General and efficient synthesis of benzoxazol-2(3H)-ones: evolution of their anti-cancer and anti-mycobacterial activities

Abstract

A novel class of benzo[d]oxazol-2(3H)-one derivatives has been synthesized and their in vitro cytotoxicity against human pancreatic adenocarcinoma and human non-small cell lung carcinoma cancer cell lines was evaluated. Many of these compounds were found to display excellent to moderate activity. Among them, 6b, 6l, 6n and 6x were identified as lead molecules. In particular, 6l and 6n were found to be potent against the pancreatic adenocarcinoma cell line whereas the 6x was found to be effective against the human non-small cell lung carcinoma cell line. Conversely, the compounds 6l–x were found to be ineffective against Mycobacterium tuberculosis. Of the various molecules, 6h showed promising anti-mycobacterial activity, with an IC₅₀ value equal to that of ciprofloxacin.

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1. Introduction

The increasing resistance of cancer and tuberculosis to current treatments obviously demands the development of novel chemical entities with improved activity profiles.^1–5 Per the latest information, it is predicted that cancer may cause over 13.1 million deaths in 2030 worldwide.⁶ The World Health Organization (WHO) estimates that 11.4 million people worldwide are infected with both Mycobacterium tuberculosis (Mtb) and HIV. Currently, there are approximately 8 million new infections and 3 million deaths attributed to M. tuberculosis annually.^7–9 Irrespective of the tireless efforts and myriad compounds assessed for anti-cancer activity,^10–12 the ambiguity about the cause of cancer, limits to its detection at an early stage, its direct relationship with the process of cell division, the metastatic nature of cancer cells and deficient drug diffusion to cancer tissues are some of the features of this disease that present hurdles in the successful treatment of cancer. A few of the highly effective drugs, like pemetrexed,¹³ methotrexate,¹⁴ 5-fluorouracil,¹⁵ etc., appearing on the market provide hope for cancer patients. As an extension of previous reports on the development of anti-cancer agents,^16–19 mainly from the benzoxazol-2(3H)-one skeleton, we report herein an additional set of compounds with significant anti-cancer activity with certain human cell lines.

Tuberculosis (TB) is a disease caused by infection with Mycobacterium tuberculosis. It is a serious public health issue, due to the high risk of person-to-person transmission, and high level of morbidity and mortality. The primary cause of death in those infected with both Mycobacterium tuberculosis and HIV is from TB and not from AIDS. Enhanced sanitation has significantly restricted the frequency of the disease. The spread of multidrug-resistant TB (MDR-TB) and the emergence of extensively-drug-resistant TB (XDR-TB) pose new challenges in the prevention, curing and management of this lethal disease.²⁰ Therefore, the development of new drugs with enhanced activity against MDR-TB and XDR-TB is highly appreciated for the management of the disease.

In particular, benzo[d]oxazol-2(3H)-ones are considered as “privileged scaffolds” in the area of pharmacological probes. They are very useful for drug discovery, as they can mimic a phenol or a catechol moiety in a metabolically stable template. This category of compounds has led to the discovery of a number of derivatives endowed with antibacterial, antifungal, analgesic, anti-inflammatory, anticonvulsant, dopaminergic, antioxidant, antitumor, and HIV-1 reverse transcriptase activity,^21–33 as well as normolipenic agents.³⁴ Usually, the functionalization of the nitrogen atom is of interest, since the electronic characteristics of this atom can strongly influence the biological activity. Recently it was reported that the alkylation of benzoxazol-2(3H)-ones and benzothiazole-2(3H)-ones gave intermediates, which are used in pharmacotherapy for their anticocaine activity, as these substituted heterocycles interact with signal receptors. Consequently, numerous methods, such as the Hofmann rearrangement of amides,³⁵ carbonylation of o-substituted aryl azides using a rhodium catalyst,³⁶ two step cyclization of o-hydroxybenzoic acids,³⁷ cyclization of arenecarbohydroxamic acid,³⁸ photochemical rearrangements of 1,2-benisoxazolinones and ³⁹ cyclization of azidoformates,⁴⁰ have been developed for the synthesis of benzoxazol-2(3H)-one derivatives. Among them, the cyclization of o-aminophenols with various carbonylating reagents, such as 1,1-carbonyldimidazole,⁴¹ chloroformates,⁴² alkyl carbonates,⁴³ triphosgene,⁴⁴ pentafluorobenzoyl chloride⁴⁵ are the most commonly used reagents for the synthesis of benzoxazol-2(3H)-ones. Many of these reagents are either moisture sensitive or hazardous in nature. In order to overcome these difficulties, there is a need to develop a simple and convenient carbonylating reagent for the synthesis of benzo[d]oxazol-2(3H)-ones. Therefore, we used ethyl imidazole-1-carboxylate (EImC) as the carbonylating agent for the synthesis of benzoxazol-2(3H)-ones from amino phenols, since it is not moisture sensitive or hazardous in nature.

In continuation of our earlier work on EImC,^46,47 herein we report an efficient and simple method to synthesize benzoxazol-2(3H)-ones using EImC as a carbonylating reagent.

2. Results and discussion

As a continuation of our interest in biologically active heterocycles, we report herein a simple and efficient method for the synthesis of benzoxazol-2(3H)-ones using EImC as a carbonylating reagent.

Benzo[d]oxazol-2(3H)-one derivatives 6a–k were synthesized from 2-aminophenol and ethyl 1H-imidazole-1-carboxylate. Initially, we performed the reaction of 2-aminophenol with ethyl 1H-imidazole-1-carboxylate in the presence of various bases and solvents, as summarized in Table 1. Among them, K₂CO₃ in THF afforded the benzo[d]oxazol-2(3H)-one 6g in 92% yield (Scheme 1).

