2024 年 72 巻 1 号 p. 61-67
Two novel series of quinazolinone-based hybrids, including quinazolinone-1,3,4-oxadiazoles (10a–l) and quinazolinone-1,3,4-oxadiazole-benzimidazoles (8a–e), were designed and synthesized and their cytotoxic activities against three human cancer cell lines, lung cancer (A549), cervical cancer (HeLa), and breast cancer (MCF-7), were evaluated. The cytotoxic assays revealed that 10i with a lipophilic 4-fluoro-phenyl moiety at the C-2 position of the quinazolinone ring displayed good cytotoxicities against the A549 and MCF-7 cell lines, while 8b–d with the thioether-linked benzimidazole moiety incorporated on the right side of the oxadiazole ring induced comparable stronger activities toward the MCF-7 cell line, relative to the simple two-heterocycle-containing hybrid 10i. These novel quinazolinone-based hybrids could be considered as lead compounds that merit further optimization and development as anti-cancer agents.
Heterocyclic compounds are known as important scaffolds for developing anticancer agents.1–4) Among such privileged moieties, quinazolinone derivatives show significant activities against different types of cancer cell lines, since they can bind with high affinity to multiple therapeutic targets such as the DNA repair enzyme system, epidermal growth factor receptor (EGFR), thymidylate enzyme, and tubulin polymerization machinery.5) Several 2,3-disubstituted quinazolin-4(3H)-one derivatives reportedly possess antitumor activities, as exemplified by compounds I6) and II7) shown in Fig. 1. Noticeably, we previously found that compounds IIIa and b displayed promising cytotoxicity towards the A549 cancer cell line, with compound IIIb (IC50 = 23.6 µM) exhibiting activity comparable to the positive control 5-fluorouracil (5-FU) (IC50 = 27.9 µM).8)
In addition, the benzimidazole heterocycle and its derivatives are also regarded as excellent pharmacophores against different types of cancer cell lines, through their inhibitory activities toward multiple targets such as topoisomerases I and II, tubulin polymerization enzymes, dihydrofolate reductase, poly adenosine diphosphate-ribose polymerase (PARP), and tyrosine kinase.9,10) Furthermore, 1,3,4-oxadiazoles have attracted wide attention due to their useful biological properties, especially their anticancer activities.11–14)
Heterocyclic hybrids have recently attracted keen interest and become significantly important in pharmaceutical research due to their enhanced biological activities, especially cytotoxicity, compared to their starting units.15) Extensive research has focused on developing novel benzimidazole,9,10) 1,3,4-oxadiazole16–20) and quinazolinone21–23) heterocycles conjugated with other heterocycles as anti-tumor agents, since these structures overcome various disadvantages of current anti-cancer drugs, such as drug resistance, toxicity, and side effects.15) Akhtar reported that the hybrid structure of 1,3,4-oxadiazole-benzimidazole IV showed good cytotoxicity against the MCF-7 cell line, with an IC50 value of 5.00 µM17) (Fig. 2). This compound also displayed inhibitory activities towards EGFR and the receptor tyrosine–protein kinase erbB2, with IC50 values of 0.08 and 0.61 µM, respectively. Compound VI,19) with the 1,3,4-oxadiazole integrated into the triazole moiety through a thioether linkage, also exhibited strong activity against the MCF-7 cell line (IC50 = 1.26–5.80 µM). Notably, compound VII,21) with the 1,3,4-oxadiazole scaffold connected to the quinazolinone ring through a mercaptoacetamide linkage, showed good cytotoxicity against the HeLa cell line, with an IC50 value of 7.52 µM. We previously reported that compound V bearing the methylthioether-linked 1,3,4-oxadiazole/benzimidazole hybrid exhibited cytotoxicities against the MCF-7 and HeLa cancer cell lines comparable to that of the positive control 5-FU, with IC50 values of 11.66 and 22.27 µM, respectively.18) Compounds V, VI, and VII share the common structural characteristic of the thioether-linked 1,3,4-oxadiazole moiety, and this structural feature in conjunction with a simple aryl group on one side and a heterocycle, especially a benzimidazole or quinazolinone ring, on the other side of the oxadiazole ring plays a critical role in contributing to the observed cytotoxic activities (Fig. 2).
