Synthesis of new tricyclic 1,2-thiazine derivatives with anti-inflammatory activity

Article abstract of DOI:10.3390/ijms22157818

New, tricyclic compounds containing a sulfonyl moiety in their structure, as potential safer COX inhibitors, were designed and synthesized. New derivatives have three conjugated rings and a sulfonyl group. A third ring, i.e., an oxazine, oxazepine or oxaz

Full text of DOI:10.3390/ijms22157818

International Journal of
Molecular Sciences
Article
Synthesis of New Tricyclic 1,2-Thiazine Derivatives with
Anti-Inflammatory Activity
1
,
2
, Zaneta Czy z˙ nikowska 3 and Berenika M. Szcz e˛ s´ niak-Si e˛ ga 1
˙
Jadwiga Maniewska * , Benita Wiatrak
1
Department of Medicinal Chemistry, Faculty of Pharmacy, Wroclaw Medical University,
Borowska 211, 50-556 Wrocław, Poland; berenika.szczesniak-siega@umed.wroc.pl
Department of Pharmacology, Faculty of Medicine, Wroclaw Medical University,
J. Mikulicza-Radeckiego 2, 50-345 Wrocław, Poland; benita.wiatrak@umed.wroc.pl
Department of Inorganic Chemistry, Faculty of Pharmacy, Wroclaw Medical University,
Borowska 211a, 50-556 Wrocław, Poland; zaneta.czyznikowska@umed.wroc.pl
Correspondence: jadwiga.maniewska@umed.wroc.pl; Tel.: +48-71-784-03-97
2
3
*
Abstract: New, tricyclic compounds containing a sulfonyl moiety in their structure, as potential
safer COX inhibitors, were designed and synthesized. New derivatives have three conjugated rings
and a sulfonyl group. A third ring, i.e., an oxazine, oxazepine or oxazocin, has been added to the
1
,2-benzothiazine skeleton. Their anti-COX-1/COX-2 and cytotoxic effects in vitro on NHDF cells,
together with the ability to interact with model membranes and the influence on reactive oxygen
species and nitric oxide, were studied. Additionally, a molecular docking study was performed
to understand the binding interaction of the compounds with the active site of cyclooxygenases.
For the abovementioned biological evaluation of new tricyclic 1,2-benzothiazine derivatives, the
following techniques and procedures were employed: the differential scanning calorimetry, the COX
colorimetric inhibitor screening assay, the MTT, DCF-DA and Griess assays. All of the compounds
studied demonstrated preferential inhibition of COX-2 compared to COX-1. Moreover, all the
examined tricyclic 1,2-thiazine derivatives interacted with the phospholipid model membranes.
Finally, they neither have cytotoxic potency, nor demonstrate significant influence on the level of
reactive oxygen species or nitric oxide. Overall, the tricyclic 1,2-thiazine derivatives are good starting
points for future pharmacological tests as a group of new anti-inflammatory agents.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Maniewska, J.; Wiatrak, B.;
Czy z˙ nikowska, Z.; Szcz e˛ s´ niak-Si e˛ ga,
˙
B.M. Synthesis of New Tricyclic
1
,2-Thiazine Derivatives with
Anti-Inflammatory Activity. Int. J.
Mol. Sci. 2021, 22, 7818. https://
doi.org/10.3390/ijms22157818
Academic Editors:
Andrea Spallarossa and
Eleonora Russo
Keywords: synthesis; tricyclic compounds; 1,2-thiazine; cyclooxygenase inhibition; model mem-
brane; DSC; molecular docking
Received: 10 June 2021
Accepted: 19 July 2021
Published: 22 July 2021
1
. Introduction
Inflammation is the body’s natural response to factors that threaten homeostasis such
as microbial infection or tissue damage resulting from trauma. Activation of the immune
system is aimed at removing pathogens or damaged cells. Under physiological conditions,
once the stimulus has been removed, the inflammation begins to cease because its initial
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published maps and institutional affil-
iations.
purpose has been achieved [1]. However, fairly frequently we are dealing with prolonged
inflammation lasting months or years, the so-called chronic inflammation. Many different
factors lead to chronic inflammation such as failure to eliminate the initial cause of cell
injury, exposure to low levels of certain irritants or foreign materials, an autoimmune disor-
der, a defect in the cells mediating inflammation, recurrent episodes of acute inflammation
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
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distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
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as well as molecular inducers of oxidative stress and mitochondrial dysfunction [2]. Al-
though chronic inflammation is not a primary cause of most diseases such as cardiovascular
disease, diabetes, rheumatoid arthritis, asthma, chronic obstructive pulmonary disease,
chronic kidney disease, inflammatory bowel disease, cancer or Alzheimer’s disease, it
contributes significantly to their pathogenesis [2–11].
There are many different inflammatory mediators such as prostaglandins, pro-inflammatory
cytokines, and chemokines, but prostaglandin E (PGE ) is among those most impor-
4
.0/).
2
2
Int. J. Mol. Sci. 2021, 22, 7818. https://doi.org/10.3390/ijms22157818
https://www.mdpi.com/journal/ijms

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tant [12]. PGE is formed as a result of the transformation of arachidonic acid by the
2
cyclooxygenase (COX). There are three isoforms of the enzyme: COX-1, COX-2, and COX-
3 [13]. COX-1 is a glycoprotein that occurs under physiological conditions (constitutive
form) in many organ tissues, such as the kidneys, stomach, intestines, ovaries, platelets, and
fulfills many protective functions there [14]. COX-2 is expressed upon induction by various
factors (e.g., cytokines, IL-1
β
, IL 6, TNF , mitogens, growth factors) in inflammation, pain
α
response, tissue damage or carcinogenesis. Moreover, this COX variant is produced in
large amounts during the adaptive processes (e.g., at the site of the wound or ulcer healing).
In recent years, the constitutive form of COX-2 has also been found in some organs, such
as the spinal cord, kidneys, vascular endothelium or the uterus [14]. The COX-3 isoform
is present mainly in the brain and spinal cord, and its role is not fully understood [15].
Overexpression of the inducible form of COX-2, associated with increased production of
PGE , plays a key role in the process of chronic inflammation [3].
2
The non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen,
meloxicam, indomethacin or nimesulide, are the best-known cyclooxygenase inhibitors.
However, they show many side effects, mainly from the gastrointestinal tract (such as
erosions, stomach ulcers or gastrointestinal bleeding), which get especially dangerous
when NSAIDs are used over the long term in conditions associated with chronic pain and
inflammation, e.g., in rheumatoid diseases. For this reason, studies are underway for the
development of new, safer painkillers and anti-inflammatory drugs.
In 2019, Rabbani published a patent review of COX-2 inhibitors. This review discusses
the structures of novel COX-2 inhibitors synthesized during the last five years [16]. Our
attention was drawn to the fact that the new COX-2 inhibitors differ from classic NSAIDs—
they have a multi-ring structure—3-, 4- or 5-membered. This prompted us to look for
second-generation COX-2 inhibitors among compounds with a polycyclic structure. As a
continuation of our previous work on new oxicam derivatives, we decided to expand their
molecule by adding a third ring to the 1,2-benzothiazine skeleton found in meloxicam-a
classic NSAID [17].
Among the structures described by Rabbani, there are the sulfone derivatives obtained
by El-Gamal [18]. El-Gamal synthesized a group of three-conjugated ring compounds with
a sulfonyl group (Figure 1). El-Gamal’s work reassured us that our designed compounds
were the right direction of research, as its sulfonyl tricyclic compounds showed promising
properties—they were COX-2 inhibitors at both the enzymatic and gene levels, with a po-
tency superior to etoricoxib, which is a selective COX-2 inhibitor. The sulfonyl group seems
to enhance the anti-inflammatory effect, as many NSAIDs incorporate it in their structure,
including piroxicam, meloxicam, nimesulide, celecoxib, rofecoxib or etoricoxib (Figure 2).
1
f
1p
Figure 1. Chemical structure of the two most potent sulfonate derivatives synthesized by El-Gamal,
named 1f and 1p [18].

