Synthesis of novel naphtho[1,2-e][1,3]oxazines bearing an arylsulfonamide moiety and their anticancer and antifungal activity evaluations

Seyed Gholamhossein Mansouri; Hassan Zali-Boeini; Kamiar Zomorodian; Bahman Khalvati; Razie Helali Pargali; Ali Dehshahri; Hadi Amiri Rudbari; Mehdi Sahihi; Zahra Chavoshpour

doi:10.1016/j.arabjc.2017.10.009

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Original article

13 (

); 1271-1282

doi:

10.1016/j.arabjc.2017.10.009

Synthesis of novel naphtho[1,2-e][1,3]oxazines bearing an arylsulfonamide moiety and their anticancer and antifungal activity evaluations

Seyed Gholamhossein Mansouri^{^a}, Hassan Zali-Boeini^{^a,⁎}, Kamiar Zomorodian^{^b}, Bahman Khalvati^{^c,^d}, Razie Helali Pargali^{^b}, Ali Dehshahri^{^d}, Hadi Amiri Rudbari^{^a}, Mehdi Sahihi^{^a}, Zahra Chavoshpour^{^a}

a Department of Chemistry, University of Isfahan, Isfahan, Iran

b Basic Sciences in Infectious Diseases Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

c Medicinal Plants Research Center, Yasuj University of Medical Sciences, Yasuj, Iran

d Department of Pharmaceutical Biotechnology, Shiraz School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

⁎Corresponding author. h.zali@chem.ui.ac.ir (Hassan Zali-Boeini)

Received: 2017-7-2, Accepted: 2017-10-29, Published: 2017-01-01

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

Abstract

A series of novel naphtho[1,2-e][1,3]oxazines bearing arylsulfonamide moiety have been synthesized via a one-pot approach and in a green reaction medium. These new naphtho[1,2-e][1,3]oxazine derivatives have been characterized by their ¹H NMR, ¹³C NMR and the X-ray single crystallography method for compound 7a. All the newly synthesized compounds were examined for their in vitro anticancer activity against breast (MCF-7), colon (HCT116), and B-CLL (Waco3-CD5) cancers. Some of these compounds such as 7j and 7l showed remarkable activities against MCF-7 (breast) and HCT116 (colon) cancers with comparable IC₅₀ (The half maximal inhibitory concentration) values as that of known drugs such as 5-fluorouracil (5-FU). In vitro antimicrobial activities of all compounds were also evaluated against five human pathogenic fungi strains and two bacteria (one gram positive and one gram negative). The best MICs (Minimum Inhibitory Concentrations) were found against the C. albicans.

Keywords

Anticancer

Naphthoxazine

Sulfonamide

Synthesis

Antifungal

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1 Introduction

The development of environmentally friendly and economically inexpensive processes for the synthesis of biologically active heterocycles using readily available and cheap reagents is an important goal of current organic synthesis (Laszlo, 1995). 1,3-Oxazine derivatives condensed with aromatic rings have gained much attention due to varied pharmacological and biological properties such as anticancer (Urbański et al., 1956; Chylińska et al., 1963; Verma et al., 2012; Bouaziz et al., 1991; Thaler et al., 2013; Benameur et al., 1996 ), antibacterial (Mathew et al., 2010; Kategaonkar et al., 2010; Mayekar et al., 2011; Didwagh and Piste, 2013; Abou-Elmagd and Hashem, 2013; Verma et al., 2012 ), antifungal (Verma et al., 2012; Bouaziz et al., 1991; Mathew et al., 2010; Kategaonkar et al., 2010; Mayekar et al., 2011 ), anti-HIV (Pedersen and Pedersen, 2000; Cocuzza et al., 2001), and anti-inflammatory (Akhter et al., 2011) activities. Naphtho[1,2-e][1,3]oxazine derivatives are also included in this category.

Owing to their medicinal importance, several methods have been reported for the preparation of these compounds in the literature. The most widely adopted route has been benefited from a Mannich-type condensation reaction between phenols, primary amines, and formaldehyde through either direct or indirect pathway. The multi-component condensation of phenols or naphthols with primary amines (or ammonia) and two equivalents of aldehydes leads to these target molecules directly (Burke, 1949; Burke et al., 1952; Burke et al., 1954; Burke and Reynolds, 1954; Dhakane et al., 2014; Sadaphal et al., 2010 ), whereas a Betti base as starting material represents an indirect approach to the synthesis of 1,3-naphthoxazines (Shi et al., 2010; Turgut et al., 2007; Sapkal et al., 2009; Verma et al., 2012 ). Another method includes using the condensation reaction of salicylaldehyde with a primary amine followed by reduction and subsequent cyclization reaction with a suitable aldehyde (Tang et al., 2011; Tang et al., 2012). Furthermore, miscellaneous approaches such as reaction of oxygen-containing dihalo compounds with primary amines (Colin and Loubinoux, 1982), rhodium-catalyzed reaction of 2-(alkenyloxy)benzylamines with a mixture of H₂/CO (Campi et al., 1994; Campi et al., 1996), neutral redox reaction of cyclic amines and 2-hydroxy aromatic aldehydes or ketones (Richers et al., 2014), and direct reaction of ortho-lithiated phenols or thiophenols with N,N-bis[(benzotriazol-1-yl)methyl]amines (Katritzky et al., 2002) have also been reported.

The prominent roles of this heterocyclic skeleton in pharmaceutical chemistry inspired chemists to develop novel synthetic methods for efficient preparation of these biologically important compounds. On the other hand, the value of sulfonamide moiety in medicinal chemistry cannot be disregarded, as it consists of a large class of drugs used broadly as pharmaceutical agents. Various biological properties such as antibacterial (Bekdemir et al., 2008), antiviral, (Romines et al., 1997) and carbonic anhydrase (CA) inhibitory (Duffel et al., 1986; Roujeinikova et al., 2016) have been reported for the sulfonamide structures as lead (Jain et al., 2013).

Over the past years, dithioimidocarbonates have been identified as useful synthons in organic synthesis and in construction of diverse Heterocycles (Zali-Boeini et al., 2015; Maybhate et al., 1991; Servi et al., 2005; Sato et al., 1981; Alvarez Ibarra, et al. 1985; Sauter et al., 1995; Sullivan et al., 1976 ).

Meanwhile, our preliminary plan was to extend the synthetic utility of 1-(amino(aryl)methyl)naphthalen-2-ol derivatives (Betti bases) by examining their applications in the synthesis of novel 1,3-naphthoxazine derivatives. We intended to develop a novel one-step and efficient method for synthesis of new 1-aryl-2,3-dihydro-1H-naphtho[1,2-e][1,3]oxazine derivatives bearing a sulfonamide moiety via the reaction of N-sulfonyldithioimidocarbonates and Betti base salts in the presence of a base in aqueous EtOH as a green solvent. After characterization of all synthesized compounds, their anticancer and antimicrobial activities were evaluated.

2 Experimental

2.1

2.1 Chemistry

Reagents and solvents were obtained from commercial suppliers and used as received unless otherwise indicated. Melting points were determined with a Stuart Scientific SMP-2 apparatus and are uncorrected. Reactions were monitored by thin-layer chromatography (TLC Silica gel 60 F254). ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) spectra were recorded on a Bruker Avance 400 MHz NMR spectrometer referenced to residual solvent protons and signals are reported in ppm (δ). The singlet peak at 3.35 ppm in ¹H NMR spectra corresponds to protons of H₂O. X-ray crystallographic data was obtained using a Bruker AXS Kappa APEX II single crystal X-ray diffraction instrument.

