Synthesis of peptidomimetic-spirostane hybrids via Ugi reaction: a versatile approach for the formation of peptide-steroid conjugates

Article abstract of DOI:10.1016/j.tet.2006.06.050

A general approach towards the preparation of peptide-steroid conjugates has been addressed. Utilizing the Ugi reaction, five peptidomimetic-steroid hybrids were achieved in good to excellent yields from carboxy- and amino-spirostanes and mono-protected α-amino acids. Diverse synthetic routes, specially focused on the formation of secosteroids, were implemented to introduce Ugi-type functionalities at different positions of the steroidal nucleus. This versatile approach is suitable for the formation of stable conjugates of steroids with other biologically relevant molecules.

Full text of DOI:10.1016/j.tet.2006.06.050

Tetrahedron 62 (2006) 8327–8334
Synthesis of peptidomimetic-spirostane hybrids via
Ugi reaction: a versatile approach for the
formation of peptide–steroid conjugates
*
Daniel G. Rivera, Orlando Pando and Francisco Coll
Center for Natural Products Study, Faculty of Chemistry, University of Havana, Havana 10400, Cuba
Received 18 May 2006; revised 11 June 2006; accepted 14 June 2006
Available online 7 July 2006
Abstract—A general approach towards the preparation of peptide–steroid conjugates has been addressed. Utilizing the Ugi reaction, five
peptidomimetic-steroid hybrids were achieved in good to excellent yields from carboxy- and amino-spirostanes and mono-protected a-amino
acids. Diverse synthetic routes, specially focused on the formation of secosteroids, were implemented to introduce Ugi-type functionalities at
different positions of the steroidal nucleus. This versatile approach is suitable for the formation of stable conjugates of steroids with other
biologically relevant molecules.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
applicability of the conjugate. By far the most commonly
used chemical linkages in steroidal conjugates (i.e., glyco-
sidic, amide, and ester) present an undeniable drawback:
the sensitivity towards chemical or enzymatic hydrolysis.
Another more stable and thereby widely exploited linkage,
such as the oxime bond, is generally fixed to the presence
of oxo-functions at the steroid.6,7
The conjugation of steroids to other chemically or biologi-
cally relevant molecules represents a valuable strategy to
generate new properties in the resulting molecular hybrid.
Similarly to the naturally occurring saponins,1 many syn-
thetic biomolecule-steroid conjugates have shown to possess
physico–chemical and biological features arising from the
junction of the two molecular entities. E.g., synthetic
sugar–steroid conjugates have been synthesized to provide
novel amphiphilic molecules capable to interact with phos-
pholipid membranes.2 Likewise, peptide–steroid conjugates
have been employed as synthetic receptors of oligopeptide
sequences,3 as protease-like artificial enzymes,4 and as
mimics of the natural cationic peptide antibiotics.5 Addition-
ally, the attachment of detectable labels (e.g., fluorescent) to
currently used steroidal hormones is an important approach
in the development of clinical immunoassays. Indeed, this
objective requires the production of novel reagents and
methodologies toward improvements in the conjugation
process.6
Herein we report on the use of the Ugi four-component
reaction (Ugi-4CR) as a versatile approach towards the
formation of peptide–steroid conjugates. The Ugi-4CR is
the one-pot condensation of a primary amine, an oxo-
component, a carboxylic acid, and an isocyanide to afford
an N-substituted dipeptide backbone8 (Scheme 1). This
and other related scaffolds arising from the Ugi-4CR, or
its variants, have found relevance in medicinal chemistry
in the last decades.9 Particularly interesting are oligomeric
peptidomimetics containing N-substituted amide bonds
(i.e., peptoids), which have shown a wide variety of biologi-
cal applications10 and a high resistance towards proteolytic
degradation.11
The formation of a chemically and metabolically stable link-
age between a steroid and a biomolecule, a bioactive com-
pound or a detectable tag is a crucial step for the potential
The Ugi-4CR has been recently utilized to assemble very
large cholane-based macrocycles tethered by highly diverse
peptoid moieties.12 However, a general conjugation process
of biomolecules to steroids utilizing this approach has not
been previously addressed. Apart from the accessible struc-
tural diversity arising from the possibility of utilizing car-
boxy, amino, isocyano, or oxo-steroids as building blocks,
this strategy provides a very straightforward method to
access stable peptide–steroid conjugates with potential bio-
logical activities.
Keywords: Steroids; Spirostanes; Steroid conjugates; Multicomponent reac-
tions; Ugi reaction.
* Corresponding author. Present address: Leibniz-Institute of Plant
Biochemistry, Weinberg 3, 06120 Halle/Saale, Germany. Tel.: +537
8702102; fax: +537 8733502; e-mail: dgr@fq.uh.cu
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.06.050

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D. G. Rivera et al. / Tetrahedron 62 (2006) 8327–8334
O
O
O
a
b
TBSO
O
O
R
R
R
OH
OAc
OAc
1, R = H
3, R = H, 59%
5, R = H, 69%
2, R = OH
4, R = OH, 66%
6, R = OH, 66%
e
c
H2N
HO
d
O
O
O
HO2C
OAc
O
O
10, 59%
7, 78%
8, 71%
N
NHBoc
CO2H
(CH2O)n
tBuNC
BnNC
(CH2O)n
H2N
CO2Me
O
N
H
O
BocHN
O
O
O
N
N
N
H
O
O
O
O
OAc
CO2Me
11, 64%
O
O
HN
N
H
N
9, 82%
Scheme 1. Reagents and conditions: (a) i: Ac2O, Py; ii: TBAF, THF; iii: PCC, CH2Cl2; (b) mCPBA/CH2Cl2; (c) from 5, i: 5% KOH, MeOH, reflux; ii: Dowex
50W, set pH 3; (d) i: MsCl, CH2Cl2, Et3N; ii: NaN3, DMPU; iii: H2, Lindlar catalyst; (e) from 6; i: H2SO4, MeOH; ii: LiOH, THF/H2O.
TBAF¼Tetrabutylammonium fluoride; DMPU¼1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidonone.
2. Results and discussion
Scheme 1 summarizes the synthesis of peptidomimetic-
spirostane hybrids functionalized on ring A. Baeyer–Villiger
oxidation of 3-oxosteroids 3 and 4 gave access to the corre-
sponding lactones, which upon ring opening processes
afforded varied secosteroids properly functionalized for the
conjugation process. Thus, treatment of lactone 5 with basic
conditions followed by acidification to pH 3 rendered the
g-lactone 7 in 78% yield after chromatography purification.
The experimental condition were established to favor the
kinetic product (5-exo-trig ring closing), thereby avoiding
the regeneration of the 3-lactone. Further replacement of
the primary hydroxyl group by an amino function allowed
accomplishing the conjugation of the N-Boc-protected
a-amino acid L-histidine in 82% yield by using tert-butyliso-
cyanide. It must be mentioned that paraformaldehyde was
always employed as the oxo-component to avoid the stereo-
isomers formation during the conjugation process.
This article focuses on the conjugation process of interesting
a-amino acids at different positions of the spirostane skele-
ton. However, we also aim to illustrate that this approach is
suitable for introducing oligopeptides either by their C- or
N-terminus at varied positions of the steroidal nucleus.
Therefore, we concentrated on functionalizing steroids
with carboxy and amino functions and taking advantage of
the wide set of commercially available isocyanides (iso-
nitriles) that can be employed to incorporate aromatic, ali-
phatic or other amino acid into the final hybrid compound.
