Design, synthesis and pharmacological evaluation of new 2-oxo-quinoline derivatives containing α-aminophosphonates as potential antitumor agents

Novel 2-oxo-quinoline derivatives containing α-aminophosphonates were synthesized as antitumor agents. Compound 5b blocked HepG2 cell cycle at G₂/M phase and induced apoptosis in mitochondrial pathway.

Abstract

A series of novel 2-oxo-quinoline derivatives containing α-aminophosphonates were designed and synthesized as antitumor agents. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay results demonstrated that some compounds exhibited moderate to high inhibitory activity against HepG2, SK-OV-3 and NCI-H460 tumor cell lines, and most compounds showed much lower cytotoxicity against HL-7702 normal cells than 5-FU and cisplatin. The action mechanism of representative compound 5b was investigated by fluorescence staining assay, flow cytometric analysis and western blot (WB) assay, which indicated that this compound induced apoptosis and G₂/M phase arrest accompanied by an increase in the production of intracellular Ca²⁺ and reactive oxygen species (ROS) and affecting associated enzymes and genes.

1. Introduction

It is well known that cancer is a global health problem and one of the main causes of mortality worldwide. In order to prevent and combat this disease, a great deal of effort has been devoted to the design and synthesis of new antitumor drugs with high efficiency and low toxicity.

Quinoline alkaloids exhibit a variety of outstanding biological activities, including inhibition of cellular proliferation and developmental changes. So the design and synthesis of new quinoline derivatives with better antitumor activity has attracted many chemists' interest. As a family of quinolines, 2-oxo-quinoline derivatives display potent proliferation inhibitory activity on tumor cells.1,2 Our previous work has also demonstrated that 2-oxo-quinoline Schiff base derivatives exhibited good antioxidant activity and obvious nontoxicity on bald mice.3,4 In addition, antioxidants are fabricated as drug candidates to counter these multifarious chronic diseases, including carcinogenesis, atherogenesis and aging,5 and many antioxidants exhibit efficient antitumor activity.6,7 For instance, the powerful antioxidant resveratrol is also deemed to be an antitumor agent.7 Moreover, 2-oxo-quinoline Schiff base derivatives (Fig. 1) show a similar structure with resveratrol and can be considered as its bioisosteric derivatives. So it is considered that 2-oxo-quinoline Schiff base derivatives may exhibit potent proliferation inhibitory activity. Furthermore, our previous work has demonstrated that the introduction of an aminophosphonate group to a pharmacophore is able to increase the antitumor activity and many aminophosphonate derivatives have exhibited potent inhibition activities against human tumors.8,9 It is thus expected that the combination of 2-oxo-quinoline Schiff base derivatives and aminophosphonate (APA) groups may lead to good antitumor activity and low toxicity. In our previous work, we have described the synthesis, antitumor activity, preliminary apoptosis-inducing and cycle-arresting effects of some 2-oxo-quinoline APA derivatives.10 However, to the best of our knowledge, the scale and mode of the synthesis are still limited. Particularly, the cytotoxicity on normal cells (i.e., toxicity) and the further apoptosis-inducing and cycle-arresting mechanism of 2-oxo-quinoline Schiff base derivatives with APA moieties have not been reported. Therefore, in the present work, as a continuation of our previous work, we introduced some APA moieties into different 2-oxo-quinoline Schiff base skeletons and evaluated the in vitro cytotoxicity of the target compounds. Moreover, the mechanism of apoptosis and cycle arrest was further deeply and systematically investigated.

Fig. 1. The chemical structures of 2-oxo-quinoline Schiff base derivatives and resveratrol.

2. Results and discussion

2.1. Chemistry

2-Oxo-quinoline derivatives bearing APA moieties (compounds 4 and 5) were synthesized as shown in Scheme 1. Firstly, 2-chloroquinoline-3-carbaldehyde derivatives 2 (2a–2d) were obtained via the Vilsmeier–Haack–Arnold reaction according to our previous work,3 which included the condensation of acetanilide derivatives 1 (1a–1d) with N,N-dimethylformamide (DMF) in the presence of phosphorus oxychloride. 2-Oxo-quinoline 3-carbaldehyde derivatives 3 (3a–3d) were then obtained in good yields by the hydrolytic reaction of 2 (2a–2d) in the presence of 70% acetic acid aqueous solution.3 The target compounds 4 (4a₁–4d₇) were synthesized by the Kabachnik–Fields reaction, which was carried out by the simultaneous condensation of compounds 3, diethyl phosphate and many kinds of amines, through a one-pot three-component synthesis method. The other target compounds 5 (5a–5d) were also synthesized by the condensation of compounds 3 with diethyl 4-aminobenzylphosphonate. The structures of target compounds 4 (4a₁–4d₇) and 5 (5a–5d) were confirmed using various spectroscopic methods, including ¹H NMR, ¹³C NMR and high-resolution mass spectrometry (HR-MS) (part 3 of the ESI‡). In order to better understand the structures of these target compounds, the crystal structures of compounds 4c₅, 4d₂ and 5c were also determined by X-ray diffraction (Fig. 2, for selected relevant parameters, see part 1 of the ESI‡). As shown in Fig. 2, the crystal structures of compounds 4c₅, 4d₂ and 5c were well consistent with the characterization data of NMR and HR-MS, indirectly confirming the structures of target compounds 4 and 5.

Scheme 1. General synthetic route to compounds 3–5. Reagents and conditions: (a) POCl₃/DMF; (b) 70% acetic acid aqueous solution; (c) diethyl phosphate, amines, CH₃CN; (d) diethyl 4-aminobenzylphosphonate, CH₃CN.

Fig. 2. The crystal structures of compounds 4c₅, 4d₂ and 5c.

2.2. Biological activity

2.2.1. Cytotoxicity test

The in vitro cytotoxicity of compounds 4 (4a₁–4d₇) and 5 (5a–5d) was evaluated by methyl thiazolyl tetrazolium (MTT) assay against HepG2 (human liver cancer cell line), SK-OV-3 (human ovarian cancer cell line), NCI-H460 (human large cell lung cancer cell line) and HL-7702 (human liver normal cell line) cell lines. Two commercial anticancer drugs 5-fluorouracil (5-FU) and cisplatin were used as positive controls. The results are shown in Table 1.

Table 1. IC₅₀ values of 2-oxo-quinoline derivatives bearing APA moieties (4 and 5) towards three selected tumor cell lines and a normal cell line for 48 h ^a .

Compounds	IC50 (μM)
	HepG2	SK-OV-3	NCI-H460	HL-7702
4a1	61.43 ± 2.14	38.30 ± 1.74	>100	>100
4a2	67.27 ± 2.35	66.75 ± 2.48	>100	>100
4a3	65.46 ± 2.64	55.95 ± 1.11	>100	>100
4a4	46.33 ± 2.02	54.11 ± 1.18	>100	>100
4a5	75.21 ± 3.74	29.98 ± 0.91	>100	>100
4a6	37.21 ± 2.23	40.95 ± 0.85	>100	>100
4a7	39.12 ± 2.11	25.81 ± 0.54	>100	>100
4b1	39.17 ± 3.12	130.26 ± 4.59	>100	>100
4b2	37.57 ± 1.45	37.22 ± 1.34	>100	>100
4b3	45.95 ± 1.78	342.40 ± 4.26	>100	>100
4b4	39.56 ± 1.54	69.37 ± 1.08	>100	>100
4b5	35.61 ± 2.73	>200	>100	>100
4b6	46.21 ± 2.11	30.33 ± 0.66	>100	>100
4b7	68.11 ± 2.64	191.26 ± 4.02	>100	>100
4c1	43.25 ± 3.35	58.21 ± 1.75	>100	>100
4c2	57.63 ± 2.75	59.96 ± 2.77	>100	>100
4c3	45.54 ± 2.85	73.011 ± 2.29	>100	>100
4c4	60.77 ± 3.01	54.66 ± 1.95	>100	>100
4c5	20.80 ± 1.65	25.13 ± 1.04	44.56 ± 2.56	>100
4c6	58.92 ± 2.27	269.696 ± 5.12	>100	>100
4c7	40.15 ± 1.88	102.11 ± 4.39	>100	>100
4d1	46.27 ± 2.66	37.23 ± 1.95	47.86 ± 2.05	>100
4d2	43.11 ± 1.17	48.89 ± 1.53	>100	>100
4d3	>100	>100	>100	>100
4d4	45.21 ± 3.14	64.95 ± 2.43	>100	>100
4d5	44.01 ± 1.31	28.23 ± 0.75	>100	>100
4d6	37.95 ± 1.98	26.25 ± 0.87	59.56 ± 2.75	>100
4d7	78.95 ± 4.45	22.36 ± 0.45	>100	>100
5a	17.85 ± 0.68	35.97 ± 0.98	57.88 ± 2.24	>100
5b	9.99 ± 0.25	34.76 ± 1.14	38.96 ± 1.88	>100
5c	17.31 ± 1.13	52.991 ± 1.61	>100	>100
5d	28.92 ± 1.47	47.92 ± 1.14	>100	>100
3a	>200	>200	>200	>100
3b	>200	>200	>200	>100
3c	>200	>200	>200	>100
3d	>200	>200	>200	>100
5-FU	31.98 ± 0.56	26.34 ± 0.57	45.44 ± 0.94	58.74 ± 2.31
Cisplatin	10.12 ± 0.71	15.60 ± 1.70	20.36 ± 0.50	15.67 ± 0.32

^aIC₅₀ values are presented as the mean ± SD (standard error of the mean) from three separate experiments.

