Lecture №19
General characteristic of hormones as
medicines, their classification, the
obtaining methods, the identification
methods, assays, storage conditions and
application.
Thyroid hormones, hormones of the
adrenal medulla, pancreas hormones.
prepared Kozachok S.S.
Hormones are biologically active substances that are
produced by glands of internal secretion in very
small quantities, and regulate all the vital processes
occurring in the body. Only sex hormones affect
more than 120 body functions.
The formation process of hormones is regulated by the
hypothalamus it is a brain region which is under the optic
tubercles.
Nowadays there are about 50 natural hormones. As drugs
there are used galena preparations and individual hormones,
which are derived from endocrine glands of a beef cattle as
well as synthetic analogs of natural hormones.
Hormones have long been classified according to their place
of origin, taking into account their physiological effect: thyroid
hormones, pancreatic hormones, adrenal hormones, etc.
Recent advances in the chemistry of hormones
Over the past decade in the chemistry of hormones there have been
many new studies. It was synthesized the hypophysis hormones,
several hypothalamic hormones. Some of them (rifatiroin,
somatostatin) are recommended for clinical use. Discover morphinesimilar hormones (enkephalins, endorphins), hormones of memory
and sleep. This is one of the ways to create new analgesics that are
not
addictive.
Create the new methods of synthesis of neurohormones with peptide
structure, and many of their structural analogs. It turned out that
some of them may affect on the heart, showing mediator or
modulator of the function, which allows using them for
atherosclerosis and myocardial infarction.
Established the chemical structure and the synthesis of insulin.
However, the chemical synthesis of this hormone has only
theoretical interest, since the yield of the final product is very small
and the cost of performing the synthesis of significant. Therefore,
insulin is extracted based on animal products or by genetic
engineering.
Tissue hormones or kinins have the importance role for
the body's vital functions. They are formed not in the
endocrine glands, but at various points in the body, where
their influence is currently required. Kinins have a peptide
structure. They regulate the basic biochemical and
physiological processes in the body and affect on many of
its functions. This gave impetus to the use of some of
kinins in human and veterinary medicine.
Modern studies of drugs of animal origin are carried out in
several directions. The components of organs and tissues
cause the greatest interest in studying, which in chemical
structure are proteins, glycosaminoglycans and nucleic
acids. They have a wide range of actions. Obtained drugs
from the thymus gland, prostate, trachea, blood vessels
and other organs of the animal materials.
Chemical classification of hormones
1.
a)
b)
c)
d)
e)
2.
a)
b)
c)
d)
Hormones that have the structure of amino-acid, amino
alcohols, polypeptides, proteins and their derivatives :
thyroid hormones and their synthetic analogues;
parathyroid hormone;
pituitary hormones;
Hormones of the pancreas;
Hormones of adrenal medulla .
Hormones that have the steroid structure :
Cortical layer of the adrenal hormones (corticosteroids) and
their semisynthetic analogs;
progestin (lutoidin) hormones and their semisynthetic
analogs;
androgenic hormones and semisynthetic anabolic drugs;
estrogenic hormones and their synthetic analogs of nonsteroidal structure.
Thyroid hormones
Thyronine derivatives of the thyroid gland have biological
activity:
In 1914, Mr. Kendall is siparated out from the thyroid gland
of a cattle thyronine tetraiodidne derivative (chemical
structure was established in 1927). This substance called
thyroxine (3,5,3',5'-tetraiodothyronine):
The presence of an asymmetric carbon atom determines the
presence of two optical isomers, of which L-thyroxine
which is 3 times more active than D-isomer.
In the 1952-1955 years , it was proved that the hormonal activity of the
thyroid gland the thyroxine does not only have, but other iodo
derivatives of the thyronine: 3,5,3'-triiodothyronine and 3,3'diiodothironine
Biosynthesis of these hormones in the body is produced by the
tyrosine and iodine, which enter to the body with food and water.
