1. 1
ANNETA P. SMYRNIOTOU
PhD THESIS – ABSTRACT
“Synthesis and study of novel inhibitors of intracellular phospholipases A2 with possible anti-
inflammatory and anticancer action”
Phospholipases A2 are enzymes that hydrolyze the sn-2 ester bond of the cellular membrane’s
phospholipids. This hydrolysis has as a result the release of free fatty acids and lysophospholipids,
precursors of eicosanoids and other bioactive molecules. Eicosanoids, as are leukotriens,
thromboxans and prostaglandins participate in a plethora of normal functions in the cell among
which is the creation of inflammation that in pathological conditions is associated to illnesses such
as cancer, autoimmune diseases and cardiac disorders. Lysophospholipids are precursors of the
platelet-activating factor (PAF) which is involved in inflammation while lysophospholipids
themselves activate blood leukocytes and in high concentrations may cause tissue damage, gastric
ulcer, tumor proliferation and metastasis. Therefore, these enzymes constitute interesting
pharmaceutical targets and synthesis of new potent inhibitors can lead to novel drugs for the cure of
contemporary diseases such as multiple sclerosis and cancer.
The superfamily of phospholipases consists of five main groups: the secreted PLA2, the
cytosolic, the Ca2+
-independent, PAF-acetylhydrolases and the lysosomal PLA2. GIVA cPLA2 and
GVIA iPLA2 are the most studied enzymes of this family and have been associated with the
development and progression of multiple diseases. cPLA2 is a cytosolic enzyme of 85 kDa with
specificity towards phospholipid substrates that contain arachidonic acid. Its active center contains
the catalytic dyad Ser/Asp while its action is regulated by the presence of Ca2+
ions. This enzyme
has been found to play a role in the pathophysiology of brain diseases (Parkinson), disorders of the
nervous system (multiple sclerosis) and in cancer. On the other hand, iPLA2 consists of two
isoforms weighted 85-90 kDa and its active center is a lipase type Gly-X-Ser-X-Gly. Regulation of
this enzyme is independent of calcium ions and it takes place either through the activity of enzyme
caspace-3 or through the attachment of an ATP molecule. iPLA2 has a great part in many
physiological cellular functions as are the growth, the proliferation and the apoptosis but mostly is
characterized by its ability to maintain the homeostasis of the phospholipid membrane. It has been
associated with the pathophysiology of various diseases such as schizophrenia, diabetes, genesis
and progression of cancer. All these data suggest that the development of inhibitors of these

2. 2
enzymes and mostly of iPLA2 is an important research target for both further investigation of the
structure and function of the enzyme and the creation of new medicines.
Thus, a plethora of inhibitors of iPLA2 is reported in the literature and the most important and
potent are bromoenol-lactone, trifluoromethyl ketones, methyl fluorophosphonic esters,
polyfluoroketones and 2-oxoamides. Below some typical structures of each category of inhibitors
are shown.
Studying of the activity of known inhibitors in the literature and their structure-activity relationship
led to the design and synthesis of 2-oxoamide derivatives and polyfluoroketone compounds as
possible inhibitors of the Ca2+
-independent phospholipase A2. In the present work the following
were synthesized:
1) 2-Oxoamide derivatives that contain a para-methoxy substituted aromatic core in a
distance of four carbon atoms from the activated carbonyl followed by an amino component
which differentiates in each derivative.
R1= But
, Et, H R2= OBut
, NH2
n= 1,2,3 R3= But
, Bn

3. 3
R4= CH(CH3)2, CH2CH(CH3)2, CH(CH3)CH2CH3
2) Polyfluoroketone compounds that contain a heterocyclic aromatic ring, a chain of four
carbon atoms and the activated carbonyl of the polyfluorogroup.
Rf= CF2, CF2CF3
The enzymatic assays of the inhibitors prepared against phospholipases GIVA cPLA2, GVIA
iPLA2 and GV sPLA2 were performed in in vitro experiments using the method of mixed micelles.
The results showed that 2-oxoamides which presented very good inhibition and good selectivity are
those that contained dipeptide norleukine – glycine and a tert-butylester or a benzylester and the
one which contained dipeptide leukine – glycine and a tert-butylester which the greatest inhibition
showed the 2-oxoamide that contained dipeptide glycine – γ-aminobutyric acid with the tert-
butylester by 99.9% and ΧΙ (50)= 0.007. Generally, polyfluoroketones showed low to medium
inhibition towards iPLA2. Pentafluoroethyl-ketone containing a methyl-protected indole and a four-
carbon chain presented the highest inhibition of 93%.