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O
O
O
O
S
S
CH3
H C
NH
3
N
O
H
N
N
OH
O
NO2
piroxicam
meloxicam
nimesulide
celecoxib
rofecoxib
Figure 2. Examples of NSAIDs with the sulfonyl group in their structures.
etoricoxib
Our new derivatives, similarly to the El-Gamal’s compounds, have three conjugated
rings and a sulfonyl group. To the 1,2-benzothiazine skeleton in which the sulfonyl group
is a part of the thiazine ring, we had added a third oxazine, oxazepine or oxazocin ring
(Figure 3). The most active of El-Gamal’s compounds had also a methyl and methoxy
substituent; therefore, it was planned to incorporate these substituents as well into the new
structures. Besides, compounds with bromine or chlorine substituents were planned to be
explored for the effects of these structural modifications on the COX-2 inhibitory activity.
Firstly, all new compounds were tested for cytotoxicity to exclude toxic compounds
from further studies. The next step was to study COX-1 and COX-2 inhibition. Because
the delayed phase of the inflammatory response has been associated with the neutrophil
infiltration and production of free radicals and neutrophil-derived oxidants such as H O ,
2
2
·
·
O2 and HO , we decided to investigate the free radical scavenging ability of the new
compounds [19,20].
(
)
n
b
a
(
)
n
n = 1, 2, 3
c
3 3
R = H, Br, Cl, CH , OCH
Figure 3. Structures of meloxicam (a), El-Gamal’s compounds (b) and the designed new compounds (c).
Since COX is a membrane-bound enzyme, the ability to penetrate membranes is
necessary for the drug–enzyme interactions, which was tested on the model biological
membranes. Additionally, a molecular modeling study was performed to determine the
binding of the new compounds to the target enzyme. In order to determine the importance

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of the tricyclic structure on COX activity, three bicyclic compounds with 1,2-benzothiazine
scaffold were also tested.
2. Results and Discussion
2.1. Chemistry
The synthetic route of new tricyclic 1,2-thiazine derivatives is shown in Scheme 1. The
conventional synthesis of the intermediates 3a–3e and 4a–4e was previously
reported [17 21 23]. However, in this work, microwave synthesis, which has not been
,
–
described before, was used to prepare compounds 3a
–
3e. A mixture of saccharine
1 with
0
appropriate 4 -substituted-2-bromoacetophenone 2a
–2e in dimethylformamide and tri-
ethylamine was exposed to microwave irradiation at 150 W for 3 min. The application
of this method significantly shortened the reaction time from 10 h in the conventional
method to 3 min. Moreover, compounds 3a
95–98%). In the next step, compounds 3a 3e were rearranged in Gabriel–Colman rear-
rangement, which resulted in compounds 4a 4e. The final compound was obtained in
reaction 1-bromo-2-chloroethane with compound 4a in acetonitrile with potassium carbon-
ate. Compounds 6a 6e were obtained in the same conditions as compound , in reaction
-bromo-3-chloropropane with appropriate compounds 4a 4e. Similarly, compound was
–3e were obtained with very high yields
(
–
–
5
–
5
1
–
7
obtained in reaction 1,4-dibromobutane with compound 4a. The chemical structure and
symbols of newly synthesized tricyclic 1,2-thiazine derivatives are shown in Table 1 and
Scheme 1.
O
O
O
O
O
O
O
R
S
R
O
S
EtONa
HCl
hν
S
NH
R
N
NH
+
Br
DMF
N(C H )
2 5 3
O
O
O H
O
2
a - 2e
3a - 3e
1
4a - 4e
R
H
2
2
2
2
a, 3a, 4a
b, 3b, 4b Br
c, 3c, 4c Cl
d, 3d, 4d CH
O
O
3
Cl
Br
S
N
2e, 3e, 4e OCH
3
O
O
5
O
O
Br
Cl
S
O
O
N
S
R
NH
O
O
O H
O
O
O
4
a - 4e
Br
S
Br
N
R
O
6a - 6e
O
7
R
H
Br
Cl
CH
6
6
6
a
b
c
6d
3
6
e
OCH
3
Scheme 1. The synthetic route of new tricyclic 1,2-thiazine derivatives.
2.2. QSAR Studies
The 3D/4D QSAR model with restricted docking protocol was used to estimate
the octanol–water partition coefficient (LogP). The lipophilicity of compounds indicates
penetration of orally available drugs through biological membranes. According to the
results, all compounds investigated are lipophilic. The values of the LogP for new tricyclic
1,2-thiazine derivatives are not exceeding 4 (Table 1). It is also worth noting that substitution
of benzene ring in the case of compounds lowers calculated lipophilicity of compounds.
5–7

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The lowest value of LogP was obtained for 6e compound. The compounds with good
oral absorption show LogP in the range from
synthesized tricyclic 1,2-thiazine derivatives falls within this range.
−
1.0 to 5.9 [24]. The LogP of the newly
Table 1. Chemical structure and symbols of new tricyclic 1,2-thiazine derivatives with LogP values.
Compound
Symbol
Chemical
Structure
LogP 1
4
a
3.43
4
c
3.96
3.81
4
d
5
3.53
3.64
6
a
6
b
2.64
2.76
2.64
6
c
6
d

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Table 1. Cont.
Compound
Symbol
Chemical
Structure
LogP 1
6
e
1.48
7
3.54
1
The octanol–water partition coefficient (logP) predicted based on the 3D/4D QSAR algorithm Cinderella’s Shoe (CiS) for model [25].
2.3. Interaction with Model Membranes
To investigate the interaction of the tricyclic 1,2-thiazine derivatives (Table 1) with
model membranes, we used the differential scanning calorimetry (DSC) method. As a
model of the phospholipid membrane, the multibilayers made of 1,2-dipalmitoyl-n-glycero-
3
-phosphatidylcholine (DPPC) in a buffer solution (pH 7.4) were used. The influence of
ten new 1,2-thiazine derivatives on the thermal properties of DPPC bilayers in pH 7.4 was
examined. For comparison, the effect of meloxicam was also registered. The addition of the
studied compounds caused the disappearance of the DPPC pretransition and concentration-
dependent shift of the main transition temperature towards lower values, accompanied by
a decrease in the transition peaks area and the broadening of the peaks (Figure 4).
Figure 4. The example thermograms obtained for DPPC mixed with meloxicam (left) and compound
6e (right) as well as for pure lipid (the first curve from the top—black color). Curves in the Figure
represent the thermograms obtained for different molar ratios (studied compound: DPPC—from the
bottom: 0.12, 0.10, 0.08, 0.06, pure lipid). The exothermic direction in this graph is downward.
m
The dependencies of the DPPC phase main transition temperature (T ) on the 1,2-
thiazine derivatives: phospholipid molar ratio obtained for mixtures of DPPC with the
studied compounds and meloxicam are shown in Figure 5. In DSC studies, meloxicam
appeared to be the least effective compound.

