Lipid-lowering (hetero)aromatic tetrahydro-1,4-oxazine derivatives with antioxidant and squalene synthase inhibitory activity

Article abstract of DOI:10.1021/jm800663w

A number of newly synthesized 2-[4-(hetero)aromatic]phenyl-2-hydroxy- tetrahydro-1,4-oxazine derivatives were found to inhibit lipid peroxidation (IC₅₀ of the most potent was 20 μM) as well as rat squalene synthase (IC₅₀ for most between 1-10 μM). Antidyslipidemic action was demonstrated in vivo: the most active compound decreased triglycerides, total cholesterol, and LDL-cholesterol of hyperlipidemic rats by 64, 67, and 82%, respectively, at 56 μmol/kg (ip). Most of the novel compounds are more active than the structurally related and reference biphenyl-morpholine, pointing to useful structural approaches for the design of antiatherosclerotic agents.

Full text of DOI:10.1021/jm800663w

J. Med. Chem. 2008, 51, 5861–5865
5861
Lipid-Lowering (Hetero)Aromatic Tetrahydro-1,4-Oxazine Derivatives with Antioxidant and
Squalene Synthase Inhibitory Activity
Angeliki P. Kourounakis,*,† Christos Charitos,‡ Eleni A. Rekka,§ and Panos N. Kourounakis§
Department of Pharmaceutical Chemistry, School of Pharmacy, UniVersity of Athens, 15771 Athens, Greece, ELPEN Pharmaceutical Co Inc.,
95 Marathonos AVenue, 19009 Pikermi, Greece, Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotelian UniVersity of
Thessaloniki, 54124 Thessaloniki, Greece
ReceiVed April 14, 2008
A number of newly synthesized 2-[4-(hetero)aromatic]phenyl-2-hydroxy-tetrahydro-1,4-oxazine derivatives
were found to inhibit lipid peroxidation (IC50 of the most potent was 20 µM) as well as rat squalene synthase
(IC50 for most between 1-10 µM). Antidyslipidemic action was demonstrated in vivo: the most active
compound decreased triglycerides, total cholesterol, and LDL-cholesterol of hyperlipidemic rats by 64, 67,
and 82%, respectively, at 56 µmol/kg (ip). Most of the novel compounds are more active than the structurally
related and reference biphenyl-morpholine, pointing to useful structural approaches for the design of
antiatherosclerotic agents.
thus isoprenoid formation is not affected. Furthermore, squalene
synthase inhibitors have been found to lower triglyceride levels
in addition to the decrease of plasma cholesterol.10 It has been
reported that several quinuclidine derivatives reduce cholesterol
and triglyceride levels, acting as squalene synthase inhibitors.10-13
We have previously shown that 2-biphenyl morpholine deriva-
tives lower cholesterol and triglyceride levels in vivo. These
compounds combined antidyslipidemic action with antioxidant
activity14 and nitric oxide releasing properties.15 The two most
active compounds of those studies were further pharmacologi-
cally characterized both in vitro16,17 and in vivo.18
1. Introduction
Atherosclerosis is recognized as a major pathologic condition
with serious social and economic consequences. Although at
present it is a problem of the western world, the World Health
Organization predicts an epidemic of atherosclerosis as develop-
ing countries adopt the western lifestyle.1 Atherosclerosis is
characterized by the accumulation of cholesterol, mainly LDL-
cholesterol in macrophages, leading to lesion formation in large-
and medium-size arteries.2,3 These lesions may develop into
atheromatic plaques, which are prone to rupture and cause
clinical events such as heart attack and stroke.4,5 Today, there
is evidence that atherosclerosis is a condition of elevated
oxidative stress in the vascular wall, expressed as lipid and
protein oxidation.6,7 Oxidation of LDL-cholesterol is an early
event in atherosclerosis, and oxidized LDL-cholesterol is
proatherogenic.8 Currently, treatment of atherosclerosis aims in
reducing blood cholesterol and triglyceride content. A temporary
interruption of cholesterol biosynthesis can lead to decreased
plasma LDL-cholesterol levels by removing LDL-cholesterol
from the circulation through upregulation of hepatic LDL
receptors. Inhibitors of cholesterol biosynthesis via inhibition
of HMGCo-Aa reductase are presently the most effective means
for blood LDL-cholesterol reduction.9 However, inhibition of
HMGCo-A reductase may block the formation of biologically
necessary isoprenoids. Squalene synthase inhibition seems to
offer a preferable alternative because squalene synthase-
catalyzed reductive dimerization of farnesyl pyrophosphate to
squalene is the first specific step in cholesterol biosynthesis,
In this investigation, we synthesized and investigated further
substituted 1,4-oxazine derivatives (compounds 2-22), which
meet the assumed structural requirements for antidyslipidemic,
antioxidant, as well as squalene synthase inhibitory activity. The
performed structural modifications involve mainly the 2-aryl
substitution of the 1,4-oxazine ring. This substituent appears to
be implicated in the antioxidant as well as the lipid lowering
ability of these compounds. Compound 1, which has been
studied earlier,14 was included as a reference.
