LECTURE № 13 Theme: Derivatives of pyridine as drugs. Derivatives of isonicotinic acid as antitubercular agents Associate prof. Mosula L.M.

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1 LECTURE № 13 Theme: Derivatives of pyridine as drugs. Derivatives of isonicotinic acid as antitubercular agents Associate prof. Mosula L.M.

2 The plan 1. Derivatives of pyridine-3-carboxylic (nicotinic) acid as drugs: Nikethamide, Nikethamide injection, Nicodine. 2. Derivatives of pyridine-4-carboxylic (isonicotinic) acid as antitubercular agents: Isoniazid, Phthivazid, Flurenizidum.

3 Hexatomic heterocycles as drugs
To hexatomic heterocycles with one heteroatom of Nitrogene belong pyridine: Completely hydrogenated pyridine (saturated heterocycle) names piperidine:

4 Structure and chemical properties of pyridine
In molecule of pyridine the atom of Nitrogene is in a condition of sp2-hybridization and gives in an aromatic sextet one p-electron. Not divided pair electrons on sp2-hybrid orbital causes properties pyridine as bases. Atom of Nitrogene with such electronic structure names pyridinic. As result big electronegativity in comparison with atom Carbon pyridinic atom of Nitrogene reduces electronic density on atoms Carbon of aromatic series. Therefore pyridine and other heterocyclic compounds with pyridinic atom of Nitrogene are electron-deficient. Its is much more difficult , rather than benzene, reacts electrophilic substitution, and electrophile takes (occupies) -position concerning atom of Nitrogene. It is oxidised more difficultly, but is easier hydrogenated.

5 pyridine β-nitropyridine
A low reactionary ability of pyridine is caused also by that in strongly acid mediums, in which occurs electrophilic substitution, pyridine exists in the proton form in kind cation pyridinium, that essentially complicates electrophilic attack.

6 Pyridine is colourless liquid (the temperature of boiling 115 °С), toxic, with a characteristic smell, mixes up with water and organic solvents. In small amounts of pyridine and its homologues are in coal pitch. Has strong bactericidal action, however because of toxicity in medicine it is not applied. Water solutions of pyridine paints litmus in dark blue colour (the basic properties); at action of acids crystal salts of pyridine are formed : pyridine base pyridine salt of pyridine

7 Homologues of pyridine easily are oxidised with formation of corresponding pyridine carboxylic acids (pyridine dicarboxylic acids); thus pyridinic cycle is not broken. For example, oxidation of 3-methylpyridine (β-pikolin) and 4-methylpyridine (γ-pikolin) to corresponding acids – nicotinic (pyridin-3-carboxylic acid or β-pyridincarboxylic acid) and isonicotinic (pyridin-4-carboxylic acid or γ-pyridincarboxylic acid) acids:

8 Drugs – derivatives of nicotinic acid
Nicotinic acid (pyridine-3-carboxylic acid, vitamin РР) has been received in 1867, but its specific vitamin action has been established only in 1937. It is a white crystal powder, slightly soluble in cold water, soluble in hot water, Shows amphoteric properties in view of presence of Nitrogene atom in pyridinic cycle (the basic properties) and mobile Hydrogene atom in carboxylic group (acid properties), therefore it is dissolved in solutions of acids and alkalis. There is a nicotinic acid in vegetables, fruit, buckwheat cereal, liver, milk, fish, yeast as transformation product nicotinamide. Release forms: powder, tablets, solution for injections.

9 The general formula of derivatives of nicotinic acid:
In the medical practice apply not only acid nicotinic, but also a number of preparations which are its derivatives: nikethamide (diethylamide nicotinic acid), nikethamide injections (cordiamine) (25 % solution), nicodine, etc. The general formula of derivatives of nicotinic acid: For preparations, derivatives of nicotinic acid, is characteric basic properties, because Hydrogene in carboxylyc group is substituted nitrogen-containing radicals.

