New multifunctional di- tert -butylphenoloctahydro(pyrido/benz)oxazine derivatives with antioxidant, antihyperlipidemic, and antidiabetic action

Article abstract of DOI:10.1021/jm400101e

Oxidative stress, inflammation, and hyperlipidemia are common factors involved in the pathophysiology of atherosclerosis and type 2 diabetes. We have previously developed multifunctional antidyslipidemic derivatives with antioxidant and antiatherogenic properties. We now report the design, synthesis, and evaluation of two such novel derivatives that incorporate a structural moiety of the antidiabetic agent succinobucol. The new compounds exhibited a much improved in vitro antioxidant and squalene synthase inhibitory activity (at lower micromolar concentrations) as well as a significant antihyperlipidemic effect, reducing plasma total cholesterol, triglycerides, and MDA by 65-90%. Compound 2 also indicated a good anti-inflammatory activity, decreasing edema by 44%, while it was further evaluated for its antidiabetic activity using a type 2 diabetes experimental mouse model. After 7 weeks of administration, it produced a significant antihyperglycemic and antihyperlipidemic activity. In conclusion, rational drug design led to a compound combining improved antioxidant, antidyslipidemic, and antidiabetic action that may serve as a potential therapeutic strategy in metabolic syndrome disorders.

Full text of DOI:10.1021/jm400101e

Article
pubs.acs.org/jmc
New Multifunctional Di-tert-butylphenoloctahydro(pyrido/benz)oxazine
Derivatives with Antioxidant, Antihyperlipidemic, and Antidiabetic
Action
Eleni Ladopoulou, Alexios N. Matralis, and Angeliki P. Kourounakis*
Department of Medicinal Chemistry, School of Pharmacy, University of Athens, 15771 Athens, Greece
ABSTRACT: Oxidative stress, inflammation, and hyperlipidemia
are common factors involved in the pathophysiology of
atherosclerosis and type 2 diabetes. We have previously
developed multifunctional antidyslipidemic derivatives with
antioxidant and antiatherogenic properties. We now report the
design, synthesis, and evaluation of two such novel derivatives
that incorporate a structural moiety of the antidiabetic agent
succinobucol. The new compounds exhibited a much improved in vitro antioxidant and squalene synthase inhibitory activity
(at lower micromolar concentrations) as well as a significant antihyperlipidemic effect, reducing plasma total cholesterol,
triglycerides, and MDA by 65−90%. Compound 2 also indicated a good anti-inflammatory activity, decreasing edema by 44%,
while it was further evaluated for its antidiabetic activity using a type 2 diabetes experimental mouse model. After 7 weeks of
administration, it produced a significant antihyperglycemic and antihyperlipidemic activity. In conclusion, rational drug design led
to a compound combining improved antioxidant, antidyslipidemic, and antidiabetic action that may serve as a potential therapeutic
strategy in metabolic syndrome disorders.
Two such derivatives that were designed to combine within
one structure antidyslipidemic, anti-inflammatory, and enhanced
antioxidant properties were synthesized and evaluated in vitro
and in vivo for their extended activity.
1. INTRODUCTION
Cardiovascular disease (CVD) is classified as the dominant cause
of mortality in Western populations. It is mainly characterized
by three pathological conditions: atherosclerosis, hypertension,
and heart failure. Metabolic syndrome is the name for a group
of concurring diseases or symptoms that raise the risk of
cardiovascular disease, including obesity, diabetes mellitus,
impaired glucose tolerance, insulin resistance, dyslipidemia (high
LDL cholesterol, low HDL cholesterol, and high triglyceride
levels in blood), and increased blood pressure. Ideally, drug
therapy must address the biological mechanisms that are common
to a number of the above diseases. Recent studies outline the role
of oxidative stress in combination with hyperlipidemia in the
pathogenesis and development of atherosclerosis as well as type 2
diabetes.1 In addition, inflammatory processes are involved in
the development of atherosclerosis, while continuous low grade
inflammation has been linked to obesity; abdominal fat tissue
appears to excrete pro-inflammatory factors that may affect other
tissues in the body as well.2
A single-targeted therapeutic approach in multifactorial
disorders such as those mentioned above is mostly considered
inadequate.3,4 On the basis of the underlying pathophysio-
logical processes, we have previously developed multifunctional
antidyslipidemic morpholine and octahydro(pyrido/benz)-
oxazine derivatives with antioxidant, anti-inflammatory, and
antiatherogenic properties.5−8 Here we incorporate in the
pharmacophore of our biphenyloctahydrobenzoxazine lead
(a, Chart 1) structural features of succinobucol, an antioxidant
2,6-di-tert-butyl-4-methylphenol derivative that was designed as
an antiatherosclerotic drug and proved to have antihypergly-
cemic activity.9
2. RESULTS
2.1. Chemistry. Both 3-hydroxy-3-aryloctahydropyridoxa-
zine derivative 1 and 2-hydroxy-2-aryloctahydrobenzoxazine 2
(Scheme 1) were formed via a spontaneous cyclization of the
relevant amino alcohol with the 4-bromoacetylbiphenyl derivative
b to the corresponding hemiketal structure in good yields
(∼80%). As verified by spectroscopic and theoretical studies, the
fused octahydro(pyrido/benz)oxazine ring systems of com-
pounds 1 and 2 adopt a chair−chair (trans) conformation.6,7
2.2. In Vitro Effect on Lipid Peroxidation. The effect of
the investigated derivatives on the nonenzymatic peroxidation
of hepatic microsomal membrane lipids after 45 min of
incubation, expressed as IC50 values, is shown in Figure 1. The
new derivatives 1 and 2 are very active antioxidants (IC50 of
3.8 and 5.9 μM, respectively) compared to lead compound a
(Chart 1) which inhibits the lipid peroxidation with an IC50 of
450 μM.6,7 Under the same experimental conditions, probucol
and 2,6-di-tert-butyl-4-methylphenol (BHT), known potent
antioxidants and structurally related to succinobucol, exhibited
IC50 values of >1 mM and 25 μM, respectively.
