Synthesis, biological properties, and acid dissociation constant of novel naphthoquinone–triazole hybrids

Article abstract of DOI:10.1016/j.bioorg.2020.104441

A series of novel 1,4-naphthoquinone–triazole hybrids, N-(3-amino-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2-(4-R-1H-1,2,3-triazol-1-yl)acetamide, was synthesized by click chemistry in the presence of sodium ascorbate and copper(II) sulfate pentahydrate in 81–94% yield. Various biological properties of the synthesized compounds including DNA binding/cleavage, antioxidant, antibacterial and antifungal properties were evaluated. The DNA binding study was performed using dsDNA and G-quadruplex DNA. All of the compounds showed fluorescence increase in the presence of DNA, regardless of the structure. Up to 2.9 and 2.5 times fluorescence increase upon incubation with double stranded or G-quadruplex DNA was detected for 5f and 5g, respectively. The docking studies performed on dsDNA and G-quadruplex structures suggested compounds’ mode of interactions were populated around the grooves. All of the compounds showed excellent DNA cleavage activity and 5e was almost degraded the plasmid DNA. The highest radical scavenging activity was obtained as 89.9% at 200 mg/L with 5d. However, the highest ferrous chelating activity was obtained as 68.1% at 200 mg/L with 5g. The compounds exhibited antimicrobial activity against Bacillus cereus, Legionella pneumophila subsp. pneumophila, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus hirae as bacteria strains and Candida albicans and Candida tropicalis as microfungus strains. The compounds exhibited antibacterial and antifungal activity in the range of 4–128 μg/mL and 16–128 μg/mL, respectively. The best antimicrobial activity was obtained with 5d and 5e with a MIC value of 4 μg/mL against Enterococcus hirae. The acid dissociation constants (pK_a) were determined potentiometrically in 20% (v/v) dimethyl sulfoxide-water hydro-organic solvent at an ionic background of 0.1 mol/L of NaCl, at 25 ± 0.1 °C. Five pK_a values were obtained for each ligand.

Full text of DOI:10.1016/j.bioorg.2020.104441

Journal Pre-proofs
Synthesis, biological properties, and acid dissociation constant of novel naph-
thoquinone–triazole hybrids
Yahya Nural, Sadin Ozdemir, Osman Doluca, Bunyamin Demir, Mustafa
Serkan Yalcin, Hasan Atabey, Beyza Kanat, Selma Erat, Hayati Sari, Zeynel
Seferoglu
PII:
S0045-2068(20)31739-9
DOI:
https://doi.org/10.1016/j.bioorg.2020.104441
YBIOO 104441
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
27 July 2020
17 September 2020
27 October 2020
Please cite this article as: Y. Nural, S. Ozdemir, O. Doluca, B. Demir, M. Serkan Yalcin, H. Atabey, B. Kanat, S.
Erat, H. Sari, Z. Seferoglu, Synthesis, biological properties, and acid dissociation constant of novel
naphthoquinone–triazole hybrids, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.
2
020.104441
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Synthesis, biological properties, and acid dissociation constant of novel
naphthoquinone–triazole hybrids
Yahya Nural1,2*, Sadin Ozdemir , Osman Doluca , Bunyamin Demir , Mustafa Serkan
3
4
2,5
6
7
4
2,8
9
10**
Yalcin , Hasan Atabey , Beyza Kanat , Selma Erat , Hayati Sari , Zeynel Seferoglu
1Department of Analytical Chemistry, Faculty of Pharmacy, Mersin University, Mersin,
3169 Turkey
3
2Advanced Technology, Research and Application Center, Mersin University, 33343 Mersin,
Turkey
3Food Processing Programme, Technical Science Vocational School, Mersin University,
Mersin, 33343 Turkey
4Department of Biomedical Engineering, Izmir University of Economics, Izmir, 35330, Turkey
5Department of Mechanical Engineering, Faculty of Engineering, Mersin University, Mersin,
3
3169 Turkey
6Department of Chemistry and Chemical Processing Technologies, Technical Science
Vocational School, Mersin University, Mersin, 33343 Turkey
7Mersin National Education Directorate, Department of Analytical Chemistry, Mersin,
Turkey
8Department of Medical Services and Techniques, Vocational School of Technical Sciences,
Mersin University, Mersin, 33343 Turkey
9Department of Chemistry, Faculty of Science and Arts, Tokat Gaziosmanpasa University,
Tokat, 60250 Turkey
10Department of Chemistry, Faculty of Science, Gazi University, Ankara, 06500 Turkey
*
*
Correspondence: yahyanural@mersin.edu.tr; ynural1805@yahoo.com
*Correspondence: znseferoglu@gazi.edu.tr
1

Abstract
A series of novel 1,4-naphthoquinone–triazole hybrids, N-(3-amino-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)-2-(4-R-1H-1,2,3-triazol-1-yl)acetamide, was synthesized by click
chemistry in the presence of sodium ascorbate and copper(II) sulfate pentahydrate in 81-94%
yield. Various biological properties of the synthesized compounds including DNA
binding/cleavage, antioxidant, antibacterial and antifungal properties were evaluated. The DNA
binding study was performed using dsDNA and G-quadruplex DNA. All of the compounds
showed fluorescence increase in the presence of DNA, regardless of the structure. Up to 2.9
and 2.5 times fluorescence increase upon incubation with double stranded or G-quadruplex
DNA was detected for 5f and 5g, respectively. The docking studies performed on dsDNA and
G-quadruplex structures suggested compounds’ mode of interactions were populated around
the grooves. All of the compounds showed excellent DNA cleavage activity and 5e was almost
degraded all of the plasmid DNA. The highest radical scavenging activity was obtained as
8
6
9.9% at 200 mg/L with 5d. However, the highest ferrous chelating activity was obtained as
8.1% at 200 mg/L with 5g. The compounds exhibited antimicrobial activity against Bacillus
cereus, Legionella pneumophila subsp. pneumophiia, Staphylococcus aureus, Pseudomonas
aeruginosa, Escherichia coli and Enterococcus hirae bacteria strains and two microfungus
Candida albicans and Candida tropicalis strains. The compounds exhibited antibacterial and
antifungal activity in the range of 4-128 μg/mL and 16-128 μg/mL, respectively. The best
antimicrobial activity was obtained with 5d and 5e with a MIC value of 4 μg/mL against
Enterococcus hirae. The acid dissociation constants (pK ) were determined potentiometrically
a
in 20% (v/v) dimethyl sulfoxide-water hydro-organic solvent at an ionic background of
0
.1ꢀmol/L of NaCl, at 25±0.1 °C. Five pK values were obtained for each ligand.
a
Keywords: Naphthoquinone; Triazole; Click chemistry; DNA-binding; DNA-cleavage;
Antioxidant; Antibacterial; Antifungal; Acid dissociation constants
2

