Synthesis and structure-activity relationships of 17β-substituted 14β- hydroxysteroid 3-(α-l-rhamnopyranoside)s: Steroids that bind to the digitalis receptor

Article abstract of DOI:10.1021/jm960880l

The preparation of 17β-substituted 14β-hydroxysteroid C-3 α-L- rhamnopyranosides is described. These derivatives have a 14β,20-ether, 14β,20-lactone, or 17β-CH₂CH₂OH, -CH₂CH₂NH₂, -CH=CHNO₂(

Full text of DOI:10.1021/jm960880l

J . Med. Chem. 1997, 40, 1439-1446
1439
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip s of 17â-Su bstitu ted
14â-Hyd r oxyster oid 3-(r-L-Rh a m n op yr a n osid e)s: Ster oid s Th a t Bin d to th e
Digita lis Recep tor
J ohn F. Templeton,*,† Yangzhi Ling,† Kirk Marat,‡ and Frank S. LaBella§
Faculty of Pharmacy and Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada, and
Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba,
Winnipeg, Manitoba R3E 0W3, Canada
Received December 30, 1996X
The preparation of 17â-substituted 14â-hydroxysteroid C-3 R-L-rhamnopyranosides is described.
These derivatives have a 14â,20-ether, 14â,20-lactone, or 17â-CH2CH2OH, -CH2CH2NH2,
-CHdCHNO2(E), -CHdCHCOOH(E), -CH(OH)CH2NO2(R), -CH(OMe)CH2NO2(R), -CH2-
CH2COOH, or -CH(OH)CH2NH2(R) group. Derivatives were assayed in a radioligand binding
assay for [3H]ouabain in membranes from canine heart muscle. The digitalis “receptor”
comprises isoenzymes of the ion-pumping enzyme, Na+,K+-ATPase. The 17â-CHdCHNO2(E),
17â-CHdCHCOOH(E), and 17â-CH(OMe)CH2NO2(R) derivatives were the most potent and
equivalent to ouabain with low-nanomolar IC50 values. The very potent binding affinity of the
disubstituted compound 17â-CH(OMe)CH2NO2(R) further demonstrates that 17â-unsaturated
substitution is not required for potent binding affinity. This observation may be of value in
the separation of cardiotonic and cardiotoxic effects. Tosylation of the 17â-CH2OH, prepared
from the 17â-CHO by lithium aluminum hydride reduction, yielded the 14â,17â-ether.
Synthesis of the 17â-CH2COOH gave the epimeric 14R,17R- and 14â,17â-lactones. Structures
have been established by NMR analysis.
In tr od u ction
duction of the aldehyde 1, into the 20-tosylate 3, a
suitable leaving group for substitution and chain exten-
sion, led to rapid intramolecular cyclization to the 14â,-
20-ether 4a (Scheme 1). Hydrolysis of the benzoate
esters in the oxide 4a with NH3/MeOH gave the rham-
nopyranoside 4b.
During our investigation into the structure-activity
relationships of 14â-hydroxy-21-norpregnanes and 14â-
hydroxypregnanes possessing digitalis-like activity the
20-amino- and 20-nitro-14â-hydroxy-3â-(R-L-rhamnopy-
ranosyloxy)-21-nor-5â-pregnane and 21-nitro-14â-hy-
droxy-3â-(R-L-rhamnopyranosyloxy)-5â-pregnane were
shown to bind strongly to the digitalis receptor of heart
muscle in a radioligand binding assay.1,2 Isosteric and
isoelectronic analogues of these amino and nitro com-
pounds, together with alcohol and carboxylic acid de-
rivatives, have been synthesized. The 20,21-disubsti-
tuted and 20-unsaturated 14â-hydroxypregnane R-L-
rhamnopyranosides have been prepared, and their
digitalis receptor binding affinity has been determined.
The nature of the 17â-pregnane side chain has been
shown to have a major influence on binding affinity.1-3
We report here on the chemical synthesis and receptor
binding affinity of these derivatives as measured in a
radioligand binding assay.4
A number of methods are available for the extension
of an aldehyde by one carbon unit.5,6 The synthetic
route to the alcohol 8 shown in Scheme 1 was chosen
because of its simplicity and the stability of other
functional groups to the reagents employed.5-10 2-Chloro-
1,3-dithiane was prepared as described by Arai and
Okai7 and converted into 1,3-dithiane 2-triphenylphos-
phonium chloride as described by Kruse et al.8 Con-
densation of the aldehyde 1 with the ylid generated from
the phosphonium salt followed by hydrolysis with HgII-
9
Cl2 gave two isomeric products (6 and 7a ) which were
separated by chromatography. The major compound
was identified as the 17â-isomer of the lactone 7a and
the minor product the 17R-isomer 6. Hydrolysis of the
17â-isomer with NH3/MeOH gave the rhamnopyrano-
side 7b. The 17â-lactone 7a was reduced with lithium
aluminium hydride (LAH), to give the 14â,21-diol 8. The
14â-hydroxyl group is ideally located for cyclization to
a 5- or 6-membered ring. The small amount of the 17R-
isomer 6 obtained may result from epimerization of the
aldehyde 1 by the sodium ethoxide used in the reaction
to produce the intermediate ketene dithioacetal. For-
mation of the 14R,17R-lactone 6 may result, after initial
epimerization at C-17, from carbonium ion formation
at C-14 induced by the acidic HgCl2/MeCN/H2O condi-
tions. C-17 epimerization decreases steric crowding of
the 14-OH and, perhaps also through anchimeric as-
sistance by the carboxyl group, favors carbonium ion
formation. The carboxylic acid would be favorably
located for intramolecular cyclization to the lactone.
While generally not reactive the sterically hindered
Resu lts a n d Discu ssion
Ch em istr y. The 17â-aldehyde 1, obtained from
degradation of the 17â-unsaturated lactone present in
the natural cardiac glycosides, is a key synthetic inter-
mediate in the synthesis of C-17 steroid derivatives.2
Modification of our earlier procedure increased the
overall yield of the 17â-aldehyde 1 from digitoxigenin
from 27% to 86%.2
Attempts to convert the 20-alcohol2 2, obtained by
lithium tri-tert-butoxyaluminum hydride (LTBAH) re-
† Faculty of Pharmacy.
‡ Department of Chemistry.
§ Department of Pharmacology and Therapeutics.
X Abstract published in Advance ACS Abstracts, April 1, 1997.
S0022-2623(96)00880-1 CCC: $14.00 © 1997 American Chemical Society

1440 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Templeton et al.
Sch em e 1a
a
(i) LTBAH/Et2O; (ii) TsCl/pyridine; (iii) NaOMe/EtOH/PhP3 + 1,3-dithiane Cl-; (iv) HgCl2/CH3CN/H2O; (v) LAH/Et2O; (vi) NH3/
MeOH.
tertiary 14-alcohol readily undergoes these intramo-
lecular cyclizations.
Dornow et al.13 reported an improved yield of aminol
on LAH reduction of a nitrol when a solution of LAH
was added to the nitrol rather than the reverse. The
nitrol 9a was therefore reduced in this way to give the
aminol 11. The silyl-protected nitrol 10 was also
reduced with LAH in refluxing Et2O to give the aminol
11 because it has been reported that the silyl derivative
can protect the nitrol group from bond cleavage.14
However, in both cases the yield of the aminol 11 was
low.
