Synthesis and Anticancer Evaluation of PCNA Inhibitor AOH1996 Analogs in Cancer Cell Cultures

Abstract

Proliferating cell nuclear antigen (PCNA) is a critical regulator of DNA replication and repair, and its cancer-associated isoforms represent promising therapeutic targets. The small molecule AOH1996 has been previously reported as a PCNA inhibitor with potent antiproliferative activity. Here, a series of novel AOH1996-based structural analogs were synthesized using structure–activity relationship (SAR) and scaffold-hopping strategies, including 1,2,3-triazole, glycine, and amide derivatives with diverse aromatic and polar substituents. The antiproliferative activity of these compounds was evaluated in MCF-7 (breast cancer) and U87 (glioblastoma) cell lines using the MTT assay. The parent compound AOH1996 exhibited the strongest cytotoxicity, reducing cell viability below 30% at 10 μM. Among the analogs, compounds 1f, 2b, 3b, 3c, and 3d demonstrated significant activity, reducing MCF-7 viability by 60–70% and U87 viability to 30–40% at 10 μM. SAR analysis revealed that electron-withdrawing or moderately lipophilic substituents on the amide side chain and aromatic extensions on the triazole ring enhanced potency, while bulky or strongly electron-donating groups diminished activity. ADMET predictions indicated that most derivatives possessed favorable drug likeness and absorption potential, but high plasma protein binding, short predicted half-lives, and potential cardiotoxicity represent limitations that will require further optimization. Several active compounds were predicted to inhibit P-glycoprotein, suggesting their potential to overcome multidrug resistance. Overall, compounds 2b and 3b showed relatively favorable predicted profiles and can serve as useful lead scaffolds for further optimization and experimental validation.

1. Introduction

Proliferating cell nuclear antigen (PCNA) is an important protein in DNA replication, repair, chromatin organization, and cell cycle regulation, making it essential for the proliferation and survival of both normal and cancer cells [1]. PCNA is a homotrimeric ring-shaped protein that encircles DNA and acts as a sliding clamp, recruiting DNA polymerases and repair proteins via specific motifs (PIP-box, APIM) that bind to its hydrophobic pocket. Inhibition of PCNA can occur through several mechanisms, each targeting its central role in DNA metabolism (

Table 1. Summary of PCNA inhibition mechanisms and their biological effects.

Inhibitor Type	Mechanism of Action	Biological Consequence
Small molecules (e.g., T2AA)	Block PIP-box/monoubiquitinated PCNA interactions	Inhibit TLS, impair DNA repair, sensitize to chemotherapy
Peptides (PIP-box/APIM mimics)	Compete for PCNA binding pocket	Disrupt replication/repair protein recruitment
Aptamers	Form stable complexes with PCNA and DNA polymerases	Block DNA synthesis at replication fork
Endogenous proteins (p21)	Bind PCNA, block polymerase activation	Inhibit DNA replication, cell cycle arrest
Genetic/pharmacologic inhibition	Disrupt PCNA-PARP1 axis, impair DNA repair/cell cycle progression	Sensitize PARP inhibitors, induce apoptosis

).

PCNA in cancer cells is frequently overexpressed, correlating with aggressive tumor behavior, poor prognosis, and resistance to therapy, thus establishing it as a promising biomarker and therapeutic target. Historically, PCNA was considered “undruggable” owing to its lack of deep binding pockets and the absence of endogenous small-molecule modulators, but recent advances in structural biology and drug discovery have led to the identification of small molecules and peptides capable of disrupting PCNA function [2]. Overexpression and cancer-specific isoforms of PCNA (notably, caPCNA) are associated with aggressive tumor phenotypes and poor treatment prognosis across multiple cancer types, including hepatocellular carcinoma, glioblastoma, pancreatic ductal adenocarcinoma, multiple myeloma, and others [3,4,5,6,7].

Small molecules, peptides, or aptamers can bind to the hydrophobic pocket of PCNA—often at the interdomain connecting loop or the PIP-box binding site—thereby preventing the association of PCNA with its protein partners, such as DNA polymerases δ and ε, or repair factors like p15 and PARP1. For example, anti-PCNA aptamers can form stable complexes with PCNA and DNA polymerases, outcompeting primer-template DNA and directly blocking the assembly of the replication machinery, which halts DNA synthesis at the replication fork [8,9]. Small-molecule inhibitors like T2AA specifically disrupt interactions between monoubiquitinated PCNA and translation synthesis (TLS) polymerases (e.g., pol η, REV1), impairing the repair of interstrand DNA cross-links and enhancing DNA double-strand breaks, especially in the presence of DNA-damaging agents such as cisplatin [10]. Peptidic inhibitors, such as those mimicking the PIP-box or APIM motifs, competitively inhibit the binding of PCNA to its partners, thereby suppressing DNA replication and repair processes and sensitizing tumor cells to genotoxic stress. Additionally, endogenous inhibitors like the C-terminal domain of p21 can bind PCNA and block its ability to activate DNA polymerase δ, leading to cell cycle arrest independent of cyclin-dependent kinase inhibition [11]. PCNA inhibition also disrupts its interaction with PARP1, a key player in the DNA damage response, further impairing DNA repair and promoting cell cycle arrest or apoptosis in cancer cells. Collectively, these mechanisms converge on the disruption of PCNA’s scaffolding function, leading to impaired DNA synthesis, defective repair, cell cycle blockade, and increased sensitivity to DNA-damaging therapies, making PCNA a promising target for cancer treatment.

Notable examples of small-molecule inhibitors of PCNA include T2 amino alcohol S-T2AA, AOH1996, PCNA-I1, LRRK2-IN-1, SAR-24, and T2AA-NEal-NTyr, each with distinct mechanisms and selectivity profiles (Figure 1) [2,10].

T2AA and its analogs inhibit PCNA/PIP-box interactions, leading to DNA replication stress and chemosensitization. AOH1996, a first in its class inhibitor, selectively targets a cancer-associated PCNA isoform (caPCNA). The development of small-molecule PCNA inhibitors, particularly AOH1996, represents a breakthrough in targeted cancer therapy. Notably, the small molecule AOH1996 has emerged as a leading candidate, demonstrating the ability to bind caPCNA, stabilize its trimeric structure, and disrupt its association with chromatin, thereby selectively inducing cancer cell death without affecting non-cancerous cells. AOH1996 and its analogs, developed through structure–activity relationship (SAR) studies and scaffold-hopping strategies, have shown enhanced potency, selectivity, and metabolic stability, with some analogs exhibiting up to nine-fold improved efficacy and increased liver microsome stability. These compounds are now in phase 1 clinical trials [12]. AOH1996 is designed to selectively bind to a cancer-associated region of PCNA, disrupting its interactions with key proteins involved in DNA replication, repair, and transcription–replication conflict resolution [4,12,13,14,15,16]. AOH1996 also exhibits anti-metastatic properties by inhibiting angiogenesis, cancer cell migration, and modulating the tumor microenvironment, including suppression of MYC expression and tumor-associated macrophage recruitment. Furthermore, AOH1996 shows promise in combination therapies, including with histone deacetylase inhibitors, KRAS inhibitors, platinum agents, and PARP inhibitors. These advances underscore the growing importance of PCNA inhibitors, and especially AOH1996, as innovative and potentially transformative agents in the fight against cancer.

Researchers have conducted extensive structure–activity relationship (SAR) studies, synthesizing more than 100 analogs by systematically altering three core structural elements: the naphthyl group, glycine linker, and the diphenyl ether segment (Figure 2).

These modifications included replacing the glycine linker with various amino acids, substituting the 1-naphthoyl group with alternative aromatic systems like quinoline and isoquinoline, and introducing a range of functional groups—such as sulfonyl, imido, sulfur, amide, halogen, or methyl groups—at different positions on the diphenyl ether. Through these strategies, analogs like AOH1160S, AOH1996S-4CH3, and AOH1996-3CH3 were identified, exhibiting equal to or greater potency than the original compound. Additional approaches, including scaffold-hopping and bioisostere replacement, produced analogs with unsymmetrical 3-methoxy-5-methyl groups and further substitutions, resulting in up to nine-fold increased potency and markedly improved metabolic stability [2].

Our scientific group synthesized a series of compounds in which the structure is based on the AOH-1996 scaffold, designed with the expectation that they could potentially modulate PCNA-associated pathways. Here, we investigate their anticancer properties in MCF-7 and U87 cell cultures and ADMET predictions of various glycine derivatives, including N-substituted glycine derivatives, triazoles formed from intermediate azides and various terminal acetylenes, and compounds with substituents other than naphthoyl and attached strained heterocycles such as aziridine and azetidine.

