Design and Multi-Level Biological Evaluation of Naphthyridine-Based Derivatives as Topoisomerase I/II-Targeted Anticancer Agents with Anti-Fowlpox Virus Activity Supported by In Silico Analysis

Abstract

Naphthyridine derivatives have emerged as privileged scaffolds with diverse pharmacological activities, particularly in anticancer and antiviral drug discovery. In this study, a series of naphthyridine-based derivatives (1–10b) was designed, synthesized, and structurally characterized using IR, ¹H/¹³C NMR, and mass spectrometry, and evaluated as dual-function antiproliferative and anti-fowlpox virus agents supported by integrated computational analyses. The synthesized compounds were screened for in vitro antiproliferative activity against HeLa, HCT-116, and MCF-7 cancer cell lines, as well as normal WI-38 lung fibroblasts. Several derivatives exhibited potent cytotoxic activity with enhanced selectivity toward cancer cells. Compound 5b showed the highest activity against HeLa cells, compound 1 was most effective against HCT-116 cells, while compounds 7 and 8 displayed remarkable activity against MCF-7 cells, with compound 7 surpassing doxorubicin and compound 8 demonstrating excellent selectivity toward normal cells. Mechanistic investigations revealed that compounds 7 and 8 acted as dual topoisomerase I/IIβ inhibitors, inducing G2/M cell cycle arrest and intrinsic apoptosis associated with caspase-9 activation and downregulation of topoisomerase II protein expression. Selected derivatives were further evaluated for antiviral activity against fowlpox virus using in ovo and in vivo SPF embryonated chicken egg models, where compounds 2 and 9a exhibited the highest therapeutic indices, comparable to ribavirin, and compound 9a markedly suppressed viral replication and titers in vivo. ADMET profiling, molecular docking, molecular dynamics simulations, and DFT calculations supported the experimental findings and identified compound 10a as the most favorable theoretical candidate. Overall, this integrated experimental–computational approach establishes naphthyridine derivatives as a rationally designed multifunctional chemotype for simultaneous anticancer and antiviral drug development.

1. Introduction

Breast cancer remains a major oncological challenge not only because of its high prevalence but also due to its adaptability, therapeutic resistance, and capacity to sustain continuous proliferation under genomic stress [1,2,3]. According to the World Health Organization (WHO, 2023–2024), breast cancer accounts for over 2.3 million new cases annually (12% of global cancer diagnoses) and is the leading cause of cancer-related mortality among women, with more than 670,000 deaths reported each year [4,5]. This burden is exacerbated by tumor heterogeneity and drug resistance, underscoring the urgent need for therapeutic strategies that directly disrupt the molecular mechanisms supporting tumor survival and progression [6,7,8,9].

DNA topoisomerases, essential regulators of DNA topology, are among the cellular systems exploited to sustain rapid DNA replication and cell division in cancer cells [10]. Topoisomerase II (Topo II) controls DNA double-strand passage events required for chromosome segregation [11], while Topoisomerase I (Topo I) relieves torsional stress through transient single-strand breaks [12]. Beyond their canonical roles, dysregulated topoisomerase activity contributes to oncogenic transcriptional programs and enhanced tumor cell survival [13]. The marked sensitivity of malignant cells to Topo II-mediated cytotoxicity highlights their dependence on these enzymes under genotoxic stress, and recent progress in dual Topo I/II inhibitor development further validates topoisomerases as key anticancer targets [14,15].

FDA-approved topoisomerase inhibitors include the camptothecin-based Topo I agents topotecan I and irinotecan II [16,17,18,19], semisynthetic derivatives of the natural alkaloid camptothecin III built on a quinoline-containing scaffold [20,21]. Structurally distinct Topo II-targeting agents include the naphthyridine-derived vosaroxin IV, which progressed to Phase III clinical evaluation (VALOR study) and received orphan drug designation [22,23], as well as classical acridine-based poisons such as amsacrine V with established clinical utility, particularly in hematological malignancies [24] (Figure 1).

The clinical effectiveness of current topoisomerase inhibitors is limited by intrinsic drawbacks, including lactone instability and rapid cleavage complex reversibility of camptothecin-derived Topo I inhibitors [25], as well as cardiotoxicity, genotoxicity, and therapy-related secondary malignancies associated with classical Topo II poisons [26,27,28].

Accordingly, there is a need for chemically robust and safer scaffolds for selective and sustained topoisomerase inhibition. In this context, naphthyridine-based heterocycles have emerged as privileged anticancer scaffolds, as their rigid fused aromatic systems facilitate DNA intercalation and enable effective inhibition of both Topo I [29,30,31,32,33] and Topo II [34].

Several naphthyridine-based compounds have been identified as effective non-camptothecin Topo I inhibitors. ARC-111 (Topovale) VI stabilized the Topo I–DNA cleavage complex and exhibited low-nanomolar antiproliferative activity against MCF-7 cells via S-phase arrest and apoptosis [35]. Related derivatives VII–IX were also reported as representative Topo I inhibitors/poisons, demonstrating substantial inhibition of Topo I-mediated DNA relaxation, with inhibition levels ranging approximately from 60% to over 80% under comparable experimental conditions, thereby confirming the susceptibility of Topo I to modulation by naphthyridine scaffolds and the importance of structural and electronic features in activity optimization [36,37,38].

Beyond Topo I, several naphthyridine-based analogues have demonstrated selective Topo II inhibition. Representative compounds X–XII exhibited inhibitory activity against Topo IIα/β with IC₅₀ values ranging from ~50 µM to submicromolar levels, in some cases comparable to reference drugs such as etoposide and doxorubicin [39,40]. Notably, more potent derivatives inhibited both Topo II isoforms and were associated with G2/M cell-cycle arrest, suppression of cell proliferation, and induction of apoptosis, further supporting the suitability of naphthyridine scaffolds for Topo II targeting [40].

In parallel, dual-targeting naphthyridine-based derivatives capable of inhibiting both Topo I and Topo II have been reported. Among these, compound XIII exhibited potent dual inhibition, with inhibition levels exceeding 80–90% at 100 µM and micromolar IC₅₀ values against both enzymes, approaching the activity of reference agents such as camptothecin and doxorubicin [41]. The chemical structures of the reported topoisomerase inhibitors are shown in Figure 2.

Beyond their anticancer applications, naphthyridine-based compounds exhibit broader biological potential against pathological systems that rely on DNA replication. Fowlpox represents a persistent global challenge in avian medicine, caused by the fowlpox virus (FPV; genus Avipoxvirus), and predominantly affects domestic poultry such as chickens and turkeys, leading to significant economic losses through reduced productivity, diminished egg yield, and increased management costs [42].

FPV is among the largest animal DNA viruses, displaying a brick-shaped morphology and a double-stranded DNA genome of 260–309 kbp encoding over 250 proteins [43]. Unlike most DNA viruses, FPV is highly self-sufficient and completes its entire replication cycle within the cytoplasm of infected epithelial cells [44].

Clinically, fowlpox occurs in two forms: a cutaneous (dry) form characterized by wart-like lesions on unfeathered areas following skin injury or insect bites, causing substantial physiological stress [45], and a more severe diphtheritic (wet) form that infects the oral and respiratory mucosa, producing necrotic plaques that may obstruct the airway and frequently result in death by asphyxiation [46]. Disease control is further complicated by the exceptional environmental stability of FPV, which can persist for months in dried scabs and spreads efficiently via direct contact, contaminated dust, and insect vectors [42]. Although live attenuated vaccines are available, the continued adaptability of FPV highlights the urgent need for alternative antiviral strategies.

In this context, naphthyridine-based heterocycles previously recognized for anticancer activity via interference with DNA-processing enzymes, including topoisomerases I and II, have also been reported to exert antiviral effects by targeting viral enzymes essential for genome processing [47]. Representative compounds XIV and XV exhibited pronounced antiviral potency in FP-based assays, with IC₅₀ values in the submicromolar-to-low micromolar range [48]. In addition, the well-established naphthyridine derivative XVI (dercitin) has been extensively employed as a reference scaffold for DNA-associated enzymatic targets, thereby validating the naphthyridine framework and supporting its evaluation against the fowlpox virus resolvase (Figure 3) [49].

Collectively, these findings further support naphthyridine-based scaffolds as versatile modulators of DNA-processing pathways with potential relevance to both anticancer and antiviral drug discovery.

Rational Design Strategy Based on Naphthyridine Reference Compounds

Guided by the privileged naphthyridine scaffold, a unified rational design strategy was adopted to optimize the framework toward topoisomerase II inhibition and interference with DNA-processing pathways. Vosaroxin (IV) was selected as the Topo II reference due to its established DNA intercalation and cleavage complex stabilization. Relative to vosaroxin, replacement of the scaffold oxygen atom with an NH₂ group increased hydrogen-bonding capacity, while substitution of the COOH moiety with a carbonyl group (=O) reduced excessive ionization. The thiazole ring was replaced by a fused 4-amino-2-oxo-1,2-dihydropyridine-3-carbonitrile system to enhance planarity and π–π stacking, with the cyano group providing favorable electronic modulation. Introduction of a chloro-phenyl substituent and replacement of the flexible pyrrolidine side chain with a rigid fused 1,4-dihydronaphthalene moiety further increased hydrophobicity and molecular rigidity, collectively rationalizing enhanced Topo II inhibitory activity.

In parallel, dercitin (XVI) served as a reference naphthyridine scaffold associated with DNA-processing enzymes. Remodeling of the substituent linked to the naphthyridine core introduced a fused 1,4-dihydronaphthalene system, increasing rigidity and planarity, while replacement of the methyl group with a chloro-phenyl moiety enhanced hydrophobic and electronic modulation. Substitution of the flexible 2-(5,6-dihydrobenzo[d]thiazol-4-yl)-N,N-dimethylethan-1-amine fragment with a compact 4-aminopyrimidin-2(1H)-one system improved hydrogen-bonding orientation and reduced conformational flexibility. Additional introduction of NH₂ and carbonyl (=O) functionalities further supported stabilization of protein–DNA assemblies and interference with viral DNA processing (Figure 4).

These modifications were intended to bias the scaffold toward distinct DNA-processing pathways rather than imply simultaneous dual-target inhibition.

2. Results and Discussion

2.1. Chemistry

Scheme 1 summarizes the reactivity of the key intermediate 4-(4-chlorophenyl)-2-oxo-1,2,5,10-tetrahydrobenzo[g]quinoline-3-carbonitrile (A) toward different nucleophilic and electrophilic reagents, affording a structurally diverse series of fused heterocycles (compounds 1–5b). Reaction with 2-cyanoacetohydrazide proceeded via nucleophilic addition-cyclocondensation to afford compound 1 bearing a naphtho[2,3-b][1,8]naphthyridine moiety.

Interaction with malononitrile occurred through nucleophilic addition to the cyano group followed by intramolecular heterocyclization, yielding compound 2 with a dihydronaphthyridone fused system. Treatment with hydrazine hydrate involved nucleophilic addition and subsequent 5-exo cyclization to give compound 3 containing a benzo[g]pyrazolo[3,4-b]quinoline moiety. Furthermore, reaction with urea or thiourea under basic conditions afforded compounds 4a and 4b, respectively, via annulation to form the corresponding amino- and thioamino-substituted heterocyclic systems. Finally, nucleophilic acyl substitution with 4-chlorobenzoyl chloride produced compound 5a, while reaction with phenyl isothiocyanate yielded compound 5b via thiocarbamoylation.

The spectroscopic data of compound A are consistent with its proposed benzo[g]quinoline framework and confirm its successful formation. Although this compound is not newly reported, its spectral features are discussed here for completeness and consistency within the present study. The IR spectrum showed a characteristic NH stretching band at 3130 cm⁻¹, aromatic C–H absorptions at 3069 and 3030 cm⁻¹, and an aliphatic C–H band at 2931 cm⁻¹. The presence of the cyano (C≡N) group was confirmed by a sharp absorption at 2222 cm⁻¹, while the carbonyl (C=O) group appeared at 1636 cm⁻¹. The bands at 1552 and 1535 cm⁻¹ were attributed to aromatic C=C stretching vibrations. The ¹H NMR spectrum displayed two singlets at δ 2.94 and 3.13 ppm (4H, 2CH₂), a multiplet at δ 7.26–7.47 ppm integrating for eight aromatic protons, and a broad exchangeable signal at δ 12.12 ppm corresponding to the NH proton. The ¹³C NMR spectrum further supported the assigned structure, showing methylene carbon signals at δ 23.38 and 27.27 ppm, a cyano carbon resonance at δ 115.84 ppm, aromatic carbon signals in the range δ 116.23–139.72 ppm, and downfield signals at δ 159.99 and 170.11 ppm attributable to the C=O and pyridine carbons, respectively.

The spectroscopic data confirmed the successful formation of compound 1. The IR spectrum showed the disappearance of the NH band of precursor A and the appearance of new absorptions at 3482–3115 cm⁻¹, consistent with the formation of two amino groups (2NH₂), while the cyano and carbonyl functionalities were retained. Accordingly, the ¹H NMR spectrum revealed the loss of the exchangeable NH signal and the emergence of two broad NH₂ signals at δ 4.75 and 5.13 ppm. The ¹³C NMR spectrum further supported the proposed structure, confirming the incorporation of C=N and C=O carbons within the annulated heterocyclic framework.

The spectral data of compound 2 confirmed the formation of the new naphthyridone framework with the introduction of a single NH₂ group. This was evidenced by the appearance of NH₂ absorptions at 3412–3130 cm⁻¹ in the IR spectrum and a broad exchangeable signal at δ 5.15 ppm in the ¹H NMR spectrum, supported by the corresponding C-NH₂ carbon resonance at δ 176.02 ppm in the ¹³C NMR spectrum. All other functional groups were retained, showing only minor spectral shifts attributable to annulation. The EI mass spectrum displayed a molecular ion peak at m/z 398.85, consistent with the proposed structure.