1
. Introduction
Click chemistry, a term introduced by Sharpless in 2001 [1], is one of the most popular strategy
for synthesis of pharmacologically active compounds. Because click chemistry offers many
advantages such as stereo- and regio-specific product, high yield, mild reaction conditions etc.,
it has rapidly become a popular tool in drug research. Click chemistry allows the reliable
synthesis of a large number of new compounds. Copper-catalyzed azide-alkyne cycloaddition
click chemistry is widely used in the synthesis of 1,4-substituted-1,2,3-triazoles as regio- and
stereo-specific. Triazoles represent an important group of pharmacophores of drug chemistry
and there are many drugs or drug candidates containing 1,2,3-triazole moiety such as
Rufinamide, Savolitinib, Seviteronel, Solithromycin, Ticagrelor, Molidustat and Tazobactam
in the markets (Fig. 1) [2-5].
H2N
N
N
F2HCO
NH
N
N
N
F
N
O
N
N
N
N
F2HCO
N
N
N
N
OH
NH2
N
N
N
F
Rufinamide
Savolitinib
Seviteronel
(Anticonvulsant)
(Anticancer)
(Anticancer)
F
F
O
O
N
HO
OH
O
N
HO
O
O
H
OMe
O
N
N
N
O
O
N
N
O
S
N
N
N
OH
N
N
O
O
N
N
N
N
O
F
N
N
O
HN
SC3H7
O
CO2H
Ticagrelor
Antiplatelet agent)
Molidustat
(Antianaemics)
Tazobactam
(Antimicrobial)
Solithromycin
(Antimicrobial)
(
Figure 1. Some pharmaceuticals based on 1,2,3-triazoles
Triazole moiety has many indispensable properties desired in drug researches such as non-
toxicity, stability under physiological conditions, H-bonds and π-π stacking interactions and
having both acidic and basic character [1-5]. It is known that 1,2,3-triazole-containing hybrids
exhibited a wide range of pharmacological activities such as antibacterial [6-9],
antimycobacterial [10], antifungal [11-13], antiviral [14], anticancer [15-17], DNA–binding
3

[18,19], DNA–cleavage [20,21], antioxidant [22-25] and multi-target inhibitors against
Alzheimer disease [25].
Naphthoquinone derivatives naturally occur in various fungi, bacteria and plants species
and play an active role in various biological processes [26]. The 1,4-naphthoquinone is one of
the privileged pharmacophore group in drug research since, a great number of naturally
occurring compounds and many synthetic drugs such as atovaquone, buparvaquone and
lapachol contain naphthoquinone core in their molecular structures (Fig. 2) [27,28]. The 1,4-
naphthoquinone derivatives are known to showed a wide range of pharmacological activities,
such as antibacterial [29-33], antifungal [29,34], antimalarial [35], antioxidant [36,37],
anticancer [38,39], DNA–binding/cleavage [40,41] activities. The synthesis of hybrids that
contain at least two pharmacophore groups is an important strategy for achieving invaluable
pharmacological activities. The binding of 1,2,3-triazole core to the molecular structure of
pharmacologically active compounds has become very popular in recent years [2-5]. Within
this framework, there are numerous studies in medicinal chemistry on the synthesis of
compounds that have both triazole and naphthoquinone moieties in their molecular structure. It
has been reported that the compounds exhibited a wide range of pharmacological activities such
as anticancer [42], anti-inflammatory [43] anti-T. cruzi [44], leishmanicidal [45] and
antimycobacterial [46] activity. In the light of this information, it was aimed to synthesize
potentially bioactive novel hybrid compounds containing naphtoquinone and 1,2,3-triazole
pharmacophore groups and to investigate a wide range of biological activities. Although many
compounds bearing 1,2,3-triazole and 1,4-naphtoquinone rings have been synthesized in
literature, there is no hybrid similar to 1,4-naphthoquinone–triazole hybrids to be reported in
present study.
4

C(CH3)3
Cl
O
O
O
O
O
O
OH
OH
OH
Atovaquone
Buparvaquone
Lapachol
(antipneumocysticy)
(Antiprotozoal)
(Anticancer)
Figure 2. Some pharmaceuticals based on naphthoquinones
In present study, novel 1,4-naphthoquinone–1,2,3-triazole hybrids were synthesized and a
wide range pharmacological activities of synthesized hybrids (DNA–binding/cleavage,
antioxidant, antibacterial and antifungal properties) were reported. Additionally, acid
dissociation constants (pK ) were determined in 20% (v/v) dimethyl sulfoxide (DMSO) were
a
also reported.
2
. Experimental
2
.1. Materials and instrumentation
The precursor chemicals purchased from Merck or Aldrich were high-grade and used without
further purification. Fourier-transform infrared spectroscopy (FTIR) spectra were recorded by
a Mattson 1000 FTIR spectrophotometer. Nuclear magnetic resonance (NMR) spectra and
decoupling experiments were recorded with a Bruker Ultrashield Plus Biospin GmbHt at 400
MHz. Chemical shifts were given in parts per million (δ) downfield from TMS as internal
standard. Spectra were determined in dimethyl sulfoxide-d . The following abbreviations were
6
used; s = singlet, d = doublet, dd = doublet of doublets, q = quartet, t = triplet, td = triplet of
doublets and m = multiplet. High resolution mass spectra (HRMS) were recorded in Waters-
LCT-Premier-XE-LTOF (TOF-MS) and Agilent 6224 TOF LC-MS instruments using
electrospray ionization technique and are reported in m/z (rel. %). Melting points were
5

determined by a Mettler Toledo MP90 device. The pH-metric titrations were performed using
a Titroline 7000 automated titrator with SI-Analytics combined with a glass pH electrode,
which could be controlled by a computer and had an automatic micro-burette.
2
.2. The synthesis of 2,3-diaminonaphthalene-1,4-dione, 2
The 2,3-diaminonaphthalene-1,4-dione 2 was prepared from 2,3-dichloronaphthalene-1,4-
dione 1 using the method specified in literatures [47,48] and structure of the compound 2 was
1
confirmed by H NMR spectroscopy, which were found to be identical with the data described
in Ref. [47,48].
2
.3. The synthesis of N-(3-amino-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2-chloroacetamide, 3
The stirred solution of 2,3-diaminonaphthalene-1,4-dione (0.190 g, 1 mmol) in chloroform (20
mL) at 0 ºC was supplemented with a solution of pyridine (0.160 g, 2 mmol) in chloroform (10
mL) and the resultant mixture was stirred for 15 min. After then, a solution of chloroacetyl
chloride (0.230 g, 2 mmol) in chloroform (20 mL) was added to the reaction medium and was
allowed the temperature to come to room temperature. Upon completion of the reaction after 6
h, the solvent was evaporated under reduced pressure and the crude product was first washed
with water and then washed several times with diethyl ether and dichloromethane (DCM),
respectively, in this way the pure product was obtained as orange powder. Yield, 0.20 g, 75%.
-1
1
m.p.: 198-200 ºC (decomp.). IR (cm ): υ 3412, 3259, 3210, 3005, 2954, 1683. H NMR (400
max
MHz, DMSO‑d ): δ 9.36 (s 1H, NH), 7.99-7.95 (m, 2H, Ar-H), 7.83 (td, 1H, J = 7.5 Hz, 1.3
6
1
3
Hz, Ar-H), 7.74 (td, 1H, J = 7.5 Hz, 1.3 Hz, Ar-H), 6.99 (s, 2H, NH ), 4.29 (s, 2H, CH ). C
2
2
NMR (100 MHz, DMSO‑d ): δ 181.6 (C=O), 177.5 (C=O), 164.6 (C=O), 144.2, 134.9, 132.5,
6
6

