Antihyperlipidemic morpholine derivatives with antioxidant activity: An investigation of the aromatic substitution

Article abstract of DOI:10.1016/j.bmc.2015.09.034

Drugs affecting more than one target could result in a more efficient treatment of multifactorial diseases as well as fewer safety concerns, compared to a one-drug one-target approach. Within our continued efforts towards the design of multifunctional molecules against atherosclerosis, we hereby report the synthesis of 17 new morpholine derivatives which structurally vary in terms of the aromatic substitution on the morpholine ring. These derivatives simultaneously suppress cholesterol biosynthesis through SQS inhibition (IC₅₀ values of the most active compounds are between 0.7 and 5.5 μM) while exhibiting a significant protection of hepatic microsomal membranes against lipid peroxidation (with IC₅₀ values for the most active compounds being between 73 and 200 μM). Further evaluation of these compounds was accomplished in vivo in an animal model of acute experimental hyperlipidemia, where it was observed that compounds reduced the examined lipidemic parameters (TC, TG and LDL) by 15-80%. In order to examine the mode of binding of these molecules in the active catalytic site of SQS, we also performed docking simulation studies. Our results indicate that some of the new compounds can be considered interesting structures in the search for new multifunctional agents of potential application in atherosclerosis.

Full text of DOI:10.1016/j.bmc.2015.09.034

Bioorganic & Medicinal Chemistry 23 (2015) 7015–7023
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antihyperlipidemic morpholine derivatives with antioxidant activity:
An investigation of the aromatic substitution
⇑
Eleni M. Ladopoulou, Alexios N. Matralis, Anastasios Nikitakis, Angeliki P. Kourounakis
Department of Medicinal Chemistry, School of Pharmacy, University of Athens, 15771 Athens, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 July 2015
Revised 17 September 2015
Accepted 20 September 2015
Available online 25 September 2015
Drugs affecting more than one target could result in a more efficient treatment of multifactorial diseases
as well as fewer safety concerns, compared to a one-drug one-target approach. Within our continued
efforts towards the design of multifunctional molecules against atherosclerosis, we hereby report the
synthesis of 17 new morpholine derivatives which structurally vary in terms of the aromatic substitution
on the morpholine ring. These derivatives simultaneously suppress cholesterol biosynthesis through SQS
inhibition (IC50 values of the most active compounds are between 0.7 and 5.5
nificant protection of hepatic microsomal membranes against lipid peroxidation (with IC50 values for the
most active compounds being between 73 and 200 M). Further evaluation of these compounds was
accomplished in vivo in an animal model of acute experimental hyperlipidemia, where it was observed
that compounds reduced the examined lipidemic parameters (TC, TG and LDL) by 15–80%. In order to
examine the mode of binding of these molecules in the active catalytic site of SQS, we also performed
docking simulation studies. Our results indicate that some of the new compounds can be considered
interesting structures in the search for new multifunctional agents of potential application in
atherosclerosis.
lM) while exhibiting a sig-
Keywords:
Multifunctional
l
Squalene synthase inhibitor
Lipid peroxidation
Lipidemic parameters
Aromatic morpholine derivatives
Docking
Ó 2015 Published by Elsevier Ltd.
1. Introduction
As oxidative stress and hypercholesterolemia/hyperlipidemia
are seriously implicated in the pathogenesis of atherosclerosis,
The widely accepted oxidation theory of atherosclerosis, the
leading cause of death in western countries, is based on the
observation that high levels of LDL-cholesterol in blood are prone
to extensive oxidative modification, believed to be initiated by
the radical-mediated peroxidation of cholesterol-esterified lipids.
This peroxidation can lead to the subsequent increased and uncon-
trolled uptake of cholesterol by macrophages, an event that trig-
gers a cascade of cellular processes that result in the formation
of fatty streaks and eventually atherosclerotic lesions in the
arterial wall.1
many antioxidants and antihyperlipidemic agents have been eval-
uated in animal models as well as in human clinical trials. Several
antioxidants, such as vitamin E and probucol, are known to lower
lipid levels and coronary heart disease incidence in several animal
models. However, the results of many epidemiological and bio-
chemical studies have been disappointing, prompting the question
of whether an antioxidant alone can be used as a therapeutic in the
prevention and/or treatment of atherosclerosis.2
On the other hand, the effective reduction of LDL levels was
shown to halt the progression of atherosclerosis and reduce CAD-
associated morbidity and mortality. HMG-CoA reductase inhibi-
tors, that is, statins, are so far the most effective medications for
serving the above purpose. However, by inhibiting cholesterol
biosynthesis at an early step, statins also decrease not only the pro-
duction of mevalonate but also of isoprenoid intermediates such as
isopentenyl pyrophosphate, farnesyl pyrophosphate and geranyl-
geranyl pyrophosphate. Some of these molecules not only serve
as lipid attachments for signaling molecules such as Ras, Rho
and GTPases, but are also important precursors for signaling
molecules that regulate protein synthesis (isopentenyl adenine),
Abbreviations: CVD, cardiovascular disease; SQS, squalene synthase; HMGCo-A,
3-hydroxy-3-methylglutaryl-CoA; FPP, farnesyl pyrophosphate; PSPP, presqualene
pyrophosphate; rt, room temperature; BSA, bovine serum albumin; NADPH,
nicotinamide adenine dinucleotide phosphate; IC50, inhibitory concentration for
50% of the reaction; TC, total cholesterol; LDL, low-density lipoprotein cholesterol;
HDL, high-density lipoprotein cholesterol; TG, triglyceride.
⇑
Corresponding author at present address: Department of Pharmaceutical
Chemistry, School of Pharmacy, University of Athens, 15771 Athens, Greece.
Tel./fax: +30 210 7274818.
E-mail address: angeliki@pharm.uoa.gr (A.P. Kourounakis).
http://dx.doi.org/10.1016/j.bmc.2015.09.034
0968-0896/Ó 2015 Published by Elsevier Ltd.

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E. M. Ladopoulou et al. / Bioorg. Med. Chem. 23 (2015) 7015–7023
mitochondrial respiration (ubiquinone) and glycosylation (dolichol).
The suppression of the above-mentioned mechanisms is responsible
for statins side (toxic) effects such as myopathy, rhabdomyolysis
and hepatotoxicity. Thus, inhibition of isoprenoid synthesis by sta-
tins could serve as a double-edged sword.3–5 Because of these side
effects by statins, squalene synthase (SQS) a downstream enzyme
of the cholesterol biosynthesis pathway, is considered an alterna-
tive target for antihyperlipidemic agents. SQS catalyzes the first
committed step that leads exclusively to the formation of choles-
terol by converting farnesyl pyrophosphate to squalene. It has been
shown that SQS inhibitors reduce hepatic cholesterol biosynthesis
and upregulate LDL receptors, without depleting cellular levels of
isoprenoids.6,7 Compared to statins, TAK-475, the first SQS inhibi-
tor that entered clinical trials, has shown a similar anti-hyperlipi-
demic effect in many experimental animal models while several
other SQS inhibitors have also shown similar or even improved
anti-hyperlipidemic properties compared to statins.7,8a,b,11,14b In
addition, in clinical trials it was proven that TAK-475 exhibited a
very significant pharmacological effect (comparable to that of sta-
tins) that was even enhanced with co-administration of a statin.8c,d
Furthermore, SQS inhibitors do not cause myotoxicity8e while
when they are coadministered with statins they decreased statin-
induced myotoxicity.9
respective bromoacetyl derivative 24–29 (Scheme 2) and subse-
quent cyclization of the corresponding hydroxyaminoketone inter-
mediate to a hemiketal structure.10–12
As verified by our theoretical studies as well as by previous
experimental studies,11,14a the fused octahydropyridino (com-
pounds 2, 5, 7, 10, 13 and 16) or octahydrobenzoxazino (com-
pounds A, 3, 6, 8, 11, 14 and 17) ring system adopts a chair–chair
(trans) conformation. The initial acetyl derivatives (19–22,
Scheme 1), were produced either via standard Pd-catalyzed
(Suzuki) cross-coupling reactions, or were commercially available
(18 and 23). The conversion of the acetyl derivatives 18–23 to
the respective bromoacetyl derivatives 24–29 was performed
either by the reaction with bromine (compounds 24 and 28) or
phenyltrimethylammonium tribromide (compounds 25, 26, 27
and 29, Scheme 1).
