Novel benzoxazine and benzothiazine derivatives as multifunctional antihyperlipidemic agents

Article abstract of DOI:10.1021/jm200763k

Atherosclerosis is a multifactorial disease with several mechanisms participating in its manifestation. To address this disorder, we applied a strategy involving the design of a single chemical compound able to simultaneously modulate more than one target

Full text of DOI:10.1021/jm200763k

ARTICLE
pubs.acs.org/jmc
Novel Benzoxazine and Benzothiazine Derivatives as Multifunctional
Antihyperlipidemic Agents
Alexios N. Matralis, Maria G. Katselou, Anastasios Nikitakis, and Angeliki P. Kourounakis*
Department of Medicinal Chemistry, School of Pharmacy, University of Athens, 15771 Athens, Greece
ABSTRACT: Atherosclerosis is a multifactorial disease with
several mechanisms participating in its manifestation. To ad-
dress this disorder, we applied a strategy involving the design of
a single chemical compound able to simultaneously modulate
more than one target. We hereby present the development of
novel benzoxazine and benzothiazine derivatives that signifi-
cantly inhibit in vitro microsomal lipid peroxidation and LDL oxidation as well as squalene synthase activity (IC50 of 5ꢀ16 μM).
Further, these compounds show antidyslipidemic and antioxidant properties in vivo, decreasing total cholesterol, LDL, triglyceride,
and MDA levels of hyperlipidemic rats by 26ꢀ74%. Finally, by determination of their in vivo concentration (up to 24 h) in target
tissues (blood/liver), it is shown that compounds reach their targets in the low micromolar range. The new compounds seem to be
interesting multifunctional molecules for the development of a new pharmacophore for disease-modifying agents useful in the
treatment of atherosclerosis.
4-dihydro-2H-benzo[1,4]oxazin-2-ol] that incorporates struc-
tural features of antioxidants (i.e., Trolox and A) as well as anti-
hyperlipidemic (B) lead compounds8,10 (Scheme 1). Moreover,
compound 2 [2-(4-biphenyl)-4-methyl-3,4-dihydro-2H-benzo-
[1,4]thiazin-2-ol] was synthesized as an isostere of compound 1.
The effect of the 2-hydroxy group on the activity of compounds 1
and 2 was also investigated; thus, compounds 3 and 4 were syn-
thesized (Scheme 1). The pharmacological activity of the newly
synthesized compounds 1ꢀ4 was investigated in vitro for anti-
oxidant and SQS inhibitory activity, while intermediates 13, 14,
and 17 were evaluated in vitro to investigate the effect of the
3-carbonyl group on lipid peroxidation. Target compounds 1 and
2 were also evaluated in vivo (antioxidant and antidyslipidemic
activity, as well as determination of their time-dependent con-
centration in target tissues, i.e., blood/liver) and proven to be
interesting molecules with combined activities useful as potential
antiatherosclerotic agents.
1. INTRODUCTION
Atherosclerosis is a severe pathology that can be directly or
indirectly implicated in approximately 50% of all mortality in
Western countries. It is considered the main contributor to the
pathogenesis of myocardial and cerebral infarction, gangrene,
and loss of function of the extremities. Over the past years, evi-
dence has accumulated suggesting that free radicals, and the
consequent lipid peroxidation they induce, could trigger most of
the factors involved in atherosclerotic vascular injuries, such as
cytotoxicity, inflammation, and formation of atheromatic plaques.1
Oxidative modification of cholesterol-esterified lipids of lipopro-
teins (LDL) can lead to their uncontrolled uptake by macro-
phages. This event triggers a cascade of cellular processes that
lead to the formation of fatty streaks and eventually athero-
sclerotic lesions in the arterial wall.2ꢀ4
Currently, treatment of atherosclerosis is aimed at reducing
blood cholesterol and triglyceride content. The widespread clinical
use of the HMGCo-A reductase inhibitors (statins) is accom-
panied by potential dose-limiting hepato- and myotoxicity, which
may be the consequence of reduced levels of essential isopren-
oid precursors, the antioxidant ubiquinone, or dolichols.5 These
levels are not affected, however, by the inhibition of the down-
stream enzyme of this pathway, squalene synthase (SQS), an
alternative target for developing lipid-lowering agents.6
2. RESULTS AND DISCUSSION
2.1. Chemistry. The synthesis of compound 1 is rather
straightforward (Scheme 2). The reaction of 2-methylamino-
phenol (6) with 4-bromoacetylbiphenyl (7) produced com-
pound 1. Compound 2 was synthesized following the synthetic
procedure depicted in Scheme 2: ethyl (4-biphenyl)-R-bromoa-
cetate (10) reacted with 2-aminobenzenethiol (11) to give the
benzothiazine derivative 12, N-methylation of which afforded 13.
