Synthesis, Evaluation, and Mechanism Study of New Tepotinib Derivatives as Antiproliferative Agents

Abstract

Inspired by the potent inhibition activity of the c-Met (mesenchymal−epithelial transition factor) inhibitor Tepotinib, a series of new Tepotinib derivatives were synthesized and evaluated for their ability to act as antiproliferative agents to find the leading compounds with good activity and limited side effects. Among them, compound 31e exhibited potent antiproliferative activity (IC₅₀ (50% inhibitory concentration) = 0.026 μΜ) against hepatic carcinoma 97H (human liver cancer cell) cells and, importantly, had very low inhibitory activity against normal cells. A mechanism study demonstrated that 31e induced G1 phase (First growth phase or G indicating gap) arrest, inhibited the phosphorylation of c-Met and its downstream signaling component, Akt (Protein Kinase B), and also inhibited the migration of hepatic carcinoma 97H cells.

1. Introduction

Cancer is a major disease that seriously threatens human health. According to statistics from the World Health Organization (WHO), an estimated 9.6 million patients died from cancer in 2018 [1]. Therefore, the development of effective anti-tumor drugs is an important task for medicinal chemists. Mesenchymal−epithelial transition factor (c-Met), the high affinity receptor for hepatocyte growth factor (HGF), is a unique subfamily of receptor tyrosine kinases (RTKs) [2]. Physiologically, HGF/c-Met signaling plays important roles in cell growth, survival, motility, and morphogenesis [3]. However, aberrant c-Met activation has been observed in a wide variety of human cancers, including liver and lung cancer, as a consequence of gene amplification or rearrangement, transcriptional regulation, as well as autocrine or paracrine ligand stimulation [4,5,6,7,8,9]. Due to its status as a proto-oncogene and the correlation of its dysregulation with a poor prognosis, c-Met is becoming a promising target for cancer therapy [10]. The present c-Met kinase inhibitors are divided into three groups according to their binding modes and structural features. Type I c-Met kinase inhibitors, such as Crizotinib A, are adenosine triphosphate (ATP)-competitive inhibitors with a U-shaped binding mode at the ATP binding site (Figure 1) [11,12]. Type II c-Met kinase inhibitors are usually multi-targeted agents (for example, Cabozantinib B) (Figure 1) [13]. Type I inhibitors are generally described as specific for c-Met kinase and more selective than class II inhibitors. However, they have limited activity against the Tyr1230His mutation that is present in certain human tumors [4]. Type III inhibitors include other atypical c-Met kinase inhibitors, such as Tivantinib C (Figure 1) [14].

In 2015, Dorsch et al. reported optimized pyridazinones as being potent and selective c-Met kinase inhibitors [15]. We found that the optimal compound, Tepotinib D (Figure 1), displays excellent in vitro potency and in vivo anti-tumor efficacy at low doses. According to the co-crystal structure of Tepotinib D with c-Met, it belongs to the type I c-Met kinase inhibitors. The review by Manjunath D. Ghate et al. described the recent advances of small molecule c-Met kinase inhibitors, including several pyridazinone derivatives, but the similar structures of our compounds have not been reported before [16]. Inspired by the interesting scaffold of Tepotinib, we decided to synthesize and evaluate new Tepotinib derivatives to find leading compounds with good activity and limited side effects.

Our design is shown in Figure 2 and includes the following steps: (a) Changing the position of nitrogen on the pyridazinone ring to provide pyrazin-2(1H)-one and pyrimidin-2(1H)-one derivatives for the study of the structure–activity relationships; (b) evaluating the anti-tumor activity of the derivatives with or without alkyl substitution on the methylene group; (c) examining the effect of fluorine substitution on pyridin-2(1H)-one on the anti-tumor activity; and (d) investigating the effect of side chains on the activity. Herein, we reported the synthesis, evaluation, and optimization of new Tepotinib derivatives as anti-tumor agents.

2. Results

2.1. Chemistry

The synthesis of target compounds 11a and 11b is summarized in Scheme 1. The conventional Suzuki coupling reaction of (3-(hydroxymethyl)phenyl)boronic acid and 2-chloro-5-fluoropyrimidine catalyzed with PdCl₂(PPh₃)₂ gave (3-(5-fluoropyrimidin-2-yl)phenyl)methanol 5 with a yield of 68%. Chlorination of 5 in the presence of thionyl chloride provided 2-(3-(chloromethyl)phenyl)-5-fluoropyrimidine 6. On the other hand, the oxidation of ketones 1a or 1b followed by cyclization provided 2a and 2b [17], which reacted with compound 6 to give 7a and 7b. Compound 10 was synthesized by the reaction of 8 and 9. Finally, compounds 11a and 11b were prepared by the reaction of 7a, 7b, and 10 under alkaline conditions.

The synthesis of compounds 18, 22, and 25 is shown in Scheme 2. CH₃OCH₂-protected 5-bromopyrimidin-2-ol 14 reacted with (3-cyanophenyl)boronic acid to provide 15, which was converted to 16 in the presence of 10% hydrogen chloride. Compound 16 reacted with intermediate 6 to provide 3-(1-(3-(5-fluoropyrimidin-2-yl)benzyl)-2-oxo-1. 2-dihydropyrimidin-5-yl)benzonitrile 17, which reacted with 10 to give target compound 18. Compound 22 was synthesized from 19 via the Mitsunobu reaction and Suzuki coupling reaction. The reductive amination of 23 with formaldehyde and sodium borohydride gave compound 24, which reacted with compound 16 under alkaline conditions to provide compound 25.

The synthesis of compounds 31a–g is summarized in Scheme 3. Intermediate 27 was prepared by the Suzuki coupling reaction of (3-acetylphenyl)boronic acid and 2-chloro-5-fluoropyrimidine. The reduction of compound 27 with sodium borohydride and then chlorination with thionyl chloride produced 29. In the presence of potassium carbonate, 29 reacted with compound 16 to give 30a, which reacted with the corresponding amino alcohols or amino thiols to produce target compounds 31a–f (the R and S configurations of 31e were prepared and are shown in the supporting information). The fluorine-containing target compound 31g was prepared with a similar procedure, starting from compound 14 and intermediate 30b.

