Recent Advances in dehydroacetic acid-clubbed pyridine conjugates: design, synthesis, characterization, molecular docking investigations and evaluation of anti-cancer efficacy

ABSTRACT

Aim

The aim of this study was to synthesize novel pyridine conjugates incorporating dehydroacetic acid via the Krohnke reaction and to evaluate their potential as anti-cancer agents. Materials & methods: The Krohnke reaction has previously been reported using acetic acid as the solvent, here we employed ethanol as the reaction medium. The trisubstituted pyridine derivatives were synthesized via a multistep procedure that involved the reaction of pyridinium bromide salts with chalcone derivatives of dehydroacetic acid. The synthesized hybrids were comprehensively characterized using a range of analytical techniques, including Nuclear Magnetic Resonance (NMR) spectroscopy, Fourier Transform Infrared (FT-IR) spectroscopy, and Mass Spectrometry (MS).

Results

These compounds were subjected to in vitro evaluation of their anti-cancer potential against MCF-7 (human breast cancer) and A-549 (human lung cancer) cell lines. The anti-cancer screening results indicate that the synthesized conjugates exhibit potent activity against MCF-7 cells and demonstrate moderate activity against A-549 cell lines.

Conclusion

The analysis indicates a binding energy of −9.3 kcal/mol for the 7a derivative, which is closely comparable to that of the standard drug, which exhibits a binding energy of −9.9 kcal/mol.

GRAPHICAL ABSTRACT

The protocol entailed the synthesis of a pyridinium bromide salt via the reaction of phenacyl bromide with pyridine, followed by its subsequent treatment with a chalcone derivative of dehydroacetic acid, yielding 2,4,6-trisubstituted pyridine derivatives.

1. Introduction

Pyridine, a heterocyclic aromatic compound characterized by the molecular formula C₅H₅N, was first isolated from coal tar and bone oil in the year 1846 [1]. In biological systems, pyridine serves a crucial function as a catalytic scaffold, particularly in enzymatic processes facilitated by pyridine nucleotides, such as NADP⁺. These nucleotides are essential cofactors involved in a diverse array of redox reactions. It has been observed that several anti-cancer agents featuring a pyridine nucleus, including Sorafenib [2], Axitinib [3], Regorafenib [4], and Apatinib [5], are currently available on the market. Additionally, a range of heterocyclic compounds containing a pyridine moiety has demonstrated significant anti-cancer potential, exhibiting efficacy comparable to that of established chemotherapeutic agents, such as topoisomerase inhibitors [6], DNA intercalators [7], and inhibitors of EGFR [8] and VEGFR [9] (Figure 1). Cancer continues to pose a major global health burden, currently standing as the second leading cause of mortality worldwide. Data from the International Agency for Research on Cancer (IARC) in 2020 indicated that approximately 19.3 million new cancer cases were diagnosed globally, leading to an estimated 9.9 million deaths attributable to the disease [10]. Projections indicate that by the year 2030, the global burden of cancer is anticipated to increase substantially, with new cases expected to exceed 24.1 million and cancer-related deaths approaching approximately 13.0 million [11,12]. Although chemotherapy continues to be a widely employed therapeutic strategy, its use is often accompanied by substantial adverse effects on healthy tissues, resulting in long-term damage to critical organs such as the heart, lungs, kidneys, and reproductive system. In contemporary medicinal chemistry research, the concept of hybridizing two or more biologically active motifs is employed to generate compounds with enhanced biological significance [13]. This strategy is implemented with the expectation of achieving enhanced biological potency by integrating active chemical entities to elicit a synergistic effect. In this study, we aim to synthesize a series of dehydroacetic acid-pyridine hybrid molecules utilizing a hybridization strategy to achieve an enhanced biological profile through Krohnke reaction [14].

Figure 1.

Pyridine scaffold present in FDA-approved anticancer therapeutics.

In a previous study, Ladani et al. [15]. reported the synthesis of a trisubstituted pyridine derivative incorporating a thiophene moiety. The hybrid was obtained via a one-pot reaction performed in acetic acid under microwave irradiation. Furthermore, Gomha et al. [16]. explored the synthesis of pyrazoles that incorporate pyridine derivatives through the Khronke reaction. The Patil and research group [17] also synthesized a series of quinoline-based pyridine derivatives and conducted a systematic evaluation of their biological activities (Figure 2). The Khronke reaction was previously conducted using acetic acid as the solvent. In the present study, ethanol has been employed as a greener and less toxic alternative, offering improved environmental compatibility. Furthermore, pyridine-linked dehydroacetic acid scaffolds have not yet been explored.

Figure 2.

Previous research on pyridine analogs.

Dehydroacetic acid (DHA) is a biologically active compound with significant utility in synthetic organic chemistry [18]. It demonstrates a wide range of pharmacological activities, encompassing anti-microbial, anti-bacterial, anti-fungal [19], anti-inflammatory [20], anti-tubercular, anti-depressant [21], anti-malarial, and anti-cancer [22] effects, among others. Phenacyl bromides have attracted considerable interest in organic synthesis due to the presence of two adjacent electrophilic centers the α-halocarbon and the carbonyl carbon within their molecular structure. This distinctive characteristic enables their participation in a wide array of selective organic transformations with various reagents [23–25]. In the present manuscript, we are focusing on the synthesis of trisubstituted pyridine derivatives using 3-cinnamoyl-4-hydroxy-6-methyl-2-oxo-2H-pyrans (chalcone analogs of DHA) [26] and phenacyl bromide [23]. The resultant hybrid compounds were further subjected to exploration of anti-cancer potential against MCF-7 and A-549 cell lines (Figure 3).