The spectral data of compound 3 confirmed its conversion into a pyrazolo-fused system. The newly formed pyrazole-linked NH₂ group was evidenced by IR absorptions at 3467 and 3129 cm⁻¹ and a broad exchangeable ¹H NMR signal at δ 6.62 ppm, while the two newly generated C=N units were confirmed by characteristic IR bands at 1613 and 1583 cm⁻¹ and corresponding ¹³C NMR resonances at δ 152.11 and 152.53 ppm. The EI mass spectrum displayed a molecular ion peak at m/z 346.82 [M⁺], consistent with the formation of the new pyrazoloquinoline framework.

In 4a and 4b, the spectral data confirmed the formation of the fused pyrimidinone framework in both compounds. In both cases, the emergence of a new NH₂ group was evidenced by broad IR absorptions at 3443–3198 cm⁻¹ (4a) and 3462–3255 cm⁻¹ (4b), along with broad exchangeable ¹H NMR signals at δ 7.06 ppm (4a) and 6.96 ppm (4b). The formation of two new C=N units was supported by characteristic IR bands at 1600 and 1584 cm⁻¹ (4a) and at 1593 cm⁻¹ (4b), together with corresponding ¹³C NMR resonances at δ 145.01 and 155.03 ppm (4a) and δ 147.50 and 155.00 ppm (4b).

The key structural distinction between the two compounds was confirmed by ¹³C NMR spectroscopy: compound 4a exhibited a urea carbonyl signal at δ 151.11 ppm, whereas compound 4b showed a characteristic thioxo (C=S) resonance at δ 182.93 ppm. In both cases, the complete disappearance of the cyano (C≡N) group confirmed its consumption during ring closure. The EI mass spectra displayed molecular ion peaks at m/z 374.09 [M⁺] for 4a and m/z 390.07 [M⁺] for 4b, consistent with their proposed structures.

The spectral data confirmed the structural modification of compounds 5a and 5b through acylation reactions. In compound 5a, benzoylation was evidenced by the complete disappearance of the NH functionality and the appearance of a new benzoyl carbonyl group, indicated by a distinct IR absorption at 1704 cm⁻¹ and a corresponding ¹³C NMR resonance at δ 163.92 ppm. The introduction of the benzoyl moiety was further supported by the expansion of the aromatic region, observed as a broadened ¹H NMR envelope at δ 7.22–7.48 ppm (13H) and extended aromatic ¹³C signals spanning δ 120.00–139.44 ppm. The cyano group remained intact, as confirmed by IR and ¹³C signals at 2222 cm⁻¹ and δ 115.00 ppm. The EI mass spectrum showed a molecular ion peak at m/z 436.10 [M⁺].

In contrast, compound 5b exhibited thiocarbamoylation features, marked by the emergence of a new NH group (IR: 3202 cm⁻¹; ¹H NMR: δ 12.42 ppm) together with the formation of a thio-carbonyl (C=S) functionality, confirmed by a strong IR band at 1084 cm⁻¹ and a characteristic ¹³C NMR signal at δ 176.53 ppm. Similar to 5a, aromatic expansion was observed in the ¹H NMR spectrum at δ 7.26–8.02 ppm (13H) and in the ¹³C NMR spectrum across δ 116.19 –140.32 ppm. The EI mass spectrum displayed a molecular ion peak at m/z 467.09 [M⁺], consistent with the proposed structure.

Scheme 2 illustrates the synthetic elaboration of compound 1 through its reactions with various active methylene and heterocumulene reagents, affording the fused derivatives 6–9b.

Reaction of compound 1 with acetylacetone in AcOH/HCl proceeded through an initial condensation step followed by intramolecular cyclization, which contains a benzo[6,7]quinolino[2,3-h][1,6]naphthyridin-5-one moiety. Treatment of 1 with ethyl cyanoacetate afforded compound 7 through a condensation–annulation process, producing a dioxo-benzoquinolino naphthyridine scaffold. Similarly, its reaction with malononitrile involved base-assisted condensation and heterocyclization, yielding compound 8 bearing a triamino oxo naphthyridine-fused system. Finally, the reactions of 1 with urea and thiourea enabled cyclocondensation onto the naphthyridine core, affording compounds 9a and 9b, which feature pyrimido[4,5-f][1,8]naphthyridine dione and thioxo-pyrimido naphthyridine moieties, respectively. Collectively, these transformations highlight the high reactivity of compound 1 toward active methylene and heterocumulene reagents, enabling the construction of richly fused heterocyclic architectures 6–9b.

The spectral data of compound 6 confirmed its formation via acetylation and ring extension relative to compound 1. The introduction of a new acetyl carbonyl was evidenced by a distinct IR absorption at 1723 cm⁻¹ and a strongly deshielded ¹³C NMR signal at δ 192.37 ppm, in addition to the original heterocyclic C=O band at 1636 cm⁻¹. The formation of an additional C=N unit was supported by new IR bands at 1600 and 1595 cm⁻¹. In the ¹H NMR spectrum, two new methyl singlets appeared at δ 2.15 and 2.25 ppm (6H), corresponding to acetyl and pyridine-bound CH₃ groups, with matching ¹³C NMR resonances at δ 20.00 and 29.72 ppm. The EI mass spectrum showed a molecular ion peak at m/z 495.97 [M⁺], confirming the increased molecular weight associated with acetylation and framework extension.

The spectral data of compound 7 confirmed its further elaboration from compound 1. The introduction of an additional NH group was evidenced by an IR absorption at 3130 cm⁻¹ and a broad exchangeable ¹H NMR signal at δ 11.26 ppm (1H), while the formation of a second carbonyl functionality was confirmed by new IR bands at 1684 and 1637 cm⁻¹ and corresponding ¹³C NMR resonances at δ 160.29 and 162.70 ppm. Perturbation of the NH₂ environment was indicated by broadened IR absorptions at 3475, 3455 and 3292–3195 cm⁻¹ and broadened exchangeable ¹H NMR signals at δ 4.16 and 4.19 ppm (4H). An additional downfield ¹³C NMR signal at δ 176.52 ppm further supported incorporation of the new amide functionality. The EI mass spectrum displayed a molecular ion peak at m/z 480.11 [M⁺], consistent with the proposed structure.

The spectral data of compound 8 confirmed the formation of a triamino, mono-oxo naphthyridine-type system. The appearance of three NH₂ groups was evidenced by multiple broad IR absorptions at 3453, 3413, 3388, 3341, 3292, and 3198 cm⁻¹ and by three broad exchangeable ¹H NMR signals at δ 6.22, 6.25, and 6.95 ppm (6H). The introduction of an additional C=N environment was supported by an IR band at 1599 cm⁻¹ and by ¹³C NMR resonances at δ 162.57 and 163.79 ppm, together with amino-substituted heterocyclic carbons at δ 159.98 and 166.28 ppm. The cyano and carbonyl functionalities were retained, as confirmed by IR/¹³C signals at 2223 cm⁻¹ and δ 115.71 ppm (C≡N) and at 1637 cm⁻¹ and δ 162.11 ppm (C=O), respectively. The EI mass spectrum showed a molecular ion peak at m/z 479.93 [M⁺], consistent with the proposed structure.

The spectral data of compounds 9a and 9b confirmed their conversion into fused pyrimido–naphthyridine systems with loss of the cyano functionality and formation of multiple heteroatomic centers. In both compounds, consumption of the nitrile group was evidenced by the disappearance of the C≡N carbon resonance in the ¹³C NMR spectra, accompanied by the emergence of two NH₂ environments, observed as multiple broad IR absorptions at 3468–3198 cm⁻¹ and broad exchangeable ¹H NMR signals at δ 4.53–6.94 ppm (9a) and δ 4.94–6.94 ppm (9b). An additional NH group was also present in 9b, as indicated by an IR band at 3131 cm⁻¹ and a downfield ¹H NMR signal at δ 13.40 ppm.

In both derivatives, the formation of new azomethine centers was supported by ν(2C=N) bands at 1598–1599 cm⁻¹ and corresponding ¹³C NMR resonances at δ 147.08 and 154.03 ppm for 9a, and at δ 158.00 and 167.49 ppm for 9b. The key structural distinction between the two compounds was clearly reflected in the carbonyl/thio-carbonyl region: compound 9a exhibited a dioxo pyrimidinone system, confirmed by two C=O absorptions at 1687 and 1637 cm⁻¹ and ¹³C NMR signals at δ 160.02 and 162.70 ppm, whereas compound 9b showed replacement of one carbonyl by a thio-carbonyl, evidenced by a single C=O band at 1637 cm⁻¹ with a ¹³C signal at δ 161.97 ppm and a new C=S group characterized by an IR absorption at 1085 cm⁻¹ and a downfield ¹³C resonance at δ 180.01 ppm.

The EI mass spectra further supported these assignments, displaying molecular ion peaks at m/z 456.11 [M⁺] for 9a and m/z 472.09 [M⁺] for 9b, consistent with cyclization and differentiation between the oxo and thioxo frameworks.

Scheme 3 illustrates the cyclocondensation of compounds 5a and 5b with hydrazine hydrate, leading to the formation of the corresponding pyrazolo[3,4-b]quinoline derivatives 10a and 10b. The reaction proceeds through hydrazine-mediated heterocyclization, while the pre-existing substituents (COPh in 5a and CSNHPh in 5b) remain unchanged throughout the transformation.

For the hydrazinolysis products 10a and 10b, the spectral changes relative to their precursors 5a and 5b are fully consistent with pyrazole ring formation on the benzo[g]quinoline core. In both derivatives, the diagnostic CN and pyridinone C=O functions of the starting materials disappear: no nitrile band is observed in the IR spectra of 10a or 10b, and no C≡N carbon signal appears in the ¹³C NMR, confirming complete consumption of the cyano group during cyclization. At the same time, the pyridinone C=O present in 5a and 5b is lost, being replaced by the new pyrazole system, while a single remaining carbonyl persists only in 10a at 1708 cm⁻¹ and δ 167.63 ppm, attributable to the benzoyl C=O.

The formation of the pyrazole-fused products 10a and 10b was confirmed by the appearance of new characteristic spectral signals corresponding to the newly generated functional groups. In the IR spectra, both compounds showed new ν(NH₂) absorption bands at 3433/3292 cm⁻¹ for 10a and 3451/3300 cm⁻¹ for 10b, while compound 10b additionally exhibited a ν(NH) band at 3198 cm⁻¹. In the ¹H NMR spectra, the newly formed pyrazole NH₂ group appeared as a broad exchangeable signal at δ 5.73 ppm for 10a and δ 6.53 ppm for 10b, whereas compound 10b showed an additional NH signal at δ 11.77 ppm attributable to the thiocarbamoyl–pyrazole linkage. In the ¹³C NMR spectra, two new resonances corresponding to the pyrazole C=N carbons were observed at δ 151.92 and 152.59 ppm for 10a and at δ 156.16 and 157.00 ppm for 10b. In addition, compound 10b displayed a characteristic thiocarbonyl carbon signal at δ 176.33 ppm, consistent with the presence of a C=S group.

These spectral features collectively confirm successful pyrazole ring formation with incorporation of NH₂ and 2C=N functionalities in compounds 10a and 10b.

Taken together, the synthetic transformations described in Scheme 1, Scheme 2 and Scheme 3 enabled the construction of a structurally diverse library of benzo[g]quinoline-based heterocycles, generated through sequential annulation, condensation, acylation, and thiocarbamoylation pathways. The strategic functionalization of the key intermediates A, 1, and 5a,b resulted in the formation of multiple fused scaffolds including naphthonaphthyridines, pyrazolo quinolines, dioxo-naphthyridines, and pyrimido-naphthyridines each incorporating pharmacophoric moieties known to enhance DNA intercalation and enzyme binding affinity. This high level of structural diversity was deliberately designed to modulate electronic properties, planarity, and hydrogen-bonding patterns within the synthesized molecules, thereby improving their potential interaction with biological targets implicated in cancer progression.

Accordingly, all synthesized derivatives (1–10b) were subsequently subjected to in vitro anticancer evaluation, aiming to identify the most promising candidates with potent cytotoxic profiles and mechanistic relevance for further biological investigations.

2.2. Biological Evaluation

2.2.1. Antiproliferative In Vitro Potency

The synthesized compounds were evaluated for their in vitro antiproliferative activity against three human cancer cell lines, namely cervical cancer (HeLa), colorectal cancer (HCT-116), and breast cancer (MCF-7), in addition to the normal human lung fibroblast cell line (WI-38) [50,51,52]. Cytotoxicity was expressed as IC₅₀ values (µM), and doxorubicin (DOX) and sorafenib (SOR) were used as reference drug (

Table 1. In vitro cytotoxic activity of the synthesized compounds against human cancer cell lines HeLa (cervical cancer), HCT-116 (colorectal cancer), and MCF-7 (breast cancer), as well as the normal WI-38 lung fibroblast cell line, expressed as IC₅₀ values (µM). Doxorubicin (DOX) and sorafenib (SOR) were used as reference drugs.

