. IR ν (cm−1): 3049
25.8 Hz), 112.3 (d, 3JC-F = 9.9 Hz), 113.7 (d, 4JC-F = 4.9 Hz), 114.0, 117.7,
118.4, 127.2(d, 3′JC-F = 9.3 Hz), 124.5, 125.8, 130.0, 133.0, 144.3, 156.6
(d, 1JC-F = 231 Hz), 163.7. Anal. calcd. for C19H16FBrN2O2: C, 56.59; H,
4.00; N, 6.95. Found: C, 56.14; H, 3.96; N, 6.89.
(C─H arom.), 2937 (C─H aliph.), 1650 (C ON), 1582 (C C). H NMR
(CDCl3) δ: 2.65 (t, 4H, 2″-H, 6″-H, J = 4.9 Hz), 3.05 (t, 4H, 3″-H, 5″-H,
J = 4.9 Hz), 3.35 (s, 2H, 2′-H), 3.92 (t, 2H, 3-H, J = 4.7 Hz), 4.23 (t, 2H,
2-H, J = 4.6 Hz), 6.75–6.90 (m, 6H, 5-H, 8-H, 2″′-H, 3″′-H, 5″′-H and
6″′-H), 7.0 (t, 1H, 6-H or 7-H, J = 7.6 Hz), 7.47 (br.s, 1H, 7-H or 6-H).
13C NMR (CDCl3): 43.2, (2x)49.6, (2x)52.7, 66.3, 76.8, (2x)115.0
1


|
4.1.5 General procedure for the synthesis of 1-(2,3-
2
3
(d, JC-F = 22 Hz), 116.8, 117.3, (2x)117.4 (d, JC-F = 7.7 Hz), 119.7,
123.6, 125.6 (d, 4JC-F = 3.3 Hz), 146.3, 147.3, 156.7 (d, 1JC-F = 239 Hz),
167.3. HRMS: (EI) calcd. for C20H22FN3O2 (M+1): 356.1774. Found:
356.1780.
dihydro-4H-benzo[b][1,4]oxazin-4-yl)-2-(4-
arylpiperazin-1-yl)ethan-1-one derivatives 10a–j, with
1-(2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-2-(4-(4-
nitrophenyl)piperazin-1-yl)ethan-1-one (10a) as a
model
1-(2,3-Dihiydro-4H-benzo[b][1,4]oxazin-4-yl)-2-(4-(4-
To a solution of 2-chloro-1-(2,3-dihydro-4H-benzo[b][1,4]oxazin-4-
yl)-ethan-1-one 9a (0.5 g, 2.4 mmol) in AcCN (30 mL) was added 1-(4-
nitrophenyl)piperazine (0.49 g, 2.4 mmol), K2CO3 (0.33 g 2,4 mmol)
and the mixture heated at 80°C for 24 h. After completion, the reaction
mixture was quenched with water (50 mL) and extracted with AcOEt
(2 × 50 mL). The combined organic layers were dried over anhydrous
Na2SO4 and concentrated under vacuum to give crude 10a which was
purified by silica gel column chromatography (AcOEt/n-hexane 2:1) to
afford pure 10a (0.52 g, 57%.) as a yellow solid. Mp: 147.2–148.1°C. IR
(trifluoromethyl)phenyl)piperazin-1-yl)ethan-1-one (10d)
Prepared from 2-chloro-1-(2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-
ethan-1-one (9a) (0.5 g, 2.4 mmol), 1-(4-(trifluoromethyl)phenyl)-
piperazine (0.54 g 2.4 mmol) and K2CO3 (0.33 g, 2.4 mmol) to give
pure 10d (0.62 g, 65%) as a light pink solid. Mp: 118.6–119.3°C. IR ν
(cm−1): 3045 (C─H arom.), 2947 (C─H aliph.), 1651 (C ON), 1583

1

(C C). H NMR (CDCl3) δ: 2.65 (t, 4H, 2″-H, 6″-H, J = 4.9 Hz), 3.22 (t,
4H, 3″-H, 5″-H, J = 5.0 Hz), 3.36 (s, 2H, 2′-H), 3.92 (t, 2H, 3-H,
J = 4.6 Hz), 4.24 (t, 2H, 2-H, J = 4.6 Hz), 6.80–6.87 (m, 4H, 5-H, 8-H, 2″
′-H and 6″′-H), 7.01 (t, 1H, 6-H or 7-H, J = 7.5 Hz), 7.40 (d, 2H, 3″′-H
and 5″′-H, J = 8.8 Hz), 8.0 (br.s, 1H, 7-H or 6-H). 13C MNR (CDCl3):
(2x)47.4, (2x)52.4, 59.9, 66.3, 76.8, 114.1, 116.8, 119.7, (2x)120.1
ν (cm−1): 3073 (C─H arom.), 2955 (C─H aliph.), 1665 (C ON), 1596

