Design, synthesis, and biological evaluation of novel Tempol derivatives as effective antitumor agents

Article abstract of DOI:10.1007/s11164-016-2560-5

Two series of novel Tempol derivatives T1–T6 based on the piperidine nitroxide Tempol and phenolic acids were designed and synthesized, and their biological evaluation is also described. The chemical structure was verified by HRMS, IR, and EPR analysis. The antitumor activity was tested against two tumor cell lines (A549 and Hela cells). Simultaneously, HK-2 cells were selected to investigate cytotoxicity and selectivity of synthetic compounds to the normal cells. The antioxidant property was also studied by DPPH radical scavenging assay and hydrogen peroxide-induced cell injury assay. The results demonstrated that most of the Tempol derivatives exhibited more active antioxidant activity than Tempol, and all synthesized Tempol derivatives exhibited more potent antitumor activity than Tempol. Among them, compound T6 displayed the highest antitumor activity (IC₅₀?=?29.4?μg/mL for A549 cells; IC₅₀?=?16.2?μg/mL for Hela cells). The results indicated that T6 exhibited efficient antitumor performance, having the potential of being excellent antitumor agents for cancer treatment.

Full text of DOI:10.1007/s11164-016-2560-5

Res Chem Intermed
DOI 10.1007/s11164-016-2560-5
Design, synthesis, and biological evaluation of novel
Tempol derivatives as effective antitumor agents
Xiao-Liang Sun1 Shi-Yu Wang1
•
•
Zhi-Min Qi2 Ning Wan2 Bang-Le Zhang2
Wei He1
•
•
•
Received: 2 February 2016 / Accepted: 22 April 2016
Ó Springer Science+Business Media Dordrecht 2016
Abstract Two series of novel Tempol derivatives T1–T6 based on the piperidine
nitroxide Tempol and phenolic acids were designed and synthesized, and their
biological evaluation is also described. The chemical structure was verified by
HRMS, IR, and EPR analysis. The antitumor activity was tested against two tumor
cell lines (A549 and Hela cells). Simultaneously, HK-2 cells were selected to
investigate cytotoxicity and selectivity of synthetic compounds to the normal cells.
The antioxidant property was also studied by DPPH radical scavenging assay and
hydrogen peroxide-induced cell injury assay. The results demonstrated that most of
the Tempol derivatives exhibited more active antioxidant activity than Tempol, and
all synthesized Tempol derivatives exhibited more potent antitumor activity than
Tempol. Among them, compound T6 displayed the highest antitumor activity
(IC50 = 29.4 lg/mL for A549 cells; IC50 = 16.2 lg/mL for Hela cells). The results
indicated that T6 exhibited efficient antitumor performance, having the potential of
being excellent antitumor agents for cancer treatment.
Keywords Piperidine nitroxide Á Phenolic acids Á Antitumor activity Á
Growth inhibition Á Antioxidant activity
Electronic supplementary material The online version of this article (doi:10.1007/s11164-016-2560-
5) contains supplementary material, which is available to authorized users.
&
&
1
Bang-Le Zhang
blezhang@fmmu.edu.cn
Wei He
weihechem@fmmu.edu.cn
Department of Chemistry, School of Pharmacy, Fourth Military Medical University,
Xi’an 710032, China
2
Department of Pharmaceutics, School of Pharmacy, Fourth Military Medical University,
Xi’an 710032, China
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X.-L. Sun et al.
Introduction
In recent years, cancer incidence and morbidity are steadily increasing even though
great progress has been made in the treatment of tumors. In 2015, an estimated
1,658,370 new cancer cases diagnosed and 589,430 cancer deaths occurred in the
US [1]. In addition to surgical intervention, current cancer treatments mainly rely on
radiation and chemotherapeutic agents. The design of antitumor agents is very
important for cancer treatment. Much evidence suggests that multiple metabolites
and metabolic by-products such as ATP, acetyl-CoA, a-ketoglutarate (a-KG), and
reactive oxygen species (ROS) can have a crucial role in tumor development [2].
Carefully considering these points will allow for the development of efficient agents
for cancer therapy.
4-Hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (Tempol, Fig. 1) is a piperidine
nitroxide analogue, which is widely used as biological agent [3–7] and has various
biological activities, such as radio-protection [8–12] and anti-proliferation proper-
ties [13]. It has been extensively studied for its antitumor activity by inducing cell
apoptosis in breast cancer and human leukemia [14]. Antitumor activity is
potentiated by pretreatment with Tempol in the colon carcinoma cell lines [15]. The
paradoxical pro-oxidant action of Tempol has accounted for provoking cancer cell
apoptosis [16]. More detailed studies indicate that Tempol can inhibit the tumor
growth, which seems to be related to the reactivity of the nitroxyl group [3, 4, 17,
18]. It suggested that we can prepared Tempol derivatives as antitumor drugs by
introduction of a piperidine nitroxide radical into pharmaceutical molecules.
