- Email: [email protected]
New family of antimicrobial agents derived from 1,4-naphthoquinone
Accepted Manuscript New family of antimicrobial agents derived from 1,4-naphthoquinone Monika Janeczko, Oleg M. Demchuk, Dorota Strzelecka, Konrad Kubiński, Maciej Masłyk PII:
S0223-5234(16)30896-0
DOI:
10.1016/j.ejmech.2016.10.034
Reference:
EJMECH 8999
To appear in:
European Journal of Medicinal Chemistry
Received Date: 27 May 2016 Revised Date:
14 October 2016
Accepted Date: 15 October 2016
Please cite this article as: M. Janeczko, O.M. Demchuk, D. Strzelecka, K. Kubiński, M. Masłyk, New family of antimicrobial agents derived from 1,4-naphthoquinone, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.10.034. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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1,4-Naphthoquinone
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New family of antimicrobial agents derived from
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Monika Janeczkoa, Oleg M. Demchukb, Dorota Strzelecka,b Konrad Kubińskia, and Maciej
a
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Masłyka*
Department of Molecular Biology, Institute of Biotechnology, The John Paul II Catholic University of Lublin, ul. Konstantynów 1i, 20-708 Lublin, Poland
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Organic Chemistry Department, Faculty of Chemistry, Maria Curie-Skłodowska University,
*
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ul. Gliniana 33, 20-614 Lublin, Poland
Corresponding Author
Dr. Maciej Masłyk, PhD, Department of Molecular Biology, Institute of Biotechnology, The
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b
John Paul II Catholic University of Lublin, ul. Konstantynów 1i, 20-708 Lublin, Poland, Tel: +48814545452, e-mail: [email protected]
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ABSTRACT:
Naphthalene-1,4-dione derivatives were synthesized and tested against selected bacterial strains.
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All the tested compounds were prepared by direct introduction of corresponding substituents into the naphthoquinone core in oxidative conditions. In this study, eight strains of bacteria (Proteus, Escherichia,
Klebsiella,
Staphylococcus,
Enterobacter,
Pseudomonas,
Salmonella,
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Enterococcus) were used for determination of antimicrobial activity of synthesized compounds with the Minimal Inhibitory Concentration (MIC) method. Additionally, selected compounds
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were tested for haemolytic activity using human erythrocytes. All naphthalene-1,4-dione derivatives exhibited significant antimicrobial activity with MIC values between 7.8 and 500 µg/ml. A majority of the synthesized compounds showed the strongest antibacterial properties towards S. aureus, with a high level of selectivity. None of the tested naphthalene-1,4-dione
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KEYWORDS
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derivatives exhibited haemolytic activity.
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Antimicrobial, naphthoquinones, Staphylococcus aureus, haemolysis
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INTRODUCTION In the recent decade, a problem of an increasing number of bacterial pathogens exhibiting
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multidrug resistance to antibiotics has been observed. According to the World Health Organization, multidrug-resistant bacteria are responsible for approximately 25 000 deaths in Europe each year. The accumulation of antibacterial agents in the environment promotes spread
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of resistant microorganisms, turning the environment into a gigantic reservoir for antibiotic resistance genes [1]. An important aspect of bacterial resistance is represented by bacteria that
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cause nosocomial infections worldwide such as Staphylococcus aureus, Pseudomonas aeruginosa, or Streptococcus pneumoniae [2-4]. One of the evident examples is the methicillin and vancomycin-resistant Staphylococcus aureus, which accounts for a high percentage of hospital-acquired infections. S. aureus has the ability to form biofilms on biomaterials, which is responsible for its resistance towards antimicrobial agents, which makes it difficult to eradicate
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the bacteria from the infected hosts [3, 5]. S. aureus is frequently isolated in association with peripheral intravascular catheters, endotracheal and tracheotomy tubing, peritoneal dialysis tubing, prosthetic joint, vascular graft infections, and corneal infections related to contact lens
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wear. Chronic infection can serve as a septic focus that can lead to osteomyelitis, acute sepsis,
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and death, particularly in immunocompromised patients [6-13]. The Joint Programming Initiative on Antimicrobial Resistance supported by 18 European countries plus Canada recommended promotion of research and development of novel antimicrobial strategies and antibacterial agents as one of the key measures that should be adopted to fight the emergence and spread of antibiotic resistance worldwide [1]. Regarding development of novel antimicrobial drugs, naphthoquinone derivatives arouse wide interest due to their diverse functions and clinical applications. This moiety is present in many
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natural compounds such as plumbagin, juglone, lawsone, menadione, and lapachol. One of the examples is vitamin K playing a very important role in regulating blood coagulation, bone metabolism, and vascular biology [14, 15]. Naphthoquinones show cytotoxic, insecticidal, anti-
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inflammatory, and antipyretic activities [16]. They are also widely used as anticancer, antimalarial, and antimicrobial agents [17-21]. The biological relevance of 1,4-naphthoquinone is dependent of quinone redox cycling that yields "reactive oxygen species" (ROS) as well as
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arylation reactions [22]. The antimicrobial activity of naphthoquinone derivatives is also known and frequently studied in various microorganisms [23, 24]. In addition to their antibacterial
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activity, naphthoquinones possess antifungal activity [25-27].
The major objective of the present study was to carry out the synthesis and biological evaluation of a new series of 1,4-naphthoquinone derivatives. The compounds obtained were tested against a panel of gram-positive and gram-negative bacterial strains. Additionally, we have verified their
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toxicity by means of a haemolytic assay of selected compounds against human erythrocytes.
Chemistry
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MATERIALS AND METHODS
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General. All reagents were purchased from Sigma-Aldrich, Strem, TCI, and Alfa Aesar chemical companies and used without further purification. Analytical thin-layer chromatography (TLC) was performed using silica gel 60 F254 precoated plates (0.25 mm thickness) with a fluorescent indicator. Visualisation of TLC plates was performed by means of UV light or either KMnO4 or I2 stains. NMR spectra were recorded on Bruker Avance 500 MHz spectrometers, and chemical shifts are reported in ppm, and calibrated to residual solvent peaks at 7.27 ppm and 77.00 ppm for 1H and 13C in CDCl3 or internal reference compounds. The following abbreviations are used
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in reporting the NMR data: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad). Coupling constants (J) are in Hz. Spectra are reported as follows: chemical shift (δ, ppm), multiplicity, integration, coupling constants (Hz). Products were purified by flash
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chromatography on silica gel 60 (230-400 mesh) using a BUCHI chromatograph. MS spectra were recorded on a Shimadzu LCMS IT-TOF spectrometer.
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General procedure I: palladium catalysed arylation of naphthoquinone (Scheme 1: cat. = Pd(OAc)2, additive = CF3CO2H, oxidant = Bz2O2, solvent = CCl4 or ArH). A test tube equipped
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with a stirring bar was charged with 1,4-naphthoquinone (47 mg, 0.3 mmol), Pd(OAc)2 (6.7mg, 10 mol%), Bz2O2 (145 mg, 0.6 mmol), TFA (115 µL, 1.5 mmol) and aromatic compounds (2 mL), the mixture was stirred at 30 oC for 24 h, and then extracted with CH2Cl2 (2x10 mL); the combined organic layers were dried over anhydrous MgSO4. The crude product was purified by
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column chromatography using a gradient hexane/acetone mixture as eluent. 2-phenylnaphthalene-1,4-dione (3): obtained according to the general procedure I. Yield: 49 mg (70 %), yellow solid, mp = 111.3- 113.8 oC (lit. mp = 108-110 oC) [28]. 1H NMR (500.13
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MHz, CDCl3): δ = 7.04 (s, 1H, CH); 7.45-7.46 (m, 3H, CH); 7.55-7.57 (m, 2H, CH); 7.73-7.75 (m, 2H, CH); 8.06-8.08 (m, 1H, CH); 8.13-8.15 (m, 1H, CH) ppm.
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C NMR (125.75 MHz,
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CDCl3): δ = 125.76, 126.20, 126.85, 128.29, 129.30, 129.86, 131.86, 132.25, 133.22, 133.64, 133.70, 133.74, 135.01, 138.45, 147.84, 184.12, 184.88 ppm.
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C NMR (DEPT 135, 125.75
MHz, CDCl3): δ = 125.93 (CH), 127.02 (CH), 128.42 (CH), 129.38 (CH), 129.99 (CH), 133.79 (CH), 133.85 (CH), 135.18 (CH) ppm. HRMS (ESI): m/z = not ionisable [M+H]+.
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2,3-diphenylnaphthalene-1,4-dione (4). obtained as a by-product in the synthesis of 3. Yield: 20 mg (22 %), yellow solid, mp = 135.7 - 138.0 oC (lit. mp = 134-135 oC) [28]. 1H NMR (500.13 MHz, CDCl3): δ = 7.06-7.08 (m, 4H, CH); 7.21-7.23 (m, 6H, CH); 7.76-7.78 (m, 2H, CH); 8.1713
C NMR (125.75 MHz, CDCl3): δ = 126.63, 127.65, 128.24, 130.55,
132.15, 133.26, 133.88, 145.78, 184.78 ppm.
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8.18 (m, 2H, CH) ppm.
C NMR (DEPT 135, 125.75 MHz, CDCl3): δ =
126.63 (CH), 127.65 (CH), 128.24 (CH), 130.56 (CH), 133.88 (CH) ppm. HRMS (ESI): m/z =
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not ionisable [M+H]+.
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2-(3,4-dimethylphenyl)naphthalene-1,4-dione (5): obtained according to the modified general procedure I. o-Xylene was used instead of benzene. Yield: 40 mg (51 %), yellow solid, mp = 156.7-158.7 oC (lit. mp = 151 - 153 oC) [29]. 1H NMR (500.13 MHz, CDCl3): δ = 1H NMR (500.13 MHz, CDCl3): δ = 2.34 (s, 3H, CH3); 2.35 (s, 3H, CH3); 7.07 (s, 1H, CH); 7.25 (d, J =
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7.88 Hz, 1H, CH); 7.34 (dd, J = 7.88, 1.89 Hz, 1H, CH); 7.37 (s, 1H, CH); 7.77-7.79 (m, 2H, CH); 8.12-8.13 (m, 1H, CH); 8.18-8.20 (m, 1H, CH) ppm. 13C NMR (125.75 MHz, CDCl3): δ = 19.77, 19.88, 125.92, 126.92, 126.97, 127.02, 129.81, 130.53, 132.14, 132.58, 133.74, 133.79, 13
C NMR (DEPT 135, 125.75 MHz, CDCl3): δ =
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134.52, 136.82, 139.20, 184.67, 185.27 ppm.
19.77 (CH3), 19.88 (CH3), 125.92 (CH), 126.98 (CH), 127.03 (CH), 129.81 (CH), 130.53 (CH),
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133.74 (CH), 133.79 (CH), 134.52 (CH) ppm. HRMS (ESI): m/z = not ionisable [M+H]+.
2-Mesityl-1,4-naphthoquinone (6): obtained in a 6.3 mmol scale according to the modified general procedure I. Mesitylene was used instead of benzene at 70 oC. Yield: 0.700 g (40 %), yellow solid, mp = 169.0 - 171.0. 1H NMR (500.13 MHz, CDCl3): δ = 2.12 (s, 6H, CH3); 2.34 (s, 3H, CH3); 6.88 (s, 1H, CH); 6.96 (bs, 2H, CH); 7.79 - 7.81 (m, 2H, CH); 8.15-8.17 (m, 2H, CH)
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ppm. 13C NMR (125.75 MHz, CDCl3): δ = 20.39, 21.14, 126.19, 127.07, 128.38, 130.55, 132.24, 133.87, 133.90, 135.45, 137.88, 150.49, 185.16 ppm.
