Design, Synthesis and Biological Evaluation of Amide derivatives of 1,3,4-oxadiazole-isoxazol-pyridine-benzimidazole as Anticancer Agents.

doi:10.21203/rs.3.rs-1496043/v1

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Design, Synthesis and Biological Evaluation of Amide derivatives of 1,3,4-oxadiazole-isoxazol-pyridine-benzimidazole as Anticancer Agents.

https://doi.org/10.21203/rs.3.rs-1496043/v1

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We have designed and synthesized a series of novel amide derivatives of 1,3,4-oxadiazole-isoxazol-pyridine-benzimidazole (10a-j), and their structures are characterized by ¹HNMR, ¹³CNMR and mass spectral data. The preliminary anticancer applications of these compounds are screened towards four types of human cancer cell lines including PC3 (prostate), A549 (lung), MCF-7 (breast) and DU-145 (prostate). The assay results revealed that many of the target compounds displayed remarkable anticancer activity. Among them, the compounds 10f, 10g, 10h and 10j are found to be more potent than rest of the compounds. In particularly, one compound 10f displayed most promising anticancer activity.

Nocodazole

Luminespib

benzimidazole

isoxazole and anticancer profile

Generally, heterocyclic compounds are considered as heart of medicinal filed due to its utility as a crucial core intermediates for discovery and development of novel chemotherapeutic entities. Specifically, nitrogen contained heterocyclic motifs is a part of many therapeutic agents [1–3]. In addition to that, nitrogen atoms bearing heterocyclic like benzimidazoles are one of the most promising hetero-aromatic scaffolds having a significant consideration in the field of modern medicinal chemistry [4, 5]. These derivatives have demonstrated a wide variety of biological applications such as DNA topoisomerase-I [6], human cytomegalovirus (HCMV) [7], antiulcer [8], anticancer [9], antimicrobial [10], antihelminthic [11], anti-inflammatory [12], anticoagulant [13] and antifungal [14]. Among the numerous benzimidazole derivatives, one of the derivative namely Nocodazole (1, Fig. 1) was a proved as a very good anticancer agent in the inhibition of tubulin polymerization [15]. In addition, 1,2-isoxazole derivatives are well established compounds with potential medicinal activity [16]. They possessed a broad range of biological activities including anti-HIV [17], antinociceptive [18], anticancer [19], antituberculosis [20], analgesic [21], anti-inflammatory [22], antimicrobial [23], anticonvulsant [24] and anthelmintic [25]. One of the compound with 1,2-isoxazole isooxazole moiety, which is approved by US Food and Drug Administration as anticancer drug candidate is Luminespib (2, Fig. 1), it was developed by pharmaceutical company Vernalis [26].

In view of numerous biological findings of both benzimidazole and 1,2-isoxazole moieties and as continuation of efforts, we have designed and developed a new bunch of amide derivatives of 1,3,4-oxadiazole-isoxazol-pyridine-benzimidazole (10a-j). Further, all compounds were characterized by ¹HNMR, ¹³CNMR and mass spectral data. Furthermore, compounds were evaluated for their preliminary anticancer applications against four human cancer cell lines including PC3 (prostate), A549 (lung), MCF-7 (breast) and DU-145 (prostate).

Chemistry

The synthetic route for the amide derivatives contained 1,3,4-oxadiazole-isoxazol-pyridine-benzimidazole (10a-j) was illustrated in Scheme 1. The key intermediate 5 was developed from intermediate 3 underwent Claisen condensation reaction with 5-bromo-1H-benzo[d]imidazole-2-carbaldehyde 4 by using of aq. NaOH in ethanol at room temperature for 12 hours. Further, this chalcone 5 is allowed to react with NH₂OH.HCl and pyridine in isopropyl alcohol with continuous stirring at reflux for 6 hours to give pure isoxazole intermediate 6. Furthermore, this intermediate 6 undergone Suzuki coupling reaction with pyridin-4-yl-4-boronic acid 7 in presence of Pd(PPh₃)₄ catalyst and triethyl amine in 1,4-dioxane at reflux for 6 hours to afford intermediate 8. Afterwords, intermediate 8 coupled with various substituted benzoyl chlorides (9a-j) by using of Cs₂CO₃ in acetonitrile and reaction mixture is stirred at room temperature for 5 hours to provide pure target compounds 10a-j. The structure of compound 10f was confirmed by ¹HNMR spectrum values indicated that the methoxy protons appeared two sets at 3.83 (s, 6H), 3.90 (s, 3H) ppm, two aryl proton observed at 7.32 (s, 2H) ppm, and the isoxazole contained proton at 7.39 (s, 1H) ppm, 1,3,4-oxadiazole proton observed at 8.29 (s, 1H) ppm. The benzimidazole and pyridine contained protons appeared at 7.70 (d, 1H, J = 8.2 Hz), 7.76–7.80 (m, 3H), 8.12 (d, 1H, J = 8.2 Hz), 8.48 (d, 2H, J = 6.1 Hz). The mass spectrm value MS (ESI): m/z 525 [M + H]⁺.

