Novel 5β-hydroxyspirostan-6-ones ecdysteroid antagonists: Synthesis and biological testing

Article abstract of DOI:10.1016/j.steroids.2005.02.024

Eight new 5β-hydroxy-spirostan-6-ones bearing hydroxy and amino functions in the A ring, i.e., 3β-OH, 3α-OH, 2β,3β-OH, 2α,3β-OH, 3β-NH₂, 2α-NH₂-3β-OH, 2β-NH₂-3β-OH, and 2β-OH-3β-NH₂, were efficiently synthesized, and their antiecdysteroid activities were evaluated on the metamorphosis bioassay of mosquito Aedes aegypti. To our knowledge, these new steroids represent the first 5β-hydroxy-spirostanes which have been tested for antiecdysteroid activity in mosquitoes. The higher antagonistic effect was found for compounds bearing the 3β-hydroxy and 2β,3β-dihydroxy functionality, which show promise as environmental friendly insecticides.

Full text of DOI:10.1016/j.steroids.2005.02.024

steroids 7 1 ( 2 0 0 6 ) 1–11
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Novel 5␤-hydroxyspirostan-6-ones ecdysteroid antagonists:
Synthesis and biological testing
Daniel G. Riveraa,∗, Fredy Leo´na, Francisco Colla,∗, Gema P. Davisonb
a
Center for Natural Products Studies, Faculty of Chemistry, University of Havana, Zapata y G, Vedado 10400, Ciudad Habana, Cuba
Center for Research and Biological Evaluation, University of Havana, Cuba
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Eight new 5␤-hydroxy-spirostan-6-ones bearing hydroxy and amino functions in the A ring,
i.e., 3␤-OH, 3␣-OH, 2␤,3␤-OH, 2␣,3␤-OH, 3␤-NH2, 2␣-NH2-3␤-OH, 2␤-NH2-3␤-OH, and 2␤-OH-
3␤-NH2, were efficiently synthesized, and their antiecdysteroid activities were evaluated
on the metamorphosis bioassay of mosquito Aedes aegypti. To our knowledge, these new
steroids represent the first 5␤-hydroxy-spirostanes which have been tested for antiecdys-
teroid activity in mosquitoes. The higher antagonistic effect was found for compounds
bearing the 3␤-hydroxy and 2␤,3␤-dihydroxy functionality, which show promise as envi-
ronmental friendly insecticides.
Received 15 November 2004
Received in revised form 24 January
2005
Accepted 2 February 2005
Available online 26 September 2005
Keywords:
© 2005 Elsevier Inc. All rights reserved.
Steroids
Antiecdysteroid activity
Spirostanones
Steroidal sapogenins
1.
Introduction
The inhibitory effect of natural brassinosteroids on the
insect moulting process has been shown in Periplaneta amer-
Considerable research effort has been devoted to studying the
actions of brassinosteroids as antagonists or agonists of ecdys-
teroids due to the structural similarity between these families
of steroidal hormones. Ecdysteroids are widely distributed in
invertebrates where they are involved in different biochemical
and metabolic processes, such as genetic expression, moult-
ing and metamorphosis, gonad maturation and reproduction
[1,2]. They have also been found in plants, where they seem
to have a defensive action, though this is not clear yet [2].
Ecdysone (1a) and 20-hydroxyecdysone (1b) are the most rep-
resentative compounds of this group. On the other hand,
brassinosteroids are considered the sixth class of phytohor-
mones due to their extremely high plant growth-promoting
activity and anti-stress effect [3], amongst which brassinolide
(2) is the more active and widely distributed brassinosteroid
in the plant kingdom.
icana [4] and Phormia terra novae [5], thereby indicating
that these steroids are capable of binding competitively to
the ecdysteroid receptor. This fact has been experimentally
demonstrated with the receptor isolated from Calliphora vic-
ina, to which brassinosteroids bound acting as antagonists of
ecdysteroids [6].
Several brassinosteroid analogues with ecdysteroid-type
activities have also been reported [7–9]. However, their struc-
tures are difficult to synthesize because of the variety of
stereochemically different hydroxy groups at similar posi-
tions. Nevertheless, elegant syntheses have provided brassi-
nosteroids and ecdysteroids for the unified task of producing
practical alternatives for plant protection by environmental
friendly insecticides [7].
We have previously reported the synthesis of novel
spirostanones, e.g., 3a, 3b and 4, which have shown high
∗
Corresponding author. Present address: Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany.
E-mail addresses: drivera@ipb-halle.de (D.G. Rivera), coll@fq.uh.cu (F. Coll).
0039-128X/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2005.02.024

2
steroids 7 1 ( 2 0 0 6 ) 1–11
Fig. 1 – Structures of ecdysteroids, brassinosteroids and spirostanic brassinosteroid analogues.
growth-promoting activity in both bioassays and field trials
[10–14] (Fig. 1). These successful results suggested that the
spiroketal side chain might be able to mimic the brassinos-
teroid side chain. Hence, the spirostanic derivatives are also
interesting because of their potential capability to surrogate
the ecdysteroid side chain and thus act as agonists or antago-
nists of these compounds. Consequently, we envisioned that
spirostanes with structural similarities to ecdysteroids, e.g.,
A/B cis ring junction and the 2␤,3␤-dihydroxy-6-oxo func-
tionality, could bind to the ecdysteroid receptor and thereby
behave as antagonists of this family of steroidal hormones.
Previous reports of the synthesis of ecdysone analogues with
a spirostane skeleton do not include any biological data and
the compounds, which were obtained, do not belong to the
5␤-series [15].
With a view to testing our steroid structural–biological
activity hypothesis, we synthesized eight new 5␤-hydroxyspir-
ostan-6-ones bearing hydroxy and amino functions on ring A.
We focused our attention on the biological importance of the
stereochemistry of these functional groups. Four of the new
compounds are A-ring analogues of the natural ecdysone (1a),
2-deoxyecdysone, 3-epiecdysone, and 2-deoxy-3-epiecdysone
as well as some of their corresponding amino derivatives.
Our approach was to exploit the accessibility of the 5␤-
hydroxy-6-oxo functionality as the common feature on the
spirostane skeleton and to introduce a variety of functions
on the A ring that could facilitate structure–activity relation-
ship (SAR) studies as well as a thorough understanding of the
antagonistic effect. To our knowledge, our target molecules
represent the first spirostanic compounds bearing the 5␤-
hydroxy-6-oxo function for which the ecdysteroid-type activ-
ity has been tested.
2.
Experimental
2.1.
Chemistry
Melting points were determined on a Stuart Scientific Appa-
ratus and are uncorrected. IR spectra were obtained on a
Nicolet 205 FT-IR spectrometer. 1H NMR and 13C NMR were
recorded on a Bruker ACF-250 spectrometer at 250.13 MHz
and 62.9 MHz for 1H and 13C, respectively, using TMS as
the internal standard. Two-dimensional spectra: 1H-1H cor-
relation spectroscopy (COSY), heteronuclear single-quantum
coherence (HSQC), heteronuclear multiple bond correlation
(HMBC), and the phase sensitive NOESY (mixing time 0.7 s)
were measured for compounds 12, 13, 14, 18, and 26 on
a Varian INOVA-500 at 499.81 MHz using the standard pulse
sequences given by the manufacture. The high resolution
ESI mass spectra were obtained on
a Bruker Apex 70e
Fourier transform ion cyclotron resonance (FT-ICR) mass
spectrometer equipped with an InfinityTM cell and a 7.0
Tesla superconducting magnet. Reactions were monitored
by thin-layer chromatography on precoated plates with sil-
ica gel (Merck), and spots were visualized with a 1% (w/v)
spray of vanillin in perchloric acid and subsequent heat-
ing. Flash column chromatography was performed on silica
gel 60 (Merck, >230 mesh). “Usual work-up” refers to dilu-
tion with water, extraction with an organic solvent, washing
the extract consecutively with 5% HCl and/or 5% NaHCO3
and brine, drying over anhydrous Na2SO4, and removal
of the solvent under reduced pressure. The solvents were
purified and dried according to standard procedures. The
starting material 5 was synthesized according to a previ-

steroids 7 1 ( 2 0 0 6 ) 1–11
3
ously reported procedure, m.p. (MeOH): 280–282 ◦C (ref [16]:
281–283 ◦C).
was then evaporated under reduced pressure. The resulting
crude product was dissolved in 120 mL of a saturated solu-
tion of NaHCCO3 in MeOH-H2O (4:1) and stirred for 1 h at room
temperature. Usual work-up (EtOAc) yielded a crude product,
which was purified by flash column chromatography (hex-
ane/EtOAc 4:1) to afford the ketol 8 (800 mg, 56%). m.p. (ace-
tone): 232–233 ◦C. IR (KBr, cm−1): 3445, 3368, 3325, 1705, 1105,
1092, 1060, 980, 900, 860. 1H NMR (CDCl3): ı = 0.77 (3 H, s, H-
18); 0.75 (3 H, s, H-19); 0.78 (3 H, d, J = 6.2 Hz, H-27); 0.98 (3 H,
d, J = 6.7 Hz, H-21); 2.78 (1 H, t, J = 12.5 Hz, H-7␣); 3.36 (1 H, t,
J = 10.8 Hz, H-26ax); 3.49 (1 H, dd, J = 10.7/3.9 Hz, H-26eq); 4.25
(1 H, m, H-3␣); 4.42 (1 H, m, H-16␣). 13C NMR (CDCl3): ı = 28.3
(C-1); 25.4 (C-2); 67.4 (C-3); 31.4 (C-4); 80.3 (C-5); 211.9 (C-6); 41.7
(C-7); 37.5 (C-8); 44.8 (C-9); 43.4 (C-10); 21.2 (C-11); 39.4 (C-12);
41.0 (C-13); 56.5 (C-14); 31.6 (C-15); 80.4 (C-16); 61.9 (C-17); 16.3
(C-18); 16.6 (C-19); 41.6 (C-20); 14.4 (C-21); 109.3 (C-22); 31.3 (C-
23); 28.7 (C-24); 30.2 (C-25); 66.9 (C-26); 17.1 (C-27). HRMS (ESI-
FT-ICR) m/z: 469.29292 [M + Na]+ (calculated for C27H42NaO5:
485.29299).