(C C). 1H NMR (CDCl3) δ: 2.74 (t, 4H, 2″-H, 5″-H, J = 5.0 Hz), 3.43–

3.46 (m, 6H, 3″-H, 5″-H and 2′-H), 4.0 (t, 2H, 2-H, J = 4.9 Hz), 4.32 (t,
2H, 3-H, J = 4.5 Hz), 6.82 (d, 2H, 2″′-H and 6″′-H, J = 9.5 Hz), 6.92 (d,
2H, 5-H and 8-H, J = 7.5 Hz), 8.11 (d, 2H, 3″′-H, 5″′-H, J = 9.5 Hz), 7.10
(t, 1H, 6-H, J = 7.3 Hz), 7.30–7.50 (br.s, 1H, 7-H). 13C NMR (CDCl3)
43.5, (2x)46.9, (2x)52.6, 66.9, 77.3, (2x)112.8, 115.3, 117.4, 120.2,
123.9, (2x)125.9, 138.5, 143.2, 146.8, 154.8, 168.0. HRMS: (EI) calcd.
for C20H22N4O4 (M+1): 383.1719. Found: 383.1699.
3
1
(c, JC-F = 32.4 Hz), 122.4, 123.6, 124.2 (c, JC-F = 272 Hz), (2x)125.9
(c, 4JC-F = 3.8 Hz), 131.7 (c, 2JC-F = 79 Hz), 146.4, 152.7, 167.3. HRMS:
(EI) calcd. for C21H22F3N3O2 (M+1): 406.1742. Found: 406.1733.
1-(2,3-Dihydro-4H-benzo[b][1,4]oxazin-4-yl)-2-(4-(2-
methoxyphenyl)piperazin-1-yl)ethan-1-one (10e)
Prepared from 2-chloro-1-(2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-
ethan-1-one (9a) (0.5 g, 2.4 mmol), 1-(2-methoxyphenyl)piperazine
(0.45 g, 2.4 mmol) and K2CO3 (0.33 g, 2.4 mmol) to give pure 10e
(0.57 g, 66%) as a dark brown solid. Mp: 101.3–102.2°C. IR ν (cm−1):
1-(2,3-Dihydro-4H-benzo[b][1,4]oxazin-4-yl)-2-(4-(4-
methoxyphenyl)piperazin-1-yl)ethan-1-one (10b)
Prepared from 2-chloro-1-(2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-
ethan-1-one (9a) (0.5 g, 2.4 mmol), 1-(4-methoxyphenyl)piperazine
(0.45 g, 2.4 mmol) and K2CO3 (0.33 g, 2.4 mmol) to give pure 10b
(0.51 g, 59%) as a brown solid. Mp: 95.0–95.7°C. IR ν (cm−1): 3063


3058 (C─H arom.), 2929 (C─H aliph.), 1675 (C ON), 1584 (C C),
1238 (C─O). 1H NMR (CDCl3) δ: 2.70 (br.s, 4H, 2″-H, 6″-H), 3.04 (br.s,
4H, 3″-H, 5″-H), 3.36 (s, 2H, 2′-H), 3.79 (s, 3H, Ar-OMe), 3.95 (t, 2H, 3-
H, J = 4.4 Hz), 4.26 (t, 2H, 2-H, J = 4.4 Hz), 6.75–7.10 (m, 7H, 5-H, 6-H
or 7-H, 8-H, 3″′-H, 4″′-H, 5″′-H, and 6″′-H), 7.42 (br.s, 1H, 7-H or 6-
H). 13C NMR (CDCl3): (2x)40.4, (2x)50.0, 53.0, 54.9, 66.2, 76.8, 110.7,
116.7, 117.7, 119.7, 120.5, 122.5, 123.7, 125.7, 134.3, 140.6, 146.3,
151.8, 167.5. HRMS: (EI) calcd. for C21H25N3O3 (M+1): 368.1974.
Found: 368.1987.