2.3. Effect on LDL Oxidation. Since oxidative modification
of LDL can trigger a cascade of cellular processes that lead
to the formation of fatty streaks and eventually atherosclerotic
Received: January 22, 2013
© XXXX American Chemical Society
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Chart 1. Design of Compounds 1 and 2 and Structures of Lead Compounds a and Succinobucol
Scheme 1. Synthetic Route Applied toward the Formation of Desired Products 1 and 2
lesions in the arterial wall, compounds 1 and 2 were also
examined for their antioxidant activities on human LDL
oxidation induced by cupric ions (CuSO4). The metal-ion-
dependent modification of LDL occurs primarily through lipid
peroxidation and subsequent derivatization of apolipoprotein B
lysine residues by reactive, lipid hydroperoxide-derived alde-
hydes.10 The susceptibility of LDL to oxidation (in the presence
or absence of our compounds) was assessed by determining
(a) the time before the oxidation products (conjugate dienes)
become detectable (lag time), (b) the rate of oxidation
(propagation time), and (c) the maximum amount of oxidation
products at 234 nm (Table 1). Figure 2 depicts the activity of
0.5 and 1 μM compounds 1 and 2 on LDL oxidation induced by
10 μM CuSO4. Oxidation was significantly and dose-dependently
inhibited by 0.5−1 μM compounds 1 and 2, as shown by an
increase in lag time (Table 1). Compound 1 prolonged the
control lag time (110 min) to 130 min at 0.5 μM (15% increase)
and to 220 min at 1 μM (100% increase). Compound 2 is shown
to be a more effective antioxidant than compound 1, prolonging the
control lag time (110 min) to 170 min at 0.5 μM (55% increase),
B
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Figure 1. Time course of lipid peroxidation of rat microsomal
membranes in the presence of various concentrations of compound 1
(a) and compound 2 (b).
Figure 2. Inhibition of LDL oxidation in the presence of 0.5 and 1 μM
compound 1 (a) and compound 2 (b).
while in the presence of 1 μM the lag time was too long to be
recorded (≫300 min, ≫100% increase). In addition, the
presence of 1 μM compound gave a remarkable decrease in
the rate of conjugate diene formation from 10.2 (nmol/min)/mg
protein (control) to 6.7 (nmol/min)/mg protein (34.3%
decrease) for 1 and 0 (nmol/min)/mg protein (100% decrease)
for 2 (Table 1). Under the same experimental conditions, the
lead compound a (Chart 1) increased the control 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%. The above results characterize
compounds 1 and 2 as very strong inhibitors of LDL oxidation.
2.4. Effect on Squalene Synthase. Both compounds 1
and 2 inhibited significantly and dose-dependently squalene
synthase activity. Inhibition of the activity of squalene synthase
from rat liver microsomes by compound 2 is shown in Figure 3.
IC50 values for 1, 2, and lead compound a are 12, 0.21, and
36 μM, respectively. It seems that the inhibitory potency
markedly increased (170-fold) by substituting the second
phenyl ring of the lead compound a with BHT’s structural
features. It is possible that this lipophilic moiety interacts more
efficiently with the hydrophobic cavity of the binding site of
SQS.
Figure 3. Representative graph showing the activity of squalene
synthase as affected by various concentrations of compound 2.
(rat) based on the reduction of experimentally induced elevated
plasma lipid and MDA levels. Experimental hyperlipi-
demia was successfully established 24 h after Triton WR 1339
administration (liberation of cholesterol from the liver induced
by Triton WR 1339 triggers the hepatic synthesis of
cholesterol),11 with an increase in plasma total cholesterol
and triglyceride levels of 260% and 580%, respectively, com-
pared to normal values (Figure 4). Compounds 1 and 2 were
2.5. In Vivo Antidyslipidemic and Antioxidant Activity.
The hypocholesterolemic/hypolipidemic as well as the anti-
oxidant activity of the new compounds was evaluated in vivo
Table 1. LDL Oxidation Parameters in Presence of 0.5 and 1 μM Compounds 1 and 2
lag phase
lag time
ta, min
% increase of lag
propagation phase, rate of oxidation,
(nmol dienes/min)/(mg LDL)
decomposition phase, amount of dienes,
(nmol dienes)/(mg LDL)
LDL sample
time ta
control
110
130
10.2
9.5
6.7
12.0
0
490.9
457.6
506.7
508.5
0
1 (0.5 μM)
1 (1.0 μM)
2 (0.5 μM)
2 (1.0 μM)
15
100
220
170
55
≫300
≫100
C
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Figure 5. Plasma MDA levels (nmol/mL) of experimentally induced
hyperlipidemic rats in the presence of compounds 1 and 2. Statistically
significant difference from control: (∗∗) P < 0.01.
