Development and Evaluation of Indole-Based Phospholipase D Inhibitors for Lung Cancer Immunotherapy

Abstract

This study explored novel immunomodulatory approaches for cancer treatment, with a specific focus on lung cancer, the leading cause of cancer-related deaths worldwide. We synthesized indole-based phospholipase D (PLD) inhibitors with various substituents to improve anticancer efficacy. Through structure–activity relationship studies, the key compound was identified that significantly inhibiting PLD, suppressing cell growth, viability, and migration in vitro, while inducing apoptosis of lung cancer cells. In silico docking studies confirmed its binding to the PLD1 active site, highlighting the role of specific residues in inhibiting PLD1 activity. The inhibitor modulated oncogenic pathways and immune evasion in lung cancer cells, showing potential for immunotherapy. In vivo experiments in a mouse model showed tumor reduction and immune response alteration. Combining these inhibitors with gemcitabine, an anticancer drug, synergistically enhanced inhibition of lung cancer cell apoptosis and proliferation. This research offers new insights into PLD inhibitor as potential cancer therapeutics.

Introduction

Phospholipase D (PLD) is a pivotal enzyme involved in numerous cellular processes, such as signal transduction, cytoskeletal organization, membrane trafficking, and gene expression regulation.¹ PLD catalyzes phospholipid hydrolysis to produce phosphatidic acid (PA),² a process fundamental to various organisms, ranging from bacteria to animals.³ PLD structure, with a core catalytic domain and regulatory domains, is highly conserved among different species.⁴ The catalytic domain contains conserved residues that are essential for enzymatic activity, while the regulatory domains mediate interactions with other proteins and lipids.³ PLD superfamily members are identified by conserved sequence regions, including the HKD motif, which is integral to PLD.^5,6 Two HKD motifs are essential for catalysis: the first catalytic histidine coordinates the substrate in a phosphohistidine intermediate, while the second histidine aids in removing the lipid headgroup and subsequently hydrolyzing PA from the intermediate.⁷ Human PLD isoforms, PLD1 and PLD2, despite similar primary sequences and ubiquitous expression,⁸ exhibit distinct regulatory and functional characteristics.⁹ PLD is implicated in various physiological and pathological conditions, including cancer, inflammation, and neurological disorders. Furthermore, PLD is a potential therapeutic target due to its involvement in cell growth, differentiation, and survival.⁹ PLD1 and PLD2 have different subcellular localizations and biochemical properties. PLD1 is primarily located in the cytosol and trans-Golgi network, while PLD2 is found in the plasma membrane and endosomes.¹⁰ Both isoforms are regulated by various signaling molecules, such as small GTPases, protein kinases, and phosphatases,¹¹ and their activity is influenced by stress, hormones, and nutrients.¹² Particularly, PLD1 is associated with tumor growth and metastasis¹³ and is elevated in several cancer types.¹⁴ PLD1 plays a pivotal role in cancer progression, enhancing cell proliferation and survival by modulating PI3K/Akt and ERK pathways.^15,16 It also facilitates cancer cell migration and invasion, influencing focal adhesion dynamics and actin cytoskeleton reorganization.¹⁷ The function of PLD1 is associated with tumor immune microenvironment including T cells and macrophages. It has been reported that targeting PLD1 induces cytotoxic T cell activation and T cell receptor-mediated signaling, and reduces the immune response mediated by T_reg cells, followed by anticancer effect in tumor microenvironment.^18,19 Moreover, targeting PLD decreased the function of macrophages and the migration of neutrophil to tumor sites.¹⁷ Furthermore, cancer cells can suppress immunogenic cell death (ICD), which can suppress the process where macrophages engulf dying cells or present them as tumor-associated antigens to trigger an adaptive immune response.²⁰ The presence of phagocytosis checkpoints, represented by “don’t eat-me” signals induced by antiphagocytic surface proteins, plays a role in facilitating tumor cells’ evasion of phagocytic clearance.²¹ Cancer cells often display increased “don’t eat-me” signals through CD24, CD47, and PD-L1,¹⁹ potentially offsetting elevated “eat-me” signals, such as calreticulin (CRT).²² CRT presence, alongside ATP and HMGB1 release, represents an essential damage-associated molecular pattern (DAMP) for triggering immunogenic cell death (ICD).²³ Moreover, PLD1 depletion in the tumor microenvironment significantly reduces tumor load and metastasis,²⁴ with PLD1 inhibition impairing macrophage functionality and neutrophil tumor migration.¹⁷

Recently, we explored PLD1 inhibition as an innovative strategy for cancer therapy, focusing on colorectal cancer (CRC).¹⁹ Furthermore, an observed upregulation of PLD in lung cancer²⁵ prompted us to evaluate a series of indole analogs for broader cancer applications, specifically lung cancer, a leading cause of cancer-related deaths worldwide.²⁶ In this study, we developed indole-based PLD inhibitors with modifications to improve their anticancer effects. The synthesized compounds were evaluated for in vitro PLD activity assay, cell growth and viability, apoptosis, transwell migration and invasion, colony formation, synergistic effect with gemcitabine, RAS activation, phagocytosis, immune cell infiltration, and extensive animal studies, revealing their potential as PLD modulators for cancer immunotherapy.

Results and Discussion

Rational Design of PLD Inhibitors

In a previous study, we synthesized and discovered a potent PLD1 inhibitor inspired by VU0155069 (Figure 1A), showing significant efficacy in vitro and in cellular models.^19,27 By integrating a pharmacophore approach with previous findings,¹⁹ we have identified the key features of VU0155069 that contribute to its pharmacological activity. (Figure 1B Top) VU0155069 exhibits several important properties: the hydrophobicity of the phenyl ring (cyan), the hydrogen donor by the amide (pink), the aromaticity contributed by the indole moiety (orange), and an additional hydrogen donor by the indole (pink). Additionally, a positive ionizable feature (red) was observed in the piperidine. VU0155069 can be divided into three primary fragments: the left fragment, which includes the aromatic ring (orange) and hydrogen donor features (pink); the right fragment, which encompasses the hydrophobic feature (cyan) and another hydrogen donor feature (pink); and the central fragment, which contains the positive ionizable feature (red). It is hypothesized that two terminal fragments are crucial pharmacophores, while the central ionizable feature produces a positive charge that may deteriorate ADME (absorption, distribution, metabolism, and excretion) properties. Additionally, intramolecular hydrogen bonding between amide and piperidine ring may reduce the size of linker between two aromatics. Based on these, the design of new PLD inhibitors should focus on eliminating the central ionizable feature and reducing the number of rotatable bonds in linker. This approach aims to improve ADME profile of newly designed compounds while preserving the functional features each terminal (Figure 1B, middle). Furthermore, to optimize the linker length between the aromatic rings, modifications were designed to retain the carbonyl group while adjusting the linker length. These modifications are intended to facilitate a comprehensive Structure–Activity Relationship (SAR) analysis (Figure 1B, bottom).

Figure 1.

Structure design of indole derivatives. (A) Representative PLD inhibitors. (B) Rational design of new PLD inhibitors.

Synthesis of Compounds

We synthesized various compounds for SAR studies, including indole derivatives with diverse substituents, such as 5-H, 5-fluoro, 5-chloro, or 5-trifluoromethyl groups and carbonyl groups linked to the indole through 0–3 carbon chains. Additionally, we introduced diverse substituted phenyl rings with substituents, such as methyl, tert-butyl, fluoro, bromo, difluoro, dichloro, trifluoromethyl, ditrifluoromethyl, dimethoxy, or dihydroxyl groups, linked to the amide group through 0–1 of carbon chain length. Scheme 1 summarizes the synthesis pathway of the target compounds, which was initiated by converting commercially available indole-2-carboxylic acid (1) or indole-3-carboxylic acid (2) through a reaction with the corresponding amine or alcohol to obtain corresponding indole-based analogs 3a–3ac. Compounds 4 were synthesized by demethylating compounds 3a, 3d, and 3w.

Scheme 1. Reagents and Conditions: (a) EDC·HCl, HOBt·H₂O, DIPEA, CH₂Cl₂, RT or DIPEA, CH₂Cl₂, RT and (b) BBr₃ in Hexane, CH₂Cl₂, RT.

Structure–Activity Relationships

We synthesized 32 compounds to evaluate their inhibition of PLD in HCT116 CRC cells at a 10 μM concentration, using VU0155069 as a reference. PLD activity was evaluated by quantifying the production of [H³] phosphatidylbutanol (PtdBut), generated through PLD-mediated transphosphatidylation, in the presence of 1-butanol. The radioactivity integrated into total phospholipids was measured, and the outcomes were expressed as the percentage of total lipid cpm incorporated into PtdBut. Compounds 3r, 3ac, and 4c achieved >50% PLD activity reduction at 10 μM (Tables 1 and S1).

Table 1. Effect of Synthetic Compounds on PLD Activity in HCT116 Cells.

We investigated the effect of various functional groups on the PLD activity through SAR analysis, revealing the significance of substituents in the indole ring, with electronegativity playing a crucial role in activity levels, specifically regarding -F, -CF₃, and -Cl substituents. PLD inhibitory activity compared to other derivatives, including 3b (3-F), 3c (4-Cl-3-NO₂), 3d (3,4-dimethoxy), 3e (4-ethynyl), 3j (3-Br), and 3k (4-tert-butyl). This comparative analysis underscores the significance of specific phenyl ring substituent patterns in enhancing PLD inhibition. Comparison between phenyl and benzyl groups showed a tendency toward higher activity of phenyl-containing compounds. For instance, compound 3b (3-fluorophenyl) outperformed compound 3h (3-fluorobenzyl) in PLD inhibition. The most favorable activity was achieved with a chain length of 2 carbons when a carbon chain of varying lengths (1–3 carbons) between the indole moiety and the amide was introduced. For instance, among compounds 3b, 3p, and 3s, the compound with 2 introduced carbons (3p) exhibited the highest activity. Furthermore, the derivative with 2 carbons (3r) also demonstrated better activity when comparing compounds 3r and 3t. Additionally, replacing the amide group with an ester, as in compound 3ac, diminished PLD inhibitory potency. Further exploration into the modification of the phenyl group with various substituents revealed that compounds 3f (3,5-diCF₃) and 4b (3,4-dihydroxy) exhibited superior.

Among the 32 synthesized compounds, 3r, 3ac, and 4c emerged as potent PLD inhibitors, underscoring the importance of specific structural modifications for enhancing inhibitory activity.

In Silico Study

In the previous study, we attempted to model the interaction between PLD1 and its inhibitor, VU0155069, using the PLD1 crystal structure (PDB: 6OHR).¹⁹ This endeavor was aimed to identify potential inhibitors exhibiting high congruence with pharmacophore features critical for effective PLD1 inhibition. The pharmacophore model involving the inhibitor-PLD1 complex highlighted key interaction features, such as hydrophobic interactions, ring aromaticity, hydrogen bond donation, and positive ionizability (Figure S1). Through our model, we conducted a rational design approach and screened 300 derivatives for their FitValues to these pharmacophore features. Based on these FitValues, we selected and synthesized the top 32 derivatives. This method was chosen because the PLD activity assay has limitations in screening a large number of compounds. The FitValue was used to quantify the alignment, with higher scores indicating better matches. Most synthesized compounds exhibited FitValues greater than 2.5, with compound 3r standing out as the most promising PLD1 inhibitor (Table S2). We employed docking models that confirmed their proper alignment and interaction with the site to further validate the binding efficacy and positional accuracy of compounds 3r, 3ac, and 4c within the PLD1 active site (Figure 2). As a result, compound 4c exhibited the highest activity in the in vitro PLD assay but displayed a binding energy (−135.35 kcal/mol) that was inferior to those of the other two derivatives, 3r (−152.72 kcal/mol) and 3ac (−170.70 kcal/mol), contrary to expectations.

Figure 2.

