Introduction

Malignant tumors represent one of the most common human diseases, and their clinical prognosis remains relatively poor. The discovery and development of new treatments for these diseases is urgently needed due to problems with currently available drugs, such as toxicities and drug resistance (Haskell, 2001), leaving ample space for the development of new therapeutic strategies. During last few years 2-substituted benzoxazole analogues have been screened for their antitumor (Ueki et al., 1993; Cheng et al., 1993; Shi et al., 1996; Hall et al., 1999; Kumar et al., 2002; Easmon et al., 2001; Michel et al., 1984), antiviral (Balani et al., 1992; Hoffman et al., 1993; Saari et al., 1992; Perrin et al., 1996; Staszewski et al., 1995; Olsen et al., 1994; Prudhomme et al., 1986), and antimicrobial activities (Arpaci et al., 2002; Ersan et al., 1997; Oren et al., 1997; Temiz et al., 1998; Sener et al., 1997; Yalcin et al., 1992; Reiner, 1982).

2-Substituted benzoxazoles have also been shown to exert analgesic (Bartsch and Erker, 1991), fungicidal, insecticidal, nematocidal (Yalcin et al., 1992), potent protease inhibitory, and anticancer activities and serve as a topoisomerase I poison (Dumez et al., 2002; Kim et al., 1996).

Bearing these results in mind we have taken up the synthesis of 2-substituted benzoxazoles due to their structural similarity with some benzoxazoles and likelihood that they will exhibit important biological effects such as antibacterial, antitumor, anticancer, and antifungal activities.

Chemistry

We have adopted a concise synthetic route for the preparation of 2-[(arylhydrazono)cyanomethyl]-5-chloro benzoxazoles (II), 2-[(arylidene)cyanomethyl]-5-halo benzoxazoles (III), and 2-[(cycloalkylidene)cyanomethyl]-5-chlorobenzoxazoles (IV). The starting synthon for these was 5-halo-2-cyano methylbenzoxazole (I). Diazocoupling of 5-chloro-2-cyanomethyl benzoxazole with the appropriate diazonium acetate gave the corresponding 2-[(arylhydrazono) cyanomethyl]-5- chlorobenzoxazoles as shown in Scheme 1, compound 1–7 (see also Table 1). Condensation of I (X = Cl, F) with the appropriate aromatic aldehyde in the presence of a catalytic amount of benzyl dimethylamine gave the corresponding 2-[(arylidene)cyanomethyl]-5-halo benzoxazoles as shown in Scheme 2, compounds 8–11 and 14 (Table 2). 2-[(cycloalkylidine) cyanomethyl]-5-chlorobenzoxazoles were prepared by condensing compound I (X = Cl) with cyclic ketone, as shown in Scheme 3, compounds 12 and 13 (Table 3, Table 4, Table 5, Table 6 ).

Scheme 1
scheme 1

 

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Table 1 Synthesis of 2-[(arylhydrazono) cyanomethyl]-5-chlorobenzoxazoles
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Scheme 2
scheme 2

 

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Table 2 Synthesis of 2-[(arylidene )cyanomethyl] -5-halobenzoxazoles
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Scheme 3
scheme 3

 

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Table 3 Synthesis of 2-[(cycloalkylidine) cyanomethyl]-5-chlorobenzoxazoles
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Table 4 Antiproliferation activity against various cancer cell lines
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Table 5 Antifungal activity
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Table 6 Antibacterial activity
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The structure of the compounds in Schemes 1, 2, and 3 were confirmed by infrared spectroscopy (IR), proton magnetic resonance (PMR), carbon-13 magnetic resonance spectroscopy (CMR), and microanalysis.

