Lecture № 10

LectureLecture №№1010Current state and development of Current state and development of chemistry as a corticosteroid drug. chemistry as a corticosteroid drug.

Cortical layer of the adrenal hormones Cortical layer of the adrenal hormones and some of their semisynthetic and some of their semisynthetic analogues. Relationship between analogues. Relationship between structure and biological activity. structure and biological activity.

Gestogen hormones and their synthetic Gestogen hormones and their synthetic analogues.analogues.

prepared Kozachok S.S.prepared Kozachok S.S.

The hormones of the adrenal of cortical layer The hormones of the adrenal of cortical layer ((corticosteroidscorticosteroids)) and and sex hormonessex hormones belong to the steroid belong to the steroid hormones. Sex hormones can be divided into hormones. Sex hormones can be divided into male sex male sex hormones (androgens), female sex hormones hormones (androgens), female sex hormones (estrogen) and luteal hormones (progestogens, or (estrogen) and luteal hormones (progestogens, or lutoid hormones). lutoid hormones).   The sThe structural bastructural basee of of the the steroid hormones is a steroid hormones is a cyclopentaneperhydrocyclopentaneperhydrophenanthrene phenanthrene hydrocarbon hydrocarbon chainchain. The general formula of steroid hormones. The general formula of steroid hormones are are: :

The chemical structure of the steroid hormones

Hormone’s group Double bond

SubstituteХ1 Х2 R

corticosteroid

en-4dien-1,4

=О -Н-ОН=О

gestogen ен-4 =О -Н

androgen ен-4 =О -Н -ОН

estrogen trien-1,3,5(don’t have

С-19)

-ОН -Н -ОН=О

CH2OH

C O

CH3

C O

- - - -OH

Steroid hormonesSteroid hormonesMethyl groups attached to the steroid cycle in the 10-th and 13-th position, are named angularly. Radical R and the hydrogen atoms (in 8, 9, 14 positions) are oriented to the space in a cis-or trans-position relative to the angularly groups. Conventionally assumed that the angularly methyl groups are located above the plane of the drawing (the bond is indicated by solid (full) line). If the other substituents are in cis-position, in the same plane as the angularly groups (β-configuration), then their relationship are denoted by the solid line, and if the trans-position (α-configuration) - dotted line. Because the structure of the steroid hormones have much in common, a lot of their analysis method are common. Steroid hormones - crystalline substances, so for them to determine the melting point it is one of the method to measure the purity and identity.

Steroid hormones and their analogues Steroid hormones and their analogues are are an optically-active substances, an optically-active substances, most of them are most of them are dexterdexter isomers (met isomers (methyhylandrostendiol - landrostendiol - levogyratelevogyrate). ). ANAND for D for the identification and confirmation of purity recommendthe identification and confirmation of purity recommendss to determine the to determine the angle of rotation of the plane of polarized light by the solution of the angle of rotation of the plane of polarized light by the solution of the compounds in organic solvents and to calculate the specific rotation.compounds in organic solvents and to calculate the specific rotation.General reaction for all steroid hormones and their synthetic analogues is General reaction for all steroid hormones and their synthetic analogues is the reaction with concentrated Hthe reaction with concentrated H22SOSO44. When to dissolve in it and to heat the . When to dissolve in it and to heat the substances give appearance a specific color, sometimes fluorescence, with substances give appearance a specific color, sometimes fluorescence, with a further addition of water, chloroform, the color changes, there is a specific a further addition of water, chloroform, the color changes, there is a specific fluorescence.fluorescence.Steroid hormones possessi keto group in 3-d position, they give the addition Steroid hormones possessi keto group in 3-d position, they give the addition reaction Steroid hormones possessing keto group in position 3, given the reaction Steroid hormones possessing keto group in position 3, given the addition reaction with elimination of water with hydroxylamine, addition reaction with elimination of water with hydroxylamine, phenylhydrazine, 2,4-dinitrophenylhydrazine, isoniazid and elimination of phenylhydrazine, 2,4-dinitrophenylhydrazine, isoniazid and elimination of water – observing the precipitation with a characteristic melting point, or water – observing the precipitation with a characteristic melting point, or there is a characteristic color (yellow, orange) : with hydroxylamine, there is a characteristic color (yellow, orange) : with hydroxylamine, phenylhydrazine, 2,4-dinitrophenylhydrazine, isoniazid - observing phenylhydrazine, 2,4-dinitrophenylhydrazine, isoniazid - observing precipitation with a characteristic melting point, or there is a characteristic precipitation with a characteristic melting point, or there is a characteristic color (yellow, orange):color (yellow, orange):

These reactions can be used for the quantity determination These reactions can be used for the quantity determination of the steroid hormones and their analogues by the of the steroid hormones and their analogues by the gravimetry method (gravimetry method ((mass of sediment formed) or (mass of sediment formed) or photometric (optical density of colored solutions).photometric (optical density of colored solutions).

For the hormone’s identification which have hydroxyl For the hormone’s identification which have hydroxyl groups in 3 or 17 position, often there is using the reaction groups in 3 or 17 position, often there is using the reaction of the esters forming (acetate, benzoate), with a of the esters forming (acetate, benzoate), with a characteristic melting point:characteristic melting point:

For the hormone’s identification and their synthetic analogues that are For the hormone’s identification and their synthetic analogues that are used as esters (acetate, propanoate), apply the hydroxamic reaction:used as esters (acetate, propanoate), apply the hydroxamic reaction:

ForFor the the identificationidentification and quantitative analysis of steroid and quantitative analysis of steroid hormones and their analogues are widely used UV hormones and their analogues are widely used UV spectroscopy of alcohol solutions. spectroscopy of alcohol solutions. The iThe identification is carried dentification is carried out out according to the location according to the location oof the maximusf the maximus and and minimumminimum on on a particular part of the spectrum, comparing with the a particular part of the spectrum, comparing with the spectrum of standard sample, spectrum of standard sample, according to the according to the relative relative between the between the optical densities in the various absorption optical densities in the various absorption maximmaximusus and calculat and calculatee the spesific absorbtion valurthe spesific absorbtion valur. The . The content of the active substance is determined by the specific content of the active substance is determined by the specific absorption rate or standard solution. absorption rate or standard solution.

