2,4-Bis{4-[(dialkylaminoalkyl)aminomethyl]phenyl}-7-substituted-7H-pyrrolo[2,3-d]pyrimidine Derivatives: Synthesis and Biological Evaluation as Novel Antiprotozoal Agents by Potentially Targeting G-Quadruplex

Abstract

A series of substituted pyrrolo[2,3-d]pyrimidines was designed, synthesized, and evaluated in vitro against two protozoan parasites: Plasmodium falciparum and Trypanosoma brucei brucei. Pharmacological studies revealed antiprotozoal activity with IC₅₀ values in the submicromolar to micromolar range. Additionally, the in vitro cytotoxicity of these new compounds was assessed using human HepG2 cells. Among them, the pyrrolopyrimidine derivative 1d emerged as the most potent antimalarial compound, exhibiting a selectivity index (SI) of 600.81 against the P. falciparum chloroquine-resistant W2 strain. For the chloroquine-sensitive 3D7 strain, the most notable selectivity index (SI) was observed for pyrrolo[2,3-d]pyrimidine 1c, with a value of approximately 123. Furthermore, compound 1b demonstrated the most interesting activity against Trypanosoma brucei brucei, with an SI of 39.52, marking it as a promising trypanocidal agent. FRET melting assays confirmed that these nitrogen-containing heterocyclic compounds bind to telomeric G-quadruplexes in P. falciparum and Trypanosoma. However, no clear correlation was found between G-quadruplex binding and antiparasitic activity or selectivity, suggesting that G-quadruplex targeting is unlikely to be the main mechanism underlying cytotoxicity.

1. Introduction

Nowadays, malaria remains one of the most challenging infectious diseases worldwide, presenting an ongoing threat to human health. Caused by Plasmodium parasites and transmitted by Anopheles female mosquitoes, malaria was responsible for 597,000 malaria deaths in 2023, with 263 million new cases of malaria [1]. The WHO Africa Region is hardest hit, shouldering an estimated 95% of the malaria burden each year [2]. In 2023, 11 countries of the WHO Africa Region bear about two thirds of the global malaria burden. These countries are part of a targeted initiative, “high burden to high impact (HBHI)” [3], to accelerate progress against this public health problem. Despite decades of global efforts, malaria eradication remains elusive, making this disease an ongoing challenge for healthcare systems, political leaders and affected populations. The pathology is particularly worrying for at-risk populations such as young children under five years old, pregnant women, and immunocompromised people. The COVID-19 pandemic has disrupted malaria control programs, reducing the impact of prevention and treatment efforts. However, efforts to combat malaria have evolved significantly over the past century, despite the recent emergence of insecticide resistance in mosquitoes, along with the growing resistance of Plasmodium parasites to antimalarial drugs, both of which led the WHO to develop control strategies [4,5]. The WHO’s Global Technical Strategy for Malaria 2016–2030 set ambitious targets, including reducing global malaria incidence and mortality rates by at least 90% by 2030 [6]. Key strategies include strengthening vector control, expanding access to effective diagnostics and treatment, investing in research and innovation, and fostering robust health systems. Innovative tools are at the forefront of current malaria control efforts. The recent introduction of the RTS,S/AS01 (RTS,S) malaria vaccine marks a significant milestone in the fight against the disease. In October 2023, WHO recommended a second vaccine, R21/Matrix-M (R21), to complement the ongoing rollout of the first malaria vaccine, resulting in sufficient vaccine supply to prevent malaria among children living in areas of risk [1]. Additionally, research into next-generation vaccines, gene-drive technologies to suppress mosquito populations, and novel insecticides offers hope for overcoming barriers posed by resistance. The ongoing struggle to control and eventually eliminate malaria serves as a testament to the importance of integrated, multidisciplinary approaches in addressing complex global health issues. Therefore, it is essential to adopt and strengthen cross-disciplinary approaches, including the development of new therapeutic strategies and the pursuit of community-based and applied research targeting various tropical diseases. One promising possibility could be the discovery of novel antimalarial treatments, particularly through the design and synthesis of quinoline-based derivatives in analogies of chloroquine (CQ), amodiaquine (AQ), mefloquine (MQ), tafenoquine (TQ), and piperaquine, that could bypass the protein systems responsible for drug efflux—mechanisms that limit the efficacy of drugs such as primaquine (PQ) and amodiaquine (AQ) (Figure 1). These efflux pumps, which serve both as natural defense systems and as key determinants of drug bioavailability and distribution, are central to this approach [7,8,9,10,11,12,13].

In our ongoing research, we have focused on developing novel nitrogen-containing heterocyclic compounds with potential applications in antiprotozoal chemotherapy [14,15,16,17,18,19,20,21,22,23]. Our previous research work includes the synthesis of several series such as 2,9-bis[(substituted-aminomethyl)phenyl]phenanthrolines (Series A–B), 2,4-bis[(substituted-aminomethyl)phenyl]quinoline, 1,3-bis[(substituted-aminomethyl)phenyl]isoquinoline, and 2,4-bis[(substituted-aminomethyl)phenyl]quinazoline derivatives (Series C), designed as new homologues to the bisquinoline A and bisacridine B [18,19,20,21]. By taking into account our experience in the field of the synthesis of new aza heterocyclic compounds, we describe herein the design and synthesis of new substituted pyrrolo[2,3-d]pyrimidines 1 that could be considered as original structural bioisoster analogues of our previously described antiprotozoal phenanthroline, quinoline, isoquinoline or quinazoline compounds [18,19,20,21]. Although some of these compounds were previously reported by our team as anti-COVID-19 agents [24], it is important to emphasize that this chemical series was originally conceived and designed with antimalarial purposes in mind. These pyrrolopyrimidine derivatives 1 were developed as novel structural analogues of the A and B series, with a specific focus on targeting Plasmodium falciparum. In particular, the molecular design was guided by the objective of promoting interactions with Plasmodium falciparum DNA G-quadruplexes, notably through π–π stacking interactions, which are known to stabilize these non-canonical nucleic acid structures and potentially disrupt key biological processes in the parasite. Following their initial evaluation and publication in the context of SARS-CoV-2, we significantly expanded the pharmacomodulation of this chemical series. This optimization effort aimed to refine their physicochemical and biological properties, as well as to explore structure–activity relationships more thoroughly. Subsequently, the entire library of compounds 1 was systematically screened against Plasmodium falciparum, allowing us to reassess their antimalarial potential in line with the original design rationale. This repositioning strategy not only highlights the versatility of the scaffold but also underscores the relevance of these derivatives as promising candidates for antimalarial drug development. G-quadruplexes, particularly in protozoan telomeres, offer an attractive therapeutic target for such ligands. Of note, P. falciparum telomerase activity is present in gametocytes and during erythrocytic transition stages. The parasite’s telomeric 3′ G-overhang contains a degenerate repeat sequence (5′GGGTTYA3′, with Y = T or C) capable of forming stable intramolecular G-quadruplexes that modulate gene expression. Previous studies have shown that G-quadruplex-binding agents are effective and fast-acting against intraerythrocytic stages of the parasite [25,26,27,28,29,30,31]. The sequence divergence between human (5′GGGTTA3′) and parasitic P. falciparum and also Trypanosoma brucei brucei G4 motifs suggests a promising path for designing selective antiprotozoal ligands. As a result, several G-quadruplex-binding drug candidates have demonstrated promising activity against the intraerythrocytic stages of the parasite in vitro [25,26,28,30]. Evidence from the literature suggests that certain G-quadruplex-binding ligands, such as PhenDC3, PDS, and quarfloxin derivatives, could potentially be repurposed as interesting antimalarial agents. Notably, quarfloxin, a phase II anticancer drug, showed potent activity against the P. falciparum 3D7 strain, with an IC₅₀ value of 0.114 μM [30]. This fluoroquinolone quarfloxin was also described as an interesting candidate against Trypanosoma brucei brucei with an IC₅₀ of 0.155 μM [32]. Furthermore, its therapeutic window against P. falciparum is at least 40 times wider than that observed in human cells (0.114 μM against 3D7 strain versus 4.44 μM in MCF10A human mammary epithelial cells [30]).

Thus, in this work, we evaluated the in vitro antiprotozoal activity of these substituted pyrrolo[2,3-d]pyrimidines 1 against both chloroquine-sensitive (3D7) and chloroquine-resistant (W2) P. falciparum strains, and assessed their cytotoxicity in human HepG2 cells. These novel quinoline-like homologues were also tested for in vitro efficacy against medically important protozoans Trypanosoma brucei brucei. For each compound, selectivity indices and cytotoxicity-to-efficacy ratios were calculated. Furthermore, the ability of these new polyaromatic ligands to stabilize parasitic telomeric G-quadruplexes was assessed using a FRET melting assay.

2. Materials and Methods

2.1. Chemistry

2.1.1. General

Commercially available reagents solvents were used without additional purification, and were purchased from Sigma-Aldrich (Darmstadt, Germany) and BLD Pharmatech GmbH (Kaiserslautern, Germany). Melting points were determined with an SM-LUX-POL Leitz hot-stage microscope (Leitz GMBH, Midland, ON, USA) and are uncorrected.

IR spectra were recorded on a NICOLET 380FT-IR spectrophotometer (Bruker BioSpin, Wissembourg, France). NMR spectra were recorded with tetramethylsilane as an internal standard using a BRUKER AVANCE 300 spectrometer (Bruker BioSpin, Wissembourg, France). Splitting patterns have been reported as follows: s = singlet; bs = broad singlet; d = doublet; t = triplet; q = quartet; dd = double doublet; qt = quintuplet; and m = multiplet.

Analytical TLC were carried out on 0.25 precoated silica gel plates (POLYGRAM SIL G/UV254) (Merck KGaA, Darmstadt, Germany) and compounds were visualized after UV light irradiation. A silica gel 60 (70–230 mesh) was used for column chromatography. Mass spectra were recorded on an ESI LTQ Orbitrap Velos mass spectrometer (ThermoFisher, Bremen, Germany). Ionization was performed using an Electrospray ion source operating in positive ion mode with a capillary voltage of 3.80 kV and capillary temperature of 250 °C. The scan type analyzed was full scan, all MS recordings were in the m/z range between 150 to 2000 m/z. No fragmentation was carried out and the resolution used for the analysis was 60,000. Crystallographic data were collected at 298 K on a Bruker APEX Duo diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) using monochromatic Mo-Kα radiation (λ = 0.71073 Å). The collected data were reduced using SAINT v8.40A software (SAINT, Bruker AXS Inc., Madison, Wisconsin, USA), and all reflections were used for unit-cell refinement. The substituted pyrrolo[2,3-d]pyrimidines 1e–l were synthesized as previously described by our team [24].

2,4-Dichloro-7-[2-(phenyl)ethyl]-7H-pyrrolo[2,3-d]pyrimidine (2e)

A suspension of 0.5 g of 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (2.66 mmol) in acetonitrile (25 mL) was treated with 0.74 g of Cs₂CO₃ (8.0 mmol, 3.0 eq.) and phenylethyl bromide (4.0 mmol, 1.5 eq.), and then stirred at 70 °C for 3.5 h. The solvent was then evaporated under vacuum. The residue was triturated in water then extracted with CH₂Cl₂. The organic phase was washed with water, dried over anhydrous sodium sulfate and activated charcoal, filtered and evaporated under vacuum. The residue was cooled and triturated with a minimum of petroleum ether and filtered on sintered glassware to give the crude product. The residue was purified by silica gel column chromatography (CH₃Cl). White crystals (40%); Mp = 63 °C; ¹H NMR δ (300 MHz, CDCl₃): 7.28–7.19 (m, 3H, H-3′, H-4′ and H-5′), 7.07–7.03 (m, 2H, H-2′ and H-6′), 6.94 (d, 1H, J = 3.60 Hz, H-6), 6.46 (d, 1H, J = 3.60 Hz, H-5), 4.47 (t, 2H, J = 7.05 Hz, CH₂), 3.11 (t, 2H, J = 7.05 Hz, CH₂).

General Procedure for 2,4-Bis(4-formylphenyl)-7-substituted-7H-pyrrolo[2,3-d]pyrimidine (3a–e)

To a solution of 4.37 mmol of 2,4-dichloro-7- substituted-7H-pyrrolo[2,3-d]pyrimidine 2a–e, 1.44 g of 4-formylphenyl boronic acid (9.63 mmol, 2.2 eq.) and 506 mg (0.437 mmol, 0.1 eq.) of tetrakis(triphenylphosphine) palladium in 45 mL of 1,2-dimethoxyethane (DME), 5 mL of 2M Na₂CO₃ aqueous solution, previously degassed for 10 min with nitrogen, were added at room temperature. Then, the mixture was warmed to reflux and stirred for 24 h under nitrogen positive pressure. The solvent was then evaporated under vacuum. The residue was extracted with CH₂Cl₂ and the organic phase was filtered on filter paper, then washed with water (20 mL × 3 times), dried over anhydrous sodium sulfate and activated charcoal, filtered and evaporated under vacuum. The residue was cooled and triturated with a minimum of EtOH and EtO₂ and filtered on sintered glassware to give the crude product. The residue was purified by silica gel column chromatography (CH₂Cl₂), then cooled and triturated again in EtOH, filtered on sintered glassware, washed with a minimum of EtOH, EtO₂ and petroleum ether and dried under pressure to give the solid product 3.

2,4-Bis(4-formylphenyl)-7-methyl-7H-pyrrolo[2,3-d]pyrimidine (3a)

Pale-yellow crystals (58%); Mp = 176 °C; ¹H NMR δ (300 MHz, CDCl₃): 10.17 (s, 1H, CHO), 10.14 (s, 1H, CHO), 8.87 (d, 2H, J = 8.10 Hz, H-2′ and H-6′), 8.46 (d, 2H, J = 8.10 Hz, H-2″ and H-6″), 8.11 (d, 2H, J = 8.10 Hz, H-3′ and H-5′), 8.05 (d, 2H, J = 8.10 Hz, H-3″, H-5″), 7.38 (d, 1H, J = 3.30 Hz, H-6), 6.87 (d, 1H, J = 3.30 Hz, H-5).

2,4-Bis(4-formylphenyl)-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (3c)

Yellow crystals (80%); Mp = 211 °C; ¹H NMR δ (300 MHz, CDCl₃): 10.20 (s, 1H, CHO), 10.13 (s, 1H, CHO), 8.81 (d, 2H, J = 8.10 Hz, H-2′ and H-6′), 8.48 (d, 2H, J = 8.10 Hz, H-2″ and H-6″), 8.15 (d, 2H, J = 8.10 Hz, H-3′ and H-5′), 8.02 (d, 2H, J = 8.10 Hz, H-3″ and H-5″), 7.77 (d, 2H, J = 9.00 Hz, H-2‴ and H-6‴), 7.66 (d, 1H, J = 3.60 Hz, H-6), 7.16 (d, 2H, J = 9.00 Hz, H-3‴ and H-5‴), 7.03 (d, 1H, J = 3.60 Hz, H-5), 3.96 (s, 3H, CH₃O). Dark yellow single crystal of 3c was obtained by slow evaporation from a methanol/chloroform solution (v/v: 20/80): triclinic, space group P-1, a = 9.206(6) Å, b =10.142(6) Å, c = 23.478(15) Å, α = 93.588(10)°, β = 90.021(12)°, γ = 90.806(9)°, V = 2188(2) ų, Z = 4, δ(calcd) = 1.316 Mg.m⁻³, FW = 433.45 for C₂₇H₁₉N₃O₃, F(000) = 904.0. Full crystallographic results were deposited at the Cambridge Crystallographic Data Centre (CCDC-2537115), UK, as shown in the Supplementary X-ray Crystallo-graphic Data [33].