Biological results and discussion

Anticancer activity

All the compounds were tested for the antiproliferation activity against four tumor cell lines. Compounds 6, 12, and 13 were found to be active against human cervical carcinoma cell line (HeLa). However, compound 11 showed much higher activity against HeLa. Compounds 1 and 8 were found to be active against all four cell lines. Compound 9 showed activity against the human cervical carcinoma and human hepatoma cell line, but did not show any activity against colon carcinoma and human breast adenocarcinoma cell lines. Compound 10 showed activity against the human breast adenocarcinoma cell line but was inactive against the other three cell lines. Compounds 4, 6, and 7 did not show any activity against the human colon carcinoma cell line (WiDr) but compounds 11, 12, and 13 were found to be active against WiDr. Compounds 6, 7, 11, 12, and 13 were active against the human hepatoma cell line (Hep G2) but compound 4 was found to be inactive against Hep G2. Similarly, compounds 4, 11 and 12 showed activity against the human breast adenocarcinoma cell line (MCF-7). However, compound 13 did not show any activity. Compound 14, which contains a fluoro substituent, was found to be significantly highly active against all four cell lines tested (HeLa, WiDr, Hepa 2, and MCF-7). Thus it may be concluded that compounds with fluoro and chloro substitutents at position 5 of the benzoxazole ring coupled with three methoxy groups in the phenyl ring exhibited much higher activity.

Antifungal activity

Three series of compounds, namely aryl hydrazono benzoxazoles (1, 4, 8, 9, and 10), arylidene benzoxazoles (11 and 14), and cycloalkylidene benzoxazoles (12 and 13), were assayed for antifungal activity against Aspergillus flavus and Aspergillus niger. Antifungal testing was carried out using the potato dextrose agar plate diffusion method. Compounds 4, 8, and 9 exhibited inhibition in the range of 60–70%. Compound 4, i.e., 2-[(4-aryl hydrazono) cyanomethyl]-5-chloro benzoxazole showed 70% inhibition against Aspergillus niger. Compounds 12 and 13 showed much higher activity (percentage inhibition). 2-[(cyclohexylidene)cyanomethyl]-5-chloro-benzoxazole (12) showed 75% inhibition against Aspergillus niger but was less active against Aspergillus flavus (60%). In contrast, compound 13 i.e., (2-[(cyclopentylidene)cyanomethyl]-5-chloro-benzoxazole showed higher activity against A. flavus but less inhibition against A. niger (60%). Compound 11, i.e., 2-[(3,4,5-trimethoxy arylidene]-5-chloro benzoxazole, showed remarkable activity against both A. flavus (85%) as well as A. niger (80%). Compound 14 with a fluoro substituent exhibited exceptional activity against both A. flavus and A. niger (>90%).

As a generalized observation, chloro and methoxy groups at the 4-position in the aromatic ring enhanced antifungal activity. Therefore, it is not surprising that the introduction of 3-methoxy groups (compound 11) further enhanced antifungal activity. The introduction of the fluoro group in place of the chloro at position 5 in the benzoxazole ring greatly enhanced antifungal activity against both A. niger and A. flavus, primarily due to the enhanced solubility of the fluoro substituent as well as due to the –I effect compared to the chloro group, which is electron withdrawing.

Antibacterial activity

All the compounds were tested for antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus, and Klebsiella pneumoniae. Compound 1 was active against Staphylococcus aureus. Compounds 6 and 7 were active against all three bacteria. Compounds 4 and 8 were not very active against Staphylococcus aureus. Compounds 9 and 10 were active against all three bacteria. Compound 11 was highly active against Pseudomonas aeruginosa and Staphylococcus aureus, but not against Klebsiella pneumoniae. Compound 12 exhibited a much higher activity than 13 against Staphylococcus aureus and Klebsiella pneumoniae. Compound 14 was significantly active against Pseudomonas aeruginosa, Staphylococcus aureus, and Klebsiella pneumoniae.

Experimental chemistry

Melting points were taken in open glass capillaries and are uncorrected. IR spectra was determined by using a Perkin-Elmer FT-IR-Spectrometer in KBr phase (γmax in cm−1). PMR spectra were recorded on a Varian 200 MHz spectrometer using TMS as an internal standard. CMR spectra were recorded on a 13C Advance Brucker 300 MHz spectrometer. Microanalyses were done at IICT, Hyderabad. Mass spectra were recorded on an Autospec fast atom bombardment (FAB+) magnet with a 7 kV accelerator voltage and 25 kV gun voltage.