2.1.1. (25R)-3␤-(t-Butyldimethylsilyloxy)-5␣-spirostane-
5,6␤-diol (6)
TBDMSCl (2.6 g, 17.5 mmol) and imidazole (1.8 g, 26 mmol)
were added to a solution of triol 5 (8.7 g, 19.5 mmol) in anhy-
drous DMF (150 mL). The reaction mixture was stirred under a
nitrogen atmosphere for 3 h and then concentrated. The usual
work-up (CHCl3) yielded a crude product, which was purified
by flash column chromatography (hexane/EtOAc 3:1) to give
6 (9.22 g, 84%). m.p. (EtOAc): 219–221 ◦C. IR (KBr, cm−1): 3418,
3378, 1075, 1043, 1024, 980, 900, 860. 1H NMR (CDCl3): ı = 0.07
(s, 3 H, (CH3)2Si); 0.09 (s, 3 H, (CH3)2Si); 0.79 (s, 3 H, H-18); 1.19 (s,
3 H, H-19); 0.79 (3 H, d, J = 6.2 Hz, H-27); 0.92 (s, 9 H, (CH3)3CSi);
0.96 (3 H, d, J = 6.7 Hz, H-21); 2.16 (dd, 1 H, J = 12.8/11.5 Hz, H-
7␣); 3.36 (1 H, t, J = 10.8 Hz, H-26ax); 3.49 (1 H, dd, J = 10.7/3.9 Hz,
H-26eq); 3.53 (m, 1 H, H-6␣); 4.09 (1 H, br m, H-3␣); 4.40 (1 H,
m, H-16␣). 13C NMR (CDCl3): ı = 31.1 (C-1); 30.3 (C-2); 67.1 (C-3);
45.5 (C-4); 75.5 (C-5); 75.2 (C-6); 34.1 (C-7); 29.8 (C-8); 45.3 (C-9);
38.8 (C-10); 20.8 (C-11); 39.9 (C-12); 40.5 (C-13); 55.7 (C-14); 32.1
(C-15); 80.2 (C-16); 61.9 (C-17); 16.3 (C-18); 16.4 (C-19); 41.5 (C-
20); 14.2 (C-21); 109.4 (C-22); 31.4 (C-23); 28.5 (C-24); 30.0 (C-25);
66.7 (C-26); 16.9 (C-27); 26.1 ((CH3)3CSi); 18.3 ((CH3)3CSi); −2.9
((CH3)2Si). HRMS (ESI-FT-ICR) m/z: 585.39509 [M + Na]+ (calcu-
lated for C33H58NaSiO5: 585.39512).
2.1.4. (25R)-3␤,5-Dihydroxy-5␤-spirostan-6-one (9)
Ketol 7 (5.0 g, 11.2 mmol) was dissolved in a 10% solution of
KOH in MeOH (200 mL). The reaction mixture was refluxed for
24 h and then neutralized with HCl 5%. The mixture was con-
centrated until half volume, poured into 500 mL of cold water,
filtered under reduced pressure, and washed with water. The
resulting product was purified by flash column chromatog-
raphy (hexane/EtOAc 3:1) to obtain, by collection of the less
hydrophilic fractions, the ketol 9 (3.70 g, 74%). m.p. (EtOH):
212–214 ◦C. IR (KBr, cm−1): 3455, 3335, 1708, 1048, 980, 900, 860.
1H NMR (CDCl3): ı = 0.77 (3 H, s, H-18); 0.78 (3 H, s, H-19); 0.78
(3 H, d, J = 6.2 Hz, H-27); 0.98 (3 H, d, J = 6.7 Hz, H-21); 2.25 (1 H,
t, J = 12.1 Hz, H-7␣); 2.44 (1 H, dd, J = 1.8/3.6 Hz, H-7␤; 3.36 (1 H,
t, J = 10.8 Hz, H-26ax); 3.49 (1 H, dd, J = 10.7/3.9 Hz, H-26eq); 4.02
(1 H, s, OH-5); 4.05 (1 H, m, H-3␣); 4.42 (1 H, m, H-16␣). 13C NMR
(CDCl3): ı = 24.9 (C-1); 27.8 (C-2); 65.5 (C-3); 37.1 (C-4); 81.0 (C-
5); 212.3 (C-6); 41.5 (C-7); 36.8 (C-8); 42.9 (C-9); 44.1 (C-10); 21.5
(C-11); 39.5 (C-12); 41.3 (C-13); 56.6 (C-14); 31.6 (C-15); 80.3 (C-
16); 62.0 (C-17); 16.3 (C-18); 17.2 (C-19); 41.4 (C-20); 14.5 (C-21);
109.3 (C-22); 31.3 (C-23); 28.7 (C-24); 30.2 (C-25); 66.9 (C-26); 17.1
(C-27). HRMS (ESI-FT-ICR) m/z: 469.29298 [M + Na]+ (calculated
for C27H42NaO5: 469.29291).
2.1.2. (25R)-3␤,5-Dihydroxy-5␣-spirostan-6-one (7)
A solution of diol 6 (9.0 g, 15.9 mmol) in CH2Cl2 (200 mL) was
added to a suspension of pyridinium chlorochromate (PCC)
(3.9 g, 18 mmol) in CH2Cl2 (80 mL) at 0 ◦C. The reaction mixture
was stirred for 20 min and then allowed to reach room temper-
ature and stirred for additional 40 min. The mixture was then
filtered through a pad of alumina, and the resulting solution
was evaporated under reduced pressure. The resulting ketol
was dried and dissolved in 150 mL of anhydrous THF. Tetra-
butylammonium fluoride (TBFA) (4 mL, 1 M in THF) was added
to the solution and the reaction mixture was stirred under
a nitrogen atmosphere for 6 h. Usual work-up (Et2O) yielded
a quantitative yield of the crude product, which was recrys-
tallized from EtOH to give the pure ketol 7 (7.14 g, 82%). m.p.
(EtOH): 268–270 ◦C. IR (KBr, cm−1): 3440, 3335, 3318, 1700, 1086,
1048, 980, 912, 868. 1H NMR (CDCl3): ı = 0.77 (3 H, s, H-18); 0.69
(3 H, s, H-19); 2.07 (1 H, dd, J = 13.0/4.2 Hz, H-7␤); 2.82 (1 H, t,
J = 13.0 Hz, H-7␣); 3.36 (1 H, t, J = 10.8 Hz, H-26ax); 3.49 (1 H, dd,
J = 10.7/3.9 Hz, H-26eq); 3.91 (1 H, br m, H-3␣); 4.40 (1 H, m, H-
16␣). 13C NMR (CDCl3): ı = 30.0 (C-1); 30.0 (C-2); 66.9 (C-3); 35.5
(C-4); 80.1 (C-5); 214.3 (C-6); 42.0 (C-7); 37.1 (C-8); 44.5 (C-9); 42.6
(C-10); 21.4 (C-11); 39.7 (C-12); 41.2 (C-13); 56.2 (C-14); 31.6 (C-
15); 80.8 (C-16); 62.1 (C-17); 16.1 (C-18); 14.1 (C-19); 41.7 (C-20);
14.4 (C-21); 109.3 (C-22); 31.4 (C-23); 28.8 (C-24); 30.3 (C-25); 67.0
(C-26); 17.1 (C-27). HRMS (ESI-FT-ICR) m/z: 469.29296 [M + Na]+
(calculated for C27H42NaO5: 469.29299).
2.1.5. (25R)-3␣,5-Dihydroxy-5␤-spirostan-6-one (10)
Ketol 8 (700 mg, 1.56 mmol) was treated in a similar way as
described for the synthesis of 9 to give 10 (496 mg, 71%). The
starting material was also collected in approximately 20%
yield, m.p. (EtOH): 206–208 ◦C. IR (KBr, cm−1): 3497, 3452, 3342,
1707, 1240, 1063, 975, 935, 878. 1H NMR (CDCl3): ı = 0.71 (3 H, s,
H-19); 0.75 (3 H, s, H-18); 0.78 (3 H, d, J = 6.2 Hz, H-27); 0.97 (3 H,
d, J = 6.7 Hz, H-21); 2.31 (1 H, t, J = 12.8 Hz, H-7␣); 2.42 (1 H, dd,
J = 13.1/4.0 Hz, H-7␤); 3.35 (1 H, t, J = 10.8 Hz, H-26ax); 3.49 (1 H,
dd, J = 10.7/3.9 Hz, H-26eq); 3.95 (1 H, s, OH-5); 4.00 (1 H, br. m,
H-3␤); 4.42 (1 H, m, H-16ct). 13C NMR (CDCl3): ı = 30.0 (C-1); 29.6
(C-2); 67.3 (C-3); 41.8 (C-4); 81.9 (C-5); 212.6 (C-6); 41.4 (C-7); 37.1
(C-8); 43.0 (C-9); 44.0 (C-10); 21.2 (C-11); 39.5 (C-12); 41.1 (C-13);
56.6 (C-14); 31.6 (C-15); 80.3 (C-16); 62.0 (C-17); 16.3 (C-18); 16.4
(C-19); 41.6 (C-20); 14.5 (C-21); 109.3 (C-22); 31.3 (C-23); 28.7 (C-
24); 30.2 (C-25); 66.9 (C-26); 17.1 (C-27). HRMS (ESI-FT-ICR) m/z:
469.29298 [M + Na]+ (calculated for C28H42NaO5: 469.29299).
