Abstract
In this study, a series of novel 2,4,6-trisubstituted quinazoline derivatives were designed, synthesized and biologically evaluated their antiproliferative activity against four human cancer cell lines (Eca-109, A549, PC-3 and MGC-803). The most of designed compounds showed considerable antiproliferative activity against the tested four cancer cell lines, while compound 28g displayed the best antiproliferative activity with the IC50 values of 1.95 μM and 2.46 μM against MGC-803 cells and Eca-109 cells, respectively. Further mechanism studies indicated that 28g significantly inhibited the cell migration and colony formation of MGC-803 cells. Besides, 28g also dose-dependently induced cellular apoptosis and cell cycle arrest at S phase in MGC-803 cells. Overall, all these studies suggested that 28g has the potential to act as a valuable lead compound for the development of antitumor agents.
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Introduction
Cancer is one of the most lethal diseases all over the world nowadays and even in the future. It has resulted in 10 million cancer deaths which account for one-sixth of deaths over the world in 2020, according to the World Health Organization (WHO). Besides, the global cancer burden is expected to be increased from 19.3 million cases in 2020 to 28.4 million cases in 2040 [1], emphasizing the emergency of effectively controlling the cancer development and treating various kinds of cancer. Regarding the increasing rate of cancer prevalence, vast efforts have been taken in the cancer treatment and great successes were made, including surgery, chemotherapy and radiotherapy. Although many new cancer treatment strategies, such as immunotherapy [2,3,4] and stem cell therapy [5,6,7], have been developed and shown promising potential against cancer, the chemotherapy is still the main strategy widely applied to the treatment of cancer.
Quinazoline, an important nitrogen-containing heterocyclic compound, has been widely applied in the development of drug candidates due to the wide range of pharmacological effects its derivatives presented, including antitumor, antiviral, antibacterial, anti-inflammatory, and antimalarial activities [8,9,10,11,12,13,14,15,16]. To date, many quinazoline-based drugs have been approved for the treatment of hypertension (Doxazosin) [17], chronic graft-versus-host disease (Tucatinib) [18] and, most importantly, cancer (Gefitinib, Erlotinib, Afatinib and Idelalisib etc.) [19,20,21]. Among all of the quinazoline derivatives, 4-anilinoquinazoline plays an important role in the development of antitumor agents as seen in EGFR inhibitors Gefitinib, Erlotinib and Afatinib which are 4-aminoquinazoline derivatives. Besides, this skeleton also can be seen in HDAC inhibitors (1) [22], microtubule inhibitors (2, 3) [23,24,25], and aurora kinase inhibitors (4, 5) [26, 27]. Aliphatic amines, especially aliphatic nitrogen heterocycles, are well-known building blocks in drug design and play a key role in improving the pharmacologic properties of new chemical entities via altering their physicochemical and ADME properties [28]. Compounds 6 and 7 are potent aliphatic nitrogen heterocycles contained LSD1 inhibitors showed good antitumor activities both in vitro and in vivo [29, 30]. Furthermore, alkynyl groups as star building blocks also have been broadly exploited in the discovery and development of drugs due to the advantages of improving metabolic stability, affinity, selectivity and the antitumor activities of drug candidates, as showed in Erlotinib and LSD1 inhibitor 8 [31, 32]. Therefore, based on the molecule hybridization strategy, we introduced propargyl group and aliphatic amine moieties into the 2-position and 4-position of the 4-anilinoquinazoline core to design and synthesize a series of 2,4,6-trisubstituted quinazoline derivatives (Fig. 1) and evaluated their biological activities in vitro.
The design strategy of 2,4,6-trisubstituted quinazoline derivatives reported in this paper
Results and discussion
Chemistry
The synthesis route of compounds 18a-18l was shown in Scheme 1. The commercially available 4-nitrophenol (9) reacted with corresponding alcohol in the presence of triphenylphosphine and diisopropyl azodicarboxylate through Mitsunobu reaction to afforded compounds 10a-10l, followed by a reduction reaction mediated by Fe powder to give compounds 11a-11l. On the other hand, commercially available compound 12 reacted with thionyl chloride to obtain its acyl chloride derivative 13, and then the latter performed a cyclization with the ammonium thiocyanate to form compound 14. Compound 15 was obtained through a nucleophilic substitution between compound 14 and 3-bromopropyne in the presence of potassium hydroxide. After compound 15 underwent an acylation reaction, the acyl chloride derivative compound 16 was obtained. Then, the nucleophilic aromatic substitution between compound 16 and compounds 11a-11l was performed to afford compounds 17a-17l. Finally, the deprotection of compound 11a-11l using trifluoroacetic acid in dichloromethane gave the target compound 18a-18l.
Reaction conditions and reagents: a) corresponding alcohol, triphenylphosphine, diisopropyl azodicarboxylate, THF, 0 °C, 3 h; b) Fe, NH4Cl, EtOH, H2O, 85 °C, 4 h; c) SOCl2, 75 °C, 2 h; d) ammonium thiocyanate, acetone, 0.5 h; e) 3-bromopropyne, KOH, H2O, rt, 2 h; f) SOCl2, DMF, 65 °C, 2 h; g) K2CO3, DMF, 80 °C, 2 h; h) THF, DCM, rt, 1 h
As shown in Scheme 2, the synthesis route of compounds 21a-21w is present. Compound 17l was reduced by the Fe powder to afford its arylamine derivatives 19, and then the latter performed an amidation reaction with appropriate carboxylic acid to give compounds 20a-20w. Finally, target compounds 21a-21w were obtained by removing the tert-butoxycarbonyl protecting group of compounds 20a-20w.
Reaction conditions and reagents: a) K2CO3, DMF, 80 °C, 1 h; b) Fe, NH4Cl, EtOH, H2O, 85 °C, 4 h; c) corresponding carboxylic acid, HATU, DIPEA, DMF, rt, 6 h; d) THF, DCM, rt, 1 h
As depicted in Scheme 3, compounds 28a-28h were synthesized. After undergoing an acylation reaction, compound 13 was obtained. Then, compound 13 reacted with ammonium hydroxide in the tetrahydrofuran to form corresponding amide 21. Further, a cyclization reaction between compound 22 and appropriate aldehyde was performed in the presence of sodium acetate and iodine to afford compounds 23a-23h. Treatment of 23a-23h with thionyl chloride gave the corresponding acyl chloride derivatives 24a-24h, followed by the nucleophilic aromatic substituting with the amino of compound 11l to afford compounds 25a-25h. Subsequent reduction then gave the desired arylamine compounds 26a-26h. Then, the amide condensation reaction was performed between compounds 26a-26h and 2-naphthoic acid, which resulted in compounds 27a-27h. Finally, the target compounds 28a-28h were obtained by deprotecting the tert-butoxycarbonyl groups of 27a-27h. The structures of all target compounds were confirmed by 1H NMR, 13C NMR and HRMS.
Reaction conditions and reagents: a) SOCl2, 75 °C, 2 h; b) NH3•H2O, THF, 0 °C, 0.5 h; c) corresponding aldehyde, C2H5ONa, iodine, DMF, 120 °C, 12 h; d) SOCl2, 65 °C, 2 h; e) K2CO3, DMF, 80 °C, 1 h; f) Fe, NH4Cl, EtOH, H2O, 85 °C, 4 h; g) 2-naphthoic acid, HATU, DIPEA, DMF, rt, 6 h; h) THF, DCM, rt, 1 h
Biological activity
Antiproliferative activity
We tested the antiproliferative activity of all the target compounds against four human cancer cell lines, the Eca-109 (human esophageal epithelial cancer cell line), A549 (human non-small cell lung cancer cell line), PC-3 (human prostate cancer cell line) and MGC-803 (human gastric cancer cell line), by MTT assay and Gefitinib as well as the 5-Fluorouracil (5-FU) was used as the positive control. The results were summarized in Tables 1–3. To investigate the effects of aliphatic amine moiety of 4-position on the antiproliferative activity, compounds 18a-18l were first designed and synthesized, and their antiproliferative activity results were shown in Table 1. Apart from 18c and 18d, all of the tested compounds showed medium to potent antiproliferative activity against these four human cancer cells. Generally, compared with straight-chain primary amine moieties, the compounds containing alicyclic amine moieties exhibited more potent antiproliferative activity. While the long chains are unfavorable for the bioactivity in the straight-chain primary amine moieties contained compounds (18a-18d), the bioactivity of big rings is superior to small rings in the alicyclic amine moieties contained compounds (18f-18i, 18k-18l). Besides, enhanced antiproliferative activities were observed when introduced a methyl into the terminal amino of compound 18a, as shown in 18e. Extending the piperidine-4yloxy moiety of compound 18i to piperidine-4ylmethoxy moiety resulted in a more potent compound 18l. Among these compounds, 18l displayed the most potent antiproliferative activity against tested cancer cells and thus was chosen for further optimization.
For many quinazoline derivatives, the substitutions of the 6-and 7-position are essential for their bioactivities [8, 22, 33,34,35]. Thus, we hypothesized whether modifying the 6-position of 18l could further improve its antiproliferative activity. To verify this hypothesis, we introduced various amide moieties into the 6-position of 18l to design and synthesize compounds 21a-21w. The antiproliferative activities of these compounds were shown in Table 2. Similarly, the alicyclic alkane moieties contained compounds also showed better activity, compared with those who had straight-chain alkane moieties in the 6-position (21a-21f). Then, diversely substituted benzamide were introduced to initially investigate the effects of aryl groups on the antiproliferative activities and resulted in 21g-21q. It can be concluded that the electron-withdrawing groups, such as cyano and nitro groups (21o-21q) are unfavorable for the bioactivities, while electron-donating groups are tolerable (21i-21m). Further introducing heteroaromatic groups showed that nitrogen-contained heterocycles also are detrimental to antiproliferative activity (21s-21t, 21v-21w). Interestingly, the substitution of a naphthalene group at the 6-position resulted in the most potent compound 21u which displayed enhanced activity against tested four cancer cell lines (IC50 = 4.91 μM, 3.82 μM, 3.62 μM and 4.98 μM against Eca-109, A549, PC-3 and MGC-803, respectively) compared with 18l. However, this round of optimization still did not achieve our desired antiproliferative activities.
