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ANTIBACTERIAL ANTIBIOTICS 1 ANTIBIOTICS The word "antibiotics" comes from the Greek anti ("against") and bios ("life"). Antibiotics are drugs that either destroy bacteria or prevent their reproduction. Antibiotics that kill bacteria are called "bactericidal" and the ones that stop the growth of bacteria are called "bacteriostatic". Some antibiotics are 'bactericidal', meaning that they work by killing bacteria. Other antibiotics are 'bacteriostatic', meaning that they work by stopping bacteria multiplying. 2 Each different type of antibiotic affects different bacteria in different ways. For example, an antibiotic might inhibit a bacterium's ability to turn glucose into energy, or its ability to construct its cell wall. When this happens, the bacterium dies instead of reproducing. Some antibiotics can be used to treat a wide range of infections and are known as 'broadspectrum' antibiotics. Others are only effective against a few types of bacteria and are called 'narrow-spectrum' antibiotics. 3 Targets for Antibiotics Fig 24.9 pg. 741 4 Antibiotics That Inhibit Cell Wall Synthesis Penicillin and Cephalosporins 1. One major class of antibiotics inhibit the synthesis of peptidoglycan. 2. Once cell wall synthesis (involving penicillin binding proteins) is inhibited, enzymatic autolysis of the cell wall can occur. 5 Antibiotic resistance Antibiotics are extremely important in medicine, but unfortunately bacteria are capable of developing resistance to them. Antibiotic-resistant bacteria are germs that are not killed by commonly used antibiotics. When bacteria are exposed to the same antibiotics over and over, the bacteria can change and are no longer affected by the drug. Bacteria have number of ways how they become antibioticresistant. For example, they possess an internal mechanism of changing their structure so the antibiotic no longer works, they develop ways to inactivate or neutralize the antibiotic. Also bacteria can transfer the genes coding for antibiotic resistance between them, making it possible for bacteria never exposed to an antibiotic to acquire resistance from those which have. 6 ANTIBIOTICS CLASSIFICATION Based on chemical structure •β-lactum antibiotics •Aminoglycoside •Tetracyclines •Macrolide antibiotics •Lincomycins •Polypeptide antibiotics •miscellaneous Based on pharmacological activity • Antifungal antibiotics • Anticancer antibiotics • Anti typhoid antibiotics • Anti diarrheal antibiotics • Antituberculor antibiotics 7 Β-LACTAMS β-lactam ring 8 HOW DO THEY WORK? 1. 2. 3. 4. The β-lactam binds to Penicillin Binding Protein (PBP) PBP is unable to crosslink peptidoglycan chains The bacteria is unable to synthesize a stable cell wall The bacteria is lysed 9 Mechanism of action of β-lactam antibiotics. Normally, a new subunit of N-acetylmuramic acid (NAMA) and Nacetylglucosamine (NAGA) disaccharide with an attached peptide side chain is linked to an existing peptidoglycan polymer. This may occur by covalent attachment of a glycine () bridge from one peptide side chain to another through the enzymatic action of a penicillinbinding protein (PBP). In the presence of a β-lactam antibiotic, this process is disrupted. The β-lactam antibiotic binds the PBP and prevents it from cross-linking the glycine bridge to the peptide side chain, thus blocking incorporation of the disaccharide subunit into the existing peptidoglycan polymer. 10 Mechanism of penicillin-binding protein (PBP)inhibition by β-lactam antibiotics. PBPs recognize and catalyze the peptide bond between two alanine subunits of the peptidoglycan peptide side chain. The β-lactam ring mimics this peptide bond. Thus, the PBPs attempt to catalyze the β-lactam ring, resulting in inactivation of the PBPs. 11 Peptidoglycan Layer 12 13 The beta lactam antibiotics include penicillins (e.g. ampicillin), cephalosporins and monobactams. They bind to and inhibit enzymes (penicillin binding proteins) involved in the transpeptidation (cross-linking) of peptidoglycan. These antibiotics have in common the four membered lactam ring. Attached to the lactam, penicillins have an additional five membered ring and cephalosporins a six membered ring. Monobactams consist of the lactam ring alone and display antibiotic activity. 