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Figure 5. Influence of meloxicam and the 1,2-thiazine derivatives on the DPPC main phase transition
temperature (T ). Bars represent standard deviations of four measurements; where no error bars are
m
shown, they were smaller than the symbols representing results.
m
The dependencies of the DPPC phase main transition temperature (T ) and the peak
transition half-height (
∆
T
1
2
) on DPPC mixed with 6e compound or meloxicam are shown
in Figures 4 and 6. Whereas, regarding the dependencies of the transition peak half-height
width (
∆
T
1
) and transition enthalpy (
∆
H) of the main phase transition of DPPC on the
2
1
,2-thiazine derivatives, the phospholipid molar ratio obtained for mixtures of DPPC with
the studied compounds and meloxicam are shown in the Supplementary Materials.
(
a)
(b)
Figure 6. The influence of
m
6e compound and Meloxicam on the DPPC: (a) main phase transition temperature (T ); (b) peak
half-height width (T
1
2
). Bars represent standard deviations of four measurements; where no error bars are shown, they were
smaller than the symbols representing results.
Kyrikou and coworkers studied the thermotropic properties of DPPC multibilayers
in the presence of oxicam derivatives (piroxicam, tenoxicam, meloxicam, lornoxicam)
before [26]. They found that oxicam derivatives under consideration caused broadening
of the main phase transition (∆T1/2) of DPPC bilayers and lowering of the main phase
transition temperature (Tm). Similar results were obtained in our former studies [27,28].
The 1,2-thiazine derivatives added to model membranes influenced the thermotropic
properties of DPPC in a concentration-dependent manner. Meloxicam, used as a reference
drug, appeared to be the least effective compound in the studies of interaction with model

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membranes. All examined compounds decreased the main transition temperature (T
m
) of
DPPC, increased transition peak width at the half-height (T
1
) by broadening the transition
2
peaks, and decreased the enthalpy (
∆
H) of DPPC main phase transition. The character of
observed changes may allow to conclude that interactions between phospholipid molecules
in the gel state became weaker in the presence of studied compounds, and that lipid polar
heads as well as hydrocarbon chains regions were affected by the studied compounds
(according to the standard interpretation of calorimetric data proposed by Jain and Wu) [29].
The results of the interaction of the 1,2-thiazine derivatives with the model membranes do
not strictly correlate with the theoretically determined LogP values. It could be assumed
that the greater the lipophilicity of the compound, the better the interaction with the
model membrane might be. However, too high lipophilicity may result in the increased
nonspecific plasma protein binding [24]. In our results, in case of parameter changes of
DPPC gel-liquid crystalline phase transition studied here, the most pronounced effects
were found in the presence of compounds 6c
(see Table 1 and Figure 5). These compounds differ only by the substituent in benzene
ring—a chlorine atom (compound 6c), a methyl group (compound 6d) or a methoxy group
, 6d and 6e, whose LogP is approximately
2
(
(
compound 6e). The difference of the interaction of compound 6e and the reference drug
meloxicam) with model membranes formed from DPPC is shown in Figure 4. The effect is
much more pronounced for compound 6e than for meloxicam.
2.4. Biological Tests
2.4.1. Viability of Cell Cultures
The viability of the Normal Human Dermal Fibroblasts (NHDF) cells was assessed af-
ter 24 h incubation with 10, 50 and 100
dependent decrease in cell viability was observed for all compounds tested (Figure 7). After
incubation with 4a 4c and 6c compounds at 10 M and 50 M, a statistically significant in-
µM tested compounds in MTT assay. A concentration-
,
µ
µ
crease in mitochondrial activity (possibly related to an increase in proliferation) was noted.
In 4a and 4c, no cytotoxic effect was observed in the entire concentration range tested. The
less than 30% decrease in culture viability was observed only in concentration of 100 µM
for compound 6b in comparison to control (i.e., no cytotoxic potential of tested compound).
Results of viability of cell cultures test showed that all new 1,2-thiazine derivatives are
non-toxic for a NHDF cell line and have no cytotoxic potencies. Thus, all new compounds
were directed to further research.
Figure 7. Effect of tested compounds on the viability of NHDF cells; * p < 0.05—significant difference
compared to control.
2.4.2. The COX Colorimetric Inhibitor Screening Assay
The impact of three bicyclic and seven tricyclic 1,2-thiazine derivatives, and meloxicam,
as reference drug, on COX-1 and COX-2 activities was tested. The IC50 values were
calculated separately for COX-1 and COX-2 activity estimations at 2 min of incubation with
the tested compounds. The selectivity of the compounds for COX-1 or COX-2 was assessed

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by calculation of the IC50 ratios. Results of COX-1 and COX-2 inhibitory activity for all
studied compounds and meloxicam are given in Table 2.
All compounds tested—both bicyclic (4a
inhibited COX-1 and COX-2 activity (Table 2). Moreover, most of the tested compounds
except 6b and 6c) showed preferential inhibition of COX-2 compared to COX-1. The 4a
4d 6e and compounds had stronger COX-2 inhibition effect than the meloxicam.
For 4a 4c 4d 6e and compounds were observed higher COX-2/COX-1 selectivity than
for meloxicam, considered to be the preferred COX-2 inhibitor. However, also for the 6a
, 4c and 4d) and tricyclic (5, 6a–6e and 7)—
(
,
4c,
,
5,
7
,
,
,
7
5
,
and 6d compounds, this ratio is close to the reference one, which shows that most of the
compounds studied are preferential COX-2 inhibitors, similarly to meloxicam.
Although compounds 4a
compounds, because they have no third conjugated ring, they all showed similar high
inhibitory activity towards COX-2. This suggests that the third ring, attached to the 1,2-
benzothiazine skeleton, is not essential for this activity. Comparing the 4a 4c and 4d
compounds with each other, it can be seen that they show very similar activity, which
, 4c and 4d have different chemical structure than 5, 6e and
7
,
indicates that the substituent on the phenyl ring (H, Cl or CH ) is irrelevant to this activity
3
in this group of compounds.
In the tricyclic group of 1,2-thiazine derivatives, most of the compounds with the
oxazepine ring (6a
–
6d) showed weaker activity than the derivative containing the ring
), suggesting that the ring size is important for enzyme
of oxazine ( ) and oxazocin (
5
7
inhibition. However, the highest COX-2 selectivity showed 6e compound, with oxazepine
ring and methoxy substituent, indicating that the effect of the substituent on the inhibitory
potency of COX-2 is also significant. However, a full understanding of the structure–
activity relationship (SAR) in the group of tricyclic 1,2-thiazine derivatives requires further
in-depth studies.
Table 2. IC50 values calculated for COX-1 and COX-2 enzymes after incubation for 2 min with the
1
,2-thiazine derivatives or meloxicam, and COX selectivity ratio (mean (SD); n = 3).
IC50 [µM]
Ratio:
COX-2/COX-1
Compound
COX-1
COX-2
4
4
a
c
91.2 (0.18)
89.0 (0.16)
89.3 (0.09)
66.1 (0.08)
105.5 (0.15)
86.9 (0.15)
89.8 (0.14)
125.7 (0.07)
115.3 (0.11)
86.1 (0.08)
83.7 (0.10)
54.6 (0.06)
55.9 (0.08)
55.3 (0.10)
56.9 (0.13)
89.9 (0.14)
95.1(0.10)
94.1 (0.12)
92.9 (0.09)
56.9 (0.06)
54.0 (0.09)
59.2 (0.12)
0.6
0.63
0.62
0.86
0.85
1.09
1.05
0.74
0.49
0.63
0.71
4
d
5
6
a
6
b
6
c
6
d
6
7
e
meloxicam
SD values are given in brackets.
2.4.3. Reactive Oxygen Species (ROS) and Nitric Oxide (NO)
The level of reactive oxygen species (ROS) was evaluated in DCF-DA assay and
nitric oxide (NO) in Griess assay, on NHDF cells. The tested compounds did not cause
a significant increase or decrease in the level of free oxygen radicals (ROS), except for
compound 4a at a concentration of 10 µM, when a statistically significant increase was
observed compared to the control (Figure 8). This increase may be due to the normal
cellular activity due to the increased cell numbers, probably resulting from proliferation.
Similarly as in ROS studies, all compounds tested showed no increase or decrease in
NO level across the range of concentrations tested (data not shown). The mean level of NO
ranged from 0.98 to 1.03 compared to the control group (1.0).