2,4-Bis(4-formylphenyl)-7-benzyl-7H-pyrrolo[2,3-d]pyrimidine (3d)

Pale-yellow crystals (36%); Mp = 210 °C; ¹H NMR δ (300 MHz, CDCl₃): 10.17 (s, 1H, CHO), 10.14 (s, 1H, CHO), 8.89 (d, 2H, J = 8.40 Hz, H-2′ and H-6′), 8.46 (d, 2H, J = 8.40 Hz, H-2″ and H-6″), 8.12 (d, 2H, J = 8.40 Hz, H-3′ and H-5′), 8.05 (d, 2H, J = 8.40 Hz, H-3″ and H-5″), 7.40–7.28 (m, 6H, H-2‴, H-3‴, H-4‴, H-5‴, H-6‴ and H-6), 6.89 (d, 1H, J = 3.60 Hz, H-5), 5.65 (s, 2H, CH₂).

2,4-Bis(4-formylphenyl)-7-[2-(phenyl)ethyl]-7H-pyrrolo[2,3-d]pyrimidine (3e)

Yellow crystals (61%); Mp = 201 °C; ¹H NMR δ (300 MHz, CDCl₃): 10.17 (s, 1H, CHO), 10.15 (s, 1H, CHO), 8.86 (d, 2H, J = 8.40 Hz, H-2′ and H-6′), 8.45 (d, 2H, J = 8.40 Hz, H-2″ and H-6″), 8.12 (d, 2H, J = 8.40 Hz, H-3′ and H-5′), 8.06 (d, 2H, J = 8.40 Hz, H-3″ and H-5″), 7.34–7.24 (m, 3H, H-3‴, H-4‴ and H-5‴), 7.20–7.16 (m, 2H, H-2‴ and H-6‴), 7.15 (1H, J = 3.60 Hz, H-6), 6.79 (d, 1H, J = 3.60 Hz, H-5), 4.69 (t, 2H, J = 7.20 Hz, CH₂), 3.28 (t, 2H, J = 7.20 Hz, CH₂).

General Procedure for 2,4-bis[(substituted-iminomethyl)]-7-substituted-7H-pyrrolo[2,3-d]pyrimidines (4a–u)

To a solution of the appropriate diamines (0.126 mmol, 2.1 eq.) in ethanol (7 mL), the 2,4-bis(4-formylphenyl)-7-substituted-7H-pyrrolo[2,3-d]pyrimidine (0.6 mmol) was added. The reaction mixture was then heated under reflux for 5 h, and then evaporated to dryness under reduced pressure. After cooling, the residue was extracted with dichloromethane (40 mL). The organic layer was dried over sodium sulfate and activated charcoal and evaporated to dryness. Products were then used without further purification.

2,4-Bis{4-[(3-dimethylaminopropyl)iminomethyl]phenyl}-7-methyl-7H-pyrrolo[2,3-d]pyrimidine (4a)

Yellow oil (98%); ¹H NMR δ (300 MHz, CDCl₃): 8.72 (d, 2H, J = 8.40 Hz, H-2′ and H-6′), 8.38 (s, 1H, CH=N), 8.37 (s, 1H, CH=N), 8.31 (d, 2H, J = 8.40 Hz, H-2″ and H-6″), 7.91 (d, 2H, J = 8.40 Hz, H-3′ and H-5′), 7.85 (d, 2H, J = 8.40 Hz, H-3″ and H-5″), 7.22 (d, 1H, J = 3.45 Hz, H-6), 6.49 (d, 1H, J = 3.45 Hz, H-5), 3.95 (s, 3H, NCH₃), 3.71–3.65 (m, 4H, 2NCH₂), 2.38 (t, 4H, J = 6.90 Hz, 2NCH₂), 2.25 (s, 12H, N(CH₃)₂), 1.94–1.82 (m, 4H, 2CH₂).

2,4-Bis{4-[(3-(4-methylpiperazin-1-yl)propyl)iminomethyl]phenyl}-7-methyl-7H-pyrrolo[2,3-d]pyrimidine (4b)

Orange-yellow oil (97%); ¹H NMR δ (300 MHz, CDCl₃): 8.62 (d, 2H, J = 8.40 Hz, H-2′ and H-6′), 8.26 (s, 2H, 2CH=N), 8.20 (d, 2H, J = 8.10 Hz, H-2″ and H-6″), 7.80 (d, 2H, J = 8.10 Hz, H-3′ and H-5′), 7.75 (d, 2H, J = 8.10 Hz, H-3″, H-5″), 7.08 (d, 1H, J = 3.60 Hz, H-6), 6.65 (d, 1H, J = 3.60 Hz, H-5), 3.8 (s, 3H, NCH₃), 3.60–3.52 (m, 4H, 2NCH₂), 2.41–2.20 (m, 20H, 10NCH₂), 2.18 (s, 6H, 2NCH₃), 1.89–1.78 (m, 4H, 2CH₂).

2,4-Bis{4-[(5-(tert-butoxycarbonylamino)-5-methoxycarbonyl)pentyl)iminomethyl]phenyl}-7-methyl-7H-pyrrolo[2,3-d]pyrimidine (4c)

Yellow oil (88%); ¹H NMR δ (300 MHz, CDCl₃): 8.69 (d, 2H, J = 8.40 Hz, H-2′ and H-6′), 8.31 (s, 1H, CH=N), 8.30 (s, 1H, CH=N), 8.27 (d, 2H, J = 8.40 Hz, H-2″ and H-6″), 7.87 (d, 2H, J = 8.40 Hz, H-3′ and H-5′), 7.80 (d, 2H, J = 8.40 Hz, H-3″ and H-5″), 7.19 (d, 1H, J = 3.60 Hz, H-6), 6.74 (d, 1H, J = 3.60 Hz, H-5), 5.17 (d, 2H, J = 8.00 Hz, 2NH), 4.29–4.27 (m, 2H, 2CH), 3.90 (s, 3H, NCH₃), 3.69 (s, 3H, COOCH₃), 3.68 (s, 3H, COOCH₃), 3.61–3.65 (m, 4H, 2NCH₂), 1.95–1.60 (m, 12H, 6CH₂). 1.39 (s, 18H, 2C(CH₃)₃).

2,4-Bis{4-[(3-dimethylaminopropyl)iminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (4m)

Yellow-orange oil (98%); Mp = 107 °C, ¹H NMR δ (300 MHz, CDCl₃): 8.66 (d, 2H, J = 8.10 Hz, H-2′ and H-6′), 8.40 (s, 1H, CH=N), 8.35 (s, 1H, CH=N), 8.31 (d, 2H, J = 8.40 Hz, H-2″ and H-6″), 7.94 (d, 2H, J = 8.10 Hz, H-3′ and H-5′), 7.83 (d, 2H, J = 8.40 Hz, H-3″ and H-5″), 7.72 (d, 2H, J = 9.00 Hz, H-2‴ and H-6‴), 7.50 (d, 1H, J = 3.60 Hz, H-6), 7.07 (d, 2H, J = 9.00 Hz, H-3‴ and H-5‴), 6.91 (d, 1H, J = 3.75 Hz, H-5), 3.88 (s, 3H, OCH₃), 3.71 (t, 2H, J = 7.20 Hz, NCH₂), 3.68 (t, J = 7.20 Hz, NCH₂), 2.43–2.35 (m, 4H, 2NCH₂), 2.27 (s, 6H, N(CH₃)₂), 2.25 (s, 6H, N(CH₃)₂), 1.93 (qt, J = 7.20 Hz, NCH₂) 1.98–1.86 (m, 4H, 2CH₂).

2,4-Bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (4n)

Yellow-orange crystals (98%); Mp = 107 °C, ¹H NMR δ (300 MHz, CDCl₃): 8.67 (d, 2H, J = 8.10 Hz, H-2′ and H-6′), 8.39 (s, 1H, CH=N), 8.35 (s, 1H, CH=N), 8.32 (d, 2H, J = 8.10 Hz, H-2″ and H-6″), 7.94 (d, 2H, J = 8.10 Hz, H-3′ and H-5′), 7.83 (d, 2H, J = 8.10 Hz, H-3″ and H-5″), 7.75 (d, 2H, J = 9.00 Hz, H-2‴ and H-6‴), 7.52 (d, 1H, J = 3.75 Hz, H-6), 7.09 (d, 2H, J = 9.00 Hz, H-3‴ and H-5‴), 6.93 (d, 1H, J = 3.75 Hz, H-5), 3.90 (s, 3H, OCH₃), 3.71–3.64 (m, 4H, 2NCH₂), 2.34–2.30 (m, 4H, 2NCH₂), 2.25 (s, 6H, N(CH₃)₂), 2.23 (s, 6H, N(CH₃)₂), 1.79–1.71 (m, 4H, 2CH₂), 1.63–1.55 (m, 4H, 2CH₂).

2,4-Bis{4-[(5-dimethylaminopentyl)iminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (4o)

Yellow-orange oil (90%); ¹H NMR δ (300 MHz, CDCl₃): 8.68 (d, 2H, J = 8.40 Hz, H-2′ and H-6′), 8.41 (s, 1H, CH=N), 8.36 (s, 1H, CH=N), 8.34 (d, 2H, J = 8.10 Hz, H-2″ and H-6″), 7.96 (d, 2H, J = 8.40 Hz, H-3′ and H-5′), 7.84 (d, 2H, J = 8.10 Hz, H-3″ and H-5″), 7.76 (d, 2H, J = 9.00 Hz, H-2‴ and H-6‴), 7.56 (d, 1H, J = 3.60 Hz, H-6), 7.12 (d, 2H, J = 9.00 Hz, H-3‴ and H-5‴), 6.98 (d, 1H, J = 3.60 Hz, H-5), 3.93 (s, 3H, OCH₃), 3.74–3.64 (m, 4H, 2NCH₂), 2.34–2.27 (m, 4H, 2NCH₂), 2.26 (s, 6H, N(CH₃)₂), 2.25 (s, 6H, N(CH₃)₂), 1.83–1.73 (m, 4H, 2CH₂), 1.57–1.52 (m, 4H, 2CH₂), 1.47–1.38 (m, 4H, 2CH₂).

2,4-Bis{4-[(4-diethylaminobutyl)iminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (4p)

Yellow-orange oil (98%); ¹H NMR δ (300 MHz, CDCl₃): 8.66 (d, 2H, J = 8.40 Hz, H-2′ and H-6′), 8.39 (s, 1H, CH=N), 8.35 (s, 1H, CH=N), 8.33 (d, 2H, J = 8.40 Hz, H-2″ and H-6″), 7.95 (d, 2H, J = 8.40 Hz, H-3′ and H-5′), 7.83 (d, 2H, J = 8.40 Hz, H-3″ and H-5″), 7.74 (d, 2H, J = 9.00 Hz, H-2‴ and H-6‴), 7.53 (d, 1H, J = 3.60 Hz, H-6), 7.10 (d, 2H, J = 9.00 Hz, H-3‴ and H-5‴), 6.94 (d, 1H, J = 3.60 Hz, H-5), 3.91 (s, 3H, OCH₃), 3.70 (t, 2H, J = 6.90 Hz, NCH₂), 3.67 (t, J = 6.90 Hz, NCH₂), 2.59–2.46 (m, 12H, 6NCH₂), 1.79–1.71 (m, 4H, 2CH₂), 1.61–1.52 (m, 4H, 2CH₂), 1.04 (t, 6H, J = 7.20 Hz, 2CH₃), 1.03 (t, 6H, J = 7.20 Hz, 2CH₃).

2,4-Bis{4-[(5-diisopropylaminopentyl)iminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (4q)

Orange oil (98%); ¹H NMR δ (300 MHz, CDCl₃): 8.67 (d, 2H, J = 8.10 Hz, H-2′ and H-6′), 8.39 (s, 1H, CH=N), 8.34 (s, 1H, CH=N), 8.32 (d, 2H, J = 8.10 Hz, H-2″ and H-6″), 7.94 (d, 2H, J = 8.10 Hz, H-3′ and H-5′), 7.83 (d, 2H, J = 8.10 Hz, H-3″ and H-5″), 7.73 (d, 2H, J = 9.00 Hz, H-2‴ and H-6‴), 7.52 (d, 1H, J = 3.60 Hz, H-6), 7.09 (d, 2H, J = 9.00 Hz, H-3‴ and H-5‴), 6.93 (d, 1H, J = 3.60 Hz, H-5), 3.90 (s, 3H, OCH₃), 3.69 (t, 2H, J = 7.20 Hz, NCH₂), 3.65 (t, J = 7.20 Hz, NCH₂), 3.05–2.96 (m, 4H, 4NCH), 2.44–2.34 (m, 4H, 2NCH₂), 1.81–1.73 (m, 4H, 2CH₂), 1.53–1.43 (m, 4H, 2CH₂), 1.38–1.23 (m, 4H, 2CH₂), 1.03–0.99 (m, 24H, 8CH₃).

2,4-Bis{4-[(4-(4-methylpiperazin-1-yl)butyl)iminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (4r)

Yellow oil (98%); ¹H NMR δ (300 MHz, CDCl₃): 8.67 (d, 2H, J = 8.40 Hz, H-2′ and H-6′), 8.40 (s, 1H, CH=N), 8.35 (s, 1H, CH=N), 8.33 (d, 2H, J = 8.40 Hz, H-2″ and H-6″), 7.94 (d, 2H, J = 8.40 Hz, H-3′ and H-5′), 7.83 (d, 2H, J = 8.40 Hz, H-3″ and H-5″), 7.75 (d, 2H, J = 9.00 Hz, H-2‴ and H-6‴), 7.55 (d, 1H, J = 3.60 Hz, H-6), 7.11 (d, 2H, J = 9.00 Hz, H-3‴ and H-5‴), 6.97 (d, 1H, J = 3.60 Hz, H-5), 3.92 (s, 3H, OCH₃), 3.74–3.64 (m, 4H, 2NCH₂), 2.66–2.34 (m, 20H, 10NCH₂), 2.29 (s, 3H, NCH₃), 2.28 (s, 3H, NCH₃), 1.82–1.70 (m, 4H, 2CH₂), 1.64–1.54 (m, 4H, 2CH₂).

