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i
Synthesis and Identification of New Aminoacetylenic Tetrahydrophthalimide Derivatives
Anticipated as COXs Inhibitor
By
Ahmed Basim
Thesis Submitted in Partial Fulfillment of the Requirements for the
Master Degree in Pharmaceutical Sciences at
University of Petra
Faculty of Pharmacy and Medical Sciences
Amman-Jordan
April 2014
ii
Synthesis and Identification of New Aminoacetylenic Tetrahydrophthalimide Derivatives
Anticipated as COXs Inhibitor
By
Ahmed Basim
Thesis Submitted in Partial Fulfillment of the Requirements for the
Master Degree in Pharmaceutical Sciences at
University of Petra
Faculty of Pharmacy and Medical Sciences
Amman-Jordan
April 2014
Advisor Professor: Signature
Prof. Zuhair Muhi-eldeen …………...
Co- Advisor Professor:
Prof. Tawfiq Arafat …………...
Examination Committee:
1. Prof. Zuhair Muhi-eldeen …………...
2. Prof. Tawfiq Arafat …………...
3. Dr. Kenza mansoor …………...
4. Dr. Ghadeer Suaifan …………...
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Abstract
Synthesis and Identification of New Aminoacetylenic Tetrahydrophthalimide Derivatives
Anticipated as COXs Inhibitor
By
Ahmed Basim
University of Petra
April 2014
Supervisor Co-Supervisor
Prof. Zuhair Muhi-eldeen Prof. Tawfiq Arafat
Aminoacetylenic Tetrahydrophthalimide derivatives were synthesized from the
reaction of cis-1,2,3,6 Tetrahydrophthalimide with 3-bromoprop-1yne to generate
2-(prop-2-yn-1-yl)-2,3,3a,4,7,7a-hexahydro-1H-isoindole-1,3-dione (AM). A
mixture of 2-(prop-2-yn-1-yl)-2,3,3a,4,7,7a-hexahydro-1H-isoindole-1,3-dione,
paraformaldehyde, cyclic amine and cuprous chloride in catalytic amount, in
peroxide free dioxane through mannich reaction yielded the desired
Aminoacetylenic compounds AM1-AM6. The IR, 1H-NMR, DSC, and elemental
iv
analysis were consistent with the assigned structures. The design of these
compounds as COX inhibitors is based on our rationalization for the important
criteria
Needed to overlap effectively with COX to induce antagonistic activity. These
criteria are: A) A basic amino group for ionic interaction. B) The aceltylenic group
for electrostatic interaction. C) The 2-butyne provides the appropriate distance
between the basic nitrogen and cis-1,2,3,6 Tetrahydrophthalimide. The docking
results show that all the designed compounds have good COX inhibition
especially AM4 had (-8.6 kcal/mol) for COX2 showing a promising approach in
managing inflammatory diseases.
v
Acknowledgments
Hereby I dedicate my thesis to a great man that I have seen none like him, the
one who taught me the meaning of dedication, commitment and sacrifice, my role
model that I’ll always look up to, to my beloved father, I owe you an eternal
gratitude.
To my mother: to immortalize her smiles, tears, kindness and unconditional love.
Many thanks for two great brothers Atheer and Ayad, for that their consultations
were always of a great deal of help.
Thanks to my dear friend Ragheed for setting clear path for me to follow and
making things easier.
I am so grateful for Ibrahim AKA: bebo, bay_bow, beez, beepzone, darabeez for
that he got us rid of the sodium with the new procedure.
Special thanks to Ubisoft®, sony®, Eiichiro Oda, Masashi Kishimoto, mohammed
rudha and Ahmed Issam for the great mental support.
Many thanks for the Petra Uni. staff especially: Prof.zuhair Muhi-eldeen, Prof.
Tawfiq Arafat, dr.kenza mansoor, dr.nidhal Al-Qinna and Dr.Ghadeer Suaifan for
their support and Jordan university staff who helped through the analysis
especially: Ayad, Omran And Rula.
Sincere gratitude to Sir Thomas Edison, Robert E. Khan, Vint Cerf, Isidor Rabi,
Dr.Ali Al-wardy, Michael Faraday, Nikola Tesla, Larry page and Sergey Brim.
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And finally I want to thank me, myself and my fellow Ahmed Basim for their
extraordinary patience with me.
1
ContentsList of figures..................................................................................................... 2
List of tables...................................................................................................... 4
List of Abbreviations.......................................................................................... 4
Chapter one.......................................................................................................... 5
Introduction........................................................................................................... 5
1.1 Inflammation ................................................................................................... 5
1.2 Prostaglandins................................................................................................ 8
1.3 Prostaglandins Biosynthesis........................................................................... 9
1.4 Cyclooxygenase Isoforms............................................................................. 17
1.5 Non-steroidal anti inflammatory drugs (NSAIDs) .......................................... 20
1.7 Aim of investigation ...................................................................................... 27
Chapter two ........................................................................................................ 29
2. Materials and methods ................................................................................... 29
2.1 Chemical synthesis and compounds......................................................... 29
2.1.1 Chemicals .............................................................................................. 29
2.1.2 Instrumentation................................................................................... 29
2.1.3 Docking method of AM1 – 6 into COX-1 and COX-2 ........................ 30
2.2 Synthesis of 2-(prop-2-yn-1-yl)-2,3,3a,4,7,7a-hexahydro-1H-isoindole-1,3-dione (AM). ......................................................................................................... 32
2.3 Synthesis of aminoacetylenic compounds (AM1-6) ...................................... 33
2.4 Aminoacetylenic compounds: ....................................................................... 34
Chapter Three .................................................................................................... 36
Results ............................................................................................................... 36
Chapter Four ...................................................................................................... 60
4. Discussion and conclusion ............................................................................. 60
4.1 Discussion .................................................................................................... 60
figure 4.1 proposed Mannich reaction. ............................................................... 61
4.2 Conclusion.................................................................................................... 66
References ......................................................................................................... 67
2
List of figures
FigureNo.
Caption PageNo.