As shown in Table 1, most of the target compounds 4 and 5 displayed much higher inhibitory activity than their corresponding 2-oxo-quinoline 3-carbaldehyde derivatives 3 (3a–3d) against the HepG2, SK-OV-3 and NCI-H460 cell lines, indicating that the introduction of α-aminophosphonates on 2-oxo-quinoline may improve the antitumor activity. It was important to note that some target compounds showed better cytotoxic inhibition against these three cell lines than 5-FU, while they displayed lower cytotoxicity on the HL-7702 normal cell line than the commercial anticancer drugs 5-fluorouracil (5-FU) and cisplatin, indicating that they may be good candidates for anti-tumour drugs. Table 1 also demonstrates that, for compound 4, both the substituents (R₁) in the 2-oxo-quinoline group and the substituents (R₂) in the α-aminophosphonate moiety had an important influence on the cytotoxic inhibition, though the influence was irregular. In addition, by the comparison of the cytotoxic inhibition activity of compounds 4 and 5, it could be also concluded that the Schiff base bond had a potent effect on the antitumor activity.

In the HepG2 assay, compounds 4c₅ and 5a–5d exhibited better cytotoxicity than the commercial anticancer drug 5-FU (IC₅₀ = 31.98 μM), with IC₅₀ of 20.80 μM, 17.85 μM, 9.99 μM, 17.31 μM and 28.92 μM, respectively. It was worth noting that compound 5b even exhibited better cytotoxic inhibition than cisplatin (IC₅₀ = 10.12 μM), implying its favourable inhibition activity on the HepG2 cell line. So compound 5b was then selected as the representative compound to study the action mechanism of these 2-oxo-quinoline APA derivatives 4 and 5.

In the SK-OV-3 assay, compounds 4a₇, 4c₅, 4d₆ and 4d₇ displayed better cytotoxicity than 5-FU (IC₅₀ = 26.34 μM), with IC₅₀ of 25.81 μM, 25.13 μM, 26.25 μM and 22.36 μM, respectively. It was obvious that compound 4d₇ showed the best cytotoxic inhibition among these compounds. What is unfortunate was that no compound exhibited better cytotoxic inhibition than cisplatin on this cell line.

In the NCI-H460 assay, compounds 4c₅ and 5b exhibited better cytotoxic inhibition than 5-FU (IC₅₀ = 45.44 μM), with IC₅₀ of 44.56 and 38.96 μM, respectively, while no compound demonstrated better cytotoxic inhibition than cisplatin in this assay.

2.2.2. Investigation of cell cycle distribution

Cell cycle checkpoints are significant control mechanisms that ensure the nonreversible performance of cell cycle events.11 Understanding of cell cycle distribution may provide important information for the regulation of the cell cycle.12 To determine the possible role of cell cycle arrest in 2-oxo-quinoline APA derivative-induced growth inhibition, HepG2 cells were treated with the representative compound 5b at different concentrations (0, 8, 10 and 12 μM) for 48 h. As shown in Fig. 3, treatment of HepG2 cells with compound 5b increased cell cycle arrest at the G₂ phase, leading to a significant increase in the G₂-phase population (34.68% (8 μM), 36.92% (10 μM) and 46.77% (12 μM)) compared with the control cells (19.04%). The G₁-phase and S-phase populations of HepG2 cells were reduced to 35.31% (8 μM), 33.91% (10 μM) and 25.75% (12 μM), and 30.01% (8 μM), 29.16% (10 μM) and 27.49% (12 μM), respectively, compared with those of the control (50.47% and 30.49%). These results evidently demonstrated that compound 5b potentially arrested the cell cycle of HepG2 cells in the G₂/M stage in a concentration-dependent manner.

Fig. 3. Cell cycle analysis of HepG2 cells treated with compound 5b. HepG2 cells were treated with different concentrations of compound 5b ((a) 0 μM, (b) 8 μM, (c) 10 μM and (d) 12 μM) for 48 h to determine the cell cycle phase distribution.

G₂/M transition is basically mediated by timed activation of distinct cyclin B1/CDK1 (CDC2) complexes which is mainly regulated by the positive regulator CDC25C phosphatase.12,13 CDC25C dephosphorylates CDK1 resulting in the activation of cyclin B1/CDK1 complexes and performance of G₂/M transition.12,13 In response to DNA damage or other alterations causing G₂/M arrest, phosphorylation of CDC25C results in its degradation and sequestration in the cytoplasm, which eventually leads to the destabilization of cyclin–CDK complexes.14 Moreover, the anti-oncogene p53, p27 and p21 proteins also play a significant role in G₂/M arrest through promoting the accumulation of the inactive cyclin B–CDK complex, which leads to the regulation of G₂/M transition.15 So the expression of these regulatory proteins including cyclin B1, CDK1 and CDC25C, as well as p53, p27 and p21 anti-oncogenes, was further evaluated by western blot assay, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as control. As shown in Fig. 4, the HepG2 cell lysates demonstrated that compound 5b dramatically decreased the expression of cyclin B1, CDK1 and CDC25C in a dose-dependent manner, accompanied by a dose-dependent increase in the levels of p53, p21 and p27 proteins compared with the control. These results clearly displayed that compound 5b potentially induced G₂/M phase cell cycle arrest in HepG2 cells, which might be one of the reasons for its antiproliferative effect.

Fig. 4. Effect of compound 5b on the expression of cyclins and associated proteins. HepG2 cells were treated with compound 5b for 48 h. Western blot analysis was carried out with antibodies against cyclin B1, CDK1 and CDC25C, and GAPDH was used as loading control.

2.2.3. Apoptosis assay study by mitochondrial membrane potential staining

Apoptosis is a key pathway leading to cell death and has been considered as another effective approach in cancer treatment.16 Its assays may provide important information for preliminary investigation of the mode of action.16–19 So it may be interesting to investigate the apoptosis-inducing effect of compound 5b. In order to investigate the apoptosis-inducing effect of target compound 5b, mitochondrial membrane potential changes were designed and detected, using the fluorescent probe JC-1. HepG2 cells treated with compound 5b at 10 μM for 12 h were stained with JC-1 and those not treated with compound 5b were used as control. The results are shown in Fig. 5. Using fluorescence microscopy, Fig. 5 shows that cells not treated with compound 5b were normally red, while cells treated with compound 5b showed strong green fluorescence and indicated typical apoptotic morphology after 12 h. This phenomenon suggested that compound 5b was able to induce apoptotic cell morphology on the HepG2 cell line.17–19

Fig. 5. Mitochondrial membrane potential staining of compound 5b on HepG2 cells. (a) Cells not treated with 5b used as control, (b) compound 5b-treated HepG2 cells at a concentration of 10 μM.

2.2.4. Apoptosis assay study by Hoechst 33258 staining

The apoptosis-inducing effect of compound 5b was also determined by Hoechst 33258 staining of HepG2 cancer cells and the results are shown in Fig. 6. As shown in Fig. 6, HepG2 cells not treated with compound 5b were normally stained blue, while those treated with 5b for 12 h showed strong blue fluorescence and had characteristic apoptotic morphologies. These observations demonstrated that compound 5b induced apoptosis of HepG2 cells, consistent with those from a previous experiment of mitochondrial membrane potential staining.

Fig. 6. Effects of compound 5b on the morphology of HepG2 cells after staining with Hoechst 33258 dye. (a) Cells not treated with 5b used as control, (b) compound 5b-treated HepG2 cells at a concentration of 10 μM.