Predicted binding mode of indole derivatives with PLD1 (PDB code: 6OHR). 3r (green), 3ac (cyan), and 4c (yellow). (A) Superimposition of active site views of PLD1 with 3r, 3ac, and 4c. (B) The binding site of PLD1 with the compound 4c, where the binding energy is −135.35 kcal/mol. (C) The binding site of PLD1 with the compound 3r, where the binding energy is −152.72 kcal/mol. (D) The binding site of PLD1 with the compound 3ac, where the binding energy is −170.70 kcal/mol. Structural simulation of the complexes shows that some residues are involved in binding with derivatives. (green: hydrogen bond, pale pink: π-alkyl interaction, deep pink: π-π T shaped interaction and cyan: carbon–halogen).

Furthermore, its interaction with amino acids was less significant than that observed for compound 3r. Intriguingly, compound 3ac demonstrated the highest binding energy value among these compounds. However, it was distinguished by adopting an inverted docking mode compared to the other two compounds. Therefore, to understand their interactions more thoroughly, we proceeded to perform molecular dynamics (MD) simulations to examine the interactions in an equilibrium state. While the docking models provided preliminary understanding of their proper alignment and interaction with the site, they are inherently limited in accounting for the dynamic movements and conformational changes of the PLD1. To address these important aspects and analyze the behavior of these complexes in an equilibrium state, we transitioned from rigid docking studies to molecular dynamics simulations. Then, we performed molecular dynamics simulations for 40–90 ns to assess the stability of complexes, including PLD1 and these compounds. Throughout the simulation, an analysis of the PLD1-3r complex revealed that the Root Mean Square Deviation (RMSD) values for PLD1 and 3r compound exhibited a maximum variation of 5.3 Å, maintaining an average of 4.7 Å, signifying a stable interaction between them (Figure 3A). The Root Mean Square Fluctuation (RMSF) graph for PLD1 residues was presented, showcasing its flexibility with a black line for PLD1 alone and a red line for its complex with 3r (Figure 3B). Areas with significant fluctuation between PLD1-3r complex and free-PLD1 residues were highlighted with blue boxes. At 90 ns, significant interactions were observed, including π–π stacking between the indole ring of 3r and the amino acid residues Leu425 and Val1034 (Figure 3C). Furthermore, the phenyl group of 3r interacted with multiple amino acids, including π–π stacking with Trp382, Phe614, and Phe789, as well as hydrophobic interactions with Trp381 and Trp640. Significantly, Trp381, Trp382, Phe614, and Trp640, identified as the active site amino acids in PLD1, interact with the substrate,²⁸ notably with Trp381 playing a key role in substrate recognition.²⁹ The 4c and 3ac complexes exhibited higher RMSD variations compared to compound 3r, with maximum variations of 7.6 and 8.3 Å, respectively, suggesting greater structural variation and potentially different dynamic behaviors (Figures S2 and S3). The RMSF graphs demonstrated that both 4c and 3ac induced specific fluctuations in PLD1 residues, suggesting that these ligands influence the PLD1 flexibility differently. For instance, 4c reduced fluctuations at Arg486, while 3ac influenced fluctuations at Ala800, Ile804, and Trp1035. Furthermore, 4c and 3ac demonstrated unique interaction patterns, with 4c forming a water-bridge with the carbonyl group and Arg486 and 3ac showing π–π stacking with Phe745 and Trp1035. The interaction patterns exhibited by 3r and other variants showed distinct characteristics. 4c formed hydrogen bonds with the hydroxyl groups of Gly788, Glu790, and Tyr856. In contrast, it lacked interactions with Trp381, Trp382, and Trp640. Meanwhile, 3ac demonstrated π–π stacking between the indole ring and Phe745, as well as between the phenyl ring and Trp1035, which were not observed in interactions with Trp382, Phe614, and Trp640.

Figure 3.

Molecular dynamics simulation results for the PLD1-3r complex (PDB code: 6OHR). (A) The RMSD analysis showed distinct trajectories for PLD1 and 3r, with PLD1 stability and 3r structural alignment on PLD1 illustrated through their respective RMSD values. (B) The RMSF data highlighted regions of PLD1 that exhibit significant flexibility and interactions with 3r, providing insights into the molecular dynamics influenced by the complex formation. (C) At 90 ns, the PLD1–3r complex was visualized, emphasizing the interactions between PLD1 and 3r through specific residue highlights and showcasing the structural integrity and interaction specifics of the complex. Interaction frequencies between PLD1 and 3r demonstrated diverse interaction types, such as hydrogen bonds, π–π stacking, hydrophobic interactions, and water bridges, indicating a dynamic interplay of molecular forces.

Stable equilibrium states with the synthesized compounds were achieved through these MD studies. The interactions between compound 4c and the PLD1 protein residues appear to be significantly influenced by the role of hydrogen bonds from the dihydroxyl group. Unlike with compound 3ac, the interactions observed between compound 3r and PLD1 protein residues were similar to those critical in the interactions between PLD1 and its inhibitors. To enhance the interaction, particularly through π–π interactions, with residues Trp381, Trp382, Leu425, Phe614, and Trp640, designing a compound that can more effectively engage with these sites may lead to improved activity.

Afterward, we analyzed the dynamic properties of ligand interactions with PLD1, focusing on compounds 3r, 4c, and 3ac. We assessed critical parameters, including the RMSF, solvent accessible surface area (SASA), polar surface area (PSA), and radius of gyration (rGyr), through MD simulations for 40–90 ns (Figures 4 and S4). The average rGyr values for 3r, 4c, and 3ac were 5.1 Å, 4.4 Å, and 4.7 Å, respectively, indicating a significant impact on PLD1 compactness (Figures 4 and S4, green lines). Moreover, MolSA analysis for these complexes showed that compounds 3r, 4c, and 3ac maintained an average MolSA at 338 Ų, 260 Ų, and 308 Ų, respectively, suggesting ligand stability without significant conformational changes (Figures 4 and S4, yellow lines). SASA analysis highlighted the extent of solvation, with 3r, 4c, and 3ac showing average SASA values of 116 Ų, 71 Ų, and 124 Ų, respectively, and significant standard deviations indicating dynamic water-mediated interactions within the PLD1 binding site (Figures 4 and S4, orange line). Despite variations in SASA, the PSA values for 3r, 4c, and 3ac remained relatively stable, averaging at 76 Ų, 166 Ų, and 64 Ų, respectively, with minimal standard deviations (Figures 4 and S4, red line). Despite the high SASA variability, this stability suggested that the observed SASA fluctuations were less attributable to polar atoms and more to the structural contributions of nonpolar atoms, such as those in benzene rings.

Figure 4.

Ligand properties of 3r in molecular dynamics simulation. In a molecular dynamics simulation lasting 40–90 ns, the ligand 3r behavior was meticulously analyzed through line graphs and histograms. Key properties, including the ligand RMSD (navy line), radius of gyration (green line), molecular surface area (yellow line), solvent-accessible surface area (orange line), and polar surface area (red line), were charted. These metrics offer insights into the ligand structural shifts, interaction potential, and hydrogen bonding capabilities. Corresponding histograms provided a statistical view of the predominant states of these properties, enhancing our understanding of the ligand dynamics within the simulation.

Identification of the Binding Residues of 3r on PLD1 Involved in PLD1 Inhibition

To investigate the interaction between PLD1 and 3r, the drug affinity responsive target stability (DARTS) assay was employed. This assay operates by detecting changes in a protein’s susceptibility to proteolysis, induced by alterations in protein conformation upon binding with small molecules.³⁰ The DARTS assay with immunoblotting showed the increased stability of PLD1 against Pronase upon 3r treatment, whereas β-tubulin showed no Pronase resistance with 3r treatment (Figure 5A). Next, we sought to examine which residues are involved in the inhibition of PLD1 activity by 3r. As shown in the molecular dynamics simulation in Figure 3, three key residues (L425, F614, F789) are involved in the binding of the 3r compound to PLD1. Thus, mutations of these residues (L425A, F614A, F789A) were performed and used for the PLD1 activity assay. The 3r compound significantly suppressed PLD activity in wild-type and L425A mutant PLD1-transfected cells, whereas it did not significantly inhibit PLD activity in F614A and F789A mutant PLD1-transfected cells (Figure 5B). Thus, it is suggested that the F614 and F789 residues in PLD1 are responsible for the interaction between the compound and PLD1. The compound 3r selectively suppressed PLD1 activity in PLD1-overexpressing A549 cells, but not PLD2 activity in PLD2-overexpressing cells (Figure 5C), suggesting selective inhibition of PLD1 by 3r. The concentration that inhibits PLD activity by 50% (IC₅₀) is 1.97 μM in A549 cells (Figure 5D). Collectively, these results suggest that the 3r compound binds to PLD1 and the binding residues are involved in the inhibition of PLD1.

Figure 5.

Compound 3r exhibits binding affinity to PLD1 and selectively inhibits its enzymatic activity. (A) Drug affinity responsive target stability (DARTS) assay for investigating the interaction between PLD1 and 3r. (B) PLD assay for identifying the binding affinity residues of 3r to PLD1. (C) Effect of 3r on the PLD activity in A549 cells overexpressing PLD isozymes. (D) IC₅₀ value for the inhibition of PLD activity by 3r in A549 cells. Results represent at least three independent experiments and are expressed as mean ± standard error of the mean (SEM). Significance levels are indicated as ***p < 0.001, ns = not significance.

Comparative Analysis of IC₅₀ Values and Cytotoxicity in A549 and HCT116 Cells

This study determined the half-maximal inhibitory concentrations (IC₅₀) of compounds 3r, 4c, and 3ac on A549 and HCT116 cell viability utilizing the WST-1 assay (Figures 6A and S5). For A549 lung cancer cells, IC₅₀s were 18 μM (3r), > 200 μM (4c), and 50 μM (3ac), showing a dose-dependent cytotoxicity, with compound 3r being the most potent (Figure S5A). HCT116 cells displayed IC₅₀s of 29 μM (3r) and >50 μM for compounds 4c and 3ac (Figure S5B), indicating lower susceptibility compared to A549 cells. Furthermore, we measured the IC₅₀ for PLD inhibition of compounds 3r, 4c, 3ac, and VU0155069, a reported PLD1 inhibitor,²⁷ in A549 cells. As shown in Figures 5D and S5, the IC₅₀ values of the compounds for PLD inhibition are followed: 1.974 μM for 3r, 4.308 μM for 4c, 3.099 μM for 3ac, and 2.499 μM for VU0155069. Interestingly, the 3r compound exhibited a more potent IC₅₀ for PLD activity in A549 lung cancer cells compared to VU0155069 under the same assay conditions. We compared the viability of 3r and VU0155069 in A549, Calu6 lung cancer cells and HCT116 colorectal cancer cells. The IC₅₀ of viability in A549 cells is followed (Figures 6A and S6A): 18.54 μM for VU0155069, 18.44 μM for 3r. The IC₅₀ values for viability in A549 cells were as follows (Figures 6A and S6A): 18.54 μM for VU0155069 and 18.44 μM for 3r. In Calu6 lung cancer cells, the IC₅₀ values were (Figure S6B): 21.39 μM for VU0155069 and 19.0 μM for 3r. This suggests that 3r and VU0155069 have a similar effect on viability in lung cancer cells. The IC₅₀ values for viability in HCT116 cells were (Figures S5B and S6C): 20.92 μM for VU0155069 and 29.12 μM for 3r. Thus, it is suggested that VU0155069 exhibits a more potent cytotoxic effect in HCT116 colorectal cancer cells than 3r. Collectively, these results indicate that the 3r compound shows a more potent effect on PLD inhibition in lung cancer cells compared to VU0155069, and both 3r and VU0155069 exhibit similar cytotoxic effects in lung cancer cells.

Figure 6.

Anticancer effect of 3r in lung cancer cells. (A) Effect of 3r on the viability of various lung cancer cells. (B) Effect of 3r on the proliferation and death of A549 cells based on IncuCyte. (C) Effect of 3r on the colony formation of A549 cells. (D) Effect of 3r on the proliferation of A549 cells based on Ki67 staining. (E) Effect of 3r on the migration and invasion of A549 cells. (F) Effect of 3r on annexin V-positive apoptosis as analyzed by flow cytometry. (G) Effect of 3r on caspase-3 activity. Scale bar: 200 μm. Results represent at least three independent experiments and are expressed as mean ± standard error of the mean (SEM). Significance levels are indicated as ***p < 0.001.