Biological Evaluation

In vitro cytotoxicity

In vitro anticancer effects of the newly developed compounds 10a-j was examined against a panel of four human cancer cell lines such as PC3 (prostate), A549 (lung), MCF-7 (breast) and DU-145 (prostate) by using of MTT assay and results were summarized in Table 1, which compared with etoposide as the positive control. Among the all compounds 10f, 10g, 10h and 10j were found to be more potent than rest of the compounds. In particularly, one compound 10f displayed most promising anticancer activity. Further, the structure activity relationships (SARs) study of the compounds indicated that the presence of electron-rich group contained compound 10f (3,4,5-trimethoxy) displayed highest anticancer activity with IC₅₀ values (PC3 = 0.26 ± 0.084µM; A549 = 0.11 ± 0.071µM; MCF-7 = 0.87 ± 0.096µM and DU-145 = 0.55 ± 0.067µM), respectively. The effect was observed in compound 10g with 3,5-dimethoxy substituent on the phenyl moiety showed slightly lower activity (PC3 = 0.98 ± 0.082µM; A549 = 1.02 ± 0.45µM; MCF-7 = 1.29 ± 0.66µM and DU-145 = 1.74 ± 0.89µM) than with 10f. Similarly, 4-methoxy group having compound 10h showed lesser activity (PC3 = 1.90 ± 0.49µM; A549 = 2.12 ± 1.37µM; MCF-7 = 2.45 ± 1.60µM and DU-145 = 1.88 ± 2.12µM) than with both 10f and 10g. Where, the weaker electron-donating group contained compound 10i (4-dimethylamino) demonstrated decreased activity. Whereas, compound 10j with 4-methyl group and showed increased activity (PC3 = 2.78 ± 1.56µM; A549 = 2.98 ± 1.48µM; MCF-7 = 2.13 ± 1.43µM and DU-145 = 2.44 ± 1.77µM) than with 10i. The antiviral activity of the compounds were weakened when an electron-withdrawing groups at phenyl ring, this effect was observed in compounds 10a (4-nitro), 10b (3,5-dinitro), 10c (4-chloro), 10d (4-bromo) and 10e (4-cyano) have shown very poor activity.

Table 1

In vitro cytotoxicity of newly developed compounds10a-j with IC₅₀ in µM.^a
Compound	^cPC3	^dA549	^eMCF-7	^fDU-145
10a	ND	7.45 ± 4.32	4.91 ± 3.12	5.23 ± 3.61
10b	9.45 ± 6.87	8.55 ± 6.22	ND	ND
10c	ND	10.3 ± 7.79	7.53 ± 6.02	8.12 ± 5.63
10d	11.20 ± 7.12	ND	ND	3.69 ± 2.58
10e	ND	4.60 ± 3.27	6.33 ± 2.90	11.98 ± 6.85
10f	0.26 ± 0.084	0.11 ± 0.071	0.87 ± 0.096	0.55 ± 0.067
10g	0.98 ± 0.082	1.02 ± 0.45	1.29 ± 0.66	1.74 ± 0.89
10h	1.90 ± 0.49	2.12 ± 1.37	2.45 ± 1.60	1.88 ± 2.12
10i	3.40 ± 2.42	3.09 ± 2.16	4.26 ± 2.96	6.44 ± 3.10
10j	2.78 ± 1.56	2.98 ± 1.48	2.13 ± 1.43	2.44 ± 1.77
Etoposide	2.39 ± 1.56	3.08 ± 0.135	2.11 ± 0.024	1.97 ± 0.45
ND = Not determine.
^aEach data represents as mean ± S.D values. From three different experiments performed in triplicates.^bcPC3: human prostate cancer cell line. ^dA549: human lung cancer cell line. ^eMCF-7: human breast cancer cell line. ^fDU-145: human prostate cancer cell line.

