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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5406_Библиотеки_им_академика_М_И_Перельмана.pdf
92 Pharmaceutical Chemistry
Antifungal agents can have the following mechanisms of action such as cell membrane
disruption, inhibition of cell division and inhibition of cell wall formation.
Cell wall formation inhibition: Antibacterial agents such as cephalosporins and
penicillins, can be used to treat bacterial infection, and are not active against fungal
cell. Many drug candidates have been discovered with potent antifungal activity in
vitro which can interfere in various steps of fungal cell wall synthesis but still these
drugs need to be improved. Many antifungal drugs have been developed to target
ǃ-glucan synthesis.
Cell membrane disruption: Antifungal agents which are responsible for cell
membrane disruption specially targeting ergosterol, either by binding with sterol,
results in pores, which causes the membrane to become loose or by the inhibition of
ergosterol biosynthesis. Structure of ergosterol is almost similar to the mammalian
cholesterol. It concludes that the ergosterol binding agents can have a cytotoxic effect
in the host tissue. Ergosterol contains two conjugated double bonds while mammalian
sterols don’t.
Cell division inhibition: Cell division inhibition can be done using nucleoside
antifungal agents. They can inhibit DNA transcription process or can target the
microtubule effects.
4.3.2 Drug classes
Antifungal agents (Fig. 4.5) are classified on the basis of their structure and mechanism as
follows:
(a) Macrolides: The examples of macrolids are nystatin and amphotericin B. These
drugs bind to sterol in the plasma membrane and are especially more selective
towards ergosterol.
(b) Cytoskeleton agent: Griseofulvin is the best known cytoskeleton agent which
inhibits microtubules. It is therefore known as fungistatic which also binds to
keratin.
(c) Antimetabolites: The examples of antimetabolites are flucytosine which inhibit
fungal RNA and DNA synthesis and are selectively converted by fungi to active
metabolites.
(d) Imidazoles: They inhibit fungal ergosterol biosynthesis; however selectively inhibit
fungal cytochrome-P
(inhibit mammalian P
enzymes. Other examples are clotrimazole, ketoconazole
450
enzymes) and miconazole.
450
(e) Triazoles: They inhibit fungal ergosterol biosynthesis. Examples: itraconazole, and
fluconazole.
(f) Echinocandins: They inhibit E-1,3-D-glucan synthesis and block cell wall synthesis.
Example: caspofungin.
(g) Allylamines: They inhibit fungal squalene metabolism. Increased level of squalene
is toxic to fungi that causes reduced ergosterol. Example: naftifine and terbinafine.
Antibiotic, Antibacterial and Antifungal Agents 93
HO
Me
HO
Me
N
Me
Me
Cl
OH
O
Me
OHOHO
Amphotericin B
OH
O
Me
OHO
OH
Nystatin
N
O
N
O
N
N
O
OH
OHOH
O
O O
HO
OH
OHOH
O
O O
NH
OH
COOH
Me
OH
2
OH
COOH
Me
HO
NH
2
F
N
N
H
Flucytosine
N
N
Me
O
Cl
HO
OH
NH
2
O
N
N
N
Clotrimazole
N
O
N
O
O
Cl
Me
Me
OMe
MeO
Cl
F
Fluconazole
Ketoconazole
Me
Ergosterol
OMe
O
Me
Griseofulvin
N
F
N
OH
N
N
N
N
Me
Me
O
N
N
COMe
H
N
O N
O
NH
OH
H
N
OH
2
OH
NH
2
HO
OH
HN
N O
O
O
OH
O
O
NH
NH
Caspofungin
Itraconazole
Cl
Me
Naftifine
N
Cl
Cl
N
O
N
Me
N
Cl
HO
HO
Cl
Cl
Miconazole
Terbinafine
Fig. 4.5 Antifungal agents
The adverse effects of antifungal agents could be fever, nausea, vomiting, elevation of
liver enzymes, and irritation at injection sites, allergic syndrome, hepatitis, enterocolitis,
muscle spasms, renal impairment, etc. Sometimes they are known for sporadic visual
disturbance and hepatotoxicity.
94 Pharmaceutical Chemistry
4.4 CHLORAMPHENICOL
The antibiotic drug, chloramphenicol is useful for the treatment
of various bacterial infections. It was synthesized by J. Controulis
et al. in 1949. Chloramphenicol was also known as Chlornitromycin
and later Parke, Davis & Co. adopted “Chloromycetin” as its
trademark. Chloramphenicol has been effective in treating
various bacterial infections caused by numerous bacteria
HO
Cl2HC
Chloramphenicol
OH
NH
O
NO
2
including Escherichia coli, Staphylococcus aureus and Streptococcus
pneumoniae. At last we can say it has a broad spectrum of activity. However, it is not active
against Pseudomonas aeruginosa.