In medical practice, there is using thyroidin, synthetic Lthyroxine and triiodothyronine as sodium salts.
Thyroidin
Thyreoidin contains hormones of L- thyroxin and L-3,5,3’triiodo thyronine.
Obtaining by the drying and grinding the thyroid
glands of a cattle.
Yellowish-white powder with faint odor that is a
characteristic of the dried animal tissues.
Not soluble in water and other solvents, soluble in
alkalis and carbonates (acid properties).
Store in a well-sealed jars of dark glass.
Apply at the hypofunction of thyroid gland, which leads
to the hypothyroidism, myxedema, cretinism, obesity
or endemic goiter. H.s.d.-0, 3 g; H.d.d.-1, 0 g.
Identification of Thyroidin
1. Protein is determined by the formation of yellow
color after boiling the drug in alkaline solution. With
further addition of diluted sulfuric acid solution there
is decolorizing and droping out the white colloidal
precipitate.
2. To determine the organically bound of iodine the
preparation is destroyed by the calcination with a
mixture of KNO3 and Na2CO3. Forming iodides are
extracted with water and identified by the oxidation
of chloramines in the HCl environment. Iodine
liberated, the chloroform layer is coloured in redviolet color.
3.
4.
Thyroidin is burned with oxygen in the flask. As an
absorption substance using a starch solution that
contains 0.2% sulfamic acid. Iodine forms during
combustion, it paints an absorbing layer in blue.
Gives reaction of the phenolic hydroxyl with FeCl3.
Quantitative determination
In thyroidin there is determined the content of
the organically iodine bound.
Spending the mineralization by perhydrol in the
presence of concentrated H2SO4 (USPh
X).Forming the iodides and partial oxidation of
iodides to iodates. Iodides is oxidized to iodates by
the solution of KMnO4:
The excess of KMnO4 is removed by NaNO2 solution:
Possible excess of the nitrates can be destroyed by an urea:
In the solution, there is only one oxidant - iodate acid in an
amount equivalents to the iodine content in thyroidin precise
mass. To add a solution of KI and iodine, that was separated,
it titrated by Na2S2O3 solution :
Thyroidin contains 0,17-0,23 % of the iodine. Ем=1/6Ам(І).
2. Mineralization is carried out in a flask
combustion with oxygen (SP XI):
RСН2І + О2 → І2 + СО2 + Н2О
 Evolving iodine is absorbed by NаОН:
І2 + 2NаОН → NaІO + NaІ + Н2О
 For the oxidation of the forming iodides in the flask to
put acetate bromide solution till yellow colour:
2Br+(СH3СOO)- + NaІ + Н2О → Br2 + NaІO + 2CH3СОOH
NaІO + NaІ + 5Br2 + 5Н2О → 2NaІO3 + 10HBr
Then to add the concentrated formic acid solution till
the colourlessing of the solution:
НСООН + Br2 → 2НBr + СО2
Bromine residual traces is pumped.
At the thyroidin combustion also forming nitrogen oxides,
which dissolves in a solution of NaOH, converting to nitrite
ions. For the delation of them to add to the reaction mixture
3% solution of sulfamic acid :
H2N-SO2OH + HNO2 → H2SO4 + N2 + Н2О
Therefore, remove all oxidants except iodate equivalent to
the content of iodine bound in the sample. After that, in the
flask to add 1 g of KI, which interacts with iodate, allocates
an equivalent amount of iodine. It is titrated by 0.005 M
Na2S2O3 (indicator - starch):
2NaІO3 + 10КІ + 6H2SO4 → 6І2 + Na2SО4 + 5K2SO4 + 6Н2О
I2 + 2 Na2S2O3 → NaІ + Na2S4O6
Parallel to do a control experiment (without burning).
Ем=1/6Ам(І).
Levothyroxine sodium (SPU)
Levothyroxinum natricum
I
I
NH2
HO
O
CH2
CH
O
H2O
C
ONa
I
I
Sodium (2S)-2-amino-3-[4-(4-hydroxy-3,5diiodidophenoxy)-3,5- diiodophenyl] propanoate
hydrate
The synthetic levogyrate isomer of thyroxine (L-thyroxine).