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CDCl3): δ = 20.39 (CH3), 21.14 (CH3), 126.19 (CH), 127.07 (CH), 128.38 (CH), 133.87 (CH),
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133.90 (CH), 137.89 (CH) ppm. Anal. Calcd. for C19H16O2: C, 82.58; H, 5.84; O, 11.58. Found: C, 81.85; H, 5.77; O, 12.38. HRMS (ESI): m/z = not ionisable [M+H]+.
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6',7'-dimethoxy-2,2'-binaphthalene-1,4-dione (7): obtained according to the modified general procedure I. 2,3-dimethoxynaphthalene (113 mg, 0.6 mmol) and CCl4 (2 mL) were used instead
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of benzene at 50 oC. Yield: 20 mg (20 %), dark purple solid, mp = 228.3 - 230.5 oC. 1H NMR (500.13 MHz, CDCl3): δ = 4.03 (s, 3H, OCH3); 4.05 (s, 3H, OCH3); 7.16 (s, 1H, CH); 7.19 (s, 1H, CH); 7.22 (s, 1H, CH); 7.54 (dd, J = 6.62, 1.89 Hz, 1H, CH); 7.78 (d, J = 8.51 Hz, 1H, CH); 7-79-7.83 (m, 2H, CH); 8.02-8.03 (bd, 1H, CH); 8.14 - 8.16 (m, 1H, CH); 8.22 - 8.24 (m, 1H, 13
C NMR (125.75 MHz, CDCl3): δ = 55.96, 55.98, 106.04, 107.08, 124.70, 125.96,
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CH) ppm.
126.54, 127.07, 128.34, 128.78, 129.03, 130.09, 132.18, 132.66, 133.83, 134.79 149.99, 150.77, 185.17 ppm.
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C NMR (DEPT 135, 125.75 MHz, CDCl3): δ = 55.96 (OCH3), 55.99 (OCH3),
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106.04 (CH), 107.08 (CH), 124.70 (CH), 125.96 (CH), 126.55 (CH), 127.07 (CH), 128.35 (CH), 133.82 (CH), 133.83 (CH), 134.79 (CH) ppm. HRMS (ESI): m/z = 345.1114 [C22H16O4+H]+,
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m/z (theor.) = 345.1121, diff. = -2.03 ppm.
General procedure II: Friedel-Crafts arylation of naphthoquinone (Scheme 1: cat. = H3PW12O40, oxidant = air, solvent = DMSO/CH3CO2H). A 50 mL flask equipped with a magnetic stirrer was charged with 1,4-naphthoquinone (1) (20 mmol; 3.16 g), aromatic compounds (10 mmol), 15 mL of CH3COOH and H3PW12O40 (288 mg; 1 mol%). The flask was
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heated at 100 oC for 24 hours. After that time, the flask was cooled down to room temperature and 15 mL of chloroform was added. The solution was washed with 20 mL of brine. The organic phase was separated and dried by MgSO4. The solvent was evaporated under reduced pressure
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and the residue was purified by column chromatography on silica gel, using a gradient hexane/acetone mixture as eluent.
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2-(2,4-dimethoxyphenyl)naphthalene-1,4-dione (8): obtained according to the modified general procedure II. Instead of acetic acid, acetone was used in reflux conditions. The product
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was crystalized from the hexane/acetone mixture. Yield: 77%, mp = 155.3-56.9 oC. 1H NMR (500.13 MHz, CDCl3): δ = 3.79 (s, 3H, CH3) 3.87 (s, 3H, CH3) 6.56-6.60 (m, 2H, CH) 7.04 (s, 1H, CH) 7.21 - 7.22 (d, J= 8.51, 1H, CH) 7.74 - 7.76 (m, 2H, CH) 8.10-8.17 (m, 2H, CH) ppm. 13
C NMR (125.75 MHz, CDCl3): δ = 55.23, 55.46, 98.79, 104.44, 115.61, 125.64, 126.66,
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131.38, 131.92, 132.44, 133.20, 133.33, 135.98, 147.15, 158.36, 162.06, 183.71, 185.15 ppm. C NMR (DEPT 135, 125.75 MHz, CDCl3): δ = 55.51 (OCH3), 55.74 (OCH3), 99.06 (CH),
104.71 (CH), 125.92 (CH), 126.95 (CH), 131.67 (CH), 133.49 (CH), 133.61 (CH), 136.26 (CH)
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ppm. HRMS (ESI): m/z = 317.0770 [C18H14O4+Na]+, m/z (theor.) = 317.0784, diff. = 4.42 ppm.
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2-(2,4,6-trimethoxyphenyl)naphthalene-1,4-dione (9): obtained according to the modified general procedure II. Instead of acetic acid, acetone was used in reflux conditions. Yield: 85%, mp = 183 oC. 1H NMR (500.13 MHz, CDCl3): δ = 3.74 (s, 6 H, OCH3) 3.87 (s, 3 H, OCH3) 6.21 (s, 2 H, CH) 6.96 (s, 1 H, CH) 7.73- 7.75 (m, 2 H, CH) 8.11 - 8.14 (m, 2 H, CH) ppm. 13C NMR (125.75 MHz, CDCl3): δ = 55.,67 56.09, 91.08, 104.88, 126.16, 127.07, 132.51, 133.08, 133.49, 133.67, 138.93, 145.14, 159.01, 162.63, 183.99, 185.70 ppm.
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C NMR (DEPT 135, 125.75
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MHz, CDCl3): δ = 55.41 (COCH), 55.83 (OCH3)3, 90.81 (CH), 125.90 (CH), 126.82 (CH), 133.24 (CH), 133.42 (CH), 138.67 (CH) ppm. HRMS (ESI): m/z = 325.1066 [C19H16O5+H]+,
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m/z (theor.) = 325.1071, diff. = -1.54 ppm
2-[4-(Dimethylamino)phenyl]naphthalene-1,4-dione (10): obtained according to the general procedure II. Yield: 35%, mp = 140.5-141.5 oC. 1H NMR (500.13 MHz, CDCl3): δ = 3.05 (s, 6H,
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CH3) 6.74-6.77 (m, 2H, CH) 7.02 (s, 1H, CH) 7.59- 7.62 (m, 2H, CH) 7.72-7.75 (m, 2H, CH) 8.08-8.11 (m, 1H, CH) 8.15-8.17 (m, 1H, CH) ppm. 13C NMR (125.75 MHz, CDCl3): δ = 40.16,
185.25, 185.51 ppm.
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11.73, 120.52, 125.71, 126.92, 130.98, 131.18, 132.33, 132.92, 133.38, 133.54, 147.43, 151.74, C NMR (DEPT 135. 125.75 MHz, CDCl3): δ = 40.07 (CH3), 111.65
(CH), 125.61 (CH), 126.84 (CH), 130.92 (CH), 131.05 (CH), 133.29 (CH), 133.45 (CH) ppm.
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HRMS (ESI): m/z = 278.1184 [C18H15NO2+H]+, m/z (theor.) = 278.1176, diff. = 2.88 ppm
2-[4-(Dimethylamino)-2-methylphenyl]naphthalene-1,4-dione (11): obtained according to the general procedure II. Yield: 37%, mp = 158.3-159.2 oC. 1H NMR (500.13 MHz, CDCl3): δ =
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2.25 (s, 3H, CH3) 3.02 (s, 6H, N(CH3)2) 6.61-6.63 (m, 2H, CH) 6.91 (s, 1H, CH) 7.11-7.13 (m, 1H, CH) 7.76-7.78 (s, 2H, CH) 8.12-8.18 (m, 2H, CH) ppm. 13C NMR (125.75 MHz, CDCl3): δ
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= 21.39, 40.31, 109.52, 114.08, 121.60, 125.94, 127.02, 131.05, 132.29, 132.62, 133.61, 133.63, 135.78, 137.56, 150.52, 151.26, 184.76, 185.42 ppm.
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C NMR (DEPT 135, 125.75 MHz,
CDCl3): δ = 21.39 (CH3), 40.31 (CH3), 109.51 (CH), 114.07 (CH), 125.93 (CH), 127.01 (CH), 131.04 (CH), 133.61 (CH), 135.77 (CH) ppm. HRMS (ESI): m/z = 292.1335 [C19H17NO2+H]+, m/z (theor.) = 292.1332, diff. = 1.03 ppm
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2-[4-(dibenzylamino)phenyl]naphthalene-1,4-dione (12): obtained according to the general procedure II. Yield: 25%, mp = 145.3 - 146.7 oC. 1H NMR (500.13 MHz, CDCl3): δ = 4.74 (s, 4H, CH2) 6.81-6.84 (m, 2H, CH) 7.01 (s, 1H, CH) 7.26-7.31 (m, 6H, CH) 7.35-7.38 (m, 4H, CH) 13
C NMR (125.75
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7.53-7.56 (m, 2H, CH) 7.73-7.76 (m, 2H, CH) 8.09-8.18 (m, 2H, CH) ppm.
MHz, CDCl3): δ = 54.05, 112.12, 121.31, 125.74, 126.53, 126.96, 127.24, 128.86, 131.20, 131.46, 132.32, 132.87, 133.43, 133.59, 137.61, 147.27, 150.83, 185.21, 185.44 ppm. 13C NMR
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(DEPT 135, 125.75 MHz, CDCl3): δ = 54.05 (CH2), 112.11 (CH), 125.73 (CH), 126.52 (CH), 127.23 (CH), 128.84 (CH), 131.18 (CH), 131.45 (CH), 133.42 (CH), 133.58 (CH) ppm. HRMS
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(ESI): m/z = 430.1804 [C30H23NO2+H]+, m/z (theor.)= 430.1802, diff. = 0.43 ppm
2-[4-(Dimethylamino)-2-methoxyphenyl]naphthalene-1,4-dione (13); obtained according to the general procedure II. Yield: 18%, mp = 144.2-145.6 oC. 1H NMR (500.13 MHz, CDCl3): δ =
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3.04 (s, 6H, N(CH3)2) 3.82 (s, 3H, OCH3) 6.27 (s, 1H, CH) 6.37-6.39 (d, J = 8.83, 1H, CH) 7.09 (s, 1H, CH) 7.20-7.21 (d, J = 8.51, 1H, CH) 7.72- 7.74 (m, 2H, CH) 8.09- 8.15 (m, 2H, CH) ppm. 13C NMR (125.75 MHz, CDCl3): δ = 40.36, 55.52, 95.51, 104.44, 110.89, 125.73, 126.86,
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132.14, 132.32, 133.02, 133.29, 133.32, 134.81, 147.47, 153.03, 158.94, 184.64, 185.55 ppm. C NMR (DEPT 135, 125.75 MHz, CDCl3): δ = 40.36 (N(CH3)2), 55.52 (CH3), 95.52 (CH),
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104.44 (CH), 125.73 (CH), 126.86 (CH), 132.13 (CH), 133.29 (CH), 133.32 (CH), 134.80 (CH) ppm. HRMS (ESI): m/z = 308.1271 [C19H17NO3+H]+, m/z (theor.)= 308.1281, diff. = -3.25 ppm
N-[4-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)-3-methoxyphenyl]acetamide (14): obtained according to the general procedure II. Yield: 483 mg (15 %), mp= 209-210 oC (dec.). 1H NMR (500.13 MHz, CDCl3): δ = 2.21 (s, 3H, NHCOCH3) 3.80 (s, 3H, OCH3) 6.89-6.91 (m, 1H, CH)
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7.04 (s, 1H, CH) 7.18-7.20 (d, J = 8.20, 1H, CH) 7.42 (s, 1H, CH) 7.59 (s, 1H, CH) 7.75-7.79 (m, 2H, CH) 8.11-8.17 (m, 2H, CH) ppm. 13C NMR (125.75 MHz, DMSO-d6): δ = 24.34, 55.72, 102.36, 110.74, 117.67, 125.73, 126.63, 131.12, 131.73, 132.25, 134.28, 134.38, 135.93, 142.22, 13
C NMR (DEPT 135, 125.75 MHz, CDCl3): δ =
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147.41, 157.46, 168.82, 183.40, 184.75 ppm.
