What Is Histamine? - Looks Simple

Home , Burimamide, Histidine, Metiamide, Timoprazole

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What is histamine? - Looks simple. Made from aminoacid histidine.

histamine histidine H H N N N N N H N H H H O OH pK 's = 5.7, 9.8 a pKa's = 1.9, 6.0, 9.3 How is it made – Handout.

What does it do? – Four different receptors known now. All part of G protein- coupled family.

H1 – immune response that causes hives, makes blood vessels leaky H2 – stimulates stomach acid secretion, H3 – found in CNS and can inhibit release of other neurotransmitters, H4 - found in bone marrow and white blood cells and regulates neutrophil release from bone marrow. It is also expressed in the colon, liver, lung, small intestine, spleen, testes, thymus, tonsils, and trachea

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Hives also known as urticaria, is a kind of skin rash with red, raised, itchy bumps. They may also burn or sting. Often the patches of rash move around.

Hives often occur following an infection or as a result of an allergic reaction such as to medication, insect bites, food, psychological stress, cold temperature, or vibrations. In half of cases the cause remains unknown. Risk factors include having conditions such as hay fever or asthma. Diagnosis is typically based on the appearance. Patch testing may be useful to determine the allergy. Prevention is by avoiding whatever it is that causes the condition. Typically they last a few days and do not leave any long -lasting skin changes . Fewer than 5% of cases last for more than six weeks. The condition frequently recurs.

Treatment is typically with antihistamines such as diphenhydramine and ranitidine. In severe cases, corticosteroids or leukotriene inhibitors may also be used. Antihistamines that block the histamine H1 receptors are the first line of therapy.

About 20% of pppeople are affected. Cases of short duration occur equally in males and females while cases of long duration are more common in females. Cases of short duration are more common among children while cases of long duration are more common among those who are middle aged. Hives have been described at least since the time of Hippocrates. The term urticaria is from the Latin urtica meaning "nettle“. 2 © Oxford University Press, 2013

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Peptic ulcer disease (PUD), is a break in the lining of the stomach, first part of the small intestine, or occasionally the lower esophagus. An ulcer in the stomach is known as a gastric ulcer while that in the first part of the intestines is known as a duodenal ulcer. Common symptoms upper abdominal pain that improves with eating. Other symptoms include belching, vomiting, weight loss, or poor appetite. About a third of older people have no symptoms. Complications may include bleeding, (15% of people) with an ulcer perforation, and blockage of the stomach.

Common causes include the bacteria Helicobacter pylori and non-steroidal anti-inflammatory drugs (NSAIDs). Other less common causes include tobacco smoking, stress due to serious illness, Crohn disease and liver cirrhosis, among others. H. pylori can be diagnosed by testing the blood for antibodies or a biopsy of the stomach.

Diet does not play an important role in either causing or preventing ulcers . Treatment includes stopping smoking , stopping NSAIDs, stopping alcohol, and medications to decrease stomach acid. The medication used to decrease acid is usually either a proton pump inhibitor (PPI) or an H2 blocker . Ulcers due to H. pylori are treated with a combination of medications such as amoxicillin, clarithromycin, and a PPI.

Peptic ulcers are present in around 4% of the population. They newly began in around 87.4 million persons worldwide in 2015. About 10% of people develop a peptic ulcer at some point in their life. They resulted in 267,500 deaths in 2015 down from 327,000 deaths in 1990.

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In the early 1960s only one class of antihistamine was known (H1 now)

First generation antihistamines

Clinically, H1-antihistamines are used to treat allergic reactions and mast cell-related disorders. Sedation is a common side effect of H1-antihistamines that readily cross thbldhe blood–bibbrain barri er; some of fh these d rugs, such as N O diphenhydramine and doxylamine, are therefore used to treat insomnia. H1-antihistamines can also reduce inflammation. diphenhydramine (Benadryl)

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Second generation antihistamines Mepyramine crosses the blood–brain barrier to a much lower degree than the first-generation

N antihistamines. Their main benefit is they N primarily affect peripheral histamine receptors (as opposed to the CNS) and therefore are less O N sedating. However, high doses can still induce the central nervous system drowsiness. The reason Mepyramine for their peripheral selectivity is that most of these compounds are zwitterionic at physiological pH (around pH 7.4). As such, they are very polar, meaning that they do not easily cross the blood– brain barrier and act mainly outside the central nervous system.

Possible synthesis

O O NH2 H N N N H C H O H N N O Cl N O N O Mepyramine

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Second generation antihistamines Ketotifen is a noncompetitive H1-antihistamine and mast cell stabilizer. It is most commonly sold ketotifen O as a salt with fumaric acid, ketotifen fumarate, and is available in two forms. In its ophthalmic form, S it is used to treat allergic conjunctivitis, or the itchy red eyes caused by allergies. In its oral form, it is used to prevent asthma attacks. Ketotifen relieves and prevents eye itchiness and/or irritation associated with most seasonal allergies. It starts working within minutes after N administering the drops. The mean elimination half life is 12 hours.

It was known that histamine stimulated stomach acid, but none of the known antihistamines inhibited acid secretion.

