Penicillin: Discovery, Development, Medicinal Chemistry

Penicillin was one of the first antibiotics discovered by A.Fleming. The accidental findings lead to treatment of worldwide spread disease as penicillin molecule inhibited the synthesis of the bacterial cell wall by mimicking the acyl-D-alanyl-D-alanine of peptide side chain and soon the new peptidoglycan (PGN) formation is stopped and bacteria cell is susceptible to osmotic rupture and therefore killing the bacteria. However, penicillins structure contains highly reactive β-lactam ring which makes it susceptible to various antibiotic resistance issues. Due to this problem different alterations to initial penicillin molecule has been made and 4 penicillin generations have been introduced with wide effectiveness against Gram- positive (Staphylococci, Streptococci) and Gram-negative (Pseudomonas Aeruginosa) bacteria.

Introduction

Penicillin was discovered in 1928 by Scottish scientist Alexander Fleming.1 The discovery and use of antibiotic agents has saved numerous lives, and earned A. Fleming — together with H. Florey and E. Chain, who devised methods for the large-scale isolation and production of penicillin — the 1945 Nobel prize in Physiology/Medicine.2 In 1928, A. Fleming carried various experiments involving the common staphylococcal bacteria. An uncovered Petri plate sitting next to an open window became contaminated with mould spores. Fleming observed that the fungal contaminant was affecting the expansion of bacteria. The fungus was found to be Penicillium notatum, and so, the antibacterial molecule that it produced was named penicillin. He found it to be effective against all Gram-positive pathogens, causing diseases like scarlatina, pneumonia, gonorrhea, meningitis, and diphtheria. Penicillin is almost an ideal antibiotic because it inhibits the synthesis of the bacterial cell wall – a component not present in human. For this reason, there is not a deleterious effect on the host, and penicillin can be given in very high doses. The sole limitation with penicillin is that the occurrence of allergies of several types.

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Penicillin molecule

The penicillin is formed by three carboxylic groups, one of them corresponding to the carboxylic acid moiety. The molecule is mostly planar: there are two hetero rings fused together to form a β-lactam group and thiazolidine ring- the core of penicillin molecule.

Another important feature of penicillins, closely associated with their biological properties, is the stereochemistry of core that impacts the efficiency of binding. (Figure 1).3 Therefore, chemical adjustments made on penicillin nuclei have to preserve this stereochemistry.

Figure 1 Core structure of penicillin, where R is a variable side chain.

Mode of action

Penicillins act by inhibiting the formation of the bacteria cell wall. Depending on a dose penicillin effect bacteria in three different ways: it may appear to be bacteriostatic, bactericidal, or lytic, depending on conditions.4 At very low doses (1/10 of bacteriostatic dose), may cause pronounced morphological changes. Cocci swell to a good size, and bacilli elongate to several times their length. Penicillin is here interfering with the ability of the bacteria to divide, and its action would seem to be bacteriostatic. Bacteriostasis is a loose term, since stationary population may result from inhibition of growth or from the killing of a fraction of each new generation. The powerful killing and lytic actions are exercised only against dividing bacteria.

Mechanism of action

Penicillin kills susceptible bacteria by inhibiting the cross-linking of peptidoglycan (PGN) layer (Figure 3).5 PGN is found outside the cytoplasmic membrane and consists chains of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues, which are covalently crosslinked via short peptides.6 Due to highly reactive β-lactam ring structure, penicillin irreversibly acylates the semipermeable membrane transpeptidase (Figure 4). The membranes of varied bacteria contain one major and few minor proteins which are readily available to bind with penicillin through a covalent bond. Penicillin is proved to be a structural analog of the acyl-D-alanyl-D-alanine terminus of the pentapeptide side chains of nascent peptidoglycan (Figure 2). Therefore, it interrupts the construction of peptide bridges and penicillin molecule blocks new PGN formation and bacteria cell is susceptible to osmotic rupture. As this process occurs the PGN is no longer capable to resist the osmotic stress and soon the bacteria is rendered inactive.Figure 3 Schematic representation of penicillin's mode of action. Peptidoglycan layer is made of polysaccharide chains of GlcNAc and MurNAc units (shown in white and black circles). Small peptides are attached to PGN and transpiptedase enzyme assembles the construction of cross-linking between peptides. First, the terminal alanine from each peptide is hydrolyzed and secondly, one alanine is joined to lysine through an amide bond. Arranged binding to the last D-alanine residues of peptides are mimicked by penicillin and therefore disable the PBP by creating covalent bonds to the catalytic serine residue.

