Anticancer, antimicrobial, and DNA protection analysis of novel 2,4-dihydroxyquinoline dyes

Article abstract of DOI:10.1016/j.dyepig.2018.04.040

A new series of 2,4-dihydroxyquinoline derived disazo dyes has been synthesized by the reaction of 5-amino-4-phenylazo-3-methyl-1H-pyrazole derivatives with 2,4-dihydroxyquinoline. The structures of the obtained dyes were identified by various spectrophotometric methods such as FT-IR, ¹H-NMR, and elemental analysis. The synthesized compounds were also examined for different biological activities, including DNA protection, antimicrobial, and anticancer activities. Compound 3b was found most effective on Gram-positive bacteria. A DNA protection assay was applied to all compounds, and it was found that compounds 3h and 3j had a high capacity for binding to DNA. Besides, compounds 3h and 3j showed cytotoxicity against HeLa and PC3 cancer cell lines. These compounds could potentially be used as drugs or drug additives based on their effects on bacterial and cancer cell lines.

Full text of DOI:10.1016/j.dyepig.2018.04.040

Accepted Manuscript
Anticancer, antimicrobial, and DNA protection analysis of novel 2,4-
dihydroxyquinoline dyes
Nesrin Şener, Hassan Jaballah Abdullah Mohammed, Serife Yerlikaya, Yasemin celik
Altunoglu, Mahmut Gür, Mehmet Cengiz Baloglu, İzzet Şener
PII:
S0143-7208(18)30872-6
10.1016/j.dyepig.2018.04.040
DYPI 6701
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 18 April 2018
Accepted Date: 20 April 2018
Please cite this article as: Şener N, Mohammed HJA, Yerlikaya S, celik Altunoglu Y, Gür M, Baloglu MC,
Şener İ, Anticancer, antimicrobial, and DNA protection analysis of novel 2,4-dihydroxyquinoline dyes,
Dyes and Pigments (2018), doi: 10.1016/j.dyepig.2018.04.040.
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ACCEPTED MANUSCRIPT
OH
N
N N
NH
N N
H3C
OH
N
N
HO
HO
X
0-5 oC
H2N
N
NH
N
N
CH3
X
DNA PROTECTION
SYNTHESIS
ANTIMICROBIAL
ANTICANCER

ACCEPTED MANUSCRIPT
Anticancer, Antimicrobial, and DNA Protection Analysis of Novel 2,4-
Dihydroxyquinoline Dyes
Nesrin ŞENERa*, Hassan Jaballah Abdullah MOHAMMEDb, Serife YERLIKAYAc,
Yasemin CELIK ALTUNOGLUc, Mahmut GÜRd, Mehmet Cengiz BALOGLUc, İzzet
ŞENERe
aDepartment of Chemistry, Faculty of Science-Arts, Kastamonu University, 37200, Kastamonu, Turkey
bDepartment of Biology, Institute of Science, Kastamonu University, 37200, Kastamonu, Turkey
c
Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Kastamonu University,
Kastamonu, Turkey
dDepartment of Forest Industrial Engineering, Faculty of Forestry, Kastamonu University, 37200, Kastamonu,
Turkey
eDepartment of Food Engineering, Faculty of Engineering and Architecture, Kastamonu University, 37200,
Kastamonu, Turkey
*Corresponding author. Tel.: 90 366 2801972; fax: 0 366 215 4969.
E-mail addresses: nsener@kastamonu.edu.tr, (N Sener).
ABSTRACT:
A new series of 2,4-dihydroxyquinoline derived disazo dyes has been synthesized by the
reaction
of
5-amino-4-phenylazo-3-methyl-1H-pyrazole
derivatives
with
2,4-
dihydroxyquinoline. The structures of the obtained dyes were identified by various
spectrophotometric methods such as FT-IR, 1H-NMR, and elemental analysis. The
synthesized compounds were also examined for different biological activities, including DNA
protection, antimicrobial, and anticancer activities. Compound 3b was found most effective
on Gram-positive bacteria. A DNA protection assay was applied to all compounds, and it was
found that compounds 3h and 3j had a high capacity for binding to DNA. Besides,
compounds 3h and 3j showed cytotoxicity against HeLa and PC3 cancer cell lines. These
compounds could potentially be used as drugs or drug additives based on their effects on
bacterial and cancer cell lines.
1

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Keywords: Diazotization, Disazo dyes, Anti-cancer, DNA protection, 2,4-
dihydroxyquinoline, Antimicrobial activity
1. INTRODUCTION:
Azo dyes are the subject of many studies due to their biological and pharmacological
activities [1–7]. Many arylazo compounds are metabolized by biological systems to their
corresponding primary amines, probably via hydrazo intermediates. Generally, current in vivo
and in vitro metabolic studies are focused on azo compounds having an aromatic ring such as
phenyl, naphthyl, pyrazole, quinoline, and their derivatives [8–10].
Quinolines and their derivatives are a significant class of nitrogen-containing
heterocycles because they include useful dyes and intermediates in organic synthesis [11].