2.1.3. (25R)-3␣,5-Dihydroxy-5␣-spirostan-6-one (8)
A solution of diisopropylazodicarboxylate (DIAD) (1.16 mL,
6.0 mmol) in 10 mL of THF was added dropwise to a stirred
solution of ketol 7 (1.4 g, 3.2 mmol), formic acid (0.40 mL) and
triphenylphosphine (1.27 g, 5.0 mmol) in THF (80 mL). The mix-
ture was stirred at room temperature for 24 h, and the solvent

4
steroids 7 1 ( 2 0 0 6 ) 1–11
CHCl3 and washed sequentially with solutions of 10%
2.1.6. (25R)-5-Hydroxy-5␤-spirost-2-en-6-one (11)
Na2SO3 (2 × 100 mL) and 10% K2CO3 (2 × 200 mL). The organic
phase was then dried over Na2SO4 and evaporated under
reduced pressure. The resultant crude product was puri-
fied by flash column chromatography (hexane/EtOAc 5:1)
to yield the epoxides 13 (224 mg, 24%) and 14 (498 mg,
48%).
p-Toluene-sulfonyl chloride (TsCl) (3.655 g, 19.5 mmol) was
added to a solution of ketol 9 (3.0 g, 7.0 mmol) in 80 mL of
anhydrous pyridine, and the reaction mixture was stirred
at room temperature in the dark for 24 h. The mixture was
poured into 300 mL of a saturated solution of KHCO3 and
filtered under reduced pressure. The resulting tosylate was
washed with cold water several times, dried, and dissolved
in 90 mL of anhydrous DMF. The solution was treated with
Li2CO3 (4.15 g, 56 mmol) and LiBr (4.65 g, 56 mmol) and refluxed
for 2 h under a nitrogen atmosphere. The reaction mixture
was poured slowly while stirring into 300 mL of 10% HCl, fil-
tered under reduced pressure, and dried to give the olefin 11
(2.36 g, 74%). m.p. (acetone): 189–192 ◦C. IR (KBr, cm−1): 3473,
3025, 1718, 980, 935, 874. 1H NMR (CDCl3): ı = 0.70 (3 H, s, H-
19); 0.75 (3 H, s, H-18); 0.78 (3 H, d, J = 6.3 Hz, H-27); 0.97 (3
H, d, J = 6.5 Hz, H-21); 2.32 (1 H, t, J = 12.8 Hz, H-7␣); 2.44 (1 H,
dd, J = 13.0/4.0 Hz, H-7␤); 3.35 (1 H, t, J = 10.8 Hz, H-26ax); 3.47
(1 H, dd, J = 10.7/3.9 Hz, H-26eq); 4.44 (1 H, m, H-16␣); 5.56 (2 H,
m, H-3 and H-2). 13C NMR (CDCl3): ı = 30.7 (C-1); 124.9* (C-2);
121.8* (C-3); 35.7 (C-4); 79.6 (C-5); 213.8 (C-6); 41.3 (C-7); 36.6
(C-8); 43.3 (C-9); 43.1 (C-10); 22.1 (C-11); 39.4 (C-12); 41.1 (C-
13); 56.5 (C-14); 31.5 (C-15); 80.4 (C-16); 61.4 (C-17); 16.4 (C-18);
16.6 (C-19); 41.6 (C-20); 14.4 (C-21); 109.3 (C-22); 31.3 (C-23);
28.7 (C-24); 30.2 (C-25); 66.8 (C-26); 17.1 (C-27). HRMS (ESI-
FT-ICR) m/z: 451.28249 [M + Na]+ (calculated for C27H40NaO4:
451.28244).
2.1.8.1. (25R)-2␣,3␣-Epoxy-5-hydroxy-5␤-spirostan-6-one
(13). m.p. (Acetone): 202–203 ◦C. IR (KBr, cm−1): 3388, 1705,
1204, 976, 916, 860. 1H NMR (CDCl3): ı = 0.70 (3 H, s, H-19); 0.76
(3 H, s, H-18); 0.80 (3 H, d, J = 6.3 Hz, H-27); 0.97 (3 H, d, J = 6.7 Hz,
H-21); 3.22–3.29 (2 H, m, H-2␤ + H-3␤); 3.34 (1 H, t, J = 10.3 Hz,
H-26ax); 3.46 (1 H, dd, J = 10.5/4.2 Hz, H-26eq); 3.95 (1 H, s,
OH-5); 4.39 (1 H, m, H-16␣). 13C NMR (CDCl3): ı = 34.2 (C-1); 48.2
(C-2); 53.4 (C-3); 37.4 (C-4); 78.9 (C-5); 213.4 (C-6); 41.5 (C-7); 37.3
(C-8); 43.9 (C-9); 41.1 (C-10); 21.6 (C-11); 39.2 (C-12); 41.1 (C-13);
56.3 (C-14); 31.5 (C-15); 80.4 (C-16); 61.9 (C-17); 16.3 (C-18);
17.5 (C-19); 41.6 (C-20); 14.4 (C-21); 109.3 (C-22); 31.4 (C-23);
28.8 (C-24); 30.2 (C-25); 66.9 (C-26); 17.0 (C-27). HRMS (ESI-
FT-ICR) m/z: 467.27680 [M + Na]+ (calculated for C27H40NaO5:
467.27683).
2.1.8.2. (25R)-2␤,3␤-Epoxy-5-hydroxy-5␤-spirostan-6-one
(14). m.p. (Acetone): 192–194 ◦C. IR (KBr, cm−1): 3418, 1711,
1228, 980, 920, 878. 1H NMR (CDCl3): ı = 0.72 (3 H, s, H-19); 0.75
(3 H, s, H-18); 0.77 (3 H, d, J = 6.3 Hz, H-27); 0.94 (3 H, d, J = 6.7 Hz,
H-21); 3.28 (1 H, br m, H-2␣); 3.35 (1 H, t, J = 10.5 Hz, H-26ax);
3.49 (1 H, dd, J = 10.5/4.2 Hz, H-26eq); 3.61 (1 H, m, H-3␣); 3.87
(1 H, s, OH-5); 4.37 (1 H, m, H-16␣). 13C NMR (CDCl3): ı = 20.4
(C-1); 53.8 (C-2); 53.2 (C-3); 22.8 (C-4); 77.6 (C-5); 210.0 (C-6);
44.0 (C-7); 36.7 (C-8); 43.2 (C-9); 43.6 (C-10); 20.8 (C-11); 39.3
(C-12); 40.9 (C-13); 56.5 (C-14); 31.5 (C-15); 80.2 (C-16); 62.0
(C-17); 16.2 (C-18); 15.1 (C-19); 41.6 (C-20); 14.4 (C-21); 109.3
(C-22); 31.4 (C-23); 28.7 (C-24); 30.2 (C-25); 66.9 (C-26); 17.1
(C-27). HRMS (ESI-FT-ICR) m/z: 467.27687 [M + Na]+ (calculated
for C27H40NaO5: 467.27683).
2.1.7. (25R)-2␤,3␤,5-Trihydroxy-5␤-spirostan-6-one (12)
NMO (2.75 g, 34.1 mmol) and a solution of OsO4 in tBuOH
(3.0 mL, 12.5 mg/mL) were added to a solution of 11 (1.35 g,
3.15 mmol) in 100 mL of the solvent mixture THF/fBuOH/H2O
(10:10:1). The reaction was stirred under a nitrogen atmo-
sphere at room temperature for 24 h and then treated with
50 mL of a saturated solution of Na2SO3. The mixture was
stirred for an additional hour and then concentrated to half
volume. The usual work-up (EtOAc) yielded a crude product,
which was purified by flash column chromatography (hex-
ane/EtOAc 3:1) to give the triol 12 (993 mg, 68%). m.p. (hep-
tane/acetone): 226–228 ◦C. IR (KBr, cm−1): 3475, 3387, 3335,
1725, 1250, 1050, 980, 920, 860. 1H NMR (CDCl3): ı = 0.71 (3 H,
s, H-19); 0.75 (3 H, s, H-18); 0.80 (3 H, d, J = 6.3 Hz, H-27); 0.97
(3 H, d, J = 6.7 Hz, H-21); 3.35 (1 H, t, J = 10.3 Hz, H-26ax); 3.48
(1 H, dd, J = 10.4/4.3 Hz, H-26eq); 4.02 (1 H, br m, H-2␣); 4.08 (1
H, m, H-3␣); 4.40 (1 H, m, H-16␣). 13C NMR (CDCl3): ı = 35.7 (C-
1); 67.9 (C-2); 69.7 (C-3); 35.4 (C-4); 81.9 (C-5); 212.8 (C-6); 42.0
(C-7); (C-8); 45.7 (C-9); 44.4 (C-10); 22.2 (C-11); 39.4 (C-12); 41.0
(C-13); 56.5 (C-14); 31.6 (C-15); 80.4 (C-16); 61.9 (C-17); 16.3 (C-
18); 16.6 (C-19); 41.6 (C-20); 14.4 (C-21); 109.3 (C-22); 31.3 (C-23);
28.7 (C-24); 30.2 (C-25); 66.9 (C-26); 17.1 (C-27). HRMS (ESI-
FT-ICR) m/z: 485.28738 [M + Na]+ (calculated for C27H42NaO6:
485.28733).