(C─H arom.), 2963 (C─H aliph.), 1666 (C ON), 1582 (C C), 1226
(C─O). 1H NMR (CDCl3) δ: 2.65 (t, 4H, 2″-H, 6″-H, J = 4.7 Hz), 3.03 (t,
4H, 3″-H, 5″-H, J = 4.7 Hz), 3.35 (s, 2H, 2′-H), 3.68 (s, 3H, Ar-OMe),
3.92 (t, 2H, 3-H, J = 4.6 Hz), 4.24 (t, 2H, 2-H, J = 4.6 Hz), 6.7–6.9 (m, 6H,
5-H, 8-H, 2″′-H, 3″′-H, 5″′-H and 6″′-H), 7.0 (t, 1H, 6-H or 7-H,
J = 7.5 Hz), 7.42 (br.s, 1H, 7-H or 6-H). 13C NMR (CDCl3) 42.1, (2x)50.6,
(2x)53.3, 55.6, 66.6, 77.3, (2x)114.4, 117.2, (2x)118.2, 120.2, 124.1,
126.2, 135.2, 145.6, 146.8, 153.9, 167.8. HRMS: (EI) calcd. for
1-(6-Fluoro-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-2-(4-(4-
nitrophenyl)piperazin-1-yl)ethan-1-one (10f)
C21H25N3O3 (M+1): 368.1974. Found: 368.1984.
Prepared from from 2-chloro-1-(6-fluoro-2,3-dihydro-4H-benzo[b]-
[1,4]oxazin-4-yl)ethan-1-one (9b) (0.5 g, 2.2 mmol), 1-(4-nitrophenyl)-
piperazine (0.45 g, 2.2 mmol) and K2CO3 (0.30 g, 2.2 mmol) to give
pure 10f (0.52 g, 60%) as a yellow solid. Mp: 192.4–193.3°C. IR ν
1-(2,3-Dihydro-4H-benzo[b][1,4]oxazin-4-yl)-2-(4-(4-
fluorophenyl)piperazin-1-yl)ethan-1-one (10c)
Prepared from 2-chloro-1-(2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-
ethan-1-one (9a) (0.5 g, 2.4 mmol), 1-(4-fluorophenyl)piperazine
(cm−1): 3043 (C─H arom.), 2946 (C─H aliph.), 1664 (C ON), 1599


14 of 17
MÉNDEZ-ROJAS ET AL.
|
1
(C C), 1496 (NO2 asym.), 1320 (NO2 sym.). 1H NMR (DMSO-d6) δ:
2.63 (br.s, 4H, 2″-H, 6″-H), 3.48 (br.s, 6H, 3″-H, 5″-H, 2′-H), 3.95 (t,
2H, 3-H, J = 3.9 Hz), 4.32 (t, 2H, 2-H, J = 3.9 Hz), 6.91 (d, 2H, 7-H and
8-H, J = 9.9 Hz), 7.03 (d, 2H, 2″′- H and 6″′-H, J = 9.1 Hz), 7.93 (d, 1H,
(C C). H NMR (DMSO-d6) δ: 2.62 (t, 4H, 2″-H, 6″-H, J = 4.4 Hz), 3.28
(br.s, 4H, 3″-H, 5″-H), 3.46 (s, 2H, 2′-H), 3.95 (t, 2H, 3-H, J = 4.0 Hz),
4.27 (t, 2H, 2-H, J = 4.3 Hz), 6.91 (dd, 2H, 7-H, 8-H, Jo = 7.4 Hz and


