Synthesis and biological evaluation of 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-ones as cisplatin sensitizers

A series of novel 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one derivatives were synthesized and their anti-cancer as well as cisplatin sensitization activities were evaluated.

Abstract

A series of novel 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one derivatives were synthesized and their anti-cancer as well as cisplatin sensitization activities were evaluated. Among them, compounds 6e and 6h exhibited significant cisplatin sensitization activity against HCT116. Hoechst staining and annexin V-FITC/PI dual-labeling studies demonstrated that the combination of 6e/6h and cisplatin can induce tumour cell apoptosis. Western blot showed that the expression of ATR downstream protein, CHK1, decreased in 6e + cisplatin and 6h + cisplatin groups compared with that in the test compound and cisplatin group. Furthermore, docking of 6e/6h into the ATR structure active site revealed that the N1 and N8 atoms in the naphthyridine ring and the hybrid atom in the oxadiazole ring are involved in hydrogen bonding with Val170, Glu168 and Tyr155. Additionally, the naphthyridine ring is also involved in π–π stacking with Trp169. Accordingly, compounds 6e and 6h can be expected to be potential cisplatin sensitizers that can participate in HCT116 cancer therapy.

Introduction

The 1,8-naphthyridinone moiety is a structural feature of many drug molecules and pharmaceutical agents and lots of compounds bearing 1,8-naphthyridinone exhibit a variety of biological properties, such as anti-tumour,1–3 anti-bacterial,4,5 and anti-inflammatory,6 CB2-selective agonist activities,7–10 etc. As far as anti-cancer activity was concerned, Ke Chen and his co-workers discovered a series of 2-aryl-1,8-naphthyridin-4(1H)-ones and 2-phenyl-1,8-naphthyridin-4-ones as anti-tumour agents through inhibiting tubulin polymerization.2,3 Indeed, HKL-1 (2-(3-methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one) is an antimitotic agent that can induce G₂/M arrest and mitotic catastrophe in human leukemia HL-60 cells.1 Furthermore, 1,3,4-oxadiazole is a commonly used moiety for pharmacophore development, which has been thoroughly investigated because of its hydrogen bonding ability within the receptor site and good metabolic profile.11 The azole group (–N C—O) of 1,3,4-oxadiazole can increase the lipophilicity of drugs, promote drug transportation through cell membranes to reach the target site, and facilitate a drug's various biological activities.12 In fact, many studies showed that the synthetic compounds bearing the 1,3,4-oxadiazole moiety have excellent anticancer activities both in vitro and in vivo.13–19 Although various substituent 1,8-naphthyridinones and 1,3,4-oxadiazoles have been developed, there are still a lot of analogues that have not been designed and synthesized (Fig. 1). Therefore, a novel molecular skeleton, 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one (Fig. 2), was designed and synthesized, which was seen as the core structure of the target compounds in this study.

Fig. 1. Chemical structures of small-molecule compounds bearing 1,8-naphthyridin-4(1H)-one or oxadiazole with anti-cancer activities.

Fig. 2. Chemical structure of 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one.

Cisplatin is a DNA-damaging agent that is commonly used in the treatment of solid tumours and is highly effective against certain cancers like metastatic testicular cancer.20–23 However, for the majority of solid tumours such as ovarian, lung, and prostatic tumours, cisplatin has only a modest benefit.24 Additionally, the drug resistance, nephrotoxicity, ototoxicity and neurotoxicity of cisplatin also limit the scope of its application.16 Recent studies have demonstrated that the poor response to those cancers and drug resistance are closely related to the DNA damage response (DDR).25–31 The DDR network is an elaborate transduction system which can sense different types of damage and coordinate a series of responses, including activation of apoptosis, cell cycle control, transcription, senescence, and DNA repair processes.32,33 One critical signalling pathway in the DDR network is the ataxia telangiectasia mutated and rad3 related/checkpoint kinase 1 (ATR/CHK1) pathway, which is activated by single-stranded DNA arising from stalled replication forks caused by replication stress or as an intermediate of DNA repair processes.34,35 Many studies showed that the cisplatin sensitivity activities can increase by the inhibition of ATR and its downstream protein CHK1.36–38

In this study, a series of novel 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one derivatives were designed and synthesized (Schemes 1 and 2), and their biological activities as anti-cancer and cisplatin sensitizer agents were evaluated. Among these compounds, 6e and 6h were discovered to have cisplatin-sensitizing effects. Hoechst staining, annexin V-FITC/PI dual-labelling, western blot and molecular docking experiments were employed to investigate the possible mechanism.

Scheme 1. Reagents and conditions: (i) diethyl 2-(ethoxymethylene)malonate, 130 °C, 2 h; (ii) Ph₂O, 250 °C, 30–40 min; (iii) (4-(methylsulfonyl)phenyl)boronic acid, Pd cat., 60 °C, 8 h; (iv) H₂NNH₂, 50 °C, 3 h; (v) RCOCl, THF, DIEA, overnight; (vi) pyridine, SOCl₂, 3 h.

Scheme 2. Reagents and conditions: (i) H₂NNH₂, 50 °C, 3 h; (ii) R₁COCl, THF, DIEA, overnight; (iii) pyridine, SOCl₂, 3 h; (iv) aryl boronic acid or aryl borate, Pd cat., 50 °C, 6 h; (v) R₂COCl, THF, DIEA, overnight; (vi) pyridine, SOCl₂, 3 h; (vii) aryl boronic acid or aryl borate, Pd cat., 50 °C, 6 h.

Results and discussion

Chemistry

In this study, a new series of 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one derivatives were synthesized. The general procedures for the preparation of the 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one derivatives 6a–6h were according to the protocol outlined in Scheme 1. The condensation product of 5-bromopyridin-2-amine with diethyl ethoxymethylenemalonate was heated under 130 °C for 2 h to provide intermediate 1. Phenyl ether was heated under stirring at 250 °C, and then 1 was added slowly. The resulting mixture was refluxed for 4 h to obtain intermediate 2. Intermediate 3 was prepared from 2 by Suzuki coupling with 4-(methylsulfonyl)phenylboronic acid and dichlorobis(triphenylphosphine)palladium(ii). Intermediate 4 was obtained by treating 3 with hydrazine hydrate in the presence of methanol at room temperature. A series of aromatic acid derivatives were reacted with SOCl₂ and DMF as catalyst and refluxed for 1 h to afford aroyl chloride derivatives, which were converted to amide intermediates 5a–5h by treatment with 4 in the presence of DIEA. Finally, the compounds 6a–6h were obtained by refluxing 5a–5h with SOCl₂ in pyridine for 3 h.