Table 1 Optimization of the reaction using different bases and solvents

Entry	Base	Temp. (°C)	Time (h)	5I^a	6g^a
a Isolated yield.b THF was used as a solvent.c DMF was used as a solvent.d Toluene was used as a solvent.
1	K2CO3	25	15	92^b	0
2	K2CO3	80	18	0	92^b
3	Cs2CO3	80	12	0	91^b
4	NaOMe	60	14	0	45^b
5	NaH	RT	7	0	89^b
6	tBuOK	RT	15	0	52^b
7	NaH	RT	8	0	68^c
8	NaH	RT	10	0	35^d

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Scheme 1 Synthesis of the benzo[d]oxazol-2(3H)-one derivatives (6a–k).

Various substituted benzoxazol-2(3H)-ones were synthesized from the corresponding 2-aminophenols and the results are summarized in Table 2. In all cases, the products were obtained in good yields. Both electron-rich and electron-deficient substrates underwent smooth cyclization to give the desired products. Base-sensitive substrates, such as halogen substituted aminophenol derivatives, were able to tolerate the reaction conditions.

Table 2 Preparation of benzo[d]oxazol-2(3H)-ones 6a–k

S. no.	Substrate	Product	Yield^a (%)
a Isolated yield.
1			88
2			82
3			91
4			84
5			87
6			70
7			90
8			78
9			88
10			72
11			85

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Compounds 6m–x were synthesized in three steps starting from 5-bromobenzo[d]oxazol-2(3H)-one (Scheme 2). In the first step, 6k was treated with isopropyl iodide in THF in the presence of K₂CO₃ to afford the isopropyl derivative 6k1. In the second step, isopropyl derivative 6k1 was treated with (2-aminophenyl)boronic acid in the presence of Pd(II)Cl₂(dppf) and potassium carbonate in an ethanol–toluene (1 : 1) system under an N₂ atmosphere to produce the amino derivative 6l. In the final step, 6l was reacted with various acid chlorides and isocyanates in dichloromethane to produce the corresponding amides (6m–v) and urea derivatives (6w–x) (Table 3).

Scheme 2 Synthesis of the amide and urea derivatives (6m–x). Reagents and conditions: (a) THF, K₂CO₃, isopropyl iodide, reflux; (b) Pd(II)Cl₂(dppf), K₂CO₃, (2-aminophenyl)boronic acid, EtOH–toluene (1 : 1), reflux; (c) RCOCl (R = alkyl or aryl); (d) RNCO, CH₂Cl₂, 25 °C.

Table 3 Preparation of amide and urea derivatives (6m–x)

Cpd no.	Amide	Yield%	Cpd no.	Amide/urea	Yield%
6l		80	6s		88
6m		91	6t		85
6n		89	6u		88
6o		90	6v		84
6p		92	6w		87
6q		88	6x		91
6r		92

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2.1. Cytotoxicity studies

All the synthesized compounds 6a–x were subjected to a WST-1 cytotoxicity assay against Panc-1 (human pancreatic adenocarcinoma) and H-460 (human non-small cell lung carcinoma) cell lines. Many of the various compounds 6a–x, except 6a, 6d, 6g, 6i, 6j, 6p, 6t and 6v were found to exhibit cytotoxicity against the above cancer cell lines, hence the % of cytotoxicity at various concentrations of the compounds 6a–x was determined and the respective IC₅₀ values for the corresponding cell lines are shown in Table 4. The observed IC₅₀ values reveal that the compounds 6b, 6h, 6l, 6m, 6n, 6u, 6w and 6x are active against both cell lines (Panc-1 as well as H-460). In particular, 6o, 6p, 6u and 6v are active against the Panc-1 cell line, whereas the compounds 6r, 6s and 6w are active against the H-460 cell line. The compounds 6c, 6f, 6k and 6q showed moderate activity against both the cell lines. The remaining compounds 6a, 6d, 6g, 6i, 6j and 6t are inactive against both of the tested cell lines. Therefore, with the exception of 6r, 6s and 6w, all of the remaining compounds are highly active against human pancreatic adenocarcinoma cell lines. The IC₅₀ values further indicate that the fluorine containing compounds are more active than the other compounds. Among the active compounds, 6b, 6l, 6n and 6x were identified as lead molecules. Out of these four molecules, 6l and 6n were found to be potent against the pancreatic adenocarcinoma cell line, whereas the 6x was found to be potent against the human pancreatic adenocarcinoma cell line (Panc-1) and human non-small cell lung carcinoma cell line (H-460) (Fig. 1 and 2).

Table 4 HTS data of compounds 6a–t (in DMSO) against Panc-1 and H-460^a

Cpd no.	Panc-1 (IC50) μg mL^−1	H-460 (IC50) μg mL^−1	Cpd no.	Panc-1 (IC50) μg mL^−1	H-460 (IC50) μg mL^−1
a Gemcitabine was used for the comparison of activity.
6a	>100	>100	6m	6 ± 0.6	7 ± 0.8
6b	6.5 ± 0.7	8.5	6n	3 ± 0.4	5 ± 0.6
6c	33 ± 1.2	58	6o	27 ± 1.2	75 ± 2.5
6d	>100	>100	6p	3 ± 0.5	>100
6e	71 ± 3.2	81	6q	76 ± 2.3	100
6f	79 ± 2.9	62	6r	15 ± 1.1	5 ± 0.6
6g	>100	>100	6s	15 ± 1.3	6 ± 0.9
6h	7 ± 0.6	8	6t	>100	>100
6i	>100	>100	6u	5 ± 0.9	27 ± 2.1
6j	>100	>100	6v	3 ± 0.4	>100
6k	22 ± 2.4	43	6w	17 ± 1.2	9 ± 102
6l	3 ± 0.5	4	6x	3 ± 0.3	3 ± 0.5
Positive control (gemcitabine) 500 nm				70	89

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Fig. 1 Viability of the Panc-1 cancer cell line with compounds 6n, 6l and 6x.