Oxidation of compound 13 with m-chloroperbenzoic acid in
chloroform gave a (unstable) sulfoxide (13a) that was rearranged
(via a Pummerer reaction) during isolation with silica gel column
As multiple mechanisms are involved in the development of
atherosclerosis (hyperlipidemia, oxidative stress, and inflamma-
tion), agents with at least two mechanisms of action may offer a
therapeutic benefitcompared tothose onlytargetinga single mech-
anism.7 We have previously developed 2-heteroaromatic8 and
polyaromatic9 morpholine derivatives that lower cholesterol and
triglyceride levels in vivo while combining in vitro antioxidant
and SQS inhibitory activity. Extending our efforts toward this
end, we designed compound 1 [2-(4-biphenyl)-4-methyl-3,
Received: June 14, 2011
Published: June 27, 2011
r
2011 American Chemical Society
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Scheme 1. Lead Structures and the Design of Target Molecules
Scheme 2. Synthetic Route for Compounds 1 and 2
chromatography to produce the 2-hydroxy compound 14. Re-
duction of 14 afforded the end product 2.
The synthesis of compounds 3 and 4 is shown in Scheme 3.
Reaction of methyl (4-biphenyl)-R-bromoacetate (15) with
2-aminophenol (5) in the presence of K2CO3 afforded the
benzoxazine derivative 16, N-methylation of which gave 17.
Reduction of 17 and 13 with calcium borohydride (Ca(BH4)2)
gave the end products 3 and 4, respectively.11
13C and 1H NMR spectra indicated that compounds 1 and 2
are in the hemiketal and thiohemiketal forms, respectively, in
solution, with no traceable amounts of the open keto alcohol or
ketothiol form. Further, as verified by our theoretical studies,
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Scheme 3. Synthetic Route for Compounds 3 and 4
and >1 mM,8 respectively. To the best of our knowledge, only a
few antioxidant compounds are reported with IC50 lower than
9 μM in this experimental protocol. The intermediate 3-
oxobenzoxa(or thia)zine derivatives 13, 14, and 17 did not exhibit
significant activity against lipid peroxidation. The altered con-
formation of the benzoxazine or benzothiazine ring in the
presence of the 3-carbonyl group of compounds 13, 14, and 17
may contribute to reduced access, retention, and interaction with
biological membranes, the site of lipid peroxidation. No apparent
correlation between antioxidant activity and lipophilicity seems to
apply among the tested compounds.
2.3. Effect on LDL Oxidation. Since LDL is considered the
major target site for antioxidants intended as antiatherosclerotic
agents, the effect of the more active derivative 1 was further
investigated on Cu2+-mediated oxidation of human LDL.13
Figure 2a depicts the activity of 5, 7.5, and 10 μM 1 on LDL
oxidation. Oxidation of LDL (56 μg/mL) was determined by
measuring conjugate diene formation at 234 nm for 300 min,
which represents the early phase peroxidation of LDL (Table 2).
As reported in Table 2, the extension of the lag time of oxidation
in the presence of antioxidant is interpreted as the resistance
capacity of LDL particles toward oxidation. Results show a typical
concentration-dependent effect of compound 1 on prolonging
the control lag time (125 min) to 168 min at 5 μM (34% increase)
and to 214 min at 7.5 μM (71% increase), decreasing the rate of
conjugate diene formation from 8.41 (nmol/min)/mg protein
(control) to 5.57 (nmol/min)/mg(34% decrease) at5 μM andto
3.14 (nmol/min)/mg (63% decrease) at 7.5 μM, respectively
(Figure 2). In the presence of 10 μM 1 the lag time was too
prolonged to be recorded, while there was an almost total
inhibition of LDL oxidation. Under the same experimental
conditions, the lead compound B increased the lag time by
65% and decreased the rate of conjugate diene formation by 7% at
10 μM, while probucol at 5 μM increased the control lag time by
22%.13 The above results render compound 1 a very strong
inhibitor of LDL oxidation, a property that may be due to the
incorporated structural similarity with known antioxidants (e.g.,
Trolox and A), favoring an increased interaction with LDL
particles.
Figure 1. Time course of lipid peroxidation as affected by various
concentrations of compounds 1 and 2.
their stable conformations include equatorial position of the
biphenyl substituent and axial position of the OH.
Compound 1 was stable at 4 °C for several weeks, after which
it appeared to degrade as verified by TLC.
2.2. In Vitro Effect on Lipid Peroxidation. The time course
of nonenzymatic lipid peroxidation, as affected by 1 and 2, is
represented in Figure 1, while IC50 values for 1ꢀ4 and inter-
mediates 13, 14, and 17 are reported in Table 1. The new benz-
oxazine derivatives 1 and 3 are very active antioxidants (IC50 of
9 and 12 μM, respectively), while the benzothiazine derivatives 2
and 4 have significant antioxidant activity (IC50 of 83 and 39 μM,
respectively) compared to lead compound B (IC50 = 450 μM).8
Under the same experimental conditions, Trolox, a known potent
antioxidant, and probucol exhibited IC50 values of 25 μM 12
2.4. Effect on Squalene Synthase. All compounds were
evaluated for the inhibition of rat microsomal SQS. The inhibitory
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Table 1. Antioxidant [Inhibition of Lipid Peroxidation (LP)], SQS Inhibitory, And Antihyperlipidemic Effects of the Investigated
Compounds and cLogP
IC50 (μM)
microsomal SQS
% decrease compared to hyperlipidemic controls (56 μmol/kg ip)
LDL/HDL
ratio
compd
X
Y
Z
inhibition of LP
inhibition
TC
LDL
TG
MDA
cLogP
1
2
3
4
OH
OH
H
O
S
CH2
CH2
CH2
CH2
CO
9
12.8
16.5
5.7
62a
58a
74b
26c
73a
38b
63a
31b
0.29b
0.45d
4.87
5.16
6.01
6.35
5.06
4.55
4.76
4.51
1.98
10.75
3.09
83
O
S
12
H
39
4.9
13
H
S
>1000
1000
422
450
14
OH
H
S
CO
17
O
CO
B
36
54a
75a
18b
51b
70a
18d
49a
0
11d
simvastatin
probucol
Trolox
>1000
24.8
a P < 0.005. b P < 0.05. c P < 0.1. d Not significant (P > 0.1) (Student’s t test).