As outlined in Scheme 2, compounds 10a–10c and 13a–13c were obtained starting from intermediate 2 and the synthesis began with hydrazine hydrate to prepare intermediate 7. 8 and 11 were obtained by the reaction of the corresponding aromatic acid derivatives with 7. Refluxing 8 and 11 with SOCl₂ in pyridine for 3 h can provide the intermediates 9 and 12. Bis(triphenylphosphine)palladium(ii) chloride catalyzed the coupling of 9 with a series of boronic acid derivatives in the presence of Na₂CO₃ to yield the compounds 10a–10c. In addition, compounds 13a–13c were obtained by a similar synthetic route with intermediate 12 and different kinds of boronic acid analogues. All synthetic compounds gave satisfactory analytical and spectroscopic data, which were consistent with their depicted structures.

In vitro inhibition evaluation of compounds

The proliferation-inhibitory effects of compounds 6a–6h, 10a–10c and 13a–13c in several tumour cell lines were evaluated primarily by the MTT assay. It was shown that cancer cells, including human non-small cell lung cancer A549, hepatocellular carcinoma HepG2, oral epidermoid carcinoma KB, breast cancer MCF-7, cervical carcinoma HeLa and colorectal carcinoma HCT116, were inhibited by the treatment with each compound with different concentrations (Tables 1 and 2). However, compared with that of cisplatin, the inhibitory effects of the compounds were slight. Comfortingly, all the compounds have lower cytotoxicity to LO2 normal cells (Table S1 of the ESI†).

Table 1. In vitro cancer cell growth inhibiting rate of compound (10 μM) alone.

Compound (10 μM)	Inhibition rate (% of control)
	A549	HepG2	KB	MCF-7	HeLa	HCT116
6a	9.51 ± 1.38	6.98 ± 1.67	5.20 ± 1.80	2.83 ± 1.11	17.53 ± 0.25	15.71 ± 1.13
6b	—	—	—	—	—	14.73 ± 0.59
6c	12.11 ± 0.50	6.65 ± 2.10	3.02 ± 0.21	4.27 ± 0.44	18.36 ± 1.02	7.83 ± 2.58
6d	—	—	—	—	—	16.22 ± 1.46
6e	8.95 ± 1.95	13.34 ± 2.12	7.50 ± 2.19	7.76 ± 0.67	2.57 ± 0.38	25.19 ± 0.65
6f	26.76 ± 0.90	11.61 ± 2.75	9.30 ± 0.66	13.07 ± 0.38	1.64 ± 0.76	17.00 ± 2.69
6g	3.59 ± 0.61	11.09 ± 2.52	7.58 ± 2.62	8.10 ± 0.97	15.68 ± 0.10	18.65 ± 1.19
6h	11.22 ± 0.95	8.94 ± 1.91	3.19 ± 1.41	10.58 ± 1.44	18.48 ± 1.25	24.44 ± 0.69
10a	—	—	—	—	—	11.83 ± 2.71
10b	12.51 ± 0.78	12.67 ± 1.84	9.73 ± 3.29	19.69 ± 0.56	13.65 ± 0.43	18.30 ± 2.66
10c	9.77 ± 1.63	14.93 ± 2.31	19.49 ± 2.88	26.90 ± 0.30	31.29 ± 0.18	21.63 ± 2.40
13a	—	—	—	—	—	19.68 ± 1.54
13b	—	—	—	—	—	20.53 ± 2.07
13c	—	—	—	—	—	18.89 ± 0.77
Cisplatin	68.52 ± 0.53	76.16 ± 3.94	42.43 ± 3.58	27.37 ± 0.56	44.16 ± 0.56	57.76 ± 1.09

Table 2. In vitro cancer cell growth inhibiting rate of compound (5 μM) alone.

Compound (5 μM)	Inhibition rate (% of control)
	A549	HepG2	KB	MCF-7	HeLa	HCT116
6a	8.17 ± 0.50	5.46 ± 0.22	4.38 ± 2.73	1.38 ± 0.65	8.58 ± 0.34	8.60 ± 2.16
6b	—	—	—	—	—	9.26 ± 1.42
6c	6.76 ± 2.01	3.49 ± 0.30	2.16 ± 0.81	2.35 ± 0.35	7.08 ± 0.04	5.61 ± 1.32
6d	—	—	—	—	—	11.34 ± 3.27
6e	7.92 ± 2.15	13.20 ± 1.76	3.70 ± 0.81	1.86 ± 0.36	1.49 ± 0.48	20.35 ± 1.25
6f	23.77 ± 0.21	11.25 ± 3.26	2.15 ± 2.10	0.86 ± 0.10	1.07 ± 0.57	16.38 ± 1.25
6g	3.11 ± 0.15	9.45 ± 2.84	2.55 ± 3.38	3.78 ± 0.50	8.47 ± 1.71	14.86 ± 2.89
6h	6.69 ± 0.67	6.90 ± 1.65	0.43 ± 0.60	6.26 ± 0.30	10.45 ± 0.34	20.44 ± 1.46
10a	—	—	—	—	—	15.86 ± 1.02
10b	7.60 ± 0.99	11.51 ± 4.45	7.35 ± 0.81	4.65 ± 3.23	4.80 ± 0.58	17.86 ± 2.56
10c	1.44 ± 0.85	5.10 ± 1.20	2.15 ± 1.06	11.95 ± 0.42	3.55 ± 1.58	12.29 ± 2.97
13a	—	—	—	—	—	11.09 ± 0.24
13b	—	—	—	—	—	10.93 ± 2.17
13c	—	—	—	—	—	12.56 ± 1.84
Cisplatin	39.77 ± 1.01	55.09 ± 3.21	24.30 ± 3.29	7.76 ± 1.35	34.99 ± 0.16	41.12 ± 0.39