The synthesis of target compounds 36a–c is listed in Scheme 4. Compound 33 was obtained by the reaction of 6 and commercially available 5-bromo-3-fluoropyridin-2(1H)-one 32. Then, the reaction with aryl boric acids 34a–c afforded the key intermediates, 35a–c, which reacted with 10 in the presence of NaOH to provide target compounds 36a–c.

The synthesis of target compound 39 is outlined in Scheme 5. Intermediate 29 reacted with 32 to produce compound 37. Compound 38 was prepared by the Suzuki coupling reaction of 37 and (3-cyanophenyl)boronic acid. Finally, the reaction of compound 38 with 10 afforded target compound 39.

2.2. Biological Evaluation

2.2.1. In Vitro Growth Inhibition of Human Cancer Cell Lines.

Hepatocellular carcinoma (HCC), a primary liver cancer with a high mortality rate, accounts for 90% of all liver cancers. It is the most common malignant tumor worldwide, especially in Asia, Africa, and southern Europe. China is the “hardest hit” by liver cancer, and the number of annual deaths from this condition in China accounts for about 50% of the total number of liver cancer deaths worldwide. C-Met overexpression, mutational activation, and amplification have been found in some types of cancer, including liver cancer [4,5]. Therefore, hepatic carcinoma 97H cells were selected for the evaluation of the anti-tumor activity of the synthesized compounds, and the results are summarized in

Table 1. Structures and antiproliferative activity of target compounds.

Comp.	R1	R2	R3	R4	IC50 of Against 97H cells (µM)
11a	CN	H	-	-	9.810 ± 0.790
11b	F	F	-	-	9.10 ± 0.670
18	CN	H	H		0.443 ± 0.004
22	CN	H	H		0.559 ± 0.018
25	CN	H	H		0.361 ± 0.012
31a	CN	H	CH3		0.186 ± 0.007
31b	CN	H	CH3		0.149 ± 0.012
31c	CN	H	CH3		0.216 ± 0.087
31d	CN	H	CH3		0.196 ± 0.056
(Rac)-31e	CN	H	CH3		0.026 ± 0.003
(R)-31e	CN	H	CH3		0.018 ± 0.006
(S)-31e	CN	H	CH3		1.834 ± 0.026
31f	CN	H	CH3		0.248 ± 0.034
31g	F	F	CH3		0.332 ± 0.044
36a		-	H	-	0.149 ± 0.035
36b		-	H	-	3.807 ± 0.045
36c		-	H	-	0.671 ± 0.056
39		-	CH3	-	0.206 ± 0.013
Tepotinib	-	-	-	-	0.013 ± 0.008

. Most compounds displayed good antiproliferative activity. Among them, compound 31e, 5-(3-cyanophenyl)-1-(1-(3-(5-((1-methylpiperidin-4-yl)methoxy)pyrimidin-2-yl)phenyl)ethyl)pyrimidin-2(1H)-one, exhibited the best result with 26 nM of the IC₅₀ value. Further study of the structure–activity relationship indicated the pyridazinone ring or pyrimidin-2(1H)-one ring plays an important role in antiproliferative activity. Compounds 11a and 11b, pyrazin-2(1H)-one derivatives, that were obtained by shifting the nitrogen from the C-1 to C-5 position of pyridazin-3(2H)-one, displayed poor activity (IC₅₀ values: 9.8 µM for 11a and 9.1 µM for 11b). Interestingly, compounds 18 and 25, which were obtained by shifting the nitrogen from the C-1 to the C-4 position, resulted in significant improvement in anti-proliferative potency (IC₅₀: 0.36–0.55 µM). Compounds 31a–g, which have a methyl substitution on the methylene carbon between pyrimidin-2(1H)-one and the benzene ring, provided 0.3606 µM to 0.026 µM of the IC₅₀ value. This indicates that the methyl substitution at this position is favorable for antiproliferative activity. Compounds Rac-31e and (R)-31e with cyclic piperidinylmethyloxy substitution showed excellent activity (IC₅₀ = 0.026 µM, 0.018 µM). However, the (S)-31e almost lost activity (IC₅₀ = 1.834 µM). The replacement of the pyridazinone moiety with 3-fluoropyridin-2(1H)-one moiety afforded compounds 36a–c. Compound 36a showed the best result of the 3-fluoropyridin-2(1H)-one derivatives, indicating that the benzonitrile group is more favorable than the other two substitutions.

2.2.2. Effect of Compound 31e on Cell Cycle Progression

To evaluate the ability of compound 31e to disrupt the regulated cell cycle distribution, we performed flow cytometry analysis to determine the arrest effect of 31e on the G1 transition, a rigorously regulated process in the cell cycle. The results in Figure 3 show that 31e can arrest cells in the G1 phase in a dose-dependent manner. When hepatic carcinoma 97H cells were treated with 10 nM of 31e for 24 h, the population of cells in the G1 phase (83.84%) increased compared to that of the vehicle (DMSO, 70.42%), along with concomitant losses in the G2/M phase. This phenomenon was more obvious at a concentration of 100 nM. A population of up to 90.88% cells was found in the G1 phase. Correspondingly, a population of only 5.78% cells was found in the G2/M phase.

2.2.3. Inhibition of the Migration of Hepatic Carcinoma 97H Cells with Compound 31e

Tumor cell migration is one of the major causes of death in cancer patients. To evaluate the inhibitory ability of 31e against tumor cell migration, hepatic carcinoma 97H cells (5 × 10⁴ cells per well) were seeded in six-well plates with or without the presence of 31e and cultured as confluent monolayers. The results shown in Figure 4 demonstrate that 31e can inhibit tumor cells’ migration. At a concentration of 10 nM, 31e displayed moderate inhibition of migration. Migration was significantly suppressed when 31e was used at a dosage of 50 nM. Tumor cells’ migration was completely suppressed with 100 nM of 31e.

2.2.4. Inhibition of Phosphorylation of c-Met and its Downstream Signaling Component, Akt, with Compound 31e

To further study the antiproliferative mechanism of compound 31e, we analyzed its effects on the phosphorylation of c-Met and its downstream signaling component, Akt, in hepatic carcinoma 97H cells, which overexpress c-Met. As shown in Figure 5A, when hepatic carcinoma 97H cells were treated with compound 31e at a concentration of 10 or 50 nM for 1 h, the phosphorylation level of Akt and c-Met was suppressed in a dose-dependent manner. When 100 nM of compound 31e was used, the phosphorylation of both c-Met and Akt was completely inhibited. When 97H cells were treated at the same concentration of compound 31e for 2 h, the inhibition of c-Met and Akt phosphorylation was more obvious (Figure 5B).