B led to 3-fold higher squalene synthase inhibitory activity
(IC50 = 12.8 μMfor1compared to 36 μM8 for B) while the isosteric
replacement of oxygen with thio (compound 2) did not alter
significantly the activity of 1 (IC50 = 16.5 μM for 2). To our sur-
prise the non-hydroxyl derivatives 3 and 4 appeared to be even
better squalene synthase inhibitors with IC50 values of 5.7 and
4.9 μM, respectively. Although the latter derivatives are more
lipophilic and thus may interact more efficiently with the
hydrophobic cavity of the binding site of SQS, the hydroxyl
group of similar derivatives has been considered a moiety that
increases interactions via hydrogen bonding.9 In this case sig-
nificant correlation between IC50 (or pIC50) and cLogP was found
(r2 = 0.9, n = 4, P = 0.06). In order to visualize these differences, we
performed rigid docking studies of compounds 1ꢀ4 using the
available X-ray crystal structure of human SQS and Autodock 4.9
The best fit complexes of SQS with derivatives 1 and 2 (magenda
and light blue structures, Figure 3) revealed a hydrogen bond
between their respective 2-hydroxyl group and GLN 212 and
lipophilic interactions of their biphenyl moiety with the hydro-
phobic cavity15 of what is considered the binding site of SQS
(oriented “diagonally” from bottom left to top right of the
protein structure in Figure 3). The more lipophilic derivatives
3 and 4 (green and yellow structures, Figure 3), lacking the 2-OH
group, bind deeper in this cavity via lipophilic interactions. This
difference in binding mode may possibly explain the unexpected
activity of derivatives 3 and 4 compared to 1 and 2. In all cases,
binding of 1ꢀ4 appears to be somewhat deeper in the hydro-
phobic cavity of SQS compared to previously experimentally and
theoretically evaluated derivatives9 (e.g., compound B, blue
structure in Figure 3).
Figure 2. (a) Effect of compound 1 on the Cu2+-induced oxidation of
LDL. (b) Representative graph showing the activity of squalene synthase
as affected by various concentrations of compound 1.
effect on enzyme activity was measured according to the method
of Amin et al.14 Compounds 1ꢀ4 inhibited squalene synthase
activity significantly and dose-dependently (Table 1), as shown in
Figure 2b, which depicts the activity of compound 1. It was shown
that aromatic replacement of the cyclohexyl ring of reference
2.5. Effect on Dyslipidemia. Compounds 1 and 2 were
administered in a single ip dose of 56 μmol/kg (in order to
allow comparison with related compounds of previous
studies)8,9 to hyperlipidemic rats (after Triton WR 1339
administration), and their effect on total cholesterol, LDL
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Table 2. Effect of 1 on the Lag Time (min) and Rate of Conjugate Diene formation in Copper-Induced Lipid Peroxidation of LDL
ARTICLE
concentration
lag time
(min)
Δt
% increase lag
time
rate of conjugate diene formation (nmol/min)/mg
protein)
% decrease of rate of conjugate diene
formation
(μM)
(min)
0
125
168
214
a
8.4
5.6
3.1
5
43
89
34
71
34
63
7.5
10
100
100
a 100% inhibition of conjugate diene formation.
Figure 3. Binding mode of compounds 1 and 2 (magenta and light
blue), 3 and 4 (green and yellow), and B (blue) at the active site of SQS.
Hydrogen bonds between the respective 2-hydroxyl groups of 1 and 2
and the amide residual of GLN 212 are highlighted in green.
Figure 5. Concentrationꢀtime profile of compounds 1 and 2 in liver
and blood after a single ip administration of 56 μmol/kg (17.8 mg/kg for
1 and 18.7 mg/kg for 2) to mice. Values are the mean ((SEM) of four to
five animals per time point.
Figure 4. Effect of compounds 1 and 2 on MDA levels of hyperlipi-
demic rats. Statistical significance is as follows: /, P < 0.05; //, P < 0.005.
2.6. In Vivo Antioxidant Activity. Compound 1 reduced
malondialdehyde (MDA) content16 in the plasma of experimen-
tally induced hyperlipidemic rats by 63%, while the benzothia-
zine derivative 2 reduced it by 31% (Table 1 and Figure 4). The
increased in vitro antioxidant and in vivo antidyslypidemic
activity of 1 relative to 2 is also reflected in the greater in vivo
antioxidant activity of this compound.