It is no longer a first-line drug in developed countries due to safety and is prone to
resistance. Thus, the bacterial resistance of many drugs is heading us towards new
discoveries. It is a frequently used antibiotic in developing countries due to low cost and
ease of manufacture.
4.4.1 Specification
Name : (2,2-Dichloro-N-{1,3-dihydroxy-1(4-nitropenyl)propan-2-yl}acetamide)
Molecular formula : C
11H12Cl2N2O5
Molecular weight : 323.1320 g/mol
Melting point : 150.5-151.5°C
Physical state : White to greyish-white or yellow-white fine crystalline powder,
needle-like.
Solubility : Polyethylene glycol and polar organic solvents.
4.4.2 Synthesis of Chloramphenicol
2
EtO
Cl2HC
Ph2HC
O
NH
O
80%
H
OH
O
H
CHCl2COOH (10 eq)
1,2-C
reflux, 1 h
NO
2H4Cl2
2
CHNH
Ph
2
MgSO4, DCM
°C, 10 h
25
N
NO
2
80% 85%
NO
2
1 mol%
S-VAPOL-B(OPh)
CHN2CO
, 0°C, 24 h
PhCH
3
NaBH
4
MeOH
0
°C, 0.5 h
3
Et
2
HO
Cl2HC
O
Chloramphenicol
74% (99% )ee
C
EtO
2
cis:trans = >100:1
OH
NH
CHPh
N
96.4% ee
2
NO
NO
2
2
Antibiotic, Antibacterial and Antifungal Agents 95
4.4.3 Mechanism of Action
Chloramphenicol is a bacteriostatic drug which is used to inhibit protein synthesis during
the bacterial growth. It inhibits peptidyl-transferase activity of the bacterial ribosome, thus
elongation of protein chain or synthesis can be prevented. It binds to protein residue
(A2451 & A2452) in the 23S-rRNA of the 50S-ribosomal subunit of bacteria, therefore is
able to prevent peptide bond formation. However, chloramphenicol and macrolides (a
class of antibiotics) both interact with ribosomes through substrate binding whereas
elongation of growing peptide is specially inhibited sterically by macrolides.
4.4.4 Side Effects
Bone marrow toxicity is the most adverse effect with the chloramphenicol treatment. It can
be in two forms: bone marrow suppression, (direct toxic effect) and aplastic anaemia,
(unpredictable, rare and generally fatal).
Gray baby syndrome (difficulty in breathing, shock,
vomiting, and swelling of the abdomen, pale or blue skin colour, limp muscles and loose
green stools) is another common side effect associated with being intravenously used.
Some common side effects include headache, fever, itching, redness, rashes, swelling of
the mouth, face, lips, tongue, difficulty in breathing, confusion, hives, chest pain and
swelling in the sore throat.
4.4.5 Avoidance of Chloramphenicol
Individuals having past history of allergy from chloramphenicol.
Individuals suffering with low level of WBC & RBC and low level of platelets.
Individuals having past history of infection like throat infection, flu and cold, etc.
Individuals under medication which decrease the bone marrow, for anaemia, liver
diseases and kidney problems.
Women breastfeeding and/or pregnant.
4.5 SULFONAMIDES
Sulfonamides are sulphur containing organic compounds having
SO
group (amides of sulfonic acids), first discovered by Gelmo
2NH2
H
N
2
in 1908 as an intermediate in the study of azo dyes. Sulfonamides
are derived from sulphanilamide and sometimes known as sulfa
drugs. These sulfa drugs interfere with the metabolic processes in
Sulfanilamides
(R group can be varied)
bacteria which require p-amino benzoic acid (PABA). The molecular structure of
sulphonamide is similar to (PABA). PABA is the main substrate of the enzyme
dihydropteroate-synthetase for the synthesis of tetrahydrofolic acid (THF) in the bacterial
organisms. Sulfanilamide is the first chemical substance that has been used to treat and
prevent bacterial infections in humans. Thus these drugs act similar to antimicrobial agents
where they inhibit the growth and activity of bacteria.
HN
O
O
S
R
96 Pharmaceutical Chemistry
Medicinal chemists thus began with the various analogues of sulphanilamide to prove
an accurate structure-activity profiling, which finally came into the following conclusions:
Unsubstituted p-Amino group is essential for activity.
Sulfonamide group and aromatic ring are essential for activity.
Aromatic ring must be p-substituted.