2. Results and Discussion
2.1. Synthesis. The 2-hydroxy-2-aryl morpholine derivatives
(1-22) were formed through a spontaneous cyclization of a
hydroxyaminoketone intermediate to a hemiketal structure,
generally in very good yields. The structures of the synthesized
compounds, as well as their lipophilicity, calculated as ClogP
values by the method of Leo-Hansch,19 are shown in Table 1.
As shown by previous studies20,21 but also by theoretical studies
(unpublished results), the stable conformation of the final
compounds includes equatorial position of the biaryl system (and
axial position of the OH) and chair or chair-chair (trans)
conformation of the fused octahydro-oxazine ring systems.
Taking the above into consideration, each final product is
considered to be a racemic mixture of two enantiomers, defined
at the chiral center of position 2 or 3 (for compounds 4, 7, 10,
13, 16, 19, and 22), respectively.
* To whom correspondence should be addressed. Phone: +30-210
7274818. Fax: +30-210 7274747. E-mail: angeliki@pharm.uoa.gr.
†
Department of Pharmaceutical Chemistry, School of Pharmacy,
University of Athens.
‡
ELPEN Pharmaceutical Co Inc.
Department of Pharmaceutical Chemistry, School of Pharmacy,
§
Aristotelian University of Thessaloniki.
a Abbreviations: SQS, squalene synthase; HMGCo-A, 3-hydroxy-3-
methylglutaryl-CoA; FPP, farnesyl pyrophosphate; BSA, bovine serum
albumin; NADPH, nicotinamide adenine dinucleotide phosphate; IC50,
inhibitory concentration (for 50% of the reaction); TC, total cholesterol;
LDL-cholesterol, low-density lipoprotein cholesterol; TG, triglyceride;
DMSO, dimethyl sulfoxide; trolox, (S)-(-)-6-hydroxy-2,5,7,8-tetrameth-
ylchroman-2-carboxylic acid; probucol, 4,4′-[(1-methylethylidene)bis(thio)]-
bis[2,6-bis(1,1-dim ethylethyl)phenol]; TBAR, 2-thiobarbituric acid reactive
material.