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Figure 8. Effect of tested compounds on ROS level on NHDF cells; * p < 0.05–significant difference
compared to control.
2.5. Molecular Docking
The binding mode of the 1,2-thiazine derivatives to the binding site of both cyclooxy-
genases was determined by using molecular docking. All data are presented in the Supple-
mentary Materials and Figure 9. The crystallographic data, despite the structural diversity,
exhibited that COX-1 and COX-2 enzymes have almost identical molecular weight and
are characterized by similar binding site [30]. Due to their amino acid sequence homology
close to 65%, the binding manner of the ligands is slightly different. The subtle differences
are responsible for selectivity of inhibitors and are adopted in the design process. The
formation of additional binding pockets including Leu352, Ser353, Tyr355, Phe518 and
Val523 of COX-2 is connected with the replacement of Ile523 with smaller side-chain Val523
residue and the conformation changes of Tyr355.
It is well known that scoring functions which are used in the docking algorithms
only give approximate values of binding energies. Hence, it was necessary to validate
them with in vitro measurements. According to the results of molecular modelling study,
all compounds tested can bind to the active center of COX-2. The colorimetric inhibitor
screening assay indicated that compounds 4a, 4c, 4d, 5, 6a, 6d, 6e and 7 had stronger
COX-2 inhibition effect than meloxicam. In the mentioned cases, the binding manner was
dependent on the structural properties of docked ligand. Most of the considered ligands
can bind similarly to the substrate arachidonic acid in the active site of cyclooxygenase
typically through H-bonding interactions with Arg120, Tyr355 and Ser350 [31
analogous mode of binding is observed in the case of classic nonsteroidal anti-inflammatory
drugs (NSAIDs) including indomethacin and diclofenac [34 35]. Some compounds similar
–33]. The
,
to the meloxicam bind in a hydrophobic pocket comprising Ser353, Leu384, Tyr385, Trp387,
Val523 and Met522 and interact with the water molecule (4a, 4c, 4d, 5 and 6e), [36].
In the case of the 4c-COX-2 complex, four hydrogen bonding interactions were found
with Arg120 (2.84 Å; 3.30 Å), Tyr355 (3.01 Å) and Ser530 (2.81Å). As shown in Figure S17 (in
Supplementary Materials), 4c can bind to the same binding pocket of COX-2 as meloxicam
including Arg120, Tyr355, Val523, Gly526, Ala527, Ser530 and Leu531, which arises due
to the conformation of Tyr 355. Similar results were obtained in the case of binding to the
COX-1, although
is located near Leu117, Arg120, Leu352, Ile523, Gly526, Ala527 and Ser530 of COX-1 and
can form one hydrogen bond with Ser530 amino acid.
∆
G of binding has higher value ( 10.0 kcal/mol). Similar to meloxicam,
−
4c

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(
a) 6e‐COX‐1
6e‐COX‐2
(
b) COX‐1
COX‐2
Figure 9. (a) The intermolecular interactions (hydrophobic as red arches and hydrogen bonds as navy blue lines). (b) Binding
mode of compound 6e and meloxicam in the active center of cyclooxygenases (meloxicam is marked in yellow, 6e is green,
water molecule is red).
As determined, the 4a and 4d compounds are similar to 4c orientation in the binding
site of COX-2 (see Supplementary Materials). Two hydrogen bonds are created between
oxygens of the sulfonyl group of both compounds and Arg120 and Tyr355 amino acid
residues. Additionally, 1,2-benzothiazine skeleton forms hydrophobic interactions with
Val116, Met522, Ala527 and Leu531. There is also H-bonding interaction, which involves
oxygen atoms of the carbonyl group of tested compounds and Ser530 of COX-2. On the
contrary, the 4a and 4d compounds’ binding to the binding pocket of COX-1 is slightly
different. Firstly, you can observe two hydrogen bonds creation with Arg120 and Tyr355.
Moreover, benzene and methylbenzene rings are exposed towards hydrophobic and polar
amino acid residues (Leu384, Tyr385, Trp387, Met522).
The addition of a third ring to the benzothiazine moiety in the tricyclic 1,2-thiazine
derivatives (compounds
of enzymes. As determined, compound
5
and 6a) affects orientation of inhibitors in the binding site
can form two hydrogen bonds with Arg120
5
and Tyr355 amino acids in the case of COX-2 binding. The benzene ring can penetrate
the hydrophobic pocket formed by Leu384, Tyr385, Trp387, Met522 and Gly526. Similar

Int. J. Mol. Sci. 2021, 22, 7818
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binding manner was observed in the binding cavity of COX-1; however, no hydrogen
bonds were formed.
The oxazepino-benzothiazine derivatives (compounds 6b, 6c, 6d and 6e) bind to the
enzymes differently than the ligands described above. The 6b and 6c compounds can bind
more strongly to COX-1, which is proven by inhibition measurements (see Supplementary
Materials). In contrast to COX-2, COX-1 can form two hydrogen bonds with oxygen atoms
of oxazepine and the sulfonyl group of 6b. In the vicinity of the bromobenzene ring, the
hydrophobic and polar amino acids Leu93, Val116, Leu359 and Tyr355 are present. The
binding mode of 6c in the active center of protein is similar; however, in this case three
hydrogen bonds with Arg120, Ser530 and Leu531 of COX-1 are formed.
The replacement of halogens with a methyl and metoxy substituent does not sub-
stantially change the binding manner in the case of binding to the COX-1 pocket. The
compounds 6d, 6e and 6a (without a substituent in the benzene ring) binding to the protein
are exposed towards mainly hydrophobic amino acid residues (Leu93, Val116, Leu117,
Ile345, Val349, Leu357, Leu359, Ile523, Ala527 and Ser530) which can be involved in the
van der Waals type of interactions. The first two inhibitors also form three hydrogen bonds
with Arg120, Ser530, Leu531 of COX-1.
In the case of interactions with COX-2, 6a and 6d interact via two hydrogen bonds
with Arg120 moiety with a distance between electronegative atoms close to 3 Å. The way
of binding is typical for the inhibitors with oxazepine-benzothiazine moiety (6a–6d).
According to the enzymatic measurements, the 6e compound is the most selective
inhibitor of COX-2 (the lowest value of the COX-2-/COX-1 ratio). As can be observed
in Figure 9, compound 6e exhibits a unique binding configuration. As can be seen, in
this case the oxazepine-benzothiazine moiety occupied the characteristic hydrophobic
pocket of COX-2 formed by Val349, Leu352, Tyr355, Ser353, Leu359, Tyr385, Trp387, Val523,
Gly526 and Ala527 and forms a hydrogen bond with water molecule (H O784 (2.96 Å)) as
2
meloxicam. On the other hand, the methoxybenzene ring penetrates the cavity created by
Ser353, Leu531, Met535 and Leu534.
The oxazocin derivative (compound
of COX-1. As can be observed, two hydrogen bonds are created between the oxygens of
the sulfonyl group of compound and Ser530 and Leu531 amino acid residues of COX-2.
7) is almost located in the same binding place
7
The hydrophobic interactions with Met113, Val116, Leu117, Arg120, Ile345, Tyr355, Leu359,
Val523, Ala527 and Ser530 were also found.
3. Materials and Methods
3.1. Chemistry
The reagents and solvents were obtained from commercial sources (Merck Life Science,
Merck KGaA, Darmstadt, Germany; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany,
Alfa Aesar, Thermo Fisher GmbH, Kandel, Germany). All the chemicals were of analytical
grade and used without further purification. Melting points were determined in open
glass capillaries using a MEL-TEMP melting-point apparatus and were uncorrected. Thin-
layer chromatography (TLC) was performed on aluminum sheets pre-coated with Merck
1
13
silica gel 60 F254, and detection was achieved under ultraviolet (UV) light. H and
C
NMR spectra were recorded on a Bruker 300 MHz spectrometer using CDCl as a solvent.
3
1
Chemical shifts for proton nuclear magnetic resonance ( H NMR) spectra are reported
in parts per million (ppm) relative to the signal of tetramethylsilane at 0 ppm (internal
standard). Splitting patterns are designated as follows: s, singlet; t, triplet; m, multiplet.
13
Chemical shifts for carbon nuclear magnetic resonance ( C NMR) spectra are reported in
parts per million (ppm) relative to the center line of the CDCl triplet at 76.9 ppm. FT-IR
3
spectra were recorded on a Perkin-Elmer Spectrum Two UATR FT-IR spectrometer. Mass
data were acquired on a Bruker Daltonics micrOTOF-Q Mass Spectrometer in a positive ion
mode with flow-injection electrospray ionization (ESI). The elemental analyses were carried
out on a Carlo Erba NA 1500 analyzer and were within ±0.4% of the theoretical value.

