Phase-I Metabolism Pt-2 – Oxidations, Reductions, Hydrolysis & Pro-drugs

Quick Orientation

Continuing Phase-I (“functionalisation”) metabolism
Focus is still on oxidation-type reactions, then reductions & hydrolyses
Reminder: whenever the lecturer says “you introduce an $\text{OH}$ , $\text{NH}$ or $\text{SH}$ ” group, that new hetero-atom IS COVALENTLY ATTACHED TO (part of) THE PARENT DRUG and becomes part of the metabolite.

Aliphatic & Alicyclic Hydroxylation

• Applies to saturated carbon chains or saturated rings (alicyclic)
• Requirements for a classic "aliphatic" case

Straight chain (≥ 3 carbons, un-branched preferred)
Cytochrome P450 (CYP) catalysed
• Possible insertion sites
$\omega$ -position = terminal carbon
$\omega{-}1$ -position = next-to-terminal carbon
Goal: stay as far as possible from bulky / electronic bulk of the molecule
• Mechanistic Sketch
CYP “oxene” abstracts a hydride (H $^-$ ) from the target carbon → leaves a carbenium (C $^+$ ).
The oxene becomes $\text{O}^{-}$ (hydroxide)
Re-combination → new C–O bond; carbon now bears one fewer H.
• Tracking hydrogens
At $\omega$ carbon you start with $\text{CH}3$ , end with $\text{CH}2\text{OH}$ (one H migrated into the hydroxyl).
Same logic for $\omega{-}1$ (start $\text{CH}_2$ → end $\text{CH}\text{OH}$ ).
• Alicyclic variant
Ring behaves like a “folded” chain; hydroxylation usually at “3” or “4” positions
Generates cis / trans pairs (stereochemistry NOT required on exams).
• Example: Acetylhexamide → 4-hydroxy metabolite recovered in vivo.

Oxidative $\text{O/N/S}$ De-alkylation (★★★ high-yield)

Recognition

• Parent must contain – $\text{O–R}$ , – $\text{N–R}$ or – $\text{S–R}$ where the R-carbon (α-carbon) possesses ≥ 1 hydrogens.
• Heteroatom = “Y” (O, N, S).
• Enzyme = CYP-450 (mostly).

Mechanistic Outline

Oxene abstracts an α-hydride → gives $\text{O}^-$ and a C $^+$ .
Re-combination → carbinolamine/hemialkoxide (two hetero-atoms on same carbon).
That intermediate is UNSTABLE; collapses:
- Lone pair on hetero-oxygen kicks down → carbonyl forms (ketone or aldehyde).
- $Y–R$ bond breaks; Y picks up H $^+$ .
Products (always TWO):
• A carbonyl fragment (ketone if α-C had 1 H, aldehyde if ≥ 2 H)
• A new nucleophile $Y–H$ (ROH, RNH, RSH).

Special Cases

• If $R$ = methyl (O-, N- or S-methyl) ⇒ oxidative demethylation

Always liberates formaldehyde ( $\text{HCHO}$ ) as the carbonyl product.
Terminology: “demethylation” ⊂ generic “de-alkylation”.
Catalysed by CYP (esp. CYP2D6, CYP3A4, etc.).
• Successive dealkylations possible (e.g.
Imipramine → Desipramine → Nor-desipramine).
• Two sides on same N can de-alkylate from either side (α-C on both sides).

Examples

Methamphetamine → Amphetamine + formaldehyde
Imipramine → Desipramine (active metabolite)
Multiple O-demethylations in morphine analogues, etc.

Oxidative De-amination by Monoamine Oxidase (MAO)

• Target: primary amines (common in neurotransmitters)
• Requires α-carbon with ≥ 1 H (usually 2)
• Enzyme: MAO-A & MAO-B (flavin cofactor)

MAO-A & MAO-B both in liver; MAO-B predominantly in brain
• Cofactor = FAD (flavin adenine dinucleotide) acts as hydride acceptor
• Products: aldehyde + $\text{NH}_3$
• Physiological relevance: dopamine, norepinephrine, serotonin catabolism

Alcohol & Aldehyde Oxidations

Alcohol → Aldehyde

Enzyme = Alcohol Dehydrogenase (ADH)
Cofactor = $\text{NAD}^+ \rightarrow \text{NADH}$ (hydride transfer onto nicotinamide ring).

Aldehyde → Carboxylic Acid

Enzyme = Aldehyde Dehydrogenase (ALDH)
Same hydride transfer to $\text{NAD}^+$ .

Ethanol Pathway

$\text{CH}3\text{CH}2\text{OH} \xrightarrow[\text{NAD}^+][]{\text{ADH}} \text{CH}3\text{CHO} \xrightarrow[\text{NAD}^+][]{\text{ALDH}} \text{CH}3\text{COOH}$
• Minor back-up route: MEOS (microsomal ethanol-oxidising system) = CYP2E1
• Disulfiram inhibits ALDH ⇒ acetaldehyde build-up ⇒ severe nausea (aversion therapy).