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ANTI-ULCER AGENTS - HISTAMINE ANTAGONISTS Ulcers •Localised erosions of the mucous membranes of the stomach and duodenum •Potentially fatal if untreated •Caused by stress, infection (H. Pylori), smoking and drugs (NSAIDS) •Agrevated by gastric acid (HCl) in the stomach •Estimated 1 out of 10 people will have one sometime in their life

Therapy of ulcers

•Old days: antacids, bland diet (crackers, milk), surgery on stomach

•New days: •Lower the levels of gastric acid a. histami ne antagoni sts and b. proton pump inhibitors •Antibacterial agents vs. H. Pylori •Herbal remedies

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Parietal cells and gastric acid release

Acetylcholine oesophagus Histamine Gastrin + M + 3 + H2 Cck2 cAMP + + + Parietal Cells

- Stomach H+ Cl

Stomach HCl Antrum Pyloric Receptors Sphincter Ion channel Duodenum Proton pump Notes •Release of gastric acid is promoted by acetylcholine, gastrin and histamine

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sulfonated tyrosine O3SO

O CO2 CO2 H Glu Glu Ala Met Phe N Pro Leu Gly Gly Trp Glu Glu Glu N Trp Asp NH3 O H CO O C CO O CO modified Glu 2 2 2 2 gastrin - peptide hormone (made by G cells)

O CH3 H3N H2 CH3 C N N H H3C O C CH H 3 2 N acetyl choline histamine

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Biosynthesis of histamine from the amino acid histidine (with vitamine B6)

H B H B H H O HO O CO2H N O N N H H NH OH H N -2 N O3PO H B H B N histidine (pKa's = 1.8, 6.1, 9.3) H N PLP - vitamine B6 (aldehyde version) H

B B H B H H O O O H C O O N C N N H H NH H NH N NH N N A similar process occurs with H B many other amino acids, including synthesis of neurotransmitters serotonin and N dopamine (which forms N PLP - vitamine B6 N epinephrine and norepinephrine). (imine version) H Glutamate can react in a similar H H manner.

B H O H H B Histamine is released when O N cells are damaged. It N increases the permeabililty H NH N of small blood vessels and NH allows white blood cells HN H into that area to defend H against infection. Allergic N reactions and irritation are histamine common side effects. H N PLP - vitamine B6 (aldehyde version) 11 H © Oxford University Press, 2013

Histamine - Properties Notes •A chemical messenger released by cells •Two possible tautomers •Acts as a local hormone •pKa for the -NH2 group = 9.80. •% ionisation at pH 7.4 = 99.6

•pKa for the imidazole ring = 5.74 •Imidazole ring is slightly ionized at blood pH

Percent conjugate acid and conjugate base plasma pH = 7.4 H N N H N H pKa1 = 5.7 pKa2 = 9.8 N N N HN H HN H HN H H H H [B:] [B:] pH - pKa = log pH - pKa = log What could make the [HB+] [HB+] pKa go higher or lower? [B:] [B:] 7.4 - 5.7 = 1.7 = log 7.4 - 9.8 = -2.4 = log The conjugate acid is [HB+] [HB+] positive and the [B:] [B:] =101.7 =50/1=98%/2% =10-2.4 = 1 / 250 = 0.4% / 99.6% conjugate base is neutral. [HB+] [HB+]

Two tautomer possibilities

BH H H H N N N N

N N N N H H H3N H3N H H3N H3N is closer to the side chain B H N How does the pKa of imidazole compare to pKa1 of histamine? N H (higher, lower or similar)? 12 © Oxford University Press, 2013

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Histamine Actions

Histamine is released by cell damage

Stimulates dilation of blood vessels with increased permeability

White blood cells escape blood vessels and access area of tissue damage

White blood cells combat infection

BUT

Histamine is also released by allergies, asthma, hay fever and insect bites 13 © Oxford University Press, 2013

Classical antihistamines

Commonly used to treat symptoms such as inflammation & itching

MeO NMe2 NMe2 O N

Mepyramine Diphenhydramine Benadryl •But no effect on gastric acid release •Casts doubt on histamine receptors being present on parietal cells •Histamine may promote gastric acid release indirectly

•SK&F propose two types of histamine receptor (H1 and H2) (SKF = Smith, Kline & French)

•H1 - responsible for classical actions of histamine •H2 - proposed as the receptor on the parietal cells in the stomach •Claim that H2 receptors are unaffected by classical antihistamines •Implies classical antihistamines are H1 specific

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Histamine used as the lead compound N

•No known H2 antagonist at the time - no lead compound N H •SK&F decide to use histamine itself as the lead compound H3N •Aim is to alter an agonist into an antagonist •Compare development of propranolol (-blocker) from adrenaline histamine = lead compound

•Need to know SAR requirements for H2 agonists •Analogues tested by their ability to promote gastric acid release

•Does not prove existence of H2 receptor S-epinephrine (adrenoline) was used as a lead compound for labetalol. SKF proposed to follow a similar strategy to find an antagonist for histamine stomach acid secretion.

OH H changes to  adrenergic antagonist O OH H

HO N N Me H2N epinephrine changes to  adrenergic (adrenoline) HO HO antagonist Epinephrine is a nonselective agonist of a variety of adrenergic receptors, Labetalol is used to lower blood pressure and including the major subtypes 1, 2, 1, 2, and 3, triggering a number of heart rate. It was the first drug created that metabolic changes. Binding to -adrenergic receptors inhibits insulin combined both alpha- and beta- adrenergic secretion by the pancreas, stimulates glycogenolysis in the liver and muscle, receptor blocking properties. It was postulated and stimulates glycolysis and inhibits insulin-mediated glycogenesis in that weak blocking of both alpha- and beta- muscle.  adrenergic receptor binding triggers glucagon secretion in the receptors could work together to decrease blood pancreas, increased adrenocorticotropic hormone (ACTH) secretion by the pressure. Labetalol was not found to cross the pituitary gland, and increased lipolysis by adipose tissue. Together, these blood-brain-barrier. effects lead to increased blood glucose and fatty acids, providing substrates for energy production within cells throughout the body.

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SAR for H1 and H2 agonists

Two nitrogen atoms are required for H1 agonist activity - hives All three nitrogen atoms are required for H2 agonist activity – stomach acid

NH3 NH3 HN HN N N

H1 Receptor H2 Receptor

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Strategies for converting agonists to antagonists •Add extra functional groups to find extra binding interactions with the binding site •Extra binding interactions may result in a different mode of binding resulting in a different induced fit for the receptor •Different induced fit may fail to activate the receptor •As a result, analogue binds but fails to activate the receptor •Antagonist is likely to bind more strongly than an agonist

Histamine HN

HN N NH3

N NH3

Receptor (Inactive) Induced Fit - Receptor 'Switched on'

Extra HN Functionality

N NH2 HN

N NH2

Receptor (Inactive) Receptor (Inactive)

Different induced fit 17 © Oxford University Press, 2013

Strategies for converting agonists to antagonists Examples - extra hydrophobic groups? Over 200 compounds were made without any antagonist using hydrophobic groups. R H H 4 H H R1 R1 N N N H H R2 H R2 N N N N N N

histamine possible changes R3 But 2 of them made SKF believe there were 2 different receptors for “hives” vs. “stomach acid”.

