Oxidative Reactions

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Chapter: Biopharmaceutics and Pharmacokinetics : Biotransformation of Drugs

Oxidative reactions are the most important and most common metabolic reactions.



Oxidative reactions are the most important and most common metabolic reactions. Almost all drugs that undergo phase I biotransformation undergo oxidation at some stage or the other. A simple reason for oxidation being a predominant reaction is that energy in animals is primarily derived by oxidative combustion of organic molecules containing carbon and hydrogen atoms.

Oxidative reactions increase hydrophilicity of xenobiotics by introducing polar functional groups such as—OH. Such a polar metabolite can thus rapidly undergo phase II reaction or is excretable by the kidneys.

Oxidation of xenobiotics is non-specifically catalysed by a number of enzymes located in the microsomes. Such enzymes require both molecular oxygen (O2) and the reducing agent NADPH to effect reaction. They are therefore referred to as the mixed-function oxidases. The overall stoichiometry of this reaction involving the substrate RH which yields the product ROH, is given by the following equation:

RH + O2 + NADPH + H+   ROH + H2O + NADP+

where NADPH = reduced nicotinamide adenine dinucleotide phosphate.

Since only one oxygen atom from the molecular oxygen (dioxygen or O2) is incorporated in the product formed, the mixed function oxidases are also called as monooxygenases. Quite often, the product of such a reaction contains a hydroxyl function; hence, the enzymes are sometimes also called as hydroxylases.

The multienzyme mixed function oxidase system, located in the endoplasmic reticulum of hepatic cells, is composed of an electron transfer chain consisting of 3 components:

1. A heme protein known as cytochrome P-450 (CYP-450), which is actually a family of enzymes. It is a terminal oxidase. Its function is to transfer an oxygen atom to the substrate RH and convert it to ROH.

2. A second enzyme, the flavoprotein known as cytochrome P-450 reductase (or cytochrome c reductase) which is NADPH-dependent. It functions as an electron carrier, catalysing the reduction of cytochrome P-450 to the ferrous form by transferring an electron from NADPH.

3. A heat stable lipid component known as phosphatidylcholine. Its function is to facilitate electron transfer from NADPH to cytochrome P-450.

Magnesium ions are also required for maximal activity of mixed function oxidases.

The most important component of mixed function oxidases is the cytochrome P-450 since it binds to the substrate and activates oxygen. The reduced form of this enzyme (Fe++) binds with carbon monoxide to form a complex that shows maximum absorption at 450 nm, hence the name. The mechanism of cytochrome P-450 catalysed metabolism of xenobiotics is depicted in the redox cycle in Fig. 5.3.

Fig. 5.3. Cytochrome P-450 oxidation-reduction cycle

The various steps in the oxidation of xenobiotics are:

1. Binding of the substrate (RH) to the oxidised form of the cytochrome P-450 (Fe+++) to form a complex.

2. A one-electron transfer from NADPH to the complex by cytochrome P-450 reductase to form reduced (Fe++) P-450—substrate complex. This step is considered as the rate-limiting step in the overall oxidation of xenobiotics.

3. The reduced enzyme-substrate complex combines with a molecule of oxygen to form a ternary complex.

4. The ternary complex combines with a second electron supplied by NADH in presence of enzyme cytochrome b5 reductase to form a ternary activated oxygen—P-450— substrate complex.

5. One atom of oxygen from the activated oxygen complex is transferred to the substrate to yield the oxidised product and the other atom forms water. The free oxidised form of cytochrome P-450 is now ready to attach to yet another molecule of substrate.

Oxidation of Aromatic Carbon Atoms (Aromatic Hydroxylation)

This reaction proceeds via formation of a reactive intermediate arene oxide (epoxide), which in most cases undergoes rearrangement to yield arenols, and in some cases catechols and glutathione conjugates.

The arene oxide intermediate is highly reactive and known to be carcinogenic or cytotoxic in some instances, e.g. epoxides of bromobenzene and benzo(a)pyrene.

