Lecture №29

1. Lecture №29 General characteristic and classification of glycosides.  Cardiac glycosides: correlation between the structure and action, role of the steric factors. Obtaining of the appropriate drugs, their properties, standardization by the usage of the different methods, special features of the storage in a pharmacy and usage in a medical practice. prepared: assist. Medvid I.I.

2. Glycoside - a natural substances that contain carbohydrates where glycosidic part of the molecule (cyclic form of sugars) is connected with an organic radical that is not a sugar (aglycone or henin). • By the nature of the sugar glycosides are divided into two groups: piranosides and furanosides. There are also α-and β-glycosides depending on the configuration of carbon connected to the aglycone. Sugar part of the molecules may contain one or more interconnected sugars (monosaccharides, disaccharides, etc.). • Pentosides - pentose glycosides, hexosides - hexose glycosides, biosides - glycosides of disaccharides. • Bound of the sugar residue with henin is made through oxygen (O-glycosides) or nitrogen (N-glycosides: ATPh, antibiotics-aminoglycosides), or sulfur (thioglycosides: sinigrin from mustard).

3. O-glycosides by the nature of aglycone are divided into: 1) phenyl glycosides contain the phenyl radical in aglycone (bearberry glycosides - Arbutin); 2) Anthraquinone glycosides contain the aglycone, anthraquinone derivative (glycosides buckthorn, rhubarb, aloe); 3) flavone glycosides, which aglycone is derivative of flavone (catechins, rutin); 4) nitrogencontaining, cyanogenic glycosides (amigdaline); 5) glucoalkaloids, glycosides, in which the sugar component bound with the remainder of alkaloid (solasodine); 6) steroid glycosides (cardiac glycosides); 7) tannins (tannin); 8) saponins.

4. Cardiac glycosides (CG) - biologically active substances contained in some types of plants or secretions of some species of frogs; in small doses can show a specific effect on the heart muscle. • Sources of cardiac glycosides extraction: different types of digitalis, spring adonis, oleander, valley, erysimum, strofant, hellebore, and others. • In the plants primary (genuinic) glycosides are contained, which are very labile and down easily (under the action of enzymes, acids, alkalis) to form secondary glycosides. The latter may hydrolyse to aglycons and sugar components. • In 1875 y. from digitalis purpurea secondary glycoside are identified - digitoxin and in 40-th years of the twentieth century – from digitalis lanata - digoxin. • The first study of the chemical structure of cardiac glycosides Windaus completed in 1915 y., then in 30-th years Jacobs and Chesnet found that they contain steroid structure.

5. Current directions in the research of CG • Search of the new natural sources of CG and their extraction. Over the past 20 years the number of selected glycosides are doubled and is more than 2500. Shown, that biologically more active are ones from CG that are more unstable in conformation and thermodynamic relation, on the base of which the "alternatives" of natural strophanthin was synthesized - number of active 19-norcardenolides. • Chemical and biochemical transformations of the natural CG to improve their pharmacological properties. Thus, created nitroethers of CG combine cardiotonic, coronarolytic and respiratory effects. • Increasing of the CG content in cultured species by agrotechnical methods and introduction of the new species. New species of the digitalis lanata contains in 15-20 times more digoxin and lanatoside C than known species of the plant. • Developing of the new, more perfect methods of selection from the raw materials of CG used in medical practice. So, original ways of getting of digitoxin, digoxin, gitoxin, strofantidin, lanatoside C, erysimine, adenoside, cordigidin are developed. Cardiotonic drug corglycone was obtained.

6. Synthesis of some CG though conducted, but due to the presence of many stagesand low output of the practical value is not received. The only industrial source of CG obtaining is plant material. The process of selection is difficult, because the plants contain enzymes that can changethe chemical structure of glycosides and these changes are not reverse. Such changes can occur under the action of light, temperature, etc.. In the plants, usually a few CG are located and a number of related substances. To separate the mixtures of glycosides using chromatographic techniques. • By the chemical structure heart (and others) glycosides are esters in which aglycone and residues of mono-, di-, tri-or tetrasugars are bounded between each other by glycosidic connection. In some primary glycosides to the sugar component the balance of acetate acid attached. • Sugars that are a part of cardiac glycosides, except glucose and ramnose are specific for these group of compounds. These sugars are 6-deoxyhexoses (L-ramnose), 2,6-deoxyhexoses (D-digitoxose) or 3-O-methyl ethers (D-cymarose, L-oleandrose). From the cardiac glycosides more than 50 carbohydrates are allocated. • Aglycons (henins) of the cardiac glycosides have a steroid structure that are derivatives of cyclopentane perhydro-phenanthrene.