J
m = 1.2 Hz), 7.06 (d, 2H, 2″′-H, 6″′-H, J = 8.7 Hz), 7.51 (d, 2H, 3″′-H,
5-H, J = 9.9 Hz), 8.06 (d, 2H, 3″′-H and 5″′-H, J = 9.0 Hz). 13C NMR
5″′-H, J = 8.7 Hz), 7.92 (br.d, 1H, 5-H, J = 10.5 Hz). 13C NMR (DMSO-
2
(DMSO-d6) δ: 43.2, (2x)46.8, (2x)52.3, 61.4, 66.3, 110.3 (d, JC-F
=
d6): (2x)47.5, (2x)52.5, 60.3, 66.1, 76.6, 110.4 (d, 2JC-F = 27.1 Hz), 112.1
2′
2′
27 Hz), 112.8 (d,
126.2, 126.9 (d,
J
= 30 Hz), (2x)113.1, 117.9 (d, 3JC-F = 8.8 Hz), (2x)
(d,
J
= 25.4 Hz), 117.9 (d, 3JC-F = 8.9 Hz), (2x)121 (c, 3″JC-F = 32 Hz),
C-F
3′
C-F
4
4″
3′
1″
J
= 11.5 Hz), 137.4, 143.1 (d, JC-F = 2.2 Hz),
126.6 (c,
J
C-F
= 3.9 Hz), 126.9 (d,
J
= 11.1 Hz), 129.3 (c,
J
C-F
=
C-F
C-F
1
2″
4′
155.1, 155.6 (d, JC-F = 234 Hz), 168.5 ppm. HRMS: (EI) calcd. for
20H21FN4O4 (M+1): 401.1625. Found: 401.1620.
274 Hz), (2x)133.1 (c,
J
= 82 Hz), 143.1 (d,
J
= 2.2 Hz), 151.5,
C-F
C-F
1
C
162.9 (d, JC-F = 250 Hz), 168.6. HRMS: (EI) calcd. for C21H21F4N3O2
(M+1): 424.1648. Found: 424.1663.
1-(6-Fluoro-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-2-(4-(4-
methoxiphenyl)piperazin-1-yl)ethan-1-one (10g)
1-(6-Fluoro-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-2-(4-(2-
methoxyphenyl)piperazin-1-yl)ehan-1-one (10j)
Prepared from 2-chloro-1-(6-fluoro-2,3-dihydro-4H-benzo[b][1,4]ox-
azin-4-yl)ethan-1-one (9b) (0.5 g, 2.2 mmol), 1-(4-methoxyphenyl)-
piperazine (0.42 g, 2.2 mmol) and K2CO3 (0.30 g, 2.2 mmol) to give
pure 10g (0.59 g, 70%) as a purple solid. Mp: 121.3–122.1°C. IR ν
Compound 10j was prepared in a similar fashion from 2-chloro-1-(6-
fluoro-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)ethan-1-one
(9b)
(0.5 g, 2.2 mmol), 1-(2-methoxyphenyl)piperazine (0.42 g, 2.2 mmol),
(cm−1): 3041 (C─H arom.), 2959 (C─H aliph.), 1665 (C ON), 1581
K2CO3 (0.30 g, 2.2 mmol) to give pure 10j (0.61 g, 73%) as a brown
solid. Mp: 125.8–126.2°C. IR ν (cm−1): 3054 (C─H arom.), 2937 (C─H

1

(C C), 1245 (C─O). H NMR (DMSO-d6) δ: 2.61 (br.s, 4H, 2″-H, 6″-H),
1


3.01 (br.s, 4H, 3″-H, 5″-H), 3.43 (s, 2H, 2′-H), 3.68 (s, 3H, Ar-OMe),
3.95 (t, 2H, 2-H, J = 3.9 Hz), 4.27 (t, 2H, 3-H, J = 4.3 Hz), 6.77–6.94 (m,
6H, 7-H, 8-H, 2″′-H, 3″′-H, 5″′-H, 6″′-H), 7.93 (d, 1H, 5-H,
J = 10.6 Hz). 13C NMR (DMSO-d6) δ: 43.1, (2x)50.1, (2x)52.3, 55.6,
61.8, 66.1, 110.4 (d, 2JC-F = 28.5 Hz), 112 (d, 2′JC-F = 22 Hz), (2x)114.7,
aliph.), 1660 (NC O), 1593 (C C), 1239 (C─O). H NMR (DMSO-d6)
δ: 2.63 (br.s, 4H, 2″-H, 6″-H), 2.97 (br.s, 4H, 3″-H, 5″-H), 3.45 (s, 2H,
2′-H), 3.77 (s, 3H, Ar-OMe,) 3.96 (t, 2H, 3-H, J = 4.3 Hz), 4.28 (t, 2H,
2-H, J = 4.2 Hz), 6.82–6.97 (m, 6H, 3″′-H, 4″′-H, 5″′-H, 6″′-H and 7-H,
8-H), 7.94 (br.d, 1H, 5-H, J = 10.7 Hz). 13C NMR (DMSO-d6): 43.2, (2x)
3
3′
117.8 (d, JC-F = 8.6 Hz), (2x)117.9, 127.0 (d,
J
C-F
= 11 Hz), 143
50.5, (2x)53.1, 55.8, 61.9, 66.1, 110.3 (d, 2JC-F = 28 Hz), 112 (d, 2′JC-F
=
3
(d, 4JC-F = 2.2 Hz), 145.8, 153.4, 155.5 (d, 1JC-F = 233 Hz), 166.7. HRMS:
23.6 Hz), 112.4, 117.9 (d, JC-F = 8.8 Hz), 118.4, 121.3, 122.9, 127
3′
4
(EI) calcd. for C21H24FN3O3 (M+1): 386.1880. Found: 386.1875.
(d,
J
C-F
= 11.5 Hz), 141.5, 143 (d, JC-F = 2.2 Hz), 152.4, 155.5
1
(d, JC-F = 234 Hz), 168.7. HRMS: (EI) calcd. for C21H24FN3O3 (M+1):
1-(6-Fluoro-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-2-(4-(4-
fluorophenyl)piperazin-1-yl)ethan-1-one (10h)
386.1880. Found: 386.1823.
Prepared from 2-chloro-1-(6-fluoro-2,3-dihydro-4H-benzo[b][1,4]ox-
azin-4-yl)ethan-1-one (9b) (0.5 g 2.2 mmol), 1-(4-fluorophenyl)pipera-
zine (0.39 g, 2.2 mmol) and K2CO3 (0.30 g, 2.2 mmol) to give pure 10h
1-(2,3-Dihydro-4H-benzo[b][1,4]oxazin-4-yl)-2-(4-(2-
fluorobenzyl)piperazin-1-yl)ethan-1-one (11a)
Prepared from 2-chloro-1-(2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)
ethan-1-one (9a) (0.5 g, 2.4 mmol), 1-(2-fluorobenzyl)piperazine
(0.46 g, 2.4 mmol) and K2CO3 (0.33 g, 2.4 mmol) to give pure 11a
(0.67 g, 77%) as brown light solid. Mp: 63.2–64.2°C. IR ν (cm−1): 3042
(0.57 g, 70%) as a brown solid. Mp: 118.3–119.2°C. IR ν (cm−1): 3051
1


