Lecture № 10

1. Lecture№10 Current state and development of chemistry as a corticosteroid drug. Cortical layer of the adrenal hormones and some of their semisynthetic analogues. Relationship between structure and biological activity. Gestogen hormones and their synthetic analogues. prepared Kozachok S.S.

2. The hormones of the adrenal of cortical layer (corticosteroids) and sex hormones belong to the steroid hormones. Sex hormones can be divided into male sex hormones (androgens), female sex hormones (estrogen) and luteal hormones (progestogens, or lutoid hormones). The structural base of the steroid hormones is a cyclopentaneperhydrophenanthrene hydrocarbon chain. The general formula of steroid hormones are:

3. The chemical structure of the steroid hormones

4. Steroid hormones • Methyl 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 indicatedby 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.

5. Steroid hormones and their analogues are an optically-active substances, most of them are dexter isomers (methylandrostendiol - levogyrate). AND for the identification and confirmation of purity recommends to determine 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. • General reaction for all steroid hormones and their synthetic analogues is the reaction with concentrated H2SO4. When to dissolve in it and to heat the substances give appearance a specific color, sometimes fluorescence, with a further addition of water, chloroform, the color changes, there is a specific fluorescence. • Steroid hormones possessi keto group in 3-d position, they give the addition reaction Steroid hormones possessing keto group in position 3, given the addition reaction with elimination of water with hydroxylamine, phenylhydrazine, 2,4-dinitrophenylhydrazine, isoniazid and elimination of water – observing the precipitation with a characteristic melting point, or there is a characteristic color (yellow, orange) : with hydroxylamine, phenylhydrazine, 2,4-dinitrophenylhydrazine, isoniazid - observing precipitation with a characteristic melting point, or there is a characteristic color (yellow, orange):

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

7. For the hormone’s identification which have hydroxyl groups in 3 or 17 position, often there is using the reaction of the esters forming (acetate, benzoate), with a characteristic melting point: • For the hormone’s identification and their synthetic analogues that are used as esters (acetate, propanoate), apply the hydroxamic reaction:

8. For theidentification and quantitative analysis of steroid hormones and their analogues are widely used UV spectroscopy of alcohol solutions. The identification is carried out according to the location of the maximus and minimum on a particular part of the spectrum, comparing with the spectrum of standard sample, according to the relative between the optical densities in the various absorption maximus and calculatethe spesific absorbtion valur. The content of the active substance is determined by the specific absorption rate or standard solution. • The substance’s indentification is confirmed by the IR-spectropy, which is compared with the pharmacopeial or standard semples. • For the identifying and determining the presence of impurities there is widely used method of TLC.

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

10. The initial products for the synthesis of corticosteroids can be natural substances of steroid structure (diosgenin, stigmasterol) and cholesterol, which is considered a precursor of corticosteroids in the body. • Cortisone is isolated in 1936 from the adrenal cortex by Kendal and Vintershteyner inUSA and Reichstein in Switzerland. • The problem of its synthesis in the fact that in nature there are no available steroidal compounds containing keto group in 11 position. Synthesis such a group can be by biochemical oxidation (using fungi, yeasts, actinomycetes, and various bacteria). This process allows us to introduce the hydroxyl in 9, 11, 14, 15, 16, 17, 21positions, in addition to the α-and β-configuration. • The total synthesis of cortisone was spent in 1951 by Woodward (USA). It includes about 30 stages that’s why have only theoretical interest.In 1956, Suvorov M.M. (VNDHFI) used solasodin - aglycone of glucoalkaloid of avian nightshade (Solanum aviculare) as the initial product for the industrial production of cortisone. Scheme of its synthesis consists of several stages.

11. Corticosteroids have a very important side-effects and thus their use as anti-inflammatory, desensitizing and anti-allergic means. In addition, they have antishock and immunosuppressive activity and therefore they are used at the transplantation of organs and tissues to suppress the rejection reaction, as well as in various autoimmune diseases. • Side effects of corticosteroids: a delay of sodium and water in the body with the appearance of an edema, increased allocation of potassium ions, increased blood pressure, hyperglycemia, osteoporosis, etc. • At the long-term treatment they can suppress thecortical adrenal function, so the treatment should not be suddenly stoped cause it may be the aggravation of the disease or condition of acute adrenal insufficiency. • Corticosteroids in ointments and drops can not be usedat the viral diseases of the eye (can form the ulcers). All dosage forms of corticosteroids stored as potent tools in a dark place. • All dosage forms of corticosteroids stored as potent tools in a dark place.