1.1 Inflammation steps 71.2 Prostaglandin E2 structure 81.3 Arachidonic acid structure 91.4 the site of phospholipase a2 action on phosphatidyl inositol to release
Arachidonic acid10
1.5 Arachidonic acid metabolism pathways 131.6 arachidonic acid conversion to PGH2 14
1.7 Conversion of PGH2 to the five principal prostaglandins 161.8 Primary structures of COX genes and COX proteins 191.9 Aspirin 201.10 2-{2-[(2,3-dimethylphenyl)amino]phenyl}propanoic acid. (anthranilic
acid compounds)21
1.11 2-[4-(2-methylpropyl)phenyl]propanoic acid 221.12 2-[2-[(2, 6- dichlorophenyl) amino] phenyl] acetic acid 221.13 2-{1-[(4-chlorophenyl) carbonyl]-5-methoxy-2-methyl-1H-indol-3-yl}
acetic acid.( indomethacin)23
1.14 4-butyl-1-(4-methylphenyl)-2-phenylpyrazolidine-3,5-dione 23
1.15 4-hydroxy-2-methyl-1,1-dioxo-N-(pyridin-2-yl)-2H-1λ�,2-benzothiazine-3-carboxamide.
24
1.16 4-hydroxy-2-methyl-N-(5-methyl-2-thiazolyl)-2H-1, 2-benzothiazine-3-carboxamide-1, 1-dioxide. (meloxicam).
24
1.17 4-(5-methyl-3-phenylisoxazol-4-yl) benzensulfonamide (valdecoxib) 262.1 Synthesis of 2-(prop-2-yn-1-yl)-2,3,3a,4,7,7a-hexahydro-1H-
isoindole-1,3-dione (AM).32
3.1 1H-NMR spectrum of (AM). 37
3.2 IR spectrum of (AM). 38
3.3 DSC thermogram of (AM). 39
3.4 1H-NMR spectrum of (AM-1). 41
3.5 IR spectrum of (AM-1). 42
3.6 1H-NMR spectrum of (AM-2). 44
3.7 IR-spectrum of (AM-2). 45
3.8 1H-NMR spectrum of (AM-3). 47
3.9 IR spectrum of (AM-3). 48
3
3.10 1H-NMR spectrum of (AM-4). 50
3.11 IR-spectrum of (AM-4). 51
3.12 DCS thermogram of (AM-4). 52
3.13 1H-NMR spectrum (AM-5) 54
3.14 IR spectrum of (AM-5). 55
3.15 1H-NMR of (AM-6). 57
3.16 IR spectrum of (AM-6). 58
3.17 DSC thermogram of (AM-6). 59
4.1 Proposed Mannich reaction 61
4.2.A Binding mode of Am6 (blue) within the COX-1 active site (gold). 644.2.B Binding mode Am4 (yellow) within the COX-2 active site (gray),
respectively.64
4
List of tables
Table No.
Caption PageNo.
4.1 Docking scores of the six compounds into the COX-1 and COX-2 active site.
65
List of Abbreviations
PG Prostaglandin
EFAs Essential fatty acids
PLA2 Phospholipase A2
DAG Diacylglycerol
IP3 Inositol triphosphate
COX-1 Cyclooxygenase 1
COX-2 Cyclooxygenase-2
PGG2 Prostaglandin G2
PGH2 Prostaglandin H2
SP Single peptide
DM Dimerization domain
EG Epidermal growth factor domain
Mb Membrane binding domain
Cat Catalytic domain
NSAINDs Non steroidal anti-inflammatory drugs
GI Gastrointestinal
DMARDS Disease modifying anti-rheumatic drugs
IR Infrared
DMSO Dimethyl sulfoxide
DSC Differential scanning calorimetry
NMR Neuclear magnetic resonance
PDB Protein data bank
δ Delta
5
Chapter one
Introduction
1.1 Inflammation
Is a non-specific defense mechanism of the body to harmful stimuli, such as
pathogens, abrasion, chemical irritations, damages cells and extreme
temperatures. The common signs and symptoms of inflammation are: redness,
pain, heat and swelling, and could also lead to loss of function in the injured area.
Inflammation is a protective attempt by the organism to remove microbes, toxins,
or foreign material at the site of injury, to prevent their spread to other tissues
and to initiate the healing process. Inflammation can be classified into acute or
chronic, where acute is the initial response of the body to the noxious stimuli
which is characterized by the movement of plasma and leukocytes to the injured
area, while the chronic inflammation leads to progressive shift in type of cells
present at the inflammation area. (Gerard, tortora, 11th edition2006)
Since the inflammation is non-specific resistance mechanism, the response of
tissue cut or burn is similar to bacterial or viral invasion, the inflammatory
response has three basic stages(Fig 1.1):
1-vasodilation and increased blood vessels permeability.
2-imigration of phagocyte from the blood into interstitial fluid and.
3-tissue repair.
6
Two immediate changes occur in the blood vessels of the inflamed tissues:
Vasodilation which allows more blood flow into the inflamed area and increased
permeability allowing antibodies and clotting factors to enter the injured area from
blood.Increased blood flow and permeability helps in the removal of microbial
toxins and dead cells.Many substances contribute to vasodilation including:
histamine, kinins, prostaglandins and leukotrienes.
Prostaglandins intensify and prolong the pain associated with inflammation,
within an hour of inflammation phagocytes and neutrophils start to migrate to the
damaged area in an attempt to destroy the invading microbes.
(Gerard, tortora, 11th edition2006)
7
Figure 1.1 inflammation steps
8
1.2 Prostaglandins
Are a group of lipid compounds that are derived enzymatically from fatty
acids and have important functions in the animal body. Every prostaglandin
contains 20 carbon carboxylic acid containing a cyclopentane ring and a hydroxyl
group (Fig1.2). (Nelson, R. J. 2005).
Figure 1.2 Prostaglandin E2 structure.
prostaglandin derived from the prostate gland. When prostaglandin was first
isolated from seminal fluid in 1935 by the Swedish physiologist Ulf von Euler
(Von Euler 1935).
They are considered as mediators and have a variety of
strong physiological effects, such as regulating the contraction and relaxation of
smooth muscle tissue. They differ from hormones in that they are not produced
at a discrete site but in every cell of the body except red blood cells.
The prostaglandins, together with the thromboxanes and prostacyclins, form
the prostanoid class of fatty acid derivatives, a subclass of eicosanoids.