4 5 NH3 4 5 NH3

H N H N 3 N 1 3 N 1 2 2 The 2-methylhistamine The 4-methylhistamine was was an H1 agonist (hives). an H2 agonist (stomach acid).

The thought was steric hindrance might restrain rotations of the aminoethyl side chain. H H H NH3 H C H C 4 5 NH3

H N 3 N 1 H N 2 N 18 © Oxford University Press, 2013

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•Next step was to try adding extra hydrophilic groups instead •Aim was to search for extra polar binding regions

N-Guanylhistamine H H Guanidinium N N group H H N N N H H Biological properties

•Partial agonist - promotes HCl release but less strongly than histamine (possible reason?)

•Prevents histamine from fully promoting the release of HCl

 •SK&F suggested that N guanylhistamine was binding to the proposed H 2 receptor, resulting in weak activation

• While present, Nguanylhistamine blocks histamine from binding

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N-Guanylhistamine - Structure and chemical properties •The guanidine group is basic and ionized •Different tautomers are possible •The positive charge can be delocalized over 3 resonance structures •There are many possible shapes

H H H N N NH2 NH2 N NH2 HN HN HN N N N NH2 NH2 NH2

H N NH2 HN

N H2N

The positive charge is more diffuse and can be further away from the imidazole ring. SKF also tried several derivatives without the imidazole ring, but none showed antagonist activity. A decision was made that the imidazole ring was necessary for receptor recognition. This proved to be a very costly mistake when a few years later competitors, Glaxo (ranitidine/Zantac) and Yamanouchi (famotidine/Pepcid) made even more active H2 antihistamines without the imidazole ring that made even more money. 20 © Oxford University Press, 2013

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Binding theory for agonists and antagonists Possible binding regions

antagonist binding region

N N NH N versus 2 H N NH3 H N NH2 imidazole ring agonist H binding region binding region imidazole ring agonist binding region binding region

•Three binding regions are proposed for the H2 receptor - an imidazole binding region (H bonds) and two polar binding regions •Two binding modes are proposed - one for agonists and one for antagonists •The imidazole binding region is common to both binding modes •Proposed that one of the polar binding regions is accessed by agonists and the other by antagonists. The antagonist polar region is further from the imidazole binding region

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Binding of histamine

antagonist binding region antagonist binding region

imidazole ring N binding region N N H N H NH3 imidazole ring NH2 binding region No interaction as an antagonist Strong interaction as an agonist

•Histamine has a short chain •Charged -nitrogen can only reach the polar agonist region •The antagonist binding region is out of range •Histamine can only bind as an agonist, so acts as a pure agonist

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Binding of N-guanylhistamine

antagonist antagonist binding region binding region

imidazole ring N N NH2 binding region N N H H N NH H 2 imidazole ring binding region NH

binding as an weak agonist binding as an antagonist receptor weakly activated H2N NH2 receptor not activated

•Positive charge on the structure is more diffuse and further out •Allows N-guanylhistamine to bind in two different modes •Structure binds as an agonist in one mode and as an antagonist in the other mode, making it a partial agonist

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Chelation binding theory - The proposal bond length H (pm) strong CH106A X N interaction H CC154A HN CN147 A N N O CO143 A H H CF134 A CS181 A X = -NH, -S O Receptor

SK&F proposed that the guanidine moiety interacts with a carboxylate ion in the antagonist binding region by means of two H-bonds and an ionic interaction

The evidence

H S NH H 2 N NH2 N NH2 HN HN HN N NH N 2 S N A B Me B' Me

Structures A, B and B’ are all partial agonists, but structure A has greater antagonist properties. What does that suggest?

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Chelation binding theory

Binding modes for analogues

H H stronger H S N interaction weaker H N N interaction HN H N N O HN H H N X O

O Receptor X = -Me, -SMe O Receptor Positive charge is localized further out Only one H-bond is possible with the leading to better interactions with the antagonist binding region. Charge is also antagonist binding region directed away from the carboxylate ion - weaker antagonist property.

The chelation binding theory was eventually disproved, but it served a purpose in explaining results and pushing the project forward on rational grounds

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less potent N NH2 N NH N H N S NH2 H S NH2

Another possibility considered was However, when the more rigid analog was altering the side chain to make it more found to be less potent it was felt that side conformationally rigid. How could it chain flexibility was necessary. Could be made more rigid? have tried different ring sizes (but we don’t know all of the compounds). Probably wanted minimal changes possible.

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Chain extension strategy Aim: To push the polar guanidine group further out and to increase the interaction with the antagonist binding region. Results H + H H + H N N 3C bridge 3C bridge H H S N N N HN + HN + + N N H H H isothiourea guanidinium ion

Partial agonist. Partial agonist Antagonist activity increases Antagonist activity decreases!

•Antagonist activity of the extended guanidine analogue increases as expected. •Isothiourea analogue might have been expected to have increased antagonist activity since the charge is further out, but it didn’t.

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There are many possible substituents, shapes and polar interactions. Remember, decisions are made by how much acid gets secreted in a rats stomach based on pH reading .

