Immunosuppressants

Question 1

What are anti-metabolites?

Answer 1

Molecules that disrupt metabolic functions within the body
Antimetabolites have two mechanisms by which it disrupts metabolic functions:

1-Affect metabolic synthesis of functional molecules.

2-Affect biochemical metabolism and recycling of functional molecules.

Immunosuppressant: Alter pyrimidine and purine biosynthesis and metabolism.

1-Methotrexate
2-Azathioprine
3-allopurinol
4-Leflunomide

Question 2

Folic acid

Answer 2

1939-1947 research of yeast extract Vitamin B to reduce tumour growth then research was focused on (Vit B-9). In 1947 clinical trials using folic acid and it is analogues (di and tri glutamates) in leukaemia patients it accelerated their condition.

Research was reconsidered because biological target was unidentified
If it is a receptor which needs an antagonist and if it is enzyme it needs an inhibitor

Folic acid has 3 important parts
1-Pteroyl ring system
2- para amino benzoic acid which is substituted with one or more glutamic acid residues.

3-glutamic acid

Question 3

How to develop anti-metabolites?

Answer 3

1-making methyl derivatives, the methyl group was added unspecifically to folic acid

2- x-methyl pteroylglutamic acid was weakly active which showed a potential to be an anti-metabolite. It showed similar effects with folic acid deficiency a reduction in blood cells formation.

3- Pteroylaspartic acid same as folic acid but with one difference the glutamic acid residue in folic acid is swapped with shorter aspartic acid. Post mortem indicated bone marrow responded to treatment.

4- Aminopterin: change to pteroyl ring with a change of oxygen to an amino group. The potential binding interactions by changing nature of hydrogen bonding and offers possibility for an ionic interaction via the amino group. When used in patient it gave remission and patient developed normal white blood cell count.

4- methylation of aliphatic amine to give methotrexate it gave less side-effects than aminopterin and enhanced potency. It gave a better side-effect profile. Remission observed but continued treatment was necessary. However methotrexate is widely used as an immunosuppressant.

Question 4

What is the role of folic acid?

Answer 4

To understand how methotrexate works biochemically, we need to know the role of folic acid.

Folic acid is involved in the denovo synthesis of purines and pyrimidines and thus in DNA and RNA.
It is metabolised on the body to give N5,N10-methylenetetrahydrofolic acid and this acid acts a CH2 donor it gives a carbon to another molecule.

The main enzyme that is involved in the transformation of folic acid in the N5,N10-methylenetetrahydrofolic acid is DIHYDROFOLATE REDUCTASE DHFR

Question 5

How does DIHYDROFOLATE REDUCTASE DHFR enzyme convert folic acid into N5,N10-methylenetetrahydrofolic acid?

Draw mechanism

Answer 5

DIHYDROFOLATE REDUCTASE DHFR reduces the pteroyl ring of folic acid to dihydrofolate and then to tetrahydrofolate.

Tetrahhyrofolate undergoes a reaction with pyroxidal enzyme and serine to form N5,N10 methylenetetrahydrofolic acid.

In that process Serine becomes glycine and CH2 of serine is given to N5,N10-methylenetetrahydrofolic acid, this molecule is really useful int he synthesis of pyrimidines specifically in the conversion of uradylic acid to thymidine via the enzyme thymdilate synthase.

Question 6

How does N5,N10-methylenetetrahydrofolic acid form pyrimidines?
Draw mechanism

Answer 6

N5,N10-methylenetetrahydrofolic acid is involved in the synthesis of pyrimidines particularly the conversion of uridylic acid to thymidine by the enzyme thymidylate synthase.

Mechanism:
The thiol group (SH) of thymidylate is able to attach to double bind of dUMP this is because it has an alpha beta unsaturated ketone in dUMP and this carbon is electron deficient and this allows bond to form between N5,N10-methylenetetrahydrofolate and dUMP and therefore the transfer of CH2 unit to dUMP to give off dTMP

Question 7

How is N5,N10-methylenetetrahydrofolic acid synthesis Purines?
Draw mechanism

Answer 7

N5,N10-methylenetetrahydrofolic acid is involved in the synthesis of purines.

