Lecture №28
Vitamins of the heterocyclic row:
derivatives of pyrimidinethiazol,
isoalloxazine, corrine. Properties,
quality requirements, storage,
application. Multivitamin drugs .
prepared ass. Medvid I.I.
Derivatives of pyrimidine and
thiazole
Molecule of pyrimidine-thiazol vitamins (vitamins B1 thiamines) consists of two heterocycles - pyrimidine (A)
and thiazole (B) connected by a methylene group:
In medical practice used thiamine hydrobromide, thiamine
hydrochloride, diphosphate ester of thiamine
hydrochloride (cocarboxylase).
Preparations of vitamin В1
Thiamine hydrobromide
(Thiamini
hydrobromidum) (SPhU)
Thiamine hydrochloride
(Thiamini
hydrochloridum) (SPhU)
3-[(4-Amino-2-methylpyrimidine-5-yl)methyl]-5-(2hydroxyethyl)-4-methylthiazole
bromide hydrobromide
3-[(4-Amino-2-methylpyrimidine-5-yl)methyl]-5-(2hydroxyethyl)-4-methylthiazole
chloride hydrochloride
Vitamin В1 is the first vitamin, which pioneered the
opening of vitaminology science .
For the first time it was identified from the rice bran
(Funk in 1912 y.). This substance in small doses cured
polyneuritis of pigeons and because its structure contains
sulfur atom, called thiamine.
Most contained in the cereal (grain) plants, yeast, egg
yolks, ox liver. Extraction from natural sources is difficult
with low output (from 1 t of yeast - 0,25 g of vitamin В1).
For the medical purpose it is received synthetically, at the
first pyrimidine and thiazole compounds should be
extracted separately, then they are condensed .
Thiamine belongs to the aminoalcohol of heterocyclic
series (availability of carbonyl and amino-groups ).
Properties of thiamines
Thiamine
hydrobromide
Thiamine
hydrochloride
The crystalline powder of
The crystalline powder of
white or white with
white or almost white or
yellowish tint with a
colorless crystals. Easily
specific smell. Easily
soluble in water, soluble in
soluble in water, little
glycerin, slightly soluble in
soluble in 96% alcohol,
96% alcohol. Hygroscopic.
practically insoluble in
ether.
This compounds are stable only in acidic environment In the
neutral and alkaline environments decompose with rupture
of thiazole cycle and formation of mercapto group.
Identification of thiamines
1.
2.
3.
Thiamine hydrobromide
IR-spectrophotometry.
Thiochrome test.
Reactions on bromides.
1.
2.
3.
Thiamine hydrochloride
IR-spectrophotometry.
Thiochrome test.
Reactions on chlorides.
Unpharmacopoeial reactions
а) With solutions of FeCl3 and K3[Fe(CN)6] a blue coloration
of Berlin blue formed.
b) At the melting with crystalline NaOH or metallic sodium
sulfide ions formed, which can be detected by the reaction
with sodium nitroprusside (red-purple coloration).
c) Thiamine can be quantitatively precipitated from the
solutions by the usage of some general alkaloid reagents
(Dragendorff, Bertran, Sheibler, picric acid, p-
Thiochrome test
Thiamine is oxidized by the alkali solution of potassium
ferricyanide with the thiochrome formation (bright
yellow substance), which is extracted by isoamyl or
butyl alcohol - alcohol layer gives blue fluorescence in
UV light (λ = 365 nm):
Quantitative determination of thiamine
hydrochloride and hydrobromide
1.
Thiamine h/br. (SPhU). Acidimetry in non-aqueous
environment in the presence of mercury (II) acetate .
Equivalent point is determined by potentiometric
method:
Е = ½ М.m.(C12H18Br2N4OS)
2.
3.
Thiamine h/chl. (SPhU). Alkalimetry in the mixture of 0,01 М
chloride acid solution and 96 % alcohol. Titrant – 0,1 М
NaOH solution. Equivalent point is determined by
potentiometric method. Volume of titrant between two
potential jumps on the titration curve is taken in the
calculation. Е = ½ М.m.
