Mohammed Nooraldeen Al-Qattan (PhD)

Transcription

1 Mohammed Nooraldeen Al-Qattan (PhD)

2 Macrolides are macro lactone rings made up of 12 or more atoms, and usually contain one or more sugars. They are bacteriostatic agents that inhibit protein synthesis The best-known example of this class is erythromycin (a metabolite isolated in 1952 from the soil microorganism Streptomyces erythreus found in the Philippines. It is one of the safest antibiotics in clinical use. The structure is composed from 14-membered macrocyclic lactone ring with attached sugars and amino sugars (the sugar residues are important for activity) Erythromycin contains a 14-membered lactone ring and two sugars, desosamine (attached at C 5 ) and cladinose (attached at C 3 ).

3 Macrolides isolated from microorganisms are generally having the following molecular characteristics: Large lactone ring (12,14,15, or 16-membered rings) A highly substituted macrocyclic lactone: aglycone moiety. A ketone group. One or two amino sugars glycosidically linked to the aglycone moiety. The presence of dimethylamino moiety on the sugar residue increase basicity aid in salt formation. Being highly lipophilic (although glycosidic linkages are hydrophilic) Being basic

4 The lactone ring usually has 12, 14, or 16 atoms and is often unsaturated Erythromycin is a very bitter, white or yellow-white, crystalline powder. It is soluble in alcohol and in the other common organic solvents but only slightly soluble in water Macrolide antibiotics are weak bases and different salts with pka range of can be formed on the amino group. Macrolides are water-insoluble molecules. Salts prepared by glucoheptonic and lactobionic salts are water soluble, whereas stearic acid and laurylsulfuric acid salts are water-insoluble. Macrolides are stable in aqueous solutions at or below room temperature. They are unstable in acidic or basic conditions or at high temperatures.

5 Aglycone Or Erythronolide moiety (3,4,6-trideoxy-3-dimethylamino-D-xylo-hexose) Glycone (2,3,6-trideoxy-3-methoxy-3-C-methyl-L-ribo-hexose)

6 The B analog is more acid stable but has only about 80% of the activity of erythromycin.

7 Erythromycin A Erythromycin B Erythromycin C C12-OH C12-H More acid stable Activity reduced to 80% of ery Cladinose OCH3 -OH Similar activity to ery Present in small amount in fermentation medium

8 Naturally occurring macrolides (erythromycin) are acid-labile, have short t 1/2 (1.5h) and narrow spectrum (Gram-positives, Staphylococci, Streptococci, Bacilli) Macrolides spectrum is similar to penicillin. It have low activity against Gram-ve bacilli due to difficult penetration through cell wall. Semi-synthetic derivatives (clarithromycin, t 1/2 =3-7h), azalides (azithromycin, t 1/2 >35h!), have improved 1. Stability 2. pharmacokinetic properties 3. spectrum (Gram-negatives, Haemophilus influenzae, atypical bacteria: Legionella, Chlamydia, Mycoplasma)

9 Erythromycin has been chemically modified with primarily two different goals in mind: (a) to increase either its water or its lipid solubility for parenteral dosage forms (b) to increase its acid stability (and possibly its lipid solubility) for improved oral absorption. Modified derivatives of the antibiotic are of two types: 1. Acid salts of the dimethylamino group of the desosamine moiety (e.g., the glucoheptonate, the lactobionate, and the stearate) 2. Esters of the 2-hydroxyl group of the desosamine (e.g., the ethylsuccinate and the propionate, available as the lauryl sulfate salt and known as the estolate).

10 The structure consists of a 12, 14, 15 or 16-membered macrocyclic lactone ring with a sugar (e.g. cladinose) and an amino sugar (e.g. desosamine) attached. The sugars are important for activity. Due to the amino sugar the molecule is weakly basic (pka=8) It is acid sensitive, thus given orally as coated tablets The acid sensitivity of erythromycin is due to the presence of a ketone and two alcohol groups which form intramolecular ketal in presence of acids Anhydroerythromycin

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12 Since neither the hemiketal nor the spiroketal exhibits significant antibacterial activity, unprotected erythromycin is inactivated substantially in the stomach. Furthermore, evidence suggests that the hemiketal may be largely responsible for the GI (prokinetic) adverse effects associated with oral erythromycin.

