Introduction

Inflammation is the reaction process of living tissues to stimuli induced by inflammatory agents, such as physical injuries, heat, microbial infections, and noxious chemical irritants, where the response of cells toward inflammation will lead to appear certain pathological indicators, such as redness, heat, swelling, and pain, sometimes with impaired physiological functions (Ruedi et al. 2008; Artis and Spits 2015). The occurrence of inflammation can be associated with a number of disorders and is prominently related to painful conditions resulting from tissue injury, cell death, cancer, and ischaemia (Lucas et al. 2006; Rock et al. 2011; Waisman et al. 2015).

Important inflammatory mediators, such as prostaglandins, prostacyclin, and thromboxane, are produced through the prostanoid biosynthetic pathway from arachidonic acid using cyclooxygenase (COX) as a key enzyme (Kurumbail et al. 1996). The COX enzyme exists in two different forms, cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2). COX-2 is associated with inflammation and the resulting pain. Thus, to inhibit the activity of the COX-2 enzyme, many non-steroidal anti-inflammatory drugs (NSAIDs) have been developed (Hawkey 1999). However, the use of such drugs results in many side effects such as, ulcerogenic, cardiovascular effects, etc. (Lanas and Chan 2017; Trelle et al. 2011). In view of occurrence of these adverse side effects, we have been interested in the development of novel non-steroidal anti-inflammatory compounds with minimum side effects.

1,3‐Oxazine and its derivatives constitute an important class of heterocyclic compounds, which occupy a unique place in medicinal chemistry because of their wide spectrum of biological properties, such as antimicrobial (Mathew et al. 2010), antitubercular (Mathew et al. 2010), anti-HIV (Cocuzza et al. 2001), antimalarial (Ren et al. 2001), anticoagulant (Riveiro et al. 2010), anticonvulsant (Kurz 2005), antitubular (Zhang et al. 2003), antithrombotic (Hsieh et al. 2005), and antitumour activities (Fig. 1a–c) (Benameur et al. 1996). They also play an important role as a monomer in the polymer industry (Wang and Ishida 2000). Further, they act as important precursors in various organic transformations (Lee et al. 2012; Kajjout et al. 2013) and in the total synthesis of natural products (Fig. 2a, b) (Ji-Yeon et al. 2011; Park et al. 2015; Jin et al. 2014). However, only a few relevant studies on the anti-inflammatory activity of 1,3-oxazine derivatives have been reported (Yan-Fei et al. 2016; Puwen et al. 2002). Moreover, the potential of naphtho[1,2-e][1,3]oxazine derivatives against the inflammatory enzyme COX-2 has remained unexplored.

Fig. 1
figure 1

1,3-Oxazines-fused bioactive heterocycles

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Fig. 2
figure 2

1,3-Oxazines as precursors for the synthesis of natural products for their preparation

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One of the factors for the lack of reports on the anti-inflammatory activity of naphtho[1,2-e][1,3]oxazines may be due to a few synthetic methods available in the literature (Harish et al. 2012; Istvan et al. 2004; Daqing et al. 2010; Maria et al. 2016). Thus, we undertook the challenge of developing new and convenient methods for synthesising naphtho[1,2-e][1,3]oxazines and studying their potential as anti-inflammatory agents. Our research group has been interested in the synthesis of bioactive heterocycles involving cascade/domino reactions, either through multicomponent reactions (MRCs) or one-pot, sequential convergent reactions (Okram et al. 2008; Prasanta et al. 2013; Thokchom et al. 2018; Leimajam et al. 2018). Thus, we report herewith an efficient and diastereoselective synthesis of a series of trans-1,3-diaryl-1H-naphtho[1,2-e][1,3]oxazines 4 based on an MCR involving aliphatic amines, aromatic aldehydes, and β-naphthol using a heterogeneous catalyst, SiO2.HClO4, with ethanol as the solvent. The potential activity of these compounds against inflammation with supporting information from in silico studies is presented in this report as a short commentary.

Experimental

Chemistry

Materials and reagent

All reagents and solvents were purchased from commercial sources, such as Merck and Aldrich, and were used as received. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on FT-NMR spectrometer using CDCl3 and d6-DMSO. Chemical shifts δ are measured in parts per million (ppm) and are relative to tetramethylsilane as the internal reference. Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, br = broad) and coupling constants (J) in Hertz. The FT-IR spectra were recorded on a Perkin-Elmer FT-IR spectrometer (KBr). Melting points were determined on a ‘Veego' capillary melting point apparatus and are uncorrected. Silica gel 60–120 was used for column separations. Chemical yields refer to pure isolated substances. Mass, COSY, and NOESY spectra were also taken in a Bruker SFO1 400 MHz.

Preparation of the catalyst, SiO2.HClO4

We started with the preparation of the heterogeneous catalyst, SiO2.HClO4. In this procedure, to a suspension of SiO2 (230–400 mesh, 11.6 g) in diethylether (40 mL), 70% aqueous perchloric acid (1.3 g, 9.4 mmol) was added. Then, the mixture was kept for overnight and the residue was heated at 100 °C for 72 h. The residue was washed several times with diethyl ether.

Preparation of 1,3-oxazine, 4a

A mixture of benzaldehyde (2 mmol), ethylamine (1 mmol), and β-naphthol (1 mmol) was refluxed in ethanol using 4 mol% SiO2.HClO4. The reaction was monitored by thin layer chromatography. After refluxing for 18 h, ethanol was evaporated using rotary vacuum evaporator. Water (50 mL) was added to the residue followed by ethyl acetate (50 mL). The ethyl acetate extract was treated with anhydrous sodium acetate and the product was isolated from column chromatography.