Based on the above findings, we have explored the anticancer potential of heterocycles.8,18,24,25) Herein, we report the design and syntheses of two series of heterocyclic hybrids, by integrating the quinazolinone pharmacophore on one side of the 1,3,4-oxadiazole structure with the other side of the heterocycle bearing (i) a methylthioether linked benzimidazole (series A) and (ii) a simple aryl group (series B) (Fig. 3), and their in vitro cytotoxicities against three human cancer cell lines, HeLa, MCF-7, and A549.
The synthetic pathway toward the two designed series A and B is depicted in Chart 1. Starting from the commercially available anthranilamide 1, the condensation reaction with aldehydes 2 took place in one step using oxygen26) (air) as an environmental highly oxidative cyclization agent to obtain the quinazolinones 3. Subsequently, the ester moiety was attached to the quinazolinone ring via a nucleophilic substitution reaction with ethyl chloroacetate under mild basic conditions (K2CO3) to afford the esters 4. Aminolysis of the ester moieties of 4 using an excess of hydrated hydrazine in ethanol afforded the corresponding acylhydrazides 5.
In our previous study,18) we reported that the 1,3,4-oxadiazole moiety of compound V (Fig. 3), bearing the thioether-linked benzimidazole on one side, played an important role in determining the cytotoxicity of the compound. We therefore expected that the substitution of the aryl group on the other side of the oxadiazole ring with an inherent anticancer scaffold such as the quinazolinone would enhance the activity of the resulting structure. Meanwhile, Alsibaee reported that among the six positions of the quinazolinone ring, the additional modifications at the C-2 and N-3 positions were significant for anticancer activities.26) Based on these rationales, we designed the series of quinazolinone-1,3,4-oxadiazole-benzimidazole conjugates (series A), in which the 1,3,4-oxadiazole moiety bearing the thioether-linked benzimidazole was connected to the quinazolinone ring at the N-3 position (Fig. 3). To investigate the structure and activity relationship (SAR) of these hybrid conjugates, we synthesized several structures of series A with combinations of different substituents R1 at the C-2 position of the quinazolinone and substituents R2 on the benzimidazole moieties. The second hybrid series (series B) with a simple aryl group on the other side of the 1,3,4-oxadiazole ring was also designed for comparison.
The synthetic pathway toward the two designed series A and B is depicted in Chart 1. Starting from the commercially available anthranilamide 1, the condensation reaction with aldehydes 2 took place in one step using oxygen27) (air) as a highly environmentally oxidative cyclization agent to obtain the quinazolinones 3. Subsequently, the ester moiety was sticked onto the quinazolinone ring via nucleophilic substitution reaction with ethyl chloroacetate under mild basic conditions (K2CO3) to afford esters 4. Aminolysis of the ester moieties of 4 using excess solution of hydrated hydrazine in ethanol provided the corresponding acylhydrazides 5.
Reaction conditions: (a) 1 (2 mmol), aldehydes 2 (1.2 equivalent (equiv.)), dimethyl sulfoxide (DMSO), 100 °C, open flask; (b) 3 (1.5 mmol), K2CO3 (3 equiv.), ClCH2COOEt (2 equiv.), N,N-dimethylformamide (DMF), 80 °C; (c) 4 (1 mmol), NH2NH2 (8 equiv.), ethanol, room temperature; (d) 5 (0.5 mmol), KOH (1 equiv.), CS2 (2 equiv.), ethanol, reflux; (e) 6 (0.25 mmol), benzimidazoles 7 (1.2 equiv.), NaOAc (1.2 equiv.), ethanol, reflux; (f) (i) 5 (0.5 mmol), aldehydes 9 (1 equiv.), ethanol, 70 °C; (ii) imine from (i) (0.5 mmol), I2 (3 equiv.), K2CO3 (5 equiv.), DMSO, 100 °C.