Int. J. Mol. Sci. 2021, 22, 7818
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3.1.1. The Microwave Synthesis of Compounds 3a–3e
The conventional synthesis and experimental data of compounds 3a–3e and 4a–4e
were previously reported [17,21–23].
A mixture of commercially available saccharine (0.92 g, 5 mmol) with 5 mmol of
0
corresponding 4 -substituted-2-bromoacetophenone 2a
–
2e (2-bromoacetophenone for 3a
;
0
0
0
2
,4 -dibromoacetophenone for 3b; 4 -chloro-2-bromoacetophenone for 3c; 4 -methyl-2-
0
bromoacetophenone for 3d; 4 -methoxy-2-bromoacetophenone for 3e) in 7 mL of dimethyl-
formamide (DMF) and 0.7 mL of triethylamine (TEA) was exposed to microwave irradiation
at 150 W for 3 min. Then, the mixture was poured over ice-cooled water (50 mL), result-
ing in the formation of a white solid, which was filtered and washed with cold water.
The solid was dried and recrystallized from ethanol to give 2-(4-substitutedphenacyl)-2H-
benzisothiazol-3-on 1,1-dioxides with 95–98% yield.
In the next step, compounds 3a
–3e were rearranged in Gabriel–Colman rearrangement,
which resulted in compounds 4a–4e [17,21–23].
3
.1.2. Synthesis and Experimental Data of Tricyclic 1,2-Benzothiazine Derivatives (
In a 100 mL round-bottom flask equipped with a reflux condenser and a magnetic
stirrer, 3 mmol of compound 4a (for final compounds 6a and ) or compound 4b (for 6b),
compound 4c (for 6c), compound 4d (for 6d) or compound 4e (for 6e) were dissolved in
5, 6a–6e and 7)
5,
7
1
0 mL of acetonitrile, and then 9 mmol of 1-bromo-2-chloroethane (for compound
5) or
1
-bromo-3-chloropropane (for compounds 6a 6e) or 1,4-dibromobutane (for compound 7
–
)
and 9 mmol of anhydrous potassium carbonate were added. The obtained suspension
was stirred at reflux for 5 h. When the reaction ended, which was controlled on TLC
plates, acetonitrile was distilled off, the residue was treated with 50 mL of chloroform and
insoluble materials were filtered off. The filtrate was then evaporated and the residue was
purified by crystallization from ethanol to give desirable products
medium yields.
5, 6a–6e and 7 with
6
,6-dioxo-1-phenyl-3,4-dihydro-[1,4] oxazino [4,3-b][1,2]benzothiazin-11-one (5)
◦
−1
Yellow powder, 56% yield, mp 182–183 C; FT-IR (cm ): 1665 (C=O), 1325, 1172
1
(
(
(
SO ). H NMR (300 MHz, CDCl )
t, J = 4.8 Hz, 2H, O-CH ), 7.43–7.51 (m, 5H, arom.), 7.65–7.75 (m, 2H, arom.), 7.89–7.92
m, 1H, arom.), 8.04–8.07 (m, 1H, arom.). C NMR (300 MHz, CDCl3)
δ
(ppm): 4.00–4.03 (t, J = 4.8 Hz, 2H, N-CH ), 4.53–4.56
2
3
2
2
13
δ
(ppm): 177.37,
1
6
57.55, 139.27, 134.17, 133.17, 132.97, 131.32, 130.17, 129.08, 128.96, 128.06, 121.53, 115.36,
7.46, 38.79. HRMS (ESI) calcd. for C H NO S [M+H]+ 328.0638; found: 328.0628. Anal.
17
13
4
calcd. for C H NO S: C, 62.37; H, 4.00; N, 4.28; found: C, 62.83; H, 4.05; N, 4.21.
17
13
4
7
,7-dioxo-1-phenyl-4,5-dihydro-3H-[1,4]oxazepino[4,3-b][1,2]benzothiazin-12-one (6a)
◦
−1
Beige powder, 52% yield, mp 171–172 C; FT-IR (cm ): 1698 (C=O), 1352, 1177 (SO2).
H NMR (300 MHz, CDCl3) (ppm): 1.98 (m, 1H, 4-CHax), 2.22–2.27 (m, 1H, 4-CHeq),
.62–2.73 (m, 1H, 5-CHax), 3.06–3.15 (m, 1H, 5-CHeq), 3.51–3.60 (m, 1H, 3-CHax), 4.04–4.12
m, 1H, 3-CHeq), 7.44–7.60 (m, 3H, arom.), 7.73–7.86 (m, 3H, arom.), 8.16–8.27 (m, 3H,
arom.). 13C NMR (300 MHz, CDCl3)
(ppm): 194.64, 189.32, 139.10, 135.06, 133.95, 133.25,
33.18, 129.71, 129.38, 128.48, 128.40, 124.56, 83.23, 50.96, 36.08, 22.81. HRMS (ESI) calcd. for
C H NO S [M+H]+ 342.0795; found: 342.0803. Anal. calcd. for C H NO S: C, 63.33; H,
1
δ
2
(
δ
1
18
15
4
18 15
4
4.43; N, 4.10; found: C, 63.44; H, 4.15; N, 4.49.
1
-(4-bromophenyl)-7,7-dioxo-4,5-dihydro-3H-[1,4]oxazepino[4,3-b][1,2]benzothiazin-12-
one (6b)
◦
−1
Beige powder, 39% yield, mp 188–189 C; FT-IR (cm ): 1706 (C=O), 1344, 1177 (SO2).
H NMR (300 MHz, CDCl3) (ppm): 1.97 (m, 1H, 4-CHax), 2.26 (m, 1H, 4-CHeq), 2.59–2.64
m, 1H, 5-CHax), 3.08–3.10 (m, 1H, 5-CHeq), 3.53–3.56 (m, 1H, 3-CHax), 4.05–4.08 (m, 1H,
1
δ
(
3
8
-CHeq), 7.60–7.62 (m, 2H, arom.), 7.76–7.86 (m, 3H, arom.), 8.04–8.07 (m, 2H, arom.),
13
.24–8.27 (m, 1H, arom.). C NMR (300 MHz, CDCl3)
δ (ppm): 193.58, 188.90, 138.92,