Figure 3.

Present research.

2. Materials and methods

Dehydroacetic acid was procured from Sigma-Aldrich. The ¹H and¹³C Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker FT-NMR spectrometer operating at 400 MHz, using tetramethylsilane (TMS) as the internal standard and CDCl₃ as the solvent. Chemical shifts (δ) are reported in parts per million (ppm), and coupling constants (J) are expressed in hertz (Hz). Mass spectral analysis of the synthesized compounds was performed using a Triple TOF 5600+ mass spectrometer (AB SCIEX) at Guru Jambheshwar University, Hisar. Infrared (IR) spectra were acquired on a Bruker Tensor 27 FT-IR spectrometer at the Central University of Punjab, Bathinda, with measurements taken in the range of 6000–400 cm^{− 1} as percent transmittance. Melting points (mp) of the synthesized compounds were determined using open capillary tubes and are reported without correction. The progress of the reactions was monitored by thin-layer chromatography (TLC) on silica gel aluminum plates (Merck Specialties Pvt. Ltd.), using a solvent system of ethyl acetate and petroleum ether as the eluent.

2.1. Experimental

2.1.1. General procedure for the preparation of substituted 3-cinnamoyl-4-hydroxy-6-methyl-2-pyrones (3)

A reaction mixture comprising (1) dehydroacetic acid (0.01 mol), an (2) aldehyde (0.01 mol), and a catalytic amount of piperidine (approximately 10 drops) in 20 mL of chloroform was refluxed for 8 to 10 hours. The reaction progress was systematically monitored at regular intervals through the application of thin-layer chromatography (TLC), facilitating the assessment of the formation of the target product. Crystals of the resulting product were formed upon the slow evaporation of chloroform, followed by the subsequent addition of ethanol. The resulting derivatives were recrystallized from ethanol to obtain the purified compounds [26].

2.1.2. Procedure for the synthesis of substituted 3-(4,6-diphenylpyridin-2-yl)-4-hydroxy-6-methyl-2H-pyran-2-one(7a-7 h)

A reaction solution was prepared by mixing chalcone of dehydroacetic acid (DHA) 3 (0.5 mmol), pyridinium bromide salt 6 (1 mmol), and ammonium acetate (5 mmol), which was then refluxed in 20 mL of ethanol for 3 to 4 hours. In this procedure, ammonium acetate served both as a base and as a cyclizing agent. After completion of the reaction, the solvent was evaporated under reduced pressure, and the resulting crude product was thoroughly washed with water and subsequently filtered. Purification was accomplished through recrystallization from ethanol, yielding the pure compound. The yields of the final conjugates ranged from 65% to 78%.

2.1.3. 4-hydroxy-3-(4-(4-isopropylphenyl)-6-phenylpyridin-2-yl)-6-methyl-2 H-pyran-2-one (7a)

Color: yellow solid; % Yield 70; M.P.: 180 °C; FT-IR (KBr, ῡ, cm⁻¹): 1685.48 (C = O); ¹H NMR (400 MHz, CDCl₃): δ 9.21 (s, 1H), 7.66 (dd, J = 144.1, 76.5 Hz, 10H), 5.87 (s, 1H), 3.01 (s, 1H), 2.22 (s, 3H), 1.32 (s, 6H); ¹³C NMR (400 MHz, CDCl₃): δ 182.03, 163.55, 162.24, 154.69, 153.77, 151.50, 147.78, 134.77, 133.39, 130.95, 129.67, 127.39, 118.19, 114.49, 106.25, 91.93, 77.40, 33.82, 23.85, 19.96; Mass spectrometry (ESI-TOF) m/z (Da): Calcd. for C₂₆H₂₃NO₃, 397.16, [M + 1^]+ obtained at 398.17.

2.1.4. 3-(6-(4-chlorophenyl)-4-(4-isopropylphenyl)pyridin-2-yl)-4-hydroxy-6-methyl-2 H-pyran-2-one (7b)

Color: yellow solid; % Yield 72; M.P.: 160 °C; FT-IR (KBr, ῡ, cm⁻¹): 1690.3 (C = O); ¹H NMR (400 MHz, CDCl₃): δ 9.19 (s, 1H), 8.32–6.97 (m, 9H), 5.87 (s, 1H), 3.01 (s, 1H), 2.23 (s, 3H), 1.34 (s, 6H); ¹³C NMR (400 MHz, CDCl₃): δ 181.48, 163.37, 162.54, 154.97, 153.64, 151.57, 147.06, 137.17, 134.66, 132.24, 129.89, 127.62, 127.42, 118.43, 114.57, 105.79, 92.23, 77.36, 34.01, 23.84, 19.99; Mass spectrometry (ESI-TOF) m/z (Da): Calcd. for C₂₆H₂₂ClNO₃, 431.12, [M + 1^]+ obtained at 432.13.

2.1.5. 3-(6-(4-bromophenyl)-4-(4-isopropylphenyl)pyridin-2-yl)-4-hydroxy-6-methyl-2 H-pyran-2-one (7c)

Color: Yellow solid; % Yield 65; M.P.: 200 °C; FT-IR (KBr, ῡ, cm⁻¹): 1690.3 (C = O); ¹H NMR (400 MHz, CDCl₃): δ 9.19 (s, 1H), 8.17–7.03 (m, 9H), 5.87 (s, 1H), 3.03 (s, 1H), 2.23 (s, 3H), 1.25 (s, 6H); ¹³C NMR (400 MHz, CDCl₃): δ 181.42, 163.35, 162.56, 155.01, 153.63, 151.57, 147.18, 134.66, 132.76, 127.96, 127.42, 125.48, 118.50, 114.57, 105.75, 92.27, 77.36, 34.01, 23.84, 20.00; Mass spectrometry (ESI-TOF) m/z (Da): Calcd. for C₂₆H₂₂BrNO₃, 475.07, [M + 1^]+ obtained at 476.08.

