Transcript 05.Antibiotics of the aromatic and heterocyclic rows

Lecture №5
Antibiotics of the aromatic row:
synthesis, stereoisomery, methods of
analysis. Antibiotics of the heterocyclic
structure: β-lactam antibiotics.
ass. Medvid I.I,
Antibiotics of the aromatic row
Chloramphenicol (Chloramphenicolum) (SPhU)
Laevomycetin (Laevomycetinum)
O
1
HN
O2N
4''
H 2'
C
C
1'
C
H
2
CHCl2
CH2OH
OH
2,2-dichloro-N-[(1′R,2′R)-2′-hydroxy-1′-(hydroxymethyl)-2′-(4"nitrophenyl)ethyl]acetamide
D-(-)-threo-1 -p-Nitrophenyl-2-dichloroacetylaminopropanediol-1,3
Laevomycetin – fine white with a grey or yellowish-green tinge
crystalline substance, odorless. Bitter taste. Slightly soluble in water,
soluble in alcohol. Solution of the substance in ethanol rotates the
plane of polarization on the right, in ethylacetone – on the left.
Leavomycetin stearate
(Laevomycetini stearas)
O
HN
O2N
H
C
OH
C
H
C
CHCl2
CH2O
C
C17H35
O
D-(-)-threo-1-p-Nitrophenyl-2-dichloroacetylaminopropanediol1,3-3 –atearate
Laevomycetin stearate - white with a yellowish or yellowishgreen tinge crystalline substance, odorless. Laevomycetin
stearatehas no taste. Practically insoluble in water, difficult
soluble in alcohol, in all solvents forms a cloudy solutions.
Leavomycetin succinate soluble
(Laevomycetini succinas solubile)
O
HN
O2N
H
C
OH
C
H
C
CHCl2
CH2O
C
(CH2)2 COONa
O
D-(-)-threo-1 -p-Nitrophenyl-2-dichloroacetylaminopropanediol1,3-3-sodium succinate
Laevomycetin succinate soluble -white or slightly
yellowish porous mass with low specific smell. Laevomycetin
succinate soluble – has a bitter taste. Very easily soluble in water,
slightly soluble in alcohol, hygroscopic.
Leavomycetin molecule has 2 asymmetric carbon atoms, and
therefore it is possible the existence of four isomers: D- and Lthreo, D- and L-erythro-, which differ by the spatial
arrangement of functional groups:
Laevomycetin is a leftrotatory threo-isomer of the D row. Mixture of D() and L(+) threo-isomers of laevomycetin is called racemic mixture,
optically inactive compound known as synthomycin (has 50% of the
laevomycetin physiological activity). Erythro-forms is not used in
medicine because it is a toxic compounds.
Obtaining of laevomycetin
Laevomycetin first was isolated in 1947 year from the
cultural liquid of actinomycetes, in 1949 year was
established the chemical structure.
The drug is extracted by the growing of actinomycetes
Streptomyces venezuelae on the medium (broth, glycerol,
molasses and mineral salts) at 23-27 °С and strong
aeration for 89 hours. After the filtering of the mycelium
fungus antibiotic is extracted and purified by the
chromatographic.
Laevomycetin - the first antibiotic, which began to obtain
by the chemical synthesis, whereas the majority antibiotic
was obtained by biosynthesis.
Synthesis of laevomycetin
Extracted drug by the interaction with NaOH is transformed in a racemic
base of laevomycetine and divided into optical isomers by using of the
different solubility of salts, formed with dextrorotatory tartaric acid, in
methanol. However, only salt of D-isomer is soluble in methanol. The
basis from solution is sedimented by the ammonia and acylated by the
methyl ester of dichloroacetic acid Cl2CHCOOCH3.
Identification
1.
2.
3.
By the physico-chemical constants: melting point, IR- and
UV-spectroscopy, TLC, specific rotation.
To the alcoholic solution of laevomycetin add calcium
chloride and zinc powder and heat. Filtrate the obtained
hot solution, after cooling add benzoylchloride. Then add
iron (ІІІ) chloride solution and chloroform, shake the
solution; aqueous layer should be pained in the color from
light violet-red to purple.
