DOI: 10.31038/JPPR.2025814

Abstract

Objective: A novel Cdk9 inhibitor 1-(2-adamantane-1-yl-1H indole-5-yl)-3-substituted thiourea derivative was designed and synthesized and their anti-gastric cancer activities were studied.

Methods: A series of target compounds 7a-7m were synthesized from amantadane formyl chloride by 6-step reactions. The structures of the target compounds were identified by 1H NMR, 13C NMR and HRMS. MTT assay was used to detect the inhibitory effect of synthetic compounds on the growth of gastric cancer cells, and Western blot assay was used to detect the regulatory effect of hit compound on downstream signaling pathways. Results: The results showed that the target compounds had certain inhibitory activity on the growth of gastric cancer cells, among which compound 7l had the best activity on the gastric cancer cell line (SGC-7901) with IC50 value of 2.26 ± 0.04 μM, and 7I had little toxicity on normal gastric epithelial cells (IC50 > 100 μM). In addition, 7I can bind to CDK9 and inhibit pSer2 expression in gastric cancer cells in a concentration dependent manner. Finally, molecular docking study showed that 7l can stably bind to the active site of CDK9 and has a high binding affinity.

Conclusion: This series of compounds has good anti-gastric cancer activity and has the significance of further study.

Keywords

Adamantane derivatives; Indole; Gastric cancer; CDK9

Gastric cancer is one of the most common cancers worldwide and ranks as the third leading cause of cancer-related deaths [1]. Middle-aged and elderly individuals are at high risk, and gastric cancer is often diagnosed at an advanced stage, with a five-year survival rate of less than 30% [2,3]. Therefore, understanding the occurrence and progression of gastric cancer, along with the potential discovery of novel diagnostic and prognostic biomarkers, is crucial for improving clinical outcomes. Clinically, the primary treatment for gastric cancer is surgical intervention, supplemented by pharmacological therapy [4]. Although chemotherapy can extend the survival of patients to some extent, conventional chemotherapeutic agents suffer from poor specificity, significant toxicity, side effects, and a tendency to induce drug resistance, all of which adversely affect treatment efficacy and patient survival [5]. Thus, identifying effective drugs with favorable therapeutic profiles for gastric cancer is of great importance. Cyclin-dependent kinases (CDKs) are a family of serine/threonine protein kinases that play vital roles in cell cycle progression and transcriptional regulation [6-9]. Recent studies have shown that CDKs are overexpressed in various cancers, leading to uncontrolled cell proliferation and drug resistance [10]. CDK9, a key member of the CDK family, is essential for stable RNA transcriptional elongation. CDK9 and cyclin T form the positive transcription elongation factor b (P-TEFb) complex, which promotes transcriptional elongation through phosphorylation of RNA polymerase II (RNAPII) [11,12]. Recent evidence indicates that CDK9 plays a critical role in numerous human cancers, including cervical, prostate, and lung cancers [13-18]. Dual-luciferase reporter assays have demonstrated that miR-613 inhibits the metastasis and progression of gastric cancer cells by downregulating CDK9 gene expression [19]. Therefore, targeting CDK9 to interfere with the development and progression of gastric cancer holds therapeutic promise. Building on previous work, this study designed and synthesized a novel series of CDK9 inhibitors (Figure 1). The influence of different substituted R groups on the antitumor activity of these thiourea compounds was investigated to identify more potent 1-(2-(adamantan-1-yl)-1H-indol-5-yl)-3-substituted thiourea derivatives. The synthetic route for target compounds 7a–7l is illustrated in Figure 1.

Figure 1: Synthetic route of compounds 7a～7l.

Materials and Methods

Chemicals and Reagents

Starting materials were purchased from Shanghai Titan Scientific Co., Ltd. All reagents were of analytical grade and used without further purification. Solvents were used as received. Thin-layer chromatography (TLC) silica gel (Qingdao Marine Chemical Factory, 60–100 mesh) and MTT powder (ST1537, Beyotime) were used.

Instruments

NMR spectra were recorded on a Bruker Avance 600 spectrometer using DMSO-d6 or CDCl3 as the solvent with TMS as the internal standard. Mass spectra were obtained using an Agilent 6230 mass spectrometer. A Thermo microplate reader was used for absorbance measurements.

Methods

MTT Assay for Cell Viability

The MTT assay was used to assess cell survival and growth. Cells were digested and seeded into 96-well plates at a density of 3,000 cells per well. After cell attachment, they were treated with the respective compounds or solvent, with cisplatin used as a positive control. After 48 hours of incubation, the medium was replaced with fresh medium containing MTT solution (1.2 mg/mL), and the cells were incubated for another 3 hours. The MTT formazan product was dissolved in DMSO, and the absorbance was measured at 492 nm using a microplate reader.

CDK9 Inhibition Assay

The ADP-Glo Kinase Assay was employed to determine the inhibitory activity of the compounds against CDK9 protein, conducted by Innovative CRO + Explorer (Beijing, China). Kinase activity was assessed in a buffer containing 50 mM Hepes, 10 mM MgCl₂, 0.01% Brij 35, 1 mM EGTA, 2 mM DTT, and ddH₂O. Test compounds were prepared in DMSO. The inhibition rate against CDK9/cyclin T1 (ATP: 20 μM) was tested for all compounds at a concentration of 1 μM.