Fig. 2 Viability of the H-460 cancer cell line with compounds 6n, 6l and 6x.

2.2. In vitro anti-mycobacterial activity

Compounds 6a–x were also screened for their in vitro anti-mycobacterial activity against M. tuberculosis H37Rv (MTB) using the agar dilution method recommended by the National Committee for Clinical Laboratory Standards for the determination of the MIC values of the synthesized compounds, along with the standard drugs isoniazid, ethambutol and ciprofloxacin. The comparative results are presented in Table 5. Based on the MIC values, we deduced the structure–activity relationship based on the influence of the substituent present on the benzo[d]oxazol-2(3H)-one, N-(2-(2,3-dihydro-3-isopropyl-2-oxobenzo[d]oxazol-5-yl)phenyl) benzamide and 1-(2-(2,3-dihydro-3-isopropyl-2-oxobenzo[d]oxazol-5-yl)phenyl)-3-phenylurea skeletons. The observed MIC values range from 3.125 to 50.0 μg mL⁻¹. Compounds 6h, 6i, 6l and 6p displayed considerable activity, whereas compounds 6a, 6j, 6n and 6o showed moderate activity. Among these derivatives, 6h showed promising activity, with an IC₅₀ value equal to that of ciprofloxacin. In addition, p-CF₃-(6n), p-methyl-(6o), m-methoxy-(6p), difluoro-(6t) and ortho-CF₃-(6x) compounds displayed significant activity. However, m-methoxy-(6p) was more effective than p-methyl-(6o). Similarly, ortho-CF₃ (6x) was more active compared to the p-CF₃ (6n) derivative.

Table 5 Anti-mycobacterial activity of compounds 6a–x

Cpd no.	C log P	MIC (μg mL^−1)	MIC (μM)
6a	1.127	12.5	15.44
6b	2.984	6.25	30.56
6c	1.871	25	6.76
6d	1.657	25	5.96
6e	0.901	25	7.20
6f	1.464	12.5	17.12
6g	1.158	6.25	21.60
6h	3.046	3.12	67.52
6i	1.657	6.25	11.92
6j	1.301	12.5	6.12
6k	2.021	25	34.24
6l	2.583	6.25	42.92
6m	4.662	50	7.80
6n	5.553	12.5	35.23
6o	4.817	12.5	30.91
6p	4.519	6.25	64.39
6q	5.382	25	18.05
6r	5.553	50	8.80
6s	3.742	50	7.00
6t	4.466	12.5	32.64
6u	4.056	25	16.33
6v	3.421	12.5	28.70
6w	4.673	50	8.10
6x	5.210	12.5	36.43
Isoniazid		0.05	0.66
Ethambutol		1.56	7.63
Ciprofloxacin		3.13	9.44

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3. Experimental section

3.1. General methods

The melting points reported in this work were recorded in capillary tubes on an Elchem lab melting point apparatus and uncorrected. ¹H and ¹³C NMR were recorded on a Bruker FT-NMR spectrometer at either 300 MHz or 400 MHz using 5 mm PABBO BB-1H tubes. ¹H NMR spectra were recorded using approximately 0.03 M solutions in CDCl₃ with TMS as an internal reference. ¹³C NMR spectra were recorded using approximately 0.05 M solutions in CDCl₃ at 100 MHz or 125 MHz. Chemical shift values were reported in parts per million (δ ppm) from the internal standard TMS. UV-visible spectra were recorded on a SYSTRONIC AU-2701 UV-Vis spectrophotometer. All reagents were purchased from Aldrich and used as received. Solvents were removed under reduced pressure on a rotavapour. Organic extracts were dried over anhydrous Na₂SO₄. Silica gel 60F₂₅₄ coated aluminum sheets were used for TLC and silica gel (230–400 mesh) was used for column chromatography. The visualization of spots on TLC plates was effected by UV illumination, exposure to iodine vapor and heating the plates dipped in KMnO₄ stain.

3.2. General procedure for the preparation of intermediates 6a–x

3.2.1. Synthesis of benzoxazolidin-2-one (6a–k). 2-Aminophenol was allowed to react with EImC (ethyl imidazole-1-carboxylate) in the presence of potassium carbonate in THF under reflux conditions for 12 h to produce the substituted benzoxazolidin-2-one.

3.2.2. Synthesis of 5-bromo-3-isopropylbenzo[d]oxazol-2(3H)-one (6k1). To a solution of 5-bromobenzo[d]oxazol-2(3H)-one (1 mmol) in THF (10 vol), K₂CO₃ (1.5 mmol) was added and stirred for 30 min, and then isopropyl iodide (1.5 mmol) was added. The resulting solution was stirred overnight at 80 °C. The excess THF was removed under reduced pressure and then the mixture was quenched with water and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with brine solution and dried over Na₂SO₄. Removal of the solvent followed by purification using silica gel column chromatography (eluted with ethyl acetate–hexane, 1 : 5 mixture) afforded the pure isopropyl derivative. Off white solid, 91% yield, m.p. 171–173 °C; ¹H NMR (400 MHz, CDCl3): δ 6.91 (d, J = 7.2 Hz, 2H), 7.20 (s, 1H), 8.99 (s, 1H); ¹³C NMR (100 MHz, DMSO): δ 154.5, 142.6, 132.7, 124.0, 115.1, 112.4, 111.0; IR: ν_max 3204, 2951, 1682, 1425, 1106, 891 cm⁻¹. HRMS: m/z 215.10752 [MH⁺].

