N-Benzyl-6-Chloro-4-Hydroxy-2-Quinolone-3-Carboxamides: Synthesis, Computational Studies, and Biological Investigation as Anticancer Agents

Abstract

Cancer remains the second leading cause of death worldwide, highlighting the urgent need for novel therapeutic agents. In this work, twenty derivatives of N-benzyl-6-chloro-4-hydroxy-2-quinolone-3-carboxamides were synthesized and spectroscopically analyzed using FT-IR, NMR (¹H and ¹³C), and elemental analysis. Substitution of benzyl moiety with o-CH₃ (8), p-OCH₃ (10), m-CH₃ (18), p-CH₃ (19), and p-CF₃ (21) demonstrated three-fold distinct cytotoxicity against human colon cancer (HCT-116) cells with IC_50s of 72.0, 100.0–112.0 µM. The cheminformatics calculations disclosed that the analogues possess diverse physicochemical properties and invariable predictions across six drug-likeness scoring models, supporting their potential cytotoxicity profile against colorectal cancer cell lines (Caco-2 and HCT-116). The docking studies against both wild-type and mutant PI3Kα clarified binding interactions, implying that particular functionalities improve efficacy and selectivity. This study provides further evidence for the therapeutic promise of quinolones in targeting cancer-specific pathways and expedites the process for developing potent anticancer agents.

1. Introduction

Cancer is the second most common cause of death worldwide and is characterized by disordered cellular growth, disintegrated signaling pathways, abnormal cellular functions, and widespread metastasis. Diverse factors contribute to cancer development, including oxidative stress, hypoxia, nutrition depletion, chemicals and radiation exposure, as well as lifestyle-related habits such as obesity and alcohol consumption [1]. Genetic abnormalities trigger signaling pathways such as (PI3K/PKB/mTOR) [2,3,4,5,6], (MAPK/ERK) [7,8], EGFR/RAS/RAF [9], and JAK/STAT [10], and hence, promote cancer development [11]. Current studies highlight the contribution of the microbiome in cancer development, paving the way for cutting-edge research [12,13,14]. And the prevalence of drug-resistant cancers emphasizes the urgent need for novel anticancer therapies. Accordingly, researchers in academia and pharmaceutical companies are making remarkable efforts to reveal and identify new drugs and drug targets for cancer treatment.

Applying ligand-based pharmacophore modeling and in silico screening of the National Cancer Institute (NCI) database, we disclosed N-benzyl-4-hydroxy-2-quinolone-3-carboxamide (1) as a lead phosphoinositide 3-kinase (PI3Kα) inhibitor with IC_50s of 1.1μM and 0.73μM against the wild-type (WT) and mutant (MUT) H1047R PI3Kα, respectively [15]. Successive lead optimization protocols identified N-phenyl-4-hydroxy-2-quinolone-3-carboxamide (2) as a selective MUT H1047R PI3Kα inhibitor demonstrating IC_50s of 9.4 μM and 2.5 μM for the WT PI3Kα and MUT H1047R PI3Kα, respectively [16]. Additionally, two quinolone compounds, Tasquinimod and Roquinimex (Linomide^®), are undergoing clinical trials as anticancer agents inciting antiangiogenetic effect [17,18] (Figure 1).

Studies declared that quinolones exhibit a sophisticated anticancer activity by triggering apoptosis, suppressing the cell cycle, disrupting the mitochondrial membrane, and ceasing angiogenesis [19,20,21,22,23,24]. We released analogues of N-phenyl-6-chloro-4-hydroxy-2-quinolone-3-carboxamide that showed a potent cytotoxicity against human colon carcinoma (HCT-116) (IC_50s = 4.9–79.5 µM) and epithelial colorectal adenocarcinoma (Caco-2) (IC_50s = 13.8–81.4 µM) cells [25]. In order to probe the effect of elongation of the carboxamide side-chain, we employed a substituted benzyl moiety. And to derive their structure–activity relationship (SAR), we attached a chloro moiety on position 6 to examine its effect on anticancer activity and to compare the activity with analogues harboring phenyl functionality. Reported studies revealed that chlorinated aromatic rings induce anticancer activity [26,27] and apoptosis [28].

Building on our previous discovery of N-phenyl-4-hydroxy-2-quinolone-3-carboxamides as PI3Kα inhibitors [16], we now describe a strategic structural modification that enhances the scaffold’s therapeutic profile. By elongating the carboxamide side-chain with a benzyl linker, we generated a new series of N-benzyl-6-chloro-4-hydroxy-2-quinolone-3-carboxamides. This systematic study integrating computational and experimental chemical biology methods shows that this single-atom change markedly improves selectivity toward human colon carcinoma HCT-116 cells, which carry mutant PI3Kα. We also defined a new, detailed SAR for the benzyl series, revealing a preference for electron-donating substituents and position-dependent effects that point to tailored interactions within the PI3Kα kinase domain. Together, these findings establish the N-benzyl quinolone scaffold as a promising chemotype for selective anticancer agent development and provide a clear framework for its further optimization and potential clinical translation.

2. Results and Discussion

2.1. Chemistry

Target compounds (8–27) were synthesized to probe the effect of substituting the quinolone scaffold with a chloro moiety at position 6, accompanied by functionalization of the carboxamide side-chain by inserting a tailored benzyl motif, on their bioactivity. Ethyl anthranilate (4) was generated by refluxing a mixture of anthranilic acid with an excess of ethanol in acidic media (Figure 2). Next, 4 and diethyl malonate (5) were reacted under reflux in a basic medium to produce the target scaffold (6). Thin-layer chromatography (TLC) was applied to monitor the reaction’s progress. Compound (7) was assembled after the ethyl anthranilate spot had disappeared on TLC. Later, 7 and an excess of the corresponding benzylamines (R-CH₂-NH₂) (7a-n) were refluxed in THF and DMF to generate the targeted compounds (8–27), as presented in

Table 1. The chemical structures of 6-chloro-4-hydroxy-2-quinolone 3-carboxamides (8–27).

Compound	R	Compound	R
8		18
9		19
10		20
11		21
12		22
13		23
14		24
15		25
16		26
17		27

. Characterization of the chemical structures of 8–27 was carried out using ¹H and ¹³C-NMR, FTIR, and elemental analysis. The physicochemical and spectroscopic data of 8–27 are presented in the experimental section. The established experimental and spectroscopic data agree with the prospective structures.

2.2. Biological Evaluation of the Synthesized Compounds

To examine the cytotoxicity of compounds (8–27), we used the human colon cancer (HCT-116), colorectal adenocarcinoma (Caco-2), and prostate cancer cell (PC-3) cell lines. Two aligned biological studies were conducted at Toledo University in the USA against PC-3 and at the University of Jordan against HCT-116 and Caco-2 for all derivatives.

The malignant human colon carcinoma cell line (HCT-116) contains both wild-type (WT) and mutant (MUT) (H1047R) PI3Kα, and it was derived from an original cancer by a cell culture protocol [29]. HCT-116 cells encode matrix metalloproteinase 9 (MMP-9), AMP-activated protein kinase (AMPK), estrogen receptor β (ERβ), and prostaglandin E₂ (PGE2) receptors [30]. HCT-116 cells express Fas receptor (tumor necrosis factor receptor superfamily 6 (TNFRSF6) or CD95) and cell death regulator protein (B-cell lymphoma 2 (Bcl-2) [31,32]. HCT-116 cells encode nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), PI3K, peroxisome proliferator-activated receptor gamma (PPAR-γ) [33], and tumor suppressor gene (p53) [34]. The extracellular-signal-regulated kinase (1/2) (ERK) (1/2)/MAPK [35,36], PI3K/AKT [36,37,38], stromal cell-derived factor-1 (SDF-1)/chemokine receptor type 4 (CXCR4) [39,40], and JAK/STAT [41,42] pathways are essential for HCT-116 function. Histone deacetylase (HDAC-1, HDAC-2, HDAC-3) [43] and DNA methyltransferases (DNMTs) are expressed in HCT-116 cells [44].

The human colorectal adenocarcinoma (Caco-2) cell line encodes EGF, EGFR, retinoic acid binding protein I, and retinol binding protein II [45,46]. Caco-2 expresses MAPK, ERK1/2, JNK, protein kinase C (PKC-α), and MMP-9 [47,48]. The PI3K/AKT pathway and histone deacetylase (HDAC-1, HDAC-2, and HDAC-3) are essential for Caco-2 growth [49]. The Caco-2 expresses WT PI3Kα [50,51] while the malignant HCT-116 carcinoma encodes both WT and MUT H1047R PI3Kα [29]. Therefore, the difference in the activity between Caco-2 and HCT-116 related to MUT (H1047R) PI3Kα.

The malignant human prostate cancer cell line (PC-3) expresses HDACs (HDAC-1, HDAC-2, and HDAC-3) [52], androgen receptor [53], EGFR [54], PPAR-γ [55], human leukocyte antigen (HLA) (HLA1 and HLA9) [56], transforming growth factor-β (TGFβ) [57], and tyrosine protein kinase (c-Met) [58]. The JAK/STAT [59], PI3K/AKT [60], nuclear factor NF-Kappa B (NF-κB) [61], and c-Met/AKT/mTOR [62] pathways are essential for PC-3 function.

Biological data demonstrated that the verified analogues exerted distinct cytotoxicity against Caco-2 and HCT-116 cells. The analogues exerted obvious selective inhibitory activity against HCT-116 (

Table 2. Cytotoxic activity and selectivity of synthesized compounds in colorectal cancer cell lines Caco-2 and HCT-116.

Compound	IC50 μM		Selectivity Fold	Compound	IC50 μM		Selectivity Fold
	Caco-2	HCT-116			Caco-2	HCT-116
LY294002	7.4	6.5	1.1	17	218	102	2.1
6	>300	>300	>1.0	18	>300	103	>2.9
8	268	72	3.7	19	>300	109	>2.8
9	290	115	2.5	20	128	97	1.3
10	>300	100	>3.0	21	>300	112	>2.7
11	>300	164	>1.8	22	>300	143	>2.1
12	272	188	>1.5	23	>300	118	>2.5
13	261	212	>1.2	24	134	82	1.6
14	>300	218	>1.4	25	156	91	1.7
15	>300	279	>1.1	26	253	77	3.3
16	>300	240	>1.3	27	278	199	1.4

Cytotoxic activity illustrated as the IC₅₀ (μM) for synthesized compounds. The selectivity fold is calculated as IC₅₀ Caco-2/IC₅₀ HCT-116. The SD for the IC₅₀ values never exceeded 5%, where n = 9, and treatment time is 48 h.

