Theme: Pharmaceutical analysis of derivatives of pyridine (pyridine-3-carboxylic acid, pyridine-4-carboxylic acid) and pyrimidine (barbituric acid) as drug substances: synthesis, properties, analysis, storage, action and use.

 

 

 

 

Derivatives of pyridine

To hexatomic heterocycles with one heteroatom of Nitrogene belong pyridine:

Completely hydrogenated pyridine (saturated heterocycle) names piperidine:

        

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) b-position

 

                                   pyridine          β-nitropyridine

concerning atom of Nitrogene. It is oxidised more difficultly, but is easier hydrogenated.

                              

 

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.

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 paint litmus in dark blue colour (the basic properties); at action of acids forms crystal salts of pyridinium:

                                     pyridine base         pyridine          salt of pyridinium

 

Homologues of pyridine easily are oxidised with formation 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) – it is possible to present oxidation by such schemes:

In medical practice apply not only acid nicotinic, but also a number of preparations which are its derivatives: nikethamide (diethylamide nicotinic acid), nikethamide injection (cordiamine) (25 % solution), nicodine, etc.

 

 

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.

In the medical practice apply not only acid nicotinic, but also a number of preparations which are its derivatives: nicotinamide, 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.

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.

 

 

 

 

 

 

 Nikethamide

(Ph Eur monograph 0233)

 

Diethylamide nicotinic acid                        

Diaethylamidum acidi nicotinici

  Nicethamidum*

                                                                                                                                                                                         

 

 

 

 

 

 

 

 

 C₁₀H₁₄N₂O   178.2   59-26-7

   

 DEFINITION

  

 Nikethamide contains not less than 99.0 per cent and not more than the equivalent of  101.0 per cent of N,N-diethylpyridine-3-carboxamide, calculated with reference to the  anhydrous substance.

  

Synthesis

 Condensation of nicotinic acid (or its chloranhydride) with diethylamine in the presence of dehydrating means (usually use phosphorus (V) oxochloride POCl₃):

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 100.0 ml with the same acid. Dilute 1.0 ml of this solution to 100.0 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 blue.

                                                                                                                      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.

 

 

 

                                                                                            yellow colour

 

 

 

Other reactions:

1. Reaction with solution of copper sulphate at presence of ammonium thiocyanate

To 5 ml 10 % solution of preparation add 5 ml solution CuSO₄; dark blue colour are formed; after addition 3 ml of ammonium thiocyanate solution NH₄SCN bright green precipitate is formed.

         Dark blue colour has a complex of preparation with copper sulphate (type [Cu (NH₃) ₄] SO₄):

                                              

 

                                     

         The bright green precipitate is complex of preparation with copper thiocyanate:

 

3. Reaction with solution 2,4-dinitrochlorbenzole in ethanol (for pyridine cycle)

2–3 drops of preparation and 0,05 g 2,4-dinitrochlorbenzole dissolve in 3 ml of 95 % alcohol and boil during 1 mines, the solution is painted in yellow colour. After cooling and addition of 1 drop of solution NaOH appears violet colouring which at the further addition of several drops of alkali solution gradually passes in brownish-red.

                   It is reaction to derivatives of pyridine, having free 2 and 6 positions concerning heteroatom of Nitrogene. Pyridine cycle splitting at action 2,4- dinitrochlorbenzole in the alkaline mediumt with formation polymethine derivative glutaconic aldehyde, which as a result of hydrolysis turns in derivative of glutaconic aldehyde, existing in two tautomeric forms.

 

 

 

 

TESTS

  

 Solution S

  

 Dissolve 2.5 g in carbon dioxide-free water R and dilute to 10 ml with the same  solvent.

 

 Appearance

  

 The substance to be examined, in liquid form or liquefied by slight heating, is clear  (2.2.1) and not more intensely coloured than reference solution Y₅ (2.2.2, Method II).

 

 pH (2.2.3)

  

 The pH of solution S is 6.0 to 7.8.

 

 Refractive index (2.2.6)

  

 1.524 to 1.526.

 

 Related substances

  

 Examine by thin-layer chromatography (2.2.27), using silica gel GF₂₅₄ R as the  coating substance.

 

 Test solution Dissolve 0.4 g of the substance to be examined in methanol R and  dilute to 10 ml with the same solvent.

 

 Reference solution (a) Dissolve 40 mg of ethylnicotinamide CRS in methanol R and  dilute to 100 ml with the same solvent.

 

 Reference solution (b) Dilute 1 ml of reference solution (a) to 10 ml with methanol R.

 

 Apply separately to the plate 10 µl of each solution. Develop over a path of 15 cm  using a mixture of 25 volumes of propanol R and 75 volumes of chloroform R. Allow  the plate to dry in air and examine in ultraviolet light at 254 nm. In the chromatogram  obtained with the test solution, any spot corresponding to ethylnicotinamide is not  more intense than the spot in the chromatogram obtained with reference solution (a)  (1.0 per cent) and any spot, apart from the principal spot and the spot corresponding  to ethylnicotinamide, is not more intense than the spot in the chromatogram  obtained with reference solution (b) (0.1 per cent).

 

 Heavy metals (2.4.8)

  

 Dilute 10 ml of solution S to 25 ml with water R. 12 ml of this solution complies with  limit test A for heavy metals (10 ppm). Prepare the standard using lead standard  solution (1 ppm Pb) R.

 

 Water (2.5.12)

  

 Not more than 0.3 per cent, determined on 2.00 g by the semi-micro determination of  water.

 

 Sulphated ash (2.4.14)

  

 Not more than 0.1 per cent, determined on 1.0 g.

  

 ASSAY

  

Acidimetry, non-aqueous titration

Dissolve 0.150 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 17.82 mg of C₁₀H₁₄N₂O.

 

 

  

 

Em (C₁₀H₁₄N₂₀) = M.m.

 

Ph Eur

 

 

 

 

 

Action and use

  

 Respiratory stimulant.

 

 Preparation

  

 Nikethamide Injection  (25%).

 

 Ph Eur

 

 

 

 

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).