Molecular Docking

Molecular docking was performed to explore the interaction mode between compound 7l and CDK9. The crystal structure of CDK9/cyclin T1 (PDB ID: 7NWK, resolution: 2.81 Å) was downloaded from the Protein Data Bank (https://www.rcsb.org/structure/7NWK). The protein structure was prepared using the Protein Preparation Wizard module in Schrödinger software (V 2023-1) under default parameters, including removal of water and solvent molecules, addition of hydrogen atoms, assignment of charges, completion of missing amino acid residues, optimization of the hydrogen bond network, and energy minimization using the OPLS4 force field. The structure of 7l was prepared using LigPrep (Schrödinger, LLC, New York, NY, 2023) under the OPLS4 force field with default parameters, including hydrogen addition, charge assignment, and generation of possible protonation states at pH 7.0 ± 2.0. Molecular docking was performed using the Ligand Docking module in Schrödinger software, docking 7l into the ATP-binding site of CDK9. The docking box was centered on the centroid of the original crystal ligand, and extra precision (XP) docking was employed. The binding energy of the 7l-CDK9 complex was calculated using the MM-GBSA module. Maestro (Version 12.5, Schrödinger) was used for visualization, and PyMOL (Version 2.5.4, Schrödinger, LLC) was used for rendering and presentation.

Experimental Methods and Results

Synthesis of Intermediate N-(o-Tolyl)adamantane-1-Carboxamide (2)

In a dried 50 mL round-bottom flask, toluene (10 mL), adamantane-1-carbonyl chloride (0.99 g, 5 mmol), o-toluidine (0.54 g, 5 mmol), and potassium carbonate (0.69 g, 5 mmol) were added successively. The mixture was stirred and heated to 80°C, and the reaction progress was monitored by TLC. After completion, the reaction mixture was cooled, filtered, washed with water, and dried to afford a crude white solid, which was recrystallized from ethanol to yield N-(o-tolyl)adamantane-1-carboxamide (1.17 g, 87%). 1H-NMR(600 MHz, CDCl₃) δ: 7.88 (d, J=8.1 Hz, 1H), 7.24-7.18 (m, 2H), 7.17 (d, J=7.5Hz, 1H), 7.05 (dt, J=1.1, 7.4 Hz, 1H), 2.26 (s, 3H), 2.11 (brs, 3H), 1.99 (d, J=2.6 Hz, 6H), 1.77 (m, 6H).

Synthesis of Intermediate 2-(Adamantan-1-yl)-1H-indole (3)

A dried 25 mL three-necked flask was charged with N-(o-tolyl)adamantane-1 carboxamide (0.285 g, 1.0 mmol) and THF (15 mL). The mixture was cooled to 0–5°C under nitrogen, and n-butyllithium (2 mL) was added dropwise. The reaction was monitored by TLC. Upon completion, the pH was adjusted to 7.0–7.5 with 5% dilute hydrochloric acid. The mixture was extracted, and the organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate=10: 1, v/v) to yield 2-(adamantan-1-yl)-1H-indole (0.166 g, 66.1%). 1H-NMR (600 MHz, CDCl₃) δ: 7.30 (d, J=7.9 Hz, 1H), 7.18 (dd, J=0.5, 8.1 Hz, 1H), 6.88 (dt, J=1.1, 7.5 Hz, 1H), 6.83-6.77 (m, 1H), 5.98 (d, J=0.6 Hz, 1H), 1.97 (d, J=2.6 Hz, 3H), 1.93 (d, J=2.9 Hz, 6H), 1.73 (brs, 6H).

Synthesis of Intermediate 2-(Adamantan-1-yl)-5-nitro-1H-indole (4)

A 25 mL three-necked flask was charged with 2-(adamantan-1-yl)-1H-indole (0.25 g, 1.0 mmol) and concentrated sulfuric acid (0.8 mL). A solution of sodium nitrate (0.085 g, 1.0 mmol) in sulfuric acid (0.8 mL) was added dropwise at 0°C. After addition, the reaction was maintained for 1–2 hours and monitored by TLC. Upon completion, a large amount of ice water was added, and a yellow solid precipitated. The solid was filtered, washed with water, and dried. The crude product was purified by column chromatography (eluent: petroleum ether/ethyl acetate=5: 1, v/v) to yield 2 (adamantan-1-yl)-5-nitro-1H-indole (0.24 g, 82.8%) as a yellow solid. 1H-NMR (600 MHz, DMSO-d6) δ: 11.65 (brs, 1H), 8.44 (d, J=2.2 Hz, 1H), 7.93 (dd, J=2.2 ,9.0 Hz, 1H), 7.44 (d, J=8.8 Hz, 1H), 6.38 (d, J=1.5 Hz, 1H), 2.07 (brs,3H), 1.98 (brs, 6H), 1.81-1.67 (m, 6H);13C-NMR (150 MHz, DMSO-d6) δ: 154.0, 140.8, 139.9, 127.7, 116.9, 116.3, 111.4, 98.2, 41.9, 36.6, 34.1, 28.2; HRMS (ESI) m/z: 297.1599 [M+H]⁺, Calt: 297.1598 [M+H]⁺.

Synthesis of Intermediate 5-Amino-2-(adamantan-1-yl)-1H-indole (5)