). These findings emphasize the significance of elongating the carboxamide side-chain by one carbon and pave the way for future investigations into the effects of inserting two or more carbons on cytotoxicity.

The activity of analogue bearing o-OCH₃ (8), m-OCH₃ (9), and p-OCH₃ (10) demonstrates that o-OCH₃(8) exerts higher activity in HCT-116 compared to those of 9 and 10, suggesting that a hydrophobic and/or Hydrogen bond mediate(s) ligand/receptor interaction. However, the activity of o-CH₃ (17), m-CH₃ (18), and p-CH₃ (19) provides further support for Hydrogen bonding at the o-position, anticipating the closeness of OCH₃ at the o-position to the NH backbone.

In contrast, the activity of o-Cl (11), m-Cl (12), and p-Cl (13) demonstrates that o-Cl (11) and m-Cl (12) exhibit preferential activity comparable to p-Cl (13), suggesting that a hydrophobic lining is close to the o- and m-positions. However, the activity of o-F (14), m-F (15), and p-F (16) reveals that the steric effect and/or hydrophobicity might guide ligand/receptor complex formation.

Interestingly, a better cytotoxic effect is displayed for electron-donating groups (EDG), represented by 8–10 and 17–19, compared to those of electron-withdrawing groups (EWG), represented by 11–13 and 14–16. However, the activity of m-CF₃ (20) and p-CF₃ (21) suggests that hydrophobicity is more preferential than electronegativity. The data might pinpoint the steric effect while comparing the cytotoxicity of 11–13, 14–16, and 20–21. The size and hydrophobicity of -CF₃ are comparable to those of -OCH₃ and -CH_3, and therefore, it exerts a similar cytotoxic profile. Indeed, the activity of m-Br (24) provides a further clue for the steric effect and hydrophobicity on the m-position. However, the activity of p-NO₂ (27) aligns with those of p-Cl (13) and p-F (16) while the activity of o-NO₂ (25), m-NO₂ (26) surpasses those of o-F (14), m-F (15), o-Cl (11), and m-Cl (12), suggesting that NO₂ mediates Hydrogen bonding and/or enhances water solubility that might improve the pharmacokinetic profile (PK), and thus, in turn, potentiate the activity. The inhibitory effect of 22 and 23 clarifies that the N-position in pyridine affects the biological activity; specifically, N at the p-position might be a Hydrogen bond acceptor, and therefore, might induce the action. Finally, the activity of 6 confirms the significance of aromatic-ring attachment to enhancing the biological inhibitory effect in both cell lines. Eventually, comparing the activity of this series with a recently reported scaffold [25] shows that these analogues exhibit (2–3) folds of selectivity against the HCT-116 cell line. The results show that elongation of the carboxamide side-chain by one carbon enhances the activity and selectivity against HCT-116 cell lines. Biological data show that the synthesized analogues incite a suppressive activity against PC-3 cells (Supplementary Materials Figure S1).

In order to assess the toxicity of analogues against human normal cells and to evaluate their selectivity against cancer cells, compound 20 was screened against an adult primary dermal fibroblast (HDFa) (PCS-201-012), yielding an IC₅₀ value of 1428 uM. Interestingly, 20 exerted ≈15-fold selective toxicity against HCT-116 and 11-fold against Caco-2. Such a finding illustrates the safety profile of the verified analogues against normal cells, and paves the way for in vivo studies.

2.2.1. The Effect of Analogues on Apoptosis of HCT-116 Cancer Cells

The flow cytometric analysis of HCT cells using Annexin V-FITC/PI staining reveals that the experimental compounds exert potent pro-apoptotic activity, successfully driving cells from a viable state into programmed cell death. The control group confirmed high baseline viability at 98.5%, while the Doxorubicin (DOX) positive control induced near-total late apoptosis at 96.3%. Among the tested samples, analogue 8 demonstrated the most aggressive cytotoxic profile, with a 93.5% total apoptotic rate, characterized by a dominant late-apoptotic population (65.0%) that mirrors the kinetics of the DOX control (Figure 3).

In the series (20–26), the compounds initiated a more gradual apoptotic transition primarily characterized by early-stage signaling; sample 24 achieved the highest early apoptotic fraction (41.9%), whereas sample 26 proved most effective at driving cells into late-stage death (26.7%) for a total apoptotic population of 60.2%. These results collectively indicate that the analogues function as robust anticancer agents by collapsing the HCT viable cell reservoir and triggering a clear progression through the early and late stages of the apoptotic cascade (Supplementary Materials Figure S4).

2.2.2. The Effect of Analogues on the Cell Cycle of HCT-116 Cancer Cells

Based on the flow cytometric analysis of the HCT cell line, the analogues induced a significant redistribution of the cell cycle, characterized by a marked depletion of the resting phase and an accumulation in the active division phases. The control group exhibited a typical proliferative profile, with 36.8% of cells in the G0/1 phase. In contrast, treated samples, particularly 24, showed a substantial reduction in this population to 26.7%. This decrease in the G0/1 reservoir was accompanied by a concomitant increase in the G2/M-phase population, which rose to 20.2% in sample 24 compared to 17.7% in the control. A slight elevation in the S-phase fraction was also observed in the 24–26 series, peaking at 10.8%.

The observed redistribution, characterized by a contraction of the G0/G1 reservoir and accumulation in active division phases, suggests that the compounds push cells out of quiescence while simultaneously blocking successful cell cycle completion. This pattern is consistent with a cytostatic mechanism that limits proliferative capacity by enforcing checkpoint activation and preventing orderly progression through S phase and mitosis. Such effects are particularly relevant in the context of anticancer strategies, as sustained G2/M arrest is frequently associated with mitotic catastrophe and enhanced sensitivity to apoptosis or combination therapies (Figure 4).

Further studies examining cyclin expression, checkpoint kinase activation, and markers of DNA damage would be valuable to delineate the precise molecular mechanisms underlying this cell cycle perturbation [63].

2.3. Cheminformatics Characterization of Chemical Compounds

2.3.1. Drug-Likeness Analysis

All synthesized compounds exhibited comparable profiles across six drug-likeness scoring models, as illustrated in Figure 5. Notably, DLS_04 and the consensus score (DLS_cons) showed the greatest variability, reflecting differences in structural features and physicochemical properties. LY294002 was included as a reference molecule for comparative assessment.

Compounds 8, 9, and 10, which each bear a chloro and a methoxy group on different phenyl rings, demonstrated broader radar coverage and consistently higher scores, particularly in DLS_04 and DLS_cons, indicating strong drug-likeness across multiple models. Similarly, compounds 11–14, which feature two chlorinated phenyl rings, displayed balanced radar profiles with favorable DLS_cons values, suggesting consistent drug-likeness within this subseries. Compounds 15–17, bearing one chlorophenyl and one fluorophenyl group, maintained the core structure but showed slight shifts in DLS_04, likely due to electronic and steric effects introduced by the fluorine substituent.

In contrast, compounds 18–21, which include chlorophenyl groups combined with methylphenyl or trifluoromethyl groups, exhibited reduced DLS_cons and more variation in DLS_04, likely reflecting increased lipophilicity and steric bulk. The more rigid structures of compounds 22 and 23, incorporating a pyridine ring in place of a phenyl, resulted in diminished drug-likeness scores, particularly in DLS_cons. Finally, compounds 24–27, containing nitro or bromo substituents alongside a chlorophenyl moiety, displayed the lowest DLS_04 and DLS_cons values, suggesting less favorable overall drug-likeness profiles within this subset.

2.3.2. Chemical Space Analysis

Using 1790 2D alvaDesc descriptors, a PCA was conducted to explore the chemical diversity of the synthesized compounds (8–27). The first two principal components (PC1 = 56.5%, PC2 = 9.8%) accounted for 66.3% of the total variance in the dataset (Figure 6). Compounds were color-mapped based on their DLS_04 drug-likeness scores, which ranged from 0.4 (low, blue) to 0.6 (high, red) for the synthesized compounds.

Compounds 8, 9, and 10 clustered tightly in the upper-right quadrant (PC1 > 0, PC2 > 0), exhibiting the highest DLS_04 scores. These molecules share similar features: chloro and methoxy substituents on distinct aromatic rings, which appear to contribute to favorable drug-likeness. Likewise, compounds 17–19, positioned nearby in the upper-left quadrant (PC1 < 0, PC2 < 0), also demonstrated elevated DLS_04 values and similar chloro/methoxy substitution patterns.

In contrast, compounds 22 and 23, which bear pyridyl moieties instead of phenyl rings, are located in the lower-left quadrant (PC1 < 0, PC2 < 0) and showed reduced DLS_04 scores (deep blue), suggesting structural rigidity and heteroaromatic substitution that negatively impacted their drug likeness. Compounds 24–27, containing nitro or bromo substituents on one aromatic ring, also occupy regions associated with lower DLS_04 scores, and show a spatial shift from the high-performing analogues. Similarly, compounds 20 and 21, which contain bulkier lipophilic groups (e.g., trifluoromethyl), are separated far to the right (PC1 ≫ 0, PC2 < 0) but also display low drug-likeness, reinforcing that increased size and lipophilicity may compromise DLS_04. Meanwhile, compounds 12–14 occupy a central region and exhibit intermediate DLS_04 values, likely reflecting a balance between substitution pattern and molecular complexity.

Overall, the PCA clearly highlighted that structural features such as halogenation pattern, ring type, and substituent polarity influence drug-likeness (DLS_04), and that small chemical modifications can shift compounds across PCA space and affect their desirability as drug-like molecules with better potential for clinical development.

2.3.3. Mapping the Synthesized Analogues with the PI3Kα Pharmacophore Model

In order to clarify the cheminformatics analysis and examine the binding ability of the synthesized analogues (8–27), mapping of the 3D coordinates of 8–27 against our pharmacophore model for active PI3Kα inhibitors was performed. This pharmacophore model, shown in Figure 7, which encloses the key functional groups required for PI3Kα binding, presented as a guide to evaluate the alignment of 8–27 to these essential binding groups. Modeling results demonstrated that 8–27 fit the pharmacophoric features of active PI3Kα inhibitors. This accordance implies that the analogues (8–27) have the fingerprint for successful PI3Kα binding and, therefore, indicates their potential inhibitory activity. Such results provide a clue for the hypothesis that the cytotoxicity of the quinolones might be due to PI3Kα inhibition, underscoring the significance of these key functionalities in molecular interaction.