2. Results and Discussion

To start with, modeling of the AOH1996 interaction with PCNA involved detailed structural studies, including crystallography of PCNA in complex with AOH1996 and its more soluble analogs, confirming binding at the PIP box cavity and adjacent pockets critical for protein–protein interactions. Scaffold-hopping and bioisostere replacement strategies further enhanced potency, selectivity, and metabolic stability, leading to analogs such as AOH-3M5Me-6F and AOH-3M5Me-6CN, which demonstrated up to nine-fold improved potency and greater liver microsome stability compared to the clinical lead [17]. Synthesis of these inhibitors typically involved combinatorial chemistry approaches, such as fragment-based design and flexible N-alkyl-glycine amide scaffolds, enabling the exploration of diverse subpockets on PCNA and the rapid generation of compound libraries for screening [18]. Following the evaluation of the established PCNA inhibitor AOH1996 for its anticancer efficacy in cell models and informed by findings from prior studies, we initiated the synthesis of novel, previously unreported potential PCNA inhibitors. Structural modifications were guided by structure–activity relationship (SAR) analyses and scaffold-hopping strategies, incorporating both alterations described in the literature and original modifications developed in this work. Figure 2 presents an overview of the structural changes introduced by other researchers [2], alongside the novel modifications to the core scaffold described herein (Scheme 1).

A series of 4-substituted 1,2,3-triazole derivatives (compounds 1a–g) was synthesized using the copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) reaction. The synthesis involved the reaction of substituted organic azide with various terminal alkynes, as depicted in Scheme 2. The CuAAC reaction proceeded regiospecifically to afford 1,4-disubstituted triazoles, with copper(I) facilitating cycloaddition between the azide and alkyne components under mild conditions. Reactions were performed strictly under an inert argon atmosphere to prevent oxidation of Cu(I) to Cu(II) (Scheme 2) (

Table 2. Assignment of R groups to compounds 1a–g.

Compound	R
1a	ClCH2CH2CH2
1b	Ph
1c	4-F-Ph
1d	4-OMe-Ph
1e	4-Et-Ph
1f	4-Ph-Ph
1g	4-n-Amyl-Ph

).

Also, a series of derivatives, designated as compounds 2a–g, were synthesized via nucleophilic substitution of the 2-chloroacetamide derivative with a range of cyclic and acyclic amines, including aziridine, azetidine, pyrrolidine, etc. (Scheme 3) (

Table 3. Assignment of R groups to compounds 2a–g.

Compound	R1	R2
2a	Me	Me
2b	CH2CH2
2c	(CH2)3
2d	Me	Bn
2e	(CH2)4
2f	(CH2)5
2g	O(CH2CH2)2

). Reactions were performed in refluxing acetonitrile using excess of amine.

The parent compound, 2-amino-N-(2-(3-methoxyphenoxy)phenyl)acetamide, exhibited no observable anticancer activity in all preliminary assays. Consequently, a new series of derivatives was synthesized by functionalizing the initial acetamide scaffold with variously substituted benzoyl chlorides, cyanuric chloride, and 3,6-dichloro-1,2,4,5-tetrazine, in an effort to enhance potential anticancer properties (Scheme 4) (

Table 4. Assignment of R groups to compounds 3a–f.

Compound	R
3a	4-NO2PhCO
3b	2-FPhCO
3c	2-ClPhCO
3d	Ph(CH2)3CO
3e	4,6-dichloro-1,3,5-triazin-2-yl
3f	6-chloro-1,2,4,5-tetrazin-3-yl

).

Moreover, the cytotoxic activity of the synthesized AOH1996 derivatives was assessed in MCF-7 (breast cancer) and U-87 (glioblastoma) cell lines after 72 h of treatment using the MTT assay (

Table 5. Cytotoxicity screening of AOH1996 and its derivatives against MCF-7 and U-87 cancer cell lines and BJ-5ta non-cancer cell line after 72 h treatment.

Compound	Concentration (μM)	Cell Viability (% ± SD)			SI (BJ-5ta)
		MCF-7	U-87	BJ-5ta	vs. MCF-7	vs. U-87
1a	10	88.9 ± 5.6	68.5 ± 2.0	72.4 ± 1.8	<2	<2
	100	13.8 ± 6.5	23.5 ± 1.1	20.2 ± 4.4	<2	<2
1f	1	72.1 ± 1.0	70.2 ± 9.0	97.7 ± 7.0	<2	<2
	10	41.2 ± 9.6	28.2 ± 5.2	83.5 ± 4.0	≥2	≥2
	100	9.2 ± 0.4	12.8 ± 1.5	56.9 ± 4.1	≥2	≥2
2b	1	63.2 ± 9.1	74.5 ± 5.4	96.3 ± 7.5	<2	<2
	10	59.1 ± 5.7	41.0 ± 2.5	73.6 ± 5.6	<2	<2
	100	4.9 ± 1.0	13.3 ± 2.1	18.2 ± 3.1	≥2	<2
2f	10	89.6 ± 3.7	82.1 ± 5.3	84.9 ± 11.1	<2	<2
	100	4.7 ± 0.2	26.3 ± 4.0	52.0 ± 6.8	≥2	≥2
2g	10	89.9 ± 5.6	88.3 ± 6.1	98.7 ± 8.0	<2	<2
	100	20.7 ± 6.5	78.3 ± 1.1	73.1 ± 5.1	≥2	<2
3a	1	93.0 ± 3.8	64.1 ± 11.1	93.5 ± 4.5	<2	<2
	10	65.3 ± 1.9	62.0 ± 12.2	82.1 ± 7.9	<2	<2
	100	26.4 ± 0.4	45.5 ± 3.9	74.2 ± 5.6	≥2	<2
3b	1	77.3 ± 4.1	50.0 ± 10.3	89.3 ± 6.0	<2	<2
	10	42.1 ± 2.9	36.7 ± 10.4	70.7 ± 2.3	<2	<2
	100	9.2 ± 1.5	23.8 ± 2.4	38.7 ± 3.5	≥2	<2
3c	1	50.3 ± 6.0	59.2 ± 2.1	78.6 ± 3.5	<2	<2
	10	28.8 ± 6.5	38.7 ± 5.3	31.0 ± 3.5	<2	<2
	100	6.6 ± 0.2	16.1 ± 3.5	38.5 ± 8.8	≥2	≥2
3d	1	62.0 ± 8.0	55.9 ± 3.2	95.0 ± 4.6	<2	<2
	10	44.5 ± 4.6	54.5 ± 3.2	88.6 ± 0.6	≥2	<2
	100	29.0 ± 10.3	56.2 ± 3.5	37.2 ± 3.6	<2	<2
3e	10	93.8 ± 2.4	99.1 ± 8.9	94.6 ± 9.3	<2	<2
	100	6.2 ± 0.1	93.0 ± 4.3	80.5 ± 6.1	≥2	<2
AOH1996	1	40.5 ± 10.2	43.4 ± 12.9	54.2 ± 6.8	<2	<2
	10	17.9 ± 8.4	25.9 ± 2.0	46.7 ± 4.7	≥2	<2
	100	12.9 ± 0.8	21.2 ± 2.0	36.1 ± 3.8	≥2	<2

Cells were treated with compounds at 1, 10, and 100 µM. Viability was determined using the MTT assay and expressed as a percentage of vehicle-treated control (DMSO, 100% viability). Data represent the mean ± standard deviation (SD) from three independent experiments performed in triplicate. The Selectivity Index (SI) was calculated as the ratio of % viability in BJ-5ta normal fibroblasts to % viability in cancer cells (MCF-7 or U-87) at the same concentration.

). The parent compound AOH1996 exhibited the strongest antiproliferative effect, reducing cell viability to below 30% at a concentration of 10 µM in both cell lines. Among the derivatives, compounds 1f, 2b, 3b, 3c, and 3d showed significant reductions in cell viability. Compounds 1f, 3b, 3c, and 3d at a concentration of 10 µM reduced MCF-7 cell viability by 60–70% and demonstrated the highest activity within the series. In the U-87 cell line, compounds 1f, 2b, 3b, and 3c at a concentration of 10 µM reduced cell viability to 30–40%. Derivatives 1a, 2f, 2g, 3a, and 3e, however, maintained a cell viability above 70% across both cell lines at a concentration of 10 µM, indicating limited antiproliferative effects. These results suggested that the incorporation of electron-withdrawing or moderately lipophilic substituents enhanced the cytotoxic potency of the AOH1996 scaffold, whereas bulky or strongly electron-donating groups diminished activity.

Importantly, evaluation in a non-cancerous cell line (BJ-5ta) demonstrated reduced cytotoxic effects for the majority of derivatives.

Based on the preliminary screening, six compounds (1f, 2b, 3b, 3c, 3d, and parent AOH1996) were selected for full dose–response analysis. As shown in Figure 3, distinct cell line-dependent activity profiles were observed. In MCF-7 breast cancer cells, several derivatives showed similar potency (3b IC₅₀ = 0.93 µM, 3c IC₅₀ = 0.85 µM, 3d IC₅₀ = 0.93 µM) to AOH1996 (IC₅₀ = 1.74 µM). By contrast, in U-87 glioblastoma cells, AOH1996 remained the most potent compound (IC₅₀ = 0.99 µM), with all derivatives showing lower potency (IC₅₀ 8.72–38.50 µM) relative to the parent compound. Among the derivatives, 3d retained the highest activity in this cell line.