In vitro inhibition evaluation of compounds in combination with cisplatin

To investigate whether compounds 6a–6h, 10a–10c and 13a–13c have activities in tumour therapy, the combined effects of each compound and cisplatin against HCT116 cells were evaluated in this study. As shown in Table 3, the inhibitory rate of cisplatin (2.5 μM) was 26.98%, while the inhibition rate of each compound (2.5 μM) was low. It is worth noting that the compounds, especially 6e and 6h, can enhance the sensitivity of tumour cells to cisplatin (p < 0.01 or p < 0.001). The inhibition rate of cisplatin increased from 26.98% to 50.81% and 54.78%, after combining with 6e and 6h, respectively. However, the cytotoxicity against LO2 normal cells did not significantly increase in the 6e/6h + cisplatin group (Table S2 of the ESI†).

Table 3. In vitro cancer cell growth inhibiting rate of compound combined with cisplatin for HCT116 cells.

	Inhibition rate (% of control)
	Cisplatin (2.5 μM)	Compound (2.5 μM)	Cisplatin (2.5 μM) + compound (2.5 μM)
	26.98 ± 2.84
6a		0.53 ± 2.17	27.19 ± 2.16
6b		4.32 ± 0.88	28.59 ± 1.63
6c		2.64 ± 1.71	29.16 ± 1.88
6d		5.37 ± 1.71	34.28 ± 2.56
6e		8.13 ± 3.63	50.81 ± 1.35***###
6f		2.06 ± 3.38	24.91 ± 2.06
6g		10.71 ± 1.29	39.46 ± 0.75
6h		7.47 ± 0.73	54.78 ± 2.70***###
10a		10.42 ± 2.08	20.13 ± 1.37
10b		13.73 ± 3.16	35.43 ± 1.99
10c		9.81 ± 5.15	29.83 ± 0.36
13a		9.87 ± 2.03	20.06 ± 2.12
13b		7.54 ± 1.26	19.57 ± 0.29
13c		10.15 ± 0.27	25.64 ± 1.82

Morphological analysis by Hoechst staining

We next explored the morphology of cancer cell nuclei by Hoechst 33258 staining. Hoechst 33258 is a membrane-permeable fluorescent dye that can stain the chromatin of cells. In cells with normal morphology, the nuclei stained by Hoechst 33258 were round and light-blue coloured, and the chromatin was stained blue, while the apoptotic cells have condensed chromatins, bright blue nuclei, and nuclear fragmentation.40,41 In this study, the treatment with cisplatin, 6e or 6h alone can induce morphological changes in HCT116 nuclei. In fact, the morphology of cancer cell nuclei could undergo further changes such as remarkable chromatin condensation, cell shrinkage, and evident reduction in the number of adherent cells (Fig. 3a). The number of nuclei was quantified and analyzed and shown in Fig. 3b, which was consistent with the MTT assay.

Fig. 3. Fluorescence microscopic appearance of Hoechst 33258-stained nuclei of HCT116 cells with the treatment of compounds 6e and 6h in combination with cisplatin. HCT116 cells were treated with 6e or 6h alone for 24 h before combination with cisplatin (2.5 μM) for 48 h. (a) i, control; ii, cisplatin (2.5 μM); iii, 6e (2.5 μM); iv, 6e (2.5 μM) + cisplatin (2.5 μM); v, 6h (2.5 μM); vi, 6h (2.5 μM) + cisplatin (2.5 μM). (b) The blue fluorescence was quantified and analyzed by T test. All data were from three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. cisplatin group; #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. compound group.

Apoptosis analysis by FCM

To further understand the morphological changes of cancer cells, annexin V-FITC/PI dual-labelling was performed by FCM. As shown in Fig. 4a, cisplatin, 6e and 6h at different concentrations can induce HCT116 cell apoptosis at different degrees. Remarkably, the percentage of annexin V-positive cells significantly increased in the 6e + cisplatin group, which indicated that cisplatin and 6e could produce synergistic apoptosis in the appropriate concentration (Fig. 4b). At the same time, the percentage of PI-positive cells was enhanced remarkably (p < 0.001) compared with that with cisplatin or 6e alone (Fig. 4c). Similar results were also observed in the 6h + cisplatin group. Based on these together with the Hoechst 33258 staining experiment results, we propose that apoptosis may be one signalling pathway for the enhanced cisplatin sensitization effects of compounds 6e and 6h.

Fig. 4. Apoptosis analysis of compounds 6e and 6h in combination with cisplatin. HCT116 cells were treated with 6e/6h alone for 24 h before combination with cisplatin for 48 h. Cell apoptosis was evaluated by flow cytometry. (a) i, control; ii, cisplatin (2.5 μM); iii, 6e (2.5 μM); iv, 6e (2.5 μM) + cisplatin (2.5 μM); v, 6h (2.5 μM); vi, 6h (2.5 μM) + cisplatin (2.5 μM). (b and c) Percentage of annexin V⁺ and PI⁺ cells analyzed of 6e and 6h. All data were from three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. cisplatin group; #p < 0.05, ##p < 0.01 and ###p < 0.001 vs. compound group.

Western blot analysis

The cisplatin sensitization mechanism initiated by 6e/6h in combination with cisplatin was explored by western blot. The ATR/CHK1 signalling pathway can regulate many common proteins, such as p53, CDC25 phosphatases, and Wee1 kinase, which further regulated G₁, S and G₂/M checkpoints. Besides, suppression of the CHK1 signalling pathway will lead to DDR damage and then may trigger DNA damage-induced apoptosis or senescence.42 It has been reported that some small-molecule compounds, such as SCH900776 (MK-8776)36 and CBP-93872,39 can enhance cisplatin sensitivity by inhibiting the phosphorylation of CHK1 and thus induce cell death.