These findings support the promise of 8–27 as PI3Kα inhibitors and motivate future in-depth biological investigation of their cytotoxic mechanisms. Examining the structural basis of binding of the prospective ligands is essential for enhancing their potency and designing more effective analogues in the future.

2.3.4. Docking and Scoring Using Native and Mutant PI3Kα

The verified compounds were designed by a structure-based modification approach of selective phosphoinositide-3-kinase (PI3Kα) inhibitors that were presented by our group and described elsewhere [16]. Accordingly, docking studies against both wild-type (WT) and mutant (MUT) PI3Kα could provide explanations for the binding affinity and selectivity of the verified compounds. The compounds (8–21) that exhibited cytotoxicity against human colon carcinoma (HCT-116) and (Caco-2) cells were docked into the WT (PDB ID: 4L23) [65] and MUT (H1047R) (PDB ID: 3HHM) PI3Kα [66] to probe the structural basis of their binding interaction. Therefore, to understand the engagement of 8–27 in the PI3Kα kinase domain, we performed induced-fit docking (IFD) [67,68,69] studies against 4L23 and 3HHM structures. IFD data showed that 8–27 occupy PI3Kαs kinase domains. The docked poses of 11–13 and 14–16 overlay the template of X6K in WT and MUT PI3Kα kinase cleft (Figure 8A,B and Figure 9A,B).

Figure 9. The 3HHM kinase domain encloses the superposed IF docked poses of (A) 11 (violet), 12 (orange), 13 (wheat), and native coordinates of (X6K) (red color); (B) 14 (yellow), 15 (green), 16 (blue), and native coordinates of (X6K) (red color). Key binding residues and ligands’ core structures are portrayed in stick model, and H atoms are hidden for clarification. Picture made by PYMOL [69].

Figure 9. The 3HHM kinase domain encloses the superposed IF docked poses of (A) 11 (violet), 12 (orange), 13 (wheat), and native coordinates of (X6K) (red color); (B) 14 (yellow), 15 (green), 16 (blue), and native coordinates of (X6K) (red color). Key binding residues and ligands’ core structures are portrayed in stick model, and H atoms are hidden for clarification. Picture made by PYMOL [69].

Reported studies revealed that MUT H1047R PI3Kα incites carcinogenesis by triggering the kinase binding cleft [66]. Indeed, a remarkable decrease in Hydrogen bonding within MUT H1047R PI3Kα assemblies was revealed by molecular dynamics (MD) simulations, MD studies illustrated a wide distance between the regulatory and catalytic subunits, revealing unlimited phosphorylation of substrates and induction of carcinogenic signaling cascades [70]. Additionally, the MUT H1047R incites the PI3Kα loops (L1 and L2) to acquire a definite hook-like conformer, featured by a positively charged profile that expedites membrane translocation. Moreover, MD studies confirmed that MUT H1047R PI3Kα shapes a narrow binding cleft [71,72].

The IFD examines the conformational changes in PI3Kα coordinates, and ligands are docked to the kinase binding site using the iduced-fit docking [67,68,69]. The ligand template and PI3Kα binding domain are then energetically treated with the Prime algorithm [69], succeeded by a redocking approach. The flexibility of PI3Kα is evaluated during docking, revealing that the verified compounds form Hydrogen bonds with residues S773, S774, A775, W780, K802, D810, Y836, E849, V851, R852, N853, S854, Q859, H917, S919, N920, and D933 (

Table 3. The IFD scores, ΔGexp, ΔΔG (Kcal/mol) and Hydrogen bonding against native (PDB ID: 4L23) and mutant PI3Kα (PDB ID: 3HHM).

CPD ID	NATIVE PI3Kα (PDB ID: 4L23)				MUTANT PI3Kα (PDB ID: 3HHM)
	Docking Score	ΔGexp	ΔΔG	Hydrogen Bonding	Docking Score	ΔGexp	ΔΔG	Hydrogen Bonding
6	−8.18	NA	NA	K802, Y836, D933	−7.74	NA	NA	Y836
8	−7.13	−5.65	1.48	V851, S854, Y836	−7.20	−5.66	1.54	S774
9	−6.89	−5.37	1.51	Y836	−7.83	−5.34	2.46	S774, H917, N920
10	−7.13	−5.46	1.67	W780, E849, V851	−8.37	−5.45	2.92	NA
11	−7.16	−5.16	2.00	E849, V851	−7.92	−5.16	2.76	S774
12	−7.53	−5.08	2.45	D933	−8.04	−5.08	2.96	S774
13	−5.60	−5.01	0.59	W780, V851, S854	−9.04	−5.01	4.03	Y836, D933
14	−7.08	−4.99	2.08	W780, V851, S854	−7.28	−4.99	2.29	S774
15	−6.53	−4.85	1.68	S774, K802	−7.82	−4.85	2.97	E849, V851
16	−6.65	−4.94	1.71	E849, V851	−8.21	−4.94	3.27	S774
17	−6.71	−5.44	1.27	V851	−6.23	−5.45	0.78	D810, Y836
18	−7.60	−5.44	2.16	E849, V851	−7.48	−5.44	2.04	S773, N920, D933
19	−5.96	−5.41	0.56	V851	−6.62	−5.41	1.21	S773, N920, D933
20	−6.52	−5.47	1.04	W780, V851	−5.82	−5.48	0.34	S774, N920
21	−8.19	−5.39	2.80	V851	−9.81	−5.39	4.42	S774, N920
22	−9.66	−5.24	4.42	E849, Q859	−8.33	−5.25	3.08	S774, N920
23	−7.30	−5.36	1.94	S774, Y836	−9.45	−5.36	4.09	S774, A775, N920
24	−7.27	−5.57	1.70	K802, Y836	−9.02	−5.58	3.44	V851, S854
25	−6.54	−5.51	1.03	V851, R852, S854	−7.16	−5.51	1.65	S774, A775
26	−7.30	−5.61	1.68	E849, V851	−6.96	−5.62	1.34	D810, Y836, V851
27	−7. 19	−5.05	2.14	V851	−6.23	−5.05	1.18	S774
LY294002	−9.62	−7.08	2.55	V851	−10.43	−7.18	3.35	V851, Y836, Q859
Error			1.83				2.48

). The ability of the derivatives to engage with primary residues implies that structural modifications of the quinolone scaffold might improve the selectivity against the mutant isoform, which contributes to cancerogenic signaling pathways. The mean errors (ΔΔG) of 1.83 and 2.48 Kcal/mol confirmed the predictability of the IFD method, where ΔΔG = ΔG_exp − Docking scores.

Other computational [73,74] and experimental studies [66,75] pinpoint the importance of these residues in PI3Kα/ligand complex formation. Analogues 8–27 showed comparable binding affinity against WT and MUT (H1047R) PI3Kα, with residues S774, S854, Q859, and D933 implying that the core structure of 8–27 might be selective binders. Differences in binding scores and interactions underscore the contribution of the position of the substituent and its electronic features in promoting affinity and selectivity, due to the substituents’ effect on binding strength. Eventually, the verified compounds showed promising anticancer activity in functional assays.

3. Discussion

This study establishes the strategic redesign of the quinolone pharmacophore from an N-phenyl to an N-benzyl scaffold as a viable path to enhance anticancer selectivity. While the synthetic chemistry utilized robust, conventional methods to ensure efficient library generation, the novelty of this work lies in the systematic exploration of this specific chemotype (N-benzyl-6-chloro-4-hydroxy-2-quinolone-3-carboxamide) and the subsequent elucidation of its unique structure–activity and structure–property relationships. The incorporation of the 6-chloro substituent, combined with the critical one-carbon elongation of the side-chain, was a hypothesis-driven design informed by our computational models, which predicted improved target engagement.

Biological evaluation confirmed the success of this strategy. The new N-benzyl series demonstrated a marked 2-3-fold selectivity for HCT-116 cells over Caco-2 cells, outperforming the previous N-phenyl analogues [22]. This phenotypic selectivity provides a key mechanistic insight. Given that the primary genetic distinction between these cell lines is the presence of the oncogenic H1047R PI3Kα mutation in HCT-116, the data strongly suggest that the N-benzyl quinolone series preferentially impairs cancer cell viability in the context of mutant PI3Kα-driven signaling. This interpretation is structurally supported by our rigorous computational workflow. The induced-fit docking (IFD) studies confirmed that active analogues, such as 8 and 20, form stable complexes within the PI3Kα kinase domain, engaging key residues like Val851 and Ser854. The reliability of this model is underscored by its ability to reproduce the crystallized pose of a known PI3Kα inhibitor and to correctly rank order the relative potency of our compounds, including the reference inhibitor LY294002.

The detailed SAR further illuminates the requirements for activity within this new chemotype. A clear preference for electron-donating groups (EDGs) like -OCH₃ and -CH₃ over electron-withdrawing groups (EWGs) was observed, indicating the binding pocket favors increased electron density. Notably, analogues bearing o-CH₃ (8), p-OCH₃ (10), m-CH₃ (18), p-CH₃ (19), and p-CF₃ (21) demonstrated approximately three-fold increased cytotoxicity against HCT-116 cells, exhibiting IC₅₀ values of 72.0, 100.0, 103.0, 109.0, and 112.0 µM, respectively. These position-specific effects revealed the pocket’s topography: the enhanced activity of the o-OCH₃ analogue (8) suggests potential for Hydrogen bonding due to the closeness of OCH₃ at the o-position to the NH backbone, while the potency of bulky, hydrophobic groups at the meta position (e.g., m-CF₃ in 20, m-Br in 24) highlights a favorable hydrophobic subpocket. The promising safety profile of compound 20, which showed ~15-fold selectivity for HCT-116 over normal human dermal fibroblasts, underscores the therapeutic potential of this scaffold for developing anticancer agents with minimal off-target toxicity.

