Crystal structure, DFT, vibrational properties, Hirshfeld surface and antitumor activity studies of 1-Octyl-1H-Indole derivatives

Wanxia Zhang; Xiaocui Tai; Xiangxiang Liu; Xiaoxiao Wang; Yunyun Huang; Chunshen Zhao; Zhaopeng Zheng; Zhixu Zhou

doi:10.25259/AJC_820_2025

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Original Article

ARTICLE IN PRESS

doi:

10.25259/AJC_820_2025

Crystal structure, DFT, vibrational properties, Hirshfeld surface and antitumor activity studies of 1-Octyl-1H-Indole derivatives

Wanxia Zhang^a,b,c, Xiaocui Tai^a,b,c, Xiangxiang Liu^a,b,c, Xiaoxiao Wang^d, Yunyun Huang^a,b,c, Chunshen Zhao^a,b,c,, Zhaopeng Zheng^d,e,*, Zhixu Zhou^a,b,c,*

aSchool of Pharmaceutical Sciences, Guizhou University, Guiyang, P. R. China.

bGuizhou Engineering Laboratory for Synthetic Drugs, Guiyang, P. R. China.

cGuizhou Provincial Key Laboratory of Innovation and Manufacturing for Pharmaceuticals, Guiyang, P. R. China.

dDepartment of Oncology, Guizhou Provincial People’s Hospital, Guiyang, P.R. China.

eDepartment of Oncology, Zhenfeng County People’s Hospital, Qianxinan, P. R. China.

* Corresponding authors: E-mail addresses: zhixuzhou@126.com (Z. Zhou), zhengzhaopeng@163.com (Z. Zheng)

Received: 2025-07-16, Accepted: 2025-11-30, Epub ahead of print: 2026-03-20,

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Abstract

Indole derivatives, recognized as privileged scaffolds in medicinal chemistry, were utilized to design ten novel compounds synthesized from 4-bromobenzenesulfonyl chloride and 5-bromoindole via a five-step route. Structural confirmation was achieved by nuclear magnetic resonance (¹H NMR, ¹³C NMR), infrared (IR), and mass spectrometry (MS). Remarkably, single-crystal X-ray diffraction (XRD) of compound 7a, supported by density functional theory (DFT) calculations, confirmed a highly consistent molecular conformation. Subsequent molecular electrostatic potential (MEP) and frontier molecular orbital (FMO) studies identified key electronic features, while Hirshfeld analysis decoded intermolecular packing forces. Molecular docking against protein target PDB: 4A2N indicated stable binding modes and significant affinity. Importantly, 7a and its analogues exhibited potent antiproliferative effects against the human breast cancer cell line MCF-7 (Michigan Cancer Foundation-7) and the human promyelocytic leukemia cell line HL-60, underscoring their potential as promising anticancer leads for further development.

Keywords

Antitumor activity

DFT

Indole derivatives

Molecular docking

Surface analysis

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1. Introduction

Indole derivatives possess a distinctive structural motif that underlies their diverse biological functions and tunable physicochemical characteristics, rendering them versatile scaffolds in medicinal chemistry, agricultural technology, and synthetic methodology.

In anticancer drug development, indole-based architectures have enabled the discovery of several mechanistically unique agents. A notable example is the indole-derived Inositol-requiring enzyme 1 alpha (IRE1α) inhibitor IA107, which allosterically regulates X-box binding protein 1 (XBP1) mRNA splicing and holds promise for treating endoplasmic reticulum stress-related cancers [1]. Selective histone deacetylase 10 (HDAC10) inhibitors built on an indole core have also emerged as prospective anticancer therapeutics, leveraging their specificity toward this epigenetic target [2]. Further expanding this portfolio, late-stage modification of osimertinib’s indole moiety yielded compound 3r, a derivative that retains potent activity against epidermal growth factor receptor (EGFR) T790M/L858R mutant and H1975 cancer cells, and demonstrates robust efficacy in vivo, positioning it as a promising lead for overcoming resistance in next-generation targeted oncology treatments [3].

Within agricultural science, indole derivatives contribute significantly to stress resilience and phytohormone monitoring. Indole-3-acetic acid (IAA), a native auxin, was shown to improve cadmium stress tolerance in sorghum, suggesting a viable strategy for safe crop cultivation in contaminated environments [4]. Complementing this, a minimally invasive sensor system was engineered for continuous, real-time tracking of IAA and other key phytohormones directly in plant tissues, enabling dynamic physiological studies [5].

Advances in synthetic chemistry further highlight the utility of indole templates. Biosynthetic studies revealed that cytochrome P450 enzymes such as MsSAS catalyze the transformation of spirocyclic oxindole alkaloids like mitraphylline in Mitragyna species, underscoring the enzymatic plasticity in indole diversification [6]. In addition, a palladium-catalyzed asymmetric Larock reaction was developed to efficiently assemble N─N axially chiral indoles, affording a library of 64 structurally diverse analogues with high enantiocontrol and functional group compatibility. This methodology provides essential chiral synthons for pharmaceutical, catalyst, and materials research [7].

Given the remarkable pharmacological and biological properties of indole derivatives, this study was designed to synthesize a series of novel derivatives featuring an indole core scaffold. The synthesis was accomplished via a five-step reaction sequence, as outlined in Scheme 1. The structures of all target compounds were fully characterized by Fourier transform infrared (FT-IR), high-resolution mass spectrometry (HRMS), and nuclear magnetic resonance (¹H NMR and ¹³C NMR). Among them, compound 7a was obtained as a single crystal, and its structure was unambiguously determined by single-crystal X-ray diffraction (SC-XRD). The ground-state geometry of 7a was further optimized using density functional theory (DFT) at the B3LYP/6-311+G(2d, p) level, demonstrating excellent agreement between experimental and theoretical data. A comprehensive investigation of 7a, including molecular electrostatic potential (MEP), frontier molecular orbitals (FMOs), vibrational frequency analysis, Hirshfeld surface analysis, and NMR spectral studies (¹H/¹³C), confirmed its high structural stability. From a series of ten indole derivatives screened in vitro, seven were identified with inhibitory rates surpassing the positive control at 100 μM. The activity of three specific analogs, 7e, 7h, and 7g, was found to be on par with cymethynil, suggesting their strong candidacy for further investigation as novel small-molecule therapeutics.

2. Materials and Methods

2.1. General remarks

All experimental materials, including chemical reagents, starting materials, and cell lines, were obtained exclusively from certified commercial suppliers for research purposes. Structural characterization of compound 7a was conducted using: a Bruker AVANCE NEO 400 MHz spectrometer (Germany) for ¹H/¹³C NMR spectra, an Agilent 6530 Q-TOF mass spectrometer for HRMS, a Bruker IFS-55V spectrometer (Germany) for FT-IR spectroscopy, and a Bruker APEX-II CCD diffractometer for single-crystal XRD analysis. High-performance liquid chromatography (HPLC) was conducted on Agilent 1100 Series, and all the results were obtained under UV 254 nm. For in vitro antitumor activity assays, human cancer cell lines were purchased from Procella Biotechnology Co., Ltd (Wuhan, China), with cell culture maintained under sterile conditions in a Class II biological safety cabinet (Model HF safe 1200 LC (A2), Shanghai, China) and a CO₂ incubator (Model HHF90 (HT), Shanghai, China) to ensure experimental reproducibility.

2.2. Experimental section

Scheme 1 shows the synthetic route of 1-octyl-1H-indole derivatives. In the process of synthesizing intermediates, some reactions need nitrogen protection, so researchers need to pay special attention to the sealing of the reaction device. At the same time, protective equipment such as goggles, lab coats, and masks must be worn throughout the experiment.

