Synthesis and electrochemical, spectral, and biological evaluation of novel 9,10-anthraquinone derivatives containing piperidine unit as potent antiproliferative agents

Article abstract of DOI:10.1016/j.molstruc.2018.07.070

The present study describes the synthesis and electrochemical, spectral and biological evaluation of new 9,10-anthraquinone derivatives containing one or two piperidine units incorporated in the different positions of the anthraquinone scaffold. The authors focused their efforts on the characterization of the obtained derivatives using the NMR and IR techniques and on the analysis of the molecules properties using the UV-VIS spectroscopy in the DMSO solution. Additionally, the electrochemical investigation of the compounds was performed using the cyclic voltammetry at the GC (glassy carbon) electrode in the DMSO solution. The primary aim of this study was to determine the antiproliferative activity of the obtained compounds. All novel anthraquinone derivatives containing piperidine unit in the structure were tested in vitro against four human cancer cell lines—HL-60, LoVo, HL-60/MX2, LoVo/Dx—which the latter two are drug-resistant. The tests also included the in vitro analyses of the antiproliferative activities against BALB/3T3 normal mouse fibroblasts cell lines. The results led to an interesting observation concerning the selectivity—all the derivatives exhibit potential anticancer activity against leukemic drug-resistant cell lines. Moreover, the most active compound 1-(piperidin-1-yl)-9,10-anthraquinone 1 inhibited also the proliferation of the colon cancer cell lines, distinctly overcoming the drug-resistance. Its activity towards the cancer cell lines was 5–8 times higher than the activity against normal fibroblasts cell line, which can suggest its selectivity towards cancer cells.

Full text of DOI:10.1016/j.molstruc.2018.07.070

Accepted Manuscript
Synthesis and electrochemical, spectral, and biological evaluation of novel 9,10-
anthraquinone derivatives containing piperidine unit as potent antiproliferative agents
Paweł Niedziałkowski, Elżbieta Czaczyk, Joanna Jarosz, Anna Wcisło, Wioleta
Białobrzeska, Joanna Wietrzyk, Tadeusz Ossowski
PII:
S0022-2860(18)30891-3
10.1016/j.molstruc.2018.07.070
MOLSTR 25475
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 11 May 2018
Revised Date: 20 July 2018
Accepted Date: 23 July 2018
Please cite this article as: Paweł. Niedziałkowski, Elż. Czaczyk, J. Jarosz, A. Wcisło, W. Białobrzeska,
J. Wietrzyk, T. Ossowski, Synthesis and electrochemical, spectral, and biological evaluation of novel
9,10-anthraquinone derivatives containing piperidine unit as potent antiproliferative agents, Journal of
Molecular Structure (2018), doi: 10.1016/j.molstruc.2018.07.070.
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Synthesis and Electrochemical, Spectral, and Biological Evaluation of Novel
9,10-Anthraquinone Derivatives Containing Piperidine Unit as Potent
Antiproliferative Agents
Paweł Niedziałkowski*1, Elżbieta Czaczyk1, Joanna Jarosz2, Anna Wcisło1, Wioleta
Białobrzeska1, Joanna Wietrzyk2, Tadeusz Ossowski1
1 Faculty of Chemistry, University of Gdansk, 63, Wita Stwosza Street, Gdansk 80-308,
Poland
2
Institute of Immunology and Experimental Therapy, Polish Academy of Science, 12, R.
Weigl Street, Wroclaw 53-114, Poland
Received: ……/Accepted …
Abstract
The present study describes the synthesis and electrochemical, spectral, and biological
evaluation of new 9,10-anthraquinone derivatives containing one or two piperidine units
incorporated in the different positions of the anthraquinone scaffold. The authors focused their
efforts on the characterization of the obtained derivatives using the NMR and IR techniques
and on the analysis of the molecules properties using the UV-VIS spectroscopy in the DMSO
solution. Additionally, the electrochemical investigation of the compounds was performed
using the cyclic voltammetry at the GC (glassy carbon) electrode in the DMSO solution.
The primary aim of this study was to determine the antiproliferative activity of the
obtained compounds. All novel anthraquinone derivatives containing piperidine unit in the
structure were tested in vitro against four human cancer cell lines—HL-60, LoVo, HL-
60/MX2, LoVo/Dx—which the latter two are drug-resistant. The tests also included the in

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vitro analyses of the antiproliferative activities against BALB/3T3 normal mouse fibroblasts
cell lines. The results led to an interesting observation concerning the selectivity—all the
derivatives exhibit potential anticancer activity against leukemic drug-resistant cell lines.
Moreover, the most active compound 1-(piperidin-1-yl)-9,10-anthraquinone 1 inhibited also
the proliferation of the colon cancer cell lines, distinctly overcoming the drug-resistance. Its
activity towards the cancer cell lines was 5–8 times higher than the activity against normal
fibroblasts cell line, which can suggest its selectivity towards cancer cells.
Keywords 9,10-anthraquinone derivatives, electrochemistry, UV-Vis, piperidine,
antiproliferative activity, cytotoxicity