General procedure for the synthesis of compounds 1, 4, 6, and 7

5-Chloro-2-cyanomethyl benzoxazole (I, 0.01 M) was taken in acetic acid (10 mL), the solution was cooled to 0°C and to this solution was added dropwise during half an hour, aryl diazonium acetate, prepared from aryl amine (0.01 M) in acetic acid (15 mL) and NaNO2 (0.015 M). The reaction was continued for a specific period as mentioned in Table 1. To the reaction mass was added water (40 mL) and the solid product was isolated after filtration and washing with water. It was then recrystallized with the appropriate solvent as mentioned in Table 1 to achieve the percentage yield also listed therein.

2-[(4′-Methoxy aryl hydrazono)cyanomethyl]-5-chlorobenzoxazole (1)

IR(KBr): 3421(NH), 2222(C≡N),1608,1553,1461(C = N,NH,C = C), 1252, 1137, 1080(C-O-C); 1H-NMR(CDCl3): δ 3.90(s,3H;OCH 3), 7.32(s,1H,benzoxazole C4-H), 7.46-7.83(m,6H,4Ar-H and benzoxazole C6,7 –H, 13.57(s,1H,NH , D2O exchangable); MS m/z: 326(M+); 13C-NMR (CDCl3) δ: 112.05, 117.28, 127.1, 119.5, 146.6, 157.2, 157.88, 131.1, 134.9, 115.09, 114.9, 112.05, 55.61;

Calculated for C16H11ClN4O2: C,58.80;H,3.36;N,17.15: found C, 58.65;H,3.30;N,17.02.

2-[(4′-Chloro aryl hydrazono)cyanomethyl]-5-chlorobenzoxazole (4)

IR(KBr): 3416(NH),2228(C≡N), 1597,1541,1445(C = N,NH,C = C),1262,1197,1134,1101(C-O-C)

1H-NMR(CDCl3):7.42(s,1H,benzoxazoleC4-H),7.48-7.8(m,6H,4-ArH and benzoxazole C6,7-H), 13.46(s,1H,NH,D2O exchangeable. MS: m/z 331(M+)

Calculated for C15H8 Cl 2 N4O: C,54.38;H,2.41;N,16.91: found C,54.20; H,2.35; N,16.78.

2-[(4′-Bromo aryl hydrazono)cyanomethyl)-5-chlorobenzoxazole (6)

IR(KBr): 3418(NH),2230(C≡N),1615,1545,1463(C = N,NH,bendingC = C); 1262,1096

(C-O-C). 1H-NMR(CDCl3): δ7.38(s,1H,benzoxazoleC4-H), 7.43–7.73( C6,7-H)

13.65(s,1H,NH,D2O exchangeable,4.03(s,3H;OCH 3). MS: m/z 376(M + 1)

Calculated for C15H8 BrClN4O: C,47.93;H,2.13;N,14.91: Found C,47.85,H,2.03,N,14.78.

2-[(Aryl hydrazono) cyanomethyl]-5-chlorobenzoxazole (7)

IR(KBr): 3415(NH),2224(C≡N),1610,1545,1476(C = N,NH bending,C = C), 1262,1136,1100(C-O-C) 1H-NMR(CDCl3): 7.37(s,1H,benzoxazoleC4-H),7.46–7.74(m,6H,4-ArH and benzoxazole C6,7-H), 13.40(s,1H,NH,D2O exchangeable. MS: m/z 297(M+)

Calculated for C15H9 ClN4 O: C,60.70;H,3.03;N,18.88: found C, 60.57;H,2.94; N,18.70.

2-[4′-Methoxy arylidene)cyanomethyl]-5-chlorobenzoxazole (8)

IR(KBr): 2230(C≡N), 1590,1534(C = N,C = C), 1175,1046(C-O-C) 1H-NMR(CDCl3): 3.91 (s,3H,OCH3), 7.47(s,1H,benzoxazole C4-H), 7.5–7.74(m,6H,4Ar-H and benzoxazole C6,7-H), 8.23(s,1H, = CH). MS: m/z 311(M + 1)

Calculated for C17H11 ClN2O2: C,65.70;H,3.54;N,9.01: found C, 65.51;H,3.37;N,8.90.