Thiamine h/chl.(SPhU addition 1). Acidimetry in non-aqueous
environment in the mixture of formic acid and acetic
anhydride. Equivalent point is determined by potentiometric
method. Е = ½ М.m.
4.
5.
6.
7.
8.
Gravimetry after precipitation of the drug by silicon tungsten
acid. Sediment composition: SiO2•12WO3• 2C12H17BrN4OS або
SiO2•12WO3• 2C12H17ClN4OS.
Alkalimetry, direct titration, indicator – bromothymol blue
orphenolphthalein. Е=М.m.
Argentometry by Fayans method, indicator – bromophenol blue.
Е=1/2 М.m.
Fluorimetry (by the intensity of thiochrome fluorescence).
Argentometry after the neutralization of the substance solution
with alkali. Analytical weight of thiamine bromide is titrated by
sodium hydroxide solution to the blue-green color, indicator –
bromothymol blue .
Then solution acidified by nitrate acid, add indicator – iron (III)
ammonium sulfate and 0,1 ml of 0,1М ammonium thiocyanate
solution – red color appears due to the formation of iron (III)
Reaction mixture is titrated by 0,1 М silver nitrate solution:
In the equivalent point excess of silver nitrate reacts with
iron (III) thiocyanate – solution becomes colorless:
The content of thiamine bromide is calculated by the
difference of volumes of silver nitrate, ammonium
thiocyanate and sodium hydroxide. Е = М.m.
Storage of the thiamine preparations
In airtight containers, that protect from the action of light. Not
allowed contact with metals to prevent the reduction to
dihydrothiamine:
Application of the thiamine
preparations
В1 plays an important role in human body. Is part of
coenzyme – cocarboxylase. While lack of vitamin B1
carbohydrate metabolism disorder arising, lactic and
pyruvic acid accumulate in the tissues, and therefore may
be neuritis and disorders of the heart. Thiamine affects on
protein and lipid metabolism, participates in the regulation
of water exchange.
Daily demand 2 mg (1 vg of the drug – 330 IU).
Avitaminosis –a beriberi disease, hypovitaminosis disorders of the nervous system.
Used for treatment of hypo- and avitaminosis of this
vitamin and diseases associated with dysfunction of the
nervous system.
Thiamine hydrobromide due to its higher molecular
weight are taken in large doses (1 mg of thiamine
hydrochloride is responsible by the activity to 1,29 mg
of thiamine hydrobromide).
Issue: Thiamine hydrochloride – dragee by 0,002 g,
amp. 5%-1,0 №10; thiamine hydrobromide – dragee by
0,0026 g №50.
Included in multivitamin preparations and vitamin of B
group (neurobex, neuron, neurovitan, neurorubine,
multi-tabs B-complex).
It is not recommended to enter at once parenterally with
B6 and B1 vitamins (delay esterification of thiamine by
phosphate acid) and B12 (enhances allergic effect of
thiamine) and mixed in one syringe vitamin B1 with
penicillin or streptomitsin (decomposition of antibiotics)
and nicotinic acid (thiamine decomposition).
Cocarboxylase
(Cocarboxylasum) CCB
Hydrochloride of thiamine diphosphate ester
CCB – freeze (lyophylization) dry white porous mass with
low specific smell and bitter-sour taste. Easily soluble in
water, sparingly soluble in ethanol.
Decomposes at the heated above 35°С.
Store in a dark place at a temperature less than 5°С.
CCB –coenzyme of enzymes involved in carbohydrate
metabolism. In the compound with proteins and magnesium
ions is a part of the carboxylase enzyme that catalyzes
carboxylation and decarboxylation of a-ketoacids.
In connection with the lack of CCB at the beri-beri diseases aketoacids (especially pyruvic and СН3СОСООН acids)
accumulate in the tissues.
Assign it foe the treatment of arrhythmia, coronary circulation
failure and other cardiovascular diseases, diabetes and various
pathological processes associated with deterioration of
carbohydrate metabolism.
