1032 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 7
Kirilyuk et al.
were measured in ethanol (Specord UV/VIS spectrophoto-
meter). 1H NMR spectra (δ, ppm) were recorded using 1.4%
solutions on a 200-MHz Varian Gemini 200 H/C spectrometer
(1b, 5a , and 6) or 600-MHz Bruker DMX 600 spectrometer
(2b). Reaction of 1a with cysteine was investigated by
recording 1H NMR spectra of the reaction mixture on a Varian
Gemini 200 H/C spectrometer in D2O at different reaction
times; starting reagent concentrations were 1a , 10 mM;
cysteine‚HCl, 40 mM; K2CO3, 30 mM.
chromatograph, equipped with a Supelcosil LC-18S column
filled with 50 mM phosphate buffer in methanol/water (3:1)
as eluent running at 1 mL/min. Absorption at 210 nm was
measured using a SPD-10A UV-vis detector to determine the
concentrations of the compounds. In this system, typical
retention times were 13 min (2a ), 10 min (2b), 7 min (1a ), 5
min (1b), and 3 min (N-acetylcysteamine). 2,4-Dinitrophenol
was added to the reaction mixtures as a standard to decrease
the injection error.
3,4-Dih yd r o-3-ch lor o-3,4,4-tr im eth yld ia zete 1,2-Diox-
id e (1b) a n d 2a ,3,4,5,6,6a -Hexa h yd r o-2a -ch lor o-6a -m eth -
ylben zod ia zete 1,2-Dioxid e (2b). A solution of 45 mL of
sodium hypochlorite (Merck, Germany) containing 12% active
chlorine (0.07 mol) was saturated with a mixture of sodium
dihydrogen phosphate and disodium hydrogen phosphate to
pH 8 and cooled to a temperature of -4 to -8 °C. A solution
of 12 mmol of 2-(hydroxyamino)-2-methylbutan-3-one oxime
(5a) acetate or 2-(hydroxyamino)-2-methylcyclohexanone oxime
(5b) acetate and 600 mg of KHCO3 in 20 mL of water was
added dropwise within 30 min under vigorous stirring main-
taining the temperature of the reaction mixture below -3 °C.
Reaction products were extracted with chloroform (3 × 30 mL);
the extract was dried with MgSO4. The solvent was removed
under vacuum, and the residue was purified by column
chromatography (silica gel, CHCl3). 1b: yield 20%, mp 140-
142 °C dec (from ethanol); IR 1560 (i, NdN); UV (ethanol, nm)
267 (ꢀ ) 9000); 1H NMR (CDCl3) 1.75 (3H, s), 1.85 (3H, s) (gem.
CH3), 2.30 (3H, s, CBr-CH3). Anal. (C5H9N2O2Cl) C, H, N;
Cl: calcd, 21.5; found, 22.8. 2b: yield 7%, mp 127-129 °C
dec (from ethanol); IR 1560 (i, NdN); UV (ethanol, nm) 265 (ꢀ
) 8200); 1H NMR (CDCl3) 1.73 (3H, s, CH3), 1.61 (2H, m,
5-CH2), 1.77 (2H, m, 4-CH2), 1.90 (1H, m), 2.25 (1H, m) (6-
CH2), 2.27 (1H, m), 2.67 (1H, m) (3-CH2). Anal. (C7H11N2O2-
Cl).
R ea ct ion of 3-Br om o-3,4-d ih yd r o-3,4,4-t r im et h yl-
d ia zete 1,2-Dioxid e (1a ) w ith Cystein e. A. A suspension
of 1a (105 mg/0.5 mmol) in 20 mL of a solution containing 300
mg of sodium dihydrogen phosphate and 245 mg (2 mmol) of
cysteine was stirred at room temperature overnight; the
resulting solution was filtered to remove cystine precipitate
and extracted with ethyl acetate. The extract was dried with
sodium sulfate. After the solvent was removed, a solid residue
(60 mg) was obtained; recrystallization from ethyl acetate gave
2-(hydroxyamino)-2-methylbutan-3-one oxime (5a ): yield 80%,
mp 100-104 °C (no mp depression was observed for mixed
probe with the sample obtained according to literature data);14a
1H NMR ((CD3)2SO) 1.17 (6H, s), 1.73 (3H, s), 5.50 (1H, s, br),
6.95 (1H, s), 10.30 (1H, s). The sample synthesized according
to the literature14a had an identical spectrum.
Nitr ic Oxid e Mea su r em en ts. NO formation was moni-
tored by ozone-mediated chemiluminescence (270 B NO chemi-
luminescence analyzer, Sievers, Boulder, CO). This method
is based on the oxidation of NO released into the gas phase
by ozone. For continuous measurements (Figures 6 and 7)
Corning flasks (25 cm2) with or without BAEC, containing 2
mL of D-PBS (with Ca2+, Mg2+), were placed into a Boekel
shaker-incubator at 37 °C; the headspace gas was forced by
vacuum directly into the reaction chamber. The measure-
ments of NO release from BAEC were performed after the cells
were washed five times with the buffer. To measure NO
release under anaerobic conditions (Figure 4), a DD solution
in D-PBS was placed into the purge vessel under constant He
flow, the thiol solution was injected through a septum, and
signal detector readings were registered at the plateau level.