10 Nicotinamide (Nicotinamidum) – amide pyridine-3-carboxylic acid:
It is a white crystal powder, freely soluble in water, alcohol, solutions of acids and alkaly. Medicinal forms: tablets, solution for injections. Vitamin РР.

11 Nikethamide Diaethylamidum acidi nicotinici
(Ph Eur monograph 0233) Diaethylamidum acidi nicotinici Diethylamide nicotinic acid Nicethamidum* C10H14N2O   178.2   DEFINITION Nikethamide contains not less than 99.0 per cent and not more than the equivalent of per cent of N,N-diethylpyridine-3-carboxamide, calculated with reference to the anhydrous substance.

12 Synthesis Condensation of nicotinic acid (or its chloranhydride) with diethylamine in the presence of dehydrating means (usually use phosphorus (V) oxochloride POCl3): Or by means of oxidation of -picoline (3-methylpyridine):

13 First identification A, B.
CHARACTERS An oily liquid or a crystalline mass, colourless or slightly yellowish, miscible with water and with alcohol. IDENTIFICATION First identification A, B. Second identification A, C, D.  A. Dissolve 0.15 g in 0.01 M hydrochloric acid and dilute to ml with the same acid. Dilute 1.0 ml of this solution to ml with 0.01 M hydrochloric acid. Examined between 230 nm and 350 nm (2.2.25) in a 2 cm cell, the solution shows a single absorption maximum, at 263 nm. The specific absorbance at the maximum is about 285. B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with nikethamide CRS. C. Alkaline hydrolysis of preparation. Heat 0.1 g with 1 ml of dilute sodium hydroxide solution R. Diethylamine is evolved progressively and is recognisable by its characteristic odour and by its turning red litmus paper R in blue.

14 Diethylamine (characteristic odour of ammonia)
D. Reaction with bromide thiocyanate solution (BrSCN). Dilute 1 ml of solution S (see Tests) to 250 ml with water R. To 2 ml of this solution add 2 ml of cyanogen bromide solution R. Add 3 ml of a 25 g/l solution of aniline R and shake. A yellow colour develops.

15 The inadmissible impurities are not presents in the test substance
yellow colour TESTS The inadmissible impurities are not presents in the test substance

16 Respiratory stimulant. Nikethamide Injection (25%).
ASSAY (BrPh). Acidimetry, non-aqueous titration Dissolve g in a mixture of 5 ml of acetic anhydride R and 20 ml of anhydrous acetic acid R. Titrate with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M perchloric acid is equivalent to mg of C10H14N2O. Em (C10H14N20) = M.m. Action and use Respiratory stimulant. Preparation Nikethamide Injection (25%). Ph Eur

17 Nikethamide Injection
Cordiamine (25 % solution of Nikethamide) DEFINITION Nikethamide Injection is a sterile solution containing 25% w/v of Nikethamide in Water for Injections. The injection complies with the requirements stated under Parenteral Preparations and with the following requirements. Content of nikethamide, C10H14N2O 24.0 to 26.0% w/v. CHARACTERISTICS A colourless solution. IDENTIFICATION Make 1 ml alkaline with 5M sodium hydroxide, extract with 5 ml of dichloromethane and evaporate the solvent. The infrared absorption spectrum of the oily residue, Appendix II A, is concordant with the reference spectrum of nikethamide (RS 249).

