L-Proline mediated synthesis of quinoxalines; evaluation of cytotoxic and antimicrobial activity

Abstract

A simple, greener and highly efficient method for the synthesis of functionalized quinoxalines has been developed employing L-proline as a catalyst in water. To the best of our knowledge this transformation is achieved for the first time using an organic catalyst. A small library of quinoxaline–sulphonamide conjugates have been synthesized using this protocol. The newly synthesized conjugates have been tested for their cytotoxicity and antimicrobial activity against several bacterial strains including one fungal strain. The majority of the compounds have exhibited significant cytotoxicity as well as antimicrobial activity. Compounds 5a, 5b and 5d were found to be promising with respect to both cytotoxicity and antimicrobial activity.

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Introduction

Quinoxalines represent as important class of biologically active compounds that are known to possess antibacterial,¹ anticancer,² and antiviral activities.³ This scaffold is present in several anticancer agents such as chloroquinoxaline sulfonamide (1), XK469 (2) and NCG555879-01 (3)⁴ and in some natural products like izumiphenazine C (4)⁵ (Fig. 1). It is also a part of various antibiotics such as levomycin, echinomycin and actinoleutin, which are known to inhibit the growth of Gram positive bacteria.⁶ In addition, quinoxaline derivatives find application in cavitands,⁷ efficient electroluminescent materials,⁸ organic semiconductors,⁹ dyes,¹⁰ etc.

Fig. 1 Biologically important quinoxalines.

Owing to extensive applications of quinoxalines several synthetic methods for their preparation both in solution¹¹ as well as in solid-phase¹² have been developed. Among them the condensation of 1,2-diamines with 1,2-diketone¹³ and oxidative cyclization of α-hydroxy ketones with 1,2-diamines¹⁴ under various conditions are widely used. Recent reports indicate the use of several catalysts such as MnO₂,^15a Ru/C in the presence of β-CD,^15b manganese oxide octahedral molecular sieves (OMS-2),^15c HgI₂,^15d RuCl₂(PPh₃)₃-TEMPO,^15e KF/Al₂O₃,^15f and Au-NPs^15g for onepot synthesis of quinoxaline from α-hydroxy ketones. However, they often suffer from one or more disadvantages such as long reaction time, use of hazardous organic solvents, unsatisfactory product yields and harsh reaction conditions.

On the other hand, organic catalysis is an emerging area of organic synthesis wherein small molecules are used to catalyze organic transformations. L-Proline is a versatile catalyst reported to catalyze many important organic reactions and asymmetric transformations such as aldol,¹⁶ Mannich,¹⁷ Michael,¹⁸ and Diels–Alder reaction.¹⁹ It is also excellent promoter for the copper-catalysed coupling reactions.²⁰ It's ease of handling, experimental simplicity, cost effectiveness and excellent solubility in water and organic solvents are some reasons for its extensive use in the development of synthetic methods. Further, use of water as a solvent is one of the greener way of organic synthesis. Therefore in order to overcome the disadvantages of previous methods and considering the advantages of L-proline as a catalyst and ecofriendly nature of water as a solvent we have developed a new method to synthesize quinoxalines. We herein report a green, simple and practical method for the synthesis of quinoxaline derivatives from hydroxy ketones and 1,2-diamines catalyzed by L-proline.

Results and discussion

Chemistry

In the beginning, a systematic study was carried out for the catalytic evaluation of L-proline towards the synthesis of quinoxalines. Initially a blank reaction was performed using benzoin and 1,2-diaminobenzene in water without any catalyst at room temperature and the completion of the reaction was monitored by TLC. It was observed that the reaction did not proceed even until 24 hours. Whereas the same reaction was executed in the presence of catalytic amounts of L-proline in water at room temperature and traces of the product were found (less than 5%). Later, this reaction was carried out under refluxing conditions and the desired transformation was observed furnishing the product in very good yield (Scheme 1). After obtaining the desired product, the amount of catalyst and the time required for the completion of reaction were evaluated. The reaction was performed using 5, 10, 20 and 30 mol% of the catalyst and was monitored for 12–16 hours. It was observed that 20 mol% of the catalyst loading provided maximum yield (87%) in 12 hours. While 5 and 10 mol% of the catalyst afforded 64% and 72% of the product even after refluxing the reaction for 16 hours. An additional increase of the catalyst loading to 30% did not improve the yield. On the contrary, the reaction slows down on adding more than 20 mol% of the catalyst (Table 1).

Scheme 1 L-Proline catalyzed synthesis of quinoxalines.