In a 250 mL three-necked flask, ethanol (100 mL), acetic acid (10 mL), water (20 mL), and iron powder (8.62 g, 0.15 mol) were added. The mixture was heated to 75°C, and 2-(adamantan-1-yl)-5-nitro-1H-indole (11.4 g, 0.038 mol) was added in portions. The reaction was maintained for 2 hours and monitored by TLC. After completion, the reaction mixture was hot-filtered, and the filtrate was concentrated under reduced pressure to remove the solvent. Water (80 mL) was added, followed by ethyl acetate (80 mL) with stirring. The pH was adjusted to 7–8 with sodium bicarbonate. The organic phase was separated, dried over anhydrous Na₂₂SO₄₄, filtered, and purified by column chromatography (eluent: petroleum ether/ethyl acetate=2: 1, v/v) to yield 5 amino-2-(adamantan-1-yl)-1H-indole (6.9 g, 67.0%) as a red solid. 1H-NMR (600 MHz, CDCl3) δ: 7.80 (brs, 1H), 7.10 (d, J=8.4 Hz, 1H), 6.86 (d, J=2.2 Hz, 1H), 6.57 (dd, J=2.2, 8.4 Hz, 1H), 6.05 (dd, J=0.7, 2.2 Hz, 1H), 3.23-3.65 (m, 2H), 2.09 (brs, 3H), 1.96 (d, J=2.38 Hz, 6H), 1.78 (q, J=12.2 Hz, 6H);13C-NMR (150 MHz, CDCl3) δ: 149.9,139.3, 130.3, 129.4, 111.6, 110.8, 105.3, 95.5, 42.6, 36.8, 33.7, 28.5; HRMS (ESI) m/z: 267.1858 [M+H]⁺, Calt: 267.1856 [M+H]⁺.

Synthesis of Intermediate 5-Isothiocyanato-2-(adamantan-1-yl)-1H-indole (6)

In a dried 100 mL three-necked flask, a solution of 5-amino-2-(adamantan-1-yl)-1H indole (4.10 g, 15.5 mmol) in toluene (20 mL) and triethylamine (3 mL) was added. Carbon disulfide (3.50 g, 46.5 mmol) was added dropwise slowly. The reaction was stirred at room temperature for 8 hours. The mixture was filtered and dried, then dissolved in dichloromethane (30 mL). Triphosgene (5.0 g, 17 mmol) was added dropwise at 0–5°C. After 2 hours, the reaction was monitored by TLC. Upon completion, the product was purified by column chromatography (eluent: petroleum ether/ethyl acetate=10: 1, v/v) to yield 5-isothiocyanato-2-(adamantan-1-yl)-1H-indole (2.89 g, 64.0%) as a white solid. 1H-NMR(600 MHz, CDCl3) δ: 7.40 (d, J=1.8 Hz, 1H ), 7.23 (d, J=8.4 Hz, 1H, ), 6.98 (dd, J=2.0, 8.4 Hz, 1H), 6.20 (d, J=1.5 Hz, 1H ), 2.14 2.08 (m, 3H), 1.99-1.94 (m, 6H), 1.85-1.74 (m, 6H); 13 C-NMR (150 MHz, CDCl3) δ: 151.4, 134.1, 131.8, 128.7, 122.6, 119.0, 117.3, 111.2, 96.8, 42.5, 36.7, 33.9, 28.4; HRMS (ESI) m/z: 309.1422 [M+H]⁺, Calt: 309.1420 [M+H]⁺.

Synthesis of 1-(2-(Adamantan-1-yl)-1H-indol-5-yl)-3-Substituted Thiourea Derivatives (7a–7l)

In a 25 mL reaction flask, 5-isothiocyanato-2-(adamantan-1-yl)-1H-indole (0.145 g, 0.5 mmol) and toluene (10 mL) were added. The appropriate amine (0.5 mmol) was added, and the mixture was heated to 80°C. The reaction was monitored by TLC. After cooling, the mixture was filtered and dried to afford the target compounds as white solids in yields of 70.0–88.6%.

7a: white solid, yield 70.1%, mp 198～200℃, 1H NMR (400 MHz, DMSO-d6) δ: 10.92 (s, 1H), 9.25 (brs, 1H), 7.27 (dd, J=3.2, 4.95 Hz, 2H), 6.83 (dd, J=1.7 8.5 Hz, 1H), 6.08 (d, J=1.71 Hz, 1H), 3.47-3.40 (m, 2H), 2.06 (brs, 3H), 1.96 (d, J=1.96 Hz, 6H), 1.76 (br, 6H), 1.54-1.41 (m, 2H), 1.32-1.19 (m, 6H), 0.87 (t, J=6.79 Hz, 3H);13C NMR (100 MHz, DMSO-d6): δ 181.0, 152.5, 151.1, 135.3, 134.6, 128.4, 116.8, 111.5, 95.8, 44.5, 42.2, 36.8, 33.9, 31.5, 29.1, 28.3, 26.5, 22.5, 14.4; HRMS (ESI) m/z: 410.2625 [M+H]⁺, Calt: 410.2624 [M+H]⁺.

7b: white solid, yield 78.4%, mp 201～203℃, 1H NMR (400 MHz, DMSO-d6) δ: 10.90 (s, 1H), 9.21 (s, 1H), 7.32 (s, 1H), 7.25 (d, J=8.44 Hz, 1H), 6.96 (brs, 1H), 6.87 (dd, J=1.7, 8.4 Hz, 1H), 6.07 (d, J=1.3 Hz, 1H), 2.06 (brs, 3H), 1.96 (brs, 6H), 1.89-1.83 (m, 2H), 1.80-1.71 (m, 6H), 1.64 (d, J=12.6 Hz, 2H), 1.54 (d, J=12.7 Hz, 1H), 1.32-1.04 (m, 5H); 13C NMR (100 MHz, DMSO-d6) δ: 180.0, 151.0, 134.4, 130.1, 128.3, 118.8, 116.4, 111.3, 95.7, 52.9, 42.2, 36.8, 33.9, 32.4, 28.4, 25.6, 25.1; HRMS (ESI) m/z: 408.2469 [M+H]⁺, Calt: 408.2468 [M+H]⁺.

