INTRODUCTION

Cancer is a one of the leading illnesses and causes of death for humans all over the World and which can be caused by abnormal cell division of normal cells [1, 2]. This is induced by genetic and epigenetic deviations, which can be divided into two types such as external (radiations, infections and tobacco) [3–5] and internal factors (hormones, mutations) [6–8]. Cancer treatments include surgery, radiation therapy, and chemotherapy, in which chemotherapy is the finest treatment for cure of various cancers [9]. Nitrogen-containing heterocycles are the best source for discovery and development of new types of chemotherapeutic agents. Among them, hetero-aromatic, imidazo[1,5-a]pyridines are the distinguished nitrogenous-bridgehead fused building blocks and highly attracted compounds due to their numerous biological application in the field of medicinal and synthetic chemistry [10–13]. They exhibit different kinds of biological properties including antiviral [14], aromatase [15], thromboxane synthetase inhibitors [16], positive inotropic agents [17], antibacterial [18], (VEGF)-receptor KDR [19], and apoptosis [20]. Alpidem (1, Scheme 1) [21] is the US FDA accepted chemotherapeutic drug bearing imidazole-pyridine skeleton as a part of the chemical structure, it is used for treatment of non-sedative agent.

Scheme
scheme 1

1.

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In addition, the N,O-five-membered ring system, namely 1,3,4-oxadiazoles, are the most privileged heterocyclic compounds, which can able to formation of hydrogen bonding with suitable target site [22, 23]. These derivatives displayed a different types of pharmacological properties like tubulin inhibitors [24], anticancer [25], antioxidant [26], HDACs [27], antibacterial [28], anti-inflammatory [29], insecticidal [30], antifungal [31], analgesics [32], antidepressant [33], antitubercular [34], antiviral [35], antidiabetic [36], antithrombotic [37], and tyrosinase [38]. The US Food and Drug Administrations (FDA) approved chemotherapeutic agent as Zibotentan (2, Scheme 1), which was utilized for treatment of various cancers [39].

Based on the biological findings of imidazole[1,5-a]pyridines and 1,3,4-oxadiazole moieties, we have design and prepared a new series of structurally modified different aryl substituted 1,3,4-oxadiazole-imidazo[1,5-a]pyridine derivatives and examined their preliminary anticancer activity against human cancer cell lines (MCF-7, A549, Colon-205 and A2780).

RESULTS AND DISCUSSION

The synthetic route for the preparation of 1,3,4oxadiazole incorporated imidazo[1,5-a]pyridine derivatives 10a–10j is outlined in Scheme 2. Intermediate 3 was coupled with 4-methyl benzoyl chloride 4 in the presence of triethyl amine in anhydrous THF. The reaction proceeded at room temperature for 3 h to afford pure compound 5. The latter underwent cyclization in POCl3 at reflux for 3 h to form 3-p-tolyl-imidazo[1,5-a]pyridine 6. This compound reacted with 3,4,5-trimethoxyphenyl acid hydrazide 7 in the presence of iodine and K2CO3 in DMSO at 110°C for 8 h to furnish 3-{4-[5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazol-2-yl]phenyl}imidazo[1,5-a]pyridine 8. Further Suzuki-coupling with various types of aryl boronic acids 9a–9j in presence of Pd(OAc)2, triphenyl phosphine and Cs2CO3 in dry acetone at 30°C for 24 h resulted in the formation of final compounds 10a–10j.

Scheme
scheme 2

2.