24.83 (CH3), 55.84 (OCH3), 103.23 (CH), 111.04 (CH), 126.02 (CH), 126.96 (CH), 130.95 (CH), 133.64 (CH), 133.74 (CH), 136.63 (CH) ppm. HRMS (ESI): m/z = 322.1070
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[C19H15NO4+H]+, m/z (theor.) = 322.1074, diff. = -1.24 ppm
General procedure III: amination of naphthoquinone (Scheme 2: cat. = none, oxidant = air, solvent = EtOH/H2O, additive = Et3N). H2O (2.5 mL) and Et3N (700 µL, 5.0 mmol) were added to a suspension of 1,4-naphthoquinone (790 mg, 5.0 mmol) and amino acid (2.5 mmol) in EtOH
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(50 mL) and the mixture was stirred at room temperature for 24 h. The solvent was evaporated, and the crude product was extracted by 100 mL of a 5% aqueous solution of Na2CO3. The extract was washed with EtOAc (50 mL), the separated aqueous layer was acidized with 36% HCl, and
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the crude product was extracted with EtOAc (100 mL). The organic layer was separated, dried over anhydrous MgSO4, and the solvent was removed under reduced pressure after filtration. The
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product was purified by crystallization from warm methanol or by column chromatography using a gradient CH2Cl2/hexane/EtOAc/MeOH mixture as eluent.
1-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)-L-proline (15): obtained according to the general procedure III; the product was not soluble in EtOAc. It was precipitated after acidisation of the extract and used without further purification. Yield: 270 mg (40 %), red solid, mp = 151.2-153.0
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C (dec.) (lit. mp = 165-168 oC)[30]. 1H NMR (500.13 MHz, DMSO-d6): δ = 1.84-1.91 (m, 1H),
1.94-1.99 (m, 1H) 2.04-2.10 (m, 1H) 2.24-2.31 (m, 1H), 3.45 (bs, 2H), 4.99 (s, 1H), 5.77 (s, 1H), 7.73 (t, J = 7.57 Hz, 1H, CH), 7.80-7.84 (t, J = 7.57 Hz, 1H, CH), 7.91-7.93 (m, 2H, CH), 12.74
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(br, 1H, CO2H) ppm. 13C NMR (125.75 MHz, DMSO-d6): δ = 21.52, 30.76, 50.58, 62.16, 69.67, 104.53, 124.46, 125.87, 130.97, 131.98, 134.13, 145.46, 148.42, 180.86, 182.32 ppm. 13C NMR (DEPT 135, 125.75 MHz, DMSO-d6): δ = 21.59 (CH2), 30.83 (CH2), 50.68 (CH2), 62.23 (CH),
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104.60 (CH), 124.54 (CH), 125.94 (CH), 132.05 (CH), 134.20 (CH) ppm. HRMS (ESI): m/z =
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272.0923 [C15H13NO4+H]+, m/z (theor.) = 272.0917, diff.= 2.21 ppm.
N-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)valine (16): obtained according to the general procedure III and purified by column chromatography using a mixture of hexane-EtOAc-MeOH (5:3:1) as eluent. Yield: 270 mg (40 %), red solid, mp = 171.1-173.1 oC (dec.). 1H NMR (500.13
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MHz, DMSO-d6): δ = 0.91 (d, J = 6.94 Hz, 3H, CH3); 0.96 (d, J = 6.62 Hz, 3H, CH3); 2.23-2.27 (m, 1H, CH); 3.93-3.96 (m, 1H, CH); 5.72 (s, 1H, CH); 6.85 (d, J = 8.51 Hz, 1H, CH); 7.76 (dt, J = 7.57, 1.26 Hz, 1H, CH); 7.85 (dt, J = 7.57, 1.26 Hz, 1H, CH); 7.94 (dd, J = 7.57, 0.95 Hz, 1H,
EP
CH); 8.01 (dd, J = 7.57, 0.95 Hz, 1H, CH), 13.22 (bs, 1H, OH) ppm.
13
C NMR (125.75 MHz,
DMSO-d6): δ = 18.24, 18.36, 29.54, 59.88, 100.66, 125.09, 125.09, 125.71, 129.86, 132.18,
AC C
132.42, 134.69, 147.35, 171.68, 180.88, 181.52 ppm.
13
C NMR (125.75 MHz, DMSO-d6): δ=
19.03 (CH3), 19.15 (CH3), 30.33 (CH), 60.67 (CH), 101.44 (CH), 125.88 (CH), 126.52 (CH), 132.98 (CH), 135.49 (CH) ppm. HRMS (ESI): m/z = 272.0929 [C15H15NO4-H]-, m/z (theor.) = 272.0928, diff. = 0.37 ppm.
12
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N-(1,4-Naphthoquinone-2-yl)-L-tryptophan (17): obtained according to the general procedure III. Yield: 570 mg (63 %), red solid, mp = 159.1 - 161.3 oC (dec.) (lit. mp = 208.0211.0 oC (dec.))[30, 31]. 1H NMR (500.13 MHz, DMSO-d6): δ = 3.23 (d, J = 5.67 Hz, 2H), 4.34
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– 4.36 (m, 1H), 5.60 (s, 1H), 6.78 – 6.83 (m, 2H), 6.90 (dt, J = 7.8, 1.0 Hz, 1H), 7.04 (d, J = 2.2 Hz, 1H), 7.17 (dd, J = 8.2, 1.0 Hz, 1H), 7.37 (d, J = 8.2 Hz, 1 H), 7.60 (dt, J = 7.6, 1.3 Hz, 1H), 7.70 (dt, J = 7.6, 1.6 Hz, 1H), 7.78–7.84 (m, 2H), 10.76 (br, 1H), 13.10 (bs, 1H) ppm. 13C NMR
SC
(125.75 MHz, DMSO-d6): δ = 25.84, 54.93, 100.60, 108.53, 111.11, 117.86, 118.17, 120.67, 123.74, 125.07, 125.63, 126.96, 129.79, 132.08, 132.45, 134.64, 135.74, 146.92, 171.85, 180.85, 13
C NMR (DEPT 135, 125.75 MHz, DMSO-d6): δ = 26.58 (CH2), 55.66 (CH),
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181.41 ppm.
101.38 (CH), 111.89 (CH), 118.64 (CH), 118.94 (CH), 121.45 (CH), 124.53 (CH), 125.87 (CH), 126.46 (CH), 132.93 (CH), 135.48 (CH) ppm. HRMS (ESI): m/z = 383.0994 [C21H16N2O4+Na]+,
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m/z (theor.) = 383.1002, diff. = 2.09 ppm.
[(1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino]-D-(phenyl)acetic
acid
(18):
obtained
according to the general procedure III. Yield: 70 mg (9%), red solid, mp = 195.3 – 199.5 oC
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(dec.). 1H NMR (500.13 MHz, DMSO-d6): δ = 4.56 (d, J = 5.99 Hz, 1H), 5.20 (s, 1H), 7.19 (t, J = 7.25 Hz, 1H), 7.27 (t, J = 7.25 Hz, 2H, CH), 7.36 (d, J = 7.57 Hz, 2H, CH) 7.71 (t, J = 7.57 Hz,
ppm.
AC C
1H, CH), 7.79 (t, J = 7.57, 1H, CH), 7.86 (d, J = 7.25 Hz, 1H, CH), 8.00 (d, J = 6.62, 2H, CH), 13
C NMR (125.75 MHz, DMSO-d6): δ = 61.30, 98.36, 101.05, 122.68, 122.97, 125.56,
125.80, 126.18, 126.38, 126.93, 127.17, 128.40, 129.07, 129.41, 131.38, 132.62, 135.33, 155.67 ppm.
13
C NMR (DEPT 135, 125.75 MHz, DMSO-d6): δ = 57.14 (CH), 101.45 (CH), 124.71
(CH), 125.28 (CH), 126.60 (CH), 127.83 (CH), 128.04 (CH), 131.83 (CH), 134.27 (CH) ppm. HRMS (ESI): m/z = 308.0921 [C18H13NO4+H]+, m/z (theor.) = 308.0917, diff. = 1.30 ppm.
13
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Methyl N-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)-L-alaninate (19). H2O (2.5 mL) and Et3N (700 µL, 5.0 mmol) were added to a suspension of 1,4-naphthoquinone (790 mg, 5.0 mmol)
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and L-alanine methyl ester hydrochloride (350 mg, 2.5 mmol) in MeOH (50 mL) and the mixture was stirred at room temperature for 24 h. The solvent was evaporated under reduced pressure and the product was purified by column chromatography using a mixture of hexane-acetone (9:1) as
SC
eluent. Yield: 63 mg (10%), yellow solid, mp = 91.4 – 93.5 oC (lit. mp = 188 oC) [32]. 1H NMR (500.13 MHz, CDCl3): δ = 1.58 (d, J = 6.94 Hz, 3H, CH3); 3.81 (s, 3H, OCH3); 4.13 (p, J = 6.94
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Hz, 1H, CH); 5.68 (d, J = 0.63 Hz, 1H, CH); 6.31 (bd, J = 6.94 Hz, 1H, NH) 7.65 (dt, J = 7.57, 1.26 Hz, 1H, CH); 7.74 (dt, J = 7.57, 1.26 Hz, 1H, CH); 8.07-8.11 (m, 2H, CH) ppm. 13C NMR (125.75 MHz, CDCl3): δ = 17.33, 50.19, 52.56, 101.74, 125.90, 126.11, 130.18, 131.95, 132.97, 134.47, 146.26, 171.76, 181.08, 182.91 ppm. 13C NMR (125.75 MHz, CDCl3): δ = 17.62 (CH3),
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50.48 (CH), 52.85 (CH), 102.05 (CH), 126.20 (CH), 126.40 (CH), 132.24 (CH), 134.76 (CH) ppm. HRMS (ESI): m/z = 260.0905 [C14H13NO4+H]+, m/z (theor.) = 260.0917, diff. = 4.61 ppm.
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Biological activities
Microbial strains. In present study, reference bacterial strains were used. Gram-positive
AC C
bacteria: Staphylococcus aureus (ATCC 6538), Enterococcus faecalis (PCM 2673), Gramnegative: Pseudomonas aeruginosa (PCM 2562), Klebsiella pneumoniae (PCM1), Escherichia coli (ATCC 8739), Enterobacter cloacae (PCM 2569), Proteus vulgaris (PCM 2668), Salmonella bongori (PCM 2552),
14
ACCEPTED MANUSCRIPT
MIC determination. Bacterial strains were inoculated in Mueller–Hinton broth (Biocorp, Poland) for 24h before performing the minimal inhibitory concentration (MIC) test and incubated at 37 °C with vigorous shaking (180 rpm). MIC was determined with the microbroth
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dilution method. Bacterial suspensions in Mueller–Hinton liquid medium at initial inoculums of 5x105 colony forming units per ml were added to polystyrene 96-well plates and exposed to the investigated naphthoquinones at adequate concentrations (range: 0.001 – 5 mg/ml) for 20 h at 37 o
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C. DMSO was used as a solvent. MICs were taken as the lowest drug concentration at which
observable growth was inhibited. Tetracycline (TET) was used as a reference compound.
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Experiments were performed in triplicate.
Haemolytic assay. The human blood samples were placed in sterile tubes containing a citrate
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dextrose solution as anticoagulant.