Condensation of the aldehyde 1 with nitromethane
in the presence of KF gave only one isomer, the 20(R)-
nitrol 9a (Scheme 2).10 This stereoselectivity may result
from steric hindrance by the 13-Me group and hydrogen
bonding between the 14â-OH and the 17â-CHO, which
orients the aldehyde so that approach of the ni-
tromethane carbanion occurs to the re face of the
carbonyl bond.
The 17â-CHdC(CH3)NO2(E) trisdigitoxoside, pre-
pared by Eberlein et al.,12 showed weak digitalis-like
activity on heart muscle. However, because of the high
reactivity of the nitroethylene group, it proved necessary
to have a methyl group at C-22 to increase the stability
of nitroethylene in order to prepare the glycoside.12
Nevertheless, despite the instability of the unsubsti-
tuted olefin, we have been able to synthesize the
unsubstituted derivative 13b by the following proce-
dure.
The nitrol 9a was acetylated to give the intermediate
nitrol acetate 12, a compound prone to elimination,
which formed the nitroethylene 13a during chromato-
graphic purification on SiO2. Attempts to hydrolyze the
benzoate groups in 13a with NH3/MeOH caused a series
of changes probably due to the reactivity of the nitro-
ethylene group.12 We therefore studied the hydrolysis
by NaOMe in more detail and found that although
hydrolysis of the benzoates took place very rapidly so
did Michael addition of MeO- at C-20. When the
nitroethylene 13a was treated with 0.25 M NaOMe/
MeOH in an ice-water bath, the 20(R)-methoxy deriva-
tive 14 was separated as the main product. Steric
requirements favor selective addition to the double bond
from the re face. When 13a was treated with 0.25 M
t-BuOK/t-BuOH for 10 min, the bulky t-BuO- group
Attempts to hydrolyze the benzoate groups in 9a with
NH3/MeOH yielded a mixture of products containing
aldehyde signals in the 1H NMR spectrum. The 2-nitrol
group can undergo a retro-aldol reaction in base.11 To
avoid this reaction the 20-hydroxyl group in 9a was
protected as the silyl ether 10. Employing the conven-
tional method (t-BuMe2SiCl/imidazole/DMF) prepara-
tion of the silyl ether failed. However, when the nitrol
9a was treated with tert-butyldimethylsilyl triflate and
triethylamine, the noncrystalline silyl ether 10 was
obtained.
In 1974, Eberlein et al.12 reported that when the 17â-
aldehyde trisdigitoxose tetraacetate obtained from digi-
toxin was condensed with nitroethane in the presence
of NaOMe/MeOH for 3 h, the 17â-(2-nitro-1-hydroxypro-
pyl) glycoside was obtained as two pairs of diastereo-
isomers.12 When similar conditions were applied to the
condensation of the 17â-aldehyde tribenzoate 1 with
nitromethane, the major product obtained was the nitrol
9b together with the 17R-epimer 9c as a minor product.
When the condensation reaction was carried out for a
longer time (16 h) the proportion of 17R-epimer 9c
increased. This result is indicative of reversible alde-
hyde condensation with nitromethane and epimeriza-
tion of the 17â-aldehyde followed by nitromethane
condensation (Scheme 3).

Steroids That Bind to the Digitalis Receptor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1441
Sch em e 2a
a
(i) MeNO2/KF; (ii) t-BuMe2SiSO3CF3/Et3N; (iii) LAH/Et2O; (iv) Ac2O/DMAP; (v) SiO2; (vi) NaOMe/MeOH; (vii) NaBH4/EtOH; (viii)
NH3/MeOH; (ix) MeNO2/NaOMe; (x) t-KOBu/t-BuOH; (xi) H2/PtO2/HOAc; (xii) d-tartaric acid.
R,â-unsaturated ester 17 (Scheme 4).15 Hydrolysis of
Sch em e 3. Enolization/Aldol/Retro-Aldol Equilibria
the esters in compound 17 gave the acid 18. The ester
17 was hydrogenated at 5 atm with PtO2 as catalyst to
give the saturated ester, which was hydrolyzed to give
the saturated acid 19. The proposed mechanism for the
phosphonate modification of the Wittig reaction sug-
gests that only the E isomer is produced under these
conditions.16
Nu clea r Ma gn etic Reson a n ce Str u ctu r a l Assign -
m en ts. Structures of all synthetic compounds were
established from the 1H and 13C NMR and are in
agreement with published data.1,2 Complete 2D assign-
ments (HSQC and COSY) were performed on com-
pounds 6, 7a , 9a -c, 13a , 14 and 18.
The structure of 4a was established by comparison
1
of the H and 13C NMR with compound 2 which showed
the expected changes to the C-20 methylene protons.
retarded Michael addition as expected and the rham-
nopyranoside 13b was obtained in 32% yield.
Compound 6 showed a relatively small NOE (ca. 2%)
from the C-13 methyl to the C-20 protons, in contrast
to 7a (4%). However, a substantial NOE was observed
from the C-13 methyl group to H-17 (6.0%), with a
corresponding NOE observed from the C-13 methyl
(3.5%). These data clearly establish that the C-17 side
chain in 6 has R stereochemistry. The C-14 signal in
the 14R,17R-isomer 6 at 85.76 ppm, unlike the 14â,17â-
isomer 7a at 87.09 ppm, clearly distinguishes between
The 21-nitro compound 15 was prepared from the
nitrol 9a via the intermediate 12 as described in ref 2.
Compound 15 was hydrogenated in glacial HOAc at 5
atm with PtO2 as catalyst to give the 21-amine which
was isolated as the hydrogen tartrate salt 16.
The aldehyde 1 reacted readily with the anion of
[(ethoxycarbonyl)methyl]diethylphosphonate to give the

1442 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Templeton et al.
Sch em e 4a
a
(i) (EtO)2POCH2COOEt/NaH; (ii) NaOMe/MeOH; (iii) H2/PtO2/EtOH.
Ta ble 1. [3H]Ouabain Radioligand Assay Potency of
17â-Substituted 14â-Hydroxypregnane
3â-(R-L-Rhamnopyranoside)sa,b
the isomers. The â-face stereochemistry of the lactone
7a was established by the observation of NOEs from
the C-13 methyl to the low-field C-20 proton (4.1%) and
from the low-field C-20 proton to the C-13 methyl group
(4.5%). Only a relatively small NOE (<2%) was ob-
served from the C-13 methyl group to H-17 which gave
further confirmation of the stereochemistry. The struc-
tures of both lactone isomers 6 and 7a were determined
unambiguously using 2D-NMR techniques.
The 21-alcohol 8 showed comparable spectra to the
21-nitro derivative 15 which is in agreement with the
structure proposed. In nitrol 9c J (17,20) is very small
(ca. 1 Hz) indicating a gauche arrangement of H-17 and
H-20. Irradiation of the C-13 methyl showed NOEs to
H-20 (8.8%), H-8 (3.4%), and H-12â (1.9%) and indicates
a â stereochemistry for the C-17 side chain.
no.