To further improve the bioactivities, we then turned our attention to the 2-position of the quinazoline core which has not been modified yet. It has been reported that 2-arylquinazoline derivatives exhibited potent bioactivities in the cancer treatment and thus broadly applied in drug discovery [36,37,38]. Therefore, we designed a series of 2-arylquinazoline derivatives based on 21u and evaluated their antiproliferative activity. As shown in Table 3, most of the compounds displayed improved activity compared with 21u. It is worth noting that in the substituted phenyl groups, the para-substitution and meta-substitution exhibited better activity than the ortho-substitution (28b-28f). Particularly, p-tolyl contained compound 28g displayed the most potent antiproliferative activity against Eca-109, A549, PC-3 and MGC-803 cell lines with the IC50 values of 2.46 μM, 3.21 μM, 3.10 μM and 1.95 μM, respectively. Therefore, 28g and MGC-803 cells were chosen for further investigating the underlying mechanism of these designed compounds.
Compound 28g inhibited the migration and colony formation of MGC-803 cells
The migration and invasion of tumour cells into the surrounding tissue and lymphatic and blood vessels is the initial step of cancer metastasis which is the leading cause of the cancer related death [39, 40]. Therefore, inhibition of the cancer cell metastasis is one of the effective strategies to treatment of cancer. Thus, we investigated the effects of compound 28g on the migration of MGC-803 cells through Transwell assay. As shown in Fig. 2, the amount of migration cells was significantly decreased after incubating with 28g with the increased concentration (0, 1, 2, 4 μM), which indicated that 28g could dose-dependent inhibit the migration of MGC-803 cells. To further evaluate the antiproliferative ability of 28g against MGC-803 cells, the colony formation assay was performed. As displayed in Fig. 2, after incubation of increased concentrations of compounds 28g for 24 h, the cell colonies of MGC-803 cells were getting small and few compared with the control group (0 μM). It demonstrated that 28g indeed could inhibit the colony formation of MGC-803 cells in a dose-dependent manner.
A Compound 28g inhibited migration of MGC-803 cells. B Quantification of the effects of 28g on the migration of MGC-803 cells. C Compound 28g inhibited the migration of MGC-803 cells. Compared with the control group, **P < 0.01, ***P < 0.001
The effects of compound 28g on the cell cycle of MGC-803 cells
We then explored the effects of compound 28g on the cell cycle distribution of MGC-803 cells using the flow cytometry assay. As depicted in Fig. 3, after the treatment of increasing concentrations of compound 28g (1, 2, 4 μM) for 24 h, the percentage of MGC-803 cells in S phase was increased significantly (25.43% to 46.79%) compared with the control group (0 μM, 23.84%), which indicated that 28g could arrest the cell cycle of MGC-803 cells at S phase in a dose-dependent manner.
A The effects of compound 28g on the cell cycle of MGC-803 cells. B Quantitative analysis of the cell cycle distribution of MGC-803 cells
The effects of compound 28g on the cell apoptosis of MGC-803 cells
Apoptosis is one kind of programmed cell death which orderly and effectively clears away the damaged cells and deregulation of apoptosis is the hallmark of cancer [41, 42]. Triggering apoptosis remains an efficient strategy for the cancer treatment [43]. To investigate whether compound 28g could affect the apoptosis of MGC-803 cells, the DAPI staining assay and flow cytometry assay was performed. Before staining using DAPI, the cells were incubated with increasing concentrations of 28g for 48 h. As shown in Fig. 4A, the number of cells with shrunken or broken nuclear was dramatically increased compared with the control group (0 μM). Further characterizing apoptosis effects of compound 28g on MGC-803 cells was performed using Annexin V-FITC/PI double staining flow cytometry assay. As depicted in Fig. 4B, C, after incubating with 28g with increasing concentrations for 48 h, the percentage of apoptotic MGC-803 cells was increased from 11.64% to 38.64% compared with the control group (0 μM, 9.21%). All these results demonstrated that compound 28g could significantly induce the cell apoptosis of MGC-803 cells in a dose-dependent manner.
A The effects of compound 28g on the nuclear morphology of MGC-803 cells. The arrows in the figure indicate the cells with shrunken or broken nuclear. B The effects of compound 28g on the apoptosis of MGC-803 cells. C Quantification of the effects of 28g on the apoptosis of MGC-803 cells. Compared with the control group, **P < 0.01, ***P < 0.001
Conclusions
In summary, a series of 2, 4, and 6-trisubstituted quinazoline derivatives were designed, synthesized and evaluated for their antiproliferative activity against four human cancer cell lines (Eca-109, A549, PC-3 and MGC-803) by MTT assay. The results manifested that most of the designed compounds exhibited moderate to excellent in vitro antiproliferative activity, while compound 28g displayed the most potent antiproliferative activity against the tested cell lines Eca-109, A549, PC-3 and MGC-803 cell lines with the IC50 values of 2.46 μM, 3.21 μM, 3.10 μM and 1.95 μM, respectively. Further mechanism studies showed that 28g could significantly inhibit the migration and colony formation of MGC-803 cells. In addition, DAPI staining assay and flow cytometry assay indicated that 28g also dose-dependently induced cellular apoptosis and cell cycle arrest at S phase in MGC-803 cells. Taken together, all of these results suggested that 28g has the potential to act as a valuable lead compound for the development of antitumor agents.
Experimental section
General procedure
All reagents and solvents were purchased from commercial sources and used without further purification. Reactions were monitored by thin-layer chromatography (TLC) on 0.25 mm silicagel plates (GF254) and visualized under UV light. 1H NMR and 13C NMR spectra were recorded on a Bruker 400 MHz and 101 MHz spectrometer, respectively. High resolution mass spectra (HRMS) were recorded on a Waters Micromass Q-T of Micromass spectrometer by electrospray ionizaton (ESI). Melting points of target compounds were determined on an X-5 micromelting apparatus.
General procedure for the synthesis of compounds 11a-11l and 16
Compounds 11a-11l and 16 were synthesized according to the published literature and the characterization data was consistent with these literature [44,45,46].
General procedure for the synthesis of compounds 17a-17l
Compound 16 (0.5 g, 1.80 mmol), compounds 11a-11l (1.80 mmol) and K2CO3 (0.27 g, 1.98 mmol) were dissolved in 10 mL DMF, and then the reaction mixture was stirred at 90 °C for 1 h. After the reaction was completed, the mixture was treated with water and extracted with ethyl acetate. Then, the organic layer was dried with anhydrous Na2SO4 and concentrated in vacuo, followed by purifying through column chromatography to afford compounds 17a-17l (yield: 79.4–90.2%).
General procedure for the synthesis of compounds 18a-18l
To a solution of compounds 17a-17l (0.81 mmol) in DCM (10 mL), the TFA (2 mL) was added. After stirring at room temperature for 1 h, the resulting mixture was diluted with water and neutralized using Na2CO3, followed by extracting with ethyl acetate. Then, the organic layer was dried with anhydrous Na2SO4 and concentrated in vacuo. Finally, compounds 18a-18l were obtained by column chromatography.
N-(4-(2-aminoethoxy)phenyl)-6-nitro-2-(prop-2-yn-1-ylthio)quinazolin-4-amine (18a)
Yellow solid, yield: 81.3%, mp: 198.5–199.3 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.52 (s, 1H), 8.45 (d, J = 9.2 Hz, 1H), 7.68 (d, J = 8.5 Hz, 3H), 7.00 (d, J = 8.3 Hz, 2H), 3.96 (d, J = 6.9 Hz, 4H), 3.16 (s, 1H), 2.92 (s, 2H). 13C NMR (101 MHz, DMSO-d6). 13C NMR (101 MHz, DMSO-d6) δ 169.43, 157.43, 155.81, 153.41, 143.21, 130.77, 127.51, 127.07, 124.56, 121.15, 114.20, 112.36, 80.41, 73.23, 70.03, 40.82, 18.63. HRMS (ESI) calcd for C19H18N5O3S [M + H]+: 396.1130, found: 396.1128.
N-(4-(3-aminopropoxy)phenyl)-6-nitro-2-(prop-2-yn-1-ylthio)quinazolin-4-amine (18b)
Faint yellow solid, yield: 80.8%, mp: 169.6–170.3 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.50 (s, 1H), 8.43 (d, J = 9.2 Hz, 1H), 7.66 (t, J = 8.0 Hz, 3H), 6.98 (d, J = 8.5 Hz, 2H), 4.06 (t, J = 6.6 Hz, 2H), 3.95 (s, 2H), 3.16 (s, 1H), 2.76 (t, J = 6.9 Hz, 2H), 1.84 (p, J = 6.7 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 169.42, 157.34, 155.75, 153.46, 143.13, 131.00, 127.41, 126.99, 124.52, 121.16, 114.11, 112.54, 80.43, 73.21, 65.57, 38.07, 31.87, 18.61. HRMS (ESI) calcd for C20H19N5O3S [M + H]+: 410.1287, found: 410.1277.
N-(4-(4-aminobutoxy)phenyl)-6-nitro-2-(prop-2-yn-1-ylthio)quinazolin-4-amine (18c)
Faint yellow solid, yield: 83.6%, mp: 156.5–157.2 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.50 (s, 1H), 8.43 (d, J = 9.1 Hz, 1H), 7.65 (d, J = 8.5 Hz, 3H), 6.97 (d, J = 8.4 Hz, 2H), 4.02–3.97 (m, 2H), 3.95 (s, 2H), 3.15 (s, 1H), 2.64 (d, J = 7.1 Hz, 2H), 1.76 (s, 2H), 1.54 (t, J = 8.1 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 169.42, 157.29, 155.68, 153.55, 143.08, 131.32, 127.34, 126.97, 124.55, 121.21, 114.11, 112.76, 80.47, 73.19, 67.54, 40.87, 28.72, 26.17, 18.59. HRMS (ESI) calcd for C21H21N5O3S [M + H]+: 424.1443, found: 424.1430.