Comp.	In Vitro Cytotoxicity IC50 (µM)
	WI-38	Hela	HCT-116	MCF-7
DOX	6.72 ± 0.5	5.57 ± 0.4	5.23 ± 0.3	4.17 ± 0.2
SOR	10.65 ± 0.8	8.04 ± 0.5	5.47 ± 0.3	7.26 ± 0.3
9a	19.41 ± 1.4	63.55 ± 3.6	39.96 ± 2.4	47.33 ± 2.8
10a	41.17 ± 2.5	56.01 ± 3.3	15.62 ± 1.3	38.27 ± 2.4
10b	58.86 ± 3.4	37.71 ± 2.4	24.68 ± 1.6	33.43 ± 2.1
3	61.74 ± 3.5	59.29 ± 3.4	32.58 ± 2.1	40.91 ± 2.6
5a	72.60 ± 3.7	21.44 ± 1.5	44.09 ± 2.6	18.14 ± 1.4
7	55.16 ± 3.2	42.84 ± 2.5	75.23 ± 3.9	3.98 ± 0.2
6	87.35 ± 4.5	34.59 ± 2.2	53.15 ± 3.1	22.48 ± 1.6
4b	43.05 ± 2.6	16.12 ± 1.3	49.87 ± 2.9	6.23 ± 0.4
8	>100	48.79 ± 2.8	68.55 ± 3.6	6.49 ± 0.4
9b	27.69 ± 1.8	73.33 ± 3.7	46.62 ± 2.7	51.76 ± 3.0
2	45.73 ± 2.7	91.43 ± 4.6	31.80 ± 2.0	69.54 ± 3.5
4a	30.22 ± 2.1	>100	84.27 ± 4.2	78.41 ± 3.9
A	76.07 ± 3.9	82.64 ± 4.1	60.53 ± 3.4	54.25 ± 3.2
5b	52.75 ± 3.1	7.56 ± 0.5	13.46 ± 1.1	8.82 ± 0.6
1	35.13 ± 2.3	11.62 ± 0.9	9.49 ± 0.7	14.19 ± 1.2

). The dose–response curves of the compounds against WI-38, HeLa, HCT-116, and MCF-7 cells are shown in Figure S57, while the corresponding cell viability data are summarized in Table S1 and Figure S60.

Cytotoxic Activity Against HeLa Cells

The cytotoxic screening against HeLa cells revealed marked differences in potency among the tested compounds. Compound 5b emerged as the most potent derivative against HeLa cells, exhibiting an IC₅₀ value of 7.56 ± 0.5 µM, which is lower than that of sorafenib (IC₅₀ = 8.04 ± 0.5 µM), although less potent than doxorubicin (IC₅₀ = 5.57 ± 0.4 µM). Compound 1 also showed notable activity with an IC₅₀ value of 11.62 ± 0.9 µM, while compounds such as 4b and 10b displayed only moderate effects. Importantly, compound 5b demonstrated high selectivity, as indicated by its significantly higher IC₅₀ value against WI-38 cells (52.75 ± 3.1 µM), suggesting a favorable therapeutic window.

Accordingly, compound 5b can be identified as the lead compound against HeLa cervical cancer cells.

Cytotoxic Activity Against HCT-116 Cells

Evaluation against colorectal carcinoma HCT-116 cells showed that compound 1 was the most active member of the synthesized series, with an IC₅₀ value of 9.49 ± 0.7 µM. Although this activity is lower than that of doxorubicin (IC₅₀ = 5.23 ± 0.3 µM), it indicates moderate cytotoxic potency within the series. Compound 5b also exhibited reasonable activity (IC₅₀ = 13.46 ± 1.1 µM), whereas most other derivatives were less active or weakly effective. Notably, compound 1 displayed reduced cytotoxicity toward WI-38 normal cells (IC₅₀ = 35.13 ± 2.3 µM), supporting a degree of selectivity toward cancer cells.

Thus, compound 1 represents the most promising lead against HCT-116 colorectal cancer cells based on potency–selectivity balance.

Cytotoxic Activity Against MCF-7 Cells

The most remarkable results were observed against breast cancer MCF-7 cells.

Compound 7 exhibited the strongest cytotoxic activity, with an IC₅₀ value of 3.98 ± 0.2 µM, outperforming doxorubicin (IC₅₀ = 4.17 ± 0.2 µM). This was followed by compound 8 (IC₅₀ = 6.49 ± 0.4 µM) and compound 4b (IC₅₀ = 6.23 ± 0.4 µM). Importantly, compounds 7 and 8 showed excellent selectivity, as their IC₅₀ values against WI-38 cells were 55.16 ± 3.2 µM and >100 µM, respectively.

Accordingly, compound 7 can be classified as the most potent anti-breast cancer agent in the series, while compound 8 represents the safest and most selective derivative toward MCF-7 cells.

Cytotoxicity Toward Normal WI-38 Cells and Selectivity Profile

Most of the synthesized compounds exhibited significantly lower cytotoxicity toward WI-38 cells compared with doxorubicin (IC₅₀ = 6.72 ± 0.5 µM), indicating improved safety profiles.

Compounds 7, 8, 5b, and 1 were particularly distinguished by their high IC₅₀ values against normal cells, reflecting enhanced selectivity toward cancer cells.

Based on the cytotoxicity results, further studies were focused on the MCF-7 breast cancer cell line. Compounds 7 and 8 were selected as lead candidates due to their superior antiproliferative activity and high selectivity. Compound 7 showed the strongest cytotoxic effect against MCF-7 cells (IC₅₀ = 3.98 µM), exceeding doxorubicin, while compound 8 exhibited slightly lower potency (IC₅₀ = 6.49 µM) but markedly reduced toxicity toward normal WI-38 cells (IC₅₀ > 100 µM). Accordingly, both compounds were advanced to topoisomerase I/II inhibition assays, cell cycle analysis, apoptosis and caspase-3 Western blot studies, together with computational investigations (molecular docking, molecular dynamics, DFT, and ADMET analyses) to confirm their potency, selectivity, and mechanistic relevance as anti-breast cancer leads.

Structure Activity Relationship (SAR) Study

A comprehensive SAR analysis was performed by correlating the systematic structural modifications of the parent compound A and key intermediate 1 with the observed antiproliferative activity and cell line selectivity. Overall, both functional group modulation and heterocyclic annulation played decisive roles in tuning anticancer potency.

Conversion of compound A into naphtho[2,3-b][1,8]naphthyridine derivatives (1 and 2) markedly enhanced cytotoxic activity, particularly against HCT-116 cells, with compound 1 outperforming compound 2 due to the presence of two amino groups, which increased hydrogen bonding capacity and electrostatic interactions. In contrast, excessive rigidification through pyrazolo fusion in compound 3 led to a general loss of activity, likely due to reduced flexibility and impaired target engagement. Introduction of a pyrimido-fused quinoline system (4a,b) significantly improved activity against MCF-7 breast cancer cells, with thione substitution in 4b conferring higher potency than the corresponding carbonyl analogue, highlighting the favorable impact of sulfur-containing functionalities. Similarly, N-functionalization revealed a strong dependence on substituent nature, where benzoylation (5a) resulted in moderate activity, while incorporation of a carbothioamide group (5b) dramatically enhanced cytotoxicity, particularly against HeLa cells, due to increased hydrogen bonding, electronic delocalization, and binding adaptability.

Further derivatization of compound 1 into polycyclic systems (6–9b) clarified the role of additional substituents. Introduction of alkyl and acetyl groups (6) reduced activity, whereas carbonyl enrichment (7) and amino group expansion (8) significantly enhanced potency and selectivity toward MCF-7 cells, with compound 7 benefiting from improved electronic distribution and compound 8 from increased electrostatic interactions without nonspecific toxicity. In pyrimido-fused derivatives, thioxo substitution (9b) consistently outperformed its dione analogue (9a), reaffirming the positive role of sulfur.

Finally, pyrazolo ring annulation via hydrazine-mediated cyclization afforded compounds 10a and 10b, where thioamide containing 10b exhibited superior activity compared to benzoylated 10a, particularly against MCF-7 and HeLa cells, underscoring once more the critical contribution of sulfur substitution to anticancer efficacy.

Overall, the SAR trends demonstrate that amino group enrichment enhances colorectal and breast cancer activity, carbonyl-rich scaffolds favor breast cancer selectivity, while sulfur-containing moieties (thione and carbothioamide) consistently improve cytotoxic potency. These insights provide a clear molecular rationale for selecting compounds 7 and 8 as primary anti-breast cancer leads.

2.2.2. Topoisomerase I and II Inhibitory Activity of Naphthyridine Derivatives 7 and 8

Based on the MTT cytotoxicity results, MCF-7 breast cancer cells were selected for further investigation, as the synthesized compounds showed their highest antiproliferative activity against this cell line. Given the reported topoisomerase-inhibitory potential of naphthyridine-based compounds [48], compounds 7 and 8 were selected as the most potent candidates for mechanistic evaluation. Their inhibitory effects on Topo I and Topo IIβ (TOP2B) expression were subsequently assessed in MCF-7 cells.

As summarized in

Table 2. In vitro inhibitory activity of the tested naphthyridine-based derivatives 7 and 8 against TOP1 and TOP2B in MCF-7 cells, expressed as IC₅₀ values (µM ± SD) and percentage inhibition.

Compound	MW (g/mol)	Topo I IC50 (µM ± SD)	Topo I % Inhibition	Topo IIβ IC50 (µM ± SD)	Topo IIβ % Inhibition
7	480.91	12.53 ± 0.43	77.240	3.49 ± 0.12	85.201
8	479.93	15.41 ± 0.53	72.041	3.77 ± 0.13	84.001
Camptothecin (Ref.)	348.35	11.565 ± 0.40	78.992	—	—
Doxorubicin (Ref.)	543.52	—	—	3.75 ± 0.13	84.058

and Figure 5a,b, both compounds exhibited dual inhibitory activity against Topo I and Topo IIβ. Compound 7 showed stronger inhibition than compound 8 against both enzymes, with IC₅₀ values of 12.53 ± 0.43 µM (Topo I) and 3.49 ± 0.12 µM (Topo IIβ). While its Topo I activity was slightly lower than that of camptothecin, compound 7 displayed comparable potency to doxorubicin against Topo IIβ. Overall, these findings identify compounds 7 and 8 as dual Topo I/Topo II inhibitors in MCF-7 cells, with compound 7 emerging as the more potent derivative. Raw experimental data are provided in Supplementary Tables S2 and S3.

2.2.3. Cell Cycle Arrest and Apoptosis of MCF-7 Cells of Naphthyridine Derivative 7

Based on enzyme inhibition assays, naphthyridine-based derivative 7 was identified as the most potent topoisomerase II inhibitor, which prompted further mechanistic studies on cell cycle progression and apoptosis in MCF-7 cells [53,54].

As summarized in Tables S4 and S5 and illustrated by flow cytometry outputs (Figure 6a,b) together with the corresponding Excel-based graphical representations (Figure 7 and Figure 8), compound 7 markedly disrupted cell cycle distribution by reducing the G0/G1 population to 37.14% (vs 54.66% in control) and inducing a pronounced G2/M arrest (44.64% vs. 12.32%).

In parallel, compound 7 significantly increased total apoptosis to 29.71% compared with 2.96% in untreated cells, predominantly through late apoptosis (21.92%) with a contribution from early apoptosis (3.29%), while necrosis remained low (4.5%), indicating that cell death occurred mainly via a programmed apoptotic pathway rather than nonspecific cytotoxicity.

Overall, these results confirm that compound 7 exerts a strong anticancer effect through G2/M cell cycle arrest and effective apoptosis induction, in agreement with its superior topoisomerase II inhibitory activity.

2.2.4. Western Blot Analysis of Topoisomerase II Suppression Induced by Naphthyridine-Based Derivative 7

The suppressive effect of naphthyridine-based derivative 7 on topoisomerase II (Topo II) protein expression was further quantified by densitometric analysis of Western blot bands in MCF-7 cells [55].

As summarized in

Table 3. Densitometric quantification of Topo II protein expression in MCF-7 cells following treatment with naphthyridine-based derivative 7, normalized to β-actin.

Sample	Western Blotting, (Topo II) Fold Change
7	0.29
Control	1.00

and Figure S61, treatment with compound 7 resulted in a marked reduction in Topo II expression, showing a relative density of 0.29 compared with the untreated control after normalization to β-actin.

This pronounced decrease at the protein level provides quantitative evidence that the potent enzymatic inhibition exerted by compound 7 is effectively translated into intracellular target suppression. Moreover, the observed downregulation of Topo II offers a mechanistic rationale for the previously reported G2/M cell cycle arrest, further supporting the role of Topo II inhibition as a central contributor to the antiproliferative activity of compound 7.

2.2.5. Western Blot Analysis of Caspase-9-Mediated Apoptotic Signaling Induced by Naphthyridine-Based Derivative 7

Western blot analysis was initially carried out to investigate the effect of naphthyridine-based derivative 7 on Topoisomerase II expression in MCF-7 cells, where a pronounced downregulation of the enzyme was observed compared to the untreated control, as previously demonstrated.

To further elucidate whether this suppression was associated with apoptosis induction, caspase-9 expression was subsequently examined [56,57].

As illustrated in Figure S62, 7-treated MCF-7 cells exhibited a markedly intensified caspase-9 immunoreactive band at approximately 37 kDa relative to the control group, whereas β-actin bands at 42 kDa remained comparable between samples, confirming equal protein loading.

Quantitative densitometric data obtained from Western blot analysis are summarized in Table S6, which presents the raw band intensity measurements of caspase-9, including band area, percentage, relative density, and adjusted density after normalization to β-actin in MCF-7 cells.

Based on this densitometric analysis, caspase-9 expression was further normalized and expressed as fold change values relative to the control, as reported in

Table 4. Normalized fold change values of caspase-9 expression in MCF-7 cells treated with naphthyridine-based derivative 7, relative to the control, as determined from Western blot densitometric analysis.

Sample	Western Blotting, (Topo II) Fold Change
7	5.21
Control	1.78

. As shown, treatment with compound 7 resulted in a pronounced increase in caspase-9 expression, reaching an optical density value of 5.21, compared to 1.78 in control cells. Collectively, the strong consistency between the Western blot images, raw densitometric analysis, normalized fold change values, and graphical presentation supports the conclusion that inhibition of Topoisomerase II by compound 7 leads to activation of the intrinsic mitochondrial apoptotic pathway, thereby providing a mechanistic basis for its cytotoxic activity.

2.2.6. Antiviral in Ovo Potency Against Fowlpox Virus

Four representative β-naphthyridine derivatives (2, 3, 4a, and 9a) were selected from the synthesized series for in ovo antiviral evaluation based on structural diversity and the presence of motifs associated with antiviral activity, allowing focused assessment within a consistent scaffold framework.