A key feature of these synthetic routes is the avoidance of
the established succinate moiety as a source of carboxy
groups. Instead, we concentrated on developing highly prac-
tical pathways that easily allow incorporating the above
mentioned functional groups for the Ugi-4CR. These
approaches are specially focused on secosteroid syntheses
via lactone-ring opening and further activation of the
Ugi-type functionality.
The 5a-hydroxylated lactone 6 allowed an alternative path-
way, in which an acid-catalyzed methanolysis led to dehy-
dration at C-5 and subsequent nucleophilic attack of the
primary hydroxyl at C-2 to yield a cyclic ether. Cleavage
of the methyl ester function on the later intermediate fur-
nished the final steroidal acid 10. The C-protected a-amino
acid L-alanine and benzylisocyanide were employed to
achieve the formation of conjugate 11 in 64% yield. The
lower isolated yield of this compound compared to 9 may
be due to the less accessible character of the b-face directed
carboxy function at C-3 because of typical steric effect.
Spirostanes were chosen because of various features that
make them amenable starting material to produce bioactive
compounds. Derivatives of these sapogenins have exhibited
a variety of biological activities depending on the function-
alization pattern incorporated in their structures, e.g., ecdy-
steroid13 and brassinosteroid14 type activities have been
reported upon introduction on rings A and B of functionali-
ties typical of these natural products. Likewise, the general
interest on these widely available steroids has been increased
due to their unquestionable potential to access analogues15
of cephalostatins16 and ritterazines.17
As depicted in Scheme 2, the introduction on ring B of
Ugi-type functionalities is accomplished by treatment of

D. G. Rivera et al. / Tetrahedron 62 (2006) 8327–8334
8329
O
O
O
O
O
H
O
H
H
NH2
CbzHN
( )4
a
CO2Me
NC
b
O
O
O
CO2H
O
CO2H
HO
HO
N
O
CO2Me
( )4
MeO2C
O
O
O
12
14, 73%
(CH2O)n
13, 81%
MeO2C
N
H
NHCbz
15, 74%
Scheme 2. Reagents and conditions: (a) i: Ac2O, Py; ii: Jones, 60 ꢀC; (b) i: H2, Pd/C 10%; ii: mCPBA; CH2Cl2.
ketol 12 with strong oxidative conditions to allow C–C bond
cleavage. Secospirostane 13 was then submitted to palla-
dium-catalyzed hydrogenation and subsequent Baeyer–
Villiger oxidation of the oxo-function at C-5 to afford the
A-ring lactone 14 properly functionalized for the further
Ugi-4CR. Thus, the carboxy functionality at C-6 was em-
ployed to achieve the conjugate 15 in 74% yield by using
3-N-Cbz-L-lysine methyl ester as the amino component
and methyl isocyanoacetate. The use of the polyfunctional
amino acid L-lysine and a functionalized isocyanide makes
of the dipeptide–secospirostane hybrid 15 an amenable scaf-
fold for further peptide coupling as well as for introducing
other biologically relevant moieties. Indeed, this was possi-
ble due to the multicomponent nature of the Ugi-4CR, which
easily allows the incorporation of multiple functionalized
building blocks in a one-pot procedure.
oxidation of the oxo-function at C-12 allowed accessing
Ugi-type functionalities via reductive ring opening with
LiAlH4 and subsequent replacement of the primary hydroxyl
at C-12 by an amino group. Benzylisocyanide was then
employed in the conjugation process of N-Boc-protected
L-serine to the aminosteroid 19, to afford the dipeptide–
secospirostane hybrid 20 in 79% yield.
Having established the value of this strategy, we focused on
extending it towards a double conjugation procedure of nat-
ural a-amino acids by either its amino or carboxy groups. In
this sense, the C-protected secosteroidal amino acid 22 was
produced by acid-catalyzed lactone-ring opening of inter-
mediate 5 followed by incorporation of the required amino
group at C-2 using a standard protocol. A sequential
Ugi-4CR/deprotection/Ugi-4CR approach enabled the easy
incorporation of two L-phenylalanine units to obtain the
conjugate 23 in 61% yield (Scheme 4). Indeed, the use of
both C- and N-protected a-amino acids as the carboxylic
and amino components of the reaction sequence, respec-
tively, confirms the versatile character of this methodology
to produce highly functionalized conjugates with very low
synthetic cost.
To accomplish the formation of steroid conjugates function-
alized on ring C, we turned to the use of hecogenin as start-
ing material of the synthetic planning shown in Scheme 3.
Protection of the hydroxyl at C-3 with tert-butyldimethyl-
silane (TBS) enabled further functionalizations on ring C
without affecting the ring A. Once more, the Baeyer–Villiger
BocHN
OH
O
HO
H2N
H
O
N
HO
HO
N
O
HO
O
O
O
(CH2O)n
BnNC
O
H
H
d
b, c
TBSO
HO
RO
H
H
H
19, 62%
H
CO2H
NHBoc
HO
HO
18, 72%
H
16, R = H
a
17, R = TBS, 84%
20, 79%
Scheme 3. Reagents and conditions: (a) TBSCl, imidazole, DMF; (b) mCPBA, CH2Cl2; (c) LiAlH4, THF; (d) i: MsCl, CH2Cl2, Et3N; ii: NaN3, DMPU; iii: H2,
Lindlar catalyst; iv: TBAF, THF, TBS¼tert-Butyldimethylsilane.
(CH2O)n
tBuNC
CO2H
NHBoc
NH
O
c)
O
O
O
O
N
O
R1
O
a
NHBoc
d
H
O
H
N
OAc
OR2
e)
CO2Me
MeO2C
OAc
N
O
O
5
21, R1 = OH, R2 = H, 88%
22, R1 = NH2, R2 = Ac, 62%
NH2
CO2Me
23, 59%
b
(CH2O)n
tBuNC
Scheme 4. Reagents and conditions: (a) i: H2SO4, MeOH, reflux; ii: NaOMe, MeOH; (b) i: MsCl, CH2Cl2, Et3N; ii: NaN3, DMPU; iii: Ac2O, Py; iv: H2, Lindlar
catalyst; (d) LiOH, THF/H2O.

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D. G. Rivera et al. / Tetrahedron 62 (2006) 8327–8334
3. Conclusions
J¼4.1/10.8 Hz, H-26eq); 4.40 (m, 1H, H-16a); 4.96 (m,
1H, H-6a). 13C NMR (CDCl3): d¼37.0 (C-1); 37.9 (C-2);
211.3 (C-3); 44.2 (C-4); 49.1 (C-5); 72.6 (C-6); 36.3
(C-7); 30.4 (C-8); 53.8 (C-9); 35.4 (C-10); 20.9 (C-11);
39.6 (C-12); 40.5 (C-13); 55.5 (C-14); 31.4 (C-15); 80.6
(C-16); 62.0 (C-17); 16.5 (C-18); 15.2 (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); 66.8 (C-26); 17.1 (C-27); 21.4 (CH3CO); 170.2
(CH3CO). HRMS (ESI-FT-ICR) m/z: 495.3084 [M+Na]+
(Calculated for C29H44NaO5: 495.3087).
As it has been demonstrated, various proteogenic amino
acids could be conjugated to spirostanes via the Ugi-4CR
to form peptidomimetic–steroid hybrids in yields ranging
from 60% to 85%. Indeed, the high value of this synthetic
approach lies on its potential applicability towards a general
conjugation strategy of varied types of molecules with
steroids. The multicomponent character of the reaction
allowed utilizing the steroid and the a-amino acids either
as the amino or the carboxylic component.