3.2.3. Synthesis of 5-(2-aminophenyl)benzo[d]oxazol-2(3H)-one (6l). To a solution of 5-bromobenzo[d]oxazol-2(3H)-one (1 mmol) in an ethanol–toluene (1 : 1) mixture (10 vol), (2-aminophenyl)boronic acid (1.2 mmol) in the presence of Pd(II)Cl₂(dppf) (0.01 mmol) and potassium carbonate (1.5 mmol) in an ethanol–toluene (1 : 1) mixture (10 vol) was added under a N₂ atmosphere. The resulting solution was stirred for 3 h at 80 °C. The excess ethanol–toluene mixture was removed under reduced pressure and then the mixture was quenched with water and extracted with ethyl acetate (2 × 100 mL). The combined organic layers were washed with brine solution and dried over Na₂SO₄. Removal of the solvent followed by purification using silica gel column chromatography (ethyl acetate + hexane, 1 : 3) afforded the pure isopropyl derivative. Brown solid, 80% yield; m.p. 226–228 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.26 (m, 4H), 6.99 (d, J = 7.0 Hz, 1H), 6.92 (s, 1H), 6.79 (d, J = 7.2 Hz, 1H), 6.79 (d, J = 7.0 Hz, 1H), 4.60 (m, 1H), 1.59 (s, 6H); ¹³C NMR (100 MHz, DMSO): δ 153.8, 146.6, 142.0, 141.9, 137.7, 130.3, 129.7, 120.9, 117.6, 114.2, 113.8, 109.8, 108.1, 46.5, 19.7. IR: ν_max 3252, 2862, 1746, 1654, 1335, 1118, 982 cm⁻¹. HRMS: m/z 269.10671 [MH⁺].

3.2.4. General procedure for the preparation of compounds 6m–v. To a solution of 5-(3-aminophenyl)benzo[d]oxazol-2(3H)-one (6l) in dichloromethane was added the corresponding acid chloride under a N₂ atmosphere. The resulting mixture was stirred for 1 h at room temperature. Upon completion, the mixture was diluted with excess DCM and the organic layer was washed with NaHCO₃ solution and then dried over Na₂SO₄. Removal of the solvent followed by purification using silica gel column chromatography (ethyl acetate–hexane, 1 : 2) afforded the desired product.

3.2.5. General procedure for the preparation of compounds 6w–x. To a solution of 5-(3-aminophenyl)benzo[d]oxazol-2(3H)-one (6l) in dichloromethane was added the corresponding isocyanate under a N₂ atmosphere. The resulting mixture was stirred for 12 h at room temperature. After completion, the mixture was diluted with dichloromethane and then washed with water. The organic layer was dried over Na₂SO₄ and concentrated in vacuo. The compound was purified using column chromatography (ethyl acetate–hexane, 1 : 2).

3.2.6. Methyl 2-oxo-2,3-dihydrobenzo[d] oxazole-5-carboxylate (6a). Off white solid, 92% yield; m.p. 142–144 °C; ¹H NMR (400 MHz, DMSO): δ 11.99 (s, 1H), 7.73 (d, J = 7.2 Hz, 1H), 7.58 (s, 1H), 7.35 (d, J = 8.8 Hz, 1H), 3.89 (s, 1H); ¹³C NMR (100 MHz, DMSO): δ 164.5, 154.6, 143.6, 129.5, 125.3, 122.4, 120.4, 107.5, 56.6; IR: ν_max 3155, 1768, 1598, 1432, 1142, 807 cm⁻¹. HRMS: m/z 194.20712 [MH⁺].

3.2.7. 5-tert-Butylbenzo[d]oxazol-2(3H)-one (6b). White solid, 94% yield; m.p. 164–166 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.99 (s, 1H), 7.29 (s, 1H), 7.18 (d, J = 7.2 Hz, 1H), 7.12 (d, J = 8.0 Hz, 1H), 1.29 (s, 9H); ¹³C NMR (100 MHz, DMSO): δ 156.4, 147.9, 141.7, 129.0, 119.6, 109.4, 107.3, 34.9, 31.5; IR: ν_max 3154, 1765, 1641, 1454, 1162, 851 cm⁻¹. HRMS: m/z 189.81236 [MH⁻].

3.2.8. 5-Chlorobenzo[d]oxazol-2(3H)-one (6c). White solid, 89% yield; m.p. 186–188 °C; ¹H NMR (400 MHz, DMSO): δ 11.85 (s, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.16 (s, 1H), 7.12 (d, J = 7.2 Hz, 1H); ¹³C NMR (100 MHz, DMSO): δ 154.9, 142.3, 129.9, 129.6, 122.8, 111.0, 110.3; IR: ν_max 3052, 1773, 1618, 1479, 1150, 707 cm⁻¹. HRMS: m/z 167.76892 [MH⁻].

3.2.9. 6-Methylbenzo[d]oxazol-2(3H)-one (6d). Off white solid, 91% yield; m.p. 147–149 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.62 (s, 1H), 7.02 (s, 1H), 6.89 (d, J = 8.2 Hz, 1H), 6.85 (d, J = 8.0 Hz, 1H), 2.29 (s, 3H); ¹³C NMR (100 MHz, DMSO): δ 156.0, 144.0, 132.8, 126.8, 124.5, 110.7, 109.6, 21.4. IR: ν_max 3269, 3082, 1632, 1498, 1269, 940 cm⁻¹. HRMS: m/z 147.80412 [MH⁻].