In summary, a series of novel amide derivatives (10a-j) contained 1,3,4-oxadiazole-isoxazol-pyridine-benzimidazole skeleton were designed and synthesized. The newly synthesized compounds were characterized by ¹HNMR, ¹³CNMR and mass spectral data. The preliminary anticancer applications of these compounds were screened towards four types of human cancer cell lines including PC3 (prostate), A549 (lung), MCF-7 (breast) and DU-145 (prostate). The assay results revealed that many of the target compounds displayed remarkable anticancer activity. Among them, the compounds 10f, 10g, 10h and 10j were found to be more potent than rest of the compounds. In particularly, one compound 10f displayed most promising anticancer activity.

All chemicals and reagents were obtained from Aldrich (Sigma–Aldrich, St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey Company, Ward Hill, MA, 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 BRUKER NMR (300 MHz, 400 MHz) instrument. Chemical shifts (d) are reported in ppm downfield from internal TMS standard. ESI spectra were recorded on Micro mass, QuattroLC using ESI + software with capillary voltage 3.98 kV and ESI modepositive ion trap detector. Melting points were determined with an electro thermal melting point apparatus, and are uncorrected.

(E)-3-(5-Bromo-1H-benzo[d]imidazol-2-yl)-1-(1,3,4-oxadiazol-2-yl)prop-2-en-1-one (5):The compound 4 (8 g, 0.035 mmol) is dissolved in 50 mL of ethanol, followed by addition of 1-(1,3,4-oxadiazol-2-yl) ethanone (3) (3.3 ml, 0.035 mmol) and 30%NaOH (10 ml). The reaction mixture is stirred at room temperature for 12 hours. After completion of reaction, solvent is evaporated and acidified with 2M HCl. Then compound was extracted in ethyl acetate and dried over with sodium sulphate, the crude product is recrystallized with methanol to give pure compound 5, dark yellow solid (9.6 g, 85% yield); mp 156–158 ^oC; ¹H NMR (DMSO-d6, 400 MHz) δ 6.91 (d, 1H, J = 15.2 Hz), 7.50 (d, 1H, J = 8.1 Hz), 7.61–7.68 (m, 2H), 8.10 (s, 1H), 8.44 (s, 1H), 8.73 (s, 1H). ¹³C NMR (DMSO-d6, 100 MHz,) δ 111.4, 117.3, 118.6, 121.7, 123.5, 132.2, 139.1, 142.6, 151.6, 152.3, 162.5, 190.5. ESI (MS) : m/z 318 (M)⁺.

2-(3-(1,3,4-Oxadiazol-2-yl)isoxazol-5-yl)-5-bromo-1H-benzo[d]imidazole (6): A mixture of compound 5 (9 g, 0.028 mmol) and NH₂OH.HCl (4 g, 0.056 mmol) is dissolved in 60 ml of 2-propanol, then 1 ml of pyridine is added and reaction mixture is stirred at reflux for 6 hours. After completion of reaction by TLC and the solvent is evaporated under reduced pressure. The precipitated product is washed with water (3×20 ml) and crude product is purified by column chromatography with ethyl acetate/hexane (4:6) to afford pure compound 6, white solid (8.3 g, 88% yield). mp 222–224 ^oC; ¹H NMR (DMSO-d6, 400 MHz) δ 7.43 (s, 1H), 7.66 (d, 1H, J = 7.9 Hz), 7.77–7.83 (m, 2H), 8.31 (s, 1H), 8.81 (s, 1H). ¹³C NMR (DMSO-d6, 100 MHz,) δ 101.3, 117.6, 118.3, 121.4, 132.5, 139.4, 142.2, 151.5, 152.6, 152.9, 153.5, 168.4. ESI (MS): m/z 331 [M]⁺.

