Benzoxazole derivatives: design, synthesis and biological evaluation

Background A new series of benzoxazole analogues was synthesized and checked for their in vitro antibacterial, antifungal and anticancer activities. Results and discussion The synthesized benzoxazole compounds were confirmed by IR, 1H/13C-NMR, mass and screened for their in vitro antimicrobial activity against Gram-positive bacterium: Bacillus subtilis, four Gram-negative bacteria: Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi and two fungal strains: Candida albicans and Aspergillus niger using tube dilution technique and minimum inhibitory concentration (MIC) was noted in µM and compared to ofloxacin and fluconazole. Human colorectal carcinoma (HCT116) cancer cell line was used for the determination of in vitro anticancer activity (IC50 value) by Sulforhodamine B assay using 5-fluorouracil as standard drug. Conclusion The performed study indicated that the compounds 1, 10, 13, 16, 19, 20 and 24 had highest antimicrobial activity with MIC values comparable to ofloxacin and fluconazole and compounds 4, 6, 25 and 26 had best anticancer activity in comparison to 5-fluorouracil.


Open Access
*Correspondence: naru2000us@yahoo.com 1 Faculty of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak 124001, India Full list of author information is available at the end of the article Background A great number of deaths are occurring throughout the world because of infectious diseases [1]. It has been observed that there is a rapid increase in multi drug resistant infections these days which are causing a rise in various public health problems. There are number of diseases which are now hard to treat with traditional antibiotics drugs and clinicians have to depend on limited drugs such as vancomycin [2]. Because of this there is an increased demand to develop newer antimicrobial agents [3]. One of the most dangerous diseases in the world is cancer and irrespective of so much medical advancement, cancer remains the second leading cause of death in developing as well as developed countries. Although chemotherapy is mostly used for treating cancer, the failure of available chemotherapeutics to treat cancer underscores the need of developing new chemical entities [4]. Human colorectal cancer (CRC) has poor prognosis and is the third most commonly diagnosed malignancies. Therapy is very much required with better efficacy, less adverse effects and improved survival rates [5]. Benzoxazole derivatives have gained a lot of importance in the past few years because of their use in intermediates for the preparation of new biological materials. Benzoxazoles are prominent in medicinal chemistry due to their wide spectrum of pharmacological activities such as antibacterial [2], antifungal [6], anticancer [7], anti-inflammatory [8], antimycobacterial [9], antihistamine [10], antiparkinson [11], inhibition of hepatitis C virus [12], 5-HT 3 antagonistic effect [13], melatonin receptor antagonism [14], amyloidogenesis inhibition [15] and Rho-kinase inhibition [16]. A number of marketed drugs (Fig. 1) are available having benzoxazole as core active moiety like, nonsteroidal anti-inflammatory drug (NSAID)flunoxaprofen, benoxaprofen, antibiotic-calcimycin, antibacterial-boxazomycin B, muscle relaxant-chloroxazone. Prompted by the above findings (Fig. 2) in the present study, we hereby report the synthesis, antimicrobial and anticancer activities of a series of benzoxazole derivatives.
The presence of IR absorption band at 3214 cm −1 in the spectral data of synthesized derivatives (26) corresponds to the group Ar-OH. The C-Br stretching of aromatic bromo compounds shows band around 705 cm −1 (19 and 20). The presence of Ar-NO 2 group in compounds (11, 12 and 13) was indicated by the appearance of asymmetric Ar-NO 2 stretches in the scale of 1347-1339 cm −1 . Arylalkyl ether category (Ar-OCH 3 ) present in the compounds 2, 3, 4, 5 and 6 shows IR absorption stretching at 3053-2835 cm −1 . In case of halogen group Ar-Cl vibration appears at 747-740 cm −1 whereas existence of Ar-F group in compounds 8, 17 and 18 was indicated by appearance of Ar-F stretches at 1383-1119 cm −1 . The presence of IR stretching at 759-660 cm −1 reflected the presence of C-S group. The presence of CO-NH group is reflected by the presence of absorption bands at 1629-1605 cm −1 whereas the absorption bands at 3213-2919 cm −1 , 1496-1452 cm −1 and 1688-1654 cm −1 corresponds to the presence of C-H, C=C and C=N group respectively. In case of 1 H-NMR spectra the presence of multiplet signals between 6.85 and 8.83 ppm reflected the presence of aromatic protons in synthesized derivatives. The compound 26 showed singlet at 4.6 ppm because of the presence of OH of Ar-OH. The appearance of singlet at 7.01-8.24 ppm, 7.49-8.26 ppm, 4.61-4.63 ppm and 4.57-4.59 ppm is due to the existence of -CONH, N=CH, N-CH 2 and CH 2 -S groups respectively. Compound 7 showed doublet around 1.22 ppm due to existence of isopropyl group at para position. Compounds 2, 3, 4, 5 and 6 showed singlet at range of 3.72-3.81 ppm due to presence of OCH 3 of Ar-OCH 3 . Finally, DMSO-d6 was used for recording the 13 C-NMR spectra of benzoxazole derivatives and it was observed that the spectral signals and proposed molecular structure of the prepared compounds showed good agreement.