Structure—Activity Relationship

The SAR analysis of the AOH1996 derivatives indicated that compounds 1f, 2b, 3b, 3c, and 3d exhibited the most pronounced antiproliferative activity, suggesting that specific structural features on both the triazole and amide moieties played key roles in modulating potency. Compound 1f, containing a benzyl-substituted triazole, demonstrated notable cytotoxicity, likely due to enhanced π–π interactions and increased lipophilicity, which facilitated membrane penetration. The aziridine-bearing derivative 2b also showed good activity, suggesting that small polar rings could significantly improve reactivity and possibly favor target binding through hydrogen-bond interactions. Among the 3-series derivatives, the presence of electron-withdrawing substituents such as fluoro (3b) and chloro (3c) groups, as well as the phenylalkyl chain (3d), significantly improved antiproliferative effects, consistent with improved electronic complementarity and optimal lipophilicity for cellular uptake. Comparison of activity in cancerous versus non-cancerous cells suggested that increased lipophilicity alone did not directly correlate with nonspecific toxicity. Derivatives such as 1f and 3d appeared to achieve improved cancer cell selectivity. By contrast, derivatives with bulky or strongly electron-donating groups (e.g., 1a, 2f, 2g, 3a, and 3e) displayed reduced anticancer activity, likely due to steric hindrance or an altered physicochemical balance that compromised target affinity or permeability. Overall, these results highlighted that moderate lipophilicity combined with electron-withdrawing substituents on the amide side chain and aromatic extensions on the triazole ring enhanced anticancer potency, making compounds 1f, 2b, 3b, 3c, and 3d the most promising scaffolds for further optimization.

3. Materials and Methods

3.1. Cell Culturing

Human breast cancer cell line MCF-7 (ATCC HTB-22^TM) and human glioblastoma cell line U87 (ATCC HTB-14^TM) were used in the experiments. These cell lines were selected as representative models of highly proliferative solid tumors in which PCNA is overexpressed and associated with tumor progression and poor prognosis [19,20]. MCF-7 is a well-characterized, hormone-responsive breast cancer model widely used in antiproliferative screening studies, whereas U87 is an aggressive glioblastoma model with high replicative activity and therapeutic resistance, and is commonly used to test novel compounds. Human foreskin fibroblast BJ-5ta (ATCC CRL-4001^TM) was used as a non-cancerous control cell line to assess selectivity. Cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA). Cell cultures were maintained in a humidified incubator at 37 °C with 5% CO₂.

The human breast cancer cell line MCF-7 (ATCC HTB-22™) and the human glioblastoma cell line U87 (ATCC HTB-14™) were originally obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and kindly provided by Prof. Daumantas Matulis (Vilnius University, Vilnius, Lithuania). Human foreskin fibroblasts BJ-5ta (ATCC CRL-4001™) were also originally obtained from ATCC and kindly provided by Prof. Helder Santos (University of Helsinki, Helsinki, Finland).

3.2. MTT Cell Viability Assay

The antiproliferative influence of the compounds was determined using the MTT assay, which measures the activity of mitochondrial dehydrogenases in metabolically active cells. Cells were seeded in 96-well plates (TPP, Trasadingen, Switzerland) at a density of 10,000 cells per well and allowed to adhere overnight. The medium was then aspirated and replaced with fresh medium containing the test compounds. Compounds were prepared by serial dilution from stock solutions (50 mM in DMSO; Fisher Scientific, Waltham, MA, USA), ensuring the final concentration of DMSO in all wells did not exceed 0.2% (v/v). Control wells included a negative control (medium with 0.2% DMSO, serving as the vehicle control) and a positive control (medium with 1% Triton X-100; Fisher Scientific, Waltham, MA, USA). The plates were incubated for 72 h. Following the 72 h treatment, 10 µL of 5 mg/mL MTT (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) solution in phosphate-buffered saline (PBS; Gibco, Grand Island, NY, USA) was added to each well, and the plates were incubated for an additional 4 h to allow for formazan crystal formation. The medium was then carefully removed, and 100 µL of DMSO was added to each well to dissolve the resulting formazan crystals. The absorbance was measured at 570 nm using a microplate reader (CLARIOstar Plus; BMG LABTECH, Ortenberg, Germany), with a 650 nm reference wavelength employed to account for background absorbance. Cell viability was calculated as a percentage relative to the vehicle control.

The IC₅₀ values were determined from dose–response curves fitted using a four-parameter logistic nonlinear regression model in Origin 2015 software (OriginLab, Northampton, MA, USA) with eight concentrations of the compounds (0.01–30 µM). IC₅₀ values are reported as the mean ± SD. All measurements were repeated at least three times.

3.3. Predicted ADMET Properties

Cell viability was calculated as a percentage of the vehicle-treated control (0.2% DMSO). All experiments were performed in triplicate and repeated at least three times. Dose–response graphs were generated (Figure 3). Data are presented as the mean ± standard deviation.

ADMET properties, including absorption, distribution, metabolism, excretion, and toxicity parameters, were predicted using the ADMETlab 3.0 online platform (https://admetmesh.scbdd.com/, accessed on 1 September 2025) based on structure-input analysis [21].

The medicinal chemistry profiles predicted by ADMETlab 3.0 indicated that all compounds satisfied the Lipinski, Pfizer, and Golden Triangle rules, suggesting acceptable drug likeness. However, the GSK rule (MW ≤ 400; logP ≤ 4) was satisfied only for compounds 2b and 3b, while others exceeded the molecular weight threshold.

The Human Intestinal Absorption (HIA) predictions indicated generally high intestinal absorption for compounds (predicted values ≥ 30% for all derivatives) and the potential for good oral bioavailability. The predicted Caco-2 permeability values for all derivatives further supported favorable membrane permeability and consistent absorption potential across the series. Evaluation of P-glycoprotein (P-gp) interactions showed that compounds 2b, 3b, and 3d had high or moderate probabilities of being P-gp inhibitors, which could help to overcome multidrug resistance in tumor cells, although the clinical relevance remains to be verified experimentally. The predicted plasma protein binding (PPB) was approximately 98% for all compounds. Such high PPB values are generally unfavorable, as they may result in lower free drug concentrations and a reduced therapeutic index. The volume of distribution (VDss) values were within the optimal range (0.04–20 L/kg) for compounds 1f and 2b. Blood–brain barrier (BBB) permeability predictions, relevant to CNS-targeted therapy (e.g., glioblastoma), indicated that compound 3c had the highest probability of BBB penetration, while compound 2b showed a low probability, suggesting limited CNS exposure.

ADMETlab 3.0 predicted that compounds 2b and 3d were likely CYP3A4 and CYP2D6 inhibitors and substrates, showing high probabilities of enzyme interaction and poor metabolic stability (HLM > 0.99), highlighting the potential for rapid hepatic metabolism and drug–drug interactions. This suggested rapid hepatic metabolism and a higher potential for drug–drug interactions. By contrast, compounds 1f, 3c, and 3b displayed low CYP interaction probabilities and lower HLM instability values (<0.3), indicating favorable metabolic stability and a lower likelihood of CYP-mediated metabolism. Regarding elimination kinetics, all compounds except 1f showed moderate plasma clearance (5–15 mL/min/kg), while compound 1f exhibited low clearance. Notably, all compounds were classified as ultra-short half-life drugs (t_1/2 < 1 h), indicating rapid systemic elimination that could challenge maintaining therapeutic exposure in vivo.

The toxicity predictions indicated that all compounds displayed moderate to high probabilities of toxicity, including AMES (mutagenic) toxicity, hepatotoxicity, and nephrotoxicity. AOH1996 showed even higher predicted AMES and hepatotoxicity probabilities than its derivatives, suggesting that the structural modifications in 2b, 3b, 3c, and 3d may have partially mitigated intrinsic toxic liability. All compounds showed high probabilities of hERG inhibition, indicating a potential cardiotoxic liability. For the FDA maximum recommended daily dose (FDAMDD), compound 1f showed the highest probability (0.735) of being FDAMDD-positive, suggesting a potentially higher tolerated dose range. Compound 2b displayed a moderate probability (0.52), while for other compounds, a narrower therapeutic window and higher toxicity risk at elevated doses were predicted.

Overall, while these ADMET profiles (

Table 6. ADMET predicted medicinal chemistry and absorption data.

	Medicinal Chemistry					Absorption
	Lipinski	Pfizer rule	GSK rule	Golden triangle		Human intestinal absorption	Caco2 permeability	P-gp substrate/inhibitor
	(MW ≤ 500; logP ≤ 5; etc.)	(logP > 3; TPSA < 75)	(MW ≤ 400; logP ≤ 4)	(200 ≤ MW ≤ 500; −2 ≤ logD ≤ 5)			(Optimal: higher than −5.15 Log unit)
1f	yes	yes	no	yes	1f	≥30%	yes	no
2b	yes	yes	yes	yes	2b	≥30%	yes	inhibitor
3c	yes	yes	no	yes	3c	≥30%	yes	no
3b	yes	yes	yes	yes	3b	≥30%	yes	inhibitor
3d	yes	yes	no	yes	3d	≥30%	yes	inhibitor
AOH1996	yes	yes	no	yes	AOH1996	≥30%	yes	no

,

Table 7. ADMET predicted distribution and metabolism probabilities.