Characterisation data of compounds 4a–4n

2-Ethyl-1,3-diphenyl-2,3-dihydro-1H-naphtho[1,2-e][1,3] oxazine, 4a

White crystal, melting point 143–145 °C. 1HNMR (400 MHz, CDCl3, δ ppm): 1.00 (3H, t, J = 8), 1.4 (2H, q, J = 9.6), 5.3 (1H, s), 5.7 (1H, s), 7–7.16 (10H, m), 7.23–7.25 (2H, d, J = 5.6), 7.37–7.35 (2H, d, J= 9.6), 7.58–7.56 (1H, d, J = 9.6), 7.62–7.60 (1H, d, J = 8.4); 13CNMR (100 MHz, CDCl3, δ ppm): 14.42, 21, 39, 58, 87, 112, 119, 124, 126, 127, 129, 130, 134, 136, 138, 139, 141, 153; IR (KBr) (ν max, cm−1): 3062, 2946, 2848, 1601, 1507, 1452, 1392, 1333, 1233, 1125, 945, 813, 700. ESI-MS m/z: 366.18, calcd. for C26H23NO (M + H)+: 365.18; CHN analysis (%): cal. C, 82.95; H, 8.41; N, 4.03; found C, 82.79; H, 8.44; N, 4.01.

2-Ethyl-1,3-bis(4-methoxyphenyl)-2,3-dihydro-1H-naphtho [1,2-e][1,3]oxazine, 4b

Yellow crystal, melting point 159–161 °C. 1HNMR (400 MHz, CDCl3, δ ppm): 1.00 (3H, t, J = 8), 1.4 (2H, q, J = 9.6), 3.7 (3H, s), 3.8 (3H, s), 5.4 (1H, s), 5.7 (1H, s), 7–7.16 (8H, m), 7.23–7.25 (2H, d, J = 5.6), 7.37–7.35 (2H, d, J = 9.6), 7.58–7.56 (1H, d, J = 9.6), 7.62–7.60 (1H, d, J = 8.4); 13CNMR (100 MHz, CDCl3, δ ppm): 14.42, 21, 39.5, 59.2, 87, 113, 118, 122, 126, 127, 129, 130, 133, 136, 137, 138, 140, 152; IR (KBr) (ν max, cm−1): 3062, 2946, 2848, 1601, 1507, 1452, 1392, 1333, 1233, 1125, 945, 813, 700. ESI-MS m/z: 426.20, calcd. for C28H27NO3 (M + H)+: 425.53; CHN analysis (%): cal. C, 79.03; H, 6.40; N, 3.29; found C, 78.01; H, 6.47; N, 3.26.

1,3-Bis(5-bromothiophen-2-yl)-2-ethyl-2,3-dihydro-1H-naphtho[1,2-e][1,3]oxazine, 4c

Brown crystal, melting point 137–138 °C. 1H NMR (400 MHz, CDCl3, δ ppm): 1.3 (3H, t, J = 4), 2.4 (2H, q, J= 8), 5.6 (1H, s), 6.1 (1H, s), 6.63–6.64 (1H, d, J = 4), 6.77–6.79 (1H, d, J = 8), 7.01–7.0 (1H, d, J = 8), 7.21–7.69 (2H, m), 7.71–7.81 (3H, m). 13C NMR (100 MHz, CDCl3, δ ppm): 18.5, 43.9, 57.9, 110, 119, 123.3, 125.4, 126.5, 127.6, 127.8, 128, 131.8, 135.8, 138.8, 142.9, 147.5. IR (KBr) (ν max, cm−1): 3789, 2927, 1899, 1379, 1299, 1195, 1026, 990, 650. ESI-MS m/z: 380.20, calcd. for C22H17Br2NOS2 (M + H)+: 532.19; CHN analysis (%): cal. C, 71.90; H, 4.87; N, 3.22; found; C, 69.97; H, 4.67; N, 3.17.

2-Ethyl-1,3-di-p-tolyl-2,3-dihydro-1H-naphtho[1,2-e][1,3] oxazine, 4d

White semi crystal, melting point 137–139 °C. 1H NMR (400 MHz, CDCl3, δ ppm): 1.14–1.16 (3H, t, J= 8), 2.3 (6H, s), 2.6–2.7 (2H, q, 4), 5.5 (1H, s), 5.7 (1H, s), 7.01–7.16 (8H, m), 7.08–7.09 (1H, d, J = 7), 7.78–7.76 (2H, d, J = 8), 7.4–7.3 (2H, d, J = 7), 7.7–7.8 (1H, d, J = 8). 13C NMR (100 MHz, CDCl3, δ ppm): 14.5, 21.2, 21.3, 39.5, 58.0, 86.0, 112.8, 119.1, 123.2, 123.3, 126.4, 128.6, 129.1, 129.5, 135.5, 136.7, 137.3, 140.5, 153.0; IR (KBr) (ν max, cm−1): 3059, 2943, 2849, 1600, 1505, 1456, 1392, 1323, 1231, 1125, 945, 812, 704. ESI-MS m/z: 394.21, calcd. for C28H27NO (M + H)+: 393.21; CHN analysis (%): cal. C, 85.46; H, 6.92; N, 3.56; found C, 85.41; H, 6.89; N, 3.45.

2-Ethyl-1,3-bis(4-fluorophenyl)-2,3-dihydro-1H-naphtho [1,2-e][1,3]oxazine, 4e

White crystalline, melting point 119–121 °C. 1H NMR (400 MHz, CDCl3, δ ppm) 1.01 (3H, t, J = 8), 1.3 (2H, q, J = 9.6), 5.4 (1H, s), 5.6 (1H, s), 7.01–7.16 (8H, m), 7.22, 7.26 (2H, d, J = 5.6), 7.38–7.34 (2H, d, J = 9.6), 7.59–7.55 (1H, d, J = 9.6), 7.63–7.59 (1H, d, J = 8.4); 13CNMR (100 MHz, CDCl3, δ ppm): 14.4, 21, 39.1, 58, 86, 113, 115, 124, 126, 127.2, 129, 131, 133.1, 136, 137.5, 139, 141, 153; 162 (d, JC-F = 43.2 Hz), 163 (d, JC-F = 43.3 Hz); IR (KBr) (ν max, cm−1): 3062, 2946, 2848, 1601, 1507, 1452, 1392, 1333, 1233, 1125, 945, 813, 700. ESI-MS m/z: 399, calcd. for C26H19F2NO: (M + H)+: 402.12; CHN analysis (%): cal. C, 77.79; H, 5.27; F, 9.46; N, 3.49; found; C, 77.75; H, 5.23; N, 3.51.