Reaction on chloride-ion after the mineralization of the
substance by the anhydrous sodium carbonate and
dissolution of the residue in the diluted nitrate acid.
4.
Hydrolysis in acidic or basic mediums with the
following identification of the formed products. Thus, at
the heating of laevomycetin with sodium hydroxide at
first yellow color appears, that transfers to the redorange (as a result of formation of acy-nitroform), after
the following heating brick-red precipitate formed and
smell of ammonia appeared:
5. Hydroxame reaction on laevomycetin esters.
6. Laevomycetin stearate at the heating with
concentrated chloride acid hydrolyzed – stearic
acid is formed, which floats to the surface in the
form of oily drops which harden when cooled:
7. By the reaction of azodyes red color formation, after
the reduction of nitro-group to amino-group with
following diazotation and azojoining:
8. In the express-analysis use reaction of
laevomycetin with copper (II) sulfate in alkali
medium in the presence of n-butanol – alcoholic
layer painted in blue-violet color due to the
formation of complex salt, which supposedly has
the following structure:
Assay
1. SPhU - Spectrophotometry. Laevomycetine content in the
substance Content of the laevomycetin in the substance is
calculated by the specific absorption.
2. Nitritometry after the previous reduction of nitro-group to
amino-group by the zinc dust in acidic medium. Е = М.m.
3. By liquid chromatography.
4.
5.
Cooper-metry, direct titration. The method is based on
the formation of soluble laevomycetin complex
compounds with copper (II) sulfate in alkaline medium
(look identification). Titrant - 0,01 М solution of cooper
(II) sulfate, indicator - murexide. Titration goes from the
purple to brownish-red color, uniform with color of the
“blind” sample. Е = 2 М.m.
Cooper-iodometry, direct titration by the substitute.
To the laevomycetin in alkali medium add cooper (II)
sulfate. Precipitate of cooper (ІІ) hydroxide is filtrated, in
the filtrate soluble cooper-laevomycetin complex is
destroyed by the action of sulfate acid with formation of
equivalent amount of cooper (II) sulfate, which is
determined by iodometry, indicator - starch. In parallel
control experiment is conducted. Е = 2 М.m.
CuSO4
Precipitate titration (argentometry or mercurymetry).
Methods based on the oxidation of laevomycetin by Н2О2
in alkali medium, as a result в 2 molecules of NaCl
formed, which are determined by argentometric method
(Folgard’s method) or mercurymetry with
diphenylcarbazone as indicator. Е = 1/2 М.m.
7. Photocolorimetry by the formation of azodye after the
reduction of of nitro-group to amino-group with following
diazotation and azojoining.
8. Iodometry. The method is based on the oxidation of the
products of alkaline hydrolysis of laevomycetin.
Experimentally determined Е = 1/6 М.m.
9. Bromatometry, reverse titration. Е = 1/4 М.m.
10. Acidimetry in non-aqueous medium after the acidic
hydrolysis.
6.
STORAGE
In airtight containers, in the protected from light place.
USAGE
Laevomycetine belongs to the broad-spectrum antibiotics,
used in the treatment of dysentery, pneumonia, whooping
cough, typhoid and other infectious diseases. Course of
treatment is 8-10 days. In pediatric practice use
laevomycetine stearate, which hasn't bitter taste.
Laevomycetine succinatecan be used for injections.
Issue: tablets, alcoholic solution, eye drops (0,25%),
ointments Levosin, Levomekol.
Side effects. Disorders of the function of hematopoietic
organs, so, at the treatment by laevomycetin blood test is
required. Can cause an overgrowth .
Antibiotics heterocyclic
structure
Penicillins
(pennames)
in the core of the penicillin's molecule is 6-aminopenicillinic acid (6-APA), which consists of the condensed
thiazolidine (А) and β-lactam (В) cycles:
COOH
O
7
H 2N
6
N1
5
CH3
2
3
4
S
CH3
Obtaining of the natural penicillins
For the obtaining of penicillin growth mouldy fungus Penicillium
notatum on the medium, which consists of corn extract, lactose and
different mineral salts. To increase the outlet of benzylpenicillin, to
the medium add 0,02 – 0,08 % of phenylacetic acid С6Н5СН2СООН
or its amide. Fermentation is conducted approximately for 70 hours
at 23-24 оС, рН = 6-6,5 and strong aeration (1 l of air on 1 l of
environment per 1 min.). After the end of fermentation the obtained
product is transformed by the scheme represented on the next slide.