	Distribution				Metabolism
	Volume distribution	BBB permeability Log BB	Plasma protein binding		Substrate CYP 2D6	Substrate CYP 3A4	Inhibitor CYP 2D6	Inhibitor CYP 3A4	Human liver microsomal instability
	(optimal: 0.04–20 L/kg)	(probability of being BBB+)	(Optimal: <90%)		(probability)	(probability)	(probability)	(probability)	(probability)
1f	0.254	0.595	98.469	1f	0.017	0.04	0.003	0.156	0.204
2b	0.057	0.003	98.442	2b	0.992	0.164	0.992	1	0.99
3c	−0.049	0.728	98.449	3c	0.002	0.001	0.35	1	0.381
3b	0.007	0.568	97.939	3b	0	0.005	0.217	1	0.403
3d	−0.014	0.435	98.396	3d	0.814	0.025	0.814	0.998	0.988
AOH1996	0.046	0.344	98.687	AOH1996	0.193	0.002	0.867	1	0.793

and

Table 8. ADMET predicted excretion and toxicity probabilities.

	Excretion			Toxicity
	CL	t 1/2		AMES toxicity	Hepatoxicity	Nephrotoxicity	hERG+	FDA maximum recommended dose
	*	**		(probability)	(probability)	(probability)	(probability)	(probability)
1f	low	ultra short	1f	0.772	0.736	0.829	0.934	0.735
2b	moderate	ultra short	2b	0.662	0.619	0.729	0.936	0.52
3c	moderate	ultra short	3c	0.643	0.673	0.395	0.818	0.229
3b	moderate	ultra short	3b	0.794	0.763	0.627	0.835	0.213
3d	moderate	ultra short	3d	0.549	0.646	0.585	0.906	0.296
AOH1996	moderate	ultra short	AOH1996	0.806	0.712	0.45	0.878	0.303

* (>15 mL/min/kg: high clearance; 5–15 mL/min/kg: moderate clearance; <5 mL/min/kg: low clearance). ** (ultra-short half-life drugs: 1/2 < 1 h; short half-life drugs: T1/2 between 1–4 h; intermediate short half-life drugs: T1/2 between 4–8 h; long half-life drugs: T1/2 > 8 h).

) highlighted significant challenges, particularly related to cardiotoxicity, high plasma binding, and rapid clearance, the derivatives possessed features—such as favorable intestinal absorption, moderate metabolic stability (for 2b and 3b), and potential for P-gp inhibition—that justify their consideration as lead scaffolds for further optimization. Future studies could focus on structural modifications or formulation strategies, including targeted or nanoformulated delivery, to mitigate these limitations while maintaining antiproliferative activity.

3.4. Chemical Synthesis

¹H (400 MHz), ¹³C (101 MHz), HSQC, and HMBC NMR spectra were recorded using a Bruker 400 spectrometer (Bruker, Leipzig, Germany). Residual values of deuterated solvents or TMS were used as internal standards. Chemical shift values are given on the δ (ppm) scale. The following symbols are used to describe the NMR spectra: s—singlet, d—doublet, t—triplet, dt—doublet of triplets, dd—doublet of doublets, td—triplet of doublets, ddd—doublet of doublet of doublets and m—multiplet. Melting points of compounds were determined in open capillaries using a Stuart SMP 10 (Cole-Parmer, Stone, Staffordshire, UK) instrument and are uncorrected. The progress of the reactions was monitored using a Shimadzu GCMS-QP2050 (Shimadzu, Kyoto, Japan) and thin-layer chromatography using TLC silica gel 60 F254 plates (Merck, Darmstadt, Germany). HRMS spectra were obtained on a mass spectrometer (Dual-ESI Q-TOF 6520 (Agilent Technologies, Santa Clara, CA, USA). Eluents included mixtures of hexane, ethyl acetate, methanol, and dichloromethane at various ratios. Vanillin, ninhydrin, UV light, and potassium permanganate were used to develop the TLC plates.

2-Azido-N-(2-(3-methoxyphenoxy)phenyl)acetamide, 2-chloro-N-(2-(3-methoxyphenoxy)phenyl)acetamide, 2-amino-N-(2-(3-methoxyphenoxy)phenyl)acetamide, and AOH1996 were prepared in our laboratory by our developed and yet unpublished procedures.

• General procedure for synthesis of N-(2-(3-methoxyphenoxy)phenyl)-2-(4-substituted-1H-1,2,3-triazol-1-yl)acetamides (1a–g):

Into a 100 mL round-bottomed flask with a reflux condenser, magnetic stirrer, and thermocouple was added 1 eq (2 g, 6.7 mmol) of starting 2-azido-N-(2-(3-methoxyphenoxy)phenyl)acetamide and 1.6 eq (10.72 mmol) of 4-substituted phenylacetylene, unsubstituted phenylacetylene, 4-ethynyl-1,1′-biphenyl, or 5-chloropent-1-yne, followed by the addition of 30 mL of dimethylformamide and 0.15 eq (1 mmol) of copper(I) iodide. The reaction mixture was heated in an oil bath at 80 °C for 24 h. The reaction was performed under an inert atmosphere of argon. After 24 h, the reaction mixture was poured into brine and extracted with MTBE. The separated organic layer was dried over anhydrous sodium sulfate, filtered, and then evaporated under reduced pressure. The resulting solid was crystallized from isopropanol or ethanol depending on the substrate.

• 2-(4-(3-Chloropropyl)-1H-1,2,3-triazol-1-yl)-N-(2-(3-methoxyphenoxy)phenyl)acetamide (1a)

White crystals, m.p. 112–115 °C (iPrOH), Yield 2 g (74%).

¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.32 (dd, J = 8.0, 1.7 Hz, 1H, H_Ar), 8.12 (s, 1H, NH), 7.45 (s, 1H, H_HAr), 7.25–7.15 (m, 1H, H_Ar), 7.15–7.02 (m, 2H, H_Ar), 6.89 (dd, J = 8.0, 1.5 Hz, 1H, H_Ar), 6.71–6.64 (m, 1H, H_Ar), 6.52–6.45 (m, 2H, H_Ar), 5.16 (s, 2H, NCH₂), 3.79 (s, 3H, OCH₃), 3.52 (t, J = 6.3 Hz, 2H, CH₂Cl), 2.85 (t, J = 7.4 Hz, 2H, CCH₂), 2.15–2.04 (m, 2H, CH₂CH₂CH₂).

¹³C NMR (101 MHz, CDCl₃) δ (ppm): 163.21 (CO), 161.03 (C_Ar), 157.23 (C_Ar), 147.32 (C_Ar), 145.55 (C_Ar), 130.35 (C_Ar), 128.59 (C_Ar), 125.20 (C_Ar), 124.24 (C_Ar), 122.61 (C_Ar), 121.06 (C_Ar), 118.34 (C_Ar), 110.21 (C_Ar), 109.55 (C_Ar), 104.42 (C_Ar), 55.45 (OCH₃), 53.60 (NCH₂), 44.08 (CH₂Cl), 31.63 (CCH₂), 22.55 (CH₂CH₂CH₂).

HRMS (ESI) m/z: [M+Na]⁺ calcd for C₂₀H₂₁ClN₄NaO₃, 423.1200; found, 423.1198 (error = −0.45 ppm).

• N-(2-(3-Methoxyphenoxy)phenyl)-2-(4-phenyl-1H-1,2,3-triazol-1-yl)acetamide (1b)

White crystals, m.p. 83–86 °C (iPrOH), Yield 1 g (50%).

¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.32 (dd, J = 8.1, 1.7 Hz, 1H, H_Ar), 8.22 (s, 1H, NH), 7.87 (s, 1H, H_HAr), 7.81–7.71 (m, 2H, H_Ar), 7.48–7.38 (m, 2H, H_Ar), 7.39–7.30 (m, 1H, H_Ar), 7.18–7.01 (m, 3H, H_Ar), 6.90 (dd, J = 8.1, 1.5 Hz, 1H, H_Ar), 6.64–6.54 (m, 1H, H_Ar), 6.48–6.38 (m, 2H, H_Ar), 5.22 (s, 2H, NCH₂), 3.68 (s, 3H, OCH₃).

¹³C NMR (101 MHz, CDCl₃) δ (ppm): 163.15 (CO), 160.93 (C_Ar), 157.27 (C_Ar), 148.57 (C_Ar), 145.49 (C_Ar), 130.27 (C_Ar), 129.99 (C_Ar), 128.83 (C_Ar), 128.71 (C_Ar), 128.43 (C_Ar), 125.86 (C_Ar), 125.29 (C_Ar), 124.34 (C_Ar), 121.23 (C_Ar), 121.01 (C_Ar), 118.66 (C_Ar), 109.90 (C_Ar), 109.48 (C_Ar), 103.99 (C_Ar), 55.30 (OCH₃), 53.72 (NCH₂).

HRMS (ESI) m/z: [M+H]⁺ calcd for C₂₃H₂₁N₄O₃, 401.1614; found, 401.1616 (error = 0.58 ppm).

• 2-(4-(4-Fluorophenyl)-1H-1,2,3-triazol-1-yl)-N-(2-(3-methoxyphenoxy)phenyl)acetamide (1c)

White crystals, m.p. 104–108 °C (iPrOH), Yield 1.5 g (54%).

¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.33 (d, J = 8.0 Hz, 1H, H_Ar), 8.12 (s, 1H, NH), 7.83 (s, 1H, H_HAr), 7.79–7.68 (m, 2H, H_Ar), 7.55–7.44 (m, 3H, H_Ar), 7.18–7.12 (m, 2H, H_Ar), 6.91 (d, J = 8.0 Hz, 1H, H_Ar), 6.65–6.52 (m, 1H, H_Ar), 6.48–6.36 (m, 2H, H_Ar), 5.23 (s, 2H, NCH₂), 3.71 (s, 3H, OCH₃).

¹³C NMR (101 MHz, CDCl₃) δ (ppm): 162.98 (CO), 160.97 (C_Ar), 157.26 (C_Ar), 147.48 (C_Ar), 145.42 (C_Ar), 134.54 (d, J = 8.6 Hz, C_Ar), 130.29 (C_Ar), 128.66 (C_Ar), 127.63 (d, J = 7.9 Hz, C_Ar), 126.23 (C_Ar), 125.33 (C_Ar), 124.39 (C_Ar), 121.13 (C_Ar), 120.69 (C_Ar), 118.64 (C_Ar), 117.82 (C_Ar), 109.89 (C_Ar), 109.44 (C_Ar), 104.08 (C_Ar), 55.33 (OCH₃), 53.78 (NCH₂).

HRMS (ESI) m/z: [M+H]⁺ calcd for C₂₃H₂₀FN₄O₃, 419.1519; found, 419.1510 (error = −2.25 ppm).

• N-(2-(3-Methoxyphenoxy)phenyl)-2-(4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl)acetamide (1d)

White crystals, m.p. 138–141 °C (iPrOH), Yield 2 g (69%).

¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.32 (dd, J = 8.1, 1.7 Hz, 1H, H_Ar), 8.20 (s, 1H, NH), 7.77 (s, 1H, H_Ar), 7.72–7.65 (m, 2H, H_Ar), 7.51–7.40 (m, 1H, H_Ar), 7.18–7.03 (m, 3H, H_Ar), 6.90 (dd, J = 8.0, 1.5 Hz, 1H, H_Ar), 6.88–6.78 (m, 1H, H_Ar), 6.65–6.54 (m, 1H, H_Ar), 6.48–6.38 (m, 2H, H_Ar), 5.20 (s, 2H, NCH₂), 3.85 (s, 3H, OCH₃), 3.69 (s, 3H, OCH₃).

¹³C NMR (101 MHz, CDCl₃) δ (ppm): 163.22 (CO), 160.94 (C_Ar), 159.79 (C_Ar), 157.27 (C_Ar), 148.48 (C_Ar), 145.48 (C_Ar), 134.04 (C_Ar), 130.26 (C_Ar), 128.72 (C_Ar), 127.19 (C_Ar), 125.26 (C_Ar), 124.34 (C_Ar), 122.69 (C_Ar), 121.22 (C_Ar), 120.16 (C_Ar), 118.65 (C_Ar), 114.22 (C_Ar), 109.48 (C_Ar), 103.98 (C_Ar), 55.33 (OCH₃), 55.31 (OCH₃), 53.72 (NCH₂).

HRMS (ESI) m/z: [M+Na]⁺ calcd for C₂₄H₂₂N₄NaO₄, 453.1539; found, 453.1554 (error = 3.37 ppm).

• 2-(4-(4-Ethylphenyl)-1H-1,2,3-triazol-1-yl)-N-(2-(3-methoxyphenoxy)phenyl)acetamide (1e)

White crystals, m.p. 121–124 °C (iPrOH), Yield 2.1 g (56%).

¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 8.49 (s, 1H, H_HAr), 8.10–8.00 (m, 1H, H_Ar), 7.77 (d, J = 7.9 Hz, 2H, H_Ar), 7.35–7.25 (m, 3H, H_Ar), 7.17–7.09 (m, 2H, H_Ar), 6.97–6.89 (m, 1H, H_Ar), 6.76 (dd, J = 8.3, 2.4 Hz, 1H, H_Ar), 6.67–6.53 (m, 2H, H_Ar), 5.44 (s, 2H, NCH₂), 3.74 (s, 3H, OCH₃), 2.63 (q, 2H, CH₂), 1.20 (t, J = 7.6 Hz, 3H, CH₃).

¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 165.21 (CO), 161.11 (C_Ar), 158.12 (C_Ar), 147.71 (C_Ar), 147.63 (C_Ar), 146.75 (C_Ar), 143.95 (C_Ar), 130.96 (C_Ar), 129.40 (C_Ar), 128.77 (C_Ar), 128.66 (C_Ar), 125.64 (C_Ar), 124.20 (C_Ar), 123.61 (C_Ar), 123.11 (C_Ar), 119.17 (C_Ar), 111.06 (C_Ar), 109.78 (C_Ar), 105.31 (C_Ar), 55.77 (OCH₃), 52.66 (NCH₂), 28.41 (CH₂), 15.98 (CH₃).

HRMS (ESI) m/z: [M+H]⁺ calcd for C₂₅H₂₅N₄O₃, 429.1927; found, 429.1915 (error = −2.72 ppm).

• 2-(4-([1,1′-Biphenyl]-4-yl)-1H-1,2,3-triazol-1-yl)-N-(2-(3-methoxyphenoxy)phenyl)acetamide (1f)

White crystals, m.p. 156–158 °C (iPrOH), Yield 2.0 g (63%).

¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.33 (dd, J = 8.1, 1.7 Hz, 1H, H_Ar), 8.19 (s, 1H, NH), 7.90 (s, 1H, H_HAr), 7.88–7.80 (m, 2H, H_Ar), 7.69–7.61 (m, 4H, H_Ar), 7.51–7.41 (m, 2H, H_Ar), 7.41–7.33 (m, 1H, H_Ar), 7.18–7.03 (m, 3H, H_Ar), 6.91 (dd, J = 8.0, 1.5 Hz, 1H, H_Ar), 6.63–6.55 (m, 1H, H_Ar), 6.48–6.40 (m, 2H, H_Ar), 5.24 (s, 2H, NCH₂), 3.67 (s, 3H, OCH₃).

¹³C NMR (101 MHz, CDCl₃) δ (ppm): 163.11 (CO), 160.96 (C_Ar), 157.27 (C_Ar), 148.31 (C_Ar), 145.47 (C_Ar), 141.16 (C_Ar), 140.48 (C_Ar), 130.28 (C_Ar), 128.93 (C_Ar), 128.87 (C_Ar), 128.70 (C_Ar), 127.53 (C_Ar), 127.49 (C_Ar), 126.98 (C_Ar), 126.25 (C_Ar), 125.30 (C_Ar), 124.36 (C_Ar), 121.19 (C_Ar), 120.97 (C_Ar), 118.65 (C_Ar), 109.88 (C_Ar), 109.52 (C_Ar), 104.01 (C_Ar), 55.30 (OCH₃), 53.77 (NCH₂).

HRMS (ESI) m/z: [M+Na]⁺ calcd for C₂₉H₂₄N₄NaO₃, 499.1746; found, 499.1753 (error = 1.38 ppm).

• N-(2-(3-Methoxyphenoxy)phenyl)-2-(4-(4-pentylphenyl)-1H-1,2,3-triazol-1-yl)acetamide (1g)

White crystals, m.p. 77–80 °C (EtOH), Yield 1.8 g (57%).

¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 8.48 (s, 1H, H_HAr), 8.03 (q, J = 5.0 Hz, 1H, H_Ar), 7.75 (d, J = 7.9 Hz, 2H, H_Ar), 7.30 (t, J = 8.2 Hz, 1H, H_Ar), 7.27 (s, 1H, H_Ar), 7.25 (s, 1H, H_Ar), 7.11 (dt, J = 9.3, 3.6 Hz, 2H, H_Ar), 6.91 (dd, J = 6.2, 3.4 Hz, 1H, H_Ar), 6.74 (dd, J = 8.3, 2.4 Hz, 1H, H_Ar), 6.62 (d, J = 2.5 Hz, 1H, H_Ar), 6.57 (dd, J = 8.1, 2.3 Hz, 1H, H_Ar), 5.42 (d, J = 2.1 Hz, 2H, NCH₂), 3.73 (s, 3H, OCH₃), 2.58 (t, J = 7.7 Hz, 2H, CH₂), 1.57 (h, J = 6.9, 6.3 Hz, 2H, CH₂), 1.29 (qt, J = 8.0, 3.5 Hz, 4H, CH₂CH₂), 0.89–0.80 (m, 3H, CH₃).

¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 165.20 (CO), 161.10 (C_Ar), 158.11 (C_Ar), 147.62 (C_Ar), 146.75 (C_Ar), 142.56 (C_Ar), 132.81 (C_Ar), 130.95 (C_Ar), 129.29 (C_Ar), 128.65 (C_Ar), 125.80 (C_Ar), 125.58 (C_Ar), 124.19 (C_Ar), 123.59 (C_Ar), 123.10 (C_Ar), 119.17 (C_Ar), 111.06 (C_Ar), 109.78 (C_Ar), 105.31 (C_Ar), 55.77 (OCH₃), 52.66 (NCH₂), 35.33 (CH₂), 31.36 (CH₂), 31.03 (CH₂), 22.44 (CH₂), 14.41 (CH₃).