4.1.2. (25R)-6b-Acetoxy-5-hydroxy-5a-spirostan-3-one
(4). Diol 2 (3.0 g, 7.16 mmol) was treated in a similar way
as described in Section 4.1.1 to give the ketol 4 (1.53 g,
66 %). Mp (MeOH): 212–213 ꢀC. 1H NMR (CDCl3):
d¼0.79 (d, 3H, J¼6.2 Hz, H-21); 0.82 (s, 3H, H-18); 0.97
(d, 3H, J¼6.2 Hz, H-27); 1.39 (s, 3H, H-19); 2.09 (s, 3H,
CH3CO); 3.37 (t, 1H, J¼10.6 Hz, H-26ax); 3.45 (dd, 1H,
J¼4.1/10.8 Hz, H-26eq); 4.41 (m, 1H, H-16a); 4.73 (m,
1H, H-6a). 13C NMR (CDCl3): d¼31.6 (C-1); 37.7 (C-2);
211 (C-3); 48.4 (C-4); 76.9 (C-5); 75.6 (C-6); 33.6 (C-7);
30.1 (C-8); 45.2 (C-9); 38.9 (C-10); 20.9 (C-11); 39.8
(C-12); 40.5 (C-13); 55.4 (C-14); 31.4 (C-15); 80.6 (C-16);
62.0 (C-17); 16.5 (C-18); 15.9 (C-19); 41.6 (C-20); 14.5
(C-21); 109.2 (C-22); 31.3 (C-23); 28.7 (C-24); 30.2
(C-25); 66.8 (C-26); 17.1 (C-27); 21.4 (CH3CO); 170.0
(CH3CO). HRMS (ESI-FT-ICR) m/z: 511.3038 [M+Na]+
(Calculated for C29H44NaO6: 511.3036).
4. Experimental
4.1. General
Melting points were determined on a Stuart Scientific appa-
1
ratus and are uncorrected. H NMR and 13C NMR were
recorded on a Bruker ACF-250 spectrometer at 250.13 MHz
1
and 62.9 MHz for H and 13C, respectively, using TMS as
an internal standard. The high resolution ESI mass spectra
were obtained from a Bruker Apex 70e Fourier transform
ion cyclotron resonance (FT-ICR) mass spectrometer equip-
ped with an InfinityÔ cell, a 7.0 tesla superconducting
magnet. Reactions were monitored by thin-layer chromato-
graphy on precoated plates with silica gel (Merck) and spots
were visualized with a 1% w/v spray of vanillin in perchloric
acid and subsequent heating. ‘Usual work-up’ refers to dilu-
tion with an organic solvent, washing the extract consecu-
tively with 5% HCl and/or 5% NaHCO3, and brine, drying
over anhydrous Na2SO4 and removal of the solvent under
reduced pressure. The solid compounds were recrystallized
from selected solvents for the melting point measurements.
Flash column chromatography was performed on silica gel
60 (Merck, >230 mesh). Solvents were purified and dried
according to standard procedures. Compounds 1, 2, and 12
were obtained from the widely available diosgenin as
described in Ref. 13.
4.1.3. (25R)-A-Homo-2a-oxa-6b-acetoxy-5a-spirostan-3-
one (5). A solution of mCPBA (2.0 g, 11.6 mmol) in
CH2Cl2 (100 mL) was added to a stirred solution of ketone
3 (2.2 g, 4.6 mmol) in CH2Cl2 (100 mL) at 0 ꢀC. The reac-
tion was allowed to reach room temperature and stirred for
4 h in the dark. The mixture was diluted with CHCl3
(150 mL) and washed sequentially with solutions of 10%
Na2SO3 (2ꢁ200 mL) and 10% NaHCO3 (2ꢁ200 mL). The
organic phase was dried over anhydrous Na2SO4 and evapo-
rated under reduced pressure. The resulting crude product
was purified by flash chromatography (hexane/EtOAc, 5:1)
to yield the lactone 5 (1.57 g, 69%). Mp (EtOH): 202–
205 ꢀC. 1H NMR (CDCl3): d¼0.77 (d, 3H, J¼6.3 Hz,
H-27); 0.79 (s, 3H, H-18); 0.97 (d, 3H, J¼6.6 Hz, H-21);
1.26 (s, 3H, H-19); 2.03 (s, 3H, CH3CO); 3.36 (t, 1H,
J¼10.8 Hz, H-26ax); 3.47 (dd, 1H, J¼4.2/10.9 Hz,
H-26eq); 4.21 (m, 1H, H-2a); 4.39 (m, 1H, H-2b); 4.41
(m, 1H, H-16a); 4.94 (m, 1H, H-6a). 13C NMR (CDCl3):
d¼41.0 (C-1); 63.9 (C-2); 175.0 (C-3); 29.0 (C-4); 52.1
(C-5); 72.1 (C-6); 36.5 (C-7); 30.0 (C-8); 53.3 (C-9); 35.7
(C-10); 21.0 (C-11); 39.6 (C-12); 40.3 (C-13); 55.4 (C-14);
31.5 (C-15); 80.7 (C-16); 62.1 (C-17); 16.5 (C-18); 15.1
(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); 66.8 (C-26); 17.2 (C-27); 170.1
(CH3CO). HRMS (ESI-FT-ICR) m/z: 511.3039 [M+Na]+
(Calculated for C29H44NaO6: 511.3036).
4.1.1. (25R)-6b-Acetoxy-5a-spirostan-3-one (3). Ac2O
(6 mL, 64 mmol) was added to a solution of compound 1
(5.8 g, 10.2 mmol) in anhydrous pyridine (60 mL) and the
reaction mixture was stirred at room temperature for 18 h.
The mixture was poured into 400 mL of cold water and
the solid was filtered under reduced pressure and washed
several times with water. The resulting crude product was
dried and dissolved in anhydrous THF (60 mL). Tetrabutyl-
ammonium fluoride (TBAF) (4 mL, 1 M in THF) was added
to the solution and the reaction mixture was stirred under
nitrogen atmosphere for 6 h. The usual work-up (Et2O)
yielded a crude product, which was dissolved in CH2Cl2
(150 mL) and added to a suspension of pyridinium chloro-
chromate (PCC) (3.8 g, 17.6 mmol) in CH2Cl2 (200 mL) at
0 ꢀC. The reaction mixture was stirred for 1 h at room
temperature and then filtered through a pad of alumina.
The solution was evaporated under reduced pressure and
the crude product was purified by flash column chromato-
graphy (hexane/AcOEt, 4:1) to afford the ketone 3 (2.72 g,
59%). Mp (MeOH): 204–206 ꢀC. 1H NMR (CDCl3):
d¼0.79 (d, 3H, J¼6.4 Hz, H-21); 0.81 (s, 3H, H-18); 0.96
(d, 3H, J¼6.3 Hz, H-27); 1.35 (s, 3H, H-19); 2.05 (s, 3H,
CH3CO); 3.36 (t, 1H, J¼10.8 Hz, H-26ax); 3.45 (dd, 1H,
4.1.4. (25R)-A-Homo-2a-oxa-6b-acetoxy-5-hydroxy-5a-
spirostan-3-one (6). Ketol 4 (1.5 g, 3.1 mmol) was treated
in a similar way as described in Section 4.1.3 to give the lac-
1
tone 6 (1.02 g, 66%). Mp (MeOH): 218–220 ꢀC. H NMR
(CDCl3): d¼0.78 (d, 3H, J¼6.3 Hz, H-27); 0.79 (s, 3H,
H-18); 0.97 (d, 3H, J¼6.4 Hz, H-21); 1.25 (s, 3H, H-19);
2.08 (s, 3H, CH3CO); 3.37 (t, 1H, J¼10.9 Hz, H-26ax);

D. G. Rivera et al. / Tetrahedron 62 (2006) 8327–8334
8331
3.47 (dd, 1H, J¼4.2/10.9 Hz, H-26eq); 4.19 (m, 1H, H-2a);
4.38 (m, 1H, H-2b); 4.40 (m, 1H, H-16a); 4.93 (m, 1H,
H-6a). 13C NMR (CDCl3): d¼36.2 (C-1); 65.1 (C-2);
169.8 (C-3); 42.9 (C-4); 76.6 (C-5); 72.1 (C-6); 31.7
(C-7); 29.7 (C-8); 44.7 (C-9); 41.5 (C-10); 20.9 (C-11);
39.7 (C-12); 40.3 (C-13); 55.4 (C-14); 31.5 (C-15); 80.7
(C-16); 61.9 (C-17); 16.5 (C-18); 15.8 (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); 66.8 (C-26); 17.1(C-27); 170.0 (CH3CO). HRMS
(ESI-FT-ICR) m/z: 527.2987 [M+Na]+ (Calculated for
C29H44NaO7: 527.2985).
