Developing potential agents against atherosclerosis: Design, synthesis and pharmacological evaluation of novel dual inhibitors of oxidative stress and Squalene Synthase activity

Article abstract of DOI:10.1016/j.ejmech.2017.06.042

For the treatment of multifactorial and complex diseases, it has become increasingly apparent that compounds acting at multiple targets often deliver superior efficacy compared to compounds with high specificity for only a single target. Based on previous studies demonstrating the important antioxidant and anti-hyperlipidemic effect of morpholine and 1,4-benzo(x/thi)azine derivatives (A-E), we hereby present the design, synthesis and pharmacological evaluation of novel dual-acting molecules as a therapeutic approach for atherosclerosis. Analogues 1–10 were rationally designed through structural modifications of their parent compounds (A-E) in order for structure-activity relationship studies to be carried out. Most compounds showed a significant inhibition against Squalene Synthase activity exhibiting at the same time a very potent multimodal antioxidant (against lipid peroxidation and as free-radical scavengers) effect, thus bringing to light the 2-aryl-1,4-benzo(x/thia)zin-2-ol scaffold as an outstanding pharmacophore for the design of potent antioxidants. Finally, the replacement of the octahydro-1,4-benzoxazine moiety of lead compound D with its respective 1,4-benzothiazine (compound 4), although conserved (anti-hypercholesterolemic) or even improved (anti-hyperlipidemic) activity, did not preserve the anti-diabetic effect of D.

Full text of DOI:10.1016/j.ejmech.2017.06.042

Accepted Manuscript
Developing potential agents against atherosclerosis: Design, synthesis and
pharmacological evaluation of novel dual inhibitors of oxidative stress and Squalene
Synthase activity
Maria G. Katselou, Alexios N. Matralis, Angeliki P. Kourounakis
PII:
S0223-5234(17)30489-0
10.1016/j.ejmech.2017.06.042
EJMECH 9537
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 November 2016
Revised Date: 13 March 2017
Accepted Date: 23 June 2017
Please cite this article as: M.G. Katselou, A.N. Matralis, A.P. Kourounakis, Developing potential agents
against atherosclerosis: Design, synthesis and pharmacological evaluation of novel dual inhibitors of
oxidative stress and Squalene Synthase activity, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.06.042.
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1
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Graphical Abstract
Developing Potential Agents against Atherosclerosis: Design, Synthesis and
Pharmacological Evaluation of Novel Dual Inhibitors of Oxidative Stress and
Squalene Synthase Activity
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Developing Potential Agents against Atherosclerosis: Design, Synthesis and
Pharmacological Evaluation of Novel Dual Inhibitors of Oxidative Stress and
Squalene Synthase Activity
Maria G. Katselou, Alexios N. Matralis and Angeliki P. Kourounakis*
Division of Medicinal Chemistry, Department of Pharmacy, School of Health
Sciences, National and Kapodistrian University of Athens, 15771 Athens, Greece.
angeliki@pharm.uoa.gr
*
Corresponding author:
As. Prof. A. Kourounakis
Division of Medicinal Chemistry,
Department of Pharmacy, School of Health Sciences, National and Kapodistrian
University of Athens, 15771 Athens, Greece
Tel./Fax: +30-210 7274818
E-mail address: angeliki@pharm.uoa.gr
Abbreviations List: CVD, Cardiovascular disease; SQS, squalene synthase; HMG-
CoA, 3-hydroxy-3-methylglutaryl-CoA; FPP, farnesyl pyrophosphate; PSPP,
presqualene pyrophosphate; rt, room temperature; BSA, bovine serum albumin;
NADPH; nicotinamide adenine dinucleotide phosphate, IC , inhibitory concentration
5
0
for 50 % of the enzymatic reaction; TC, total cholesterol; LDL, low-density
lipoprotein cholesterol; HDL, high-density lipoprotein cholesterol; TG, triglyceride;
NSAID, non-steroidal anti-inflammatory drug;

2
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ABSTRACT
For the treatment of multifactorial and complex diseases, it has become increasingly
apparent that compounds acting at multiple targets often deliver superior efficacy
compared to compounds with high specificity for only a single target. Based on
previous studies demonstrating the important antioxidant and anti-hyperlipidemic
effect of morpholine and 1,4-benzo(x/thi)azine derivatives (A-E), we hereby present
the design, synthesis and pharmacological evaluation of novel dual-acting molecules
as a therapeutic approach of atherosclerosis. Analogues 1-10 were rationally designed
through structural modifications of their parent compounds (A-E) in order for
structure-activity relationship studies to be carried out. Most compounds showed a
significant inhibition against Squalene Synthase activity exhibiting at the same time a
very potent multimodal antioxidant (against lipid peroxidation and as free-radical
scavengers) effect, thus bringing to light the 2-aryl-1,4-benzo(x/thia)zin-2-ol scaffold
as an outstanding pharmacophore for the design of potent antioxidants. Finally, the
replacement of the octahydro-1,4-benzoxazine moiety of lead compound D with its
respective 1,4-benzothiazine (compound 4), although conserved (anti-
hypercholesterolemic) or even improved (anti-hyperlipidemic) activity, did not
preserve the anti-diabetic effect of D.
Keywords: Multi-target; morpholine; benzoxazine; benzothiazine; antioxidant;
squalene synthase; hyperlipidemia; triglycerides; antiatherogenic index;

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1
.
Introduction
Hyperlipidemia and oxidative stress are common factors involved in the
pathophysiology of atherosclerosis and type-2 diabetes, two of the major causes of
death and morbidity in developed countries [1-3]. The benefits of lowering cholesterol
and triglyceride levels have been clearly demonstrated by standard drug therapies,
including statins, fibrates, nicotinic acid, and bile acid sequestrants. In addition,
antioxidants that protect LDL from oxidation, such as probucol, vitamin E and C, may
help to reduce atherogenesis and prevent CVD. However, their therapeutic effects
were not remarkable compared to anti-hyperlipidemic agents; statins, the most widely
lipid-modifying drugs, reduce plasma LDL by 30-45% at standard dosage and also
plasma TG although less effectively. In contrast, fibrates reduce plasma TG more
effectively but not LDL. In humans, statins are well tolerated at standard dosages, but
high doses may be associated with adverse effects, such as elevated liver enzymes and
rhabdomyolysis. This is in part the result of the reduction of levels of isoprenoid
intermediates, and their metabolites (e.g. coenzyme Q10 and dolichols), of the
cholesterol biosynthetic pathway [4]. This has stimulated the search for agents
inhibiting cholesterol synthesis further downstream in its biosynthetic pathway, in
order to spare these important non-sterol isoprenoid intermediates.
Our research for the development of effective lipid-lowering compounds focuses on
an alternative point of inhibition of cholesterol biosynthesis, the enzyme Squalene
Synthase (SQS) which catalyzes the conversion of two molecules of farnesyl
pyrophosphate to squalene. SQS is located downstream of HMG-CoA reductase and
its inhibition should thus be free of isoprenoid depletion issues [5,6]. Several
morpholine and 1,4-benzo(x/thi)azine derivatives with significant SQS inhibitory
activity have been described (Chart 1) [7-11]. The therapeutic potential of these
series of SQS inhibitors has been broadened by adding antioxidant activity as well,
leading to a potentially superior synergistic effect against the development of
atherosclerosis. This has been achieved through a rational design approach, by
incorporating appropriate structural features of known antioxidant lead compounds
into our SQS-inhibitory pharmacophore. Accordingly, a similar strategy has been
followed for the development of xanthine oxidase inhibitors, antibacterial and
antifungal as well as antitumor derivatives, that were endowed also with antioxidant