The substance’s indentification is The substance’s indentification is confirmed by the IR-confirmed by the IR-spectropy, which is compared with the spectropy, which is compared with the pharmacopeial pharmacopeial or standard semplesor standard semples..For the identifying and determining the presence of impurities For the identifying and determining the presence of impurities there is widely used method of TLC.there is widely used method of TLC.

Corticosteroid and their synthetic analoguesCorticosteroid and their synthetic analoguesCortical layer of the human and animals adrenal Cortical layer of the human and animals adrenal glands producesglands produces corticosteroids (Latin "cortex" - corticosteroids (Latin "cortex" - crust).crust). Corticosteroids are the regulators of metabolic Corticosteroids are the regulators of metabolic processes and are divided into two groups: processes and are divided into two groups: mineralocorticoids (regulate the exchange of mineralocorticoids (regulate the exchange of electrolytes and water in the body) and steroids electrolytes and water in the body) and steroids (regulate the exchange of carbohydrates and (regulate the exchange of carbohydrates and proteins).proteins). They are used at the sharp adrenal insufficiency They are used at the sharp adrenal insufficiency (Addison's disease), as well as addisonizmi.(Addison's disease), as well as addisonizmi. Natural hormones - hydrocortisone, cortisone, Natural hormones - hydrocortisone, cortisone, desoxycorticosterone (Dox), etc.desoxycorticosterone (Dox), etc. The source of the more than 40 adrenal The source of the more than 40 adrenal corticosteroids is beef cattle. corticosteroids is beef cattle.

The initial products for the synthesis of The initial products for the synthesis of corticosteroids can be natural substances of steroid corticosteroids can be natural substances of steroid structure (diosgenin, stigmasterol) and cholesterol, structure (diosgenin, stigmasterol) and cholesterol, which is considered a precursor of corticosteroids which is considered a precursor of corticosteroids in the body.in the body. CortisoneCortisone is is isolated in 1936 from the adrenal cortex isolated in 1936 from the adrenal cortex byby Kendal and Kendal and Vintershteyner inVintershteyner in USUSAA and Reichstein in Switzerland. and Reichstein in Switzerland.

The problem of its synthesis in the fact that in nature there are no The problem of its synthesis in the fact that in nature there are no available steroidal compounds containing keto group in 11 position. available steroidal compounds containing keto group in 11 position. Synthesis Synthesis such a group can besuch a group can be by by biochemical oxidation (using biochemical oxidation (using fungi, yeasts, actinomycetes, and various bacteria). This process fungi, yeasts, actinomycetes, and various bacteria). This process allows us to introduce the hydroxyl in 9, 11, 14, 15, 16, 17, 21allows us to introduce the hydroxyl in 9, 11, 14, 15, 16, 17, 21 positionpositionss, in addition to the α-, in addition to the α- and and ββ-configuration. -configuration. The total synthesis of cortisone was spent in 1951 by Woodward The total synthesis of cortisone was spent in 1951 by Woodward (USA). It includes about 30 stages that’s why have only theoretical (USA). It includes about 30 stages that’s why have only theoretical interest.interest.In 1956, Suvorov M.M. (VNDHFI) used solasodin - aglycone of In 1956, Suvorov M.M. (VNDHFI) used solasodin - aglycone of glucoalkaloid of avian nightshade (Solanum aviculare) as the initial glucoalkaloid of avian nightshade (Solanum aviculare) as the initial product for the industrial production of cortisone. Scheme of its product for the industrial production of cortisone. Scheme of its synthesis consists of several stages.synthesis consists of several stages.

Corticosteroids have a very important side-effects and thus their use Corticosteroids have a very important side-effects and thus their use as anti-inflammatory, desensitizing and anti-allergic means. In as anti-inflammatory, desensitizing and anti-allergic means. In addition, they addition, they havehave antishock and immunosuppressive activity and antishock and immunosuppressive activity and therefore therefore they are they are used used atat the transplantation of organs and tissues the transplantation of organs and tissues to suppress the rejection reaction, as well as in various autoimmune to suppress the rejection reaction, as well as in various autoimmune diseases. diseases. Side effects of corticosteroids: a delay of sodium and water in the Side effects of corticosteroids: a delay of sodium and water in the body with the appearance of an edema, increased allocation of body with the appearance of an edema, increased allocation of potassium ions, increased blood pressure, hyperglycemia, potassium ions, increased blood pressure, hyperglycemia, osteoporosis, etc.osteoporosis, etc. At the lAt the long-term treatment ong-term treatment they can they can suppresssuppress the the cortical cortical adrenal adrenal function, so function, so the the treatment should not be treatment should not be suddenly stoped causesuddenly stoped cause it it may be the aggravation of the disease or condition of acute adrenal may be the aggravation of the disease or condition of acute adrenal insufficiency. insufficiency.

Corticosteroids in ointments and drops Corticosteroids in ointments and drops can notcan not be be usedused at theat the viral viral diseases of the eye (can form diseases of the eye (can form the the ulcers). ulcers). All dosage forms of corticosteroids stored as potent tools in a dark All dosage forms of corticosteroids stored as potent tools in a dark place. place. All dosage forms of corticosteroids stored as potent tools in a dark All dosage forms of corticosteroids stored as potent tools in a dark place.place.

Natural corticosteroids

Hydrocortisone Cortisone

Desoxycorticosterone (Dox),

CH3

CH3

O

COCH2OH

OHHO

CH3

CH3

O

COCH2OH

OHO

O

CH3

CH3COCH2OH

Chemical signs of corticosteroidsChemical signs of corticosteroids1.1. Derivatives of pregnaneDerivatives of pregnane2.2. In the In the 1717-th position there is -th position there is

oxyacetyl oxyacetyl –– СОСНСОСН22ОН ОН ((reducing propertiesreducing properties))

3.3. In the 17 In the 17 αα oxyoxy (-ОН) (-ОН) group group ((forfor glucocorticosteroidsglucocorticosteroids))

4.4. In theIn the 11 11 oxooxo (=О) (=О) oror о оxyxy (-ОН) (-ОН) groupgroup

5.5. In theIn the 4 – 4 – double bonddouble bond, , in the in the separate remedy there is also in separate remedy there is also in the 1-st position.the 1-st position.