The flow cytometric analysis of HCT cells using Annexin V-FITC/PI staining reveals that the verified analogues exert potent pro-apoptotic activity, successfully pushing cells from a viable state into programmed cell death. Analogue 8 displayed the most aggressive cytotoxic profile with a 93.5% total apoptotic rate, characterized by a dominant late-apoptotic population (65.0%) that reflects the kinetics of the DOX control. Analogues 20–26 produced a more gradual apoptotic transition, primarily characterized by early-stage signaling; analogue 24 achieved the highest early apoptotic fraction (41.9%), whereas analogue 26 proved most effective at driving cells into late-stage death (26.7%) for a total apoptotic population of 60.2%.

The flow cytometric analysis of the HCT cell line showed that the analogues induced a significant redistribution of the cell cycle, characterized by a marked depletion of the resting phase and an accumulation in the active-division phases. The control group exhibited a typical proliferative profile, with 36.8% of cells in the G0/1 phase. In contrast, treated samples, particularly 24, showed a substantial reduction in this population to 26.7%. This decrease in the G0/1 reservoir was accompanied by a concomitant increase in the G2/M-phase population, which rose to 20.2% in sample 24 compared to 17.7% in the control. A slight elevation in the S-phase fraction was also observed in the 24–26 series, peaking at 10.8%.

The observed redistribution, characterized by a contraction of the G0/G1 reservoir and accumulation in active-division phases, suggests that the compounds push cells out of quiescence while simultaneously blocking successful cell cycle completion. Such effects are particularly relevant in the context of anticancer strategies, as sustained G2/M arrest is frequently associated with mitotic catastrophe and enhanced sensitivity to apoptosis or combination therapies.

Cheminformatics analysis offered a complementary view of drug-likeness, revealing clear structure–property relationships. PCA of 2D descriptors showed that methoxy-substituted analogues (8–10) clustered in a region of high drug-likeness, whereas more rigid pyridyl derivatives (22–23) and bulky lipophilic analogues occupied less favorable regions. This pattern was confirmed by DLS_04 and DLS_cons scores: methoxybenzyl derivatives scored highest, chlorobenzyl and fluorobenzyl analogues remained acceptable, and drug-likeness progressively declined for methyl/trifluoromethylbenzyl, pyridyl, and bromo/nitrobenzyl derivatives. Overall, excessive rigidity, size, and lipophilicity were associated with poorer drug-likeness within this series.

The convergence of evidence from our multi-tiered computational strategy, encompassing pose reproduction, benchmark compound validation, internal SAR trend prediction, and pharmacophore compliance, provided a reliable and compelling rationale for the proposed mechanism of action. The compounds presented, particularly the selective leads, represent high-priority candidates, and their submission for external enzymatic validation is a defined and immediate goal of our ongoing research.

In conclusion, this study establishes the N-benzyl-6-chloro-4-hydroxy-2-quinolone-3-carboxamide scaffold as a novel and promising chemotype for anticancer development. The strategic elongation of the carboxamide side-chain yielded derivatives with remarkable selectivity for HCT-116 cells, a profile mechanistically linked through our integrated computational approach to the potential preferential targeting of mutant PI3Kα signaling. The convergence of this phenotypic screening, selectivity profiling, and multi-tiered computational validation provides a compelling and rational foundation for the proposed mechanism of action. The elucidated SAR and drug-like property landscape offer a robust, actionable framework for the further optimization of these compounds. Consequently, lead candidates such as 8 and 20 are nominated as high-priority for subsequent investigation, including direct in vitro enzymatic validation and in vivo efficacy studies, to fully realize their therapeutic potential.

4. Materials and Methods

4.1. Chemistry

All the previously mentioned chemicals were of analytical grade and highly purified; hence, they were utilized without further purification.

All the chemicals and solvents used in this project have been purchased from the corresponding companies (SD Fine-Chem Limited (SDFCL, Mumbai, India), (Acros Organics, Belgium, WI, USA), (Sigma-Aldrich, St. Louis, MO, USA), (Fluka, Everett, Washington, DC, USA), (Sharlau, Barcelona, Spain), (Tedia, Fairfield, OH, USA), (Gainland Chemical Company (GCC), Deeside, Clwyd, UK), and (BBC Chemicals, Portland Place, London, UK): n-hexane (95%), anhydrous tetrahydrofuran (THF), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and petroleum ether (M-Tedia); ethanol (C₂H₅OH) and chloroform (CHCl₃) (Emsure Company, Jamshedpur, Jharkhand, India); methanol (CH₃OH) and sodium bicarbonate (NaHCO₃) (Sigma-Aldrich); ethyl acetate (C₄H₈O₂) and anhydrous sodium sulfate (Na₂SO₄) (Fisher Scientific, Waltham, MA, USA); sulfuric acid (H₂SO₄) and sodium ethoxide (C₂H₅ONa) (Honeywell/Fluka, Charlotte, NC, USA); acetone 99.8% (LABCHEM, Zelienople, PA, USA), hydrochloric acid (HCl) 35.4% (Alpha-Chemika, Mumbai, India), silicone liquid, and calcium chloride (BBC Chemicals, Blackburn, Lancashire, UK); 3-Trifluromethylbenzylamine, 4-trifluromethyl benzyl amine, 2-methoxy benzyl amine, 3-methoxy benzyl amine, 4-methoxybenzylamine, 2-chlorobenzyl amine, 3-chlorobenzylamine, 4-chlorobenzylamine, 2-flurobenzylamine, 3-flurobenzylamine, 4-flurobenzyl, 3-bromobenzylamine, 2-methylbenzylamine, 3-methylbenzylamine, 4-methylbenzylamine, 2-nitrobenzylamine, 3-nitrobenzylamine, 4-nitrobenzylamine, pyridine-3-ylmethylamine, and pyridine-4-ylmethylamine (Sigma-Aldrich, St. Louis, MO, USA).

Thin-layer chromatography (TLC) was performed on 20 × 20 cm and 0.20 mm thick pre-coated aluminum sheets with fluorescent silica gel (Macherey-Nagel, Düren, Germany) and visualized using UV light (254/366nm, Terra Universal Company, Fullerton, CA, USA). Evaporation of ordinary solvents was carried out using a Rota vapor model R-215 (Buchi, Meierseggstrasse, Flawil, Switzerland) linked to a vacuum pump v-700 and a heating water bath (Hei-VAP value digital, Heidolph, Schwabach, Germany). The melting point was measured using a Gallenkamp melting point apparatus. Hot plates and magnetic stirrers were obtained from Thermo Scientific Cimarec, Waltham, MA, USA. Shimadzu IR Affinity FTIR spectrophotometer was used to record Infrared (IR) spectra, Kyoto, Japan; samples were mixed with potassium bromide (Sigma-Aldrich, St. Louis, MO, USA) and pressed into a disc. ¹H and ¹³C Nuclear Magnetic Resonance (NMR) spectra were analyzed by Bruker NanoBay 400 MHz spectrophotometer, Houston, TX, USA, (the Hashemite University) and Bruker AMX 500, Billerica, MA, USA (500 MHz for ¹ H nucleus and 125 MHz ¹³C nucleus at magnet strength (B0) of 14.1 Tesla) for spectrophotometer at 293 K using standard Bruker software (Bruker, Faellanden, Switzerland AG, version 4) (the University of Jordan); chemical shifts are expressed in δ (ppm) and (J) coupling constant values are presented in Hz (Hertz) using TMS internal reference; DMSO and/or NaOD were/was used to dissolve the samples. Elemental analyses (the Hashemite University) were conducted using a Euro Vector (Pavia, Italy) elemental analyzer, model EUROEA3000 A.

4.2. Synthesis of the Targeted Compounds

4.2.1. Ethyl 2-Amino-5-chlorobenzoate (4)

A mixture of 2-amino-5-chlorobenzoic acid (3) (5.00 g, 29.14 mmol) and an excess amount of absolute ethanol (450 mL) was prepared with drop-wise addition of sulfuric acid (H₂SO₄) (10 mL) in an ice bath. Reaction was refluxed in an oil bath at temperature (90–95 °C). Completion of the reaction was accomplished by the formation of a new spot on TLC after 7 days. The excess of the solvent was evaporated in a rotary evaporator at (50 °C) and the residual acid was extracted with sodium bicarbonate solution (5%) and chloroform four times. And extraction with chloroform and water (20 mL) three times followed in order to remove any byproducts. Then, the organic layer was dried by the addition of anhydrous sodium sulphate and filtered, followed by evaporation under reduced pressure.

4.2.2. Ethyl 6-Chloro-4-hydroxy-2-quinolone 3-carboxylate (6)

Ethyl 2-amino-5-chlorobenzoate (4) (5.32 g, 26.62 mmol) was dissolved in (37.2 mL) DMSO followed by addition of diethyl malonate (5) (40.40 mL, 266.02 mmol) and sodium ethoxide (5.43 g, 79.80 mmol). This mixture was refluxed at (130–140 °C) for 5 days. The absence of a (4) spot on TLC confirmed the completion of the reaction. The reaction mixture was cooled in an ice bath, rinsed with (10 mL) water and acidified with (0.3 M) HCl until pH 4.5 was reached. The resulting precipitate was filtered and washed with water, THF, and methanol, then dried in a vacuum oven at (70 °C). Beige powder (6.48 g) with yield 91%, R_f = 0.7 (CHCl₃); m.p: decomposes at 239 °C; ¹H-NMR (400 MHz, DMSO-d₆): δ = 1.20 (t, J = 6.8 Hz, 3H, -CH₃), 4.11 (q, J = 6.8 Hz, 2H, -OCH₂), 6.89 (d, J = 8.8 Hz, 1H, Ar-H), 6.97 (d, J = 2.8 Hz, 1H, Ar-H), 7.72 (s, 1H, Ar-H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆): δ = 15.0 (1C), 58.6 (1C), 101.98 (1C), 119.1 (1C), 124.1 (1C), 124.2 (1C), 125.3 (1C), 127.9 (1C), 149.7 (1C), 172.9 (2C), 173.7 (1C) ppm; IR (KBr disc): 3010 (C-H), 2931 (C-H), 2854 (C-H), 1747 (C=O), 1678 (C=O), 1602–1477 (C=C aromatic) cm⁻¹. Anal. Calcd. for C₁₂H₁₀ClNO₄: C, 53.85; H, 3.77; N, 5.23. Found: C, 54.00; H, 3.94; N, 5.09.