33.2 (C-7); 30.3 (C-8); 41.4 (C-9); 36.8 (C-10); 20.9
(C-11); 39.8 (C-12); 40.2 (C-13); 56.6 (C-14); 31.6 (C-15);
80.6 (C-16); 62.0 (C-17); 16.3 (C-18); 17.2 (C-19); 41.7
(C-20); 14.5 (C-21); 109.4 (C-22); 31.4 (C-23); 28.8 (C-24);
29.1 (C-25); 66.9 (C-26); 17.1 (C-27). HRMS (ESI-FT-
ICR) m/z: 446.3272 [M+H]+ (Calculated for C27H44NO4:
446.3270).
4.1.7. Peptide-steroid conjugate 9. A solution of amine
8 (700 mg, 1.6 mmol) and paraformaldehyde (48 mg,
1.6 mmol) in MeOH (60 mL) were stirred at room tempera-
ture for 1 h to accomplish the formation of the corresponding
imine. N-Boc-L-histidine (402 mg, 1.6 mmol) and tert-butyl-
isocyanide (0.18 mL, 1.6 mmol) were then added and the re-
action mixture was stirred for 6 h at room temperature. The
solution was concentrated under reduced pressure and then,
the usual work-up (CHCl3) yielded a crude product. Flash
column chromatography purification (CHCl3/MeOH, 20:1)
furnished the conjugate 9 (1.01 g, 82%). Mp (AcOEt):
226–227 ꢀC. 1H NMR (CDCl3): d¼0.77 (s, 3H, H-18);
0.77 (d, 3H, J¼6.5 Hz, H-27); 0.94 (s, 3H, H-19); 0.93 (d,
3H, J¼6.8 Hz, H-21); 1.32 (s, 9H, (CH3)3CNH); 1.43 (s,
9H, (CH3)3C); 3.36 (t, 1H, J¼11.0 Hz, H-26ax); 3.46 (dd,
1H, J¼4.1/11.0 Hz, H-26eq); 3.57 (m, 2H, H-2); 4.40 (m,
1H, H-16); 3.72 (s, 3H, OCH3); 4.02–3.94 (m, 6H, CH2);
4.25–4.18 (m, 2H, CH2); 4.45 (m, 1H, NCH); 4.54 (m, 1H,
H-6a); 6.74 (m, 1H, CH-imidazole); 7.32 (m, 1H, CH-imi-
dazole). 13C NMR (CDCl3): d¼14.5 (CH3); 16.3 (CH3);
17.1 (CH3); 17.2 (CH3); 20.9 (CH2); 28.8 (CH2); 28.5
(CH3); 28.3 (CH3); 29.1 (CH); 30.3 (CH); 31.4 (CH2);
31.6 (CH2); 33.2 (CH2); 35.1 (CH2); 36.8 (C); 39.8 (CH2);
40.2 (C); 41.4 (CH); 41.7 (CH); 42.3 (CH2); 45.6 (CH2);
46.2 (CH); 49.6 (CH2); 50.6 (CH2); 51.8 (CH3); 56.6
(CH); 57.9 (CH2); 62.0 (CH); 66.9 (CH2); 79.6 (C); 79.8
(CH); 80.6 (CH); 109.4 (C); 155.7 (CO); 168.5 (CO); 169.6
(CO); 178.0 (CO). HRMS (ESI-FT-ICR) m/z: 818.5044
[M+Na]+ (Calculated for C44H69NaN5O8: 818.5045).
4.1.5. (25R)-2,3-Seco-2-hydroxy-3-carboxy-5a-spiro-
stan-6b-yl (7). Lactone 5 (1.5 g, 3.1 mmol) was dissolved
in a 2% solution of KOH in MeOH (70 mL). The reaction
mixture was refluxed for 3 h, then acidified with resin
Dowex 50W (H+ form) until pH 3 and stirred at 0 ꢀC for
2 h. The resin was then filtered off and the filtrate was evapo-
rated under reduced pressure to give a white crude product.
Recrystallization from AcOEt afforded the pure g-lactone 7
(1.17 g, 78%). Mp (AcOEt): 202–203 ꢀC. 1H NMR
(CDCl3): d¼0.75 (s, 3H, H-18); 0.77 (d, 3H, J¼6.3 Hz,
H-27); 0.91 (s, 3H, H-19); 0.94 (d, 3H, J¼6.8 Hz, H-21);
3.35 (t, 1H, J¼11.0 Hz, H-26ax); 3.46 (dd, 1H, J¼4.1/
10.9 Hz, H-26eq); 3.65 (m, 2H, H-2); 4.39 (m, 1H, H-16);
4.53 (m, 1H, H-6a). 13C NMR (CDCl3): d¼35.0 (C-1);
58.4 (C-2); 178.1 (C-3); 42.2 (C-4); 46.2 (C-5); 79.9
(C-6); 33.1 (C-7); 30.3 (C-8); 41.3 (C-9); 36.8 (C-10);
20.9 (C-11); 39.6 (C-12); 40.2 (C-13); 56.5(C-14); 31.6
(C-15); 80.6 (C-16); 62.0 (C-17); 16.3 (C-18); 17.4 (C-19);
41.7 (C-20); 14.5 (C-21); 109.4 (C-22); 31.4 (C-23); 28.8
(C-24); 29.0 (C-25); 66.9 (C-26); 17.1 (C-27). HRMS
(ESI-FT-ICR) m/z: 469.2932 [M+Na]+ (Calculated for
C27H42NaO5: 469.2930).