3.2.10. 5-Nitrobenzo[d]oxazol-2(3H)-one (6e). Off white solid, 85% yield; m.p. 180–182 °C; ¹H NMR (400 MHz, DMSO): δ 7.92 (d, J = 8.4 Hz, 1H), 7.78 (s, 1H), 6.94 (d, J = 7.2 Hz, 3H); ¹³C NMR (100 MHz, DMSO): δ 168.2, 156.5, 146.2, 136.3, 120.0, 109.5, 100.2. IR: ν_max 3382, 1709, 1657, 1279, 1163 cm⁻¹. HRMS: m/z 178.88412 [MH⁻].

3.2.11. 5-Chloro-6-nitrobenzo[d]oxazol-2(3H)-one (6f). Yellow solid, 93% yield; m.p. 190–192 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.62m (s, 1H), 7.21 (s, 1H), 7.20 (d, J = 7.2 Hz, 1H); ¹³C NMR (100 MHz, DMSO): δ 154.9, 142.3, 130.0, 129.6, 122.8, 111.0, 110.3. IR: ν_max 3208, 1752, 1621, 1365, 851 cm⁻¹. HRMS: m/z 215.60584 [MH⁻].

3.2.12. Benzo[d]oxazol-2(3H)-one (6g). Off white solid, 92% yield; m.p. 134–136 °C; ¹H NMR (400 MHz, CDCl3): δ 8.59 (s, 1H), 7.21 (m, 1H), 7.19 (m, 1H), 7.15 (m, 1H); ¹³C NMR (100 MHz, DMSO): 156.2, 143.8, 129.4, 124.1, 122.6, 110.1, 110.1. IR: ν_max 3511, 1735, 1629, 1479, 1262, 953 cm⁻¹. HRMS: m/z 136.09812 [MH⁻].

3.2.13. 5-Phenylbenzo[d]oxazol-2(3H)-one (6h). Off white solid, 91% yield; m.p. 196–198 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.58 (d, J = 7.2 Hz, 2H), 7.42 (t, J = 7.0 Hz, 2H), 7.30 (t, J = 6.9 Hz, 1H), 7.15 (s, 1H), 7.10 (s, 2H); ¹³C NMR (100 MHz, DMSO): δ 153.5, 145.3, 142.6, 140.9, 137.3, 131.3, 129.2, 121.2, 117.4, 114.1, 112.8, 109.8, 107.6. IR: ν_max 3222, 1766, 1634, 1493, 1310, 1090 cm⁻¹. HRMS: m/z 212.31874 [MH⁻].

3.2.14. 5-Methylbenzo[d]oxazol-2(3H)-one (6i). White solid, 90% yield; m.p. 157–159 °C; ¹H NMR (400 MHz, CDCl₃): δ 9.45 (s, 1H), 7.10 (d, J = 7.2 Hz, 1H), 7.01 (s, 1H), 6.90 (d, J = 6.9 Hz, 1H), 2.39 (s, 3H); ¹³C NMR (100 MHz, DMSO): δ 156.6, 143.6, 128.5, 125.3, 122.4, 120.4, 107.5, 16.1. IR: ν_max 3108, 1763, 1643, 1465, 955 cm⁻¹. HRMS: m/z 148.04874 [MH⁻].

3.2.15. 6-Fluorobenzo[d]oxazol-2(3H)-one (6j). White solid, 85% yield; m.p. 145–147 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.89 (s, 1H), 7.01 (m, 2H), 6.91 (t, J = 7.2 Hz, 1H); ¹³C NMR (100 MHz, DMSO): δ 156.8, 144.4, 134.2, 122.8, 114.1, 110.7, 109.6. IR: ν_max 3061, 1771, 1625, 1471, 1258, 942 cm⁻¹. HRMS: m/z 154.21096 (MH⁻).

3.2.16. 5-Bromobenzo[d]oxazol-2(3H)-one (6k). Off white solid, 91% yield, m.p. 172–174 °C ¹H NMR (400 MHz, CDCl₃): δ 6.91 (d, J = 6.8 Hz, 2H), 7.20 (s, 1H), 8.99 (s, 1H); ¹³C NMR (100 MHz, DMSO): δ 154.5, 142.6, 132.7, 124.0, 115.1, 112.4, 111.0. IR: ν_max 3161, 1769, 1632, 1378, 1123, 824 cm⁻¹. HRMS: m/z 215.21798 [MH⁻].

3.2.17. 5-(3-Aminophenyl)-3-isopropylbenzo[d] oxazol-2(3H)-one (6l). Brown solid, 80% yield; m.p. 226–228 °C; ¹H NMR (400 MHz, CDCl₃): δ 7.26 (m, 4H), 6.99 (d, J = 7.0 Hz, 1H), 6.92 (s, 1H), 6.79 (d, J = 7.2 Hz, 1H), 6.79 (d, J = 7.0 Hz, 1H), 4.60 (m, 1H), 1.59 (s, 6H); ¹³C NMR (100 MHz, DMSO): δ 153.8, 146.6, 142.0, 141.9, 137.7, 130.3, 129.7, 120.9, 117.6, 114.2, 113.8, 109.8, 108.1, 46.5, 19.7. IR: ν_max 3252, 2862, 1746, 1654, 1335, 1118, 982 cm⁻¹. HRMS: m/z 227.21892 [MH⁻].