4.2.3. N-(2-Methoxybenzyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (8)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and 2-methoxybenzylamine (7b) (2.50 mL, 7.32 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF. The mixture was refluxed and heated at (120–130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the inner flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and methanol, then drying in a vacuum oven at (70 °C). Beige powder (0.70 g) with yield 52%, R_f = 0.4 (n-C₆H₁₄: EtOAc) (6:4); m.p: decomposes at 230 °C; ¹H-NMR (400 MHz, DMSO-d₆): δ = 3.82 (s, 3H, -OCH₃), 4.49 (d, J = 3.2 Hz, 2H, -CH₂), 6.86–6.88 (m, 2H, Ar-H), 6.94–6.99 (m, 2H, Ar-H), 7.23–7.25 (m, 2H, Ar-H), 7.92 (s, 1H, Ar-H) 10.74 (m, 1H, -NH), 11.00 (s, 1H, -NH), 11.74 (s, 1H, -OH) ppm; ¹³C-NMR (100 MHz, DMSO-d₆): δ = 37.0 (1C), 55.8 (1C), 97.8, (1C), 110.8 (1C), 117.5 (1C), 120.6 (2C), 123.4 (1C), 125.0 (1C), 125.2 (1C), 128.1 (1C), 131.2 (1C), 139.7 (1C),151.9 (1C), 157.4 (1C), 166.2 (1C). 169.8 (1C) 176.1 (1C) ppm; IR (KBr disc): 3394 (O-H), 3278 (N-H), 3059 (C-H), 2945 (C-H), 2833 (C-H), 1639 (C=O), 1602- 1496 (C=C aromatic) cm⁻¹. Anal. Calcd. for C₁₈H₁₅ClN₂O₄: C, 60.26; H, 4.21; N, 7.81. Found: C, 59.51; H, 4.67; N, 7.42.

4.2.4. N-(3-Methoxybenzyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (9)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and 3-methoxybenzylamine (7c) (2.50 mL, 7.32 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF. The mixture was refluxed and heated at (120–130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the inner flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and methanol, then drying in a vacuum oven at (70 °C). White powder (0.57 g) with yield 43%, R_f = 0.4 (n-C₆H₁₄: EtOAc) (6:4); m.p: 230–233 °C; ¹H-NMR (400 MHz, DMSO-d₆ + NaOD): δ = 3.74 (s, 3H, -OCH₃), 4.46 (s, 2H, -CH₂), 6.77 (dd, J = 2Hz, 8Hz, 1H, Ar-H), 6.90–6.95 (m, 3H, Ar-H), 7.03–7.06 (m, 1H, Ar-H), 7.20–7.24 (m, 1H, Ar-H), 7.78 (s, 1H, Ar-H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆ + NaOD): δ = 42.0 (1C), 55.4 (1C), 100.9 (1C), 112.0 (1C), 113.4 (1C), 119.6 (1C), 119.8 (1C), 124.3 (1C), 124.6 (1C), 128.5 (1C), 129.7 (2C), 143.3 (1C), 148.9 (1C), 159.7 (1C), 171.9 (1C), 173.9 (1C), 175.8 (1C) ppm; IR (KBr disc): 3203 (N-H), 2970 (C-H), 2900 (C-H), 2837 (C-H), 1643 (C=O), 1589–1490 (C=C aromatic) cm⁻¹. Anal. Calcd. for C₁8H₁₅ClN₂O₄: C, 60.2 6; H, 4.21; N, 7.81. Found: C, 59.11; H, 4.50; N, 7.54.

4.2.5. N-(4-Methoxybenzyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (10)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and 4-methoxybenzylamine (7a) (3.50 mL, 10.24 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF. The mixture was refluxed and heated at (120–130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the inner flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and methanol, then drying in a vacuum oven at (70 °C). Beige powder (0.86 g) with yield 65%, R_f = 0.4 (n-C₆H₁₄: EtOAc) (6:4); m.p: >360 °C; ¹H-NMR (400 MHz, DMSO-d₆): δ = 3.76 (s, 3H, -CH₃), 4.41 (s, 2H, -CH₂), 6.76 (d, J = 6.4 Hz, 2H, Ar-H), 7.19–7.23 (m, 2H, Ar-H), 7.44 (d, J = 8.4 Hz, 2H, Ar-H), 7.91 (s, 1H, Ar-H), 10.74 (s, 1H, -NH), 11.79 (s, 1H, -OH) ppm; ¹³C-NMR (100 MHz, DMSO-d₆): δ = 41.6 (1C), 55.4 (1C), 98.3 (1C), 114.1 (2C), 117.4 (1C), 123.4 (1C), 125.1 (1C), 125.2 (1C), 129.4 (2C), 131.4 (1C), 132.3 (1C), 137.8 (1C), 158.5 (2C), 169.5 (1C) ppm; IR (KBr disc): 3155 (N-H), 2987 (C-H), 2939 (C-H), 2831 (C-H), 1656 (C=O), 1614–1512 (C=C aromatic) cm⁻¹. Anal. Calcd. for C₁₈H₁₅ClN₂O₄: C, 60.26; H, 4.21; N, 7.81. Found: C, 59.61; H, 4.45; N, 7.22.

4.2.6. N-(2-Chlorobenzyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (11)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and 2-chlorobenzylamine (7d) (5.80 mL, 18.73 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF. The mixture was refluxed and heated at (120-130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the inner flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and methanol, then drying in a vacuum oven at (70 °C). Off-white powder (0.50 g) with yield 37%, R_f = 0.5 (n-C₆H₁₄: EtOAc) (6:4); m.p: 298–300 °C; ¹H-NMR (400 MHz, DMSO-d₆): δ = 4.57 (s, 2H, -CH₂), 7.23–7.29 (m, 3H, Ar-H), 7.40–7.46 (m, 3H, Ar-H), 7.94 (s, 1H, Ar-H), 10.73 (s, 2H, -NH), 11.89 (s, 1H, -OH) ppm; ¹³C-NMR (100 MHz, DMSO-d₆): δ = 40.2 (1C), 98.3 (1C), 117.5 (1C), 123.3 (1C), 125.1 (1C), 127.5 (1C), 129.0 (1C), 129.5 (1C), 129.7 (1C), 131.4 (1C), 132.8 (1C), 137.4 (1C), 137.6 (1C), 166.3 (1C), 169.8 (2C) ppm. IR (KBr disc): 3383 (O-H), 3269 (N-H), 3061 (C-H), 1635 (C=O), 1606–1463 (C=C aromatic) cm⁻¹. Anal. Calcd. for C₁₇H₁₂Cl₂N₂O₃: C, 56.22; H, 3.33; N, 7.71. Found: C, 55.51; H, 3.42; N, 7.13.

4.2.7. N-(3-Chlorobenzyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (12)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and 3-chlorobenzylamine (7e) (5.80 mL, 18.73 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF. The mixture was refluxed and heated at (120–130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the inner flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and methanol, then drying in vacuum oven at (70 °C). Beige powder (0.37 g) with yield 27%, R_f = 0.4 (n-C₆H₁₄: EtOAc) (6:4); m.p: 256–259 °C; ¹H-NMR (400 MHz, DMSO-d₆ + NaOD): δ = 4.49 (s, 2H, -CH₂), 6.95 (d, J = 8.8 Hz, 1H, Ar-H), 7.03 (dd, J = 2.8 Hz, 8.8 Hz, 1H, Ar-H), 7.24 (d, J = 2.4 Hz, 1H, Ar-H), 7.26–7.34 (m, 3H, Ar-H), 7.78 (s, 1H, Ar-H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆): δ = 41.4 (1C), 100.8 (1C), 119.6 (1C), 124.3 (1C), 126.3 (1C), 126.6 (1C), 127.3 (2C), 128.5 (1C), 130.5 (1C), 133.4 (2C), 144.6 (1C), 148.9 (1C), 172.0 (1C), 173.9 (1C), 175.9 (1C) ppm; IR (KBr disc): 3217 (N-H), 2976 (C-H), 2891 (C-H), 2839 (C-H), 1672 (C=O), 1597–1471 (C=C aromatic) cm⁻¹. Anal. Calcd. for C₁₇H₁₂Cl₂N₂O₃: C, 56.22; H, 3.33; N, 7.71. Found: C, 55.68; H, 3.55; N, 7.49

4.2.8. N-(4-Chlorobenzyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (13)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and 4-chlorobenzylamine (7f) (10.40 mL, 33.58 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF. The mixture was refluxed and heated at (120–130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the inner flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and acetone, then drying in a vacuum oven at (70 °C). White powder (0.99 g) with yield 73%, R_f = 0.5 (n-C₆H₁₄: EtOAc) (6:4); m.p: 285–288 °C; ¹H-NMR (400 MHz, DMSO-d₆ + NaOD): δ = 4.47 (s, 2H, -CH₂), 6.95 (d, J = 8.8 Hz, 1H, Ar-H), 7.03 (d, J = 8.8 Hz, 1H, Ar-H), 7.31–7.37 (m, 4H, Ar-H), 7.78 (s, 1H, Ar-H) ppm. ¹³C-NMR (100 MHz, DMSO-d₆ + NaOD): δ = 41.4 (1C), 100.9 (1C), 119.7 (1C), 124.2 (1C), 128.5 (3C), 129.5 (3C), 131.2 (1C), 140.7 (2C), 148.8 (1C), 172.0 (1C), 173.7 (1C), 175.8 (1C) ppm. IR (KBr disc): 3381 (O-H), 3226 (N-H), 3070 (C-H), 2974 (C-H), 2893 (C-H), 1654 (C=O), 1597–1490 (C-C aromatic) cm⁻¹. Anal. Calcd. for C₁₇H₁₂Cl₂N₂O₃: C, 56.22; H, 3.33; N, 7.71. Found: C, 55.47; H, 3.01; N, 7.98.