Therefore, the expression of CHK1 in HCT116 cancer cells was examined in this study. The results showed that phosphorylated CHK1 was upregulated by cisplatin treatment (Fig. 5), which may be attributed to the DNA-damage agent cisplatin, leading to the DDR in cancer cells. However, CHK1 expression was decreased in 6e + cisplatin and 6h + cisplatin groups in a concentration-dependent manner, indicating that 6e and 6h can inactivate the CHK1 induced by cisplatin treatment and then achieve synergistic anti-tumour effects. The expression of CHK1 can recover to normal levels in 6e (2.5 μM) + cisplatin and 6h (5 μM) + cisplatin groups.

Fig. 5. Compounds 6e (a) and 6h (b) in combination with cisplatin inhibited CHK1 expression. HCT116 cells were treated with 6e or 6h alone for 24 h before combination with cisplatin (2.5 μM) for 48 h. CHK1 expression was quantified by densitometry analysis using ImageJ. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. cisplatin group.

Binding mode of 6e and 6h into ATR

Based on the above western blot result, we suspected that the 6e and 6h might interact with the ATR signaling pathway. Therefore, in an attempt to explain the cisplatin sensitization effects of 6e and 6h, molecular docking to ATR43 was performed in our research and the results showed that the N1 and N8 atoms in the naphthyridine ring and the hybrid atoms in the oxadiazole ring are involved in H-bonding interactions with Val170, Glu168 and Tyr155 (Fig. 6). Additionally, the naphthyridine ring is also involved in π–π stacking with Trp169. Besides the key binding of the skeleton mentioned above, the sulfone group also forms a hydrogen bond with Gly175. Moreover, the methylphenyl ring is involved in hydrophobic interaction with Lys117, Leu122 and Pro159.

Fig. 6. Theoretical binding mode of 6e (a) and 6h (b) to the ATR active binding pocket.

The biological activities of the tested compounds could be correlated to structure variations. Regarding 6a–6h, it was found that the replacement of phenyl with a pyridine group resulted in the decrease in the sensitization activity, which can be ascribed to the decline of hydrophobic interaction with Lys117 and Leu122 of the ATR kinase pocket. Furthermore, exploration of the impact of the substitution on the different position of the phenyl group demonstrated that 4-modification is more beneficial than 3-position (6e and 6h). With regard to 4-substituted compound counterparts in 6f, 6g, and 6h, the sensitivity activities were decreased in the order –CH₃ > –F > –Cl.

Conclusions

In summary, a series of 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one derivatives were synthesized and their anti-tumour activities in vitro against A549, HepG2, KB, MCF-7, HeLa, and HCT116 cells were tested in this research. Regrettably, all compounds have only slight activities. However, the anti-tumour activities initiated by the compounds in combination with cisplatin were measured and the results showed that compound 6e and 6h exhibited good cisplatin sensitization. Hoechst staining and annexin V-FITC/PI dual-labeling studies demonstrated that the combination of 6e/6h (2.5 μM) and cisplatin (2.5 μM) can significantly induce apoptosis of HCT116 cancer cells.

ATR is the most important protein in the DDR network and western blot showed that cisplatin treatment led to significant upregulation of CHK1 expression in HCT116 cancer cells, which indicated that the DNA-damage agent cisplatin can cause HCT116 cells to initiate DDR, produce drug resistance and ultimately induce parts of tumour cell survival. However, CHK1 expression was decreased in the 6e + cisplatin and 6h + cisplatin groups, suggesting that 6e and 6h can inhibit the DDR response and increase the sensitivity of tumour cells to cisplatin. Furthermore, molecular docking was employed in our study and docking of 6e/6h into the ATR structure active site revealed that the N1 and N8 atoms in the naphthyridine ring and the hybrid atom in the oxadiazole ring are involved in hydrogen bonding with Val170, Glu168 and Tyr155. Additionally, the naphthyridine ring is also involved in π–π stacking with Trp169. Taken together, 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one derivatives 6e and 6h deserve further investigation as potential cisplatin-sensitizing agents for HCT116 cancer cells.

Experimental

Materials and instruments

Unless specified otherwise, all chemicals and reagents used in this study were of analytical grade and used without any purification. The reactions were monitored by thin layer chromatography (TLC) on pre-coated silica GF254 plates (Qingdao Ocean Chemical Factory, China). Melting points (uncorrected) were determined with an SGW X-4 melting point apparatus (Shanghai Precision & Scientific Instrument Co., Ltd, China). Magnetic resonance spectra (NMR) were detected on a Bruker AC-E400 spectrometer operating at a field strength of 400 MHz at room temperature with TMS and solvent signals allotted as internal standards. Chemical shifts are reported in ppm (δ) with references. Standard and peak multiplicities are designated as follows: s, singlet; d, doublet; dd, double doublets; t, triplet; q, quartet; brs, broad singlet; and m; multiplet. Mass spectrometry (MS) data were obtained with an ESI-QTOF spectrometer. The purity of all the compounds was evaluated by an HPLC system (Dionex Ultimate 3000, USA) and was found to be higher than 98%.

Synthesis of diethyl 2-(((5-bromopyridin-2-yl)amino)methylene)malonate (1)

The condensation product of 5-bromopyridin-2-amine (1.73 g, 10 mmol) with diethyl ethoxymethylene-malonate (2.16 g, 10 mmol) was heated under 130 °C in an open system for 2 h and then cooled to room temperate followed by recrystallization in ethanol to obtain 1 as a yellow solid (2.6 g). Yield: 75.7%. ¹H NMR (400 MHz, CDCl₃) δ = 11.12 (d, J = 12.5 Hz, 1H), 9.07 (d, J = 12.7 Hz, 1H), 8.39 (d, J = 2.2 Hz, 1H), 7.75 (dd, J = 8.6 Hz, J = 2.4 Hz, 1H), 6.78 (d, J = 8.6 Hz, 1H), 4.50–4.12 (m, 4H), 1.36 (dt, J = 15.9 Hz, J = 7.1 Hz, 6H).