To the separation of the different types of penicillin and
following purification of the penicillin sodium salt, this salt is
transformed into the penicillin salt with organic bases or purified by
chromatographic method. Water solution of benzylpenicillin sodium
salt is evaporated to the dry state at the temperature – 40оС in the
vacuum (0,1-0,2 mm of mercury column): ice, without melting,
transformed in pair, а preparation remained dry (liophillic drying).
For the manifestation of biological activity of great importance is βlactam cycle, which is very unstable in acidic and alkaline
environments. Penicillin is of the early era of antibiotics and
because of its wide usage now 80-90 % strains of staphylococci
developed resistance to it, producing the enzyme penicillinase,
which split the β-lactam cycle. Account this and the fact that
penicillins are the least toxic in the comparison with other
antibiotics, conduct a search of semi-synthetic penicillins on the
basis of 6-APA.
Semi-synthetic penicillins are an acylderivatives of 6-APA.
General formula of penicillin’s medicines
COOR1
O
O
R
C
CH3
N
N
H
S
CH3
Three generations of penicillins:
• Natural (benzylpenicillin Na, К or Novocain salt,
phenoxymethylpenicillin, Bicillin (1,3,5))
• Semi-synthetic penicillins (oxacillin, ampicillin,
amoxicillin, dicloxacillin, carbenicillin and others)
• Semi-synthetic antibiotics + inhibitor of -lactamases
(amoxiclav (augmentine), timentin).
Benzylpenicillin potassium (sodium) salt
Benzylpenicillinum kalicum (natricum) SPhU
H
O
H
N
7
N1
6
5
COOK(Na)
3
4
S
H
CH3
2
CH3
H
O
• Sodium (2S,5R,6R) – 3,3 – dimethyl – 7 – oxo – 6
[(phenylacetyl)amino] – 4 – thio – 1 –
azabicyclo[3.2.0]heptane – 2 – carboxylate
• Sodium salt of 6 – phenylacetylaminopenicilanic acid
Benzylpenicillin Novocain salt
Benzylpenicillinum novocainum
H
O
COO
CH3
N
H
N
CH3
S
H
-
*
H
O
C2H5
* H2N
C
O
O
C
H2
C
H2
N+
H
*H2O
C2H5
• Novocain salt of 6 –phenylacetylaminopenicilanic acid
Phenoxymethylpenicillin (ospen)
Phenoxymethylpenicillinum
H
O
COOH
CH3
N
H
N
S
O
H
O
CH3
H
6 – phenylacetylaminopenicilinic acid
• Bicillin - 1 (N,N’ – dibenzylethyldiammonium salt of
benzylpenicillin
• Bicillin - 3 (a mixture of equal parts of potassium (sodium)
benzylpenicillin salt, benzylpenicillin and bicillin-1Novocain salt)
• Bicillin – 5 (a mixture of 1part of benzylpenicillin Novocain salt
and 4 parts of bicillin – 1)
Bicillin-1(Вicillinum-1)
Benzathine Benzylpenicillin
(Benzathine Benzylpenicillinum)
COO
H
O
-
CH3
N
H
N
CH3
S
*
H
H
O
2
*
C
H2
+
N
H2
C
H2
C
H2
+
N
H2
C
H2
N,N′ – dibenzylethylenediammonium salt of benzylpenicillin
Oxacillin sodium salt
Oxacillinum natrium
H
O
COONa
CH3
N
*H2O
H
N
S
H
N
CH3
H
O
O
CH3
Sodium salt of 3-phenyl-5-methyl-4isoxazolylpenicillin monohydrate
Ampiox (ampicillin and oxacillin sodium salts)
Ampicillin trihydrate (SPhU)
Ampicillinum trihydricum
H
O
NH2
2'
7
1'
H
N
6
H
N
5
COOH
CH3
2
1
3
4
S
*3H2O
CH3
H
O
(2S,5R,6R)-6-[[(2′R)-2′-amino-2′-phenylacetyl]amino]-3,3dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2carboxylic acid trihydrate
Also pharmacopoeial preparation is Ampicillin sodium
salt.