17â
IC50, nM, ( SE
4b
7b
8a
9c
11
14â,20-ether
2200 ( 320
NAc
14â,20-lactone
-CH2CH2OH
660 ( 70
4730 ( 420
3220 ( 250
8.3 ( 2.2
30 ( 7.5
1890 ( 220
13 ( 2.2
230 ( 30
45.5 ( 8.2
-CH(OH)CH2NO2(R)
-CH(OH)CH2NH2(R)
-CHdCHNO2(E)
-CH(OMe)CH2NO2(R)
-CH2CH2NH2
13b
14
16d
18
-CHdCHCOOH(E)
-CH2CH2COOH
-CH2CH2NO2
19
20e
a
IC50 represents the concentration that inhibits binding of
[3H]ouabain by 50%. Digitoxin IC50 ) 8 nM. c Not active, >100
b
µM. Hydrogen tartrate salt. e See ref 2.
d
The silyl derivative 10 shows a value of 1.5 Hz for J
(17,20) indicating a gauche arrangement of H-20 and
H-17. Irradiation of H-20 resulted in a 1.6% NOE of
the C-13 methyl group, while irradiation of the C-13
methyl group resulted in a 5.5% NOE of H-20, clearly
indicating that H-20 is anti to C-16 as opposed to anti
to C-13. Irradiation of the C-13 methyl also resulted
in a 2.4% NOE of the low-field C-20 Me3Si. Irradiation
of the low-field Me3Si resulted in a 0.9% NOE of the
C-13 methyl. The NOEs observed between the C-13
methyl and the C-20 Me3Si indicate that the C-20
t-BuMe2Si group must be anti to H-17. With the
location of H-20 already established, this is sufficient
to determine the C-20 stereochemistry as R.17 NOE for
this compound was determined in benzene-d6 to sepa-
rate the side chain proton shifts. The 20(R) configura-
tion was therefore established for 9a (and 9b) based on
the silyl ether 10.
The J (20,21) coupling of 13.3 Hz in the nitroethylene
13a indicates a trans rather than a cis arrangement of
H-20 and H-21. This assignment was confirmed by the
absence of a detectable NOE between the two protons.
Irradiation of H-21 resulted in a 1.7% NOE to H-17,
while irradiation of the C-13 methyl group resulted in
a 4.2% NOE to H-20. These data, along with a J (17,-
20) of 11.5 Hz suggest that the C-17 side chain adopts
a conformation in which H-17 is anti to H-20. This
analysis establishes the E stereochemistry for 13a .
A value of 1.5 Hz was observed for J (17,20) in the
methoxy derivative 14. Irradiation of the C-13 methyl
resulted in NOEs to H-20 (13.5%) and C-20 (3.65%).
Irradiation of the C-20 methoxy resulted in NOEs to
H-20 (6.1%) and the C-13 methyl (1.5%). These data
establish that H-20 is anti to C-16 and that the C-20
methoxyl is anti to H-17. The C-20 stereochemistry is
thus R. NOE measurements involving the C-21 protons
were not possible because of overlap with the solvent
peak.
The E geometry of the 20,21-double bond in 17 and
18 was evident from the coupling constant of the doublet
(J ) 15.5 Hz) for H-21. In the unsaturated acid 18 the
15.5 Hz value for J (20,21) and the lack of any observ-
able NOE between these protons clearly indicate a trans
arrangement of H-20 and H-21.15 Irradiation of H-21
resulted in a 3% NOE to H-17, while irradiation of H-17
resulted in a 7.4% NOE to H-21. Irradiation of the C-13
methyl group resulted in a 7.5% NOE to H-20. J (17,-
20) is 10.7 Hz. This coupling and the NOE data suggest
that the side chain adopts a conformation in which H-17
is anti to H-20 as seen in 13a .
Ra d ioliga n d Recep tor Bin d in g a n d Str u ctu r e-
Activity Rela tion sh ip s. The relatively weak binding
affinity obtained for the cyclic ether 4b (see Table 1)
nevertheless shows that, as has been observed previ-
ously, neither the unsaturated lactone1,2 nor the 14â-
hydroxyl group of the natural cardiac glycosides are
essential for strong receptor binding.3 Similarly the
binding affinity values of 14, 19, and 20 show that an
unsaturated system at C-17 is not required for strong
receptor binding although considered necessary for
strong positive inotropy3,18 (see below). Examination of
3â-rhamnopyranosyl-14â-amino-5â-pregnan-20(R)-ol
(LND 623) indicates that the saturated pregnane side
chain is adequate for strong inotropic activity.19 The
lack of significant affinity observed for the 6-membered
14â,17â-lactone ring 7b shows that this substituent

Steroids That Bind to the Digitalis Receptor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1443
interferes with receptor binding, more so than the
smaller, and less polar, 5-membered ether ring in 4b.
permit differentiation between cardiotonic and car-
diotoxic effects.3
The derivatives which are isosteric with the naturally
occurring unsaturated lactone ring, 13b and 18, show
binding affinity approaching that of the natural cardiac
glycosides (ca. 8 nM). Thomas et al.20 demonstrated
that bioisosteres of the unsaturated lactone ring such
as the esters -CHdCHCOOMe(E) showed positive
inotropy while the acid -CHdCH-COOH(E) was inac-
tive. The nitroethylene 13b and the unsaturated car-
boxylic acid 18 show very strong binding affinity. In
both compounds 13b and 18, the NMR results clearly
demonstrate that the C-17 side chain and its polar
substituent are orientated so that C-21 eclipses H-17.21
This places the polar C-21 substituent in a similar
location to that of the C-23 carbonyl in the cardiac
glycosides and supports the proposal that optimum
receptor binding requires a polar group in this location
and a positive center at C-20.3 Thomas et al.3 correlated
positive inotropy with the size of the positive charge
induced at C-20 based on its chemical shift which places
13b (C-20 138.92 ppm) among the potentially least
potent inotropic compounds. However, while consis-
tently the 20-methyl glycoside analogue of 13b, synthe-
sized by Eberlein et al.,12 showed only weak cardiotonic
activity, this may be due to steric factors which do not
permit the 17-substituent to take up the most favorable
conformation. The saturated analogues, 19 and 20, of
18 and 13b, respectively, while binding strongly to the
receptor, bind somewhat more weakly than 13b and 18.
Thomas et al.3,18 have shown that inotropic activity was
almost completely abolished upon saturation of the C-20
double bond in the 17â-acrylate, whereas these results
indicate that receptor binding affinity is much less
affected.
The saturated 21-nitro-20(R)-methoxy-substituted de-
rivative 14, unlike the corresponding 21-nitro-20(R)-
alcohol 9c, binds strongly, comparable to the unsubsti-
tuted 21-nitro derivative 20. The 21-amino-20(R)-
alcohol 11, like the 21-nitro-20(R)-alcohol 9c, shows
weak binding affinity. The 21-amino derivative 16
shows weak binding compared with the much stronger
binding affinity of the 21-nitro derivative 20. The 21-
alcohol 8a binds more strongly than the 21-amino
derivative 16 but less strongly than the 21-nitro deriva-
tive 20. Differences in receptor binding may result from
competition between the hydrogen-bonding donor mol-
ecules 8a and 16 and the acceptor molecule 20. These
results are in agreement with the demonstration by
Repke and co-workers22 that the 5â,14â-androstane-
3â,14-diol is the basic steroid structure of the cardiac
glycosides possessing cardiotonic activity.