13 Methods for protecting erythromycin from acidcatalyzed ketal formation: 1. the hydroxyl groups are changed to methoxy groups as in clarithromycin which has improved acid-stability and oral absorption. Responsible for basic properties of macrolides

14 2. Increasing the member atoms of the macrolide (e.g. 15-membered ring of azithromycin). 3. Formation of salts with fatty acids

15 The free bases of erythromycins are moderately soluble in water Water solubility can be improved by salt formation with some organic acids such as glucoheptonic and lactobionic acids to be used for parenteral administrations Glucoheptonic Lactobionic

16 Water solubility can be decreased if salts are prepared with fatty acids as stearate, estolate and laurylsulfate salts Erythromycin stearate is a very insoluble salt form of erythromycin. The water insolubility helps: 1. To increase stability toward acids 2. To increase oral absorption 3. To mask bitter taste

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18 Erythromycin ethylsuccinate is a mixed double-ester prodrug in which one carboxyl of succinic acid esterifies the C-2 hydroxyl of erythromycin and the other esterifies ethanol. This prodrug is 1. Used to mask bitter taste for oral suspension 2. Acid sensitive 3. Slightly water soluble in water 4. slowly hydrolyzed after absorption to free erythromycin

19 Erythromycin estolate, erythromycin propionate lauryl sulfate: is the lauryl sulfate salt of the 2-propionate ester of erythromycin. It is acid stable and absorbed as the propionate ester. The ester undergoes slow hydrolysis in vivo. Only the free base binds to bacterial ribosomes. 2-propionate ester of the aminosugar Lauryl sulfate

20 The 14-membered ring macrolides are synthesized in bacteria from propionic acid units so that every second carbon of erythromycin, for example, bears a methyl group and the rest of the carbons are oxygen bearing (with one exception [H]) Two carbons bear so-called extra oxygen atoms [O] introduced later in the biosynthesis (not present in a propionic acid unit), and two hydroxyls are glycosylated

21 ETHYLSUCCINATE : Erythromycin ethyl succinate is a mixed double-ester prodrug in which one carboxyl of succinic acid esterifies the C-2 hydroxyl of erythromycin and the other ethanol. This prodrug is frequently used in an oral suspension for pediatric use largely to mask the bitter taste of the drug.

22 Most naturally occurred macrolides have undesirable chemical and biological characteristics Modifications has been made to design macrolides with improved: potency, spectrum, stability, pharmacokinetics and toxicity. The macrolides can be classified into generations: - 1 st generation: Picromycin and Erythromycin - 2 nd generation : - 14-membered ring: Clarithromycin, Roxithromycin, Dirithromycin, and Flurithromycin - 15-membered ring: Azithromycin 3 rd generation (ketolides) : 14-membered ring this gen has no 3-Ocladinose sugare) - Telithromycin

23 Structural modifications have been made to erythromycin to solve acid-instability and to mask its bitter taste. Semisynthetic macrolides such as 1. Clarithromycin: is similar to erythromycin with the C 6 hydroxyl in erythromycin has been converted to an methoxy. Due to this modification, clarithromycin has - The spectrum is slightly improved compared to erythromycin (but less active) - Greater acid stability than erythromycin. - Does not cause cramp in GIT - Higher blood concentrations. - More lipophilicity - Longer half-life - Concentration in lungs is remarkably higher than that in the liver, thus recommended for RTI Clarithromycin

24 2. Roxithromycin: The addition of hydroxylamine to the ketone to form oxime - Increased acid stability by reducing intermolecular ketalization - Diminished antimicrobial activity compared to erythromycin and clarithromycin - 9-oxime decreased its affinity for P-450, which reflects reduced interaction with hepatic mono-oxygenases and reduces interaction with metabolism of other drugs - higher tissue distribution and a longer half-life At 37 C and ph of 7.4 Roxitrhomycin log P = 2.61 is higher than Erythromycin log P = 1.70, which explains the higher tissue penetration of Roxithromycin Omura, Satoshi, ed. Macrolide antibiotics: chemistry, biology, and practice. Academic press, Roxithromycin

25 3. Azithromycin: nitrogen atom has been introduced to expand a 14- membered ring to 15-membered azalide ring. Removal of the 9-keto group coupled with incorporation of a weakly basic tertiary amine nitrogen function into the macrolide ring increases the stability. Which offers: - Spectrum is slightly broader than erythromycin and clarithromycin. It is more active vs Gram-ve and less active vs Gram+ve bacteria. - Stability toward acids (no ketal formation no abdominal cramp) - Longer half life (once daily dosage) - Increased lipophilicity - Greater tissue penetration the ring expansion and the introduction of tertiary amine do not extremely alter the structure-activity relationships between azithromycin and erythromycin, but the alteration of hydrophilicity and pks by two ionizable nitrogen groups of azithromycin seem to have an effect on the antibacterial spectrum and pharmacokinetics Azithromycin

26 Erythromycin Clarithromycin Roxithromycin Azithromycin

27 Erythromycin 9-oxime is currently the intermediate to synthesize 2 nd generation erythromycin analogues - Azithromycin (erythromycin 9-oxime derivative) - Roxithromycin (erythromycin 9-oxime ether) - Clarithromycin (6-O-methylerythromycin) The hemi-ketal erythromycin is used as intermediate to synthesize flurithromycin

28 - less active on Gram+ve - More active on Gram ve - Acid stable

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30 The main product of liver metabolism of erythromycin is the N-demethylated analogues. Macrolides inhibit CYP3A4 isoform of the cytochrome P450 oxidase family and lead to potentiation of co-administered drugs.