2.1.6. 4-hydroxy-3-(4-(4-isopropylphenyl)-6-(4-methoxyphenyl)pyridin-2-yl)-6-methyl-2 H-pyran-2-one(7d)

Color: Yellow solid; % Yield 75; M.P.: 140 °C; FT-IR (KBr, ῡ, cm⁻¹): 1690.3 (C = O); ¹H NMR (400 MHz, CDCl₃): δ 9.10 (s, 1H), 8.03–6.90 (m, 9H), 5.83 (s, 1H), 3.87 (s, 3H), 2.99 (s, 1H), 2.20 (s, 3H), 1.31 (s, 6H); ¹³C NMR (400 MHz, CDCl₃): δ 182.26, 163.62, 161.89, 154.20, 153.76, 151.41, 147.17, 134.83, 127.89, 127.56, 125.25, 117.31, 115.03, 113.48, 106.52, 91.72, 76.77, 55.49, 33.98, 23.84, 19.91; Mass spectrometry (ESI-TOF) m/z (Da): Calcd. for C₂₇H₂₅NO₄, 427.17, [M + 1^]+ obtained at 428.18.

2.1.7. 3-(4-(4-chlorophenyl)-6-phenylpyridin-2-yl)-4-hydroxy-6-methyl-2 H-pyran-2-one (7e)

Color: Yellow solid; % Yield 70; M.P.: 220 °C; FT-IR (KBr, ῡ, cm⁻¹): 1691.27 (C = O); ¹H NMR (400 MHz, CDCl₃): δ 9.03 (s, 1H), 8.22–7.07 (m, 10H), 5.93 (s, 1H), 2.28 (s, 3H); ¹³C NMR (400 MHz, CDCl₃): δ 181.59, 163.39, 162.62, 154.93, 152.63, 147.69, 137.42, 133.57, 130.99, 130.46, 127.38, 126.61, 121.30, 117.96, 105.84, 92.26, 76.74, 20.03; Mass spectrometry (ESI-TOF) m/z (Da): Calcd. for C₂₃H₁₆ClNO₃, 389.08, [M + 1^]+ obtained at 390.08.

2.1.8. 3-(4,6-Bis(4-chlorophenyl) pyridin-2-yl)-4-hydroxy-6-methyl-2 H-pyran-2-one (7f)

Color: solid; Yellow % Yield 62; M.P.: 230 °C; FT-IR (KBr, ῡ, cm⁻¹): 1690.3 (C = O); ¹H NMR (400 MHz, CDCl₃): δ 9.02 (s, 1H), 8.32–6.92 (m, 9H), 5.94 (s, 1H), 2.27 (s, 3H); 13C NMR (400 MHz, CDCl3): δ 180.83, 163.20, 162.93, 155.23, 152.42, 147.13, 137.33, 132.55, 132.01, 130.95, 129.91, 127.90, 127.41, 121.56, 118.08, 105.30, 92.64, 20.06; Mass spectrometry (ESI-TOF) m/z (Da): Calcd. for C₂₃H₁₅Cl₂NO₃, 423.04, [M + 1^]+ obtained at 424.04.

2.1.9. 3-(6-(4-bromophenyl)-4-(4-chlorophenyl)pyridin-2-yl)-4-hydroxy-6-methyl-2 H-pyran-2-one (7 g)

Color: Yellow solid; % Yield 78; M.P.: 240 °C; FT-IR (KBr, ῡ, cm⁻¹): 1694.16 (C = O); ¹H NMR (400 MHz, CDCl₃): δ 9.03 (s, 1H), 8.49–6.35 (m, 9H), 5.94 (s, 1H), 2.27 (s, 3H); ¹³C NMR (400 MHz, CDCl₃): δ 180.78, 163.20, 155.26, 152.41, 147.23, 137.31, 132.88, 132.02, 130.54, 130.40, 128.09, 127.41, 125.50, 121.63, 118.07, 105.27, 92.68, 77.36, 20.06; Mass spectrometry (ESI-TOF) m/z (Da): Calcd. for C₂₃H₁₅BrClNO₃, 466.99, [M + 1^]+ obtained at 467.01.

2.1.10. 3-(4-(4-chlorophenyl)-6-(4-methoxyphenyl) pyridin-2-yl)-4-hydroxy-6-methyl-2 H-pyran-2-one (7 h)

Color: Yellow solid; % 65; M.P.: 210 °C; FT-IR (KBr, ῡ, cm⁻¹): 1693.19 (C = O), ¹H NMR (400 MHz, CDCl₃): δ 8.96 (s, 1H), 8.17–6.62 (m, 10H), 5.91 (s, 1H), 3.91 (s, 3H), 2.26 (s, 3H); ¹³C NMR (400 MHz, CDCl₃): δ 181.95, 163.46, 162.39, 162.00, 154.57, 152.71, 147.15, 137.57, 132.02, 130.91, 130.36, 128.08, 125.54, 120.42, 117.00, 106.13, 92.02, 76.71, 20.00; Mass spectrometry (ESI-TOF) m/z (Da): Calcd. for C₂₄H₁₈ClNO₄, 419.09, [M + 1^]+ obtained at 420.10.