In order to separate erythrocytes from plasma, the samples were centrifuged at 500×g for 10 minutes at 4 °C and the supernatant was discarded. Next, the erythrocytes were resuspended with
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PBS buffer (10 mM phosphate, pH 7.5; 150 mM NaCl) and centrifuged as previously. The washing procedure was repeated until a transparent supernatant was obtained. The washed
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erythrocytes were finally resuspended in PBS buffer to a final concentration of 2%. Simultaneously, appropriate concentrations (in the range: 1 - 250 µg/ml) of the examined compounds were prepared in a final volume of 50 µl DMSO. The compounds prepared in this way were mixed with 450 µl of 2% erythrocyte suspension followed by incubation for 1h at 37 °C. Then, the samples were centrifuged at 5000×g for 10 minutes and absorbance at wavelength 415 nm was measured.
15
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RESULTS AND DISCUSSION Keeping in mind the known biological activity of quinone derivatives, a series of
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naphthoquinones substituted by the aryl group, which can be easily prepared with the simple one-step modular synthesis of 2-arylnaphthaquinones, were assessed as potential antimicrobial agents. The antimicrobial activity of unsubstituted naphthoquinones (1, 2) was determined as a
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reference.
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Chemistry
Depending on the desired substitution pattern, different sub-classes of 2-arylonaphthoquinones 3-14 were obtained according to the complementary synthetic approaches. The coupling of weakly activated aromatics with naphthoquinone leading to 2-arylonaphthoquinones 3-7 was performed in palladium trifluoroacetate-catalysed conditions according to the modified
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procedure presented in the literature (cat. = Pd(OAc)2, additive = CF3CO2H, oxidant = Bz2O2, solvent = CCl4 or ArH, t = 60 oC) (Scheme 1) [33]. Despite the low yields observed in some of these cases, application of cationic palladium catalysis yields products with substituents in
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positions that do not correspond to the usual reactivity of aromatics undergoing the electrophilic
AC C
substitution reaction. Sites of molecules thus activated by the substitution effects are left accessible to further transformation or biochemical interactions. In all cases, the palladiumcatalysed reaction always yields less sterically hindered substitution products.
16
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ACCEPTED MANUSCRIPT
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Scheme 1. Synthesis of 2-arylnaphthoquinones (3-14)
Quinones 8-14, bearing more electron-rich aryl substituents, were obtained in coupling reactions
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of naphthoquinone with corresponding reactive aromatics according to the Friedel-Crafts protocol in Brönsted acid-mediated conditions (Scheme 1) (cat. = H3PW12O40, oxidant = air, solvent DMSO/CH3CO2H, additive - none). These reactions follow the SE2Ar mechanism furnishing products substituted in predictable positions [34].
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Finally, the substituted aminonaphthoquinones 15-19 were prepared by a modified Katritzky procedure starting from naphthoquinone and corresponding D or L-amino acids or esters (Scheme 2) (cat. = none, oxidant = air, solvent = EtOH/H2O, additive = Et3N) [31]. For
EP
comparison, 2-(phenylamino)naphthalene-1,4-dione (2) was obtained in a reaction catalysed by copper acetate (Scheme 2) (cat. = Cu(OAc)2, solvent = CH3CO2H, oxidant = air) according to the
AC C
procedure described in the literature [35].
Scheme 2. Synthesis of 2-aminonaphthoquinones (2, 15-19)
17
ACCEPTED MANUSCRIPT
All new compounds were fully characterised while the structure of the known compounds were confirmed by comparison of experimental and physical data from the literature. In such a way, a
AC C
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function groups was prepared for the biological tests.
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series of 19 naphthoquinones substituted in positions 2 and 3 and bearing a wide pattern of
18
AC C
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ACCEPTED MANUSCRIPT
Figure 1. Assessed naphthoquinones 19
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Biology Certain alkyl-, alkenyl- and amino-1,4-naphthoquinones have already been assessed as
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prospective building blocks for the construction of numerous biologically important compounds [31, 32, 36, 37]. At the same time, the much more readily available and significantly more stable arylo-1,4-naphthoquinones still require more comprehensive biological studies.
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All the synthesized compounds (Figure 1) were tested against a panel of microorganisms and the MIC values were calculated. Each of the examined naphthoquinone derivatives exhibited
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certain antimicrobial activity with predominant MIC values between 7.8 and 500 µg/ml. In terms of the most promising bacterial target of the naphthoquinone derivatives, compounds 4, 6, 8, 11, 13, 14, 15, 17, and 19 had the highest potency towards S. aureus with MIC values between 7.8 and 62.5 µg/ml (Table 1). The anti-S. aureus activities of these chemicals are higher or at
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least at the same level in comparison with the reference compounds 1 and 2. The other analysed strains, especially E. faecalis and P. vulgaris, were less susceptible to the effect of the tested compounds.
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Notably, 2-arylonaphthoquinone without additional pharmacophore groups (3) is less active in comparison with compounds substituted with additional groups (e.g. 6, 8, 11). Substitution of the
AC C
phenyl ring with methyl or methoxy groups results in changes in the activity depending on the position of the substituents. Addition of two methyl moieties in positions 3 and 4 of the phenyl ring (5) does not influence the antimicrobial activity significantly. On the other hand, when the phenyl is substituted with three methyl groups at positions 2, 4, and 6 (6), the activity against S. aureus drastically increases. In the case of the 2,4-dimetoxyphenyl derivative (8), we obtained a 16-fold increase in the activity in comparison with the 2,4,6-trimetoxyphenyl derivative (9).
20
ACCEPTED MANUSCRIPT
Interestingly, the presence of the sole methyl group in chemical 11 can result in a 16-fold increase in the anti-S. aureus activity in comparison with compound 10. Deniz and coworkers showed that S. aureus was also susceptible to heteroatom-substituted 1,4-
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naphthoquinones containing a piperazine ring. They showed that 2-[1-Piperonylpiperazin-1-yl]3-chloro-1,4-naphthoquinone and 2-(1-Ethylsulfanyl)-3-(1-N-diphenylmethylpiperazin-1-yl)-1,4naphthoquinone inhibited S. aureus growth at a MIC value of 62.6 µg/ml [26]
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Since the amino group plays a key role in many bioactive compounds, we have tested several compounds based on the aminonaphthoquinone core. Formally, products of oxidative
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condensation of naphthoquinone with natural amino acids satisfy our assumption that antibacterial activities of substituted naphthoquinones bearing an amino group are significantly high with the exception of valine- (16) and phenylalanine- (18) substituted naphthoquinones. Medina and coworkers show in their study that amino- and hydroxyl- substituted 1,4-
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naphthoquinones induce oxidative stress and inhibit the growth of S. aureus at a concentration of 50 µg/ml [38].
E. faecalis
P. aeruginosa
K. pneumoniae
E. coli
E. cloacae
P. vulgaris
S. bongori
31.2
125
125
250
125
250
n.a.
125
2
62.5
n.a.
250
250
250
250
n.a.
250
3
n.a.
n.a.
250
n.a.
n.a.
n.a.
n.a.
250
4
62.5
n.a.
500
500
250
500
500
500
5
500
n.a.
500
500
500
500
500
500
6
15.6
n.a.
500
500
500
500
500
500
7
500
n.a.
500
500
500
500
n.a.
500
AC C
1
compound
S. aureus
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Table 1. Antibacterial activity of 1,4-naphthoquionones expressed as minimal inhibitory concentration (µg/ml).
21
n.a.
250
250
250
250
n.a.
250
9
250
n.a.
250
250
250
250
n.a.
250
10
125
n.d.
250
62.5
125
250
250
125
11
7.8
n.a.
250
250
250
250
n.a.
250
12
250
250
250
250
250
250
n.a.
250
13
7.8
n.a.
250
250
250
250
n.a.
250
14
15.6
n.a.
250
250
250
250
n.a.
250
15
31.2
250
250
250
250
250
500
250
16
500
500
500
500
500
500
500
500
17
62.5
500
500
500
500
500
500
500
18
250
500
500
500
500
500
n.a.
500
19
62.5
250
500
500
250
500
500
500
31.2 15.6 15.6 0.5 TET 7.8 n.d.: not determined; n.a.: no activity;
7.8
7.8
3.9
SC
15.6
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8
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ACCEPTED MANUSCRIPT
Additionally, the representatives of the most active compounds, namely 8, 11, and 14, were examined in terms of their haemolytic activity against human erythrocytes. As presented in
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Figure 2, all the examined compounds used in an 8-fold higher amount than the MIC values induce haemolysis of no more than 2 % of erythrocytes. For comparison, the well-known 1,4naphthoquinone derivative, vitamin K3 menadione (2-methyl-1,4-napthoquinone), inhibits the
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growth of S. aureus at a similar level as our best compounds with a MIC value of 3 µg/ml [39]. In contrast, menadione caused haemolysis of at least 3.5 % of erythrocytes at a concentration of
AC C
5 µg/ml [40], while compound 11 at a higher concentration (60 µg/ml) damaged merely 2 % of blood cells (Figure 2). This clearly indicates that our compounds are less toxic to human blood cells.
22
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Figure 2. Haemolytic activity of compounds 8, 11, and 14. Tetracycline (TET) and chloramphenicol (CAM) were used as a negative control while Triton X-100 (1%) was used as a positive control. Compounds and antibiotics were tested in 8-fold MIC values.
CONCLUSIONS
On the basis of the activities of the presented 1,4-naphthoquinones, further studies to determine their pharmacological properties would be needed in order to define the usefulness of the
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synthesized compounds as antibacterial agents targeting S. aureus, i.e. one of the most prevalent microorganisms in nosocomial infections.
To conclude, our results show several compounds to be potent and selective anti-S. aureus
AC C
properties.
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agents, which can be a starting point for further improvement in respect of anti-bacterial
The phenomenon of drug resistance forces exploration of novel anti-bacterial agents that overcome these mechanisms. Derivatives of 1,4-naphthoquinones appear to be proper chemicals fulfilling the expectations. Here we present at least six compounds (6, 8, 11, 13, 14, 15) showing anti-bacterial activity at the common antibiotic level. Since the chemicals show no toxicity, they can be taken under consideration as potential anti-bacterial drugs.
23
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ACKNOWLEDGEMENTS The
financial
support
from
the
Polish
National
Science
Centre
grant
number
SC
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2012/05/B/ST5/00362 is gratefully acknowledged.
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[29] S. Zhang, F. Song, D. Zhao, J. You, Tandem oxidation-oxidative C-H/C-H cross-coupling: synthesis of arylquinones from hydroquinones, Chem Commun (Camb), 49 (2013) 4558-4560. [30] P. ShresthaDawadi, S. Bittner, M. Fridkin, S. Rahimipour, On the synthesis of
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naphthoquinonyl heterocyclic amino acids, Synthesis-Stuttgart, (1996) 1468-&.
Synthesis-Stuttgart, (2010) 2011-2016.
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[31] A. Katritzky, L. Huang, R. Sakhuja, Efficient Syntheses of Naphthoquinone-Dipeptides,
[32] V.K. Tandon, D.B. Yadav, R.V. Singh, A.K. Chaturvedi, P.K. Shukla, Synthesis and biological evaluation of novel (L)-alpha-amino acid methyl ester, heteroalkyl, and aryl substituted 1,4-naphthoquinone derivatives as antifungal and antibacterial agents, Bioorg Med
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Chem Lett, 15 (2005) 5324-5328.
[33] Z. She, Y. Shi, Y. Huang, Y. Cheng, F. Song, J. You, Versatile palladium-catalyzed C-H olefination of (hetero)arenes at room temperature, Chem Commun, 50 (2014) 13914-13916.
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[34] K. Kapłon, O.M. Demchuk, M. Wieczorek, K.M. Pietrusiewicz, Brönsted acid catalyzed direct oxidative arylation of 1,4-naphthoquinone, Curr Chem Lett, 3 (2014) 23-36.
