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Dr Jess Healy: PHAY2003 Cardiovascular drugs (Medicinal chemistry) Lecture 1 What makes a good drug? A good drug must have high affinity for its macromolecular target (i.e. bind tightly). The human body is complex (containing numerous microenvironments) and for a drug to orally bioavailable it needs to fulfil a number of criteria:

• It must be soluble • Be stable under a range of pH’s (1.5-8.0) • It must avoid excessive metabolism in the kidneys and liver • Be able to cross the cell membrane and reach its target organ • It must also avoid partitioning into undesirable places like the brain or

the foetus The physical and chemical properties of the drug can be used to predict the behaviour of the drug in the body. This has resulted in a set of rules for medicinal chemists called ‘Lipinksi’s Rule of 5’. Drugs which fulfil some or all of these criteria are more likely to be orally bioavailable. Lipinski’s Rule of 5

• MW < 500 Da • LogP < 5 • H-bond donors < 5 • H-bond acceptor < 10

Log P = is a measure of the distribution of a drug between water and octanol phases. These solvents act as a model for the cytosol and the lipid bilayer. P = !"#$ !"#!$!%/ !"#$ !"#$% LogP is one of the most important properties for predicting the behavior of a drug in the body. For a drug to be orally bioavailable we need a balance between lipid and aqueous solubility.

H-bond = Is a non-covalent interaction/bond which can form between a H-bond donor and an acceptor. The donor is a H-atom attached to an electronegative atom (e.g. N, O, F). The acceptor is a neighboring electronegative atom which has a lone pair of electrons. An image showing a H-bond between 2 water molecules is shown below:

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Ionisation is also a very important factor and can affect the membrane permeability of a drug. Ionisation = protonation /deprotonation resulting in a charged molecule.

• A base is protonated resulting in a positive charge. • An acid is deprotonated resulting in a negative charge. • Many drugs contain acidic and basic functional groups and depending

of the pKa of the acidic or basic group it will be ionised to different extents in different parts of the body (due to differences in pH e.g. stomach pH = 1.4-2.0 but in the duodenum the pH = 4.4-6.6)

Most drugs are absorbed by passive diffusion across the cell membrane and the un-ionised or uncharged form of the molecule will be preferentially absorbed. Therefore the extent of ionisation of a drug at physiologically relevant pH’s can be used to predict whether a drug will be efficiently absorbed or not. The equilibrium between ionised and un-ionised compound is described by the acidity constant Ka or pKa = -log10Ka. When the pKa = pH then the compound is 50% ionized.

Remember: the lower the pKa the stronger an acid the compound is. Part I: ACE inhibitors See lecture by RH about the biological basis of disease and pharmacology. Review amino acid structures. Renin angiotensin system: brief overview. This pathway is involved in the regulation of blood volume, blood pressure and electrolyte balance. It consists of 2 enzymes: RENIN and ANGIOTENSIN CONVERTING ENZYME (ACE).

δ+

O

H Hδ-

°° °°Lone pair

OH

H

°°°°

H-bond

% ionised = 100 1 + 10(pKa - pH)

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Development of the first ACE inhibitor, Captopril: Captopril was the first ACE inhibitor, and was approved for human use by the FDA in 1981. The story of the development of this drug began in the 1960’s: The initial leads were peptides isolated from snake venom (Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro). These were found to be potent inhibitors of ACE. But because of their polarity and peptidic structure, no oral activity was observed. A C-terminal proline (Pro) residue was found to be key to inhibition. Inspiration for the development of the next generation of inhibitors came from a related enzyme Carboxypeptidase A: Both Carboxypeptidase A and ACE hydrolyse peptide bonds, and use an arginine, a glutamate and a zinc ion in their active sites to catalyse the hydrolysis of the amide bond. The most important parts of the substrates for Carboxypeptidase A (CPDA) and ACE are shown below:

Angiotensinogen CO2HAsp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu Val-Ile10 11

Renin

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe His-Leu

Angiotensin I

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe

Angiotensin II

AngiotensinConverting

Enzyme(ACE)

Angiotensin II Receptor

binds to

Activation

Vasocontriction and water retention ∴ Increased BP!

inhibition of pathway at any of these points ⟼

Lower BP!