2.2. Antioxidant activity
The effect of the investigated derivatives on the non enzymatic
peroxidation of hepatic microsomal membrane lipids after 45 min
of incubation, expressed as IC50 values, is shown in Table 1. Under
the same experimental conditions, 2,6-di-tert-butyl-4-methylphe-
nol (BHT) and probucol, two known antioxidants with
In several previous studies we have developed multifunctional
antihyperlipidemic and antioxidant agents based on the morpho-
line structure.10–13 Herein, as part of our lead optimization efforts,
we synthesized new morpholine analogs with a variable aromatic
substituent at position-2 or -3 of the morpholine ring (Chart 1). The
17 new derivatives were evaluated for their antioxidant and SQS
inhibitory activity in vitro, while in silico docking studies of
selected compounds on SQS shed some light on their inhibitory
interactions. Finally, all derivatives were investigated in vivo for
their antihypercholesterolemic/antihyperlipidemic activity.
antiatherosclerotic activity, exhibited IC50 values of 25
>1 mM, respectively. Furthermore, lead compound A inhibited
lipid peroxidation with an IC50 value of 450
M.10–12,14b The time
lM and
l
course of lipid peroxidation, as affected by several concentrations
of one of the most active compounds, 9, is depicted in Figure 1.
We speculate that compounds 1–17 exhibit their antioxidant
activity through the abstraction of their hemiketalic hydroxyl group
(on the morpholine ring) after a radical attack. The intermediate
radical formed can subsequently be stabilized by resonance with
the adjacent aromatic substituent.11b Substitution of the second
ring of the biphenyl group of lead compound A with an electron
acceptor (NO2, compounds 4, 6) or an electron donor (OH,
compounds 7, 8) or a group with a potent inductive effect
[C(CH3)3, compounds 9, 11] increases antioxidant activity of the
respective compounds. The exhibition of higher antioxidant
activity seems to be also favored by the replacement of the
biphenyl group of A with more lipophilic structural moieties; the
replacement of the second phenyl group with cyclohexyl group
led to a 2.5-fold increase in antioxidant activity (IC50 values of 1
2. Results and discussion
2.1. Chemistry
Target compounds 1–17 (Chart 1) were synthesized through the
reaction of the appropriate aminoalcohol (2-methylaminoethanol,
2-piperidinemethanol or 2-methylaminocyclohexanol) with the
and
3 are 190 lM and 180 lM, respectively), while the
replacement of the biphenyl with an extended and planar
aromatic system (phenanthrene) led also to similar results (IC50
values of 15, 16 and 17 are 174 lM, 198 lM and 191 lM,
respectively). In contrast, replacement of biphenyl’s second
OH
Ar
HBr
OH
Ar
HBr
OH
Ar
HBr
O
N
O
N
O
N
CH3
CH3
phenyl with its isostere thiazole, significantly decreased
1,4,9,12,15
2,5,7,10,13,16
A,3,6,8,11,14,17
antioxidant activity (IC50 values of 12, 13 and 14 are 600
lM,
1000 M and >1000 M, respectively). From these results it is
l
l
concluded that lipophilicity probably contributes to the increased
access, retention, and interaction of these compounds with
biological membranes, the site of lipid peroxidation, although no
apparent correlation between antioxidant activity and
lipophilicity seems to apply among the tested compounds.
Furthermore, in an attempt to elucidate in part the mechanism of
antioxidant activity of this series of compounds, we evaluated
most of them in the DPPH assay. None of the compounds tested
exhibited any significant activity (results not shown). Reference
compound A was also inactive in the DPPH assay, while probucol
Ar:
A
NO2
OH
1,2,3
7,8
4,5,6
S
N
exhibited an IC50 value of 234 l
M11b under the same
experimental conditions. The ability of a compound to interact
with the stable free radical DPPH is considered an indicator of its
reducing potential. Thus, 1–17 seem to exhibit their activity
against lipid peroxidation through other antioxidant mechanism(s).
9,10,11
12,13,14
15,16,17
Chart 1. Structures of substituted hydroxymorpholins.

E. M. Ladopoulou et al. / Bioorg. Med. Chem. 23 (2015) 7015–7023
7017
O
OH
OH
O
O
(a)
(b)
+
Br
B
X
X
X
H3C
H3C
Br
X= NO2
X= OH
X= tert-butyl
19 X= NO2
20
21 X= tert-butyl
I
25 X= NO2
26
27 X= tert-butyl
X= OH
X= OH
Br
S
S
O
OH
O
O
(d)
S
N
(c)
+
B
H3C
OH
H3C
N
N
Br
I
22
28
O
O
O
(d)
(b)
H3C
Br
24
29
18
O
H3C
Br
23
Scheme 1. General method for the synthesis of intermediates 19–29. Reagents and conditions: (a) Pd(OAc)2, Bu4NBr, K2CO3, H2O, Ar, 70 °C, 1.5-2 h; (b) phenyltrimethy-
lammonium tribromide, THF/H2O, room temperature, 1.5 h; (c) Pd(PPh3)3, Na2CO3, toluene, reflux; (d) Br2, CHCl3, room temperature, 2.5 h.
studies.11,12,14b,15 All compounds were initially tested for SQS inhi-
bitory activity at a concentration of 10 lM (% inhibition of SQS,
Table 1). Full dose–response inhibition curves (IC50, Fig. 2) were
subsequently determined for the most active (15–17) or fairly
active (1–3) compounds. From the results depicted in Table 1 we
observe that the substitution of biphenyl of the lead compound A
with various groups (–NO2, –OH and –C(CH3)3) leads to a better
Table 1
Antioxidant (inhibition of lipid peroxidation—LP), SQS inhibitory activity and
antihyperlipidemic effects of compounds 1–17
Compound
Inhibition of Inhibition of
LP IC50 (lM) SQS at 10 lM
% decrease
compared to
hyperlipidemic
ClogPh
controls (56
kg ip)
lmol/
inhibitory activity of SQS. Compound A at 10
activity by 13%, while exhibited an IC50 value of 36
l
M inhibited SQS
TC
LDL-C TG
l
M.11,12,14 On
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
190
700
180
73
539
57
134
135
91
410
214
600
1000
>1000
174
198
191
450e
>1000e
—
0% (>100)g
9% (80)g
18% (42)g
29%
43%
n.d
7%
61%
61%
40%
44a
80b
51b
41b
59a
62a
40a
54a
45b
64a
17d
15d
21d
23b
58a
40b
49a
36a
62a
23c
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d
52a
53a
20c
34a
70a
20c
44a
31b
20c
43b
48b
47c
59b
15d
46b
58b
62b
69a
66a
59a
49a,e
3.81
4.61
5.24
2.82
3.62
4.25
3.21
3.85
4.91
5.71
6.34
1.63
2.43
3.07
3.54
4.34
4.97
4.51
the other hand, replacement of the second phenyl of biphenyl with
the non aromatic cyclohexyl group (1–3) or its isostere thiazole
(12–14), led to a very significant decrease in activity. The IC50 val-
ues for 1, 2 and 3 are >100
lM, 80
lM, and 42
lM, respectively,
while compounds 12, 13 and 14 at 50
l
M inhibited SQS activity
by 30%, 35% and 29%, respectively.
This is in accordance with other studies on a series of 3-hydroxy-
3-biaryl quinuclidines16 as well as phenoxypropylamines17 that
demonstrated an improved SQS inhibitory activity for compounds
with planar biaryl substitution while a directional requirement
for the rigid biaryl side chain was identified. It is hypothesized that
aryl units may act as isosteres for isoprenyl subunits in the farnesyl
chain of the endogenous enzyme substrate farnesyl pyrophosphate
67%
30%f
35%f
29%f
48% (8.8)
87% (0.7)
76% (5.5)
13% (36)
—
17
A
54a,e 51b,e
18b,e 18d,e
Probucol
Simvastatin
11d,e 10.75
0 4.48
75a
70a
1.0
—
IC50 = 91 µM
Control
75 μM
n.d: not determined.
a
P <0.005.
P <0.01.
P <0.05.
b
c
d
Not significant (P >0.1) (Student’s test).
Data from Ref. 14b.
% inhibition at 50 lM.
0.5
0.0
e
f
100 μM
150 μΜ
g
h
IC50 in
lM.
Values refer to the respective base.