2.2.5. Apoptosis assay study by acridine orange/ethidium bromide (AO/EB) staining

To further characterize the cell apoptosis induced by compound 5b, AO/EB staining was performed to evaluate the accompanying changes in morphology. The cytotoxicity of compound 5b was evaluated in HepG2 cells following treatment with 10 μM 5b for 24 h. HepG2 cells not treated with 5b were used as control. The results (Fig. 7) showed that the morphology of the 5b-treated HepG2 cells changed significantly. The cell nuclei were stained yellow green or orange, and the morphology showed pycnosis, membrane blebbing and cell budding characteristic of apoptosis. These phenomena were associated with cell apoptosis, indicating that compound 5b was able to induce the apoptosis of HepG2 cells.

Fig. 7. Compound 5b-induced apoptosis in HepG2 cells was determined by AO/EB staining and photographed via fluorescence microscopy. (a) Cells not treated with compound 5b for 24 h used as control, (b) cells treated with compound 5b for 24 h at a concentration of 10 μM.

2.2.6. Apoptosis assay study by flow cytometry

The apoptosis ratios induced by compound 5b in HepG2 tumor cells were quantitatively assayed by flow cytometry and the results are shown in Fig. 8. The results contained the differentiation of live cells (annexin V^–/PI^–), early apoptotic cells (annexin V⁺/PI^–), late apoptotic cells (annexin V⁺/PI⁺), and necrotic cells (annexin V^–/PI⁺). Treatment of HepG2 cells with different concentrations (8, 10 and 12 μM) of compound 5b led to the increment of apoptotic cell population, from 8.41% in the control to 34.57% (8 μM) (i.e., 15.49% early apoptotic cells and 19.08% late apoptotic cells), 43.62% (10 μM) (i.e., 9.24% early apoptotic cells and 34.38% late apoptotic cells) and 54.90% (12 μM) (i.e., 11.83% early apoptotic cells and 43.07% late apoptotic cells), in treated cells. The results demonstrated that the apoptosis of HepG2 cells treated with compound 5b increased gradually with concentration and compound 5b may suppress cell proliferation by inducing apoptosis.

Fig. 8. Apoptosis ratio detection of compound 5b ((b) 8 μM, (c) 10 μM, (d) 12 μM) against HepG2 cells by annexin V/PI assay. The control (a) was used for comparison.

2.2.7. ROS generation assay

Previous work has proven that the generation of intracellular reactive oxygen species (ROS) may lead to the induction of apoptosis.20,21 To investigate the role of ROS in 5b-induced apoptosis in HepG2 cells, the ROS levels were investigated by fluorescence microscopy using 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and 2,7-dichlorofluorescein diacetate (DCFH-DA) as fluorescent probes. As shown in Fig. 9, for DAPI staining, HepG2 cells treated with compound 5b displayed stronger fluorescence in the cytoplasm, while the fluorescence of the untreated control cells was weak and spread over the cells. For DCFH-DA staining, HepG2 cells treated with compound 5b exhibited stronger green fluorescence, indicating that 5b significantly up-regulated generation of ROS and induced apoptosis of HepG2 cells. This phenomenon indicated that compound 5b significantly increased the intracellular level of ROS and was generally considered as a cue for the induction of apoptosis.17–19

Fig. 9. Compound 5b (10 μM) affected the levels of intracellular ROS in HepG2 cells.

2.2.8. Intracellular Ca²⁺ release

Intracellular calcium played an important role in inducing cell apoptosis and the overload of intracellular calcium can induce cell apoptosis.22,23 Our above results have demonstrated that compound 5b can induce apoptosis of HepG2 cells. To determine the role of calcium signaling in 5b-induced apoptosis, HepG2 cells were treated with 5b for 24 h and Ca²⁺ was synchronously detected by fluorescence microscopy with a calcium indicator dye DCFH-DA (Fig. 10). As shown in Fig. 10, treatment with 5b led to an increment of Ca²⁺ in the HepG2 cells. The results implied that 5b-induced apoptosis might be associated with its induction of Ca²⁺ increment.

Fig. 10. Compound 5b (10 μM) caused the elevation of levels of intracellular Ca²⁺ in HepG2 cells.

2.2.9. Caspase-dependent apoptosis in HepG2 cells

As a physiological program of cellular death, apoptosis plays a vital role during normal development and in cellular homeostasis. It is well known that both Fas-dependent and mitochondria-dependent apoptotic pathways are considered as major pathways directly causing neuronal apoptosis, which are associated with expression of Bax, Bcl-2 and cytochrome c proteins.24,25 To test the mechanism of 5b-induced apoptosis in HepG2 cells, the expression of Bax, Bcl-2 and cytochrome c was also investigated by western blotting assay. As shown in Fig. 11, treatment of HepG2 cells with 5b resulted in an elevation in the expression of Bax and reduction in the expression of Bcl-2 accompanied by the release of mitochondrial cytochrome c into the cytosol. The results suggested that 5b induced apoptosis by regulating the levels of Bcl-2 family proteins, Bax and Bcl-2.

Fig. 11. Effects of compound 5b on the levels of cytochrome c, Bcl-2, Bax, caspase-9 and caspase-3. HepG2 cells were treated with compound 5b for 24 h at different concentrations. An equal amount of protein was loaded onto SDS-PAGE gel for western blot analysis as described in the Experimental section. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

Caspases are a family of cysteinyl aspartate specific proteases involved in apoptosis and are dichotomized into groups of initiators (caspases 8, 9 and 10) and executioners (caspases 3, 6 and 7).19 The caspase cascade, which is initiated by the proteolysis of inactive procaspases, is propagated by the cleavage of downstream caspases and substrates such as poly(ADP-ribose) polymerase (PARP). To determine whether caspases were activated in 5b-induced apoptosis, the expression of caspase-9, -3 and PARP was investigated by western blot assay (Fig. 11). As shown in Fig. 11, treatment of HepG2 cells with compound 5b led to a significant increment in the expression of caspase-9, -3 and PARP compared to the control. These results indicated that compound 5b might induce apoptosis through a mitochondrial mediated pathway and the caspase cascade.

3. Experimental section

3.1. Chemistry

All chemicals (reagent grade) were commercially available and used without further purification. NMR spectra were obtained using a BRUKER AVANCE AV500 spectrometer using tetramethylsilane (TMS) as the internal standard. Mass spectra were collected on a BRUKER ESQUIRE HCT spectrometer. GelRed nucleic acid stain was purchased from Biotium.

3.1.1. General procedure for compound 4 (4a₁–4d₇)

2-Oxo-quinoline 3-carbaldehyde derivatives 3 (1 mmol), primary amines (1.5 mmol), diethyl phosphate (1 mmol) and 3 mL acetonitrile were mixed in a pressure tube and reacted at 105 °C for 4 h. After the reaction, the mixture was cooled to room temperature and filtered to yield compound 4 as a white or yellow powder.

4a₁: Yield 54.29%, ¹H NMR (500 MHz, DMSO) δ 11.87 (s, 1H), 8.05 (d, J = 3.7 Hz, 1H), 7.65 (d, J = 7.7 Hz, 1H), 7.49 (t, J = 7.7 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 4.48 (d, J = 21.9 Hz, 1H), 4.14–4.05 (m, 2H), 3.96–3.84 (m, 2H), 2.40 (ddd, J = 25.7, 13.6, 7.0 Hz, 2H), 1.36 (dt, J = 14.0, 7.0 Hz, 2H), 1.26 (ddd, J = 16.7, 10.7, 5.3 Hz, 5H), 1.08 (t, J = 7.0 Hz, 3H), 0.82 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 161.98, 161.93, 138.40, 137.70, 137.64, 130.60, 129.88, 128.18, 122.42, 119.55, 119.53, 115.37, 62.92, 62.87, 62.42, 62.36, 53.20, 51.96, 47.67, 47.55, 40.48, 40.31, 40.23, 40.14, 39.98, 39.81, 39.64, 39.48, 31.86, 20.17, 16.79, 16.74, 16.63, 16.58, 14.26. ESI-HRMS m/z calc for C₁₈H₂₇N₂O₄P [M + H]⁺: 367.1787; found: 367.1782.

