Synthesis and preliminary bioactivity evaluation of new pregnane brassinosteroid-like compounds

Article abstract of DOI:10.1139/v05-051

Seven new pregnane compounds bearing some representative A- and B-ring brassinosteroid functions, as well as hydrogen bond donor and acceptor ones on the D ring, were efficiently synthesized. The obtained compounds did not show remarkable plant growth-pro

Full text of DOI:10.1139/v05-051

1084
Synthesis and preliminary bioactivity evaluation of
new pregnane brassinosteroid-like compounds
Daniel G. Rivera and Francisco Coll
Abstract: Seven new pregnane compounds bearing some representative A- and B-ring brassinosteroid functions, as
well as hydrogen bond donor and acceptor ones on the D ring, were efficiently synthesized. The obtained compounds
did not show remarkable plant growth-promoting activity in the radish hypocotyl elongation and cotyledon expansion
bioassays, however, introducing oxygen and amino functions on the D ring led to an enhancement of the bioactivity.
The 16β-functionalized pregnane brassinosteroid-like compounds were slightly more active than the 16α-functionalized
ones.
Key words: steroids, brassinosteroids, pregnane analogues.
Résumé : On a réalisé des synthèses efficaces de sept nouveaux dérivés du prégnane portant des fonctions représentati-
ves des cycles A et B des brassinostéroïdes, ainsi que des fonctions pouvant donner ou accepter des liaisons hydrogènes
sur le cycle D. Les composés obtenus ne présentent pas d’activité remarquable pour la promotion de la croissance
dans des bioessais d’élongation de l’hypocotyle du radis et de l’expansion du cotylédon, toutefois l’introduction de
fonctions oxygénées et aminées sur le cycle D conduit à une augmentation de leur bioactivité. Les composés du pré-
gnane fonctionnalisés en 16β, de type brassinostéroïde, sont légèrement plus actifs que ceux fonctionnalisés en 16α.
Mots clés: stéroïdes, brassinostéroïdes, analogues du prégnane.
[Traduit par la Rédaction] Rivera and Coll 1092
Introduction
biological point of view, its advantage is due to the greater
synthetic accessibility starting from the readily available
5,6-epoxide derivatives (12, 13).
Since their discovery (1), the brassinosteroids have com-
manded much interest in both the chemical and biological
communities (2, 3) because of their extremely high plant
growth-promoting activity and anti-stress effect (4). In view
of their challenging steroidal structures and the presence of
different stereochemical arrays of hydroxy groups, this class
of plant hormones has also been the subject of elegant syn-
thetic and biosynthetic studies (2, 3).
Nevertheless, the fascinating goal of achieving more syn-
thetically accessible bioactive compounds continues to be a
promising approach for improving the benefits of these hor-
mones in agriculture. Thus, a highlight of this program has
been the development of interesting analogues with drastic
modifications either in the side chains or the ring system (3,
5–8), which has also facilitated the structure–activity rela-
tionship (SAR) studies and a thorough understanding of their
mode of action (9–11).
Whereas brassinolide (1) is endowed with the unique fea-
ture of having a 6-oxo-7-oxa lactone function on ring B, a
number of bioactive analogues bearing the 5α-hydroxyl group
(e.g., 2) have been reported and the effect of this function on
the brassinosteroid-type activity is well documented (5, 12)
(Fig. 1). Whilst this later function certainly does not bring
out noteworthy improvements over the natural one from the
Additionally, despite the fact that most active natural and
synthetic brassinosteroids are known to be those with cam-
pestane, ergostane, and stigmastane side chains, Kohout and
co-workers have reported a set of analogues with androstane
(14–16) and pregnane (17–19) side chains (e.g., 3), some of
them showing activity in the bean second internode bioassay.
Herein, we report the synthesis and a preliminary evalua-
tion of the biological activity of seven pregnane brassinosteroid-
like compounds. In connection with ongoing projects towards
the development of brassinosteroid analogues, our approach
was to exploit the great accessibility of the 2α,3α,5α-trihyd-
roxy-6-oxo functionality and to introduce a variety of func-
tions on ring D of the pregnane skeleton. The potential
advantages of this approach should be realized in the further
possible construction of the natural brassinosteroid side
chain from 20-oxo derivatives (20), thereby achieving a
striking array of structural features not found in any natural
or synthetic brassinosteroid.
Results and discussion
The scope of our strategy is focused on the influence in
the activity of modified D rings bearing either hydrogen
Received 23 March 2004. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on 14 September 2005.
D.G. Rivera1,2 and F. Coll. Center for Natural Products Studies, Faculty of Chemistry, University of Havana, Zapata y G, Vedado,
10400 Habana, Cuba.
1Present address: Institute of Plant Biochemistry, Weinberg 3, D-06120, Halle (Saale), Germany.
2Corresponding author (e-mail: drivera@ipb-halle.de).
Can. J. Chem. 83: 1084–1092 (2005)
doi: 10.1139/V05-051
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Fig. 1. Structures of some natural and synthetic brassinosteroids.
bond acceptors or donors. With a view toward achieving this
goal, an efficient plan, which could generate A- and B-ring
brassinosteroid functionalities, as well as functional and
stereochemical diversity on ring D, was chosen. Moreover,
encouraged by the fact that D-ring functionalized brassino-
steroid analogues have not been studied, mainly because this
ring is not recognized as one of the subsites of interaction in
the brassinosteroid-receptor complex (11), we envisioned
not only generating a set of brassinosteroid-like pregnanes,
but also establishing the starting point of a more promising
strategy towards D-ring functionalized brassinosteroid ana-
logues.
Our approach for functionalizing rings A and B took ad-
vantage of the simple synthetic pathway to achieve the 5α-
hydroxy-6-oxo functionality from the ∆5 double bond via the
acid-catalyzed epoxide opening (13). The attainment of the
key ∆2 double bond was accomplished by selective tosyl-
ation of the 3β-OH group and subsequent dehydrotosylation
(without previous purification) to afford the precursor ∆2-5α-
hydroxy-6-oxo system. Additionally, we did not detect the
formation of the 2β,3β-diol functionality by using the com-
mon osmium tetroxide – N-methylmorpholine N-oxide
(NMO) procedure, which had been previously described in
the osmium-catalyzed asymmetric dihydroxylation of both
the ∆2-5α-hydroxy-6-oxo and ∆22 systems on stigmastane
analogues (12).
Scheme 1 summarizes the initial pathway to synthesize
the target brassinosteroid-like compounds. Ketol 4 was ob-
tained following a reported procedure (13) and represents
the starting material of our synthetic route. The D-ring
unfunctionalized analogue 7 was synthesized by a conven-
tional procedure, which involved the reduction of the conju-
gated ∆16-olefin by catalytic hydrogenation and the previously
described method for functionalizing ring A in 46% yield
from ketol 4. The emphasized importance of this analogue is
beyond its synthesis, since it is certainly necessary for SAR
studies. Alternatively, the pentahydroxylated analogue 9 was
obtained in good yield through the simultaneous ∆2- and
∆16-dihydroxylation processes, though several equivalents of
NMO and noncatalytic amounts of OsO4 as well as long re-
precursors 10 and 11 were successfully obtained by treat-
ment of intermediate 8 with NaOMe–MeOH and H2O2–
NaOH–MeOH, respectively. Final dihydroxylation led to the
desired analogues 12 and 13.