Summary Table – Oxidations (need to know enzymes)

Aliphatic / Alicyclic hydroxylation → CYP
Aromatic hydroxylation (from part 1) → CYP
$\text{O/N/S}$ de-alkylation → CYP
Oxidative de-amination → MAO (FAD)
Alcohol → Aldehyde → ADH (NAD $^+$ )
Aldehyde → Acid → ALDH (NAD $^+$ )

Reductions (Phase-I but opposite direction)

• Enzyme generically called “Reductase”; cofactor $\text{NADH}$ or $\text{NADPH}$ (hydride DONOR)

Carbonyl Reductions

Ketone $\rightarrow$ secondary alcohol
Aldehyde $\rightarrow$ primary alcohol (less common; more commonly it gets oxidised)

Nitro Reductions

$\text{NO}2$ \xrightarrow[\text{Reductase, 2 e^-}]{\text{NADH}} $\text{NH}2$ (primary amine)
~90 % cases are AROMATIC nitro groups
Aliphatic nitro may accumulate toxic nitroso intermediate

Azo Reductions

$\text{R–N}=N–\text{R'}$ + 4 × $\text{NADH}$ $\rightarrow$ $\text{R–NH}2 + \text{R'–NH}2$
Typically both R & R' are aromatic
Classic pro-drug: Prontosil (inactive) → Sulfanilamide (active sulfa antibiotic)

Azido Reductions (rare)

$\text{R–N}3$ → $\text{R–NH}2 + \tfrac{1}{2} \text{N}_2$ gas

Sulfur-containing reductions (thio-S→SH) occur but not examined.

Hydrolytic Reactions (Water, NOT redox)

General Features

Cofactor: $\text{H}_2\text{O}$
Bonds cleaved: C=O attached to heteroatom (O, N, S).
Enzymes & Typical Ease
- Esterase > Thioesterase > Amidase > Carbamate hydrolase > Urease
- Blood & liver rich in esterases ⇒ esters hydrolyse fastest.

Groups & Products

Functional group	Enzyme	Products after hydrolysis
Ester $\text{R–C(=O)–OR'}$	Esterase	Carboxylic acid + Alcohol
Thio-ester $\text{R–C(=O)–SR'}$	Thio-esterase	Carboxylic acid + Thiol
Amide $\text{R–C(=O)–NR'R''}$	Amidase (peptidase/protease in peptides)	Carboxylic acid + Amine

Mechanistic mnemonic

Break the C–Y bond (Y = O, N, S)
Give Y the H of water, give carbonyl the OH.

Relative Lability

\text{Ester} > \text{Thio-ester} > \text{Carbonate} > \text{Amide} > \text{Carbamate} > \text{Urea}

Example: Cocaine

Two ester sites ⇒ very short $t_{1/2}$ ; cleavage at either ester de-activates the drug.

Pro-drug Strategy via Esterification

• Esterify existing $\text{OH}$ (or $\text{COOH}$ ) in the active drug

Inactive / less active; ↑ lipophilicity; masks unpleasant taste; or provides depot release.
• In vivo esterases regenerate the parent drug + benign acid/alcohol fragment.

Depot Example: Haloperidol Decanoate

Parent antipsychotic has phenolic $\text{OH}$ .
Esterified with decanoic acid (10-C saturated chain) ⇒ \text{O–C(=O)–(CH2)8CH_3}
Very lipophilic, dissolved in oil; injected intramuscularly.
Slow leaching from “depot” up to ≈ 3 weeks; once in plasma, esterase → active haloperidol + decanoic acid.

Other Reasons for Pro-drugs

Improve oral absorption (mask polarity)
Targeted release (intestinal, CNS, etc.)
Bypass first-pass metabolism
Reduce GI irritation

Minor / Additional Notes

• GI tract & even gut microbiota possess enzymes (reductions, hydrolyses) capable of metabolising drugs before hepatic portal circulation (e.g. extensive L-DOPA metabolism).
• Flavin-containing mono-oxygenases (FMO) exist but play smaller role (covered in skipped section).
• Several sections (de-halogenation, sulfoxide formation, etc.) were explicitly marked “not examinable”.

One-Page Enzyme Cheat-Sheet

CYP450: most oxidations (hydroxylation, de-alkylation, de-halogenation)
FMO: some S/N oxidations (not tested)
MAO-A/B: oxidative de-amination (primary amines)
ADH: alcohol → aldehyde
ALDH: aldehyde → acid
Reductase + NADH: carbonyl, nitro, azo reductions
Esterase / Amidase / Thio-esterase: hydrolysis

Ethical / practical angle: Disulfiram therapy demonstrates how manipulating metabolism can modify behaviour; understanding CYP & MAO isoforms underpins drug–drug interaction management.