Scaffold‐Hopping of Natural Alkaloid Rutaecarpine: Synthesis and Anticancer‐Apoptosis and Autophagy Effects of Indole‐Based Analogs

ABSTRACT

Natural products have been recognized as important molecular skeletons in drug discovery due to their structural diversity and promising bioactivity. In this study, a scaffold‐hopping strategy was applied to Rutaecarpine, an indolo[2',3':3,4]pyrido[2,1‐b]quinazolinone alkaloid with antiproliferative activity. A novel series of 2‐arylamino‐3‐indolylethyl‐amines/alcohols was prepared by an explored synthetic approach with a Buchwald‐Hartwig cross‐coupling key step to introduce diverse aryl/heteroaryl amines. The compounds showed notable antiproliferative properties against MCF‐7, T‐47D, A‐549, AGS, SNU‐1, HL‐60, and MOLT‐4 cell lines. The most potent compound, 11a, induced nuclear condensation, mitochondrial membrane depolarization, and autophagosome formation. Western blot analysis confirmed a significant reduction in expression of Pro‐caspase‐3 and BCL‐2. The in vitro results collectively implicate the investigated molecule's significant effect on anticancer properties with simultaneous activation of intrinsic apoptosis and autophagy in cancer cells. SwissADME analysis indicated favorable drug‐likeness properties. This work demonstrates the value of a natural product‐inspired scaffold‐hopping strategy in identifying promising new anticancer molecules with important pharmacological properties.

A natural product‐inspired scaffold‐hopping strategy is applied to Rutaecarpine to design a new series of 2‐arylamino‐3‐indolylethyl amines/alcohols as potential anticancer agents. They are prepared via a synthetic approach of Buchwald–Hartwig cross‐couplings. The compounds exhibit significant antiproliferative activity against various cancer cell lines, with the lead molecule inducing mitochondrial dysfunction, apoptosis, and autophagy. Favourable drug‐likeness properties are supported by SwissADME analysis.

1. Introduction

Natural products have long served as a vital source of biologically relevant molecules, significantly impacting fields such as medicinal chemistry and drug discovery [1]. These compounds, derived from plants, microorganisms, and other living organisms, exhibit a remarkable diversity in structure and function, which has been honed through millions of years of evolution. Natural products often possess unique biological activities that make them invaluable starting templates for developing new therapeutic agents [2, 3]. Many natural compounds have been identified as effective agents for the treatment of various diseases, including cancer and infectious diseases, due to their ability to interact with biological targets in specific and potent ways [1, 2].

Rutaecarpine, an indolo[2',3':3,4]pyrido[2,1‐b]quinazolinone alkaloid extracted from the traditional Chinese herb Evodia rutaecarpa [4], has garnered attention for its potential as an antiproliferative agent. Rutaecarpine was found to exhibit promising cancer cell antiproliferative properties [5, 6], with a half‐maximal inhibitory concentration (IC₅₀) value of 26 µM against cervical cancer cells (HeLa) [7] (Figure 1A).

FIGURE 1.

(A) Our previous study on Rutacarpine inspired scaffold hopping anticancer analog design and discovery; (B) Rutacarpine inspired design and identification of novel 2,3‐disubstituted indole analogs with anticancer activity (This work).

Scaffold hopping is a powerful strategy in medicinal chemistry that involves the systematic replacement of the core structure of a bioactive molecule with a different scaffold while retaining its essential biological activity [8, 9, 10]. In the context of natural products, scaffold hopping enables the transformation of complex, often synthetically challenging, natural backbones into synthetically more accessible or drug‐like frameworks. This approach facilitates the creation of a bioactivity potential new chemical pool with structural diversity [9]. In our previous study, we employed a scaffold‐hopping strategy on the natural alkaloid Rutaecarpine to develop novel scaffold‐hopped acyclic analogs of Rutaecarpine with enhanced antiproliferative activity (Figure 1A). Notably, the analog 1A1 demonstrated more pronounced anticancer activity and selectivity compared to Rutaecarpine and an anticancer drug 5‐FU, underscoring the utility of scaffold hopping‐based natural product‐inspired design in anticancer drug discovery [11].

In this study, we report an iterative scaffold‐hopping strategy‐based study of Rutaecarpine that led to the development of new anticancer analogs 2‐arylamino‐3‐indolylethyl‐amines/alcohols. The scaffold hopping design involved a unique combination of secondary scaffold hopping (2°, ring‐opening) with the incorporation of drug‐privileged motifs (Figure 1B). The investigated molecules contain the structural features of an indole ethyl amine or alcohol and a variety of aromatic or heteroaromatic motifs. A structure‐activity relationship of our previously studied [11] Rutaecarpine analogs indicated the importance of the 4‐methoxyphenyl motif for anticancer activity, and therefore, this motif was considered for most of the present investigated structural new analog molecules. A synthetic approach was developed, with Buchwald‐Hartwig cross‐coupling as a key step that enabled assembly of various privileged drug or bioactive motifs for the preparation of new analogs of Rutaecarpine. The synthesized analogs exhibited in vitro antiproliferative activity. The compound 11a induced features of programmed cell death, including nuclear condensation, disruption of mitochondrial membrane potential, and accumulation of autophagosomes, indicative of concurrent activation of intrinsic apoptosis and autophagy pathways. Western blot analysis further validates these findings, demonstrating cleavage of procaspase‐3 and downregulation of Bcl‐2. In silico Absorption, Distribution, Metabolism, and Excretion (ADME) evaluations revealed favorable physicochemical and pharmacokinetic profiles across the series. Collectively, these findings underscore the effectiveness of iterative scaffold‐hopping combined with structural simplification strategies in the development of natural product‐inspired analogs with improved drug‐like properties and promising therapeutic potential.

2. Results and Discussion

2.1. Chemistry

For the synthesis of scaffold‐hopped analogs of Rutaecarpine, 2‐arylamino‐3‐indolylethyl‐amines/alcohols, a retrosynthetic analysis was performed, and the Buchwald‐Hartwig cross‐coupling reaction was considered as a key step that can facilitate the assembly of various aromatic and heteroaromatic amines as designed. The synthetic approach includes (Scheme 1) base‐mediated amidation of tryptamine using aroyl chloride, followed by C2‐bromination of the indole ring using N‐bromosuccinimide (NBS), yielding compound 2 in an overall yield of 48%. Initial attempts at Buchwald–Hartwig cross‐coupling of the 2‐bromoindole derivative 2 with a series of aromatic and heteroaromatic amines resulted in poor conversions, with either low yields or, in some cases, no product formation. To avoid this limitation, the indole N‐H was protected with a Boc group to suppress undesired side reactions, affording compound 3. Subsequent Buchwald–Hartwig coupling of Boc‐protected 2‐bromoindole derivative 3 with various aromatic and heteroaromatic amines was carried out under palladium catalysis in the presence of phosphorus‐based ligands and base. After systematic evaluation, the combination of Pd₂(dba)₃, Xantphos, and Cs₂CO₃ was identified as optimal, to afford products 4a–j in 30%–77% yield. The N‐Boc deprotection was done using trifluoroacetic acid (TFA), providing the analog 5a in 30% yield.

SCHEME 1.

Synthesis of 4‐methoxy‐N‐(2‐(2‐(arylamino)‐indol‐3‐yl)ethyl)benzamide: Reagents and conditions: (i) 4‐methoxybenzoyl chloride (1 equiv.), TEA (1.5 equiv.), CH₂Cl₂ (0.35 M), 15–20°C, 1 h; (ii) NBS (1.1 equiv.), AcOH (0.16 M), 25–30°C, 2.5 h; (iii) (Boc)₂O (1.1 equiv.), 4‐dimethylaminopyridine (DMAP, 10 mol%), CH₂Cl₂ (0.12 M), 15–20°C, 30 min; (iv) (Hetero)aryl amine (1.3 equiv.), Pd₂dba₃ (4 mol%), Xantphos (9 mol%), Cs₂CO₃ (1.6 equiv.), Dioxane (0.15 M), 105–108°C, 15 h; (v) TFA (20 equiv.), CH₂Cl₂ (0.14 M), 20–25°C, 12 h.

To explore the synthesis of 2‐arylamino‐3‐indolylethylalcohols, tryptophol (indole‐3‐ethanol) was selected as the starting material. Initial attempts at C2‐bromination of tryptophol using the reaction conditions established in Scheme 1 (step ii) resulted in the formation of multiple side products. For selective C2‐bromination, the hydroxyl group was protected as a tert‐butyldiphenylsilyl (TBDPS) ether (Scheme 2), thereby masking its reactivity and enabling selective functionalization. Subsequent bromination of the indole core was performed using NBS, affording intermediate 8 in an excellent yield of 82%. The free N–H of the indole was then protected with a tert‐butyloxycarbonyl (Boc) group to prevent undesired side reactions during cross‐coupling. The doubly‐protected intermediate was subjected to Buchwald–Hartwig cross‐coupling with various aromatic and heteroaromatic amines under optimized catalytic conditions, yielding 10a–c in 52%–66% yields. The TBDPS protecting group was cleaved using tetrabutylammonium fluoride (TBAF) to obtain the desired analogs 11a–c, in 78%–85% yields (Scheme 2).

SCHEME 2.

