Synthesis of cytotoxic 6E-hydroximino-4-ene steroids: Structure/activity studies

Article abstract of DOI:10.1021/jm010867n

In an effort to determine the pharmaceutical utility and the structural requirements for activity against various tumor cell lines, several 6E-hydroximino-4-ene steroids with different side chains and degrees of unsaturation on ring A were synthesized in our laboratory. Evaluation of the synthesized compounds for cytotoxicity against P-388, A-549, HT-29, and MEL-28 tumor cells revealed that some important structural features are required for activity. The presence of a cholesterol-type side chain, which appears to play a major role in determining the biological activity, the existence of a ketone functionality at C-3, and an elevated degree of oxidation on ring A all result in higher bioctivity than other structural motifs.

Full text of DOI:10.1021/jm010867n

2612
J . Med. Chem. 2001, 44, 2612-2618
Syn th esis of Cytotoxic 6E-Hyd r oxim in o-4-en e Ster oid s: Str u ctu r e/Activity
Stu d ies
Noe´ Deive, J aime Rodr´ıguez,* and Carlos J ime´nez
Departamento de Quı´mica Fundamental, Facultad de Ciencias, Universidad de A Corun˜a, Campus da Zapateira,
15071 A Corun˜a, Spain
Received March 2, 2001
In an effort to determine the pharmaceutical utility and the structural requirements for activity
against various tumor cell lines, several 6E-hydroximino-4-ene steroids with different side
chains and degrees of unsaturation on ring A were synthesized in our laboratory. Evaluation
of the synthesized compounds for cytotoxicity against P-388, A-549, HT-29, and MEL-28 tumor
cells revealed that some important structural features are required for activity. The presence
of a cholesterol-type side chain, which appears to play a major role in determining the biological
activity, the existence of a ketone functionality at C-3, and an elevated degree of oxidation on
ring A all result in higher bioactivity than other structural motifs.
In tr od u ction
Natural products derived from marine organisms
have become an important source of biologically active
compounds. Especially abundant are those systems with
a steroidal skeleton, which are usually isolated in large
quantities from marine sponges.1 Despite the enormous
number of steroids found to have unusual and intriguing
structures,2 marine steroids with an oxime group have
F igu r e 1. Natural 6E-Hydroximino-3-oxo-4-ene steroids from
the sponges C. apion and C. alloclada.
not been reported in the literature to date, with com-
pounds 1 and 2 representing the first examples known
(see Figure 1). These 6E-hydroximino-3-oxo-4-ene ste-
roids were discovered during the chemical study of two
morphospecies of the sponges Cinachyrella alloclada
and Cynachirella apion, and their structures were
deduced by extensive use of 1D- and 2D-NMR studies.3
Our interest in this type of compound was sparked
by the selective cytotoxic activity shown by 1 (see Figure
1) (IC50 of 1.25 µg/mL against P-388, A-549, HT-29, and
2.5 µg/mL toward MEL-28 tumor cells) and the lack of
activity of 2 (IC50 >10 µg/mL).3 Furthermore, this group
of steroids was reported to show a high affinity for
human placental aromatase and to function as a com-
petitive inhibitor of this enzyme.4
available key precursors 3â-acetoxycholest-4-en-6-one
(6a ), (24R)-3â-acetoxy-24-ethylcholest-4-en-6-one (6b),
and 3â-acetoxygorgost-4-en-3-one (6c), obtained from
cholesterol, â-sitosterol, and gorgosterol,5 respectively.
This synthetic route was undertaken using the following
standard steps: acetylation followed by epoxidation of
the starting materials and subsequent oxidation of
compounds 4a -c6 to give the corresponding 3-acetoxy-
5-hydroxy-6-one steroids 5a -c. The elimination of the
hydroxyl group at C-5 with thionyl chloride in pyridine
afforded compounds 6a -c, which, after removal of the
acetate with alcoholic KOH and upon treatment with
hydroxylamine hydrochloride followed by allylic oxida-
tion with aqueous CrO3, provided the 6E-hydroximino-
4-en-3-oxo analogues.7,8
Due to the lack of activity found in compounds 2 and
8 (see IC50 values in Table 1), we decided to proceed by
investigating the influence of the absence of a side chain
(Scheme 2). Wolff-Kishner reduction of (+)-dehydroiso-
androsterone (9) led to the formation of 5-androsten-
3â-ol (10), which was subsequently converted into oxime
11 by the application of the methodology already
discussed for Scheme 1.
To explore the structure/activity relationships of this
type of compound as cytotoxic agents, the naturally
occurring steroids 1 and 2 were prepared along with
some derivatives with different structural features (side
chains and differing degrees of unsaturation on ring A).
Resu lts a n d Discu ssion
Ch em istr y. The initial studies for the synthesis of
this oxime-steroid system were based on the methodol-
ogy developed by Holland.4 Using as a model the active
natural oxime 1, we first investigated the variation of
the side chain and observed the effect of these changes
on the cytotoxicity. In this respect we prepared several
systems, namely cholesterol-like, sitosterol-like, and
gorgosterol-like side chains. As depicted in Scheme 1,
the synthesis of these compounds began from the readily
Compound 11 still showed a mild cytotoxicity (IC50
) 5 µg/mL against all the tumor cells tested), and so
we decided to prepare compounds with different degrees
of oxidation at C-17 with the aim of inducing a higher
or selective cytotoxic effect. In this way, analogue 14a
was prepared by protection of the keto group at C-17 of
9 as the ketal to give compound 12,9 which was
subjected to the synthetic strategy outlined in Scheme
1. Thus, after epoxidation and oxidation (steps a-c in
* Corresponding author. Tel: +34 981 167000 (2173). Fax: +34 981
167065. E-mail: jaimer@udc.es.
10.1021/jm010867n CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/10/2001

Synthesis of Cytotoxic 6E-Hydroximino-4-ene Steroids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16 2613
Sch em e 1a
a
Reagents and conditions: (a) Ac2O, Py (99%); (b) MCPBA, CH2Cl2 (97%); (c) CrO3, H2O (90%); (d) SOCl2, Py (92%); (e) KOH, MeOH
(82%); (f) NH2OH.HCl, EtOH, H2O (97%); (g) CrO3, H2O (70%).