In Ovo Cytotoxicity and Antiviral Evaluation

Antiviral activity against fowlpox virus (FPV) was evaluated in SPF embryonated chicken eggs using non-cytotoxic concentrations. All tested compounds exhibited high CC₅₀ values, indicating low embryotoxicity and acceptable safety margins (

Table 5. Cytotoxic concentration (CC₅₀), inhibitory concentration (IC₅₀), and therapeutic index (TI) of the tested synthetic compounds and ribavirin against fowlpox virus (FPV) in SPF embryonated chicken eggs.

Compound	CC50	IC50	TI%
2	>400	≤4	100
3	>800	≤9	88.8
4a	>500	≤7	71.4
9a	>200	≤2	100
Ribavirin	>300	≤3	100

CC₅₀: Cytotoxicity Concentration fifty. IC₅₀: The Inhibitory Concentration. TI %: The Therapeutic Index.

). Compound 3 showed the highest CC₅₀ value (>800), followed by 4a (>500), 2 (>400), and 9a (>200).

At sub-cytotoxic concentrations, all compounds inhibited FPV replication on the chorioallantoic membrane, with IC₅₀ values ranging from ≤2 to ≤9a. Compound 9a displayed the strongest antiviral activity (IC₅₀ ≤ 2), followed by compound 2 (IC₅₀ ≤ 4), while compounds 4a and 3 showed comparatively lower potency. Evaluation of the therapeutic index (TI = CC₅₀/IC₅₀) identified compounds 2 and 9a as the most favorable candidates (TI = 100), comparable to the reference antiviral drug ribavirin [58].

Overall, despite the high safety profile of compound 3, its lower antiviral potency reduced its therapeutic index, whereas compound 9a emerged as the most potent and selective FPV inhibitor under non-cytotoxic in ovo conditions.

In Vivo Antiviral Activity

The in vivo antiviral efficacy of the selected compounds was further evaluated in 10–12-day-old SPF embryonated chicken eggs using non-cytotoxic concentrations determined from the in vitro assays. Antiviral activity was assessed based on embryo survival, reduction in viral infectivity, and suppression of pock lesion formation on the chorioallantoic membrane (CAM), with ribavirin employed as a reference control.

FPV infectivity was evaluated across serial virus dilutions (10¹–10⁶) by monitoring the number of CAMs exhibiting pock lesions. As shown in

Table 6. In vivo antiviral activity of selected synthetic compounds against fowlpox virus (FPV) compared with ribavirin at different viral dilutions, expressed as the number of surviving embryos per total inoculated eggs.

Virus Dilution	FPV Only	Cpd. 2	Cpd. 3	Cpd. 4a	Cpd. 9a	Ribavirin
101	5/5	5/5	5/5	5/5	5/5	5/5
102	5/5	5/5	5/5	5/5	5/5	5/5
103	5/5	5/5	5/5	5/5	4/5	5/5
104	5/5	1/5	3/5	3/5	1/5	3/5
105	5/5	0/5	1/5	2/5	0/5	1/5
106	4/5	0/5	0/5	0/5	0/5	0/5
Virus titer	4.0	1.4	2.0	2.2	1.4	1.8

, FPV infection alone resulted in pronounced CAM lesions and active viral replication, confirming the pathogenic nature of the virus. This was further corroborated by gross morphological examination of the CAM, which revealed extensive pock lesion formation and severe tissue damage in virus-only infected embryos (Figure 9a).

At lower viral dilutions (10¹–10³), all treated groups exhibited complete embryo survival, indicating effective protection at higher viral loads. In contrast, differences in antiviral efficacy became evident at higher dilution levels (10⁴–10⁶), where compounds 2 and 9a demonstrated superior protective effects compared to compounds 3 and 4a, as evidenced by a marked reduction in CAM pock lesion formation.

Morphological examination of the CAM further supported the in vivo antiviral findings. FPV-only infected embryos exhibited extensive pock lesion formation and severe tissue damage (Figure 9a,b). In contrast, treatment with compound 2 resulted in a noticeable reduction in lesion number with improved preservation of CAM morphology (Figure 9c). Notably, embryos treated with compound 9a showed marked suppression of pock lesions and well-preserved CAM integrity, indicating a pronounced protective effect against FPV infection (Figure 9d).

Viral titer analysis further substantiated these observations, revealing a pronounced reduction in FPV infectivity in embryos treated with compounds 2 and 9a, which exhibited the lowest viral titers (1.4). Collectively, these results confirm the superior in vivo antiviral efficacy of compound 9a, followed by compound 2, against FPV infection.

Structure–Activity Relationship (SAR) Analysis

Structure–activity relationship analysis revealed a clear correlation between antiviral efficacy and specific structural features within the β-naphthyridine series. Despite sharing a common core scaffold, variations in functional group composition and spatial orientation exerted a pronounced influence on biological performance. Compounds 2 and 9a consistently demonstrated superior antiviral activity across in vitro, in ovo, and in vivo assays, as reflected by low IC₅₀ values, high therapeutic indices, enhanced embryo survival, significant viral titer reduction, and pronounced suppression of CAM lesions (Figure 9).

The enhanced activity of compound 9a may be attributed to the presence of multiple hydrogen-bond donor and acceptor functionalities, including amino and carbonyl groups, in combination with an extended conjugated system and balanced molecular polarity. These features are likely to facilitate favorable interactions with viral or host targets while supporting efficient membrane permeability. Similarly, the strong antiviral activity of compound 2 may be rationalized by the presence of an electron-withdrawing cyano group in conjunction with hydrogen-bonding motifs, which could enhance binding affinity and stabilize ligand–target interactions.

In contrast, compounds 3 and 4a, despite retaining the same core scaffold, lack the optimal combination or spatial arrangement of these key functional groups, resulting in reduced inhibitory potency and lower therapeutic indices. These findings underscore the crucial role of functional group selection and heteroatom positioning in regulating antiviral activity within the β-naphthyridine framework, providing valuable guidance for the rational design of more potent FPV antiviral agents.

2.3. Computational Studies

2.3.1. In Silico Elucidation of ADMET

In silico ADMET evaluation of dihydronaphtho[2,3-b][1,8]naphthyridine derivatives 2, 7, 8, and 9a was performed using SwissADME and pkCSM [59,60,61] to predict their drug-likeness and pharmacokinetic behavior. As summarized in

Table 7. Calculated physicochemical characteristics of the promising dihydronaphtho[2,3-b][1,8]naphthyridines 2, 7, 8, and 9a.

Compd.	Violations a	MW b	nHBD c	nHBA d	nRB e	TPSA (Å2) f	MLogP g
Rule	-	≤500	≤5	≤10	≤10	≤140	≤4.15
2	0 (Lipinski & Veber)	398.84	2	3	1	95.56	3.51
7	0 (Lipinski) & 1 (Veber, TPSA > 140)	480.91	3	4	1	143.58	3.24
8	0 (Lipinski) & 1 (Veber, TPSA > 140)	479.92	3	4	1	149.63	3.17
9a	0 (Lipinski & Veber)	456.88	3	4	1	132.68	3.71

^a Violations from Lipinski and Veber Rules; ^b Molecular Weight; ^c Number of Hydrogen Bond Donor; ^d Number of Hydrogen Bond Acceptor; ^e Number of Rotatable Bond; ^f Calculated Lipophilicity (MLog Po/w); ^g Topological Polar Surface Area.

, compounds 2 and 9a fully satisfied both Lipinski’s and Veber’s rules with molecular weights of 398.84 and 456.88 Da, TPSA values of 95.56 and 132.68 Ų, and MLogP values of 3.51 and 3.71, respectively. In contrast, compounds 7 (MW = 480.91 Da, TPSA = 143.58 Ų) and 8 (MW = 479.92 Da, TPSA = 149.63 Ų) exhibited a single Veber violation due to elevated TPSA, which may adversely affect membrane permeability, although their lipophilicity (MLogP = 3.24 and 3.17, respectively) remained within the optimal range for oral drugs.

Compounds 2 and 9a exhibited high human intestinal absorption (HIA), whereas compounds 7 and 8 were predicted to have low HIA. All derivatives were anticipated to be P-glycoprotein substrates, which may help to lower intestinal permeability. None of the chemicals were projected to pass the blood–brain barrier, as represented by their negative BBB permeability values (−0.659 for 2, −0.852 for 7, −0.850 for 8, and −1.162 for 9a), in keeping with the boiled-egg model illustrated in Figure 10, implying limited central nervous system exposure.

Distribution analysis demonstrated low steady-state volumes of distribution (VDss), ranging from −1.052 to −1.494, indicating a preference for plasma localization rather than extensive tissue distribution (

Table 8. Anticipated ADMET profile of dihydronaphtho[2,3-b][1,8]naphthyridine 2, 7, 8, and 9a using pkCSM.

Properties (Predicted Value)	Compound
	2	7	8	9a
Absorption
BBB	NO	NO	NO	NO
HIA	High	Low	Low	High
P-glycoprotein Substrate	Yes	Yes	Yes	Yes
Distribution
VDss (human)	−1.052	−1.287	−1.494	−1.436
Fraction unbound (human)	0.201	0.246	0.265	0.201
BBB permeability	−0.659	−0.852	−0.85	−1.162
Metabolism
CYP2D6 substrate	No	No	No	No
CYP3A4 substrate	Yes	Yes	Yes	Yes
CYP1A2 inhibitor	Yes	Yes	No	No
CYP2C19 inhibitor	Yes	Yes	No	Yes
CYP2C9 inhibitor	Yes	Yes	Yes	Yes
CYP2D6 inhibitor	No	No	No	No
CYP3A4 inhibitor	Yes	No	No	No
Excretion and Toxicity
Total Clearance	−0.089	−0.184	−0.172	−0.313
Renal OCT2 substrate	No	No	No	No
AMES toxicity	Yes	Yes	Yes	Yes
Max. tolerated dose (human)	0.386	0.383	0.332	0.377
hERG I inhibitor	No	No	No	No
hERG II inhibitor	Yes	Yes	Yes	Yes
Oral Rat Acute Toxicity (LD50)	2.964	2.638	2.689	2.551
Oral Rat Chronic Toxicity (LOAEL)	0.869	0.667	0.688	0.807
Hepatotoxicity	Yes	Yes	Yes	Yes
Skin Sensitization	No	No	No	No
T. Pyriformis toxicity	0.285	0.285	0.285	0.285
Minnow toxicity	1.438	0.165	2.085	1.593

). The fraction unbound values were moderate for all compounds (0.201–0.265), signifying that a reasonable proportion of the administered dose remains pharmacologically available in systemic circulation.

Metabolic profiling indicated that all compounds are substrates of CYP3A4 but not CYP2D6. Compounds 2 and 7 displayed broader CYP inhibition profiles, inhibiting CYP1A2, CYP2C19, and CYP2C9, however compounds 8 and 9a demonstrated reduced CYP inhibition liabilities, particularly toward CYP1A2 and CYP2C19, suggesting a comparatively lower risk of drug–drug interactions. None of the tested compounds were predicted to inhibit CYP2D6.

Regarding excretion and toxicity, all derivatives had poor total clearance values (−0.089 to −0.313), suggesting persistent systemic exposure. Interestingly, none of the substances were anticipated to inhibit hERG I channels, whereas all were predicted to inhibit hERG II channels. The acute oral toxicity (LD₅₀) values in rats ranged from 2.551 to 2.964 mol/kg, whereas the chronic toxicity (LOAEL) values ranged from 0.667 to 0.869 mol/kg. All compounds were anticipated to be AMES-positive and hepatotoxic, emphasizing the need for additional structural optimization and experimental toxicity testing. However, none of the substances were expected to elicit cutaneous sensitization or operate as renal OCT2 substrates.

2.3.2. Molecular Docking Simulation

Based upon the amazing in vitro inhibitory potencies against Topoisomerase I (Topo I) and Topoisomerase IIβ (Topo IIβ), a docking simulation of potential dihydronaphtho[2,3-b][1,8]naphthyridine 7 and 8 was conducted using Molecular Operating Environment (MOE-Dock) software version 2024.0601 [62,63,64] to determine the anticipated binding affinities with Topo I and Topo IIβ, (PDB IDs: 1T8I and 4G0U, respectively) [65,66]. The X-ray crystallographic structures of Topo I and Topo IIβ, along with their natural ligands, camptothecin and amsacrine, were obtained from the Protein Data Bank. The original ligands, camptothecin and amsacrine, were first redocked into their receptors to validate the docking procedures. This resulted in small RMSD values of 0.86 and 0.72 Å between the docked poses and the co-crystallized ligands, as well as energy scores of −10.28 and −10.61 kcal/mol, respectively.

With high energy scores of −10.52 and −10.18 kcal/mol, respectively, dihydronaphtho[2,3-b][1,8]naphthyridine 7 and 8 were well-placed within Topo I, as seen in Figure 11A,B, respectively. The hexahydrobenzo[6,7]quinolino[2,3-h][1,6]naphthyridine scaffold in both derivatives was fitted well through arene–arene interactions with DNA nucleotides represented in TGP11 and DA113, with additional interaction with DT10 in 7. The key amino acid Arg364 sidechain afforded H-bonding with the amino group at p-6 (distances: 2.33 and 2.44 Å for 7 and 8, respectively). The backbone of Thr718 displayed two H-bonds with the nitrogen of the cyano group at p-3 in compound 7 (distances: 2.78 and 2.99 Å).

During docking within Topo IIβ, derivatives 7 and 8 demonstrated promising energy scores of −9.79 and −10.24 kcal/mol, respectively. The amino group at p-6 of hexahydrobenzo[6,7]quinolino[2,3-h][1,6]naphthyridine scaffold shared H-bond donors with the Arg503 backbone (distances: 2.38 and 3.23 Å for 7 and 8, respectively). Meanwhile, the hexahydrobenzo[6,7]quinolino[2,3-h][1,6]naphthyridine scaffold in both compounds fitted well via arene–arene interactions with DNA nucleotides DA12 and DG13. Furthermore, the centroid of 4-chlorophenyl in 8 exhibits an arene–H interaction with Gln778 (Figure 12A,B).

