Synthesis and biological evaluation of benzothiazin-4-ones: a possible new class of acetylcholinesterase inhibitors

Article abstract of DOI:10.1080/14756366.2018.1543286

A series of nineteen benzothiazin-4-ones from N-(3-aminopropyl) piperidine, 4-(2-aminoethyl)morpholine or 1-(2-aminoethyl)piperidine, aliphatic or aromatic aldehyde and thiosalicylic acid, were synthesized in good yields by multicomponent one-pot reactions. The solvent was toluene and this efficient procedure afforded the desired heterocycles in 5 h. Identification and characterization were achieved by NMR and GC–MS techniques. In vitro AChE activities of all compounds were evaluated in cerebral cortex and hippocampus of rats and in general, the results in cortex were more promising than hippocampus. The benzothiazinone 5Bd showed the best AChE inhibition activity IC₅₀ 8.48 μM (cortex) and IC₅₀ 39.80 μM (hippocampus). The cytotoxicity of seven compounds in MCR-5 human fibroblast cell by SRB test in 24 h were evaluated and 5Bd suggest preliminary safety, showing no cytotoxicity at 100 μM. Finally, these important findings could be a starting point for the development of new AChE inhibitors agents and will provide the basis for new studies.

Full text of DOI:10.1080/14756366.2018.1543286

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and biological evaluation of
benzothiazin-4-ones: a possible new class of
acetylcholinesterase inhibitors
Gabriele A. Berwaldt, Daniela P. Gouvêa, Daniel S. da Silva, Adriana M. das
Neves, Mayara S. P. Soares, Juliana H. Azambuja, Geonir M. Siqueira, Roselia
M. Spanevello & Wilson Cunico
To cite this article: Gabriele A. Berwaldt, Daniela P. Gouvêa, Daniel S. da Silva, Adriana M. das
Neves, Mayara S. P. Soares, Juliana H. Azambuja, Geonir M. Siqueira, Roselia M. Spanevello &
Wilson Cunico (2019) Synthesis and biological evaluation of benzothiazin-4-ones: a possible new
class of acetylcholinesterase inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry,
34:1, 197-203, DOI: 10.1080/14756366.2018.1543286
To link to this article: https://doi.org/10.1080/14756366.2018.1543286
© 2018 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group
Published online: 27 Nov 2018.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 34, NO. 1, 197–203
https://doi.org/10.1080/14756366.2018.1543286
SHORT COMMUNICATION
Synthesis and biological evaluation of benzothiazin-4-ones: a possible new class
of acetylcholinesterase inhibitors
a
a
Gabriele A. Berwaldt , Daniela P. Gouvea , Daniel S. da Silvaa,b, Adriana M. das Nevesa, Mayara S. P. Soaresb,
^
Juliana H. Azambujab, Geonir M. Siqueiraa, Roselia M. Spanevellob and Wilson Cunicoa
a
ꢀ
ꢀ
^
ꢀ
^
ꢀ
Laboratorio de Quımica Aplicada a Bioativos, Centro Ciencias Quımicas, Farmaceuticas e de Alimentos, Universidade Federal de Pelotas, Predio
b
ꢀ
ꢀ
~
~
ꢀ
ꢀ
~
^
32, Laboratorio 410, Campus Universitario S/N, Capao do Leao, RS, CEP 96160-000, Brazil; Laboratorio de Neuroquımica, Inflamac¸ao e Cancer,
Centro de Ciencias Quımicas Farmaceuticas e de Alimentos, Universidade Federal de Pelotas, Predio 29, Sala 303, Campus Universitario S/N,
Capao do Leao, RS, CEP 96160-000, Brazil
^
ꢀ
^
ꢀ
ꢀ
~
~
ABSTRACT
ARTICLE HISTORY
Received 14 August 2018
Revised 26 October 2018
Accepted 29 October 2018
A series of nineteen benzothiazin-4-ones from N-(3-aminopropyl) piperidine, 4-(2-aminoethyl)morpholine
or 1-(2-aminoethyl)piperidine, aliphatic or aromatic aldehyde and thiosalicylic acid, were synthesized in
good yields by multicomponent one-pot reactions. The solvent was toluene and this efficient procedure
afforded the desired heterocycles in 5 h. Identification and characterization were achieved by NMR and
GC–MS techniques. In vitro AChE activities of all compounds were evaluated in cerebral cortex and hippo-
campus of rats and in general, the results in cortex were more promising than hippocampus. The benzo-
thiazinone 5Bd showed the best AChE inhibition activity IC50 8.48 lM (cortex) and IC50 39.80 lM
(hippocampus). The cytotoxicity of seven compounds in MCR-5 human fibroblast cell by SRB test in 24 h
were evaluated and 5Bd suggest preliminary safety, showing no cytotoxicity at 100 mM. Finally, these
important findings could be a starting point for the development of new AChE inhibitors agents and will
provide the basis for new studies.
KEYWORDS
Benzothiazinone;
propylpiperidine;
acetylcholinesterase;
fibroblast cells
Introduction
with benzene at 5 and 6 positions of thiazinone ring11. These sub-
stances represent a class of compounds that have a great scien-
tific interest due to their chemical and biological properties such
as antibacterial, antifungal, anti-hypertensive, anti-inflammatory,
antirheumatic, aldose reductase inhibitor, antioxidant, anti-HIV,
Alzheimer’s disease (AD) is a progressive and neurodegenerative
disorder and the main cause of dementia affecting older people.
The hallmark pathological abnormalities of AD are the formation
of amyloid plaques that are due to accumulation of extracellular
Ab and neurofibrillary tangles formed by Tau protein1–3. However,
the cognitive dysfunction in this neurodegenerative disease also
has been associated with loss of cholinergic neurons in many
anti-malarial and anti-helminthic activities12–17
.
Therefore, the aims of this study were the application of the
multicomponent one-pot strategy to obtain benzothiazinones, the
evaluation of the in vitro AChE activity on cerebral cortex and
hippocampus of rats and cytotoxicity effect against MCR-5 human
fibroblast cells. The compound design was based in structure of
the neurotransmitter acetylcholine by mimetic its functional
groups, as shown in Figure 1.
brain regions4–6
.
Acetylcholinesterase (AChE) is a crucial and the most efficient
enzyme of the central nervous system with the active site located
near at the bottom of a deep and narrow gorge7. The principal
biological role of the AChE is termination of impulse transmission
at cholinergic synapses by rapid hydrolysis of the acetylcholine
neurotransmitter8. AChE inhibitors are the most promising
approaches for treating the symptoms of AD. These drugs are cap-
able to prevent the degradation of acetylcholine and increase the
level of this neurotransmitter in the cholinergic synapses improv-
ing cognitive deficits6,9. However, the adverse effects, as nausea,
vomiting, bradycardia and weight loss, associated with AChE
inhibitors therapy have limited their clinical efficacy10.
Experimental
Chemistry
General
Reagents and solvents were use as obtained from commercial
suppliers without further purification. Reaction progress was
monitored by thin-layer chromatography (TLC) using hexane:ethyl
acetate 3:1 mixture as eluent and/or by Shimadzu Gas
Chromatograph GC-2010 (HP-1 column crosslinked methyl silox-
ane, 30 m ꢀ 0.32 mm ꢀ0.25 lm: Column head pressure, 14 psi,
In this context, the research in this field is required in order to
trigger the synthesis of AChE inhibitors compounds with better
pharmacological profile and therapeutic efficacy. The biological
potential of heterocycles have been widely reported in the litera-
program: Ti ¼ 60 ꢁC; ti ¼ 2.0 min; rate 10.0 ꢁC minꢂ1; Tf ¼ 280 ꢁC;
1
ture. Thiazinones are six-membered heterocycles containing nitro- tf ¼ 40.0 min; Inj. ¼ 250 ꢁC; Det. ¼ 280 ꢁC). H and 13C NMR spectra
gen, sulfur and carbonyl group. Benzothiazinones has a fusion were recorded on a Bruker DRX 400 spectrometer (1H at 400 MHz
CONTACT Wilson Cunico
wjcunico@yahoo.com.br; Roselia M. Spanevello
rspanevello@gmail.com
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

198
G. A. BERWALDT ET AL.
2.9%); 281 (4.6%); 207 (2.4%); 191 (1.6%); 113 (40.9%); 100 (100%);
91 (8.3%) 70 (4.8%); 56 (10.9%); 44 (7.7%).