4a₂: Yield 75.82%, ¹H NMR (500 MHz, DMSO) δ 11.96 (s, 1H), 8.08 (d, J = 3.7 Hz, 1H), 7.57 (d, J = 7.7 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.19–7.14 (m, 1H), 6.85 (d, J = 8.3 Hz, 2H), 6.62 (d, J = 8.5 Hz, 2H), 6.11 (dd, J = 10.4, 6.2 Hz, 1H), 5.26 (dd, J = 24.5, 10.4 Hz, 1H), 4.11 (dq, J = 14.2, 7.1 Hz, 2H), 4.02–3.87 (m, 2H), 2.09 (s, 3H), 1.22 (t, J = 7.1 Hz, 3H), 1.08 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 161.68, 161.64, 145.11, 144.99, 138.47, 137.65, 137.60, 130.80, 130.13, 129.78, 128.12, 126.32, 122.56, 119.40, 119.38, 115.50, 113.86, 63.22, 63.16, 62.92, 62.86, 48.28, 47.05, 40.50, 40.43, 40.34, 40.26, 40.17, 40.00, 39.84, 39.67, 39.50, 20.45, 16.77, 16.73, 16.58, 16.54. ESI-HRMS m/z calc for C₂₁H₂₅N₂O₄P [M + Na]⁺: 423.1450; found: 423.1447.

4a₃: Yield 70.24%, ¹H NMR (400 MHz, DMSO) δ 12.00 (s, 1H), 8.10 (d, J = 3.7 Hz, 1H), 7.59 (d, J = 7.7 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.17 (t, J = 7.5 Hz, 1H), 6.93 (t, J = 7.8 Hz, 1H), 6.57 (s, 1H), 6.50 (d, J = 8.1 Hz, 1H), 6.39 (d, J = 7.4 Hz, 1H), 6.25 (dd, J = 10.2, 6.2 Hz, 1H), 5.30 (dd, J = 24.4, 10.3 Hz, 1H), 4.12 (dq, J = 14.2, 7.1 Hz, 2H), 4.04–3.85 (m, 2H), 2.13 (s, 3H), 1.22 (t, J = 7.1 Hz, 3H), 1.09 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 161.66, 161.61, 147.46, 147.32, 138.46, 138.35, 137.73, 137.67, 130.86, 130.14, 129.24, 128.16, 122.60, 119.39, 119.36, 118.69, 115.51, 114.43, 110.74, 63.26, 63.19, 62.97, 62.90, 48.02, 46.47, 40.57, 40.36, 40.15, 39.95, 39.74, 39.53, 39.32, 21.78, 16.79, 16.73, 16.60, 16.54. ESI-HRMS m/z calc for C₂₁H₂₅N₂O₄P [M – H]^–: 399.1474; found: 399.1493.

4a₄: Yield 63.62%, ¹H NMR (500 MHz, DMSO) δ 11.87 (s, 1H), 8.05 (d, J = 3.7 Hz, 1H), 7.65 (d, J = 7.7 Hz, 1H), 7.49 (t, J = 7.7 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 4.48 (d, J = 21.9 Hz, 1H), 4.14–4.05 (m, 2H), 3.96–3.84 (m, 2H), 2.40 (ddd, J = 25.7, 13.6, 7.0 Hz, 2H), 1.36 (dt, J = 14.0, 7.0 Hz, 2H), 1.26 (ddd, J = 16.7, 10.7, 5.3 Hz, 5H), 1.08 (t, J = 7.0 Hz, 3H), 0.82 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 161.66, 161.62, 147.44, 147.33, 138.48, 137.76, 137.71, 130.87, 129.99, 129.36, 128.16, 122.60, 119.39, 119.36, 117.79, 115.53, 113.65, 63.26, 63.21, 62.99, 62.93, 47.97, 46.73, 40.47, 40.30, 40.14, 39.97, 39.80, 39.63, 39.47, 16.76, 16.72, 16.58, 16.53. ESI-HRMS m/z calc for C₂₀H₂₃N₂O₄P [M + Na]⁺: 409.1293; found: 409.1293.

4a₅: Yield 73.01%, ¹H NMR (400 MHz, DMSO) δ 12.13 (s, 1H), 8.13 (d, J = 3.4 Hz, 1H), 8.03 (d, J = 9.3 Hz, 2H), 7.95 (dd, J = 9.4, 5.6 Hz, 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.25–7.17 (m, 1H), 6.86 (d, J = 9.3 Hz, 2H), 5.47 (dd, J = 22.6, 9.4 Hz, 1H), 4.21–3.88 (m, 4H), 1.21 (t, J = 7.0 Hz, 3H), 1.11 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 161.37, 161.31, 153.69, 153.58, 138.63, 138.45, 138.39, 137.66, 131.28, 128.59, 128.41, 126.45, 122.77, 119.18, 119.15, 115.65, 112.43, 63.46, 63.39, 63.36, 63.29, 47.73, 46.17, 40.60, 40.39, 40.18, 39.98, 39.77, 39.56, 39.35, 16.78, 16.72, 16.59, 16.54. ESI-HRMS m/z calc for C₂₀H₂₂N₃O₆P [M + Na]⁺: 454.1144; found: 454.1163.

4a₆: Yield 79.13%, ¹H NMR (400 MHz, DMSO) δ 12.00 (s, 1H), 8.10 (d, J = 3.7 Hz, 1H), 7.59 (d, J = 7.7 Hz, 1H), 7.50 (t, J = 7.7 Hz, 1H), 7.32 (d, J = 8.2 Hz, 1H), 7.22–7.14 (m, 1H), 6.96–6.85 (m, 2H), 6.71 (ddd, J = 6.8, 5.2, 2.8 Hz, 2H), 6.37 (dd, J = 10.3, 6.3 Hz, 1H), 5.25 (dd, J = 24.4, 10.3 Hz, 1H), 4.13 (dq, J = 14.2, 7.1 Hz, 2H), 4.05–3.84 (m, 2H), 1.23 (t, J = 7.0 Hz, 3H), 1.09 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 161.68, 161.62, 156.67, 154.36, 144.10, 143.95, 138.49, 137.78, 137.71, 130.92, 129.82, 128.19, 122.62, 119.35, 119.31, 115.88, 115.66, 115.54, 114.60, 114.53, 63.27, 63.20, 62.99, 62.92, 48.53, 46.98, 40.60, 40.39, 40.19, 39.98, 39.77, 39.56, 39.35, 16.79, 16.74, 16.59, 16.54. ESI-HRMS m/z calc for C₂₀H₂₂FN₂O₄P [M + Na]⁺: 427.1199; found: 427.1204.

4a₇: Yield 80.64%, ¹H NMR (400 MHz, DMSO) δ 12.13 (s, 1H), 8.13 (d, J = 3.4 Hz, 1H), 8.03 (d, J = 9.3 Hz, 2H), 7.95 (dd, J = 9.4, 5.6 Hz, 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.25–7.17 (m, 1H), 6.86 (d, J = 9.3 Hz, 2H), 5.47 (dd, J = 22.6, 9.4 Hz, 1H), 4.21–3.88 (m, 4H), 1.21 (t, J = 7.0 Hz, 3H), 1.11 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 161.52, 161.47, 150.77, 150.64, 138.56, 138.03, 137.97, 131.07, 129.29, 128.28, 126.88, 126.75, 126.71, 124.19, 122.68, 119.27, 119.24, 117.62, 117.30, 115.59, 112.99, 99.99, 63.35, 63.29, 63.16, 63.09, 47.66, 46.10, 40.60, 40.39, 40.18, 39.97, 39.76, 39.56, 39.35, 16.76, 16.71, 16.59, 16.53. ESI-HRMS m/z calc for C₂₁H₂₂F₃N₂O₄P [M + Na]⁺: 477.1167; found: 477.1165.

4b₁: Yield 38.61%, ¹H NMR (500 MHz, DMSO) δ 11.87 (s, 1H), 8.05 (d, J = 3.7 Hz, 1H), 7.65 (d, J = 7.7 Hz, 1H), 7.49 (t, J = 7.7 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 4.48 (d, J = 21.9 Hz, 1H), 4.14–4.05 (m, 2H), 3.96–3.84 (m, 2H), 2.40 (ddd, J = 25.7, 13.6, 7.0 Hz, 2H), 1.36 (dt, J = 14.0, 7.0 Hz, 2H), 1.26 (ddd, J = 16.7, 10.7, 5.3 Hz, 5H), 1.08 (t, J = 7.0 Hz, 3H), 0.82 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 161.86, 161.82, 137.44, 137.39, 136.41, 131.83, 131.39, 129.77, 127.65, 119.50, 119.47, 115.29, 62.87, 62.82, 62.39, 62.34, 53.21, 51.97, 47.65, 47.53, 40.52, 40.35, 40.18, 40.02, 39.85, 39.68, 39.52, 31.85, 20.83, 20.18, 16.80, 16.76, 16.64, 16.60, 14.28. ESI-HRMS m/z calc for C₁₉H₂₉N₂O₄P [M + H]⁺: 381.1943; found: 381.1953.