HRMS (ESI) m/z: [M+H]⁺ calcd for C₂₈H₃₁N₄O₃, 471.2396; found, 471.2399 (error = −0.60 ppm).

• General procedure for substitution of 2-chloro-N-(2-(3-methoxyphenoxy)phenyl)acetamide with various amines (2a–g):

Into a 100 mL round-bottomed flask with a reflux condenser and magnetic stirrer was added 1 eq (6.87 mmol) of starting 2-chloro-N-(2-(3-methoxyphenoxy)phenyl)acetamide and 2.1 eq (4.4 mmol) of a nitrogen atom-containing nucleophile (Table 3), followed by the addition of 30 mL of acetonitrile. The reaction mixture was refluxed for 3 h. After the reaction was finished, the acetonitrile was evaporated under reduced pressure. The resulting solid was extracted with methylene chloride, washed with brine, dried over sodium sulfate, and then evaporated once more under reduced pressure to give a solid, which was crystallized from isopropanol or cyclohexane depending on the substrate.

• 2-(Dimethylamino)-N-(2-(3-methoxyphenoxy)phenyl)acetamide (2a)

White crystals, m.p. 54–56 °C (cyclohexane), Yield 1.7 g (83%).

¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.64 (s, 1H, NH), 8.46 (m, 1H, H_Ar), 7.26–7.13 (m, 2H, H_Ar), 7.06 (m, 1H, H_Ar), 6.99 (m, 1H, H_Ar), 6.64 (m, 1H, H_Ar), 6.58–6.50 (m, 2H, H_Ar), 3.77 (s, 3H, OCH₃), 3.01 (s, 2H, NCH₂), 2.19 (s, 6H, CH₃).

¹³C NMR (101 MHz, CDCl₃) δ (ppm): 169.00 (CO), 161.03 (C_Ar), 158.41 (C_Ar), 144.80 (C_Ar), 130.24 (C_Ar), 130.13 (C_Ar), 124.84 (C_Ar), 124.10 (C_Ar), 120.97 (C_Ar), 119.65 (C_Ar), 109.52 (C_Ar), 108.73 (C_Ar), 103.64 (C_Ar), 63.72 (NCH₂), 55.39 (OCH₃), 45.72 (CH₃).

HRMS (ESI) m/z: [M+H]⁺ calcd for C₁₇H₂₁N₂O₃, 301.1552; found, 301.1557 (error = 1.60 ppm).

• 2-(Aziridin-1-yl)-N-(2-(3-methoxyphenoxy)phenyl)acetamide (2b)

White crystals, m.p. 75–78 °C (cyclohexane), Yield 1.5 g (63%).

¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.47 (s, 1H, NH), 8.49 (dd, J = 8.1, 1.6 Hz, 1H, H_Ar), 7.26–7.12 (m, 2H, H_Ar), 7.06 (td, J = 7.8, 1.6 Hz, 1H, H_Ar), 6.96 (dd, J = 8.1, 1.5 Hz, 1H, H_Ar), 6.69–6.62 (m, 1H, H_Ar), 6.59–6.52 (m, 2H, H_Ar), 3.77 (s, 3H, OCH₃), 3.02 (s, 2H, NCH₂CO), 1.74–1.68 (m, 2H, CH₂), 1.28–1.20 (m, 2H, CH₂).

¹³C NMR (101 MHz, CDCl₃) δ (ppm): 168.70 (CO), 161.02 (C_Ar), 158.11 (C_Ar), 145.16 (C_Ar), 130.26 (C_Ar), 129.93 (C_Ar), 124.60 (C_Ar), 124.24 (C_Ar), 120.91 (C_Ar), 119.06 (C_Ar), 109.94 (C_Ar), 108.99 (C_Ar), 103.99 (C_Ar), 64.24 (NCH₂CO), 55.40 (OCH₃), 27.74 (CH₂).

HRMS (ESI) m/z: [M+Na]⁺ calcd for C₁₇H₁₈N₂NaO₃, 321.1215; found, 321.1214 (error = −0.35 ppm).

• 2-(Azetidin-1-yl)-N-(2-(3-methoxyphenoxy)phenyl)acetamide (2c)

White crystals, m.p. 91–94 °C (iPrOH), Yield 1.7 g (73%).

¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.56 (s, 1H, NH), 8.47 (dd, J = 8.2, 1.6 Hz, 1H, H_Ar), 7.26–7.12 (m, 2H, H_Ar), 7.05 (td, J = 7.6, 7.2, 1.6 Hz, 1H, H_Ar), 7.00 (dd, J = 8.1, 1.7 Hz, 1H, H_Ar), 6.65 (ddd, J = 8.3, 2.4, 0.9 Hz, 1H, H_Ar), 6.61–6.52 (m, 2H, H_Ar), 3.78 (s, 3H, OCH₃), 3.20–3.12 (m, 6H, CH₂CH₂CH₂ and NCH₂CO), 1.96 (p, J = 7.0 Hz, 2H, CH₂CH₂CH₂).

¹³C NMR (101 MHz, CDCl₃) δ (ppm): 168.72 (CO), 161.09 (C_Ar), 158.56 (C_Ar), 144.46 (C_Ar), 130.33 (C_Ar), 130.28 (C_Ar), 124.99 (C_Ar), 124.04 (C_Ar), 120.81 (C_Ar), 119.89 (C_Ar), 109.21 (C_Ar), 108.67 (C_Ar), 103.39 (C_Ar), 63.28 (NCH₂CO), 55.76 (CH₂CH₂CH₂), 55.40 (OCH₃), 17.49 (CH₂CH₂CH₂).

HRMS (ESI) m/z: [M+H]⁺ calcd for C₁₈H₂₁N₂O₃, 313.1552; found, 313.1564 (error = 3.77 ppm).

• 2-(Benzyl(methyl)amino)-N-(2-(3-methoxyphenoxy)phenyl)acetamide (2d)

Yellow oil, Yield 2 g (78%).

¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.86 (s, 1H, NH), 8.50 (dd, J = 8.1, 1.6 Hz, 1H, H_Ar), 7.28–7.10 (m, 7H, H_Ar), 7.04 (td, J = 7.8, 1.6 Hz, 1H, H_Ar), 7.01–6.93 (m, 1H, H_Ar), 6.68 (ddd, J = 8.3, 2.3, 1.0 Hz, 1H, H_Ar), 6.63–6.56 (m, 2H, H_Ar), 3.75 (s, 3H, OCH₃), 3.57 (s, 2H, PhCH₂), 3.13 (s, 2H, NCH₂CO), 2.18 (s, 3H, NCH₃).

¹³C NMR (101 MHz, CDCl₃) δ (ppm): 169.11 (CO), 161.09 (C_Ar), 158.16 (C_Ar), 144.94 (C_Ar), 137.86 (C_Ar), 130.35 (C_Ar), 130.05 (C_Ar), 128.76 (C_Ar), 128.44 (C_Ar), 127.41 (C_Ar), 124.59 (C_Ar), 123.97 (C_Ar), 120.49 (C_Ar), 118.93 (C_Ar), 110.00 (C_Ar), 109.10 (C_Ar), 104.02 (C_Ar), 62.30 (NCH₂CO), 61.33 (PhCH₂), 55.39 (OCH₃), 42.99 (NCH₃).

HRMS (ESI) m/z: [M+H]⁺ calcd for C₂₃H₂₅N₂O₃, 377.1865; found, 377.1852 (error = −3.49 ppm).

• N-(2-(3-Methoxyphenoxy)phenyl)-2-(pyrrolidin-1-yl)acetamide (2e)

White crystals, m.p. 88–91 °C (iPrOH), Yield 1.5 g (67%).

¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.71 (s, 1H, NH), 8.49 (dd, J = 8.2, 1.6 Hz, 1H, H_Ar), 7.24–7.15 (m, 2H, H_Ar), 7.06 (td, J = 7.8, 1.6 Hz, 1H, H_Ar), 7.01 (dd, J = 8.1, 1.6 Hz, 1H, H_Ar), 6.62 (ddd, J = 8.3, 2.4, 0.9 Hz, 1H, H_Ar), 6.54–6.45 (m, 2H, H_Ar), 3.77 (s, 3H, OCH₃), 3.22 (s, 2H, NCH₂CO), 2.54–2.45 (m, 4H, CH₂CH₂CH₂CH₂), 1.64 (dd, J = 10.0, 3.3 Hz, 4H, CH₂CH₂CH₂CH₂).

¹³C NMR (101 MHz, CDCl₃) δ (ppm): 169.38 (CO), 161.00 (C_Ar), 158.63 (C_Ar), 144.17 (C_Ar), 130.59 (C_Ar), 130.25 (C_Ar), 125.17 (C_Ar), 124.06 (C_Ar), 120.79 (C_Ar), 120.13 (C_Ar), 108.76 (C_Ar), 108.43 (C_Ar), 102.97 (C_Ar), 59.53 (NCH₂CO), 55.37 (OCH₃), 54.30 (CH₂CH₂CH₂CH₂), 23.97 (CH₂CH₂CH₂CH₂).