2.7. Pharmacokinetic Studies. The time course of the blood
and liver concentrations of compounds 1 and 2 after ip injection
in mice is shown in Figure 5, while corresponding pharmacoki-
netic/analytical parameters of both compounds are presented
in Table 3. The HPLC method for 1 and 2 was linear in the
range 0.1ꢀ14 μg/mL (before extraction), and recovery over
the whole range was more than 95% for both liver and whole
blood samples. Intraperitoneal injection of 1 and 2 at equimolar
doses (56 μmol/kg in mice) showed an initial concentration of
2ꢀ5 μg/g in blood and 20ꢀ37 μg/g in liver. For both com-
pounds these levels decreased within approximately 2 h, after
which concentration levels (approximately 0.15ꢀ3 μg/g tissue)
cholesterol, LDL/HDL ratio, and triglyceride plasma levels
(mg/dL plasma) was investigated. Both compounds were
found to reduce the examined parameters by 26ꢀ74%
(Table 1). Under the same experimental conditions and at
the same dose, probucol and simvastatin reduced plasma total
cholesterol by 18 and 75%, LDL cholesterol by 18% and 70%,
and triglycerides by 11% and 0%, respectively. The lead
compound B decreased the above parameters by 54%, 51%,
and 49%, respectively.8 Compound 1 had a remarkable effect,
greater than the lead compound B, on all lipidemic indices,
comparable to that of simvastatin in total cholesterol and LDL-
cholesterol parameters and even greater on triglyceride levels
while the ratio LDL/HDL that is considered of interest
remained significantly lower (0.29 ( 0.06) compared to
control values (0.58 ( 0.02) (P < 0.05). Although compound
2 was somewhat less active than compound 1, both compounds
were more active than probucol, a known antioxidant molecule
that has been used as an antiatherosclerotic agent.
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Table 3. Pharmacokinetic/Analytical Parameters of 1 and 2
after a Single ip Administration of 56 μmol/kg (17.8 mg/kg
for 1 and 18.7 mg/kg for 2) to Mice
(21.5 Ci/mmol), NADPH, FPP, and BSA were purchased from Sigma-
Aldrich (Germany). For the in vivo experiments, Wistar male rats
(200ꢀ250 g) or SKH2 mice (26ꢀ29 g) were kept in a controlled
temperature room (22 ( 2 °C), having free access to chow and tap
water, under a 12 h light/dark cycle.
1
2
3.2. Synthesis. Melting points were determined with a digital
Electrothermal IA 9000 series apparatus and are uncorrected. 1H
NMR and 13C NMR spectra were recorded with a Br€ucker Avance
DRX 400 (400 MHz) and DPX 200 (200 MHz) spectrometer, res-
pectively. The mass spectrum of 1 was obtained on an API 2000 mass
spectrometer. Purity of tested compounds was established by elemental
analyses performed by the Service Central de Microanalyse, France
(analysis of C, H) and is g95%.
parameter
blood liver blood
liver
0
AUC(5 ꢀ24h) (μg h/g)
2.0
14.3
99.8
7.0
9.7
56.1
404.2
7.2
3
AUMC(5 ꢀ24h) a (μg h/g)
16.6
8.4
71.9
7.4
0
3
0
AUMC/AUC(5 ꢀ24h) (h)
concentration (μg/g) at t = 5 min
1.6
20.1
2.9
3.0
37.1
12.0
concentration (μg/g) at t = 0.5 h
0.4
1.4
0
0
AUC(5 ꢀ24h)(liver)/AUC(5 ꢀ24h)(blood)
7.1
5.8
2-(4-Biphenyl)-4-methyl-3,4-dihydro-2H-benzo[1,4]oxazin-2-ol (1).
To a solution of 2-hydroxyphenylmethylamine 6 (2.03 mmol) in dry
DMF were added NaHCO3 (2.13 mmol) and 4-bromoacetylbiphenyl 7
(2.23 mmol). After stirring at RT (4 h), EtOAc was added and the
mixture was filtered, washed with water and brine, dried, and concen-
trated in vacuum. The product was purified by silica gel flash chroma-
tography (PE/EtOAc, 5:1) to afford 1 as a brown oil (40%) stable for a
few weeks. 1H NMR (400 MHz, CDCl3) δ (ppm): 2.95 (s, 3H, NCH3),
3.07 (d, J = 11.15 Hz, 1H, 3-Hax benzoxazine), 3.15 (d, J = 10.96 Hz, 1H,
3-Heq benzoxazine), 4.33 (brs, 1H, ꢀOH), 6.80ꢀ6.83 (m, 2H, 5,7-H
benzoxazine), 6.92ꢀ6.98 (m, 2H, 6,8-H benzoxazine), 7.24ꢀ7.33 (m,
1H, biphenyl-H), 7.38ꢀ7.42 (t, J = 7.43 Hz, 2H, biphenyl-H), 7.56 (d,
J = 7.24 Hz, 2H, biphenyl-H), 7.60 (d, J = 8.22 Hz, 2H, biphenyl-H), 7.70
(d, J = 8.41 Hz, 2H, biphenyl-H). 13C NMR (200 MHz, CDCl3) δ
(ppm): 38.50, 59.28, 94.89, 112.95, 117.34, 120.37, 121.83, 126.49 (2C),
127.16 (2C), 127.21 (2C), 127.50, 128.81 (2C), 134.90, 138.90, 140.69,
141.79, 143.26. C21H19NO2, Mr = 318.14, ESI-MS (m/z): 318.29 (M +
LODb (μg/g)
LOQc (μg/g)
0.06
0.05
0.20
0.02
0.01
0.04
0.21
0.07
a Area under the (first) moment curve. b Estimated limit of detection.
