A novel pseudo-five-component synthesis, molecular docking analysis, ADMET and drug likeness studies of new 3,4-dihydro-2H-pyrrole derivatives as potent HIF-1α inhibitors

Abstract

New poly-substituted 3,4-dihydro-2H-pyrrole derivatives were synthesized via a novel one-pot pseudo-five-component reaction in fairly high yields (68–85%). The structures of the products were elucidated from their FT-IR, ¹H NMR, ¹³C NMR and mass spectral data. A NOESY experiment was used to verify the stereochemistry of the single stereoisomer resolved from the reaction mixture. Insights into vibrations, geometry and energetics of the products were obtained using DFT calculations. Given the well-established role of the HIF-1α protein in breast cancer progression, its active site was selected for molecular docking studies. The products demonstrated favorable binding scores (ranging from −9.4 to −10.5 kcal mol⁻¹) and specific interactions with amino acid residues within the active pocket of HIF-1α. Drug-likeness predictions reveal that the products adhere to Lipinski's rules and none of them trigger pan-assay interference, suggesting their potential as candidates for further development. In silico ADMET profiling for the products exhibited promising properties across critical parameters. The synthesized products are promising candidates for subsequent in vitro evaluation as potential HIF-1α inhibitors.

New poly-substituted 3,4-dihydro-2H-pyrrole derivatives were synthesized via a novel one-pot pseudo-five-component reaction in fairly high yields (68–85%).

Introduction

Cancer remains a global health challenge, with breast cancer accounting for approximately one in every eight cancer diagnoses and an estimated 2.3 million new cases in women annually as of 2022.¹ Malignancies such as these constitute a leading cause of morbidity worldwide, representing a significant portion of global cancer cases and mortality. The prevalence of many cancer types continues to rise, particularly in transitioning nations.^2,3 A critical factor in the progression and therapy resistance of solid tumours is tumour hypoxia, which activates Hypoxia-Inducible Factor-1α (HIF-1α). HIF-1α is a master transcriptional regulator that drives angiogenesis, metastasis, and cell survival, making it a well-validated therapeutic target in oncology, with significant implications for breast, renal, liver, and pancreatic cancers, among others.⁴ Inhibiting HIF-1α offers a promising strategy to disrupt tumour growth and improve treatment outcomes across various cancer types. Consequently, there is a pressing need to discover novel compounds that can selectively target HIF-1α and inhibit cancer cell proliferation while sparing healthy cells.^5,6

Pyrrole and 2H-pyrrole rings are commonly occurring frameworks present in numerous natural products and small molecule chemotherapeutics, exhibiting a wide range of pharmacological properties.^7,8 These structures are highly valued by synthetic chemists due to their broad utility in medicinal chemistry and materials science. They have been recognized as natural products⁹1 and biologically active compounds¹⁰2, making them privileged building blocks for the development of pharmaceutical products (Fig. 1).^11–13 Compounds featuring a 2H-pyrrole core have demonstrated a diverse range of biological activities (Fig. 1), including antitumor 3, anti-infective 4, antimicrobial 5, immunomodulatory 6, antihypertensive 7, and antiviral 8 properties.^14–19

Fig. 1. Biologically active and natural 3,4-dihydro-2H-pyrrole derivatives.

Even simple 3,4-dihydro-2H-pyrroles are significant compounds due to their excellent flavouring and odorant characteristics, such as a cooked-rice flavour 9 (Fig. 2).^20,21 In addition to this, they also exhibit potential antiviral²²10 and antifungal 11 activities (Fig. 2).²³ They have also been identified as structural motifs in the bioactive alkaloid²⁴12 and in the optically active tryptophan analogue²⁵13. Taken together, the diverse biological and sensory properties of the 3,4-dihydro-2H-pyrrole derivatives, illustrated in Fig. 2, highlight the synthetic and medicinal values of this scaffold and provide a strong impetus for the development of new analogues. 3,4-Dihydro-2H-pyrroles compounds can be strategically designed to specifically target and interact with pathways and molecular targets that are associated with cancer. These compounds have the potential to inhibit the growth of cancer cells and induce apoptosis by selectively modulating these pathways involved in tumour growth, development, and survival.¹⁷

Fig. 2. Simple 3,4-dihydro-2H-pyrroles.

In recent years, there has been limited research focused on the synthesis of 3,4-dihydro-2H-pyrrole ring system. Song, Jiao and co-workers have developed an intramolecular C(sp³)–H bond amination of aralkyl azides 14 and employed it in the synthesis of 3,4-dihydro-2H-pyrroles. It is believed that the reaction goes through the intermediacy of a benzylium cation forms through oxidation of the aralkyl azide.²⁶ Cui and co-workers conducted the intermolecular coupling of β-amino alcohols 15 with allylic alcohols 16 under catalysis of Fe-PNP complex to yield 3,4-dihydro-2H-pyrroles. Fe-PNP is assumed to catalyze the dehydrogenation of the two substrates, which subsequently undergo condensation, followed by cyclization of the resulting 2H-pyrrolenine intermediate.²⁷ Cao successfully synthesized 3,4-dihydro-2H-pyrroles through tandem Heck reactions of γ,δ-unsaturated oxime esters 17 with cyclopropanol 18.²⁸ Song et al. reported a novel [2 + 2 + 1] dimerization annulation of styrenes 19 and trimethylsilyl azide 20via Schmidt rearrangement, providing a straightforward electrochemical access to 3,4-dihydro-2H-pyrroles.²⁹ Trivedi and co-workers based their route to 3,4-dihydro-2H-pyrroles on the reaction of β-aminoalcohols 21 with ketones 22.³⁰ Mechanistically, this reaction involves imine formation, Ir-mediated dehydrogenation, intramolecular cyclodehydration, and controlled hydrogen transfer steps. Yoo successfully synthesized 3,4-dihydro-2H-pyrroles through the 1,3-dipolar cycloaddition reaction of acrylamides 23 with the nitrile ylide generated from its precursor 24.³¹ Selander et al. envisaged oxime esters as electrophilic partners for iron catalysis. Reductive cleavage of the oxime N–O bond by iron generates a key iminyl radical from γ,δ-unsaturated oxime 25, which through an intramolecular C–N bond formation followed by coupling of the resulting cyclic radical with a silyl enol ether 26 gives the desired 3,4-dihydro-2H-pyrroles.³² Bergner and co-workers based their route to 3,5-disubstituted-3,4-dihydro-2H-pyrrole-2-carbonitriles on one-pot reaction of α,β-unsaturated carbonyl compounds 27 with aminoacetonitrile hydrochloride 28.³³ This synthetic method is indeed a development of the protocol pioneered by Katritzky et al. by studying the reaction of enones with aminomalonic acid ester hydrochloride.³⁴ Investigations of this group showed that 3,4-dihydro-2H-pyrroles are reasonably stable but upon oxidation to 2H-pyrroles thermally rearrange to 1H-pyrroles. Menino et al. designed a sequence of reactions starting from simple materials to achieve 5-amino-3,4-dihydro-2H-pyrrole derivatives. In the key step of this synthetic route, the acrylonitrile derivative 29 is added to benzophenone-Schiff base of a glycinate ester 30 in a Michael addition manner. Acid-catalyzed hydrolytic deprotection of the amino group of the resulting seco adduct 31 followed by its addition to the nitrile group yields the 5-amino-3,4-dihydro-2H-pyrroles³⁵ (Scheme 1).