  

 TESTS

  

 Acidity or alkalinity

  

 pH, 6.0 to 8.0, Appendix V L.

 

 Related substances

  

 Carry out the method for thin-layer chromatography, Appendix III A, using silica gel  GF₂₅₄ as the coating substance and a mixture of 25 volumes of propan-1-ol  and 75  volumes of chloroform as the mobile phase. Apply separately to the plate 10 µl of  each of the following solutions. For solution (1) dilute 1 ml of the injection to 5 ml  with methanol . Solutions (2) and (3) contain 0.05% w/v and 0.005% w/v respectively  of ethylnicotinamide EPCRS in methanol . After removal of the plate, allow it to dry in  air and examine under ultraviolet light (254 nm). Any spot corresponding to  ethylnicotinamide in the chromatogram obtained with solution (1) is not more intense  than the spot in the chromatogram obtained with solution (2) (1%) and any other  secondary spot is not more intense than the spot in the chromatogram obtained with  solution (3) (0.1%).

  

 ASSAY

  

 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. Refraktometry (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                  From here

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.

         n_о = 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.

         Storage. The list of strong substances. In densely corked container, in the place protected from light.

 

Action and use

  

 Respiratory stimulant.

 

 

 

Nicodine                                                    SP X

Nicodinum

  Bilamidum

Cholamidum

    С₇Н₈N₂O₂ М m. = 152,15 g/mol

                                                                                                        

Not less than 98,0 %

The chemical name: N-oxymethylamide pyridine-3-carboxylic acid, N- oxymethylamide nicotinic acid.

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.

Identification

1. 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 blue).

                                                                                                             characteristic odour

 

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; there is an orange-red colouring.

(The reaction mechanism is described by consideration diethylamide nicotinic acid).

        

 

3. Decomposition of preparation with the next revealing of formaldehyde with disodium salt of chromotropic acid

To 5 ml 2 % solution of disodium salt chromotrope acid add 5 ml of concentrated sulphatic acid H₂SO₄ and 1–2 mg of preparation; there is a red-violet colour are formed (at the expense of formation aurin dye).

         The scheme of decomposition of preparation with formaldehyde formation:

Reaction of revealing of formaldehyde with disodium salt of chromotropic acid:

                                          aurin dye

                         (red-violet colour)

 

 

 

4. Melting point 147 to 149 °С.

 

 

 

Tests

1. A transparency and colourityty of solution. 4 % solution of preparation should be transparent and colourless.

2. Acidity. 0,4 g preparation dissolve in 10 ml fresh-boiling and cooled water, add 3 drops of solution of methyl red; the appeared pink colouring should pass in yellow from additioo more than 0,8 ml of solution of 0,05 M sodium hydroxide NaOH.

3. The general impurity of chlorides, sulphates, heavy metals, Arsenic – within standards.

4. Sulphatic ashes from 0,5 g preparation should not exceed 0,1 %.

 

Assay

1. 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).

 

 

I₂ + 2Na₂S₂O₃ = 2NaI + Na₂S₄O₆

 

E_m (С₇Н₈N₂O₂) = М m/2.

Storage. In densely corked container, in protected from light and humidity a place, at temperature not above 20 °С.

 

Application. Cholagogue,  disinfectant agent.

 

The release form: tablets 0,5 g.

 

 

 

 

 

 

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:

however they are badly transferred by an organism, that considerably limited their application.

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).

Hydrazones of isonicotinic acid:

Hydrazones – interaction products hydrazide 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.

 

 

 

 

Isoniazid

General Notices

 

 

 (Ph Eur monograph 0146)

Izoniazidum

  Nicozid

 

 

 

 

 

 

 

 

 

 

 C₆H₇N₃O   137.1  54-85-3

 

  

 DEFINITION

  

 Isoniazid contains not less than 99.0 per cent and not more than the equivalent of  101.0 per cent of pyridine-4-carbohydrazide, calculated with reference to the dried  substance.

  

Synthesis

         Synthesis  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.

The synthesis scheme:

          4-methylpyridine        pyridine-4-carboxylic acid   methyl ester  of isonicotinic acid        isoniazid

                   (γ-picoline)

 

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.

 

Other reactions (SPU):

1. 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 colouring and gas vials are allocated.

It is possible to present occurring processes by such reactions.

1. Formation of blue precipitate of salt Cu^{2 +} with enol form of isoniazid with the next oxidation of the hydrazide rest to free nitrogen N₂ and reduction Cu^{2 +} to Cu ⁺ (precipitate Cu₂O of green colour).

 

              

                                                                    blue precipitate                     green  precipitate

 

2. Formation of complex salt Cu^{2 +} with two molecules of isoniazid (in enol form) blue colour with the subsequent oxidation of the hydrazide rest to free nitrogen N₂ and reduction Cu^{2 +} to Cu ⁺ (precipitate Cu₂O).

2. Reaction with 2,4-dinitrochlorbenzole in ethanol (for pyridine cycle).

To several crystals of preparation and 0,05 g 2,4-dinitrochlorbenzole, 3 ml of 95 % alcohol and boil during 1–1,5 minutes. After cooling add 2 drops of solution sodium hydroxide NaOH; there is a red-brown colouring which quickly passes in red-brown.

         The essence of process consists that atom of Chlorine Cl of molecule 2,4-dinitrochlorbenzole attacks atom N in pyridine cycle, adjoins the rest of dinitrobenzene with formation pyridinium salt. At addition of alkali there is a bond breaking in pyridine cycle with formation derivative of glutaconic aldehyde, painted in brown or red colour (the mechanism 1) or quinoid structure (the mechanism 2).

         The mechanism 1.

 

         The mechanism 2.

 

3. Reaction of “silver mirror” (reducing properties of the hydrazine rest)

0,01 g preparation dissolve in 2 ml of water and add 1 ml of ammoniac solution silver nitrate AgNO₃; there is a yellowish precipitate, which at heating on a water heater darkens and on test tube walls “the silver mirror” is formed.

                   

 

4. Preparation pyrolysis (not pharmacopoeial reaction)

At heating of isoniazid in a dry test tube with waterless sodium carbonate Na₂CO₃ arises a characteristic unpleasant smell of pyridine.