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 2. Under the same
10.1021/jm800663w CCC: $40.75
2008 American Chemical Society
Published on Web 08/28/2008

5862 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Brief Articles
Table 1. Structure of the Examined Derivatives and Their Lipophilicity (ClogP)
compd
R1
R2
R3
H
R4
ClogP
1
2
3
4
5
6
7
8
CH3
CH3
CH3
CH2CH2CH2CH2
phenyl
4.72
2.94
4.37
3.74
3.84
5.27
4.64
3.23
4.66
4.03
3.22
4.65
4.02
3.79
5.23
4.59
3.94
5.37
4.74
3.22
4.65
4.02
H
2-thienyl
2-thienyl
2-thienyl
CH2CH2CH2CH2
CH2CH2CH2CH2
H
H
CH3
CH3
H
2-bromo-5-thienyl
2-bromo-5-thienyl
2-bromo-5-thienyl
2-benzothiazolyl
2-benzothiazolyl
2-benzothiazolyl
2-fluorophenyl
2-fluorophenyl
2-fluorophenyl
4-chlorophenyl
4-chlorophenyl
4-chlorophenyl
4-bromophenyl
4-bromophenyl
4-bromophenyl
4-fluorophenyl
4-fluorophenyl
4-fluorophenyl
CH2CH2CH2CH2
CH2CH2CH2CH2
H
H
CH3
CH3
H
9
CH2CH2CH2CH2
10
11
12
13
14
15
16
17
18
19
20
21
22
CH2CH2CH2CH2
H
H
CH3
CH3
H
CH2CH2CH2CH2
CH2CH2CH2CH2
H
H
CH3
CH3
H
CH2CH2CH2CH2
CH2CH2CH2CH2
CH3
H
H
H
CH3
CH2CH2CH2CH2
CH2CH2CH2CH2
CH3
H
H
H
CH3
CH2CH2CH2CH2
CH2CH2CH2CH2
H
Table 2. Antioxidant, SQS Inhibitory, and Antidyslipidemic Effects of
the Investigated Compounds
% decrease compared to
inhibition of lipid
hyperlipidemic controls
peroxidation IC50
inhibition of
compd
(µM)
SQS IC50 (µM) TG
TC
LDL-C
1
2
3
4
5
6
7
8
450
87
20
174
51
36
5
10
7
13
1
7
2
6
5
40
42
10
9
9
15
6
4
3
23
28
10
49a
38a
47a
29a
35a
26a
37a
44a
0
54a
48a
45a
43a
43a
35a
36a
46a
0
51a
61b
46a
46a
26
160
164
113
202
187
182
490
390
111
180
178
220
200
139
330
420
162
45a
35a
53a
24
9
10
11
12
13
14
15
16
17
18
19
20
21
22
46a
64c
25
55a
67b
18
41a
82c
47a
11
Figure 1. Representative graph of the time course of lipid peroxidation
as affected by various concentrations of compound 3.
15
5
is favored by resonance effects of the conjugated biaryl
substituent because we have found that 2-hydroxy-2-phenyl-4-
methyl morpholine and 2-hydroxy-2-t-butyl-4-methyl-morpho-
line had a weak (IC50 > 1 mM) or negligible effect on lipid
peroxidation, respectively (data not shown). Although, in
general, the lipophilic character of antioxidants contributes to
the offered inhibition of lipid peroxidation, because lipophilic
compounds may acquire an easy access to biological mem-
branes, it seems that lipophilicity, given as ClogP values, is
not a determining factor in this series of derivatives.
70c
41a
52a
51a
7
63b
11a
44a
47a
23
57b
29a
35a
36a
53b
6
17
17
18a
94c
95c
23
36a
33a
11
41a
27
a p < 0.05, b p < 0.01, c p < 0.001 (Student’s t test)
experimental conditions, trolox and probucol, known potent
antioxidants, exhibited IC50 values of 25 µM and >1 mM,
respectively. The time course of lipid peroxidation, as affected
by several concentrations of the most active compound 3, is
depicted in Figure 1. Most of the studied derivatives demon-
strated significant antioxidant activity, higher than that of the
reference compound 1. Thus, the performed structural modifica-
tions, primarily the addition of a thienyl group (compounds 2-7)