1.3 Prostaglandins
Prostaglandins are found in most tissues and organs. They are produced by
almost all nucleated cells. They are
act upon platelets, endothelium
the cell from the essential fatty
There are three series of prostaglandins (PGE) (PGE1, PGE2 and PGE3). The
most abundant series in human is the series PGE2. The precursor for this series
of prostaglandin is arachidonic acid. Arachidonic acid
essential fatty acid present in the phospholipids of of
body's cells.
The biological active derivatives of arachidonic acid are called eicosanoids
including prostaglandins and leukotrienes.
Figure1.3 Arachidonic acid structure
s Biosynthesis
found in most tissues and organs. They are produced by
almost all nucleated cells. They are autocrine and paracrine lipid mediators that
endothelium, uterine and mast cells. They are synthesized in
essential fatty acids (EFAs).
There are three series of prostaglandins (PGE) (PGE1, PGE2 and PGE3). The
most abundant series in human is the series PGE2. The precursor for this series
of prostaglandin is arachidonic acid. Arachidonic acid (fig1.3) is an unsaturated
ial fatty acid present in the phospholipids of of membranes
The biological active derivatives of arachidonic acid are called eicosanoids
including prostaglandins and leukotrienes.
Arachidonic acid
Figure1.3 Arachidonic acid structure
9
found in most tissues and organs. They are produced by
lipid mediators that
. They are synthesized in
There are three series of prostaglandins (PGE) (PGE1, PGE2 and PGE3). The
most abundant series in human is the series PGE2. The precursor for this series
is an unsaturated
membranes of the
The biological active derivatives of arachidonic acid are called eicosanoids
10
Arachidonic acid is present mainly as esterified in the phospholipid pool of cell
membrane. It is often esfterified to the hydroxyl on C2 of
glycerophospholipids,especially phosphatidyl inositol(tanaka 2003) (Figure 1.4)
Arachidonate is released from phospholipids by hydrolysis catalyzed by
phospholipase A2. This enzyme hydrolyzes the ester linkage between a fatty
acid and the hydroxyl at carbon 2 of the glycerol backbone, releasing the fatty
acid arachidonate and a lysophospholipid as products (sekharudu et all., 1992;
Diwan,2008).
Figure 1.4 The site of phospholipase A2 action on phosphatidyl inositol to release
arachidonic acid
11
There are many phospholipase A2 (PLA2s) enzymes which subject to activation
through different signal cascades. They represent a large family of distinct
enzymes whose products are important for signal transduction processes,
eicosanoid and platelet-activating factor formation, membrane remodeling and
lipid metabolism.At least three different PLA2s exist in mammalian cells, each of
which have different characteristics. Which are: PLA2s types are: the secretory
PLA2 have low molecular weights, require Ca+2 concentration for activity, and it is
secreted into the extracellular space.The other type is called cytosolic PLA2
which has a molecular mass of 85Kda, requires increases in intracellular Ca+2 for
phosphorylation of the enzyme and translocation to intracellular membranes but
does not require Ca+2 for its catalytic activity.
The third type is Ca+2 independent PLA2 and has a molecular mass of 80 KDa
and it doesn’t require Ca+2 for activity. In some cell types, several lines of
evidence suggest that the different PLA2 isoenzymes are distinct in terms of
signaling processes but there is cross talk between them as found for the product
of secretory PLA2 to activate cytosolic PLA2. Nevertheless, the cytosolic
phospholipase A2 seems to be the most important in the synthesis of arachidonic
acid (Huwiler et al., 1997; McHowat et al.,2001; Cummings et al., 2004a;
McHowat and creer, 2004; Cumming et al., 2004b).
Cytosolic phospholipase A2 is activated by phosphorylation. There are widely
variable stimuli that can release prostaglandins from cell, and the type of stimuli
varies with the cell type. This occurs in response to signal transduction events
when thrombin stimulates platelets,bradykinin stimulate fibroblasts, and antigen-
12
antibody stimulation for mast cells. General cell damage as well triggers
prostaglandins activation in some cell types (miller, 2006).
Phospholipase C enzyme is another source for arachidonic acid.
As a part of phosphatidyl inositol signal cascade, phosphatidyl inositol is
phosphorylated to phosphatidyl 4,5 biphosphate. Cleavage of phosphatidyl 4, 5
biphosphate by phospholipase C yields Diacylglycerol (DAG) and inositol
triphosphate (IP3). Arachidonate released from diacylglycerol is then catalyzed
by diacylglycerol lipase (Tang et al., 2006).
Arachidonic acid is a substrate for cyclic pathway. This pathway is
branches mainly into either leukotrienes or prostaglandins and thromboxanes.
The earlier branch is catalyzed by lipooxygenase while the later by
cyclooxygenase (COX). (Figure 1.5)
13
Figure 1.5 Arachidonic acid metabolism pathways.
14
Since the research is about COX inhibitor the latter branch will be the field of
interest since it’s catalyzed by cyclooxygenase. Also denoted as prostaglandin
H2 synthase or prostaglandin endoperoxide synthase.The enzyme has two
activities, the cyclooxygenase and peroxidase. Arachidonic acid is catalyzed by
this enzyme in two steps. First of which is the conversion to prostaglandin G2
(PGG2) by the addition of oxygen (cyclooxygenase activity), and then to
prostaglandin H2 (PGH2), (peroxidase activity). (thuresson et al., 2001), (Figure
1.6)
Figure 1.6 Arachidonic acid conversion to PGH2
15
PGH2 is unstable and rapidly metabolized to other biologically active form of
prostaglandins (Fig 1.7). The conversion is catalyzed by various enzymes like
isomerases, synthases and reductases into the subsequent prostaglandins are
PGD2, PGF2α, PGE2, PGI2(prevents formation of the platelet plug involved in
primary hemostasis) and Thromboxane A2 (which stimulates activation of new
platelets as well as increases platelet aggregation and it’s also vasoconstrictor
which is important in tissue injury and inflammation). The factor which determines
the type of prostaglandin to be formed from PHG2 is the type tissue and the
availability of specific enzymes. ( Moriuchi et al., 2008)
16
Figure 1.7: Conversion of PGH2 to the five principal prostaglandins
17
1.4 Cyclooxygenase Isoforms
Cyclooxygenases exist in three distinct isoforms, a constitutive form (COX-1),
inducible form (COX-2) and COX-3.The COX-1 enzyme is expressed in resting
cells of most tissues, functioning as a housekeeping enzyme, and is responsible
for maintaining homeostasis (gastric and renal integrity) and normal production of
prostaglandins.COX-2 is predominantly found in brain, kidney and endothelial
cells, but is virtually absent in most other tissues. However, COX-2 expression is
significantly up regulated as a part of various acute and chronic inflammatory
conditions, and in neoplastic tissues. (Sharma, S. K., 2012).