7c: white solid, yield 80.1%, mp 221～223℃, 1H NMR (400 MHz, DMSO-d6) δ: 10.91 (s, 1H), 9.66 (s, 1H), 9.35 (brs, 1H), 7.50 (d, J=7.8 Hz, 2H), 7.42 (s, 1H), 7.33-7.25 (m, 3H), 7.13-7.07 (m, 1H), 6.98 (dd, J=1.8, 8.5Hz, 1H), 6.10 (d, J=1.6Hz, 1H), 2.07 (brs, 3H), 1.97 (s, 6H), 1.84-1.66 (m, 6H); 13C NMR (100 MHz, DMSO-d6) δ: 180.4, 151.0, 140.3, 134.5, 130.5, 128.7, 128.3, 124.6, 124.3, 119.0, 116.6, 111.1, 95.8, 42.3, 36.8, 33.9, 28.4; HRMS (ESI) m/z: 402.1999 [M+H]⁺, Calt: 402.1998 [M+H]⁺.

7d: white solid, yield 78.8%, mp 225～226℃; 1H NMR (400 MHz, DMSO-d6) δ: 10.91 (s, 1H), 9.54 (s, 1H), 8.87 (brs, 1H), 7.42 (d, J=1.5 Hz, 1H), 7.30-7.24 (m, 2H), 7.22 (dd, J=1.5, 6.9 Hz, 1H), 7.19-7.09 (m, 2H), 6.98 (dd, J=1.9, 8.5 Hz, 1H), 6.10 (d, J=1.7 Hz, 1H), 2.24 (s, 3H), 2.07 (brs, 3H), 1.97 (d, J=2.0 Hz, 6H), 1.82 1.72 (m, 6H); 13C NMR (100 MHz, DMSO-d6) δ: 181.1, 151.0, 138.8, 135.3, 134.6, 130.6, 128.7, 128.3, 126.6, 126.3, 119.2, 116.9, 111.2, 95.8, 42.3, 36.8, 33.9, 28.4, 18.4;HRMS (ESI) m/z: 416.2152 [M+H]⁺,Calt: 416.2155 [M+H]⁺.

7e: white solid, yield 75.6%, mp 234～236℃;1H NMR (400 MHz, DMSO-d6) δ: 10.91 (s, 1H), 9.62 (s, 1H), 9.27 (brs, 1H), 7.41 (s, 1H), 7.32-7.24 (m, 3H), 7.19 (t, J=8.1 Hz, 1H), 6.97 (dd, J=1.8, 8.5 Hz, 1H), 6.92 (d, J=7.5 Hz, 1H), 6.09 (d, J=1.5 Hz, 1H), 2.28 (s, 3H), 2.07 (brs, 3H), 1.97 (brs, 6H), 1.83-1.63 (m, 6H);13C NMR (100 MHz, DMSO-d6) δ: 180.3, 151.0, 140.2, 137.9, 134.5, 130.5, 128.5, 128.2, 125.3, 124.9, 121.5, 119.1, 116.6, 111.1, 95.8, 42.3, 36.8, 33.9, 28.4, 21.5; HRMS (ESI) m/z: 416.2150 [M+H]⁺,Calt: 416.2155 [M+H]⁺.

7f: white solid, yield 79.5%, mp 227～228℃; 1H NMR (600 MHz, DMSO-d6) δ: 10.92 (brs, 1H), 9.59 (brs, 1H), 9.26 (brs, 1H), 7.41 (s, 1H), 7.35 (d, J=8.0 Hz, 2H), 7.27 (d, J=8.4 Hz, 1H), 7.11 (d, J=8.0 Hz, 2H), 6.97 (d, J=7.7 Hz, 1H), 6.09 (s, 1H), 2.28 (s, 3H), 2.07 (brs, 3H), 1.97 (brs, 6H), 1.81-1.70 (m, 6H); 13C NMR (150 MHz, DMSO-d6) δ: 180.4, 151.0, 137.7, 134.5, 133.8, 130.5, 129.1, 128.2, 124.6, 119.1, 116.6, 111.1, 95.7, 42.3, 36.8, 33.9, 28.4, 21.0;HRMS (ESI) m/z: 416.2151 [M+H]⁺,Calt: 416.2155 [M+H]⁺.

7g: white solid, yields 80.1%, mp 244～246℃; 1H NMR (400 MHz, DMSO-d6) δ: 10.90 (s, 1H), 9.50 (s, 1H), 9.14 (brs, 1H), 7.41 (d, J=0.9 Hz, 1H), 7.33 (d, J=8.9 Hz, 2H), 7.27 (d, J=8.4 Hz, 1H), 6.97 (dd, J=1.8, 8.5 Hz, 1H), 6.88 (d, J=8.9 Hz, 2H), 6.09 (d, J=1.5 Hz, 1H), 3.74 (s, 3H), 2.07 (brs, 3H), 1.97 (brs, 6H), 1.82-1.71 (m, 6H);13C NMR (100 MHz, DMSO-d6) δ: 180.7, 156.8, 151.0, 134.5, 133.1, 130.5, 128.3, 126.7, 119.1, 116.7, 113.9, 111.1, 95.7, 55.7, 42.3, 36.8, 33.9, 28.4;HRMS (ESI) m/z: 432.2106 [M+H]⁺,Calt: 432.2104 [M+H]⁺.

7h: white solid, yields 73.1%, mp 208～210℃; 1H NMR (600 MHz, DMSO-d6) δ: 10.93 (s, 1H), 9.70 (brs, 1H), 9.31 (brs, 1H), 7.46 (dd, J=5.1, 8.6 Hz, 2H), 7.40 (s, 1H), 7.27 (d, J=8.6 Hz, 1H), 7.14 (t, J=8.8 Hz, 2H), 6.96 (dd, J=1.4, 8.5 Hz, 1H), 6.09 (d, J=1.5 Hz, 1H), 2.07 (brs, 3H), 1.97 (brs, 6H), 1.80-1.72 (m, 6H); 13C NMR (150 MHz, DMSO-d6) δ: 180.7, 159.5 (d, J=238.5 Hz), 151.0, 136.7, 134.6, 130.3, 128.3, 126.9, 119.0, 116.6, 115.2(d, J=21.0 Hz), 111.2, 95.8, 42.2, 36.8, 33.9, 28.4; (ESI) m/z: 420.1904 [M+H]⁺,Calt: 420.1904 [M+H]⁺.