2,4-Bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-7-benzyl-7H-pyrrolo[2,3-d]pyrimidine (4s)

Yellow-orange oil (87%); ¹H NMR δ (300 MHz, CDCl₃): 8.75 (d, 2H, J = 8.40 Hz, H-2′ and H-6′), 8.38 (s, 1H, CH=N), 8.37 (s, 1H, CH=N), 8.32 (d, 2H, J = 8.40 Hz, H-2″ and H-6″), 7.93 (d, 2H, J = 8.40 Hz, H-3′ and H-5′), 7.87 (d, 2H, J = 8.40 Hz, H-3″ and H-5″), 7.35–7.28 (m, 5H, H-arom), 7.23 (d, 1H, J = 3.60 Hz, H-6), 6.82 (d, 1H, J = 3.60 Hz, H-5), 5.57 (s, 2H, ArCH₂), 3.72–3.65 (m, 4H, 2NCH₂), 2.33 (t, 4H, J = 7.20 Hz, 2NCH₂), 2.24 (s, 6H, N(CH₃)₂), 2.23 (s, 6H, N(CH₃)₂), 1.83–1.72 (m, 4H, 2CH₂), 1.64–1.53 (m, 4H, 2CH₂).

2,4-Bis{4-[(5-diisopropylaminopentyl)iminomethyl]phenyl}-7-benzyl-7H-pyrrolo[2,3-d]pyrimidine (4t)

Yellow oil (98%); ¹H NMR δ (300 MHz, CDCl₃): 8.73 (d, 2H, J = 8.40 Hz, H-2′ and H-6′), 8.34 (s, 1H, CH=N), 8.33 (s, 1H, CH=N), 8.30 (d, 2H, J = 8.40 Hz, H-2″ and H-6″), 7.91 (d, 2H, J = 8.40 Hz, H-3′ and H-5′), 7.85 (d, 2H, J = 8.40 Hz, H-3″ and H-5″), 7.31–7.28 (m, 5H, H-arom), 7.20 (d, 1H, J = 3.60 Hz, H-6), 6.79 (d, 1H, J = 3.60 Hz, H-5), 5.53 (s, 2H, ArCH₂), 3.66 (t, 2H, J = 6.90 Hz, NCH₂), 3.64 (t, J = 6.90 Hz, NCH₂), 3.02–2.92 (m, 4H, 4NCH), 2.40–2.32 (m, 4H, 2NCH₂), 1.78–1.72 (m, 4H, 2CH₂), 1.49–1.20 (m, 8H, 4CH₂), 0.98 (d, 12H, J = 6.60 Hz, 4CH₃), 0.97 (d, 12H, J = 6.60 Hz, 4CH₃).

2,4-Bis{4-[(4-dimethylaminobutyl)iminomethyl]phenyl}-7-[2-(phenyl)ethyl]-7H-pyrrolo[2,3-d]pyrimidine (4u)

Yellow oil (97%); ¹H NMR δ (300 MHz, CDCl₃): 8.73 (d, 2H, J = 8.40 Hz, H-2′ and H-6′), 8.37 (s, 2H, 2CH=N), 8.31 (d, 2H, J = 8.40 Hz, H-2″ and H-6″), 7.91 (d, 2H, J = 8.40 Hz, H-3′ and H-5′), 7.88 (d, 2H, J = 8.40 Hz, H-3″ and H-5″), 7.29–7.19 (m, 3H, H-3‴, H-4‴ and H-5‴), 7.15 (d, 2H, J = 8.10 Hz, H-2‴ and H-6‴), 6.99 (d, 1H, J = 3.60 Hz, H-6), 6.69 (d, 1H, J = 3.60 Hz, H-5), 4.58 (t, 2H, J = 7.20 Hz, ArCH₂), 3.69 (t, 4H, J = 6.90 Hz, 2NCH₂), 3.21 (t, 2H, J = 7.20 Hz, NCH₂), 2.33 (t, 4H, J = 6.90 Hz, 2NCH₂), 2.24 (s, 12H, 2N(CH₃)₂), 1.77 (qt, 4H, J = 6.90 Hz, 2CH₂), 1.58 (qt, 4H, J = 6.90 Hz, 2CH₂).

General Procedure for 2,4-Bis[(substituted-aminomethyl)]-7-substituted-7H-pyrrolo[2,3-d]pyrimidines (1a–u)

To a solution of 2,4-bis[(substituted-iminomethyl)]-7- substituted-7H-pyrrolo[2,3-d]pyrimidines 4 (0.4 mmol) in methanol (10 mL), sodium borohydride (3.2 mmol, 8 eq.) was added portion-wise at 0 °C. The reaction mixture was then stirred at room temperature for 2 h. Then it was evaporated to dryness under reduced pressure. After cooling, the residue was triturated in water and extracted with dichloromethane (40 mL). The organic layer was separated, dried over sodium sulfate and activated charcoal and evaporated to dryness to give the 2,4-bis[(substituted-aminomethyl)-7-substituted-7H-pyrrolo[2,3-d]pyrimidine 1.

2,4-Bis{4-[(3-dimethylaminopropyl)aminomethyl]phenyl}-7-methyl-7H-pyrrolo[2,3-d]pyrimidine (1a)

Yellow oil (98%); ¹H NMR δ (300 MHz, CDCl₃): 8.63 (d, 2H, J = 8.10 Hz, H-2′ and H-6′), 8.24 (d, 2H, J = 8.10 Hz, H-2″ and H-6″), 7.51 (d, 2H, J = 8.10 Hz, H-3′, H-5′), 7.45 (d, 2H, J = 8.10 Hz, H-3″ and H-5″), 7.19 (d, 1H, J = 3.60 Hz, H-6), 6.79 (d, 1H, J = 3.60 Hz, H-5), 3.94 (s, 3H, NCH₃), 3.90 (s, 2H, NCH₂), 3.88 (s, 2H, NCH₂), 2.74–6.65 (m, 4H, 2NCH₂), 2.37–2.30 (m, 4H, 2NCH₂), 2.23 (s, 6H, N(CH₃)₂), 2.22 (s, 6H, N(CH₃)₂), 1.80–1.69 (m, 4H, 2CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 158.8 (C-2), 158.0 (C-4), 154.4 (C-7a), 143.7 (C-4″), 143.0 (C-4′), 139.1 (C-1′), 138.8 (C-1″), 131.2 (C-6), 130.4 (C-2″ and C-6″), 129.7 (C-2′ and C-6′), 129.5 (C-3′, C-5′, C-3″ and C-5″), 115.1 (C-4a), 101.6 (C-5), 59.4 (NCH₂), 55.1 (NCH₂), 49.2 (NCH₂), 46.9 (N(CH₃)₂), 32.4 (NH₃), 29.3 (CH₂); ESI MS m/z [M+H]⁺ Calculed for C₃₁H₄₄N₇: 514.3658, Found: 514.3664.

2,4-Bis{4-[(3-(4-methylpiperazin-1-yl)propyl)aminomethyl]phenyl}-7-methyl-7H-pyrrolo[2,3-d]pyrimidine (1b)

Yellow oil (95%); ¹H NMR δ (300 MHz, CDCl₃): 8.60 (d, 2H, J = 8.10 Hz, H-2′ and H-6′), 8.21 (d, 2H, J = 8.10 Hz, H-2″ and H-6″), 7.48 (d, 2H, J = 8.10 Hz, H-3′ and H-5′), 7.41 (d, 2H, J = 8.10 Hz, H-3″, H-5″), 7.16 (d, 1H, J = 3.60 Hz, H-6), 6.76 (d, 1H, J = 3.60 Hz, H-5), 3.91 (s, 3H, NCH₃), 3.86 (s, 2H, NCH₂), 3.84 (s, 2H, NCH₂), 2.71–2.64 (m, 4H, 2NCH₂), 2.57–2.34 (m, 20H, 10NCH₂), 2.24 (s, 6H, 2NCH₃), 1.75–1.68 (m, 4H, 2CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 158.8 (C-2), 158.1 (C-4), 154.4 (C-7a), 143.8 (C-4″), 143.2 (C-4′), 139.2 (C-1′), 138.8 (C-1″), 131.2 (C-6), 130.4 (C-2″ and C-6″), 129.7 (C-2′ and C-6′), 129.4 (C-3′, C-5′, C-3″ and C-5″), 115.1 (C-4a), 101.6 (C-5), 58.4 (NCH₂), 56.5 (NCH₂pip.), 55.1 (NCH₂), 54.6 (NH₂pip.), 49.4 (NCH₂), 47.4 (NCH₃), 32.4 (NCH₃), 28.3 (CH₂); ESI MS m/z [M+H]⁺ Calculed for C₃₇H₅₄N₉: 624.4502, Found: 624.4487.

2,4-Bis{4-[(5-(tert-butoxycarbonylamino)-5-methoxycarbonyl)pentyl)aminomethyl]phenyl}-7-methyl-7H-pyrrolo[2,3-d]pyrimidine (1c)

Yellow oil (75%); ¹H NMR δ (300 MHz, CDCl₃): 8.62 (d, 2H, J = 8.40 Hz, H-2′ and H-6′), 8.23 (d, 2H, J = 8.40 Hz, H-2″ and H-6″), 7.53 (d, 2H, J = 8.40 Hz, H-3′ and H-5′), 7.48 (d, 2H, J = 8.40 Hz, H-3″ and H-5″), 7.20 (d, 1H, J = 3.60 Hz, H-6), 6.78 (d, 1H, J = 3.60 Hz, H-5), 5.14 (d, 2H, J = 7.80 Hz, 2NH), 4.31–4.27 (m, 2H, 2CH), 3.94 (s, 3H, NCH₃), 3.90 (s, 4H, 2NCH₂), 3.74 (s, 3H, COOCH₃), 3.73 (s, 3H, COOCH₃), 3.19 (bs, 2H, 2NH), 2.70 (t, 4H, J = 7.20 Hz, 2NCH₂), 1.87–1.43 (m, 12H, 6CH₂). 1.40 (s, 18H, 2C(CH₃)₃); MALDI-TOF MS m/z [M+H]⁺ Calculed for C₄₅H₆₄N₇O₈: 830.4816, Found: 830.4827.

2,4-Bis{4-[(5-amino-5-(methoxycarbonyl)pentyl)aminomethyl]phenyl}-7-methyl-7H-pyrrolo[2,3-d]pyrimidine (1d)

To a solution of 2,4-bis{4-[(5-(tert-butoxycarbonylamino)-5-methoxycarbonyl)pentyl)aminomethyl]phenyl}-7-methyl-7H-pyrrolo[2,3-d]pyrimidine 1c (0.08 mmol) in 6 mL of dichloromethane, 1.0 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature for 24 h, then neutralized with 65 mL of a saturated aqueous solution of potassium carbonate and extracted with 15 mL of dichloromethane. The organic layer was washed with water, then brine and dried with anhydrous sodium sulphate. The solvent was removed under reduced pressure to give the 2,4-bis{4-[(5-amino-5-(methoxycarbonyl)pentyl)aminomethyl]phenyl}-7-methyl-7H-pyrrolo[2,3-d]pyrimidine 1d. Yellow oil (82%); ¹H NMR δ (300 MHz, CDCl₃): 8.64 (d, 2H, J = 8.40 Hz, H-2′ and H-6′), 8.24 (d, 2H, J = 8.40 Hz, H-2″ and H-6″), 7.52 (d, 2H, J = 8.40 Hz, H-3′ and H-5′), 7.46 (d, 2H, J = 8.40 Hz, H-3″ and H-5″), 7.23 (d, 1H, J = 3.60 Hz, H-6), 6.83 (d, 1H, J = 3.60 Hz, H-5), 3.98 (s, 3H, NCH₃), 3.91 (s, 2H, NCH₂), 3.89 (s, 2H, NCH₂), 3.73 (s, 3H, COOCH₃), 3.72 (s, 3H, COOCH₃), 3.50–3.44 (m, 2H, 2CH), 2.73–2.65 (m, 4H, 2NCH₂), 1.83–1.43 (m, 12H, 6CH₂); ESI MS m/z [M+H]⁺ Calculed for C₃₅H₄₈N₇O₄: 630.3768, Found: 630.3769.

2,4-Bis{4-[(3-dimethylaminopropyl)aminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (1m)

Orange oil (80%); ¹H NMR δ (300 MHz, CDCl₃): 8.57 (d, 2H, J = 8.10 Hz, H-2′ and H-6′), 8.22 (d, 2H, J = 8.10 Hz, H-2″ and H-6″), 7.70 (d, 2H, J = 8.70 Hz, H-2‴ and H-6‴), 7.51 (d, 2H, J = 8.10 Hz, H-3′ and H-5′), 7.43 (d, J = 3.60 Hz, H-6), 7.40 (d, 2H, J = 8.10 Hz, H-3″ and H-5″), 7.04 (d, 2H, J = 8.70 Hz, H-3‴ and H-5‴), 6.89 (d, 1H, J = 3.60 Hz, H-5), 4.74 (bs, 2H, 2NH), 3.87 (s, 2H, NCH₂), 3.85 (s, 3H, OCH₃), 3.83 (s, 2H, NCH₂), 2.73–2.63 (m, 4H, 2NCH₂), 2.33 (t, 2H, J = 7.20 Hz, NCH₂), 2.30 (t, 2H, J = 7.20 Hz, NCH₂), 2.21 (s, 6H, N(CH₃)₂), 2.19 (s, 6H, N(CH₃)₂), 1.73–1.64 (m, 4H, 2CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 159.6 (C-2), 159.4 (C-4), 158.7 (C-4‴), 153.9 (C-7a), 143.9 (C-4″), 143.3 (C-4′), 139.1 (C-1‴), 138.7 (C-1′), 132.3 (C-1″), 130.4 (C-2″ and C-6″), 130.1 (C-6), 129.8 (C-2′ and C-6′), 129.6 (C-3″ and C-5″), 129.4 (C-3′ and C-5′), 128.9 (C-4a), 126.5 (C-2‴ and C-6‴), 115.8 (C-3‴ and C-5‴), 103.0 (C-5), 59.4 (NCH₂), 56.9 (OCH₃), 55.2 (NCH₂), 49.2 (NCH₂), 49.1 (NCH₂), 46.8 (N(CH₃)₂), 29.3 (CH₂); ESI MS m/z [M+H]⁺ Calculed for C₃₇H₄₈N₇O: 606.3920, Found: 606.3904.

2,4-Bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (1n)

Yellow oil (61%); ¹H NMR δ (300 MHz, CDCl₃): 8.60 (d, 2H, J = 8.10 Hz, H-2′ and H-6′), 8.26 (d, 2H, J = 8.10 Hz, H-2″ and H-6″), 7.78 (d, 2H, J = 9.00 Hz, H-2‴ and H-6‴), 7.56 (d, 2H, J = 8.10 Hz, H-3′ and H-5′), 7.52 (d, J = 3.90 Hz, H-6), 7.44 (d, 2H, J = 8.10 Hz, H-3″ and H-5″), 7.12 (d, 2H, J = 9.00 Hz, H-3‴ and H-5‴), 6.97 (d, 1H, J = 3.90 Hz, H-5), 3.94 (s, 2H, NCH₂), 3.92 (s, 3H, OCH₃), 3.87 (s, 2H, NCH₂), 2.73–2.66 (m, 4H, 2NCH₂), 2.30 (t, 2H, J = 7.20 Hz, NCH₂), 2.27 (t, 2H, J = 7.20 Hz, NCH₂), 2.22 (s, 6H, N(CH₃)₂), 2.21 (s, 6H, N(CH₃)₂), 1.58–1.47 (m, 8H, 4CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 159.5 (C-2), 159.4 (C-4), 158.7 (C-4‴), 153.9 (C-7a), 143.8 (C-4″), 143.0 (C-4′), 139.1 (C-1‴), 138.8 (C-1′), 132.3 (C-1″), 130.5 (C-2″ and C-6″), 130.2 (C-6), 129.8 (C-2′ and C-6′), 129.6 (C-3″ and C-5″), 129.5 (C-3′ and C-5′), 128.8 (C-4a), 126.5 (C-2‴ and C-6‴), 115.8 (C-3‴ and C-5‴), 103.1 (C-5), 61.0 (NCH₂), 56.9 (OCH₃), 55.0 (NCH₂), 50.6 (NCH₂), 50.5 (NCH₂), 46.8 (N(CH₃)₂), 29.3 (CH₂), 26.9 (CH₂); ESI MS m/z [M+H]⁺ Calculed for C₃₉H₅₂N₇O: 634.4233, Found: 634.4245.