3-(2-morpholinoethyl)-2-(p-tolyl)-2,3-dihydro-4H-benzo[e][1,3]thia-
zin-4-one 5Ac
Yield: 81%; oil; 1H NMR (600 MHz, CDCl3) d (ppm, JH–H ¼ Hz): 8.12
(dd, 1H, H7, Ar, 3J ¼ 7.8, 4J ¼ 1.3); 7.27 (td, 1H, H9, Ar, 3J ¼ 7.8,
4J ¼ 1.4); 7.21 (td, 1H, H10, Ar, 3J ¼ 7.8, 4J ¼ 1.1); 7.14 (d, 2H, H19,
3J ¼ 8.1); 7.08 (d, 1H, H8, Ar, 3J ¼ 7.7); 7.03 (d, 2H, H18, 3J ¼ 8.0);
5.94 (s, 1H, H2); 4.26 (dt, 1H, H11a, 2J ¼ 13.9, 3J ¼ 5.9); 3.69–3.59
(m, 4H, H15); 3.31 (dt, 1H, H11b, 2J ¼ 13.8, 3J ¼ 6.8); 2.74 (dt, 1H,
H12a, 2J ¼ 13.5, 3J ¼ 6.9); 2.64 (dt, 1H, H12b, 2J ¼ 12.8, 3J ¼ 5.8);
2.52 (s, 4H, H14); 2.25 (s, 3H, CH3). 13C NMR (150 MHz, CDCl3) d
(ppm): 164.0 (C4); 138.0 (Ar); 135.9 (Ar); 133.0 (Ar); 131.9 (Ar);
129.7 (Ar); 129.0 (Ar); 129.0 (Ar); 127.4 (Ar); 126.1 (Ar); 126.0 (Ar);
66.7 (C15); 62.4 (C2); 56.9 (C12); 53.6 (C14); 45.2 (C11); 20.9 (CH3).
MS m/z: 368 (Mþ, 2.9%); 254 (3.3%); 136 (4.0%); 113 (42.7%); 98
(5.2%); 100 (100%); 70 (6.1%); 56 (10.9%); 42 (3.8%).
Figure 1. Design of benzothiazinones in comparison with acethylcholine.
and 13C at 100 MHz), on a Bruker Avance 600 spectrometer (1H at
600 MHz and 13C at 150 MHz), or on a Bruker Avance III 600 MHz
(1H at 600 MHz and 13C at 150 MHz), in CDCl3 or DMSO containing
TMS as an internal standard. The mass spectra were obtained on a
Shimadzu GCMS-QP2010SE with
a split-splitless injector and
equipped with a RDX–SMS capillary column (30 m ꢀ 0.25 mm
ꢀ0.25 lm); helium was used as the carrier gas (56 kPa).
3-(2-morpholinoethyl)-2-(4-nitrophenyl)-2,3-dihydro-4H-ben-
zo[e][1,3]thiazin-4-one 5Ad
General procedure for the synthesis of benzothiazinones 5Aa–g,
5Ba–f and 5Ca–f
To a flask with a Dean–Stark apparatus are add 70 ml of toluene,
1 mmol of an aliphatic amine 1A–C and 1 mmol of corresponding
aldehyde (2a–g). The 1 mmol thiosalicylic acid 4 is add after
15 min in a preheated (50 ꢁC) reaction mixture due its low solubil-
ity. The mixture is maintain in reflux of toluene for 5 h. The
1
Yield: 79%; oil; H NMR (600 MHz, CDCl3) d (ppm, JH–H ¼ Hz): 8.13 (dd,
3
4
3
1H, H7, Ar, J¼ 7.8, J¼ 1.4); 8.08 (d, 2H, H19, J¼ 8.8); 7.31 (td, 1H,
3
4
3
4
H9, Ar, J¼ 7.6, J¼ 1.5); 7.25 (td, 1H, H10, Ar, J¼ 7.7, J¼ 1.2); 7.09
3
4
3
(dd, 1H, H8, Ar, J¼ 7.7, J¼ 0.9); 7.46 (d, 2H, H18, J¼ 8.7); 6.11 (s,
1H, H2); 4.27 (dt, 1H, H11a, 2J¼ 14.1, 3J¼ 5.5); 3.67 (dtd, 4H, H15,
2J¼ 15.5, 2J¼ 10.9, 3J¼ 4.5); 3.40 (ddd, 1H, H11b, 2J¼ 13.6, 3J¼ 7.5,
3J¼ 5.4); 2.78 (dt, 1H, H12a, J¼ 13.1, J¼ 7.4, J¼ 5.4); 2.68 (dt, 1H,
2
3
3
H12b, J¼ 13.2, J¼ 5.4); 2.55 (s, 4H, H14). 13C NMR (150 MHz, CDCl3)
d (ppm): 163.7 (C4); 147.4 (C20); 146.7 (Ar); 132.3 (Ar); 131.9 (Ar);
129.9 (Ar); 128.9 (C17); 127.6 (Ar); 127.0 (Ar); 126.7 (Ar); 123.5 (Ar);
66.7 (C15); 61.9 (C2); 57.1 (C12); 53.6 (C14); 45.4 (C11). MS m/z: 399
(Mþ, 2,6%); 313 (0.4%); 286 (0.5%); 245 (0.1%); 136 (3.6%); 113
(17.4%); 100 (100%); 90 (2.0%); 70 (3.4%); 56 (8.8%); 42 (2.1%).
2
3
organic layer is wash with
a saturated solution of NaOH
(3 ꢀ 30 ml), dry with MgSO4 and the solvent is remove. The crude
products are purified by column chromatography using silica and
hexane:ethyl acetate (9:1) as eluent.
2-butyl-3-(2-morpholinoethyl)-2,3-dihydro-4H-benzo[e][1,3]thiazin-
4-one 5Aa
1
Yield: 77%; oil; H NMR (600 MHz, CDCl3) d (ppm, JH–H ¼ Hz): 8.07 (dd,
2-(4-methoxyphenyl)-3-(2-morpholinoethyl)-2,3-dihydro-4H-ben-
zo[e][1,3]thiazin-4-one 5Ae
1H, H7, Ar, 3J¼ 7.8, 4J¼ 1.1); 7.34 (td, 1H, H9, Ar, 3J¼ 7.6, 4J¼ 1.4);
2
3
3
1
7.26 (dd, 2H, H8, H10, Ar, J¼ 12.3, J¼ 4.5) 4.53 (dd, 1H, H2, J¼ 9.6,
Yield: 50%; oil; H NMR (600 MHz, CDCl3) d (ppm, JH–H¼Hz): 8.12 (d,
3J¼ 5.4); 4.27 (dt, 1H, H11a, 2J¼ 13.7, 3J¼ 6.0); 3.22 (dt, 1H, H11b,
3
3
4
1H, H7, Ar, J¼ 7.8); 7.28 (td, 1H, H9, Ar, J¼ 7.5, J¼ 1.5); 7.21 (t, 1H,
2J¼ 13.7, J¼ 6.8); 3.82–3.66 (m, 4H, H15); 2.69 (dt, 2H, H12a, H12b,
3
H10, Ar, 3J¼ 7.6); 7.18 (d, 2H, H19, Ar, 3J¼ 8.7); 7.09 (d, 1H, H8, Ar,
2J¼ 12.2, 3J¼ 6.2); 2.62–2.54 (m, 4H, H14); 1.80–1.94 (m, 2H, H17);
1.45–1.38 (m, 2H, H18); 1.30–1.21 (m, 2H, H19); 0.86 (t, 3H, H20,
3J¼ 7.2). 13C NMR (150 MHz, CDCl3) d (ppm): 162.8 (C4); 134.0 (Ar);
131.8 (Ar); 129.9 (Ar); 129.0 (Ar); 127.8 (Ar); 125.8 (Ar); 66.8 (C15); 61.8
(C2); 57.0 (C12); 53.7 (C14); 45.6 (C11); 34.5 (C17); 29.0 (C18); 21.9
(C19); 13.8 (C20). MS m/z: 281 (Mþ-53, 0.5%); 248 (0.6%); 220 (1.0%);
113 (47.8%); 100 (100%); 83 (1.6%) 70 (4.5%); 56 (8.9%); 44 (7.5%).