Monosubstituted benzene derivatives can be hydroxylated at ortho-, meta- or para-positions but para-hydroxylated product is most common, e.g. conversion of acetanilide to paracetamol, and phenylbutazone to oxyphenbutazone.

Such a reaction is favoured if the substituent is an activating group (electron rich) like the amino group. Deactivating or electron withdrawing groups such as carboxyl and sulphonamide retard or prevent aromatic hydroxylation, e.g. probenecid.

Oxidation of Olefins

Oxidation of nonaromatic carbon-carbon double bonds is analogous to aromatic hydroxylation i.e. it proceeds via formation of epoxides to yield 1,2-dihydrodiols. A better known example of olefinic oxidation is conversion of carbamazepine to carbamazepine-10,11-epoxide; the latter is converted to corresponding trans-10,11-dihydrodiol.

Olefinic hydroxylation differs from aromatic hydroxylation in that their epoxides are stable and detectable which also indicate that they are not as reactive as aromatic epoxides. However, an important example where the olefin epoxide is highly reactive is that of aflatoxin B1. It is known as the most potent carcinogen (causes hepatic cancer).

Oxidation of Benzylic Carbon Atoms

Carbon atoms attached directly to the aromatic rings (benzylic carbon atoms) are hydroxylated to corresponding carbinols. If the product is a primary carbinol, it is further oxidised to aldehydes and then to carboxylic acids, e.g. tolbutamide. A secondary carbinol is converted to ketone.

Oxidation of Allylic Carbon Atoms

Carbon atoms adjacent to olefinic double bonds (are allylic carbon atoms) also undergo hydroxylation in a manner similar to benzylic carbons, e.g. hydroxylation of hexobarbital to 3'-hydroxy hexobarbital.

Oxidation of Carbon Atoms Alpha to Carbonyls and Imines

Several benzodiazepines contain a carbon atom (C-3) alpha to both carbonyl (C=O) and imino (C=N) functions which readily undergoes hydroxylation, e.g. diazepam.

Oxidation of Aliphatic Carbon Atoms (Aliphatic Hydroxylation)

Alkyl or aliphatic carbon atoms can be hydroxylated at two positions - at the terminal methyl group (called as -oxidation) and the penultimate carbon atom (called as -1 oxidation) of which the latter accounts for the major product, e.g. valproic acid. Hydroxylation at other carbon atoms in long chain compounds is less common.

Terminal hydroxylation of methyl group yields primary alcohol which undergoes further oxidation to aldehyde and then to carboxylic acid quite rapidly. Penultimate carbon atom can be secondary or tertiary of which the latter type is also equally reactive, e.g. ibuprofen.

The ω-1 oxidations of secondary and tertiary penultimate carbons yield corresponding alcohols. The secondary alcohol products seldom undergo further oxidation to ketones as the latter are less hydrophilic. Further oxidation of tertiary alcohol products is improbable.

Hydroxylation of aliphatic side chains attached to an aromatic ring generally occurs at benzylic methylene groups (1') which can be considered as ω -1 oxidation, e.g. parbendazole.

Oxidation of Alicyclic Carbon Atoms (Alicyclic Hydroxylation)

Cyclohexane (alicyclic) and piperidine (nonaromatic heterocycle) rings are commonly found in a number of molecules, e.g. acetohexamide and minoxidil respectively. Such rings are generally hydroxylated at C-3 or C-4 positions.

Oxidation of Carbon-Heteroatom Systems

Biotransformation of C-N, C-O and C-S systems proceeds in one of the two ways –

1. Hydroxylation of carbon atom attached to the heteroatom and subsequent cleavage at carbon-heteroatom bond, e.g. N-, O- and S- dealkylation, oxidative deamination and desulphuration.

