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Dr Jess Healy: PHAY2003 Cardiovascular drugs (Medicinal chemistry) Lecture 2 !Angiotensin II receptor (AGIIR) blockers: In the first lecture we looked at the developed of ACE inhibitors. These inhibited the enzymatic activity of ACE, i.e. prevented the conversion of angiotensin I to angiotensin II. In this lecture we will look at the development of angiotensin II receptor blockers, which prevent angiotensin II binding to its receptor. Binding of angiotensin II to its receptor triggers a cellular response, which ultimately leads to vasoconstriction and increased blood pressure. Both strategies (i.e. inhibition of ACE or blocking the angiotensin II receptor) have the same effect and result in decreased blood pressure. Efforts to develop AGIIR began in the 1970’s. The first inhibitors were peptidomimetics of angiotensin II – competing with angiotensin II for the receptor binding site. An example Saralasin is shown below. The structural changes relative to angiotensin II are shown in blue. The N-terminal Asp (aspartic acid) is changed to a N-methylglycine and the C-terminal Phe is an isoleucine (Ile). These peptides, however, were 1) highly polar and charged, 2) had a short lifetime and duration of action, 3) were not orally bioavailable and 4) although weak antagonists of AGIIR the peptides also showed partial agonist activity. Agonist = a substance which on binding to a receptor initiates a physiological response (e.g. angiotensin II binds to AGIIR and results in increased blood pressure) Antagonist = a substance which interferes with or inhibits the action of another (e.g. interferes with the action of angiotensin II on AGIIR)

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The next break through came in the 1980’s, when it was noticed that imidazole-5-acetic acid analogues (1) block the angiotensin II receptor and have an antihypertensive effect. An example S8038 (1) is shown to the left: S8038 is a weak antagonist of AGIIR.

But did not show the partial agonist behaviour of Saralasin and related peptides – a promising lead compound for the development of a drug. Comparing S8308 and angiotensin II (using computational modelling) it was noticed that they contain some similar structural features. See below: 1)

HN

O OH

NO

NH

NH

NO

HN

O

NH

OH

O

HN

O

NH

HN

NHH2N

ONH2O

HOO

Angiotensin II

HN

O OH

NO

NH

NH

NO

HN

O

NH

OH

O

HN

O

NH

HN

NHH2N

O

OHN

Saralasin

Asp Arg Val Tyr Ile His Pro Phe

HN

O OH

NO

NH

NH

NO

HN

O

NH

OH

O

HN

O

NH

HN

NHH2N

ONH2O

HOO

Angiotensin II

N

NO2

NCl

O

O-• ionized carboxylate correlates with the C-terminal of Angiotensin II

S-8038

N

NO2

NCl

O

O-S-8038

1

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2)

3)

!

HN

O OH

NO

NH

NH

NO

HN

O

NH

OH

O

HN

O

NH

HN

NHH2N

ONH2O

HOO

Angiotensin II

N

NO2

NCl

O

O-• ionized carboxylate correlates with the C-terminal of Angiotensin II• the imidazole mimic the His residue

S-8038

HN

O OH

NO

NH

NH

NO

HN

O

NH

OH

O

HN

O

NH

HN

NHH2N

ONH2O

HOO

Angiotensin II

N

NO2

NCl

O

O-

• ionized carboxylate correlates with the C-terminal of Angiotensin II• the imidazole mimic the His residue• the n-butyl group mimics the Ile side chain

S-8038

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4)

S8308 is a moderate inhibitor of AGIIR with an IC50 = 15 µ M. It is relatively polar and the carboxylic will be charged at physiological pH (see previous lecture). The MW is also low (MW= 350.78). If we consider Lipinksi’s rules (1st lecture) then to optimise this lead we can increase the MW and improve lipid solubility (hydrophobicity). See below: Optimisation of lead: 1) The first step taken was to replace the NO2 group with a different functional group (nitro groups are well known to be metabolically unstable). A carboxylic acid was found to work well. Moving this from position 2 to position 4 on the benzene ring was found to be optimum. This resulted in a 10 fold increase in affinity for AGIIR. BUT the compound is still very polar and will be charged at physiological pH (2 carboxylic acid groups!).

