Design, synthesis, and antiproliferative investigation of novel quinazolinones incorporating 1,3,4-oxadiazole and 1,2,4-triazole thioether moieties

Abstract In the current study, a series of 1,3,4-oxadiazole and 1,2,4-triazole derivatives linked to quinazolinone were designed and synthesized by using Phase Transfer Catalysis. The structures of the synthesized compounds were confirmed by their IR, 1H NMR, 13C NMR, and HR-ESI-MS spectroscopic data, and their in vitro antiproliferative activities against A549, Hela, MCF-7 as well as L929 were detected by using CCK-8 assay. The results demonstrated that most of the compounds showed better growth inhibitory effect than 5-FU on A549, Hela, and MCF-7 cell lines. Especially, 2-(5-((3-bromophenyl)thio)-1,3,4-oxadiazole-2-yl)quinazolin-4(3H)-one (8o), 4-amino-5-((4-nitrophenyl)thio)-4H-1,2,4-triazol-3-yl)quinazolin-4(3H)-one (9m) and 2-(4-amino-5-(o-tolylthio)-4H-1,2,4-triazol-3-yl)quinazolin-4(3H)-one (9b) showed good antiproliferative activity against A549 cells with IC50 value of 3.46, 2.96, and 3.44 μM, respectively. 2-(4-Amino-5-((3,5-difluorophenyl)thio)-4H-1,2,4-triazol-3-yl)quinazolin-4(3H)-one (9h) and 4-amino-5-((4-nitrophenyl)thio)-4H-1,2,4-triazol-3-yl)quinazolin-4(3H)-one (9m) had obvious inhibitory effects on Hela cells with IC50 values of 1.76 and 3.91 μM, respectively. 2-(4-Amino-5-((2-chlorophenyl)thio)-4H-1,2,4-triazol-3-yl)quinazolin-4(3H)-one (9i) showed high inhibitory activity against MCF-7 cells with IC50 value of 2.72 μM. Meanwhile, all compounds showed lower cytotoxicity to L929 cells than positive control drug 5-FU. In development, according to the above preliminary observation, these novel quinazolinones incorporating 1,3,4-oxadiazole and 1,2,4-triazole thioether moieties could become potential molecular templates for searching new antitumor agents. Graphical Abstract