In recent years, there has been a revival of interest in the medical properties of
phenolic acids because they exhibit a broad spectrum of activity including
antioxidant, antibacterial, antifungal, antitumor, and antiinflammatory properties. It
has been reported that caffeic acid [19, 20] and ferulic acid [20] can induce
apoptosis in tumor cells and exerts anticarcinogenic effects. Oxidative stress and
free radical damage are recognized to induce malignant transformation and the
occurrence of cancer. It has been reported that the supplementation with
antioxidants can inhibit the carcinogenesis [21]. Tempol and phenolic acids have
a great effect on the antioxidant activities and anti-free radical effect. However, so
Fig. 1 Structure of 4-hydroxy-2,2,6,6,tetramethylpiperidine-N-oxyl (Tempol) and phenolic acids used to
design and synthesize Tempol derivatives
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Design, synthesis and biological evaluation of novel…
far, Tempol and phenolic acid used as starting materials to prepare new conjugated
derivatives as antitumor agents has not been paid much attention.
To explore the potential of this idea in the antitumor agents design and improve
antitumor potency of Tempol, in this paper, we firstly designed and synthesized two
series of novel Tempol derivatives as antitumor agents based on piperidine nitroxide
Tempol and phenolic acids (Fig. 1). The antitumor activity of Tempol derivatives
against A549 and Hela tumor cells was investigated, and the antioxidant capacity
was evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay
and hydrogen peroxide-induced cell injury assay.
Experimental
General
4-Hydroxy-2,2,6,6-tetramethyl-piperidinooxy (Tempol) was purchased from Sigma
(St. Louis, MO, USA). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) free radical was
purchased from TCI (Shanghai, China). All of the different phenolic acids, as well
as other reagents and solvents were purchased from Energy Chemical (Shanghai,
China). A549 (human adenocarcinoma lung cells), Hela (human cervical carcinoma
cells), and HK-2 (human proximal tubular epithelial cells) cell lines were procured
from the culture collection of the Chinese Academy of Science (Shanghai, China).
Cell culture media, Opti-MEM, and fetal bovine serum (FBS) were purchased from
Gibco (Carlsbad, CA, USA). The progress of the reactions was monitored by thin-
layer chromatography on silica gel plates from Kehao (Xi’an, China). Column
chromatography was performed with silica gel (200–300 or 300–400 mesh, Kehao).
1H NMR spectroscopy were recorded in CDCl3 or CD3OD solution on a Bruker
Spectrospin 400 (400 MHz) instrument. EPR spectra were recorded on a JES-
FA200 EPR spectrometer (JEOL, Japan, Measurement conditions: frequency
9.0514 GHz, normal pressure and temperature, CH2Cl2 as solvent). Fourier
transform infrared (FT-IR) spectroscopy was performed on a Paragon 1000
(Perkin-Elmer) instrument using KBr plates. High-resolution mass spectra (HRMS)
were measured with ESI.
General procedure for preparation of Tempol derivatives T1–T3
Preparation of 2a–2c
Gallic acid (1a, 7.53 g, 40 mmol) and acetic anhydride (22.7 mL, 240 mmol) were
combined in a 100 mL round-bottom flask. The slurry was stirred with a magnetic
stirrer and catalytic amount of sulfuric acid (500 lL) was added. The reaction
mixture was stirred at 75 °C for 1.5 h, and the slurry became a clear yellow
solution. The mixture was further stirred for 20 min at room temperature, and
60 mL of water was added. After stirring for 2.5 h, a white crystalline product was
isolated by filtration and washed with water (20 mL 9 3). The product was dried in
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X.-L. Sun et al.
a vacuum drying oven overnight to obtain the acetyl-protected gallic acid 2a (8.7 g).
2b, 2c were synthesized using similar method as 2a.
2a (73 %) was obtained as a white crystalline solid. mp 166–167 °C (lit [22].
166–168 °C); 1H NMR (400 MHz, CDCl3): d 2.31 (s, 9H, –OCH3), 7.87 (s, 2H, Ar–
H); IR (KBr, cm-1) mmax: 2983, 2955, 2930, 1778, 1693, 1433, 1372, 1322, 1183,
1056, 899.