(C─H arom.), 2941 (C─H aliph.), 1665 (C ON), 1612 (C C). H NMR
(DMSO-d6) δ: 2.62 (t, 4H, 2″-H, 6″-H, J = 4.7 Hz), 3.08 (t, 4H, 3″-H, 5″-
H, J = 4.5 Hz), 3.44 (s, 2H, 2′-H), 3.95 (t, 2H, 3-H, J = 4.2 Hz), 4.27 (t, 2H,
2-H, J = 4.3 Hz), 6.85–7.10 (m, 6H, 7-H, 8-H, 2″′-H, 3″′-H, 5″′-H and
6″′-H), 7.93 (d, 1H, 5-H, J = 10.7 Hz). 13C NMR (DMSO-d6): 43.5, (2x)
(C─H arom.), 2938 (C─H aliph.), 1661 (CON), 1585 (C C). 1H NMR

(CDCl3): 2,46 (br.s., 4H, 2″-H, 6″-H or 3″-H, 5″-H), 2.51 (br.s., 4H,
3″-H, 5″-H or 2″-H, 6″-H), 3.27 (s, 2H, 1″′-H), 3.51 (s, 2H, 2′-H), 3.90
(t, 2H, 3-H, J = 4.7 Hz), 4.22 (t, 2H, 2-H, J = 4.7 Hz), 6.75–7.35 (m, 7H,
5-H, 6-H or 7-H, 8-H, 3″″-H, 4″″-H, 5″″-H, 6″″-H), 7.91 (br.s, 1H, 7-H
or 6-H). 13C NMR (CDCl3): 26.4, (2x)52.2, (2x)52.6, 54.6, 66.2, 76.8,
114.7 (d, 2JC-F = 22.5 Hz), 116.6, 119.6, 123.3 (d, 4JC-F = 3.8 Hz), 123.6,
123.9, 124.0 (d, 2′JC-F = 14.8 Hz), 125.6, 128.3 (d, 3JC-F = 8.2 Hz), 131.1
49.5, (2x)52.8, 61,7, 66.1, 110.4 (d, 2JC-F = 29.0 Hz), 112.0 (d, 2′JC-F
=
2″
3
25.0 Hz), 115.7 (d,
J
= 21.4 Hz) (2x),117.6 (d, JC-F = 7.7 Hz), (2x)
C-F
3′
3″
4
117.9 (d,
J
= 9.3 Hz), 126.9 (d,
J
C-F
= 11.0 Hz), 143.0 (d, JC-F
=
C-F
4′
1
2.2 Hz), 148.3 (d,
J
C-F
= 1.6 Hz), 155.5 (d, JC-F = 234 Hz), 156.5 (d,
1′
J
= 235 Hz), 168.7. HRMS: (EI) calcd. for C20H21F2N3O2 (M+1):
C-F
1
374.1680. Found: 374.1604.
(d, 3′JC-F = 4.4 Hz), 146.2, 160.9 (d, JC-F = 246 Hz), 167.4. HRMS: (EI)
calcd. for C21H24FN3O2 (M+1): 370.1931. Found: 370.1926.
1-(6-Fluoro-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-2-(4-(4-
(trifluoromethyl)phenyl)piperazin-1-yl)ethan-1-one (10i)
1-(6-Fluoro-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-2-(4-(2-
fluorobenzyl)piperazin-1-yl)ethan-1-one (11b)
Prepared from 2-chloro-1-(6-fluoro-2,3-dihydro-4H-benzo[b][1,4]ox-
azin-4-yl)ethan-1-one (9b) (0.5 g, 2.2 mmol), 1-(4-(trifluoromethyl)-
phenyl)piperazine (0.50 g, 2.2 mmol) and K2CO3 (0.30 g, 2.2 mmol) to
give pure 10i (0.62 g, 67%) as a light red solid. Mp: 127.3–128.1°C. IR ν
Compound 11b was prepared in a similar fashion from 2-chloro-1-(6-
fluoro-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)ethan-1-one
(9b)
(0.5 g, 2.2 mmol), 1-(2-fluorobenzyl)piperazine (0.42 g, 2.2 mmol),
(cm−1): 3055 (C─H arom.), 2941 (C─H aliph.), 1660 (N─C O), 1614
K2CO3 (0.30 g, 2.2 mmol) to give pure 11b (0.58 g, 69%) as brown