4
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activity [12-14]. Continuing our search to this end, in the present study we designed
and synthesized, using as lead compounds A-C (Chart 2), three novel stilbenyl
derivatives (1-3), aiming at exploring the effect of the increase of conjugation and
lipophilicity in antioxidant as well as SQS inhibitory activity. Further, we replaced the
octahydro-1,4-benzoxazine moiety of lead compound D, a BHT derivative that has
previously shown a remarkable antioxidant, anti-hyperlipidemic and anti-diabetic
effect [15], with the respective 1,4-benzothiazine ring of the lead compound E (with
antioxidant, anti-hyperlipidemic and anti-inflammatory activity [10]) in order to
investigate the impact of this replacement on the pharmacological profile of
compound 4. Furthermore, compounds 5-10 were synthesized for the study of
structure-activity relationships (Chart 2) in order to refine the key structural features
that provide optimal multifunctional activity.
2
.
Results and Discussion
2
.1.
Chemistry
Compounds 1-3 were synthesized according to the procedure depicted in Scheme 1a.
In brief, the acetyl derivative 11 formed via a Heck cross-coupling reaction, reacted
with phenyltrimethylammonium tribromide (PTT) to afford the α-bromoacetyl
derivative 12, which upon reaction with the appropriate aminoalcohol (2-
methylaminethanol, 2-piperidinemethanol and trans-2-methylaminocyclohexanol) [7-
9
] afforded the hemiketalic oxazine analogues 1-3. For the synthesis of 4-7 (Scheme
1
b), intermediates 13-15 were first synthesized via a Suzuki cross-coupling reaction,
followed by α-bromination by either bromine or PTT to produce 16-18. Reaction of
6-19a with benzothiazole at high temperature and subsequent hydrolysis of the
tertiary benzothiazolium salts (20-23) provided the hemiketalic 1,4-benzothiazines
1
2
4-27, according to the procedure previously developed by our group [11]. Reduction
1
of the formyl group of 24-27 with BH :THF produced the final products 4-7. H NMR
3
spectra of compounds 24-27 revealed the existence of two rotamers in various
proportions, Z and E, resulting from the hindered free rotation around the amide bond.
This is consistent with other 4-formyl-1,4-benzothiazin-2-ol derivatives previously
described [11]. Finally, the synthesis of the hemiketalic 1,4-benzoxazine derivatives 8

5
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and 9 as well as the open form of the reference compound E (compound 10) is rather
straightforward and is represented in Scheme 1c.
2
.2.
Biological studies
2
.2.1. Antioxidant activity. Inhibition of in vitro lipid peroxidation.
The antioxidant activity of all compounds 1-10 was assayed based on the protection
2
+
offered against Fe /ascorbate-induced lipid peroxidation of hepatic rat microsomal
membranes. The results are presented in Table 1 (expressed as IC values), while the
5
0
time course of lipid peroxidation as affected by several concentrations of
representative compounds 2 and 4 is depicted in Figure 1. From the results it appears
that the elongation of the biphenyl group of lead compounds A-C with the insertion of
a vinyl group between the two phenyls (compounds 1-3), led to a very significant (3
to 10-fold) increase in antioxidant activity. This may be attributed to the more
extended aromatic system, compared to the biphenyl, thereby stabilizing more
effectively, via resonance, the intermediate free radical derived by the abstraction of
the hemiketalic hydroxyl group after radical attack (Figure 2).
Regarding the antioxidant activity of 1,4-benzothiazine-BHT derivative 4, it appears
that the replacement of the octahydro-1,4-benzoxazine part of lead compound D with
the 1,4-benzothiazine ring did not provoke any further increase in activity (IC values
5
0
of compounds 4 and D are 6.5 and 5.9 µΜ [15], respectively). However, compound 4
exhibited a 13-fold higher antioxidant activity when compared to the respective
biphenyl reference compound E, an effect that was attributed to the incorporated BHT
group (IC values of compounds 4 and E are 6.5 and 83 µΜ [10], respectively).
5
0
Removal of the two tert-butyl groups from 4 slightly decreased its antioxidant activity
compound 5, IC = 15 µΜ) while the absence of the whole second phenyl/BHT
(
5
0
moiety (compound 7) afforded very similar activity (IC = 8.2 µΜ). Replacement of
5
0
the benzothiazine ring of 7 with its isostere 1,4-benzoxazine (compound 8) also
conserved its antioxidant activity (IC = 7.6 µΜ for 8), while no further significant
5
0
increase was observed by further aromatic substitution of the benzoxazine ring
compound 9, IC = 4.9 µΜ). Finally, no antioxidant effect was observed by the open
(
5
0
form of the hemiketalic 1,4-benzothiazine derivative E, compound 10 (IC > 1 mΜ).
5
0

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Almost all compounds (except for 10) revealed a superior antioxidant effect than
probucol (IC > 1mM), and most of them exhibited similar (2, 3, 5, 6) or even higher
5
0
(4, 7, 8, 9) antioxidant activity than the widely known antioxidant (with a stilbenyl
structure) trans-resveratrol (IC = 15 µΜ), under the same experimental conditions.
5
0
From the above-mentioned results, the very potent antioxidant activity of the 2-aryl-
,4-benzo(x/thi)azin-2-ol scaffold has emerged, as an ideal springboard for the design
of very active molecules against oxidative stress.
1
2
.2.2. Radical scavenging effect. In vitro interaction with the free radical DPPH.
In order to investigate in part the mechanism through which 1-9 exhibit antioxidant
activity, their interaction with the stable free radical DPPH was evaluated. DPPH
radical scavenging activity evaluation is a standard assay offering a rapid technique
for screening radical scavenging activity (especially against nitrogen-centered free
radicals) of specific compounds or plant extracts. The result of this interaction is
recorded in Table 1 (expressed as IC values after 30 min of incubation with DPPH),
5
0
while the effect of various concentrations of 4 and 5 at 30 and 60 min of incubation is
depicted in Figure 3.
By analyzing the results shown in Table 1, it seems that derivatives bearing the 1,4-
benzothiazine scaffold, 4-7 exhibit a remarkable interaction with DPPH compared to
the respective saturated 1,4-oxazine reference derivatives A-D and compounds 1-3
(
IC50 > 400 µΜ). Thus, the 1,4-benzothiazine ring may constitute an appropriate
structural feature in terms of providing hydrogen atoms/donating electrons and
subsequently scavenging free radicals. In this series of compounds, the incorporation
of the antioxidant BHT into the structure of reference compound E increased
significantly DPPH activity (IC values of E and 4 are 271 and 34 µΜ, respectively),
5
0
while removal of the tert-butyl groups from 4 (compound 5) had no detrimental effect
on activity (IC = 44 µΜ for compound 5). However, DPPH activity was decreased
5
0
approximately 5.5-fold by removing the whole second phenyl/BHT moiety from 4
compound 7, IC = 183 µΜ), allowing us to attribute the antioxidant efficacy of 4,
(
5
0
regarding its potent scavenging ability, not only to the 2-aryl-1,4-benzothiazin-2-ol
structure but also to the incorporated phenol group. Furthermore, it can be observed
that the second phenyl group of the lead compound E does not have any significant

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ACCEPTED MANUSCRIPT
contribution to its free-radical scavenging properties as both 7 (IC = 183 µΜ) and its
5
0
isostere 8 (IC = 152 µΜ) exhibit higher activity than E (IC = 271 µΜ). Finally, no
5
0
50
apparent activity was observed by compound 9 or the open form of E, compound 10
IC > 400 µΜ). Compounds 4 and 5 had higher activity in this assay compared to
(
5
0
trans-resveratrol (ΙC50 = 96 µΜ), while all 1,4-benzothiazine derivatives showed
better efficacy than probucol (IC = 234 µΜ).
5
0
Thus, the antioxidant activity against lipid peroxidation exhibited by 4-8 could be
partially attributed to their ability to rapidly (and at low concentrations) scavenge high
concentrations of free radicals (Figure 3), in combination with an appropriate
lipophilicity (Table 1) that enables them to approach and partition into lipid bilayers.
2
.2.3. In vitro Squalene Synthase inhibitory activity.
The SQS inhibitory potency of selected derivatives was evaluated by determining
their IC50 values (Table 1) in vitro in the rat microsomal SQS activity assay (as
previously described) [7-11,16]. The effect of various concentrations of 1 and 5
against SQS activity is depicted in Figure 4.
The extension of the biphenyl group of A to that of a stilbenyl led to a 5-fold increase
in SQS inhibitory activity (IC50 = 7 µΜ for 1 against IC50 = 33 µΜ for A). We
attribute this significant difference to: a) the increase in lipophilicity (Table 1), as
well as the elongation that was offered by this structural modification, may orient the
stilbenyl aromatic group deeper in the active site of SQS that consists of a large
hydrophobic cavity [9] and b) the stilbenyl group may be able to better simulate,
compared to the biphenyl, the physiological substrate of the enzyme, farnesyl
pyrophosphate (FPP), according to Biller’s hypothesis which proposed that aryl units
may act as isosteres for isoprenyl subunits in the farnesyl chain of FPP [17].
In the 1,4-benzothiazine series, the incorporation of the bulky antioxidant BHT
moiety (compound 4) seemed to be detrimental for activity (IC > 50 µΜ). Removal
5
0
of the tert-butyl groups (compound 5) improved by 2-fold the inhibitory activity
against SQS compared to reference compound E (IC values of 8.5 and 16.5 µΜ for
5
0
5
and E, respectively), while replacing the hydroxyl group with trifluoromethyl
(
compound 6) did not significantly alter activity (IC = 14 µΜ for 6). Removal of the
50