Properties. Fine crystalline powder, almost white or slightly
brownish-yellow color. Very slightly soluble in the water,
slightly soluble in 96% ethanol, practically insoluble in the
ether. Soluble in dilute alkali.
Liothyronine Sodium (SPU)
Liothyroninum natriсum
I
I
NH2
HO
O
CH2
CH
O
C
ONa
I
Sodium (2S)-2-amino-3-[4-(4-hydroxy-3-iodophenoxy)-3,5diiodophenyl] propanoate
Synthetic analogue of triiodothyronine to 5 times stronger
than L-thyroxine.
Properties. The white or slightly colored powder. Very
slightly soluble in the water, slightly soluble in 96%
ethanol. Soluble in dilute alkali.
Identification of sodium salts
Levothyroxine
Liothyronine
1. Specific optical rotation
1. Specific optical rotation
2. IR spectroscopy
2. IR spectroscopy
3. TLC
3. UV - spectroscopy
4. To the substance to add some drops of the concentrated H2SO4 and
heat – forming the steam of the violet color (iodine).
5. After mineralization (under the action of the diluted H2SO4 ) it gives
reaction of sodium.
Assay
1. Both drugs were determined by liquid chromatography.
2. Combustion in a flask with oxygen, using for the absorption
NaOH. Than Iodometry (see thyroidin).
3. The method is based on the dehalogen at the heating with
zinc powder in alkaline medium:
I
HO
I
O
C
H2
H
C
COOH + 2 Zn + 9 KOH
NH2
I
HO
I
O
C
H2
H
C
COOK + 4 KI + 2 K2[Zn(OH)4] + H2O
NH2
Then to do argentometric titration educated iodide ions (indicator sodium eosinate). Еm=1/4М.m.
4КІ + 4AgNO3 → 4AgI + 4KNO3
Storager – in the airtight containers. In the dark
place, at 2-8 0С.
Remedy of the synthetic analogs of thyreodine:
L - thyroxine (levothyroxine) (Pharmak) 50 mg
and 100 mg № 50. Triiodothyronine
(Borschagovsky Chemical Plant ) - liotironine
Novotiral
(levothyroxine
+
liotironine)
Tiratrikol (Triakan) - 3,5,3 '- triiodotiacetate acid a physiological metabolite of liotironine.
Iodtirox - levothyroxine + potassium chloride
Antithyroid drugs (for treatment of toxic goiter) merkazolil (tiamazol) Metizol.
Hormones of adrenal medulla (catecholamines and
their synthetic analogues)
Adrenal glands - paired glands, which are composed
of two layers: cortical and medullary (inner).
Medulla layer produces the hormone of adrenaline
and noradrenaline accompanying to it, and the
cortical layer - about 40 different hormones, which
are called corticosteroids.
Adrenaline was first identified by a russian scientist
Cybulski M.O. at the end of the XIX century, and in 1903
established its structure, which is confirmed by synthesis.
Raw material for production of adrenaline and
corticosteroids is a adrenal (gland) of a beef cattle. So from
45 kg of the adrenal glands receive only 9.5 grams of pure
adrenaline. Therefore, in the present preparations of adrenal
hormones are obtained only synthetically ways.
Despite there are several
hydrophilic groups in the
molecule, the adrenaline
practically insolubles in
water according to the
formation of betaine (inner
salt, zwitter-ion).
From aqueous solutions of salts
(hydrochloride, hydrotartrate) it
can be settled down the base of
adrenaline with ammonia,
filtered, dried and used for the
determining of the specific
rotation.
In medical practice, there are using of adrenaline
(epinephrine)
hydrochloride,
epinephrine
tartrate,
noradrenaline hydrotartrate and their synthetic analogs mezaton (phenylephrine h/ch) izadrin (isoprenaline h/ch)
berotek (fenoterol h/br), salbutamol, verapamil h/ch.