As can be noted, this initial synthetic planning allowed us
to only access one of the faces of ring D, since the expected
stereoselectivity was achieved in the introduction of epoxy,
methoxy, and hydroxy functions giving an α configuration
for substituents at C-16 and C-17. Clearly, to achieve stereo-
chemical diversity on ring D, it was critical to implement re-
actions that could enable us to obtain 16β-substituted
pregnanes. With the 16,17-epoxide previously obtained, the
oxirane ring opening was the first attempt to introduce dif-
ferent functionalities. However, in our hands, this system
was extremely sensitive, even to mild acidic and basic condi-
tions. Finally, this task was accomplished efficiently by per-
forming the successful Mitsunobu reaction (21) for the
epimerization of the 16α-hydroxy function, also making it
possible to access nonoxygenated functions previously intro-
duced at different sites of the steroidal moiety (22).
Two examples, highlighted in Scheme 2, illustrate the
possibility of conveniently functionalizing the β face of ring
D. Ketol 4 was initially tosylated and subsequently submit-
ted to the ∆16-dihydroxylation procedure affording the tosylate
14 in excellent overall yield. Further dehydrotosylation and
selective protection of the secondary 16α-OH group with
tert-butyldimethylsilyl (TBDMS) ether (23) gave the key
intermediate 15. The established ∆2-dihydroxylation proce-
dure and isopropylidene protection of the resulting diol suc-
ceeded in obtaining the acetonide 16 by using the 2,2-
dimethoxypropane – pyridinium p-toluenesulfonate (PPTS)
system without affecting the silicon protecting group (24).
Selective removal of the TBDMS group (23) by using
tetrabutylammonium fluoride (TBFA) gave the acetonide 17
in 33% overall yield, representing an advanced intermediate
on the synthetic pathway towards the 16β-functionalized de-
rivatives.
Epimerization of C-16 was accomplished by using the
known DIAD–Ph3P–HCO2H system and subsequent cleav-
age of the 16β-formyloxy group, following a protocol al-
ready applied to a sort of equatorial hydroxyl at different
sites of the steroidal moiety (5, 13, 25, 26). Final removal of
the isopropylidene ketal led to the target compound 21 in
61% yield from 17.
We were also attracted to introducing amine functions on
ring D, since nitrogen-containing-brassinosteroid analogues
have been previously tested and included in the currently ac-
action times were required because of the deactivation of the
16
∆
double bond.
At this stage, we planned an efficient route, which
avoided the use of protecting groups, and consisted of first
introducing the D-ring functions and performing the ∆2-
dihydroxylation as the last step. The conjugated character of
the ∆16 double bond allowed us to achieve this goal since
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Can. J. Chem. Vol. 83, 2005
Scheme 1. Reagents and conditions: (a) 10% Pd(C), MeOH; (b) (i) TsCl, Py, (ii) LiBr, Li2CO3, DMF; (c) OsO4, NMO, THF–t-BuOH–
H2O; (d) NaOMe, MeOH; (e) NaOH, H2O2, MeOH, 0 °C.
cepted SAR studies (11, 27, 28). Thus, a simple mesylation
of the 16α-OH group followed by azide displacement af-
forded the intermediate 19, which upon reduction of the
azide group and subsequent deprotection of the 2α,3α-diol
functionality led to the final 16β amino derivative 22 in 46%
yield from 17.
Evidently, synthetically challenging is the introduction of
the 16α amino group from the function 16α,17α-diol of com-
pound 17. However, having previously established the value
of the Mitsunobu reaction to access the β face of ring D, the
replacement of the 16α-OH group by azide with the net re-
tention of configuration was fulfilled by utilizing methane-
sulfonic acid as the acidic–nucleophilic component of the
Mitsunobu reaction and consecutive azide displacement of
the resulting 16β mesylate (23). The isopropylidene removal
procedure used for achieving the epimer 22 similarly suc-
ceeded affording the final 16α amino derivative 23 in 36%
yield from 17. Of particular interest is the the prospect of in-
troducing a variety of functions on ring D of pregnane
brassinosteroid-like compounds. Whilst these could not be
considered as brassinosteroid analogues since they lack the
classical side-chain functionalities, the planning strategy
used in the present work was intended to serve as the open-
ing issue for accessing D-ring-functionalized brassinosteroid
analogues. Moreover, considering that these additional D-
ring functions could somehow present a high influence on
the final active conformation of the hormone, the present ap-
proach might have a further outstanding impact on the pres-
ent way of focussing the SAR studies.
1
The H and 13C NMR assignments of the obtained com-
pounds were achieved by a combination of 1D and 2D ex-
periments. The 1H NMR features of the 2α,3α-diol
functionality are usually the easiest to be assigned from the
H-2 and H-3 coupling patterns even in the presence of the
5α-OH. Additionally, the stereochemistry of the function-
alized carbons was unequivocally demonstrated by perform-
ing 1D NOE difference experiments, in which the
enhancements on H-16, H-2, and H-3 upon irradiation of the
two β-angular methyl groups CH3-18 and CH3-19 confirmed
the α stereochemistry of the functions at these positions. The
β stereochemistry of the functions at C-16 on compounds 21
and 22 was similarly demonstrated because of the lack of
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Rivera and Coll
1087
Scheme 2. Reagents and conditions: (a) (i) TsCl, Py, (ii) OsO4, NMO, THF–t-BuOH–H2O; (b) (i) LiBr, Li2CO3, DMF, (ii) TBDMSCl,
imidazole, DMF; (c) (i) OsO4, NMO, THF–t-BuOH–H2O, (ii) Me2C(OMe)2, PPTS, DMF; (d) TBFA, THF; (e) (i) HCO2H, Ph3P,
DIAD, THF, (ii) NaHCO3, MeOH–H2O; (f) (i) MsCl, TEA, CH2Cl2, (ii) NaN3, DMPU; (g) (i) MeSO3H, Ph3P, DIAD, THF, (ii) NaN3,
DMPU; (h) AcOH, MeOH–H2O; (i) (i) H2, PtO2, AcOEt, (ii) AcOH, MeOH-H2O. DIAD = diisopropylazodicarboxylate, DMPU = 1,3-
dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidonone.
enhancement on H-16 upon irradiation of CH3-18. Interest-
ingly, the coupling pattern of H-16 in the 16α-functionalized
pregnanes showed the lack of coupling with one of the C-15
protons, suggesting a dihedral angle near 90° between H-16β
and H-15α.
