Term
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Definition
DNA alkylating agents are compounds that are very reactive or form reactive intermediates that interact with proteins or nucleic acids
nitrogen mustards
aziridines
methanesulfonates
nitrosoureas
methylhydrazines
platinum complexes |
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Term
| what is an alkylating agent? |
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Definition
[image]
alkylating agent attachment of an allkyl group to a nucleophile (e.g. DNA or a side chain on a protein) |
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Term
most reactive nucleophilic sites on DNA
[image] |
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Definition
[image]
N7-guanine > N3-adenine > N7-adenine > N3-guanine > N1-adenine and N3-cytosine and O6-guanine
the most predominant site of DNA alkylation is the N7 position of guanine
the reason being is the electron donation of the 2 amino group on to the N7 position
the next most likely site of alkylation is the N3 position of adenine
the reason again is the electron donation of the 6 amino group on the N3 nitrogen
the least likely site of alkylation of purine and pyrimidine bases is the O6 of guanine.
the reason is due to the greater electronegativity of the O6 groups |
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Term
| compounds capable of bis-alkylation can form: |
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Definition
[image]
types of alkylation of DNA and proteins
in the case of a bifunctional alkylating group, reaction can occur with two nucleophilic groups
Thus, 1) monoalkylation of DNA, 2) intrastrand alkylation by a bifunctional alkylating agent, 3) interstrand bis-alkylation by a bifuncctional alkylating agent, and 4) bis-alkylation that occurs with a single strand of DNA and a side chain of a protein
[image]
a representation of DNA interstrand alkylation caused by a bifunctional alkylating agent
notice that the charge on the purine bases is positive |
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Term
| results of DNA alkylation |
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Definition
prevention of DNA replication and RNA transcription
leads to DNA fragmentation by hydrolytic and repair enzymes
mispairing of base pairs (nucleotides) by alteration of normal hydrogen bonding which can lead to alteration of amino acid structure in proteins and altered protein function
fragmentation of DNA:
[image]
DNA fragments are produced by the action of the DNA repair enzymes that attempt to remove the altered purine or pyrimidine bases
cross-linking of DNA by a bifunctional alkylating agent inhibits synthesis and transcription:
[image]
mispairing of nucleotides caused by DNA alkylation alters the normal hydrogen bonding between base pairs:
[image] |
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Term
| mispairing of nucleotides caused by alkylating agents |
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Definition
[image]
normally, cytosine base pairs with guanine to form 3 H-bonds
[image]
upon alkylation of the 6O of guanine, the ene tautomer is favored
this results in only 2 H-bonds rather than the normal 3 H-bonds between C-G
the unshared electron pair of the N3 of cytosine is repulsed by the unshared electron pair of N1 of guanine
the 6-O alkylated guanine can now mis-pair with thymidine |
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Term
nitrogen mustards (aziridine-mediated alkylators)
[image] |
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Definition
[image]
chemically: bis-beta-chloroethylamines
KEY TO REACTIVITY IS THE FORMATION OF AN AZIRIDINIUM ION:
[image]
biological nucleophiles: DNA bases, SH, NH2, COO-, H2PO3-, H2O
the nitrogen mustards get their name from mustard gas (sulfur instead of nitrogen)
the mustards produce intese skin reactions and burns
the key to their reactivity is the formation of a 3-membered aziridinium ion
the reason that this is so reactive is due to the ring strain of the 3 membered ring and the position charge on the aziridine ring nitrogen
attachment of a nucleophile (e.g. a DNA base) opens up the ring and covalently attaches a nucleophile to the beta carbon of the aziridine
the same reaction can occur with the other beta-chloroethyl group
in addition to reacting with DNA bases, the aziridine ring can react with nucleophile side chains on proteins (SH, NH2, OH) to produce an altered protein structure
thus, nitrogen mustards can be considered as bis (bifunctional) alkylating agents |
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Term
| structure activity relationship of nitrogen mustards |
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Definition
[image]
aromatic nitrogen mustards are more stable b/c electron pair is delocalized by resonance (not as electron rich as the alkyl nitrogen mustards) |
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Term
| stability issues with nitrogen mustards: why are they formulated as HCL salt |
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Definition
formulated as HCl salt to prevent aziridinium ion formation and decomposition
[image]
OH is not a good leaving group
[image]
nitrogen is protonated (the unshared electron pair "tied-up")
the nitrogen mustards are highly reactive and must be formulated at an acidic pH
this ties up the unshared electron pair on nitrogen and prevents the compound from reacting with itself
at higher pH values the free base form of the nitrogen mustard forms the species (aziriidnium ion) that can alkylate biological nucleophiles
non-specific alkylation is also possible, resulting in numerous ADRs of these compounds
the stability of nitrogen mustards is an issue in neutral or basic solution
in these cases the highly strained aziridinium ion reacts with water to deompose the nitrogen mustard |
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Term
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Definition
[image]
used as hydrochloride salt pKa = 6.1
use is limited due to non-specific alkylation
IV administration (rapidly forms reactive species at pH 7.4)
severe vesicant: flush skin or eyes with water; followed by treatment with 2% sodium thiosulfate
[image]
mechlorethamine is a nitrogen mustard that produces non-specific alkylation
this compound is highly reactive
the reason is that the N-methyl group on nitrogen increases the electron density on N making it a better nucleophile
thus, it forms the highly reactive aziridinium ion more readily
