Design,synthesis,and biological evaluation of quinazoline derivatives containing piperazine moieties as antitumor agents

Abstract

A series of novel quinazoline derivatives containing piperazine analogs are synthesized via substitution reactions with 6,7-disubstituted 4-chloroquinazoline and benzyl piperazine (amido piperazine). Potent antiproliferative activities are observed against A549, HepG2, K562, and PC-3 with N-(3-chlorophenyl)-2-(4-(7-methoxy-6-(3-morpholino-propoxy)quinazoline-4-yl)piperazine-1-yl)acetamidename C9 showing excellent activity. This active derivative was screened for cell migration ability, proliferation effects, and apoptosis against A549 and PC-3 cells, with the result showing biological activity almost equal to that of the control gefitinib.

Introduction

It has been reported that the quinazoline unit is of great value as a chemical skeleton with diverse pharmaceutical and physiological utility, including antimicrobial, ¹ antihyperlipidemic, ² anticonvulsant, ³ antihypertensive, ⁴ and anti-inflammatory ⁵ activities. Therefore, many quinazolines have contributed to the quest for an ultimate antitumor chemotherapeutic agent. Moreover, several small molecules such as gefitinib (Figure 1), erlotinib, and lapatinib, which containing quinazoline analogs, were designed to inhibit epidermal growth factor receptor (EGFR) kinase activity. Among them, gefitinib—an oral small molecule agent that inhibits EGFR tyrosine phosphorylation, was approved by the US Food and Drug Administration (FDA) for locally advanced or metastatic non-small cell lung cancer therapy—is involved in cellular signal-transduction pathways that regulate essential functions such as proliferation, differentiation, and apoptosis.^6–14 However, its efficient use is seriously hampered by EGFR mutation. Even though tumors were sensitive to gefitinib, tumor regrowth occurs after several months and all of the mechanisms of the resistance have not been clarified. Thus, it is necessary to exploit novel antitumor drugs with high efficiency and low toxicity due to the various side effects of gefitinib. ¹⁵

Figure 1.

The structure of gefitinib.

Over past decades, a large number of piperazine heterocyclic derivatives have attracted significant attention as chemotherapeutic drugs. Numerous studies have illustrated that the introduction of piperazine moieties can influence the physicochemical properties and enhance the bioactivity of compounds. It is known that piperazine is versatile and privileged in drug discovery,^16–20 for example, in ciprofloxacin for the treatment of bacterial infections, trifluoperazine for psychosis, cetirizine for the treatment of allergies, and tanitinib for the treatment of acute myeloid leukemia. A series of 4-amino-2H-benzo(h)chromen-2-one analogs containing the piperazine moiety were reported by Chen in 2019, and bioassays showed that these compounds exhibited potent antagonistic potency against androgen receptors (AR; inhibition >50%), and exhibited potent AR binding affinities as well as displaying higher activities than finasteride toward LNCaP cells (AR-rich) versus PC-3 cells (AR-deficient). ²¹

Based on the active substructure combination theory, the piperazine ring was utilized in this research. Thus, two series of novel 4-piperazine quinazoline derivatives were designed and synthesized. The preliminary antitumor activity of these new compounds was also evaluated in vitro. In addition, the primary apoptotic effect mechanism induced by a representative compound C9 was also investigated by flow cytometry.

Results and discussion

Synthesis

The synthetic route for the preparation of the novel quinazoline derivatives containing a piperazine moiety is summarized in Scheme 1. The key intermediate 7 was synthesized in six steps (a to f). 3-Hydroxy-4-methoxybenzaldehyde (1) was chosen as the starting material and was reacted with 1-bromo-3-chloropropane followed by reaction with NH₂OH·HCl, AcOH, and AcONa to form 3-(3-chloropropoxy)-4-methoxybenzonitrile (3). This was subjected to nitration using nitric acid and the product 4 reacted with morpholine under potassium iodide (KI) catalysis to give intermediate 5. This was converted to the quinazoline 7 by reduction with hydrazine hydrate followed by ring closure using formic acid and chlorination. ²² As shown in route 1, intermediate 7 was reacted with a series of monosubstituted piperazines 8 to give the target compounds C1–C5. In addition, intermediate 7 was reacted with a series of amide-containing monosubstituted piperazines 9 to give the target compounds C6–C10 (route 2). All the target compounds were characterized, and the structures were confirmed by ¹H NMR, ¹³C NMR, and mass spectrometry. Taking compound C9 as a representative example, two singlets appeared at 8.41 and 3.91 ppm in CD₃OD attributed to the quinazoline –N=CH and –OCH₃, respectively. In the ¹³C NMR spectrum, a signal due to C=O was observed at 169.5 ppm.