Nitrogen adjacent to SO
group must be primary or secondary.
2
R-group can be varied at nitrogen adjacent to sulphonamide.
Nowadays use of the antibiotic drugs has reduced drastically due to increased drug
resistance and new more effective antibiotics. However, sulfonamides are still in use to
treat or prevent burning infection and urinary tract infections. They are also used in the
treatment of hypertension, gout, certain forms of malaria and diabetes mellitus.
Gerhard Domagk (1932), a German bacteriologist and pathologist first observed the
antibacterial effects of sulphonamides (prontosil—a red dye) on
mice. Later, French researchers proved that prontosil contains sulphanilamide (
Streptococcus infections in
p-amino
benzene sulphonamide) group as an active group which was also a metabolite of Prontosil.
Sulphonamides were used widely till 1940s and sulfanilamide powder was used in first-aid
kits for the treatment of open wounds during World War-II, however sulfanilamide tablets
were used orally for the treatment of intestinal infections. Thus, sulphonamides are
relatively less toxic derivatives so the production of penicillin is reduced.
H2N
Prontosil (Red Dye)
NH
N
N
2
SO2NH
2
In 1940s numerous sulfonamides were derived from sulphanilamide, viz., sulfadiazine
(used for urinary tract and intestinal tract infections), sulfathiazole (used for bacterial
infections) and sulfamethazine (used for urinary tract infections). The main problems with
these classes of drugs are that they develop bacterial resistance after being exposed to the
drugs. Thus, the interest of common people in these drugs is decreasing day by day.
Several sulfonamides and antibiotics are used in combination to treat a wide range of
bacterial infections including pneumonia, malaria, skin burns and HIV/AIDS, etc.
Trisulfapyrimidine (mixture of sulfadiazine, sulfamethazine, and sulfamerazine) is an
antibiotic drug specially used to treat vaginal infections.
In general, sulfonamides inhibit the multiplication and growth of bacteria without
harming the host. Sulphonamides usually interfere with folic acid (folate) synthesis (folic
acid is an essential component of vitamin B present in all living cells). Most bacteria used
simpler starting materials to synthesize in house folic acid while folic acids containing
natural diet supplements are necessary for humans and animals. Combination therapy of
trimethoprim (known as dihydrofolate reductase inhibitor) and sulfamethoxazole are
Antibiotic, Antibacterial and Antifungal Agents 97
showing much greater inhibition of folic acid synthesis, thus this combination may be
useful in the treatment of some systemic infections and urinary tract infections.
Sulphonamides contain sulfone group which is majorly responsible for folic acid
synthesis inhibition. They usually tend to accumulate and are retained for long periods
inside the inflammed tissue and skin. Thus, sulfones derivative, dapsone is very useful in
the treatment of leprosy. Sulfonamides do
not work for cold, flu, and other infections
caused by the viruses.
4.5.1 Synthesis
4.5.2 Classification of Sulfonamide Drugs (Table 4.4)
Table 4.4: Classification of sulfonamide drugs
(There is no evidence of cross reactivity between arylamine sulphonamides and non-arylamine sulfonamides)
Arylamine Sulfonamides
(Allergic cross reactivity within this group is possible)
Sulfonamide containing Medications
Non-Arylamine Sulfonamides
(No allergic cross reactivity between groups)
Sulfonamide Antibiotics
(Sulfamethoxazole, Sulfadiazine, Sulfacetamide,
Sulfasalazine)
Sulfonamide Antiretrovirals
(Amprenavir, Fosampranavir)
Carbonic Anhydrase Inhibitors
(Acetazolamide, Dorzolamide)
Sulfonylureas
(Gliclazide, Glimepiride, Glibenclamide, Gliperizide)
Loop Diuretics
(Frusemide, Bumetanide,
Chlorthalidone, Chlorothiazide, Diazoxide,
Hydrochlorothiazide, Indapamide, Metolazone,
Torsemide)
Thiazide Diuretics
(Hydrochlorothiazide, Indapamide, Chlorthalidone,
Metolazone, Diazoxide)
Anti-inflammatory
(Celecoxib, Valdecoxib,
Sulfasalazine)
Sulfonamides moiety without benzene ring
(Dapsone, Sotalol, Sumatriptan, Topiramate,
Probenecid)
98 Pharmaceutical Chemistry
4.5.3 Bacterial Resistance
Both chromosomal and R-factor-mediated resistance to sulfonamides have been attributed
to altered forms of dihydropterate synthetase (for which sulfonamides have a lowered
affinity). Competency of sulfonamides results in overproduction of p-Amino benzoic acid
(PABA) that can preclude inhibition of dihydropterate synthetase. Cross-resistance
between sulfonamides is common.
