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

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.


Introduction
Cancer, defined as uncontrolled, rapid and pathological proliferation of cells, is the second leading cause of death, with more than 18 million cases worldwide annually [1]. Pancreatic cancer is predicted to become the second cause of cancer-related deaths by 2030, behind lung cancer [2]. Pancreatic ductal adenocarcinoma is the most common pancreatic cancer type, accounting for more than 90% of cases with a five-year survival rate of less than 9% [3,4]. Current therapy suffers from major limitations due to severe side-effects and multidrug resistance, thereby a continued search to find new and safer anticancer drugs and innovate the development of new cancer treatments are required [5,6].
More than 75% of drugs approved by the FDA and currently available on the market are nitrogen-containing heterocycles due to their ability to easily form hydrogen bonding, dipole-dipole interactions, hydrophobic effects, van der Waals forces and π-stacking interactions with biological targets [7]. The naturally occurring purines play vital roles in numerous life processes [8]. Over the past two decades, purines and their isosteres have appeared as important pharmacophores interacting with the synthesis and functions of nucleic acids and enzymes [9]. To date, 22 pyrimidine-fused bicyclic heterocycles have been approved for clinical use in the treatment of different cancers [10]. Among pyrimidinefused bicyclic heterocycles, the pyrrolo [2,3-d]pyrimidine core can be considered as an isosteric replacement of the biologically relevant purine heterocycle and is hence an important pharmacophore widely used in the field of medicinal chemistry and drug design primarily due to its anticancer properties [11][12][13]. Pyrrolo [2,3-d]pyrimidine derivatives exhibited cytotoxic effects in lung and colon cancer cell lines through activation of the mitochondrial apoptotic pathway [14][15][16]. Aryl-substituted pyrrolopyrimidines showed potent inhibition of the membrane bound epidermal growth factor receptor tyrosine kinase (EGFR) and angiogenic inhibitors against human vascular endothelial growth factor receptor-2 (VEGFR-2) that represent important targets in cancer therapy [17][18][19][20][21][22][23][24]. Over the past years, some pyrrolo [2,3-d]pyrimidine derivatives were identified as inhibitors of Mer receptor and Src non-receptor tyrosine kinases, isoform of protein kinase B (Akt), mitotic checkpoint kinase (Mps1), Janus kinase 2 (JAK2), and phosphoinositide 3 kinase (PI3K) with promising anticancer activity [25][26][27][28][29][30][31][32][33]. Design strategy in development of purine derivatives as cytostatic agents for kinase inhibition revealed that introduction of cyclic amines improved the activity by forming an additional hydrogen bond to kinase hinge [9,34,35]. In addition, prevalence of halogenated drugs showed that halogen bonds contribute to the stability of protein-ligand complexes [36]. Pyrrolo [2,3-d]pyrimidine sulfonamides were recently found to act as cytotoxic agents in hypoxia via inhibition of transmembrane carbonic anhydrases [37]. A 4-(benzylamino)-pyrrolo [2,3-d]pyrimidine derivative exerted potent antitumour effects in vivo and induced mitotic cell blockade by impairing both mitotic microtubule organization and dynamics [38]. To overcome multidrug resistance in cancer patients, pyrrolo [2,3-d]pyrimidines and purine derivatives with high lipophilicity and molecular weight were developed as potent and selective inhibitors of multidrug resistance-associated protein 1 (MRP1, ABCC1) associated with non-response to chemotherapy in different cancers [39]. Several heterocyclic dimers such as bis-purines [40] and bis-benzimidazoles [41] linked in a head-to-head [42] or head-totail manner [43] with acylic and cyclic spacers, have been reported to exhibit anticancer properties by noncovalent interactions with the minor groove of DNA [44].
To develop a less toxic and more environmentally friendly synthetic method, optimization of the syntheses of target compounds by application of ultrasonic waves and microwaves was performed. Computational analysis was used to elucidate kinetic and thermodynamic aspects of the investigated reactions and identify the precise mechanistic role of the Cu(I) catalyst. Antiproliferative evaluations of bis-purines and bis-pyrrolo [2,3d]pyrimidines and apoptotic mechanism of the selected compound with best antiproliferative effect were also investigated.
