This section is a continuation of a previous article on drug metabolism. This article focusses on structural changes that occur when drugs undergo biotransformation, as well as its importance in drug design and medicinal chemistry.

Following entry to the body, drug molecules undergo metabolic transformations to derivatives known as metabolites. These metabolic transformations are sometimes referred to as biotransformations. The body generally identifies drugs as foreign substances. Enzymes such as the Cytochrome P450 superfamily are heavily involved in the metabolism of foreign (xenobiotic) substances. The human body is armed with numerous metabolic enzymes that mediate the conversion of xenobiotic compounds to more excretable compounds.

Most small drug molecules are relatively lipophilic. Many drugs are modified or broken down such that they are readily more excretable. Often these drugs are more hydrophilic than their parent drug.  Metabolites may possess a different level of activity as the starting compound or none at all. Moreover, metabolites may possess different biological activity or may be toxic to the patient. A parent drug may be inactive but is eventually converted to the active metabolite in the body. These drugs are referred to as prodrugs. Prodrug molecules take advantage of drug metabolism. Prodrugs were discussed in greater detail in a previous article (see link under Further Reading).

Phase I

Drug metabolism can be divided into the Phase I reactions (functionalisation) and the Phase II reactions (conjugation). Phase I biotransformations include oxidation, reduction, and hydrolysis reactions. Often these reactions involve the introduction of polar functional groups such as –OH to drug molecules to make them more hydrophilic. This can be done by directly introducing a functional group or by converting an existing functional group in a drug to more hydrophilic ones. The metabolites of Phase I reactions often undergo Phase II reactions where small, ionisable, and polar compounds are attached to the new functional groups. Phase I and Phase II reactions complement each other in the modification and excretion of xenobiotic compounds.

Oxidative Biotransformations

Oxidative processes are the most common. The Cytochrome P450 (CYP450) superfamily of enzymes is involved in this type of reactions. These enzymes are haemoproteins (i.e. they contain haem and iron). These enzymes involve molecular oxygen (O2). CYP450 enzymes belong to a class of enzymes known as monooxygenases. The reader is directed to the first article for a more detailed discussion of the CYP450 enzymes. The general formula is shown below:

\[ R – H + O_2 + NADPH + H^{+} \stackrel{CYP450}{\Rightarrow} R – OH + NADP + H_{2}O\]

Carbons that are activated or easily accessible are readily oxidised. For instance, unhindered methyl substituents are often oxidised to form alcohols. The alcohol can also undergo further oxidation to the carboxylic acid. Carbon atoms in the alpha position to a heteroatom can also undergo oxidation.

Aromatic hydroxylation of arenes to arenols can also happen. In the case of benzene, the para position is often favoured. An example of a drug that undergoes aromatic hydroxylation is phenobarbital.


Some of the main oxidative reactions are shown below. The CYP450 enzymes have rich chemistry and the diagram shown below is a small subset of reactions they can catalyse. As mentioned earlier, some metabolites may undergo further oxidations.

CYP450 Enzymes

Drug metabolism is often considered during drug design. For instance, a drug containing a benzene group may undergo Phase I reactions. Most commonly, hydroxylation of the ring occurs. The para position of benzene is usually favoured. This biotransformation can be deterred by replacing the hydrogen at the para position and using fluorine as a bioisostere. The role of fluorine as a bioisostere has been briefly introduced in a previous article.

Para Hydroxylation

On average, the bond dissociation energy of the C-F is around 425 kJ mol-1. As a result, C-F bonds generally resist metabolism. Furthermore, due to the similar van der Waals radii of hydrogen and fluorine, replacement of hydrogen with the fluorine bioisostere does not exert significant steric demand at binding sites. A very similar strategy was used in the design of the cholesterol absorption inhibitor, ezetimibe from a lead compound.

J. Med. Chem., 2004, 47 (1), pp 1–9.


Reductive Biotransformations

Reductive drug metabolism reactions can occur with drugs containing carbonyl, azo, and nitro groups. These reactions are catalysed by various enzymes. For example, the reduction of ketones is facilitated by reductases. Carbonyl groups are reduced to alcohols whereas nitro and azo groups are reduced to amino derivatives. Hydroxyl and amino functional groups can readily undergo Phase II conjugation reactions. The oral contraceptive levonorgestrel, colloquially known as ‘the morning after pill’, is an example of a drug that goes through reductive Phase I biotransformations.


Other less encountered reductive pathways of biotransformations include N-oxide reduction to tertiary amines and the reduction of sulfoxide groups to thioethers. The bioprecursor prodrug, sulindac has a sulfoxide functional group that is converted to the active NSAID by enzymes in the body.


Hydrolytic Biotransformations

Hydrolytic reactions are catalysed by enzymes found in plasma, in certain tissues, and certain organs. Esters and amide linkages in drugs can undergo hydrolysis. Esterases and amidases are hydrolytic enzymes. Ester prodrugs in particular take advantage of hydrolytic enzymes. The biotransformation of aspirin to salicylic acid is a well-known example of ester hydrolysis.