2.2. Anti-cancer activity

The cell lines were cultured in a suitable growth medium supplemented with 2 mM L-glutamine and 10% fetal bovine serum (FBS). For the current screening assay, 5,000 cells were inoculated into each well of 96-well microtiter plates, achieving a final volume of 100 µL per well. Following cell inoculation, the microtiter plates were incubated at 37 °C in a humidified atmosphere comprising 5% CO₂ and 95% air, with a relative humidity of 100%, for 24 hours prior to the addition of the experimental compounds. The test compounds were initially dissolved in an appropriate solvent at a concentration of 100 mg/mL, then diluted to 1 mg/mL using distilled water, and stored at sub-zero temperatures until further use. At the time of drug treatment, an aliquot of the frozen stock solution (1 mg/mL) was thawed and subsequently diluted with complete medium containing the test article to prepare working solutions at concentrations of 100 μg/mL, 200 μg/mL, 400 μg/mL, and 800 μg/mL. From each of these dilutions, 10 μL was added to the corresponding wells of a microtiter plate, which already contained 90 μL of culture medium, resulting in final drug concentrations of 10 μg/mL, 20 μg/mL, 40 μg/mL, and 80 μg/mL, respectively. Following the addition of the test compounds, the plates were incubated under standard conditions for 48 hours. The assay was subsequently terminated by the addition of cold trichloroacetic acid (TCA). For in situ fixation, 50 µL of cold 30% (w/v) TCA was gently added to each well, achieving a final TCA concentration of 10%, and the plates were incubated at 4 °C for 60 minutes. Thereafter, the supernatant was carefully removed, and the plates were washed five times with tap water and allowed to air dry completely. A 50 µL aliquot of Sulforhodamine B (SRB) solution (0.4% w/v in 1% acetic acid) was added to each well, and the plates were incubated at room temperature for 20 minutes to allow staining. Following incubation, the unbound dye was aspirated, and the wells were washed five times with 1% acetic acid to remove residual stain. The plates were then air-dried thoroughly. Subsequently, the protein-bound dye was solubilized using 10 mM Trizma base, and absorbance was measured at 540 nm using a microplate reader, with a reference wavelength of 690 nm for background correction. Percent growth was calculated on a plate-by-plate basis for test wells relative to control wells. Percent Growth was expressed as the ratio of average absorbance of the test well to the average absorbance of the control wells * 100. Percentage growth at each drug concentration was derived from six absorbance readings: the baseline value at time zero (Tz), the absorbance of untreated control wells (C), and the absorbances of drug treated wells at the four test concentrations (Ti). The 50 % growthinhibitory concentration (GI₅₀) was defined as the drug level that reduced the net protein accumulation-quantified by SRB staining-in treated cells by 50 % relative to controls during the incubation period. Each metric was reported only when its activity threshold was achieved; if the threshold lay outside the concentration range examined, the result was expressed as greater than the highest or less than the lowest concentration tested [27,28].

2.3. Molecular docking investigations

Molecular docking was performed in PyRx 0.8 tool using the epidermal growth factor receptor tyrosine kinase (EGFR; PDB ID 1M17) retrieved from the RCSB Protein Data Bank. Prior to docking, all crystallographic water molecules and nonprotein heteroatoms were deleted, and polar hydrogen atoms were added to the receptor. Ligand structures were sketched in ChemDraw Professional 16.0, saved in SDF format, energyminimised within PyRx, and converted to PDBQT files. Docking results were inspected and illustrated with BIOVIA Discovery Studio Visualizer [29].

2.4. ADMET studies

ADMETlab 3.0 is a freely accessible, user-friendly web-based platform designed to enhance the success rate of drug discovery by proactively identifying chemical entities with a high risk of failure during clinical development. This objective is achieved through the evaluation of compounds against a rigorously curated database comprising 288,967 entries, thereby increasing the likelihood of identifying promising drug candidates. In the present study, ADMETlab 3.0 was utilized to predict a comprehensive range of physicochemical, pharmacokinetic, and toxicological properties of the synthesized compounds [30].

3. Results and discussion

3.1. Chemistry

The present study focuses on the synthesis of pyridine-conjugated dehydroacetic acid derivatives employing the Krohnke reaction. This reaction was first introduced by F. Kröhnke in the 1960s, who extensively demonstrated its versatility in synthesizing various substituted pyridines from acyl derivatives and chalcones. The synthetic pathway for the target compounds is depicted in (Figure 4). The methodology entails the synthesis of 3a-b chalcones through the reaction of DHA 1 and aldehyde 2 in chloroform, facilitated by piperidine as a catalyst, under reflux conditions for a duration of night. Concurrently, pyridinium bromide salt 6 was synthesized via the reaction of 2-bromo-1-(5-methyl-1-phenyl-1H-pyrazol-4-yl) ethanone 4 with an equivalent amount of pyridine 5 in dry benzene under reflux conditions. The resulting compound displayed ¹H-NMR peaks at δ 6.16 and 7.25–7.83, which correspond to the CH₂ and aromatic protons, respectively. In the subsequent step, compounds 3a-b underwent smooth cyclocondensation with pyridinium bromide 6 [23] in the presence of ammonium acetate, utilizing refluxing ethanol as the solvent yielded the trisubstituted pyridine derivatives (7a-7 h) in good yields. This reaction has been previously reported utilizing acetic acid as the solvent. In this work, ethanol has been employed as a more environmentally friendly alternative, owing to its relatively lower toxicity. We are considering 4-hydroxy-3-(4-(4-isopropylphenyl)-6-phenylpyridin-2-yl)-6-methyl-2 H-pyran-2-one (7a) derivative with 70% yield as a reference compound to explain spectra of all synthesizing hybrids. The ¹H NMR spectrum revealed a singlet at δ 3.01 ppm, which is indicative of the methine (CH) proton associated with the isopropyl group. Additionally, a singlet at δ 2.22 ppm was identified as the methyl group of the dehydroacetic acid moiety, while a singlet at δ 1.32 ppm corresponds to the two equivalent methyl groups of the isopropyl substituent. Charactersitic peak of pyridine at δ 9.21 (s, 1 H) indicate that next carbon is connected to DHA and at δ 5.87 (s, 1 H) is appeared for dehydroacetic acid. One multiplet is appeared at δ 7.66 ppm for 9 H of benzene rings and 1 H of pyridine ring. All compounds are yellowish in color. The characteristic peaks observed in the ¹³C NMR spectrum at 114.49 and 118.19 ppm correspond to the C-3 and C-5 carbon atoms of the pyridine ring, respectively, which are bonded to hydrogen atoms. The mass spectrum of the product 7a displayed a molecular ion peak m/z = 398.1785. FTIR spectrum of 7a hybrid exhibits a prominent absorption band at v 1689 cm⁻¹ which is attributed to the presence of a carbonyl group. The physical data for compounds (7a-7h) are presented in Table 1.