Enter by i/m, i/v, s/c way by 0,05-0,1 g 1 time per day.
Issue: sealed ampoules by 0,05 g of sterile powder, which
before usage (ex tempore) is dissolved in 0,5% novocaine
solution or 0,9% sodium chloride solution.
Derivatives of pterin
Pterin vitamins, which include folic acid (vitamin Вс),
contained in green leaves of spinach, parsley, lettuce, in
legume and cereal crops (wheat, rye, corn), as well as in
yeast, liver.
The basis of chemical structure of the data vitamins is a
pteridine kernel which is a condensed system of pyrimidine
(A) and pyrazine (B) cycles. Derivative of pteridine 2amino-4-oxypteridine is called pterin.
Folic acid
(Acidum folicum), vitamin Вс (SPhU)
(2S)-2-[[4-[[(2-Amino-4-oxy-pteridine-6yl)methyl]amino]benzoyl]amino]pentadioc acid
Name of the vitamin is derived from the Latin word folium – leaf.
Folic acid is in nature both in free state and as polyglutaminates
in which there are additional glutamic acid residues.
Extraction of folic acid
(method of А.V. Truchanova and B.А. Kirsanova)
Condensed equimolar quantity of 2,5,6-tri-amino-4oxypirimidine hydrochloride, p-aminobenzoyl-L(+)glutamic acid and 2,3-dibromopropionic aldehyde:
Storage of folic acid
In airtight containers, in the place protected from light.
Properties of folic acid
Crystalline yellowish or orange powder.
Practically not soluble in water and most organic
solvents (different from other vitamins). Soluble in
dilute acids (the presence of nitrogen atoms of
amine nature) and alkali (availability of free
carboxyl groups). Decomposing under the action
of light, hygroscopic.
Folic acid molecule consists of three main parts: 2amino-4-oxypteridine (pterin), p-aminobenzoic
acid and associated with this acid residue of
glutamic acid.
Folic acid is an amphoteric compounds: basic
properties is caused by the nitrogen atoms of the
pteridine molecule, acidic - the carboxyl groups
and hydroxyl group in position 4.
Identification of folic acid
By the physico-chemical constants: specific rotation, by
liquid chromatography method, TLC.
Unpharmacopeial reactions:
a) at the adding of potassium permanganate to the drug
solution in hydrochloric acid (with following selection of
its excess by hydrogen peroxide) p-aminobenzoylglutamic
and pterine-6-carboxylic (pteric) acid are produced. The
last has a much stronger blue fluorescence than folic acid,
and its use for fluorimetric quantification of the drug.
b) determination of UV-spectral characteristics of folic acid.
c) due to its acidic properties folic acid with salts of heavy
metals forms insoluble colored complexes; with CuSO4 –
green precipitate, with Co(NO3)3 – dark yellow precipitate,
with FeCl3 – red-yellow precipitate. The general formula of
these salts:
Assay of folic acid
1.
2.
Liquid chromatography.
Polarographic method. Is used the folic acid ability to
recover in the medium of sodium carbonate to 7,8dyhydrofolic acid which is easily oxidized to folic even by
atmospheric oxygen, polarographic cell is constantly
blowing by nitrogen.
3.
Photocolorimetric method. In the core of method is folic
acid oxidation by potassium permanganate with the
following formation of p-aminobenzoylglutamic acid, its
diazotation and azoconnection with N-(1-naphthyl)ethylenediamine dyhydrochloride. As a result formed
colored in purple color azo dye, the intensity of its
coloration in a solution is determined by photocolorimeter
at =550 nm.
Application of folic acid
Folic acid is involved in the process of blood formation, so it is
called antianemic factor. Together with vitamin В12 it stimulates
erythropoiesis, is involved in the synthesis of amino-acids
(methionine, serine, etc.), nucleic acids, purine and pyrimidine
metabolism and in choline metabolism. In the body is redused to
tetrahydrofolic acid - coenzyme that participates in various
metabolic processes.
Daily demand of the folic acid for a healthy human is 0,2 - 1 mg.
Its deficiency leads to anemia.