evaluated by the method of carrageenan-induced paw edema
which is a nonspecific inflammation maintained by the release
of histamine and serotonin and later by prostaglandins.14,15
The examined compound was administered ip at a dose of
300 μmol/kg right after the injection of carrageenan. Compound
2 reduced the increase in edema by 44%. Naproxen, a well-
known nonsteroidal anti-iflammatory drug, has been reported
to give a 51% edema decrease,16 while BHT was inactive at the
same dose and experimental conditions. The results demonstrate
a significant anti-inflammatory activity of compound 2, compar-
able to that of naproxen at the same dose.
2.7. In Vivo Antidiabetic Activity. In the type 2 diabetes
experimental animal model used, insulin resistance was induced
by a high fat diet (HFD), in addition to multiple low doses of
streptozotocin (STZ) that produces, via β cell death, a mild
impairment of insulin secretion that is similar to that of the
later stage of type 2 diabetes.17,18 The most potent antioxidant
and antidyslipidemic compound 2 was evaluated for its activity
in this type 2 diabetes animal model.
Figure 4. Percent reduction of total cholesterol and triglyceride plasma
levels of experimentally induced hyperlipidemic rats after ip administra-
tion of 56 μmol/kg compounds 1 and 2. Statistically significant
difference from control: (∗∗∗) P < 0.005.
administered in a single ip dose of 56 μmol/kg (in order to
allow comparison with related compounds of previous studies)
to hyperlipidemic rats. Both compounds were found able to
reduce the examined parameters in the plasma of hyper-
lipidemic rats by 68−90%. Specifically, compound 1 decreased
both total cholesterol and triglyceride levels by 68%, while
compound 2 decreased the above lipid parameters by 90% and
76%, respectively (Figure 4). Under the same experimental
conditions and at the same molar dose, probucol and
simvastatin reduced plasma total cholesterol by 18% and
75% and triglycerides by 11% and 0%, respectively.6,7 Lead
compound a (2-biphenyloctahydrobenzoxazine, Chart 1)
decreased the above parameters by 54% and 49%, respectively.5
Both compounds show a greater effect on all lipidemic indices
compared to that of simvastatin, probucol, and compound a.
It appears from the results that the hypocholesterolemic/
hypolipidemic activity of probucol (which contains in its
structure two structural moieties of BHT) is low in this experi-
mental protocol. This finding agrees with the lack of effect of
the antioxidant BHT on plasma levels of total cholesterol and
triglyceride in rabbits12 and the modest reduction of plasma
total cholesterol and triglyceride levels, at the same dose of
56 μmol/kg, by 29% and 26%, respectively, in the Triton WR
1339 induced hyperlipidemia experimental model by 3,5-di-tert-
butyl-4-hydroxybenzoic acid (BHA), the benzoic acid derivative
of BHT.13 Thus, the incorporation of the BHT structure into
lead compound a (which showed very good hypolipidemic
activity) contributed to a significant synergistic hypocholester-
olemic/hypolipidemic effect. Furthermore, compounds 1 and 2
reduced malondialdehyde (MDA) content in the plasma of
experimentally induced hyperlipidemic rats by 63% and 64%,
respectively (Figure 5). Thus, the increased in vitro antioxidant
activity of these compounds is also confirmed in vivo.
2.7.1. Body Weight and Food Consumption. As shown in
Figure 6a, significantly higher body weights were observed
between the 14th and the 35th day for group B (HFD-STZ)
compared to group C (HFD-STZ and 2). After the 35th day
Figure 6. Body weight (a) in the obesity−diabetes type 2 mouse model
after treatment with saline (group B, control) or with compound 2
(group C) and (b) in group A (fed only HFD). Statistically significant
different values between the two groups: (∗) P < 0.05, (∗∗) P < 0.01,
and (∗∗∗) P < 0.005.
2.6. In Vivo Anti-Inflammatory Activity. The anti-
inflammatory activity of the most active compound 2 was
D
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and until the end of the experiment, no differences were
observed in body weight gain between the two groups because
of significant body weight loss of group B (reflecting a negative
effect of STZ on this group). Compound 2-treated rats showed
a healthier appearance compared to control group B, while no
signs of toxicity were observed. Furthermore, it seems that food
intake of 2-treated rats (group C) was similar or somewhat
larger (10%) than that of the control group (group B) (average
food consumption of 110 (mg/g BW)/day, results not shown).
Treatment with compound 2 preserved the average body
weight of mice at initial levels, as seen when compared also with
mice of group A (HFD without STZ) in which a statistically
significant increase of 23% in average body weight, between the
first and the 50th day, was observed (Figure 6b).