MÉNDEZ-ROJAS ET AL.
15 of 17
|
light solid. Mp: 69.8–70.1°C. IR ν (cm−1): 3082 (C─H arom.), 2944
4.2 Biological assays
|
(C─H aliph.), 1658 (C ON), 1585 (C C). 1H NMR (DMSO-d6): 2.40
(br.s, 4H, 2″-H, 6″-H or 3″-H and 5″-H), 2.47 (br.s, 4H, 3″-H, 5″-H or
2″-H, 6″-H), 3.35 (s, 2H, 1″′-H), 3.51 (s, 2H, 2′-H), 3.91 (t, 2H, 3-H,
J = 4.1 Hz), 4.24 (t, 2H, 2-H, J = 4.2 Hz), 6.89 (d, 2H, 3″″-H, 6″″-H,
J = 6.7 Hz.), 7.14 (d, 1H, 7-H or 8-H, J = 8.4 Hz), 7.18 (d, 1H, 8-H or 7-H,
J = 6.9 Hz), 7.30 (t, 1H, 4″″-H or 5″″-H, J = 6.7 Hz), 7.39 (t, 1H, 5″″-H
or 4″″-H, J = 6.9 Hz), 7.89 (br.s, 1H, 5-H). 13C NMR (DMSO-d6): 29.5,