Antibiotic resistance

Antibiotic resistance occurs when bacteria change in response to the use of these medicines.8 Process of resistance is accelerated by the misuse and overuse of antibiotics, as well as poor infection prevention and control. Currently, at least 700,000 individuals die each year due to drug-resistant infections, counting 230,000 individuals who die from multidrug-resistant tuberculosis. Some bacteria have developed resistance to antibiotics that were once commonly used to treat them. Staphylococcus aureus and Neisseria gonorrhoeae are now mostly resistant to benzylpenicillin. In the past, these infections were usually controlled by penicillin. Therefore, resistance to β-lactams mainly are caused by different mechanisms including 1) the production of β-lactamases, which destroy the β-lactam ring; 2) the presence of altered penicillin-binding proteins (PBPs), which have lower affinity for β-lactams; and 3) a reduced permeability of the outer membrane or active efflux of the drug from the periplasmic space.7,11Figure 2 Penicillin G structure compared to D-alanyl-D-alanine.

Increased drug efflux

Drug efflux is a key mechanism of resistance in Gram-negative bacteria.9,10 Efflux pumps permit the microorganisms to control their internal environment and remove toxic substances, antimicrobial agents, metabolites, and quorum sensing signal molecules, without drug alteration or degradation.

The acylamino side-chain can open the β-lactam ring

The acyl-amino side chain on β-lactam ring can lead to its opening by acting as an internal nucleophile.12 In this particular case a strongly strained intermediate will form that leads to the opening of the ring. This process leads to inactivation of penicillin and is commonly known as the ‘self-destruct’ mechanism. (Figure 5)

Figure 4 Mechanism of action of β -lactam antibiotics on bacterial transpeptidases and dd -carboxypeptidases. Nucleophilic attack of the PBP-Ser hydroxyl on the β -lactam ring carbonyl results in an opening of the β-lactam ring.

Hydrolysis of the β-lactam ring in a penicillin

Due to extremely reactive β-lactam ring, a penicillin can react with water under acidic conditions, where such conditions would be met in the stomach. β-lactam ring breaks in hydrolysis reaction (Figure 6).6 The reaction is a nucleophilic acyl substitution reaction, which in turn proceeds to form a penicilloic acid. As formed acid has an open ring system it does not have the desired antibiotic activity in the body. Therefore, missuse of antibiotic leads to loss of antibiotic effectiveness.

Penicillin generations

Penicillins are bactericidal β-lactam antibiotics. To produce more stable penicillins different alterations to side- chains are made. The spectrum of antibacterial activity varies with each class of the penicillin family.12 Following a wide use of the “natural penicillins”, penicillinase-producing strains also emerged among Gram-positive species.

This suggested pharmacology research to develop semisynthetic, beta-lactamase-resistant penicillins (i.e. second-generation penicillins): oxacillin, methicillin, and dicloxacillin, also defined as anti-staphylococcal penicillins. Their ability is to resist penicillinases present in staphylococci.14 As second generation had relatively narrow spectrum of activity there was a need for broader coverage antibiotics to fight against Gram-negative organisms.

In the 1960s, the third generation and broad-spectrum penicillins also known as aminopenicillins were introduced. Amoxicillin and ampicillin are the main examples of this group and unlike their predecessors, third-generation penicillins proved to be more effective against a wider group of Gram-negative bacteria (including Haemophilus influenzae, Escherichia coli, Salmonella spp., and Shigella spp.), due to their higher stability to penicillinases. The last generation of penicillins which includes carboxypenicillins and ureidopenicillins further broadened the spectrum of penicillin coverage against Gram-negative bacteria and displayed potent activity against Pseudomonas aeruginosa. 6

Penicillins face enzyme-catalyzed degradation in vivo. Amidases catalyze the conversion of the amide to an amine. Such amidases are useful in industry for the production of 6-Aminopenicillanic acid. This compound is used as a precursor for many semisynthetic penicillins.

1st generation

Naturally derived from the mold, Penicillium chrysogenum. Antibiotic molecular structure stays relatively same with minor modifications made on side chains.13 Antibiotic inhibits synthesis of bacteria cell wall causing bacteria death. Bacteria that are not resistant to first-generation penicillins are Streptococcus pneumoniae, different groups of streptococci, and non-penicillinase-producing staphylococcus. Figure 5 Explained mechanism how acylamino side- chain opens the β-lactam ring

Figure 6 Mechanism of nucleophilic acyl substitution reaction of penicillin which forms a penicilloic acid.