To prevent a lack of folic acid at the unbalanced or poor nutrition
take 20-50 mcg inside daily, during pregnancy - 400 micrograms
per day and lactation - 300 mcg.
Issue: tabl. by 0,001 g and 0,005 g № 50.
Used for increased erythropoiesis, in certain types of anemia,
including anemia and leukopenia caused by drugs and ionizing
radiation, chronic gastroenteritis and tuberculosis of intestine.
Derivatives of isoalloxazine
Isoalloxazine –heterocyclic system, which consists of
condensed pyrazine, pyrimidine and benzene cycles, that is
derived from benzopteridine. Pyrimidine nucleus of
isoalloxazine has character of lactam cycle because it
contains two keto-groups :
Benzopteridine
Isoalloxazine
Detection of vitamin properties of flavin associated with the
presence in the molecule extremely labile group with two
conjugated double bonds in isoalloxazine nucleus. This
group of atoms causes the redox properties of riboflavin.
Riboflavin (Riboflavinum)
vitamin В2 (SPhU)
7,8-Dimethyl-10-[(2S,3S,4R)2,3,4,5-tetrahydroxy-pentyl]
benzo[g]pteridine2,4(ЗH,10H)-dion,
or 6,7-dimethyl-9-(1’-Dрibityl)-isoalloxazine
At the first isoalloxazine vitamins (vitamin B2) was
allocated from whey (because it is also called "laktoflavin).
Name of vitamin B2 "Riboflavin" comes from the fact that it
has a residue of polyhydric alcohols of the ribose original,
and its solutions are yellow (Latin flavus - yellow).
Riboflavin is very common in plant and in animal
products: contained in the milk serum, liver, kidney,
brewer's and baker yeast; in grains - millet, barley; in
vegetables - spinach and tomatoes.
For extraction of 1 g of the drug should be processed 5,4
tons of whey, so now vitamin B2 is synthesizeed from Dribose, о-4-xylidine and barbituric acid.
Characters. Yellow or orange-yellow crystalline powder.
Very few soluble in water, hardly soluble in 96% alcohol,
ether, acetone, chloroform. Easily soluble in chloride
(forms salts) and acetic acid. Solutions decompose under
the influence of light, especially in the presence of alkali.
Detects polymorphism.
Identification of riboflavin
1.
2.
By the physico-chemical constants: the specific rotation,
infrared spectroscopy, TLC.
Solution of the substance in the light that passes through
it, has a pale greenish-yellow color, and in reflected light
- an intense yellow-green fluorescence, which
disappears at the adding of mineral acid or alkali (in
acidic medium on the light forms riboflavin forms
lumichrome (6,7-dymethylalloxazine) - a colorless
substance that breaks down into ethanol and chloroform
with the appearance of blue fluorescence, in alkaline
medium on light riboflavin forms lumiflavin (6,7,9trimethylalloxazine), which solution has the same color
and fluorescence as riboflavin, but soluble in
chloroform).
3.
At the riboflavin reduction by sodium hydrosulfite and
fluorescence and coloration of aqueous solution disappear (a
colorless compound formed - leukoriboflavin):
Unpharmacopoeial reaction:
a) at the adding of sulfate concentrated acid to riboflavin red
coloration appears, which becomes yellow after the adding of
water.
b) with a silver nitrate solution orange-red coloration forms
(presence of imide group).
4.
Test on purity
Determined lyumiflavin that formed as a result of
changes in the chemical structure of matter under the
influence of light and alkaline environment.
Determination of the lyumiflavin impurity based on its
solubility in chloroform (in chloroform riboflavin is
insoluble) - color of the filtrate should not exceed the
standard(SPhU).
Determination of the lyumiflavin impurity conducted by
thin layer chromatography(SPhU, additions).
Storage
In airtight containers, in the place protected from light.
Assay of riboflavin
1.
2.
3.
4.
5.
UV spectrophotometry (SPhU). Determination of the
optical density is conducted at the weakened
transmitting light in aqueous solution, acidified by
acetic acid at =444 nm. Riboflavin content is
calculated using the specific absorption rate, which
equals 328.