+
1
2
32.2, 130.1, 125.8, 125.6, 111.7, 43.0. HRMS (ESI-TOF-MS): calcd. for C H ClN O [MH]
12 9 2 3
65.0380; found 265.0371.
2
.4. The synthesis of N-(3-amino-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2-azidoacetamide, 4
The stirred solution of the compound 3 (0.270 g, 1 mmol) in dimethylformamide (DMF) (10
mL) was supplemented with a solution of sodium azide (0.100 g, 1.5 mmol) in DMF (10 mL)
and the resultant mixture was stirred at 75 °C for 18 h. After completion of the reaction, the
mixture was quenched with saturated aqueous sodium bicarbonate, and extracted with ethyl
acetate. The crude mixture was purified by column chromatography (ethyl acetate:hexane / 1:2)
to have the pure product 4 (0.220 g, 81%) as brown powder. m.p.: 192-194 ºC (decomp.). IR
-1
1
(
cm ): υ 3413, 3288, 3204, 3067, 2948, 2114, 1690. H NMR (400 MHz, DMSO‑d ): δ 9.24
m
a
x
6
(s 1H, NH), 7.99-7.96 (m, 2H, Ar-H), 7.85-7.81 (m, 1H, Ar-H), 7.76-7.72 m, 1H, Ar-H), 7.05
1
3
(
s, 2H, NH ), 4.04 (s, 2H, CH ). C NMR (100 MHz, DMSO‑d ): δ 181.6 (C=O), 177.6 (C=O),
2 2 6
1
66.2 (C=O), 144.4, 134.9, 132.4, 132.2, 130.1, 125.8, 125.6, 111.6, 50.8. HRMS (ESI-TOF-
+
MS): calcd. for C H N O [MH] 272.0785; 272.0784.
1
2
9
5
3
2
.5. General procedure for the synthesis of 1,4-naphthoquinone–triazole hybrids, 5a–h
The novel 1,4-naphthoquinone–triazole hybrids 5a–h were synthesized by click chemistry. The
stirred solution of the compound 4 (1 mmol) in DMF (20 mL) was supplemented with sodium
ascorbate (0.6 mmol), CuSO .5H O (0.3 mmol), corresponding alkyne compound (1.5 mmol)
4
2
and 4 mL deionized water, respectively. Resultant mixture was stirred at room temperature for
6 h and then, the mixture was quenched with saturated aqueous sodium bicarbonate, and
1
extracted with ethyl acetate. The crude product was purified by column chromatography (ethyl
acetate:hexane / 1:2).
7

2
1
.5.1. N-(3-amino-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2-(4-(pentan-2-yl)-1H-1,2,3-triazol-
-yl)acetamide, 5a
-1
Black powder. Yield, 0.33 g, 89%. m.p.: 172-174 ºC. IR (cm ): υ 3424, 3309, 3255, 3066,
max
1
2
956, 2931, 1689, 1663, 1614. H NMR (400 MHz, DMSO‑d ): δ 9.54 (s 1H, NH), 7.99-7.96
6
(
m, 2H, Ar-H), 7.85-7.81 (m, 2H, Ar-H and triazole C-H), 7.74 (td, 1H, J = 7.5 Hz, 0.7 Hz, Ar-
H), 7.05 (s, 2H, NH ), 5.28 (s, 2H, C(O)CH ), 290-2.85 (m, 1H, C2H), 1.66-1.57 (m, 1H, C3H),
2
2
1
.53-1.44 (m, 1H, C4H), 1.30-1.20 (m, 2H, C3-H’, C4H’), 1.21 (d, 3H, J = 7.3 Hz, C1H ), 0.86
3
1
3
(
t, 3H, J = 7.3 Hz, C5H ). C NMR (100 MHz, DMSO‑d ): δ 181.5 (C=O), 134.8, 132.4, 132.1,
3
6
1
30.0, 125.7, 125.5, 122.2, 51.6, 38.7, 30.0, 20.4, 19.6, 13.8. HRMS (ESI-TOF-MS): calcd. for
+
C H N O [MH] 368.1723; found 368.1711.
1
9
21
5
3
2
1
.5.2. N-(3-amino-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2-(4-cyclopropyl-1H-1,2,3-triazol-
-yl)acetamide, 5b
-1
Orange powder. Yield, 0.31 g, 91%. m.p.: 230-232 ºC (decomp.). IR (cm ): υ 3354, 3263,
max
1
3
7
0
211, 3143, 3071, 3005, 2950, 1670, 1613. H NMR (400 MHz, DMSO‑d ): δ 9.53 (s 1H, NH),
6
.99-7.96 (m, 2H, Ar-H), 7.86-7.80 (m, 2H, Ar-H and triazole C-H), 7.73 (td, 1H, J = 7.5 Hz,
.8 Hz, Ar-H), 7.04 (s, 2H, NH ), 5.26 (s, 2H, C(O)CH ), 1.99-1.93 (m, 1H, C1H), 0.92-0.88
2
2
1
3
(
m, 2H, C2H, C3H), 0.74-0.70 (m, 2H, C2H’, C3H’). C NMR (100 MHz, DMSO‑d ): δ 181.5
6
(C=O), 177.5 (C=O), 164.2 (C=O), 148.6, 144.3, 134.8, 132.4, 132.1, 130.0, 125.7, 125.5,
+
1
3
22.1, 111.4, 51.6, 7.4 (2 x C), 6.4. HRMS (ESI-TOF-MS): calcd. for C H N O [MH]
17 15 5 3
38.1253; found 338.1245.
2
1
.5.3. N-(3-amino-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2-(4-cyclopentyl-1H-1,2,3-triazol-
-yl)acetamide, 5c
8