Table 1 Condensation of benzoin and 1,2-diaminobenzene in water at different catalyst (L-proline) concentrations

Entry	Catalyst (mol%)	Time (h)	Yield^a (%)
a Isolated yield.
1	—	24	Nil
2	5	12	62
3	5	16	64
4	10	12	71
5	10	16	72
6	20	12	87
7	20	16	87
8	30	12	82
9	30	16	86

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With the optimized conditions in hand, the reaction was performed with different set of substituents to explore the scope and generality of the present protocol. The quinoxaline derivatives were synthesized using two hydroxy ketones namely benzoin (2a) and furoin (2b) with varying 1,2-diamines. The diamines used possessed both ring activating as well as deactivating substituents and the results of these observations are summarized in Table 2. From the results it can be concluded that the electronic factors of 1,2-diamine influences the progress of the reaction. Electron donating substituents such as methyl (entry 3 and 8) provided excellent yields of the corresponding products. In presence of weak ring deactivating groups such as fluoro and dichloro (entry 2, 5, 7 and 10) the reaction progressed smoothly and the product was obtained in good yields. This trend was also observed in the absence of substituents on the diamine moiety. However, in case of ring deactivating groups such as nitro (entry 4 and 9) the reaction was slower and the yields were also comparable very lower.

Table 2 L-Proline mediated synthesis of quinoxalines from hydroxy ketone with 1,2-diamines

Entry	1,2-Diamine (1)	Hydroxyketone (2)	Product (3a–h)	Time (h)	Yield^a (%)
a Isolated yield.
1				12	87
2				12	83
3				12	88
4				14	65
5				13	85
6				12	85
7				12	82
8				12	86
9				15	67
10				13	82

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Plausible mechanism

The plausible mechanism for the formation of quinoxaline ring from 1,2-diaminobenzene and α-hydroxy ketone is shown in Fig. 2. The reaction proceeds with the formation of an iminium ion resulting from the condensation of L-proline and α-hydroxy ketone. The iminium ion gets converted to an alkene by reorganisation of proton from α-position. The alkene formed is electron rich and readily adds on oxygen from atmospheric air to form a diradical. Further rearrangement of α-hydroxyl group generates α-keto peroxide. The peroxy oxygen is then ionized with the help of lone pair of proline and leaves to form diketo imine which is hydrolysed to form 1,2-diketobenzene. The condensation of 1,2-diketobenzene and 1,2-diaminobenzene results in the formation of functionalized quinoxalines.

Fig. 2 Plausible mechanism of quinoxaline formation.

Controlled experiments

In order to determine whether the reaction is proceeding through the mechanism anticipated, a series of experiments have been performed. Firstly, the reaction was performed using other secondary amines such as piperidine, pyrrolidine and morpholine as a catalyst. The reaction was found to proceed under the similar reaction conditions. However, the reaction times were longer and the yields are lower compared to the proline catalyzed reaction. Similarly, another reaction involving heating of benzoin with proline in water without the addition of 1,2-diamine has been carried out in presence of air. Formation of benzil, the oxidized product of benzoin was observed under these conditions. Since the mechanism depicts the formation of H₂O₂, it was of interest to test its effect on the reaction. In this context, a reaction was carried out using benzoin, 1,2-diamine, proline and H₂O₂ under inert conditions (in presence of nitrogen). Surprisingly, the reaction did not proceed how ever on removal of nitrogen gas the reaction proceeded to completion. This indicates the necessity of air which serves as a source of oxygen (Table 3).

Table 3 Controlled experiments to determine mechanism of L-proline catalyzed synthesis of quinoxalines

S. no.	Catalyst (mol%)	In presence of	Time (h)	Yield^a (%)
a Isolated yield.
1	Piperidine (20 mol%)	Air	16	82
2	Pyrrolidine (20 mol%)	Air	16	80
3	Morpholine (20 mol%)	Air	16	72
4	L-Proline (20 mol%)	Air	12	87
5	L-Proline (20 mol%) + H2O2	Nitrogen	12	No reaction
6	L-Proline (20 mol%) + H2O2	Air	12	87

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Since quinoxaline structure finds many applications in pharmaceutical research, it was of interest to develop a library of quinoxaline conjugates. In continuation to our research to develop newer anticancer²¹ and antimicrobial agents,²² the protocol developed was applied to synthesize a set of quinoxaline derivatives (5a–i). The synthetic route to access these derivatives is outlined in Scheme 2. As shown, the quinoxaline derivative 3i obtained from the reaction of furoin with nitro-1,2-diamine was hydrogenated in presence of Pd/C to yield amino derivative (4).²³ This amino derivative was then reacted with substituted sulphonyl chlorides in pyridine to yield final conjugates 5a–i. These derivatives were later evaluated for their cytotoxic as well as antimicrobial activities.

Scheme 2 Reagents and condition: (i) H₂, Pd/C, MeOH, rt, 6 h (ii) aryl sulfonyl chloride, pyridine, rt, 2 h.

Biology

Antiproliferative activity. These quinoxalin–sulphonamide conjugates 5a–i were investigated for their antiproliferative activity against three human cancer cell lines namely Hela (cervical cancer), DU145 (prostate cancer) and A549 (non small cell lung cancer). Nocodazole was employed as reference standard and the IC₅₀ values are summarized in Table 4. All the compounds (5a–i) have exhibited moderate to good cytotoxicity with the IC₅₀ values ranging between 5.0 and 38.9 µM. Notably, the compounds 5a and 5b have shown significant activity on all the cell lines examined.