led consistently to a considerable improvement of their anti-
oxidant profile. This could be due to an extension of the
conjugated system and/or to the presence of the sulfur atom
that contributes to this action. It seems that antioxidant activity
2.3. Inhibition of Rat Microsomal Squalene Synthase.
Inhibition of the activity of squalene synthase from rat liver
microsomes by the test compounds expressed as IC50 values is
shown in Table 2. All compounds inhibited squalene synthase
activity significantly and dose-dependently as shown in Figure
2, which depicts the activity of the most active compound 6
and the activity of compound 20. Thienyl, benzothiazolyl, as
well as 4-bromophenyl substitution seem to improve the activity
in this series of derivatives. Closer examination of the set of
compounds (1, 12, 21, 15, 18), as well as (11, 20, 14, 17) and
(3, 6), indicates that halogen substitution in the distal aromatic
ring affects activity, which goes in parallel with the lipophilic

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5863
and in vivo activity. Other parameters such as in vivo distribu-
tion and pharmacokinetics may disrupt the relative in vivo-in
vitro order of activity of these compounds. Thus reduced
lipophilicity together with increased antioxidant activity appear
as more significant factors determining in vivo antihyperlipi-
demic action.
In conclusion, a new class of multiple acting molecules that
holds promise for the treatment of atherosclerosis seems to be
emerging. Together with recent studies, the observed structure-
activity relationships in this series of substituted 1,4-oxazine
derivatives are expected to contribute to the design of novel
molecules with improved activity profile that may be useful as
potential antiatherogenic agents.
Figure 2. Representative graph showing the activity of squalene
synthase as affected by various concentrations of compound 6 ([) and
20 (0).
3. Materials and Methods
character and resonance effect of the halogen. This observation
is in accordance with studies in a series of 3-hydroxy-3-biaryl
quinuclidines, demonstrating that the best squalene synthase
inhibitory activity was expressed by compounds with planar
biaryl substitution and, furthermore, that there is a directional
requirement for the rigid biaryl side chain.13 As far as the
mechanism of this inhibitory activity is concerned, it is
considered that the biaryl substituent may act as an isoster for
isoprenyl subunits of the farnesyl chain in cholesterol biosynthesis.
2.4. Antidyslipidemic Activity. The in vivo antidyslipidemic
activity of these derivatives is also demonstrated in Table 2.
Hyperlipidemia was successfully established 24 h after Triton
WR 1339 administration as shown by the increase in plasma
total cholesterol, LDL-cholesterol, and triglyceride levels that
was found to be 93%, 77%, and 84%, respectively, compared
to normal values. Almost all test compounds were found able
to reduce the examined parameters in the plasma of hyperlipi-
demic rats, with the exception of some derivatives, which had
a marginal or no effect on triglycerides (compounds 9, 13, 18,
and 19), on total cholesterol (compounds 9, 13, and 19) and on
LDL-cholesterol (compounds 19, 13, and 22). Under the same
experimental conditions, probucol and simvastatin (used as
antidyslipidemic drugs), at the same dose, reduced plasma total
cholesterol by 18 and 75%, LDL-C by 11 and 70%, and
triglycerides by 18 and 0%, respectively.
Most compounds had a similar or lower effect than 1 on
triglyceride concentration. However, four compounds, namely
11, 14, 21, and 22, were significantly more effective than the
reference compound. Compounds 11 and 14 were also more
active in total cholesterol reduction compared to most of the
other compounds, which presented activity similar to 1.
Furthermore, compounds 2, 11, and 14 were more effective than
1 in LDL-cholesterol reduction. The consistently increased effect
of derivatives 11 and 14 followed by 22, 21, and 2, concerning
the in vivo reduction of lipid parameters, indicates useful
combinations of structural features toward increased activity.
2-Bromo-substitution on the thienyl ring (compounds 5, 6,
7) resulted in an overall decrease in antidyslipidemic activity,
compared to 2, 3, and 4. This may indicate that an increase in
the bulk and/or a decrease in the electron density of the thienyl
group do not favor activity.
3.1. Materials. 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, Fischer-
344 male rats (180-220 g) were used. Animals were kept in a
controlled temperature room (22 ( 2 °C), having free access to
laboratory chow and tap water, under a 12 h light/dark cycle.