Concerning the enhanced antiviral activity in vitro and in vivo of compounds 2 and 9a against fowlpox virus (FPV), a docking study was performed on these derivatives against fowlpox virus resolvase (PDB ID: 6P7A) [67] to anticipate their binding affinities and interactions that influence potential activity (Figure 13A,B). NH at p-1 in 2 and NH₂ at p-6 in 9a afforded two H-bond donors with the sidechains of Asp7 and Glu60 (distances: 3.03, 2.94 Å for 2 and 2.40, 2.57 Å for 9a, respectively). Moreover, the nitrogen of the cyano group at p-3 in 2 showed an H-bond acceptor with Thr92 (distance: 3.17 Å).

2.3.3. Molecular Dynamics Simulations

To further validate the docking results and investigate the dynamic behavior and binding stability of compound 7, molecular dynamics (MD) simulations were performed. MD simulations provide a time-dependent description of protein–ligand interactions under physiologically relevant conditions, allowing assessment of conformational stability, binding persistence, and flexibility of both the ligand and the protein environment. Accordingly, MD analyses were conducted for compound 7 in complex with DNA topoisomerase I and DNA topoisomerase II to elucidate its dynamic binding features.

Molecular Dynamics Simulation of the Topo I-Compound 7 Complex

The dynamic stability of the Topoisomerase I-compound 7 complex was evaluated through a comprehensive 100 ns molecular dynamics simulation, incorporating protein–ligand RMSD, RMSF, interaction persistence, and detailed interaction profiling (Figure 14A–F).

Analysis of the protein–ligand RMSD (Figure 14A) revealed an initial equilibration phase during the first 0–5 ns, where the protein backbone RMSD increased from approximately 1.0 Å to ~3.5 Å, reflecting structural relaxation from the docked conformation. A transient increase in protein RMSD was observed around 35–40 ns, reaching a maximum of ~6.8–7.0 Å, which is attributed to localized conformational adjustments within flexible regions rather than global structural destabilization. Following ~45 ns, the protein RMSD converged and fluctuated steadily around 4.5–5.0 Å until the end of the simulation, indicating attainment of a dynamically equilibrated and structurally stable protein conformation. In parallel, the ligand RMSD (Lig fit Prot) exhibited a brief equilibration period within the first 5–10 ns, followed by stable fluctuations in the range of 2.8–3.5 Å, remaining consistently lower than the protein RMSD and showing no abrupt deviations, thereby confirming that compound 7 maintained a stable binding orientation without dissociation from the binding pocket.

Further insight into the flexibility of the complex was obtained through RMSF analysis (Figure 14B,C). The protein RMSF profile demonstrated generally low fluctuations across most residues, with average RMSF values of 0.8–1.5 Å, indicating a rigid and stable protein core upon ligand binding. A pronounced RMSF peak was detected near the C-terminal region (residue index ~470–500), reaching approximately 6.2–6.4 Å; however, this region is distal from the binding pocket and is known to possess intrinsic flexibility, suggesting no adverse impact on active-site integrity. Importantly, residues directly involved in ligand binding exhibited notably reduced RMSF values (<1.0 Å), reflecting restricted mobility and stabilization induced by ligand engagement. Consistently, ligand RMSF analysis revealed limited atomic fluctuations, predominantly within 0.6–0.9 Å, with only minor flexibility observed for peripheral substituents, confirming that compound 7 retained a stable conformation within the Topo I binding pocket throughout the simulation.

The persistence and nature of protein–ligand interactions were subsequently evaluated via contact timeline analysis (Figure 14D). The total number of contacts remained stable over the entire simulation, fluctuating mainly between 2 and 5 interactions, with no prolonged loss of contacts, indicating sustained ligand engagement. Residue-wise analysis identified Ala351, Lys532, Thr718, and Asn722 as key residues maintaining persistent interactions with compound 7, while Arg364, Trp416, Ile427, Met428, and Asp533 contributed additional transient stabilizing contacts. The absence of extended contact-free intervals further confirmed that the ligand remained tightly bound within the Topo I active site, in full agreement with the RMSD and RMSF findings.

The detailed ligand–protein interaction analysis provides insight into the intermolecular contacts stabilizing compound 7 within the Topoisomerase I binding pocket throughout the MD simulation (Figure 14E,F). The two-dimensional interaction map (Figure 14E) illustrates that the ligand establishes a network of persistent polar and water-mediated interactions involving its heterocyclic nitrogen atoms and carbonyl functionalities. Notably, the nitrile nitrogen of compound 7 formed a stable water-mediated hydrogen bond with Asn722, which was maintained for approximately 54% of the simulation time, highlighting a major polar anchoring interaction. In addition, the ligand’s carbonyl oxygen and amino groups participated in water-bridged hydrogen bonds with Thr718 and Lys532, exhibiting occupancies of approximately 22–42%, thereby reinforcing ligand stabilization within the binding cavity. Hydrophobic interactions were also observed between the chlorophenyl moiety of compound 7 and Ala351, contributing favorable van der Waals contacts and hydrophobic packing.

Complementary quantitative analysis of protein–ligand contacts (Figure 14F) further supports these observations, demonstrating that Asn722, Thr718, Lys532, and Ala351 contribute the highest interaction fractions during the 100 ns simulation through a combination of hydrogen bonds, water bridges, and hydrophobic interactions, while ionic contributions were negligible. The combined presence of hydrogen bonding, water-mediated interactions, and hydrophobic contacts reflects a well-balanced and highly stable binding mode that supports both specificity and binding persistence.

Therefore, the convergence of RMSD profiles, low residue-wise and ligand atomic fluctuations, persistent interaction networks, and high interaction occupancies provides compelling evidence for the dynamic stability and robust binding of compound 7 within the active site of human DNA topoisomerase I. The strong consistency among all MD descriptors validates the docking predictions and supports a stable, mechanistically plausible binding mode under physiologically relevant conditions. Based on these encouraging results, the study was subsequently extended to investigate the binding behavior of compound 7 toward DNA topoisomerase II, to explore its potential dual-target inhibitory profile.

Molecular Dynamics Simulation of the Topo II–Compound 7 Complex

The dynamic behavior and binding stability of compound 7 toward DNA topoisomerase II were investigated through a 100 ns molecular dynamics simulation using multiple complementary trajectory analyses (Figure 15A–F).

Evaluation of the protein–ligand RMSD profiles (Figure 15A) revealed a rapid equilibration phase within the first ~5 ns, followed by stable fluctuations of the protein backbone predominantly within ~3.0–4.0 Å over the remainder of the simulation. In parallel, the ligand RMSD stabilized early and fluctuated mainly between ~2.5 and 3.2 Å, remaining consistently lower than the protein RMSD and exhibiting no abrupt increases, indicating that compound 7 retained a persistent binding orientation within the Topo II binding pocket without dissociation.

Residue-wise flexibility analysis further supported complex stability (Figure 15B,C). The protein RMSF profile showed moderate fluctuations across most residues (~0.8–2.0 Å), with higher RMSF peaks reaching ~4.0–4.8 Å in flexible loop or terminal regions distant from the binding site. Importantly, residues directly involved in ligand binding displayed reduced RMSF values (generally ≤1.5 Å), reflecting restricted mobility and local stabilization induced by ligand engagement. Ligand RMSF analysis demonstrated uniform atomic fluctuations mainly within ~2.0–2.9 Å, with slightly higher but smooth variations compared to the Topo I complex, consistent with a stable yet adaptable ligand conformation inside the Topo II active site.

The persistence of protein–ligand interactions throughout the simulation was confirmed by contact timeline analysis (Figure 15D). After an initial equilibration period, sustained interaction patterns were observed across the entire trajectory, with a noticeable increase in the number of contacts during the late stage (~80–100 ns), suggesting progressive stabilization of the bound ligand. Residue-wise contact analysis identified Glu477, Arg503, Glu522, His775, Gln778, Met782, Asn786, Gln789, LYS814, Asp815, and Ala816 as recurrent contributors to ligand binding, while additional transient contacts involving Ile565, Arg729, His774, Gly812, and Ala779 provided auxiliary stabilization. The absence of extended contact-free intervals confirmed that compound 7 remained continuously associated with the Topo II binding site.

Detailed residue-level interaction mapping (Figure 15E) revealed that polar and solvent-assisted interactions primarily mediated ligand stabilization. The amino and carbonyl groups of compound 7 formed water-mediated hydrogen bonds with interaction occupancies of approximately 34–46%, establishing persistent polar anchoring within the binding cavity. The aromatic framework, including the chlorophenyl moiety, exhibited substantial solvent exposure, indicating a limited contribution of hydrophobic enclosure and suggesting that binding is mainly governed by water-bridged hydrogen-bond networks rather than direct hydrophobic packing.

Quantitative interaction fraction analysis (Figure 15F) corroborated these observations, showing that Arg503 contributed the highest interaction fraction predominantly through water bridges, while His775 exhibited notable hydrophobic contributions. Additional stabilizing interactions were observed for Lly505 and Glu477, which combined water-mediated contacts with minor hydrogen-bonding interactions. Moderate interaction frequencies were also detected for Asp815 and Ala816, whereas hydrophobic contributions were limited to residues such as His774 and Met782 with relatively low occupancies. Ionic interactions were negligible across all residues, confirming that electrostatic forces do not play a dominant role in the binding of compound 7 to Topoisomerase II.

From previous data, the molecular dynamics simulation results demonstrate that compound 7 forms a stable and persistent complex with DNA topoisomerase II under solvated conditions. The convergence of protein–ligand RMSD profiles, controlled residue-wise and ligand atomic fluctuations, and the absence of ligand dissociation throughout the 100 ns simulation collectively confirm the dynamic stability of the complex. Persistent protein–ligand contacts, particularly those dominated by water-mediated hydrogen bonds involving key residues such as Arg503, His775, Lys505, Glu477, Asp815, and Ala816 further support a reliable binding mode. Although hydrophobic contributions were limited and direct ionic interactions were negligible, the predominance of solvent-assisted interactions enabled compound 7 to maintain a stable yet adaptable orientation within the Topo II binding pocket. Taken together, these findings indicate that the interaction of compound 7 with Topoisomerase II is robust and dynamically stable, albeit more flexible in nature.

Comparative MD Analysis of Compound 7 Toward Topo I and Topo II

A comparative MD analysis indicated that compound 7 forms a more rigid and structurally stabilized complex with DNA topoisomerase I, as evidenced by lower ligand RMSD values, reduced binding-site flexibility, and persistent interactions supported by hydrogen bonds, water bridges, and hydrophobic contacts. In contrast, binding to topoisomerase II was characterized by slightly higher ligand flexibility and a predominantly water-mediated interaction profile, reflecting a more dynamic yet stable binding mode. Notably, this increased dynamic adaptability likely facilitates more effective enzymatic disruption under cellular conditions, consistent with the superior biological activity observed for Topo II. Accordingly, while Topo I represents the more tightly stabilized in silico target, Topo II emerges as the biologically preferred target of compound 7.

2.3.4. Quantum Chemical Calculations

Quantum chemical calculations based on DFT were performed for compounds 1–10b to investigate their electronic properties, reactivity, and stability using key descriptors such as HOMO-LUMO energies, energy gap (ΔE), global reactivity indices, dipole moment, and charge-transfer parameters (

Table 9. DFT-calculated frontier molecular orbital energies, HOMO-LUMO gap, and global quantum chemical reactivity descriptors of the investigated compounds.

CPD	LUMO	HOMO	ΔE	A	I	X	η	S or σ	ω	ΔN max	ΔN
1	−0.17061	−0.26343	0.09282	0.17061	0.26343	0.21702	0.04641	21.54708037	1.014817505	2.338073691	73.07670761
2	−0.1744	−0.20055	0.02615	0.1744	0.20055	0.187475	0.013075	76.48183556	2.688097562	7.169216061	260.5172084
3	−0.18082	−0.27027	0.08945	0.18082	0.27027	0.225545	0.044725	22.3588597	1.137407424	2.521464505	75.73454444
4a	−0.16657	−0.27095	0.10438	0.16657	0.27095	0.21876	0.05219	19.16075877	0.916956076	2.095803794	64.96685189
4b	−0.16108	−0.23963	0.07855	0.16108	0.23963	0.200355	0.039275	25.4614895	1.02207832	2.550668364	86.56454488
5a	−0.19135	−0.27382	0.08247	0.19135	0.27382	0.232585	0.041235	24.25124288	1.311889953	2.820237662	82.0591124
5b	−0.17716	−0.24043	0.06327	0.17716	0.24043	0.208795	0.031635	31.61055793	1.378073401	3.300063221	107.3368895
6	−0.18207	−0.2011	0.01903	0.18207	0.2011	0.191585	0.009515	105.0972149	3.857573539	10.06752496	357.7727273
7	−0.16343	−0.27191	0.10848	0.16343	0.27191	0.21767	0.05424	18.43657817	0.873529294	2.006544985	62.52147861
8	−0.16549	−0.25149	0.086	0.16549	0.25149	0.20849	0.043	23.25581395	1.010885584	2.424302326	78.97104651
9a	−0.16037	−0.26117	0.1008	0.16037	0.26117	0.21077	0.0504	19.84126984	0.881428431	2.090972222	67.35347222
9b	−0.17048	−0.21018	0.0397	0.17048	0.21018	0.19033	0.01985	50.37783375	1.824962665	4.794206549	171.5282116
10a	−0.20589	−0.21383	0.00794	0.20589	0.21383	0.20986	0.00397	251.8891688	11.0935062	26.43073048	855.1813602
10b	−0.20131	−0.21252	0.01121	0.20131	0.21252	0.206915	0.005605	178.412132	7.638504411	18.45807315	605.9843889

). Overall, compounds 10a and 10b exhibited the most reactive electronic profiles, followed by compounds 6 and 9b, while the remaining derivatives showed moderate to weak electronic reactivity and higher stability.

