Synthesis and Identification of New Aminoacetylenic ... · Synthesis and Identification of New Aminoacetylenic Tetrahydrophthalimide Derivatives Anticipated as COXs Inhibitor By Ahmed

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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

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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 …………...

iii

Abstract



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.

vi

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


3.7 IR-spectrum of (AM-2). 45



3


3.11 IR-spectrum of (AM-4). 51

3.12 DCS thermogram of (AM-4). 52

3.13 1H-NMR spectrum (AM-5) 54


3.15 1H-NMR of (AM-6). 57


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).



of prostaglandin is arachidonic acid. Arachidonic acid (fig1.3) is an unsaturated

ial fatty acid present in the phospholipids of of membranes


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



is an unsaturated

membranes of the


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-

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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



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


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).


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


45

Figure 3.7: IR-spectrum of (AM-2).

AM-3: 2-[4-(azepan-1-yl)but

dione


of Aminoacetylenic compounds 1

Yielded 2.85g 95%.


(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


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).


, (Fig 3.9).

46

1H-isoindole-1,3-


protons of cyclic amine), 3.63-4.11

H stretch), 1702 (C=O stretch), 1190

47


48


AM-4: 2-[4-(piperidin-1-

1,3-dione



Yielded 2.7g 94.4%.


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


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-


ons of cyclic amine), 4.05 (s, 1H,


C (71.3%), H (7.74%), N (9.78%).

50


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


of Aminoacetylenic compounds 1

Yielded 2.9g 96.34%.


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


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-


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


AM-6: 2-[4-(pyrrolidin-1

1,3-dione



Yielded 2.6g 95.58%.


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



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-


3.56 (s, 2H, CH2-


57

Figure 3.15: 1H-NMR of (AM-6).

58


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

References

A Qinna, N., A Muhi-eldeen, Z., Ghattas, M., M Alhussainy, T., Al-Qaisi, J., & Z

Matalka, K. (2012). Non-selective inhibition of cyclooxygenase enzymes by

aminoacetylenic isoindoline 1, 3-diones. Inflammation & Allergy-Drug Targets

(Formerly Current Drug Targets-Inflammation & Allergy), 11(5), 369-374.

Baker Jawabrah Al-Hourani a,c, Sai Kiran Sharma a,b, Mavanur Suresh b, Frank

Wuest, Novel 5-substituted 1H-tetrazoles as cyclooxygenase-2 (COX-2)

inhibitors, Bioorganic & Medicinal Chemistry Letters, 22, 2235–2238, 2012.

Botting R. "COX-1 and COX-3 inhibitors". Thromb. Res. 110 (5-6): 269–72, 2003

Chandrasekharan, N. V., Dai, H., Roos, K. L. T., Evanson, N. K., Tomsik, J.,

Elton, T. S., & Simmons, D. L. (2002). COX-3, a cyclooxygenase-1 variant

inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning,

structure, and expression. Proceedings of the National Academy of

Sciences,99(21), 13926-13931.

Chandrasekharan, N. V., & Simmons, D. L. (2004). The

cyclooxygenases.Genome biology, 5, 2004-5.

Cummings, B. S., Gelasco, A. K., Kinsey, G. R., Mchowat, J., & Schnellmann, R.

G. (2004). Inactivation of endoplasmic reticulum bound Ca2+-independent

phospholipase A2 in renal cells during oxidative stress. Journal of the American

Society of Nephrology, 15(6), 1441-1451.

68

Cummings, B. S., McHowat, J., & Schnellmann, R. G. (2004). Role of an

endoplasmic reticulum Ca2+-independent phospholipase A2 in cisplatin-induced

renal cell apoptosis. Journal of Pharmacology and Experimental

Therapeutics,308(3), 921-928.

Diwan J.J. synthesis of Eicosanoids,

http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/prostag.htm,2008.