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1. Introduction
Anthraquinone derivatives—both natural and synthetic—have a wide range of
applications. For many years these compounds have also been used for the medical purposes
[1,2]. The 9,10-anthraquinone scaffold occurs in the antitumor drugs called anthracycline
antibiotics. Anthracyclines are commonly used in the treatment of myeloid leukemia,
malignant lymphomas, and breast cancer [3–5]. The glycosylated derivatives of
anthraquinone such as daunorubicin, doxorubicin, and carminomycin—are the most active
anthracycline antibiotics used in the treatment of many tumors. These molecules have been
already in use for many decades [6–8]. Anthracyclines are composed of the tetracyclic planar
ring while anthracenediones usually contain three rings in the structure [9]. Mitoxantrone and
ametantrone belong to a group of anthracenediones synthetic derivatives. These compounds
are derivatives of the 9,10-anthraquinone substituted in the 1 and 4 amino group position.
[10]. Despite of the structural differences, anthracenediones show similar biological activity
and disadvantages like anthracyclines, therefore they are commonly called the anthracycline-
like drugs [11].
The mechanism of action, antitumor activity, and cardiotoxicity of anthracyclines and
anthracenediones have been extensively discussed and depend on many factors [12–14]. The
biological activity of these compounds results from the possibility of the DNA intercalation
leading to the inhibition of many cellular processes [15], interstrand DNA cross-linking
[16,17], free radical formation leading to DNA damage [18], DNA alkylation [19], and
inhibition of the enzyme topoisomerase II [20]. Taking into consideration the above-
mentioned factors, one can conclude that the antitumor activity of anthraquinone derivatives
strongly depends on the chemical structure and redox activities.
Previous studies prove that the biological activities of an anthracyclines and
anthracenediones are correlated with their redox properties [21]. The anthracycline

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electrochemical mechanism of action has been described elsewhere [22]. However, due to the
fact that the biological properties of molecules depends on many factors, the electrochemical
parameters of examined compound cannot be treated as only one determinant of their
biological properties [22]. For instance, the investigations performed on relations between
redox properties of anthraquinone derivatives and their cytotoxicity toward the murine
leukemia P388 cell line indicate that the action of examined compounds result rather from the
DNA affinity than the redox activity [23]. Therefore, electrochemical studies of the
anthraquinone derivatives are particularly crucial.
Despite the biological activity, anthraquinone derivatives due to their optical and
electrochemical properties have also found many applications in the development of the
optical and electrochemical chemosensors utilized in the molecular recognition of organic
molecules and inorganic species [24–27].
The piperidine ring is a structural unit present in many alkaloids [28]. Both natural and
synthetic derivatives of the piperidine possess a wide range of biological activities. The
biological activity of piperidine derivatives depends on the properties of substituents attached
directly to the nitrogen atom in the scaffold or attached to the carbon atoms [29]. The
substituted piperidines are exceptionally interesting due to their potential biological activities,
like antiviral [30], antibacterial [31], antifungal [32], antitumor [33], anti-HIV [34], and many
others.
The current study describes the synthesis of new 9-10 anthraquinone containing
piperidine derivatives, their antiproliferative activities against HL-60, HL-60/MX2, LoVo,
LoVo/Dx cancer cell lines and BALB/3T3 normal fibroblasts, and their electrochemical and
spectral characteristics.

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2. Experimental
2.1. Materials and Methods
2.2. General Information
All reagents and solvents were purchased from Sigma Aldrich, POCh, and were used
without further purification. The flash column chromatography was performed on the silica
gel 60 (Merck 0.040–0.063 mm, 230–400 mesh). The reactions were monitored using the
1
thin-layer chromatography on the percolated Merck silica gel 60 F254. H NMR spectra were
recorded on the on the Bruker AVANCE III spectrometer (500 MHz) using CDCl3 as a
solvent and TMS (tetramethyl silane) as the internal standard. The following abbreviations
were used: s = singlet, d = doublet, dd = doublet of doublet, t = triplet, dt = doublet of triplet,
13
p = pentet. H NMR spectra were recorded on the Bruker AVANCE III spectrometer (700
1
MHz). The representative of H NMR spectra of compound 1 were recorded on the Bruker
AVANCE III spectrometer (500 MHz) using Bruker standard software (TopSpin 3.2).
Infrared spectra (IR) were recorded on the Bruker Model IFS66 spectrometer using KBr
pellets. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectra
were recorded using the Bruker Biflex III instrument. Shimadzu LC-10A HPLC equipped
with an analytical reversed-phase column (Phenomenex Luna C8 column 100 Å, 3 ꢀm, 150 ×
4.60 mm) was used to determine the purity of the studied compounds. The eluents: (A) –
aqueous solution of 0.1% trifluoroacetic acid (TFA) and (B) – acetonitrile with 0.1% TFA. A
linear gradient was applied from 0% to 100% B at the flow rate of 1.2 mL/min. The total run
time was 20 min with UV detection at 254 and 226 nm. Melting points were determined using
Bushi 565 melting point apparatus. UV-Vis absorption spectra were recorded on the Perkin
Elmer Lambda 650 spectrophotometer in the 280–900 nm range using 1.0 cm quartz cuvettes.