Introduction
Cancer is the uncontrolled division or abnormal growth of cells that spread or invade other parts of the body with serious health consequences. [1][7] It was reported that these compounds are potential biologically active compounds, for instance, a number of 1,3,4-oxadiazole and 1,2,4-triazole derivatives were reported to possess a wide range of biological activities such as antiproliferative, [8][9][10][11][12] antimicrobial, [13,14] anticonvulsant, [15,16] anti-inflammatory, [17,18] antioxidant, [19][20][21] and antiviral effects. [22,23]Besides, it was also reported that the quinazolinone derivatives possess anticancer, [24][25][26] antituberculosis, [27] antioxidant, [28] anti-inflammatory, [29] and antibacterial activities. [30]In addition, heterocyclic rings that are connected by electron donor elements (O, S), enhance the affinity between the receptor and the ligand and improve the biological activity. [31]NA double-strand breaks (DSBs) are the most lethal form of DNA damage and a major cause of cytotoxicity from ionizing radiation and many chemotherapeutic agents.Without timely and proper repair, double-strand breaks can result in chromosomal deletions, fusions, and ectopics, leading to cell death.[32] In fact, organisms have developed a series of defense pathways to overcome these DNA damages, mainly including base excision repair (BER) and homologous recombination repair (HRR).[33] Breast cancer susceptibility gene (BRCA1/2) played a critical role in a variety of molecular processes related to DNA metabolism, including HRR and mediating the replication stress response.When the BRCA gene is mutated, the DNA repair pathway will depend on poly-ADP-ribose polymerase-1 (PARP-1) enzyme, and PARP inhibitor makes DNA replication unable to proceed smoothly, resulting in blocked BER.At the same time, mutations in BRCA1/2 cause HRD to fail to proceed normally, and the two steps together cause cell death, which is called the synthetic lethal effect.However, the presence of BRCA in normal cells can still repair DNA allowing cell survival, so PARP inhibitor is a targeted drug that can selectively kill BRCA mutated cells.[34] Based on these findings, a pharmacophore query in which the presence of three structural features was imposed: (1) an aromatic ring, (2) a carboxamide moiety with at least one NH group locked into the desired anti-conformation, (3) a side chain extending into the deep pocket located in the auto-modification domain of PARP-1.Therefore, it is likely that 2-substituted quinazolinones have potential antitumor activity using PARP-1 as the drug target (Figure 1). [35]uinazolinone derivatives have been shown to have strong COX-2 inhibitory activity, with half maximal inhibitory concentration (IC 50 ) values and selectivity indices similar to the control drug celecoxib. [36]A new class of antitubercular agents named 2-alkylsulfanyl-5-(3,5-dinitrophenyl)-1,3,4-oxadiazoles, and its structure-activity relationships are described.These compounds possessed excellent activity against mycobacterium tuberculosis, including the clinically isolated multidrug resistant (MDR) and extensively drug-resistant (XDR) strains, with no cross resistance with first or second-line anti-TB drugs. [37]Meanwhile, ribavirin (RBV) is a class of antiviral drugs with a 1,2,4-triazole structure.Ribavirin has been shown to significantly reduce the risk of virus recurrence in patients with hepatitis C virus (HCV) infection after treatment. [38]ased on the above reports, a series of new 1,3,4-oxadiazole and 1,2,4-triazole derivatives linked to quinazolinone were designed and synthesized, all the structures of synthesized compounds were confirmed by their infrared spectroscopy (IR), nuclear magnetic resonance ( 1 H and 13 C), and high resolution electrospray ionization mass spectroscopy (HR-ESI-MS) spectroscopic data, and their in vitro antiproliferative activities against human lung cancer cell (A549), human cervix adenocarcinoma cell (Hela), human breast cancer cell line (MCF-7) and mouse fibroblast cell (L929) were detected by using cell counting kit-8 (CCK-8) assay.
As shown in Scheme 1, the preparation of intermediate 3 was obtained by the reaction of compounds 1 and 2 according to the reported procedure. [25]The intermediates 4 and 5 were gained by the reported literature method. [39,40]The final newly compounds 8a-q, 9a-m were obtained by phase transfer catalysis through the reaction between 6/7 with halide derivatives in the presence of tetrabutylammonium bromide (TBAB, a phase transfer catalyst) and toluene in H 2 O at room temperature for 8 h, and were purified by silica gel column chromatography and their structure were characterized by IR, 1 H NMR, 13 C NMR, and HR-ESI-MS.All synthesized compounds were depicted in Table S1 (Supplemental Materials).
The structures of the final products were qualitatively analyzed by IR, 1 H NMR, 13  In the 1 H NMR spectrum of compound 8a, the nine proton signals at d 8.01, 7.59, 7.51, 7.39, and 7.33 ascribed to the aromatic protons in quinazolinone structure and benzene unit connecting S-alkyl.The signal at d 4.60 (s, 2H) was assigned to the typical thio-methylene, implying that intermediate 6 occurred the reaction with the bromobenzyl, and finally obtain the compound 8a.Furthermore, HR-ESI-MS also provided this structure an additional certification by giving a molecular ion at m/z 337.0759 For quinazolinone derivatives incorporating 1,2,4-triazole thioether moieties 9a-m, we selected compound 9a as an example to confirm the structures of this series.In the IR spectrum of 9a, the peaks at 3421 and 3048 cm À1 corresponding to NH and CONH stretching were observed.Significantly, the band at 3380 cm À1 revealed the existence of NH 2 group.The C ¼ O and C ¼ C at 1666 and 1536 cm À1 , respectively, were also observed.In its 13   Antiproliferative evaluation 5-Fluorouracil (5-FU) as positive control was used to determine the antiproliferative activities of target compounds 8aq and 9a-m against three human cancer cells A549, Hela, MCF-7, and a normal cell L929 by CCK-8 method.The inhibitory effects of these compounds on cell proliferation were measured at five different concentrations, and their IC 50 values were calculated as shown in Table S2 (Supplemental Materials).
All target compounds 8a-q and 9a-m showed separate degrees of antitumor effects for selected cancer cell lines.Some compounds showed stronger antiproliferative activities than 5-FU.In A549 cells, compound 9m showed the best inhibitory activity with IC 50 value of 2.96 lM, which is higher than that of 5-FU (IC 50 ¼8.13lM).Meanwhile, compounds 8b-d, 8f-g, 8k-p, 9b-c, and 9h-j showed highly effective activities with IC 50 values ranging from 3.44 to 9.96 lM.Compound 9l showed weak inhibitory activity with IC 50 value of more than 50 lM, and the other compounds all exhibited moderate antitumor activity with IC 50 value of 10.84 to 45.23 lM.In Hela cells, compounds 9h showed the strongest inhibitory activity with IC 50 value of 1.76 lM, and showed stronger activity than 5-FU (IC 50 ¼17.21lM).At the same time, compounds 8a, c, e-h, 9eÀi, and m displayed highly effective activities with IC 50 values ranging from 3.91 to 9.92 lM.Compounds 8i-j showed weak inhibitory activity with IC 50 value of more than 50 lM.The other compounds all exhibited moderate antitumor activity with IC 50 value of 10.20-44.80lM.In MCF-7 cells, compound 9i showed the best inhibitory activity with IC 50 value of 2.72 lM, and showed better activity than 5-FU (IC 50 ¼12.26lM).Compounds 8d, e, k, o, q showed weak inhibitory activity with IC 50 value of more than 50 lM, and compounds 8a-c, l, n, 9c-d, and f exhibited moderate antitumor activity with IC 50 value of 14.75-34.15lM.The other compounds all showed highly inhibitory activities with IC 50 values ranging from 5.00 to 9.37 lM.In normal mouse cell line L929, except compound 8c showed the strongest inhibitory activity with IC 50 value of 2.57 lM, the cytotoxicity of all other compounds was weaker than that of 5-FU.The results indicated that 1,3,4-oxadiazole and 1,2,4-triazole scaffold linked to quinazolinone derivatives had little inhibition on the activity of normal mouse cell L929 and had a certain degree of impact against human cancer cell lines A549, Hela, and MCF-7, and the different substituent groups could Scheme 1. Synthetic routes of target compounds 8a-q and 9a-m.
affect the degree of inhibition on the cell lines.These above findings are of great significance for further study of the structure-activity relationship.