2b (46 %) was obtained as a white solid. mp 105–106 °C (lit [23]. 104–106 °C);
1H NMR (400 MHz, CDCl3): d 2.24 (s, 3H, –OCH3), 2.25 (s, 3H, –OCH3), 3.63 (s,
2H, –CH2–), 7.14–7.20 (m, 3H, Ar–H); IR (KBr, cm-1) mmax: 2939, 2922, 2854,
1770, 1708, 1508, 1373, 1261, 1211, 1184, 1114, 1014, 906.
2c (76 %) was obtained as a white solid. 1H NMR (400 MHz, CDCl3): d 2.33 (s,
0
0
6H, –OCH3), 7.32 (d, J = 8.4 Hz, 1H, Ar–H5 ), 7.94 (d, J = 2.0 Hz, 1H, Ar–H2 ),
0
8.02 (dd, J1 = 8.4 Hz, J2 = 2.0 Hz, 1H, Ar–H6 ); IR (KBr, cm-1) mmax: 2985, 2945,
2854, 1766, 1685, 1373, 1242, 1164, 1049, 914, 779.
Preparation of 3a–3c
To the solution of 2a (2.96 g, 10 mmol) in dry dichloromethane (25 mL), SOCl2
(0.88 mL, 12 mmol) and DMF (0.2 mL, 2.5 mmol) were added dropwise. The
mixture was refluxed for 3 h. After the solvent was evaporated under reduced
pressure, 3a (3.1 g, 98 %) was obtained as a white solid. mp 105–107 °C (lit [24].
106–107 °C). 3b, 3c were synthesized using similar method as 3a. 3b (98 %), 3c
(99 %) were obtained as a yellow oil, which used without purification for the next
reaction.
Preparation of 4a–4c
4-Hydroxy-2,2,6,6-tetramethyl-piperidinooxy (Tempol, 1.85 g, 10.73 mmol) and
pyridine (1.2 mL, 15 mmol) were dissolved in dry dichloromethane (15 mL) and
the mixture was stirred for 1 h at room temperature. 3a (3.1 g, 9.5 mmol) dissolved
in dry dichloromethane (15 mL) was added to the above mixture at 0 °C. Then, the
reaction mixture was stirred for 3 h at room temperature and monitored by TLC.
The reaction mixture was repeatedly washed with saturated copper sulfate solution
and the organic phase was dried under magnesium sulfate. After filtering, the
solvent was removed in a vacuum. The residue was purified by flash chromatog-
raphy on silica gel with petroleum ether/ethyl acetate (100/1 to 20/1) to give 4a
(2.32 g). 4b, 4c were synthesized using a similar method as 4a.
4a (53 %) was obtained as a red solid. mp 101–102 °C; IR (KBr, cm-1) mmax
:
2975, 2937, 2869, 1739, 1463, 1365, 1236, 1180, 1031, 889; HRMS (ESI?) Calcd
for [M ? Na]?: 473.1662 m/z. Found: 473.1659 m/z.
4b (56 %) was obtained as a red solid. mp 86–87 °C; IR (KBr, cm-1) mmax: 2977,
2935, 2854, 1770, 1731, 1504, 1369, 1261, 1203, 1180, 1110, 1010, 902; HRMS
(ESI?) Calcd for [M ? Na]?: 429.1763 m/z. Found: 429.1750 m/z.
4c (67 %) was obtained as a red solid. mp 118–119 °C; IR (KBr, cm-1) mmax
:
2977, 2939, 2854, 1770, 1708, 1612, 1504, 1373, 1288, 1195, 1164, 1114, 1091,
1010, 910, 763, 736.
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Design, synthesis and biological evaluation of novel…
Typical procedure of preparation of T1–T3
Guanidine hydrochloride (764 mg, 8 mmol) was added to a solution of 4a (450 mg,
1 mmol) in 20 mL mixed solvent (dichloromethane/methanol = 5/1). The mixture
was refluxed for 48 h and monitored by TLC. The solvent was evaporated and the
residue was purified by flash chromatography on silica gel with dichloromethane/
ethyl acetate (20/1 to 2/1) to give T1 (150 mg, 43 %) as a brownish red solid. mp
78–79 °C; IR (KBr, cm-1) mmax: 3371, 2977, 2935, 2873, 1701, 1612, 1465, 1319,
1226, 1180, 1033, 771; ESR: giso = 2.0044, AN = 15.39 Gs (CH2Cl2); HRMS
(ESI-) Calcd for [M–H]-: 323.1369 m/z. Found: 323.1386 m/z. T2, T3 were
synthesized by the method as T1.