2.7.2. Blood Glucose Levels. Figure 7 depicts the time
course of glycemic values in nonfasting HFD-STZ mice after
Figure 8. Plasma levels of total cholesterol (TC), LDL cholesterol
(LDL), HDL cholesterol (HDL), trigelycerides (TG) and the ratio of
HDL/LDL in the type 2 diabetes mouse model after treatment with
(a) saline (group B) or (b) with compound 2 (group C). Inserted bar
graph: MDA levels of groups B and C on day 50.
of TC and LDL of group C were reduced by 51% and 80%
compared to control group B, while on day 50 the above
parameters were reduced by 70% and 95%, respectively.
Interestingly, the ratio HDL/LDL, which is considered of
importance, differed substantially between the two groups. The
HDL/LDL ratio significantly increased in group C compared
to control (group B) in the time course of the experiment.
Compound 2 also exhibited a strong antioxidant activity in vivo,
decreasing plasma MDA levels by ∼50% as measured at the end
of the experiment, i.e., day 50 (Figure 8, inserted bar graph).
Thus, the potent antioxidant and hypolipidemic effect shown
by compound 2 in the experimentally induced hyperlipidemic
rat model mentioned previously is also confirmed in this type 2
diabetes experimental animal model.
Figure 7. Blood glucose levels in the obesity−diabetes type 2 mouse
model after treatment with saline (group B, control) or with compound
2 (group C) as well as in group A (HFD). Statistically significant
difference between groups (or from day 0, inserted graph): (∗) P <
0.05, (∗∗) P < 0.01, (∗∗∗) P < 0.005.
treatment with compound 2 (group C) or saline (control,
group B). Glucose levels of mice in group A (HFD without
STZ) increased only marginally between the first and the
50th day (Figure 7, inserted bar graph). In contrast, after STZ
administration (group B) blood glucose levels progressively
increased, reaching above 400 mg/dL on the 50th day. Long-
term treatment of mice with compound 2 (group C) fully pre-
vented this marked, STZ-induced rise in blood glucose levels.
2.7.3. Plasma Lipid and MDA Levels. Since there is a strong
correlation among dyslipidemia, oxidative stress, and type 2
diabetes, plasma concentrations of total cholesterol (TC), LDL
cholesterol (LDL), HDL cholesterol (HDL), triglycerides
(TG), and MDA levels were measured on days 22, 43, and
50 (Figures 8). In the control group B, TC, HDL, and LDL
levels progressively increased during the time course of the
experiment while TG concentrations increased only up to
day 43. In contrast, treatment with compound 2 (group C)
significantly decreased TC and LDL levels while maintaining at
initial levels HDL and TG levels. Specifically, on day 43, levels
3. DISCUSSION
Vascular complications associated with type 2 diabetes confer
significant morbidity and mortality. Atherosclerosis develops
much earlier and progresses more rapidly in subjects with
diabetes. The clustering of cardiovascular risk factors associated
with type 2 diabetes is mainly responsible for accelerated
atherosclerotic disease. Dyslipidemia is considered a prominent
cardiovascular risk factor in treatment guidelines. Lipid-modifying
therapy has an important role in attenuating diabetes-related
macrovascular atherosclerotic disease, with statins being the
predominant choice for therapeutic intervention.1,19 However,
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apart from inhibiting HMG-CoA reductase, statins also suppress
the production of mevalonate, a precursor of several non-sterol
products that are vital for diverse cellular functions. Both known
major side effects of statins (hepatotoxicity and myotoxicity)
have been associated with this inhibition of synthesis of certain
non-sterol products.20−22 Furthermore, many clinical studies
indicate that use of intensive-dose statin therapy compared
with moderate-dose statin therapy is associated with a higher
incidence of new-onset diabetes, albeit with fewer major
cardiovascular events.23,24 Although a potential mechanism to
explain this phenomenon is currently lacking, it has been
suggested that statin-induced myopathy may be associated with
the development of muscle insulin resistance.25 Lead structure
a (Chart 1) is an inhibitor of squalene synthase, an enzyme that
catalyzes the first commited step in the de novo cholesterol
biosynthesis pathway. Squalene synthase inhibitors decrease
circulating LDL cholesterol via an increased expression of
hepatic LDL receptors, in a manner similar to statins. However,
they leave other non-sterol products of mevalonate metabolism
unaffected and therefore have at least one theoretical advantage
compared with statins, as safer hypocholesterolemic and anti-
atherosclerotic agents.22,26 The design of compounds 1 and 2
was based on the incorporation of a structural moiety of the
antiatherosclerotic and antidiabetic agent succinobucol (a
probucol derivative) into the structure of the antidyslipidemic
squalene synthase inhibitor, compound a (Chart 1). The BHT
moiety was selected from succinobucol’s structure because of
its potent antioxidant effect against lipid peroxidation. Com-
pounds 1 and 2 exhibited a very strong hypocholesterolemic/
hypolipidemic effect, decreasing plasma concentrations of total
cholesterol and triglycerides by 68−90% in hyperlipidemic rats
(Figure 4).
Reactive oxygen species (ROS) are implicated in the damage
of various cell types, including endothelial (in atherosclerosis)
and pancreatic β cells (in diabetes), acting by various
mechanisms, e.g., via lipid peroxidation.27,28 LDL oxidation is
a crucial event in the initiation and development of athero-
sclerosis leading to abnormalities in endothelium function and
the formation of atherosclerotic lesions. Moreover, oxidative
stress, among others, is proposed to be responsible for the
progressive loss of β cells due to the particularly high sensitivity
of these cells to excessive ROS.29,30 Compounds 1 and 2 were
shown to be potent inhibitors of in vitro lipid peroxidation
at very low concentrations (IC50 values of 1 and 2 are 3.8 and
5.9 μM, respectively, compared to reference compounds a
and BHT with IC50 values of 450 and 25 μM, respectively).