8
ACCEPTED MANUSCRIPT
second phenyl of E slightly decreased activity (compound 7, IC = 28 µΜ) while
5
0
further replacement of the sulfur atom of the 1,4-benzothiazine ring of 7 with oxygen
led to a further decrease (compound 8, IC = 50 µΜ and compound 9, IC > 50 µΜ).
5
0
50
Methylthiolaminoketone derivative 10 (the "open" form of E) did not show any
significant activity against SQS (IC50 > 50 µΜ), confirming the important
contribution of the 1,4-benzothiazine ring in reference compound E and derivatives 5
and 6, for SQS inhibitory activity.
2
.2.4. Anti-inflammatory activity. In vitro COX-1 and COX-2 inhibitory activity.
Compounds 2-4 were screened for their potential anti-inflammatory activity with
regard to the inhibition offered in the activity of the inflammatory enzymes COX-1
and COX-2. Neither isoform of COX was significantly inhibited by these compounds
(at 50 µΜ). Of note, lead compound A (at 50 µΜ) inhibited COX-1 and COX-2
activities by 38% and 100%, respectively, and E at 20 µΜ by 89% and 100%,
respectively. Furthermore, the known NSAID indomethacin (at 20 µΜ) decreased the
activity of COX-1 and COX-2 by 100% and 80%, respectively, while diclofenac (at
2
0 µM) inhibited the activity of COX-2 by 100%.
2
.2.5. In vivo Anti-hyperlipidemic and anti-diabetic effect of compound 4.
In a previous study, we showed a very potent in vivo anti-hyperlipidemic, anti-obesity
and anti-diabetic activity of compound D, designed via the incorporation of structural
features of the anti-hyperlipidemic compound C and the anti-diabetic agent
succinobucol [15]. With this in mind, in the present study we focused our attention on
investigating the in vivo pharmacological profile of 4 and the effect of the
replacement of the octahydro-1,4-benzoxazine scaffold of D with the respective 1,4-
benzothiazine. We used the streptozotocin-induced type-2 diabetes experimental
animal model (that was previously used to investigate the activity of compound D) in
which insulin resistance was induced by a high fat diet (HFD) and a mild impairment
of insulin secretion [through β-cell death, via multiple low doses of streptozotocin
(STZ)]. This combination of HFD and STZ provoked severe hypercholesterolemia
and hyperlipidemia, while the impairement is considered similar to that of the later
stage of type-2 diabetes [15,18,19].

9
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2
.2.5.1. Body weight and glucose levels.
Figures 5 and 6 depict body weight and glucose level changes of mice of groups A
HFD), B (HFD+STZ) and C (HFD+STZ+Compound 4). Administration of
(
compound 4 (group C) did not exhibit an improved body weight profile compared to
th
either group A or B (until the 23 day where the experimental conditions are similar).
Unlike compound 4, parent compound D had shown an "anti-obesity" effect under
similar experimental conditions [15] in which the increase in body weight was
attenuated compared to the corresponding group B. As shown in Figure 5, significant
differences of body weight were observed between groups A and B (or C) after the
th
th
2
8
day and until the end of the experiment (53 day) despite similar food
consumption (average food consumption was 140-150 mg/g BW/day) of all groups
data not shown). This indicates a possible negative effect of STZ on group B (and C).
(
Regarding glucose levels of groups A and B (Figure 6), a rapid increase was noticed
th
in mice of group B (control) and C (compound 4) between the 27 day (end of STZ
th
administration) and the 53 day (end of experiment), indicating the establishment of
progressive diabetes. Administration of 4 (group C) did not protect from increased
glucose levels in contrast to previously studied compound D which fully maintained
blood glucose at normal levels after STZ administration [15].
2
.2.5.2. Plasma lipid levels.
Inserted graphs of Figure 7 depict the anti-hypercholesterolemic (TC and LDL-C
levels) and anti-hyperlipidemic (TG levels) effect of 4. Lipid parameters were
th
measured before the administration of STZ (22 day) and at the end of the experiment
th
(
53 day). Graphs a and b of Figure 7 show that after 22 days of HFD administration,
plasma levels of TC and TG are similar for groups A-C. A statistically significant
increase of both parameters was observed in group A (HFD) at the end of the
experiment compared to day 22 (~50% increase of TC and ~120% increase of TG
levels). The combination of the administration of HFD and of STZ (group B) induced
th
a further increase of TC and TG levels by 90% and 200% (53 day), respectively,
compared to day 22, apparently due to the established diabetes and the subsequent
insulin resistance in mice of group B. In contrast, administration of compound 4
(group C) for 53 days significantly decreased TC as well as TG levels by 31% and

1
0
ACCEPTED MANUSCRIPT
5
5%, respectively, compared to group B. A remarkable decrease of plasma LDL
levels by 65% was also observed compared to group B (Figure 7c) without affecting
HDL levels (data not shown). Furthermore, the antiatherogenic index HDL/LDL ratio
increased 3-fold by 4 (group C) compared to control (group B) in the time course of
the experiment. The octahydro-1,4-benzoxazine-BHT derivative D, under the same
experimental conditions and at the same dose, decreased TC and LDL levels by 70%
and 95%, respectively, and increased the HDL/LDL ratio by 7.5 times, while it did
not affect TG and HDL levels [15]. Thus, compound 4 exhibited a very interesting,
although not higher than its parent D, anti-hypercholesterolemic effect (decrease of
TC and LDL-C levels), and a much higher anti-hyperlipidemic (decrease of TG
levels) activity without however providing anti-diabetic activity. The anti-
hypercholesterolemic/anti-hyperlipidemic effect of 1,4-benzothiazine derivative E
(whose structure is incorporated in 4) has also been previously demonstarted in
animal models of both acute (Triton WR-1339-induced) [10] and HFD-induced
hyperlipidemia [20].
3
. Conclusion
In the present study, the design, synthesis and evaluation of novel bifunctional agents
against pathogenic mechanisms of atherosclerosis, bearing a superior antioxidant and
SQS inhibitory activity than previously studied lead compounds A-E, is described.
Combining the results of this study with those of previous ones [10,11], the 2-aryl-
1
,4-benzo(x/thi)azin-2-ol moiety is emerging as an appropriate structural starting
point, endowed with significant antioxidant (against lipid peroxidation activity) and
potent radical scavenging properties, for the design of potent compounds against
oxidative stress. Finally, compound 4 preserves the anti-hypercholesterolemic effect
of D in the experimental animal model of type-2 diabetes, exhibits a much better anti-
hyperlipidemic effect than D, but does not exert any anti-diabetic activity. The
balanced biological profile of 1-8 renders them interesting structural substrates for
further modifications towards the design of multi-targeted compounds against
atherosclerosis.
4
. Experimental protocols
4
.1. Materials

1
1
ACCEPTED MANUSCRIPT
All commercially available chemicals are of the appropriate purity and purchased
3
from standard sources. [ H] FPP (21.5 Ci/mmol), NADPH, FPP, and BSA were
purchased from Sigma-Aldrich (Germany). For the in vivo experiment, SKH-2 male
mice (25-28 g) were used. Animals were kept in a controlled temperature room (22 ±
o
2
C), having free access to laboratory chow and tap water, under a 12 h light/dark
cycle and according to EC guidelines.
.2. Synthesis
4
Melting points (mp) were determined with a digital Electrothermal IA 9000 series
apparatus and are uncorrected. Infrared (IR) spectra were recorded on a Perkin-Elmer
1
FT-IR spectrometer (Spectrum RXI). H NMR spectra were obtained with a Bruker
1
3
Avance DRX 400 (400 MHz) spectrometer and C NMR spectra with a Bruker
Avance DPX 200 (200 MHz) spectrometer. Elemental analyses were performed by
the Service Central de Microanalyse, France (analysis of C, H).
4
4
.2.1. Synthesis of compounds 1-3
.2.1.1. (Ε)-p-acetyl-stilbene (11) [21]
In a three-necked-flask 0.90 mmol of 4-bromoacetophenone, 1.8 mmols of styrene,
.026 mmol of Pd(PPh ) , 3.29 mmols of Et N, 0.074 mmol of LiBr and anhydrous
0
3
4
3
DMF (3 mL) were added. The mixture was stirred and heated for 48 h at 130°C under
argon. It was then cooled to room temperature, diluted with cold water, and extracted
with EtOAc. The organic phase was dried over Na SO and concentrated in vacuum.
2
4
The residue was purified by silica gel flash chromatography eluted with petroleum
ether/dichloromethane (3:2) to afford the title compound as a white solid. Yield 30%,
o
mp 134-137 C.
1
H-NMR (400 MHz, CDCl ), δ(ppm): 2.39 (s, 3H, CH ), 7.07 (d, J = 16.24 Hz, 1H,
3
3
trans H), 7.17 (d, J = 16.43 Hz, 1H, trans H), 7.24 (t, 1H, 4΄-Η), 7.32 (t, 2H, 3΄-H και
΄-H), 7.48 (d, J = 7.63 Hz, 2H, 2΄-H and 6΄-H), 7.52 (d, J = 8.41 Hz, 2H, 2-Η and 6-
Η), 7.89 (d, J = 8.22 Hz, 2H, 3-Η and 5-Η).
.2.1.2. (E)-p-(α-bromoacetyl)-stilbene (12) [22]
5
4
To a stirring solution of 1 mmol of the respective acetyl-derivative 11 in anhydrous
THF (2 mL), 1.1 mmol phenyltrimethylammonium tribromide was added in small
portions over a period of 20 min. The reaction mixture was then stirred for 1h at rt and
cold water was added. After filtering the mixture, the solid was washed with water
and dried. The residue was purified by silica gel flash chromatography eluted with