Amoxicillin trihydrate (SPhU) Amoxicillinum
trihydricum
amoxyl, gramox, ospamox, hiconcil, Phemoxinsolutab
H
O
NH2
CH3
N
H
N
S
H
HO
COOH
CH3
H
O
2S,5R,6R)-6-[[(2′R)-2′-amino-2′-(4′′′-hydroxyphenyl)-acetyl]amino]-3,3dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid
trihydrate
Vampiox (amoxicillin trihydrate + cloxacillin + Lactobacillus)
Dependence between the chemical structure
and biological action of penicillins
1 – character of the radical which derermines
the level of bounding with proteins;
2 – Substitute in о-position of
the phenyl radical influences
on the stability to the penicillinase;
3 – character of the bounding of the phenyl
radical with methyl group
determines the acidic stability of the penicillin;
4 – substitute of the Hydrogen atom in the methyl group в determines the
spectrum of penicillin action;
5 – splitting of the β-lactam bound conducts to the disappearance of
antibiotic properties and appearance of the allergic action;
6 – substitute in the carboxylic group gives the possibility to obtain the
penicillin salty forms;
P – penicillinase splits β-lactam cycle;
А – amylase splits amide bound.
CHARACTERS
Medical drugs of the natural and semisynthetic penicillins -white crystalline
substance, odorless, bitter taste. Sodium and
potassium benzylpenicillin salts
are
hygroscopic and easily soluble in water.
Benzylpenicillin
Novocain
salt,
phenoxymethylpenicillin and ampicillin few
soluble in water.
Water or alcohol solutions penicillins rotate
plane polarized beam to the right.
Identification
By the physico-chemical constants: IR- and UVSpectroscopy, TLC.
Reaction with formaldehyde in the presence of
concentrated sulfate acid (Marki reagent). Reaction is
distinguished, because each penicillin produces at these
conditions characteristic color (benzylpenicillins reddish-brown color, amoxicillin - dark yellow, etc.).
Substances give reactions on potassium, sodium and
Novocain.
Unpharmacopoeial reactions:
а) reaction of copper (II) (green) or iron (III) (red color)
penicilloinhydroxamates
formation
after
the
hydroxylaminolysis (β-lactam cycle):
b) reaction with chromotropic acid in the presence of
concentrated sulfate acid, which is distinguished, because
each penicillin produces at these conditions characteristic
color (benzylpenicillins - brown color, amoxicillin – violet
ets.);
c) determination of the organically bound sulfur (dry and wet
pyrolysis);
d) determine the melting temperature of the N-ethylpiperidine
salt of benzylpenicillin (for natural penicillins);
e) reaction on aliphatic amino-group (ampicillin, amoxicillin)
– at the heating with a ninhydrin solution observed purple
color .
f) Vitali – Moren’s reaction.
Reaction of the azo-dye of amoxicillin formation (red color)
Penicillins due to the Sulfur atom have the reducing
properties and can reduce silver from the Tollense reagent,
mercury from the Nesler reagent ets..
Assay
By the liquid chromatography (SPhU).
Microbiological methods of the diffusion in agar
(reproducing of the results - 5-10 %).
Spectrophotometric determination of the semi-synthetic
penicillins.
Chemical methods in two phases:
а) determination of the penicillins amount;
b) identify the content of the relevant drug.
The amount of penicillins for the medical drugs of natural
penicillins determine by iodometric method, the essence
of which is that the products of alkaline hydrolysis of
penicillin can be oxidized by iodine in the presence of
acetic buffer with рН = 4,5. Е = М.m/8
For determination of the benzylpenicillin content use
gravimetric method by the reaction of N-ethylpiperidine salt
formation:
Amount of penicillins in the semi-synthetic compounds
determine by the alkalimetry, reverse titration, with control
experiment, indicator –phenolphthalein. Е = М.m.