Scheme 1. Examples of the previously reported synthesis of 3,4-dihydro-2H-pyrroles.

Most of the previously developed routes require complex metal catalysts, elaborately prepared starting materials, long reaction times and involve multi-step reactions. Thus, it is worth addressing these disadvantages by carrying out the synthesis in a multicomponent method. Multicomponent reactions hold a unique place in modern synthetic strategy due to their ability to efficiently produce diverse and complex products from three or more simple starting materials.³⁶ Although typically conducted in one pot, these reactions may also proceed over multiple steps. Domino reactions represent a particularly intriguing subclass, generating products directly in a single step.³⁷ These features make multicomponent reactions, and domino reactions in particular, practically straightforward, enabling rapid and simple access to diverse product libraries by varying the starting materials. These typically atom–economic reactions fall under the fold of green chemistry, as they eliminate the need for isolating and purifying intermediates, thereby reducing pollution.³⁸

In this background, we explain here, for the first time, a novel pseudo-five-component route (5CR) to synthesizing some new fully substituted 3,4-dihydro-2H-pyrroles. Our design was inspired by observing that, under specific conditions, the four-component synthesis of nicotinonitriles 32 (following the protocol of Silva and co-workers)³⁹ can be accompanied by the formation of 3,4-dihydro-2H-pyrroles 33via a pseudo-five-component reaction. By elucidating their structure, formation mechanism, and key yield-influencing factors, we present here an efficient domino-synthesis of 3,4-dihydro-2H-pyrroles (Scheme 2). Additionally, we explored the potential medicinal properties of these compounds through molecular docking studies.

Scheme 2. The previously reported 4CR produces nicotinonitriles 32 and the pseudo-5CR, presented here, yields 3,4-dihydro-2H-pyrroles (33).

Results and discussion

Synthesis and characterization

At the outset of this investigation, the reaction of dehydroacetic acid, 3-nitrobenzaldehyde (Ia), ammonium acetate (II), and ethyl cyanoacetate (34) was selected as the model. In order to find the optimal conditions, the effect of various parameters on efficiency of the trial reaction in terms of yield and reaction time was explored. As Table 1 shows, none or moderate yields of the model product 33a are obtained in the absence of any catalyst in different solvents (entries 1–6). Following these tests, ethanol (absolute) was determined as the best solvent (entry 4) after AcOH (entry 7) for running the synthesis of 33a. The low yield observed in EtOH/H₂O (1 : 1) is attributed to the poor solubility of dehydroacetic acid in the aqueous mixture, which limits its availability for the reaction. In contrast, EtOH provides complete dissolution of all starting materials, leading to a significantly higher yield (50%, entry 4).

Table 1. Optimization of the reaction conditions for the synthesis of 33a.

Ent.	Solvent a	Catalyst (mol%)	T (°C)	Time (h)	Yieldc (%)
1	H2O	—	25	24	—
2	H2O	—	70	24	—
3	EtOH\H2O b	—	70	24	19
4	EtOH	—	70	24	50
5	Acetone	—	70	24	13
6	DMF	—	70	24	32
7	AcOH	—	70	2	77
8	EtOH	Et3N (100)	70	24	—
9	EtOH	Piperidine (100)	70	24	—
10	EtOH	DABCO (100)	70	24	—
11	EtOH	Na2HPO4 (100)	70	24	—
12	EtOH	[BMIm]BF4 (220)	70	24	58
13	EtOH	Citric acid (100)	70	6	42
14	EtOH	[BMIm]HSO4 (210)	70	6	37
15	EtOH	p-TSA·H2O (100)	70	6	32
16	EtOH	H2SO4 (100)	70	2	Trace
17	EtOH	AcOH (100)	70	2	73
18	EtOH	AcOH (200)	70	2	85
19	EtOH	AcOH (200)	80	2	85
20	EtOH	AcOH (200)	60	2	81

^a5 mL per 1 mmol of dehydroacetic acid.

^bA 1/1 v/v mixture.

^cYields of isolated product.

The supremacy of AcOH reflects its dual role as solvent and promoter. Subsequent screening experiments with organic and inorganic bases (entries 8–11) did not produce 33a, confirming the requirement for acid catalysis in this reaction. As a matter of fact, a variety of acids with moderate strengths showed reasonable to good efficiencies in catalyzing the synthesis (12–15). However, strong acids are not suitable, because of forming multiple byproducts (entry 16). Remarkably, the reaction proceeds cleanly in ethanolic solution of AcOH, delivering the 3,4-dihydro-2H-pyrrole 33a in fairly high yield within a short reaction time (entry 18). The mixed EtOH/AcOH system was selected over pure AcOH because it provides a milder pH (3.5–4 vs. 2.4), prevents the slight formation of side product 32 and gives the highest yield (85%, entry 18).

This work also offers good atom economy, since most atoms from the starting materials are efficiently incorporated into the final product. Moreover, all six derivatives precipitated directly from the reaction mixture and were purified simply by recrystallization from ethanol, eliminating the need for column chromatography.

Table 1 is reflecting the importance of pH and type of acid on efficiency of the reaction. Bases and strong acids inhibit or severely impede the formation of 33a. Under such unfavourable conditions, the reaction predominantly yields 32a alongside numerous byproducts. It is also noteworthy that employing acidic ammonia sources, such as NH₄NO₃ and NH₄Cl, instead of NH₄OAc resulted in negligible yields of the product 33a.

After optimizing the reaction conditions, we set out to explore the substrate scope of the method by using different aldehydes and malononitrile instead of ethyl cyanoacetate. According to Table 2, malononitrile and ethyl cyanoacetate exhibit nearly equal reactivity in the reaction, while electron-deficient aldehydes (33a–33c) produce higher yields within shorter reaction times.

Table 2. Synthesis of the products 33a–33f.