Although quinoline itself has few applications, its derivatives are used in diverse applications,
including pharmaceuticals, and are even available as drugs today [12]. Quinoline derivatives
have shown activity against many types of cancer. For example, chelidonine, chelerythrine,
and sanguinarine derivatives are effective against lung and pancreatic cancers [13,14], and a
berberine derivative is effective against colon cancer [15]. Derivatives of these compounds,
lavendamycin, lophocereine, and streptonigrin, have been used as antitumor antibiotics [16-
18]. Moreover, many quinoline derivatives were investigated for their antimalarial, anti-
inflammatory, antiasthmatic, antibacterial, and antihypertensive activities [19, 20]. In
addition, many studies showed species that exhibit DNA binding and can protect DNA [21–
23]. It was suggested that quinolines can chelate Fe(II) and Fe(III) and inhibit free radical
production in the Fenton reaction [24] and that quinolines can also intercalate DNA duplexes
and react with free radicals to protect DNA from oxidative damage; covalent binding of
quercetin to DNA and protein has also been reported [25–27].
A study on the biological properties and DNA interaction with quinoline derived azo
dyes has been published [28]. This presents the in vitro activities of nine 3-
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hetarylazoquinolin-2,4-dioles against eight clinical pathogenic bacteria and two fungi, with
the expectation that they might exhibit biological activity and be useful in the design of
biologically active azoquinolines. In addition, to gain further insight into their biological
activities, the nature of plasmid DNA protection of the compounds was also studied [28].
Numerous studies have been performed to examine the biological properties of
quinoline derivatives. However, the study of azo dyes derived quinoline in this area has been
limited. For this purpose, we have prepared a novel series of 2,4-dihydroxyquinoline derives
disazo dyes by reacting 5-amino-4-phenylazo-3-methyl-1H-pyrazole derivatives with 2,4-
dihydroxyquinoline. The structures of the obtained dyes were characterized by various
1
spectrophotometric methods such as FT-IR, H-NMR, and elemental analysis. Biological
activity studies were performed to investigate the antimicrobial, anticancer and DNA
protective properties of these novel compounds.
2. EXPERIMENTAL
2.1. Synthesis of 2-arylhydrazono-3-ketiminobutyronitriles 1(a–m) and 5-amino-4-arylazo-3-
methyl-1H-pyrazoles 2(a–m)
The 2-arylhydrazone-3-ketiminobutyronitrile compounds 1(a–m), and 5-amino-4-
arylazo-3-methyl-1H-pyrazoles, compounds 2(a–m), were prepared according to the literature
procedures [29, 30]. The general route of the reactions is outlined in Scheme 1.
CN
C
NH
NC
H
NH2
CH3
NaNO2/HCl/C2H5OH
NH2NH2.H2O
N
NH2
NH
N
NH2
C
N
NH
+
X
CH3
X
X
H3C
2 (a-m)
N
1 (a-m)
a; X: H
d; X: p-Cl
g; X: m-OCH3
h; X: m-Cl
j; X: o-NO2
k; X: o-OCH3
l; X: o-Cl
m; X: o-CH3
b; X: p-NO2
c; X: p-OCH3
e; X: p-CH3
f; X: m-NO2 i; X: m-CH3
Scheme 1. Synthesis of Compounds 1(a–m) and 2(a–m)
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2.2. Synthesis of 3-(3'-methyl-4'-(arylazo-1'-H-5-ylazo) pyrazole)-2,4-dihydroxyquinoline
3(a–m)
2.2.1. Synthesis of 3-(3'-methyl-4'-(phenylazo-1'-H-5-ylazo) pyrazole-2,4-dihydroxyquinoline
(3a)
A solution of 5-amino-4-phenylazo-3-methyl-1H-pyrazole (2.5 mmol) (2a) in
concentrated hydrochloric acid (3 mL) (ratio 1:1) and glacial acetic acid (6 mL) was prepared
and stirred magnetically in an ice-salt bath. Then, a solution of NaNO2 (2.5 mmol) in water (2
mL) was added dropwise to this mixture and stirred for 2 hours. In another beaker, a solution
of 2,4-quinolinediol (2.5 mmol) was dissolved in sodium hydroxide solution (2.5 mmol) in
water (5 mL). The obtained solution of 2,4-quinolinediol was then vigorously stirred, while
adding dropwise over 15 min to the prepared diazonium salt, continuing magnetic stirring for
2 hours in the ice-salt bath. The resulting dark brown product was precipitated with water,
filtered, dried, and crystallized from DMSO-water mixtures (ratio 4:1). Yield: 63% mp:
decomposition >325°C FT-IR (cm-1) υmax: 3178 (-NH), 3092 (aromatic C-H), 2950 (aliphatic
1
C-H), 1657 (C=O), 1546 and 1460 (stretching and bending N=N); H-NMR (DMSO-d6)
(ppm): 2.59 (s, 3H pyrazole –CH3), 7.17–8.21 (9H, aromatic C-H), 11.13–11.20 (s, O-H),
11.26–11.32 (b, N-H), 11.49–11.57 (s, O-H). Elemental analysis: Anal. Calc. for
C19H15N7O2: C, 61.12%; H, 4.05%; N, 26.26%. Found: C, 61.63%; H, 4.41%; N, 26.95%.
The other compounds named as 3(a-m) were also synthesized using the same method
described for the synthesis of compound 3a. The general reaction route is as outlined in
Scheme 2.
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X
X
OH
N
OH
N
N
N
H2N
HN
CH3COOH / HCl / NaNO2
+
N
N
N
N
CH3
OH
N
OH
HN
CH3
N
2 (a-m)
3 (a-m)
a; X: H
b; X: p-NO2
c; X: p-OCH3
d; X: p-Cl
e; X: p-CH3
f; X: m-NO2 i; X: m-CH3
g; X: m-OCH3
h; X: m-Cl
j; X: o-NO2
k; X: o-OCH3
l; X: o-Cl
m; X: o-CH3
Scheme
2.