3.2.18. 3-Fluoro-N-(3-(3-isopropyl-2-oxo-2,3-dihydrobenzo[d]oxazol-5-yl)phenyl)benzamide (6m). White solid, 91% yield; m.p. 148–150 °C; ¹H NMR (400 MHz, DMSO): δ 10.42 (s, 1H), 8.05 (s, 1H), 7.85 (m, 3H), 7.60 (m, 3H), 7.45 (m, 4H), 7.39 (d, J = 7.0 Hz, 1H), 4.59 (m, 1H), 1.51 (d, J = 6.8 Hz, 6H); ¹³C NMR (100 MHz, DMSO): δ 164.1, 164.1, 163.1, 160.7, 152.9, 141.6, 140.3, 139.3, 137.1, 137.0, 136.4, 130.8, 130.6, 130.5, 129.1, 123.8, 122.6, 120.5, 119.4, 119.0, 118.6, 118.4, 114.5, 114.3, 109.9, 107.9,46.1. IR: ν_max 3367, 1750, 1663, 1483, 1259, 1120, 986 cm⁻¹. HRMS: m/z 391.10712 [MH⁺].

3.2.19. N-(3-(3-Isopropyl-2-oxo-2,3-dihydro-benzo[d]oxazol-5-yl)phenyl)-3-(trifluoromethyl) benzamide (6n). White solid, 89% yield; m.p. 207–209 °C; ¹H NMR (400 MHz, DMSO): δ 10.65 (s, 1H), 7.92 (s, 1H), 7.84 (m, 2H), 7.72 (m, 3H), 7.54 (s, 1H), 7.42 (m, 3H), 7.33 (d, J = 7.2 Hz, 1H), 4.55 (m, 1H), 1.50 (d, J = 6.8 Hz, 6H); ¹³C NMR (100 MHz, DMSO): δ 165.7, 152.9, 141.6, 140.5, 139.4, 136.4, 136.1, 132.6, 130.7, 130.0, 129.3, 128.5, 126.3, 126.0, 125.7, 125.1, 122.6, 122.4, 120.5, 118.5, 118.1, 110.0, 108.0, 107.9, 46.1, 19.2. IR: ν_max 3327, 1760, 1677, 1556, 1437, 1122, 984 cm⁻¹. HRMS: m/z 441.19872 [MH⁺].

3.2.20. N-(3-(3-Isopropyl-2-oxo-2,3-dihydro-benzo[d]oxazol-5-yl)phenyl)-4-methyl-benzamide (6o). Off white solid; 90% yield, m.p. 184–186 °C; ¹H NMR (400 MHz, DMSO): δ 10.29 (s, 1H), 8.08 (s, 1H), 7.93 (d, J = 7.0 Hz, 2H), 7.85 (d, J = 8.0 Hz, 1H), 7.61 (s, 1H), 7.44 (m, 6H), 4.60 (m, 1H), 2.40 (s, 3H), 1.51 (d, J = 6.8 Hz, 6H); ¹³C NMR (100 MHz, DMSO): δ 165.3, 152.9, 141.6, 140.2, 139.7, 136.5, 131.9, 130.7, 129.2, 129.1, 128.9, 127.6, 122.2, 120.5, 119.3, 118.9, 109.9, 107.9, 64.8, 46.1, 40.1, 20.9, 19.3, 15.1. IR: ν_max 3358, 2986, 1748, 1661, 1542, 1259, 983 cm⁻¹. HRMS: m/z 387.10872 [MH⁺].

3.2.21. N-(3-(3-Isopropyl-2-oxo-2,3-dihydrobenzo[d]oxazol-5-yl)phenyl)-3-methoxybenzamide (6p). Off white solid; 92% yield, m.p. 236–238 °C; ¹H NMR (400 MHz, DMSO): δ 10.35 (s, 1H), 8.08 (s, 1H), 7.85 (d, J = 7.2 Hz, 1H), 7.61 (m, 3H), 7.45 (m, 5H), 7.19 (d, J = 7.0 Hz, 1H), 4.61 (m, 1H), 3.89 (s, 3H), 1.55 (d, J = 6.8 Hz, 6H); ¹³C NMR (100 MHz, DMSO): δ 165.3, 159.1, 152.9, 141.6, 140.3, 139.6, 136.4, 136.2, 130.7, 129.5, 129.1, 122.4, 120.5, 119.8, 119.4, 119.0, 117.3, 112.9, 109.9, 107.9, 55.3, 46.1, 40.1, 19.3. IR: ν_max 3363, 1748, 1663, 1546, 1480, 1259, 1051 cm⁻¹. HRMS: m/z 403.19875 [MH⁺].

3.2.22. 3-Bromo-N-(3-(3-isopropyl-2-oxo-2,3-dihydrobenzo[d]oxazol-5-yl)phenyl)benzamide (6q). White solid; 88% yield, m.p. 214–216 °C; ¹H NMR (400 MHz, DMSO): δ 10.25 (s, 1H), 8.04 (s, 1H), 7.94 (d, J = 7.0 Hz, 2H), 7.82 (m, 1H), 7.74 (d, J = 8.2 Hz, 2H), 7.58 (s, 1H), 7.42 (m, 4H), 4.51 (m, 1H), 1.51 (d, J = 7.0 Hz, 6H); ¹³C NMR (100 MHz, DMSO): δ 166.5, 164.5, 152.9, 141.6, 140.3, 139.4, 136.4, 133.8, 131.6, 131.3, 131.2, 130.7, 129.7, 129.1, 125.3, 122.5, 120.5, 119.4, 119.0, 109.9, 107.9, 46.1, 19.3. IR: ν_max 3350, 1744, 1665, 1484, 1258, 983 cm⁻¹. HRMS: m/z 451.206542 [MH⁺].