7i: white solid, yields 79.1%, mp 245～246℃; 1H NMR (600 MHz, DMSO-d6) δ: 10.94 (brs, 1H), 9.78 (brs, 1H), 9.44 (brs, 1H), 7.52 (d, J=8.4 Hz, 2H), 7.41 (brs, 1H), 7.35 (d, J=8.8 Hz, 2H), 7.27 (d, J=8.4 Hz, 1H), 6.96 (d, J=8.0 Hz, 1H), 6.09 (s, 1H), 2.07 (brs, 3H), 1.97 (brs, 6H), 1.81-1.72 (m, 6H); 13C NMR (150 MHz, DMSO d6) δ: 180.4, 151.1, 139.4, 134.6, 128.7, 128.4, 128.2, 126.0, 125.8, 119.0, 116.6, 111.2, 95.8, 42.2, 36.8, 33.9, 28.4; (ESI) m/z: 436.1605 [M+H]⁺,Calt: 436.1609 [M+H]⁺.

7j: white solid, yield 82.3%, mp 218～220℃; 1H NMR (400 MHz, DMSO-d6) δ: 10.93 (s, 1H), 9.95 (s, 1H), 9.71 (brs, 1H), 7.78 (d, J=8.4Hz, 2H), 7.65 (d, J=8.6 Hz, 2H), 7.43 (s, 1H), 7.28 (d, J=8.4 Hz, 1H), 6.99 (dd, J=1.7, 8.5 Hz, 1H), 6.10 (d, J=1.5Hz, 1H), 2.07 (brs, 3H), 1.97 (d, J=2.0 Hz, 6H), 1.82-1.69 (m, 6H);13C NMR (100 MHz, DMSO-d6) δ: 180.3, 151.1, 144.3, 134.6, 130.3, 128.3, 125.7, 123.5, 118.8, 116.5, 111.2, 95.8, 42.2, 36.8, 33.9, 28.4; (ESI) m/z: 470.1872 [M+H]⁺,Calt: 470.1872 [M+H]⁺.

7k: white solid, yield: 88.6%; mp 242-243℃; 1H NMR (600 MHz, DMSO-d6) δ: 10.78-10.94 (m, 1H), 9.42 (brs, 1H), 8.77 (brs, 1H), 7.40 (s, 1H), 7.26 (d, J=8.4 Hz, 1H), 7.10 (d, J=7.8 Hz, 1H), 7.01 (s, 1H), 6.98-6.92 (m, 2H), 6.14-5.94 (m, 1H), 2.25 (s, 3H), 2.19 (s, 3H), 2.05 (brs, 3H), 1.96 (brs, 6H), 1.82-1.70 (m, 6H);13C NMR (150 MHz, DMSO-d6) δ: 151.0, 135.8, 135.1, 134.6, 131.2, 129.4, 128.7, 128.6, 128.3, 126.9, 119.2, 116.9, 95.8, 42.3, 36.8, 33.9, 28.4, 21.0, 18.3;(ESI) m/z: 430.2310 [M+H]⁺,Calt: 430.2311 [M+H]⁺.

7l: white solid, yield 72.8%, mp 228～230℃;1H NMR (400 MHz, DMSO-d6) δ: 10.94 (s, 1H), 9.85 (brs, 1H), 8.96 (brs, 1H), 7.52 (d, J=6.7 Hz, 1H), 7.42 (s, 1H), 7.31-7.23 (m, 2H), 7.07-7.01 (m, 1H), 6.97 (dd, J=1.8, 8.5Hz, 1H), 6.11 (d, J=1.6 Hz, 1H), 2.07 (brs, 3H), 1.97 (d, J=2.2 Hz, 6H), 1.83-1.69 (m, 6H);13C NMR (100 MHz, DMSO-d6) δ: 181.6, 161.7, 156.2, 151.1 (d, J=25.0 Hz), 134.7, 131.0, 130.2 (d, J=25.0 Hz), 129.4, 128.7, 128.3, 124.9, 119.0, 116.8, 111.3, 111.1, 104.5, 95.8, 42.2, 36.8, 33.9, 28.4; (ESI) m/z: 438.1812 [M+H]⁺,Calt: 438.1810 [M+H]⁺.