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Structure of newly prepared compounds was analysed by 1H NMR, 13C NMR and mass spectral data

The anticancer activity profile of newly prepared compounds 10a–10j against breast cancer (MCF-7), lung cancer (A549), colon cancer (Colon-205) and ovarian cancer (A2780) cell lines was examined by employing MTT assay, and compared with reference drug candidate as etoposide. These obtained results are summarized in Table 1. Among all the synthesized compounds, six derivatives 10a, 10f, 10g, 10h, 10i and 10j demonstrated more potent anticancer activity than etoposide. Further, these compounds were studied for SARs profile, the results indicated that the compound 10a with 3,4,5-trimethoxyphenyl ring shows good inhibitory anticancer activity (MCF-7 0.19±0.038 µM; A549 1.66±0.28 µM; Colo-205 1.98±0.77 µM and A2780 0.55±0.014 µM) to compare with reference drug. Compounds 10b (R = 4-methoxyphenyl) and 10c (R = 4-methylphenyl) show very lower activity than 10a. In addition, compounds 10c and 10d bearing strong electron-withdrawing substituents display moderate activity. Interestingly, heteroaromatic compound 10h with pyrazole skeleton exhibit excellent activity among all the tested cancer cell lines (MCF-7 0.012±0.005 µM; A549 0.43±0.017µM; Colo-205 0.038±0.002µM and A2780 0.77±0.018µM). Similarly, compound 10j having 3-methylpyrazole motif demonstrates slightly decreased activity (MCF-7 0.83±0.017µM; A549 0.33±0.038µM; Colo-205 0.10±0.018µM and A2780 0.23±0.063µM) than imidazo[1,5-a]pyridine 10h. When replacing pyrazole skeleton with isoxazole ring, compound 10i shows better activity (MCF-7 1.88±0.72 µM; A549 1.35±0.82 µM; Colo-205 0.66±0.013 µM and A2780= 0.89±0.056µM), whereas compound 10g bearing thienyl unit exhibit lowest activity (MCF-7 2.18±1.60 µM; Colo-205 2.55±1.56 µM and A2780 2.19±1.25 µM) compared to compounds 10f, 10h, and 10j. Moreover, compound 10f with pyridine substituent displays slightly improved activity (MCF-7 1.37±0.33 µM; A549 1.28±0.82 µM; Colo-205 1.63±0.66µM and A2780= 1.07±0.045 µM).

Table 1. Anticancer activity of compounds 10a–10j towards human cancer cell linesa
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All chemicals and reagents were obtained from Aldrich (Sigma–Aldrich, USA), Lancaster (Alfa Aesar, USA) and were used without further purification. Reactions were monitored by TLC, performed on silica gel glass plates containing 60 F-254, and visualization on TLC was achieved by UV light or iodine indicator. 1H and 13C NMR spectra were recorded on a 300 MHz spectrometer. Chemical shifts are reported in ppm downfield from internal TMS standard. ESI spectra were recorded on Micro mass, Quattro LC spectrometer equipped with ESI mode positive ion trap detector. Melting points were determined with an electro thermal melting point apparatus, and were uncorrected.

4-Methyl-(N-pyridin-2-yl)methylbenzamide (5). To a stirred solution of (pyridin-2-yl)metha namine 3 (15 g, 138.7 mmol) in anhydrous THF (200 mL) was added triethylamine (57.9 mL, 416.1 mmol) followed by 4-methylbenzoylchloride 4 (4.8 mL, 166.4 mmol) at 0°C. The reaction mixture was stirred at room temperature for 3 h. After the reaction completed, THF was removed under vacuum to afford crude product, which was further purified by column chromatography with ethyl acetate–hexane (2 : 3) to afford pure compound 5. Yield 21.6 g (69%). 1H NMR spectrum, δ, ppm: 2.46 s (3H), 4.70 d (2H, J = 5.0 Hz), 6.83 t (1H, J = 8.3, 6.7 Hz), 7.20 d. d (1H, J = 6.7, 6.0 Hz), 7.32 d (2H, J = 8.3 Hz), 7.64 d (2H, J = 8.3 Hz), 8.33 s (1H), 8.36 d (1H, J = 4.5 Hz). 13C NMR spectrum, δC, ppm: 23.2, 49.5, 124.6, 125.3, 129.5, 130.4, 131, 139.6, 141.3, 151.1, 160.3, 167.8. Mass spectrum (ESI), m/z: 227 [M + H]+.