Synthesis of ethyl 6-bromo-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylate (2)

Phenyl ether (30 mL) was heated under stirring at 250 °C. Diethyl 2-(((5-bromopyridin-2-yl)amino)methylene)malonate (10 g, 29.1 mmol) was slowly added, and the resulting mixture was refluxed for 30–40 min. After the mixture was cooled to room temperature, the resulting precipitate was collected by filtration, washed with petroleum ether, and recrystallized from DMF to obtain 2 as a white solid (7.8 g). Yield: 90.0%. ¹H NMR (400 MHz, CDCl₃) δ = 9.38 (d, J = 1.4 Hz, 1H), 9.04 (s, 1H), 7.97 (dd, J = 9.3 Hz, J = 2.0 Hz, 1H), 7.67 (d, J = 9.3 Hz, 1H), 4.43 (q, J = 7.1 Hz, 2H), 1.42 (t, J = 7.1 Hz, 3H).

Synthesis of ethyl 6-(4-(methylsulfonyl)phenyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylate (3)

4-(Methylsulfonyl)phenylboronic acid (2.53 g, 12.65 mmol) was added to a solution of intermediate 2 (10.54 mmol) in a 1,4-dioxane/H₂O mixture (5 : 1, 20 mL), followed by Na₂CO₃ (3.32 g, 31.62 mmol) and dichlorobis(triphenylphosphine)palladium(ii) (220 mg, 0.32 mmol). The mixture was then heated and stirred at 60 °C under a nitrogen atmosphere for 8 h. After being cooled to room temperature, the mixture was diluted with DCM (120 mL), washed with H₂O (3 × 40 mL), and dried (Na₂SO₄). The solvent was then evaporated under reduced pressure, and the residue was purified by chromatography to afford 3 as a white solid (3.7 g). Yield: 94.1%, mp, 233.5–234.5 °C. ¹H NMR (400 MHz, CDCl₃) δ = 9.52 (s, 1H), 9.08 (s, 1H), 8.21 (d, J = 9.0 Hz, 1H), 8.13 (d, J = 8.2 Hz, 2H), 7.92 (d, J = 9.1 Hz, 1H), 7.88 (d, J = 8.3 Hz, 2H), 4.44 (q, J = 7.1 Hz, 2H), 3.13 (s, 3H), 1.44 (t, J = 7.1 Hz, 3H). ESI-QTOF-MS: 395.0766 (C₂₃H₁₆N₄O₄SNa, M + Na⁺), anal. calcd for C₁₈H₁₆N₂O₅S: 372.0780.

Synthesis of 6-(4-(methylsulfonyl)phenyl)-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carbohydrazide (4)

3 (2.68 mmol) obtained by a known method was suspended in methanol (15 mL) and to this mixture hydrazine monohydrate (1 mL) was added dropwise at room temperature for 3 h. The precipitate was separated from the reaction mixture by means of suction filtration to give 4 (600 mg). Yield: 62.3%. ¹H NMR (400 MHz, DMSO-d₆) δ = 9.77 (s, 1H), 9.42 (s, 1H), 9.05 (s, 1H), 8.61 (dd, J = 9.1 Hz, J = 1.7 Hz, 1H), 8.14 (q, J = 8.4 Hz, 4H), 8.03 (d, J = 9.1 Hz, 1H), 4.71 (s, 2H), 3.31 (s, 3H).

Synthesis of compounds 5a–5h

An appropriate aromatic acid (0.63 mmol) was reacted with SOCl₂ (4 mL) and DMF as a catalyst and refluxed for 1 h to prepare acid chlorides. The product was used as such in the next step. A solution of 4 (150 mg, 0.42 mmol) and DIEA (162 mg, 1.26 mmol) in THF (3 mL) was added to a stirred solution of acid chlorides in 30 mL of THF at 0 °C. The mixture was stirred at room temperature overnight. The white precipitate was recovered by filtration, washed three times with 5 mL portions of THF and dried overnight under vacuum.

General procedure for the synthesis of compounds 6a–6h

To a mixture of the appropriate intermediate (5a–5h, 0.32 mmol) in SOCl₂ (5 mL), pyridine (50 mg, 0.64 mmol) was added dropwise. The reaction mixture was then gradually heated to the reflux temperature and maintained there for 3 h. The remaining SOCl₂ was removed by rotary evaporation and poured into a saturated NaHCO₃ solution to acidify to pH 7. Then the aqueous layer was extracted with CH₂Cl₂, and the combined organic layers were dried over Na₂SO₄, filtered and concentrated. The products (6a–6h) were purified by column chromatography.

6-(4-(Methylsulfonyl)phenyl)-3-(5-phenyl-1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one (6a)

Yellow solid: yield 53.0%, mp 301.1–302.0 °C. ¹H NMR (400 MHz, DMSO-d₆): δ = 9.49 (s, 1H), 9.16 (s, 1H), 8.65 (d, J = 9.0 Hz, 1H), 8.19 (d, J = 8.1 Hz, 2H), 8.13 (d, J = 7.6 Hz, 4H), 8.07 (d, J = 9.1 Hz, 1H), 7.67 (s, 3H), 3.32 (s, 3H). ESI-QTOF-MS: 445.0891 (C₂₃H₁₇N₄O₄S, M + H⁺), anal. calcd for C₂₃H₁₆N₄O₄S: 444.0892.

6-(4-(Methylsulfonyl)phenyl)-3-(5-(pyridin-3-yl)-1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one (6b)

Yellow solid: yield 49.1%, mp 297.7–299.2 °C. ¹H NMR (400 MHz, DMSO-d₆) δ = 9.49 (s, 1H), 9.30 (s, 1H), 9.19 (s, 1H), 8.84 (s, 1H), 8.65 (d, J = 8.5 Hz, 1H), 8.49 (d, J = 6.8 Hz, 1H), 8.16 (d, J = 14.9 Hz, 4H), 8.07 (d, J = 8.1 Hz, 1H), 7.70 (s, 1H). 3.32 (s, 3H). ESI-QTOF-MS: 446.0847 (C₂₂H₁₆N₅O₄S M + H⁺), anal. calcd for C₂₂H₁₅N₅O₄S, 445.0845.