-1
Green powder. Yield, 0.32 g, 86%. m.p.: 246-248 ºC (decomp.). IR (cm ): υ 3458, 3347,
max
1
3
1
1
2
280, 3115, 3067, 2950, 2867, 1688, 1666, 1628. H NMR (400 MHz, DMSO‑d ): δ 9.55 (s
6
H, NH), 7.99-7.94 (m, 2H, Ar-H), 7.85-7.79 (m, 2H, Ar-H and triazole C-H), 7.75-7.72 (m,
H, Ar-H), 7.07 (s, 2H, NH ), 5.28 (s, 2H, C(O)CH ), 3.18-3.06 (m, 1H, C1H), 2.06-1.94 (m,
2
2
1
3
H, C2H, C5H), 1.70-1.56 (m, 6H, C2H’, C3H, C3H’, C4H, C4H’ C5H’). C NMR (100 MHz,
DMSO‑d ): δ 181.5 (C=O), 177.5 (C=O), 164.2 (C=O), 144.3, 134.8, 132.4, 132.1, 130.0,
6
1
25.7, 125.5, 122.2, 111.5, 51.6, 36.1, 32.7 (2 x C), 24.6 (2 x C). HRMS (ESI-TOF-MS): calcd.
+
for C H N O [MH] 366.1566; found 366.1550.
1
9
19
5
3
2
.5.4. N-(3-amino-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2-(4-(cyclohexylmethyl)-1H-1,2,3-
triazol-1-yl)acetamide, 5d
-1
Green powder. Yield, 0.37 g, 94%. m.p.: 202-204 ºC (decomp.). IR (cm ): υ 3465, 3356,
max
1
3
1
1
1
0
1
3
3
281, 3109, 3062, 2925, 2852, 1688, 1669, 1625. H NMR (400 MHz, DMSO‑d ): δ 9.55 (s
6
H, NH), 8.03-7.93 (m, 2H, Ar-H), 7.85-7.81 (m, 2H, Ar-H and triazole C-H), 7.75-7.72 (m,
H, Ar-H), 7.06 (s, 2H, NH ), 5.29 (s, 2H, C(O)CH ), 2.57-2.45 (m, 2H, C1H, C1H’), 1.65-
2
2
.53 (m, 6H, C2H, C3H, C4H, C5H, C6H, C7H), 1.23-1.06 (m, 3H, C4H’, C5H’, C6H’), 0.97-
1
3
.86 (m, 2H, C3H’, C7H’). C NMR (100 MHz, DMSO‑d ): δ 181.6 (C=O), 177.6 (C=O),
6
64.3 (C=O), 144.3, 134.9, 132.5, 132.2, 130.1, 125.8, 125.6, 123.9, 123.8, 111.5, 51.6, 37.6,
+
2.7, 32.4 (2 x C), 26.0, 25.6 (2 x C). HRMS (ESI-TOF-MS): calcd. for C H N O [MH]
2
1
23
5
3
94.1879; found 394.1869.
2
.5.5.
N-(3-amino-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2-(4-(2-bromoethyl)-1H-1,2,3-
triazol-1-yl)acetamide, 5e
9

-1
Brown powder. Yield, 0.36 g, 88%. m.p.: 182-184 ºC (decomp.). IR (cm ): υ 3320, 3232,
max
1
3
7
168, 3017, 2966, 1686, 1646, 1618. H NMR (400 MHz, DMSO‑d ): δ 9.57 (s 1H, NH), 7.99-
6
.96 (m, 3H, Ar-H and triazole C-H), 7.85-7.81 (m, 1H, Ar-H), 7.75-7.72 (m, 1H, Ar-H), 7.05
(
s, 2H, NH ), 5.33 (s, 2H, C(O)CH ), 3.75 (t, 2H, J = 6.5 Hz, CH ), 3.23 (t, 2H, J = 6.5 Hz,
2 2 2
1
3
CH ). C NMR (100 MHz, DMSO‑d ): δ 181.5 (C=O), 177.6 (C=O), 164.1 (C=O), 144.3,
2
6
1
34.9, 132.5, 132.2, 130.1, 125.8, 125.6, 124.3, 124.2, 111.5, 51.7, 32.6, 29.0. HRMS (ESI-
+
TOF-MS): calcd. for C H BrN O [MH] 404.0358; found 404.0344.
1
6
14
5
3
2
1
.5.6. Ethyl 1-(2-((3-amino-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-2-oxoethyl)-1H-
,2,3-triazole-4-carboxylate, 5f
-1
Brown powder. Yield, 0.32 g, 85%. m.p.: 162-164 ºC (decomp.). IR (cm ): υ 3459, 3383,
max
1
3
1
7
1
1
6
3
241, 3149, 3016, 2969, 1712, 1673, 1641, 1621. H NMR (400 MHz, DMSO‑d ): δ 9.66 (s
6
H, NH), 8.72 (s, 1H, triazole C-H), 7.99-7.97 (m, 2H, Ar-H), 7.85-7.81 (m, 1H, Ar-H), 7.76-
.72 (m, 1H, Ar-H), 7.08 (s, 2H, NH ), 5.43 (s, 2H, C(O)CH ), 4.32 (q, 2H, J = 6.9 Hz, CH ),
2
2
2
1
3
.31 (t, 3H, J = 6.9 Hz, CH ). C NMR (100 MHz, DMSO‑d ): δ 181.5 (C=O), 177.5 (C=O),
3
6
63.6 (C=O), 160.2 (C=O), 144.3, 138.6, 134.8, 132.4, 132.1, 130.5, 130.0, 125.7, 125.5, 111.2,
+
0.4, 51.9, 14.1. HRMS (ESI-TOF-MS): calcd. for C H N O [MH] 370.1151; found
1
7
15
5
5
70.1143.
2
.5.7.
N-(3-amino-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2-(4-phenyl-1H-1,2,3-triazol-1-
yl)acetamide, 5g
-1
Green powder. Yield, 0.30 g, 81%. m.p.: 164-166 ºC (decomp.). IR (cm ): υ 3456, 3349,
max
1
3
8
274, 3131, 3066, 2956, 1687, 1668, 1624. H NMR (400 MHz, DMSO‑d ): δ 9.62 (s 1H, NH),
6
.55 (s, 1H, triazole C-H), 7.99-7.97 (m, 2H, Ar-H), 7.88-7.81 (m, 2H, Ar-H), 7.83 (td, 1H, J =
1
0

7
7
.6 Hz, 0.7 Hz, Ar-H), 7.73 (td, 1H, J = 7.6 Hz, 0.7 Hz, Ar-H), 7.48-7.44 (m, 2H, Ar-H), 7.36-
.32 (m, 1H, Ar-H), 7.09 (s, 2H, NH ), 5.42 (s, 2H, C(O)CH ). 13C NMR (100 MHz,
2
2
DMSO‑d ): δ 181.6 (C=O), 177.6 (C=O), 164.1 (C=O), 146.2, 144.4, 134.9, 132.5, 132.2,
6
1
30.8, 130.1, 128.9 (2 x C), 127.8, 125.8, 125.6, 125.1 (2 x C), 122.9, 111.5, 51.9. HRMS (ESI-
+
TOF-MS): calcd. for C H N O [MH] 374.1253; found 374.1262.
2
0
15
5
3
2
.5.8. N-(3-amino-1,4-dioxo-1,4-dihydronaphthalen-2-yl)-2-(4-phenethyl-1H-1,2,3-triazol-1-
yl)acetamide, 5h
-1
Brown powder. Yield, 0.33 g, 82%. m.p.: 177-179 ºC (decomp.). IR (cm ): υ 3365, 3207,
max
1
3
7
7
2
154, 3025, 2953, 1695, 1668, 1639, 1615. H NMR (400 MHz, DMSO‑d ): δ 9.54 (s 1H, NH),
6
.99-7.97 (m, 2H, Ar-H), 7.85-7.81 (m, 2H, Ar-H and triazole C-H), 7.75-7.72 (m, 1H, Ar-H),
.30-7.24 (m, 4H, Ar-H), 7.20-7.16 (m, 1H, Ar-H), 7.05 (s, 2H, NH ), 5.29 (s, 2H, C(O)CH ),
2
2
1
3
.94 (s, 4H, 2 x CH ). C NMR (100 MHz, DMSO‑d ): δ 181.6 (C=O), 177.6 (C=O), 164.3
2
6
(
C=O), 144.3, 141.2, 134.9, 132.5, 132.2, 130.1, 128.30 (2 x C), 128.27 (2 x C), 125.9, 125.8,
25.6, 123.6, 123.5, 111.6, 51.7, 34.9, 27.0 HRMS (ESI-TOF-MS): calcd. for C H N O
3
1
2
2
19
5
+
[
MH] 402.1566; found 402.1559.
2
.6. DNA Binding
Double
stranded
DNA
structure
(dsDNA)
was
prepared
by
mixing
and
5
5
ʹ-GACGTGTCGAAAGAGCTCCGATTA-3ʹ
ʹ-TAATCGGAGCTCTTTCGACACGTC-3ʹ oligonucleotides in Tris HCl buffer (pH 7.4).
The mixture was heated up to 50 °C and left to cool down to room temperature. For G-
quadruplexes, 5ʹ-AGGGTTAGGGTTAGGGTTAGGG-3ʹ oligonucleotide was incubated in
Tris HCl buffer containing 50 mM KCl. G-quadruplexes were heated up to 95 °C and left to
1
1