250 μM
750 μM
0
10
20
30
40
50
2.3. Squalene synthase inhibitory activity
Incubation Time (min)
Inhibition of the activity of SQS, from rat liver microsomes, by
the test compounds was carried out according to previous
Figure 1. Representative graph of the time course of lipid peroxidation as affected
by various concentrations of compound 9.

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E. M. Ladopoulou et al. / Bioorg. Med. Chem. 23 (2015) 7015–7023
interactions deeper in the hydrophobic cavity with its second
100
75
50
25
0
and third condensed aromatic (phenanthrene) groups. These moi-
eties (Fig. 3b) are found to occupy approximately the same area
(i.e., deep in the cavity) as the naphthyl group of CP-320473. Thus,
these extended hydrophobic interactions in the ‘bulky’ phenan-
threnyl-containing compounds 15–17, may explain the higher
binding and inhibitory capacity of the former compounds com-
pared to 1–3 and the rest of the compounds. It should also be men-
tioned that the reduced activity of 1–3 may be due to the fact that
they do not show the same alignment with the planar lipophilic
group of phenanthrene, probably causing a disruption in the orien-
tation of hydrophobic interactions or even steric hindrance
(Fig. 3a).
IC50 = 0.7µM
-3
-2
-1
0
1
2
3
2.5. Antihyperlipidemic activity
Log (μΜ)
The in vivo antihyperlipidemic activity of compounds 1–17 is
demonstrated in Table 1. Acute experimental hyperlipidemia
was successfully established 24 h after Triton WR1339 adminis-
tration.10–12,14b,20 All compounds were found able to reduce (at
Figure 2. Representative graph showing the activity of squalene synthase as
affected by various concentrations of compound 16.
(FPP).18 Moreover, the replacement of biphenyl with the more
planar and extended aromatic system of phenanthrene led to
derivatives with significantly improved inhibitory activity against
SQS. This overall increase in lipophilicity seems to favor a better
interaction between the molecule and the enzyme, as we will see
below in Docking Studies paragraph (Section 2.4). The IC50 values
a dose of 56 lmol/kg) TC levels by 15–80% and TG levels by
15–70% in the plasma of hyperlipidemic rats. Furthermore, com-
pounds 1–3 and 15–17 decreased LDL levels by 20–62%. The most
active compounds are the cyclohexyl-phenyl derivative 2 and the
phenanthrenyl derivative 15 which decreased TC by 80% and 58%,
LDL by 62% and 52% as well as TG levels by 70% and 69%, respec-
tively. Although these compounds have similar antihyperlipi-
demic activity in vivo, they remarkably differ in terms of their
of compounds 15, 16 and 17 are 8.8 lM, 0.7 lM, and 5.5 lM,
respectively.
SQS inhibitory activity (IC50 values of 2 and 15 are 80
lM and
2.4. Docking studies
8.8 M, respectively). This variability between in vitro and
l
in vivo activity for compounds 2 and 15 may be attributed to
other parameters such as (a) compound 2 may exert its antihy-
perlipidemic activity through additional mechanisms other than
SQS inhibition, a topic that deserves further investigation, and
(b) the two compounds may have different degrees of distribution
and pharmacokinetics as they differ in volume, lipophilicity and
other molecular characteristics. In contrast, the increased SQS
inhibitory activity of the phenanthrene derivatives 15–17 and
the decreased SQS inhibitory activity of thiazole derivatives
12–14 are reflected in their respective high (15–17) and low
(12–14) antihypercholesterolemic effect in vivo (Table 1).
Regarding the substituted biphenyl derivatives 4–11, they
reduced, with the exception of compound 11, the examined lipid
parameters in the plasma of hyperlipidemic rats (ca. 20–64%)
with activities more or less comparable to those of reference
compound A. Under the same experimental conditions, probucol
and simvastatin (used as reference antihyperlipidemic drugs), at
the same dose, reduced plasma TC by 18% and 75%, LDL by
18% and 70%, and TG by 11% and 0%, respectively. Furthermore,
lead compound A decreased the above mentioned lipidemic
parameters by 54%, 51%, and 49%, respectively.10–12,14b
To elucidate the binding mode of the test compounds in the
active site of SQS and to further explain their variable inhibitory
against SQS activity, we performed rigid docking studies using an
available X-ray crystal structure (1EZF) of SQS with a bound inhi-
bitor (CP-320473, Fig. 3). This theoretical study was performed
using the software Autodock 4. The procedure followed has been
previously described.11a Figure
visualization of compounds
3
depicts the binding mode
1
and 16. The choice of these
compounds for docking studies was based on the fact that the
molecules with these aromatic substituents (1–3 and 15–17)
exhibited
a very different degree of inhibitory against SQS
activity (Table 1).
As has been previously reported, the active site of SQS consists
of a large lipophilic cavity, having two charged surfaces on oppo-
site ‘‘entrance” walls. One of these surfaces is composed of two
conserved aspartate-rich motifs (80DTLED84 and 219DELED223) that
bind the diphosphates of two substrate farnesyl pyrophosphate
(FPP) molecules via Mg2+-ion bridging.11,19 Docking of compounds
1 and 16 revealed a hydrogen bond between the hemiketalic
hydroxyl group of the morpholine ring of both compounds and
the side chain carboxyl group of ASP80 (Fig. 3).
This interaction may be considered significant for our com-
pounds inhibitory activity assuming that the binding of phosphate
groups of the physiological substrate (FPP) to the 80DTLED84
sequence of the catalytic site of SQS is hindered. Furthermore, in
both cases hydrophobic residues such as VAL175, ALA176, VAL
179, LEU 211, GLY208 and LEU183 surround the hydrophobic aro-
matic side chains of the compounds (Fig. 3). These aminoacids are
also in close proximity to the tert-butyl and naphthyl groups of the
co-crystalized ligand CP-320473 suggesting similar hydrophobic
interactions. In specific, the tert-butyl part of CP-320473 is found
in the same area as the phenyl group of compound 1 and the first
aromatic ring of phenanthrene of compound 16, respectively. In
addition, however, compound 16 exhibits further hydrophobic
2.6. Conclusion
In summary, as part of our lead optimization efforts, we synthe-
sized new morpholine analogs that are able to inhibit significantly
lipid peroxidation as well as SQS activity in vitro. Furthermore,
docking simulations were carried out to investigate potential dif-
ferences in interactions of two representative compounds, an
active (16) and inactive (1), with the active site of SQS. The antihy-
percholesterolemic/ antihyperlipidemic effect of all compounds
was also confirmed in vivo. Thus, the dual-action profile of these
molecules renders them interesting derivatives in the study of
structure–activity relationships of such potential anti-atheroscle-
rosis agents.

E. M. Ladopoulou et al. / Bioorg. Med. Chem. 23 (2015) 7015–7023
7019
(a)
OHO
O
OH
O
Cl
O
NH
N
O
(b)
CP-320473
Figure 3. Theoretical ‘best fit’ binding mode of compounds 1 (a) and 16 (b) at the active site of squalene synthase (SQS), as shown by docking studies. Lipophilic interactions
of the aromatic moiety as well as hydrogen bond of the hemiketalic OH with ASP80 can be seen in both cases. The binding mode of co-crystalized (1EZF) CP-320473 (green
wire structure) is also included for comparison.
3.2.1. trans-2-Methylaminocyclohexanol (31)
3. Experimental section
3.1. Materials
To a solution of cyclohexene oxide (30) (55.7 mmol), synthe-
sized as previously descibed,21 in 20 mL methanol, 15 mL of an
aqueous solution (40%) of methylamine was added and the mix-
ture was stirred for 19 h at rt. The product was isolated by vacuum
distillation (bp4 66–69 °C).21 Yield 70%.
All commercially available chemicals are of the appropriate
purity and purchased from standard sources. [3H] FPP (21.5 Ci/
mmol), NADPH, FPP, and BSA were purchased from Sigma–Aldrich
(Germany). For the in vivo experiments, Wistar male rats
(200–250 g) were used. Animals were kept in a controlled temper-
ature room (22 2 °C), having free access to laboratory chow and
tap water, under a 12 h light/dark cycle.