HN

O OH

NO

NH

NH

NO

HN

O

NH

OH

O

HN

O

NH

HN

NHH2N

ONH2O

HOO

Angiotensin II

N

NO2

NCl

O

O-

• the aromatic ring was thought to lie in the direction of the N-terminus of the peptide - but was not thought to be very important for binding to the receptor

S-8038

N

NO2

NCl

O

O-

S-8038

• Moderate inhibitor (IC50 = 15 µM)• Relatively hydrophilic or polar• Also small (MW = 350.78)• Need to improve lipid solubility or make more hydrophobic

• Increased potency (IC50 = 1.2 µM)• but still very polar hydrophilic or polar• Still need to improve lipid solubility or make more hydrophobic

N

NCl

O

OH

HO2C

N-terminal C-terminal

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2) To decrease the polarity the carboxylic acid group on the imidazole ring was changed to an alcohol/hydroxyl functional group. No significant drop in affinity was observed.

3) The aromatic ring was thought to be a good position to build off (i.e. add additional functional groups) to increase affinity, lipid solubility and MW. Addition of a phenyl ring with a carboxylic acid in the 2-position was achieved by amide bond formation. This was found to result in a further increase in affinity for AGIIR. They explored a number of functional groups with the acidic group at the 2 position was found to be essential for binding and potency.

4) A biphenyl ring with a carboxylic acid in the 2 position was found to have similar affinity to the amide linked analogue above: IC50 = 0.27 µM. The carboxylic acid in the 2 or ortho position restricts rotation around the C-C bond between the 2 benzene rings. BUT due to the charged carboxylic acid, this compound was not orally bioavailable. Moving the carboxylic acid to the 3 or 4 positions resulted in a drop in affinity.

N

NO2

NCl

O

O-

S-8038

• Moderate inhibitor (IC50 = 15 µM)• Relatively hydrophilic or polar• Also small (MW = 350.78)• Need to improve lipid solubility or make more hydrophobic

N

NCl

OH

HO2C

• No significant change (IC50 = 1.7 µM)• Relatively hydrophilic or polar - less charged• Need to improve lipid solubility or make more hydrophobic

N

NO2

NCl

O

O-

S-8038

• Moderate inhibitor (IC50 = 15 µM)• Relatively hydrophilic or polar• Also small (MW = 350.78)• Need to improve lipid solubility or make more hydrophobic

N

NCl

OH

O

NHCO2H

• Improved potency (IC50 = 0.12 µM)• BUT IV administration only

Explored lots of analogues but found that an acidic group was essential for potency

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5) The carboxylic acid group appeared to be essential – but a solution was needed to allow oral administration. This problem was overcome by using a bioisostere to replace the carboxylic acid. Replacement of the carboxylica acid with a tetrazole resulted in Losartan and orally bioavailable AGIIR inhibitor.

The bioisostere strategy provides an alternative to the prodrug strategy described in the first lecture.

• A bioisostere can be defined as: ‘Compounds or groups that possess near-equal molecular shapes and volumes, approximately the same distribution of electrons, and which exhibit similar physical properties’ • i.e the replacement functional group has similar shape and charge and importantly in medicinal chemistry still exhibits the desired biological activity.

The tetrazole functional group (shown in red above) can be used as a replacement for carboxylic acids.

• Tetrazole is more lipid soluble or hydrophobic than the carboxylic acid fuctional group.

N

NO2

NCl

O

O-

S-8038

• Moderate inhibitor (IC50 = 15 µM)• Relatively hydrophilic or polar• Also small (MW = 350.78)• Need to improve lipid solubility or make more hydrophobic

N

NCl

OH

CO2H

N

NCl

OH

CO2H

N

NCl

OH

CO2H

IC50 = 0.27 µM IC50 = 11 µM IC50 = 1.1 µM

N

NO2

NCl

O

O-

S-8038

• Moderate inhibitor (IC50 = 15 µM)• Relatively hydrophilic or polar• Also small (MW = 350.78)• Need to improve lipid solubility or make more hydrophobic