5. Reaction with p-dimethylaminobenzaldehyde after alkaline hydrolysis (for hydrazine, not pharmacopoeial reaction)

         The essence of this reaction consists that hydrazine, formed at alkaline hydrolysis of isoniazide, interacted with p–dimethylaminobenzaldehyde with formation of product, which at presence of chloride acid HCl forms compound of                    quinoid structure of yellow-orange colour

 

6. Reaction with alkaline solution of sodium nitroprusside (not pharmacopoeial reaction)

With alkaline solution of sodium nitroprusside Na₂ [Fe (CN) ₅NO] arises intensive orange colouring, which passes in cherry, and then – in yellow at acidifying by means of HCl.

 

  

 TESTS

  

 Solution S

  

 Dissolve 2.5 g in carbon dioxide-free water R and dilute to 50 ml with the same  solvent.

 

 Appearance of solution

  

 Solution S is clear (2.2.1) and not more intensely coloured than reference solution  BY₇ (2.2.2, Method II).

 

 pH (2.2.3)

  

 The pH of solution S is 6.0 to 8.0.

 

 Hydrazine and related substances

  

 Examine by thin-layer chromatography (2.2.27), using silica gel GF₂₅₄  R as the  coating substance.

 

 Test solution Dissolve 1.0 g of the substance to be examined in a mixture of equal  volumes of acetone R and water R and dilute to 10.0 ml with the same mixture of  solvents.

 

 Reference solution Dissolve 50.0 mg of hydrazine sulphate R in 50 ml of water R  and dilute to 100.0 ml with acetone R. To 10.0 ml of this solution add 0.2 ml of the  test solution and dilute to 100.0 ml with a mixture of equal volumes of acetone R and  water R.

 

 Apply separately to the plate 5 µl of each solution and develop over a path of 15 cm  using a mixture of 10 volumes of water R, 20 volumes of acetone R, 20 volumes of  methanol R and 50 volumes of ethyl acetate R. Allow the plate to dry in air and  examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with  the test solution, apart from the principal spot, is not more intense than the spot in  the chromatogram obtained with the reference solution (0.2 per cent). Spray the plate  with dimethylaminobenzaldehyde solution R1. Examine in daylight. An additional  spot, corresponding to hydrazine, appears in the chromatogram obtained with the  reference solution. Any corresponding spot in the chromatogram obtained with the  test solution is not more intense than the spot corresponding to hydrazine in the  chromatogram obtained with the reference solution (0.05 per cent).

 

 Heavy metals (2.4.8)

  

 2.0 g complies with limit test C for heavy metals (10 ppm). Prepare the standard  using 2 ml of lead standard solution (10 ppm Pb) R.

 

 Loss on drying (2.2.32)

  

 Not more than 0.5 per cent, determined on 1.00 g by drying in an oven at 100 °C to  105 °C.

 

 Sulphated ash (2.4.14)

  

 Not more than 0.1 per cent, determined on 1.0 g.

  

 ASSAY

  

 Bromatometry, direct titration.

Dissolve 0.250 g in water R and dilute to 100.0 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  0.0167 M potassium bromate, shaking continuously, until the red colour disappears.

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):

E_m (С₆Н₇N₃O) = М. м./4

k(KBrО₃)= 6

 

 1 ml of 0.0167 M potassium bromate is equivalent to 3.429 mg of C₆H₇N₃O.

 

  Ph Eur

 

 

Other method (SPU):

Iodometry at presence of sodium hydrogencarbonate, back titration.

Nearby 0,1 g (exact навеска) preparation bring in a conic flask with volume of 500 ml with the ground in stopper, dissolve in 100 ml of water, add 2 g sodium hydrogencarbonate NaHCO₃, 50 ml 0,05 mol/l of iodine solution I₂ and leave on 30 minutes at 38–40 °C in a dark place. After that put on 10 minutes in an ice cooler and then add in the small portions of a mix of 20 ml concentrated HCl and waters (1:2) (at solution cooling). Excess of iodine I₂ titrate with 0,1 M sodium thiosulphate Na₂S₂O₃ in the presence of starch (before disappearance of dark blue colouring). In parallel spend control experience.

 

I₂ + 2Na₂S₂O₃ = 2NaI + Na₂S₄O₆

E_m (С₆Н₇N₃O) = М m./4

 

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.

 

 

  Ph Eur

 

 

Action and use

  

 Antituberculous.

 

 Preparations

  

 Isoniazid Injection

 

 Isoniazid Tablets

 

 Ph Eur

 

 

 

 

 

 

Isoniazid Injection

DEFINITION

  

 Isoniazid Injection is a sterile solution of Isoniazid in Water for Injections.

 

 The injection complies with the requirements stated under Parenteral Preparations  and with the following requirements.

 

 Content of isoniazid, C₆H₇N₃O

  

 95.0 to 105.0% of the stated amount.

  

 IDENTIFICATION

  

  A. Using a rotary evaporator, evaporate a volume of the injection containing 50 mg of Isoniazid to dryness, extract the residue with two 10 ml quantities of ethanol (96%), filter and evaporate the combined ethanol extracts to dryness. The infrared absorption spectrum of the residue, Appendix II A, is concordant with the reference spectrum of isoniazid (RS 196).

 

  B. To a volume containing 25 mg of Isoniazid add 5 ml of ethanol (96%), 0.1 g of sodium tetraborate and 5 ml of a 5.0% w/v solution of 1-chloro-2,4-dinitrobenzene in ethanol (96%), evaporate to dryness on a water bath, heat for a further 10 minutes and dissolve the residue in 10 ml of methanol . A reddish purple colour is produced.

  

 TEST

  

 Acidity

  

 pH, 5.6 to 6.0, Appendix V L.