Inhibition of Oncogenic Properties by 3r in Lung Cancer Cells

We further investigated the effects of compound 3r on the viability of various lung cancer cells. Compound 3r showed similar IC₅₀ of viability in A549, HCC44, H460, and HCC15 lung cancer cells (Figure 6A), suggesting that 3r has a cytotoxic effect on various lung cancer cells. Moreover, compound 3r significantly suppressed proliferation of A549 cells and increased death of A549 cells in a time-dependent manner a decreased cell growth and increased cell death rate in a time-dependent manner (Figures 6B and S7A). In addition, 3r significantly inhibited colony formation, Ki67-positive proliferation, migration and invasion of A549 cells (Figures 6C-6E and S7B). Furthermore, the compound 3r induced apoptosis of A549 cells using flow cytometry and caspase-3 activity assay (Figures 6F, 6G, and S7C). Taken together, these data suggest that compound 3r has antioncogenic properties against lung cancer cells.

Modulation of Oncogenic Signaling and Immune Evasion Mechanisms in Lung Cancer

Activating mutations in the epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma (KRAS)³¹ are key oncogenic factors in lung cancer. PLD1 upregulation is critical for tumorigenesis induced by mutant H-RAS,³² influencing p-ERK and RAS expression.³³ EGF-induced RAS activation stimulates downstream mitogenic signaling pathways, including PI3K/Akt and Raf/ERK pathways, which are involved in cancer progression.^34,35 We investigated whether compound 3r impacts EGFR-RAS-mediated signaling pathways. Compound 3r markedly reduced phospho-Akt, phospho-ERK, and phospho-IkBα levels, suggesting suppression of EGFR-RAS mitogenic pathways by 3r compound (Figure 7A). Moreover, compound 3r inhibited KRAS activation in KRAS-transfected cells (Figure 7B). Therefore, pharmacologically inhibiting PLD1 with compound 3r might reduce the activation of RAS effector molecules. KRAS mutations in cancer cells increase the expression of “don’t eat-me” signals, such as CD24, CD47, and PD-L1, but decrease the expression of “eat-me” signals, such as CRT, followed by the suppression of macrophage-mediated phagocytosis of cancer cells.³⁶⁻³⁸ Using the cancer genome atlas (TCGA) database, we found that the expression of KRAS is positively correlated with PLD1 and PD-L1 but negatively correlated with CRT in lung cancer (Figure 7C). Treating A549 cells with compound 3r reduced the expression of “don’t eat-me” signals and enhanced that of “eat-me” signals, as demonstrated by flow cytometry (Figures 7D and S8). Then, we investigated whether compound 3r affects macrophage-induced phagocytosis of cancer cells. CFSE (carboxyfluorescein succinimidyl ester)-labeled A549 cells were treated with compound 3r and then cocultured with THP-1 human monocytes activated with PMA (phorbol myristate acetate) for 36 h for differentiation into macrophages, followed by flow cytometry analysis. Compound 3r significantly increased the proliferation of CD11b⁺CFSE⁺ macrophages (Figure 7E). Therefore, PLD1 inhibition by compound 3r enhanced macrophage-induced phagocytosis of cancer cells by regulating “don’t eat-me” and “eat-me” signals.

Figure 7.

PLD1 inhibition by compound 3r suppresses the RAS signaling pathway and increases phagocytosis of lung cancer cells. (A) Effect of 3r on the activation of indicated RAS downstream molecules in EGF-stimulated A549 cells. (B) Effect of 3r on RAS activation. (C) Analysis of the correlation between indicated gene expressions based on TCGA databases. (D) Effect of 3r on the levels of “do not eat-me” signals and “eat-me” signals in A549 cells, as analyzed by flow cytometry. (E) Effect of 3r on the macrophage-induced phagocytosis of A549 cells, as analyzed by flow cytometry. The populations of CFSE⁺ CD11b⁺ macrophages were quantified. The results, representative of at least three independent experiments, are presented as the mean ± standard error of the mean (SEM). **p < 0.01, ***p < 0.001.

Enhanced Efficacy of 3r and Gemcitabine Combination Therapy in Inhibiting Lung Cancer Cell Proliferation and Increasing Apoptosis

We explored the effects of compound 3r in combination with gemcitabine, an anticancer drug used in the clinic,³⁹ on cell proliferation and apoptosis to assess their enhanced therapeutic potential against lung cancer. We found that this combination significantly reduced cell proliferation and increased apoptosis compared to either drug alone using real-time imaging and fluorescence (Figure 8A). Additionally, combined treatment significantly increased apoptosis of A549 cells, as assessed by PI staining and apoptosis assay (flow cytometry, cleaved PARP, and caspase-3 activity) (Figures 8B–D and S9B–C). Furthermore, we found a synergistic effect of compound 3r and gemcitabine using Synergyfinder analysis (a Bliss δ score of 20.06 for A549 cells) (Figures 8E and S9D). Our findings indicate that coadministration of a PLD1 inhibitor and αPD-L1 significantly enhances the antitumor immune response in the tumor microenvironment, outperforming monotherapy.¹⁹ This synergy suggests combination therapy as a viable strategy to prevent resistance development.⁴⁰ Furthermore, this study demonstrates that the combined treatment’s efficacy exceeds the sum of individual effects, allowing for lower dosages and reducing potential side effects. Hence, compound 3r significantly potentiated the anticancer effect of gemcitabine in lung cancer cells.

Figure 8.

Effect of the combination of 3r and gemcitabine on the proliferation and apoptosis of A549 cells. (A) A549 cells were treated with a single drug or their combination, and cell proliferation and death were measured using Incucyte. A549 cells were treated with the indicated drug and analyzed by (B) propidium iodide (PI) staining, (C) apoptosis assay based on the cleavage of PARP (poly ADP-ribose polymerase), and (D) caspase-3 activity assay. (E) The Bliss Index, assessing drug–drug synergy between 3r and gemcitabine, was analyzed using SynergyFinder. The results are representative of at least three independent experiments and presented as the mean ± standard deviation of the mean (SEM). ***p < 0.001.

In Vivo Antitumor Effect of 3r

Next, we examined whether 3r exerts suppression of tumor formation using a syngeneic mouse model. Before conducting in vivo experiments, the ADME properties of compound 3r were evaluated. In human liver microsomes, 57.3% of 3r remained after a 30 min incubation, indicating a relatively stable (Table S3). Similarly, in human plasma stability tests, 97.4% of 3r persisted after 30 min, and 94.6% remained after 120 min (Table S4). Based on these favorable microsomal and plasma stability results, we advanced to in vivo experiments in oral administration. After LLC mouse lung cells were subcutaneously injected into C57BL/6 mice, the drug was administered orally every other day. Compound 3r significantly attenuated tumor formation (Figure 9A and 9B) and also recovered the size and weight of the spleen, which were increased in vehicle-treated mice bearing tumors (Figure S10B). Differential spleen metrics reveal varying levels of tumor immunogenicity. We further monitored the body weight of normal mice treated with 3r to determine if 3r is selectively killing cancer cells or harming healthy cells as well. The compound 3r was administered orally to C57BL/6 mice without cancer cells. As shown in Figure S10A, the compound has no effect on the weight of the normal mice compared to untreated groups, suggesting that compound 3r does not affect normally healthy cells but selectively induces the death of cancer cells. Compound 3r reduced proliferation and enhanced apoptosis as analyzed by IHC using antibodies to Ki67 and active caspase 3 (Figure 9C). In addition, 3r decreased the levels of RAS and its downstream molecules such as p-ERK, p-Akt, and p-IKBα, suggesting inhibition of RAS activation signaling pathways by 3r (Figure 9D). Moreover, compound 3r reduced the levels of ’don’t eat-me’ signals such as CD47, CD24, and PD-L1, but increased the expression of ’eat-me’ signals such as calreticulin (Figure 9E). 3r also increased the population of CD80⁺ pro-inflammatory M1 macrophages, which are involved in the killing of cancer cells in the tumor microenvironment, whereas compound 3r decreased the population of CD163⁺ anti-inflammatory M2 macrophages, which are involved in cancer cell survival (Figure 9F). Furthermore, compound 3r increased the population of cytotoxic CD8⁺ T cells and the population of CD4⁺ and IL-17⁺ T_h17 cells, which are involved in the killing of cancer cells, but decreased the population of T_reg cells, which are involved in cancer cell survival (Figures 9G and S10C). These results suggest that PLD1 inhibition by 3r induces immunotherapeutic effects via activation of immune cells and suppression of oncogenic RAS pathways in vivo. We further investigated whether the antitumorigenic effect of 3r is dependent on immune cells. Thus, we aimed to examine the efficacy of 3r in a tumor model using immune-deficient nude mice with depleted T cells. Interestingly, 3r significantly suppressed tumor formation in the immune-deficient nude mice injected with LLC lung cancer cells (Figures S10D and S10E). We also confirmed that compound 3r treatment showed reduced proliferation, enhanced apoptosis, and reduced activation of RAS signaling pathways as analyzed by IHC (Figures S10F and S10G). Collectively, these results suggest that PLD1 inhibition by 3r compound exerts antitumorigenic effect through both immune and nonimmune response in vivo.

Figure 9.

PLD1 inhibition led to tumor suppression, modulated RAS signaling, and enhanced immune activation. (A) Change curves of tumor volume in mice during treatment. (B) The average weight of tumors in each mice group after the experiment. Immunohistochemistry (IHC) analysis of (C) Ki67, cleaved caspase-3, (D) RAS signaling, and (E) phagocytosis checkpoint proteins in tumor tissues. Scale bar: 50 μm. Populations of (F) CD80⁺ and CD163⁺ cells and infiltration of (G) CD8⁺ T cells in tumor tissues, as analyzed by immunofluorescence. Scale bar: 20 μm. Representative images were selected from at least four distinct fields. Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001.

Conclusions

This study presents a significant advance in cancer therapeutics through the development and evaluation of indole-based PLD inhibitors. Our research highlights the inhibitory effects of these compounds on PLD activity, especially in lung cancer cells, underscoring their potential as effective cancer treatments. Through in silico studies, we identified compounds to be synthesized and subsequently discovered inhibitors of PLD1 based on a PLD enzyme activity assay. Further analysis through docking studies and molecular dynamics (MD) simulations confirmed stable equilibrium states and critical interactions at the active site between the derivatives and PLD1. Notably, compound 3r demonstrates significant interactions with crucial amino acids in the active site of PLD1, which are essential for substrate interaction and recognition. Compound 3r binds to PLD1, and the residues in the binding of 3r to PLD1 are responsible for inhibition of PLD1 activity, suggesting that 3r inhibits PLD1 activity through its interaction with PLD1. This finding underscores the potential of 3r as a targeted inhibitor of PLD1, contributing valuable insights into the development of therapeutic agents. In vitro studies confirmed the impact of these compounds on cell growth, viability, apoptosis, and migration in lung cancer cell lines, particularly A549 cells. The synthesized compounds effectively inhibited PLD activity, leading to a dose-dependent reduction in cell viability and enhanced cytotoxic effects. The IC₅₀ values and cytotoxicity analysis further emphasized the superior efficacy of compound 3r, making it a prime candidate for further investigation. The comparative analysis of IC₅₀ values in different cell lines further validated the selective efficacy of these compounds, particularly 3r, against lung cancer cells compared with colorectal cancer cells. Compound 3r has a more potent effect on PLD1 inhibition than that of the reported PLD1 inhibitor, VU0155069.