c Estimated limit of quantification.
remained fairly stable up to 24 h, which did not allow for an
accurate extrapolation in order to determine the total AUC. The
pharmacokinetic parameters that were calculated based on
noncompartmental analysis17 are shown in Table 3. The liver
concentrations for both compounds were consistently higher than
their blood levels, a phenomenon that may favor their activity on
SQS. Although in vitro activities are not always directly corre-
lated with in vivo efficacies, it is shown here that both 1 and 2
reach their target sites (i.e., liver (for SQS activity) and blood (for
protection against LDL oxidation)) at low micromolar levels, a
range more or less “comparable” to their in vitro IC50 values.
2.8. Conclusion. We designed, synthesized, and evaluated
novel compounds 1ꢀ4 in order to develop more active com-
pounds aimed toward atherosclerosis. Several pharmacophore
moieties found in antioxidant and antidyslipidemic lead com-
pounds are intergrated in the structures of compounds 1 and 3,
while compounds 2 and 4 are derived from the isosteric replace-
ment of the oxygen of compounds 1 and 3 with sulfur. Com-
pound 1 is a powerful in vitro and in vivo inhibitor of lipid per-
oxidation and a potent antidyslipidemic agent, while compound
2 exhibited significant, although not higher than 1, antioxidant
and antidyslipidemic activity. The mechanism of the antidyslipi-
demic activity of these compounds is considered to proceed via
squalene synthase inhibition: all compounds inhibited the activ-
ity of this enzyme significantly. Further, we determined the sys-
temic exposure for compounds 1 and 2 during the time course of
the in vivo experiment, which was in the lower micromolar range
for both target tissues, blood and liver.
H)+. Elemental analysis (C, H, N): C21H19NO2 0.2H2O. Calculated C
3
78.48, H 6.10, N 4.36. Found C 78.58, H 6.09, N 4.36.
2-(4-Biphenyl)-4-methyl-3,4-dihydro-2H-benzo[1,4]thiazin-2-ol (2).
Benzothiazine derivative 14 (0.32 mmol) was dissolved in dry THF
under argon, and BH3 THF (1M, 4.7 mL) was added dropwise and
3
stirred at RT (24 h). Water was added and the mixture extracted with
CH2Cl2 and washed with water and saturated NaHCO3 solution. The
residue was purified by silica gel flash chromatography (PE/DCM, 1:1)
to afford 2 as a yellow semisolid (48%). 1H NMR (400 MHz, CDCl3) δ
(ppm): 3.05 (s, 3H, ꢀNCH3), 3.23 (d, J = 11.74 Hz, 1H, 3-H
benzothiazine), 3.56 (d, J = 11.94 Hz, 1H, 3-H benzothiazine), 4.04 (brs,
1H, ꢀOH), 6.09 (t, J = 7.34 Hz, 1H, 7-H benzothiazine), 6.88 (d, J = 8.61
Hz, 1H, 5-H benzothiazine), 7.06ꢀ7.07 (m, 2H, 6,8-H benzothiazine),
7.30ꢀ7.32 (m, 1H, biphenyl-H), 7.39 (t, J = 7.54 Hz, 2H, biphenyl-H),
7.54ꢀ7.59 (2d, 4H, biphenyl-H), 7.70 (d, J = 8.22 Hz, 2H, biphenyl-H).
13C NMR (200 MHz, CDCl3) δ (ppm): 40.77, 64.16, 82.06, 113.36,
119.89, 125.38, 126.38 (2C), 126.64, 127.16 (4C), 127.49, 128.81 (2C),
130.28, 139.59, 140.55, 141.23, 143.28. Elemental analysis (C, H):
C21H19NOS 0.5H2O. Calculated C 73.65, H 5.89. Found C 73.99,
Structurally diverse benzoxazine derivatives (but very different
from the compounds described here) have been shown to have anti-
oxidant10,18 or neuroprotective activity,19,20 although no benzothia-
zine derivative has been previously evaluated for antioxidant activity.
To our knowledge, this is the first time that 1,4-benzoxazine and 1,4-
benzothiazine structures are shown to combine, by design in one
structure, antioxidant, protective against LDL oxidation, and antidy-
slipidemic activity. Compounds 1ꢀ4 seem to be interesting multi-
functional molecules for the development of a new pharmacophore
for disease-modifying agents useful in the treatment of atherosclerosis.
3
H 6.02.