2.2.5. Selectivity of 31e towards Other Cancer Cells and Normal Human Cells

To evaluate the selectivity of 31e towards other cancer cells and normal human cells, the antiproliferative activities against Pc9 (human non-small cell lung cancer cells), Hela (human epithelial cervical cancer cells), SJSA1 (human osteosarcoma cells), LO2 (human normal cells), and HLF (human embryonic lung fibroblast cells) were examined. As shown in

Table 2. The inhibitory activity of compound 31e against cancer cells (97H, Pc9, Hela, SJSA1), human normal cells (LO2 and HLF), and c-Met enzyme.

IC50 (μM)
Comp.	Pc9	Hela	SJSA1	LO2	HLF	C-Met
31e	42.9 ± 0.13	>50	>50	>50	>50	0.032 ± 0.06
Tepotinib	8.105 ± 0.012	11.22 ± 0.32	12.17 ± 0.35	10.33 ± 0.46	9.44 ± 0.15	0.004 ± 0.001

, compound 31e barely inhibited the cancer cells (Pc9, Hela, SJSA1) and normal cells (LO2, HLF), demonstrating that it exclusively inhibits 97H cancer cells. The reason for this remains to be further investigated.

3. Experimental Section

3.1. Chemistry

¹H-NMR and ¹³C-NMR spectra were recorded on a Bruker AVANCE 400 or 500 spectrometer (Karlsruhe, Germany). Chemical shifts of protons are reported in parts per million downfield from tetramethylsilane. Peaks are labeled as single (s), broad singlet (br), doublet (d), triplet (t), double doublet (dd), doublet of triplets (dt), or multiplet (m). The high-resolution mass spectra were analyzed on a SHIMADZU LCMS-IT-TOF mass spectrometer. The purity of the synthesized compounds was determined by high-performance liquid chromatography (HPLC) (Agilent, Palo Alto, CA, USA) with a TC-C18 column (250 mm × 4.6 mm, 5 μm) using methanol (1/1000 diethylamine)/water mobile phase (0.50 mL/min). Melting points were determined in open capillary tubes on a MPA100 Optimelt automated melting point system (Stanford Research Systems, San Francisco, CA, USA,). All chemicals were purchased from Sigma-Aldrich and Alfa Aesar chemical companies (Shanghai, China) and were used without further purification.

Synthesis of 5-(3-cyanophenyl)-1-(3-(5-((3-(dimethylamino)propyl)thio)pyrimidin-2-yl)benzyl)pyrazin-2(1H)-one (11a). In a 200 mL two-necked round-bottomed flask provided with a magnetic stirrer and condenser, 50 mmol of thiourea 9 were dissolved in 60 mL of absolute ethanol. Compound 8 (3.161 mg, 20 mmol) was added in one portion in the thiourea solution. After heating at reflux for 12 h, the reaction mixture was concentrated by rotary evaporation under reduced pressure. The desired compound 10 was obtained as a white solid and was further used without purification [18].

NaOH (135 mg, 3.375 mmol) in 0.5 mL water was added to a mixture of 7a (287.25 mg, 0.75 mmol) and 10 (444 mg) in DMF (5 mL) under nitrogen, and the mixture was stirred at room temperature for 15 min. Then, the mixture was stirred at 60 °C under nitrogen for 8 h. The reaction mixture was allowed to cool to room temperature. The aqueous phase was extracted with dichloromethane (30 mL × 3). The combined organic layer was washed with H₂O (15 mL) and brine (10 mL), and then dried over anhydrous Na₂SO₄, filtered, and evaporated in vacuo. The residue was purified by flash chromatography over silica gel (DCM/MeOH = 40:1–10:1) to give 5-(3-cyanophenyl)-1-(3-(5-((3-(dimethylamino)propyl)thio)pyrimidin-2-yl)benzyl)pyrazin-2(1H)-one (11a) (224 mg, 62%) as a white solid. m.p. 106.3–107.9 °C. ¹H-NMR (400 MHz, CDCl₃) δ 8.74 (s, 1H), 8.46–8.39 (m, 2H), 8.32 (s, 1H), 8.01 (s, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.64 (s, 1H), 7.58 (d, J = 7.7 Hz, 1H), 7.55–7.47 (m, 3H), 5.27 (s, 2H), 3.03 (t, J = 7.2 Hz, 2H), 2.43 (t, J = 7.0 Hz, 2H), 2.24 (s, 6H), 1.91–1.76 (m, 2H); ¹³C-NMR (101 MHz, CDCl₃) δ 161.18, 157.41, 155.35, 149.17, 138.08, 136.67, 134.95, 131.70, 131.28, 130.82, 129.79, 129.70, 129.08, 128.52, 128.04, 125.10, 118.62, 113.06, 57.87, 52.29, 45.34, 31.60, 27.04. HRMS (ESI) calculated for C₂₇H₂₆N₆OS [M + H]⁺: 483.1962, found: 483.1947. Purity: 98.3% (by HPLC).

5-(3,5-difluorophenyl)-1-(3-(5-((3-(dimethylamino)propyl)thio)pyrimidin-2-yl)benzyl)pyrazin-2(1H)-one (11b). Compound 11b was prepared via a similar procedure of 11a. White solid. m.p. 143.7–145.2 °C. Yield: 65%. ¹H-NMR (500 MHz, CDCl₃) δ 8.74(s, 2H), 8.46–8.39 (m, 2H), 8.30 (s, 1H), 7.56 (s, 1H), 7.54–7.46 (m, 2H), 7.25–7.18 (m, 2H), 6.82–6.65 (m, 1H), 5.25 (s, 2H), 3.03 (t, J = 7.2 Hz, 2H), 2.42 (t, J = 7.0 Hz, 2H), 2.24 (s, 6H), 1.92–1.76 (m, 2H). ¹³C-NMR (126 MHz, CDCl₃) δ 164.52 (J_CF = 247.5 Hz), 163.42 (J_CF = 246.25 Hz), 161.21, 157.45, 155.37, 148.97, 138.74 (J_CF = 10.0 Hz), 138.10, 134.92, 131.64 (J _CF = 3.0 Hz), 130.81, 130.76, 129.76, 128.49, 128.00, 125.03, 107.79 (J_CF = 26.25 Hz), 107.78 (J_CF = 13.75 Hz), 103.21 (J_CF = 26.25 Hz), 57.89, 52.24, 45.38, 31.65, 27.11. HRMS (ESI) calculated for C₂₆H₂₅N₅OF₂S [M + H]⁺: 494.1821, found: 494.1823. Purity: 99.7% (by HPLC).