Figure 4.

Synthetic strategy for the construction of pyridine-based conjugates.

Table 1.

Physical data of compounds 7a-7h

Entry	Product	R1	R2
1	7a	CH(CH3)2	H
2	7b	CH(CH3)2	Cl
3	7c	CH(CH3)2	Br
4	7d	CH(CH3)2	OCH3
5	7e	Cl	H
6	7f	Cl	Cl
7	7g	Cl	Br
8	7h	Cl	OCH3

3.1.1. Plausible mechanism for the synthesis of dehydroacetic acid-conjugated pyridine derivatives

Novel pyridine derivatives were synthesized through the Krohnke reaction [31], which entails the condensation of chalcones derived from DHA with a range of pyridinium phenacyl bromide salts. The mechanism proceeds via the formation of intermediate B, which arises from the addition of the chalcone to the methyl pyridinium bromide derivative A. Intermediate C is formed via the tautomerization of compound B, which facilitates a subsequent nucleophilic attack by ammonia on the electrophilic carbonyl carbon. Subsequently, an imine intermediate (D) is formed via a dehydration process. This imine, upon deprotonation, is converted into the enamine species (E). Further deprotonation of E facilitates aromatization, accompanied by the elimination of water, ultimately yielding the desired pyridine derivative (F) (Figure 5).

Figure 5.

Mechanism for the synthesis of dehydroacetic acid hybrids.

3.2. Anti-cancer activity

The newly synthesized hybrids were evaluated for their anti-cancer activity. Amongst the synthesized compounds, five derivatives were studied against two cancer cell lines: MCF-7 (human estrogen receptor-positive breast cancer cells) and A-549 lung cancer cell line where adriamycin was taken as a reference drug or positive control using SRB assay.

3.2.1. Michigan cancer foundation-7 (MCF-7) cell line

Breast cancer represents the most common malignancy among women and constitutes a considerable public health challenge due to its profound effects on the global population. Mitigating this burden requires ongoing molecular-level research aimed at enhancing prognostic precision and formulating targeted therapeutic strategies. The MCF-7 cell line, which has been extensively employed for over four decades, serves as a robust in vitro model in breast cancer research [32]. It has been widely utilized by numerous scientific communities for the investigation of tumor biology and the assessment of potential therapeutic agents [33]. The expression of estrogen receptors in these cells establishes them as a valuable model for investigating hormone-responsive breast cancers and evaluating the efficacy of receptor-targeted therapeutic interventions [34]. Owing to their well-defined characteristics and consistent reproducibility, MCF-7 cells remain a cornerstone model in breast cancer research. Among the synthesized compounds, five derivatives were selected for evaluation against the MCF-7 cell line, with Adriamycin employed as the reference standard. Among the compounds evaluated, derivative 7a, which possesses an isopropyl substituent, demonstrated an average growth inhibition of 38% against the MCF-7 breast cancer cell line. A modest enhancement in cytotoxic activity was observed with increasing concentrations of the compound, ranging from 10 to 80 µg/mL. Similarly, compound 7c demonstrated a growth inhibition of 46.4% across the tested concentration range of 10 to 80 µg/mL. Furthermore, compounds 7e, 7f, and 7 h exhibited growth inhibitions of 47.3%, 49.8%, and 46.0%, respectively, within the same concentration range. In contrast, the standard drug exhibited a control growth value of −29.2% under identical experimental conditions. The five derivatives 7a, 7c, 7e, 7f, and 7 h demonstrated GI₅₀ values of 59 µg/ml, 75 µg/ml, 48 µg/ml, 70 µg/ml and 49 µg/ml, respectively, against the MCF-7 cell line. The GI₅₀ value represents the concentration of a compound required to inhibit 50% of cellular proliferation, serving as a critical indicator of its antiproliferative potency. The GI₅₀ data indicate that derivatives 7 h and 7e exhibit significant cytotoxic activity against MCF-7 cell lines compared to derivatives 7a, 7c, and 7f. The results of the anti-cancer activity against MCF-7 cell lines are comprehensively presented in Table 2. The growth curves and morphological screening images of the compounds, highlighting the comparative analysis, are illustrated in Figures S1 and S2.

Table 2.

Demonstrating the % control growth for compounds (4a-4 h) against anti-cancer efficacy with MCF-7 cell line.