The growth-promoting activity was evaluated using the
radish (Raphanus sativus L.) hypocotyl elongation and coty-
ledon expansion bioassays (29). Thus, the bioactivities were
assessed in terms of increments of hypocotyl length or coty-
ledon weight with respect to the untreated control plants re-
ferred to as 100%.
As should be expected from the fact that these compounds
lack the brassinosteroid functionalities in the side chain, the
results of plant growth-promoting activity in the radish bio-
assays are far away from being considered as remarkable.
However, analysis of Table 1 highlights some tendencies that
might be seen as promising. Thus, all D-ring functionalized
compounds were more active than the D-ring unfunction-
alized pregnane 7. Compound 22, bearing the function
16β,17α-diol, was found to be the most active in the
hypocotyl elongation bioassay.
Interestingly, the presence of hydroxy functions provoked
a higher bioactivity than for epoxy and methoxy, which is in
agreement with the classical behavior in bioassays reported
for these functions at different sites of the steroidal moiety
or the side chain. Furthermore, similar results were found in
a comparison between hydroxy and amino groups at the
same position, since the 16,17-dihydroxy derivatives 9 and
21 clearly were more active than the 16-amino-17-hydroxy
ones (22, 23). In both series of analogues, the epimerization
of the function at C-16 provoked a moderate enhancement of
activity either in the hypocotyl elongation or cotyledon ex-
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Can. J. Chem. Vol. 83, 2005
Table 1. Results of the radish bioassays.
Elongation of hypocotyls (%)a
Weight of cotyledons (%)a
Conc.
Conc.
Conc.
Conc.
Conc.
Conc.
Compound
0.1 ppm
0.01 ppm
0.001 ppm
0.1 ppm
0.01 ppm
0.001 ppm
7
9
105 5
124 6
117 5
111 5
128 6
120 5
117 5
105 5
123 6
116 5
112 5
126 6
121 5
116 5
103 5
123 6
116 5
110 5
127 6
119 5
116 5
104 4
115 5
110 5
124 6
117 6
110 5
104 4
102 4
114 5
108 5
121 6
115 6
110 5
105 4
103 4
114 5
109 5
118 6
114 6
109 5
104 4
12
13
21
22
23
aControl experiment (untreated plants) refers to 100%.
pansion bioassays. As can be noted, the general tendency
was that the tested compounds elicited higher activity in the
hypocotyl elongation bioassay than in the cotyledon expan-
sion one, with the only exception being the epoxy derivative
13.
3␤,5-Dihydroxy-5␣-pregna-6,20-dione (5)
Pd/C 10% (210 mg) was added to a solution of 4 (1.5 g,
4.3 mmol) in 80 mL of MeOH. The mixture was treated suc-
cessively with hydrogen and vacuum and finally stirred un-
der a hydrogen atmosphere for 24 h. The reaction was
monitored on TLC by the absence of UV absorption. The
suspended catalyst was filtered off and the resulting solution
was evaporated under reduced pressure to give the ketol 5
(1.44 g, 96%); mp (MeOH) 192–195 °C. IR (KBr, cm–1):
Conclusions
1
Seven new pregnane brassinosteroid-like compounds were
efficiently synthesized and their plant growth-promoting ac-
tivities evaluated. Either the introduction of oxygen or amino
functions on ring D led to an increment in the bioactivity in
the radish bioassays. Further experiments to compare the bi-
ological activity of the reported compounds with that of nat-
ural brassinosteroids are required.
3547, 3445, 1705, 1702, 1080, 1039. H NMR (CDCl3) δ:
0.59 (s, 3H, H-18), 0.80 (s, 3H, H-19), 2.03 (s, 3H, H-21),
2.50 (t, 1H, J = 8.9 Hz, H-17α), 3.87 (br m, 1H, H-3α). 13C
NMR (CDCl3) δ: 29.7 (C-1), 29.7 (C-2), 66.6 (C-3), 35.2
(C-4), 79.8 (C-5), 213.9 (C-6), 41.4 (C-7), 37.1 (C-8), 44.0
(C-9), 42.2 (C-10), 21.2 (C-11), 38.6 (C-12), 13.7 (C-13),
56.2 (C-14), 23.9 (C-15), 22.5 (C-16), 63.3 (C-17), 13.2 (C-
18), 13.7 (C-19), 210.1 (C-20), 31.3 (C-21). Anal. calcd. for
C21H32O4: C 72.4, H 9.3; found: C 72.7, H 9.6.
Experimental
5-Hydroxy-5␣-pregn-2-ene-6,20-dione (6)
Chemistry
Tosyl chloride (1.14 g, 6 mmol) was added to a solution
of ketol 5 (1.4 g, 4.0 mmol) in 50 mL of pyridine and the re-
action was stirred at room temperature in the dark for 24 h.
After completion as indicated by TLC, the mixture was
poured into 300 mL of 10% NaHCO3 and the resulting sus-
pension was stirred for another hour and filtered under
reduced pressure. The obtained tosylate was dried and dis-
solved in 60 mL of anhydr. DMF. Li2CO3 (2.2 g) and LiBr
(2.5 g) were added to this solution and the resulting mixture
was refluxed under a nitrogen atmosphere for 2 h, poured
slowly with stirring into 300 mL of 10% HCl, and filtered
under reduced pressure. The solid was washed with water,
dried, and recrystallized from acetone to give the olefin 6
(1.17 g, 88%); mp (acetone) 156 to 157 °C. IR (KBr, cm–1):
Melting points were determined on a Stuart Scientific ap-
paratus and are uncorrected. IR spectra were obtained on a
Nicolet 205 FT-IR spectrometer. H NMR and 13C NMR
1
were recorded on a Bruker ACF-250 spectrometer at
250.13 and 62.9 MHz, respectively, using TMS as the inter-
nal standard. Two dimensional spectra: H–1H correlation
1
spectroscopy (COSY), heteronuclear single-quantum coher-
ence (HSQC), and heteronuclear multiple bond correlation
(HMBC) were measured for the final compounds on a
Varian INOVA-500 spectrometer operating at 499.81 MHz
using the standard pulse sequences given by the manufac-
turer. Elemental analyses were performed with a Leco
CHNS-932 instrument. Reactions were monitored by thin-
layer chromatography (TLC) on precoated plates with silica
gel (Merck) and spots were visualized with a 0.01 g/mL so-
lution of vanillin in perchloric acid and subsequent heating.
Flash column chromatography was performed on silica gel
60 (Merck, >230 mesh). “Usual work-up” refers to dilution
with water, extraction with an organic solvent, washing the
extract consecutively with 5% HCl and (or) 5% NaHCO3
and brine, drying over anhydr. Na2SO4, and removal of the
solvent under reduced pressure. The solvents were purified
and dried according to recommended procedures.
