Structure-based design and synthesis of novel potent Na+,K +-ATPase inhibitors derived from a 5α,14α-androstane scaffold as positive inotropic compounds

Article abstract of DOI:10.1021/jm030830y

The design, synthesis, and biological properties of novel inhibitors of the Na⁺,K⁺-ATPase as potential positive inotropic compounds are reported. Following our model of superposition between cassaine and digitoxigenin, digitalis-like

Full text of DOI:10.1021/jm030830y

3644
J . Med. Chem. 2003, 46, 3644-3654
Str u ctu r e-Ba sed Design a n d Syn th esis of Novel P oten t Na +,K+-ATP a se
In h ibitor s Der ived fr om a 5r,14r-An d r osta n e Sca ffold a s P ositive In otr op ic
Com p ou n d s
Sergio De Munari,†,§ Alberto Cerri,† Mauro Gobbini,*,† Nicoletta Almirante,†,§ Leonardo Banfi,† Giulio Carzana,†
Patrizia Ferrari,‡ Giuseppe Marazzi,† Rosella Micheletti,‡ Antonio Schiavone,‡ Simona Sputore,† Marco Torri,†
Maria Pia Zappavigna,† and Piero Melloni†,§
Departments of Medicinal Chemistry and Cardiovascular Pharmacology, Prassis Istituto di Ricerche Sigma-Tau,
Via Forlanini 3, 20019 Settimo Milanese (MI), Italy
Received March 26, 2003
The design, synthesis, and biological properties of novel inhibitors of the Na+,K+-ATPase as
potential positive inotropic compounds are reported. Following our model of superposition
between cassaine and digitoxigenin, digitalis-like activity has been elicited from a non-digitalis
steroidal structure by suitable modifications of the 5R,14R-androstane skeleton. The strong
hydrophobic interaction of the digitalis or cassaine polycyclic cores can be effectively obtained
with the androstane skeleton taken in a reversed orientation. Thus, oxidation of C-6 and
introduction in the C-3 position of the potent pharmacophoric group recently introduced by
us, in the 17 position of the digitalis skeleton, namely, O-(ω-aminoalkyl)oxime, led to a series
of substituted androstanes able to inhibit the Na+,K+-ATPase, most of them with an IC50 in
the low micromolar level, and to induce a positive inotropic effect in guinea pig. Within this
series, androstane-3,6,17-trione (E,Z)-3-(2-aminoethyl)oxime (22b, PST 2744) induced a strong
positive inotropic effect while being less arrhythmogenic than digoxin, when the two compounds
were compared at equiinotropic doses.
In tr od u ction
the digitalis field. Although some non-digitalis-like
compounds are inhibitors of the Na+,K+-ATPase, dis-
place 3H-ouabain binding from the Na+,K+-ATPase
digitalis receptor, and some of them induce positive
inotropic effects (cassaine, canrenone, and chlormadi-
none acetate: Figure 1),2 their low potency, poor selec-
tivity, and poor therapeutic ratio, have hampered their
development as inotropic agents.
Among inotropic agents, digoxin (Figure 1) is the most
prescribed digitalis cardiac glycoside for the treatment
of congestive heart failure. Through the inhibition of the
Na+,K+-ATPase, which promotes the outward transport
of Na+ and the inward transport of K+, Na+ concentra-
tion inside the cell is increased and, as a consequence,
Ca2+ is introduced by exchange with Na+. The final
result is a higher concentration of Ca2+ available to
activate contraction during systole. Digoxin can alleviate
symptoms, increase exercise tolerance, and reduce
hospitalization. A very well-known drawback of digitalis
drugs is their arrhythmogenic side effect. Evidence of
digitalis toxicity emerges at 2-3-fold higher serum
concentration than the therapeutic dose, such as dis-
turbances of conduction and cardiac arrhythmia which
are characteristics of digitalis toxicity.1
The selective ability of the natural digitalis com-
pounds to inhibit Na+,K+-ATPase is strictly related to
their 5â,14â-androstane three-dimensional structure.