Photocolorimetry.
Fluorimetry.
Alkalimetry by substituent after the interaction with
silver nitrate solution. Е = М.m.
Periodate oxidation (Malaprad’s reaction). Methodic is
based on the ribityl fragment of riboflavin molecule
oxidation with formation of formic acid.
Formic acid, which eliminated as a result of reaction, is
titrated by alkalimetry method :
НСООН + NaOH → НСООNa + Н2О
According to another method after the action of periodate
to the solution is added sodium iodide and sulphate acid:
5 NaI + NaIO3 + 3 H2SO4 → 3 I2 + 3 Na2SO4 + 3 H2O
Iodine that allocated by the reaction is titrated by the
standard solution of sodium thiosulfate.
6. Estarification byconcentrated sulfate acid. Due to the
hydroxyl groups are formed by mono-, di-, tri- and
tetrasulfoxyl esters are formed. Then by potentiometric
titration with KOH solution determine an excess of sulfate
acid. The reaction proceeds in a stechiometric ratio 1 : 3.
Application
Plays an important physiological role, because is a part of
the oxidativ cell enzymes as riboflavin-phosphate ester.
Daily demand – 2 mg. Hyporiboflavinose is characterized
by deterioration of appetite, weight loss, sores in the corners
of mouth; ariboflavinose - conjunctivitis, clouding of the
cornea and lens its..
Vitamin В2 is used orally in doses of 5-10 mg per day
(depending on the degree of disease) at ariboflavinose,
conjunctivitis, irytah, keratitis, radiation sickness and others.
Issue: tabl. by 0,002 g; 0,005 g; 0,01 g; 0,01% eye drops; is
a part of riboflavin mononucleotide (riboflavin-5'monophosphate sodium) – amp. 1%-1,0 №10.
Corrine derivatives
Corrine vitamins (group В12) were found in natural
products of the animal origin, mainly in the internal
organs. So, the richest source of vitamin B12 are fish
entrails, high content of this vitamin is in the whale liver
and the highest content meat is in the meat of molluscs. In
human and animal B12 is synthesized by microflora of
intestine and accumulates in the liver, kidney, intestine
walls.
Vitamin В12 (cyanocobalamine) is extracted from the
waste of the production of streptomycin and
chlortetracycline antibiotics. Another source - sewage
(waste products of actinomycetes, bacteria, blue-green
algae), from this sourse vitamin B12 is allocated by
adsorption or extraction.
Cyanocobalamin
(Cyanocobalaminum) vitamin В12
(SPhU)
α-(5,6-dimethylbenzimidazole-1yl)cobamide cyanide
Corrine
Cyanocobalamin molecule consists of two main parts.
First
nucleotide
containing
5,6dimethylbenzimidazole
associated
with
Dribofuranose, which, in turn, linked by etheric bound
with phosphate acid. Nucleotide connected with
macrocyclic corrine system (second part) by peptide
bond. Nitrogen atom of 5,6-dimethylbenzimidazole in
position 3 bounded by coordination bond with cobalt
atom. Cobalt forms a chelate compound with cyanogroup and with nitrogen atoms and of hydrogenated
pyrrole cycles of corrine system.
Positive charge of cobalt ion is neutralized by
negatively charged phosphate acid anion. Therefore,
cyanocobalamin is not only a chelating compound, but
an internal salt.
Properties of cyanocobalamin
Crystalline powder with dark red color, odorless,
hygroscopic. Sparingly soluble in water, soluble in 95 %
alcohol, practically insoluble in ether, chloroform,
acetone. The anhydrous substance is very hygroscopic
At the heating above 300°С decomposes. Stable at рН=46. Easily decomposed in an alkaline environment.
СN-group in the molecule of vitamin В12 can be replace
by
the
ОН-group
(oxycobalamin)
or
NO2
(nitrocobalamin). Both products are converting to a group
of vitamin В12.