  

 ASSAY

  

 Dilute a quantity containing 0.4 g of Isoniazid to 250 ml with water . To 25 ml of the  solution in a glass-stoppered flask add 25 ml of 0.05M bromine VS and 5 ml of  hydrochloric acid, shake for 1 minute, allow to stand for 15 minutes, add 1 g of  potassium iodide and titrate with 0.1M sodium thiosulphate VS using starch mucilage  as indicator. Repeat the operation without the injection. The difference between the  titrations represents the amount of bromine required. Each ml of 0.05M bromine VS  is equivalent to 3.429 mg of C₆H₇N₃O.

  

 STORAGE

  

 Isoniazid Injection should be protected from light.

 

 

 

 

 

 

Isoniazid Tablets

DEFINITION

  

 Isoniazid Tablets contain Isoniazid.

 

 The tablets comply with the requirements stated under Tablets and with the following  requirements.

 

 Content of isoniazid, C₆H₇N₃O

  

 95.0 to 105.0% of the stated amount.

  

 IDENTIFICATION

  

  A. Shake a quantity of the powdered tablets containing 0.1 g of Isoniazid with 10 ml of ethanol (96%) for 15 minutes, centrifuge and decant the supernatant liquid. Extract the residue with two further 10-ml quantities of ethanol (96%) and evaporate the combined extracts to dryness. The infrared absorption spectrum of the residue, Appendix II A, is concordant with the reference spectrum of isoniazid (RS 196).

 

  B. Shake a quantity of the powdered tablets containing 1 mg of Isoniazid with 50 ml of ethanol (96%) and filter. To 5 ml of the filtrate add 0.1 g of sodium tetraborate and 5 ml of a 5% w/v solution of 1-chloro-2,4-dinitrobenzene in ethanol (96%), evaporate to dryness on a water bath and continue heating for a further 10 minutes. To the residue add 10 ml of methanol  and mix. A reddish purple colour is produced.

  

 TEST

  

 Dissolution

  

 Comply with the dissolution test for tablets and capsules, Appendix XII D, using as  the medium 900 ml of water  and rotating the basket at 100 revolutions per minute.  Withdraw a sample of 10 ml of the medium. Measure the absorbance of the filtered  sample, suitably diluted if necessary, at the maximum at 263 nm, Appendix II B.  Calculate the total content of isoniazid, C₆H₇N₃O, in the medium taking 307 as the  value of A (1%, 1 cm) at the maximum at 263 nm.

  

 ASSAY

  

 Weigh and powder 20 tablets. Dissolve a quantity of the powder containing 0.4 g of  Isoniazid as completely as possible in water , filter and wash the residue with  sufficient water  to produce 250 ml. To 50 ml of the resulting solution add 50 ml of  water , 20 ml of hydrochloric acid and 0.2 g of potassium bromide and titrate with  0.0167M potassium bromate VS determining the end point electrometrically. Each ml  of 0.0167M potassium bromate VS is equivalent to 3.429 mg of C₆H₇N₃O.

  

 STORAGE

  

 Isoniazid Tablets should be protected from light.

 

 

                                                  

 

Ftivazide                                           SP X

  Vanicide

Vanillaberon

Phthivazidum

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-metoxy-4-oxybenzylidenehydrazide pyridine-4-carboxylic acid hydrate or 3-metoxy-4-oxybenzylidenehydrazide isonicotinic acid hydrate.

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.

 

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).

To several crystals (0,05 g) of preparation and 0,05 g 2,4-dinitrochlorbenzole, 3 ml of 95 % alcohol and boil during 1–1,5 minutes. After cooling add 2 drops of solution sodium hydroxide NaOH; there is a red-brown colouring, the caused formation of compound with quionoid structure (see the mechanism reaction for isoniazid).

 

3. Reaction of alcoholic solution of preparation with alkali

0,05 g preparation dissolve at weak heating in 10 ml of 95 % alcohol and cool. At addition of 1 drop of solution  NaOH light yellow colouring of solution passes in the orange-yellow. At the subsequent addition of 1 drop diluted HCl a solution becomes yellow colouring, and then at further acidifying solution is painted in orange-yellow colour.

Reaction confirms amphoteric properties of phthivazid.

                                                                                                  orange-yellow colour

 

4. Interaction with phosphotungstic and phosphomolybdic acids (reaction for vanilline)

Phosphotungstic and phosphomolybdic acids allocate precipitates of complex salts. Dissolution phosphorous molybdate of phthivazid in alkalis leads to occurrence of intensive green colouring; its heating with camphor and concentrated sulphatic acid H₂SO₄ – to occurrence of violet colouring.

        

 

Tests

1. Hydrazide of isonicotinic acid (isoniazid, specific inadmissible impurity)

0,5 g preparation shake up about 30 ml of ice water and filter. To filtrate add 0,5 ml of solution HCl and 1 drop 0,1 M solution of sodium nitrite NaNO₂. The test taken in 3 minutes, should give a dark blue stain on iodide-starched paper.

         In the presence of impurity isoniazid there is reaction:

         Then this impurity is absence – the dark blue stain on the iodide-starched paper is formed:

2NaNO₂ + 2KI + 4HCl à I₂ + 2NO + 2KCl + 2NaCl + 2H₂O

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.

3. The general impurity of chlorides, sulphates, heavy metals – within standards.

4. Sulphatic ashes from 0,5 g preparation should not exceed 0,1 %.

5. Weight loss at drying. Nearby 0,5 g preparation (exact of shot) dry at 120 °C to constant weight. Loss in weight should not exceed 7 %.

 

Assay

1. 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).

E_m (С₁₄Н₁₃N₃O₃×Н₂О) = М m.

 

Storage. The list of strong substances. In densely corked container.

 

Application. An antitubercular preparation.

Release forms: powder, tablets on 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).

 

 

       

                                            Flurenizidum                        (prof. L.I.Petruh, Lviv)

           M = 299,33 g/mol

                                                                  

The chemical name: N – (9-fluoreneilidene)-N ‘-isonicotinhydrazide.

 

Synthesis

 Condensation 9-fluorenone with isoniazid under the scheme:

             9-fluorenone            isoniazid                                   flurenizidum

 

 

 

CHARACTERS

  

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.