In order to optimize the CuAAC reaction, the reactions of bis-alkynes (4a−4d) and heterocyclic azides (3a and 3c−3e) were carried out using different catalysts and reaction conditions to give 6-substituted bis-and mono-purines and 7-deazapurines (Table 1).  Based on the known protocols [51] for CuAAC reaction that include the in situ generation of Cu(I) from a Cu(II) salt or the alternative direct utilization of a Cu(I) source using the combination of CuI/DIPEA/HOAc, which has been found to be a highly efficient catalytic system for this reaction [52], we performed an optimization of the CuAAC reaction using methods A-C.
The most common catalytic system, CuSO 4 in the presence of metallic copper as a reducing agent (in aqueous t-BuOH), was initially chosen in method A. For better performance, we then investigated the application of ultrasound as a green alternative for energy efficient processes in method B. Chemical transformations induced by ultrasound have been described previously [53,54], and it was discovered that ultrasonic irradiation generates a large number of cavitation bubbles, which cause an increase in the local temperature within the reaction mixture and eventually enable the crossing of the activation energy barrier [54]. Finally, copper(I) iodide in the presence of N,N-diisopropylethylamine (DIPEA) and acetic acid (HOAc) was employed in method C. Comparing the applied synthetic methods A-C in CuAAC reactions of 6-chloro-7-deazapurine azide derivative (3a) with all selected bis-alkynes, it can be observed that bis-triazole dimers 5a-5d were obtained in all methods, while when methods A and C were used with 4,4 -bis(propynyloxy)-1,1biphenyl (4b) and 1,6-heptadiyne (4c) the corresponding mono-triazole analogues 5f and 5g were also obtained (Scheme 1, Table 1).
In the 6-piperidinyl-and 6-pyrrolidinylpurine series, the CuI/DIPEA/HOAc catalytic system using method C was the least selective, yielding both bis-(9a-9d and 10a-10d) and mono-heterocycles 9e-9g and 10f. The use of Cu(II) salt as a catalyst in method A afforded only bis-heterocycles in most cases, with exceptions for 5g and 9e when small amounts of mono-heterocycles were isolated. However, these reactions were extremely slow and were carried out over 7 days. It can be observed that ultrasound irradiation employed in method B significantly reduced the reaction time to 1.5 h. We may assume that acoustic cavitation in the heterogeneous CuSO 4 /Cu(0) system facilitated mass transfer and surface activation [55] and ultimately accelerated the CuAAC reaction. An additional advantage of the ultrasoundassisted reactions was the exclusive formation of dimeric heterocyclic analogues with improved yields (in the range of 61-82%) compared to reactions without ultrasound irradiation in method A (yields of 28-60%) and method C (yields of 24-64%). In the case of the adenine series, only the ultrasound-assisted reaction (method B) of 2-azidoethyladenine 3e with bis-alkyne 4a afforded bis-adenine 11a with 1,4-bis(oxymethylene)phenyl linker in low yield (Scheme 2), while 3e could not react with 4b in any conditions. When this reaction was performed using CuI, as catalyst, only mono-adenine 11e was obtained. Also, only CuAAC reactions of azide 3e with aliphatic bis-alkynes 4c and 4d in method C afforded mono-adenine 11g with propyl chain at C-4 of 1,2,3-triazole, and both bis-11d and mono-adenine 11h in low yields, respectively. Reactions of adenine azide derivative 3e and bis-alkynes (4a-4d) did not afford the target products using other applied synthetic methods, indicating the influence of 6-aminopurine on its lower reactivity compared to its 6-piperidino-(3c) and 6-pyrrolidino-substituted (3d) purine congeners.