4.1.6. (25R)-2,3-Seco-2-amino-3-carboxy-5a-spirostan-
6b-yl (8). Mesyl chloride (0.44 mL, 3.7 mmol) was added
dropwise at 0 ꢀC to a solution of lactone 7 (1.1 g,
2.4 mmol) in anhydrous CH2Cl2 (80 mL) and Et3N
(3.8 mL, 30 mmol). The reaction mixture was stirred at
0 ꢀC for 1 h and then washed with brine (2ꢁ100 mL). The
organic phase was dried over anhydrous Na2SO4 and con-
centrated under reduce pressure. The resulting crude product
was dissolved in 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-
pyrimidonone (DMPU, 30 mL) and the solution was treated
with NaN3 (403 mg, 7.2 mmol). The reaction mixture was
stirred vigorously under an argon atmosphere at 50 ꢀC for
48 h and then, the usual work-up (EtOAc) yielded a crude
product, which was dissolved in 150 mL of absolute
EtOH. Lindlar catalyst (350 mg) was added to the solution
and the mixture was treated successively with hydrogen
and vacuum, and finally stirred under hydrogen atmosphere
for 36 h. The catalyst was removed by filtration and the
resulting solution was evaporated under reduced pressure
to give a crude product. Flash column chromatography puri-
fication (CHCl3/MeOH/Et3N, 10:1:0.5) furnished the amine
8 (758 mg, 71%). Mp (MeOH): 195–197 ꢀC. 1H NMR
(CDCl3): d¼0.76 (s, 3H, H-18); 0.77 (d, 3H, J¼6.4 Hz,
H-27); 0.90 (s, 3H, H-19); 0.94 (d, 3H, J¼6.8 Hz, H-21);
3.36 (t, 1H, J¼11.1 Hz, H-26ax); 3.46 (dd, 1H, J¼4.2/
11.0 Hz, H-26eq); 3.54 (m, 2H, H-2); 4.41 (m, 1H, H-16);
4.53 (m, 1H, H-6a). 13C NMR (CDCl3): d¼35.1 (C-1);
57.9 (C-2); 178.0 (C-3); 42.3 (C-4); 46.2 (C-5); 79.8 (C-6);
4.1.8. (25R)-2,3-Seco-6b-acetoxy-2(5)-oxa-5a-spirostan-
3-oic acid (10). Fuming H2SO4 (2 mL) was added to a solu-
tion of lactone 6 (1.0 g, 1.9 mmol) in MeOH (50 mL) and the
reaction mixture was stirred at reflux with the appearance of
a precipitate after 10 min. The solid was then filtered under
reduced pressure, washed with cold MeOH (2ꢁ20 mL) and
dissolved in a mixture of THF/H2O (2:1, 300 mL). LiOH
(210 mg, 5.0 mmol) was added and the reaction mixture
was stirred at 0 ꢀC for 2 h and then acidified with aqueous
10% NaHSO4 to pH 3. The usual work-up (AcOEt) gave
a product, which was purified by flash column chromato-
graphy (CHCl3/MeOH/AcOH, 15:1:0.1) to afford the acid
1
10 (583 mg, 59%). Mp (heptane/AcOEt): 255–256 ꢀC. H
NMR (CDCl3): d¼0.77 (d, 3H, J¼6.8 Hz, H-21); 0.77 (s,
3H, H-18); 0.95 (d, 3H, J¼6.9 Hz, H-27); 0.97 (s, 3H,
H-19); 2.05 (s, 3H, CH3CO); 2.41 (d, 1H, J¼13.1 Hz,
H-4); 2.63 (d, 1H, J¼13.1 Hz, H-4); 3.35 (t, 1H, J¼
10.9 Hz, H-26ax); 3.44 (dd, 1H, J¼4.1/10.8 Hz, H-26eq);
3.90 (m, 2H, H-2); 4.36 (m, 1H, H-16a); 5.21 (m, 1H,
H-6a). 13C NMR (CDCl3): d¼37.9 (C-1); 63.9 (C-2);
169.3 (C-3); 36.4 (C-4); 83.8 (C-5); 70.5 (C-6); 30.8
(C-7); 29.3 (C-8); 45.7 (C-9); 46.6 (C-10); 22.7 (C-11);
40.6 (C-12); 39.8 (C-13); 56.7 (C-14); 31.6 (C-15); 80.7
(C-16); 62.2 (C-17); 16.4 (C-18); 15.1 (C-19); 41.6 (C-20);
14.5 (C-21); 109.4 (C-22); 31.5 (C-23); 28.6 (C-24); 30.4

8332
D. G. Rivera et al. / Tetrahedron 62 (2006) 8327–8334
(C-25); 66.7 (C-26); 17.2 (C-27); 169.7 (CH3CO). HRMS
(ESI-FT-ICR) m/z: 503.3009 [MꢂH]ꢂ (Calculated for
C29H43O7: 503.3008).
(100 mL). The reaction mixture was treated successively
with hydrogen and vacuum and finally stirred under hydro-
gen atmosphere for 24 h. The catalyst was removed by filtra-
tion and the resulting solution was evaporated under reduced
pressure to give a crude product. This product was dissolved
in CH2Cl2 (80 mL) and treated with mCPBA (1.55 g,
9.0 mmol) in a similar way as described in Section 4.1.3 to
give the lactone 14 (1.54 g, 73%). Mp (MeOH): 199–
200 ꢀC. 1H NMR (CDCl3): d¼0.78 (d, 3H, J¼6.2 Hz,
H-27); 0.81 (s, 3H, H-18); 0.96 (d, 3H, J¼6.9 Hz, H-21);
1.28 (s, 3H, H-19); 3.34 (t, 1H, J¼10.8 Hz, H-26ax); 3.46
(dd, 1H, J¼10.8/4.0 Hz, H-26eq); 4.35 (m, 1H, H-16). 13C
NMR (CDCl3): d¼24.7 (C-1); 39.9 (C-2); 29.2 (C-3); 41.2
(C-4); 176.1 (C-5); 172.5 (C-6); 40.8 (C-7); 41.6 (C-8);
50.9 (C-9); 79.1 (C-10); 35.6 (C-11); 36.7 (C-12); 48.2
(C-13); 55.1 (C-14); 31.4 (C-15); 80.4 (C-16); 61.8 (C-17);
16.2 (C-18); 22.3 (C-19); 42.2 (C-20); 14.6 (C-21); 109.0
(C-22); 31.6 (C-23); 28.8 (C-24); 30.3 (C-25); 66.8 (C-26);
17.2 (C-27). HRMS (ESI-FT-ICR) m/z: 461.2906 [MꢂH]ꢂ
(Calculated for C27H41O6: 461.2903).
4.1.9. Peptide-steroid conjugate 11. A solution of L-alanine
methyl ester hydrochloride (139 mg, 1.0 mmol), paraform-
aldehyde (30 mg, 1.0 mmol), and triethylamine (0.14 mL,
1.0 mmol) in MeOH (60 mL) were stirred at room tempera-
ture for 1 h to accomplish the formation of the corresponding
imine. Acid 10 (500 mg, 1.0 mmol) and benzylisocyanide
(0.12 mL, 1.0 mmol) were then added and the reaction mix-
ture was stirred for 10 h at room temperature. The solution
was concentrated under reduced pressure and then, the usual
work-up (CHCl3) yielded a crude product. Flash column
chromatography purification (CHCl3/MeOH, 20:1) fur-
nished the pure conjugate 11 (471 mg, 64%). Mp (MeOH):
1
211–214 ꢀC. H NMR (CDCl3): d¼0.77 (d, 3H, J¼6.8 Hz,
H-21); 0.77 (s, 3H, H-18); 0.94 (d, 3H, J¼6.7 Hz, H-27);
1.14 (s, 3H, H-19); 1.53 (d, 3H, J¼7.2 Hz, (CH3)CHN);
2.03 (s, 3H, CH3CO); 3.35 (t, 1H, J¼10.8 Hz, H-26ax);
3.44 (dd, 1H, J¼4.1/10.9 Hz, H-26eq); 3.83 (s, 3H,
OCH3); 3.92 (m, 2H, H-2); 4.15–4.08 (m, 2H, NCH);
4.29–4.26 (m, 2H, CH2); 4.39 (m, 1H, H-16a); 4.50–4.56
(m, 2H, CH2); 5.23 (m, 1H, H-6a); 7.13 (m, 5H, Ph). 13C
NMR (CDCl3): d¼14.5 (CH3); 15.1 (CH3); 16.4 (CH3);
17.2 (CH3); 22.7 (CH2); 28.6 (CH2); 29.3 (CH); 30.4
(CH); 30.6 (CH3); 30.8 (CH2); 31.5 (CH2); 31.6 (CH2);
36.4 (CH2); 37.9 (CH2); 39.8 (C); 40.6 (CH2); 41.6 (CH);
44.8 (CH2); 45.7 (CH); 46.6 (C); 53.7 (CH3); 55.7 (CH2);
56.7 (CH); 62.2 (CH); 63.9 (CH2); 66.7 (CH2); 70.5 (CH);
80.7 (CH); 83.8 (C); 109.4 (C); 126.8 (CH); 127.3 (CH);
128.1 (CH); 131.2 (CH); 168.9 (CO); 169.6 (CO); 169.9
(CO); 174.2 (CO). HRMS (ESI-FT-ICR) m/z: 759.4199
[M+Na]+ (Calculated for C42H60NaN2O9: 759.4197).
