Medicinal Chemistry of Antifungal Agents

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Medicinal Chemistry of Antifungal Agents
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Introduction

• Long before Pasteur and Köch works on pathogenic
  bacteria Gruby and Schönlein have been studying a
  pathogenic fungi, Trichophyton schöenleinii
  (1839).
• The causative microorganism for thrush,Candida
  albicans was discovered in the same year.
• Gruby applied the causative fungi for favus on a
  kids scalp to induce the infection successfully.
  
• But medical mycology was neglected under the
  shadow of medical bacteriology, maybe for the
  non-malignancy of the fungal diseases.

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Types of Fungal Infections (Mycoses)

• The term fungi is a general term that includes
  both yeasts and molds.
• Fungal infections fall into two distinct
  categories
• Superficial Mycoses, Dermatophytoses.
• Deep-Seated Mycoses, Systemic Mycoses.

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Superficial Mycoses

• Contagious skin infections that are limited to
  the epidermal region.
• Most common fungal infections in the form of
  skin, hair and nail lesions.
• Caused by a relatively homogenous group of fungi,
  dermatophytoses, specialized saprophytic molds
  that unusually digest keratin of the skin.

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Systemic Mycoses

• Non-contagious infection that invade the skin,
  lungs and lymphatic tissue.
• In the case of neonates who have acquired the
  etiological agent of candidiasis from their
  mother it is contagious.
• There are different systemic mycosis, e.g.,
  histoplasmosis, blastomycosis, sporotrichosis and
  coccidiomycosis.
• The causative agents are free-living saprophytes.

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Fungal Cell Structure and Targets

• Like mammalian cells, fungi are eukaryotes with
  many similar biochemical structures, especially
  cell membranes to mamalian cells.
• DNA is organized into chromosomes within the cell
  nucleus and have distinct cytoplasmic organelles
  including endoplasmic reticulum, Golgi apparatus,
  mitochondria, and storage vacuoles.
• This homology to mammalian cells also extends to
  biosynthetic pathways, where fungi share similar
  mechanisms for DNA replication and protein
  synthesis.

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• The similarity of fungal and mammalian cells
  creates a number of problems for designing drugs
  that are selectively toxic to fungal cells but
  not the human host.

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Fungal Cell Membrane

• Lipid bilayars form an unstable structure which
  cannot retain its shape and functions. Sterols
  lie within the lipid bilayer and act as
  stiffening agents.
• Sterols, account for approximately 25 of the
  weight of the cell membrane.
• Whereas mammalian cell membranes contain
  primarily cholesterol, ergosterol is the
  predominant sterol in many pathogenic fungi.
• This difference in sterol content has been
  exploited as the target of selective antifungal
  drug action.

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Fungal Cell Wall

• The fungal cell wall is critical for cell
  viability and pathogenicity.
• Beyond serving as a protective shell and
  providing cell morphology, the fungal cell wall
  is a critical site for exchange and filtration of
  ions and proteins, as well as metabolism and
  catabolism of complex nutrients.
• Because mammalian cells lack a cell wall, it also
  represents an ideal and specific target for
  antifungal therapy.

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• Structurally, the fungal cell wall is composed of
  a complex network of proteins and
  polycarbohydrates that varies in composition
  depending on the fungal species.
• Disruption of this protein/carbohydrate matrix
  results in a structurally-defective cell wall,
  rendering the fungal cell sensitive to osmotic
  lysis.

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Agents Acting on The Cell WallGlucan synthesis
inhibitors

• Because of lacking the cell wall, interruption
  in the fungal cell wall biosynthesis would be
  safe for the mammalian host.
• They do this by inhibiting the enzyme 1,3-beta
  glucan synthase.
• Inhibition of this enzyme results in depletion of
  glucan polymers in the fungal cell, resulting in
  an abnormally weak cell wall unable to withstand
  osmotic stress.

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• Echinocandin and Pneumocandin were herbal
  compounds discovered in 1970s able to block cell
  wall synthesis.
• They are peptide compounds with a long lypophil
  chain that inhibit ß-1,3-d-glucan synthase, an
  enzyme resposible for the synthesis of ß-glucan
  polymers.
• A recently FDA approved drug of this category is
  Caspofungin (pneumocandin) which is used to treat
  aspergillosis.