3.2.2. General procedure for the cross-coupling reactions (19–21)
To a three-necked flask 2 mmol of arylhalide, 2.2 mmol of 1-(4-
acetyl-phenyl)-boronic acid, 0.2 mol % of Pd(OAc)2, 5 mmol of
powdered K2CO3 and 2 mmol of Bu4NBr were added. The flask
was flushed with argon and via a rubber septum, water (2.2 mL)
was added and the resulting suspension was stirred, degassed
and heated for 2 h at 70 °C under argon. It was then cooled to room
temperature, diluted with water, and extracted with EtOAc. The
solution was dried (Na2SO4), the solvent was removed in vacuum,
and the crude product purified by silica gel column chromatogra-
phy to yield the desired product.22
3.2. Synthesis
Melting points (mp) were determined with a digital Elec-
trothermal IA 9000 series apparatus and are uncorrected. Infra-
red (IR) spectra were recorded on
a Perkin–Elmer FT-IR
spectrometer (Spectrum RXI). 1H NMR spectra and 2D-NMR
(COSY and HMQC) were obtained with a Bruker Avance DRX
400 (400 MHz) spectrometer and 13C 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).
3.2.2.1. 1-(40-Nitro-4-biphenyl)ethanone (19).
flash chromatography eluting with EtOAc–petroleum ether 1:3,
yield 95%, mp 154.5–155.5 °C.23 1H NMR (CDCl3): d 2.57 (s, 3H,
CH3), 7.62 (dd, J1 = 8.20 Hz, J2 = 1.59 Hz, 2H, 3,5-H biphenyl), 7.67
Purified by

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E. M. Ladopoulou et al. / Bioorg. Med. Chem. 23 (2015) 7015–7023
(dd, J1 = 9.05 Hz, J2 = 2.08 Hz, 2H, 20,60-H biphenyl), 7.98 (dd,
J1 = 8.19 Hz, J2 = 1.47 Hz, 2H, 2,6-H biphenyl), 8.23 (dd,
J1 = 9.05 Hz, J2 = 2.08 Hz, 2H, 30,50-H biphenyl).
3.2.4.2.
(26).
2-Bromo-1-[40-hydroxy-4-biphenyl]-ethanone
Purified by flash chromatography eluting with CH2Cl2–
petroleum ether 1:3, yield 84%, mp 142–144 °C. 1H NMR (CDCl3):
d 4.45 (s, 2H, CH2), 6.92 (d, J = 8. 81 Hz, 2H, 30,50-H biphenyl),
7.52 (d, J = 8.61 Hz, 2H, 20,60-H biphenyl), 7.64 (d, J = 8.22 Hz, 2H,
3,5-H biphenyl), 8.01 (d, J = 8.22 Hz, 2H, 2,6-H biphenyl). Anal.
(C14H11BrO2ꢁ0.4H2O), Calcd: C, 56.34; H, 3.99. Found: C, 56.31; H,
3.75.
3.2.2.2. 1-(40-Hydroxy-4-biphenyl)ethanone (20).
Purified
by flash chromatography eluting with EtOAc–petroleum ether
1:2, yield 99%, mp 200–202.5 °C.24 1H NMR (CDCl3): d 2.13 (s, 3H,
CH3), 5.80 (d, J = 8.56 Hz, 2H, 3,5-H biphenyl), 6.39 (d, J = 8.56 Hz,
2H, 20,60-H biphenyl), 6.53 (d, J = 8.55 Hz, 2H, 2,6-H biphenyl),
6.87 (d, J = 8.56 Hz, 2H, 30,50-H biphenyl), 8.25 (s, 1H, OH).
3.2.4.3.
(27).
2-Bromo-1-[40-tert-butyl-4-biphenyl]-ethanone
Purified by flash chromatography eluting with
3.2.2.3. 1-(40-tert-Butyl-4-biphenyl)ethanone (21).
Purified
CH2Cl2–petroleum ether 1:3, yield 84%, mp 93.4–96.8 °C.27
1H NMR (CDCl3): d 1.38 (s, 9H, C(CH3)3), 4.49 (s, 2H, CH2),
7.50–7.63 (m, 4H, 30,50,2,60-H biphenyl), 7.73 (d, J = 8.19 Hz,
2H, 3,5-H biphenyl), 8.07 (d, J = 8.43 Hz, 2H, 2,6-H biphenyl).
by flash chromatography eluting with EtOAc–petroleum ether
1:3, yield 81%, mp 130.3–132 °C.23 1H NMR (CDCl3): d 1.31 (s, 9H,
C(CH3)3), 2.57 (s, 3H, CH3), 7.44 (d, J = 8.61 Hz, 2H, 30,50-H biphenyl)
7.51 (d, J = 8.41 Hz, 2H, 20,60-H biphenyl), 7.56 (d, J = 8.61 Hz, 2H,
3,5-H biphenyl), 7.96 (d, J = 8.61 Hz, 2H, 2,6-H biphenyl).
3.2.4.4. 2-Bromoacetylphenanthrene (29).
Purified by flash
chromatography eluting with CH2Cl2–petroleum ether 1:2, yield
67%, mp 141–144.6 °C.28 1H NMR (CDCl3): d 4.56 (s, 2H, CH2),
7.62–7.68 (m, 2H, 7,8-H phenanthrene), 7.77 (s, 2H, 4,5-H phenan-
threne), 7.88 (dd, J1 = 1.50 Hz, J2 = 7.00 Hz, 1H, 6-H Ar phenan-
3.2.2.4. 2-(4-Acetyl-phenyl)thiazole (22).
Purified by flash
chromatography eluting with EtOAc–petroleum ether 3:2, yield
57%, mp 107–109 °C.25 1H NMR (CDCl3): d 2.60 (s, 3H, CH3),
7.40–8.20 (m, 6H, Ar-H).
threne),
8.15
(dd,
J1 = 1.88 Hz,
J2 = 8.71 Hz, 1H,
1-H
phenanthrene), 8.47 (ds, J = 1.45 Hz, 1H, 3-H phenanthrene), 8.65
(d, J = 8.19 Hz, 1H, 9-H phenanthrene), 8.70 (d, J = 8.70 Hz, 1H,
10-H phenanthrene).
3.2.3. General procedure for the preparation of bromo-acetyl
derivatives 24 and 28
Bromine (5.5 mmol) in dry chloroform (20 mL) was added
dropwise to a stirred solution of the corresponding acetyl deriva-
tive 4-cyclohexyl-a-acetophenone (18) or 2-(4-acetyl-phenyl)thia-
zole (22) (5 mmol) in dry chloroform (10 mL). After the addition of
bromine, the solution was stirred at room temperature for 2.5 h.
The solution was washed with H2O, 5% NaHCO3, and H2O, dried
(Na2SO4) and concentrated. The residue was subjected to flash col-
umn chromatography on silica gel to give the desired product.
3.2.5. General procedure for the preparation of the final products
1–17
The final products (Chart 1, Scheme 2) were obtained by the
reaction of 2.2 mmol of 2-methylaminoethanol, 2-piperidine-
methanol or trans-2-methylaminocyclohexanol (31) with 1.0 mmol
of 4-cyclohexyl-a-bromoacetophenone (compounds 1, 2, 3) or
2-bromo-1-[40-nitro-4-biphenyl]ethanone (compounds 4, 5, 6)
or 2-bromo-1-[40-hydroxy-4-biphenyl]ethanone (compounds 7, 8) or
2-bromo-1-[40-tert-butyl-4-biphenyl]ethanone (compounds 9, 10,
11) or 2-(4-bromoacetyl-phenyl)thiazole (compounds 12, 13, 14)
or 2-bromoacetylphenanthrene (compounds 15, 16, 17) in anhydrous
acetone (40 mL) at room temperature with stirring for 20–24 h.
Acetone was then distilled off, ether was added to the residue,
the mixture was washed with saturated sodium chloride solution,
3.2.3.1. 4-Cyclohexyl-a-bromoacetophenone (24).
Purified
by flash chromatography eluting with CH2Cl2–petroleum ether
2:3, yield 75%.26 1H NMR (CDCl3): d 1.25–1.32 (m, 1H, 4-Hax cyclo-
hexyl), 1.35–1.49 (m, 4H, 2ꢀ 3-H and 2ꢀ 5-H cyclohexyl), 1.78 (m,
1H, 4-Heq cyclohexyl), 1.88 (m, 4H, 2ꢀ 2-H and 2ꢀ 6-H cyclohexyl),
2.55–22.61 (m, 1H, 1-Hax cyclohexyl), 4.45 (s, 2H, CH2), 7.33 (d,
J = 8.47 Hz, 2H, 3,5-H Ar), 7.92 (d, J = 8.40 Hz, 2H, 2,6-H Ar).