14 penicillins Penicillin is the oldest known antibiotic in the whole world and what is really surprising that it was discovered by total coincidence. It was discovered by Alexander Fleming in the late nineteenth century when he was trying to multiply the penicillum fungi in the lab. Penicillin is still used till now for the treatment of many bacterial and fungal infections and of course it was developed immensely since that time . There are now different categories of penicillin but the core of these categories is the natural penicillin or what is known the penicillin G. natural penicillin is effective against most of the gram positive microorganisms like streptococcus and staphylococcus. It is also effective against some gram negative bacteria and it is 15 mainly used for treatment of oral cavity infections. The second category of penicillin antibiotic is penicillinase resistant antibiotics. Oxacillin and cloxacillin are prominent examples for this group. This group does not target a wide range of microorganisms like the natural penicillin but it targets a specific group of bacteria which produces the beta lactamase enzyme. The lactamase producing bacteria are resistant to natural penicillin. The third category of penicillin antibiotics is the aminopenicillins. Amoxicillin is the best example for this group and it is widely used today. This is group is very potent against gram negative bacteria like Ecoli and Hemophelus influenza. The best feature of this group of penicillin antibiotics is that they can tolerate the gastric acid. This means that they could be administered orally without being degraded through the acid medium of the stomach. 16 The fourth group is called the extended penicillin antibiotics, although this group is effective against many gram - negative microorganisms but they are still inactive against beta- lacatam producing bacteria like the aminopinicillins . The four categories of penicillin are still used till now to combat different types of microorganisms and every category is used to perform its specific role. All penicillin drugs act in the same way by affecting the bacterial cell wall. Suppression the formation of the bacterial cell wall will cause the bacteria to die and omit the effect of the microorganism on the body, the best feature of penicillin is that it develops the least side effects on the patient. 17 Penicillin • Prevents crosslinking of peptidoglycan, gram +/some • Beta-lactam nucleus – Resistance through beta-lactamases • In low concentrations,kills gram + cells, gram (-) in higher doses • Allergic reactions 18 History of penicillins In 1875 Pasteur and Joubert discovered that certain moulds could produce toxic substances which killed bacteria. Unfortunately, these substances were also toxic to humans and of no clinical value. However, they did demonstrate that moulds could be a potential source of antibacterial agents. In 1928, Fleming noted that a bacterial culture which had been left several weeks open to the air had become infected by a fungal colony. Of more interest was the fact that there was an area surrounding the fungal colony where the bacterial colonies were dying. He correctly concluded that the fungal colony was producing an antibacterial agent which was spreading into the surrounding area. Recognizing the significance of this, he set out to culture and identify the fungus and showed it to be a relatively rare species of Penicillium. It has since been suggested that the Penicillium spore responsible for the fungal colony originated from another laboratory in the building and that the spore was carried by air currents and eventually blown through the window of Fleming's laboratory. 19 Fleming spent several years investigating the novel antibacterial substance and showed it to have significant antibacterial properties and to be remarkably non-toxic to humans. Unfortunately, the substance was also unstable and Fleming was unable to isolate and purify the compound. He therefore came to the conclusion that penicillin was too unstable to be used clinically. The problem of isolating penicillin was eventually solved in 1938 by Florey and Chain by using a process known as freeze-drying which allowed isolation of the antibiotic under much milder conditions than had previously been available. By 1941, Florey and Chain were able to carry out the first clinical trials on crude extracts of penicillin and achieved spectacular success. Further developments aimed at producing the new agent in large quantities were developed in the United States such that by 1944, there was enough penicillin for casualties arising from the D-Day landings. 