3J¼ 7.7); 6.75 (d, 2H, H18, Ar, J¼ 8.7); 5.94 (s, 1H, H2); 4.25 (dt, 1H,
3
H11a, 2J¼ 13.6, 3J¼ 5.9); 3.72 (s, 3H, OCH3); 3.72–3.66 (m, 4H, H15);
3.33 (dt, 1H, H11b, 2J¼ 13.8, 3J¼ 6.8); 2.74 (dt, 1H, H12a, 2J¼ 13.4,
3J¼ 6.9); 2.64 (dt, 1H, H12b, J¼ 12.8, J¼ 5.8); 2.53 (s, 4H, H14). 13C
NMR (150 MHz, CDCl3) d (ppm): 164.0 (C4); 159.4 (Ar); 131.9 (Ar);
130.8 (Ar); 129.7 (Ar); 127.4 (Ar); 127.4 (Ar); 126.0 (Ar); 113.7 (Ar); 66.7
(C15); 62.2 (C2); 56.9 (C12); 55.1 (OCH3); 53.6 (C14); 45.1 (C11). MS m/
z: 384 (Mþ, 4.4%); 351 (0.6%); 270 (6.0%); 245 (0.1%); 113 (38.0%);
121 (11.5%); 100 (100%); 91 (2.2%); 70 (4.5%); 56 (11.1%); 42 (2.2%).
2
3
3-(2-morpholinoethyl)-2-phenyl-2,3-dihydro-4H-benzo[e][1,3]thia-
zin-4-one 5Ab
Yield: 81%; oil; 1H NMR (400 MHz, CDCl3) d (ppm, JH–H ¼ Hz): 8.06
(dd, 1H, H7, Ar, 3J ¼ 7.8, 4J ¼ 1.5); 7.23–7.10 (m, 7H, H9, H10, H18,
H19, H20, Ar); 7.01 (dd, 1H, H8, Ar, 3J ¼ 7.6, 4J ¼ 1.1) 5.89 (s, 1H,
2-(4-fluorophenyl)-3-(2-morpholinoethyl)-2,3-dihydro-4H-ben-
zo[e][1,3]thiazin-4-one 5Af
Yield: 88%; oil; 1H NMR (600 MHz, CDCl3) d (ppm, JH–H¼Hz): 8.12
(dd, 1H, H7, Ar, 3J ¼ 7.8, 4J ¼ 1.3); 7.28 (td, 1H, H9, Ar, 3J ¼ 7.6,
4J ¼ 1.5); 7.25–7.20 (m, 3H, H10, H19, Ar); 7.09 (dd, 1H, H8, Ar,
3J ¼ 7.7, 4J ¼ 0.9); 6.94–6.87 (m, 2H, H18, Ar); 5.96 (s, 1H, H2); 4.25
(dt, 1H, H11a, 2J ¼ 14.0, 3J ¼ 5.7); 3.68 (dtd, 4H, H15, 2J ¼ 15.7,
2
3
H2,); 4.18 (dt, 1H, H11a, J ¼ 13.9, J ¼ 5.8); 3.65–3.54 (m, 4H, H15);
3.24 (dt, 1H, H11b, 2J ¼ 13.9, 3J ¼ 6.4); 2.68–2.48 (m, 2H, H12a,
H12b); 2.45–2.37 (m, 4H, H14). 13C NMR (100 MHz, CDCl3) d (ppm):
163.9 (C4); 139.1 (Ar); 132.9 (Ar); 131.9 (Ar); 129.8 (Ar); 129.1 (Ar);
128.4 (Ar); 128.1 (Ar); 127.4 (Ar); 126.2 (Ar); 126.2 (Ar); 67.0 (C15); 2J ¼ 11.1, 3J ¼ 4.6); 3.34 (dt, 1H, H11b, 2J ¼ 13.7, 3J ¼ 6.8); 2.73 (dt,
2
3
2
3
62.5 (C2); 57.1 (C12); 53.7 (C14); 45.3 (C11). MS m/z: 354 (Mþ, 1H, H12a, J ¼ 13.3, J ¼ 6.8); 2.63 (dt, 1H, H12b, J ¼ 12.9, J ¼ 5.9);

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
199
2.52 (s, 4H, H14). 13C NMR (150 MHz, CDCl3) d (ppm, JC–F ¼Hz): 4J ¼ 2.0); 7.20 (td, 1H, H10, Ar, 3J ¼ 7.7, 4J ¼ 1.2); 7.12 (d, 2H, H19,
1
3
163.8 (C4); 162.3 (d, C20, J ¼ 247.8); 134.9 (d, C17, J ¼ 3.0); 132.7 3J ¼ 8.1); 7.08 (dd, 1H, H8, Ar, 3J ¼ 7.7, 4J ¼ 0.7); 7.03 (d, 2H, H18,
(Ar); 132.0 (Ar); 129.8 (Ar); 129.0 (Ar); 127.9 (d, C19, J ¼ 8.3); 127.5 3J ¼ 8.0); 5.95 (s, 1H, H2); 4.30 (dt, 1H, H11a, 2J ¼ 13.3, 3J ¼ 6.5);
3
2
2
3
2
(Ar); 126.3 (Ar); 115.2 (d, J ¼ 21.8); 61.9 (C2); 66.8 (C15); 57.0 (C12); 3.04 (dt, 1H, H11b, J ¼ 13.6, J ¼ 6.9); 2.72 (dt, 1H, H12a, J ¼ 12.5,
53.6 (C14); 45.3 (C11). MS m/z: 372 (Mþ, 5,2%); 150 (1.1%); 136 3J ¼ 7.9); 2.47–2.56 (m, 5H, H12b, H14); 2.03–1.98 (m, 2H, H13a,
(7.9%); 114 (8.5%); 113 (50.0%); 109 (11.1%); 100 (100%); 86 (6.6%); H13b); 2.24 (s, 3H, CH3); 1.67 (dt, 4H, H15, 2J ¼ 11.2, 3J ¼ 5.7);
1.51–1.48 (m, 2H, H16). 13C NMR (150 MHz, CDCl3) d (ppm): 164.1
70 (6.2%); 56 (15.4%); 42 (3.4%).
(C4); 137.9 (Ar); 135.9 (Ar); 133.0 (Ar); 131.9 (Ar); 129.6 (Ar); 129.1
(Ar); 127.4 (Ar); 126.0 (Ar); 126.0 (Ar); 61.6 (C2); 55.1 (C13); 53.9
(C14); 46.4 (C11); 25.0 (C15); 24.5 (C12); 23.8 (C16); 20.9 (CH3). MS
m/z: 380 (Mþ, 11.1%); 347 (2.6%); 289 (3.1%); 192 (0.5%); 164
(0.9%); 136 (9.3%); 112 (11.7%); 105 (11.4%); 98 (100%); 84 (13.7%);
70 (6.0%); 55 (6.5%); 41 (10.0%).