Synthesis of tert‐butyl 3‐(2‐hydroxyethyl)‐2‐(arylamino)‐indole‐1‐carboxylate: Reaction conditions: (i) TBDPSCl (1.1 equiv.), imidazole (2 equiv.), 4‐dimethylaminopyridine (DMAP, 10 mol%), CH₂Cl₂ (0.5 M), 25‐30°C, 2 h; (ii) NBS (1.1 equiv.), CH₂Cl₂ (0.05 M), 25–30°C, 30 min; (iii) (Boc)₂O (1.1 equiv.), CH₂Cl₂ (0.05 M), 25–30°C, 30 min; (iv) (Hetero)aryl amine (1.3 equiv.), Pd₂dba₃ (4 mol%), Xantphos (9 mol%), Cs₂CO₃ (1.6 equiv.), Dioxane (0.15 M), 105–108°C, 15 h; (v) tetrabutylammonium fluoride (TBAF) (2 equiv.), tetrahydrofuran (THF) (0.17 M).

The synthesized compounds were characterized by spectroscopic (¹H and ¹³C nuclear magnetic resonance [NMR], and Fourier‐transform infrared [FT‐IR]) and high‐resolution mass‐spectrometric (HRMS) methods (Figure 2).

FIGURE 2.

Synthesized 2,3‐disubstituted indole derivatives, scaffold hopped and acyclic analogs of Rutaecarpine; The yield represents the overall isolated yield% calculated considering all the steps mentioned in Schemes 1 and 2.

2.2. Biological Evaluation

2.2.1. Antiproliferative Activity

Synthesized iterative‐scaffold‐hopped analogs of Rutaecarpine were evaluated for their cytotoxic potential using 10 µM concentration against numerous human cancer cell lines, including breast adenocarcinoma (MCF‐7), breast ductal carcinoma (T‐47D), lung adenocarcinoma (A549), gastric adenocarcinoma (AGS), gastric carcinoma (SNU‐1), acute promyelocytic leukemia (HL‐60), and acute lymphoblastic leukemia (MOLT‐4), with doxorubicin (10 µM) as a positive standard (Table 1). Among the analog series molecules 2‐arylamino‐3‐indolylethyl‐amines/alcohols, compound 11a demonstrated pronounced cytotoxicity against the SNU‐1 cancer cells. It showed characteristic concentration‐dependent antiproliferative activity. The IC₅₀ of 11a in SNU‐1 cells was found to be 16.29 ± 2.80 µM (Figure 3). On the other hand, the parent natural product Rutaecarpine was known to display cancer cells antiproliferative activities with an IC₅₀ value >20 µM [1, 11, 12]. In addition, compound 11a exhibited minimal cytotoxicity toward normal gastric epithelial cells (Hs 738.St/Int), demonstrating less than 30% inhibition even at a relatively high concentration of 250 µM. This observation highlights the compound's favorable selectivity, showing 42‐fold higher selectivity against cancerous cells compared to noncancerous cells.

TABLE 1.

Percentage growth inhibition of the synthesized compounds in different human cancer cell lines, 10 µM, 48 h.

Compound	Percentage growth inhibition in different human cancer cell lines, 10 µM, 48 h
	MCF7	T47D	A549	AGS	SNU‐1	HL60	MOLT4
4a	10.25	12.45	7.21	3.08	7.20	11.25	21.22
4b	15.88	19.88	9.93	1.81	8.19	8.77	8.71
4c	18.19	28.35	11.70	0.00	17.08	6.80	22.34
4d	22.49	27.66	7.71	0.00	8.80	1.71	22.10
4e	20.41	27.74	16.46	3.45	4.34	4.30	14.88
4f	19.03	16.51	14.37	3.45	4.91	6.02	7.36
4g	16.55	24.20	8.11	0.00	4.65	8.57	3.95
4h	14.29	22.99	6.80	10.24	13.94	1.70	0.00
4i	14.60	23.51	11.83	8.43	0.00	0.00	0.00
4j	12.47	28.95	15.19	32.37	11.27	12.01	20.63
5a	7.63	12.88	16.18	0.00	10.35	6.63	23.46
11a	7.28	20.14	3.99	19.85	50.10	7.49	33.70
11b	10.25	24.29	5.80	3.35	15.87	2.73	17.11
11c	2.22	10.20	0.86	1.18	18.95	8.20	14.56
Doxorubicin (10 µM)	75.29	71.20	72.40	70.99	78.26	72.80	79.50

FIGURE 3.

(A) Concentration‐dependent inhibition of cancer cells for compound 11a to determine the half‐maximal inhibitory concentration (IC₅₀) value after 48 h of treatment. (B, C) Graphical and pictorial representation of the cytotoxicity of 11a against the normal gastric cell, Hs 738.St/Int.

2.2.2. Effect on Nuclear Morphology, Mitochondrial Membrane Integrity, and Autophagosome Formation

To understand the anticancer potential of compound 11a with bio‐functional properties, we investigated its impact on cancer cells' nuclear morphology, mitochondrial membrane potential, and autophagosome formation. The experiments were done using the gastric cancer cell line SNU‐1.

Nuclear morphology alterations, such as chromatin condensation, membrane blebbing, and apoptotic body formation, reflect nuclear disassembly characteristic of cells undergoing programmed death. A nuclear staining study using 4’,6‐diamidino‐2‐phenylindole (DAPI) staining agent in SNU‐1 cells was done, treating with compound 11a at increasing concentrations of 10, 20, and 50 µM for 48 h treatment. The study indicated the investigated molecule's effect on distinct morphological changes in a concentration‐dependent manner. Compared to the untreated control, treated cells showed a significant increase in nuclear condensation and membrane blebbing (Figure 4a).

FIGURE 4.

Effects of compound 11a in SNU‐1 cells: (a) Increased nuclear condensation and membrane blebbing in nuclear morphology study (with DAPI stain); (b) Mitochondrial membrane integrity disruption (with Rhodamine 123 stain); (c) Enhanced formation of autophagosome—acidic vacuole in the cytoplasm (Acridine orange stain).

Mitochondrial membrane integrity is crucial for maintaining cellular homeostasis, and its loss is a hallmark of intrinsic apoptotic signaling. The mitochondrial membrane potential (ΔΨm) was assessed using Rhodamine 123, a cationic dye staining agent that accumulates selectively only in intact mitochondria. SNU‐1 cells treated with compound 11a showed a progressive reduction in green fluorescence intensity with increasing concentration, with the highest (50 µM) treatment exhibiting the faintest intensity signal (Figure 4b). This suggests that compound 11a disrupts mitochondrial membrane potential, thereby initiating mitochondrial‐mediated apoptosis.

Autophagosome formation, another critical cellular response, was evaluated using Acridine Orange staining. Autophagy plays a dual role in cancer, acting either as a survival mechanism or a form of programmed cell death. Enhanced formation of acidic vacuole‐like structures in the cytoplasm, marked by bright orange puncta in AO‐stained cells, indicates the induction of autophagy. Upon treatment of SNU‐1 cells with compound 11a, a significant increase in autophagosome formation was observed compared to the untreated control (Figure 4c), indicating an upregulation of the autophagic pathway. This suggests that compound 11a not only induces apoptosis but also modulates autophagic flux, contributing to its anticancer activity.

Collectively, these findings demonstrate that compound 11a interferes with multiple stress responses in SNU‐1 cells, including nuclear fragmentation, mitochondrial dysfunction, and autophagy induction, supporting its potential as an anticancer agent.

2.2.3. Effect on the Protein Expression of Pro‐caspase‐3, BCL‐2, Beclin1, and SQSTM1

Furthermore, the involvement of key protein markers associated with apoptosis and autophagy pathways, Pro‐caspase‐3, BCL‐2, Beclin1, and SQSTM1, was examined using the western blotting technique. Pro‐caspase‐3 serves as a pivotal precursor protein that, upon activation, is cleaved into caspase‐3, thereby initiating apoptotic cell death. BCL‐2, an anti‐apoptotic protein, promotes cell survival by inhibiting the activation of downstream caspases. Beclin1 plays a central role in orchestrating the initiation of autophagosome formation, whereas SQSTM1 functions as an autophagic substrate and receptor that binds ubiquitinated cargo for degradation, making it a widely used marker for monitoring autophagic activity. In SNU‐1 cells treated with increasing concentrations of compound 11a showed a marked decrease in Pro‐caspase‐3 expression, converting to active caspase‐3, and a pronounced downregulation of BCL‐2 were observed. Concurrently, an increase in Beclin1 expression and a reduction in SQSTM1 levels were noted, collectively demonstrating that compound 11a induces autophagy in addition to triggering apoptosis. These findings suggest that compound 11a significantly enhances apoptotic signaling in SNU‐1 cells, likely through modulation of intrinsic cell death pathways (Figure 5).

FIGURE 5.

Effect of compound 11a in expression and quantification of apoptosis and autophagy markers: Pro‐caspase‐3, BCL‐2, and SQSTM1 (all three are downregulated) and Beclin 1 (upregulated) using western blotting. The corresponding uncropped/full‐size image for the blot is available in the Supporting Information. Statistical significance was determined through one‐way analysis of variance (ANOVA), where *p < 0.05, **p < 0.01, and ***p < 0.001, n = 3.

