Exploration of novel 6,8,9-trisubstituted purine analogues: synthesis, in vitro biological evaluation, and their effect on human cancer cells

Cancer, a leading global cause of mortality, demands continuous advancements in therapeutic strategies. This study focuses on the design and synthesis of a novel series of purine derivatives, specifically 6-(substituted phenyl piperazine)-8-(4-phenoxyphenyl)-9-cyclopentyl purine derivatives (5–11). The motivation behind this endeavor lies in addressing acquired resistance mechanisms in cancer cells, a significant hurdle in current treatment modalities. The synthesis, starting from 4,6-dichloro-5-nitropyrimidine, involves a multi-step process, resulting in seven new purine derivatives. Biological evaluation against human liver, colon, and breast cancer cells (Huh7, HCT116, and MCF7, respectively) was performed using the SRB assay. Among the synthesized analogs, compounds 5 and 6, exhibited notable cytotoxic activity, surpassing clinically used positive controls 5-Fluorouracil and Fludarabine in terms of efficacy. This research underscores the potential of purine derivatives with a phenyl group at the C-8 position as a scaffold for developing compounds with improved anticancer properties. The findings offer insights for future exploration and development of novel agents in cancer pharmaceutical research.


Introduction
Cancer, a pervasive cause of death on a global scale, accounted for approximately 10 million fatalities in the year 2020, as reported by GLOBOCAN statistics [1].Existing treatment options encompass chemotherapy, hormone therapy, immunotherapy, and γ-radiation.However, the emergence of acquired resistance mechanisms in cancer cells poses a significant obstacle to effective disease management, compounded by the adverse effects of the drugs employed.Consequently, the exploration of novel compounds with anticancer properties becomes imperative for advancing cancer pharmaceutical research.
Analogues of purine nucleosides, and purine/pyrimidine nucleobase have found utility in the therapeutics of cancer.Purine analogues play vital roles in many cellular bioprocesses including but not limited to cell growth, proliferation, and division [2].Consequently, these molecules assume substantial importance in cancer therapy.These analogues function as antimetabolites due to targeting nucleobases and nucleosides, which serve as precursors in the cellular metabolism of nucleic acids.Multiple nucleoside analogues have been employed in the area of cancer therapeutics.These derivatives hinder the activity of ribonucleotide reductase, impeding cellular synthesis of DNA and apoptosis or senescence induction in cancer cells [3][4][5].For instance, Pentostatin, Cladribine, and Fludarabine were approved for clinical use in hematological malignancies (Figure 1) [6][7][8].Fludarabine, a purine derivative employed in chronic lymphocytic leukaemia (CLL) treatment, exhibits favorable effects on melanoma, breast, and colon carcinoma [9][10][11].Investigating different fludarabine complexes, platinum variants displayed enhanced cytotoxicity, particularly against cisplatin-resistant cells, B-cell lymphoma, and CLL, attributed to their ten-fold higher cellular uptake.Notably, platinum complexes exhibited greater Abstract: Cancer, a leading global cause of mortality, demands continuous advancements in therapeutic strategies.This study focuses on the design and synthesis of a novel series of purine derivatives, specifically 6-(substituted phenyl piperazine)-8-(4-phenoxyphenyl)-9-cyclopentyl purine derivatives (5)(6)(7)(8)(9)(10)(11).The motivation behind this endeavor lies in addressing acquired resistance mechanisms in cancer cells, a significant hurdle in current treatment modalities.The synthesis, starting from 4,6-dichloro-5-nitropyrimidine, involves a multi-step process, resulting in seven new purine derivatives.Biological evaluation against human liver, colon, and breast cancer cells (Huh7, HCT116, and MCF7, respectively) was performed using the SRB assay.Among the synthesized analogs, compounds 5 and 6, exhibited notable cytotoxic activity, surpassing clinically used positive controls 5-Fluorouracil and Fludarabine in terms of efficacy.This research underscores the potential of purine derivatives with a phenyl group at the C-8 position as a scaffold for developing compounds with improved anticancer properties.The findings offer insights for future exploration and development of novel agents in cancer pharmaceutical research.selectivity for cancer cells over nonmalignant ones, contrasting with the interaction pattern of palladium complexes with isolated Calf thymus DNA [12].Moreover, In preclinical studies, cladribine, a purine analogue, has demonstrated the ability to enhance the intracellular uptake of cytarabine in leukemic blasts, suggesting a potential synergistic effect between cladribine and cytarabine [13].The utilization of cladribine as adjuvant therapy has been a focus of extensive research by the Polish Adult Leukemia Group, conducting several of the most significant studies on the subject [14,15].
Another notable example is Gemcitabine, which has demonstrated success in lung cancer treatment (Figure 1).This drug enters the cell through nucleoside transporters, gets activated by deoxycytidine kinase, and subsequently incorporates itself into cellular genetic material.Gemcitabine exhibits efficacy against various other cancer types, including ovarian cancer (when used alongside triapine or hydroxyurea) [16].Furthermore, the use of gemcitabine in conjunction with lobaplatin interventional therapy was shown to have the potential to enhance the cure rate of locally advanced cervical cancer (LACC) by decreasing the levels of VEGF and MMP-9 in patients' serum [17].Additionally, 5-Fluorouracil (5-FU) serves as another widely employed therapeutic agent for cancer.Although significant advancements have been made in enhancing its anticancer activity over the years, drug resistance remains a significant impediment associated with this molecule (Figure 1) [18].
Compound 3 is intended to be obtained by the reaction of 2 and cyclopentylamine in the presence of NEt 3 via nucleophilic aromatic substitution reaction (in step "b").By heating pyrimidine derivatives and cyclopentyl amine and 2 in ethanol at 125 °C in a sealed tube, the targeted compounds were reached as a result of nucleophilic aromatic substitution.In the next step "c", thanks to the free amine group in the structure of compound (3), when treated with 4-phenoxybenzaldehyde under p-TSA catalysis, a cyclization was obtained as a result of a second addition to the corresponding purine ring (4) on the corresponding imine structures.As a result of the reaction of 3 with 4-phenoxybenzaldehyde in the presence of p-TSA in DMF at 80 °C, compound 4 was obtained.
As a result, seven new purine derivatives were synthesized, and their purity checks were controlled by TLC and melting point determination.Their chemical structures were characterized by using spectroscopic techniques such as 1 H, 13 C NMR, mass, and elemental analyses.