18 The maintenance cordiamine (Х, %) calculate by means of formula:
ASSAY (BrPh). Dilute 5 ml to 500 ml with water . To 5 ml of the solution add 5 ml of 1M hydrochloric acid and dilute to 500 ml with water . Measure the absorbance of the resulting solution at the maximum at 263 nm, Appendix II B. Calculate the content of C10H14N2O taking 282 as the value of A (1%, 1 cm) at the maximum at 263 nm. Other method. Refractometry (SP X). On a prism of refractometer put some drops of water and on a scale find index of refraction. Wipe a prism dry, put on it some drops of the examinee solution (cordiamine) and find index of refraction, which is defined by 3–4 times, taking each time a new portion of preparation, for calculation take medial number from all definitions. The maintenance cordiamine (Х, %) calculate by means of formula: n = n0 + CF

19 Where n – index of refraction.of preparation;
n0 – index of refraction.of water; F – refractometric factor (for cordiamine F = 0,002). Example of calculation of concentration diethylamide nicotinic acid in cordiamine. nо = 1,333; n = 1,383 It is possible to calculate the maintenance of operating substance (in g) in 1ml injection solution: Maintenance С10Н14N2O in 1 ml of preparation should be 0,240–0,258 g.

20 Respiratory stimulant.
Storage The list of strong substances. In densely corked container, in the place protected from light. Action and use Respiratory stimulant.

21 Nicodinum Bilamidum Cholamidum С7Н8N2O2 М m. = 152,15 g/mol
Nicodine SP X Nicodinum Bilamidum Cholamidum С7Н8N2O М m. = 152,15 g/mol Not less than 98,0 % The chemical name: N-oxymethylamide pyridine-3-carboxylic acid, N- oxymethylamide nicotinic acid .

22 Condensation amide nicotinic acid with formaldehyde:
Synthesis Condensation amide nicotinic acid with formaldehyde: CHARACTERS White fine-crystalline powder, without a smell. Melting point 147–149 °С. Soluble in water, difficultly soluble in 95 % alcohol, practically insoluble on ether.

23 Alkaline hydrolysis of preparation
Identification Alkaline hydrolysis of preparation Heat to boiling 0.1 g with 5 ml of sodium hydroxide solution - allocated NН3 (characteristic odour and by its turning red litmus paper R in blue). 2. Reaction with solution 2,4-dinitrochlorbenzole in ethanol (for pyridine cycle) To 0,1 g of preparation and 0,05 g 2,4-dinitrochlorbenzole, 5 ml of 95 % alcohol and boil during 2–3 mines before full dissolution., the solution is painted in yellow colour. After cooling add 0,5 ml of solution sodium hydroxide NaOH; orange-red colour are formed.

24 : 3. Decomposition of preparation with the next revealing of formaldehyde by means of disodium salt of chromotropic acid orange-red colour

25 Formaldehyde identification by means of disodium salt of chromotropic acid:
red-violet colour (aurin dye) 4. Melting point 147 to 149 °С.

26 Iodometry, back titration, after alkaline hydrolysis
Tests The inadmissible impurities are not presents in the test substance Assay Iodometry, back titration, after alkaline hydrolysis Nearby 0,1 g of test substance dissolve in 20 ml of water in a flask with volume of 500 ml with the ground in stopper, moistened with solution KI. To solution add 20 ml (excess) 0,05 M of solution I2, 7 ml 30 % solution NaOH and stand in a dark place for 20 minutes. Flask contents are diluted with 100 ml of water, add 50 ml diluted HCl, cool to a room temperature and allocated iodine I2 titrate with 0,1 M solution sodium thiosulphate Na2S2O3 (as indicator - starch solution).

27 I2 + 2Na2S2O3 = 2NaI + Na2S4O6 Em (С7Н8N2O2) = М.m/2

28 Cholagogue, disinfectant agent. The release form: tablets (0,5 g).
Storage In densely corked container, in protected from light and humidity a place, at temperature not above 20 °С. Action and use Cholagogue, disinfectant agent. The release form: tablets (0,5 g).

29 Isonicotinic acid (pyridine-4-carboxylic acid):
Derivatives of isonicotinic acid Isonicotinic acid (pyridine-4-carboxylic acid): underlies chemotherapeutic means with antitubercular action, which synthesis has begun in the USSR in 50th years ХХ centuries. Among them: isoniazid, phthivazid, flurenizidum (prof. Petruh L. I at the Lviv national medical university is introduced), etc. The first preparations in this area were thiosemicarbazone:

30 In due course high physiological activity has been revealed in derivatives of isonicotinic acid. Such derivatives concern: hydrazide of isonicotinic acid (However it in small doses slow-acting, and in big doses – is toxic).