Computational Analysis
Computational analysis was performed to identify precise molecular mechanisms underlying the conversion of azides and alkynes into the matching triazoles, and to reveal the role of the Cu(I) catalysts on the reaction outcomes. For that purpose, we employed a series of DFT calculations on several model systems ( Figure 2) with the aim of providing enough structural and electronic features to help interpreting the yields and product distributions observed experimentally. To demonstrate the necessity to employ the metal catalyst in the CuAAC reaction, we have initially investigated the uncatalyzed conversion between model azide m2 and neutral model alkyne m1 0 , which proceeds in accordance with Figure 3. Bringing reactants into the reactive complex is unfavorable and endergonic by 6.9 kcal mol −1 , from which it takes additional 21.6 kcal mol −1 to reach the transition state corresponding to the concerted formation of both C-N bonds. The latter gives the final triazole in a single step in a very exergonic fashion, linked with the total reaction free energy of ∆G R = −51.0 kcal mol −1 . Still, although thermodynamically favorable, the overall reaction has a rather high kinetic barrier, ∆G ‡ = 28.5 kcal mol −1 , which renders it as very much unlikely under normal conditions, and emphasizes the necessity to employ a suitable catalyst. Although alkynes are very weakly acidic systems, and their C-H acidity is typically related with pK a values over 20 in water [56], one could still find a suitable base to initiate the deprotonation [57] and convert alkyne m1 0 into its anionic form m1 -, with the idea to increase the electrophilicity towards azide and facilitate the reaction. Interestingly, this does not occur ( Figure S40), as it takes 7.0 kcal mol −1 to form the reactive complex, and, although the kinetic barrier is reduced by 2.6 kcal mol -1 to ∆G ‡ = 25.9 kcal mol −1 , the reaction is thermodynamically significantly less feasible, as the reaction free energy is made less exergonic by 11.6 kcal mol −1 to ∆G R = −39.4 kcal mol −1 . The latter will likely prevail and the reaction with deprotonated m1will be less favorable. This suggests that attempts to improve the reaction outcomes with strong bases will likely fail.
Introducing Cu(I) ions in the system can result in their complexation with reagents, as both acetylenes and azides are well capable of forming organometallic complexes. Therefore, we proceeded by analyzing potential 1:1 complex with both reagents, which might turn useful in clarifying the role of the metal in the catalysis and identifying which reagent is being activated. The calculated interaction energies ( Figure S41) show that Cu(I) ions are much more efficient in binding alkynes (∆G INT = −11.9 kcal mol −1 for m1 0 ) than azides (∆G INT = −5.6 kcal mol −1 for m2). This is rationalized by electronic distribution as m1 0 contains more electronic density within its two carbons (−0.34 |e|) than m2 within three of its nitrogen atoms (−0.24 |e|), thus the observed trend is predominantly electrostatic in nature.
With this in mind, we analyzed a situation where the cycloaddition reaction occurs with alkyne binding the Cu(I) ion ( Figure 4). There, bringing reactants into the reactive complex is exergonic and favorable (−2.8 kcal mol −1 ), while the reaction again proceeds in one step with the kinetic barrier of ∆G ‡ = 19.7 kcal mol −1 , which represents a considerable 8.8 kcal mol −1 reduction from the uncatalyzed reaction. This offers a product complex with Cu(I) binding around alkyne carbons, from which it takes only 1.1 kcal mol −1 to detach Cu(I) and allow the final triazole. The obtained reaction profile is highly feasible, while the obtained reduction in the kinetic activation roughly translates into 6-7 orders of magnitude higher rate constant, which is significant and underlines the crucial role of the metal catalyst in facilitating the reaction. On the other hand, if Cu(I) ions would be able to overcome an initially more favorable placement around alkyne and form a complex with azide prior to reaction ( Figure S42), the formation of the reactive complex in that case, would, as anticipated, be less favorable by 5.8 kcal mol −1 and even endergonic (+3.0 kcal mol −1 ). In addition to this negative aspect, the obtained product complex featuring triazole with azide-bound Cu(I) is exceedingly exergonic (-64.4 kcal mol −1 ), which will hinder further reaction progress. Specifically, such a stable product complex would increase the energy requirement to finalize the reaction from 1.1 kcal mol −1 in the previous case to 13.4 kcal mol −1 here. Both of the mentioned aspects will expectedly predominate over formally slightly lower kinetic barrier, by 1.3 kcal mol −1 to ∆G ‡ = 18.4 kcal mol −1 , and make such a conversion less likely. As it was the case with the uncatalyzed reaction, converting alkyne into its deprotonated form m1again does not promote the reaction ( Figure S43). First, both processes become less exergonic here, with the overall reaction free energy reduced by 10.7 kcal mol −1 to ∆G R = −40.3 kcal mol −1 . On top of that, when Cu(I) is bound to the anionic alkyne, the kinetic barrier becomes increased to ∆G ‡ = 19.8 kcal mol −1 , and even further to ∆G ‡ = 22.0 kcal mol −1 when the reaction occurs with the azide-bound metal. All of this again eliminates the necessity to employ any catalytic base in the reaction, let alone that it could, on its own, undergo the complexation reaction with the Cu(I) ions, thus further interfere with the reaction progress in an undesired way.