cool down to room temperature before use. The stock solutions containing DNA solutions were
prepared at 50 μM.
The samples for fluorescence measurement were prepared for each compound at 10 μM in
Tris HCl (pH 7.4) containing 5% DMSO. The DNA stock solutions were added to the sample
to achieve respective equivalent concentrations. For all titrations, up to three consecutive
fluorescence measurements were performed with 15 minutes intervals. Only the measurements
after the fluorescence reached equilibrium were taken into account.
All fluorescence measurements were performed at 360 nm excitation and emissions
between 380 and 700 nm with 10 nm excitation and emission slits using Jasco FP8300 in quartz
cuvettes.
Docking studies of the compounds with DNA structures associated topologies were
performed using Autodock Vina and in-house Python script to automate the process. To
represent the dsDNA and the G-quadruplex, PDB files with accession numbers, 1BNA and
1
XAV were used, respectively. After removal of water molecules, removal of non-polar
hydrogens and calculation of Kollman charges, the PDB files were saved as PDBQT. Docking
studies were performed with a grid size of 26 Å for all axes and centred at coordinates 0,0,0 for
1
2
BNA and with a grid of 18, 18 and 40 Å for x, y and z axes and centred at coordinates 14.78,
0.976 and 8.807 for 1XAV. The exhaustiveness was set to 64. Docking studies were performed
on a workstation with 32 cores and 128 GB ECC Ram.
2
.7. DNA cleavage activity
The DNA cleavage activity was tested by agarose gel electrophoresis, which was studied by
incubation at 37 °C as follows: pBR322DNA (0.1 μg/μL) in Tris–HCl 50 mM and NaCl buffer
(18 mM; pH:7.2) was reacted with 1,4-naphthoquinone–triazole hybrids 5a–h and then the
mixture was incubated for 2 h. After that the samples were electrophoresed for 120 min at 80
1
2

V in 0.9% agarose gel and Tris-boric acid-EDTA buffer. After 120 min electrophoresis, bands
were monitored under UV-A light and photographed [49].
2
.8. DPPH Scavenging Activity
DPPH radical scavenging activities of the 1,4-naphthoquinone–triazole hybrids 5a–h were
performed according to the Blois method [50]. For this test, 2 mL of 0.004% DPPH solution
was taken to each tube and then 0.5 mL of 5a–h was added into each tube. The tubes were
shaken quickly and kept at room temperature for 30 minutes in the dark for incubation. After
incubation, absorbances were then measured at 517 nm. The ability to scavenge DPPH free
radical was calculated by the following equation:
%
Inhibition Activity = [A -A )/A ] × 100
0 1 0
A = Control absorbance and A = the absorbance value of the solution containing
0
1
compounds and DPPH after 30 min. The IC50 values were calculated using linear regression
analysis and used to indicate antioxidant capacity.
2
.9. Chelating Activity
The iron chelating activities of the 1,4-naphthoquinone–triazole hybrid compounds 5a–h were
determined with the method specified by Dinis et al. [51]. Different concentrations of 5a–h
solutions were separately put into the test tubes and FeCl (0.05 mL of 2 mM) solution was
2
added to each tubes. The reaction was started by adding 0.1 mL of 5 mM ferrozine
(
C H N NaO S ). After the total volume was completed to 2.5 mL with the solvent used, the
20 13 4 6 2
solution was mixed quickly and kept for 10 minutes at room temperature. Then absorbance
values were read at 562 nm. Without adding 5a–h, 50 µL FeCl (2 mM) and 2.35 mL solvent
2
were added to 100 µL of ferrozine (5 mM) and measured spectrophotometrically at 562 nm.
1
3

For blanc, 0.05 mL of FeCl was added and final volume was completed to 2.5 mL with solvent.
2
The same procedures were applied for the standard (EDTA). The chelating activity of the
2
+
compounds for Fe was calculated using the following equation:
Chelating activity (%) = (Acontrol - Asample)/Acontrol×100;
where; Acontrol is the absorbance of the control reaction, and Asample represents the
absorbance obtained in the presence of compounds or EDTA. In addition, IC50 values were
calculated using linear regression analysis to determine iron chelating ability.
2
.10. Antimicrobial activity
Six different bacteria strains (Bacillus cereus, Legionella pneumophila subsp. pneumophiia
ATCC 33152), Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC
027), Escherichia coli (ATCC 10536) and Enterococcus hirae (ATCC 10541)) and two
(
9
microfungus (Candida albicans and Candida tropicalis (ATCC 750)) were used to investigate
antimicrobial activity. The minimum inhibition concentrations (MICs) of the tested compounds
8
9
were evaluated by two-fold serial dilution method. Microorganisms (about 10 -10 colony
forming units (CFU/mL) were inoculated in medium containing different concentrations of 5a–
h. Culture media were then incubated at 37 °C and 120 rpm for 24 hours in a shaker [52].
2
.11. Determination of Acid Dissociation Constants
The pH values were measured by model TitroLine® 7000 automatic titrator using a combined
glass electrode. The glass electrode was calibrated using standard buffer solutions of pH 4.01,
pH 7.00 and pH 10.01 according to the procedure described elsewhere [53,54] and
determination of pK values was performed by modification of a literature method [55,56]. To
a
maintain the ionic strength at a desired value a high concentrated solution of NaCl was used for
1
4

all titrations. Potentiometric titrations were performed in a double-walled glass titration cell at
5.0±0.1 °C using a thermostat and the titration cell was stirred at a constant rate throughout
2
-3
the titration using a magnetic stirrer. The ligand 5a–h solutions were prepared as 1.10 mol/L
in DMSO, and 0.025 mol/L NaOH, 0.1 mol/L HCl and 1.0 mol/L NaCl stock solutions were
prepared in deionized water. To determine the pK values of 5a–h in 20% (v/v) DMSO-water
a
hydro-organic solvent, titration cell was supplemented with 10 mL of the 5a–h ligand solutions,
1
mL of the HCl solution and 5 mL of the NaCl solution from previously prepared stock
solutions. After that, the titration cell was filled to 50.00 mL with deionized water, The pKw
+
-
value, which is defined as –log[H ][OH ] for the aqueous system, was obtained as 14.49±0.07
at the ionic strength employed. The pK values of 5a–h were calculated from the potentiometric
a
data using HYPERQUAD.
3
. Results and discussion
3
.1. Synthesis and characterization
The synthesis of 1,4-naphthoquinone–triazole hybrids 5a–h was performed by click chemistry
in the presence of copper(II) sulfate pentahydrate and sodium ascorbate as catalyst in 81-94%
yield. The 2,3-diaminonaphthalene-1,4-dione 2 was prepared from 2,3-dichloronaphthalene-
1
,4-dione 1 according to a literature method [47,48]. The 2,3-diaminonaphthalene-1,4-dione 2
was reacted with chloroacetyl chloride, then the resultant product 3 was reacted with sodium
azide to prepare the desired intermediate 4, N-(3-amino-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)-2-azidoacetamide. To obtain the targeted products 5a–h, the N-(3-amino-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)-2-azidoacetamide 4 was reacted with various alkyne compounds in
the presence of sodium ascorbate (0.6 equivalent) and CuSO .5H O (0.3 equivalent) in DMF /
4
2
water (5:1 v/v) at room temperature [17]. 5a–h were obtained with a good to excellent yield
1
5