Sch em e 2a
Ta ble 1. In Vitro Antitumor Activities (IC50 in µg/mL) of 1 and
Its Analogues
compd
P-388
A-549
HT-29
MEL-28
1
2
1.25
10
1.25
10
1.25
10
2.5
10
7a
10
10
10
10
7c
10
10
10
10
8
5
5
5
5
1.25
1.25
0.25
0.25
0.5
5
5
5
5
5
5
5
5
5
1.25
1.25
0.25
0.25
0.25
5
5
5
5
5
a
11
14a
14b
20a
20b
21a
21b
22
24
Reagents and conditions: (a) DEG, NH2NH2‚H2O, K2CO3
(66%); (b) Scheme 1 (30%).
Sch em e 3a
1.25
1.25
0.125
0.125
0.125
5
1.25
1.25
0.125
0.125
0.125
5
Scheme 1), deprotection of the ketal occurred. The
presence of two keto groups, at C-6 and C-17, meant
that the synthesis to obtain the oxime at position 6 was
regioselective because an oxime at C-17 was not de-
tected. The synthesis of the 17â-ol analogue 14b was
also undertaken,4 thus completing the series of different
compounds lacking a side chain in the steroidal skeleton
(Scheme 3).
a
Reagents and conditions: (a) HOCH2CH2OH, adipic acid, Ph
Me (60%); (b) steps a-b as in Scheme 1 (96%); (c) CrO3, H2O (90%);
(d) steps d-g as in Scheme 1 (51%).
When the cytotoxic tests of 14a and 14b were per-
formed, we found that there was no increase in the
activity (see IC50 values in Table 1) in comparison to
compound 1. For this reason, the next step in this
structure/activity relationship study involved a different
approach consisting of the preparation of a number of
6E-hydroximino-4-ene steroids with different degrees of
oxidation in ring A. At this point, and with the IC50
values of the previously synthesized analogues in mind,
we decided to keep the cholesterol-type side chain in
order to maintain a good level of cytotoxicity.
Cholesterol (3a ) was thus converted into 16 by tosy-
lation followed by epoxidation, oxidation, and dehydro-
tosylation.10 Subsequent osmium-catalyzed dihydroxy-
lation of the ∆2 double bond gave, after chromatographic
purification, diols 2R,3R,5R- and 2â,3â,5R-trihydroxy-
cholestan-6-ones 17 and 18, respectively (Scheme 4).
Protection of the hydroxyl groups at C-2 and C-3 as
acetates allowed us to follow the same sequence of
reactions to obtain the ∆4 double bond and the oxime
group at C-6. Deprotection of the acetates with KOH/
EtOH, followed by an allylic oxidation with aqueous
CrO3, afforded the desired 6E-hydroximino-2-hydroxy-
4-en-3-oxo epimers 21a and 21b in good yields. Surpris-
ingly, the use of MnO2 as the oxidizing agent with 20b
afforded the 6E-hydroximino-2-hydroxy-3-oxo-1,4-diene
22. However, the diasteroisomer 20a under the same
conditions yielded the expected oxidation product at the
allylic position (21a ), but compound 22 was not detected
at all. The proton coupling constants of compounds 17-
21 allowed the unequivocal assignment of the configu-
rations at C-2 and C-3. At present, compounds 21a , 21b,
and 22 have proven to be the most potent cytotoxic 6E-
hydroxymino-4-ene steroids found in this work.
A final analogue, compound 24, was synthesized (see
Scheme 5) from 16 in order to study the effect on the
cytotoxicity of a 2,4-diene conjugated with the oxime at
C-6. Thus, elimination of the hydroxyl group at C-5 was
performed with LiBr in DMF under reflux, and the
resulting ketone, 23, was converted into 24 by the
standard procedure.
Biologica l Activities. All these novel 6E-hydrox-
imino-4-ene steroids were studied in vitro on P-388,

2614 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16
Deive et al.
Sch em e 4a
a
Reagents and conditions: (a) TsCl, Py (98%); (b) MCPBA, CH2Cl2 (97%); (c) CrO3, H2O (90%); (d) LiBr, DMF, reflux (93%); (e) OsO4,
NMO, THF, t-BuOH, H2O, (65%); (f) Ac2O, Py (70%); (g) SOCl2, Py (90%); (h) KOH, MeOH (90%); (i) NH2OH‚HCl, EtOH, H2O (95%); (j)
CrO3, H2O (70%); (k) MnO2, CHCl3 (50%).
Sch em e 5a
side chains, respectively) and in compounds with no side
chain at C-17 (11, 14a , and 14b presented IC50 values
of 5 µg/mL).
Additional relevant evidence can be deduced by
considering the overall skeleton in the derivatives with
a ketone at C-3, which apparently plays a key role in
increasing the activity given that the change from a keto
to a hydroxyl group at this position results in loss of
the activity by a factor of ∼10 (see the IC50 values of
pairs 1/7a , 20a /21a , and 20b/21b). Besides, compound
22, which has a higher level of oxidation on ring A,
shows a very promising level of selective cytotoxicity
because the IC50 value for the cell lines A-549 and MEL-
28 for compound 22 was 0.125 µg/mL, while the values
for the remaining cell lines HT-29 and P-388 are higher
by 100% (0.25 µg/mL) and 400% (0.5 µg/mL), respec-
tively.
a
Reagents and conditions: (a) LiBr, DMF, reflux (90%); (b)
NH2OH‚HCl, EtOH, H2O (94%).
A-549, HT-29, and MEL-28 tumor cells. The results,
expressed as IC50 values in µg/mL, are reported in Table
1. Compounds 8, 11, 14a , 14b, and 24 presented
moderate activity (5 µg/mL) in comparison to that of the
“parent” natural compound 1 (IC50 1.25 µg/mL for all
the cell lines, 2 showed a very mild activity). However,
the cytotoxicity data for analogues 21a , 21b , and 22
showed an increase in the cytotoxic activity, and these
compounds were 10 times more potent in comparison
to 1, while compounds 20a and 20b offered similar IC50
values to that of 1.
Con clu sion s
In summary, we have described the chemical synthe-
sis of a new series of 6E-hydroximino-4-ene steroids and
have found some important features needed for cyto-
toxicity. As outlined in Figure 2, these first structure/
cytotoxicity investigations have demonstrated that one
important feature is the presence of a cholesterol-type
side chain, which appears to play a major role in
The importance of the presence of a cholesterol-like
side chain (compounds 1, 20a , 20b, 21a , 21b, and 22)
is illustrated by the lack of significant cytotoxicity in
compounds 2 and 8 (sitosterol-like and gorgosterol-like
F igu r e 2. Pattern of nonselective cytotoxicity of 6E-hydroximino-4-ene steroids. a: Higher oxidation degree implies higher
cytoxicity. b: Required side chain for cytoxicity.