3-p-Tolylimidazo[1,5-a]pyridine (6). A mixture of compound 5 (20 g, 88.4 mmol) and 100 mL of POCl3 was stirred at reflux for 3 h. After the reaction completed, the mixture was poured into cold water and neutralized with NaHCO3 solution. The water layer was extracted three times with ethyl acetate. The combined organic phases were dried over anhydrous Na2SO4 and evaporated under vacuum. The residue thus obtained was purified by column chromatography using ethyl acetate–hexane (4 : 6) to give compound 6. Yield 13.1 g (71%), white solid. 1H NMR spectrum, δ, ppm: 8.06 d (1H, J = 7.3 Hz), 7.51 d (2H, J = 8.1 Hz), 7.32 s (1H), 7.29 d (1H, J = 9.0 Hz), 7.14 d (2H, J = 7.9 Hz), 6.49 d. d (1H, J = 6.4, 6.2 Hz), 6.33 d. d (1H, J = 7.3, 7.1 Hz), 2.27 s (3H). 13C NMR spectrum, δC, ppm: 23.7, 114.9, 124.4, 125.3, 128.2, 129.4, 130.1, 130.7, 131.5, 140.2, 141.7, 146.5. Mass spectrum (ESI), m/z: 209 [M + H]+.

3-{4-[5-(3,4,5-Trimethoxyphenyl)[1,3,4]oxadiazol-2-yl]phenyl}imidazo[1,5-a]pyridine (8). A clean and dry 500 mL pressure tube was charged with compound 6 (11 g, 52.8 mmol), I2 (33.4 g, 132.2 mmol) and DMSO (200 mL). The vial was sealed and the resulting mixture was stirred at 110°C for 6 h. After completion of the reaction, 3,4,5-trimethoxybenzohydrazide 7 (14.3 g, 63.3 mmol) and K2CO3 (43.7 g, 316.8 mmol) were added. The resulting mixture was stirred at 110°C for 6 h. After the reaction completed, the mixture was treated with 300 mL of water and extracted with MTBE (3 × 200 mL). The extract was washed with 10% Na2S2O3 solution (w/w), dried over anhydrous Na2SO4 and concentrated. The crude product was purified by column chromatography (ethyl acetate–hexane, 1 : 1). Yield 13.9 g (62%), white solid, mp 207–209°C. 1H NMR spectrum, δ, ppm: 3.77 s (6H), 3.86 s (3H), 7.28 s (2H), 7.36 d. d (1H, J = 8.0, 1.3 Hz), 7.42–7.44 m (1H), 7.74 d (1H, J = 7.3 Hz), 7.81 d (2H, J = 7.9 Hz), 8.30 d (2H, J = 7.9 Hz), 8.43 s (1H), 8.55 t (1H, J = 8.3). 13C NMR spectrum, δC, ppm: 58.4, 62.9, 113.6, 114.9, 124.4, 125.3, 126.7, 127.2, 128.2, 129.4, 130.8, 131.4, 132.2, 140.2, 141.3, 146.5, 154.8, 165.9, 167.5. Mass spectrum (ESI), m/z: 429 [M + H]+.

1-(3,4,5-Trimethoxyphenyl)-3-{4-[5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazol-2-yl]phenyl}imidazo[1,5-a]pyridine (10a). To a solution of compound 8 (500 mg, 1.16 mmol) and 3,4,5-trimethoxyphenyl iodide 9a (687 mg, 2.33 mmol) in dry acetone (60 mL) were added Pd(OAc)2 (13 mg, 0.058 mmol, 5 mol %), triphenylphosphine (60 mg, 0.232 mmol, 20 mol %) and Cs2CO3 (756 mg, 2.32 mmol). The reaction mixture was stirred at 30°C for 24 h. After completion of the reaction, the mixture was diluted with water and extracted with ethyl acetate. The combined organic extracts were dried with anhydrous Na2SO4 and concentrated under vacuum to afford crude product which was purified by column chromatography (hexane–ethyl acetate, 7 : 3). Yield 242.6 mg (35% yield), white solid, mp 328–330°C. 1H NMR spectrum, δ, ppm: 3.77 s (6H), 3.80 s (3H), 3.87 s (3H), 3.94 s (6H), 7.12 s (2H), 7.27 s (2H), 7.36 d. d (1H, J = 8.0, 1.3 Hz), 7.42–7.45 m (1H), 7.68 d (1H, J = 7.2 Hz), 7.80 d (2H, J = 8.0 Hz), 8.31 d (2H, J = 8.0 Hz), 8.56 d. d (1H, J = 8.3, 1.01 Hz). 13C NMR spectrum, δC, ppm: 58.4, 59.7, 62.1, 62.7, 113.6, 114.2, 115.9, 124.4, 125.3, 125.7, 127.2, 128.8, 129.4, 130.1, 133.8, 139.4, 139.8, 140.2, 141.3, 146.5, 154.8, 155.7, 165.6, 167.5. Mass spectrum (ESI), m/z: 595 [M + H]+.