|
4.2.1 AChE inhibition assay
Human blood plasma was generously gifted by Dr. Cecilia Tapia from
blood bank of Davila Clinic (Recoleta, Chile). 5,5′-Dithiobis-(2-nitro-
benzoic acid) (DTNB), acetylthiocholine chloride (ATC), dimethyl
sulfoxide (DMSO), and donepezil were obtained from Sigma–Aldrich
(St. Louis, MO, USA). All other reagents used were of analytical grade
from Merck (Darmstadt, Germany). The cholinesterase inhibition assays
were performed according to Ellman's method with some modifications
to the 96-well multiplate. The assays were carried out in a total volume
of 200 μL per well containing human blood plasma 180 μg protein/mL,
DTNB 250 μM, synthesized compound in DMSO, ATC 250 μM in
phosphate buffer (PB) 0.1 M pH 8.0. The general procedure was as
follows: Human blood plasma, DTNB, and the synthesized compound
solution in PB 0.1 M pH 8.0 were mixed and incubated at 25°C for
10 min. After incubation the substrate ATC was added to the mixture.
The production of the yellow anion (5-thio-2-nitrobenzoic acid) was
recorded for 10 min measuring the absorbance at 412 nm using Biotek-
Synergy MX microplate reader (Biotek Instruments Inc., Winoosky, VT,
USA). DMSO 5% v/v was used as positive control (100% activity) and
donepezil as method control of inhibition on cholinesterase activity. A
screening wasfirst performed with all synthesized compounds prepared
at a final concentration of 100 μM. Compounds that displayed more
than 50% of inhibition on cholinesterase activity were then used to
obtain cholinesterase inhibition curves from 100 to 0.01 μM to obtain
IC50 value for each drug. Data were plotted using GraphPad Prism 7.01
(GraphPad, San Diego, CA). All obtained values correspond to the
mean DS of three independent assays in triplicate.
2
43.2, (2x)52.8, (2x)54.9, 61.9, 66.1, 110.3 (d, JC-F = 28.8 Hz), 112 (d,
2′
2″
3
J
= 23.8 Hz), 115.6 (d,
J
C-F
= 22.1 Hz), 118 (d, JC-F = 9.4 Hz),
C-F
4
2″′
3
124.6 (d, JC-F = 3.9 Hz), 125 (d,
J
= 14.4 Hz), 127 (d, JC-F
=
C-F
3
3′
8.3 Hz), 129.5 (d, JC-F = 8.3 Hz), 132.1 (d,
J
= 4.4 Hz), 143
C-F
4′
1
1′
(d,
J
= 2.2 Hz), 155.5 (d, JC-F = 234 Hz), 161.3 (d,
J
=
C-F
C-F
244 Hz), 168.7. HRMS: (EI) calcd. For C21H23F2N3O2 (M+1):
388.1837. Found: 388.1817.
1-(2,3-Dihydro-4H-benzo[b][1,4]oxazin-4-yl)-2-(3-(4-
fluorophenyl)-1H-pyrazol-1-yl)ethan-1-one (12a)
Prepared from 2-chloro-1-(2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-
ethan-1-one (9a) (0.5g, 2.4 mmol), 3-(4-fluorophenyl)-1H-pyrazole
(0,38 g 2.4 mmol) and K2CO3 (0.33 g, 2.4 mmol) to give pure 12a
(0.58 g, 73%) as a white solid. Mp: 181.1–181.5°C. IR ν (cm−1): 3051
1


(C─H arom.), 2984 (C─H aliph.), 1667 (C ON), 1584 (C C). H NMR
(DMSO-d6) δ: 3.93 (t, 2H, 3-H, J = 4.1 Hz), 4.34 (t, 2H, 2-H, J = 4.3 Hz),
5.42 (s, 2H, CH2-pyrazole), 6.75 (d, 1H, 4″-H, J = 1.8 Hz), 6.88 (d, 1H, 8-
H or 5-H, J = 7.0 Hz), 6.92 (d, 1H, 5-H or 8-H, J = 7.7 Hz), 7.07 (t, 1H, 6-
H or 7-H, J = 6.9 Hz), 7.23 (t, 2H, 2″′-H or 6″′-H, J = 8.9 Hz), 7.75 (d,
1H, 5″-H, J = 2.2 Hz), 7.83 (dd, 2H, 3″′-H and 5″′-H, Jo = 8.6 Hz,
J
m = 5.7 Hz), 7.85–8.0 (br.s, 1H, 7-H or 6-H). 13C NMR (DMSO-d6):
2
42.8, 54.2, 70.4, 103.3, (2x)116 (d, JC-F = 21.6 Hz), 117.4, 118.8,
3
4
120.4, 124.3, 126.1, (2x)127.5 (d, JC-F = 8.3 Hz), 130.4 (d, JC-F
=
|
1
4.2.2 Kinetic studies
2.8 Hz), 134.1, 136.2, 149.7, 160.3 (d, JC-F = 201 Hz), 166.3.
HRMS: (EI) calcd. for
338.1307.
C19H16FN3O2 (M+1): 338.1305. Found:
The kinetic studies of ATC, ATC-donepezil, ATC-7a, and ATC-7d were
performed according to Ellman's method[23] with some modifications to
the 96-well multiplate. The assays were carried out in a total volume of
200 μL per well containing human blood plasma 180 μg protein/mL,
DTNB 250 μM, synthesized compound in DMSO at IC50 concentration or
DMSO 5% v/v, ATC 0.0–500.0 μM in PB 0.1 M pH 8.0. The general
procedure was as follows: Human blood plasma, DTNB, and the
synthesized compounds 7a, 7d or donepezil at IC50 concentration in PB
0.1 M pH 8.0 were mixed and incubated at 25°C for 10 min. After
incubation, substrate ATC from 1.0 to 500.0 μM was added into the
mixture. The reaction was recorded for 10 min measuring the absorbance
at 412 nm using a Biotek-Synergy MX microplate reader. Data were
plotted using GraphPad Prism 7.0 software (GraphPad Software Inc.) to
obtain theMichaelis–Mentenconstant (Km), maximal velocity (Vmax), and Ki
values. All values were expressed in μM and Vmax as mol/min/mg protein
and correspond to the mean SD of three independent assays in triplicate.
1-(6-Fluoro-2,3-dihydro-4H-benzo[b][1,4]oxazin-4-yl)-2-(3-(4-
fluorophenyl)-1H-pyrazol-1-yl)ethan-1-one (12b)
Prepared from 2-chloro-1-(6-fluoro-2,3-dihydro-4H-benzo[b][1,4]ox-
azin-4-yl)ethan-1-one (9b) (0.5 g, 2.2 mmol), 3-(4-fluorophenyl)-1H-
pyrazole (0.35 g, 2.2 mmol), K2CO3 (0.30 g, 2.2 mmol) to give pure 12b
(0.58 g, 75%) as a white solid. Mp: 127.3–128.3°C. IR ν (cm−1): 3045