The lipophilic character of compounds 1 and 2 (ClogP values
of 6.67 and 7.30, respectively) may facilitate their presence
in biological membranes, adding to the strong antioxidant
properties of the di-tert-butylphenol moiety and contributing to
the enhanced protection against lipid peroxidation. Further-
more, both 1 and 2 provide an effective protection against
LDL oxidation in vitro, prolonging the initiation of LDL lipid
peroxidation and significantly decreasing the rate of conjugate
diene formation at 0.5 and 1 μM. The incorporation of the
antioxidant and lipophilic structure of BHT in the lead com-
pound a (Chart 1) may also favor a better interaction of the new
compounds (i.e., compared to compound a and probucol) with
LDL particles and a more efficient protection from oxidation.
The enhanced antioxidant activity of the new compounds
is also exhibited in vivo, as they decrease plasma MDA levels in
hyperlipidemic rats by ∼65%. This activity of compound 2 is
furthermore accompanied by a significant anti-inflammatory
activity in vivo, reducing carrageenan-induced edema by 44%.
Succinobucol (or AGI-1067, Chart 1), the monosuccinic acid
ester of probucol, was designed to retain probucol’s antioxidant
properties with reduced adverse effects that were attributed to
its spiroquinone metabolite. Unlike probucol, AGI-1067 inhibits
human aortic smooth muscle cell proliferation and the
progression of atherosclerosis, lowering LDL levels (in animals)
while maintaining (or even elevating) HDL levels. The potential
mechanism of action of succinobucol on glycemic improvement
is uncertain. It has been hypothesized that its antioxidant and
anti-inflammatory actions may reduce tissue damage and
inflammation from oxidative stress and free-radical accumulation
that are implicated in pancreatic β-cell destruction, insulin
resistance, and subsequent development of diabetes.9,31−33
Taking into consideration the promising antioxidant,
hypolipidemic, and anti-inflammatory activities of compound 2,
it was evaluated for its antidiabetic effect using a mouse
model of type 2 diabetes with reduced β-cell mass, obtained
by the combined administration of multiple low doses of
streptozotocin (STZ) and high fat diet (HFD).17,18 HFD
increased significantly the body weight in mice of group A
(23% increase of average body weight at 50 days) , as well as in
control mice (group B) until the 23th day (19% increase). The
subsequent administration of streptozotocin in control mice
(group B) at days 23−27 led to the progressive loss of body
weight to initial levels on day 50 (Figure 6a). However,
administration of compound 2 (group C) maintained body
weight (during the time course of the experiment) without
affecting the feeding pattern of the animals. Furthermore, at the
end of the experiment, accumulation of visceral fat mass in
control mice (group B) was macroscopically observed despite
the restoration (from day 21 to day 50) of the average body
weight at initial levels. In contrast, no such fat accumulation
was observed in mice treated with compound 2 (group C). The
above-mentioned differences may be attributed to the potential
effect of compound 2 on lipid metabolism or a different lipolytic
pathway. Noteworthy, obesity has recently been found to be
characterized by a low grade inflammatory process involving
stimulated adipocytes, macrophages, and other regulatory
immune cells, resulting in the excretion of proinflammatory
compounds from the fat tissue.2 An additional effect observed in
such activated fat tissues is a reduced insulin sensitivity of the
fat cells, contributing to diabetes type 2. Several compounds
with anti-inflammatory action have been shown to reduce
obesity in animal models.34 The apparent “antiobesity” activity of
compound 2 is accompanied by a very strong antihyperglycemic
effect, conserving glucose levels of group C at physiological levels
in relation to the increased glucose levels of control group B
(Figure 7). Thus, administration of compound 2 inhibited the
pathological increase of glucose levels and forecoming diabetes
compared to control group (group B). Further, the potent
antioxidant and hypolipidemic activity of compound 2 was also
confirmed in this experimental animal model of type 2 diabetes.
The marked antidiabetic activity of compound 2 is possibly
explained by the attenuation of prolonged oxidative-stress-
induced tissue damage and inflammation as well as the protection
of pancreatic β cells against oxidative stress. The molecular
mechanism of its antihypercholesterolemic/antihyperlipidemic
activity is considered to be via squalene synthase inhibition.