N5,N10-methylenetetrahydrofolic acid is converted to
N10-formyltetrahydrofolic acid it is still acting as a single carbon CH2 donor but in a different oxidation state.

It plays a crucial role in the formation of purine ring and therefore affects guanine and adenosine synthesis

The synthesis of purines is less affected than the synthesis of pyrimidines.

Question 8

How does methotrexate work, mechanism of action?

Answer 8

Methotrexate is a competitive inhibitor of dihydrofolate reductase

Reduces formation of dihydrofolate, tetrahydrofolate and methylenetetrahydrofolic acid so it can stop the synthesis of purines and pyrimidines.

Methotrexate has a much stronger binding affinity than folic acid but it is still reversible. This strong binding is associated with an additional binding site at it is amino group NH2 which so either working by changing the hydrogen bonding interactions or allowing ionic interactions at that position.

Just like folic acid it is metabolised further to in vivo to give polyglutamates which increase it is size and shape and traps it within cells prolonging it is action as it makes it much larger as a molecule.

Question 9

Draw SAR of methotrexate?

Question 10

What are the rescue therapies used in methotrexate toxicity

Draw SAR of rescue therapy

Answer 10

Leucovorin

Because methotrexate is toxic, rescue therapy is needed sometimes. This is because leucovorin provides an alternative CH2 donor so leucovorin is metabolised to N5-formyltetrahydrofolic acid then further to
N10-formyltetrahydrofolic acid and can be further metabolised to N5,N10-methylenetetrahydrofolic acid. So it can provide a carbon unit donor to both pyrimidines and purines, so Overcoming the effects of methotrexate if somebody revived a high dose of methotrexate.

Question 11

What are the other inhibitors of dihydrofolate reductase?

Answer 11

Trimethoprim used as an anti-bacterial, exploits the fact that microbial dihydrofolate reductase and mammalian dihydrofolate reductase are subtly different so it only binds to bacterial form.

Combination of Trimethoprim and
Sulfamethoxazole given together in the co-drug co-trimoxazole.

This is useful because the synthesis pathways are attacked in 2 different areas; sulphonamide affecting the synthesis of folic acid and dihydrofolate reductase inhibitor reducing the synthesis from folic acid to di and tetrahydrofolate.

Pyrimethamine is used in the treatment of malaria again it exploits the difference between dihydrofolate reductase in mammals and in plasmodium

Question 12

Summary

Answer 12

AI:

Methotrexate is a commonly used medication for the treatment of rheumatoid arthritis (RA). Its pharmacology and mechanism of action involve multiple pathways that contribute to its effectiveness in managing the symptoms and progression of RA.

Methotrexate belongs to a class of drugs known as disease-modifying antirheumatic drugs (DMARDs). It exerts its therapeutic effects through several mechanisms:

1. Inhibition of folate metabolism: Methotrexate acts by inhibiting the enzyme dihydrofolate reductase, which is involved in the metabolism of folate. By doing so, it disrupts the synthesis of nucleic acids, such as DNA and RNA, which are essential for cell division and proliferation. This mechanism primarily affects rapidly dividing cells, including immune cells involved in the inflammatory response seen in RA.
2. Anti-inflammatory effects: Methotrexate has direct anti-inflammatory properties. It reduces the production of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and interleukin-6 (IL-6). These cytokines play a significant role in the inflammatory process in RA, contributing to joint damage and pain. By suppressing their production, methotrexate helps to reduce inflammation and alleviate RA symptoms.
3. Immunomodulatory effects: Methotrexate also modulates the immune system by affecting the activity and function of immune cells. It reduces the activation of T cells, which are involved in the immune response and contribute to the inflammation in RA. Methotrexate can also enhance the production of anti-inflammatory substances, such as adenosine, which further helps to dampen the immune response and decrease inflammation.
4. Suppression of synovial hyperplasia: In RA, there is abnormal proliferation of synovial tissue in the joints, leading to joint inflammation and damage. Methotrexate helps to suppress this synovial hyperplasia by inhibiting the growth and division of synovial cells, thereby reducing joint inflammation and preventing further joint destruction.