• Potent inhibitor (IC50 = 0.019 µM)• Less polar• Bigger (MW = 422.92)• OH in place of charge CO2-

• Tetrazole of bioistere of CO2-

N

NCl

OH

NNNHN

2

3 4

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• The NH in tetrazole is a weak acid and has a pKa of 6. • The NH will be 90% ionized at physiological pH (see previous lecture

and try to calculate for yourself!). • However this charge can be distributed or delocalized around the ring

(resonance) Summary of SAR of Losartan:

One of the major metabolites of Losartan is a derivative in which the OH has been oxidized to a carboxylic acid. This metabolite is also an inhibitor of AGIIR and has a 40-fold higher affinity than Losartan for its target. Other AGIIR inhibitors: If we consider again what components of angiotensin II S8038 was thought to mimic 1) the carboxylic acid was thought to mimic the C-terminal carboxylate of the phenylalanine residue in angiotensin II 2) the imidazole was thought to mimic the histidine side chain 3) the n-butyl functional group was proposed to mimic the isoleucine side chain 4) it was first thought that the nitro-benzyl functional group pointed towards the N-terminus of angiotensin II but was not very important for binding. An alternate hypothesis suggested that this aromatic ring was a mimic of the tyrosine side chain of angiotensin II. The development of the next generation of AGIIR inhibitors was based on these ideas/ this pre-existing SAR knowledge.

NNN-N

R NNNN

R NNNN

R

• Potent inhibitor (IC50 = 0.019 µM)• Less polar• Bigger (MW = 422.92)

N

NCl

OH

NNNHN

SAR Summary:• n-butyl chain can be shortened without significantly effecting potency• Potency can be improved by increasing MW and hydrophobicity (addition of aromatic ring)• Imidazole carboxylate could be replaced with OH• An acidic substituent on aromatic ring was found to be essential. • Substitution at the ortho position was found to be optimal• This is likely to restrict rotation• A bioisostere strategy was employed to replace CO2H with a less polar group - tetrazole• This improved potency and importantly increased the lipophlicity.• Compound now orally bioavailable• NB this compound is a potent antagonist of AT-II receptor and does not show partial agonist activity of peptide inhibitor.

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1) Eprosartan has 10-fold higher affinity for AGIIR than Losartan. In this case the nitro group was replaced by an acidic group as before (better mimic of the tyrosine side chain). The most significant change is the addition of a thiophene functional group – which more effectively mimics the C-terminal phenylalanine residue.

Other sartans have also been developed, and as can be seen below the biphenyl ring tetrazole core has been retained. The alterations in structure are in the top portion of the molecule, in which the His/Phe mimics have been varied.

A prodrug strategy has also been employed for a number of sartans (shown below). The ester groups are shown in blue. In vivo esterases hydrolyse the ester group releasing the active carboxylic acid (as in lecture 1).

N

NO2

NCl

O

O-

S-8038

Proposed to mimic Tyr

Proposed to mimic Phe

• Acidic group added - as before• Major change addition of thiophene group to provide a better mimic for Phe• Potent antagonist IC50 = 0.0015 µM• 10 fold more potent that Losartan

N

NCl

CO2H

S

-O2C

N

NCl

OH

NNNHN

N

NNNHN

O

CO2-N

N

NNNHN

O N

N

NNNHN

N

N

Losartan Irbesartan TelmisartanValsartan

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Rank ordering of affinity of sartans (by affinity for AGIIR): Azilsartan, Candesartan, Olmesartan > Irbesartan, Eprosartan > Telmisartan, Valsatan and Losartan It is also important to note that there are 2 subtypes of AGIIR –1 and 2 Type 1 is found in the brain, neuronal, vascular, renal, hepatic, adrenal and myocardial tisses. Type 2 is thought to play a role in growth and development. All available and approved drugs which target the angiotensin II receptor are 10,000 fold selective for receptor type 1 over type 2.

Candesartan cilexitil Olmesartan medexomil

N

N

O

NNNHN

O O

O

O O

N

NC

O

NNNHN

O

OO

OOH

N

N

O

NH

CON

O O O

OO

O

Azilsartan medexomil

Candesartan Olmesartan Azilsartan