20 Penicillins were used widely and often carelessly, so that the evolution of penicillinresistant bacteria became more and more of a problem. The fight against these penicillin-resistant bacteria was promoted greatly when, in 1976, Beechams discovered a natural product called clavulanic acid which has proved highly effective in protecting penicillins from the bacterial enzymes which attack penicillin. STRUCTURE OF PENICILLIN Penicillin contains a highly unstable-looking bicyclic system consisting of a fourmembered lactam ring fused to a fivemembered thiazolidine ring. The skeleton of the molecule suggests that it is derived from the amino acids cysteine and valine (Fig.), and this has been established. 21 STRUCTURE OF PENICILLIN O S C NH CH CH CH3 C CH3 O C N CH COOH Site of penicillinase action. Breakage of the β lactam ring. 22 STRUCTURE O R= C CH2 H S Me R Benzyl penicillin (Pen G) R= O H H N CH2 Phenoxymethyl penicillin (Pen V) N Acyl side chain Me 6-Aminopenicillanic acid (6-APA) O CO2H Thiazolidine ring b-Lactam ring Side chain varies depending on carboxylic acid present in fermentation medium .For example, corn steep liquor was used as the medium when penicillin was first mass-produced in the United States and this gave penicillin G (R=benzyl). This was due to high levels of phenylacetic acid (PhCH2CO2H) present in the medium. CH2 CO2H Penicillin G present in corn steep liquor OCH2 CO2H Penicillin V (first orally active penicillin) 23 Mechanism of action - bacterial cell wall synthesis NAM NAG NAM NAG L-Ala L-Ala D-Glu D-Glu L-Lys Gly Gly Gly Gly Gly L-Lys D-Ala D-Ala D-Ala D-Ala SUGAR BACKBONE Gly Gly Gly Gly Gly PENICILLIN TRANSPEPTIDASE D-Alanine NAM NAG NAM L-Ala L-Ala D-Glu D-Glu L-Lys D-Ala Gly Gly Gly Gly Gly Cross linking L-Lys NAG SUGAR BACKBONE Gly Gly Gly Gly Gly D-Ala 24 CONSTITUTION OF PENICILLIN Molecular formula of penicillin is C9H11N2O4SR presence of carboxylic group: They form mono salts indicating the presence of a carboxylic group Penicillins are not found to posses a free amino group , thiol group Hydrolysis: penicillins are hydrolysed by hot dilute inorganic acids , they lose one carbon atom in the Form of carbondioxide and they are also degraded to the equimolar amounts of an amine, PENICILLAMINE, and an aldehyde PENILLOALDEHYDES. All the penicillins are degraded to the Same amine but different aldehydes because the fragement comes in the aldehyde portion. C9H11N2O4SR + 2H2O CO2 + C5H11NO2S + C3H4NO2R PENICILLAMINE PENILLOALDEHYDE Now since the fragment R comes in the aldehyde portion, all the penicillins give the same amine but different aldehydes. Structure of D-Penicillamine : C5H11NO2S.this ha been elucidated on the basisof following facts: Molecular formula of Penicillamine is C5H11NO2S. As Penicillamine respond to the indigo colour reaction with ferric chloride solution and Also gives colour reaction with sodium nitroprusside , this shows that DPenicillamine is probably a substituted cysteine. From electrometric titrations ,it is revealed that there are three pka values 1.8, 7.9,and 10.5 corresponding to the carboxylic , α-amino and thiol groups respectively. The presence Of these groups also reveals that D-penicillamine is probably substituted cysteine. Moreover, the chemical reactions are parrell to those of cysteine . For example like cysteine D-penicillamine also reacts with acetone to yield an isopropylidene derivative. The latter Compound does not contain a free amino or thiol group and is reconverted into pencillamine On hydrolysis. These reactions reveal that these groups,i.e, amino and thiol group , are Attached to adjacent carbon atoms.further oxidation of penicillamine with brominewater Yields a sulphonic acid(this reaction is characterstic of a thiol) When kuhn-roth method for the determination of methy side chain is applied to D-PENICILLAMINE It gives a very low value (~ 0.2molecules): this low value reveals that D-PENICILLAMINE contains An isopropyl end-group and not a methyl end-group. so D-PENICILLAMINE is β,β-dimethylcysteine which has been confirmed by its synthesis. NaOH ClCH2COCL (CH3)2CHCHCO2H (CH3)2CHCHCO2H -HCL NH2 NHCOCH2Cl DL-valine (CH3CO)2O C (H3C)2C CHCO2H (CH3)2C SH H2S BOIL NHCOCH3 CHCO2H 1.H2S S N C C (H3C)2C + 2.-H 3.