2.2.1. Synthesis of intermediate 5-bromo-1-octyl-1H-indole (5)

Under an ice-salt bath, N,N-dimethylformamide (DMF, solvent), sodium hydride (5.1 g, 212 mmol, 60%), 5-bromoindole (30 g, 153 mmol), and n-octyl bromide (39.65 mL, 229.5 mmol) were added into the reaction bottle in turn, and the reaction was monitored by thin-layer chromatography (TLC). Quenching with water, extracting with petroleum ether (PE), drying with anhydrous sodium sulfate, concentrating to obtain crude oil, and purifying to obtain compound 5 (43.3 g, 91.7% yield). ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 7.73(d, J = 1.9 Hz, 1H), 7.26 (dd, J = 8.7, 1.9 Hz, 1H), 7.19 (d, J = 8.6 Hz, 1H), 7.08 (d, J= 3.1 Hz, 1H), 6.41 (d, J = 3.2 Hz, 1H), 4.06 (t, J = 7.1 Hz, 2H), 1.80 (p, J = 7.1 Hz, 2H), 1.26 (ddd, J = 12.8, 6.3, 3.4 Hz, 10H), 0.86 (t, J = 6.8 Hz, 3H).

2.2.2. General procedure for synthesis of 2a-2c

A 250 mL three-necked round-bottom flask was charged with tetrahydrofuran (80 mL). To this solution, p-bromobenzenesulfonyl chloride (78.3 mmol) was added with stirring. The reaction mixture was then cooled to 0°C using an ice-salt bath and maintained at this temperature. Subsequently, a 40% aqueous solution of dimethylamine (391.5 mmol) and triethylamine (156.6 mmol) was added dropwise and sequentially. The progress of the reaction was monitored by TLC, which indicated completion after 0.5 h of stirring. The reaction was quenched with water, resulting in the formation of a white solid. The solid was collected by suction filtration and washed with petroleum ether to afford the crude product 2a.

N,N-Dimethyl-4-bromobenzenesulfonamide ( 2a). White solid, yield 97.8%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 7.72-7.62 (m, 4H), 2.72 (s, 6H).

4-(4-bromophenylsulfonyl)morpholine ( 2b). White solid, yield 96.3%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 7.73 – 7.67 (m, 4H), 3.28 – 3.23 (m, 4H), 1.83 – 1.78 (m, 4H).

1-(4-bromophenylsulfonyl)pyrrolidine ( 2c). White solid, yield 96.4%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 7.72 – 7.68 (m, 2H), 7.63 – 7.60 (m, 2H), 3.76 – 3.73 (m, 4H), 3.02 – 2.99 (m, 4H).

2.2.3. General procedure for synthesis of 3a-3c

A 500 mL three-necked flask was charged with N, N-dimethyl-4-bromobenzenesulfonamide (76.5 mmol) and tetrahydrofuran (150 mL) under a nitrogen atmosphere. The reaction mixture was cooled to –78°C, and the flask was purged with nitrogen three times to ensure an inert atmosphere. Subsequently, triisopropyl borate (229.5 mmol) was added dropwise, followed by the slow addition of n-butyllithium (191.3 mmol) after 15 min. After completion of the reaction, the mixture was quenched with saturated aqueous NH₄Cl and extracted with ethyl acetate (EA). The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure to afford compound 3a.

(4-(N,N-Dimethylaminosulfonyl)phenyl)boric acid ( 3a). White solid, yield 63.3%. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) = 8.37 (s, 2H), 8.01 (d, J = 7.9 Hz, 2H), 7.70 (d, J = 7.9 Hz, 2H), 2.61 (s, 6H).

4-(morpholinesulfonyl)phenylboronic acid ( 3b). White solid, yield 60.3%. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) = 8.45 (s, 2H), 8.04 (d, J = 8.0 Hz, 2H), 7.71 (d, J = 8.0 Hz, 2H), 3.64 - 3.61 (m, 4H), 2.86 - 2.85 (m, 4H).

4-(sulfonylpyrroline)phenylboronic acid ( 3c). White solid, yield 61.9%. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) = 8.42 (s, 2H), 8.00 (d, J = 8.0 Hz, 2H), 7.77 (d, J = 8.0 Hz, 2H), 3.15 - 3.12 (m, 4H), 1.63 - 1.61 (m, 4H).

2.2.4. General procedure for synthesis of 6a-6c

In a 250 mL single-necked flask, 100 mL of a solvent mixture (1,4-dioxane/water = 10:1, v/v) was introduced, followed by the addition of intermediate 5 (0.746 mmol) and intermediate 3a (11.5 mmol), tetrakis(triphenylphosphine)palladium(0), and cesium carbonate (1.492 mmol). To minimize catalyst deactivation, the catalyst was introduced last into the reaction vessel. After complete consumption of the starting materials, the reaction mixture was allowed to cool naturally to room temperature. A suitable amount of water was then added, and the mixture was extracted with methyl tert-butyl ether. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to afford a black oily residue. Purification by column chromatography using a 10:1 mixture of PE and EA as the eluent yielded compound 6a.

N,N-dimethyl-4-(1-octyl-1H-indole-5-yl)benzenesulfonamide ( 6a). White solid, yield 57.2%. ¹H NMR (400 MHz, CDCl₃) δ(ppm) = 7.88 (d, J = 1.7 Hz, 1H), 7.84 - 7.79 (m, 4H), 7.46 (dd, J = 9.3, 7.6 Hz, 2H), 7.16 (d, J = 3.1 Hz, 1H), 6.56 (d, J = 3.1 Hz, 1H), 4.15 (t, J = 7.1 Hz, 2H), 2.76 (s, 6H), 1.90-1.84 (m, 2H), 1.34-1.25 (m, 10H), 0.87 (t, J = 6.8 Hz, 3H).

4-((4-(1-octyl-1H-indole-5-yl)phenyl)sulfonyl)morpholine ( 6b). White solid, yield 55.8%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 7.89 – 7.86 (m, 3H), 7.80 – 7.76 (m, 2H), 7.46 (d, J = 1.8 Hz, 1H), 7.42 (d, J = 7.4 Hz, 1H), 7.15 (d, J = 3.1 Hz, 1H), 6.56 (dd, J = 3.1, 0.7 Hz, 1H), 4.14 (t, J = 7.1 Hz, 2H), 3.31 – 3.28 (m, 4H), 1.87 (q, J = 7.0 Hz, 2H), 1.80 – 1.77 (m, 4H), 1.33 – 1.25 (m, 10H), 0.89 – 0.85 (m, 3H).

1-octyl-5-(4-(pyrrolidine-1-ylsulfonyl)phenyl)-1H-indole ( 6c). White solid, yield 58.3%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 7.88 (d, J = 1.6 Hz, 1H), 7.80 (s, 4H), 7.46 – 7.43 (m, 2H), 7.16 (d, J = 3.1 Hz, 1H), 6.57 (d, J = 3.1 Hz, 1H), 4.15 (t, J = 7.1 Hz, 2H), 3.76 (s, 4H), 3.06 (dd, J = 4.2, 2.4 Hz, 4H), 1.90 – 1.83 (m, 2H), 1.34 – 1.25 (m, 10H), 0.89 – 0.85 (m, 3H).

2.2.5. Synthesis of intermediate 4-((5-(4-(morpholinosulfonyl)phenyl)-1-octyl-1H-indole-3-yl) methyl)piperazine-1-carboxylic acid tert-butyl ester (7j)

Glacial acetic acid (15mL), intermediate 6b (2 g, 3.1 mmol), N-Boc-piperazine (3.61 g, 19.38 mmol) and 37% aqueous formaldehyde solution (1.4 mL, 19.1 mmol) were added to the reaction flask in sequence. Heating to 60°C, TLC monitoring. Quenching with water, EA extraction, anhydrous Na₂SO₄ drying, evaporation of organic layer, and column chromatography (PE: EA = 20:1) to obtain compound 7j as a crystalline white solid. (1.86 g, 64.8% yield). ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 7.94 (d, J = 1.8 Hz, 1H), 7.81 (s, 4H), 7.48 - 7.39 (m, 2H), 7.10 (s, 1H), 4.11 (t, J = 7.2 Hz, 2H), 3.81 - 3.74 (m, 6H), 3.44 (t, J = 5.0 Hz, 4H), 3.07 (dd, J = 5.8, 3.5 Hz, 4H), 2.48 (t, J = 5.0 Hz, 4H), 1.85 (t, J = 6.7 Hz, 2H), 1.44 (s, 9H), 1.34 - 1.25 (m, 10H), 0.87 (t, J = 6.7 Hz, 3H).