N-(4-((5-aminopentyl)oxy)phenyl)-6-nitro-2-(prop-2-yn-1-ylthio)quinazolin-4-amine (18d)
Faint yellow solid, yield: 82.1%, mp: 130.4–131.2 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.54 (s, 1H), 8.46 (d, J = 9.3 Hz, 1H), 7.74–7.60 (m, 3H), 6.98 (d, J = 8.4 Hz, 2H), 4.00 (d, J = 6.7 Hz, 2H), 3.96 (s, 2H), 3.19–3.12 (m, 1H), 2.65 (t, J = 6.0 Hz, 2H), 1.80–1.67 (m, 2H), 1.47 (s, 4H).13C NMR (101 MHz, DMSO-d6) δ 169.43, 157.41, 155.77, 153.50, 143.19, 131.00, 127.48, 127.08, 124.59, 121.23, 114.14, 112.59, 80.45, 73.22, 67.53, 40.56, 30.82, 28.49, 22.82, 18.60. HRMS (ESI) calcd for C22H23N5O3S [M + H]+: 438.1600, found: 438.1592.
N-(4-(2-(methylamino)ethoxy)phenyl)-6-nitro-2-(prop-2-yn-1-ylthio)quinazolin-4-amine (18e)
Faint yellow solid, yield: 81.1%, mp: 91.8–92.4 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.52 (s, 1H), 8.45 (d, J = 9.2 Hz, 1H), 7.68 (d, J = 8.6 Hz, 3H), 7.00 (d, J = 8.4 Hz, 2H), 4.06 (s, 2H), 3.95 (s, 2H), 3.16 (s, 1H), 2.87 (d, J = 6.5 Hz, 2H), 2.37 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 169.44, 157.50, 155.76, 153.38, 143.22, 130.71, 127.54, 127.07, 124.54, 121.14, 114.19, 112.30, 80.39, 73.23, 67.19, 50.15, 35.91, 18.66. HRMS (ESI) calcd for C20H19N5O3S [M + H]+: 410.1287, found: 410.1282.
N-(4-(azetidin-3-yloxy)phenyl)-6-nitro-2-(prop-2-yn-1-ylthio)quinazolin-4-amine (18f)
Faint yellow solid, yield: 86.1%, mp: 207.9-208.6 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.56 (d, J = 2.5 Hz, 1H), 8.50 (dd, J = 9.2, 2.4 Hz, 1H), 7.73 (d, J = 9.0 Hz, 3H), 7.02–6.85 (m, 2H), 5.10 (p, J = 5.7 Hz, 1H), 4.36 (dd, J = 11.3, 6.6 Hz, 2H), 3.97 (d, J = 2.5 Hz, 2H), 3.97–3.92 (m, 2H), 3.16 (q, J = 2.7 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 169.36, 157.47, 153.30, 152.81, 143.27, 131.94, 127.59, 127.12, 124.67, 121.13, 114.62, 112.26, 80.32, 73.27, 67.47, 52.45, 18.66. HRMS (ESI) calcd for C20H17N5O3S [M + H]+: 408.1130, found: 408.1121.
(R)-6-nitro-2-(prop-2-yn-1-ylthio)-N-(4-(pyrrolidin-3-yloxy)phenyl)quinazolin-4-amine (18g)
Faint yellow solid, yield: 85.5%, mp: 164.5–164.9 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.71–8.21 (m, 1H), 8.09–7.34 (m, 3H), 7.23–6.74 (m, 2H), 4.94 (s, 1H), 3.17 (s, 1H), 2.08 (s, 2H), 1.95 – 1.55 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 169.40, 157.48, 153.93, 153.37, 143.29, 131.12, 127.60, 127.15, 124.60, 121.15, 115.32, 112.29, 80.38, 76.78, 73.24, 51.24, 44.38, 31.57, 18.60. HRMS (ESI) calcd for C21H19N5O3S [M + H]+: 422.1287, found: 422.1271.
(S)-6-nitro-2-(prop-2-yn-1-ylthio)-N-(4-(pyrrolidin-3-yloxy)phenyl)quinazolin-4-amine (18h)
Faint yellow solid, yield: 85.5%, mp: 162.1–162.9 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.48 (d, J = 9.3 Hz, 1H), 7.70 (t, J = 9.3 Hz, 3H), 6.98 (d, J = 8.5 Hz, 2H), 4.92 (s, 1H), 3.96 (s, 2H), 3.14 (d, J = 14.1 Hz, 2H), 2.99 (d, J = 9.8 Hz, 2H), 2.89 (s, 1H), 2.13–1.81 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 169.40, 157.48, 153.93, 153.37, 143.29, 131.12, 127.60, 127.15, 124.60, 121.15, 115.32, 112.29, 80.38, 76.78, 73.24, 51.24, 44.38, 31.57, 18.60. HRMS (ESI) calcd for C21H19N5O3S [M + H]+: 422.1287, found: 422.1272.
6-nitro-N-(4-(piperidin-4-yloxy)phenyl)-2-(prop-2-yn-1-ylthio)quinazolin-4-amine (18i)
Faint yellow solid, yield: 89.4%, mp: 117.3–118.0 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.45 (d, J = 9.3 Hz, 1H), 7.80–7.62 (m, 3H), 7.04 (d, J = 8.5 Hz, 2H), 4.56 (dq, J = 8.9, 4.1 Hz, 1H), 3.96 (s, 2H), 3.26–3.16 (m, 1H), 3.12 (s, 2H), 2.87 (s, 2H), 2.04 (s, 2H), 1.70 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 169.40, 157.42, 153.89, 153.35, 143.24, 130.94, 127.54, 127.08, 124.55, 121.18, 115.64, 112.28, 80.36, 73.25, 71.16, 41.77, 29.37, 18.69. HRMS (ESI) calcd for C22H21N5O3S [M + H]+: 436.1443, found: 436.1428.
6-nitro-N-(4-(2-(piperazin-1-yl)ethoxy)phenyl)-2-(prop-2-yn-1-ylthio)quinazolin-4-amine (18j)
Faint yellow solid, yield: 89.4%, mp: 101.2–101.6 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.53 (s, 1H), 8.45 (d, J = 9.2 Hz, 1H), 7.67 (d, J = 8.6 Hz, 3H), 6.99 (d, J = 8.4 Hz, 2H), 4.10 (s, 2H), 3.95 (s, 2H),3.49 (s, 2H) 3.15 (s, 1H), 2.73 (d, J = 27.1 Hz, 4H), 2.44 (s, 4H). 13C NMR (101 MHz, DMSO-d6) δ 169.43, 157.43, 155.62, 153.39, 143.21, 130.76, 127.52, 127.07, 124.51, 121.16, 114.22, 112.33, 80.40, 73.22, 65.60, 57.08, 53.45, 45.24, 18.64. HRMS (ESI) calcd for C23H24N6O3S [M + H]+: 465.1709, found: 465.1690.
N-(4-(azetidin-3-ylmethoxy)phenyl)-6-nitro-2-(prop-2-yn-1-ylthio)quinazolin-4-amine (18k)
Faint yellow solid, yield: 86.7%, mp: 197.2–198.1 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.57 (s, 1H), 8.49 (d, J = 9.3 Hz, 1H), 7.72 (d, J = 8.6 Hz, 3H), 7.05 (d, J = 8.4 Hz, 2H), 4.17 (s, 2H), 4.04 (s, 2H), 3.97 (s, 2H), 3.82 (s, 2H), 3.39 (s, 1H), 3.20 (s, 1H). 13C NMR (101 MHz, DMSO-d6) δ 169.41, 157.51, 155.45, 153.38, 143.28, 131.09, 127.58, 127.15, 124.59, 121.16, 114.37, 112.26, 80.42, 73.24, 67.57, 47.85, 31.02, 18.63. HRMS (ESI) calcd for C21H19N5O3S [M + H]+: 422.1287, found: 422.1272.
6-nitro-N-(4-(piperidin-4-ylmethoxy)phenyl)-2-(prop-2-yn-1-ylthio)quinazolin-4-amine (18l)
Faint yellow solid, yield: 81.2%, mp: 144.1–144.7 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.55 (s, 1H), 8.47 (d, J = 9.2 Hz, 1H), 7.70 (d, J = 8.7 Hz, 3H), 7.00 (d, J = 8.4 Hz, 2H), 3.97 (s, 2H), 3.90 (d, J = 6.1 Hz, 2H), 3.31 (d, J = 12.5 Hz, 2H), 3.16 (d, J = 2.9 Hz, 1H), 2.90 (t, J = 12.6 Hz, 2H), 2.06 (s, 1H), 1.93 (d, J = 13.8 Hz, 2H), 1.48 (q, J = 12.8 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 169.42, 157.50, 155.72, 153.38, 143.29, 130.78, 127.58, 127.14, 124.56, 121.17, 114.22, 112.30, 80.41, 73.25, 71.49, 43.07, 33.36, 25.61, 18.64. HRMS (ESI) calcd for C19H18N5O3S [M + H]+: 450.1600, found: 450.1589.
General procedure for the synthesis of compound 19
A mixture of compound 17l (1.00 g, 1.82 mmol), Fe powder (0.71 g, 12.74 mmol) and NH4Cl (0.29 g, 5.46 mmol), 5 mL H2O and 15 mL EtOH in a flask was heated at 85 °C for 4 h. After the completion of the reaction, the residual Fe powder was removed under reduced pressure. Then the resulting liquid was extracted with ethyl acetate and water. Finally, the organic layer was dried with anhydrous Na2SO4 and concentrated in vacuo, followed by column chromatography to afford compound 19 (yield: 79.3%).
General procedure for the synthesis of compounds 20a-20w
A mixture of corresponding carboxylic acid (1.44 mmol), HATU (0.55 g, 1.44 mmol), 10 mL DMF and DIPEA (224 μL, 1.44 mmol) in 25 mL flask was stirred at room temperature for 10 min. Then, compound 19 (0.50 g, 0.96 mmol) was added and reacted at room temperature for an additional 6 h. After the consumption of starting materials, the resulting mixture was diluted with water and extracted with ethyl acetate. Then, the organic layer was dried with anhydrous Na2SO4 and evaporated under reduced pressure, followed by column chromatography to afford compounds 20a-20w (yield: 81.2–90.3%).
General procedure for the synthesis of compounds 21a-21w
To a solution of compounds 20a-20w (0.53 mmol) in DCM (10 mL), the TFA (2 mL) was added. After stirring at room temperature for 1 h, the resulting mixture was diluted with water and neutralized using Na2CO3, followed by extracting with ethyl acetate. Then, the organic layer was dried with anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified by column chromatography to afford compounds 21a-21w.