Scheme 1.

Synthetic routes to the target compounds C1–C5 and C6–C10.

Biological activity

The antitumor activities of the synthesized compounds against four cell lines were evaluated using gefitinib as the control. The IC₅₀ values indicated that the compounds exerted antitumor effects, with varying degrees of antitumor activity against the four cell lines. As shown in Table 1, the IC₅₀ values ranged from 35.56 ± 9.39 to 8.24 ± 1.40 µM against A549, from 14.57 ± 5.52 to 5.62 ± 3.23 µM against PC-3, from 56.42 ± 0.14 to 8.63 ± 0.86 µM against HepG2, and from 159.3 ± 14.28 to 15.02 ± 0.88 µM against K562, being typically better than gefitinib (IC₅₀ values were 5.475 ± 1.06 µM against A549, 5.99 ± 1.47 µM against PC-3, 31.48 ± 10.23 µM against HepG2, and 13.67 ± 1.44 µM against K562). The cytotoxicities of the target compounds against the normal cell line NRK-52E in vitro are shown in the Supporting Information Figure S1). The result showed that the survival rate of the cells at low concentration was close to 100%, and the cytotoxicity of compound C9 was the weakest among these compounds.

Table 1.

The IC₅₀ values of compounds C1 to C10 against four cancer cell lines.

IC50 (µM) a
Compound	A549	PC-3	HepG2	K562
Gefitinib b	5.475 ± 1.06	5.99 ± 1.47	31.48 ± 10.23	13.67 ± 1.44
C1	13.37 ± 6.40	8.19 ± 1.53	24.69 ± 2.73	70.69 ± 10.24
C2	24.85 ± 4.06	14.57 ± 5.52	32.23 ± 3.20	60.94 ± 13.41
C3	13.53 ± 2.35	8.51 ± 3.20	43.45 ± 3.85	15.02 ± 0.88
C4	15.34 ± 7.35	6.44 ± 2.31	29.07 ± 5.65	62.96 ± 13.17
C5	13.94 ± 6.38	5.62 ± 3.23	22.29 ± 2.81	45.52 ± 2.55
C6	8.99 ± 6.29	15.26 ± 5.12	51.29 ± 13.08	17.63 ± 8.20
C7	12.96 ± 1.80	13.72 ± 4.32	31.05 ± 5.53	159.3 ± 14.28
C8	22.51 ± 1.24	13.42 ± 4.87	8.63 ± 0.86	50.00 ± 5.13
C9	8.24 ± 1.40	7.66 ± 1.27	38.62 ± 2.41	21.96 ± 3.51
C10	35.56 ± 9.39	10.42 ± 1.97	40.64 ± 13.18	62.56 ± 5.36

a

The average of three trials.

b

Commercial gefitinib was used as a positive control.

To further investigate the cell migration ability of compound C9 with the A549 and PC-3 cell lines, wound healing assays were examined with gefitinib and C9 at concentrations of 5 µM for 24 h, respectively. Representative micrographs showed that compound C9 efficiently inhibited A549 and PC-3 migration in comparison with dimethyl sulfoxide (DMSO) in a time-dependent manner. Furthermore, the migration ratios of A549 ranged from 13.39% to 53.18% and from 12.40% to 51.54%, and the migration ratio of PC-3 from 5.50% to 16.74% and from 3.03% to 12.60% after being cultured with gefitinib and C9, respectively (Figure S2).

To evaluate the cell proliferation effects of C9, we performed colony formation on the A549 and PC-3 cell lines. The results showed that the colonies presented significant corresponding downregulated trends on A549 and PC-3 cells when stimulated by a range of C9 doses (1.25, 2.5, 5, 10, and 20 µM). It was obvious that there was very little colony at 20 µM, and the effect of C9 was in a dose-dependent manner. As Figure S3 shows, the clonogenic ratio of A549 was from 3.87% to 14.53% and that of PC-3 was from 3.60% to 46.13% after being cultured with C9 at different concentrations (Figure S3).