6.6. In the In the 33 position position - - oxooxo (=О) (=О) group.group.

Characteristics and general identification reaction of the Characteristics and general identification reaction of the ccorticosteroidsorticosteroids

Adrenal cortex hormones and their synthetic analogs - a white Adrenal cortex hormones and their synthetic analogs - a white crystalline substance, which sometimes have a yellowish or cream crystalline substance, which sometimes have a yellowish or cream color, and odorless.color, and odorless. They are practicaly insoluble in water, soluble in the most organic They are practicaly insoluble in water, soluble in the most organic solvents. Desoxycorticosterone acetate and cortisone acetate readily solvents. Desoxycorticosterone acetate and cortisone acetate readily soluble in chloroform.soluble in chloroform. Corticosteroids and their analogues are dextergyrate isomers.Corticosteroids and their analogues are dextergyrate isomers. The steroid structure is confirmed by:The steroid structure is confirmed by:

а) а) Boscot reactionBoscot reaction: : corticosteroids are dissolved in the mixture of corticosteroids are dissolved in the mixture of 88% Н88% Н33РОРО44 and concentratedand concentrated СН СН33СООН, СООН, the solution heatedthe solution heated 2 2 minutes atminutes at 100 100ººС С and leave and leave for 1 hour at room temperaturefor 1 hour at room temperature, , and and then it is diluted bythen it is diluted by СНСН33СООН. СООН. Under the UV lamp (Bah lamp) Under the UV lamp (Bah lamp) fluorescence occurs.fluorescence occurs.

b) reaction with concentrated sulfuric acid.b) reaction with concentrated sulfuric acid.

αα--KKetonicetonic group group – – reducing propertiesreducing propertiesі (і (oxidizing to oxidizing to carboxilic group.carboxilic group.

а) а) Felling reaction Felling reaction ((copper-tartrate reagentcopper-tartrate reagent). ). At the heating At the heating of the mixture of the alcohol substance’s solution and of the mixture of the alcohol substance’s solution and Felling reagent on the waterbathe CuFelling reagent on the waterbathe Cu22O is settled downO is settled down. .

bb) ) Except Felling reagent as a oxidizing agent Tollens Except Felling reagent as a oxidizing agent Tollens reagent can be usedreagent can be used ( (ammonia solution of silver nitrateammonia solution of silver nitrate) – ) – reaction ofreaction of ““silver mirrorsilver mirror”, ”, Zonnenshten reagent Zonnenshten reagent - - НН33Р0Р044 • 12Мо0 • 12Мо033 • 2Н • 2Н2200 ), ), Iron (III) saltIron (III) salt..

c) Gorega reaction. At the oxidation of corticosteroids with ethanolic solution of triphenyltetrazolium chloride in the presence of a solution of tetrametylamine hydroxide, as a reducing product there is formed red color of formazans:

The reaction is used for the identification and quantification by a spectrophotometry.d) At the oxidation of potassium periodate there is released 17-carboxylic acid, formaldehyde, which can be associated by chromotropic acid (aurine dye).

C

HO

C

O

O

H

C

HO

OH

O

NN

N N

C6H5

C6H5

C6H5

NHN

N N

C6H5

C6H5

C6H5

Cl-++

+

Carbonyl (keto) group in the 3-d position: the reaction of Carbonyl (keto) group in the 3-d position: the reaction of association with the elimination of water with association with the elimination of water with hydroxylamine, phenylhydrazine, 2,4-hydroxylamine, phenylhydrazine, 2,4-dinitrophenylhydrazine, isoniazid - observed precipitation dinitrophenylhydrazine, isoniazid - observed precipitation with a characteristic melting point, or there is a with a characteristic melting point, or there is a characteristic color.characteristic color.Esteric group:Esteric group:a) hydroxamic testa) hydroxamic testb) alkaline hydrolysis with subsequent identification of the b) alkaline hydrolysis with subsequent identification of the hydrolysis products.hydrolysis products. Identification of the covalently bounded fluorite. In fluorine Identification of the covalently bounded fluorite. In fluorine continuing ccontinuing corticosteroids the present of fluorine is the present of fluorine is identified after the mineralization (to burn with oxygen in identified after the mineralization (to burn with oxygen in flask) with zirconium-alizarine complex. flask) with zirconium-alizarine complex.

(Desoxycorticosteroni acetas) Desoxycorticosterone acetate*, DOXA

Pregnane-4-оl-21-dione-3,20-21-acetate or 21-acetoxypregnane-4-dion-3,20

Desoxycorticosterone is a natural mineral-corticosteroid, does not contain -OH group in 11-th position. Extracted from cholesterol.

O

CH3

CH3COCH2OCOCH3

Identification of DOXAIdentification of DOXA1.1. On the steroid systemOn the steroid system – – Boscot reactionBoscot reaction. . A violet color wiyh a A violet color wiyh a

fluorescencefluorescence..2.2. With concentratedWith concentrated HH22SOSO44 – – cherry color with greenish-brown cherry color with greenish-brown

fluorescence. After adding chloroform and shaking, the lower fluorescence. After adding chloroform and shaking, the lower layer is painted in yellow top - green (a steroid cycle).layer is painted in yellow top - green (a steroid cycle).

3.3. On the oxyacetylic groupOn the oxyacetylic group, , which is acetylatedwhich is acetylated::а) а) with Felling reagentwith Felling reagent – – forming the red sedimentforming the red sediment CuCu22OObb) ) With Tollens reagentWith Tollens reagent – – metallic silver settled down metallic silver settled down4.4. On the ester group On the ester group – – hydroxamic reactionhydroxamic reaction ( (red-brown colorred-brown color))5.5. On the keto-group in 3-th position – the oxym formation, On the keto-group in 3-th position – the oxym formation,

phenylhydrazine, 2,4phenylhydrazine, 2,4--dinitrophenylhydrazonedinitrophenylhydrazone..6.6. Acetylic group after hydrolysis Acetylic group after hydrolysis КОН КОН is identified according to is identified according to

the formation ethylacetatethe formation ethylacetate ( (specific smellspecific smell):):