5-(3-cyanophenyl)-1-(3-(5-((3-(dimethylamino)propyl)thio)pyrimidin-2-yl)benzyl)pyrimidin-2(1H)-one (18). Compound 18 was prepared via a similar procedure of 11a. White oil. Yield: 77.1%. ¹H-NMR (500 MHz, CDCl₃) δ 8.82 (d, J = 3.2 Hz, 1H), 8.69 (s, 2H), 8.42 (s, 1H), 8.38 (d, J = 7.6 Hz, 1H), 7.99 (d, J = 3.2 Hz, 1H), 7.65 (s, 1H), 7.60 (t, J = 7.1 Hz, 2H), 7.55–7.46 (m, 3H), 5.28 (s, 2H), 3.01 (t, J = 7.2 Hz, 2H), 2.40 (t, J = 7.0 Hz, 2H), 2.21 (s, 6H), 1.86–1.76 (m, 2H). ¹³C-NMR (126 MHz, CDCl₃) δ 164.70, 161.23, 157.43, 155.73, 145.11, 138.15, 135.10, 134.64, 131.48, 131.11, 130.93, 130.32, 130.14, 129.83, 129.37, 128.58, 128.18, 118.25, 116.63, 57.95, 54.44, 45.43, 31.67, 27.17. HRMS (ESI) calculated for C₂₇H₂₆N₆OS [M + H]⁺: 483.1962, found: 483.1942. Purity: 98.3% (by HPLC).

Synthesis of 5-(3-cyanophenyl)-1-(3-(5-(3-(dimethylamino)propoxy)pyrimidin-2-yl)benzyl)pyrimidin-2(1H)-one (22). Compound 21a (157.85 mg, 0.55 mmol) and triphenylphosphine (328 mg, 1.25 mmol) were added successively to a suspension of 16 (116.75 mg, 0.5 mmol) in THF (5 mL) under nitrogen. A solution of diisopropyl azodicarboxylate (104.49 mg, 0.6 mmol) in THF (1 mL) was then slowly added dropwise with ice cooling. The resultant solution was stirred at room temperature for 12 h. The aqueous phase was extracted with dichloromethane (30 mL × 3). The combined organic layer was washed with H₂O (20 mL) and brine (10 mL), and then dried over anhydrous Na₂SO₄, filtered, and evaporated in vacuo. The residue was purified by flash chromatography over silica gel (DCM/MeOH = 40:1–10:1) to give 22 [14]. Yellow solid. m.p. 122.3–123.6 °C. Yield: 45%. ¹H-NMR (500 MHz, CDCl₃) δ 8.84 (d, J = 3.0 Hz, 1H), 8.45 (s, 2H), 8.40–8.33 (m, 2H), 7.91 (d, J = 3.0 Hz, 1H), 7.66 –7.57 (m, 3H), 7.56–7.47 (m, 3H), 5.29 (s, 2H), 4.18 (t, J = 6.2 Hz, 2H), 2.53 (t, J = 6.2 Hz, 2H), 2.31 (s, 6H), 2.07–2.00 (m, 2H). ¹³C-NMR (126 MHz, CDCl₃) δ 164.56, 156.67, 155.65, 151.81, 144.80, 143.87, 138.53, 134.73, 134.60, 131.43, 130.25, 130.20, 130.09, 129.76, 129.26, 128.14, 127.78, 118.14, 116.54, 113.67, 67.04, 55.84, 54.31, 45.37, 27.17. HRMS (ESI) calculated for C₂₇H₂₆N₆O₂ [M + H]⁺: 467.2190, found: 467.2185. Purity: 96.784% (by HPLC).

Synthesis of 5-(3-cyanophenyl)-1-(3-(5-((1-methylpiperidin-4-yl)methoxy)pyrimidin-2-yl)benzyl)pyrimidin-2(1H)-one (25). 2-(3-(chloromethyl)phenyl)-5-((1-methylpiperidin-4-yl)methoxy)pyrimidine 24 (99 mg, 0.3 mmol, 1 eq.) and potassium carbonate (124 mg, 0.9 mmol, 3 eq.) were added to a suspension of 16 (77 mg, 0.33 mmol) in dry DMF (2 mL), and the mixture was stirred at 80 °C for 12 h. The reaction mixture was allowed to cool to room temperature. The aqueous phase was extracted with dichloromethane (10 mL × 3). The combined organic layer was washed with H₂O (5 mL) and brine (10 mL), and then dried over anhydrous Na₂SO₄, filtered, and evaporated in vacuo. The residue was purified by flash chromatography over silica gel (DCM/MeOH = 30:1–10:1) to give compound 25 [15]. White solid. m.p. 116.7–118.2 °C. Yield: 48%. ¹H-NMR (500 MHz, CDCl₃) δ 8.84 (d, J = 2.9 Hz, 1H), 8.44 (s, 2H), 8.39–8.33 (m, 2H), 7.89 (d, J = 2.6 Hz, 1H), 7.65–7.60 (m, 2H), 7.60–7.56 (m, 1H), 7.56–7.47 (m, 3H), 5.29 (s, 2H), 3.96 (d, J = 5.6 Hz, 2H), 2.98 (d, J = 11.1 Hz, 2H), 2.34 (s, 3H), 2.04 (t, J = 11.8 Hz, 2H), 1.89–1.81 (m, 3H), 1.54–1.46 (m, 2H). ¹³C-NMR (126 MHz, CDCl₃) δ 164.56, 156.70, 155.63, 151.85, 144.72, 143.82, 138.52, 134.73, 134.61, 131.43, 130.25, 130.22, 130.08, 129.77, 129.26, 128.15, 127.77, 118.13, 116.53, 113.68, 73.28, 55.16, 54.30, 46.21, 35.17, 28.69. HRMS (ESI) calculated for C₂₉H₂₈N₆O₂ [M + H]⁺: 493.2347, found: 493.2351. Purity: 97.8% (by HPLC).