In summarizing this part, we can emphasize that obtained reaction profiles clearly support the role of the metal catalyst in facilitating the conversion and demonstrate that Cu(I) ions act by binding and activating the alkyne for a successful reaction with nucleophilic azides. Also, the revealed kinetic and thermodynamic aspects seem to suggest that increasing the reaction temperature will likely improve the reaction outcomes, which is found in excellent agreement with a general trend that method B at higher temperatures with ultrasound irradiation offers better reaction yields than method A. Still, to understand different reaction outcomes between methods A and B, where reactive Cu(I) ions are generated in situ, and the method C, where these are directly employed, we must not forget that first two approaches contain both Cu(II) ions and elementary Cu(0) within the solution, which could, at certain cases, impact the reaction and allow different yields, whether higher or lower. To investigate this possibility, we have repeated the analysis considering alternative copper oxidation states.
When Cu(0) is concerned, it more favorably binds to the azide (−20.5 kcal mol −1 , Figure S41), which even surpasses the most exergonic interaction that charged Cu(I) makes with alkyne by 8.6 kcal mol −1 , likely being the result of the solvation effect. In that scenario ( Figure S44), as expected, the formation of the reactive complex is highly exergonic (−13.7 kcal mol −1 ), yet leading to a very high barrier of ∆G ‡ = 33.0 kcal mol −1 , which makes this process as unfeasible and renders any impact of Cu(0) on the reaction outcomes as insignificant. We note in passing that a much less stable reactive complex involving the alkyne-bound Cu(0) would proceed in a stepwise fashion with both C-N bonds formed separately ( Figure S44) and the rate-limiting second step. On the other hand, Cu(II) ions reveal a very interesting trend as their ability to complex reactants is precisely identical for both alkyne and azide at ∆G R = −13.0 kcal mol −1 ( Figure S41), which makes both options viable.
However, the reactive complex with the alkyne-bound Cu(II) is by 6.2 kcal mol −1 more favorable, which directs the reactivity towards the stepwise triazole formation ( Figure S45), where both N-C bonds are created under similar kinetic requirements. Still, the process to form the second bond is a bit more demanding and defines the rate-limiting step, yet the overall activation energy is only ∆G ‡ = 8.3 kcal mol −1 , which hints at a possible impact of Cu(II) ions on the overall conversion. However, one must emphasize that this reaction is again associated with a rather high energy cost of 12.7 kcal mol −1 to move from the reactive complex and detach Cu(II) to allow the final triazole, which might hinder any positive effect, let alone the availability of Cu(II) to undertake the reaction in the first place. Which of these aspects will prevail in solution and whether the impact of Cu(II) will be significant or insignificant at all, is difficult to say, and may likely depend on a particular reaction condition and different electronic and structural features of the employed reactants. As such, using Cu(II) salts in a combination with elementary copper, in order to generate Cu(I) in situ, may and likely will generate slightly different conditions for the alkyne-azide cycloaddition reaction than when Cu(I) salts are directly used, which justifies why different trends are experimentally detected and no wide-ranging conclusions can be made in this respect.
Lastly, a somewhat general trend emerging from experiments is that 6-chloro derivatives are typically more reactive than their 6-amino analogues when reaction times or yields are compared. At first, such differences appear surprising given a large distance of 6-substituents from the reacting triazole to exhibit any direct impact, or the fact that triazole is separated from the aromatic fragment by two methylene units for any indirect influence of these substituents through resonance/inductive effects. Therefore, a plausible explanation for the observed reactivity differences could be linked with their abilities to form complexes with metal catalysts ( Figure S46). Namely, electron-withdrawing chlorine depletes the electron density from the aromatic skeleton, which diminishes the complex formation with Cu(I). In contrast, electron-donating amines consistently increase the tendency to complex Cu(I) in solution in both pyrimidine and purine derivatives, which could reduce the catalytic efficiency of the metal, thus somewhat lower reactivity of 6-amino derivatives.