7. Monosaccharides, which more often are a part of cardiac glycosides D-glucoseL-ramnoseD-digitoxose AcetyldigitoxoseD-cymaroseL-oleandrose

8. By the chemical structure aglycones can be divided into two groups, which are different according to the structure of attached in the position 17 lactone cycle. Cardiac glycosides containing fivemember lactone ring are called cardenolides and, as such, containing sixmember lactone ring with two double bonds - bufadienolides: In position 3 to aglyconesthe sugar componentis attached. RadicalR – СН3or СОН, and Х1, Х2, Х3 – Н or ОН.

9. Cardenolides are found in the various types of digitalis (Digitalis), strophanth (Strophanthus), valley(Convallaria), erysimum (Erysimum), oleander (Nerium oleander), spring adonis (Adonis vernaris).

10. Bufadienolides are a part of hellebore (Helléborus), squill (Scillae bulbus), and also are found in frogs (their venom contains bufohenins having steroid structure with sixmember lactone cycle).

11. Connect between the structure and action of cardiac glycosides • Carrier of biological activity is aglycone. The sugar component that is attached in position 3 to the aglycone influences on the rate of absorption, respectively, on the duration of action. The bigger quantity of sugar residues in the glycoside molecule cases their bigger activity. • Specific action of glycoside on the heart is caused by the presence of lactone cycle in the aglycone molecule in position 17 and OH-group in position 14. On the cardiotonic action substitute in position 10 has a great influence. For the most of aglycones this is methyl or aldehyde group. Aldehyde group oxidation to carboxylic group significantly weakens the effect on the heart muscle. • Replacing of the aglycons steroid cycle by the derivatives of benzene, naphthalene, and also like lactone cycle by other radicals and even the change of character of the connection between steroid nucleus and lactone, leads to the loss of physiological activity.This indicates the specificity of the molecular structure of cardiac glycosides and aglycones and on the complexity of obtaining themby synthetic way.

12. The chemical structure and obtaining of CG • In medical practice use different galen, neogalen preparations (extracts, tinctures), which contain CG and preparations of individual CG, which by their chemical structure are cardenolides. • InAddition 1 toSPhU digitoxin, digoxin, quabaine are included. • From the leaves of various kinds of digitalis extract: Celanide (Сеlanidum) (digilanide or lanatoside С) - primary glycosideof the Digitalis lanata. Tabl. by 0,00025 g (0,25 mg); 0,05 % solutionin fl. by 10 ml (0,5 mgin 1 ml) for internalusage and 0,02 % solutionin amp. by 1 ml. Digitoxin anddigoxin – secondary glycosides in digitalis purpurea and lanata(Digitalis purpurea L., Digitalis lanata). Digoxin: tabl. by 0,25 mgand by 0,1 mgfor children; 0,025% solutionin amp. by 1 ml. Digitoxin – tabl. by 0,1 mgandsuppositories by 0,15 mg. Digalen-neo.Neo-galenicpreparation from the leaves ofDigitalis ferniginea. Amp. by 1 mlf/in.; flaconsby 15 ml of preparation, for internalusage.

13. Chemical content of the primary digitalis glycosides Secondary digitalis glycosides of both species after the loss of these products of hydrolysis consist of aglycones and sugar part, which is the same in all three kinds of secondary glycosides.

14. From the seeds of various kinds of strophant extract: Strophanthin-К(Strophanthinus К) –mixture of cardiac glycosides isolated from strophanth Kombe (Strophanthus Kombe Oliver). Issue: amp. by 1 ml of 0,025% solutionfori/vori/minjections. Quabaine (Ouabainum) (Strophanthin-G) – glycoside from Strophanthus gratus. Ampoules by 1 mlof250 mcg/мml solutionfori/vinjections. • From the leaves of Convallaria majalis extract: Corglycone(Corglусоnum) - the sum of not less than five glycosides. Ampoules by 1 ml of 0,06% solution fori/vinjections. Convallatoxin (Convallatoxinum) –the main glycoside of lilies. Ampoules by 1 ml of 0,03% aqueous solution of the substancefor i/v njections. • Adoniside(Аdonisidum) – neo-galenic preparationof spring adonis grass (Аdonis vernalis).Flacons by 15 ml. • FromErysimum - erysimine, cardiovalene –the sum of glycosides.