Synthesis of Cytotoxic 6E-Hydroximino-4-ene Steroids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16 2615
at 0 °C. A solution of m-chloroperbenzoic acid (0.58 g, 3.36
mmol) in 20 mL of CHCl3 was added dropwise to the reaction
mixture, and the solution was stirred for 20 h. Na2SO3 (5%,
100 mL) was added to the mixture with cooling (ice/water
bath), and the mixture was kept at this temperature for 6 h.
The final aqueous phase was extracted twice with CHCl3 (20
mL) and, after removal of the solvent, a mixture of 3â-acetoxy-
5R,6R-epoxycholestane (4a ) and 3â-acetoxy-5â,6â-epoxycholes-
tane was obtained in a 2:1 ratio. 4a : 1H NMR (200 MHz,
CDCl3) δH: 4.95 (H-3, 1H, m); (2.90, H-6, 1H, d, J ) 4.4 Hz);
2.03 (OAc, 3H, s); 1.08 (H-19, 3H, s); 0.92 (H-21, 3H, d, J )
5.4 Hz); 0.86 (H-26, H-27, 6H, d, J ) 5.9 Hz); 0.64 (H-18, 3H,
s). 13C NMR (50 MHz, CDCl3) δC: 170.4 (OAc, s); 71.5 (C-3,
d); 59.2 (C-6, d). EIMS (70 eV, m/z %): 444 (M, 2); 384 (M -
CH3COOH, 42); 366 (M - CH3COOH - H2O, 44).
3â-Acetoxy-5r-h yd r oxych olesta n -6-on e (5a ). A solution
of chromium trioxide (0.40 g, 4.0 mmol) in 1.2 mL of water
was added dropwise to a solution of a mixture 2:1 of 3â-
acetoxy-5R,6R-epoxycholestane (4a ) and 3â-acetoxy-5â,6â-ep-
oxycholestane (0.50 g, 1.12 mmol) in 20 mL of methyl ethyl
ketone at 0 °C. This addition was repeated at room temper-
ature, and the solution was stirred for 20 min. The reaction
mixture was poured into water (90 mL), and the resulting
precipitate was filtered off under reduced pressure, washed
with water, and subjected to chromatography (hexane/ethyl
acetate, 7:3) to give 5a (0.47 g, 90%): 1H NMR (200 MHz,
CDCl3) δH: 5.04 (H-3, 1H, m); 2.75 (H-4â, 1H, t, J ) 12.2 Hz);
2.02 (OAc, 3H, s); 1.05 (H-19, 3H, s); 0.94 (H-21, 3H, d, J )
5.9 Hz); 0.86 (H-26, H-27, 6H, d, J ) 6.4 Hz); 0.64 (H-18, 3H,
s). 13C NMR (50 MHz, CDCl3) δC: 210.0 (C-6, s); 171.1 (OAc,
s); 81.3 (C-5, s); 71.7 (C-3, d). EIMS (70 eV, m/z %): 460 (M,
2); 400 (M - CH3COOH, 34); 382 (M - CH3COOH - H2O, 3).
3â-Acetoxych olest-4-en -6-on e (6a ). Thionyl chloride (0.36
mL, 4.9 mmol) was added dropwise to a solution of 3â-acetoxy-
5R-hydroxycholestan-5-one (5a , 0.46 g, 1.0 mmol) in 15 mL of
dry pyridine at 0 °C. The mixture was stirred for 45 min and
then poured into water (80 mL). The resulting precipitate was
extracted with ethyl acetate (2 × 30 mL), and the combined
extracts were washed (10% HCl followed by brine), dried, and
evaporated under reduced pressure. The residue was subjected
to chromatography (hexane/ethyl acetate, 8:2) to give 3â-
acetoxycholest-4-en-6-one (6a , 0.41 g, 92%). 1H NMR (200
MHz, CDCl3) δH: 6.07 (H-4, 1H, d, J ) 2 Hz); 5.34 (H-3, 1H,
m); 2.56 (H-7â, 1H, dd, J ) 0.2 and 11.7 Hz); 2.07 (OAc, 3H,
s); 1.02 (H-19, 3H, s); 0.92 (H-21, 3H, d, J ) 6.4 Hz); 0.87 (H-
26, H-27, 6H, d, J ) 6.8 Hz); 0.70 (H-18, 3H, s). 13C NMR (50
MHz, CDCl3) δC: 203.0 (C-6, s); 171.8 (OAc, s); 148.0 (C-5, s);
128.6 (C-4, d). EIMS (70 eV, m/z %): 442 (M, 36); 400 (M -
CH3COH, 100).
6E-Hydr oxim in och olest-4-en -3â-ol (7a). 3â-acetoxycholest-
4-en-6-one (6a , 0.40 g, 0.90 mmol) was dissolved in a 10%
methanolic potassium hydroxide solution (20 mL) under argon.
The mixture was stirred at room temperature for 1 h, and then
the solvent was evaporated under reduced pressure to a
volume of 10 mL. This solution was poured into ice/water (50
g), and the product was extracted with ethyl acetate (2 × 20
mL). The combined extracts were washed with saturated brine,
dried, and evaporated under reduced pressure. The residue
was subjected to chromatography (hexane/ethyl acetate, 7:3)
to give 3â-hydroxycholest-4-en-6-one (0.32 g, 82%).
determining the biological activity. The existence of a
ketone functionality at C-3 and an elevated degree of
oxidation on ring A result in higher bioactivity than
other structural motifs.
Compound 22 was selected for NCI’s 60 human tumor
cell line panel. The results showed IC50 values in a range
of 10-5 to 10-6 µM but did not produce any pattern of
differential cytotoxicity of sufficient interest to warrant
further study based upon the NCI screen.