N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)acetamide (21a)
Faint yellow solid, yield: 83.2%, mp: 143.5–144.1 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.25 (s, 1H), 9.88 (s, 1H), 8.60 (d, J = 2.2 Hz, 1H), 7.79 (dd, J = 9.0, 2.1 Hz, 1H), 7.69–7.63 (m, 2H), 7.57 (d, J = 8.9 Hz, 1H), 6.98–6.90 (m, 2H), 3.93 (d, J = 2.7 Hz, 2H), 3.82 (d, J = 6.3 Hz, 2H), 3.11 (t, J = 2.6 Hz, 1H), 3.02 (d, J = 11.9 Hz, 2H), 2.56 (t, J = 12.2 Hz, 2H), 2.11 (s, 3H), 1.79–1.93 (s, 1H), 1.74 (d, J = 13.2, Hz, 2H), 1.29–1.16 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 168.32, 162.99, 156.95, 155.27, 146.76, 135.87, 131.66, 127.13, 126.65, 124.19, 114.07, 113.18, 112.64, 80.92, 72.88, 72.23, 44.70, 35.29, 28.39, 23.77, 18.30. HRMS (ESI) calcd for C25H27N5O2S [M + H]+: 462.1964, found: 462.1949.
N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)propion-amide (21b)
Faint yellow solid, yield: 84.6%, mp: 129.6–130.2 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.16 (s, 1H), 9.88 (s, 1H), 8.65 (d, J = 2.2 Hz, 1H), 7.77 (dd, J = 9.0, 2.2 Hz, 1H), 7.65 (d, J = 8.9 Hz, 2H), 7.57 (d, J = 8.9 Hz, 1H), 6.94 (d, J = 8.9 Hz, 2H), 3.92 (d, J = 2.7 Hz, 2H), 3.81 (d, J = 6.3 Hz, 2H), 3.11 (t, J = 2.6 Hz, 1H), 2.96 (dd, J = 11.9, 3.5 Hz, 2H), 2.46 (dd, J = 12.0, 2.5 Hz, 2H), 2.39 (q, J = 7.5 Hz, 2H), 1.85–1.70 (m, 1H), 1.70 (d, J = 13.3 Hz, 2H), 1.25–1.15 (m, 2H), 1.13 (d, J = 7.5 Hz, 3H).13C NMR (101 MHz, DMSO-d6) δ 171.98, 162.96, 156.97, 155.39, 146.79, 135.84, 131.55, 127.24, 126.68, 124.27, 114.06, 113.17, 112.52, 80.92, 72.86, 72.72, 45.68, 36.06, 29.70, 29.32, 18.29, 9.59. HRMS (ESI) calcd for C26H29N5O2S [M + H]+: 476.2120, found: 476.2107.
N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)butyramide (21c)
Faint yellow solid, yield: 84.6%, mp: 148.7–149.3 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.16 (s, 1H), 9.88 (s, 1H), 8.65 (d, J = 2.2 Hz, 1H), 7.77 (dd, J = 9.0, 2.2 Hz, 1H), 7.65 (d, J = 8.9 Hz, 2H), 7.57 (d, J = 8.9 Hz, 1H), 6.94 (d, J = 8.9 Hz, 2H), 3.92 (d, J = 2.7 Hz, 2H), 3.81 (d, J = 6.3 Hz, 2H), 3.11 (t, J = 2.6 Hz, 1H), 2.96 (dd, J = 11.9, 3.5 Hz, 2H), 2.46 (dd, J = 12.0, 2.5 Hz, 2H), 2.39 (q, J = 7.5 Hz, 2H), 1.89–1.79 (m, 1H), 1.70 (dd, J = 13.3, 3.4 Hz, 2H), 1.25–1.15 (m, 2H), 1.13 (d, J = 7.5 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 171.22, 162.86, 156.94, 155.23, 146.56, 136.21, 131.80, 126.82, 126.48, 124.01, 114.04, 113.27, 112.58, 80.93, 72.87, 72.45, 54.85, 38.17, 35.86, 29.15, 18.54, 18.29, 13.66. HRMS (ESI) calcd for C27H31N5O2S [M + H]+: 490.2277, found: 490.2262.
N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)cyclo-propanecarboxamide (21d)
Faint yellow solid, yield: 81.6%, mp: 136.1–138.7 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.63 (s, 1H), 9.93 (s, 1H), 8.71 (d, J = 2.2 Hz, 1H), 7.84 (dd, J = 8.9, 2.1 Hz, 1H), 7.67 (d, J = 8.9 Hz, 2H), 7.57 (d, J = 8.9 Hz, 1H), 7.00–6.89 (m, 2H), 3.93 (d, J = 2.7 Hz, 2H), 3.81 (d, J = 6.3 Hz, 2H), 3.11 (t, J = 2.7 Hz, 1H), 2.97 (s, 2H), 2.60–2.51 (m, 2H), 1.94–1.86 (m, 1H), 1.89-1.79 (m, 1H), 1.72 (d, J = 12.6 Hz, 2H), 1.19 (qd, J = 12.1, 4.1 Hz, 2H), 0.85 (t, J = 5.2 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) δ 171.69, 162.92, 156.95, 155.29, 146.67, 136.00, 131.68, 126.98, 126.63, 124.13, 114.06, 113.23, 112.54, 80.93, 72.86, 72.50, 45.27, 35.78, 29.17, 18.29, 14.39, 7.17. HRMS (ESI) calcd for C27H29N5O2S [M + H]+: 488.2120, found: 488.2108.
N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)cyclo-butanecarboxamide (21e)
Faint yellow solid, yield: 83.8%, mp: 133.5–134.3 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.06 (s, 1H), 9.92 (s, 1H), 8.67 (s, 1H), 7.78 (d, J = 9.1 Hz, 1H), 7.65 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.9 Hz, 1H), 6.95 (d, J = 8.5 Hz, 2H), 3.93 (s, 2H), 3.81 (d, J = 6.3 Hz, 2H), 3.29 (d, J = 8.3 Hz, 2H), 3.14 (q, J = 2.2 Hz, 1H), 2.97 (d, J = 11.9 Hz, 2H), 2.47 (s, 1H), 2.29 (q, J = 9.5 Hz, 2H), 2.15 (q, J = 10.5, 9.9 Hz, 2H), 1.98 (q, J = 9.2 Hz, 1H), 1.84 (q, J = 10.8, 10.0 Hz, 2H), 1.71 (d, J = 12.7 Hz, 2H), 1.19 (tt, J = 12.3, 6.0 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 172.87, 162.98, 156.96, 155.38, 146.82, 135.81, 131.55, 127.35, 126.67, 124.27, 114.07, 113.15, 112.61, 80.91, 72.87, 72.69, 45.59, 39.42, 35.97, 29.56, 24.60, 18.29, 17.78. HRMS (ESI) calcd for C28H31N5O2S [M + H]+: 502.2277, found: 502.2263.
N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)cyclo-pentanecarboxamide (21f)
Faint yellow solid, yield: 79.1%, mp: 142.7–143.1 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.89 (s, 1H), 8.68 (d, J = 2.1 Hz, 1H), 7.77 (dd, J = 8.8, 2.1 Hz, 1H), 7.65 (d, J = 8.6 Hz, 2H), 7.57 (d, J = 8.9 Hz, 1H), 6.95 (d, J = 8.6 Hz, 2H), 3.93 (d, J = 2.6 Hz, 2H), 3.85 (d, J = 6.3 Hz, 2H), 3.23–3.08 (m, 3H), 2.86 (p, J = 7.9 Hz, 1H), 2.78–2.65 (m, 2H), 1.92–1.67 (m, 9H), 1.58 (dt, J = 8.5, 3.9 Hz, 2H), 1.34 (qd, J = 12.7, 4.0 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 174.37, 162.89, 156.98, 155.19, 146.78, 135.98, 131.71, 127.33, 126.66, 124.34, 114.07, 113.16, 112.45, 80.91, 72.91, 71.89, 45.04, 43.99, 34.34, 30.06, 27.10, 25.70, 18.28. HRMS (ESI) calcd for C29H33N5O2S [M + H]+: 516.2433, found: 516.2419.
N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)benzamide (21g)
Faint yellow solid, yield: 85.4%, mp: 131.0–131.9 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H), 10.05 (s, 1H), 8.89 (s, 1H), 8.07 (d, J = 7.6 Hz, 2H), 8.03 (d, J = 9.0 Hz, 1H), 7.70 (d, J = 8.5 Hz, 2H), 7.61 (dd, J = 15.1, 8.0 Hz, 2H), 7.53 (t, J = 7.4 Hz, 2H), 6.91 (d, J = 8.5 Hz, 2H), 3.95 (d, J = 2.6 Hz, 2H), 3.80 (d, J = 6.3 Hz, 2H), 3.15 (d, J = 2.6 Hz, 1H), 3.04 (d, J = 12.1 Hz, 2H), 2.64–2.54 (m, 2H), 1.92–1.82 (m, 1H), 1.75 (d, J = 12.8 Hz, 2H), 1.24 (qd, J = 12.7, 2.4 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 165.37, 163.34, 156.96, 155.22, 147.11, 135.70, 134.38, 131.66, 128.39, 128.31, 127.65, 126.49, 124.13, 114.48, 114.02, 113.12, 80.91, 72.94, 72.32, 44.91, 39.90, 35.25, 28.50, 18.32. HRMS (ESI) calcd for C30H29N5O2S [M + H]+: :524.2120, found: 524.2108.