3.2.3.2. 2-(4-Bromoacetyl-phenyl)thiazole (28).
Purified by
OH
flash chromatography eluting with CH2Cl2–petroleum ether 20:1,
yield 40%, mp 111.1–113 °C.25 1H NMR (CDCl3): d 4.47 (s, 2H,
CH2), 7.43–8.13 (m, 6H, Ar-H).
OH
Ar
O
N
NH
CH3
HBr
3.2.4. General procedure for the preparation of bromo-acetyl
derivatives 25–27 and 29
CH3
1, 4, 9, 12, 15
OH
To a solution of the acetyl derivative 1-(40-nitro-4-biphenyl)
ethanone (19) or 1-(40-hydroxy-4-biphenyl)ethanone (20) or
1-(40-tert-butyl-4-biphenyl)ethanone (21) or 2-acetylphenan-
threne (23) (5 mmol) in 10 mL of anhydrous tetrahydrofuran,
phenyltrimethylammonium tribromide (5 mmol) was added in
portions over a 30 min period. A white precipitate forms immedi-
ately. The mixture was stirred at room temperature for 1.5–2 h and
then cold water was added. The crystalline precipitate was filtered,
washed with water, dried, and was subjected to flash column chro-
matography on silica gel to give the desired product.
H
OH
O
N
O
(a)
Ar
N
HBr
Br
24-29
Y
2, 5, 7, 10, 13, 16
OH
Ar
O
N
(b)
OH
HBr
O
CH3
NH
CH3
3.2.4.1. 2-Bromo-1-[40-nitro-4-biphenyl]-ethanone (25).
Puri-
3, 6, 8, 11, 14, 17
30
31
fied by flash chromatography eluting with CH2Cl2–petroleum ether
3:1, yield 79%, mp 175–175.8 °C.27 1H NMR (CDCl3): d 4.49 (s, 2H,
CH2), 7.74–7.81 (m, 4H, 3,5,20,60-H biphenyl), 8.13 (d, J = 8.19 Hz,
2H, 2,6-H biphenyl), 8.36 (d, J = 8.56 Hz, 2H, 30,50-H biphenyl).
Scheme 2. General method for the synthesis of final compounds 1–17. Reagents
and conditions: (a) (i) acetone, room temperature, 20–24 h; (ii) HBr/Et2O;
(b) CH3NH2, room temperature, 19 h.

E. M. Ladopoulou et al. / Bioorg. Med. Chem. 23 (2015) 7015–7023
7021
dried (K2CO3), and the products were isolated as hydrobromides
recrystallized by acetone–diethyl ether.10–12
(t, J = 11.83 Hz, 1H, 1-Hax oxazine), 7.76 (d, J = 8.22 Hz, 2H, 2,6-H
biphenyl), 7.84 (d, J = 8.02 Hz, 2H, 3,5-H biphenyl), 7.86 (d,
J = 8.41 Hz, 2H, 20,60-H biphenyl), 8.37 (d, J = 8.61 Hz, 2H, 30,50-H
biphenyl), 10.37 (br s, 1H, NH). Anal. (C20H23BrN2O4ꢁ0.3 H2O),
Calcd: C, 54.43; H, 5.41. Found: C, 54.43; H, 5.42.
3.2.5.1. 2-(4-Cyclohexylphenyl)-4-methylmorpholin-2-ol hydro-
bromide (1).
Yield 38%, mp 208.5–209 °C. 1H NMR (DMSO-
d6): d 1.13–1.38 (m, 5H, 2,3,4,5,6-Hax cyclohexyl), 1.60–1.71 (m,
5H, 2,3,4,5,6-Heq cyclohexyl), 2.00 (s, 1H, OH), 2.35–2.45 (m, 1H,
1-Hax cyclohexyl), 2.70 (s, 3H, NCH3), 3.02 (d, J = 11.53 Hz, 1H,
3-Hax oxazine), 3.13 (dt, J1 = 3.11 Hz, J2 = 12.08 Hz, 1H, 5-Hax oxa-
zine), 3.37 (m, 1H, 5-Heq oxazine), 3.39 (d, J = 12.07 Hz, 1H, 3-Heq
oxazine), 3.89 (dd, J1 = 3.29 Hz, J2 = 12.63 Hz, 1H, 6-Heq oxazine),
4.19 (dt, J1 = 2.74 Hz, J2 = 12.08 Hz, 1H, 6-Hax oxazine), 7.19 (d,
J = 8.42 Hz, 2H, 3,5-H Ar), 7.39 (d, J = 8.23 Hz, 2H, 2,6-H Ar), 9.74
(br s, 1H, NH). 13C NMR (DMSO-d6): d 26.00 (4-C cyclohexyl),
26.75 (3,5-C cyclohexyl), 34.36 (2,6-C cyclohexyl), 43.45 (1-C
cyclohexyl), 43.96 (N-CH3), 52.07 (5-C oxazine), 57.19 (6-C oxa-
zine), 59.84 (3-C oxazine), 94.03 (2-C oxazine), 126.16 (3,5-C Ar),
126.85 (2,6-C Ar), 139.15 (1-C Ar), 148.77 (4-C Ar). Anal.
(C17H26BrNO2ꢁ0.3H2O), Calcd: C, 56.44; H, 7.41. Found: C, 56.44;
H, 7.37.
3.2.5.6. 2-[4-(40-Nitrophenyl)phenyl]-4-methyloctahydro-1,4-
benzoxazin-2-ol hydrobromide (6).
Yield 60%, mp 165.6–
166.1 °C. 1H NMR (CDCl3): d 1.10–1.45 (m, 4H, 8-Hax, 5-Hax and
2ꢀ 7-H oxazine), 1.74 (t, J = 14.67 Hz, 2H, 2ꢀ 6-H oxazine), 1.87
(d, 1H, J = 10.36 Hz, 8-Heq oxazine), 2.18 (m, 1H, 5-Heq oxazine),
2.68 (s, 3H, NCH3), 2.97 (br s, 1H, 4a-Hax oxazine), 3.15 (br s, 1H,
3-Hax oxazine), 3.52 (br d, 1H, 3-Heq oxazine), 4.01 (br t, 1H,
8a-Hax oxazine), 7.65 (d, J = 8.22 Hz, 2H, 2,6-H biphenyl), 7.83 (d,
J = 8.21 Hz, 2,3,5-H biphenyl), 7.93 (d, J = 8.61 Hz, 2H, 20,60-H biphe-
nyl), 8.27 (d, J = 8.60 Hz, 2H, 30,50-H biphenyl), 9.96 (br s, 1H, NH).
Anal. (C21H25BrN2O4), Calcd: C, 55.39; H, 5.68. Found: C, 55.40; H,
5.74.
3.2.5.7.
[2,1-c]oxazin-3-ol hydrobromide (7).
3-[4-(40-Hydroxyphenyl)phenyl]octahydro-1,4-pyrido
Yield 80%, mp 196.9–
3.2.5.2. 3-(4-Cyclohexylphenyl)octahydro-1,4-pyrido[2,1-c]ox-
198.3 °C. 1H NMR (CDCl3 + DMSO-d6): d 1.96 (m, 2H, 8-Hax and
9-Hax oxazine), 2.14 (d, J = 10.37 Hz, 2H, 2ꢀ 7-H oxazine), 2.25–
2.28 (m, 1H, 8-Heq oxazine), 2.35–2.49 (m, 1H, 9-Heq oxazine),
2.88 (s, 1H, OH), 3.33 (t, J = 11.93 Hz, 1H, 6-Hax oxazine), 3.58–
3.64 (m, 3H, 6-Heq, 4-Hax and 4-Heq oxazine), 3.71 (m, 1H, 9a-Hax
oxazine), 4.19 (d, J1 = 12.52 Hz, 1H, 1-Heq oxazine), 4.55 (t,
J = 11.83 Hz, 1H, 1-Hax oxazine), 7.21 (d, J = 8.60 Hz, 2H, 30,50-H
biphenyl), 7.73 (d, J = 8.21 Hz, 2H, 3,5-H biphenyl), 7.87 (d,
J = 8.21 Hz, 2H, 2,6-H biphenyl), 7.96 (d, J = 8.02 Hz, 2H, 2,60-H
biphenyl), 9.56 (br s, 1H, NH). Anal. (C20H24BrNO3), Calcd: C,
59.12; H, 5.95. Found: C, 58.81; H, 6.05.
azin-3-ol hydrobromide (2).