2. Oxidation of the heteroatom itself, e.g. N- and S-oxidation.

Oxidation of Carbon-Nitrogen Systems

1. N-Dealkylation: Alkyl groups attached directly to nitrogen atom in nitrogen bearing compounds are capable of undergoing N-dealkylation reactions, e.g. secondary and tertiary aliphatic and aromatic amines, tertiary alicyclic amines and N-substituted amides and hydrazines. Since N-dealkylation of amines yield amines and amides yield amides, the reaction is said to undergo without any change in the state of oxidation. It is however the removed alkyl group that is oxidised. Mechanism of N-dealkylation involves oxidation of α- carbon to generate an intermediate carbinolamine which rearranges by cleavage of C-N bond to yield the N-dealkylated product and the corresponding carbonyl of the alkyl group (a primary alkyl is transformed to aldehyde and a secondary alkyl to ketone).

Tertiary nitrogen is more rapidly dealkylated in comparison to secondary nitrogen because of its higher lipid solubility. Thus, one alkyl from a tertiary nitrogen compound is removed rapidly and the second one slowly. Small alkyl groups like methyl, ethyl, n-propyl, isopropyl, etc. are removed rapidly. N-dealkylation of t-butyl group is not possible because the -carbon cannot be hydroxylated. Such groups are, however, removed via initial hydroxylation of one of the methyl groups to alcohol.

Tertiary nitrogen attached to different alkyl groups undergoes dealkylation by removal of smaller alkyl group first. A representative example of each of the chemical classes of compounds capable of undergoing N-dealkylation is given below.

Secondary aliphatic amines e.g. methamphetamine.

Tertiary aliphatic amines e.g. imipramine.

Secondary and tertiary aromatic amines are rare among therapeutic agents.

Tertiary alicyclic amines (nonaromatic heterocycle) e.g. hexobarbital.

Amides e.g. diazepam.

Hydrazines e.g. iproniazid.

2. Oxidative Deamination: Like N-dealkylation, this reaction also proceeds via the carbinolamine pathway but here the C-N bond cleavage occurs at the bond that links amino group to the larger portion of the drug molecule.

Thus, oxidative deamination is converse of N-dealkylation in terms of product formed — the carbonyl product retains a large portion of the parent structure and the amines formed are simple, e.g. NH3. Actually speaking, both the reactions are same, the criterion for differentiation being the relative size of the products.

Primary aliphatic amines readily undergo deamination, e.g. amphetamine, while secondary and tertiary amines are deaminated only when bulky groups are attached to nitrogen, e.g. propranolol.

Primary amine metabolites formed by N-dealkylation or decarboxylation also undergo deamination. Deamination is inhibited by the absence of -hydrogen, e.g. phentermine. Aromatic and alicyclic amines are resistant to deamination.

3. N-Oxide Formation: N-oxides are formed only by the nitrogen atoms having basic properties. Thus, amines can form N-oxides but amides cannot. Generally, the tertiary amines yield N-oxides. Four categories of tertiary amines that form N-oxides are—

(i) Aliphatic amines e.g. imipramine.

(ii) Alicyclic amines e.g. nicotine.

(iii) Nitrogen atoms of aromatic heterocycles e.g. trimethoprim.

(iv) Amines attached to aromatic rings e.g. N,N-dimethyl aniline.

The N-oxide products are highly water-soluble and excreted in urine. They are, however, susceptible to reduction to the corresponding amine.

4. N-Hydroxylation: Converse to basic compounds that form N-oxides, N-hydroxy formation is usually displayed by non-basic nitrogen atoms such as amide nitrogen, e.g. lidocaine.

N-hydroxylation of amides often leads to generation of chemically reactive intermediates capable of binding covalently with macromolecules, e.g. paracetamol. Paracetamol is safe in therapeutic doses since its reactive metabolite imidoquinone is neutralized by glutathione. However, in high doses, the glutathione level becomes insufficient and significant covalent tissue binding thus occurs resulting in hepatotoxicity. Phenacetin is also toxic for the same reason.

Non-basic aromatic amines also undergo hydroxylation. A primary aromatic amine is further converted to nitroso derivative, e.g. dapsone.