1
2
ACCEPTED MANUSCRIPT
petroleum ether/dichloromethane (7:4) to afford the respective bromoacetyl derivative
o
as white solid. Yield 58%, mp 137-138 C.
1
H-NMR (400 MHz, CDCl ), δ(ppm): 4.39 (s, 2H, CH ), 7.07 (d, J = 16.43 Hz, 1H,
3
2
trans H), 7.19 (d, J = 16.43 Hz, 1H, trans H), 7.26 (t, 1H, 4΄-H), 7.33 (t, 2H, 3΄-H and
΄-H), 7.49 (d, J = 7.83 Hz, 2H, 2΄-H and 6΄-H), 7.55 (d, J = 8.48 Hz, 2H, 2-H and 6-
Η), 7.92 (d, J = 8.48 Hz, 2H, 3-Η and 5-Η).
.2.1.3. General procedure for the synthesis of stilbenyl-morpholine derivatives 1-3.
5
4
To a stirring solution of the appropriate aminoalcohol (2-methylaminethanol, 2-
piperidinemethanol or trans-2-methylaminocyclohexanol) (22 mmols) in dry acetone
(30 mL), 10 mmols of (E)-p-(α-bromoacetyl)-stilbene 12 in dry acetone (50 mL) were
added dropwise and the resulting mixture was stirred for 20-24 hours at rt.
Subsequently, acetone was removed and ether was added to the residue. The mixture
was washed with saturated aqueous solution of sodium chloride (NaCl) and the
organic phase was dried over anhydrous potassium carbonate (K CO ). The products
2
3
were obtained as hydrobromide salts which were recrystallized from acetone and
ether.
2
-(trans-4-Stilbenyl)-4-methyl-morpholin-2-ol, Hydrobromide (1)
o
White solid. Yield 61%, mp 189-190 C.
1
H-NMR (400 MHz, CDCl ), δ(ppm): 2.66 (s, 3H, N-CH ), 2.80-2.81 (m, 1H, 5-H ),
3
3
ax
2
1
2
1
.89 (s, 1H, OH), 3.01 (m, 1H, 5-H ), 3.24 (d, J = 11.93 Hz, 1H, 3-H ), 3.27 (d, J =
eq ax
1.93 Hz, 1H, 3-H ), 3.73 (dd, J = 3.13 Hz, J = 12.82 Hz, 1H, 6-Η ), 4.39 (dt, J =
eq
1
2
eq
1
.51 Hz, J = 12.52 Hz, 1H, 6-H ), 6.82-7.35 (11H, Arom. H and trans Η), 10.2 (brs,
2
ax
H, NH).
1
3
C-NMR (50 MHz, CDCl ), δ(ppm): 44.04 (N-CH ), 52.51 (5-C morpholine), 56.94
3
3
(6-C morpholine), 60.65 (3-C morpholine), 93.87 (2-C morpholine), 125.6 – 139.45
(Arom. C and 2xC trans).
.
Elemental analysis: (C H BrNO
4 H O), Calculated(%) C: 50.90, H: 6.74
2
1
9
22
2
Found(%) C: 50.89, H: 6.75.
3
-(trans-4-Stilbenyl)octahydropyridino[2,1-c][1,4]oxazin-3-ol, Hydrobromide (2)
o
White solid. Yield 43%, mp 217-218 C.
1
H-NMR (400 MHz, CDCl ), δ(ppm): 1,32 (s, 1H, OH), 1.70-2.02 (m, 6H, 8,9-H
3
ax
and 2 x 7-H και 8-H ), 2.36-2.40 (m, 1H, 9-H ), 2.56-2.68 (m, 1H, 6-H , 2.83-2.89
eq
eq
ax)
(
t, 1H, 6-H ), 3.20-3.24 (m, 1H, 9a-Η ), 3.34-3.46 (m, 1H, 4-H ), 3.83 (dd, J = 3.13
eq ax ax 1

1
3
ACCEPTED MANUSCRIPT
Hz, J = 13.30 Hz, 1H, 1-H ), 4.16 (d, J = 8.61 Hz, 1H, 4-H ),4.46 (t, J = 12.71 Hz,
2
eq
eq
1
H, 1-H ), 6.98-7.58 (11H, Arom. H and trans Η), 11.35 (brs, 1H, NH).
ax
1
3
C-NMR (50 MHz, CDCl ), δ(ppm): 21.85 (8-C oxazine), 22.33 (7-C oxazine), 24.02
3
(9-C oxazine, 53.79 (6-C oxazine), 60.90 (4-C oxazine), 61.45 (9a-C oxazine), 62.38
(1-C oxazine), 94.41 (3-C oxazine), 126.34 – 139.27 (Arom. C and 2xC trans).
.
Elemental analysis: (C H BrNO 2.5 H O), Calculated(%) C: 57.27, H: 6.77,
2
2
26
2
2
Found(%) C: 57.24, H: 6.78.
2
-(trans-4-Stilbenyl)-4-methyloctahydro-1,4-benzoxazin-2-ol, Hydrobromide (3)
o
White solid. Yield 52%, mp 70-80 C.
1
H-NMR (400 MHz, CDCl ), δ(ppm): 1.26-1.45 (m, 5H, 2 x 5-Η, 2 x 7-Η and 8-Η ),
3
ax
1
.61 (brs, 1H, OH), 1.79 (m, 2H, 2x 6-H), 1.94 (d, J = 10.76 Hz, 1H, 8-H ), 2.08-2.11
eq
(
m, 1H, 4α-H ), 2.70 (s, 3H, NCH ), 2.86-2.93 (m, 1H, 3-H ), 3.58 (d, 1H, 3-H ),
ax 3 ax eq
4
.46 (m, 1H, 8α-H ), 6.99-7.64 (11H, Arom. H and trans Η), 11.52 (brs, 1H, NH).
ax
1
3
C-NMR (50 MHz, CDCl ), δ(ppm): 23.93 (6,7-C oxazine), 24.52 (5-C oxazine),
3
2
4.91 (8-C oxazine), 31.02 (NCH ), 61.21 (3-C oxazine), 67.69 (4a-C oxazine), 69.6
3
(8a-C oxazine), 94.00 (2-C oxazine), 125.7-139.13 (Arom. C and 2xC trans).
.
Elemental analysis: (C H BrNO
3 H O), Calculated (%)C: 57.03, H: 7.07,
2
2
3
28
2
Found(%) C: 57.12, H: 7.08.
4
.2.2. Synthesis of 1,4-Benzothiazine derivatives 4-7
4
.2.2.1. General procedure for the synthesis of acetyl derivatives 13-15
In a three-necked-flask, a solution of 2 mmols of 4-acetyl-phenyl-boronic acid in
toluene (8 mL), an aqueous solution of saturated Na CO and a solution of 2.2 mmols
2
3
of arylbromide in absolute EtOH (4 mL) were mixed. 0.04 mmol of Pd(PPh ) were
3
4
gradually added under argon and the mixture was stirred for 5 h at 110°C. After
cooling to rt, the mixture was diluted with cold water, and extracted with EtOAc. The
organic phase was then washed with 1N HCl, saturated NaCl solution and water,
dried over Na SO and concentrated in vacuum. The residue was purified by silica gel
2
4
flash chromatography to afford compounds 13-15.
-Acetyl-3΄,5΄-di-tert-butyl-4΄-hydroxybiphenyl (13) [23,24]
4
Purified by silica gel flash chromatography eluted with petroleum
ο
ether/dichloromethane (3:1). White solid, yield 81%, mp 144-146 C.