Test on purity
Measure the optical density of solutions of natural penicillins at a
wavelength of 264, 280 and 325 nm. Specific impurities determined
by the liquid chromatography, residual amounts of the organic
solvents - gas chromatography. Pyrogens, abnormal toxicity and
sterility are also determined.
Storage
In airtight containers, in dry, dark place at the room temperature.
Usage
Natural penicillins affect on the gram-positive bacteria and used to
treat pneumonia, gonorrhea, syphilis, septic infections, diphtheria,
scarlet fever. They can not be received per os, because in acidic
environments inactivation occurs (semi-synthetic penicillins and
phenoxymethylpenicillin are stable in acid solutions). Penicillins
destroy under the action of penicillinase, semi-synthetic analogs
resistant to it and have a broader spectrum of activity .
Side effects. Safest. Penicillins may cause gastro-intestinal disorders,
very rare - allergic reactions.
Cephalosporins (cephems)
In the structure of cephalosporin is condensed system
consisting of -lactam and dihydrothiazine cycles.
Cephalosporin are derivatives of 7-aminocephalosporanic
acid (7-ACA) (cefalotin, cephalopirin, cefuroxime, sodium
cefotaxime) and 7-amino-desacetoxycephalosporanic acid
(7-АDCA) (cefalexin monohydrate, cefazolin sodium,
sodium ceftriaxon, cefixime, cefaloridine).
• Mouldy fungi Cephalosporium sort and actinomycetes
produce natural antibiotic - cephalosporin C, which did
not found application in medicine because of low
activity. Cephalosporin C is a source of obtaining of
the semi-synthetic cephalosporins.
• In the medical practice use the modern semi-synthetic
cephalosporins of four generations (classification by
Kharkevich).
The general formula of cephalosporin group of
medicines:
COOH
O
CH2R2
N
R1HN
S
Cefazolin sodium (SPhU)
(Cefazolinum natricum), kefzol , reflin
COONa
O
3''
N
2''
8
O
N
1''
N
N
4''
5''
C
H2
C
2
N
H
1
N
7
3
H2
C
S
3'
4'
N
N
2'
5'
S
1'
6
4
S
H
H
5
Sodium (6R,7R)-3-[[(5′-methy-1′,3′,4′-thiadiazol-2′-yl)
sulphanyl]methyl]-8-oxo-7-[(1"Н-tetrazol-1"-ylacetyl)amino]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylate
CH3
Cefalexin monohydrate (SPhU)
(Cefalexinum monohydricum)
ospexin, lexin
COOH
O
8
NH2 O
C
2'
H
C
1'
N
H
1
N
7
3
*H2O
6
4
S
H
H
CH3
2
5
(6R,7R)-7-[[(2′R)-2′-amino-2′-phenylacetyl]amino]-3methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylic acid monohydrate
Cefixime (SPhU)
(Cefiximum), cefix, loprax
H2C
NH2
COOH
O
3''
2''
COOH
N
N
O
4''
S
1''
5''
C
C
2'
1'
2
8
O
N
H
H
C
1
N
7
CH2
3
* 3 H2O
6
4
S
H
H
5
(6R,7R)-7-[[(2′R)-2′-(2′′-aminothiazol-4"-yl)-2′[(carboxymethoxy)imino]acetyl]amino]-3-ethenyl-8oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic
acid trihydrate
Cefotaxime sodium (SPhU)
(Cefotaximum natricum)
cefabol, cafantral
CH3
NH2
O
3''
2''
N
COONa
O
N
O
C
C
2'
1'
8
4''
S
1''
5''
2
N
H
H2
C
O
C
1
N
7
3
O
6
4
S
H
H
5
Sodium (6R,7R)-3-[(acetyloxy)methyl]-7-[[(2Z)-2-(2aminothiazol-4-yl)-2-(methoxyimino)acetyl]amino]8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylate.