4-fluoro-N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)benzamide (21h)
Faint yellow solid, yield: 87.6%, mp: 191.6–192.4 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.62 (s, 1H), 9.96 (s, 1H), 8.82 (d, J = 2.3 Hz, 1H), 8.14 (dd, J = 8.6, 5.4 Hz, 2H), 7.94 (dd, J = 9.0, 2.1 Hz, 1H), 7.69 (d, J = 8.6 Hz, 2H), 7.64 (d, J = 8.9 Hz, 1H), 7.42 (t, J = 8.7 Hz, 2H), 6.98 (d, J = 8.6 Hz, 2H), 3.95 (d, J = 2.7 Hz, 2H), 3.88 (d, J = 6.2 Hz, 2H), 3.28 (d, J = 12.3 Hz, 2H), 3.15 (t, J = 2.6 Hz, 1H), 2.93–2.84 (m, 2H), 2.05 (s, 1H), 1.97–1.88 (m, 2H), 1.55–1.42 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 164.31, 163.44, 162.93(d, J = 250.4 Hz), 156.99, 155.15, 147.25, 135.46, 131.72, 130.82(d, J = 2.8 Hz), 130.43(d, J = 9.1 Hz), 128.63, 126.61, 124.25, 115.54(d, J = 21.6 Hz), 114.59, 114.14, 113.05, 80.90, 72.96, 71.47, 43.01, 33.43, 25.65, 18.32. HRMS (ESI) calcd for C30H28FN5O2S [M + H]+: 542.2026, found: 542.2012.
4-chloro-N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)benzamide (21i)
Faint yellow solid, yield: 82.7%, mp: 144.5–145.3 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.65 (s, 1H), 9.95 (s, 1H), 8.81 (d, J = 2.2 Hz, 1H), 8.08 (d, J = 8.3 Hz, 2H), 7.93 (dd, J = 8.9, 2.1 Hz, 1H), 7.70–7.66 (m, 3H), 7.66–7.61 (m, 2H), 6.96 (d, J = 8.6 Hz, 2H), 3.95 (d, J = 2.6 Hz, 2H), 3.82 (d, J = 6.3 Hz, 2H), 3.15 (d, J = 2.7 Hz, 1H), 3.03 (dd, J = 12.3, 3.3 Hz, 2H), 2.57 (td, J = 12.3, 2.6 Hz, 2H), 1.93–1.83 (m, 1H), 1.75 (d, J = 13.2, Hz, 2H), 1.31–1.18 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 164.33, 163.54, 156.99, 155.39, 147.33, 136.63, 135.29, 133.11, 131.52, 129.56, 128.57, 126.66, 124.27, 114.63, 114.11, 113.05, 80.89, 72.92, 72.46, 45.13, 35.48, 28.82, 18.33. HRMS (ESI) calcd for C30H28ClN5O2S [M + H]+: 558.1730, found: 558.1716.
4-bromo-N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)benzamide (21j)
Faint yellow solid, yield: 90.7%, mp: 156.7–157.2 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.97 (s, 1H), 10.18 (s, 1H), 8.94 (s, 1H), 8.18 (d, J = 9.1 Hz, 1H), 8.03 (d, J = 8.2 Hz, 2H), 7.75 (d, J = 8.5 Hz, 2H), 7.62 (dd, J = 12.7, 8.6 Hz, 3H), 6.85 (d, J = 8.6 Hz, 2H), 3.96 (d, J = 2.6 Hz, 2H), 3.80 (d, J = 6.2 Hz, 2H), 3.29 (d, J = 12.1 Hz, 2H), 3.15 (d, J = 2.7 Hz, 1H), 2.94–2.85 (m, 2H), 2.07–1.97 (s, 1H), 1.90 (d, J = 13.3 Hz, 2H), 1.48 (q, J = 12.5 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 164.31, 163.29, 156.84, 154.83, 146.93, 135.75, 133.35, 131.98, 131.27, 129.79, 126.35, 125.43, 124.49, 123.86, 114.38, 113.94, 113.18, 80.92, 72.95, 71.24, 42.84, 33.10, 25.22, 18.33. HRMS (ESI) calcd for C30H28BrN5O2S [M + H]+: 602.1225, found: 602.1209.
4-methyl-N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)benzamide (21k)
Faint yellow solid, yield: 88.6%, mp: 126.7–127.4 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 10.04 (s, 1H), 8.88 (s, 1H), 7.99 (d, J = 7.8 Hz, 3H), 7.70 (d, J = 8.6 Hz, 2H), 7.63 (d, J = 8.9 Hz, 1H), 7.37 (d, J = 7.9 Hz, 2H), 6.96 (d, J = 8.7 Hz, 2H), 3.95 (d, J = 2.6 Hz, 2H), 3.82 (d, J = 6.3 Hz, 2H), 3.15 (s, 1H), 3.00 (d, J = 11.7 Hz, 2H), 2.55 (d, J = 10.9 Hz, 2H), 2.40 (s, 3H), 1.91–1.81 (m, 1H), 1.79–1.69 (m, 2H), 1.21 (qd, J = 11.2, 10.2, 3.6 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 165.21, 163.32, 156.98, 155.32, 147.10, 141.78, 135.74, 131.62, 131.53, 128.98, 128.42, 127.68, 126.51, 124.18, 114.44, 114.08, 113.10, 80.92, 72.92, 72.47, 45.12, 35.60, 28.95, 20.98, 18.28. HRMS (ESI) calcd for C31H31N5O2S [M + H]+: 538.2277, found: 538.2261.
4-methoxy-N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)benzamide (21l)
Faint yellow solid, yield: 84.7%, mp: 137.6–138.4 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.45 (s, 1H), 9.95 (s, 1H), 8.82 (d, J = 2.3 Hz, 1H), 8.06 (d, J = 8.5 Hz, 2H), 7.95 (dd, J = 9.0, 2.1 Hz, 1H), 7.68 (d, J = 8.6 Hz, 2H), 7.63 (d, J = 8.9 Hz, 1H), 7.10 (d, J = 8.5 Hz, 2H), 6.95 (d, J = 8.6 Hz, 2H), 3.95 (d, J = 2.6 Hz, 2H), 3.86 (s, 3H), 3.82 (d, J = 6.4 Hz, 2H), 3.15 (t, J = 2.6 Hz, 1H), 3.00 (d, J = 11.9 Hz, 2H), 2.60–2.51 (m, 2H), 1.91–1.82 (m, 1H), 1.78–1.69 (m, 2H), 1.21 (qd, J = 12.2, 4.0 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 164.78, 163.27, 162.01, 156.97, 155.34, 147.09, 135.77, 131.57, 129.58, 128.57, 126.51, 126.41, 124.21, 114.39, 114.08, 113.68, 113.08, 80.92, 72.92, 72.53, 55.38, 45.28, 35.66, 29.08, 18.32. HRMS (ESI) calcd for C31H31N5O3S [M + H]+: 554.2226, found: 554.2210.
4-ethoxy-N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)benzamide (21m)
Faint yellow solid, yield: 88.9%, mp: 138.7–139.6 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.45 (s, 1H), 9.96 (s, 1H), 8.88–8.80 (m, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.99–7.92 (m, 1H), 7.69 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.9 Hz, 1H), 7.07 (d, J = 8.4 Hz, 2H), 6.95 (d, J = 8.5 Hz, 2H), 4.12 (q, J = 6.9 Hz, 2H), 3.95 (d, J = 2.7 Hz, 2H), 3.82 (d, J = 6.3 Hz, 2H), 3.15 (d, J = 2.7 Hz, 1H), 3.04 (d, J = 12.1 Hz, 2H), 2.64–2.53 (m, 2H), 1.92–1.82 (m, 1H), 1.76 (d, J = 12.7 Hz, 2H), 1.37 (t, J = 6.9 Hz, 3H), 1.25 (qd, J = 12.6, 2.5 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 164.78, 163.24, 161.30, 156.97, 155.28, 147.05, 135.84, 131.64, 129.59, 128.50, 126.49, 126.24, 124.18, 114.33, 114.17, 114.07, 113.09, 80.91, 72.93, 72.33, 63.38, 44.75, 35.24, 28.50, 18.32, 14.52. HRMS (ESI) calcd for C32H33N5O3S [M + H]+: 568.2382, found: 568.2368.
N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)-4-(trifluoromethyl)benzamide (21n)
Faint yellow solid, yield: 87.8%, mp: 179.8–180.4 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 10.00 (s, 1H), 8.86 (s, 1H), 8.25 (d, J = 8.1 Hz, 2H), 7.97 (d, J = 7.7 Hz, 3H), 7.68 (dd, J = 15.7, 8.7 Hz, 3H), 6.98 (d, J = 8.6 Hz, 2H), 3.96 (s, 2H), 3.89 (s, 2H), 3.28 (d, J = 12.3 Hz, 2H), 3.17 (d, J = 3.0 Hz, 1H), 2.92–2.82 (m, 2H), 2.02–1.92 (m, 1H), 1.89 (d, J = 5.5 Hz, 2H), 1.45 (qd, J = 13.0, 4.0 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 164.24, 163.60, 156.99, 155.16, 147.36, 138.20, 135.22, 131.71, 131.36(d, J = 32.0 Hz), 128.55, 128.41, 126.70, 125.49(q, J = 3.6 Hz), 125.21(d, J = 273.5 Hz), 124.23, 114.61, 114.13, 113.07, 80.88, 72.95, 71.49, 43.18, 33.51, 25.83, 18.33. HRMS (ESI) calcd for C31H28F3N5O2S [M + H]+: :592.1994, found: 592.1978.
4-cyano-N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)benzamide (21o)
Faint yellow solid, yield: 89.4%, mp: 155.6-157.3 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.84 (s, 1H), 9.99 (s, 1H), 8.84 (s, 1H), 8.20 (d, J = 8.0 Hz, 2H), 8.08 (d, J = 8.1 Hz, 2H), 7.94 (d, J = 9.1 Hz, 1H), 7.66 (t, J = 8.8 Hz, 3H), 6.96 (d, J = 8.7 Hz, 2H), 3.95 (d, J = 2.6 Hz, 2H), 3.82 (d, J = 6.3 Hz, 2H), 3.15 (d, J = 2.7 Hz, 1H), 2.99 (d, J = 12.0 Hz, 2H), 2.55 (s, 2H), 1.87–1.77 (m, 1H), 1.73 (d, J = 12.7 Hz, 2H), 1.19 (qd, J = 12.3, 4.2 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 164.00, 163.67, 156.99, 155.40, 147.43, 138.41, 135.08, 132.54, 131.48, 128.47, 126.71, 124.27, 118.24, 114.70, 114.08, 114.03, 113.06, 80.88, 72.94, 72.57, 45.38, 35.75, 29.23, 18.34. HRMS (ESI) calcd for C31H28N6O2S [M + H]+: 549.2072, found: 549.2061.