2.2.6. General procedure for synthesis of 7a-7k

To a reaction flask were added acetic acid (10 mL), intermediate 6a (1.2 mmol), morpholine (4.8 mmol), and a 37% aqueous formaldehyde solution (4.8 mmol). The reaction mixture was heated to 60°C and monitored by TLC. Upon completion, the reaction was quenched with water and extracted with EA. The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by step gradient chromatography (from PE: EA = 10:1 to EA: MeOH = 50:1) to obtain compound 7a.

N,N-dimethyl-4-(3-(morpholinomethyl)-1-octyl-1H-indole-5-yl)benzenesulfonamide (7a). White powder, yield 64.2%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 7.97 (d, J = 1.8 Hz, 1H), 7.85 - 7.79 (m, 4H), 7.47 (dd, J = 8.5, 1.8 Hz, 1H), 7.40 (d, J = 8.6 Hz, 1H), 7.10 (s, 1H), 4.11 (t, J = 7.1 Hz, 2H), 3.72 (dd, J = 9.2, 4.5 Hz, 6H), 2.76 (s, 4H), 2.52 (d, J = 5.3 Hz, 4H), 1.90 - 1.80 (m, 4H), 1.33 - 1.24 (m, 10H), 0.87 (t, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ (ppm) = 147.15, 136.49, 132.86, 130.48, 128.56, 128.23, 127.63, 121.17, 118.67, 111.22, 110.02, 67.10, 53.92, 53.60, 46.49, 38.04, 31.75, 30.26, 29.18, 29.16, 27.00, 22.61, 14.07. HRMS(ESI-MS) m/z: calcd for C₂₉H₄₁O₃N₃S 511.2869, found 512.41620 [M+H] ⁺. Purity was determined by reverse phase HPLC and found to be 98.95%.

N,N-dimethyl-4-(3-((4-hydroxypiperidine-1-yl)methyl)-1-octyl-1H-indole-5-yl) benzenesulfonamide ( 7b). Brown red solid, yield 61.9%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 11.68 (s, 1H), 7.84 (s, 6H), 7.49 (q, J = 8.2 Hz, 2H), 4.37 (s, 1H), 4.17 (s, 2H), 3.26 (s, 3H), 2.76 (s, 6H), 1.95 (d, J = 63.0 Hz, 10H), 1.29 (d, J = 25.7 Hz, 10H), 0.87 (t, J = 6.2 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ (ppm) = 146.57, 136.15, 133.38, 133.20, 131.88, 128.90, 128.26, 127.93, 121.80, 117.46, 110.80, 102.04, 60.91, 51.65, 47.05, 46.65, 38.06, 31.73, 30.21, 29.85, 29.15, 29.13, 26.97, 22.58, 14.05. HRMS(ESI-MS) m/z: calcd for C₃₀H₄₃O₃N₃S 525.3025, found 526.30829 [M+H] ⁺. Purity was determined by reverse phase HPLC and found to be 98.37%.

4-((4-(1-octyl-3-(thiomorpholinemethyl)-1H-indole-5-yl)phenyl)sulfonyl)morpholine ( 7c). Pale yellow solid, yield 62.7%. ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) = 12.56 (s, 1H), 7.87 - 7.79 (m, 6H), 7.55 - 7.48 (m, 2H), 4.42 (d, J = 4.3 Hz, 2H), 4.19 (t, J = 7.3 Hz, 2H), 3.78 (t, J = 4.8 Hz, 6H), 3.64 (t, J = 13.6 Hz, 2H), 3.08 - 2.97 (m, 6H), 2.62 (d, J = 14.8 Hz, 2H), 1.89 (s, 2H), 1.33 - 1.25 (m, 10H), 0.87 (t, J = 6.7 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ (ppm) = 151.38, 141.21, 138.51, 137.23, 135.54, 133.84,133.43, 132.80, 126.42, 123.50, 116.30, 107.28, 70.53, 57.15, 55.92, 51.16, 51.12, 49.86, 36.35, 34.90, 33.80, 33.75, 31.40, 29.21, 28.69, 27.24, 19.12. HRMS(ESI-MS) m/z: calcd for C₃₁H₄₃O₃N₃S₂ 569.2746, found 570.31573 [M+H] ⁺. Purity was determined by reverse phase HPLC and found to be 98.52%.

4-((4-(3-(morpholinomethyl)-1-octyl-1H-indole-5-yl)phenyl)sulfonyl)morpholine ( 7d). White solid, yield 64.2%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 12.96 (s, 1H), 7.83 (s, 6H), 7.52 (d, J = 6.1 Hz, 2H), 4.41 (s, 2H), 4.22 (d, J = 23.3 Hz, 4H), 3.96 (s, 2H), 3.78 (t, J = 4.6 Hz, 4H), 3.40 (s, 2H), 3.06 (t, J = 4.7 Hz, 4H), 2.94 (s, 2H), 1.89 (s, 2H), 1.26 (s, 10H), 0.88 (q, J = 4.8, 4.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ (ppm) = 146.79, 136.26, 133.58, 133.02, 132.10, 128.81, 128.43, 127.94, 122.07, 117.14, 110.99, 101.06, 66.13, 63.84, 52.01, 50.88, 47.11, 46.08, 31.71, 30.21, 29.12, 26.96, 22.56, 14.02. HRMS(ESI-MS) m/z: calcd for C₃₁H₄₃O₄N₃S 553.7620, found 554.34827 [M+H] ⁺. Purity was determined by reverse phase HPLC and found to be 98.84%.

N-ethy-N-((1-octyl-5-(4-(pyrrolidine-1-yl)sulfonyl)phenyl)-1H-indole-3-yl)methyl) ethylamine ( 7e). White solid, yield 60.7%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 12.13 (s, 1H), 7.96 - 7.89 (m, 3H), 7.83 - 7.77 (m, 3H), 7.54 - 7.46 (m, 2H), 4.40 (d, J = 5.0 Hz, 2H), 4.18 (t, J = 7.3 Hz, 2H), 3.35 - 3.28 (m, 4H), 3.25 - 3.16 (m, 2H), 3.11 - 3.02 (m, 2H), 1.87 (d, J = 7.2 Hz, 2H), 1.83 - 1.77 (m, 4H), 1.47 (t, J = 7.2 Hz, 6H), 1.34 - 1.24 (m, 10H), 0.89 - 0.85 (m, 3H). ¹³C NMR (101 MHz, CDCl₃) δ (ppm) = 146.2, 136.1, 135.1, 133.1, 132.1, 128.6, 128.1, 127.7, 121.9, 116.7, 110.9, 101.9, 47.9, 47.1, 45.4, 31.7, 30.2, 29.7, 29.15, 26.9, 25.3, 22.6, 14.1, 8.9. HRMS(ESI-MS) m/z: calcd for C₃₁H₄₅O₂N₃S 523.3232, found 524.34806 [M+H] ⁺. Purity was determined by reverse phase HPLC and found to be 96.45%.

1-((1-octyl-5-(4-(pyrrolidine-1-ylsulfonyl)phenyl)-1H-indole-3-yl)methyl) piperidine-4-ol ( 7f). Pale yellow solid, yield 64.9%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 11.73 (s, 1H), 7.91 - 7.80 (m, 6H), 7.52 - 7.45 (m, 2H), 4.35 (s, 2H), 4.21 - 4.13 (m, 3H), 3.29 (d, J = 6.4 Hz, 8H), 2.43 (s, 2H), 2.23 (s, 4H), 1.87 (s, 4H), 1.26 (d, J = 5.2 Hz, 10H), 0.87 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ (ppm) = 146.40, 146.29, 136.21, 134.67, 133.27, 131.91, 128.72, 127.87, 121.77, 117.39, 110.75, 102.05, 60.91, 51.52, 48.02, 46.99, 46.54, 31.90, 30.18, 29.78, 29.12, 26.94, 25.24, 22.56, 20.55, 14.03. HRMS(ESI-MS) m/z: calcd for C₃₂H₄₅O₃N₃S 551.3182, found 552.32452 [M+H] ⁺. Purity was determined by reverse phase HPLC and found to be 97.00%.