Penicillin G and penicillin V

Originally this molecule was extracted from P. notatum.6 Penicillin G, where R = an ethyl phenyl group (Figure 2), is the most potent of all penicillin derivatives it is also known as benzylpenicillin.14 Penicillin’s overall shape is similar to a half-open book. The bicyclic ring system has large, torsional strain and angle strain. Unlike typical tertiary amides, the carbonyl group of the strained four-membered ring is very reactive and susceptible to nucleophilic attack. Considering resonance forms of β-lactam ring, amide resonance is diminished. For steric reasons the bonds to the nitrogen cannot be planar as the opening of the four-membered ring relieves strain. However, benzylpenicillin may be broken down in the stomach by gastric acids and is poorly and irregularly absorbed into the bloodstream, therefore, it is suggested to administer antibiotic intravenously or intramuscularly. Phenoxymethylpenicillin (Penicillin V), have enhanced acid stability as they have electron-withdrawing side chain (Figure 7).

2nd generation

The penicillinase-resistant second-generation penicillins are semisynthetically modified from naturally occurring penicillins.15 A bulky group attached to the amino acid side chain provides steric hindrance which interferes with the enzyme attachment which would deactivate the penicillins i.e. methicillin.

Methicillin

This antibiotic initially had phenol group of benzylpenicillin distributed along with methoxy groups (Figure 8 top). By having methoxy groups steric hindrance around the amide bond was reduced, however, that lead to reduced affinity for staphylococcal β-lactamses. Therefore, as soon as this drug entered clinical use it failed as methicillin-resistant staphylococcus aureus strains were isolated.16 This reactivity lead to creation of additional penicillin-binding protein from another species, which proved its resistance and supplied no antibiotic properties in the body. Certain external factors are affecting methicillin resistance for example NaCl concentration, pH, medium composition, osmolarity, and temperature. By exploiting some of these factors, strains, exhibiting heterogenous methicillin resistance, can be detected.

3rd generation

The aminopenicillins (Figure 8 middle) referred to as third-generation penicillins are semisynthetically modified from natural penicillins.17 3rd generation penicillins have core structure of β-lactam ring fused to thiazolidine. Powerful electron-attracting groups attached to the amino acid side chain prevent acid attack. Finally, if the polar character is increased as in ampicillin, there is a greater activity against Gram-negative bacteria.18 Figure 7 Structure of Penicillin V.

Ampicillin and amoxicillin

Broad-spectrum antibiotics such as amoxicillin and ampicillin both have -NH2 groups attached to the carbon that is α to the carbonyl group on the side chain (Figure 8 middle); both compounds being orally active. As well as this, the presence of the electron-withdrawing amino group increases acid stability. Ampicillin is poorly absorbed by the gut due to ionisation of both the amino and carboxylic groups. Oral absorption of amoxicillin is, in contrast, much higher.

Ampicillin in addition of sulbactam sodium gives a coverage against penicillinase-resistant bacteria.20 These drugs are used to treat mild to severe infections due to susceptible organisms. Ampicillin is administered orally and when combined with sulbactam provides a powerful indication against Gram-positive and Gram-negative bacteria.19 However, reactions indicated ampicillin being more prone to cause hepatic injuries rather than its combination with sulbactam.

β-lactamse inhibitor- clavulanic acid work by binding irreversibly to the catalytic site of an organism’s penicillinase enzyme, which causes resistance to the original β-lactam ring. When amoxicillin is coupled with clavulanic acid, the drugs bactericidal activity is broadened and its antibiotic resistance is reduced even more. However, both ampicillin and amoxicillin are poorly absorbed through the gut wall as their amino and carboxlic groups gets ionised. Therefore, this problem can be alleviated by using a protecting group to mask the polar groups. As the drug gets absorbed this protective group can be removed metabolically.

4th generation

The newest generation of penicillins further broaden the spectrum of penicillin coverage against Gram-negative bacteria and display potent activity against Pseudomonas aeruginosa.20 23 These extended-spectrum penicillins are more resistant to inactivation by extended-spectrum β-lactamases (ESBLs) which are produced by Gram-negative bacteria and because they more readily penetrate the outer membranes of these Gram-negative organisms. By placing a bulky group on the acylamino side chain, degradation of the drug by β-lactamases is minimized. However, if a steric shield is too bulky, the penicillin is not able to bind to transpeptidase.

Carbenicillin, piperacillin and tazobactam

Carbenicillin shows a broad spectrum due to hydrophilic carboxylic acid group on the side chain. Specific stereochemistry is crucial as only one out of two enantiomers is active.21 In-vitro studies of piperacillin and tazobactam have resulted in β-lactamase inhibitor with an extremely low toxicity, a wide range of inhibition and a weak induction of β-lactamases.22

Figure 8 Penicillin structure alterations in different generations. Top: 2nd generation; middle: generalized 3rd generation penicillin structure where R1 is variable; bottom: generalized ureidopenicilin molecule where R1 is a variable side chain. 3rd and 4th generation R1s are not the same.

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