+H+ CH3 C N O H C SH CH3 Azalactone 2,5-trimethyl-2thiazoline-4-carboxylic acid HCL(boil) pyridine (CH3)2C SH CHCO2H NH2 DL-penicillamine From the above synthesis, the recemic miture DL is obtained.for resolution of this racemic mixture , this is treated with formic acid to yield the formyl derivative which is then resolved by means of brucine . The formyl group is removed by hydrolysis. HCO2H (CH3)2C SH CHCO2H NH2 CH3)2C CHCHO2H SH NHCHO DL-form DL-formyl derivative 1.resolution by brucine 2.HCL 3.pyridine (CH3)2C SH CHCO2H NH2 D-penicillamine The D- penicillamine odtained from the resoltuion of the recemic mixture has been found to be identical with the natural penicillamine. When the penicillamine is treated with diazomethane , it is converted into its methyl ester. The lattes compound when treated with aqueous solution of mercuric chloride yields the methyl ester of penicillamine .these reactions reveal that the carboxylic group in penicillin itself . Structure of penilloaldehyde: This has been elucidated on the basis of the following facts.: The general formula of penilloaldehyde has been found to be C3H4NO2R . When hydrolysed vigorously , all penilloaldehydes yield a substitued acetic acid and Amino acetaldehyde . This reaction reveals that penilloaldehyde are RCOOH + NH2CH2CHO RCONHCH2CHO (or C3H4NO2R) + H2O acyl derivatives of aminoacetaldehyde. substitued aminoacetaldehyde penilloaldehydes acetic acid The above structure of penilloaldehyde has been confirmes by its synthesis from the Corresponding acid chloride and amino acetal. RCOCl + NH2CH2CH(OC2H5)2 RCONHCH2CH(OC2H5)2 HCL RCONHCH2CHO penilloaldehydes In this point it was stated that acid hydrolysis of penicillinyields penicillamine,penilloaldehyde And corbondioxide. The formation of co2 molecule is indicative of the fact that some unstable acid is formed as an intermediate which undergoes ready decarboxylation to yield carbon dioxide .such an acid forme as an intermediate in the hydrolysis of penicillin. RCONHCHCHO COOH penaldic acid CO2 + RCONHCH2CHO penilloaldehyde Mode of lincking of penicillamine and penilloaldehyde.: now the next question arises How are the two fragments ,penicillamine and penilloaldehyde, linked in penicillin … So that it may explain tha formation of panaldic acid that is to explain the formation of co2 ? Tha mode of linkage of these fragments has been established on the basis of the following facts; When penicillin is hydrolysed with dil alkali or with the enzyme penicillinase it It yields penicilloic acid which is a dicarboxylic acid and readily eliminates a molecule of co2 to yield a monocarboxylic acid, penilloic acid . this suggest that in penicilloic acid one of the carboxylic groups is in the β-position with respect to anelectron-attractin g group. The structure of penillonic acid has been proved on the basis of the fact that it,when hydrolysed with aqueous mercuric chloride,yields pwnicillamine and penilloaldehyde. This type of hydrolysis is characteristic of compounds having a thiazolidine ring. The presence of thiazolidine type of nucleus in penicillin has been proved by the fact that it possesses neither free amino group nor a free thiol group .thus penilloic acid could be represented as •Because this structure would yield the desired products SH S ROCHNH2CHC C(CH3)2 HN CH(CH3)2 H2O HgCl2 CHCO2H Penilloic acid (A ) H2N + RCONHCH2CHO CHCO2H penicillamine penilloaldehyde If A is the structure of penilloic acid , then the structure of penicilloic acid would be represented as S R.CO.NH.CH HOOC CH HN C(CH3)2 CHCOOH PENICILLOIC ACID CO2 + (A) The structure B of penicilloic acid has been confirmed by the fact that penicillin when treated with methanol yields methyl penicilloate which on hydrolysis with aq. Mercuric chloride yields methyl penaldate S R.CO.NH.CH CH C(CH3)2 CH3OH PENICILLIN CO2H3C HN CHCO2H Methl penicilloate H2O CH(CH3)2 HgCl2 HO2CHCNH2 penicillamine + RCONHCHCHO COOCH3 methl penaldate Structure of penicillin: From the above evidences , two structures C , D are possible for penicillin . But penicillin on treatment with dil acid undergoes molecular rearrangement readly to yield penillic acid . Therefore , the chemical evidence could not decide about the correct structure C (or) D of penicillin. However , the physical methods could decide about the correct structure of penicillin. S N H3CR N CH O HC ROCHNHC HN oxazolone structure C(CH3) CH(CH3) CO CO HC N CHCO2H CHCO2H (C) beta lactum structure (D) Definite evidence of the existing of the β-lactum ring in penicillin was obtained by desulphurisation of the benzylpenicillin with raney nickel to yield desthiobenzylpenicillin (E) Which on hydrolysis by acid yields desthiopenicilloic acid (F) or on boiling with benzylamine in Dioxane solution yields benzylaminde of desthiobenzyopenicilloic acid(G). S C6H5H2COC.HN HC B OC CH3 A N C.