Nevertheless, the goal of synthesizing potent Na+,K+-
ATPase inhibitors structurally not based on the 5â,14â-
androstane steroidal skeleton has been actively pursued,
but not yet achieved by medicinal chemists working in
In recent years, we proposed a new three-dimensional
model for the binding mode of cassaine at the digitalis
receptor site, based on the structural and conforma-
tional similarities between cassaine and 14,15-secodigi-
toxigenin analogues.3 This model was corroborated by
the good inhibitory activity on the Na+,K+-ATPase of
the novel synthesized compounds, thus demonstrating
that a suitable functionalization of the lipophilic per-
hydrophenanthrene (trans, trans, trans) nucleus can
result in potent inhibitors of the Na+,K+-ATPase that
do not possess the structural characteristics peculiar of
the digitalis structure.4
Following these results, we further explored chemical
series structurally unrelated to cardiac glycosides, but
still possessing high inhibitory potency and selectivity
on the Na+,K+-ATPase, with the aim of finding novel
inotropic agents endowed with lower proarrhythmogenic
effects than classical digitalis compounds.
As recognized by Repke,5 the perhydrophenanthrene
nucleus acts as the minimal pharmacophoric lead
structure for bimolecular recognition, a significant
contribution to the binding energy being given by the
hydrophobic interaction in the alpha region of the
polycyclic core. Recently, San Feliciano’s group and our
group reported that inhibition of Na+,K+-ATPase and
* Corresponding author: Mauro Gobbini, Department of Medicinal
Chemistry, Prassis Istituto di Ricerche Sigma-tau, Via Forlanini 3,
20019 Settimo Milanese (MI), Italy. Tel.: ++39.0233500388. Fax:
++39.0233500408. E-mail: mauro.gobbini@prassis.it
† Department of Medicinal Chemistry.
‡ Department of Cardiovascular Pharmacology.
§ Present addresses: S.D.M.: Department of Medicinal Chemistry,
Novuspharma SpA, Via Ariosto 23, 20091 Bresso (MI), Italy. N.A.:
Nicox Research Institute Srl, Via Ariosto 21, 20091 Bresso (MI), Italy;
P.M.: Newron Pharmaceuticals, Via R. Lepetit 34, 21040 Gerenzano
(VA), Italy.
10.1021/jm030830y CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/23/2003

Novel Potent Na+,K+-ATPase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3645
F igu r e 1. Structures of compounds with digitalis activity.
Sch em e 1a
positive inotropic activity, although with weak potency,
can be obtained from smaller scaffolds substituted with
the appropriate side chains, namely, hydroindene ami-
dinohydrazones and perhydroindene O-aminoalkyl-
oximes;7 these weak potencies could arise from the
reduced hydrophobic interaction of the scaffolds.
The result that 3â-(dimethylaminoalkoxy)androst-5-
en-17-one derivatives inhibit the Na+,K+-ATPase with
IC50 in the millimolar range (unpublished data) sug-
gested to us the idea that an easily accessible classical
steroid skeleton like that of prasterone (3â-hydroxyan-
drost-5-en-17-one 2, Scheme 1), beside providing a good
support for strong hydrophobic interactions, could serve
also as a manageable starting base for chemical elabo-
ration. This would allow the introduction of the very
powerful pharmacophoric group O-(ω-aminoalkyl)oxime,
already investigated for the classical digitalis skeleton,8
seco-D steroidal derivatives,4 and the perhydroindene
derivatives mentioned above. Moreover, we considered
that the 5R,14R-androstane scaffold would be worth
exploiting also in a reversed orientation (see Figure 2),
facilitating the introduction and matching of some of
the functional groups of cassaine, thus extending our
model for the binding mode at the digitalis receptor site.
In this superposition, rings A, B, and C of androstane
are superposed to rings C, B, and A of cassaine,
respectively, and the functionalized C-3 and C-6 carbon
of androstane coincide with C-13 and C-7 of cassaine,
respectively. In this new orientation, the two axial
methyl groups of androstane are now oriented toward
the hydrophobic alpha region, although none of them
coincide with the 14R-methyl of cassaine.
a
Reagents and conditions. a: BH3‚THF, THF, -10 °C to room
temperature; then NaBO3‚4H2O, water, room temperature; b:
NaBrO3, RuO2‚2H2O, EtOAc/water, room temperature; c: NBS,
acetone/water/Py, room temperature; d: act. MnO2, CH2Cl2/
acetone/water, 45 °C.