Table 4 Anticancer activity of the synthesized compounds against various cancer cell lines expressed in µM

IC50^a (µM)
Compound	R1	R2	HeLa^b	DU145^c	A549^d
a IC50: the amount of the compounds concentration required to reduce the cells to half of its original number.b HeLa: human cervical cancer cell line.c DU145: human prostate cancer cell line.d A549: non small cell lung cancer cell line; Noc: nocodazole.
5a	OMe	H	6.7 ± 1.0	12.3 ± 1.1	8.4 ± 0.6
5b	OMe	OMe	5.0 ± 1.1	11.1 ± 0.1	7.4 ± 1.0
5c	NO2	H	26.8 ± 1.4	38.9 ± 1.6	25.7 ± 1.2
5d	F	H	14.1 ± 1.7	26.5 ± 1.6	33.6 ± 1.5
5e	CF3	H	13.2 ± 0.4	16.7 ± 0.3	16.0 ± 0.6
5f	Cl	Cl	15.3 ± 0.3	32.6 ± 0.4	20.7 ± 0.9
5g	Br	H	27.9 ± 0.7	23.6 ± 0.6	15.8 ± 0.9
5h	H	Cl	19.2 ± 1.2	21.4 ± 0.8	16.0 ± 0.4
5i	H	NO2	18.1 ± 1.0	15.8 ± 1.1	32.1 ± 1.6
Noc	—	—	0.9 ± 0.9	1.2 ± 0.7	1.3 ± 0.7

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In order to determine the effect of substituent on the cytotoxicity the quinoxalin–sulphonamide conjugates have been synthesized with various substitutions on the phenyl ring attached to sulphonamide (Scheme 2). The compounds 5a and 5b possessing ring activating substituents such as methoxy group on phenyl ring have significantly inhibited the growth of HeLa and A549 cells with IC₅₀ of 6.7; 5.0 µM and 8.4; 7.4 µM respectively. While the same compounds have shown moderate growth inhibition on the DU145 cell line (IC₅₀: 5a = 12.3 and 5b = 11.1 µM). In comparison, 5c and 5e functionalized with ring deactivating substituents such as nitro and trifluoromethyl on phenyl ring exhibited lower cytotoxicity with IC₅₀ values of 26.8 and 13.2 µM respectively against HeLa cells. Based on these observations it is clear that substitution at C4 position on the phenyl ring plays an important role with respect to the activity. The presence of electropositive groups on the phenyl ring significantly enhances the cytotoxicity than electronegative groups such as halogen and nitro. However, there is an insignificant effect on the activity by the substitution at C₃ position of the phenyl ring.

Antimicrobial activity. The synthesized quinoxalin–sulphonamide conjugates 5a–i were also tested for their activity against various Gram positive bacteria like Bacillus subtilis MTCC121, Staphylococcus aureus MTCC 96, staphylococcus MLS-16 MTCC2940, Micrococcus luteus MTCC2470 and Gram negative bacteria like Escherichia coli MTCC739, Pseudomonas aeruginosa MTCC2453, Klebsiella planticola MTCC530 taking chloramphenicol as a positive control, and the results are summarized in Table 5. As evident from the results, most of the conjugates have shown activity against all the strains tested. Interestingly, the compounds have shown selectivity towards Gram positive bacteria. Some of the conjugates 5b, 5c and 5d have exhibited excellent antibacterial activity (MIC 10 µg mL⁻¹) comparable to and sometimes even better than the standard against the pathogens B. subtilis, S. MLS and M. luteus. However, other conjugates have displayed moderate activity against both Gram positive and Gram negative bacteria. Furthermore, all the conjugates were tested against a fungal strain, Candida albicans (MTCC3017). All the compounds have shown moderate activity against it with MIC values ranging between 20 and 80 µg mL⁻¹. Compounds 5b, 5c and 5d were found to be most effective from the series displaying MIC 20 µg mL⁻¹, however the compounds have not superseded the standard nistatin (MIC 12 µg mL⁻¹).

Table 5 Antimicrobial activity of the compounds 5a–i and values are expressed in MIC (µg mL⁻¹)^a

Compound	R1	R2	B s^a	S c^a	M l^a	S a^a	E c^b	P a^b	K p^b	C a^c
a a = gram positive bacteria; b = gram negative bacteria; c = fungal strain; B s^a = Bacillus subtilis MTCC121; S c^a = Staphylococcus MLS-16 MTCC2940; M l^a = Micrococcus luteus MTCC2470; S a^a = Staphylococcus aureus MTCC 96; E c^b = Escherichia coli MTCC739; P a^b = Pseudomonas aeruginosa MTCC2453; K p^b = Klebsiella planticola MTCC530; C a^c = Candida albicans MTCC3017.
5a	OMe	H	40	10	40	40	80	80	80	40
5b	OMe	OMe	10	10	10	25	30	40	30	20
5c	NO2	H	10	10	10	15	25	30	25	20
5d	F	H	10	16	10	25	40	40	40	20
5e	CF3	H	40	40	40	40	>100	>100	>100	80
5f	Cl	Cl	25	50	20	30	80	80	40	30
5g	Br	H	10	50	20	40	40	40	80	25
5h	H	Cl	25	25	20	25	80	40	80	30
5i	H	NO2	40	40	40	50	>100	>100	>100	80
Chloramphenicol			20	15	10	12	14	20	25	—
Nistatin			—	—	—	—	—	—	—	12