X H H N + H HN N N + + HN N X N H H + N H

many, many possibilities

+ H + H HN N HN N

N H N H N X X N + + H H

possibilities O O O

R O OPO OSO R O R O R O possibilities X X NH 2 NH2 NH2 R NH3 H R H R H R N N H R N N N N N N H R N N H H H H H H H H H H X = -SCH , -CH 3 3 X = -NH2 28 © Oxford University Press, 2013

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Chain extension strategy Proposed binding for 3C extension analogues

H + H H + H N N 3C bridge 3C bridge H H N N S N HN + + HN + N N H H H Stronger Weaker interaction interaction

O O O O

Receptor Receptor

Is there a different form of hydrogen bonding taking place?

Compare 2C bridged analogues. X = NH, S H Stronger X N interaction H HN N N O H H

O Receptor 29 © Oxford University Press, 2013

Summarize effects

Antagonist NH2 binding region HN

NH2 HN HN N Imidazole ring N NH2 binding region HN Agonist binding region NH2 Good binding as an antagonist Partial binding as an agonist

NNH Partial agonists with good antagonist HN activity (X= Me or SMe) N HH

NNH HN N HH Stronger interaction

O O

Receptor X=NH ,SMe,Me 2 30 © Oxford University Press, 2013

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NNH Partial agonists with good antagonist HN activity (X= Me or SMe) N HH X

NNH HN N HH Stronger in terac tion

O O

Receptor X=NH2,SMe,Me

NH2 NH2 S HN NH X HN 2 HN

N N

Agonist Agonist binding region binding region Poor binding as an antagonist Good binding as an antagonist

Emphasis now switches to the types of binding interactions at the polar binding regions 31 © Oxford University Press, 2013

Distinguishing between the polar binding regions 1 Strategy Replace the ionic guanidine group with a neutral H-bonding group

2 Rationale May allow a distinction to be made between the two polar binding regions. Ionic bonding is known to be crucial for the agonist binding region It may not be crucial for the antagonist binding region

3 Method Replace the basic guanidine moiety with a neutral thiourea group

Thiourea SK&F 91581 HN H NNH2 No agonist activity N Very weak antagonist S

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Distinguishing between the polar binding regions

Comparison between the thiourea and guanidine groups Similarities - Planarity, geometry, size, polarity, H-bonding ability Differences - Thiourea is neutral while guanidine is basic and ionised

Thiourea Guanidine H H N NH2 N NH2 R R e- withdrawing S NH2 Neutral Basic R = 2C = weak antagonist R = 3C = better antagonist

Conclusions - •Agonist polar region involves ionic and H-bonding interactions •Antagonist polar region may not require ionic interactions. H-bonding may be sufficient

33 © Oxford University Press, 2013

Chain extension (again), and addition of N-methyl group Strategy

Extend the carbon bridge to 4 carbons (another CH2 in the side chain) Pushes thiourea group further out May increase the interaction with the antagonist binding region

Results N S What does methyl do? Chain extension Discovery of burimamide 1. more hydrophobic? CH3 2. desolvation effect N N N (less water to slough off) Properties of burimamide H H •100 times more active as an antagonist compared to N-guanylhistamine

•No antagonist activity at H1 receptors •Activity still too low for oral use

Conclusions •Chain extension leads to a pure antagonist with good activity •Chain extension allows a better overlap of the thiourea group with the antagonist binding region •N-methyl effect (hydrophobic pocket or desolvation?) •Establishes the existence of H receptors 2 34 © Oxford University Press, 2013

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The imidazole ring Structures – more complicated than appears at first look

B H H H

N N N N    

    N R N R N R N R

BH H H H I III II

•Imidazole ring can exist as two tautomers (I) and (II) as well as an intermediate with two resonance forms (III) •Which of these is preferred? •Which nitrogen atom in III is more positive? (Depends on what “R” is)

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The imidazole ring basicity

Percent ionization of imidazole ring at physiological pH  7.4

H H H N N e- withdrawing N e- donating S

CH3 H N N N N NH3 N

H H H H Histamine (pKa  5.74) Imidazole (pKa  6.80) Burimamide (pKa = 7.25) Ionization  2% of imidazole ring Ionization  25% Ionization  40% of imidazole ring (H is reference group) Conclusions •The imidazole ring of histamine is not ionized when it interacts with the imidazole binding region

•The ionized form of burimamide is unlikely to bind well

•Decreasing the basicity and ionization of the imidazole ring in burimamide closer to that of histamine may increase the binding interactions to the imidazole binding region

•An extra methyl on the the end thiourea group made it a stronger antagonist (probably tried many variations)

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Varying basicity of the imidazole ring Strategy Convert the side chain of burimamide to a more e-withdrawing group

Thiaburimamide – S has slightly longer bonds, slightly more polar

(Kp = partition coefficient, more later)

H pK = 6.25 N S a e- withdrawing Increase in antagonist activity S CH3 Non-ionised imidazole is favoured N N N Ionization  7% H H Compare:

H H H

N N N S e- withdrawing e- donating

CH3 H N N N N NH3 N

Tautomer studies of the imidazole ring

B H H H

N N N N    

    N R N R N R N R

BH H H H I III II Tautomer I vs tautomer II H inductive N effect of •Favoured tautomer for histamine is I  histamine •Inductive effect decreases with distance  R •N is less basic than N N •N is more likely to be protonated •Favoured tautomer for thiaburimamide is also tautomer I

Strategy •Increase the basicity of N relative to N to further increase the percentage population of tautomer I vs tautomer II •Add an electron-donating group to the imidazole ring closer to N than to N

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Tautomer studies e- donating H CH3 Metiamide N S

S CH3 N N N e- withdrawing H H Notes •10 fold increase in antagonist activity versus burimamide •Electron-donating effect of methyl group is more significant at N •Increases basicity of N •Favours tautomer I over tautomer II

•But, increases in pKa to 6.80 •Increase in ionization to 20% •Increase in the population of tautomer (I) outweighs the increase in population of the ionised structures (III) •Unacceptable side effects - kidney damage Alternative rationales •The increases in activity for thiaburimamide and metiamide may be due to a conformational effect •The thioether link increases the length and flexibility of the side chain •This may lead to increased binding •The methyl substituent may orientate the side chain into the active conformation – i.e. the methyl group acts as a conformational blocker 39 © Oxford University Press, 2013

Substituted oxygen for sulfur in the side chain to make it more electron withdrawing

H bonds? Me H H H N O H H O N N S H H bond? H H S N S H N O O Me N Me N N N N N O N Oxaburimamide H H H H possible change in shape because of possible desolvation cost due to H bonding with N-H? stronger H bonds with water?