this compound is a severe vesicant producing intense burns on the skin
flushing the skin with water will hydrolyze the mustard
additional treatment with sodium thiosulfate will form a highly water soluble species that can be washed from the skin |
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Term
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Definition
[image]
aromatic nitrogen mustard - less reactive due to delocalization of unshared electron pair on nitrogen and thus the aziridinium ion does not form as readily
orally active
L-amino acid side chain (designed to be taken into cells more readily); no appreciable difference in uptake between L-isomer and racemate
the amino acid side chain increases its water solubility |
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Term
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Definition
[image]
orally active-absorption increased with food
extensively protein bound
metabolized in the liver to an active metabolite by beta oxidation
[image]
melphalan and chlorambucil are aromatic mustards; these compounds are less reactive compared to metchlorethamine
the reason is the unshared electron pair on the mustard nitrogen is delocalized by resonance
thus, the aziridinium ion does not form as readily
evidence of this is shown by the fact that these compounds can be taken orally
chlroambucil is metabolized to active metabolite by beta-oxidation (a metabolic process very similar to fatty acid metabolism) |
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Term
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Definition
[image]
BINDS TO TUBULIN AND TO TUBULIN AND MICROTUBULE-ASSOCIATED PROTEINS
CAUSES MICROTUBULE DISASSEMBLY AND ANTIMIOTIC EFFECTS
[image]
estramustine is actually a prodrug
during absorption, it is hydrolyzed to the active drug
it contains a bis-chloroethyl carbamate group
due to the delocalization of the unshared electron pair on nitrogen on to the adjacent carbonyl group, it does not form appreciable amounts of aziridinium ion
although estramustine resembles the beta-chloroethylamines, it is not an alkylating agent
estramustine is used for metastatic or hormone refractory prostate cancer
estramustine undergoes extensive metabolism by CYP3A4 and 1A2
[image]
instead of acting as an alkylating agent, estramustine binds to microtubules and with tubulin |
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Term
metabolism of estramustine
[image] |
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Definition
[image]
the carbamate group of estramustine can be hydrolyzed by the action of estrases to estradiol
estradiol and estrone are metabolically interconvertible due to the action of 17 beta hydroxy steroid dehydrogenase |
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Term
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Definition
[image]
cyclophosphamide is a phosphoramide
it was originally designed to be hydrolyzed by phosphoramidases which have a higher concentration in tumor cells
the compound can be considered a prodrug that undergoes initial activation by P450
the ring is hydroxylated adjacent to the ring nitrogen
this species is in equilibrium with its ring opened aldophosphoramide form
these species pass by passive diffusion into the tumor cells where they are activated by a base catalyzed mechanism
during this process, the alpha-beta-unsaturated aldehyde (acrolein) and the phosphoramide mustard are formed
the phosphoramide mustard is charged and is trapped in the cancer cell
the negative charge on the oxygen can be delocalized on to the other oxygen atom of the phosphoramide
this balances the charge and allows the nitrogen to form a 3 membered aziridine species that acts as an alkylating agent
this phosphoramide can also be hydrolyzed by phosphoramidases to give the bis-beta-chlroethane derivative
this species can also form a highly reactive aziridinium ion and serve as a bifunctional alkylating agent
the additional product of activation, acrolein, can react with biological nucleophiles to produce toxic adverse effects |
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Term
| adverse effects of acrolein generated by the breakdown of cyclophosphamide (HEMORRHAGIC CYSTITIS) |
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Definition
[image]
arcolein is highly toxic to the bladder and kidney
this is caused by reaction with SH containing proteins with the alpha, beta-unsaturated aldehyde (acrolein)
to offset this effect, the compound, mesna, can be administered
this compound is a thiol sulfonate
the thiol of mesna reacts to produce a water soluble sulfonic acid that is readily excreted
[image]
mesna - a mercaptosulfonic acid that concentrates in bladder can be used with cyclophosphamide to decrease toxicity |
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Term
metabolism of cyclophosphamide
[image] |
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Definition
[image]
the products of cyclophosphamide activation can be converted to the inactive metabolites by the action of alcohol and aldehyde dehydrogenase
additionally, cyclophosphamide can undergo N-dealkylation on either chain
the product of this reaction is chloroacetaldehyde, which is highly nephrotoxic and neurotoxic
it can be further oxidized by the action of aldehyde dehydrogenase to chloroacetic acid |
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Term
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Definition
[image]
ifosfamide is a related analog of cyclophosphamide
note that the second 2-chloroethyl chain is on the ring nitrogen
it is bioactivated in a similar fashion to cyclophsophamide
it can form a phosphormamide mustard that is the active alkylating species
also, the aldehyde acrolein is produced in the bioactivation of ifosfamide |
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Term
metabolism of ifosfamide
[image] |
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Definition
[image]
bladder toxicity:
> amounts of chloroacetaldehyde generated
higher doses are used than with cyclophsophamide
concentrates in renal system
acrolein also generated
may be combined with mesna (SH reagent that reacts with acrolein)
the toxicity of ifosfamide is greater than cyclophosphamide due to greater N-dealkylation to produce chloroacetaldehyde (45% as compared to 10%)
mesna can be administered to react with the acrolein that is generated in the bioactivation of ifosfamide |
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Term