1
4
ACCEPTED MANUSCRIPT
1
H-NMR (400 MHz, CDCl ), δ(ppm): 1.52 (s, 18H, 2xC(CH ) ), 2.65 (s, 3H, CH ),
3
3 3
3
5
8
4
.39 (s, 1H, OH), 7.46 (s, 2H, 2΄-Η and 6΄-Η), 7.65 (d, J=8.54 Hz, 2H, 2-Η and 6-Η),
.03 (d, J=8.54 Hz, 2H, 3-Η and 5-Η).
-Acetyl-4΄-hydroxybiphenyl (14) [25,26]
Purified by silica gel flash chromatography eluted with petroleum ether/ethyl acetate
ο
(
2:1). White solid, yield 82%, mp 86-87 C.
1
H-NMR (400 MHz, DMSO-d ), δ(ppm): 2.54 (s, 3H, CH ), 6.80 (d, J = 8.6 Hz, 2H,
6
3
2
-H and 6-Η), 7.51 (d, J = 8.6 Hz, 2H, 3-H and 5-Η), 7.65 (d, J = 8.41 Hz, 2H, 2΄-H
and 6΄-Η), 7.9 (d, J = 8.41 Hz, 2H, 3΄-H and 5΄-Η), 9.65 (s, 1H, OH).
-Acetyl-4΄-trifluoromethylbiphenyl (15) [27,28]
4
Purified by silica gel flash chromatography eluted with petroleum
ο
ether/dichloromethane (2:5). Colorless liquid, yield 91%, mp 122-124 C.
1
H-NMR (400 MHz, CDCl ), δ(ppm): 2.59 (s, 3H, CH ), 7.63 (d, J=8.62 Hz, 2H, 2-Η
3
3
and 6-Η), 7.66 (m, 4H, 2΄-Η and 5΄-Η, 3΄-Η and 5΄-Η), 8.00 (d, J=8.41 Hz, 2H, 3-Η
and 5-Η).
4
.2.2.2. Synthesis of a-bromoacetyl derivatives 16-18
Compounds 16-18 were synthesized through standard bromination methods using
bromine (for 16 and 18) or PTT (compound 17) as previously mentioned [9].
4
-α-Bromoacetyl-3΄,5΄-di-tert-butyl-4΄-hydroxybiphenyl (16) [23]
Purified by silica gel flash chromatography eluted with petroleum
ο
ether/dichloromethane (3:1). White solid, yield 71%, mp 144-145 C.
1
H-NMR (400 MHz, CDCl ), δ(ppm): 1.50 (s, 18H, , 2xC(CH ) ), 4.49 (s, 2H, CH ),
3
3 3
2
5
.41 (s, 1H, OH), 7.31 (s, 2H, 2΄-Η and 6΄-Η), 7.71 (d, J = 8.56 Hz, 2H, 2-Η and 6-Η),
.10 (d, J = 8.56 Hz, 2H, 3-Η and 5-Η).
8
1
3
C-NMR (200 MHz, CDCl ), δ(ppm): 30.3 (2xC(CH ) ), 30.85 (CH ), 34.49
3
3 3
2
(2xC(CH ) ), 124.2 (2΄-C and 6΄-C), 127.1 (2-C and 6-C), 129.5 (3-C and 5-C), 136.4
3 3
(
1΄-C), 136.5 (4-C), 147.7 (1-C), 154.6 (4΄-C), 166.09 (2-C), 192 (C=O).
-α-Bromoacetyl-4΄-hydroxybiphenyl (17)
4
Purified by silica gel flash chromatography eluted with petroleum
ο
ether/dichloromethane (3:1). White solid, yield 91%, mp 142-144 C.
1
H-NMR (400 MHz, CDCl ), δ(ppm): 4.41 (s, 2H, CH ), 5.33 (s, 1H, OH), 6.88 (d, J
3
2
=
8.61 Hz, 2H, 3΄-H and 5΄-Η), 7.47 (d, J = 8.60 Hz, 2H, 2΄-H and 6΄-Η), 7.59 (d, J =
8
.22 Hz, 2H, 2-H and 6-Η), 7.97 (d, J = 8.41 Hz, 2H, 3-H and 5-Η).

1
5
ACCEPTED MANUSCRIPT
.
Elemental analysis: (C H BrO 0.4 H O), Calculated (%): C: 56.34, H: 3.99, Found
1
4
11
2
2
(%):C: 56.31, H: 3.75.
4
-α-Bromoacetyl-4΄-trifluoromethylbiphenyl (18)
Purified by silica gel flash chromatography eluted with petroleum
ο
ether/dichloromethane (3:1). White solid, yield 71%, mp 102 C.
1
H-NMR (400 MHz, CDCl ), δ(ppm): 4.41 (s, 2H, CH ), 7.64 (d, J = 8.56 Hz, 2H, 2-
3
2
Η and 6-Η), 7.67 (m, 4H, 2΄-H and 6΄-H, 3΄-Η and 5΄-Η), 8.03 (d, J = 8.56 Hz, 2H, 3-
Η and 5-Η).
4
.2.2.3. General procedure for the synthesis of tertiary benzothiazolium salts 20-23
10].
To a stirring solution of 5 mmols of acylbromides 16-19a in absolute EtOH (4 mL),
[
7
.5 mmols of benzothiazole was added and the reaction mixture was stirred for 5-24 h
ο
at 100 C. After cooling to rt it was triturated with anhydrous Et O. The respective
2
solid residue was then filtered, washed with Et O and dried in vacuum.
2
3
-[p-(3΄,5΄-di-tert-Butyl-4΄-hydroxyphenyl)phenacyl]benzothiazolium bromide (20)
o
1
White solid, yield 81%, mp 209 C. H-NMR (400 MHz, DMSO-d ), δ(ppm): 1.37 (s,
6
1
8H, , 2xC(CH ) ), 6.66 (brs, 1H, OH), 6.82 (s, 2H, CH ), 7.35 (s, 2H, 2΄-Η and 6΄-
3 3 2
Η), 7.67 (d, J = 8.56 Hz, 2H, 3-H and 5-Η), 7.72 (m, 2H, 5-H and 6-Η benzothiazole),
.08 (d, J = 8.56 Hz, 2H, 2-H και 6-Η), 8.18 (dd, J = 1.34 Hz, J = 8.20 Hz, 1H, 7-H
8
1
2
benzothiazole), 8.34 (dd, J = 1.4 Hz, J = 8.20 Hz, 1H, 4-H benzothiazole), 10.90 (s,
1
2
1
H, 2-Η benzothiazole).
-[(4΄-Hydroxyphenyl)phenacyl]benzothiazolium bromide (21)
3
o
1
Off-white solid, yield 70%, mp 260-261 C. H-NMR (400 MHz, DMSO-d ), δ(ppm):
6
6
2
.73 (s, 2H, CH ), 6.85 (d, J = 8.6 Hz, 2H, 3΄-Η and 5΄-Η), 7.60 (d, J = 8.61 Hz, 2H,
2
΄-Η and 6΄-Η), 7.63-7.83 (m, 5H, 3-H and 5-Η biphenyl and 5-H, 6-Η
benzothiazole), 8.06 (d, J = 8.61 Hz, 2H, 2-H and 6-Η), 8.31 (dd, J = 1.42 Hz, J =
1
2
8
.61 Hz, 1H, 7-H benzothiazole), 8.51 (dd, J = 1.4 Hz, J = 8.32 Hz, 1H, 4-H
1 2
benzothiazole), 9.76 (brs, 1H, OH), 10.58 (s, 1H, 2-Η benzothiazole).
.
Elemental analysis: (C H BrNO S 3 H O), Calculated (%): C: 52.50, H: 4.58,
21
16
2
2
Found (%): C: 52.50, H: 4.52.
-[(4΄-Trifluoromethylphenyl)phenacyl]benzothiazolium bromide (22)
3
o
1
White solid, yield 75%, mp 255-257 C. H-NMR (400 MHz, DMSO-d ), δ(ppm):
6
6
.74 (s, 2H, CH ), 7.83 (d, J = 8.8 Hz, 4H, 3-H and 5-Η, 2΄-Η and 6΄-Η), 7.99 (m, 4H,
2