For the syntheses of the hybrid 1,3,4-oxadiazole-quinazolinone-benzimidazole derivatives (8a–e) (series A, Chart 1), acylhydrazides 5 were condensed with CS2 under basic conditions (KOH in ethanol)28) to obtain the quinazolinone-based 1,3,4-oxadiazole hybrids 6 bearing a free thiol functionality at the C-2′ position of the oxadiazole ring. The benzimidazole pharmacophore was then conjugated to the molecule through the methylthioether linker via a nucleophilic substitution reaction with 2-(chloromethyl)-benzimidazole derivatives 7 under mild basic conditions (sodium acetate),18) to afford the desired 1,3,4-oxadiazole-quinazolinone-benzimidazole hybrids (8a–e) with reasonable total yields over five steps (23–30%) (Table 1).
 | |||||||
|---|---|---|---|---|---|---|---|
| Compd. | R1 | R2 | R3 | Yield (%)a) | IC50 (µM) | ||
| A549 | HeLa | MCF-7 | |||||
| 8a | 4-F-Ph | Cl | — | 30 | >100 | 97.44 ± 8.50 | >100 |
| 8b | 4-OMe-Ph | H | — | 28 | >100 | 73.47 ± 3.55 | 6.88 ± 1.57 |
| 8c | 4-OMe-Ph | Cl | — | 30 | >100 | >100 | 12.56 ± 3.56 |
| 8d | 4-F-Ph | H | — | 24 | 61.97 ± 1.15 | 79.86 ± 6.40 | 2.65 ± 0.22 |
| 8e | 2-Furyl | H | — | 23 | 20.63 ± 1.65 | >100 | 41.91 ± 2.04 |
| 10a | 4-OMe-Ph | — | 3,4,5-(OMe)3-Ph | 31 | 81.12 ± 3.72 | 27.12 ± 0.36 | 26.46 ± 1.73 |
| 10b | 4-OMe-Ph | — | 2,5-(OMe)2-Ph | 31 | 32.82 ± 0.76 | 30.06 ± 0.24 | 44.22 ± 6.88 |
| 10c | 4-OMe-Ph | — | 4-Pyridyl | 29 | >100 | >100 | >100 |
| 10d | 4-Me-Ph | — | 2,4-(OMe)2-Ph | 30 | >100 | >100 | 79.21 ± 3.84 |
| 10e | 2-Furyl | — | 2,4-(OMe)2-Ph | 24 | 65.02 ± 4.12 | 80.09 ± 0.51 | 51.54 ± 0.80 |
| 10f | 2-Furyl | — | 2,5-(OMe)2-Ph | 25 | 74.56 ± 3.74 | >100 | >100 |
| 10g | 2-Furyl | — | 3,4,5-(OMe)3-Ph | 22 | 70.40 ± 2.68 | 52.55 ± 0.63 | 69.61 ± 3.43 |
| 10h | 2-Furyl | — | 4-NO2-Ph | 20 | >100 | 71.19 ± 3.55 | >100 |
| 10i | 4-F-Ph | — | 2,4-(OMe)2-Ph | 26 | 11.71 ± 0.26 | 30.74 ± 1.43 | 11.90 ± 2.92 |
| 10j | 4-F-Ph | — | 3,4,5-(OMe)3-Ph | 25 | 90.38 ± 3.66 | >100 | >100 |
| 10k | 4-F-Ph | — | 2,5-(OMe)2-Ph | 26 | 86.65 ± 4.75 | 88.33 ± 2.45 | 54.98 ± 2.84 |
| 10l | 4-F-Ph | — | 4-Pyridyl | 25 | >100 | 98.57 ± 2.47 | >100 |
| 5-FUb) | — | — | — | — | 25.50 ± 2.77 | 19.25 ± 1.50 | 35.75 ± 1.03 |
a) Total isolated yield. b) 5-Fluorouracil (5-FU) was used as a positive control. Data are presented as mean ± standard deviation (S.D.) (n = 3); Ph: phenyl.
The second series (series B) was prepared by the condensation of acylhydrazides 5 with different aromatic aldehydes 9, using iodine as the oxidant under mild basic conditions (K2CO3),18) to generate the desired quinazolinone-1,3,4-oxadiazole hybrids (10a–l) with acceptable total yields (20–31% over four steps, Table 1). All R1 and R2 substituents equally tolerated the reaction conditions of the synthetic pathway with only slight differences in the total yields of the final products.