4-((1-octyl-5-(4-(pyrrolidine-1-ylsulfonyl)phenyl)-1H-indole-3-yl)methyl)morpholine ( 7g). Pink solid, yield 60.7%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 12.88 (s, 1H), 7.93 - 7.78 (m, 6H), 7.55 - 7.47 (m, 2H), 4.43 (s, 2H), 4.21 (d, J = 23.7 Hz, 4H), 3.94 (d, J = 11.6 Hz, 2H), 3.43 - 3.26 (m, 6H), 2.97 (s, 2H), 1.80 (s, 6H), 1.34 - 1.25 (m, 10H), 0.89 - 0.85 (m, 3H). ¹³C NMR (101 MHz, CDCl₃) δ (ppm) = 146.27, 136.15, 134.78, 133.47, 132.12, 128.89, 128.03, 127.80, 121.95, 117.19, 110.86, 101.17, 36.81, 51.72, 50.67, 48.02, 47.04, 31.70, 30.19, 29.65, 29.11, 26.94, 25.24, 22.56, 14.02. HRMS(ESI-MS) m/z: calcd for C₃₁H₄₃O₃N₃S 537.3025, found 538.30841 [M+H] ⁺. Purity was determined by reverse phase HPLC and found to be 97.15%.

2-(4-((1-octyl-5-(4-(pyrrolidine-1-ylsulfonyl)phenyl)-1H-indol-3-yl)methyl) piperazine-1-yl)ethane-1-ol ( 7h). Pale yellow solid, yield 64.8%. ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 13.08 (s, 1H), 8.07 (s, 1H), 7.87 (s, 4H), 7.72 (s, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.46 (d, J = 8.5 Hz, 1H), 4.53 (s, 2H), 4.17 (s, 2H), 4.01 (s, 4H), 3.81 (s, 2H), 3.61 (s, 2H), 3.28 (s, 4H), 1.82 (d, J = 36.4 Hz, 11H), 1.27 (s, 10H), 0.88 (d, J = 3.8 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ (ppm) = 146.11, 136.24, 134.24, 133.42, 121.69, 128.43, 127.96, 127.89, 121.81, 118.62, 110.64, 101.25, 55.77, 47.99, 46.99, 31.93, 31.76, 30.25, 29.70, 29.36, 29.18, 29.16, 26.95, 25.16, 22.69, 22.60, 14.12, 14.08. HRMS(ESI-MS) m/z: calcd for C₃₃H₄₈O₃N₄S 580.3447, found 581.35065 [M+H] ⁺. Purity was determined by reverse phase HPLC and found to be 96.95%.

4-((1-octyl-5-(4-(pyrrolidine-1-ylsulfonyl)phenyl)-1H-indole-3-yl)methyl)piperazine-1-carboxylic acid tert-butyl ester ( 7i). White solid, yield 61.4%. ¹H NMR (400 MHz, Chloroform-d) δ (ppm) = 7.94 (d, J = 1.8 Hz, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.81 - 7.76 (m, 2H), 7.47 (dd, J = 8.6, 1.8 Hz, 1H), 7.40 (d, J = 8.5 Hz, 1H), 7.10 (s, 1H), 4.11 (t, J = 7.2 Hz, 2H), 3.76 (s, 2H), 3.44 (t, J = 5.0 Hz, 4H), 3.33 - 3.28 (m, 4H), 2.48 (t, J = 5.0 Hz, 4H), 1.84 (d, J = 7.1 Hz, 2H), 1.82 - 1.78 (m, 4H), 1.44 (s, 9H), 1.33 - 1.25 (m, 10H), 0.89 - 0.85 (m, 3H). ¹³C NMR (101 MHz, CDCl₃) δ (ppm) = 154.81, 146.95, 136.46, 134.40, 130.57, 128.97, 128.52, 128.00, 127.60, 121.18, 118.60, 111.33, 110.01, 79.56, 53.56, 52.86, 47.97, 46.49, 31.75, 30.26, 29.70, 29.18, 29.15, 28.44, 27.00, 25.26, 22.60, 14.07. HRMS(ESI-MS) m/z: calcd for C₃₆H₅₂O₄N₄S 636.3709, found 637.37616 [M+H] ⁺. Purity was determined by reverse phase HPLC and found to be 97.81%.

2.2.7. Synthesis of 4-((4-(1-octyl-3-(piperazine-1-ylmethyl)-1H-indole-5-yl) phenyl) sulfonyl) morpholine (7k)

Methanol (30 mL), intermediate 7j (0.5 g, 0.8 mmol) and concentrated hydrochloric acid (0.7 mL, 8.0 mmol) were sequentially added to the reaction flask. TLC monitoring. Quenching with water, EA extraction, concentration and purification to obtain the hydrochloride of compound 7k as a crystalline solid (0.36 g, 85.1% yield). ¹H NMR (400 MHz, CDCl₃) δ (ppm) = 7.88 (d, J = 1.7 Hz, 1H), 7.79 (s, 4H), 7.51 - 7.48 (m, 1H), 7.44 (d, J = 8.6 Hz, 1H), 6.94 (s, 1H), 4.37 (d, J = 20.2 Hz, 2H), 4.10 (t, J = 7.1 Hz, 3H), 3.78 (t, J = 4.7 Hz, 7H), 3.06 (dq, J = 7.7, 4.5, 3.6 Hz, 6H), 1.84 (t, J = 7.1 Hz, 2H), 1.68 (d, J = 20.3 Hz, 2H), 1.28 (s, 10H), 0.88 (d, J = 3.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ (ppm) = 147.57, 136.68, 132.28, 130.40, 129.88, 128.33, 127.69, 127.25, 121.04, 118.43, 114.69, 110.09, 77.41, 77.09, 76.78, 66.19, 46.46, 46.09, 31.76, 30.40, 29.17, 27.00, 22.61, 20.93, 15.12, 14.44, 14.09. HRMS(ESI-MS) m/z: calcd for C₃₁H₄₂O₃N₄S 552.3134, found 551.28711 [M+H] ⁺. Purity was determined by reverse phase HPLC and found to be 97.25%.

2.3. X-ray crystal structure determination

The crystal of compound 7a can be obtained by dissolving compound 7a in acetonitrile and slowly volatilizing acetonitrile at room temperature. Choose a transparent crystal whose length, width, and height are all 0.20 mm. It is randomly installed on a glass fiber to collect data. Single-crystal XRD data were collected at 296(2) K on a Bruker APEX II diffractometer equipped with a graphite-monochromated Mo-Kα radiation source (λ = 0.71073 Å), utilizing φ and ω scan modes. Diffraction data were collected by the full matrix least square method, and then these data were enriched by the program SHEXL-2018/3 [8], and the structure of the final compound was directly determined by the program SHELX-2018/3 [9]. The crystal structure of the compound 7a is kept in the Cambridge Crystal Data Center (CCDC: 2350474).

2.4. DFT calculation

In this study, the software package Gaussian 16 [10] and B3LYP/6-311+G (2d, p) [11] are used for DFT calculation, and DFT calculations were performed on the ground-state structures in the gas phase. According to the optimized structure, we used the program of GuassView 6.0 to obtain the geometric, electronic, and energy parameters of the compound 7a [12].