HRMS (ESI) m/z: [M+H]⁺ calcd for C₁₉H₂₃N₂O₃, 327.1709; found, 327.1715 (error = 1.93 ppm).

• N-(2-(3-Methoxyphenoxy)phenyl)-2-(piperidin-1-yl)acetamide (2f)

White crystals, m.p. 89–92 °C (iPrOH), Yield 1.5 g (80%).

¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.90 (s, 1H, NH), 8.50 (dd, J = 8.2, 1.6 Hz, 1H, H_Ar), 7.28–7.14 (m, 2H, H_Ar), 7.04 (td, J = 7.8, 1.6 Hz, 1H, H_Ar), 6.98 (dd, J = 8.2, 1.6 Hz, 1H, H_Ar), 6.64 (ddd, J = 8.3, 2.3, 1.0 Hz, 1H, H_Ar), 6.58–6.50 (m, 2H, H_Ar), 3.77 (s, 3H, OCH₃), 3.03 (s, 2H, NCH₂CO), 2.42 (t, J = 5.0 Hz, 4H, CH₂CH₂CH₂CH₂CH₂), 1.45–1.30 (m, 6H, CH₂CH₂CH₂CH₂CH₂).

¹³C NMR (101 MHz, CDCl₃) δ (ppm): 169.29 (CO), 161.02 (C_Ar), 158.40 (C_Ar), 144.58 (C_Ar), 130.41 (C_Ar), 130.25 (C_Ar), 124.86 (C_Ar), 123.99 (C_Ar), 120.66 (C_Ar), 119.50 (C_Ar), 109.36 (C_Ar), 108.67 (C_Ar), 103.50 (C_Ar), 62.92 (NCH₂CO), 55.37 (OCH₃), 54.84 (CH₂CH₂CH₂CH₂CH₂), 26.01 (CH₂CH₂CH₂CH₂CH₂), 23.57 (CH₂CH₂CH₂CH₂CH₂).

HRMS (ESI) m/z: [M+H]⁺ calcd for C₂₀H₂₅N₂O₃, 341.1865; found, 341.1852 (error = −3.86 ppm).

• N-(2-(3-Methoxyphenoxy)phenyl)-2-morpholinoacetamide (2g)

White crystals, m.p. 114–117 °C (iPrOH), Yield 1.8 g (78%).

¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.72 (s, 1H, NH), 8.48 (dd, J = 8.2, 1.6 Hz, 1H, H_Ar), 7.26–7.14 (m, 2H, H_Ar), 7.07 (td, J = 7.8, 1.6 Hz, 1H, H_Ar), 6.99 (dd, J = 8.1, 1.5 Hz, 1H, H_Ar), 6.64 (ddd, J = 8.3, 2.3, 1.1 Hz, 1H, H_Ar), 6.57–6.50 (m, 2H, H_Ar), 3.77 (s, 3H, OCH₃), 3.51 (t, J = 4.6 Hz, 4H, CH₂OCH₂), 3.10 (s, 2H, NCH₂CO), 2.48 (t, J = 4.6 Hz, 4H, CH₂NCH₂).

¹³C NMR (101 MHz, CDCl₃) δ (ppm): 168.13 (CO), 161.10 (C_Ar), 158.39 (C_Ar), 144.43 (C_Ar), 130.40 (C_Ar), 130.26 (C_Ar), 125.07 (C_Ar), 124.33 (C_Ar), 120.72 (C_Ar), 119.75 (C_Ar), 109.16 (C_Ar), 108.53 (C_Ar), 103.35 (C_Ar), 66.83 (CH₂OCH₂), 62.47 (NCH₂CO), 55.41 (OCH₃), 53.65 (CH₂NCH₂).

HRMS (ESI) m/z: [M+H]⁺ calcd for C₁₉H₂₃N₂O₄, 343.1658; found, 343.1649 (error = −2.57 ppm).

• General procedure for amide formation from 2-amino-N-(2-(3-methoxyphenoxy)phenyl)acetamide and various substituted benzoyl chlorides (3a–f):

Into a 100 mL round-bottomed flask with a reflux condenser, magnetic stirrer, thermocouple, and dropping funnel was added 1 eq (7.4 mmol) of starting 2-amino-N-(2-(3-methoxyphenoxy)phenyl)acetamide and 30 mL of methylene chloride. Then, the reaction mixture was cooled to −50 °C, and 1.05 eq (7.7 mmol) of the corresponding benzoyl chloride was added. Subsequently, 1.07 eq (7.9 mmol) of triethylamine was slowly added to the reaction mixture, maintaining the same temperature. After triethylamine addition, the reaction mixture was stirred for another hour and then for 24 h at room temperature. After the reaction ended, the reaction mixture was washed with water, then with saturated potassium carbonate solution and brine until a pH value of 8–9 was reached. The organic layer was dried over sodium sulfate and evaporated under reduced pressure to give a solid, which was crystallized from isopropanol.

• N-(2-((2-(3-Methoxyphenoxy)phenyl)amino)-2-oxoethyl)-4-nitrobenzamide (3a)

Yellowish crystals, m.p. 134–136 °C (iPrOH), Yield 2.5 g (80%).

¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.56 (s, 1H, NHCH₂CONH), 8.30 (dd, J = 7.9, 1.8 Hz, 1H, H_Ar), 8.22–8.14 (m, 2H, H_Ar), 7.95–7.87 (m, 2H, H_Ar), 7.67 (t, J = 5.4 Hz, 1H, NHCH₂CONH), 7.22–7.13 (m, 1H, H_Ar), 7.14–7.01 (m, 2H, H_Ar), 6.90 (dd, J = 7.9, 1.7 Hz, 1H, H_Ar), 6.67–6.59 (m, 1H, H_Ar), 6.54–6.47 (m, 2H, H_Ar), 4.30 (d, J = 5.2 Hz, 2H, CH₂CO), 3.73 (s, 3H, OCH₃).

¹³C NMR (101 MHz, CDCl₃) δ (ppm): 167.15 (CH₂CO), 165.86 (CO), 161.04 (C_Ar), 157.47 (C_Ar), 149.74 (C_Ar), 145.75 (C_Ar), 138.75 (C_Ar), 130.37 (C_Ar), 129.05 (C_Ar), 128.41 (C_Ar), 124.99 (C_Ar), 124.17 (C_Ar), 123.72 (C_Ar), 121.24 (C_Ar), 118.45 (C_Ar), 110.35 (C_Ar), 109.25 (C_Ar), 104.75 (C_Ar), 55.38 (OCH₃), 44.82 (CH₂CO).

HRMS (ESI) m/z: [M+H]⁺ calcd for C₂₂H₂₀N₃O₆, 422.1352; found, 422.1365 (error = 3.05 ppm).

• 2-Fluoro-N-(2-((2-(3-methoxyphenoxy)phenyl)amino)-2-oxoethyl)benzamide (3b)

White crystals, m.p. 93–95 °C (iPrOH), Yield 2.5 g (80%).

¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.43 (s, 1H, NHCH₂CONH), 8.41 (dd, J = 8.1, 1.6 Hz, 1H, H_Ar), 7.99 (td, J = 7.9, 1.9 Hz, 1H, H_Ar), 7.53–7.46 (m, 2H, H_Ar and NHCH₂CONH), 7.29–7.20 (m, 1H, H_Ar), 7.20–7.13 (m, 2H, H_Ar), 7.13–7.07 (m, 1H, H_Ar), 7.04 (td, J = 7.8, 1.6 Hz, 1H, H_Ar), 6.92 (dd, J = 8.1, 1.5 Hz, 1H, H_Ar), 6.64–6.57 (m, 1H, H_Ar), 6.52–6.45 (m, 2H, H_Ar), 4.30 (dd, J = 5.4, 1.3 Hz, 2H, CH₂CO), 3.73 (s, 3H, OCH₃).

¹³C NMR (101 MHz, CDCl₃) δ (ppm): 166.86 (CH₂CO), 163.82 (d, J = 3.0 Hz, CO), 162.01 (C_Ar), 160.95 (C_Ar), 159.54 (C_Ar), 157.66 (C_Ar), 145.20 (C_Ar), 133.77 (d, J = 9.4 Hz, C_Ar), 132.07 (d, J = 1.9 Hz, C_Ar), 130.24 (C_Ar), 129.55 (C_Ar), 124.78 (d, J = 3.3 Hz, C_Ar), 124.52 (C_Ar), 124.40 (C_Ar), 121.06 (C_Ar), 118.63 (C_Ar), 116.09 (d, J = 24.6 Hz, C_Ar), 110.00 (C_Ar), 109.29 (C_Ar), 104.07 (C_Ar), 55.33 (OCH₃), 44.89 (CH₂CO).

HRMS (ESI) m/z: [M+H]⁺ calcd for C₂₂H₂₀FN₂O₄, 395.1407; found, 395.1409 (error = 0.48 ppm).

• 2-Chloro-N-(2-((2-(3-methoxyphenoxy)phenyl)amino)-2-oxoethyl)benzamide (3c)

Yellow oil, Yield 2.5 g (74%).

¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.47 (s, 1H, NHCH₂CONH), 8.36 (dd, J = 8.1, 1.7 Hz, 1H, H_Ar), 7.59–7.52 (m, 1H, H_Ar), 7.40–7.31 (m, 2H, H_Ar), 7.28–7.23 (m, 1H, H_Ar), 7.23–7.15 (m, 2H, H_Ar and NHCH₂CONH), 7.10 (td, J = 7.8, 1.5 Hz, 1H, H_Ar), 7.03 (td, J = 7.8, 1.7 Hz, 1H, H_Ar), 6.88 (dd, J = 8.1, 1.5 Hz, 1H, H_Ar), 6.69–6.61 (m, 1H, H_Ar), 6.57–6.50 (m, 2H, H_Ar), 4.26 (d, J = 5.4 Hz, 2H, CH₂CO), 3.73 (s, 3H, OCH₃).

¹³C NMR (101 MHz, CDCl₃) δ (ppm): 166.90 (CO), 166.79 (CO), 160.99 (C_Ar), 157.45 (C_Ar), 145.69 (C_Ar), 133.89 (C_Ar), 131.65 (C_Ar), 130.85 (C_Ar), 130.32 (C_Ar), 130.32 (C_Ar), 130.25 (C_Ar), 129.14 (C_Ar), 127.03 (C_Ar), 124.59 (C_Ar), 124.08 (C_Ar), 121.18 (C_Ar), 118.13 (C_Ar), 110.55 (C_Ar), 109.56 (C_Ar), 104.62 (C_Ar), 55.36 (OCH₃), 44.67 (CH₂CO).

HRMS (ESI) m/z: [M+H]⁺ calcd for C₂₂H₂₀ClN₂O₄, 411.1112; found, 411.1123 (error = 2.77 ppm).

• N-(2-((2-(3-Methoxyphenoxy)phenyl)amino)-2-oxoethyl)-4-phenylbutanamide (3d)

White crystals, m.p. 106–109 °C (iPrOH), Yield 2.7 g (90%).

¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.38–8.31 (m, 2H, NHCH₂CONH + H_Ar), 7.30–7.05 (m, 7H, NHCH₂CONH + H_Ar), 7.03 (td, J = 7.8, 1.7 Hz, 1H, H_Ar), 6.89 (dd, J = 8.1, 1.5 Hz, 1H, H_Ar), 6.69–6.62 (m, 1H, H_Ar), 6.57–6.50 (m, 2H, H_Ar), 6.31 (t, J = 5.3 Hz, 1H, H_Ar), 4.03 (d, J = 5.4 Hz, 2H, NCH₂CO), 3.74 (s, 3H, OCH₃), 2.60 (t, J = 7.5 Hz, 2H, CH₂CH₂CH₂CO), 2.18 (dd, J = 8.3, 6.8 Hz, 2H, CH₂CH₂CH₂CO), 1.98–1.86 (m, 2H, CH₂CH₂CH₂).

¹³C NMR (101 MHz, CDCl₃) δ (ppm): 173.37 (CH₂CH₂CO), 167.27 (CO), 161.06 (C_Ar), 157.56 (C_Ar), 145.54 (C_Ar), 141.31 (C_Ar), 130.36 (C_Ar), 129.30 (C_Ar), 128.48 (C_Ar), 128.39 (C_Ar), 125.99 (C_Ar), 124.54 (C_Ar), 124.19 (C_Ar), 121.08 (C_Ar), 118.32 (C_Ar), 110.44 (C_Ar), 109.47 (C_Ar), 104.54 (C_Ar), 55.38 (OCH₃), 44.26 (NCH₂CO), 35.39 (CH₂CH₂CH₂CO), 35.15 (CH₂CH₂CH₂CO), 26.90 (CH₂CH₂CH₂).

HRMS (ESI) m/z: [M+H]⁺ calcd for C₂₅H₂₇N₂O₄, 419.1971; found, 419.1977 (error = 1.47 ppm).

• 2-((4,6-Dichloro-1,3,5-triazin-2-yl)amino)-N-(2-(3-methoxyphenoxy)phenyl)acetamide (3e)

White crystals, m.p. 160–164 °C (iPrOH), Yield 0.67 g (22%).

¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 9.56 (s, 1H, NH), 9.24 (t, J = 6.2 Hz, 1H, CH₂NH), 8.00–7.87 (m, 1H, H_Ar), 7.19 (t, J = 8.3 Hz, 1H, H_Ar), 7.11–6.99 (m, 2H, H_Ar), 6.88–6.80 (m, 1H, H_Ar), 6.68–6.60 (m, 1H, H_Ar), 6.53–6.45 (m, 1H, H_Ar), 6.47–6.41 (m, 1H, H_Ar), 4.09 (d, J = 6.1 Hz, 2H, CH₂CO), 3.66 (s, 3H, OCH₃).

¹³C NMR (101 MHz, DMSO-d₆) δ (ppm): 169.89 (NCNH), 169.16 (CCl), 167.10 (CO), 161.05 (C_Ar), 158.20 (C_Ar), 147.53 (C_Ar), 130.83 (C_Ar), 129.88 (C_Ar), 125.61 (C_Ar), 124.32 (C_Ar), 123.85 (C_Ar), 119.46 (C_Ar), 110.72 (C_Ar), 109.55 (C_Ar), 104.94 (C_Ar), 55.74 (OCH₃), 44.59 (CH₂CO).

HRMS (ESI) m/z: [M+H]⁺ calcd for C₁₈H₁₆Cl₂N₅O₃, 420.0630; found, 420.0633 (error = 0.66 ppm).

• 2-((6-Chloro-1,2,4,5-tetrazin-3-yl)amino)-N-(2-(3-methoxyphenoxy)phenyl)acetamide (3f)

Orange crystals, m.p. 166–169 °C (iPrOH), Yield 0.62 g (32%).

¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.38 (d, J = 8.1 Hz, 1H, CH₂NH), 8.11 (s, 1H, CONH), 7.24–7.11 (m, 2H, H_Ar), 7.11–7.03 (m, 1H, H_Ar), 6.95–6.88 (m, 1H, H_Ar), 6.72–6.63 (m, 2H, H_Ar), 6.53–6.46 (m, 2H, H_Ar), 4.37 (d, J = 5.5 Hz, 2H, CH₂CO), 3.78 (s, 3H, OCH₃).

¹³C NMR (101 MHz, CDCl₃) δ (ppm): 165.39 (CO), 161.19 (C_Ar), 161.09 (C_Ar), 157.37 (C_Ar), 145.27 (C_Ar), 130.48 (C_Ar), 128.96 (C_Ar), 124.96 (C_Ar), 124.49 (C_Ar), 121.08 (C_Ar), 118.55 (C_Ar), 110.06 (C_Ar), 109.51 (C_Ar), 104.29 (C_Ar), 102.09 (C_Ar), 55.45 (OCH₃), 45.42 (CH₂CO).

HRMS (ESI) m/z: [M+H]⁺ calcd for C₁₇H₁₆ClN₆O₃, 387.0972; found, 387.0977 (error = 1.18 ppm).

4. Conclusions

This study demonstrates that rational modification of the AOH-1996 scaffold yields several promising candidates with potent anticancer activity in MCF-7 and U-87 cell lines. Structure–activity relationship (SAR) analysis revealed that derivatives incorporating electron-withdrawing or moderately lipophilic substituents—particularly on the amide side chain and triazole ring—significantly enhanced cytotoxic potency, while bulky or strongly electron-donating groups diminished activity. Notably, compounds 1f, 2b, 3b, 3c, and 3d exhibited the most pronounced antiproliferative effects against the MCF-7 cell line with IC₅₀ values ranging from 0.65 µM to 11.4 µM, and 3d was also effective against the U-87 cell line, with an IC₅₀ of 8.72 µM, highlighting the importance of specific aromatic and polar functionalities for optimal activity.

ADMET predictions indicated that most derivatives possessed favorable drug likeness and high intestinal absorption, with compounds 2b and 3b best satisfying stringent drug-likeness criteria. Several active compounds were predicted to inhibit P-glycoprotein, suggesting their potential to overcome multidrug resistance, although high plasma protein binding and rapid systemic elimination may limit the therapeutic index. Metabolic stability varied, with 1f, 3b, and 3c showing lower CYP interaction probabilities and more favorable profiles, while 2b and 3d may be prone to rapid hepatic metabolism. All compounds presented moderate to high predicted toxicity, including potential cardiotoxicity, which emphasizes the need for careful optimization. It is noteworthy that the parental compound AOH1996 showed even higher predicted toxicity (including AMES mutagenicity and hepatotoxicity), indicating that the structural modifications in the derivatives may have partially mitigated inherent liabilities. Taken together, these derivatives—particularly 2b and 3b—represent promising lead scaffolds for further preclinical development, with the understanding that future studies should focus on structural optimization or formulation strategies to overcome ADMET-related limitations while retaining antiproliferative activity. Collectively, these findings align with recent advances in naphthalene- and 2-quinolone-based PCNA-targeting anticancer agents, where fine-tuning of electronic and lipophilic properties is critical for maximizing potency and selectivity [22].

The most promising analogs—particularly 2b and 3b—combined strong antiproliferative activity with acceptable ADMET profiles, supporting their prioritization for further preclinical evaluation as potential compounds in cancer therapy.