R11,R12,R14- Steroids in the treatment of asthma and copd 1

mechanisms,steroid families ,glucacorticoids

Why are steroids important?

Respiratory Theme

• Biosynthesis

• Adrenocorticoids

• Pharmaceutical derivatives used in respiratory diseases

Endocrinology Theme

• Adrenal diseases

• Sex Hormones

Cardiovascular Theme

• Statins

Note - some molecules aren’t steroids by structure but work the same way

What is the key structure of a steroid?

• 6,6,6,5 Ring

• A modified tail which differs across different types of steroids

What are the ways to discuss the steroid structures?

1. Lettered: A, B, C, D

• A is a six membered the ring bearing the oxygen

2. Numbered

• Starts from the A ring, at the top of the skeleton, works its way round, then goes to the side chains last

• Side chains are at position 17; they can have alpha or beta positions= chiral

• up solid - beta; away dashed- alpha

How does the arrangement of steroid rings affect their structure and function?

• Steroids contain four fused rings (A, B, C and D): three 6-membered rings and one 5-membered ring.

• The B/C and C/D ring junctions are trans in the typical steroid structure.

• The A/B ring junction can be either cis or trans, affecting the overall 3D shape of the steroid.

Ring arrangement	Structure	Examples
Cis A/B	Rings are bent, giving a less flat structure	Bile acids
Trans A/B	Rings form a more extended, relatively flat structure	Many adrenocorticoids and sex hormones

Key point: The cis or trans arrangement changes the steroid's 3D shape, which influences how it interacts with receptors and its biological function.

How are steroids made ?

Multi-step biosynthesis ( the mevalonate pathway) - makes multiple products along the way, more than one way of making the molecules

• Starting material is acetyl coenzyme A

• Step one – synthesis of mevalonate

• Step two – synthesis of isopentenyl pyrophosphate

• Step three – synthesis of squalene

• Step four – cyclisation to lanosterol

• Step five – synthesis of cholesterol

This always goes up in multiples of 5 carbons

Describe the first step - synthesis of mevalonate from acetyl-CoA?

• uses 3 molecules of acetyl-CoA

• 2 part: claisen and aldol reaction which occur at the acetyl-CoA synthase

• HMG-CoA reductase removes mevalonate from the enzyme

Describe the Claisen ester condensation in steroid biosynthesis?

1. Enol is formed - enzyme acts as a base and removes a hydrogen from the carbon (CH3) on one of the acetyl-CoA molecules; this makes an enol, which is electron-rich and can attack an electrophile

2. Nucleophilic attack - the enol attacks teh electrophilic carbon (C=O) of a second acetyl-CoA molecule, making a bond between teh carbons ( tetrahedral intermediate )

3. Elimination of CoA - the tetrahedral intermediate collapses, reforming the (C=O) bond. The CoA-containing group leaves as CoA-SH because it is a good leaving group. This produces acetoacetyl-CoA, a four-carbon β-keto thioester.

4. Two acetyls are joined together

Describe the mechanism of the aldol reaction in steroid biosynthesis.

Step 1: Formation of the enol - The third acetyl-CoA molecule is converted into its enol/enolate form by an enzyme. This makes the α-carbon nucleophilic (electron-rich), allowing it to attack an electrophile.

Step 2: Nucleophilic attack (aldol addition)

• The enol/enolate attacks the electrophilic carbonyl carbon (C=O) of acetoacetyl-CoA, formed in the previous Claisen condensation reaction.

• A new carbon–carbon bond forms, joining the third acetyl-CoA to acetoacetyl-CoA.

• The carbonyl oxygen is protonated (gains H⁺), forming an OH group.

• This produces a six-carbon intermediate containing three acetyl units.

Step 3: Hydrolysis

• One thioester bond is broken by hydrolysis (addition of water).

• This releases CoA-SH and forms a carboxylic acid group (COOH).

• The final product is HMG-CoA, which contains six carbon atoms

Describe the synthesis of isopentenyl pyrophosphate from mevalonate (step2)

1. First phosphorylation: Mevalonate reacts with ATP, which donates a phosphate group to the hydroxyl group at carbon 5, forming 5-phosphomevalonate. ATP is converted to ADP.

2. Second phosphorylation: Another ATP molecule donates a second phosphate group, forming 5-pyrophosphomevalonate (mevalonate-5-diphosphate). ATP is again converted to ADP.