2-(3-(1,3,4-Oxadiazol-2-yl)isoxazol-5-yl)-5-(pyridin-4-yl)-1H-benzo[d]imidazole (8): A mixture of compound 6 (7 g, 0.022 mmol), Pd(PPh₃)₄ (2.5 g, 0.0022 mmol) in 1,4-dioxane (90 mL), pyridin-4-yl-4-boronic acid (7) (2.2ml, 0.022 mmol) and Et₃N (3.1 ml, 0.108 mmol) is stirred at reflux for 6 hours. The reaction mixture is cooled to room temperature and the solvent is evaporated through vacuum. The residue is dissolved in ethyl acetate (100 mL) and water (80 mL) is added. The organic phase is separated and dried over anhydrous sodium sulfate. The crude compound is purified by column chromatography by using ethyl acetate/hexane (1:1) to afford compound 8, off white solid (5.9 g, 82% of yield); mp 240–242 ^oC; ¹H NMR (DMSO-d6, 400 MHz) δ 7.45 (s, 1H), 7.68 (d, 1H, J = 8.3 Hz), 7.79–7.86 (m, 3H), 8.30 (s, 1H), 8.51 (s, 1H), 8.63 (d, 2H, J = 6.5 Hz), 8.81 (s, 1H). ¹³C NMR (DMSO-d6, 100 MHz,) δ 101.4, 115.4, 120.4, 123.5, 128.3, 137.4, 138.5, 139.4, 145.6, 150.2, 151.4, 152.3, 152.7, 153.4, 168.6. MS (ESI): m/z 331 [M + H]⁺.

(2-(3-(1,3,4-Oxadiazol-2-yl)isoxazol-5-yl)-5-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)(4-nitrophenyl)methanone (10a): The compound 8 (200 mg, 0.60 mmol) is dissolved in 10 mL of acetonitrile, followed by addition of 4-nitrobenzoyl chloride (9a) (112 mg, 0.60 mmol), and Cs₂CO₃ (320 mg, 1.2 mmol). The reaction mixture is stirred at room temperature for 5 hours, till the completion of the reaction and it is monitored by TLC. The reaction mixture is washed with water and extracted with dichloromethane, dried over anhydrous Na₂SO₄ and the crude product was purified by column chromatography with ethyl acetate/hexane (6:4) to obtain pure compound 10a, white solid (196.2 mg, 68% yield); mp 263–265 ^oC; ¹H NMR (DMSO-d6, 300 MHz) δ 7.40 (s, 1H), 7.68 (d, 1H, J = 8.3 Hz), 7.84–7.90 (m, 5H), 8.10–8.21 (m, 3H), 8.27 (s, 1H), 8.49 (d, 2H, J = 6.3 Hz). ¹³C NMR (DMSO-d6, 100 MHz,) δ 101.3, 111.5, 120.3, 121.5, 123.4, 128.2, 130.3, 132.5, 137.4, 138.4, 142.2, 145.1, 150.5, 151.2, 152.3, 152.6, 153.4, 165.4, 165.8, 168.5. MS (ESI): m/z 480 [M + H]⁺.

(2-(3-(1,3,4-Oxadiazol-2-yl)isoxazol-5-yl)-5-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)(3,5-dinitrophenyl)methanone (10b): This compound 10b is prepared by following the method described for the preparation of the compound 10a, employing 8 (200 mg,0.60 mmol) with 3,5-dinitrobenzoyl chloride (9b) (138 mg, 0.60 mmol), Cs₂CO₃ (320 mg, 1.2 mmol) and the crude product is purified by column chromatography with ethyl acetate/hexane (6:4) to afford pure compound 10b, off white solid (216.8 mg, 68% yield); mp 310–312 ^oC; ¹H NMR (DMSO-d6, 300 MHz) δ 7.39 (s, 1H), 7.69 (d, 1H, J = 8.2 Hz), 7.81–7.89 (m, 3H), 8.12 (s, 1H), 8.29 (s, 1H), 8.50 (d, 2H, J = 6.5 Hz), 8.59 (s, 1H), 8.76 (s, 2H). ¹³C NMR (DMSO-d6, 100 MHz,) δ 101.3, 111.4, 116.2, 120.4, 120.7, 123.5, 128.3, 132.5, 135.4, 138.6, 142.2, 145.6, 150.3, 151.4, 152.3, 152.6, 153.4, 165.2, 165.6, 168.5. MS (ESI): m/z 525 [M + H]⁺.