Identification

1. Reaction with concentrated nitric acid (for fluorene ring)

To several crystals (0,01 g) preparations on hour glass add some drops (0,03 ml) concentrated НNO₃; there is orange-red colouring, which disappears at addition of water of 0,04 ml.

Depending on conditions, according to the literature, various nitroderivatives can be formed: 2-nitro- (most easily), 2,7-dinitro-, 2,3,7-trinitro-, 2,3,6,6-tetranitroderivatives of fluorenes. These derivatives are applied as sensitizers of electrophotographic layers.

In general it is possible to present the reaction mechanism such equation:

                                                              orange-red colouring

 

2. Reaction with alkaline solution of copper (ІІ) sulphate

To 0,1 g substances in test tube add 1,0 ml of 96 % alcohol R, 0,5 ml of water R, 0,08 ml of solution NaOH and 0,15 ml of solution copper sulphate R CuSO₄; the bluish-green precipitate,which passes in brown-green, is formed and gas vials are allocated.

Explanation the reaction mechanism.

Enol form of preparation forms salt with ions Cu^{2 +}:

 

                                                                                     bluish-green precipitate

 

Allocation of gas vials testifies about oxidation of hydrazono-group=N-NH – to free nitrogen N₂:

3. Reaction with 2,4-dinitrochlorbenzole in ethanol (for pyridine cycle).

To 0,1 g preparation in a test tube add some crystals 2,4-dinitrochlorbenzole, 3 ml of 95 % alcohol and boil during 1,5 minutes. After cooling add 0,08 ml of solution sodium hydroxide NaOH; there is a brownish-red colouring,which quickly passes in red-brown.

The mechanism of reaction the general for all derivatives of pyridine (see isoniazid, phthivazid, etc.).

4. Uv-spectroscopy

The Uv-spectrum of absorption of substance solution in ethanol should have maximum absorption at 250 nanometers and 260 nanometers.

5. IR-spectroscopy

The IR-spectrum test substance, received in disks with KBr, should correspond to spectrum SPS flurenizidum.

 

Assay

1. Acidimetry, non-aqueous titration

Nearby 0,15 g (exact of shot) preparation bring in conic flask with volume of 100 ml, dissolve in 5 ml of ice CH₃COOH, add 40 ml of anhydrous chloroform CHCl₃, 8 drops of crystal violet solution and titrate (from microburet) with 0,1 M perchloric acid HClO₄ before change of colouring from red-brown to  grey-green.

In parallel spend control experience (transition of colouring from violet to dark blue).

 

 

 

E_m = М m.

 

Storage. In densely corked container.

 

Application. Antitubercular, antimicrobial, antichlamydial agent.

         Release forms: tablets on 0,05 g and 0,15 g (Tabulettae Flurenizidi 0,05 aut 0,15).

 

 

 

 

 

Derivatives of pyrimidine

            In the structure of many natural and synthetic drugs is pyrimidine – hexatomic heterocycle with two atoms of Nitrogene which are in position 1,3:

            Completely hydrogenated cycle of pyrimidine names hexahydropyrimidine:

           

In medical practice the synthetic preparations, which contains hexahydropyrimidine cycle with three oxogroups –С=О in position 2,4,6 – derivatives of barbituric acid are widely applied:

barbituric acid

barbituric acid

 is derivative of uracil (1,2,3,4-tetrahydropyrimidine cycle with two hydroxy-groups in position 2 and 4):

    Uracil

            Both groups of preparations play the important role in medicine as sleeping medicines (barbiturates) and for treatment of malignant tumour (derivatives of uracil).

            Derivatives of pyrimidine concern uracil, thymine and cytosine – the pyrimidine bases, which are a part РNA (ribonucleic acid) and DNA, playing the important role in the course of synthesis of protein and transfer of the genetic information.

 

Drugs – derivatives of barbituric acid

Barbituric acid represents cyclic ureide – a product of condensation of carbamide (urea) with the dibasic malonic acid:

The chemical name of barbituric acid – 2,4,6-trioxohexahydropyrimidine .

Derivatives of barbituric acid are products of condensation of a carbamide with derivatives of malonic acid:

                             

          Carbamide               derivatives of malonic acid                     derivatives of barbituric acid

                                                 

            As the condensation product contains the closed system with two atoms of the Nitrogene in position (1 and 3), therefore barbiturates consider as derivatives of pyrimidine.

Barbiturates are cyclic imides used as hypnotics and (in the case of phenobarbital) as anticonvulsants. They are derivatives of barbituric acid (which is not pharmacologically active) and differ only in their substituents on the 5-position of the ring.

            The hypnagogue action of barbiturates was revealed in the early ХХ-th century by Fisher and Mering. In the 1904 Fisher was synthesized  barbital, after that were synthesized many barbiturates and was fixed relationship between structure and action.

1. The hypnagogue action is characteristic for derivatives of barbituric acid, which have in the position 5,5 alkyl, aryl or other radicals.

2. The impact surface and valid time of barbiturates are increased at increase of lenght of hydrocarbonic chain in the alkyl substitute in the position 5,5 until 5-6 atoms of Carbon. Then lenght of hydrocarbonic chain in the barbiturates is more? Such drugs have stimulant action.

3. The pharmacological effect at hydrocarbonic chain branching, presence of unsaturated bonds, alkoholic hydroxyl –ОН, atom of halogen (especially Br) is intensified.

4. Then action of barbiturates is more intensified the hypnagogue effect is shot.

5. With one phenyl radical (C6H5) on the 5-position of the ring to intensify action, not change duration of action, but then second phenyl radical on the 5-position is presents the hypnagogue action is decreased.

6. Then alkyl radical is on the 1- or 3-position (nearly imide group) valid time of drugs is abbreviated.

7. The change of hydrogen on the 1-position on the rest of aromatic acid (for example, benzoic acid) add to drug antiepileptic action (benzonal).

8. Then atoms of hydrogen of imide groups (1- and 3-positions of the ring) are substituted such drug can make convulsions.

9. Derivatives of thiobarbituric acid (atom of sulphur on the 2-position of the ring ) have more intensive and short-time action unlike oxygen analogue of barbiturates. 