In order to investigate the induction of apoptosis in A549 and PC-3 cells, the Hoechst staining assay was conducted in nuclear morphology by fluorescent microscopy. As shown in Figure S4, it was easy to observe the distinct pattern of morphological changes, including cell shrinkage, fragmentation of the nucleus, and chromatin condensation after culturing with gefitinib and C9 at different concentrations (1.25, 2.5, 5, 10, and 20 µM) in A549 and PC-3 cell lines for 24 h (Figure S4).

Flow cytometry

Apoptosis assays can provide preliminary information on the mechanism of growth inhibition of tumor cells. Therefore, the apoptotic effects of C9 against the A549 and PC-3 cell lines were investigated. The results are given in Figure 2. After treatment of A549 and PC-3 cell lines for 24 h with gefitinib and C9 at different concentrations (5, 10, and 20 µM), the apoptosis ratio of A549 (including the early and late apoptosis rates) was from 41.9% to 57.4% with gefitinib and from 16.4% to 39.8% with C9. Furthermore, the apoptosis ratios of PC-3 was from 35.3% to 43.8% with gefitinib and from 28.7% to 38.7% with C9, being remarkably influenced by gefitinib and C9 in a dose-dependent manner.

Figure 2.

C9 and gefitinib increase the apoptosis of A549 and PC-3 cells. (a, b) Annexin/PI statin of A549 and (c, d) Annexin/PI statin of PC-3.

Binding mode analysis

In order to further evaluate these promising compounds and to guide structure–activity relationship (SAR) studies, the interaction effect between compound C9 and the EGFR crystal structure (3W2S.pdb) was investigated. Compound C9 was inserted into the active site of EGFR kinase for molecular docking. All docking runs were applied in Surflex-Dock using Sybyl-X 2.0. The binding mode of compound C9 and EGFR kinase is depicted in Figure 3. As shown in Figure 3(a), compound C9 was potently bound to the active site of EGFR kinase by hydrophobic interactions, and the binding mode was stabilized by two hydrogen bonds. The enzyme surface model is shown in Figure 3(b), which revealed that the target molecule C9 was well embedded in the active pocket of EGFR kinase. This molecular docking result, along with the biological assay data, suggested that compound C9 was a potential inhibitor of EGFR kinase.

Figure 3.

(a) Compound C9 (colored by atom—carbon: gray, nitrogen: dark blue, oxygen: red, chlorine: green, and hydrogen: blue) is bound into EGFR. The dotted lines show the hydrogen bonds. (b) The surface model structure of binding of compound C9 with the EGFR complex is shown.

Conclusion

In conclusion, a series of quinazoline derivatives containing a piperazine moiety have been designed and synthesized. Furthermore, the biological activities of these compounds were evaluated according to the Thiazolyl Blue Tetrazolium Bromide (MTT) method, wound healing assay, clonogenic survival assay, Hoechst staining, and flow cytometry. Compound C9 with excellent activity may represent a promising lead candidate for EGFR-inhibiting therapeutics.

Experimental

Materials and methods

All reagents were purchased from commercial sources as analytical grade and were used without further purification. All reactions were monitored by thin-layer chromatography (TLC). ¹H NMR and ¹³C NMR spectra were performed on a Bruker Ascend 400 NMR (Bruker, Germany) and JEOL-ECX 500 NMR spectrometers (JEOL, Japan) using methanol (CD₃OD) as the solvent and tetramethylsilane as the internal standard. Chemical shifts are expressed in ppm (parts per million) and coupling constants are given in Hz (Hertz). Mass spectra (MS) were recorded on an Agilent 1100 MSD-Trap-VL series with an electron spray ionization (ESI) source. High-resolution mass spectra (HRMS) were recorded on Thermo Scientific Q Exactive series with an ESI source.