(2-(3-(1,3,4-Oxadiazol-2-yl)isoxazol-5-yl)-5-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)(4-chlorophenyl)methanone (10c): This compound 10c is prepared by following the method described for the preparation of the compound 10a, employing 8 (200 mg,0.60 mmol) with 4-chlorobenzoyl chloride (9c) (0.08 ml, 0.60 mmol), Cs₂CO₃ (320 mg, 1.2 mmol) and the crude product is purified by column chromatography with ethyl acetate/hexane (6:4) to afford pure compound 10c, off white solid (220.6 mg, 78% yield); mp 270–272 ^oC; ¹H NMR (DMSO-d6, 300 MHz) δ 7.41 (s, 1H), 7.49 (d, 2H, J = 8.4 Hz), 7.67 (d, 1H, J = 8.0 Hz), 7.85–7.96 (m, 5H), 8.16 (d, 1H, J = 8.0 Hz), 8.30 (s, 1H), 8.49 (d, 2H, J = 6.3 Hz. ¹³C NMR (DMSO-d6, 100 MHz,) δ 101.2, 111.4, 120.5, 123.4, 128.2, 129.3, 129.9, 132.5, 134.2, 137.4, 138.4, 142.2, 145.5, 150.2, 151.5, 152.6, 152.6, 153.2, 165.6, 168.6. MS (ESI): m/z 469 [M]⁺.

(2-(3-(1,3,4-Oxadiazol-2-yl)isoxazol-5-yl)-5-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)(4-bromophenyl)methanone (10d): This compound 10d is prepared by following the method described for the preparation of the compound 10a, employing 8 (200 mg,0.60 mmol) with 4-bromobenzoyl chloride (9d) (132 mg, 0.60 mmol), Cs₂CO₃ (320 mg, 1.2 mmol) and the crude product was purified by column chromatography with ethyl acetate/hexane (6:4) to afford pure compound 10d, off white solid, (201.7 mg, 65% yield); mp: 276–278 ^oC; ¹H NMR (DMSO-d6, 300 MHz) δ 7.40 (s, 1H), 7.55 (d, 2H, J = 8.5 Hz), 7.68 (d, 1H, J = 8.1 Hz), 7.86–7.98 (m, 5H), 8.17 (d, 1H, J = 8.1 Hz), 8.31 (s, 1H), 8.50 (d, 2H, J = 6.4 Hz). ¹³C NMR (DMSO-d6, 100 MHz) δ 101.2, 111.3, 120.4, 123.4, 123.5, 128.3, 130.5, 132.3, 132.6, 137.2, 138.4, 143.4, 145.6, 150.2, 151.4, 152.5, 152.8, 153.5, 165.3, 168.5. MS (ESI): m/z 514 [M + 2H]⁺.

(2-(3-(1,3,4-Oxadiazol-2-yl)isoxazol-5-yl)-5-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)(4-cyanophenyl)methanone (10e): This compound 10e is prepared by following the method described for the preparation of the compound 10a, employing 8 (200 mg,0.60 mmol) with 4-cyanobenzoyl chloride (9e) (99 mg, 0.60 mmol), Cs₂CO₃ (320 mg, 1.2 mmol) and the crude product is purified by column chromatography with ethyl acetate/hexane (6:4) to afford pure compound 10e, off white solid, (286.2 mg, 69% yield); mp 280–282 ^oC; ¹H NMR (DMSO-d6, 300 MHz) δ 7.40 (s, 1H), 7.70 (d, 1H, J = 8.2 Hz), 7.78–7.99 (m, 7H), 8.18 (d, 1H, J = 8.2 Hz), 8.30 (s, 1H), 8.51 (d, 2H, J = 6.3 Hz). ¹³C NMR (DMSO-d6, 100 MHz,) δ 101.3, 111.3, 111.6, 119.4, 120.5, 123.4, 128.3, 129.2, 132.5, 133.2, 137.4, 138.4, 142.2, 145.5, 150.3, 151.4, 152.3, 152.6, 153.5, 165.2, 168.5. MS (ESI): m/z 460 [M + H]⁺.