3-cyano-N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)benzamide (21p)
Faint yellow solid, yield: 87.5%, mp: 167.0-167.8 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H), 10.29 (s, 1H), 8.99 (s, 1H), 8.57 (d, J = 10.9 Hz, 1H), 8.37 (d, J = 7.9 Hz, 1H), 8.10 (d, J = 8.0 Hz, 2H), 7.79 (t, J = 7.8 Hz, 1H), 7.73 (d, J = 8.5 Hz, 2H), 7.63 (d, J = 9.0 Hz, 1H), 6.95 (d, J = 9.0 Hz, 2H), 3.96 (d, J = 2.6 Hz, 2H), 3.81 (d, J = 6.4 Hz, 2H), 3.13 (t, J = 2.7 Hz, 1H), 2.95 (dt, J = 12.1, 3.3 Hz, 2H), 2.46 (d, J = 12.0, Hz, 2H),1.87–1.77 (m, 1H), 1.70 (d, J = 13.1 Hz, 2H), 1.23–1.11 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 163.56, 163.46, 156.99, 155.35, 147.22, 135.48, 135.09, 132.52, 131.69, 131.27, 129.86, 127.95, 126.56, 124.10, 118.28, 114.60, 114.06, 113.22, 111.59, 80.92, 72.90, 72.77, 45.74, 36.12, 29.78, 18.34. HRMS (ESI) calcd for C31H28N6O2S [M + H]+: 549.2072, found: 549.2056.
4-nitro-N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)benzamide (21q)
Faint yellow solid, yield: 82.3%, mp: 261.6-262.3 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.00 (s, 1H), 10.10 (s, 1H), 8.92 (s, 1H), 8.41 (d, J = 8.4 Hz, 2H), 8.30 (d, J = 8.4 Hz, 2H), 8.02 (d, J = 8.9 Hz, 1H), 7.70 (d, J = 8.5 Hz, 2H), 7.65 (d, J = 8.9 Hz, 1H), 6.96 (d, J = 8.5 Hz, 2H), 3.96 (d, J = 2.6 Hz, 2H), 3.81 (d, J = 6.4 Hz, 2H), 3.16 (d, J = 2.9 Hz, 1H), 3.01–2.92 (m, 2H), 2.50–2.43 (m, 2H), 1.86–1.78 (m, 1H), 1.71 (d, J = 12.5 Hz, 2H), 1.24–1.17 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 163.73, 163.69, 156.99, 155.43, 149.23, 147.40, 140.12, 135.20, 131.54, 129.17, 128.32, 126.67, 124.22, 123.60, 114.79, 114.08, 113.12, 80.88, 72.92, 72.77, 45.75, 36.12, 29.79, 18.33. HRMS (ESI) calcd for C30H28N6O4S [M + H]+: 569.1971, found: 569.1956.
N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)furan-2-carboxamide (21r)
Faint yellow solid, yield: 86.3%, mp: 144.7–145.5 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.76–10.42 (m, 1H), 9.97 (s, 1H), 8.78 (s, 1H), 7.99 (s, 2H), 7.68 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.9 Hz, 1H), 7.44 (d, J = 3.5 Hz, 1H), 6.96 (d, J = 8.5 Hz, 2H), 6.75 (dd, J = 3.5, 1.7 Hz, 1H), 3.95 (d, J = 2.6 Hz, 2H), 3.81 (d, J = 6.3 Hz, 2H), 3.15 (s, 1H), 2.97 (dd, J = 9.4, 6.2 Hz, 2H), 2.50 (s, 2H), 1.89–1.79 (m, 1H), 1.72 (d, J = 11.8 Hz, 2H), 1.18 (qd, J = 12.2, 4.1 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 163.48, 156.94, 156.24, 155.41, 147.32, 147.21, 145.91, 134.88, 131.51, 128.38, 126.61, 124.19, 114.87, 114.54, 114.11, 113.08, 112.19, 80.89, 72.91, 72.71, 45.60, 35.98, 29.59, 18.33. HRMS (ESI) calcd for C28H27N5O3S [M + H]+: 514.1913, found: 514.1901.
N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)nicotinamide (21s)
Faint yellow solid, yield: 86.0%, mp: 165.4–166.1 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 1H), 10.15 (s, 1H), 9.24 (d, J = 2.2 Hz, 1H), 8.98 (s, 1H), 8.79 (d, J = 4.8 Hz, 1H), 8.46 (d, J = 7.9 Hz, 1H), 8.09 (d, J = 9.1 Hz, 1H), 7.73 (d, J = 8.5 Hz, 2H), 7.61 (dd, J = 14.2, 8.6 Hz, 2H), 6.95 (d, J = 8.7 Hz, 2H), 3.95 (d, J = 2.6 Hz, 2H), 3.82 (d, J = 6.3 Hz, 2H), 3.13 (s, 1H), 2.97 (d, J = 12.4 Hz, 2H), 1.89–1.79 (m, 1H), 1.72 (d, J = 12.5 Hz, 2H), 1.18 (qd, J = 12.2, 4.1 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 163.97, 163.58, 156.98, 155.14, 152.28, 148.67, 147.34, 135.37, 135.24, 131.73, 130.04, 128.37, 126.73, 124.23, 123.57, 114.45, 114.16, 113.07, 80.90, 72.94, 71.30, 42.82, 33.10, 25.22, 18.34. HRMS (ESI) calcd for C29H28N6O2S [M + H]+: 525.2072, found: 525.2060.
N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)imidazo[1,2-a]pyridine-6-carboxamide (21t)
Faint yellow solid, yield: 88.5%, mp: 193.6–194.5 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.74 (s, 1H), 9.98 (s, 1H), 9.37 (s, 1H), 8.84 (d, J = 2.2 Hz, 1H), 8.14 (s, 1H), 7.96 (dd, J = 8.9, 2.1 Hz, 1H), 7.83 (dd, J = 9.6, 1.8 Hz, 1H), 7.76–7.70 (m, 3H), 7.70–7.62 (m, 2H), 6.98 (d, J = 8.6 Hz, 2H), 3.96 (d, J = 2.7 Hz, 2H), 3.89 (d, J = 6.2 Hz, 2H), 3.31 (s, 2H), 3.16 (t, J = 2.7 Hz, 1H), 2.98–2.87 (m, 2H), 2.13–2.03 (s, 1H), 1.98–1.90 (m, 2H), 1.50 (qd, J = 11.0, 3.2 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 163.50, 163.42, 156.97, 155.16, 147.26, 144.62, 135.36, 134.60, 131.73, 129.56, 129.01, 126.71, 124.26, 124.22, 123.04, 119.72, 116.27, 114.42, 114.15, 113.10, 80.90, 72.95, 71.46, 43.14, 33.48, 25.78, 18.33. HRMS (ESI) calcd for C31H29N7O2S [M + H]+: 564.2181, found: 564.2172.
N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)-2-naphthamide (21u)
Faint yellow solid, yield: 89.8%, mp: 144.9–145.5 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.79 (s, 1H), 10.14–9.75 (m, 1H), 8.88 (s, 1H), 8.69 (s, 1H), 8.12 (d, J = 8.1 Hz, 3H), 8.02 (dd, J = 13.3, 8.3 Hz, 2H), 7.67 (q, J = 7.1, 5.5 Hz, 5H), 6.97 (d, J = 8.5 Hz, 2H), 3.97 (d, J = 2.6 Hz, 2H), 3.82 (d, J = 6.3 Hz, 2H), 3.17 (d, J = 3.0 Hz, 1H), 3.06–2.95 (m, 2H), 2.50 (s, 2H), 1.91–1.81 (q, J = 7.6 Hz, 1H), 1.74 (d, J = 12.9 Hz, 2H), 1.21 (qd, J = 12.3, 4.0 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 165.48, 163.45, 157.01, 155.36, 147.28, 135.61, 134.34, 132.08, 131.73, 131.57, 128.96, 128.62, 128.12, 128.08, 127.91, 127.66, 126.88, 126.65, 124.33, 124.27, 114.54, 114.10, 113.11, 80.93, 72.95, 72.43, 45.05, 35.43, 28.74, 18.36. HRMS (ESI) calcd for C34H31N5O2S [M + H]+: 574.2276, found: 574.2264.
N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)quinoline-6-carboxamide (21v)
Faint yellow solid, yield: 85.4%, mp: 142.7–143.1 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.96 (s, 1H), 10.06 (s, 1H), 9.04 (d, J = 4.2 Hz, 1H), 8.93 (s, 1H), 8.77 (s, 1H), 8.56 (d, J = 8.3 Hz, 1H), 8.36 (d, J = 8.8 Hz, 1H), 8.18 (d, J = 8.8 Hz, 1H), 8.04 (d, J = 8.9 Hz, 1H), 7.83–7.58 (m, 4H), 6.97 (d, J = 8.5 Hz, 2H), 3.96 (d, J = 2.6 Hz, 2H), 3.84 (t, J = 6.6 Hz, 2H), 3.17 (d, J = 2.8 Hz, 1H), 3.04 (d, J = 12.4 Hz, 2H), 2.58 (t, J = 12.2 Hz, 2H), 1.89 (s, 1H), 1.76 (d, J = 12.6 Hz, 2H), 1.25 (q, 11.6 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 164.97, 163.48, 157.01, 155.32, 152.30, 148.81, 147.24, 137.17, 135.62, 132.23, 131.64, 129.18, 128.56, 128.35, 127.96, 127.08, 126.63, 124.20, 122.28, 114.55, 114.11, 113.15, 80.92, 72.93, 72.36, 44.84, 35.29, 28.53, 18.35. HRMS (ESI) calcd for C33H30N6O2S [M + H]+: 575.2229, found: 575.2213.