1
3550, 3020, 1705, 1702, 1086, 1048. H NMR (CDCl3) δ:
0.56 (s, 3H, H-18), 0.66 (s, 3H, H-19), 1.97 (dd, 1H, J =
12.8, 4.2 Hz, H-7β), 2.04 (s, 3H, H-21), 2.50 (t, 1H, J =
8.9 Hz, H-17α), 2.75 (t, 1H, J = 12.6 Hz, H-7α), 5.60 (m,
2H, H-3 + H-2). 13C NMR (CDCl3) δ: 30.0 (C-1), 125.2 (C-
2), 122.4 (C-3), 34.5 (C-4), 77.7 (C-5), 211.0 (C-6), 42.2 (C-
7), 36.7 (C-8), 44.9 (C-9), 42.2 (C-10), 20.7 (C-11), 38.6 (C-
12), 43.7 (C-13), 56.2 (C-14), 23.9 (C-15), 22.5 (C-16), 63.3
(C-17), 13.2 (C-18), 13.7 (C-19), 210.1 (C-20), 31.3 (C-21).
Anal. calcd. for C21H30O3: C 76.3, H 9.15; found: C 76.5, H 9.1.
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2␣,3␣,5-Trihydroxy-5␣-pregnane-6,20-dione (7)
5-Hydroxy-16␣-methoxy-5␣-pregn-2-ene-6,20-dione (10)
A freshly prepared solution of sodium (1.24 g, 54 mmol)
in 30 mL of dry MeOH was added to a solution of the enone
8 (2.0 g, 5.4 mmol) in 100 mL of MeOH. The reaction mix-
ture was stirred for 8 h under a nitrogen atmosphere, after
which time TLC analysis (hexane–EtOAc, 3:1) revealed that
some starting material had not been consumed. The solvent
was removed under reduced preasure and the usual work-up
(Et2O) yielded a product, which was purified by flash col-
umn chromatography (hexane–EtOAc, 4:1) to furnish 10
(1.5 g, 70%); mp (acetone) 158–160 °C. IR (KBr, cm–1):
3335, 3020, 1705, 1702, 1224, 1086, 1048. 1H NMR (CDCl3)
δ: 0.57 (s, 3H, H-18), 0.66 (s, 3H, H-19), 2.15 (s, 3H, H-21),
2.54 (d, 1H, J = 6.3 Hz, H-17α), 2.75 (t, 1H, J = 12.5 Hz, H-
7α), 4.00 (br s, 1H, OH), 3.16 (s, 3H, OCH3), 4.29 (t, 1H,
J = 6.5 Hz, H-16β), 5.60 (m, 2H, H-3 + H-2). 13C NMR
(CDCl3) δ: 30.0 (C-1), 125.2 (C-2), 122.4 (C-3), 34.5 (C-4),
77.7 (C-5), 211.0 (C-6), 42.2 (C-7), 36.7 (C-8), 44.9 (C-9),
42.2 (C-10), 20.7 (C-11), 38.6 (C-12), 44.7 (C-13), 54.0 (C-
14), 31.5 (C-15), 81.2 (C-16), 71.2 (C-17), 14.4 (C-18), 14.5
(C-19), 207.9 (C-20), 31.6 (C-21), 57.2 (OCH3). Anal.
calcd. for C22H32O4: C 73.3, H 8.95; found: C 73.2, H 8.8.
General dihydroxylation procedure
NMO (1.05 g, 9.0 mmol) and a solution of OsO4 in t-
BuOH (9 mL, 12.5 mg/mL) were added to a solution of 6
(1.0 g, 3.0 mmol) in 60 mL of the solvent mixture THF–t-
BuOH–H2O (10:10:1). The reaction was stirred under a ni-
trogen atmosphere at room temperature for 16 h and then
treated with 50 mL of a saturated solution of Na2SO3. The
mixture was stirred for an additional hour and concentrated
to half volume. The usual work-up (AcOEt) yielded a prod-
uct that was purified by flash column chromatography
(CHCl3–MeOH, 10:1) to give the triol 7 (884 mg, 81%); mp
(heptane–acetone) 205 to 206 °C. IR (KBr, cm–1): 3477,
3420, 3335, 1715, 1697, 1121, 1080, 1034. 1H NMR
(CDCl3) δ: 0.56 (s, 3H, H-18), 0.73 (s, 3H, H-19), 1.97 (dd,
1H, J = 12.9, 4.6 Hz, H-7β), 2.04 (s, 3H, H-21), 2.50 (t, 1H,
J = 8.9 Hz, H-17α), 2.69 (t, 1H, J = 12.5 Hz, H-7α), 3.78–
3.84 (br m, 1H, H-2β), 4.12 (m, 1H, H-3β). 13C NMR
(CDCl3) δ: 30.1 (C-1), 67.4 (C-2), 69.5 (C-3), 34.1 (C-4),
79.3 (C-5), 211.0 (C-6), 41.2 (C-7), 36.9 (C-8), 44.2 (C-9),
45.2 (C-10), 21.0 (C-11), 38.5 (C-12), 44.5 (C-13), 56.2 (C-
14), 24.0 (C-15), 22.7 (C-16), 63.3 (C-17), 13.3 (C-18), 14.6
(C-19), 209.8 (C-20), 31.4 (C-21). Anal. calcd. for
C21H32O5: C 69.2, H 8.85; found: C 69.1, H 8.9.
2␣,3␣,5-Trihydroxy-16␣-methoxy-5␣-pregna-6,20-dione
(12)
Olefin 10 (600 mg, 1.7 mmol) was treated following the
general dihydroxylation procedure (NMO (596 mg,
5.1 mmol), OsO4–t-BuOH (5 mL, 12.5 mg/mL)). Purifica-
tion by flash column chromatography (CHCl3–MeOH, 9:1)
gave the triol 12 (470 mg, 72%); mp (heptane–acetone) 222–
224 °C. IR (KBr, cm–1): 3547, 3447, 3416, 3335, 1705,
1698, 1245, 1121, 1086. 1H NMR (CDCl3) δ: 0.56 (s, 3H, H-
18), 0.72 (s, 3H, H-19), 2.15 (s, 3H, H-21), 2.54 (d, 1H, J =
6.3 Hz, H-17α), 2.73 (t, 1H, J = 12.5 Hz, H-7α), 3.17 (s, 3H,
OCH3), 3.74–3.81 (br m, 1H, H-2β), 4.09 (m, 1H, H-3β),
4.17 (br s, 1H, OH), 4.29 (t, 1H, J = 6.5 Hz, H-16β). 13C
NMR (CDCl3) δ: 30.2 (C-1), 67.4 (C-2), 69.5 (C-3), 34.1 (C-
4), 79.3 (C-5), 210.9 (C-6), 41.0 (C-7), 36.4 (C-8), 44.2 (C-
9), 45.2 (C-10), 20.7 (C-11), 38.6 (C-12), 44.8 (C-13), 53.9
(C-14), 31.4 (C-15), 81.1 (C-16), 71.1 (C-17), 14.5 (C-18),
14.6 (C-19), 208.3 (C-20), 31.5 (C-21), 57.1 (OCH3). Anal.
calcd. for C22H34O6: C 67.0, H 8.7; found: C 66.85, H 8.8.