1
6
ACCEPTED MANUSCRIPT
3
΄-Η and 5΄-Η biphenyl, 5-H και 6-Η benzothiazole), 8.16 (d, J = 8.21 Hz, 2H, 2-H
and 6-Η), 8.33 (dd, J = 1.35 Hz, J = 7.82 Hz, 1H, 7-H benzothiazole), 8.51 (dd, J =
1
2
1
1
.3 Hz, J = 7.54 Hz, 1H, 4-H benzothiazole), 10.56 (s, 1H, 2-Η benzothiazole).
2
3
-Phenacyl-benzothiazolium bromide (23)
o
1
White solid, yield 75%, mp 241-243 C. H-NMR (400 MHz, DMSO-d ), δ(ppm):
6
6
.73 (s, 2H, CH ), 7.59 (t, J= 7.73 Hz, 2H, 2,6-H phenyl), 7.72 (t, J= 7.43 Hz, 1H, 4-
2
H phenyl), 7.76-7.83 (m, 2H, 5,6-H benzothiazole), 8.04 (d, J= 7.44 Hz, 2H, 3,5-H
phenyl), 8.32 (d, J= 7.63 Hz, 1H, 7-H benzothiazole), 8.52 (d, J= 8.80 Hz, 1H, 4-Η
benzothiazole), 10.58 (s, 1H, 2-Η benzothiazole).
4
.2.2.4. General procedure for the synthesis of 4-formyl-1,4-benzothiazin-2-ol
derivatives 24-27 [10,11]
To a solution of 2 mmols of 3-(arylacyl)-substituted benzothiazole bromide salts 20-
2
3 in 150-200 mL MeOH, 2.2 mmols of an aqueous solution of ΝaOH was added
dropwise under argon at rt. The mixture was further stirred for 2-4.5 hours. Then,
50-200 mL cold water was added, the mixture was extracted with dichloromethane
and the organic layers were dried over Na SO and concentrated in vacuum, affording
1
2
4
the intermediate products 24-27.
-Formyl-2-(3΄,5΄-di-tert-butyl-4΄-hydroxybiphenyl)]-3,4-dihydro-2Η-1,4-
benzothiazin-2-ol (24)
Yellow solid, yield 91%, mp 132 C. H-NMR (400 MHz, CDCl / DMSO-d ),
4
o
1
3
6
δ(ppm): 1.36 (s, 18H, 2xC(CH ) ), 3.50 (d, J = 12.71 Hz, 2/3 x 1H, 3-H
3
3
benzothiazine), 3.70 (d, J = 13 Hz, 1/3 x 1H, 3-H benzothiazine), 3.90 (d, J = 13 Hz,
1/3 x 1H, 3-H benzothiazine), 4.52 (d, J = 12.71 Hz, 2/3 x 1H, 3-H benzothiazine),
7.03-7.61 (m, 10H, Arom. H), 8.16 (s, 1/3 x 1H, CHO), 8.73 (s, 2/3 x 1H, CHO).
4-Formyl-2-(4΄-hydroxybiphenyl)-3,4-dihydro-2Η-1,4-benzothiazin-2-ol (25)
o
1
Yellow solid, yield 85%, mp 149-150 C. H-NMR (400 MHz, DMSO-d ), δ(ppm):
6
1
.65 (s, 1H, 4΄-ΟΗ), 3.48 (d, J = 13.11 Hz, 2/3 x 1H, 3-H benzothiazine), 3.68 (d, J =
3.69 Hz, 1/3 x 1H, 3-H benzothiazine), 3.93 (d, J = 13.86 Hz, 1/3 x 1H, 3-H
1
benzothiazine), 4.52 (d, J = 13.11 Hz, 2/3 x 1H, 3-H benzothiazine), 7.08-7.16 (m,
4H, 5,6,7,8-H benzothiazine), 7.34-7.46 (m, 2H, 2΄, 3΄, 5΄,6΄-H biphenyl), 7.55 (d, J =
8.61 Hz, 2H, 2-H και 6-Η biphenyl), 7.61 (d, J = 8.8 Hz, 2H, 3-H και 5-Η biphenyl),
8.2 (s, 1/3 x 1H, CHO), 8.73 (s, 2/3 x 1H, CHO), 9.52 (s, 1H, OH).

1
7
ACCEPTED MANUSCRIPT
4
-Formyl-2-(4΄-trifluoromethylbiphenyl)-3,4-dihydro-2Η-1,4-benzothiazin-2-ol (26)
o
1
Orange solid, yield 91%, mp 155-156 C. H-NMR (400 MHz, DMSO-d ), δ(ppm):
6
3
.53 (d, J = 12.91 Hz, 2/3 x 1H, 3-H benzothiazine), 3.68 (d, J = 13.5 Hz, 1/3 x 1H, 3-
H benzothiazine), 3.96 (d, J = 13.89 Hz, 1/3 x 1H, 3-H benzothiazine), 4.53 (d, J =
2.91 Hz, 2/3 x 1H, 3-H benzothiazine), 7.07 - 7.18 (m, 3H, Arom. H), 7.24 (s, 1H,
OH), 7.37-7.86 (m, 9H, Arom. H), 8.4 (s, 1/3 x 1H, CHO), 8.75 (s, 2/3 x 1H, CHO).
-Formyl-2-phenyl-3,4-dihydro-2H-1,4-benzothiazin-2-ol (27)
1
4
o
1
White solid, yield 60%, mp 150-151 C. H-NMR (400 MHz, DMSO-d ), δ(ppm):
6
1
.69 (brs, ¼ x 1H, OH), 3.26 (brs, ¾ x 1H, OH), 3.39 (d, J= 13.49 Hz, ¾ x 1H, 3-H
benzothiazine), 3.68 (d, J= 13.50 Hz, ¼ x 1H, 3-H benzothiazine), 3.88 (d, J= 13.79
Hz, ¼ x 1H, 3-H benzothiazine), 4.95 (d, J= 13.21 Hz, ¾ x 1H, 3-H benzothiazine),
7
.11-7.26 (m, 4H, 5,6,7,8-H benzothiazine), 7.41 (t, J= 7.34 Hz, 1H, 4-H phenyl),
.46 (t, J= 7.48 Hz, 2H, 3,5-H phenyl), 7.75 (d, J= 7.04 Hz, 2H, 2,6-Η phenyl), 8.26
7
(s, ¼ x 1H, CHO), 8.82 (s, ¾ x 1H, CHO).
4
.2.2.5. General procedure for the synthesis of the final compounds 4-7
The respective 4-formyl-2-aryl-1,4-benzothiazin-2-ol derivatives 24-27 (1 mmol)
were dissolved in anhydrous THF (5 mL) under argon and BH :THF (1M, 7 mL) was
3
added dropwise. The mixture was stirred for 1 hour, under argon, at rt. Water was
added, the mixture was washed with water and saturated NaHCO solution and dried
3
over MgSO . The residue was purified by silica gel chromatography to afford the final
4
products.
4
-Methyl-2-(3΄,5΄-di-tert-butyl-4΄-hydroxy-4-biphenyl)-3,4-dihydro-2Η-1,4-
benzothiazine-2-ol (4)
Purified by silica gel flash chromatography eluted with petroleum
ο
ether/dichloromethane (2:1). Yellow solid, yield 87%, mp 163-164 C.
1
H-NMR (400 MHz, CDCl ), δ(ppm): 1.52 (s, 18H, 2xC(CH ) ), 3.12 (s, 3H, N-CH ),
3
3 3
3
3
.29 (d, J = 11.74 Hz, 1H, 3-H benzothiazine), 3.64 (d, J = 11.86 Hz, 1H, 3-H
benzothiazine), 4.01 (s, 1H, OH benzothiazine), 5.30 (s, 1H, OH biphenyl), 6.84-6.96
m, 2H, Arom. H benzothiazine), 7.10-7.16 (m, 2H, Arom. H benzothiazine), 7.43 (s,
(
2
H, 2΄-Η and 6΄-Η biphenyl), 7.59 (d, J = 8.32 Hz, 2H, 2-H and 6-Η), 7.75 (d, J =
8
.31 Hz, 2H, 3-H and 5-H biphenyl).