2,4-Bis{4-[(5-dimethylaminopentyl)aminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (1o)

Yellow oil (53%); ¹H NMR δ (300 MHz, CDCl₃): 8.58 (d, 2H, J = 8.10 Hz, H-2′ and H-6′), 8.25 (d, 2H, J = 8.10 Hz, H-2″ and H-6″), 7.77 (d, 2H, J = 9.00 Hz, H-2‴ and H-6‴), 7.54 (d, 2H, J = 8.10 Hz, H-3′ and H-5′), 7.50 (d, J = 3.60 Hz, H-6), 7.42 (d, 2H, J = 8.10 Hz, H-3″ and H-5″), 7.10 (d, 2H, J = 9.00 Hz, H-3‴ and H-5‴), 6.95 (d, 1H, J = 3.60 Hz, H-5), 3.91 (s, 5H, NCH₂ and OCH₃), 3.86 (s, 2H, NCH₂), 2.73–2.63 (m, 4H, 2NCH₂), 2.27 (t, 2H, J = 7.20 Hz, NCH₂), 2.23 (t, 2H, J = 7.20 Hz, NCH₂), 2.22 (s, 6H, N(CH₃)₂), 2.21 (s, 6H, N(CH₃)₂), 1.61–1.48 (m, 8H, 4CH₂), 1.46–1.34 (m, 4H, 2CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 159.4 (C-2), 159.3 (C-4), 158.7 (C-4‴), 153.8 (C-7a), 143.7 (C-4″), 143.0 (C-4′), 139.0 (C-1‴), 138.7 (C-1′), 131.5 (C-1″), 130.4 (C-2″ and C-6″), 130.1 (C-6), 129.8 (C-2′ and C-6′), 129.6 (C-3″ and C-5″), 129.4 (C-3′ and C-5′), 128.9 (C-4a), 126.6 (C-2‴ and C-6‴), 115.9 (C-3‴ and C-5‴), 103.1 (C-5), 61.2 (NCH₂), 56.9 (OCH₃), 55.2 (NCH₂), 50.6 (NCH₂), 46.9 (NCH₃), 31.5 (CH₂), 29.0 (CH₂), 26.6 (CH₂); ESI MS m/z [M+H]⁺ Calculed for C₄₁H₅₆N₇O: 662.4546, Found: 662.4524.

2,4-Bis{4-[(4-diethylaminobutyl)aminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (1p)

Yellow-orange oil (64%); ¹H NMR δ (300 MHz, CDCl₃): 8.57 (d, 2H, J = 7.80 Hz, H-2′ and H-6′), 8.23 (d, 2H, J = 7.80 Hz, H-2″ and H-6″), 7.73 (d, 2H, J = 9.00 Hz, H-2‴ and H-6‴), 7.52 (d, 2H, J = 7.80 Hz, H-3′ and H-5′), 7.45 (d, J = 3.60 Hz, H-6), 7.41 (d, 2H, J = 7.80 Hz, H-3″ and H-5″), 7.06 (d, 2H, J = 9.00 Hz, H-3‴ and H-5‴), 6.92 (d, 1H, J = 3.60 Hz, H-5), 3.89 (s, 2H, NCH₂), 3.86 (s, 3H, OCH₃), 3.84 (s, 2H, NCH₂), 2.66–2.40 (m, 18H, 8NCH₂ and 2NH), 1.55–1.48 (m, 8H, 4CH₂), 1.02 (t, 6H, J = 6.90 Hz, 2CH₃), 1.00 (t, 6H, J = 6.90 Hz, 2CH₃); ¹³C NMR δ (75 MHz, CDCl₃): 158.3 (C-2), 158.0 (C-4), 157.4 (C-4‴), 152.6 (C-7a), 138.0 (C-4″), 137.6 (C-4′), 131.0 (C-1‴), 129.2 (C-1′), 129.1 (C-2″ and C-6″), 128.9 (C-1″), 128.6 (C-2′ and C-6′), 128.5 (C-3″ and C-5″), 128.3 (C-3′, C-5′ and C-6), 125.2 (C-2‴ and C-6‴), 114.6 (C-4a), 114.5 (C-3‴ and C-5‴), 101.7 (C-5), 55.6 (OCH₃), 53.6 (NCH₂), 53.4 (NCH₂), 52.7 (NCH₂), 52.6 (NCH₂), 49.1 (NCH₂), 49.0 (NCH₂), 46.7 (NCH₂), 46.6 (NCH₂), 27.9 (CH₂), 24.6 (CH₂), 24.5 (CH₂), 11.2 (CH₃); ESI MS m/z [M+2H]⁺ Calculed for C₄₃H₆₁N₇O: 691.4937, Found: 691.4910.

2,4-Bis{4-[(5-diisopropylaminopentyl)aminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (1q)

Yellow oil (73%); ¹H NMR δ (300 MHz, CDCl₃): 8.58 (d, 2H, J = 7.50 Hz, H-2′ and H-6′), 8.24 (d, 2H, J = 7.80 Hz, H-2″ and H-6″), 7.75 (d, 2H, J = 8.70 Hz, H-2‴ and H-6‴), 7.53 (d, 2H, J = 7.50 Hz, H-3′ and H-5′), 7.48 (d, J = 3.60 Hz, H-6), 7.41 (d, 2H, J = 7.80 Hz, H-3″ and H-5″), 7.08 (d, 2H, J = 8.70 Hz, H-3‴ and H-5‴), 6.93 (d, 1H, J = 3.60 Hz, H-5), 3.89 (s, 5H, NCH₂ and OCH₃), 3.86 (s, 2H, NCH₂), 3.04–2.96 (m, 4H, 4NCH), 2.71–2.57 (m, 4H, 2NCH₂), 2.39–2.32 (m, 4H, 2NCH₂), 1.58–1.50 (m, 4H, 2CH₂), 1.49–1.35 (m, 4H, 2CH₂), 1.34–1.23 (m, 4H, 2CH₂), 1.02–0.98 (m, 24H, 8CH₃); ¹³C NMR δ (75 MHz, CDCl₃): 159.5 (C-2), 159.3 (C-4), 158.6 (C-4‴), 153.7 (C-7a), 143.9 (C-4″), 143.4 (C-4′), 139.0 (C-1‴), 138.8 (C-1′), 131.9 (C-1″), 130.5 (C-2″ and C-6″), 130.1 (C-6), 129.8 (C-2′ and C-6′), 129.6 (C-3″ and C-5″), 129.4 (C-3′ and C-5′), 128.9 (C-4a), 126.5 (C-2‴ and C-6‴), 115.8 (C-3‴ and C-5‴), 103.1 (C-5), 56.9 (NCH), 55.2 (NCH₂), 50.9 (NCH₂), 49.9 (OCH₃), 46.7 (NCH₂), 32.8 (CH₂), 31.5 (CH₂), 26.6 (CH₂), 22.0 (CH₃); ESI MS m/z [M+H]⁺ Calculed for C₄₉H₇₂N₇O: 774.5798, Found: 774.5769.

2,4-Bis{4-[(4-(4-methylpiperazin-1-yl)butyl)aminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (1r)

Yellow-orange oil (82%); ¹H NMR δ (300 MHz, CDCl₃): 8.57 (d, 2H, J = 8.10 Hz, H-2′ and H-6′), 8.24 (d, 2H, J = 8.10 Hz, H-2″ and H-6″), 7.76 (d, 2H, J = 9.00 Hz, H-2‴ and H-6‴), 7.53 (d, 2H, J = 8.10 Hz, H-3′ and H-5′), 7.50 (d, J = 3.60 Hz, H-6), 7.42 (d, 2H, J = 8.10 Hz, H-3″ and H-5″), 7.09 (d, 2H, J = 9.00 Hz, H-3‴ and H-5‴), 6.95 (d, 1H, J = 3.60 Hz, H-5), 3.90 (s, 5H, NCH₂ and OCH₃), 3.86 (s, 2H, NCH₂), 2.72–2.63 (m, 4H, 2NCH₂), 2.58–2.34 (m, 20H, 10NCH₂), 2.28 (s, 3H, NCH₃), 2.27 (s, 3H, NCH₃), 1.60–1.52 (m, 4H, 2CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 158.3 (C-2), 158.0 (C-4), 157.4 (C-4‴), 152.6 (C-7a), 137.9 (C-4″), 137.5 (C-4′), 130.9 (C-1‴), 129.2 (C-1′, C-2″ and C-6″), 128.8 (C-1″), 128.5 (C-2′, C-6′, C-3″ and C-5″), 128.3 (C-3′, C-5′ and C-6), 125.2 (C-2‴ and C-6‴), 114.5 (C-4a, C-3‴ and C-5‴), 101.7 (C-5), 58.4 (NCH₂), 55.6 (OCH₃), 55.0 (NCH₂), 53.2 (NCH₂), 53.1 (NCH₂), 49.2 (NCH₂), 49.0 (NCH₂), 46.0 (N(CH₃)₂), 28.0 (CH₂), 24.7 (CH₂); ESI MS m/z [M+H]⁺ Calculed for C₄₅H₆₂N₉O: 744.5077, Found: 744.5051.

2,4-Bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-7-benzyl-7H-pyrrolo[2,3-d]pyrimidine (1s)

Yellow oil (92%); ¹H NMR δ (300 MHz, CDCl₃): 8.66 (d, 2H, J = 8.10 Hz, H-2′ and H-6′), 8.25 (d, 2H, J = 8.10 Hz, H-2″ and H-6″), 7.53 (d, 2H, J = 8.10 Hz, H-3′ and H-5′), 7.46 (d, 2H, J = 8.10 Hz, H-3″ and H-5″), 7.35–7.30 (m, 5H, H-arom), 7.19 (d, 1H, J = 3.60 Hz, H-6), 6.82 (d, 1H, J = 3.60 Hz, H-5), 5.56 (s, 2H, ArCH₂), 3.90 (s, 2H, NCH₂), 3.88 (s, 2H, NCH₂), 2.70 (t, 2H, J = 6.60 Hz, NCH₂), 2.68 (t, 2H, J = 6.60 Hz, NCH₂), 2.30–2.24 (m, 4H, 2NCH₂), 2.23 (s, 3H, NCH₃), 2.22 (s, 3H, NCH₃), 1.57–1.51 (m, 4H, 2CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 157.7 (C-2), 157.0 (C-4), 153.0 (C-7a), 142.3 (C-4″), 141.7 (C-4′), 138.0 (C-1‴), 137.5 (C-1′), 137.3 (C-1″), 129.1 (C-3‴ and C-5‴), 128.8 (C-2‴ and C-6‴), 128.7 (C-4‴), 128.5 (C-2″ and C-6″), 128.3 (C-2′ and C-6′), 128.2 (C-3″ and C-5″), 127.9 (C-6, C-3′ and C-5′), 113.8 (C-4a), 101.0 (C-5), 59.9 (ArCH₂), 53.8 (NCH₂), 49.3 (NCH₂), 49.2 (NCH₂), 47.9 (NCH₂), 45.5 (N(CH₃)₂), 45.4 (N(CH₃)₂), 28.0 (CH₂), 25.5 (CH₂); ESI MS m/z [M+H]⁺ Calculed for C₃₉H₅₂N₇: 618.4284, Found: 618.4216.

2,4-Bis{4-[(5-diisopropylaminopentyl)aminomethyl]phenyl}-7-benzyl-7H-pyrrolo[2,3-d]pyrimidine (1t)

Yellow oil (76%); ¹H NMR δ (300 MHz, CDCl₃): 8.66 (d, 2H, J = 8.10 Hz, H-2′ and H-6′), 8.26 (d, 2H, J = 8.10 Hz, H-2″ and H-6″), 7.54 (d, 2H, J = 8.10 Hz, H-3′ and H-5′), 7.46 (d, 2H, J = 8.10 Hz, H-3″ and H-5″), 7.37–7.32 (m, 5H, H-arom), 7.22 (d, 1H, J = 3.60 Hz, H-6), 6.84 (d, 1H, J = 3.60 Hz, H-5), 5.60 (s, 2H, ArCH₂), 3.92 (s, 2H, NCH₂), 3.89 (s, 2H, NCH₂), 3.06–2.98 (m, 4H, 4NCH), 2.69 (t, 2H, J = 6.60 Hz, NCH₂), 2.67 (t, 2H, J = 6.60 Hz, NCH₂), 2.42–2.35 (m, 4H, 2NCH₂), 1.63–1.54 (m, 4H, 2CH₂), 1.47–1.41 (m, 4H, 4CH₂), 1.36–1.31 (m, 4H, 4CH₂), 1.02 (d, 12H, J = 6.60 Hz, 4CH₃), 0.98 (d, 12H, J = 6.60 Hz, 4CH₃); ¹³C NMR δ (75 MHz, CDCl₃): 157.7 (C-2), 157.0 (C-4), 153.1 (C-7a), 142.5 (C-4″), 142.0 (C-4′), 137.9 (C-1‴), 137.5 (C-1′), 137.3 (C-1″), 129.1 (C-3‴ and C-5‴), 128.9 (C-2‴ and C-6‴), 128.6 (C-4‴), 128.4 (C-2″ and C-6″), 128.2 (C-2′ and C-6′), 128.1 (C-6), 127.9 (C-3′, C-5′ C-3″ and C-5″), 113.8 (C-4a), 101.0 (C-5), 53.9 (ArCH₂), 49.5 (NCH₂), 49.4 (NCH₂), 49.0 (NCH₂), 47.9 (NCH), 45.5 (NCH₂), 31.1 (CH₂), 30.1 (CH₂), 25.2 (CH₂), 20.4 (CH₃); ESI MS m/z [M+H]⁺ Calculed for C₄₉H₇₂N₇: 758.5849, Found: 758.5822.

