Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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 Chloramphenicol (Chloramphenicolum) (SPhU) Laevomycetin (Laevomycetinum) O 1 HN O2N 4'' H 2' C C 1' C H 2 CHCl2 CH2OH OH 2,2-dichloro-N-[(1′R,2′R)-2′-hydroxy-1′-(hydroxymethyl)-2′-(4"nitrophenyl)ethyl]acetamide D-(-)-threo-1 -p-Nitrophenyl-2-dichloroacetylaminopropanediol-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. Leavomycetin stearate (Laevomycetini stearas) O HN O2N H C OH C H C CHCl2 CH2O C C17H35 O D-(-)-threo-1-p-Nitrophenyl-2-dichloroacetylaminopropanediol1,3-3 –atearate Laevomycetin stearate - white with a yellowish or yellowishgreen tinge crystalline substance, odorless. Laevomycetin stearatehas no taste. Practically insoluble in water, difficult soluble in alcohol, in all solvents forms a cloudy solutions. Leavomycetin succinate soluble (Laevomycetini succinas solubile) O HN O2N H C OH C H C CHCl2 CH2O C (CH2)2 COONa O D-(-)-threo-1 -p-Nitrophenyl-2-dichloroacetylaminopropanediol1,3-3-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. Leavomycetin molecule has 2 asymmetric carbon atoms, and therefore it is possible the existence of four isomers: D- and Lthreo, D- and L-erythro-, which differ by the spatial arrangement of functional groups: Laevomycetin is a leftrotatory threo-isomer of the D row. Mixture of D() and L(+) threo-isomers of laevomycetin is called racemic mixture, optically inactive compound known as synthomycin (has 50% of the laevomycetin physiological activity). Erythro-forms is not used in medicine because it is a toxic compounds. Obtaining of laevomycetin Laevomycetin first was isolated in 1947 year from the cultural liquid of actinomycetes, in 1949 year was established the chemical structure. The drug is extracted by the growing of actinomycetes Streptomyces venezuelae on the medium (broth, glycerol, molasses and mineral salts) at 23-27 °С and 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. Synthesis of laevomycetin Extracted drug by the interaction with NaOH is transformed in a racemic base of laevomycetine and divided into optical isomers by using of the different solubility of salts, formed with dextrorotatory tartaric acid, in methanol. However, only salt of D-isomer is soluble in methanol. The basis from solution is sedimented by the ammonia and acylated by the methyl ester of dichloroacetic acid Cl2CHCOOCH3. Identification 1. 2. 3. By the physico-chemical constants: melting point, IR- and UV-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. 4. Hydrolysis in acidic or basic mediums with 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 redorange (as a result of formation of acy-nitroform), after the following heating brick-red precipitate formed and smell of ammonia appeared: 5. Hydroxame reaction on laevomycetin esters. 6. 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: 7. By the reaction of azodyes red color formation, after the reduction of nitro-group to amino-group with following diazotation and azojoining: 8. In the express-analysis use reaction of laevomycetin with copper (II) sulfate in alkali medium in the presence of n-butanol – alcoholic layer painted in blue-violet color due to the formation of complex salt, which supposedly has the following structure: Assay 1. SPhU - Spectrophotometry. Laevomycetine content in the substance Content of the laevomycetin in the substance is calculated by the specific absorption. 2. Nitritometry after the previous reduction of nitro-group to amino-group by the zinc dust in acidic medium. Е = М.m. 3. By liquid chromatography. 4. 5. 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 of cooper (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 titration by the substitute. To the laevomycetin in alkali medium add cooper (II) sulfate. Precipitate of cooper (ІІ) hydroxide is 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. CuSO4 Precipitate titration (argentometry or mercurymetry). Methods based on the oxidation of laevomycetin by Н2О2 in alkali medium, as a result в 2 molecules of NaCl formed, which are determined by argentometric method (Folgard’s method) or mercurymetry with diphenylcarbazone as indicator. Е = 1/2 М.m. 7. Photocolorimetry by the formation of azodye after the reduction of of nitro-group to amino-group with following diazotation and azojoining. 