New family of antimicrobial agents derived from 1,4-naphthoquinone

Article abstract of DOI:10.1016/j.ejmech.2016.10.034

Naphthalene-1,4-dione derivatives were synthesized and tested against selected bacterial strains. All the tested compounds were prepared by direct introduction of corresponding substituents into the naphthoquinone core in oxidative conditions. In this stu

Full text of DOI:10.1016/j.ejmech.2016.10.034

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
10.1016/j.ejmech.2016.10.034
EJMECH 8999
DOI:
Reference:
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.
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ACCEPTED MANUSCRIPT

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New family of antimicrobial agents derived from
1,4-Naphthoquinone
Monika Janeczkoa, Oleg M. Demchukb, Dorota Strzelecka,b Konrad Kubińskia, and Maciej
Masłyka*
a Department of Molecular Biology, Institute of Biotechnology, The John Paul II Catholic
University of Lublin, ul. Konstantynów 1i, 20-708 Lublin, Poland
b Organic Chemistry Department, Faculty of Chemistry, Maria Curie-Skłodowska University,
ul. Gliniana 33, 20-614 Lublin, Poland
*Corresponding Author
Dr. Maciej Masłyk, PhD, Department of Molecular Biology, Institute of Biotechnology, The
John Paul II Catholic University of Lublin, ul. Konstantynów 1i, 20-708 Lublin, Poland, Tel:
+48814545452, e-mail: maciekm@kul.pl
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ABSTRACT:
Naphthalene-1,4-dione derivatives were synthesized and tested against selected bacterial strains.
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,
Enterococcus) were used for determination of antimicrobial activity of synthesized compounds
with the Minimal Inhibitory Concentration (MIC) method. Additionally, selected compounds
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
derivatives exhibited haemolytic activity.
KEYWORDS
Antimicrobial, naphthoquinones, Staphylococcus aureus, haemolysis
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INTRODUCTION
In the recent decade, a problem of an increasing number of bacterial pathogens exhibiting
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
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
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
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
wear. Chronic infection can serve as a septic focus that can lead to osteomyelitis, acute sepsis,
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-
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
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
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
toxicity by means of a haemolytic assay of selected compounds against human erythrocytes.
MATERIALS AND METHODS
Chemistry
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
13
for 1H and C 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
chromatography on silica gel 60 (230-400 mesh) using a BUCHI chromatograph. MS spectra
were recorded on a Shimadzu LCMS IT-TOF spectrometer.
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
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
o
mL), the mixture was stirred at 30 C 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
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
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1
mg (70 %), yellow solid, mp = 111.3- 113.8 C (lit. mp = 108-110 C) [28]. H NMR (500.13
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. 13C NMR (125.75 MHz,
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. 13C 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); 8.17-
8.18 (m, 2H, CH) ppm. 13C 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. 13C 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 =
not ionisable [M+H]+.
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 =
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,
134.52, 136.82, 139.20, 184.67, 185.27 ppm. 13C NMR (DEPT 135, 125.75 MHz, CDCl3): δ =
19.77 (CH3), 19.88 (CH3), 125.92 (CH), 126.98 (CH), 127.03 (CH), 129.81 (CH), 130.53 (CH),
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
o
general procedure I. Mesitylene was used instead of benzene at 70 C. 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. 13C NMR (DEPT 135, 125.75 MHz,
CDCl3): δ = 20.39 (CH3), 21.14 (CH3), 126.19 (CH), 127.07 (CH), 128.38 (CH), 133.87 (CH),
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]+.
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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1
of benzene at 50 C. Yield: 20 mg (20 %), dark purple solid, mp = 228.3 - 230.5 C. H 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,
CH) ppm. 13C NMR (125.75 MHz, CDCl3): δ = 55.96, 55.98, 106.04, 107.08, 124.70, 125.96,
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. 13C NMR (DEPT 135, 125.75 MHz, CDCl3): δ = 55.96 (OCH3), 55.99 (OCH3),
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]+,
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 C 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
and the residue was purified by column chromatography on silica gel, using a gradient
hexane/acetone mixture as eluent.
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 C. H 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.
13C NMR (125.75 MHz, CDCl3): δ = 55.23, 55.46, 98.79, 104.44, 115.61, 125.64, 126.66,
131.38, 131.92, 132.44, 133.20, 133.33, 135.98, 147.15, 158.36, 162.06, 183.71, 185.15 ppm.
13C 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)
ppm. HRMS (ESI): m/z = 317.0770 [C18H14O4+Na]+, m/z (theor.) = 317.0784, diff. = 4.42 ppm.
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. 13C 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]+,
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,
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,
11.73, 120.52, 125.71, 126.92, 130.98, 131.18, 132.33, 132.92, 133.38, 133.54, 147.43, 151.74,
185.25, 185.51 ppm. 13C 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.
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 =
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
= 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. 13C 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
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1
procedure II. Yield: 25%, mp = 145.3 - 146.7 C. H 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)
7.53-7.56 (m, 2H, CH) 7.73-7.76 (m, 2H, CH) 8.09-8.18 (m, 2H, CH) ppm. 13C NMR (125.75
MHz, CDCl3): δ = 54.05, 112.12, 121.31, 125.74, 126.53, 126.96, 127.24, 128.86, 131.20,
13
131.46, 132.32, 132.87, 133.43, 133.59, 137.61, 147.27, 150.83, 185.21, 185.44 ppm. C NMR
(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
(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 =
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)
13
ppm. C NMR (125.75 MHz, CDCl3): δ = 40.36, 55.52, 95.51, 104.44, 110.89, 125.73, 126.86,
132.14, 132.32, 133.02, 133.29, 133.32, 134.81, 147.47, 153.03, 158.94, 184.64, 185.55 ppm.
13C NMR (DEPT 135, 125.75 MHz, CDCl3): δ = 40.36 (N(CH3)2), 55.52 (CH3), 95.52 (CH),
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
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according to the general procedure II. Yield: 483 mg (15 %), mp= 209-210 C (dec.). H 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,
147.41, 157.46, 168.82, 183.40, 184.75 ppm. 13C NMR (DEPT 135, 125.75 MHz, CDCl3): δ =
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
[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
(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
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
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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oC (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
(br, 1H, CO2H) ppm. 13C NMR (125.75 MHz, DMSO-d6): δ = 21.52, 30.76, 50.58, 62.16, 69.67,
13
104.53, 124.46, 125.87, 130.97, 131.98, 134.13, 145.46, 148.42, 180.86, 182.32 ppm. C NMR
(DEPT 135, 125.75 MHz, DMSO-d6): δ = 21.59 (CH2), 30.83 (CH2), 50.68 (CH2), 62.23 (CH),
104.60 (CH), 124.54 (CH), 125.94 (CH), 132.05 (CH), 134.20 (CH) ppm. HRMS (ESI): m/z =
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
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,
13
CH); 8.01 (dd, J = 7.57, 0.95 Hz, 1H, CH), 13.22 (bs, 1H, OH) ppm. 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,
132.42, 134.69, 147.35, 171.68, 180.88, 181.52 ppm. 13C 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.
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N-(1,4-Naphthoquinone-2-yl)-L-tryptophan (17): obtained according to the general
o
procedure III. Yield: 570 mg (63 %), red solid, mp = 159.1 - 161.3 C (dec.) (lit. mp = 208.0-
211.0 oC (dec.))[30, 31]. 1H NMR (500.13 MHz, DMSO-d6): δ = 3.23 (d, J = 5.67 Hz, 2H), 4.34
– 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
(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,
181.41 ppm. 13C NMR (DEPT 135, 125.75 MHz, DMSO-d6): δ = 26.58 (CH2), 55.66 (CH),
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]+,
m/z (theor.) = 383.1002, diff. = 2.09 ppm.
[(1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino]-D-(phenyl)acetic acid (18): obtained
o
according to the general procedure III. Yield: 70 mg (9%), red solid, mp = 195.3 – 199.5 C
(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,
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),
ppm. 13C 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. 13C 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)
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
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
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,
13
134.47, 146.26, 171.76, 181.08, 182.91 ppm. C NMR (125.75 MHz, CDCl3): δ = 17.62 (CH3),
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.
Biological activities
Microbial strains. In present study, reference bacterial strains were used. Gram-positive
bacteria: Staphylococcus aureus (ATCC 6538), Enterococcus faecalis (PCM 2673), Gram-
negative: 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
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
oC. 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.
Experiments were performed in triplicate.
Haemolytic assay. The human blood samples were placed in sterile tubes containing a citrate
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
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
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
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
reference.
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
procedure presented in the literature (cat. = Pd(OAc)2, additive = CF3CO2H, oxidant = Bz2O2,
o
solvent = CCl4 or ArH, t = 60 C) (Scheme 1) [33]. Despite the low yields observed in some of
these cases, application of cationic palladium catalysis yields products with substituents in
positions that do not correspond to the usual reactivity of aromatics undergoing the electrophilic
substitution reaction. Sites of molecules thus activated by the substitution effects are left
accessible to further transformation or biochemical interactions. In all cases, the palladium-
catalysed reaction always yields less sterically hindered substitution products.
16