5-Hydroxy-5␣-pregna-2,16-diene-6,20-dione (8)
Ketol 4 (6.0 g, 17.4 mmol) was submitted to tosylation
(TsCl, 3.30 g, 26.1 mmol) and subsequent dehydrotosylation
(Li2CO3 (6.8 g), LiBr (7.2 g)) in a similar way as described
in the synthesis of 6 to furnish the olefin 8 (5.32 g, 80%);
mp (acetone) 157 to 158 °C. IR (KBr, cm–1): 3550, 3020,
1
1707, 1660, 1583, 1060, 1029. H NMR (CDCl3) δ: 0.86 (s,
3H, H-18), 0.72 (s, 3H, H-19), 2.25 (s, 3H, H-21), 2.32 (dd,
1H, J = 12.6, 4.5 Hz, H-7β), 2.79 (t, 1H, J = 12.8 Hz, H-7α),
5.63 (m, 2H, H-3 + H-2), 6.68 (dd, 1H, J = 3.4, 1.8 Hz, H-
16). 13C NMR (CDCl3) δ: 30.1 (C-1), 125.4 (C-2), 122.3 (C-
3), 34.3 (C-4), 78.0 (C-5), 210.7 (C-6), 42.4 (C-7), 35.6 (C-
8), 45.5 (C-9), 42.4 (C-10), 20.7 (C-11), 46.5 (C-13), 56.1
(C-14), 31.9 (C-15), 143.9 (C-16), 155.0 (C-17), 15.7 (C-
18), 14.4 (C-19), 196.6 (C-20), 27.0 (C-21). Anal. calcd. for
C21H28O3: C 76.8, H 8.6; found: C 76.85, H 8.5.
16␣,17␣-Epoxy-5-hydroxy-5␣-pregn-2-ene-6,20-dione (11)
A solution of enone 8 (2.0 g, 6 mmol) in 180 mL of
MeOH was cooled to 0 °C and treated successively with
12 mL of 30% H2O2 and 4 mL of cold, aqueous 4 mol/L
NaOH. The mixture was stirred and kept at 0–5 °C for 24 h.
The reaction was monitored on TLC by the absence of UV
absorption. The usual work-up (Et2O) yielded a white crude
solid, which was purified by flash column chromatography
(hexane–EtOAc, 5:1) to give the epoxide 11 (1.5 g, 72%);
mp (acetone) 158 to 159 °C. IR (KBr, cm–1): 3550, 3020,
2␣,3␣,5,16␣,17␣-Pentahydroxy-5␣-pregna-6,20-dione (9)
Olefin 8 (500 mg, 1.5 mmol) was treated following the
general dihydroxylation procedure (NMO, 1.05 mg, 9 mmol;
OsO4–t-BuOH, 9 mL, 12.5 mg/mL) for 48 h. Purification by
flash column chromatography (CHCl3–MeOH, 5:1) afforded
the pentol 9 (365 mg, 65%); mp (EtOAc) 238 to 239 °C. IR
1
(KBr, cm–1): 3337, 3414, 3439, 1697, 1240, 1162, 1048. H
NMR (CD3OD) δ: 0.70 (s, 3H, H-18), 0.73 (s, 3H, H-19),
2.07 (s, 3H, H-21), 2.71 (t, 1H, J = 12.5 Hz, H-7α), 3.74–
3.82 (br m, 1H, H-2β), 4.10 (m, 1H, H-3β), 4.50 (d, 1H, J =
7.3 Hz, H-16β). 13C NMR (CD3OD) δ: 30.7 (C-1), 66.7
(C-2), 69.3 (C-3), 33.4 (C-4), 79.0 (C-5), 211.0 (C-6), 40.2
(C-7), 35.5 (C-8), 43.2 (C-9), 44.6 (C-10), 19.5 (C-11), 34.5
(C-12), 47.0 (C-13), 48.9 (C-14), 27.9 (C-15), 74.0 (C-16),
77.2 (C-17), 16.5 (C-18), 14.5 (C-19), 216.5 (C-20), 29.7
(C-21). Anal. calcd. for C21H32O5: C 63.6, H 8.1; found: C
63.5, H 8.3.
1
1705, 1700, 1092, 1036. H NMR (CDCl3) δ: 0.95 (s, 3H,
H-18), 0.66 (s, 3H, H-19), 2.06 (s, 3H, H-21), 2.75 (t, 1H,
J = 12.6 Hz, H-7α), 3.77 (d, 1H, J = 2.5 Hz, H-16β), 5.60
(m, 2H, H-3 + H-2). 13C NMR (CDCl3) δ: 30.0 (C-1), 125.2
(C-2), 122.4 (C-3), 34.5 (C-4), 77.7 (C-5), 211.0 (C-6), 42.2
(C-7), 36.7 (C-8), 44.9 (C-9), 42.2 (C-10), 20.7 (C-11), 38.6
(C-12), 41.6 (C-13), 44.8 (C-14), 27.3 (C-15), 60.4 (C-16),
70.8 (C-17), 15.3 (C-18), 14.0 (C-19), 205.1 (C-20), 25.9
© 2005 NRC Canada

1090
Can. J. Chem. Vol. 83, 2005
(C-21). Anal. calcd. for C21H28O4: C 73.2, H 8.2; found: C
73.1, H 8.3.
by flash column chromatography (hexane–AcOEt, 3:1) to
give the olefin 15 (6.6 g, 79%); mp (acetone) 171–173 °C.
IR (KBr, cm–1): 3481, 3382, 3020, 1698, 1702, 1163, 1086,
1
16␣,17␣-Epoxy-2␣,3␣,5-trihydroxy-5␣-pregna-6,20-dione
(13)
1048. H NMR (CDCl3) δ: 0.07 (s, 3H, (CH3)2Si), 0.09 (s,
3H, (CH3)2Si), 0.69 (s, 3H, H-18), 0.66 (s, 3H, H-19), 0.92
(s, 9H, (CH3)3CSi), 2.05 (s, 3H, H-21), 4.56 (d, 1H, J =
7.0 Hz, H-16β), 5.63 (m, 2H, H-3 + H-2). 13C NMR (CDCl3)
δ: 30.1 (C-1), 125.1 (C-2), 122.4 (C-3), 34.5 (C-4), 77.9 (C-
5), 211.6 (C-6), 42.2 (C-7), 36.6 (C-8), 44.7 (C-9), 42.0 (C-
10), 20.7 (C-11), 38.9 (C-12), 43.7 (C-13), 49.2 (C-14), 27.0
(C-15), 75.2 (C-16), 77.4 (C-17), 13.1 (C-18), 13.7 (C-19),
210.3 (C-20), 31.3 (C-21), 26.1 ((CH3)3CSi), 18.3
((CH3)3CSi), –2.9 ((CH3)2Si). Anal. calcd. for C27H44O5Si:
C 68.0, H 9.3; found: C 68.5, H 9.5.