•Less potent than burimamide despite the side chain being more electron withdrawing, should be less ionized

Possible explanations •The ether link is smaller and less flexible •The eth er may b e i nvol ved i n a ‘b ad’ h yd rogen b ond •There may be an energy penalty involved in desolvating the oxygen prior to binding

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From metiamide to cimetidine

•The side effects of metiamide (decrease in white blood count) may be due to the thiourea group •The thiourea group is not a natural functional group •Replacing thiourea with a natural functional group may remove the side effects

H S O N

R CH 3 R CH3 R CH3 N N N N N N

H H H H H H

Thiourea - Toxic side effects, few out Urea Guanidinium, of 700 patients had granulocytopenia Drop in activity (positive side chain) (low white blood cells)  5% as active Drop in acti vit y, (  5% as active) but no agonist activity! Conclusions •First guanidine analogue to be a pure antagonist •The longer 4C chain pushes the guanidine unit beyond the agonist binding region, but not beyond the antagonist binding region

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From metiamide to cimetidine wrong compounds Binding interactions for the 4C extended guanidine

an antagonist an antagonist + binding region binding region H2N

NH2 NH + H S H N N

N N S imidazole ring imidazole ring + binding region agonist binding region agonist NH binding region binding region NH2

Binding as an antagonist H2N + was good Not binding as an agonist was good

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From metiamide to cimetidine Strategy: •Retain the guanidine group, but try to remove the positive charge (use dipoles instead) •Guanidine is a natural group present in the amino acid arginine •Increase activity of drug by making the guanidine group neutral •Add a strong electron-withdrawing group to decrease basicity (many tried)

•(NO2 and CN), both showed similar antagonist activity

O O H CH 3 H H H H H N N N N N NH2

S CH3 R CH3 R CH3 R CH3 N N N N N N N N N

H H H H H H H H pK  11.5 pK  8 pKa  12.5 a a pKa  7.5 O N H O H C H N H N CH N N O S body pH  7.4 3

R CH3 R CH3 R CH3 R CH3 N N N N N N N N

H H H H H H H H pKa  7 pKa  -0.4 pKa  -0.9 pKa  -1.2

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Electron-withdrawing cyano group Cimetidine (Tagamet)

Properties •Comparable activity to metiamide •Fewer sideeffects

•Inhibits H2-receptors and lowers levels of gastric acid released •Marketed in 1976, greatly reduced surgeries •Biggest selling prescription drug until ranitidine (1981) •Metabolically pretty stable •Inhibits cytochrome p450 enzymes (side effect) •Drug-drug interactions with diazepam, lidocaine and warfarin (side effect) •More recent studies suggest it may help chronic tendenitis of the shoulder and as a supplemental drug in colorectal cancer

H H OH O H H H H CH H O CH 2 Fe+4 CH 2 N N 2 N N Fe+4

N R N R N R N R Fe+3 metabolites further oxidation possible 44 © Oxford University Press, 2013

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Cimetidine (Tagamet) •The cyanoguanidine group acts as a bio-isostere for the thiourea group

H N H C N N N S

S CH S CH3 3 N N N N N N

H H H H

•Both groups are planar and of similar geometry

•Both groups are polar but essentially neutral

•Both groups have high di pol e moments

•Both groups have low partition coefficients (Kp values, more water soluble)

•The cyanoguanidine group is weakly acidic and weakly basic - amphoteric

•The cyanogaunidine group is not ionised at pH 7.4 45 © Oxford University Press, 2013

New Consideration - Possible tautomers of the cyanoguanidine group (Cimetidine = Tagamet)

•The favoured tautomer is the imino tautomer

N N N H C C H C N N N

R CH3 R CH3 R CH3 N N N N N N

H H H H I II III Amino Tautomer Imino Tautomer Amino Tautomer

•The electron-withdrawing effect of the CN group is an inductive effect •The inductive effect is felt most at the neigggghbouring nitrogen •The neighboring nitrogen is least likely to form a bond to hydrogen •The imino tautomer allows pi resonance with the cyano substituent (and N lone pairs below)

46 © Oxford University Press, 2013

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The cyanoguanidine group – possible conformational isomers (and configurational?)

Drawn with "CN" on the right (same as CH3). All are presumed to be flat. steric interaction N N N N Z C E C E C Z C N E N E N Z N Z R H H H H CH R CH N N 3 3 N N N N N N

H CH3 R CH steric 3 R H H H (Z,E)interaction (E,E) (E,Z) (Z,Z) Drawn with "CN" on the left (same as R). All are presumed to be flat. steric interaction N N N N C E C E C Z C Z Z N E N E N Z N

R H H H H CH3 R CH3 N N N N N N N N

H CH3 R CH3 R H H H (Z,E) steric interaction (E,E) (E,Z) (Z,Z) •Notes •The E, E and Z,Z conformations are not favoured because of steric interactions (X-ray and NMR evidence support this) •Bad news for the chelation bonding theory because Z,Z conformation is not favored •Chelation to the one carboxylate group requires the E,E or the Z,Z conformation 47 © Oxford University Press, 2013

The cyanoguanidine group – new idea as to possible binding modes

Old idea for interaction at active site had 2 H bonds. steric interaction - not favored N Z C N Z

R CH3 N N (Z,Z) H H

H bonds Hbonds

O O

48 © Oxford University Press, 2013

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Rigidification nitropyrrole analogue

O CH H O 3 N E N H N S Z N N H

•Unable to adopt the E,E or Z,Z conformation •Strongest analogue of cimetidine (antagonist) •Locked into the active conformation •Can only interact with two separate H-bond acceptors in the antagonist binding region

49 © Oxford University Press, 2013

Desolvation theory – would an increase in hydrophobic character help activity?