Exp er im en ta l Section
Gen er a l. All 1H and 13C NMR survey spectra were recorded
on a Bruker AM300 spectrometer, while homonuclear correla-
tion (COSY), heteronuclear correlation (HSQC), and nuclear
Overhauser effect (NOE) difference spectra were recorded on
a Bruker AMX500 instrument as described previously.26 All
J values are reported in hertz (Hz). Samples were ca. 50 mM
solutions in 5 mm tubes in the solvents reported. For samples
in CDCl3 the residual CHCl3 peak in the solvent (δH 7.26 ppm,
δC 77.0 ppm) was used as the internal reference; for other
solvents Me4Si was used as an internal reference. All spectra
were run at 300 K. Carbon spectra were classified as to
multiplicity with the DEPT technique.27 Complete H and 13C
1
NMR assignments are reported for the tribenzoate 13a and
R-L-rhamnopyranoside 14; for the remaining compounds only
data relevant to the C-17 side chain are given. Reactions were
monitored by TLC on silica gel plates (Merck type 60H) and
visualized by dipping in 4% H2SO4 in EtOH followed by
heating at 120-150 °C. Flash column chromatography (FCC)
was carried out on silica gel (Merck type 60 for column
chromatography) in the solvent systems indicated. LP refers
to petroleum fractions with bp 35-60 °C. Solutions were dried
using anhydrous Na2SO4. Melting points were measured on
a Kofler type hot-stage apparatus and are uncorrected.
3-[(Tr i-O-ben zoyl-r-L-r h a m n op yr a n osyl)oxy]-17â-(h y-
d r oxym eth yl)-5â-a n d r osta n e-3â,14â-d iol (2). To a stirred
solution of 128 (200 mg, 0.25 mmol) in dry Et2O (20 mL) was
added LTBAH (254 mg, 1.3 mmol) for 2 h when TLC (25%
acetone/LP) showed no starting material. CH2Cl2 was added
and the mixture washed with 1 M HCl. After evaporation FCC
of the residue on elution with 25% acetone/LP gave the
noncrystalline 2 (145 mg, 57%) which was sufficiently pure
by TLC and 1H NMR for use in the following reaction: 1H
NMR (CDCl3) δ 1.00 and 1.02 (2s, 6H, 10-, 13-Me), 3.43 (dd, J
) 2.5, 10.3, 1H, 20-H), 3.72 (d, J ) 10.3, 1H, 20-H); 13C NMR
(CDCl3) δ 21.31 or 21.71 (16), 51.28 (17), 14.75 (18), 62.31 (20).
3â-[(Tr i-O-b e n zoyl-r-L-r h a m n op yr a n osyl)oxy]-17â-
m eth ylen e-5â-a n d r osta n e 14â,20-Ep oxid e (4a ). 2 (130 mg,
0.17 mmol) was treated with p-toluene sulfonyl chloride (100
mg, 0.53 mmol) in pyridine (2 mL) and allowed to stand at 20
°C for 16 h; CH2Cl2 (50 mL) was added and the mixture washed
with 1 M HCl, saturated NaHCO3, and water. After evapora-
tion FCC (elution with 10% acetone/LP) gave 4a (70 mg,
1
54%): mp 207-210 °C (Et2O/LP); H NMR (CDCl3) δ 0.99 (s,
3H, 13-Me), 1.09 (s, 3H, 10-Me), 3.47 (d, J ) 7.5, 1H, 20-H),
3.97 (ddd, J ) 3.2, 3.2, 7.3, 1H, 20-H); 13C NMR (CDCl3) δ
25.65 (16), 46.16 (17), 15.32 (18), 73.37 (20). Anal. (C47H54O9)
C, H.
3â-(r-L-R h a m n op yr a n osyloxy)-17â-m et h ylen e-5â-a n -
d r osta n e 14â,20-Ep oxid e (4b). 4a (90 mg, 0.12 mmol) in
MeOH (5 mL) was treated with 10% NH3/MeOH (5 mL) at 0
°C for 14 h. After evaporation, the residue on FCC (elution
with 7.5% MeOH/CH2Cl2) gave 4b (45 mg, 85%): mp 249-
251.5 °C (MeOH/acetone/LP); 1H NMR (CD3OD) δ 0.97 and
1.00 (2s, 6H, 10-, 13-Me), 3.48 (d, J ) 7.3, 1H, 20-H), 3.95 (m,
2H, 20-H); 13C NMR (CD3OD) δ 26.47 (16), 47.41 (17), 15.65
(18), 74.17 (20). Anal. (C26H42O6) C, H.
14r-Hydr oxy-3â-[(tr i-O-ben zyoyl-r-L-r h am n opyr an osyl)-
oxy]-5â,17r-p r egn a n e-21-ca r boxylic Acid 14,21-La cton e
(6) a n d 14â-Hyd r oxy-3â-[(tr i-O-ben zoyl-r-L-r h a m n op y-
r a n osyl)oxy]-5â-p r egn a n e-21-ca r boxylic Acid 14,21-La c-
ton e (7a ). To the 1,3-dithaine-2-triphenylphosphonium chlo-
ride7,8 (180 mg, 0.36 mmol) in absolute ethanol (2.5 mL) under
Ar was added with vigorous stirring 0.25 M ethanolic NaOEt
(1.8 mL, 0.45 mmol). The reaction mixture immediately
became bright yellow, and a precipitate formed. After 5 min
a solution of 1 (240 mg, 0.31 mmol) suspended in absolute
ethanol (l mL) was added in one portion. The solid dissolved
slowly on stirring which was continued for 2 h when TLC (20%
acetone/LP) showed that reaction was complete. Water (100
mL) was added, the mixture extracted with CH2Cl2, and the
organic layer washed with water, dried, and evaporated. The
Con clu sion s
Pregnane 3â-R-L-rhamnopyranoside derivatives have
been synthesized from digitoxigenin. Some derivatives
show receptor binding affinity comparable to the natu-
rally occurring cardiac glycosides as measured in a
radioligand binding assay. Unsaturation at C-20 is not
required for strong receptor binding affinity. Some
synthetic pregnane glycosides have been shown to
possess an improved margin of safety compared with
the naturally occurring cardiac glycosides.23-25 Phar-
macological studies of compounds 13b, 14, 18, and 20
may provide structure-activity relationship evidence to

1444 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Templeton et al.
residue, without further purification, was taken up in CH3-
CN/water (5:l) (12 mL), HgIICl2 (400 mg, 1.50 mmol) added,
and the mixture stirred for 18 h when TLC indicated that
hydrolysis was complete.9 The reaction mixture was filtered
through a Celite pad which was washed with acetonitrile and
on evaporation the residue was taken up in CH2Cl2 and dried.
After evaporation the residue on FCC (elution with 25%
EtOAc/LP) gave fractions (23 mg) which on recrystallization
Further elution gave 9c (25 mg, 8%): mp 196-197 °C (acetone/
LP); 1H NMR (CD3OD) δ 0.95 (s, 10-Me), 1.12 (s, 13-Me), 4.23
(ddd, J ) 1.9, 9.9, 9.9, 20-H), 4.32 (d, J ) 9.9, 21-HB), 4.55
(dd, J ) 2.0, 9.9, 21-HA); 13C NMR (CD3OD) δ 24.43 (16), 51.97
(17), 20.47 (18), 72.21 (20), 82.85 (21). Anal. (C27H4509N‚H2O)
C, H, N.