4.2.9. N-(2-Fluorobenzyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (14)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and 2-fluorobenzylamine (7g) (9.70 mL, 30.63 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF. The mixture was refluxed and heated at (120–130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and methanol, then drying in a vacuum oven at (70 °C). White powder (0.64 g) with yield 50%, R_f = 0.4 (n-C₆H₁₄: EtOAc) (6:4); m.p: 255–258 °C; IR (KBr disc): ¹H-NMR (400 MHz, DMSO-d₆ + NaOD): δ = 4.53 (s, 2H, -CH₂), 6.95 (d, J = 8.8 Hz, 1H, Ar-H), 7.03 (d, J = 8.8 Hz, 1H, Ar-H), 7.12–7.17 (m, 2H, Ar-H), 7.25–7.29 (m, 1H, Ar-H), 7.44 (d, J = 7.6 Hz, 1H, Ar-H), 7.79 (s, 1H, Ar-H) ppm. ¹³C-NMR (125 MHz, DMSO-d₆ + NaOD): δ = 35.6 (1C), 100.8 (1C), 115.1 (1C, ²F_CF = 20 Hz), 119.5 (1C), 124.3 (2C), 124.6 (1C), 124.7 (1C, ⁴F_CF = 2.5 Hz), 128.3 (1C, ²F_CF = 23.7 Hz), 128.4 (1C), 128.6 (1C, ³F_CF = 7.5 Hz), 129.9 (1C, ³F_CF =5 Hz), 149.0 (1C), 160.5 (1C, ¹F_CF = 241 Hz), 172.0 (1C), 173.9 (1C), 175.9 (1C). IR (KBr disc): 3246 (N-H), 3072 (C-H), 2962 (C-H), 2899 (C-H), 1664 (C=O), 1597–1490 (C=C aromatic) cm⁻¹. Anal. Calcd. for C₁₇H₁₂ClFN₂O₃: C, 58.89; H, 3.49; N, 8.08. Found: C, 59.43; H, 3.02; N, 7.55.

4.2.10. N-(3-Fluorobenzyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (15)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and 3-fluorobenzylamine (7h) (6.00 mL, 18.95 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF. The mixture was refluxed and heated at (120–130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the inner flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and methanol, then drying in a vacuum oven at (70 °C). White powder (0.72 g) with yield 56%, R_f = 0.4 (n-C₆H₁₄: EtOAc) (6:4); m.p: 260–263 °C; ¹H-NMR (400 MHz, DMSO-d₆ + NaOD): δ = 4.51 (s, 2H, -CH₂), 6.95 (d, J = 8.8 Hz, 1H, Ar-H), 7.03–7.06 (m, 2H, Ar-H), 7.19 (dd, J = 7.6 Hz, 20.8Hz, 2H, Ar-H), 7.33 (d, J = 6.4 Hz, 1H, Ar-H), 7.78 (s, 1H, Ar-H) ppm; ¹³C-NMR (125 MHz, DMSO-d₆ + NaOD): δ = 41.5 (1C), 100.9 (1C), 113.3 (1C, ²F_CF = 20.07 Hz), 114.1 (1C, ²F_CF = 21.3 Hz), 119.8 (1C), 123.6 (1C, ⁴F_CF = 2.5 Hz), 124.2 (1C), 124.2 (1C), 124.5 (1C), 128.6 (1C), 130.5 (1C, ³F_CF = 8.8 Hz), 144.9 (1C, ³F_CF = 6.3 Hz), 148.8 (1C), 162.7 (1C, ¹F_CF = 241 Hz), 172.0 (1C), 173.8 (1C), 175.9 (1C); IR (KBr disc): 3244 (N-H), 3147 (C-H), 3045 (C-H), 2987 (C-H), 2904 (C-H), 1681 (C=O), 1643–1490 (C=C aromatic) cm⁻¹. Anal. Calcd. for C₁₇H₁₂ClFN₂O₃: C, 58.89; H, 3.49; N, 8.08. Found: C, 58.73; H, 3.43; N, 7.85.

4.2.11. N-(4-Fluorobenzyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (16)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and 4-fluorobenzylamine (7i) (3.60 mL, 11.37 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF. The mixture was refluxed and heated at (120–130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the inner flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and methanol, then drying in a vacuum oven at (70 °C). White powder (0.95 g) with yield 73%, R_f = 0.4 (n-C₆H₁₄: EtOAc) (6:4); m.p: 276–279 °C; ¹H-NMR (400 MHz, DMSO-d₆ + NaOD): δ = 4.45 (s, 2H, -CH₂), 6.92–6.96 (m, 1H, Ar-H), 7.02–7.13 (m, 3H, Ar-H), 7.37 (t, J = 7.2 Hz, 2H, Ar-H), 7.78 (s, 1H, Ar-H) ppm; ¹³C-NMR (125 MHz, DMSO-d₆ + NaOD): δ = 41.3 (1C), 100.8 (1C), 115.2 (2C, ²F_CF = 20.0 Hz), 120.4 (1C), 123.6 (1C), 124.3 (1C), 124.4 (1C), 128.8 (1C), 129.6 (2C, ³F_CF = 7.5 Hz), 137.6 (1C), 147.7 (1C), 161.3 (1C, ¹F_CF = 240 Hz), 171.7 (1C), 173.0 (1C), 175.8 (1C) ppm; IR (KBr disc): 3385 (O-H), 3244 (N-H), 3143 (C-H), 2976 (C-H), 2893 (C-H), 1676 (C=O), 1633–1510 (C=C aromatic) cm⁻¹. Anal. Calcd. for C₁₇H₁₂ClFN₂O₃: C, 58.89; H, 3.49; N, 8.08. Found: C, 58.53; H, 3.53; N, 8.39. 14

4.2.12. N-(2-Methylbenzyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (17)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and o-tolymethanamine/2-methylbenzylamine (7l) (4.00 mL, 11.11 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF. The mixture was refluxed and heated at (120–130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the inner flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and methanol, then drying in a vacuum oven at (70 °C). Beige powder (0.80 g) with yield 63%, R_f = 0.4 (n-C₆H₁₄: EtOAc) (6:4); m.p: 240–242 °C; ¹H-NMR (400 MHz, DMSO-d₆ + NaOD): δ = 2.23 (s, 3H, -CH₃), 4.46 (s, 2H, -CH₂), 7.02–7.48 (m, 6H, Ar-H), 7.89 (s, 1H, Ar-H), 10.84 (s, 1H, NH), 11.07 (s, 1H, NH), 11.90 (s, 1H, NH) ppm. ¹³C-NMR (100 MHz, DMSO-d₆ + NaOD): δ = 19.1 (1C), 42.1 (1C), 100.8 (1C), 120.5 (1C), 124.3 (1C), 124.7 (2C), 126.1 (1C), 126.8 (2C), 127.8 (1C), 128.4 (1C), 130.2 (1C), 135.9 (1C), 139.2 (1C), 171.6 (1C), 173.6 (1C), 175.7 (1C) ppm. IR (KBr disc): 3204 (N-H), 2972 (C-H), 2906 (C-H), 1683 (C=O), 1631–1467 (C=C aromatic) cm⁻¹. Anal. Calcd. for C₁₈H₁₅ClN₂O₃: C, 63.07; H, 4.41; N, 8.17. Found: C, 62.85; H, 4.61; N, 7.92.

4.2.13. N-(3-Methylbenzyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (18)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and m-tolymethanamine/3-methylbenzylamine (7k) (4.00 mL, 11.11 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF. The mixture was refluxed and heated at (120–130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the inner flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and methanol, then drying in a vacuum oven at (70 °C). Beige powder (0.80 g) with yield 63%, R_f = 0.4 (n-C₆H₁₄: EtOAc) (6:4); m.p: 238–240 °C; ¹H-NMR (400 MHz, DMSO-d₆ + NaOD): δ = 2.28 (s, 3H, -CH3), 4.45 (s, 2H, -CH₂), 6.93 (d, J = 8.8 Hz, 1H, Ar-H), 7.02 (d, J = 6.4 Hz, 2H, Ar-H), 7.13–7.14 (m, 2H, Ar-H), 7.17–7.21 (m, 1H, Ar-H) 7.77 (s, 1H, Ar-H) ppm. ¹³C-NMR (100 MHz, DMSO-d₆ + NaOD): δ = 21.5 (1C), 42.1 (1C), 100.8 (1C), 119.3 (1C), 124.3 (1C), 124.8 (1C), 124.9 (2C), 127.3 (1C), 128.3 (2C), 128.5 (1C), 137.6 (1C), 141.6 (1C), 149.1 (1C), 171.9 (1C), 174.1 (1C), 175.8 (1C) ppm. IR (KBr disc): 3204 (N-H), 2972 (C-H), 2906 (C-H), 1683 (C=O), 1631–1467 (C=C aromatic) cm⁻¹. Anal. Calcd. for C₁₈H₁₅ClN₂O₃: C, 63.07; H, 4.41; N, 8.17. Found: C, 62.64; H, 4.78; N, 8.37.

4.2.14. N-(4-Methylbenzyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (19)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and 4-methylbenzylamine (7j) (8.10 mL, 22.49 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF. The mixture was refluxed and heated at (120–130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the inner flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and methanol, then drying in a vacuum oven at (70 °C). Beige powder (1.04 g) with yield 81%, R_f = 0.4 (n-C₆H₁₄: EtOAc) (6:4); m.p: 236–239 °C; ¹H-NMR (400 MHz, DMSO-d₆ + NaOD): δ = 2.27 (s, 3H, -CH3), 4.44 (s, 2H, -CH₂), 6.95 (d, J = 8.4 Hz, 1H, Ar-H), 7.03 (d, J = 6.0 Hz, 1H, Ar-H), 7.12 (d, J = 7.2 Hz, 2H, Ar-H), 7.23 (d, J = 7.2 Hz, 2H, Ar-H) 7.78 (s, 1H, Ar-H) ppm; ¹³C-NMR (100 MHz, DMSO-d₆ + NaOD): δ = 21.1 (1C), 41.9 (1C), 100.9 (1C), 119.5 (1C), 124.3 (2C), 124.6 (1C), 127.7 (2C), 128.4 (1C), 129.2 (2C), 135.7 (1C), 138.4 (1C), 148.9 (1C), 171.9 (1C), 173.9 (1C), 175.8 (1C) ppm; IR (KBr disc): 3300 (O-H), 3205 (N-H), 3070 (C-H), 2964 (C-H), 1680 (C=O), 1645–1512 (C=C aromatic) cm⁻¹. Anal. Calcd. for C₁₈H₁₅ClN₂O₃: C, 63.07; H, 4.41; N, 8.17. Found: C, 63.46; H, 3.97; N, 7.77.