31 Ketolides are erythromycin derivatives. They have improved antibacterial activity, thus active against a significant number of erythromycin- resistant microorganisms. Ketolides are structurally different from macrolides in three ways: 1. The presence of 3-keto group in place of L-cladinose moiety: the sugar is essential for antimicrobial activity of macroldies, however, the removal of the sugar is compensated by change in macro-lacton ring (C11/C12). 3-keto group improves activity against erythromycin resistant strains 2. The C6-OH is changed to methoxy to prevent ketal formation (similar to clarithromycin) 3. Large aromatic N-substituted carbamate is extended at C11/C12 which improves interaction to ribosome, thus activity against resistant strains. Douthwaite, S. "Structure activity relationships of ketolides vs. macrolides." Clinical Microbiology and Infection 7.s3 (2001):

32 Telithromycin: is a semi-synthetic derivative of erythromycin classified as ketolide where - the cladinose sugar has been replaced with keto-group - The carbamate ring has been fused to macrocyclic ring - The two hydroxyls (which causes ketal) have been masked, one as methoxy and the other as part of carbamate ring - Imidazolyl and pyridyl rings inked to the macrolide nucleus through a butyl chain. - It binds to domain II and V of 23S RNA of 50S (i.e. active against resistant methylated domain V. - Metabolized by P450 several drug-drug interaction Acid stability OH changed to methoxy OH involved in carbamate ring Telithromycin

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34 Telithromycin Solithromycin Pyridine ring Aniline ring Imidazole ring triazole ring C2-H C2-F

35 3-O-caldinose is necessary for antibacterial activity. removal of caldinose forms 3-descladinosyl erythromycin derivatives which are all inactive analogues such as: - 3-OH - 3-O-acetyl - 3-O-(substituted)benzoyl - 3-keto derivatives Picomycin was the earliest discovered macrolide and showed low antibacterial activity Note ketolides have 3-keto but with extra C11,C12- carbamate side chain that increases binding to ribosome

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37 The macrolides inhibit bacterial protein biosynthesis by binding to the 23S rrna in the polypeptide exit tunnel adjacent to the peptidyl transferase center in the 50S ribosomal subunit This prevents the growing peptide from becoming longer than a few residues, resulting in the dissociation of peptidyl trna molecules. Macrolides, clindamycin, lincomycin, and chloramphenicol bind in the same vicinity, leading to extensive crossresistance between them.

38 Most of the 14-member-ring macrolides, which include erythromycin and its related compounds, have three structural components: the lactone ring, the desosamine sugar, and the cladinose sugar. The reactive groups of the desosamine sugar and the lactone ring mediate all the hydrogen-bond interactions of erythromycin and its second generation derivatives clarithromycin and roxithromycin with the peptidyl transferase cavity. The 2 -OH group of the desosamine sugar appears to form hydrogen bonds at three positions: N6 and N1 of A2041Dr (A2058Ec) and N6 of A2042Dr (A2059Ec). The hydrogen bonds between 2 -OH and N1 and N6 of A2041Dr (A2058Ec) explain why this nucleotide is essential for macrolide binding, thus shedding light on the two most common ribosomal resistance mechanisms against macrolides: the N6 dimethylation of A2041Dr (A2058Ec) by the erythromycin resistance family of methylases31, and rrna mutations changing nucleotide identity at this position32. The dimethylation of the N6 group would not only add a bulky substituent causing steric hindrance for the binding, but will prevent the formation of hydrogen bonds to the 2 -OH group. Schlünzen, Frank, et al. "Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria." Nature (2001):

39 The macrolide binding site is located on the large ribosomal subunit inside the nascent peptide exit tunnel near the peptidyl transferase center. Its proximity to the peptidyl transferase center explains the inhibitory effect of some macrolides on peptide bond formation. The sugar residues attached at the C5 position of the lactone ring protrude towards the peptidyl transferase center.

40 Exit tunnel for growing polypeptide Harms, Jörg M., et al. "Alterations at the peptidyl transferase centre of the ribosome induced by the synergistic action of the streptogramins dalfopristin and quinupristin." BMC biology 2.1 (2004): 4.

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42 The spectrum of antibacterial activity for erythromycin is similar to penicillin. 1. Active against Gram +ve cocci and bacilli 2. Active against Gram ve cocci (especially Neisseria spp) 3. Low activity against Gram-ve bacilli Unlike penicillins, macrolides are also active against Mycoplasma, Chlamydia, Campylobacter and Legionella spp.

43 Lower binding to bacterial ribosome due to: 1. Methylation of a specific guanine residue of rrna reduce affinity to erythromycin but not telithromycin 2. Adenine to guanine mutation in rrna 1000 fold reduction in affinity of erythromycin and clarithromycin to 23S rrna Active efflux (expel) of macrolide from the cell Lack of penetration mechanism as in some Gram-ve bacteria