Comparing to BIS-PP2, it can be observed that structural modifications in novel bisand mono-pyrrolo[2,3-d]pyrimidine and purine derivatives reduced their cytotoxic effects on normal HFF-1 cells to a lesser extent. Compounds with best antiproliferative activities also exhibited inhibitory effects on normal HFF-1 cells.
Overall, it can be observed that the linker between the heterocycle scaffolds in the symmetrical 7-deazapurines and bis-purines had a significant impact on antitumor activity ( Figure 5). Compounds with aliphatic linkers (5c-7c, 5d-7d, 9c, 10c and 9d-11d) exhibited moderate activity or were deprived of any antitumor activities, while introducing the 4,4bis(oxymethylene)biphenyl linker (5b, 9b and 10b) caused an enhancement of tumor cell growth inhibition. The influence of heterocyclic scaffold was also observed showing that series of cyclic 6-amino bis-purines (9a−9d and 10a−10d) were generally more active than the corresponding cyclic 4-amino bis-pyrrolo[2,3-d]pyrimidine (6a−6d and 7a−7d) analogues. Comparison of the antiproliferative activity of all purine derivatives showed that cyclic amines in purine derivatives improved the activity relative to adenine derivatives that did not exhibit inhibitory activities.

Apoptosis Detection
Annexin V assay was performed to determine if the antiproliferative activity of compound 5f exhibiting the most pronounced potency in ductal adenocarcinoma cancer cell line (CFPAC-1) could be attributed to induction of apoptosis. Obtained data are presented in Table 3 and Figure 6.   Table 3 shows percentages of cells in different stages of apoptosis. It can be observed that compound 5f showed pro-apoptotic effect in CFPAC-1 cells as early as 48 h after treatment with both 2 × IC 50 (1.58 µM) and 5 × IC 50 (3.95 µM) concentrations. Following 48-h treatment, both concentrations of compound 5f showed pro-apoptotic activity, where 5 × IC 50 concentration induced decrease in cell viability by 28.4% and increase in the percentage of cells that underwent early apoptosis by 21.21%. After 72 h of treatment, a significant decrease in cell viability was noticed in both treatments concomitantly with a profound increase in early apoptotic cells after 2 × IC 50 and 5 × IC 50 treatments by 24.08% and 33.35%, respectively, followed by a dramatic rise in the late apoptotic/primary necrotic cells by 26.14% that occurred after 5 × IC 50 treatment. Collectively, these results show that compound 5f induces apoptosis and primary necrosis in CFPAC-1 cells in a concentrationand time-dependent manner.

General
All the solvents and chemicals were purchased from Aldrich (St. Louis, MO, USA) and Acros (Geel, Belgium). Thin layer chromatography was performed on pre-coated silica gel 60F-254 plates (Merck, Kenilworth, NJ, USA ) while glass column slurry-packed under gravity with silica gel (0.063-0.2 mm Fluka, Seelze, Germany) was employed for column chromatography. Melting points of compounds were determined using a Kofler micro hot stage. 1 [47] were prepared according to known procedures.

General Procedure for the N-Alkylation of Compounds
The corresponding heterocyclic base 1a-1e was dissolved in dry DMF (8 mL), K 2 CO 3 was added (1.2 eq) and the reaction mixture was stirred for 1 h. 1,2-Dibromoethane (1.2 eq) was added to the mixture and stirred for 24 h at room temperature. The solvent was evaporated to dryness and the residue was purified by column chromatography.

General Procedure for the Synthesis of Azidoethyl Derivatives
The corresponding 2-bromoethyl derivative 2a-2f was dissolved in acetone. NaN 3 (4 eq) dissolved in water (~3 mL) was added dropwise to the reaction mixture and stirred under reflux overnight. The solvent was evaporated to dryness and the residue dissolved in ethyl-acete (60 mL) and extracted with brine. The organic layer was dried over MgSO 4 , filtered and evaporated.