Compounds 10b–10j were prepared similarly.

1-(4-Methoxyphenyl)-3-{4-[5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazol-2-yl]phenyl}imidazo[1,5-a]pyridine (10b). Yield 279.5 mg (45%), white solid, mp 335–337°C. 1H NMR spectrum, δ, ppm: 3.77 s (6H), 3.87 s (3H), 3.91 s (3H), 7.19 d (2H, J = 8.2 Hz), 7.29 s (2H), 7.36 d. d (1H, J = 8.0, 1.3 Hz), 7.43–7.45 m (1H), 7.62–7.69 m (3H), 7.82 d (2H, J = 8.1 Hz), 8.31 d (2H, J = 8.1 Hz), 8.57 d. d (1H, J = 8.3, 1.07 Hz). 13C NMR spectrum, δC, ppm: 57.2, 58.4, 62.9, 113.6, 114.9, 116.3, 124.4, 125.3, 125.7, 127.2, 128.4, 129.8, 130.1, 131.4, 132.5, 139.9, 140.1, 141.2, 141.7, 146.5, 154.5, 162.6, 165.9, 167.5. Mass spectrum (ESI), m/z: 535 [M + H]+.

4-(3-{4-[5-(3,4,5-Trimethoxyphenyl)1,3,4-oxadiazol-2-yl]phenyl}imidazo[1,5-a]pyridin-1-yl)benzonitrile (10c). Yield 325.6 mg (53%), white solid, mp 370–372°C. 1H NMR spectrum, δ, ppm: 3.77 s (6H), 3.87 s (3H), 7.29 s (2H), 7.36 d. d (1H, J = 8.0, 1.3 Hz), 7.44–7.47 m (1H), 7.67 d (1H, J = 7.3 Hz), 7.82–7.94 m (4H), 8.33–8.44 m (4H, J = 8.1 Hz), 8.60 d. d (1H, J = 8.4, 1.07 Hz). 13C NMR spectrum, δC, ppm: 58.4, 62.9, 113.6, 114.9, 115.5, 120.2, 124.4, 125.3, 127.5, 128.7, 129.4, 130.8, 131.4, 132.3, 133.5, 134.2, 139.4, 140.7, 141.2, 142.3, 146.5, 154.5, 165.6, 167.6. Mass spectrum (ESI), m/z: 530 [M + H]+.

3-{4-[5-(3,4,5-Trimethoxyphenyl)-1,3,4-oxadiazol-2-yl]phenyl}-1-(4-nitrophenyl)imidazo [1,5-a]pyridine (10d). Yield 342.8 mg (53%), white solid, mp 363–365°C. 1H NMR spectrum, δ, ppm: 3.77 s (6H), 3.87 s (3H), 7.28 s (2H), 7.36 d. d (1H, J = 8.0, 1.3 Hz), 7.45–7.48 m (1H), 7.68 d (1H, J = 7.5 Hz), 7.85–7.98 m (4H), 8.35–8.47 m (4H, J = 8.1 Hz), 8.64 d. d (1H, J = 8.5, 1.1 Hz). 13C NMR spectrum, δC, ppm: 58.4, 62.9, 113.6, 114.9, 122.5, 124.4, 125.7, 126.3, 127.2, 128.4, 129.8, 130.4, 131.6, 132.5, 139.9, 140.1, 140.8, 141.3, 146.5, 154.8, 165.6, 167.5, 168.7. Mass spectrum (ESI), m/z: 550 [M + H]+.