CytotoxicityAll of the synthesized hybrid heterocyclic compounds 8a–e and 10a–l were tested for their cytotoxicities against three human cancer cell lines, including lung cancer (A549), cervical cancer (HeLa), and breast cancer (MCF7). The assay results, summarized in Table 1, indicated that 8a, 10c–d, 10f, 10h, 10j, and 10l showed very weak or no activity against all tested cancer cell lines. In contrast, 10a and 10b exhibited equal cytotoxicities for two cancer cell lines, HeLa and MCF-7 and A549 and HeLa, respectively, which were as potent as 5-FU. Notably, 10i exhibited good cytotoxicities against the three tested cell lines, especially on A549 (IC50 = 11.71 µM) and MCF-7 (IC50 = 11.90 µM), which were 2.2-fold and 3-fold higher than those of 5-FU [IC50 = 25.5 µM (A549) and 35.75 µM (MCF-7)], respectively. Comparable or stronger activities towards one cell line with respect to that of 5-FU were found for the hybrid structures of the three heterocyclic scaffolds 8b, 8c, and 8e, in which 8e (IC50 = 20.63 µM) showed activity against A549 comparable to that of 5-FU, while 8b (IC50 = 6.88 µM) and 8c (IC50 = 12.56 µM) displayed almost 5.2- and 2.8-fold increases in activity against MCF-7 compared to 5-FU, respectively. Compound 8d (IC50 = 2.65 µM) exhibited 13.5-fold higher activity against the MCF-7 cell line compared to 5-FU, and its cytotoxicity was not only much greater than that of the reported quinazolinone conjugated oxadiazole VII (IC50 = 82.18 µM),21) but also better than that of the thioether-linked oxadiazole-benzimidazole hybrid V (IC50 = 11.66 µM),16) and increased to a level comparable to that of VIb, with the 1,3,4-oxadiazole incorporated with the triazole moiety through a methylthioether linkage (IC50 = 1.26 µM)19) (Fig. 4).
The SAR analyses of 8a–e and 10a–l revealed that the cytotoxicities of these compounds depend essentially on the group (R1) attached at position C-2 of the quinazolinone ring and the group incorporated on the right side of the oxadiazole heterocycle. Among 10a–l bearing the substituted aryl groups on the right side of the 1,3,4-oxadiazole moiety, the furan heterocycle at the C-2 position of the quinazolinone ring in 10e–h led to very weak or no activity towards the three tested cancer cell lines. In addition, the presence of electron withdrawing groups such as the 4-pyridyl in 10c and 10l or the 4-NO2 on the benzene ring in 10h on one side of the oxadiazole scaffold probably decreased the desired activity. However, the substitution of the furyl group with the 4-OMe-Ph group in 10a and 10b led to an increase in activity against HeLa and MCF-7 cells [10a: IC50 = 27.12 µM (HeLa) and 26.46 µM (MCF-7)] and all three tested cancer cell lines [10b: IC50 = 32.82 µM (A549), 30.06 µM (HeLa) and 44.22 µM (MCF-7)], respectively. Notably, the introduction of the lipophilic substituent, 4-F-Ph, at the C-2 position of the quinazolinone ring in 10i enhanced its cytotoxicities against the three tested cancer cell lines, especially with A549 (IC50 = 11.71 µM) and MCF-7 (IC50 = 11.90 µM), which were 2.8- and 3.7-fold higher than those of 10b. Furthermore, the positions of the methoxy groups on the benzene ring apparently affected the cytotoxic activity, as observed in the cases of 10j bearing the 3,4,5-(OMe)3-Ph moiety and 10k with the 2,5-(OMe)2-Ph group attached on one side of the oxadiazole ring. These findings were also in accordance with the previous report, in which the coexistence of the 4-Cl-Ph at the C-2 position of the quinazolinone ring and the 4-CH3-Ph or 4-Cl-Ph group attached on one side of the oxadiazole ring was found to be crucial for the observed strong antimicrobial activities,29) while the 3,4,5-(OMe)3-Ph moiety attached at the C-2 position of the quinazolinone was highly necessary for the anticancer activity.30)