	Human Breast Cancer Cell Line MCF-7, % Control Growth, (µg/ml)
	Experiment 1				Experiment 2				Experiment 3				Average Values
Compounds	10	20	40	80	10	20	40	80	10	20	40	80	10	20	40	80
7a	85.7	82.7	49.6	37.9	92.1	72.6	52.5	33.0	97.1	88.5	61.5	43.0	91.6	81.2	54.5	38.0
7c	83.2	93.5	58.3	43.4	85.5	75.3	65.5	43.0	92.7	92.6	89.2	52.9	87.1	87.1	71.0	46.4
7e	58.3	47.1	40.2	47.0	56.9	54.8	35.8	42.3	61.5	68.7	57.2	52.6	58.9	56.9	44.4	47.3
7f	68.6	61.4	40.3	39.1	78.3	69.9	57.9	56.1	83.9	72.0	59.4	54.2	77.0	67.8	52.5	49.8
7h	43.3	49.3	37.5	43.2	65.0	64.5	44.0	37.5	61.3	64.2	57.5	57.3	56.5	59.4	46.3	46.0
ADR	−63.6	−65.1	−62.3	−36.3	−57.7	−55.4	−52.2	−27.4	−50.7	−53.6	−47.4	−23.9	−57.4	−58.0	−54.0	−29.2

3.2.2. Human lung adenocarcinoma (A549) cell line

Lung cancer remains one of the most prevalent malignancies in humans and continues to be the leading cause of cancer-related mortality globally. Current therapeutic approaches primarily include surgical resection, radiotherapy, and chemotherapy. Among these modalities, chemotherapy serves as a cornerstone of clinical management; however, its therapeutic efficacy is frequently compromised by significant limitations, including systemic toxicity, severe adverse effects, and the common development of drug resistance. As a result, lung cancer continues to contribute to substantial mortality, with 5-year overall survival rates in many regions remaining below 15%. These challenges highlight the urgent need for the discovery and development of novel therapeutic agents that offer enhanced efficacy and improved safety profiles for the effective prevention and treatment of lung cancer and related malignancies [35]. Among the synthesized derivatives, five conjugates were evaluated for their efficacy against A549 cell lines, using Adriamycin as a standard reference drug. Among the synthesized derivatives, the 7a compound featuring an isopropyl substituent demonstrated an average growth inhibition of 87% against the A549 cell line. A pronounced increase in biological activity was observed with escalating concentrations of the compounds, ranging from 10 to 80 µg/mL. Notably, compound 7c demonstrated a growth inhibition of 90.3% within this concentration range. Additionally, compounds 7e, 7f, and 7 h exhibited control growth of 100.7%, 106.4%, and 99.9%, respectively, at increasing concentrations ranging from 10 to 80 µg/mL. In contrast, the standard drug demonstrated a control growth rate of −52.2%. The synthesized pyridine derivatives exhibited GI₅₀ values greater than 80 µg/mL against A549 lung cancer cell lines, indicating substantially lower potency compared to the reference drug. In contrast, the standard chemotherapeutic agent Adriamycin (ADR) showed a GI₅₀ value below 10 µg/mL under identical conditions. Therefore, relative to Adriamycin, the synthesized hybrids demonstrated only moderate anticancer activity against breast cancer cell lines. The anti-cancer activity data against MCF-7 cell lines are comprehensively presented in Table 3. Comparative analysis through growth curves and morphological screening of the tested compounds is illustrated in Figure S3 and S4. Table 4 presents the percentage growth inhibition of compounds (7a-7h) against the human lung adenocarcinoma (A549) cell line. Table 5 summarizes the in vitro cytotoxicity data of the test compounds, with drug concentrations (µg/mL) derived from graphical analysis.

Table 3.

In vitro test results of the test compounds (drug concentrations (µg/ml) calculated from graph).

MCF-7	LC50	TGI	GI50*
7a	NE	>80	59
7c	NE	>80	75
7e	NE	>80	48
7f	NE	>80	70
7h	NE	>80	49
ADR	<10	<10	<10

Table 4.

Demonstrating the % control growth for compounds (7a-7 h) against human lung adenocarcinoma (A549) cell line

	Human Lung Cancer Cell Line A-549, % Control Growth, (µg/ml)
	Experiment 1				Experiment 2				Experiment 3				Average Values
Compounds	10	20	40	80	10	20	40	80	10	20	40	80	10	20	40	80
7a	150.7	117.0	138.9	102.8	49.5	118.8	88.4	82.0	93.4	98.7	93.5	78.3	97.9	111.5	106.9	87.7
7c	128.4	99.8	129.4	123.1	67.1	95.4	52.0	86.1	65.7	90.6	132.9	61.7	87.1	95.3	104.7	90.3
7e	123.4	91.8	84.0	143.5	106.1	29.2	58.3	72.9	80.4	71.1	116.4	85.6	103.3	64.0	86.2	100.7
7f	72.7	87.1	67.4	131.8	69.8	65.9	19.3	86.5	77.4	85.7	114.3	100.8	73.3	79.6	67.0	106.4
7h	101.7	57.6	34.5	119.5	83.9	54.4	24.1	82.0	90.1	64.4	90.4	98.1	91.9	58.8	49.7	99.9
ADR	2.0	−40.7	−40.3	−52.3	−20.9	−54.5	−59.4	−51.8	−40.4	−40.1	− 41.3	− 52.5	− 19.8	− 45.1	− 47.0	−52.2

Table 5.