O

CH3

CH3C

O

CH2

O C CH3

O

KOH

O

CH3

CH3C

O

CH2

OH

+ CH3COOK

2CH3COOK + H2SO4 + 2C2H5OH 2CH3COOC2H5 + K2SO4 + 2H2O

Assay of DOXAAssay of DOXA UV-spectrometryUV-spectrometry.. Photocolorimetric determination Photocolorimetric determination of theof the 0,5% 0,5% oil oil

solution for injection after heating with solution for injection after heating with concentrated concentrated HH33POPO44. . Application Application           For the treatment of Addison's disease,           For the treatment of Addison's disease, ggipoipocortocyzmcortocyzm, myasthenia gravis, asthenia, etc. , myasthenia gravis, asthenia, etc.           Injected intramuscularly           Injected intramuscularly of the of the oily solution oily solution of 5 mg 3 times a week, or in of 5 mg 3 times a week, or in the the severe cases severe cases ofof 10 mg daily. 10 mg daily.                     ProducedProduced - amp. 1,0 - 0,5% - amp. 1,0 - 0,5% ooilil solution solution, tab. , tab. 5 mg sublingual application. 5 mg sublingual application.

GGlucocorticoidlucocorticoidThey regulate the body carbohydrate and protein metabolism they have little effect on the exchange of electrolytes and water.

Their side effects are much more important as functional.

From natural glucocorticosteroids the practical value acquired cortisone and hydrocortisone, which is produced from solasodinu that is located in the bird nightshade (Solanum aviculare).The synthetic analogues are widely used - prednisolone, dexamethasone and their derivatives, which active in smaller doses and have less side effects.

Divided into three groups: 1. Glucocorticosteroids which do not contain halogen atoms in molecules; 2. Fluorine compounds; 3. Compounds with the atoms of chlorine and fluorine or only with chlorine atoms in the molecules.

(Cortisoni acetas) Cortisone acetate*

Pregnane-4-diol-17α,21-trion-3,11,20-21-аcetate or 21-acetoxy- 17α-oxypregnane-4-trion-3,11,20

It is a semisynthetic substance, natural hormone cortisone. It was first identified in 1937 as a drug first used in the late 40's, when obtained synthetically from solasodyne.

O

CH3

CH3COCH2OCOCH3

O OH

Identification of Identification of Cortisone acetateCortisone acetate1.1. Boscot reactionBoscot reaction. . A violet fluorescenceA violet fluorescence..2.2. Basic hydrolysisBasic hydrolysis ( (heating with heating with КОНКОН solution solution). ). At the At the

addition of the concentrated Haddition of the concentrated H22SOSO44 appearance the fruit appearance the fruit smell of ethyl acetatesmell of ethyl acetate..

3.3. On the oxyacetylic groupOn the oxyacetylic group, , which is acetylated herewhich is acetylated here::а) а) With Felling reagentWith Felling reagent – – formation red sedimentformation red sediment CuCu22OObb) ) With Tollns reagentWith Tollns reagent – – metallic silver settled down metallic silver settled down 5. On ester group 5. On ester group – – hydroxamic reactionhydroxamic reaction ( (darkreddarkred colorcolor))4.4. On keto group in 3-th positionOn keto group in 3-th position – – formation of formation of

phenylhydrazone phenylhydrazone ((bright-yellow colorbright-yellow color), 2,4-), 2,4-dinitrophenylhydrazonedinitrophenylhydrazone ( (melting temperaturemelting temperature 240 240ººС).С).

5.5. Remedy solution in the concentratedRemedy solution in the concentrated HH22SOSO44 in UV-ligt gives in UV-ligt gives intensive yellow fluorescence.intensive yellow fluorescence.

Assay of Assay of of of Cortisone acetateCortisone acetate UVUV-- spectrophotometry spectrophotometry in alcohol solution atin alcohol solution at λλ=238 =238 nmnm

in the comparing with the standard solutionin the comparing with the standard solution..

ApplicationApplicationWhen Addison's disease, rheumatism, rheumatoid When Addison's disease, rheumatism, rheumatoid arthritis, asthma, and leukemia, mononucleosis, in arthritis, asthma, and leukemia, mononucleosis, in the the dermatology - dermatology - atat neurodermatitis, eczema and neurodermatitis, eczema and other allergic diseases. other allergic diseases. TakingTaking inside first inside firstlyly byby 0,1-0,2 g, and then 0,1-0,2 g, and then byby 25 mg 25 mg per day in pill or injected intramuscularly at 25-50 mg per day in pill or injected intramuscularly at 25-50 mg as a suspension. as a suspension.

ProducedProduced: Table. 25-50 mg: Table. 25-50 mg;; vials vials onon 10 ml 10 ml suspension (1 ml - 25 mg suspension (1 ml - 25 mg ccortisone acetateortisone acetate). ).

Hydrocortisone acetateHydrocortisone acetate ((Hydrocortisonum)Hydrocortisonum)

1111,17,17αα,21-,21-trioxypregnanetrioxypregnane-4--4-diondion-3,20-3,20Natural primary hormone Natural primary hormone more more active active thanthan cortisone. cortisone. Applied in the form of microcrystalline suspension (Suspensio Applied in the form of microcrystalline suspension (Suspensio Hydrocortisoni 25%) for the Hydrocortisoni 25%) for the injectioninjection in the joint cavity of 0,2-1 ml in the joint cavity of 0,2-1 ml atat rheumatoid arthritis, bursitis, etc. tendovaginitah 1 time per week. rheumatoid arthritis, bursitis, etc. tendovaginitah 1 time per week. Available in 5 ml vials. Available in 5 ml vials. For the treatment of inflammatory and allergic skin diseases, eczema, For the treatment of inflammatory and allergic skin diseases, eczema, neurodermatitis - hydrocortisone cream 1% (Unguentum neurodermatitis - hydrocortisone cream 1% (Unguentum Hydrocortisoni 1%). Produced in tubes of 10 g. Hydrocortisoni 1%). Produced in tubes of 10 g. Aerosol "oAerosol "oxycxycort - oxytetracycline g / x 0,3 g, hydrocortisone, 0,1 g. ort - oxytetracycline g / x 0,3 g, hydrocortisone, 0,1 g. Ointment "LoOintment "Loccoida", "Latoida", "Latycycort - 0.1% hydrocortisone 17-N-butyrate, ort - 0.1% hydrocortisone 17-N-butyrate, tubtubee 15 15 g. g.Ointment "PimafuOintment "Pimafuccort - hydrocortisone, neomycin, natamycin. ort - hydrocortisone, neomycin, natamycin.