1
8
ACCEPTED MANUSCRIPT
1
3
C-NMR (50 MHz, CDCl ), δ(ppm): 30.33 (2xC(CH ) ), 34.47 (2xC(CH ) ), 40.77
3
3 3
3 3
(
N-CH ), 64.21 (3-C benzothiazine), 82.07 (2-C benzothiazine), 113.37 – 143.30
3
(Arom. C), 153.70 (4΄-C biphenyl).
Elemental analysis: (C H NO S), Calculated (%) C: 75.45, H: 7.64, Found (%) C:
2
9
35
2
7
5.16, H: 8.08.
-Methyl-2-(4΄-hydroxybiphenyl)-3,4-dihydro-2Η-1,4-benzothiazin-2-ol (5)
4
Purified by silica gel flash chromatography eluted with petroleum
ο
ether/dichloromethane (2:1). Yellow solid, yield 67%, mp 164-165 C.
1
H-NMR (200 MHz, CDCl ), δ(ppm): 3.11 (s, 3H, N-CH ), 3.28 (d, J = 11.86 Hz, 1H,
3
3
3
-H benzothiazine), 3.63 (d, J = 11.86 Hz, 1H, 3-H benzothiazine), 4.00 (s, 1H, OH
benzothiazine), 4.91 (s, 1H, OH biphenyl), 6.93 (d, J = 8.56 Hz, 2H, 2΄-H and 6΄-H),
.13 (m, 4H, Arom. H benzothiazine), 7.51 (d, J = 8.56 Hz, 2H, 3΄-H and 5΄-H), 7.59
d, J = 8.44 Hz, 2H, 2-H and 6-H biphenyl), 7.74 (d, J = 8.31 Hz, 2H, 3-H και 5-H
biphenyl).
7
(
1
3
C-NMR (50 MHz, DMSO-d ), δ(ppm): 41.6 (N-CH ), 63.96 (3-C benzothiazine),
6
3
8
0.51 (2-C benzothiazine), 112.85 - 143.75 (Arom. C), 157.71 (4΄-C biphenyl).
.
Elemental analysis: (C H NO S 0.75 H O), Calculated (%)C: 69.97, H: 6.15,
2
1
19
2
2
Found(%) C: 69.48, H: 5.70.
-Methyl-2-(4΄-trifluromethyl-4-biphenyl)-3,4-dihydro-2Η-1,4-benzothiazine-2-ol (6)
4
Purified by silica gel flash chromatography eluted with petroleum
ο
ether/dichloromethane (7:2). Orange solid, yield 59%, mp 133-134 C.
1
H-NMR (400 MHz, DMSO-d ), δ(ppm): 2.91 (s, 3H, N-CH ), 3.29 (d, J = 12.52 Hz,
6
3
1
H, 3-H benzothiazine), 3.59 (d, J = 12.52 Hz, 1H, 3-H benzothiazine), 6.59 (t, J =
.43 Hz, 7-Η benzothiazine), 6.71 (s, 1H, OH), 6.74 (d, J = 8.21 Hz, 2H, 5-Η
7
benzothiazine), 6.91 (d, J = 7.63 Hz, 2H, 8-Η benzothiazine), 6.96 (t, J = 8.41 Hz, 1H,
6
-Η benzothiazine), 7.65 (d, J = 8.61 Hz, 2H, 3-Η and 5-Η biphenyl), 7.70 (d, J =
.41 Hz, 2H, 2-Η and 6-Η biphenyl), 7.74 (d, J = 8.42 Hz, 2H, 2΄-H and 6΄-Η), 7.84
8
(d, J = 8.21 Hz, 2H, 3΄-H and 5΄-H biphenyl).
1
3
C-NMR (50 MHz, DMSO-d ), δ(ppm): 41.00 (N-CH ), 63.82 (3-C benzothiazine),
6
3
8
0.49 (2-C benzothiazine), 112.92 – 144.15 (Arom. C and CF3).
.
Elemental analysis: (C H NOSF 0.83 THF), Calculated (%) C: 65.82, H: 4.52, N:
2
2
18
3
3
.49, Found (%) C: 66.22, H: 5.38, N: 3.08.
4
-Methyl-2-phenyl-3,4-dihydro-2H-benzothiazin-2-ol (7)

1
9
ACCEPTED MANUSCRIPT
Purified by silica gel flash chromatography eluted with petroleum
ether/dichloromethane (4:1). Yellow oil, yield 64%.
1
H-NMR (400 MHz, CDCl ) δ(ppm): 3.02 (s, 3H, N-CH ), 3.17 (d, J= 11.93 Hz, 1H,
3
3
3
-H benzothiazine), 3.52 (d, J= 11.74 Hz, 1H, 3-H benzothiazine), 3.91 (s, 1H, OH),
.78 (t, J= 7.44 Hz, 1H, 7-H benzothiazine), 6.85 (d, J= 8.41 Hz, 1H, 5-H
6
benzothiazine), 7.04 (d, J= 7.63 Hz, 2H, 3,5-H phenyl), 7.23-7.29 (m, 1H, 4-H
phenyl), 7.33-7.37 (m, 2H, 6,8-Η benzothiazine), 7.63 (d, J= 7.04 Hz, 2H, 2,6-Η
phenyl).
1
3
C-NMR (50 MHz, CDCl ) δ(ppm): 35.42 (N-CH ), 60.51 (3-C benzothiazine),
3
3
9
1
7.05 (2-C benzothiazine), 126.60 (5,7-C benzothiazine), 127.13 (2,6-C phenyl),
27.59 (6,8-C benzothiazine), 127.80 (4-C phenyl), 128.86 (3,5-C phenyl), 135.82
(8a-C benzothiazine), 140.20 (1-C phenyl), 142.36 (4a-C benzothiazine).
.
Elemental analysis: (C H NOS 0.25 Η Ο), Calculated (%) C: 68.79, H: 5.98, Found
1
5
15
2
(%) C: 68.51, H: 5.63.
4
.2.3. Synthesis of final compounds 8 and 9
Compounds 8 and 9 were synthesized via the reaction of 2-methylaminophenol or 4-
phenyl-2-methylaminophenol with phenylacetyl bromide 19a or 4-biphenylacetyl
bromide 19b, as previously described [10].
4
-Methyl-2-phenyl-3,4-dihydro-2H-benzoxazin-2-ol (8)
Purified by silica gel flash chromatography eluted with petroleum ether/ethyl acetate
(7:1). Brown oil, yield 45%.
1
H NMR (200MHz, CDCl ) δ(ppm): 3.25 (s, 3H, N-CH ), 3.40 (d, J= 11.90 Hz, 1H,
3
3
3
-H), 3.74 (d, J= 11.80 Hz, 1H, 3-H), 4.14 (s, 1H, OH), 7.01 (t, J= 7.50 Hz, 1H, 7-H
benzoxazine), 7.07 (d, J= 8.40 Hz, 1H, 5-H benzoxazine), 7.27 (d, J= 7.6 Hz, 2H, 3,5-
H phenyl), 7.46-7.53 (m, 1H, 6-H benzoxazine), 7.55-7.59 (m, 3H, 8-H benzoxazine
and 2-H phenyl), 7.86 (d, J= 8.4 Hz, 1H, 6-H phenyl).
1
3
C NMR (50MHz, CDCl ) δ(ppm): 41.18 (Ν-CH ), 66.27 (3-C benzoxazine), 102.77
3
3
(
2-C benzoxazine), 132.32 (8-C benzoxazine), 132.93 (6,7-C benzoxazine), 133.32
2,6-C phenyl), 133.52 (5-C benzoxazine), 134.66 (3,5-C phenyl), 141.54 (4-C
(
phenyl), 145.95 (1-C phenyl), 148.12 (8a-C benzoxazine), 148.13 (4a-C
benzoxazine).
.
Elemental analysis: (C H NO 0.75 H O), Calculated (%) C: 70.65, H: 6.24 Found
1
5
15
2
2
(%) C: 70.70, H: 6.60.