2.2.4. In Silico Drug‐likeness Evaluations

In silico evaluation of drug‐likeness, encompassing physicochemical properties, ADME characteristics, pharmacokinetics, and medicinal chemistry structural alerts, was performed for Rutaecarpine analogs 4a–j, 5a, and 11a–c using the SwissADME web tool (Table 2–3). The majority of the compounds exhibited favorable physicochemical profiles within acceptable ranges for drug‐like molecules. The predicted bioavailability scores, indicative of >10% oral bioavailability in rats or measurable Caco‐2 cell permeability, were optimal across the series. Notably, 50% compounds were predicted to be non‐substrates of P‐glycoprotein (P‐gp), suggesting a lower likelihood of active efflux‐mediated pharmacokinetic liabilities. Furthermore, none of the compounds triggered structural alerts in the PAINS or Brenk filters, with the exception of compound 4 h, which flagged a single PAINS alert. These findings collectively support the drug‐likeness and favorable ADME profiles of the synthesized Rutaecarpine analogs.

TABLE 2.

Selected physicochemical properties and bioavailability scores.

Compound	Physicochemical properties						Bioavailability score
	HBA, a HBD b (≤10, ≤5)	MLOGP c (≤4.15)	No. of rotatable bonds (≤13)	TPSA d (Å2) (20–130 Å2)	Veber filter	ESOL e (>‐6)
4a	8.2	2.2	13	125.8	No. 1 violation: Rotors>10	−6.6	0.17
4b	7.2	3.0	14	109.2	No. 1 violation: Rotors>10	−7.1	0.55
4c	4.2	4.9	10	81.6	Yes	−8.1	0.17
4d	4.2	4.4	10	81.6	Yes	−7.5	0.17
4e	5.2	4.3	10	81.6	Yes	−7.1	0.17
4f	6.2	3.3	13	100.0	No. 1 violation: Rotors>10	7.0	0.55
4g	11.6	4.7	11	81.5	Yes	−7.2	0.17
4h	5.2	3.4	12	94.0	No. 1 violation: Rotors>10	−7.1	0.55
4i	11.5	3.9	11	94.4	Yes	−7.4	0.55
4j	5.2	3.9	10	94.5	Yes	−7.4	0.55
5a	6.3	1.6	10	110.3	Yes	−5.1	0.55
11a	5.2	4.3	7	63.4	Yes	−5.6	0.55
11b	6.2	2.3	10	91.1	Yes	−5.5	0.55
11c	7.2	1.4	9	107.7	Yes	−5.0	0.55

Selected physicochemical properties and Bioavailability scores of investigated compounds 4a–j, 5a, and 11a–c, along with recommended ranges. HBA ^a : Hydrogen bond acceptors, HBD ^b : Hydrogen bond donors, MLOGP ^c : Moriguchi Log P, TPSA ^d : Topological polar surface area, ESOL ^e : Estimated Solubility.

TABLE 3.

Selected pharmacokinetics‐relevant properties and structural alert filters.

Compound	Pharmacokinetics‐relevant properties			Structural alerts
	GI a absorption	BBB b permeant	P‐gp c substrate	PAINS d	Brenk
4a	Low	No	No	0 alert	0 alert
4b	Low	No	Yes	0 alert	0 alert
4c	Low	No	Yes	0 alert	0 alert
4d	Low	No	Yes	0 alert	0 alert
4e	Low	No	Yes	0 alert	0 alert
4f	Low	No	Yes	0 alert	0 alert
4g	Low	No	Yes	0 alert	0 alert
4h	High	No	Yes	1 alert: anil_di_alk_A	0 alert
4i	Low	No	No	0 alert	0 alert
4j	Low	No	No	0 alert	0 alert
5a	High	No	No	0 alert	0 alert
11a	High	No	No	0 alert	0 alert
11b	High	No	No	0 alert	0 alert
11c	High	No	No	0 alert	0 alert

GI ^a : Gastrointestinal, BBB ^b : blood–brain barrier, P‐gp ^c : Permeability glycoprotein, PAINS ^d : Pan Assay Interference Compounds.

The Boiled Egg model, provided by SwissADME, is a widely accepted graphical tool for predicting the passive gastrointestinal (GI) absorption and brain penetration potential of small molecules based on their physicochemical properties. In this study, the synthesized scaffold‐hopped analogs of Rutaecarpine were evaluated using this model to assess their oral bioavailability potential. Compound 11a was found to lie within the white region of the Boiled Egg diagram (Figure 6), which corresponds to a high probability of passive absorption through the GI tract. This favorable positioning indicates that the molecular modifications enabled to obtaining of favorable physicochemical characteristics essential for efficient intestinal permeability, such as balanced lipophilicity and polarity. Moreover, none of the compounds entered the yellow (yolk) region, suggesting limited blood‐brain barrier penetration, which may be beneficial for therapeutic applications targeting peripheral tissues while minimizing central nervous system side effects. Overall, the Boiled Egg analysis supports the drug‐likeness and oral bioavailability potential of the compound 11a.

FIGURE 6.

Boiled Egg diagram (SwissADME) showed high GI absorption of 11a (white region). No compound in the yellow (yolk) region suggested limited blood‐brain barrier (BBB) penetration.

2.2.5. Structure–Activity Relationship

Structure–activity relationship (SAR) analysis was established for 14 synthesized 2‐arylamino‐3‐indolylethyl‐amine/alcohol derivatives (Table 1). The antiproliferative activity data were measured as percentage growth inhibition (PGI). Based on their PGI values, the compounds were categorized as active (PGI > 50%), weakly active (PGI = 30%–50%), or inactive (PGI < 30%). Among the synthesized derivatives, only compound 11a exhibited significant antiproliferative activity against the SNU‐1 gastric cancer cell line, with an IC₅₀ of 16.29 ± 2.80 µM and 50.1% inhibition at 10 µM, whereas all other analogs displayed negligible activity under the same concentration.

Matched‐pair comparison between compound 11a and its closest structural analogs revealed that the presence of a free alkyl hydroxyl group on the indole ring, in combination with di‐fluoro substitutions on the phenyl ring on the side chain, is critical for activity. This substitution pattern possibly provides a unique structural requirement for a balance of hydrophilicity and lipophilicity. In contrast, replacement of the free alkyl hydroxyl group on the C‐3 position of indole with an ethyl‐4‐methoxybenzamide moiety and C‐2 modification of indole with other functional group(s) resulted in a marked loss of potency (Table 1). In silico physicochemical profiling, particularly in terms of topological polar surface area (TPSA) and aqueous solubility, indicated that the inactive molecules generally possessed higher TPSA values and lower solubility, which could hinder cellular uptake and contribute to their diminished biological activity (Table 2).

3. Conclusions

In this study, a novel iterative‐scaffold‐hopping strategy was employed on the natural alkaloid Rutaecarpine, leading to the design, synthesis, and discovery of a new class of 2‐arylamino‐3‐indolylethyl‐amines/alcohols as potential anticancer agents. This “Nature‐to‐New” approach not only preserved key bioactive elements of the natural scaffold but also enabled the introduction of structural diversity to enhance biological activity. Using a synthetically accessible and modular route, 14 structurally diverse analogs were synthesized and evaluated against a panel of cancer cell lines. Among them, compound 11a demonstrated the most promising activity against the SNU‐1 gastric cancer cell line. The compound 11a was found to exhibit a significant effect in SNU‐1 cells with nuclear condensation, membrane blebbing, mitochondrial membrane integrity disruption, and autophagosome formation. Significant interference with apoptotic proteins was observed. Chemoinformatic analysis of the compounds confirmed favorable drug‐likeness properties, including compliance with Lipinski's Rule of Five, and optimal lipophilicity—supporting their potential bioavailable drug candidates. Taken together, these findings highlight the value of scaffold‐hopping combined with structural simplification as an effective strategy in drug discovery and demonstrate that rational modifications of NP‐frameworks can lead to the identification of novel chemotypes with potent anticancer properties.

4. Experimental

4.1. General

All commercially available reagents and solvents were used as received without further purification unless otherwise noted. Anhydrous solvents were obtained using an MBRAUN MB SPS‐800 solvent purification system. Solvent removal was performed under reduced pressure using a Büchi rotary evaporator. Analytical thin‐layer chromatography (TLC) was carried out on Merck silica gel 60 F254 aluminum‐backed plates (0.2 mm) and visualized under UV light (254 nm), with additional visualization by iodine or ninhydrin staining as appropriate. Flash column chromatography was conducted using neutral spherical silica gel (100–200 mesh). NMR spectra were recorded on JEOL spectrometers operating at 500 or 600 MHz for ¹H and at 126 or 151 MHz for ¹³C. Chemical shifts (δ) are reported in parts per million (ppm) relative to the residual solvent peak. Coupling constants (J) are reported in hertz (Hz), and multiplicities are designated as s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), td (triplet of doublets), dt (doublet of triplets), and m (multiplet). ¹³C NMR chemical shifts are also reported in ppm relative to the solvent peak. High‐resolution mass spectrometry (HRMS) was performed on an Agilent Q‐TOF instrument. Infrared (IR) spectra were obtained using a PerkinElmer FTIR spectrometer equipped with an ATR accessory.

4.2. Chemistry

4.2.1. General Procedure for Synthesis of N‐(2‐(2‐bromo‐1H‐indol‐3‐yl)ethyl)‐4‐methoxybenzamide (2) (Scheme 1, steps i and ii)

To a stirred solution of tryptamine (4.0 g, 25 mmol) in CH₂Cl₂ (54 mL) was added triethylamine (5.2 mL, 37.5 mmol) at 0–5°C under a nitrogen atmosphere. After stirring for 15 min., a solution of 4‐methoxybenzoyl chloride (3.39 mL, 25 mmol) in CH₂Cl₂ (18 mL) was added dropwise at 0–5°C. The resulting mixture was stirred at room temperature (25–30°C) for 1 h. The reaction was quenched with water and washed sequentially with EtOAc and dilute aqueous HCl (pH ∼5). The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to afford N‐(2‐(1H‐indol‐3‐yl)ethyl)‐4‐methoxybenzamide as a white solid, which was used in the next step without further purification.