(2-(3-(1,3,4-Oxadiazol-2-yl)isoxazol-5-yl)-5-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)(3,4,5-trimethoxyphenyl)methanone (10f): This compound 10f is prepared by following the method described for the preparation of the compound 10a, employing 8 (200 mg,0.60 mmol) with 3,4,5-trimethoxybenzoyl chloride (9f) (138 mg, 0.60 mmol), Cs₂CO₃ (320 mg, 1.2 mmol) and the crude product is purified by column chromatography with ethyl acetate/hexane (6:4) to afford pure compound 10e, white solid (210.3 mg, 66% yield); mp 300–302 ^oC; ¹H NMR (DMSO-d6, 300 MHz) δ 3.83 (s, 6H), 3.90 (s, 3H), 7.32 (s, 2H), 7.39 (s, 1H), 7.70 (d, 1H, J = 8.2 Hz), 7.76–7.80 (m, 3H), 8.12 (d, 1H, J = 8.2 Hz), 8.29 (s, 1H), 8.48 (d, 2H, J = 6.1 Hz). ¹³C NMR (DMSO-d6, 100 MHz,) δ 57.4, 57.8, 101.3, 105.5, 111.5, 120.6, 123.2, 128.3, 132.4, 135.4, 138.2, 140.3, 142.2, 145.6, 150.3, 151.4, 152.3, 152.7, 153.5, 154.6, 165.5, 168.6. MS (ESI): 525 [M + H]⁺.

(2-(3-(1,3,4-Oxadiazol-2-yl)isoxazol-5-yl)-5-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)(3,5-dimethoxyphenyl)methanone (10g): This compound 10g is prepared by following the method described for the preparation of the compound 10a, employing 8 (200 mg,0.60 mmol) with 3,5-dimethoxybenzoyl chloride (9g) (120 mg, 0.60 mmol), Cs₂CO₃ (320 mg, 1.2 mmol) and the crude product is purified by column chromatography with ethyl acetate/hexane (6:4) to afford pure compound 10g, white solid (176.5 mg, 59% yield); mp 304–306 ^oC; ¹H NMR (DMSO-d6, 300 MHz) δ 3.80 (s, 6H), 6.62 (s, 1H), 7.12 (s, 2H), 7.40 (s, 1H), 7.69 (d, 1H, J = 8.3 Hz), 7.75–7.79 (m, 3H), 8.11 (d, 1H, J = 8.3 Hz), 8.30 (s, 1H), 8.49 (d, 2H, J = 6.2 Hz. ¹³C NMR (DMSO-d6, 100 MHz,) δ 57.2, 101.4, 102.5, 111.5, 113.4, 120.4, 123.5, 128.2, 132.5, 135.3, 138.4, 142.2, 145.3, 150.5, 151.5, 152.6, 152.8, 153.5, 161.3, 165.4, 168.7. MS (ESI): m/z 495 [M + H]⁺.

(2-(3-(1,3,4-Oxadiazol-2-yl)isoxazol-5-yl)-5-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)(4-methoxyphenyl)methanone (10h): This compound 10h is prepared by following the method described for the preparation of the compound 10a, employing 8 (200 mg,0.60 mmol) with 4-methoxybenzoyl chloride (9h) (0.085 ml, 0.60 mmol), Cs₂CO₃ (320 mg, 1.2 mmol) and the crude product is purified by column chromatography with ethyl acetate/hexane (6:4) to afford pure compound 10h, white solid (188.3 mg, 67% yield); mp 290–292 ^oC; ¹H NMR (DMSO-d6, 300 MHz) δ 3.89 (s, 3H), 7.40 (s, 1H), 7.49 (d, 2H, J = 7.8 Hz), 7.70 (d, 1H, J = 8.2 Hz), 7.77–7.86 (m, 5H), 8.10 (d, 1H, J = 8.2 Hz), 8.30 (s, 1H), 8.50 (d, 2H, J = 6.1 Hz). ¹³C NMR (DMSO-d6, 100 MHz,) δ 57.4, 101.3, 111.5, 115.4, 120.5, 122.7, 128.2, 130.6, 132.4, 137.4, 138.5, 142.3, 145.5, 150.4, 151.3, 152.5, 152.7, 153.5, 160.3, 165.4, 168.6. MS (ESI): m/z 465 [M + H]⁺.

