Synthesis, characterisation, cytotoxicity and radioprotective effect of novel chiral nitronyl nitroxyl radicals

Article abstract of DOI:10.3184/030823409X12474221035163

Nitroxyl compounds have been previously investigated as potential radioprotection drugs. To develop new radioprotectors, two kinds of novel chiral nitronyl nitroxyl radicals: Z-tert-butyl 2-(4, 5-dihydro-4, 4, 5, 5-tetramethyl- 3-oxido-1H-imidazol-3-ium-1

Full text of DOI:10.3184/030823409X12474221035163

JOURNAL OF CHEMICAL RESEARCH 2009
AUGUST, 511–514
RESEARCH PAPER 511
Synthesis, characterisation, cytotoxicity and radioprotective effect of
novel chiral nitronyl nitroxyl radicals
Xiang-Yang Qina, Gui-Rong Dingb, Xiao-Wu Wangc, Juan Tanb, Guo-Zhen Guob and Xiao-Li Suna
aDepartment of Chemistry, bDepartment of Radiation Medicine, Fourth Military Medical University, Xi'an, Shanxi 710032, P.R. China
Nitroxyl compounds have been previously investigated as potential radioprotection drugs. To develop new
radioprotectors, two kinds of novel chiral nitronyl nitroxyl radicals: L-tert-butyl 2-(4, 5-dihydro-4, 4, 5, 5-tetramethyl-
3-oxido-1H-imidazol-3-ium-1-oxyl-2-yl) pyrrolidine-1-carboxylate (L-NNP) and L-tert-butyl 2-[(4-(4, 5–dihydro-4,4,5,5–
tetramethyl–3–oxido-1H-imidazol-3-ium-1-oxyl-2-yl)-2-methoxyphenoxy)methyl] pyrrolidine-1-carboxylate (L-NNVP)
have been synthesised. The cytotoxic and radioprotective effects of these two compounds were then evaluated in
rat glioma C6 cells.
Keywords: chiral nitronyl nitroxyl radicals, cytotoxicity, radioprotective effect
In recent years, stable nitronyl nitroxyl radicals, have been
extensively studied as a unique and interesting class of
antioxidants, for protection against oxidative damage.1-3
Cellular and in vivo pharmacological studies of stable
nitroxyls proved that they can be reduced to hydroxylamines
or oxidised to oxo-ammonium cations by electron-transfer
reactions.3,4 The redox transformations among the oxidation
states of nitroxyl, hydroxylamine, and the oxoammonium
cation are shown in Fig. 1.5,6 Unlike other antioxidants which
protect against radiation damage through stoichiometric
reactions, nitroxyls can participate in redox reactions and
self-replenish, thereby giving a catalytic protective activity.7
At the same time, nitroxyls readily cross the blood–brain
barrier and permeate the cell membrane.8 These key
features indicate the potential of nitroxyls against radiation-
induced injury.
O
N
R
Oxoammonium Cation
e
N
e
e
O
N
+
+
O
R
2e
+
N
O
O
e
N
R
Nitroxyl Radical
Hydroxylamine
N
OH
Fig. 1 The redox transformation of the various oxidation
So far, Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-
N-oxyl) has been shown to be one of the most promising
radiation protectors.9-12 In 2004, a phase II study of Tempol
was initiated.13 However, the results have not yet been
reported.
states of nitroxyls.
O
N
R
R
N
O
Stable nitroxyls include two types: TEMPO (2,2,6,6-
tetramethylpiperidine-N-oxyl) and NIT (nitronyl nitroxyl)
(Fig.2).14 At present, NIT radicals for radioprotective effects
have not been well characterised. The structure of nitroxyls
has an importance influence on their radioprotective
activity.5,15 In addition, biological molecules can discriminate
between enantiomers.16 To search for more effective radiation
protectors with minimal cytotoxicity, two kinds of new chiral
NIT nitroxyl compounds (L-NNP and L-NNVP), containing
pyrrolidine moieties originating from natural L-proline, have
been synthesised (Fig.3). Their cytotoxicity and protective
effects were evaluated by MTT17 (3-[4, 5-dimethylthiazol-2-
yl]-2, 5-diphenpyltetrazolium bromide) assay in C6 cells.
