LECTURE LECTURE № № 1313

Theme: Theme: Derivatives of pyridine as Derivatives of pyridine as drugs. Derivatives of isonicotinic drugs. Derivatives of isonicotinic

acid as antitubercular agentsacid as antitubercular agents

Associate prof. Mosula L.M.Associate prof. Mosula L.M.

The planThe plan

1. 1. Derivatives of pyridine-3-carboxylic Derivatives of pyridine-3-carboxylic (nicotinic) acid(nicotinic) acid as drugs: Nikethamide, as drugs: Nikethamide, Nikethamide injection, Nicodine.Nikethamide injection, Nicodine.

2. 2. Derivatives of pyridine-4-carboxylic Derivatives of pyridine-4-carboxylic (isonicotinic) acid(isonicotinic) acid as antitubercular as antitubercular agents: Isoniazid, Phthivazid, agents: Isoniazid, Phthivazid, Flurenizidum.Flurenizidum.

Hexatomic heterocycles as drugsHexatomic heterocycles as drugs

N 1

2

3

4

5

6

NH 1

2

3

4

5

6

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) -

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

is easier hydrogenated.

N

KNO3

H2SO4

N

NO2

3000C

N

H

+

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.

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 :

N

H

+OH

_+

+ HOH

N

+ HCl

N

H

+ Cl_

pyridine base pyridine salt of pyridine

N

CH3

[O]

N

CO

OH

12

3

1

2

3

N

[O]

N12

3

1

2

3

CH3

4

COOH4

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:

Drugs – derivatives of nicotinic acidNicotinic 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: 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.

N

C

O

NH2

Nikethamide(Ph Eur monograph 0233)

Diaethylamidum acidi nicotiniciDiethylamide nicotinic acid

N

C

O

NC2H5

C2H5

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.

Nicethamidum*

N

C

O

NC2H5

C2H5N

C

O

OH + HNC2H5

C2H5

POCl3- H2O

N

C

O

OH

N

CH3

[O]

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):

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 in blue.

N

C

O

NC2H5

C2H5

+ NaOHt 0C

N

C

O

ONa + HN(C2H5)2

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.

_

N

R

+ BrSCN

N

R

SCN

+Br

C

HC

O H

HC

CR

C OH

H

+ NH2SCN

+ HBr

2HOH

CH C CH

R

CH CO

HHONH2R + + H2N R

_

NHR CH C CH

R

CH CH

N R

2H2O

yellow colour

TESTSThe inadmissible impurities are not presents in the test substance

ASSAY(BrPh). 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.

Action and use Respiratory stimulant.

Preparation Nikethamide Injection (25%).

Ph Eur

N

O

NC2H5

C2H5

HClO4

N+

H

O

NC2H5

C2H5* ClO4-+

Nikethamide InjectionCordiamine (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).

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

Fnn%С 0

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

%25002,0

333,1383,1%С

It is possible to calculate the maintenance of operating substance (in g) in 1ml injection solution:

100, 0

F

nngХggХ 25,0

100002,0333,1383,1,

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 BilamidumCholamidum

N

CO

N

CH2OH

H

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:

N

CO

N

CH2OH

H

N

C

O

NH2+ H C

O

H

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.

Identification1. 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).

N

CO

N

CH2OH

H

N

CO

ONa+ H C

O

H

+ NH3+ NaOH

t 0C

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.

:

N

CO

N

CH2OH

H

H C

O

H

t 0C

N

CO

NH2 +

N

R

+

Cl

NO2

NO2

1

2

3

45

6C2H5OH

t 0CN

R

NO2

NO2

+ Cl_

NaOH- NaCl

N

R

NO2

NO2

+ Cl_

OH_

NO2

NO2

NH CH C CH

R

CH CO

HNO2

NO2

NH2

CH C CH

R

CH CO

H

OH

++ HOH

orange-red colour3. Decomposition of preparation with the next revealing of formaldehyde by means of disodium salt of chromotropic acid

Formaldehyde identification by means of disodium salt of chromotropic acid:

HO

HO

SO3Na

SO3Na

H H

NaO3S

NaO3S

OH

OHH C H

O

H2O

HO

HO

SO3H

SO3H

CH2

HO3S

HO3S

OH

OH

+ H2SO4(conc)_

[O]

[O]

H2O_

HO

HO

SO3H

SO3H

CH

HO3S

HO3S

O

OH

red-violet colour (aurin dye)

4. Melting point 147 to 149 °С.

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).