4.5.4 Mechanism of Action
Most of animal population gradually developed resistance with regular use of antibiotics.
Plasmid mediated sulphonamide-resistance in intestinal Gram-negative bacteria is often
linked with tetracycline and ampicillin resistance.
Trimethoprim was specially used to inhibit the reduction of dihydrofolic acid (DHF) to
tetrahydrofolic acid (THF) by forming a complex with the dihydrofolate reductase catalyst
used in the conversion. THF is an essential scaffold in the synthetic pathway of thymidine
and it interferes with this pathway that inhibits the synthesis of bacterial-DNA.
Tetrahydrofolate is an essential component for the synthesis of purine and thymidylate
which is regulated by the dihydrofolate reductase (DHFR) in the cell. It is very important
for cell growth and cell proliferation.
presence of dihydrofolate reductase, however a lack of DHFR was observed in the mutant
cells.
The affinity of trimethoprim for the bacterial dihydrofolate reductase is multiple times
greater as compared to the human dihydrofolate reductase. Dihydropteroate synthetase is
an enzyme (which is used upstream in the same pathway) can be inhibited by
sulfamethoxazole. Therefore, trimethoprim and sulfamethoxazole can be used in
combination due to their synergistic effects in different steps under the same pathway. In
this combination, less resistant development was observed.
Sulfonamides are the mimic of PABA, thus they can inhibit bacterial DNA synthetic
pathway by mimicking PABA. Sulphonamides bound to the active site of bacterial
dihydropteroate synthetase with a greater affinity as PABA does, thus preventing PABA from
binding and as a result synthesis of dihydropteroate is inhibited. Therefore, sulfonamides
act as competitive inhibitors of PABA due to close structural resemblance (Fig. 4.6).
On the basis of the above mechanism it is concluded that sulfonamides have the same
mechanism of action however the differences are evident with respect to pharmacokinetics,
biological spectrum, concentration, activity, etc.
Many nucleic acid syntheses are performed in the
4.5.5 Bioactivation of Sulphonamides
A number of biochemical processes take place in metabolism of antimicrobial drugs
sulphonamide. In humans, large amount of the sulphonamides (viz., sulfamethoxazole) is
metabolized in the liver by N-acetyltransferase to the nontoxic N4-acetylated which is later
eliminated via urine, however approximately 15–20% of the sulphonamide drug is
glucuronidated at the N1-nitrogen (metabolite of sulphonamides) and excreted renally.
H2N S
Sulfamethoxazole
HN
HN
H2N
N
O
Dihydropterin sulfamethoxazole
(Inhibit dihydropteroate synthetase)
H2N
dihydrofolate reductase)
O
O
N
HN
N
N
(Inhibit the enzyme
O
HN S
NH
2
N
Trimethoprim
OMe
O
HN
OMe
OMe
Antibiotic, Antibacterial and Antifungal Agents 99
HN
H2N
Dihydropteroate synthetase
O
N
O
N
N
O
Pteridine di-phosphate p-Amino benzoic acid
O
OHN
P OH
O
H2N
Dihydrofolate
H2N
Dihydrofolate
H2N
Cofactors
O
OH
P
OH
HN
N
synthetase
HN
N
reductase
HN
HN
N
H2N
HNHN
N
O
O
O
Dihydropteroate
ATP,L-Glutamate
ADP
HNHN
N
Dihydrofolic acid or
(Pteridine-PABA-Glutamate)
NADPH
+
NADP
HN
NH
Tetrahydrofolic acid
OH
O
HN
HN
O
OH
O
O
HO
O
O
HO
HO
O
O
HO
Thymidine Purines
DNA DNA, RNA t-RNA, Proteins
Methionine
Fig. 4.6: Bacterial synthesis of tetrahydrofolate and formation of its essential amino acid constituents
N4-nitrogen is also involved in glucuronidation and forms N4-metabolites which are very
unstable unlikely N1-metabolite. About 10% of sulfamethoxazole is metabolised to a
100 Pharmaceutical Chemistry
reactive hydroxyl amine intermediate by an action of several enzymes, viz., cyclooxygenase
(COX), CYP2C9 (cytochrome-P450) and myeloperoxidase (MPO). Thus, hydroxyl amine
intermediate gets unstable which further auto-oxidizes to the highly reactive nitrosoderivative known as nitroso-sulfamethoxazole. Later, nitroso-sulfamethoxazole can be self
acetylated, forms complex with glutathione, reduces back to the hydroxylamine derivative
and eliminated. Nitroso-sulfamethoxazole can also react covalently to form hapten-protein
complex with the cysteine-residue of the cellular surface proteins including albumin,
immunoglobulins, serum proteins, splenocytes, skin keratinocytes and circulating
peripheral blood mononuclear cells. The hydroxylamine derivative shows adverse
reactions to the human host which includes nephritis, lupus erythematous, thrombocytopenia, hepatitis and the sulphonamide hypersensitivity syndrome.