Synthesis
of
3-(3'-methyl-4'-(arylazo-1'-H-5-ylazo)
pyrazole-2,4-
dihydroxyquinoline derivatives.
2.2.2.
Synthesis
of
3-(3'-methyl-4'-(4-nitrophenylazo-1'-H-5-ylazo)
pyrazole-2,4-
dihydroxyquinoline (3b)
Brown product, Yield: 67% mp: 238°C FT-IR (cm-1) υmax: 3269 (-NH), 3099
(aromatic C-H), 2981 (aliphatic C-H), 1656 (C=O), 1560 and 1470 (stretching and bending
1
N=N), 1506 and 1332 (stretching and bending -NO2); H-NMR (DMSO-d6) (ppm): 2.59 (s,
3H pyrazole–CH3), 7.11–8.42 (8H, aromatic C-H), 11.12–11.16 (s, O-H), 11.18–11.40 (b, N-
H), 11.46–11.50 (s, O-H). Elemental analysis: Anal. Calc. for C19H14N8O4: C, 54.55%; H,
3.37%; N, 26.78%. Found: C, 55.02%; H, 3.32%; N, 27.05%.
2.2.3. Synthesis of 3-(3'-methyl-4'-(4-methoxyphenylazo-1'-H-5-ylazo) pyrazole-2,4-
dihydroxyquinoline (3c)
Red product, Yield: 71% mp: decomposition >330°C FT-IR (cm-1) υmax: 3185 (-NH),
3092 (aromatic C-H), 2981, 2857 (aliphatic C-H), 1653 (C=O), 1546 and 1459 (stretching and
1
bending N=N), 1100 (C-O); H-NMR (DMSO-d6) (ppm): 2.59 (s, 3H pyrazole -CH3), 5.74
(s, 3H p-OCH3), 7.12–7.79 (8H, aromatic C-H), 11.21–11.23 (s, O-H), 11.32–11.33 (s, O-H).
Elemental analysis: Anal. Calc. for C20H17N7O3: C, 59.25%; H, 4.25%; N, 24.30%. Found:
C, 59.63%; H, 4.20%; N, 24.75%.
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2.2.4. Synthesis of 3-(3'-methyl-4'-(4-chlorophenylazo-1'-H-5-ylazo) pyrazole-2,4-
dihydroxyquinoline (3d)
Dark red product, Yield: 68% mp: 316°C FT-IR (cm-1) υmax: 3207 (-NH), 3077
(aromatic C-H), 2950 (aliphatic C-H), 1653 (C=O), 1546 and 1460 (stretching and bending
N=N); 1H-NMR (DMSO-d6) (ppm): 2.63 (s, 3H pyrazole -CH3), 7.13–8.20 (8H, aromatic C-
H), 11.16–11.18 (s, O-H), 11.27–11.29 (s, O-H). Elemental analysis: Anal. Calc. for
C19H14ClN7O2: C, 55.96%; H, 3.46%; N, 24.04%. Found: C, 55.53%; H, 3.61%; N, 24.71%.
2.2.5. Synthesis of 3-(3'-methyl-4'-(4-methylphenylazo-1'-H-5-ylazo) pyrazole-2,4-
dihydroxyquinoline (3e)
Bright orange product, Yield: 71% mp: decomposed >340°C FT-IR (cm-1) υmax
:
3179 (-NH), 3093 (aromatic C-H), 2981, 2951 (aliphatic C-H), 1659 (C=O), 1546 and 1469
1
(stretching and bending N=N); H-NMR (DMSO-d6) (ppm): 2.55 (s, 3H pyrazole -CH3),
5.74 (s, 3H p-CH3), 7.12–7.79 (8H, aromatic C-H), 11.20–11.22 (s, O-H), 11.32–11.34 (s, O-
H). Elemental analysis: Anal. Calc. for C20H17N7O2: C, 62.01%; H, 4.42%; N, 25.31%.
Found: C, 62.71%; H, 4.35%; N, 24.95%.
2.2.6.
Synthesis
of
3-(3'-methyl-4'-(3-nitrophenylazo-1'-H-5-ylazo)
pyrazole-2,4-
dihydroxyquinoline (3f)
Bright light brown product, Yield: 73% mp: decomposed >325°C FT-IR (cm-1) υmax
:
3201 (-NH), 3071 (aromatic C-H), 2981 (aliphatic C-H), 1654 (C=O), 1545 and 1460
1
(stretching and bending N=N), 1528 and 1348 (stretching and bending -NO2); H-NMR
(DMSO-d6) (ppm): 2.55 (s, 3H pyrazole -CH3), 7.18–8.55 (8H, aromatic C-H), 8.97–9.01 (b,
-NH), 11.02–11.12 (s, O-H), 11.33–11.39 (s, O-H). Elemental analysis: Anal. Calc. for
C19H14N8O4: C, 54.55%; H, 3.37%; N, 26.78%. Found: C, 55.72%; H, 3.42%; N, 26.02%.
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2.2.7. Synthesis of 3-(3'-methyl-4'-(3-methoxyphenylazo-1'-H-5-ylazo) pyrazole-2,4-
dihydroxyquinoline (3g)
Bright red, Yield: 74% mp: decomposed >280°C FT-IR (cm-1) υmax: 3173 (-NH),
3065 (aromatic C-H), 2981, 2863 (aliphatic C-H), 1661 (C=O), 1547 and 1498 (stretching and
bending N=N), 1100 (C-O); 1H-NMR (DMSO-d6) (ppm): 2.62 (s, 3H pyrazole -CH3), 3.92
(s, 3H m-OCH3), 7.05–7.99 (8H, aromatic C-H), 11.13–11.19 (s, O-H), 11.28–11.35 (s, N-H),
11.41–11.48 (s, O-H). Elemental analysis: Anal. Calc. for C20H17N7O3: C, 59.25%; H,
4.25%; N, 24.30%. Found: C, 59.82%; H, 4.43%; N, 23.87%.