2-(4-(Methylsulfonyl)phenyl)-3-(2-morpholinoethyl)-2,3-dihydro-
4Hbenzo[e][1,3]thiazin-4-one 5Ag
Yield: 78%; oil; 1H NMR (600 MHz, CDCl3) d (ppm, JH–H¼Hz): 8.13
(d, 1H, H7, Ar, 3J ¼ 7.0); 7.79 (d, 2H, H19, Ar, 3J ¼ 8.4); 7.47 (d, 2H,
H18, Ar, 3J ¼ 8.2); 7.31 (td, 1H, H9, Ar, 3J ¼ 7.5, 4J ¼ 1.2); 7.25 (td,
3
4
3
1H, H10, J ¼ 7.3, J ¼ 0.7); 7.09 (d, 1H, H8, Ar, J ¼ 7.5); 6.09 (s, 1H,
2
3
2-(4-nitrophenyl)-3-(3-(piperidin-1-yl)propyl)-2,3-dihydro-4H-ben-
zo[e] [1,3]thiazin-4-one 5Bd
H2); 4.31 (dt, 1H, H11a, J ¼ 13.8, J ¼ 5.7); 3.73–3.64 (m, 4H, H15);
3.37 (dt, 1H, H11b, 2J ¼ 13.9, 3J ¼ 6.7); 2.99 (s, 3H, H21); 2.80 (dt,
Yield: 52%; oil; 1H NMR (600 MHz, CDCl3) d (ppm, JH–H¼Hz): 8.12
2
3
2
3
1H, H12a, J ¼ 13.1, J ¼ 6.7); 2.71 (dt, 1H, H12b, J ¼ 12.9, J ¼ 6.2);
2.57 (s, 4H, H14). 13C NMR (150 MHz, CDCl3) d (ppm): 163.7 (C4);
145.7 (Ar); 140.1 (Ar); 132.3 (Ar); 132.0 (Ar); 129.9 (Ar); 128.7 (Ar);
127.5 (Ar); 127.4 (Ar); 127.0 (Ar); 126.6 (Ar); 66.5 (C15); 61.7 (C2);
56.8 (C12); 53.5 (C14); 45.3 (C11); 44.2 (C21).
(d, 1H, H7, Ar, 3J ¼ 7.9); 8.06 (d, 2H, H19, Ar 3J ¼ 8.7); 7.41 (d, 2H,
3
H18, Ar, J ¼ 8.7); 7.28 (t, 1H, H9, Ar, 3J ¼ 7.4); 7.23 (t, 1H, H10, Ar,
3J ¼ 7.6); 7.07 (d, 1H, H8, Ar, 3J ¼ 7.7); 6.23 (s, 1H, H2); 4.38 (ddd,
2
3
3
2
1H, H11a, J ¼ 13.6, J ¼ 6.7, J ¼ 6.1); 3.08 (dt, 1H, H11b, J ¼ 12.7,
3J ¼ 7.9); 2.78 (dt, 1H, H12a, 2J ¼ 12.7, 3J ¼ 7.9); 2.69–2.43 (m, 5H,
H12b, H14); 2.13–1.96 (m, 2H, H13b, H13b); 1.68 (dt, 4H, H15,
2J ¼ 11.1, 3J ¼ 5.5); 1.51–1.48 (m, 2H, H16). 13C NMR (150 MHz,
CDCl3) d (ppm): 163.8 (C4); 146.7 (C20); 132.3 (Ar); 132.0 (Ar); 129.9
(Ar); 129.0 (Ar); 127.5 (Ar); 127.0 (Ar); 126.6 (Ar); 123.6 (Ar); 61.2
(C2); 54.9 (C13); 54.0 (C14); 46.7 (C11); 25.1(C15); 24.5 (C12); 23.8
(C16). MS m/z: 411 (Mþ, 5.0%); 378 (0.7%); 355 (0.7%); 327 (1.4%);
281 (5.1%); 207 (14.1%); 191 (1.7%); 163 (2.2%); 136 (5.6%); 112
(9.0%); 98 (100%); 84 (11.6%); 70 (4.7%); 55 (5.5%); 41 (6.4%).
2-butyl-3-(3-(piperidin-1-yl)propyl)-2,3-dihydro-4H-benzo[e][1,3]-
thiazin-4-one 5Ba
Yield: 50%; oil; 1H NMR (600 MHz, CDCl3) d (ppm, JH–H¼Hz): 8.07
3
3
4
(d, 1H, H7, Ar, J ¼ 7.8); 7.34 (td, 1H, H9, Ar, J ¼ 7.6, J ¼ 1.3); 7.23
(t, 2H, H8, H10, Ar, 3J ¼ 7.6) 4.58 (dd, 1H, H2, 3J ¼ 8.4, 3J ¼ 6.5);
2
3
2
4.24 (dt, 1H, H11a, J ¼ 13.5, J ¼ 6.3); 3.05 (dt, 1H, H11b, J ¼ 13.5,
3J ¼ 7.1); 2.62 (dt, 1H, H12a, 2J ¼ 12.6, 3J ¼ 7.9); 2.56–2.34 (m, 5H,
H12b, H14); 1.93 (dt, 2H, H13a, H13b, 2J ¼ 13.5, 3J ¼ 6.5); 1.63 (td,
4H, H15, 2J ¼ 11.2, 3J ¼ 5.6); 1.88–1.81 (m, 2H, H17); 1.49–1.45 (m,
2H, H18); 1.33–1.22 (m, 2H, H19); 0.86 (t, 3H, H20, 3J ¼ 7.2). 13C
NMR (150 MHz, CDCl3) d (ppm): 162.7 (C4); 133.9 (Ar); 131.6 (Ar);
129.8 (Ar); 129.2 (Ar); 127.7 (Ar); 125.8 (Ar); 61.3 (C2); 55.3 (C13);
54.1 (C14); 46.6 (C11); 34.5 (C17); 28.8 (C18); 25.5 (C15); 25.0 (C12);
24.1 (C16); 21.8 (C19); 13.7 (C20). MS m/z: 346 (Mþ, 1.3%); 290
(7.8%); 164 (3.9%); 136 (6.5%); 124 (6.4%); 112 (17.7%); 98 (100%);
84 (19.5%); 70 (6.1%); 56 (3.5%); 41 (8.3%).
2-(4-methoxyphenyl)-3-(3-(piperidin-1-yl)propyl)-2,3-dihydro-4H-
benzo[e][1,3]thiazin-4-one 5Be
Yield: 47%; oil; 1H NMR (600 MHz, CDCl3) d (ppm, JH–H¼Hz): 8.12
(dd, 1H, H7, Ar, 3J ¼ 7.8, 4J ¼ 1.2); 7.27 (td, 1H, H9, Ar, 3J ¼ 7.4,
4J ¼ 1.4); 7.20 (t, 1H, H10, Ar, 3J ¼ 7.6); 7.15 (d, 2H, H19, Ar,
3
3
3J ¼ 8.8); 7.08 (d, 1H, H8, Ar, J ¼ 7.7); 6.75 (d, 2H, H18, Ar, J ¼ 8.7);
5.96 (s, 1H, H2); 4.28 (dt, 1H, H11a, 2J ¼ 13.1, 3J ¼ 6.4); 3.79 (t, 1H,
H11b, 3J ¼ 4.8); 3.72 (s, 3H, OCH3); 3.05 (dt, 1H, H12a, 2J ¼ 13.9,
3J ¼ 7.1); 2.65 (dt, 1H, H12b, 2J ¼ 12.5, 3J ¼ 8.0); 2.01–1.93 (m, 2H,
H13a, H13b); 2.50–2.39 (m, 4H, H14); 1.63 (dt, 4H, H15, 2J ¼ 11.2,
3J ¼ 5.6); 1.46 (s, 2H, H16). 13C NMR (150 MHz, CDCl3) d (ppm):
163.9 (C4); 159.3 (Ar); 133.0 (Ar); 131.8 (Ar); 130.9 (Ar); 129.6 (Ar);
129.1 (Ar); 127.4 (Ar); 127.4 (Ar); 126.0 (Ar); 113.7 (Ar); 61.5 (C2);
55.3 (C13); 55.1 (OCH3); 54.1 (C14); 46.5 (C11); 25.4 (C15); 24.8
(C12); 24.0 (C16). MS m/z: 396 (Mþ, 8.9%); 363 (1.9%); 312 (1.0%);
289 (2.1%); 278 (1.2%); 257 (1.5%); 136 (3.9%); 121 (9.0%); 98
(100%); 84 (11.6%); 70 (4.7%); 55 (4.6%); 41 (5.5%).