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Electrochemical experiments were performed using the Autolab potentiostat (model
PGSTAT-128N) with a three electrodes configuration. The working electrode was a glassy
carbon electrode of 3 mm diameter, the counter electrode was a platinum wire, and the
reference electrode was an Ag/AgCl in 0.1 M NaCl reference electrode. All measurements
were recorded at the room temperature in the degassed solution of DMSO containing 0.1 M
tetrabutylammonium perchlorate (TBAP) and 2 mmol of the examined compounds 1–4, at the
100 mV/s scan rate.
2.3. Antiproliferative activity in vitro
2.4. Cell lines
The cell lines used in these studies were obtained from: (1) American Type Culture
Collection (ATCC; Rockville, Maryland, USA): human promyelocytic leukemia HL-60 and
HL-60/MX2 – its variant resistant to mitoxantrone with multidrug cross-resistance (the
mechanism of drug resistance seems to be related to the differential activity of topoisomerase
II), and mouse embryonic fibroblast: BALB/3T3; (2) received as a gift from prof. Borowski,
Gdansk University of Technology, Poland: LoVo colon carcinoma and LoVo/Dx—its variant
resistant to doxorubicin with multidrug cross-resistance; the mechanism of drug resistance
depends on the expression of MDR1 gene product—P-glycoprotein (P-gp).
The cell lines were cultured in vitro in the Cell Culture Collection of the Institute of
Immunology and Experimental Therapy, Wroclaw, Poland. All lines were maintained under
the standard conditions as we described in details below.
LoVo and Lovo/Dx cell lines were cultured in the mixture of OptiMEM and RPMI 1640
(1:1) medium (IIET, Wroclaw, Poland) supplemented with 5% fetal bovine serum (Thermo
Fisher Scientific, Waltham, MA, USA), 2 mM L-glutamine, 1 mM sodium pyruvate (Sigma
Aldrich) and 10 µg/100 mL doxorubicin (Sigma Aldrich, Germany) for LoVo/Dx.

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HL-60 and HL-60/MX2 were cultured in RPMI-1640 + GlutaMAX (Gibco, Darmstadt,
Germany) medium (1:1) containing 10% fetal bovine serum, 3.5 g/L glucose (Sigma Aldrich)
and 1 mM sodium pyruvate (Sigma Aldrich, Germany).
Murine embryonic fibroblast cells were cultured in Dulbecco medium (Gibco, Darmstadt,
Germany) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 2 mM
L-glutamine (Sigma Aldrich, Germany).
All culture media contained antibiotics: 100 U/mL penicillin (Sigma Aldrich, Germany)
and 100 µg/mL streptomycin (Polfa-Tarchomin, Poland). All cell lines were cultured during
the entire experiment in a humid atmosphere at 37 °C and 5% CO2.
2.5. Antiproliferative assay in vitro
24 hours before applications of the tested compounds, the cells were plated in 96-well
plates at a density of 104 cells per well, and cultured in 37 °C in a humid atmosphere saturated
with 5% CO2. Subsequently, the cells were exposed during 72 hours to the various
concentrations of the tested compounds. Test solutions of the test compounds (10.0 mg/mL)
were prepared ex tempore for each test by dissolving the compounds in the dimethyl sulfoxide
(DMSO). After that, the tested solutions were diluted to reach final concentrations in the
range from 100 to 0.1 µg/mL. The cells were also exposed to the reference drugs: cisplatin
(Accord, London, UK), doxorubicin, and mitoxantrone (both from Sigma Aldrich, Germany).
The antiproliferative effect in vitro was determined using SRB or MTT (leukemia cell lines)
method [35–37]. The optical densities of the samples were measured on the Synergy H4
photometer (BioTek Instruments, Winooski, VT, USA) at 540 (SRB) or 570 nm (MTT). The
results were calculated as an inhibitory dose 50% (ID50)—the concentration of the tested
compound which inhibits proliferation of the cells by 50% as compared to the control
untreated cells, calculated according to the published software [38]. The compounds during