N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(prop-2-yn-1-ylthio)quinazolin-6-yl)quinoxaline-6-carboxamide (21w)
Faint yellow solid, yield: 86.4%, mp: 143.7–144.2 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H), 10.03 (s, 1H), 9.09 (d, J = 5.4 Hz, 2H), 8.89 (d, J = 19.9 Hz, 2H), 8.49–8.41 (m, 1H), 8.28 (d, J = 8.8 Hz, 1H), 8.07–7.99 (m, 1H), 7.68 (t, J = 9.5 Hz, 3H), 6.96 (d, J = 8.5 Hz, 2H), 3.96 (d, J = 2.6 Hz, 2H), 3.82 (d, J = 6.4 Hz, 2H), 3.17 (d, J = 2.5 Hz, 1H), 3.00 (d, J = 11.9 Hz, 2H), 2.55 (d, J = 11.5 Hz, 2H), 1.91–1.81 (s, 1H), 1.74 (d, J = 13.0 Hz, 2H), 1.21 (qd, J = 12.2, 3.8 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 164.33, 163.59, 157.01, 155.37, 147.37, 147.18, 146.79, 143.53, 141.54, 135.44, 135.33, 131.54, 129.54, 128.85, 128.74, 128.50, 126.67, 124.26, 114.70, 114.09, 113.09, 80.91, 72.95, 72.51, 45.25, 35.64, 29.04, 18.34. HRMS (ESI) calcd for C32H29N7O2S [M + H]+: 576.2181, found: 576.2167.
General procedure for the synthesis of compound 22
Compound 13 (4.00 g, 20.00 mmol) was dissolved in THF (20 mL), and then the NH3•H2O was dropped slowly at 0 °C and stirred for an additional 30 min. The precipitate was separated by filtration, washed with water for three times and dried in the oven to afford compound 22 (yield: 89.3%).
General procedure for the synthesis of compounds 23a-23h
Compound 22 (3.00 g, 16.56 mmol), corresponding aldehyde (1.68 g, 16.56 mmol), C2H5ONa (1.09 g, 16.56 mmol) and iodine (5.25 g, 20.70 mmol) was dissolved in DMF (20 mL), and then the mixture was stirred at 120 °C for 12 h. After the consumption of starting materials, the resulting mixture was slowly dropped into a saturated sodium sulfite solution and stirred for 5 min. Then, the precipitate was separated by filtration, washed with water for three times and dried in the oven to afford compound 23a-23h (yield: 40.2–55.6%).
General procedure for the synthesis of compounds 24a-24h, 25a-25h, 26a-26h and 27a-27h
The procedure for the synthesis of compounds 24a-24h, 25a-25h, 26a-26h and 27a-27h was similar with compounds 16, 17l, 19 and 20u.
General procedure for the synthesis of compounds 28a-28h
To a solution of compounds 27a-27h (0.53 mmol) in DCM (10 mL), the TFA (2 mL) was added. After stirring at room temperature for 1 h, the resulting mixture was diluted with water and neutralized using Na2CO3, followed by extracting with ethyl acetate. Then, the organic layer was dried with anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified by column chromatography to afford compounds 28a-28h.
N-(2-phenyl-4-((4-(piperidin-4-ylmethoxy)phenyl)amino)quinazolin-6-yl)-2-naphthamide (28a)
Faint yellow solid, yield: 89.2%, mp: 178.5–179.1 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 9.90 (s, 1H), 9.01–8.95 (m, 1H), 8.90 (s, 1H), 8.72 (s, 1H), 8.57 (s, 1H), 8.48–8.39 (m, 2H), 8.13 (q, J = 10.4, 7.2 Hz, 3H), 8.07–8.04 (m, 1H), 7.88 (dd, J = 17.1, 8.7 Hz, 3H), 7.71–7.64 (m, 2H), 7.51 (d, J = 5.9 Hz, 2H), 7.06 (d, J = 8.5 Hz, 2H), 3.93 (d, J = 6.2 Hz, 2H), 3.33 (d, J = 10.9 Hz, 2H), 2.93 (d, J = 11.6 Hz, 2H), 2.05–2.15 (m, 1H), 1.97 (d, J = 13.8 Hz, 2H), 1.53 (q, J = 13.5, 12.6 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 165.53, 158.11, 157.66, 154.81, 147.46, 138.44, 136.39, 134.36, 132.51, 132.10, 131.74, 129.99, 128.98, 128.36, 128.33, 128.32, 128.21, 128.13, 127.93, 127.74, 127.67, 126.89, 124.35, 123.76, 114.18, 114.06, 71.68, 43.40, 33.86, 26.26. HRMS (ESI) calcd for C37H33N5O2 [M + H]+: 580.2713, found: 580.2700.
N-(2-(2-fluorophenyl)-4-((4-(piperidin-4-ylmethoxy)phenyl)amino)quinazolin-6-yl)-2-naphthamide (28b)
Faint yellow solid, yield: 87.8%, mp: 188.4–189.1 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.89 (s, 1H), 9.93 (s, 1H), 9.01 (s, 1H), 8.73 (s, 1H), 8.13 (s, 3H), 8.05 (d, J = 7.0 Hz, 2H), 7.83 (dd, J = 31.1, 8.6 Hz, 3H), 7.70 (d, J = 19.1 Hz, 3H), 7.51 (d, J = 41.7 Hz, 3H), 6.94 (d, J = 8.4 Hz, 2H), 3.82 (s, 2H), 3.10 (d, J = 11.8 Hz, 2H), 2.66 (t, J = 12.3 Hz, 2H), 1.96–1.86 (m, 1H), 1.79 (d, J = 13.1 Hz, 2H), 1.34–1.22 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 165.58, 159.72 (d, J = 236.6 Hz), 154.99, 147.06, 139.19, 136.83, 134.38, 132.19, 132.09, 131.71, 131.46, 131.36, 129.93, 129.86, 128.98, 128.30, 128.22, 128.15, 128.04, 127.96, 127.68, 126.91, 126.88, 124.37, 124.34, 123.87, 114.11, 113.86 (d, J = 21.2 Hz), 113.73, 72.04, 44.31, 34.62, 27.60. HRMS (ESI) calcd for C37H32FN5O2 [M + H]+: 598.2618, found: 598.2604.
N-(2-(3-fluorophenyl)-4-((4-(piperidin-4-ylmethoxy)phenyl)amino)quinazolin-6-yl)-2-naphthamide (28c)
Faint yellow solid, yield: 89.2%, mp: 171.2–173.0 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.87 (s, 1H), 9.96 (s, 1H), 8.99 (s, 1H), 8.72 (s, 1H), 8.27 (d, J = 7.8 Hz, 1H), 8.12 (d, J = 3.6 Hz, 3H), 8.10 (s, 2H), 8.05 (d, J = 7.6 Hz, 1H), 7.91 (d, J = 8.9 Hz, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.68 (q, J = 6.3 Hz, 2H), 7.56 (q, J = 7.4 Hz, 1H), 7.36–7.30 (m, 1H), 7.04 (d, J = 8.5 Hz, 2H), 3.84 (d, J = 6.3 Hz, 2H), 3.00 (d, J = 11.9 Hz, 2H), 2.49 (s, 2H), 1.91–1.81 (m, 1H), 1.74 (d, J = 12.7 Hz, 2H), 1.28–1.19 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 165.55, 163.56(d, J = 243.2 Hz), 157.75, 156.90, 155.15, 147.25, 141.22(d, J = 7.7 Hz), 136.71, 134.37, 132.14, 132.10, 131.74, 130.35(d, J = 8.0 Hz), 128.96, 128.45, 128.21, 128.13, 128.05, 127.92, 127.67, 126.88, 124.35, 124.02, 123.66, 116.80 (d, J = 21.4 Hz), 114.24, 114.12, 113.92, 113.85(d, J = 21.2 Hz), 72.51, 45.23, 35.62, 29.00. HRMS (ESI) calcd for C37H32FN5O2 [M + H]+: 598.2618, found: 598.2604.
N-(2-(4-fluorophenyl)-4-((4-(piperidin-4-ylmethoxy)phenyl)amino)quinazolin-6-yl)-2-naphthamide (28d)
Faint yellow solid, yield: 87.3%, mp: 190.2–190.8 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.86 (s, 1H), 9.91 (s, 1H), 8.97 (s, 1H), 8.73 (s, 1H), 8.46 (d, J = 7.2 Hz, 2H), 8.12 (q, J = 8.3, 7.8 Hz, 4H), 8.04 (d, J = 7.5 Hz, 1H), 7.88 (d, J = 8.8 Hz, 1H), 7.82 (d, J = 8.5 Hz, 2H), 7.66 (s, 2H), 7.33 (t, J = 8.7 Hz, 2H), 7.04 (d, J = 8.4 Hz, 2H), 3.87–3.84 (m, 2H), 3.11 (d, J = 11.9 Hz, 2H), 2.66 (t, J = 12.1 Hz, 2H), 1.98–1.88 (m, 1H), 1.81 (d, J = 12.7 Hz, 2H), 1.32 (d, J = 13.1 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 165.54, 162.26 (d, J = 248.2 Hz), 157.71, 157.24, 154.96, 147.40, 136.38, 134.94 (d, J = 2.5 Hz), 134.37, 132.32, 132.09, 131.74, 129.95 (d, J = 8.9 Hz), 128.97, 128.29, 128.21, 128.13, 128.02, 127.94, 127.68, 126.90, 124.34, 123.91, 115.29 (d, J = 21.6 Hz), 114.19, 114.04, 113.95, 72.01, 44.11, 34.53, 27.31. HRMS (ESI) calcd for C37H32FN5O2 [M + H]+: 598.2618, found: 598.2601.
N-(2-(2-chlorophenyl)-4-((4-(piperidin-4-ylmethoxy)phenyl)amino)quinazolin-6-yl)-2-naphthamide (28e)
Faint yellow solid, yield: 91.8%, mp: 185.3–186.1 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.93 (s, 1H), 9.01 (s, 1H), 8.73 (s, 1H), 8.13 (s, 3H), 8.05 (d, J = 7.3 Hz, 2H), 7.83 (dd, J = 27.7, 8.7 Hz, 3H), 7.70 (d, J = 18.0 Hz, 3H), 7.59–7.53 (m, 1H), 7.46 (s, 2H), 6.95 (d, J = 8.5 Hz, 2H), 3.89–3.83 (m, 2H), 3.25 (d, J = 11.9 Hz, 2H), 2.84 (t, J = 12.3 Hz, 2H), 2.06–1.96 (m, 1H), 1.89 (d, J = 13.3 Hz, 2H), 1.48–1.38 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 165.58, 159.71, 157.37, 154.86, 147.06, 139.20, 136.87, 134.38, 132.30, 132.09, 131.69, 131.46, 131.36, 129.85, 128.98, 128.98, 128.30, 128.22, 128.15, 127.97, 127.68, 126.91, 124.35, 123.86, 114.14, 113.84, 113.73, 71.49, 43.10, 33.45, 25.78. HRMS (ESI) calcd for C37H32ClN5O2 [M + H]+: 614.2323, found: 614.2308.