3-{4-[5-(3,4,5-Trimethoxyphenyl)-1,3,4-oxadiazol-2-yl]phenyl}-1-p-tolylimidazo[1,5-a]pyridine (10e). Yield 291.7 mg (65%), white solid, mp 353–355°C. 1H NMR spectrum, δ, ppm: 2.39 s (3H), 3.78 s (6H), 3.88 s (3H), 7.28 s (2H), 7.32–7.40 m (3H), 7.44–7.55 m (3H), 7.63 d (1H, J = 7.3 Hz), 7.86 d (2H, J = 7.7 Hz), 8.31 d (2H, J = 7.7 Hz), 8.61 d. d (1H, J = 8.4, 1.1 Hz). 13C NMR spectrum, δC, ppm: 23.7, 58.4, 62.1, 113.6, 114.9, 124.4, 125.3, 126.7, 127.5, 128.4, 129.8, 130.4, 131.2, 132.4, 133.5, 139.9, 140.1, 141.2, 142.3, 146.5, 155.5, 165.6, 167.5. Mass spectrum (ESI), m/z: 519 [M + H]+.

3-{4-[5-(3,4,5-Trimethoxyphenyl)-1,3,4-oxadiazol-2-yl]phenyl}-1-(pyridin-4-yl)imidazo[1,5-a]pyridine (10f). Yield 331.4 mg (56%), white solid, mp 368–370°C. 1H NMR spectrum, δ, ppm: 3.78 s (6H), 3.88 s (3H), 7.29 s (2H), 7.38 d (1H, J = 8.0 Hz), 7.47–7.58 m (3H), 7.65 d (1H, J = 7.4 Hz), 7.86–7.95 m (4H), 8.31 d (2H, J = 7.7 Hz), 8.66 d. d (1H, J = 8.6, 1.2 Hz). 13C NMR spectrum, δC, ppm: 58.4, 62.9, 113.6, 114.9, 119.3, 124.4, 125.3, 125.7, 127.2, 128.4, 129.4, 130.1, 132.8, 139.9, 140.1, 140.8, 141.3, 146.5, 152.4, 155.8, 165.6, 167.6. Mass spectrum (ESI), m/z: 506 [M + H]+.

3-{4-[5-(3,4,5-Trimethoxyphenyl)-1,3,4-oxadiazol-2-yl]phenyl}-1-(thien-2-yl)imidazo[1,5-a]pyridine (10g). Yield 316.8 mg (53%), white solid, mp 338–340°C. 1H NMR spectrum, δ, ppm: 3.78 s (6H), 3.88 s (3H), 7.30 s (2H), 7.36–7.43 m (2H), 7.47–7.59 m (3H), 7.66 d (1H, J = 7.5 Hz), 7.84 d (2H, J = 7.9 Hz), 8.33 d (2H, J = 7.9 Hz), 8.65 d. d (1H, J = 8.5, 1.2 Hz). 13C NMR spectrum, δC, ppm: 58.4, 62.9, 113.6, 114.9, 124.4, 125.3, 126.7, 127.2, 127.7, 128.3, 129.4, 130.8, 131.1, 132.4, 135.4, 139.9, 140.2, 141.3, 146.5, 155.8, 165.6, 167.6. Mass spectrum (ESI), m/z: 511 [M + H]+.