2-phenyl-3-(3-(piperidin-1-yl)propyl)-2,3-dihydro-4H-benzo[e][1,3]-
thiazin-4-one 5Bb
Yield: 52%; oil; 1H NMR (400 MHz, CDCl3) d (ppm, JH–H¼Hz): 8.14
(dd, 1H, H7, Ar, 3J ¼ 7.8, 4J ¼ 1.5); 7.28–7.19 (m, 7H, H9, H10, H18,
H19, H20, Ar); 7.08 (dd, 1H, H8, Ar, 3J ¼ 7.8, 4J ¼ 1.0); 6.05 (s, 1H,
H2); 4.32 (ddd, 1H, H11a, 2J ¼ 13.4, 3J ¼ 6.8, 3J ¼ 5.3); 3.04 (dt, 1H,
H11b, 2J ¼ 13.5, 3J ¼ 6.2); 2.64 (dt, 1H, H12a, 2J ¼ 12.5, 3J ¼ 8.0);
2.51–2.30 (m, 5H, H12b, H14); 2.03–1.84 (m, 2H, H13a, H13b); 1.59
(dt, 4H, H15, 2J ¼ 11.1, 3J ¼ 5.7); 1.45 (s, 2H, H16). 13C NMR
(100 MHz, CDCl3) d (ppm): 163.9 (C4); 139.3 (Ar); 132.8 (Ar); 131.8
(Ar); 129.7 (Ar); 129.4 (Ar); 128.4 (Ar); 128.0 (Ar); 127.3 (Ar); 126.1
(Ar); 126.0 (Ar); 61.9 (C2); 55.3 (C13); 54.2 (C14); 46.8 (C11); 25.9
(C15); 25.1 (C12); 24.3 (C16). MS m/z: 366 (Mþ, 12.7%); 347 (3.4%);
289 (4.4%); 136 (5.8%); 124 (7.4%); 118 (9.1%); 112 (12.7%); 98
(100%); 84 (15.6%); 70 (4.3%); 55 (6.1%); 41 (6.0%).
2-(4-fluorophenyl)-3-(3-(piperidin-1-yl)propyl)-2,3-dihydro-4H-ben-
zo[e][1,3]thiazin-4-one 5Bf
Yield: 65%; oil; 1H NMR (600 MHz, CDCl3) d (ppm, JH–H¼Hz): 8.12
(dd, 1H, H7, Ar, 3J ¼ 7.8, 4J ¼ 0.9); 7.27 (td, 1H, H9, Ar, 3J ¼ 7.4,
4J ¼ 1.3); 7.29–7.19 (m, 3H, H10, H19, Ar); 7.08 (d, 1H, H8, Ar,
3
3J ¼ 7.7); 6.09 (t, 2H, H18, Ar, J ¼ 8.6); 6.02 (s, 1H, H2); 4.30 (dt, 1H,
H11a, 2J ¼ 12.9, 3J ¼ 6.2); 3.06 (dt, 1H, H11b, 2J ¼ 13.4, 3J ¼ 7.2);
2.64 (dt, 1H, H12a, 2J ¼ 12.6, 3J ¼ 7.9); 2.56–2.33 (m, 5H, H12b,
H14); 2.00–1.89 (m, 2H, H13a, H13b); 1.61(dt, 4H, H15, 2J ¼ 11.2,
3-(3-(Piperidin-1-yl)propyl)-2-(p-tolyl)-2,3-dihydro-4H-benzo[e][1,3]-
thiazin-4-one 5Bc
Yield: 43%; oil; 1H NMR (600 MHz, CDCl3) d (ppm, JH–H¼Hz): 8.12 3J ¼ 5.6); 1.47–1.46 (m, 2H, H16). 13C NMR (150 MHz, CDCl3) d
(dd, 1H, H7, Ar, 3J ¼ 7.9, 4J ¼ 1.3); 7.26 (dt, 1H, H9, Ar, 3J ¼ 7.6, (ppm, JC–F¼Hz): 163.8 (C4); 162.4 (d, C20, 1J ¼ 247.6); 135.1 (d,

200
G. A. BERWALDT ET AL.
3
C17, J ¼ 3.1); 132.7 (Ar); 131.9 (Ar); 129.7 (Ar); 129.2 (Ar); 127.9 (d, 7.42 (d, 2H, H18, Ar, 3J ¼ 8.7); 7.28 (td, 1H, H9, Ar, 3J ¼ 7.5,
C19, 3J ¼ 8.3); 127.4 (Ar); 126.2 (Ar); 115.3 (d, 2J ¼ 21.8); 61.5 (C2); 4J ¼ 1.5); 7.22 (dd, 1H, H10, Ar, 2J ¼ 11.0, 3J ¼ 4.1); 7.07 (d, 1H, H8,
3
2
3
Ar, J ¼ 7.6); 6.27 (s, 1H, H2); 4.29 (dt, 1H, H11a, J ¼ 14.0, J ¼ 5.5);
3.28 (dt, 1H, H11b, 2J ¼ 13.9, 3J ¼ 5.9); 2.77 (ddd, 1H, H12a,
2J ¼ 13.2, 3J ¼ 7.3, 3J ¼ 5.9); 2.64 (dt, 1H, H12b, 2J ¼ 11.2, 3J ¼ 5.5);
2.48 (s, 4H, H14); 1.63–1.47 (m, 4H, H16); 1.44 (m, 2H, H16,
2J ¼ 11.3, 3J ¼ 5.6). 13C NMR (150 MHz, CDCl3) d (ppm): 163.7 (C4);
147.5 (C20); 147.07 (Ar); 132.2 (Ar); 132.2 (Ar); 129.9 (Ar); 129.1
(Ar); 127.0 (Ar); 126.6 (Ar); 123.4 (Ar); 61.2 (C2); 54.9 (C13); 54.0
(C14); 46.7 (C11); 25.1 (C15); 24.5 (C12); 23.8 (C16). MS m/z: 397
(Mþ, 1.7%); 267 (0.3%); 163 (1.2%); 136 (3.5%); 111 (7.5%); 98
(100%); 70 (4.6%); 55 (5.3%); 44 (3.5%).
55.2 (C13); 54.2 (C14); 46.8 (C11); 25.7 (C15); 25.0 (C12); 24.2 (C16).
MS m/z: 384 (Mþ, 6.2%); 351 (1.8%); 289 (1.7%); 259 (1.1%); 164
(1.1%); 151 (1.0%); 136 (13.8%); 124 (6.8%); 112(11.6%); 98 (100%);
84 (14.7%); 69 (4.7%); 55 (5.4%); 41 (7.8%).