Exp er im en ta l Section
Biologica l Ma ter ia ls. In h ibition of Cell Gr ow th by
Cou n tin g Cells. This form of assay employs 24-well multi-
dishes of 16 mm diameter.11 The tumor cell lines employed
are as follows: P-388 (ATCC CCL 46), suspension culture of
a lymphoid neoplasm from a DBA/2 mouse; A-549 (ATCC CCL
185), monolayer culture of a human lung carcinoma; HT-29
(ATCC HTB-38), monolayer culture of a human colon carci-
noma; and MEL-28 (ATCC HTB-72). Cells were maintained
in their logarithmic phase of growth in Eagle’s minimum
essential medium, with Earle’s balanced salts, with nones-
sential amino acids, with 2.0 mM L-glutamine, without sodium
bicarbonate (EMEM/neaa), supplemented with 5% fetal calf
serum (FCS), 10-2 M sodium bicarbonate and 0.1 g/L penicillin
G + 0.1 g/L streptomycin sulfate. For each experiment, the
cells were harvested from subconfluent cultures using trypsin
and resuspended in fresh medium before plating. P-388 cells
were seeded into wells of 16 mm diameter at 1 × 104 cells per
well in 1 mL aliquots of EMEM 5% FCS containing different
concentrations of the sample to be tested. A separate set of
cultures (without the drug) was seeded as a growth control to
ensure that cells remained in the exponential phase of growth.
All determinations were carried out in duplicate. After 3 days
of incubation at 37 °C and 5% CO2 in an atmosphere with 98%
humidity, an approximate value of IC50 was determined by
comparing the growth in wells with drug to the growth in the
control wells. A-549, HT-29, and MEL-28 cells were seeded
into wells of 16 mm diameter at 1 × 104 cells per well in 1 mL
aliquots of EMEM 5%FCS containing different concentrations
of the sample to be tested. A separate set of cultures (without
drug) was seeded as a growth control to ensure that cells
remained in the exponential phase of growth. All determina-
tions were carried out in duplicate. After 3 days of incubation
at 37 °C and 5% CO2 in an atmosphere of 98% humidity, the
cells were stained with 0.1% crystal violet. An approximate
IC50 value was determined by comparing the growth in wells
with drug to the growth in the control well.
To quantify the activity after completion of the incubation
time, the cells were trypsinized and counted in a Coulter
Counter ZM. All counts (net cells per well) represent the
average of duplicate wells. The percentage growth (% G) is
relative to cultures without drug. The results of these assays
were used to generate dose-response curves from which more
precise IC50 values were determined (sample concentration
that produces 50% cell growth inhibition).
Ch em istr y. Nuclear magnetic resonance spectra (proton
and carbon) were recorded on a Bruker AC 200F or AMX 500
spectrometer, using CDCl3 as the solvent and internal stan-
dard. Multiplicities of 13C signals were obtained by DEPT.
Microanalyses were performed on a Carlo Erba Instruments
CHNS-O 1108, and the results were within (0.4% (for C, H,
N) of the theoretical values. Medium-pressure chromato-
graphic separations were carried out on silica gel 60 (230-
400 mesh).
A solution of 3â-hydroxycholest-4-en-6-one (0.29 g, 0.65
mmol) in ethanol (15 mL) was treated with a solution of
hydroxylamine hydrochloride (0.34 g, 4.9 mmol) in 50% aque-
ous ethanol (4 mL) and trihydratated sodium acetate (0.40 g,
2.9 mmol) in 50% aqueous ethanol (11 mL). The resulting
mixture was stirred at room temperature for 24 h, and the
solvent was removed under reduced pressure. The residue was
diluted with water (20 mL) and extracted with ethyl acetate
(20 mL). The extract was dried and evaporated, and the
residue was subjected to chromatography (hexane/ethyl ace-
tate, 7:3) to give 6E-hydroximinocholest-4-en-3â-ol (7a , 0.29
g, 97%). 1H NMR (200 MHz, CDCl3) δH: 5.78 (H-4, 1H, s); 4.18
(H-3, 1H, m); 3.32 (H-7â, 1H, dd, J ) 4.4 Hz and 10.3 Hz);
0.98 (H-19, 3H, s); 0.92 (H-21, 3H, s); 0.87 (H-26, H-27, 6H, d,
3â-Acetoxy-5r,6r-ep oxych olesta n -6-on e (4a ). Choles-
terol (0.50 g, 1.29 mmol) and acetic anhydride/pyridine (1:1,
10 mL) were stirred at room temperature for 24 h. After
removal of the solvents, the residue was dissolved in 20 mL
of ethyl acetate and the solution was washed with NaHCO3
(20 mL) and 5% HCl (20 mL) and dried over anhydrous Na2-
SO4. The resulting organic phase was evaporated under
vacuum to give 3â-acetoxy-5-cholestene (0.52 g, 94%). This
compound (0.52 g, 1.19 mmol) was dissolved in 20 mL of CHCl3

2616 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16
Deive et al.
J ) 6.8 Hz); 0.68 (H-18, 3H, s). 13C NMR (50 MHz, CDCl3) δC:
158.0 (C-6, s); 146.6 (C-5, s); 122.5 (C-4, d); 67.5 (C-3, d). EIMS
(70 eV, m/z %): 415 (M, 4); 398 (M - OH, 27).
(C-5, s); 120.8 (C-6, d); 71.9 (C-3, d). EIMS (70 eV, m/z %):
274 (M, 21); 259 (M - CH3, 16).
6E-Hyd r oxim in oa n d r ost-4-en -3-on e (11). 1H NMR (200
MHz, CDCl3) δH: 6.50 (H-4, 1H, s); 3.45 (H-7â, 1H, dd, J )
0.8 and 11.8 Hz); 2.48 (H-2, 2H, m); 2.10 (H-1, 2H, m); 1.15
(H-19, 3H, s); 0.75 (H-18, 3H, s). 13C NMR (50 MHz, CDCl3)
δC: 201.3 (C-3, s); 162.2 (C-6, s); 155.5 (C-5, s); 122.5 (C-4, d);
54.7; 51.4; 40.7; 40.0; 38.8; 38.1; 34.8; 33.6; 33.0; 30.0; 25.3;
20.9; 20.4; 17.3 (C-19, q); 16.5 (C-18, q). EIMS (70 eV, m/z %):
301 (M +, 13); 284 (M - OH, 19); 270 (M - NOH, 25); 258 (M
- NOH - H2O, 78).