Prod.a	Ar	Y	Yieldb (%)	Reaction time (h)
33a	3-NO2·C6H4	CO2Et	85	2
33b	3-Cl·C6H4	CO2Et	70	2
33c	3-Br·C6H4	CN	75	2
33d	4-Et·C6H4	CN	68	3
33e	4-Me·C6H4	CN	71	3
33f	Ph	CN	69	3

^aReaction conditions: dehydroacetic acid (1 mmol), arylaldehyde (2 mmol), malononitrile or ethyl cyanoacetate (1 mmol), NH₄OAc (3 mmol), and AcOH (2 drops, 200 mol%) in absolute EtOH (5 mL) at 70 °C.

^bYields of the isolated products.

Aromatic aldehydes bearing strong electron-donating groups at the para position—such as –OCH₂Ph, –OMe, –F, –OH, –Cl, and –NMe₂—consistently failed to produce the corresponding 3,4-dihydro-2H-pyrroles under the model reaction conditions. In contrast, benzaldehyde and the arylaldehydes with electron-withdrawing groups (–NO₂, –Cl, –Br) at the meta position or weak electron-donating alkyl substituents (–Et, –Me) gave the target products in fairly high yields. This clear electronic trend provides valuable insight into the reaction mechanism.

The ¹H NMR spectrum of 33a displays peaks at appropriate chemical shifts corresponding to its distinct protons (vide infra). The singlet peak with the integral of one proton in the down-field region of this spectrum at δ 15.44 is assigned to the phenolic proton of the molecule. The large chemical shift of this proton can be attributed to the synergy of intramolecular H-bonding, ring current effect, and conjugation involving the OH lone pair and the lactonic carbonyl group (Fig. 3a).

Fig. 3. (a) The preferred conformation of 33a and its ring current. (b) The spatial dipole–dipole correlations displayed by the NOESY spectrum of 33a.

In aromatic region of this spectrum, protons from two 3-nitrophenyl rings are observed at distinct chemical shifts, ranging from δ 8.43 to 7.80. Downfield in this region are observed a multiplet at δ 8.43–8.40, a doublet of doublets at δ 8.31(J 7.9 and 1.5 Hz) and a broad singlet at δ 8.25, each with the integral of one proton, attributing to 4-H, 6-H and 2-H of one 3-nitrophenyl ring, respectively. This ring is proposed to be the substituent at the 2-position of the dihydropyrrole ring. A triplet signal with the integral of one proton and J value of 8.0 Hz in up-field of the aromatic region at δ 7.82 is attributed to 5-H of the other 3-nitrophenyl ring. This spectrum also shows a broad singlet at δ 8.18, a doublet at δ 7.97 (J 7.8 Hz), and a multiplet at δ 7.88–7.87, each integrating to one proton signal. Two characteristic singlet peaks of this spectrum at δ 5.98 and δ 5.89 are ascribed to 5-H proton of pyran-2-one ring and 2-H proton of dihydropyrrole ring, respectively. Other characteristic peaks of the molecule are observed in the aliphatic region of the spectrum. Notably, the dd peak at δ 4.42, with J values of 11.8 and 5.0 Hz, is assigned to the 4-H proton of the dihydropyrrole ring. The dd peak at δ 4.08 with J values of 19.9 and 5.0 Hz is attributed to CH_A proton of the two diastereotopic CH₂ protons in the benzyl moiety. The CH_B proton of this group has resonated at δ 3.53 as a dd peak with J values of 11.4 and 19.9 Hz. The diastereotopic CH₂ protons of the ester group appear as a multiplet in the δ 4.01–3.92 range. Further evidences in support of the elucidated structure were obtained from ¹³C NMR spectrum of 33a, where all carbon signals appeared in the expected chemical shift range. The two lines in downfield of this spectrum at δ 184.3 and 173.8 are the signals of carbons at C-4 of pyran ring and the esteric carbonyl group. There are additional downfield signals at δ 164.2 and 164.1, arising from the resonances of lactone and imine carbons. The signals at δ 147.9 and 147.8 are due to the aromatic carbons bearing the nitro substituents. Up-field side of the aromatic region has displayed lines at δ 106.4 and 95.3, corresponding to signals of C-5 and C-4 carbons of the pyran ring, along with a signal at δ 114.1 due to carbon of the cyano group. Of importance to notice are the 7 lines have been appeared in the aliphatic region of the spectrum at δ 63.8, 60.7, 55.6, 42.2, 33.2, 19.3 and 13.3. Among these lines, those at δ 63.8, 55.6 and 42.2 are readily assigned to C-2, C-3 and C-4 of the dihydropyrrole ring and that at δ 33.2 is attributed to aliphatic carbon of the 3-nitrobenzyl moiety. The number of aliphatic signals suggests that two originate from the carbonyl groups of the two 3-nitrobenzaldehyde molecules.

To corroborate the proposed 3,4-dihydro-2H-pyrrole structure and gain insights into the spatial arrangement of its substituents, the NOESY spectrum of compound 33a was acquired in DMSO-d₆ (SI Fig. S1). As summarized in Fig. 3b, the NOESY spectrum of compound 33a exhibits cross-peaks between the 2-H and 4-H protons, as well as between the 6-H and 7-H protons. These through-space dipole–dipole interactions indicate a cis–cis configuration for the 3-nitrophenyl, 3-nitrobenzyl, and ester substituents on the dihydropyrrole ring.

The reaction appears to proceed via two parallel Knoevenagel condensations of the arylaldehyde (I) with dehydroacetic acid 34 and the cyanomethylene component 35, yielding intermediates 36 and 37. A conjugate addition of these intermediates forms adduct 38, which then undergoes an exo-trig cyclization to form product 33. This cyclization step is an intramolecular Michael reaction in which the aryl ring acts as the electron-accepting moiety, followed by rearomatization.

The proposed mechanism is supported by the observation that the pre-prepared intermediate 36 alternatively affords product 33 upon reaction with an arylaldehyde, ammonium acetate, and 35 (Fig. 4). When electron-rich aldehydes such as 2,4-dimethoxybenzaldehyde were employed, only the corresponding intermediate 37 was isolated from the reaction mixture, indicating that the formation of 38 is reversible and proceeds to the final product only when the aryl ring is electron-deficient. This result strongly supports the electron-accepting role of the aromatic ring in directing the intramolecular Michael addition. Presumably, cyclization of 38 would face a higher energy barrier if it proceeded via an endo-trig pathway leading to annulation of a six-membered tetrahydropyridine ring, likely due to the E-configuration of the alkene moiety in 38. Moreover, formation of a tetrahydropyridine ring may be disfavoured by stabilization of the imine π-system through a ring current interaction with the pyran moiety (Fig. 3a).

Fig. 4. The mechanism proposed for the reaction.