T2 (38 %) was obtained as a pale yellow solid. mp 139–140 °C; IR (KBr, cm-1
)
max: 3394, 2977, 2939, 2854, 1731, 1604, 1519, 1365, 1284, 1242, 1145, 1045,
m
798; ESR: giso = 2.0044, AN = 15.53 Gs (CH2Cl2); HRMS (ESI-) Calcd for [M–
H]-: 321.1576 m/z. Found: 321.1576 m/z.
T3 (90 %) was obtained as a yellow solid. mp 191–192 °C; IR (KBr, cm-1) mmax
:
3309, 2977, 2939, 2854, 1708, 1604, 1442, 1292, 1218, 1176, 1091, 767; ESR:
giso = 2.0044, AN = 15.61 Gs (CH2Cl2); HRMS (ESI-) Calcd for [M–H]-:
307.1420 m/z. Found: 307.1420 m/z.
General procedure for preparation of Tempol derivatives T4–T6
Preparation of 6a–6c
Acetic anhydride (0.59 mL, 6.25 mmol) was added slowly to a solution of ferulic
acid (5a, 970 mg, 5 mmol) and DMAP (62 mg, 0.5 mmol) in pyridine (5 mL) at
0 °C. After stirring for 1.5 h, the mixture was poured into ice water. The aqueous
phase was acidified with hydrochloric acid (2 M) to pH 2 and then extracted with
ethyl acetate (3 9 150 mL). The combined organic layers were dried over Mg2SO4.
After filtering and concentrating in vacuo, the residue was treated with petroleum
ether/ethyl acetate (95/5; v/v; 10 mL) to give 6a (1.04 g, 88 %) as a white powder.
mp 198–199 °C (lit [25, 26]. 197–200 °C); 1H NMR (400 MHz, CD3OD): d 2.26 (s,
3H, –OCH3), 3.84 (s, 3H, –OCH3), 6.84 (d, J = 16.0 Hz, 1H, –CH=CH–), 7.05 (d,
0
J = 8.4 Hz, 1H, Ar–H6 ), 7.18 (dd, J1 = 8.4 Hz, J2 = 2.0 Hz, 1H, Ar–H5 ), 7.30 (d,
0
-1
0
J = 1.6 Hz, 1H, Ar–H2 ), 7.65 (d, J = 16.0 Hz, 1H, –CH=CH–); IR (KBr, cm
max: 2977, 2944, 2873, 1761, 1684, 1632, 1507, 1261, 1223, 1199, 1034, 855. 6b
and 6c were synthesized using a similar method as 6a.
)
m
1
6b (97 %) was obtained as a white solid. H NMR (400 MHz, CD3OD): d 2.28
(s, 3H, –OCH3), 6.46 (d, J = 16.0 Hz, 1H, –CH=CH–), 7.15 (d, J = 8.4 Hz, 2H,
0
0
Ar–H3,5 ), 7.64 (d, J = 8.8 Hz, 2H, Ar–H2,6 ), 7.68 (d, J = 15.6 Hz, 1H, –CH=CH–);
IR (KBr, cm-1) mmax: 2972, 2935, 2854, 1743, 1677, 1508, 1209, 1170, 916, 838.
6c (89 %) was obtained as a white solid. 1H NMR (400 MHz, CDCl3): d 2.31 (s,
3H, –OCH3), 2.32 (s, 3H, –OCH3), 6.39 (d, J = 16.0 Hz, 1H, –CH=CH–), 7.26 (d,
0
J = 2.8 Hz, 1H, Ar–H6 ), 7.39 (d, J = 2.0 Hz, 1H, Ar–H5 ), 7.43 (dd, J1 = 8.4 Hz,
0
-1
0
J2 = 2.0 Hz, 1H, Ar–H2 ), 7.72 (d, J = 16.0 Hz, 1H, –CH=CH–); IR (KBr, cm
max: 2952, 2925, 2854, 1755, 1674, 1631, 1434, 1261, 1190, 1118, 827.
)
m
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X.-L. Sun et al.
Preparation of 7a–7c
Thionyl chloride (0.88 mL, 12 mmol) was added to a suspension of 6a (2.36 g,
10 mmol) in dry chloroform (25 mL) and DMF (0.2 mL). The mixture was refluxed
for 4 h. After the solvent and excess thionyl chloride were removed under reduced
pressure, 7a (2.45 g, 92 %) was obtained as a pale yellow solid. This material was
used without further purification. mp 129–130 °C (lit [25]. 129–131 °C). 7b, 7c
were synthesized using similar method as 7a. 7b (99 %) and 7c (98 %) were
obtained as a white powder and used without further purification.