2,4-Bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-7-[2-(phenyl)ethyl]-7H-pyrrolo[2,3-d]pyrimidine (1u)

Yellow oil (90%); ¹H NMR δ (300 MHz, CDCl₃): 8.65 (d, 2H, J = 8.10 Hz, H-2′ and H-6′), 8.25 (d, 2H, J = 8.10 Hz, H-2″ and H-6″), 7.52 (d, 2H, J = 8.10 Hz, H-3′ and H-5′), 7.47 (d, 2H, J = 8.10 Hz, H-3″ and H-5″), 7.32–7.27 (m, 3H, H-3‴, H-4‴ and H-5‴), 7.18 (d, 2H, J = 8.10 Hz, H-2‴ and H-6‴), 6.99 (d, 1H, J = 3.60 Hz, H-6), 6.72 (d, 1H, J = 3.60 Hz, H-5), 4.60 (t, 2H, J = 7.20 Hz, ArCH₂), 3.90 (s, 2H, NCH₂), 3.24 (t, 2H, J = 7.20 Hz, NCH₂), 2.70 (t, 4H, J = 6.60 Hz, 2NCH₂), 2.28 (t, 4H, J = 6.60 Hz, 2NCH₂), 2.23 (s, 6H, N(CH₃)₂), 2.22 (s, 6H, N(CH₃)₂), 1.57–1.53 (m, 8H, 4CH₂); ¹³C NMR δ (75 MHz, CDCl₃): 157.4 (C-2), 156.8 (C-4), 152.7 (C-7a), 142.4 (C-4″), 141.7 (C-4′), 138.4 (C-1‴), 138.0 (C-1′), 137.6 (C-1″), 129.2 (C-4‴), 129.1 (C-3‴ and C-5‴), 128.9 (C-2‴ and C-6‴), 128.6 (C-2″ and C-6″), 128.4 (C-2′ and C-6′), 128.2 (C-3″, C-5″), 128.1 (C-3′ and C-5′), 126.7 (C-6), 113.9 (C-4a), 100.0 (C-5), 59.7 (ArCH₂), 53.8 (NCH₂), 53.7 (NCH₂), 49.3 (NCH₂), 49.2 (NCH₂), 45.5 (N(CH₃)₂), 45.4 (N(CH₃)₂), 36.6 (CH₂), 28.0 (CH₂), 25.6 (CH₂); ESI MS m/z [M+H]⁺ Calculed for C₄₀H₅₄N₇: 632.4440, Found: 632.4416.

General Procedure for 2,4-Bis[(substituted-aminomethyl)]-7-substituted-7H-pyrrolo[2,3-d]pyrimidine oxalate salts (1a–u·m(COOH)₂)

To a solution of compounds 1 (0.3 mmol) in isopropanol (11 mL), oxalic acid (2.4 mmol, 8 eq.) was added. The reaction mixture was heated under reflux for 30 min. The precipitate was filtered, washed with isopropanol then with diethyl ether and dried under reduced pressure to give the ammonium oxalate salts, which were then crystallized using 2-PrOH–H₂O (80/20, v/v) as solvent.

2,4-Bis{4-[(4-dimethylaminopropyl)aminomethyl]phenyl}-7-methyl-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1a·4(COOH)₂)

Pale-yellow crystals (53%); ¹H NMR δ (300 MHz, DMSO-d₆): 8.64 (d, 2H, J = 7.80 Hz, H-2′ and H-6′), 8.37 (d, 2H, J = 7.80 Hz, H-2″ and H-6″), 7.78–7.65 (m, 5H, H-6, H-3′, H-5′, H-3″ and H-5″), 6.99–6.97 (m, 1H, H-5), 4.27 (s, 2H, NCH₂), 4.24 (s, 2H, NCH₂), 3.96 (s, 3H, NCH₃), 3.08–2.99 (m, 8H, 4NCH₂), 2.71 (s, 12H, 2N(CH₃)₂), 2.06–1.99 (m, 4H, 2CH₂).

2,4-Bis{4-[(3-(4-methylpiperazin-1-yl)propyl)aminomethyl]phenyl}-7-methyl-7H-pyrrolo[2,3-d]pyrimidine (oxalate salt (1b·6(COOH)₂)

Beige crystals (45%); ¹H NMR δ (300 MHz, DMSO-d₆): 8.65 (d, 2H, J = 7.50 Hz, H-2′ and H-6′), 8.38 (d, 2H, J = 7.50 Hz, H-2″ and H-6″), 7.79–7.66 (m, 5H, H-6, H-3′, H-5′, H-3″ and H-5″), 6.98 (d, 1H, J = 3.60 Hz, H-5), 4.29 (s, 2H, NCH₂), 4.25 (s, 2H, NCH₂), 3.96 (s, 3H, NCH₃), 3.09–2.97 (m, 14H, 7NCH₂), 2.74–2.62 (m, 16H, 5NCH₂ and 2NCH₃), 1.87–1.84 (m, 4H, 2CH₂).

2,4-Bis{4-[(5-amino-5-(methoxycarbonyl)pentyl)aminomethyl]phenyl}-7-methyl-7H-pyrrolo[2,3-d]pyrimidine (oxalate salt (1d·4(COOH)₂)

Yellow crystals (57%); ¹H NMR δ (300 MHz, DMSO-d₆): 8.65 (d, 2H, J = 8.10 Hz, H-2′ and H-6′), 8.38 (d, 2H, J = 8.10 Hz, H-2″ and H-6″), 7.77–7.73 (m, 3H, H-6, H-3′ and H-5′), 7.67 (d, 2H, J = 8.10 Hz, H-3″ and H-5″), 6.98–6.97 (m, 1H, H-5), 4.31 (s, 2H, NCH₂), 4.27 (s, 2H, NCH₂), 4.04–4.02 (m, 2H, 2CH), 3.97 (s, 3H, NCH₃), 3.83 (s, 6H, 2COOCH₃), 2.98–2.96 (m, 4H, 2NCH₂), 1.82–1.40 (m, 12H, 6CH₂).

2,4-Bis{4-[(4-dimethylaminopropyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1e·4(COOH)₂)

Beige crystals (70%); ¹H NMR δ (300 MHz, DMSO-d₆): 8.57 (d, 2H, J = 7.80 Hz, H-2′ and H-6′), 8.40 (d, 2H, J = 7.80 Hz, H-2″ and H-6″), 8.20 (d, 1H, J = 3.75 Hz, H-6), 8.01 (d, 2H, J = 7.80 Hz, H-2‴ and H-6‴), 7.79 (d, 2H, J = 7.80 Hz, H-3′ and H-5′), 7.71–7.64 (m, 4H, H-3″, H-5″, H-3‴ and H-5‴), 7.48 (t, 1H, J = 7.80 Hz, H-4‴), 7.23 (d, 1H, J = 3.75 Hz, H-5), 4.28 (s, 2H, NCH₂), 4.22 (s, 2H, NCH₂), 3.09–2.97 (m, 8H, 4NCH₂), 2.69 (s, 6H, N(CH₃)₂), 2.68 (s, 6H, N(CH₃)₂), 2.09–1.98 (m, 4H, 2CH₂).

2,4-Bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1f·4(COOH)₂)

White crystals (89%); ¹H NMR δ (300 MHz, DMSO-d₆): 8.57 (d, 2H, J = 7.80 Hz, H-2′ and H-6′), 8.40 (d, 2H, J = 7.80 Hz, H-2″ and H-6″), 8.21 (d, 1H, J = 3.75 Hz, H-6), 8.01 (d, 2H, J = 7.80 Hz, H-2‴ and H-6‴), 7.79 (d, 2H, J = 7.80 Hz, H-3′ and H-5′), 7.71–7.62 (m, 4H, H-3″, H-5″, H-3‴ and H-5‴), 7.49 (t, 1H, J = 7.80 Hz, H-4‴), 7.24 (d, 1H, J = 3.75 Hz, H-5), 4.29 (s, 2H, NCH₂), 4.23 (s, 2H, NCH₂), 3.05–2.94 (m, 8H, 4NCH₂), 2.71 (s, 6H, N(CH₃)₂), 2.69 (s, 6H, N(CH₃)₂), 1.73–1.65 (m, 8H, 4CH₂).

2,4-Bis{4-[(4-dimethylaminopentyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1g·4(COOH)₂)

Pale-yellow crystals (76%); ¹H NMR δ (300 MHz, DMSO-d₆): 8.58 (d, 2H, J = 7.50 Hz, H-2′ and H-6′), 8.39 (d, 2H, J = 7.50 Hz, H-2″ and H-6″), 8.19–8.17 (m, 1H, H-6), 8.01 (d, 2H, J = 7.50 Hz, H-2‴ and H-6‴), 7.78 (d, 2H, J = 7.50 Hz, H-3′ and H-5′), 7.71–7.64 (m, 4H, H-3″, H-5″, H-3‴ and H-5‴), 7.50 (t, 1H, J = 7.50 Hz, H-4‴), 7.23–7.21 (m, 1H, H-5), 4.28 (s, 2H, NCH₂), 4.22 (s, 2H, NCH₂), 2.94–2.91 (m, 8H, 4NCH₂), 2.69 (s, 6H, N(CH₃)₂), 2.68 (s, 6H, N(CH₃)₂), 1.66–1.63 (m, 8H, 4CH₂), 1.37–1.35 (m, 4H, 2CH₂).

2,4-Bis{4-[(4-diethylaminobutyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1h·4(COOH)₂)

White crystals (80%); ¹H NMR δ (300 MHz, DMSO-d₆): 8.56 (d, 2H, J = 7.80 Hz, H-2′ and H-6′), 8.39 (d, 2H, J = 7.80 Hz, H-2″ and H-6″), 8.18 (d, 1H, J = 3.60 Hz, H-6), 8.01 (d, 2H, J = 7.80 Hz, H-2‴ and H-6‴), 7.79 (d, 2H, J = 7.80 Hz, H-3′ and H-5′), 7.71–7.64 (m, 4H, H-3″, H-5″, H-3‴ and H-5‴), 7.48 (t, 1H, J = 7.80 Hz, H-4‴), 7.22 (d, 1H, J = 3.60 Hz, H-5), 4.27 (s, 2H, NCH₂), 4.21 (s, 2H, NCH₂), 3.10–2.94 (m, 16H, 8NCH₂), 1.75–1.66 (m, 8H, 4CH₂), 1.22–1.14 (m, 12H, 4CH₃).

2,4-Bis{4-[(5-diisopropylaminopentyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1i·4(COOH)₂)

Pale-yellow crystals (71%); ¹H NMR δ (300 MHz, DMSO-d₆): 8.56 (d, 2H, J = 7.80 Hz, H-2′ and H-6′), 8.39 (d, 2H, J = 7.80 Hz, H-2″ and H-6″), 8.18–8.17 (m, 1H, H-6), 8.01 (d, 2H, J = 7.80 Hz, H-2‴ and H-6‴), 7.80 (d, 2H, J = 7.80 Hz, H-3′ and H-5′), 7.70–7.64 (m, 4H, H-3″, H-5″, H-3‴ and H-5‴), 7.49 (t, 1H, J = 7.80 Hz, H-4‴), 7.23–7.22 (m, 1H, H-5), 4.29 (s, 2H, NCH₂), 4.23 (s, 2H, NCH₂), 3.61–3.53 (m, 4H, 4CH), 3.00–2.89 (m, 8H, 4NCH₂), 1.75–1.61 (m, 8H, 4CH₂), 1.44–1.35 (m, 4H, 2CH₂), 1.27–1.23 (m, 24H, 8CH₃).

2,4-Bis{4-[(3-(4-methylpiperazin-1-yl)propyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1j·6(COOH)₂)

White crystals (74%); ¹H NMR δ (300 MHz, DMSO-d₆): 8.57 (d, 2H, J = 7.80 Hz, H-2′ and H-6′), 8.41 (d, 2H, J = 7.80 Hz, H-2″ and H-6″), 8.19–8.17 (m, 1H, H-6), 8.02 (d, 2H, J = 7.80 Hz, H-2‴ and H-6‴), 7.81 (d, 2H, J = 7.80 Hz, H-3′ and H-5′), 7.71–7.64 (m, 4H, H-3″, H-5″, H-3‴ and H-5‴), 7.49 (t, 1H, J = 7.80 Hz, H-4‴), 7.23–7.22 (m, 1H, H-5), 4.32 (s, 2H, NCH₂), 4.25 (s, 2H, NCH₂), 3.05–2.96 (m, 14H, 7NCH₂), 2.71–2.63 (m, 16H, 5NCH₂ and 2NCH₃), 1.87–1.85 (m, 4H, 2CH₂).

2,4-Bis{4-[(4-(4-methylpiperazin-1-yl)butyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1k·6(COOH)₂)

Beige crystals (71%); ¹H NMR δ (300 MHz, DMSO-d₆): 8.56 (d, 2H, J = 7.65 Hz, H-2′ and H-6′), 8.39 (d, 2H, J = 7.65 Hz, H-2″ and H-6″), 8.18 (s, 1H, J = 3.60 Hz, H-6), 8.01 (d, 2H, J = 7.80 Hz, H-2‴ and H-6‴), 7.80 (d, 2H, J = 7.65 Hz, H-3′ and H-5′), 7.70–7.64 (m, 4H, H-3″, H-5″, H-3‴ and H-5‴), 7.48 (t, 1H, J = 7.80 Hz, H-4‴), 7.22 (s, 1H, J = 3.60 Hz, H-5), 4.30 (s, 2H, NCH₂), 4.24 (s, 2H, NCH₂), 3.02–2.94 (m, 14H, 7NCH₂), 2.73–2.70 (m, 10H, 5NCH₂), 2.62 (s, 3H, NCH₃), 2.60 (s, 3H, NCH₃), 1.72–1.66 (m, 4H, 2CH₂), 1.59–1.52 (m, 4H, 2CH₂).

2,4-Bis{4-[(5-methylhexyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1l·2(COOH)₂)

Pale-yellow crystals (78%); ¹H NMR δ (300 MHz, DMSO-d₆): ¹H NMR δ (300 MHz, DMSO-d₆): 8.57 (d, 2H, J = 7.80 Hz, H-2′ and H-6′), 8.40 (d, 2H, J = 7.80 Hz, H-2″ and H-6″), 8.17–8.15 (m, 1H, H-6), 8.02 (d, 2H, J = 7.80 Hz, H-2‴ and H-6‴), 7.78 (d, 2H, J = 7.80 Hz, H-3′ and H-5′), 7.71–7.64 (m, 4H, H-3″, H-5″, H-3‴ and H-5‴), 7.49 (t, 1H, J = 7.80 Hz, H-4‴), 7.21–7.20 (m, 1H, H-5), 4.29 (s, 2H, NCH₂), 4.23 (s, 2H, NCH₂), 3.01–2.91 (m, 4H, 2NCH₂), 1.69–1.60 (m, 4H, 2CH₂), 1.58–1.53 (m, 2H, 2CH), 2.69 (s, 6H, N(CH₃)₂), 2.68 (s, 6H, N(CH₃)₂), 1.66–1.63 (m, 8H, 4CH₂), 1.57–1.53 (m, 2H, 2CH), 1.38–1.32 (m, 4H, 2CH₂), 1.21–1.16 (m, 4H, 2CH₂), 0.90–0.85 (m, 12H, 4CH₃).