To a solution of N‐(2‐(1H‐indol‐3‐yl)ethyl)‐4‐methoxybenzamide (1.0 g, 3.2 mmol) in AcOH (20 mL) was added a solution of NBS (660 mg, 3.52 mmol) in AcOH dropwise at room temperature. The reaction mixture was stirred at 25–30°C for 2.5 h, then concentrated under reduced pressure to remove AcOH. The residue was diluted with CH₂Cl₂, washed with water, and extracted again with CH₂Cl₂. The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (100–200 mesh) using EtOAc/hexane as eluent to afford N‐(2‐(2‐bromo‐1H‐indol‐3‐yl)ethyl)‐4‐methoxybenzamide (2) as a white solid (48% yield).

4.2.1.1. N‐(2‐(2‐bromo‐1H‐indol‐3‐yl)ethyl)‐4‐methoxybenzamide (2)

Purification by column chromatography (EtOAc‐hexane, 33:67 V/V) provided 2; white solid (isolated yield, 48% over 2 steps, 0.447 g); ¹H NMR (600 MHz, DMSO‐d ₆) δ 11.59 (s, 1H), 8.45 (t, J = 5.8 Hz, 1H), 7.77 (d, J = 8.9 Hz, 2H), 7.53 (d, J = 7.9 Hz, 1H), 7.24 (d, J = 8.1 Hz, 1H), 7.05 (t, J = 7.2 Hz, 1H), 6.97 (t, J = 7.5 Hz, 1H), 6.94 (d, J = 8.8 Hz, 2H), 3.77 (s, 3H), 3.39 (q, J = 6.8 Hz, 2H), 2.86 (t, J = 7.5 Hz, 2H) ppm; ¹³C{¹H} NMR (151 MHz, DMSO‐d ₆) δ 166.3, 162.0, 136.8, 129.5, 127.9, 127.6, 122.0, 119.7, 118.4, 114.0, 112.1, 111.3, 109.2, 55.8, 40.6, 25.5 ppm; IR ν _max 3358.1, 2928.9, 1605.8,1500.6, 1176.6, 1030.0, 739.7 cm⁻¹, HRMS (ESI+) m/z calculated for C₁₈H₁₈BrN₂O₂ ⁺ [M+H]⁺: 373.0546, found 373.0563.

4.2.2. General Procedure for Synthesis of tert‐Butyl 2‐bromo‐3‐(2‐(4‐methoxybenzamido)ethyl)‐1H‐indole‐1‐carboxylate (3) (Scheme 1, step iii)

To a round‐bottom flask charged with N‐(2‐(2‐bromo‐1H‐indol‐3‐yl)ethyl)‐4‐methoxybenzamide (2) (1.36 g, 3.64 mmol) and 4‐dimethylaminopyridine (DMAP, 44 mg, 0.36 mmol, 10 mol%) vacuum was applied, followed by purging with nitrogen. The mixture was stirred at 15–20°C for 5 min and then cooled to 0–5°C for an additional 5 min. Di‐tert‐butyl dicarbonate (Boc₂O, 920 µL) was added dropwise at 0–5°C, and the reaction mixture was stirred at 15–20°C for 1 h. Upon completion, the reaction was diluted with EtOAc and washed with water. The aqueous layer was extracted once more with EtOAc. The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude residue was purified by silica gel column chromatography (100–200 mesh) using EtOAc/hexane as eluent to afford tert‐butyl 2‐bromo‐3‐(2‐(4‐methoxybenzamido)ethyl)‐1H‐indole‐1‐carboxylate (3) as a white solid in 93% yield.

4.2.2.1. tert‐Butyl 2‐bromo‐3‐(2‐(4‐methoxybenzamido)ethyl)‐1H‐indole‐1‐carboxylate (3)

Purification by column chromatography (EtOAc‐hexane, 25:75 V/V) provided 3; white solid (isolated yield, 93%, 1.10 g); ¹H NMR (600 MHz, CDCl₃) δ 8.06 (d, J = 8.3 Hz, 1H), 7.63 (d, J = 8.9 Hz, 2H), 7.55 (d, J = 7.3 Hz, 1H), 7.30–7.27 (m, 1H), 7.21 (t, J = 7.5 Hz, 1H), 6.86 (d, J = 8.8 Hz, 2H), 6.17 (s, 1H), 3.82 (s, 3H), 3.71 (q, J = 6.5 Hz, 2H), 3.08 (t, J = 6.7 Hz, 2H), 1.69 (s, 9H) ppm; ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 167.3, 162.2, 149.2, 136.6, 129.0, 128.8, 126.9, 124.8, 123.2, 120.6, 118.4, 115.4, 113.8, 109.6, 85.2, 55.5, 39.3, 28.3, 25.4 ppm; IR ν _max 3730.3, 3628.1, 3282.8, 2931.8, 1730.2 1631.8, 1604.8, 1249.9, 1153.4, 1097.5, 750.3 cm⁻¹; HRMS (ESI+) m/z calculated for C₂₃H₂₆BrN₂O₄ ⁺ [M+H]⁺: 473.1070, found: 473.1076.

4.2.3. General Procedure for Synthesis of tert‐Butyl 2‐(((hetro)aryl)amino)‐3‐(2‐(4‐methoxybenzamido)ethyl)‐1H‐indole‐1‐carboxylates (4a–j) (Scheme 1, step iv)

A sealed tube was charged with tert‐butyl 2‐bromo‐3‐(2‐(4‐methoxybenzamido)ethyl)‐1H‐indole‐1‐carboxylate (3) (1.0 mmol), Pd₂(dba)₃ (37 mg, 0.04 mmol, 4 mol%), Xantphos (52 mg, 0.09 mmol, 9 mol%), and Cs₂CO₃ (520 mg, 1.6 mmol). Solid (hetero)aryl amines (1.3 mmol) were added at this stage. The tube was degassed and purged with argon three times. In the case of liquid (hetero)aryl amines, these were added after purging. Anhydrous 1,4‐dioxane (4 mL) was added under argon, and the sealed tube was stirred at 105–108°C for 15 h. After cooling to room temperature, the reaction mixture was diluted with EtOAc and washed with water. The aqueous phase was extracted again with EtOAc, and the combined organic layers were dried over anhydrous Na₂SO₄, brine solution, filtered, and concentrated under reduced pressure. The crude residue was purified by column chromatography on silica gel (100–200 mesh) or neutral alumina using EtOAc/hexane or EtOAc/CH₂Cl₂ as eluent to afford the corresponding tert‐butyl 2‐(((hetero)aryl)amino)‐3‐(2‐(4‐methoxybenzamido)ethyl)‐1H‐indole‐1‐carboxylates (4a–j) in 43%–77% yields.

4.2.3.1. tert‐Butyl 2‐((4,6‐dimethoxypyrimidin‐2‐yl)amino)‐3‐(2‐(4‐methoxybenzamido)ethyl)‐1H‐indole‐1‐carboxylate (4a)

Purification by column chromatography on neutral alumina (EtOAc‐hexane, 15:85 V/V) provided 4a; off white solid (isolated yield, 77%, 104 mg); ¹H NMR (600 MHz, CDCl₃) δ 8.08 (d, J = 8.2 Hz, 1H), 7.60 (d, J = 7.7 Hz, 1H), 7.50 (d, J = 8.9 Hz, 2H), 7.29 (t, J = 8.46,7.7 Hz, 1H), 7.23 (t, J = 7.5 Hz, 1H), 7.16 (s, 1H), 6.80 (d, J = 8.9 Hz, 2H), 6.31 (t, J = 5.6 Hz, 1H), 5.47 (s, 1H), 3.80 (s, 3H), 3.73 (t, 2H), 3.71 (s, 6H), 3.06 (t, J = 6.6 Hz, 2H), 1.51 (s, 9H) ppm; ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 172.4, 167.4, 162.0, 160.7, 150.2, 134.3, 130.2, 128.6, 128.3, 127.1, 124.5, 122.9, 118.8, 115.3, 113.6, 113.5, 84.1, 81.4, 55.4, 53.9, 39.4, 28.1, 24.1 ppm; IR ν _max 3730.3, 3628.1, 1724.4, 1631.8, 1577.8, 1359.8 1145.7, 675.1 cm⁻¹; HRMS (ESI+) m/z calculated for C₂₉H₃₄N₅O₆ ⁺ [M+H]⁺: 548.2504, found: 548.2510.