H H O

H R R G R O G G Desolvation Binding H Energy penalty Energy released (Cost) (Gain) O H H

hydrophobic hydrophilic

•A guanidine unit is highly polar and highly solvated •Solvated water must be removed prior to binding •An energy penalty is involved •The ease of desolvation may affect strength of binding and activity •A urea group is more hydrophilic than a cyanoguanidine group •May exp lai n l ower acti vit y of th e urea anal ogue

50 © Oxford University Press, 2013

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Reminder – partition coefficient at specific pH values

[neutral compound]1-octanol [all forms compound]1-octanol log Poct/H2O = log log Doct/H2O = log [neutral compound]water [all forms compound]water

The partition-coefficient (P) or distribution-coefficient (D) is the ratio of concentrations of a compound in a mixture of two immiscible phases at equilibrium.

These ratios are a measure of the difference in solubility of the compound in two phases (water and 1-octanol).

The partition-coefficient generally refers to the concentration ratio of un-ionized species of compound whereas the distribution-coefficient refers to the concentration ratio of all species of the compound (ionized plus un-ionized) and depends on pH.

The distribution coefficient is a partition coefficient at a specific pH value.

The partition coefficient measures how hydrophilic or hydrophobic a chemical substance is. Hydrophobic drugs with high octanol/water partition coefficients are mainly distributed to hydrophobic areas such as lipid bilayers of cells, while low octanol/water partition coefficients are found primarily in aqueous regions such as blood serum.

51 © Oxford University Press, 2013

Solubility and partition coefficients of straight chain functional groups in 100 g water

OH OH OH OH C5H11OH C6H13OH C7H15OH C8H17OH C9H19OH CH3 0.0058 mol 0.0008 mol 0.0058 mol 0.0008 mol 100% 100% 0.11 mol 0.030 mol solubility = 100% 2.6g 0.6g 0.09g 0.6g 0.09g miscible miscible miscible 7.9g +1.51 +2.03 +2.62 +2.97 +4.02 log Poct/H2O = -0.74 -0.38 +0.25 +0.84

O O O O O O O O

C C C C C C C C C H OH C H OH H OH H3C OH C2H5 OH C3H7 OH C4H9 OH C5H11 OH 6 13 7 15

100% 100% 100% 100% 4.9g 1.1g 0.2g 0.07g solubility = miscible miscible miscible miscible (guess)

+1.39 +1.92 +2.6 +3.05 log Poct/H2O = -0.54 -0.32 +0.33 +0.79 (guess)

O O O O O O O O

C H H H H H H 100% 100% 100% 100% soluble (?) 100% 27g 6g solubility = miscible miscible miscible miscible miscible log P = +0.45 +0.88 +1.3 oct/H2O +0.35 +0.59 (guess) -0.24 +0.29 +0.84 web site with lots of partition coef. values: https://www.nist.gov/sites/default/files/documents/srd/jpcrd367.pdf On-line partition coefficient calculator: http://www.molinspiration.com/services/logp.html

52 © Oxford University Press, 2013

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Reminder [compound]1-octanol log Poct/H2O = log [compound]water

There is good correlation of calculated partition coefficients with experimental partition coefficients

log D

1.5

1.0

What might a distribution coefficient curve 0.5 look like for the following compound? O 0.0 pH 2 4 6 81012 14 H2N -0.5 OH -1.0

Desolvation theory - hydrophobic analogues H CH3 N X

S CH3 N N N Strategy •Increase the hydrophobic character of the planar aminal system H H •Implies less solvation by water Aminal system (Z) •Implies less of an energy penalty associated with desolvation •Implies easier binding at active site and a stronger activity

Result •Antagonist activity of analogues increases as hydrophobic character increases Lipinski’s rules •Lipinski's rules state (rules of thumb) •No more than 5 hydrogen bond donors (the total number of N-H and O-H) •No more than 10 hydrogen bond acceptors (all N and O atoms) •A molecular mass less than 500 daltons (some say < 300) •An 1-octanol/water partition coefficient , log P < 5 (some say log P < 3) •There are many exceptions to Lipinski's Rules. •Lipinski said, in general, an orally active drug has no more than one violation and Veber’s rules •Polar surface area (all the O and N < 140A2 and < 90A2 for the BBB) and •The number of rotatable bonds (< 7-10) has been found to better discriminate between compounds that are orally active and those that are not

Veber’s paper: J. Med. Chem. 2002, 445, 2615-2623 54 https://pdfs.semanticscholar.org/8493/576a7927322ebb79c353bc1c1100a186eb50.pdf© Oxford University Press, 2013

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Desolvation theory - hydrophobic analogues Result •Antagonist activity of analogues increases as hydrophobic character increases

log (Act)

NCN ‘Outlier’ HN NHMe NNO CHNO2 2 . S 6.0 . HN NHMe HN NHMe. HN NHMe . S NNO2 HN NH2 . NCN . . HN NH2

HN NH2 O 5.0 . N

O HN N O . H N HN NHMe HN N . H O 4.0 . HN NH2

Desolvation theory - hydrophobic analogues vs. activity

NO2

R CH3 N N H H log A = 5.5 log P = -1.4 O

CN NO2 O N N S N R CH R CH 3 R CH3 R CH3 3 R N N N N H H N N N N H H N N H H H H H H log A = 5.0 log A = 4.6 log A = 6.1 log A = 5.8 log A = 6.0 log P = -0.95 log P = -1.35 log P = -0.8 log P = -0.8 log P = -0.7

O CN NO2 O N N S N R R R R R N NH2 N NH N NH H 2 2 N NH N N H H H 2 H H log A = 4.1 log A = 3.9 log A = 5.3 log A = 5.4 log A = 5.3 log P = -1.6 log P = -1.5 log P = -1.15 log P = -0.9 log P = -1.1