The synthesis of intermediate 7

To a solution of 1 in N, N-dimethylformamide (DMF) (40 mL), K₂CO₃ (15.20 g, 0.11 mol) and 1-bromo-3-chloropropane (18.89 g, 0.12 mol) were added. The mixture was stirred and refluxed at 85 °C for 4 h. After completion of the reaction, as monitored by TLC, the solution was added to cold water and the mixture filtered and concentrated under reduced pressure to give the ether 2 (yield 80%). Ether 2 (15.50 g, 0.068 mol), NH₂OH·HCl (10 g, 0.136 mol), and AcONa (11.12 g, 0.136 mol) were dissolved in AcOH (40 mL), and the solution was heated at 105 °C for 8 h. After completion of the reaction, as monitored by TLC, the solution was added to saturate sodium chloride (200 mL), and the mixture filtered and concentrated to give the nitrile 3 (yield 90%). Intermediate 3 (17.26 g, 0.076 mol) was added to a mixture of AcOH (50 mL) and Ac₂O (50 mL) keeping the temperature between 0 and 5 °C, and then HNO₃ (40 mL) was added dropwise to the solution. The mixture was stirred and refluxed at room temperature (RT) for 72 h, and then the solution was added to cold water. Ammonia was added to adjust the pH to 7, and then the mixture was filtered and dried to obtain the nitro compound 4 (yield 68%). To a solution of 4 (14.16 g, 0.063 mol) in DMF (40 mL), KI (0.57 g, 0.0035 mol) and morpholine (10.90 g, 0.126 mol) were added. The mixture was stirred and refluxed at 70 °C for 10 h. After the reaction was finished, the mixture was added to cold water, and the solid precipitate was filtered and concentrated to give compound 5 (yield 56%). This intermediate 5 (12.11 g, 0.038 mol) and FeCl₃ (0.38 g, 0.002 mol) were dissolved in a mixture of methanol and water (3:1, 120 mL) followed by 80% N₂H₄·H₂O (10 mL), and the mixture was heated under reflux (3 h) and then concentrated at 40 °C. Water (37.5 mL), HCl (36%–38%, 45 mL), and HCOOH (>88%, 60 mL) were added to the filtrate until the temperature of reaction mixture reached RT. The mixture was then refluxed at 130 °C for 3.5 h. After completion of the reaction, as evidenced by TLC, the water (75 mL) was added to the mixture until the temperature of the reaction mixture reached RT, after which NaOH was added to adjust the pH to 7. A yellow and white solid was obtained by extracting with CHCl₃ (3 mL × 200 mL) and drying the organic layer with anhydrous MgSO₄. Ethyl acetate (20 mL) was added into the solid and the solution heated to boiling. The residue was filtered and dried to obtain white solid (6) (yield 58%) which could be further converted into key intermediate 7 by reduction with thionyl chloride (SOCl₂) in DMF (1.0 g, yield 94%) at 84 °C.

General procedure for the preparation of C1–C5

Taking compound C1 as a representative example, 4-(3-((4-chloro-7-methoxyquinazolin-6-yl)oxy)propyl)morpholine (7) (0.50 g, 1.48 mmol), 1-benzylpiperazine (0.31 g, 1.78 mmol), and i-PrOH (15 mL) were added in turn to a flask and refluxed for 4 h. After completion of the reaction, as indicated by TLC, the solvent was evaporated to give a yellowish-brown oily substance. This substance was dissolved in methanol and isolated by column chromatography (CHCl₃: MeOH = 15:1). Pure compound was evaporated to give the yellow oily substance (0.08 g, 0.17 mmol).

General procedure for the preparation of C6–C10

Taking compound C6 as a representative example, 4-(3-((4-chloro-7-methoxyquinazolin-6-yl)oxy)propyl)morpholine (0.50 g, 1.48 mmol), N-phenyl-2-piperazinylacetamide (0.39 g, 1.78 mmol), and i-PrOH (15 mL) were added to a flask and refluxed for 4 h. After completion of the reaction, as indicated by TLC, the solvent was evaporated to give a yellowish-brown oily substance. This substance was dissolved in methanol and isolated by column chromatography (CHCl₃: MeOH = 15:1). The pure compound was evaporated to give the brownish black oil substance (0.10 g, 0.19 mmol).