The integration of an additional heterocycle on the right-hand side of the 1,3,4-oxadiazole (series A) enhanced the activity of the resulting hybrid structures. For example, the activity of 8e towards the A549 cell line was increased over 3-fold compared to the other structurally similar compounds in series B, such as 10e–h. The same results were also observed for 8c (IC50 = 12.56 µM) and 8b (IC50 = 6.88 µM), which showed 2- and 3.8-fold increases in cytotoxicity compared to that of 10a (IC50 = 26.46 µM) against the MCF-7 cell line, respectively. Remarkably, 8d (IC50 = 2.65 µM), with a methylthioether benzimidazolyl moiety on the right-hand side of the oxadiazole and a 4-F-Ph group at the C-2 position of the quinazolinone, displayed 4.5-fold stronger cytotoxicity against the MCF-7 cell line as compared to that of 10i (IC50 = 11.90 µM), containing a simple aryl group attached to the oxadiazole ring. However, the concomitant presence of two halogen groups like –Cl and –F just led to the complete loss of activity towards all tested cancer cell lines, as observed in 8a. Taken together, we concluded that the basic scaffolds of quinazolinone, 1,3,4-oxadiazole, and benzimidazole are necessary for the observed cytotoxic activities. Furthermore, the enhanced activities of 8b–e compared to their analogs in series B are attributable to the sulfur atom of the thioether linkage, which would improve the liposolubility of the hybrids and lead to good tissue permeability.31)
As summarized in Fig. 5, the two-heterocycle hybrid quinazolinone-1,3,4-oxadiazole 10i, bearing the simple aryl group 2,4-(OMe)2-Ph on the right side of the oxadiazole ring, exhibited good cytotoxicities towards the A549 and MCF-7 cell lines, while the tri-heterocycle conjugates quinazolinone-1,3,4-oxadiazole-benzimidazoles 8b–d displayed comparable to much better activities compared to the hybrid 10i on the MCF-7 cell line. These observations suggest that these novel hybrid structures could serve as lead compounds towards the tested cancer cell lines. The 1,3,4-oxadiazole derivatives such as compound VI,19) as well as the quinazolinone32) and benzimidazole33) scaffolds, are reportedly inhibitors of EGFR, an attractive target in cancer therapy due to its involvement in cell proliferation and differentiation.34,35) This rationale-based design suggested that further investigations of the inhibitory effects of the synthesized derivatives, especially 8d, on the EGFR activity may present an excellent platform for developing quinazolinone-based conjugate antitumor agents.
Two novel series of quinazolinone-based hybrids, including twelve two-heterocycle-containing quinazolinone-1,3,4-oxadiazole hybrids (10a–l) and five three-heterocycle-containing quinazolinone-1,3,4-oxadiazole-benzimidazole conjugates (8a–e), have been successfully designed and synthesized in reasonable total yields starting from the commercially available anthranilamide. Cytotoxicity evaluations of the synthesized compounds revealed that the quinazolinone-1,3,4-oxadiazole 10i with the simple aryl group attached to the oxadiazole heterocycle exhibited good cytotoxicities against the A549 and MCF-7 cell lines, while the analogues 8b–d with the thioether-linked benzimidazole moiety incorporated on the right side of the oxadiazole ring displayed comparable or stronger activities, compared to the simple two-heterocycle-containing hybrid 10i, toward the MCF-7 cell line. These results suggest that the quinazolinone-based 1,3,4-oxadiazole-benzimidazole hybrids represent potent structural hybrid motifs for developing novel anti-cancer agents.