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each experiment were tested in triplicates for each concentration. Each experiment was
repeated 3–5 times.
2.6. General Procedure for the Synthesis Compounds (1 - 4)
1-(Piperidin-1-yl)-9,10-anthraquinone (1, C19H17NO2)
To the solution of 0.5 g 1-chloro-9,10-anthraquinone (2.06 mmol) in 60 mL toluene, the 177
mg of piperidine (2.05 mmol) was added. The reaction mixture was stirred in 80 °C oil bath
for 48 h. After the reaction was finished, the resulting mixture was evaporated under the
reduced pressure to remove the solvent. The obtained residue was dissolved in 150 mL of the
dichloromethane and washed with water (2 × 100 mL), the organic layer was dried with
MgSO4, filtered, and concentrated. The crude product was purified using silica gel column
chromatography with dichloromethane:methanol mixture (5:0.1). The product was obtained as
a red solid. For the compounds (1) and (3) the crystal structures were described in the
previous papers [39,40]. Single crystals were grown during slow evaporation from the
mixture of methanol and dichloromethane solution at the room temperature (m.p. 112–114
°C). Compounds (2-4) were obtained in the same procedure.
Yield: m = 540 mg, 90%.
TLC (SiO2): CH2Cl2; Rf = 0.286.
1H NMR (CDCl3, 500 MHz): δ (ppm): 1.68-1.72 (p, 2H, -HN-CH2-CH2-CH2-CH2-CH2-,
J1=6.5 Hz, J1=5.5 Hz, J1=5.0 Hz, J1=6.5 Hz, J2=6.0 Hz, J2=5.2 Hz, J2=6.7 Hz, J3=6.0 Hz,
J3=5.6 Hz, J4=5.8 Hz); 1.87-1.91 (p, 4H, -HN-CH2-CH2-CH2-CH2-CH2-, J1=6.0 Hz, J1=5.0
Hz, J1=5.5 Hz, J1=6.0 Hz, J2=5.5 Hz, J2=5.2 Hz, J2=5.7 Hz, J3=5.5 Hz, J3=5.3 Hz, J4=5.4 Hz);
3.18-3.20 (t, 4H, -HN-CH2-CH2-CH2-CH2-CH2-, J1=5.0 Hz, J1=5.0 Hz, J2=5.0 Hz); 7.42-7.43

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(d, 1H, H-2 Ar, J2=8.5 Hz); 7.60-7.63 (t, 1H, H-3 Ar, J1=7.5 Hz, J1=7.5 Hz, J2=7.75); 7.71-
7.74 (dt, 1H, H-6 Ar, J1=0.5 Hz, J1=7.5 Hz, J1=7.5 Hz, J2=7.5 Hz); 7.76-7.80 (dt, 1H, H-7 Ar,
J1=1.0 Hz, J1=7.5 Hz, J1=7.5 Hz, J2=7.5 Hz); 7.89-7.91 (d, 1H, H-4 Ar, Hz, J1=7.5); 8.23-8.25
(dd, 1H, H-5 Ar, J1=0.5 Hz, J1=1.0 Hz, J2=7.7); 8.29-8.31 (dd, 1H, H-8 Ar, J1=0.5 Hz, J1=1.0
Hz, J2=7.7).
13C NMR (CDCl3, 700 MHz): δ (ppm): 184.3 (C=O); 181.68 (C=O); 154.09; 136.50; 135.57;
134.11; 133.94; 132.84; 132.36; 127.29; 126.39; 125.06; 122.19; 119.88; 54.03; 29.70; 29.35;
26.08.
IR (KBr) (cm-1): 2943, 2808, 1667, 1648, 1580, 1424, 1312, 1260, 1240, 1132, 1081, 896,
708.
MALDI TOF MS: m/z 292.1 [M+H]+, (MW = 291.34).
RP-HPLC: Rt = 8.15 - gradient elution 0–100% (A–B) in 20 min.
Melting point: 112 – 114 °C.
1,4-Di(piperidin-1-yl)-9,10-anthraquinone (2, C24H26N2O2)
Yield: m = 664 mg; 97.0%.
TLC (SiO2): CH2Cl2:MeOH (5:0.1); Rf = 0.481.
1H NMR (CDCl3, 500 MHz): δ (ppm): 1.64-1.69 (p, 4H, -HN-CH2-CH2-CH2-CH2-CH2-,
J1=6.5 Hz, J1=5.0 Hz, J1=6.0 Hz, J1=6.0 Hz, J2=6.7 Hz, J2=5.5 Hz, J2=6.0 Hz, J3=5.6 Hz,
J3=5.7 Hz, J4=5.6 Hz); 1.81-1.86 (p, 8H, -HN-CH2-CH2-CH2-CH2-CH2-, J1=6.5 Hz, J1=4.5
Hz, J1=5.0 Hz, J1=6.0 Hz, J2=5.5 Hz, J2=4.7 Hz, J2=5.5 Hz, J3=5.1 Hz, J3=5.1 Hz, J4=5.1 Hz);
3.10-3.12 (t, 8H, -HN-CH2-CH2-CH2-CH2-CH2-, J1=5.0 Hz, J1=4.5 Hz, J2=4.7 Hz); 7.38 (s,
2H, H-2 Ar, H-3 Ar); 7.65-7.67 (t, 1H, H-7 Ar, J1=J2=3.5 Hz); 7.67-7.69 (t, 1H, H-3 Ar,
J1=3.5 Hz, J1=3.0 Hz, J2=3.2 Hz); 8.18-8.20 (d, 2H,H-5 Ar, J2=9.0 Hz); 8.32-8.33- (d, 2H. H-
8 Ar, J2=8.0 Hz).