Density functional theory calculations (DFT)

The electronic structure and physicochemical properties of the synthesized compounds 33a–33f were investigated using Density Functional Theory (DFT) calculations at the B3LYP/6-311++G(d,p) level to provide insights into their chemical profiles. The fully optimized geometries, confirmed as local minima by the absence of imaginary frequencies, are provided in SI Fig. S2, with corresponding energies, dipole moments, and polarizabilities listed in SI Table S1. Analysis of the frontier molecular orbitals (FMOs) offers a quantitative perspective on the electronic characteristics of the series (SI Table S2 and Fig. S3). Notably, compound 33b exhibits the narrowest HOMO–LUMO energy gap (3.057 eV), indicating a propensity for higher chemical reactivity and kinetic softness within the series, which may facilitate interactions with biological targets. In contrast, compound 33a shows the highest calculated electronegativity (χ = 7.37674), suggesting a pronounced electrophilic character. To validate the computational model and the assigned molecular structures, experimental infrared vibrational frequencies were compared with scaled DFT-calculated values. A strong linear correlation was observed for most vibrational modes (SI Table S3 and Fig. S4), confirming the reliability of the optimized structures. A notable deviation was observed for high energy stretches such as the O–H vibration in 33a (experimental: 3434 cm⁻¹; calculated: 3819 cm⁻¹), a common discrepancy attributed to the anharmonicity of such bonds and intermolecular hydrogen bonding in the solid state. This comprehensive computational study not only validates the structure of the products but also provides a foundational electronic profile of these novel 3,4-dihydro-2H-pyrroles.

Molecular docking studies

Therapeutic targeting of hypoxia-inducible factor-1α (HIF-1α) represents a promising strategy for diseases including breast cancer. To evaluate the potential of our novel synthesized 3,4-dihydro-2H-pyrroles 33 as HIF-1α inhibitors, we conducted molecular docking studies to simulate their binding interactions. For these simulations, the high resolution (2.6 Å) crystal structure of the human HIF-1α in complex with an inhibitor (PDB ID: 3KCX) was selected as a biologically relevant model (SI Fig. S5).⁴⁰ Prior to docking, heteroatoms, crystallographic water molecules, and the native ligand (clioquinol) were removed. The binding site was defined based on the location of the co-crystallized ligand and further characterized using the CASTp server. This analysis identified a major binding pocket in chain A with a volume of 171.3 ų and a surface area of 328.7 Ų, lined with key residues including ILE281, ARG-238, PHE-207, HIS-279, and ASP-201. The docking grid was centered on the geometric centroid of this cavity (coordinates: X = 22, Y = 34, Z = 10) with dimensions of 20 × 31 × 10 Å, ensuring complete coverage of the binding site. The docking parameters were benchmarked to ensure the predicted binding poses aligned closely with the known crystallographic orientation of ligands in this site. Molecular docking of the synthesized derivatives was performed using AutoDock Vina.⁴¹ All compounds showed favorable predicted binding affinities, ranging from −9.4 to −10.5 kcal mol⁻¹, with compound 33e exhibiting the most favorable score (−10.5 kcal mol⁻¹) (SI Table S4). The lowest-energy conformation of 33e was optimized using Density Functional Theory (DFT) at the B3LYP/6-311++G(d,p) level prior to detailed docking analysis. The results suggest that 33e engages in extensive interactions within the binding pocket, forming two conventional hydrogen bonds and multiple hydrophobic, pi-alkyl, and alkyl interactions with residues including ASP201, ILE281, PRO197, LEU188, and PRO221. Additional van der Waals contacts were observed with residues such as PHE207, HIS279, ARG238, and GLN239 within the pocket. The specific interacting residues are listed in SI Fig. S6, and a detailed 2D interaction diagram is presented in Fig. 5 (for further see Fig. S6–S11). The amino acid residues involved in bonding and non-bonding interactions with 33e include PHE207, HIS279, TRP296, TYR93, THR196, GLN239, GLN147, PHE100, ARG238, HIS199, ASP201, TYR102, ILE281, PRO197, LEU188 and LEU186. The predicted binding mode of 33e shows strong complementarity with the computationally identified active site and is consistent with the general binding orientation observed for known ligands of this target. These in silico results suggest a potential role for these derivatives in modulating HIF-1α activity. This promising preliminary computational analysis inspires future experimental investigation into biological evaluation of compounds 33.

Fig. 5. Binding interactions of compound 33e within the active pocket of HIF-1α protein.

Drug-likeness prediction

The concept of drug likeness characterizes the similarity of novel compounds to known drugs by evaluating a complex interplay of molecular and structural properties. Key properties influencing this assessment include hydrophobicity, electronic distribution, hydrogen bonding capacity, molecular weight, pharmacophoric features, bioavailability, reactivity, toxicity, and metabolic stability. Lipinski's rule of five is a standard method for predicting oral drug potential based on solubility and permeability. Compounds violating more than one criterion of this rule often exhibit poor absorption or permeation. Analysis of the products 33a–33f using the SwissADME web serve^42,43 revealed that all compounds except 33a, 33b, and 33c adhere to Lipinski's rule, as their parameter values fall within acceptable ranges, suggesting favorable absorption potential (Table 3). Furthermore, all the synthesized compounds complied with the Pfizer rule (also known as the 3/75 rule), which suggests a lower developmental attrition risk for molecules with fewer than three rings, a molecular weight ≤ 600, and a calculated log P ≤7.5. These compounds occupy a desirable region in physicochemical space, indicating their suitability as lead compounds. Pharmacokinetic evaluations predicted that the tested compounds 33a–33f would be well absorbed via the gastrointestinal (GI) tract following oral administration, although P-glycoprotein (P-gp) efflux might contribute to the elimination of 33d, 33e, and 33f. Within medicinal chemistry, Pan-Assay Interference Structure (PAINS) filters are employed to identify substructures associated with instability, reactivity, or toxicity.^44,45 Significantly, none of the compounds 33a–33f triggered PAINS alerts, providing further support for their potential as viable drug candidates. The bioavailability score estimates how well a compound is absorbed when administered orally. As shown in Table 3, only 33a is predicted to have low oral bioavailability, whereas 33b–33f are forecasted as promising oral drug candidates. The Synthetic Accessibility (SA) score gauges the practical feasibility of synthesizing drug-like molecules. All the synthesized compounds demonstrated favorable SA scores, indicating structures amenable to straightforward synthesis (Table 3).

Table 3. Physicochemical, pharmacokinetics, and medicinal chemistry properties of the compounds 33a–33f^a.