2.5. Antitumor activity assay

This study utilized the human breast cancer cell line MCF-7 and the human acute promyelocytic leukemia cell line HL-60, with methotrexate (MTX), 5-fluorouracil (5-FU), and cymethynil serving as positive control drugs. The in vitro inhibitory rates of the target compounds 7a-7k were determined using the CCK-8 assay. The experimental procedure was conducted as follows: Upon reaching the logarithmic growth phase, cells were uniformly seeded into 96-well plates and pre-incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 12 h. Subsequently, test compounds were diluted to concentrations of 50 μM and 100 μM, and 100 μL of the respective drug solution was added to each well (the blank control and vehicle control groups received an equivalent volume of culture medium only). Each drug concentration was tested in quintuplicate (n = 5). The plates were then returned to the incubator (37°C, 5% CO₂) for a 72-h treatment period. Following the initial incubation period, Cell Counting Kit-8 (CCK-8) solution (10 μL per well) was introduced to the culture plates under low-light conditions. After an additional 1.5-h incubation, absorbance measurements were conducted at 450 nm using a microplate reader. For data analysis, the highest and lowest optical density (OD) values within each treatment group were excluded. The inhibitory rate for each compound was calculated based on the remaining three intermediate OD values, and the mean in vitro inhibitory rate for each compound was then derived.

3. Results and Discussion

3.1. Synthesis and characterization

Structural confirmation of compound 7a, synthesized as outlined in Scheme 1, was achieved utilizing ¹H NMR, ¹³C NMR, FT-IR spectroscopy, and HRMS.

3.2. Crystallographic analysis

To elucidate the structural features of compound 7a, single-crystal XRD analysis was performed. The compound crystallizes in the monoclinic space group P2₁/c, and its unit cell size is: a = 11.9343(14) Å, b = 8.1882(10) Å, c = 28.772(4) Å, V = 2784.2(6) Å ³, Z = 4, density (calculated) = 1.221 mg m^-3. The molecular structure of compound 7a, depicted in the ORTEP diagram (Figure 1), crystallizes in the monoclinic space group P2₁/c. Corresponding crystallographic refinement data have been presented in Table 1.

Crystal and DFT-optimized structures of compound 7a.

Table 1. Crystal data and parameters for structure refinement of compound 7a.

Compound	7a
CCDC	2350474
Empirical formula	C₂₉H₄₁N₃O₃S
Formula weight	511.71
Temperature/K	296(2)
Crystal system	monoclinic
Space group	P 21/c
a/Å	11.9343(14)
b/Å	8.1882(10)
c/Å	28.772(4)
α/°	90
β/°	98.001(3)
γ/°	90
Volume/Å³	2784.2(6)
Z	4
ρ_calcg/cm³	1.221
μ/mm^-1	0.150
F(000)	1104
Crystal size/mm³	0.20× 0.20× 0.20
Radiation λ (Å)	MoKα (λ = 0.71073)
2θ range for data collection/°	2.080 ° to 24.998 °
Index ranges	-14 ≤ h ≤ 14, –9 ≤ k ≤9, –34≤ l ≤33
Reflections collected	58836
Independent reflections	4895 [R_int = 0.1274, R_sigma = 0.0858]
Data/restraints/parameters	4895/255/391
Goodness-of-fit on F²	1.016
Final R indexes [I>=2s (I)]	R₁ = 0.0989, wR₂ = 0.2384

Bond lengths, angles, and torsion angles for compound 7a Supplementary material (Table S1) show good agreement with the DFT-optimized structure, both falling within expected ranges. It is normal that there is a slight difference between these two values. This difference may be due to the interactions between single crystals, while the DFT optimized structure only calculates one molecule, which may be the reason for the difference between them.

Supplementary material

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Molecular stacking and intramolecular hydrogen bonding in the crystal structure of compound 7a have been depicted in Figure 2. Examination of Figure 2(b) specifically shows the presence of three intermolecular C-H⋯π interactions. They are C(1)-H(1A)…Cg(3) (2.939 Å), C(19)-H(19A)…Cg(1)(2.958 Å) and C(24A)-H(24B)…Cg(4) (2.999Å). Cg(3), Cg(1) and Cg(4) are the ring centers of C3-C4-C5-C6-C7-C8, N3-C12-C13-C15-C16 and C9-C10-C11-C12-C13-C14, respectively. As illustrated in Figure 2(c), compound 7a exhibits two intramolecular hydrogen bonding interactions: C(1)-H(1B)···O(1) (2.424) and C(2)-H(2B)···O(2) (2.456 Å). Comprehensive hydrogen bond parameters are provided in Table 2. These two hydrogen bond interactions contribute to the stability of compound 7a. The synergistic effect of these interactions critically defines the macroscopic crystal properties. The network of C-H⋯π interactions significantly enhances the three-dimensional structural cohesion and packing density, thereby increasing the lattice energy and thermal stability. Concurrently, the intramolecular C-H⋯O hydrogen bonds rigidify the molecular conformation, reducing internal flexibility and pre-organizing the molecule for efficient packing. This combination of inter- and intra-molecular forces collectively results in a stable crystal lattice with potential implications for its mechanical behavior, dissolution rate, and overall physicochemical stability.

(a) Crystal structure stacking diagram of compound 7a; (b) C-H...π diagram; (c) Intramolecular hydrogen bonds diagram.

Table 2. Hydrogen bonds for compound 7a (Å, °)

D—H…A	d(D–H))	d(H···A)	d(D···A)	∠DHA
C(1)-H(1B)…O(1)	0.96	2.42	2.871(10)	108
C(2)-H(2B)…O(2)	0.96	2.46	2.893(10)	107

3.3. Conformational stability

A molecule often has multiple conformations due to the rotation of some bonds, and different conformations have different contributions to the structural stability of compounds. Molecular conformation directly impacts the stability and physicochemical properties of compounds, reflecting the fundamental principle that structure dictates functionality [13]. Therefore, it is necessary to calculate the conformational analysis of compound 7a to understand its physical and chemical properties [14]. Consequently, we used Spartan 08 [15] program and MMFF [16] molecular mechanical force field to search and analyze the initial conformation of compound 7a. Using Gaussian 16, we performed DFT/B3LYP/6-311+G(2d,p) [17] geometry optimizations and frequency calculations on all identifiable conformers of 7a. The relative Gibbs free energy (ΔG) of each conformer governs its equilibrium abundance. Table 3 quantifies these populations via Boltzmann statistics (Pi, %), listing ΔG values and employing R = 8.314 J·mol⁻¹·K⁻¹ [18]. These conformational analysis results provide a molecular-level foundation for interpreting the crystal structure of 7a. The predominant population of a specific low-energy conformer suggests that the molecule crystallizes in this particular geometry, which in turn pre-organizes the molecular structure and dictates the specific set of intermolecular interactions (such as hydrogen bonds, van der Waals contacts) that are sterically and electrostatically feasible.

Table 3. Gibbs free energy (G), relative Gibbs free energy (ΔG) ^a, and Boltzmann weighting factor (Pi%) ^b of the conformers of compound 7a.

Conformer	G (kcal mol^-1)	△G (kcal mol^-1)	Pi%
1-1	-1203342.7430	0	86.49
1-2	-1203341.5200	1.2230	10.73
1-3	-1203340.7230	2.0199	2.76
1-4	-1203337.8510	4.8927	0.02

^a Related to the most stable conformer.

^b Boltzmann weighting factor (Pi%) based on △G.

Compound 7a has four relatively stable conformations, which are shown in Figure 3, namely 1-1 (86.49%), 1-2 (10.73%), 1-3 (2.76%), and 1-4 (0.02%). By analyzing these conformations, it can be seen that the conformations of compound 7a are slightly different. Rotational freedom about two critical bonds, the indole N-to-methylene C bond and the methylene C-to-morpholine N bond, accounts for the subtle conformational differences observed in 7a.

Relatively stable conformers of compound 7a.