31 Hydrazones of isonicotinic acid:
Hydrazones – are the products of interaction hydrazide of isonicotinic acid with aldehydes: They not have free hydrazine group (Н2N–NH2), therefore are less toxic, show high therapeutic activity against a tubercular stick, are well transferred by an organism. In the medical practice are used such drugs: isoniazid, phthivazid and flurenizidum.

32 Isoniazid Nicozid C6H7N3O 137.1 54-85-3 DEFINITION
General Notices (Ph Eur monograph 0146) Izoniazidum Tubazidum Nicozid C6H7N3O   137.1   DEFINITION Isoniazid contains not less than 99.0 per cent and not more than the equivalent of per cent of pyridine-4-carbohydrazide, calculated with reference to the dried substance.

33 of isonicotinic acid isoniazid
Synthesis Synthesis of methyl ester of isonicotinic acid and its condensation with hydrazine Initial substance for synthesis is isonicotinic acid (which receive oxidation picolinic fractions of coal pitch) from which receive methylester, and then it is condensed with hydrazine H2N–NH2 with formation hydrazide. 4-methylpyridine pyridine-4-carboxylic acid methyl ester of isonicotinic acid isoniazid (γ-picoline)

34 CHARACTERS A white, crystalline powder or colourless crystals, freely soluble in water, sparingly soluble in alcohol. IDENTIFICATION First identification A, B. Second identification A, C.  A. Melting point (2.2.14): 170 °C to 174 °C.  B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with isoniazid CRS.  C. Dissolve 0.1 g in 2 ml of water R and add 10 ml of a warm 10 g/l solution of vanillin R. Allow to stand and scratch the wall of the test tube with a glass rod. A yellow precipitate is formed, which, after recrystallisation from 5 ml of alcohol (70 per cent V/V) R and drying at 100 °C to 105 °C, melts (2.2.14) at 226 °C to 231 °C.

35 Other reactions (SPU): Reaction with solution of copper (ІІ) sulphate
0,1 g preparation dissolve in 5 ml of water and add 4–5 drops of solution copper (ІІ) sulphate CuSO4; the blue precipitate is formed; at stirring the solution is painted in blue colour. At heating solution and precipitate get light green, and then yellow-green colour is formed and gas vials are allocated. It is possible to present occurring processes by such reactions. 1. Formation of blue precipitate of salt Cu2 + with enol form of isoniazid with the next oxidation of the hydrazide rest to free nitrogen N2 and reduction Cu2 + to Cu + (precipitate Cu2O of green colour). blue precipitate green precipitate

36 The inadmissible impurities are not presents in the test substance.
ASSAY (BrPh). Bromatometry, direct titration Dissolve g in water R and dilute to ml with the same solvent. To 20.0 ml of the solution add 100 ml of water R, 20 ml of hydrochloric acid R, 0.2 g of potassium bromide R and 0.05 ml of methyl red solution R. Titrate dropwise with M potassium bromate, shaking continuously, until the red colour disappears. 1 ml of M potassium bromate is equivalent to mg of C6H7N3O. KBrО3 + 5KBr + 3H2SO4 → 3Br2 + 3K2SO4 + 3H2O In the end point excess drop of potassium bromate KBrО3 reacts with new formed KBr; bromine Br2 is formed, and red colour disappears (disappears colour of indicator): KBrО3 + 5KBr + 3H2SO4 → 3Br2 + 3K2SO4 + 3H2O Em = М. m./4; k(KBrО3)= 6

37 Storage The list of strong substances. In densely corked container from dark glass, in the place protected from light; ampoules – at temperature to +10 C. Action and use Antituberculous. Preparations Isoniazid Injection Isoniazid Tablets Ph Eur