Log (activity) = 2.0 log P +7.4 Greater activity than expected ? Lower activity than expected ? 56 © Oxford University Press, 2013

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Dipole moment theory – dipole-dipole interactions

•A dipole-dipole interaction takes places between the drug and the binding site on

approach of the drug Approach and orientation Strong hydrogen bonding O2N

H •The dipoles line up and orientate the N NMe drug R H O2N

H •Good interaction with the binding site N NMe R H occurs if the binding groups are positioned correctly with respect to the Receptor Receptor binding regions - results in good activity surface surface

Dipole ideal H-Bonding regions Moments

Approach and orientation Weak hydrogen bonding O •Poor interaction occurs if the binding N N groups are not positi oned correctl y with H O respect to the binding regions - leads to H N R N N poor activity H H N R less than ideal Receptor Receptor surface surface

QSAR study including dipole-dipole interactions

•The orientation of the dipole is more important than its strength •Log (activity) = 9.12 cos  +0.6logP -2.71

30o  Ideal dipole orientation X Observed dipole H orientation NN RH

•Activity increases as hydrophobicity increases (log P) •The ideal angle of the dipole moment = 30o •At 30o, = 0o and cos  = 1 •At 30o, Log (activity) = 9.12 + 0.6 log P - 2.71 •When dipole moment does not equal 30o, cos < 1 and activity falls

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O O N O C O N N N N O N N O H CH3 H CH N N H H H 3 N N N N N N N N R H H R H R H R H  = 13o R  = 2o  =-6o  =27o o  = 13.1  = 33  = 13.1  = 16.7  = 14.2  = 15.1 higher lower activity lower higher higher activity activity activity activity

log A = 9.12 cos  + 0.6 log P - 2.7

A = activity  = deviation of angle of dipole moment from vertical (receptor site?) P = partition coefficient (related to solubility in water vs. 1-octanol

H N C N N

Cimetidine S H N N N

H CH3

Ranitidine (Zantac, Glaxo) Glaxo merged with SmithKline O Ranitidine H N N Cimetidine C 4 3 O N N N 5 2 S H S H N N O N N N 1 H CH 3 H CH3 •Contains a nitroketeneaminal group (other flat groups will work too) •Different heterocyclic ring, has a furan ring •Fewer side effects •10 times more active •Longer duration of action

•Replacing sulfur with methylene (CH2) reduces activity •Placing sulfur next to ring reduces activity •Replacing furan with a more hydrophobic ring lowers activity •252,5-disubstitution pattern works best •The methyl groups on the nitrogen can be varied without lowering activity •That same N,N-dimethylaminomethylene substituent on Cimetidine lowers activity •Methyl substitution at position 3 on the furan ring eliminates activity •Methyl substitution at position 4 increases activity (indicates a different sort of interaction than Cimitadine) •Introduced in 1981, was making over $7,000,000 per day •Took over from Cimetidine as the most widely sold prescription drug in the world 60 © Oxford University Press, 2013

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Famotidine (Pepcid)

O sulfonylamidine NH2 H N Famotidine group S Cimetidine C NH 3 2 4 N N N O 2 5 S H S H H2N N N N S N N N guanidine group 1 H CH thiazole 3 H CH3 ring •Different heterocyclic ring, thiazole ring •30 times more active than Cimetidine •Side chain of sulfonylamidine group, but not essential, needs to have a dipole moment and be able to hydrogen bond, more variety possible •Side chain length can be 4-5 atoms

•Replacing S with CH2 in side chain increases activity •Methyl group at position 4 decreases activity •3 of the 4 hydrogen atoms on the guinidine group are required for activity •Has no effect on cytochrom P450 and does not appear interact with other drugs •Pepcid is also used with dogs and cats with acid reflux •Pepcid is used with H1 antagonists for severe urticaria •Relief of heartburn, acid indigestion, and acid reflux (GERD) •Part of a multidrug regimen for Helicobacter pylori eradication (in combination with antibiotics) •Given to surgery patients before operations to reduce the risk of aspiration pneumonitis. •It has even been used as a co-drug in treatment-resistant schizophrenia

•About 80% of stomach ulcers are healed in 4-6 weeks of treatment with all of these drugs 61 © Oxford University Press, 2013

Parietal Cells, the Proton Pump and Inhibitors (PPI)

M3 H 2 Cck2

ATP ADP + Pi Receptors

Proton pump

H+ K+ Ion channels - Cl Canaliculus HCl Lumen of the stomach The proton pump •Pumps protons out of the parietal cell and potassium ions back in (H+, outside) / (H+, inside)  3,000,000 / 1 [highest ion differential in the body] •Requires energy - provided by hydrolysis of ATP to ADP, catalysed by ATPase •The proton pump is also called H+/K+-ATPase •Chloride ions depart through a separate ion channel •HCl is formed in the canaliculus •The potassium ions exit the parietal cell as counter ions for the chloride ions and are then pumped back in •A separate potassium ion channel is used for K+ ions leaving the cell

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Proton Pump Inhibitors

methylsulfinyl methylsulfinyl 'linker' H O N O F 'linker' H O N O CH pyridine N S 3 pyridine N S F N H3C N benzamidazole benzamidazole

H3C O O Pantoprazole H3C O O Omeprazole H3C H3C

methylsulfinyl methylsulfinyl 'linker' H O 'linker' H N O N

pyridine N S pyridine N S

N N benzamidazole benzamidazole

O CH 3 Lansoprazole O CH3 Rabeprazole

F3C

H3C O

•All of these are prodrugs, minimal side effects until they reach their target •Activated by the strongly acidic conditions (pH < 4) found in the canaliculae of parietal cells in the stomach wall 63 © Oxford University Press, 2013

Mechanism of inhibition

OCH OCH3 OCH3 3

+ B N H N N H H H S N S N pH < 4 N S O O O N N H N OCH OCH3 3 B H H OCH3 BH spiro intermediate