N
O
TEMPO radical
NIT radical
Fig. 2 Two types of nitroxyls.
O
O
N
N
N
O
N
N
O
O
N
O
Boc
Boc
Results and discussion
L-NNVP
L-NNP
According to Ullman's pioneering work,18 any aldehyde may
give rise to a nitronyl nitroxyl. Therefore, the first step is to
obtain chiral aldehydes. As shown in Scheme 1, the precursor
of L-NNP, aldehyde (1) [L-tert-butyl 2-formylpyrrolidine-1-
carboxylate] was obtained by rapid oxidation of L-N-Boc-
prolinol under mild conditions with trichloroisocyanuric
acid in the presence of catalytic TEMPO.19 The precursor
of L-NNVP, aldehyde (2) {L-tert-butyl 2-[(4-formyl-2-
methoxyphenoxy) methyl] pyrrolidine-1-carboxylate} was
obtained by an intermolecular dehydration reaction between
L-N-Boc- prolinol and vanillin.20-22 To purify aldehyde (2),
the side-products triphenylphosphine oxide and diethyl
Fig. 3 The structure of chiral nitroxyls (L-NNP and L-NNVP).
hydrazinedicarboxylate were precipitated by ether and
then filtered off. The filtrate was evaporated under reduced
pressure and the crude product was purified by chromatography
on silica.
The chiral aldehydes as described above were subsequently
subjected to condensation with 2,3-bis(hydroxylamino)-
2,3-dimethylbutane to generate the corresponding N, N'-
dihydroxyimidazolidines as stable white solids from methanol
solution at room temperature. The target compounds, L-NNP
and L-NNVP, were obtained after NaIO4 was used to
oxidise N, N'-dihydroxyimidazolidines at 0°C as shown in
* Correspondent. E-mail: qinxiangyang@fmu.edu.cn

512 JOURNAL OF CHEMICAL RESEARCH 2009
Cl
O
N
O
O
Cl
N
N Cl ,TEMPO(0.01 mol equiv)
CHO
OH
N
N
CH2Cl2, r.t.
Boc
Boc
(1)
CHO
CHO
PPh3/DEAD
THF, r.t.
OH
+
HO
O
N
N
O
O
Boc
Boc
(2)
Scheme 1
L-NNP
OH
N
NHOH
NHOH
NaIO4, 0 0C
CH2Cl2
R
H
CH3OH
r.t.
RCHO
+
N
OH
L-NNVP
Scheme 2
Scheme 2. One of the key steps in the synthesis of nitronyl
nitroxyl radicals was the oxidation of the N, N'-dihydroxy-
imidazolidines. While PbO2 was usually used as an oxidant,
it is not only time-consuming but also it is difficult to observe
the progress of the reaction due to the colour of PbO2.
However, when NaIO4 was used as an oxidant, observation
became much easier despite the overoxidation product
obtained. Thus, we used aqueous NaIO4 as the best choice
of oxidant to give the corresponding chiral nitronyl nitroxyl
radicals, provided that the amount of NaIO4 and the time of the
oxidation reaction was strictly controlled and the reaction was
carried out in an immiscible solvent such as dichloromethane
in which the product was soluble.
ESR spectroscopy is the best tool for the study of free
radicals, due to its sensitivity and accuracy. As shown in
Fig. 4, the ESR spectra of the two types of radicals show five
major lines in the ratio 1:2:3:2:1, which is expected for
coupling of two identical nitrogens.