We resolved to check the possibility of substituting
the cassaine skeleton with the androstane skeleton in
the reversed orientation by functionalizing a series of
3-keto androstane derivatives, bearing either oxo or
hydroxy groups in positions 6 and 17, and by adding
the above-mentioned O-(ω-aminoalkyl)oxime groups at
position 3.
require a variable and flexible length of the amino-chain
substituent to maximize the binding.
Ch em istr y. The compounds listed in Table 1 were
synthesized from the corresponding ketones of general
formula 1 (Figure 3), except for compound 22e that was
prepared by acetylation of 22b, and the appropriate
O-(aminoalkyl)hydroxylamines dihydrochlorides in a
THF/water solution at room temperature (see Experi-
mental Section).
The best superposition was expected to be obtained
with the E isomers of 3[)NO(CH2)nNH2] (with n ) 2 or
n ) 3) androstane derivatives, considering that a
possible rearrangement in the receptor pocket could

3646 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
De Munari et al.
F igu r e 3. Structures of androstane ketones.
F igu r e 2. Model of the superposition between cassaine (blue)
and E isomer of compound 22b (red).
Ta ble 1. Structure and Na+,K+-ATPase Inhibition for
Compounds 22a -q
F igu r e 4. Structure of digitoxigenin.
3,17-dione11 1b, and 6R,17â-dihydroxyandrostan-3-one12
1c are reported; these known compounds were obtained
by oxidation of the common intermediate 3 at positions
3, 6, and 17 (1a ) or by regioselective oxidation, at
positions 3 and 17 (1b) or at position 3 only (1c), using
the appropriate reagents. Androstane-3â,6R,17â-triol13
3 was obtained by hydroboration, and concomitant
reduction of the 17 keto group, of the commercially
available 3â-hydroxyandrost-5-en-17-one 2, with BH3‚
THF complex in THF solution and NaBO3 in THF/water
for the oxidative step. Compound 3 was obtained in 75%
yield after crystallization from methanol/ethyl acetate;
from the mother liquor the previously unknown 5â-
androstane-3â,6â,17â-triol 4 was isolated in 12% yield
by flash chromatography on silica gel. Compound 3 was
then oxidized with catalytic RuO2 and NaBrO314 in ethyl
acetate/water to give androstane-3,6,17-trione 1a in 95%
yield; the same compound could be obtained by reaction
with J ones reagent, although in lower yield (74%) and
with some amounts of a 5,6-seco-5-oxo-6-carboxylic acid
derivative. To obtain 6R-hydroxyandrostane-3,17-dione
1b, compound 3 was reacted with NBS in acetone in
the presence of pyridine; 1b was isolated in 85% yield.
Finally, 6R,17â-dihydroxyandrostan-3-one 1c, was ob-
tained in low yield (30% after flash chromatography)
by reaction with activated MnO2 in CH2Cl2/acetone/
water, together with the other monoketones in position
6 or in position 17 and the starting material. However,
despite the low yield, this reaction is more favorable
when compared, in terms of time and effort, with a more
“classical” path with protection and deprotection steps.