More recently, a novel COX-1 splice variant termed as COX-3 has been
reported. COX-3 is an enzyme that is encoded by the COX-1 gene, with
difference that COX-3 retains an intron that is not retained in COX-1. (Botting R.
2003, Baker Jawabrah, 2012).
Two smaller COX-1 derived proteins, partial COX-1a (PCOX-1a) and partial
COX-1b (PCOX-1b) variants have been found. Both derivatives have been
shown not to be involved in prostaglandins synthesis and their function still need
to be identified. (Chandraskharan et al 2002).
Each enzyme has four domains: the dimerization domain,epidermal growth factor
domain, membrane binding domain and the catalytic domain.The catalytic
domain has the cyclooxygenase active site and peroxidase active site. The
cyclooxygenase active site is long, narrow channel of largely hydrophobic
character that opens in the membrane binding domain.
18
The channel extend approximately 25 A into globular catalytic domain and about
8A wide. A single amino acid difference in position 523 (isoleucine in COX-1,
Valine in COX2) make a crucial difference between COX-1 and COX-2 which
make the channel wider in COX-2 than in COX-1. This may play an important
role in selectivity of COX-2 inhibitors. (Chandraskharan et al 2004).
19
Figure 1.8
(a) Human COX-1 and COX-2 genes and the mRNAs they encode (shown as white bars below
the genes). Black boxes in the genes and white boxes in the mRNAs denote exons; numbers
above each gene are exon numbers while numbers within the white boxes indicate the size of
each exon in nucleotides; single lines in the genes indicate introns and untranslated regions of
first and last exons (the latter being shown as gray boxes in the mRNAs). (b) Human COX
proteins. Numbers denote amino-acid residues; the exons encoding each domain are shown on
bars below the proteins; important residues are indicated as shown in the key (and with letters in
the single-letter amino-acid code, with a subscript number indicating the residue number).
1.5 Non-steroidal anti inflammatory drugs (NSAIDs)
Classification
NSAIDs classification may expressed in the following derivatives
2008)
1- Salicylic acid compounds
It is one of the most widely used drugs f
as: platelet aggregation inhibitor and supportive in cancer
1.9).
The drug suffers from the side effects associated with local GI
to its acidic functionality.
Figure 1.9 Aspirin
steroidal anti inflammatory drugs (NSAIDs)
sification may expressed in the following derivatives
Salicylic acid compounds
It is one of the most widely used drugs for its multipurpose application such
as: platelet aggregation inhibitor and supportive in cancer treatment
The drug suffers from the side effects associated with local GI
to its acidic functionality.
spirin
20
sification may expressed in the following derivatives: (Muhi-eldeen
or its multipurpose application such
treatment (Figure
The drug suffers from the side effects associated with local GI irritation, due
2- Anthranilic acid (mefenates) ex: mefenamic
An isoster of salicylic acid d
group (figure 1.10). The
acid is about 2-3 times of aspirin.
Figure 1.10: 2-{2-[(2,3-dimethylphenyl)amino]phenyl}propanoic acid
compounds)
Anthranilic acid (mefenates) ex: mefenamic
An isoster of salicylic acid due to the replacement of acetyl group by an amino
. The analgesic and anti-inflammatory activity
3 times of aspirin.
dimethylphenyl)amino]phenyl}propanoic acid. (Anthranilic
21
group by an amino
inflammatory activity of anthranilic
Anthranilic acid
3- Arylacetic acid ex: ibuprofen
Eight times the activity of aspirin as anti
Figure 1.11: 2-[4-(2-methylpropyl
Diclofenac
High potency as anti-
leukotrienes (Cyclooxygenase
Figure 1.12: 2-[2-[(2, 6-
Arylacetic acid ex: ibuprofen
Eight times the activity of aspirin as anti-inflammatory agent (Fig. 1.11)
methylpropyl) phenyl]propanoic acid
-inflammatory agent, inhibit prostaglandin synthesis and
(Cyclooxygenase and lipooxygenase inhibitor) (Fig.1.12)
- dichlorophenyl) amino] phenyl] acetic acid
22
1.11).
inflammatory agent, inhibit prostaglandin synthesis and
1.12).
4- Indole acetic acid
Potent anti-inflammatory and analgesic activity with cyclooxygenase and
lipooxygenase inhibitory activity (Fig. 1.13)
Figure 1.13: 2-{1-[(4-chlorophenyl) carbonyl]
acid.( indomethacin)
5- Pyrazolidine ex: phenylbutazone
Phenylbutazone is highly toxic and not in use currently
Figure 1.14
4-butyl-1-(4-methylphenyl)
Indole acetic acid
inflammatory and analgesic activity with cyclooxygenase and
ipooxygenase inhibitory activity (Fig. 1.13).
chlorophenyl) carbonyl]-5-methoxy-2-methyl-1H-
Pyrazolidine ex: phenylbutazone
nylbutazone is highly toxic and not in use currently (Fig. 1.14).
R= H, phenylbutazone
R= OH,oxyphenylbutazone
methylphenyl)-2-phenylpyrazolidine-3,5-dione
23
inflammatory and analgesic activity with cyclooxygenase and
-indol-3-yl} acetic
.
6- Enolic anti-inflammatory agents
Figure 1.15: 4-hydroxy-2
carboxamide.
New enolic class of non
action against the inducible isoform
which is implicated in inflammatory response, than a
(COX-1), COX2 inhibitors provide less gastric
uses.
Figure 1.16: 4-hydroxy-
carboxamide-1, 1-dioxide
inflammatory agents
2-methyl-1,1-dioxo-N-(pyridin-2-yl)-2H-1λ�,2-benzothiazine
New enolic class of non-steroidal anti-inflammatory drug with a greater inhibitory
action against the inducible isoform of cyclooxygenase (COX-
which is implicated in inflammatory response, than against constitutive
), COX2 inhibitors provide less gastric and kidney damage on long term
-2-methyl-N-(5-methyl-2-thiazolyl)-2H-1, 2-benzothiazine
dioxide. (meloxicam).