The study also explored the modulation of oncogenic signaling and immune evasion mechanisms. Compound 3r significantly reduced the expression of “don’t eat-me” signals while upregulating “eat-me” signals, enhancing macrophage phagocytic activity. Moreover, the combination of compound 3r and gemcitabine synergistically enhanced their anticancer efficacy, underscoring the potential for integrated treatment strategies that leverage the unique properties of PLD inhibitors. In vivo studies in a syngeneic mouse model further confirmed the therapeutic value of compound 3r, demonstrating significant tumor suppression and immunomodulatory effects: an increase in the population of pro-inflammatory M1 macrophages and cytotoxic CD8⁺ T cells, and a decrease in the population of anti-inflammatory M2 macrophages and T_reg cells. Interestingly, the 3r compound exerts an antitumorigenic effect through both immune and nonimmune responses. This research also shows that the effectiveness of the combined therapy surpasses the aggregate effects of the treatments when used separately, enabling the use of reduced doses and diminishing potential side effects. In conclusion, this comprehensive study elucidates the potential of the indole-based PLD inhibitors, particularly compound 3r, as novel cancer therapeutics. Our findings highlight the importance of targeted PLD1 inhibition in lung cancer, thereby paving the way for novel and efficacious approaches in cancer immunotherapy. The synthesis of these compounds, coupled with their promising biological evaluations, SAR analysis, and in vivo efficacy, makes them potential candidates for future clinical applications in cancer treatment. These findings could offer the opportunity for improved cancer immunotherapy, particularly in lung cancer.

Experimental Section

General Chemistry Information

All chemicals were obtained from commercial suppliers and used without further purification. All reactions were monitored by thin-layer chromatography (TLC) on precoated silica gel 60 F254 (mesh) (E. Merck, Mumbai, India), and spots were visualized under UV light (254 nm). Flash column chromatography was performed with silica (Merck EM9385, 230–400 mesh). ¹H and ¹³C nuclear magnetic resonance (NMR, Varan Unity Inova) spectra were recorded at 400 and 100 MHz, or at 500 and 125 MHz. Proton and carbon chemical shifts are expressed in ppm relative to internal tetramethylsilane, and coupling constants (J) are expressed in Hertz. Splitting patterns were presented as s, singlet; d, doublet; t, triplet; q, quartet; dd, double of doublets; dt, double of triplet; m, multiplet; br, broad. LC-MS (Liquid chromatography–mass spectrometry) spectra were recorded by electrospray ionization (ESI) probe using a Shimadzu LC-MS2010 instrument with an Agilent C18 column, 50*4.6 mm, 5 mm particle size; mobile phase: 0.1% formic acid in H₂O/0.1% formic acid in CH₃CN (1:9) over 10 min; flow rate: 0.2 mLmin^–1; scan mode (0–500 amuz^–1). All compounds are >95% pure by HPLC analysis. The detected ion peaks were (M+z)/z in positive where M represents the molecular weight of the compound and z represents the charge (number of protons). High-resolution ESI-MS measurements were performed on a Micromass Quadrupole-Time of Flight (Q-TOF) Acquity UPLC-Mass System at Yonsei University; positive mode.

Representative Procedure for the Preparation of 3

A mixture of indole-2-carboxylic acid or indole-3-carboxylic acid 1 (1 equiv), corresponding amine or alcohol 2 (1 equiv), 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC·HCl, 2 equiv), 1-Hydroxybenzotriazole hydrate (HOBt·H₂O, 2 equiv) and N,N-Diisopropylethylamine (DIPEA, 3 equiv) in CH₂Cl₂ (0.1 M) was added simultaneously in a capped and sealed vial and stirred at room temperature for overnight. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (EtOAc: hexane = 1:5–1:1 or 2–5% MeOH in CH₂Cl₂) to afford the compound 3a - 3ac.

N-(3,4-Dimethoxyphenyl)-1H-indole-3-carboxamide (3a)

The title compound was obtained by column chromatography (3% MeOH in CH₂Cl₂). Brown solid (95.1 mg, 25.5%). MP: 235–238 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 11.67 (s, 1H), 9.56 (s, 1H), 8.24 (d, 1H, J = 1.4 Hz), 8.18 (d, 1H, J = 7.5 Hz), 7.46 (d, 1H, J = 2.2 Hz), 7.44 (d, 1H, J = 7.8 Hz), 7.28 (dd, 1H, J = 8.7, 2.2 Hz), 7.17–7.08 (m, 2H), 6.88 (d, 1H, J = 8.7 Hz), 3.74 (s, 3H), 3.70 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.4, 148.9, 144.8, 136.6, 133.8, 128.7, 126.8, 122.5, 121.5, 121.0, 112.4, 112.3, 112.0, 111.0, 105.4, 56.1, 55.8; RP-HPLC (254 nm): 99.07%; ESI (m/z) 297 (MH⁺), 319 (MNa⁺), 295 (MH^–); HRMS (ESI) calculated for C₁₇H₁₆N₂O₃ (MH⁺) 297.1239, found 297.1232.

N-(3-Fluorophenyl)-2-(1H-indol-3-yl)acetamide (3b)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:1). Pale pink solid (247.8 mg, 54.0%). MP: 111–114 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.92 (s, 1H), 10.32 (s, 1H), 7.63–7.53 (m, 2H), 7.34–7.22 (m, 4H), 7.04 (d, 1H, J = 7.6 Hz), 6.96 (t, 1H, J = 7.4 Hz), 6.85–6.77 (m, 1H), 3.71 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.54, 163.74, 161.35, 141.59, 141.48, 136.52, 130.79, 130.69, 127.59, 124.38, 121.45, 119.06, 118.86, 115.17, 115.14, 111.82, 110.00, 109.79, 108.66, 106.31, 106.05, 34.25; RP-HPLC (254 nm): 98.14%; ESI (m/z) 269 (MH⁺), 291 (MNa⁺); HRMS (ESI) calculated for C₁₆H₁₃FN₂O (MH⁺) 269.1090, found 269.1087.

N-(4-Chloro-3-nitrophenyl)-2-(1H-indol-3-yl)acetamide (3c)

The title compound was obtained by column chromatography (2% MeOH in CH₂Cl₂). Yellow solid (275.0 mg, 72.8%). MP: 165–168 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.93 (s, 1H), 10.64 (s, 1H), 8.41 (d, 1H, J = 2.2 Hz), 7.79 (dd, 1H, J = 8.9, 2.3 Hz), 7.65 (d, 1H, J = 8.8 Hz), 7.56 (d, 1H, J = 7.9 Hz), 7.33 (d, 1H, J = 8.1 Hz), 7.26 (s, 1H), 7.05 (t, 1H, J = 7.5 Hz), 6.96 (t, 1H, J = 7.4 Hz), 3.75 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 171.0, 147.6, 139.6, 136.5, 132.4, 127.5, 124.5, 124.3, 121.4, 119.0, 118.9, 118.6, 115.7, 111.8, 108.2, 34.2; RP-HPLC (254 nm): 97.89%; ESI (m/z) 328 (MH^–); HRMS (ESI) calculated for C₁₆H₁₂ClN₃O₃ (MH⁺) 330.0645, found 330.0638.

N-(3,4-Dimethoxyphenyl)-2-(1H-indol-3-yl)acetamide (3d)

The title compound was obtained by column chromatography (2% MeOH in CH₂Cl₂). White solid (470.2 mg, 53.1%). MP: 159–163 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.89 (s, 1H), 9.94 (s, 1H), 7.59 (d, 1H, J = 7.8 Hz), 7.36–7.29 (m, 2H), 7.23 (s, 1H), 7.12–7.01 (m, 2H), 6.96 (t, 1H, J = 7.4 Hz), 6.83 (d, 1H, J = 8.7 Hz), 3.69–3.65 (m, 7H); ¹³C NMR (100 MHz, DMSO-d₆) δ 169.7, 148.9, 145.0, 136.5, 133.5, 127.6, 124.2, 121.4, 119.1, 118.8, 112.4, 111.7, 111.2, 109.1, 104.6, 56.1, 55.7, 34.1; RP-HPLC (254 nm): 96.51%; ESI (m/z) 311 (MH⁺), 333 (MNa⁺), 309 (MH^–); HRMS (ESI) calculated for C₁₈H₁₈N₂O₃ (MH⁺) 311.1396, found 311.1391.

N-(4-Ethynylphenyl)-2-(1H-indol-3-yl)acetamide (3e)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:3). White solid (267.9 mg, 54.5%). MP: 163–166 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.90 (s, 1H), 10.25 (s, 1H), 7.55 (d, 1H, J = 8.0 Hz), 7.50 (dd, 4H, J = 8.2 Hz, 93.8 Hz), 7.34 (d, 1H, J = 8.0 Hz), 7.24 (s, 1H), 7.05 (t, 1H, J = 7.4 Hz), 6.96 (t, 1H, J = 7.4 Hz), 4.03 (s, 1H), 3.73 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.4, 140.4, 136.5, 132.8, 132.8, 127.6, 124.4, 121.5, 119.3, 119.3, 119.1, 118.9, 116.4, 111.8, 108.8, 84.0, 80.2, 34.3; RP-HPLC (254 nm): 99.64%; ESI (m/z) 275 (MH⁺), 297 (MNa⁺); HRMS (ESI) calculated for C₁₈H₁₄N₂O (MH⁺) 275.1184, found 275.1176.

N-(3,5-Bis(trifluoromethyl)phenyl)-2-(1H-indol-3-yl)acetamide (3f)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:2). White solid (131.8 mg, 19.1%). MP: 159–162 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.94 (s, 1H), 10.72 (s, 1H), 8.27 (s, 2H), 7.68 (s, 1H), 7.57 (d, 1H, J = 7.6 Hz), 7.34 (d, 1H, J = 8.0 Hz), 7.28 (s, 1H), 7.05 (t, 1H, J = 7.6 Hz), 6.96 (t, 1H, J = 7.4 Hz), 3.78 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 171.31, 141.62, 136.56, 131.64, 131.32, 130.99, 130.66, 127.57, 125.00, 124.56, 122.29, 121.49, 119.05, 118.94, 116.21, 111.84, 108.07, 34.31; RP-HPLC (254 nm): 96.30%; ESI (m/z) 387 (MH⁺); HRMS (ESI) calculated for C₁₈H₁₂F₆N₂O (MH⁺) 387.0932, found 387.0928.

N-(2-Fluorobenzyl)-2-(1H-indol-3-yl)acetamide (3g)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:1). White solid (255.7 mg, 79.3%). MP: 155–158 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.88 (s, 1H), 8.41 (t, 1H, J = 5.6 Hz), 7.53 (d, 1H, J = 8.0 Hz), 7.32 (d, 1H, J = 5.2 Hz), 7.26 (d, 1H, J = 5.2 Hz), 7.24 (t, 1H, J = 6.2 Hz), 7.18 (d, 1H, J = 2.0 Hz), 7.14 (d, 1H, J = 10.0 Hz), 7.09 (t, 1H, J = 6.0 Hz), 7.05 (t, 1H, J = 8.8 Hz), 6.94 (t, 1H, J = 7.4 Hz), 4.29 (d, 2H, J = 5.6 Hz), 3.57 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 171.35, 161.66, 159.23, 136.52, 129.93, 129.89, 129.26, 129.18, 127.64, 126.65, 126.50, 124.64, 124.60, 124.26, 121.38, 119.11, 118.69, 115.54, 115.33, 111.74, 109.17, 36.47, 36.43, 33.01; RP-HPLC (254 nm): 99.48%; ESI (m/z) 283 (MH⁺), 305 (MNa⁺); HRMS (ESI) calculated for C₁₇H₁₅FN₂O (MH⁺) 283.1247, found 283.1242.

