The Medicinal Chemistry of 5-Fluorouracil (5FU)

Question 1

What is 5-FU?

Answer 1

• An antimetabolite (like MTX)
• Pro-drug; transformed to 5-Fluro-dUMP in the body which inhibits TS
• Impeded biosynthesis of precursor ting required to make key components that cancer cells require for growth/development
• Inhibits Thymidilate (dTMP, conjugate base of thymidine nucleotide) production by inhibiting TS; thymidylate synthase

Question 2

How is thymidilate (dTMP) normally synthesised?

Answer 2

• dTMP biosynthesised from dUMP by TS
• TS introduces methyl group (reductive methylation) into dTMP by transferring it from the cofactor, N5, N10-methylene tetrahydrofolate (THF)
• N5, N10-methylene tetrahydrofolate (THF) converted to DHF as a result
• DHF recycled by DHFR (dihydrofolate reductase); site of action for MTX, which depletes cell of THF (DHF can’t be reduced back to THF as a result)

Question 3

What is the mechanism of action for normal TS catalysis of dUMP > dTMP?

Answer 3

Nucleophilic attack:
1) Thiolate anion (negative) of TS side chain attacks C=C bond of pyrimidine ring of dUMP
2) This results in the attack of the methylene group of N5, N10-methylene THF co-factor from dUMP (C=C e-?)
3) Rando floater H+ is picked up by N of N5, N10-methylene THF
»> TS enzyme, dUMP and N5, N10-methylene THF are thus covalently linked together (both anchored to TS)

Elimination (regenerate C=C of dUMP):

4) H is lost from the pyrimidine ring of dUMP (e- reforms C=C)
5) Results in Thiolate anion regeneration too; dUMP-TS bond is cleaved

Second Elimination (dUMP still attached to folate cofactor)

6) H is lost from folate cofactor, forming N=C in now-DHF cofactor
7) N of DHF has too many bonds; DHF detaches from dUMP by C formally of (H2)C-N picking up H+ floater to form completed methyl group (transfer complete from THF)

Completed reaction:

• Methylated dUMP (dTMP)
• DHF
• TS recycled

Question 4

What is the mechanism of action of 5-FU?

Answer 4

Enzyme picks up and anchors 5-Fluoro-dUMP instead of dUMP

• Nucleophilic attack (as before but w/5-F-dUMP)
1) Thiolate anion (negative) of TS side chain attacks C=C bond of pyrimidine ring of 5-F-dUMP
2) Resulting in attack of the methylene group of N5, N10-methylene THF co-factor by 5-F-dUMP (C=C e-?)
3) Rando floater H+ is picked up by N of N5, N10-methylene THF
»> TS enzyme, 5-F-dUMP and N5, N10-methylene THF are thus covalently linked together (both anchored to TS)

• Elimination (regenerate C=C)
4) Doesn’t happen! Can’t perform elimination to regnerate C=C as can’t lose F as F+ (formally H/H+ in standard dUMP)
> Thus 5-F-dUMP - TS bond is not cleaved either (no thiolate anion regeneration), can’t proceed to second elimination
»> Everything (TS, N5, N10-methylene THF and 5-F-dUMP) stays ‘glued’ together covalently bonded
»> Irreversible inhibition of Thymidilate Synthase (TS)