4-(4-Benzylpiperazin-1-yl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]quinazoline (C1)

Yield 11%, 0.08 g; yellow oily substance; ¹H NMR (CD₃OD, 500 MHz): δ 8.44 (s, 1H), 7.23–7.35 (m, 5H), 7.12 (s, 2H), 4.12 (t, J = 6.0 Hz, 2H), 3.94 (s, 3H), 3.68–3.70 (m, 8H), 3.58 (s, 2H), 2.49–2.63 (br, m, 10H), 2.03–2.06 (br, m, 2H); ¹³C NMR (CD₃OD, 125 MHz): δ 163.6, 155.5, 152.0, 148.2, 137.1, 129.3, 128.1, 127.2, 110.6, 105.8, 104.5, 66.9, 66.3, 62.6, 55.4, 55.3, 53.5, 52.6, 49.2, 25.6; ESI-MS: m/z 478.2 [M + H]⁺. HRMS (ESI) m/z calcd for C₂₇H₃₅N₅O₃ [M + H]⁺ 478.2813, found: 478.2808.

4-(4-(2,6-Dichlorobenzyl)piperazin-1-yl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]quinazoline (C2)

Yield 7%, 0.05 g; yellow oily substance; ¹H NMR (CD₃OD, 500 MHz): δ 8.43 (s, 1H), 7.35 (d, J = 8.0 Hz, 2H), 7.22 (t, J = 8.0 Hz, 1H), 7.11 (s, 1H), 7.10 (s, 1H), 4.12 (t, J = 6.0 Hz, 2H), 3.94 (s, 3H), 3.82 (s, 2H), 3.63–3.69 (m, 8H), 2.74 (t, J = 4.6 Hz, 4H), 2.49–2.57 (m, 6H), 2.03–2.06 (br, m, 2H); ¹³C NMR (CD₃OD, 125 MHz): δ 163.6, 155.5, 152.0, 148.2, 148.1, 136.8, 133.7, 129.4, 128.3, 110.9, 105.8, 104.6, 66.9, 66.3, 56.0, 55.4, 55.3, 53.5, 52.7, 49.4, 25.7; ESI-MS: m/z 546.4 [M + H]⁺. HRMS (ESI) m/z calcd for C₂₇H₃₃Cl₂N₅O₃ [M + H]⁺ 546.2033, found: 546.2026.

4-(4-(2-Methylbenzyl)piperazin-1-yl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]quinazoline (C3)

Yield 17%, 0.12 g; yellow oily substance; ¹H NMR (CD₃OD, 500 MHz): δ 8.38 (s, 1H), 6.91–7.13 (br, m, 6H), 3.99 (t, J = 6.0 Hz, 2H), 3.86 (s, 3H), 3.61 (t, J = 4.6 Hz, 4H), 3.41–3.51 (br, m, 4H), 3.39 (s, 2H), 2.46–2.49 (m, 6H), 2.27–2.39 (br, m, 7H), 1.94–1.97 (br, m, 2H); ¹³C NMR (CD₃OD, 125 MHz): δ 163.5, 163.4, 155.2, 152.1, 147.9, 137.4, 135.6, 130.2, 129.9, 127.2, 125.4, 110.7, 106.0, 104.3, 67.0, 66.4, 60.5, 55.4, 55.3, 53.5, 52.8, 49.4, 25.7, 18.4; ESI-MS: m/z 492.5[M + H]⁺. HRMS (ESI) m/z calcd for C₂₈H₃₇N₅O₃ [M + H]⁺ 492.2969, found: 492.2963.

4-(4-(3-Methylbenzyl)piperazin-1-yl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]quinazoline (C4)

Yield 15 %, 0.11 g; yellow oily substance; ¹H NMR (CD₃OD, 500 MHz): δ 8.39 (s, 1H), 7.12 (t, J = 7.5 Hz, 1H), 7.04–7.08 (m, 2H), 6.98–7.00 (m, 2H), 6.94 (s, 1H), 4.02 (t, J = 6.0 Hz, 2H), 3.88 (s, 3H), 3.63 (t, J = 2.1 Hz, 4H), 3.58 (s, 4H), 3.42 (s, 2H), 2.40–2.53 (m, 6H), 2.25–2.40 (br, m, 7H), 1.96–1.99 (br, m, 2H); ¹³C NMR (CD₃OD, 125 MHz): δ 163.4, 155.2, 152.1, 148.1, 148.0, 137.7, 137.1, 129.9, 128.0, 127.9, 126.4, 110.8, 105.9, 104.3, 66.9, 66.4, 62.7, 55.4, 55.3, 53.5, 52.7, 49.2, 25.8, 20.4; ESI-MS: m/z 492.5 [M + H]⁺. HRMS (ESI) m/z calcd for C₂₈H₃₇N₅O₃ [M + H]⁺ 492.2969, found: 492.2966.