Int. J. Mol. Sci. 2021, 22, 7818
14 of 19
1
35.17, 133.27, 132.63, 131.84, 131.23, 129.43, 128.54, 128.27, 124.62, 83.14, 50.90, 35.95, 22.76.
HRMS (ESI) calcd. for C H BrNO S [M+H]+ 419.9900; found: 419.9906. Anal. calcd. for
18
14
4
C H BrNO S: C, 51.44; H, 3.36; N, 3.33; found: C, 51.68; H, 3.45; N, 3.27.
18
14
4
1
-(4-chlorophenyl)-7,7-dioxo-4,5-dihydro-3H-[1,4]oxazepino[4,3-b][1,2]benzothiazin-12-
one (6c)
◦
Beige powder, 19% yield, mp 168–170 C; FT-IR (cm-1): 1709 (C=O), 1348, 1174 (SO ).
H NMR (300 MHz, CDCl3)
2
1
δ (ppm): 1.99 (m, 1H, 4-CHax), 2.28 (m, 1H, 4-CHeq), 2.60–2.64
(m, 1H, 5-CHax), 3.08–3.11 (m, 1H, 5-CHeq), 3.53–3.56 (m, 1H, 3-CHax), 4.05–4.08 (m, 1H,
3
8
-CHeq), 7.42–7.45 (m, 2H, arom.), 7.76–7.85 (m, 3H, arom.), 8.12–8.15 (m, 2H, arom.),
13
.24–8.26 (m, 1H, arom.). C NMR (300 MHz, CDCl3)
δ (ppm): 193.38, 188.92, 139.75,
1
38.92, 135.17, 133.27, 132.19, 131.17, 129.43, 128.84, 128.27, 124.62, 83.14, 50.90, 35.95, 22.75.
HRMS (ESI) calcd. for C H ClNO S [M+H]+ 376.0405; found: 376.0401. Anal. calcd. for
18
14
4
C H ClNO S: C, 57.52; H, 3.75; N, 3.73; found: C, 57.60; H, 3.50; N, 3.73.
18
14
4
7
,7-dioxo-1-(p-tolyl)-4,5-dihydro-3H-[1,4]oxazepino[4,3-b][1,2]benzothiazin-12-one (6d)
◦
Beige powder, 46% yield, mp 204–206 C; FT-IR (cm-1): 1701 (C=O), 1336, 1175 (SO ).
2
1
H NMR (300 MHz, CDCl3) δ (ppm): 1.97 (m, 1H, 4-CHax), 2.55 (m, 1H, 4-CHeq), 2.43 (s,
3
4
2
1
2
H, CH ), 2.68–2.72 (m, 1H, 5-CHax), 3.09–3.15 (m, 1H, 5-CHeq), 3.54–3.60 (m, 1H, 3-CHax),
3
.06–4.11 (m, 1H, 3-CHeq), 7.28–7.29 (m, 2H, arom.), 7.76–7.86 (m, 3H, arom.), 8.09–8.12 (m,
1
3
H, arom.), 8.25–8.28 (m, 1H, arom.). C NMR (300 MHz, CDCl3)
44.20, 139.14, 134.99, 133.11, 131.21, 129.84, 129.34, 129.19, 128.41, 124.53, 83.20, 50.96, 36.02,
2.74, 21.72. HRMS (ESI) calcd. for C H NO S [M+H]+ 356.0951; found: 356.0976. Anal.
δ (ppm): 194.23, 189.40,
19
17
4
calcd. for C H NO S: C, 64.21; H, 4.82; N, 3.94; found: C, 64.21; H, 4.69; N, 3.89.
19
17
4
1
-(4-methoxyphenyl)-7,7-dioxo-4,5-dihydro-3H-[1,4]oxazepino[4,3-b][1,2]benzothiazin-
1
2-one (6e)
◦
−1
Beige powder, 50% yield, mp 144–145 C; FT-IR (cm ): 1709 (C=O), 1338, 1173 (SO2).
H NMR (300 MHz, CDCl3) (ppm): 1.97 (m, 1H, 4-CHax), 2.26 (m, 1H, 4-CHeq), 2.71
1
δ
(
m, 1H, 5-CHax), 3.16 (m, 1H, 5-CHeq), 3.57 (m, 1H, 3-CHax), 3.87 (s, 3H, OCH ), 4.09 (m,
3
1
H, 3-CHeq), 6.92–6.96 (m, 2H, arom.), 7.74–7.82 (m, 3H, arom.), 8.18–8.26 (m, 3H, arom.).
1
3
C NMR (300 MHz, CDCl3)
δ (ppm): 193.09, 189.42, 163.57, 139.06, 134.98, 133.11, 132.16,
1
29.33, 128.40, 126.44, 124.52, 113.72, 83.13, 55.48, 50.96, 36.96, 22.64. HRMS (ESI) calcd. for
C H NO S [M+H]+ 372.0900; found: 372.0897. Anal. calcd. for C H NO S: C, 61.44; H,
19
17
5
19 17
5
4.61; N, 3.77; found: C, 61.02; H, 4.63; N, 3.45.
8
,8-dioxo-1-phenyl-3,4,5,6-tetrahydro-[1,4]oxazocino[4,3-b][1,2]benzothiazin-13-one (7)
◦
−1
Beige powder, 12% yield, mp 198–201 C; FT-IR (cm ): 1703 (C=O), 1343, 1172 (SO2).
H NMR (300 MHz, CDCl3) (ppm): 1.39–1.42 (m, 1H, 5-CHax), 1.83–1.95 (m, 3H, 5-CHeq
1
δ
i
4
3
CH ), 2.13–2.23 (m, 1H, 6-CHax), 2.68–2.77 (m, 2H, 6-CHeq i 3-CHax), 4.07–4.11 (m, 1H,
2
1
3
-CHeq), 7.43–7.58 (m, 3H, arom.), 7.73–7.80 (m, 3H, arom.), 8.28–8.34 (m, 3H, arom.).
C
NMR (300 MHz, CDCl3)
30.19, 129.05, 128.62, 128.29, 124.97, 79.01, 48.19, 30.04, 23.92, 30.39. HRMS (ESI) calcd. for
C H NO S [M+H]+ 356.0951; found: 356.0947. Anal. calcd. for C H NO S: C, 63.85; H,
δ (ppm): 194.98, 188.81, 137.34, 135.08, 134.40, 133.34, 133.00,
1
19
17
4
19 17
4
5.36; N, 3.92; found: C, 63.80; H, 5.22; N, 4.06.
3.2. Interaction with Model Membranes
3.2.1. Chemicals
Tris-EDTA buffer solution (pH 7.4) and 1,2-dipalmitoyl-n-glycero-3-phosphatidyl-
choline (DPPC) were purchased from Sigma-Aldrich, Merck KGaA, Darmstadt, Germany.
None of the compounds studied were soluble in water, so their chloroform (P.P.H. STAN-
LAB, Lublin, Poland, analytical grade) solutions were used for calorimetric experiments.