2-Ethyl-1,3-di-o-tolyl-2,3-dihydro-1H-naphtho[1,2-e][1,3] oxazine, 4f

White crystal, melting point 133–135 °C. 1H NMR (400 MHz, CDCl3, δ ppm): 1.14–1.17 (3H, t, J = 8), 2.3 (6H, s), 2.6–2.7 (2H, q, 4), 5.6 (1H, s), 5.7 (1H, s), 7.08–7.45 (10H, m), 7.78–7.76 (2H, d, J= 8), 7.88–7.87 (2H, d, J = 4). 13C NMR (100 MHz, CDCl3, δ ppm) 14.5, 21.2, 21.3, 39.5, 58.0, 86.0, 112.8, 119.1, 123.2, 123.3, 126.4, 128.6, 129.1, 129.5, 135.5, 136.7, 137.3, 140.5, 153.0. IR (KBr) (ν max, cm−1): 3058, 2943, 2849, 1601, 1505, 1456, 1392, 1323, 1231, 1125, 945, 810, 701. ESI-MS m/z: 394.21, calcd. for C28H27NO (M + H)+: 393.21; CHN analysis (%): cal. C, 85.46; H, 6.92; N, 3.56; found; C, 84.89; H, 6.78; N, 3.42.

2-Ethyl-1,3-di(thiophen-2-yl)-2,3-dihydro-1H-naphtho[1,2-e][1,3] oxazine, 4g

Yellow crystal solid. Melting point 125–126 °C. 1H NMR (400 MHz, CDCl3, δ ppm): 1.3 (3H, t, J = 4), 2.4 (2H, q, J = 8), 5.6 (1H, s), 6.1 (1H, s), 6.61–6.62 (1H, d, J= 4), 6.77–6.79 (1H, d, J= 8), 7.02–7.04 (1H, d, J = 8), 7.21–7.69 (6H, m), 7.71–7.81 (3H, m); 13C NMR (100 MHz, CDCl3, δ ppm): 18.5, 43.9, 57.9, 110, 119, 123, 125, 126.5, 127.2, 127.6, 128.2, 131.8, 135.8, 138.8, 142.9, 147.5; IR (KBr) (ν max, cm−1): 3788, 2924, 1897, 1375, 1289, 1196, 1029, 996, 650. ESI-MS m/z: 406.13, calcd. for C24H23NOS2 (M + H)+: 405.12; CHN analysis (%): cal. C, 69.99; H, 5.07; N, 3.71; found; C, 68.91; H, 5.15; N, 4.10.

1,3-Bis(4-chlorophenyl)-2-ethyl-2,3-dihydro-1H-naphtho [1,2-e][1,3]oxazine, 4h

White crystal, melting point 117–118 °C. 1HNMR (400 MHz, CDCl3, δ ppm): 1.00 (3H, t, J = 8), 1.4 (2H, q, J = 9.6), 5.3 (1H, s), 5.7 (1H, s), 7–7.16 (8H, m), 7.23–7.25 (2H, d, J = 5.6), 7.37–7.35 (2H, d, J = 9.6), 7.58–7.56 (1H, d, J= 9.6), 7.62–7.60 (1H, d, J = 8.4); 13CNMR (100 MHz, CDCl3, δ ppm): 14.42, 21, 39, 58, 87, 112, 119, 124, 126, 127, 129, 130, 134, 136, 138, 139, 141, 153; IR (KBr) (ν max, cm−1): 3062, 2946, 2848, 1601, 1507, 1452, 1392, 1333, 1233, 1125, 945, 813, 700. ESI-MS m/z: 435.10, calcd. for C26H21Cl2NO (M + H)+: 434.36; CHN analysis (%): cal. C, 49.36; H, 3.20; N, 2.62; found C, 50.01; H, 2.98; N, 2.69.

2-Ethyl-1,3-bis(4-nitrophenyl)-2,3-dihydro-1H-naphtho[1,2-e][1,3]oxazine, 4i

White powder, melting point 153–155 °C. 1H NMR (400 MHz, CDCl3, δ ppm): 1.1 (3H, t, J = 8), 1.4 (2H, q, J = 9.6), 5.3 (1H, s), 5.7 (1H, s), 7–7.16 (8H, m), 7.23–7.25, (2H, d, J= 5.6), 7.37–7.35 (2H, d, J = 9.6), 7.58–7.56 (1H, d, J = 9.6), 7.62–7.60 (1H, d, J = 8.4); 13C NMR (100 MHz, CDCl3, δ ppm): 14.5, 21, 39, 57, 87, 112, 117, 122, 124, 126, 128, 130, 132, 134, 137, 139, 141, 153; IR (KBr) (ν max, cm−1): 3052, 2936, 2841, 1601, 1507, 1452, 1392, 1333, 1233, 1125, 945, 813, 699. ESI-MS m/z: 456.15, calcd. for C26H21N3O5 (M + H)+: 455.15; CHN analysis (%): cal. C, 68.56; H, 4.65; N, 9.23; found; C, 67.67; H, 4.78; N, 8.98.