The reaction probably proceeds via highly reactive Cys34 with electrophiles. It is still a
choice of discovery whether specific T-cells are involved as a receptor or not. But it is
believed that T-cells are involved in the process since hapten-protein antigens stimulate
CD8+ effector and CD4+ regulatory T-cells from the hypersensitive patients, but still needs
more study (Fig. 4.7). The bioactivation of the drugs in this case is still a choice of research
as regards to the relationship of drug metabolism to the immune response. Recent studies
postulated that hapten-protein antigen complex cells are self responsible to synthesize
sulfamethoxazole metabolites which finally induce the T-cell response to the drug. It was
anticipated that sulfamethoxazole may be intracellularly activated by dendritic cells to the
reactive and immunogenic nitroso-sulfamethoxazole. Sulfamethoxazole and its nitroso
and hydroxylamine metabolites help in stimulation of human T-cells and generate a T-cell
clone which shows three recognition patterns: i) 13% stimulation by sulfamethoxazole,
ii) 44% stimulation by sulfamethoxazole metabolite, and iii) 43% stimulation by both
sulfamethoxazole and its metabolites.
4.5.6 Metabolism and Excretion
Trimethoprim (TMP) usually absorbed from the gastrointestinal tract however takes 2
hours to achieve peak blood levels. Tissue levels often exceed those of plasma, and the
concentration of trimethoprim in urine may be 100 times more than the plasma. It readily
enters the cerebrospinal fluid (CSF) if inflammation is present. Trimethoprim has half-life
of approximately 11 hours however sulfamethoxazole (SMX) with t
can be used frequently with a ratio of 1 : 5 (TMP : SMX). After oral administration, in
combination of these drug takes 1-4 hours to achieve peak drug levels in plasma. At this
stage, TMP-SMX ratio is approximately 1 : 20 in plasma which results in most effective
drug having synergetic effect against the deadly susceptible pathogens. Trimethoprim
have much greater lipid solubility, thus the above ratio influenced its larger volume
distribution. Both these drugs specially bind to plasma proteins (45 & 66%, respectively)
which are metabolized in the liver. After 24 hours of oral administration approximately
40-60% of both the parent drugs and their respective metabolites are excreted through the
kidney. Both drugs can also cross the placenta and are found in breast milk.
= 10 hours, and both
1/2
Antibiotic, Antibacterial and Antifungal Agents 101
O
C
H
NH
3
NH
2
NH
2
CH
3
N-Acetyltransferase
O
S
O
O
N
N
H
N-Acetylsulfamethoxazole
(Detoxification product, 45-70%)
P450
HO
Adverse reaction
Oxidation
Cell damage,
Dimerization,
Protein heptanation
Nitrososulfamethoxazole
CYP2C9/
MPO/COX
-10%
NH
CH
O
S
O
Sulfamethoxazole-
O
N
N
H
hydroxylamine
O
N
CH
O
S
O
O
N
N
H
O
S
N
N
O
H
Sulfamethoxazole
3
Reduction
Glutathione
Cysteine
3
CH
3
UDP-Glucuronosyl
O
CYP2C9
Hydroxylation
CH
transferase
O
S
O
O
N
N
OH
O
HOOC
OH
OH
Sulfamethoxazole- -N
glucuronide (Detoxification
product 5-15%)
NH
2
OH
O
S
O
O
N
N
H
5-Hydroxysulfamethoxazole (5-10%)
3
Protein-SH Antigen formation
Hypersensitivity
Adverse drug reactions
T-cell response
T-Cell recognition
Transport to cell surface
Protein complex on
cell membrane
(Unstable mercapto
intermediate)
Protein
S
OH
N
CH
3
O
S
O
O
N
N
H
by MHC Class II
Sulfamethoxazole-
protein adduct
Protein
O
OHS
N
CH
3
Internalization
into endosomes
O
S
O
O
N
N
H
Degradation to
peptide fragments
Fusion of vesicles with
peptide fragments with vesicle
containing MHC Class II
Fig. 4.7: Bioactivation of sulfamethoxazole (reactive metabolite formation, metabolite specific immune response
and tissue damage or hypersensitivity)