OCH3 OCH3

OCH3

N N S N S S N N Proton HN N N Proton S S Pump Pump H HO N BH H

irreversible B inhibition B OCH3 OCH (covalent binding) H 3 OCH3 sulphenic acid intermediate H O BH Pyridinium sulphenamide structure 64 © Oxford University Press, 2013

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Design of omeprazole (Losec) The lead compound N S

NH2 CMN 131 •Originally an antiviral drug •Inhibited gastric acid secretion (side effect) •Liver toxicity due to the thioamide group (side effect) •Many derivatives were made to try and overcome this Modification of the thiourea group N

S N

H 77/67 HN •Also inhibited gastric acid secretion (desired effect)

•The pyridine ring and bridging CH2S moiety are important to activity •Many “heterocycle–X-Y-heterocycle” two atom bridges were tried

•Left side pyridine and -CH2-S- bridge worked best, but right heterocycle was changed benzimidazole

Design of omeprazole (Losec)

Modify the imidazole ring N benzimidazole

S N

pyridine HN H 124/26

Increase in activity due to the benzimidazole ring Sulfoxide metabolite had increased activity over H 124/26

Drug metabolism studies N O

S N

HN Timoprazole •Timoprazole formed by oxidation of sulfur on of H124/26 •Timoprazole was the active drug (prodrug) •Pyridinylmethylsulfinyl benzimidazole structure •Side effect – inhibited iodine uptake by the thyroid gland, so no clinical trials •More analogs were synthesized to get around the iodine problem

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Design of omeprazole (Losec)

N O

S N O

CH3 HN OCH3 Picoprazole CH3

•Potent antisecretory properties over long periods of time •No toxic side effects on the thyroid •No other serious side effects using animal studies •Picoprazole was found to be the most effective antisecratory compound ever tested in humans •At this point (1977) the proton pump was discovered and identified as the target of picoprazole •Various substituents were tried on the pyridine ring to make it more basic/nucleophilic

Add substituents to the heterocyclic rings

Design of omeprazole (Losec) Substituents varied on the pyridine ring

H3C N O

H3C S N O O

CH3 HN OCH3 H 159/69 CH3 •Substituents which increase the basicity/nucleophilicity of the pyridine ring are good for activity •Consistent with the mechanism of activation •Methyl substituents at the meta position have an inductive effect •Methoxy substituent is more effective at para position than meta position because of resonance effect

H C H C H3C H3C 3 3 N N N N

H C H C H3C H3C 3 3 O R O R O R O R

CH CH CH3 CH3 3 3

•H159/69 is potent but chemically too labile (unstable in vivo)

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Design of omeprazole (Losec) Substituents varied on the benzimidazole ring

•Substituents were varied to get the right balance of potency, chemical stability and synthetic accessibility •Omeprazole was found to have the best balance

H3C N O

H3C S N O O

CH3 HN CH3 Omeprazole •Launched in 1988 by Astra •World’s biggest selling drug (over $6 billion in 2000 alone, > $16,000,000 per day) •Patents ran out in 2001 (Europe and US)

Esomeprazole (Nexium) •Omeprazole has an asymmetric centre •The S-enantiomer has better potency and pharmacokinetic profile •Undergoes less metabolism than the “R” enantiomer so maintains higher levels of the drug in the body •Example of chiral switching, started a new patent •The “R” enantiomer was approved in the U.S. In 2009 as Dexlansoprazole (another patent)

H3C N O

H3C S N O O

CH3 HN CH3 Nexium "S" enantiomer of Omeprazole

H2 Metabolites C HO N O N O

H C S N H3C S N 3 O O O O CH HN CH3 HN 3 CH CH3 3

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Synthesis of Omeprazole

H3C H3C N HS N N

H3C O NaOH H3C S N O O HN CH 3 O

CH3 Cl CH3 HN CH3 pyridine benzimidazole O portion portion Cl OH O

meta chloroperbenzoic acid (mCPBA)

H3C N O

H3C S N O O

CH3 HN CH3 Omeprazole

Features of Helicobacter pylori

•Spiral, curved bacterium •Grows best in oxygen concentrations of 5% •Naturally present in the stomachs of many people •Attaches to a sugar molecule on the surface of the cells lining the stomach wall •The organism secretes proteins and toxins that inflame the stomach lining •Responsible for the recurrence of ulcers •The organism is protected by the mucus layer •A pH gradient across the mucus layer means that the pH is near neutral at the stomach lining •The helicobacter pylori bacteria was discovered in the contents of Otzi's stomach (5000 year old frozen ice man). Helicobacter pylori, is a gram-negative, microaerophilic bacterium found usually in the stomach. It was identified in 1982 and found that it was present in a person with chronic gastritis and gastric ulcers, conditions not previously believed to have have a microbial cause. It is also linked to the development of duodenal ulcers and stomach cancer. Over 80% of individuals infected with the bacterium are asymptomatic, and it may play an important role in the natural stomach ecology.

More than 50% of the world's population harbor H. pylori in their upper gastrointestinal tract. H. pylori's helical shape is thought to have evolved to penetrate the mucoid lining of the stomach.

Individuals infected with H. pylori have a 10 to 20% lifetime risk of developing peptic ulcers and a 1 to 2% risk of acquiring stomach cancer. 72 © Oxford University Press, 2013

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Features of Helicobacter pylori

Stomach contents pH = 2

Mucus layer pH = 4

H. pylori pH = 7

•Bacterium produces urease enzyme, releases basic ammonia •Neutralises any acid in the local environment of the bacteria

Treatment of Helicobacter pylori

•Triple therapy of a proton pump inhibitor and two antibiotics •Antibiotics work better at a higher pH than is normally present in stomach •The proton pump inhibitor is present to raise the pH

Example

OH NH H3C 2 N O H N S H3C S N O O2N N O O N HO CH3 HN CH CH3 O 3 Omeprazole Amoxicillin CO2H Metronidazole