General characteristic and classification of glycosides.

1
Lecture ?12

• General characteristic and classification of
  glycosides.  Cardiac glycosides correlation
  between the structure and action, role of the
  steric factors. Obtaining 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 tha t 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 a-and ß-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 stages and 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
  change the 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 carohydrates 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-glucose L-ramnose
D-digitoxose
Acetyldigitoxose D-cymarose
L-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 aglycones the sugar
  component is attached. Radical R ??3 or ???,
  and ?1, ?2, ?3 ? or ??.

9

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

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• 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 them by synthetic way.

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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.
• In Addition 1 to SPhU digitoxin, digoxin,
  quabaine are included.
• From the leaves of various kinds of digitalis
  extract
• Celanide (??lanidum) (digilanide or lanatoside ?)
  - primary glycoside of the Digitalis lanata.
  Tabl. by 0,00025 g (0,25 mg) 0,05 solution in
  fl. by 10 ml (0,5 mg in 1 ml) for internal usage
  and 0,02 solution in amp. by 1 ml.
• Digitoxin and digoxin secondary glycosides in
  digitalis purpurea and lanata (Digitalis purpurea
  L., Digitalis lanata). Digoxin tabl. by 0,25 mg
  and by 0,1 mg for children 0,025 solution in
  amp. by 1 ml. Digitoxin tabl. by 0,1 mg and
  suppositories by 0,15 mg.
• Digalen-neo. Neo-galenic preparation from the
  leaves of Digitalis ferniginea. Amp. by 1 ml
  f/in. flacons by 15 ml of preparation, for
  internal usage.

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Chemical content of the primary digitalis
glycosides
Kind of digitalis Primary glycosides Products of hydrolysis Secondary glycosides
Digitalis purpurea Purpureaglycoside ? Purpureaglycoside ? Glucose Glucose Digitoxin Gitoxin
Digitalis lanata Digilanide ? Digilanide ? Digilanide ? (celanide) ??3???? Glucose ??3???? Glucose ??3???? Glucose Digitoxin Gitoxin DIgoxin
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-? (Strophanthinus ?) mixture of
  cardiac glycosides isolated from strophanth Kombe
  (Strophanthus Kombe Oliver). Issue amp. by 1 ml
  of 0,025 solution for i/v or i/m injections.
• Quabaine (Ouabainum) (Strophanthin-G) glycoside
  from Strophanthus gratus. Ampoules by 1 ml of 250
  mcg/?ml solution for i/v injections.
• From the leaves of Convallaria majalis extract
• Corglycone (Corgl???num) -the sum of not less
  than five glycosides. Ampoules by 1 ml of 0,06
  solution for i/v injections.
• Convallatoxin (Convallatoxinum) the main
  glycoside of lilies. Ampoules by 1 ml of 0,03
  aqueous solution of the substance for i/v
  njections.
• Adoniside (?donisidum) neo-galenic preparation
  of spring adonis grass (?donis vernalis). Flacons
  by 15 ml.
• From Erysimum - erysimine, cardiovalene the sum
  of glycosides.

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Chemical content of the medical drugs from
cardiac glycosides
Medical preparation Aglycon Sugar part
Digitoxin Digoxin Celanide Quabaine Convallatoxin Corglycone ? K-strophanthin-? ?-strophanthoside Digitoxigenine Digoxigenine Digoxigenine Quabagenine(strophanthidol) Strophanthidine Strophanthidine Strophanthidine 3 molecules of D-digitoxose 3 molecules of D-digitoxose D-glucose, acetyldigitoxose, 2 molecules of D-digitoxose L-ramnose L-ramnose D-glucose, D-cymarose Two molecules of D-glucose, D-cymarose
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Digitoxin (Digitoxinum)(SPhU)

• 3ß-(?-2,6-dideoxy- ß-D-ribo-hexopyranosyl
  hexopyranosyl -(1?4)-?-2,6-dideoxy-
  ß-D-ribo-hexopyranosyl-(1?4)-?-2,6-dideoxy-
  ß-D-ribo-hexopyranosyl)oxy-14-hydroxy-5ß,14ß-card-
  20(22)-enolide