 

 

Chemical properties of barbituric acid and barbiturates (derivatives of barbituric acid)

 

Barbiturates contaiitrogen atoms, but the lone pair on the nitrogen is not available for reaction with protons, so barbiturates are not basic.

            Barbituric acid and its derivatives have the acid nature. Thus barbituric acid in 5–6 times is stronger then acetic acid. 5-Monosubstituted of barbituric acid (for example, 5-ethylbarbituric acid) – enough strong acids, and 5,5-disubstituted of barbituric acid (for example, 5,5-diethylbarbituric acid) – very weak acid.

            Acid properties of these compounds are caused keto-enol tautomerism of  barbituric acid – at the expense of Hydrogene atoms of methylene groups  –СН₂–.

 

 

 

 

 

                                                                              

 

           

Besides, at the expense of Hydrogene atoms of imide groups-NH – it is possible imido-imidolnic tautomerism:

           

 

 

 

 

 

 

 

For barbiturates, in which Hydrogene atoms of methylene groups are substituted on radicals, it is possible only imido-imidolnic tautomerism (lactam-lactim tautomerism).

 

 

Thus it is necessary to notice, that unlike barbituric acids its derivatives in water solutions almost not dissociates; at presence of ions ОН^–– they dissociates as acids also are capable to give salts with metals:

 

 

 

 

 

 

 

Barbituric acid and its salts not have medical properties and consequently are not drugs.

 

General formula of barbiturates (imide form):

 

 

 

 

 

 

 

General formula of Na-salts (imidol-form):

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 1

 

 

Chemical structure of preparations – derivatives of barbituric acid

 

Preparation (drug)	Substituent				Formula (name)	Duration of action
	R (1)		R1 (5)	R2 (5)
Barbiturates
Barbital   (Barbitalum, Barbitone, Venoral, Diethyl- barbituric acid)		–	–C2H5	–C2H5	5,5- diethyl- barbituric acid	Long hypnagogue
Phenobarbital (Phenobarbitalum, Luminal)		–	–C2H5	–C6H5	5-ethyl-5-phenyl-barbituric acid or 5-ethyl-5-phenylpyrimidine-2,4,6(1H,3H,5H)-trione	Long hypnagogue, antiepileptic agent
Benzobarbital (Бензобарбитал–Международное наименование)  Benzonal (Benzonalum)			–C2H5	–C6H5	1-benzoil-5-ethyl-5-phenylbarbituric acid	Not hypnagogue action, antiepileptic agent
Sodium salts of barbiturates
Barbital sodium (Barbitone sodium, Barbitalum-Natrium)	–		–C2H5	–C2H5	sodium 5,5-diethylbarbiturate	Long hypnagogue
Hexobarbital (Hexenal, (Hexenalum)	–CH3		–CH3		1,5-dimethyl-5 (cyclohexene-1¢–yl)-barbiturate sodium or 1,5-dimethyl-5 (tetrahydrobenzene-1¢–yl)-barbiturate sodium	Short-term hypnagogue, narcotic (intravenously narcosis)
Amytal Sodium (amobarbital sodium), Barbamyl (Barbamylum, Sodium amital)	–		–C2H5		Sodium 5-ethyl-5-isoamyl-barbiturate	Average hypnagogue
Tiopental sodium (Thiopentalum-Natrium)	–		–C2H5		sodium derivative of 5-ethyl-5-[(1RS)-1-methylbutyl]-2-thioxo-2,3-dihydropyrimidine-4,6(1H,5H)-dione or Sodium 5-ethyl-5(2′-amyl)-2-thyobarbiturate	Strong hypnagogue, short-term, narcotic

Synthesis of barbiturates

Derivatives of barbituric acid received by condensation of urea and corresponding esters of malonic acid. Therefore synthesis consists of two stages.

                  1. Synthesis corresponding esters of malonic acid:

 

 

 

 

 

2. Condensation of ester with urea in the presence of Na-alcoholate in solution of absolute alcohol.

For example, synthesis of barbital it is possible to present by the scheme:

 

 

 

 

 

 

 

 

 

 

Properties (the description and solubility) are induced in separate table 2.

 

Identification

1. Formation of the painted complexes with salts of heavy metals (AgNO₃, Co (NO₃) ₂ in the presence of CaCl₂, CuSO₄ in the presence of КНСО₃ and К₂СО₃, CuSO₄, etc.) – see table 2.

To notice, pharmacopoeial (SPU) reaction (a) for all barbiturates (except tiopental-sodium) is  reaction with solution Co (NO₃) ₂ and CaCl₂ with formation of violet-dark blue colouring and precipitate. Therefore this is group reaction. Reaction should is spent in the neutral medium that precipitates Me (OH) _n did not drop out¯.

 

a) SPU. Identification of barbiturates (except N-replaced). Formation of the painted complex with solution of cobalt (ІІ) nitrate at presence calcium chloride

About 5 mg of an investigated substance dissolve in 3 ml CH₃OH, add 0,1 ml of a solution which contains 100g/l cobalt (ІІ) nitrate Co (NO₃) ₂ and 100 g/l calcium chloride CaCl₂ R, mix and add at stirring of a solution of 0,1 ml NaOH diluted; there is a violet-dark blue colouring and precipitate is formed.

                                                                                                         violet-dark blue colouring

                                                                                                        and precipitate

b) SPU. Formation of the painted complex with solution of copper (ІІ) sulphate (CuSO₄) in the presence of alkali (solution 10 g/l NaOH R) and potassium carbonate (К₂СО₃ R) and hydrogencarbonate (КНСО₃)

0,1 г a preparation shake up about 1 ml of a during 1–2 mines, add 0,2 ml of a solution калий гидрогенкарбоната Р and калий carbonate, 0,1 ml of a solution купрум (ІІ) sulphate Р; instantly there is the deposit of is which is not changing at standing:

                                                     pale-lilac colour of precipitate

! This reaction use for difference of one barbiturate from another as in each case precipitates and complexes of various colour (see table 2) are formed.