4-(4-(4-Methylbenzyl)piperazin-1-yl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]quinazoline (C5)

Yield 14%, 0.10 g; yellow oily substance; ¹H NMR (CD₃OD, 500 MHz): δ 8.59 (s, 1H), 8.42 (s, 1H), 7.85 (t, J = 9.9 Hz, 2H), 7.45 (t, J = 8.2 Hz, 2H), 7.06 (s, 2H), 4.10 (t, J = 5.7 Hz, 2H), 3.93 (s, 3H), 3.74 (br, s, 6H), 3.65 (t, J = 4.6 Hz, 4H), 3.29 (s, 3H), 2.80 (t, J = 4.6 Hz, 4H), 2.54 (t, J = 7.3 Hz, 2H), 2.46 (s, 3H), 2.01–2.04 (br, m, 2H); ¹³C NMR (CD₃OD, 125 MHz): δ 163.6, 155.4, 152.0, 148.2, 148.0, 136.9, 133.9, 129.3, 128.7, 110.9, 105.9, 104.4, 66.9, 66.4, 62.4, 55.4, 55.3, 53.5, 52.6, 49.2, 25.7, 19.9; ESI-MS: m/z 492.5 [M + H]⁺. HRMS (ESI) m/z calcd for C₂₈H₃₇N₅O₃ [M + H]⁺ 492.2969, found: 492.2962.

2-(4-(7-Methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)piperazin-1-yl)-N-phenylacetamide (C6)

Yield 13%, 0.10 g; yellow oily substance; ¹H NMR (CD₃OD, 500 MHz): δ 8.43 (s, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 9.2 Hz, 2H), 7.06 (s, 1H), 7.04 (s, 2H), 4.09 (t, J = 6.0 Hz, 2H), 3.92 (s, 3H), 3.72 (s, 4H), 3.64 (t, J = 4.9 Hz, 4H), 3.23 (s, 2H), 2.76 (t, J = 4.5 Hz, 4H), 2.54 (t, J = 7.5 Hz, 2H), 2.45 (s, 4H), 2.45–2.52 (m, 4H), 2.02–2.03 (br, m, 2H); ¹³C NMR (CD₃OD, 125 MHz): δ 169.3, 163.5, 155.4, 152.0, 148.2, 148.0, 137.9, 128.6, 124.8, 120.0, 110.9, 105.9, 104.3, 67.0, 66.3, 61.6, 55.4, 55.3, 53.5, 52.9, 49.3, 25.7; ESI-MS: m/z 521.4 [M + H]⁺. HRMS (ESI) m/z calcd for C₂₈H₃₆N₆O₄ [M + H]⁺ 521.2871, found: 521.2863.

2-(4-(7-Methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)piperazin-1-yl)-N-(o-methylphenyl)acetamide (C7)

Yield 13%, 0.10 g; yellow oily substance; ¹H NMR (CD₃OD, 500 MHz): δ 8.46 (s, 1H), 7.69 (d, J = 8.3 Hz, 1H), 7.03–7.18 (m, 5H), 4.12 (t, J = 6.2 Hz, 2H), 3.94 (s, 3H), 3.67–3.74 (m, 8H), 3.26 (s, 2H), 2.82 (t, J = 4.6 Hz, 4H), 2.56 (t, J = 7.6 Hz, 2H), 2.47 (s, 4H), 2.27 (s, 3H), 2.03–2.06 (br, m, 2H); ¹³C NMR (CD₃OD, 125 MHz): δ 169.5, 163.6, 155.5, 152.0, 148.3, 148.1, 135.4, 130.2, 130.1, 126.3, 125.3, 123.0, 111.0, 105.9, 104.3, 67.3, 66.3, 61.4, 55.4, 55.3, 53.5, 53.0, 49.6, 25.7, 16.9; ESI-MS: m/z 535.5 [M + H]⁺. HRMS (ESI) m/z calcd for C₂₉H₃₈N₆O₄ [M + H]⁺ 535.3027, found: 535.3019.

