الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Inflammation is a protective mechanism employed by tissues against endogenous and exogenous antigens, induced by microbial infection or tissue injury and is characterized by
redness, edema, fever, pain and loss of function.(200) Traditional (NSAIDs) interact with both COX-1 and COX-2 and therefore their long term administrations often cause gastrointestinal
(GI), renal and hepatic side effects.(201-204) Discovery of selective COX-2 inhibitors provide good
anti-inflammatory agents with improved therapeutic potency and reduced side effects.
However,some coxibs suffered from market withdrawal owing to increasing cardiovascular disorders of
selective COX-2 inhibition, due to imbalance in the COX pathway.(205-207)
In addition, the
inhibition of COX-1/COX-2-mediated metabolism of arachidonic acid can result in an increased
formation of leukotrienes (LTs) via the lipoxygenase (LOX) pathway.(73) LTs are known to be
involved in the progression of inflammation, osteoarthritis, and asthma.(208-210) Consequently,
combined LOX/COX inhibition could provide anti-inflammatory and analgesic effects with the
advantage of reduced adverse effects.(211)
Moreover, the anti-inflammatory research focus also shifted towards signaling pathways,
for instance; (TNF-α, iNOS) inhibitors and increase expression of anti-inflammatory IL-10.
On the other hand, literature survey revealed that various pyrimidine derivatives have
been reported as anti-inflammatory agents by blocking COX, LOX and signaling pathway.
Motivated by the aforementioned facts and continuation of our ongoing effort endowed
with discovering and exploring new structural candidates that might be of value for the
development of novel, potent anti-inflammatory agents with better safety profile this
investigation was directed towards design and synthesis of new various substituted pyrimidine
derivatives.
The target structures (TS-1-4) were designed as potential multitargeted antiinflammatory agents.
The present thesis comprises the following chapters:
Chapter 1: Introduction It includes an introductory part on inflammation and the different pre-inflammatory
mediators and different strategies to treat inflammatory disorder also; it illustrates literature
survey on various biologically active pyrimidine derivatives exhibiting anti-inflammatory
activity, focusing on the recent researches.
Chapter 2: Research objectives It illustrates the aim of the present work and the rational upon which the newly suggested
target structures were designed.
<Chapter 3: results and discussion It deals with results and discussion of the synthesis of the target compounds, their
biological evaluation and the utilized molecular modeling methods.
This chapter is divided into
three parts:3.1. Chemistry<It discusses the theoretical concepts of the experimental methods adopted for the
synthesis of the target compounds with the reference to the available literature. It also
investigates the structure elucidation of the synthesized compounds by elemental analyses and
varies spectroscopic methods (IR, 1H-NMR, 13C-NMR and mass spectra).
It includes the
following six schemes.
Scheme 1:
It outlines the chemical pathway for the preparation of key intermediates Ethyl 4-(4-
Substituted phenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylates (1a,b) and
their acylation with chloroacetyl chloride to ethyl-3-(2-chloroacetyl)-4-(substituted phenyl)-6-
methyl-2-oxo-1,2,3,4- tetrahydropyrimidine-5-carboxylates (2a,b).
Scheme 2:
It presents the synthetic pathway for one pot reaction of ethylacetoacetate, 4-hydroxybenzaldehyde or vanillin with urea in ethanol containing catalytic amount of HCl to
afford ethyl-4-(4-hydroxy-3-substituted phenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylates (4a,b) which underwent alkylation by propargyl bromide to afford ethyl-6-methyl4-(3-substituted-4-(prop-2-yn-1-yloxy)phenyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylates (5a,b).
It also illustrates the In situ preparation of diazotizated substituted aromatic
amines which further reacted with sodium azide to afford 4-substituted azidobenzens (6a-c)
which subsequently underwent 1,3-dipolar cycloaddition reaction with (5a,b) to produce the
target ethyl 6-methyl-4-(3-substituted-4-((1-substituted-1H-1,2,3-triazol-4- yl)methoxy)phenyl)-
2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylates (7a-f).