Results and Discussion

Structure-Activity Relationship (SAR) Discussion

The target compounds were synthesized starting from adamantane-1-carbonyl chloride and o-toluidine to yield N-(o-tolyl)adamantane-1-carboxamide (compound 2), which was then reacted with n-butyllithium to give 2-(adamantan-1-yl)-1H-indole (compound 3). Nitration of 3 afforded 2-(adamantan-1-yl)-5-nitro-1H-indole (compound 4), which was reduced to 5-amino-2-(adamantan-1-yl)-1H-indole (compound 5). Treatment of 5 with triphosgene yielded 5-isothiocyanato-2 (adamantan-1-yl)-1H-indole (compound 6). Reaction of 6 with various amines gave the target compounds 7a–7l, whose structures were confirmed by 1H NMR, 13C NMR, and HRMS. The in vitro inhibitory activities of the synthesized compounds against the gastric cancer cell line SGC-7901 were evaluated using the MTT assay, and the results are summarized in Table 1. The effects of different R groups (alkyl, cycloalkyl, substituted aryl, and fused aromatic rings) on the inhibitory activity against SGC-7901 were investigated. According to Table 1, the order of potency for R=hexyl, cyclohexyl, phenyl, and naphthyl was 7c (phenyl) > 7b (cyclohexyl) > 7a (hexyl). For monosubstituted phenyl derivatives (7d–7j), the order was 4-Cl (7i) > 4-F (7h) > 4-OCH3 (7g) > 4-CF3 (7j) > 2-CH3 (7d) > 3-CH3 (7e) > 4-CH3 (7f). For disubstituted phenyl derivatives (7k–7l), 2,4-diCl (7l) > 2,4-diCH3 (7k). Compound 7l exhibited the most potent activity, with an IC50 value of 2.26 ± 0.04 μM. Moreover, 7l showed low toxicity toward normal gastric epithelial cells (IC50>100 μM).

Table 1: Structure, kinase and cell inhibitory activity of the targeted compounds.

Analysis of the Binding Mode of 7l with CDK9

Molecular docking studies were performed to predict the binding mode of 7l with Compound Structure SGC-7901 (IC50/μM) CDK9 inhibiton (@ 1μM) R 7a 15.35 ± 0.23 50.1% 7c 10.11 ± 0.12 58.1% 7e 14.56 ± 0.20 52.3% 7g O 8.45 ± 0.07 73.8% 7i Cl 7.35 ± 0.11 79.5% 7k 13.18 ± 0.47 54.1% 7m 24.16 ± 0.14 45.9% 7b 21.24 ± 0.34 40.2% 7d 12.10 ± 0.45 54.3% 7f 17.23 ± 0.11 49.7% 7h F 8.09 ± 0.37 75.5% 7j CF3 9.76 ± 0.20 60.1% 7l F F 2.26 ± 0.04 85.6% CDK9. The results indicated that 7l binds within the ATP-binding pocket of CDK9, and its three-dimensional structure fits well into the active site (Figure 2A). As shown in Figure 2B, the indole core and thiourea moiety of 7l form stable interactions with key residues of CDK9. The hydrogen atom on the indole nitrogen acts as a hydrogen bond donor, forming a strong hydrogen bond (1.80 Å) with the carbonyl oxygen of GLU107 in the hinge region, indicating that the core of 7l is well-anchored in the hinge region of the CDK9 active site. Additionally, the hydrogen atoms on the thiourea moiety provide further opportunities for interactions with amino acid residues in the active site. The docking results showed that a hydrogen atom on the thiourea group acts as a hydrogen bond donor, forming a hydrogen bond (2.20 Å) with GLY28 of CDK9. Furthermore, the binding free energy of 7l with CDK9, calculated using the MM-GBSA method, was –32.86 kcal/mol, further confirming the high binding affinity of 7l for CDK9.

Through a molecular hybridization strategy, twelve new 1-(2-(adamantan-1-yl) 1H-indol-5-yl)-3-substituted thiourea derivatives (7a–7l) were synthesized and characterized by 1H NMR, 13C NMR, and HRMS. The MTT and CDK9 inhibition assay demonstrated that these compounds inhibit the growth of gastric cancer cells, with compound 7l exhibiting the most potent activity. Molecular docking studies indicated that 7l binds stably to the active site of CDK9 with high binding affinity. Future work will focus on further evaluation of 7l.

Figure 2: Binding pattern of compound 7l to CDK9. A. Schematic representation of the surface of 7l binding to CDK9; B. The three-dimensional interaction diagram of 7l binding to the protein.

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DOI: 10.31038/JPPR.2025813

Abstract

Kinases represent one of the most prominent classes of drug targets in current medicinal chemistry research. They are frequently over expressed in various diseases such as cancer, inflammation, or autoimmune disorders, playing critical roles in their pathophysiology. Since the early 1980s, numerous potent kinase inhibitors have been developed and approved. However, kinase inhibitors often face challenges including drug resistance and off-target effects. To overcome these limitations, novel strategies are required. Since 2013, several research groups have proposed converting potent kinase inhibitors into PROTAC molecules, leveraging cellular machinery to degrade target proteins. Results demonstrate that PROTACs significantly enhance biological effects compared to their parent inhibitors. This review focuses on recent advances in PROTAC technology applied to receptor tyrosine kinases (RTKs), with particular emphasis on compounds reported since 2018.

Keywords

Receptor tyrosine kinase; Targeted protein degradation; PROTAC; Review

Kinases are macromolecules involved in signal transduction and pathway regulation within biological systems, primarily functioning through two mechanisms: (1) Catalytic function: transferring phosphate groups from high-energy donor molecules (e.g., ATP) to specific substrates; (2) Non-catalytic functions: such as scaffold roles and allosteric regulation mediated by protein interactions. Protein kinases mainly include receptor tyrosine kinases (RTKs), non-receptor tyrosine kinases (NRTKs), and serine-threonine kinases (STKs), among other families. Dysregulated kinase activity contributes to diseases such as acute myeloid leukemia [1], melanoma [2], breast cancer [3], and prostate cancer, making them hot targets for small-molecule drug discovery. Although existing small-molecule inhibitors [4] have shown significant efficacy in clinical treatments, they possess limitations including lack of selectivity toward highly homologous kinases and the emergence of drug resistance. The rapidly developing technology of Proteolysis-Targeting Chimeras (PROTACs) is considered a promising strategy to overcome these challenges [5]. PROTACs are bifunctional molecules that induce the formation of a complex between the target protein (protein of interest, POI; here, a kinase) and an E3 ubiquitin ligase (UL). This complex mediates polyubiquitination of the POI, leading to its recognition and degradation by the ubiquitin-proteasome system (UPS) (Figure 1). Since 2013, reports on kinase-targeting PROTAC molecules have grown exponentially. This article reviews recent progress in PROTACs targeting protein kinases, which may facilitate the development of novel therapies to overcome drug resistance in cancer treatment.