2.2.8. Synthesis of 3-(3'-methyl-4'-(3-chlorophenylazo-1'-H-5-ylazo) pyrazole-2,4-
dihydroxyquinoline (3h)
Bright orange, Yield: 68% mp: decomposed >310°C FT-IR (cm-1) υmax: 3151 (-NH),
3065 (aromatic C-H), 2981 (aliphatic C-H), 1661 (C=O), 1559 and 1464 (stretching and
1
bending N=N); H-NMR (DMSO-d6) (ppm): 2.55 (s, 3H pyrazole -CH3), 7.10–8.21 (8H,
aromatic C-H), 11.08–11.24 (s, O-H), 11.24–11.31 (s, N-H), 11.47–11.61 (s, O-H).
Elemental analysis: Anal. Calc. for C19H14ClN7O2: C, 55.96%; H, 3.46%; N, 24.04%.
Found: C, 55.74%; H, 3.83%; N, 24.68%.
2.2.9. Synthesis of 3-(3'-methyl-4'-(3-methylphenylazo-1'-H-5-ylazo) pyrazole-2,4-
dihydroxyquinoline (3i)
Claret red, Yield: 72% mp: decomposed >312°C FT-IR (cm-1) υmax: 3194 (-NH),
3064 (aromatic C-H), 2981, 2867 (aliphatic C-H), 1659 (C=O), 1592 and 1461 (stretching and
1
bending N=N); H-NMR (DMSO-d6) (ppm): 2.55 (s, 3H pyrazole -CH3), 2.64 (s, 3H m-
CH3), 7.01–8.10 (8H, aromatic C-H), 11.11–11.20 (s, O-H), 11.25–11.33 (s, N-H), 11.39–
11.54 (s, O-H). Anal. Calc. for C20H17N7O2: C, 62.01%; H, 4.42%; N, 25.31%. Found: C,
62.65%; H, 4.34%; N, 24.74%.
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2.2.10.
Synthesis
of
3-(3'-methyl-4'-(2-nitrophenylazo-1'-H-5-ylazo)
pyrazole-2,4-
dihydroxyquinoline (3j)
Bright claret red, Yield: 61% mp: 328°C FT-IR (cm-1) υmax: 3200 (-NH), 3070
(aromatic C-H), 2981 (aliphatic C-H), 1660 (C=O), 1545 and 1469 (stretching and bending
N=N), 1504 and 1349 (stretching and bending -NO2); 1H-NMR (DMSO-d6) (ppm): 2.56 (s,
3H pyrazole -CH3), 7.08–8.13 (8H, aromatic C-H), 11.12–11.21 (s, O-H), 11.28–11.36 (s, N-
H), 11.49–11.59 (s, O-H). Elemental analysis: Anal. Calc. for C19H14N8O4: C, 54.55%; H,
3.37%; N, 26.78%. Found: C, 54.82%; H, 3.40%; N, 26.52%.
2.2.11. Synthesis of 3-(3'-methyl-4'-(2-methoxyphenylazo-1'-H-5-ylazo) pyrazole-2,4-
dihydroxyquinoline (3k)
Bright orange, Yield: 75% mp: decomposed >193°C FT-IR (cm-1) υmax: 3220 (-NH),
3068 (aromatic C-H), 2981, 2863 (aliphatic C-H), 1658 (C=O), 1591 and 1469 (stretching and
bending N=N), 1101 (C-O); 1H-NMR (DMSO-d6) (ppm): 2.55 (s, 3H pyrazole -CH3), 3.96
(s, 3H o-OCH3), 7.02–8.00 (8H, aromatic C-H), 11.14–11.18 (s, O-H), 11.27–11.30 (s, O-H).
Elemental analysis: Anal. Calc. for C20H17N7O3: C, 59.25%; H, 4.25%; N, 24.30%. Found:
C, 59.55%; H, 4.32%; N, 24.02%.
2.2.12. Synthesis of 3-(3'-methyl-4'-(2-chlorophenylazo-1'-H-5-ylazo) pyrazole-2,4-
dihydroxyquinoline (3l)
Orange, Yield: 67% mp: decomposed >265°C FT-IR (cm-1) υmax: 3226 (-NH), 3062
(aromatic C-H), 2981 (aliphatic C-H), 1654 (C=O), 1560 and 1459 (stretching and bending
N=N); 1H-NMR (DMSO-d6) (ppm): 2.55 (s, 3H pyrazole -CH3), 7.12–7.99 (8H, aromatic C-
H), 11.11–11.20 (s, O-H), 11.26–11.33 (g, -NH), 11.40–11.51 (s, O-H). Elemental analysis:
Anal. Calc. for C19H14ClN7O2: C, 55.96%; H, 3.46%; N, 24.04%. Found: C, 55.69%; H,
3.43%; N, 24.47%.