2-butyl-3-(2-(piperidin-1-yl)ethyl)-2,3-dihydro-4H-benzo[e][1,3]thia-
zin-4-one 5Ca
1
Yield: 57%; oil; H NMR (600 MHz, CDCl3) d (ppm, JH–H¼Hz): 8.07 (dd,
1H, H7, Ar, 3J¼ 8.3, 4J¼ 1.4); 7.36 (td, 1H, H9, Ar, 3J¼ 7.5, 4J¼ 1.4);
7.26 (dd, 2H, H8, H10, Ar, 2J¼ 11.2, 3J¼ 4.3); 4.63 (dd, 1H, H2,
3J¼ 9.6, 3J¼ 5.4); 4.29 (ddd, 1H, H11a, 2J¼ 13.3, 3J¼ 7.4, 3J¼ 5.6);
2
3
2-(4-methoxyphenyl)-3-(2-(piperidin-1-yl)ethyl)-2,3-dihydro-
4Hbenzo[e][1,3]thiazin-4-one 5Ce
3.32 (dt, 1H, H11b, J¼ 13.6, J¼ 6.7); 2.60 (s, 4H, H14); 2.78–2.68 (m,
2H, H12a, H12b); 1.97–1.79 (m, 2H, H17); 1.72–1.61 (m, 4H, H15);
1.50–1.39 (m, 2H, H18); 1.33–1.22 (m, 2H, H19); 0.86 (t, 3H, H20,
3J¼ 7.2). 13C NMR (150 MHz, CDCl3) d (ppm): 162.7 (C4); 134.1 (Ar);
131.7 (Ar); 129.8 (Ar); 129.0 (Ar); 127.8 (Ar); 125.8 (Ar); 61.8 (C2); 54.7
(C14); 57.1(C12); 45.7 (C11); 34.5 (C17); 28.9 (C18); 25.5 (C15); 23.8
(C16); 21.9 (C19); 13.8 (C20). MS m/z: 332 (Mþ, 0.8%); 304 (0.1%); 276
(0.5%); 248 (0.9%); 192 (0.4%); 136 (2.2%); 111 (35.2%); 112 (7.0%); 98
(100%); 84 (3.6%); 70 (4.4%); 55 (7.4%); 42 (7.7%).
Yield: 72%; oil; 1H NMR (600 MHz, CDCl3) d (ppm, JH–H¼Hz): 8.13
(dd, 1H, H7, Ar, 3J ¼ 7.8, 4J ¼ 1.2); 7.27 (td, 1H, H9, Ar, 3J ¼ 7.4,
4J ¼ 1.5); 7.21 (td, 1H, H10, Ar, 3J ¼ 7.7, 4J ¼ 1.2); 7.17 (d, 2H, H19,
Ar, 3J ¼ 8.7); 7.09 (dd, 1H, H8, Ar, 3J ¼ 7.7, 4J ¼ 0.8); 6.75 (d, 2H,
H18, Ar, 3J ¼ 8.8); 6.03 (s, 1H, H2); 4.31 (ddd, 1H, H11a, 2J ¼ 13.6,
3J ¼ 7.0, J ¼ 5.1); 3.26 (dt, 1H, H11b, J ¼ 13.9, J ¼ 7.1); 3.73 (s, 3H,
OCH3); 2.74 (ddd, 1H, H12a, 2J ¼ 12.9, 3J ¼ 7.1, 3J ¼ 5.8); 2.62 (dt,
1H, H12b, 2J ¼ 12.8, 3J ¼ 5.8); 2.49 (s, 4H, H14); 1.67–1.53 (m, 4H,
H15); 1.50–1.40 (s, 2H, H16). 13C NMR (150 MHz, CDCl3) d (ppm):
163.9 (C4); 159.3 (Ar); 133.2 (Ar); 131.8 (Ar); 131.0 (Ar); 129.7 (Ar);
129.0 (Ar); 127.4 (Ar); 127.4 (Ar); 126.0 (Ar); 113.7 (Ar); 62.0 (C2);
57.2 (C12); 55.1 (OCH3); 54.6 (C14); 45.3 (C11); 25.7 (C15); 24.0
(C16). MS m/z: 382 (Mþ, 5.2%); 349 (0.3%); 243 (0.9%); 136 (1.8%);
121 (6.1%); 111 (24.8%); 98 (100%); 84 (10.1%); 70 (3.9%); 55
(6.2%); 44 (3.1%).
3
2
3
2-phenyl-3-(2-(piperidin-1-yl)ethyl)-2,3-dihydro-4H-benzo[e][1,3]-
thiazin-4-one 5Cb
Yield: 62%; oil; 1H NMR (600 MHz, CDCl3) d (ppm, JH–H¼Hz): 8.15
(dd, 1H, H7, Ar, 3J ¼ 7.8, 4J ¼ 1.3); 7.30-7.20 (m, 7H, H9, H10, H18,
H19, H20, Ar); 7.10 (d, 1H, H8, Ar, 3J ¼ 7.7); 6.09 (s, 1H, H2); 4.38
(dt, 1H, H11a, 2J ¼ 13.7, 3J ¼ 5.8); 3.28 (dt, 1H, H11b, 2J ¼ 13.9,
3J ¼ 7.1); 2.83 (dt, 1H, H12a, 2J ¼ 12.7, 3J ¼ 6.8); 2.69 (ddd, 1H,
H12b, 2J ¼ 12.5, 3J ¼ 7.0, 3J ¼ 5.3); 2.56 (s, 4H, H14); 1.71–1.52 (m,
4H, H15); 1.55–1.43 (m, 2H, H16). 13C NMR (150 MHz, CDCl3) d
(ppm): 163.9 (C4); 139.1 (Ar); 133.0 (Ar); 131.9 (Ar); 129.0 (Ar);
128.3 (Ar); 128.0 (Ar); 127.4 (Ar); 126.1 (Ar); 62.1 (C2); 56.8 (C12);
54.4 (C14); 45.3 (C11); 25.4 (C15); 23.8 (C16). MS m/z: 352 (Mþ,
2.8%); 268 (0.6%); 136 (3.8%); 112 (4.6%); 111 (21.9%); 98 (100%);
84 (7.2%); 70 (4.1%); 55 (5.5%); 44 (6.4%).
2-(4-fluorophenyl)-3-(2-(piperidin-1-yl)ethyl)-2,3-dihydro-4H-ben-
zo[e][1,3]thiazin-4-one 5Cf
Yield: 88%; oil; 1H NMR (600 MHz, CDCl3) d (ppm, JH–H¼Hz): 8.12
(dd, 1H, H7, Ar, 3J ¼ 7.8, 4J ¼ 0.9); 7.28–7.25 (m, 1H, H9, Ar);
7.24–7.17 (m, 3H, H10, H19, Ar); 7.08 (d, 1H, H8, Ar, 3J ¼ 7.7); 6.89
(t, 2H, H18, Ar, 3J ¼ 8.6); 6.08 (s, 1H, H2); 4.29 (dt, 1H, H11a,
2J ¼ 13.7, 3J ¼ 5.7); 3.26 (dt, 1H, H11b, 2J ¼ 13.8, 3J ¼ 6.9); 2.72
(ddd, 1H, H12a, 2J ¼ 12.9, 3J ¼ 7.2, 3J ¼ 6.3); 2.59 (dt, 1H, H12b,
2J ¼ 12.9, 3J ¼ 5.8); 1.61 (s, 4H, H14); 1.46–1.39 (m, 2H, H16). 13C
NMR (150 MHz, CDCl3) d (ppm, JC–F¼Hz): 163.8 (C4); 162.3 (d, C20,
3-(2-(Piperidin-1-yl)ethyl)-2-(p-tolyl)-2,3-dihydro-4H-benzo[e][1,3]-
thiazin-4-one 5Cc
1J ¼ 247.6); 135.1 (d, C17, J ¼ 3.2); 132.9 (Ar); 131.9 (Ar); 129.8 (Ar);
3
Yield: 61%; oil; 1H NMR (600 MHz, CDCl3) d (ppm, JH–H¼Hz): 8.12
(dd, 1H, H7, Ar, 3J ¼ 7.8, 4J ¼ 1.3); 7.26 (dt, 1H, H9, Ar, 3J ¼ 7.6,
4J ¼ 1.6); 7.20 (td, 1H, H10, Ar, 3J ¼ 7.7, 4J ¼ 1.2); 7.12 (d, 2H, H19,
3J ¼ 8.1); 7.08 (dd, 1H, H8, Ar, 3J ¼ 7.7, 4J ¼ 0.8); 7.03 (d, 2H, H18,
3J ¼ 8.0); 6.03 (s, 1H, H2); 4.34 (dt, 1H, H11a, 2J ¼ 13.4, 3J ¼ 7.4);
129.2 (Ar); 127.9 (d, C19, 3J ¼ 8.3); 127.4 (Ar); 126.2 (Ar); 115.2 (d,
2J ¼ 21.8); 61.8 (C2); 57.5 (C12); 54.7 (C14); 45.5 (C11); 25.9 (C15);
24.1 (C16). MS m/z: 370 (Mþ, 1.8%); 286 (0.5%); 136 (3.3%); 123
(0.8%); 111 (15.8%); 98 (100%); 70 (3.8%); 55 (5.3%); 44 (3.8%).