5-(3-cyanophenyl)-1-(1-(3-(5-((2-(dimethylamino)ethyl)thio)pyrimidin-2-yl)phenyl)ethyl)pyrimidin-2(1H)-one (31a). Compound 31a was prepared via a similar procedure of 11a. Yellow oil. Yield: 66%. ¹H NMR (400 MHz, CDCl₃) δ 8.78 (d, J = 3.3 Hz, 1H), 8.74 (s, 2H), 8.48 (s, 1H), 8.42 (d, J = 7.5 Hz, 1H), 7.71 (d, J = 3.3 Hz, 1H), 7.63–7.53 (m, 3H), 7.52–7.45 (m, 3H), 6.35 (q, J = 6.9 Hz, 1H), 3.08 (t, J = 7.1 Hz, 2H), 2.60 (t, J = 7.1 Hz, 2H), 2.28 (s, 6H), 1.90 (d, J = 7.0 Hz, 3H). ¹³C-NMR (126 MHz, CDCl₃) δ 164.01, 161.33, 157.54, 155.44, 142.40, 138.86, 138.26, 134.89, 131.45, 130.97, 130.31, 130.15, 129.84, 129.32, 128.64, 126.92, 118.22, 116.76, 113.72, 58.36, 56.32, 45.37, 32.02, 19.19. HRMS (ESI) calculated for C₂₇H₂₆N₆OS [M + H]⁺: 483.1962, found: 483.1949. Purity: 98% (by HPLC).

5-(3-cyanophenyl)-1-(1-(3-(5-((3-(dimethylamino)propyl)thio)pyrimidin-2-yl)phenyl)ethyl)pyrimidin-2(1H)-one (31b). Compound 31b was prepared via a similar procedure of 11a. Yellow oil. Yield: 75%. ¹H NMR (500 MHz, CDCl₃) δ 8.78 (d, J = 3.2 Hz, 1H), 8.73 (s, 2H), 8.49 (s, 1H), 8.42 (d, J = 7.6 Hz, 1H), 7.70 (d, J = 3.2 Hz, 1H), 7.63–7.58 (m, 1H), 7.57–7.46 (m, 5H), 6.36 (q, J = 6.9 Hz, 1H), 3.03 (t, J = 7.2 Hz, 2H), 2.42 (t, J = 7.0 Hz, 3H), 2.23 (s, 6H), 1.91 (d, J = 7.0 Hz, 3H), 1.88–1.77 (m, 2H). ¹³C-NMR (126 MHz, CDCl₃) δ 164.04, 161.27, 157.50, 155.49, 142.43, 138.82, 138.32, 134.88, 131.49, 131.02, 130.33, 130.31, 130.18, 129.86, 129.34, 128.66, 126.92, 118.24, 116.83, 113.74, 57.97, 56.37, 45.46, 31.70, 27.18, 19.21. HRMS (ESI) calculated for C₂₈H₂₈N₆OS [M + H]⁺: 497.2118, found: 497.2108. Purity: 97.1% (by HPLC).

Synthesis of 5-(3-cyanophenyl)-1-(1-(3-(5-(3-(dimethylamino)propoxy)pyrimidin-2-yl)phenyl)ethyl)pyrimidin-2(1H)-one (31c). A solution of 3-(dimethylamino)propan-1-ol (113.3 mg, 1.1 mmol, 2 eq.) in dry DMF( 5 mL) was added to a suspension of NaH (43.56 mg, 3.3 eq.) in dry DMF (5 mL) at 0 °C. After 30 mins, 5-(3-cyanophenyl)-1-(1-(3-(5-fluoropyrimidin-2-yl)phenyl)ethyl)pyrimidin-2(1H)-one 30a (210 mg, 0.55 mmol, 1 eq.) was added. After the reaction mixture was stirred for 5 h, the reaction was quenched with water (15 mL) and the mixture was extracted with dichloromethane (20 mL × 3). The combined organic layer was dried over anhydrous Na₂SO₄ and filtered. After the solvent was removed in vacuo, the crude product was purified by column chromatography (DCM/MeOH = 50:1–10:1) to afford 31c as a white solid [19]. m.p. 68.3–69.9 °C. Yield: 62%. ¹H-NMR (400 MHz, CDCl₃) δ 8.78 (d, J = 3.4 Hz, 1H), 8.46 (s, 2H), 8.42 (s, 1H), 8.35 (d, J = 7.7 Hz, 1H), 7.73 (d, J = 3.4 Hz, 1H), 7.64–7.58 (m, 1H), 7.56–7.42 (m, 5H), 6.35 (q, J = 7.0 Hz, 1H), 4.21 (t, J = 6.1 Hz, 2H), 2.75 (t, J = 7.3 Hz, 2H), 2.47 (s, 6H), 2.24–2.09 (p, 2H), 1.90 (d, J = 7.0 Hz, 3H). ¹³C-NMR (101 MHz, CDCl₃) δ 163.98, 156.99, 155.48, 151.75, 144.00, 142.53, 138.71, 138.62, 134.92, 131.44, 130.32, 130.19, 129.77, 129.44, 129.35, 128.22, 126.55, 118.28, 116.76, 113.69, 66.75, 56.44, 55.79, 44.79, 26.49, 19.24. HRMS (ESI) calculated for C₂₈H₂₈N₆O₂ [M + HCOO]⁻: 525.2256, found: 525.2238. Purity: 98.3% (by HPLC).