6E-Hyd r oxim in och olest-4-en -3-on e (1). A solution of 6E-
hydroximinocholest-4-en-3â-ol (7a , 0.28 g, 0.67 mmol) in 5 mL
of pyridine was added dropwise to the chromium trioxide/
pyridine complex prepared by the addition of CrO3 (0.49 g, 4.9
mmol) to 5 mL of pyridine at 0 °C. The reaction mixture was
stirred at room temperature for 1 h and then diluted with 10
mL of ethyl acetate. The resulting precipitate was filtered off,
and the filtrate was washed (10% HCl, 10% NaHCO3, brine),
dried with anhydrous Na2SO4, and evaporated under reduced
pressure. The residue was subjected to chromatography (hex-
ane/ethyl acetate, 7:3) to give 6E-hydroximinocholest-4-en-3-
3â-Acetoxy-17,17-eth ylen ed ioxoa n d r ost-5-en e (12). A
mixture of 3â-hydroxyandrost-5-en-17-one (9, 0.87 g, 3.0
mmol), adipic acid (0.23 g, 1.6 mmol), and ethylene glycol (9
mL, 161 mmol) was dissolved in 40 mL of benzene. A Liebig
condenser and a Dean Stark apparatus were fitted to the
reaction vessel, and the reaction temperature was raised at
100 °C. After 28 h, the mixture was washed with saturated
NaHCO3 and water and dried over Na2SO4. The mixture was
filtered and the solvent removed under reduced pressure to
give a white solid, which was purified by flash chromatography
(hexane:EtOAc, 8:2) to afford 3â-hydroxy-17,17-ethylenedioxo-
5-androstene (0.60 g, 60%). This compound was further
elaborated in a way to that for 3a -c (i.e., acetylation and
epoxidation).
3r-Acetoxy-5r-h yd r oxya n d r osten -6,17-d ion e (13). Spec-
tral data similar to those reported in the literature.12
6E-Hyd r oxim in oa n d r ost-4-en -3,17-d ion e (14a ). 1H NMR
(200 MHz, CDCl3) δH: 6.53 (H-4, 1H, s); 3.57 (H-7â, 1H, dd, J
) 4.4 and 11.2 Hz); 2.50 (H-2, 2H, m); 2.09 (H-1, 2H, m); 1.17
(H-19, 3H, s); 0.92 (H-18, 3H, s). 13C NMR (50 MHz, CDCl3)
δC: 220.0 (C-17, s); 201.1 (C-3, s); 161.7 (C-6, s); 154.7 (C-5,
s); 122.8 (C-4, d); 51.5; 51.1; 47.6; 38.7; 35.7; 34.7; 33.5; 32.3;
31.0; 28.4; 21.7; 20.2; 16.6 (C-19, q); 13.7 (C-18, q). EIMS (70
eV, m/z %): 315 (M, 16); 298 (M - OH, 30); 272 (M - NOH -
H2O, 83).
1
one (0.19 g, 70%). H NMR (200 MHz, CDCl3) δH: 9.71 (OH,
1H, br s); 6.54 (H-4, 1H, s); 3.43 (H-7â, 1H, dd, J ) 3.7 and
16.0 Hz); 2.54 (H-2, 2H, m); 2.04 (H-1, 2H, m); 1.19 (H-19, 3H,
s); 0.91 (H-21, 3H, d, J ) 6.4 Hz); 0.86 (H-26, H-27, 6H, d, J
) 6.8 Hz); 0.70 (H-18, 3H, s). 13C NMR (50 MHz, CDCl3) δC:
201.6 (C-3, s); 162.8 (C-5, s); 155.3 (C-6, s); 122.4 (C-4, d); 56.5
(C-14, s); 55.9 (C-17, d); 51.0 (C-9, d); 42.4 (C-13, s); 39.4 (C-
24, t); 39.3 (C-12, t); 38.6 (C-10, s); 36.0 (C-22, t); 35.6 (C-20,
d); 34.7 (C-1, t); 33.5 (C-2, t); 32.5 (C-8, d); 29.5 (C-7, t); 28.1
(C-16, t); 28.0 (C-25, q); 24.0 (C-15, t); 23.7 (C-23, t); 22.8 (C-
26, q); 22.5 (C-27, q); 20.8 (C-11, t); 18.6 (C-21, q); 16.5 (C-19,
q); 11.9 (C-18, q). EIMS (70 eV, m/z %): 413 (M, 21); 396 (M
- OH, 42); 370 (M - NOH - H2O, 100).
Compounds 2 and 8 were obtained in a similar way, and
for this reason, only experimental details for the synthesis of
compound 1 are reported.
6E-Hydr oxim in o-24-eth ylch olest-4-en -3-on e (2). 1H NMR
(200 MHz, CDCl3) δH: 10.43 (OH, 1H, br s); 6.43 (H-4, 1H, s);
3.42 (H-7â, 1H, dd, J ) 3.7 and 16.0 Hz); 2.53 (H-2, 2H, m);
2.05 (H-1, 2H, m); 1.13 (H-19, 3H, s); 0.93 (H-21, 3H, d, J )
6.3 Hz); 0.86 (H-29, 3H, t; d, J ) 6.6 Hz); 0.84/0.81 (H-26, H-27,
6H, d, J ) 6.6 Hz); 0.70 (H-18, 3H, s). 13C NMR (50 MHz,
CDCl3) δC: 201.0 (C-3, s); 162.3 (C-5, s); 156.0 (C-6, s); 122.6
(C-4, d); 56.6 (C-14, s); 55.9 (C-17, d); 51.2 (C-9, d); 46.1 (C-24,
t); 42.5 (C-13, s); 39.3 (C-12, t); 38.7 (C-10, s); 36.2 (C-20, d);
34.8 (C-1, t); 33.8 (C-22, t); 33.6 (C-2, t); 32.7 (C-8, d); 29.6
(C-7, t); 29.0 (C-25, q); 28.1 (C-16, t); 26.4 (C-23, t); 24.0 (C-
15, t); 23.1 (C-28, t); 20.8 (C-11, t); 19.0 (C-26, q); 19.6 (C-27,
q); 18.7 (C-21, q); 16.6 (C-19, q); 12.3 (C-29, q); 12.0 (C-18, q).
EIMS (70 eV, m/z %): 441 (M, 17); 425 (M - OH, 23); 399 (M
- NOH - H2O, 78).
17â-Hyd r oxy-6E-h ydr oxim in oa n d r ost-4-en -3-on e (14b).