3. Decarboxylation and elimination: The molecule undergoes ATP-dependent decarboxylation, releasing CO₂, followed by elimination of the hydroxyl group and formation of a C=C double bond. This produces isopentenyl pyrophosphate (IPP), a five-carbon molecule.

Note: this reaction requires two molecules of ATP to install two phophates produces ipp

Describe the synthesis of squalene via condensation (step 3) - to make monoterpenes geranyl phosphate

The formation of geranyl pyrophosphate (GPP) involves enzymatic isomerisation, formation of an allylic carbocation, electrophilic addition and elimination.

1. Enzymatic isomerisation: Isopentenyl pyrophosphate (IPP) is converted into dimethylallyl pyrophosphate (DMAPP) by rearranging the position of the C=C double bond. This reaction is reversible equilibrium favoured forwardly

2. Formation of an allylic carbocation: DMAPP loses its pyrophosphate group (PPi), which acts as a leaving group, producing a positively charged allylic carbocation.

3. Resonance stabilisation: The positive charge is delocalised across the allylic system through resonance, making the carbocation more stable.

4. Electrophilic addition: The allylic carbocation acts as an electrophile, while the C=C double bond of another IPP molecule acts as a nucleophile. The double bond attacks the carbocation, forming a new C–C bond and joining the two five-carbon units.

5. Elimination: A proton (H⁺) is removed from the intermediate, allowing a new C=C double bond to form, producing geranyl pyrophosphate (GPP).

Describe the synthesis of farnesyl phosphate - step 3

Part 1: Formation of farnesyl pyrophosphate (FPP)

1. Formation of an allylic carbocation: Geranyl pyrophosphate (GPP, C₁₀) loses its pyrophosphate group (PPi), which acts as a leaving group, forming an allylic carbocation.

2. Resonance stabilisation: The positive charge is delocalised through resonance, stabilising the allylic carbocation.

3. Electrophilic addition: The allylic carbocation acts as an electrophile, while the C=C double bond of isopentenyl pyrophosphate (IPP, C₅) acts as a nucleophile. The double bond attacks the carbocation, forming a new carbon–carbon bond and extending the carbon chain.

4. Elimination: A proton (H⁺) is removed, allowing a new C=C double bond to form, producing farnesyl pyrophosphate (FPP, C₁₅).

Daescribe the final synthesis step of squalene using farsenyl phosphate

Part 2: Formation of squalene

1. Condensation: Two molecules of farnesyl pyrophosphate (C₁₅) combine in a head-to-head condensation reaction catalysed by squalene synthase.

2. Reduction: NADPH provides reducing power, allowing the intermediate to be converted into squalene (C₃₀), with the release of pyrophosphate groups.

3. Final product: Squalene is a 30-carbon molecule that acts as a precursor for cholesterol and other steroids

Describe the cyclisation of squalene to lanosterol and cholesterol (step 4 and 5 )

The conversion of squalene into cholesterol involves epoxidation, cyclisation and further structural modifications.

1. Epoxidation: Squalene (C₃₀) is converted into 2,3-oxidosqualene by the enzyme squalene epoxidase, which introduces an epoxide (a three-membered ring containing oxygen).

2. Cyclisation: The enzyme oxidosqualene cyclase converts oxidosqualene into lanosterol by folding the molecule and forming four fused carbon rings, producing the characteristic steroid ring structure.

3. Formation of cholesterol: Lanosterol undergoes further modifications, including removal of three carbon atoms, rearrangement of double bonds and reduction reactions, to produce cholesterol (C₂₇).

what is the steroid families that you can make using cholesterol?

describe the synthesis of pregnenolone from choletsrol ( loss of a side chain)

• Stimulated by adrenocorticotropic hormone (ACTH)

•1st step – multi-hydroxylation by cytochrome P450 monooxygenase - introduces OH groups on the side chain (requires NADPH and O2)

•2nd step – cleavage of the bond between hydroxyl groups by desmolase

Describe the synthesis of progesterone from pregnenolone

•1st step – oxidation of the ring alcohol to a ketone

•2nd step – isomerisation of the double bond it moves form the B ring to the A ring ,common double bond in the 4,5-ring allows extended conjugation and different types of reactions

How are corticoids synthesised ( adrenocorticoids)

• In the adrenal cortex

• The levels are determined by feedback

2 types:

Glucocorticoids

• Responsible for the synthesis of carbohydrates from proteins

• Involved in the deposition of glycogen in the liver

• Very important in inflammatory processes

• useful drug targets

Mineralocorticoids

• Control the electrolyte balance (diuresis)

• Promote retention of sodium and chloride

• Promote loss of potassium

• used less

Describe the synthesis of cortisol from progesterone

Step 1 – Hydroxylation at carbon 17 (C17):

• Progesterone undergoes hydroxylation at the 17α-position.