In conclusion, in the present study a multitarget-directed
approach led us to rationally design compounds 1 and 2, which
are characterized by a unique multimodal profile. In particular,
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2 × 7-H), 1.92−2.01 (m, 2H, 8-Heq, 9-Heq), 2.56−2.62 (dt, J1 = 13.45
Hz, J2 = 2.11 Hz, 1H, 6-Hax), 2.79−2.83 (1H, d, J = 12.72 Hz, 4-Hax),
3.17 (m, 1H, 9a-Hax), 3.31−3.33 (m, 1H, 6-Heq), 3.44 (d, J = 12.71 Hz,
1H, 4-Heq), 3.78 (dd, J2 = 13.40 Hz, J1 = 3.42 Hz, 1H, 1-Heq), 4.39−
4.46 (dd, J1 = 13.30 Hz, J2 = 11.01 Hz, 1H, 1-Hax), 5.17 (s, 1H, 2-OH),
6.71 (s, 1H, 4′-OH), 7.24 (s, 2H, 2′-H and 6′-H biphenyl), 7.55 (d, J =
1.96 Hz, 2H, 3-H and 5-H biphenyl), 7.57 (d, J = 1.86 Hz, 2H, 2-H
and 6-H biphenyl), 11.38 (brs, 1H, -NH). 13C NMR (200 MHz,
CDCl3) δ (ppm): 22.07 (6-C), 22.36 (7-C), 24.14 (8-C), 30.30 (6 C,
6 × -C(CH3)3), 34.45 (2 C, 2 × -C(CH3)3), 55.23 (5-C), 61.25 (1-C),
61.43 (4-C), 62.70 (8α-C), 94.53 (3-C), 124.00 (2′,6′-C biphenyl),
126.23 (2,6-C biphenyl), 126.96 (3,5-C biphenyl), 131.65 (1′-C
biphenyl), 136.31 (3′,5′-C biphenyl), 137.95 (4-C biphenyl), 143.18
(1-C biphenyl), 153.81 (4′-C biphenyl). C28H39NO3, ESI-MS (m/z)
(M + H)+, calculated: 438.3. Found: 438.2. Anal. Calcd for C28H40BrNO3
(%): C, 64.86; H, 7.78. Found (%): C, 65.09; H, 7.84.
compound 2 was able (a) to potently inhibit in vitro lipid
peroxidation and LDL oxidation, (b) to inhibit in vitro squalene
synthase activity in the submicromolar scale, (c) to decrease
in vivo lipidemic parameters and oxidative stress, (d) to reduce
in vivo the increase in edema induced by carrageenan, and (e) to
suppress the onset and progress of diabetes mellitus with a
potential antiobesity effect as well. To the best of our knowledge,
this is the first time that a squalene synthase inhibitor combines
simultaneously hypolipidemic, antioxidant, and antidiabetic
activity. The very potent and appropriately balanced activities
of the new compounds render them interesting multifunctional
molecules useful in the treatment of metabolic syndrome
disorders.
4. EXPERIMENTAL SECTION
2-(3′,5′-Di-tert-butyl-4′hydroxy-4-biphenyl)-4-methylocta-
hydro-1,4-benzoxazin-2-ol Hydrobromide (2). Yield 80%, mp =
Materials. All commercially available chemicals are of the appropriate
purity and purchased from standard sources. For the in vivo experiments,
Wistar male rats (200−250 g) and SKH-2 male mice were used. Animals
were kept in a temperature controlled room (22 2 °C), having free
access to laboratory chow and tap water, under a 12 h light/dark cycle.
Synthesis. Melting points (mp) were determined with a digital
Electrothermal IA 9000 series apparatus and are uncorrected. 1H NMR
and 13C NMR spectra were recorded with a Bruker Avance DRX 400
(400 MHz) and DPX 200 (200 MHz) spectrometer, respectively.
Mass spectra were obtained on a 4000 QTRAP MS/MS spectrometer.
Purity of tested compounds was established by elemental analyses
performed by the Service Central de Microanalyse, France (analysis of
C, H) and is ≥95%.
4-Acetyl-3′,5′-di-tert-butyl-4′-hydroxybiphenyl (a). To a
stirring solution of 4-acetylphenylboronic acid (6.2 mmol, 1.0 g) in
25 mL of toluene were added a saturated aqueous solution of Na2CO3
(12.5 mL), a solution of 4-bromo-2,6-bis-tert-butylphenol (6.78 mmol,
1.9 g) in 12 mL of absolute ethanol, and 0.12 mmol (0.14 g) of
Pd(PPh3)4, followed by 4 h of reflux in the presence of argon. After
the reaction mixture reached room temperature, ice was added and
was extracted with ethyl acetate. The organic layer was washed with
1 N HCl, water, and saturated NaCl aqueous solution and dried over
Na2SO4. The mixture was separated via column chromatography
(flash) (petroleum ether/dichloromethane, 3/1). Yield 66%, mp =
143.5−146.0 °C.35 1H NMR (400 MHz, CDCl3) δ (ppm): 1.52 (s,
18H, 2 × -C(CH3)3), 2.65 (s, 3H, -CH3), 5.39 (s, 1H, -OH), 7.46 (s,
2H, 2′-H and 6′-H biphenyl), 7.65 (d, J = 8.54 Hz, 2H, 2-H and 6-H
biphenyl), 8.03 (d, J = 8.54 Hz, 2H, 3-H and 5-H biphenyl).
4-Bromoacetyl-3′,5′-di-tert-butyl-4′-hydroxybiphenyl (b).