2,4-Bis{4-[(3-dimethylaminopropyl)aminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1m·4(COOH)₂)

Beige crystals (81%); ¹H NMR δ (300 MHz, DMSO-d₆): 8.54 (d, 2H, J = 7.80 Hz, H-2′ and H-6′), 8.37 (d, 2H, J = 7.80 Hz, H-2″ and H-6″), 8.04 (d, 1H, J = 3.60 Hz, H-6), 7.86 (d, 2H, J = 8.40 Hz, H-2‴ and H-6‴), 7.78 (d, 2H, J = 7.80 Hz, H-3′ and H-5′), 7.66 (d, 2H, J = 7.80 Hz, H-3″ and H-5″), 7.21 (d, 2H, J = 8.40 Hz, H-3‴ and H-5‴), 7.15 (d, 1H, J = 3.60 Hz, H-5), 4.26 (s, 2H, NCH₂), 4.21 (s, 2H, NCH₂), 3.89 (s, 3H, OCH₃), 3.09–2.97 (m, 8H, 4NCH₂), 2.68 (s, 6H, N(CH₃)₂), 2.67 (s, 6H, N(CH₃)₂), 2.06–1.99 (m, 4H, 2CH₂).

2,4-Bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1n·4(COOH)₂)

Yellow crystals (85%); ¹H NMR δ (300 MHz, DMSO-d₆): ¹H NMR δ (300 MHz, DMSO-d₆): 8.55 (d, 2H, J = 7.80 Hz, H-2′ and H-6′), 8.39 (d, 2H, J = 7.80 Hz, H-2″ and H-6″), 8.07–8.05 (m, 1H, H-6), 7.86 (d, 2H, J = 8.40 Hz, H-2‴ and H-6‴), 7.76 (d, 2H, J = 7.80 Hz, H-3′ and H-5′), 7.64 (d, 2H, J = 7.80 Hz, H-3″ and H-5″), 7.21 (d, 2H, J = 8.40 Hz, H-3‴ and H-5‴), 7.17–7.15 (m, 1H, H-5), 4.23 (s, 2H, NCH₂), 4.18 (s, 2H, NCH₂), 3.89 (s, 3H, OCH₃), 2.97–2.93 (m, 4H, 2NCH₂), 2.86–2.84 (m, 4H, 2NCH₂), 2.62 (s, 6H, N(CH₃)₂), 2.59 (s, 6H, N(CH₃)₂), 1.69–1.66 (m, 8H, 4CH₂).

2,4-Bis{4-[(5-dimethylaminopentyl)aminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1o·4(COOH)₂)

Beige crystals (51%); ¹H NMR δ (300 MHz, DMSO-d₆): ¹H NMR δ (300 MHz, DMSO-d₆): 8.54 (d, 2H, J = 7.80 Hz, H-2′ and H-6′), 8.39 (d, 2H, J = 7.80 Hz, H-2″ and H-6″), 8.10–8.08 (m, 1H, H-6), 7.87 (d, 2H, J = 8.40 Hz, H-2‴ and H-6‴), 7.79 (d, 2H, J = 7.80 Hz, H-3′ and H-5′), 7.67 (d, 2H, J = 7.80 Hz, H-3″ and H-5″), 7.23–7.18 (m, 3H, H-3‴, H-5‴ and H-5), 4.28 (s, 2H, NCH₂), 4.22 (s, 2H, NCH₂), 3.88 (s, 3H, OCH₃), 2.97–2.93 (m, 8H, 4NCH₂), 2.71 (s, 6H, N(CH₃)₂), 2.70 (s, 6H, N(CH₃)₂), 1.66–1.62 (m, 8H, 4CH₂), 1.36–1.33 (m, 4H, 2CH₂).

2,4-Bis{4-[(4-diethylaminobutyl)aminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1p·4(COOH)₂)

Beige crystals (77%); ¹H NMR δ (300 MHz, DMSO-d₆): ): ¹H NMR δ (300 MHz, DMSO-d₆): 8.54 (d, 2H, J = 7.80 Hz, H-2′ and H-6′), 8.37 (d, 2H, J = 7.80 Hz, H-2″ and H-6″), 8.06–8.04 (m, 1H, H-6), 7.87 (d, 2H, J = 8.20 Hz, H-2‴ and H-6‴), 7.79 (d, 2H, J = 7.80 Hz, H-3′ and H-5′), 7.67 (d, 2H, J = 7.80 Hz, H-3″ and H-5″), 7.21 (d, 2H, J = 8.40 Hz, H-3‴ and H-5‴), 7.16–7.15 (m, 1H, H-5), 4.26 (s, 2H, NCH₂), 4.21 (s, 2H, NCH₂), 3.89 (s, 3H, OCH₃), 3.08–2.95 (m, 16H, 8NCH₂), 1.73–1.71 (m, 8H, 4CH₂), 1.23–1.15 (m, 12H, 4CH₃).

2,4-Bis{4-[(5-diisopropylaminopentyl)aminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1q·4(COOH)₂)

Beige crystals (46%); ¹H NMR δ (300 MHz, DMSO-d₆): 8.55–8.53 (m, 2H, H-2′ and H-6′), 8.39–8.37 (m, 2H, H-2″ and H-6″), 8.10–8.08 (m, 1H, H-6), 7.88–7.67 (m, 6H, H-2‴, H-6‴, H-3′, H-5′, H-3″ and H-5″), 7.24–7.18 (m, 3H, H-3‴, H-5‴ and H-5), 4.30 (s, 2H, NCH₂), 4.23 (s, 2H, NCH₂), 3.88 (s, 3H, OCH₃), 3.60–3.57 (m, 4H, 4CH), 2.98–2.96 (m, 8H, 4NCH₂), 1.69–1.67 (m, 8H, 4CH₂), 1.51–1.48 (m, 4H, 2CH₂), 1.27–1.25 (m, 24H, 8CH₃).

2,4-Bis{4-[(4-(4-methylpiperazin-1-yl)butyl)aminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1r·6(COOH)₂)

Beige crystals (71%); ¹H NMR δ (300 MHz, DMSO-d₆): ¹H NMR δ (300 MHz, DMSO-d₆): 8.55 (d, 2H, J = 7.80 Hz, H-2′ and H-6′), 8.38 (d, 2H, J = 7.80 Hz, H-2″ and H-6″), 8.06 (d, 1H, J = 3.60 Hz, H-6), 7.86 (d, 2H, J = 8.40 Hz, H-2‴ and H-6‴), 7.81 (d, 2H, J = 7.80 Hz, H-3′ and H-5′), 7.68 (d, 2H, J = 7.80 Hz, H-3″ and H-5″), 7.21 (d, 2H, J = 8.40 Hz, H-3‴ and H-5‴), 7.17 (d, 1H, J = 3.60 Hz, H-5), 4.29 (s, 2H, NCH₂), 4.23 (s, 2H, NCH₂), 3.88 (s, 3H, OCH₃), 3.02–2.94 (m, 14H, 7NCH₂), 2.76–2.74 (m, 10H, 5NCH₂), 2.62 (s, 3H, NCH₃), 2.61 (s, 3H, NCH₃), 1.72–1.67 (m, 4H, 2CH₂), 1.58–1.53 (m, 4H, 2CH₂).

2,4-Bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-7-benzyl-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1s·4(COOH)₂)

Pale-yellow crystals (75%); ¹H NMR δ (300 MHz, DMSO-d₆): 8.62 (d, 2H, J = 7.80 Hz, H-2′ and H-6′), 8.35 (d, 2H, J = 7.80 Hz, H-2″ and H-6″), 7.82–7.81 (m, 1H, H-6), 7.76 (d, 2H, J = 7.80 Hz, H-3′ and H-5′), 7.68 (d, 2H, J = 7.80 Hz, H-3″ and H-5″), 7.43–7.28 (m, 5H, H-phenyl), 7.00–6.98 (m, 1H, H-5), 5.63 (s, 2H, ArCH₂), 4.25 (s, 2H, NCH₂), 4.22 (s, 2H, NCH₂), 3.00–2.94 (m, 8H, 4NCH₂), 2.67 (s, 12H, 2N(CH₃)₂), 1.73–1.71 (m, 8H, 4CH₂).

2,4-Bis{4-[(5-diisopropylaminopentyl)aminomethyl]phenyl}-7-benzyl-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1t·4(COOH)₂)

Yellow crystals (78%); ¹H NMR δ (300 MHz, DMSO-d₆): 8.64 (d, 2H, J = 7.80 Hz, H-2′ and H-6′), 8.36 (d, 2H, J = 7.80 Hz, H-2″ and H-6″), 7.88 (d, 1H, J = 3.60 Hz, H-6), 7.77 (d, 2H, J = 7.80 Hz, H-3′ and H-5′), 7.69 (d, 2H, J = 7.80 Hz, H-3″ and H-5″), 7.44–7.28 (m, 5H, H-phenyl), 7.02 (d, 1H, J = 3.60 Hz, H-5), 5.63 (s, 2H, ArCH₂), 4.27 (s, 2H, NCH₂), 4.23 (s, 2H, NCH₂), 3.59–3.53 (m, 4H, 4CH), 2.98–2.92 (m, 8H, 4NCH₂), 1.68–1.65 (m, 8H, 4CH₂), 1.41–1.38 (m, 4H, 2CH₂), 1.26–1.23 (m, 24H, 8CH₃).

2,4-Bis{4-[(4-dimethylaminobutyl)aminomethyl]phenyl}-7-[2-(phenyl)ethyl]-7H-pyrrolo[2,3-d]pyrimidine oxalate salt (1u·4(COOH)₂)

Pale-yellow crystals (76%); ¹H NMR δ (300 MHz, DMSO-d₆): 8.62 (d, 2H, J = 7.80 Hz, H-2′ and H-6′), 8.37 (d, 2H, J = 7.80 Hz, H-2″ and H-6″), 7.75 (d, 2H, J = 7.80 Hz, H-3′ and H-5′), 7.73 (d, 1H, J = 3.60 Hz, H-6), 7.68 (d, 2H, J = 7.80 Hz, H-3″ and H-5″), 7.26–7.15 (m, 5H, H-phenyl), 6.94 (d, 1H, J = 3.60 Hz, H-5), 4.67 (t, 2H, J = 7.20 Hz, CH₂), 4.31–4.26 (m, 4H, 2NCH₂), 3.26 (t, 2H, J = 7.20 Hz, CH₂), 3.11–3.05 (m, 8H, 4CH₂), 2.78 (s, 12H, 2N(CH₃)₂), 1.73–1.67 (m, 8H, 4CH₂).

2.2. Biological Evaluation

2.2.1. In Vitro Antiplasmodial Activity

Derivatives 1 oxalate salts were dissolved in DMSO and then diluted in sterile water in order to obtain a range of concentration from 40 nM to 40 mM for the first screening against culture-adapted Plasmodium falciparum reference strains, 3D7 and W2. The former strain is susceptible to CQ but displays a decreased susceptibility to MQ; the latter is considered resistant to CQ. These two strains were obtained from the collection of the National Museum of Natural History (Paris, France). The parasites were cultivated in RPMI medium (Sigma-Aldrich, Lyon, France) supplemented with 0.5% Albumax I (Life Technologies Corporation, Paisley, UK), hypoxanthine (Sigma-Aldrich), and gentamicin (Sigma-Aldrich) with human erythrocytes and were incubated at 37 °C in a candle jar, as described previously [34]. The P. falciparum drug susceptibility test was carried out in 96-well flat-bottom sterile plates in a final volume of 250 µL. After 48 h incubation period with the drugs, quantities of DNA in treated and control cultures of parasites in human erythrocytes were quantified using the SYBR Green I (Sigma-Aldrich) fluorescence-based method [35,36]. Briefly, after incubation, plates were frozen at −20 °C until use. Plates were then thawed for 2 h at room temperature, and 100 µL of each homogenized culture were transferred to a well of a 96-well flat-bottom sterile black plate (Sigma-Aldrich, Lyon, France) that contained 100 µL of the SYBR Green I lysis buffer (2xSYBR Green, 20 mM Tris base pH 7.5, 5 mM EDTA, 0.008% w/v saponin, 0.08% w/v Triton X-100). Negative controls treated with solvent (typically DMSO or H₂O) and positive controls (CQ and MQ) were added to each set of experiments. Plates were incubated for 1 h at room temperature and then read on a fluorescence plate reader (Tecan Trading AG, Switzerland) using excitation and emission wavelengths of 485 and 535 nm, respectively. The concentrations at which the screening drug or antimalarial can inhibit 50% of parasitic growth (IC₅₀) were calculated from a sigmoid inhibition model Emax with an estimate of IC₅₀ by non-linear regression (IC Estimator version 1.2) and were reported as means calculated from three independent experiments [37].

2.2.2. In Vitro Antitrypanosomal Activity

The effects of the tested compounds 1 oxalate salts on the growth of Trypanosoma brucei brucei were assessed using an Alamar Blue^® assay described by Räz et al. [38] Trypanosoma brucei brucei AnTat 1.9 (IMTA, Antwerpen, Belgium) was cultured in MEM with Earle’s salts, supplemented according to the protocol of Baltz et al. [39] with the following modifications: 0.5 mM mercaptoethanol (Sigma-Aldrich, Darmstadt, Germany), 1.5 mM L-cysteine (Sigma-Aldrich, Darmstadt, Germany), 0.05 mM bathocuproine sulfate (Sigma-Aldrich, Darmstadt, Germany), and 20% heat-inactivated horse serum (Gibco™ - Thermo Fisher Scientific, Illkirch-Graffenstaden, France) at 37 °C and 5% CO₂. Samples were incubated at an average density of 2000 parasites/well in sterile 96-wells plates (Thermo Fisher Scientific, Illkirch-Graffenstaden, France) with various concentrations of compounds dissolved in 0.9% NaCl. All doses were tested in duplicate. Appropriate controls treated with solvents 0.9% NaCl or DMSO or with suramin, pentamidine, eflornithine, and fexinidazole (reference drugs purchased from Sigma Aldrich (Darmstadt, Germany), and Fluorochem Ltd (Hadfield, Derbyshire, UK) were added to each set of experiments. After 69 h incubation period at 37 °C, 10 µL of the viability marker Alamar Blue (Fisher) was added to each well, and the plates were incubated for 5 h. The plates were read in a PerkinElmer ENSPIRE (Rodgau, Germany) microplate reader using an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The IC₅₀ was defined as the concentration of drug necessary to inhibit by 50% the activity of Trypanosoma brucei brucei compared to the control. IC₅₀ values were calculated using a nonlinear regression analysis of dose–response curves performed using GraphPad Prism 10.0 software (GraphPad Software, San Diego, CA, USA). IC₅₀ values were calculated from three independent experiments.