Yield 60%, mp 192.5–194 °C.
1H NMR (CDCl3): d 1.36–1.41 (m, 6H, 8,9-Hax oxazine and 2ꢀ
4-H, 3,5-Hax cyclohexyl), 1.73–1.94 (m, 8H, 8,9-Heq oxazine and
3,5-Heq, 2ꢀ 2-H, 2ꢀ 6-H cyclohexyl), 2.05–2.18 (m, 2H, 2ꢀ 7-H
oxazine), 2.43–2.5 (m, 2H, OH and 1-Hax cyclohexyl), 2.63–2.72
(m, 1H, 6-Hax oxazine), 2.89 (t, J = 12.35 Hz, 1H, 4-Hax oxazine),
3.22-3.29 (m, 1H, 6-Heq oxazine), 3.42-3.45 (m, 1H,
9a-Hax
oxazine), 3.52 (d, J = 12.72 Hz, 1H, 4-Heq oxazine), 3.90 (dd,
J1 = 3.40 Hz, J2 = 13.41 Hz, 1H, 1-Heq oxazine), 4.51-4.57 (m, 1H,
1-Hax oxazine), 7.22 (d, J = 8.25 Hz, 2H, 3,5-H Ar), 7.56 (d, J =
8.26 Hz, 2H, 2,6-H Ar), 11.50 (br s, 1H, NH). Anal. (C20H30BrNO2ꢁ0.2
H2O), Calcd: C, 60.06; H, 7.58. Found: C, 59.86; H, 7.80.
3.2.5.8. 2-[4-(40-Hydroxyphenyl)phenyl]-4-methyloctahydro-1,
4-benzoxazin-2-ol hydrobromide (8).
Yield 48%, mp 139.9–
3.2.5.3. 2-(4-Cyclohexyl-phenyl)-4-methyl-octahydro-1,4-ben-
140.6 °C. 1H NMR (DMSO-d6): d 1.20–1.60 (m, 4H, 8-Hax, 5-Hax
and 2ꢀ 7-H oxazine), 1.75–1.95 (m, 3H, 2ꢀ 6-H and 8-Heq oxazine),
2.15–2.32 (m, 2H, 3-Hax and 4a-Hax oxazine and s, 3H, NCH3), 2.70
(s, 1H, OH), 2.91 (d, J = 11.35 Hz, 1H, 3-Heq oxazine), 3.93 (m, 1H,
8a-Hax oxazine), 6.36 (s, 1H, OH biphenyl), 6.99 (d, J = 8.61 Hz, 2H,
3,5-H biphenyl), 7.62 (d, J = 8.61 Hz, 2H, 2,6-H biphenyl), 7.68 (s, 4H,
20,6,30,50-H biphenyl), 9.71 (br s, 1H, NH). Anal. (C21H26BrNO3ꢁ1.2H2O),
Calcd: C, 56.97; H, 6.49. Found: C, 57.00; H, 6.82.
zoxazin-2-ol hydrobromide (3).
Yield 78%, mp 189–190 °C.
1H NMR (CDCl3): d 1.18–1.47 (m, 11H, 2ꢀ 2-H, 2ꢀ 3-H, 2ꢀ 4-H,
2ꢀ 5-H, 2ꢀ 6-H cyclohexyl and 8-Hax oxazine), 1.66–1.69 (m, 2H,
2ꢀ 7-H oxazine), 1.90–1.99 (m, 2H, 2ꢀ 6-H oxazine), 2.06–2.08
(m, 1H, 8-Heq oxazine), 2.10–2.23 (m, 2H, 2ꢀ 5-H oxazine), 2.33
(m, 1H, 1-Hax cyclohexyl), 2.43 (s, 1H, OH), 2.72 (ds, J = 4.41 Hz,
3H, NCH3), 2.90–2.93 (m, 2H, 3-Hax and 4a-Hax oxazine), 3.54 (d,
J = 12.72 Hz, 1H, 3-Heq oxazine), 4.41–4.50 (m, 1H, 8
a
-Hax oxazine),
7.15 (d, J = 7.33 Hz, 2H, 3,5-H Ar), 7.49 (d, J = 7.09 Hz, 2H, 2,6-H Ar),
11.60 (br s, 1H, NH). Anal. (C21H32BrNO2ꢁ0.5H2O), Calcd: C, 60.14;
H, 7.71. Found: C, 59.89; H, 7.60.
3.2.5.9. 2-[4-(40-tert-Butylphenyl)phenyl]-4-methylmorpholin-
2-ol hydrobromide (9).
Yield 30%, mp 189–191.1 °C. 1H
NMR (DMSO-d6): d 1.59 (s, 9H, C(CH3)3), 2.82 (s, 1H, OH), 3.13 (s,
3H, NCH3), 3.34–3.37 (m, 2H, 3-Hax and 5-Hax oxazine), 3.67–
3.72 (m, 2H, 5-Heq and 3-Heq oxazine), 4.26 (d, J = 11.93 Hz, 1H,
6-Heq oxazine), 4.77 (t, J = 11.93 Hz, 1H, 6-Hax oxazine), 7.62–8.02
(m, 8H, 20,60,30,50,2,6,3,5-H biphenyl). Anal. (C21H28BrNO2ꢁ0.2H2O),
Calcd: C, 61.55; H, 6.98. Found: C, 61.54; H, 6.90.
3.2.5.4. 2-[4-(40-Nitrophenyl)phenyl]-4-methylmorpholin-2-ol
hydrobromide (4).
Yield 40%, mp 166.4–166.9 °C. 1H NMR
(CDCl3 + DMSO-d6): d 2.63 (s, 1H, OH), 2.96 (s, 3H, NCH3), 3.20–
3.90 (m, 4H, 2ꢀ 5-H and 2ꢀ 3-H oxazine), 4.09 (d, 1H,
J = 12.52 Hz, 6-Heq oxazine), 4.63 (t, 1H, J = 12.52 Hz, 6-Hax
oxazine), 7.76–7.86 (m, 6H, 3,5,2,6,20,60-H biphenyl), 8.35 (d,
J = 8.61 Hz, 2H, 3,50-H biphenyl), 11.28 (br s, 1H, NH). Anal.
(C17H19BrN2O4ꢁ0.5 H2O), Calcd: C, 50.54; H, 4.98. Found: C, 50.54;
H, 4.99.
3.2.5.10. 3-[4-(40-tert-Butylphenyl)phenyl]octahydro-1,4-pyrido
[2,1-c]oxazin-3-ol hydrobromide (10).
Yield 65%, mp 191.1–
192.5 °C. 1H NMR (DMSO-d6): d 1.62 (s, 9H, C(CH3)3), 1.87–1.98 (m,
2H, 8-Hax and 9-Hax oxazine), 2.03–2.13 (m, 2H, 7-Hax and 7-Heq
oxazine), 2.18–2.24 (m, 1H, 8-Heq oxazine), 2.31–2.42 (m, 1H, 9-
Heq oxazine), 2.84 (s, 1H, OH), 3.25–3.35 (m, 1H, 6-Hax oxazine),
3.37–3.67 (m, 3H, 6-Heq, 4-Hax, 4-Heq oxazine), 3.65–3.73 (m, 1H,
9a-Hax oxazine), 4.16 (dd, J1 = 12.61 Hz, J2 = 3.23 Hz, 1H, 1-Heq oxa-
zine), 4.50 (t, J = 12.72 Hz, 1H, 1-Hax oxazine), 7.74 (d, J = 8.42 Hz,
2H, 20,60-H biphenyl) 7.81 (d, J = 8.41 Hz, 2H, 30,50-H biphenyl),
7.91 (d, J = 8.42 Hz, 2H, 3,5-H biphenyl), 7.96 (d, J = 8.42 Hz, 2H,
2,6-H biphenyl). Anal. (C24H32BrNO2), Calcd: C, 64.57; H, 7.23.
Found: C, 64.25; H, 7.35.
3.2.5.5. 3-[4-(40-Nitrophenyl)phenyl]octahydro-1,4-pyrido[2,1-
c]oxazin-3-ol hydrobromide (5).