4b₂: Yield 75.87%, ¹H NMR (400 MHz, DMSO) δ 11.88 (s, 1H), 8.01 (d, J = 3.6 Hz, 1H), 7.38–7.27 (m, 2H), 7.21 (d, J = 8.3 Hz, 1H), 6.85 (d, J = 8.2 Hz, 2H), 6.61 (d, J = 8.3 Hz, 2H), 6.12 (dd, J = 10.4, 6.2 Hz, 1H), 5.26 (dd, J = 24.5, 10.5 Hz, 1H), 4.11 (p, J = 7.2 Hz, 2H), 4.02–3.82 (m, 2H), 2.31 (s, 3H), 2.08 (s, 3H), 1.22 (t, J = 7.0 Hz, 3H), 1.07 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 161.58, 161.53, 145.14, 144.99, 137.44, 137.37, 136.46, 132.05, 131.60, 129.99, 129.76, 127.57, 126.27, 119.35, 119.32, 115.39, 113.87, 63.20, 63.13, 62.93, 62.86, 48.37, 46.82, 40.59, 40.38, 40.17, 39.96, 39.75, 39.54, 39.34, 20.78, 20.46, 16.79, 16.74, 16.60, 16.54. ESI-HRMS m/z calc for C₂₂H₂₇N₂O₄P [M + Na]⁺: 437.1606; found: 437.1616.

4b₃: Yield 71.80%, ¹H NMR (500 MHz, DMSO) δ 11.89 (s, 1H), 8.02 (d, J = 3.7 Hz, 1H), 7.36 (s, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 6.92 (t, J = 7.8 Hz, 1H), 6.56 (s, 1H), 6.49 (dd, J = 8.1, 1.8 Hz, 1H), 6.39 (d, J = 7.4 Hz, 1H), 6.21 (dd, J = 10.3, 6.2 Hz, 1H), 5.29 (dd, J = 24.4, 10.3 Hz, 1H), 4.12–3.86 (m, 4H), 2.31 (s, 3H), 2.13 (s, 3H), 1.25 (t, J = 7.1 Hz, 1H), 1.21 (d, J = 7.0 Hz, 3H), 1.08 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 161.55, 161.51, 147.46, 147.35, 138.32, 137.49, 137.44, 136.47, 132.06, 131.62, 130.00, 129.20, 127.60, 119.36, 119.34, 118.66, 115.41, 114.48, 110.79, 63.19, 63.14, 62.94, 62.89, 48.00, 46.76, 40.49, 40.42, 40.32, 40.25, 40.16, 39.99, 39.82, 39.66, 39.49, 21.77, 20.77, 16.77, 16.73, 16.59, 16.54. ESI-HRMS m/z calc for C₂₂H₂₇N₂O₄P [M – H]^–: 413.1681; found: 413.1681.

4b₄: Yield 71.72%, ¹H NMR (500 MHz, DMSO) δ 11.89 (s, 1H), 8.03 (d, J = 3.7 Hz, 1H), 7.35 (s, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.04 (dd, J = 8.4, 7.4 Hz, 2H), 6.71 (d, J = 7.8 Hz, 2H), 6.56 (t, J = 7.3 Hz, 1H), 6.32 (dd, J = 10.2, 6.3 Hz, 1H), 5.30 (dd, J = 24.4, 10.2 Hz, 1H), 4.13–3.86 (m, 4H), 2.31 (s, 3H), 1.27–1.24 (m, 1H), 1.22 (t, J = 7.0 Hz, 3H), 1.08 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 161.56, 161.52, 147.45, 147.34, 137.53, 137.48, 136.47, 132.10, 131.65, 129.85, 129.34, 127.61, 119.35, 119.32, 117.76, 115.43, 113.67, 63.23, 63.18, 62.99, 62.93, 47.98, 46.73, 40.46, 40.38, 40.29, 40.21, 40.12, 40.05, 39.96, 39.79, 39.62, 39.46, 20.76, 16.76, 16.72, 16.57, 16.53. ESI-HRMS m/z calc for C₂₁H₂₅N₂O₄P [M + Na]⁺: 423.1450; found: 423.1451.

4b₅: Yield 76.32%, ¹H NMR (400 MHz, DMSO) δ 12.05 (s, 1H), 8.06 (d, J = 3.4 Hz, 1H), 8.02 (d, J = 9.3 Hz, 2H), 7.96 (dd, J = 9.3, 5.6 Hz, 1H), 7.43 (s, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 8.4 Hz, 1H), 6.86 (d, J = 9.3 Hz, 2H), 5.47 (dd, J = 22.6, 9.4 Hz, 1H), 4.16–3.90 (m, 4H), 2.34 (s, 3H), 1.22 (t, J = 7.0 Hz, 3H), 1.11 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 161.27, 161.21, 153.72, 153.61, 138.23, 138.17, 137.63, 136.63, 132.53, 131.85, 128.47, 127.81, 126.43, 119.14, 119.12, 115.55, 112.42, 63.42, 63.35, 63.29, 47.73, 46.18, 40.60, 40.39, 40.18, 39.97, 39.76, 39.56, 39.35, 20.77, 16.78, 16.73, 16.59, 16.54. ESI-HRMS m/z calc for C₂₁H₂₄N₃O₆P [M – H]^–: 444.1344; found: 444.1357.

4b₆: Yield 84.63%, ¹H NMR (400 MHz, DMSO) δ 11.98 (s, 1H), 8.03 (d, J = 3.5 Hz, 1H), 7.42–7.37 (m, 3H), 7.34 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.17 (dd, J = 9.7, 6.1 Hz, 1H), 6.85 (d, J = 8.7 Hz, 2H), 5.37 (dd, J = 23.7, 9.7 Hz, 1H), 4.16–3.88 (m, 4H), 2.33 (s, 3H), 1.22 (t, J = 7.0 Hz, 3H), 1.10 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 161.42, 161.37, 150.79, 150.66, 137.80, 137.74, 136.55, 132.32, 131.74, 129.16, 127.70, 126.88, 126.73, 126.69, 124.20, 119.23, 119.20, 117.58, 117.27, 116.95, 115.49, 113.00, 63.31, 63.25, 63.16, 63.09, 47.66, 46.11, 40.60, 40.39, 40.18, 39.98, 39.77, 39.56, 39.35, 20.77, 16.77, 16.72, 16.59, 16.53. ESI-HRMS m/z calc for C₂₁H₂₄FN₂O₄P [M + Na]⁺: 441.1355; found: 441.1368.

4b₇: Yield 59.59%, ¹H NMR (500 MHz, DMSO) δ 11.76 (s, 1H), 8.00 (d, J = 3.6 Hz, 1H), 7.25 (d, J = 8.9 Hz, 1H), 7.18 (d, J = 2.5 Hz, 1H), 7.14 (dd, J = 8.9, 2.6 Hz, 1H), 4.48 (d, J = 21.8 Hz, 1H), 4.13–4.05 (m, 2H), 3.94–3.85 (m, 2H), 3.79 (s, 3H), 2.49–2.31 (m, 2H), 1.35 (dd, J = 14.0, 7.0 Hz, 2H), 1.30–1.22 (m, 8H), 1.08 (t, J = 7.0 Hz, 3H), 0.83 (t, J = 7.2 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 161.52, 161.47, 154.73, 137.33, 137.28, 132.91, 130.20, 120.11, 119.90, 116.69, 109.52, 62.91, 62.85, 62.44, 62.38, 55.93, 53.21, 51.97, 47.66, 47.54, 40.48, 40.41, 40.31, 40.24, 40.15, 39.98, 39.81, 39.65, 39.48, 31.85, 20.17, 16.79, 16.75, 16.64, 16.60, 14.27, 8.41. ESI-HRMS m/z calc for C₂₂H₂₄F₃N₂O₄P [M + Na]⁺: 491.1323; found: 491.1328.

