New C2- and N3-Modified Thieno[2,3-d]Pyrimidine Conjugates with Cytotoxicity in the Nanomolar Range
Authors: Mavrova Anelia Ts. , Yancheva Denitsa , Dimov Stefan , Rangelov Miroslav , Wesselinova Diana , Naydenova Emilia 
Published: July 2021

Aims:
The aim of the current study was to develop and explore a series of new cytotoxic agents based on the
conjugation between the thieno[2,3-d]pyrimidine moiety and a second pharmacophore at the C2 or N3 position.

Background:
As the thieno[2,3-d]pyrimidine core is a bioisostere of the 4-anilinoquinazoline, various new thienopyrimidine
derivatives were synthesized by modifying the structure of the clinically used anticancer quinazoline EGFR
inhibitors of the first generation – gefitinib, and second-generation – dacomitinib and canertinib. It was reported that
some thieno[2,3-d]pyrimidine derivatives showed improved EGFR inhibitory activity. On the other hand, the benzimidazole
heterocycle is present as a pharmacophore unit in the structure of many clinically used chemotherapeutic agents.
Some 2-aminobenzimidazole derivatives, possessing anticancer activity, demonstrated EGFR inhibition and the benzimidazole
derivative EGF816 is currently in the second phase of clinical trials.

Objective:
The objectives of the study were the design of a novel series thieno[2,3-d]pyrimidines, synthesis of the
compounds and investigation of their effects towards human cancer HT-29, MDA-MB-231, HeLa, HepG2 and to normal
human Lep3 cell lines. (American Type Culture Collection, ATCC, Rockville, MD, USA).

Methods:
The synthetic protocol implemented cyclocondensation of 2-amino-thiophenes and nitriles in an inert medium, aza-
Michael addition to benzimidazole derivatives and nucleophylic substitution at the N3 place. MTS test was used in order to
establish the cytotoxicity of the tested compounds. SAR analysis and in silico assessment of the inhibitory potential towards
human oncogenic V599EB-Raf were performed using Molinspiration tool and Molecular Operating environment software.

Results:
The MTS test data showed that almost all studied thieno[2,3-d]pyirimidines (9-13, 21-22 and 25) manifest high
inhibitory effect on cell proliferation at nanomolar concentrations, whereas compounds 9 (IC50 = 130 nM) and 10 (IC50 =
261 nM) containing amino acid moiety, and 21 (IC50 = 108 nM) possessing two thienopyrimidine moieties attached to a
1,3-disubstituted benzimidazole linker, revealed many times lower toxicity against Lep3 cells compared to the cancer cells.
Thienopyrimidines 11-13 possessed high selectivity against HeLa cells. Compound 13 showed high inhibitory activity
against MDA-MB-231 and HepG2, with IC50 1.44 nM and 1.11 nM respectively. To outline the possible biological target of
the studied coumpounds, their potential to interact with human oncogenic V599EB-Raf was explored by a docking study. As
a result, it was suggested that the benzimidazolyl and glycyl fragments could enhance the binding ability of the new compounds
by increasing the number of hydrogen bond acceptors and by stabilizing the inactive form of the enzyme.

Conclusion:
The thienopyrimidines tested in vitro for human cancer HT-29, MDA-MB-231, HeLa, HepG2 and normal
human Lep3 cell lines demonstrated cytotoxicity in the nanomolar range. It was established that compounds 9, 10 and
21 showed many times lower toxicity against normal Lep3 cells that can provide a high selectivity towards all four
cancer cell lines at small concentrations. Based on the analysis of the structure-activity relationship, the observed
trends in the cytotoxicity could be related to the lipophilicity and the topological polar surface area of the tested compounds.
The docking study on the potential of the new thieno[2,3-d]pyrimidine-4-ones to interact with mutant V599EBRaf
showed that the compounds might be able to stabilize the enzyme in its inactive form.