2.1.9. (25R)-2␣,3␤,5-Trihydroxy-5␤-spirostan-6-one (15)
A solution of epoxide 13 (180 mg, 0.4 mmol) in 40 mL of ace-
tone was treated with 1.2 mL of water and 0.5 mL of 70%
HClO4. The reaction mixture was stirred at 50 ◦C for 2 h and
then, concentrated under reduced pressure. The usual work-
up (EtOAc) yielded a crude product, which was purified by
flash column chromatography (CHCl3/MeOH 10:1) to give the
triol 15 (149 mg, 81%). m.p. (heptane/acetone): 259–260 ◦C. IR
(KBr, cm−1): 3477, 3420, 3335, 1715, 1121, 1080, 1034, 976, 916,
860. 1H NMR (CDCl3): ı = 0.69 (3 H, s, H-19); 0.75 (3 H, s, H-
18); 0.81 (3 H, d, J = 6.3 Hz, H-27); 0.96 (3 H, d, J = 6.7 Hz, H-21);
3.36 (1 H, t, J = 10.5 Hz, H-26ax); 3.49 (1 H, dd, J = 10.4/4.2 Hz,
H-26eq); 4.09 (1 H, m, H-3␣); 4.07 (1 H, m, H-2␤); 4.41 (1 H,
m, H-16␣). 13C NMR (CDCl3): ı = 35.2 (C-1); 68.7 (C-2); 69.4
(C-3); 35.8 (C-4); 81.7 (C-5); 212.6 (C-6); 42.0 (C-7); 37.3 (C-
8); 44.9 (C-9); 44.4 (C-10); 22.1 (C-11); 39.4 (C-12); 41.1 (C-13);
56.5 (C-14); 31.6 (C-15); 80.4 (C-16); 61.9 (C-17); 16.3 (C-18);
16.6 (C-19); 41.6 (C-20); 14.4 (C-21); 109.3 (C-22); 31.3 (C-23);
28.7 (C-24); 30.2 (C-25); 66.9 (C-26); 17.1 (C-27). HRMS (ESI-
FT-ICR) m/z: 485.28740 [M + Na]+ (calculated for C27H42NaO6:
485.28734).
2.1.8. (25R)-2,3-Epoxy-5-hydroxy-5␤-spirostan-6-one
(13, 14)
A solution of mCPBA (0.77 g, 4.5 mmol) in 30 mL of CH2Cl2
was added to
a stirred solution of the olefin 11 (1.0 g,
2.25 mmol) in 60 mL of CH2Cl2 at 0 ◦C in the dark. The
reaction was allowed to reach room temperature and
stirred for 4 h. The mixture was diluted with 100 mL of

steroids 7 1 ( 2 0 0 6 ) 1–11
5
(C-10); 22.2 (C-11); 39.4 (C-12); 41.0 (C-13); 56.4 (C-14); 31.6 (C-
15); 80.4 (C-16); 61.9 (C-17); 16.3 (C-18); 16.6 (C-19); 41.6 (C-20);
14.4 (C-21); 109.3 (C-22); 31.3 (C-23); 28.7 (C-24); 30.2 (C-25); 66.9
(C-26); 17.1 (C-27). HRMS (ESI-FT-ICR) m/z: 510.29440 [M + Na]+
(calculated for C27H41NaN3O5: 510.29438).
2.1.10. (25R)-2␣-Amino-3␤,5-dihydroxy-5␤-spirostan-6-
one (16)
A solution of epoxide 14 (300 mg, 0.68 mmol) in 60 mL of the
mixture solvent CH3CN/H2O (9:1) was treated with CeCl3·7H2O
(85 mg, 0.34 mmol) and NaN3 (45 mg 0.68 mmol). The reaction
mixture was refluxed for 3 h, and then, TLC analysis revealed
that all of the starting material had been consumed. The usual
work-up (CHCl3) yielded a crude product, which was analyzed
by 1H NMR confirming the structure of the azide. This was dis-
solved in 50 mL of absolute EtOH, and Lindlar catalyst (200 mg)
was added to the solution. The reaction mixture was treated
successively with hydrogen and vacuum and finally stirred
under a hydrogen atmosphere for 36 h, at which point TLC
analysis revealed that all of the azide had been consumed.
The catalyst was removed by filtration, and the resulting solu-
tion was evaporated under reduced pressure to give a crude
product, which was purified by flash column chromatogra-
phy (CHC3/MeOH/Et3N 8:1:0.5) to afford the pure aminoketol
16 (200 mg, 64%). m.p. (heptane/acetone): 221 ◦C (decomp). IR
2.1.12. (25R)-2␤-Amino-3␤,5-dihydroxy-5␤-spirostan-6-
one (18)
Lindlar catalyst (100 mg) was added to a solution of azide 17
(300 mg, 0.6 mmol) in 50 mL of absolute EtOH. The mixture was
treated successively with hydrogen and vacuum and finally
stirred under a hydrogen atmosphere for 36 h, at which point
TLC analysis revealed that all of the azide had been con-
sumed. The catalyst was removed by filtration, and the result-
ing solution was evaporated under reduced pressure to give a
crude product, which was purified by flash column chromatog-
raphy (CHCl3/MeOH/Et3N 10:1:0.5) to afford the aminoketol
18 (259 mg, 91%). m.p. (heptane/acetone): 253–255 ◦C. IR (KBr,
cm−1): 3337, 3315, 3298, 1722, 1245, 1050, 978, 920, 860. 1H NMR
(CDCl3): ı = 0.71 (3 H, s, H-19); 0.75 (3 H, s, H-18); 0.81 (3 H,
d, J = 6.4 Hz, H-27); 0.97 (3 H, d, J = 6.6 Hz, H-21); 3.35 (1 H, t,
J = 10.4 Hz, H-26ax); 3.49 (1 H, dd, J = 10.4/4.3 Hz, H-26eq); 4.01
(1 H, br m, H-2␣); 4.09 (1 H, m, H-3␣); 4.40 (1 H, m, H-16␣). 13C
NMR (CDCl3): ı = 35.7 (C-1); 67.4 (C-2); 69.8 (C-3); 35.5 (C-4); 81.7
(C-5); 212.2 (C-6); 42.0 (C-7); 37.2 (C-8); 45.7 (C-9); 44.4 (C-10);
22.2 (C-11); 39.4 (C-12); 41.0 (C-13); 56.4 (C-14); 31.6 (C-15); 80.4
(C-16); 61.9 (C-17); 16.3 (C-18); 16.6 (C-19); 41.6 (C-20); 14.4 (C-
21); 109.3 (C-22); 31.3 (C-23); 28.7 (C-24); 30.2 (C-25); 66.9 (C-26);
17.1 (C-27). HRMS (ESI-FT-ICR) m/z: 462.31419 [M + H]+ (calcu-
(KBr, cm−1): 3395, 3381, 3345, 1721, 1210, 1048, 980, 924, 865. 1
H
NMR (CDCl3): ı = 0.68 (3 H, s, H-19); 0.75 (3 H, s, H-18); 0.80 (3
H, d, J = 6.4 Hz, H-27); 0.97 (3 H, d, J = 6.7 Hz, H-21); 3.35 (1 H, t,
J = 10.3 Hz, H-26ax); 3.48 (1 H, dd, J = 10.5/4.1 Hz, H-26eq); 4.06 (1
H, m, H-3␣); 3.91 (1 H, m, H-2␤); 4.41 (1 H, m, H-16␣). 13C NMR
(CDCl3): ı = 35.5 (C-1); 67.3 (C-2); 69.7 (C-3); 35.4 (C-4); 81.7 (C-5);
212.6 (C-6); 42.1 (C-7); 37.2 (C-8); 45.7 (C-9); 44.4 (C-10); 22.2 (C-
11); 39.4 (C-12); 41.0 (C-13); 56.5 (C-14); 31.6 (C-15); 80.4 (C-16);
61.9 (C-17); 16.3 (C-18); 16.6 (C-19); 41.6 (C-20); 14.4 (C-21); 109.3
(C-22); 31.3 (C-23); 28.7 (C-24); 30.2 (C-25); 66.9 (C-26); 17.1 (C-
27). HRMS (ESI-FT-ICR) m/z: 462.31410 [M + H]+ (calculated for
lated for C27H44NO5: 462.31414).
C
27H44NO5: 462.31414).