4c₁: Yield 59.59%, ¹H NMR (500 MHz, DMSO) δ 11.76 (s, 1H), 8.00 (d, J = 3.6 Hz, 1H), 7.25 (d, J = 8.9 Hz, 1H), 7.18 (d, J = 2.5 Hz, 1H), 7.14 (dd, J = 8.9, 2.6 Hz, 1H), 4.48 (d, J = 21.8 Hz, 1H), 4.13–4.05 (m, 2H), 3.94–3.85 (m, 2H), 3.79 (s, 3H), 2.49–2.31 (m, 2H), 1.35 (dd, J = 14.0, 7.0 Hz, 2H), 1.30–1.22 (m, 8H), 1.08 (t, J = 7.0 Hz, 3H), 0.83 (t, J = 7.2 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 161.52, 161.47, 154.73, 137.33, 137.28, 132.91, 130.20, 120.11, 119.90, 116.69, 109.52, 62.91, 62.85, 62.44, 62.38, 55.93, 53.21, 51.97, 47.66, 47.54, 40.48, 40.41, 40.31, 40.24, 40.15, 39.98, 39.81, 39.65, 39.48, 31.85, 20.17, 16.79, 16.75, 16.64, 16.60, 14.27, 8.41. ESI-HRMS m/z calc for C₁₉H₂₉N₂O₅P [M + H]⁺: 397.1892; found: 397.1908.

4c₂: Yield 79.27%, ¹H NMR (500 MHz, DMSO) δ 11.86 (s, 1H), 8.02 (d, J = 3.7 Hz, 1H), 7.25 (d, J = 9.0 Hz, 1H), 7.13 (dd, J = 8.9, 2.6 Hz, 1H), 7.07 (d, J = 2.7 Hz, 1H), 6.85 (d, J = 8.3 Hz, 2H), 6.61 (d, J = 8.5 Hz, 2H), 6.06 (dd, J = 10.1, 6.6 Hz, 1H), 5.26 (dd, J = 24.6, 10.2 Hz, 1H), 4.12 (dq, J = 14.2, 7.1 Hz, 2H), 4.01–3.86 (m, 2H), 3.76 (s, 3H), 2.09 (s, 3H), 1.23 (t, J = 7.0 Hz, 3H), 1.08 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 161.22, 161.18, 154.78, 145.06, 144.94, 137.19, 137.14, 132.97, 130.46, 129.77, 126.28, 120.16, 119.96, 119.94, 116.83, 113.81, 109.33, 63.19, 63.14, 62.95, 62.90, 55.90, 48.33, 47.09, 40.50, 40.42, 40.33, 40.26, 40.17, 40.00, 39.83, 39.67, 39.50, 20.45, 16.77, 16.73, 16.59, 16.55. ESI-HRMS m/z calc for C₂₂H₂₇N₂O₅P [M + H]⁺: 431.1736; found: 431.1742.

4c₃: Yield 74.36%, ¹H NMR (500 MHz, DMSO) δ 11.87 (s, 1H), 8.04 (d, J = 3.6 Hz, 1H), 7.25 (d, J = 8.9 Hz, 1H), 7.14 (dd, J = 8.9, 2.6 Hz, 1H), 7.09 (d, J = 2.7 Hz, 1H), 6.92 (t, J = 7.8 Hz, 1H), 6.56 (s, 1H), 6.48 (d, J = 8.1 Hz, 1H), 6.39 (d, J = 7.4 Hz, 1H), 6.17 (dd, J = 10.0, 6.5 Hz, 1H), 5.29 (dd, J = 24.5, 10.0 Hz, 1H), 4.11 (dq, J = 14.2, 7.1 Hz, 2H), 4.01–3.87 (m, 2H), 3.77 (s, 3H), 2.13 (s, 3H), 1.24 (dt, J = 14.1, 7.0 Hz, 4H), 1.09 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 161.20, 161.16, 154.80, 147.39, 147.27, 138.35, 137.25, 137.20, 132.99, 130.46, 129.23, 120.20, 119.97, 118.67, 116.85, 114.44, 110.70, 109.36, 63.21, 63.16, 62.99, 62.93, 55.91, 48.03, 46.79, 40.48, 40.41, 40.32, 40.24, 40.15, 39.98, 39.82, 39.65, 39.48, 21.77, 16.77, 16.72, 16.59, 16.55. ESI-HRMS m/z calc for C₂₂H₂₇N₂O₅P [M + Na]⁺: 453.1555; found: 453.1574.

4c₄: Yield 77%, ¹H NMR (500 MHz, DMSO) δ 11.88 (s, 1H), 8.04 (d, J = 3.6 Hz, 1H), 7.26 (d, J = 8.9 Hz, 1H), 7.14 (dd, J = 8.9, 2.7 Hz, 1H), 7.09 (d, J = 2.7 Hz, 1H), 7.05 (dd, J = 8.3, 7.5 Hz, 2H), 6.70 (d, J = 8.1 Hz, 2H), 6.57 (t, J = 7.3 Hz, 1H), 6.28 (dd, J = 9.9, 6.6 Hz, 1H), 5.30 (dd, J = 24.5, 10.0 Hz, 1H), 4.15–4.09 (m, 2H), 4.02–3.88 (m, 2H), 3.76 (s, 3H), 1.27–1.24 (m, 1H), 1.22 (t, J = 7.0 Hz, 3H), 1.09 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 161.21, 161.17, 154.81, 147.38, 147.27, 137.31, 137.26, 132.97, 130.30, 129.36, 120.24, 119.96, 119.94, 117.78, 116.88, 113.62, 109.36, 63.26, 63.20, 63.04, 62.98, 55.90, 48.03, 46.79, 40.44, 40.27, 40.11, 39.94, 39.77, 39.60, 39.44, 16.75, 16.71, 16.58, 16.53. ESI-HRMS m/z calc for C₂₁H₂₅N₂O₅P [M + Na]⁺: 439.1399; found: 439.1403.

4c₅: Yield 80.08%, ¹H NMR (400 MHz, DMSO) δ 12.03 (s, 1H), 8.07 (d, J = 3.4 Hz, 1H), 8.02 (d, J = 9.3 Hz, 2H), 7.94 (dd, J = 9.1, 6.1 Hz, 1H), 7.29 (d, J = 8.8 Hz, 1H), 7.18 (dt, J = 8.1, 2.6 Hz, 2H), 6.85 (d, J = 9.2 Hz, 2H), 5.46 (dd, J = 22.7, 9.1 Hz, 1H), 4.18–3.93 (m, 4H), 3.79 (s, 3H), 1.22 (t, J = 7.0 Hz, 3H), 1.11 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 160.91, 160.85, 154.89, 153.69, 153.58, 137.98, 137.93, 137.63, 133.13, 128.91, 126.43, 120.71, 119.76, 119.73, 117.00, 109.47, 63.43, 63.38, 63.31, 55.95, 47.82, 46.27, 40.60, 40.39, 40.18, 39.97, 39.77, 39.56, 39.35, 16.78, 16.72, 16.60, 16.55. ESI-HRMS m/z calc for C₂₁H₂₄N₃O₇P [M + Na]⁺: 484.1250; found: 484.1273.

4c₆: Yield 86.00%, ¹H NMR (400 MHz, DMSO) δ 11.90 (s, 1H), 8.03 (d, J = 3.6 Hz, 1H), 7.26 (d, J = 9.0 Hz, 1H), 7.15 (dd, J = 8.9, 2.6 Hz, 1H), 7.09 (d, J = 2.6 Hz, 1H), 6.90 (t, J = 8.9 Hz, 2H), 6.74–6.66 (m, 2H), 6.33 (dd, J = 9.9, 6.7 Hz, 1H), 5.24 (dd, J = 24.4, 10.0 Hz, 1H), 4.15–3.84 (m, 4H), 3.77 (s, 3H), 1.23 (t, J = 7.0 Hz, 3H), 1.08 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 161.21, 161.16, 156.64, 154.79, 154.33, 144.05, 143.90, 137.31, 137.25, 132.99, 130.14, 120.31, 119.90, 119.87, 116.88, 115.87, 115.65, 115.43, 115.06, 114.99, 114.53, 114.45, 109.28, 63.23, 63.17, 63.02, 62.95, 55.90, 48.57, 47.02, 40.61, 40.40, 40.19, 39.98, 39.77, 39.56, 39.35, 16.79, 16.74, 16.60, 16.54. ESI-HRMS m/z calc for C₂₁H₂₄FN₂O₅P [M + Na]⁺: 457.1305; found: 457.1323.