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The red dotted line denotes the bond that is broken in the enzymatic reaction.

An observation was made that D-2-benzylsuccinic acid (1) was a potent inhibitor of a related enzyme carboxypeptidase A.

As you can see this inhibitor mimics the C-terminal end of its substrate. The addtional carboxylic acid functional group was thought to form an electrostatic interaction with the Zn in the enzyme active site. This compound was used as a starting point for the design of a

potent and selective ACE inhibitor: This compound (2) was found to be a weak inhibitor of ACE. The proline residue (highlighted in the blue box) mimics the C-terminal amino acid in the inhibitors isolated from snake venom. The carboxylate interacts with the Zn atom in the enzyme active site. An additional CH2 group was added to put the Zn chelating functional group in the correct position (CPDA

cleaves 1 amino acid whereas ACE cleaves 2 amino acids). Optimisation of lead compound and structure activity relationships: To develop the lead compound a number of structural modifications were investigated (see figure on next page): 1) it was found that replacement of the proline with any other amino acid resulted in a drop in affinity for ACE. This trend was also observed for the peptide inhibitors isolated from snake venom. 2) Addition of a CH3 group next to the proline amide bond (see figure below) provided a better mimic the peptide substrate/peptide inhibitors. The stereochemistry of this CH3 was found to be important for binding to ACE (and mimics the stereochemistry found naturally in amino acids). The potency of this compound (3) was improved over the lead compound. 3) Addition of a better Zn chelator (i.e. replacement of CO2

- with SH) resulted in a dramatic increase in affinity. This inhibitor was called Captopril (4), and inhibited ACE with a Ki of 1.7 nM. (Ki = the concentration of inhibitor required to produce half maximum inhibition)

O

O-O

O-

Zn2+

NO

-OO

Zn2+

O

O-

Lead

NH

OHN

O

Zn2+

NH O

O-O

R

HN

Zn2+NH

N

O

HN

O

O-

CPDA ACE

1

2

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Why does ACE not hydrolyze Captopril??? Summary of key interaction formed between Captopril and ACE:

Captopril is a potent inhibitor of ACE and is orally bioavailable BUT the thiol results in number of side effects (e.g. skin rash and taste disturbances). Captopril also has a short duration of action. 2nd generation ACE inhibitors (Dicarboxylate class): Drugs that mimic the transition state of the enzyme-catalysed reaction are often potent inhibitors. (transition state is a high energy intermediate formed on the pathway from reactant/substrate to product) To mimic the transition state of the hydrolysis of angiotensin I Enalaprilat was designed:

NO

-OO

Zn2+

O

O-N

O-OO

Zn2+

O

O-

Lead

Improved potency

Stereochemistry important!

NO

-OO

Zn2+

HS CAPTOPRIL

1st approved ACE inhibitor

NO

-OO

Zn2+

HS

H2N

NH3

O

OH

H2N

HNN

O

OHOH

O

NNH

NH2

Different orientation

hydrophobic pocket

potential to extend off here

2 3

4

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Product of reaction:

The phenylethyl group (5) mimics the phenylalanine residue in the substrate/transition state and forms interactions with a hydrophobic pocket. The carboxylic acid group is attached to a tetrahedral carbon – provides a mimic of the tetrahedral intermediate formed during the hydrolysis reaction. This chelates the Zn in the enzyme active site (less effective chelator of Zn than SH). The secondary amine mimics the amide bond of the peptide substrate. The proline and adjacent CH3, which mimics the alanine residue, were found to be essential

as before. Enalaprilat, however, is not orally bioavailable – charged at physiological pH (pH>pKa).

Ionisation of the carboxylic acid increases the basicity of the adjacent NH – Why? This NH is likely to be protonated and charged in the intestine.