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[35] C. Lisboa, V. Santos, B. Vaz, N. de Lucas, M. Eberlin, S. Garden, C-H Functionalization of 1,4-Naphthoquinone by Oxidative Coupling with Anilines in the Presence of a Catalytic Quantity of Copper(II) Acetate, J Org Chem, 76 (2011) 5264-5273. [36] Y.S. Kim, S.Y. Park, H.J. Lee, M.E. Suh, D. Schollmeyer, C.O. Lee, Synthesis and cytotoxicity of 6,11-dihydro-pyrido- and 6,11-dihydro-benzo[2,3-b]phenazine-6,11-dione derivatives, Bioorg Med Chem, 11 (2003) 1709-1714.
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[37] P. Ravichandiran, D. Premnath, S. Vasanthkumar, Synthesis, molecular docking and antibacterial evaluation of new 1,4-naphthoquinone derivatives contains carbazole-6,11-dione moiety, J Chem Biol, 7 (2014) 93-101.
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[38] L.F. Medina, P.F. Hertz, V. Stefani, J.A. Henriques, A. Zanotto-Filho, A. Brandelli,
Aminonaphthoquinone induces oxidative stress in Staphylococcus aureus, Biochem Cell Biol, 84 (2006) 720-727.
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[39] P.M. Schlievert, J.A. Merriman, W. Salgado-Pabón, E.A. Mueller, A.R. Spaulding, B.G. Vu, O.N. Chuang-Smith, P.L. Kohler, J.R. Kirby, Menaquinone analogs inhibit growth of
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bacterial pathogens, Antimicrob Agents Chemother, 57 (2013) 5432-5437.
[40] S.M. Qadri, M. Eberhard, H. Mahmud, M. Föller, F. Lang, Stimulation of ceramide formation and suicidal erythrocyte death by vitamin K(3) (menadione), Eur J Pharmacol, 623
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(2009) 10-13.
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ACCEPTED MANUSCRIPT Highlights • A series of naphthalene-1,4-dione derivatives has been synthesized. • The compounds show significant antimicrobial activity. • The chemicals exhibit the highest potency towards Staphylococcus aureus.
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• The compounds show no toxicity towards erythrocytes.
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• The novel 1,4-naphthoquinones can be considered as useful antimicrobial agents.
S0223-5234(16)30896-0
DOI:
10.1016/j.ejmech.2016.10.034
Reference:
EJMECH 8999
To appear in:
European Journal of Medicinal Chemistry
Received Date: 27 May 2016 Revised Date:
14 October 2016
Accepted Date: 15 October 2016
Please cite this article as: M. Janeczko, O.M. Demchuk, D. Strzelecka, K. Kubiński, M. Masłyk, New family of antimicrobial agents derived from 1,4-naphthoquinone, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.10.034. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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1,4-Naphthoquinone
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New family of antimicrobial agents derived from
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Monika Janeczkoa, Oleg M. Demchukb, Dorota Strzelecka,b Konrad Kubińskia, and Maciej
a
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Masłyka*
Department of Molecular Biology, Institute of Biotechnology, The John Paul II Catholic University of Lublin, ul. Konstantynów 1i, 20-708 Lublin, Poland
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Organic Chemistry Department, Faculty of Chemistry, Maria Curie-Skłodowska University,
*
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ul. Gliniana 33, 20-614 Lublin, Poland
Corresponding Author
Dr. Maciej Masłyk, PhD, Department of Molecular Biology, Institute of Biotechnology, The
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b
John Paul II Catholic University of Lublin, ul. Konstantynów 1i, 20-708 Lublin, Poland, Tel: +48814545452, e-mail: [email protected]
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ABSTRACT:
Naphthalene-1,4-dione derivatives were synthesized and tested against selected bacterial strains.
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All the tested compounds were prepared by direct introduction of corresponding substituents into the naphthoquinone core in oxidative conditions. In this study, eight strains of bacteria (Proteus, Escherichia,
Klebsiella,
Staphylococcus,
Enterobacter,
Pseudomonas,
Salmonella,
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Enterococcus) were used for determination of antimicrobial activity of synthesized compounds with the Minimal Inhibitory Concentration (MIC) method. Additionally, selected compounds
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were tested for haemolytic activity using human erythrocytes. All naphthalene-1,4-dione derivatives exhibited significant antimicrobial activity with MIC values between 7.8 and 500 µg/ml. A majority of the synthesized compounds showed the strongest antibacterial properties towards S. aureus, with a high level of selectivity. None of the tested naphthalene-1,4-dione
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KEYWORDS
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derivatives exhibited haemolytic activity.
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Antimicrobial, naphthoquinones, Staphylococcus aureus, haemolysis
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INTRODUCTION In the recent decade, a problem of an increasing number of bacterial pathogens exhibiting
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multidrug resistance to antibiotics has been observed. According to the World Health Organization, multidrug-resistant bacteria are responsible for approximately 25 000 deaths in Europe each year. The accumulation of antibacterial agents in the environment promotes spread
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of resistant microorganisms, turning the environment into a gigantic reservoir for antibiotic resistance genes [1]. An important aspect of bacterial resistance is represented by bacteria that
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cause nosocomial infections worldwide such as Staphylococcus aureus, Pseudomonas aeruginosa, or Streptococcus pneumoniae [2-4]. One of the evident examples is the methicillin and vancomycin-resistant Staphylococcus aureus, which accounts for a high percentage of hospital-acquired infections. S. aureus has the ability to form biofilms on biomaterials, which is responsible for its resistance towards antimicrobial agents, which makes it difficult to eradicate
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the bacteria from the infected hosts [3, 5]. S. aureus is frequently isolated in association with peripheral intravascular catheters, endotracheal and tracheotomy tubing, peritoneal dialysis tubing, prosthetic joint, vascular graft infections, and corneal infections related to contact lens
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wear. Chronic infection can serve as a septic focus that can lead to osteomyelitis, acute sepsis,
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and death, particularly in immunocompromised patients [6-13]. The Joint Programming Initiative on Antimicrobial Resistance supported by 18 European countries plus Canada recommended promotion of research and development of novel antimicrobial strategies and antibacterial agents as one of the key measures that should be adopted to fight the emergence and spread of antibiotic resistance worldwide [1]. Regarding development of novel antimicrobial drugs, naphthoquinone derivatives arouse wide interest due to their diverse functions and clinical applications. This moiety is present in many
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natural compounds such as plumbagin, juglone, lawsone, menadione, and lapachol. One of the examples is vitamin K playing a very important role in regulating blood coagulation, bone metabolism, and vascular biology [14, 15]. Naphthoquinones show cytotoxic, insecticidal, anti-
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inflammatory, and antipyretic activities [16]. They are also widely used as anticancer, antimalarial, and antimicrobial agents [17-21]. The biological relevance of 1,4-naphthoquinone is dependent of quinone redox cycling that yields "reactive oxygen species" (ROS) as well as
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arylation reactions [22]. The antimicrobial activity of naphthoquinone derivatives is also known and frequently studied in various microorganisms [23, 24]. In addition to their antibacterial
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activity, naphthoquinones possess antifungal activity [25-27].
The major objective of the present study was to carry out the synthesis and biological evaluation of a new series of 1,4-naphthoquinone derivatives. The compounds obtained were tested against a panel of gram-positive and gram-negative bacterial strains. Additionally, we have verified their
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toxicity by means of a haemolytic assay of selected compounds against human erythrocytes.
Chemistry
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MATERIALS AND METHODS
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General. All reagents were purchased from Sigma-Aldrich, Strem, TCI, and Alfa Aesar chemical companies and used without further purification. Analytical thin-layer chromatography (TLC) was performed using silica gel 60 F254 precoated plates (0.25 mm thickness) with a fluorescent indicator. Visualisation of TLC plates was performed by means of UV light or either KMnO4 or I2 stains. NMR spectra were recorded on Bruker Avance 500 MHz spectrometers, and chemical shifts are reported in ppm, and calibrated to residual solvent peaks at 7.27 ppm and 77.00 ppm for 1H and 13C in CDCl3 or internal reference compounds. The following abbreviations are used
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in reporting the NMR data: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad). Coupling constants (J) are in Hz. Spectra are reported as follows: chemical shift (δ, ppm), multiplicity, integration, coupling constants (Hz). Products were purified by flash
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chromatography on silica gel 60 (230-400 mesh) using a BUCHI chromatograph. MS spectra were recorded on a Shimadzu LCMS IT-TOF spectrometer.
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General procedure I: palladium catalysed arylation of naphthoquinone (Scheme 1: cat. = Pd(OAc)2, additive = CF3CO2H, oxidant = Bz2O2, solvent = CCl4 or ArH). A test tube equipped
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with a stirring bar was charged with 1,4-naphthoquinone (47 mg, 0.3 mmol), Pd(OAc)2 (6.7mg, 10 mol%), Bz2O2 (145 mg, 0.6 mmol), TFA (115 µL, 1.5 mmol) and aromatic compounds (2 mL), the mixture was stirred at 30 oC for 24 h, and then extracted with CH2Cl2 (2x10 mL); the combined organic layers were dried over anhydrous MgSO4. The crude product was purified by
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column chromatography using a gradient hexane/acetone mixture as eluent. 2-phenylnaphthalene-1,4-dione (3): obtained according to the general procedure I. Yield: 49 mg (70 %), yellow solid, mp = 111.3- 113.8 oC (lit. mp = 108-110 oC) [28]. 1H NMR (500.13
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MHz, CDCl3): δ = 7.04 (s, 1H, CH); 7.45-7.46 (m, 3H, CH); 7.55-7.57 (m, 2H, CH); 7.73-7.75 (m, 2H, CH); 8.06-8.08 (m, 1H, CH); 8.13-8.15 (m, 1H, CH) ppm.
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C NMR (125.75 MHz,
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CDCl3): δ = 125.76, 126.20, 126.85, 128.29, 129.30, 129.86, 131.86, 132.25, 133.22, 133.64, 133.70, 133.74, 135.01, 138.45, 147.84, 184.12, 184.88 ppm.
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C NMR (DEPT 135, 125.75
MHz, CDCl3): δ = 125.93 (CH), 127.02 (CH), 128.42 (CH), 129.38 (CH), 129.99 (CH), 133.79 (CH), 133.85 (CH), 135.18 (CH) ppm. HRMS (ESI): m/z = not ionisable [M+H]+.
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2,3-diphenylnaphthalene-1,4-dione (4). obtained as a by-product in the synthesis of 3. Yield: 20 mg (22 %), yellow solid, mp = 135.7 - 138.0 oC (lit. mp = 134-135 oC) [28]. 1H NMR (500.13 MHz, CDCl3): δ = 7.06-7.08 (m, 4H, CH); 7.21-7.23 (m, 6H, CH); 7.76-7.78 (m, 2H, CH); 8.1713
C NMR (125.75 MHz, CDCl3): δ = 126.63, 127.65, 128.24, 130.55,
132.15, 133.26, 133.88, 145.78, 184.78 ppm.
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8.18 (m, 2H, CH) ppm.
C NMR (DEPT 135, 125.75 MHz, CDCl3): δ =
126.63 (CH), 127.65 (CH), 128.24 (CH), 130.56 (CH), 133.88 (CH) ppm. HRMS (ESI): m/z =
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not ionisable [M+H]+.
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2-(3,4-dimethylphenyl)naphthalene-1,4-dione (5): obtained according to the modified general procedure I. o-Xylene was used instead of benzene. Yield: 40 mg (51 %), yellow solid, mp = 156.7-158.7 oC (lit. mp = 151 - 153 oC) [29]. 1H NMR (500.13 MHz, CDCl3): δ = 1H NMR (500.13 MHz, CDCl3): δ = 2.34 (s, 3H, CH3); 2.35 (s, 3H, CH3); 7.07 (s, 1H, CH); 7.25 (d, J =
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7.88 Hz, 1H, CH); 7.34 (dd, J = 7.88, 1.89 Hz, 1H, CH); 7.37 (s, 1H, CH); 7.77-7.79 (m, 2H, CH); 8.12-8.13 (m, 1H, CH); 8.18-8.20 (m, 1H, CH) ppm. 13C NMR (125.75 MHz, CDCl3): δ = 19.77, 19.88, 125.92, 126.92, 126.97, 127.02, 129.81, 130.53, 132.14, 132.58, 133.74, 133.79, 13
C NMR (DEPT 135, 125.75 MHz, CDCl3): δ =
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134.52, 136.82, 139.20, 184.67, 185.27 ppm.