4c₇: Yield 74.08%, ¹H NMR (400 MHz, DMSO) δ 11.96 (s, 1H), 8.04 (d, J = 3.6 Hz, 1H), 7.39 (d, J = 8.7 Hz, 2H), 7.27 (d, J = 8.9 Hz, 1H), 7.15 (ddd, J = 13.0, 7.7, 2.6 Hz, 3H), 6.84 (d, J = 8.6 Hz, 2H), 5.36 (dd, J = 23.8, 9.4 Hz, 1H), 4.15–3.89 (m, 4H), 3.78 (s, 3H), 1.22 (t, J = 7.0 Hz, 3H), 1.10 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 161.06, 161.01, 154.84, 150.74, 150.61, 137.55, 137.49, 133.05, 129.59, 126.89, 126.70, 124.20, 120.49, 119.83, 119.80, 117.57, 117.25, 116.93, 112.96, 109.36, 63.32, 63.26, 63.19, 63.12, 55.91, 47.73, 46.18, 40.60, 40.39, 40.18, 39.97, 39.76, 39.56, 39.35, 16.77, 16.72, 16.60, 16.54. ESI-HRMS m/z calc for C₂₁H₂₄F₃N₂O₅P [M + Na]⁺: 507.1273; found: 507.1252.

4d₁: Yield 43.26%, ¹H NMR (400 MHz, DMSO) δ 11.80 (s, 1H), 7.92 (d, J = 3.4 Hz, 1H), 7.17 (s, 1H), 6.83 (s, 1H), 6.10 (s, 2H), 4.44 (d, J = 21.4 Hz, 1H), 4.08 (ddd, J = 10.2, 8.7, 5.3 Hz, 2H), 3.97–3.82 (m, 2H), 2.50–2.34 (m, 2H), 1.40–1.22 (m, 10H), 1.08 (t, J = 7.0 Hz, 3H), 0.83 (t, J = 7.2 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 174.78, 161.75, 161.69, 150.38, 143.78, 137.65, 137.58, 135.35, 130.11, 126.35, 113.81, 113.78, 105.58, 102.20, 95.38, 62.85, 62.78, 62.41, 62.34, 53.17, 51.62, 47.63, 47.47, 31.78, 30.84, 29.56, 29.51, 29.46, 29.31, 29.18, 29.06, 27.02, 25.59, 22.58, 20.18, 16.82, 16.77, 16.66, 16.61, 14.42, 14.30. ESI-HRMS m/z calc for C₁₉H₂₇N₂O₆P [M + H]⁺: 411.1585; found: 411.1519.

4d₂: Yield 66.81%, ¹H NMR (500 MHz, DMSO) δ 11.85 (s, 1H), 7.92 (d, J = 3.5 Hz, 1H), 7.08 (s, 1H), 6.85 (d, J = 8.3 Hz, 2H), 6.82 (s, 1H), 6.60 (d, J = 8.5 Hz, 2H), 6.07 (s, 2H), 6.02 (dd, J = 10.1, 6.5 Hz, 1H), 5.20 (dd, J = 24.3, 10.1 Hz, 1H), 4.10 (dq, J = 14.2, 7.1 Hz, 2H), 4.01–3.83 (m, 2H), 2.09 (s, 3H), 1.22 (t, J = 7.1 Hz, 3H), 1.08 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 161.44, 161.40, 150.49, 145.13, 145.01, 143.88, 137.47, 137.42, 135.47, 129.74, 126.68, 126.18, 113.79, 113.70, 113.68, 105.44, 102.22, 95.45, 63.10, 63.05, 62.89, 62.83, 48.14, 46.90, 40.49, 40.42, 40.33, 40.25, 40.16, 39.99, 39.83, 39.66, 39.49, 20.45, 16.77, 16.72, 16.59, 16.54. ESI-HRMS m/z calc for C₂₂H₂₅N₂O₆P [M + Na]⁺: 467.1348; found: 467.1381.

4d₃: Yield 77.29%, ¹H NMR (400 MHz, DMSO) δ 11.94 (s, 1H), 7.95 (d, J = 3.3 Hz, 1H), 7.10 (s, 1H), 6.89 (dd, J = 15.8, 7.0 Hz, 3H), 6.70 (dd, J = 8.9, 4.5 Hz, 2H), 6.30 (dd, J = 9.8, 6.6 Hz, 1H), 6.08 (s, 2H), 5.19 (dd, J = 24.2, 10.0 Hz, 1H), 4.16–4.06 (m, 2H), 3.94 (ddt, J = 25.1, 10.0, 7.5 Hz, 2H), 3.06 (d, J = 7.2 Hz, 1H), 1.21 (q, J = 7.2 Hz, 6H), 1.09 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 161.46, 161.41, 156.62, 154.31, 150.58, 144.14, 143.98, 143.94, 137.63, 137.57, 135.54, 130.11, 126.31, 115.84, 115.63, 114.53, 114.46, 113.69, 113.66, 105.47, 102.28, 95.51, 63.18, 63.12, 62.99, 62.92, 48.40, 46.84, 45.82, 40.56, 40.35, 40.14, 39.93, 39.73, 39.52, 39.31, 35.59, 31.75, 30.84, 29.45, 29.17, 29.04, 27.01, 25.60, 22.57, 16.78, 16.73, 16.60, 16.54, 14.41, 8.89. ESI-HRMS m/z calc for C₂₂H₂₅N₂O₆P [M – H]^–: 443.1372; found: 443.1727.

4d₄: Yield 77.32%, ¹H NMR (500 MHz, DMSO) δ 11.87 (s, 1H), 7.94 (d, J = 3.4 Hz, 1H), 7.09 (s, 1H), 7.04 (dd, J = 8.3, 7.5 Hz, 2H), 6.82 (s, 1H), 6.70 (d, J = 7.8 Hz, 2H), 6.56 (t, J = 7.3 Hz, 1H), 6.25 (dd, J = 9.9, 6.6 Hz, 1H), 6.07 (s, 2H), 5.23 (dd, J = 24.2, 9.9 Hz, 1H), 4.10 (dq, J = 14.2, 7.1 Hz, 2H), 4.02–3.84 (m, 2H), 1.22 (t, J = 7.1 Hz, 3H), 1.08 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 161.43, 161.38, 150.54, 147.49, 147.38, 143.92, 137.56, 137.52, 135.52, 129.32, 126.57, 117.65, 113.70, 113.68, 113.59, 105.48, 102.25, 95.48, 63.13, 63.08, 62.95, 62.89, 47.83, 46.58, 40.49, 40.42, 40.33, 40.25, 40.16, 39.99, 39.82, 39.66, 39.49, 16.76, 16.72, 16.59, 16.54. ESI-HRMS m/z calc for C₂₁H₂₃N₂O₆P [M + Na]⁺: 453.1191; found: 453.1169.

4d₅: Yield 73.71%, ¹H NMR (400 MHz, DMSO) δ 12.02 (s, 1H), 8.08–7.93 (m, 4H), 7.18 (s, 1H), 6.84 (s, 3H), 6.10 (s, 2H), 5.40 (dd, J = 22.4, 9.2 Hz, 1H), 4.13–3.91 (m, 4H), 1.20 (t, J = 7.0 Hz, 3H), 1.11 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 161.16, 161.10, 153.75, 153.65, 150.89, 144.09, 138.24, 138.18, 137.54, 136.93, 135.78, 126.44, 125.10, 113.55, 113.52, 112.29, 105.67, 102.38, 95.51, 63.34, 63.32, 63.28, 63.25, 47.62, 46.06, 40.59, 40.38, 40.17, 39.97, 39.76, 39.55, 39.34, 16.78, 16.72, 16.60, 16.54, –14.99. ESI-HRMS m/z calc for C₂₁H₂₂N₃O₈P [M + Na]⁺: 498.1042; found: 498.1064.

4d₆: Yield 85.70%, ¹H NMR (400 MHz, DMSO) δ 11.98 (s, 1H), 7.94 (d, J = 3.2 Hz, 1H), 7.38 (d, J = 8.6 Hz, 2H), 7.15–7.06 (m, 2H), 6.88–6.79 (m, 3H), 6.08 (s, 2H), 5.29 (dd, J = 23.5, 9.4 Hz, 1H), 4.13–4.07 (m, 2H), 4.02–3.86 (m, 3H), 1.26–1.18 (m, 6H), 1.09 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 161.32, 150.72, 144.01, 137.81, 135.65, 126.71, 125.77, 113.63, 112.94, 105.56, 102.32, 95.52, 63.27, 63.20, 63.15, 63.08, 47.54, 45.97, 40.54, 40.33, 40.12, 39.91, 39.70, 39.50, 39.29, 29.02, 22.56, 16.76, 16.71, 16.59, 16.53. ESI-HRMS m/z calc for C₂₁H₂₂FN₂O₆P [M + Na]⁺: 471.1097; found: 471.1118.

