THE SYNTHESIS OF
O
ꢀSUBSTITUTED 3ꢀOXIMES
265
of side labeled compounds were formed. Hence, the (415 mg, 1.09 mmol) in pyridine (5 ml) was stirred at
synthesis of isomers of the labeled steroid ( ) was 60 for 6 h. The solvent was removed in a vacuum,
achieved by the selective hydrogenation of 1,4ꢀbisdeꢀ and the residue was coevaporated with toluene (
hydrosteroid (
) to ([1,2ꢀ3H]IV) by a modified proceꢀ 30 ml). Ethyl acetate and water were added to the
dure [6] with the subsequent interaction of the resultꢀ resulting residue, the water phase was extracted with
ing labeled steroid with ꢀ(3ꢀcarboxypropyl)hydroxyꢀ ethyl acetate, and the combined organic layers were
lamine. After chromatographic purification and washed with 5% HCl and water and dried over Na2SO4
separation, the individual isomers (Zꢀ[1,2ꢀ3H]
) and A mixture of isomeric 3ꢀ ꢀ(3ꢀcarboxypropyl)oximes
Eꢀ[1,2ꢀ3H]
) were finally isolated in 43 and 36% II) was dissolved in methanol (5 ml) and kept for 0.5 h
yields, respectively. The molar radioactivities of both at 20 with 10 ml of an ethereal solution of diazoꢀ
labeled preparations were equal (50 Ci/mmol). The methane (from 460 mg of nitrosomethylurea and
treatment of acid (Zꢀ[1,2ꢀ3H]
) with diazomethane 0.92 ml of 45% KOH). The solvents were evaporated
yielded the individually labeled 3ꢀ
bonyl)propyl]oxime ( ꢀVI).
V
°С
3
×
I
O
.
V
O
(
V
(
°C
V
Oꢀ[3ꢀ(methoxycarꢀ in a vacuum, and the residue was chromatographed on
Z
a column filled with Kieselgel 60 (0.063–0.100 mm)
(Merck). Elution with a petroleum ether–acetone
mixture of 99 : 1 to 96 : 4 gave individual 3ꢀ
(methoxycarbonyl)propyl]oximes: (EꢀIII), mp 73–
75 (hexane) (229 mg, 42%) and (ZꢀIII), mp 101–
103
(215 mg, 40%). The 1Нꢀ and 13C NMR spectra
of these compounds are given in the table.
Oꢀ[3ꢀ
EXPERIMENTAL
°
C
One and twoꢀdimensional NMR spectra ( , ppm)
δ
°C
were registered in CDCl3 at 30°C on a Bruker
DRXꢀ500 spectrometer (Germany) with working freꢀ
quencies of 500.13 and 125.76 MHz for protons and
13C nuclei, respectively. The signals of residual CHCl3
3
Z
ꢀ(3ꢀCarboxypropoxyimino)ꢀ ꢀmethylꢀ16
cyclohexanopregnaꢀ1,4ꢀdienꢀ20ꢀone ꢀII). Ester
III) (70 mg, 0.14 mmol) and NaOH (110 mg,
6
α
α
,17
α
ꢀ
(
Z
1
(7.27 ppm) for the H NMR spectra and CDCl3
(Zꢀ
(77 ppm) for the 13C NMR spectra were used as referꢀ
ences. When measuring twoꢀdimensional spectra, the
standard Bruker programs were applied. The spectra
were decoded using the COSY, HSQC, and HMBC
procedures. The configuration of oximes was deterꢀ
mined by the use of 2DꢀNOESY spectroscopy.