20(R)-(ter t-Bu t yld im et h ylsiloxy)-21-n it r o-3â-[(t r i-O-
ben zoyl-r-L-r h am n opyr an osyl)oxy]-5â-pr egn an -14â-ol (10).
To a solution of 9a (200 mg, 0.24 mmol) in CH2Cl2 (10 mL)
containing Et3N (261 mg, 2.6 mmol), cooled in an ice-water
bath under an Ar atmosphere, was added slowly tert-bu-
tyldimethylsilyl triflate (283 mg, 1.1 mmol), and stirring was
continued for 1.5 h when TLC (20% acetone/LP) indicated
reaction was complete. CH2Cl2 (150 mL) was added, and the
organic layer was washed with water to give a residue which
on FCC (elution with 12% acetone/LP) gave the noncrystalline
1
yielded 6 (12 mg, 5%): mp 205-207 °C (Et2O/LP); H NMR
(CDCl3) δ 1.05 (s, 3H, 10-Me), 1.38 (s, 3H, 13-Me), 2.31 (d, J
) 18.2, 1H, 20-H), 2.48 (t, J ) 9.5, 1H), 2.90 (dd, J ) 9.3, 18.1,
1H, 20-H); 13C NMR (CDCl3) δ 33.49 or 35.62 or 37.36 (16,
20), 43.28 (17), 18.77 (18), 177.14 (21). Anal. (C48H54O10) C,
H.
Further elution yielded fractions (143 mg) which gave 7a
(92 mg, 37%): mp 214-216 °C (Et2O/LP); 1H NMR (CDCl3) δ
1.08 (s, 6H, 10-, 13-Me), 2.41 (d, J ) 19.7, 1H, 20-H), 2.87 (dq,
J ) 2, 4, 19, 1H, 20-H); 13C NMR (CDCl3) δ 27.48 (16), 42.14
(17), 14.63 (18), 31.07 or 32.29 (20). Anal. (C48H54O10) C, H.
14â-H yd r oxy-3â-(r-L-r h a m n op yr a n osyloxy)-5â-p r eg-
n a n e-21-ca r boxylic Acid 14,21-La cton e (7b). 7a (55 mg,
0.069 mmol) was stirred in MeOH (7 mL) and l0% NH3/MeOH
(5 mL) for 16 h under Ar when TLC (12% MeOH/CH2Cl2)
indicated that hydrolysis was complete. On evaporation the
residue was immediately recrystallized to give 7b (22 mg,
66%): mp 271-274 °C (acetone/LP); 1H NMR (DMSO-d6) δ
0.94 and 0.95 (2s, 6H, 10-, 13-Me), 2.35 (d, J ) 19.2, 1H, 20-
H), 2.77 (dq, J ) 2.4, 4.8, 18.9, 1H, 20-H), 4.46 (d, J ) 4.5,
ROH), 4.64 (dd, J ) 5.3, ROH), 4.69 (dd, J ) 5.3, ROH); 13C
NMR (DMSO-d6) δ 26.78 (16), 41.43 (17), 14.18 (18), 38.24 (20),
170.73 (21). Anal. (C27H42O7) C, H.
1
10 (116 mg, 51%) which was sufficiently pure by TLC and H
and 13C NMR for use in the following reaction: 1H NMR
(CDCl3) δ 0.93 (s, 3H, 13-Me), 1.03 (s, 3H, 10-Me), 4.48 (m,
3H, 20-H, 21-H2); 13C NMR (CDCl3) δ 19.41 (16), 52.68 (17),
14.90 (18), 39.90 (20), 79.12 (21).
21-Am in o-3â-(r-L-r h a m n op yr a n osyloxy)-5â-p r egn a n e-
14â,20(R)-d iol (11). F r om 9a : To a stirred solution, under
Ar, of 9a (500 mg, 0.60 mmol) in dry Et2O (50 mL) under reflux
was added LAH (600 mg, 16 mmol) in dry Et2O (100 mL) over
15 min, and reflux was continued for a further 75 min when
TLC [CHCl3:MeOH:Et3N (100:40:3)] indicated that reaction
was complete. To the stirred, cooled (ice bath) mixture were
added slowly water (0.4 mL), 15% NaOH (0.4 mL), and again
water (1.2 mL), and was stirring continued for a further 20
min to form
a
granular precipitate.29 The residue from
evaporation of the Et2O was extracted by 40 min reflux with
MeOH (3 × 50 mL) and filtered through silica gel, and the
filtrate was evaporated and subjected to FCC. Elution with
CHCl3:MeOH:Et3N (100:40:3) gave 11 (50 mg, 17%): mp 225-
229 °C (MeOH/Et2O); 1H NMR (CD3OD) δ 0.96 (s, 3H, 10-Me),
1.04 (s, 3H, 13-Me), 2.80 (m, 2H, 21-H2), 3.93 (m, 1H, 20-H);
13C NMR (CD3OD) δ 19.44 (16), 53.81 (17), 15.38 (18), 67.60
(20), 45.71 (21). Anal. (C27H47O7N‚2.5H2O) C, H, N.
F r om 10: 10 (190 mg, 0.18 mmol) was added to a solution
of LAH (400 mg, 10.5 mmol) in Et2O (45 mL) and the mixture
refluxed for 1.5 h. Workup as above gave 11 (27 mg, 30%):
mp 224-228 °C (MeOH/Et2O).
3â-(r-L-R h a m n op yr a n osyloxy)-5â-p r e gn a n e -14â,21-
d iol (8). To a solution of 7a (170 mg, 0.22 mmol) in dry Et2O
(15 mL) was slowly added a solution of LAH (180 mg, 4.7
mmol) in dry Et2O (30 mL). After refluxing under Ar for 1 h
the stirred mixture was cooled in an ice-water bath, and water
(0.2 mL), 15% NaOH (0.18 mL), and again water (0.5 mL) were
added to give a granular precipitate.29 After evaporation the
residue was extracted by reflux with MeOH (3 × 45 mL) for
30 min. The mixture was filtered through Celite and evapo-
rated. The residue on FCC (elution with 12% MeOH/CH2Cl2)
gave 8 (31 mg, 30%): mp 227-230 °C (MeOH/acetone); 1H
NMR (CD3OD) δ 0.94 and 0.95 (2s, 6H, 10-, 13-Me), 3.43 (m,
1H, 21-H), 3.58 (m, 1H, 21-H); 13C NMR (CD3OD) δ 28.44 (16),
47.26 (17), 15.92 (18), 38.68 (20), 62.45 (21). Anal. (C27H46O7)
C, H.