(2-(3-(1,3,4-Oxadiazol-2-yl)isoxazol-5-yl)-5-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)(4-(dimethylamino)phenyl)methanone (10i): This compound 10i is prepared by following the method described for the preparation of the compound 10a, employing 8 (200 mg,0.60 mmol) with 4-(dimethylamino)benzoyl chloride (9i) (110 mg, 0.60 mmol), Cs₂CO₃ (320 mg, 1.2 mmol) and the crude product is purified by column chromatography with ethyl acetate/hexane (6:4) to afford pure compound 10i, off white solid (205.3 mg, 71% yield); mp: 294–296 ^oC; ¹H NMR (DMSO-d6, 300 MHz) δ 2.90 (s, 6H), 7.41 (s, 1H), 7.51 (d, 2H, J = 7.9 Hz), 7.69 (d, 1H, J = 8.1 Hz), 7.75–7.84 (m, 5H), 8.11 (d, 1H, J = 8.1 Hz), 8.31 (s, 1H), 8.51 (d, 2H, J = 6.2 Hz). ¹³C NMR (DMSO-d6, 100 MHz,) δ 43.5, 101.5, 111.5, 113.2, 120.2, 123.5, 128.5, 130.4, 132.5, 137.2, 138.4, 141.2, 145.5, 150.2, 151.4, 151.7, 152.3, 152.6, 153.5, 165.6, 168.5. MS (ESI): m/z 478 [M + H]⁺.

(2-(3-(1,3,4-Oxadiazol-2-yl)isoxazol-5-yl)-5-(pyridin-4-yl)-1H-benzo[d]imidazol-1-yl)(p-tolyl)methanone (10j):This compound 10j is prepared by following the method described for the preparation of the compound 10a, employing 8 (200 mg,0.60 mmol) with 4-methylbenzoyl chloride (9j) (0.06 ml, 0.60 mmol), Cs₂CO₃ (320 mg, 1.2 mmol) and the crude product is purified by column chromatography with ethyl acetate/hexane (6:4) to afford pure compound 10j, off white solid (200.5 mg, 74% yield); mp 281–283 ^oC; ¹HNMR (DMSO-d6, 300 MHz) δ 2.41 (s, 3H), 7.39 (d, 2H, J = 7.7 Hz), 7.41 (s, 1H), 7.70 (d, 1H, J = 8.0 Hz), 7.74–7.86 (m, 5H), 8.10 (d, 1H, J = 8.0 Hz), 8.30 (s, 1H), 8.50 (d, 2H, J = 5.9 Hz). ¹³C NMR (DMSO-d6, 100 MHz,) δ 27.2, 101.3, 111.5, 120.4, 123.4, 128.5, 129.1, 130.4, 132.5, 137.4, 138.4, 142.2, 142.6, 145.3, 150.3, 151.4, 152.5, 152.8, 153.5, 165.5, 168.6. MS (ESI): m/z 449 [M + H]⁺.

MTT assay:

Individual wells of a 96-well tissue culture micro titer plate are inoculated with 100 µL of complete medium containing 1×10⁴ cells. The plates are incubated at 37 ^oC in a humidified 5% CO₂ incubator for 18 hours 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 are added to each well and incubated at 37 ^oC for 24 hours. Then the medium is discarded and replaced with 10 µL MTT dye. Plates are incubated at 37 ^oC for 2 hours. The resulting formazan crystals were solubilized in 100 µL extraction buffer. The optical density (O.D) is 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%.

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Schemes 1 is available in the Supplementary Files section.

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Schme1.png
Scheme 1: Synthesis of amide derivatives of 1,3,4-oxadiazole-isoxazol-pyridine-benzimidazoles
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Design, Synthesis and Biological Evaluation of Amide derivatives of 1,3,4-oxadiazole-isoxazol-pyridine-benzimidazole as Anticancer Agents.

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