Aspirin to Salicylic Acid

Phase II

Phase I functionalisations don’t always give rise to pharmacologically inactive, nontoxic, or sufficiently hydrophilic metabolites. Phase II reactions often render metabolites more hydrophilic by attaching small, ionisable, and polar molecules to certain functional groups in the drug or the drug’s metabolites. The resulting metabolites are generally inactive and readily excretable.

Phase II reactions are mostly conjugation reactions. These reactions are typically catalysed by transferaseenzymes. Other phase II reactions such as methylation and acetylation often serve to put an end to or diminish pharmacological activity. The different Phase II reactions will now be discussed.

Glucuronic Acid Conjugation

Glucuronic Acid

Glucuronidation is the most common Phase II reaction. D-Glucuronic acid is derived from D-Glucose. Carboxylic acids, alcohols, phenols, and hydroxylamine can undergo glucuronidation. Glucuronosyl transferases (UGTs) are the transferases involved with the reaction. Structures of some drugs and the site of glucuronidation are shown below: naproxen & desipramine.

Naproxen & Desipramine

Sulfate Conjugation

Sulfate conjugation of xenobiotics is less common compared to glucuronidation. These reactions mainly occur with phenols and sometimes aromatic amines, N-hydroxy compounds, and alcohols. The β-adrenergic bronchodilator, salbutamol is known to undergo sulfate conjugation.


GSH Conjugation

Glutathione (GSH)

Glutathione (GSH) is a tripeptide that is involved in the detoxification of reactive electrophilic compounds or intermediates. Glutathione S-Transferases (GSH S-Transferase) are the enzymes that catalyse the conjugation of this molecule with drugs. In general, compounds that react with GSH do so through a nucleophilic attack on an electrophilic carbon with a good leaving group or through addition to electron-deficient double bonds. Reactive electrophiles are toxic due to their ability to form covalent bonds with important macromolecules such as cellular proteins and nucleic acids. Depletion of GSH is associated with the toxicity observed with paracetamol (acetaminophen) overdose.

Paracetamol can be converted to NAPQI (N-acetyl-p-benzoquinone imine) during Phase I reactions by CYP450 enzymes. NAPQI is a highly reactive compound. At safe doses of the drug, Phase II GSH conjugation reactions convert toxic NAPQI to a non-toxic metabolite. At unsafe doses of paracetamol, excess NAPQI is generated. GSH is eventually depleted and NAPQI begins reacting with nucleophilic groups in cellular macromolecules. This reaction is detrimental to the organism.


Drug Design & Drug Discovery

Having knowledge of drug metabolism is of great value to medicinal chemists. During drug design, molecules may be designed such that they don’t form toxic metabolites. This knowledge can also be used as a guide to assist researchers in making molecules have optimal pharmacokinetic properties. Studies of metabolism can be useful in drug discovery as well. For example, the known potassium channel-opening vasodilator, cromakalim was discovered through these studies. Compound I was discovered to have less activity in vitro than in vivo which suggests that this compound was converted to a more active compound in living systems. The more active metabolite, cromakalim was eventually discovered.


Drug Metabolism Studies

Patient safety is of utmost importance. Releasing unsafe compounds to the market can lead to undesirable side effects and could potentially cost lives. Withdrawing drugs from the market can be costly to pharmaceutical companies. Drug metabolism studies must be performed before a drug is approved for clinical use. These studies involve the identification of all metabolites. The metabolites are characterised and their structures and stereochemistry are identified. Furthermore, metabolites are studied and assessed for biological activity and toxicity for safety reasons.

Experiments and studies may be in silico, in vitro, in vivo or combinations thereof. Certain computer software packages can predict a drug’s metabolites. It is important to mention that interspecies variation must be taken into account when selecting an animal model for in vivo studies. Metabolic differences across species can vary greatly.

Isotopic labelling studies are also common. This involves the synthesis of drugs that are labelled with isotopes. This type of study is particularly useful in metabolite identification and characterisation. The isotopes used may be ‘stable’ or radioactive isotopes (radioisotopes). The drug is labelled with isotopes such as carbon-13 (13C), carbon-14 (14C), deuterium (D or 2H), and tritium (T or 3H).

Isotopes are generally incorporated to molecules during organic synthesis. Synthetic routes are designed accordingly to allow the use of commercially available isotope-labelled building blocks to make the drug. Synthetic routes may be designed through retrosynthetic analysis.

Drugs and metabolites which contain ‘stable’ isotopes are studied using spectroscopic techniques such as nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). Both 14C and 3H undergo β decay. Metabolites containing these radioisotopes are detected by measurement of β radiation. Blood samples from in vivo experiments involving radioisotopes can take advantage of high performance liquid chromatography (HPLC). A radioactivity detector may be used to study the different metabolites.

The study of drug metabolism and drug design are closely related. Having knowledge of both can lead to the development of safe and clinically useful drugs.

Further Reading

  • Drug Metabolism I
  • Prodrugs
  • Drug metabolism in silico– the knowledge-based expert system approach. Historical perspectives and current strategies.
    • Drug Discovery Today: Technologies. 2013, 10 (1), pp e147-e153.