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Conclusion

In conclusion, we have developed a simple, efficient and ecofriendly method for the synthesis of quinoxalines from 1,2-diamines and α-hydroxy ketones using cost-effective and readily available catalyst L-proline. To the best of our knowledge this transformation has not been reported with an organic catalyst. The advantages of this method over previous reports include its simplicity of operation, cleaner reactions, higher yields, shorter reaction times and use of inexpensive catalyst. The mild reaction condition makes this methodology an alternative procedure to the conventional acid or base-catalyzed processes for the synthesis of quinoxalines and has practical applicability. Further, using this protocol a short library of quinoxaline–sulphonamide conjugates have been developed. The conjugates were found to be cytotoxic and effectively inhibit the growth of many bacterial strains.

Experimental

General remarks

Melting points were determined with an electrothermal melting point apparatus and are uncorrected. Infrared (IR) spectra were recorded on Perkin-Elmer model 683 or 1310 spectrometers with sodium chloride optics. ¹H NMR spectra were recorded on an Avance 300 MHz spectrometer (Bruker, Fallanden, Switzerland) and ¹³C NMR spectra were recorded on a UNITY 300 MHz (Varian, Switzerland). Chemical shifts (d) are reported in ppm, downfield from internal TMS standard. Mass spectra were recorded using a quadruple ion trap mass spectrometer (Thermo Finnign, San Jose, CA, USA) equipped with an electro spray source.

Representative experimental procedure for the synthesis quinoxalines (3a–j)

In a 50 mL round bottom flask 1,2-diamine (1 mmol) and α-hydroxy ketone (1 mmol) were taken in water (5 mL). Catalytic amount (20 mol%) of L-proline was added and the reaction mixture was refluxed for 12 hours. The progress of the reaction was monitored by TLC. After completion of the reaction, the mixture was cooled to room temperature. The precipitated solid was collected by filtration, washed with water and recrystallized using methanol.

2,3-Diphenylquinoxaline (3a)

White solid; Mp: 126–127 °C; ¹H NMR (300 MHz CDCl₃): δ 7.31–7.4 (m, 6H), 7.52–7.57 (m, 4H), 7.76–7.82 (m, 2H), 8.17–8.22 (m, 2H); ¹³C NMR (75 MHz CDCl₃): δ 127.50, 128.13, 128.40, 129.14, 129.35, 138.33, 140.38, 152.57; ESI-MS: m/z = 283 (M + H)⁺.

6-Fluoro-2,3-diphenylquinoxaline (3b)

Brown solid; Mp: 100–102 °C; ¹H NMR (500 MHz CDCl₃): δ 7.32–7.40 (m, 6H), 7.49–7.52 (m, 4H), 7.53–7.57 (m, 1H), 7.81 (dd, J = 2.74 & 9.30 Hz, 1H), 8.16–8.19 (m, 1H); ¹³C NMR (75 MHz CDCl₃): δ 112.52, 112.78, 120.20, 120.51, 128.32, 128.90, 129.08, 129.80, 129.86, 131.20, 131.34, 138.44, 138.70, 138.82, 141.88, 142.05, 152.84, 154.24, 161.19, 164.54; ESI-MS m/z = 301 (M + H)⁺.

6-Methyl-2,3-diphenylquinoxaline (3c)

Brown solid; Mp: 120–122 °C; ¹H NMR (300 MHz CDCl₃): δ 2.59 (s, 3H), 7.32 (d, J = 6.79 Hz, 6H), 7.49 (d, J = 6.79 Hz, 4H), 7.59 (dd, J = 1.51, 8.68 Hz, 1H), 7.90 (s, 1H), 8.03 (d, J = 8.49 Hz, 1H); ¹³C NMR (75 MHz CDCl₃): δ 20.78, 126.79, 127.04, 127.52, 127.58, 128.76, 131.18, 138.07, 138.45, 139.27, 140.02, 151.20, 151.95; ESI-MS: m/z = 297 (M + H)⁺.

6-Nitro-2,3-diphenylquinoxaline (3d)

Brown solid; Mp: 140–142 °C; ¹H NMR (300 MHz CDCl₃): δ 7.34–7.46 (m, 6H), 7.54–7.58 (m, 4H), 8.28 (d, J = 9.25 Hz, 1H), 8.54 (dd, J = 2.45 & 9.25 Hz, 1H), 9.09 (d, J = 2.45 Hz, 1H); ¹³C NMR (75 MHz CDCl₃): δ 123.28, 125.60, 128.43, 129.60, 129.74, 129.78, 129.86, 130.72, 137.97, 138.04, 139.94, 143.55, 147.84, 155.66, 156.29; ESI-MS: m/z = 328 (M + H)⁺.