Conclusion
This research involved creating new purine compounds that had a phenyl group attached to the C-8 position of the purine ring.These compounds were synthesized as a novel set of derivatives called 6-(substituted phenyl piperazine)-8-(4-phenoxyphenyl)-9-cyclopentyl purine derivatives (5)(6)(7)(8)(9)(10)(11).The researchers then tested the biotoxic effects of these compounds against various types of cancer cells (including liver, colon, and breast cancer) and the obtained cytotoxicity values were compared to the activity of 5-Fluorouracil (5-FU), Fludarabine, and Cladribine, three clinically used anticancer agents that are frequently used as positive controls in cytotoxicity assays.The results revealed that 5 and 6 exhibited auspicious cytotoxic activity on liver cancer cells with lower IC 50 values than clinical reference drugs 5-FU and Fludarabine.These results indicated that by using these molecules as scaffolds, new compounds can be synthesized with improved performance for future perspectives.

General
To determine the melting points, an Electrothermal 9100 capillary melting point apparatus was employed.NMR spectra were recorded using a VARIAN Mercury 400 FT-NMR spectrometer operating at frequencies of 400 MHz for 1 H and 100.6 MHz for 13 C. TMS served as the internal standard for both 1 H NMR and 13 C NMR spectra, with chemical shifts given in ppm and coupling constants in Hz.Mass spectra were obtained using the ESI+ method on a Waters Micro-mass ZQ instrument.Elemental analyses (C, H, N) were conducted on a Leco CHNS 932 instrument, and the determined values were within ±0.4% of the theoretical values.Silica gel 60 (particle size: 40-63mm) was employed for column chromatography.Chemical reagents obtained from reputable suppliers such as Merck, Fluka, Sigma, and Aldrich were utilized for the synthesis process.4.2.Chemistry experimental procedures 5-Amino-4,6-dichloropyrimidine (2) was synthesized according to the reduction methodology reported in the literature [19].

NCI-60 Sulphorhodamine B (SRB) assay [20]
Cancer cells, (2000-5000 cells per well) were seeded into 96-well plates.After 24 h of incubation, the cells were exposed to escalating concentrations of the compounds (2.5µM to 40µM) for a duration of 72 h.Then the cells were fixed using 10% ice-cold trichloroacetic acid (TCA) in the dark at + 4 °C for one h.The plates were stained with a solution of 0.4% sulphorhodamine B (SRB) in a 1% acetic acid solution.The bound SRB stain was dissolved to measure the absorbance using a 10 mM Tris-Base solution, and the optical density (OD) values were recorded at a wavelength of 515 nm.

Figure 2 .
Figure2. % Growth Inhibition of compounds 5-11 on cancer cells.Cell lines were treated with the compounds for 72 h in triplicate with increasing concentrations (40µM-2.5µM).NCI-SRB analysis was executed to explore the effect of the compounds on cellular growth. .
The human cancer cell lines were cultured in standard Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin.

Table .
The cytotoxicity of compounds 5-11 was evaluated in vitro.