24
benzothiazine-3-
inflammatory drug with a greater inhibitory
-2) (Fig. 1.15),
gainst constitutive enzyme
and kidney damage on long term
benzothiazine-3-
25
Structural differences from feldene (piroxicam) involve the replacement of the 2-
amino pyridine group at 3- carboxamide position of the benzothiazine-1,1 dioxide
system by 5-methyl-1, 3- thiazole, which resulted in that selectivity between the
two COX isozymes (Fig. 1.16).
26
7- Compounds free of carboxylic group: celecoxib,
valdicoxib.
Figure 1.17: 4-(5-methyl-3-phenylisoxazol-4-yl) benzensulfonamide (valdecoxib)
Coxib drugs are with potent anti-inflammatory and analgesic activity.
The structural features that lead to selectivity are 1, 2 diarylsubstitution on a
central hetero or carbocyclic ring system with a characteristic methanesulfonyl,
sulfonamide, azido, methanesulfonamide, or tetrazole pharmacophore group on
one of the aryl rings that plays an important role in COX-2 selectivity.( Zarghi
2011) (Fig. 1.17).
COX-2 inhibitor under normal condition expressed in GI tract at undetectable
level, but this expression rises significantly in the presence of colorectal
malignancies, COX-2 role and angiogenesis are under investigation, prolong use
of coxibs are associated with cardiovascular problems.
27
1.7 Aim of investigation
Reviewing the various structural features of non steroidal compounds with COX1
or COX2 inhibitory activity and the good results of COXs inhibitory activity of the
synthesized and published compounds namely aminoacetylenic N-( 4-t-amino-2
butynyl ) phthalimide. These observations promoted our interest to synthesize
aminoacetylenic tetrahydrophthalimide to generate new and novel anti-
inflammatory agents, these new derivatives provide greater lipophilic properties
relative to our previous compounds, the structure flexibility gained from
tetrahydrophthalimide may provide an effective overlap with COX enzymes.
Structural features of COX1,COX2 selective and non selective ones are
associated with gastric ulceration or cardiovascular problem respectively, The
current research directed to have compounds free of acidic groups such as
carboxyl, sulfonamide or sulphone, namely 2-(prop-2-yn-1-yl)-2,3,3a,4,7,7a-
hexahydro-1H-isoindole-1,3-dione
These derivatives are basic in nature and has less gastric ulceration, with
analgesic and antipyretic activity. The selective structural properties and ability to
overlap and inhibit the inflammatory enzymes COX1 or COX2 represent a novel
approach that has to be tested.
The proposed COX inhibitor
Where R =
proposed COX inhibitor
,
,
,
28
29
Chapter two
2. Materials and methods
2.1 Chemical synthesis and compounds
2.1.1 Chemicals
The following chemicals and material were used: cis-1,2,3,6
Tetrahydrophthalimide 96% (SIGMA ALDRICH), propargyl bromide (SIGMA-
ALDRICH), piperidine 99% reagent plus (SIGMA), 2-methylpiperidine 99%(alpha
Aesar), 2,6- Dimethylpiperidine, pyrollidine 98%, N-Methylpiperazine 99%
(ALDRICH) , Hexamethyleneimine 98% (Alfa Aesar) , Ethanol ( Gianland
chemical company), 1,4-Dioxan (FullTime), Chloroform , Paraformaldehyde
(BDH chemicals) , Cuprous chloride.
2.1.2 Instrumentation.
Infrared spectra (IR): FTIR Bruker.
H1 NMR was required with the aid of Varian 300 MHz spectrometer and DMSO-
d6 as solvent. The analysis was indicated by EuroEA elemental analyser. The
result obtained had a maximum deviation of (3.6%) from the theoretical value,
which is considered within the acceptable variation range in results (± 4.4%).
This variation range is set according to the accuracy of EuroEA elemental
analyser device in Faculty of Pharmacy in the University of Jordan.
30
2.1.3 Docking method of AM1 – 6 into COX-1 and COX-2
The 3D structures of COX-1 and COX-2 were downloaded from the protein data
bank (PDB, ID: 3N8Z and 3NT1) and then all water molecules were removed .
Partial charges were assigned to all atoms using Kollman united atom model that
exists in the Autodock Tool software. Preparations of the COX-1 and COX-2
active site were completed by creating a grid box of a 50 x 50 x 50 Å size with a
grid spacing of 0.375 Å using Autogrid (part of the Autodock software package).
The active site was identified using the COX1 and COX2 co-crystallized ligands.
After preparing the protein structures, the ligand preparation process was
initiated by building their own 3D structures using the Maestro software. A
minimization process was carried out on each ligand structure using OPLS force
field. Subsequently, Gasteiger-Marsili model was employed to assign partial
charges for all prepared ligands. Tertiary amines of all ligands were set as
protonated. The ligands were separately docked into the previously prepared
active site using Autodock (version 4.2). Whereas the protein structure was
treated as a rigid entity, the ligand structures were treated as flexible and a
conformational sampling process was carried out using Lamarckian Genetic
Algorithm. The Autodock scoring function was then used to score all docked
poses. The Autodock scoring function includes different terms for van der Waals,
hydrogen bond, electrostatic interactions, and the ligand internal energy.
Compound 5 has two tertiary amine groups in the piperazine ring where one of
31
them is expected to be protonated at physiological pH. Thus, two forms of
compound 5 were built with one of the tertiary amine groups is protonated and
every one was then docked separately in the COX active site.
32
2.2 Synthesis of 2-(prop-2-yn-1-yl)-2,3,3a,4,7,7a-hexahydro-1H-
isoindole-1,3-dione (AM).
A mixture of cis-1,2,3,6-Tetrahydrophthalimide (2g), potassium carbonate K2CO3
(2.18gm), in 20ml acetonirtile, added to it mixture of propargyl bromide and 10ml
acetonirtile then refluxed and stirred for 80 minutes. After 80 minute the mixture
cooled and filtered. The solvent was removed under reduced pressure then add
30 ml chloroform and 20 ml water and separate the chloroform layer in
separatory fennel and removed the chloroform under reduced pressure to afford
the desired white powder (Fig 2.1). The structure was confirmed through:
1H- NMR (DMSO- d6): δ, 3.15(s, 1H), 4.05(s, 1H, CH2-C), 5.90 (s, 2H, CH=CH).