1,3-Diphenyl-2-propyl-2,3-dihydro-1H-naphtho[1,2-e][1, 3]oxazine, 4j

White-brown amorphous solid, melting point 135–137 °C. 1H NMR (400 MHz, CDCl3, δ ppm): 0.89 (3H, t, J = 8), 1.6 (2H, t, J = 9.6), 2.5 (2H, m, J = 8), 5.3 (1H, s), 5.7 (1H, s), 7–7.16 (3H, m), 7.18–7.10 (3H, m), 7.23–7.25 (2H, d, J = 5.6), 7.37–7.35 (2H, d, J= 9.6), 7.58–7.56 (1H, d, J = 9.6), 7.57–7.60 (4H, m), 7.62–7.60 (1H, d, J = 8.4); 13C NMR(100 MHz, CDCl3, δ ppm): 14.42, 21, 39, 58, 87, 112, 119, 122, 123, 124, 125, 126, 127, 128, 129, 138, 139, 141, 153; IR (KBr) (ν max, cm−1): 3062, 2946, 2848, 1601, 1507, 1452, 1392, 1333, 1233, 1125, 945, 813, 700. ESI-MS m/z: 380.20, calcd. for C27H25NO (M + H)+: 379.19; CHN analysis (%): cal. C, 84.25; H, 5.72; N, 4.68; found; C, 83.98; H, 5.56; N, 4.53.

1,3-Bis(4-chlorophenyl)-2-propyl-2,3-dihydro-1H-naphtho [1,2-e][1,3]oxazine, 4k

White crystal solid, melting point 162–164 °C. 1H NMR (400 MHz, CDCl3, δ ppm): 0.89 (3H, t, J = 8), 2.5 (2H, t, J = 9.4), 2.5 (2H, t, J = 8), 5.4 (1H, s), 5.7 (1H, s), 7–7.16 (4H, m), 7.23–7.25 (2H, d, J = 5.6), 7.26–7.28 (2H, d, 5.7), 7.30–7.32 (2H,d, J = 8), 7.34–7.36 (2H, d, J = 8), 7.58–7.56 (1H, d, J = 9.6), 7.62–7.60 (1H, d, J = 8.4); 13C NMR(100 MHz, CDCl3, δ ppm): 14, 21, 39, 58, 87, 112, 119, 122, 123, 124, 125, 126, 127, 128, 129, 138, 140, 141, 153; IR (KBr) (ν max, cm−1): 3061, 2916, 2848, 1601, 1504, 1451, 1391, 1332, 1232, 1125, 945, 813, 704. ESI-MS m/z: 448.12, calcd. for C27H23Cl2NO (M + H)+: 447.12; CHN analysis (%): cal. C, 72.33; H, 5.17; N, 3.12; found; C, 71.89; H, 4.97; N, 3.34.

1,3-Bis(4-nitrophenyl)-2-propyl-2,3-dihydro-1H-naphtho [1,2-e][1,3]oxazine, 4l

White solid, melting point 139–140 °C. 1H NMR (400 MHz, CDCl3, δ ppm): 0.89 (3H, t, J = 8), 2.5 (2H, t, J = 9.4), 2.5 (2H, t, J = 8), 5.4 (1H, s), 5.7 (1H, s), 7–7.16 (4H, m), 7.23–7.25 (2H, d, J = 5.6), 7.26–7.28 (2H, d, 5.7), 7.30–7.32 (2H, d, J = 8), 7.34–7.36 (2H, d, J = 8), 7.58–7.56 (1H, d, J = 9.5), 7.62–7.60 (1H, d, J = 8.3); 13C NMR(100 MHz, CDCl3, δ ppm): 14, 21, 39, 58, 87, 112, 119, 122, 123, 124,125, 126, 127, 128, 129, 138, 140, 141, 153; IR (KBr) (ν max, cm−1): 3061, 2915, 2848, 1601, 1504, 1451, 1391, 1332, 1232, 1225, 945, 814, 705. ESI-MS m/z: 470.17, calcd. for C27H23N3O5 (M + H)+: 469.16; CHN analysis (%): cal. C, 69.07; H, 4.94; N, 8.95; found; C, 67.98; H, 4.55; N, 7.98.

1,3-Bis(4-methoxyphenyl)-2-propyl-2,3-dihydro-1H-naphtho[1,2-e][1,3]oxazine,4m

White crystalline solid, melting point 152–153 °C. 0.89 (3H, t, J = 8), 2.5 (2H, t, J = 9.4), 2.5 (2H, t, J = 8), 3.8 (6H, s), 5.4 (1H, s), 5.7 (1H, s), 7–7.16 (4H, m), 7.23–7.25 (2H, d, J = 5.6), 7.37–7.35 (2H, d, J = 9.6), 7.22–7.24 (2H, d, J = 5.5), 7.36–7.34 (2H, d, J = 9.4), 7.58–7.56 (1H, d, J = 9.6), 7.62–7.60 (1H, d, J = 8.4); 13CNMR (100 MHz, CDCl3, δ ppm): 10.42, 21.55, 44.2, 58.2, 60.6, 78.95, 85.49, 113.01, 120.05, 123.29, 126.40, 126.55, 127.13, 127.68, 128.05, 128.05, 128.13, 128.13, 128.60, 128.60, 129.08, 129.52, 129.52, 133.21, 138.25, 143.30, 152.90, 159.80; IR (KBr) (ν max, cm−1): 3052, 2945, 2847, 1621, 1506, 1451, 1394, 1334, 1233, 1125, 945, 813, 708. ESI-MS m/z: 440.22, calcd. for C29H29NO3 (M + H)+: 439.21; CHN analysis (%): cal. C, 79.24; H, 6.65; N, 3.19; found; C, 78.99; H, 6.53; N, 2.96.