2-(4-(7-Methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)piperazin-1-yl)-N-(p-methylphenyl)acetamide (C8)

Yield 16%, 0.13 g; yellow oily substance; ¹H NMR (CD₃OD, 500 MHz): δ 8.74 (s, 1H), 7.44 (d, J = 8.6 Hz, 2H), 7.16 (d, J = 11.5 Hz, 2H), 7.11 (d, J = 8.6 Hz, 2H), 4.16 (t, J = 6.0 Hz, 2H), 3.96 (s, 3H), 3.80 (s, 4H), 3.69 (t, J = 4.6 Hz, 4H), 3.25 (s, 2H), 2.80 (t, J = 4.6 Hz, 4H), 2.61 (t, J = 7.5 Hz, 2H), 2.53 (s, 4H), 2.28 (s, 3H), 2.05–2.08 (br, m, 2H); ¹³C NMR (CD₃OD, 125 MHz): δ 169.3, 163.7, 155.6, 152.0, 148.3, 148.1, 135.2, 134.0, 128.9, 120.2, 110.9, 105.8, 104.5, 66.9, 66.2, 61.5, 55.4, 55.3, 53.4, 52.8, 49.3, 25.6, 19.6; ESI-MS: m/z 535.5 [M + H]⁺. HRMS (ESI) m/z calcd for C₂₉H₃₈N₆O₄ [M + H]⁺ 535.3027, found: 530.3018.

N-(3-Chlorophenyl)-2-(4-(7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)piperazin-1-yl)acetamide (C9)

Yield 15%, 0.12 g; yellow oily substance; ¹H NMR (CD₃OD, 500 MHz): δ 8.41 (s, 1H), 7.74 (t, J = 2.5 Hz, 1H), 7.40–7.42 (m, 2H), 7.17–7.21 (m, 1H), 6.97–7.03 (m, 2H), 4.06 (t, J = 6.0 Hz, 2H), 3.91 (s, 3H), 3.63–3.69 (m, 8H), 3.21 (s, 2H), 2.74 (t, J = 7.4 Hz, 4H), 2.43–2.51 (m, 6H), 1.99–2.02 (br, m, 2H); ¹³C NMR (CD₃OD, 125 MHz): δ 169.5, 163.5, 155.3, 152.0, 148.1, 148.0, 139.4, 134.0, 129.8, 123.8, 119.7, 117.9, 110.9, 105.9, 104.3, 67.0, 66.3, 61.6, 55.4, 55.3, 53.4, 52.8, 49.3, 25.7; ESI-MS: m/z 555.4 [M + H]⁺. HRMS (ESI) m/z calcd for C₂₈H₃₅ClN₆O₄ [M + H]⁺ 555.2481, found: 555.2474.

2-(4-(7-Methoxy-6-(3-morpholinopropoxy)quinazolin-4-yl)piperazin-1-yl)-N-(2-nitrophenyl)acetamide (C10)

Yield 8%, 0.07 g; yellow oily substance; ¹H NMR (CD₃OD, 500 MHz): δ 8.66 (d, J = 7.3 Hz, 1H), 8.44 (s, 1H), 8.14 (d, J = 6.6 Hz, 1H), 7.66 (t, J = 7.1 Hz, 1H), 7.22 (t, J = 7.8 Hz, 1H), 7.11 (d, J = 9.6 Hz, 2H), 4.13 (t, J = 6.2 Hz, 2H), 3.94 (s, 3H), 3.84 (m, 4H), 3.67 (t, J = 4.6 Hz, 4H), 3.29 (s, 2H), 2.82 (t, J = 4.6 Hz, 4H), 2.57 (t, J = 7.6 Hz, 2H), 2.48 (s, 4H), 2.03–2.06 (br, m, 2H); ¹³C NMR (CD₃OD, 125 MHz): δ 170.7, 163.6, 155.5, 152.0, 148.3, 148.1, 137.3, 135.3, 125.5, 123.7, 121.9, 110.9, 106.0, 104.6, 67.2, 66.3, 61.7, 61.5, 55.4, 53.4, 52.9, 49.5, 25.7; ESI-MS: m/z 566.4 [M + H]⁺. HRMS (ESI) m/z calcd for C₂₈H₃₅N₇O₆ [M + H]⁺ 566.2722, found: 566.2716.