Preparation of 8a–8c
8a, 8b, and 8c were synthesized using similar methods as 4a. 8a (85 %) was
obtained as a pale red solid. mp 142–143 °C; IR (KBr, cm-1) mmax: 2974, 2933,
2854, 1766, 1704, 1635, 1506, 1259, 1197, 1155, 1124, 1031, 831; HRMS (ESI?)
Calcd for [M ? Na]?: 413.1814 m/z. Found: 413.1800 m/z. 8b (69 %) was obtained
as a red solid. mp 104–105 °C; IR (KBr, cm-1) mmax: 2952, 2925, 2854, 1774, 1708,
1635, 1506, 1199, 1012, 910, 835; HRMS (ESI?) Calcd for [M ? Na]?:
383.1709 m/z. Found: 383.1694 m/z. 8c (69 %) was obtained as a pale red solid.
mp 115–116 °C; IR (KBr, cm-1) mmax: 2975, 2937, 2854, 1774, 1712, 1635, 1506,
1371, 1205, 1178, 1112, 1012, 900; HRMS (ESI?) Calcd for [M ? Na]?:
441.1763 m/z. Found: 441.1754 m/z.
Typical procedure of preparation of T4–T6
8a (2.34 g, 6 mmol) was dissolved in a 20 mL mixed solvent (dichloromethane/
methanol = 6/1) at room temperature. Potassium tert-butoxide (741 mg, 6.6 mmol)
was added and the mixture was stirred for 15 min at 0 °C. Then, ice water was
added into the reaction mixture. The product was extracted using dichloromethane
(3 9 100 mL), dried over sodium sulfate, and concentrated under reduced pressure.
The crude product was purified by flash chromatography using hexane and ethyl
acetate as eluent (100/1 to 10/1) to give T4 (1.08 g). T5 was synthesized by the
method as T4. T6 was synthesized by the method as T1.
T4 (52 %) was obtained as a golden solid. mp 183–184 °C; IR (KBr, cm-1) mmax
:
3413, 2974, 2925, 2854, 1704, 1635, 1515, 1157, 1027, 985, 848; ESR:
giso = 2.0049, AN = 15.67 Gs (CH2Cl2); HRMS (ESI?) Calcd for [M ? Na]?:
371.1709 m/z. Found: 371.1718 m/z.
T5 (34 %) was obtained as a golden solid. mp 178–179 °C; IR (KBr, cm-1) mmax
:
3394, 2977, 2937, 2854, 1704, 1631, 1604, 1585, 1515, 1240, 1166, 1045, 833;
ESR: giso = 2.0049, AN = 15.47 Gs (CH2Cl2); HRMS (ESI-) Calcd for [M–H]-:
317.1627 m/z. Found: 317.1615 m/z.
T6 (56 %) was obtained as a brownish red solid. mp 90–91 °C; IR (KBr, cm-1
)
max: 3371, 2974, 2931, 2869, 1704, 1685, 1635, 1604, 1515, 1446, 1272, 1157,
m
1114, 979, 813, 736; ESR: giso = 2.0044, AN = 15.64 Gs (CH2Cl2); HRMS (ESI-)
Calcd for [M–H]-: 333.1576 m/z. Found: 333.1577 m/z.
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Design, synthesis and biological evaluation of novel…
Biological studies on cell growth inhibition
A549 cells and Hela cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM, Sigma) supplemented with 10 % fetal bovine serum (FBS) in T25 culture
flasks (Nunc, Denmark) and were incubated at 37 °C in a humidified atmosphere
containing 5 % CO2. HK-2 cell was cultured in Dulbecco’s modified eagle essential
medium and F-12 nutrient mixture (DMEM/F-12) (Hyclone, Thermo Scientific,
Barrington, IL) supplemented with 10 % fetal bovine serum (FBS) in T75 culture
flasks (Nunc, Denmark) and were incubated at 37 °C in a humidified atmosphere
containing 5 % CO2. Cells were regularly passaged by 0.1 % trypsin (EDTA
0.02 % and trypsin 0.1 %) in PBS (pH 7.4).
The antitumor activity and selectivity of Tempol derivatives was evaluated by
MTT assays through determining the inhibition effect on the proliferation of A549
cells, Hela cells or HK-2 cells. Tempol and Dox were used as the positive control.
The in vitro antitumor activities of each Tempol derivatives was determined using
3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction
assay following the literature procedures [27, 28]. Nearly 2 9 104 cells/well were
plated in 96-well plate. The concentration of compounds was set as 5–100 lg/mL.
After 24 h of incubation, test samples were added to the wells containing the
appropriate tumor cells and incubated for an additional 24 h. Twenty microliters of
MTT (5 mg/mL) solution was added, and the cells were incubated for another 3 h.