(81-94%) after isolation using flash chromatography (Scheme 1). The structures of 5a–h were
1
13
fully characterized by various analytical techniques such as H NMR, C NMR, DEPT, COSY,
HMQC, FT-IR and HRMS (See supplementary part). In the FT-IR spectra of 5a–h, after
formation of the triazole ring in the 1,4-naphthoquinone–triazole hybrids 5a–h, the
characteristic strong band of the azide group of the intermediate compound 3, which appeared
−
1
1
at 2114 cm , disappeared. In the H NMR spectra of 5a–h, proton of the triazole ring were
1
observed as a singlet in the range of 8.72 –7.79 ppm depending on inductive effect. In the H
NMR spectra of compound 4, the signal observed as singlet at 4.04 ppm was assigned to the
1
protons of C(O)CH group. After formation of the triazole ring, in the H NMR spectra of 5a–
2
1
3
h, the corresponding signal was observed as singlet in the range of 5.43–5.26 ppm. In the C
NMR spectra of 5a–h, although the NMR solution was prepared as very concentrated and the
NMR measurement time was extended, the carbon peak belonging to the quaternary carbon
atom in the triazole ring was sometimes observed in the NMR spectrum and sometimes not
1
3
observed. In the C NMR spectra of 5a, some of the C=O peaks were by no means observed.
The shift values of the all carbon peaks originating from alkyne compounds have been found
to be appropriate.
O
O
O
O
O
H
N
H
N
Cl
N3
Cl
Cl
48
NH2
NH2
Cl
Cl
NaN3
O
O
R
DMF, 75 o
C
CHCl3, rt, 6 h
NH2
NH2
18 h
O
O
O
O
1
2
3
4
CuSO4.5H2O,
Sodium ascorbate,
DMF:H2O (5:1), rt,
16 h
O
N
H
N
N
N
O
R
NH2
O
5
Compound
R =
5a
5b
5c
5d
5e
5f
5g
5h
O
Br
O
Yield (%)
89
91
86
94
88
85
81
82
Scheme 1. Synthesis of the naphthoquinone–triazole hybrids 5a–h
1
6

3
.2. DNA Binding Activity
The potential of 5a–h as DNA-binding fluorescent agents were analyzed through fluorescence
change in the presence of dsDNA and G-quadruplex DNA. While all components showed
absorbance at low wavelengths, the emission maxima varied between 400 and 440 depending
on the compound. While all compounds showed very low fluorescence, an increase in
fluorescence was apparent for all compounds, except for 5b (Fig. S65). The fluorescence
increase was most dramatic for 5f, 5g and 5h. For these compounds, the titrations of dsDNA
and G-quadruplex resulted in an increase in fluorescence (Fig. S66). Surprisingly, a shift in the
emission maxima was apparent only for 5g in the presence of dsDNA with a new maximum at
4
50 nm. The increase in intensity was tested up to 1:10 equivalent for dsDNA and 3:40
equivalent for G-quadruplex. The most dramatic increases in fluorescence emission in the
presence of dsDNA was detected for 5f, and in the presence of G-quadruplex it was detected
for 5g with up to 2.9 and 2.5 times increase, respectively. While all three compounds, showed
fluorescence increase for the both DNA topologies, 5g showed the highest difference in
fluorescence emission in the presence of difference DNA-topologies as it showed a
fluorescence increase of only 1.3 times increase for equal amount of dsDNA in comparison to
G-quadruplex. This could be interpreted as 5g having higher potential as a topology specific
fluorescent probe among the compounds tested.
The docking studies have showed that affinity of the compounds varied between -7.0 and
-8.3 kcal/mol for G-quadruplex topology, 1XAV (Table S1). For all fluorescently active
compounds, 5f–h, grooves of the G-quadruplex remained as the main target (Figure S67.d-f).
Docking positions also revealed formation of hydrogen bonds by all compounds and it may be
associated to change in fluorescence properties in the presence of DNA structures (Table S2).
In the case of duplex DNA, higher affinities were observed in general and minor groove was
determined as the only target by the docking studies (Figure S67.a-c). For all compounds, the
1
7

affinities towards the duplex form of DNA have ranged between -8.5 and -10.8 kcal/mol. In all
cases, the highest affinities for both DNA structures belonged to 5g. Subsequently, a dramatic
increase in fluorescence was detected in both dsDNA and G-quadruplex of 5g.
3
.3. DNA Cleavage Activity
The DNA cleavage activity of 5a–h was performed by agarose gel electrophoresis. DNA
cleavage was examined by relaxation of super coiled circular form into nicked circular form
and linear form. When circular DNA is run to electrophoresis, relatively fast move will be
monitored for the uncleaved super coiled form. If one strand cleavage is observed, the super
coiled form relax to generate slower moving open circular form. If double strand cleavage is
occurred, linear form produces and moves between super coiled and open circular forms. The
results of DNA cleavage activity of 5a–h are presented in Fig. 3. After the gel electrophoresis,
it can clearly be seen in Fig. 3 that all tested compounds 5a–h exhibited nuclease activity,
whereas DNA+DMSO did not exhibit cleavage activity in Lane 2. The compound 5a showed
single strand cleaved DNA activity, whereas 5b, 5f and 5g demonstrated double strand cleaved
DNA activities in Lane 4 Lane 6 and Lane 8, respectively. Among the test compounds,
especially 5c, 5d, 5e and 5h showed excellent cleaved DNA activities in Lane 5, Lane 7, Lane
9
and Lane 10, respectively. The plasmid DNA was cleaved into the unidentified small DNA
fragments with 5c, 5d and 5h. And also, 5e was nearly degraded the plasmid DNA into the
indistinguishable particles. As a result, the 1,4-naphthoquinone-triazole hybrids 5a–h may be
applied in medicine industries after further studies.
1
8