4.2.3.2. tert‐Butyl 3‐(2‐(4‐methoxybenzamido)ethyl)‐2‐((3,4,5‐trimethoxyphenyl)amino)‐1H‐indole‐1‐carboxylate (4b)

Purification by column chromatography on silica gel (100–200 mesh) (EtOAc‐hexane, 27:73 V/V) provided 4b; off white solid (isolated yield, 70%, 100 mg); ¹H NMR (600 MHz, CDCl₃) δ 7.98–7.94 (m, 1H), 7.63 (d, J = 8.9 Hz, 2H), 7.61–7.59 (m, 1H), 7.33 (s, 1H), 7.27 (m, 2H), 6.88 (d, J = 8.9 Hz, 2H), 6.27 (s, 1H), 6.04 (s, 2H), 3.83 (s, 3H), 3.78 (s, 3H), 3.73 (s, 6H), 3.63 (q, J = 6.6 Hz, 2H), 2.90 (t, J = 6.8 Hz, 2H), 1.65 (s, 9H) ppm; ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 167.1, 162.1, 154.0, 151.4, 140.1, 135.3, 133.2, 132.4, 129.2, 128.7, 127.0, 123.5, 123.3, 118.2, 115.3, 113.7, 107.8, 94.0, 85.0, 61.1, 56.1, 55.5, 39.5, 28.3, 24.2 ppm; IR ν _max 3455.5, 3382.2, 2979.1, 2939.6, 1733.1, 1648.2, 1606.7, 1486.2, 1261.5, 1140.0 730.1 cm⁻¹; HRMS (ESI+) m/z calculated for C₃₂H₃₈N₃O₇ ⁺ [M+H]⁺: 576.2704, found: 576.2707.

4.2.3.3. tert‐Butyl 2‐((2,4‐Dichlorophenyl)amino)‐3‐(2‐(4‐methoxybenzamido)ethyl)‐1H‐indole‐1‐carboxylate (4c)

Purification by column chromatography on silica gel (100–200 mesh) (EtOAc‐CH₂Cl₂, 2:98 V/V) provided 4c; off white solid (isolated yield, 46%, 63 mg); ¹H NMR (600 MHz, CDCl₃) δ 8.07 (d, J = 8.2 Hz, 1H), 7.65 (d, J = 7.6 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.34–7.32 (m, 2H), 7.28 (t, J = 7.4 Hz, 1H), 6.97 (dd, J = 8.8, 2.4 Hz, 1H), 6.89 (s, 1H), 6.87 (d, J = 8.7 Hz, 2H), 6.46 (d, J = 8.8 Hz, 1H), 6.14 (t, J = 6.3 Hz, 1H), 3.84 (s, 3H), 3.65 (q, J = 6.6 Hz, 2H), 2.89 (t, J = 6.9 Hz, 2H), 1.54 (s, 9H) ppm; ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 166.1, 161.1, 149.2, 139.2, 133.1, 130.8, 128.2, 127.6, 127.2, 126.7, 125.7, 123.6, 123.4, 122.2, 120.1, 117.7, 114.4, 113.8, 112.6, 111.6, 83.7, 54.4, 38.4, 27.1, 23.1 ppm; IR ν _max 3456.5, 3382.2, 2978.1, 2940.5, 1734.0, 1648.2, 1486.2, 1259.5, 1139.0, 760.0 cm⁻¹; HRMS (ESI+) m/z calculated for C₂₉H₃₀Cl₂N₃O₄ ⁺ [M+H]⁺: 554.1608, found: 554.1611.

4.2.3.4. tert‐Butyl 2‐((4‐Chlorophenyl)amino)‐3‐(2‐(4‐methoxybenzamido)ethyl)‐1H‐indole‐1‐carboxylate (4d)

Purification by column chromatography on silica gel (100–200 mesh) (EtOAc‐CH₂Cl₂, 2:98 V/V) provided 4d; off white solid (isolated yield, 43%, 55 mg); ¹H NMR (600 MHz, DMSO‐D ₆) δ 8.42 (t, J = 5.8 Hz, 1H), 8.01 (d, J = 8.3 Hz, 1H), 7.92 (s, 1H), 7.72 (d, J = 8.9 Hz, 2H), 7.67 (d, J = 6.7 Hz, 1H), 7.26 (t, J = 7.3 Hz, 1H), 7.20 (t, J = 7.4 Hz, 1H), 7.08 (d, J = 8.9 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 6.52 (d, J = 9.0 Hz, 2H), 3.75 (s, 3H), 3.37 (q, J = 5.9 Hz 2H), 2.77 (t, J = 7.7 Hz, 2H), 1.31 (s, 9H) ppm; ¹³C{¹H} NMR (151 MHz, DMSO‐D ₆) δ 166.2, 161.9, 149.8, 146.8, 134.4, 132.8, 129.45, 129.2, 128.6, 127.3, 124.6, 123.0, 121.7, 119.4, 115.8, 115.2, 114.5, 113.9, 83.7, 55.8, 40.5, 28.0, 24.5 ppm; IR ν _max 3327.2, 2980.0, 1719.5, 1605.7, 1491.0, 1251.8, 1140.9, 1028.0, 840.9, 745.5, 607.5 cm⁻¹; HRMS (ESI+) m/z calculated for C₂₉H₃₁ClN₃O₄ ⁺ [M+H]⁺: 520.1998, found: 520.2000.

4.2.3.5. tert‐Butyl 2‐((4‐Fluorophenyl)amino)‐3‐(2‐(4‐methoxybenzamido)ethyl)‐1H‐indole‐1‐carboxylate (4e)

Purification by column chromatography on silica gel (100–200 mesh) (EtOAc‐CH₂Cl₂, 2:98 V/V) provided 4e; off white solid (isolated yield, 69%, 86 mg); ¹H NMR (600 MHz, CDCl₃) δ 7.96–7.91 (m, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.54 (dd, J = 7.2, 1.8 Hz, 1H), 7.32 (s, 1H), 7.25–7.21 (m, 2H), 6.86 (d, J = 8.7 Hz, 2H), 6.71 (d, J = 8.6 Hz, 1H), 6.44 (d, J = 2.6 Hz, 1H), 6.32 (dd, J = 8.6, 2.6 Hz, 1H), 6.26 (s, 1H), 3.82 (s, 3H), 3.81 (s, 3H), 3.76 (s, 3H), 3.59 (q, J = 6.8 Hz, 2H), 2.83 (t, J = 6.8 Hz, 2H), 1.63 (s, 9H) ppm; ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 167.1, 162.1, 157.7, 151.1, 140.4, 135.3, 133.4, 129.0, 128.7, 127.0, 123.7, 123.3, 118.2, 117.4, 116.0, 115.4, 113.7, 108.9, 84.9, 55.5, 39.4, 28.3, 24.0 ppm; ¹⁹F NMR (565 MHz, CDCl₃) δ ‐123.43 ppm; IR ν _max 3326.3, 2979.1, 1720.53, 1605.7, 1503.5, 1251.8, 1140.9, 826.5, 746.4 cm⁻¹; HRMS (ESI+) m/z calculated for C₂₉H₃₁FN₃O₄ ⁺ [M+H]⁺: 504.2293, found: 504.2294.

4.2.3.6. tert‐Butyl 2‐((3,4‐Dimethoxyphenyl)amino)‐3‐(2‐(4‐methoxybenzamido)ethyl)‐1H‐indole‐1‐carboxylate (4f)

Purification by column chromatography on silica gel (100–200 mesh) (EtOAc‐CH₂Cl₂, 4:96 V/V) provided 4f; brown solid (isolated yield, 67%, 91 mg); ¹H NMR (600 MHz, CDCl₃) δ 7.94 (dd, J = 6.8, 2.5 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.54 (dd, J = 6.3, 2.7 Hz, 1H), 7.32 (s, 1H), 7.25–7.21 (m, 2H), 6.86 (d, J = 8.8 Hz, 2H), 6.71 (d, J = 8.6 Hz, 1H), 6.44 (d, J = 2.6 Hz, 1H), 6.32 (dd, J = 8.6, 2.6 Hz, 1H), 6.26 (s, 1H), 3.82 (s, 3H), 3.80 (s, 3H), 3.76 (s, 3H), 3.57 (q, J = 6.8 Hz, 2H), 2.83 (t, J = 6.8 Hz, 2H), 1.63 (s, 9H) ppm; ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 167.0, 162.0, 151.4, 149.8, 144.0, 137.6, 136.3, 133.0, 129.4, 128.7, 127.0, 123.3, 123.2, 117.8, 115.2, 113.6, 112.3, 108.4, 106.6, 102.4, 84.9, 56.4, 55.9, 55.4, 39.5, 28.3, 23.9 ppm; IR ν _max cm⁻¹ 3359.0, 2980.0, 1719.5, 1605.7, 1496.7, 1457.2, 1251.8, 1139.9, 1027.1, 841.9, 746.4, 608.5; HRMS (ESI+) m/z calculated for C₃₁H₃₆N₃O₆ ⁺ [M+H]⁺: 546.2599, found: 546.2602.