Properties. Adrenaline and noradrenaline hydrotartratis white or white with a yellowish white crystalline substance,
odorless. It easily solubles in water, practically insoluble in
ether and chloroform, practically insoluble in ethanol. Like
other phenols, these compounds are soluble in alkaline
solutions, can be oxidized. Under the influence of light and
oxygen they form a colored oxidation products. They have
amphoteric properties. As diatom phenol it is easily
oxidized and is a good reducing agent. Natural adrenaline levogyrate,
synthetic
racemate.
Adrenaline tartrate
(Adrenalinі tartras)(SPhU 1.1)
Epinephrine bitartrate*
HO
OH
HO
HO
C
H
C
H2
N
H
CH3 *
HOOC
H
C
C
H
COOH
OH
(1R)-1 -(3,4-dihydroxyphenyl)-2-(methylamino) ethanol
hydrogen (2R,ЗR)-2,3-dihydroxy butandioate
Noradrenaline hydrotartrate(Noradrenalini
hydrotartras)
Levarterenol bitartrate*
HO
OH
HO
HO
C
H
C
H2
NH2 *
HOOC
H
C
C
H
COOH
* H2O
OH
(-)-1-(3,4-Dioxyphenyl)-2-amino ethanol
hydrotartrate
Synthesis scheme of remedies of
adrenaline and noradrenaline
The gotten racemate according the synthesis are separated
by the tartratic acid, using different solubility of the
hydrotartrates in alcohol. Levogyrate tartrate isomer is not
soluble in alcohol, dexter levogyrate - soluble.
Identification
1.
The specific rotation after transfering to the chloride salt, ultraviolet
and infrared spectroscopy.
To the aqueous solution of a substance to add diethoxy
tetrahydrofuran in the concentrated acetate acid and heated. To a
cooled solution to add dimethyl amino benzaldehyde solution in a
mixture of hydrochloric and acetate acids. The obtaining and the
control solution have the same yellow color.
Reaction on tartrate: a substance is heated with potassium bromide
and resorcin in the presence of concentrated sulfuric acid,
appearance a dark blue color. After cooling to add water, color
changes to red:
2.
3.
HO
H
C
O
COOH
+
HOOC
C
H
C
2
H2SO4
OH
H
OH
COOH +
COOH
H2SO3 +
H 2O ;
OH
OH
OH
C
CH
OH
COOH
COOH
+
2
C
O
OH
[O]
KBr
- H2O
H
.
HO
OH
HO
O
4.
Oxidized by 0,1М iodine solution in the buffer solutions with рН
3,56 and 6,5. Adrenaline an this condition forms adrenochrome,
which gives dark-red color of solution (рН 3,56) (SPU) or red-violet
(рН 6,5) color.
Noradrenaline forms noradrenochrome (red-violet color) only
in the solution with рН 6,5.
5.
a)
b)
c)
d)
e)
Not pharmacopeial reaction:
Due to the presence of two phenolic hydroxyl epinephrine
and norepinephrine with a solution of FeCl3 form an emeraldgreen color, which transfers by the adding drops of ammonia
solution to a cherry-red, then an orange-red colors.
On the tartrate ion reaction with a potassium salt.
With Tollens reagent (silver is reduced from the amino
solution).
With Felling reagent (orange-red sediment Cu2O).
Color reaction with 1,2-dinitrobenzene - blue-violet color.
HO
CH NH CH3 O
OH
OH
HO
O
CH NH CH3
+ H2
OH
O
OH
KO
NH
NO2
NO2
H2
NO2
KOH
N
OK
N+
O-
f)
In alkaline solution of adrenaline is oxidized by
atmospheric oxygen to form adreno lutain - there is a
yellow-green fluorescence.
g)
At the heating with NaOH or KOH adrenaline and noradrenalin
have hydroamino splitting to form 3,4-dioxyacetophen and
methylamine or ammonia:
Purity tests
By the UV- spectrometry it is determined in adrenaline
tartrate adrenalon in norepinephrine hydrotartratis noradrenalon; Noradrenaline in adrenaline tartrate is
determined by TLC.