3.2.23. N-(3-(3-Isopropyl-2-oxo-2,3-dihydro-benzo[d] oxazol-5-yl)phenyl)-4-(trifluoromethyl)benzamide (6r). Off white solid; 92% yield, m.p. 216-218 °C; ¹H NMR (400 MHz, DMSO): δ 10.35 (s, 1H), 8.41 (d, J = 7.2 Hz, 2H), 8.04 (s, 1H), 8.19 (d, J = 7.0 Hz, 2H), 7.59 (d, J = 8.2 Hz, 2H), 7.57 (m, 1H), 7.52 (s, 1H), 7.45 (m, 4H), 4.52 (m, 1H), 1.51 (d, J = 6.8 Hz, 6H); ¹³C NMR (100 MHz, DMSO): δ 164.4, 152.9, 141.6, 140.3, 139.3, 138.6, 136.4, 131.5, 131.2, 130.8, 129.2, 128.6, 125.3, 125.2, 122.7, 122.5, 120.5, 119.4, 119.0, 109.9, 107.9, 46.1, 19.3. IR: ν_max 3334, 2740, 1765, 1642, 1437, 1274, 984, 725 cm⁻¹. HRMS: m/z 441.19742 [MH⁺].

3.2.24. N-(3-(3-Isopropyl-2-oxo-2,3-dihydro-benzo[d]oxazol-5-yl)phenyl)cyclobutane carboxamide (6s). White solid; 88% yield, m.p. 224–226 °C; ¹H NMR (400 MHz, DMSO): δ 10.25 (s, 1H), 7.91 (s, 1H), 7.65 (d, J = 7.2 Hz, 1H), 7.65 (d, J = 6.8 Hz, 1H), 7.55 (s, 1H), 7.39 (m, 4H), 4.60 (m, 1H), 3.22 (m, 1H), 2.23 (m, 2H), 2.15 (m, 2H), 1.98 (m, 1H), 1.81 (m, 1H), 1.45 (d, J = 6.8 Hz, 6H); ¹³C NMR (100 MHz, DMSO): 172.9, 152.9, 141.5, 140.3, 139.8, 136.5, 130.5, 129.1, 121.7, 120.5, 118.1, 117.6, 109.3, 107.8, 46.0, 24.5, 19.2, 17.7. IR: ν_max 3353, 2945, 1750, 1676, 1435, 1218, 984 cm⁻¹. HRMS: m/z 351.11972 [MH⁺].

3.2.25. 1-(2-Fluorophenyl)-3-(3-isopropoxy-1-isopropyl-4-(3,5-dimethyl-2H-pyrrol-2-yl)-1H-pyrazol-5-yl)urea (6t). Off white solid; 85% yield, m.p. 258–260 °C; ¹H NMR (400 MHz, DMSO): δ 10.50 (s, 1H), 8.00 (s, 1H), 7.79 (m, 2H), 7.60 (s, 1H), 7.48 (m, 4H), 7.39 (d, J = 6.8 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 4.60 (m, 1H), 1.51 (d, J = 6.8 Hz, 6H); ¹³C NMR (100 MHz, DMSO): δ 164.6, 162.2, 162.1, 161.9, 160.8, 160.7, 158.3, 158.2, 152.9, 141.6, 140.5, 139.2, 136.3, 131.7, 131.6, 130.7, 129.3, 122.6, 121.6, 121.5, 120.5, 118.7, 111.9, 111.7, 109.9, 107.9, 104.9, 104.6, 104.4, 46.1, 19.3. IR: ν_max 3353, 2945, 1750, 1676, 1435, 1218, 984 cm⁻¹. HRMS: m/z 407.00981 [MH⁻].

3.2.26. 2,6-Difluoro-N-(3-(3-isopropyl-2-oxo-2,3-dihydrobenzo[d]oxazol-5-yl)phenyl) benzamide (6u). Off white solid; 88% yield, m.p. 228–230 °C; ¹H NMR (400 MHz, DMSO): δ 10.89 (s, 1H), 7.96 (s, 1H), 7.76 (m, 1H), 7.59 (m, 2H), 7.48 (m, 3H), 7.36 (d, J = 6.8 Hz, 1H), 7.26 (t, J = 7.2 Hz, 2H), 4.51 (m, 1H), 1.51 (d, J = 6.8 Hz, 6H); ¹³C NMR (100 MHz, DMSO): δ 160.0, 159.9, 158.2, 157.5, 157.5, 152.9, 141.6, 140.7, 139.0, 136.3, 132.1, 130.8, 129.5, 123.0, 120.5, 118.3, 117.9, 115.3, 115.1, 112.2, 111.9, 110.0, 108.0, 46.1, 19.2. IR: ν_max 3325, 2932, 1743, 1676, 1466, 1256, 1008 cm⁻¹. HRMS: m/z 409.10942 [MH⁺].

3.2.27. 3-Chloro-N-(3-(3-isopropyl-2-oxo-2,3-dihydrobenzo[d]oxazol-5-yl)phenyl)propanamide (6v). White solid; 84% yield, m.p. 208–210 °C; ¹H NMR (400 MHz, DMSO): δ 10.2 (s, 1H), 7.88 (s, 1H), 7.66 (d, J = 6.8 Hz, 1H), 7.56 (s, 1H), 7.42 (m, 4H), 4.59 (m, 1H), 3.85 (t, J = 7.2 Hz, 2H), 2.89 (t, J = 7.2 Hz, 2H), 1.57 (d, J = 6.8 Hz, 6H); ¹³C NMR (100 MHz, DMSO): δ 168.0, 152.9, 141.6, 140.4, 139.4, 136.4, 130.7, 129.3, 122.0, 120.5, 118.0, 117.6, 109.9, 107.9, 64.8, 46.0, 40.7, 19.2, 15.1. IR: ν_max 3318, 2970, 1733, 1696, 1437, 1236, 984, 791 cm⁻¹. HRMS: m/z 359.10872 [MH⁺].