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3.2.2. Differential Scanning Calorimetry (DSC)
Calorimetric measurements were performed using a differential scanning calorimeter
DSC 214 Polyma (Netzsch GmbH & Co., Selb, Germany) equipped with an Intracooler
IC70 (Netzsch GmbH & Co., Selb, Germany) in the Laboratory of Elemental Analysis and
Structural Research (Faculty of Pharmacy, Wroclaw Medical University, Wroclaw, Poland).
For each sample, 2 mg of phospholipid (DPPC) were dissolved in the appropriate amount
of chloroform stock solution (5 mM) of the compounds studied (the compound: DPPC
molar ratios in the samples were: 0.06, 0.08, 0.10, 0.12). The solvent was then evaporated
under a stream of nitrogen gas. After that, the residual solvent was evacuated under
vacuum (Rotary evaporator, Büchy Poland, Warsaw, Poland) for 2 h. In this process, the
phospholipid was transferred onto the dry film on the inner surface of the Eppendorf tube.
Samples were hydrated by 20 µL of Tris–EDTA buffer (pH 7.4). Hydrated mixtures of
DPPC, compounds studied and buffer, closed in Eppendorf tubes, were heated (Labnet
◦
Dry Bath, Labnet International Inc.) to the temperature higher by 10 C than the main
phase transition temperature of the phospholipid used (DPPC) and vortexed (neoVortex,
neoLab) until homogeneous dispersion was obtained. Then, the samples were transferred
®
into aluminum sample pans of the Concavus type (Netzsch GmbH & Co., Selb, Germany)
and sealed. A pan of the same type, filled with 20 µL of Tris–EDTA buffer (pH 7.4),
was employed as a reference. Measurements of the DPPC main phase transition were
◦
performed using the heat-flow measurement method at a heating rate of 1 C per minute
◦
over a temperature range of 30–50 C in a nitrogen dynamic atmosphere (25 mL/min).
Data were analyzed off-line using Netzsch Proteus 7.1.0 (Netzsch GmbH & Co., Selb,
®
Germany) analysis software. The transition enthalpies were stated in [J/g]. The measured
heat was normalized per gram of lipid. The apparatus was calibrated using standard
samples from calibration set 6.239.2–91.3 supplied by Netzsch (Netzsch GmbH & Co., Selb,
Germany). All samples were weighed on a Sartorius CPA225D-0CE analytical balance
(
Sartorius AG, Gottingen, Germany) with a resolution of 0.01 mg.
3.3. Biological Assay
3.3.1. Cell Line
The study was carried out using the NHDF cell line obtained from ATCC (Manassas,
VA, USA). These cells are commonly used to determine the cytotoxicity of new compounds.
◦
Cells were cultured at 37 C in a humidified 5% CO /95% air atmosphere incubator and
2
passaged twice a week.
Cell Culture Media
The cells were cultivated in DMEM without phenol red supplemented with 10% fetal
bovine serum (FBS), 2 mM L-glutamine, 1.25
gentamicin. Prepared culture medium was stored at 4–8 C for up to one month.
µ
g/mL amphotericin B and 100
µg/mL
◦
Tested Compounds for Viability and ROS/NO Studies
The 1,2-thiazine derivatives were dissolved in DMSO to a stock concentration of
◦
1
0 mM. All prepared stock solutions were stored at
−
20 C for up to 6 months. For the
experiment, the compounds tested were used in the concentration range of 10, 50 and
00 M. To achieve the working concentrations, all compounds were dissolved in the
1
µ
medium, and the final DMSO concentration did not exceed 1%. The abovementioned
tested compounds were tested on NHDF cells.
3.3.2. Viability of Cell Cultures
The viability of the cells was assessed after 24 h incubation with 10–100 µM tested
compounds in MTT assay. After incubation, cell culture with a 1 mg/mL solution of MTT
◦
for 2 h at 37 C, the formazan crystals were dissolved in isopropanol. The plates were
then shaken for 30 min, and the absorbance was measured at 570 nm using VariuScan
microplate reader.

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3.3.3. Reactive Oxygen Species (ROS) and Nitric Oxide (NO)
Levels of reactive oxygen species (ROS) and nitric oxide (NO) were evaluated in
0
0
DCF-DA (2 ,7 -dichlorofluorescein diacetate) and Griess (cat. No. G7921; Thermo Fisher
Scientific, Waltham, MA, USA) assays, respectively. In these assays, the cells were treated
with tested compounds only for 1 h. After that time, the 50 µL of supernatant were
transferred into new plates to assess the NO level. The remaining supernatant was removed,
◦
the cell culture was washed, and the 25
µ
M DCF-DA solution was added for 1 h at 37 C.
At the same time, the Griess reagent was added to the collected supernatant into new
plates for 20 min at RT in the dark. The plates with DCF-DA solution were analyzed
with the Varioskan LUX microplate reader (λex = 485 nm and λem = 535 nm). The NO
level was analyzed by the measurement of the absorbance at 548 nm using VariuScan
microplate reader.
3.3.4. The COX Colorimetric Inhibitor Screening Assay
The ready-to-use Cayman kit (COX Colorimetric Inhibitor Screening Assay Kit cat.
No. 701050; Cayman Chemical Company, Ann Arbor, MI, USA) was applied for evaluating
the COX peroxidase activity for all tested compounds. The COX colorimetric kit includes
ovine COX-1 and human recombinant COX-2 isoenzymes. The compounds were dissolved
in ethanol to get the final concentration of 100 µM. Each tested solution of compounds was
transferred into a 96-well plate in three repetitions and then incubated for 2 min at RT with
prepared reagents according to the manufacturing procedure. This assay measured the
peroxidase component of COXs, which is evaluated as monitoring the oxidized form of N,
N, N’, N’-tetramethyl-p-phenylenediamine (TMPD). The absorbance was measured using
a VariuScan microplate reader at 590 nm. The data were calculated according to 3 steps.
First, the average absorbance of all the samples was determined. Second, the absorbance of
the background wells was subtracted from the absorbance of 100% of the starting COX-1
and COX-2 activity and the test compound wells. Finally, each sample of test compounds
is subtracted from the sample with 100% initial COX-1 or COX-2 activity, then divided by
the given sample by 100% initial activity and multiplied by 100 to obtain percent inhibition.
The calculated value was defined as the IC50 (the concentration at which 50% inhibition of
enzyme activity occurred for COX-1 and COX-2). The ratios of IC50 values (COX-2/COX-1)
were calculated to determine the selectivity of inhibition of cyclooxygenases. Meloxicam
was used as a reference compound because of its structural similarity to the compounds
tested and relative selectivity towards COX-2.
3.3.5. Statistical Analysis
All results are presented as mean
±
SEM (standard error of the mean) relative to the
control (E/E ), where E is the culture with the tested substance and E is the negative
0
0
control (without tested compounds).
Statistical significance was calculated compared to the control. The normal distribu-
tion, using the Shapiro–Wilka test, was checked for all biological assays calculations. In
the next step, Levene’s test was calculated to assess the equality of variances for a variable.
The parametric test was also used to evaluate statistical significance for data. The p < 0.05
was set for significant.
3.4. Molecular Docking
The geometry optimization of designed compounds structures was performed at the
B3LYP/6–31++G** level of theory with polarizable continuum model (PCM) including
solvent effects using Gaussian 09 program [31 33]. Docking package AutoDock4.2 and a
–
standard protocol were used to predict the binding mode and the free energy of binding.
The semi-empirical force field includes six pair-wise evaluations of energy, and the confor-