2-[(4′-Hydroxy arylidene)cyanomethyl]-5-chlorobenzoxazole (9)

IR (KBr): 2222(C≡N), 1594,1565(C = N,C = C),1288,1175,1047(C-O-C) 6.01(brs,1H,Ar-4′OH,exchangeable on D2O shake 1H-NMR(CDCl3): 7.38(s,1H,benzoxazole C4-H),7.47–7.67(m,6H,4 Ar-H and benzoxazole C6,7-H), 8.21(s,1H, = CH). MS:m/z:297(M + 1)

Calculated for C16H9 ClN2O2: C,64.75; H,3.03; N,9.44: Found c,64.60;H, 2.95; N,9.26.

2-[(Arylidene)cyanomethyl]-5-chlorobenzoxazole (10)

IR (KBr): 2226(C≡N), 1592,1513(C = N,C = C),1260,1180,1047(C-O-C) 1H-NMR(CDCl3): 7.5 (s,1H,benzoxazole C4-H), 7.58–8.06(m,7H,5 Ar-H and benzoxazole C6,7-H), 8.32(s,1H, = CH)

MS:m/z 280(M+)

Calculated for C16H9 ClN2O: C, 68.44;H,3.20;N,9.98: found C,68.24; H,3.09; N,9.79.

2-[(3′,4′,5′-Trimethoxy arylidene)cyanomethyl]-5-chlorobenzoxazole (11)

IR (KBr): 2229 (C≡N), 1576,1501 (C = N,C = C),1246,1128,1036(C-O-C) 1H-NMR(CDCl3): 3.95(s,9H,-OCH 3),7.48(s,1H,benzoxazole C4-H), 7.50–7.73(m,4H,2Ar-H and benzoxazole

C6,7-H),8.21(s,1H, = CH) MS:m/z 371 (M + 1) 13C-NMR(CDCl3) δ: 160.42,153.36,149.58,14923, 142.69,130.77,127.07,126.31, 120.31,115.18,114.51, 111.37,108.87, 108.14,56.31.

Calculated for C19H15ClN2O4; C, 61.53; H, 4.04; N, 7.55; Found C, 61.40; H, 4.19; N, 7.38.

2-[3′,4′,5′-Trimethoxy arylidene) cyanomethyl] -5-fluorobenzoxazole (14)

IR (KBr): 2232(C≡N), 1580,1492(C = N,C = C), 1230(Ar-F), 1246,1128,1036(C-O-C).

1H-NMR(CDCl3): 4.0(s,9H,-OCH3),7.36(s,1H,benzoxazole C4-H), 7.5–7.82(m,4H,2Ar-H and benzoxazole C6,7-H), 8.25(s,1H, = CH) 13C-NMR(CDCl3) δ: 161.32, 160.78,159.40,153.36, 149.33,147.00,142.49,127.07,115.18, 113.61,111.02, 110.94,108.17,106.67, 56.30 MS: m/z 355(M + 1)

Calculated for C19H15FN2O4: C, 64.4; H,4.23;N,7.90: found C,64.21; H,4.37; N, 7.63.

2-[(Cyclopentylidene)cyanomethyl]-5-chlorobenzoxazole (12)

IR (KBr): 2226 (C≡N), 1605,1552,1446,(C = N,C = C),1256, 1060(C-O-C) 1H-NMR(CDCl3): 1.60–1.95(m,4H,cyclopentyl-C3,4-H2); 2.93–3.16(two t, each 2H,J = 6.4,7.2 Hz, cyclopentyl-C2,5-H 2); 7.26(s,1H,benzoxazole-C4-H); 7.31–7.47(m,2H,benzoxazole-C6,7-H) MS: m/z 259(M + 1)

Calculated for C14H11ClN2O: C,64.99;H,4.25; N,10.83: found C,64.79;H,4.42; N,10.64.

2-[Cyclohexylidene)cyanomethyl]-5-chlorobenzoxazole (13)

IR (KBr) 2222(C≡N), 1620,1549,1449(C = N,C = C), 1263,1060(C-O-C) 1H-NMR(CDCl3): 1.63–1.88(m,6H,Cyclohexyl-C3,4,5-H 2); 2.78–3.19 (two t,each 2H, J = 6.5,6.2 Hz,cyclohexyl-C2,6-H 2); 7.28(s,1H,benzoxazole C4-H) 7.33–7.5 (m,2H,benzoxazole C6,7-H); MS: m/z 272,(M + 1)

Calculated for C15 H13ClN2O: C, 66.0; H,4.77;N,10.27: found C, 66.12; H, 4.61; N,10.32.