1,3-Bis(4-fluorophenyl)-2-propyl-2,3-dihydro-1H-naphtho [1,2-e][1,3]oxazine, 4n

White crystal solid, melting point 140–141 °C. 1H NMR (400 MHz, CDCl3, δ ppm): 0.89 (3H, t, J = 8), 2.5 (2H, t, J = 9.4), 2.5 (2H, t, J = 8), 5.4 (1H, s), 5.7 (1H, s), 7–7.16 (4H, m), 7.23–7.25 (2H, d, J = 5.6), 7.37–7.35 (2H, d, J = 9.6), 7.22–7.24 (2H, d, J = 5.5), 7.36–7.34 (2H, d, J = 9.4), 7.58–7.56 (1H, d, J = 9.6), 7.62–7.60 (1H, d, J = 8.4); 13C NMR (100 MHz, CDCl3, δ ppm): 14.32, 39.52, 58.27, 84.95, 111.49, 119.02, 123.05, 123.29, 126.40, 126.55, 126.13, 127.68, 128.05, 128.05, 128.13, 128.12, 128.64, 128.60, 129.07, 129.52, 129.52, 133.21, 138.25, 143.30, 152.90, 162 (d, JC-F = 43.4 Hz), 163 (d, JC-F = 43.5 Hz); IR (KBr) (ν max, cm−1): 3061, 2915, 2848, 1601, 1504, 1451, 1391, 1332, 1232, 1225, 945, 814, 705. ESI-MS m/z: 416.18, calcd. for C27H23F2NO (M + H)+: 415.17; CHN analysis (%): cal. C, 78.05; H, 5.58; N, 3.37; found; C, 77.89; H, 5.32; N, 3.76.

Pharmacological screening

Erythrocyte suspension preparation

Fresh whole blood (5 mL) was collected from healthy volunteers into heparinized tubes. It was then centrifuged at 3000 × rpm for 10 min. The volume of normal saline equivalent to that of the supernatant was used to dissolve the red blood pellets. The volume of the dissolved red blood pellets obtained was measured and reconstituted as a 40% v/v suspension with isotonic buffer solution (10 mM sodium phosphate buffer, pH 7.4). The buffer solution contained 0.2 g of NaH2PO4, 1.15 g of Na2HPO4 and 9 g of NaCl in 1 L of distilled water. The reconstituted red blood cells (RBCs) (resuspended supernatant) were used as such.

Heat induced haemolysis

Samples of the extract used were dissolved in isotonic phosphate buffer solution. A set of five centrifuge tubes containing, respectively, 5 mL graded doses of the extracts (100, 200, 400, 600, and 800 μg/mL) were arranged in quadruplicate sets (four sets per dose). Two sets of control tubes contained 5 mL of the vehicle and 5 mL of 200 μg/mL of diclofenac, respectively. Human red blood cells, HRBC, suspension (0.1 mL) was added to each of the tubes and mixed gently. A pair of the tubes was incubated at 54 °C for 20 min in a regulated water bath. The other pair was maintained at −10 °C in a freezer for 20 min. Afterward, the tubes were centrifuged at 1300 × g for 3 min and the haemoglobin content of the supernatant was estimated using Spectronic 21D (Milton Roy) Spectrophotometer at 540 nm.

The percent inhibition of haemolysis by the extract was calculated as:

$$\% \,{\mathrm{Inhibition}}\,{\mathrm{of}}\,{\mathrm{Haemolysis}} = 1 - \frac{{OD2 - OD1}}{{OD3 - OD1}} \times 100,$$

where OD1 = absorbance of the test sample unheated. OD2 = absorbance of the test sample heated. OD3 = absorbance of the control sample heated. From the % inhibition, the results are then expressed as IC50 value.

Hypotonicity induced haemolysis

Samples of the extract used in this test were dissolved in distilled water (hypotonic solution). The hypotonic solution (500 µL) containing graded doses of the extracts (10, 20, 40, 80, and 100 μg/mL) were put into duplicate pairs (per dose) of the centrifuge tubes. Isotonic solution (500 µL) containing graded doses of the extracts (10–100 μg/mL) were also put into duplicate pairs (per dose) of the centrifuge tubes. Control tubes contained 500 µL of the vehicle (distilled water) and 500 µL of 20 μg/mL of diclofenac sodium, respectively. Erythrocyte suspension (10 µL) was added to each of the tubes and mixed gently. The mixtures were incubated for 1 h at room temperature (37 °C), and afterward, centrifuged for 3 min at 1300 × g. Absorbance (OD) of the haemoglobin content of the supernatant was estimated at 540 nm using Spectronic 21D (Milton Roy) spectrophotometer. The percentage heamolysis was calculated by assuming the heamolysis produced in the presence of distilled water as 100%.

The percent inhibition of haemolysis by the extract was calculated thus:

$$\% \,{\mathrm{Inhibition}}\,{\mathrm{of}}\,{\mathrm{Haemolysis}} = 1 - \frac{{OD2 - OD1}}{{OD3 - OD1}} \times 100,$$

where OD1 = absorbance of the test sample in an isotonic solution. OD2 = absorbance of the test sample in a hypotonic solution. OD3 = absorbance of the control sample in a hypotonic solution.

Inhibition of albumin denaturation

The reaction mixture of 1% aqueous solution of bovine serum albumin (Sigma) and test extract at different concentration (10–100 μg/mL) was taken in a centrifuge tube and pH was adjusted to 6.8 using 1N HCl. It was incubated at 37 °C for 20 min followed by heating at 57 °C for 20 min. The solution was cooled and absorbance was taken at 660 nm. The results are then expressed as IC50 value.

Molecular docking analysis

COX-2 crystal structure (PDB ID: 1PXX) was downloaded from the Protein Data Bank website (http://www.rcsb.org/structure/1PXX) and imported in the molecular docking software Molegro Virtual Docker 6.0 (MVD 6.0). The active site residue Tyr385, Gly526, and Ser530 were set as the search space binding site (X: −21.51, Y: 18.21, Z: −1.82) and these residues were made flexible and soften with a tolerance of 1.0 and strength of 0.90. On the other hand, the 3D geometrically optimised conformer of compounds 4h and 4c was imported in the MVD 6.0. The scoring function was set for the Grid Score (MolDock) with 0.30 Å as the grid resolution. Further, the compounds 4h and 4c were set for Internal ES (ElectroStatic), Sp2–Sp2 torsions and H-bond (Hydrogen bond) evaluation. The docking search algorithm was set for MolDock Simplex Evolution (MSE) for 20 numbers of runs. The MSE parameters were further set for 1200 maximum iterations and population size of 50. Root mean squared deviation, RMSD, threshold was set at 2.00 calculated by auto-morphisms for better accuracy and reliability. The best orientation of compounds 4h and 4c was carried forward for the molecular interaction analysis with the COX-2 enzyme.