Assay of adrenaline and noradrenaline
hydrotatrates (SPhU)
1.
Acidimetry in nonaqueous medium. Titrate in the
present of the concentrated acetic acid, the indicator
- crystal violet (Е=М.m).
2.
3.
4.
The content of drugs in solutions for injections it is
determined by photocolorimetry (color reaction with
Iron-citrate reagent, 1,2-dinitrobenzene).
UV- spectrometry.
Alkalimetry. Ем = М.m./2.
Storage
In the dark place in airtight container. Easily oxidized
when exposed to light and oxygen, and therefore
stabilizing the solutions for injection of adrenaline
and noradrenaline hydrotartratis by the adding of the
reductant - 0,1% sodium metabisulfite (disulfite)
Na2S2O5.
Application
Adrenaline and noradrenaline hydrotartratis used as
adrenomimetic (vasoconstrictor) remedy. They are prescribed
an the collapse, a sharp decrease in blood pressure as a
result of injuries, poisonings, during surgery to reduce
bleeding
and
blood
loss.
Adrenaline hydrotartrate is injected hypodermic as 0.18%
solution of 0,1-0,5 ml, norepinephrine hydrotartratis intravenous 0.2% solution. In oculus medical practice (dilates
the
pupil)
using
0,1%
solution
of
adrenaline.
Phenylephrine hydrochloride
(Phenylephrini hydrocloridum) (SPhU)
Mesaton (Mesatonum)
HO
OH
C
H
C
H2
N
H
CH3 * HCl
(1R)-1-(3-Hydroxyphenyl)-2-((methylamino) ethanol hydrochloride
Synthetic analogue of the adrenaline.
CHARACTERS. Appearance. White or almost white, crystalline powder.
 Solubility. Slightly soluble in water, sparingly soluble in methanol, slightly
soluble in ethanol (96 per cent). It dissolves in dilute mineral acids and in
dilute solutions of alkali hydroxides.
Identification of Phenylephrine
hydrochloride
1.
2.
3.
4.
Specific optical rotation, melting point, ІR-spectroscopy.
Gives reaction of chloride.
With CuSO4 at present of NaOH mezaton forms the complex of a
violet color, which , unlike ephedrine, insoluble in ether.
Not pharmacopeial reaction: with FeCl3 (on the
phenol hydroxyl) – violet color.
ASSAY
1.
2.
(SPhU). Alkalimetry. Substance is dissolved in a mixture of 0.1 M
HCl and 96% ethanol and titrated by 0.1 M ethanolic solution of
NaOH potentiometrically. At the calculation to taken the volume of
titrant between the two potential jumps on the titration curve. Em =
M.m.
Return bromatometry. Indicator – starch. Parallel to do control test.
Ем = 1/6 М.m.
Acidimetry in nonaqueous medium, a direct titration
at the present of mercury (II) acetate. Еm = М.m.
3. Alkalimetry. Еm = М.m.
4. Argentometry. Еm = М.m.
5. Mercurimetry. Еm = М.m.
2.
Application
Alpha-adrenoceptor agonist, Adrenomimetic
(vasoconstrictor) agent.
Release. Solution for injection 1% 1,0 № 10:
mezaton-, Mesafeton-Darnisa. Nazol-baby - a drop
of 0,125% 15 ml. Combination preparations:
Farmacitron (10 mg), Coldrex (10mg), KOLDFLU,
rinza, TeraFlu, AntiFlu, Anticataral.
Isadrine (Isadrinum)
hydrochloride*
H
C
HO
CH3
C
H2
N
H
CH
* HCl
CH3
OH
HO
1-(3,4-dioxyphenyl)-2-isopropilaminoethanol h/ch
Synthetic analogue of adrenalіne .