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2.2.13. Synthesis of 3-(3'-methyl-4'-(2-methylphenylazo-1'-H-5-ylazo) pyrazole-2,4-
dihydroxyquinoline (3m)
Light brown, Yield: 68% mp: decomposed >130°C FT-IR (cm-1) υmax: 3346 (-OH)
3242 (-NH), 3067 (aromatic C-H), 2982, 2851 (aliphatic C-H), 1592 and 1470 (stretching and
1
bending N=N); H-NMR (DMSO-d6) (ppm): 2.51 (s, 3H pyrazole -CH3), 2.61 (s, 3H o-
CH3), 7.13–7.99 (8H, aromatic C-H), 11.15–11.19 (s, O-H), 11.40–11.46 (s, O-H). Elemental
analysis: Anal. Calc. for C20H17N7O2: C, 62.01%; H, 4.42%; N, 25.31%. Found: C, 62.53%;
H, 4.35%; N, 25.78%.
2.3. DNA Protective Activity Test
Plasmid pUC19 (pDNA) was used for DNA protective activity tests. Plasmid DNA
isolation was implemented with a Thermo Fisher Scientific GeneJET Plasmid Miniprep Kit.
The reaction comprised 1 µL of pDNA (300 µg/µL), 2 µL of Fenton’s reagent (30 mM H2O2,
50 mM ascorbic acid, and 80 mM FeCl3), and 3 µL of each compound at two concentrations
(5 mg/mL and 10 mg/mL). The reaction mixture was then made up to 20 ??L with double-
distilled water. The negative control included only 1 µL of pDNA and 19 µL of distilled
water, while the positive control comprised 1 µL of pDNA, 2 µL of Fenton’s reagent, and 17
µL of distilled water. The prepared mixtures were incubated for 60 min at 37°C. Samples
were then run on 0.8% agarose gel with DNA loading dye (Thermo Fisher Scientific, USA)
and analyzed using ultraviolet light. The analysis was performed three times, and the DNA
band density was assessed using gel image analysis software (Quantum, Vision-Capt., Vilber
Lourmat SAS, France) [31, 32].
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2.4. Determination of Cytotoxicity
2.4.1. Cell Culture Maintenance
HeLa (cervical cancer cell line) and PC3 (prostate cancer cell line) cells were provided
by the Department of Genetics and Bioengineering at Kastamonu University. Culture media
were prepared in DMEM solution containing 1% penicillin-streptomycin (Pen-Strep) and
10% fetal bovine serum (FBS). This medium was stored at +4°C. Cancer cell lines were
grown in an incubator at 37°C with 5% carbon dioxide. Test cells were grown in 96-well cell
culture plates, and stock cells were proliferated in 25 cm2 sterile Corning flasks. Cells were
seeded during their logarithmic growth phase [33].
2.4.2. Preparation of Chemicals
Diluted stock chemicals were prepared at a concentration of 1 M (molar). Chemicals
were dissolved in DMSO (dimethyl sulfoxide)/water at a ratio of 1:5 and then centrifuged at
10,000 × g for 2 min. Supernatants were filtered with a 0.22 µm pore size filter and the
chemical solutions obtained were kept at -20°C and protected from sunlight.
2.4.3. MTT-Based Cytotoxicity Assay
Approximately 10000 PC3 and HeLa cells were seeded in 96-well plates and
proliferated for 24 h. Cells were incubated with variable doses of compounds 3h and 3j at
concentrations of 1, 25, 50, and 100 µM, and 10 and 25 mM for 24, 48, and 72 h after
entering their logarithmic growth phase. The medium was refreshed with DMEM containing
0.5% FBS + 0.5 mg/mL MTT at 37°C with 5% Carbon dioxide for 4 h after the exposure
time. The MTT-formazan crystals were dissolved by treating the cells with 3% SDS + 40 mM
HCl/isopropanol for 15 min. The absorbance of samples was calculated at 570 nm [32, 34].
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2.5. Antimicrobial Activity Analysis
Antimicrobial activity analysis was performed using the disc diffusion method. Nine
different microorganisms were tested against compounds 3(a–m). As an antibacterial drug,
5µg of Levofloxacin antimicrobial susceptibility disks (Thermo Fisher Scientific, USA) were
used to compare the antibacterial effect of new synthesized compounds. Bacterial colonies
were incubated for 24 h at 37°C after inoculation into 20 mL nutrient broth medium. Sterile
saline solution (0.9%) was added to the bacterial suspension, and the turbidity was calibrated
to 0.5 McFarland. The prepared bacterial suspension was inoculated into 100 mm petri plates
containing nutrient agar medium. The stock solutions of compounds 3(a-m) were prepared by
dissolving in 1 mL DMSO (M = 0.1mol \ L). Sterile discs were saturated with compounds that
were dissolved in DMSO and left to dry at room temperature. After drying, the discs
containing compounds and disks containing Levofloxacin antibiotic were transferred to petri
plates and incubated for 24 h at 37°C. The disc diffusion experiment was performed three
times, and the observed inhibition zones were measured in mm.
3. RESULTS AND DISCUSSION
3.1. Characterization of synthesized compounds
FT-IR spectra were recorded in the region 4000–200 cm−1 on a Bruker Alpha FT-IR
spectrometer. The infrared absorption values of all the compounds are listed in Table 1. The
IR absorptions of the synthesized compounds were observed as follows: vibration of N-H
group at 3220–3173 cm−1, stretching vibration of aromatic C-H at 3099–3064 cm−1, stretching
vibration of aliphatic C-H at 2981–2857 cm−1, stretching vibration of C=O (tautomeric keto
form) at 1660–1653 cm−1, and stretching and bending vibration of N=N at 1560–1545 cm-1
1470–1459 cm−1. Also, vibration of C-O (OCH3) in compounds 3c, 3g, and 3k was observed
at 1100 and 1101 cm−1. In addition, stretching and bending vibrations of the NO2 group
(compounds 3b, 3f, and 3i) were observed at 1528–1504 and 1349–1332 cm−1, respectively.