Analysis of global reactivity descriptors indicated that higher electronic reactivity was associated with lower HOMO-LUMO gaps, increased softness, stronger electrophilicity, and enhanced charge-transfer capability. However, this electronic reactivity did not directly correlate with biological performance. Notably, compounds 7 and 8 demonstrated superior anticancer activity despite their moderate electronic reactivity, suggesting that a balanced combination of stability and reactivity favors selective interaction with cancer-related targets.

In contrast, compounds 2 and 9a displayed enhanced antiviral activity against fowlpox virus, which may be attributed to favorable charge-transfer characteristics and moderate electrophilicity. Collectively, these results emphasize that biological activity is target-dependent and governed by an optimal balance between electronic reactivity and molecular selectivity rather than maximal electronic reactivity alone.

Frontier Molecular Orbitals and Electrostatic Potential Analysis

HOMO/LUMO distributions and electrostatic potential (ESP) maps were analyzed to elucidate key electronic features, with detailed data provided in the Supplementary Information (Tables S7 and S8). HOMO density was mainly localized on the conjugated heterocyclic cores, whereas LUMO density was concentrated on electron-deficient regions, indicating preferred donor–acceptor sites. ESP analysis confirmed pronounced charge separation, supporting the calculated reactivity descriptors and rationalizing the observed target-dependent biological activity.

Quantum Chemical Interpretation of Target-Dependent Structure–Activity Relationships

In addition to global DFT descriptors, an extended electronic structure analysis was performed for five representative compounds (10a, 7, 8, 9a, and 2) to gain deeper mechanistic insight into their target-specific biological behavior. This advanced approach included density of states (DOS), reduced density gradient (RDG) and noncovalent interaction (NCI) analyses, electron localization function (ELF) mapping, and optimized geometries.

These compounds were selected as they represent the most electronically favorable benchmark (10a), the most potent Topo I/II inhibitors (7 and 8), and the most active antiviral candidates against fowlpox virus (9a and 2).

Among the series, compound 10a displayed the most favorable theoretical electronic profile, establishing it as an electronic benchmark rather than the most biologically selective candidate. In contrast, compounds 7 and 8 emerged as the most effective Topo I/II inhibitors, where balanced electronic distribution, molecular rigidity, and functional group orientation favored DNA intercalation and enzyme inhibition. Conversely, compounds 9a and 2 demonstrated superior antiviral activity, attributed to enhanced charge-transfer capability and pronounced electrostatic polarization supporting interaction with viral DNA-processing enzymes.

Frontier molecular orbital analysis revealed consistent donor–acceptor patterns, with HOMO localization on amino-substituted heteroaromatic regions and LUMO concentration on carbonyl-, cyano-, and heterocyclic nitrogen-containing π-systems. Complementary ESP, DOS, and noncovalent interaction analyses identified heterocyclic nitrogen atoms and carbonyl groups as dominant hydrogen-bond acceptors, while amino functionalities acted as hydrogen-bond donors.

Collectively, these findings demonstrate that maximal electronic reactivity does not necessarily translate into optimal biological performance, and that target-specific activity is governed by a balanced interplay between electronic reactivity, molecular selectivity, and functional group orientation, explaining the distinct anticancer (7, 8) and antiviral (9a, 2) profiles observed in this study (Figure 16, Figure 17, Figure 18, Figure 19 and Figure 20; Tables S7 and S8).

Overall DFT Interpretation

DFT analysis of the four selected compounds (7 and 8 as anticancer agents, and 9a and 2 as antiviral candidates) showed that biological activity is primarily driven by molecular planarity, polarized electronic distribution, and functional group orientation rather than by absolute global reactivity parameters. The anticancer compounds displayed electronic features supportive of Topo I/II inhibition, whereas the antiviral compounds exhibited charge distribution and hydrogen-bonding patterns consistent with effective fowlpox virus inhibition.

3. Methods and Materials

3.1. Chemistry

Scheme 1, Scheme 2 and Scheme 3 summarize the synthetic pathways adopted for preparing the designed compounds 1–10b. The strategy commenced with the key intermediate 4-(4-chlorophenyl)-2-oxo-1,2,5,10-tetrahydrobenzo[g]quinoline-3-carbonitrile (A), which was previously prepared and reported [68]. For clarity and reproducibility, its synthesis and physicochemical characterization are also provided in Section 3. Related heterocyclic transformations toward benzo-fused quinoline and naphthyridine scaffolds have been previously reported, supporting the feasibility of the present synthetic approach [69]. Owing to its multifunctional reactive sites, intermediate A enabled further derivatization to afford the final target compounds 1–10b. All spectroscopic characterization data and corresponding spectra (IR, MS, and NMR) are provided in the Supplementary Material (Figures S1–S59).

4-(4-Chlorophenyl)-2-oxo-1,2,5,10-tetrahydrobenzo[g]quinoline-3-carbonitrile (A)

A solution of tetralone (1.46 g, 10 mmol) was prepared in absolute ethanol (30 mL), followed by the addition of ammonium acetate (excess, 3.9 g, 50 mmol). Subsequently, ethyl (E)-3-(4-chlorophenyl)-2-cyanoacrylate (2.35 g, 10 mmol) was added portion-wise to the reaction mixture. The resulting mixture was heated under reflux for 3–5 h, and the progress of the reaction was monitored by TLC. During heating, a solid material gradually separated, which was collected by filtration, thoroughly washed with cold ethanol, and recrystallized from ethanol to afford compound A as yellow crystals; yield: 79%; MP 242–244 °C. IR (KBr, ν max cm⁻¹): 3130 (NH), 3069, 3030 (CH–Ar), 2931 (CH–aliphatic), 2222 (CN), 1636 (C=O), 1552, 1535 (C=C, Ar).¹H NMR (DMSO-d₆, 400 MHz, δ ppm): 2.94, 3.13 (s, 4H, 2CH₂), 7.26 –7.47 (m, 8H, Ar-H), 12.12 (br s, 1H, NH, exchangeable).¹³C NMR (DMSO-d₆, 100 MHz, δ ppm): 23.38 C-7; 27.27 C-8; 115.84 CN; 116.23 C-2; 124.48 C-10; 127.48 C-11; 128.21 C-12; 128.91 C-13; 129.04 C-16, C-20; 135.72 C-18; 139.37 C-9; 139.72 C-14; 159.99 C-1; 170.11 C-3 ppm. Anal. Calcd for C₂₀H₁₃ClN₂O: C, 72.18; H, 3.94; Cl, 10.65; N, 8.42; O, 4.81%. Found: C, 72.20; H, 3.96; N, 8.42%.

1,4-Diamino-5-(4-chlorophenyl)-2-oxo-1,2,6,11-tetrahydronaphtho[2,3-b][1,8]naphthyridine-3-carbonitrile (1)

A mixture of intermediate A (3.32 g, 10 mmol) and cyanoacetohydrazide (0.99 g, 10 mmol) was dissolved in absolute ethanol (12 mL), followed by the addition of a few drops of triethylamine. The reaction mixture was heated under reflux for 36 h, then allowed to cool to room temperature. The precipitated product was filtered off, washed thoroughly with water, and dried. Recrystallization from ethanol furnished compound 1 as yellow crystals; yield 90%; MP 286–288 °C. IR (KBr, ν max cm⁻¹): 3482, 3423, 3255, 3115 (2NH₂), 3075, 3055 (CH-Ar), 2926 (CH aliphatic), 2211 (CN), 1639 (C=O), 1617 (C=N), 1515, 1449 (C=C Ar).¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 4.14, 4.27 (2s, 4H, 2CH₂); 4.53, 5.39 (br s, 4H, 2NH₂, exchangeable); 7.25–7.47 (m, 8H, Ar-H).¹³C NMR (100 MHz, DMSO-d₆) δ (ppm): 24.19 C-6; 28.00 C-11; 83.07 C-3; 114.31, 115.24, 115.71, 122.52, 127.57, 128.10, 128.80, 128.92 Ar-CH; 135.05 C-4′-Cl; 145.04 C-4; 154.00 C-11a; 156.05 C-5a; 160.02 C-2=O; 167.52 C-4a; 175.00 C-NH₂ ppm. MS (EI): m/z 413.3 [M⁺], consistent with C₂₃H₁₆ClN₅O; base peak at m/z 391. Anal. Calcd for C₂₃H₁₆ClN₅O: C, 66.75; H, 3.90; N, 16.92%. Found: C, 66.74; H, 3.88; N, 16.90%.

4-Amino-5-(4-chlorophenyl)-2-oxo-1,2,6,11-tetrahydronaphtho[2,3-b][1,8]naphthyridine-3-carbonitrile (2)

A solution of intermediate A (3.32 g, 10 mmol) in absolute ethanol (10 mL) was treated with triethylamine (5 mL), followed by the addition of malononitrile (0.66 g, 10 mmol). The reaction mixture was heated under reflux for 6 h, then cooled to room temperature and poured into cold water. Acidification with dilute hydrochloric acid induced complete precipitation of the product. The solid was filtered, washed thoroughly with water, dried, and recrystallized from ethanol to afford compound 2 as purple crystals; yield 72%; MP = 277–279 °C. IR (KBr, ν max cm⁻¹): 3412, 3130 (NH₂), 3292 (NH), 3068, 3030 (CH Ar), 2929 (CH aliphatic), 2222 (CN), 1636 (C=O), 1605 (C=N), 1552, 1534 (C=C Ar). ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 4.03, 4.26 (2s, 4H, 2CH₂); 5.15 (br s, 2H, NH₂, exch.); 7.26–7.47 (m, 8H, Ar-H); 12.42 (br s, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆) δ (ppm): 22.11 C-10; 26.33 C-6; 83.00 C-3; 115.71 CN; 116.14, 119.43, 124.32, 125.96, 126.54, 127.36, 128.08, 128.78, 128.89 Ar-CH; 129.37, 129.53, 129.94 C-ortho, meta of p-Cl-Ph; 134.91, 135.57, 135.79, 139.28, 139.58 Ar-C quaternary; 147.12 C-5; 149.78 C-10a; 154.00 C-5a; 166.68 C-2=O; 176.02 C-4-NH₂ ppm. MS (EI): m/z 398.85 [M⁺], consistent with C₂₃H₁₅ClN₄O; base peak at m/z 391.4. Anal. Calcd for C₂₃H₁₅ClN₄O (398.85): C, 69.26; H, 3.79; N, 14.05%. Found: C, 69.24; H, 3.77; N, 14.02%.

4-(4-Chlorophenyl)-10,11-dihydro-5H-benzo[g]pyrazolo[3,4-b]quinolin-3-amine (3)

A mixture of intermediate A (3.32 g, 10 mmol) and hydrazine hydrate (1.21 mL, 25 mmol) in absolute ethanol (5 mL) was heated under reflux for 4 h. The reaction mixture was then allowed to stand overnight at room temperature and subsequently poured onto crushed ice to ensure complete precipitation. The resulting solid was collected by filtration and recrystallized from dry benzene to afford compound 3 as yellow crystals; yield 68%; MP 290–292 °C. IR (KBr, ν max cm⁻¹): 3467, 3129 (NH₂), 3291 (NH), 3067, 3030 (Ar-CH), 2935 (aliphatic CH), 1613, 1583 (2C=N), 1551, 1534 (C=C Ar). ¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 3.02, 3.05 (2s, 4H, 2CH₂); 6.02 (br s, 1H, NH); 6.62 (br s, 2H, NH₂, exchangeable); 7.56–8.08 (m, 8H, Ar-H).¹³C NMR (100 MHz, DMSO-d₆) δ (ppm): 22.27 C-10; 26.07 C-6; 117.23, 124.48, 127.48, 128.21, 128.91, 129.04, 131.55 Ar-CH; 133.40, 134.12, 135.20, 135.72, 139.15, 139.80, 140.22 Ar-C quaternary; 152.11 C-3; 152.53 C-3a; 154.64 C-5a; 158.68 C-4 ppm. MS (EI): m/z 346.82 [M⁺], base peak at m/z 274.4. Anal. Calcd for C₂₀H₁₅ClN₄: C, 69.26; H, 4.36; N, 16.15%. Found: C, 69.24; H, 4.41; N, 16.14%.

General procedure for the synthesis of compounds 4a,b (urea/thiourea derivatives)

A mixture of intermediate A (3.32 g, 10 mmol) and the appropriate reagent, urea or thiourea (0.60 and 0.76 g, 10 mmol), in absolute ethanol (10 mL) containing sodium ethoxide (0.68 g, 10 mmol) was heated under reflux for 20 h. The reaction mixture was then allowed to cool to room temperature, poured onto ice-cold water (50 mL), and neutralized with dilute hydrochloric acid. The precipitated solid was collected by filtration, washed with water, dried, and recrystallized from ethanol to afford the corresponding products 4a,b as crystalline solids (yields and melting points are given below for each compound).

4-Amino-5-(4-chlorophenyl)-6,11-dihydrobenzo[g]pyrimido[4,5-b]quinolin-2(1H)-one (4a)

Off-white crystals; yield 88%; MP 297–299 °C. IR (KBr, ν max cm⁻¹): 3443, 3198 (NH₂), 3287 (NH), 3066, 3030 (Ar–CH), 2964, 2929 (aliphatic CH), 1636 (C=O), 1600, 1584 (2C=N), 1553, 1536 (C=C Ar).¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 3.94, 4.24 (2s, 4H, 2CH₂); 7.06 (br s, 2H, NH₂, exch.); 7.26–8.02 (m, 8H, Ar-H); 12.28 (br s, 1H, NH). ¹³C NMR (100 MHz, DMSO-d₆) δ (ppm): 21.07 C-10; 26.27 C-6; 115.84 C-4a; 116.23, 124.48, 127.48, 128.21, 128.91, 129.04 Ar-CH; 135.72 C-4′-Cl; 139.37, 139.72 Ar-C quaternary; 145.01 C-11a; 146.86 C-5; 151.11 C-2=O; 155.03 C-4; 163.03 C-5a ppm. MS (EI): m/z 374.09 [M⁺], base peak at m/z 274.4. Anal. Calcd for C₂₁H₁₅ClN₄O: C, 67.29; H, 4.03; N, 14.95%. Found: C, 67.31; H, 4.06; N, 14.95%.