Figure 3. DNA cleavage activities of the compounds 5a–h. Lane 1, pBR 322 DNA; Lane 2,
pBR 322 DNA + DMSO; Lane 3, pBR 322 DNA + 250 μg/mL of 5a; Lane 4, pBR 322 DNA
+
250 μg/mL of 5b; Lane 5, pBR 322 DNA + 250 μg/mL of 5c; Lane 6, pBR 322 DNA + 250
μg/mL of 5g; Lane 7 pBR 322 DNA + 250μg/mL of 5e; Lane 8, pBR 322 DNA + 250 μg/mL
of 5f; Lane 9, pBR 322 DNA + 250 μg/mL of 5d; Lane 10, pBR 322 DNA + 250 μg/mL of 5h
3
.4 DPPH Scavenging and Chelating Activity
The antioxidant activities of 1,4-naphthoquinone-triazole hybrids 5a–h were determined using
,2-diphenyl-1-picrylhydrazyl (DPPH) free radical. The DPPH process is one of the most
2
powerful procedures for measuring the quantity of radical scavenging compounds active by
chain-breaking operations [57]. DPPH scavenging activity measurement is also a fast, easy,
economic and suitable method to check the antioxidant activity of the compounds. Fig. 4 is
depicted the radical scavenging ability of 5a–h, compared with Trolox and Ascorbic acids. All
tested compounds exhibited well scavenging radical ability. As seen in Fig. 4, the radical
scavenging activities were concentration dependent. When the concentration of 5a, 5b, 5c, 5d,
5
e, 5f, 5g and 5h was increased from 25 mg/L to 50 mg/L, the scavenging activities were
increased from 27.1% to 30.1%, from 31.4% to 59.9%, from 30.6% to 36.8%, from 41.8% to
5
3
3.4%, from 29.6% to 52.9%, from 33.5% to 47.6%, from 26.2% to 39.1%, and from 32.9% to
7.6%, respectively. The DPPH scavenging activities at 100 mg/L of 5b, 5d, 5e, 5f, and 5h
were obtained as 75.6%, 83.7%, 61.1%, 59.4%, and 49.8%, respectively. The highest
scavenging activity was obtained as 89.9% at 200 mg/L with 5d. Additionally, IC50 values of
5
a, 5b, 5c, 5d, 5e, 5f, 5g and 5h were determined as 223.6 mg/L, 48.9 mg/L, 89.2 mg/L, 40.4
mg/L, 47.9 mg/L, 77.9 mg/L, 125.8 mg/L and 107.2 mg/L, respectively. The antioxidant
abilities of the compounds 5a–h may be due to their redox characteristics, which give them to
+
treat as reducing agents or H donor and free radical scavengers. These results showed that the
synthesized compounds may be better candidate for the future studies to develop new
antioxidant agents.
1
9

Figure 4. % Radical scavenging activity of the 1,4-naphthoquinone-triazole hybrids 5a–h
Ferrous Chelating Activity: Most of the reactive oxygen species (ROS) are created as by-
product throughout the electron transport system and other metabolic activities and ROS are
also generated by metal catalyzed oxidation reactions. The transition metal Fe(II) ions have the
ability to maintain the generation of free radicals by loss or gain of electrons. So, the reducing
of the generation of ROS can be achieved by the chelation of toxic metal ions with chelating
agents [58]. The 5a–h have been tested for their ferrous chelating activities. The ferrous
chelating assay of the compounds 5a–h indicated that all compounds 5a–h exhibited chelating
activities and as presented in Fig. 5, chelating activities were concentration dependent.
Chelating activity of 5a–h was compared with the standard (EDTA) material. The ferrous
chelating activities at 25 mg/L were obtained as 26.1%, 18.8%, 24.3%, 24.7%, 22.6%, 30.1%,
2
6.4%, and 21.1% for 5a, 5b, 5c, 5d, 5e, 5f, 5g and 5h, respectively. The ferrous chelating
method displayed that the compounds 5a–h also exhibited chelating ability in the order of 5g >
f > 5a > 5c > 5e > 5b > 5d > 5h at concentration of 100 mg/L. The highest ferrous chelating
5
activity was obtained as 68.1% with 5g at concentration of 200 mg/L. Moreover, the IC50
values of 5a, 5b, 5c, 5d, 5e, 5f, 5g and 5h were found as 153.2 mg/L, 163.7 mg/L, 169.6 mg/L,
2
0

3
08.1 mg/L, 136.4 mg/L, 130.4 mg/L, 121.8 mg/L and 710.9 mg/L, respectively. The chelating
agents are used for neutralizing iron overload in the body in chelation therapy for treatment of
several diseases such as Alzheimer, Parkinson, Thalassemia [59,60]. According to present
findings, especially 5e, 5f and 5g can be used in chelation therapy after further investigations.
Figure 5. Chelating activity of the 1,4-naphthoquinone-triazole hybrids 5a–h
3
.5. Antimicrobial activities
The increase of antibiotic tolerance in microorganisms especially bacteria has become a
significant attention for successful treatment and diagnosis of infectious illnesses. Over the past
few decades, major investigations have been realized on the synthesis and development of new
medicines for combating multi-drug tolerance in microorganisms [61]. The results of the
antimicrobial activity of 5a–h and standard antibiotics are demonstrated in Table 1. All
compounds tested 5a–h exhibited antimicrobial activity against all tested microorganisms with
MICs ranging between 4 and 128 μg/mL. Only one substituent has been changed in the
molecular structure of the 1,4-naphthoquinone-triazole hybrids 5a–h. Compound 5b, which
contains the cyclopropyl group as a substituent, exhibited the best activity among the others
2
1

against E. coli and S. aureus strains with a MIC value of 16 μg/mL and 8 μg/mL, respectively.
Compared to ampicillin (MIC value = 0.5 μg/mL and 1 μg/mL, respectively), all compounds
showed moderate antibacterial activity against E. coli strain but 5b showed noteworthy
antibacterial activity against S. aureus strains. The 5d, which contains the cyclohexylmethyl
group as a substituent, and 5b exhibited the best activity among the others against B. cereus and
P. aeruginosa strains with a MIC value of 8 μg/mL. Compared to ampicillin (MIC value = 1
μg/mL, 0.5 μg/mL, respectively), 5b and 5d exhibited remarkable antibacterial activity against
B. cereus strain. All compounds exhibited antibacterial activity against L. pneumophila subsp.
Pneumophiia strain with a MIC value in the range of 16-64 μg/mL. The 5e, which contains the
2
-bromoethyl as a substituent, and 5d exhibited better antibacterial activity against E. hirae
strain with a MIC value of 4 μg/mL and E. hirae was the most sensitive microorganism among
the test compounds. Compound 5d exhibited the best antifungal activity among the others
against C. albicans and C. tropicalis strains with a MIC value of 32 μg/mL and 16 μg/mL,
respectively. Compared to Fluconazole (MIC value = 0.5 μg/mL), 5a–h exhibited moderate
antifungal activity against the tested fungus strains. As a result of antimicrobial activity study,
it was observed that especially 5d could be an important antimicrobial agent after some
modifications to its molecular structure.
Table 1. The MIC values (μg/mL) of 5a–h against the microbial strains (MIC: The minimal
inhibitory concentrations)
Compound E. coli
B.
S.
P.
E. L. pneumophila C.
C.
cereus aureus aeruginosa hirae
subsp.
albicans tropicalis
pneumophiia
5
a
128
32
64
32
32
32
64
32
5
5
5
5
5
5
5
b
c
d
e
f
16
64
32
8
16
8
16
32
64
16
1
8
8
16
8
16
32
32
16
0.5
-
16
16
4
16
16
16
16
16
32
64
0.5
-
128
128
32
64
128
64
128
-
64
64
16
32
64
32
64
-
16
16
16
32
32
16
1
32
4
128
128
128
0.5
-
32
16
16
0.5
-
g
h
Ampicillin
Fluconazole
-
-
0.5
0.5
2
2

