Synthesis, physical properties, and cytotoxicity of Nitroxyl-Aziridine hybrid

Article abstract of DOI:10.1002/hlca.201000076

Nitroxyl-aziridine hybrid, a candidate for a magnetic resonance imaging (MRI) probe and an anticancer drug, was synthesized by aziridine formation reaction of nitroxyl-introduced aldehyde and guanidinium ylide in good diastereoselectivity. The relative co

Full text of DOI:10.1002/hlca.201000076

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Synthesis, Physical Properties, and Cytotoxicity of Nitroxyl – Aziridine Hybrid
by Takuya Kumamotoa), Kazuya Suzukia), Sang-kook Kimb), Katsuyoshi Hoshinob),
Masahiro Takahashic), Hiromi Satod), Hiroki Iwatad), Koichi Uenod), Masahiro Fukuzumia), and
Tsutomu Ishikawa*a)
a) Department of Medicinal Organic Chemistry, Graduate School of Pharmaceutical Sciences, Chiba
University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan
(phone/fax: þ 81-43-290-2910; e-mail: benti@p.chiba-u.ac.jp)
b) Graduate School of Advanced and Integrated Science, Chiba University, 1-33 Yayoi-cho, Inage-ku,
Chiba 263-8522, Japan
c) Graduate School of Applied Chemistry and Biotechnology, Chiba University, 1-33 Yayoi-cho,
Inage-ku, Chiba 263-8522, Japan
d) Department of Geriatric Pharmacology and Therapeutics, Graduate School of Pharmaceutical
Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8875, Japan
Nitroxyl – aziridine hybrid, a candidate for a magnetic resonance imaging (MRI) probe and an
anticancer drug, was synthesized by aziridine formation reaction of nitroxyl-introduced aldehyde and
guanidinium ylide in good diastereoselectivity. The relative configuration at aziridine C(2)ꢀC(3) bond
of the major diastereoisomer was determined to be cis by X-ray crystallographic analysis. Application of
chiral guanidinium ylide resulted in the formation of the corresponding optically active aziridine in 84%
ee. Reversible one-electron redox potential and quantitative spin yield of the hybrid were observed in
cyclic voltammogram and electron spin resonance, respectively. However, cytotoxicity of the hybrid
against cancer cell lines used was not observed.
Introduction. – Nitroxyl compounds such as TEMPO (¼2,2,6,6-tetramethylpiper-
idin-1-yloxyl; 1) and PROXYL (¼2,2,5,5-tetramethylpyrrolidin-1-yloxyl; 2) (Fig. 1,a)
are well-known stable radical species and widely used for synthetic and medicinal
purpose, e.g., as spin labels [1], spin traps [2], oxidizing catalysts/reagents [3],
antioxidants [4], and magnetic resonance imaging (MRI) contrast agents [5]. Because
selective accumulation of 1 in hamster and mice melanotic melanomas [6] was
observed, design, synthesis, and cytotoxicities were examined for the hybrids of nitroxyl
and antimelanomic drugs such as nitrosourea, so-called spin-labeled N-ethylnitroso-
ureas (SLENU) [7]1). Recent successful application of SLENU as noninvasive real-
time MRI probes based on high permeability of nitroxyl compounds for blood – brain
barrier [9] implies the importance of the series of these compounds. On the other hand,
aziridines play an important role in cytotoxicity of anticancer drugs such as thiotepa
[10] and mitomycins [11]. Thus, a nitroxyl – aziridine hybrid is a very interesting
candidate for not only a spin-labeled compound but also an anti-cancer drug. We have
developed a new synthetic method for 3-aryl (or 3-alkenyl)-aziridine-2-carboxylates 3
from aromatic (or olefinic) aldehydes and guanidinium salt 4 in the presence of a base
1
)
For an example of synthesis of spin-labeled analogs of antitumor podophyllotoxin glycoside, see [8].
ꢀ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