6-(4-(Methylsulfonyl)phenyl)-3-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one (6c)

Yellow solid: yield 60.3%, mp 320.9–321.6 °C. ¹H NMR (400 MHz, DMSO-d₆) δ = 9.51 (s, 1H), 9.20 (s, 1H), 8.89 (d, J = 5.3 Hz, 2H), 8.67 (d, J = 9.5 Hz, 1H), 8.19 (d, J = 8.4 Hz, 2H), 8.14 (d, J = 8.3 Hz, 2H), 8.09 (d, J = 9.1 Hz, 1H), 8.05 (d, J = 5.4 Hz, 2H), 3.32 (s, 3H). ESI-QTOF-MS: 446.0848 (C₂₂H₁₆N₅O₄S, M + H⁺), anal. calcd for C₂₂H₁₅N₅O₄S, 445.0845.

6-(4-(Methylsulfonyl)phenyl)-3-(5-(pyridin-2-yl)-1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one (6d)

Yellow solid: yield 42.2%, mp 291.2–292.3 °C. ¹H NMR (400 MHz, DMSO-d₆): δ = 9.48 (d, J = 2.0 Hz, 1H), 9.11 (s, 1H), 8.83 (d, J = 4.8 Hz, 1H), 8.67 (d, J = 2.1 Hz, 1H), 8.64 (d, J = 2.2 Hz, 1H), 8.27 (d, J = 7.8 Hz, 1H), 8.18 (d, J = 8.5 Hz, 4H), 8.09 (dd, J = 9.5 Hz, J = 5.3 Hz, 2H), 7.67 (dd, J = 7.5 Hz, J = 5.0 Hz, 1H). 3.32 (s, 3H). ESI-QTOF-MS: 446.0847 (C₂₂H₁₆N₅O₄S, M + H⁺), anal. calcd for C₂₂H₁₅N₅O₄S, 445.0845.

6-(4-(Methylsulfonyl)phenyl)-3-(5-(m-tolyl)-1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one (6e)

Yellow solid: yield 47.4%, mp 311.2–312.9 °C. ¹H NMR (400 MHz, DMSO-d₆): δ = 9.49 (d, J = 1.9 Hz, 1H), 9.15 (s, 1H), 8.64 (dd, J = 9.1 Hz, J = 2.1 Hz, 1H), 8.19 (d, J = 8.5 Hz, 2H), 8.13 (d, J = 8.4 Hz, 2H), 8.06 (d, J = 9.1 Hz, 1H), 7.94 (s, 1H), 7.92 (d, J = 7.8 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H), 3.32 (s, 3H), 2.45 (s, 3H). ESI-QTOF-MS: 459.1051 (C₂₄H₁₉N₄O₄S, M + H⁺), anal. calcd for C₂₄H₁₈N₄O₄S, 458.1049.

3-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)-6-(4-(methylsulfonyl)phenyl)-1,8-naphthyridin-4(1H)-one (6f)

Yellow solid: yield 55.0%, mp 320.0–320.9 °C. ¹H NMR (400 MHz, DMSO-d₆): δ = 9.49 (s, 1H), 9.16 (s, 1H), 8.65 (dd, J = 9.1 Hz, J = 1.9 Hz, 1H), 8.18 (d, J = 8.3 Hz, 2H), 8.13 (d, J = 7.6 Hz, 4H), 8.07 (d, J = 9.1 Hz, 1H), 7.74 (d, J = 8.5 Hz, 2H), 3.31 (s, 3H). ESI-QTOF-MS: 479.0505 (C₂₃H₁₆ClN₄O₄S, M + H⁺), anal. calcd for C₂₃H₁₅ClN₄O₄S, 478.0503.

3-(5-(4-Fluorophenyl)-1,3,4-oxadiazol-2-yl)-6-(4-(methylsulfonyl)phenyl)-1,8-naphthyridin-4(1H)-one (6g)

Yellow solid: yield 50.4%, mp 307.3–308.2 °C. ¹H NMR (400 MHz, DMSO-d₆): δ = 9.49 (d, J = 2.0 Hz, 1H), 9.16 (s, 1H), 8.65 (dd, J = 9.1 Hz, J = 2.2 Hz, 1H), 8.21–8.19 (m, 1H), 8.18 (d, J = 2.2 Hz, 2H), 8.13 (d, J = 8.5 Hz, 2H), 8.07 (d, J = 9.1 Hz, 1H), 7.51 (t, J = 8.9 Hz, 2H), 3.32 (s, 3H). ESI-QTOF-MS: 463.0800 (C₂₃H₁₆FN₄O₄S, M + H⁺), anal. calcd for C₂₃H₁₅FN₄O₄S, 462.0798.

6-(4-(Methylsulfonyl)phenyl)-3-(5-(p-tolyl)-1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one (6h)

Yellow solid: yield 59.2%, mp 300.0–301.2 °C. ¹H NMR (400 MHz, DMSO-d₆): δ = 9.48 (d, J = 1.7 Hz, 1H), 9.13 (s, 1H), 8.64 (dd, J = 9.1 Hz, J = 2.0 Hz, 1H), 8.18 (d, J = 8.4 Hz, 2H), 8.13 (d, J = 8.4 Hz, 2H), 8.05 (d, J = 9.1 Hz, 1H), 8.00 (d, J = 8.0 Hz, 2H), 7.45 (d, J = 8.0 Hz, 2H), 3.32 (s, 3H), 2.42 (s, 3H). ESI-QTOF-MS: 459.1051 (C₂₄H₁₉N₄O₄S M + H⁺), anal. calcd for C₂₄H₁₈N₄O₄S, 458.1049.

Synthesis of 6-bromo-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carbohydrazide (7)

Using a method similar to that of 4, compound 7 was synthesized as a pink solid (340 mg). Yield: 23.6%. ¹H NMR (400 MHz, DMSO-d₆): δ = 9.73 (s, 1H), 9.23 (d, J = 1.8 Hz, 1H), 9.02 (s, 1H), 8.32 (dd, J = 9.3 Hz, J = 2.1 Hz, 1H), 7.84 (d, J = 9.3 Hz, 1H), 4.79 (s, 2H).

Synthesis of compounds 8 and 11

Using a method similar to that of 5a, compounds 8 and 11 were synthesized as brown solids. Yield: 76.1% and 90.0%, respectively.