3â-[(Tr i-O′-Ben zoyl-r-L-r h am n opyr an osyl)oxy]-21-n itr o-
5â-p r egn a n e-14â,20(R)-d iol (9a ). 1 (1.19 g, 1.3 mmol),
freshly distilled nitromethane (400 mg, 6.4 mmol), and KF (94
mg, 1.0 mmol) were stirred in dry 2-PrOH (20 mL) for 16 h.10
The mixture was diluted with CH2Cl2 (100 mL) and washed
with water. The residue from evaporation on FCC (elution
with 20% acetone/LP) gave fractions of the noncrystalline 9a 2
(460 mg, 38%) which was sufficiently pure by TLC (25%
acetone/LP) and 1H and 13C NMR for use in the following
reactions: 1H NMR (CDCl3) δ 1.04 (s, 3H, 10-Me), 1.11 (s, 3H,
13-Me), 4.23 (m, H, 21-H), 4.42 (dd, J ) 8.6, 11.4, 1H, 21-H),
4.52 (dd, J ) 4.2, 8.7, 1H, 20-H); 13C NMR (CDCl3) δ 27.48
(16), 42.14 (17), 14.63 (18), 31.29 or 32.07 (20).
14â-Hydr oxy-21-n itr o-3â-[(tr i-O-ben zoyl-r-L-r h am n opy-
r a n osyl)oxy]-5â-p r egn a -20,21-d ien e (13a ). 9a (250 mg,
0.30 mmol), Ac2O (1 mL), and 4-(N,N-dimethylamino)pyridine
(10 mg) in dry Et2O (5 mL) were stirred for 14 h to give 12.
Et2O (50 mL) was added and the mixture washed with excess
aqueous NaHCO3 and water. The residue on FCC (elution
with 20% acetone/LP) gave 13a (113 mg, 46%): mp 242-244
°C (acetone/Et2O); 1H NMR (CDCl3) δ 0.91 (s, 3H, 10-Me), 1.04
(s, 3H, 13-Me), 4.08 (br s, 1H, 3-H), 5.10 (d, J ) 1.2, 1H, 1′-H),
5.63 (dd, J ) 1.8, 3.4, 1H, 2′-H), 5.87 (dd, J ) 3.4, 10.1, 1H,
3′-H), 5.67 (t, J ) 9.9, 1H, 4′-H), 4.23 (m, 1H, 5′-H), 1.35 (d, J
) 6.3, 1H, 6′-H), 6.85 (d, J ) 13.3, 1H, 21-H), 7.25-8.12 (m,
16H, 20-H, aromatic H); 13C NMR (CDCl3) δ 30.47 (1), 26.47
(2), 73.22 (3), 29.64 (4), 35.29 (5) (interchangeable numbers),
26.52 (6), 20.78 (7), 41.86 (8), 35.80 (9) (interchangeable
numbers), 36.45 (10), 21.24 (11), 38.26 (12), 50.04 (13), 85.92
(14), 32.85 (15), 26.83 (16), 49.72 (17), 15.82 (18), 23.80 (19),
137.73 (20), 148.35 (21), 95.80 (1′), 70.17 (2′), 71.56 (3′), 71.99
(4′), 66.93 (5′), 17.70 (6′). Anal. (C48H55O11N) C, H, N.
14â-H yd r oxy-21-n it r o-3â-(r-L-r h a m n op yr a n osyloxy)-
5â-p r egn -20-en e (13b). 13a (272 mg, 0.33 mmol) was added
in one portion to a vigorously stirring solution of 0.256 M
t-BuOK/t-BuOH at 20 °C, and stirring was continued for 10
min. The reaction mixture was then adjusted to pH 7-8
(indicator paper) with 1 M HCl in MeOH and evaporated to
give a residue which on FCC (elution with 7.5% MeOH/CH2-
Cl2) gave 13b (54 mg, 32%): mp 215-216 °C (MeOH/H2O);
1H NMR (CD3OD) δ 0.95 (s, 3H, 10-Me), 0.88 (s, 3H, 13-Me),
6.99 (d, J ) 13.3, 1H, 21-H), 7.53 (dd, J ) 11.4, 13.4, 1H, 20-
H); 13C NMR (CD3OD) δ 27.86 (16), 51.06 (17), 15.40 (18),
138.92 (20), 149.80 (21). Anal. (C27H43O8N) C, H, N.
21-Nit r o-3â-(r-L-r h a m n op yr a n osyloxy)-5â-p r egn a n e-
14â,20(R)-d iol (9b) a n d 21-Nitr o-3â-(r-L-r h a m n op yr a n o-
syloxy)-5â,17r-p r egn a n e-14â,20(R)-d iol (9c). To a solution
of 1 (470 mg, 0.60 mmol) and freshly distilled nitromethane
(400 mg, 6.6 mmol) in MeOH (10 mL) was added slowly 4 M
NaOMe/MeOH (3.0 mL, 12 mmol). After stirring for 3.5 h at
20 °C, the mixture was carefully adjusted to pH 8 (indicator
paper) with 1 M HCl in MeOH while cooled in an ice-water
bath. EtOAc (150 mL) was added and the mixture washed
with aqueous NaHCO3 and water. After evaporation, the
residue was crystallized to give 9b (61 mg, 19%): mp 195-
1
196 °C (acetone/LP); H NMR (CD3OD) δ 0.96 and 1.06 (2s,
6H, 10-, 13-Me), 4.32-4.49 (3H, 20-H, 21-H2); 13C NMR (CD3-
OD) δ 19.64 (16), 53.36 (17), 15.21 (18), 68.84 (20), 81.67 (21).
Anal. (C27H45O9N‚H2O) C, H, N.
The mother liquor on FCC (elution with 7.5% MeOH/CH2-
Cl2) gave 9b (140 mg, 44%): mp 194-196 °C (acetone/LP).
14â-H yd r oxy-21-n it r o-3â-(r-L-r h a m n op yr a n osyloxy)-
5â-p r egn -20-en e (13b). 13a (272 mg, 0.33 mmol) was added

Steroids That Bind to the Digitalis Receptor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10 1445
in one portion to a vigorously stirred solution of 0.256 M
t-BuOK/t-BuOH (27 mL) at 20 °C and stirring continued for
10 min. The reaction mixture was then adjusted to pH 7-8
(indicator paper) with 1 M HCl in MeOH and evaporated to
give a residue which on FCC (elution with 7.5% MeOH/CH2-
Cl2) gave 13b (54 mg, 54%): mp 215-216 °C (MeOH/H2O).
20(R)-Meth oxy-21-n itr o-3â-(r-L-r h a m n op yr a n osyloxy)-
5â-p r egn a n -14â-ol (14). 13a (140 mg, 0.17 mmol) was stirred
in 0.25 M NaOMe/MeOH (5 mL) in an ice-water bath for 3.45
h when TLC (7.5% MeOH/CH2Cl2) showed formation of one
major component. The reaction mixture was neutralized to
pH 7-8 (indicator paper) with 1 M HCl and evaporated to give
a residue which on FCC (elution with 7.5% MeOH/CH2Cl2)
bath for 45 min. Workup and FCC (elution with 10% MeOH/
CH2Cl2) gave 19 (54 mg, 18%): mp 102-105 °C (acetone/LP);
1H NMR (CD3OD) δ 0.94 (s, 3H, 13-Me), 0.95 (s, 3H, 10-Me),
2.13 (m, 1H, 21-H), 2.33 (m, 1H, 21-H); 13C NMR (CD3OD) δ
29.19 (16), 50.84 (17), 15.70 (18), 31.18 or 34.28 (20), 31.18 or
34.28 (21). Anal. (C28H46O8) C, H.
