3NU0: Design, Synthesis, Biological Evaluation and X-ray Crystal Structure of Novel Classical 6,5,6-TricyclicBenzo[4,5]thieno[2,3-d]pyrimidines as Dual Thymidylate Synthase and Dihydrofolate Reductase Inhibitors

Classical antifolates (4-7) with a tricyclic benzo[4,5]thieno[2,3-d]pyrimidine scaffold and a flexible and rigid benzoylglutamate were synthesized as dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. Oxidative aromatization of ethyl 2-amino-4-methyl-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxylate (+/-)-9 to ethyl 2-amino-4-methyl-1-benzothiophene-3-carboxylate 10 with 10% Pd/C was a key synthetic step. Compounds with 2-CH(3) substituents inhibited human (h) TS (IC(5)(0) =0.26-0.8 muM), but not hDHFR. Substitution of the 2-CH(3) with a 2-NH(2) increases hTS inhibition by more than 10-fold and also affords excellent hDHFR inhibition (IC(5)(0) = 0.09-0.1 muM). This study shows that the tricyclic benzo[4,5]thieno[2,3-d]pyrimidine scaffold is highly conducive to single hTS or dual hTS-hDHFR inhibition depending on the 2-position substituents. The X-ray crystal structures of 6 and 7 with hDHFR reveal, for the first time, that tricyclics 6 and 7 bind with the benzo[4,5]thieno[2,3-d]pyrimidine ring in the folate binding mode with the thieno S mimicking the 4-amino of methotrexate.
PDB ID: 3NU0Download
MMDB ID: 90789
PDB Deposition Date: 2010/7/6
Updated in MMDB: 2017/11
Experimental Method:
x-ray diffraction
Resolution: 1.35  Å
Source Organism:
Similar Structures:
Biological Unit for 3NU0: monomeric; determined by author and by software (PISA)
Molecular Components in 3NU0
Label Count Molecule
Protein (1 molecule)
Dihydrofolate Reducatase(Gene symbol: DHFR)
Molecule annotation
Chemicals (5 molecules)
* Click molecule labels to explore molecular sequence information.

Citing MMDB