2.1.1

2.1.1 General procedure for the synthesis of 1-(amino(aryl)methyl)naphthalen-2-ol hydrochlorides (Betti base hydrochlorides, 3a-e)

The Betti bases (2a-e) required for the synthesis of [1,3]naphthoxazines were obtained by the Betti reaction through a known method (Betti, 1941) as follows: In a 50 mL round-bottomed flask was placed a cold solution of β-naphthol (1) (7.2 g, 50 mmol) in 95 per cent alcohol (10 mL). To this solution was added benzaldehyde derivative (100 mmol) and EtOH (95%, 10 mL) saturated with ammonia at room temperature. The flask was stoppered and allowed to stand for 2 h. Then the excess ammonia was removed. After about 12 h, the condensation product (2a-e), which separated as white needle crystals, was filtered with suction and washed with EtOH (95%, 50 mL). The condensation product thus obtained was introduced into a 250 mL round-bottomed flask arranged for steam distillation and treated four times its volume with a solution of HCl (20%). The mixture was steam distilled about two hours. Meanwhile, an abundant flocculent precipitate of light pink or white needles was separated. The mixture in the flask was cooled thoroughly and filtered with suction. The yields of 3a-e were 84–91%.

2.1.2

2.1.2 General procedure for the synthesis of arylsulfonamides (5a-d)

Arylsulfonyl chloride (50 mmol) and chloroform (20 mL) were placed in a 100 mL round-bottomed flask. To the mixture an aqueous solution of ammonia (25%, 20 mL) was added and the reaction mixture was stirred for 2 h at room temperature. Then, the precipitated product was filtered and washed with cold water. The crude product was recrystallized using EtOH to give pure sulfonamides (5a-d) as white crystals in excellent yields (91–98%).

2.1.3

2.1.3 General procedure for the synthesis of dimethyl arylsulfonylcarbonimidodithioates (6a-d)

Dimethyl arylsulfonylcarbonimidodithioates (6a-d) were synthesized according to a reported procedure (Maybhate et al. 1991) starting from aryl sulfonamides 5a-d with slight modifications as follows: A solution of arylsulfonamide5a-d (14 mmol) in DMF (10 mL) was cooled in an ice-bath. To this, a solution of NaOH (20 M, 0.8 mL, 16 mmol) was added with vigorous stirring followed by addition of CS₂ (0.7 g, 9 mmol). After 20 min a second lot of NaOH solution (20 M, 0.4 mL, 8 mmol) and CS₂ (0.35 g, 4.5 mmol) was added to the reaction mixture followed by a third portion of NaOH solution (20 M, 0.4 mL, 8 mmol) and CS₂ (0.35 g, 4.5 mmol) after an interval of 10 min. The ice-bath was removed and the mixture was stirred at r.t. for 2 h. The mixture was cooled in an ice-bath and dimethyl sulfate (3.73 g, 29 mmol) was added dropwise during 10 min. The reaction mixture was stirred for 2 h at r.t. and poured into H₂O (50 mL). The precipitated solid was filtered, washed with H₂O, and dried under vacuum. The crude product was recrystallized where necessary from a suitable solvent to obtain compounds 6a-d as odoriferous white solids in good yields (75–86%).

2.1.4

2.1.4 General procedure for the synthesis of (E)-N-(1-aryl-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide derivatives (7a–o)

A mixture of 1-(amino(aryl)methyl)naphthalen-2-ol hydrochloride 3 (1 mmol), dimethyl arylsulfonylcarbonimidodithioate6 (1 mmol) and Na₂CO₃ (0.318 g, 3 mmol) in H₂O/EtOH (1: 3; 5 mL) was refluxed for 2 h. The reaction mixture was cooled to room temperature and neutralized by addition of aqueous HCl (10%). After some dilution with water, the liquor was decanted and the residue after purification by column chromatography using silica gel and a mixture of EtOAc-Hexane (1:1) as eluent gave pure compounds 7a–o as white or off-white solids in moderate to good yields (43–78%).

2.1.5

2.1.5 Spectral data (¹H-NMR and ¹³C-NMR spectra) of the synthesized compounds

2.1.5.1

2.1.5.1 (E)-N-(1-phenyl-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7a)

M.p.: 247–249 °C (decomp.); yield: 62%; ¹H NMR (400 MHz, DMSO-d₆): δ 10.18 (s, 1H), 8.03 (d, J = 9.0 Hz, 1H), 8.00–7.96 (m, 1H), 7.91 (d, J = 7.1 Hz, 2H), 7.80 (d, J = 8.7 Hz, 1H), 7.61–7.47 (m, 5H), 7.35–7.25 (m, 4H), 7.22 (d, J = 7.4 Hz, 2H), 6.34 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 152.1, 145.0, 143.2, 141.1, 131.9, 131.0, 130.7, 129.1, 128.8, 128.7, 128.4, 127.8, 126.9, 126.3, 125.8, 123.0, 115.8, 114.1, 52.0; IR (cm⁻¹, KBr): 3294, 3059, 1630, 1415, 1350, 1272, 1084; Anal. Calcd. for C₂₄H₁₈N₂O₃S: C, 69.55; H, 4.38; N, 6.76; S, 7.74 Found: C, 62.59; H, 4.41; N, 6.71; S, 7.77.

2.1.5.2

2.1.5.2 (E)-4-methyl-N-(1-phenyl-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7b)

M.p.: 260–262 °C (decomp.); yield: 78%; ¹H NMR (400 MHz, DMSO-d₆): δ 10.08 (s, 1H), 8.04 (d, J = 9.0 Hz, 1H), 8.00–7.97 (m, 1H), 7.81–7.76 (m, 3H), 7.56–7.47 (m, 2H), 7.36 (d, J = 9.0 Hz, 1H), 7.34–7.25 (m, 5H), 7.21 (d, J = 7.1, 2H), 6.33 (s, 1H), 2.35 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ 152.1, 145.1, 142.1, 141.1, 140.3, 130.9, 130.7, 129.2, 129.0, 128.7, 128.40, 128.36, 127.8, 126.9, 126.4, 125.8, 123.0, 115.8, 114.1, 52.0, 20.9; IR (cm⁻¹, KBr): 3240, 3140, 1643, 1431, 1311, 1288, 1153; Anal. Calcd. for C₂₅H₂₀N₂O₃S: C, 70.07; H, 4.70; N, 6.54; S, 7.48 Found: C, 70.09; H, 4.68; N, 6.51; S, 7.47.

2.1.5.3

2.1.5.3 (E)-4-chloro-N-(1-phenyl-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7c)

M.p.: 244–246 °C; yield: 70%; ¹H NMR (400 MHz, DMSO-d₆): δ 10.27 (s, 1H), 8.04 (d, J = 9.0 Hz, 1H), 8.00–7.96 (m, 1H), 7.92 (d, J = 8.6 Hz, 2H), 7.81–7.77 (m, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.55–7.47 (m, 2H), 7.37 (d, J = 9.0 Hz, 1H), 7.34–7.26 (m, 3H), 7.21 (d, J = 7.2 Hz, 2H), 6.34 (s, 1H). ¹³C NMR (100 MHz, DMSO-d₆): δ 152.1, 145.1, 142.0, 141.2, 136.6, 131.0, 130.7, 129.0, 128.9, 128.7, 128.5, 128.4, 128.3, 127.7, 126.9, 125.8, 123.1, 115.8, 114.0, 52.1; IR (cm⁻¹, KBr): 3225, 3085, 1643, 1477, 1431, 1219, 1157; Anal. Calcd. for C₂₄H₁₇ClN₂O₃S: C, 64.21; H, 3.82; N, 6.24; S, 7.14 Found: C, 64.27; H, 3.80; N, 6.21; S, 7.11.