Antimicrobial activity
The screening of antibacterial and antifungal activity of the synthesized derivatives was done by tube dilution method [21] and the results are shown in Table 2 as well as Figs. 3 and 4. The study revealed that the prepared derivatives showed moderate to good antimicrobial activity against various microbial strains used. Particularly, compounds 1, 10, 13, 16, 19, 20 and 24 have shown better antimicrobial activity than the standards ofloxacin and fluconazole. Compound 10 (MIC bs = 1.14 × 10 −3 µM) was found to be most effective against B. subtilis. Compound 24 (MIC ec = 1.40 × 10 −3 µM) was found to be active against E. coli, compound 13 (MIC pa = 2.57 × 10 −3 µM) against P. aeruginosa, compounds 19 and 20 (MIC st = 2.40 × 10 −3 µM) against S. typhi, compound 16 (MIC kp = 1.22 × 10 −3 µM) against K. pneumonia. The results of antifungal activity indicated that compound 19 (MIC an = 2.40 × 10 −3 µM) was most potent against A. niger and compound 1 (MIC ca = 0.34 × 10 −3 µM) was most effective against C. albicans. The other derivatives showed average to poor antimicrobial activity against all seven species.

Anticancer activity
Human colorectal carcinoma [HCT-116 (ATCC CCL-247)] cancer cell line was used for evaluating the anticancer activity of the prepared benzoxazole compounds using Sulforhodamine B (SRB) assay [22]. 5-Fluorouracil was used as standard drug and the results are shown in Table 2. The results indicated that the compound 6 (IC 50 = 24.5 µM) exhibited the best anticancer activity in comparison with the standard drug (IC 50 = 29.2 µM) whereas the compounds 4 and 26 displayed IC 50 values closer to the reference drug (39.9 µM and 35.6 µM, respectively).

SAR (structure activity relationship) studies
The structure-activity relationship of the synthesized benzoxazole derivatives with their antibacterial and anticancer activity results is summarized in Fig. 5.
• The substitution of aromatic aldehydes with dimethoxy (compound 4) and tri-methoxy groups (compound 6) improved the anticancer activity of prepared derivatives.