Conclusion
In conclusion, a general synthesis route of new 1,3,4-oxadiazole derivatives and 1,2,4-triazole derivatives linked quinazolinone were designed by phase transfer catalysis, and finally, we successfully synthesized 17 new 1,3,4-oxadiazole derivatives 8a-q and 13 new 1,2,4-triazole derivatives 9a-m, the structures of which were confirmed by IR, 1 H NMR, 13 C NMR, and HR-ESI-MS spectroscopic data, and CCK-8 method was used to detect their in vitro antiproliferative activities against human cancer cell lines A549, Hela, MCF-7, and a normal cell line L929.The results showed that compound 9m had the strongest inhibitory activity on A549 cells with IC 50 value of 2.96 lM.Compound 9h could effectually suppress the multiplication of Hela cells with IC 50 value of 1.76 lM.Compound 9i displayed the best growth inhibitory effect against MCF-7 cells with IC 50 value of 2.72 lM, and all compounds except compound 8c showed less toxicity to normal mouse cells than 5-FU.These results indicated this series of 1,3,4-oxadiazole and 1,2,4-triazole derivatives linked to quinazolinone has a certain anticancer activity and meanwhile possesses little inhibitory effect on normal cells.This discovery will lead to the development of new compounds bearing 1,3,4-oxadiazole and 1,2,4-triazole scaffold which is more effective in inhibiting the proliferation of cancer cells.

General methods
The reagents used in the experiments were all supplied from Tianjin Hengshan, Tianjin Jiangtiantongyi and Energy Chemical without further purification.The reaction process was monitored by thin layer chromatography (TLC).The silica gel GF254 for TLC and silica gel (200-300 mesh) for column chromatography were provided by Qingdao Marine Chemical Company.All melting points were determined on the X-4 precision microscopic melting point analyzer and had no modification. 1H NMR (400 MHz) and 13 C NMR (100 MHz) were recorded on Bruker Avance III 400 MHz spectrometer.Chemical shifts are shown in d values (ppm) and the coupling constants are expressed in J values (Hz).Infrared spectra were recorded on the Nicolet Avatar 370 spectrometer with KBr as diluents.The high-resolution mass spectra were measured with the Waters Xevo G2 QTof (ESI) mass spectrometer.The Supplemental Materials contain sample 1 H and 13 C NMR, IR and mass spectra for the products 8 and 9 (Figures S1-S120).

Experimental procedure for the synthesis of intermediate 6
To a solution of intermediate 5 (1.0 eq.) and water (20 mL), dilute hydrochloric acid was added dropwise to pH 3 and continued for 30 min at room temperature.The mixture was filtered and then washed with water to give the crude product, which was further purified by recrystallization from benzene (20 mL) to obtain the intermediate 6.

Experimental procedure for the synthesis of intermediate 7
To a solution of intermediate 5 (1.0 eq.) and ethanol in a round bottle (100 mL) at room temperature, hydrazine hydrate (10 mL, 80%) was added dropwise.After stirring for 8 h at 80 C, the mixture was poured into ice water (100 mL), neutralized to pH 3 using dilute hydrochloric acid and stirred for 10 min to form a yellow solid.After removal of the solvents under reduced pressure at room temperature, the solid was recrystallized from benzene (20 mL) to obtain 7. Experimental procedure for the synthesis of 8a-q and 9a-m To a solution of thiol 6/7 (1.0 eq.), TBAB (0.1 eq.) in toluene (5 mL) and water (10 mL), bromobenzyl (0.25 mmol) dissolved in toluene (5 mL) was added and then stirred at room temperature for 8 h.After the reaction completed, the mixture was filtered, and then washed with water.
C NMR, and HR-ESI-MS.As an example, compound 8a showed sharp peaks at 3365 and 3051 cm À1 due to NH and CONH stretching in its IR spectrum.Meanwhile, the characteristic absorption bands at 1661 and 1552 cm À1 revealed the existence of C ¼ O and C ¼ C in the structure.The 13 C NMR spectrum showed 17 carbon resonances, including an amide carbonyl (d 171.35).

Figure 1 .
Figure 1.The structure required for PARP inhibitors and the structure of 2-substituted quinazolinone.