Hydrocortisone acetate (Hydrocortisoni acetas) (SPhU) Hydrocortisonе acetate

11β,17α-dihydroxy-3,20-dioxypregn-4-en-21-yl аcetate Characteristics. Crystalline powder of white or nearly white.

Practically insoluble in water, slightly soluble in ethanol and methylene chloride. Melts at about 220 º C with decomposition.

O

CH3

CH3COCH2OCOCH3

HO OH

Identification of Identification of Hydrocortisone acetateHydrocortisone acetate 1.1. Using physical-chemical constantUsing physical-chemical constant: : IRIR--spectroscopyspectroscopy, Т, ТLCLC..2.2. Remedy sRemedy solution olution in ethanol within ethanol with H H22SOSO44 gives an intense gives an intense

brownish-red color with a green fluorescence. The resulting brownish-red color with a green fluorescence. The resulting solution is added to water and stirred, the solution solution is added to water and stirred, the solution decolorized, and the fluorescence disappears (a steroid decolorized, and the fluorescence disappears (a steroid cycle).cycle).

3.3. Substance gives a characteristic response to acetyl.Substance gives a characteristic response to acetyl.4.4. Not pharmacopeial reactionNot pharmacopeial reaction: :

a) a) BosBosccotot reaction reaction. Violet fluorescence. . Violet fluorescence. b) On theb) On the o oxyxyaacetyliccetylic group which is acetylated, the reaction: group which is acetylated, the reaction: with triphenyltetrazolium chloride in alkaline medium - red with triphenyltetrazolium chloride in alkaline medium - red color (formazan formation); color (formazan formation); with with Felling and Tollens Felling and Tollens reagentsreagents.. c) Onc) On the the ester group - hydroxamic reaction (dark cherry ester group - hydroxamic reaction (dark cherry color) color) d) Ond) On the keto group in the keto group in the 3-d the 3-d position: position:   the formation ofthe formation of phenylhydrazone (yellow color); phenylhydrazone (yellow color);  with isoniazid in the presence of HCl - yellow in color (this  with isoniazid in the presence of HCl - yellow in color (this reaction prednisolone and dexamethasonereaction prednisolone and dexamethasone don’t give don’t give). ).

Assay of Hydrocortisone acetateAssay of Hydrocortisone acetate

UV spectrophotometry. The content of active ingredient UV spectrophotometry. The content of active ingredient calculated calculated by the by the usingusing of of the specific absorption index. the specific absorption index. Application Apply Apply at the same at the same diseasesdiseases that that hydrocortisone. hydrocortisone. FFor the or the injectioninjection in the joint cavity in the joint cavity atat rheumatoid arthritis, bursitis rheumatoid arthritis, bursitis there is there is used hydrocortisone used hydrocortisone suspension for injection 2,5% suspension for injection 2,5% (Suspensio Hydrocortisoni acetatis 2,5% pro (Suspensio Hydrocortisoni acetatis 2,5% pro injectionibus) in the amp. 2 ml. injectionibus) in the amp. 2 ml. For the treatment of inflammatory and allergic skin For the treatment of inflammatory and allergic skin diseases, eczema, neurodermatitis apply ointment 1%, diseases, eczema, neurodermatitis apply ointment 1%, which is produced in tubes of 5 g ointment Giowhich is produced in tubes of 5 g ointment Gioxysxyson "- on "- together with oxytetracycline together with oxytetracycline hh//ch.ch. In ophthalmic practice using 0,5% eye ointment, which In ophthalmic practice using 0,5% eye ointment, which can not be used for viral and fungal diseases of the eye. can not be used for viral and fungal diseases of the eye.

Prednisolone (Prednisolonum) (SPhU 1.2)

11β,17,21-trihydropregn-1,4-dien-3,20-dionor 1,2-dehydrohydrocortisone

Characteristics. Crystalline powder of white or nearly white color. Hygroscopic. Very slightly soluble in water, soluble in ethanol and methylene chloride. It has polymorphism.

O

CH3

CH3COCH2OH

HO OH

Identification of Identification of PrednisolonePrednisolone1.1. Pharmacopeial reactionPharmacopeial reaction : : IRIR--spectroscopyspectroscopy, Т, ТLCLC..2.2. Remedy solution in ethanol with a concentratedRemedy solution in ethanol with a concentrated HH22SOSO44 forming the forming the

pink colorpink color ( (steroid cyclesteroid cycle).).3.3. With diphenylamine at the present ofWith diphenylamine at the present of HH22SOSO44 andand СН СН33СООН СООН it it

forms green colorforms green color..4.4. Boscot reactionBoscot reaction. . A violetA violet fluorescence fluorescence..5.5. On theOn the o oxyxyaacetyliccetylic group which is acetylated, the reaction: group which is acetylated, the reaction:

with triphenyltetrazolium chloride in alkaline medium - red with triphenyltetrazolium chloride in alkaline medium - red color (formazan formation); color (formazan formation); with with Felling and Tollens Felling and Tollens reagentsreagents..

6.6. OnOn the keto group in the keto group in the 3-d the 3-d position: position:   the formation ofthe formation of phenylhydrazone phenylhydrazone at the present of H at the present of H22SOSO44 (yellow color); (yellow color);  with isoniazid in the presence of HCl – with isoniazid in the presence of HCl – solution doesn’t solution doesn’t change. change.

Assay of Assay of PrednisolonePrednisolone UV spectrophotometry. Method of specific absorption. UV spectrophotometry. Method of specific absorption.

ApplicationApplication It was the first received rst received by Hercoh by Hercoh in 1955 in 1955 The introduction of the double bond in The introduction of the double bond in the first the first position position inin molecule of molecule of

hydrocortisone enhances the action hydrocortisone enhances the action byby 3-5 3-5 times times. . It iIt is used for rheumatism, infectious nonspecific arthritis, asthma, s used for rheumatism, infectious nonspecific arthritis, asthma, eczema, neurodermatitis, etc. eczema, neurodermatitis, etc.

