Summary:
Cardiovascular diseases, such as coronary disease, hypertension and heart
attack, are the leading causes of death in contemporary societies. The majority
of cardiovascular diseases and thereby mortality are caused by atherosclerosis
which is the result of hyperlipidaemia, i.e. the elevated as well as oxidized
levels of LDL-cholesterol and lipids in plasma. Atherosclerosis is also a
result of oxidative stress and a chronic inflammation which frequently turns
into acute thrombosis as a result of atherosclerotic plaque rupture.
Furthermore, the combination of atherosclerosis (hyperlipidaemia and
dyslipidaemia), obesity and diabetes mellitus leads to the metabolic syndrome
disorder.
During the last decade, medicinal chemistry research has been expanding to a
multi-target drug design approach since addressing a multiplicity of targets is
considered to be an asset in the treatment of a wide range of disorders
providing superior therapeutic effects and side effect profile compared to the
action of a selective ligand. Towards this direction, our laboratory has
applied a multifunctional drug design approach in order to develop agents
against atherosclerosis and diabetes. In this context, new benzothiazine and
morpholine derivatives were designed, synthesized and pharmacologically
evaluated as far as their potential activities on the mechanisms involved in
atherosclerosis and the metabolic syndrome process. These new derivatives were
designed to combine in their structure moieties with both antioxidant and
hypolipidaemic / antidyslipidaemic properties.
The new compounds were synthesized employing new methods developed in our
laboratory, as well as modifications of existing methods, while their
structures were confirmed by 1H-NMR και 13C-NMR spectroscopy, as well as
elemental analysis.
According to theoretical measurements of their lipophilicity and other
parameters of Lipinski’s “Rule of Five”, all the new derivatives are able to
cross biological membranes, which is an important property for their in vivo
action.The pharmacological activity of the new synthesized compounds was
studied in vitro in individual assays that simulate mechanisms involved in
atherosclerosis. The most potent derivative was further evaluated for its
activity in vivo in an experimental mouse model of hyperlipidaemia-type 2
diabetes mellitus. Thus, the new molecules were examined for their in vitro
antioxidant activity via inhibition offered against lipid peroxidation of rat
microsomal membranes induced by Fe2+ / ascorbic acid, as well as their
interaction with the stable free radical DPPH (1,1-diphenyl-2-picrylhydrazyl
radical). All the new compounds exhibited very significant protection against
lipid peroxidation with IC50 values between 7 and 110 μΜ. The combination of
specific structural characteristics of the new compounds theoretically favors
their interaction with biological membranes and stabilizes potential free
radicals. The benzothiazine derivatives potently interacted with DPPH (IC50
values between 34 και 171 μΜ), while the morpholine compounds proved to be
inactive in scavenging free radical DPPH in the examined concentrations.The
potential hypocholesterolaemic activity of some of the new compounds was
evaluated by the inhibition of squalene synthase, an enzyme that plays a
crucial role in the biosynthesis of cholesterol. The inhibition of this enzyme
may not provoke the side effects of statins, such as rabdomyolysis,
hepatotoxicity and diabetes type 2. Results showed significant inhibition of
this enzyme with IC50 values ranging from 6.8 to 14 μΜ. In the above in vitro
assays the novel compounds proved to be more potent than the lead compounds (A,
B, C, E and resveratrol), a fact that highlights and confirms the success of
their rational design. Some of the new derivatives were studied for their
ability to inhibit the two isoforms of cycloxygenase (COX-1 και COX-2) in
vitro. Most of them exhibited good activity on either one or / and both COX
isoforms in the examined concentration (50 μΜ).The in vitro studies of the
newly synthesized molecules confirmed their ability to act as multifunctional
agents, since they combine remarkable antioxidant, antihyperlipidaemic and
anti-inflammatory activity. Due to the particularly interesting multifunctional
activity of lead compound B against pathogenic mechanisms of atherosclerosis
(oxidative stress, LDL-lipid peroxidation, anti-inflammatory activity,
hypercholesterolaemia, hyperlipidaemia), which is explained by the
pharmacokinetic behavior in vivo in mice, as seen in the past, and also because
of its remarkable antidiabetic action presented in an experimental
obesity-diabetes model, B was further evaluated in an experimental model of
type 2 diabetes mellitus (transgenic C57BL/KsJ-db/db mice) and hyperlipidaemia
for the investigation of its mechanism of action as antidyslipidaemic and
antidiabetic agent. Results showed a decrease (54%) in blood glucose levels of
transgenic diabetic mice that received this agent (B) compared to the control
group, a fact that demonstrates and confirms the antidiabetic action of
compound B. Furthermore, a decrease by 43% in total cholesterol and 48% in
triglyceride levels of the treated group (with B) versus the control group was
measured, as well as a decrease in oxidative stress levels (malondialdehyde
levels) by 22%.The most potent in vitro agent, compound 10, was further studied
in vivo using an experimental model of streptozotocin-induced-type 2 diabetes
mellitus in combination with a high-fat diet so that its potential action as an
antihyperlipidaemic and antidiabetic agent would be assessed. Compound 10 did
not prevent the increase in glucose levels, although administration led to
reduction of total cholesterol (31%), triglycerides (55%) and LDL-cholesterol
(65%). Furthermore, is seems to have the potential to protect against
atherogenesis as estimated by the antiatherogenic index values of HDL/LDL and
ΑΙΡ. These results, as well as the remarkable in vitro activities of 10, render
this molecule an interesting multifunctional - antioxidant, anti-inflammatory
and hypolipidaemic – agent for the treatment of a multifactorial disease, such
as atherosclerosis. Finally, lead compound E was further investigated after
being intraperitoneally administered to mice. The aim was to examine some basic
pharmacokinetic properties, such as the duration of its presence and
concentration in plasma and liver (target tissues) after intraperitoneal
injection (i.p.) in mice. This study was carried out employing an analytical
method using an isocratic-phase High Performance Liquid Chromatography (HPLC)
after respective validation studies. The analysis of blood and liver samples at
predetermined time points after administration showed the presence of E in
detectable amounts as soon as within 5 minutes, as well as 24 hours after i.p.
administration. These results demonstrate and confirm that compound E reaches
its target tissues, i.e. blood (for protection of LDL oxidation) and liver (for
SQS inhibition) in the lower micromolar range, concentration that is comparable
to its in vitro IC50 value.