5-(3-cyanophenyl)-1-(1-(3-(5-(2-(dimethylamino)ethoxy)pyrimidin-2-yl)phenyl)ethyl)pyrimidin-2(1H)-one (31d). Compound 31d was prepared via a similar procedure of 31c. White solid. m.p. 53.3-55.0 °C. Yield: 74%. ¹H-NMR (400 MHz, CDCl₃) δ 8.80 (d, J = 3.3 Hz, 1H), 8.51 (s, 2H), 8.45 (s, 1H), 8.38 (d, J = 7.7 Hz, 1H), 7.72 (d, J = 3.4 Hz, 1H), 7.64–7.58 (m, 1H), 7.57–7.42 (m, 5H), 6.38 (q, J = 7.0 Hz, 1H), 4.24 (t, J = 5.4 Hz, 2H), 2.83 (t, J = 5.4 Hz, 2H), 2.40 (s, 6H), 1.91 (d, J = 7.0 Hz, 3H). ¹³C-NMR (101 MHz, CDCl₃) δ 163.94, 156.94, 155.45, 151.78, 144.05, 142.52, 138.67, 138.59, 134.89, 131.40, 130.27, 130.15, 129.73, 129.44, 129.32, 128.19, 126.53, 118.24, 116.72, 113.66, 66.88, 58.10, 56.40, 45.90, 19.18. HRMS (ESI) calculated for C₂₇H₂₆N₆O₂ [M + HCOO]⁻: 511.2099, found: 511.2074. Purity: 98% (by HPLC).

5-(3-cyanophenyl)-1-(1-(3-(5-((1-methylpiperidin-4-yl)methoxy)pyrimidin-2-yl)phenyl)ethyl)pyrimidin-2(1H)-one (31e). Compound 31e was prepared via a similar procedure of 31c. Yellow solid. m.p. 98.2–99.9 °C. Yield: 76%. ¹H-NMR (500 MHz, DMSO-d₆) δ 9.04 (d, J = 3.2 Hz, 1H), 8.72 (d, J = 3.2 Hz, 1H), 8.64 (s, 2H), 8.33 (s, 1H), 8.27–8.18 (m, 2H), 8.03 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 7.7 Hz, 1H), 7.65 (t, J = 7.8 Hz, 1H), 7.59–7.45 (m, 2H), 6.02 (q, J = 7.0 Hz, 1H), 4.04 (d, J = 5.8 Hz, 2H), 2.80 (d, J = 11.1 Hz, 2H), 2.16 (s, 3H), 1.92 (d, J = 7.2 Hz, 3H), 1.87 (d, J = 11.3 Hz, 2H), 1.78–1.67 (m, 3H), 1.36–1.27 (m, 2H). ¹³C-NMR (101 MHz, CDCl₃) δ 163.97, 156.82, 155.49, 151.98, 143.94, 142.52, 138.66, 134.92, 131.44, 130.30, 130.17, 129.77, 129.44, 129.34, 128.21, 126.52, 118.26, 116.75, 113.71, 73.39, 56.42, 55.30, 46.35, 35.26, 28.81, 19.21. HRMS (ESI) calculated for C₃₀H₃₀N₆O₂ [M + HCOO]⁻: 551.2412, found: 551.2402. Purity: 98.9% (by HPLC).

5-(3-cyanophenyl)-1-(1-(3-(5-(2-morpholinoethoxy)pyrimidin-2-yl)phenyl)ethyl)pyrimidin-2(1H)-one (31f). Compound 31f was prepared via a similar procedure of 31c. White solid. m.p. 82.3–83.9 °C. Yield: 68%. ¹H-NMR (400 MHz, CDCl₃) δ 8.78 (d, J = 3.4 Hz, 1H), 8.47 (s, 2H), 8.43 (s, 1H), 8.35 (d, J = 7.7 Hz, 1H), 7.72 (d, J = 3.4 Hz, 1H), 7.62–7.56 (m, 1H), 7.56–7.42 (m, 5H), 6.35 (q, J = 7.0 Hz, 1H), 4.25 (t, J = 5.5 Hz, 2H), 3.79–3.66 (m, 4H), 2.85 (t, J = 5.5 Hz, 2H), 2.68–2.50 (m, 4H), 1.89 (d, J = 7.0 Hz, 3H). ¹³C-NMR (101 MHz, CDCl₃) δ 163.95, 157.05, 155.45, 151.72, 144.13, 142.50, 138.70, 138.56, 134.91, 131.42, 130.28, 130.15, 129.75, 129.46, 129.33, 128.21, 126.56, 118.25, 116.72, 113.68, 66.87, 66.77, 57.53, 56.39, 54.17, 19.21. HRMS (ESI) calculated for C₂₉H₂₈N₆O₃ [M + HCOO]⁻: 553.2205, found: 553.2181. Purity: 99.6% (by HPLC).

5-(3,5-difluorophenyl)-1-(1-(3-(5-((1-methylpiperidin-4-yl)methoxy)pyrimidin-2-yl)phenyl)ethyl)pyrimidin-2(1H)-one (31g). Compound 31g was prepared via a similar procedure of 31c. White solid. m.p. 86.9–88.6 °C. Yield: 55%. ¹H-NMR (500 MHz, CDCl₃) δ 8.76 (d, J = 3.2 Hz, 1H), 8.43 (s, 2H), 8.42 (s, 2H), 8.35 (d, J = 7.7 Hz, 1H), 7.67 (d, J = 3.3 Hz, 1H), 7.51 (t, J = 7.7 Hz, 1H), 7.47–7.39 (m, 1H), 6.82–6.69 (m, 3H), 6.35 (q, J = 6.9 Hz, 1H), 3.95 (d, J = 5.7 Hz, 2H), 3.00 (d, J = 11.2 Hz, 2H), 2.35 (s, 3H), 2.08 (t, J = 11.5 Hz, 2H), 1.93–1.77 (m, 6H), 1.60–1.49 (m, 2H). ¹³C-NMR (126 MHz, CDCl₃) δ 163.92, 163.665(J_CF = 248.75 Hz), 163.565(J_CF = 248.75 Hz), 156.88, 155.54, 151.94, 143.92, 142.42, 138.69, 138.65, 136.67 (J_CF = 10.0 Hz), 129.74, 129.36, 128.18, 126.59, 116.74 (J_CF = 2.2 Hz),108.83 (J_CF = 12.5 Hz), 108.825(J_CF = 26.25 Hz), 103.41 (J_CF = 25.2 Hz), 73.28, 56.39, 55.20, 46.15, 35.17, 28.61, 19.18. HRMS (ESI) calculated for C₂₉H₂₉N₅O₂F₂ [M + HCOO]⁻: 562.2271, found: 562.2248. Purity: 99.4% (by HPLC).