Experimental part
The analytical grade chemicals procured from commercial sources were used as such without further purification. Thin-layer chromatography on 0.25 mm silica gel (Merck) plates was performed for monitoring the progress of reaction, using chloroform and methanol as mobile phase in ratio of 9:1 and exposure to iodine vapours helped in observing the spots. Open capillary tube was used for determining the melting points of synthesized compounds. Bruker 12060280, software: OPUS 7.2.139.1294 spectrometer was used for recording infrared spectrum (ATR). Bruker Avance III 600 NMR spectrometer was used for recording 1 H and 13 C NMR spectra in appropriate deuterated solvents and are expressed in parts per million (ppm) downfield from tetramethylsilane (internal standard). NMR data are given as multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet) and number of protons. Perkin-Elmer 2400 C, H and N analyzer was utilized for the elemental analysis of the new synthesized compounds. All the compounds gave C, H and N analysis within ± 0.4% of the theoretical results. Mass spectra were obtained on Waters Micromass Q-ToF Micro instrument. The physicochemical and spectral data of the prepared compounds helped in their characterization.
Step 2: Synthesis of benzo[d]oxazole-2-thiol (II) A mixture of 2-aminophenol (1.1 g) in methanol (15 ml) was prepared to which potassium hydroxide (0.7 g) in water (3 ml) was added, followed by the addition of carbon-di-sulfide (0.9 ml). Resulting solution was refluxed at 65 °C for 5 h. After the completion of reaction, the mixture was poured in water, which was neutralized with concentrated hydrochloric acid. Solid separated was filtered and washed with hexane, recrystallized with ethanol and dried to afford the pure compound (Yield: 90%). MP: 168-170 °C.

13
Step 4: and anhydrous potassium carbonate (1 g) in dry acetone (15 ml) was prepared to which ethyl chloroacetate (1.2 ml) was added and the mixture was stirred for 8 h at room temperature. The reaction was monitored by TLC (TLC System: chloroform: methanol/9:1, R f : 0.65). The resulting solution was then evaporated and solid obtained was suspended in cold water with stirring, which was then filtered, washed with water, recrys-tallized with ethanol and dried to give desired product IV (Yield: 3.1 g, 85%). MP: 163-165 °C.

Spectral data of intermediates and final compounds (1-26)
Intermediate

Biological study
Antimicrobial activity Tube dilution method [21] was used for the determination of minimum inhibitory concentration (MIC) of the synthesized derivatives  [23]. Dimethyl sulfoxide (DMSO) was used for the preparation of stock solution of test and standard compounds. The concentrations of 50, 25, 12.5, 6.25, 3.125 and 1.562 µg/ml were obtained by doing further progressive dilutions. The samples were incubated at 37 ± 1 °C for 24 h (bacteria), at 25 ± 1 °C for 7 days (A. niger) and at 37 ± 1 °C for 48 h (C. albicans), respectively and the results were recorded in terms of MIC. The lowest concentration of the compounds under evaluation that showed no signs of microbial growth of in the tube was the MIC. A control was performed with the test medium supplemented with DMSO at the same dilutions as used in the study to ensure that the solvent had no effect on the bacterial growth.
Anticancer activity Human colorectal carcinoma [HCT-116 (ATCC (American Type Culture Collection) CCL-247)] cancer cell line was used for the determination of anticancer activity of the prepared derivatives using 2-(3-diethyl-amino-6-diethylazaniumylidene-xanthen-9-yl)-5-sulfobenzene-sulfonate (SRB) assay. In this study, trichloroacetic acid was used for fixing the cells and then staining was done for 30 min with 0.4% (w/v) sulforhodamine B mixed with 1% acetic acid. Five washes of 1% acetic acid solution helped in discarding the unbound dye and protein-bound dye was extracted with 10 mM unbuffered tris base solution for confirmation of optical density at 570 nm in a computer-interfaced, 96-well microtiter plate reader [22].

Conclusion
A series of new benzoxazole derivatives was prepared and its chemical structure was confirmed by 1 H/ 13 C NMR, Mass and IR studies. All the derivatives were further evaluated for antibacterial, antifungal and anticancer activity and it was observed that the compounds 1, 10, 13, 16, 19, 20 and 24 displayed the best activity against various microbial species in comparison to reference drug ofloxacin and fluconazole. In case of anticancer activity, the compound 4 was the most active whereas compounds 6 and 26 had activity closer to the reference drug, 5-fluorouracil.