3
.6. Acid Dissociation Constants
In order to continue drug research studies on a molecule, pK value(s) which is one of the most
a
important physicochemical parameters must be determined because it provides critical data
related to acidity, degree of ionization, solubility and hydrogen bonding capacity of the
compounds [62,63]. The pK values of 5a–h were determined potentiometrically at 25.0±0.1
a
°
C, 0.1 mol / L ionic strength of NaCl in 20% (v/v) DMSO-water hydro-organic solvent system
using a literature method [54,55]. The calculated pK values are given in Table 2, and titration
a
curves of the ligands 5a–h and distribution curve of 5a for symbolized all ligands 5a–h are
presented in Fig. 6. All obtained logβ values and distribution curves were submitted as
supporting data (Fig. S68).
Table 2. pK values of 5a–h (20% (v/v) DMSO-water, 25.0 ±0.1 ºC, I= 0.1 mol/L by NaCl)
a
Ligand pKa1
pKa2
pKa3
pKa4
pKa5
5
a
2.85±0.01 6.25±0.02 10.43±0.02
3.06±0.03 5.25±0.02 10.81±0.03
2.13±0.02 6.66±0.02 10.47±0.02
2.85±0.01 6.25±0.02 10.43±0.03
10.86±0.01 11.39±0.03
5
5
5
5
5
5
5
b
c
d
e
f
10.99±0.05 11.36±0.07
11.05±0.03 11.46±0.06
10.89±0.03 11.36±0.06
10.38±0.05 11.21±0.08
10.67±0.05 11.17±0.08
10.92±0.02 11.31±0.06
10.68±0.03 11.29±0.047
2.60±0.03 6.69±0.03
9.83±0.05
2.93±0.02 7.07±0.03 10.29±0.03
3.07±0.01 5.72±0.01 10.39±0.03
2.71±0.01 7.27±0.02 10.04±0.02
g
h
2
3

100
LH3
LH5
L
LH4
80
60
40
20
0
LH
LH2
2
6
10
pH
Figure 6. Titration curves of 5a–h and distribution curve of 5a for symbolized all ligands.
(25.0±0.1 °C, 0.1 mol/L ionic strength of NaCl in 20% (v/v) DMSO-water)
According to the results, five protonated species were obtained as LH, LH , LH , LH and
2
3
4
LH calculated five pK values. Full protonated form of the ligands is showed in Fig. 7 and four
5
a
protonated species are related nitrogen atoms and a protonated species is related oxygen in the
ligands.
H
O
N
H
H
N 2 NH
N
1
3
O
4
NH3
R
5
OH
5
+
Figure 7. Full protonated form of the ligands 5a–h (LH )
5
In our previous study [17], pyrrolidine linked to 1,2,3-triazole derivatives were examined
and dissociation properties of 1,2,3-triazole moieties were explained clearly. The 1,2,3-triazole
includes diazinyl group (-N=N-) and tertiary imine nitrogen. While one of two nitrogen atoms
in diazinyl groups is very acidic, the other is very basic. Thus, calculated pK values (between
a1
2
.85-3.07) in present study are related to acidic diazinyl nitrogen. Additionally, other diazinyl
nitrogen is very basic and it is related to pK values (between 10.61 – 10.97). The third nitrogen
a3
2
4

atom in 1,2,3-triazole ring is the most basic atoms due to electronic effects and related pKa5
values varied between 11.01 – 11.54. In present study, proton affinity of nitrogen atoms in the
ligands were calculated theoretically using semi-empirical methods such as modified neglect of
diatomic overlap (MNDO). The formation heats (Hf) and the total energies (TE) of the ligands
and mono-protonated species were calculated. In addition, the proton affinity of each nitrogen
atom (PA) in the ligands was found using formation heats in the following equation and given
in Table 3.
+
PA = 367.2 + ΔH (B) - ΔH (BH )
fº
fº
where; PA is the proton affinity of B types; ΔH (B) is the formation heat of B molecule;
fº
+
+
+
ΔH (BH ) is the formation heat of BH molecule, and 367.2 is the formation heat of H [64].
fº
Table 3. The calculated formation heat (H ), total energy (TE) and proton
f
affinity (PA) values with MNDO methods for 5a for symbolized all of the
ligands and their mono-protonated forms.
MNDO
Species
T.E. (kcal/mol)
Hf
kcal/mol)
-6.50
PA
(
5
a
-94591.29
-94717.18
-94756.19
-94769.26
-94747.62
-
1
N-H+
N-H2+
N-H2+
N-H2+
194.28
155.27
142.21
163.84
166.42
205.45
218.49
196.86
2
3
4
According to Table 3, the proton affinity of nitrogen in position 3 was higher than the other
nitrogen atoms and proton affinity of the nitrogen in position 1 was lower than the others. The
nitrogen atom in position 3 was the most basic atom and the most acidic atom was the nitrogen
in position 1. Therefore, the first protonated atom was the nitrogen atom in position 3 which
2
5

present in the molecular structures of the ligands. According to the calculation results, the
protonation order for nitrogen atoms in the ligand was as 3N, 2N, 4N and 1N. Quinoline
molecules behave as an aromatic cycle due to delocalization of π electrons. Thus, other two pKa
values (pK and pK ) related to phenolic oxygen and aniline nitrogen atoms. Therefore,
a2
a4
attention was paid in this study to explain the pK values of 1,4-naphthoquinone-triazole hybrid
a
ligands because of their pharmacological importance.
4
. Conclusion
The synthesis of the novel 1,4-naphthoquinone-triazole hybrid compounds 5a–h containing two
very valuable pharmacophore groups was demonstrated and their various biological properties
including DNA binding / cleavage, antioxidant and antimicrobial activities were evaluated in
present study. The 5a–h exhibited high DPPH scavenging and ferrous chelating activities. A
form of DNA interaction was apparent due to fluorescence increase for all compounds in the
presence of the duplex DNA. While the level of increase was limited up to 2.9 times, this was
measured in presence of as low as 1:10 equivalent DNA. Moreover, in spite of the toplogy of
the DNA used, dsDNA or G-quadruplex, the fluorescence was enhanced for all tested
compounds, 5f, 5g, 5h. This result indicating the potential of these compounds as fluorescent
probes; however, fluorescence enhancement may be overshadowed by the DNA cleavage
activity. Especially 5c, 5d, 5e and 5h showed high DNA cleavage activity. All tested
compounds displayed antibacterial and antifungal activities. Among all the compounds, 5d
demonstrated the most efficient antimicrobial activity. Present findings on biological activities
of the compounds should pass through further pharmacological and toxicological tests and
probably will conveniently be used in further antioxidant, antimicrobial and anticancer
medicine development. Further new drug researches are recommended to be performed on such
2
6

1
,4-naphthoquinone-triazole hybrids. The pK values containing critical information about the
a
compounds will provide significant contributions to further pharmacological studies on these
types of compounds.
Acknowledgements
The authors thank the Scientific and Technological Research Council of Turkey (TÜBİTAK,
project grant 119Z033) for financial support.
Conflict of Interest: The authors declare no conflict of interest.
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