2.2.3. Cytotoxicity Evaluation

A cytotoxicity evaluation was performed using the method reported by Mosmann [40] with slight modifications to determine the CC₅₀ and using doxorubicin as a cytotoxic reference compound. These assays were performed in human HepG2 cells (ATCC® HB-8065™, Manassas, VA, USA). These cells are a commonly used human hepatocarcinoma-derived cell line that has characteristics like those of primary hepatocytes. These cells express many hepatocyte-specific metabolic enzymes, thus enabling the cytotoxicity of tested product metabolites to be evaluated. Briefly, cells in 100 µL of complete RPMI medium (RPMI supplemented with 10% FCS, 1% L-glutamine (200 mM), penicillin (100 U/mL), and streptomycin (100 µg/mL)) were inoculated at 37 °C into each well of 96-well plates in a humidified chamber in 6% CO₂. After 24 h, 100 µL of medium with the test compound at various concentrations dissolved in DMSO (final concentration less than 0.5% v/v) were added, and the plates were incubated for 72 h at 37 °C. Duplicate assays were performed for each sample. Each well was microscopically examined for precipitate formation before the medium was aspirated from the wells. After aspiration, 100 µL of MTT solution (0.5 mg/mL in medium without FCS) were then added to each well. Cells were incubated for 2 h at 37 °C. The MTT solution was removed and DMSO (100 µL) was added to dissolve the resulting blue formazan crystals. Plates were shaken vigorously (300 rpm) for 5 min. The absorbance was measured at 570 nm with 630 nm as the reference wavelength in a BIO-TEK ELx808 Absorbance Microplate Reader (BioTek Instruments, Winooski, VT, USA). DMSO was used as blank and doxorubicin (Sigma Aldrich) as the positive control. Cell viability was calculated as percentage of control (cells incubated without compound). The CC₅₀ was determined from the dose–response curve using TableCurve 2D V5.0 software (Systat Software, Palo Alto, CA, USA).

2.3. FRET Melting Experiments

Compounds 1 oxalate salts were tested for the subsequent FRET melting experiments. These were performed with dual-labeled oligonucleotides mimicking the Plasmodium telomeric sequences FPf1T (FAM-5′(GGGTTTA)3-GGG3′-TAMRA) and FPf8T [FAM-5′(GGGTTCA)3GGG3′-TAMRA], the human telomeric sequence F21T (FAM-(GGGTTA)3-GGG3′-TAMRA), and the human duplex sequence FdxT (FAM5′-TATAGCTATA-hexaethyleneglycol-TATAGCTATA3′-TAMRA) [22,23,41]. The oligonucleotides were pre-folded in 10 mM lithium cacodylate buffer (pH 7.2), with 10 mM KCl and 90 mM LiCl (K+ condition). The FAM emissions were recorded at 516 nm using a 492 nm excitation wavelength in the absence and presence of a single compound as a function of temperature (25 to 95 °C) in 96-well microplates by using a Stratagene MX3000P real-time PCR device (Agilent Technologies, Santa Clara, CA, USA) at a rate of 1 °C∙min⁻¹. Data were normalized between 0 and 1, and the required temperature for half denaturation of oligonucleotides corresponding to an emission value of 0.5 was taken as the Tm. Each experiment was performed in duplicate with 0.2 µM of labelled oligonucleotide and 2 µM of compound under K⁺ condition. For each compound, three independent experiments were carried out.

3. Results and Discussion

3.1. Chemistry

These reported 2,4-bis[(substituted-aminomethyl)-pyrrolo[2,3-d]pyrimidines 1a–u were synthesized from 2a–e which were synthesized from commercially available 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (Scheme 1 and Scheme 2). The first step was to prepare the N-substituted pyrrolo[2,3-d]pyrimidines 2a–e (Scheme 2). The pyrrolo[2,3-d]pyrimidine was alkylated at the NH pyrrole moiety by using sodium hydride and methyl iodide in tetrahydrofuran leading to 2a [42]. N-arylation of this 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine under the Chan-Lam conditions using arylboronic acid, copper (II) acetate and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or triethylamine led to the preparation of compounds 2b–c [24,43]. Nucleophilic substitution of this same pyrrolo[2,3-d]pyrimidine with benzyl bromide or phenylethyl bromide in the presence of cesium carbonate led to compounds 2d–e [44,45]. The bis(4-formylphenyl)-phenyl-7H-pyrrolo[2,3-d]pyrimidines 3a–e were prepared by a direct double-Suzuki-Miyaura cross-coupling reaction of derivatives 2a–e with the 4-formylphenylboronic acid performed in the presence of Pd(PPh₃)₄ as a catalyst, and sodium carbonate as the base [16,18,19,20,21,22,23,24]. The reaction of various primary substituted aminoalkylamines with these dialdehydes 3a–e yielded the di-imines 4a–u, which were then immediately reduced into the 2,4-bis[(substituted-aminomethyl)-7-phenyl-7H-pyrrolo[2,3-d]pyrimidines 1a–u using sodium borohydride in methanol, as previously described by our team [16,18,19,20,21,22,23,24]. Complete characterization of the synthesized compounds, including ¹H NMR, ¹³C NMR, and mass spectrometric analyses, was systematically carried out on the corresponding free-base forms 1. These novel heterocyclic derivatives 1 were then converted into their ammonium oxalate salts by treatment with oxalic acid in isopropanol under reflux conditions (Scheme 1). These oxalate salts have been found to be less hygroscopic than hydrochloride salts and also soluble in water.

Table 1. Physical properties of ammonium oxalate salts 1a–d and 1m–u.

Compound		Salt a	mp (°C) b	% Yield c
1a	Pale-yellow crystals	4 (COOH)2	179–181	53
1b	Beige crystals	6 (COOH)2	203–205	45
1c	Pale-yellow crystals	2 (COOH)2	172–174	72
1d	Yellow crystals	4 (COOH)2	210–213	57
1m	Beige crystals	4 (COOH)2	183–185	81
1n	Yellow crystals	4 (COOH)2	134–136	85
1o	Beige crystals	4 (COOH)2	137–139	51
1p	Beige crystals	4 (COOH)2	143–145	77
1q	Beige crystals	4 (COOH)2	92–94	46
1r	Beige crystals	6 (COOH)2	204–206	71
1s	Pale-yellow crystals	4 (COOH)2	205–207	75
1t	Yellow crystals	4 (COOH)2	132–135	78
1u	Pale-yellow crystals	4 (COOH)2	169–171	76

^a The stoichiometry and composition of the salts were determined using elemental analyses, and the obtained values were within ±0.4% of the theoretical values. ^b Crystallization solvent: 2-PrOH–H₂O. ^c The yields only included conversions into ammonium oxalates.

summarizes the physical properties of the 1a–u oxalates. The ¹H NMR, ¹³C NMR and ESI-MS spectral copies of the novel synthesized derivatives are enclosed in the Supplementary Materials (Figures S1–S33).

3.2. Biological Evaluation

3.2.1. In Vitro Antimalarial Activity

These novel 2,4-bis[(substituted-aminomethyl)-pyrrolo[2,3-d]pyrimidine derivatives 1a–u oxalate salts were evaluated for their in vitro antimalarial activity by incubation with CQ-resistant P. falciparum W2 strain (IC₅₀ CQ = 0.40 μM, IC₅₀ MQ = 0.016 μM) and the 3D7 strain, which is CQ sensitive and has decreased sensitivity to MQ (IC₅₀ CQ = 0.11 μM, MQ = 0.06 μM). As shown in

Table 2. In vitro sensitivity of P. falciparum and Trypanosoma brucei brucei strains to compounds 1a–u oxalate salts and cytotoxicity of these compounds in HepG2 cells.

Compound	P. falciparum Strains IC50 Values (μM) a		Trypanosoma brucei brucei IC50 Values (μM) b	Cytotoxicity to HepG2 Cells CC50 Values (μM) c
	W2	3D7	Trypanos Antat 1.9
CQ d	0.40 ± 0.04	0.11 ± 0.01	n.d. f	30
MQ d	0.016 ± 0.002	0.06 ± 0.003	n.d. f	n.d. f
Pentamidine e	n.d. f	n.d. f	0.0002 ± 0.00006	2.3 ± 0.50
Suramin e	n.d. f	n.d. f	0.03 ± 0.003	n.d. f
Fexinidazole e	n.d. f	n.d. f	0.59 ± 0.039	n.d. f
Eflornithine e	n.d. f	n.d. f	15.19 ± 0.64	n.d. f
1a	4.46 ± 0.60	1.95 ± 0.69	0.55 ± 0.05	16.42 ± 1.40
1b	0.13 ± 0.05	17.83 ± 1.49	1.00 ± 0.10	39.52 ± 2.31
1c	4.36 ± 1.70	0.61 ± 0.24	7.05 ± 0.66	75.00 ± 1.52
1d	0.074 ± 0.01	>40	5.04 ± 0.73	44.46 ± 1.05
1e	2.71 ± 0.31	2.21 ± 0.11	0.55 ± 0.07	1.65 ± 0.24
1f	1.94 ± 0.16	1.47 ± 0.37	0.38 ± 0.02	3.54± 0.33
1g	1.25 ± 0.08	0.53 ± 0.14	0.72 ± 0.03	1.00 ± 0.13
1h	0.70 ± 0.15	2.47 ± 0.19	n.d. f	2.33 ± 0.10
1i	0.76 ± 0.07	0.93 ± 0.15	n.d. f	2.05 ± 0.10
1j	1.01 ± 0.16	n.d. f	0.88 ± 0.07	10.8 ± 0.52
1k	1.58 ± 0.10	n.d. f	0.69 ± 0.04	1.35 ± 0.18
1l	7.71 ± 1.61	0.089 ± 0.15	n.d. f	16.64 ± 0.61
1m	2.35 ± 0.22	0.97 ± 0.12	n.d. f	0.49 ± 0.13
1n	n.d. f	1.57 ± 0.41	n.d. f	2.83 ± 0.21
1o	1.19 ± 0.30	0.59 ± 0.08	n.d. f	2.09 ± 0.16
1p	0.91 ± 0.17	0.61 ± 0.09	n.d. f	6.36 ± 0.43
1q	0.57 ± 0.10	1.27 ± 0.24	n.d. f	1.18 ± 0.37
1r	1.67 ± 0.34	0.59 ± 0.16	n.d. f	2.07 ± 0.12
1s	1.04 ± 0.17	1.38 ± 0.21	n.d. f	1.52 ± 0.18
1t	0.71 ± 0.20	n.d. f	n.d. f	2.35 ± 0.43
1u	3.78 ± 0.52	n.d. f	n.d. f	1.79 ± 0.12

^a Values were measured against CQ-resistant and mefloquine-sensitive W2 strain and the CQ-sensitive and MQ decreased sensitivity 3D7 strain. The IC₅₀ (µM) values correspond to the means +/− standard deviations from three independent experiments. ^b IC₅₀ values were measured against the slender bloodstream trypomastigotes of Trypanosoma brucei brucei AnTat 1.9 strain. The IC₅₀ (µM) values correspond to the means +/− standard deviations from 3 independent experiments with each concentration tested in duplicate in all experiments. ^c CC₅₀ values were measured against HepG2 cells. The CC₅₀ (µM) values correspond to the means +/− standard deviations from three independent experiments. ^d CQ and MQ were used as antiplasmodial compounds of reference. ^e Suramin, pentamidine, fexinidazole and eflornithine were used as antitrypanosomal compounds of reference. ^f n.d.: not determined.

, these new pyrrolo[2,3-d]pyrimidine derivatives 1 exhibited IC₅₀ values ranging from 0.074 to 7.71 μM against W2, and from 0.089 to 17.83 μM against 3D7 P. falciparum strains.

In subseries 1a–d, the first observation to note is that replacing the dimethylamine motif (compound 1a) with a methylated piperazine (compound 1b) on both side chains led to an increase in antimalarial activity against the W2 strain; i.e., IC₅₀ = 4.46 μM for 1a versus 0.13 μM for 1b. Still in this series of pyrrolopyrimidines substituted in position 7 with a methyl group, it can be noted that the deprotection of the Boc group in compound 1c, leading to derivative 1d, shows an improvement in parasitic activity with an IC₅₀ of 4.36 and 0.074 μM, respectively. Concerning the sub-series of compounds substituted in position 7 with a phenyl group, initial results show that the lengthening of the alkyl dimethyl amino chains slightly improves the antimalarial activity against the W2 strain with the IC₅₀ decreasing from 2.71 to 1.25 μM (derivatives 1e–g). As with subseries 1a–b, replacing the dimethylamino functions with methylpiperazine units (1j versus 1e, and 1k versus 1f) leads to a slight increase in the antiprotozoal activity; i.e., IC₅₀ 1.01 and 2.71 μM for 1j and 1e, then for IC₅₀ 1.58 and 1.94 μM for 1k and 1f. When comparing compounds 1f and 1l, which differ in the absence of nitrogen atoms at the ends of the aminoalkyl side chains, there is a very strong decrease in the antimalarial activity of derivative 1l (IC₅₀ = 7.71 μM) in comparison with that of 1f, which is 1.94 μM. The introduction of a methoxy group at position 4 of the phenyl ring of the 7-(phenyl)-7H-pyrrolo[2,3-d]pyrimidine system slightly decreases the IC₅₀ against the CQ-resistant P. falciparum W2 strain, except for derivative 1r compared to 1k. Moreover, replacing this phenyl ring at position 7 of the polyheterocyclic system with a benzyl group, when comparing derivatives 1s and 1f and then 1t and 1i, does not result in any significant change in IC₅₀ values (e.g., IC₅₀ = 0.71 μM for 1t versus 0.76 μM for 1i), while replacing with a phenethyl motif at position 7 decreases the activity; i.e., IC₅₀ = 3.78 and 1.94 μM for 1u and 1f, respectively.

Among these newly reported 2,4-bis[(substituted-aminomethyl)-pyrrolo[2,3-d]pyrimidines, derivative 1l with (5-methylhexyl)aminomethyl side chains exhibited the most potent activity against the 3D7 strain with an IC₅₀ of 0.089 μM, in the same range of activity order as the reference compound, mefloquine (IC₅₀ = 0.06 μM). Also, surprisingly, this compound 1l, which was highly active against the 3D7 strain, proved to be only moderately active against the W2 strain.

Comparing derivatives 1a and 1b, it can be noted that replacing a dimethylamine group with a methylpiperazine on the side chains results in a loss of activity on strain 3D7; i.e., IC₅₀ = 1.95 and 17.83 μM for 1a and 1b, respectively. Moreover, deprotection of the Boc group in 1c (IC₅₀ = 0.61 μM) leading to compound 1d results in a total loss of antiprotozoal activity against 3D7 strain, IC₅₀ > 40 μM for 1d. In the sub-series in which the pyrrolopyrimidine ring is substituted by a phenyl group, the lengthening of the alkyl chain between the two amine functions of the side chains (compounds 1e–g) leads to an improvement in antiplasmodial activity with an IC₅₀ between 2.21 and 0.53 μM. Surprisingly, replacing the (4-dimethylaminobutyl)aminomethyl chains (compound 1f) with homologous carbon chains (5-methylhexyl)aminomethyl (compound 1l) results in a marked improvement in activity; i.e., IC₅₀ = 1.47 μM for 1f versus 0.089 μM for 1l. The introduction of a methoxy group at position 4 of the phenyl group located at position 7 of the heterocyclic system (subseries 1m-1r) appears to give results substantially similar to those obtained for the subseries 1e-1l, with Ian C₅₀ between 1.57 and 0.59 μM. Similarly, replacing the phenyl ring in compound 1f with a benzyl group, leading to 1s, gives a similar antimalarial activity against the 3D7 strain; i.e., IC₅₀ = 1.47 and 1.38 μM for 1f and 1s, respectively.

