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Lecture № 5

Lecture № 5. Antibiotics of the aromatic row: synthesis, stereoisomery, methods of analysis. Antibiotics of the heterocyclic structure: β -lactam antibiotics. ass. Medvid I.I,. Antibiotics of the aromatic row.

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Lecture № 5

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  1. Lecture №5 Antibiotics of the aromatic row: synthesis, stereoisomery, methods of analysis. Antibiotics of the heterocyclic structure: β-lactam antibiotics. ass. Medvid I.I,

  2. Antibiotics of the aromatic row Chloramphenicol (Chloramphenicolum) (SPhU) Laevomycetin (Laevomycetinum) 2,2-dichloro-N-[(1′R,2′R)-2′-hydroxy-1′-(hydroxymethyl)-2′-(4"-nitrophenyl)ethyl]acetamide D-(-)-threo-1 -p-Nitrophenyl-2-dichloroacetylamino- propanediol-1,3 Laevomycetin – fine white with a grey or yellowish-green tinge crystalline substance, odorless. Bitter taste. Slightly soluble in water, soluble in alcohol. Solution of the substance in ethanol rotates the plane of polarization on the right, in ethylacetone – on the left.

  3. Leavomycetin stearate (Laevomycetini stearas) D-(-)-threo-1-p-Nitrophenyl-2-dichloroacetylaminopropanediol-1,3-3 –atearate Laevomycetin stearate -white with a yellowish or yellowish-green tinge crystalline substance, odorless.Laevomycetin stearatehas no taste. Practically insoluble in water, difficult soluble in alcohol, in all solvents forms a cloudy solutions.

  4. Leavomycetinsuccinatesoluble (Laevomycetini succinas solubile) D-(-)-threo-1 -p-Nitrophenyl-2-dichloroacetylaminopropanediol-1,3-3-sodium succinate Laevomycetin succinate soluble -white or slightly yellowish porous mass with low specific smell. Laevomycetin succinate soluble – has a bitter taste. Very easily soluble in water, slightly soluble in alcohol, hygroscopic.

  5. Leavomycetin molecule has2 asymmetric carbon atoms, and therefore it is possible the existence of four isomers: D- and L-threo, D- and L-erythro-, which differ by the spatial arrangement of functional groups: Laevomycetinisa leftrotatorythreo-isomer ofthe Drow. Mixture ofD(-) andL(+) threo-isomers oflaevomycetin iscalled racemic mixture, opticallyinactive compound known assynthomycin (has 50% of the laevomycetin physiological activity). Erythro-formsis not used in medicine because it is a toxiccompounds.

  6. Obtaining oflaevomycetin • Laevomycetinfirst was isolated in 1947 yearfrom the cultural liquid of actinomycetes, in 1949 year was established the chemical structure. • The drug is extractedby the growing of actinomycetes Streptomyces venezuelae on the medium (broth, glycerol, molasses and mineral salts) at 23-27 °Сand strong aeration for 89 hours. After the filtering of the mycelium fungus antibiotic is extracted and purified by the chromatographic. • Laevomycetin - the first antibiotic, which began to obtain by the chemical synthesis, whereas the majority antibiotic was obtained by biosynthesis.

  7. Synthesis of laevomycetin

  8. Extracted drug by the interaction withNaOHis transformedin a racemic base of laevomycetineand divided into optical isomers by using of the different solubilityof salts, formed withdextrorotatory tartaric acid, in methanol. However, only salt of D-isomer is soluble in methanol. The basis from solution is sedimented by the ammonia andacylated by the methyl ester of dichloroacetic acid Cl2CHCOOCH3.

  9. Identification • By the physico-chemical constants: melting point, IR- andUV-spectroscopy, TLC, specific rotation. • To the alcoholic solution of laevomycetin add calcium chloride and zinc powder and heat. Filtrate the obtained hot solution, after cooling add benzoylchloride. Then add iron (ІІІ) chloride solution and chloroform, shake the solution; aqueous layer should be pained in the color from light violet-red to purple. • Reaction on chloride-ion after the mineralization of the substance by the anhydrous sodium carbonate and dissolution of the residue in the diluted nitrate acid.