Int. J. Mol. Sci. 2021, 22, 7818
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mational entropy lost upon binding was used. The binding affinity of ligand is directly
related to the Gibbs energy of binding which can be expressed as follows:
ꢀ
ꢁ
ꢂ
ꢃ
ligand−ligand
ligand−ligand
cox−cox
cox−cox
∆
G = V
− V
+ V
− V
bound
unbound
bound
unbound
ꢀ
ꢁ
(
1)
cox−ligand
cox−ligand
+
V
− V
+ ∆S
con f
bound
unbound
The V term is the sum of dispersion, hydrogen bonding, electrostatics, and desolvation
energies according to the following equation:
!
!
2
−
r
ꢂ
ꢃ
ij
Aij
Bij
Cij
Dij
qiq
j
(
2
V = Wvdw
+
−
+ Whbond E(t)
−
+ Welec
ꢂ
ꢃ
+ Wsol
S V + S V e 2σ
)
(2)
∑
∑
∑
∑
i
i
j j
12
6
12
10
r
r
r
r
e r rij
ij
i,j
ij
ij
i,j
ij
ij
i,j
i,j
The crystal structures of COX-1 (PDB ID: 4O1Z) and COX-2 (PDB ID: 4M11) with
meloxicam were taken from the Protein Data Bank [35]. The polar hydrogen atoms and
solvent parameters were added to the chain A of cyclooxygenases, and Gasteiger charges
for each of the atoms have been assigned. The binding site was defined using a grid of
6
0
×
60
×
60 point with 0.375 Å spacing. The grid center was established in the active
site according to crystalized inhibitor location. The validation protocol was performed
by docking of meloxicam into the crystal structures of cyclooxygenases and the compar-
ison of its position with crystal structure. Binding modes of designed compounds were
visualized using Chimera with UCSF Chimera, developed by the Resource for Biocomput-
ing, Visualization, and Informatics at the University of California and LIGPLOT v.4.5.3
programs [36,37].
4. Conclusions
This study presents the synthesis and biological evaluation of new 1,2-thiazine deriva-
tives designed as new anti-inflammatory agents with potential use in pain and inflamma-
tion therapy. Their cytotoxic effects as well as anti-COX-1/COX-2 activity on NHDF cells,
together with the ability to interact with model membranes and the influence on reactive
oxygen species and nitric oxide, were studied. Additionally, a molecular docking study
was performed to understand the binding interaction of the compounds with the active site
of both cyclooxygenases. According to the results of molecular modeling and the in vitro
study, most of the compounds bind more strongly to the active center of COX-2 than COX-1
(4a, 4c, 4d, 5, 6a, 6d, 6e and 7). The examined ability of the 1,2-thiazine derivatives to
penetrate lipid bilayers may indicate potential modulation of the activity of the membrane-
bound cyclooxygenase. Actually, according to the results of the COX colorimetric inhibitor
screening assay, all of the compounds studied demonstrated preferential inhibition of
COX-2 compared to COX-1. Compound 6e showed the highest COX-2 selectivity, and what
is worth noting, higher than meloxicam, considered to be the preferred COX-2 inhibitor.
Moreover, all the examined bi- and tricyclic 1,2-thiazine derivatives interacted with the
phospholipid model membranes, and their calculated LogP ranged between 1.48 and 3.96,
which may indicate high bioavailability. Finally, the new compounds demonstrate no
influence on the level of reactive oxygen species or nitric oxide and have no cytotoxic
potencies. Overall, the new 1,2-thiazine derivatives are good starting points for future
pharmacological tests as a group of new anti-inflammatory agents.

Int. J. Mol. Sci. 2021, 22, 7818
18 of 19
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10
1
13
.
3390/ijms22157818/s1, Figure S1: H NMR spectrum of 5 in CDCl , Figure S2: C NMR spectrum
3
1
13
of 5 in CDCl , Figure S3: H NMR spectrum of 6a in CDCl , Figure S4: C NMR spectrum of
3
3
1
13
6
a in CDCl , Figure S5: H NMR spectrum of 6b in CDCl , Figure S6: C NMR spectrum of 6b
3
3
1
13
in CDCl , Figure S7: H NMR spectrum of 6c in CDCl , Figure S8: C NMR spectrum of 6c in
CDCl , Figure S9: H NMR spectrum of 6d in CDCl , Figure S10: C NMR spectrum of 6d in
CDCl , Figure S11: H NMR spectrum of 6e in CDCl , Figure S12: C NMR spectrum of 6e in
CDCl , Figure S13: H NMR spectrum of 7 in CDCl , Figure S14: C NMR spectrum of 7 in CDCl3,
3
3
1
13
3
3
1
13
3
3
1
13
3
3
Figure S15: The effect of 1,2-thiazine derivatives in comparison to the meloxicam on the transition half
height (T1/2) of the main phase transition of DPPC, Figure S16: The effect of 1,2-thiazine derivatives
in comparison to the meloxicam on the transition enthalpy (
∆H) of the main phase transition of
DPPC, Table S1: Values of DPPC phase transition temperatures (T ) with studied compounds,
M
Table S2: Values of DPPC transition half height (T
DPPC transition enthalpy changes (
1
2
) with studied compounds, Table S3: Values of
H) with studied compounds, Figure S17: The intermolecular
∆
interactions between COX-1 and COX-2 and designed compounds, Table S4: Free energy of binding
and intermolecular interactions of new 1,2-benzothiazine derivatives with COX-1, Table S5: Free
energy of binding and intermolecular interactions of new 1,2-benzothiazine derivatives with COX-2.
˙
Author Contributions: Conceptualization, J.M. and B.M.S.-S.; methodology, J.M., B.M.S.-S., Z.C.
˙
˙
and B.W.; software, J.M., B.M.S.-S., Z.C. and B.W.; validation, J.M., B.M.S.-S., Z.C. and B.W.; formal
˙
˙
analysis, J.M., B.M.S.-S., Z.C. and B.W.; investigation, J.M., B.M.S.-S., Z.C. and B.W.; resources, J.M.,
B.M.S.-S., Z.C. and B.W.; data curation, J.M., B.M.S.-S., Z.C. and B.W.; writing—original draft prepa-
ration, J.M., B.M.S.-S., Z.C. and B.W.; writing—review and editing, J.M. and B.M.S.-S.; visualization,
J.M., B.M.S.-S., Z.C. and B.W.; supervision, J.M. and B.M.S.-S.; project administration, J.M. All authors
˙
˙
˙
˙
have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Ministry of Health subvention according to the number
SUB.D070.21.094 from the IT Simple system of Wroclaw Medical University.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the article and in the
Supplementary Materials.
Acknowledgments: 1H NMR: 13C NMR, FT-IR, ESI-MS and DSC measurements were carried out
in the Laboratory of Elemental Analysis and Structural Research, Faculty of Pharmacy, Wroclaw
Medical University. Calculations for molecular modeling were carried out in Wroclaw Center of
Networking and Supercomputing (WCSS), Wroclaw University of Technology. The authors would
like to thank Monika Konopka for proofreading parts of this article.
Conflicts of Interest: The authors declare no conflict of interest.
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