In our model, as stated above and shown in Figure 2,
the optimal superposition between the basic chains of
cassaine and our compounds would be reached with the
E isomers of the oxime group. However, the products
obtained from the coupling reactions between the keto
steroids 1a -c and 1f-i and the appropriate hydroxyl-
Na+,K+-
ATPase
inhibition,
b
compd
R2
othera
6
17
IC50
,
µM
22a
22b
22c
22d
22e
22f
22g
22h
22i
22j
22k
22l
22m
22n
22o
22p
22q
(E,Z) dNO(CH2)2N(CH3)2
(E,Z) dNO(CH2)2NH2
(E) dNO(CH2)2NH2
(E) dNO(CH2)2NH2
(E,Z) dNO(CH2)2NHAc
(E,Z) dNO(CH2)3NH2
(E,Z) dNO(CH2)2NH2
(E,Z) dNO(CH2)2N(CH3)2
(E,Z) dNO(CH2)2NH2
(E) dNO(CH2)2NH2
(Z) dNO(CH2)2NH2
(E) dNO(CH2)3NH2
(Z) dNO(CH2)3NH2
(E,Z) dNO(CH2)2NH2
(E,Z) dNO(CH2)2NH2
(E,Z) dNO(CH2)2NH2
(E,Z) dNO(CH2)2NH2
oxo
oxo
oxo
oxo
oxo
oxo
oxo
oxo
12.0
0.2
2R-Me oxo
4R-Me oxo
oxo
25.0
6.0
>10.0c
0.8
oxo
R-OH oxo
1.2
R-OH â-OH >100.0d
R-OH â-OH
R-OH â-OH
R-OH â-OH
R-OH â-OH
R-OH â-OH
â-OH oxo
â-OH â-OH
oxo
oxo
1.6
2.0
12.0
2.0
3.0
2.5
10.0
0.6
â-OH
R-OH
4.5
cassaine
digitoxigenin
digoxin
5.0e
0.5
0.4
R and R1 are hydrogens where not otherwise indicated.
Concentrations able to inhibit 50% of enzyme activity; mean of
a
b
two or three experiments. c 35% inhibition at 10 µM. 34% inhibi-
tion at 100 µM. e Estimated from the reported relative potency of
ca. 0.1 in comparison to digitoxigenin (inhibition assays on
different enzymes).5,21 Compounds were isolated as free bases or
as salts (see Experimental Section).
d
The required starting ketones of formula 1a -f and
1i were obtained as described in the following schemes,
while compounds 1g and 1h were prepared as described
in the literature.9 In Scheme 1, the syntheses of
androstane-3,6,17-trione10a 1a , 6R-hydroxyandrostane-

Novel Potent Na+,K+-ATPase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3647
Sch em e 2a
a
Reagents and conditions. a: NHMe2‚HCl, paraformaldehyde, CH3CN, reflux; b: CH3I, CH3CN, room temperature; c: 3 N HCl, dioxane,
80 °C; d: H2, PtO2, EtOAc, room temperature; e: IBX, CH3CN, reflux.
Sch em e 3a
a
Reagents and conditions. a: NaBH4, CeCl3, MeOH, -5 °C to room temperature; b: 0.5 N HCl, THF, room temperature; c: LiCuMe2,
Me3SiCl, Et2O/THF, -10 to 0 °C; d: IBX, CH3CN, reflux.
amines were mixtures of E and Z isomers in a ratio of
about 1:1. For compounds derived from ketone 1a all
efforts to separate the two isomers had poor success and
attempts to prepare the pure E and Z isomers of 22b
and 22f (see below) by oxidation of the corresponding
pure derivatives 22j-m were unsuccessful. In an at-
tempt to bypass this problem, we decided to prepare the
analogous ketones 1d and 1e, so that the methyl group
in position 2R or 4R, respectively, could orient the
coupling reaction to give only a single isomer with the
oxime chain opposite to the methyl group. The hypoth-
esis was supported by molecular mechanics conforma-
tional searching (MacroModel, v. 7.2)15a on the E and Z
isomers of 22b, 22c, and 22d .15b The preparation of
ketone 1d is reported in Scheme 2. 6-Methoxy-17â-
hydroxyandrostane-4,6-diene 516 was reacted with di-
methylamine hydrochloride and paraformaldehyde in
acetonitrile to give a mixture of 6 and 7 which on
treatment with methyl iodide in acetonitrile gave the
exomethylene derivative 7 in 83% yield from 5. Hy-
drolysis of 7 with 3 N HCl in dioxane gave the diketo
derivative 8 in 55% yield. This compound was hydro-
genated over PtO2 in ethyl acetate to give a mixture of
keto/hydroxy derivatives 9 that was oxidized with
o-iodoxybenzoic acid (IBX)17 in acetonitrile to give the
desired 2R-methylandrostane-3,6,17-trione 1d in 53%
yield from 8. In Scheme 3, compound 5 was reacted with
NaBH4 in MeOH in the presence of CeCl3 to give 10
(93% yield) which was in turn treated with 0.5 N HCl
in THF to give the R,â-unsaturated ketone 11 in 70%
yield. 11 was alkylated in position 4 with LiCuMe2 and
Me3SiCl in Et2O/THF to give the 4R-Me derivative 12
in 36% yield. Finally, the required 4R-methylandros-
tane-3,6,17-trione 1e was obtained in 76% yield by
oxidation with IBX in acetonitrile. The reaction between
ketones 1d or 1e and 2-aminoethoxyamine gave only
the pure isomers 22c and 22d , respectively, as expected.