Olefin 11 (860 mg, 2.5 mmol) was treated following the
general dihydroxylation procedure (NMO (978 mg,
7.5 mmol), OsO4–t-BuOH (7 mL, 12.5 mg/mL)). Purifica-
tion by flash column chromatography (CHCl3–MeOH, 10:1)
gave the epoxytriol 13 (766 mg, 81%); mp (heptane–ace-
tone) 213–215 °C. IR (KBr, cm–1): 3477, 3420, 3335, 1700,
1697, 1121, 1080, 1034. 1H NMR (CDCl3) δ: 0.95 (s, 3H, H-
18), 0.73 (s, 3H, H-19), 2.06 (s, 3H, H-21), 2.70 (t, 1H, J =
12.5 Hz, H-7α), 3.77 (d, 1H, J = 2.5 Hz, H-16β), 3.78–3.84
(br m, 1H, H-2β), 4.12 (m, 1H, H-3β). 13C NMR (CDCl3) δ:
30.1 (C-1), 67.4 (C-2), 69.5 (C-3), 34.1 (C-4), 79.3 (C-5),
211.0 (C-6), 41.2 (C-7), 36.9 (C-8), 44.2 (C-9), 45.2 (C-10),
21.0 (C-11), 38.5 (C-12), 41.6 (C-13), 44.8 (C-14), 27.3 (C-
15), 60.5 (C-16), 70.6 (C-17), 15.3 (C-18), 14.0 (C-19),
205.0 (C-20), 25.8 (C-21). Anal. calcd. for C21H30O6: C
66.65, H 8.0; found: C 66.6, H 8.05.
16␣-(tert-Butyldimethylsilyloxy)-5,17␣-dihydroxy-2␣,3␣-
isopropylidenedioxy-5␣-pregna-6,20-dione (16)
Olefin 15 (6.5 g, 13.6 mmol) was treated following the
general dihydroxylation procedure (NMO (4.6 g, 39 mmol),
OsO4–t-BuOH (30 mL, 12.5 mg/mL)) until completion as
indicated by TLC (minor impurities were also detectable).
The usual work-up (AcOEt) afforded a product that was
dried, dissolved in anhydr. DMF (150 mL), and treated with
PPTS (0.8 g) and 2,2-dimethoxypropane (30 mL). Activated
K2CO3 (500 mg) was added to the solution after 24 h, and
the mixture was stirred overnight at room temperature. The
usual work-up (CH2Cl2) yielded a product that was purified
by flash column chromatography (hexane–AcOEt, 2:1) to
give the colorless syrup 16 (4.93 g, 61%); mp (MeOH) 206–
209 °C. IR (KBr, cm–1): 3461, 1703, 1699, 1116, 1089,
5,16␣,17␣-Trihydroxy-3␤-tosyloxy-5␣-pregna-6,20-dione
(14)
Tosyl chloride (6.6 g, 34.6 mmol) was added to a solution
of ketol 4 (8.0 g, 23.1 mmol) in 120 mL of pyridine and the
reaction was stirred at room temperature in the dark for
24 h. After completion as indicated by TLC, the mixture was
poured into 500 mL of 10% NaHCO3, stirred for another
hour, and filtered under reduced pressure. The resulting
tosylate was treated following the general dihydroxylation
procedure (NMO (7.7 g, 66 mmol), OsO4–t-BuOH (40 mL,
12.5 mg/mL)) for 48 h. Purification by flash column chro-
matography (CHCl3–MeOH, 8:1) afforded the triol 14
(9.38 g, 76%); mp (AcOEt) 219 °C (decomp). IR (KBr, cm–1):
1
1056, 1041. H NMR (CDCl3) δ: 0.08 (s, 3H, (CH3)2Si),
0.09 (s, 3H, (CH3)2Si), 0.67 (s, 3H, H-18), 0.75 (s, 3H, H-
19), 0.90 (s, 9H, (CH3)3CSi), 1.47 (s, 3H, CH3-ketal), 1.34
(s, 3H, CH3-ketal), 2.05 (s, 3H, H-21), 4.08–4.15 (br m, 1H,
H-2β), 4.31 (m, 1H, H-3β), 4.56 (d, 1H, J = 7.0 Hz, H-16β).
13C NMR (CDCl3) δ: 28.2 (C-1), 73.7 (C-2), 74.3 (C-3),
33.8 (C-4), 79.5 (C-5), 211.8 (C-6), 41.0 (C-7), 36.4 (C-8),
44.2 (C-9), 45.2 (C-10), 20.7 (C-11), 38.6 (C-12), 43.7 (C-
13), 49.2 (C-14), 27.2 (C-15), 75.2 (C-16), 77.4 (C-17), 13.1
(C-18), 14.2 (C-19), 210.3 (C-20), 31.3 (C-21), 26.0
((CH3)3CSi), 18.3 ((CH3)3CSi), –2.9 ((CH3)2Si), 108.2 (O-
C-O), 26.9 + 28.4 (CH3 ketal). Anal. calcd. for C31H54O7Si:
C 55.7, H 9.6, Si 4.95; found: C 55.8, H 9.7, Si 5.1.
1
3419, 3402, 1695, 1702, 1360, 1175, 1119, 1104. H NMR
(CDCl3) δ: 0.70 (s, 3H, H-18), 0.76 (s, 3H, H-19), 2.05 (s,
3H, H-21), 2.41 (s, 3H, CH3-Ts), 2.70 (t, 1H, J = 12.4 Hz,
H-7α), 4.51 (d, 1H, J = 7.1 Hz, H-16β), 4.84 (br m, 1H, H-
3α), 7.75 (d, 2H, J = 8.3 Hz, Ts), 7.30 (d, 2H, J = 8.5 Hz,
Ts). 13C NMR (CDCl3) δ: 24.8 (C-1), 25.4 (C-2), 75.6.6 (C-
3), 35.7 (C-4), 79.4 (C-5), 212.6 (C-6), 41.9 (C-7), 36.8 (C-
8), 42.09 (C-9), 43.8 (C-10), 21.3 (C-11), 39.2 (C-12), 47.0
(C-13), 48.9 (C-14), 27.9 (C-15), 74.1 (C-16), 77.2 (C-17),
16.5 (C-18), 16.9 (C-19), 216.5 (C-20), 29.7 (C-21), 21.3
(CH3-Ts), 134.2, 127.5, 129.8, 144.6 (6C-Ts). Anal. calcd.
for C28H38O8S: C 62.9, H 7.2, S 6.00; found: C 63.1, H 7.2,
S 6.2.
5,16␣,17␣-Trihydroxy-2␣,3␣-isopropylidenedioxy-5␣-
pregna-6,20-dione (17)
TBAF (3 mL, 1 mol/L in THF) was added to a solution of
acetonide 16 (4.9 g, 8.9 mmol) in anhydr. THF (100 mL).
