Novel Bis- and Mono-Pyrrolo[2,3- d]pyrimidine and Purine Derivatives: Synthesis, Computational Analysis and Antiproliferative Evaluation

Abstract

Novel symmetrical bis-pyrrolo[2,3-d]pyrimidines and bis-purines and their monomers were synthesized and evaluated for their antiproliferative activity in human lung adenocarcinoma (A549), cervical carcinoma (HeLa), ductal pancreatic adenocarcinoma (CFPAC-1) and metastatic colorectal adenocarcinoma (SW620) cells. The use of ultrasound irradiation as alternative energy input in Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) shortened the reaction time, increased the reaction efficiency and led to the formation of exclusively symmetric bis-heterocycles. DFT calculations showed that triazole formation is exceedingly exergonic and confirmed that the presence of Cu(I) ions is required to overcome high kinetic requirements and allow the reaction to proceed. The influence of various linkers and 6-substituted purine and regioisomeric 7-deazapurine on their cytostatic activity was revealed. Among all the evaluated compounds, the 4-chloropyrrolo[2,3-d]pyrimidine monomer 5f with 4,4'-bis(oxymethylene)biphenyl had the most pronounced, although not selective, growth-inhibitory effect on pancreatic adenocarcinoma (CFPAC-1) cells (IC₅₀ = 0.79 µM). Annexin V assay results revealed that its strong growth inhibitory activity against CFPAC-1 cells could be associated with induction of apoptosis and primary necrosis. Further structural optimization of bis-chloropyrrolo[2,3-d]pyrimidine with aromatic linker is required to develop novel efficient and non-toxic agent against pancreatic cancer.

Keywords: DFT calculations; antiproliferative activity; apoptosis; pancreatic adenocarcinoma; purine; pyrrolo[2,3-d]pyrimidines; ultrasound.

Figure 1

Design of symmetric bis-pyrrolo[2,3-d]pyrimidines (5a–5d, 6a–6d, 7a–7d) and bis-purines (9a–9d, 10a–10d, 11a–11d).

Scheme 1

Reagents and conditions: (i) BrCH₂CH₂Br, K₂CO₃, DMF, rt, 24 h; (ii) NaN₃, acetone, reflux, 24 h; (iii) Method A: 1M CuSO₄, Cu(0), t-BuOH:H₂O = 1:1, DMF, rt, 120 h; Method B: 1M CuSO4, Cu(0), t-BuOH:H₂O = 1:1, DMF, 50 °C, US, 45 min, 1000 W; Method C: CuI, DIPEA, AcOH, CH₂Cl₂, rt., 72 h; (iv) piperidine/pyrrolidine, H₂O, MW, 400 W, 100 °C, 10 min.

Scheme 2

Reagents and conditions: (i) BrCH₂CH₂Br, K₂CO₃, DMF, rt, 24 h; (ii) NaN₃, acetone, reflux, 24 h; (iii) Method A: 1M CuSO₄, Cu(0), t-BuOH:H₂O = 1:1, DMF, rt, 120 h; Method B: 1M CuSO4, Cu(0), t-BuOH:H₂O = 1:1, DMF, 50 °C, US, 45 min, 1000 W; Method C: CuI, DIPEA, AcOH, CH₂Cl₂, rt., 72 h.

Figure 2

Structure of model alkyne and azide studied computationally.

Figure 3

The uncatalyzed direct conversion of alkyne m1⁰ and azide m2 into triazole. Relative Gibbs free energies for isolated reactants (R), reactant complex (RC), transition state (TS) and isolated product (P) correspond to the DMF solution (in kcal mol⁻¹).

Figure 4

Cu(I)-catalyzed conversion of alkyne m1⁰ and azide m2 into triazole with Cu(I) bound to alkyne. Relative Gibbs free energies for isolated reactants (R), reactant complex (RC), transition state (TS), product complex (PC) and isolated product (P) correspond to the DMF solution (in kcal mol⁻¹).

Figure 5

Structure–activity relationship for antiproliferative activity of bis- and mono-pyrrolo[2,3-d]pyrimidine and purine derivatives.

Figure 6

Detection of apoptosis induced by compound 5f in ductal adenocarcinoma cancer cell line CFPAC-1 using Annexin V assay after 48 (A) and 72 (B) hours of treatment at concentrations of 2 × IC₅₀ (1.58 µM) and 5 × IC₅₀ (3.95 µM). Cells were visualized by fluorescence microscope using 40× magnifications. Shown here are bright-field images and late apoptotic/primary necrotic cells.