3.4. Molecular electrostatic potential

Molecular electrostatic potential (MEP) maps molecular interaction landscapes, providing critical insights for predicting reaction sites, activity patterns, and feasible reaction pathways [19][20]. The MEP diagram of a molecule can reflect the overall shape of a molecule, the potential distribution of the molecule, and the potential area occupied by different potentials in the molecule [21]. Figure 4 depicts the MEP map of compound 7a, calculated at the B3LYP/6-311+G(2d,p) level. The color codes of these maps range from –6.0 e ⁻² and + 6.0 e ⁻². Red, the color code near –6.0 e ⁻² represents the negative potential, that is, the area where the electron cloud density is concentrated, which is related to the electrophilic reaction. Blue represents a positive potential, that is, an area with relatively low electron cloud density, which is related to the nucleophilic reaction of molecules [22]. The N atom and the methylene group connected to the indole ring are mainly positively charged regions, which have strong electron absorption ability. According to Figure 4, in this study, the negatively charged region of compound 7a is mainly distributed around two O atoms, namely the O atom of the sulfonyl group and the O atom of the morpholine ring, which can donate electrons and may cause nucleophilic reaction. The pronounced electrostatic complementarity revealed by the MEP map is crucial for the crystal engineering of compound 7a. It directly facilitates the formation of specific and directional intermolecular interactions, such as C–H⋯O hydrogen bonds, which are critical for constructing a stable supramolecular architecture with enhanced lattice energy and predictable solid-state properties.

Molecular electrostatic potential diagram of the conformer 1-4.

3.5. Frontier molecular orbitals

The different energy levels of molecular orbitals are determined by the electron clouds around the molecules. Electron occupancy distinguishes the highest occupied molecular orbital (HOMO) from the lowest unoccupied molecular orbital (LUMO) in these frontier orbitals [23]. The electrophilic reaction and nucleophilic reaction are closely related to HOMO and LUMO. Therefore, the atom possessing the maximal electron density in the HOMO is predicted to be the primary site for electrophilic attack, whereas nucleophilic species are anticipated to react at the atom with the highest electron density in the LUMO. FMOs are widely used, such as analyzing the physical and chemical properties of dye-sensitized solar cells [24], nonlinear optical materials [25] and organic solar cells [26]. DFT-derived HOMO/LUMO energies and orbital energy gaps provided insights into the conformational features of the target compounds. The HOMO and LUMO values of compound 7a are shown in Figure 5, and the energy values of its four conformational isomers are -5.7549 eV and -1.4144 eV, respectively, and the energy difference between them is 4.3405 eV. The frontier molecular orbital (FMO) energy gap is considered as an important factor to determine hardness. The hardness parameter η measures molecular stability against electron redistribution, characterizing both the difficulty of electron transfer and resistance to orbital distortion. This metric is calculated through frontier orbital energies: η = (ε_LUMO-ε_HOMO )/2 [27]. The hardness of compound 7a is η=2.17025 eV. The relatively large HOMO-LUMO energy gap (4.3405 eV) and consequent high hardness value (η = 2.17025 eV) for compound 7a indicate significant electronic rigidity and high kinetic stability. This implies that the crystalline solid possesses low chemical reactivity and high stability against external perturbations, which are desirable properties for its potential application as a solid-state material.

3.6. Vibrational analysis

In the research, researchers can identify compounds by absorption peaks and wavelengths characterized by the IR spectrum, and study the structure of molecules. At the same time, the IR spectrum can also characterize compounds [28]. Experimental and DFT-calculated infrared spectra of compound 7a are presented in Figure 6, with the theoretical spectrum computed at B3LYP/6-311+G(2d,p) using a wavenumber scaling coefficient of 0.9618 [29]. Figure 6 displays compound 7a’s experimental (red curve) and computed (blue curve) infrared spectra. Corresponding vibrational frequencies, including experimental measurements and scaled DFT values with mode assignments, have been compiled in Table S2.

Theoretical and experimental IR spectra of compound 7a.

3.6.1. -CH₂- vibration (alkane)

The range of -CH₂- stretching vibration of alkanes is 2926-2850 cm^-1. By analyzing the infrared spectrum, we can know that -CH₂- alkane has telescopic vibration at 2923.29 cm^-1, and the calculated telescopic vibration value ranges from 2977.32 cm^-1 to 2828.10 cm^-1, which belongs to the normal range. The bending vibration range of -CH₂- (alkane) is from 1485 cm^-1 to 1440 cm^-1. We can also know that -CH₂- alkane has bending vibration at 1479 cm^-1, and the calculated bending vibration value was 1459.22-1417.96 cm^-1_.

3.6.2. C-N vibration

The C-N telescopic vibration of alkanes belongs to the range of 1300-1000 cm^-1. Experimental IR data indicate a C-N stretching vibration at 1111.9 cm⁻¹, while computed values range from 1080.45 to 1111.68 cm⁻¹.

3.6.3. C=C vibration

The C=C (aromatic ring) telescopic vibration is 1675-1500 cm^-1. We can clearly see from the infrared spectrum that the C=C telescopic vibration of compound 7a is obvious at 1588.99 cm^-1. The corrected theoretical values are 1546.50 cm^-1, 1568.88 cm^-1and 1587.90 cm^-1, respectively.

3.6.4. C-O-C vibration

The telescopic vibration of C-O-C is 1150-1050 cm^-1. Compound 7a exhibits an experimental C-O-C stretch at 1160.82 cm⁻¹, while scaled computational values for this vibration are calculated at 1086.69 and 1088.01 cm⁻¹.

3.6.5. C-S vibration

The telescopic vibration of C-S is 600-200 cm^-1. It can be clearly observed from the infrared spectrum that the C-S telescopic vibration of compound 7a is 549.02 cm^-1. The corrected theoretical value is 545.75 cm^-1, which belongs to the normal range.

3.6.6. Ar-H vibration

Aromatic systems exhibit both in-plane and out-of-plane C-H rocking vibrations. Generally, these vibrations can be observed in the range of 1250-950 cm^-1 and 900-650 cm^-1 [30]. The in-plane C-H deformation mode of compound 7a appears at 945.48 cm⁻¹ experimentally, with scaled theoretical counterparts at 912.33 cm⁻¹. Out-of-plane bending vibrations observed at 800.61 cm⁻¹ correspond to scaled DFT frequencies of 783.08, 822.58, and 823.78 cm⁻¹.

3.7. Hirshfeld surface analysis

Hirshfeld surface analysis provides information about intermolecular contacts and is usually a common method for analyzing crystals [31]. We performed Hirshfeld surface and 2D fingerprint analyses on compound 7a using Crystal Explorer 21.5 to examine intermolecular interactions. As shown in Figure 7a, the dnorm Hirshfeld curvature of compound 7a is represented by a red-blue-white color scheme. Analysis of the 2D fingerprint plots (Figure 7b-f) reveals that H⋯H (67.8%), C⋯H (9.0%), and O⋯H (7.9%) interactions predominantly facilitate crystal packing in compound 7a. These non-covalent contacts, particularly hydrogen bonding (O⋯H/H⋯O = 13.8% combined), critically stabilize the molecular arrangement as evidenced in Figure 7. The stability of the crystal is secured by extensive van der Waals forces, primarily through H⋯H contacts, with directional O⋯H/H⋯O hydrogen bonds providing critical rigidity, thereby enhancing the overall structural cohesion.

(a) Hirshfeld surface analysis mapped with dnorm of compound 7a. (b-f) Resolution of 2D fingerprint plots into specific interactions. Both in units of Ångströms (Å): (b) H⋯O, (c) O⋯H, (d) H⋯H, (e) H⋯C, (f) C⋯H contacts.

3.8. ¹H NMR and ¹³C NMR spectral analysis

Theoretical ¹H and ¹³C NMR spectra were computed at the B3LYP/6-311+G(2d,p) level to elucidate compound 7a’s structure. Chemical shift predictions employed the gauge independent atomic orbital (GIAO) approach, with computational results benchmarked against experimental data [32]. As shown in Figure 8, the linear correlation between the DFT/B3LYP-calculated values and experimental values was strong, confirming the reliability of the theoretical model in structural analysis. The chemical shift ranges vary significantly depending on the hybridization state of carbon atoms: sp³-hybridized carbons typically appear at 20–200 ppm (such as saturated carbons or those bonded to heteroatoms), sp²-hybridized carbons at 120–240 ppm (such as alkenes, aromatic carbons, or carbonyls), and sp-hybridized carbons at 30–70 ppm (such as alkynes or nitriles) [33]. Additionally, conjugation effects can further downfield-shift the sp² carbon resonances. For Compound 7a, all carbon and heteroatom (N, O, S) consistency between calculated and experimental ¹H/¹³C NMR data (Figure 8) validated the proposed structure.