Compd	MW (g mol−1)	HBA	HBD	TPSA (Å2)	Consensus log Po/w*	MR	GI absorption	BBB permeant	P-gp substrate	Lipinski	Pfizer	PAINS (alert)	Bioavailability score	Synthetic accessibility score
33a	546.48	11	1	204.53	4.06	147.68	Low	No	No	No	Yes	0	0.17	5.20
33b	525.38	7	1	112.89	3.21	140.05	High	No	No	No	Yes	0	0.55	4.98
33c	567.23	6	1	110.38	3.17	134.28	High	No	No	No	Yes	0	0.55	4.78
33d	465.54	6	1	110.38	1.79	138.43	High	No	Yes	Yes	Yes	0	0.55	4.74
33e	437.49	6	1	110.38	1.23	128.82	High	No	Yes	Yes	Yes	0	0.55	4.35
33f	409.44	6	1	110.38	0.90	118.88	High	No	Yes	Yes	Yes	0	0.55	4.27

^aMW: molecular weight; HBA: no of H-bond acceptors; HBD: no of H-bond donors; MR: molar refractivity; TPSA: topological polar surface area; GI: gastrointestinal; BBB: blood–brain barrier; P-gp: P glycoprotein.

In silico predicted ADMET properties

The development of novel therapeutics relies critically on robust pharmacological and toxicological profiling. Such knowledge streamlines development timelines and enhances the likelihood of success. Key indicators used to characterize drug candidates are the ADMET properties (Absorption, Distribution, Metabolism, Excretion, and Toxicity). For the synthesized derivatives 33a–33f, these properties were evaluated using the ADMET Lab 2.0 platform.⁴⁶ Intestinal absorption potential was assessed using established cellular models. CaCo-2 cells, derived from human colon epithelium, are a standard model for predicting human gut uptake. Madin–Darby Canine Kidney (MDCK) cells offer a complementary model for rapid permeability screening.⁴⁷ The predicted permeability values across both models were highly favorable for all compounds (Table 4), indicating a strong potential for efficient intestinal absorption and suggesting they are candidates for oral administration. The predicted human intestinal absorption (HIA) was high for most derivatives and further supports this profile. Analysis of distribution properties revealed that all compounds exhibit low predicted blood–brain barrier (BBB) penetration (CBrain/CBlood < 1).⁴⁸ This is a desirable characteristic for non-CNS targeted anticancer agents, as it minimizes the potential for neurotoxic side effects. The volume of distribution (VD) values were relatively low (0.58–1.26 L kg⁻¹), suggesting these compounds are primarily retained within the systemic circulation rather than extensively distributing into tissues. Plasma protein binding (PPB) was predicted to be high (>94% for all compounds). While high PPB can influence a drug's half-life and free concentration, it is a common feature of many successful drugs and will be a focus of subsequent optimization. Notably, in silico analysis predicted a lack of interaction with P-glycoprotein (P-gp) for all compounds, meaning they are not anticipated to be substrates or inhibitors of this key efflux transporter. This is a positive early indicator, as it suggests a lower risk of transporter-mediated drug resistance or interactions. Initial toxicological screening did not raise major structural alerts (PAINS). In summary, the comprehensive in silico ADMET profiling of compounds 33a–33f reveals promising drug-like properties, including favorable permeability, high predicted oral absorption, low BBB penetration, and no predicted P-gp liability. These computational results position these novel 3,4-dihydro-2H-pyrroles as promising preclinical candidates. It is emphasized that these are predictive data, and the compounds should be regarded as pro-drug candidates requiring further experimental validation.

ADMET profiles of the compounds 33a–33f^a.

Comp.	CaCo-2 permeability	MDCK	PGP-inhibitor	P-glycoprotein substrate (PGP substrate)	Human intestinal absorption (HIA)	Plasma protein binding (PPB)	Volume of distribution (VD)	Blood brain barrier (BBB) penetration (c. brain/c. blood)
33a	−4.696	0.00021	−0.004	0.222	−0.009	97.25%	1.263	+ +0.83
33b	−4.758	0.00018	0.003	0.051	−0.005	95.36%	0.888	−0.47
33c	−4.757	0.00022	0.001	0.042	0.272	96.11%	0.789	−0.44
33d	−4.775	0.00016	0.018	0.061	−0.005	93.36%	0.993	+0.52
33e	−4.781	0.00017	0.039	0.034	−0.005	92.51%	0.583	+0.55
33f	−4.816	0.00017	0.011	0.031	−0.006	91.24%	0.667	+ +0.69

^ahERG toxicity is due to cardiac side effects. H-HT refers to human hepatotoxicity.

Comp.	AMES toxicity	Carcinogenicity	Eye corrosion	Eye irritation	Toxicity
					hERG	H-HT	LD50	Respiratory toxicity
33a	+0.534	+0.472	−0.013	−0.475	0.085	−0.408	409.843	+ + +0.979
33b	−0.011	−0.186	0.004	0.209	0.03	0.19	452.973	+ + +0.983
33c	0.006	0.227	+0.083	0.204	0.004	0.095	522.437	+ + +0.995
33d	0.019	0.157	0.034	0.101	0.008	0.153	588.513	+ + +0.994
33e	0.012	0.129	0.004	0.100	0.003	0.158	602.120	+ + +0.995
33f	0.015	0.232	0.003	0.112	0.003	0.125	555.963	+ + +0.995

Metabolism											Elimination
	CYP2C19 inhibitor	CYP2C19 substrate	CYP2C9 inhibitor	CYP2C9 substrate	CYP2D6 inhibitor	CYP2D6 substrate	CYP3A4 inhibitor	CYP3A4 substrate	CYP1A2 inhibitor	CYP1A2 substrate	T1/2	CL
33a	0.147	+ +0.769	+ +0.944	+0.646	++0.942	++0.881	−0.121	−0.227	+ +0.807	+ +0.715	0.530	2.666
33b	−0.365	+ +0.81	+ +0.903	+ +0.128	++0.958	++0.733	++0.871	−0.108	+ +0.664	+ +0.849	0.428	3.919
33c	−0.311	−0.497	+ +0.784	+ +0.069	++0.928	++0.765	++0.744	0.099	−0.358	+ +0.585	0.537	4.621
33d	0.168	+ +0.777	+ +0.765	−0.08	++0.864	++0.877	−0.468	0.094	−0.375	+ +0.849	0.434	3.756
33e	+0.557	−0.454	+ +0.777	−0.109	++0.862	++0.863	−0.419	−0.242	−0.472	+ +0.883	0.535	4.648
33f	+0.564	−0.479	+ +0.791	−0.069	++0.931	++0.844	−0.515	0.074	−0.405	+ +0.666	0.512	4.436

The in silico toxicity profile of the 3,4-dihydro-2H-pyrrole derivatives 33a–33f reveals a promising overall safety prediction, supporting their further investigation as candidate molecules. Computational analysis indicates several favorable safety aspects across the series. All compounds are predicted to have a very low potential for respiratory toxicity. Importantly, they also shown eligible inhibition of the hERG channel, a critical indicator for a low risk of drug-induced cardiac arrhythmia. Furthermore, predictions for eye irritation and corrosion are predominantly negative, suggesting a favorable ocular safety profile. Acute oral toxicity (LD₅₀) values fall within a moderate and acceptable range for early-stage compounds. Regarding genotoxicity predictions, the interpretation requires careful consideration of the model's output format. Compounds 33b–33f display negative scores for AMES and carcinogenicity in this assessment. Compound 33a presents a distinct profile, showing positive scores in these endpoints which warrant specific investigation. It is crucial to emphasize that these computational results are probabilistic and serve primarily to prioritize compounds for definitive in vitro assays, such as the experimental AMES test, which is a standard and necessary step in development. In conclusion, the computational toxicity assessment highlights a robust preliminary safety profile for this scaffold, particularly concerning cardiac, respiratory, and ocular endpoints (Table 4).