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Echinocandin
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Pneumocandin(Caspofungin)
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Drugs Affecting Ergosterol Biosynthesis

• Biosynthesis of Ergosterol in Fungi
• Ergosterol is synthesized from squalene, an
  ethylenic hydrocarbon through several steps, with
  lanosterol as an steroid intermediate.
• In this biosynthetic pathway there are four
  target enzymes for antifungal drug therapy
• Squalene epoxidase
• 14a-demethylase
• ?14-reductase
• ?7, ?8-isomerase

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Squalene Epoxidase Inhibitors

• Allylamines and Related Compounds
• They were discovered by Random Screening in order
  to find antifungal agents.
• They are only effective against nail and skin
  dermatophytes (narrow spectrum of activity).
• They cause a decrease in fungal membrane
  ergosterol and an increase in squalene level
  which is toxic for the fungi.

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Allylamine and

• Mammalian cells have the same enzyme in the
  cholesterol biosynthesis pathway with less
  affinity toward this inhibitors.
• Ki for squalene epoxidase in Candida albicans is
  0.03µM and for rat liver enzymes is 77µM (Drug
  Terbinafine).
• Some are oral antifungal agents.

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• Naftifine The first antifungal allylamine,
  against tinea.
• Terbinafine Stronger, against tinea and
  onychomycosis (oral).
• Butenafine Against Candida albicans in
  tolnaftate resistant cases.
• Tolnaftate Against dermatophytosis caused by
  tricophytone, mycrosporum and epidermophytone. In
  artificial nail preparations.

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14a-demethylase Inhibitors

• In superficial and deep-seated mycoses.
• High bioavailability after oral absorption and
  broad spectrum of antifungal activity.
• 14a-demethylase is an enzyme in mammalian
  cholesterol synthesis too, but it needs a more
  concentration of drug to be inhibited.
• IC50 for C.albicans demethylase is 10-9 and for
  human enzyme is 10-6 .

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Mechanism of 14a-Demethylase Inhibition with
Azole Compounds

• N3 of azole antifungals is basic and bonds to
  iron atom in the heme of CYP450, where the
  activated oxygen is bonded normally.
• The other parts of the drug bonds to specific
  sites of the enzyme.

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Toxic Effect of Azole on Fungal Cell Membrane
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Azole Antifungals

• Clotrimazole In the treatment of tinea and
  candidiasis.
• Butoconazole In the treatment of vaginal
  candidiasis.

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Azole Antifungals

• Econazole In the treatment of tinea and
  candidiasis.
• Miconazole Against severe systemic mycosis
  coccidiomycosis (parentheral). Very
  broad-spectrum.

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Azole Antifungals

• Ketoconazole Broad spectrum.
• Orally for systemic mycoses systemic
  candidiasis, coccidiomycosis, thrush and
  blastomycosis.
• Inhibits the human demethylase too and causes an
  decrease in the concentration of testosterone and
  corticosterone in human.
• Inhibits other P450s in human so increases the
  plasma concentration of cyclosporin, phenytoin or
  terfenadine.

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SAR of Azole Antifungals

• A basic imidazole or 1,2,4-triazole with a pKa of
  6.5-6.8 is essential for antifungal activity.
• N3 of imidazole and N4 of imidazole of triazole
  binds to P450 iron.
• The most active ones have two or three aromatic
  rings, at least one of them is substituted with
  halogens or other nonpolar groups
  (2,4-dichlorophenyl 1,4-dichlorophenyl, or
  2,4-difluorophenyl).
• The most active azoles have fluore in the
  structure.
• Ring substitution at other positions makes the
  azole inactive.
• The big nonpolar part resembles the steroid
  molecule in binding to the enzyme.

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?14-reductase Inhibitors

• Amorolfine the only morpholino compound is such
  an inhibitor.
• In the treatment of dermatophytosis.

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?7, ?8-isomerase Inhibitors

• Amorolfine inhibits ?7, ?8-isomerase too.

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Polyene Membrane disrupters

• Macrocyclic lactones with
• hydrophilic parts a number of hydroxyl groups on
  the ring and a deoxy amino hexose (mycosamine).
• hydrophobic ring with a number of double bonds.
• They belong to two different groups
• With a 26 member ring Natamycin
• With a 38 member ring Amphotericin B and
  Nystatin
• They were introduced to cure the deep-seated
  mycoses.

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Mechanism of Antifungal Activity of Polyenes

• Polyene antifungals act by binding to ergosterol
  in the fungal cell membrane.
• This binding results in the membrane formation of
  pores that increase permeability to proteins and
  monovalent especially K and divalent cations,
  eventually leading to cell death.
• Polyenes attach with higher affinity to
  ergosterol containing membranes than cholesterole
  containing ones.