Scheme 3:It describes the condensation of D-glucose with phenyl hydrazine in water containing
catalytic amount of acetic acid to afforded (3S, 4R, 5R)-5,6-bis(2-phenylhydrazono)hexane1,2,3,4-tetraol (8). Boiling compound 8 with copper sulphate in water afforded (3S, 4R, 5R)-1-
(2-phenyl-2H-1,2,3-triazol-4-yl)butane-1,2,3,4-tetraol (9).
Treatment of 9 with HIO4 in water
gave the corresponding 2-phenyl-2H-1,2,3-triazole-4-carbaldehyde (10).
Scheme 4:
It illustrates the preparation of the target (6-Methyl-4-(2-phenyl-2H-1,2,3-triazol-4-yl)-2-
thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)(Phenyl)methanone (11) by heating a mixture of
compound 10, thiourea and benzoylacetone in acetonitrile containing catalytic amount of HCl. In
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addition, one pot reaction of compound 10 with urea or thiourea and acetoacetanilide or its 4-
chloro derivative in acetonitrile containing catalytic amount of HCl gave 6-methyl-2-oxo-(orthioxo)-N-substituted phenyl-4-(2-phenyl-2H-1,2,3-triazol-4-yl)-1,2,3,4- tetrahydropyrimidine5-carboxamides 12a-c
Scheme 5:
It describes the condensation of aryl methyl ketones with 10 in aqueous ethanolic NaOH
(40%) to yield the corresponding chalcones (E)-1-Aryl-3-(2-phenyl-2H-1,2,3-triazol-4-yl)prop2-en-1-ones (13a-e).
Treatment of compounds 13a-e with guanidinium chloride in absolute
ethanol containing anhydrous sodium acetate afforded the target 4-Aryl-6-(2-phenyl-2H-1,2,3-
triazol-4-yl)pyrimidin-2-amines (14a-e).>
Scheme 6:
It outlines the condensation of aryl methyl ketones with the appropriate aryl aldehydes in
aqueous ethanolic NaOH (40%) to yield (E)-1,3-Diarylprop-2-en-1-ones (15a-g).
In addition,
reaction of 15a-g with guanidinium chloride in absolute ethanol containing anhydrous sodium
acetate gave the corresponding 4,6-Diaryl pyrimidin-2-amines (16a-g).
Furthermore, Acylation
of later compounds with acetic anhydride produced N-(4,6-Diaryl pyrimidine-2-yl)acetamides
(17a-g).
3.2. Biological screening
This part describes the biological investigation of the newly synthesized compounds in
the following aspects.
<3.2.1. In vitro COX inhibition assay
All synthesized compounds were subjected to In vitro COX-1/COX-2 inhibition assay
using an ovine COX-1/human recombinant COX-2 assay kit.
The half-maximal inhibitory
concentrations (IC50 μM) as well as selectivity index (SI) values were determined.
The results revealed that all tested compounds were more active than diclofenac sodium
and indomethacin against COX-2 (IC50= 0.1-0.5 μM). Compounds 7c, 7f and 12b (IC50= 0.1
μM) displayed almost half the potency of celecoxib against COX-2 (IC50= 0.049 μM) while the
rest of the tested compounds showed around 10-45% the activity of celecoxib.
Moreover, all tested compounds were less potent COX-1 inhibitors (IC50= 3.98-15.21
μM) compared to diclofenac sodium (IC50=3.8 μM) and indomethacin (IC50= 0.04 μM).
In terms
of COX-2 selectivity, all tested compounds had higher selectivity than diclofenac sodium and
indomethacin.
3.2.2. In vitro15-LOX inhibitory assay
All synthesized compounds have been also tested for their ability to inhibit lipoxygenase
enzyme (15-LOX) using meclofenamate sodium as reference drug.
Compounds 2b, 13e, 14a,c>
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17e and 17f showed inhibitory activity (IC50= 4.62, 5.11, 3.11, 3.98, 4.23 and 3.97 μM,
respectively) higher than meclofenamate sodium (IC50= 5. 64 μM) while compound 17c (IC50=
5. 64 μM) showed equal inhibitory potency to meclofenamate sodium.