5-(3-cyanophenyl)-1-(3-(5-((3-(dimethylamino)propyl)thio)pyrimidin-2-yl)benzyl)-3-fluoropyridin-2(1H)-one (36a). Compound 36a was prepared via a similar procedure of 11a. Yellow solid. m.p. 116.3–117.9 °C. Yield: 72%. ¹H-NMR (400 MHz, CDCl₃) δ 8.74 (s, 1H), 8.43 (s, 1H), 8.41–8.37 (m, 1H), 7.64 (s, 1H), 7.62–7.52 (m, 3H), 7.52–7.49 (m, 2H), 7.45–7.41 (m, 1H), 7.37 (dd, J = 9.8, 2.3 Hz, 1H), 5.36 (s, 2H), 3.03 (t, J = 7.2 Hz, 2H), 2.45 (t, J = 7.0 Hz, 2H), 2.26 (s, 6H), 1.93–1.80 (m, 2H). ¹³C-NMR (126 MHz, CDCl₃) δ 161.35, 157.48, 155.62 (J_CF = 26.25 Hz), 152.58 (J_CF = 251.25 Hz), 137.92, 137.05, 135.88, 131.10, 130.72, 130.68, 130.23, 130.16(J_CF = 3.75 Hz), 130.06, 129.66, 129.42, 128.25, 127.85, 119.54 (J_CF = 17.5 Hz), 118.29, 116.57 (J_CF = 5.0 Hz), 113.46, 57.86, 52.50, 45.31, 31.65, 27.03. HRMS (ESI) calculated for C₂₈H₂₆N₅OFS [M + H]⁺: 500.1915, found: 500.1898. Purity: 98.1% (by HPLC).

1-(3-(5-((3-(dimethylamino)propyl)thio)pyrimidin-2-yl)benzyl)-5,5′-difluoro-[3,3′-bipyridin]-6(1H)-one (36b). Compound 36b was prepared via a similar procedure of 11a. White solid. m.p. 128.3–129.9 °C. Yield: 75%. ¹H-NMR (400 MHz, CDCl₃) δ 8.74 (s, 2H), 8.47–8.36 (m, 4H), 7.51 (d, J = 5.1 Hz, 2H), 7.45–7.42 (m, 1H), 7.40–7.33 (m, 2H), 5.37 (s, 2H), 3.04 (t, J = 7.2 Hz, 2H), 2.44 (t, J = 7.0 Hz, 2H), 2.25 (s, 6H), 1.91–1.79 (m, 2H). ¹³C-NMR (126 MHz, CDCl₃) δ 161.29, 159.52 (J_CF = 257.50 Hz), 157.52, 157.45, 155.59 (J_CF = 26.25 Hz), 152.66 (J_CF = 252.50 Hz), 142.80 (J_CF = 3.75 Hz), 137.96, 137.20 (J_CF = 23.75Hz), 135.75, 130.77, 130.73, 130.36 (J_CF = 5.0 Hz), 129.65, 128.26, 127.88, 120.01 (J_CF = 18.75 Hz), 119.33 (J_CF = 18.75 Hz), 113.96 (J_CF = 6.25 Hz), 57.91, 52.48, 45.40, 31.69, 27.16. HRMS (ESI) calculated for C₂₆H₂₅N₅OF₂S [M + H]⁺: 494.1821, found: 494.1807. Purity: 99.7% (by HPLC).

1-(3-(5-((3-(dimethylamino)propyl)thio)pyrimidin-2-yl)benzyl)-3-fluoro-5-(pyrimidin-5-yl)pyridin-2(1H)-one (36c). Compound 36c was prepared via a similar procedure of 11a. White solid. 128.9–129.8 °C. Yield: 78%. ¹H-NMR (400 MHz, CDCl₃) δ 9.16 (s, 1H), 8.75 (s, 2H), 8.74 (s, 2H), 8.45–8.37 (m, 2H), 7.51 (d, J = 5.1 Hz, 2H), 7.44 (s, 1H), 7.36 (dd, J = 9.5, 2.3 Hz, 1H), 5.37 (s, 2H), 3.04 (t, J = 7.2 Hz, 2H), 2.45 (t, J = 7.0 Hz, 2H), 2.26 (s, 6H), 1.93–1.80 (m,2H). ¹³C-NMR (126 MHz, CDCl₃) δ 161.06, 157.52, 157.25, 155.40 (J_CF = 25.7 Hz), 153.68, 151.64, 137.82, 135.46, 131.62 (J_CF = 10.2 Hz), 130.63, 130.16 (J_CF = 4.6 Hz), 129.50, 128.71 (J_CF = 12.5 Hz), 128.14, 127.73, 118.68 (J_CF = 18.2 Hz), 111.65 (J_CF = 5.5 Hz), 57.73, 52.35, 45.21, 31.49, 26.96. HRMS (ESI) calculated for C₂₅H₂₅N₆OFS [M + H]⁺: 477.1867, found: 477.1857. Purity: 98.9% (by HPLC).

5-(3-cyanophenyl)-1-(1-(3-(5-((3-(dimethylamino)propyl)thio)pyrimidin-2-yl)phenyl)ethyl)-3-fluoropyridin-2(1H)-one (39). Compound 39 was prepared via a similar procedure of 11a. Colorless oil. Yield: 82%. ¹H-NMR (400 MHz, CDCl₃) δ 8.75 (s, 2H), 8.49 (s, 1H), 8.40 (d, J = 7.6 Hz, 1H), 7.60–7.43 (m, 6H), 7.32 (dd, J = 9.8, 2.3 Hz, 1H), 7.22 (s, 1H), 6.62 (q, J = 7.0 Hz, 1H), 3.04 (t, J = 7.2 Hz, 2H), 2.47 (t, J = 7.0 Hz, 2H), 2.27 (s, 6H), 1.91–1.81 (m, 5H). ¹³C-NMR (101 MHz, CDCl₃) δ 161.36, 157.45, 155.42(J_CF = 25.0 Hz), 152.15(J_CF = 251.0 Hz), 139.61, 137.97, 137.32, 130.98, 130.76, 130.21, 130.03, 130.01, 129.53, 129.40, 128.20, 127.26 (J_CF = 5.0 Hz), 126.64, 119.00 (J_CF = 18.0 Hz), 118.33, 116.57 (J_CF = 6.0 Hz), 113.37, 57.88, 53.78, 45.34, 31.65, 27.08, 19.23. HRMS (ESI) calculated for C₂₉H₂₈N₅OFS [M + H]⁺: 514.2071, found: 514.2066. Purity: 99% (by HPLC).