6,7-Dichloro-2,3-diphenylquinoxaline (3e)

Brown solid; Mp: 141–143 °C; ¹H NMR (300 MHz CDCl₃): δ 7.31–7.39 (m, 6H), 7.48–7.52 (m, 4H), 8.29 (s, 2H); ¹³C NMR (75 MHz CDCl₃): δ 127.63, 128.62, 129.05, 133.44, 137.61, 139.17, 153.77; ESI-MS: m/z = 351 (M + H)⁺.

2,3-Di(furan-2-yl)quinoxaline (3f)

Brown solid; Mp: 134–136 °C; ¹H NMR (300 MHz CDCl₃): δ 6.56–6.58 (m, 2H), 6.65 (dd, J = 0.56 & 3.58 Hz, 2H), 7.63 (dd, J = 0.56 & 1.70 Hz, 2H), 7.74–7.78 (m, 2H), 8.11–8.17 (m, 2H); ¹³C NMR (75 MHz CDCl₃): δ 111.85, 112.93, 129.02, 130.33, 138.46, 140.51, 142.55, 144.15; m/z = 263 (M + H)⁺.

6-Fluoro-2,3-di(furan-2-yl)quinoxaline (3g)

Brown solid; Mp: 106–108 °C; ¹H NMR (300 MHz CDCl₃): δ 6.56–6.58 (m, 2H), 6.66 (dd, J = 3.02, 9.80 Hz, 2H), 7.49–7.56 (m, 1H), 7.62–7.65 (m, 2H), 7.75 (dd, J = 3.02 & 9.06 Hz, 1H), 8.10–8.15 (m, 1H); ¹³C NMR (75 MHz CDCl₃): δ 111.23, 111.61, 112.01, 112.27, 112.76, 119.92, 123.64, 130.33, 136.74, 140.46, 140.95, 142.25, 143.66, 148.74, 149.52; ESI-MS: m/z = 281 (M + H)⁺.

2,3-Di(furan-2-yl)-6-methylquinoxaline (3h)

Brown solid; Mp: 122–124 °C; ¹H NMR (300 MHz CDCl₃): δ 6.61 (d, J = 16.42 Hz, 4H), 7.59 (t, J = 8.87 Hz, 3H), 7.89 (s, 1H), 7.99 (d, J = 8.49 Hz, 1H); ¹³C NMR (75 MHz CDCl₃): δ 21.26, 111.31, 111.95, 112.20, 127.20, 127.87, 132.14, 138.33, 139.96, 140.45, 141.07, 141.82, 143.32, 143.45, 150.20; ESI-MS: m/z = 277 (M + H)⁺.

2,3-Di(furan-2-yl)-6-nitroquinoxaline (3i)

Brown solid; Mp: 152–154 °C; ¹H NMR (300 MHz CDCl₃): δ 6.60–6.63 (m, 2H), 6.87 (dd, J = 3.58 & 16.80 Hz, 2H), 7.67 (dd, J = 0.94 & 5.09 Hz, 2H), 8.23 (d, J = 9.253 Hz, 1H), 8.50 (dd, J = 2.45 & 9.253 Hz, 1H), 9.01 (d, J = 2.45 Hz, 1H); ¹³C NMR (75 MHz CDCl₃): δ 111.28, 111.40, 113.44, 114.28, 122.39, 123.93, 129.37, 137.92, 141.78, 142.93, 143.45, 143.95, 144.41, 146.64, 148.89, 148.98; ESI-MS: m/z = 308 (M + H)⁺.

6,7-Dichloro-2,3-di(furan-2-yl)quinoxaline (3j)

Brown solid; Mp: 135–137 °C; ¹H NMR (300 MHz CDCl₃): δ 6.58–6.59 (m, 2H), 6.73 (d, J = 3.50 Hz, 2H), 7.63 (d, J = 1.06 Hz, 2H), 8.24 (s, 2H); ¹³C NMR (75 MHz CDCl₃): δ 111.46, 113.26, 128.73, 133.72, 138.42, 142.49, 143.96, 149.50; ESI-MS: m/z = 330 (M + H)⁺.

2,3-Di(furan-2-yl)quinoxalin-6-amine (4)

6-Nitro-2,3-di(furan-2-yl)quinoxaline in methanol (20 mL) was hydrogenated in the presence of 5% Pd/C at room temperature for 3 h. After completion of the reaction, the mixture was filtered over celite. The filtrate was collected and concentrated.