[3H]Ou a ba in Ra d ioliga n d Bin d in g Assa y. [3H]Ouabain
binding to dog heart microsomes was determined in the
presence of steroids added in 5 µL of ethanol as previously
described.4
Ack n ow led gm en t. We thank the Medical Research
Council of Canada and the Heart and Stroke Founda-
tion of Manitoba for financial support. The Bruker
AM300 and AMX500 instruments were funded by the
Natural Sciences and Engineering Council of Canada
with additional support from the Manitoba Research
Council (AM300), The University of Manitoba Research
Board (AM300), The University of Manitoba (AMX500),
The University of Winnipeg (AMX500), and Lakehead
University (AMX500). We are grateful to Willson
Caetano for assistance with the experimental work. F.
S. LaBella is a Career Investigator of the Medical
Research Council of Canada.
1
yielded 14 (40 mg, 28%): mp 212-213 °C (MeOH/Et2O); H
NMR (CD3OD) δ 0.97 and 1.00 (2s, 6H, 10-, 13-Me), 3.94 (br
s, 1H, 3-H), 4.76 (d, J ) 1.2, 1H, 1′-H), 3.76 (dd, J ) 1.5, 3.1,
1H, 2′-H), 3.68 (m, 1H, 3′-H) (overlapping signals), 3.37 (t, J
) 9.4, 1H, 4′-H), 3.68 (m, 1H, 5′-H) (overlapping signals), 1.23
(d, J ) 6.2, 1H, 6′-H), 4.10 (m, 1H, 20-H), 4.56 (m, 1H, 21-H),
4.73 (m, 1H, 21-H); 13C NMR (CD3OD) δ 31.66 (1), 27.52 (2),
73.62 (3), 30.89 (4), 38.17 (5), 27.87 (6), 22.14 (7), 41.16 (8),
36.42 (9), 36.36 (10), 22.63 (11), 41.13 (12), ∼50 (13), 85.59
(14), 33.39 (15), 20.26 (16), 53.58 (17), 15.25 (18), 24.39 (19),
79.70 (20), 78.03 (21), 99.84 (1′), 72.93 (2′), 72.53 (3′), 74.09
(4′), 70.00 (5′), 17.98 (6′). Anal. (C28H47O9N) C, H, N.
21-Am in o-3â-(r-L-r h a m n op yr a n osyloxy)-5â-p r egn a n -
14â-ol d -Hyd r ogen Ta r tr a te (16). 15 (300 mg), prepared
from 9a as described in ref 2, in glacial HOAc (30 mL)
containing PtO2 (150 mg) was shaken with H2 at 5 atm for 16
h when TLC [CHCl3:MeOH:Et3N (100:30:1)] indicated reaction
was complete. The mixture was filtered through a Celite pad
and evaporated. The residue was triturated with Et2O, to give
the 21-amine (270 mg, 95%) as a pale yellow noncrystalline
powder, showing one component on TLC and no extraneous
signals in the 1H NMR. The 21-amine (100 mg, 0.20 mmol)
and d-tartaric acid (30 mg, 0.20 mmol) were dissolved in MeOH
(2 mL), and Et2O was added slowly until the solution became
turbid, to give 16 (83 mg, 53%): mp 203-206 °C; 1H NMR
(5% D2O/CD3OD) δ 0.93 (2s, 6H, 10-, 13-Me), 2.81 (m, 1H, 21-
H), 2.96 (m, 1H, 21-H), 4.42 (s, 2H, tartrate); 13C NMR (5%
D2O/CD3OD) δ ca. 27.5 (16), 47.91 (17), 15.59 (18), 32.32 (20),
40.57 (21), 74.00 and 176.97 (tartrate). Anal. (C31H53O12N‚
4H2O) C, H, N.
Su p p or tin g In for m a tion Ava ila ble: Tables containing
1H and 13C NMR spectral data (5 pages). Ordering information
is given on any current masthead page.
Refer en ces
(1) Templeton, J . F.; Ling, Y.; Zeglam, T. L.; Marat, K.; LaBella, F.
S. Synthesis of 20R and 20â-Acetamido-, Amino-, Nitro-, and
Hydroxy-Derivatives of 14-Hydroxy-5â,14â-pregnane 3â-Glyco-
sides: Pregnanes that Bind to the Digitalis Receptor. J . Chem.
Soc., Perkin Trans. 1 1992, 2503-2517 and references therein.
(2) Templeton, J . F.; Ling, Y.; Zeglam, T. H.; Marat, K.; LaBella, F.
S. Synthesis of 20-Hydroxy-, 20-Amino-, 20-Nitro-, 14-Hydroxy-
21-nor-5â,14â-pregnane C-3 Glycosides: Structure-Activity Re-
lationships of Pregnanes that Bind to the Digitalis Receptor. J .
Med. Chem. 1993, 36, 42-45.
(3) Thomas, R.; Gray, P.; Andrews, J . Digitalis: Its Mode of Action,
Receptor, and Structure-Activity Relationships. Adv. Drug Res.
1990, 19, 311-562.
(4) Hnatowich, M.; LaBella, F. S. Endogenous Digitalis-like Fac-
tors: Comparison of in vitro Biological and Immunological
Activities of Peptide and Steroid Candidates. Eur. J . Pharmacol.
1984, 30, 385-394.
(5) Martin, S. F. Synthesis of Aldehydes, Ketones, and Carboxylic
Acids from Lower Carbonyl Compounds by C-C Coupling Reac-
tions. Synthesis l979, 633-663.
(6) Grobel, B. T.; Seebach, D. Umpolung of the Reactivity of
Carbonyl Compounds through Sulfur-Containing Reagents.
Synthesis 1977, 357-402.
(7) Arai, K.; Oki, M. Evidence for Ionic Dissociation of 2-Chloro-
1,3-dithiane in Various Solvents. Tetrahedron Lett. 1975, 2183-
2186.
14â-Hyd r oxy-3â-[(tr i-O-ben zoyl-r-L-r h a m op yr a n osyl)-
oxy]-5â-a n d r osta n e-17â-a cr ylic Acid Eth yl Ester (17).
[(Ethoxycarbonyl)methyl]diethylphosphonate (500 mg, 2.23
mmol) in diglyme (5 mL) was added over 5 min to NaH (120
mg, 50% oil suspension, 2.5 mmol) suspended in diglyme (3
mL) and the mixture stirred at 20 °C for 20 min until H2
evolution had ceased.16 A solution of 1 (500 mg, 0.655 mmol)
in diglyme (5 mL) was slowly added and the mixtured stirred
for a further 20 min. TLC (25% acetone/LP, developed twice)
showed that no starting material remained. EtOAc (100 mL)
was added and the mixture washed with water to give a
residue which on FCC (elution with 15% acetone/LP) gave the
noncrystalline 17 (450 mg, 80%) which was sufficiently pure
(8) Kruse, C. G.; Broekhof, N. L. J . M.; Wijsman, A.; van der Gen,
A. Synthetic Applications of 2-Chloro-1,3-dithiane, Preparation
of Ketene Dithioacetals. Tetrahedron Lett. 1977, 885-888.
(9) Corey, E. J .; Shulman, J . I. The Application of Lithium Reagents
fron (1-Methyl)alkylphosphonate Esters to the Synthesis of
Ketones. J . Org. Chem. 1979, 35, 777-780.
1
by TLC and H and 13C NMR for use in the following reactions.