(a) Correlation between experimental and calculated 1H NMR chemical shifts for compound 7a. (b) Correlation between experimental and calculated 13C NMR chemical shifts for compound 7a.

3.9. Molecular docking analysis

Molecular docking is a computational simulation technique used to predict intermolecular interactions, primarily applied in drug design and the study of protein-ligand binding mechanisms [34]. Its primary objective is to predict the binding mode, affinity, and stability between small molecules (such as drug candidates) and biomacromolecules (such as proteins).

In this study, compound 7a was docked into the binding site of the target protein (PDB: 4A2N) to predict its binding conformation. Figure 9(a) and (b) illustrate the 2D and 3D binding modes, respectively, of compound 7a with the key active-site residues of 4A2N. Additionally, the binding energy, interaction type, and bond length for compound 7a with the target protein have been listed in Table S3.

(a) 2D Binding pose of compound 7a with active site residues of target protein (4A2N). (b) 3D Binding pose: compound 7a at the active site of the target protein.

The four distinct molecular interaction profiles between compound 7a and protein 4A2N have been systematically tabulated in Table S3. In terms of hydrogen bonding, C-12 formed a hydrogen bond with HIS126(ND1) at 3.396 Å with a binding energy of -1.1 kcal mol^-1, where the slightly elongated distance (beyond the optimal 2.5-3.0 Å range) suggested a weaker interaction. A parallel hydrogen bond was observed between O-18 and HIS126(N) with an identical distance (3.396 Å) but a lower binding affinity (-0.84 kcal mol^-1). Significantly, O-194 established the most robust hydrogen bond with TYR179(OH), exhibiting an optimal distance of 2.60 Å and a substantial binding energy of -2.78 kcal mol^-1. Additionally, a π-hydrogen interaction occurred between the 5-ring and ALA105(CA) with a distance of 4.234 Å and a binding energy of -0.84 kcal mol^-1.

Comprehensive analysis revealed that the binding of compound 7a to 4A2N was predominantly mediated by a hydrogen-bond network, with the strong hydrogen bond interaction with tyrosine 179 (TYR179) (-2.78 kcal mol^-1) playing a pivotal role. The histidine 126 (HIS126) residue participated through two distinct hydrogen bonds, implying its potential as a crucial pharmacophoric element. The cumulative interaction energy reached approximately -5.56 kcal mol^-1, indicating a moderate binding affinity that was characteristic of reversible molecular recognition events. This interaction profile provided valuable insights for subsequent structure-activity relationship optimization.

3.10. In vitro antitumor activity analysis

Cellular proliferation, assessed at the cellular level, plays a pivotal role in biomedical research and serves as a key indicator for evaluating the inhibitory effects of pharmacological agents on cancer cells. This study employed the CCK-8 assay to investigate the anti-proliferative activity of compounds 7a-7k against MCF-7 cells, laying the groundwork for further exploration of their potential anti-tumor properties. Methotrexate (MTX), 5-FU, and cymethynil were utilized as positive control drugs. It is noteworthy that cysmethynil (2-(1-octyl-5-m-tolyl-1H-indol-3-yl)acetamide), a well-characterized isoprenylcysteine carboxyl methyltransferase (ICMT) inhibitor[35], possesses a structural scaffold notably similar to that of the compounds 7a-7k synthesized in this study.

Table 4 summarizes the inhibitory rates of compounds 7a-7k against both MCF-7 and HL-60 cells at concentrations of 50 μM and 100 μM. At 50 μM, compound 7a, along with the positive controls MTX and 5-FU, exhibited inhibition rates of 13.02%, 79.39%, and 63.42%, respectively, against the human breast cancer cell line MCF-7 (Michigan Cancer Foundation-7). Corresponding inhibition rates against the human promyelocytic leukemia cell line HL-60 were 27.86%, 84.27%, and 81.91%. When the concentration was increased to 100 μM, the inhibitory activities of 7a, MTX, and 5-FU against MCF-7 cells reached 34.16%, 63.64%, and 84.51%, respectively, while inhibition rates against HL-60 cells were 78.85%, 86.30%, and 85.18%. Although compound 7a demonstrated only moderate anti-proliferative potency compared to MTX, its dose-dependent inhibition profile suggests potential anti-tumor characteristics meriting further investigation.

Table 4. Inhibitory rates of compound 7a-7k against MCF-7 and HL-60 cells in vitro at concentrations of 50 μM and 100 μM.

Compound	MCF-7		HL-60
Compound	50 μM	100 μM	50 μM	100 μM
7a	13.02	34.16	27.86	78.85
7b	79.30	90.53	98.12	88.35
7c	14.28	11.46	4.08	52.51
7d	89.38	90.02	3.97	95.47
7e	99.68	99.66	98.35	98.13
7f	99.26	96.45	94.49	90.94
7g	91.28	90.80	97.08	93.30
7h	99.96	98.34	98.70	92.02
7i	24.59	26.67	28.72	19.68
7k	22.68	25.17	13.79	15.26
Cymethynil	99.46	99.83	98.11	99.35
MTX	63.42	63.64	84.27	86.30
5- FU	79.39	84.51	81.91	85.18

Note: MTX represents methotrexate; 5-FU represents 5-fluorouracil.

The collective analysis of the screening data for the 7a-7k compound series led to the successful identification of several promising candidates, most notably 7e, 7h, and 7g. These compounds exhibited potent and broad-spectrum antiproliferative activity against both MCF-7 and HL-60 cancer cell lines. Remarkably, their efficacy surpassed that of the conventional clinical drugs 5-FU and MTX under most tested conditions and was comparable to that of the reference ICMT inhibitor cysmethynil. Comparative structural analysis of these three compounds with the other seven revealed that compounds 7e, 7g, and 7h uniquely feature a pyrrolidine ring as the amine component in their sulfonamide linkage. This distinct structural feature is likely a key structural determinant responsible for their superior inhibitory potency in vitro. These findings suggest that compounds 7e, 7h, and 7g represent promising candidates for development as potential small-molecule therapeutics in oncology.

4. Conclusions

In this study, a series of ten indole derivatives was designed and synthesized. Single crystals of a representative compound, 7a, were successfully cultivated via slow evaporation of acetonitrile, enabling precise structural elucidation. The molecular structure was further investigated using DFT calculations at the B3LYP/6-311+G(2d,p) level and Hirshfeld surface analysis. The excellent agreement between the computational results and the experimental crystallographic data confidently confirmed the solid-state structure. Subsequent evaluation of the in vitro anti-tumor activity of compounds 7a-7k identified three lead molecules, 7e, 7h, and 7g, which exhibited potent and broad-spectrum anti-proliferative efficacy against both MCF-7and HL-60 cell lines. Notably, the potent activity of the structural analog cysmethynil, an established ICMT inhibitor, provides a compelling benchmark for the 7a-7k series. Given their promising efficacy, compounds 7e, 7h, and 7g emerge as compelling small-molecule candidates worthy of further investigation. Collectively, this work provides a valuable framework for the development of novel indole-based broad-spectrum anticancer agents.

Acknowledgments

We want to thank the National Natural Science Foundation of China (82460682), the Science and Technology Fund of Guizhou Provincial Health Department (gzwjkj2020-1-238) and the Guizhou Province High level Innovative Talents Project - Thousand level Talents (gzwjrs2024-004) for financial support.

CRediT authorship contribution statement

Wanxia Zhang: Writing - original draft, Data curation, Methodology, Writing - review & editing. Xiaocui Tai: Writing - review & editing. Xiangxiang Liu: Writing - review & editing. Xiaoxiao Wang: Writing - review & editing. Yunyun Huang: Writing - review & editing. Chunshen Zhao: Writing - review & editing. Zhixu Zhou: Supervision, Software, Writing - review & editing. Zhaopeng Zheng: Supervision, Software, Writing - review & editing.