2.1.5.4

2.1.5.4 (E)-N-(1-(p-tolyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7d)

M.p.: 271–273 °C (decomp.); yield: 54%; ¹H NMR (400 MHz, DMSO-d₆): δ 10.10 (s, 1H), 8.02 (d, J = 9.0 Hz, 1H), 7.99–7.95 (m, 1H), 7.90 (d, J = 7.0 Hz, 2H), 7.79–7.75 (m, 1H), 7.61–7.47 (m, 5H), 7.32 (d, J = 9.0 Hz, 1H), 7.13–7.07 (m, 4H), 6.29 (s, 1H), 2.23 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ 152.1, 145.0, 143.2, 138.2, 137.8, 131.9, 131.0, 130.6, 129.5, 128.8, 128.7, 128.4, 127.7, 126.8, 126.3, 125.8, 123.0, 115.7, 114.2, 51.8, 20.6; IR (cm⁻¹, KBr): 3245, 3078, 1650, 1446, 1423, 1223, 1130; Anal. Calcd. for C₂₅H₂₀N₂O₃S: C, 70.07; H, 4.70; N, 6.54; S, 7.48 Found: C, 70.10; H, 4.73; N, 6.50; S, 7.45.

2.1.5.5

2.1.5.5 (E)-4-methyl-N-(1-(p-tolyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7e)

M.p.: 203–205 °C; yield: 43%; ¹H NMR (400 MHz, DMSO-d₆): δ 10.08 (s, 1H), 8.01 (d, J = 9.0 Hz, 1H), 7.96 (d, J = 7.1 Hz, 1H), 7.76 (d, J = 8.0 Hz, 3H), 7.53–7.46 (m, 2H), 7.33 (d, J = 9.0 Hz, 1H), 7.29 (d, J = 8.0 Hz, 2H), 7.11–7.06 (m, 4H), 6.25 (s, 1H), 2.34 (s, 3H), 2.22 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ 151.9, 145.3, 141.7, 140.6, 138.6, 137.5, 130.8, 130.4, 129.4, 129.0, 128.7, 128.5, 127.6, 126.8, 126.5, 125.6, 123.0, 115.9, 114.4, 52.0, 20.9, 20.6; IR (cm⁻¹, KBr): 3244, 3067, 1651, 1435, 1219, 1161, 1134; Anal. Calcd. for C₂₆H₂₂N₂O₃S: C, 70.57; H, 5.01; N, 6.33; S, 7.25 Found: C, 70.61; H, 5.03; N, 6.30; S, 7.21.

2.1.5.6

2.1.5.6 (E)-4-chloro-N-(1-(p-tolyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7f)

M.p.: 224–226 °C (decomp.); yield: 55%; ¹H NMR (400 MHz, DMSO-d₆): δ 10.22 (s, 1H), 7.89 (d, J = 8.7 Hz, 2H), 7.79–7.72 (m, 3H), 7.47–7.38 (m, 2H), 7.34 (d, J = 8.5 Hz, 2H), 7.23 (d, J = 8.9 Hz, 1H), 6.99–6.92 (m, 4H), 6.07 (s, 1H), 2.21 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ 150.8, 146.9, 143.4, 140.9, 136.2, 135.1, 130.4, 129.2, 129.1, 128.9, 128.6, 128.5, 127.8, 127.0, 126.7, 124.7, 122.9, 116.3, 115.3, 53.6, 20.6; IR (cm⁻¹, KBr): 3290, 3074, 1651, 1423, 1335, 1219, 1011; Anal. Calcd. for C₂₅H₁₉ClN₂O₃S: C, 64.86; H, 4.14; N, 6.05; S, 6.93 Found: C, 64.80; H, 4.11; N, 6.07; S, 6.98.

2.1.5.7

2.1.5.7 (E)-N-(1-(4-isopropylphenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)-4-methylbenzenesulfonamide (7g)

M.p.: 255–257 °C; yield: 51%; ¹H NMR (400 MHz, DMSO-d₆): δ 10.07 (s, 1H), 8.03 (d, J = 9.0 Hz, 1H), 8.00–7.96 (m, 1H), 7.82–7.77 (m, 3H), 7.56–7.47 (m, 2H), 7.38–7.31 (m, 3H), 7.18 (d, J = 8.3 Hz, 2H), 7.12 (d, J = 8.3 Hz, 2H), 6.29 (s, 1H), 2.81 (sep, J = 6.9 Hz, 1H), 2.35 (s, 3H), 1.13 (dd, J = 6.9 Hz, 1.9 Hz, 6H); ¹³C NMR (100 MHz, DMSO-d₆): δ 152.1, 148.6, 145.1, 142.0, 140.4, 138.6, 130.9, 130.6, 129.2, 128.7, 128.4, 127.8, 126.94, 126.87, 126.4, 125.8, 123.0, 115.8, 114.3, 51.7, 33.0, 23.7, 23.6, 20.9; IR (cm⁻¹, KBr): 3240, 3090, 1643, 1419, 1304, 1161, 1134; Anal. Calcd. for C₂₈H₂₆N₂O₃S: C, 71.46; H, 5.57; N, 5.95; S, 6.81 Found: C, 71.49; H, 5.52; N, 5.97; S, 6.85.

2.1.5.8

2.1.5.8 (E)-4-chloro-N-(1-(4-isopropylphenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7h)

M.p.: 262–264 °C; yield: 55%; ¹H NMR (400 MHz, DMSO-d₆): δ 10.22 (s, 1H), 8.05 (d, J = 9.0 Hz, 1H), 8.01–7.97 (m, 1H), 7.92 (d, J = 8.6 Hz, 2H), 7.81 (d, J = 7.7 Hz, 1H), 7.61 (d, J = 8.6 Hz, 2H), 7.56–7.48 (m, 2H), 7.37 (d, J = 9.0 Hz, 1H), 7.18 (d, J = 8.3 Hz, 2H), 7.12 (d, J = 8.3 Hz, 2H), 6.31 (s, 1H), 2.81 (sep, J = 6.9 Hz, 1H), 1.13 (dd, J = 6.9 Hz, 1.6 Hz, 6H); ¹³C NMR (100 MHz, DMSO-d₆): δ 152.3, 148.6, 145.0, 142.0, 138.6, 136.7, 131.0, 130.6, 128.9, 128.7, 128.5, 128.4, 127.8, 126.95, 126.88, 125.8, 123.1, 115.7, 114.2, 51.8, 33.0, 23.65, 23.57; IR (cm⁻¹, KBr): 3225, 3080, 1643, 1477, 1311, 1219, 1134; Anal. Calcd. for C₂₇H₂₃ClN₂O₃S: C, 66.05; H, 4.72; N, 5.71; S, 6.53 Found: C, 66.01; H, 4.76; N, 5.67; S, 6.57.