14â-Hyd r oxy-3â-(r-L-r h a m n op yr a n osyloxy)-5â-a n d r os-
ta n e-17â-a cr ylic Acid (18). 17 (357 mg, 0.44 mmol) was
taken up in 0.25 M NaOMe/MeOH (27.5 mL) and cooled in an
ice-water bath for 45 min when TLC (10% MeOH/CH2Cl2)
indicated that reaction was complete. The mixture was
adjusted to pH 7-8 (indicator paper) with 1.4 M HCl. After
evaporation, the residue was purified by FCC (on elution with
10% MeOH/CH2Cl2) which gave 18 (67 mg, 28%): mp 121-
(10) Wollenberg, R. H.; Miller, S. J . Nitroalkane Synthesis.
A
Convenient Method for Aldehyde Reductive Nitromethylation.
Tetrahedron Lett. 1978, 3219-3222.
(11) March, J . Advanced Organic Chemistry, 4th ed.; J ohn Wiley and
Sons: New York, 1992; p 939.
(12) Eberlein, W.; Heider, J .; Machleidt, H. Steroidal Cardenolides:
II. Replacement of the Butenolide Ring of Cardiac Glycosides
by Unbranched Open Chain π-Eectron Systems. Chem. Ber.
1974, 107, 1275-1284.
(13) Dornow, A.; Gellrich, M. Aliphatic Nitro Compounds. X. Reduc-
tion of Aliphatic Nitrocompounds with Lithium Aluminium
Hydride. Ann. 1955, 594, 177-184.
(14) Colvin, E. W.; Seebach, D. Silyl Nitronates: Improved Nitro-
aldol Reactions and Reductive Routes to 2-Aminoalcohols. J .
Chem. Soc. Chem. Commun. 1978, 689-690.
(15) Boutagy, J . S.; Thomas, R. Cardenolide Analogues. 2. Synthesis
of C17â-unsaturated Esters and Derivatives. Aust. J . Pharm.
Sci. 1972, NS1, 67-75.
1
124 °C (118 °C shrinking) (acetone/LP); H NMR (CD3OD) δ
0.95 (s, 3H, 10-Me), 0.85 (s, 3H, 13-Me), 2.32 (m, 1H, 17R-H),
7.19 (dd, J ) 10.6, 15.5, 1H, 20-H); 13C NMR (CD3OD) δ 28.05
(16), 55.52 (17), 14.60 (18), 157.22 (20), 120.11 (21). Anal.
(C28H44O8‚2H2O) C, H.
14â-Hyd r oxy-3â-(r-L-r h a m n op yr a n osyloxy)-5â-a n d r os-
ta n e-17â-p r op ion ic Acid (19). 17 (480 mg, 0.56 mmol) in
absolute EtOH (20 mL) containing 10% Pd/C as catalyst was
shaken with H2 at 5 atm for 14 h. The mixture was filtered
through Celite and the residue from evaporation dissolved in
0.25 M NaOMe/MeOH (16 mL) and cooled in an ice-water

1446 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 10
Templeton et al.
(16) Wadsworth, D. H.; Schupp, O. E.; Seus, E. J .; Ford, J . A. The
Stereochemistry of the Phosphonate Modification of the Wittig
Reaction. J . Org. Chem. 1965, 30, 680-685.
(17) Marat, K.; Templeton, J . F.; Ling, Y. NMR Study of Conforma-
tion and Configuration in C-20-Substituted 5â,14â-Pregnanes,
5â-Pregn-14-enes and 21-Nor-5â,14â-Pregnanes. Magn. Reson.
Chem. 1993, 31, 17-22.
(18) Thomas, R.; Boutagy, J .; Gelbart, A. Cardenolide Analogs. V.
Cardiotonic Activity of Semisynthetic Analogs of Digitoxigenin.
J . Pharmacol. Exp. Ther. 1974, 191, 219-231.
(19) Maxient, J . M.; Berrebi-Bertrand, I.; Lelievre, L. G. Inhibition
of Cardiac (Na+,K+)-ATPase isozymes by LND 623. Biochem.
Pharmacol. 1991, 42, S223-S224.
(20) Smith, P.; Brown, L.; Boutagy, J .; Thomas, R. Cardenolide
Analogues. 14. Synthesis and Biological Activity of Glucosides
of C17â-Modified Derivatives of Digitoxigenin. J . Med. Chem.
1982, 25, 1222-1226.
(21) Hintsche, R.; Megges, R.; Pfeiffer, D.; Portius, H. J .; Schonfeld,
W.; Repke, K. R. H. Biological Potency and Solution Conforma-
tion of Cardenolides Determined by a New 1H NMR Method.
Eur. J . Med. Chem. 1985, 20, 9-15.
(24) Maxient, J . M.; Bertrand, I. B.; Lelievre, L. G.; Fenard, S.
Efficacy and Safety of the Novel Na+,K+-ATPase Inhibitor 20R
14â-Amino 3â-rhamnosyl 5â-pregnan 20â-ol in a Dog Model of
Heart Failure. Arzneim.-Forsch. 1992, 1301-1305.
(25) Weiland, J .; Schwabe, K.; Hubler, D.; Schonfeld, W.; Repke, K.
R. H. Glycosidation of Chlormadinol Acetate Alters its Actions
on Na+,K+-transporting ATPase and Cardiac Contractility: A
Contribution to the Endogenous Digitalis Problem. J . Enzyme
Inhib. 1987, 2, 31-36.
(26) Templeton, J . F.; Ling, Y.; Lin, W,; Pitura, R. J .; Marat, K.;
Bridson, J . Novel Insertion, Rearrangement and Addition Prod-
ucts from Dihalocarbene Reaction with 5(10)-Unsaturated Ste-
roids. J . Chem. Soc., Perkin Trans. 1 1994, 1149-1158.
(27) Doddrell, D. M.; Pegg, D. P.; Bendall, M. T. Distortionless
Enhancement of NMR Signals by Polarization Transfer. J .
Magn. Reson. 1982, 48, 323-327.
(28) The aldehyde 1 was prepared as described in ref 2 except for
the following modifications which increased the overall yield
from 27% to 86%. Evomonoside tribenzoate was prepared as in
ref 2 and used without FCC purification. Workup of the LTBAH
product was carried out by washing with 1 M HCl instead of
NaHCO3 to avoid formation of an emulsion. Reaction time for
the NaIO4 cleavage was shortened to 30 min; longer reaction
time markedly decreased the yield.
(22) Schonfeld, W.; Weiland, J .; Lindig, C.; Masnyk, M.; Kabat, M.
M.; Kurek, A.; Wicha, J .; Repke, K. H. R. The Lead Structure in
Cardiac Glycosides is 5â,14â-androstane-3â,14-diol. Arch. Phar-
macol. 1985, 329, 414-426.
(23) Smyth, D. D.; Templeton, J . F.; Kumar, V. P. S.; Yan, Y.;
Widajewicz, W.; LaBella, F. S. Digitaloid Pregnanes Promote
Potassium-sparing Diuresis in the Guinea Pig. Can. J . Phar-
macol. Physiol. 1992, 70, 723-727.
(29) Micovic, V. M.; Mihailovic, M. L. J . The Reduction of Acid Amides
with Lithium Aluminum Hydride. J . Org. Chem. 1953, 18,
1190-1200.
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