IR: 3300, (acetylenic C-H stretch), 2150, (C, C triple bond stretch), 1700, (C=O
stretch), 1600, (Ar C=C stretch), 1200, (N-C stretch).
Elemental analysis (C11H11N1O2):Calc: C (69.83%), H (5.86%), N (7.4%).
Found: C (66.2%), H (5.8%), N (8.3%).
Figure 2.1: Synthesis of 2-(prop-2-yn-1-yl)-2,3,3a,4,7,7a-hexahydro-1H-isoindole-1,3-
dione (AM).
33
2.3 Synthesis of aminoacetylenic compounds (AM1-6)
A mixture of 2-(prop-2-yn-1-yl)-2,3,3a,4,7,7a-hexahydro-1H-isoindole-1,3-dione
(2g), paraformaldehyde ( 0.5 g ), cyclic amines around (0.01mol ) and cuprous
chloride catalytic amount (0.03 g), in peroxide-free dioxane 35ml was refluxed for
1 hour. Filtered and evaporated under reduced pressure afforded compounds
(AM1 -6).
2.4 Aminoacetylenic compounds
AM-1: 2-[4-(2-methylpiperidin
isoindole-1,3-dione
AM-2: 2-[4-(2,6-dimethylpiperidin
isoindole-1,3-dione
AM-3: 2-[4-(azepan-1-yl)but
dione
Aminoacetylenic compounds:
methylpiperidin-1-yl)but-2-yn-1-yl]-2,3,3a,4,7,7a-hexahydro
dimethylpiperidin-1-yl)but-2-yn-1-yl]-2,3,3a,4,7,7a-
yl)but-2-yn-1-yl]-2,3,3a,4,7,7a-hexahydro-1H
34
hexahydro-1H-
-hexahydro-1H-
1H-isoindole-1,3-
AM-4: 2-[4-(piperidin-1-
1,3-dione
AM-5: 2-[4-(4-methylpiperazin
isoindole-1,3-dione
AM-6: 2-[4-(pyrrolidin-1
1,3-dione
-yl)but-2-yn-1-yl]-2,3,3a,4,7,7a-hexahydro-1H
methylpiperazin-1-yl)but-2-yn-1-yl]-2,3,3a,4,7,7a-hexahydro
1-yl)but-2-yn-1-yl]-2,3,3a,4,7,7a-hexahydro-1H
35
1H-isoindole-
hexahydro-1H-
1H-isoindole-
36
Chapter Three
Results
AM: 2-(prop-2-yn-1-yl)-2,3,3a,4,7,7a-hexahydro-1H-isoindole-1,3-dione
1H- NMR (DMSO- d6): δ, 3.15(s, 1H), 4.05(s, 1H, CH2-C), 5.90 (s, 2H, CH=CH),
(Fig 3.1).
IR: 3300, (acetylenic C-H stretch), 2150, (C, C triple bond stretch), 1700, (C=O
stretch), 1600, (Ar C=C stretch), 1200, (N-C stretch), (Fig 3.2).
Elemental analysis (C11H11N1O2): Calc: C (69.83%), H (5.86%), N (7.4%).
Found: C (66.2%), H (5.8%), N (8.3%).
37
Figure 3.1: 1H-NMR spectrum of (AM).
38
Figure 3.2: IR spectrum of (AM).
39
Figure 3.3: DSC thermogram of (AM).
AM-1: 2-[4-(2-methylpiperidin
isoindole-1,3-dione
The titled compound was prepared following the general
of aminoacetylenic compounds 1
Yielded: 2.8g, 93.3%.
1H-NMR (DMSO- d6): δ
4H, C-CH2-N), 5.86 (d, 2H, CH=CH), (Fig
IR Spectra: 2905 (C-H stretch),
(N-C stretch), (Fig 3.5)
methylpiperidin-1-yl)but-2-yn-1-yl]-2,3,3a,4,7,7a-hexahydro
The titled compound was prepared following the general procedure for synthesis
of aminoacetylenic compounds 1-6.
2.8g, 93.3%.
d6): δ, 1.17-1.72 (m, 11H protons of cyclic amine), 3.15
N), 5.86 (d, 2H, CH=CH), (Fig 3.4).
H stretch), 2845 (C-H stretch), 1700 (C=O stretch), 1280
40
hexahydro-1H-
procedure for synthesis
of cyclic amine), 3.15-3.6 (s,
stretch), 1700 (C=O stretch), 1280
41
Figure 3.4: 1H-NMR spectrum of (AM-1).
42
Figure 3.5: IR spectrum of (AM-1).
AM-2: 2-[4-(2,6-dimethylpiperidin
isoindole-1,3-dione
The titled compound was prepared following the general procedure for synthesis
of aminoacetylenic compounds 1
Yielded 3g, 95.59%.
1H-NMR (DMSO- d6): δ,
4H, C-CH2-N), 4.12 (s, 1
IR Spectra: 2910 (C-H stretch), 2830 (
(C-N stretch), (Fig 3.7).
dimethylpiperidin-1-yl)but-2-yn-1-yl]-2,3,3a,4,7,7a-
compound was prepared following the general procedure for synthesis
of aminoacetylenic compounds 1-6.
d6): δ, 1.16-1.66, (m, 12H protons of cyclic amine), 3.18, 4 (s,
N), 4.12 (s, 1H, CH2-C), 5.83 (s, 2H, CH=CH), (Fig 3.6).
H stretch), 2830 (C-H stretch), 1700 (C=O stretch), 1312
3.7).
43
-hexahydro-1H-
compound was prepared following the general procedure for synthesis
amine), 3.18, 4 (s,
3.6).
stretch), 1700 (C=O stretch), 1312
44
Figure 3.6: 1H-NMR spectrum of (AM-2).
45
Figure 3.7: IR-spectrum of (AM-2).
AM-3: 2-[4-(azepan-1-yl)but
dione
The titled compound was prepared following the general procedure for synthesis
of Aminoacetylenic compounds 1
Yielded 2.85g 95%.
1H-NMR (DMSO- d6): δ,
(s, 4H, CH2-C), 5.82 (s, 2H, CH=CH
IR Spectra: 2920 (C-H stretch), 2825 (C
(N-C stretch), (Fig 3.9).
yl)but-2-yn-1-yl]-2,3,3a,4,7,7a-hexahydro-1H
The titled compound was prepared following the general procedure for synthesis
f Aminoacetylenic compounds 1-6.
d6): δ, 1.18-1.62 (m, 12H, protons of cyclic amine
C), 5.82 (s, 2H, CH=CH), (Fig 3.8).