Figure 1: PROTACs induce protein ubiquitination and degradation.

Receptor tyrosine kinases (RTKs) are cell surface receptors with a similar molecular structure, comprising an extracellular ligand-binding domain, a transmembrane helix, and an intracellular region containing a tyrosine kinase domain. Genetic mutations can alter RTK activity, expression levels, cellular distribution, and regulation, potentially leading to cancers, diabetes, inflammation, and severe bone or vascular diseases. Recently, FMS-like tyrosine kinase 3 (FLT3) [6], tropomyosin receptor kinase C (TrkC) [7], anaplastic lymphoma kinase (ALK) [8,9], and epidermal growth factor receptor (EGFR) [10] have been reported to be degraded by their corresponding PROTAC molecules. Their structures are shown in Figure 2, and biological activities are summarized in Table 1.

Figure 2: Reported RTKs degrader.

Table 1: Reported RTKs degrader.

PROTACs Degrading FLT3

FLT3 is expressed in hematopoietic stem cells and plays a crucial role in normal hematopoiesis; its dysfunction often leads to blood disorders. Acute myeloid leukemia (AML) is a common malignant tumor of the hematopoietic system, with FLT3 gene mutations accounting for 30% of AML cases, among which internal tandem duplication (ITD) mutations are the most common. Patients carrying FLT3-ITD mutations exhibit elevated white blood cell levels and poor prognosis [1,11-13]. Therefore, FLT3/ITD is a promising target for AML treatment, and maximal sustained inhibition of this signaling is crucial for clinical response [11]. Based on this background, numerous PROTACs targeting FLT3 for degradation have emerged. The Burslem group reported a PROTAC 1a [6,11] composed of AC220 [14,15] as the warhead, VHL as the E3 ligase ligand, and connected by a PEG linker of appropriate length. This PROTAC exhibited similar cellular inhibitory activity and selectivity to AC220 in vitro but showed 3.5-fold greater inhibition of cell proliferation in MOLM-14 and MV4-11 cell lines and induced better apoptosis in leukemia cells at low doses. 1a induced FLT3-ITD degradation in vivo; in MV4-11 xenograft models, administration at 30 mg·kg⁻¹ every 24 hours for three days reduced FLT3 levels in tumors by 60%. These results suggest that degradation of FLT3-ITD may provide a useful therapeutic approach for AML.

PROTACs Degrading TrkC

Trk kinases are a family of protein tyrosine kinases with high affinity for neurotrophin (NT) growth factors, which are essential for neuronal differentiation and survival in the peripheral and central nervous systems. TrkC is overexpressed in many human tumors, particularly in neuroblastoma [12], glioblastoma [12], breast cancer [16], and melanoma [2]. Its aberrant activation promotes cell growth and metastasis in certain forms of tumorigenesis [3], making effective reduction of TrkC expression significant for treating these cancers. In 2019, Zhao and Burgess reported the first PROTAC 1b degrading TrkC [7]. This PROTAC uses a bivalent peptide (isoleucine-tyrosine-tyrosine-isoleucine) analog as the warhead, which binds TrkC with submicromolar affinity and facilitates good cellular internalization [17,18]. The warhead is connected to pomalidomide via a PEG linker. 1b degraded TrkC in Hs578t cells at concentrations of 1-10 μM, with a DC₅₀ of 0.1-1.0 μM [7]. This PROTAC demonstrates the applicability of the technology to reduce TrkC levels and provides insights for developing more PROTACs, such as those using FDA-approved inhibitors like larotrectinib or entrectinib as warheads [19,20], potentially yielding highly effective molecules.

PROTACs Degrading ALK

ALK is part of the insulin receptor family. Although its exact function in mammalian cells is not fully understood, various forms of ALK fusion proteins are known to drive oncogenesis in multiple cancers. For example, the NMP-ALK fusion is commonly found in anaplastic large cell lymphoma (ALCL) [21,22]. Numerous studies indicate that inhibiting ALK activity suppresses the proliferation of cancer cells driven by ALK fusions [23,24]. Thus, developing ALK-targeted PROTACs appears to be a promising approach to enhance the efficacy of approved ALK inhibitors. The Kang group reported a PROTAC TD-004 [8] using ceritinib as the warhead [25] and connected to a VHL ligand via a long linker containing an isopropyl chain and an amide bond. TD-004 degraded approximately 90% of NMP-ALK fusion protein in SU-DHL-1 cells, with IC₅₀ values of 58 nM and 180 nM in ALK-positive SU-DHL-1 and H3122 cell lines, respectively, while showing an IC₅₀ > 1000 nM in ALK-low A549 cells, indicating significant selectivity [8]. In H3122 xenograft models, daily administration of 58 mg·kg⁻¹ for 14 days significantly reduced tumor volume without causing notable weight loss. Besides TD-004, Zhang et al. [26] and Powell et al. [27] reported other ceritinib-based PROTACs using pomalidomide as the E3 ligase ligand. These PROTACs differed mainly in linker length and composition, exhibiting stronger anti-proliferative activity in vitro than TD-004 but lacking in vivo validation. These examples illustrate that even with the same warhead, variations in linker length, composition, and E3 ligase ligand can significantly impact PROTAC bioactivity. Besides NMP-ALK, another ALK fusion protein, EML4-ALK, was targeted by a degrader reported by the Liu group. This multivalent PROTAC 2d consists of multiple warheads and E3 ligase ligands attached to gold nanoparticles [9]. Due to the strong affinity of gold nanoparticles for thiols, modified ceritinib and pomalidomide were easily conjugated to the nanoparticle surface to form multivalent PROTACs. Cellular analysis showed that incubation with NCI-H2228 cells for 24 hours reduced EML4-ALK levels by 80%, with prominent anti-proliferative activity (IC₅₀ = 4.8 μM), while negligible cytotoxicity was observed in ALK-negative A549 cells. Although in vivo efficacy requires further validation, the multivalency offered by gold nanoparticles provides an effective strategy to bring warheads and E3 ubiquitin ligases into proximity, facilitating ternary complex formation.