Synthesis of 6-bromo-3-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one (9)

To a mixture of 8 (0.94 mmol) in SOCl₂ (5 mL), pyridine (160 mg, 2 mmol) was added dropwise. The reaction mixture was then gradually heated to the reflux temperature and maintained there for 3 h. The remaining SOCl₂ was removed by rotary evaporation and poured into a saturated NaHCO₃ solution to acidify to pH 7. Then the aqueous layer was extracted with CH₂Cl₂, and the combined organic layers were dried over Na₂SO₄, filtered, and concentrated. The product 9a was purified by column chromatography to obtain a yellow solid (250 mg, yield 65.9%). ¹H NMR (400 MHz, DMSO-d₆): δ = 9.27 (d, J = 2.2 Hz, 1H), 9.14 (s, 1H), 8.35 (dd, J = 9.3 Hz, J = 2.2 Hz, 1H), 8.13 (d, J = 8.6 Hz, 2H), 7.88 (d, J = 9.3 Hz, 1H), 7.73 (d, J = 8.6 Hz, 2H).

Synthesis of 6-bromo-3-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one (12)

Using a method similar to that of 9a, compound 9b was synthesized as a yellow solid (280 mg, yield 40.8%). ¹H NMR (400 MHz, DMSO-d₆): δ = 9.26 (d, J = 1.8 Hz, 1H), 9.12 (s, 1H), 8.34 (dd, J = 9.3 Hz, J = 2.2 Hz, 1H), 8.17 (dd, J = 8.8 Hz, J = 5.4 Hz, 2H), 7.87 (d, J = 9.3 Hz, 1H), 7.50 (t, J = 8.9 Hz, 2H).

General procedure for the synthesis of compounds 10a–10c and 13a–13c

Appropriate boronic acid or boronic acid ester (0.089 mmol) was added to a solution of intermediate 9a–9b (0.074 mmol) in a 1,4-dioxane/H₂O mixture (5 : 1, 5 mL), followed by Na₂CO₃ (23 g, 0.223 mmol) and dichlorobis(triphenylphosphine)palladium(ii) (3 mg, 0.004 mmol). The mixture was then heated and stirred at 60 °C under a nitrogen atmosphere for 8 h. After being cooled to room temperature, the mixture was diluted with DCM, washed with H₂O, and dried (Na₂SO₄). The solvent was then evaporated under reduced pressure, and the residue was purified by chromatography to afford compounds 10a–10c. Compounds 13a–13c were synthesized by a similar method.

3-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-6-(pyridin-3-yl)-1,8-naphthyridin-4(1H)-one (10a)

Yellow powder: yield 80.4%, mp 305.5–306.5 °C. ¹H NMR (400 MHz, DMSO-d₆): δ = 9.47 (s, 1H), 9.16 (s, 1H), 9.10 (s, 1H), 8.73 (d, J = 4.8 Hz, 1H), 8.65 (d, J = 8.9 Hz, 1H), 8.33 (d, J = 8.0 Hz, 1H), 8.13 (d, J = 8.5 Hz, 2H), 8.06 (d, J = 9.4 Hz, 1H), 7.74 (d, J = 8.5 Hz, 2H), 7.67–7.56 (m, 2H). ESI-QTOF-MS: 402.0682 (C₂₁H₁₃ClN₅O₂ M + H⁺), anal. calcd for C₂₁H₁₂ClN₅O₂, 401.0680.

4-(6-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)-5-oxo-5,8-dihydro-1,8-naphthyridin-3-yl)benzonitrile (10b)

Yellow powder: yield 80.1%, mp 292.5–293.5 °C. ¹H NMR (400 MHz, DMSO-d₆): δ = 9.48 (s, 1H), 9.16 (s, 1H), 8.64 (d, J = 8.9 Hz, 1H), 8.16–8.10 (m, 2H), 8.08 (s, 1H), 8.07–8.04 (m, 1H), 7.74 (d, J = 8.4 Hz, 2H). ESI-QTOF-MS: 426.0683 (C₂₃H₁₃ClN₅O₂, M + H⁺), anal. calcd for C₂₃H₁₂ClN₅O₂, 425.0680.

3-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)-6-phenyl-1,8-naphthyridin-4(1H)-one (10c)

Yellow powder: yield 81.3%, mp 238.9–239.2 °C. ¹H NMR (400 MHz, DMSO-d₆): δ = 9.39 (d, J = 1.9 Hz, 1H), 9.13 (s, 1H), 8.60 (dd, J = 9.1 Hz, J = 2.1 Hz, 1H), 8.12 (d, J = 8.5 Hz, 2H), 8.02 (d, J = 9.1 Hz, 1H), 7.88 (t, J = 8.0 Hz, 2H), 7.73 (d, J = 8.5 Hz, 2H), 7.61 (t, J = 7.4 Hz, 2H), 7.53 (t, J = 7.3 Hz, 1H). ESI-QTOF-MS: 401.0730 (C₂₂H₁₄ClN₄O₂, M + H⁺), anal. calcd for C₂₂H₁₃ClN₄O₂, 400.0727.

3-(5-(4-Fluorophenyl)-1,3,4-oxadiazol-2-yl)-6-(pyridin-3-yl)-1,8-naphthyridin-4(1H)-one (13a)

Yellow powder: yield 82.4%, mp 306.2–307.1 °C. ¹H NMR (400 MHz, DMSO-d₆): δ = 9.47 (s, 1H), 9.15 (s, 1H), 9.10 (s, 1H), 8.73 (s, 1H), 8.65 (d, J = 8.9 Hz, 1H), 8.33 (d, J = 7.3 Hz, 1H), 8.19 (s, 1H), 8.06 (d, J = 9.1 Hz, 1H), 7.62 (s, 1H), 7.51 (t, J = 8.6 Hz, 2H). ESI-QTOF-MS: 386.0978 (C₂₁H₁₃FN₅O₂, M + H⁺), anal. calcd for C₂₁H₁₂FN₅O₂, 385.0975.