In vitro test results of the test compounds (drug concentrations (µg/ml) calculated from graph)

MCF-7	LC50	TGI	GI50*
7a	NE	NE	>80
7c	NE	NE	>80
7e	NE	NE	>80
7f	NE	NE	>80
7h	NE	NE	>80
ADR	63	<10	<10

3.2.3. Structure activity relationship (SAR)

The Structure-Activity Relationship (SAR) analysis is employed to elucidate the impact of molecular structural variations, specifically positioning of substituents, on the biological activity of compounds. Among the synthesized derivatives, five conjugates were selected for evaluation of their anti-cancer activity against A-549 and MCF-7 cell lines, with Adriamycin used as the standard compound. Among the compounds assessed, derivative 7a, which contains an isopropyl substituent, demonstrated significant activity, achieving an average growth inhibition of 38% against the MCF-7 breast cancer cell line and 87% against the A549 lung carcinoma cell line. A concentration-dependent increase in activity was observed within the tested range of 10–80 µg/mL, suggesting a modest enhancement in growth inhibition correlating with increasing concentrations of the compound. The MCF-7 cell line exhibited superior results in comparison to the A549 cell line. The 7f derivatives, characterized by the presence of two chloro substituents, displayed the lowest efficacy against both cell lines when compared to the standard drug.

3.3. Molecular docking

Molecular docking is a widely utilized computational strategy in medicinal chemistry, serving as a cornerstone in rational drug design by elucidating the principles of molecular recognition. It is primarily employed to predict the binding affinity between enzyme and ligand molecules through the estimation of binding energies. The crystal structure of the target protein (PDB ID: 1M17) [36] was retrieved from the RCSB Protein Data Bank and utilized for molecular docking studies. The protein purification process entailed the removal of all crystallographic water molecules and heteroatoms, as well as the elimination of all protein chains except for the A-chain, which harbors the active site. Following this, polar hydrogen atoms were incorporated into the refined structure. The prepared protein structure was subsequently saved in the PDBQT format using Biovia Discovery Studio. The two-dimensional structures of the ligands were sketched using ChemDraw Professional 16.0 and subsequently converted into three-dimensional conformations using the Avogadro software package. Molecular docking studies were performed using PyRx 0.8 software. The prepared protein was imported and converted into a macromolecule format, while the ligand structure was also loaded into the workspace. Both the macromolecule and ligand were selected for further processing, and their corresponding PDBQT files were generated for docking analysis. The grid box was defined by selecting both the protein and the ligand, followed by advancing through the setup process. Upon its generation, the grid box was meticulously adjusted to encompass the active site region, ensuring optimal alignment with the docking parameters. The grid dimensions are specified as 120 Å in the X-direction, 78 Å in the Y-direction, and 25 Å in the Z-direction. The docking process started by clicking the forward button. Following the completion of forward docking, the binding energy value was determined. A lower binding energy value generally signifies stronger intermolecular interactions, reflecting a higher binding affinity between the ligand and the target enzyme, and suggesting an enhanced potential for biological activity of the compounds. Molecular docking was conducted to determine interaction force scores between the ligands and protein active sites. Epidermal Growth Factor Receptor (EGFR) tyrosine kinase (PDB ID: 1M17) was selected as the protein target for anti-cancer studies against MCF-7 cell lines, with Adriamycin serving as the standard reference drug. The docking data revealed that the synthesized derivatives displayed binding affinities comparable to Adriamycin, indicating favorable interactions with the target protein. In the molecular docking studies with anticancer protein targets, derivative 7a, featuring isopropyl substituents, demonstrated a binding energy score of −9.3 kcal/mol. This is closely comparable to the binding energy of the standard drug, which exhibited a score of −9.9 kcal/mol. The amino acid residues forming the binding pocket for these derivatives include ARG 807A, PRO 675A, LEU 837A, ARG 808A, ALA 731A, LEU 723A, ILE 735A, GLU 738A, PRO 853A, ASP 831A, PHE 699A etc (Figure S5 & S6). Table S1 presents the molecular docking results for the synthesized compounds, detailing the binding energies (kcal/mol) of the eight pyridine derivatives (7a-7 h), which range from −7.5 to −9.3 kcal/mol. The complete dataset is available in the supplementary file. Among the synthesized derivatives, compound 7a demonstrated 38% growth inhibition against the MCF-7 cell line. This biological activity was further corroborated by molecular docking studies, which revealed a binding energy of −9.3 kcal/mol, closely comparable to the binding energy of the standard drug (−9.9 kcal/mol).