 

2. Fusion with alkalis

At fusion with sodium hydroxide NaOH molecules of barbiturates collapse, forming salts corresponding disubstituted of acetic acid derivatives, ammonia NH₃ (product of decomposition of urea) and sodium carbonate Na₂CO₃.

 

 

If a product of fusion to dissolve in water and to acidify by diluted HCl (or H₂SO₄), allocation of vials of gas CO₂ is observed:

Na₂CO₃ + 2HCl = 2NaCl + H₂O + CO₂­

Also there will be a characteristic smell corresponding disubstituted acid (for example, diethylacetic acid has a smell rancid butter (oil), 2-phenylbutyric acid  – an acacia smell)

3. Reactions of condensation with aldehydes and the concentrated sulphatic acid

At heating with formaldehyde НСНО and concentrated sulphatic acid H₂SO₄ (the Mark reactant) the products painted in various colour are formed: phenobarbital and benzonal – pink colouring (for phenylacetic acid); hexenal – dark red colouring with green fluorescence.

            From steam-dimethylaminobenzaldehyde and concentrated H₂SO₄:

 

 

 

 

barbital – yellow colouring; barbamyl – red-brown colouring and green fluorescence.

 

4. Interaction with solution of chloride acid (for sodium salts of barbiturates – barbital-sodium, hexenal, barbamyl).

At interaction of a solution of a preparation with HCl there is a reaction of neutralisation with formation precipitate of barbiturate. The precipitate of barbiturate filter, wash out water, dry and identify on fusion temperature, and in a filtrate find out Sodium ions.

 

 

 

 

 

 

 

 

 

 

Reactions for definition of functional group (in position 1 and 5,5)

1. Reactioitration (for phenylic radical –C₆H₅) (phenobarbital and benzonal)

At heating of preparation, containing in a molecule benzoic cycle, with concentrated sulphatic acid H₂SO₄ and solution of sodium nitrate NaNO₃ (or a mix conc. HNO₃ and H₂SO₄) occurs nitration in meta-position with formation of nitroderivative yellow colour.

 

                                        

 

 

 

 

 

                                                                                          nitroderivative yellow colour

2. Reaction for benzoate-ion after alkaline hydrolysis of  preparation (benzonal).

            SPU. To 1 ml of solution add 0,5 ml solution of iron(ІІІ) chloride R1; the pale yellow precipitate, soluble in ether R is formed

            3. Reaction for double bound (hexenal) –decolouration solution of potassium permanganate KMnO₄ or bromic water Br₂.

 

 

 

 

 

 

 

 

  4. Reactions for revealing Sulphur (thiopental sodium)

     a) At preparation heating at presence sodium hydroxide NaOH and lead acetate (CH₃COO) ₂Pb is formed a black precipitate – lead sulphide PbS.

              

                                                      black precipitate

b) At mineralization of  preparation with mix for sintering (mix Na₂CO₃ and NaNO₃) Sulphur passes in anions SO₄^{2 –} which reveals by means of solution BaCl₂:

SO₄^{2 –} + Ba^{2 +} → BaSO₄ ↓

                                                                                     white precipitate

 

5. Reaction with silver nitrate AgNO₃ in the medium of soda Na₂CO₃

            At interaction with ions Ag ⁺ are formed one-substituted (soluble in water) and two- substituted (insoluble in water) Silver salts. In the presence of Na₂CO₃ are at first formed Na-salt, and then Ag-salt in positions 4 and 6.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Tests for cleanliness

            Specific impurities – semisynthesis products

1. Barbital-ethylbarbituric acid

Preparation dissolve at boiling in water; the solution should be colourless and transparent. The cooled solution filter. The filtrate should not give pink colouring from addition of 1 drop of methyl orange solution.

2. Phenobarbital – phenylbarbituric acid

Preparation dissolve in 10 % solution anhydrous sodium carbonate Na₂CO₃; the solution should be colourless and transparent.

Preparation dissolve at boiling during 1 mines in water, cool and filter. The filtrate should not give pink-orange colouring from addition of 1 drop of methyl red solution.

3. Barbamyl, thyopental-sodium, hexenal – methanol

Preparation dissolve in water, add diluted sulphatic acid H₂SO₄, stir up during 2–3 mines and filter. To filtrate add potassium permanganate solution KMnO₄ in H₃PO₄, mix and leave for 10 minutes. Then add drops the sated solution of sodium sulphite Na₂SO₃ to solution discoloration,  solution of sodium salt of  chromotrope acid, concentrated sulphatic acid H₂SO₄ and mix.

Colouring of test solution should not exceed standard colouring.

 

Assay of barbiturates

         For quantitative definition of barbiturates various methods are used:

            1. Titrimetric:

                        a) acid- base titration in water, aqueous-alcoholic and non-aqueous mediums;

                        b) argentometry;

                        c) bromatometry;

                        d) iodo-chlorometry (for barbiturates with nonsaturated bonds, for example, hexenal).

            2. Gravimetry.

            3. Fotocolorimetry.

 

            1. Alkalimetry, non-aqueous titration

            This method is applied to quantitative definition of barbital, phenobarbital, benzonal.

Shot of preparation dissolve in a mix dimethylformamide (DMFA) and benzene С₆Н₆ (1:3), preliminary neutralised on thymol dark blue in DMFA (protophilic solvent, strengthens acid properties of barbiturates) and titrate with sodium methylate (sodium methoxide) CH₃ONa or sodium hydroxide NaOH in the mix of methanol CH₃OH and benzene C₆H₆ before occurrence of dark blue colouring.

            This method is based on ability of barbiturates to tautomeric transformations and formation imidolicor the aci-form having acid character, under the scheme:

           

 

 

a) Titrant – sodium methylate CH₃ONa:

 

 

E_m = М m

            b) Titrant – sodium hydroxide NaOH:

 

E_m = М m.

            2. Br. Ph. (The British Pharmacopoeia, 1998). Alkalimetry, non-aqueous substitute? titration (phenobarbital, etc.)

            0,100 g substance dissolve in 5 ml pyridine R, add 0,5 ml of thymolphthaleine solution R,   10 ml 87 g/l silver  nitrate AgNO₃ in pyridine R and titrate with 0,1 M sodium hydroxide NaOH in the ethanol before not disappearing blue colouring.