3-(5-(4-Fluorophenyl)-1,3,4-oxadiazol-2-yl)-6-(pyridin-4-yl)-1,8-naphthyridin-4(1H)-one (13b)

Yellow powder: yield 76.2%, mp 260.1–262.1 °C. ¹H NMR (400 MHz, DMSO-d₆): δ = 9.53 (d, J = 1.8 Hz, 1H), 9.15 (s, 1H), 8.78 (d, J = 5.1 Hz, 2H), 8.65 (dd, J = 9.1 Hz, 2.0, 1H), 8.18 (dd, J = 8.7 Hz, J = 5.4 Hz, 2H), 8.06 (d, J = 9.1 Hz, 1H), 7.94 (d, J = 5.8 Hz, 2H), 7.50 (t, J = 8.8 Hz, 2H). ESI-QTOF-MS: 386.0978 (C₂₁H₁₃FN₅O₂, M + H⁺), anal. calcd for C₂₁H₁₂FN₅O₂, 385.0975.

4-(6-(5-(4-Fluorophenyl)-1,3,4-oxadiazol-2-yl)-5-oxo-5,8-dihydro-1,8-naphthyridin-3-yl)benzonitrile (13c)

Yellow powder: yield 86.1%, mp 339.0–340.5 °C. ¹H NMR (400 MHz, DMSO-d₆): δ = 9.48 (s, 1H), 9.16 (s, 1H), 9.03 (s, 1H), 8.63 (d, J = 8.9 Hz, 1H), 8.22–8.15 (m, 2H), 8.12 (d, J = 8.4 Hz, 2H), 8.08 (s, 1H), 8.05 (d, J = 8.9 Hz, 1H). ESI-QTOF-MS: 410.1047 (C₂₁H₁₃FN₅O₂, M + H⁺), anal. calcd for C₂₁H₁₂FN₅O₂, 409.0975.

Cell lines and culture

A549, HepG2, KB, MCF-7, HeLa, and HCT116 cancer cells were maintained in Dulbecco's minimum essential medium (DMEM, Gibco, USA) or Roswell Park Memorial Institute 1640 (RPMI 1640, Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Auckland, NZ), 100 U mL^–1 penicillin and 100 U mL^–1 streptomycin in a humidified atmosphere containing 5% CO₂ at 37 °C. In addition, the human normal hepatocyte cell line LO2 was introduced in this study to evaluate the cytotoxicity of 3-(1,3,4-oxadiazol-2-yl)-1,8-naphthyridin-4(1H)-one derivatives.

Cell viability assay by MTT

Cells were seeded into 96-well plates 24 h before experimental manipulation. Then, cells were treated with the compounds. Cells were treated with compound alone for 24 h before combination with cisplatin (2.5 μM) for 48 h. After incubation, 20 μL of a 5 mg mL^–1 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution was added, and the plates were incubated for an additional 2–4 h at 37 °C. The medium was subsequently removed, and DMSO was then added. The OD₅₇₀ was measured using a Spectra MAX M5 microplate spectrophotometer (Molecular Devices, CA, USA). For the combination treatment, cells were treated with compounds alone for 24 h before combination with cisplatin (2.5 μM) for 48 h.

Morphological analysis by Hoechst staining

The morphological changes were evaluated by Hoechst 33258 staining. HCT116 cells were treated with 6e/6h alone for 24 h before combination with cisplatin (2.5 μM) for 48 h. Then, cells were fixed with 4% paraformaldehyde and stained with a solution of Hoechst 33258 dye (5 μg mL^–1). Next, the nuclear morphology of cells was observed by fluorescence microscopy (Zeiss, Axiovert 200, Germany), and the number of nuclei were counted and analyzed.

Apoptosis analysis by FCM

To detect the apoptosis effect of 6e/6h combined with cisplatin, an annexin V-FITC/PI apoptosis detection kit was used. Briefly, HCT116 cells were treated with 6e/6h alone for 24 h before combination with cisplatin (2.5 μM) for 48 h. The cells were then harvested and washed with cold PBS. After centrifugation, the cells were stained with annexin V-FITC and PI and analyzed by flow cytometry (Becton-Dickinson, USA).

Western blot analysis

HCT116 cells were collected by scraping and lysis in RIPA buffer (50 mM Tris HCL, 1% NP-40, 0.1% SDS, 150 nM NaCl, 1 mM EDTA and 0.5% sodium deoxycholate) containing protease and phosphatase inhibitors. The total protein concentrations were measured with an Enhanced BCA Protein Assay Kit (Beyotime® Biotechnology, China). Equal amounts of protein were loaded onto 12% SDS-PAGE gels (Willget Biotech, Shanghai, China) and electro-transferred to Millipore 0.2 μm PVDF membranes. After washing, the membranes were blocked with 5% skimmed milk and incubated with a primary antibody (anti-CHK1 mouse mAb) at 4 °C overnight. Antibodies were detected with a horseradish peroxidase (HRP)-conjugated secondary antibody and developed with an enhanced chemiluminescence detection kit (Luminata Crescendo Western HRP Substrate or Immobilon Western Chemiluminescent HRP Substrate, Millipore Corporation, Billerica, MA, USA). The membranes were probed for GAPDH to confirm equal loading. In this study, the anti-CHK1 primary antibody (220064) was a product of Zen Bioscience Co., Ltd. (Chengdu) and the GAPDH primary antibody (AF5718) was purchased from R&D Systems, Inc. (Minneapolis, MN).

Molecular docking

Structure-based docking studies were carried out by using GOLD V3.0.1. 3D conformations of the 2 molecules were generated and minimized using the molecular mechanics (MM2) method and the Hamiltonian approximations Austin model 1 (AM1) method available in the MOPAC2009 software. The RESP charges were assigned to the molecules in 3 steps: first, ESP charges of the molecule were calculated (HF/6-31G* OPT ESP) based on the structure using Gaussian 03; then the restrained ESP fitting was conducted using the Antechamber program in AMBER; finally, the obtained RESP charges were assigned to the Sybyl mol2 files of each molecule. The kinase domain of ATR was built by homology modelling with PI3K γ as template. A sphere of 10 Å around the binding site in the ATR kinase domain was defined as the docking site. The RMSD of early termination was set to 1.5 Å. GA parameters were set to GOLD default and for each molecule, 30 dockings were performed.

Conflicts of interest

The authors declare no conflict of interest.