In several experiments, up to 10-7 M CuSO4, Fe2(SO4)3, or 0.1
mM neocuproine (Sigma) was added. Alternatively, NO
production was measured under the same conditions by the
oxyhemoglobin method16 using a Beckman DU 640 spectro-
photometer. Concentration of oxyhemoglobin was 5 µM, and
absorbance was registered at 401 nm. A differential extinction
coefficient of 39 mM-1 cm-1 was used for calculations. In
separate experiments, the concentration of oxyhemoglobin was
increased up to 8 µM or SOD (40 U/mL) was added.
Cell Cu ltiva tion a n d Toxicity Stu d ies. Cells of the
BAEC line BKEz-7,17 passages 12-22, were cultivated in
Corning flasks (25 cm2) as previously described.18 The cyto-
toxicity of DD 1a and 2a ,b was tested in a concentration range
of 0.05-6 mM. After 24 h of incubation with DD, percentages
of viable cells were determined using the neutral red viability
test.19
Stu d ies of Va sor ela xa tion P r op er ties of 2a . Diazetine
dioxide 2a was tested regarding its vasorelaxant action on rat
mesenterial vessels in a concentration range from 2 × 10-8 to
1.5 × 10-4 M in an accumulative mode.20 Briefly, ring
segments of mesenteric small arteries (diameter, <300 µm;
length, 2 mm) were dissected from male MOL-Wistar rats
(Møllegaard, Denmark) and mounted in a dual myograph for
small vessels (J .P. Trading, Denmark). The vessels were
equilibrated for 30 min at 37 °C with incubation medium
(gassed with 5% CO2/95% O2, pH 7.4) containing (mM): NaCl,
119; KCl, 4.7; KH2PO4, 1.18; MgSO4, 1.17; NaHCO3, 25; CaCl2,
2.5; EDTA, 0.026; glucose, 5.5. The arteries were precon-
tracted with 10 µM PGF2R prior to addition of DD. In separate
experiments, glutathione (0.5 mM) was added to the incubation
medium.
B. Solid cysteine was occasionally added by portions (0.5-1
mg/h) during 120 h to a stirred suspension of 1a (105 mg, 0.5
mmol) in an aqueous solution of Na2HPO4 (300 mg in 10 mL).
The reaction mixture was treated as described above, and the
residue obtained after extract evaporation was separated by
chromatography on a preconditioned TLC plate (Kieselgel 60,
Merck, Germany), eluent chloroform/methanol (10:1). The
products were washed out with methanol yielding 40 mg of
5a (60%, identified as described above) and 12 mg (20%) of 6:
Ack n ow led gm en t. This work was supported by
BMBF (WTZ X224.5 and BEO 0310015B), Russian
Foundation of Basic Research (96-03-33269 and 95-04-
12506a), DFG (SFB 507), and Grant 2.95.30 of the
Gottlieb Daimler- und Karl Benz-Stiftung. The authors
would like to thank Dr. R. Winter and B. Schlegel for
their collaboration in the NMR measurements and J .
Eichhorst for her assistance in the vasorelaxation
experiments.
1
mp 84-87 °C; H NMR (CDCl3) 1.38 (6H, s), 1.91 (3H, s) (1H
NMR data and melting point are in agreement with literature
data15); 13C NMR (CDCl3) 10.5, 28.5, 73.3, 162.7.
Sp in -Tr a p p in g Exp er im en ts. EPR spectra were recorded
using a Bruker ECS 106 spectrometer. A solution of gluta-
thione (2 mM), 2a (1 mM), and DMPO (200 mM) was placed
into a flat quartz cell; EPR spectra were recorded after 5 and
10 min. Instrument settings were modulation frequency, 100
kHz; modulation amplitude, 0.1 mT; field set, 347.5 mT; scan
range, 10 mT; microwave power, 10 mW. SOD (Boehringer
Mannheim, 50 U/mL) and catalase (Sigma, 1 kU/mL) were
added to prevent formation of the hydroxyl radical adduct.
Spectrum simulation was performed using the EPR simulation
program, version 1.4 (Bruker, Germany).
Refer en ces
(1) (a) Ullman, E. F.; Singh, P. 3,3,4,4-Tetramethyl-1,2-diazetine
1,2-dioxide, a useful low-energy triplet quencher. J . Am. Chem.
Soc. 1972, 94, 5077-5078. (b) Volodarsky, L. B.; Tikhonova, L.
A. Formation of furoxanes and 1,2-diazetine-1,2-dioxides upon
R-hydroxylaminooximes oxidation. Khim. Geterotsykl. Soedin.
1975, 6, 748-752 (in Russian).
Rea ction Kin etic Mea su r em en ts w ith HP LC. Reaction
mixtures were analyzed by HPLC using a Shimadzu LC-10A