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Definition
[image]
[image]
may impart unique activity compared to other alkylating agents (cyclophosphamide, cisplatin, or carmustine)
DNA cross linking agent causes DNA breaks (single and double) more durable than other alkylating agents
bendamustine is another aromatic mustard
unique activity: the compound seems to produce greater single and double strand DNA breaks than other alkylating agents
approved for B-cell non-Hodgkin lymphoma and chronic lymphocytic leukemia |
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Term
melphalan formulated as HCl salt
[image] |
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Definition
[image]
in the case of melphalan, it can be formulated as the HCl to improve water solubility
under neutral conditions, it readily forms the aziridinium ion which can function as a bifunctional alkylating agent
this compound can be administered by the oral route which indicates that it is much less reactive
similar to all nitrogen mustards, it can undergo hydrolysis in neutral or basic solution
once hydrolyzed to the diOH species, it no longer functions as an alkylating agent
in acidic conditions, the NH3+ is protonated and there will not be free electrons to act as a nucleophile and the aziridinium ion is not formed |
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Term
| structural comparison of nitrogen mustards |
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Definition
[image]
this shows the structures of the 4 types of nitrogen mustards or related compound (cyclophosphamide)
the presence of the COOH group on bendamustine and chlorambucil enhances their water solubilities
bendamustin, chlorambucil, and melphalan are aromatic nitrogen mustards. as such, due to electron delocalization on the aromatic ring, they are less reactive than an aliphatic nitrogen mustard (e.g. mechlorethanamine)
in the truest sense, cyclophosphamide is not a nitrogen mustard
more appropriately, it is a cyclic phosphoramide that is activated to a reactive phosphoramide mustard |
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Term
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Definition
[image]
MUST BE ACTIVATED BY OXIDATIVE DESULFURATION
aziridine is less reactive than the protonated aziridinium ion
several species could cross link DNA (one possible reaction is shown):
[image]
aziridines are highly reactive species due to the ring strain
they are readily attacked by nucleophiles.
in the case of thiotepa, it is a thiophosphoramide. as such, it is much less reactive than an aziridine
the reason is that the unshared electron pair on nitrogen can be delocalized by resonance
the compound is activated to a phosphoramide
the reason is that the more electronegative oxygen facilitates the attack of a nucleophile on the 3 membered aziridine ring by making the ring carbons more electrophilic (i.e. more susceptible to attack by nucleophiles)
the compound can function as a bifunctional alkylating agents by reaction on 2 of the 3 membered rings |
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Term
| methanesulfonates: busulfan (granulocytic leukemia) |
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Definition
[image]
busulfan can serve as a bifunctional alkylating agent by attack of 2 nucleophilic groups on the carbons containing the sulfonate leaving groups
[image]
administered orally and IV
undergoes slow hydrolysis in water
[image]
in addition to alkylation of DNA, busulfan can undergo slow hydrolysis with water to give a diol (2 OH) which is biologically inactive
much of busulfan is subject to sulfur stripping in that the compounds can react with endogenous SH compounds such as Cys-SH groups and glutathione:
[image]
busulfan can undergo a process known as sulfur stripping
in this case, certain thiol-containing proteins displace one of the fulfonate groups
the sulfur from thiol containing proteins then can displace the other sulfonate group
this creates a cyclic sulfonium species (not the positive charge on sulfur)
this charge can be eliminated by reaction with biological nucleophiles |
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Term
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Definition
[image]
the nitrosoureas are another type of alkylating agent that must undergo bioactivation
nitrosoureas typically contain a 2-chloroethyl or methyl group on the nitrogen bearing the nitroso group
decomposition of nitrosoureas: thermal decomposition pathway
[image]
the thermal decomposition of nitrosoureas generates an isocyanate and an azohydroxide |
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Term
| fate of decomposition products of nitrosoureas |
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Definition
AZOHYDROXIDE:
[image]
the azohydroxide that is generated by thermal decomposition of a nitrosourea can decompose to nitrogen and a chloroethyl cation
this cation can serve as a biological alkylating agent (bis-alkylating agent) or undergo reaction with water (to form an alcohol) or Cl- (to form dichloroethane)
it can also rearrange to react with water to produce acetaldehyde
ISOCYANATE:
[image]
isocyanates that are generated by decomposition of nitrosoureas can react with amino groups on Lys side chains of proteins
the amino groups are said to be carbamoylated which alters protein structure
may lead to inhibition of several types of DNA repair enzymes
the isocyanate that is generated by decomposition of nitrosoureas, is probably responsible for adverse effects by carbamoylating amino acid side chains that are contained in proteins
the isocyanate carbon is electropositive (due to the electron withdrawing effects of the oxygen and nitrogen), thus it is readily attacked by biological nucleophiles (e.g. the amino groups of Lys)
note that this produces a urea function on the side chain nitrogen. the protein structure is therefore oltered by carbamoylation |
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Term
| alternative mechanism for DNA alkylation by nitrosoureas (reaction with water) |
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Definition
[image]
an alternative activation of a nitrosourea can exist in its tautomeric enol tautomer
the tautomer can be attacked by water that gives a species that breaks down to yield a carbamic acid and an azohydroxide
carbamic acids are unstable and eliminate carbon dioxide to yield amines
the other product of this decomposition is an azohydroxide
as mentioned earlier, the azohydroxide breaks down to yield nitrogen and acation