19.77 (CH3), 19.88 (CH3), 125.92 (CH), 126.98 (CH), 127.03 (CH), 129.81 (CH), 130.53 (CH),
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133.74 (CH), 133.79 (CH), 134.52 (CH) ppm. HRMS (ESI): m/z = not ionisable [M+H]+.
2-Mesityl-1,4-naphthoquinone (6): obtained in a 6.3 mmol scale according to the modified general procedure I. Mesitylene was used instead of benzene at 70 oC. Yield: 0.700 g (40 %), yellow solid, mp = 169.0 - 171.0. 1H NMR (500.13 MHz, CDCl3): δ = 2.12 (s, 6H, CH3); 2.34 (s, 3H, CH3); 6.88 (s, 1H, CH); 6.96 (bs, 2H, CH); 7.79 - 7.81 (m, 2H, CH); 8.15-8.17 (m, 2H, CH)
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ppm. 13C NMR (125.75 MHz, CDCl3): δ = 20.39, 21.14, 126.19, 127.07, 128.38, 130.55, 132.24, 133.87, 133.90, 135.45, 137.88, 150.49, 185.16 ppm.
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CDCl3): δ = 20.39 (CH3), 21.14 (CH3), 126.19 (CH), 127.07 (CH), 128.38 (CH), 133.87 (CH),
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133.90 (CH), 137.89 (CH) ppm. Anal. Calcd. for C19H16O2: C, 82.58; H, 5.84; O, 11.58. Found: C, 81.85; H, 5.77; O, 12.38. HRMS (ESI): m/z = not ionisable [M+H]+.
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6',7'-dimethoxy-2,2'-binaphthalene-1,4-dione (7): obtained according to the modified general procedure I. 2,3-dimethoxynaphthalene (113 mg, 0.6 mmol) and CCl4 (2 mL) were used instead
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of benzene at 50 oC. Yield: 20 mg (20 %), dark purple solid, mp = 228.3 - 230.5 oC. 1H NMR (500.13 MHz, CDCl3): δ = 4.03 (s, 3H, OCH3); 4.05 (s, 3H, OCH3); 7.16 (s, 1H, CH); 7.19 (s, 1H, CH); 7.22 (s, 1H, CH); 7.54 (dd, J = 6.62, 1.89 Hz, 1H, CH); 7.78 (d, J = 8.51 Hz, 1H, CH); 7-79-7.83 (m, 2H, CH); 8.02-8.03 (bd, 1H, CH); 8.14 - 8.16 (m, 1H, CH); 8.22 - 8.24 (m, 1H, 13
C NMR (125.75 MHz, CDCl3): δ = 55.96, 55.98, 106.04, 107.08, 124.70, 125.96,
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CH) ppm.
126.54, 127.07, 128.34, 128.78, 129.03, 130.09, 132.18, 132.66, 133.83, 134.79 149.99, 150.77, 185.17 ppm.
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C NMR (DEPT 135, 125.75 MHz, CDCl3): δ = 55.96 (OCH3), 55.99 (OCH3),
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106.04 (CH), 107.08 (CH), 124.70 (CH), 125.96 (CH), 126.55 (CH), 127.07 (CH), 128.35 (CH), 133.82 (CH), 133.83 (CH), 134.79 (CH) ppm. HRMS (ESI): m/z = 345.1114 [C22H16O4+H]+,
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m/z (theor.) = 345.1121, diff. = -2.03 ppm.
General procedure II: Friedel-Crafts arylation of naphthoquinone (Scheme 1: cat. = H3PW12O40, oxidant = air, solvent = DMSO/CH3CO2H). A 50 mL flask equipped with a magnetic stirrer was charged with 1,4-naphthoquinone (1) (20 mmol; 3.16 g), aromatic compounds (10 mmol), 15 mL of CH3COOH and H3PW12O40 (288 mg; 1 mol%). The flask was
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heated at 100 oC for 24 hours. After that time, the flask was cooled down to room temperature and 15 mL of chloroform was added. The solution was washed with 20 mL of brine. The organic phase was separated and dried by MgSO4. The solvent was evaporated under reduced pressure
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and the residue was purified by column chromatography on silica gel, using a gradient hexane/acetone mixture as eluent.
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2-(2,4-dimethoxyphenyl)naphthalene-1,4-dione (8): obtained according to the modified general procedure II. Instead of acetic acid, acetone was used in reflux conditions. The product
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was crystalized from the hexane/acetone mixture. Yield: 77%, mp = 155.3-56.9 oC. 1H NMR (500.13 MHz, CDCl3): δ = 3.79 (s, 3H, CH3) 3.87 (s, 3H, CH3) 6.56-6.60 (m, 2H, CH) 7.04 (s, 1H, CH) 7.21 - 7.22 (d, J= 8.51, 1H, CH) 7.74 - 7.76 (m, 2H, CH) 8.10-8.17 (m, 2H, CH) ppm. 13
C NMR (125.75 MHz, CDCl3): δ = 55.23, 55.46, 98.79, 104.44, 115.61, 125.64, 126.66,
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131.38, 131.92, 132.44, 133.20, 133.33, 135.98, 147.15, 158.36, 162.06, 183.71, 185.15 ppm. C NMR (DEPT 135, 125.75 MHz, CDCl3): δ = 55.51 (OCH3), 55.74 (OCH3), 99.06 (CH),
104.71 (CH), 125.92 (CH), 126.95 (CH), 131.67 (CH), 133.49 (CH), 133.61 (CH), 136.26 (CH)
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ppm. HRMS (ESI): m/z = 317.0770 [C18H14O4+Na]+, m/z (theor.) = 317.0784, diff. = 4.42 ppm.
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2-(2,4,6-trimethoxyphenyl)naphthalene-1,4-dione (9): obtained according to the modified general procedure II. Instead of acetic acid, acetone was used in reflux conditions. Yield: 85%, mp = 183 oC. 1H NMR (500.13 MHz, CDCl3): δ = 3.74 (s, 6 H, OCH3) 3.87 (s, 3 H, OCH3) 6.21 (s, 2 H, CH) 6.96 (s, 1 H, CH) 7.73- 7.75 (m, 2 H, CH) 8.11 - 8.14 (m, 2 H, CH) ppm. 13C NMR (125.75 MHz, CDCl3): δ = 55.,67 56.09, 91.08, 104.88, 126.16, 127.07, 132.51, 133.08, 133.49, 133.67, 138.93, 145.14, 159.01, 162.63, 183.99, 185.70 ppm.
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C NMR (DEPT 135, 125.75
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MHz, CDCl3): δ = 55.41 (COCH), 55.83 (OCH3)3, 90.81 (CH), 125.90 (CH), 126.82 (CH), 133.24 (CH), 133.42 (CH), 138.67 (CH) ppm. HRMS (ESI): m/z = 325.1066 [C19H16O5+H]+,
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m/z (theor.) = 325.1071, diff. = -1.54 ppm
2-[4-(Dimethylamino)phenyl]naphthalene-1,4-dione (10): obtained according to the general procedure II. Yield: 35%, mp = 140.5-141.5 oC. 1H NMR (500.13 MHz, CDCl3): δ = 3.05 (s, 6H,
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CH3) 6.74-6.77 (m, 2H, CH) 7.02 (s, 1H, CH) 7.59- 7.62 (m, 2H, CH) 7.72-7.75 (m, 2H, CH) 8.08-8.11 (m, 1H, CH) 8.15-8.17 (m, 1H, CH) ppm. 13C NMR (125.75 MHz, CDCl3): δ = 40.16,
185.25, 185.51 ppm.
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11.73, 120.52, 125.71, 126.92, 130.98, 131.18, 132.33, 132.92, 133.38, 133.54, 147.43, 151.74, C NMR (DEPT 135. 125.75 MHz, CDCl3): δ = 40.07 (CH3), 111.65
(CH), 125.61 (CH), 126.84 (CH), 130.92 (CH), 131.05 (CH), 133.29 (CH), 133.45 (CH) ppm.
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HRMS (ESI): m/z = 278.1184 [C18H15NO2+H]+, m/z (theor.) = 278.1176, diff. = 2.88 ppm
2-[4-(Dimethylamino)-2-methylphenyl]naphthalene-1,4-dione (11): obtained according to the general procedure II. Yield: 37%, mp = 158.3-159.2 oC. 1H NMR (500.13 MHz, CDCl3): δ =
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2.25 (s, 3H, CH3) 3.02 (s, 6H, N(CH3)2) 6.61-6.63 (m, 2H, CH) 6.91 (s, 1H, CH) 7.11-7.13 (m, 1H, CH) 7.76-7.78 (s, 2H, CH) 8.12-8.18 (m, 2H, CH) ppm. 13C NMR (125.75 MHz, CDCl3): δ
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CDCl3): δ = 21.39 (CH3), 40.31 (CH3), 109.51 (CH), 114.07 (CH), 125.93 (CH), 127.01 (CH), 131.04 (CH), 133.61 (CH), 135.77 (CH) ppm. HRMS (ESI): m/z = 292.1335 [C19H17NO2+H]+, m/z (theor.) = 292.1332, diff. = 1.03 ppm
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2-[4-(dibenzylamino)phenyl]naphthalene-1,4-dione (12): obtained according to the general procedure II. Yield: 25%, mp = 145.3 - 146.7 oC. 1H NMR (500.13 MHz, CDCl3): δ = 4.74 (s, 4H, CH2) 6.81-6.84 (m, 2H, CH) 7.01 (s, 1H, CH) 7.26-7.31 (m, 6H, CH) 7.35-7.38 (m, 4H, CH) 13
C NMR (125.75
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7.53-7.56 (m, 2H, CH) 7.73-7.76 (m, 2H, CH) 8.09-8.18 (m, 2H, CH) ppm.