1H NMR (200 MHz, CDCl3) δH: 6.38 (H-4, 1H, s); 3.68 (H-17,
1H, m); 3.43 (H-7â, 1H, dd, J ) 4.4 and 11.2 Hz); 2.48 (H-2,
2H, m); 2.13 (H-1, 2H, m); 1.16 (H-19, 3H, s); 0.80 (H-18, 3H,
s). 13C NMR (50 MHz, CDCl3) δC: 201.1 (C-3, s); 162.1 (C-6,
s); 155.4 (C-5, s); 122.7 (C-4, d); 81.5 (C-17, d); 51.3; 51.2; 42.9;
38.7; 36.1; 34.8; 33.6; 32.7; 30.4; 29.1; 23.2; 20.5; 16.6 (C-19,
q); 11.0 (C-18, q). EIMS (70 eV, m/z %): 317 (M, 15); 300 (M
- OH, 18).
6E-Hyd r oxim in ogor gost -4-en -3-on e (8). 1H NMR (200
Hz, CDCl3) δH: 6.36 (H-4, 1H, s); 3.43 (H-7â, 1H, dd, J ) 3.9
and 11.2 Hz); 2.48 (H-2, 2H, m); 2.07 (H-2, 2H); 1.14 (H-19,
3H, s); 0.94 (H-21, 3H, d, J ) 6.3 Hz); 0.88 (H-26, H-27, 6H, d,
J ) 6.8 Hz); 0.77 (H-30, 3H, d, J ) 6.3 Hz); 0.71 (H-18, 3H, s);
0.46 (H-28, 1H, m); 0.21 (H-22, 1H, m); -0.12 (H-28, 1H, m).
13C NMR (50 MHz, CDCl3) δC: 200.6 (C-3, s); 161.9 (C-6, s);
156.4 (C-5, s); 122.8 (C-4, d); 56.5; 55.8; 51.3; 42.5; 39.2; 39.0;
38.8; 36.1; 34.8; 33.7; 33.6; 32.8; 32.0; 31.4; 30.5; 29.6; 29.3;
28.0; 24.0; 20.8; 20.5 (C-26, q); 18.8 (C-21, q); 17.6 (C-29, q);
16.6 (C-19, q); 15.4; 11.9 (C-18, q). EIMS (70 eV, m/z %): 453
(M, 3); 410 (M - NOH - H2O, 13).
An d r ost-5-en -3â-ol (10). Hydrazine monohydrate (0.40 mL,
8.25 mmol) was added dropwise to a solution of dehydroisoan-
drosterone (9, 0.50 g, 1.73 mmol) and K2CO3 (0.20 g, 1.45
mmol) diluted in 15 mL of diethylene glycol. The resulting
mixture was heated to 125 °C and stirred for 5 h with a Liebig
condenser fitted to the reaction vessel. The excess hydrazine
monohydrate was distilled off, and the temperature was then
raised to 200 °C. The solution was stirred for 15 h at this
temperature. The reaction mixture was cooled to room tem-
perature and poured into water (100 mL). The resulting
precipitate was extracted with diethyl ether (3 × 40 mL), and
the combined extracts were washed with water, dried, and
evaporated under reduced pressure. The residue was subjected
to chromatography (hexane/ethyl acetate, 7:3) to give androst-
5-en-3â-ol (10, 0.32 g, 66%). 1H NMR (200 MHz, CDCl3) δH:
5.37 (H-6, 1H, d, J ) 6.0 Hz); 3.50 (H-3, 1H, m); 1.02 (H-19,
3H, s); 0.72 (H-18, 3H, s). 13C NMR (50 MHz, CDCl3) δC: 140.7
5r-Hyd r oxych olest-2-en -6-on e (16). A solution of choles-
terol (3a , 1.5 g, 3.9 mmol) in 20 mL of dry pyridine was treated
with p-toluenesulfonyl chloride, and the mixture was stirred
at room temperature for 22 h. The reaction mixture was poured
into 5% aqueous NaHCO3 (100 mL) and was left to stand for
1 h. The solid was filtered off, washed with water, dried, and
subjected to chromatography (hexane/ethyl acetate, 9:1) to give
3â-p-toluenesulfonyloxycholest-5-ene (2.0 g, 95%). This com-
pound was subjected to epoxidation and oxidation, as shown
in Scheme 1, to afford 3â-p-toluenesulfonyloxy-5R-hydroxy-
cholestan-6-one. A solution of 3â-p-toluenesulfonyloxy-5R-
hydroxycholestan-6-one (1.54 g, 2.68 mmol) in 40 mL of dry
DMF was treated with LiBr (0.63 g, 7.25 mmol) under argon,
and the solution was refluxed in the dark with stirring for 2
h. The reaction mixture was poured into 250 mL of water and
extracted with ethyl acetate (2 × 40 mL). The combined
extracts were washed (5% HCl and 5% NaHCO3) and dried,
and the solvent evaporated under reduced pressure. The
residue was subjected to chromatography (hexane/ethyl ace-
tate, 8:1) to give 5R-hydroxycholest-2-en-6-one (16, 1.00 g,
1
83%). H NMR (200 MHz, CDCl3) δH: 5.62 (H-2, H-3, 2H, m);
2.68 (H-7â, 1H, dd, J ) 4.4 and 12.7 Hz); 0.92 (H-21, 3H, d, J
) 6.3 Hz); 0.87 (H-26, H-27, 6H, d, J ) 6.3 Hz); 0.71 (H-19,
3H, s); 0.65 (H-18, 3H, s). 13C NMR (50 MHz, CDCl3) δC: 211.5
(C-6, s); 125.8 (C-3, d); 122.9 (C-2, d); 78.0 (C-5, s). EIMS (70
eV, m/z %): 400 (M, 20); 382 (M - H2O, 6).
2r,3r,5r-Tr ih yd r oxych olesta n -6-on e (17) a n d 2â,3â,5r-
Tr ih yd r oxych olesta n -6-on e (18). N-Methylmorpholine N-
oxide (10.0 g, 85.3 mmol) and tetrabutylammonium hydrogen

Synthesis of Cytotoxic 6E-Hydroximino-4-ene Steroids
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16 2617
sulfate (5.18 g, 15.2 mmol) were dissolved in a mixture of 37
mL of THF (free of peroxides), 43 mL of t-BuOH, and 9 mL of
water. To the resulting solution was added osmium tetroxide
(0.25 g, 0.98 mmol) followed by the dropwise addition of a
solution of 5R-hydroxycholest-2-en-6-one (16, 1.00 g, 2.49
mmol) in 20 mL of THF. The mixture was stirred for 86 h at
0 °C in the dark under an argon atmosphere. The reaction
mixture was poured into 5% Na2SO3 (300 mL), stirred for 1 h,
and extracted with CH2Cl2 (2 × 150 mL). The combined
extracts were evaporated, and the residue was redissolved in
400 mL of ethyl acetate, washed (5 M H2SO4, saturated
NaHCO3 and saturated brine), and dried over MgSO4. The
solvent was evaporated under reduced pressure and the
residue subjected to chromatography (CH2Cl2/dimethyl ketone,
90:10) to give 2R,3R,5R-trihydroxycholestan-6-one (17, 0.31 g,
29%) and 2â,3â,5R-trihydroxycholestan-6-one (18, 0.40 g, 36%).