4-Amino-5-(4-chlorophenyl)-6,11-dihydrobenzo[g]pyrimido[4,5-b]quinoline-2(1H)-thione (4b)

Pale yellow crystals; yield 82%; MP > 300 °C. IR (KBr) cm⁻¹ ν max: 3462, 3255 cm⁻¹ (ν NH₂), 3296 cm⁻¹ (ν NH), 3065 cm⁻¹ (ν CH- Ar), 2942 cm⁻¹ (ν CH-Alip), 1593 cm⁻¹ (ν 2C=N), 1547, 1524 cm⁻¹ (ν C=C Ar), 1089 cm⁻¹ (ν C=S).¹H-NMR (DMSO-d₆, 400 MHz,) δ = 4.06, 4.24 (s, 4H, 2CH₂); 6.96 (brs, 2H, NH₂ exchangeable); 7.26–7.47 (m, 8H, Ar-H); 14.25 (brs, 1H, NH exchangeable) ppm.¹³C-NMR (DMSO-d₆, 100 MHz) δ = 23.57 C-10; 26.61 C-6; 110.03 C-4a; 124.48, 127.48, 128.21, 128.91, 129.04 Ar-CH; 135.72 C-4′-Cl; 139.37, 139.72 Ar-C quaternary; 145.11 C-5; 147.50 C-11a; 155.00 C-4; 162.93 C-5a; 182.93 C-2=S ppm. MS (EI): m/z 390.07 [M⁺], base peak at m/z 274.4. Anal. Calcd for C₂₁H₁₅ClN₄S: C, 64.53; H, 3.87; N, 14.33%. Found: C, 64.52; H, 3.87; N, 14.35%.

General procedure for the synthesis of compounds 5a,b.

A solution of intermediate A (2.32 g, 10 mmol) in dry ethanol (8 mL) was treated with triethylamine (5 mL), followed by the addition of the appropriate electrophile benzoyl chloride (1.16 mL, 10 mmol) and phenyl isothiocyanate (1.15 mL, 10 mmol). The reaction mixture was stirred at room temperature for 25 h, then poured into ice-cold water and acidified with dilute hydrochloric acid to induce complete precipitation. The obtained solid was collected by filtration, washed thoroughly with water, dried, and recrystallized from acetic acid to afford the corresponding derivatives 5a and 5b. Yields, melting points, and spectral data are provided below for each compound.

1-Benzoyl-4-(4-chlorophenyl)-2-oxo-1,2,5,10-tetrahydrobenzo[g]quinoline-3-carbonitrile (5a)

Dark brown powder; yield 92%; MP > 300 °C. IR (KBr) cm⁻¹ ν max: 3068, 3030 cm⁻¹ (ν CH- Ar), 2926 cm⁻¹ (ν CH-Alip), 2222 cm⁻¹ (ν CN), 1704, 1636 cm⁻¹ (ν 2C=O), 1552, 1534 cm⁻¹ (ν C=C Ar). ¹H-NMR (DMSO-d₆, 400 MHz,) δ = 3.10, 3.66 (s, 4H, 2CH₂); 7.16–7.52 (m, 13H, Ar-H) ppm. 13C-NMR (DMSO-d₆, 100 MHz) δ = 22.39 C-10; 26.29 C-6; 115.00 CN; 120.00, 125.48, 126.73, 127.81, 128.98, 129.25, 129.52, 130.09, 133.58 Ar-CH; 136.00, 139.44 Ar-C quaternary; 160.00 C-2=O; 163.92 C=O phenyl; 169.06 C-4. ppm. MS (EI): m/z 436.10 [M⁺], base peak at m/z 274.4. Anal. Calcd for C₂₇H₁₇ClN₂O₂: C, 74.23; H, 3.92; N, 6.41%. Found: C, 74.25; H, 3.92; N, 6.41%.

4-(4-Chlorophenyl)-3-cyano-2-oxo-N-phenyl-5,10-dihydrobenzo[g]quinoline-1(2H)-carbothioamide (5b)

Dark yellow powder; yield 77%; MP > 300 °C. IR (KBr) cm⁻¹ ν max: 3202 cm⁻¹ (ν NH), 3067, 3033 cm⁻¹ (ν CH- Ar), 2964 cm⁻¹ (ν CH-Alip), 2222 cm⁻¹ (ν CN), 1637 cm⁻¹ (ν C=O), 1552, 1535 cm⁻¹ (ν C=C Ar), 1084 cm⁻¹ (ν C=S). ¹H-NMR (DMSO-d₆, 400 MHz,) δ = 3.02, 3.13 (s, 4H, 2CH₂); 7.26–8.02 (m, 13H, Ar-H); 12.42 (brs, 1H, NH exchangeable) ppm.¹³C-NMR (DMSO-d₆, 100 MHz) δ = 21.55 C-10; 25.19 C-6; 115.73 CN; 116.19, 122.98, 124.84, 127.88, 128.03, 128.16, 128.59, 129.24, 129.39, 129.47, 129.56, 129.86, 131.63 Ar-CH; 135.68, 136.09, 139.79, 140.08, 140.32 Ar-C quaternary; 160.29 C-2=O; 169.00 C-4; 176.53 C=S ppm. MS (EI): m/z 467.09 [M⁺], base peak at m/z 274.4. Anal. Calcd for C₂₇H₁₈ClN₃O_S: C, 69.30; H, 3.88; N, 8.98%. Found: C, 69.32; H, 3.85; N, 8.98%.

3-Acetyl-4,6-diamino-14-(4-chlorophenyl)-2-methyl-8,13-dihydrobenzo[6,7]quinolino[2,3-h][1,6]naphthyridin-5(6H)-one (6)

A mixture of compound 1 (4.13 g, 10 mmol) and acetylacetone (1.03 mL, 10 mmol) in glacial acetic acid (10 mL) containing a few drops of concentrated hydrochloric acid was heated under reflux for 4 h. Progress of the reaction was monitored by TLC. Upon completion, the reaction mixture was allowed to cool to room temperature, and the resulting precipitate was collected by filtration, washed thoroughly with ethanol, and dried to afford compound 6 as yellow crystals; yield 80%; MP 288–290 °C. IR (KBr) cm⁻¹ ν max: 3442, 3290 cm⁻¹ (ν 2NH₂), 3068, 3030 cm⁻¹ (ν CH- Ar), 2955, 2928 cm⁻¹ (ν CH-Alip), 1723, 1636 cm⁻¹ (ν 2C=O), 1600, 1595 cm⁻¹ (ν 2C=N), 1552, 1535 cm⁻¹ (ν C=C Ar). ¹H-NMR (DMSO-d₆, 400 MHz,) δ = 2.15, 2.25 (s, 6H, 2CH₃); 4.10, 4.25 (s, 4H, 2CH₂); 5.50, 6.40 (brs, 4H, 2NH₂ exchangeable); 7.26–8.02 (m, 8H, Ar-H) ppm. ¹³C-NMR (DMSO-d₆, 100 MHz) δ = 20.00 CH3-pyridine; 22.22 C-10; 26.35 C-6; 29.72 CH3-CO; 127.57, 128.01, 128.14, 128.33, 128.59, 129.02, 129.47, 131.63 Ar-CH; 134.11, 134.86, 135.21, 139.75, 140.08 Ar-C quaternary; 143.83 C-5; 150.20 C-12a; 153.39 C-5a; 158.15 C-4; 160.32 C-2=O; 162.33 C-10a; 165.21 C-11a; 192.37 C=O-CH3 ppm. MS (EI): m/z 495.97 [M⁺], base peak at m/z 274.4. Anal. Calcd for C₂₈H₂₂ClN₅O₂: C, 67.81; H, 4.47; N, 14.12%. Found: C, 67.80; H, 4.49; N, 14.12%.

4,6-Diamino-14-(4-chlorophenyl)-2,5-dioxo-1,2,5,6,8,13-hexahydrobenzo[6,7]quinolino [2,3-h][1,6]naphthyridine-3-carbonitrile (7)

Compound 1 (4.13 g, 10 mmol) was suspended in glacial acetic acid (15 mL), followed by the addition of ethyl cyanoacetate (1.8 mL, 10 mmol) and a few drops of concentrated hydrochloric acid. The mixture was heated under reflux for 4 h, during which the progress of the reaction was checked by TLC. Upon completion, the reaction mixture was cooled to room temperature, allowing a solid product to separate out. The precipitated material was isolated by filtration, thoroughly washed with ethanol, and dried to give compound 7 as yellow crystals; yield 68%; MP 279–281 °C. IR (KBr) cm⁻¹ ν max: 3475, 3455, 3292, 3195 cm⁻¹ (ν 2NH₂), 3130 cm⁻¹ (ν NH), 3068, 3030 cm⁻¹ (ν CH- Ar), 2929 cm⁻¹ (ν CH-Alip), 2222 cm⁻¹ (ν CN), 1684, 1637 cm⁻¹ (ν 2C=O), 1597 cm⁻¹ (ν C=N), 1552, 1535 cm⁻¹ (ν C=C Ar). ¹H-NMR (DMSO-d₆, 400 MHz,) δ = 4.13, 4.21 (s, 4H, 2CH₂); 4.16, 4.19 (brs, 4H, 2NH₂ exchangeable); 7.24–7.94 (m, 8H, Ar-H); 11.26 (brs, 1H, NH exchangeable) ppm. ¹³C-NMR (DMSO-d₆, 100 MHz) δ = 21.00 C-10; 25.00 C-6; 70.00 C-3; 115.71 CN; 114.78, 115.31, 116.18, 123.00, 124.85, 127.89, 128.04, 128.59, 129.27, 129.39, 129.47, 129.55 Ar-CH; 135.69, 136.10, 136.21, 139.77 Ar-C quaternary; 140.07 C-4a; 140.30 C-5; 150.66 C-5a; 160.29 C-4=O; 162.70 C-2=O; 176.52 C-11a ppm. MS (EI): m/z 480.11 [M⁺], base peak at m/z 274.4. Anal. Calcd for C₂₆H₁₇ClN₆O₂: C, 64.94; H, 3.56; 7.37; N, 17.48%. Found: C, 64.96; H, 3.58; N, 17.48%.

2,4,6-Triamino-14-(4-chlorophenyl)-5-oxo-5,6,8,13-tetrahydrobenzo[6,7]quinolino[2,3-h][1,6]naphthyridine-3-carbonitrile (8)

Compound 1 (4.13 g, 10 mmol) was combined with malononitrile (0.66 g, 10 mmol) in glacial acetic acid (0.73 mL) containing a few drops of concentrated hydrochloric acid. The reaction mixture was heated under reflux for 4 h, and its progress was monitored by TLC. After completion, the mixture was allowed to cool to room temperature, leading to the formation of a solid product. The precipitate was collected by filtration, washed thoroughly with ethanol, and dried to afford compound 8 as yellow crystals; yield 88%; MP > 300 °C. IR (KBr) cm⁻¹ ν max: 3453, 3413, 3388, 3341, 3292, 3198 cm⁻¹ (ν 3NH₂), 3069, 3030 cm⁻¹ (ν CH- Ar), 2963, 2929 cm⁻¹ (ν CH-Alip), 2223 cm⁻¹ (ν CN), 1637 cm⁻¹ (ν C=O), 1599 cm⁻¹ (ν 2C=N), 1552, 1535 cm⁻¹ (ν C=C Ar). ¹H-NMR (DMSO-d₆, 400 MHz,) δ = 4.06, 4.25 (s, 4H, 2CH₂); 6.22, 6.25, 6.95 (brs, 6H, 3NH₂ exchangeable); 7.12–8.08 (m, 8H, Ar-H) ppm. ¹³C-NMR (DMSO-d₆, 100 MHz) δ = 23.33 C-10; 27.12 C-6; 75.28 C-3; 115.71 CN; 116.14, 119.43, 124.32, 125.96, 126.54, 127.36, 128.08, 128.78, 128.89 Ar-CH; 129.37, 129.53, 129.94 C-ortho, meta of p-Cl-Ph; 134.91, 135.57, 135.79, 139.28, 139.58 Ar-C quaternary; 147.12 C-5; 154.13 C-5a; 159.98 C-10a; 162.11 C-2=O; 162.57 C-4; 163.79 C-11a; 166.28 C-4-NH₂ ppm. MS (EI): m/z 479.93 [M⁺], base peak at m/z 274.4. Anal. Calcd for C₂₆H₁₈ClN₇O: C, 65.07; H, 3.78; N, 20.43%. Found: C, 65.05; H, 3.78; N, 20.42%.

General procedure for the synthesis of compounds 9a,b (urea/thiourea derivatives)

A mixture of intermediate 1 (4.13 g, 10 mmol) and the appropriate reagent, urea or thiourea (0.60 and 0.76 g, 10 mmol), in absolute ethanol (10 mL) containing sodium ethoxide (0.68 g, 10 mmol) was heated under reflux for 12 h. The reaction mixture was then allowed to cool to room temperature, poured onto ice-cold water (50 mL), and neutralized with dilute hydrochloric acid. The precipitated solid was collected by filtration, washed with water, dried, and recrystallized from ethanol to afford the corresponding products 9a,b as crystalline solids (yields and melting points are given below for each compound).