Overall, the pharmacology and mechanism of action of methotrexate in treating RA involve its inhibitory effects on folate metabolism, anti-inflammatory properties, immunomodulatory effects, and suppression of synovial hyperplasia. These effects work together to reduce inflammation, alleviate symptoms, slow down the progression of joint damage, and improve the overall outcomes for individuals with RA.

It is important to note that methotrexate is a potent medication that requires careful monitoring and management by a healthcare professional. Regular monitoring of blood counts, liver function, and kidney function is necessary to ensure its safe and effective use in treating RA.

Question 13

Azathioprine

Answer 13

Azathioprine is an anti metabolite that belongs to a class of drugs called immunosuppressants. It is used to treat various autoimmune diseases, such as rheumatoid arthritis, Crohn’s disease, and ulcerative colitis. Azathioprine works by suppressing the immune system, which helps to reduce inflammation and prevent the immune system from attacking the body’s own tissues. It is often used in combination with other medications to manage these conditions. It is important to note that azathioprine can have side effects and should be taken under the supervision of a healthcare professional with monitoring of therapeutic and toxic monitoring parameters and regular LFT and Blood tests.

Anti-metabolites are molecules that distrust metabolic function within the body, affect biochemical synthesis of functional molecules and affects biochemical metabolism and recycling.

Immunosuppressants alter pyrimidine and purine biosynthesis and metabolism.

Azathioprine is a pro-drug of mercaptopurine and has sustained release therefore slower or reduced metabolism.

Question 14

What is the mechanism of action of azathioprine?

Answer 14

Azathioprine is activated by GLUTATHIONE conjugation to give mercaptopurine which can be activated or deactivated by further metabolism.
Two pathways of metabolism

1-Xanthine oxidase metabolism leads to thiouric acid which is inactive.

2-Thiopurine S-methyl transferase we end up with inactive methyl mercaptopurine by introducing a methyl group to the sulphur in the thiol group.

TMPT activity can be altered—pharmacogenomic effect increased activity of azathioprine which leads to build up and toxicity because in some patient this enzyme is under expressed and that means more of azathioprine is fed down the active metabolite route leading to higher levels and activity leading to toxicity.

Question 15

Azathioprine mechanism of action

Answer 15

First step: Glutathione is a tripeptide that has a thiol group which makes it activite.
GS- which is able to attack the imidazole ring and that occurs because the imidazole ring of azathioprine has a low electron density at the carbon and that occurs because we have an electron withdrawing group at the ring.
We get an attack on the carbon by the nucleophile and the electrons are pushed up into and out of the ring into the nitro group but can be fed back into the ring to aromatise the ring leading to loss of one of the thiol groups and that loss is the loss of mercaptopurine so the splitting of the azathioprine molecule. Mercaptopurine can exist in two forms isomers one with thiol own with double bond to the sulphur.

Question 16

How is mercaptopurine deactivated?

Answer 16

Mercaptopurine can be deactivated by undergoing further metabolism and is taken through purine salvage pathway.

This is occurs via the enzyme HPRT Hypoxanthine guanine phosphoribosyltransferase enzyme which introduces a ribose group to the mercaptopurine giving us Thioinosine mono-phosphate which can be further metabolised to give us Thioguanine monophosphate which can be further phosphorylated to give Thio-deoxyguanine triphosphate (DNA unit TdGTP) this molecule is able to incorporate itself into DNA but it is a false nucleotide so it can’t undergo same binding interactions or activities that we normally expect from our DNA or RNA.
Cells must replicate before effect of incorporation is seen.

Major role as a drug molecule is affecting the purine salvage pathway it acts as false substrate.

Question 17

How does mercaptopurine inhibit the de-novo purine synthesis?

Answer 17

Mercaptopurine undergoes further metabolism to inhibit de-novo purine synthesis.

Thiopurine S-methyl transferase TPMT converts TIMP to MeTIMP

Thiopurine S-methyl transferase can deactivate mercaptopurine and it can do this to ioinosine monophosphate and guanine monophosphate and it makes the methyl derivative of both substrates. Methyl thioguanine monophosphate substrate doesn’t matter but the methyl thioinosine monophosphate acts as an enzyme inhibitor and it inhibit the synthesis of ribose unit in the early stage of de-novo-purine synthesis.

methyl thioinosine monophosphate and methyl thioguanine monophosphate.