17: 1H NMR (200 MHz, CDCl3) δH: 4.19 (H-3, 1H, m); 3.96
(H-2, 1H, m); 2.71 (H-4R, 1H, t, J ) 12.2 Hz); 0.92 (H-21, 3H,
d, J ) 6.3 Hz); 0.86 (H-26, H-27, 6H, d, J ) 6.3 Hz); 0.78 (H-
19, 3H, s); 0.65 (H-18, 3H, s). 13C NMR (50 MHz, CDCl3) δC:
CDCl3) δH: 6.06 (H-4, 1H, s); 4.25 (H-2, 1H, dd, J ) 5.9 and
6.3 Hz); 3.42 (H-7â, 1H, dd, J ) 4.4 and 10.7 Hz); 2.47 (H-1R,
1H, dd, J ) 5.9 and 8.3 Hz); 0.95 (H-19, 3H, s); 0.92 (H-21,
3H, d, J ) 6.3 Hz); 0.87 (H-26, H-27, 6H, d, J ) 6.3 Hz); 0.72
(H-18, 3H, s). 13C NMR (50 MHz, CDCl3) δC: 199.6 (C-3, s);
163.8 (C-6, s); 157.4 (C-5, s); 120.4 (C-4, d); 68.7 (C-2, d); 56.4;
55.9; 49.0; 42.9; 41.2; 39.4; 39.3; 39.1; 36.0; 35.7; 33.9; 30.6;
28.1; 28.0; 24.0; 23.8; 22.8 (C-26, q); 22.7 (C-27, q); 22.5 (C-21,
q); 18.6 (C-19, q); 12.1 (C-18, q). EIMS (70 eV, m/z %): 411 (M
- H2O, 20); 384 (M - NOH - Me, 28).
2-Hydr oxy-6E-h ydr oxim in och olesta-1,4-dien -3-on e (22).
A mixture of 6E-hydroximinocholest-4-en-2R,3R-diol (20b, 0.10
g, 0.23 mmol) and activated MnO2 (0.39 g, 4.55 mmol) in dry
chloroform (25 mL) was shaken vigorously at room tempera-
ture for 17 h. The reaction was subjected to chromatography
(hexane/ethyl acetate, 1:1) to give 2-hydroxy-6-hydroximino-
1
cholest-1,4-dien-3-one (22, 0.050 g, 50%). H NMR (200 MHz,
CDCl3) δH: 6.72 (H-1, 1H, s); 6.42 (H-4, 1H, s); 3.47 (H-7â,
1H, dd, J ) 4.4 and 11.2 Hz); 0.95 (H-19, 3H, s); 0.92 (H-21,
3H, d, J ) 6.3 Hz); 0.87 (H-26, H-27, 6H, d, J ) 6.8 Hz); 0.73
(H-18, 3H, s). 13C NMR (50 MHz, CDCl3) δC: 181.8 (C-3, s);
163.7 (C-6, s); 156.3 (C-5, s); 146.4 (C-2, s); 124.1 (C-1, d); 121.5
(C-4, d); 56.1; 56.0; 49.8; 44.5; 42.8; 39.4; 39.2; 36.0; 35.7; 33.2;
30.4; 28.1; 28.0; 24.1; 23.8; 23.3; 22.8 (C-26, q); 22.5 (C-27, q);
19.9 (C-21, q); 18.6 (C-19, q); 12.0 (C-18, q). EIMS (70 eV, m/z
%): 427 (M, 65); 410 (M - H2O, 48).
211.5 (C-6, s); 79.4 (C-5, s); 71.0 (C-3, d); 68 (C-2, d). EIMS
+
(70 eV, m/z %): 434 (M, 35); 416 (M + - H2O, 16); 398 (M
-
2H2O, 9); 380 (M - 3H2O, 3). 18: 1H NMR (200 MHz, CDCl3/
CD3OD) δH: 3.35 (H-3, 1H, m); 3.10 (H-2, 1H, m); 2.71 (H-4R,
1H, t, J ) 12.2 Hz, 12.7 Hz); 0.95 (H-19, 3H, s); 0.86 (H-21,
3H, d, J ) 6.3 Hz); 0.82 (H-26, H-27, 6H, d, J ) 6.3 Hz); 0.60
(H-18, 3H, s). 13C NMR (50 MHz, CDCl3/CD3OD 4:1) δC: 214.6
(C-6, s); 79.0 (C-5, s); 69.8 (C-3, d); 68.1 (C-2, d). EIMS (70 eV,
m/z %): 434 (M, 37); 416 (M - H2O, 7); 398 (M - 2H2O, 7).
6E-Hyd r oxim in och olesta -2,4-d ien e (24). A solution of
5R-hydroxycholest-2-en-6-one (16, 0.1 g, 0.25 mmol) in 10 mL
of dry DMF was treated with LiBr (0.63 g, 0.67 mmol) under
an argon atmosphere. The reaction mixture was refluxed in
the dark with stirring for 2 h and then poured into 25 mL of
water. The product was extracted with ethyl acetate (2 × 10
mL), and the combined extracts were washed (5% HCl and
5% NaHCO3), dried, and evaporated under reduced pressure.
The residue was subjected to chromatography (hexane/ethyl
acetate, 8:1) to give 2,4-cholestadien-6-one (23, 0.086 g, 90%).
