LECTURE № 13
Theme: Derivatives of pyridine as
drugs. Derivatives of isonicotinic
acid as antitubercular agents
Associate prof. Mosula L.M.
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.
Hexatomic heterocycles as drugs
To hexatomic heterocycles with one heteroatom of
Nitrogene belong pyridine:
4
5
3
6
2
N
1
Completely hydrogenated pyridine (saturated heterocycle)
names piperidine:
4
5
3
6
2
N
H 1
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.
NO2
KNO3
N
H2SO4
3000C
N
β-nitropyridine
pyridine
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.
+
N
H
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 :
_
OH
+ HOH
+ HCl
+
+
N
N
+
N
Cl
H
H
pyridine base
_
pyridine
salt of pyridine
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 4methylpyridine (γ-pikolin) to corresponding acids – nicotinic (pyridin3-carboxylic acid or β-pyridincarboxylic acid) and isonicotinic
(pyridin-4-carboxylic acid or γ-pyridincarboxylic acid) acids:
O
3 C
3 CH3
[O]
4
4
3
3
[O]
2
2
N1
OH
COOH
CH3
N
1
2
2
N 1
N
1
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: 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-3carboxylic acid:
O
C
NH2
N
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)
Diaethylamidum acidi nicotinici
Diethylamide nicotinic acid
Nicethamidum*
O
C
N
C2H5
C2H5
N
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-3carboxamide, 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):
O
O
C2H5
C
OH
+
HN
C2H5
C
POCl3
N
- H2O
C2H5
N
N
Or by means
methylpyridine):
of
oxidation
-picoline
of
O
CH3
C
[O]
N
C2H5
OH
N
(3-
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
O
C
N
O
C2H5
+ NaOH
C
t 0C
ONa
C2H5
N
N
+ 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.
R
_ 2HOH
Br
+ BrSCN
+
N
N
R
NH2 + HO
CH
C
R
HC
C
C
C
O
SCN
R
H
C
R
H
+ NH2SCN
OH
+ HBr
H
O
CH
CH
+ H2N
C
R
H
_ 2H O
2
R
R
NH
CH
C
CH
CH
C
H
N
R
yellow colour
TESTS
The 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).
O
O
N
N
C2 H5
+ HClO4
+
C2 H5
C2 H5
N
N
C2 H5
* ClO4-
H
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
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).
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
n  n0
С% 
F
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
1,383  1,333
С% 
 25%
0,002
It is possible to calculate the maintenance of operating substance (in
g) in 1ml injection solution:
1,383 1,333
Х,g 
 0,25g
n  n0
Х,g 
0,002100
F  100
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
O
C
H
N
N
С7Н8N2O2
CH2OH
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:
O
O
NH2 +
H
H
N
C
H
N
C
O
C
N
CH2OH
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 in blue).
O
C
O
H
N
N
C
0
t C
O
ONa
+ NaOH
CH2OH
N
+ NH3
+ H C
H
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.
R
1
NO2
6
2
+
+
N
C2H5OH
3
5
N
R
R
Cl
_
Cl
t 0C
_
+
N
NaOH
- NaCl
Cl
NO2
NO2
4
_
OH
NO2
:
NH
CH
C
NO2
NO2
R
O
CH
CH
C
NH 2
H
NO2
NO2
+ HOH
+
OH
R
CH
C
O
CH
CH
C
H
NO2
NO2
orange-red colour
3. Decomposition of preparation with
the next revealing of
formaldehyde by means of disodium salt of chromotropic
acid
O
C
O
H
N
N
C
t 0C
O
NH2
CH2OH
N
+
H
C
H
Formaldehyde identification by means of disodium salt of
chromotropic acid:
SO3Na
HO
NaO3S
H
H
O
+
H
C
OH
H2SO4(conc)
_ HO
H
HO
OH
SO3Na
[O]
HO
O
H2O
OH
HO
SO3H
CH2
HO3S
red-violet colour (aurin dye)
4. Melting point 147 to 149 °С.
OH
OH
HO
SO3H
HO3S
CH
HO
HO3S
2
NaO3S
SO3H
_
SO3H
HO3S
[O]
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).