Question 18

How is xanthine oxidase inhibited in order to stop the production of thiouric acid?

Answer 18

Allopurinol reduces the metabolism of mercaptopurine and enhances its activity by inhibiting the xanthine oxidase enzyme. It does this by stopping the conversion of xanthine oxidase into thiouric acid.

Allopurinol is an isomer of a purine HYPOXANTHINE five membered ring nitrogens are differently positioned pyrazolopyrimidine. It was not effective in enhancing mercaptopurine activity but it showed effect in the biosynthesis of Uric acid and therefore useful in the treatment of gout.

Question 19

What is the biochemistry of gout?

Answer 19

Uric acid is synthesised form
The breakdown of adenine and guanine both undergo deaminase reactions with adenine being further metabolised by xanthine oxidase to give xanthine.
Xanthine is further metabolised by xanthine oxidase to give Uric acid.

Uric acid is a weak acid with low solubility and exist as monosodium salt. If Uric acid is increased in production or reduced metabolism this leads to crystallisation of Uric acid in the body in joints and this causes pain.

Allopurinol stops the production of Uric acid because it inhibits xanthine oxidase so it reduces the production of Uric acid this leads to build up of xanthine and hypoxanthine but they are harmless and can be readily excreted.

Question 20

Allopurinol mechanism of action?

Answer 20

Allopurinol is metabolised in the body to oxypurinol and that has a longer half life and it is active and oxypurinol has more effect.

There is another way we can inhibit Xanthine oxidase febuxostat. It works via its binding in the channel leading to active site and blocks entry of substances binds to both oxidised and reduced form of enzyme it makes it better than allopurinol because it is very selective and very active. It is not a competitive inhibitor it blocks site of entry to channel.

AI:

The mechanism of action of allopurinol involves inhibiting an enzyme called xanthine oxidase. Xanthine oxidase is responsible for the conversion of hypoxanthine and xanthine into uric acid, which is a waste product that can build up in the body and lead to conditions like gout.

Allopurinol is a structural analog of hypoxanthine, and it works by binding to the active site of xanthine oxidase. By doing so, it prevents the enzyme from converting hypoxanthine and xanthine into uric acid. This inhibition of xanthine oxidase activity leads to a decrease in the production of uric acid.

In addition to inhibiting the production of uric acid, allopurinol also has another important mechanism of action. It is converted into a metabolite called oxypurinol, which is also an inhibitor of xanthine oxidase. Oxypurinol has a longer half-life than allopurinol and is responsible for the prolonged effects of the medication.

By reducing the production of uric acid, allopurinol helps to lower the levels of urate in the body. This can prevent the formation of urate crystals in the joints and other tissues, which are responsible for the inflammation, pain, and swelling seen in gout.

It is important to note that allopurinol does not provide immediate relief from gout attacks. It takes time for the medication to lower the levels of uric acid in the body and prevent future attacks. Therefore, it is usually prescribed as a long-term treatment for gout, rather than a short-term solution for acute attacks.

Question 21

Leflunomide

Answer 21

Leflunomide works by blocking de-novo synthesis of pyrimidines. That leads to lymphocytes being unable to replicate as quickly as needed in inflammation. Lymphocytes require a very large increase in pyrimidine biosynthesis and they are not able to support this need from the salvage pathways.

Leflunomide is not the cleanest enzyme inhibitor as it also has activity against some types of tyrosine kinases abs COX when used at higher levels.

70% of Leflunomide activity comes from the metabolite teriflunomide. This metabolism can give two isomers with the Z form being most active at 12 nanomola inhibitor.

Because teriflunomide Z isomer is most active a lot of development to its SAR is done to explore better activity.

Cyano group is essential for activity can’t be removed and the methyl group has limited option for changes but a cyclopropyl is equally active.
The amide is essential if we change the amide to oxygen or carbon it leads to loss of activity because the NH part of the amide is important for the hydrogen bonding interaction.