3.2.3.In vitro determination of the level of expression of COX-2, iNOS, IL-10 and TNF-
in LPS-challenged monocytes: LPS treated monocytes showed increase in COX-2 expression, and such increase was
greatly attenuated in cells treated with compounds 7b, 14a and 17f.
Moreover the latter
compounds were even more potent than the reference celecoxib and diclofenac. In addition,
iNOS expression was greatly enhanced upon treating the monocytes with LPS and such effect
was efficiently diminished upon treatment with compounds 7a,c,e, 12b, 13e and 17f. The tested
compounds were more potent than celecoxib and diclofenac. With regard to anti-inflammatory
cytokine IL-10, compounds 7c, 14c, 12b, 14a and 17f were capable of inducing its expression
higher than celecoxib, while compounds 7a, b, e, 13e, and 17e were capable of inducing IL-10
expression more than diclofenac. Interestingly, 17f was able to suppress both expression levels
of COX-2 and iNOS as well as upregulate expression of IL-10 and reversed all these parameters
to normal ranges after treating LPS-stimulated monocytes with its EAIC for 72 h.
Exposing the
human monocytes to LPS significantly increased TNF- production (272.15 pg/ml) Adding the
EAICs of 7a,b,e, 12b, 14a and 17e to the LPS-stimulated monocytes reduced the TNF-
production compared to those cells stimulated by LPS alone, in the following order: 7a, b, e>
14a, 17e> diclofenac, 12b, 7c> 17f, 13e, 14c> celecoxib. 3.2.4. In vivo anti-inflammatory activity:
Six compounds (7a,b, 12b, 14a, and 17e and 17f) were selected to investigate their In
vivo anti-inflammatory activity using formalin-induced rat paw edema protocol as an acute
inflammation model. All compounds showed % inhibition of edema after 4 h lower than that of
standard diclofenac sodium. Interestingly, compound 17e exhibited slightly higher % inhibition
of edema than that of the standard celecoxib (67.73% vs 64.8 %, respectively). Furthermore, 17f
displayed % inhibition of edema, which is comparable to that of celecoxib (63.7%). Moreover,
compounds 7a,b and 12b showed moderate % inhibition (40.73%, 46.17 % and 51.3 %,
respectively)
3.3. Molecular Modeling
This part includes:
3.3.1. Docking study
Compounds 7e, 12b and 14a, 17f were selected for molecular docking studies into the
binding site of COX-2 and 15-LOX enzymes, respectively, to develop an insight into the putative
intermolecular interactions and explore the possible binding pattern behind the inhibitory
activities of these compounds. The docking results indicated that, they adopted similar binding
modes to those of the co-crystallized ligands as well as remarkable interactions with crucial
amino acid residues. This was in concordance with their In vitro biological results (IC50 values).
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3.3.2. In silico prediction of physicochemical properties, pharmacokinetic profile and
toxicity
The most active compounds were subjected to molecular properties prediction by Swiss
ADME in order to compute physicochemical descriptors as well as to predict ADME parameters,
and drug like nature.
Toxicity of the most active compounds was also evaluated using Lazar
toxicity prediction.
Chapter 4: Experimental
This chapter is divided into three parts: 4.1. Chemistry>
<This part illustrates the detailed practical procedures adopted for the synthesis of the
intermediates as well as the target compounds.
In addition, it includes the physical characters,
elemental analysis, IR,
1H
-NMR,
13C-NMR and mass spectra for the newly synthesized
compounds.4.2.
Biological screening
This part describes the materials and methodology adopted for In vitro and In vivo
biological anti-inflammatory evaluation. 4.3.
Molecular Modeling
This part involved the practical computational steps utilized for docking the most active
compounds into active site of the target enzymes (COX-2/15-LOX).
Moreover, prediction of
activity as well as physiochemical properties, pharmacokinetic and toxicity profile.