N-(3-Fluorobenzyl)-2-(1H-indol-3-yl)acetamide (3h)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:1). White solid (292.2 mg, 90.6%). MP: 115–118 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.89 (s, 1H), 8.44 (t, 1H, J = 5.6 Hz), 7.53 (d, 1H, J = 8.0 Hz), 7.33 (d, 1H, J = 8.0 Hz), 7.27 (t, 1H, J = 7.4 Hz), 7.19 (d, 1H, J = 1.6 Hz), 7.06 (d, 1H, J = 7.6 Hz), 7.05 (s, 1H), 7.02 (d, 1H, J = 8.4 Hz), 7.01 (t, 1H, J = 20.8 Hz), 6.94 (t, 1H, J = 7.4 Hz), 4.26 (d, 2H, J = 6.0 Hz), 3.57 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 171.39, 163.83, 161.41, 143.22, 143.15, 136.55, 130.56, 130.48, 127.61, 124.30, 123.56, 123.53, 121.40, 119.08, 118.71, 114.28, 114.07, 113.92, 113.71, 111.76, 109.16, 42.12, 33.12.; RP-HPLC (254 nm): 99.89%; ESI (m/z) 283 (MH⁺), 305 (MNa⁺); HRMS (ESI) calculated for C₁₇H₁₅FN₂O (MH⁺) 283.1247, found 283.1241.

N-(4-Fluorobenzyl)-2-(1H-indol-3-yl)acetamide (3i)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:1). White solid (203.2 mg, 42.0%). MP: 144–147 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.87 (s, 1H), 8.41 (t, 1H, J = 5.6 Hz), 7.51 (d, 1H, J = 8.0 Hz), 7.32 (d, 1H, J = 8.0 Hz), 7.24 (d, 1H, J = 6.0 Hz), 7.22 (d, 1H, J = 6.0 Hz), 7.17 (d, 2H, J = 1.6 Hz), 7.08 (d, 1H, J = 7.6 Hz), 7.05 (t, 1H, J = 3.4 Hz), 7.03 (d, 1H, J = 7.6 Hz), 6.94 (t, 1H, J = 7.2 Hz), 4.22 (d, 2H, J = 6.0 Hz), 3.55 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 171.2, 162.7, 160.3, 136.5, 136.3, 136.3, 129.6, 129.6, 127.6, 124.3, 121.4, 119.1, 118.7, 115.4, 115.2, 111.7, 109.2, 41.9, 33.1; RP-HPLC (254 nm): 99.28%; ESI (m/z) 283 (MH⁺), 305 (MNa⁺); HRMS (ESI) calculated for C₁₇H₁₅FN₂O (MH⁺) 283.1247, found 283.1240.

N-(3-Bromophenyl)-2-(1H-indol-3-yl)acetamide (3j)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:3). White solid (352.2 mg, 62.5%). MP: 123–126 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.92 (s, 1H), 10.27 (s, 1H), 7.95 (t, 1H, J = 1.7 Hz), 7.56 (d, 1H, J = 7.8 Hz), 7.50–7.46 (m, 1H), 7.32 (d, 1H, J = 8.1 Hz), 7.25–7.16 (m, 3H), 7.07–7.01 (m, 1H), 6.98–6.92 (m, 1H), 3.71 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 170.5, 141.3, 136.5, 131.1, 127.5, 126.0, 124.3, 121.9, 121.7, 121.4, 119.0, 118.8, 118.1, 111.8, 108.6, 34.2; RP-HPLC (254 nm): 98.23%; ESI (m/z) 329 (MH⁺), 351 (MNa⁺); HRMS (ESI) calculated for C₁₆H₁₃BrN₂O (MH⁺) 329.0290, found 329.0291.

N-(4-(tert-Butyl)phenyl)-2-(1H-indol-3-yl)acetamide (3k)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:3). White solid (385.8 mg, 75.5%). MP: 152–155 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.88 (s, 1H), 10.00 (s, 1H), 7.59 (d, 1H, J = 7.2 Hz), 7.51 (d, 2H, J = 8.4 Hz), 7.34 (d, 1H, J = 7.6 Hz), 7.27 (d, 2H, J = 8.0 Hz), 7.23 (s, 1H), 7.05 (t, 1H, J = 7.2 Hz), 6.96 (t, 1H, J = 7.0 Hz), 3.70 (s, 2H), 1.22 (s, 9H); ¹³C NMR (100 MHz, DMSO-d₆) δ 169.9, 145.7, 137.3, 136.6, 127.7, 125.7, 125.7, 124.3, 121.4, 119.3, 119.3, 119.1, 118.8, 111.8, 109.1, 34.4, 34.2, 31.6, 31.6, 31.6; RP-HPLC (254 nm): 97.52%; ESI (m/z) 307 (MH⁺), 329 (MNa⁺); HRMS (ESI) calculated for C₂₀H₂₂N₂O (MH⁺) 307.1810, found 307.1808.

2-(1H-Indol-3-yl)-N-(3-methylbenzyl)acetamide (3l)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:1). White solid (166.2 mg, 52.3%). MP: 121–124 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.88 (s, 1H), 8.36 (t, 1H, J = 5.7 Hz), 7.56 (d, 1H, J = 7.9 Hz), 7.33 (d, 1H, J = 8.1 Hz), 7.19 (s, 1H), 7.13 (t, 1H, J = 7.5 Hz), 7.05 (t, 1H, J = 7.4 Hz), 7.02–6.92 (m, 4H), 4.21 (d, 2H, J = 5.9 Hz), 3.56 (s, 2H), 2.18 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 171.2, 139.9, 137.7, 136.6, 128.5, 128.1, 127.7, 127.6, 124.7, 124.3, 121.4, 119.2, 118.7, 111.7, 109.3, 42.5, 33.2, 21.4; RP-HPLC (254 nm): 99.31%; ESI (m/z) 279 (MH⁺), 301 (MNa⁺); HRMS (ESI) calculated for C₁₈H₁₈N₂O (MH⁺) 279.1497, found 279.1494.

2-(1H-Indol-3-yl)-N-(3-(trifluoromethyl)benzyl)acetamide (3m)

The title compound was obtained by column chromatography (EtOAc: hexane = 2:1). White solid (396.7 mg, 65.8%). MP: 127–130 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.86 (s, 1H), 8.46 (t, 1H, J = 5.2 Hz), 7.55–7.47 (m, 5H), 7.31 (d, 1H, J = 8.4 Hz), 7.17 (s, 1H), 7.03 (t, 1H, J = 7.4 Hz), 6.92 (t, 1H, J = 7.4 Hz), 4.32 (d, 2H, J = 6.0 Hz), 3.56 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 171.49, 141.72, 136.60, 131.75, 129.92, 129.65, 129.61, 129.30, 128.98, 127.61, 126.04, 124.32, 124.03, 123.99, 123.95, 123.92, 123.89, 123.86, 123.82, 123.78, 123.34, 121.40, 118.99, 118.72, 111.77, 109.10, 42.19, 33.16; RP-HPLC (254 nm): 98.91%; ESI (m/z) 333 (MH⁺), 355 (MNa⁺); HRMS (ESI) calculated for C₁₈H₁₅F₃N₂O (MH⁺) 333.1215, found 333.1206.

2-(1H-Indol-3-yl)-N-(4-(trifluoromethyl)benzyl)acetamide (3n)

The title compound was obtained by column chromatography (EtOAc: hexane = 2:1). White solid (349.2 mg, 61.4%). MP: 154–158 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.86 (s, 1H), 8.46 (t, 1H, J = 5.4 Hz), 7.60 (d, 2H, J = 8.0 Hz), 7.51 (d, 1H, J = 7.6 Hz), 7.40 (d, 2H, J = 8.0 Hz), 7.32 (d, 1H, J = 8.0 Hz), 7.18 (d, 1H, J = 2.0 Hz), 7.04 (t, 1H, J = 7.2 Hz), 6.94 (t, 1H, J = 7.0 Hz), 4.32 (d, 2H, J = 6.0 Hz), 3.57 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 171.46, 145.10, 136.57, 128.27, 127.97, 127.66, 127.64, 126.13, 125.53, 125.49, 125.45, 125.42, 124.33, 123.43, 121.40, 119.08, 118.72, 111.77, 109.10, 42.31, 33.12; RP-HPLC (254 nm): 97.71%; ESI (m/z) 333 (MH⁺); HRMS (ESI) calculated for C₁₈H₁₅F₃N₂O (MH⁺) 333.1215, found 333.1210.

N-(2,3-Dichlorobenzyl)-2-(1H-indol-3-yl)acetamide (3o)

The title compound was obtained by column chromatography (EtOAc: CH₂Cl₂ = 1:9). White solid (250.5 mg, 65.9%). MP: 161–164 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.87 (s, 1H), 8.43 (t, 1H, J = 5.7 Hz), 7.54 (d, 1H, J = 7.9 Hz), 7.48 (dd, 1H, J = 6.9, 2.5 Hz), 7.33 (d, 1H, J = 8.0 Hz), 7.25–7.18 (m, 3H), 7.05 (t, 1H, J = 7.5 Hz), 6.95 (t, 1H, J = 7.4 Hz), 4.33 (d, 2H, J = 5.8 Hz), 3.60 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 171.5, 139.6, 136.5, 132.0, 130.3, 129.3, 128.2, 127.6, 127.6, 124.3, 121.4, 119.0, 118.7, 111.7, 109.0, 41.3, 33.0; RP-HPLC (254 nm): 99.36%; ESI (m/z) 355 (MNa⁺), 331 (MH^–); HRMS (ESI) calculated for C₁₇H₁₄Cl₂N₂O (MH⁺) 333.0561, found 333.0555.

N-(3-Fluorophenyl)-3-(1H-indol-3-yl)propanamide (3p)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:2). White solid (363.8 mg, 79.4%). MP: 128–131 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.76 (s, 1H), 10.12 (s, 1H), 7.63 (d, 1H, J = 12.0 Hz), 7.55 (d, 1H, J = 7.6 Hz), 7.32 (d, 1H, J = 8.0 Hz), 7.29 (t, 1H, J = 5.2 Hz), 7.29 (s, 1H), 7.11 (s, 1H), 7.05 (t, 1H, J = 7.4 Hz), 6.96 (t, 1H, J = 7.4 Hz), 6.84–6.81 (m, 1H), 3.02 (t, 2H, J = 7.6 Hz), 2.69 (t, 2H, J = 7.4 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 171.8, 162.6, 141.5, 136.7, 130.7, 127.4, 122.6, 121.4, 118.8, 118.6, 115.2, 114.0, 111.8, 109.8, 106.2, 37.8, 21.1; RP-HPLC (254 nm): 98.89%; ESI (m/z) 283 (MH⁺), 305 (MNa⁺); HRMS (ESI) calculated for C₁₇H₁₅FN₂O (MH⁺) 283.1247, found 283.1239.

N-(3-Bromophenyl)-3-(1H-indol-3-yl)propanamide (3q)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:2). White solid (469.5 mg, 86.3%). MP: 125–128 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.75 (s, 1H), 10.07 (s, 1H), 7.97 (s, 1H), 7.54 (d, 1H, J = 7.6 Hz), 7.47 (d, 1H, J = 7.6 Hz), 7.32 (d, 1H, J = 8.0 Hz), 7.22 (t, 1H, J = 7.8 Hz), 7.20 (d, 1H, J = 5.6 Hz), 7.11 (s, 1H), 7.04 (t, 1H, J = 7.4 Hz), 6.96 (t, 1H, J = 7.2 Hz), 3.01 (t, 2H, J = 7.2 Hz), 2.68 (t, 2H, J = 7.6 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 171.8, 141.3, 136.7, 131.1, 127.4, 126.0, 122.6, 122.0, 121.8, 121.4, 118.8, 118.6, 118.2, 114.0, 117.8, 37.8, 21.1; RP-HPLC (254 nm): 98.03%; ESI (m/z) 343 (MH⁺), 365 (MNa⁺); HRMS (ESI) calculated for C₁₇H₁₅BrN₂O (MH⁺) 343.0446, found 343.0439.