The N-hydroxy dapsone can oxidize ferrous form of haemoglobin to ferric form and cause methemoglobinaemia. A secondary aromatic amine yields a nitrone subsequent to formation of secondary hydroxylamine which is further hydrated to primary hydroxylamine.

N-hydroxylation is also possible at basic nitrogen, e.g. primary and secondary amines. Phentermine, a primary aliphatic amine, cannot undergo deamination as the -carbon does not contain hydrogen and the drug is therefore N-hydroxylated.

Oxidation of Carbon-Sulphur Systems

1. S-Dealkylation: The mechanism of S-dealkylation of thioethers (RSR’) is analogous to N-dealkylation i.e. it proceeds via -carbon hydroxylation. The C-S bond cleavage results in formation of a thiol (RSH) and a carbonyl product, e.g. 6-methyl mercaptopurine.

2. Desulphuration: This reaction also involves cleavage of carbon-sulphur bond (C=S or thiono). The product is the one with C=O bond. Such a desulphuration reaction is commonly observed in thioamides (RCSNHR’) such as thiopental.

Desulphuration also occurs with compounds containing P=S bonds such as the organophosphate pesticides, e.g. parathion.

3. S-Oxidation: Apart from S-dealkylation, thioethers can also undergo S-oxidation reactions to yield sulphoxides which may be further oxidised to sulphones (RSO2R). Several phenothiazines, e.g. chlorpromazine, undergo S-oxidation.

Oxidation of Carbon-Oxygen Systems

O-Dealkylation: This reaction is also similar to N-dealkylation and proceeds by α-carbon hydroxylation to form an unstable hemiacetal or hemiketal intermediate which spontaneously undergoes C-O bond cleavage to form alcohol (arenol or alkanol) and a carbonyl moiety.

The O-containing functional groups (analogous to amines and amides, the substrates which undergo N-dealkylation) are ethers and esters. However, only the ethers undergo O-carbonyl moiety.-dealkylation reaction. Aliphatic ether drugs are rare and aromatic ethers (phenolic) are common. Methyl ethers are rapidly dealkylated in comparison to longer chain ethers such as the one containing n-butyl group. The reaction generally leads to formation of active metabolites, e.g. phenacetin to paracetamol, and codeine to morphine.

Oxidation of Alcohol, Carbonyl and Carboxylic Acid

These reactions are mainly catalysed by non-microsomal enzymes, dehydrogenases. Primary and secondary alcohols and aldehydes undergo oxidation relatively easily but tertiary alcohols, ketones and carboxylic acids are resistant since such a reaction involves cleavage of C-C bonds.

Primary alcohols are rapidly metabolised to aldehydes (and further to carboxylic acids) but oxidation of secondary alcohols to ketones proceeds slowly. This is because –

1. Primary alcohols are better substrates for dehydrogenases due to their acidic nature, and

2. Their oxidation products, carboxylic acids, are more water-soluble than ketones.

Since primary alcohols are inactivated rapidly, drugs bearing such groups are rare. In case of ethanol, oxidation to acetaldehyde is reversible and further oxidation of the latter to acetic acid is very rapid since acetaldehyde is highly toxic and should not accumulate in the body. Compounds with two primary alcohol functions are oxidised stepwise and not simultaneously. With secondary alcohols, the rate of oxidation increases with an increase in alkyl chain length. Compounds with both primary and secondary alcohol groups are oxidised preferentially at the primary group.

Miscellaneous Oxidative Reactions

a. Oxidative Aromatisation/Dehydrogenation: An example of metabolic aromatisation of drugs is nifedipine.

b. Oxidative Dehalogenation: This reaction is common with halogen containing drugs such as chloroform. Dehalogenation of this drug yields phosgene which may result in electrophiles capable of covalent binding to tissues.

Oxidative ring cleavage, oxidation of arenols to quinones, etc. are other oxidative reactions.

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