2.1.13. (25R)-3␤-Azido-5-hydroxy-5␣-spirostan-6-one (19)
Ketol 7 (1.0 g, 2.2 mmol) was treated in a similar way as
described for the synthesis of 17 to give the azide 19 (595 mg,
58%). m.p. (EtOH): 194 ◦C (decomp.). IR (KBr, cm−1): 3235, 2090,
1710, 1048, 980, 912, 860. 1H NMR (CDCl3): ı = 0.77 (3 H, s, H-18);
0.70 (3 H, s, H-19); 2.08 (1 H, dd, J = 13.0/4.4 Hz, H-7␤; 2.78 (1 H, t,
J = 13.0 Hz, H-7␣); 3.36 (1 H, t, J = 10.8 Hz, H-26ax); 3.49 (1 H, dd,
J = 10.7/3.9 Hz, H-26eq); 3.69 (1 H, br m, H-3␣); 4.41 (1 H, m, H-
16␣). 13C NMR (CDCl3): ı = 30.0 (C-1); 30.2 (C-2); 66.0 (C-3); 35.7
(C-4); 80.1 (C-5); 214.3 (C-6); 42.0 (C-7); 37.1 (C-8); 44.5 (C-9); 42..6
(C-10); 21.4 (C-11); 39.7 (C-12); 41.2 (C-13); 56.2 (C-14); 31.6 (C-
15); 80.8 (C-16); 62.1 (C-17); 16.1 (C-18); 14.1 (C-19); 41.7 (C-20);
14.4 (C-21); 109.3 (C-22); 31.4 (C-23); 28.8 (C-24); 30.3 (C-25); 67.0
(C-26); 17.1 (C-27). HRMS (ESI-FT-ICR) m/z: 494.29946 [M + Na]+
2.1.11. (25R)-2␤-Azido-3␤,5-dihydroxy-5␤-spirostan-6-one
(17)
Methanesulfonic acid (195 ␮L, 3.0 mmol, 2.2 eq.) was added to
a stirred solution of triol 12 (600 mg, 1.33 mmol) and triph-
enylphosphine (1.05 g, 4.0 mmol, 3 eq.) in dry THF (60 mL).
The temperature was raised to 40 ◦C, and a solution of DIAD
(770 ␮L, 4 mmol, 3 eq.) in 5 mL of THF was added dropwise
over a 10 min period under an argon atmosphere. The reac-
tion mixture was stirred vigorously for 24 h at 40 ◦C, and then,
the volatiles were evaporated off under reduced pressure. The
crude product was purified by flash column chromatography
to give a pure product which was analyzed by 1H NMR con-
firming the structure of the mesylate. This compound was
carefully dried and dissolved in DMPU (30 mL). NaN3 (112 mg,
2.0 mmol) was added, and the resulting mixture was stirred
vigorously under an argon atmosphere at 50 ◦C for 48 h. TLC
analysis of a mini work-up revealed the formation of minor
impurities. The usual work-up (EtOAc) yielded a crude prod-
uct, which was purified by flash column chromatography (hex-
ane/EtOAc 3:1) to afford, by collection of the more hydrophilic
fractions, the azide 17 (320 mg, 51%). m.p. (acetone): 218 ◦C
(decomp). IR (KBr, cm−1): 3337, 3315, 2095, 1718, 1050, 980, 920,
860. 1H NMR (CDCl3): ı = 0.72 (3 H, s, H-19); 0.75 (3 H, s, H-18);
0.81 (3 H, d, J = 6.4 Hz, H-27); 0.97 (3 H, d, J = 6.7 Hz, H-21); 3.35 (1
H, t, J = 10.3 Hz, H-26ax); 3.48 (1 H, dd, J = 10.4/4.3 Hz, H-26eq);
3.90 (1 H, br m, H-2␣); 4.06 (1 H, m, H-3␣); 4.41 (1 H, m, H-16␣).
13C NMR (CDCl3): ı = 35.2 (C-1); 66.1 (C-2); 69.3 (C-3); 35.6 (C-
4); 81.6 (C-5); 212.7 (C-6); 42.1 (C-7); 37.2 (C-8); 45.7 (C-9); 44.4
(calculated for C27H41NaN3O4: 494.29949).
2.1.14. (25R)-3␤-(t-Butoxycarbonylamino)-5-hydroxy-5␣-
spirostan-6-one (20)
Lindlar catalyst (300 mg) was added to a solution of azide
19 (700 mg, 1.5 mmol) in 100 mL of absolute EtOH. The mix-
ture was treated successively with hydrogen and vacuum and
finally stirred under a hydrogen atmosphere for 36 h. The cat-
alyst was then removed by filtration and the resulting solution
was evaporated under reduced pressure to give a crude prod-
uct, which was dissolved in dioxan (60 mL) and treated with
triethylamine (560 ␮m, 4.0 mmol) and di-tert-butyl dicarbon-
ate (436 mg, 2.0 mmol). The reaction mixture was stirred for
20 h and then, concentrated to half volume under reduced
pressure. The usual work-up (CHCl3) yielded a crude product,

6
steroids 7 1 ( 2 0 0 6 ) 1–11
which was purified by flash column chromatography (hex-
ane/EtOAc 2:1) to give the ketol 20 (620 mg, 76%). m.p. (MeOH):
224–227 ◦C. IR (KBr, cm−1): 3400, 3235, 1715, 1710, 1078, 980,
912, 860. 1H NMR (CDCl3): ı = 0.78 (3 H, s, H-18); 0.70 (3 H, s, H-
19); 1.40 (9 H, s, (CH3)3C); 2.82 (1 H, t, J = 13.0 Hz, H-7␣); 3.36 (1 H,
t, J = 10.8 Hz, H-26ax); 3.49 (1 H, dd, J = 10.8/4.0 Hz, H-26eq); 3.85
(1 H, br m, H-3␣); 4.40 (1 H, m, H-16␣); 4.69 (1 H, br m, NHCO).
13C NMR (CDCl3): ı = 30.0 (C-1); 30.2 (C-2); 66.6 (C-3); 34.7 (C-
4); 80.1 (C-5); 214.3 (C-6); 42.0 (C-7); 37.1 (C-8); 44.5 (C-9); 42.6
(C-10); 21.4 (C-11); 39.7 (C-12); 41.2 (C-13); 56.2 (C-14); 31.6 (C-
15); 80.8 (C-16); 62.1 (C-17); 16.1 (C-18); 14.1 (C-19); 41.7 (C-20);
14.4 (C-21); 109.3 (C-22); 31.4 (C-23); 28.8 (C-24); 30.3 (C-25); 67.0
(C-26); 17.1 (C-27); 28.3, 2 × 28.5 ((CH3)3C); 79.2 ((CH3)3C); 155.8
(NCO). HRMS (ESI-FT-ICR) m/z: 546.37169 [M + H]+ (calculated
for C32H52NO6: 546.37163).
25); 66.9 (C-26); 17.1 (C-27). HRMS (ESI-FT-ICR) m/z: 451.28249
[M + Na]+ (calculated for C27
40NaO4: 451.28243).
H
2.1.17. (25R)-2␤,3␤,5-Trihydroxy-5␣-spirostan-6-one (23)
AgAcO (965 mg, 5.85 mmol) and I2 (890 mg, 3.5 mmol) were
added consecutively to a solution of the olefin 22 (1.0 g,
2.33 mmol) in 50 mL of AcOH. The reaction was stirred vig-
orously at room temperature until the I2 had been completely
consumed (approximately 1 h), and then, 1 mL of a solution
of 96% AcOH was added. The mixture was refluxed under a
nitrogen atmosphere for 3 h, diluted with EtOAc (150 mL), and
filtered to eliminate the inorganic salts. The resulting solu-
tion was washed successively with solutions of 10% Na2SO3
(2 × 100 mL) and 10% KOH (2 × 100 mL), dried over Na2SO4, and
concentrated under reduced pressure. The resulting crude
product was dissolved in 60 mL of a solution of 5% KOH in
MeOH and refluxed for 30 min. The usual work-up (EtOAc)
yielded a crude product, which was purified by flash col-
umn chromatography (hexane/EtOAc 3:1) to give the triol
23 (660 mg, 61%). m.p. (heptane/acetone): 251–253 ◦C. IR (KBr,
cm−1): 3487, 3390, 3337, 1705, 1235, 1050, 978, 920, 860. 1H NMR
(CDCl3): ı = 1.00 (3 H, s, H-19); 0.77 (3 H, s, H-18); 0.78 (3 H,
d, J = 6.4 Hz, H-27); 0.97 (3 H, d, J = 6.7 Hz, H-21); 2.78 (1 H, t,
J = 12.6 Hz, H-7␣); 3.36 (1 H, t, J = 10.3 Hz, H-26ax); 3.48 (1 H, dd,
J = 10.4/4.3 Hz, H-26eq); 3.96–4.07 (2 H, m, H-2␣ + H-3␣); 4.40 (1
H, m, H-16␣). 13C NMR (CDCl3): ı = 30.5 (C-1); 68.7 (C-2); 67.9 (C-
3); 36.0 (C-4); 80.1 (C-5); 212.9 (C-6); 41.5 (C-7); 38.3 (C-8); 43.4
(C-9); 44.3 (C-10); 22.0 (C-11); 39.5 (C-12); 41.0 (C-13); 56.5 (C-14);
31.6 (C-15); 80.3 (C-16); 61.9 (C-17); 16.3 (C-18); 16.6 (C-19); 41.5
(C-20); 14.4 (C-21); 109.3 (C-22); 31.3 (C-23); 28.7 (C-24); 30.2 (C-
25); 66.9 (C-26); 17.1 (C-27). HRMS (ESI-FT-ICR) m/z: 485.28732
[M + Na]+ (calculated for C27H42NaO6: 485.28733).
2.1.15. (25R)-3␤-Amino-5-hydroxy-5␤-spirostan-6-one
(21)
Ketol 20 (600 mg, 1.1 mmol) was dissolved in a solution of 10%
KOH in MeOH (50 mL). The reaction mixture was refluxed for
24 h, neutralized with 5% HCl, and then, concentrated under
reduced pressure. The crude product was purified by flash
column chromatography (hexane/EtOAc 3:1) to give a pure
compound, which was analyzed by 1H NMR confirming the
epimerization of C-5 as indicated by the coupling pattern of
the now equatorial 3␣-H (minor amounts of the starting mate-
rial were also isolated). The resulting ketol was dissolved in
CH2Cl2 (30 mL) and treated with trifluoroacetic acid (100 ␮L).