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Toxicities of Polyenes

• Amphotericin B may also induce oxidative damage
  in fungal cells and has been reported to
  stimulate of host immune cells.
• Stimulation of the host immune cells by
  amphotericin B causes release of inflammatory
  cytokines by circulating monocytes resulting in
  fever, chills, rigor, nausea, vomiting, myalgias,
  arthralgias, and headache during intravenous
  infusions.
• At higher concentrations, amphotericin B binds to
  cholesterol in mammalian cell membranes leading
  to various organ toxicities, most importantly
  nephrotoxicity.

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Amphotericin B
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• Amphotericin B forms a transmembrane channel
  after interaction with mmembrane steroles.
• Hydrogen bonding between hydroxyl, carboxyl and
  amino groups of the drug and membrane makes the
  channel stable.
• It is used in lethal fungal infections such as
  coccidimycosis and histoplasmosis.

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Mechanism of Antifungal Activity of Amphotericin B
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Possible molecular assemblage formed in
biomembrane by amphotericn B, sterol and
phospholipid
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The effect of Amphotericin B on the Fungal Cell
Membrane
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The effect of Amphotericin B on the Fungal Cell
Membrane
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Nystatain
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Nystatain

• The first polyene entered the clinic without
  systemic absorption.
• The macrocyclic ring contains 38 atoms.
• Locally used in the treatment of dermal and GI
  infections caused by Candida albicans.

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• Schematic representation of the main stages of
  nystatin interaction with a model system of
  membranes. After adsorption to the membrane
  interface (top), the antibiotics self-associate
  in a pore structure (bottom), when the surface
  concentration is higher than a critical value.
  The structures formed in the biological membranes
  are most probably mixed antibiotic-sterol
  aggregates.

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Natamycin

• It has a macrocyclic ring with 26 atoms.
• It is used in the treatment of fungal infections
  caused by candida, aspergilus, cefalosporium,
  penicillinum and fusarium spp.

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Nucleoside Antifungals

• DNA and protein synthesis have historically been
  difficult targets for the development of
  selectively-toxic antifungal therapy, as fungal
  and mammalian cells share remarkable homology in
  DNA replication and RNA translation.
• However, advances in molecular biology and
  functional genomics are beginning to highlight
  important differences between mammalian and
  fungal cells that could be exploited for the
  development of new antifungal therapies.
• For the time being, only one class of agents in
  clinical use targets DNA/RNA synthesis
  Antimetabolites

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Antimetabolites

• This class has only one example, flucytosine or
  5-fluorocytosine (5-FC).
• Flucytosine was originally developed in the
  1950's as a potential antineoplastic agent.
  Although ineffective against tumors it was later
  found to have antifungal activity.
• This small molecule is transported into
  susceptible fungal cells by a specific enzyme
  cytosine permease and converted in the cytoplasm
  by cytosine deaminase to 5-fluorouracil (5-FU)- a
  pyrimidine anti-metabolite used as chemotherapy
  for many types of colorectal cancer

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Mechanism of Antifungal Action

• 5-FU is phosphorylated and incorporated into RNA
  where it causes miscoding and halts protein
  synthesis.
• Additionally, phosphorylated 5-FU is converted to
  its deoxynucleoside, which inhibits DNA synthesis
  by blocking the functions of a key enzyme in DNA
  replication- thymidylate synthetase.

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Mechanism of Adverse Effects of 5-FC

• Flucytosine can be converted to 5-FU by bacteria
  residing in the gastrointenstinal tract.
• Not surprisingly, the most common adverse effects
  seen with flucytosine are similar to 5-FU
  chemotherapy (diarrhea, nausea and vomiting, bone
  marrow suppression) but at reduced intensity.

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Miscellaneous Agents

• Griseofulvin
• Isolated from Penicillium griseofulvum.
• Griseofulvin inhibits fungal cell mitosis by
  disrupting mitotic spindle formation-a critical
  step in cellular division.
• It is used orally for superficial mycoses, enters
  in the structure of the precursors of keratin.
• It doesnt have local activity.

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Miscellaneous Agents

• Fatty Acids
• Sebum, the naturally occurring fatty acid on the
  human skin is a natural antifungal agent and a
  part of the immune system.
• Fatty acids or their salts have antifungal
  activity
• Sodium or zinc caprylate.
• Undecylenic acid.