N

CO

N

CH2OH

H

N

CO

ONa+ H C

O

H

+ NH3+ NaOH

H C

O

H

I2 + 2NaOH NaIO + NaI + H2O

+ NaIO + NaOH H CO

ONa

+ NaI + H2O+1 -1

NaI + NaIO + H2SO4 I2 + Na2SO4 + H2O

I2 + 2Na2S2O3 = 2NaI + Na2S4O6

Em (С7Н8N2O2) = М.m/2

StorageIn 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).

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:

N

O OH

R CH N NH C

S

NH2

In due course high physiological activity has been revealed in derivatives of isonicotinic acid. Such derivatives concern:

N

C

O

NH NH2

hydrazide of isonicotinic acid(However it in small doses slow-acting, and in big doses – is toxic).

N

C

O

NH N CR

H

Hydrazones of isonicotinic acid:

Hydrazones – are the products of interaction hydrazide of isonicotinic acid with aldehydes:

N

C

O

NH N CR

H

N

C

O

NH NH2

+ C R

O

H- H2O

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.

IsoniazidGeneral Notices (Ph Eur monograph 0146)

Izoniazidum Tubazidum Nicozid

N

CO

NH NH2

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 of methyl ester of isonicotinic acid and its

condensation with hydrazineInitial 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.

N

CO

NH NH2

N

CH3

N

CO

OH

N

CO

OCH3

[O] HOCH3

H2SO4-H2O

H2N-NH2

- CH3OH

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):Reaction with solution of copper (ІІ) sulphate0,1 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).

N

C

O

HN NH2

N

C

OH

N NH2

CuSO4

N

C

O

OH

Cu2ON2H2O

N

C

O-

N NH2

Cu2+

2

+ +t

blue precipitate green precipitate

TESTSThe inadmissible impurities are not presents in the test substance.

ASSAY (BrPh). 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. 1 ml of 0.0167 M potassium bromate is equivalent to 3.429 mg of C6H7N3O.

KBrО3 + 5KBr + 3H2SO4 → 3Br2 + 3K2SO4 + 3H2O

N

C

O

NH

NH22Br2

N

O

NH

NH2

Br

Br

2HBr

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

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

Phthivazid SP X

Phthivazidum VanicideVanillaberonFtivazidum Vanizide

N

CO

NH N CH OH

OCH3

. H2O

С14Н13N3O3Н2О 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.

N

CO

NH NH2

N

CO

NH N CH OH

OCH3

C OH

OCH3

O

H

+-H2O

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.

Identification1.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)

N

CO

OH

N

CO

NH N CH OH

OCH3

C OH

OCH3

O

H

+HOHHClt 0C

+ H2N-NH2 +

3. Reaction of alcoholic solution of preparation with alkali Reaction confirms amphoteric properties of phthivazid.

orange-yellow colour

N

CONH N CH ONa

OCH3N

CONH N CH OH

OCH3

N

CO

NH N CH OH

OCH3

H

+

Cl-

HClNaOH

HCl

Tests1. Hydrazide of isonicotinic acid (isoniazid, specific inadmissible

impurity)In the presence of impurity of isoniazid there is reaction:

N

CO

NH NH2

+ NaNO2 + HCl

N

CO

N N N+ _

+ NaCl + H2O

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

5NaNO2 + 2KIО3 + 2HCl → I2 + 2KCl + 5NaNO3 + H2O

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.

C

OH

H3CO

O H

+ NaOH

C

ONa

H3CO

O H

+ H2O

AssayAcidimetry, 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).

N

CONH N CH OH

OCH3

+ HClO4

N

CONH N CH OH

OCH3

CH3COOH

H

+

ClO4. _

Em = М.m.

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).

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

N NH C

O

N

1

23

4

5

6 7

8

9

M = 299,33 g/mol

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

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.

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).

Thanks for attention!