2.1.5.9

2.1.5.9 (E)-N-(1-(4-isopropylphenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)-4-methoxybenzenesulfonamide (7i)

M.p.: 228–230 °C; yield: 46%; ¹H NMR (400 MHz, DMSO-d₆): δ 10.01 (s, 1H), 8.04 (d, J = 9.0 Hz, 1H), 8.00–7.97 (m, 1H), 7.84 (d, J = 8.9 Hz, 2H), 7.82–7.79 (m, 1H), 7.56–7.48 (m, 2H), 7.38 (d, J = 9.0 Hz, 1H), 7.18 (d, J = 8.2 Hz, 2H), 7.12 (d, J = 8.2 Hz, 2H), 7.05 (d, J = 8.9 Hz, 2H), 6.28 (s, 1H), 3.81 (s, 3H), 2.81 (sep, J = 6.9 Hz, 1H), 1.13 (dd, J = 6.9 Hz, 1.8 Hz, 6H); ¹³C NMR (100 MHz, DMSO-d₆): δ 161.7, 152.0, 148.5, 145.1, 138.7, 135.0, 130.9, 130.6, 128.7, 128.5, 128.4, 127.8, 126.9 126.8, 125.8, 123.0, 115.9, 114.4, 113.8, 55.5, 51.7, 33.0, 23.65, 23.61; IR (cm⁻¹, KBr): 3244, 3086, 1651, 1427, 1292, 1257, 1184;; Anal. Calcd. for C₂₈H₂₆N₂O₄S: C, 69.11; H, 5.39; N, 5.76; S, 6.59 Found: C, 69.08; H, 5.36; N, 5.79; S, 6.54.

2.1.5.10

2.1.5.10 (E)-N-(1-(4-chlorophenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7j)

M.p.: 226–228 °C; yield: 63%; ¹H NMR (400 MHz, DMSO-d₆): δ 10.20 (s, 1H), 8.03 (d, J = 9.0 Hz, 1H), 8.00–7.96 (m, 1H), 7.90–7.87 (m, 2H), 7.78–7.74 (m, 1H), 7.59–7.47 (m, 5H), 7.37 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 9.0 Hz, 1H), 7.21 (d, J = 8.5 Hz, 2H), 6.35 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 151.9, 145.4, 143.2, 140.3, 132.9, 131.8, 130.9, 130.7, 129.0, 128.8, 128.75, 128.69, 128.4, 127.8, 126.4, 125.8, 122.9, 115.9, 113.7, 51.5; IR (cm⁻¹, KBr): 3240, 3082, 1643, 1473, 1284, 1184, 1084; Anal. Calcd. for C₂₄H₁₇ClN₂O₃S: C, 64.21; H, 3.82; N, 6.24; S, 7.14 Found: C, 64.25; H, 3.82; N, 6.22; S, 7.13.

2.1.5.11

2.1.5.11 (E)-N-(1-(4-chlorophenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)-4-methylbenzenesulfonamide (7k)

M.p.: 249–251 °C (decomp.); yield: 47%; ¹H NMR (400 MHz, DMSO-d₆): δ 10.05 (s, 1H), 8.04 (d, J = 8.9 Hz, 1H), 7.98 (d, J = 7.2 Hz, 1H), 7.77 (d, J = 7.8 Hz, 2H), 7.75–7.71 (m, 1H), 7.55–7.46 (m, 2H), 7.39–7.34 (d, J = 8.4 Hz, 3H), 7.30 (d, J = 7.8 Hz, 2H), 7.21 (d, J = 8.2 Hz, 2H), 6.36 (s, 1H), 2.34 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ 152.1, 145.2, 142.2, 140.0, 139.9, 133.0, 130.95, 130.91, 129.2, 129.1, 128.9, 128.8, 128.3, 127.9, 126.4, 125.9, 122.9, 115.9, 113.6, 51.3, 20.9; IR (cm⁻¹, KBr): 3294, 3047, 1635, 1411, 1377, 1234, 1080; Anal. Calcd. for C₂₅H₁₉ClN₂O₃S: C, 64.86; H, 4.14; N, 6.05; S, 6.93 Found: C, 64.82; H, 4.13; N, 6.08; S, 6.95.

2.1.5.12

2.1.5.12 (E)-4-chloro-N-(1-(4-chlorophenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7l)

M.p.: 242–244 °C (decomp.); yield: 75%; ¹H NMR (400 MHz, DMSO-d₆): δ 10.25 (s, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.82 (d, J = 8.9 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.66 (d, J = 8.5 Hz, 2H), 7.45–7.33 (m, 2H), 7.20 (d, J = 8.5 Hz, 2H), 7.15 (m, 1H), 7.12 (d, J = 8.5 Hz, 2H), 6.96 (d, J = 8.5 Hz, 2H), 5.97 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ 149.9, 148.4, 144.6, 144.5, 134.1, 130.7, 130.0, 129.6, 128.8, 128.6, 128.5, 128.4, 127.8, 127.1, 126.7, 124.0, 122.6, 116.8, 115.5, 54.3; IR (cm⁻¹, KBr): 3290, 3067, 1635, 1488, 1250, 1153, 1080; Anal. Calcd. for C₂₄H₁₆Cl₂N₂O₃S: C, 59.64; H, 3.34; N, 5.80; S, 6.63 Found: C, 59.62; H, 3.31; N, 5.85; S, 6.61.

2.1.5.13

2.1.5.13 (E)-N-(1-(4-chlorophenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)-4-methoxybenzenesulfonamide (7m)

M.p.: 235 °C (decomp.); yield: 65%; ¹H NMR (400 MHz, DMSO-d₆): δ 10.10 (s, 1H), 8.00 (d, J = 9.0 Hz, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.80–7.73 (m, 3H), 7.53–7.45 (m, 2H), 7.36–7.30 (m, 3H), 7.17 (d, J = 8.2 Hz, 2H), 6.96 (d, J = 8.6 Hz, 2H), 6.28 (s, 1H), 3.79 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ 161.4, 151.6, 145.9, 141.0, 135.4, 132.5, 130.7, 130.4, 128.8, 128.7, 128.6, 128.5, 127.6, 125.5, 122.8, 116.1, 114.1, 113.5, 55.4, 51.9; IR (cm⁻¹, KBr): 3290, 3070, 1643, 1497, 1338, 1257, 1084; Anal. Calcd. for C₂₅H₁₉ClN₂O₄S: C, 62.69; H, 4.00; N, 5.85; S, 6.69 Found: C, 62.66; H, 4.04; N, 5.81; S, 6.73.

2.1.5.14

2.1.5.14 (E)-4-chloro-N-(1-(4-methoxyphenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7n)

M.p.: 193–195 °C; yield: 48%; ¹H NMR (400 MHz, DMSO-d₆): δ 10.20 (s, 1H), 8.01 (d, J = 9.0 Hz, 1H), 7.99–7.95 (m, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.76 (d, J = 7.5 Hz, 1H), 7.55 (d, J = 8.5 Hz, 2H), 7.53–7.45 (m, 2H), 7.34 (d, J = 9.0 Hz, 1H), 7.08 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H), 6.24 (s, 1H), 3.69 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ 158.9, 151.9, 145.3, 142.3, 136.3, 133.7, 130.9, 130.3, 128.7, 128.52, 128.49, 128.2, 127.6, 125.6, 123.0, 115.8, 114.4, 114.2, 55.0, 51.9; IR (cm⁻¹, KBr): 3240, 3086, 1643, 1438, 1254, 1161, 1080; Anal. Calcd. for C₂₅H₁₉ClN₂O₄S: C, 62.69; H, 4.00; N, 5.85; S, 6.69 Found: C, 62.65; H, 4.02; N, 5.87; S, 6.66.