(CH3) H2C RANEY Ni COOH BENZYLPENICILLIN C6H5CH2CO.NH.CH OC CH2 CH(CH3)2 N CH.COOH desthiobenzylpenicilln(E) C6H5CH2NH2 C6H5CH2CO.NH.CH CH2 COOH HN CH(CH3)2 CH.COOH desthiobenzylpenicilloic acid (G) C6H5CH2CO.NH.CH CH2 (H3C)2HC NH CO.NH.CH2.C6H3 benzylamide of desthiobenzylpenicilloic acid (H) CH.COOH The compounds G , H can be obtained by the desulphation of benzyl penicilloic acid and Its benzyl amide respectively. However it was not possible to decide between th two possible structure of penicillami On chemical evidenca alone , because penicillin readly undergoes moleclar rearrangem Eg; on treatment with dil acid , penicillin rearranges to yield penillic acid penicillin HCL CHOO S H C C N C(CH3)2 N C R PENILLIC ACID CH.COOH CLASSIFICATION OF PENICILLINS NATURAL PENICILLINS BENZYL PENICILLIN(PENICILLIN-G) PENTYL PENICILLIN(PENICILLIN-F) 3-PENTYL PENICILLIN(PENICILLIN-K) PHENOXY METHYL PENICILLIN(PENICILLIN-V) 39 Semi-synthetic Penicillin • Ampicillin • Amoxicillin • Carbenicillin • Methicillin 40 Penicillins 41 Figure 20.6 Cephalosporins 42 Cephalosporins • Semisynthetic B-lactams derived from chemical side chains added to 7-aminocephalosporanic acid. • Generally more resistant to Blactamases. 43 Other Inhibitors of Cell Wall Synthesis • Cephalosporins – 2nd, 3rd, and 4th generations more effective against gram-negatives 44 Figure 20.9 Other Inhibitors of Cell Wall Synthesis • Polypeptide antibiotics – Bacitracin • Topical application • Against gram-positives – Vancomycin • Glycopeptide • Important "last line" against antibiotic resistant S. aureus 45 The Cephalosporins (generalized) *Not effective vs. Enterococcus or Listeria 1st Generation Gram (+) 2nd Generation Decreasing Gram (+) and Increasing Gram (-) 3rd Generation Gram (-), but also some GPC 4th Generation Gram (+) and Gram (-) 46 Discovery and structure of cephalosporin C The second major group of β-lactam antibiotics to be discovered were the cephalosporins. The first cephalosporin was cephalosporin C— isolated in 1948 from a fungus obtained from sewer waters on the island of Sardinia. Although its antibacterial properties were recognized at the time, it was not until 1961 that the structure was established. It is perhaps hard for modern chemists to appreciate how difficult and painstaking structure determination could be, even in the post-war period. The advent of NMR spectroscopy in the sixties and seventies has revolutionized the field so that if a new fungal metabolite is discovered today, its structure can be worked out in a matter of days rather than a matter of years. . 47 The structure of cephalosporin C (Fig. 10.41) has similarities to that of penicillin in that it has a bicyclic system containing a four-membered (βlactam ring. However, this time the (3-lactam ring is fused with a sixmembered dihydrothiazine ring. This larger ring relieves the strain in the bicyclic system to some extent, but it is still a reactive system A study of the cephalosporin skeleton reveals that cephalosporins can be derived from the same biosynthetic precursors as penicillin, i.e. cysteine and valine (Fig. 10.42). 48 Properties of cephalosporin C The properties of cephalosporin C can be summarized as follows. • Difficult to isolate and purify due to a highly polar side-chain. • Low potency (one-thousandth of penicillin G). • Not absorbed orally. • Non-toxic. • Low risk of allergenic reactions. • Relatively stable to acid hydrolysis compared to penicillin G. • More stable than penicillin G to penicillinase (equivalent to oxacillin). • Good ratio of activity against Gram-negative bacteria and Grampositive bacteria. 49 Cephalosporin C has few clinical uses, is not particularly potent and at first sight seems rather uninteresting. However, its importance lies in its potential as a lead compound to something better. This potential resides in the last property mentioned above. Cephalosporin C may have low activity, but the antibacterial activity which it does have is more evenly directed against Gram-negative and Gram-positive bacteria than is the case with penicillins. By modifying Cephalosporin C we might be able to increase the potency whilst retaining the breadth of activity against both Grampositive and Gram-negative bacteria. Another in-built advantage of Cephalosporin C over penicillin is that it is already resistant to acid hydrolysis and to penicillinase enzymes. Cephalosporin C has been used in the treatment of urinary tract infections since it is found to concentrate in the urine and survive the body's hydrolytic enzymes. 50 Monobactams • Monobactams: Aztreonam • Spectrum: ONLY Gram negative aerobic bacteria • Lack of Coverage: – Some resistant P. aeruginosa, E. cloacae, and C. freundii – Acinetobacter sp., Stenotrophomonas sp. • Pharmacokinetics: – Well distributed into tissues, esp. inflamed tissues – Excretion: renal clearance • Adverse reactions: – Skin rash – Very low cross-reactivity with Beta-Lactam class – highest risk in patient allergic to ceftazidime. 51