Binding affinity calculation

Binding affinity was calculated for compounds 4h and 4c based on the coefficients of various energy terms (H-bond, E-Intra (vdw), E-Solvation etc) using multiple linear regression (MLR) equations. The MLR equation is further calibrated with a set of more than 200 structurally diverse complexes obtained from the Protein Data Bank with known binding affinities expressed in kJ/mol.

Density functional theory (DFT) study

DFT study was carried out for compounds 4h and 4c estimating the band energy gap (HOMO/LUMO gap). The calculation was carried out using the Gaussian (R) 09 System (Gaussian, Inc, USA). The molecular orbital energies including the HOMO and LUMO band gap energies were calculated at DFT/B3LYP level using the 6–31 G basis set with a net charge of zero and a single spin.

Results and discussion

Chemistry

Our initial trial experiments to prepare dihydro-1,3-oxazine 4a by an MCR protocol involving benzaldehyde (2a), ethylamine (3a), and β-naphthol (1) without using a catalyst or solvent at 100 °C led to unsuccessful results. Refluxing and stirring a mixture of 2a, 3a, and β-naphthol using solvents, such as THF, acetonitrile, dichloromethane, ethanol, and methanol, did not give any useful results. But, surprisingly, when the three components were refluxed in ethanol directly with SiO2.HClO4 for 18 h, the formation of dihydro-1,3-oxazine 4a was observed (Scheme 1). The reaction mixture was filtered to recover the SiO2.HClO4. The filtrate was extracted using ethyl acetate and water and dried over anhydrous Na2SO4. The pure compound 4a was isolated by column chromatography (SiO2). The yield of the solid white product was 90%.

Scheme 1
scheme 1

Synthesis of naphtho[1,2-e][1,3]oxazine

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For optimisation, we tested this MCR by varying the reaction conditions and parameters, i.e, the catalytic system, solvent, temperature, refluxing/stirring time, etc. (Table 1). Screening of various solvents with the catalyst was initially evaluated, guided by the template reaction between benzaldehyde, ethylamine, and β-naphthol (Scheme 1).

Table 1 Optimization of the solventa
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The shortest time period for the reaction was 18 h in SiO2.HClO4/ethanol with a 90% product yield being obtained. Thus, ethanol was the most suitable solvent. It was also observed that acetonitrile gave a 90% yield of the product, but the reaction was longer. Using THF, dichloromethane, and methanol also resulted in longer times with lower yields being obtained. We next, examined the quantity of the catalyst (Table 2). Refluxing the reaction mixture in ethanol for 18 h with 4 mol% of SiO2.HClO4 was found to be optimum; increasing the catalytic amount did not improve the yield.

Table 2 Optimisation of the catalyst amounta
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After optimising the reaction conditions, we next investigated the generality of this reaction for the synthesis of a wide variety of 1,3-oxazine derivatives and the results are summarised in (Table 3). A wide range of structurally varied aldehydes and amines were reacted with β-naphthol to give the corresponding dihydro-1,3-oxazine derivatives in good yields. From Table 3, it can be clearly seen that the reaction required strong ring activating groups such as p-OMe, p-Me and o-Me in order to proceed smoothly to give the desired products. Interestingly, the p-MeO substituted benzaldehyde and simple benzaldehyde gave the highest yields. Similarly, when we used ring-deactivating groups, such as –NO2, –F, –Cl-substituted benzaldehydes, moderate yields of the products were obtained. However, except for the o-Me substituted benzaldehyde, all the other reactions of substrates with o-, m- and 2,4-disubstituted electron donating or with withdrawing substituents failed, either giving back the starting materials or inseparable products. In the case of the amines, the reaction proceeded smoothly with primary amines only; bulky amines such as aromatic amines failed to give the corresponding products.

Table 3 Substrate scope for the synthesis of trans-1,3-diaryl-1H-naphtho[1,2-e][1,3]oxazines 4a
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Probable mechanism

The plausible mechanism is shown in Scheme 2, even though we were unable to prove experimentally the formation of the intermediates. The mechanism involves the formation of an imine intermediate with dehydration leading to the final naphtho-fused oxazine. Probably, the role of SiO2.HClO4 is to activate the benzaldehyde and the intermediates possessing carbonyl functional groups.

Scheme 2
scheme 2

Tentative mechanism of naphtho[1,2-e][1,3]oxazine synthesis

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Since these oxazines contain two chiral centres, there are possibilities for the formation of the syn- or anti-product. Theoretically, we expected the anti-product rather than the syn product. We thought of confirming the conformations of the structures by single-crystal X-ray studies; however, the literature (Zhang 2009; Yong et al. 2008; Cristina et al. 2001) had already revealed the anti-configuration based on single-crystal structures. The physical properties of the reported compounds were compared with our newly synthesised compounds and their spectral and analytical data were found to be in agreement with our proposed structures. As a representative example, the spectral and analytical data of 2-ethyl-1,3-Bis(4-fluorophenyl)-2,3-dihydro-1H-naphtho[1,2-e][1,3]oxazine (4e) have analysed along with the NOESY and COSY spectra (see the Supporting Information). The proton NMR spectrum showed peaks at 1.01 (t, J = 8.0, 3H), 1.30 (q, J = 9.6, 2H), 5.40 (s, 1H), 5.60 (s, 1H), 7.01–7.16 (m, 8H), 7.22–7.26 (d, J = 5.6, 2H), 7.38–7.34 (d, J = 9.6, 2H), 7.59–7.55 (d, J = 9.6, 1H), 7.63–7.59 (d, J = 8.4, 1H) ppm. The 13C NMR spectrum exhibited signals at 14.4, 21, 39.1, 58.1, 86.2, 113.1, 115.0, 124.2, 126.3, 127.2, 129.0, 131.2, 133.1, 136.1, 137.5, 139.2, 141.0, 153.1, 162 (d, JC-F = 43.2 Hz), 163 (d, JC-F = 43.5 Hz). The structure was further confirmed by the mass spectrum (ESI-MS) with a prominent mass ion peak at m/z: 399, calcd. for C26H19F2NO: 402.12 (M + H)+.