The UV-vis absorption spectra of the two compounds
L-NNP and L-NNVP were determined in dichloromethane at
room temperature. They presented absorption in the UV at
320 nm and 360 nm respectively, arising from π–π* transitions
of the ONCNO units.23
The IR spectra of L-NNP and L-NNVP are noteworthy in
that the characteristic band was observed at 1372 nm and
1358 nm, arising from the N–O stretching frequency.24
Experimental
1H NMR and 13C NMR spectra were recorded on a Bruker AV-300
NMR spectrometer (400 MHz) (Bruker, Karlsruhe, Germany), with
CDCl3 as solvent and TMS as internal reference. Optical rotation
values were measured at 20°C on a PerkinElmer 343 polarimeter
(PerkinElmer Bodenseewerk, Überlingen, Germany). High
resolution mass spectroscopy (HRMS) was carried out on a Varian
7.0T ESI-FTICR-MS (Varian. USA) instrument. IR was recorded on
a Bruker tensor27 (Bruker optics, German) instrument. Absorption
spectra were recorded on a Jasco V-570 UV/vis/NIR (Jasco,
Japan) spectrophotometer. The EPR spectra were obtained using a
Bruker ESP 300E spectrometer on solutions of radicals dissolved
in dichloromethane. Elemental analyses were carried out using a
Perkin–Elmer analyser model 240C (PerkinElmer, America).
2,3-bis(hydroxylamino)-2,3-dimethylbutane was prepared by the
published method.25 N-Boc-L-prolinol was prepared in our laboratory.
All other chemical reagents were purchased from the Nanjing Tianzun
Zezhong Chemical Limited Company (Nanjing, China). THF was
distilled under N2 from Na/benzophenone and CH2Cl2 was distilled
from CaH2. The other chemical reagents were used without further
purification.
L-NNVP
L-NNP
Fig.4 ESR spectra of nitroxyls in dichloromethane.

JOURNAL OF CHEMICAL RESEARCH 2009 513
Synthesis of L -tert-butyl 2-formylpyrrolidine-1-carboxylate
To mixture of N-Boc-L-prolinol (1.9 g, 9.5 mmol) and
68.7, 60.0, 55.9, 46.9, 28.4, 23.6, 22.6. Anal. Calcd for C18H25NO5: C,
64.46; H, 7.51; N, 4.18. Found: C, 64.29; H, 7.37; N, 4.05%.
a
trichloroisocyanuric acid (2.3 g, 10 mmol), CH2Cl2 (20 mL) was
added, and the mixture was stirred and maintained at 0°C for
5 min, followed by addition of TEMPO (0.015 g, 0.1 mmol).
Then the mixture was warmed to room temperature and stirred for
20 min and filtered on Celite. The precipitate was washed with
CH2Cl2 (10 mL ¥ 2). The combined organic phases were washed
with 15 mL of a saturated solution of Na2CO3 and then by 0.1 N HCl
(20 mL) and brine (20 mL). The organic phase was dried over
Na2SO4, and stripped of solvent. The crude product was purified by
column chromatography on silica gel using ethyl acetate/petroleum
ether (1:10) as eluant, giving the product as a colourless liquid
(1.68 g, 88.8%). MS(m/z): 222.11 (MNa+); [a]D20 = –102.3 (c = 1.1,
CH2Cl2); IR(KBr)cm-1: 1396, 1363, 2977, 1478 (n CH3), 1165, 1123,
912, 858, 772 (n C–C ring of pyrrolidine), 2711, 2810, 1736 (n
CHO), 1255, 1696 (n ester bond and amide bond); 1H NMR(CDCl3):
d 9.47 (d, 1H, J = 2.7 Hz, –CHO), 4.21 (m, 1H, –CH), 3.47 (t, 2H,
–CH2), 2.00 (m, 2H, –CH2), 1.87 (m, 2H, –CH2), 1.42 (s, 9H, –CH3);
13C NMR d(CDCl3): 200.4, 155.8, 80.6, 65.0, 46.7, 28.2, 26.7, 23.9;
Anal. Calcd for C10H17NO3: C, 60.28; H, 8.60; N, 7.03. Found: C,
60.17; H, 8.66; N, 6.89%.