2.1.5.15

2.1.5.15 (E)-4-methoxy-N-(1-(4-methoxyphenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7o)

M.p.: 182–184 °C; yield: 55%; ¹H NMR (400 MHz, DMSO-d₆): δ 10.02 (s, 1H), 8.01–7.97 (d, J = 9.0 Hz, 1H), 7.97–7.94 (m, 1H), 7.79 (d, J = 8.8 Hz, 2H), 7.78–7.74 (m, 1H), 7.53–7.47 (m, 2H), 7.33 (d, J = 9.0 Hz, 1H), 7.08 (d, J = 8.6 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 6.82 (d, J = 8.6 Hz, 2H), 6.21 (s, 1H), 3.79 (s, 3H), 3.68 (s, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ 161.4, 158.8, 151.7, 145.6, 135.4, 134.0, 130.8, 130.2, 128.64, 128.60, 128.5, 128.2, 127.5, 125.4, 123.0, 116.0, 114.7, 114.1, 113.6, 55.4, 55.0 51.9; IR (cm⁻¹, KBr): 3271, 3067, 1643, 1496, 1261, 1153, 1084; Anal. Calcd. for C₂₆H₂₂N₂O₅S: C, 65.81; H, 4.67; N, 5.90; S, 6.76 Found: C, 65.85; H, 4.63; N, 5.96; S, 6.72.

3 Biology

3.1

3.1 In vitro investigation of antimicrobial activity

3.1.1

3.1.1 Microorganisms

The antimicrobial activities of the synthetic compounds against four American Type Culture Collection (ATCC) microorganisms, including Candida albicans (ATCC 10261), A. fumigatus (ATCC 14110), Staphylococcus aureus (ATCC 700698), and Escherichia coli (ATCC 25912) were determined. Moreover, three clinical isolates of dermatophytes including Epidermophyton flocossum (SUCC 93-552), Microsporumcanis (SUCC 93-560) and Trichophytonrubrum (SUCC 93-565) were also tested in this study. The susceptibility of all isolated bacteria and fungi against selected antibiotics were examined by microdilution methods (Clinical and Laboratory Standards Institute, 2006a, 2006b, 2006c ).

3.1.2

3.1.2 Determination of minimum inhibitory concentration

MICs were determined using broth microdilution method recommended by the CLSI with some modifications (Clinical and Laboratory Standards Institute, 2006a, 2006b, 2006c). Briefly, for determination of antifungal activities against fungi, serial dilutions of the synthesized compounds 7a-o (0.25–256.0 µg/ml) were prepared in 96-well microtiter plate using RPMI-1640 media (Sigma, St. Louis, USA) buffered with MOPS (Sigma, St. Louis, USA). To determine the antibacterial activities, serial dilutions of the synthesized compounds 7a–o (0.25–256.0 µg/ml) were prepared in Muller-Hinton Broth media (Merck, Darmstadt, Germany). The test fungi or bacteria strains were suspended in the media and the cell densities were adjusted to 0.5 McFarland standards at 530 nm wavelength using a spectrophotometric method (this yields stock suspension of 1–5 × 106 cells/ml for yeast and 1–1.5 × 108 cells/ml for bacteria). One hundred microliters of the working inoculums was added to the microtiter plates which were incubated in a humid atmosphere at 30 °C for 24–48 h (fungi) or at 37 °C for 24 h (bacteria). Two hundred microliters of the uninoculated medium was included as a sterility control (blank). In addition, growth controls (medium with inoculums but without drugs) were also included. The growth in each well was compared with that of the growth control well. MICs were visually determined and defined as the lowest concentration of the synthetic compounds or antibiotics that prevents any visible growth (MIC₉₀) of microorganisms (both bacteria and fungi). For fungi MIC₅₀ were also determined and defined as the lowest concentration of the compounds that inhibits about half, or 50% growth of fungal cells than that of the controls. Each experiment was performed in triplicate. The reference antibiotics, fluconazole (Sigma, St. Louis, MO, USA), griseofulvin (sigma) and ciprofloxacin (sigma) were used as positive controls against saprophytes, dermatophytes and bacteria, respectively.

In addition, media from wells with fungi showing no visible growth were further cultured on Sabouraud Dextrose Agar (Merck, Darmstadt, Germany) and from wells with bacteria showing no visible growth on Muller-Hinton agar (Merck, Darmstadt, Germany) to determine the minimum fungicidal concentration (MFC) and minimum bactericidal concentration (MBC). MBCs and MFCs were determined as the lowest concentration yielding no more than 4 colonies, which corresponds to a mortality of 99.9% of the microbes in the initial inoculums.

3.2

3.2 Determination of cytotoxicity

3.2.1

3.2.1 Cell line and culture conditions

The human breast (MCF-7), human colon (HCT116) and human B-CLL (Waco3-CD5) cancer cell lines were used in the study. We used RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin for cell growth under the condition of 37 °C in 5% CO₂.

Peripheral blood mononuclear cells (PBMC) were obtained by venipuncture from healthy adult donors and mononuclear cells were isolated by Ficol1-Hypaque gradient separation. Lymphocytes were washed and resuspended in RPMI medium supplemented with 10% fetal bovine serum. RPMI-1640 medium was used for B-CLL and DMEM medium for others. 5-Fluorouracil (5-FU) and Rituximab were employed as positive control drugs. Rituximab (Mabthera) drug was obtained from Roche Company and 5-FU was purchased from Merck Company.

3.2.2

3.2.2 Cytotoxic activity

3.2.2.1

3.2.2.1 MTT assay

The MTT assay was used to evaluate the cytotoxicity of novel synthesized derivatives (7a–o) on cell viability (Liuet al., 1997). When cells were in exponentially growing phase, seeded at a concentration of 10,000 cells/well on 96 tissue culture plate overnight. After the culture medium replaced with a fresh one, a solution of synthesized compounds in DMSO was added to reach various concentrations. After 48 h, the drug solutions discarded and cells were washed with PBS. Then 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml, in PBS) was added into each well and cultured for another 4 h. Subsequently, the supernatant replaced with DMSO to soften the formed formazan. In parallel, we used negative control, blank and positive control (5-FU and Rituximab) in the same conditions. At the final step, absorbance at the wavelength of 540 nm was read by a microplate reader. Data was calculated and expressed as the 50% inhibitory concentrations (IC₅₀) and presented as mean ± SD, n = 3. The MTT assay was also carried out on the non-cancer cells of PBMC as descried for the cancer cell lines. As for the PBMC cells, lymphocytes were washed, resuspended in RPMI medium supplemented with 10% fetal bovine serum and plated (1 × 10⁶ cells/mL) in the presence of phytohemagglutinin. After incubation for 24 h at 37 °C and 5% CO₂, the cells were treated with a solution of synthesized compounds in DMSO in various concentrations. (0.1–20 mmol L21) for 48 h. Cytotoxicity of violacein was determined by the MTT assay.

4 Results and discussion

4.1

4.1 Chemistry

A new route for the synthesis of novel 1-aryl-2,3-dihydro-1H-naphtho[1,2-e][1,3]oxazine derivatives bearing a sulfonamide moiety was accomplished in five steps starting from readily available chemicals in EtOH-H₂O as an environmentally benign medium with good to excellent yields. The synthetic approach is outlined in Scheme 1.

Synthetic approach for the preparation of Betti bases (A), N-sulfonyldithioimidocarbonates (B), and 1-aryl-2,3-dihydro-1H-naphtho[1,2-e][1,3]oxazine derivatives (C).