4d₇: Yield 75.49%, ¹H NMR (400 MHz, DMSO) δ 11.94 (s, 1H), 7.95 (d, J = 3.3 Hz, 1H), 7.10 (s, 1H), 6.89 (dd, J = 15.8, 7.0 Hz, 3H), 6.70 (dd, J = 8.9, 4.5 Hz, 2H), 6.30 (dd, J = 9.8, 6.6 Hz, 1H), 6.08 (s, 2H), 5.19 (dd, J = 24.2, 10.0 Hz, 1H), 4.17–4.06 (m, 2H), 4.03–3.83 (m, 2H), 3.06 (d, J = 7.2 Hz, 1H), 1.21 (q, J = 7.2 Hz, 6H). ¹³C NMR (101 MHz, DMSO) δ 161.03 (d, J = 5.4 Hz), 154.84 (s), 150.68 (d, J = 12.7 Hz), 137.52 (d, J = 6.0 Hz), 133.05 (s), 129.59 (s), 126.79 (d, J = 19.3 Hz), 120.49 (s), 119.82 (d, J = 3.1 Hz), 117.57 (s), 117.25 (s), 116.93 (s), 112.96 (s), 109.36 (s), 63.22 (dd, J = 14.0, 6.9 Hz), 55.91 (s), 47.73 (s), 46.18 (s), 40.60 (s), 40.39 (s), 40.18 (s), 39.97 (s), 39.76 (s), 39.56 (s), 39.35 (s), 16.66 (dd, J = 17.6, 5.4 Hz). ESI-HRMS m/z calc for C₂₂H₂₂F₃N₂O₆P [M + Na]⁺: 521.1065; found: 521.1089.

3.1.2. General procedure for compound 5 (5a–5d)

2-Oxo-quinoline 3-carbaldehyde derivatives 3 (1 mmol), diethyl 4-aminobenzylphosphonate (1.5 mmol) and 5 mL toluene were mixed in a pressure tube and reacted at 120 °C for 4 h. After the reaction, the mixture was cooled to room temperature and filtered to yield compound 5 as a pale yellow powder.

5a: Yield 70.81%, ¹H NMR (400 MHz, DMSO) δ 11.68 (s, 1H), 7.72 (d, J = 2.6 Hz, 1H), 6.86 (s, 1H), 6.69 (t, J = 7.7 Hz, 1H), 6.60 (s, 1H), 6.33 (s, 1H), 6.26 (d, J = 8.0 Hz, 1H), 6.16 (d, J = 7.3 Hz, 1H), 5.98–5.90 (m, 1H), 5.85 (s, 2H), 5.01 (dd, J = 24.2, 10.0 Hz, 1H), 3.93–3.82 (m, 2H), 3.79–3.62 (m, 2H), 1.91 (s, 3H), 0.99 (t, J = 7.0 Hz, 3H), 0.86 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 161.44, 161.38, 150.52, 147.49, 147.35, 143.91, 138.31, 137.56, 137.49, 135.49, 129.20, 126.69, 118.57, 114.40, 113.71, 113.68, 110.68, 105.46, 102.25, 95.46, 63.14, 63.08, 62.94, 62.87, 47.92, 46.36, 46.03, 21.78, 16.78, 16.73, 16.60, 16.54. ESI-HRMS m/z calc for C₂₁H₂₃N₂O₄P [M + H]⁺: 399.1474; found: 399.1476.

5b: Yield 63.13%, ¹H NMR (400 MHz, DMSO) δ 12.10 (s, 1H), 8.79 (s, 1H), 8.59 (s, 1H), 7.67 (s, 1H), 7.42 (dd, J = 8.4, 1.7 Hz, 1H), 7.34 (dd, J = 8.5, 2.3 Hz, 2H), 7.25 (dd, J = 12.0, 8.3 Hz, 3H), 3.97 (dq, J = 14.2, 7.1 Hz, 4H), 3.27 (d, J = 21.5 Hz, 2H), 2.36 (s, 3H), 1.19 (t, J = 7.0 Hz, 6H). ¹³C NMR (101 MHz, DMSO) δ 161.91, 155.52, 150.44, 150.40, 138.36, 137.77, 133.82, 131.91, 131.19, 131.13, 130.97, 130.88, 129.41, 126.80, 121.47, 121.44, 119.29, 115.60, 61.90, 61.84, 40.60, 40.40, 40.19, 39.98, 39.77, 39.56, 39.35, 32.93, 31.59, 20.87, 16.72, 16.66. ESI-HRMS m/z calc for C₂₂H₂₅N₂O₄P [M – H]^–: 411.1474; found: 411.1549.

5c: Yield 64.65%, ¹H NMR (500 MHz, DMSO) δ 12.06 (s, 1H), 8.80 (s, 1H), 8.64 (s, 1H), 7.46 (d, J = 2.7 Hz, 1H), 7.34 (dd, J = 8.4, 2.3 Hz, 2H), 7.30 (d, J = 8.9 Hz, 1H), 7.24 (dd, J = 8.9, 2.8 Hz, 3H), 3.96 (dd, J = 15.1, 7.1 Hz, 4H), 3.26 (d, J = 21.5 Hz, 2H), 1.18 (t, J = 7.0 Hz, 6H). ¹³C NMR (126 MHz, DMSO) δ 190.33, 161.58, 155.45, 154.95, 150.38, 150.35, 137.62, 135.01, 131.19, 131.14, 131.02, 130.94, 127.13, 122.03, 121.45, 121.43, 119.94, 116.98, 110.97, 61.90, 61.85, 55.99, 40.52, 40.45, 40.36, 40.28, 40.19, 40.02, 39.85, 39.69, 39.52, 32.85, 31.78, 16.70, 16.66. ESI-HRMS m/z calc for C₂₂H₂₅N₂O₅P [M + Na]⁺: 451.1399; found: 451.1404.

5d: Yield 70.98%, ¹H NMR (400 MHz, d-DMSO) δ 12.08 (s, 1H), 8.74 (s, 1H), 8.56 (s, 1H), 7.38 (d, J = 8.7 Hz, 2H), 7.32 (dd, J = 8.4, 2.3 Hz, 2H), 7.20 (d, J = 8.0 Hz, 2H), 6.15 (d, J = 8.2 Hz, 2H), 3.97 (dd, J = 8.0, 7.2 Hz, 4H), 3.26 (d, J = 21.5 Hz, 2H), 1.19 (t, J = 7.0 Hz, 6H). ¹³C NMR (101 MHz, DMSO) δ 189.85, 161.90, 155.37, 153.66, 152.08, 144.53, 142.07, 140.40, 131.17, 122.90, 121.34, 113.87, 113.23, 107.29, 106.73, 103.01, 102.65, 95.37, 61.83, 32.81, 31.44, 16.71. ESI-HRMS m/z calc for C₂₂H₂₃N₂O₆P [M + H]⁺: 443.1372; found: 443.1391.

3.2. Biological assays

The detailed procedures for other experimental methods are described in the ESI (part 2). The materials, instrumentation, and methods for the cytotoxicity assay, cell cycle analysis, cell apoptosis analysis, western blot and transfection assays were described in our previous work.26

4. Conclusions

In summary, we have designed and synthesized a set of 2-oxo-quinoline APA derivatives (4 and 5) and evaluated the cytotoxicity of the target compounds on three cancer cell lines (HepG2, SK-OV-3 and NCI-H460). The target compounds displayed evident anticancer activity with low cytotoxicity against normal HL-7702 cells. The investigation into the cell apoptosis-inducing effect of representative compound 5b on HepG2 cells showed that the antitumor activity of this compound may rely on the apoptosis of cancer cells by regulation of the levels of Bax, Bcl-2 and cytochrome c, intracellular Ca²⁺ release and ROS generation, activation of caspase-9 and caspase-3 and subsequent cleavage of PARP. The cell cycle study revealed that the antitumor activity of compound 5b might be exerted by G₂/M phase arrest that was accompanied by the expression of p53, p21 and p27 proteins. The above results confirmed that the rational design of 2-oxo-quinoline APA derivatives as novel antitumor agents was feasible.