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such as NaH and N,N,N’,N’-tetramethylguanidine (TMG) [12]. Optically active 3 can
be effectively obtained, when chiral guanidinium salt 5 instead of achiral 4 is used
(Fig. 1,b). We herein report synthesis, physical properties, and cytotoxicity of
nitroxyl – aziridine hybrid.
Fig. 1. Structures of a) stable nitroxyls 1 and 2, b) aziridine 3 and guanidinium salts 4 and 5
Results and Discussion. – 3-Formyl-2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrol-1-
yloxyl (6) was selected as a nitroxyl-transferring olefinic aldehyde, and it was
synthesized according to the procedure reported in [13] (Scheme): treatment of
commercially available 2,2,6,6-tetramethylpiperidin-4-one (7) with Br2 in AcOH gave
dibromo derivative 8 as a mixture of cis- and trans-diastereoisomers. Favorskii-type
reaction of 8 in the presence of N,O-dimethylhydroxylamine gave the Weinreb amide 9.
After oxidation of the secondary amine part of 9 with H2O2 in the presence of tungsten
(W) catalyst to nitroxyl 10, treatment of which with diisobutylaluminum hydride
(DIBAL) afforded the key olefinic aldehyde 6. Reaction of 6 and guanidinium salt 4 in
the presence of a base gave the desired aziridine (ꢁ)-11. Yield and diastereoselectivity
Scheme. Synthesis of Nitroxyl-Aziridine Hybrid 11

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of (ꢁ)-11 were dependent on the base and the solvent used: NaH in DMF gave 80%
yield and an 8 :1 ratio of the diastereoisomers; TMG in THF led to 33% yield and a
3.5 :1 ratio. A more polar diastereoisomer was obtained as major isomer in each case.
The relative configuration at the C(2)ꢀC(3) bond of the major aziridine was
determined to be cis by X-ray crystallographic analysis2), as expected from the results
of other olefinic aldehydes such as cyclohexenecarbaldehyde [12c] (Scheme).
Application of chiral guanidinium salt (S,S)-5 gave the corresponding optically active
(ꢀ)-cis-11 in 84% ee with chemical yield and diastereoselectivity similar to those with
achiral 4 when NaH was used as a base. The absolute configuration of (ꢀ)-cis-11 was
tentatively determined as (2R,3R) based on former results with benzaldehyde [12a].
Cyclic voltammograms (CV) of the nitroxyl – aziridine hybrid (ꢁ)-cis-11 showed
one-electron redox process for the nitroxyl/N-oxoammonium couple similar to those of
other nitroxyls 6 and 10 (Fig. 2,a). The peak separation (DEp ¼ 0.065 – 0.070 V)
estimated from the oxidation (Epa) and the reduction (Epc) peak currents confirmed the
reversible one-electron redox process of these nitroxyls3)4). The durability of the
nitroxyl (ꢁ)-cis-11 against electrochemical redox cycling was also tested. The resulting
eight-time repetitive voltammograms displayed reproducible patterns (Fig. 2,b),
indicating a good durable nature of the redox couple. Electron spin resonance
(ESR) spectrum for the nitroxyl (ꢁ)-cis-11 in benzene solution at room temperature
exhibited triplet resonance in j Dms j¼ 1 region (Fig. 3). The g value of 2.0059 and
nitrogen hyperfine splitting (aN ¼ 1.4 mT) of (ꢁ)-cis-11 are typical of nitroxyl
Fig. 2. a) CV of 6 (dot), 10 (dash), and (ꢁ)-cis-11 (line) measured in MeCN at a Pt disk electrode;
b) redox cycling of (ꢁ)-cis-11
2
)
)
CCDC-720905 contains the supplementary crystallographic data for this article. These data can be
obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
Epa and Epc values (V vs. SCE) were estimated as 0.940 and 0.870 for 6, 0.879 and 0.814 for 10, and
0.830 and 0.763 for (ꢁ)-cis-11, respectively. Calculated E1/2 (¼(Epa þ Epc)/2) and DEp (¼Epa ꢀ Epc)
values were 0.905 and 0.070 for 6, 0.847 and 0.065 for 10, and 0.797 and 0.067 for (ꢁ)-cis-11,
respectively.
3
4
)
For recent example of experimental and theoretical studies of the redox potentials of cyclic
nitroxyls, see [14].