They They are produced in are produced in ttable. able. By By 5 mg and 5 mg and solution for injectionsolution for injection. 30 mg . 30 mg amp. 1amp. 1ml number 3. ml number 3.

For the treatment of inflammatory and allergic skin diseases, eczema, For the treatment of inflammatory and allergic skin diseases, eczema, neurodermatitis applneurodermatitis applyingying ointment 0.5%, which is produced in tubes ointment 0.5%, which is produced in tubes by by 10 and10 and 2020 g. g.

Ointment PredniOintment Predniccarb-Darniarb-Darnicca - prednisolone 0.5%, urea 10%, Trilon B a - prednisolone 0.5%, urea 10%, Trilon B 1%. 1%.

Ointment "dermozolon" contains 0.5% prednisolone, and 3% Ointment "dermozolon" contains 0.5% prednisolone, and 3% ccliolioquiquinononene. .

Ointment "aurobin" - contains 20 grams of 0,04 g Ointment "aurobin" - contains 20 grams of 0,04 g pprednisolonerednisolone and 0,4 g and 0,4 g lidocaine. In ophthalmic practice using 0.5% eye drops lidocaine. In ophthalmic practice using 0.5% eye drops which which containcontainss prednisolone acetate prednisolone acetate solution solution. .

Prednisolone sodium phosphate (Prednisolonі natrii phosphas) (SPhU)

11β,17α,-dihydroxi-3,20-dioxopregna- 1,4-dien-21-yl disodium phosphate

Characteristics. Crystalline powder of white or nearly white. Hygroscopic. Easily soluble in water, very slightly soluble in ethanol.

O

CH3

CH3C

HO OH

CH2

O

O P

O

ONa

ONa

Identification PrednisoloneIdentification Prednisolone sodium phosphatesodium phosphatePharmacopeial reactionPharmacopeial reaction : : 1.1. UV- and IRUV- and IR--spectroscopy, TLCspectroscopy, TLC..2.2. Solution of the drug in ethanol with the conc. HSolution of the drug in ethanol with the conc. H22SOSO44 forms a red forms a red

color (a steroid cycle), in UV light - a red-brown fluorescence. color (a steroid cycle), in UV light - a red-brown fluorescence. After adding water, the solution becomes colorless, and in UV light After adding water, the solution becomes colorless, and in UV light - yellow-green fluorescence.- yellow-green fluorescence.

3.3. OnOn the keto group in the keto group in the 3-d the 3-d position: position:   the formation ofthe formation of phenylhydrazone phenylhydrazone at the present of H at the present of H22SOSO44 (yellow (yellow color); color);

4.4. After mineralization After mineralization – – reaction A on Sodiumreaction A on Sodium ( (with potassium with potassium hydroxoantimonate hydroxoantimonate – – white sedimentwhite sediment) ) and reaction B on phosphateand reaction B on phosphate ((with with molibdenovanadium reagent molibdenovanadium reagent – – yrllow coloryrllow color).).

ASSAYASSAY:: UV spectrophotometry. Method of specific absorptionUV spectrophotometry. Method of specific absorption..ApplicationApplication:: solution for injectionsolution for injection 30 30 mg/mlmg/ml ampamp. 1 . 1 mlml, № 3 (1 , № 3 (1 mlml

solution contains solution contains 40,32 40,32 mg pmg prednisolone sodium phosphate on the on the account account of of 30 30 mg [rednisolonemg [rednisolone..

Hydro corticosteroid with Hydro corticosteroid with fluorine atomfluorine atom

Corticosteroids - the first drugs which proved the possibility of Corticosteroids - the first drugs which proved the possibility of increasing the efficiency increasing the efficiency according toaccording to the replacement of nitrogen the replacement of nitrogen to to fluorine fluorine atoms in their molecules. atoms in their molecules. FFluoriluorinnee a atom is minimal andtom is minimal and more relative more relative to the nitrogen atom (to the nitrogen atom (VVan der Waals radian der Waals radiusus for these for these atoms are respectively 1.35 and 2.1 angstroms) and atoms are respectively 1.35 and 2.1 angstroms) and have have moremore strongerstronger electron acceptorelectron acceptor and lipophilic properties. Therefore, the and lipophilic properties. Therefore, the introduction of fluorite atoms does not cause significant spatial introduction of fluorite atoms does not cause significant spatial complications that would change the conformation of molecules andcomplications that would change the conformation of molecules and also doesn’talso doesn’t interfere on interfere on their interaction with their interaction with the the active sites and active sites and receptor receptor of of substrates. substrates.

Important the energy of the C-F is greater than the energy of the C-Important the energy of the C-F is greater than the energy of the C-H (470 and 410 kJ / mol, respectively). This leads to thermal and H (470 and 410 kJ / mol, respectively). This leads to thermal and chemical stability chemical stability of the of the flfluorine derivativesuorine derivatives, in many cases, , in many cases, it it causes causes slowingslowing of the of the metabolism and leadmetabolism and leadss to the to the unchangedunchanged extractionextraction from the body. This contributes to their high from the body. This contributes to their high lipophilicity, which increases the solubility of fllipophilicity, which increases the solubility of fluorine derivativesuorine derivatives in lipids and facilitate their transport in lipids and facilitate their transport toto biological systems. biological systems.

The iThe introduction ntroduction of of fluorite atom in a molecule fluorite atom in a molecule increases the activity of prednisolone, enhances the increases the activity of prednisolone, enhances the anti-inflammatory and antiallergic actions when anti-inflammatory and antiallergic actions when administered orally (dexamethasone, triamcinolone). administered orally (dexamethasone, triamcinolone).

The iThe introduction ntroduction inin corticosteroids molecules of two corticosteroids molecules of two fluorifluorinne atoms reduces the absorptione atoms reduces the absorption and such and such compounds are used as local anti-inflammatory, compounds are used as local anti-inflammatory, antiallergic and funds against itch antiallergic and funds against itch ((floucynoloneacetonidefloucynoloneacetonide, , flumetasonflumetasonpivalatpivalatee, , hhalomealomettaasson) on)

As a local anti-inflammatory, antiallergic means and As a local anti-inflammatory, antiallergic means and means means that are that are used against itching and prednisolone used against itching and prednisolone derivatives with one atom of fluoriderivatives with one atom of fluorinne, which are e, which are difficult absorbed (difficult absorbed (triamcinoloneacettriamcinoloneacetonide, betametazon,onide, betametazon, fluoocortolone, ingacortfluoocortolone, ingacort) )

Dexamethasone ( Dexamethasonum)Dexona, Dexasone

9α-Fluoro-16α-methylprednisolone or 11,17α,21-trioxy-16α-methyl- 9α-fluoropregnadien-1,4-dione-3,20

It is synthesized from dihydropregnenolone, which is one of the intermediate transformation products of solasodine in progesterone.