Synthesis of L–NNP; General procedure for synthesis of nitronyl
nitroxyl radicals
A solution of L-tert-butyl 2-formylpyrrolidine-1-carboxylate (0.5 g,
2.5 mmol) and 2,3-bis (hydroxylamino)-2,3-dimethylbutane (0.37 g,
2.5 mmol) in methanol (20 mL) was stirred at room temperature for
6 h (reaction periods of 24 h were used for aldehyde (2)). After the
reaction was completed, the methanol was removed and the residue
was suspended in 40 mL dichlormethane, aqueous NaIO4 (0.53 g,
2.5 mmol in 20 mL) was added dropwise over a period of 5 min
at 0°C, and the mixture was stirred for a further 2 min at 0°C. The
organic layer was dried over anhydrous Na2SO4. The deep red solution
was then evaporated. The crude product was purified by column
chromatography on silica gel using absolute ether/petroleum/acetone
(3:1:0.5) as eluent, giving a deep red solid product (445.3 mg,
54.6%); MS(m/z): 349.19 (MNa+); IR(KBr)cm-1: 1372, 1345, 1156
(n NO), 1396, 1372, 2882, 2979 (n CH3), 1696, 1282, 1251 (n ester
bond and amide bond), 1112, 915, 858, 776 (n C–C ring of pyrrolidine);
CH2Cl2
UV(lꢀmax ): 333 (ONCNO, π→π*, e = 1.6 ¥ 104 mol-1·cm-1), 530,
571 (n→π*). EPR (CH2Cl2): g factor, 2.0032; aN (2N), 7.57 G. Anal.
Calcd for C16H28N3O4: C, 58.87; H, 8.65; N, 12.87. Found: C, 58.68;
H, 8.51; N, 12.72%. As anticipated, nitronyl nitroxyl radicals showed
no NMR signal.
Synthesis of L-tert-butyl 2-((4-formyl-2-methoxyphenoxy) methyl)
pyrrolidine-1-carboxylate
Triphenyl phosphine (1.5 g, 6 mmol), vanillin (0.9 g, 6 mmol) and
N-Boc-L-prolinol (1.33 g, 6.6 mmol) were dissolved in dry THF
(45 mL) with vigorous stirring under an atmosphere of nitrogen
at 0°C. A solution of diethyl azodicarboxylate (DEAD) (1 g,
6 mmol) in dry THF (10 mL) was added dropwise over a period
of 1 h at 0°C. The mixture was warmed to room temperature and
stirred for 12 h. THF was removed under reduced pressure. Ether
was added to the residue to precipitate triphenylphosphine oxide
and diethyl hydrazinedicarboxylate, which were then filtered off.
The filtrate was evaporated. The crude product was purified by
column chromatography on silica gel using hexane/acetone (5:1)
as eluant, giving a colourless oil product (1.66 g, 82.3%). MS(m/z):
L-NNVP
Dark blue oil product; MS (m/z): 485.24 (MNa+); IR(KBr)cm-1:1358,
1125, 1168 (n NO), 1391, 2976, 2876 (n CH3), 1232, 1026, 909
(n C–C ring of pyrrolidine), 1692, 1327, 1267 (n ester bond and amide
bond), 1599, 1491, 1530, 1455, 867, 810 (n benzene ring); UV-vis
CH2Cl2
(lꢀmax ): 289 (benzene ring, π→π*), 342 (e = 4.4 ¥ 103 mol-1·cm-1),
368 (ONCNO, π→π*), 560—630 (n→π*). EPR (CH2Cl2): g factor,
2.0032; aN (2N), 7.68 G. Anal. Calcd for C24H36N3O6: C, 62.32; H,
7.84; N, 9.08. Found: C, 62.19; H, 7.92; N, 9.11%.
Cytotoxicity study
20
358.16(MNa+); [a]D = –67.2 (c = 1.1, CH2Cl2); IR(KBr)cm-1:
Rat glioma C6 cells were cultured at 37°C under a humidified
atmosphere of 5% CO2 in DMEM (Dulbecco's Modified Eagle
Media) medium supplemented with 10% fetal serum. To study the
cytotoxicity of the compounds, cells were dispersed and seeded in
96-well plates with 1 ¥ 103 cells/well overnight. Two compounds
with different concentrations were then added. PBS (phosphate buffer
solution) was used as control. Four replicates were used for each
concentration. After 72 h exposure to the compounds, cell viability
was determined by the MTT assay.