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Fig. 3. ESR (X-Band, 9.4560 GHz) Spectrum of (ꢁ)-cis-11 in benzene (0.1 mm) at room temperature
radical5). The spin yield for (ꢁ)-cis-11 was estimated to be 98.2% by integration of the
ESR signal. The quantitative spin yield means the radical is robust even at room
temperature and in air.
Cytotoxicities of the nitroxyl-aziridine hybrid (ꢁ)-cis-11, as well as of aldehyde 6
and amide 10 were examined; however, none of these compounds exhibited the
growth-inhibitory effect against cancer cell lines used (MCF-7, HeLa, and Caki-1).
Conclusions. – Nitroxyl – aziridine hybrid 11 was synthesized via aziridine
formation reaction of nitroxyl-introduced olefinic aldehyde 6 and guanidinium salts 4
and 5. The relative configuration of the major diastereoisomer of aziridine 11 was
determined to be cis. Existence of stable radical in this hybrid was confirmed with CV
and ESR; however, no inhibitory effect for cancer cell proliferation was observed.
Trials for application of the hybrid as MRI probe and as chiral catalyst of
enantioselective oxidation reaction are now in progress6).
Experimental Part
General. Anh. DMF and THF were used as purchased from Kanto Chemical and Wako Chemical,
resp. Column chromatography (CC): Kanto Chemical silica gel 60 spherical. TLC: Merck Art 5715 DC-
Fertigplatten Kieselgel 60 F254. M.p.: Yanagimoto MPSI melting point apparatus; uncorrected. IR
Spectra: Attenuated Total Reflectance (ATR) system on a JASCO FT/IR-300E spectrophotometer, ˜n in
cmꢀ1. 1H-NMR (400 MHz) Spectra: in CDCl3 on a JEOL JNM-ECP-400; chemical shifts in d [ppm] rel.
to Me4Si as internal reference; coupling constants J in Hz. MS: JEOL JNM MS-GCMATE for EI-MS,
and JEOL JNM HX-110 for HR-FAB-MS, resp.
CV of nitroxyls 6, 10, and (ꢁ)-cis-11 (2 mm) was carried out in MeCN (Kanto Chemical,
spectroscopic grade) soln. containing 0.1m tetrabutylammonium perchlorate (Tokyo Kasei, > 98%)
under N2 using a BAS model ALS 750A electrochemical analyzer. A BAS working Pt disk electrode (area
0.021 cm2) and a BAS counter Pt plate were immersed in the main compartment of a two-compartment
5
)
The g value of 2.00594 and nitrogen hyperfine splitting (aN ¼ 1.45 mT) of 12 (Scheme 1) was
reported in [15].
For examples of the application of chiral nitroxyl derivatives to asymmetric oxidation of racemic
alcohols and amines, see [16].
6
)