15. Chemical contentof the medical drugs from cardiac glycosides

16. Digitoxin (Digitoxinum)(SPhU) 3β-[(О-2,6-dideoxy- β-D-ribo-hexopyranosyl -(1→4)-О-2,6-dideoxy- β-D-ribo-hexopyranosyl-(1→4)-О-2,6-dideoxy- β-D-ribo-hexopyranosyl)oxy-14-hydroxy-5β,14β-card-20(22)-enolide

17. Digoxin (Digoxinum)(SPhU) 3β-[(О-2,6-dideoxy-β-D-ribo-hexopyranosyl-(1→4)-О-2,6-dideoxy-β-D-ribo-hexopyranosyl-(1→4)-О-2,6-dideoxy-β-D-ribo-hexopyranosyl)-oxy-12β,14-dihydroxy-5β-card-20(22)-enolide

18. Quabaine(Ouabainum) (SPhU)StrophanthinG (Strophanthinum G) 3β-[(6-deoxy- α-L-mannopyranosyl)oxy]-1,5,11,14,19-pentahydroxy-(1β,3β,5β,11α)-card-20(22)-enolideoctahydrate

19. Celanide(Сеlanidum) (digilanideorlanatoside С) Colorless or white crystalline powder. Very few soluble in water, few soluble in alcohol. Act on the heart similar to other glycosides of digitalis, showing rapid effect and has relatively low capacity for accumulation.

20. Convallatoxin(Convallatoxinum) Crystal. white powder. Moderately soluble in water and in ethanol. According to the chemical structure and therapeutic effect is similar to strophanthin and has a high activity - in 1 g contains about 75 000 FUA or 10 600 CUA. The cumulative effect is very few expressed.

21. Properties of the CG pharmacopoeial preparations • Digitoxin-white or almost white powder. Practically insoluble in water, easily soluble in the mixture ofequal volumes of methanol and methylene chloride, few soluble in 96% alcoholand methanol. • Digoxin –white or almost white powder or colorless crystals. Practically insoluble in water, easily soluble in the mixture ofequail volumes of methanol and methylene chloride, few soluble inalcohol. • Quabaine – crystalline white powder orcolorless crystals. Moderately soluble in water and ethanol, practically insoluble in ether andethylacetate. Assay Spectrophotometryin the visible part of spectrum. The content of all three drugs in the substance is calculated, taking into account the results of measurements of optical density and concentration of the studied solution and solution of PhSE of the appropriate drug (standard method).

22. Identification of the pharmacopoeial preparations of CG • IR-spectroscopy (digoxin, digitoxin) –spectrum should be identical to the spectrum of PhSE drug. • TLC (digoxin, digitoxin, quabaine). • Cedde reaction (on the fivemember lactone cycle) with alkaline solution of 3,5-dinitrobenzoic acid (digitoxin, digoxin) – purple color. • Raymond'sreaction (on the fivemember lactone cycle) with alkaline solution ofdinitrobenzene (quabaine)– blue color. • Keller-Kilianireaction(on deoxysugars) (digoxin, digitoxin). • Solution of quabaine substanceinH2SO4conc. – pink color, which quickly becomes red, this solution in the UV-light gives a green fluorescence (steroid cycle). • Reaction on deoxysugars (quabaine). After the acidic hydrolysis with Fehling’s reagent - red sediment.

23. Purity test of the pharmacopoeial preparations of CG • Digitoxin. Impurities(gitoxin, otherglycosides) – TLC. • Digoxin. Impurities(gitoxin, digitoxin and otherglycosides) – TLC. • Quabaine. Impurities (gitoxin, digitoxin and otherglycosides) – TLC. Alkaloids andStrophenthin-К – with solution of tanninacid precipitate will not formed. Storage All three drugs remain in the protected dark place. Poisonous substances. List А.

24. Generalchemical reactions for CG identification Reactions onsteroid cycle • Liberman-Burkhard reaction:a small amount of substance is dissolved in a few drops of ice acetate acid and mixed with a mixture of acetic anhydride and concentrated sulfate acid. Slowly the coloration appears, which goes from pink to green or blue. This reaction glycosides give, which are in the processing by strong acids capable to dehydration(corglycone, strophenthin-К). • Rosenheimreaction:to the chloroform solution of substance add 96% trichloracetic acid - color appears, which gradually changes from pink to lilac and blue. This reaction is typical for steroids containing diene group or can form it under the influence of reagent. • Steroid cycle in cardenolides can be detected by fluorimetric method by the usage as a reagent mixture of phosphate and sulfate acids with iron (III) chloride; iron perchlorate solution in sulfate acid and so on.

