Green sonosynthesis of novel pyrazolone-fused heterocycles catalyzed by a hydroxyl-functionalized ionic liquid: Discovery of a potent anticancer lead

2.1. Experimental Section

All chemical solvents and reagents were obtained from commercial suppliers and used as received without further purification. Reaction progress was monitored by thin-layer chromatography (TLC) throughout the synthetic procedures. All reactions were performed under ambient air atmosphere. Melting points were measured on an Electrothermal IA9100 apparatus and are reported without correction. Structural characterization of the compounds was carried out using standard spectroscopic techniques. Fourier transform infrared (FT-IR) spectra were recorded on a Shimadzu DR-8001 spectrometer with potassium bromide disks. Nuclear magnetic resonance (NMR) spectra (¹H and ¹³C) were acquired using a BRUKER 400 MHz spectrometer, while mass spectra were obtained with a JEOL JMS-600 instrument. The ionic liquid catalyst [DABCO-EtOH][AcO] was prepared according to established literature procedures [41]. For biological evaluation, the in vitro cytotoxic activities of compounds 5-9 were assessed against three human cancer cell lines: HepG-2 (hepatocellular carcinoma), MCF-7 (breast carcinoma), and A-549 (lung carcinoma) using the MTT assay method, with doxorubicin serving as the positive control. Complete physical and spectroscopic data for all synthesized compounds have been presented in Tables 1 and 2. The starting material, 1-phenyl-3,5-pyrazolidinedione (1), was synthesized following previously reported methods [42].

2.2. Synthetic procedures

3.1. Chemistry

In line with our objective to explore structure-activity relationships in bioactive heterocycles, we report the synthesis of novel pyrano[2,3-c]pyrazole analogues and their more complex fused derivatives. Our approach utilized 1-phenylpyrazolidine-3,5-dione (1) as a key building block, chosen for its two highly reactive sites: the nucleophilic N-2 nitrogen and the active methylene group at C-4. The key intermediate, 1-phenyl-4-(thiophen-2-ylmethylene)pyrazolidine-3,5-dione (4), was efficiently constructed via a Knoevenagel condensation between the active methylene group of 1 and thiophen-2-carbaldehyde. This critical C–C bond-forming reaction was catalyzed by the hydroxyl-functionalized ionic liquid [DABCO-EtOH][OAC] under ultrasonic irradiation (Scheme 1). This environmentally friendly synthetic approach successfully afforded the target product in an excellent yield of 92.65%. As outlined in (Scheme 2), we propose a catalytic mechanism in which the ionic liquid enhances the reaction by forming hydrogen bonds and stabilizing key transition states. The structure of the newly synthesized Knoevenagel adduct (4) was unambiguously verified through a combination of spectroscopic techniques and elemental analysis. The FT-IR spectrum displayed characteristic absorption bands corresponding to the key functional groups, notably an N-H stretch at 3235 cm^-1 and a C=O stretch at 1669 cm^-1. Further structural confirmation came from the ¹H NMR spectrum, which featured two diagnostically relevant singlets: one at δ 8.48 ppm for the olefinic proton (CH=C) and another at δ 10.23 ppm for the pyrazolone N-H proton. The ¹³C NMR spectrum provided conclusive evidence for the molecular framework, with distinct signals at δ 133.28 ppm for the exocyclic carbon (CH=C) and at δ 163.89 and 166.01 ppm for the two carbonyl carbons of the pyrazolidinedione ring. Together, this comprehensive analytical data firmly supports the assigned structure of compound 4, confirming its role as the key intermediate for further synthetic elaboration.

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Scheme 1.

Synthesis of compound (4) via [DABCO-EtOH][OAC]-catalyzed Knoevenagel condensation.

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Scheme 2.

Proposed catalytic mechanism of [DABCO-EtOH][OAC] in the synthesis of compound (4).