1396, 1366, 2974, 2878 (n CH3), 1167, 1136, 1024, 910 (n C–C ring
of pyrrolidine), 1688, 2833, 2723 (n CHO), 1688, 1342, 1310, 1270
(n ester bond and amide bond), 1587, 1510, 866, 811, 731 (n benzene
ring); 1H NMR(CDCl3): d 9.80 (s, 1H, –CHO), 7.37 (d, 1H, J = 3.0 Hz,
–ArH), 7.28 (s, 1H, –ArH), 7.04 (d, 1H, J = 6.0 Hz, – ArH), 4.27 (d,
2H, –CH2), 4.04 (m, 1H, –CH), 3.87(s, 3H, –CH3), 3.38 (t, 2H, –CH2),
2.06 (m, 2H, –CH2), 1.83 (m, 2H, –CH2), 1.43 (s, 9H, –CH3); 13C NMR
d(CDCl3): 190.98, 154.8, 153.9, 149.8, 129.9, 126.5, 111.8, 109.0, 79.9,
Fig. 5 The cell morphology.The typical photographs were taken at 48 h after 12 Gy radiation with or without L-NNVP (15.63 mg/ml)
or L-NNP(125 mg/ml) pretreatment. Bar: 40mm.
Table 1 Radioprotective effects of L-NNP and L-NNVP at the different radiation dosages(n=4)
–
Compd
L-NNVP
L-NNP
PBS
Drug concentration/mg·mL-1
OD values (c SD)
8Gy
10Gy
12Gy
125
62.5
31.25
15.63
125
62.5
31.25
15.63
0.47 0.17
0.54 0.09
0.56 0.005a
0.58 0.11a
0.49 0.06
0.57 0.07
0.55 0.06
0.74 0.07a
0.48 0.04
0.74 0.04
0.70 0.04
0.89 0.08a
0.75 0.07
0.69 0.17
0.77 0.02a
0.84 0.13a
0.72 0.07
0.64 0.08
0.69 0.05a
0.71 0.09a
0.70 0.06a
0.68 0.05a
0.64 0.02a
0.61 0.09
0.59 0.07
0.61 0.08
0.55 0.08
a(P<0.05), PBS was used as control (n = 8).

514 JOURNAL OF CHEMICAL RESEARCH 2009
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4
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It was found that L-NNP and L-NNVP at concentration less than 125
mg mL-1 and 250 mg mL-1 respectively had no effect on cell viability.
Radioprotective effects study
To study the radioprotective effects of L-NNP and L-NNVP, cells
were dispersed and seeded in 96-well plates with 1 ¥ 103 cells/well
overnight. Fifteen minutes prior to irradiation, two compounds at four
different concentrations (according to the results from compound
cytotoxicity) were added to the medium (final concentrations were
15.625, 31.25, 62.5 and 125 mg mL-1). The cells were irradiated with
60Co g-rays for 8, 10 and 12 Gy respectively. The dose rate used
was about 4.64Gy min-1. Cell viability was determined by the MTT
assay. PBS was used as control. Four replicates were used for each
concentration.
Treatment of cells with L-NNP and L-NNVP before irradiation at
low concentrations (125, 62.5, 31.25 and 15.625mg mL-1) effectively
decreased g-rays irradiation-induced cell death. With lower
concentrations (31.25 and 15.625mg mL-1), L-NNVP and L-NNP
exhibited better radioprotective effects for lower dose irradiation
(8 and 10 Gy) whereas with higher concentrations (125, 62.5mg mL-1),
L-NNP and L-NNVP exhibited better radioprotective effects for
higher dose irradiation (12 Gy). The experimental results are listed
in Table 1.
At 24 h and 48 h after irradiation at the different radiation dosages,
the cell morphology was observed under an inverted microscope.
The micrograph after 48 h at 12 Gy radiation dosages is shown in
Fig. 5. As shown in morphology results, in the PBS group, only a few
cells survived after radiation. However, the survival rate obviously
increased when cells were treated with L-NNVP or L-NNP before
irradiation (Fig. 5). These results suggest that the nitroxyl compounds
L-NNVP or L-NNP provide radioprotective effects in C6 cells.
7
8
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This work was supported by the National Science Foundation
of China (No.20672141; No. 30670492).
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Received 9 May 2009; accepted 14 June 2009
Paper 09/10573 doi: 10.3184/030823409X12474221035163
Published online: 10 August 2009
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