Experimental biology

Antitumor activity

The in vitro antitumor activity of the compounds was carried out by the [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] (MTT) method to estimate the effect of each compound on cell growth. The principle behind this assay depends upon the reduction of the tetrazolium salt. The yellow-colored tetrazolium MTT is reduced by metabolically active cells in part by the action of dehydrogenase enzymes to generate reducing equivalents such as nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH). The resulting intracellular purple zones were solubilized and quantified by using a spectrophotometer. The [3-(4,5-215 dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide (MTT) was dissolved in phosphate buffer solution (PBS) at a concentration of 5 mg/mL. Then, 50 μL of the MTT solution was added to each well of 96-well culture plates, containing the 100 μL medium and incubated at 37°C for 4 h. The medium was then removed carefully without disturbing the purple-colored formazon crystals. Then, 50 mL of dimethyl sulfoxide (DMSO) was added to each well and mixed thoroughly to dissolve the formazon crystals . The plates were then read on a microplate reader at a wavelength of 570 nm. The readings were presented as an optical density.

Four cell lines were used: the human hepatoma cell line HEPG-2, the human cervical carcinoma cell line HeLa, the human colon carcinoma cell line WiDr, and the human breast adenocarcinoma cell line MCF-7.

Antibacterial activity

The antibacterial activity of the compounds was determined by the disc diffusion method. In this technique, sterile discs of 5 mm diameter of filter paper (Whatmann no. 1), impregnated with the test compounds (10 μg/mL of ethanol), were placed on nutrient agar plates at 37°C for 24 h. The inhibition zones around the dried impregnated discs were measured after 24 h. The activity was classified as “highly active” (diameter = 20–25 mm), “active” (diameter = 15–20 mm) or “slightly active” (diameter = 5–10 mm). A diameter of less than 5 mm was regarded as “inactive”.

Antifungal activity

The antifungal activity of these compounds was tested by the agar plate diffusion method against the two human pathogenic fungal strains Aspergillus flavus and Aspergillus niger. One milliliter of each compound was poured into a Petri dish containing about 20 mL of molten potato dextrose agar. As the medium solidified the Petri dishes were inoculated separately with the fungal isolates and kept at 27°C for 48 h. All the values (percentage inhibition) were recorded.

Summary

Benzoxazoles and their analogues are known to possess various biological effects such as antitumor, antiviral, and antimicrobial activities. 2-Substituted benzoxazoles have also been shown to exert analgesic, fungicidal, insecticidal, nematocidal, potent protease inhibitory, and anticancer activities and to act as a topoisomerase I poison. The cyano function present in antibiotics like cephactrile, cefmetazole, and toyomycin are needed to enhance biological activity.

Based on these structural findings and in an effort to synthesize new biologically active molecules that may be less toxic and possess anticancer, antiviral, and antimicrobial activity, we have taken up the synthesis of three series of compounds, namely 2-[(arylhydrazono) cyanomethyl]-5-chloro benzoxazoles, 2-[(arylidene)cyanomethyl]-5-halo benzoxazoles, and 2-[(cycloalkylidene) cyanomethyl]-5-chlorobenzoxazoles. These were then tested for their anticancer, antifungal, and antimicrobial activities.

Fluro and chloro substitution at position 5 of the benzoxazole ring coupled with 3-methoxy groups in the phenyl ring exhibited much higher activity. Compound 14, containing a fluoro substituent, was found to be significantly highly active against all four cell lines tested (HeLa, WiDr, Hepa 2, and MCF-7). As a generalized observation, chloro and methoxy groups at the 4-position in the aromatic ring enhanced antifungal activity; therefore, it is not surprising that the introduction of 3-methoxy groups (compound 11) further enhanced antifungal activity. The introduction of a fluoro group in place of the chloro substituent at position 5 in the benzoxazole ring greatly enhanced antifungal activity against both Aspergillus niger and Aspergillus flavus.

Therefore, it was observed that compound 11 and 14 were found to possess both anticancer activity and remarkable antifungal and antibacterial activities.