8. Iodometry. The method is based on the oxidation of the products of alkaline hydrolysis of laevomycetin. Experimentally determined Е = 1/6 М.m. 9. Bromatometry, reverse titration. Е = 1/4 М.m. 10. Acidimetry in non-aqueous medium after the acidic hydrolysis. 6. STORAGE In airtight containers, in the protected from light place. USAGE Laevomycetine belongs to the broad-spectrum antibiotics, used in the treatment of dysentery, pneumonia, whooping cough, typhoid and other infectious diseases. Course of treatment is 8-10 days. In pediatric practice use laevomycetine stearate, which hasn't bitter taste. Laevomycetine succinatecan be used for injections. Issue: tablets, alcoholic solution, eye drops (0,25%), ointments Levosin, Levomekol. Side effects. Disorders of the function of hematopoietic organs, so, at the treatment by laevomycetin blood test is required. Can cause an overgrowth . Antibiotics heterocyclic structure Penicillins (pennames) in the core of the penicillin's molecule is 6-aminopenicillinic acid (6-APA), which consists of the condensed thiazolidine (А) and β-lactam (В) cycles: COOH O 7 H2N 6 N1 5 CH3 2 3 4 S CH3 Obtaining of the natural penicillins For the obtaining of penicillin growth mouldy fungus Penicillium notatum on the medium, which consists of corn extract, lactose and different mineral salts. To increase the outlet of benzylpenicillin, to the medium add 0,02 – 0,08 % of phenylacetic acid С6Н5СН2СООН or its amide. Fermentation is conducted approximately for 70 hours at 23-24 оС, рН = 6-6,5 and strong aeration (1 l of air on 1 l of environment per 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 mm of mercury column): ice, without melting, transformed in pair, а preparation remained dry (liophillic drying). For the manifestation of biological activity of great importance is βlactam cycle, which is very unstable in acidic and alkaline environments. Penicillin is of the early era of antibiotics and because of its wide usage now 80-90 % 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 search of semi-synthetic penicillins on the basis of 6-APA. Semi-synthetic penicillins are an acylderivatives of 6-APA. General formula of penicillin’s medicines COOR1 O O R C CH3 N N H S CH3 Three generations of penicillins: • Natural (benzylpenicillin Na, К or Novocain salt, phenoxymethylpenicillin, Bicillin (1,3,5)) • Semi-synthetic penicillins (oxacillin, ampicillin, amoxicillin, dicloxacillin, carbenicillin and others) • Semi-synthetic antibiotics + inhibitor of -lactamases (amoxiclav (augmentine), timentin). Benzylpenicillin potassium (sodium) salt Benzylpenicillinum kalicum (natricum) SPhU H O H N 7 N1 6 5 COOK(Na) 3 4 S H CH3 2 CH3 H O • 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 Benzylpenicillin Novocain salt Benzylpenicillinum novocainum H O COO CH3 N H N CH3 S H - * H O C2H5 * H2N C O O C H2 C H2 N+ H *H2O C2H5 • Novocain salt of 6 –phenylacetylaminopenicilanic acid Phenoxymethylpenicillin (ospen) Phenoxymethylpenicillinum H O COOH CH3 N H N S O H O CH3 H 6 – phenylacetylaminopenicilinic acid • Bicillin - 1 (N,N’ – dibenzylethyldiammonium salt of benzylpenicillin • Bicillin - 3 (a mixture of equal parts of potassium (sodium) benzylpenicillin salt, benzylpenicillin and bicillin-1Novocain salt) • Bicillin – 5 (a mixture of 1part of benzylpenicillin Novocain salt and 4 parts of bicillin – 1) Bicillin-1(Вicillinum-1) Benzathine Benzylpenicillin (Benzathine Benzylpenicillinum) COO H O - CH3 N H N CH3 S * H H O 2 * C H2 + N H2 C H2 C H2 + N H2 C H2 N,N′ – dibenzylethylenediammonium salt of benzylpenicillin Oxacillin sodium salt Oxacillinum natrium H O COONa CH3 N *H2O H N S H N CH3 H O O CH3 Sodium salt of 3-phenyl-5-methyl-4isoxazolylpenicillin monohydrate Ampiox (ampicillin and oxacillin sodium salts) Ampicillin trihydrate (SPhU) Ampicillinum trihydricum H O NH2 2' 7 1' H N 6 H N 5 COOH CH3 2 1 3 4 S *3H2O CH3 H O (2S,5R,6R)-6-[[(2′R)-2′-amino-2′-phenylacetyl]amino]-3,3dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2carboxylic acid trihydrate Also pharmacopoeial preparation is Ampicillin sodium