3.2.28. 1-(2-Fluorophenyl)-3-(3-(3-isopropyl-2-oxo-2,3-dihydrobenzo[d]oxazol-5-yl)phenyl) urea (6w). White solid; 87% yield, m.p. 180–182 °C; ¹H NMR (400 MHz, DMSO): δ 9.50 (s, 1H), 8.15 (s, 1H), 7.95 (d, J = 6.8 Hz, 1H), 7.95 (d, J = 8.2 Hz, 1H), 7.65 (m, 4H), 7.52 (d, J = 7.6 Hz, 1H), 7.35 (m, 5H), 4.52 (m, 1H), 1.51 (d, J = 6.8 Hz, 6H); ¹³C NMR (100 MHz, DMSO): 152.9, 152.4, 141.5, 140.6, 139.9, 136.6, 136.2, 132.8, 130.7, 129.4, 125.8, 125.6, 125.3, 123.7, 122.6, 121.0, 120.5, 117.2, 116.8, 109.9, 107.9, 46.0, 19.2. IR: ν_max 3368, 2981, 1741, 1705, 1541, 1316, 1184, 752 cm⁻¹. HRMS: m/z 406.10742 [MH⁺].

3.2.29. 1-(3-(3-Isopropyl-2-oxo-2,3-dihydrobenzo[d]oxazol-5-yl)phenyl)-3-(2-(trifluoromethyl)phenyl)urea (6x). White solid; 91% yield, m.p. 191–193 °C; ¹H NMR (400 MHz, DMSO): δ 9.19 (s, 1H), 8.59 (s, 1H), 8.15 (t, 1H), 7.72 (s, 1H), 7.59 (s, 1H), 7.49 (m, 1H), 7.35 (m, 4H), 7.25 (t, J = 8.2 Hz, 1H), 7.15 (t, J = 7.2 Hz, 1H), 7.02 (m, 1H), 4.61 (m, 1H), 1.50 (d, J = 6.8 Hz, 6H); ¹³C NMR (100 MHz, DMSO): δ 153.2, 152.9, 152.2, 150.8, 141.5, 140.6, 139.9, 136.6, 130.7, 129.4, 127.5, 127.4, 124.4, 122.5, 122.4, 120.9, 120.6, 120.5, 117.2, 116.7, 115.0, 114.8, 109.9, 108.0, 46.0, 19.2. IR: ν_max 3370, 2930, 1741, 1705, 1542, 1357, 1184, 781 cm⁻¹. HRMS: m/z 454.09872 [MH⁻].

4. Procedure adopted for the cytotoxicity studies

All the synthesised compounds 6a–x were subjected to a WST-1 cytotoxicity assay. The cancer cells used in this study, such as Panc-1 (human pancreatic adenocarcinoma) and H460 (human non-small cell lung carcinoma), were obtained from ATCC (American Type Culture Collection). Cells were maintained in DMEM (Dulbecco’s modified Eagle’s medium) containing 10% heat inactivated fetal bovine serum and kept in a humidified 5% CO₂ incubator at 37 °C. Logarithmically growing cells were plated at a density of 5 × 10³ cells per well in a 96-well tissue culture grade micro-plate and allowed to recover overnight. The cells were challenged with varying concentrations of compounds for 48 h. Control cells received standard media containing dimethylsulfoxide vehicle at a concentration of 0.2%. After 48 h of incubation, cell toxicity was determined using the CCK-8 (Cell Counting Kit-8) reagent (Dojindo Molecular Technologies, Inc, Maryland, Japan); (WST-1 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)]-2H-tetrazolium monosodium salt assay). In accordance with the manufacturer’s instructions,⁴¹ 5 μL CCK-8 reagent was added per well and the plates were incubated for 2 h. The cytotoxicity of all the compounds was determined by measuring the absorbance on a Tecan Sapphire multi-fluorescence micro-plate reader (Tecan, Germany, GmbH) at a wavelength of 450 nm corrected to 650 nm and normalized to controls. Each independent experiment was performed three times and the results are shown in Table 4.

5. Conclusions

In summary, we developed the general procedure for the synthesis of benzo[d]oxazol-2(3H)-ones (6a–k) in good to excellent yields from 2-aminophenols by applying ethylimidazole-1-carboxylate under mild reaction conditions. Novel classes of N-(2-(3-isopropyl-2-oxo-2,3-dihydrobenzo[d]oxazol-5-yl)phenyl) benzamides (6m–v) and 1-(2-(3-isopropyl-2-oxo-2,3-dihydrobenzo[d]oxazol-5-yl)phenyl)-3-phenylurea derivatives (6w–x) were synthesized from 5-bromo-benzo[d]oxazol-2(3H)-one. The cytotoxicity of these molecules was tested against Panc-1 (human pancreatic adenocarcinoma) and H460 (human non-small cell lung carcinoma) cell lines using a WST-1 cytotoxicity assay. Many of these compounds were found to display excellent to moderate activity. Among them, 6b, 6l, 6n and 6x were identified as lead molecules. In particular, 6l and 6n were found to be potent against the pancreatic adenocarcinoma cell line, whereas 6x was found to be effective against the human non-small cell lung carcinoma cell line. Of the various molecules, 6h showed promising anti-mycobacterial activity, with an IC₅₀ value equal to that of ciprofloxacin.

Acknowledgements

The authors are thankful to VIT University Vellore, India, Birla Institute of Technology & Science, Pilani, Hyderabad Campus and Piramal Life Sciences, Mumbai for providing the facilities to carry out research work.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra07123a

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