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Table 1. FT-IR values of the synthesized compounds
Compounds
νOH
νNH
νAromatic-H
νAliphatic-H
νC=O
νN=N
νNO2
νC-O
1546
1460
1560
1470
1546
1459
1546
1460
-
3178
3269
3185
3207
3179
3201
3173
3151
3194
3200
3220
3226
3242
3092
3099
3092
3077
3093
3071
3065
3065
3064
3070
3068
3062
3067
2950
2981
1657
1656
1653
1653
1659
1654
1661
1661
1659
1660
1658
1654
-
-
3a
3b
3c
3d
3e
3f
1506
1332
-
2981
2857
-
-
-
-
1100
-
2950
2981
2951
1546
1469
-
1545
1460
1547
1498
1559
1464
1592
1461
1545
1469
1591
1469
1528
1348
-
2981
2981
2863
-
-
-
1100
1101
3g
3h
3i
-
2981
2981
2867
1504
1349
-
-
2981
-
-
-
-
3j
2981
2863
-
3k
3l
1560
1459
1592
1470
-
2981
2982
2851
3346
3m
1H-NMR spectra of all the compounds were recorded on a Bruker spectrometer
(Germany) at 400 MHz at room temperature in DMSO-d6. All the 1H-NMR data are listed in
Table 2. As seen in Table 2, the protons of the -CH3 group attached to the pyrazole ring give
a singlet peak at 2.51–2.62 ppm. The substituted phenyl rings at the -o, -m, and -p positions
are observed as singlet peaks at 3.92–5.74 ppm for the -OCH3 protons for compounds 3c, 3g,
and 3k. The substituted phenyl rings at the -o, -m, and -p positions are observed as singlet
peaks at 2.51–5.74 ppm for the -CH3 protons for compounds 3e, 3i, and 3m. Aromatic protons
are observed at 7.02–8.55 ppm. The protons of the -OH groups of the quinolinediol were
recorded at 11.02–11.61 ppm, and -NH protons are observed at 8.97–11.40 ppm. Integration
ratios in the NMR results are consistent with the number of protons.
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Table 2. 1H-NMR (
δ
, ppm, in DMSO-d6) values of synthesized compounds
Compounds
δ Aliphatic-H
δ Aromatic-H
δ X-H
11,17 (s, -OH)
11,29 (s, -NH)
11,52 (s, -OH)
3a
2,59 (s, 3H pyrazole -CH3)
2,59 (s, 3H pyrazole -CH3)
7,17–8,21 (9H, aromatic)
7,11–8,42 (8H, aromatic)
11,14 (s, -OH)
11,29 (s, -NH)
11,53 (s, -OH)
3b
2,59 (s, 3H pyrazole -CH3)
5,74 (s, 3H p-OCH3)
11,22 (s, -OH)
11,33 (s, -OH)
3c
3d
3e
3f
7,12–7,79 (8H, aromatic)
7,13–8,20 (8H, aromatic)
7,12–7,79 (8H, aromatic)
11,17 (s, -OH)
11,17 (s, -OH)
2,63 (s, 3H pyrazole -CH3)
2,55 (s, 3H pyrazole -CH3)
5,74 (s, 3H p-CH3)
11,21 (s, -OH)
11,33 (s, -OH)
8,99 (s, -NH)
11,07 (s, -OH)
11,36 (s, -OH)
2,55 (s, 3H pyrazole -CH3)
7,18–8,55 (8H, aromatic)
7,05–7,99 (8H, aromatic)
7,10–8,21 (8H, aromatic)
7,15–8,10 (8H, aromatic)
11,16 (s, -OH)
11,32 (s, -NH)
11,45 (s, -OH)
3g
3h
3i
2,62 (s, 3H pyrazole -CH3)
3,92 (s, 3H m-OCH3)
11,16 (s, -OH)
11,28 (s, -NH)
11,59 (s, -OH)
2,55 (s, 3H pyrazole -CH3)
11,16 (s, -OH)
11,29 (s, -NH)
11,47 (s, -OH)
2,55 (s, 3H pyrazole -CH3)
2,64 (s, 3H m-CH3)
11,17 (s, -OH)
11,32 (s, -NH)
11,54 (s, -OH)
3j
2,56 (s, 3H pyrazole -CH3)
7,08–8,13 (8H, aromatic)
7,02–8,00 (8H, aromatic)
7,12–7,99 (8H, aromatic)
7,13–7,99 (8H, aromatic)
2,55 (s, 3H pyrazole -CH3)
3,96 (s, 3H o-OCH3)
11,16 (s, -OH)
11,29 (s, -OH)
3k
3l
11,16 (s, -OH)
11,30 (s, -NH)
11,46 (s, -OH)
2,55 (s, 3H pyrazole -CH3)
2,51 (s, 3H pyrazole -CH3)
2,61 (s, 3H o-CH3)
11,17 (s, -OH)
11,43 (s, -OH)
3m
1
The FT-IR and H-NMR results for the obtained compounds revealed that the
structures of the materials differ between the solid state and solution. The FT-IR results
showed amide-bound carbonyl at 1660–1653 cm-1, but the hydroxyl groups were not
observed. Thus, it was concluded that the materials obtained were in solid keto form. In the
1H-NMR spectra, the signal belonging to the -OH group was observed at 11.02–11.61 ppm,
and thus it was proposed that the substances favor the enol form in DMSO solvents. In
addition, quinolone derivatives 3(a-m) are also enol tautomeric forms in biological activities
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studies because of using DMSO for these applications. The keto and enol forms of the
compounds are shown in Scheme 3.