2-(4-Biphenyl)-4-methyl-3,4-dihydro-2H-1,4-benzoxazine (3).Ca(BH4)2
was prepared in situ by the addition of NaBH4 (2.90 mmol) to a suspension
of CaCl2 (1.24 mmol) in dry THF (5 mL) at 0 °C.11 Compound 17
(0.48 mmol) was added and the mixture refluxed for 30 h. The reaction
mixture was then treated with brine, extracted with CH2Cl2, dried, and con-
centrated in vacuum. The residue was purified by silica gel flash chromatog-
raphy (PE/DCM, 3:1) to afford 3 as a white solid (57%): mp = 112ꢀ
113.5 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 2.88 (s, 3H, ꢀNCH3),
3.20ꢀ3.27 (m, 1H, 3-H benzoxazine), 3.31ꢀ3.38 (m, 1H, 3-H benzoxazine),
5.21 (d, J = 8.32 Hz, 1H, 2-H benzoxazine), 6.65ꢀ6.71 (m, 2H, 5.7-H
benzoxazine), 6.84ꢀ6.89 (m, 2H, 6,8-H benzoxazine), 7.29ꢀ7.35 (m, 1H,
biphenyl-H), 7.38ꢀ7.45 (m, 4H, biphenyl-H), 7.50ꢀ7.58 (m, 4H, biphenyl-
H). 13C NMR (200 MHz, CDCl3) δ (ppm): 38.66, 55.97, 75.30, 112.52,
116.26, 118.39, 121.68, 126.81, 126.93, 127.13, 127.36, 128.71, 128.82,
3. EXPERIMENTAL SECTION
3.1. Materials. All commercially available chemicals are of
the appropriate purity and purchased from standard sources. [3H]FPP
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131.94, 135.87, 137.99, 138.48, 139.68, 140.06, 140.75, 144.78. Elemental
7-H benzoxazine), 7.33 (d, J = 8.32 Hz, 2H, biphenyl-H), 7.40ꢀ7.44
(m, 3H, biphenyl-H), 7.47 (d, J = 8.32 Hz, 2H, biphenyl-H). Elemental
analysis (C, H): C21H19NO 0.25H2O. Calculated C 82.45, H 6.27. Found C
3
82.07, H 6.21.
analysis (C, H): C21H17NOS 0.1CH2Cl2. Calculated C 73.94, H 5.09,
3
2-(4-Biphenyl)-4-methyl-3,4-dihydro-2H-1,4-benzothiazine (4). Com-
pound 13 (0.45 mmol) and Ca(BH4)2 were reacted under the
same conditions described for 3 to give 4 as a white solid (50%):
mp = 130.5ꢀ131.5 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 2.94 (s,
3H, ꢀNCH3), 3.55ꢀ3.63 (m, 2H, 2 ꢁ 3-H benzothiazine), 4.40ꢀ4.43
(2d, J = 4.60 Hz, 1H, 2-H benzothiazine), 6.65 (t, J = 7.44 Hz, 1H, 7-H
benzothiazine), 6.71 (d, J = 8.22 Hz, 1H, 5-H benzothiazine), 7.01 (t, J =
7.82 Hz, 1H, 6-H benzothiazine), 7.07 (d, J = 7.63 Hz, 1H, 8-H
benzothiazine), 7.27ꢀ7.31 (m, 1H, biphenyl-H), 7.37ꢀ7.40 (m, 4H,
biphenyl-H), 7.52 (d, J = 7.82 Hz, 4H, biphenyl-H). 13C NMR (200
MHz, CDCl3) δ (ppm): 40.17, 43.64, 58.56, 112.65, 118.05, 125.84,
126.96, 127.08(2C), 127.48 (4C), 128.34 (2C), 128.81 (2C), 138.53,
Found C 73.74, H 5.16.
2-(4-Biphenyl)-2-hydroxy-4-methyl-4H-benzo[1,4]thiazin-3-one (14).
A stirred solution of 2-(4-biphenyl)-4-methyl-2H-1,4-benzothiazin-3-
(4H)-one 13 (0.9 mmol) in dry CHCl3 was treated with m-chloroper-
benzoic acid, 77% (0.9 mmol), and the mixture was stirred for 3 h at room
temperature.26 The solution was washed with water and saturated NaH-
CO3 solution. The organic layer was dried and concentrated in vacuum.
The residue was purified by silica gel flash chromatography (n-hexane/
ethyl acetate, 8:1) to afford 14 as a yellow solid (64%): mp = 138.0ꢀ
140.5 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 3.57 (s, 3H, ꢀNCH3),
5.13 (brs, 1H, ꢀOH), 6.90ꢀ6.96 (m, 2H, 5,6-H benzothiazine), 7.11
(t, J = 7.83 Hz, 1H, 7-H benzothiazine), 7.23ꢀ7.26 (m, 2H, 8-H benzo-
thiazine and biphenyl-H), 7.30ꢀ7.37 (m, 4H, biphenyl-H), 7.41ꢀ7.44 (m,
140.62, 140.91, 143.96. Elemental analysis (C, H): C21H19NS 0.25H2O.
3
4H, biphenyl-H). Elemental Analysis (C, H): C21H17NO2S 0.5 hexane.