 

           

 

 

 

 

3. Alkalimetry in the aqueous-alcoholic medium

            The method is suitable for quantitative definition of any barbitrate, having acid character. As titrant use solution sodium hydroxide NaOH, as indicator – thymolphthaleine.

            Shot of preparation dissolve in neutralised on thymolphthaleine alcohol С₂Н₅ОН (for solubility improvement of barbiturates  and preventions of hydrolysis formed sodium salt).

 

 

E_m = М m.

            4. Acidimetry in the water medium

This method is suitable for quantitative definition of barbiturates sodium salts, having basic character (barbital-sodium, barbamyl, hexenal). As titrant use chloride acid HCl, as indicator – methyl orange or methyl red.

Na-salts of barbiturates are hydrolyzed in water solutions, forming the alkaline medium (рН 77) and consequently them it is possible titrate with acids, for example, chloride acid HCl in the presence of methyl orange or methyl red as indicator (before pink colouring).

 

 

E_m = М m.

Thus titrates by means of acid and the free alkali NaOH, arising at hydrolysis of  preparation:

 

At quantitative definition of thiopental sodium by means of acidimetry define the total maintenance of Sodium (titrate with sulphatic acid H₂SO₄ in the presence of methyl red as indicator).

5. Argentometry

1. Method of  Fialkov and employees (benzonal)

Shot of  substance (the acid or salt form) dissolve in 5 % anhydrous solution of sodium carbonate Na₂CO₃ and titrate with nitrate AgNO₃ without the indicator on occurrences of not disappearing dregs (the two-substituted Ag-salt).

TITRATE TO OPALESCENCE, whe formed monosilver salt we stoped titration – end point!

Proceeding processes it is possible explanes so.

At first barbiturate it is dissolved in sodium carbonate Na₂CO₃ with formation one- substituted Na-salt, which reacts with silver nitrate AgNO₃ with formation soluble one- substituted Ag-salt. Then soluble Na-Ag-salt is formed.

In equivalence point excess of titrant AgNO₃ destroys Na-Ag-salt and the insoluble two- substituted Ag-salt is formed, that specifies in the titration end.

 

 

Em = M.m.

 

6. Bromatometry, back titration, with iodometric finishing

The method is used for quantitative definition of barbiturates with nonsaturated bound, for example, hexenal.

This method is based on bromination substance in a place of double bound.

To defined volume of aliquout an investigated water solution of substance (for example, hexenal) in a flask with the ground in glass stopper add excess of standard solution bromide-bromata (solution КBrO₃, KBr), chloride acid R, close a stopper, maintain during 30 mines, periodically mixing and leave for 15 minutes.

KBrO₃ + 5KBr + 6HCl = 3Br₂ + 6KCl + 3H₂O

Allocated bromine Br₂ reacts with hexenal with formation dibromderivative:

 

 

Not reacted bromine Br₂ reacts with potassium iodide KI with formation iodine I₂:

Br₂ + 2KI = I₂ + 2KBr

The allocated iodine titrate with standard solution of sodium thiosulphate Na₂S₂O₃ in the presence of starch before disappearance of dark blue colouring (add starch towards the end of titration):

I₂ + 2Na₂S₂O₃ = 2NaI + Na₂S₄O₆

   I₂ + 2е 2I ^–

2S₂O₃^{2 –} – 2е S₄O₆^{2 –}

In parallel spend control experience.

Е_m (hexenal) =М.м./2

            7. Iodo-chlorometry, back titration

This method is used for quantitative definition of barbiturate with nonsaturated bound (for example, hexenal).

To certain volume of investigated solution (for example, hexenal) add excess of standard solution of iodochloride ICl (reacts with hexenal in a place of double bound in cyclohexenyl group):

 

Not reacted iodochloride ICl define by means of iodometry: add potassium iodide KI, excess of  iodochloride reacts with KI with formation of iodine I₂, which titrate with standard solution of sodium thiosulphate Na₂S₂O₃ (as indicator – starch solution).

ICl + KI = I₂ + KCl

I₂ + Na₂S₂O₃ = 2NaI + Na₂S₄O₆

In parallel spend control experience.

Е_m = М m./2

8. Gravimetry

Gravimetric method usually apply for quantitative definitions of barbiturates Na-salts (for example, thiopental sodium), and also at the analysis of medicinal mixes.

The method essence consists that to water solution of preparation add diluted chloride acid HCl.

 

The received acid form (thiopental-acid) extract by means of chloroform (5 times in the small portions). All chloroformic extraction connect, chloroform distillates, and the rest dry at 70°C to constant weight (mass) and weigh.

 

Storage. Group of strong preparations. In dense corked container. Hygroscopic preparations – in the dry, cool place, protected from light.

Phenobarbital and benzonal – in banks of dark glass, in the place protected from light.

Hexenal and thiopental sodium – in glass bottles on 0,5–1,0 g, which are hermetically closed by rubber stoppers, are fitted by aluminium caps; in the dry, cool place protected from light. As the stabilizer to hexenal add 0,05–0,25 % sodium hydroxide NaOH, to thiopental sodium –      5–6 % of sodium carbonate Na₂CO₃.

Water solutions of barbiturates Na-salts easily hydrolyze, therefore them prepare on a physiological solution in aseptic conditions directly ahead of the use (ex tempore).

Application (see table 2)

Sedative and hypnagogue:

    a) Long action – barbital, phenobarbital, barbital sodium;

    b) Average duration – barbamyl;

    c) Shot-term action – hexenal, thiopental sodium.

Protiepileptic (anticonvulsant) means: phenobarbital and benzonal (hypnagogue action has not).

For intravenous narcosis: hexenal and thiopental sodium.

At long application and high doses of barbiturates can be a poisoning, therefore their application should be supervised by the doctor.

In case of barbiturates poisoning applied stimulators of the central nervous system – strychnine, corasole, etc. Subsequently it has been established, that the antagonist of barbiturates is bemegride.