The old media were removed and 150 lL of DMSO was added per well. The
reduced crystal in violet was completely dissolved in DMSO, and then the
absorbance of the solution was determined at 490 nm using a microtiter plate reader
(Bio-Rad Laboratories, Richmond, CA, USA). The samples were assayed in
independent experiments to calculate either percent inhibition or IC50 values. All of
the tested compounds were dissolved in 0.1 % DMSO, and the drug solution was
diluted from the stock solution for the MTT assay.
Evaluation of antioxidant activity by DPPH radical scavenging assay
The antioxidant activity of the Tempol derivatives, based on the scavenging activity
of the stable 2,20-diphenyl-1-picrylhydrazyl (DPPH) free radical, was determined by
the method described by Yen et al. [29] and Yokozawa et al. [30]. The Tempol
derivatives, Tempol solutions (60 lL) in methanol at different concentrations were
added to a 240 lL 1.6 9 10-4 mol/L solution of DPPH in methanol, total volume
of the reactive mixture is 1.5 mL, previously prepared daily and protected from
light. The reaction mixture was incubated at room temperature in the dark for
30 min, and then the absorbance of the reactive mixture was recorded using
spectrophotometer at 517 nm. Inhibition of the DPPH free radical in percent was
calculated in following way:
Scavenging effect ð%Þ ¼ ½1 À ðAs À ArÞ=ðAm À A0ފ  100;
where Am the absorbance of the control reaction (containing all reagents except the
test compound), As the absorbance of the test compound, A0 the absorbance of the
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X.-L. Sun et al.
blank control, and Ar the absorbance of the drug control group (containing all
reagents except the DPPH). Compound concentration providing 50 % inhibition
(IC50) was calculated from the graph plotting inhibition percentage against solution
concentration. Tests were carried out in triplicate. Five different concentration
(0.05, 0.09, 0.19, 0.38, 0.75 mmol/L) of each Tempol derivatives and control
studied have been assayed in order to check the linearity of response and to establish
the antioxidant activity values in the adequate linear range. Methanol was tested
against DPPH radical and this resulted in null effect on the absorbance at 517 nm.
Effect of Temple derivatives on protecting HK-2 cells against hydrogen
peroxide-induced cell injury
Considering the complexity of antioxidant activity in biological systems, hydrogen
peroxide (H2O2)-induced cell injury model was further used to evaluate the
antioxidant activity of these compounds. The protective effects of the compounds
on HK-2 cells against hydrogen peroxide-induced cell injury were assessed by MTT
assay as reported literature procedure [31, 32]. Tempol was used as the positive
control. HK-2 cells were cultured at 37 °C in a humidified atmosphere containing
5 % of CO2. Cells were seeded into 96 well cell culture plates at a density of
8 9 103 cells/well in DMEM/F12 medium. Following protocols were carried out
12 h after cells were seeded. H2O2 was newly prepared in phosphate buffered saline
(PBS) on the day of application to cultures at a final concentration of 400 lM.
Tempol and Tempol derivatives were dissolved in DMSO and diluted with DMEM/
F12. The HK-2 cells were preincubated with samples for 2 h before the H2O2 was
added. A fresh solution of MTT (5 mg/mL) prepared in PBS (pH 7.4) was added to
the 96-well cell culture plates 3 h after H2O2 was added. The plates were incubated
in the CO2 incubator for 3 h, and the formazan was lysed with DMSO. The plates
were analyzed in a multi-well-plate reader (Bio-Rad Laboratories, Richmond, CA,
USA) at 490 nm. The samples were assayed in sextuplicate.
Statistical analysis
Results are expressed as mean SD. Differences between experimental groups
were evaluated by using analysis of variance (ANOVA). The statistical significance
of the difference between groups was evaluated by one-way ANOVA and T test.
Values of P \ 0.05 were accepted as significant.