Substrate

NH

OHN

O

Zn2+NH

N

O

HN

O

O-

H2O

NH

OHN

O

Zn2+NH

N

O

HN

O

O-

Transition state

NH

HN

O

Zn2+NH

N

O

HN

O

O-

-O OH

Enalaprilat

NO

-OONH

O O-

• 2 carboxylates - charged at physiological pH.

pKa of carboxylic acid = 3.5

At pH 7.4 (blood) - these will be deprotonated

% ionised = 100 1 + 10(pKa - pH)

100

1+ 10 (3.5-7.4)= 100%

NO

-OONH

Zn2+

O O-

Enalaprilat

10 fold more potent than Captopril!

5

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To overcome these problems a prodrug strategy was employed. This involves the addition of a functional group to mask the charge of the carboxylic acid (6). The carboxylic acid is masked with an ethyl ester. This lowers the pKa of the amine. The amine is therefore uncharged in the intestine. Enalapril is orally bioavailable.

N.B. Enalapril has 1000 fold lower affinity for ACE than Enalaprilat. The uncharged ester is no longer a good chelator of Zn! In vivo, however, esterases hydrolyse the ester group releasing the active drug.

Mechanism of ester hydrolysis:

Other members of the dicarboxylate class of ACE inhibitors include Quinapril, Ramipril and Lisinopril shown below. In Quinapril and Ramipril a larger more hydrophobic bicyclic ring system is used instead of proline. This explores the hydrophobic pocket highlighted in

Enalapril

• Ethyl ester prodrug• pKa of amine now ~5.5 uncharged in the intestine!

NO

-OONH

O O6

Enalaprilat

NO

-OONH

O O-

•2 carboxylates - charged at physiologicalpH.• Ionization of carboxylate increasesbasicity of NH. pKa = 8.02 likely to be charged in the intestine.• IV administration only!

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the X-ray crystal structure of ACE with captopril shown earlier. A prodrug strategy is required as with Enalapril above.

Lisinopril contains a primary amine (derived from the amino acid lysine) instead of a methyl group. Interestingly Lisinopril is orally bioavailable, therefore a prodrug strategy is not required. Why? Lisinopril is more polar than the other derivatives in this class – therefore would expect it not to be

orally bioavailable. BUT in the intestine Lisinopril is neutral i.e. the charges balance!

Summary of SAR: • The lead compound (2) was inspired by benzylsuccinic acid (1) - an

inhibitor of a related enzyme Carboxypeptidase A and peptide inhibitors isolated snake venom. Compound 2 was optimized and developed, this lead to 1st approved ACE inhibitor Captopril.

• The key features of Captopril are: o the thiol group which chelates Zinc o the proline which mimics the terminal amino acid of the snake

venom peptide inhibitors o the proline carboxylic acid which forms a electrostatic interaction

with a lysine residue in the enzyme active site o the methyl group which mimics the isoleucine residue of the snake

venom peptide inhibitors (stereochemistry the same as natural amino acids)

• The dicarboxylate class of inhibitors were designed to mimic the tetrahedral intermediate or the transition state of the enzyme catalyzed reaction.

• In these compounds the carboxylate chelates the zinc c.f. the SH group in Captopril

• The proline was found to be essential as before, with the carboxylate

NO

-OONH

O O

NO

-OONH

O O

NO

-OONH

O O-

NH2

Quinapril Ramipril Lisinopril

NO

-OONH2+

O O-

NH3+

No net charge!

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forming an electrostatic interaction with a positively charged lysine residue in the enzyme active site

• The phenyethyl functional group mimics the essential phenylalanine residue in angiotensin I – binds in a hydrophobic tunnel in the enzyme active site

• This class of inhibitors need to be administered as prodrugs (highly charged at physiologically relevant pH) – here the carboxylate group is masked by converting it to an ethyl ester. The active drug is released in vivo: hydrolysis by esterase’s. One exception is Lisinopril which is neutral at physiological pH.