Duggan, K. C., Walters, M. J., Musee, J., Harp, J. M., Kiefer, J. R., Oates, J. A.,

& Marnett, L. J. (2010). Molecular basis for cyclooxygenase inhibition by the non-

steroidal anti-inflammatory drug naproxen. Journal of biological

chemistry,285(45), 34950-34959.

Ferrero‐Miliani, L., Nielsen, O. H., Andersen, P. S., & Girardin, S. E. (2007).

Chronic inflammation: importance of NOD2 and NALP3 in interleukin‐1β

generation. Clinical & Experimental Immunology, 147(2), 227-235.

Gasteiger, J., & Marsili, M. (1980). Iterative partial equalization of orbital

electronegativity—a rapid access to atomic charges. Tetrahedron, 36(22), 3219-

3228.

Gerard J.; Tortora;: Principles of Anatomy and Physiology 11th Edition.

Harvison, P.J., Guengerich, F.P., Rashed, M. S., &Nelson, S.D.(1988).

Cytochrome P-450 isozyme selectivity in the oxidation of

acetoaminophen.Chemical research in toxicology, 1(1), 47-52.

69

Huwiler, A., Staudt, G., Kramer, R. M., & Pfeilschifter, J. (1997). Cross-talk

between secretory phospholipase A< sub> 2</sub> and cytosolic phospholipase

A< sub> 2</sub> in rat renal mesangial cells. Biochimica et Biophysica Acta

(BBA)-Lipids and Lipid Metabolism, 1348(3), 257-272.

Jorgensen, W. L., & Tirado-Rives, J. (1988). The OPLS [optimized potentials for

liquid simulations] potential functions for proteins, energy minimizations for

crystals of cyclic peptides and crambin. Journal of the American Chemical

Society, 110(6), 1657-1666

Kitchen, D. B., Decornez, H., Furr, J. R., & Bajorath, J. (2004). Docking and

scoring in virtual screening for drug discovery: methods and applications. Nature

reviews Drug discovery, 3(11), 935-949.

Maestro. Version 9.2 Schrodinger, LLC, New York, NY, 2011.

McHowat J., Tappia P.S., Liu S., McCrory R., and panagia V., Redistribution and

abnormal activity of phospholipase A2 isoenzymes in postinarct congestive heart

failure, Am J physiol Cell Physiol, 280, 573-580, 2001.

McHowat, J., & Creer, M. H. (2004). Catalytic features, regulation and function of

myocardial phospholipase A2. Current Medicinal Chemistry-Cardiovascular &

Hematological Agents, 2(3), 209-218.

Miller, S. B. (2006, August). Prostaglandins in health and disease: an overview.

In Seminars in arthritis and rheumatism (Vol. 36, No. 1, pp. 37-49). WB

Saunders.

70

Moriuchi, H., Koda, N., Okuda-Ashitaka, E., Daiyasu, H., Ogasawara, K., Toh, H.,

... & Watanabe, K. (2008). Molecular characterization of a novel type of

prostamide/prostaglandin F synthase, belonging to the thioredoxin-like

superfamily. Journal of biological chemistry, 283(2), 792-801.

Morris, G. M., Goodsell, D. S., Halliday, R. S., Huey, R., Hart, W. E., Belew, R.

K., & Olson, A. J. (1998). Automated docking using a Lamarckian genetic

algorithm and an empirical binding free energy function. Journal of computational

chemistry, 19(14), 1639-1662.

Morris, G. M., Huey, R., Lindstrom, W., Sanner, M. F., Belew, R. K., Goodsell, D.

S., & Olson, A. J. (2009). AutoDock4 and AutoDockTools4: Automated docking

with selective receptor flexibility. Journal of computational chemistry,30(16),

2785-2791.

Muhi-eldeen Z., Essentials of medicinal chemistry update, 2ed Edition,

2010/2011.

Nelson, R. J. (2005). An introduction to behavioral endocrinology . Sinauer

Associates.

Ogorochi, T., Ujihara, M., & Narumiya, S. (1987). Purification and Properties of

Prostaglandin H‐E Isomerase from the Cytosol of Human Brain: Identification as

Anionic Forms of Glutathione S‐Transferase. Journal of neurochemistry, 48(3),

900-909.