3.2. Biological Assay

3.2.1. Cell lines and Culture

The cell lines, 97H (human liver cancer cell), Pc9 (human non-small cell lung cancer), HELA (human epithelial cervical cancer cell line), SJSA1 (human osteosarcoma cells), LO2 (human normal liver cells), and HLF (human embryonic lung fibroblast), used in this study were purchased from the Guangzhou ginny ou Biotechnology Co. Ltd. (Guangzhou, China). Cell lines were cultivated in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin.

3.2.2. Kinase Inhibition Assay.

The c-Met enzymes and the Z’-LYTE Kinase Kit were purchased from Invitrogen. The tested compounds were used in concentrations of 0.001 to 10 mM. Briefly, 1 μL of an inhibitor or 5% DMSO, 2 μL of enzyme, and 2 μL of substrate/ATP mix were added to a 384-well low volume plate L and incubated at room temperature for 60 min. Then, 5 μL of ADP-Glo™ Reagent was added, and the mixture was incubated at room temperature for 40 min. Finally, 10 μL of Kinase Detection Reagent was added, and the mixture was incubated at room temperature for 30 min. The plate was measured on a multifunction microplate reader (Molecular Devices, Flex Station 3), and the luminescence was recorded (integration time 0.5–1 s). Curve fitting and data presentations were performed using Graph Pad Prism version 5.0 (GraphPad Inc., La Jolla, CA, USA). Every experiment was repeated at least three times.

3.2.3. MTT (Thiazolyl Blue Tetrazolium Bromide) Assay.

For the cytotoxicity assay, the cells grown in the logarithmic phase were seeded into 96-well plates (5 × 10³ cells/well) for 24 h, and then exposed to different concentrations of the test compounds for 48 h. After the attached cells had been incubated with 5 mg/mL MTT (Sigma, St. Louis, MO, USA) for another 4 h, the suspension was discarded and subsequently the dark blue crystals (formazan) were solubilized in dimethyl sulfoxide (DMSO). Then, the absorbance was measured at 570 nm using a multifunction microplate reader (Molecular Devices, Flex Station 3) (Thermo Fisher Scientific, San Jose, CA, USA), and each experiment was performed at least in triplicate. The cytotoxic effects of each compound were expressed as the IC₅₀ values, which represents the drug concentration required to cause 50% tumor cell growth inhibition. This was calculated with GraphPad Prism Software version 5.02 (GraphPad Inc., La Jolla, CA, USA).

3.2.4. Western Blot Analysis.

97H cells seeded in 60-mm dishes at a density of 5 × 10⁵ cells/well were incubated with or without compound 31e at the indicated concentrations for 4 h. After incubation, the cells were washed twice with ice-cold PBS, and then lysed in RIPA lysis buffer (Radio Immunoprecipitation Assay) containing 150 mM NaCl, 50 mM Tris (pH 7.4), 1% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, and 1 mM EDTA (ethylenediaminetetraacetic acid) (Beyotime, Shanghai, China). The lysates were incubated at 0 °C for 30 min, and vortexed every 10 min intermittently, then the total proteins were harvested by centrifuging at 12,500 g for 15 min. The protein concentrations were determined by a BCA (bicinchoninic acid) Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA), and then the protein extracts were reconstituted in loading buffer containing 62 mM Tris-HCl, 2% SDS (sodium dodecyl sulfate), 10% glycerol, and 5% β-mercaptoethanol (Beyotime, Shanghai, China), and boiled at 100 °C for 3 min. An equal amount of the proteins (40 μg) was separated by 8%–12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to nitrocellulose membranes (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). After blocking with 5% non-fat dried milk in TBS containing 1% Tween-20 for 90 min at room temperature, the membranes were incubated overnight with specific primary antibodies (Abcam, London, England) at 4 °C. After three washes in TBST, the membranes were incubated with the appropriate HRP-conjugated (Horseradish Peroxidase-conjugated) secondary antibodies at room temperature for 2 h. The blots were developed with enhanced chemiluminescence (Pierce, Rockford, IL, USA) and detected by an LAS4000 imager (GE Healthcare, Waukesha, WI, USA).

3.2.5. Cell Cycle Analysis

97H cells were seeded in 6-well plates (3 × 10⁵ cells/well) and incubated in the presence or absence of compound 31e at the indicated concentrations for 24 h. Then, cells were harvested by centrifugation and fixed in 70% ice-cold ethanol overnight. The ethanol was removed the next day, and then the cells were resuspended in the ice-cold PBS and treated with RNAse A (Keygen Biotech, Nanjing, China) at 37 °C for 30 min, followed by incubation with the DNA staining solution propidium iodide (PI) (Keygen Biotech, Nanjing, China) at 4 °C for 30 min. About 10,000 events were detected by flow cytometry (Epics XL, Beckman Coulter, Fullerton, CA, USA) at 488 nm. The data regarding the number of cells in different phases of the cell cycle were analyzed by EXPO32 ADC analysis software (Beckman Coulter, Fullerton, CA, USA).

3.2.6. Anti-Cell-Migration Study

97H cells were plated in a 6-well culture dish at 5 × 10⁴ cells/dish and grown for 24 h, and the non-migrated cells were scraped off the upper surface of the membrane with a 10 μL pipette. The medium was then replaced with 10% serum DMEM medium and treated with compound 31e at the indicated concentrations for another 36 h. After washing with phosphate buffer solution (PBS), the cell images were immediately detected by a Zeiss LSM 570 laser scanning confocal microscope (Carl Zeiss, Jena, Germany).

4. Conclusions

We synthesized a series of new Tepotinib derivatives to evaluate their ability to act as antiproliferative agents. The optimal compound, 31e, in which pyridazinone is replaced with the pyrimidin-2(1H)-one moiety and methyl substitution on the methylene moiety of Tepotinib is introduced, exhibited potent antiproliferative activity (IC₅₀: 0.026 μΜ) against hepatic carcinoma 97H cells. The mechanism study indicated that 31e induces G1 phase arrest and inhibits the phosphorylation of c-Met and its downstream signaling component, Akt, in a dose-dependent manner. Overall, the current study demonstrates that 31e is a promising leading compound in the development of anticancer agents. Further structural optimization and the evaluation of its in vivo anti-tumor activity are in progress.