38 M (phthivazid hydrate)= 289,29 g/mol M (anhydrous)= 271,28 g/mol
Phthivazid SP X Phthivazidum Vanicide Vanillaberon Ftivazidum Vanizide С14Н13N3O3Н2О M (phthivazid hydrate)= 289,29 g/mol M (anhydrous)= 271,28 g/mol Not less than 98,0 % The chemical name. 3-methoxy-4-oxybenzylidenehydrazide pyridine-4-carboxylic acid hydrate or 3-metoxy-4-oxybenzylidenehydrazide isonicotinic acid hydrate.

39 Condensation of isoniazid with vanilline under the scheme:
Synthesis Condensation of isoniazid with vanilline under the scheme: isoniazid vanilline phthivazid CHARACTERS Light yellow or yellow fine-crystalline powder with a weak smell of vanillin, without taste. Very slightly soluble in water, slightly soluble in 95 % alcohol, freely soluble in ice acetic acid, inorganic acids and alkalis.

40 1.Acid hydrolysis of preparation and vanillin detection
Identification 1.Acid hydrolysis of preparation and vanillin detection 0,05 g preparation heat up about 10 ml of diluted НCl; there is a strong smell of vanillin. smell of vanillin 2. Reaction with 2,4-dinitrochlorbenzole in ethanol (for pyridine cycle) (see isiniazid)

41 3. Reaction of alcoholic solution of preparation with alkali
Reaction confirms amphoteric properties of phthivazid. orange-yellow colour

42 5NaNO2 + 2KIО3 + 2HCl → I2 + 2KCl + 5NaNO3 + H2O
Tests 1. Hydrazide of isonicotinic acid (isoniazid, specific inadmissible impurity) In the presence of impurity of isoniazid there is reaction: Then this impurity is absence - the dark blue stain on the iodide-starched paper is formed: 5NaNO2 + 2KIО3 + 2HCl → I2 + 2KCl + 5NaNO3 + H2O

43 2. Vanilline (specific inadmissible impurity)
0,8 g preparation shake up about 40 ml of water within 2 minutes and filter not dissolved precipitate. 12,5 ml of filtrate, diluted with water to 25 ml, from addition of 2 drops of 0,05 M NaOН at presence phenolphthaleine should be painted in pink colour.

44 Acidimetry, non-aqueous titration
Assay Acidimetry, non-aqueous titration To 0,15 g of test substance add 5 ml of ice CH3COOH and 40 ml of anhydrous chloroform CHCl3, 8 drops of crystal violet solution (as indicator) and titrate (by microburet) with 0,1 M perchloric acid HClO4 before change of colour from red-brown to grey-green. In parallel spend control experience (change colour from violet to dark blue). Em = М.m.

45 The list of strong substances. In densely corked container.
Storage The list of strong substances. In densely corked container. Action and use An antitubercular agent. Release forms: powder, tablets (0,1; 0,3 and 0,5 g) (Tabulettae Phthivazidi 0,1; 0,3 aut 0,5) (tablets of light yellow or yellow colour, with a weak smell of vanillin).

46 Flurenizidum (ukranian drug, prof. L.I.Petruh, Lviv) M = 299,33 g/mol The chemical name: N - (9-fluoreneilidene)-N '-isonicotinhydrazide.

47 CHARACTERS Storage Action and use
Fine-crystalline powder with crystals needlelike forms or plate (lamellar) powder yellow or greenish-yellow colour, without a smell. Soluble in acetic acid, slowly soluble in chloroform, practically insoluble in water, slightly soluble in alcohol. Storage In the densely corked container. Action and use Antitubercular, antimicrobial, antichlamydial agent. Release forms: tablets (0,05 g or 0,15 g) (Tabulettae Flurenizidi 0,05 aut 0,15).

48 Thanks for attention!