To a stirring solution of 4-acetyl-3′,5′-di-tert-butyl-4′-hydroxybiphenyl
(1.85 mmol, 0.6 g) in 10 mL of anhydrous chloroform was added
dropwise a solution of Br2 (2.03 mmol) in 10 mL anhydrous
chloroform. After 2 h of being stirred, the reaction mixture was washed
with water, 5% NaHCO3, water and dried over Na2SO4. The mixture
was separated via column chromatography (flash) (petroleum ether/
dichloromethane, 3/1). Yield 68%, mp = 144.5−145.5 °C.36 1H NMR
(400 MHz, CDCl3) δ (ppm): 1.50 (18H, s, 2 × -C(CH3)3), 4.49 (2H,
s, -CH2), 5.41 (1H, s, -OH), 7.31 (2H, s, 2′-H and 6′-H biphenyl),
7.71 (2H, d, J = 8.56 Hz, 2-H and 6-H biphenyl), 8.10 (2H, d, J = 8.56
Hz, 3-H and 5-H biphenyl).
1
163.5−164.0 °C. H NMR (400 MHz, CDCl3) δ (ppm): 1.32−1.54
(m, 20H, 2 × 7-H and 2 × -C(CH3)3), 1.71−2.15 (5H, m, 5-Hax,
8-Hax, 2 × 6-H, 8-Heq), 2.20−2.30 (m, 1H, 5-Heq), 2.82 (s, 3H, N-CH3),
3.01 (m, 2H, 3-Hax and 4α-Hax), 3.67 (d, J = 12.63 Hz, 1H, 3-Heq), 4.52
(m, 1H, 8α-Hax), 5.30 (s, 1H, 2-OH), 6.29 (s, 1H, 4′-OH), 7.37 (s, 2H,
2′-H and 6′-H biphenyl), 7.55 (d, J = 8.19 Hz, 2H, 3-H and 5-H
biphenyl), 7.69 (d, J = 7.85 Hz, 2H, 2-H and 6-H biphenyl), 11.62
1
(brs, 1H, -NH). H NMR (400 MHz, DMSO-d6) δ (ppm): 1.12−1.47
(m, 4H, 8-Hax, 2 × 7-H, 5-Hax), 1.42 (s, 18H, 2 × -C(CH3)3), 1.75−1.93
(m, 3H, 2 × 6-H, 8-Heq), 2.22−2.24 (bd, J = 10.93 Hz, 1H, 5-Heq), 2.75
(s, 3H, -NCH3), 3.05−3.07 (m, 1H, 4a-Hax), 3.21 (t, J = 10.92 Hz, 1H,
3-Hax), 3.57 (d, J = 12.29 Hz, 1H, 3-Heq), 4.08 (t, J = 10.24 Hz, 1H,
8a-Hax), 7.13 (s, 1H, 2-OH), 7.31 (s, 2H, 2′-H and 6′-H biphenyl), 7.42
(s, 1H, 4′-OH), 7.60 (s, 4H, 2-H and 6-H, 3-H and 5-H biphenyl),
9.93 (brs, 1H, -NH). 13C NMR (200 MHz, DMSO-d6) δ (ppm): 23.82
(6,7-C), 24.50 (5-C), 24.71 (8-C), 30.77 (6 C, 6 × -C(CH3)3), 31.07
(2 C, 2 × -C(CH3)3), 35.07 (N-CH3), 61.43 (3-C), 66.26 (4α-C), 70.28
(8α-C), 94.14 (2-C), 123.64 (2,6-C biphenyl), 126.67 (3, 5, 2′, 6′-C
biphenyl), 131.69 (1′-C biphenyl), 139.42 (1-C biphenyl), 140.07 (3′,5′-
C biphenyl), 142.62 (4-C biphenyl), 154.40 (4′-C biphenyl).
C29H41NO3, ESI-MS (m/z) (M + H)+, calculated: 452.3. Found:
452.2. Anal. Calcd for C29H42BrNO3·1.5H2O (%): C, 62.23; H, 8.12.
Found (%): C, 62.20; H, 7.87.
In Vitro Lipid Peroxidation. Heat-inactivated hepatic microsomes
from untreated rats were prepared as described.5 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 (50−1 μM) 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.7 Lipid peroxida-
tion of aliquots was assessed spectrophotometrically (535 nm against
600 nm) as TBAR. Both 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.
Isolation and in Vitro LDL Oxidation. Blood was collected
from a normolipidemic volunteer. EDTA was used as anticoagulant
(1 mg/mL blood). After low-speed centrifugation (3100 rpm, 20 min,
20 °C) of whole blood to obtain plasma, LDL was isolated from the
plasma by discontinuous density gradient ultracentrifugation.37 Briefly,
the density of plasma was increased to 1.019 with KBr and then was
centrifuged at 40 102 rpm at 13 °C for 10 h. After the top layers
containing chylomicrometer and very-low-density lipoprotein (VLDL)
were removed, the density of remaining plasma fractions was increased
to 1.063 with KBr and they were recentrifuged at 40 102 rpm at 13 °C
for an additional 10 h. The LDL fraction in the top of the tube was
collected and dialyzed against three changes of phosphate buffer (pH 7.4)
in the dark at 4 °C to remove KBr and EDTA. The solution of LDL in
PBS was stored at 4 °C and used within 3 weeks.
General Procedure for the Preparation of the Final Products.