this cation can then serve as an alkylating agent |
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Term
| cross-linked DNA by the 2-chloroethyl cation |
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Definition
[image]
shown in this diagram is an example of bis-alkylation involving the 2 chloroethyl cation (generated by the decomposition of the nitrosourea)
the cation can react with the N7 position of guanine and the Cl group is readily displaced by the N3 nucleophilic nitrogen of adenine
the carbon to which the Cl is attached is electropositive due to the electron withdrawing effect of the Cl. thus, it is readily displaced by a nucleophile |
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Term
specific nitrosoureas: carmustine and lomustine
[image] |
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Definition
[image]
lipophilic and cross the BBB
carmustine used IV and as Gliadel wafers
lomustine used orally
Gliadel wafers:
added to surgical cavity after resection of the tumor
allows for a controlled release of carmustine
the wafer is a polymer consiting of bis-p-carboxyphenoxypropane (polifeprosan) 20 and sebacic acid 80
[image]
[image]
the key to release of carmustine from giadel wafers is the hydrolysis of the anhydride linkage of the polymer to 2 molecules of 2 different acids
once the anhydride linkage is hydrolyzed, the drug is slowly released giving a more prolonged action
carmustine contains chloroethyl groups on both nitrogens of the nitrosourea. it can be used in a dosage form that is formulated in a polymeric matrix. this is used in cases of certain brain tumors. after removal of the tumor the polymeric dosage form is placed in the resection cavity and upon hydrolysis of the polymer, the compound is released from the delivery device. this leads to a prolonged action of the drug. |
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Term
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Definition
[image]
specific for islet cell carcinoma
FORMATION OF THE REACTIVE METHYLATING SPECIES:
[image]
streptozocin is a nitrosourea derived from D-glucose
note that it has a methyl substitution on the N bearing the nitroso group
thus, upon activation it yields an isocyanate and a methyl cation
the methyl cation serves as the alkylating species
steptozocin is a hydrophilic hitrosourea that is specific for islet cell carcinoma
ONLY FORMS ONE ALKYLATING AGENT
reactive methyl group produced on decomposition of streptozocin can methylate purine or pyrimidine bases of DNA
[image]
the methyl carbonium ion (cation) that is generated from streptozocin can alkylate purine or pyrimidine bases
in this case, it serves as a mono-alkylating species since there is no leaving group (e.g. 2-chloroethyl) on the other end of the cation |
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Term
| nitrosoureas related to lomustine |
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Definition
[image]
decreasing the log P increased toxicity
lomustine is a highly lipophilic compound that readily penetrates the BBB |
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Term
methylhydrazines: procarbazine
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Definition
[image]
methyl hydrazines can also act as alkylating agents (again by bioactivation)
formation of methyl carbonium ion (methylating species) from procarbazine:
[image]
procarbazine is actually a prodrug for the highly reactive methylcarbonium ion
the compound is a hydrazine that undergoes N-hydroxylation by P450
the resulting N-OH compound undergoes loss of water to form a diazo CH3N=N-R intermediate
the diazo intermediate undergoes benzylic carbon oxidation by P450
remember - a benzylic carbon is the carbon alpha (adjacent) to the phenyl ring
the resulting carbinoldiazo species loses methyl diazene
this species loses a proton to form methyldiazonium which in turn loses nitrogen and forms a highly reactive carbonium ion
the methylcarbonium ion can alkylate the N7 position of guanine bases in DNA
ultimately, there is a mis-pairing of bases in DNA, leading to altered amino acids and a disruption of protein function |
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Term
triazines: dacarbazine and temozolamide
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Definition
triazenes (dacarbazine and temozolamide) are able to yield methyl diazonium (a highly reactive species and can generate a methyl carbonium ion)
hydrazines are similar to amines, but with 2 adjacent nitrogens
like amines hydrazines are basic functional groups
dacarbazine DNA methylating agent:
[image]
[image]
dacarbazine is a DNA methylating agent
it is a triazene (note 3 adjacent nitrogens in which there is unsaturation (double bond))
it is activated by P450 to yield methyl diazonium
methyl diazonium is highly reactive (note the positive charge on nitrogen)
it can be attacked by a nucleophile to displace nitrogen gas or it can break down to yield a methyl carbonium ion
in either case, alkylation occurs
in this case the methyl group is attached to a nucleophilic site on a prurine or pyrimidine base
metastatic malignant melanoma and Hodgkin's disease:
[image]
shown here are sites of alkylation by the methyl carbonium ion that is generated by bioactivation of dacarbazine |
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Term
metabolism of procarbazine
[image] |
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Definition
procarbazine is administered orally
extensively metabolized
70% is excreted renally in 24 hours
[image]
procarbazine generates an aldehyde metabolite either by bioactivation or by benylic carbon oxidation
the resulting aldehyde is converted to a carboxylic acid
two take home messages - 1) benzylic carbon oxidation is oxidation of the carbon adjacent to the benzene ring and 2) aldehyde dehydrongenase oxidizes an aldehyde to a carboxylic acid |
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Term
disadvantages of dacarbazine
[image] |
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Definition
poor water solubility (IV administration necessary)
photosensitivity (protect IV bags from light)
[image]
dacarbazine is broken by UV light to liberate dimethyl amine and a dizaonium species
the diazonium species reacts with itself
thus, it no longer functions as an alkylating agent |
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Term
temozolamide (prodrug)
[image] |
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Definition
[image]
structural comparison between dacarbazine and temozolamide:
[image]
temozolamide is a prodrug of dacarbazine
the triazene structure is incorporated in the ring structure in temozolamide, thus, it is more stable to UV light
activation of themozolammide - generation of a methylcarbonium ion (methylating species):
[image]
BIOACTIVATION IS NON ENZYMATIC
BONE MARROW TOXICITY
the activation of temozolamide is shown above
not that it is not enzymatically activated
the reactive product that is liberated is the methyl carbonium ion (the alkylating species) |
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Term
platinum compounds: cisplatin
[image] |
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Definition
[image]
two amine groups
two leaving groups
THESE COMPOUNDS ARE ELECTRON DEFICIENT AND REACT WITH ELECTRON RICH NUCLEOPHILES (DNA PRUINE BASES SUCH AS GUANINE)
ELECTRON DEFICIENT, BUT A NET CHARGE OF ZER ON PT DUE TO THE ELECTRON RELEASING EFFECTS OF THE CL GROUPS
platinum compounds have a zero net charge and a square geometry
they contain 2 groups (usually amino groups) that facilitate the binding of the compounds to the pohsphate backbone of DNA and 2 leaving groups (Cl or some type of oxygen species)
platinum compounds MUST BE BIOACTIVATED THROUGH WATER
generation of the di-aquo species:
[image]
in this case, the activation of cisplatin is shown
it passes into the cell and then is converted to its diaquo form by water
not that the oxygen derived from water has a positive charge
activation and fate of cisplatin:
[image]
cisplatin can enter the cell via the copper transporter (CTR1) or possibly by passive diffusion
in the cytoplasm where the Cl ion concentration is much less, cisplatin undergoes hydrolysis to form the activated di-aquo form
this species can react with glutathione or metallothionein in the cytoplasm to inactivate the reactive species
alternatively, an efflux transporter can pump the activated form outside the cell
these events may consume the reactive form of cisplatin prioir to its reaching the nucleus to react with DNA
these degradation pathways mop up the activated form of cisplatin and may explain resistance to cisplatin |
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Term
| intrastrand cross linking by Pt complexes |
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Definition
[image]
platinum compounds produce DNA intrastrand cross links
the key here is displace the positively charge water species from platinum by the purine base on DNA |
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Term
cisplatin is highly nephrotoxic
amifostine can be used to offset the nephrotoxicity |
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Definition
[image]
cisplatin is highly nephrotoxic
the compound amifostine can be used to offset this toxicity
in the case of amifostine it reacts with platinum to form a water soluble salt that is ionized at the pH of the kidney
resistance does occur to cisplatin and may be attributed to a number of factors:
1) cisplatin requires a transporter to enter the cell and low levels of the transporter can cause resistance
2) the reactive intermediate reacts with thiols such as glutathione. thus, resistance can occur if glutathione levels are high
3) the reactive form of cisplatin is used up (reaction with biological nucleophiles) prior to its ability to react with DNA |
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Term
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Definition
less extensively bound to plasma proteins than cisplatin
less side effects compared to cisplatin
[image]
the leaving group is the oxygen atom contained on the diester function
it can form the same reactive species (the di-aquo form) as cisplatin |
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Term
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Definition
[image]
produces intrastrand cross links at G-G, A-G, and guanines separated by one nucleotide G-X-G
due to hydrophobic character produces much less bend in DNA compared to cisplatin
less ADRs than cisplatin
decomposes in base
should not be administered with compound that increase the pH of the solution (hydrolysis of the oxalic portion)
oxaliplatin is much like carboplatin
the leaving groups are the oxygens of the oxalic acid ester that is attached to the platinum atom
these types of compounds (oxaliplatin and carboplatin) can be hydrolyzed in basic solution
INTRASTRAND CROSS LINKING like other platinum complexes |
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Term
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Definition
analogs of NATURALLY occurring compounds that interfere with their FORMATION or UTILIZATION
most antimetabolites interfere with DNA or RNA
inhibit key enzymes in essential biosynthetic reactions
incorporate into nucleic acids, which inhibits their normal function and trigger apoptosis
pyrimidine antagonists:
fluorouracil (5-FU)
floxuridine
capecitabine
pemetrexed
inhibitors of dihydrofolate reductase:
methotrexate
pralatrexate
purine antagonists:
mercaptopurine (6-MP)
thioguanine
DNA polymerase and chain elongation inhibitors:
cytarabine
gemcitabine
fludarabine
cladribine
clofarabine
nelarabine
antimetabolites prevent the synthesis or use of cellular metabolites:
nearly all of structurally related to the metabolites that they antagonize
many are enzyme inhibitors
may bind to the active site or an allosteric site on the enzyme |
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Term
| sites of intervention by antimetabolites |
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Definition
[image]
shown above are the possible sites of intervention by an antimetabolite
1) inhibition of key enzymes in the synthesis of precursors in the biosynthesis of purine or purine bases
2) inhibition of ribonucleotide reductase. this is a key enzyme that converts a ribonucleotide to a deoxyribonucleotide. if this enzyme is inhibited then deoxyribonucleotides can be synthesized that are needed for DNA synthesis
3) blocking DNA polymerase or chain elongation by becoming incorporated into DNA or RNA (actually false nucleotides) |
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Term
purine antagonists: mercaptopurine (6MP)
[image] |
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Definition
[image]
used orally or IV as the sodium salt (pKa of the SH group is about 8.6)
[image]
6MP is a purine antimetabolite
it can exist in the thiocarbonyl or thiol tautomer
in the thiol tautomer it is weakly acidic and can be used as its sodium salt
mercaptopurine requires bioactivation:
[image]
6MP is a substrate for the enzyme hypoxanthing-guanine phosphoribosyl transferase
this enzyme attaches a ribose phosphate to the N9 nitrogen of 6MP to give the bioactivated form of 6MP (thioinosinic acid)
thioinosinic acid is actually an antimetabolite of inosinic acid (note the difference C=S vs. C=O)
thioinosinic acid is an INHIBITOR OF A KEY REGULATORY ENZYME IN PURINE BIOSYNTHESIS (PHOPHORIBOSYL PYRROPHOSPHATE AMIDOTRANSFERASE)
this enzyme attaches the amido nitrogen to the sugar to produce phosphoribosylamine
this amine is a required starting material for the synthesis of purine bases needed for DNA synthesis
inhibition of this enzyme thus decreases the production of purine bases required for the synthesis of DNA
6MP can also inhibit the synthesis of AMP and GMP as shown below:
[image]
additionally, thioinosinic acid can inhibit pathways leading to AMP and GMP
the net result is the inhibition of AMP and GMP (both needed for DNA synthesis)
summary sites of inhibition by 60thioinosinic acid (activated 6-MP)
[image]
the above summarizes the action of 6MP
1) it must undergo bioactivation to thioinosinic acid
2) thioinosinnic acid is an inhibitor of the key regulatory enzyme in the biosynthesis of purines (phosphoribosylamidotransferase)
3) thioinosinic acid inhibits the synthesis of AMP and GMP by blocking the conversion of inosinic acid to AMP and GMP
mis-incorporation of thionisinic acid into DNA and RNA leads to a complex series of events that can cause cell death
[image]
6MP BIOACTIVATED TO THIOINOSINIC ACID
THIOINOSINIC ACID INHIBITS THE SYNTHESIS OF GMP AND AMP
THE DI AND TRIPHOSPHATES ARE ALSO INCORPORATED IN RNA AND DNA TO PREVENT CHAIN ELONGATION
6MP can also serve as a substrate for thiopurine methyltransferases (TPMT)
[image]
6MP and its activated form, thioinosinic acid, serve as substrates for thiopurine methyl transferase
this enzyme methylates the sulfur at the 6 position to yield S-methyl metabolites.
6-thiomethylmercaptopurine cannot be converted into its nucleotide form (i.e. it is not a substrate for hypoxanine-guanine phosphoribosyl transferase)
the S-methyl metabolite of thioinosinic acid is an active metabolite that acts much like that of thioinosinic acid |
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Term
| polymorphisms in thiopurine methyl transferase (TPMT) |
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Definition
poor metabolizers (PM) only 10% of the population:
decreased levels of S-Me TP nucleotide
increased levels of thioinosinic acid
[image]
extensive metabolizers (EM) 90% of the population:
decreased levels of thioinosinic acid
increased levels of S-Me TP nucleotide
less risk of ADRs compared to PM
PM run the risk of accumulation of thioinosinic acid which may produce adverse effects |
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Term
6MP and thioinosinic acid are substrates for xanthine oxidase
co administration of allopurinol (xanthine oxidase inhibitor) allows dose of 6MP to be reduced by 1/2 |
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Definition
[image]
6MP and thioinosinic acid are substrates for xanthine oxidase
this is an enzyme that oxidizes the purine ring at positions 2 and 8 to lead to inactive metabolites
the compound allopurinol (used in gout) is an inhibitor of xanthine oxidase
thus, in the presence of allopurinol, the dose of 6MP should be reduced to avoid toxic effects |
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Term
azathioprine (prodrug of 6MP)
[image] |
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Definition
protects against degradation by:
1) xanthine oxidase
2) thiopurine methyl transferase (TPMT)
[image]
azathioprine is actually a prodrug of 6MP
it protects 6MP from initial action of xanthine oxidase and TPMP
the compound is activated by attack of SH containing compounds (e.g. glutathione) to produce an intermediate in which sulfur is attached to the carbon adjacent to the nitro group
this species readily breaks down to generate 6MP |
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Term
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Definition
[image]
thioguanine acts much like 6MP
it is activated by the same process as 6MP
the nucleotide that is produced inhibits phophoribosylamido transferase and inhibits the conversion of inosinic acid to AMP and GMP |
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Term
| inhibitors of thymidylic acid biosynthesis |
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Definition
pyrimidine antimetabolites:
5-fluorouracil
floxuridine
capecitabine
[image]
5-fluorouracil, floxuridine, and capecitabine are pyrimidine antagonists or antimetabolites
note that floxuridine is a 2'deoxyribonucleoside and capecitabine is a 5'deoxyribonucleoside
all of these compounds can be considered prodrugs
the 5-F group of these compounds is essential for their action
F has approximately the same size as H and does not sterically affect these antimetabolites |
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Term
| bioactivation of 5-FU and floxuridine |
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Definition
[image]
the activation of 5-FU and floxuridine is shown
5-FU is converted first to nucleoside monophosphate and then to diphosphate
at the diP stage, the enzyme ribonucleotide reductase converts the ribonucleotide to the deoxyribonucleotide
a phosphatase then converts the deoxyribonucleotide to a monophosphate
in the case of floxuridine, it is bioactivated by simply phosphorylation to its deoxyribonucleotide form
RIBONUCLEOTIDE REDUCTASE WILL ONLY REDUCE THE RIBONUCLEOTIDE WHEN IT IS IN THE DIPHOSPHATE FORM
ACTIVATED FORM IS DEOXY AND ONE PHOSPHATE GROUP |
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Term
| conversion of dUMP to 5-methylene-dUMP |
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Definition
[image]
UMP is the natural metabolite
deoxy-UMP attaches to the S of the enzyme (thymidylate synthase)
the complex attacks folate which serves as a one carbon donor; this is broken down and the intermediate is still bound to thymidylate synthase
KEY TO THE REACTION IS BASE CATALYZED REMOVAL OF A PROTON
[image]
the synthesis of thymidylate is completed by a transfer of a hydride to the 5-exocyclic methylene group of uracil by tetrahydrofolate
as the hydride is transferred to the exocyclic carbon thymidylate synthase is detached
the resulting product is thymidylate and dihydrofolate
THYMIDYLATE IS REQUIRED FOR DNA SYNTHESIS