2
3
2
3.26 (dt, 1H, H11b, J ¼ 14.0, J ¼ 7.1); 2.80 (dt, 1H, H12a, J ¼ 12.7,
3J ¼ 7.1); 2.67 (ddd, 1H, H12b, 2J ¼ 12.6, 3J ¼ 7.2, 3J ¼ 5.2); 2.57 (s,
4H, H14); 2.25 (s, 3H, CH3); 1.67–1.57 (m, 4H, H15); 1.44–1.48 (m,
2H, H16). 13C NMR (150 MHz, CDCl3) d (ppm): 163.9 (C4); 137.9
(Ar); 136.0 (Ar); 133.2 (Ar); 131.9 (Ar); 129.7 (Ar); 129.0 (Ar); 129.0
(Ar); 127.4 (Ar); 126.0 (Ar); 126.0 (Ar); 62.1 (C2); 56.8 (C12); 54.4
(C14); 45.3 (C11); 25.4 (C15); 23.8 (C16); 20.9 (CH3). MS m/z: 366
(Mþ, 3.1%); 282 (0.6%); 136 (2.3%); 112 (6.6%); 111 (23.7%); 98
(100%); 84 (8.7%); 70 (4.0%); 55 (5.5%); 44 (4.2%).
Effects in vitro of benzothiazinones in the brain AChE activity
Ten rats male Wistar (60 day-old) were obtained from the Central
Animal House of Federal University of Pelotas (Brazil). All animals
procedures were approved by the Ethics Committee and Animal
Experimentation of the institution (protocol number CEEA 9220).
The animals were submitted to euthanasia and cerebral cortex
and hippocampus were removed and homogenized in the 10 mm
Tris–HCl (pH 7.4) buffer. The proteins levels were determined by
Bradford method (1976) using bovine albumin as standard. The
benzothiazinones were dissolved in methanol and different con-
centrations (1, 5, 10, 25, 50, 100 and 250 mM) were tested in the
2-(4-nitrophenyl)-3-(2-(piperidin-1-yl)ethyl)-2,3-dihydro-4H-benzo[e]
[1,3]thiazin-4-one 5Cd
Yield: 93%; oil; 1H NMR (600 MHz, CDCl3) d (ppm, JH–H¼Hz): 8.12 enzymatic assay. The AChE activity was determined according to
(dd, 1H, H7, Ar, 3J ¼ 7.8, 3J ¼ 1.3); 8.06 (d, 2H, H19, Ar 3J ¼ 8.8); the method of Elmann et al.18 using acetylthiocholine (AcSCh) as

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
201
Scheme 1. Synthetic route to obtain benzothiazin-4-ones.
substrate. The specific AChE activity was expressed in lmol of
AcSCh/h/mg of protein.
Results and discussion
Benzothiazinones derivatives have attracted continuing interest
over the years because of their diverse biological activities12. A lit-
erature survey reveals that several benzothiazin-4-ones have been
prepared based on different synthetic routes: a) from classic pri-
mary amine, aldehyde or ketone and thiosalicylic acid as reported
by Zarghi et al.17 using p-toluenesulfonic acid catalyst in toluene
reflux or as reported by Kamel et al.20 using sodium sulfate in
dioxane or as reported by Kitsiou et al.21 and Silverberg et al.11
using propylphosphonic acid anhydride (T3P) as catalyst; b) from
2-aminothiophenol and chloroacetic acid as reported by Shaikh
et al.16; c) from aromatic carboxylic acid with ammonium isothio-
cyanate as reported by Peng et al.22
In this work, the compounds were synthesized using the ther-
mal heating methodology and the reaction conditions previously
described by our research group23, however, all reactions were
monitored by TLC and/or GC analysis. Novel nineteen 1,3-benzo-
thiazin-4-ones (5Aa–g, 5Ba–f and 5Ca–f) were synthesized from
one-pot reactions between different amines 1A–C, different alde-
hydes 2a–g and thiosalicylic acid 4 (Scheme 1). All reactions were
carried out in toluene reflux and a Dean–Stark apparatus was
used for water removal.
Cytotoxicity evaluation the benzothiazinones in
fibroblast culture
The cultures of MRC-5 (human fibroblast) were treated with ben-
zothiazinones to according to the method of Skehan et al.19 At
first, all compounds were solubilized in Dimethyl Sulfoxide
(DMSO) 0,1% and were prepared and tested in DMEM (Dulbecco’s
Modified Eagles’s Medium) at final concentrations of 50, 100 and
250 mm. The MRC-5 were seeded at 5 ꢀ 103 cells/well in DMEM/
5% FBS (fetal bovine serum) in 96-well plates. Cultures were
exposed to compounds for 24 h and the control cells were treated
with vehicle.
After 24 h, the cells were washed and added trichloroacetic
acid 50% for 45 min in the fridge for cell fixation. After, were car-
ried out five washes with distilled water to full removal of the
acid. A solution of B sulfarodamina (SRB) 0.4% acetic acid was
added followed by 30 min incubation to stain proteins. Then,
washings were made 5 of the wells with 1% acetic acid for com-
plete removal of uncomplexed dye with proteins. Finally, the
plates were solubilized and read in a spectrophotometer at
530 nm. B sulforodamina binds to the amino terminal portions of
the cells that were fixed with trichloroacetic acid, which is quanti-
fied spectrophotometrically. The results are presented in percent-
age of cell proliferation, considering the proliferation of control
group as 100%.
We observed that is important the complete formation of
imine intermediate before addiction of thiosalicylic acid, once
when all three reactants were put together in a vessel, a small
proportion of by-product oxathiolone was observed in GC (data
not shown). The complete formation of benzothiazinone was
observed in five hours and it is important to note that this time is
lower than generally find in literature for reactions with thiosaly-
clic acid (20–48 h)17,20,21
.
Statistical analysis
All compounds were obtained in regular to good yields and
have been properly purified and characterized. In general, the
yields obtained after purification varied from moderate to excel-
Results were analyzed by using one-way analysis of variance
(ANOVA) followed by Tukeyꢀs post hoc test for multiple compari-
sons in the software Graphpad Prism 5. All results were expressed lent and the methodology was considerate efficient once all pro-
as mean standard error (SEM) and the differences between mean posed products were obtained. All compounds were identified
values were considered significant when P< 0.05.
and characterized by mass spectrometry (GC–MS) and by nuclear

202
G. A. BERWALDT ET AL.
5Ba
5Bd
5Cd
5
4
3
2
1
0
5
4
3
2
1
0
5
4
3
2
1
0
IC50: 87.9
*
m
M
IC50: 8.5
m
M
IC50: 121.3 mM
*
*
*
**
**
***
***
***
***
***
***
***
***
r
r
r
e
H
H
H
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M
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M
M
M
M
M
M
M
te
te
t
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
O
O
a
a
1
5
0
0
1
5
5
0
0
0
1
5
0
5
W
W
Wa
Me
10
25
50
Me
10
2
5
Me
10
25
50
00
10
25
10
25
1
2
5Ba
5Cd
5Bd
10
8
10
8
4
3
2
1
0
IC50: 73.3
**
m
M
IC50: 39.8
m
M
IC50: 119.1 mM
*
6
6
***
4
4
**
***
***
***
2
2
0
0
r
r
r
H
H
H
O
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
te
te
te
a
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
O
O
a
a
1
5
0
0
1
5
0
0
1
5
0
0
5
W
W
W
Me
10
25
50
Me
10
25
50
Me
10
25
50
10
25
10
25
10
2
Figure 2. In vitro effect of 5Ba, 5Bd and 5Cd in the AChE activity in cerebral cortex and hippocampus of rats. One-way ANOVA followed by Tukeyꢀs post hoc test.