General procedure for the synthesis of quinoxalin-6-amine sulphonamides

To a round bottom flask containing pyridine (5 mL) 2,3-di(furan-2-yl)quinoxalin-6-amine (4.1 mmol) and corresponding benzene sulphonyl chlorides (1.5 mmol) were added. The reaction mixture was stirred for 3 hours and the progress of the reaction was monitored by TLC. After completion of the reaction, aqueous CuSO₄·5H₂O solution was added and the product was extracted using ethyl acetate (3 × 20 mL). The combined organic layer was dried over anh. Na₂SO₄ and concentrated on rotavapor. The crude product was purified by column chromatography using hexane and ethyl acetate (7 : 3) as eluent.

N-(2,3-Di(furan-2-yl)quinoxalin-6-yl)-4-methoxybenzenesulfonamide (5a)

Yellow solid; Mp: 230–232 °C; ¹H NMR (300 MHz DMSO d₆): δ 3.78 (s, 3H), 6.56 (s, 2H), 6.61 (d, J = 2.64, 1H), 6.88 (dd, J = 2.07 & 8.87 Hz, 2H), 7.43 (s, 1H), 7.59–7.65 (m, 3H), 7.83–7.87 (m, 3H), 7.93 (dd, J = 2.64 & 8.87 Hz, 1H), 10.43 (brs, 1H); ¹³C NMR (75 MHz DMSO d₆): δ 54.11, 110.31, 110.48, 111.28, 111.52, 112.53, 112.80, 113.57, 122.62, 127.42, 127.69, 128.26, 129.43, 129.69, 135.74, 138.53, 139.47, 142.19, 142.45, 148.94, 149.20; ESI-MS: m/z 448 (M + H)⁺: HRMS (ESI) m/z for C₂₃H₁₈O₅N₃S calculated m/z: 448.09617, found m/z: 448.09330.

N-(2,3-Di(furan-2-yl)quinoxalin-6-yl)-3,4-dimethoxybenzenesulfonamide (5b)

Yellow solid; Mp: 245–247 °C; ¹H NMR (300 MHz DMSO d₆): δ 3.84 (s, 6H), 6.57–6.61 (m, 4H), 6.85–6.88 (m, 1H), 7.40 (s, 1H), 7.49–7.51 (m, 1H), 7.60–7.65 (m, 3H), 7.84 (s, 1H), 7.89–7.92 (m, 1H), 10.57 (brs, 1H); ¹³C NMR (75 MHz DMSO d₆): δ 55.06, 108.60, 109.56, 110.95, 111.47, 112.03, 114.41, 120.06, 123.46, 128.70, 130.09, 136.41, 139.26, 140.10, 142.92, 143.19, 147.90, 151.56; IR (KBr) (ν_max/cm⁻¹): ν = 3446, 2934, 1620, 1508, 1156, 1138, 1018, 913 cm⁻¹; ESI-MS: m/z 478 (M + H)⁺: HRMS (ESI) m/z for C₂₄H₂₀O₆N₃S calculated m/z: 478.10673, found m/z: 478.10325.

N-(2,3-Di(furan-2-yl)quinoxalin-6-yl)-4-nitrobenzenesulfonamide (5c)

Yellow solid; Mp: 260–262 °C; ¹H NMR (300 MHz DMSO d₆): δ 6.56–6.63 (m, 3H), 7.61–7.65 (m, 3H), 7.70–7.78 (m, 1H), 7.81–7.86 (m, 2H), 7.89–7.94 (m, 1H), 8.19–8.23 (m, 1H), 8.34–8.39 (m, 1H), 8.70–8.72 (m, 1H), 11.10 (brs, 1H); ¹³C NMR (75 MHz DMSO d₆): δ 110.77, 111.36, 111.92, 114.91, 120.71, 123.19, 126.14, 128.72, 129.63, 131.40, 137.85, 139.60, 140.20, 140.56, 142.77, 143.05, 143.30, 146.79, 149.20; IR (KBr) (ν_max/cm⁻¹): ν = 3246, 3095, 1619, 1571, 1527, 1353, 1165, 1123, 912 cm⁻¹; ESI-MS: m/z 463 (M + H)⁺: HRMS (ESI) m/z for C₂₂H₁₅O₆N₄S calculated m/z: 463.07068, found m/z: 463.06729.

N-(2,3-Di(furan-2-yl)quinoxalin-6-yl)-4-fluorobenzenesulfonamide (5d)

Yellow solid; Mp: 235–237 °C; ¹H NMR (300 MHz DMSO d₆): δ 6.58–6.61 (m, 4H), 7.10–7.16 (m, 2H), 7.58–7.63 (m, 3H), 7.86–7.94 (m, 4H), 10.73 (s, 1H); ¹³C NMR (75 MHz DMSO d₆): δ 110.43, 110.52, 110.90, 111.52, 113.57, 114.74, 115.03, 122.69, 128.24, 128.34, 134.13, 135.74, 138.26, 139.35, 139.45, 141.15, 142.50, 144.81, 149.05; IR (KBr) (ν_max/cm⁻¹): ν = 3251, 2924, 1620, 1491, 1325, 1229, 1088, 1018, 912 cm⁻¹; ESI-MS: m/z 436 (M + H)⁺: HRMS (ESI) m/z for C₂₂H₁₅O₄N₃FS calculated m/z: 436.07618, found m/z: 436.07273.