H stretch), 2825 (C-H stretch), 1702 (C=O stretch), 1190
, (Fig 3.9).
46
1H-isoindole-1,3-
The titled compound was prepared following the general procedure for synthesis
protons of cyclic amine), 3.63-4.11
H stretch), 1702 (C=O stretch), 1190
47
Figure 3.8: 1H-NMR spectrum of (AM-3).
48
Figure 3.9: IR spectrum of (AM-3).
AM-4: 2-[4-(piperidin-1-
1,3-dione
The titled compound was prepared following the general procedure for synthesis
of aminoacetylenic compounds 1
Yielded 2.7g 94.4%.
1H-NMR (DMSO- d6): δ,
CH2-C), 5.64 (s, 2H, CH=CH),(Fig 3.10).
IR Spectra: 2938 (C-
(N-C stretch), 875 (C-H bend),
Elemental analysis (C17
Found: C (68.6%), H (6.07%), N (8.07%).
-yl)but-2-yn-1-yl]-2,3,3a,4,7,7a-hexahydro-1H
The titled compound was prepared following the general procedure for synthesis
f aminoacetylenic compounds 1-6.
d6): δ, 1.92, 2.14 (m, 4H, protons of cyclic amine), 4.05 (s, 1
C), 5.64 (s, 2H, CH=CH),(Fig 3.10).
-H stretch), 2843 (C-H stretch), 1690 (C=O stretch), 1250
H bend), (Fig 3.11).
17H22N2O2):Calc.: C (71.3%), H (7.74%), N (9.78%)
Found: C (68.6%), H (6.07%), N (8.07%).
49
1H-isoindole-
The titled compound was prepared following the general procedure for synthesis
ons of cyclic amine), 4.05 (s, 1H,
H stretch), 1690 (C=O stretch), 1250
C (71.3%), H (7.74%), N (9.78%).
50
Figure 3.10: 1H-NMR spectrum of (AM-4).
51
Figure 3.11: IR-spectrum of (AM-4).
52
Figure 3.12: DCS thermogram of (AM-4).
AM-5: 2-[4-(4-methylpiperazin
isoindole-1,3-dione
The titled compound was prepared following the general procedure for synthesis
of Aminoacetylenic compounds 1
Yielded 2.9g 96.34%.
1H-NMR (DMSO- d6): δ,
4h, C-CH2-N), 3.63 (s, 1
IR Spectra: 2947 (C-H stretch), 2832 (C
3.14).
methylpiperazin-1-yl)but-2-yn-1-yl]-2,3,3a,4,7,7a-hexahydro
The titled compound was prepared following the general procedure for synthesis
f Aminoacetylenic compounds 1-6.
d6): δ, 2.12- 2.5 (m, 9H protons of cyclic amine), 3.6
N), 3.63 (s, 1H, CH2-C), 5.88 (s, 2H, CH=CH), (Fig 3.13).
H stretch), 2832 (C-H stretch), 1707 (C=O stretch)
53
exahydro-1H-
The titled compound was prepared following the general procedure for synthesis
ons of cyclic amine), 3.6-4.12 (s,
C), 5.88 (s, 2H, CH=CH), (Fig 3.13).
stretch), (Fig
54
Figure 3.13: 1H-NMR spectrum (AM-5)
55
Figure 3.14: IR spectrum of (AM-5).
AM-6: 2-[4-(pyrrolidin-1
1,3-dione
The titled compound was prepared following the general procedure for synthesis
of aminoacetylenic compounds 1
Yielded 2.6g 95.58%.
1H-NMR (DMSO- d6): δ,
C), 4.11 (s, 2H, CH2-C), 5.85 (d
IR Spectra: 2936 (C-H stretch), 284
(N-C stretch), 870 (C-H bend)
1-yl)but-2-yn-1-yl]-2,3,3a,4,7,7a-hexahydro-1H
The titled compound was prepared following the general procedure for synthesis
of aminoacetylenic compounds 1-6.
d6): δ, 2.16 (m, 4H, protons of cyclic amine), 3.56 (s, 2
C), 5.85 (d, 2H, CH=CH), (Fig 3.15).
H stretch), 2840 (C-H stretch), 1720 (C=O
H bend), (Fig 3.16).
56
1H-isoindole-
The titled compound was prepared following the general procedure for synthesis
3.56 (s, 2H, CH2-
H stretch), 1720 (C=O stretch), 1253
57
Figure 3.15: 1H-NMR of (AM-6).
58
Figure 3.16: IR spectrum of (AM-6).
59
Figure 3.17: DSC thermogram of (AM-6).
60
Chapter Four
4. Discussion and conclusion
4.1 Discussion
The designed compounds were prepared as mentioned earlier in the synthesis of
2-(prop-2-yn-1-yl)-2,3,3a,4,7,7a-hexahydro-1H-isoindole-1,3-dione (AM). AM was
generated through nucleophilic displacement of the bromine located at 3-
bromoprop-1-yne. The mannich reaction of 2-(prop-2-yn-1-yl)-2,3,3a,4,7,7a-
hexahydro-1H-isoindole-1,3-dione(AM) with paraformaldehyde, appropriate cyclic
amine and catalytic amount of cuprous chloride, generate the desired
compounds (AM1-AM6). The structures were verified through IR, 1H-NMR, as
illustrate in chapter three. Elemental analysis for some compounds were
consistent with the assigned structures.
The proposed mechanism for mannich reaction is outlined in figure 4.1.
In order for mannich reaction to proceed, a reactive immonium cations
intermediates should be formed from the condensation of the formaldehyde and
the appropriate amines (Shiff base formation). The attack of the carbanion in 2-
(prop-2-yn-1-yl)-2,3,3a,4,7,7a-hexahydro-1H-isoindole-1,3-dione cuprous salt on
the Schiff base; generates the desired mannich products (AM1-AM6), (Fig 4.1).
61
figure 4.1 proposed Mannich reaction.