PROTACs Degrading EGFR

Activating mutations in EGFR are closely associated with non-small cell lung cancer (NSCLC). However, even with FDA-approved third-generation EGFR-TKIs like osimertinib, resistance eventually develops, reducing therapeutic efficacy. In April 2020, Zhang et al. [28] reported a series of PROTACs selectively targeting mutant EGFR. One of the most potent compounds, 1g, selectively degraded EGFRL858R/T790M with a DC₅₀ of 5.9 nM while sparing the wild-type protein. In December of the same year, Zhao et al. [29] reported a series of VHL-based PROTACs, among which compound P3 showed potent anti-proliferative activity in HCC827 and H1975 cells with IC₅₀ values of 0.83 nM and 203.01 nM, respectively, and DC₅₀ values of 0.51 nM and 126.2 nM for EGFRdel19 and EGFRL858R/T790M, respectively. Besides inhibiting EGFR signaling, P3 significantly induced apoptosis, arrested the cell cycle, and inhibited colony formation. In June 2021, Qu et al. [30] reported SIAIS125 and SIAIS126—two PROTACs composed of the EGFR inhibitor canertinib and pomalidomide connected by linkers of different lengths. These degraders selectively degraded EGFRL858R/T790M in H1975 cells and EGFREx19del in PC9 cells for up to 72 hours, also inducing significant apoptosis, cell cycle arrest, and growth inhibition. They did not degrade the EGFREc19del/T790M mutant in PC9BRca1 cells or wild-type EGFR in A549 lung cancer cells. Since the first report of kinase-targeted PROTACs in 2013, many research groups have proposed converting potent kinase inhibitors into PROTACs. This technology, which harnesses cellular machinery to degrade proteins, has propelled several compounds to the forefront of drug development, offering advantages for enhancing therapeutic efficacy. PROTACs may yield superior biological outcomes compared to parent inhibitors; for instance, FLT3-PROTACs induce apoptosis more effectively in leukemia cells, and BCR-ABL degraders exhibit longer-lasting inhibition of BCR-ABL and its downstream signaling than dasatinib. PROTACs may achieve higher selectivity than ATP-competitive kinase inhibitors, as seen with CST620 selectively targeting CDK6. They can degrade proteins that have developed resistance to inhibitors due to mutations, such as the aforementioned EGFR degraders. PROTACs can utilize allosteric inhibitors as POI recruiters to enhance selectivity and reduce side effects of parent inhibitors; for example, GMB-475, using GNF-2 as the warhead, degrades the target kinase while abolishing its non-kinase functions (scaffold roles), deepening our understanding of the protein’s role in signaling networks. PROTACs can degrade membrane-bound proteins associated with various diseases, such as JAK degraders JP-1 and JP-2. PROTACs synthesized using reversible covalent inhibitors as POI recruiters may retain the reversible covalent binding characteristics of the parent inhibitors, slowing displacement by competitors. Rapid synthesis methods for PROTACs have been reported, reducing the time cost of degrader synthesis and facilitating the design of degraders for other targets.

Compared to small molecules, PROTACs offer numerous advantages. However, developing in vivo effective PROTACs remains a challenge for medicinal chemists. Although PROTACs can be viewed as combinations of POI ligands, linkers, and E3 ubiquitin ligase ligands, the aforementioned reports confirm that random combinations do not yield预设 effects. In the design and synthesis of PROTACs, the choice and structural modification of the POI ligand, the chemical composition and length of the linker, and the selection of the E3 ligase ligand can significantly impact their efficacy. Therefore, in-depth structure-activity relationship studies are necessary to discover the most active structures, which may require considerable time. Another critical challenge is the in vivo evaluation of PROTACs. Their large molecular weight places them outside the realm of traditional small molecules, and the flexibility and chemical composition of the linker make them susceptible to in vivo environmental interference, leading to poor stability, as seen with MT802. However, optimization has yielded PROTACs with improved pharmacokinetic parameters and orally available degraders, addressing stability issues to a great extent. In 2019, the first PROTACs entered clinical trials, and several kinase-targeted candidates are expected to follow soon. To date, the use of thalidomide and its analogs as E3 ligase ligands remains the most common approach. However, these agents can lead to degradation of lymphoid transcription factors like IKZF1 and IKZF3, potentially affecting the hematopoietic system. Moreover, cereblon (CRBN) is not essential in most cancer cell lines, and mutations in this ligase could confer resistance to PROTACs. Thus, the discovery of novel E3 ligase ligands is extremely important. In summary, PROTAC technology is a suitable tool for generating active compounds for treating various diseases. Currently, most reported PROTACs are based on well-established positive compounds and commonly used FDA-approved drugs, somewhat limiting the technology’s full potential. This strategy will likely be applied in the coming years to target currently undruggable targets, playing a crucial role in revealing new clinical targets and providing treatments for many diseases. Therefore, although this rapidly growing research field is still in its early stages, it offers encouraging directions for both biological understanding and the future of medicinal chemistry.

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