N-(3,5-Bis(trifluoromethyl)phenyl)-3-(1H-indol-3-yl)propanamide (3r)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:2). White solid (49.2 mg, 7.8%). MP: 158–161 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.77 (s, 1H), 10.57 (s, 1H), 8.25 (s, 1H), 7.70 (s, 2H), 7.55 (d, 1H, J = 7.8 Hz), 7.32 (d, 1H, J = 8.1 Hz), 7.13 (d, 1H, J = 1.7 Hz), 7.05 (t, 1H, J = 7.5 Hz), 6.96 (t, 1H, J = 7.4 Hz), 3.04 (t, 2H, J = 7.5 Hz), 2.73 (t, 2H, J = 7.5 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 172.54, 141.50, 136.69, 131.65, 131.32, 131.00, 130.67, 127.38, 125.02, 122.71, 122.31, 121.40, 119.07, 119.04, 118.70, 118.63, 116.18, 113.73, 111.82, 37.86, 20.99; RP-HPLC (254 nm): 99.74%; ESI (m/z) 401 (MH⁺); HRMS (ESI) calculated for C₁₉H₁₄F₆N₂O (MH⁺) 401.1089, found 401.1083.

N-(3-Fluorophenyl)-4-(1H-indol-3-yl)butanamide (3s)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:3). White solid (342.9 mg, 74.9%). MP: 99–102 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.75 (s, 1H), 10.06 (s,1H), 7.61 (d, 1H, J = 12.0 Hz), 7.50 (d, 1H, J = 7.6 Hz), 7.32 (d, 1H, J = 8.0 Hz), 7.28 (s, 1H), 7.28 (d, 1H, J = 10.0 Hz), 7.10 (s, 1H), 7.04 (t, 1H, J = 7.4 Hz), 6.94 (t, 1H, J = 7.4 Hz), 6.83–6.80 (m, 1H), 2.72 (t, 2H, J = 7.4 Hz), 2.36 (t, 2H, J = 7.4 Hz), 1.99–1.92 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 172.1, 162.6, 141.5, 136.8, 130.6, 127.6, 122.8, 121.3, 118.7, 118.6, 115.2, 114.4, 111.8, 109.7, 106.2, 36.6, 26.2, 24.7; RP-HPLC (254 nm): 98.16%; ESI (m/z) 297 (MH⁺), 319 (MNa⁺); HRMS (ESI) calculated for C₁₈H₁₇FN₂O (MH⁺) 297.1403, found 297.1397.

N-(3,5-Bis(trifluoromethyl)phenyl)-4-(1H-indol-3-yl)butanamide (3t)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:2). White solid (52.6 mg, 8.6%). MP: 145–149 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.76 (s, 1H), 10.51 (s, 1H), 8.25 (s, 1H), 7.69 (s, 2H), 7.50 (d, 1H, J = 7.8 Hz), 7.32 (d, 1H, J = 8.1 Hz), 7.12 (d, 2H, J = 1.9 Hz), 7.04 (t, 1H, J = 7.5 Hz), 6.94 (t, 1H, J = 7.4 Hz), 2.74 (t, 2H, J = 7.4 Hz), 2.41 (t, 2H, J = 7.4 Hz), 2.03–1.92 (m, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 172.80, 141.55, 136.77, 131.61, 131.28, 130.96, 130.63, 127.59, 125.03, 122.84, 122.32, 121.27, 119.04, 119.01, 118.73, 118.55, 116.08, 114.22, 111.77, 36.61, 25.94, 24.61; RP-HPLC (254 nm): 99.75%; ESI (m/z) 415 (MH⁺); HRMS (ESI) calculated for C₂₀H₁₆F₆N₂O (MH⁺) 415.1245, found 415.1243.

5-Fluoro-N-(4-fluorophenyl)-1H-indole-2-carboxamide (3u)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:3). White solid (199.7 mg, 65.7%). MP: 232–236 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 11.86 (s, 1H), 10.31 (s, 1H), 7.85–7.77 (m, 2H), 7.49–7.42 (m, 2H), 7.39 (s, 1H), 7.24–7.17 (m, 2H), 7.07 (td, 1H, J = 9.3, 2.5 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 159.91, 159.81, 158.82, 157.53, 156.51, 135.64, 135.61, 133.99, 133.46, 127.55, 127.45, 122.48, 122.40, 115.85, 115.63, 114.09, 114.00, 113.12, 112.86, 106.42, 106.19, 104.29, 104.24; RP-HPLC (254 nm): 99.12%; ESI (m/z) 273 (MH⁺); HRMS (ESI) calculated for C₁₅H₁₀F₂N₂O (MH⁺) 273.0839, found 273.0831.

N-(3,5-Difluorophenyl)-5-fluoro-1H-indole-2-carboxamide (3v)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:7). White solid (21.7 mg, 6.7%). MP: 259–262 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 11.91 (s, 1H), 10.53 (s, 1H), 7.58–7.51 (m, 2H), 7.47 (dd, 1H, J = 10.0, 2.5 Hz), 7.45–7.41 (m, 1H), 7.39 (s, 1H), 7.08 (td, 1H, J = 9.2, 2.3 Hz), 6.94 (t, 1H, J = 9.3 Hz); ¹³C NMR (150 MHz, DMSO-d₆) δ 163.78, 163.68, 162.17, 162.07, 160.34, 158.59, 157.04, 142.06, 141.97, 141.88, 134.34, 132.91, 129.45, 127.50, 127.43, 126.68, 114.81, 114.63, 114.29, 114.22, 113.71, 113.63, 113.46, 108.33, 108.17, 106.62, 106.46, 105.08, 105.05, 103.39, 103.35, 103.23, 103.19, 100.66, 100.63, 99.35, 99.18, 99.01, 87.16, 60.27, 21.32, 21.24, 14.57; RP-HPLC (254 nm): 99.43%; ESI (m/z) 289 (MH⁺); HRMS (ESI) calculated for C₁₅H₉F₃N₂O (MH⁺) 291.0745, found 291.0738.

N-(3,4-Dimethoxyphenyl)-5-fluoro-1H-indole-2-carboxamide (3w)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:1). Yellow solid (592.6 mg, 84.4%). MP: 221–224 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 11.79 (s, 1H), 10.10 (s, 1H), 7.46–7.39 (m, 3H), 7.37–7.32 (m, 2H), 7.05 (td, 1H, J = 9.3, 2.4 Hz), 6.92 (d, 1H, J = 8.7 Hz), 3.74 (s, 3H), 3.71 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 159.64, 158.53, 156.98, 149.07, 145.76, 133.99, 133.95, 132.86, 127.72, 127.65, 114.11, 114.04, 112.97, 112.81, 112.80, 112.56, 106.40, 106.25, 105.96, 103.94, 103.90, 56.27, 55.95; RP-HPLC (254 nm): 99.74%; ESI (m/z) 315 (MH⁺), 337 (MNa⁺), 313 (MH^–); HRMS (ESI) calculated for C₁₇H₁₅FN₂O₃ (MH⁺) 315.1145, found 315.1143.

5-Fluoro-N-(3-(trifluoromethyl)phenyl)-1H-indole-2-carboxamide (3x)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:5). White solid (182.3 mg, 50.6%). MP: 209–212 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 11.92 (s, 1H), 10.56 (s, 1H), 8.25 (s, 1H), 8.12 (d, 1H, J = 8.3 Hz), 7.63 (t, 1H, J = 8.0 Hz), 7.54–7.44 (m, 4H), 7.12 (td, 1H, J = 9.4, 2.5 Hz); ¹³C NMR (150 MHz, DMSO-d₆) δ 160.31, 158.58, 157.03, 149.92, 140.23, 134.25, 133.17, 130.49, 130.30, 130.24, 130.09, 129.88, 129.67, 127.59, 127.52, 125.58, 124.09, 123.78, 120.43, 120.41, 120.38, 120.36, 117.76, 117.75, 116.71, 116.68, 116.66, 116.63, 114.26, 114.19, 113.47, 113.30, 111.98, 111.95, 111.93, 111.90, 109.96, 109.93, 109.90, 109.88, 106.58, 106.43, 104.90, 104.87; RP-HPLC (254 nm): 99.28%; ESI (m/z) 323 (MH⁺), 321 (MH^–); HRMS (ESI) calculated for C₁₆H₁₀F₄N₂O (MH⁺) 323.0808, found 323.0798.

N-(3,5-Bis(trifluoromethyl)phenyl)-5-chloro-1H-indole-2-carboxamide (3y)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:7). White solid (52.6 mg, 47.8%). MP: 282–285 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 12.07 (s, 1H), 10.87 (s, 1H), 8.54 (s, 2H), 7.84 (d, 2H, J = 7.6 Hz), 7.52–7.45 (m, 2H), 7.27 (d, 1H, J = 8.8 Hz); ¹³C NMR (150 MHz, DMSO-d₆) δ 161.98, 150.97, 136.26, 131.72, 131.51, 131.30, 131.09, 129.05, 128.22, 126.89, 125.31, 125.23, 125.09, 123.28, 121.60, 121.48, 114.75, 113.37, 113.34, 107.78, 107.67, 107.64, 107.62, 107.59, 107.56, 52.44; RP-HPLC (254 nm): 98.11%; ESI (m/z) 407 (MH⁺), 405 (MH^–); HRMS (ESI) calculated for C₁₇H₉ClF₆N₂O (MH^–) 405.0229, found 405.0230.

Methyl 4-((1H-Indole-2-carboxamido)methyl)benzoate (3z)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:2). White solid (284.9 mg, 29.8%). MP: 215–219 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 11.63 (s, 1H), 9.13 (t, 1H, J = 6.1 Hz), 7.93 (d, 2H, J = 8.3 Hz), 7.61 (d, 1H, J = 8.0 Hz), 7.47 (d, 2H, J = 8.2 Hz), 7.43 (d, 1H, J = 8.2 Hz), 7.22–7.13 (m, 2H), 7.03 (t, 1H, J = 7.4 Hz), 4.59 (d, 2H, J = 6.0 Hz), 3.82 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 166.5, 161.7, 145.7, 136.9, 131.8, 129.7, 129.7, 128.6, 127.7, 127.5, 127.5, 123.8, 121.9, 120.2, 112.7, 103.1, 52.4, 42.4; RP-HPLC (254 nm): 98.48%; ESI (m/z) 307 (MH^–); HRMS (ESI) calculated for C₁₈H₁₆N₂O₃ (MH⁺) 309.1239, found 309.1232.

N-(3,4-Dimethoxybenzyl)-5-fluoro-1H-indole-2-carboxamide (3aa)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:1). White solid (544.3 mg, 48.8%). MP: 210–213 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 11.68 (s, 1H), 8.97 (t, 1H, J = 5.9 Hz), 7.41–7.34 (m, 2H), 7.13 (s, 1H), 7.00 (td, 1H, J = 9.3, 2.4 Hz), 6.93 (s, 1H), 6.89–6.80 (m, 2H), 4.40 (d, 2H, J = 5.9 Hz), 3.70 (s, 3H), 3.69 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ 161.27, 158.45, 156.91, 149.21, 148.38, 133.95, 133.71, 132.46, 127.75, 127.68, 119.98, 114.02, 113.96, 112.58, 112.40, 112.33, 112.03, 106.25, 106.10, 103.12, 103.09, 56.10, 55.97, 42.56; RP-HPLC (254 nm): 98.16%; ESI (m/z) 327 (MH^–); HRMS (ESI) calculated for C₁₈H₁₇FN₂O₃ (MH⁺) 329.1301, found 329.1295.

N-(3,5-Bis(trifluoromethyl)phenyl)-5-(trifluoromethyl)-1H-indole-2-carboxamide (3ab)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:3). White solid (91.2 mg, 47.5%). MP: 289–292 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 12.32 (s, 1H), 10.97 (s, 1H), 8.55 (s, 2H), 8.23 (s, 1H), 7.84 (s, 1H), 7.70–7.63 (m, 2H), 7.55 (d, J = 8.6 Hz, 1H); ¹³C NMR (150 MHz, DMSO-d₆) δ 60.41, 141.30, 138.87, 133.07, 131.57, 131.36, 131.14, 130.92, 126.70, 126.50, 124.90, 124.69, 122.89, 121.65, 121.44, 120.91, 120.88, 120.65, 120.62, 120.22, 120.19, 116.91, 116.88, 116.86, 113.92, 106.20; RP-HPLC (254 nm): 99.09%; ESI (m/z) 439 (MH^–); HRMS (ESI) calculated for C₁₈H₉F₉N₂O (MH^–) 439.0493, found 439.0493.