The metabolic fate and elimination kinetics of the novel 3,4-dihydro-2H-pyrrole derivatives 33a–33f were evaluated in silico to obtain an initial pharmacokinetic profile. The analysis focused on interactions with major cytochrome P450 (CYP) enzymes, which govern the metabolism of most drugs. The computational profile indicates that the core scaffold is predominantly processed by the CYP2C9 and CYP3A4 isoforms, as evidenced by consistently high substrate scores across the series. This is a common and well-characterized elimination route for oral drugs, facilitating the prediction and management of potential drug–drug interactions. A particularly favorable feature is the lack of strong predicted inhibition of CYP3A4 for most derivatives, which reduces concerns over interactions with the many therapeutics metabolized by this enzyme. The predicted half-life (T1/2) values are short and uniform (∼0.4–0.5 h), suggesting rapid and predictable systemic clearance. Coupled with the estimated clearance (CL) rates, this profile indicates a low risk of drug accumulation. Collectively, these in silico results reveal a manageable and promising metabolic profile for the new 3,4-dihydro-2H-pyrroles. The data provide a strong rationale for prioritizing the lead compounds, especially 33e and 33f, for the essential next phase of experimental validation. In vitro metabolic stability assays in human liver microsomes and dedicated CYP inhibition studies are now warranted to confirm these predictions and to guide the final selection of candidates for further development (Table 4).

Experimental and computational methods

All the chemicals including dehydroacetic acid (3-acetyl-4-hydroxy-6-methyl-2H-pyran-2-one) were purchased from Merck and Aldrich chemical companies and used without further purification. FT-IR spectra were recorded on a Shimadzu 8400 FT-IR spectrometer in KBr pellets. ¹H and ¹³C NMR spectra were obtained by using Bruker DRX-400 and DRX-300 AVANCE spectrometers. The chemical shifts were measured in ppm relative to TMS as the internal standard using the signal of the deuterated solvent. The Mass spectra were recorded on an Agilent Mass spectrometer (Agilent Technology 5973) operating at 70 eV. Melting points were measured using a Buchi melting point B-540 instrument in open capillaries and are uncorrected. In silico ADMET (Adsorption, Distribution, Metabolism, Excretion, and Toxicity) analysis was conducted by utilizing an online available server ADMET Lab 2.0.⁴⁶ AutoDock Vina and AutoDock Tools have been utilized to perform molecular docking of the synthesized novel compounds.^49,50 The 2D interactions between the molecule and receptor, as well as the docking consequence, ligand contacts, and surface study, were visualized using the Discovery Studio software.

General procedure for the synthesis of 3,4-dihydro-2H-pyrrole derivatives 33a–33f

A mixture of dehydroacetic acid (0.16 g, 1 mmol), arylaldehyde (2 mmol), ammonium acetate (0.07 g, 3 mmol), malononitrile (0.06 g, 1 mmol) or ethyl cyanoacetate (0.11 g, 1 mmol) and two drops of acetic acid was magnetically stirred in EtOH (5 mL) at 70 °C for 2 h. Progress of the reaction was monitored by TLC on silica gel 60 F₂₅₄ using ethyl acetate:n-hexane (in 2 : 1 v/v ratio) as eluent. After completion of the reaction, the mixture was cooled at room temperature and the product was allowed to precipitate. The crude solid product was filtered, recrystallized in ethanol (95%) and then dried under air at 50 °C. NMR and Mass spectra of the products are available (SI Fig. S12–S35).

Ethyl 3-cyano-5-(4-hydroxy-6-methyl-2-oxo-2-pyran-3-yl)-4-(3-nitrobenzyl)-2-(3-nitrophenyl)-3,4-dihydro-2H-pyrrole-3-carboxylate 33a (see S12–S15)

White powder, yield 85%, m p: 208–210 °C, FT-IR (cm⁻¹): 3090 (arom. C–H Str.), 1710 (C O Str.), 1577 (C N Str.), 1534 and 1350 (NO₂ Str.), 3434 (O–H Str.), 2923 (aliph. C–H Str.). ¹H NMR (400 MHz, DMSO-d₆); δ ppm 15.44 (1H, s, OH), 8.43–8.40 (1H, m, Ar–H), 8.31 (1H, dd, J 7.9 and 1.5 Hz, Ar–H), 8.25 (1H, s, Ar–H), 8.18 (1H, s, Ar–H), 7.97 (1H, d, J 7.8 Hz, Ar–H), 7.88–7.87 (2H, m, Ar–H), 7.82 (1H, t, J 8.0 Hz, Ar–H), 5.98 (1H, s, pyran 5-H), 5.89 (1H, s, pyrrole 2-H), 4.42 (1H, dd, J 11.6, 5.0 Hz, pyrrole 4-H), 4.08 (1H, dd, J 19.9, 5.0 Hz, Ar-CH_A), 3.53 (1H, dd, J 11.6, 19.9 Hz, Ar-CH_B), 4.01–3.92 (2H, m, O–CH₂), 2.16 (3H, s, pyran 5-CH₃), 0.84 (3H, t, J 7.1 Hz, CH₃). ¹³C NMR (100 MHz, DMSO-d₆); δ ppm 184.3 (pyran C-4), 173.8, 164.2, 164.1, 147.9, 147.8, 138.0, 136.0, 134.5, 133.8, 131.2, 131.0, 129.6, 125.2, 123.9, 123.0, 122.2, 114.1 (CN), 106.4, 95.3 (pyran C-3), 63.8, 60.7, 55.6, 42.2, 33.2, 19.3, 13.3. MS (EI, 70 eV) [C₂₇H₂₂N₄O₉]; m/z (%) 546.4 (0.07, [M]⁺), 300.3 (27), 246.3 (33), 218.2 (43), 201.2 (51), 178.2 (100). Anal. calculated for (C₂₇H₂₂N₄O₉): C 59.34; H 4.06; N 10.25%. Found: C 59.31; H 4.11; N 10.29%.