4.2.3.7. tert‐Butyl 2‐((2,4‐Difluorophenyl)amino)‐3‐(2‐(4‐methoxybenzamido)ethyl)‐1H‐indole‐1‐carboxylate (4 g)

Purification by column chromatography on silica gel (100–200 mesh) (EtOAc‐CH₂Cl₂, 3:97 V/V) provided 4 g; brown solid (isolated yield, 30%, 39 mg); ¹H NMR (600 MHz, CDCl₃) δ 8.03 (d, J = 8.9 Hz, 1H), 7.61 (d, J = 8.9 Hz, 3H), 7.32–7.26 (m, 2H), 6.86 (d, J = 8.8 Hz, 3H), 6.75 (s, 1H), 6.67–6.61 (m, 1H), 6.51 (td, J = 9.3, 5.6 Hz, 1H), 6.23 (s, 1H), 3.83 (s, 3H), 3.63 (q, J = 6.8 Hz, 2H), 2.89 (t, J = 6.8 Hz, 2H), 1.56 (s, 9H) ppm; ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 167.2, 162.1, 157.1, 157.0, 155.5, 152.7, 151.0, 150.5, 133.9, 132.9, 129.6, 129.5, 128.7, 128.7, 128.6, 128.5, 126.8, 124.3, 123.3, 123.2, 118.5, 115.5, 113.9, 113.8, 113.6, 113.5, 111.5, 111.0, 110.9, 104.3, 84.9, 55.4, 39.6, 28.2, 28.0, 23.9 ppm; IR ν _max 3347.5, 2933.7, 1724.3, 1606.7, 1501.6, 1251.8, 1137.0, 842.9, 747.4, 606.6 cm⁻¹; HRMS (ESI+) m/z calculated for C₂₉H₃₀F₂N₃O₄ ⁺ [M+H]⁺: 522.2199, found: 522.2205.

4.2.3.8. tert‐Butyl 3‐(2‐(4‐Methoxybenzamido)ethyl)‐2‐((4‐morpholinophenyl)amino)‐1H‐indole‐1‐carboxylate (4 h)

Purification by column chromatography on silica gel (100–200 mesh) (EtOAc‐CH₂Cl₂, 5:95 V/V) provided 4 h; off white solid (isolated yield, 35%, 49 mg); ¹H NMR (600 MHz, CDCl₃) δ 7.93 (dd, J = 6.9, 2.5 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.52 (dd, J = 6.4, 2.6 Hz, 1H), 7.28 (s, 1H), 7.25–7.19 (m, 2H), 6.86 (d, J = 8.8 Hz, 2H), 6.81 (d, J = 9.1 Hz, 2H), 6.77 (d, J = 8.9 Hz, 2H), 6.28 (s, 1H), 3.84 (d, J = 4.8 Hz, 4H), 3.83 (s, 3H), 3.57 (d, J = 5.4 Hz, 2H), 3.06–3.02 (m, 4H), 2.80 (t, J = 6.8 Hz, 2H), 1.62 (s, 9H) ppm; ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 167.0, 162.0, 151.3, 146.3, 137.1, 136.4, 133.0, 129.5, 128.7, 127.0, 123.2, 118.4, 118.3, 117.8, 117.3, 115.2, 113.9, 113.8, 113.5, 113.4, 106.6, 84.8, 67.0, 55.4, 50.4, 39.5, 28.4, 28.3, 28.2, 23.8 ppm; IR ν _max 3356.2, 3296.4, 2939.57, 1709.9, 1651.0, 1608.6, 1508.3, 1310.6, 1252.7, 1141.8, 768.6, 516.9, 478.3 cm⁻¹; HRMS (ESI+) m/z calculated for C₃₃H₃₉N₄O₅ ⁺ [M+H]⁺: 571.2915, found: 571.2924.

4.2.3.9. tert‐Butyl 3‐(2‐(4‐Methoxybenzamido)ethyl)‐2‐(quinolin‐3‐ylamino)‐1H‐indole‐1‐carboxylate (4i)

Purification by column chromatography on silica gel (100–200 mesh) (EtOAc‐CH₂Cl₂, 50:50 V/V) provided 4i; off white solid (isolated yield, 34%, 45 mg); ¹H NMR (600 MHz, CDCl₃) δ 8.64 (d, J = 2.8 Hz, 1H), 8.02 (d, J = 7.5 Hz, 1H), 7.98 (d, J = 9.4 Hz, 1H), 7.69 (d, J = 6.8 Hz, 1H), 7.56 (d, J = 8.9 Hz, 2H), 7.52 (d, J = 9.9 Hz, 1H), 7.48 (t, J = 6.9 Hz, 1H), 7.41 (t, J = 6.8 Hz, 1H), 7.33–7.29 (m, 3H), 7.13 (d, J = 2.8 Hz, 1H), 6.82 (d, J = 8.9 Hz, 2H), 6.19 (t, J = 5.4 Hz, 1H), 3.80 (s, 3H), 3.64 (q, J = 6.4 Hz, 2H), 2.92 (t, J = 7.1 Hz, 2H), 1.57 (s, 9H) ppm; ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 167.2, 162.2, 150.9, 143.8, 138.1, 133.8, 133.4, 129.2, 128.9, 128.7, 128.6, 127.3, 126.8, 126.6, 124.3, 123.5, 118.8, 115.5, 113.7, 110.6, 85.1, 55.4, 39.4, 28.3, 24.4 ppm; IR ν _max 3374.5, 2978.1, 1728.2, 1640.4, 1607.7, 1503.5, 1255.6, 1140.9, 752.2, 615.3 cm⁻¹; HRMS (ESI+) m/z calculated for C₃₂H₃₃N₄O₄ ⁺ [M+H]⁺: 537.2496, found: 537.2507.

4.2.3.10. tert‐Butyl 3‐(2‐(4‐Methoxybenzamido)ethyl)‐2‐(quinolin‐6‐ylamino)‐1H‐indole‐1‐carboxylate (4j)

Purification by column chromatography on silica gel (100–200 mesh) (EtOAc‐CH₂Cl₂, 50:50 V/V) provided 4j; yellowish solid (isolated yield, 40%, 53 mg); ¹H NMR (600 MHz, CDCl₃) δ 8.67 (s, 1H), 8.03 (d, J = 6.6 Hz, 1H), 7.96 (d, J = 9.1 Hz, 1H), 7.82 (d, J = 8.3 Hz, 1H), 7.68 (d, J = 7.1 Hz, 1H), 7.53 (d, J = 8.9 Hz, 2H), 7.35–7.25 (m, 4H), 7.12 (s, 1H), 6.83 (s, 1H), 6.79 (d, J = 7.4 Hz, 2H), 6.21 (s, 1H), 3.80 (s, 3H), 3.65 (q, J = 6.8 Hz, 2H), 2.94 (t, J = 7.0 Hz, 2H), 1.55 (s, 9H) ppm; ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 167.1, 162.1, 150.8, 147.8, 144.4, 142.7, 134.5, 133.7, 130.8, 129.5, 128.7, 126.8, 123.3, 121.8, 118.6, 115.4, 113.7, 113.6, 110.9, 108.3, 84.8, 55.4, 39.4, 28.3, 24.3 ppm; IR ν _max 3395.7, 2975.2, 1718.6, 1647.2, 1606.7, 1497.7, 1314.5, 1135.1, 746.4, 618.2 cm⁻¹; HRMS (ESI+) m/z calculated for C₃₂H₃₃N₄O₄ ⁺ [M+H]⁺: 537.2496, found: 537.2511.

4.2.4. Procedure for Synthesis of N‐(2‐(2‐((4,6‐dimethoxypyrimidin‐2‐yl)amino)‐1H‐indol‐3‐yl)ethyl)‐4‐methoxybenzamide (5a) (Scheme 1, step v)

tert‐Butyl 2‐((4,6‐dimethoxypyrimidin‐2‐yl)amino)‐3‐(2‐(4‐methoxybenzamido)ethyl)‐1H‐indole‐1‐carboxylate (343 mg, 0.63 mmol) was charged in a round‐bottom flask, evacuated, and purged with argon. The reaction mixture was stirred at 0–5°C for 8 min., followed by dropwise addition of TFA (960 µL, 12.6 mmol, 20 equiv.) at the same temperature. The mixture was then stirred at room temperature (25–30°C) for 12 h. After completion, the reaction was concentrated under reduced pressure, and the residue was diluted with EtOAc. The resulting mixture was washed with saturated aqueous NaHCO₃ and extracted with EtOAc. The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude product was purified by column chromatography on neutral alumina using 15% EtOAc in hexane as the eluent to afford N‐(2‐(2‐((4,6‐dimethoxypyrimidin‐2‐yl)amino)‐1H‐indol‐3‐yl)ethyl)‐4‐methoxybenzamide (5a) as a pale solid in 30% yield.

4.2.4.1. N‐(2‐(2‐((4,6‐Dimethoxypyrimidin‐2‐yl)amino)‐1H‐indol‐3‐yl)ethyl)‐4‐methoxybenzamide (5a)

Purification by column chromatography on silica gel (100–200 mesh) (EtOAc‐hexane, 30:70 V/V) afforded 5a; off white solid (isolated yield, 30%, 33 mg); ¹H NMR (600 MHz, DMSO‐d ₆) δ 10.84 (s, 1H), 9.41 (s, 1H), 8.35 (t, J = 5.6 Hz, 1H), 7.79 (d, J = 8.7 Hz, 2H), 7.51 (d, J = 7.7 Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H), 6.99 (t, J = 6.8 Hz, 1H), 6.96–6.93 (m, 3H), 5.61 (s, 1H), 3.82 (s, 6H), 3.80 (s, 3H), 3.46–3.43 (m, 2H), 2.93 (t, J = 7.6 Hz, 2H) ppm; ¹³C{¹H} NMR (151 MHz, DMSO‐d ₆) δ 172.2, 166.2, 161.9, 160.3, 133.3, 132.4, 129.4, 127.8, 127.5, 120.4, 118.9, 117.9, 113.9, 111.2, 100.7, 80.4, 55.8, 54.2, 40.6, 24.5 ppm; IR ν _max 3730.3, 3628.1, 3342.6, 2935.7, 1641.4, 1568.1, 1357.9 cm⁻¹; HRMS (ESI+) m/z calculated for C₂₄H₂₆N₅O₄ ⁺ [M+H]⁺: 448.1979, found: 448.1981.