OH
N
N
N
X
N
N
OH
HN
CH3
N
enol tautomeric form
O
H
N
N
N
X
N
N
H
O
HN
CH3
N
keto tautomeric form
Scheme 3. Keto and enol forms of the synthesized compounds
3.2. DNA Protective Activities
Oxidative processes caused the reappearance of reactive oxygen species (ROS) [35].
ROS such as hydroxyl radicals, hydrogen peroxide, and superoxide anions can damage DNA
and cause cancer and neurodegenerative diseases [36–38]. The protective ability of the
chemicals in the study was analyzed using Fenton’s reagent. This has a detrimental effect on
cellular ingredients including DNA via hydroxyl radicals (Fe2+ + H2O2 → Fe3+ + OH− + •OH).
A plasmid nicking assay was used to analyze the protective role of compounds 3(a–m) in the
study. Two concentrations (5 and 10 mg/mL) of 13 different chemicals were tested (Figure
1). Compounds 3a, 3b, 3c, and 3f showed no protective activity with plasmid DNA at any of
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the tested concentrations of compounds. Compound 3a exhibited insignificant protective
activity. However, compounds 3d, 3h, 3j, 3k, and 3l protected a supercoiled form of plasmid
DNA (pDNA) to different extents. In addition, the protective activities of compounds 3h and
3j were noticeably higher than others, and this can be correlated with their anticancer activity.
Therefore, these two compounds were chosen for studying anticancer activity. The DNA
protective activity of compounds 3d, 3h, and 3j can be explained by their binding affinity to
the negatively charged DNA due to their high positive charge. Many studies report the use of
compounds that can increase the rate of neutralization of the molecular binding affinity to
DNA [39, 40]. Compound 3j, containing a nitro group, attracts electrons from the ring via a
resonance effect, while compound 3h, which contains chlorine, attracts electrons from the
ring by an inductive effect. Thus, we can say that the nitrogen bonded ring is more positive
than the chlorinated ring. Besides, compound 3j had a higher positive electric charge than
compound 3h, which can explain the much higher protective activity of compound 3j at a
concentration of 10 mg/mL. Compound 3e contains a methyl group, which activates by
donating electrons to the ring to which it belongs, so that the group to which it is connected
becomes negatively charged. This compound disrupted the whole of pDNA, which might be
explained by the combined activity of compound 3e and Fenton reagents. It has been reported
that there are significant effects on the binding of different groups of molecules to the bond
between the molecule and DNA. For example, in different studies, quinoline Schiff base
ligands and their Cu(II) complexes were synthesized and their biological activities were
tested. The results were suggested that the Cu(II) complexes exhibited better activity than
their corresponding ligands against HeLa and HL-60 cells. Moreover, it was reported that
most active compound was CuL3, and this was explained by the difference in its terminal
functional group (3,4-dimethylpyrryl heterocycle) relative to those of CuL1and CuL2 [41].
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Figure 1. DNA protective activity analysis using plasmid DNA with compounds (3a–m), A,
B: (+) control, C: (-) control.
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3.3. Antimicrobial Activities of Quinoline Derivatives
Nine bacterial species were used to study the antimicrobial activities of quinoline
derivatives by the disk diffusion method (Figure 2). Antimicrobial analysis revealed that only
compound 3b formed inhibition zones against Proteus vulgaris, Staphylococcus hominis,
Staphylococcus epidermidis, Streptococcus hemolyticus, Klebsiella pneumoniae,
Staphylococcus aureus (ATCC 25923), and Staphylococcus aureus, with inhibition zones of
12, 15, 16, and 17 mm. Compound 3b seems to affect mainly Gram-positive bacteria, but this
compound had no effect on Escherichia coli and Serratia marcescens, which are Gram-
negative. The other compounds had no effect on any of the tested microorganisms.
Antimicrobial activity of Levofloxacin against to selected bacteria was also examined and evaluated as
control. K. pneumonia and S. hominis were bacteria strains which were not affected from
Levofloxacin. However, compound 3b had an antimicrobial effect on these bacteria strains. In
addition, the effect of compound 3b was higher than Levofloxacin against to S. epidermis. Other
bacteria strains were more susceptible to Levofloxacin than compound 3b. Therefore, it can be
concluded that compound 3b might play important roles for different antibiotics which could
not influence bacteria strains. In some special cases, compound 3b can have a potential to
replace weak antibiotics. Staphylococcus aureus (ATCC 25923) causes many health
problems, such as sepsis, osteomyelitis, endocarditis, wound infection, pneumonia, and toxic
shock syndrome in humans, and it displays resistance to some drugs including methicillin and
vancomycin [42–44]. In addition, Staphylococcus epidermidis is one of the five most common
organisms that cause nosocomial infections [42]. In a recent study, two compounds (p-F and
p-NO2) of a series of (5-(2-chloroquinolin-3-yl)-3-(aryl)-4,5-dihydro-1H-pyrazol-1-yl)
(pyridin-4-yl) methanones indicated excellent activity with MIC values of 250 and 100 µg/mL
against Staphylococcus aureus [45], which was consistent with the activity of compound 3b,
containing an NO2 group, against S. aureus in the current study. Furthermore, 3-(6-
nitrobenzothiazol-2-ylazo)quinoline-2,4-diol and 3-(6-chlorobenzothiazol-2-ylazo)quinoline-
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2,4-diol were synthesized, and their antibacterial effects against the same bacteria were
investigated under similar conditions [46]. A similar study was also conducted by Foroumadi
and his study group in 2003. According to this study, a series of N-[5-(5-nitro-2-thienyl)-
1,3,4-thiadiazole-2-yl] piperazinyl quinolones was synthesized and analyzed for antibacterial
activity. They reported that these compounds had better activity than the reference
quinolones, such as ciprofloxacin, norfloxacin, and enoxacin, against tested Gram-positive
organisms [47]. Considering that the most common human pathogens related to infections are
Gram-positive bacteria, inhibition of these bacteria by compound 3b is a promising result
with respect to the treatment of this group of bacteria.