3.4. ADMET studies

The ADMET predictions provided by ADMETlab 3.0 offer valuable insights into the pharmacokinetic behavior and safety profiles of the synthesized compounds, facilitating a rational evaluation of their drug-likeness and therapeutic potential. Tables S7–S10 are provided in the supplementary file. The properties of absorption, distribution, metabolism, excretion, and toxicity (ADMET) are essential parameters that significantly impact the design, development, and safety evaluation of pharmaceuticals, agrochemicals, food additives, consumer products, and industrial chemicals. The results of the ADMET study were discussed against the optimal parameter ranges reported in the literature [37]. The physicochemical parameters evaluated (Table S2) included the number of hydrogen bond acceptors (nHA) and donors (nHD), aqueous solubility (logS), distribution coefficient at pH 7.4 (logD_7.4), partition coefficient (logP), and topological polar surface area (TPSA). Absorption-related properties (Table S3) encompassed Caco-2 cell permeability and human intestinal absorption (HIA), while distribution parameters (Table S4) included plasma protein binding (PPB), volume of distribution (VD), and blood-brain barrier (BBB) permeability. The metabolic profile (Table S5) was predicted based on cytochrome P450 (CYP) enzyme inhibition. Excretion characteristics were assessed through predicted half-life (T₁/₂) and clearance (CL). Toxicological endpoints included human hepatotoxicity (H-HT), Ames mutagenicity (AMES), skin sensitization potential (SkinSen), and hERG channel inhibition. Quantitative data were reported for parameters such as logS, logD₇.₄, logP, TPSA, Caco-2 permeability, PPB, VD, CL, T₁/₂, H-HT, AMES, SkinSen, and hERG inhibition, while categorical descriptors were employed for the remaining ADMET parameters. The calculated logS values indicated that compounds 7f and 7 g exhibit the lowest solubility, as evidenced by their markedly negative logS values when compared to standard drugs characterized by low logS values and high aqueous solubility. The calculated logD_7.4 and logP values of the synthesized pyridine conjugates fall within the range of 3 to 6, indicating low aqueous solubility, high membrane permeability, and moderate to high metabolic stability. These values are notably higher when compared to the reference drug. In particular, the logP values exceeding 3 suggest poor water solubility of the conjugates relative to Adriamycin. The Caco-2 permeability values for the synthesized compounds ranged from approximately −4.5 to −4.8, indicating moderate to high permeability across the intestinal epithelial cell monolayer, with the exception of Adriamycin, which displayed a slightly more negative value. Most of the compounds exhibited high human intestinal absorption (HIA) and significant plasma protein binding (PPB), suggesting favorable oral bioavailability and a correspondingly low concentration of unbound drug in plasma. Among the synthesized hybrids, compounds 7e, 7f, and 7 g are predicted to exhibit poor blood-brain barrier (BBB) permeability, which may be advantageous in minimizing potential central nervous system (CNS)-related side effects. The predicted volume of distribution (VD), expressed in L/kg, ranges from 0.008 to 0.433, with higher VD values suggesting an increased distribution of the compounds into tissues. Clearance rates exhibited variability, with Adriamycin demonstrating the highest clearance and compounds 7f and 7 g exhibiting the lowest. This variation significantly influences their persistence within the body. The toxicity profiles of the synthesized compounds and Adriamycin were predicted using ADMETlab descriptors, including hERG channel blockade (hERG), human hepatotoxicity (H-HT), Ames mutagenicity (AMES), and skin sensitization potential (SkinSen). Hepatotoxicity evaluations reveal that compounds such as Adriamycin and 7a exhibit a comparatively lower potential for inducing liver toxicity. hERG channel blockade, a recognized indicator of cardiotoxicity, is significant for all synthesized compounds, including Adriamycin. The LD₅₀ values indicate that compounds such as 7f and the standard drug exhibit lower acute toxicity.

4. Conclusion

The trisubstituted pyridine-dehydroacetic acid conjugates were synthesized in good yield through a Kröhnke condensation of DHA-derived chalcone analogues (3-cinnamoyl-4-hydroxy-6-methyl-2-oxo-2 H-pyrans) with various phenacyl bromides. Comprehensive structural characterization of the resulting hybrids was achieved by ¹H and ¹³C NMR spectroscopy, FT-IR analysis, and high-resolution mass spectrometry (HRMS). In vitro evaluation revealed that these compounds possess significant cytotoxicity against MCF-7 (breast) and A549 (lung) carcinoma cell lines, with analogue 7a demonstrating the greatest antiproliferative potency. Moreover, 7a exhibited superior binding affinities in molecular docking studies against key oncogenic targets and displayed a favorable in silico ADMET profile. docking and ADMET simulations and the experimental bioactivity data highlights the potential of these novel pyridine hybrids particularly compound 7a as promising therpaeautics value. The concordance between the between the docking and ADMET simulation results and the experimental bioactivity data underscores the potential of these novel pyridine hybrids particularly compound 7a as promising therapeutic candidates.

Article highlights

• Trisubstituted pyridine-appended dehydroacetic acid scaffolds were efficiently synthesized via a Krohnke condensation between 3-cinnamoyl-4-hydroxy-6-methyl-2-oxo-2 H-pyrans (DHA-derived chalcone analogues) and phenacyl bromides.

• Structural elucidation of all hybrid compounds was accomplished using ¹H and ¹³C NMR spectroscopy, FT-IR spectroscopy, and high-resolution mass spectrometry (HRMS).

• The synthesized compounds exhibited noteworthy anti-cancer activity against MCF7 (breast) and A549 (lung) cancer cell lines.

• Compound 7a emerged as the most potent analogue, superior docking affinity toward key oncogenic targets, and a favorable insilico ADMET profile.

• Molecular docking and ADMET simulations reinforced the experimental trends, underscoring the therapeutic promise of the newly synthesized pyridine hybrids, particularly 7a, as prospective anticancer agents.

Author contributions

Ginna Kumari contributed to the investigation, software implementation, and the drafting of the original manuscript. Priyanka Rani was responsible for formal analysis. Sudeep Dhillon and Mamta Chahal also carried out the formal analysis. Deepak Kumar Aneja provided scientific supervision. Swati Rani and Jai Devi conducted the computational studies. Mayank Kinger provided overall supervision of the research and guided the manuscript development.

Financial disclosure

The authors declare no financial relationships with any organization or entity that could be perceived as having a financial interest in, or potential conflict with, the subject matter or materials presented in this manuscript.

Competing interest disclosure

The authors declare no competing interests or relevant affiliations with any organization or entity that could influence the subject matter or materials presented in this manuscript.

Data availability statement

The data supporting the findings of this study are provided in the supplementary materials accompanying this article.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/17568919.2025.2533051