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13C NMR (CDCl3, 700 MHz): δ (ppm): 182.90(C=O); 160.86 (C=O); 148.73; 146.03; 139.50;
139.36; 134.54; 132.54; 126.81; 126.21; 124.50; 122.83; 121.03; 121.30; 54.29; 51.06; 49.77;
46.89; 26.45; 26.32; 25.53; 25.09; 24.73; 24.23.
IR (KBr) (cm-1): 3428, 2932, 2850, 1641, 1574, 1442, 1371, 1264, 1204, 1120, 799, 725.
MALDI TOF MS: m/z 376.3 [M+2H]+, (MW = 374.47).
RP-HPLC: Rt = 9.72 gradient elution 0–100% (B–A) in 20 min.
Melting point: 114 – 116 °C.
1,5-Di(piperidin-1-yl)-9,10-anthraquinone (3, C24H26N2O2)
Yield: m = 657 mg; 96%.
TLC (SiO2): CH2Cl2:MeOH (5:0.1); Rf = 0.387.
1H NMR (CDCl3, 500 MHz): δ (ppm):
1.67-1.72 (p, 4H, -HN-CH2-CH2-CH2-CH2-CH2-, J1=5.5 Hz, J1=6.5 Hz, J1=6.5 Hz, J1=6.0 Hz,
J2=6.0 Hz, J2=6.5 Hz, J2=6.2 Hz, J3=6.2 Hz, J3=6.3 Hz, J4=6.3 Hz); 1.84-1.90 (p, 8H, -HN-
CH2-CH2-CH2-CH2-CH2-, J1=6.0 Hz, J1=5.5 Hz, J1=6.0 Hz, J1=6.0 Hz, J2=5.7 Hz, J2=5.7 Hz,
J2=6.0 Hz, J3=5.7 Hz, J3=5.8 Hz, J4=5.8 Hz); 3.17-3.19 (t, 8H, -HN-CH2-CH2-CH2-CH2-CH2-,
J1=J2=5.5 Hz); 7.30-7.31 (d, 2H, H-2 Ar, H-4 Ar, J1=8.0 Hz); 7.57-7.60 (t, 2H, H-3 Ar, H-7
Ar, J1=7.0 Hz, J1=7.5 Hz, J2=7.2 Hz); 7.84-7.86 (d, 2H, H-6 Ar, H-8 Ar, Hz, J1=8.0 Hz).
13C NMR (CDCl3, 700 MHz): δ (ppm): 182.67 (C=O); 153.28; 138.74; 133.89; 123.04;
121.77; 119.52; 53.83; 26.07; 24.14.
IR (KBr) (cm-1): 3434, 2940, 2855, 2813, 1648, 1582, 1423, 1381, 1226, 895, 710.
MALDI TOF MS: m/z 376.3 [M+2H] +, (MW = 374.47).
RP-HPLC: Rt = 7.07 gradient elution 0–100% (A–B) in 20 min.
Melting point: 207–208 °C.
1,8-Di(piperidin-1-yl)-9,10-anthraquinone (4, C24H26N2O2)

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Yield: m = 357 mg; 26 %.
TLC (SiO2): CH2Cl2; Rf = 0.56.
1H NMR (CDCl3, 500 MHz): δ (ppm): 1.64-1.70 (p, 4H, -HN-CH2-CH2-CH2-CH2-CH2-,
J1=5.5 Hz, J1=6.5 Hz, J1=7.0 Hz, J1=5.5 Hz, J2=6.0 Hz, J2=6.75 Hz, J2=6.2 Hz, J3=6.3 Hz,
J3=6.5 Hz, J4=6.4 Hz); 1.84-1.89 (p, 8H, -HN-CH2-CH2-CH2-CH2-CH2-, J1=5.5 Hz, J1=6.5
Hz, J1=6.5 Hz, J1=5.5 Hz, J2=6.0 Hz, J2=6.5 Hz, J2=6.0 Hz, J3=6.2 Hz, J3=6.2 Hz, J4=6.2 Hz);
3.13-3.15 (t, 8H, -HN-CH2-CH2-CH2-CH2-CH2-, J1=J2=6.0 Hz); 7.36-7.37 (d, 2H, H-4 Ar, H-
5 Ar, J1=7.0 Hz);
7.52-7.55 (t, 2H, H-3 Ar, H-6 Ar, J1=J2=7.0 Hz); 7.79-7.80 (d, 2H, H-2 Ar, H-7 Ar, J1=6.0
Hz).
13C NMR (CDCl3, 700 MHz): δ (ppm): 185.45(C=O); 185.45(C=O); 153.30; 153.31; 135.56;
135.06; 132.78; 132.58; 127.29; 126.38; 125.06; 125.07; 119.87; 119.43; 54.33; 26.34; 24.31.
IR (KBr) (cm-1): 3421, 2936, 2851, 2806, 1667, 1582, 1447, 1309, 1245, 1201, 887, 745.
MALDI TOF MS: m/z 375.2 [M+H] +, (MW = 374.47).
RP-HPLC: Rt = 9.01 gradient elution 0–100% (A–B) in 20 min
Melting point: 175–177 °C.
3. Results and Discussion
3.1. Synthesis of piperidine and 9,10-anthraquinone derivatives
The present procedure describes the new synthesis of the series of piperidine and 9,10-
anthraquinone derivatives. The different synthetic procedure of compounds 1, 3 and 4 is
described in literature, however this work describe the easier synthetic method where products
are obtained with high yields.