The Betti bases 3a-e required for the synthesis of 1,3-naphthoxazines were obtained by the Betti reaction through a known method (Betti, 1941). Also, N-sulfonyldithioimidocarbonates 6a-d were synthesized according to a reported procedure starting from arylsulfonamides 5a-d (Maybhate et al., 1991). Finally, in the reaction of bis-nucleophiles3a-e and N-sulfonyldithioimidocarbonates 6a-d in the presence of Na₂CO₃ and a mixture of EtOH-H₂O as a reaction medium, 1-aryl-2,3-dihydro-1H-naphtho[1,2-e][1,3]oxazine derivatives 7a–o containing an arylsulfonamide moiety were obtained in moderate to good yields (43–78%). The results are summarized in Fig. 1.

One-step preparation of 1-aryl-2,3-dihydro-1H-naphtho[1,2-e][1,3]oxazine derivatives. (aIsolated yields;) (bReaction conditions: 1-(amino(aryl)methyl)naphthalen-2-ol hydrochloride 3 (1 mmol), dimethyl arylsulfonylcarbonimidodithioate6 (1 mmol) and Na2CO3 (3 mmol) in H2O/EtOH (1: 3, 5 mL), reflux condition, 2 h.)

The structure of compounds 7a–o were proved by ¹H NMR, and ¹³C NMR spectra, alongside the X-ray single crystallography method. ¹H NMR spectrum of 7a revealed the presence of a singlet at 10.17 ppm which could be assigned to NH and a singlet at 6.34 ppm attributed to the only aliphatic C—H of the compound 7a. Absolute assignment of compound 7a was achieved by single-crystal X-ray crystallography (Fig. 2, CCDC 1405842). Single crystals of compound 7a was obtained thru slow evaporation of a solution of 7a in acetonitrile.

ORTEP representation of 7a. Displacement ellipsoids are drawn at the 50% probability level and H atoms are shown as small spheres of arbitrary radii.

The crystallographic data reveal that compound 7a crystallized in a monoclinic system, space group P2₁/n. The asymmetric unit of 7a contains a whole molecule (Fig.3). All bond lengths and angles are in the normal ranges and comparable with the reported ones (Hursthouse et al., 2007, Laufer et al., 2009, 2012 ).

A representation of intramolecular and intermolecular hydrogen bonds in molecule of 7a. H atoms have been omitted for clarity except hydrogens that participate in hydrogen bonds.

The hydrogen-bonding pattern of compound 7a can be described using graph theory (Chang et al., 1995, Tinhofer et al., 1999). For this structure, there are a intramolecular hydrogen bond of the N—H⋯O type (dashed line a in Fig. 3), which is described as S(6) ring in the first level graph set (Fig. 3).

There are also two intermolecular hydrogen bonds of O⋯H—C and O⋯H—N types in the structure that create a dimer (dashed line b and c in Fig. 2) (Table 1). According to graph theory, a R²₂(7) ring is formed by two intermolecular hydrogen bonds (Fig. 3). According to the hydrogen bonding classification (Steiner, 2002; Steiner, 1999), these intramolecular and intermolecular hydrogen-bonding are moderate electrostatic interactions.

Table 1 Intramolecular and intermolecular hydrogen bond geometries (Å, °) for 7a.

D—H⋯A	D—H	H⋯A	D⋯A	D—H⋯A
N(1)—H(1)⋯O(3)^#	0.800(18)	2.543(18)	3.2175(19)	142.9(16)
N(1)—H(1)⋯O(2)	0.800(18)	2.235(17)	2.8462(18)	133.6(16)
C(11)—H(11)⋯O(2)^#	0.98	2.38	3.2780(18)	151.6

# Symmetry transformations used to generate equivalent atoms: −x − 1/2, y + 1/2, −z + 3/2.

The selected crystal structure data are presented in Table 2.

Table 2 Selected bond lengths (Å) and angles (°) for 7a.

Bond	Bond length (Å)	Bonds	Bond angle (°)
S(1)—O(3)	1.4294(13)	C(18)—N(1)—C(11)	124.49(12)
S(1)—O(2)	1.4401(12)	C(18)—N(2)—S(1)	119.35(11)
S(1)—N(2)	1.6109(15)	N(1)—C(11)—C(10)	107.43(12)
S(1)—C(25)	1.7691(17)	N(1)—C(11)—C(12)	111.12(12)
N(1)—C(18)	1.3213(19)	C(18)—O(1)—C(1)	119.35(11)
N(1)—C(11)	1.4723(19)	N(2)—C(18)—O(1)	112.49(13)
N(2)—C(18)	1.307(2)	N(2)—S(1)—C(25)	105.26(7)
O(1)—C(18)	1.3532(18)	N(1)—C(18)—O(1)	117.78(14)
O(1)—C(1)	1.4003(19)

5 Biology

5.1

5.1 Antimicrobial activity

In the present study, the antibacterial and antifungal effects of newly synthesized compounds on two bacteria (one gram positive and one gram negative) and various fungi were analyzed (Table 2).

Although Verma et al. (2012) reported antibacterial activity for their naphthoxazine derivatives, none of the examined compounds in this study exhibited antibacterial activity against the tested bacteria at the concentrations up to 256 µg/mL. The difference in antibacterial activities between their compounds and ours might be attributed to presence of an aryl group in their compounds instead of an arylsulfonamide moiety. Antibacterial inactivity of our synthesized compounds 7a–o also might be due to lack of an essential p-amino substituent in the aromatic ring at R² position, which is quite necessary for the antibacterial role of arylsulfonamides.

Among several tested fungi, the compounds 7a–o only exhibited notable inhibitory activities against Candida albicans (yeasts) with MICs ranging from 32-256 µg/mL. Compounds7c and 7l with a chloro substituent as an electron withdrawing group at the R² position exhibited the most anti Candida activity with MIC of 32 µg/mL. A comparison between 7c, 7f, 7l, and 7n which have a chloro substituent at R² position and respectively an H, CH₃, Cl and/or OCH₃ at R¹ position, revealed that existence of an electron withdrawing group at R¹ position enhances antifungal activity of the final product compared to electron donating groups. Moreover, comparison of 7b and 7g as well as 7c and 7h revealed that placement of a bulky group such as isopropyl at R¹ position might decrease the anti-Candida activity of the title compounds. We found that, placement of a halogen such as chloro as an electron withdrawing group in R² position leads to more effective compounds in terms of antifungal activity. As shown in Table 3, lack of a substituent at para position of arylsulfonamide moiety (compounds 7a, 7d, and 7j) results in lessening the inhibitory activity against the Candida albicans and the best result was obtained when there is a methyl group at this position (7e). These data show that, this class of compounds could be regarded as fungistatic antifungal agents.

Table 3 Investigation of antimicrobial activities of compounds 7a–o.