4,6-Diamino-14-(4-chlorophenyl)-8,13-dihydronaphtho[2,3-b]pyrimido[4,5-f][1,8]naphthyridine-2,5(1H,6H)-dione (9a)

Dark brown powder; yield 65%; MP > 300 °C. IR (KBr) cm⁻¹ ν max: 3440, 3421, 3292, 3198 cm⁻¹ (ν 2NH₂), 3131 cm⁻¹ (ν NH), 3069, 3030 cm⁻¹ (ν CH- Ar), 2930 cm⁻¹ (ν CH-Alip), 1687, 1637 cm⁻¹ (ν 2C=O), 1598 cm⁻¹ (ν 2C=N), 1552, 1535 cm⁻¹ (ν C=C Ar). ¹H-NMR (DMSO-d₆, 400 MHz,) δ = 4.05, 4.20 (s, 4H, 2CH₂); 4.53 (brs, 2H, NH₂-N exchangeable); 6.94 (brs, 2H, NH₂-C=N exchangeable); 6.96–7.47 (m, 8H, Ar-H); 8.62 (s, 1H, CH pyrimidinone); 11.10 (brs, 1H, NH exchangeable) ppm. ¹³C-NMR (DMSO-d₆, 100 MHz) δ = 23.39 C-10; 27.16 C-6; 95.69 C-3; 114.79, 115.31, 116.20, 122.99, 124.84, 127.87, 128.02, 128.59, 129.28, 129.41, 129.48, 129.56 Ar-CH; 135.68, 136.12, 136.25, 139.78 Ar-C quaternary; 146.78 C-5; 147.08 C-10a; 153.44 C-5a; 154.03 C-11a; 160.02 C-4=O; 162.70 C-2=O ppm. MS (EI): m/z 456.11 [M⁺], base peak at m/z 274.4. Anal. Calcd for C₂₄H₁₇ClN₆O₂: C, 63.09; H, 3.75; N, 18.39%. Found C, 63.11; H, 3.75; N, 18.42%.

4,6-Diamino-14-(4-chlorophenyl)-2-thioxo-2,6,8,13-tetrahydronaphtho[2,3-b]pyrimido [4,5-f][1,8]naphthyridin-5(1H)-one (9b)

Dark yellow powder; yield 69%; MP > 300 °C. IR (KBr) cm⁻¹ ν max: 3468, 3442, 3292, 3198 cm⁻¹ (ν 2NH₂), 3131 cm⁻¹ (ν NH), 3069, 3065, 3030 cm⁻¹ (ν CH- Ar), 2963 cm⁻¹ (ν CH-Alip), 1637 cm⁻¹ (ν C=O), 1599 cm⁻¹ (ν 2C=N), 1552, 1535 cm⁻¹ (ν C=C Ar), 1085 cm⁻¹ (ν C=S). ¹H-NMR (DMSO-d₆, 400 MHz,) δ = 3.96, 4.20 (s, 4H, 2CH₂); 4.94 (brs, 2H, NH₂ exchangeable); 6.94 (brs, 2H, NH₂-C=N exchangeable); 6.96–7.48 (m, 8H, Ar-H); 8.67 (s, 1H, CH pyrimidine thione); 13.40 (brs, 1H, NH exchangeable) ppm. ¹³C-NMR (DMSO-d₆, 100 MHz) δ = 23.06 C-10; 26.03 C-6; 96.09 C-3; 110.97, 115.31, 116.18, 122.50, 124.85, 127.89, 128.04, 128.59, 129.27, 129.39, 129.47, 129.55 Ar-CH; 130.40, 131.39, 135.69, 136.10 Ar-C quaternary; 145.57 C-5; 156.53 C-5a; 158.00 C-11a; 161.97 C-4=O; 166.23 C-10a; 167.49 C-12a; 180.01 C-2=S ppm. MS (EI): m/z 472.09 [M⁺], base peak at m/z 274.4. Anal. Calcd for C₂₄H₁₇ClN₆OS: C, 60.95; H, 3.62; N, 17.77%. Found C, 60.94; H, 3.66; N, 17.77%.

General procedure for the synthesis of compounds 10a,b.

The appropriate precursor 5a and 5b (4.36 and 4.67 g, 10 mmol) was dissolved in absolute ethanol (10–15 mL), followed by the addition of hydrazine hydrate (1.25 mL, 25 mmol). The reaction mixture was heated under reflux for 4–6 h, and its progress was monitored by TLC. After completion, the mixture was allowed to cool to room temperature, then poured into ice-cold water to induce precipitation of the product. The separated solid was collected by filtration, washed thoroughly with water and a small amount of cold ethanol, and dried. Recrystallization from ethanol afforded the corresponding hydrazide derivatives 10a and 10b in good purity. Yields, melting points, and full spectral data for each compound are given below.

(3-Amino-4-(4-chlorophenyl)-5,10-dihydro-11H-benzo[g]pyrazolo[3,4-b]quinolin-11-yl)(phenyl)methanone (10a)

Dark brownish red powder; yield 84%; MP > 300 °C. IR (KBr) cm⁻¹ ν max: 3433, 3292 cm⁻¹ (ν NH₂), 3069, 3030 cm⁻¹ (ν CH- Ar), 2929 cm⁻¹ (ν CH-Alip), 1708 cm⁻¹ (ν C=O), 1605, 1615 cm⁻¹ (ν 2C=N), 1552, 1535 cm⁻¹ (ν C=C Ar). ¹H-NMR (DMSO-d₆, 400 MHz,) δ = 3.02, 3.88 (s, 4H, 2CH₂); 6.72 (brs, 2H, NH₂ exchangeable); 7.25–7.70 (m, 13H, Ar-H) ppm. ¹³C-NMR (DMSO-d₆, 100 MHz) δ = 24.93 C-10; 28.24 C-6; 110.58, 115.31, 116.18, 122.50, 124.85, 127.89, 128.04, 128.59, 129.27, 129.39, 129.47, 129.55, 131.63, 133.50 Ar-CH; 134.12, 135.69, 136.10, 139.15, 139.73 Ar-C quaternary; 151.92 C-3; 152.59 C-3a; 159.63 C-4; 167.63 C-1=O ppm. MS (EI): m/z 450.12 [M⁺], base peak at m/z 274.4. Anal. Calcd for C₂₇H₁₉ClN₄O: C, 71.92; H, 4.25; N, 12.43%. Found C, 71.93; H, 4.29; N, 12.43%.

3-Amino-4-(4-chlorophenyl)-N-phenyl-5,10-dihydro-11H-benzo[g]pyrazolo[3,4-b]quinoline-11-carbothioamide (10b)

Dark brownish yellow powder; yield 76%; MP > 300 °C. IR (KBr) cm⁻¹ ν max: 3451, 3300 cm⁻¹ (ν NH₂), 3198 cm⁻¹ (ν NH), 3068, 3031 cm⁻¹ (ν CH- Ar), 2930 cm⁻¹ (ν CH-Alip), 1579, 1607 cm⁻¹ (ν 2C=N), 1552, 1535 cm⁻¹ (ν C=C Ar), 1084 cm⁻¹ (ν C=S). ¹H-NMR (DMSO-d₆, 400 MHz,) δ = 3.02, 3.11 (s, 4H, 2CH₂); 6.53 (brs, 2H, NH₂ exchangeable); 7.07–7.74 (m, 13H, Ar-H); 11.77 (brs, 1H, NH exchangeable) ppm. ¹³C-NMR (DMSO-d₆, 100 MHz) δ = 24.45 C-10; 28.66 C-6; 119.46, 122.98, 124.84, 125.10, 127.88, 128.03, 128.16, 128.59, 129.24, 129.39, 129.47, 129.56, 131.00 Ar-CH; 134.12, 135.68, 136.09, 139.79, 140.08 Ar-C quaternary; 156.16 C-3; 157.00 C-3a; 158.16 C-4; 176.33 C-2=S ppm. MS (EI): m/z 481.11 [M⁺], base peak at m/z 274.4. Anal. Calcd for C₂₇H₂₀ClN₅S: C, 67.28; H, 4.18; N, 14.53%. Found C, 67.29; H, 4.22; N, 14.53%.

3.2. Biological Activity

3.2.1. Assessment of Antiproliferative Activity

The antiproliferative properties of the synthesized naphthyridine derivatives were examined using HeLa [70], HCT-116 [71], MCF-7 [72], and WI-38 [73] cell models employing the MTT [74] assay. Cells were treated with increasing concentrations of the compounds for 48 h, after which metabolic activity was assessed by measuring absorbance at 570 nm. Half-maximal inhibitory concentrations (IC₅₀) were determined from nonlinear regression of the dose–response profiles and were compared with reference drugs, doxorubicin and sorafenib. All methodological parameters were detailed in the Supplementary Information.

3.2.2. Topoisomerase I and II Inhibitory Activity of Derivatives 7 and 8

Human topoisomerase I (Topo I) and topoisomerase II (Topo IIβ) levels were determined using commercial sandwich ELISA kits (TOP1: Biomatik, Kitchener, ON, Canada; TOP2: SAB/Signalway Antibody, St. Charles, MO, USA). Absorbance was measured at 450 nm, and enzyme concentrations were calculated from the corresponding standard calibration curves. Full experimental details are provided in the Supplementary Materials [75,76,77,78].

3.2.3. Cell Cycle Arrest and Apoptosis of MCF-7 Cells of Naphthyridine Derivative 7

After MCF-7 cells were treated with naphthyridine derivative 7, cell cycle progression and apoptosis were evaluated using Annexin V-FITC/PI dual labeling and PI-based DNA content analysis, respectively. The stained samples were analyzed using a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA), and the obtained data were processed using Kaluza analysis software v1.2 (Beckman Coulter, Brea, CA, USA). The Supplementary Information contains comprehensive experimental procedures, including staining processes, instrument parameters, and data acquisition settings [79,80].

3.2.4. Western Blot Analysis of Topoisomerase II Suppression Induced by Naphthyridine-Based Derivative 7

Western blot analysis was performed to evaluate the effect of the naphthyridine-based derivative 7 on topoisomerase II (Topo II) protein expression. Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and detected using an alkaline phosphatase-based colorimetric system. Topo II expression levels were quantified by densitometric analysis relative to the corresponding loading control and untreated cells. Full experimental details are provided in the Supplementary Materials [81,82,83,84].

3.2.5. Western Blot Analysis of Caspase-9-Mediated Apoptotic Signaling Induced by Naphthyridine-Based Derivative 7

Caspase-9 protein expression was evaluated by Western blot analysis following treatment with the naphthyridine-based derivative 7. Proteins were separated by SDS–PAGE, transferred to nitrocellulose membranes, and detected using an alkaline phosphatase-based colorimetric system. Densitometric analysis was performed relative to the corresponding loading control, while full experimental details are provided in the Supplementary Materials [85,86].

3.2.6. Antiviral In Ovo Potency Against Fowl Pox Virus

The antiviral activity of synthetic compounds against fowlpox virus (FPV) was evaluated in ovo using 10–12-day-old specific pathogen-free embryonated chicken eggs. Virus–compound mixtures were inoculated onto the chorioallantoic membrane (CAM), and eggs were incubated at 37 °C. FPV infectivity was assessed based on pock lesion formation on the CAM, and viral titers were calculated and compared with untreated virus controls. Detailed experimental procedures are provided in the Supplementary Materials [87,88,89].

3.3. In Silico Analyses

3.3.1. ADMET Prediction

In silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of the targeted compounds were predicted using the freely available online tools admetSAR 1.0 and SwissADME. These platforms were employed to estimate drug-likeness, pharmacokinetic behavior, and toxicity-related parameters, including Lipinski’s and Veber’s rules, gastrointestinal absorption, blood–brain barrier permeability, cytochrome P450 interactions, and toxicity alerts. The predicted ADMET profiles were used to support the experimental findings and to assess the suitability of the investigated compounds for further development [90,91].

3.3.2. Molecular Docking Study

Molecular docking studies were performed to investigate the binding modes of selected naphthyridine derivatives toward DNA topoisomerase I [92], DNA topoisomerase IIβ [93], and fowlpox virus resolvase [76]. The X-ray crystal structures were obtained from the Protein Data Bank (PDB IDs: 1T8I, 4G0U, and 6P7A, respectively). Ligands were prepared and optimized using MOE-Dock (version 2024.0601), and proteins were prepared using the Protonate 3D protocol. Docking was carried out using the Triangle Matcher placement method and London dG scoring function. The docking protocol was validated by redocking the co-crystallized ligands prior to docking of the investigated compounds. Detailed experimental procedures are provided in the Supplementary Materials.

3.3.3. Molecular Dynamics Simulation

Molecular dynamics (MD) simulations were carried out for 100 ns using the Desmond software package (Desmond software package (Schrödinger Release 2021-4, Schrödinger LLC, New York, NY, USA)) [94,95,96]. Protein–ligand complexes obtained from molecular docking were prepared and simulated under NPT conditions (300 K, 1 atm) using the OPLS 2005 force field [97] and TIP3P water model [98], with 0.15 M NaCl added to mimic physiological conditions. Simulation stability was evaluated by monitoring protein and ligand RMSD. Full methodological details are provided in the Supplementary Information.

3.3.4. Quantum Chemical Calculations

DFT calculations were performed using Gaussian 09 [99] at the B3LYP/6-311G++(d,p) level [100,101]. Full computational details are provided in the Supplementary Information.

4. Conclusions

In this study, a series of naphthyridine-based derivatives was successfully designed, synthesized, and biologically evaluated using integrated experimental and computational approaches. Several compounds exhibited potent and selective antiproliferative activity against human cancer cell lines, with compounds 7 and 8 emerging as the most promising anticancer leads. Both compounds acted through dual topoisomerase I/IIβ inhibition; notably, compound 7 induced pronounced G2/M cell cycle arrest and caspase-9-mediated apoptosis, while compound 8 combined strong antiproliferative potency with an excellent safety and selectivity profile. In parallel, selected β-naphthyridine derivatives demonstrated significant antiviral activity against fowlpox virus (FPV) in both in ovo and in vivo models, with compounds 2 and 9a showing superior efficacy and favorable therapeutic indices comparable to ribavirin. Computational investigations, including ADMET profiling, molecular docking, molecular dynamics simulations, and comprehensive DFT analyses, provided mechanistic insight into the observed biological activities and supported the experimental findings. Overall, these results highlight the naphthyridine scaffold as a versatile platform for the development of dual-function anticancer and antiviral agents and provide a solid basis for further optimization and preclinical exploration.