Computational Details
All molecular geometries were optimized using the B97D functional together with the 6-31+G(d) basis set for non-metals and the Stuttgart-Dresden (SDD) effective core potentials [61] for the inner electrons of copper atoms and its associated double-ζ basis set for the outer ones, in line with our earlier work on the copper-catalyzed organic reactions [62] and other literature recommendations [63,64]. To account for the solvent effects, during geometry optimization, we included the implicit SMD solvation model corresponding to DMF (ε = 37.219). Thermal corrections were extracted from the matching frequency calculations, so that all presented results correspond to differences in the Gibbs free energies at room temperature and normal pressure. The choice of such computational setup was prompted by its success in reproducing various features of different organic [65][66][67], organometallic [68,69] and biological systems [70,71], being particularly accurate for relative trends among similar reactants, which is the focus here. All transition state structures were located using the scan procedure, employing both 1D and 2D scans, the latter specifically utilized to probe the possibility for concerted mechanisms. Apart from the visualization of the obtained negative frequencies, the validity of all transition state structures was validated by performing IRC calculations in both directions and identifying the matching reactant and product structures connected by the inspected transition state. All calculations were performed using the Gaussian 16 software [72].

Proliferation Assay
Cells were seeded onto 96-well microtiter plates at a seeding density of 3000 cells/well for carcinoma cell lines, and 5000 cells/well for normal human fibroblasts. The next day, cells were treated with test agents in five different concentrations (0.01-100 µM) and further incubated for 72 h. DMSO (solvent) was tested for potential cytotoxic effect but it did not exceed 0.1%. Following 72 h incubation, the MTT assay was performed and measured absorbances were transformed into percentage of cell growth as described previously [73]. Results were obtained from three independent experiments. IC 50 values were calculated using linear regression analysis and results were statistically analyzed by ANOVA, Tukey post-hoc test (p < 0.05).

Apoptosis Detection
Cells were seeded into 8-well chambers (Lab-tek II Chamber Slides, Thermo Fisher Scientific, Waltham, MA, USA) in a concentration of 2 × 10 4 cells per well and treated with 2 × IC 50 and 5 × IC 50 concentrations of selected compounds for 48 and 72 h. Staining of the cells was performed by Annexin-V-FITC Staining kit (Santa Cruz Biotechnology, Dallas, TX, USA) according to the manufacturer s instructions. Cells were visualised by fluorescent microscope (Olympus, Tokyo, Japan) at magnification of 40×.
DFT calculations confirmed that the investigated copper-catalyzed cycloaddition is a feasible process, and revealed that triazoles are favorably formed in a concerted and highly exergonic fashion, ∆G R = −51.0 kcal mol −1 for the studied model case. Still, the uncatalyzed reaction is associated with a high kinetic barrier of ∆G ‡ = 28.5 kcal mol −1 , which renders it as very unlikely. Once Cu(I) ions are present, they bind to the alkyne and increase its electrophylicity towards the azide, which reduces kinetic requirements to ∆G ‡ = 19.7 kcal mol −1 , thereby allowing the conversion to occur under normal conditions. The obtained reaction profiles agree that higher temperatures and ultrasound irradiation will improve the reaction outcomes, while eliminate the option to use strong bases with the prospect to activate alkynes through terminal C-H deprotonation. Lastly, different trends among reactions where catalytic Cu(I) ions are generated in situ or directly introduced could be ascribed to the presence of Cu(II) ions in the former, but this depends on their availability and other conditions that might be operative when a large variety of reagents is employed as was the instance here. Lastly, a somewhat general tendency of 6-amino derivatives to offer lower reaction yields over 6-chloro analogues is likely related to their ability to more strongly bind Cu(I) in solution, therefore hindering its catalytic efficiency.
Our findings encourage further structural optimization of purine and fused heterocycle scaffolds, such as chloropyrrolo [2,3-d]pyrimidine connected through aromatic unit, as a promising chemical entity for development of novel efficient and non-toxic agent against pancreatic cancer.