MHz, CDCl3): δ = 54.05, 112.12, 121.31, 125.74, 126.53, 126.96, 127.24, 128.86, 131.20, 131.46, 132.32, 132.87, 133.43, 133.59, 137.61, 147.27, 150.83, 185.21, 185.44 ppm. 13C NMR
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(DEPT 135, 125.75 MHz, CDCl3): δ = 54.05 (CH2), 112.11 (CH), 125.73 (CH), 126.52 (CH), 127.23 (CH), 128.84 (CH), 131.18 (CH), 131.45 (CH), 133.42 (CH), 133.58 (CH) ppm. HRMS
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(ESI): m/z = 430.1804 [C30H23NO2+H]+, m/z (theor.)= 430.1802, diff. = 0.43 ppm
2-[4-(Dimethylamino)-2-methoxyphenyl]naphthalene-1,4-dione (13); obtained according to the general procedure II. Yield: 18%, mp = 144.2-145.6 oC. 1H NMR (500.13 MHz, CDCl3): δ =
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3.04 (s, 6H, N(CH3)2) 3.82 (s, 3H, OCH3) 6.27 (s, 1H, CH) 6.37-6.39 (d, J = 8.83, 1H, CH) 7.09 (s, 1H, CH) 7.20-7.21 (d, J = 8.51, 1H, CH) 7.72- 7.74 (m, 2H, CH) 8.09- 8.15 (m, 2H, CH) ppm. 13C NMR (125.75 MHz, CDCl3): δ = 40.36, 55.52, 95.51, 104.44, 110.89, 125.73, 126.86,
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132.14, 132.32, 133.02, 133.29, 133.32, 134.81, 147.47, 153.03, 158.94, 184.64, 185.55 ppm. C NMR (DEPT 135, 125.75 MHz, CDCl3): δ = 40.36 (N(CH3)2), 55.52 (CH3), 95.52 (CH),
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104.44 (CH), 125.73 (CH), 126.86 (CH), 132.13 (CH), 133.29 (CH), 133.32 (CH), 134.80 (CH) ppm. HRMS (ESI): m/z = 308.1271 [C19H17NO3+H]+, m/z (theor.)= 308.1281, diff. = -3.25 ppm
N-[4-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)-3-methoxyphenyl]acetamide (14): obtained according to the general procedure II. Yield: 483 mg (15 %), mp= 209-210 oC (dec.). 1H NMR (500.13 MHz, CDCl3): δ = 2.21 (s, 3H, NHCOCH3) 3.80 (s, 3H, OCH3) 6.89-6.91 (m, 1H, CH)
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7.04 (s, 1H, CH) 7.18-7.20 (d, J = 8.20, 1H, CH) 7.42 (s, 1H, CH) 7.59 (s, 1H, CH) 7.75-7.79 (m, 2H, CH) 8.11-8.17 (m, 2H, CH) ppm. 13C NMR (125.75 MHz, DMSO-d6): δ = 24.34, 55.72, 102.36, 110.74, 117.67, 125.73, 126.63, 131.12, 131.73, 132.25, 134.28, 134.38, 135.93, 142.22, 13
C NMR (DEPT 135, 125.75 MHz, CDCl3): δ =
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147.41, 157.46, 168.82, 183.40, 184.75 ppm.
24.83 (CH3), 55.84 (OCH3), 103.23 (CH), 111.04 (CH), 126.02 (CH), 126.96 (CH), 130.95 (CH), 133.64 (CH), 133.74 (CH), 136.63 (CH) ppm. HRMS (ESI): m/z = 322.1070
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[C19H15NO4+H]+, m/z (theor.) = 322.1074, diff. = -1.24 ppm
General procedure III: amination of naphthoquinone (Scheme 2: cat. = none, oxidant = air, solvent = EtOH/H2O, additive = Et3N). H2O (2.5 mL) and Et3N (700 µL, 5.0 mmol) were added to a suspension of 1,4-naphthoquinone (790 mg, 5.0 mmol) and amino acid (2.5 mmol) in EtOH
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(50 mL) and the mixture was stirred at room temperature for 24 h. The solvent was evaporated, and the crude product was extracted by 100 mL of a 5% aqueous solution of Na2CO3. The extract was washed with EtOAc (50 mL), the separated aqueous layer was acidized with 36% HCl, and
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the crude product was extracted with EtOAc (100 mL). The organic layer was separated, dried over anhydrous MgSO4, and the solvent was removed under reduced pressure after filtration. The
AC C
product was purified by crystallization from warm methanol or by column chromatography using a gradient CH2Cl2/hexane/EtOAc/MeOH mixture as eluent.
1-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)-L-proline (15): obtained according to the general procedure III; the product was not soluble in EtOAc. It was precipitated after acidisation of the extract and used without further purification. Yield: 270 mg (40 %), red solid, mp = 151.2-153.0
11
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o
C (dec.) (lit. mp = 165-168 oC)[30]. 1H NMR (500.13 MHz, DMSO-d6): δ = 1.84-1.91 (m, 1H),
1.94-1.99 (m, 1H) 2.04-2.10 (m, 1H) 2.24-2.31 (m, 1H), 3.45 (bs, 2H), 4.99 (s, 1H), 5.77 (s, 1H), 7.73 (t, J = 7.57 Hz, 1H, CH), 7.80-7.84 (t, J = 7.57 Hz, 1H, CH), 7.91-7.93 (m, 2H, CH), 12.74
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(br, 1H, CO2H) ppm. 13C NMR (125.75 MHz, DMSO-d6): δ = 21.52, 30.76, 50.58, 62.16, 69.67, 104.53, 124.46, 125.87, 130.97, 131.98, 134.13, 145.46, 148.42, 180.86, 182.32 ppm. 13C NMR (DEPT 135, 125.75 MHz, DMSO-d6): δ = 21.59 (CH2), 30.83 (CH2), 50.68 (CH2), 62.23 (CH),
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104.60 (CH), 124.54 (CH), 125.94 (CH), 132.05 (CH), 134.20 (CH) ppm. HRMS (ESI): m/z =
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272.0923 [C15H13NO4+H]+, m/z (theor.) = 272.0917, diff.= 2.21 ppm.
N-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)valine (16): obtained according to the general procedure III and purified by column chromatography using a mixture of hexane-EtOAc-MeOH (5:3:1) as eluent. Yield: 270 mg (40 %), red solid, mp = 171.1-173.1 oC (dec.). 1H NMR (500.13
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MHz, DMSO-d6): δ = 0.91 (d, J = 6.94 Hz, 3H, CH3); 0.96 (d, J = 6.62 Hz, 3H, CH3); 2.23-2.27 (m, 1H, CH); 3.93-3.96 (m, 1H, CH); 5.72 (s, 1H, CH); 6.85 (d, J = 8.51 Hz, 1H, CH); 7.76 (dt, J = 7.57, 1.26 Hz, 1H, CH); 7.85 (dt, J = 7.57, 1.26 Hz, 1H, CH); 7.94 (dd, J = 7.57, 0.95 Hz, 1H,
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CH); 8.01 (dd, J = 7.57, 0.95 Hz, 1H, CH), 13.22 (bs, 1H, OH) ppm.
13
C NMR (125.75 MHz,
DMSO-d6): δ = 18.24, 18.36, 29.54, 59.88, 100.66, 125.09, 125.09, 125.71, 129.86, 132.18,
AC C
132.42, 134.69, 147.35, 171.68, 180.88, 181.52 ppm.
13
C NMR (125.75 MHz, DMSO-d6): δ=
19.03 (CH3), 19.15 (CH3), 30.33 (CH), 60.67 (CH), 101.44 (CH), 125.88 (CH), 126.52 (CH), 132.98 (CH), 135.49 (CH) ppm. HRMS (ESI): m/z = 272.0929 [C15H15NO4-H]-, m/z (theor.) = 272.0928, diff. = 0.37 ppm.
12
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N-(1,4-Naphthoquinone-2-yl)-L-tryptophan (17): obtained according to the general procedure III. Yield: 570 mg (63 %), red solid, mp = 159.1 - 161.3 oC (dec.) (lit. mp = 208.0211.0 oC (dec.))[30, 31]. 1H NMR (500.13 MHz, DMSO-d6): δ = 3.23 (d, J = 5.67 Hz, 2H), 4.34
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– 4.36 (m, 1H), 5.60 (s, 1H), 6.78 – 6.83 (m, 2H), 6.90 (dt, J = 7.8, 1.0 Hz, 1H), 7.04 (d, J = 2.2 Hz, 1H), 7.17 (dd, J = 8.2, 1.0 Hz, 1H), 7.37 (d, J = 8.2 Hz, 1 H), 7.60 (dt, J = 7.6, 1.3 Hz, 1H), 7.70 (dt, J = 7.6, 1.6 Hz, 1H), 7.78–7.84 (m, 2H), 10.76 (br, 1H), 13.10 (bs, 1H) ppm. 13C NMR
SC
(125.75 MHz, DMSO-d6): δ = 25.84, 54.93, 100.60, 108.53, 111.11, 117.86, 118.17, 120.67, 123.74, 125.07, 125.63, 126.96, 129.79, 132.08, 132.45, 134.64, 135.74, 146.92, 171.85, 180.85, 13
C NMR (DEPT 135, 125.75 MHz, DMSO-d6): δ = 26.58 (CH2), 55.66 (CH),
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181.41 ppm.
101.38 (CH), 111.89 (CH), 118.64 (CH), 118.94 (CH), 121.45 (CH), 124.53 (CH), 125.87 (CH), 126.46 (CH), 132.93 (CH), 135.48 (CH) ppm. HRMS (ESI): m/z = 383.0994 [C21H16N2O4+Na]+,
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m/z (theor.) = 383.1002, diff. = 2.09 ppm.
[(1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino]-D-(phenyl)acetic
acid
(18):
obtained
according to the general procedure III. Yield: 70 mg (9%), red solid, mp = 195.3 – 199.5 oC
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(dec.). 1H NMR (500.13 MHz, DMSO-d6): δ = 4.56 (d, J = 5.99 Hz, 1H), 5.20 (s, 1H), 7.19 (t, J = 7.25 Hz, 1H), 7.27 (t, J = 7.25 Hz, 2H, CH), 7.36 (d, J = 7.57 Hz, 2H, CH) 7.71 (t, J = 7.57 Hz,
ppm.
AC C
1H, CH), 7.79 (t, J = 7.57, 1H, CH), 7.86 (d, J = 7.25 Hz, 1H, CH), 8.00 (d, J = 6.62, 2H, CH), 13
C NMR (125.75 MHz, DMSO-d6): δ = 61.30, 98.36, 101.05, 122.68, 122.97, 125.56,
125.80, 126.18, 126.38, 126.93, 127.17, 128.40, 129.07, 129.41, 131.38, 132.62, 135.33, 155.67 ppm.
13
C NMR (DEPT 135, 125.75 MHz, DMSO-d6): δ = 57.14 (CH), 101.45 (CH), 124.71
(CH), 125.28 (CH), 126.60 (CH), 127.83 (CH), 128.04 (CH), 131.83 (CH), 134.27 (CH) ppm. HRMS (ESI): m/z = 308.0921 [C18H13NO4+H]+, m/z (theor.) = 308.0917, diff. = 1.30 ppm.
13
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Methyl N-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)-L-alaninate (19). H2O (2.5 mL) and Et3N (700 µL, 5.0 mmol) were added to a suspension of 1,4-naphthoquinone (790 mg, 5.0 mmol)
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and L-alanine methyl ester hydrochloride (350 mg, 2.5 mmol) in MeOH (50 mL) and the mixture was stirred at room temperature for 24 h. The solvent was evaporated under reduced pressure and the product was purified by column chromatography using a mixture of hexane-acetone (9:1) as
SC
eluent. Yield: 63 mg (10%), yellow solid, mp = 91.4 – 93.5 oC (lit. mp = 188 oC) [32]. 1H NMR (500.13 MHz, CDCl3): δ = 1.58 (d, J = 6.94 Hz, 3H, CH3); 3.81 (s, 3H, OCH3); 4.13 (p, J = 6.94
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Hz, 1H, CH); 5.68 (d, J = 0.63 Hz, 1H, CH); 6.31 (bd, J = 6.94 Hz, 1H, NH) 7.65 (dt, J = 7.57, 1.26 Hz, 1H, CH); 7.74 (dt, J = 7.57, 1.26 Hz, 1H, CH); 8.07-8.11 (m, 2H, CH) ppm. 13C NMR (125.75 MHz, CDCl3): δ = 17.33, 50.19, 52.56, 101.74, 125.90, 126.11, 130.18, 131.95, 132.97, 134.47, 146.26, 171.76, 181.08, 182.91 ppm. 13C NMR (125.75 MHz, CDCl3): δ = 17.62 (CH3),
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50.48 (CH), 52.85 (CH), 102.05 (CH), 126.20 (CH), 126.40 (CH), 132.24 (CH), 134.76 (CH) ppm. HRMS (ESI): m/z = 260.0905 [C14H13NO4+H]+, m/z (theor.) = 260.0917, diff. = 4.61 ppm.