Reactions were monitored by TLC on 0.2 mm pre-coated silica gel 60 F254 plates (Merck, Germany). 1H- and 13C-NMR spectra were measured with Bruker Avance 500 MHz and ECA400II 400 MHz (JEOL, Japan) spectrometers. High resolution electrospray ionization (HRESI)-MS observations were performed on a Sciex OS 1.2 mass spectrometer and on an LTQ-Orbitrap XL-ETD (Thermo Scientific, U.S.A.). Purities of the compounds tested for cytotoxicity were evaluated by using a 1290 analytical HPLC (Agilent Technologies, U.S.A.). Fourier transform (FT)-IR was conducted using the KBr pellet method on a Thermo Nicolet 6700 and on an FT/IR-460 Plus spectrometers (JASCO, Japan). Chemical shifts are given in parts per million (ppm) relative to tetramethylsilane (Me4Si, δ = 0); J values are given in Hertz. All chemicals and solvents used in this study were of analytical grade.
General Procedures for the Syntheses of Quinazolinone-1,3,4-oxadiazole-benzimidazole Conjugate Derivatives (8a–e)A mixture of 6 (0.25 mmol), benzimidazoles 736) (1.2 equiv.), and NaOAc (1.2 equiv.) in ethanol (3 mL) was refluxed at 78 °C for 2 h. After solvent evaporation under reduced pressure, the residue was dissolved in ethyl acetate (100 mL). The organic layers were then washed with a saturated aqueous solution of NH4Cl, water, and brine, and dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give the desired quinazolinone-1,3,4-oxadiazole-benzimidazole derivatives (8a–e).
General Procedures for the Syntheses of Quinazolinone-1,3,4-oxadiazole Conjugate Derivatives (10a–l)A mixture of the acylhydrazides 5 (0.5 mmol) and aldehydes 9 (1 equiv.) in absolute ethanol (5 mL) was refluxed at 70 °C for 3–5 h. After cooling to room temperature, the excess solvent was evaporated under reduced pressure to obtain the intermediate imines, which were used for the next step without further purification.
The obtained intermediate imines (0.5 mmol), I2 (3 equiv.), and K2CO3 (5 equiv.) were dissolved in dimethyl sulfoxide (DMSO) (1.5 mL). The reaction mixture was stirred at 100 °C for 2–6 h. After cooling to room temperature, the excess I2 was quenched with an aqueous saturated solution of Na2S2O3 (20 mL), and the resulting mixture was extracted with ethyl acetate (5 × 30 mL). The combined ethyl acetate layers were washed with water and brine, and dried over anhydrous Na2SO4, and the solvent was evaporated. Purification of the crude products by silica gel column chromatography afforded the desired quinazolinone-1,3,4-oxadiazole conjugate derivatives (10a–l).
Cytotoxicity AssayAll synthesized compounds were dissolved in DMSO to make 10 mM stock solutions. Serial dilutions were prepared in culture medium. The positive control, 5-FU, was dissolved in DMSO to make a 10 mM stock solution and then stored at −20 °C until use. The cytotoxic activities of the synthesized compounds were evaluated against human cancer cell lines (A549, HeLa, and MCF-7), using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay with some modifications.37) The human cancer cell lines were cultured in α-minimum essential medium (α-MEM), supplemented with 1% antibiotic antimycotic solution and 10% fetal bovine serum, at 37 °C in 5% CO2 atmosphere. Cells at 80–90% confluence were harvested and centrifuged at 3000 rpm for 3 min. The supernatant was discarded and the cell pellet was resuspended in fresh medium. Aliquots (100 µL) of the cells were seeded in 96-well plates (1 × 104 cells/well) and incubated for 24 h. The cells were then washed with phosphate-buffered saline (PBS), and 8a–e, 10a–l, and the positive control, 5-FU, were added to the wells at final concentrations of 6.25, 12.5, 25, 50, and 100 µM. After a 72 h incubation, the cells were washed with PBS, and a 100 µL aliquot of medium containing MTT solution (5 mg/mL) was added to each well and further incubated for 3 h. The absorbance was recorded using a microplate reader at 570 nm. Percentage of proliferation inhibition was calculated using the following formula:
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The IC50 values of the compounds required to inhibit 50% of the growth of the human cancer cell lines were calculated based on the relationship between the concentrations and the inhibition percentages, using the GraphPad Prism 5.0 software. Each experiment was performed in triplicate, and all data are presented as mean ± standard deviation (S.D.).
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI Grant 22H02777 to H.M.).
The authors declare no conflict of interest.
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