3.2. Synthesis. Melting points (mp) were determined with a
MEL-TEMPII (Laboratory Devices, USA) apparatus and are
uncorrected. 1H NMR spectra were obtained with a BRUKER AC-
200 MHz spectrometer. Elemental analyses were performed with
a Perkin-Elmer 2400 CHN analyzer (analysis of C, H, and N) or
by the Service Central de Microanalyse, France (analysis of C, H)
and are reported in Supporting Information.
3.2.1. General Procedure for the Preparation of the Final
Products. The final products (Table 1) were obtained by the
reaction of 3 mmol of 2-methylaminoethanol, trans-2-methylamino-
cyclohexanol,22 or 2-piperidinemethanol23 with 1.2 mmol of 2-(4-
bromoacetylphenyl)thiophene (compounds 2, 3, 4) or 2-bromo-5-
(4-bromoacetylphenyl)thiophene (compounds 5, 6, 7) or 2-(4-
bromoacetylphenyl)benzothiazole (compound 8, 9, 10) or 4-(2′-
fluorophenyl)-bromoacetophenone (compound 11, 12, 13) or 4-(4′-
chlorophenyl)-bromoacetophenone (compound 14, 15, 16) or 4-(4′-
bromophenyl)-bromoacetophenone (compound 17, 18, 19) or 4-(4′-
fluorophenyl)-bromoacetophenone (compound 20, 21, 22) in
anhydrous acetone (40 mL) at room temperature with stirring for
15 h. Acetone was then distilled off, ether was added to the residue,
the mixture was washed with saturated sodium chloride solution,
dried (K2CO3), and the products were isolated as hydrobromides.
2-[4-(2-Thienyl)phenyl]-4-methylmorpholin-2-ol hydrobro-
1
mide (2). Yield 30%, mp 175-177 °C. H NMR (CDCl3) δ 2.65
(s, 3H), 2.84-3.27 (m, 5H), 3.75 (dd, J1 ) 12.9 Hz, J2 ) 3.5 Hz,
1H), 4.30 (dt, J1 ) 12.9 Hz, J2 ) 3.3 Hz, 1H), 6.80-7.40 (m, 7H),
10.0 (bs, 1H). Anal. (C15H18BrNO2S·0.5 H2O) C, H, N.
2-[4-(2-Thienyl)phenyl]-4-methyl-octahydro-1,4-benzoxazin-
2-ol hydrobromide (3). Yield 87%, mp 76-78 °C. 1H NMR
(CDCl3) δ 1.29-2.1 (m, 9H), 2.74 (d, J ) 5.0 Hz, 1H), 2.81 (d, J
) 5.0 Hz, 3H), 2.96-3.04 (t, J ) 14.0 Hz, 1H), 3.61-3.66 (dd, J1
) 15.0 Hz, J2 ) 2.0 Hz, 1H), 4.50 (m, 1H), 7.05-7.30 (m, 5H),
7.65 (d,
(C19H24BrNO2S·2.2H2O) C, H, N.
J
)
8.0 Hz, 2H), 11.2 (bs, 1H). Anal.
3-[4-(2-Thienyl)phenyl]-octahydro-1,4-pyrido[2,1-c]oxazin-3-
ol hydrobromide (4). Yield 99%, mp 189-190 °C. Anal.
(C18H22BrNO2S·H2O) C, H.
The effect of almost all compounds on one lipid parameter
seems to correlate well with its effect on the other lipid
parameters measured, indicating a more or less consistent anti-
dyslipidemic activity. This activity seems also to be somewhat
related with reduced lipophilicity of the compounds of this
series. Although we estimate that the basic mode of antidys-
lipidemic action of these compounds is via inhibition of squalene
synthase, no direct correlation can be derived between in vitro
2-[4-(2-Bromo-5-thienyl)phenyl]-4-methylmorpholin-2-ol hy-
drobromide (5). Yield 57%, mp 168-170 °C. Anal.
(C15H17Br2NO2S·H2O) C, H, N.