12. Natural corticosteroids Hydrocortisone Cortisone Desoxycorticosterone (Dox),

13. Chemical signs of corticosteroids • Derivatives of pregnane • In the 17-th position there is oxyacetyl –СОСН2ОН (reducing properties) • In the 17 αoxy(-ОН) group (forglucocorticosteroids) • In the 11 oxo (=О) or оxy (-ОН) group • In the 4 – double bond, in the separate remedy there is also in the 1-st position. • In the 3 position - oxo (=О) group.

14. Characteristics and general identification reaction of the corticosteroids • Adrenal cortex hormones and their synthetic analogs - a white crystalline substance, which sometimes have a yellowish or cream color, and odorless. • They are practicaly insoluble in water, soluble in the most organic solvents. Desoxycorticosterone acetate and cortisone acetate readily soluble in chloroform. • Corticosteroids and their analogues are dextergyrate isomers. • The steroid structure is confirmed by: а) Boscot reaction: corticosteroids are dissolved in the mixture of 88% Н3РО4and concentrated СН3СООН, the solution heated 2 minutes at 100ºС and leave for 1 hour at room temperature, and then it is diluted byСН3СООН. Under the UV lamp (Bah lamp) fluorescence occurs. b) reaction with concentrated sulfuric acid.

15. α-Ketonic group – reducing propertiesі (oxidizing to carboxilic group. а) Felling reaction (copper-tartrate reagent). At the heating of the mixture of the alcohol substance’s solution and Felling reagent on the waterbathe Cu2O is settled down. b) Except Felling reagent as a oxidizing agent Tollens reagent can be used (ammonia solution of silver nitrate) – reaction of“silver mirror”, Zonnenshten reagent - Н3Р04 • 12Мо03 • 2Н20), Iron (III) salt.

16. 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).

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

18. (Desoxycorticosteroni acetas) Desoxycorticosterone acetate*, DOXA Pregnane-4-оl-21-dione-3,20-21-acetate or 21-acetoxypregnane-4-dion-3,20 Desoxycorticosterone is anatural mineral-corticosteroid, does not contain -OH group in 11-th position. Extracted from cholesterol.

19. Identification of DOXA • On the steroid system – Boscot reaction. A violet color wiyh a fluorescence. • With concentratedH2SO4 – cherry color with greenish-brown fluorescence. After adding chloroform and shaking, the lower layer is painted in yellow top - green (a steroid cycle). • On the oxyacetylic group, which is acetylated: а) with Felling reagent – forming the red sedimentCu2O b) With Tollens reagent – metallic silver settled down • On the ester group – hydroxamic reaction (red-brown color) • On the keto-group in 3-th position – the oxym formation, phenylhydrazine, 2,4-dinitrophenylhydrazone. • Acetylic group after hydrolysis КОН is identified according to the formation ethylacetate (specific smell):

21. Assay of DOXA UV-spectrometry. Photocolorimetric determination of the 0,5% oil solution for injection after heating with concentrated H3PO4. Application           For the treatment of Addison's disease, gipocortocyzm, myasthenia gravis, asthenia, etc.           Injected intramuscularly of the oily solution of 5 mg 3 times a week, or in the severe cases of 10 mg daily. Produced - amp. 1,0 - 0,5% oil solution, tab. 5 mg sublingual application.

22. Glucocorticoid • They 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, whichactive in smaller doses and have less side effects. • Divided into three groups: 1. Glucocorticosteroids which do not contain halogen atoms in molecules; 2. Fluorinecompounds; 3. Compounds with the atoms of chlorine and fluorine or only with chlorine atoms in the molecules.

23. (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.

24. Identification of Cortisone acetate • Boscot reaction. A violet fluorescence. • Basic hydrolysis (heating with КОН solution). At the addition of the concentrated H2SO4appearance the fruit smell of ethyl acetate. • On the oxyacetylic group, which is acetylated here: а) With Felling reagent – formation red sedimentCu2O b) With Tollns reagent – metallic silver settled down 5. On ester group – hydroxamic reaction (darkredcolor) • On keto group in 3-th position – formation of phenylhydrazone (bright-yellow color), 2,4-dinitrophenylhydrazone (melting temperature 240ºС). • Remedy solution in the concentratedH2SO4in UV-ligt gives intensive yellow fluorescence.

25. Assay of of Cortisone acetate UV- spectrophotometryin alcohol solution atλ=238 nmin the comparing with the standard solution. Application • When Addison's disease, rheumatism, rheumatoid arthritis, asthma, and leukemia, mononucleosis, in the dermatology - at neurodermatitis, eczema and other allergic diseases. • Taking inside firstlyby 0,1-0,2 g, and then by 25 mg per day in pill or injected intramuscularly at 25-50 mg as a suspension. • Produced: Table. 25-50 mg; vials on 10 ml suspension (1 ml - 25 mg cortisone acetate).