• An –OH group is added at the α-position (pointing away from us).

• This forms 17α-hydroxyprogesterone.

Step 2 – Hydroxylation at carbon 21 (C21):

• 17α-hydroxyprogesterone undergoes hydroxylation at carbon 21, on the side chain.

• An –OH group is added to the side chain.

• This forms 11-deoxycortisol.

Step 3 – Hydroxylation at carbon 11 (C11):

• 11-deoxycortisol undergoes hydroxylation at the 11β-position.

• An –OH group is added in the β-position (pointing towards us).

• This produces cortisol.

- Enzymes involved: All three hydroxylation reactions are catalysed by cytochrome P450 enzymes, which require NADPH and oxygen (O₂).

What are glucocorticoids uses in ?

Glucocorticoids have a role in

• rheumatoid arthritis

• skin diseases

• respiratory diseases

Cortisol (hydrocortisone) has significant mineralocorticoid activity

Need to minimise mineralocorticoid activity to design effective drugs

what structural components are needed for a glucocorticoid?

• C21 pregnane skeleton (2-carbon side chain)

• 17 beta COCH2OH side chain

• 11beta hydroxyl

• keto at the 3 position (generally)

• 4-5 double bond

• 17-alpha hydroxyl (generally)

Generally - still important but some don’t have these characteristics

what is structural components are needed for a mineralocorticoid?

Mineralocorticoids

• C21 pregnane skeleton

• 17 BETA COCH2OH side chain

• keto at position 3 (generally)

• 4-5 double bond

Where do glucocorticoids work /bind at?

Nuclear receptor

• N-terminus

• Central DNA-binding domain

• C-terminal ligand-binding domain

Ligand-binding domain

• All steroid receptors have 11α-helices and 4β-strands

• Glucocorticoids bind in an area formed from :

-α-helices 3, 4, 5, 6, 7

-β-strands 1 and 2

• All key binding interactions are via H-bonding

note - changing any part of steroid ,changes binding cause nearly everything has a n interaction

what is cortisol, and what is it used for?

• Highly effective, particularly for skin diseases

• But oral use shows mineralocorticoid systemic side effects

note - need to find a way to reduce mineralocorticoid activity

`What changes are made to make synthetic corticosteroids - 1 ?

Introduction of a 1,2 double bond

• Changes the shape of the A ring, making it flatter

• Increases glucocorticoid

• Decreases mineralocorticoid

What changes are made to make synthetic corticosteroids - 2?

Introduction of a 9α-fluoro substituent

• increases lipophilicity,stays longer in lung tissue

• increases glucocorticoid (10x)

• also increases mineralocorticoid (300 - 600x)

• Chloro also works

• Also reduces metabolism, so increases duration of action

What changes are made to make synthetic corticosteroids - 3 ?

Introduction of a 9α-fluoro substituent and a position 16 substituent:

• Substituent can be a or b

• Substituent can be Methyl or hydroxyl - chnages ability to bond

•  increased glucocorticoid (overall)

•  decreased mineralocorticoid

• Believed to reduce ability to bind at mineralocorticoid receptor

Introduced in 1958

What can you do to improve pharmacokinetics?

Formulation as esters

• For inhalation and topical use

• Generally the dipropionate

• Increases lipophilicity , reduces activity ( once in the body and cleaved it is highly effective )

• Easily cleaved in situ

What does introducing a substituent at position 16 affect?

Introduction of a substituent at position 16

• Possible to combine the changes to the A ring and a substituent at the 16 position

• No halogen

• Formulated as an acetal to enhance lipophilicity

Active drug, not a prodrug – 21-OH free

What does modifying the side chain affect?

Modification of side chain

• In combination with the 3 other changes

• Major synthetic change to side chain - Strong electrostatic interaction with Asn 564

• Additional incorporation of fluorine at 6α - increases glucocorticoid binding only ,6α methyl also works here but less good

Examples of ante-drugs (stable in lung but not in. circulation)

drug design sar of glucocorticoids