O

CH3

CH3COCH2OH

HO OH

CH3

F

Identification of DIdentification of Dexamethasoneexamethasone 1.1. IRIR--, UV-spectroscopy, UV-spectroscopy, Т, ТLC.LC.2.2. Remedy sRemedy solution olution in the concentratedin the concentrated H H22SOSO44 stays coloeless and at stays coloeless and at

the action of UV lights the the action of UV lights the fluorescencefluorescence is not observed is not observed. . 3.3. On theOn the o oxyxyaacetyliccetylic group, the reaction: group, the reaction:

with triphenyltetrazolium chloride with triphenyltetrazolium chloride and KOH solutionand KOH solution - red color - red color (formazan formation); (formazan formation); with with Felling and Tollens Felling and Tollens reagentsreagents..

4.4. OnOn the keto group in the keto group in the 3-d the 3-d position: position:   at the heating of the remedy in the alcohol solution withat the heating of the remedy in the alcohol solution with phenylhydrazphenylhydraziinene hydrochloride and sulfuric acid solution during 5 hydrochloride and sulfuric acid solution during 5 minutes there is apparentminutes there is apparent yellow color; yellow color;  with isoniazid in the presence of HCl –  with isoniazid in the presence of HCl – solution is staying colorlesssolution is staying colorless. .

5.5. Organically connected FluorOrganically connected Fluorineine is converted into fluoride ions is converted into fluoride ions by the by the mineralization , mineralization , fluoride ions have the ability decolorizefluoride ions have the ability decolorize the the alizarin alizarin red lacquer red lacquer ((alizarinatalizarinate e zirconium). Alizarin red lacquer is a complex zirconium). Alizarin red lacquer is a complex compound of zirconium with alizarin. compound of zirconium with alizarin. AtAt interacting with the F interacting with the F-- ions ions Alizarin red Alizarin red is decolorized is decolorized to a pale yellow color, due to formation to a pale yellow color, due to formation of a stable colorlessof a stable colorlessinging complex Na complex Na22 [ZrF [ZrF66].].

ASSAY of DASSAY of Dexamethasoneexamethasone UV-spectroscopyUV-spectroscopy Application

It has a strong anti-inflammatory and antiallergic actions. It is good tolerated, does not delay water in the body. Its efficiency the the 0.5 mg of dexamethasone corresponds to 3.5 mg of prednisolone, 15 mg of hydrocortisone, 17.5 mg of cortisone. Using inside on 2-3 mg, the treatment is stopped step by step, as with other glucocorticosteroids. Producing tablets on 0.5 mg, Eye drops 0.1%, solution for injection 0,4% in Amp. 1 and 2 ml № 5.

Triamcinolone (Triamcinolonum)Polcortolone, Ketalong

9α-Fluoro-16α- methylprednisolone оr 11,16α 17α,21- trioxy- 9α- fluoropregnadien -1,4-dione-3,20

Applying in the same cases that dexamethasone. IT is tolerated better than other glucocorticoids. Using inside of 4-8-16 mg on a day. Producing tablets on 4 mg; 0.1% ointment (ftorocort).

O

CH3

CH3COCH2OH

HO OH

OH

F

Triamcinolone acetonide (Triamcinoloni acetonidum) Kenaloge 40

Triamcinolone 16α,17α-acetonide Producing: suspension for injection 40 mg/ml in amp. 1 ml №5; 0,1

% ointment (“Fokort-Darnycya); “TrimistineDarnycya” ointment contains 0,025 % triamcinolone acetonide , 0,5 % miramistine.

F

CH3

CH3

O

HOCOCH2OH

O C

O

CH3

CH3

Fluocinoline acetonide (Fluocinoloni acetonidum) Synaflan

6α,9α-difluoro-16α-oxyprednisolone- 16α,17α-acetonide оr 6α-fluortriamcinolonacetonide

Applied externally as 0,025% ointment of Sinaflan or Flucynar, tube on 15g. Ointment “Flucynar N "- Fluocinoline acetonide 0,25 mg/g and

neomycin sulfate 5 mg/g, tube on 15g.

O

CH3

CH3COCH2OH

HO O

O

F

F

CCH3

CH3

Flumethasone pivalate (Flumethasoni pivalas)

6α,9α-difluoro-16α- methylprednisolone - 21-trimethylacetate (pivalate) оr 6α-fluorodexametasone-21-pivalate

It is included in the ointment of "Lorinden A" (with salicylic acid) and Lorinden C "(with clioquinol).

O

CH3

CH3COCH2OCOC(CH3)3

HO OH

CH3

F

F

F

CH3

CH3

O

HOCOCH2OH

OH

CH3Cl

F

CH3COCH2OCOC2H5

OCOC2H5

O

CH3

HO

CH3

F

Cl

CH3

CH3

O

HOCOCH2OCOC2H5

OCOC2H5

CH3

Halomethasone Betamethasone dipropionate (SPhU)

Beclomethasone dippropionate

Halomethasone Halomethasone ((SicortenSicorten) ) is used as is used as 0,5% 0,5% ointmentointment..Betamethasone dipropionateBetamethasone dipropionate is used as suspension for is used as suspension for injection in amp. injection in amp. 1 1 mlml №1 №1 and and №5 (№5 (DiprospanDiprospan, , FlosteronFlosteron, , CelestonCeleston) ) andand tabltabl. 0,5 . 0,5 mgmg №30 ( №30 (CelestonCeleston).).Betamethasone is in the following complex ointmentBetamethasone is in the following complex ointment ““CelestodermCelestoderm-В” (0,1%), “-В” (0,1%), “Celestoderm with Celestoderm with garamycingaramycin” ” and and ““CelestodermCelestoderm” (” (with gentamicinwith gentamicin), “), “BetasalikBetasalik” ” andand ““DiprosalikDiprosalik” (” (with salicylic acidwith salicylic acid), “Т), “Тridermriderm” (” (with with hentamicin sulfate and hentamicin sulfate and clotrimazolclotrimazol).).Betamethasone dipropionate is used as aerosol for Betamethasone dipropionate is used as aerosol for inhalationinhalation at asthma at asthma ((AldecynAldecyn, , BeconazolBeconazol, , BeclazonBeclazon). ). OintmentOintment “ “CandermCanderm” (” (with gentamicin with gentamicin sulfate and sulfate and clotrimazoleclotrimazole).).