Calculated C 78.34, H 6.12. Found C 78.48, H 6.12.
3
2-Hydroxyphenylmethylamine (6)21. A solution of 2-aminophenol 5
(18 mmol) in dry DMF was treated with NaHCO3 (19 mmol) and CH3I
(22 mmol) and stirred at room temperature for 5 h. After the usual
workup the residue was purified by silica gel flash chromatography
(petroleum ether/ethyl acetate, 5:1) to afford 6 as a white solid (50%):
mp = 102.5ꢀ105 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 2.61 (s,
3H, CH3), 4.65 (brs, 1H, NH), 6.29ꢀ6.34 (m, 2H, 3,5-H Ar),
6.54ꢀ6.58 (m, 2H, 4,6-H Ar), 9.07 (s, 1H, ꢀOH).
Calculated C 73.81, H 6.21, Found C 73.62, H 6.30.
Methyl (4-Biphenyl)-R-bromoacetate (15)27. Compound 15 was
synthesized according to the literature.27 Yellow oil (96%). H NMR
1
(400 MHz, CDCl3) δ (ppm): 3.83 (s, 3H, ꢀOCH3), 5.42 (d, J =
2.56 Hz, 1H, ꢀCHBr), 7.38ꢀ7.51 (m, 3H, biphenyl-H), 7.54ꢀ7.66
(m, 6H, biphenyl-H).
2-(4-Biphenyl)-2H-1,4-benzoxazine-3(4H)-one (16). A mixture of
2-aminophenol 5 (5.30 mmol), methyl (4-biphenyl)-R-bromoacetate
15 (4.82 mmol), K2CO3 (5.78 mmol), and acetone (25 mL) was re-
fluxed for 2 h.27 The reaction mixture was diluted with water and
extracted with EtOAc. The organic phase was washed with water, dried,
and concentrated in vacuum, and cold CHCl3 was added to the residue.
The precipitated solid was triturated with acetone, filtered off, and dried
to afford 16 as a white solid (31%): mp = 250 °C (dec). 1H NMR (400
MHz, DMSO-d6) δ (ppm): 5.74 (s, 1H, 2-H benzoxazine), 6.28ꢀ6.55
(m, 1H, 6-H benzoxazine), 6.85ꢀ6.87 (m, 3H, 5,7,8-H benzoxazine),
6.93ꢀ6.95 (m, 1H, biphenyl-H), 7.38 (d, J = 7.34 Hz, 2H, biphenyl-H),
7.52ꢀ7.60 (m, 6H, biphenyl-H), 10.90 (s, 1H, NH).
Ethyl (4-Biphenyl)acetate (9)22. Compound 9 was synthesized ac-
cording to the procedure described previously.22 Colorless oil (89%). 1H
NMR (400 MHz, CDCl3) δ (ppm): 1.21 (t, J = 7.05 Hz, 3H, ꢀCH3),
3.60 (s, 2H, ꢀCH2CO), 4.12 (q, J = 7.05 Hz, 2H, OCH2), 7.26ꢀ7.31
(m, 3H, biphenyl-H), 7.36ꢀ7.39 (m, 2H, biphenyl-H), 7.49ꢀ7.54 (m,
4H, biphenyl-H).
Ethyl (4-Biphenyl)-R-bromoacetate (10)23. Compound 10 was syn-
thesized according to the procedure described previously.23 Dark yellow
oil (91%). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.25 (t, J = 7.14 Hz,
3H, ꢀCH3), 4.20 (q, J = 7.24 Hz, 2H, ꢀOCH2), 5.33 (s, 1H, ꢀCHBr),
7.29ꢀ7.34 (m, 1H, biphenyl-H), 7.37ꢀ7.42 (m, 2H, biphenyl-H),
7.51ꢀ7.58 (m, 6H, biphenyl-H).
2-(4-Biphenyl)-4-methyl-2H-1,4-benzoxazin-3(4H)-one (17). 2-(4-
Biphenyl)-2H-1,4-benzothiazin-3(4H)-one 16 (1.9 mmol) and methyl
iodide (2.8 mmol) were reacted under the same conditions25 described
2-(4-Biphenyl)-2H-1,4-benzothiazin-3(4H)-one (12). A solution of
ethyl (4-biphenyl)-R-bromoacetate 10 (2.7 mmol) and 2-aminobenze-
nethiol 11 (4.1 mmol) in dry DMF was stirred at room temperature for
25 h.24 The reaction mixture was then poured into iceꢀwater. The
precipitated solid was filtered off, triturated with acetonitrile, filtered off,
and dried to afford 12 as a white solid (70%): mp = 235ꢀ236.5 °C. 1H
NMR (400 MHz, CDCl3) δ (ppm): 4.68 (s, 1H, 2-H benzothiazine),
6.81 (d, J = 8.02 Hz, 1H, 8-H benzothiazine), 6.96 (dt, J1 = 1.17 Hz, J2 =
7.62 Hz, 1H, 7-H benzothiazine), 7.11 (dt, J1 = 1.18 Hz, J2 = 7.83 Hz, 1H,
6-H benzothiazine), 7.25ꢀ7.29 (m, 2H, 5-H benzothiazine and biphe-
nyl-H), 7.34 (d, J = 7.83 Hz, 2H, biphenyl-H), 7.38 (d, J = 8.22 Hz, 2H,
biphenyl-H), 7.45ꢀ7.48 (m, 4H, biphenyl-H), 8.55 (s, 1H, NH).