You can change left side of the molecule the aromatic ring: aromatic ring is important for binding.
You can get activity with methyl, hexyl and cyclohexyl but less activity than just an aromatic ring. We can substitute the aromatic ring and best way is to introduce a lipophilic group but if you put a polar hydrophilic group you get a loss of activity. Position of substitute matter because if it is at a para position it works best.
Para> Meta> Ortho.

Question 22

Mechanism of action of Leflunomide

Answer 22

Reversible inhibitor of dihydroorotate dehydrogenase-rate limiting step.

Leflunomide blocks the fourth step of de-novo pyrimidine synthesis.
This synthetic pathway starts from ATP and can be converted to 2 separate building blocks carbamyl phosphate and aspartate these come together to form N-C aspartate which is then cyclised into dihydroorotate.

The next step is inhibited by Teriflunomide. Conversion of dihydroorotate to orotate via dihydroorotate dehydrogenase.
Dihydroorotate dehydrogenase works together with co-factor of ubiquitin in the normal synthetic route orotate is further transformed into OMP which itself works as a precursor for UMB and the rest of the pyrimidines.

Question 23

Summary of immunosuppressants

Answer 23

Azathioprine is a pro-drug which affects the purine salvage pathway and leads to incorrect DNA activity through incorporation of false nucleotide

Allopurinol reduces the formation of Uric acid and is an effective treatment for gout

Leflunomide effects the pyrimidine de novo synthetic pathway reducing synthesis of uridine.

Question 24

NSAIDs-1
What are prostaglandins?

Answer 24

Prostaglandins act as inflammatory mediators, they are 20 amino carbon fatty acids, they belong to the family of eicosanoids. They are derived from cell membrane phospholipids through enzyme metabolism, catalysed by phospholipase A2.

General name PGXy
y=number of double bonds

Numbering of carbons starts from the carboxylic acid alpha chain down to beta chain. It has 20 carbons PGX X is based on the substituent at 9 and 11

PGD2, PGE2, PGF2 prostaglandins that have the highest inflammatory effect.

Question 25

How do prostaglandins form in the body? Draw the diagram

Answer 25

Breakdown of cell membrane phospholipids this is achieved by phospholipase A2 this gives arachidonic acid. Corticosteroid can inhibit this step as they can inhibit the induction of the enzyme and the arachidonic acid is not further processed.

Arachidonic acid is transformed by COX enzyme into Prostaglandin G2 PGG2 (Leukotrienes follow a different path then arachidonic acid). This step can be inhibited by NSAIDs as they inhibit COX enzyme and stop the conversion of arachidonic acid into PGG2.

PGG2 once formed is highly unstable and is rapidly converted to Prostaglandin H2 PGH2 which is a common precursor of other prostaglandins TXA2 thromboxane and PGI2 prostacyclin.

Since NSAIDs work so early in the pathway they have a wide range of effects on the normal biochemistry of the body.

Question 26

What is COX?

Answer 26

Membrane embedded protein although majority of it is in the cytoplasm. It is important that it’s embedded because arachidonic acid is formed in the membrane and it is highly hydrophobic. The enzyme has a highly hydrophobic channel from the membrane which means the arachidonic acid can’t pass down into the cytoplasm, thr active site is a Haem which allows the formation of radicals.

Question 27

What are the isoforms of COX enzyme?

Answer 27

2 major isoforms of COX; COX-1 & COX-2

COX-1 is consecutive enzyme found in all cells
COX-2 released in inflammation
COX-3 found the brain believed to be target for paracetamol and that is why paracetamol acts as an analgesic and anti-pyretic and not as an anti inflammatory drug.

There is a high homology between COX1&2 because they both have a hydrophobic channel, catalytic site, acylation site and an arginine that allows binding of carboxylic acids.

They have the same biological activity but COX-2 is less selective and can metabolise other fatty acids as well as arachidonic acid.

Differences between COX-1&2
Isoleucine residues in the COX-1 at 434 and 523 altered to smaller valine in COX-2 cause that allows COX-2 to form additional binding pocket which leads to it is selectivity for certain molecules.
Modifications at C and N terminus means numbering is different between the two isoforms.