The unambiguous structural orientation of the diastereomeric compounds were confirmed from the COSY and NOESY spectra of representative compound 4e. From the analysis of the COSY spectrum, we found that the protons of the methyl carbon correlate with the protons of the methylene carbon. There were no cross peaks in the chemical shift region for the protons at 2 and 4 (Fig. 3). The absence of cross peaks in this region provided additional information that no coupling occurs between the two chiral protons and thus suggests the anti-conformation of the product. Moreover, the region in the chemical shift range 6.81–7.92 ppm represents the correlations between different aromatic protons. From the NOESY spectrum, no cross peaks were observed and two distinct singlets were present at 5.41 and 5.93 ppm, which is possible with the anti-conformation. Thus, from all these observations and by analogy, we have confirmed that the prepared compounds are anti-1,3-oxazines.

Fig. 3
figure 3

anti-configuration of 1,3-oxazine, 4e

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Pharmacological screening

The prepared anti-1,3-oxazine derivatives 4 were then screened against inflammation. Most of the compounds at doses 10–100 µg/mL showed protection of erythrocytes against heat-induced lysis. The IC50 value of lysis was different for each compound tested (Table 4). Some compounds showed more protective effects against haemolysis as compared with standard NSAIDs drugs such as diclofenac (IC50 = 30.14 µg/mL). Oxazines 4a, 4c, 4d, 4h, and 4n were more active than the diclofenac standard. Among the tested compounds, 4h showed the best activity with IC50 = 4.807 µg/mL.

Table 4 Anti-inflammatory activity of 1,3-oxazine derivatives 4a–n against heat-induced haemolysis, hypotonicity-induced haemolysis, and albumin denaturationa
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The tested compounds (10–100 µg/mL) showed protective effects against isotonic buffer and water-induced lysis. The IC50 values of lysis inhibition of the synthesised compounds were higher than that obtained for diclofenac, except oxazine (4h). 4h showed the highest activity in the hypotonicity-induced haemolysis assay with lower IC50 of 7.37 µg/mL and IC50 of 4.807 µg/mL in heat induced haemolysis (Table 4). The albumin denaturation inhibitory activity assay results are presented in Table 4. In the albumin denaturation assay, 4c showed the highest activity with IC50 = 5.5 µg/mL amongst the synthesised compounds. However, 4c showed weaker inhibitory activity as compared with NSAIDs diclofenac sodium (IC50 = 2.89 µg/mL) in preventing albumin denaturation.

There are many ways of assessing the anti-inflammatory activity, and yet there is no report that a particular test is better than the other test. Inflammation is a vascular phenomenon and also involves denaturation of protein. The ability of a substance to inhibit haemolysis and denaturation of protein indicates a good candidate for anti-inflammatory agent. Considering this, we have adopted only haemolysis (heat induced and Hypotonicity induced) and albumin denaturation in the present study.

In clinical study, due to their capacity in inhibiting protein denaturation, most of the pharmacological candidate used for the anti-inflammatory and pain-relief management are NSAIDs. 4c showed the highest activity among all the other compounds in albumin denaturation but weaker than NSAIDs Diclofenac. Though it has IC50 comparatively near to that of diclofenac. Compound 4h is more active in preventing haemolysis but not in albumin denaturation.

Compounds 4c and 4h have different physical and chemical properties. 4c is 1,3-Bis(5-bromothiophen-2-yl)-2-ethyl-2,3-dihydro-1H-naphtho[1,2-e][1,3]oxazine having molecular formula C22H17Br2NOS2 and molecular weight 532.19 g/mol, while 4h is 1,3-Bis(4-chlorophenyl)-2-ethyl-2,3-dihydro-1H-naphtho [1,2-e][1,3]oxazine having molecular formula C26H21Cl2NO and molecular weight 434.36 g/mol. Morphologically 4c is brown crystalline compound, whereas 4h is white crystalline compound. Due to the differences in their physico-chemical, morphological, and spatial arrangement, 4c and 4h may have different interacting mechanism in inhibiting the denaturation of protein, lysis of RBC membranes and haemolysis. The properties of 4h might help in preventing the excessive accumulation of fluid within the cell or by preventing the discharge of lysosomal enzymes rendering its highest activity in the haemolytic assay than 4c.

The synthesised oxazine derivatives at a concentration of 10–100 µg/mL showed protection against lysis induced by heat as well as hypotonic solution. By either preventing the discharge of lysosomal enzymes or by stabilising the lysosomal membranes, NSAIDs showed their beneficial effects as anti-inflammatory agents (Feirrali et al. 1992). The lysis of the membranes, complemented by haemolysis and oxidation of haemoglobin, is caused by contact of RBCs to injurious components, such as hypotonic medium, heat, methyl salicylate, or phenylhydrazine (Kurumbail et al. 1996). Since HRBC membranes are similar to lysosomal membrane components (Halliwell and Whiteman 2004) the inhibition of hypotonicity and heat-induced lysis of RBC membrane was used as a measure of the mechanism of anti-inflammatory activity of the tested principle. The haemolytic effect of the hypotonic solution is associated with excessive accumulation of fluid within the cell, which leads to rupturing of the membrane. RBC membrane injury will render the cell more inclined to secondary destruction through free-radical-promoted lipid peroxidation (Ricciotti and FitzGerald 2011). The pathogenesis of many diseases including arthritis, stroke, and cancer is connected with inflammation (Mizushima 1966).