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cell. In the auxiliary compartment, which is separated from the main one by a sintered glass frit, was
immersed a KCl agar bridge connected to a TOA reference saturated calomel electrode (SCE). CW X-
Band ESR spectrum for (ꢁ)-cis-11 in benzene in 5-mm o.d. ESR quartz tubes was acquired on a JEOL
JES-TE200 spectrometer with 100-kHz field modulation. Spectra were obtained using single mode
cavity, with the oscillating magnetic field perpendicular (TE102) to the swept magnetic field. The g value
was referenced using MnO dispersed in MnO2. Spin concentration of each sample was determined both
by careful integration of the ESR signal standardized with that of 4-hydroxy-2,2,6,6-tetramethylpiper-
idin-1-yloxyl (TEMPOL).
(3-{1-Benzyl-3-[(tert-butoxy)carbonyl]aziridin-2-yl}-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-
yl)oxidanyl (11). Under Ar, DMF (1.5 ml) was added to a mixture of NaH (60%, 75 mg, 1.87 mmol), 6
(150 mg, 0.89 mmol), and 4 (461 mg, 1.16 mmol) at ꢀ 208, and the whole mixture was stirred at the same
temp. for 5 min. MeCN (40 ml) and SiO2 (12 g) were added, and the whole mixture was stirred at r.t. for
12 h, then filtered off. The precipitate was washed with MeCN. The filtrate and the washings were
combined and evaporated in vacuo. The residue was purified by CC (SiO2; hexane/AcOEt 10 :1) to give
(ꢁ)-cis-11 (235 mg, 71%) and (ꢁ)-trans-11 (39 mg, 9%).
Data of (ꢁ)-cis-11. Yellow needles (recryst. from hexane/CH2Cl2). M.p. 110 – 1118. Rf (hexane/
AcOEt 2 :1) 0.49. IR: 1729. 1H-NMR 1.40 (br. s, 9 H); 2.27 (br. s, 1 H); 3.74 (br. s, 1 H); 4.00 (br. s, 1 H);
7.22 – 7.41 (m, 5 H); signals derived from H-atoms on the dihydro-tetramethylpyrrole part and of C(2) of
the aziridine part were not detected due to paramagnetism of cis- and trans-11. EI-MS: 371 (15, Mþ), 341
(19), 285 (31), 270 (62), 242 (76), 91 (100). Anal. calc. for C22H31N2O3: C 71.13, H 8.41, N 7.54; found: C
71.11, H 8.62, N 7.55.
Data of (ꢁ)-trans-11. Colorless oil. Rf (hexane/AcOEt 2 :1) 0.60. IR: 1735. 1H-NMR 1.41 (br. s, 9 H);
2.05 (br. s, 1 H); 4.18 (br. s, 2 H); 6.90 – 7.60 (m, 5 H). HR-FAB-MS 371.2348 ([M þ H]þ, C22H31N2O3þ ;
calc. 371.2335).
(3-{(2R,3R)-1-Benzyl-3-[(tert-butoxy)carbonyl]aziridin-2-yl}-2,2,5,5-tetramethyl-2,5-dihydro-1H-
pyrrol-1-yl)oxidanyl ((ꢀ)-cis-11). Under Ar, DMF (0.3 ml) was added to a mixture of NaH (60%, 7 mg,
0.18 mmol), 6 (22 mg, 0.13 mmol), and (S,S)-5 (81 mg, 0.15 mmol) at ꢀ 208, and the whole mixture was
stirred at the same temp. for 1 h and at r.t. for 15 min. MeCN (4 ml) and SiO2 (0.4 g) were added, and the
whole mixture was stirred at r.t. for 24 h, then filtered off. The precipitate was washed with MeCN. The
filtrate and the washings were combined and evaporated in vacuo. The residue was purified by CC (SiO2;
hexane/AcOEt 3 :1) to give (ꢀ)-cis-11 (38 mg, 78%) and trans-11 (3 mg, 7%).
Data of (ꢀ)-cis-11. Yellow oil. [a]2D3 ¼ ꢀ35.9 (c ¼ 0.7, CHCl3). Chiral HPLC: (DAICEL CHIR-
ALCEL OD-H; hexane/iPrOH 100 :1; flow rate, 1.0 ml/min; detection, 254 nm): 12.3 (92%) and 15.1
(8%) min.
Ms. Yumi Nakai, Analytical Instruments Division, JEOL Ltd., is acknowledged for recording the
ESR spectrum and helpful discussions. We also thank Special Funds for Education and Research
(Development of SPECT Probes for Pharmaceutical Innovation) from the Ministry of Education,
Culture, Sports, Science and Technology, Japan.
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Received February 24, 2010