O
O
C
C
H
CH2OH
N
O
H
NaIO + NaI + H2O
O
+1
+ NaIO + NaOH
C
H
-1
+ NaI + H2O
C
ONa
H
NaI + NaIO + H2SO4
+ NH3
+ H C
N
I2 + 2NaOH
H
ONa
+ NaOH
N
O
I2 + Na2SO4 + H2O
I2 + 2Na2S2O3 = 2NaI + Na2S4O6
Em (С7Н8N2O2) = М.m/2
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).
Derivatives of isonicotinic acid
Isonicotinic acid (pyridine-4-carboxylic acid):
OH
O
N
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:
R
CH
N
NH
C
S
NH2
In due course high physiological activity has been
revealed in derivatives of isonicotinic acid. Such
derivatives concern:
O
C
NH
NH2
N
hydrazide of isonicotinic acid
(However it in small doses slow-acting, and in big doses – is toxic).
Hydrazones of isonicotinic acid:
O
C
R
NH
N
C
H
N
Hydrazones – are the products of interaction hydrazide of
isonicotinic acid with aldehydes:
O
O
C
C
NH
NH2
NH
O
H
N
C
H
C
+
N
R
R
- H2O
N
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.
Isoniazid
General Notices
(Ph Eur monograph 0146)
Izoniazidum
Tubazidum
O
C
NH
Nicozid
NH2
N
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 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.
O
O
OCH3
OH
[O]
N
4-methylpyridine
N
O
C
C
CH3
HOCH3
H2N-NH2
H2SO4
-H2O
- CH3OH
N
pyridine-4-carboxylic acid methyl ester
of isonicotinic acid
C
NH
N
isoniazid
(γ-picoline)
NH2
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) 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).
O
O-
OH
C
C
HN
NH2
O
C
N
NH2
C
N
CuSO4
NH2
OH
Cu2+
t
+ N2
+ Cu2O
H2O
N
N
N
2
blue precipitate
N
green
precipitate
TESTS
The 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
O
O
N
N
H
NH2
+ 2 Br2 + H2O
OH
N
+ N2 +
4 HBr
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
Phthivazidum
Vanicide
Vanillaberon
Ftivazidum
Vanizide
SP X
O
C
NH
N
CH
.
OH H2O
OCH3
N
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.
Synthesis
Condensation of isoniazid with vanilline under the scheme:
O
O
C
NH
NH2
+
O
C
NH
C
OH
isoniazid
OCH3
CH
OH
-H2O
H
N
N
OCH3
N
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.
O
C
O
C
NH
N
CH
OH
+HOH
HCl
t 0C
OCH3
O
OH
+ H2N-NH2 +
C
OH
H
N
OCH3
N
smell of vanillin
2. Reaction with 2,4-dinitrochlorbenzole in ethanol (for pyridine
cycle) (see isiniazid)
3. Reaction of alcoholic solution of preparation with alkali
Reaction confirms amphoteric properties of phthivazid.
C
N
O
NH N CH
O
C
OH NaOH
NH N CH
HCl
OCH3
N
ONa HCl
OCH3
C
O
NH N CH
+
N Cl-
OH
OCH3
H
orange-yellow colour
Tests
1. Hydrazide of isonicotinic acid (isoniazid, specific inadmissible
impurity)
In the presence of impurity of isoniazid there is reaction:
O
O
C
NH
NH2
C
N
+ NaNO2 + HCl
N
+
N
_
N
+ NaCl + H2O
N
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.
O
C
H
O
C
+ NaOH
H3CO
+ H2O
H3CO
OH
H
ONa
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).
C
O
NH N CH
OH
+ HClO4
CH3COOH
C
OCH3
+
N
N
H
Em = М.m.
O
NH N CH
OH
OCH3
. ClO_4
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)
6
7
5
8
O
9
N
NH
C
N
1
4
3
2
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 greenishyellow 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!