In order to confirm the anti-inflammatory effects of compounds 4c and 4h, molecular docking studies were carried out against the COX-2 enzyme, which is responsible for inflammation and pain. Therefore, targeting the active site of COX-2 followed by inhibiting the enzyme leads to the reduction of pain and inflammation. In this study, the docking scores of compounds 4c and 4h docked against the active site of COX-2 are shown in Table 5. Compound 4c had an overall total score of −252.57 kJ mol−1 compared with −198.08 kJ mol−1 for compound 4h and −73.26 kJ mol−1 for diclofenac, a known inhibitor of the COX-2 enzyme. Diclofenac is known to inhibit both COX-1 and COX-2. In fact, diclofenac is similar in COX-2 selectivity to celecoxib-a selective COX-2 inhibitor (Gerarldf et al. 2001). Table 6 shows the binding affinities of the investigated compounds. Oxazine 4c has a binding affinity of −38.53, while that of compound 4h is −35.81 compared with −29.36 for diclofenac. These results suggest that oxazines 4c and 4h are good anti-inflammatory compounds because when it comes to binding affinity, lower the negative score, more stability. Hence more negative value, more binding affinity. Figure 4a, b depicts the steric and hydrophobic interactions of compounds 4c and 4h at the active site of COX-2. Compound 4c showed molecular interactions with Ser353, Tyr385, Ser530, Phe381, Trp387, Met522, Phe518, Val523, Arg513, His90, Leu352, Val349, Arg120, and 527, while compound 4h showed molecular interactions with Tyr 355, Ser 353, His90, Arg513, Ala527, Pro528, Leu531, Arg120, Val116, Val349, Met522, Phe518, Leu352, and Val523. Thus, the heavy molecular interactions suggest a strong binding affinity. Figure 5 depicts the favoured electrostatic interactions of oxazines 4c and 4h in the electronegative (green colour) and electropositive (yellow colour) regions. In addition, Fig. 6 shows the overall energy maps suggesting both the compounds reside in an energy pocket which is favoured with various energy parameters, such as hydrogen bond acceptor and donor-favoured regions, etc. Lastly, Figs. 7 and 8 represent the HOMO and LUMO energies of compounds 4c and 4h. The band gap energy of compound 4c is 0.16767, while that of compound 4h is 0.16321, which indicates a higher reactivity of compound 4c (Table 7).

Table 5 Docking Scores of the investigated compounds
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Table 6 Binding affinities of the investigated compounds
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Fig. 4
figure 4

2D interaction map of compounds 4c (a) and 4h (b) at the active site of the COX-2 enzyme

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Fig. 5
figure 5

Electrostatic interactions maps of compounds 4c (a) (green colour) and 4h (b) (yellow colour) at the active site of the COX-2 enzyme

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Fig. 6
figure 6

Energy maps of compounds 4c (a) (green colour) and 4h (b) (yellow colour) at the active site of the COX-2 enzyme

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Fig. 7
figure 7

Molecular orbitals depicting the HOMO energies of compounds 4c (a) and 4h (b)

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Fig. 8
figure 8

Molecular orbitals depicting the LUMO energies of compounds 4c (a) and 4h (b)

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Table 7 HOMO and LUMO parameters of Compounds 4h and 4c optimised at DFT/B3LYP 6-31G basis set
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The COX-2 crystal structure (PDB ID: 1PXX) was downloaded from the Protein Data Bank website (http://www.rcsb.org/structure/1PXX) and imported in the molecular docking software Molegro Virtual Docker 6.0 (MVD 6.0). The active site residues Tyr385, Gly526, and Ser530 were set as the search space binding site (X: −21.51, Y: 18.21, Z: −1.82) and these residues were made flexible and softened with a tolerance of 1.0 and a strength of 0.90. On the other hand, the 3D geometrically optimised conformers of compounds 4c and 4h were imported in MVD 6.0. The scoring function was set for the Grid Score (MolDock) with 0.30 Å as the grid resolution. Further, compounds 4c and 4h were set for Internal ES (ElectroStatic), sp2–sp2 torsions and H-bonds (hydrogen bond) evaluation. The docking search algorithm was set for MSE for 20 runs. The MSE parameters were further set for 1200 maximum iterations and population size of 50.

The RMSD threshold was set at 2.00 calculated by auto-morphisms for better accuracy and reliability. The best orientations of compounds 4c and 4h were carried forward for the molecular interaction analysis with the COX-2 enzyme.

Binding affinities were calculated for compounds 4c and 4h based on the coefficients of various energy terms (H-bond, E-Intra (vdw), E-solvation etc.) using MLR equations. The MLR equation is further calibrated with a set of more than 200 structurally diverse complexes obtained from the Protein Data Bank with known binding affinities expressed in kJ/mol.

The DFT studies were carried out for compounds 4c and 4h estimating the band energy gap (HOMO/LUMO). Calculations were carried out using the Gaussian (R) 09 System (Gaussian, Inc, USA). The molecular orbital energies including the HOMO and LUMO band gap energies were calculated the DFT/B3LYP level using the 6–31 G basis set with a net charge of zero and a single spin.

Conclusion

In conclusion, we have synthesised a series of dihydro-1,3-oxazine derivatives using SiO2.HClO4 as an effective heterogeneous catalyst using β-naphthol, aliphatic amines, and aromatic aldehydes. The anti-inflammatory activities of the prepared compounds were evaluated and observed significant results. The in silico studies revealed that compounds 4c and 4h docked at the active site of the COX-2 enzyme with favourable docking scores and binding affinities compared with diclofenac. Further, in vivo testing of the anti-inflammatory activities of compounds 4c and 4h as anti-inflammatory agents are under investigation.