N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(o-tolyl)quinazolin-6-yl)-2-naphthamide (28f)
Faint yellow solid, yield: 85.2%, mp: 189.3–189.9 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.88 (s, 1H), 9.86 (s, 1H), 9.00 (s, 1H), 8.75 (s, 1H), 8.13 (d, J = 10.2 Hz, 3H), 8.05 (d, J = 7.4 Hz, 2H), 7.84 (dd, J = 17.7, 8.0 Hz, 2H), 7.79–7.61 (m, 4H), 7.30 (q, J = 7.9, 7.5 Hz, 3H), 6.97 (d, J = 8.4 Hz, 2H), 3.92–3.78 (m, 2H), 3.24–3.09 (m, 2H), 2.71 (t, J = 12.6 Hz, 2H), 2.52 (s, 3H), 2.01–1.91 (m, 1H), 1.82 (d, J = 12.7 Hz, 2H), 1.35 (dd, J = 22.3, 11.4 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 165.54, 161.46, 157.60, 155.01, 147.26, 139.33, 136.53, 136.46, 134.37, 132.41, 132.10, 131.74, 130.77, 130.03, 128.97, 128.44, 128.25, 128.12, 127.93, 127.67, 126.89, 125.45, 124.36, 124.24, 114.15, 113.92, 113.36, 71.86, 43.85, 34.25, 26.94, 21.14. HRMS (ESI) calcd for C38H35N5O2 [M + H]+: 594.2869, found: 594.2856.
N-(4-((4-(piperidin-4-ylmethoxy)phenyl)amino)-2-(p-tolyl)quinazolin-6-yl)-2-naphthamide (28g)
Faint yellow solid, yield: 88.3%, mp: 200.4–200.9 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.84 (s, 1H), 9.86 (s, 1H), 8.95 (d, J = 2.1 Hz, 1H), 8.72 (s, 1H), 8.33 (d, J = 7.9 Hz, 2H), 8.17–8.11 (m, 3H), 8.10–8.04 (m, 2H), 7.87 (dd, J = 9.0, 3.7 Hz, 3H), 7.68 (q, J = 6.6 Hz, 2H), 7.31 (d, J = 7.9 Hz, 2H), 7.05 (d, J = 8.5 Hz, 2H), 3.92 (d, J = 6.1 Hz, 2H), 3.31 (d, J = 12.5 Hz, 2H), 2.91 (dd, J = 13.6, 10.7 Hz, 2H), 2.39 (s, 3H), 2.13–2.03 (m, 1H), 1.95 (d, J = 13.7 Hz, 2H), 1.59–1.44 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 165.52, 158.20, 157.57, 154.72, 147.53, 139.62, 136.17, 135.77, 134.36, 132.61, 132.10, 131.76, 128.97, 128.87, 128.28, 128.21, 128.14, 128.07, 127.94, 127.78, 127.68, 126.91, 124.34, 123.68, 114.19, 114.04, 113.96, 71.44, 42.95, 33.34, 25.51, 20.96. HRMS (ESI) calcd for C38H35N5O2 [M + H]+: 594.2869, found: 594.2853.
N-(2-(furan-2-yl)-4-((4-(piperidin-4-ylmethoxy)phenyl)amino)quinazolin-6-yl)-2-naphthamide (28h)
Faint yellow solid, yield: 86.3%, mp: 192.2–193.0 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.82 (s, 1H), 9.84 (s, 1H), 8.93 (s, 1H), 8.71 (s, 1H), 8.12 (d, J = 5.4 Hz, 3H), 8.05 (t, J = 7.9 Hz, 2H), 7.92–7.87 (m, 3H), 7.85 (d, J = 8.8 Hz, 1H), 7.71–7.62 (m, 2H), 7.18 (d, J = 3.3 Hz, 1H), 7.02 (d, J = 8.5 Hz, 2H), 6.69–6.65 (m, 1H), 3.83 (d, J = 6.3 Hz, 2H), 2.99 (d, J = 11.9 Hz, 2H), 2.55 (s, 2H), 1.94–1.80 (m, 1H), 1.79–1.69 (m, 2H), 1.21 (qd, J = 12.1, 3.9 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 165.54, 157.29, 154.94, 152.96, 151.96, 147.21, 144.86, 136.26, 134.36, 132.40, 132.09, 131.75, 128.97, 128.34, 128.13, 128.11, 128.07, 127.92, 127.67, 126.89, 124.35, 123.46, 114.28, 114.13, 114.06, 112.48, 112.04, 72.57, 45.32, 35.70, 29.14. HRMS (ESI) calcd for C35H31N5O3 [M + H]+: 570.2505, found: 570.2492.
Materials and methods
Materials
All tested compounds were dissolved in DMSO to make a 10 μM stock solution. RPMI-1640, DAPI and MTT were purchased from Solarbio. Annexin VFITC/PI Apoptosis Detection Kit and Cell Cycle Detection Kit were purchased from Keygen Biotech. Fetal Bovine Serum (FBS) and Trypsin were purchased from Beyotime Biotechnology. All the other reagents used were of analytical grade.
Cell culture
Eca-109 (human esophageal epithelial cancer cell line), A549 (human non-small cell lung cancer cell line), PC-3 (human prostate cancer cell line)) and MGC-803 (human gastric cancer cell line) were purchased from American Type Culture Collection (ATCC, Shanghai, China). These four human cancer cell lines were maintained in RPMI-1640 medium which was supplemented with 10 % fetal bovine serum (FBS) and were cultured at 37 °C in a humidified atmosphere containing 5% CO2.
MTT assay
The antiproliferative activity of individual compounds against four cancer cell lines was tested in vitro by MTT assay. Generally, the cells (1500–3300 cells/well) were cultured in 96-well plates for 24 h and then incubated in triplicate with different concentrations of individual compounds or DMSO contained medium (200 μL) for an additional 72 h. Then, 20 μL MTT solution (5%) was added to each well at 37 °C in a humidified atmosphere containing 5% CO2 for 4 h. After that, the MTT-containing medium was removed and, following by adding DMSO (150 μL/well) to dissolve the formazan crystals.
The absorbance was measured by the microplate reader at the wave of 490 nm and the IC50 values were calculated using GraphPad Prism 8.0.1. The results were Mean ± SD of three independent experiments.
Colony formation assay
MGC-803 cells were seeded into the 6-well plate with a density of 800 cells/well and then the cells were cultures at 37 °C in a humidified atmosphere containing 5% CO2 for 24 h. Next, the medium was replaced by different concentrations of 28g contained medium. After incubating for 7 days, the culture medium was discarded and the plates were washed with PBS twice. Then, the cells were fixed with 4% paraformalclehyde for 20 min, followed by staining with 0.1% crystal violet. After that, the crystal violet solution was removed and carefully washed with water. After the plate is naturally dried, images were photographed and processed using Photoshop.
Transwell assay
MGC-803 cells were seeded into Corning®Costar® Transwell® cell culture chamber with porous membrane. The upper chambers were placed into a 24-well plate (the lower chambers). The FBS-free medium, different concentrations of 28g and 6000-8000 MGC-803 cells were added in each upper chamber, while 500 μL 20% FBS contained medium was added into the lower chambers. After 24 h period of incubation at 37 °C, the medium was discarded and the migration cells were fixed with 4% paraformalclehyde for 20 min. Then, the cells were stained with 0.1% crystal violet solution, followed by carefully washed with water, naturally dried and photographed.
Cell cycle analysis
The distribution of cell cycle status was detected using the cell cycle detection kit (Keygen Biotech, China) and flow cytometer, according to the manufacturer’s instructions. Briefly, the cells in the logarithmic growth phase were trypsinized, centrifuged, resuspended and seeded in the 6-well plate with a concentration of 7 × 104 cells/well. After 24 h period of incubation at 37 °C, the cells were harvested and treated with various concentrations of compound 28g for another 24 h. Then, the cells were harvested, washed with PBS and incubated with 1 mL DNA staining solution and 10 μL permeabilization solution under dark conditions. Finally, the samples were subjected to the flow cytometer (BD, Biosciences, San Jose, CA, USA) for detecting the percentage of cell cycle phases. The data was analyzed using ModfitLT 5.
DAPI staining assay
The cells in the logarithmic growth phase were trypsinized, centrifuged, resuspended and seeded in the 6-well plate with a concentration of 7 × 104 cells/well. After 24 h period of incubation at 37 °C, the culture medium was replaced with indicated concentrations of 28g for an additional 48 h. Then, the medium was removed and the plates were washed with PBS twice, followed by fixed with 1 mL 4% paraformalclehyde and stained with 1 mL DAPI solution (10 μg/mL) in the dark for 10 min. After discarding the DAPI solution, the cells were washed with PBS twice and photographed using a fluorescence microscope.
Cell apoptosis analysis
Cell apoptosis was detected by Annexin V-FITC/PI apoptosis detection kit (Keygen Biotech, China) and flow cytometer, according to the manufactory instructions. Briefly, MGC-803 cells were seeded in the 6-well plate with a concentration of 7 × 104 cells/well and cultured at 37 °C for 24 h. Then, the culture medium was replaced with indicated concentrations of 28g for an additional 48 h. After that, the cells were harvested, washed with PBS and resuspended in binding buffer (500 μL), followed by incubation with Annexin V-FITC (5 μL) and PI (5 μL) under dark conditions. Cell apoptosis at different stages was detected by flow cytometry and data was analyzed using Flowjo V10.
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This work was supported by the National Natural Science Foundation of China (No. U21A20416).
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Wang, H., Wang, T., Chi, L. et al. Design, synthesis and biological evaluation of novel 2,4,6-trisubstituted quinazoline derivatives as potential antitumor agents. Med Chem Res 32, 1832–1850 (2023). https://doi.org/10.1007/s00044-023-03114-x
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DOI: https://doi.org/10.1007/s00044-023-03114-x