another key point, dihydrofolate must be reduced to tetrahydrofolate (regeneration of tetrahydrofolate for the synthesis of more thymidylate)
this is accomplished by the action of dihydrofolate reductase
7,8 DHF MUST BE REDUCED TO TETRAHYDROFOLATE SO THAT N5,N10 TETRAHYDROFOLATE CAN BE REGNERATED AND SERVE AS A ONE CARBON DONOR TO DUMP |
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Term
| synthesis of thymidylate (dTMP) blocked by 5FU and related compounds |
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Definition
[image]
the above shows the steps involved in the biosynthesis of thymidylic acid
thymidylate synthase attacks the 6 position of pyrmidine kicking out the electron pair on the oxygen at the 4 position
N5,N10 methylene tetrahydrofolate is a required cofactor in this reaction
this cofactor donates 1C (one carbon donation) to attach uracil to the cofactor
THE KEY TO REACT IN THE SYNTHESIS OF THYMIDYLIC ACID IS THE BASE CATALYZED REMOVAL OF THE PROTON AT POSITION 5
removal of this proton and a shift of the electron pair generates an exocyclic methylene group on uracil at position 5 (this carbon comes from the cofactor N5,N10-methylenetetrahydrofolate)
the cofactor is converted to tetrahydrofolate
note that thymidylate synthase is still attached to the 6 position of uracil
[image]
the above shows the inhibition of thymidylate synthase by 5FU
note that the 5F group cannot be removed by the base. THIS IS THE KEY IN THE INHIBITION OF THYMIDYLATE SYNTHASE
the ternary complex (5-FU, TS, and N5,N10 methylenetetrahydrofolate) cannot be broken down
thus, the enzyme is inhibited
the net result is the synthesis of thymidylate is inhibited, the DNA, RNA, and protein synthesis is altered |
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Term
| summary of the effects of 5FU and related analogs |
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Definition
[image]
the above shows the summary of effects by the activated form of 5FU
note that it can be incorporated into DNA to alter normal DNA function |
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Term
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Definition
[image]
oral prodrug of 5FU that is active in a variety of tumor types
approved in the US for metastatic breast and colorectal cancer
capecitabine is an oral prodrug
its activation is somewhat different: the butyl carbamate group increases its lipophilicity and it enters cells more readily
note that it is a 5'deoxyribonucleotide
[image]
THYMIDINE PHOSPHORYLASE IS HIGHLY EXPRESSED IN TUMOR CELLS ALLOWING HIGHER CONCENTRATION OF DRUG IN TUMOR CELLS
capecitabine is first acted on by esterases to cleave the carbamate linkage
the products of this reaction are an alcohol, carbon dioxide, and an amine
the amine is then oxidized at the 4 position by cytidine deaminase
note the compound is now oxidized at the 4 position in a similar fashion as 5FU
the sugar is then cleaved by the action of thymidine phosphorylase. this enzyme is present in tumor cells to a much greater level than normal cells
the phosphorylase reaction thus yields 5FU
the generated 5FU is then activated in the normal fasion |
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Term
| folate based thymidylate synthase inhibitors |
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Definition
compounds that can recognize the folate binding site on thymidylate synthase
[image]
premetrexed is a folate based compound that binds to the folate binding site on thymidylate synthase
this compound inhibits the action of tetrahydrofolate in the completion of thymidylate synthesis
remember that the final step is a transfer of a hydride from tetrahydrofolate to the exocyclic methylene group of uracil
if this step is inhibited then the completion of thymidylate synthesis cannot be completed |
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Term
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Definition
[image]
acts on multiple tartes
gains entry into cancer cells by the reduced folate carrier
undergoes polyglutamation in cancer cells
inhibits thymidylate synthase (decreased dTMP production)
inhibits glycineamide ribonucleotide tranformylase (decreased AMP and GMP production)
premetrexed acts by inhibiting thymidylate synthesis and by inhibition of a key enzyme in the synthesis of AMP and GMP
it is transported into the cells, where it undergoes polyglutamation
note the glutamate portion on this structure
the ionized glutamate groups at pH 7.4 traps the compound in the cell
[image]
molecule enters through a folate carrier and undergoes polyglutamation (more glutamic acids are added) and the carboxy groups will be ionized in the cell and traps the drug in the cell |
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Term
methotrexate - inhibitor of dihydrofolate reductase
[image] |
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Definition
[image]
INDIRECT INHIBITOR OF THYMIDYLATE SYNTHASE - therefore synthesis of dTMP and DNA is inhibited
INHIBITS GLYCINAMIDE RIBONUCLEOTIDE TRANSFORMYLASE - inhibits the formation of AMP and GMP (purine nucleotides required for DNA synthesis)
methotrexate is a folate analog
note the presence of the 4 amino group and the methyl group on the side chain
it functions to inhibit:
1) dihydrofolate reductase, thus thymidylate synthesis is inhibited
2) it blocks a key regulatory step in the synthesis of purines AMP and GMP (glycinamide formyl transferase)
[image]
note that methotrexate inhibits both AMP, GMP, and thymidylate synthesis
methotrexate can be given oral, IV, SC, intrathecal
sodium salt is actively transported by carrier facilitated transport
polyglutamation in the cancer cell traps the drug
[image] |
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Term
| binding of 7,8-dihydrofolate and methotrexate to the active site of DHRF |
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Definition
[image]
the key to methotrexate's action as an inhibitor of dihydrofolate reductase is the amino group at the 4 position of the pteridine ring
in this slide the reduction of dihydrofolate to tetrahydrofolate by dihydrofolate reductase requires a hydride transfer from the cofactor NADPH
the nitrogen at the 5 position of dihydrofolate is protonated and binds to an Asp group on the active site of the reductase
this position the double bond at the 5,6 position can accept the hydride from the cofactor
thus, duhydrofolate is converted to tetrahydrofolate
[image]
in the case of methotrexate, the 4 amino group (by resonance) renders the N1 nitrogen most basic.