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The first synthesis of compound 1 was described by Piscunow [41]. The compound 3 was
obtained in very difficult synthesis and authors do not give detailed analyzes of the obtained
product.
Arai [42] mentioned about the synthesis of 1 in reaction of 1-chloro-9,10-
anthraquinone with piperidine, without detailed analysis and synthetic procedure. Park [43]
described synthesis of compound 3 obtained in another way than presented in this work,
where 1,5-dichloroanthracene-9,10-dione and piperidine reacted in DMSO at 80 °C obtaining
3 with yield 78,7%. In the synthesis proposed by authors of this work 1,5-bis(tosyloxy-9,10-
anthraquinone and piperidine were used as substrate in toluene obtaining desired product with
yield of 96%. Additionally Umadevi in the paper [44] only explain that compound 3 was
obtained in their laboratory without any details of synthesis. The synthesis of compound 4
was described by Beletskaya [45] where authors show only synthesis without properties of
obtained compound. Beletskaya obtained compound 4 in the synthesis of 1,8-dichloro-9,10-
anthraquinone and piperazine in dioxane used as solvent in the presence of two catalysts
BINAP and dppf. In this method compound 4 was obtained with yield 31% and 36%
respectively of used catalyst. The main advantage of the procedure proposed by authors of
this work is no necessary to use catalyst in the reaction. In the case of compound 4 yield of
reaction obtained by the authors as well by Beletskaya is very low due to the steric hindrance
occurring in this compound.
In this work 1-chloro-9,10-anthraquinone was used in the substitution reaction with
piperidine to obtain the compound 1, with 90.0% yield. Compounds 2, 3 and 4 were obtained
with 97.0%, 96.0%, 26.0% yield, respectively, using 1,4-bis(tosyloxy)-9,10-anthraquinone,
1,5-bis(tosyloxy)-9,10-anthraquinone, and 1,5-dichloro-9,10-anthraquinone as the substrates.
The yields of this reaction is very high with the exception of compound 4. In the case of
substitution by piperidine in 9,10-anthraquinone moiety the steric hindrance occurring

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between the carbonyl group and two piperidine substituents. All of these tosyloxy derivatives
of the 9,10-anthraquinone were obtained as 1,5-Bis(tosyloxy-9,10-anthraquinone using the
procedure described elsewhere [46]. The crystal structure of the compound 1 and 3 has been
described in papers [39,40].
The crystal structure of 1-(Piperidin-1-yl)-9,10-anthraquinone 1 directly indicate that
the piperidine ring have a chair conformation and the planes of piperidine ring and the
9,10-anthraquinone ring are inclined at a dihedral angle of 38.7 (1) .
In the crystal structure of 1,5-Di(piperidin-1-yl)-9,10-anthraquinone the piperidine
ring have also a chair conformation and is inclined at a dihedral angle of 37.5 (1)° to the
9,10-antchraquinone ring system.
In both crystal structure of compound 1 and 3 the adjacent molecules are linked
through C–H···π and π – π [centroid–centroid distance = 3.782 (1) Å is for compound 1 and
3.806 (1) Å for compound 3, forming in both cases a layer parallel to the bc plane [39,40].
The purity of each derivative was above 99.8% and was determined using the high-
performance liquid chromatography (HPLC) technique. The HPLC chromatograms are placed
in the Supporting information. All syntheses were performed in the toluene in 80 °C using
mono- or di-substituted chloro 9,10-anthraquinone and piperidine. The route of the synthesis
is presented below (Scheme 1).

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Scheme 1 The synthetic route for the compounds 1–4.
The representative of 1H NMR spectra of 1-(Piperidin-1-yl)-9,10-anthraquinone 1 was
1
recorded in CDCl3 solution. H NMR spectrum of compound 1 is presented in Fig. 1. The
1H NMR signals of the piperidine units present in the structure of all studied compounds are
in the range of 1.64 to 3.20 ppm and gives two pentet and one triplet. The protons from
anthraquinone moiety have different chemical shifts and depends on the location of piperidine
substituents.

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Fig. 1. The representative of 1H NMR spectra of compound 1.
3.2. UV-VIS Studies
All UV-VIS measurements of the studied compounds were carried out in the DMSO
solution at the room temperature. The shape of the absorption band of all 9,10-anthraquinone
derivatives is associated with the amino substituents and the benzenoid character of the
molecules [46]. Fig. 2 shows the absorption spectra for all studied compounds. The shapes of
the absorption bands for compounds 1, 3, and 4 observed in the visible region are close to
each other. However, the intensity of the absorption band is highest for the compound 3 where
9,10-anthraquinone moiety is substituted with the piperidine in the positions 1 and 5. The
intensity of the band of compound 3 is probably associated with the influence of amino
substituents and changes caused on the π electrons of the aromatic system of the molecular
structure [44,47].
The position and the number of the piperidine present in the anthraquinone moiety
directly influence the shape and position of the bands in the obtained spectra. The previous
investigation proved that the substitution of the 9,10-anthraquinone moiety in the position 1
and 4 causes the appearance of a two-headed band [48]. Two-headed band occurs in the