At the heating of cyanocobalamin solution with HCl
ammonium
chloride,
5,6-dimethylbenzimidazole,
aminoisopropanol, cobalt chloride,
cyanic acid are
formed.
Antianemic action associated with the presence of 5,6dimethylbenzimidazole in the molecule structure.
Identification of cyanocobalamine
Determination of UV-spectral characteristics.
TLC.
Cobalt ions are determined after the mineralization by
alloying with potassium hydrosulfate and interaction
with sodium 1-nitroso-2-naphthol-3,6-disulfonate - a red
color of internally complex salts of cobalt appeares.
Cobalt ion can be detected after evaporation and
roasting of 0,25 mg of cyanocobalamin with 10 mg of
potassium sulfate and 2 drops of 15 % sulfate acid.
Saturated solution of ammonium thiocyanate in
acetone is added to the residue; blue-green coloration
appears :
Со2+ + 2 NH4SCN → Со(SCN)2 + 2 NH4+
Detection of cyano-group. Analytical weight of
cyanocobalamin is heated in the test-tube with oxalic
acid, under the action of this acid cyanic acid
eliminates, which is detected with filtration paper
soaked by the solution of benzidine and copper (ІІ)
acetate, as a result blue complex compound formes.
Assay
The method of UV-spectrophotometry.
Storage
In airtight containers, in the place protected from light.
Application of cyanocobalamin
 Vitamin В12 is a growth factor necessary for normal
hematopoiesis and maturation of erythrocytes. He participates in
the formation of choline, methionine, nucleic acids, shows a
positive effect on liver function and nervous system.
 Daily demand for healthy person – 10-20 mcg.v This quantity of
vitamin is not produced in the intestine and should be
complemented by the products of animal origin
 Apply at the malignant anemia, various forms of anemia, liver
disease, nervous system, skin diseases and others.
 Issue: solution for injection 200 mcg (0,02%) or 500 mcg
(0,05%) 1,0 №10.
Multivitamin drugs
In nature vitamins commonly found in the form of various
combinations. Herbal products often contain vitamins of group
B, vitamin C and others. The combination of vitamins is also
found in animal products.
. In many cases, vitamins mutually reinforce physiological
effects that they have. For example, the effect of vitamin P on
vascular permeability amplified by ascorbic acid, mutually
amplifying effect on hematopoiesis of folic acid and
cyanocobalamin.
In some cases, toxicity of vitamins is reduced at their combined
application. For example, vitamin D toxicity is reduceing by
vitamin A. For example, vitamin D toxicity is reduceing by
vitamin A. Vitamins can also detect antagonistic effect (nicotinic
acid slows the lipotropic action of choline).
Vitamins participating actively in various biochemical processes,
if they are associated and show a stronger complex biological
action that is served for the combined usage in both cases for the
preventive and therapeutic purposes.
Multivitamin drugs have different compositions (some
vitamins alone or in combination with microelements) and
issued in the form of various dosage forms: tablets,
“effervescent” tablets (upsavit, supradine) tablets for
chewing (multi-tabs, jungle), beans (sob) , syrups
(multivitamol), gels (Kinder-biovital), solution for injection
(nerviplex (В1, В6, В12)).
Ukrainian multivitamin drugs: revit (А, В1, В2, С), hexavit
(revit+ В6, РР), undevit (hexavit+ Е, Р, Вс, В3), decamevit
(А, В1, В2, В6, В12, РР, С, Е, Р, Вс, methionine), complevit
(В1, В2, В6, В12, РР, С, Е, Вс), quadevit (decamevit + В3,
glutamic acid, fitine, KCl. CuSO4), vitam (В1, В2, В6, РР,
В3,mefenamic, Fe, Zn, Mn, Cu, Co, Cr).
Multivitamin preparations of the foreign origin: Vitrum
vitamins (Kids, Junior, Beauty, Prenatal, Energy, Cardio,
etc..) Centrum, multi-tabsy, Duovit, Pikovit, Jungle, Unicap,
Pregnavit, Oligovit, Theravit, Supradyn, Kinder-biovital and
others.
Thank you for
attention!