The reaction mixture was stirred under a nitrogen atmo-
sphere for 6 h and then concentrated. The usual work-up
(Et2O) yielded the crude product in quantitative yield, which
was recrystallized from MeOH to furnish the acetonide 17
(3.49 g, 90%); mp (MeOH) 211 to 212 °C. IR (KBr, cm–1):
3414, 3339, 1700, 1697, 1205, 1142, 1118. 1H NMR
(CDCl3) δ: 0.69 (s, 3H, H-18), 0.74 (s, 3H, H-19), 1.48 (s,
3H, CH3-ketal), 1.34 (s, 3H, CH3-ketal), 2.07 (s, 3H, H-21),
2.72 (t, 1H, J = 12.4 Hz, H-7α), 4.08–4.15 (br m, 1H, H-2β),
4.31 (m, 1H, H-3β), 4.50 (d, 1H, J = 7.3 Hz, H-16β). 13C
NMR (CDCl3) δ: 28.4 (C-1), 73.6 (C-2), 74.0 (C-3), 33.8 (C-
4), 79.4 (C-5), 211.7 (C-6), 41.0 (C-7), 36.4 (C-8), 44.2 (C-
9), 45.2 (C-10), 20.5 (C-11), 34.5 (C-12), 47.0 (C-13), 48.9
(C-14), 27.9 (C-15), 74.1 (C-16), 77.2 (C-17), 16.5 (C-18),
16␣-(tert-Butyldimethylsilyloxy)-5,17␣-dihydroxy-5␣-
pregn-2-ene-6,20-dione (15)
Li2CO3 (12.0 g) and LiBr (14.5 g) were added to a solu-
tion of tosylate 14 (9.3 g, 17.5 mmol) in 100 mL of anhydr.
DMF. The reaction mixture was refluxed under a nitrogen
atmosphere for 2 h and followed by TLC until completion,
then poured slowly with stirring into 600 mL of cool water,
and filtered under reduced pressure. The resulting solid was
washed with water, dried and dissolved in 80 mL of anhydr.
DMF. TBDMSCl (2.6 g, 17.5 mmol) and imidazole (1.8 g,
26 mmol) were added to the solution and the reaction mix-
ture was stirred under a nitrogen atmosphere for 3 h. The
usual work-up (CHCl3) yielded a product that was purified
© 2005 NRC Canada

Rivera and Coll
1091
14.7 (C-19), 216.4 (C-20), 29.8 (C-21). Anal. calcd. for
C24H36O7: C 66.0, H 8.3; found: C 66.0, H 8.25.
mation of two products, one being the more polar and the
major steroidal product. The usual work-up (EtOAc) yielded
a crude product, which was purified by flash column chro-
matography (hexane–AcOEt, 3:1) to afford, by collection of
the more hydrophilic fractions, the azide 19 (1.1 g, 73%);
mp (MeOH) 207–210 °C. IR (KBr, cm–1): 3335, 3322, 2091,
1702, 1698, 1168, 1110. 1H NMR (CDCl3) δ: 0.78 (s, 3H, H-
18), 0.74 (s, 3H, H-19), 1.49 (s, 3H, CH3-ketal), 1.34 (s, 3H,
CH3-ketal), 2.08 (s, 3H, H-21), 4.08–4.15 (br m, 1H, H-2β),
4.24 (dd, 1H, J = 8.1, 5.7 Hz, H-16α), 4.32 (m, 1H, H-3β).
13C NMR (CDCl3) δ: 28.5 (C-1), 73.6 (C-2), 74.1 (C-3),
33.6 (C-4), 79.5 (C-5), 212.0 (C-6), 41.0 (C-7), 36.4 (C-8),
44.1 (C-9), 45.2 (C-10), 20.5 (C-11), 34.5 (C-12), 46.1 (C-
13), 53.1 (C-14), 28.0 (C-15), 68.1 (C-16), 76.5 (C-17), 15.0
(C-18), 14.6 (C-19), 216.1 (C-20), 29.8 (C-21). Anal. calcd.
for C24H35O6N3: C 62.5, H 7.6, N 9.1; found: C 62.3, H 7.5,
N 9.2.
5,16␤,17␣-Trihydroxy-2␣,3␣-isopropylidenedioxy-5␣-
pregna-6,20-dione (18)
To a stirred solution of triol 17 (700 mg, 1.6 mmol), for-
mic acid (300 µL), and triphenylphosphine (635 mg,
2.49 mmol) in THF (50 mL) was added dropwise a solution
of DIAD (570 µL, 2.94 mmol) in 7 mL of THF. The reaction
mixture was stirred at room temperature for 24 h, after
which time TLC analysis revealed that all the starting mate-
rial had been consumed. Then the volatiles were evaporated
off under reduced pressure and the resultant crude was dis-
solved in 80 mL of a saturated solution of NaHCO3 in
MeOH–H2O (4:1) and stirred for 1 h at room temperature.
The usual work-up (Et2O) yielded a crude product, which
was purified by flash column chromatography (hexane–
AcOEt, 3:1) to give the triol 18 (497 mg, 71%); mp (EtOH)
215 to 216 °C. IR (KBr, cm–1): 3459, 3401, 3346, 1700,
1696, 1204, 1145, 1074. 1H NMR (CDCl3) δ: 0.77 (s, 3H, H-
18), 0.74 (s, 3H, H-19), 1.47 (s, 3H, CH3-ketal), 1.34 (s, 3H,
CH3-ketal), 2.09 (s, 3H, H-21), 4.09–4.16 (br m, 1H, H-2β),
4.32 (m, 1H, H-3β), 4.41 (dd, 1H, J = 8.1, 5.9 Hz, H-16α).
13C NMR (CDCl3) δ: 28.5 (C-1), 73.6 (C-2), 74.0 (C-3),
33.7 (C-4), 79.4 (C-5), 211.8 (C-6), 41.0 (C-7), 36.4 (C-8),
44.2 (C-9), 45.2 (C-10), 20.5 (C-11), 34.5 (C-12), 46.1 (C-
13), 53.0 (C-14), 28.1 (C-15), 73.7 (C-16), 77.0 (C-17), 15.1
(C-18), 14.5 (C-19), 216.2 (C-20), 29.7 (C-21). Anal. calcd.
for C24H36O7: C 66.0, H 8.3; found: C 66.2, H 8.4.
16␤-Amino-2␣,3␣,5,17␣-tetrahydroxy-5␣-pregna-6,20-
dione (22)
Lindlar catalyst (200 mg) was added to a solution of azide
19 (1.0 g, 2.1 mmol) in 60 mL of absolute EtOH. The mix-
ture was treated successively with hydrogen and vacuum and
finally stirred under a hydrogen atmosphere for 36 h, when
TLC analysis revealed that all the azide had been consumed.