Properties. White crystalline powder, freely solubles in water.
Melting point 167-169 °C. Under the action of atmospheric oxygen
and
light,
easily
oxidized
and
becomes
pink.
Adrenomimetic tool. Unlike adrenaline, greatly expands the
bronchial tubes, slightly vasoconstrictor. Using at bronchial asthma.
Administered by inhalation of 1 ml of 0.5% solution.
Identification of isadrine
IR- and UV-spectroscopy.
Chloride’s reaction.
With FeCl3 – green color, at the adding NaHCO3 transfers to a
dark blue, аnd than to a red.
4. The formation of the isopropyladrenochrome. The drug is
dissolved in the two buffer solutions, whith pH 3.5 and 6, 5.
When mixing these solutions with 0.05 M iodine solution
and bleaching of the iodine excess after 5 minutes. the first
solution becomes intensely red and the second one - a redviolet (unlike noradrenaline).
5. With Tollens and Felling reagents.
6. With HNO2 – dirty-violet color.
7. Azoconection reaction (on phenolyc radical) - cherry-red
coloration.
8. With a basic CuSO4 solution – the complex has dark green color.
1.
2.
3.
ASSAY
 Kjeldahl method. Еm = М.m.
 Acidimetry in nonaqueous medium. Еm = М.m.
Hormones of the pancreas.
Insulin.
Insulin is a hormone that is produced by Langerhans islets of the
pancreas (from Lat. insula - island).
It has the huge influense on the carbonate exchanging reaction in our
body. Using inside insulin the level of suger in blood extremely
reduces. Insulin is used for the treatment of diabetes.
Insulin is obtained by the extraction from the pancreas of beef cattle,
or by genetic engineering. This crystalline powder, readily soluble in
water. Ebel was first extracted insulin in 1925.
Pharmaceutical agent is insulin injection (Insulinum pro
injectionibus), which is prepared by dissolving the crystalline powder
of insulin in water, acidified with HCl. For the preservation of insulin
was added to a solution of 0.3% solution trikrezola.
Remedy is an insulin for injection (Insulinum pro injectionibus),
which is prepared by dissolving the crystalline powder of insulin in
water, acidified with HCl. For the preservation of insulin there was
added to a solution of 0.3% tricresol solution.
The drug is a clear, colorless or slightly yellowish
liquid acid reaction (pH 2,5-3,5), with the smell of
preservative.
According to the chemical structure insulin is a
macromolecular compounds of protein nature. The empirical
formula of its is С254H337N65S6. Molecular weight - 5733.5.
In 1945-1956 Sanger for the first time established the
chemical structure of insulin. At the oxidation of the formic
acid HCOOOH it is divided into two monomeric peptides - A
peptide composed of 21 aminoacids, peptide B - with 30
aminoacids.
Placement of the aminoacids in the insulin molecule doesn’t
have the order. This protein has an unique structure.
Animals insulin has the same structure of the peptide B, but
slightly different structure of peptide A. Since pig’s insulin has
in 8-th position threonine (instead of alanine), and in position
10-th isoleucine (instead of valine).
For identification to the remedy to add by drops the
diluted NaOH solution – forming the flakes sediment,
which dissolves at the acidifying.
Insulin is easily destroyed by digestive enzymes,
so it does not take per os.
It is injected hypodermic, inner muscular. 1 МО = 0,045
mg of the crystalline insulin. Insulin syringes 100 AU/ml
and 40 AU/ml.
Released in sterile vials on 5 and 10 ml, sealed with
rubber caps. 10 ml of liquid = 20 or 40 MO.
The drug remains active for 1.5 years when stored in a
cool place.
Insulin is a short action (Farmasulin H humanity, Himulin
regular humanity, Humudar R humanity, pork) and the
midle actopn (Farmasulin HL humanity, Himulin NPH,
Humodar B, B-insulin, pig.).
Thank you for
attention