3,5-Bis(trifluoromethyl)phenyl 5-Fluoro-1H-indole-2-carboxylate (3ac)

The title compound was obtained by column chromatography (EtOAc: hexane = 1:1). White solid (249.5 mg, 34.0%). MP: 173–177 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 12.33 (s, 1H), 8.20 (s, 2H), 8.08 (s, 1H), 7.52–7.45 (m, 2H), 7.41 (s, 1H), 7.18 (td, 1H, J = 9.3, 2.5 Hz); ¹³C NMR (150 MHz, DMSO-d₆) δ 159.40, 158.70, 157.15, 151.72, 135.32, 132.45, 132.23, 132.00, 131.78, 127.56, 127.24, 127.17, 126.09, 124.45, 124.42, 124.28, 122.47, 120.66, 120.44, 120.42, 120.39, 115.27, 115.09, 114.74, 114.68, 110.64, 110.60, 106.92, 106.77; RP-HPLC (254 nm): 99.31%; ESI (m/z) 390 (MH^–).

Representative Procedure for the Preparation of 4

A mixture of corresponding indole based derivatives 3 (1 equiv) in CH₂Cl₂ (0.1 M) was stirred at 0 °C for 30 min. A 1 M solution of boron tribromide in hexane (3 equiv) was added dropwise to the reaction mixture, followed by stirring from 0 °C to room temperature for 6 h. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (EtOAc: hexane = 1:5–1:1 or 5% ∼ 10% MeOH in CH₂Cl₂) to afford the compound 4a - 4c.

N-(3,4-Dihydroxyphenyl)-1H-indole-3-carboxamide (4a)

The title compound was obtained by column chromatography (EtOAc: CH₂Cl₂ = 2:1). Pale pink solid (21.1 mg, 11.5%). MP: 220–225 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 11.60 (s, 1H), 9.35 (s, 1H), 8.86 (s, 1H), 8.50 (s, 1H), 8.18 (d, 1H, J = 2.8 Hz), 8.14 (d, 1H, J = 7.6 Hz), 7.41 (d, 1H, J = 7.9 Hz), 7.28 (d, 1H, J = 2.4 Hz), 7.16–7.06 (m, 2H), 6.90 (dd, 1H, J = 8.5, 2.4 Hz), 6.63 (d, 1H, J = 8.5 Hz); RP-HPLC (254 nm): 95.29%; ESI (m/z) 269 (MH⁺), 267 (MH^–); HRMS (ESI) calculated for C₁₅H₁₂N₂O₃ (MH⁺) 269.0926, found 269.0922.

N-(3,4-Dihydroxyphenyl)-2-(1H-indol-3-yl)acetamide (4b)

The title compound was obtained by column chromatography (5% MeOH in CH₂Cl₂). White solid (50.6 mg, 37.2%). MP: 214–218 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.85 (s, 1H), 9.69 (s, 1H), 8.89 (s, 1H), 8.53 (s, 1H), 7.58 (d, 1H, J = 7.9 Hz), 7.32 (d, 1H, J = 8.0 Hz), 7.20 (s, 1H), 7.13 (s, 1H), 7.04 (t, 1H, J = 7.5 Hz), 6.95 (t, 1H, J = 7.4 Hz), 6.76 (d, 1H, J = 8.5 Hz), 6.59 (d, 1H, J = 8.4 Hz), 3.63 (s, 2H); ¹³C NMR (100 MHz, DMSO-d₆) δ 169.3, 145.3, 141.5, 136.5, 131.9, 127.6, 124.1, 121.3, 119.1, 118.7, 115.6, 111.7, 110.7, 109.3, 108.3, 34.1; RP-HPLC (254 nm): 96.75%; ESI (m/z) 305 (MNa⁺), 281 (MH^–); HRMS (ESI) calculated for C₁₆H₁₄N₂O₃ (MH⁺) 283.1083, found 283.1075.

N-(3,4-Dihydroxyphenyl)-5-fluoro-1H-indole-2-carboxamide (4c)

The title compound was obtained by column chromatography (5% MeOH in CH₂Cl₂). White solid (107.6 mg, 59.1%). MP: 269–272 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 11.73 (s, 1H), 9.91 (s, 1H), 8.99 (s, 1H), 8.69 (s, 1H), 7.46–7.36 (m, 2H), 7.30 (d, 2H, J = 12.3 Hz), 7.03 (td, 1H, J = 9.3, 1.9 Hz), 6.98 (dd, 1H, J = 8.5, 1.7 Hz), 6.68 (d, 1H, J = 8.5 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ 159.25, 158.75, 156.44, 145.33, 142.20, 134.04, 133.78, 131.12, 127.62, 127.52, 115.65, 113.96, 113.87, 112.73, 112.47, 112.07, 109.43, 106.27, 106.04, 103.66, 103.61; RP-HPLC (254 nm): 95.16%; ESI (m/z) 287 (MH⁺), 285 (MH^–); HRMS (ESI) calculated for C₁₅H₁₁FN₂O₃ (MH^–) 285.0675, found 285.0675.

In Silico Study

Molecular docking simulations were conducted using the CHARM force field in Discovery Studio 2022 (BIOVIA).⁴¹ The protein structure of PLD1 was obtained from the protein databank⁴² (PDB ID: 6OHR(43)) and structurally refined by removing the water molecules and adding hydrogen atoms to the entire protein. The catalytic domain in PLD1 was used as the binding site for ligands. Molecular docking analyses were performed to generate ten binding poses per compound and to drive the binding energy of the most probable prospective binding mode with Ligandfit and CDOCKER docking scores. Molecular dynamics (MD) simulations were carried out utilizing Desmond 5.5. in the Maestro program,⁴⁴ employing the OPLS3e force field.⁴⁵ The PLD1-ligand complexes were situated in an orthorhombic box and supplemented with water molecules using the TIP3P model, which was generated within a buffer distance of 10 Å. The simulation systems of PLD1-ligand complexes have 20,052, 20,054, and 20,053 water molecules, respectively. For a more realistic simulation environment, monovalent ions (Na⁺ and Cl^–) were added to make a neutral system and emulate a physiological concentration of 0.15 M, where each system has 63 Na⁺ and 56 Cl^– atoms. The simulations adhered to the NPT ensemble to ensure consistency in the number of particles, temperature (set at 300 K), and pressure (maintained at 1.01325 bar). Long-range electrostatic interactions were evaluated using the particle-mesh Ewald method,⁴⁶ setting a cutoff at 9 Å for both van der Waals and short-range electrostatic interactions. The Nose-Hoover thermostats regulated the simulation’s temperature,⁴⁷ while the Martina-Tobias-Klein method managed the pressure.⁴⁸ Equations of motion were integrated via a RESPA integrator,⁴⁹ with an inner time step of 2.0 fs for both bonded and nonbonded interactions within the short-range cutoff. Following Desmond’s default equilibration protocol, a 100 ns simulation run was executed, storing conformation and energy data at 4 ps intervals. Further analysis incorporated a 50 ns MD simulation from 40 to 90 ns, by removing the initial 40 ns and last 10 ns.

Plasma Stability

We utilized Procaine (Sigma-Aldrich, P9879) and Enalapril (Sigma-Aldrich, CDS020548) as reference compounds, with Chlorpropamide (TRC, C424800) serving as the internal standard. Human plasma (Biochemed, BC23024PSC, Lot number), containing 3.8% sodium citrate, and mouse plasma (Biochemed, 029-APSC-MP), with sodium citrate, were used as biological matrices. Human and mouse plasma samples, spiked with compound A2998 and another compound to a final concentration of 10 μM, were incubated at 37 °C for varying time intervals (0, 30, and 120 min). At each designated time point, the plasma-containing tubes were removed, and an acetonitrile solution containing the internal standard was added. The samples were then vortexed for 5 min and centrifuged at 15,000 rpm for 5 min at 4 °C. The quantitative analysis of the remaining substrate was performed using a Nexera XR system (Shimadzu, Japan) coupled with a TSQ vantage mass spectrometer (Thermo, USA). Separation was achieved using a Kinetex C18 column (2.1 × 100 mm, 2.6 μm particle size; Phenomenex, USA). The mobile phases consisted of 0.1% formic acid in distilled water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). Gradient elution was employed for compound separation. Detection and quantification were carried out in Multiple Reaction Monitoring (MRM) mode using Xcalibur software (version 4.4). These experiments were performed at the DGMIF new drug development center (Daegu, Korea).

Microsomal Stability

Verapamil (TRC, V125000) was used as a reference compound along with an NADPH Regeneration system (Promega, V9510), chlorpropamide (TRC, C424800) as an internal standard, potassium phosphate buffer (pH 7.4, Corning, #451201), human liver microsomes (Corning, #452117), and mouse liver microsomes (Corning, #452701). Two types of liver microsomes (human and mouse, 0.5 mg/mL) were preincubated with 0.1 M phosphate buffer solution (pH 7.4), compound A2998, and another compound at a concentration of 1 μM at 37 °C for 5 min. The NADPH Regeneration system solution was then added, and the mixture was incubated at 37 °C for 30 min. To terminate the reaction, an acetonitrile solution containing the internal standard chlorpropamide was added, followed by centrifugation at 15,000 rpm for 5 min at 4 °C. The supernatant was collected and injected into the LC-MS/MS system for analysis. The remaining substrate quantity from the above reaction was analyzed using the Nexera XR system (Shimadzu, Japan) coupled with the TSQ vantage (Thermo, USA). Chromatographic separation was achieved using a Kinetex C18 column (2.1 × 100 mm, 2.6 μm particle size; Phenomenex, USA). The mobile phase consisted of 0.1% formic acid in distilled water (A) and 0.1% formic acid in acetonitrile (B), with gradient elution conditions. Quantitative analysis was performed in the MRM (Multiple Reaction Monitoring) mode using Xcalibur software (version 4.4). These experiments were performed at the DGMIF new drug development center (Daegu, Korea).

Cell Lines and Cell Culture

Lung cancer cell lines (A549, H358, Calu6, HCC44, H460, HCC15, and LLC), along with HCT116 and HEK293T cells, were obtained from the American Type Culture Collection (ATCC). These were cultured in either Dulbecco’s Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI) 1640 medium (both from Corning), supplemented with 10% fetal bovine serum (FBS) (Corning) and 1% penicillin/streptomycin. The cultures were maintained at 37 °C in a humidified atmosphere with 5% CO₂.

Phospholipase D Activity Assay

To investigate the PLD activity assay, two different methods were employed. First, cells were labeled with [H³] myristic acid, and PLD (phospholipase D) activity was evaluated by quantifying the production of [H³] phosphatidylbutanol. This compound is a result of PLD-mediated transphosphatidylation. The assessment was carried out in the presence of 1-butanol, in accordance with methods described previously.⁵⁰ Second, cells were treated with the compounds for 1 h and quantified using the Amplex RED Phospholipase D assay kit (Invitrogen). The fluorescence intensity was measured with an M200 PRO microplate reader (Tecan, Männedorf, Switzerland) using an excitation wavelength of 540 nm and an emission wavelength of 590 nm. Drug affinity responsive target stability (DARTS) Assay. A549 cells were lysed using a lysis buffer containing protease and phosphatase inhibitors. The supernatant from the cell lysates was incubated with compounds at the indicated concentrations at room temperature (RT) for 1h, followed by proteolysis with Pronase (5 μg/mL) for 10 min at RT. To quench proteolysis, 5x sodium dodecyl sulfate (SDS) sample loading buffer was added to each sample and boiled at 100 °C for 5 min. Samples were analyzed by immunoblotting with primary antibodies (PLD1, β-tubulin) according to the manufacturer’s instructions.