Biological activity

The antitumor activities of the products against A549, K562, HepG2, and PC-3 were tested at five concentrations (0.625, 1.25, 2.5, 5, and 10 µM). Each test was done five times and assessed according to the MTT assay. The cell cultures were obtained from the Kunming Cell Bank. PC-3 was cultured at 37 °C and 5% CO₂ in Dulbecco’s modified eagle medium supplemented with 10% fetal bovine serum and 1% penicillin streptomycin. A549, K562, and HepG2 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with the same mixture. The cells were inoculated in a 96-well plate in 180 µL aliquots at 3 × 10⁴ cells/mL and cultivated for 1 day. The plates were further incubated for 48 h, and the assay was terminated by the addition of 20 µL of 5 mg/mL MTT. Incubation with the dye was continued for 4 h, after which time the medium was removed and the dye was extracted from the cells with 150 µL of DMSO. The absorbance was measured with a Microplate Reader (TECAN infinite M200 pro, TECAN, Switzerland) at 490 nm wavelength. The growth percentage was calculated on a plate by plate basis for test wells relative to control wells. The sensitivity of the cancer cells to each test compound was expressed in terms of IC₅₀, indicated as mean values ± SD for three independent experiments. Statistical analysis was performed using GraphPad Prism (version 7) for the Student’s test. Values of p < .05 were considered significant.

The wound healing assay was as follows. Cells were seeded in a six-well plate and grown for 24 h to 80% to 90% confluence. Cells were washed twice with phosphate-buffered saline (PBS), and the monolayers were scraped with a micropipette tip to create a uniform scratch. Next, cells were washed with PBS again to remove the detached cells. The compounds were added in the RPMI medium at 5 µM. DMSO was used as a control. Digital images of the wounded monolayers were obtained using a photomicroscope (Ti-S, Nikon, Japan) at 0, 6, 12, and 24 h. The unfilled scratched zones were quantified by Java’s ImageJ software.^23,24

Antiproliferative activity was measured by the clonogenic survival assay after treating with the test compound. Briefly, cells were seeded in a six-well plate at 3 × 10⁵ cells/well and were stimulated by a range of compound doses (1.25, 2.5, 5, 10, and 20 µM). The plates were further incubated for 24 h and the cells were plated in six wells at 1000 cells per well cultured for 12 days, and the number of surviving colonies (defined as a colony with >50 cells) was counted. The survival fraction was calculated as the number of colonies divided by the number of cells seeded times plating efficiency. Three independent experiments were performed.^25,26

Hoechst staining of the cultured cells was used to observe apoptotic nuclei by evaluation of nuclear morphology using a fluorescence inverted microscope. Cells were fixed in 4% paraformaldehyde solution for 10 min at RT. Then the cells were incubated with Hoechst 33258 dye for 20 min. Next, the cells were rinsed with precooling PBS and were observed under a fluorescence inverted microscope with 20 µL of antifluorescent quencher after air-drying. Typical nuclei showed non-condensed chromatin distributed over the whole nucleus. In contrast, apoptotic nuclei were identified by nuclear fragmentation of condensed chromatin. ²⁷

Flow cytometry

To evaluate cell apoptosis, a double staining trial with propidium iodide (PI) and fluorescein isothiocyanate isomer (FITC)-conjugated Annexin V (Solabio, Beijing, China) was performed. Cells were seeded in a six-well plate at 1 × 10⁶ cells/well and were stimulated by a range of compound doses (5, 10, and 20 µM). The original culture medium was collected and the cells were cultured with trypsin, which were subsequently washed twice in binding buffer. Next, 5 µL of Annexin V-FITC and 5 µL of PI were adopted for dying cells for 15 min at RT. The apoptotic A549 and PC-3 cells were observed via flow cytometry (BD Accuri™ C6, BD Biosciences, USA) equipment.^28,29

Supplemental Material

JCR_Supplementary_Information-Revised – Supplemental material for Design, synthesis, and biological evaluation of quinazoline derivatives containing piperazine moieties as antitumor agents

Supplemental material, JCR_Supplementary_Information-Revised for Design, synthesis, and biological evaluation of quinazoline derivatives containing piperazine moieties as antitumor agents by Wen Li, Shu-Yi Chen, Wei-Nan Hu, Mei Zhu, Jia-Min Liu, Yi-Hong Fu, Zhen-Chao Wang and Gui-Ping OuYang in Journal of Chemical Research