3,4-Dichloro-N-(2,3-di(furan-2-yl)quinoxalin-6-yl)benzenesulfonamide (5e)

Yellow solid; Mp: 236–238 °C; ¹H NMR (300 MHz DMSO d₆): δ 6.63 (d, J = 9.90 Hz, 4H), 7.65 (t, J = 7.33 Hz, 4H), 7.78 (t, J = 7.33 Hz, 2H), 7.96 (d, J = 8.80 Hz, 1H), 8.01 (s, 1H), 11.01 (brs, 1H);¹³C NMR (75 MHz DMSO d₆): δ 110.42, 110.50, 110.98, 111.58, 114.24, 122.78, 124.97, 127.13, 128.43, 129.89, 131.46, 135.51, 135.96, 137.79, 138.07, 139.33, 139.67, 141.24, 142.53, 142.84, 149.00, 149.09; IR (KBr) (ν_max/cm⁻¹): ν = 3247, 2924, 1619, 1491, 1327, 1157, 1068, 1022, 919 cm⁻¹; ESI-MS: m/z 486 (M + H)⁺: HRMS (ESI) m/z for C₂₂H₁₄O₄N₃Cl₂S calculated m/z: 486.00766, found m/z: 486.00459.

N-(2,3-Di(furan-2-yl)quinoxalin-6-yl)-4-(trifluoromethyl)benzenesulfonamide (5f)

Yellow solid; Mp: 255–257 °C; ¹H NMR (300 MHz DMSO d₆): δ 6.56–6.75 (m, 4H), 7.60–7.81 (m, 5H), 7.85–8.01 (m, 2H), 8.05–8.25 (m, 2H), 10.86 (brs, 1H); ¹³C NMR (75 MHz DMSO d₆): δ 28.52, 111.01, 111.09, 111.70, 112.24, 115.12, 122.91, 122.95, 123.51, 128.51, 128.55, 128.98, 129.16, 129.41, 136.64, 138.43, 139.84, 140.02, 140.36, 141.93, 143.05, 143.31, 149.56; IR (KBr) (ν_max/cm⁻¹): ν = 3247, 2924, 1619, 1535, 1488, 1347, 1250, 1160, 919 cm⁻¹; ESI-MS: m/z 486 (M + H)⁺: HRMS (ESI) m/z for C₂₃H₁₅O₄N₃F₃S calculated m/z: 486.07299, found m/z: 486.06939.

4-Bromo-N-(2,3-di(furan-2-yl)quinoxalin-6-yl)benzenesulfonamide (5g)

Yellow solid; Mp: 220–222 °C; ¹H NMR (300 MHz DMSO d₆): δ 6.56–6.62 (m, 4H), 7.57–7.66 (m, 5H), 7.76–7.84 (m, 3H), 7.90–7.94 (m, 1H), 10.81 (brs, 1H); ¹³C NMR (75 MHz DMSO d₆): δ 111.68, 110.75, 111.24, 111.82, 114.20, 123.04, 126.29, 127.36, 128.57, 131.02, 136.15, 137.41, 138.40, 139.68, 139.85, 141.50, 142.70, 142.98, 149.27, 149.34; IR (KBr) (ν_max/cm⁻¹): ν = 3086, 2853, 1618, 1571, 1527, 1489, 1343, 1218, 1087, 914 cm⁻¹; ESI-MS: m/z 495 (M + H)⁺: HRMS (ESI) m/z for C₂₂H₁₅O₄N₃BrS calculated m/z: 495.99612, found m/z: 495.99265.

3-Chloro-N-(2,3-di(furan-2-yl)quinoxalin-6-yl)benzenesulfonamide (5h)

Yellow solid; Mp 216–218 °C; ¹H NMR (300 MHz DMSO d₆): δ 6.57–6.63 (m, 4H), 7.42–7.54 (m, 2H), 7.63 (d, J = 7.93, 3H), 7.78–7.83 (m, 2H), 7.89–7.95 (m, 2H), 10.91 (s, 1H); ¹³C NMR (75 MHz DMSO d₆): δ 110.53, 110.61, 111.09, 111.69, 114.13, 122.86, 123.82, 125.25, 128.44, 129.36, 131.58, 133.24, 135.99, 138.12, 139.47, 139.71, 139.95, 141.34, 142.59, 142.90, 149.09, 149.17; IR (KBr) (ν_max/cm⁻¹): ν = 3241, 2977, 1735, 1618, 1420, 1324, 1250, 1019, 1162, 911 cm⁻¹; ESI-MS: m/z 452 (M + H)⁺: HRMS (ESI) m/z for C₂₂H₁₅O₄N₃ClS calculated m/z: 452.04663, found m/z: 452.04381.