4.1.12. Peptide–steroid conjugate 15. 3-N-Cbz-L-lysine
methyl ester hydrochloride (596 mg, 1.7 mmol), paraform-
aldehyde (51 mg, 1.7 mmol), triethylamine (023 mL,
1.7 mmol), steroidal acid 14 (800 mg, 1.7 mmol), and
methyl isocyanoacetate (0.2 mL, 1.7 mmol) were reacted in
MeOH (80 mL) in a similar way as described in Section
4.1.9. Flash column chromatography purification (CHCl3/
MeOH, 15:1) furnished the pure conjugate 15 (1.1 g, 74%).
Mp (heptane/AcOEt): 219–223 ꢀC. 1H NMR (CDCl3):
d¼0.77 (d, 3H, J¼6.2 Hz, H-27); 0.84 (s, 3H, H-18); 0.95
(d, 3H, J¼6.9 Hz, H-21); 1.21 (s, 3H, H-19); 3.34 (t,
1H, J¼10.8 Hz, H-26ax); 3.46 (dd, 1H, J¼10.8/4.0 Hz,
H-26eq); 3.68 (s, 3H, OCH3); 3.72 (s, 3H, OCH3); 4.17 (m,
2H, CH2); 4.26 (m, 1H, NCH); 4.39 (m, 1H, H-16); 5.10 (s,
2H, OCH2); 7.32 (m, 5H, Ph–Cbz). 13C NMR (CDCl3):
d¼14.7 (CH3); 16.4 (CH3); 17.2 (CH3); 22.5 (CH3); 24.9
(CH2); 28.6 (CH2); 28.8 (CH2); 29.2 (CH2); 29.6 (CH2);
29.8 (CH2); 30.4 (CH); 30.7 (CH2); 31.4 (CH2); 31.6 (CH2);
35.6 (CH2); 36.8 (CH2); 39.5 (CH2); 40.2 (CH2); 41.3 (CH);
41.4 (CH2); 42.2 (CH); 44.4 (CH2); 44.8 (CH); 45.2 (CH2);
48.5 (C); 50.5 (CH); 54.9 (CH); 61.8 (CH); 66.7 (CH2);
66.9 (CH2); 79.1 (C); 80.6 (CH); 109.0 (C); 127.7 (CH);
127.9 (CH); 128.4 (CH); 136.5 (C); 157.0 (CO); 169.4
(CO); 171.1 (CO); 174.9 (CO); 175.2 (CO); 176.1 (CO).
HRMS (ESI-FT-ICR) m/z: 890.4780 [M+H]+ (Calculated
for C47H69N3NaO12: 890.4779).
4.1.10. (25R)-5,6-Seco-5-oxo-3-spirostan-6-oic acid (13).
Ketol 12 (2.5 g, 5.6 mmol) was dissolved in anhydrous pyri-
dine (50 mL) and treated with Ac2O (3 mL, 32 mmol) in
a similar way as described in Section 4.1.1. The resulting
crude product was dissolved in acetone (100 mL) and treated
with 5 mL of Jones reagent. The reaction mixture was stirred
at reflux for 2 h, concentrated until half volume and poured
into 400 mL of cold water. The solid was filtered under
reduced pressure, washed several times with water and
then recrystallized from MeOH/H2O (60 mL, 2:1) to afford
the pure ketoacid 13 (2.03 g, 81%). Mp (MeOH/H2O): 182–
185 ꢀC. 1H NMR (CDCl3): d¼0.77 (d, 3H, J¼6.4 Hz, H-27);
0.80 (s, 3H, H-18); 0.95 (d, 3H, J¼6.7 Hz, H-21); 1.11 (s,
3H, H-19); 3.33 (t, 1H, J¼10.8 Hz, H-26ax); 3.46 (dd, 1H,
J¼10.8/4.0 Hz, H-26eq); 4.34 (m, 1H, H-16a); 5.84 (m,
1H, J¼10.0/1.4 Hz, H-4); 6.75 (m, 1H, J¼10.0 Hz, H-3).
13C NMR (CDCl3): d¼24.7 (C-1); 39.9 (C-2); 146.6 (C-3);
128.5 (C-4); 207.9 (C-5); 172.4 (C-6); 40.4 (C-7); 41.8
(C-8); 51.3 (C-9); 35.6 (C-10); 35.5 (C-11); 36.1 (C-12);
48.0 (C-13); 55.1 (C-14); 31.4 (C-15); 80.2 (C-16); 61.4
(C-17); 16.2 (C-18); 18.2 (C-19); 42.2 (C-20); 14.6 (C-21);
109.0 (C-22); 31.6 (C-23); 28.8 (C-24); 30.3 (C-25); 66.8
(C-26); 17.2 (C-27). HRMS (ESI-FT-ICR) m/z: 443.2797
[MꢂH]ꢂ (Calculated for C27H39O5: 443.2796).
4.1.13. (25R)-3b-(tert-Butyldimethylsilyloxy)-5a-spiro-
stan-12-one (17). TBSCl (2.23 g, 17.5 mmol) and imidazole
(1.23 g, 18.1 mmol) were added to a solution of hecogenin
16 (5.2 g, 12.1 mmol) in anhydrous DMF (80 mL). The reac-
tion mixture was stirred under nitrogen atmosphere for 3 h
and then, the usual work-up (CHCl3) yielded a crude prod-
uct, which was purified by flash column chromatography
(hexane/AcOEt, 3:1) to give the ketone 17 (5.79 g, 84%).
1
Mp (EtOH): 187–189 ꢀC. H NMR (CDCl3): d¼0.07 (s,
3H, (CH3)2Si); 0.09 (s, 3H, (CH3)2Si); 0.76 (s, 3H, H-18);
1.17 (s, 3H, H-19); 0.78 (d, 3H, J¼6.4 Hz, H-27); 0.92 (s,
9H, (CH3)3CSi); 0.94 (d, 3H, J¼6.5 Hz, H-21); 3.36 (t,
1H, J¼10.9 Hz, H-26ax); 3.49 (dd, 1H, J¼10.8/3.9 Hz,
H-26eq); 3.54 (br m, 1H, H-3a); 4.40 (m, 1H, H-16a). 13C
NMR (CDCl3): d¼36.2 (C-1); 27.2 (C-2); 71.1 (C-3); 33.8
4.1.11. (25R)-5,6-Seco-A-homo-5(10)-oxa-5-oxo-spiro-
stan-6-oic acid (14). Pd/C 10% (800 mg) was added to a so-
lution of ketoacid 13 (2.0 g, 4.5 mmol) in absolute EtOH

D. G. Rivera et al. / Tetrahedron 62 (2006) 8327–8334
8333
(C-4); 44.4 (C-5); 28.1 (C-6); 31.4 (C-7); 34.3 (C-8); 55.4
(C-9); 36.1 (C-10); 37.7 (C-11); 213.2 (C-12); 55.0 (C-13);
55.6 (C-14); 31.2 (C-15); 79.2 (C-16); 53.5 (C-17); 16.1
(C-18); 11.9 (C-19); 42.2 (C-20); 13.2 (C-21); 109.2
(C-22); 31.4 (C-23); 28.8 (C-24); 30.2 (C-25); 66.8 (C-26);
17.1 (C-27); 26.1 ((CH3)3CSi); 18.3 ((CH3)3CSi); ꢂ2.9
((CH3)2Si). HRMS (ESI-FT-ICR) m/z: 567.3848 [M+Na]+
(Calculated for C33H56NaSiO4: 567.3846).