17
Digoxin (Digoxinum)(SPhU)

• 3ß-(?-2,6-dideoxy-ß-D-ribo-hexopyranosyl-(1?4
  )-?-2,6-dideoxy-ß-D-ribo-hexopyranosyl-(1?4)-?-2,6
  -dideoxy-ß-D-ribo-hexopyranosyl)-oxy-12ß,14-dihydr
  oxy-5ß-card-20(22)-enolide

18
Quabaine (Ouabainum) (SPhU)Strophanthin G
(Strophanthinum G)

3ß-(6-deoxy- a-L-mannopyranosyl)oxy-1,5,11,1
4,19-pentahydroxy-(1ß,3ß,5ß,11a)-card-20(22)-enoli
de octahydrate
19
Celanide (??lanidum) (digilanide or lanatoside ?)

• 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.

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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.

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Properties of the CG pharmacopoeial preparations

• Digitoxin - white or almost white powder.
  Practically insoluble in water, easily soluble in
  the mixture of equail volumes of methanol and
  methylene chloride, few soluble in 96 alcohol
  and methanol.
• Digoxin white or almost white powder or
  colorless crystals. Practically insoluble in
  water, easily soluble in the mixture of equail
  volumes of methanol and methylene chloride, few
  soluble in alcohol.
• Quabaine crystalline white powder or colorless
  crystals. Moderately soluble in water and
  ethanol, practically insoluble in ether
  andethylacetate.
• Assay
• Spectrophotometry in 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

1. IR-spectroscopy (digoxin, digitoxin) spectrum
   should be identical to the spectrum of PhSE drug.
2. TLC (digoxin, digitoxin, quabaine).
3. Cedde reaction (on the fivemember lactone cycle)
   with alkaline solution of 3,5-dinitrobenzoic acid
   (digitoxin, digoxin) purple color.
4. Raymond's reaction (on the fivemember lactone
   cycle) with alkaline solution of dinitrobenzene
   (quabaine) blue color.
5. Keller-Kiliani reaction (on deoxysugars)
   (digoxin, digitoxin).
6. Solution of quabaine substance in H2SO4 conc.
   pink color, which quickly becomes red, this
   solution in the UV-light gives a green
   fluorescence (steroid cycle).
7. Reaction on deoxysugars (quabaine). After the
   acidic hydrolysis with Fehlings reagent - red
   sediment.

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Purity test of the pharmacopoeial preparations of
CG

• Digitoxin. Impurities (gitoxin, other glycosides)
  TLC.
• Digoxin. Impurities (gitoxin, digitoxin and other
  glycosides) TLC.
• Quabaine. Impurities (gitoxin, digitoxin and
  other glycosides) TLC. Alkaloids and
  Strophenthin-? with solution of tannin acid no
  precipitate formed .
• Storage
• All three drugs remain in the protected dark
  place. Poisonous substances. List ?.

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General chemical reactions for CG identification

• Reactions on steroid cycle
• Liberman-Burkhard reactiona 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-?).
• Rosenheim reactionto 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.

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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-dinitrobenzene reddish-purple coloration
  appears.
• Cedde reaction - in alkaline medium with
  3,5-dinitrobenzoic caid purple 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.

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Mechanism of the Baljet reaction (Balje)
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Reaction on the sixmember lacton cycle of
bufadienolides

• Glycoside is dissolved in chloroform and
  add gradually saturated solution of antimony
  (III) chloride, at the heating dark-red color
  appears.
• Reactions of sugar component
• After the acidic hydrolysis can be used inherent
  sugar reactions, based on their reductive
  properties
• With Fehling reagent formation of the red
  sediment ?u2O
• With Tollense reagent silver mirror reaction.
  
• Specific for 2-deoxysugars contained in the
  molecules of most cardiac glycosides, are
• Keller - Kiliani reactionglycoside 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.

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• 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.

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• 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 are incompatible with acids and
  compounds, which give acidic reaction of the
  environment. Hydrolysis by glycoside bound takes
  place. Reaction goes without any visible changes
  (with ascorbic acid and other vitamins with
  acidic ?? of environment).
• Cardiac glycosides are incompatible with alkalis
  and compounds, which give alkaline reaction of
  the environment(NaHCO3, barbital-sodium and
  others).

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Assay of CG

• 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.

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Storage and aplication of CG

• 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).

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Thank you for attention!