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spectra of the compound 2 where two piperidine substituents are present in the position of 1
and 4 of the 9,10-anthraquinone.
Fig. 2. UV-VIS absorption spectra for the studied compounds 1–4 in the DMSO solution.
The values of the absorption maxima and the logarithm of the molar absorption coefficient,
for examined compounds, range from logε = 3.50 (λ = 522 nm) for compound 1 to logε = 3.75
(λ = 516 nm) for the compound 3 (Table 1). The insightful analysis of the data presented in
Table 1 in relation to compound 1 where 9,10-anthraquinone moiety is substituted with one
piperidine moiety allow for the following conclusions: The hyperchromic effect (increase of
absorbance intensity) is observed in all absorption spectra in comparison to compound 1. The
absorbance intensity is highest for the compound 3. The bathochromic effect (change of
spectral band to a longer wavelength) is observed for compound 2, where 9,10-anthraquinone
moiety is substituted with the piperidine in the positions 1 and 4. Whereas the hypsochromic

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effect (change of spectral band to a shorter wavelength) is observed for compounds 3 and 4
where the piperidine moiety is substituted in position 1, 5 and 1, 8 respectively.
Table 1
The values of absorption maxima (λ), molar extinction coefficients (ε), and the logarithm
of the molar absorption coefficient (logε) for the examined compounds 1–4.
DMSO
εmax (dm3/mol · cm)
3150
Compound
λmax (nm)
522
logεmax
3.50
1
2
3
4
570/596
516
3980/3920
5660
3.60/3.59
3.75
502
3440
3.53
3.3. Electrochemical studies
Cyclic voltammograms of the investigated 9,10-anthraquinone derivatives containing
the piperidine units in the structure 1–4 were studied in the potential range from 0.0 to −2.0 V
in the degassed 0.1 M TBAP/DMSO solution at glassy carbon versus Ag/AgCl (Silver/Silver
Chloride Electrode) electrode.
The reduction of all quinones in the aprotic media takes place by two one-electron
steps (Figure 2). The first step leads to the formation of a semiquinone radical anion (Q−•)
while the second step corresponds to the formation of quinone dianion (Q2−). Usually, the first
step is entirely reversible and the second step is quasi-reversible or irreversible [21,49–52].
For all investigated compounds, two reduction peaks Epc1 and Epc2 are observed in the range
of −1.00 V – −1.10 V, and −1.51 V – 1.64 V, respectively (Table 2).

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The cyclic voltammograms obtained for all compounds directly indicate the presence
of two principal one-electron reduction steps. For the compounds 1–4 the first reduction step
is fully reversible and the second reduction process is practically irreversible. The
electrochemical investigation performed in the potential range from 0.0 to −1.25 V (dotted
line, Fig. 3), prove that the first reduction process is fully reversible. Above-mentioned
statement is a result of the calculated values of ꢁE1 which are summarized in Table 2.
In all of the investigated compounds in the anodic part of obtained voltammograms,
one additional oxidation peak Epa3 about −0.7 V is present. The mechanism of this oxidation
process is rather complex, but this phenomenon was discussed previously [53,54]. The
appearance of this peak is related to the oxidation of the protonated dianion.

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Fig. 3. Cyclic voltammograms of the studied compounds 1–4 on a glassy carbon
electrode in 0.1 M TBAP in the DMSO solution in the range of −0.25 to −2.0 V (solid
line) and in the range of −0.25 to −1.25 V (dashed line), at 100 mV/s scan rate.
Table 2
The values of cathodic Epc and anodic Epa peak potentials and peak potential separation,
∆Ep = (Epa − Epc) obtained for all investigated compounds in the DMSO solution.
Cathodic peak
potential (V)
Anodic peak potential
(V)
Peak separation (mV)
Compound
Epc1
Epc2
Epa1
Epa2
Epa3
ꢀE1
97
ꢀE2
176
100
193
210
−1.004
−1.074
−1.105
−1.070
−1.509 −0.907 −1.333 −0.705
−1.522 −0.978 −1.422 −0.749
−1.640 −1.008 −1.447 −0.780
−1.570 −0.977 −1.360 −0.723
1
2
3
4
96
97
93
3.4. Biological Evaluation
Determination of the biological effect of the synthesized compounds towards
cancerous and normal cell lines was one of the crucial goals of the study.
Promyelocytic leukemia cell line and its mitoxantrone-resistant subline (HL-60 and
HL-60/MX2, respectively), as well as colon adenocarcinoma cells and its subline resistant to
doxorubicin (LoVo and LoVo/Dx), were used. The preliminary cytotoxicity evaluation of all
compounds was also performed towards the normal mouse embryonic fibroblasts
(BALB/3T3) cell line. As a reference compounds, three antitumor drugs were applied:
cisplatin, doxorubicin, and mitoxantrone. Table 1 summarizes the IC50 values (in µg/mL) of
the tested compounds. The antiproliferative effect in vitro was determined using SRB or MTT
(leukemia cell lines) method and software described previously [55–58].