N-(2,3-Di(furan-2-yl)quinoxalin-6-yl)-3-nitrobenzenesulfonamide (5i)

Yellow solid; Mp 260–262 °C; ¹H NMR (300 MHz DMSO d₆): δ 6.57–6.61 (m, 4H), 7.61–7.72 (m, 4H), 7.89 (s, 1H), 7.97 (d, J = 9.06 Hz, 1H), 8.23 (d, J = 7.36 Hz, 1H), 8.37 (d, J = 8.12 Hz, 1H), 8.78 (s, 1H), 11.01 (brs, 1H); ¹³C NMR (75 MHz DMSO d₆): δ 110.16, 110.24, 110.68, 111.29, 113.92, 119.84, 122.47, 125.83, 128.22, 129.50, 130.84, 135.54, 137.30, 138.83, 139.30, 140.83, 142.48, 142.82, 146.20, 148.58, 148.67; IR (KBr) (ν_max/cm⁻¹): ν = 3249, 1618, 1569, 1526, 1468, 1352, 1228, 1164, 1081, 911 cm⁻¹; ESI-MS: 463 [M + H]⁺: HR-MS (ESI) m/z for C₂₂H₁₅O₆N₄S calculated m/z: 463.07068, found m/z: 463.06717.

Materials and methods

Cell culture and reagents

All the cell lines used in this study were obtained from the American Type Culture Collection (ATCC). DU145 (human prostate carcinoma epithelial) cells have cultured in Eagle's minimal essential medium (MEM) containing nonessential amino acids, 1 mM sodium pyruvate, and 10% FBS. HeLa (human epithelial cervical cancer) and A549 (human lung carcinoma epithelial) were grown in Dulbecco's modified Eagle's medium (DMEM) containing nonessential amino acids and 10% FBS. All the cells maintained under humidified atmosphere of 5% CO₂ at 37 °C. Cells were trypsinized when sub confluent from T75 flasks per 90 mm dishes and seeded on to 96 well test plates at a concentration of 1 × 10⁴ cells per mL in complete medium, treated with compounds at desired concentrations and harvested as required.²⁴

MTT assay

Cell proliferation and viability was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The pale yellow colored tetrazolium salt (MTT) reduces to a dark blue water-insoluble formazan by metabolically active cells and which is measured quantitatively after soluble in DMSO. The absorbance of the soluble formazan is directly proportional to the number of viable cells. Cells were seeded at a density of 1 × 10⁴ cells in 200 µL of medium per well of 96-well plate. The 96-well microtiter plates were incubated for 24 h prior to addition of the experimental compounds. Cells were treated with vehicle alone (0.4% DMSO) or compounds (drugs were dissolved in DMSO previously) at different concentrations (1, 10 and 25 µM) of test compounds for 48 hours. The assay was completed with the addition of MTT (5%, 10 µL) and incubated for 60 min at 37 °C. The supernatant was aspirated and plates were air dried and the MTT-formazan crystals dissolved in 100 µL of DMSO. The optical density (O.D.) was measured at 560 nm using TECAN multimode reader. The growth percentage of each treated well of 96 well plate have been calculated based on test wells relative to control wells. The cell growth inhibition was calculated by generating dose response curves as a plot of the percentage of surviving cells versus drug concentration. Antiproliferative activity of the cancer cells to the test compounds was expressed in terms of IC₅₀ value, which defines as a concentration of compound that produced 50% absorbance reduction relative to control.²⁵

Antimicrobial susceptibility testing

All the synthesized compounds were dissolved in pure DMSO of 2 mg mL⁻¹. Empty sterilized disks of 6 mm were impregnated with test compounds in the range from 1 to 80 mg per disk and placed in triplicate in the medium inoculated with fresh bacterial strain (1 × 10² to 10⁷ cfu mL⁻¹) on the freshly prepared sterile Mueller Hilton agar plates.²⁶ Subsequently these plates were incubated at 35 °C for 24 h for zone of inhibition, if any, around the disks. The lowest concentration (higher dilution) of the test compound required to arrest the growth of tested strains was defined as minimum inhibitory concentrations (MICs). 20 µL of DMSO was loaded on sterile disc and placed on the culture. After incubation for 24 h, observed no zone of inhibition around the disc, indicated that DMSO has no inhibitory effect on the tested strains and which was used as a negative control. Chloramphenicol was employed as the reference standard. The method followed for antifungal bioassay was similar to that followed for antibacterial assay where the medium was potato dextrose agar 39 g L⁻¹. The equivalent amount of solvent (DMSO) did not exhibit any activity in the assay. The treated compounds and the controls were kept in an incubator at 28 ± 2 °C for 48 h. All the compounds were tested for minimum inhibitory concentration (MIC). Nistatin was employed as positive control.

Acknowledgements

The authors (KSB, SMAH, AB and SF) are thankful to CSIR and UGC, New Delhi for financial support and XIIth Five Year plan project “Affordable Cancer Therapeutics (ACT)”

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra08615e

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