Ethyl 3-cyano-5-(4-hydroxy-6-methyl-2-oxo-2-pyran-3-yl)-4-(3-chlorobenzyl)-2-(3-chlorophenyl)-3,4-dihydro-2H-pyrrole-3-carboxylate 33b (see S16–S19)

White powder, yield 70%, m p: 220–222 °C, FT-IR (cm⁻¹): 3067 (arom. C–H Str.), 1698 (C O Str.), 1579 (C N Str.), 3436 (O–H Str.), 2921 (aliph. C–H Str.). ¹H NMR (400 MHz, DMSO-d₆); δ ppm 15.26 (1H, s, OH), 7.62 (1H, d, J 8.2 Hz, Ar–H), 7.58 (1H, t, J 7.7 Hz, Ar–H), 7.51–7.50 (2H, m, Ar–H), 7.43–7.41 (2H, m, Ar–H), 7.37–7.34 (2H, m, Ar–H), 5.86 (1H, s, pyran 5-H), 5.73 (1H, s, pyrrole 2-H), 4.15 (1H, dd, J 11.9 and 5.2 Hz, pyrrole 4-H), 4.02–3.93 (3H, m, Ar–CH_A and O–CH₂), 3.43 (1H, dd, J 19.9 and 11.5 Hz, Ar–CH_B), 2.14 (3H, s, pyran 5-CH₃), 0.85 (3H, t, J 7.1 Hz, CH₃). ¹³C NMR (100 MHz, DMSO-d₆); δ ppm 184.4, 173.8, 164.3, 164.0, 138.4, 136.3, 133.6, 133.4, 131.3, 131.1, 130.2, 128.9, 128.1, 127.1, 126.6, 126.0, 114.3 (CN), 106.5, 95.2 (pyran C-3), 63.5, 60.8, 55.7, 42.3, 33.4, 19.3, 13.4. MS (EI 70 eV) [C₂₇H₂₂Cl₂N₂O₅]; m/z (%) 528.3 (1.4, [M]⁺, ³⁷Cl, ³⁷Cl), 526.3 (5.6, [M]⁺, ³⁷Cl, ³⁵Cl), 524.4 (7, [M]⁺, ³⁵Cl, ³⁵Cl), 451.3 (11), 374.3 (16), 328.3 (25), 288.3 (67), 254.3 (21), 178.2 (100). Anal. calculated for (C₂₇H₂₂Cl₂N₂O₅): C 61.73; H 4.22; N 5.33%. Found: C 61.79; H 4.31; N 5.16%.

4-(3-Bromobenzyl)-2-(3-bromophenyl)-5-(4-hydroxy-6-methyl-2-oxo-2-pyran-3-yl)-3,4-dihydro-2H-pyrrole-3,3-dicarbonitrile 33c (see S20–S23)

White powder, yield 75%, m p: 213–215 °C, FT-IR (cm⁻¹): 3036 (arom. C–H Str.), 1700 (C O Str.), 1576 (C N Str.), 3431 (O–H Str.), 2921 (aliph. C–H Str.). ¹H NMR (400 MHz, DMSO-d₆); δ ppm 15.46 (1H, s, OH), 7.82 (1H, br s, Ar–H), 7.77 (2H, br s, Ar–H), 7.73 (1H, d, J 7.7 Hz, Ar–H), 7.65–7.58 (3H, m, Ar–H), 7.53 (1H, t, J 7.7 Hz, Ar–H), 6.01 (1H, s, pyran 5-H), 5.89 (1H, s, pyrrole 2-H), 4.35 (1H, dd, J 11.9 and 4.2 Hz, pyrrole 4-H), 4.10 (1H, d, J 19.8 Hz, Ar-CH_A), 3.54 (1H, dd, J 11.9 and 19.8 Hz, CH_B), 2.15 (3H, s, pyran 5-CH₃). ¹³C NMR (100 MHz, DMSO-d₆); δ ppm 184.3, 173.7, 164.3, 161.6, 138.1, 136.0, 135.7, 133.6, 132.5, 131.6, 131.5, 131.4, 130.6, 127.0, 126.8, 122.2, 112.0, 111.2, 106.3, 95.5, 60.0, 44.7, 41.6, 31.8, 19.3. MS (EI 70 eV) [C₂₅H₁₇ Br₂N₃O₃]; m/z (%) 567.3 (0.05, [M]⁺, ⁷⁹Br, ⁸¹Br), 373.2 (3.7), 371.2 (3.9), 334.1 (16), 332.1 (11) 234.2 (23), 178.2 (100), 153.2 (15), 126.2 (34). Anal. Calculated for (C₂₅H₁₇Br₂N₃O₃): C 52.94; H 3.02; N 7.41%. Found: C 53.01; H 3.11; N 7.27%.

4-(4-Ethylbenzyl)-2-(4-ethylphenyl)-5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-3,4-dihydro-2H-pyrrole-3,3-dicarbonitrile 33d (see S24–S27)

White powder, yield 68%, m p: 235–237 °C, FT-IR (cm⁻¹): 3030 (arom. C–H Str.), 1708 (C O Str.), 1576 (C N Str.), 3431 (O–H Str.), 2923 (aliph. C–H Str.). ¹H NMR (400 MHz, DMSO-d₆); δ ppm 15.35 (1H, s, OH), 7.55 (2H, d, 8.0 Hz, Ar–H), 7.53 (2H, d, 8.1 Hz, Ar–H), 7.48 (2H, d, J 8.1 Hz, Ar–H), 7.41 (2H, d, J 8.0 Hz, Ar–H), 5.97 (1H, s, pyran 5-H), 5.89 (1H, s, pyrrole 2-H), 4.31 (1H, dd, J 11.8 and 5.1 Hz, pyrrole 4-H), 4.05 (1H, d, J 19.9 Hz, Ar-CH_A), 3.54 (1H, dd, J 11.8, 19.9 Hz, Ar-CH_B), 2.76–2.67 (4H, m, 2CH₂), 2.18 (3H, s, pyran 5-CH₃), 1.27 (3H, t, J 7.5 Hz, CH₃), 1.25 (3H, t, J 7.5 Hz, CH₃). ¹³C NMR (100 MHz, DMSO-d₆); δ ppm 184.3, 173.8, 164.1, 161.7, 146.5, 145.1, 133.0, 130.5, 128.7, 128.6, 128.1, 127.7, 112.4 (CN), 111.7 (CN), 106.4, 95.2 (Pyran C-3), 60.6, 45.2, 42.0, 32.4, 27.9, 27.9, 27.8, 19.3, 15.3, 15.2. MS (EI 70 eV) [C₂₉H₂₇N₃O₃]; m/z (%) 466.5 (4.2, [M + 1]⁺), 465.5 (6.7, [M]⁺), 321.3 (19), 282.3 (100), 254.2 (45), 167.2 (40). Anal. Calculated for (C₂₉H₂₇N₃O₃): C 74.82; H 5.85; N 9.03%. Found: C 74.90; H 5.89; N 8.89%.