3-{4-[5-(3,4,5-Trimethoxyphenyl)-1,3,4-oxadiazol-2-yl]phenyl}-1-(1H-pyrazol-4-yl)imidazo[1,5-a]pyridine (10h). Yield 266.3 mg (46%), white solid, mp 344–346°C. 1H NMR spectrum, δ, ppm: 3.78 s (6H), 3.88 s (3H), 7.30 s (2H), 7.33 d. d (1H, J = 8.1, 1.3 Hz), 7.42–7.44 m (1H), 7.64 d (1H, J = 7.6 Hz), 7.85–7.90 m (3H), 8.28–8.35 m (3H), 8.64 d. d (1H, J = 8.5, 1.2 Hz), 9.11 br. s (1H). 13C NMR spectrum, δC, ppm: 58.4, 62.9, 113.6, 114.9, 124.3, 125.4, 126.7, 127.2, 128.4, 129.8, 130.4, 131.3, 136.6, 139.9, 140.2, 141.3, 141.7, 146.5, 155.5, 166.2, 167.7. Mass spectrum (ESI), m/z: 495 [M + H]+.

1-(Isoxazol-4-yl)-3-{4-[5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazol-2-yl]phenyl}imidazo[1,5-a]pyridine (10i). Yield 270.5 mg (47%), white solid, mp 334–336°C. 1H NMR spectrum, δ, ppm: 3.79 s (6H), 3.88 s (3H), 7.30 s (2H), 7.33 d. d (1H, J = 8.1, 1.3 Hz), 7.42–7.44 m (1H), 7.61–7.67 m (2H), 7.86–7.93 m (3H), 8.34 d (2H, J = 7.8 Hz), 8.67 d. d (1H, J = 8.5, 1.2 Hz). 13C NMR spectrum, δC, ppm: 58.4, 62.9, 113.6, 114.9, 124.4, 125.3, 126.7, 127.2, 128.4, 129.8, 130.1, 130.8, 139.9, 140.2, 141.7, 146.5, 153.5, 154.6, 155.5, 165.6, 167.7. Mass spectrum (ESI), m/z: 496 [M + H]+.

3-{4-[5-(3,4,5-Trimethoxyphenyl)-1,3,4-oxadiazol-2-yl]phenyl}-1-(3-methyl-1H-pyrazol-4-yl)imidazo[1,5-a]pyridine (10j). Yield 210.6 mg (36%), white solid, mp 356–358°C. 1H NMR spectrum, δ, ppm: 1.96 s (3H), 3.79 s (6H), 3.88 s (3H), 7.30 s (2H), 7.35 d. d (1H, J = 8.1, 1.3 Hz), 7.43–7.45 m (1H), 7.66–7.70 m (2H), 7.85 d (2H, J = 7.9 Hz), 8.34 d (2H, J = 7.8 Hz), 8.67 d. d (1H, J = 8.5, 1.2 Hz), 8.95 br. s (1H). 13C NMR spectrum, δC, ppm: 15.1, 58.4, 62.9, 113.6, 114.9, 124.4, 125.3, 125.7, 127.2, 128.8, 129.8, 130.4, 131.3, 135.2, 139.9, 140.2, 141.3, 147.2, 148.5, 155.8, 165.9, 167.6. Mass spectrum (ESI), m/z: 509 [M + H]+.

MTT assay. Each data represents as mean ±SD values from three different experiments performed in triplicates. Individual wells of a 96-well tissue culture microtiter plate were inoculated with 100 µL of complete medium containing 1×104 cells. The plates were incubated at 37°C in a humidified 5% CO2 incubator for 18 h prior to the experiment. After medium removal, 100 µL of fresh medium containing the test compounds and etoposide at different concentrations such as 0.5, 1, and 2 µM were added to each well and incubated at 37°C for 24 h. Then the medium was discarded and replaced with 10 µL MTT dye. Plates were incubated at 37°C for 2 h. The resulting formazan crystals were solubilized in 100 µL extraction buffer. The optical density (OD) was read at 570 nm with micro plate reader (Multi-mode Varioskan Instrument-Themo Scientific). The percentage of DMSO in the medium never exceeded 0.25%.

CONCLUSIONS

In conclusion, we described a new library of 1,3,4-oxadiazole incorporated imidazo[1,5-a]pyridine derivatives. Anticancer activity of newly prepared compounds was investigated by employing MTT assay. These obtained results displayed significant anticancer activity compared with clinical drug etoposide.