Yield 45%, mp 189.8–
190.2 °C. 1H NMR (DMSO-d6): d 1.70–1.80 (m, 2H, 8-Hax, 9-Hax
oxazine), 1.89–1.92 (m, 2H, 2ꢀ 7-H oxazine), 2.00–2.02 (m, 1H,
8-Heq oxazine), 2.18–2.21 (m, 1H, 9-Heq oxazine), 2.66 (s, 1H, OH),
3.17 (q, 1H, J = 10.69 Hz, 6-Hax oxazine), 3.26–3.31 (m, 1H, 6-Heq
oxazine), 3.36–3.60 (m, 3H, 2ꢀ 4-H and 9a-Hax oxazine),
3.97 (dd, J1 = 12.72 Hz, J2 = 3.13 Hz, 1H, 1-Heq oxazine), 4.33

7022
E. M. Ladopoulou et al. / Bioorg. Med. Chem. 23 (2015) 7015–7023
3.2.5.11. 2-[4-(40-tert-Butylphenyl)phenyl]-4-methyloctahydro-
1,4-benzoxazin-2-ol hydrobromide (11). Yield 70%, mp
3.2.5.15. 2-(2-Phenanthrenyl)-4-methylmorpholin-2-ol hydro-
bromide (15).
Yield 27%, mp 130–135 °C. 1H NMR (DMSO-
166.8–168.8 °C. 1H NMR (DMSO-d6): d 1.14–1.38 (m, 4H, 2ꢀ 7-H,
5-Hax, 8-Hax oxazine and s, 9H, C(CH3)3), 1.59–1.86 (m, 4H, 2ꢀ 6-
H, 8-Heq oxazine and OH), 2.15 (br d, 1H, 5-Heq oxazine), 2.66 (s,
3H, NCH3), 2.95 (br s, 1H, 4a-Hax oxazine), 3.13 (br d, 1H, 3-Hax oxa-
zine), 3.50 (d, J = 12.23 Hz, 1H, 3-Heq oxazine), 3.96–4.30 (m, 1H,
8a-Hax oxazine), 7.40 (d, J = 7.82 Hz, 2H, 20,60-H biphenyl) 7.53 (d,
J = 7.82 Hz, 2H, 30,50-H biphenyl), 7.55 (d, J = 8.07 Hz, 2H, 3,5-H
biphenyl), 7.64 (d, J = 7.82 Hz, 2H, 2,6-H biphenyl). Anal.
(C25H34BrNO2ꢁ5H2O), Calcd: C, 54.85; H, 8.04. Found: C, 54.85; H, 8.04.
d6): d 2.74 (s, 3H, NCH3), 3.13–3.26 (m, 2H, 3-Hax and 5-Hax),
3.56 (m, 2H, 3-Heq and 5-Heq), 4.01 (dd, J1 = 3.13 Hz, J2 = 12.70 Hz,
1H, 6-Heq), 4.28 (dt, J1 = 2.16 Hz, J2 = 12.52 Hz, 1H, 6-Hax), 7.58–
7.71 (m, 3H, 1,7,8-H Ar), 7.78–7.86 (m, 2H, 4,6-H Ar), 7.94 (d,
J = 7.62 Hz, 1H, 5-H Ar), 8.10 (s, 1H, 3-H Ar), 8.79 (d, J = 8.21 Hz,
1H, 9-H Ar), 8.84 (d, J = 8.81 Hz, 1H, 10-H Ar), 9.91 (br s, 1H, NH).
13C NMR (DMSO-d6): d 43.56 (NCH3), 52.15 (5-C), 57.39 (6-C),
59.80 (3-C), 94.26 (2-C), 123.62 (10-C Ar), 124.75 (9-C Ar),
126.03 (1-C Ar), 127.40 (3-C Ar), 127.53 (8-C Ar), 127.65 (7-C Ar),
127.87 (4-C Ar), 129.01 (5-C Ar), 129.20 (6-C Ar), 129.87 (5
a-C
3.2.5.12.
hydrobromide (12).
2-[4-(2-Thiazolyl)phenyl]-4-methylmorpholin-2-ol
Yield 27%, mp 151.1–153.5 °C. 1H NMR
Ar), 130.40 (9 -C Ar), 131.54 (3 -C Ar), 132.28 (9b-C Ar), 139.89
a
a
(2-C Ar). Anal. (C19H20BrNO2ꢁH2O), Calcd: C, 57.97; H, 5.36. Found:
(CDCl3 + DMSO-d6): d 2.10 (d, J = 3.91 Hz, 3H, NCH3), 2.37 (t,
J = 11.25 Hz, 1H, 3-Hax oxazine), 2.66 (d, J = 11.89 Hz, 2H, 3-Heq
oxazine and OH), 3.05 (dd, J1 = 4.65 Hz, J2 = 10.00 Hz, 1H, 2ꢀ 5-H
oxazine), 3.24 (d, J = 12.84 Hz, 1H, 6-Heq oxazine), 3.74 (dt,
J1 = 2.45 Hz, J2 = 12.70 Hz, 1H, 6-Hax oxazine), 6.74 (d, J = 3.43 Hz,
1H, 5-H thiazole), 6.94 (d, J = 8.56 Hz, 2H, 3,5-H phenyl), 7.22 (d,
J = 3.18 Hz, 1H, 4-H thiazole), 7.34 (d, J = 8.55 Hz, 2H, 2,6-H phe-
nyl), 9.58 (br s, 1H, NH). 13C NMR (DMSO-d6): d 43.54 (NCH3),
52.07 (5-C oxazine), 57.36 (6-C oxazine), 59.50 (3-C oxazine),
94.02 (2-C oxazine), 121.41 (5-C thiazole), 126.52 (3,5-C thiazole),
127.17 (2,6-C phenyl), 133.98 (4-C phenyl), 143.23 (1-C phenyl),
144.4 (4-C thiazole), 166.92 (2-C thiazole). Anal. (C14H17BrN2
O2Sꢁ2.75H2O), Calcd: C, 41.48; H, 4.99. Found: C, 41.33; H, 5.43.
C, 58.17; H, 5.15.
3.2.5.16.
azin-3-ol hydrobromide (16).
3-(2-Phenanthrenyl)octahydro-1,4-pyrido[2,1-c]ox-
Yield 50%, mp 205–207 °C. 1H
NMR (DMSO-d6): d 1.38–1.49 (m, 2H, 9-Hax and 8-Hax), 1.68–1.88
(m, 4H, 2ꢀ 7-H, 9-Heq and 8-Heq), 2.43 (s, 1H, OH), 2.93 (q,
J = 21.85 Hz, 1H, 6-Hax), 3.19-3.22 (m, 2H, 6-Heq and 4-Hax), 3.36-
3.46 (m, 1H, 9a-Hax), 3.52 (d, J = 11.94 Hz, 1H, 4-Heq), 3.95 (dd,
J1 = 3.11 Hz, J2 = 12.42 Hz, 1H, 1-Heq), 4.02–4.08 (m, 1H, 1-Hax),
7.57–7.68 (m, 3H, 1,7,8-H Ar), 7.81–7.88 (m, 2H, 4,6-H Ar), 7.95
(d, J = 7.60 Hz, 1H, 5-H Ar), 8.12 (s, 1H, 3-H Ar), 8.80 (d,
J = 8.02 Hz, 1H, 9-H Ar), 8.86 (d, J = 8.79 Hz, 1H, 10-H Ar), 9.82 (br
s, 1H, NH). Anal. (C22H24BrNO2), Calcd: C, 63.77; H, 5.84. Found:
C, 64.04; H, 5.82.
3.2.5.13. 3-[4-(2-Thiazolyl)phenyl]octahydro-1,4-pyrido[2,1-c]
oxazin-3-ol hydrobromide (13).
Yield 59%, mp 194.7–
3.2.5.17. 2-(2-Phenanthrenyl)-4-methyloctahydro-1,4-benzox-
azin-2-ol hydrobromide (17).