31.2 (C-15); 79.8 (C-16); 51.7 (C-17); 20.3 (C-18); 12.4
(C-19); 43.7 (C-20); 14.1 (C-21); 109.2 (C-22); 31.6
(C-23); 28.8 (C-24); 31.2 (C-25); 67.5 (C-26); 17.4 (C-27).
HRMS (ESI-FT-ICR) m/z: 450.3586 [M+H]+ (Calculated
for C27H48NO4: 450.3583).
4.1.16. Peptide–steroid conjugate 20. Steroidal amine 19
(800 mg, 1.8 mmol), paraformaldehyde (53 mg, 1.8 mmol),
N-Boc-L-serine (365 mg, 1.8 mmol), and benzylisocyanide
(0.22 mL, 1.8 mmol) were reacted in MeOH (100 mL) in
a similar way as described in Section 4.1.7. Flash column
chromatography purification (CHCl3/MeOH, 15:1) fur-
nished the conjugate 20 (1.10 g, 79%). Mp (AcOEt): 231–
4.1.14. (25R)-12,13-Seco-3b-(tert-butyldimethylsilyloxy)-
5a-spirostane-12,13-diol (18). Ketone 17 (4.0 g,
7.35 mmol) was treated with mCPBA (2.0 g, 11.6 mmol)
for 36 h in a similar way as described for the synthesis of
5 to give the corresponding lactone. This crude product
was dissolved in dry THF (100 mL) and added dropwise at
0 ꢀC to a solution of LiAlH4 (570 mg, 15 mmol) in dry
THF (100 mL). The reaction mixture was stirred at room
temperature under nitrogen atmosphere for 20 h. An aque-
ous 5% NaOH solution (100 mL) was added slowly to the
reaction mixture and the stirring was continued for 30 min.
The resulting white powder was then filtered off and washed
with AcOEt (3ꢁ100 mL). The collected organic phase
was washed with brine (100 mL), dried over anhydrous
Na2SO4 and concentrated under reduced pressure to give a
crude product. Flash column chromatography purification
(CHCl3/MeOH, 10:1) afforded the compound 18 (2.97 g,
72%). Mp (AcOEt): 231–233 ꢀC. 1H NMR (CDCl3):
d¼0.08 (s, 3H, (CH3)2Si); 0.09 (s, 3H, (CH3)2Si); 0.80 (d,
3H, J¼6.7 Hz, H-27); 0.82 (s, 3H, H-19); 0.92 (s, 9H,
(CH3)3CSi); 1.04 (d, 3H, J¼6.2 Hz, H-21); 1.10 (s, 3H,
H-18); 3.36 (t, 1H, J¼10.9 Hz, H-26ax); 3.41 (2H, m,
H-12); 3.46 (dd, 1H, J¼11.0/4.0 Hz, H-26eq); 3.53 (br m,
1H, H-3a); 4.41 (m, 1H, H-16a). 13C NMR (CDCl3):
d¼37.9 (C-1); 29.0 (C-2); 70.8 (C-3); 35.8 (C-4); 44.8
(C-5); 30.6 (C-6); 32.8 (C-7); 34.3 (C-8); 52.1 (C-9); 37.8
(C-10); 38.0 (C-11); 64.5 (C-12); 78.5 (C-13); 48.6 (C-14);
31.2 (C-15); 79.8 (C-16); 51.7 (C-17); 20.3 (C-18); 12.4
(C-19); 43.7 (C-20); 14.1 (C-21); 109.2 (C-22); 31.6 (C-23);
28.8 (C-24); 31.2 (C-25); 67.5 (C-26); 17.4 (C-27); 26.1
((CH3)3CSi); 18.3 ((CH3)3CSi); ꢂ2.9 ((CH3)2Si). HRMS
(ESI-FT-ICR) m/z: 587.4107 [M+Na]+ (Calculated for
C33H60NaSiO5: 587.4108).
1
233 ꢀC. H NMR (CDCl3): d¼0.78 (s, 3H, H-19); 0.81 (d,
3H, J¼6.7 Hz, H-27); 1.04 (d, 3H, J¼6.6 Hz, H-21); 1.12
(s, 3H, H-18); 1.43 (s, 9H, (CH3)3C); 3.36 (t, 1H, J¼
10.9 Hz, H-26ax); 3.39 (m, 2H, H-12); 3.44 (dd, 1H,
J¼10.8/4.1 Hz, H-26eq); 3.55 (br m, 1H, H-3a); 3.67–3.62
(m, 2H, CH2OH); 4.25–4.23 (m, 1H, NCH); 4.28–4.32 (m,
4H, CH2); 4.40 (m, 1H, H-16a); 7.13 (m, 5H, Ph). 13C
NMR (CDCl3): d¼17.4 (CH3); 12.7 (CH3); 14.2 (CH3);
20.2 (CH3); 28.5 (CH3); 28.8 (CH2); 31.2 (CH2); 31.3
(CH+CH2); 31.6 (CH2); 32.3 (CH2); 32.6 (CH2); 34.5 (CH);
36.7 (CH2); 37.0 (CH2); 37.9 (C); 38.2 (CH2); 43.6 (CH);
44.9 (CH); 45.3 (CH2); 46.8 (CH); 50.5 (CH); 51.6
(CH); 52.5 (CH); 62.9 (CH2); 63.3, (CH2); 66.8 (CH2);
67.5 (CH2); 70.3 (CH); 78.6 (C); 79.5 (C); 80.0 (CH);
109.3 (C); 126.8 (CH); 127.3 (CH); 128.2 (CH); 131.1
(CH); 155.8 (CO); 168.7 (CO); 170.1 (CO). HRMS
(ESI-FT-ICR) m/z: 806.4933 [M+Na]+ (Calculated for
C44H69NaN3O9: 806.4932).