4.2.5. Procedure for Synthesis of 3‐(2‐((tert‐Butyldiphenylsilyl)oxy)ethyl)‐1H‐indole (7) (Scheme 2, step i)

4.2.5.1. Procedure for Synthesis of 2‐Bromo‐3‐(2‐((tert‐butyldiphenylsilyl)oxy)ethyl)‐1H‐indole (8) (Scheme 2, steps i and ii)

The synthesis of compound 8 was carried out following a literature‐reported protocol with slight modifications. Tryptophol (6) (1575 mg, 9.78 mmol), imidazole (1.33 g, 19.56 mmol, 2 equiv.), and DMAP (120 mg, 0.98 mmol, 10 mol%) were added to a two‐neck round‐bottom flask under an argon atmosphere. Anhydrous CH₂Cl₂ (20 mL) was added, and the reaction mixture was stirred at 0–5°C for 10 min. TBDPSCl (2.8 mL, 10.76 mmol, 1.1 equiv) was then added dropwise at 0–5°C. The mixture was stirred at room temperature (25–30°C) for 2 h. The reaction was diluted with EtOAc and washed once with water. The aqueous layer was extracted again with EtOAc, and the combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (100–200 mesh) using EtOAc–hexane (3:97, v/v) as the eluent to afford 3‐(2‐((tert‐butyldiphenylsilyl)oxy)ethyl)‐1H‐indole (7) as an off‐white oil in 98% yield.

To a two‐neck round‐bottom flask containing compound 7 (3.52 g, 8.8 mmol) in CH₂Cl₂ (172 mL) under an argon atmosphere was added NBS (1.72 g, 9.7 mmol, 1.1 equiv.) in five portions over 25 min at room temperature. The mixture was stirred at 25–30°C for an additional 10 min. The reaction was quenched by dilution with EtOAc and washed once with water. The aqueous layer was extracted again with EtOAc, and the combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (100–200 mesh) using EtOAc–hexane as the eluent to give 2‐bromo‐3‐(2‐((tert‐butyldiphenylsilyl)oxy)ethyl)‐1H‐indole (8) as a white solid in 82% yield.

4.2.5.2. 2‐Bromo‐3‐(2‐((tert‐butyldiphenylsilyl)oxy)ethyl)‐1H‐indole (8)

Purification by column chromatography on silica gel (100–200 mesh) (EtOAc‐hexane, 2:98 V/V) provided 8; white solid (isolated yield, 82%, 0.98 g); ¹H NMR (600 MHz, CDCl₃) δ 7.92 (s, 1H), 7.63–7.60 (m, 4H), 7.40–7.38 (m, 2H), 7.33 (t, J = 7.3 Hz, 5H), 7.24 (d, J = 7.3 Hz, 1H), 7.14–7.11 (m, 1H), 7.04–6.98 (m, 1H), 3.85 (t, J = 7.4 Hz, 2H), 2.99 (t, J = 7.4 Hz, 2H), 1.02 (s, 9H) ppm; ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 136.1, 135.7, 133.9, 129.6, 128.1, 127.7, 122.3, 120.0, 118.6, 112.6, 110.3, 108.7, 63.4, 28.5, 26.9, 19.2 ppm; IR ν _max 3054.3, 2986.8, 1264.4, 732.0, 709.1 cm⁻¹.

4.2.6. Procedure for Synthesis of tert‐Butyl 2‐Bromo‐3‐(2‐((tert‐butyldiphenylsilyl)oxy)ethyl)‐1H‐indole‐1‐carboxylate (9) (Scheme 2, step iii)

tert‐Butyl 2‐bromo‐3‐(2‐((tert‐butyldiphenylsilyl)oxy)ethyl)‐1H‐indole‐1‐carboxylate (9) was synthesized from 2‐bromo‐3‐(2‐((tert‐butyldiphenylsilyl)oxy)ethyl)‐1H‐indole (8) (1358 mg, 3.64 mmol) following the general procedure used for the Boc protection described for compound 3 (Scheme 1, step iii). The crude product was purified by silica gel column chromatography (100–200 mesh) using 2% EtOAc in hexane as the eluent to afford compound 9 in 98% yield.

4.2.6.1. tert‐Butyl 2‐Bromo‐3‐(2‐(4‐methoxybenzamido)ethyl)‐1H‐indole‐1‐carboxylate (9)

Purification by column chromatography on silica gel (100–200 mesh) (EtOAc‐hexane, 2:98 V/V) provided 9; off white oil (isolated yield, 98%, 1.41 g); ¹H NMR (600 MHz, CDCl₃) δ 8.02 (d, J = 8.2 Hz, 1H), 7.60 (d, J = 8.0, 4H), 7.41–7.38 (m, 2H), 7.34–7.32 (m, 4H), 7.24–7.21 (m, 2H), 7.12 (t, J = 8.0, 1H), 3.85 (t, J = 7.3 Hz, 2H), 3.00 (t, J = 7.3 Hz, 2H), 1.68 (s, 9H), 1.01 (s, 9H) ppm; ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 149.3, 136.5, 135.7, 133.8, 129.7, 129.3, 127.7, 120.2, 118.6, 115.2, 109.7, 84.8, 62.6, 28.9, 28.3, 26.9, 19.2 ppm; IR ν _max 3053.4, 2987.8, 1264.4, 732.0, 703.1 cm⁻¹.

4.2.7. General Procedure for Synthesis of tert‐Butyl 3‐(2‐((tert‐Butyldiphenylsilyl)oxy)ethyl)‐2‐((hetero)arylamino)‐1H‐indole‐1‐carboxylates (10a–c) (Scheme 2, step iv)

4.2.7.1. tert‐Butyl 3‐(2‐((tert‐Butyldiphenylsilyl)oxy)ethyl)‐2‐((hetro)arylamino)‐1H‐indole‐1‐carboxylates (10a–c)

Compounds 10a–c were synthesized from tert‐butyl 2‐bromo‐3‐(2‐((tert‐butyldiphenylsilyl)oxy)ethyl)‐1H‐indole‐1‐carboxylate (9) and the corresponding (hetero)aryl amines following the general C–N coupling procedure described for the synthesis of compounds 4a–j (Scheme 1, step iv). The crude products were purified by flash column chromatography on silica gel (100–200 mesh) using 2% EtOAc in hexane as the eluent to afford compounds 10a–c in 52%–66% yields.

4.2.7.2. tert‐Butyl 3‐(2‐((tert‐Butyldiphenylsilyl)oxy)ethyl)‐2‐((2,4‐difluorophenyl)amino)‐1H‐indole‐1‐carboxylate (10a)

Purification by column chromatography on silica gel (100–200 mesh) (EtOAc‐hexane, 1:99 V/V) provided 10a; off white semi‐solid (isolated yield, 59%, 92 mg); ¹H NMR (600 MHz, CDCl₃) δ 8.05 (d, J = 8.3 Hz, 1H), 7.54 (dd, J = 7.9, 1.4 Hz, 4H), 7.40–7.37 (m, 2H), 7.29 (t, J = 7.4 Hz, 4H), 7.27–7.23 (m, 2H), 7.15 (t, J = 7.5 Hz, 1H), 6.85 (ddd, J = 11.3, 8.4, 2.8 Hz, 1H), 6.59 (dddd, J = 9.1, 8.0, 2.8, 1.4 Hz, 1H), 6.42–6.35 (m, 2H), 3.80 (t, J = 7.1 Hz, 2H), 2.83 (t, J = 7.1 Hz, 2H), 1.50 (s, 9H), 0.99 (s, 9H) ppm; ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 155.6, 151.3, 150.2, 135.5, 134.0, 133.6, 132.4, 130.1, 129.5, 128.7, 127.5, 124.0, 122.7, 118.6, 115.2, 114.8, 111.7, 110.7, 103.8, 84.2, 63.0, 28.0, 27.2, 26.8, 19.1 ppm; ¹⁹F NMR NMR (565 MHz, CDCl₃) δ ‐122.72, ‐129.97 ppm; IR ν _max 3386.1, 2929.9, 2857.6, 1723.4, 1627.0, 1136.1, 698.2 cm⁻¹.

4.2.7.3. tert‐Butyl 3‐(2‐((tert‐Butyldiphenylsilyl)oxy)ethyl)‐2‐((4,6‐dimethoxypyrimidin‐2‐yl)amino)‐1H‐indole‐1‐carboxylate (10c)

Purification by column chromatography on silica gel (200–400 mesh) (EtOAc‐hexane, 1.5:98.5 V/V) provided 10c; off white semi‐solid (isolated yield, 52%, 84 mg); ¹H NMR (600 MHz, CDCl₃) δ 8.11 (d, J = 8.3 Hz, 1H), 7.58 (dd, J = 6.7 Hz, 8.0, 1.4 Hz, 4H), 7.39–7.36 (m, 3H), 7.29 (t, J = 7.5 Hz, 4H), 7.27–7.23 (m, 2H), 7.14 (t, J = 7.5 Hz, 1H), 5.53 (s, 1H), 3.85 (t, J = 6.7 Hz, 2H), 3.73 (s, 6H), 2.97 (t, J = 6.7 Hz, 2H), 1.43 (s, 9H), 1.05 (s, 9H) ppm; ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 172.2, 160.4, 150.2, 135.6, 134.4, 133.5, 130.9, 129.7, 128.4, 127.8, 127.7, 123.8, 122.4, 118.3, 115.0, 111.6, 83.4, 81.4, 81.4, 64.1, 53.8, 28.0, 27.0, 19.2 ppm; IR ν _max 3287.7, 2918.4, 2851.8, 1726.3, 1574.9, 1367.6, 1143.8, 700.2 cm⁻¹.