Results and discussion
Chemistry
The synthesis of Tempol derivatives T1–T6 was outlined in Figs. 2 and 3. Acetic
anhydride was used as a hydroxyl protection reagent to protect the hydroxyl group
of the phenolic acids, and acetyl-protected phenolic acid (2a–2c, 6a–6c) was
successfully prepared. After the reaction of 2a–2c or 6a–6c with 1.1 equiv of thionyl
123

Design, synthesis and biological evaluation of novel…
Fig. 2 Synthesis of Tempol derivatives T1–T3. Reagents and conditions: (i) Ac2O, H2SO4, 85 °C, 1.5 h;
(ii) SOCl2, DMF/CH2Cl2, reflux 3 h; (iii) pyridine/CH2Cl2, rt, 5 h; (iv) guanidine hydrochloride, CH2Cl2,
40 °C, 48 h
Fig. 3 Synthesis of Tempol derivatives T4–T6. Reagents and conditions: (i) Ac2O, DMAP, pyridine, rt,
1.5 h; (ii) SOCl2, DMF/CHCl3, reflux 4 h; (iii) Pyridine/CH2Cl2, rt, 5 h; (iv) potassium tert-butylate,
CH2Cl2/CH3OH, 0 °C, 15 min for T4, T5; guanidine hydrochloride, CH2Cl2, 40 °C, 48 h for T6
chloride in the presence of a catalytic amount DMF in CHCl3 or CH2Cl2 at refluxing
temperature, 3a–3c and 7a–7c was obtained in a good yield. Then chloride of
acetyl-protected phenolic acid (3a–3c, 7a–7c) and Tempol was carried out in the
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X.-L. Sun et al.
presence of a stoichiometry amount pyridine in CH2Cl2 at room temperature to gave
4a–4c and 8a–8c. Finally, target compounds T1–T6 were successfully prepared in a
moderate yield using potassium tert-butylate or guanidine hydrochloride as the
deacetylase reagent. All synthesized target Tempol derivatives T1–T6 were purified
by column chromatography, and confirmed by the melting point, IR, ESR, and
HRMS analyses.
Antitumor activity of Tempol derivatives
The anti-proliferative activities of Tempol derivatives T1–T6 were tested against
two tumor cells A549 and Hela cells using Tempol and Doxorubicin (Dox) as the
positive control. As shown in Figs. 4 and 5, all of the Tempol derivatives T1–T6
with a concentration range of 5–100 lg/mL exhibited a dose dependent manner
against the tumor cells growth. Remarkably, all of the Tempol derivatives were
more potent than Tempol. The same results are also confirmed in Tables 1 and 2.
Among them, T1 and T6 showed stronger growth inhibition for A549 cells with
IC50 values of 42.3, 29.4 lg/mL, respectively. Other Tempol derivatives showed
growth inhibition with IC50 values of 99.8 (T2), 225.3 (T3), 142.0 (T4), and 72.7
(T5) lg/mL, respectively. Among them, T4 and T6 showed stronger growth
inhibition for Hela cells with IC50 values of 30.1, 16.2 lg/mL, respectively. Other
Tempol derivatives showed growth inhibition with IC50 values of 363.3 (T1), 62.4
(T2), 388.7 (T3), and 64.8 (T5) lg/mL, respectively. These results indicated that
introduction of phenolic acids to Tempol derivatives can improve the antitumor
activities significantly. Furthermore, the antitumor activity of these Tempol
derivatives (T1–T6) was correlated with their structure. Tempol derivatives T2,
with a longer linker moiety, showed markedly increased antitumor activity
Fig. 4 The antitumor effect of Tempol derivatives T1–T6 against A549 cells determined by MTT assay.
Each bar value represents the mean SD (n = 6)
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Design, synthesis and biological evaluation of novel…
Fig. 5 The antitumor effect of Tempol derivatives T1–T6 on the growth of Hela cells determined by
MTT assay. Each bar value represents the mean SD (n = 6)
Table 1 The value of IC50
against A549 cells
Entry
Tempol derivatives
IC50 (lg/mL)
1
2
3
4
5
6
7
8
T1
42.3 5.8**,##
99.8 8.7**,##
225.3 18.0**,##
142.0 7.8**,##
72.7 9.3**,##
29.4 4.8**,#
1637.9 246.8##
16.2 2.8
T2
T3
T4
T5
Each value is presented as
mean SD (n = 6).
T6
** P \ 0.01, compared with
Tempol
Dox
#
Tempol. P \ 0.05,
##
P \ 0.01, compared with Dox
Table 2 The value of IC50
against Hela cells
Entry
Tempol derivatives
IC50 (lg/mL)
1
2
3
4
5
6
7
8
T1
363.3 25.1**,##
62.4 5.0**,##
388.7 24.5**,##
30.1 0.6**,##
64.8 3.0**,##
16.2 0.7**,##
559.7 15.4##
1.0 0.1
T2
T3
T4
T5
Each value is presented as
mean SD (n = 6).
T6
** P \ 0.01, compared with
Tempol
Dox
##
Tempol. P \ 0.01, compared
with Dox
compared with T3. Tempol derivatives T6, with an hydroxyl group at the C-3
position, showed markedly increased antitumor activity compared with T4 and T5.