26. Hydrocortisone acetate (Hydrocortisonum) • 11,17α,21-trioxypregnane-4-dion-3,20 • Natural primary hormone more active than cortisone. • Applied in the form of microcrystalline suspension (Suspensio Hydrocortisoni 25%) for the injection in the joint cavity of 0,2-1 ml at rheumatoid arthritis, bursitis, etc. tendovaginitah 1 time per week. Available in 5 ml vials. • For the treatment of inflammatory and allergic skin diseases, eczema, neurodermatitis - hydrocortisone cream 1% (Unguentum Hydrocortisoni 1%). Produced in tubes of 10 g. Aerosol "oxycort - oxytetracycline g / x 0,3 g, hydrocortisone, 0,1 g. Ointment "Locoida", "Latycort - 0.1% hydrocortisone 17-N-butyrate, tube 15 g. • Ointment "Pimafucort - hydrocortisone, neomycin, natamycin.

28. Hydrocortisone acetate (Hydrocortisoniacetas) (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.

29. Identification of Hydrocortisone acetate • Using physical-chemical constant: IR-spectroscopy, ТLC. • Remedy solution in ethanol with H2SO4 gives an intense brownish-red color with a green fluorescence. The resulting solution is added to water and stirred, the solution decolorized, and the fluorescence disappears (a steroid cycle). • Substance gives a characteristic response to acetyl. • Not pharmacopeial reaction: a) Boscot reaction. Violet fluorescence. b) On the oxyacetylic group which is acetylated, the reaction: with triphenyltetrazolium chloride in alkaline medium - red color (formazan formation); with Felling and Tollens reagents.c) Onthe ester group - hydroxamic reaction (dark cherry color) d) On the keto group in the 3-d position: the formation of phenylhydrazone (yellow color);  with isoniazid in the presence of HCl - yellow in color (this reaction prednisolone and dexamethasone don’t give).

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

31. Prednisolone (Prednisolonum)(SPhU 1.2) 11β,17,21-trihydropregn-1,4-dien-3,20-dion or 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.

32. Identification of Prednisolone • Pharmacopeial reaction : IR-spectroscopy, ТLC. • Remedy solution in ethanol with a concentratedH2SO4forming the pink color (steroid cycle). • With diphenylamine at the present ofH2SO4and СН3СООН it forms green color. • Boscot reaction. A violet fluorescence. • On the oxyacetylic group which is acetylated, the reaction: with triphenyltetrazolium chloride in alkaline medium - red color (formazan formation); with Felling and Tollens reagents. • On the keto group in the 3-d position: the formation of phenylhydrazone at the present of H2SO4 (yellow color);  with isoniazid in the presence of HCl – solution doesn’t change.

33. Assay of Prednisolone UV spectrophotometry. Method of specific absorption. Application It was the first received by Hercoh in 1955 The introduction of the double bond in the first position in molecule of hydrocortisone enhances the action by 3-5 times. It is used for rheumatism, infectious nonspecific arthritis, asthma, eczema, neurodermatitis, etc. They are produced in table. By 5 mg and solution for injection. 30 mg amp. 1ml number 3. For the treatment of inflammatory and allergic skin diseases, eczema, neurodermatitis applying ointment 0.5%, which is produced in tubes by 10 and20 g. Ointment Prednicarb-Darnica - prednisolone 0.5%, urea 10%, Trilon B 1%. Ointment "dermozolon" contains 0.5% prednisolone, and 3% clioquinone. Ointment "aurobin" - contains 20 grams of 0,04 g prednisolone and 0,4 g lidocaine. In ophthalmic practice using 0.5% eye drops which contains prednisolone acetate solution.

34. Prednisolonesodium phosphate (Prednisolonі natrii phosphas)(SPhU) 11β,17α,-dihydroxi-3,20-dioxopregna- 1,4-dien-21-yldisodium phosphate Characteristics. Crystalline powder of white or nearly white. Hygroscopic. Easily soluble in water, very slightly soluble in ethanol.