salt. Amoxicillin trihydrate (SPhU) Amoxicillinum trihydricum amoxyl, gramox, ospamox, hiconcil, Phemoxinsolutab H O NH2 CH3 N H N S H HO COOH CH3 H O 2S,5R,6R)-6-[[(2′R)-2′-amino-2′-(4′′′-hydroxyphenyl)-acetyl]amino]-3,3dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid trihydrate Vampiox (amoxicillin trihydrate + cloxacillin + Lactobacillus) Dependence between the chemical structure and biological action of penicillins 1 – character of the radical which derermines the level of bounding with proteins; 2 – Substitute in о-position of the phenyl radical influences on the stability to the penicillinase; 3 – character of the bounding of the phenyl radical with methyl group determines the acidic stability of the penicillin; 4 – substitute of the Hydrogen atom in the methyl group в determines the spectrum of penicillin action; 5 – splitting of the β-lactam bound conducts to the disappearance of antibiotic properties and appearance of the allergic action; 6 – substitute in the carboxylic group gives the possibility to obtain the penicillin salty forms; P – penicillinase splits β-lactam cycle; А – amylase splits amide bound. CHARACTERS Medical drugs of the natural and semisynthetic 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. Identification By the physico-chemical constants: IR- and UVSpectroscopy, TLC. Reaction with formaldehyde in 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.). Substances give reactions on potassium, sodium and Novocain. Unpharmacopoeial reactions: а) reaction of copper (II) (green) or iron (III) (red color) penicilloinhydroxamates formation after the hydroxylaminolysis (β-lactam cycle): b) reaction with chromotropic acid in the presence of concentrated sulfate acid, which is distinguished, because each penicillin produces at these conditions characteristic color (benzylpenicillins - brown color, amoxicillin – violet ets.); c) determination of the organically bound sulfur (dry and wet pyrolysis); d) determine the melting temperature of the N-ethylpiperidine salt of benzylpenicillin (for natural penicillins); e) reaction on aliphatic amino-group (ampicillin, amoxicillin) – at the heating with a ninhydrin solution observed purple color . f) Vitali – Moren’s reaction. Reaction of the azo-dye of amoxicillin formation (red color) Penicillins due to the Sulfur atom have the reducing properties and can reduce silver from the Tollense reagent, mercury from the Nesler reagent ets.. Assay By the liquid chromatography (SPhU). Microbiological methods of the diffusion in agar (reproducing of 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 determine by 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 For determination of the benzylpenicillin content use gravimetric method by the reaction of N-ethylpiperidine salt formation: Amount of penicillins in the semi-synthetic compounds determine by the alkalimetry, reverse titration, with control experiment, indicator –phenolphthalein. Е = М.m. Test on purity Measure the optical density of solutions of natural penicillins at a wavelength of 264, 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. Cephalosporins (cephems) In the structure of cephalosporin is condensed system consisting of -lactam and dihydrothiazine cycles. Cephalosporin are derivatives of 7-aminocephalosporanic acid (7-ACA) (cefalotin, cephalopirin, cefuroxime, sodium cefotaxime) and 7-amino-desacetoxycephalosporanic acid (7-АDCA) (cefalexin monohydrate, cefazolin sodium, sodium ceftriaxon, cefixime, cefaloridine). • Mouldy fungi Cephalosporium sort and actinomycetes produce natural 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). The general formula of cephalosporin group of medicines: COOH O CH2R2 N R1HN S Cefazolin sodium (SPhU) (Cefazolinum natricum), kefzol , reflin COONa O 3'' N 2'' 8 O N 1'' N N 4'' 5'' C H2 C 2 N H 1 N 7 3 H2 C S 3' 4' N N 2' 5' S 1' 6 4 S H H 5 Sodium (6R,7R)-3-[[(5′-methy-1′,3′,4′-thiadiazol-2′-yl) sulphanyl]methyl]-8-oxo-7-[(1"Н-tetrazol-1"-ylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylate CH3 Cefalexin monohydrate (SPhU) (Cefalexinum monohydricum) ospexin, lexin COOH O 8 NH2 O C 2' H C 1' N H 1 N 7 3 *H2O 6 4 S H H CH3 2 5 (6R,7R)-7-[[(2′R)-2′-amino-2′-phenylacetyl]amino]-3methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylic acid monohydrate Cefixime (SPhU) (Cefiximum), cefix, loprax H2C NH2 COOH O 3'' 2'' COOH N N O 4'' S 1'' 5'' C C 2' 1' 2 8 O N H H C 1 N 7 CH2 3 * 3 H2O 6 4 S H H 5 (6R,7R)-7-[[(2′R)-2′-(2′′-aminothiazol-4"-yl)-2′[(carboxymethoxy)imino]acetyl]amino]-3-ethenyl-8oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid trihydrate Cefotaxime sodium (SPhU) (Cefotaximum natricum) cefabol, cafantral CH3 NH2 O 3'' 2'' N COONa O N O C C 2' 1' 8 4'' S 1'' 5'' 2 N H H2 C O C 1 N 7 3 O 6 4 S H H 5 Sodium (6R,7R)-3-[(acetyloxy)methyl]-7-[[(2Z)-2-(2aminothiazol-4-yl)-2-(methoxyimino)acetyl]amino]8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylate. CH3 Ceftriaxone sodium (SPhU) (Ceftriaxonum natricum) O 5''' CH3 NH2 O 3'' 2'' N COONa O N O C C 2' 1' 1'' 5'' 2 8 4'' S ONa 4''' N H S 1 N 7 3 6 4 S H H2 C H * 3,5 H2O N 6''' 3''' N 1''' N 2''' CH3 5 Disodium (6R,7R)-7-[[(2′R)-2′-(2′′-aminothiazol-4"-yl)2′-[(methoxyimino)acetyl]amino]-3-[[(2′′′-methyl6′′′-oxido-5′′′-oxo-2′′′,5′′′-dihydro-1′′′,2′′′,4′′′-triazin3′′′-іл)sulphanyl]methyl]-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylate Ways of introducti on Generations І Cefazolin (kefzol, reflin) Cefalotin (ceporin) Cefapirin Parenteral Cefaloridin (i/v; i/m) Cafradin Enteral (per os) Cefalexin (ospecsin, lexin) Cefadroxil (duracef, cefangin-KMP) Cefradine ІІ ІІІ ІV Cefuroxime (zinacef) Cafamandole (cafambol) Cefoxitine Cefonocide Cefprozyl Cefmetazole Cefotetan Ceforanid Ceftriaxone Cefepime Cefotaxime (cefabol, (maxipime) cefantral) Cefoxitine Ceftrizoxime Cefoperazone(hepac ef-KMP, cefobit) Ceftazidime (forum, ceftum) Ceftizoxime Moxalactal Cefaclor (biclorKMP) Cefuroxime (zinnate) Loracarbef Cefprozyl Cefixime (cefix, loprax) Cefpodroxime (cefodox) Proxetyl Ceftibutene (cedex) Cefetamethe pivoxyl Identification By the physico-chemical constants: IR and UV spectrophotometry, thin layer chromatography. The reaction with Marki reagent. Reaction is distinguished because each of them forms a characteristic color (cafalexin -light yellow color, which becomes dark yellow; ceftriaxon sodium salt - greenish-yellow, which turns into yellow, cefotaxime sodium salt - bright yellow, which turns into brown). The presence of the β-lactam cycle causes the formation of cooper (II) or iron (III) hydroxamates. With the mixture of acids H2SO4 and HNO3 cefalexin becomes yellow, cefalotin -olive-green, which transforms into a red-brown . Sodium salts give an appropriate reactions on sodium cation. CHARACTERS Medicinal substances - white, sometimes yellowish powders. Sparingly soluble in water (except for sodium salts of cefalexin ceftriaxone, cefatoxime), hard soluble in alcohol. Some of them have a characteristic smell and sensitive to light. Optically active substances of the cephalosporin droup rotate the plane polarization to the right. Assay By liquid chromatography (SPU). Chemical methods (like penicillins). Biological methods. Physico-chemical methods (spectrophotometry, photocolorimetry). Assay cefalexin Acidimetry in non-aqueous environment. Equivalent point is determined by potentiometric method. Storage In airtight containers, вin dry, protected from light place at the room temperature. Usage Cephalosporins have a more broader spectrum than penicillins, and less toxicity. The difference in the chemical structure of penicillins and cephalosporins leads to the resistance to staphylococcus penicillinase and greater resistance to acids. Therefore prescribe cephalosporins are prescribed to the treatment of penicillin stable infections. Medications of cephalosporin’s froup used in the treatment of acute and chronic respiratory diseases, urinary tract, genitals, with postoperative and other infections. Other antibiotics of heterocyclic structure The structure that connects β-lactam and oxazolidine cycles –clavulanic acid: 3-(2-Hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptane-2-carboxylic acid It is used as an inhibitor of β-lactamase of gram-positive and gram-negative bacteria with penicillin and cephalosporins, increasing their effect. Also it is opened a new antibiotics of heterocyclic structure: cephamycin, thienamycin and others. Thank you for attention !