Question 28

Draw the COX-1&2 binding pocket and label it

Answer 28

Active site- Heme
Isoleucine 523
Arginine 120
Hydrophobic channel

COX-1 has a hydrophobic channel that arachidonic acid passes through to reach active sites.
There two important residues the arginine for binding a carboxylic acid like arachidonic acid or NSAIDs and Isoleucine at 523.

COX-2 has a Valine at 523 reveals an additional hydrophilic site pocket that can be exploited for additional binding interactions.

Question 29

Role of COX in the biosynthesis of PGG2? Draw mechanism

Answer 29

COX cyclooxygenase has 2 roles in the synthetic pathway

1-Endoperoxide synthase (oxidation and cyclisation)

2-peroxidase activity (conversion of peroxide into alcohol=reduction)

NSAIDs block this biosynthesis step of conversion of arachidonic acid into PGG2.

1-COX enzyme facilitate the removal of a hydrogen from the arachidonic acid because this is enzyme catalysed, this removal is very stereoselective.

2-Removal of hydrogen results int he formation of a radical which can rearrange through resonance meaning radical position is in the optimum place for next reaction. Use single headed arrow to show movement of one electron not a lone pair.

3-Once radical is rearranged it reacts with a molecule of oxygen, forming a bond between the arachidonic acid and oxygen.

4-The resulting peroxide contains a radical and reacts again with arachidonic acid through addition to the double bond on the alpha chain.

5-this leads to cyclisation reaction between alpha and beta chain to generate a 5-memebered ring

6-Further rearrangement relocates the radical and allowing for introduction of second molecule of oxygen, here COX exerts it’s second action of introducing a peroxide and then converting it to an alcohol that gives us prostaglandin G2. PPG2 is highly unstable.

Question 30

What is the SAR of salicylates?

Answer 30

Salicylates were the first drug class to act on COX. They’re aeffective but have many side effects.
SAR helps improve Salicylates and reduce their side effects but change is quite limited.

1-COOH carboxylic acid can be changed to an NH2 amide this will act as an analgesic but not as an anti-inflammatory.

2-OH hydroxyl or phenol group is essential for activity and if it is removed activity is decreased.

3-COOH and OH need to be in ortho relationship to each other.

4- Changes can be made to the ring; substitution of the ring para to the hydroxyl group. For example diflunisal.

Question 31

How does aspirin inhibit COX?

Answer 31

Aspirin has a unique mechanism of action differs to other salicylates.

1-Aspirin undergoes a reaction with serine 529 allowing irreversible inhibition of the enzyme.
This reaction is facilitated by two other amino acid residues in the enzyme. There are two tyrosines; Tyrosine 385 and tyrosine 348.

2-Tyrosine 385 forms a hydrogen bond with the acetyl portion of aspirin. This positions it in the correct place for reaction with serine.

3-Hydrogen bond is facilitated by a second hydrogen bond to make it stronger through tyrosine 348.
That docking allows the serine hydroxyl group to undergo a transesterfication reaction.

4- The nucleophilicity of the hydroxyl group is improved by the carboxylate anion of aspirin extracting a proton from the serine.

5- The oxygen is then able to attack the electron poor base of the carbonyl. You get formation of a tetrahedral intermediate and subsequent breaking of the bond between the acetyl group and the rest of aspirin.

6- The serine residue is acetylated. This acetylated residue blocks the access of arachidonic acid into active site. It’s irreversible and it can only be overcome via synthesis of a new COX enzyme by the cell.

Question 32

NSAIDs-2

Answer 32

Competitive non-selective NSAIDs e.g, Ibuprofen.

Largest class of NSAIDs, Non-selective and affect both COX-1&2 equally.

They act by a competitive mechanism which means they are reversible.
They compete for arachidonic acid binding site and stop arachidonic Avon’s the natural substrate from binding.

If the conc of arachidonic acid is increased the effect of NSAIDs will be reduced.