The reaction mixture was stirred for 10 min, then, diluted with
CHCl3, and washed with saturated Na2CO3 (3 × 50 mL) and
brine (50 mL). The organic layer was then concentrated, and
the product was recrystallized from heptane/acetone to afford
the pure aminoketol 21 (313 mg, 64%). m.p. (heptane/acetone):
243–244 ◦C. IR (KBr, cm−1): 3435, 3367, 1720, 1110, 1048, 980,
900, 860. 1H NMR (CDCl3): ı = 0.77 (3 H, s, H-18); 0.77 (3 H, s,
H-19); 0.78 (3 H, d, J = 6.4 Hz, H-27); 0.98 (3 H, d, J = 6.7 Hz, H-21);
3.36 (1 H, t, J = 10.8 Hz, H-26ax); 3.49 (1 H, dd, J = 10.7/3.9 Hz, H-
26eq); 3.95 (1 H, m, H-3␣); 4.41 (1 H, m, H-16␣). 13C NMR (CDCl3):
ı = 25.0 (C-1); 27.7 (C-2); 64.8 (C-3); 37.2 (C-4); 81.1 (C-5); 212.2 (C-
6); 41.5 (C-7); 36.8 (C-8); 42.9 (C-9); 44.1 (C-10); 21.5 (C-11); 39.5
(C-12); 41.3 (C-13); 56.6 (C-14); 31.6 (C-15); 80.3 (C-16); 62.0 (C-
17); 16.3 (C-18); 17.2 (C-19); 41.4 (C-20); 14.5 (C-21); 109.3 (C-22);
31.3 (C-23); 28.7 (C-24); 30.2 (C-25); 66.9 (C-26); 17.1 (C-27). HRMS
(ESI-FT-ICR) m/z: 446.31928 [M + H]+ (calculated for C27H44NO4:
446.31920).
2.1.18. (25R)-3␤-Azido-2␤,5-dihydroxy-5␣-spirostan-6-one
(24)
Ketol 23 (900 mg, 1.9 mmol) was treated in a similar way as
described for the synthesis of 19 to give the azidoketol 24
(541 mg, 57%). m.p. (acetone): 216–218 ◦C. IR (KBr, cm−1): 3405,
3358, 2094, 1705, 1200, 1050, 980, 920, 860. 1H NMR (CDCl3):
ı = 1.01 (3 H, s, H-19); 0.77 (3 H, s, H-18); 0.79 (3 H, d, J = 6.4 Hz,
H-27); 0.97 (3 H, d, J = 6.7 Hz, H-21); 2.80 (1 H, t, J = 12.5 Hz, H-
7␣); 3.36 (1 H, t, J = 10.4 Hz, H-26ax); 3.49 (1 H, dd, J = 10.4/4.4 Hz,
H-26eq); 3.83 (1 H, br m, H-3␣); 4.08 (1 H, m, H-2␣); 4.41 (1 H,
m, H-16␣). 13C NMR (CDCl3): ı = 30.5 (C-1); 68.8 (C-2); 66.5 (C-3);
36.3 (C-4); 80.0 (C-5); 213.0 (C-6); 41.4 (C-7); 38.2 (C-8); 43.3 (C-
9); 44.2 (C-10); 21.9 (C-11); 39.6 (C-12); 41.0 (C-13); 56.5 (C-14);
31.5 (C-15); 80.4 (C-16); 61.9 (C-17); 16.3 (C-18); 16.6 (C-19); 41.5
(C-20); 14.4 (C-21); 109.3 (C-22); 31.3 (C-23); 28.7 (C-24); 30.2 (C-
25); 66.9 (C-26); 17.1 (C-27). HRMS (ESI-FT-ICR) m/z: 462.31419
[M + H]+ (calculated for C27H44NO5: 462.31414.
2.1.16. (25R)-5-Hydroxy-5␣-spirost-2-en-6-one (22)
Ketol 7 (3.0 g, 6.7 mmol) was subjected to tosylation (TsCl,
1.26 g, 10.0 mmol) and subsequent dehydrotosylation (Li2CO3,
3.5 g; LiBr, 4.2 g) in a similar way as described for the syn-
thesis of 11 to give the olefin 22 (2.29 g, 80%). m.p. (acetone):
197–198 ◦C. IR (KBr, cm−1): 3550, 3020, 1707, 1060, 1029, 980,
900, 860. 1H NMR (CDCl3): ı = 0.76 (3 H, s, H-18); 0.72 (3 H, s,
H-19); 0.97 (3 H, d, J = 6.7 Hz, H-21); 2.20 (1 H, dd, J = 12.6/4.5 Hz,
H-7␤); 2.71 (1 H, t, J = 12.8 Hz, H-7␣); 5.61 (1 H, m, H-2); 5.66 (1 H,
m, H-3). 13C NMR (CDCl3): ı = 34.5 (C-1); 125.4 (C-2); 122.3 (C-3);
30.1 (C-4); 77.9 (C-5); 211.1 (C-6); 42.7 (C-7); 36.8 (C-8); 45.5 (C-
9); 42.2 (C-10); 20.8 (C-11); 39.6 (C-12); 40.8 (C-13); 56.2 (C-14);
31.5 (C-15); 80.4 (C-16); 62.0 (C-17); 16.3 (C-18); 17.2 (C-19); 41.4
(C-20); 14.6 (C-21); 109.3 (C-22); 31.3 (C-23); 28.7 (C-24); 30.2 (C-
2.1.19. (25R)-3␤-(t-Butoxycarbonylamino)-2␤,5-dihydroxy-
5␣-spirostan-6-one (25)
Azidoketol 24 (600 mg, 1.2 mmol) was treated in a similar way
as described for the synthesis of 20 to give the ketol 25 (374 mg,
54%). m.p. (heptane/acetone): 232–235 ◦C. IR (KBr, cm−1): 3434,
3320, 1705, 1245, 1065, 980, 920, 860. 1H NMR (CDCl3): ı = 1.02 (3
H, s, H-19); 0.78 (3 H, s, H-18); 0.79 (3 H, d, J = 6.4 Hz, H-27); 0.97 (3
H, d, J = 6.7 Hz, H-21); 1.42 (9 H, s, (CH3)3C); 3.36 (1 H, t, J = 10.3 Hz,
H-26ax); 3.48 (1 H, dd, J = 10.4/4.4 Hz, H-26eq); 3.95 (1 H, br m,

steroids 7 1 ( 2 0 0 6 ) 1–11
7
H-3␣); 4.09 (1 H, m, H-2␣); 4.41 (1 H, m, H-16␣); 4.68 (1 H, br
m, NHCO). 13C NMR (CDCl3): ı = 30.5 (C-1); 68.7 (C-2); 67.9 (C-3);
36.0 (C-4); 80.1 (C-5); 212.9 (C-6); 41.5 (C-7); 38.3 (C-8); 43.4 (C-9);
44.3 (C-10); 22.0 (C-11); 39.5 (C-12); 41.0 (C-13); 56.5 (C-14); 31.6
(C-15); 80.3 (C-16); 61.9 (C-17); 16.3 (C-18); 16.6 (C-19); 41.5 (C-
20); 14.4 (C-21); 109.3 (C-22); 31.3 (C-23); 28.7 (C-24); 30.2 (C-25);
66.9 (C-26); 17.1 (C-27); 3 × 28.5 ((CH3)3C); 79.1 ((CH3)3C); 155.3
(NCO). HRMS (ESI-FT-ICR) m/z: 562.35659 [M + H]+ (calculated
for C32H52NO7: 562.35653).
A/B cis ring junction). In this sense, our goal took advantage of
several features of the 5␣-hydroxy-6-oxo system as a precur-
sor functionality, e.g., its great synthetic accessibility starting
from the readily available ꢀ5-olefins and the known net reten-
tion of the configuration of the functional groups in the A ring
upon alkaline epimerization of C-5 [18].
Scheme 1 summarizes the strategy for functionalizing
rings A and B from triol 5, which was obtained from com-
mercially available diosgenin according to a reported proce-
dure [16]. Synthesis of the key intermediate 6 proceeded via
selective protection of the equatorial 3␤-OH group with tert-
butyldimethylsilyl (TBDMS) ether [19]. PCC oxidation of the
6␤-OH group and subsequent removal of the TBDMS group by
using TBFA gave the previously reported ketol 7 [19], repre-
senting an advanced intermediate on the synthetic pathway
towards the target 5␤-hydroxyspirostanones. Epimerization of
C-5 was successfully accomplished by refluxing compound
7 in a strongly alkaline methanolic solution. In this process,
which required remarkably drastic reaction conditions [18,20],
the final equilibrium furnished the thermodynamically more
stable 5␤-OH derivative 9 in 74% yield.
Ketol 10, which is key for SAR studies, was obtained via
two consecutive epimerization steps of C-3 and C-5. Com-
pound 7 was treated with DIAD/Ph3P/HCOOH in THF followed
by cleavage of the formyloxy group in 56% overall yield, and the
subsequent above described inversion of the C-5 configura-
tion was performed. The importance of performing the useful
Mitsunobu reaction as the first step of this synthetic pathway
should be noted. This method has been described as leading
to equatorial hydroxy groups, while the reversed route would
lead to unsatisfactory results since the 3␤-OH possesses an
axial orientation in the 5␤-series.