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Building upon the successfully synthesized Knoevenagel adduct (4), we proceeded to further functionalize the molecule via a Mannich reaction to introduce aminomethyl functionality, a modification known to often enhance biological activity and molecular diversity. Subjecting compound (4) to reaction conditions with formaldehyde and piperidine in ethanol under ultrasonic irradiation efficiently afforded the novel 1-phenyl-2-(piperidin-1-ylmethyl)-4-(thiophen-2-ylmethylene)pyrazolidine-3,5-dione (5), in an excellent yield of 90% (Scheme 3). The structure of the newly synthesized compound 5 was clearly confirmed using comprehensive spectroscopic analysis, with all data consistent with the proposed molecular framework. The ¹H NMR spectrum clearly indicated the successful introduction of the piperidinyl methyl unit, showing a set of multiplet signals between δ 1.59 and 1.66 ppm (6H) along with triplets at δ 2.21 and 2.37 ppm (2H each), which correspond to the five methylene protons of the piperidine ring. A key singlet at δ 4.43 ppm (2H) confirmed the presence of the –N–CH₂–N– bridge, serving as a diagnostic marker for the Mannich reaction. Further support came from the ¹³C NMR spectrum, which displayed signals at δ 26.55, 28.69, 43.77, and 60.14 ppm for the piperidinyl carbons, while the pivotal –N–CH₂–N– carbon appeared at δ 70.59 ppm. Mass spectrometry provided additional confirmation, showing a molecular ion peak [M]⁺ at m/z 367.47, matching the molecular formula C₂₀H₂₁N₃O₂S. The high relative abundance (98%) of this signal attests to the compound purity and stability. Together, these results firmly establish the structure of compound 5 and validate the effectiveness of the sonication-assisted synthetic route.

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Scheme 3.

Synthesis of compound (5).

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Building on the successful synthesis of compound 5, we proceeded to develop more building complex fused heterocyclic systems to evaluate their potential biological activity. Treatment of compound 5 with malononitrile in ethanol, catalyzed by [DABCO–EtOH][OAc] under ultrasonic irradiation, efficiently produced the novel amino carbonitrile derivative 6 (Scheme 4). This transformation follows a convergent multicomponent pathway, likely initiated by a Knoevenagel condensation and culminating in an intramolecular cyclization that annulates a pyran ring onto the pyrazolone core, yielding a pyrano[2,3-c]pyrazole system. The structure and purity of this key compound 6 were confirmed by TLC and detailed spectral analysis. The FT-IR spectrum confirmed successful cyclization through the disappearance of the exocyclic C=CH bond and the emergence of three diagnostic bands: broad absorptions at 3419 and 3197 cm^-1, corresponding to the asymmetric and symmetric stretches of the primary amino (NH₂) group, and a sharp peak at 2211 cm^-1 for the nitrile (C≡N) group. Further evidence came from the ¹H NMR spectrum, which displayed three key singlets: one at δ 4.17 ppm for the –N–CH₂–N– protons, a second at δ 3.78 ppm for the pyran ring methine proton (CH), and a third broad singlet at δ 6.14 ppm for the NH₂ group, which exchanged with D₂O. The ¹³C NMR spectrum provided definitive confirmation, with signals at δ 60.65 ppm (pyran CH), δ 70.59 ppm (N–CH₂–N), and δ 119.83 ppm (C≡N). Together, these data conclusively support the structure of 6 as 6-amino-3-oxo-1-phenyl-2-(piperidin-1-ylmethyl)-4-(thiophen-2-yl)-1,2,3,4-tetrahydropyrano[2,3-c]pyrazole-5-carbonitrile. The primary amine in 6 then served as a versatile handle for further structural elaboration. Sonication of 6 with chloroacetyl chloride smoothly afforded the chloroacetyl derivative 7 (Scheme 4) via straightforward N-acylation. This product, 7-(chloromethyl)-1-phenyl-2-(piperidin-1-ylmethyl)-4-(thiophen-2-yl)-1,2,4,6-tetrahydropyrazolo[4’,3’:5,6]pyrano[2,3-d] pyrimidine-3,5-dione (7), represents a significant increase in molecular complexity, featuring a chloromethyl substituent on a newly formed pyrimidine ring within a novel polycyclic framework. This chloromethyl group serves as a highly useful electrophilic site for subsequent nucleophilic substitution reactions, as demonstrated in the following synthetic steps.

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Scheme 4.

Synthesis of fused pyran and pyrimidine derivatives 6 and 7.