Figure 2. Antimicrobial activity of Levofloxacin and compound 3b. 5µg and 0.1mg\mL
concentrations were used for Levofloxacin and compound 3b, respectively.
3.4 Cytotoxic Activities of Compounds 3h and 3j
The cytotoxic activities of quinoline derivatives were tested against HeLa (cervical
cancer cell line) and PC3 (prostate cancer cell line) cells using an MTT cell viability test. Six
different concentrations (1, 25, 50, and 100 µM and 10 and 25 mM) of compounds 3h and 3j
were used for 24, 48, and 72 hours on HeLa (Figure 3) and PC3 (Figure 4) cell lines. Control
cells were not treated with any of the compounds. An inverted microscope was used to
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visualize the cell morphology. The survival rate of HeLa cells was found to be 40% after 24 h
of treatment with compounds 3j and 3h. This dropped to 20% after 72 h of treatment with
compound 3j. It can be concluded that the activity of compound 3j might be dose and time
dependent for HeLa cell lines. However, the activity of compound 3h can also be considered
as dose dependent for the same cell line because of the survival rate reduction with 25 mM of
this compound.
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Figure 3. Cell morphology (left) and MTT cell viability assay (right) of HeLa cancer cell line
after treatment with different concentrations of compounds 3h and 3j.
On the other hand, the survival rate of PC3 cells decreased to 60% after 24 h of
treatment with compound 3h. However, there was no further drop in their survival rate after
48 and 72 h of treatment with compound 3h. In addition, compound 3j at a concentration of
25 mM had cytotoxic effects on these cells after 24, 48, and 72 h of treatment.
Many quinoline derivatives are widely used as pharmaceuticals, including antimalarial
(quinine), antibacterial, antiviral, antifungal, local anesthetic, cardiotonic, and anticancer [48].
Quinoline derivatives operate via different mechanisms including inhibition of tubulin [49],
inhibition of cell migration [48], inhibition of carbonic anhydrase [50], inhibition of c-Met
kinase [51], upregulation of superoxide dismutase activity, and regulation of free radicals [52,
53]. One study evaluated the activities of certain indeno[11,2-c] quinoline derivatives against
six cancer cell types, including HeLa and PC-3 cells, and 9-methoxy-6-(piperazin-1-yl)-11H-
indeno[1,2-c]quinolin-11-one O-3-aminopropyl oxime (22b) was the most potent compound
against the growth of HeLa cells, with a GI50 value of 0.52 µM [54]. Thieno (39,29:4,5)
thieno(2,3)-quinolone derivatives were synthesized, and their cytotoxic effect were studied in
cancer cell lines including breast (MCF7) and cervical (HeLa) cells in another study. All the
tested compounds showed considerable inhibitory activities against all the tested cell lines
[55]. Besides, 1:1 Schiff base copper complexes of quinoline-2-carboxaldehyde exhibited
dose dependent proapoptotic and antiproliferative activities against PC-3 and LNCaP prostate
cancer cell lines. In particular, quinoline thiosemicarbazone (195; FPA-137) inhibited
proteasome activity in these cell types [56].
Our results are consistent with previous studies on the cytotoxic activities of a variety
of quinoline derivatives on human breast and prostate cancer cell lines. The anticancer
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potential of compound 3j is emerging according to the results of the current study. In addition,
compounds 3j and 3h at a concentration of 25 mM had cytotoxic effects on these cancer cell
lines.
Figure 4. Cell morphology (left) and MTT cell viability assay (right) of PC3 prostate cancer
cell line after treatment with different concentrations of compounds 3h and 3j.
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4. CONCLUSION
In this study, derivatives of 3-(3'-methyl-4'-(arylazo-1'-H-5-ylazo) pyrazole-2,4-
dihydroxyquinoline compounds were synthesized, and their structural characterization was
1
performed by FT-IR and H-NMR spectroscopy, and elemental analysis. The antimicrobial
and anticancer activities of the compounds were also investigated. Compound 3b had a
promising effect on Gram-positive bacteria. In addition, the obtained compounds were also
investigated for DNA protective activity; compounds 3h and 3j possessed high DNA binding
affinity. Furthermore, the activity of compound 3j was observed to be dose and time
dependent, while the activity of compound 3h was considered to be dose dependent for HeLa
cell lines. Compounds 3h and 3j exhibited cytotoxic effects on PC3 cell lines. This might be
explained by the presence of different functional groups in these compounds. Besides, the in
vivo effects of these compounds should be evaluated for further evidence of their cytotoxic
effect on cancer cells.
Acknowledgements: We would like to thank the Central Research Laboratory of Kastamonu
University for their contribution.
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Highlights
1. 2,4-dihydroxyquinoline derived disazo dyes are synthesized.
2. The structure of the compounds are confirmed.
3. The synthesized compounds are examined for different biological activities, including
DNA protection, antimicrobial, and anticancer.