Drug Affinity Responsive Target Stability (DARTS) Assay

A549 cells were lysed using a lysis buffer containing protease and phosphatase inhibitors. The supernatant from the cell lysates was incubated with compounds at the indicated concentrations at room temperature (RT) for 1h, followed by proteolysis with Pronase (5 mg/mL) for 10 min at RT. To quench proteolysis, 5x sodium dodecyl sulfate (SDS) sample loading buffer was added to each sample and boiled at 100 °C for 5 min. Samples were analyzed by immunoblotting with primary antibodies (PLD1, β-tubulin) according to the manufacturer’s instructions.

Cell Growth, Viability, and Death Assay

A549 and H358 cells were seeded at a density of 5,000 cells per well in 96-well plates and incubated for 24 h. After incubation, cells were treated with CellTox green dye (Promega) and various chemical compounds. Imaging was performed using phase contrast and green fluorescence (400 ms exposure) channels on the IncuCyte Zoom (Essen BioScience), an automated imaging system. Image analysis was conducted using the IncuCyte Zoom Basic Analyzer. To assess cell viability, we employed the WST-1 assay in accordance with the manufacturer’s instructions (DoGenBio, Seoul, Korea). Briefly, cells were seeded in 96-well plates at a density of 5 × 10³ cells/well in a total volume of 100 μL of media and incubated at 37 °C for 24 h. Subsequent to this incubation, cells were exposed to the drugs for 48 h. The absorbance for the WST-1 assay was measured at 450 nm using an Infinite M200 PRO microplate reader (Tecan, Männedorf, Switzerland). All experiments to assess cell viability were performed in triplicate. The IC₅₀, defined as the drug concentration causing 50% inhibition of cell growth compared to control, was calculated.

Cell Apoptosis Assay

Apoptosis in A549 cells was detected using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s instructions. Cell analysis was conducted with BD FACSAria III (BD Biosciences), and subsequent data analysis was performed using FlowJo v10.0 (BD Biosciences). For immunofluorescence assays, cells were directly stained with Hoechst 33342/PI (Invitrogen) and imaged with an LSM710 system (Carl Zeiss). In the caspase-3 activity assay, cells were cultured in 96-well plates at a density of approximately 40%. Drugs (3r and Gemcitabine) and Ac-DEVD-CHO, a caspase-3 inhibitor, were then administered to the corresponding wells at 10 μM. After 48 h, the wells were washed with PBS and incubated at room temperature with 100 μL of PBS containing 1 μM of NucView 488 caspase-3 substrate for 30 min. Subsequently, the cells were stained with 1 μM Hoechst 33342. The fluorescence of Hoechst 33342 and NucView 488 was quantified using a FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany). Multicolor images were captured using the Lionheart FX Automated Microscope (BioTek, Winooski, VT, USA).

Transwell Migration and Invasion Assay

The cells were seeded at a density of 5 × 10⁴ cells/ml in serum-free medium into 8 μm pore transwell polycarbonate chambers (SPL Life Sciences) for the migration assay and into chambers precoated with 100 μL of Matrigel (BD Biosciences) for the invasion assay. After incubation for 24 h, nonmigrated cells were removed using cotton swabs. Cells that migrated or invaded to the bottom of the inset were fixed with 4% paraformaldehyde (PFA) for 20 min and then stained with crystal violet for 10 min. The stained cells were enumerated using a light microscope.

Colony-Forming Assay

A colony formation assay was conducted to assess the extended proliferative capacity of cells. Cells were seeded into 6-well plates at a density of 500 to 1,000 cells per well. Forty-8 h postseeding, compound 3r was administered. Subsequently, the cells were incubated for an additional 48 h. The culture medium was replaced every 3 days, and the cells were maintained for 10 days. Following the incubation period, cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 20 min. Staining was performed using crystal violet (Sigma) for 30 min. The number of colonies formed was quantified using ImageJ software.

RAS Activation Assay

Active RAS (RAS-GTP) was harvested from the specified cell lines following 24 h of treatment with 3r. Cell lysis was performed using a lysis buffer supplemented with a protease inhibitor, followed by centrifugation at 13,000 rpm for 10 min at 4 °C. Subsequently, 800 μg of the cell lysates were incubated overnight at 4 °C with beads coated with the fusion protein GST-Raf1-RBD. After three washes with washing buffer, proteins bound to the beads were eluted using 5x SDS sample buffer. The eluted samples were then heated at 100 °C for 5 min and subsequently analyzed by immunoblotting to detect RAS.

Immunoblot Analysis

Proteins were extracted from the cells using passive buffer (Millipore) supplemented with a 1 x protease and phosphatase inhibitor cocktail. Samples were denatured by boiling at 100 °C for 5 min. The denatured samples were then separated by SDS-PAGE and transferred onto a PVDF membrane (Merk Millipore). After blocking in TBS-T buffer (Tris-buffered saline containing Tween-20) with 5% BSA, the membranes were probed with the corresponding primary antibodies at 4 °C: anti-pEGFR (3777S), anti-EGFR (sc-03), anti-pAkt (9271S), anti-Akt (9272S), anti-pERK (sc-7383), anti-ERK (sc-514302), anti-pIkBa (sc-8404), anti-IkBa (sc-1643), anti-GFP (sc-9996), and anti-KRAS (sc-30). Following overnight incubation, the membranes were washed five times with TBS-T for 10 min each and subsequently incubated with either peroxidase-labeled antimouse or antirabbit IgG. Detection was performed using a chemiluminescent solution (Merk Millipore) and images were captured using ImageJ.

Immunohistochemistry

After deparaffinization for 1 h at 65 °C, the tissue sections were rehydrated sequentially from xylene, xylene: ethanol (EtOH), 100% EtOH, to 70% EtOH. Subsequently, the samples underwent antigen retrieval using citrate buffer and were blocked with 0.3% hydrogen peroxide and 2.5% horse serum, respectively. The sections were then incubated with primary antibodies overnight at 4 °C. On the second day, the samples were incubated with an HRP-conjugated secondary antibody (Vector Laboratories) and ABC reagent (Vector Laboratories) at room temperature for 30 min. Staining was performed using DAB (Vector Laboratories), followed by counterstaining with Mayer’s hematoxylin.

Immunofluorescence Analysis

To identify intracellular proteins, immunofluorescence staining was employed using primary antibodies and corresponding secondary antibodies labeled with fluorescence. The primary antibodies used included anti-Ki67 (ab16667), anti-CD80 (A16039), anti-CD163 (ab87099), anti-CD8 (ab217344), anti-CD4 (sc-13573), anti-Granzyme B (ab53097), anti-Perforin (ab16074), anti-IL17 (sc-374218), and anti-FoxP3 (sc-53876). The secondary antibodies utilized were Alexa Fluor 488 goat antirabbit and rat IgG (A-11006, A-11008); Alexa Fluor 594 goat antimouse, rabbit, and rat IgG (A-11005, A-11012, A-11007). The cell nuclei were stained with 1 μM Hoechst 33342. Image acquisition was performed using the LSM710 system (Carl Zeiss), and Zen3.4 (lite) software was employed for subsequent image analysis.

In Vitro Phagocytosis Assay

Cells were treated with 3r in RPMI containing 10% FBS for 48 h and then labeled with 1 μM carboxyfluorescein succinimidyl ester (CFSE). Subsequently, a total of 5 × 10⁵ differentiated THP-1 cells were incubated with an equal number of CFSE-labeled cells in RPMI medium without FBS for 2 h. After washing with PBS, the cells were incubated with human BD Fc block (BD Biosciences) to prevent nonspecific binding. Additionally, the cells were incubated with PerCP-Cy5.5-conjugated anti-CD11b antibody (BD Biosciences) for 20 min at 4 °C, followed by analysis using flow cytometry.

Animal Study

A lung cancer syngeneic model was established in 6-week-old C57BL/6 male mice. A total of 3 × 10⁵ LLC cells, suspended in 50 μL of PBS, were subcutaneously injected into the flanks of the mice. Seven days’ postinjection, the mice were randomized into two groups, and each group was treated as follows for 3 weeks: in the vehicle group, mice were per orally (PO) administered a vehicle daily; in the 3r group, mice were PO administered 10 mg/kg of 3r every other day. Tumor volume in mice with subcutaneously injected LLC cells was measured every 2 days starting from day 7 postinjection. External tumor dimensions were obtained using precision calipers, and the tumor volume (TV) was calculated using the formula: TV = (W × L²)/2, where W is the width and L is the length of the tumor. All mice were housed at a temperature of 25 °C with a 12:12-h light/dark cycle. All animal procedures were conducted in accordance with institutional guidelines for animal research and were approved by the Institutional Animal Care and Use Committee of Yonsei University (IACUC-202309–1721–01).

Statistical Analysis

All experiments were conducted in triplicate and repeated a minimum of three times. The results are expressed as the mean ± standard deviation (SD). P-values for statistical analyses were computed using the appropriate tests in GraphPad Prism 6. For comparisons between two groups, an unpaired Student’s t test was utilized. In the analysis of more than two groups, a two-way analysis of variance (ANOVA) was performed, followed by Sidak’s multiple comparison test. The SynergyFinder software was employed to calculate synergy scores for the proposed drug combinations using four different reference models: Bliss, Loewe, HSA, and ZIP. Synergy scores below −10 indicated a potential antagonistic interaction between the drugs; scores from −10 to 10 were indicative of an additive interaction, while scores above 10 suggested a potential synergistic interaction.⁵¹

Glossary

Abbreviations

Akt

Protein kinase B

CRC

colorectal cancer

EGFR

epidermal growth factor receptor

ERK

Extracellular Signal-Regulated Kinase

FoxP3

Forkhead box P3

HMGB1

High mobility group box1 protein

ICD

immunogenic cell death

IkBα

Inhibitor of nuclear factor kappa B

IL

Interleukin

KRAS

Kirsten Rat Sarcoma;

LLC mouse

Lewis Lung Carcinoma mouse

MD

molecular dynamics

PD-L1

Programmed Death-Ligand 1

PI staining

Propidium Iodide staining

PI3K

Phosphoinositide 3-Kinase

PLD

Phospholipase D

PSA

polar surface area

RAS

rat sarcoma

Raf

Rapidly accelerated fibrosarcoma

RMSD

Root Mean Square Deviation

RMSF

the Root Mean Square Fluctuation

SAR

Structure–Activity Relationships

SASA

solvent accessible surface area

p-ERK

Phosphorylated Extracellular Signal-Regulated Kinase

rGyr

radius of gyration

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.4c00750.

• Synthesized compounds were tested for PLD activity (Table S1); Fitvalue of synthesized compounds based on generated pharmacophore (Table S2); Human liver microsomal stability of 3r (Table S3); Plasma stability of 3r (Table S4); 3D structure-based pharmacophore model of VU0155069 in complex with PLD1 based on the X-ray crystal structure of PLD1 (Figure S1); Molecular dynamics simulation results for the PLD1-4c complex (Figure S2); Molecular dynamics simulation results for the PLD1-3ac complex (Figure S3); Ligand properties of 4c and 3ac (Figure S4); Effect of various compounds on the viability and PLD activity in cancer cells (Figure S5); Comparative Analysis of IC₅₀ values for 3r and VU0155069 in lung cancer cells and colorectal cancer cells (Figure S6); Effect of the 3r on the inhibition of proliferation in A549 lung cancer cells (Figure S7); Investigating the impact of 3r on phagocytosis checkpoint protein levels utilizing flow cytometry (Figure S8); Potentiation effect of coadministration of 3r and gemcitabine in A549 Cells (Figure S9); In vivo suppression of tumor growth via PLD1 inhibition (Figure S10) (PDF)

• NMR spectra (¹H and ¹³C) and HPLC and HRMS spectrogram data of all target compounds SMILES formula of each tested compound and associated biochemical data (CSV)

• Binding mode and molecular dynamics of compound 3r with 6OHR (PDB)

• Binding mode and molecular dynamics of compound 3ac with 6OHR (PDB)

• Binding mode and molecular dynamics of compound 4c with 6OHR (PDB)

Author Contributions

^# D.S. and S.H.L. contributed equally to this work.

The authors declare no competing financial interest.