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The planned incorporation of the reactive chloromethyl group in compound 7 provided a versatile handle for further molecular diversification via nucleophilic substitution. Indeed, sonication of 7 with aniline efficiently yielded the secondary amine derivative (8), via a straightforward nucleophilic aromatic substitution reaction (Scheme 5). Spectral analysis provided clear confirmation of the compound 8 structure. The ¹H NMR spectrum offered definitive proof of the successful transformation, primarily through the disappearance of the –CH₂Cl singlet and the emergence of new aromatic signals accounting for five protons, which confirmed the introduction of the anilino moiety. Building on this result, we then performed a Mannich-type cyclization on compound 8 (Scheme 5), a step designed to enhance molecular complexity and further investigate the potential of this pharmacophore. Sonication with formaldehyde resulted in the facile formation of a seventh ring, yielding the novel polycyclic system 1,8-diphenyl-2-(piperidin-1-ylmethyl)-4-(thiophen-2-yl)-1,2,4,7,8,9 hexahydroimidazopyrazolopyranopyrimidinedione (9). This reaction proceeds via an intramolecular Mannich condensation reaction, where the formaldehyde bridge connects the secondary amine of the anilino group and the adjacent carbon, forming a new imidazole ring. The formation of this complex building was confirmed clearly through spectroscopic analysis. The FT-IR spectrum of compound 9 revealed a critical change: the disappearance of N–H stretching bands near 3307 cm^-1, consistent with the consumption of the secondary amine group during cyclization. Further evidence came from the ¹H NMR spectrum, where the N–H proton signal was absent, and two new distinctive singlets appeared at δ 3.67 and 3.78 ppm, each integrating for two protons. These signals correspond to the diastereotopic methylene protons of the newly formed imidazole ring, confirming the successful cyclization and final structure of compound 9. The successful preparation of these highly complex polycyclic frameworks 8 and 9 highlights the versatility of the original pyrazolone scaffold and underscores the efficiency of using sequential sonochemical reactions to build molecular diversity. With their multiple nitrogen atoms and varied ring systems, these final compounds represent particularly promising candidates for further investigation as potential anticancer agents.

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Scheme 5.

Intramolecular Mannich cyclization of derivative (8) to form polycyclic system (9).

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3.2. Cytotoxicity activities

The in vitro cytotoxic activities of the synthesized compounds (5–9) were evaluated against three human cancer cell lines: HepG-2 (liver carcinoma), MCF-7 (breast carcinoma), and A-549 (lung carcinoma), using the MTT assay. The cells were treated with a range of compound concentrations (1, 2, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, and 500 µg/mL) for 48 h. Following this incubation, the half-maximal inhibitory concentration (IC₅₀) values, expressed in µg/mL, were calculated from the resulting dose-response curves and have been summarized in Table 3. Our in vitro cytotoxicity results against HepG-2, MCF-7, and A-549 cell lines identified a compelling structure-activity relationship, demonstrating that strategic molecular modifications directly enhance biological potency. While the initial Mannich base derivative (5) exhibited only modest activity, the subsequent annulation of the pyran ring to form the amino carbonitrile (6) resulted in a dramatic enhancement in potency, reducing IC₅₀ values by approximately 5-6 fold across all cell lines. This suggests that the rigid, planar pyrano[2,3-c] pyrazole core significantly improves interaction with cellular targets. However, the most profound leap in efficacy was achieved with compound (8), which emerged as the standout candidate with exceptional, broad-spectrum cytotoxicity (IC₅₀ = 1.93 - 2.42 µg/mL), rivaling the potency of the standard drug doxorubicin. This remarkable activity can be directly attributed to its unique chemical architecture: the nucleophilic substitution of the chloromethyl group in 7 with aniline installed a flexible phenylaminomethyl side chain. This moiety likely enhances cellular penetration and provides critical pharmacophoric elements for optimal target binding, potentially through additional hydrogen bonding or π-π stacking interactions. The subsequent cyclization of 8 to form the imidazo-fused polycycle (9) slightly reduced potency, indicating that while the Mannich cyclization successfully constructs a complex scaffold, the specific conformational constraint or the loss of the secondary amine functionality in 9 may be less favorable for target engagement than the flexible aniline side chain in 8. Consequently, the structural evolution from the simple pyrazolone to the multi-ring system 8 demonstrates a masterful application of synthetic chemistry to develop a highly promising anticancer lead.