2-[4-(2-Bromo-5-thienyl)phenyl]-4-methyl-octahydro-1,4-ben-
zoxazin-2-ol hydrobromide (6). Yield 90%, mp 113-115 °C. 1H
NMR (CDCl3) δ 1.20-2.28 (m, 8H), 2.75 (m, 2H), 2.81 (d, J )
5.0 Hz, 3H), 3.00 (d, J ) 14.0 Hz, 1H), 3.67 (d, J ) 14.0 Hz, 1H),
4.46-4.58 (m, 1H), 7.00-7.06 (m, 2H), 7.49-7.52 (d, J ) 8.0

5864 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Brief Articles
Hz, 2H), 7.64-7.67 (d, J ) 8.0 Hz, 2H), 11.5 (bs, 1H). Anal.
(C19H23Br2NO2S·H2O) C, H, N.
3H), 3.70 (m, 1H), 3.85-4.05 (m, 1H), 4.25 (t, J ) 11.6 Hz, 1H),
7.10-7.70 (m, 8H), 10.7 (bs, 1H). Anal. (C20H23BrFNO2) C, H.
3-[4-(2-Bromo-5-thienyl)phenyl]-octahydro-1,4-pyrido[2,1-
c]oxazin-3-ol hydrobromide (7). Yield 99%, mp 166-167 °C. 1H
NMR (CDCl3 with drops of DMSO-d6) δ 1.21-1.82 (m, 7H),
2.40-3.00 (m, 3H), 3.20-3.40 (m, 2H), 3.70-3.77 (dd, J1 ) 13.0
Hz, J2 ) 3.0 Hz, 1H), 4.26 (t, J ) 11.0 Hz, 1H), 6.88-6.96 (m,
2H), 7.38 (d, J ) 8.0 Hz, 2H), 7.65 (d, J ) 8.0 Hz, 2H), 11.05 (bs,
1H). Anal. (C18H21Br2NO2S) C, H.
3.2.2. Preparation of Intermediate and Starting Materials.
Synthesis and characterization of the following starting materials
and intermediate bromoacetylphenyl derivatives, trans-2-methyl-
amino-cyclohexanol,22 2-(4-acetylphenyl)thiophene (23),24,25 2-(4-
bromoacetylphenyl)thiophene (24), 2-bromo-5-(4-bromoacetylphe-
nyl)thiophene (25), 2-(acetylphenyl)benzothiazole (26),26 2-(4-
bromoacetylphenyl)benzothiazole (27),27 4-(2′-fluorophenyl)aceto-
phenone (28),28 4-(4′-fluorophenyl)acetophenone (29),29 4-(4′-
chlorophenyl)acetophenone (30),30 4-(2′-fluorophenyl)-bromoac-
etophenone (31),28 4-(4′-fluorophenyl)bromoacetophenone (32),
4-(4′-chlorophenyl)-bromoacetophenone (33),31 and 4-(4′-bro-
mophenyl)-bromoacetophenone (34)32 are reported in Supporting
Information.
2-[4-(2-Benzothiazolyl)phenyl]-4-methylmorpholin-2-ol hy-
1
drobromide (8). Yield 68%, mp 110-115 °C. H NMR (CDCl3)
δ 2.55 (s, 3H), 3.10-3.55 (m, 5H), 4.05 (dd, J1 ) 11.6 Hz, J2 )
3.2 Hz, 1H), 4.36 (dt, J1 ) 12.0 Hz, J2 ) 3.1 Hz, 1H), 7.40-8.20
(m, 8H), 9.8 (bs, 1H). Anal. (C18H19BrN2O2S) C, H.
2-[4-(2-Benzothiazolyl)phenyl]-4-methyl-octahydro-1,4-ben-
zoxazin-2-ol hydrobromide (9). Yield 90%, mp 90-91 °C. Anal.
(C22H25BrN2O2S·2H2O) C, H, N.