Progestin hormonesProgestin hormones Pregnancy hormones Pregnancy hormones are are called progestins (Latin for "gestatio" called progestins (Latin for "gestatio" - pregnancy). These hormones are produced in - pregnancy). These hormones are produced in thethe yellow yellow body. During pregnancy woman's body begins to operate a body. During pregnancy woman's body begins to operate a new endocrine gland - the corpus luteum. new endocrine gland - the corpus luteum. The cThe corpus luteum orpus luteum is is formformeded in ovar in ovary at the place of the follicle y at the place of the follicle rupture, rupture, if the if the pragnancy starts the cpragnancy starts the corpus luteumorpus luteum is is tiltilll the end of it, but if the end of it, but if the pragnancy does not start the pragnancy does not start , it does not develop. , it does not develop. Corpus luteum secretes progesterone hormone, which helps to Corpus luteum secretes progesterone hormone, which helps to create conditions for the implantation of a fertilized egg and create conditions for the implantation of a fertilized egg and the correct development of pregnancy (lowers the excitability the correct development of pregnancy (lowers the excitability of the muscles of the uterus).of the muscles of the uterus).Progestins are needed in woman's body for pregnancy Progestins are needed in woman's body for pregnancy maintenance.maintenance.As drugs progestins As drugs progestins are are used used at theat the case casess of insufficient of insufficient of the of the corpus luteum function corpus luteum function atat the prevention and treatment of the prevention and treatment of threatened abortivethreatened abortive and interrupt them and interrupt them atat amenorrhea, amenorrhea, infertility, bleeding associated with ovarian dysfunction, etc. infertility, bleeding associated with ovarian dysfunction, etc.

Chemical Chemical featurefeature of of ProgestinProgestin

1.1. Derivatives of Derivatives of pregnanepregnane

2.2. OxogroupOxogroup (=О) (=О) oror tribond in the 20-th tribond in the 20-th positionposition

3.3. Double bond in the 4-Double bond in the 4-th positionth position;;

4.4. Oxogroup Oxogroup (=О) (=О) in the in the 3-d position3-d position

Progesterone (Progesteronum)

Pregnen – 4 - dion-3,20 Characters. White crystalline powder. Practically insoluble in

water, soluble in ethanol and ether, readily soluble in chloroform.      Extracted from solasodine or cholesterol.

O

CH3

CH3COCH3

Identifiation of ProgesteroneIdentifiation of Progesterone 1. IR- and UV-spectroscopy, TLC.2. A solution of progesterone in H2SO4 conc. after

water addition becomes yellow with a green fluorescence. After adding chloroform color disappears (a steroid cycle).

3. At the heating of the alcohol solution of progesterone with m-dinitrobenzene and NaOH appearance pink color that transfers to red-brown.

4. Fro the identification to determine the composition temperature 2,4-dinitrophenylhydrazone, obtaining at the quatitation detarmination (keto group the 3-d position).

5. Boscot reaction – a blue fluorescencefluorescence.6. At the heating progesterone with NH2OH*HCl and CH3COONa settled

down oxime’s sediment which is identified by the melting point.7. The double bond id identified by the interaction of the remedy solution in

СН3СООН with bromine water or with KMnO4 (colorlessing).

ASSAY of ASSAY of ProgesteroneProgesterone Gravimetry by the interaction products with 2,4-

dinitrophenylhydrazine or spectrophotometry in ethanol solution at λ = 241 nm.

Storage and applicationIn the tightly stoppered container, protecting from light.

Progestin drug.     Injecting by 5-25 mg intravenous. They don’t apply at disorders of liver function, hepatitis, cancer of the breast and genital organs, tendency to thrombosis.    Applied in the form of oil solutions for injection 1% and 2,5% 1,0 ml № 5; caps "Utrogestan" 100 mg № 30 and 1% gel, "Progestogel" tube on 80 g.

Pregnine (Praegninum)Ethisterone*

Pregnen-4-іn-20-оl-17β-оn-3 оr 17α-ethynyltestosterone Characters. White or almost yellowish crystalline powder, odorless.

Practically insoluble in water, very slightly soluble in ethanol and ether, slightly soluble in chloroform.

O

CH3

CH3C CH

OH

Identification of Identification of Pregnine

1. IR-spectroscopy, TLC.2. At the dissolving of pregnine in H2SO4 conc. and

the addition of water appears crimson color with a green fluorescence. After adding chloroform and mixing of the lower layer is painted in orange, the top - almost colorless (steroid cycle).

3. Determine the oxime’s melting point (keto group in position 3).

4. At the Boscot reaction there is a green-purple color of the solution, which after 60 minutes changes to red, and after the addition of CH3COOH observed an intense yellow-pink fluorescence.

ASSAY ofASSAY of Pregnine 1. Alkalimetry by the substitution. To a solution of the substance in

tetrahydrofuran, to add a solution of AgNO3 HNO3 is liberated it is titrated by a NaOH solution . Indicator is a bromocresol green or potentiometrically, Ем= М :

2. Spectrophotometry in ethanol solution at λ = 241 nm in comparison with the standard.

Storage and applicationIn the tightly stoppered container, protecting from light.

Progestin agent. 5-6 times less active than progesterone, but remains the activity at the using sublingual tablets. Producing table. 10 mg.

Oxyprogesterone capronate Norethisterone

Allylestrenol Norgestrel

CH3

CH3

O

COCH3

OCO(CH2)4CH3

CH3

O

C

OH

CH

CH2C

OH

O

CHH3CCH3

CH2CH=CH2

OH

Thanx for attentionThanx for attention!!