  10. Hydrolysis in acidic or basic mediumswith the following identification of the formed products. Thus, at the heating of laevomycetin with sodium hydroxide at first yellow color appears, that transfers to the red-orange (as a result of formation ofacy-nitroform), after the following heating brick-red precipitate formed and smell of ammoniaappeared:

  11. Hydroxame reaction on laevomycetin esters. • Laevomycetin stearate at the heating with concentrated chloride acid hydrolyzed – stearic acid is formed, which floats to the surface in the form of oily drops which harden when cooled:

  12. By the reaction of azodyes red color formation, after the reduction of nitro-group to amino-group with following diazotationand azojoining:

  13. In the express-analysisuse reaction of laevomycetin with copper (II) sulfate in alkali mediumin the presence ofn-butanol – alcoholic layer painted in blue-violet color due to the formation of complex salt, which supposedly has the following structure:

  14. Assay • SPhU - Spectrophotometry. Laevomycetine content in the substance Content of the laevomycetin in the substance is calculated by the specific absorption. • Nitritometryafter the previous reductionof nitro-group to amino-group by the zinc dustin acidic medium. Е = М.m. • By liquid chromatography.

  15. Cooper-metry,direct titration.The method is based on the formation of soluble laevomycetin complex compounds with copper (II) sulfate in alkaline medium (look identification). Titrant-0,01 М solution ofcooper(II) sulfate, indicator - murexide. Titration goes from the purple to brownish-red color, uniform with color of the “blind” sample. Е = 2 М.m. • Cooper-iodometry, direct titrationby the substitute.To the laevomycetin in alkali medium add cooper (II) sulfate. Precipitate of cooper (ІІ) hydroxideis filtrated, in the filtrate soluble cooper-laevomycetin complex is destroyed by the action of sulfate acid with formation of equivalent amount of cooper (II) sulfate, which is determined by iodometry, indicator - starch. In parallel control experiment is conducted. Е = 2 М.m.

  16. CuSO4

  17. Precipitate titration (argentometryormercurymetry). Methodsbasedonthe oxidationof laevomycetin by Н2О2in alkali medium, as a result в 2 molecules ofNaCl formed, which are determined by argentometric method (Folgard’s method) or mercurymetrywith diphenylcarbazone as indicator.Е = 1/2 М.m. • Photocolorimetryby the formation of azodye after the reduction of of nitro-group to amino-group with followingdiazotation and azojoining. • Iodometry. The method is based on the oxidation of the products of alkaline hydrolysis of laevomycetin. ExperimentallydeterminedЕ = 1/6 М.m. • Bromatometry, reverse titration. Е = 1/4 М.m. • Acidimetry in non-aqueous medium after the acidic hydrolysis.

  18. STORAGE • In airtight containers, in the protected from light place. USAGE • Laevomycetinebelongs tothebroad-spectrumantibiotics, usedin thetreatment ofdysentery, pneumonia, whoopingcough, typhoidandotherinfectiousdiseases. Courseoftreatmentis 8-10 days. In pediatric practiceuse laevomycetinestearate, which hasn't bitter taste. Laevomycetinesuccinatecan be used for injections. Issue: tablets, alcoholic solution, eye drops (0,25%), ointmentsLevosin, Levomekol. • Side effects. Disorders of the function of hematopoietic organs, so,at the treatment by laevomycetin blood test is required. Can cause an overgrowth.

  19. Antibiotics heterocyclic structure Penicillins (pennames) in the core of the penicillin's molecule is 6-amino-penicillinic acid (6-APA), which consistsof the condensed thiazolidine (А) and β-lactam (В) cycles:

  20. Obtaining of the natural penicillins For the obtaining of penicillin growth mouldy fungusPenicilliumnotatum on the medium, which consists ofcorn extract, lactoseanddifferent mineral salts. To increase the outlet of benzylpenicillin, to the medium add 0,02 – 0,08 % of phenylacetic acid С6Н5СН2СООН or its amide. Fermentationis conducted approximately for 70 hoursat 23-24 оС, рН = 6-6,5 and strong aeration (1 l of airon 1 l of environmentper 1 min.). After the end of fermentation the obtained product is transformed by the scheme represented on the next slide. To the separation of the different types of penicillin and following purification of the penicillin sodium salt, this salt is transformed into the penicillin salt with organic bases or purified by chromatographic method. Water solution of benzylpenicillin sodium salt is evaporated to the dry state at the temperature – 40оС in the vacuum (0,1-0,2 mmof mercurycolumn): ice, without melting, transformed in pair, а preparationremained dry (liophillicdrying).

  21. For the manifestation of biological activity of great importance isβ-lactamcycle, which is very unstable in acidic and alkaline environments. Penicillin is of the early era of antibiotics and because of its wide usage now80-90 %strains of staphylococci developed resistance to it, producing the enzyme penicillinase, which split the β-lactam cycle. Account this and the fact that penicillins are the least toxic in the comparison with other antibiotics, conduct a searchof semi-synthetic penicillins on the basis of 6-APA. • Semi-synthetic penicillinsare an acylderivativesof 6-APA.