3.3. In Vitro Lipid Peroxidation. Heat-inactivated hepatic
microsomes from untreated rats were prepared as described.14 The
incubation mixture contained microsomal 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 µM to 1 mM) dissolved
in DMSO. The reaction was initiated by addition of a freshly
prepared FeSO4 solution (10 µM), and the mixture was incubated
at 37 °C for 45 min. Lipid peroxidation of aliquots was assessed
spectrophotometrically (535 against 600 nm) as TBAR.14 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.
3.4. In Vitro Squalene Synthase Activity Assay. SQS activity
was evaluated by determining the amount of [3H] FPP converted
to squalene as previously described.33,16 The assay was performed
in 1 mL of 50 mM phosphate buffer, pH 7.4, containing 10 mM
MgCl2, 0.5 mM NADPH, rat liver microsomes (18 µg protein/mL),
various concentrations of the test compounds dissolved in ethanol,
and [3H] FPP (0.5 µM, 0.27 Ci/mmol) in a glass screw-cap tube.
All components except [3H] FPP were preincubated for 10 min
(37 °C). The reaction was initiated by the addition of [3H] FPP.
After 10 min (37 °C), the reaction was terminated by the addition
of 1 mL 15% KOH in ethanol. The mixture was incubated at 65
°C for 30 min, extracted with 5 mL of petroleum ether, which was
subsequently washed with 2 mL distilled water. Then 1.5 mL of
the upper organic phase was counted with 3 mL of scintillation
liquid using a Beckman scintillation counter. Each assay was
performed in triplicate, 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.
2-[4-(2-Benzothiazolyl)phenyl]-octahydro-1,4-pyrido[2,1-c]ox-
1
azin-3-ol hydrobromide (10). Yield 23%, mp 215-217 °C. H
NMR (CDCl3) δ 1.20-2.20 (m, 7H), 2.40-3.00 (m, 2H), 3.30-3.60
(m, 2H), 3.90 (d, J ) 12.0, 1H), 4.21 (d, J ) 12.1, 1H), 4.60 (t, J
)
12.4, 1H), 7.10-8.20 (m, 8H), 11.4 (bs, 1H). Anal.
(C21H23BrN2O2S·0.6CH2Cl2) C, H, N.
2-[4-(2-Fluorophenyl)phenyl]-4-methyl-morphonyl-2-ol hy-
drobromide (11). Yield 40%, mp 135-136 °C. Anal.
(C17H19BrFNO2) C, H, N.
2-[4-(2-Fluorophenyl)phenyl]-4-methyl-octahydro-1,4-benzox-
azine-2-ol hydrobromide (12). Yield 89%, mp 105-106 °C. Anal.
(C21H25BrFNO2 ·0.5H2O) C, H, N.
3-[4-(2-Fluorophenyl)phenyl]-octahydro-1,4-pyrido[2,1-c]ox-
azine-3-ol hydrobromide (13). Yield 99%, mp 150-151 °C. Anal.
(C20H23BrFNO2 ·3H2O) C, H.
2-[4-(4-Chlorophenyl)phenyl]-4-methyl-morphonyl-2-ol hy-
drobromide (14). Yield 31%, mp 168-169 °C. 1H NMR (CDCl3
+ DMSO-d6) δ 2.28 (s, 3H), 2.50 (m, 1H), 2.84 (m, 4H), 3.45 (dd,
J1 ) 12.6 Hz, J2 ) 3.3 Hz, 1H), 3.90 (dt, J1 ) 12.1 Hz, J2 ) 2.9
Hz, 1H), 6.82-7.14 (m, 9H). Anal. (C17H19BrClNO2 ·0.3H2O) C,
H, N.
2-[4-(4-Chlorophenyl)phenyl]-4-methyl-octahydro-1,4-ben-
zoxazine-2-ol hydrobromide (15). Yield 79%, mp 141-142 °C.
1HNMR (CDCl3 + DMSO-d6) δ 1.20-1.50 (m, 4H), 1.70-1.85
(m, 3H), 2.05-2.10 (m, 1H), 2.60 (m, 1H), 2.75 (s, 3H), 2.87-3.08
(m, 2H), 3.45, (d, J ) 12.3 Hz, 1H), 4.26 (dt, J1 ) 11.0 Hz, J2 )
3.9 Hz, 1H),7.34-7.60 (m, 8H), 10.7 (bs, 1H). Anal.
(C21H25BrClNO2 ·0.8H2O) C, H, N.