The configuration of C-5 in compounds 9 and 10 was
deduced by performing 1D NOE difference experiments in
which the absolute lack of enhancement of the A-ring proton
signals upon irradiation of H-19, with the obvious exception of
OH-5␤, confirmed the A/B cis ring junction. In the C-5 epimer-
ization procedures, the stereochemistry of the OH at C-3 was
retained as indicated in the 1H NMR spectra of ketols 9 and
10. The small width of the equatorial H-3 signal in 9 as well
as the broad multiplet assigned to the axial H-3 in 10, justify
the unequivocal axial and equatorial orientations of the OH-3␤
and OH-3␣ groups, respectively.
The formation of the ꢀ2 double bond was accomplished
by tosylation of the 3␤-OH group and subsequent dehydro-
tosylation without prior purification to give the olefin 11.
The 2␤,3␤,5␤-trihydroxy-6-oxo compound 12 was obtained by
using the common osmium tetroxide/NMO dihydroxylation
procedure. We did not detect the formation of the 2␣,3␣-diol
functionality. This finding was consistent with some previous
reports of hydroxylation of this system [20].
2.1.20. (25R)-3␤-Amino-2␤,5-dihydroxy-5␤-spirostan-6-
one (26)
Ketol 25 (300 mg, 0.5 mmol) was treated in a similar way
as described for the synthesis of 21 to give the aminoketol
26 (147 mg, 59%). m.p. (heptane/acetone): 250–251 ◦C. IR (KBr,
cm−1): 3360, 3328, 3254, 1717, 1250, 1045, 980, 922, 860. 1H
NMR (CDCl3): ı = 0.72 (3 H, s, H-19); 0.77 (3 H, s, H-18); 0.80
(3 H, d, J = 6.4 Hz, H-27); 0.97 (3 H, d, J = 6.6 Hz, H-21); 3.36 (1
H, t, J = 10.4 Hz, H-26ax); 3.49 (1 H, dd, J = 10.4/4.4 Hz, H-26eq);
3.97–4.10 (2 H, m, H-32 + H-3␣); 4.41 (1 H, m, H-16␣). 13C NMR
(CDCl3): ı = 35.5 (C-1); 68.7 (C-2); 67.9 (C-3); 35.7 (C-4); 81.9 (C-5);
212.0 (C-6); 42.0 (C-7); 37.3 (C-8); 45.4 (C-9); 44.3 (C-10); 22.0 (C-
11); 39.5 (C-12); 41.1 (C-13); 56.5 (C-14); 31.5 (C-15); 80.3 (C-16);
61.9 (C-17); 16.3 (C-18); 16.7 (C-19); 41.6 (C-20); 14.4 (C-21); 109.3
(C-22); 31.3 (C-23); 28.7 (C-24); 30.2 (C-25); 66.9 (C-26); 17.1 (C-
27). HRMS (ESI-FT-ICR) m/z: 462.31410 [M + H]+ (calculated for
C
27H44NO5: 462.31413).
2.2. Biological activity
For toxicity testing, the procedures reported by Streloke and
Ko¨pp [17] were followed. Mosquitoes (Aedes aegypti) were kept
in 250 mL glass beakers filled and aerated at least 1 day before
larvae were introduced. Water loss due to evaporation was
low, but vessels were refilled with aerated tap water as nec-
essary. To collect first-instar larvae, freshly laid egg masses
were hatched in small plastic Petri plates for 24 h. Only lar-
vae that had left the egg mass within this time were used for
experiments. Twenty larvae were placed in each test vessel.
The day of dosing was considered as day 0. During the first
two days, larvae were fed 1 mg food per larva every second
day. On approximately day 3, daily feeding commenced. Con-
trol adult emergence began on day 7 and lasted approximately
2 days. Experiments were terminated on day 25, after which
no additional emergence was observed. If 20 mosquitoes were
found at termination, that number was set as 100% emer-
gence. Results were considered valid if control emergence was
greater than 90% [17]. Statistical analysis of the data was car-
ried out utilizing the Duncan test as implemented in the Stat-
graphics program.
We also wanted to introduce epoxide functions on the A
ring, since these functional groups are known to be a source
of additional stereochemical diversity upon opening of the
oxirane ring. Epoxidation of the ꢀ2 double bond with mCPBA
afforded a mixture of ␣ and ␤ orientated epoxides 13 and 14,
respectively. The simple trans diaxial acid catalyzed opening
of epoxide 13 gave the triol 15. The cerium(III) chloride pro-
moted ring opening [21] of epoxide 14 afforded the 2␣-azido-
3␤-hydroxy derivative, which upon consecutive reduction of
3.
Results and discussion
3.1.
Synthesis and structural elucidation
Originally, to facilitate the generation of stereochemical diver-
sity on ring A, we were encouraged to address a synthetic
strategy that allowed both functionalizing the ring A at an
early stage (i.e., A/B trans ring junction) and in a later time (i.e.,

8
steroids 7 1 ( 2 0 0 6 ) 1–11
Scheme 1 – Reagents and conditions: (a) TBDMSCl, imidazole, DMF (84%); (b) (i) PCC, CH2C12, (ii) TBFA, THF (82%); (c) (i)
HCO2H, Ph3P, DIAD, THF, (ii) NaHCO3, MeOH-H2O (82%); (d) KOH 10%, MeOH, reflux (71–74%); (e) (i) TsCl, Py, (ii) LiBr, Li2CO3,
DMF, reflux (74%); (f) OsO4, NMO, THF-tBuOH-H2O (68%); (g) mCPBA/CH2Cl2 (72%); (h) HClO4 70%, H2O-acetone (81%); (i) (i)
NaN3, CeCl3·7H2O, CH3CN-H2O, (ii) Lindlar catalyst, H2 (64%). DIAD: diisopropylazodicarboxylate; TBFA:
tetrabutylammonium fluoride; NMO: N-methylmorpholine N-oxide.
the azido group, led to the corresponding aminoketol 16 in
64% yield from epoxide 14.
Woodward dihydroxylation [23] led to the successful synthesis
of triol 23 in overall 49% yield from 7. The azidoketols 19 and 24
were then synthesized by submitting ketols 7 and 23 to a pro-
cedure similar to that previously used to obtain compound 17.
It should be noted that only the equatorial hydroxyls reacted in
this procedure as described in the original report [22], though
in our hand the procedure gave lower yields than the original.
In every case, none of the axial hydroxy groups were affected,
and solely the starting material and minor elimination prod-
ucts were detected after chromatographic purification.
As shown in Scheme 2, introduction of the azido group
at C-3 was carried out in the 5␣-series looking for selectiv-
ity for the equatorial 3␤-OH. Thus, after reduction of the azido
group and Boc protection of the corresponding amine afford-
ing compounds 20 and 25, epimerization of C-5 and subse-
quent removal of the Boc protecting group led to the final
aminoketols 21 and 26 in overall 38% and 16% yield from 7,
respectively.
2D NMR experiments were performed on the final com-
pounds allowing the unequivocal assignments. In general,
the lack of a downfield shift in the CH3-19 signal of the 5␤-
series did not provide enough evidence for the stereochem-
istry of the functions at C-2 and C-3. These were determined
by NOESY analyses of the steroidal nucleus. For example, in
epoxide 13, the NOE cross-peaks between OH-5␤, H-4␤ and H-
3␤ confirmed the stereochemistry 2␣,3␣. NOESY experiments
In order to further assess the influence on the antiecdys-
teroid effect of modified A rings bearing different hydrogen
bond acceptor/donor functions, e.g., OH and NH2, we extended
our design to cover other types of structural features. Three
examples, highlighted in Scheme 2, illustrate the possibility
of introducing additional amino groups on the ␤ face of ring A
from the corresponding alcohols. As it can be noted, we also
focused on functionalizing the A ring in a step prior to the C-5
epimerization, making use of the replacement of one hydroxy
group for an amino via a Mitsunobu reaction as the key step
of this synthetic route.
The first attempt to assess the scope of this synthetic
pathway involved the selective replacement of 3␤-OH in triol
12 by azide with net retention of the configuration. This
was accomplished by utilizing methanesulfonic acid as the
acidic/nucleophilic component of the Mitsunobu reaction and
consecutive azide displacement [22]. Final reduction of the
azido group of 17 by hydrogenation over Lindlar catalyst
afforded the aminoketol 18 in 65% yield.
After assessing the success and selectivity of this proce-
dure in our hands, it was applied to additionally introduce the
3␤-amino function in order to obtain the amino analogues of
ketols 9 and 12. For this, the classical tosylation and elim-
ination procedures to give olefin 22, followed by the Prevost-

steroids 7 1 ( 2 0 0 6 ) 1–11
9
Scheme 2 – Reagents and conditions: (a) (i) MeSO3H, Ph3P, DIAD, THF, (ii) NaN3, DMPU (51–57%); (b) Lindlar catalyst, H2 (91%);
(c) (i) Lindlar catalyst, H2, (ii) Boc2O, Et3N, dioxan (76%); (d) (i) KOH 10%, MeOH, reflux, (ii) CF3CO2H, CH2Cl2 (59–64%); (e) (i)
TsCl, Py, (ii) LiBr, Li2CO3, DMF, reflux (80%); (f) (i) AgAcO2, I2, AcOH-H2O, reflux, (ii) KOH 5%, MeOH (61%). DMPU:
1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidonone.
of epoxide 14, ketol 12, and aminoketols 18 and 26 corrobo-
rated the assigned ␤ stereochemistry of the A-ring functional
groups. Additionally, the 1H NMR spectra of the 2␤,3␣ func-
tionalized compounds 15 and 16 showed a coupling pattern
typical of vicinal equatorial protons, thus confirming the trans
diaxial orientation of the functional groups at C-2 and C-3.
cerning the influence of the nature and the stereochemistry
of the A-ring functional groups on antiecdysteroid activity.