In Scheme 4, 1b was reacted with ethylene glycol in
the presence of pTSA in refluxing toluene, to give the
protected diketone 13 in 99% yield; oxidation of the
6R-hydroxy group with IBX in DMSO gave 14 in 94%
yield. 14 was reduced with NaBH4 in MeOH to obtain
15 in 97% yield; deprotection with 1 N HCl in dioxane

3648 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
De Munari et al.
Sch em e 4a
a
Reagents and conditions. a: ethylene glycol, pTSA, toluene, reflux; b: IBX, DMSO, room temperature; c: NaBH4, MeOH, 0 °C; d: 3
N HCl, dioxane, room temperature.
Sch em e 5a
a
Reagents and conditions. a: ethylene glycol, pTSA, toluene, reflux; b: mCPBA, CH2Cl2, 0 °C to room temperature; c: 4% H2SO4,
MeOH, reflux; d: ethylene glycol, pTSA, toluene, reflux; e: MeLi, Et2O/THF, -5 °C; f: pTSA, acetone, room temperature.
Sch em e 6a
gave the required 6â-hydroxyandrostane-3,17-dione 1f
in 60% yield.
17R-Hydroxyandrostane-3,6-dione 1i was synthesized
as reported in Scheme 5 by using a protection/depro-
tection approach starting from the known 17R-benzoate
1618 (prepared from testosterone in a slightly different
manner than reported) because a more straightforward
path, such as the Mitsunobu inversion of the 17â-
hydroxy group in compound 1h (Figure 3), was impos-
a
Reagents and conditions. a: 2-O-(dimethylaminoethyl)- or 3-O-
sible to achieve, even on differently protected deriva-
(aminopropyl)- or 2-O-(aminoethyl)hydroxylamine‚2HCl, THF/
water, room temperature.
tives. From 16, the ethylenedioxy derivative 17 was
obtained by reaction with ethylene glycol in the presence
of pTSA, in toluene at reflux temperature (66% yield);
this compound was oxidized with mCPBA in CH2Cl2 at
room temperature to give a mixture of isomeric epoxides
18a ,b in a roughly equal ratio and 97% yield. A small
amount of the crude mixture was purified by flash
chromatography to give the two pure epoxides 18a and
18b. By acidic treatment (4% H2SO4 in MeOH, reflux
temperature) each isomer gave the diketo derivative 19;
therefore, the reaction was carried out on the crude
mixture to give 19 in 60% yield. Attempts to deprotect
the 17R-hydroxy group with basic hydrolysis in various
conditions caused extensive degradation of the starting
compound or isomerization at the 5 position giving
mixtures of 5R/5â derivatives; on the other hand, we
found the benzoate group resistant to acidic hydrolysis.
For these reasons, we were forced to protect again the
keto groups as ethylenedioxy derivatives obtaining 20
in quantitative yield. This derivative was then reacted
with MeLi in Et2O/THF at -5 °C to give the 17R-
hydroxy derivative 21 in 83% yield. Finally, 21 was
deprotected with pTSA in acetone to give the desired
diketo derivative 1i in 57% yield.