4.2.8. General Procedure for Synthesis of tert‐Butyl 2‐(((Hetero)aryl)amino)‐3‐(2‐hydroxyethyl)‐1H‐indole‐1‐carboxylates (11a–c)

A solution of the corresponding tert‐butyl 3‐(2‐((tert‐butyldiphenylsilyl)oxy)ethyl)‐2‐((hetero)arylamino)‐1H‐indole‐1‐carboxylate (10a–c) (1 equiv.) in tetrahydrofuran (THF) was stirred at 0–5 °C for 10 min., followed by the addition of TBAF (1 M in THF, 2 equiv.) at the same temperature. The reaction mixture was stirred at 25–30 °C for 30 min. and then diluted with EtOAc and water. The aqueous phase was extracted three times with EtOAc, and the combined organic layers were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude residue was purified by column chromatography on silica gel (100–200 mesh) using EtOAc–hexane as the eluent to afford tert‐butyl 2‐(((hetero)aryl)amino)‐3‐(2‐hydroxyethyl)‐1H‐indole‐1‐carboxylates (11a–c) in 78%–85% yields.

4.2.8.1. tert‐Butyl 2‐((2,4‐Difluorophenyl)amino)‐3‐(2‐hydroxyethyl)‐1H‐indole‐1‐carboxylate (11a)

Purification by column chromatography on silica gel (200–400 mesh) (EtOAc‐hexane, 10:90 V/V) provided 11a; clear sticky liquid (isolated yield, 78%, 122 mg); ¹H NMR (600 MHz, CDCl₃) δ 8.06 (d, J = 9.0 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.29 (td, J = 7.7, 1.5 Hz, 1H), 7.27 (dd, J = 7.5, 1.3 Hz, 1H), 6.87 (ddd, J = 11.1, 8.4, 2.8 Hz, 1H), 6.69–6.64 (m, 2H), 6.50 (td, J = 9.3, 5.5 Hz, 1H), 3.82 (t, J = 6.6 Hz, 2H), 2.84 (t, J = 6.6 Hz, 2H), 1.55 (s, 9H) ppm; ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 156.2, 151.7, 150.4, 134.0, 133.2, 129.8, 128.7, 124.2, 123.1, 118.4, 115.5, 115.5, 110.8, 110.8, 104.2, 84.7, 62.0, 28.2, 27.5 ppm; ¹⁹F NMR NMR (565 MHz, CDCl₃) δ ‐121.83, ‐129.32 ppm; IR ν _max 3383.2, 3281.9, 2979.1, 2938.6, 1720.5, 1515.1, 1136.1, 736.8 cm⁻¹; HRMS (ESI+) m/z calculated for C₂₁H₂₃F₂N₂O₃ ⁺ [M+H]⁺: 389.1671, found: 389.1681.

4.2.8.2. tert‐Butyl 3‐(2‐Hydroxyethyl)‐2‐((3,4,5‐trimethoxyphenyl)amino)‐1H‐indole‐1‐carboxylate (11b)

Purification by column chromatography on silica gel (200–400 mesh) (EtOAc‐hexane, 11:89 V/V) provided 11b; off white sticky solid (isolated yield, 85%, 97 mg); ¹H NMR (600 MHz, CDCl₃) δ 7.97 (dd, J = 6.2, 3.0 Hz, 1H), 7.49 (dd, J = 5.7, 3.3 Hz, 1H), 7.26 (s, 1H), 7.24 (s, 1H), 6.02 (s, 2H), 3.81 (t, J = 6.4 Hz, 2H), 3.77 (s, 3H), 3.75 (s, 6H), 2.86 (t, J = 6.6 Hz, 2H), 1.62 (s, 9H) ppm; ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 154.0, 151.2, 140.5, 135.5, 133.4, 132.2, 129.4, 123.5, 123.1, 118.1, 115.4, 107.4, 93.7, 84.9, 61.9, 61.2, 56.2, 28.4, 27.8 ppm; IR ν _max 3364.9, 2975.3, 1715.7, 1600.9, 1126.4, 734.9 cm⁻¹; HRMS (ESI+) m/z calculated for C₂₄H₃₁N₂O₆ ⁺ [M+H]⁺: 443.2177, found: 443.2184.

4.2.8.3. tert‐Butyl 2‐((4,6‐Dimethoxypyrimidin‐2‐yl)amino)‐3‐(2‐hydroxyethyl)‐1H‐indole‐1‐carboxylate (11c)

Purification by column chromatography on silica gel (200–400 mesh) (EtOAc‐hexane, 22:78 V/V) provided 11c; white solid (isolated yield, 79%, 81 mg); ¹H NMR (600 MHz, CDCl₃) δ 8.13 (d, J = 8.3 Hz, 1H), 7.54 (d, J = 7.0 Hz, 1H), 7.32 (ddd, J = 8.4, 7.2, 1.3 Hz, 1H), 7.27–7.24 (m, 1H), 7.04 (s, 1H), 5.56 (s, 1H), 3.92 (q, J = 5.8 Hz, 2H), 3.78 (s, 6H), 3.01 (t, J = 6.0 Hz, 2H), 2.51 (s, 1H), 1.52 (s, 9H) ppm; ¹³C{¹H} NMR (151 MHz, CDCl₃) δ 172.5, 161.3, 150.0, 134.6, 130.5, 128.0, 124.6, 122.7, 118.8, 115.5, 114.0, 84.0, 81.2, 61.8, 54.0, 28.2, 27.5 ppm; IR ν _max 3467.1, 3336.9, 2954.0, 2867.2, 1700.3, 1569.1, 1358.9, 1188.2, 755.1 cm⁻¹; HRMS (ESI+) m/z calculated for C₂₁H₂₇N₄O₅ ⁺ [M+H]⁺: 415.1976, found: 415.1987.

5. Biological Evaluation

5.1. Cell Culture and Growth Conditions

The cell lines used for this study are AGS, SNU‐1, HL60, MOLT4, MCF7, T47D, and A549. The former four cell lines were procured from ATCC, MCF7 and T47D from Sigma, and A549 from NCCS. SNU‐1, MOLT4, T47D, and A549 were maintained in RPMI medium (Gibco), AGS in F12K(Gibco), HL60 in IMDM (Gibco), and MCF7 in EMEM (Sigma). All the cell lines were supplemented with 10% fetal bovine serum (FBS) except for HL60 (20% FBS). A controlled environment of 37°C temperature, 95% humidity, and 5% CO₂ gas environment was also provided.

5.2. In Vitro Cytotoxicity Assay

The cytotoxicity study was performed using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay according to the protocol described by Chatterjee et al. (PMID: 40538534). Briefly, the cells were seeded in the 96‐well plates and incubated. For the initial activity test, all the test compounds, along with doxorubicin, were dissolved in DMSO and introduced at a 10 µM concentration for each well and incubated for 24 h. MTT dye (Sigma, 2.5 mg/mL in phosphate‐buffered saline [PBS]) was added to each well 3 h before the termination time. The absorbance was measured at a wavelength of 570 nm. For the IC50 calculation, the SNU‐1 cells were treated with nine two‐fold serial dilution concentrations starting from a 250 µM concentration of SKG‐69‐12. The IC50 value was calculated using GraphPad Prism 8.0.1 (244).

5.3. DAPI Staining

SNU‐1 cells were seeded in a 12‐well plate and treated with SKG‐69‐12. After treatment, the cells were trypsinized, washed with PBS, fixed with methanol–acetic acid solution overnight, and evenly poised in a pre‐chilled slide. After drying, the cells were stained with DAPI solution (1 µg/mL in PBS), mounted using coverslips, and visualized under a fluorescence microscope (Zeiss Axiocam 202 mono) (PMID: 36033689).

5.4. Acridine Orange Staining

SNU‐1 cells were seeded in a 6‐well plate and treated with 11a. After treatment, the cells were incubated with acridine orange (1 µg/mL) for 15 min. and washed once with PBS. Autophagy was determined using a High Content Screening system (CellInsight CX7 LZR Pro HCS Platform, Thermo Fisher Scientific) (PMID: 40538534).

5.5. Rhodamine 123 Staining

After treatment with 11a, SNU‐1 cells were stained with Rhodamine 123 (5 µM in each well) and washed once with PBS. The cells were visualized using a High Content Screening system (CellInsight CX7 LZR Pro HCS Platform, Thermo Fisher Scientific) (PMID: 25977334).

5.6. Preparation of Cell Lysate and Western Blot Assay

After the treatment was completed, the cells were trypsinized and washed. Next, the cells were lysed with RIPA buffer to extract the protein and quantified using Bradford reagent. Next, the sample is loaded on the wells of SDS‐PAGE and transferred to the polyvinylidene difluoride membrane. The membrane was blocked with 5% bovine serum albumin and probed with primary and secondary antibodies, and the signal was developed using the 3,3'‐diaminobenzidine detection system.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File 1: cbdv70888‐sup‐0001‐SuppMat.docx

Data Availability Statement

The authors have nothing to report.