The final products 1 and 2 were obtained by the reaction of 3 mmol
of 2-piperidinemethanol or trans-2-methylaminocyclohexanol with
1.2 mmol of 4-bromoacetyl-3′,5′-di-tert-butyl-4′-hydroxybiphenyl (b)
in anhydrous acetone (40 mL) at room temperature with stirring for
24 h. Acetone was then distilled off. Ether was added to the residue,
and the mixture was washed with saturated NaCl aqueous solution and
dried over K2CO3. The products were isolated as hydrobromide salts
and purified by recrystallization (acetone/ethyl ether).
3-(3′,5′-Di-tert-butyl-4′-hydroxy-4-biphenyl)octahydropyrido-
[2,1-c][1,4]oxazin-3-ol Hydrobromide (1). Yield 77%, mp = 197.2−
199.2 °C. 1H NMR (400 MHz, CDCl3) δ (ppm): 1.35 (s, 18H,
2 × -C(CH3)3), 1.49−1.52 (m, 2H, 8-Hax, 9-Hax), 1.75−1.85 (m, 2H,
G
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Journal of Medicinal Chemistry
Article
Human LDL (56 μg protein/mL) in PBS was incubated at 37 °C in
the absence or presence of various concentrations of compounds 1
and 2 (in 10% DMSO−water) for 5 min before the start of oxidation.
Oxidation was initiated by the addition of 10 μM CuSO4. The
formation of conjugate dienes was determined spectrophotometrically
every 10 min for 5 h as the increase in absorbance at 234 nm and
calculated using the extinction coefficient of 29 500.38
In Vitro Squalene Synthase Activity Assay. SQS activity was
evaluated by determining the amount of [3H]FPP converted to
squalene as previously described.7,39
ABBREVIATIONS USED
■
CVD, cardiovascular disease; SQS, squalene synthase; HMGCo-A,
3-hydroxy-3-methylglutaryl-CoA; FPP, farnesyl pyrophosphate;
PSPP, presqualene pyrophosphate; rt, room temperature;
EtOAc, ethyl acetate; PE, petroleum ether; DCM, dichloro-
methane; 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; MDA, malondialdehyde; STZ,
streptozotocin; HFD, high fat diet
In Vivo Evaluation of Antidyslipidemic and Antioxidant
Activity. An aqueous solution of Triton WR 1339 was given ip to
rats (200 mg/kg),11 and 1 h later, the test compounds (56 μmol/kg),
finely suspended/dissolved with the help of Tween-80 (of a final
concentration of ∼1%) 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) and triglyceride (TG)
levels, using commercially available kits, as well as MDA content,
by measuring the complex of MDA with N-methyl-2-phenyl indole
(586 nm).40 Values are the mean from 8 to 10 rats (per compound),
while all standard errors are within 12% of the respective reported values.
Determination of in Vivo Anti-Inflammatory Effects. As a
measure of the in vivo anti-inflammatory effect of compound 2, the
inhibition of phlogistic-induced paw edema was determined. Healthy
2- to 4-month-old male SKH-2 mice were used in groups of five
animals weighing 20−30 g. A single dose of 0.3 mmol/kg test
compound was administered ip right after the injection of 0.05 mL of
the phlogistic agent carrageenan (2% w/v solution in saline) id into
the right foot paw, the left paw serving as control. The percentage of
edema inhibition is calculated for each animal by the swelling caused
by the phlogistic agent after 3.5 h (given as percentage of weight
increase of right hind paw in comparison to noninjected left hind
paw), as previously described.7,14,15
Evaluation of Compound 2 in a Type 2 Diabetes Experimental
Mouse Protocol. The 7- to 8-week-old male SKH-2 mice were
acclimated for 1 week and allowed water and normal rodent chow
ad libitum. Subsequently they were divided in three groups: group A,
HFD (high fat diet); group B, HFD + STZ (high fat diet +
streptozotocin); group C, HFD + STZ + compound 2. From day 1 to
day 50 mice of all groups were allowed ad libitum a high-fat diet (fat =
35.5% w/w). Streptozotocin was dissolved in 0.1 M citrate buffer
(pH 4.5) and injected ip after 4 h of fasting to groups B and C in
(multiple) low doses (35 mg/kg/day × 5 days) from day 23 to day
27.17,18,41 Compound 2 was administered ip at a dose of 56 μmol/kg
twice daily during the whole experimental period (i.e., for 50 days) to
group C, while similarly, saline was injected to group B.
Glucose Levels. Blood glucose concentration was determined
every 5−10 days with a hand-held glucometer (Precision Xtra Plus,
Abbott). Blood samples were applied directly to the glucose strip from
nonfasted mice to measure nonfasting levels of blood glucose.
Body Weight and Food Consumption. Body weights were
measured, and food consumption was estimated once weekly.
Lipidemic and Oxidative Parameters. For lipid analysis, blood
was drawn on days 23, 43, and 50 of the experiment. Total cholesterol,
HDL cholesterol, LDL cholesterol, TG, and MDA plasma levels were
determined using commercially available kits or as described earlier
above.
Protein Determination. The protein content of microsomal and
LDL fractions was determined according to Lowry’s method.42
Statistical Analysis. Data are expressed as the mean SD. Where
indicated, statistical comparisons were made using Student’s t test, and
a statistically significant difference was inferred if P < 0.05.
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AUTHOR INFORMATION
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Corresponding Author
*Phone/fax: +30-210 7274818. E-mail: angeliki@pharm.uoa.gr.
Notes
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