3.2.2. In Vitro Activity Against Trypanosoma brucei brucei

Some of these new synthesized heterocyclic compounds 1 oxalate salts were then evaluated against Trypanosoma brucei brucei. Pentamidine, suramin, fexinidazole, and eflornithine were used here as reference drugs. These preliminary screening data are presented in Table 2. The tested pyrrolo[2,3-d]pyrimidines 1 oxalate salts were active against Trypanosoma brucei brucei with IC₅₀ values ranging from 0.38 to 7.05 μM. When the dimethylamino endings of the side chains are replaced by methylpiperazines in the N-methyl pyrrole subseries, the antitrypanosomal activity is reduced by almost twofold; thus, the IC₅₀ of 1a was observed at 0.55 μM, while that of 1b was found at 1.00 μM. In the N-phenyl pyrrole subseries (compounds 1e–k), the overall results show submicromolar activity against Trypanosoma brucei brucei with IC₅₀ from 0.38 to 0.88 μM.

3.2.3. Cytotoxicity and Selectivity Index

In order to evaluate the selectivity of the action, the cytotoxicity of the newly synthesized antiparasitic pyrrolo[2,3-d]pyrimidines 1 oxalate salt was assessed in vitro using the human HepG2 cell line. This hepatocarcinoma-derived cell line is widely used due to its expression of many metabolic enzymes specific to hepatocytes. The objective of the assay was to determine the effect of the metabolic activation of the tested pyrrolo[2,3-d]pyrimidines on cell viability [41,42]. The 50% cytotoxic concentrations (CC₅₀) were measured, and selectivity indexes (SI)—calculated as the ratio of cytotoxic activity to antiparasitic activity (SI = CC₅₀/IC₅₀)—were determined. The cytotoxicity results and corresponding SI values are summarized in

Table 3. Selectivity indexes of compounds 1a–u oxalate salts.

Compound			Selectivity Index a
	HepG2/W2	HepG2/3D7	HepG2/Tryp.
CQ	75	272	n.d. b
Pentamidine	n.d. b	n.d. b	17.45
1a	3.68	8.42	29.85
1b	304.00	2.22	39.52
1c	17.20	122.95	10.64
1d	600.81	>1.11	8.82
1e	0.61	0.75	3.00
1f	1.82	2.41	9.31
1g	0.80	1.89	1.39
1h	3.33	0.94	n.d. b
1i	2.70	2.20	n.d. b
1j	10.70	n.d. b	12.27
1k	0.85	n.d. b	1.96
1l	2.16	187.00	n.d. b
1m	0.21	0.51	n.d. b
1n	n.d. b	1.80	n.d. b
1o	1.76	3.54	n.d. b
1p	6.99	10.43	n.d. b
1q	2.07	0.93	n.d. b
1r	1.24	3.51	n.d. b
1s	1.46	1.10	n.d. b
1t	3.31	n.d. b	n.d. b
1u	0.47	n.d. b	n.d. b

^a SI was defined as the ratio between the CC₅₀ value on HepG2 cells and the IC₅₀ value against the P. falciparum W2 or 3D7 or Trypanosoma brucei brucei strains. ^b n.d.: not determined.

. Our novel 2,4-disubstituted pyrrolo[2,3-d]pyrimidine derivatives 1a–u revealed cytotoxicity against these HepG2 cells with CC₅₀ values ranging from 0.49 to 39.52 µM.

For the Plasmodium falciparum W2 strain, the calculated selectivity indexes (SIs) ranged from 0.21 to 600.81. In the case of the chloroquine-sensitive 3D7 strain, the SIs ranged from 0.51 to 122.95. Based on these values, 2,4-bis{4-[(5-amino-5-(methoxycarbonyl)pentyl)aminomethyl]phenyl}-7-methyl-7H-pyrrolo[2,3-d]pyrimidine 1d oxalate salt was identified as a promising antiplasmodial candidate, with a SI value of 600.81 against the W2 strain. In addition, compound 1b, the 2,4-bis{4-[(3-(4-methylpiperazin-1-yl)propyl)aminomethyl]phenyl}-7-methyl-7H-pyrrolo[2,3-d]pyrimidine, was identified as another promising antiplasmodial derivative with a SI value of 304.0 against the W2 strain. This same compound 1b also demonstrates the most promising SI on the Trypanosoma brucei brucei strain, with a ratio of 39.52 between the CC₅₀ value on HepG2 cells and the IC₅₀ value against the Trypanosoma brucei brucei strain.

For the CQ sensitive strain 3D7, the most interesting SI was observed for pyrrolo[2,3-d]pyrimidine 1c, with a SI value of approximately 123.

3.3. FRET Melting Experiments

Given the potential of P. falciparum telomeres as promising targets for heterocyclic aromatic derivatives [18,46], we further investigated the ability of these novel biologically active compounds 1 oxalate salts to stabilize P. falciparum telomeric chromosomal G-quadruplexes. This study was carried out using a FRET melting assay to evaluate the effectiveness with which these new polyaromatic derivatives 1 could stabilize G-quadruplex structures formed by oligonucleotide sequences derived from P. falciparum, Trypanosoma brucei brucei and human telomeres. Two fluorescently labeled P. falciparum telomeric sequences (FPf1T and FPf8T), as well as a Trypanosoma brucei brucei chromosomal sequence (FtrypBT) and a human telomeric sequence (F21T), were employed in the assay.

To determine the G4 selectivity of the novel pyrrolo[2,3-d]pyrimidine compounds 1a–u oxalate salts relative to double-stranded DNA, we performed FRET melting experiments using a duplex control sequence, FdxT. We also included the reference G4 ligand PhenDC3 (inactive against P. falciparum) and the standard antimalarial drugs CQ and MQ for comparison. Currently, there are no commercially available antimalarial drugs described as G-quadruplex stabilizers. It is important to note that neither CQ nor MQ were specifically designed to target secondary nucleic acid structures such as G-quadruplexes, and there is no strong evidence that G-quadruplex stabilization is a significant component of their antimalarial mechanism of action. However, in these FRET-based fusion experiments, the inclusion of reference antimalarials such as CQ and MQ is essential to establish a point of comparison with our designed compounds. These clinically used agents provide well-characterized standards, allowing for a more accurate assessment of the relative ability of the tested molecules to stabilize DNA structures, such as G-quadruplexes. CQ and MQ have been extensively studied for their antiplasmodial activity, but their primary mechanisms of action differ from those generally associated with G-quadruplex stabilization. CQ acts primarily by interfering with heme detoxification within the digestive vacuole of Plasmodium falciparum. Regarding MQ, although its mechanism of action is not fully understood, several targets have been identified [17]. Therefore, the use of CQ and MQ in FRET fusion assays is not intended to demonstrate G-quadruplex targeting per se, but rather to provide pharmacological reference points. Their evaluation in FRET allows for comparison with newly synthesized compounds 1, highlighting whether the observed effects are specific and significant compared to established antimalarials that act via different biological pathways.

To assess selectivity, we calculated the ΔT_m, defined as the difference in melting temperature (Tm) of the G-quadruplex (FPf1T, FPf8T, FtrypBT, F21T) or duplex (FdxT) sequences in the presence and absence of each derivative. The ΔTm values, summarized in

Table 4. FRET melting values for compounds 1a–u oxalate salts (2 μM) with FPf1T, FPf8T, FtryBT, F21T and FdxT (0.2 µM) in K⁺ conditions.

Compound	ΔTm (°C) a		ΔTm (°C) a		ΔTm (°C) a		ΔTm (°C) a		ΔTm (°C) a
	FPf1T		FPf8T		FtrypBT		F21T		FdxT
PhenDC3	24.6	±0.1	24.7	±0.2	19.2	±0.2	26.3	±0.1	0.1	±0.2
CQ	1.9	±0.1	2.4	±1.2	n.d. b		2.4	±1.1	n.d. b
MQ	3.1	±0.5	6.6	±2.3	n.d. b		2.6	±0.5	n.d. b
1a	10.3	±0.3	10.6	±0.9	9.4	±0.5	10.9	±0.4	−0.2	±0.1
1b	10.1	±1.0	10.1	±0.2	8.4	±0.3	10.4	±0.6	0.1	±0.3
1c	12.1	±0.6	10.3	±0.6	8.2	±0.5	10.6	±0.4	0.4	±0.1
1d	11.3	±0.2	9.7	±0.2	8.1	±0.7	10.1	±0.8	0.3	±0.1
1e	23.6	±0.9	23.5	±0.3	20.5	±0.2	22.8	±0.4	1.2	±0.3
1f	25.1	±0.9	25.1	±1.1	21.9	±0.5	23.7	±1.4	1.5	±0.1
1g	32.3	±0.2	29.1	±0.6	24.1	±0.7	27.7	±1.3	6.01	±0.9
1h	30.2	±0.2	27.5	±0.2	23.1	±0.6	28.7	±1.3	6.0	±0.5
1i	37.8	±0.1	34.9	±0.8	30.5	±0.4	31.9	±0.3	8.5	±0.4
1j	19.1	±0.6	20.0	±0.7	17.3	±0.5	19.6	±1.3	0.7	±0.3
1k	19.8	±3.4	21.0	±1.1	17.4	±1.4	20.4	±1.9	0.6	±0.3
1l	1.2	±0.6	1.3	±0.8	2.8	±0.8	4.0	±0.7	0.2	±0.2
1m	32.5	±0.1	28.2	±0.7	6.42	±0.8	30.1	±0.1	4.9	±0.1
1n	33.2	±1.2	29.7	±0.4	8.0	±2.4	30.2	±0.2	5.5	±0.1
1o	35.2	±0.7	30.4	±1.1	7.5	±1.4	32.0	±0.2	6.4	±0.2
1p	35.3	±0.71	29.5	±1.6	8.7	±0.5	31.9	±0.24	5.7	±0.8
1q	37.8	±0.2	38.1	0.87	36.5	±0.5	34.5	±0.0	12.8	±0.1
1r	29.4	±0.8	29.6	±0.9	27.6	±0.7	27.6	±0.7	5.71	±0.3
1s	30.6	±0.4	29.9	±0.9	5.14	±1.1	25.11	±0.3	4.5	±0.2
1t	30.9	±0.2	26.3	±2.3	4.56	±0.7	24.9	±0.3	2.8	±0.3
1u	7.46	±0.7	5.6	±1.2	-15.1	±1.2	4.11	±0.1	0.1	±0.1

^a ΔT_m of FPf1T, FPf8T, FtryBT, F21T and FdxT (0.2 μM) were recorded in 10 mM lithium cacodylate (pH 7.2), 10 mM KCl, 90mM LiCl. PhenDC3 was tested at 0.5 μM, whereas CQ and MQ were tested at 1 μM. Error margins correspond to SD of three replicates. ^b n.d.: not determined.

, reveal that the selected ligands 1 produced ΔTm values ranging from 1.20 to 38.1 °C at a concentration of 2 µM.

The most effective ligands for stabilizing the four G-quadruplex sequences were compounds 1i, 1q, and 1r (Table 4). Among these nitrogen-containing heterocyclic ligands, the most potent on the three parasitic G-quadruplex sequences (FPf1T, FPf8T, FtrypBT) and the human sequence F21T was 2,4-bis{4-[(5-diisopropylaminopentyl)aminomethyl]phenyl}-7-(4-methoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidine (compound 1q), with ΔTm values ranging from 34.5 to 38.1 °C. Within each sub-series, the pyrrolo[2,3-d]pyrimidine-based compounds that demonstrated the strongest stabilization profiles were those bearing an aromatic ring at position 7 of the polyheterocyclic skeleton, such as a phenyl, 4-methoxyphenyl or benzyl substituents (derivatives 1e–t) in comparison with the 7-methyl substituted compounds 1a–d which exhibited moderated stabilization of the G-quadruplexes. Surprisingly, the 7-phenylethyl substituted pyrrolopyrimidine derivative 1u was found to have a low stabilization profile. Moreover, the 2,4-bis{4-[(5-methylhexyl)aminomethyl]phenyl}-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine analog 1l, whose structure contains no nitrogen atoms at the ends of the side chains, also exhibited a very low ΔT_m on the different G-quadruplex sequences; i.e., 1.2 to 4.0 °C.

Overall, the ligands 1e–t exhibited superior G-quadruplex stabilization compared to the reference ligand PhenDC3. FRET assays further confirmed that most of these compounds had binding affinity for the double-stranded DNA sequence.

4. Conclusions

In this study, we reported the design, synthesis, in vitro antiprotozoal activities, and cytotoxicity toward human cells of a novel series of 2,4-bis{4-[(dialkylaminoalkyl)aminomethyl]phenyl}-7-substituted-7H-pyrrolo[2,3-d]pyrimidine derivatives. These newly synthesized pyrrolo[2,3-d]pyrimidines were evaluated for their in vitro activity against Plasmodium falciparum (chloroquine-resistant W2 and chloroquine-sensitive 3D7 strains) and Trypanosoma brucei brucei. Cytotoxicity assays were also performed using the human HepG2 cell line to evaluate selectivity. Several of these compounds exhibited promising in vitro antiplasmodial activity, with IC₅₀ values ranging from 0.074 to 7.71 µM against both P. falciparum strains. Notably, the 2,4-bis{4-[(3-(4-methylpiperazin-1-yl)propyl)aminomethyl]phenyl}-7-methyl-7H-pyrrolo[2,3-d]pyrimidine derivative 1d emerged as the most potent antimalarial candidate, with a SI of 600.81 against the W2 strain. For the chloroquine-sensitive 3D7 strain, the most notable SI was observed for pyrrolo[2,3-d]pyrimidine 1c, with a value of approximately 123. Evaluation against Trypanosoma brucei brucei revealed IC₅₀ values from 0.38 to 7.05 µM, highlighting the potential of this novel series on several protozoan targets. Among these, derivative 1b was also identified as the most effective trypanocidal compound, with a SI of 39.52. A structure–activity relationship (SAR) analysis was conducted to better understand the molecular features contributing to antiparasitic activity. Given the growing interest in telomeric G-quadruplexes as potential drug targets, particularly in P. falciparum and Trypanosoma, we also explored whether these heterocyclic derivatives could stabilize such structures. FRET melting assays confirmed the binding of these nitrogen-containing heterocycles to parasitic telomeric G-quadruplexes. Interestingly, derivatives substituted at position 7 of the heterocyclic skeleton by a phenyl, a 4-methoxypheny or a benzyl ring showed the most pronounced stabilization effects. However, no direct correlation was observed between G-quadruplex binding and antiparasitic potency or selectivity, suggesting that G-quadruplex targeting is probably not the main mechanism of cytotoxicity.

Given their promising biological profiles, further investigation into their mechanisms of action is required—particularly with regard to their potential to inhibit β-hematin formation or disrupt apicoplast function. Overall, these new 2,4-bis{4-[(dialkylaminoalkyl)aminomethyl]phenyl}-7-substituted-7H-pyrrolo[2,3-d]pyrimidine derivatives represent a valuable scaffold for medicinal chemistry efforts in the search for new antiprotozoal agents.