in order to bind to the active site of dihydrofolate, the side chain of methotrexate is orientated away from the cofactor
thus, the 7,8 double bond of methotrexate is not reduced by NADPH
the compound thus serves to inhibit the reduction of dihydrofolate to tetrahydrofolate
the reduced cofactor is necessary for the synthesis of hymidylate |
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Term
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Definition
metabolism of methotrexate by P450 produces a 7OH metabolite
this OH group can H bond with the side chain nitrogen at 10 leading to less water soluble metabolite that may crystallize out in the kidney tubules
[image]
in severe cases of toxicity, leucovorin (N5-formyl FH4) can be administered
this compound is actually 5-formyl tetrahydrofolate
it can form methylene tetrahydrofolate WITHOUT THE AID OF DIHYDROFOLATE REDUCTASE
thus, methylenetetrahydrofolate is available for thymidylate synthesis
[image] |
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Term
| carboxylpeptidase used IV for methotrexate induced nephrotoxicity |
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Definition
use of glucarpidase for the treatment of (high dose) methotrexate induced nephrotoxicity
[image] |
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Term
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Definition
[image]
refractory peripheral T-cell lymphoma
inhibits dihydrofolate reductase
inhibits folylglutamyl synthase
enters the cell by the reduced folate carrier-1
intracellularly, it is polyglutamated and competes for the folate binding site on dihydrofolate reductase
thus, DNA, RNA, and protein synthesis is decreased |
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Term
| antimetabolites - DNA polymerase/DNA chain elongation inhibitors |
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Definition
[image]
nucleosides are taken up by the nucleoside transporter protein
activated by formation of triphosphates by specific kinases
by complex mechanism incorporated in DNA to prevent chain elongation and alteration in DNA repair
the antimetabolites shown here are all pyrimidine or purine nucleosides or nucleotides
you will notice that in all cases, the 2-OH group of ribose has been modified, either as an epimer (arabinose) or a fluoro or difluoro group
this prevents the substance or its activated form from being a substrate for ribonucleotide reductase
normal DNA synthesis:
[image]
in normal DNA synthesis the purine or pyrimidine deoxytrinucleotides are incorporated in the growing chain by DNA polymerase
the 3'OH of the last base added acts on a nucleoside triphosphate to introduce the next base that is complementary (A-T or C-G) on the DNA template strand
pyrrophosphate is generated in this reaction and the hydrolysis of the pyrrophosphate drives this reaction
chain terminators:
[image]
in the case of chain terminators, these compounds resemble the normal substrate trinucleotides; however, once incorporated into the growing chain they are unable to react with other nucleotides
thus, chain growth is terminated
chain terminators must satisfy three conditions:
1) they must be recognized by the DNA template. that is, they must resemble very closely the normal nucleoside triphsophates
2) they must be incorporated in the growing strand
3) once incorporated into the growing chain, they are unable to react with another deoxyribonucleotide. thus chain growth is terminated, leading eventually to cell death |
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Term
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Definition
[image]
cytarabine liposomal: depofoam delivery system that is administered intrathecally once every 2 weeks
cytarabine is an arabinose derivative that is activated to its triphosphate form |
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Term
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Definition
fludarabine is a nucleotide that undergoes hydrolysis upon administration
it then uses the nucleoside transporter to gain entry into the cells where it is converted to the triphosphate by deoxycytodine kinase
it is incorporated into DNA to prevent chain elongation
may inhibit DNA polymerase other enzymes
this results in decreased DNA synthesis and cell growth |
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Term
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Definition
NELARABINE IS A PRODRUG OF ARA G WITH IMPROVED WATER SOLUBILITY
[image]
ara-GTP competes with GTP for incorporation into DNA (leads to apoptosis and cell death)
narlarbine is a prodrug that is converted to ara-G (sugar is arabinose) by the action of adenosine deaminase
it gains entry into the cell by the nucleoside transporter where it is converted to the arabinosetrinucleotide by the action of kinases
this antimetabolite them competes with GTP for incorporation into DNA, leading to apoptosis and cell death
the methyl group makes the compound more water soluble, this is unusual b/c usually methyl makes a drug more lipophlic
it is more water soluble b/c ara-G is more likely to undergo intramolecular H-bonding and will decrease water solubility |
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