The catalyst was removed by filtration and the resulting so-
lution was evaporated under reduced pressure to give the
corresponding amine, which was dissolved in 60 mL of the
solvent mixture MeOH–H2O (5:1). Acetic acid (4 mL) was
added and the reaction mixture was refluxed for 1 h. The
solvent was concentrated until half volume and partitioned
between 10% NaOH (200 mL) and EtOAc (300 mL). The
aqueous layer was extracted successively with EtOAc (3 ×
100 mL), dried over anhydr. Na2SO4, and concentrated un-
der reduced pressure. Recrystallization from EtOAc gave the
pure amine 22 (522 mg, 63%); mp (EtOAc) 227–230 °C. IR
(KBr, cm–1): 3428, 3391, 3326, 3311, 1700, 1204, 1158,
2␣,3␣,5,16␤,17␣-Pentahydroxy-5␣-pregna-6,20-dione (21)
Acetic acid (2 mL) was added to a solution of 18
(400 mg, 0.9 mmol) in 20 mL of the solvent mixture
MeOH–H2O (5:1) and the reaction mixture was refluxed for
1 h. The usual work-up (AcOEt) yielded a white product,
which was recrystallized from AcOEt to afford the pure
pentaol 21 (282 mg, 86%); mp (AcOEt) 241 to 242 °C. IR
(KBr, cm–1): 3447, 3421, 3329, 3301, 1701, 1697, 1240,
1
1116. H NMR (CDCl3) δ: 0.78 (s, 3H, H-18), 0.73 (s, 3H,
1
1162, 1186, 1048. H NMR (CDCl3) δ: 0.78 (s, 3H, H-18),
H-19), 2.10 (s, 3H, H-21), 3.74–3.81 (br m, 1H, H-2β), 4.09
(m, 1H, H-3β), 4.17 (br s, 1H, OH), 4.33 (dd, 1H, J = 7.9,
5.9 Hz, H-16α). 13C NMR (CDCl3) δ: 30.2 (C-1), 67.4 (C-2),
69.5 (C-3), 34.1 (C-4), 79.3 (C-5), 210.9 (C-6), 41.0 (C-7),
36.4 (C-8), 44.2 (C-9), 45.2 (C-10), 20.7 (C-11), 35.3 (C-
12), 46.1 (C-13), 53.2 (C-14), 28.2 (C-15), 73.8 (C-16), 76.9
(C-17), 15.1 (C-18), 14.5 (C-19), 216.2 (C-20), 29.8 (C-21).
Anal. calcd. for C21H33O6N: C 63.8, H 8.4, N 3.5; found: C
63.7, H 8.3, N 3.65.
0.72 (s, 3H, H-19), 2.09 (s, 3H, H-21), 2.70 (t, 1H, J =
12.5 Hz, H-7α), 3.78–3.84 (br m, 1H, H-2β), 4.11 (m, 1H,
H-3β), 4.41 (dd, 1H, J = 8.0, 5.9 Hz, H-16α). 13C NMR
(CDCl3) δ: 30.1 (C-1), 67.4 (C-2), 69.5 (C-3), 34.1 (C-4),
79.3 (C-5), 211.0 (C-6), 41.2 (C-7), 36.9 (C-8), 44.2 (C-9),
45.2 (C-10), 21.0 (C-11), 34.5 (C-12), 46.1 (C-13), 53.4 (C-
14), 28.2 (C-15), 73.8 (C-16), 77.1 (C-17), 15.0 (C-18), 14.5
(C-19), 216.2 (C-20), 29.7 (C-21). Anal. calcd. for
C21H32O5: C 63.6, H 8.1; found: C 63.5, H 8.2.
16␣-Azido-5,17␣-dihydroxy-2␣,3␣-isopropylidenedioxy-
5␣-pregna-6,20-dione (20)
16␤-Azido-5,17␣-dihydroxy-2␣,3␣-isopropylidenedioxy-
5␣-pregna-6,20-dione (19)
Methanesulfonic acid (570 µL, 8.8 mmol) was added to a
stirred solution of acetonide 17 (1.74 g, 4.0 mmol) and
triphenylphosphine (3.14 g, 12.0 mmol) in dry THF
(120 mL). The temperature was raised to 40 °C and a solu-
tion of DIAD (2.33 mL, 12 mmol) in 15 mL of THF was
added dropwise over a 10 min period under an argon atmo-
sphere. The reaction mixture was stirred vigorously for 24 h
at 40 °C under an argon atmosphere, and then the volatiles
were evaporated off under reduced pressure. The crude prod-
uct was purified by flash column chromatography to give a
Mesyl chloride (0.558 g, 4.9 mmol) was added dropwise
to a stirred solution of acetonide 17 (1.44 g, 3.3 mmol) in
dry CH2Cl2 (50 mL) and Et3N (1.0 mL, 7.3 mmol) at 0 °C
under a nitrogen atmosphere. The resulting suspension was
stirred at this temperature for 1 h, allowed to reach room
temperature, and then poured into 200 mL of 10% NaHCO3.
The usual work-up (CH2Cl2) yielded a product, which was
carefully dried and dissolved in DMPU (50 mL). NaN3
(322 mg, 4.95 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 for-
1
pure product, which was analyzed by H NMR confirming
the structure of the mesylate. This later was submitted to
© 2005 NRC Canada

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Can. J. Chem. Vol. 83, 2005
azide displacement as described for the synthesis of 19. Pu-
rification by flash column chromatography yielded the azide
20 (1.12 g, 61%); mp (MeOH) 205 to 206 °C. IR (KBr, cm–1):
3368, 3312, 2091, 1701, 1162, 1130, 1058. 1H NMR
(CDCl3) δ: 0.70 (s, 3H, H-18), 0.74 (s, 3H, H-19), 1.48 (s,
3H, CH3-ketal), 1.34 (s, 3H, CH3-ketal), 2.06 (s, 3H, H-21),
2.70 (t, 1H, J = 12.5 Hz, H-7α), 4.08–4.15 (br m, 1H, H-2β),
4.28 (d, 1H, J = 7.1 Hz, H-16β), 4.32 (m, 1H, H-3β). 13C
NMR (CDCl3) δ: 28.5 (C-1), 73.6 (C-2), 74.1 (C-3), 33.8 (C-
4), 79.6 (C-5), 211.9 (C-6), 41.0 (C-7), 36.4 (C-8), 44.2 (C-
9), 45.2 (C-10), 20.5 (C-11), 34.5 (C-12), 47.1 (C-13), 48.9
(C-14), 27.7 (C-15), 67.9 (C-16), 76.3 (C-17), 16.4 (C-18),
14.7 (C-19), 216.3 (C-20), 29.8 (C-21). Anal. calcd. for
C24H35O6N3: C 62.5, H 7.6, N 9.1; found: C 62.1, H 7.5, N
9.05.
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Acknowledgments
28. C. Brosa, M. Amorós, M. Molist, and X. Hernández. Tetrahe-
We are grateful to Dr. Carlos. S. Pérez from the Univer-
sity of Havana, and Dr. Andrea Porzel from the Institute of
Plant Biochemistry, Halle (Saale) Germany, for the spectro-
scopic measures. We also gratefully acknowledge Ing.
Deysma C. Herrera for experimental assistance.
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© 2005 NRC Canada