  22. Generalformulaof penicillin’s medicines Three generations of penicillins: • Natural (benzylpenicillinNa,К orNovocainsalt, phenoxymethylpenicillin, Bicillin(1,3,5)) • Semi-synthetic penicillins (oxacillin,ampicillin, amoxicillin, dicloxacillin, carbenicillin and others) • Semi-syntheticantibiotics + inhibitor of-lactamases (amoxiclav (augmentine), timentin).

  23. Benzylpenicillin potassium (sodium) saltBenzylpenicillinum kalicum (natricum) SPhU • Sodium (2S,5R,6R) – 3,3 – dimethyl – 7 – oxo – 6 [(phenylacetyl)amino] – 4 – thio – 1 – azabicyclo[3.2.0]heptane – 2 – carboxylate • Sodium salt of 6 – phenylacetylaminopenicilanic acid

  24. BenzylpenicillinNovocainsaltBenzylpenicillinum novocainum • Novocain salt of 6 –phenylacetylaminopenicilanic acid

  25. Phenoxymethylpenicillin (ospen)Phenoxymethylpenicillinum 6 – phenylacetylaminopenicilinic acid • Bicillin - 1 (N,N’ – dibenzylethyldiammonium salt of benzylpenicillin • Bicillin - 3 (a mixture of equal parts of potassium (sodium)benzylpenicillin salt, benzylpenicillinand bicillin-1Novocain salt) • Bicillin – 5 (a mixture of 1part of benzylpenicillin Novocain salt and 4 parts ofbicillin – 1)

  26. Bicillin-1(Вicillinum-1) Benzathine Benzylpenicillin(Benzathine Benzylpenicillinum) N,N′ – dibenzylethylenediammoniumsalt of benzylpenicillin

  27. Oxacillin sodium salt Oxacillinum natrium Sodium salt of 3-phenyl-5-methyl-4-isoxazolylpenicillin monohydrate Ampiox(ampicillin and oxacillinsodium salts)

  28. Ampicillintrihydrate(SPhU) Ampicillinum trihydricum (2S,5R,6R)-6-[[(2′R)-2′-amino-2′-phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid trihydrate Also pharmacopoeial preparation is Ampicillin sodium salt.

  29. Amoxicillintrihydrate (SPhU) Amoxicillinum trihydricumamoxyl,gramox, ospamox, hiconcil, Phemoxin-solutab 2S,5R,6R)-6-[[(2′R)-2′-amino-2′-(4′′′-hydroxyphenyl)-acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid trihydrate Vampiox (amoxicillintrihydrate + cloxacillin + Lactobacillus)

  30. Dependence between the chemical structureand biological action of penicillins 1 – character of the radical which derermines the level of bounding with proteins; 2 – Substitute inо-position of the phenylradicalinfluences on thestability to the penicillinase; 3 – character of the boundingof the phenyl radical with methyl group determinesthe acidic stability of the penicillin; 4 – substituteof the Hydrogen atom in the methyl group в determines the spectrum of penicillin action; 5 – splitting of theβ-lactamboundconducts to the disappearance of antibiotic properties and appearance of the allergic action; 6 – substitutein the carboxylic group gives the possibility to obtain the penicillin salty forms; P – penicillinasesplitsβ-lactamcycle; А – amylasesplitsamidebound.

  31. CHARACTERS • Medical drugs of the natural and semi-synthetic penicillins -white crystalline substance, odorless, bitter taste. Sodium and potassium benzylpenicillin salts are hygroscopic and easily soluble in water. Benzylpenicillin Novocain salt, phenoxymethylpenicillin and ampicillin few soluble in water. • Water or alcohol solutions penicillins rotate plane polarized beam to the right.

  32. Identification • By the physico-chemical constants: IR- andUV-Spectroscopy, TLC. • Reaction with formaldehydein the presence of concentrated sulfate acid (Marki reagent). Reaction is distinguished, because each penicillin produces at these conditions characteristic color(benzylpenicillins-reddish-brown color, amoxicillin - dark yellow, etc.). • Substancesgive reactions on potassium, sodium and Novocain. • Unpharmacopoeial reactions: а) reactionof copper(II) (green) or iron (III) (red color) penicilloinhydroxamates formation after the hydroxylaminolysis(β-lactam cycle):

  33. b) reaction with chromotropic acidin the presence of concentrated sulfate acid, whichis distinguished, because each penicillin produces at these conditions characteristic color(benzylpenicillins- brown color, amoxicillin – violet ets.); c) determination of the organicallyboundsulfur (dryandwetpyrolysis); d) determinethemeltingtemperatureoftheN-ethylpiperidine salt of benzylpenicillin(fornaturalpenicillins); e) reaction on aliphaticamino-group (ampicillin, amoxicillin) – at the heatingwithaninhydrinsolutionobservedpurplecolor. f) Vitali – Moren’s reaction.