Declaration of competing interest

There are no conflicts of interest.

Declaration of generative AI and AI-assisted technologies in the writing process

The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

Supplementary data

Supplementary material to this article can be found online at https://dx.doi.org/10.25259/AJC_820_2025.

References

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Show Sections

[1] Liu Y., Avathan Veettil A.K., Gasper R., Jiang M., Wagner L., Hastürk O., Wu P.. Harnessing indole scaffolds to identify small-molecule IRE1α inhibitors modulating XBP1 mRNA splicing. Nature communications. 2025;16:8531. https://doi.org/10.1038/s41467-025-64291-4
[Google Scholar]

[2] Tarawneh A.H., Al-Trawneh S.A., Yesiloglu T.Z., Zessin M., Robaa D., Barinka C., Schutkowski M., Sippl W., Ross S.A.. Novel selective indole based histone deacetylase 10 inhibitors as anticancer therapeutics. Scientific Reports. 2025;15:33307. https://doi.org/10.1038/s41598-025-02774-6
[Google Scholar]

[3] Feng R., Zhen Y.Q., Wu D., Sun L., Xu J.B., Li X., Zhang L., Gao F.. Late-stage modification of complex drug: Base-controlled Pd-catalyzed regioselective synthesis and bioactivity of arylated osimertinibs. Science Advances. 2024;10:eadl0026. https://doi.org/10.1126/sciadv.adl0026
[Google Scholar]

[4] Bibi A., Ullah M.S., Mahmood A., Shahzad M., Javaid M.M., Nadeem M.A., Altihani F.A., Hashem M., Qari S.H.. Indole-3-acetic acid improves growth, physiology, photosynthesis, and ion balance under cadmium stress in Sorghum bicolor. Scientific Reports. 2025;15:33971. https://doi.org/10.1038/s41598-025-10900-7
[Google Scholar]

[5] Bukhamsin A.H., Shetty S.S., Fakeih E., Martinez M.S., Lerma C., Mundummal M., Wang J.Y., Kosel J., Al-Babili S., Blilou I., Salama K.N.. In vivo dynamics of indole- and phenol-derived plant hormones: Long-term, continuous, and minimally invasive phytohormone sensor. Science Advances. 2025;11:eads8733. https://doi.org/10.1126/sciadv.ads8733
[Google Scholar]

[6] McDonald A., Nakamura Y., Schotte C., Titchiner G., Lau K., Alam R., Lopes A.A., Buell C.R., O’Connor S.E.. Enzymatic epimerization of monoterpene indole alkaloids in kratom. Nature Chemical Biology 2025:s41589-s41025. https://doi.org/10.1038/s41589-025-01970-9
[Google Scholar]

[7] Wang J., Pan D., Wang F., Yu S., Huang G., Li X.. Pd-catalyzed asymmetric Larock reaction for the atroposelective synthesis of N─N chiral indoles. Science Advances. 2024;10:eado4489. https://doi.org/10.1126/sciadv.ado4489
[Google Scholar]

[8] Sheldrick G.M.. SHELXT–Integrated space-group and crystal-structure determination. Acta Crystallographica Section A: Foundations and Advances. 2015;71:3-8. https://doi.org/10.1107/S2053273314026370
[Google Scholar]

[9] Sheldrick G.M.. Crystal structure refinement with SHELXL. Acta Crystallographica. Section C, Structural Chemistry. 2015;71:3-8. https://doi.org/10.1107/S2053229614024218
[Google Scholar]

[10] Gaussian 16, Revision B.01Frisch M. J., Trucks G. W., Schlegel H. B., Scuseria G. E., Robb M. A., Cheeseman J. R., Scalmani G., Barone V., Petersson G. A., Nakatsuji H., Li X., Caricato M., Marenich A. V., Bloino J., Janesko B. G., Gomperts R., Mennucci B., Hratchian H. P., Ortiz J. V., Izmaylov A. F., Sonnenberg J. L., Williams-Young D., Ding F., Lipparini F., Egidi F., Goings J., Peng B., Petrone A., Henderson T., Ranasinghe D., Zakrzewski V. G., Gao J., Rega N., Zheng G., Liang W., Hada M., Ehara M., Toyota K., Fukuda R., Hasegawa J., Ishida M., Nakajima T., Honda Y., Kitao O., Nakai H., Vreven T., Throssell K., Montgomery J. A. Jr, ., Peralta J. E., Ogliaro F., Bearpark M. J., Heyd J. J., Brothers E. N., Kudin K. N., Staroverov V. N., Keith T. A., Kobayashi R., Normand J., Raghavachari K., Rendell A. P., Burant J. C., Iyengar S. S., Tomasi J., Cossi M., Millam J. M., Klene M., Adamo C., Cammi R., Ochterski J. W., Martin R. L., Morokuma K., Farkas O., Foresman J. B., Fox D. J.. Wallingford CT: Gaussian, Inc.; 2016.

[11] Becke A.D.. Density-functional thermochemistry. III. The role of exact exchange. The Journal of Chemical Physics. 1993;98:5648-5652. https://doi.org/10.1063/1.464913
[Google Scholar]

[12] Panicker C.Y., Ambujakshan K.R., Varghese H.T., Mathew S., Ganguli S., Nanda A.K., Alsenoy C.V.. FT‐IR, FT‐Raman and DFT calculations of 3‐{[(4‐fluorophenyl)methylene]amino}‐2‐phenylquinazolin‐4(3H)‐one. Journal of Raman Spectroscopy. 2009;40:527-536. https://doi.org/10.1002/jrs.2159
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[35] Go M.L., Leow J.L., Gorla S.K., Schüller A.P., Wang M., Casey P.J.. Amino derivatives of indole as potent inhibitors of isoprenylcysteine carboxyl methyltransferase. Journal of Medicinal Chemistry. 2010;53:6838-6850. https://doi.org/10.1021/jm1002843
[Google Scholar]

Crystal structure, DFT, vibrational properties, Hirshfeld surface and antitumor activity studies of 1-Octyl-1H-Indole derivatives

Abstract

Keywords

Antitumor activity

DFT

Indole derivatives

Molecular docking

Surface analysis

1. Introduction

2. Materials and Methods

2.1. General remarks

2.2. Experimental section

2.2.1. Synthesis of intermediate 5-bromo-1-octyl-1H-indole (5)

2.2.2. General procedure for synthesis of 2a-2c

2.2.3. General procedure for synthesis of 3a-3c

2.2.4. General procedure for synthesis of 6a-6c

2.2.5. Synthesis of intermediate 4-((5-(4-(morpholinosulfonyl)phenyl)-1-octyl-1H-indole-3-yl) methyl)piperazine-1-carboxylic acid tert-butyl ester (7j)

2.2.6. General procedure for synthesis of 7a-7k

2.2.7. Synthesis of 4-((4-(1-octyl-3-(piperazine-1-ylmethyl)-1H-indole-5-yl) phenyl) sulfonyl) morpholine (7k)

2.3. X-ray crystal structure determination

2.4. DFT calculation

2.5. Antitumor activity assay

3. Results and Discussion

3.1. Synthesis and characterization

3.2. Crystallographic analysis

3.3. Conformational stability

3.4. Molecular electrostatic potential

3.5. Frontier molecular orbitals

3.6. Vibrational analysis

3.6.1. -CH2- vibration (alkane)

3.6.2. C-N vibration

3.6.3. C=C vibration

3.6.4. C-O-C vibration

3.6.5. C-S vibration

3.6.6. Ar-H vibration

3.7. Hirshfeld surface analysis

3.8. 1H NMR and 13C NMR spectral analysis

3.9. Molecular docking analysis

3.10. In vitro antitumor activity analysis

4. Conclusions

Acknowledgments

CRediT authorship contribution statement

Declaration of competing interest

Declaration of generative AI and AI-assisted technologies in the writing process

Supplementary data

References

Suggested read for related articles:

3.6.1. -CH₂- vibration (alkane)

3.8. ¹H NMR and ¹³C NMR spectral analysis