RCSB Protein Data Bank. http://www.pdb.org/.

71

Roy, S., Bagchi, D., & Raychaudhuri, S. P. (Eds.). (2012). Chronic Inflammation:

Molecular Pathophysiology, Nutritional and Therapeutic Interventions. CRC

Press.

Sanner, M. F., Python: A programming language for software integration and

development. Journal of Molecular Graphics and Modelling 1999, 17, pp57-61.

Sekharudu, C., Ramakrishnan, B., Huang, B., Jiang, R. T., Dupureur, C. M., Tsai,

M. D., & Sundaralingam, M. (1992). Crystal structure of the Y52F/Y73F double

mutant of phospholipase A2: Increased hydrophobic interactions of the phenyl

groups compensate for the disrupted hydrogen bonds of the tyrosines.Protein

Science, 1(12), 1585-1594

Sharma, S. K., Al-Hourani, B. J., Wuest, M., Mane, J. Y., Tuszynski, J., Baracos,

V., ... & Wuest, F. (2012). Synthesis and evaluation of fluorobenzoylated di-and

tripeptides as inhibitors of cyclooxygenase-2 (COX-2).Bioorganic & medicinal

chemistry, 20(7), 2221-2226.

Sidhu, R. S., Lee, J. Y., Yuan, C., & Smith, W. L. (2010). Comparison of

cyclooxygenase-1 crystal structures: cross-talk between monomers comprising

cyclooxygenase-1 homodimers. Biochemistry, 49(33), 7069-7079.

Smith, W. L. (1986). Prostaglandin biosynthesis and its compartmentation in

vascular smooth muscle and endothelial cells. Annual review of physiology,48(1),

251-262.

72

Tanaka, T., Iwawaki, D., Sakamoto, M., Takai, Y., Morishige, J. I., Murakami, K.,

& Satouchi, K. (2003). Mechanisms of accumulation of arachidonate in

phosphatidylinositol in yellowtail. European Journal of Biochemistry, 270(7),

1466-1473.

Tang, X., Edwards, E. M., Holmes, B. B., Falck, J. R., & Campbell, W. B. (2006).

Role of phospholipase C and diacylglyceride lipase pathway in arachidonic acid

release and acetylcholine-induced vascular relaxation in rabbit aorta. American

Journal of Physiology-Heart and Circulatory Physiology, 290(1), H37-H45.

The PyMOL Molecular Graphic System, Version 1.2r3pre, Schrodinger, LLC.

Thuresson, E. D., Lakkides, K. M., Rieke, C. J., Sun, Y., Wingerd, B. A., Micielli,

R., ... & Smith, W. L. (2001). Prostaglandin Endoperoxide H Synthase-1 THE

FUNCTIONS OF CYCLOOXYGENASE ACTIVE SITE RESIDUES IN THE

BINDING, POSITIONING, AND OXYGENATION OF ARACHIDONIC

ACID.Journal of Biological Chemistry, 276(13), 10347-10357.

Von Euler US (1935). "Über die spezifische blutdrucksenkende Substanz des

menschlichen Prostata- und Samenblasensekrets" . Wien Klin

Wochenschr14 (33): 1182–3.

Weiner, S. J., Kollman, P. A., Case, D. A., Singh, U. C., Ghio, C., Alagona, G., ...

& Weiner, P. (1984). A new force field for molecular mechanical simulation of

nucleic acids and proteins. Journal of the American Chemical Society, 106(3),

765-784.

73

Zarghi, A., & Arfaei, S. (2011). Selective COX-2 inhibitors: a review of

their structure-activity relationships. Iranian journal of pharmaceutical

research: IJPR, 10(4), 655.

74

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

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Synthesis and Identification of New Aminoacetylenic ... · Synthesis and Identification of New Aminoacetylenic Tetrahydrophthalimide Derivatives Anticipated as COXs Inhibitor By Ahmed