4.2.15. N-(3-(Trifluoromethyl) benzyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (20)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and 3-(trifluoromethyl) benzylamine (7l) (4.00 mL, 12.39 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF. The mixture was refluxed and heated at (120–130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the inner flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and methanol, then drying in a vacuum oven at (70 °C). White powder (0.31 g) with yield 21%, R_f = 0.7 (EtOAc: n-C6H14) (8:2); m.p: 248–251 °C; 1H-NMR (400 MHz, DMSO-d6 + NaOD): δ = 4.55 (s, 2H, -CH₂), 6.95 (d, J = 8.8 Hz, 1H, Ar-H), 7.03–7.05 (dd, J = 8.8 Hz, 1H, Ar-H), 7.54 (m, 1H, Ar-H), 7.61 (m, 2H, Ar-H), 7.77 (s, 1H, Ar-H) ppm. ¹³C-NMR (125 MHz, DMSO-d₆ + NaOD): δ = 41.5 (1C), 100.9 (1C), 119.9 (1C), 123.4 (1C), 123.8 (1C), 124.2 (2C), 124.4 (1C), 124.7 (1C, q, ¹F_CF = 270 Hz), 128.6 (1C), 129.4 (1C, ²F_CF = 31 Hz), 129.7 (1C), 131.7 (1C), 143.3 (1C), 148.7 (1C), 171.9 (1C), 173.5 (1C), 175.7 (1C) ppm; IR (KBr disc): 3223 (N-H), 3047 (C-H), 2981 (C-H), 1656 (C=O), 1598–1473 (C=C aromatic) cm⁻¹. Anal. Calcd. for C₁₈H₁₂ClF₃N₂O₃: C, 54.49; H, 3.05; N, 7.06. Found: C, 54.89; H, 3.54; N, 7.00.

4.2.16. N-(4-(Trifluoromethyl) benzyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (21)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and 4-(trifluoromethyl) benzylamine (7m) (4.00 mL, 11.11 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF. The mixture was refluxed and heated at (120–130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the inner flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and methanol, then drying in a vacuum oven at (70 °C). White powder (0.052 g) with yield 4.0%, R_f = 0.6 (EtOAc: n-C₆H₁₄) (8:2); m.p: decomposes at 287 °C; ¹H-NMR (400 MHz, DMSO-d₆ + NaOD): δ = 2.15 (m, 3H, -CH₃) 4.46 (s, 1H, -CH₂), 6.88–7.89 (m, 7H, Ar-H), 11.06 (s, 1H, -NH), 11.75 (s, 1H, -OH) ppm. ¹³C-NMR (125 MHz, DMSO-d₆ + NaOD): δ = 41.7 (1C), 101.0 (1C), 119.9 (1C), 124.2 (1C), 124.4 (1C), 124.7 (1C, q, ¹F_CF = 270 Hz), 125.4 (2C), 127.4 (1C, q, ²F_CF = 31 Hz), 128.3 (3C), 128.7 (1C), 146.6 (1C), 148.7 (1C), 172.0 (1C), 173.6 (1C), 175.8 (1C) ppm; IR (KBr disc): 3205 (N-H), 2964 (C-H), 2904 (C-H), 1660 (C=O), 1600–1469 (C=C aromatic) cm⁻¹. Anal. Calcd. for C₁₈H₁₂ClF₃N₂O₃: C, 54.49; H, 3.05; N, 7.06. Found: C, 53.87; H, 2.58; N, 7.47.

4.2.17. N-(Pyridine-3-ylmethyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (22)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and pyridine-3-ylmethanamine (7n) (5.80 mL, 18.68 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF. The mixture was refluxed and heated at (120–130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the inner flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and methanol, then drying in a vacuum oven at (70 °C). Off-white powder (0.65 g) with yield 53%, R_f = 0.4 (n-C₆H₁₄: EtOAc) (3:7); m.p: 245–248 °C; ¹H-NMR (400 MHz, DMSO-d₆ + NaOD): δ = 4.51 (s, 2H, -CH₂), 6.96 (d, J = 8.4 Hz, 1H, Ar-H), 7.04 (d, J = 8.8 Hz, 1H, Ar-H), 7.31–7.34 (m, 1H, Ar-H), 7.75–7.79 (m, 2H, Ar-H), 8.41 (s, 1H, Ar-H), 8.56 (s, 1H, Ar-H) ppm. ¹³C-NMR (100 MHz, DMSO-d₆ + NaOD): δ = 39.6 (1C), 100.8 (1C), 119.6 (1C), 123.9 (1C), 124.3 (1C), 124.6 (1C), 128.5 (1C), 135.7 (1C), 137.3 (1C), 147.9 (1C), 149.0 (1C), 149.1 (2C), 172.1 (1C), 173.9 (1C), 175.9 (1C) ppm. IR (KBr disc): 3381 (O-H), 3145 (N-H), 3068 (C-H), 3008 (C-H), 1665 (C=O), 1608–1475 (C=C aromatic) cm⁻¹. Anal. Calcd. for C₁₆H₁₂ClN₃O₃: C, 58.28; H, 3.67; N, 12.74. Found: C, 57.77; H, 3.99; N, 12.23.

4.2.18. N-(Pyridine-4-ylmethyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (23)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and pyridine-4-ylmethanamine (7o) (3.50 mL, 11.27 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF. The mixture was refluxed and heated at (120–130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the inner flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and methanol, then drying in a vacuum oven at (70 °C). White powder (0.60 g) with yield 49%, R_f = 0.5 (n-C₆H₁₄: EtOAc) (3:7); m.p: 239–242 °C; ¹H-NMR (400 MHz, DMSO-d₆ + NaOD): δ = 4.51 (s, 2H, -CH₂), 6.95 (d, J = 8.8 Hz, 1H, Ar-H), 7.03 (d, J = 8.4 Hz, 1H, Ar-H), 7.35 (d, J = 5.2 Hz, 2H, Ar-H), 7.80 (s, 1H, Ar-H), 8.44 (d, J = 5.2 Hz, 2H, Ar-H) ppm. ¹³C-NMR (100 MHz, DMSO-d₆ + NaOD): δ = 39.5 (1C), 100.8 (1C), 119.6 (1C), 122.9 (2C), 124.3 (1C), 124.4 (1C), 124.6 (1C), 128.5 (1C), 149.1 (1C), 149.6 (2C), 151.1 (1C), 172.2 (1C), 174.0 (1C), 176.0 (1C) ppm. IR (KBr disc): 3242 (N-H), 3163 (C-H), 2993 (C-H), 1653 (C=O), 1598–1415 (C=C aromatic) cm⁻¹. Anal. Calcd. for C₁₆H₁₂ClN₃O₃: C, 58.28; H, 3.67; N, 12.74. Found: C, 58.31; H, 3.27; N, 12.89.

4.2.19. N-(3-Bromobenzyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (24)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and 3-bromobenzylamine (7f) (10.40 mL, 33.58 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF. The mixture was refluxed and heated at (120–130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the inner flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and acetone, then drying in a vacuum oven at (70 °C). White powder (0.99 g) with yield 73%, R_f = 0.5 (n-C₆H₁₄: EtOAc) (6:4); m.p: 292–294 °C; ¹H-NMR (400 MHz, DMSO-d₆ + NaOD): δ = 4.48 (s, 2H, -CH₂), 7.11–7.28 (m, 3H, Ar-H), 7.31 (d, J = 8.0 Hz, 1H, Ar-H), 7.39 (d, J = 8.0 Hz, 1H, Ar-H), 7.47 (s, 1H, Ar-H), 7.84 (s, 1H, Ar-H) ppm. ¹³C-NMR (100 MHz, DMSO-d₆ + NaOD): δ = 41.3 (1C), 100.9 (1C), 122.0 (2C), 124.0 (1C), 124.8 (1C), 126.7 (2C), 129.6 (1C), 131.1 (1C), 130.9 (2C), 131.5 (2C), 144.7 (1C), 150.5 (1C), 170.2 (1C), 175.8 (1C), 181.0 (1C) ppm. IR (KBr disc): 3381 (O-H), 3226 (N-H), 3070 (C-H), 2974 (C-H), 1654 (C=O), 1597–1471 (C=C aromatic) cm⁻¹. Anal. Calcd. for C₁₇H₁₂BrClN₂O₃: C, 50.09; H, 2.97; N, 6.87. Found: C, 50.54; H, 3.60; N, 7.31.

4.2.20. N-(2-Nitro benzyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (25)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and 2-nitrobenzylamine HCl (7p) (4.19 g, 11.20 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF; also, NaHCO₃ (0.94 g, 11.20 mmol) was added to neutralize HCl. The mixture was refluxed and heated at (120–130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the inner flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and Ethanol, then drying in a vacuum oven at (70 °C). White powder (1.03 g) with yield 73.9%, R_f = 0.6 (ETOAc: n-C₆H₁₄) (8:2); m.p: 290–293 °C; ¹H-NMR (400 MHz, DMSO-d₆ + NaOD): δ = 4.13 (s, 2H, -CH₂), 6.07–6.6 (m, 2H, Ar-H), 7.08 (d, J = 8.0 Hz, 2H, Ar-H), 7.34–7.66 (m, 2H, Ar-H), 7.83 (s, 1H, Ar-H) ppm. ¹³C-NMR (100 MHz, DMSO-d₆ + NaOD): δ = 41.3 (1C), 100.6 (1C), 119.5 (1C), 119.6 (1C), 124.3 (2C), 124.4 (2C), 124.5 (2C), 128.5 (1C), 128.6 (1C), 148.9 (2C), 174.4 (1C), 174.7 (1C), 176.4 (1C) ppm. IR (KBr disc): 3145 (C-H), 3001 (N-H), 2910 (C-H), 1708 (C=O), 1660 (C=O), 1600- 1471 (C=C aromatic), 1379, 1342 (NO₂) cm⁻¹. Anal. Calcd. for C₁₇H₁₂ClN₃O₅: C, 54.63; H, 3.24; N, 11.24. Found: C, 54.09; H, 3.30; N, 11.78.