The design of these compounds as COX inhibitors was based on the
rationalization for the important criteria required to overlap effectively with COX
and to induce antagonistic activity. These criteria are:
A basic amino group for ionic interaction, the aceltylenic group is for electrostatic
interaction, the 2-butyne provides the appropriate distance between the basic
nitrogen and cis-1,2,3,6 Tetrahydrophthalimide. Molecular docking results
supported our assumption in the design of new and novel COX inhibitor.
According to Duggan and his co-workers, NSAIDs bind to two key groups of
amino acids in the COX-1 and COX-2 active site: Ser530 and Tyr385 in the top
62
of the catalytic pocket or Arg120 and Tyr355 in the bottom of the pocket. For
instance, diclofenac binds to the first group residues (top) and flurbiprofen
interacts to the latter group residues (bottom), mainly via electrostatic interactions
that are made between polar heads of such residues and the carboxylate group
in these NSAID structures. The NSAIDs hydrophobic groups also have a role in
binding via making van der Waals interactions with the hydrophobic residues
lining the active pocket.
Binding energies of the best docked pose of all six compounds are shown in
Table 4.1, along with the flurbiprofen and celecoxib scoring as a reference. All
compounds seem to fit well in the COX-1 as well as in the COX-2 active site
(they have favorable binding energies). Interestingly, Am6 has shown a
remarkable binding energy (-8.6 kcal/mol) in the COX-1 active site, which is very
close to the diclofenac’s binding energy (-8.7 kcal/mol). As shown in Figure4.2.A,
Am6 also possesses a similar binding mode to diclofenac where the best pose
seems to pleasantly fit in the COX-1 catalytic pocket and to make the same
hydrogen bonding interactions that is made by diclofenac. These interactions are
made by the ligand protonated amino group and the cyclic carbonyl group.
Obviously, these interactions are assisted by the existence of the acetylenic
group which acts as an anchor; via placing these two polar groups at the correct
position for binding. In addition to these electrostatic interactions, extensive
hydrophobic interactions are made between the ligand carbon atoms and the
side chains of Tyr348, Val349, Le352, Phe381, Leu384, Trp387, and Leu534.
63
Although Am6 obtained a very good score in the COX-2 active site, Am4 seems
to have the best affinity towards the enzyme active site. Figure 4.2.B shows how
Am4 nicely fits in the COX-2 pocket, making many van der Waal contacts with the
surrounding hydrophobic residues. In terms of electrostatics, interestingly, two unusual
and strong hydrogen bonding interactions are made between the protonated nitrogen
and the backbone amide of Leu352 and Ser353, and another week hydrogen bond are
made between the cyclic carbonyl and the Ser530 hydroxyl group in the top of the
catalytic pocket.
64
Figure 4.2. (A) Binding mode of Am6 (blue) within the COX-1 active site (gold). (B) Binding mode
Am4 (yellow) within the COX-2 active site (gray), respectively. Yellow dotted lines indicate for
hydrogen bonding.
Tyr385
65
Table 4.1: Docking scores of our six compounds into the COX-1 and COX-2 active site. Docking
scores of flurbiprofen and celecoxib are also shown.
Molecule
Autodock Score
in COX-1
(kcal/mol)
Autodock Score
in COX-2
(kcal/mol)
Am1 -7.8 -7.9
Am2 -7.7 -8.0
Am3 -7.7 -8.1
Am4 -7.9 -8.6
Am5 -7.7 (resonance-1)
-7.9 (resonance-2)
-7.9 (resonance-1)
-7.9 (resonance-2)
Am6 -8.5 -8.4
Flurbiprofen -8.7 -8.0
Celecoxib -8.5 -10.6
66
4.2 Conclusion
Following the docking experiments for our compounds into the COX-1 and COX-
2 active sites, the best docked pose of each compound was studied. Whereas
Am6 seems to have the best affinity towards the COX-1 enzyme, (Am4)
possesses the best fit in the COX-2 catalytic pocket. Their binding modes appear
to be familiar and similar to those shown by different NSAIDS; apart from the
unusual hydrogen bonding made by (Am4) with the backbone amide chain of
Leu352 and Ser353.
The amino group and the cyclic carbonyl group are the key functional groups and
have a direct role in binding with the COX enzymes, whereas the acetylenic
group appears to the anchor role; probably by rigidifying the compound structure
and hence forcing it to adopt the active conformation. To sum up, the in silico
findings presented in this work indicate that the synthesized compounds have the
required complementary shape and electrostatics interaction needed for COX
inhibition. This is strongly supported by the in vitro results presented in the
previous work for structurally similar synthetic compounds (i.e. Aminoacetylenic
Isoindoline 1,3-Diones) which were proven to have an inhibition activity against
both COX-1 and COX-2 enzymes.
67
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الملخص
تصنیع و تعریف مشتقات جدیدة لمركبات أمینوأستیلینیك تیتراھایدروفثالیماید
) COXs(المتوقع فاعلیتھا كمثبطات انزیمات
احمد باسم العطار
2014, جامعة البترا
تحت اشراف االستاذ الدكتور زھیر محي الدین و االستاذ الدكتور توفیق عرفات
). AM(ینیھ تم تصنیعھا من تفاعل تیتراھایدروفثالیماید مع بروموبروبین لتعطي مشتقات تیتراھایدروفثالیماید االستیل
سایكلك امین مع كمیھ من الكبروس كلوراید للتحفیز على التفاعل , بارافورمال دھاید, )AM(المزیج المكون من
).AM1-AM6(بوجود الدایوكسین من خالل تفاعلھا سویا لتنتج المركبات المرغوب فیھا
متسقھ مع التركیب الكیمیائي Elemental analysisو , DSCجھاز ال ,IR,NMRكانت قیاسات اجھزة ال
.للمركبات
ھذه المعاییر ھي . COXو یستند تصمیم المركبات على معاییر ھامة الالزمة لتتداخل مع فعالیة انزیم
A) المجموعھ االساسیھ للربط االمینیھ االیونیھB) المجموعھacetylinic ل تداخل مع المستقبالت .C) 2-
butyne النتائج تظھر ان جمیع . توفیر المسافھ المناسبھ بین النیتروجین و مجموعة ال تیتراھایدروفثالیماید
تظھر نھجا ) مول/كیلو كالوري AM4 )-8.6خصوصا COXالمركبات المصممة لدیھا قابلیة جیدة لتثبیط انزیم
.بیةواعدا في معالجھ األمراض االلتھا