2.75 mmol) in methanol (6 ml) were kept for 20 h at
room temperature, and ether (20 ml) and 10% HCl
were added. The organic layer was washed with water,
dried over Na2SO4, and the solvent was removed in a
vacuum. The residue was chromatographed on a colꢀ
umn filled with Kieselgel 60. Elution with a 78 : 20 : 2
petroleum ether–acetone–methanol system led to the
Tris(triphenylphosphine)rhodium chloride (Fluka)
was used as a catalyst. Radioactivity was measured on an
LKB1215 scintillation counter with the efficiency of triꢀ
tium registration near 30% in a dioxane scintillator. A
MultiChrom 1.5 system (ZAO Ampersend, Russia)
was used for the collection and processing of chroꢀ
matographic data. Plates coated with SorbfilꢀUV silica
gel (Russia) were used for TLC. The optimal condiꢀ
tions for carrying out the reactions were found by the
use of a 1% tritium–protium mixture according to the
procedures in [8–12]. The stability and distribution of
the label were tested by the method in [8].
isolation of (ZꢀII), yield 56 mg, mp 223–225
°C
(ether–hexane).
3
Z
ꢀ(3ꢀMethoxycarbonylpropoxyimino)ꢀ
,17
ꢀcyclohexanoꢀ[1,2ꢀ3H]pregnꢀ4ꢀenꢀ20ꢀone
Zꢀ[1,2ꢀ3H]VI
. ( ) A solution of 1,4ꢀbisdehydroprecurꢀ
6
α
ꢀmethylꢀ
16
(
α
α
) a
sor (ZꢀIII) (2.2 mg, 0.0044 mmol) and tris(tripheꢀ
nylphosphine)rhodium chloride (2.2 mg, 0.0024 mmol)
in benzene (0.3 ml) was placed in an ampoule, frozen
in liquid nitrogen, evacuated, filled with a protium–
tritium (1 : 1 ratio) mixture up to a pressure of 400 hPa,
and kept under stirring for 19 h at room temperature.
The ampoule content was frozen again in liquid nitroꢀ
gen and evacuated. The labile tritium was removed by
the threeꢀtime evaporation of the reaction mixture
with methanol (5 : 1). Analysis was carried out by
HPLC (Fig. 1).
Labeled preparations were analyzed and purified
by HPLC on a Reprosil pur C18aq, 5
column at a flow rate of 1 ml/min in a system of
methanol–5 mM H3PO4 9 : 1. Retention times (min):
µ
m,
4 150ꢀmm
×
6.08 for
ZꢀIII), 10.86 for
preparative purification was carried out on a Kromasil
100C18, 7 m, 4.6 150ꢀmm column, flow rate
(
IV), 6.75 for
(ZꢀII), 7.53 for (ZꢀV), 9.83 for
(
(ZꢀVI), and 12.21 min for (EꢀVI). The
To remove the soluble catalyst, the reaction mixꢀ
ture was applied to a SorbfilꢀUV plate, which was
developed with a 7 : 3 hexane–acetone system. The
zone containing the target labeled substance was cut
out, and the reaction products were eluted with methꢀ
µ
×
1 ml/min; systems: (A) 1 : 1 methanol–10 mM
H3PO4, (B) methanol, 50% B for 5 min, then a linear
gradient from 50 to 80% B for 10 min; retention time
(min): 8.41 (ZꢀIII), 9.52 (ZꢀVI), 5.49 (
ZꢀII), and 6.19
anol (
rated. The analysis and the preparative purification of
ꢀ(3ꢀMethoxycarbonylpropoxyimino)ꢀ the preparation were carried out by HPLC. The radioꢀ
,17 ꢀcyclohexanopregnaꢀ1,4ꢀdieneꢀ
chemical purity of (Zꢀ[1,2ꢀ3H]VI) was 95–97% after
20ꢀones (ЕꢀIII) and ZꢀIII). A mixture of ꢀ(3ꢀcarboxyꢀ the chromatography; yield 30–35%; molar radioactivꢀ
propyl)hydroxylamine hydrochloride [13] (304 mg, ity, 25 Ci/mmol. The labeled steroid (VI) was stored as
1.95 mmol) and 1,4ꢀdidehydrosteroid ( ) [12] a solution in methanol at 10–15
5
×
10 ml). The eluates were filtered and evapoꢀ
(Zꢀ
V).
3
E
ꢀ
and
ꢀmethylꢀ16
3Z
α α
6
α
O
I
°С.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36
No. 2
2010