The greatest inhibitory effect on the development of the
first-instar larvae was elicited by ketols 9 and 12, which bear
the naturally occurring 3␤-hydroxy and 2␤,3␤-dihydroxy func-
tions, respectively. This successful result demonstrated that
the above mentioned compounds are somehow capable of
binding the ecdysteroid receptor and also ratified our assump-
tion that spirostanones bearing A- and B-ring structural sim-
ilarities to the natural ecdysteroids could compete for the
active site, thereby behaving as antagonists of the natural
compounds.
3.2.
Biological activity
The antiecdysteroid activities of compounds 9, 10, 12, 15, 16,
18, 21, and 26 were evaluated on the metamorphosis bioassay
of the mosquito A. aegypti following a previously developed
procedure [17]. Analysis of the moulting inhibitory effect as
shown in Figs. 2 and 3 allowed us to arrive at conclusions con-
Moreover, analysis of Fig. 2 also highlights some tenden-
cies, which provide additional insight into a better under-

10
steroids 7 1 ( 2 0 0 6 ) 1–11
of these compounds. A. aegypti were incapable of complet-
ing their larval molt and pupation, even at 0.01 mg/mL. Com-
pounds 9 and 12 possessed the desired effect on lepidopteran
target insects, in which the initiation of a precocious molt
quickly leads to death [24]. Additionally, these findings were
consistent with our experiences from previous experiments
[9] where mortality was also high and linked to emergence.
Most insects, however, did not expire in the process of emer-
gence; the highest mortality resulted prior to the emergence
period during the larval and pupal stages.
4.
Conclusions
Fig. 2 – Inhibitory effect on the development of the
first-instar Aedes aegypti larvae under static exposure to
different concentrations of compounds 9, 10, 12, 15, 16, 18,
21, and 26.
We synthesized eight new 5␤-hydroxy-spirostan-6-ones bear-
ing hydroxy and amino groups on ring A. The high antiecdys-
teroid activities elicited by ketols 9 and 12 in the metamor-
phosis bioassay of the mosquito A. aegypti make these com-
pounds very good candidates for further applications as envi-
ronmental friendly insecticides and support the potential use
of steroidal sapogenins as steroidal hormones biomimetics.
standing of the underlying mechanisms of this process. For
example, for ketol 10 and aminoketols 21 and 26, which
bear the 3␣-hydroxy and 3␤-amino functions, respectively,
moulting was not affected in any of the treated groups
with the exception of the one at 0.1 mg/mL, representing
the higher exposure concentration. At this concentration,
time required for ≥80 of the fourth instar-larvae to molt (to
adult) was delayed by 24 and 48 h, respectively, compared
to the other treatments and controls. Thus, the antagonistic
effect was almost null. These intriguing results are in agree-
ment with those found in nature where the ecdysone and 2-
desoxyecdysone derivatives are the most active and prevalent,
whereas the 2-desoxy-3-epiecdysone is less common [1,3].
On the other hand, the effect of the stereochemistry of
the hydroxy function at C-2 did not have much effect on the
antiecdysteroid activity compared to that of the function at
C-3, at least in our model system. Where ketol 15 displayed a
moderate antagonistic effect at all the tested concentrations.
However, introduction of amino groups at C-3, either with ␣ or
␤ stereochemistry, led to an unsuccessful antagonist effect of
compounds 16 and 18.
Acknowledgments
We are grateful to Dr. Pedro Ortiz from the University of
Havana and Dr. Andrea Porzel from the Institute of Plant Bio-
chemistry for the spectroscopic determinations. HRMS (ESI-
FT-ICR) was provided by the Institute of Plant Biochemistry,
Halle (Saale), Germany. We gratefully acknowledge Dr. Ju¨ rgen
Schmidt for the HRMS spectra.
r e f e r e n c e s
[1] Koolman J. Ecdysteroids. Zoolog Sci 1990;7:563–80.
[2] Koolman J. Ecdysone: from chemistry to mode of action.
Stuttgart New York: George Thieme-Verlag; 1989. pp.
152–60.
[3] Khripach VA, Zhabinskii VN, de Groot AE. Brassinosteroids:
a new class of plant hormones. Academic Press; 1999.
[4] Richter K, Adam G, Vorbrodt HM. Inhibiting effect of
22S,23S-homobrassinolide on the moult of the cockroach
Periplaneta Americana. J Appl Entomol 1987;103:532–44.
[5] Hetru C, Rousel JP, Mori K, Nakatani Y. Antiecdysteroid
activity of brassinosteroids. CR Acad Sc Paris 1986;302
Serie 11(7):417–20.
For the more active compounds 9 and 12, delayed develop-
ment resulting in mortality of first-instar larvae was enhanced
in a dose-dependent manner with increasing concentrations,
demonstrating the competitive profile of the inhibitory effect
[6] Lehmann M, Vorbrodt HM, Adam G, Koolman J.
Antiecdysteroid activity of brassinosteroids. Experientia
1988;44:355–6.
[7] Brosa C, Nusimovich S, Peracaula R. Synthesis of new
brassinosteroids with potential activity as
antiecdysteroids. Steroids 1994;59:463–7.
[8] Voigt B, Porzel A, Adam G, Golsch D, Adam W, Wagner C,
et al. Synthesis of 2,24-diepicastasterone and
3,24-diepicastasterone as potential brassinosteroid
metabolites of the cockroach Periplaneta Americana. Collect
Czech Chem Commun 2002;67(1):91–102.
[9] Davison GP, Restrepo R, Martinez G, Coll F, Leon OS.
Effects of a brassinosteroid analogue to mosquito larvae.
Ecotoxicol Environ Saf 2003;56:419–24.
[10] Marquardt V, Adam G, Coll F, Alonso E. Preparation of
brassinosteroid analogues of spirotanes as plant growth
regulators. German East 1988;DD 273:638 [CA 112: 217353].
Fig. 3 – Rates of successful emergence and dead pupae in
Aedes aegypti after 19 days of static exposure to various
concentrations of compounds 9, 10, 12, 15, 16, 18, 21, and
26 in first-instar larvae. Error bars indicate S.E.

steroids 7 1 ( 2 0 0 6 ) 1–11
11
[11] Coll F, Jomarron I, Robaina C, Alonso E, Cabrera MT.
[18] Yates P, Stiver S. Studies of the synthesis of 5-hydroxy
steroids and related 6-keto steroids. Can J Chem
1987;65:2203–16.
[19] Kocienski PJ. Protecting groups. Stuttgart: Thieme-Verlag;
2000. pp. 33–36.
[20] Brosa C, Soca L, Terciabas E, Ferrer JC, Alsina A. New
synthetic brassinosteroids: a 5␣-Hydroxy-6-ketone analog
with strong plant growth promoting activity. Tetrahedron
1998;54:12337–48.
[21] Sabitha G, Babu RS, Rajkumar M, Jadav JS. Cerium (III)
chloride promoted highly regioselective ring opening of
epoxides and aziridines using NaN3 in acetonitrile: A facil
synthesis of 1,2-azidoalcohols and 1,2-azidoamines. Org
Lett 2002;4:343–5.
Polyhydroxyspirostanones as growth plants regulators. PTC
Int Appl 1995;WO 97 13:780 [CA 126: 343720].
[12] Iglesias-Arteaga MA, Gil R, Perez CS, Coll F. Spirostanic
analogues of teasterone. Synthesis, characterisation and
biological activity of laxogenin, (23S)-hydroxylaxogenin
and 23-ketolaxogenin. J Chem Soc Perkin Trans 1
2001;3:261–6.
[13] Iglesias-Arteaga MA, Perez CS, Coll F. Spirostanic
analogues of castasterone. Steroids 2002;67(3/4):159–64.
[14] Robaina C, Izquierdo I, Alonso E, Coll F, Coll D, Zullo MAT.
Synthesis and biological activity of three new 5-hydroxy
spirostanic brassinosteroid analogues. J Braz Chem Soc
2003;14:466–9.
[15] Novosel’skaya IL, Gorovits MB, Abubakirov NS. Analogs of
ecdysone from steroid sapogenins II.
[22] Davis AP, Dresen S, Lawless JL. Mitsunobu reactions with
methanesulfonic acid: The replacement of equatorial
hydroxyl groups by azide with net retention of
configuration. Tetrahedron Lett 1997;38:4305–8.
[23] Woodward RB, Brutcher FV. Cis-hydroxylation of a
synthetic steroid intermediate with iodine, silver acetate
and wet acetic acid. J Am Chem Soc 1958;80:209–
11.
(25R)-5␣-Spirost-7-ene-3␤,5,14␣-triol-6-one. Chem Nat Prod
1975;11:268–71.
[16] Dawidar AWZ. Hydroxylation of ꢀ5-steroids with
N-bromosuccinimide to 5␣,6␤-diols. Z Naturforsch
1980;35:102–6.
[17] Streloke M, Ko¨pp H. Long term toxicity test with
Chironomus riparius: development and validation of a new
test system. Mitt a. d. Biol Bundesanst. Berlin, Vienna:
Blackwell Wissenschaftsverlag 1995: 315–324.
[24] Dhadialla TS, Carlson GS, LeDat P. New insecticides with
ecdysteroidal and juvenile hormone activity. Annu Rev
Entomol 1998;43:545–69.