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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
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].
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
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
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
series of 19 naphthoquinones substituted in positions 2 and 3 and bearing a wide pattern of
function groups was prepared for the biological tests.
18

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Figure 1. Assessed naphthoquinones
19

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Biology
Certain alkyl-, alkenyl- and amino-1,4-naphthoquinones have already been assessed as
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.
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
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
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.
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
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

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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-
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)-1,4-
naphthoquinone inhibited S. aureus growth at a MIC value of 62.6 µg/ml [26]
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
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-
naphthoquinones induce oxidative stress and inhibit the growth of S. aureus at a concentration of
50 µg/ml [38].
Table 1. Antibacterial activity of 1,4-naphthoquionones expressed as minimal inhibitory concentration (µg/ml).
31.2 125 125 250 125 250 n.a. 125
62.5 n.a. 250 250 250 250 n.a. 250
n.a. n.a. 250 n.a. n.a. n.a. n.a. 250
62.5 n.a. 500 500 250 500 500 500
500 n.a. 500 500 500 500 500 500
15.6 n.a. 500 500 500 500 500 500
500 n.a. 500 500 500 500 n.a. 500
1
2
3
4
5
6
7
21

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15.6 n.a. 250 250 250 250 n.a. 250
8
250 n.a. 250 250 250 250 n.a. 250
125 n.d. 250 62.5 125 250 250 125
9
10
11
12
13
14
15
16
17
18
19
7.8
250 250 250 250 250 250 n.a. 250
7.8 n.a. 250 250 250 250 n.a. 250
n.a. 250 250 250 250 n.a. 250
15.6 n.a. 250 250 250 250 n.a. 250
31.2 250 250 250 250 250 500 250
500 500 500 500 500 500 500 500
62.5 500 500 500 500 500 500 500
250 500 500 500 500 500 n.a. 500
62.5 250 500 500 250 500 500 500
TET 7.8
31.2 15.6 15.6 0.5 7.8 7.8 3.9
n.d.: not determined; n.a.: no activity;
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
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 1,4-
naphthoquinone derivative, vitamin K3 menadione (2-methyl-1,4-napthoquinone), inhibits the
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
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
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
agents, which can be a starting point for further improvement in respect of anti-bacterial
properties.
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
2012/05/B/ST5/00362 is gratefully acknowledged.
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