2
0
ACCEPTED MANUSCRIPT
2
-([1,1'-Biphenyl]-4-yl)-4-methyl-6-phenyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-2-ol
9)
Purified by silica gel flash chromatography eluted with petroleum ether/ethyl acetate
(
(6:1). Brown semisolid, yield 50%.
1
H NMR (400MHz, CDCl ) δ(ppm): 2.79 (s, 3H, N-CH ), 2.86 (s, 2H, CH ), 3.44 (s,
3
3
2
1
H, OH), 7.11-7.14 (m, 1H, 5-Η benzoxazine), 7.19-7.22 (m, 1H, 8-Η benzoxazine),
.27-7.30 (m, 2H, 7-H benzoxazine and 4-Η phenyl), 7.33-7.38 (m, 5H, 3,5,3’,4’,5’-H
7
biphenyl), 7.46-7.53 (m, 6H, 2,3,5,6-Η phenyl, 2,6-Η biphenyl), 7.65 (d, J= 8.52 Hz,
2
H, 2’,6’-H biphenyl).
1
3
C NMR (50MHz, CDCl ) δ(ppm): 29.59 (Ν-CH ), 36.66 (3-C benzoxazine), 96.94
3
3
(2-C benzoxazine), 113.67 (5-C benzoxazine), 118.22 (8-C benzoxazine), 123.02 (7-C
benzoxazine), 126.82 (3,5-C biphenyl), 126.97 (2’,6’-C biphenyl), 127.01 (2,6-C
phenyl), 127.15 (2,6-C biphenyl), 127.36 (4’-C biphenyl), 127.54 (4-C phenyl),
1
28.77 (3’,5’-C biphenyl), 128.84 (3,5-C phenyl), 129.46 (6-C benzoxazine), 130.05
1-C biphenyl), 136.53 (4-C biphenyl), 137.17 (1’-C biphenyl), 140.41 (1-C phenyl),
42.07 (8a-C benzoxazine), 142.11 (4a-C benzoxazine).
(
1
Elemental analysis: (C H NO ), Calculated (%) C: 82.42, H: 5.89 Found (%) C:
2
7
23
2
8
2.30, H: 5.89.
4
.2.4. Synthesis of final compound 10
4
.2.4.1. Intermediates 28-30 were synthesized according to the previously described
procedures [29,30].
.2.4.2. 1-(4-Biphenylyl)-2-[Ν-methyl-2-(methylthio)phenylamin]ethanone (10)
4
To a mixture consisting of 2.02 mmol of 30 and 1.48 mmol of NaHCO in 1 mL of
3
dry DMF, 1.35 mmol of 4-bromoacetylbiphenyl 19b diluted in 2 mL dry DMF was
added dropwise. The resulting mixture was stirred at rt for 3h and for further 2h at 60
o
C. After the addition of water (20 mL) the mixture was extracted with
dichloromethane and the organic phase was dried (MgSO ) and concentrated in
4
vacuum. The oily residue was subjected to silica gel flash chromatography eluted with
cyclohexane/dichloromethane (6:1) affording the desired compound. Yellow solid,
o
yield 43%, mp 113-115 C.

2
1
ACCEPTED MANUSCRIPT
1
H NMR (400MHz, CDCl ) δ(ppm): 2.34 (s, 3H, S-CH ), 2.90 (s, 3H, N-CH ), 4.49
3
3
3
(
s, 2H, CH ), 7.02 (d, J= 7.04 Hz, 1H, 6-H phenyl), 7.07-7.08 (m, 2H, 4,5-Η phenyl),
2
7
.20 (d, J= 7.82 Hz, 1H, 3-H phenyl), 7.33 (t, J= 7.24 Hz, 1Η, 4’-Η biphenyl), 7.40 (t,
J= 7.44 Hz, 2H, 3’,5’-Η biphenyl), 7.55 (d, J= 7.63 Hz, 2H, 2’,6’-H biphenyl), 7.59
(d, J= 8.22 Hz, 2H, 2,6-Η biphenyl), 7.99 (d, J= 8.02 Hz, 2H, 3,5-H biphenyl).
1
3
C NMR (50MHz, CDCl ) δ(ppm): 14.61 (S-CH ), 41.22 (N-CH ), 61.82 (CH ),
3
3
3
2
1
12.26 (2-C phenyl), 121.21 (6-C phenyl), 124.22 (4-C phenyl), 125.21 (5-C phenyl),
25.57 (3-C phenyl), 127.14 (2,6-C biphenyl), 127.24 (2’,6’-C biphenyl), 128.28 (4’-
1
C biphenyl), 128.68 (3’,5’-C biphenyl), 128.92 (3,5-C biphenyl), 134.45 (4-C
biphenyl), 139.81 (1’-C biphenyl), 145.68 (1-C phenyl), 149.61 (1-C biphenyl),
1
96.13 (C=Ο).
Elemental analysis: (C H NOS), Calculated (%) C: 76.04, H: 6.09, Found (%) C:
2
2
21
7
4
4
5.57, H: 5.94.
.3. Bilogical methods
.3.1. In vitro microsomal lipid peroxidation
Heat-inactivated hepatic microsomes from untreated rats were prepared as described
previously [31]. The inhibitory effect of compounds on lipid peroxidation was
assessed spectrophotometrically (535 against 600 nm) as TBAR material [7-11].
4
.3.2. In vitro interaction with the stable radical DPPH
Compounds, dissolved in absolute EtOH, at concentrations of 10-400 µΜ, were added
to an equal volume of an ethanolic solution of DPPH (final concentration 200 µΜ) at
room temperature. Absorbance (517 nm) was recorded at 30 and 60 min.
4
.3.3 In vitro Squalene Synthase activity assay
3
SQS activity was evaluated by determining the amount of [ H]FPP converted to
squalene as previously described [8-11,15].
4
.3.4. In vitro evaluation of Cycloxygenase activity (COX-1 and COX-2)
The effect of compounds on COX-1 and COX-2 activity was measured using ovine
COX-1 and human recombinant COX-2 enzymes included in the “COX Inhibitor
Screening Assay” kit by Cayman Chemical Co. (Ann Arbor, MI, USA). The assay
directly measures PGF produced by SnCl reduction of COX-derived PGH . The
2
a
2
2
prostanoid product is quantified via enzyme immunoassay using a broadly specific
antibody that binds to all major prostaglandin compounds.

2
2
ACCEPTED MANUSCRIPT
The inhibitory activity of the test compounds (at a final concentration of 50 µΜ) was
-
9
measured in the presence of 100 µΜ arachidonic acid (substrate) and 10 IU/mL of
the respective COX isoform.
4
.3.5. In vivo evaluation of compound 4 in a type 2 diabetes experimental mouse
protocol [15].
-8 week old male SKH-2 mice were acclimated for 1 week and allowed water and
7
normal rodent chew ad libitum. Subsequently they were divided in 3 groups:
Group A: HFD (High Fat Diet)
Group B: HFD + STZ (High Fat Diet + Streptozotocin)
Group C: HFD + STZ + compound 4
From day 1 to day 53 mice of all groups were allowed ad libitum a high-fat diet
(HFD, fat = 35.5% w/w).
Streptozotocin was dissolved in 0.1 M citrate buffer (pH 4.5) and injected i.p. after 4
h fasting to groups B and C in multiple (five) low doses (35 mg/Kg/day x 5 days),
from day 23 to day 27.
Compound 4 was administered at a dose of 56 µmol/Kg, twice a day, during the
whole experimental procedure (53 days) to group C. Saline (equal volume) was
administered to groups A and B.
4
.3.5.1. Glucose levels
Glucose concentration was determined every 5 to 10 days with a hand-held
glucometer (Precision Xtra Plus, Abbott). Blood samples were applied directly to the
glucose-measurement strip from non fasted mice, to measure non fasting levels of
blood glucose.
4
.3.5.2. Body weight and food consumption
Body weights were measured and food consumption was estimated once weekly.
.3.5.3. Lipidemic parameters
4
For lipid analysis blood was drawn on day 22 and 53 of the experiment. Total
cholesterol, HDL-cholesterol, LDL-cholesterol and Triglyceride plasma levels were
determined as previously described and using commercially available kits [11,12].
4
.4. Protein determination
The protein content of microsomal fractions was determined according to Lowry’s
method [32].
4
.5. Statistical analysis

2
3
ACCEPTED MANUSCRIPT
Data are expressed as mean ± SD. Where indicated, statistical comparisons were
made using Student’s t-test and a statistically significant difference was inferred if
P<0.05.
Appendix A. Supplementary data.
Supplementary data related to this article (NMR spectra of the final products 1-10).
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Chart 1. Morpholine and 1,4-Benzo(x/thi)azine lead compounds with Antioxidant
and Squalene Synthase inhibitory activity.

2
8
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Chart 2. Design of the novel derivatives 1-10.
OH
R3
R2
O
OH
R3
R2
O
N
N
R1
A-C
1-3
R1
A, 1: R = CH , R , R = H
1
3
2
3
B, 2: R R = -(CH ) -, R = H
1
2
2 4
3
C, 3: R = CH , R R = -(CH ) -
1
3
2
3
2 4
OH
S
N
OH
E
S
N
OH
OH
O
4
N
OH
D
OH
S
N
OH
R
X
N
5
6
: R = OH
: R = CF3
Ar
R
7
8
9
: X= S, R= H, Ar = H
: X= O, R= H, Ar = H
: X= O, R= Phenyl, Ar=Phenyl
S O
N
1
0