5-(4-Hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-4-(4-methylbenzyl)-2-(4-methylphenyl)-3,4-dihydro-2H-pyrrole-3,3-dicarbonitrile 33e (see S28–S31)

White powder, yield 80%, m p: 217–219 °C, FT-IR (cm⁻¹): 3031 (arom. C–H Str.), 1709 (C O Str.), 1576 (C N Str.), 3446 (O–H Str.), 2921 (aliph. C–H Str.). ¹H NMR (400 MHz, DMSO-d₆); δ ppm 15.32 (1H, s, OH), 7.48 (2H, d, J 8.0 Hz, Ar–H), 7.46 (2H, d, J 8.0 Hz, Ar–H), 7.41 (2H, d, J 8.0 Hz, Ar–H), 7.34 (2H, d, J 8.0 Hz, Ar–H), 5.93 (1H, s, pyran 5-H), 5.87 (1H, s, pyrrole 2-H), 4.26 (1H, dd, J 11.9 and 5.2 Hz, pyrrole 4-H), 4.00 (1H, br dd, J 5.2 and 20.1 Hz, Ar–CH_A), 3.49 (1H, br dd, J 20.1 and 11.9 Hz, Ar–CH_B), 2.39 (3H, s, pyran 5-CH₃), 2.36 (3H, s, Ar–CH₃), 2.14 (3H, s, Ar-CH₃). ¹³C NMR (100 MHz, DMSO-d₆); δ ppm 184.3, 173.8, 164.1, 161.7, 140.4, 139.0, 132.7, 130.2, 129.80, 129.76, 128.0, 127.6, 112.4, 111.6, 106.4, 95.2, 60.6, 45.3, 42.0, 32.4, 20.8, 20.7, 19.3. MS (EI 70 eV) [C₂₇H₂₃N₃O₃]; m/z (%) 438.4 (6.7, [M + 1]⁺), 437.4 (7.8, [M]⁺), 307.3 (24), 268.3 (100), 254.2 (36), 168.2 (25). Anal. Calculated for (C₂₇H₂₃N₃O₃): C 74.13; H 5.30; N 9.60%. Found: C 74.24; H 5.36; N 9.52%.

4-Benzyl-5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-2-phenyl-3,4-dihydro-2H-pyrrole-3,3-dicarbonitrile 33f (see S32–S35)

White powder, yield 65%, m p: 232–234 °C, FT-IR(cm⁻¹): 3063 (arom. C–H Str.), 1701 (C O Str.), 1582 (C N Str.), 3433 (O–H Str.), 2921 (aliph. C–H Str.). ¹H NMR (400 MHz, DMSO-d₆); δ ppm 15.43 (1H, s, OH), 7.70–7.52 (10H, m, Ar–H), 6.04 (1H, s, pyran 5-H), 5.90 (1H, s, pyrrole 2-H), 4.37 (1H, dd, J 4.4 and 11.8 Hz, pyrrole 4-H), 4.10 (1H, d, J 19.6 Hz, Ph-CH_A), 3.58 (1H, dd, J 19.6 and 11.8 Hz, Ph-CH_B), 2.18 (3H, s, pyran 5-CH₃). ¹³C NMR (100 MHz, DMSO-d₆); δ ppm 184.9 (pyran C-4), 174.3, 164.7, 162.2, 136.3, 133.8, 131.2, 130.0, 129.8, 128.8, 128.3, 112.8, 112.1, 106.9, 95.9, 61.4, 45.6, 42.9, 32.9, 19.8. MS (EI 70 eV) [C₂₅H₁₉N₃O₃]; m/z (%) 410.2 (1.2, [M+1]⁺), 409.3 (2.8, [M]⁺), 293.3 (33), 254.3 (100), 154.2 (48), 127.3 (35), 103.2 (62). Anal. Calculated for (C₂₅H₁₉N₃O₃): C 73.34; H 4.68; N 10.26%. Found: C 73.39; H 4.59; N 10.32%.

Conclusions

A novel, stereoselective route to poly-substituted 3,4-dihydro-2H-pyrroles is reported for the first time, involving a new pseudo-five-component reaction of simple starting materials. The structures of the synthesized compounds were confirmed by FT-IR, ¹H NMR, ¹³C NMR, mass spectrometry, and elemental analysis. Despite possessing three stereogenic centres, the reaction yielded only one diastereomer. 2D-NOESY spectrum of 33a revealed a cis–cis configuration for this model product. DFT calculations provided insights into the molecular geometry and validated the observed IR bands.

Given the crucial role of HIF-1α protein in tumor progression and therapy resistance across various cancers, molecular docking studies were performed to evaluate the potential interaction of the synthesized compounds with its active site. The compounds demonstrated promising binding modes and favourable affinities with key residues of HIF-1α, among them compound 33e showed the most negative binding energy. These in silico results warrant further investigation into their potential as HIF-1α inhibitors. Drug-like properties were assessed using the SwissADME web server. The majority of compounds adhered to Lipinski's rule of five, indicating a likelihood of good oral absorption. All compounds were predicted to have high CaCo-2 permeability, suggesting potential for intestinal absorption, and were classified as both P-glycoprotein substrates and inhibitors. Additionally, compound 33a was predicted to be non-mutagenic, non-carcinogenic, and unable to cross the blood–brain barrier, suggesting a potentially favourable safety profile. It's computed binding affinity for HIF-1α was comparable to that of 33e, highlighting it as a promising scaffold for further optimization. This study successfully introduces a new pseudo-five-component synthesis and provides a comprehensive in silico profile of the synthesized 3,4-dihydro-2H-pyrroles, identifying them as valuable candidates for subsequent experimental validation. The computational findings presented herein establish a foundation for future in vitro and in vivo studies aimed at developing novel HIF-1α-targeted therapies. We anticipate that this synthetic strategy will be of significant interest to both synthetic and medicinal chemists working on anticancer drug discovery.

Author contributions

Professor Kurosh Rad-Moghadam: supervision, conceptualization, methodology, writing, review and editing. Associate professor Seyyed Morteza Mehrdad; supervision. Nesa Roshani (PhD student): investigation, writing the original drafts, and data curation.

Conflicts of interest

There are no conflicts to declare.

Data availability

All data generated or analysed during this study are included in this published article and its supplementary information (SI) file.

Supplementary information: for additional UV-vis spectra, complementary experimental procedures, the EDS mapping of Cu/NSCNC, NMR and mass spectra. See DOI: https://doi.org/10.1039/d6ra04116g.