			Minimum inhibitory concentration (MIC) (µg/ml sample)
			Microorganisms
			E. coli		S. aureus		C. albicans			A. fumigatus			E. floccosum			M. canis			T. rubrum
Entry	R¹	R²	MIC₉₀	MBC	MIC₉₀	MBC	MIC₅₀	MIC₉₀	MFC	MIC₅₀	MIC₉₀	MFC	MIC₅₀	MIC₉₀	MFC	MIC₅₀	MIC₉₀	MFC	MIC₅₀	MIC₉₀	MFC
7a	H	H	G	G	G	G	32	128	G	G	G	G	G	G	G	G	G	G	G	G	G
7b	H	CH₃	G	G	G	G	128	256	G	G	G	G	G	G	G	G	G	G	G	G	G
7c	H	Cl	G	G	G	G	16	32	G	G	G	G	G	G	G	G	G	G	G	G	G
7d	Me	H	G	G	G	G	256	G	G	G	G	G	G	G	G	G	G	G	G	G	G
7e	Me	CH₃	G	G	G	G	8	64	G	G	G	G	G	G	G	G	G	G	G	G	G
7f	Me	Cl	G	G	G	G	16	64	G	G	G	G	256	G	G	128	G	G	G	G	G
7g	i-Pr	CH₃	G	G	G	G	256	G	G	G	G	G	G	G	G	G	G	G	G	G	G
7h	i-Pr	Cl	G	G	G	G	256	G	G	G	G	G	G	G	G	G	G	G	G	G	G
7i	i-Pr	OCH₃	G	G	G	G	32	64	G	G	G	G	G	G	G	G	G	G	G	G	G
7j	Cl	H	G	G	G	G	32	256	G	G	G	G	256	G	G	256	G	G	G	G	G
7k	Cl	CH₃	G	G	G	G	64	64	G	G	G	G	G	G	G	G	G	G	G	G	G
7l	Cl	Cl	G	G	G	G	16	32	G	G	G	G	G	G	G	G	G	G	G	G	G
7m	Cl	OCH₃	G	G	G	G	32	64	G	G	G	G	G	G	G	G	G	G	G	G	G
7n	OMe	Cl	G	G	G	G	32	64	G	G	G	G	256	G	G	G	G	G	G	G	G
7o	OMe	OCH₃	G	G	G	G	32	128	G	G	G	G	256	G	G	G	G	G	G	G	G
FCZ			–	–	–	–	2	4	G	2	8	G	–	–	–	–	–	–	–	–	–
GF			–	–	–	–	–	–	–	–	–	–	0.5	1	G	0.5	2	G	1	2	G
CIPX			0.025	G	0.5	G	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–

G: >256 µg/mL; FCZ: Fluconazole; GF: Griseofulvin; CIPX: Ciprofloxacin.

5.2

5.2 Cytotoxic activity

All the compounds were tested in vitro against a 3-cell line panel consisting of HCT116, ATCC No.: CCL-247 (human colon cancer), MCF-7, ATCC No.: HTB-22 (human breast cancer), and Waco3-CD5, NCBI No.: C547 (human chronic lymphocytic leukemia) and their IC₅₀ values were obtained. The MTT assay was also carried out on the non-cancer cells of PBMC as descried for the cancer cell lines. Results of IC₅₀ experiment demonstrated that all of the compounds prepared in this study showed more inhibitory effect on colon and breast cancer cell lines compared to B-CLL cell line. Among the tested compounds, 7j and 7l showed the most cytotoxic effects on breast and colon cancer cell lines whereas their cytotoxicity on Waco3-CD5 was negligible. The lowest cytotoxic effects were achieved by the compound 7f which showed the lowest toxicity on all the cell lines tested. The presence of electron withdrawing chloro group at either R¹ or R² position improved the anticancer activity in these structures, but the combination of CH₃ and Cl at R¹ and R² positions might decrease the cytotoxicity of these compounds (Table 4). The results of cytotoxicity assay on the non-cancer cells of PBMC demonstrated that the compounds prepared in this investigation had less toxic effects on non-cancer cells in comparison with the cancer cell lines. The compounds of 7g and 7i exhibited the lowest toxic effects on PBMC cells whereas the 7a, 7n and 7o showed the highest toxicity on PBMC cells. However, all of the compounds prepared in this study showed higher toxicity on PBMC cells compared with 5FU. The high toxicity of rituximab on PBMC cell could be explained by the fact that the phytohemagglutinin used for the preparation of the cells results in the over expression of CD20 protein on the surface of the cells which acts as the receptor for rituximab. Therefore, the normal cells with the receptor for the cytotoxic drug could be affected.

Table 4 Cytotoxicity evaluation of new compounds 7a-o against three different cancer cell lines and PBMC cells.

		PBMC	Breast	Colon	B-CLL
		Normal	(MCF-7)	(HCT116)	(Waco3-CD5)
Compd.	R¹		IC₅₀(µM)
7a	H	62.20 ± 2.9	35.11 ± 4.3	28.56 ± 2.4	53.56 ± 3.3
7b	H	80.56 ± 1.7	47.85 ± 0.6	54.93 ± 1.0	91.29 ± 0.6.0
7c	H	94.01 ± 3.4	38.37 ± 2.3	34.36 ± 2.3	88.11 ± 1.8
7d	CH₃	78.62 ± 1.8	37.11 ± 3.1	21.18 ± 1.8	76.15 ± 4.6
7e	CH₃	83.31 ± 3.9	37.14 ± 2.5	22.14 ± 1.1	78.12 ± 0.1
7f	CH₃	106.74 ± 2.8	96.31 ± 7.0	93.05 ± 1.7	95.57 ± 0.9
7g	Isopropyl	127.92 ± 3.6	62.25 ± 6.1	71.92 ± 0.2	92.97 ± 0.2.7
7h	Isopropyl	93.47 ± 4.2	46.87 ± 3.8	68.28 ± 4.6	89.96 ± 3.6
7i	Isopropyl	129.1 ± 2.9	46.06 ± 0.5	60.41 ± 2.4	108.39 ± 5.9
7j	Cl	82.02 ± 3.7	16.14 ± 1.4	19.72 ± 2.6	49.11 ± 2.9
7k	Cl	91.13 ± 3.1	81.21 ± 2.3	80.04 ± 1.9	94.07 ± 3.8
7l	Cl	79.29 ± 4.6	15.22 ± 1.7	18.72 ± 1.5	51.01 ± 1.0
7m	Cl	72.51 ± 2.9	36.02 ± 2.2	21.61 ± 4.6	57.36 ± 2.4
7n	OCH₃	58.70 ± 2.6	34.79 ± 0.1	26.73 ± 3.8	76.11 ± 2.0
7o	OCH₃	53.52 ± 3.8	35.90 ± 2.4	64.19 ± 3.1	82.18 ± 2.4
5-FU^a	–		14.24 ± 1.2	18.21 ± 0.7	–
Rituximab^a	–	30.16 ± 3.7	–	–	17.36 ± 2.1

a 5-FU and Rituximab were used as standard anticancer drugs.

As shown in Table 4, the cytotoxic effects of 7j and 7l against breast cancer cell line as well the 7d, 7e, 7j, 7l, and 7m against colon cancer cells were comparable with the standard anticancer drug 5-FU. However, cytotoxic activities of the compounds 7a–o against human B-CLL (Waco3-CD5) were lower than the corresponding standard drug Rituximab (with minimal IC₅₀ approximately 3 folds higher than the positive control).

6 Conclusion

In this study we reported the synthesis, characterization and anticancer activities evaluation of new 1-aryl-2,3-dihydro-1H-naphtho[1,2-e][1,3]oxazine derivatives bearing an arylsulfonamide moiety. The target compounds were obtained from the reaction of N-sulfonyldithioimidocarbonates and Betti bases in the presence of Na₂CO₃ as a cheap base and in aqueous ethanol as a green medium. To study the potential anticancer and antifungal activities of the synthesized compounds, the screening tests were performed for three cancer cell lines and five fungi strains. The best anticancer activity were presented by 7j and 7l against all three kinds of cancer cell lines [HCT116, ATCC No.: CCL-247 (human colon cancer), MCF-7, ATCC No.: HTB-22 (human breast cancer), and Waco3-CD5, NCBI No.: C547 (human B-CLL)]. The results showed that, all the compound were less toxic on the normal cells of PBMC rather than the cancer cell lines which make them great candidates for further investigations. However, The MTT test could not be able to show the mechanism by which the compounds induce cytotoxicity on different cells including normal or cancer cell lines. Therefore, it is suggested to carry out the investigations on the mechanism of cytotoxic activity of the compounds to determine the potential role of different functional groups on the cytotoxic mechanism.