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Biological activities
Microbial strains. In present study, reference bacterial strains were used. Gram-positive
AC C
bacteria: Staphylococcus aureus (ATCC 6538), Enterococcus faecalis (PCM 2673), Gramnegative: Pseudomonas aeruginosa (PCM 2562), Klebsiella pneumoniae (PCM1), Escherichia coli (ATCC 8739), Enterobacter cloacae (PCM 2569), Proteus vulgaris (PCM 2668), Salmonella bongori (PCM 2552),
14
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MIC determination. Bacterial strains were inoculated in Mueller–Hinton broth (Biocorp, Poland) for 24h before performing the minimal inhibitory concentration (MIC) test and incubated at 37 °C with vigorous shaking (180 rpm). MIC was determined with the microbroth
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dilution method. Bacterial suspensions in Mueller–Hinton liquid medium at initial inoculums of 5x105 colony forming units per ml were added to polystyrene 96-well plates and exposed to the investigated naphthoquinones at adequate concentrations (range: 0.001 – 5 mg/ml) for 20 h at 37 o
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C. DMSO was used as a solvent. MICs were taken as the lowest drug concentration at which
observable growth was inhibited. Tetracycline (TET) was used as a reference compound.
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Experiments were performed in triplicate.
Haemolytic assay. The human blood samples were placed in sterile tubes containing a citrate
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dextrose solution as anticoagulant.
In order to separate erythrocytes from plasma, the samples were centrifuged at 500×g for 10 minutes at 4 °C and the supernatant was discarded. Next, the erythrocytes were resuspended with
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PBS buffer (10 mM phosphate, pH 7.5; 150 mM NaCl) and centrifuged as previously. The washing procedure was repeated until a transparent supernatant was obtained. The washed
AC C
erythrocytes were finally resuspended in PBS buffer to a final concentration of 2%. Simultaneously, appropriate concentrations (in the range: 1 - 250 µg/ml) of the examined compounds were prepared in a final volume of 50 µl DMSO. The compounds prepared in this way were mixed with 450 µl of 2% erythrocyte suspension followed by incubation for 1h at 37 °C. Then, the samples were centrifuged at 5000×g for 10 minutes and absorbance at wavelength 415 nm was measured.
15
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RESULTS AND DISCUSSION Keeping in mind the known biological activity of quinone derivatives, a series of
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naphthoquinones substituted by the aryl group, which can be easily prepared with the simple one-step modular synthesis of 2-arylnaphthaquinones, were assessed as potential antimicrobial agents. The antimicrobial activity of unsubstituted naphthoquinones (1, 2) was determined as a
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reference.
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Chemistry
Depending on the desired substitution pattern, different sub-classes of 2-arylonaphthoquinones 3-14 were obtained according to the complementary synthetic approaches. The coupling of weakly activated aromatics with naphthoquinone leading to 2-arylonaphthoquinones 3-7 was performed in palladium trifluoroacetate-catalysed conditions according to the modified
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procedure presented in the literature (cat. = Pd(OAc)2, additive = CF3CO2H, oxidant = Bz2O2, solvent = CCl4 or ArH, t = 60 oC) (Scheme 1) [33]. Despite the low yields observed in some of these cases, application of cationic palladium catalysis yields products with substituents in
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positions that do not correspond to the usual reactivity of aromatics undergoing the electrophilic
AC C
substitution reaction. Sites of molecules thus activated by the substitution effects are left accessible to further transformation or biochemical interactions. In all cases, the palladiumcatalysed reaction always yields less sterically hindered substitution products.
16
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ACCEPTED MANUSCRIPT
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Scheme 1. Synthesis of 2-arylnaphthoquinones (3-14)
Quinones 8-14, bearing more electron-rich aryl substituents, were obtained in coupling reactions
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of naphthoquinone with corresponding reactive aromatics according to the Friedel-Crafts protocol in Brönsted acid-mediated conditions (Scheme 1) (cat. = H3PW12O40, oxidant = air, solvent DMSO/CH3CO2H, additive - none). These reactions follow the SE2Ar mechanism furnishing products substituted in predictable positions [34].
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Finally, the substituted aminonaphthoquinones 15-19 were prepared by a modified Katritzky procedure starting from naphthoquinone and corresponding D or L-amino acids or esters (Scheme 2) (cat. = none, oxidant = air, solvent = EtOH/H2O, additive = Et3N) [31]. For
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comparison, 2-(phenylamino)naphthalene-1,4-dione (2) was obtained in a reaction catalysed by copper acetate (Scheme 2) (cat. = Cu(OAc)2, solvent = CH3CO2H, oxidant = air) according to the
AC C
procedure described in the literature [35].
Scheme 2. Synthesis of 2-aminonaphthoquinones (2, 15-19)
17
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All new compounds were fully characterised while the structure of the known compounds were confirmed by comparison of experimental and physical data from the literature. In such a way, a
AC C
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function groups was prepared for the biological tests.
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series of 19 naphthoquinones substituted in positions 2 and 3 and bearing a wide pattern of
18
AC C
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ACCEPTED MANUSCRIPT
Figure 1. Assessed naphthoquinones 19
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Biology Certain alkyl-, alkenyl- and amino-1,4-naphthoquinones have already been assessed as
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prospective building blocks for the construction of numerous biologically important compounds [31, 32, 36, 37]. At the same time, the much more readily available and significantly more stable arylo-1,4-naphthoquinones still require more comprehensive biological studies.
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All the synthesized compounds (Figure 1) were tested against a panel of microorganisms and the MIC values were calculated. Each of the examined naphthoquinone derivatives exhibited
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certain antimicrobial activity with predominant MIC values between 7.8 and 500 µg/ml. In terms of the most promising bacterial target of the naphthoquinone derivatives, compounds 4, 6, 8, 11, 13, 14, 15, 17, and 19 had the highest potency towards S. aureus with MIC values between 7.8 and 62.5 µg/ml (Table 1). The anti-S. aureus activities of these chemicals are higher or at
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least at the same level in comparison with the reference compounds 1 and 2. The other analysed strains, especially E. faecalis and P. vulgaris, were less susceptible to the effect of the tested compounds.
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Notably, 2-arylonaphthoquinone without additional pharmacophore groups (3) is less active in comparison with compounds substituted with additional groups (e.g. 6, 8, 11). Substitution of the
AC C
phenyl ring with methyl or methoxy groups results in changes in the activity depending on the position of the substituents. Addition of two methyl moieties in positions 3 and 4 of the phenyl ring (5) does not influence the antimicrobial activity significantly. On the other hand, when the phenyl is substituted with three methyl groups at positions 2, 4, and 6 (6), the activity against S. aureus drastically increases. In the case of the 2,4-dimetoxyphenyl derivative (8), we obtained a 16-fold increase in the activity in comparison with the 2,4,6-trimetoxyphenyl derivative (9).
20
ACCEPTED MANUSCRIPT
Interestingly, the presence of the sole methyl group in chemical 11 can result in a 16-fold increase in the anti-S. aureus activity in comparison with compound 10. Deniz and coworkers showed that S. aureus was also susceptible to heteroatom-substituted 1,4-
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naphthoquinones containing a piperazine ring. They showed that 2-[1-Piperonylpiperazin-1-yl]3-chloro-1,4-naphthoquinone and 2-(1-Ethylsulfanyl)-3-(1-N-diphenylmethylpiperazin-1-yl)-1,4naphthoquinone inhibited S. aureus growth at a MIC value of 62.6 µg/ml [26]
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Since the amino group plays a key role in many bioactive compounds, we have tested several compounds based on the aminonaphthoquinone core. Formally, products of oxidative
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condensation of naphthoquinone with natural amino acids satisfy our assumption that antibacterial activities of substituted naphthoquinones bearing an amino group are significantly high with the exception of valine- (16) and phenylalanine- (18) substituted naphthoquinones. Medina and coworkers show in their study that amino- and hydroxyl- substituted 1,4-
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naphthoquinones induce oxidative stress and inhibit the growth of S. aureus at a concentration of 50 µg/ml [38].
E. faecalis
P. aeruginosa
K. pneumoniae
E. coli
E. cloacae
P. vulgaris
S. bongori
31.2
125
125
250
125
250
n.a.
125
2
62.5
n.a.
250
250
250
250
n.a.
250
3
n.a.
n.a.
250
n.a.
n.a.
n.a.
n.a.
250
4
62.5
n.a.
500
500
250
500
500
500
5
500
n.a.
500
500
500
500
500
500
6
15.6
n.a.
500
500
500
500
500
500
7
500
n.a.
500
500
500
500
n.a.
500
AC C
1
compound
S. aureus
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Table 1. Antibacterial activity of 1,4-naphthoquionones expressed as minimal inhibitory concentration (µg/ml).
21
n.a.
250
250
250
250
n.a.
250
9
250
n.a.
250
250
250
250
n.a.
250
10
125
n.d.
250
62.5
125
250
250
125
11
7.8
n.a.
250
250
250
250
n.a.
250
12
250
250
250
250
250
250
n.a.
250
13
7.8
n.a.
250
250
250
250
n.a.
250
14
15.6
n.a.
250
250
250
250
n.a.
250
15
31.2
250
250
250
250
250
500
250
16
500
500
500
500
500
500
500
500
17
62.5
500
500
500
500
500
500
500
18
250
500
500
500
500
500
n.a.
500
19
62.5
250
500
500
250
500
500
500
31.2 15.6 15.6 0.5 TET 7.8 n.d.: not determined; n.a.: no activity;
7.8
7.8
3.9
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15.6
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8
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ACCEPTED MANUSCRIPT
Additionally, the representatives of the most active compounds, namely 8, 11, and 14, were examined in terms of their haemolytic activity against human erythrocytes. As presented in
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Figure 2, all the examined compounds used in an 8-fold higher amount than the MIC values induce haemolysis of no more than 2 % of erythrocytes. For comparison, the well-known 1,4naphthoquinone derivative, vitamin K3 menadione (2-methyl-1,4-napthoquinone), inhibits the
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growth of S. aureus at a similar level as our best compounds with a MIC value of 3 µg/ml [39]. In contrast, menadione caused haemolysis of at least 3.5 % of erythrocytes at a concentration of
AC C
5 µg/ml [40], while compound 11 at a higher concentration (60 µg/ml) damaged merely 2 % of blood cells (Figure 2). This clearly indicates that our compounds are less toxic to human blood cells.
22
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Figure 2. Haemolytic activity of compounds 8, 11, and 14. Tetracycline (TET) and chloramphenicol (CAM) were used as a negative control while Triton X-100 (1%) was used as a positive control. Compounds and antibiotics were tested in 8-fold MIC values.
CONCLUSIONS
On the basis of the activities of the presented 1,4-naphthoquinones, further studies to determine their pharmacological properties would be needed in order to define the usefulness of the
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synthesized compounds as antibacterial agents targeting S. aureus, i.e. one of the most prevalent microorganisms in nosocomial infections.
To conclude, our results show several compounds to be potent and selective anti-S. aureus
AC C
properties.
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agents, which can be a starting point for further improvement in respect of anti-bacterial
The phenomenon of drug resistance forces exploration of novel anti-bacterial agents that overcome these mechanisms. Derivatives of 1,4-naphthoquinones appear to be proper chemicals fulfilling the expectations. Here we present at least six compounds (6, 8, 11, 13, 14, 15) showing anti-bacterial activity at the common antibiotic level. Since the chemicals show no toxicity, they can be taken under consideration as potential anti-bacterial drugs.
23
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ACKNOWLEDGEMENTS The
financial
support
from
the
Polish
National
Science
Centre
grant
number
SC
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2012/05/B/ST5/00362 is gratefully acknowledged.
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ACCEPTED MANUSCRIPT Highlights • A series of naphthalene-1,4-dione derivatives has been synthesized. • The compounds show significant antimicrobial activity. • The chemicals exhibit the highest potency towards Staphylococcus aureus.
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• The compounds show no toxicity towards erythrocytes.
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• The novel 1,4-naphthoquinones can be considered as useful antimicrobial agents.