4.2.21. N-(3-Nitro benzyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (26)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and 3-nitrobenzylamine HCl (7q) (4.19 g, 11.20 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF; also, NaHCO₃ (0.94 g, 11.20 mmol) was added to neutralize HCl. The mixture was refluxed and heated at (120–130 °C) for 72 h. As the reaction proceeded, solid precipitate of the amide product was formed on the inner flask wall. Completion of reaction was confirmed by disappearance of (6) spot on TLC. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and Ethanol, then drying in a vacuum oven at (70 °C). Beige powder (0.93 g) with yield 66.7%, R_f = 0.6 (EtOAc) (10); m.p: 280–282 °C; ¹H-NMR (400 MHz, DMSO-d₆ + NaOD): δ = 4.73 (s, 2H, -CH₂), 7.38 (s, 1H, Ar-H), 7.66 (d, J = 9.0 Hz, 2H, Ar-H), 7.83 (d, J = 9.0 Hz, 2H, Ar-H), 8.24 (d, J = 9.0 Hz, 2H, Ar-H), 10.73 (s, 1H, Ar-H), 12.03 (s, 1H, Ar-H), 16.9 (s, 1H, Ar-H) ppm. ¹³C-NMR (100 MHz, DMSO-d₆ + NaOD): δ = 41.3 (1C), 100.6 (1C), 119.5 (1C), 119.6 (1C), 124.3 (2C), 124.4 (2C), 124.5 (2C), 128.5 (1C), 128.6 (1C), 148.9 (2C), 174.4 (1C), 174.7 (1C), 176.4 (1C) ppm. IR (KBr disc): 3288 (N-H), 3169 (C-H), 3093 (C-H), 3059 (C-H), 2941 (C-H), 1660 (C=O), 1645–1481 (C=C aromatic), 1371, 1348 (NO₂) cm⁻¹. Anal. Calcd. for C₁₇H₁₂ClN₃O₅: C, 54.63; H, 3.24; N, 11.24. Found: C, 54.93; H, 3.00; N, 10.68.

4.2.22. N-(4-Nitro benzyl)-6-chloro-4-hydroxy-2-quinolone-3-carboxamide (27)

A mixture of ethyl 6-chloro-4-hydroxy-2-quinolone 3-carboxylate (6) (1.00 g, 3.74 mmol) and 4-nitrobenzylamine HCl (7q) (4.19 g, 11.20 mmol) was dissolved in (100 mL) THF with the addition of a few drops of DMF; also, NaHCO₃ (0.94 g, 11.20 mmol) was added to neutralize HCl. Then, the reaction proceeded as described in 4.2.4. The solid precipitate was isolated by suction filtration at RT, followed by washing with water, THF, and Ethanol, then drying in a vacuum oven at (70 °C). Beige powder (0.93 g) with yield 66.7%, R_f = 0.6 (EtOAc) (10); m.p: 286–288 °C; ¹H-NMR (400 MHz, DMSO-d₆ + NaOD): = 4.73 (s, 2H, -CH₂), 7.02–7.12 (m, 4H, Ar-H), 7.47 (s, 1H, Ar-H), 7.55–8.04 (m, 2H, Ar-H) ppm. ¹³C-NMR (100 MHz, DMSO-d₆ + NaOD): δ = 41.3 (1C), 100.6 (1C), 119.5 (1C), 119.6 (1C), 124.3 (2C), 124.4 (2C), 124.5 (2C), 128.5 (1C), 128.6 (1C), 148.9 (2C), 174.4 (1C), 174.7 (1C), 176.4 (1C) ppm. IR (KBr disc): 3288 (N-H), 3169 (C-H), 3093 (C-H), 3059 (C-H), 1660 (C=O), 1645–1481 (C=C aromatic), 1371, 1348 (NO₂) cm⁻¹. Anal. Calcd. for C₁₇H₁₂ClN₃O₅: C, 54.63; H, 3.24; N, 11.24. Found: C, 55.12; H, 2.73; N, 11.57.

4.3. Biological Studies: Reagents and Dyes

The Dulbecco’s modified Eagle’s medium (DMEM) was used to culture all the cancer cells grown in the lab. DMEM was purchased from GE Healthcare Life Sciences, HyClone Laboratories (Logan, UT, USA). To detach cells, 0.25% trypsin was used and was purchased from Corning Life Sciences (VWR International, Ltd., Radnor, PA, USA), as was 2.2 mM EDTA lysis buffer. Phosphate-buffered saline (PBS) was purchased from Media Tech, Inc. (Manassas, VA, USA). Dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Calbiochem EMD Millipore (Billerica, MA, USA) [1].

4.3.1. Cell Line and Culture Conditions

Prostate cancer, PC-3 and colon cancer HCT-116 cells were a gift from the late Dr. Gary Kruh, University of Illinois at Chicago. The cells were grown as an adherent monolayer in a cell culture flask in culture medium containing DMEM, supplemented with 4.5 g of glucose, 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The cells were maintained in an incubator at 37 °C with 5% CO2 and a relative humidity of 95% [76,77].

4.3.2. Cytotoxicity Assay

The Dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was performed to identify the hits at various drug concentrations (0 and 10 µM). Hit screening was performed in HCT-116 and PC-3 cells. Briefly, 3000 cells/well were seeded in a 96-well plate and allowed to attach overnight. The following day, the cells were treated with the test compounds (0 and 10 µM). Hits were identified as compounds with an IC₅₀ of less than 10 µM. The identified hits were further tested in both HCT-116 and PC-3 cells at various concentrations between 0 and 10 µM. Compound H10 was identified as a lead and was further chosen for hit to lead optimization against colon and prostate cancer.

4.4. Computational Methods

4.4.1. Chemical Structures

The chemical structures of the synthesized compounds were drawn using ChemDraw (version 15) [78]. The two-dimensional (2D) coordinates were exported and saved in Structure Data File (SDF) format. The 2D structure of the reference compound LY294002 (CID: 3973) was retrieved from PubChem [79] in SDF format. All molecular structures, including synthesized compounds and the reference, were standardized using the “Wash Molecules” utility in MOE (Molecular Operating Environment) [64] to ensure consistency in 2D coordinates, protonation states, tautomers, deleting stereochemistry and aromaticity. The final standardized structures were saved in SDF format for downstream cheminformatics analyses.

4.4.2. Molecular Descriptors

Molecular descriptors were calculated using alvaDesc (version 1.0.22) [80,81,82,83]. The preprocessed structures of synthesized compounds and the reference molecule were imported in SDF format, containing 2D coordinates. These structures were previously standardized using MOE’s “Wash Molecules” function. From alvaDesc’s descriptor calculation interface, a total of 3874 2D descriptors were selected for computation. These included a comprehensive set of 2D descriptor families, covering diverse structural and physicochemical properties relevant to cheminformatics analyses.

4.4.3. Principal Component Analysis

Principal Component Analysis (PCA) was performed, using alvaDesc’s “Analysis Module” (1), to explore and visualize the chemical space of the synthesized compounds and identify patterns in their structural diversity and drug-likeness profiles in order to investigate these patterns in the light of observed biological activities. The analysis was conducted using a subset of 1790 two-dimensional (2D) molecular descriptors, selected from a total of 3874 calculated descriptors generated by alvaDesc. Descriptor selection included several filtering steps such as excluding non-informative descriptors that had zero, constant or near constant values across all compounds. This retained the most informative set of descriptors that captured topological, constitutional, and physicochemical characteristics.

4.4.4. Docking and Scoring

Molecular docking is a computational method that explores ligand interaction with its target by speculating on the adopted pose and location of the ligand within the target’s binding domain(s). Scoring is the process of calculating the ligand’s binding energy based on the preferential binding pose. Diverse scoring algorithms can be used to quantify docking scores as representative of the binding affinities or free energies of the ligand–receptor engagement. More negative docking scores imply better binding. In this study, we employed the induced-fit docking (IFD) program in Maestro and evaluated the ligand’s binding affinities against potential targets in terms of IFD scores.

4.4.5. Ligand Preparation for Docking

The ligands’ chemical structures were prepared as follows: (1) we produced 3D assemblies of all ligands based on the coordinates of the co-crystallized ligand (X6K) in 4L23 using “Build” script in Maestro [69]; (2) the ligands’ 3D coordinates were energetically prepared by the “ligprep” algorithm in Maestro [69]. LigPrep investigated stereoisomerism, tautomerism, ring conformations, and ionization state. LigPrep produces assorted chemical and structural characteristics from a single structure.

4.4.6. Protein Preparation

The x-ray assemblies of human WT PI3Kα (PDB ID: 4L23) [65] and MUT (H1047R) PI3Kα (PDB ID: 3HHM) [66] were adopted from the Protein Data Bank (PDB) repository. The protein preparation algorithm in Maestro [69] was used to fill up the missing residues, cap the N- and C-termini, minimize the Hydrogen atoms, and optimize the protein’s Hydrogen-bonding network. Later, the proteins’ side-chains were further energetically treated to decrease steric clashes.

4.4.7. Induced-Fit Docking

Induced-fit docking (IFD) was employed to calculate the binding energies of the analogues to receptors embedded in an in-house drug target database [69]. The co-ligand X6K/4L23 was identified as a centroid in the binding domain. The Van der Waals scaling factors for receptors and ligands were adjusted to 0.5 to furnish appropriate flexibility for the best docked-ligand poses. Other parameters were used as default. The ligand pose with the most negative XP Glide binding score was recorded. Docking scores were illustrated in terms of Kcal/mol, where a more negative docking score implies a better binder.

5. Conclusions

We have successfully synthesized and structurally characterized twenty analogues of N-benzyl-6-chloro-4-hydroxy-2-quinolone-3-carboxamides, revealing their promise as anticancer agents. The analogues exhibited three-fold potency of cytotoxicity against HCT-116, with considerable IC₅₀ values for preliminary suppressive activity. This cheminformatics analysis highlighted the relationships between molecular descriptor-based chemical diversity, predicted drug-likeness, and cytotoxicity trends across colorectal cancer cell lines Caco-2 and HCT-116. Distinctly, the flexible benzyl carboxamide side-chain induced selectivity against HCT-116. These findings agree with reported work on the potential of quinolone-based compounds to induce anticancer activity, particularly by modulating molecular mechanisms attributed in cellular proliferation and metastasis. Docking studies supported the hypothesis of selective targeting of PI3Kα, particularly the mutant isoform, paving the way for further development. The cheminformatics analysis and pharmacophore mapping results reinforced the therapeutic promise of the released analogues, opening avenues for subsequent development of more effective anticancer agents.