ꢃ
ꢃꢃ
ꢃꢃꢃ
P< 0.05, P< 0.01 and
P< 0.001 compared to the water control group (n ¼ 4–5).
All benzothiazinones 5B presented inhibitory effect in AChE
activity in both cerebral structures. Benzothiazinones 5Ba inhib-
ited the AChE activity since 10 mM (27.1%) in cerebral cortex (IC50
87.9 mM) and since 100 mM (46.2%) in hippocampus (IC50
73.3 mM). Similar results were obtained for benzothiazinone 5Bb
that inhibited the AChE activity since 10 mM (47.2%) in cerebral
cortex (IC50 8.5 mM) and in hippocampus at concentrations of
50 mM (50%) (IC50 39.8 mM) (Figure 2). Additionally, compound
5Bf showed IC50 equal to 61.7 mM in hippocampus, however did
not in cerebral cortex (IC50>250 mM) (data not show).
150
100
50
DMSO
50mM
100
250
m
m
M
M
***
**
***
***
***
On the other hand, it was verified that in general benzothiazi-
nones 5A did not demonstrated relevant inhibitory effects in
AChE activity in both cerebral structures, except compound 5Ad
that have IC50 70.59 mM and statistical difference since 100 mM
(46.4% of inhibition) in hippocampus (data not shown). In general,
benzothiazinones 5C showed better AChE inhibition results than
compounds 5A. From the six tested compounds, 5Cd showed
similar IC50 for both cerebral cortex and hippocampus (121.3 and
119.1 mM, respectively).
0
a
O
d
d
S
5B
5C
5B
DM
Figure 3. Cytotoxicity of benzothiazinones 5Ba, 5Bc and 5Cd in MCR-5 human
fibroblast cell. The results were expressed at percentage of cell proliferation, con-
sidering DMSO control group as 100%.
DMSO group.
ꢃꢃꢃ
P< 0.001 when compared with
magnetic resonance (NMR) of 1H and 13C. The H2/C2 (CH of ben-
The in vitro effect and IC50 values in AChE inhibition in cerebral
zothiazinone ring) and carbonyl group (C4) are the classic signals cortex and hippocampus of rats for all compounds are show in
that confirm the cyclization. The H2 was assigned as a singlet at supplementary information section. Compounds that did not
5.89–6.27 ppm (aryl substituents, b–g) and as a doublet of dou- exceed the 50% of inhibition at 250 mM (the highest concentra-
blets at 4.53–4.58 ppm (butyl, a), while the C4 resonates at typical tion tested), could not be calculate.
amide bond at 162.7–164.1 ppm. The other signals agree with the
The benzothiazinones were prepared from three different ali-
proposal structures.
phatic amines: 1A 4-aminoethylmorpholine; 1B N-(3-aminopropyl)-
After the synthesis, the in vitro effect of all benzothiazinones piperidine); and 1C 1-(2-aminoethyl)piperidine). Two heterocycles
was evaluated as AChE activity inhibitors. For this purpose, it was (morpholine and piperidine) and two carbon link chains (two or
used hippocampus and cerebral cortex of rats, brain structures three methylenes) were studies and these differences show
that play an important role in cognitive functions. Compounds importance for the activity. Compounds 5A did not present
5Aa–g, 5Ba–f and 5Ca–f were dissolved in methanol and different expressive inhibition rates, when compared with 5B and 5C and
concentrations were prepared. It is important to note that controls suggest that the extra oxygen in morpholine ring decrease the
were performed in methanol and water and our results showed inhibitory activity of compounds 5A. Compounds 5B demon-
no difference between them in the in vitro AChE activity, so, no strated better activities than compounds 5C. This fact suggests
methanol interference was observed. Figure 2 shows the in vitro that the extension of carbon chain link between nitrogen of
AChE analyses in all concentrations tested for compounds that piperidine and nitrogen of benzothiazinone ring is important once
have IC50 values in both cerebral cortex and hippocampus (5Ba, three methylene carbons (propylene moiety) improve the activity
5Bd and 5Cd).
of compounds 5B.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
203
Assessing the different aldehydes varied in the synthesis, the
compounds with the best activities (5Ba, 5Bd and 5Cd) were der-
ivate from valeraldehyde 2a or 4-nitrobenzaldehyde 2d. It is
important to note that compound 5Ad also show moderate
results in hippocampus suggesting that the NO2 group (d) plays
an important role in the AChE inhibition.
Finally, three benzothiazinones with the best AChE inhibition
activity were selected to evaluate the cytotoxic effect in MCR-5
human fibroblasts cells compared to DMSO control (Figure 3). The
most active compounds in the AChE study 5Ba and 5Bd were
noncytotoxic at 100 M. Unfortunately compound 5Cd showed tox-
icity at the lowest concentration tested 50 mM.
7. a. Silman I, Sussman JL. Acetylcholinesterase: How is struc-
ture related to function. Chem Biol Interact 2008;175:3–10.;
ꢂ
ꢂ
b. Akıncıoglu A, Kocaman E, Akıncıoglu H, Salmas RE,
_
€
€
Durdagi S, Gulc¸in I, Supuran CT, Goksu S. The synthesis of
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spectroscopic properties of 2,3-diphenyl-1,3-thiaza-4-one
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Therefore, these findings encourage the sequence of studies
with benzothiazinone 5Bd exploring chemical changes and trying
to improve the AChE inhibition without cytotoxicity.
Conclusions
In summary, novel benzothiazin-4-ones were efficient synthesized
by one-pot multicomponent reactions in moderate to excellent
yields through conventional heating methodology for 5 h. The
novel compounds were fully identified and characterized by 1H,
13C NMR and by GC-MS. In addition, benzothiazinone 5Bd showed
the most significant in vitro AChE activity in cerebral cortex of rats
(IC50 8.48 lM) and low cytotoxicity in human fibroblast cell. These
preliminary results will guide further investigation on heterocyclic
benzothiazinones aiming to the development of a new potent
AChE inhibitors agent.
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towards the discovery of novel carbamates as potent AChE
inhibitors for the potential treatment of Alzhiemer’s disease.
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Acknowledgements
The authors thank UFPel, FAPERGS (proc. 11/2068-7 and proc. 16/
2551-0000 2452) and CNPq (proc. 308791/2015-0) for finan-
cial support.
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SGLT2 inhibitors. Bioorg Med Chem Lett 2011;21:2472–5.
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activity of novel triazole incorporated benzothiazinone
derivatives as antitubercular and antioxidant agent. Bioorg
Med Chem Lett 2016;26:561–9.
Funding
ꢀ
ꢀ
17. Zarghi A, Zebardast T, Daraie B, et al. Design and synthesis
of new 1,3-benzthiazinan-4-one derivatives as selective
cyclooxygenase (COX-2) inhibitors. Bioorg Med Chem 2009;
17:5369–73.
Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico
~
ꢁ
(proc. 308791/2015-0); Fundac¸ao de Amparo a Pesquisa do Estado
do Rio Grande do Sul (proc. 11/2068-7 and proc. 16/2551-
0000 2452).
18. Ellman GL, Courtney KD, Andres V, et al. A new and rapid
colorimetric determination of acetylcholinesterase activity.
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