  34. Reaction oftheazo-dye of amoxicillin formation (redcolor) Penicillinsdue to the Sulfur atom have the reducing properties and can reduce silver from the Tollense reagent, mercury from the Nesler reagent ets..

  35. Assay • By the liquid chromatography (SPhU). • Microbiological methods of the diffusion in agar (reproducingof the results - 5-10 %). • Spectrophotometric determination of the semi-synthetic penicillins. • Chemical methods in two phases: а) determination of the penicillins amount; b) identify the content of the relevant drug. The amount of penicillins for the medical drugs of natural penicillins determineby iodometric method, the essence of which is that the products of alkaline hydrolysis of penicillin can be oxidized by iodine in the presence of acetic buffer with рН = 4,5. Е = М.m/8

  36. For determination of the benzylpenicillincontent use gravimetric method by the reaction of N-ethylpiperidine salt formation: Amount ofpenicillins in the semi-synthetic compounds determine by the alkalimetry, reverse titration, with control experiment, indicator –phenolphthalein. Е = М.m.

  37. Test on purity Measure the optical density of solutions of natural penicillins at a wavelength of264, 280 and 325 nm. Specific impurities determined by the liquid chromatography, residual amounts of the organic solvents - gas chromatography. Pyrogens, abnormal toxicity and sterility are also determined. Storage In airtight containers, in dry, dark place at the room temperature. Usage Natural penicillins affect on the gram-positive bacteria and used to treat pneumonia, gonorrhea, syphilis, septic infections, diphtheria, scarlet fever. They can not be received per os, because in acidic environments inactivation occurs (semi-synthetic penicillins and phenoxymethylpenicillin are stable in acid solutions). Penicillins destroy under the action of penicillinase, semi-synthetic analogs resistant to it and have a broader spectrum of activity. Side effects. Safest. Penicillins may cause gastro-intestinal disorders, very rare - allergic reactions.

  38. Cephalosporins (cephems) In the structure of cephalosporin is condensed system consisting of-lactam anddihydrothiazine cycles. Cephalosporin are derivativesof 7-aminocephalosporanic acid (7-ACA) (cefalotin, cephalopirin, cefuroxime,sodiumcefotaxime) and 7-amino-desacetoxycephalosporanic acid (7-АDCA) (cefalexinmonohydrate,cefazolin sodium,sodiumceftriaxon,cefixime, cefaloridine).

  39. The general formula of cephalosporin group of medicines: • Mouldy fungi Cephalosporium sort andactinomycetesproducenatural antibiotic - cephalosporin C, which did not found application in medicine because of low activity. Cephalosporin C is a source of obtaining of the semi-synthetic cephalosporins. • In the medical practice use the modern semi-synthetic cephalosporins of four generations (classification by Kharkevich).

  40. Cefazolin sodium (SPhU)(Cefazolinum natricum), kefzol, reflin Sodium (6R,7R)-3-[[(5′-methy-1′,3′,4′-thiadiazol-2′-yl) sulphanyl]methyl]-8-oxo-7-[(1"Н-tetrazol-1"-yl-acetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

  41. Cefalexinmonohydrate (SPhU)(Cefalexinum monohydricum)ospexin, lexin (6R,7R)-7-[[(2′R)-2′-amino-2′-phenylacetyl]amino]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid monohydrate

  42. Cefixime (SPhU) (Cefiximum), cefix, loprax (6R,7R)-7-[[(2′R)-2′-(2′′-aminothiazol-4"-yl)-2′-[(carboxymethoxy)imino]acetyl]amino]-3-ethenyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid trihydrate

  43. Cefotaximesodium (SPhU)(Cefotaximum natricum)cefabol, cafantral Sodium (6R,7R)-3-[(acetyloxy)methyl]-7-[[(2Z)-2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetyl]amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate.

  44. Ceftriaxonesodium (SPhU)(Ceftriaxonum natricum) Disodium (6R,7R)-7-[[(2′R)-2′-(2′′-aminothiazol-4"-yl)-2′-[(methoxyimino)acetyl]amino]-3-[[(2′′′-methyl-6′′′-oxido-5′′′-oxo-2′′′,5′′′-dihydro-1′′′,2′′′,4′′′-triazin-3′′′-іл)sulphanyl]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate

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