A solution of 23 (0.030 g, 0.08 mmol) in ethanol (3 mL) was
treated with a solution of hydroxylamine hydrochloride (0.041
g, 0.60 mmol) in 50% aqueous ethanol (1 mL) and trihy-
dratated sodium acetate (0.048 g, 0.34 mmol) in 50% aqueous
ethanol (3 mL). The resulting mixture was stirred at room
temperature for 24 h. The solvent was removed under reduced
pressure, and the residue diluted with water (3 mL) and
extracted with ethyl acetate (3 mL). The extract was dried,
evaporated, and subjected to chromatography (hexane/ethyl
acetate, 7:3) to give 6E-hydroximinocholesta-2,4-diene (24). 1H
NMR (200 MHz, CDCl3) δH: 6.32 (H-4, 1H, d, J ) 4.9 Hz);
5.88 (H-3, H-2, 2H, m); 3.42 (H-7â, 1H, dd, J ) 0.5 and 11.2
Hz); 0.94 (H-19, 3H, s); 0.90 (H-21, 3H, d, J ) 6.3 Hz); 0.87
(H-26, H-27, 6H, d, J ) 6.3 Hz); 0.68 (H-18, 3H, s). 13C NMR
(50 MHz, CDCl3) δC: 157.7 (C-6, s); 138.6 (C-5, s); 127.2 (C-4,
d); 123.3 (C-3, d); 119.5 (C-2, d); 56.5; 56.1; 52.0; 42.6; 39.6;
39.5; 37.5; 36.8; 36.1; 35.7; 33.1; 29.1; 28.1; 28.0; 24.2; 23.8;
22.8; 22.5; 21.3; 18.7 (C-19, q); 16.8; 11.9 (C-18, q). EIMS (70
eV, m/z %): 397 (M, 34); 380 (M - OH, 29).
2â,3â-Dih yd r oxy-6E-h yd r oxim in och olest-4-en e (20a ).
1H NMR (200 MHz, CDCl3) δH: 5.63 (H-4, 1H, br s); 4.21 (H-3,
1H); 4.15 (H-2, 1H, m); 3.30 (H-7â, 1H); 0.92 (H-19, 3H, s);
0.91 (H-21, 3H, d, J ) 6.8 Hz); 0.87 (H-26, H-27, 6H, d, J )
6.8 Hz); 0.68 (H-18, 3H, s). 13C NMR (50 MHz, CDCl3) δC:
159.2 (C-6, s); 143.2 (C-5, s); 124.0 (C-4, d); 67.7 (C-3, d); 66.4
(C-2, d). EIMS (70 eV, m/z %): 431 (M, 6); 414 (M - OH, 22);
413 (M - H2O, 5); 400 (M - NOH, 5); 382 (M - H2O - NOH,
6).
2r,3r-Dih yd r oxy-6E-h yd r oxim in och olest-4-en e (20b).
1H NMR (200 MHz, CDCl3) δH: 5.93 (H-4, 1H, d, J ) 5.4 Hz);
4.17 (H-3, 1H, t, J ) 4.4 Hz); 3.89 (H-2, 1H, dt, J ) 3.9 and
12.7 Hz); 3.34 (H-7â, 1H, dd, J ) 4.4 and 10.2 Hz); 1.02 (H-
19, 3H, s); 0.92 (H-21, 3H, d, J ) 6.8 Hz); 0.88 (H-26, H-27,
6H, d, J ) 6.3 Hz); 0.68 (H-18, 3H, s). 13C NMR (50 MHz,
CDCl3) δC: 159.0 (C-6, s); 146.3 (C-5, s); 122.5 (C-4, d); 66.8
(C-3, d); 65.9 (C-2, d). EIMS (70 eV, m/z %): 431 (M, 10); 414
(M - OH, 47); 413 (M - H2O, 11); 400 (M - NOH, 13); 396 (M
- H2O - OH, 30); 382 (M - H2O - NOH, 11).
2r-Hyd r oxy-6E-h yd r oxim in och olest-4-en -3-on e (21b).
A solution of 6E-hydroximinocholest-4-en-2R,3R-diol (20b,
0.050 g, 0.11 mmol) in 4 mL of pyridine was added dropwise
to the chromium trioxide/pyridine complex prepared by the
addition of CrO3 (0.49 g, 4.9 mmol) to 4 mL of pyridine at 0
°C. The reaction mixture was stirred at room temperature for
1 h and then diluted with 10 mL of ethyl acetate. The resulting
precipitate was filtered off, and the filtrate was washed (10%
HCl, 10% NaHCO3, and brine), dried with anhydrous Na2SO4,
evaporated under reduced pressure, and subjected to chroma-
tography (hexane/ethyl acetate, 1:1) to give 2R-hydroxy-6-
Ack n ow led gm en t. This work was financially sup-
ported by grants from the Xunta de Galicia (XUGA
10301A97, PGIDT99PXI10304A, and PGIDT00MAR-
10301PN) and from CICYT (MAR99-0287). We also
thank Biomar and NCI for performing the pharmaco-
logical assays.
1
hydroximinocholest-4-en-3-one (21b, 0.030 g, 60%). H NMR
(200 MHz, CDCl3) δH: 6.24 (H-4, 1H, s); 4.35 (H-2, 1H, dd, J
) 5.4 and 7.8 Hz); 3.40 (H-7â, 1H, dd, J ) 3.9 and 11.2 Hz);
2.41 (H-1R, 1H, dd, J ) 5.4 and 7.3 Hz); 1.13 (H-19, 3H, s);
0.92 (H-21, 3H, d, J ) 6.3 Hz); 0.87 (H-26, H-27, 6H, d, J )
6.3 Hz); 0.71 (H-18, 3H, s). 13C NMR (50 MHz, CDCl3) δC:
199.9 (C-3, s); 162.8 (C-6, s); 156.8 (C-5, s); 119.9 (C-4, d); 69.6
(C-2, d); 56.4; 55.9; 51.8; 42.5; 40.9; 39.4; 39.1; 36.0; 35.7; 32.6;
29.7; 29.6; 28.1; 28.0; 23.9; 23.8; 22.8 (C-26, q); 22.5 (C-27, q);
20.7 (C-21, q); 18.6 (C-19, q); 17.3; 11.9 (C-18, q). EIMS (70
eV, m/z %): 411 (M - H2O, 10); 384 (M - NOH - Me, 63).
Su p p or tin g In for m a tion Ava ila ble: NMR spectra and
elemental analysis data for some of the compounds reported
here are available free of charge via the Internet at http://
pubs.acs.org.
Refer en ces
2â-Hyd r oxy-6E-h yd r oxim in och olest-4-en -3-on e (21a ).
Treatment of 6E-hydroximinocholest-4-en-2â,3â-diol (20a ) with
CrO3/pyridine or MnO2 gave, in both cases, 2â-hydroxy-6-
hydroximinocholest-4-en-3-one (21a ). 1H NMR (200 MHz,
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2618 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 16
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