3-[4-(4-Chlorophenyl)phenyl]-octahydro-1,4-pyrido[2,1-c]ox-
azine-3-ol hydrobromide (16). Yield 73%, mp 202-204 °C. Anal.
(C20H23BrClNO2 ·H2O) C, H, N.
2-[4-(4-Bromophenyl)phenyl]-4-methyl-morphonyl-2-ol hy-
drobromide (17). Yield 61%, mp 178.4-179 °C. Anal.
(C17H19Br2NO2 ·0.5H2O) C, H.
2-[4-(4-Bromophenyl)phenyl]-4-methyl-octahydro-1,4-ben-
zoxazine-2-ol hydrobromide (18). Yield 46%, mp 227-229 °C.
1H NMR (CDCl3 + DMSO-d6) δ 1.20-2.00 (m, 8H), 2.80 (s, 3H),
3.49 (m, 1H), 3.67 (m, 2H), 3.90, (m, 1H), 4.51 (m, 1H), 7.41-7.73
(m, 8H), 11.62 (bs, 1H). Anal. (C21H25Br2NO2) C, H.
3-[4-(4-Bromophenyl)phenyl]-octahydro-1,4-pyrido[2,1-c]ox-
azine-3-ol hydrobromide (19). Yield 93%, mp 196-198 °C
(decom 169 °C). 1H NMR (CDCl3 + DMSO-d6) δ 1.20-2.02 (m,
7H), 3.06-3.33 (m, 4H), 3.65 (m, 1H), 3.83 (d, J ) 12.3 Hz, 1H),
4.05 (t, J ) 12.5 Hz, 1H), 7.03-7.52 (m, 8H), 10.55 (bs, 1H).
Anal. (C20H23Br2NO2 ·0.5H2O) C, H.
3.5. In Vivo Evaluation of Antihypercholesterolemic and
Antihyperlipidemic Activity. An aqueous solution of Triton WR
1339 was given ip to rats (200 mg/kg), and 1 h later, the test
compounds (56 µmol/kg), dissolved in saline or saline only were
administered ip. After 24 h, blood was taken from the aorta and
used for the determination of plasma total cholesterol (TC), LDL-
cholesterol (LDL-C), and triglyceride (TG) levels, using com-
mercially available kits.15 Levels of plasma lipids were determined
in duplicate, while values presented are the mean from 8-10 rats
(per compound). All standard errors are within 10% of the respective
reported values.
Acknowledgment. Part of this work was supported by
ELPEN Pharm. Co. Inc, Greece.
2-[4-(4-Fluorophenyl)phenyl]-4-methyl-morphonyl-2-ol hy-
drobromide (20). Yield 40%, mp 120-122 °C. Anal.
(C17H19BrFNO2) C, H, N.
Supporting Information Available: Methods for preparation
of intermediate and starting materials, 1HNMR data for compounds
4, 5, 9, 11, 12, 13, 16, 17, 19, 20, and 21, as well as elemental
analysis data of all final and some intermediate compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
2-[4-(4-Fluorophenyl)phenyl]-4-methyl-octahydro-1,4-benzox-
azine-2-ol hydrobromide (21). Yield 41%, mp 140-142 °C. Anal.
(C21H25BrFNO2 ·0.5H2O) C, H, N.
3-[4-(4-Fluorophenyl)phenyl]-octahydro-1,4-pyrido[2,1-c]ox-
azine-3-ol hydrobromide (22). Yield 98%, mp 157-157.5 °C. 1H
NMR (CDCl3 + DMSO-d6) δ 1.40-2.27 (m, 8H), 3.20-3.55 (m,

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5865
synthase and lipid biosynthesis. Eur. J. Pharmacol. 2006, 535, 34–
42.
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