25. Reactions on the fivemember lactone cycle of cardenolides • Legal reaction - the interaction in an alkaline medium with sodium nitroprusside red coloration appears and gradually fades. • Raymond's reaction-in alkaline medium with m-dinitrobenzenereddish-purple coloration appears. • Cedde reaction - in alkaline medium with 3,5-dinitrobenzoic caidpurple color appears. • Baljet reaction (Balje) -with alkaline picric acid solution orange-red color appears. The disadvantage of the above reactions is that almost all glycosides with close structure give the same color. Therefore, this reaction can not be used to identify individual glycosides. Therefore, Reichstein proposed for recognition of individual glycosides use concentrated or 84% sulfuric acid, which gives color reactions with various glycosides, which are held in time and color change.

26. Mechanism of the Baljet reaction (Balje)

27. Reaction onthe sixmember lacton cycleof bufadienolides Glycoside is dissolved in chloroform and add gradually saturated solution of antimony (III) chloride, at the heating dark-red color appears. Reactions ofsugar component • After the acidic hydrolysis can be used inherent sugar reactions, based on their reductive properties: • With Fehling reagent –formation of the red sedimentСu2O; • With Tollense reagent – “silver mirror“ reaction. • Specific for 2-deoxysugars contained in the molecules of most cardiac glycosides, are: • Keller - Kilianireaction:glycoside solution in concentrated acetate acid containing iron (III) chloride thicken on concentrated sulfate acid. On the boundary of two layers dark red or brown ring appears, the top layer paints in blue or blue-green color. The reaction occurs only when deoxysugar is in the free state or takes extreme position in the molecule of glycosides.

28. Febba and Levy reaction (for those glycosides, in which 2-deoxysugars from both sides linked with other sugars) - with trichloroacetic acid and p-nitrophenylhydrazine. • Pezets reaction: at the heating of glycoside with xanthydrol or anthrone in the presence of concentrated acetic acid with the following addition of several drops of sulfate acid or phosphate acid red or green, blue-green coloring appears. During the reaction under the action of concentrated acids, sugar component forms furfural or its derivatives, which are condensed with xanthydrol or anthrone.

29. Identification of the medicinal substances from CG groups can be done by the usage of specific rotation. Perspective is also a technique, based on the building of chromatographic diagrams that show the dependence of Rf from the system of solvents. IR- and UV-spectroscopy are also used. Investigation of the good quality of CG Particular attention is drawn to the presence of impurities of extraneous glycosides. This applies primarily to the preparations derived from plants that contain more similar in structure CG. For the determination of impurities using TLC, determining of the related glycosides on the chromatogram by the Rf value and the nature of the fluorescence spots in UV-light after the treatment by appropriate reagents (chloramine B, m-dynitrobenzene).

30. Incompatibility of CG • Cardenolides areincompatible with acids and compounds, which give acidic reaction of the environment. Hydrolysis by glycoside bound takes place. Reaction goes without any visible changes (withascorbic acid and other vitamins with acidic рН of environment). • Cardiac glycosidesare incompatible with alkalis and compounds, which give alkaline reaction of the environment(NaHCO3, barbital-sodiumand others).

31. Assay ofCG • Perform by spectrophotometric, photocolorimetric methods by the products of interaction in an alkaline environment with nitroderivatives of aromatic series.Qualitative and quantitative assessment of CG also are conducted by the high effective liquid chromatography (HELC), which allow to determine not only the main but also related glycosides. • Biological control is used to determine the lowest doses of standard and investigated substances that cause systolic shutdown of the heart of experimental animals. Then calculate the content of frog (FUA), cat (CUA), pigeon (PUA) units in 1 g of envestigated substance, in one tablet or 1 ml. • Some of the CG and their dosage forms can be defined by polarographic method. The advantage of this method is that the determination made by the reduction of double bond, conjugate with the carbonyl group of lacton cycle. This system is known, is one of the factors that determine the biological activity of cardiac glycosides. Polarography also is combined with the previous chromatographic separation of CG.

32. Storage and aplication ofCG • CG and their drugs are stored in tightly corked container that protect from light and moisture (corglycon – not more than +5 º C). Such conditions would prevent their hydrolytic cleavage. • As cardiotonic means at the acute and chronic circulatory failure or cardiovascular failure. They differ in strength, duration, speed of action, influence on the central nervous system. • Medical drugs of CG are more effective at the i/v introduction. For this the drugs are pre-dissolved in 20-40% glucose solution or isotonic solution to the concentration of 0,025%. • Higher single doses of individual CG are 0,5 mg, daily - 1,0 mg. Overdose causes severe dysfunction of cardiac activity. This requires them to be ascribed to toxic substances. You must consider the ability of CG gradually accumulate in the body (the degree of accumulation).

33. Thankyou for attention!