Sub classes of NSAIDs:

1- Arylalkanoic acids (most common)
2- N-arylanthranilic acids
3- Enolic acids

Question 33

SAR of competitive non-selective NSAIDs?

Answer 33

1- Carboxylic acid or an enol or hydroxamic acid for binding for binding at specific arginine, arginine 120. Acidic group can be masked but it must be easily metabolised to ensure this interaction.
Acidic group allows it to accumulate in areas of inflammation.

2- Carbon spacer between acidic group and flat surface, must be one carbon spacer only if carbon spaced is elongated, activity will be reduced. This can be a branched spacer too so it can have a substitution but that branching can be a Methyl group only.

3- Flat surface can be aromatic or heteroaromatic and this mimics the two double bonds in arachidonic acid.

4- There is another lipophilic area which is not in the same plane as the aromatic group. It can be attached by spacer or it can be fused with the first lipophilic area. This is thought to mimic the third double bond of arachidonic acid.

Question 34

How do NSAIDs-2 bind to COX-1?

Answer 34

Arachidonic acid binds at arginine 120, so the hydrogen can react with the COX enzyme to form PPG2, this hydrogen needs to be in the correct position in the active site (heme) for reaction to occur.

When NSAIDs bind it competes for the carboxylic acid binding site with the arachidonic acid and it blocks the binding site so arachidonic acid can’t bind. NSAIDs form an ionic interaction that mimics arachidonic acid I kind interaction with Argnine 120.

Question 35

Indomethacin

Answer 35

Indometacin was first example of arylalkanoic acid structures.

SAR of Indometacin:

1- Carboxylic acid
2- Carbon spacer
3- 2 lipophilic centres not in the same plane as each other.

Carboxylic acid group necessary for activity the more acidic the better. One carbon chain could have a methyl substituents.

Nitrogen not essential for activity.

The indole ring could be highly substituted. Substitution could be enhanced by addition of O-methyl groups Ome, F, Me.

Second lipophilic area best if it is aromatic ring because both Herero aromatic rings and alkyl chains were less active.

If this ring is substituted in the para position with lipophilic electron withdrawing groups like F, Cl and CF3 this could enhance the activity.

This SAR led to the discovery of sulindac.

No longer has indole group but still has rigidity due to double hind acts as a bioisostere.
Presence of double bond means you have 2 isomers and it’s the Z form of that double bond which gives the most active drug molecule.

Sulphone addition to sulindac allows enhanced water solubility.

Question 36

Diclofenac

Answer 36

Diclofenac:
Anti-inflammatory and analgesic

1- Better activity than Indometacin

2- Member of the subclass of aryl and heteroarylacetic acids.

3- SAR consists of all active elements.

4- Chloro substituents play a key role in restricting rotation and ensuring the two lipophilic areas are co-plannar.

5- Proposed mechanism of action is also inhibition of Lipoxygenases and thus decrease the production of leukotrienes.

Question 37

Profens-Arylalkanoic acids

Answer 37

Member of sub class of aryl and heteroarylproprionic acids

Methyl substituents makes the molecule chiral:

1- Iso-butyl giving the Ibu
2- Proprionic acid (substituted spacer) giving the Pro
3- Phenyl giving the Fen

Profens have propionic acid, methyl substituents on the carbon spacer and that makes the molecule chiral.

Racemate but S form is most active
R form undergoes in vivo conversion to the active S form.

Propioniv acid is not easily changed and leads to substantial loss of activity, the aromatic portion could be readily varied like in Flurbiprofen, Ketoprofen and Naproxen.

Question 38

N-Arylanthranilic acids: fenamic acids

Answer 38

Nitrogen bioisosteres of the salicylic acids. Conform to the basic pharmacophore for activity.

SAR differs by the addition of ortho substituents like methyl group in mefenamic acid which forced the ring to be in different planes by restricting rotation.

Question 39

Enolic acids- Oxicams

Answer 39

Piroxicam and Meloxicam
Non-carboxylic acid NSAIDs
Similar anti-inflammatory properties but lower analgesics effect.
Inhibits COC through adopting a conformation similar to the peroxy radical precursor of prostaglandin G2.

SAR:
1- Primary carboxyamide and this should be substituted with a Hetero aryl ring.