In addition, we also used HK-2 cells, the human proximal tubular epithelial cells, as
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X.-L. Sun et al.
Fig. 6 The cytotoxicity of Tempol derivatives T1–T6 for HK-2 cells determined by MTT assay. Each
bar value represents the mean SD (n = 6)
the model to evaluate the cytotoxicity and selectivity of the compounds for normal
cells. As shown in Fig. 6, all of the Tempol derivatives T1–T6 with the
concentration range of 5–100 lg/mL, exhibited lower cytotoxicity than Doxoru-
bicin (Dox) for normal cells.
Antioxidant activity of Tempol derivatives
Antioxidant activity of Tempol derivatives are closely related to their bioactivities,
such as the inhibition of mutagenesis and tumor cell growth, which is often
associated with the termination of free radical transmission in biological systems
[33, 34]. Thus, the antioxidant capacity of Tempol derivatives was investigated by
DPPH radical scavenging assay and hydrogen peroxide-induced cell injury assay.
The method is based on the reduction of DPPH in the presence of a hydrogen
donating antioxidants, and the non-radical form DPPH-H was eventually obtained
by the reaction. The reduction of DPPH radical occurs with a decrease in its
absorbance at 517 nm induced by Tempol derivatives. The antioxidant activity of
Tempol derivatives was tested by DPPH radical scavenging and is shown in Fig. 7
and Table 3. All of the Tempol derivatives T1–T6 with concentration range of
0.01–0.15 lmol/mL showed dose–response relationship (Fig. 7), and the antioxi-
dant activity of Tempol derivatives were more potent than Tempol (IC50 = 21.5 -
lM) except for T5 (IC50 = 123.9 lM). The IC50 values of Tempol derivatives T1,
T2, T3, T4, T6 were 7.8, 7.4, 9.6, 19.7, and 10.2 lM, respectively.
Since biological systems are complex, we made further evaluation of the
antioxidant properties of Tempol derivatives by hydrogen peroxide-induced cell
injury assay in HK-2 cells. As shown in Fig. 8, compared with negative control, the
cell viability of H2O2-injury group was significantly reduced. Compared with H2O2-
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Design, synthesis and biological evaluation of novel…
Fig. 7 Antioxidant activity of Tempol derivatives measured by DPPH radical scavenging assay. Each
bar value is presented as mean SD (n = 3)
Table 3 The IC50 of
antioxidant activity measured by
DPPH radical scavenging assay
Entry
Tempol derivatives
IC50 (lM)
1
2
3
4
5
6
7
T1
7.8 1.0**
7.4 0.5**
9.6 0.8**
19.7 3.4
T2
T3
T4
T5
123.9 24.3**
10.2 0.9**
21.5 2.4
Each value is presented as
mean SD (n = 3).
** P \ 0.01, compared with
Tempol
T6
Tempol
Fig. 8 The protective effect of Tempol derivatives T1–T6 for the injury of HK-2 cells induced by H2O2.
Each bar value represents the mean SD (n = 6). **P \ 0.01, compared with H2O2-injury group
injury group, Tempol and T1–T6 could significantly improve the cell viability, and
exhibited a dose dependent manner against the injury protection of HK-2 cells and
improved the cell viability to 90 % at 5 lg/mL. Like the antitumor activity, the
substituent on the benzene ring influences the antioxidant activity of Tempol
derivatives. Tempol derivatives T1 and T6 with a hydroxyl group at the C-3
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X.-L. Sun et al.
position of the benzene ring showed markedly increased antioxidant activity
compared with other compounds. This might be related to their quick reduction to
hydroxylamines in the presence of OH groups.
Conclusions
In summary, two series of novel Tempol derivatives were synthesized successfully,
their antitumor activity evaluated by the MTT assay, and their antioxidant activity
investigated using the DPPH radical scavenging assay and hydrogen peroxide-
induced cell injury assay, respectively. The antitumor results showed that all of the
Tempol derivatives exhibited more potent antitumor activity than Tempol and most
of the Tempol derivatives exhibited better antioxidant activity than Tempol. The
results showed that Tempol derivatives designed by introduction of phenolic acids
are promising lead compounds in antitumor chemotherapy. Among them, Tempol
derivatives T6 is the promising derivative and can be selected as effective antitumor
agent for further investigation.
Acknowledgments This work was supported by ‘‘Significant New Drug Creation and Manufacture
Program’’ of National Science & Technology (Grant No. 2014ZX09J14104-06C) and National Natural
Science Foundation of China (Grant No. 21272272) and ‘‘Excellent Young Talents Support Program’’ of
Fourth Military Medical University.
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