196.3 °C. 1H NMR (CDCl3 + DMSO-d6): d 1.33–1.49 (m, 2H, 8-Hax
and 9-Hax oxazine), 1.55 (d, J = 13.45 Hz, 2H, 2ꢀ 7-H oxazine),
1.67 (d, J = 13.20 Hz, 1H, 8-Heq oxazine), 2.75 (q, J = 9.66 Hz, 1H,
9-Heq oxazine), 2.90 (d, J = 9.90 Hz, 1H 6-Hax oxazine), 3.00 (s, 1H,
OH), 3.06 (d, J = 11.98 Hz, 1H, 6-Heq oxazine), 3.13–3.21 (m, 1H,
4-Hax oxazine), 3.61 (dd, J1 = 3.43 Hz, J2 = 12.72 Hz, 2H, 4-Heq and
9a-Hax oxazine), 4.00 (dd, J1 = 1.47 Hz, J2 = 11.13 Hz, 2H, 2ꢀ 1-H
oxazine), 7.22 (d, J = 3.18 Hz, 1H, 5-H thiazole), 7.46 (d,
J = 8.31 Hz, 2H, 3,5-H phenyl), 7.60 (d, J = 3.18 Hz, 1H, 4-H thia-
zole), 7.72 (d, J = 8.32 Hz, 2H, 2,6-H phenyl), 10.30 (br s, 1H, NH).
13C NMR (DMSO-d6): d 21.69 (7-C oxazine), 22.08 (8-C oxazine),
23.90 (6-C oxazine), 53.87 (9-C oxazine), 59.51 (2-C oxazine),
61.25 (10-C oxazine), 61.85 (5-C oxazine), 94.39 (3-C oxazine),
121.42 (5-C thiazole), 126.51 (3,5-C phenyl), 127.21 (2,6-C phenyl),
133.96 (4-C phenyl), 143.14 (1-C phenyl), 144.39 (4-C thiazole),
166.92 (2-C thiazole). Anal. (C17H21BrN2O2Sꢁ2H2O), Calcd: C,
46.75; H, 5.32. Found: C, 47.11; H, 5.73.
Yield 50%, mp 220–221 °C. 1H
NMR (DMSO-d6): d 1.16–1.52 (m, 5H, 2ꢀ 5-H, 2ꢀ 7-H and 8-Hax),
1.73–1.84 (m, 2H, 2ꢀ 6-H), 1.92 (d, J = 10.49 Hz, 1H, 8-Heq), 2.20–
2.23 (m, 1H, 4a-Hax), 2.72 (s, 3H, NCH3), 3.03–3.10 (m, 1H, 3-
Hax), 3.63 (d, J = 12.12 Hz, 1H, 3-Heq), 4.08 (dt, J1 = 3.88 Hz,
J2 = 10.67 Hz, 1H, 8 -Hax), 7.59–7.69 (m, 3H, 1,7,8-H Ar), 7.81–
a
7.88 (m, 2H, 4,6-H Ar), 7.95 (d, J = 7.60 Hz, 1H, 5-H Ar), 8.11 (s,
1H, 3-H Ar), 8.80 (d, J = 8.02 Hz, 1H, 9-H Ar), 8.86 (d, J = 8.79 Hz,
1H, 10-H Ar), 9.98 (br s, 1H, NH). Anal. (C23H26BrNO2ꢁ0.2 H2O),
Calcd: C, 63.88; H, 6.07. Found: C, 63.95; H, 6.08.
4. In vitro lipid peroxidation
Heat-inactivated hepatic microsomes from untreated rats were
prepared as described.11 The incubation mixture contained micro-
somal fraction (corresponding to 2.5 mg of hepatic protein per mL
or 4 mM fatty acid residues), ascorbic acid (0.2 mM) in Tris–HCl/
KCl buffer (50 mM/150 mM, pH 7.4) and the studied compounds
(10
l
M to 1 mM) dissolved in DMSO. The reaction was initiated
M), and the
3.2.5.14. 2-[4-(2-Thiazolyl)phenyl]-4-methyloctahydro-1,4-ben-
by addition of a freshly prepared FeSO4 solution (10
l
zoxazin-2-ol hydrobromide (14).
Yield 57%, mp 201.2–
203.3 °C. 1H NMR (CDCl3 + DMSO-d6): d 1.31 (m, 2H, 8-Hax and
5-Hax oxazine), 1.78–1.89 (m, 2H, 2ꢀ 7-H oxazine), 1.96 (d,
J = 12.22 Hz, 1H, 8-Heq oxazine), 2.17 (d, J = 11.25 Hz, 1H, 6-Hax
oxazine), 2.76 (d, J = 4.40 Hz, 3H, NCH3), 2.97–3.05 (m, 1H, 6-Heq
oxazine), 3.15 (d, J = 11.00 Hz, 1H, 3-Hax oxazine), 3.41 (d,
J = 11.74 Hz, 1H, 3-Heq oxazine), 4.17 (t, J = 10.46 Hz, 1H, 8a-Hax
oxazine), 7.44 (d, J = 3.18 Hz, 1H, 5-H thiazole), 7.66 (d,
J = 8.31 Hz, 2H, 3,5-H phenyl), 7.81 (d, J = 3.18 Hz, 1H, 4-H thia-
zole), 7.92 (d, J = 8.31 Hz, 2H, 2,6-H phenyl), 10.46 (br s, 1H, NH).
13C NMR (DMSO-d6): d 22.50 (7-C oxazine), 23.00 (5-C oxazine),
24.10 (6-C oxazine), 24.92 (8-C oxazine), 31.65 (NCH3), 61.68 (3-
C oxazine), 66.37 (10-C oxazine), 70.17 (9-C oxazine), 94.39 (2-C
oxazine), 121.42 (5-C thiazole), 126.51 (3,5-C phenyl), 126.86
(2,6-C phenyl), 133.96 (4-C phenyl), 143.14 (1-C phenyl), 144.40
(4-C thiazole), 166.92 (2-C thiazole). Anal. (C18H23BrN2 O2S), Calcd:
C, 52.56; H, 5.64. Found: C, 52.17; H, 5.65.
mixture was incubated at 37 °C for 45 min. Lipid peroxidation of
aliquots was assessed spectrophotometrically (535 against
600 nm) as TBAR. All compounds and solvents were found not to
interfere with the assay. Each assay was performed in duplicate,
and IC50 values represent the mean concentration of compounds
that inhibits the peroxidation of control microsomes by 50% after
45 min of incubation. All standard errors are within 10% of the
respective reported values.10–12,14b
5. In vitro squalene synthase activity assay
SQS activity was evaluated by determining the amount of [3H]
FPP converted to squalene as previously described.15 The assay
was performed in 1 mL of 50 mM phosphate buffer, pH 7.4, con-
taining 10 mM MgCl2, 0.5 mM NADPH, rat liver microsomes
(18 lg protein/mL), various concentrations of the test compounds

E. M. Ladopoulou et al. / Bioorg. Med. Chem. 23 (2015) 7015–7023
7023
dissolved in ethanol, and [3H] FPP (0.5
lM, 0.27 Ci/mmol) in a glass
Acknowledgement
screw-cap tube. All components except [3H] FPP were preincu-
bated for 10 min at 37 °C. The reaction was initiated by the addi-
tion of [3H] FPP. After 10 min at 37 °C, the reaction was
terminated by the addition of 1 mL 15% KOH in ethanol. The mix-
ture was incubated at 65 °C for 30 min, extracted with 2.5 mL pet-
roleum ether, which was subsequently washed with 1 mL distilled
water. Then 1.5 mL of the upper organic phase was counted with
2.5 mL of scintillation liquid using a Beckman scintillation counter.
Each assay was performed in duplicate, and IC50 values represent
the mean concentration of compounds that inhibits the activity
of the enzyme by 50%. All standard errors are within 10% of the
respective reported values.11,12,14b,15
The authors acknowledge the Hellenic Society of Atherosclero-
sis for financial support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.09.034.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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Autodock Tools were used in order to set up the docking runs.
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containing the co-crystalized ligand CP-320473. The docking
search space was defined as the lattice containing the co-crystal-
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was then used in order to generate the appropriate grid lattice files
for every candidate ligand to be used in the subsequent docking
runs.
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Algorithm search method. The protein structure for this procedure
was kept rigid, while the ligand (compound) structures were
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ters included: 300 binding modes as the initial algorithm popula-
tion and 250.000 as the maximum number of energy evaluations.
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case). Default docking parameters (e.g., random number generator,
energy outside the grid, number of retries, output format, cluster
analysis) were used.11a,29,30
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8. Protein determination
The protein content of microsomal fractions, used in lipid per-
oxidation and squalene synthase inhibitory assay, was determined
according to Lowry’s method.31
9. Statistical analysis
31. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193,
265.
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.