CH3
Ceftriaxone sodium (SPhU)
(Ceftriaxonum natricum)
O
5'''
CH3
NH2
O
3''
2''
N
COONa
O
N
O
C
C
2'
1'
1''
5''
2
8
4''
S
ONa
4'''
N
H
S
1
N
7
3
6
4
S
H
H2
C
H
* 3,5 H2O
N
6'''
3'''
N 1'''
N 2'''
CH3
5
Disodium (6R,7R)-7-[[(2′R)-2′-(2′′-aminothiazol-4"-yl)2′-[(methoxyimino)acetyl]amino]-3-[[(2′′′-methyl6′′′-oxido-5′′′-oxo-2′′′,5′′′-dihydro-1′′′,2′′′,4′′′-triazin3′′′-іл)sulphanyl]methyl]-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylate
Ways of
introducti
on
Generations
І
Cefazolin
(kefzol, reflin)
Cefalotin
(ceporin)
Cefapirin
Parenteral Cefaloridin
(i/v; i/m) Cafradin
Enteral
(per os)
Cefalexin
(ospecsin, lexin)
Cefadroxil
(duracef,
cefangin-KMP)
Cefradine
ІІ
ІІІ
ІV
Cefuroxime
(zinacef)
Cafamandole
(cafambol)
Cefoxitine
Cefonocide
Cefprozyl
Cefmetazole
Cefotetan
Ceforanid
Ceftriaxone
Cefepime
Cefotaxime (cefabol, (maxipime)
cefantral)
Cefoxitine
Ceftrizoxime
Cefoperazone(hepac
ef-KMP, cefobit)
Ceftazidime (forum,
ceftum)
Ceftizoxime
Moxalactal
Cefaclor (biclorKMP)
Cefuroxime
(zinnate)
Loracarbef
Cefprozyl
Cefixime (cefix,
loprax)
Cefpodroxime
(cefodox)
Proxetyl
Ceftibutene (cedex)
Cefetamethe
pivoxyl
Identification
By the physico-chemical constants: IR and UV
spectrophotometry, thin layer chromatography.
The reaction with Marki reagent. Reaction is distinguished
because each of them forms a characteristic color
(cafalexin -light yellow color, which becomes dark
yellow; ceftriaxon sodium salt - greenish-yellow, which
turns into yellow, cefotaxime sodium salt - bright yellow,
which turns into brown).
The presence of the β-lactam cycle causes the formation
of cooper (II) or iron (III) hydroxamates.
With the mixture of acids H2SO4 and HNO3 cefalexin
becomes yellow, cefalotin -olive-green, which transforms
into a red-brown .
Sodium salts give an appropriate reactions on sodium
cation.
CHARACTERS
Medicinal substances - white, sometimes yellowish
powders. Sparingly soluble in water (except for sodium salts
of cefalexin ceftriaxone, cefatoxime), hard soluble in
alcohol. Some of them have a characteristic smell and
sensitive to light. Optically active substances of the
cephalosporin droup rotate the plane polarization to the
right.
Assay
By liquid chromatography (SPU).
Chemical methods (like penicillins).
Biological methods.
Physico-chemical methods (spectrophotometry,
photocolorimetry).
Assay
cefalexin
Acidimetry in non-aqueous environment. Equivalent
point is determined by potentiometric method.
Storage
In airtight containers, вin dry, protected from light place at
the room temperature.
Usage
Cephalosporins have a more broader spectrum than
penicillins, and less toxicity.
The difference in the chemical structure of penicillins
and cephalosporins leads to the resistance to
staphylococcus penicillinase and greater resistance to
acids. Therefore prescribe cephalosporins are
prescribed to the treatment of penicillin stable
infections.
Medications of cephalosporin’s froup used in the
treatment of acute and chronic respiratory diseases,
urinary tract, genitals, with postoperative and other
infections.
Other antibiotics of heterocyclic
structure
The structure that connects β-lactam and oxazolidine cycles
–clavulanic acid:
3-(2-Hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptane-2-carboxylic acid
It is used as an inhibitor of β-lactamase of gram-positive
and gram-negative bacteria with penicillin and
cephalosporins, increasing their effect.
Also it is opened a new antibiotics of heterocyclic structure:
cephamycin, thienamycin and others.
Thank you for
attention !