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Table 1
Antiproliferative activity of the novel 9,10-anthraquinone derivatives 1-4 towards human
cancer and murine normal fibroblast cell lines.
IC50 ± SD (µg/mL)
Compound
HL-60
HL-60/MX2
IR
LoVo
LoVo/Dx
IR
BALB/3T3
3.67 ± 1.34
14.60 ± 9.65
43.20 ± 16.90
39.71 ± 3.12
0.25 ± 0.08
0.005 ± 0.002
n.t.
2.50 ± 0.96
5.85 ± 2.16
30.3 ± 24.2
34.2 ± 9.76
0.40 ± 0.28
0.07 ± 0.04
n.t.
0.68 3.29 ± 0.21 2.31 ± 0.68 0.70 16.04 ± 6.00
1
0.4
0.70
0.86
1.6
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
2
3
n.a.
4
1.33 ± 0.3 1.36 ± 0.34 1.02
1.03 ± 0.39
cisplatin
mitoxantrone
doxorubicin
DMSO
14
0.06 ± 0.02 0.37 ± 0.18 6.17 IC50 < 0.001
n.t. 0.19 ± 0.06 4.72 ± 0.88 24.84 0.18 ± 0.05
-
-
-
-
-
-
-
The IC50 value is defined as the concentration of a compound that corresponds to a
50% growth inhibition. Human promyelocytic leukemia (HL-60) and its mitoxantrone-
resistant subline (HL-60/MX2); human colon adenocarcinoma cell line (LoVo) and
doxorubicin-resistant subline (LoVo/Dx); normal murine embryonic fibroblast cell line
(BALB/3T3) were subjected to tests. Data are expressed as the mean ± SD. The IR values
were calculated for each compounds using the formula: RI = IC50 for the drug-resistant cell
line/IC50 for the appropriate drug-sensitive cell line.
n.t. – not tested
n.a. – not active in the range of concentrations tested
The definition of the IR value is described elsewhere [59].
The most active was the compound 1 containing one piperidine unit in the structure.
The activity of the compounds 2, 3, and 4 containing two piperidine units in the structure is

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lower than in the case of the compound 1 and depends on the substitution of the piperidine in
the 9,10-anthraquinone ring.
The activity of the compound 1 is also interesting concerning the analysis of the drug-
resistant cell lines. Notably, this compound revealed higher antiproliferative activity against
LoVo/Dx cancer cell line than towards the doxorubicin-sensitive LoVo cells. These results
suggest that compound 1 preferentially inhibits the proliferation of the doxorubicin-resistant
colon adenocarcinoma cells.
All of the tested novel 9,10-anthraquinone derivatives containing piperidine unit
distinctly overcome the multidrug resistance of the HL60/MX2 cell line, much stronger than
cisplatin and mitoxantrone, what confirms their high efficiency towards the leukemic cells.
Among tested compounds, compound 2 seems to be interesting due to the lowest IR value for
HL-60/MX2 cell line (0.4 µg/mL). Compound 2 is a derivative of 9,10-anthraquinone
substituted by two piperidine units in the position 1 and 4 of the scaffold. One should also
emphasize that the mitoxantrone has also substituents in the position 1 and 4 of the structure.
Importantly, all tested compounds revealed very low (like compound 1) or no cytotoxicity
towards the normal mice fibroblasts what can suggest its selective cytotoxicity towards cancer
cells.
4. Conclusion
The present study describes the synthesis and characterization of the series of novel
9,10-anthraquinone derivatives containing piperidine units. The UV-VIS experiments
confirmed the previous investigations which shown the unusual behavior of the 9,10-
anthraquinone derivative containing substituent in the positions 1 and 4 during its presence in
the DMSO solution. The electrochemical investigation confirmed that the reduction of the

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investigated compounds in the non-aqueous media takes place by two reversible one-electron
steps.
The antiproliferative activities of the studied compounds were evaluated in vitro using
drug-sensitive and drug-resistant human cancer cell lines. The results of the conductebut thed
tests show that the compound 1 containing one piperidine substituent in the structure is the
most active drug from the molecules subjected to analysis. Additionally, all of the studied
compounds distinctly overcame the multidrug resistance of the tested cancer cell lines.
Acknowledgements
This study was financed by the University of Gdansk within the project supporting young
scientists and Ph.D. students (No. 538-8210-B319-14 and No. 538-8210-B722-17) and by the
State Funds for Scientific Research, of Department of Analytical Chemistry, University of
Gdansk.
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List of scheme and figures.
Scheme 1 The synthetic route for the compounds 1–4.
Fig. 1. The representative of 1H NMR spectra of compound 1.
Fig. 2. Cyclic voltammograms of the studied compounds 1–4 on a glassy carbon electrode

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Synthesis and Electrochemical, Spectral, and Biological Evaluation of Novel 9,10-
Anthraquinone Derivatives Containing Piperidine Unit as Potent Antiproliferative
Agents
Paweł Niedziałkowski*1, Elżbieta Czaczyk1, Joanna Jarosz2, Anna Wcisło1, Wioleta
Białobrzeska1, Joanna Wietrzyk2, Tadeusz Ossowski1
Highlights
Synthesis of Novel piperidine and 9,10-Anthraquinone Derivatives
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•
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1H NMR and UV-VIS studies of obtained compounds
Electrochemical studies of of obtained compounds
Biological evaluation of piperidine and 9,10-Anthraquinone Derivatives