(C─H arom.), 2968 (C─H aliph.), 1675 (N─C O), 1603 (C C).
1H NMR (DMSO-d6) δ: 3.94 (t, 2H, 3-H, J = 4.2 Hz), 4.32 (t, 2H, 2-H,
J = 4.3 Hz), 5.46 (s, 2H, CH2-pyrazole), 6.76 (d, 1H, 4″-H, J = 2.2 Hz),
6.94 (d, 2H, 7-H, 8-H, J = 7.7 Hz), 7.24 (t, 2H, 2″′-H, 6″′-H, J = 8.9 Hz),
7.75 (d, 1H, 5″-H, J = 2.2 Hz), 7.83 (dd, 2H, 3″′-H, 5″′-H, Jo = 8.6 Hz,
J
m = 5.7 Hz), 7.95 (br.s, 1H, 5-H). 13C NMR (DMSO-d6) δ: 42.7, 54.4,
2
2′
65.5, 103.4, 110 (d, JC-F = 29.9 Hz), 112.4 (d,
J
= 24.3 Hz), (2x)
C-F
2″
3
3′
115.9 (d,
J
C-F
= 21.6 Hz), 118.2 (d, JC-F = 9.4 Hz), 126.5 (d,
J
=
C-F
11.1 Hz), (2x)127.4 (d, 3″JC-F = 8.3 Hz), 130.4 (d, 4JC-F = 3.3 Hz), 134.1,
|
4.3 Molecular docking studies
4′
1
143.2 (d,
J
C-F
= 2.9 Hz), 149.8, 155.6 (d, JC-F = 234 Hz), 162.1
1
The series of indole benzoxazinones derivatives were docked into the
available crystal structure of recombinant human acetylcholinesterase
(d, JC-F = 244 Hz), 166.8. HRMS: (EI) calcd. for C19H15F2N3O2
(M+1): 356.1211. Found: 356.1207.

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MÉNDEZ-ROJAS ET AL.
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enzyme (rhAChE, PDB ID: 4EY7) protonated at pH 7.4. The water
molecule (wat 931) bound in the crystallized structure has been shown
to participate in ligand binding[25] and was thus maintained in all
docking simulations.
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4.0 following the standard docking procedure for rigid proteins.
Docking simulations were performed with a Lamarckian genetic
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60 × 60 × 60 points in the x, y, and z directions was built centered on
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ligand-binding site. Grid spacing of 0.375 Å was used. Default settings
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ACKNOWLEDGMENT
[19] H. Pessoa-Mahana, P. Silva-Matus, C. D. Pessoa-Mahana, H. Chung,
P. Iturriaga-Vásquez, G. Quiroz, P. Möller-Acuña, G. Zapata-Torres, C.
Saitz-Barría, R. Araya-Maturana, M. Reyes-Parada, Arch. Pharm.
(Weinheim) 2016, 349, 1.
This work was supported by FONDECYT Grants 1130347, 1170269
to HP-M and 1150615 to PI-V.
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CONFLICT OF INTEREST
The authors have declared no conflict of interest.
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Claudio Méndez-Rojas
http://orcid.org/0000-0001-9890-630X
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How to cite this article: Méndez-Rojas C, Quiroz G, Faúndez
M, et al. Synthesis and biological evaluation of potential
acetylcholinesterase inhibitors based on a benzoxazine core.
Arch Pharm Chem Life Sci. 2018;1–17.
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