2- Aryl rings with meta substituents also can perform well.

3- Alkyl substituents reduce activity.

4- In the sulfonamide group a methyl substituents is the best.

How does it meet original SAR? Where is the acid?
The molecule contains an Enol functional group which means it can lose a proton ( sign if an acid from the alcohol to form ketone.
Keto-enol tautomerism.

These molecules have a Pka of 4-6 with heteroaryl substitution being most acidic making it effective.

This means that physiological PH the proton can be lost from the alcohol. Allowing the keto-enol tautomerism, its able to do this as there are 2 tautomeric ketones available, meaning it is very stable.

Question 40

NSAIDS-3 Coxibs

Answer 40

*Selectively inhibit COX-2 which is expressed as a result of inflammation rather than inhibiting COX-1 which is a consecutive enzyme.

*Competitive inhibitors and at COX-1 they are reversible so increasing the conc of arachidonic acid will overcome Coxibs activity but at COX-2 they are irreversible so increasing conc of arachidonic acid doesn’t help.

Question 41

How do Coxibs bind to COX-2?

Answer 41

Ability to bind to the hydrophilic side-pocket we get selective molecules for COX-2 but they are too large for binding into COX-1

AI:

The mechanism of action of coxibs at COX-2 involves selectively inhibiting the enzyme’s activity. COX-2 is responsible for the synthesis of prostaglandins that promote inflammation, pain, and fever. By specifically targeting COX-2, coxibs prevent the production of these pro-inflammatory prostaglandins, thereby reducing inflammation and pain.

Coxibs are designed to bind to the active site of the COX-2 enzyme, which is the region where chemical reactions occur. By binding to this active site, coxibs block the enzyme’s ability to convert arachidonic acid into prostaglandins. This selective inhibition of COX-2 allows for a reduction in inflammation and pain without significantly affecting the COX-1 enzyme, which is responsible for producing prostaglandins that maintain normal physiological functions.

By specifically targeting COX-2, coxibs aim to provide relief from pain and inflammation associated with conditions like arthritis, while minimizing the risk of side effects associated with non-selective NSAIDs that inhibit both COX-1 and COX-2.

It’s important to note that coxibs may still have some effect on COX-1 at higher doses or prolonged use, which can increase the risk of adverse effects on the gastrointestinal system and blood clotting.

Question 42

SAR of Coxibs? Draw the structure

Answer 42

1- Cis-stilbene double bond that holds the two aromatic rings in the correct orientation for binding. That double bond isn’t just a double bond is held between two ring structures.

2- Ring structure can be any hetroaromatic or aromatic ring. It is possible for it to be 4,5 or 6 membered ring it simply acts as scaffold to present molecule in the correct orientation for binding.

3- Aromatic ring is substituted by sulfur but the sulfur must be in the correct oxidation state so Sulfone or Sulfonamide it matters because it enables specific hydrogen binding interactions.

4- If we change binding site you can get molecules that are selective for COX-1 instead of COX-2, this can occur due to differences in oxidation state of sulfur.

5- Sulfone or Sulfonamide because Sulfoxides and Sulfides can give COX-1 selective molecules.

Question 43

How Coxibs bind to COX-1&2?

Answer 43

Coxibs are large molecules that fit in the hydrophobic channel and basically restrict the entry of the arachidonic acid. Unlike other NSAIDs they don’t form an ionic interaction with arginine.

At COX-2 the sulfur side group of Coxibs fit into the hydrophilic side chain of COX-2 and wedges the drug molecule into the enzyme. This stops arachidonic acid being able to bind at its normal binding site at arginine-120 and this can’t be overcome by increasing the conc of arachidonic acid. This inhibition is irreversible.
The importance of hydrogen bonding interactions of the sulfur group because it is being held within the side pocket and interacting with amino acids.

Question 44

Toxicity

Answer 44

The 2 most selective COX-2 inhibitors that made it to market have both been withdrawn due to toxicity problems. Valdecoxib increases risk of cardiovascular problems. Remaining Coxibs are less selective but have sufficient selectivity to make them highly used NSAIDs.