4.1.17. Methyl (25R)-2,3-seco-2,6b-dihydroxy-5a-spiro-
stan-3-oate (21). H2SO4 (30%, 4 mL) was added dropwise
to a solution of lactone 5 (2.0 g, 4.1 mmol) in MeOH
(100 mL) and the reaction mixture was stirred at reflux for
8 h. The usual work-up (AcOEt) yielded a crude product,
which dissolved in a 1 M solution of NaOMe in MeOH
(200 mL). The reaction mixture was stirred for 2 h and
then concentrated under reduced pressure. Flash column
chromatography purification (CHCl3/MeOH, 20:1) afforded
the ester 21 (1.74 g, 88%). Mp (heptane/AcOEt): 242–
1
243 ꢀC. H NMR (CDCl3): d¼0.76 (s, 3H, H-18); 0.77 (d,
4.1.15. (25R)-12,13-Seco-12-amino-5a-spirostane-3b,13-
diol (19). Diol 18 (2.5 g, 4.4 mmol) was dissolved in
CH2Cl2 (100 mL) and treated in a similar way as described
in Section 4.1.6 to give a crude product that was identified by
1H NMR analysis as the expected amine. This product was
dissolved in THF (150 mL) and tetrabutylammonium fluo-
ride (TBAF) (4 mL, 1 M in THF) was added. The reaction
mixture was stirred under nitrogen atmosphere for 6 h and
then the usual work-up (Et2O) yielded a crude product. Flash
column chromatography purification (CHCl3/MeOH/Et3N,
10:1:0.5) afforded the amine 19 (1.24 g, 62%). Mp (hep-
tane/AcOEt): 218–220 ꢀC. 1H NMR (CDCl3/CD3OD,
95:5): d¼0.79 (s, 3H, H-19); 0.81 (d, 3H, J¼6.5 Hz,
H-27); 1.01 (d, 3H, J¼6.4 Hz, H-21); 1.08 (s, 3H, H-18);
3.36 (t, 1H, J¼10.9 Hz, H-26ax); 3.37 (m, 2H, H-12); 3.44
(dd, 1H, J¼10.8/4.0 Hz, H-26eq); 3.54 (br m, 1H, H-3a);
4.41 (m, 1H, H-16a). 13C NMR (CDCl3/CD3OD, 95:5):
d¼37.2 (C-1); 32.1 (C-2); 70.2 (C-3); 36.9 (C-4); 44.8
(C-5); 31.2 (C-6); 32.7 (C-7); 34.5 (C-8); 52.4 (C-9); 37.8
(C-10); 38.0 (C-11); 62.8 (C-12); 78.3 (C-13); 50.4 (C-14);
3H, J¼6.5 Hz, H-27); 0.81 (s, 3H, H-19); 0.94 (d, 3H, J¼
6.6 Hz, H-21); 3.36 (t, 1H, J¼11.0 Hz, H-26ax); 3.46 (dd,
1H, J¼4.1/10.9 Hz, H-26eq); 3.72 (m, 2H, H-2); 3.85 (m,
1H, H-6a); 4.41 (m, 1H, H-16a). 13C NMR (CDCl3):
d¼38.8 (C-1); 57.7 (C-2); 173.9 (C-3); 28.3 (C-4); 46.1
(C-5); 71.2 (C-6); 33.8 (C-7); 30.5 (C-8); 41.1 (C-9); 36.8
(C-10); 20.8 (C-11); 39.7 (C-12); 40.2 (C-13); 56.5 (C-
14); 31.6 (C-15); 80.5 (C-16); 62.1 (C-17); 16.3 (C-18);
17.3 (C-19); 41.5 (C-20); 14.5 (C-21); 109.3 (C-22); 31.4
(C-23); 28.8 (C-24); 29.0 (C-25); 66.9 (C-26); 17.1 (C-27).
HRMS (ESI-FT-ICR) m/z: 501.3192 [M+Na]+ (Calculated
for C28H46NaO6: 501.3194).
4.1.18. Methyl (25R)-2,3-seco-6b-acetoxy-2-amino-5a-
spirostan-3-oate (22). Compound 21 (1.6 g, 3.3 mmol) was
dissolved in dry CH2Cl2 (100 mL) and submitted to mesyla-
tion (0.6 mL of MsCl and 6.2 mL of Et3N) and subsequent
nucleophilic replacement with NaN3 (650 mg, 10 mmol) in
a similar way as described in Section 4.1.6 to give the
expected azide (identified by ESIMS analysis). This

8334
D. G. Rivera et al. / Tetrahedron 62 (2006) 8327–8334
intermediate was dissolved in pyridine (60 mL) and treated
with Ac2O (3 mL) exactly as described in Section 4.1.1.
The resulting acetate was dissolved in absolute ethanol
(100 mL) and submitted to catalytic hydrogenation
(400 mg of Lindlar catalyst) as described in Section 4.1.6
to afford the crude amine 22. Flash column chromatography
purification (CH2Cl2/MeOH/Et3N, 20:1:0.1) furnished the
pure amine 22 (1.06 g, 62%). Mp (MeOH): 217–218 ꢀC.
1H NMR (CDCl3): d¼0.77 (s, 3H, H-18); 0.77 (d, 3H,
J¼6.6 Hz, H-27); 0.82 (s, 3H, H-19); 0.96 (d, 3H, J¼
6.6 Hz, H-21); 2.02 (s, 3H, CH3CO); 3.36 (t, 1H,
J¼11.0 Hz, H-26ax); 3.45 (dd, 1H, J¼4.0/10.9 Hz,
H-26eq); 3.60 (m, 2H, H-2); 4.38 (m, 1H, H-6a); 4.41 (m,
1H, H-16a). 13C NMR (CDCl3): d¼38.2 (C-1); 53.6 (C-2);
173.8 (C-3); 28.4 (C-4); 46.5 (C-5); 72.6 (C-6); 36.3
(C-7); 30.4 (C-8); 53.8 (C-9); 35.4 (C-10); 20.9 (C-11);
39.6 (C-12); 40.5 (C-13); 55.5 (C-14); 31.4 (C-15); 80.6
(C-16); 62.0 (C-17); 16.5 (C-18); 15.2 (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); 66.8 (C-26); 17.1 (C-27); 21.3 (CH3CO); 170.0
(CH3CO). HRMS (ESI-FT-ICR) m/z: 542.3458 [M+Na]+
(Calculated for C30H49NO6Na: 542.3456).
Acknowledgements
We are grateful to the Leibniz-Institute of Plant Biochemis-
try, Halle/Saale, Germany, for providing the HRMS (ESI-
FT-ICR) determinations.
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1
234 ꢀC. H NMR (CDCl3): d¼0.78 (s, 3H, H-18); 0.77 (d,
3H, J¼6.4 Hz, H-27); 0.85 (s, 3H, H-19); 0.95 (d, 3H, J¼
6.6 Hz, H-21); 1.32–1.35 (s, 18H, (CH3)3CNH); 1.44 (s,
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3.62 (m, 2H, H-2); 3.71 (s, 3H, CH3O); 4.12–4.27 (m, 4H,
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7.21–7.25 (m, 10H, Ph). 13C NMR (CDCl3): d¼14.4 (CH3);
15.4 (CH3); 16.6 (CH3); 17.1 (CH3); 21.1 (CH2); 21.2
(CH3); 28.1 (CH2); 28.3 (CH3); 28.5 (CH3); 28.8 (CH2);
30.2 (CH); 30.7 (CH2); 31.3 (CH2); 31.4 (CH2); 35.7 (C);
36.7 (CH2); 38.0 (CH2); 39.8 (CH2); 40.7 (C); 41.5 (CH);
44.0 (CH2); 44.2 (CH2); 44.5 (CH2); 45.8 (CH); 45.9
(CH); 46.1 (CH); 53.2 (CH3); 53.3 (CH); 53.9 (CH2); 55.2
(CH); 62.1 (CH); 66.8 (CH2); 72.8 (CH); 79.5 (C); 80.4
(CH); 109.3 (C); 120.5 (CH); 121.1 (CH); 121.4 (CH);
127.1 (CH); 128.8 (CH); 129.4 (C); 155.8 (CO); 168.6
(CO); 168.8 (CO); 169.2 (CO); 169.5 (CO); 170.1 (CO);
170.3 (CO); 174.8 (CO). HRMS (ESI-FT-ICR) m/z:
1162.7036 [M+Na]+ (Calculated for C65H97N5O12Na:
1162.7033).
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