Elemental analysis (C, H): C20H15NOS. Calculated C 75.68, H 4.76,
Found C 75.35, H 4.71.
1
for 13 to give 17 as a yellow semisolid (80%): H NMR (400 MHz,
CDCl3) δ (ppm): 3.48 (s, 3H, -NCH3), 4.64 (s, 1H, 2-H benzothiazine),
6.96 (t, J = 7.58 Hz, 1H, 6-H benzothiazine), 7.02 (d, J = 8.07 Hz, 1H,
5-H benzothiazine), 7.17 (m, 1H, 7-H benzothiazine), 7.24ꢀ7.36 (m,
6H, 8-H benzothiazine and biphenyl-H), 7.42 (d, J = 8.07 Hz, 2H,
biphenyl-H), 7.46 (d, J = 7.58 Hz, 2H, biphenyl-H).
3.3. In Vitro Microsomal Lipid Peroxidation. Heat-inactivated
hepatic microsomes from untreated rats were prepared as described
previously. The inhibitory effect of compounds on lipid peroxidation
was assessed spectrophotometrically (535 against 600 nm) as TBAR
material.8
3.4. Isolation and in Vitro LDL Oxidation. Blood was collected
from a normolipidemic human volunteer, and LDL was isolated by
discontinuous density gradient (using KBr) ultracentrifugation.28
LDL (56 μg of protein/mL) in PBS was incubated at 37 °C in the
absence or presence of various concentrations of 1 (in 10% DMSOꢀ
H2O). Oxidation was initiated by 10 μM CuSO4. Conjugated dienes
were determined every 10 min for 5 h as the increase in absorbance at
234 nm and calculated using the extinction coefficient of 29 500.13
3.5. In Vitro Squalene Synthase Activity Assay. SQS activity
was evaluated by determining the amount of [3H]FPP converted to
squalene as previously described.9,14
2-(4-Biphenyl)-4-methyl-2H-1,4-benzothiazin-3(4H)-one (13). An
excess of KOH (4.1 mmol) was added to a solution of 2-(4-biphenyl)-
2H-1,4-benzoxazin-3(4H)-one 12 (1.1 mmol) in dry acetone.25 The
mixture was refluxed, while methyl iodide (1.7 mmol) in dry acetone was
added dropwise. The mixture was then stirred at 60 °C for 15 min.
Acetone was filtered, evaporated in vacuum, and diethyl ether was added.
The organic layer was washed with water and brine, dried, and concen-
trated in vacuum. The residue was purified by silica gel flash chroma-
tography (petroleum ether/dichloromethane, 2:3) to afford 13 as a
yellow solid (84%): mp = 170ꢀ171.5 °C. 1H NMR (400 MHz, CDCl3)
δ (ppm): 3.37 (s, 3H, ꢀNCH3), 5.69 (s, 1H, 2-H benzoxazine),
6.69ꢀ6.76 (m, 2H, 5,6-H benzoxazine), 6.89ꢀ6.92 (m, 1H, 8-H
benzoxazine), 6.96ꢀ6.99 (m, 2H, biphenyl-H), 7.01ꢀ7.04 (m, 1H,
3.6. Docking Studies. Preparation of ligands, protein crystal
structure (1EZF), and docking runs were performed with Spartan04
(Wavefunction Inc., Irvine, CA, 2004), Chimera, and Autodock
4/Autodock Tools29,30 and according to previous methodology.9
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3.7. In Vivo Evaluation of Antidyslipidemic and Antiox-
idant Activity. Triton WR 13398 was given ip to rats (200 mg/kg),
and 1 h later the test compounds (56 μmol/kg) were given. After 24 h,
blood was collected from the aorta and used for the determination of
lipidemic parameters, using commercially available kits, and MDA con-
tent, by measuring the complex of MDA with N-methyl-2-phenylindole
(586 nm).16 Values are the means from six to eight rats, while all
standard errors are within 10% of the respective values.
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’ AUTHOR INFORMATION
Corresponding Author
*Phone: +30-210 7274818. Fax: +30-210 7274747. E-mail:
angeliki@pharm.uoa.gr.
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The authors acknowledge the Hellenic Society of Athero-
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’ ABBREVIATIONS USED
SQS, squalene synthase; HMGCo-A, 3-hydroxy-3-methylglutar-
yl-CoA; FPP, farnesyl pyrophosphate; PSPP, presqualene pyro-
phosphate; RT, room temperature; EtOAc, ethyl acetate; PE,
petroleum ether; DCM, dichloromethane; BSA, bovine serum
albumin; NADPH, nicotinamide adenine dinucleotide phos-
phate; IC50, inhibitory concentration (for 50% of the reaction);
TC, total cholesterol; LDL-C, low-density lipoprotein cholester-
ol; HDL-C, high-density lipoprotein cholesterol; TG, triglycer-
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