In vitro evaluations of the compounds 7a–o revealed that they presented only notable antifungal activities against Candida albicans (yeasts). The most of compounds 7a–o showed moderate antifungal activities but among them compounds 7c, 7e, 7f, 7i, 7l, 7m, and 7n exhibited the highest activities. These results are suggesting that the synthesized compounds can be good nominees for future investigations to find new anticancer and antifungal compounds.

Acknowledgements

We are thankful to the University of Isfahan Research Council and Shiraz University of Medical Sciences Research Council for financial support of this work.

Conflict of interest

The authors have declared no conflicts of interest.

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Appendix A

Supplementary material

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.arabjc.2017.10.009.

Appendix A

Supplementary material

Supplementary data 1

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Introduction

Experimental

Chemistry

Biology

In vitro investigation of antimicrobial activity
Determination of cytotoxicity

Results and discussion

Chemistry

Biology

Antimicrobial activity
Cytotoxic activity

Conclusion

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[1] Abou-Elmagd W.S.I., Hashem A.I., . Synthesis of 1-amidoalkyl-2-naphthols and oxazines derivatives with study of their antibacterial and antiviral activities. Med. Chem. Res.. 2013;22:2005-2013.
[Google Scholar]

[2] Akhter M., Habibullah S., Misbahul Hasan S., MumtazAlam M., Akhter N., Shaquiquzzaman M., . Synthesis of some new 3,4-dihydro-2H-1,3-benzoxazines under microwave irradiation in solvent-free conditions and their biological activity. Med. Chem. Res.. 2011;20:1147-1153.
[Google Scholar]

[3] Alvarez Ibarra C., Cereceda J.A., Ortiz P., Vicente A., Quiroga M.L., . A novel synthesis of 2,3-dihydrooxazoles from dimethyl N-ethoxycarbonyl methyliminodithiocarbonate and aromatic aldehydes. Tetrahedron Lett.. 1985;26:243-246.
[Google Scholar]

[4] Benameur L., Bouaziz Z., Nebois P., Bartoli M.-H., Boitard M., Fillion H., . Synthesis of furnaphth[1,3]oxazine and furo[1,3]oxazinoquinoline derivatives as precursors for an o-quinonemethide structure and potential antitumor agents. Chem. Pharm. Bull.. 1996;44:605-608.
[Google Scholar]

[5] Bernstein J., Davis R.E., Shimoni L., Chang N.-L., . Patterns in hydrogen bonding: functionality and graph set analysis in crystals. Angew. Chem Int. Ed. Engl.. 1995;34:1555-1573.
[Google Scholar]

[6] Betti M., . β-Naphtholphenylaminomethane. Org. Synth. Coll.. 1941;1:381.
[Google Scholar]

[7] Bouaziz Z., Riondel J., Mey A., Berlion M., Villard J., Fillion H., . Synthesis of some naphthoxazinecarbolactone derivatives with in vitro cytotoxic and antifungal activities. Eur. J. Med. Chem.. 1991;26:469-472.
[Google Scholar]

[8] Burke W.J., . 3,4-Dihydro-1,3,2H-benzoxazines. Reaction of p-substituted phenols with N,N-dimethylolamines. J. Am. Chem. Soc.. 1949;71:609-612.
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[9] Burke W.J., Reynolds R.J., . Condensation of 2-naphthol with acetaldehyde ammonia. J. Am. Chem. Soc.. 1954;76:1291-1293.
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[10] Burke W.J., Kolbezen M.J., Stephens C.W., . Condensation of naphthols with formaldehyde and primary amines. J. Am. Chem. Soc.. 1952;74:3601-3605.
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[Google Scholar]

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[Google Scholar]

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[35] Modak J.K., Liu Y.C., Supuran C.T., Roujeinikova A., . Structure-activity relationship for sulfonamide inhibition of Helicobacter pylori α-carbonic anhydrase. J. Med. Chem.. 2016;59:11098-11109.
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Synthesis of novel naphtho[1,2-e][1,3]oxazines bearing an arylsulfonamide moiety and their anticancer and antifungal activity evaluations

Abstract

Abstract

Keywords

Anticancer

Naphthoxazine

Sulfonamide

Synthesis

Antifungal

1 Introduction

2 Experimental

2.1 Chemistry

2.1.1 General procedure for the synthesis of 1-(amino(aryl)methyl)naphthalen-2-ol hydrochlorides (Betti base hydrochlorides, 3a-e)

2.1.2 General procedure for the synthesis of arylsulfonamides (5a-d)

2.1.3 General procedure for the synthesis of dimethyl arylsulfonylcarbonimidodithioates (6a-d)

2.1.4 General procedure for the synthesis of (E)-N-(1-aryl-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide derivatives (7a–o)

2.1.5 Spectral data (1H-NMR and 13C-NMR spectra) of the synthesized compounds

2.1.5.1 (E)-N-(1-phenyl-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7a)

2.1.5.2 (E)-4-methyl-N-(1-phenyl-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7b)

2.1.5.3 (E)-4-chloro-N-(1-phenyl-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7c)

2.1.5.4 (E)-N-(1-(p-tolyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7d)

2.1.5.5 (E)-4-methyl-N-(1-(p-tolyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7e)

2.1.5.6 (E)-4-chloro-N-(1-(p-tolyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7f)

2.1.5.7 (E)-N-(1-(4-isopropylphenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)-4-methylbenzenesulfonamide (7g)

2.1.5.8 (E)-4-chloro-N-(1-(4-isopropylphenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7h)

2.1.5.9 (E)-N-(1-(4-isopropylphenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)-4-methoxybenzenesulfonamide (7i)

2.1.5.10 (E)-N-(1-(4-chlorophenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7j)

2.1.5.11 (E)-N-(1-(4-chlorophenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)-4-methylbenzenesulfonamide (7k)

2.1.5.12 (E)-4-chloro-N-(1-(4-chlorophenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7l)

2.1.5.13 (E)-N-(1-(4-chlorophenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)-4-methoxybenzenesulfonamide (7m)

2.1.5.14 (E)-4-chloro-N-(1-(4-methoxyphenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7n)

2.1.5.15 (E)-4-methoxy-N-(1-(4-methoxyphenyl)-1H-naphtho[1,2-e][1,3]oxazin-3(2H)-ylidene)benzenesulfonamide (7o)

3 Biology

3.1 In vitro investigation of antimicrobial activity

3.1.1 Microorganisms

3.1.2 Determination of minimum inhibitory concentration

3.2 Determination of cytotoxicity

3.2.1 Cell line and culture conditions

3.2.2 Cytotoxic activity

3.2.2.1 MTT assay

4 Results and discussion

4.1 Chemistry

5 Biology

5.1 Antimicrobial activity

5.2 Cytotoxic activity

6 Conclusion

Acknowledgements

Conflict of interest

References

Appendix A

Supplementary material

Appendix A

Supplementary material

Supplementary data 1

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2.1.5 Spectral data (¹H-NMR and ¹³C-NMR spectra) of the synthesized compounds