35. Identification Prednisolonesodium phosphate Pharmacopeial reaction : • UV- and IR-spectroscopy, TLC. • Solution of the drug in ethanol with the conc. H2SO4 forms a red color (a steroid cycle), in UV light - a red-brown fluorescence. After adding water, the solution becomes colorless, and in UV light - yellow-green fluorescence. • On the keto group in the 3-d position: the formation of phenylhydrazone at the present of H2SO4 (yellow color); • After mineralization – reaction A on Sodium (with potassium hydroxoantimonate – white sediment) and reaction B on phosphate (with molibdenovanadium reagent – yrllow color). ASSAY:UV spectrophotometry. Method of specific absorption. Application:solution for injection 30 mg/mlamp. 1 ml, № 3 (1 mlsolution contains 40,32 mg prednisolonesodium phosphateon the account of 30 mg [rednisolone.

37. Hydro corticosteroid with fluorine atom • Corticosteroids - the first drugs which proved the possibility of increasing the efficiency according to the replacement of nitrogen to fluorine atoms in their molecules. Fluorine atom is minimal and more relative to the nitrogen atom (Van der Waals radius for these atoms are respectively 1.35 and 2.1 angstroms) and have more strongerelectron acceptor and lipophilic properties. Therefore, the introduction of fluorite atoms does not cause significant spatial complications that would change the conformation of molecules and also doesn’tinterfere on their interaction with the active sites and receptor of substrates. • 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 chemical stability of the fluorine derivatives, in many cases, it causes slowing of the metabolism and leadsto the unchanged extraction from the body. This contributes to their high lipophilicity, which increases the solubility of fluorine derivatives in lipids and facilitate their transport to biological systems.

38. The introduction of fluorite atom in a molecule increases the activity of prednisolone, enhances the anti-inflammatory and antiallergic actions when administered orally (dexamethasone, triamcinolone). • The introduction in corticosteroids molecules of twofluorine atoms reduces the absorptionand such compounds are used as local anti-inflammatory, antiallergic and funds against itch (floucynoloneacetonide, flumetasonpivalate, halometason) • As a local anti-inflammatory, antiallergic means and means that are used against itching and prednisolone derivatives with one atom of fluorine, which are difficult absorbed (triamcinoloneacetonide, betametazon,fluoocortolone, ingacort)

39. 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.

40. Identification of Dexamethasone • IR-, UV-spectroscopy, ТLC. • Remedy solution in the concentrated H2SO4stays coloeless and at the action of UV lights the fluorescence is not observed. • On the oxyacetylic group, the reaction: with triphenyltetrazolium chloride and KOH solution - red color (formazan formation); with Felling and Tollens reagents. • On the keto group in the 3-d position: at the heating of the remedy in the alcohol solution with phenylhydrazine hydrochloride and sulfuric acid solution during 5 minutes there is apparent yellow color;  with isoniazid in the presence of HCl – solution is staying colorless. • Organically connected Fluorine is converted into fluoride ions by the mineralization , fluoride ions have the ability decolorizethe alizarin red lacquer (alizarinate zirconium). Alizarin red lacquer is a complex compound of zirconium with alizarin. At interacting with the F-ions Alizarin red is decolorized to a pale yellow color, due to formation of a stable colorlessing complex Na2 [ZrF6].

41. ASSAY of Dexamethasone UV-spectroscopy Application • It has a strong anti-inflammatory and antiallergic actions. It is good tolerated, does not delaywater in the body. Its efficiency 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.

43. 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).

44. Triamcinoloneacetonide(Triamcinoloni acetonidum) Kenaloge 40 Triamcinolone16α,17α-acetonide Producing: suspensionfor injection 40 mg/mlin amp. 1 ml №5; 0,1 %ointment (“Fokort-Darnycya);“TrimistineDarnycya” ointmentcontains 0,025 % triamcinoloneacetonide, 0,5 % miramistine.

46. Fluocinolineacetonide (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 "- Fluocinolineacetonide0,25 mg/gandneomycin sulfate 5 mg/g, tube on 15g.

47. 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).

49. HalomethasoneBetamethasone dipropionate (SPhU) Beclomethasonedippropionate

50. Halomethasone (Sicorten) is used as 0,5% ointment. • Betamethasone dipropionateis used as suspension for injection in amp. 1 ml №1 and №5 (Diprospan, Flosteron, Celeston) andtabl. 0,5 mg №30 (Celeston). • Betamethasone is in the following complex ointment “Celestoderm-В” (0,1%), “Celestoderm with garamycin” and “Celestoderm” (with gentamicin), “Betasalik” and “Diprosalik” (with salicylic acid), “Тriderm” (with hentamicin sulfate and clotrimazol). • Betamethasone dipropionate is used as aerosol for inhalationat asthma (Aldecyn, Beconazol, Beclazon). Ointment “Canderm” (with gentamicin sulfate and clotrimazole).