Scheme 6 reports the general coupling reaction be-
tween the ketosteroids 1a -i and the appropriate (O-
aminoalkyl)hydroxylamine dihydrochlorides8 in a THF/
water solution at room temperature, to give 22a -d ,

Novel Potent Na+,K+-ATPase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3649
Sch em e 7a
Ta ble 2. Inotropic and Toxic Effects in Anesthetized Guinea
Pig
a
Emax
b
c
% increase
EDmax
ED80
dead/ lethal dose/
compd in +dP/dtmax µmol/kg µmol/kg treated
ED80
22b
22f
22g
22j
22l
22n
digoxin
190
129
207
158
99
17.90
18.20
54.60
4.13
7.37
111.20
0.97
4.84
6.55
5.30
0.97
1.58
14.80
0.41
7/8
1/3
1/5
3/3
1/3
20.2
n.c.d
17.8
9.6
n.c.
9.2
154
127
1/3
10/10
3.2
a
b
Maximal increase in +dP/dtmax
.
Dose inducing maximum
positive inotropic effect. c Inotropic potency: dose increasing +dP/
d
dtmax by 80%, calculated from dose-response curves. Not calcu-
lated.
model predicts that a beta hydroxyl group should have
a lower inhibitory potency than an alpha hydroxyl group
because the 6â stereochemistry places the hydrophilic
hydroxy group in a hydrophobic region of the pharma-
cophore, while a 6R is located in a similar position to
the keto group. In agreement with this prediction the
6â-hydroxy derivative has an inhibitory potency of 1
order of magnitude less than the corresponding 6-keto
derivative (22n vs 22b), while the 6R-hydroxy analogue
gave a lower decrease (22g vs 22b). The reduction of
the keto group in position 17 gave compounds with lower
potency: 17â-hydroxy derivatives showed a slightly
lower inhibition when compared to the corresponding
17-keto derivatives (22p vs 22b, 22i vs 22g, and 22o
vs 22n ), while the reduction to the 17R-hydroxy group
resulted in a strikingly lower inhibitory potency (22q
vs 22b).
On the basis of our model, a higher inhibition was
predicted for the E isomer of oximes compared with the
corresponding Z isomer. In the case of 6R,17â-dihydroxy
derivatives (22j-m ), it was possible to separate the E
and Z isomers. The compounds with the aminoethoxy
chain showed noticeable difference in the inhibitory
potency between E (22j) and Z (22k ) isomers, the E
being the more potent of the two as predicted by the
model. Comparison of the derivatives with the amino-
propoxy chain (22l and 22m ) shows a smaller difference
in the Na+,K+-ATPase inhibition than those with the
aminoethoxy chain (22j and 22k ). Among the amino-
propoxy derivatives the E,Z isomeric difference may not
be relevant because increased conformational freedom
in the chain may allow the amino group to reach an
appropriate conformation in the Z isomer (22m ) also.
As described above, since it was impossible to achieve
a separation of the isomers of compounds having 6,17-
diketo groups such as 22b, we introduced a methyl
group in the equatorial position 2R to give 22c, the
derivative corresponding to the Z isomer of 22b (22c is
actually an E isomer, because the substituted C-2 has
a higher priority than C-4 in this case); the same applies
for compound 22d , where an equatorial 4R-methyl group
was introduced to give the E derivative. Our prediction
proved to be correct because 22d was more potent than
22c, even though the presence of the methyl group
caused a relative decrease of inhibition when compared
with the E,Z analogue 22b. Compound 22f showed that
an augmented chain length brought about a reduced
activity (22f vs 22b). By comparing the length of the
alkoxyimino chain in position 3, differences were not
found in the dihydroxy E derivatives (22j vs 22l); on
a
Reagents and conditions. a: trifluoroacetic anhydride, THF,
room temperature; then 5% NaHCO3, MeOH, 35 °C; b: flash
chromatography; c: K2CO3, MeOH/water, room temperature.
22f-q (Table 1). It is worth noting the very good
regioselectivity of this reaction that, when in the pres-
ence of three or two keto groups, led to the derivative
at the 3 position with only traces of dioxime side
products. Compound 22e was obtained by acetylation
(acetic anhydride/pyridine in methylene chloride) of 22b.
As mentioned above, the oximes were obtained as
mixtures of E and Z isomers that proved to be insepa-
rable by crystallization or flash chromatography. In the
case of 16R,17â-diols (22j,k and 22l,m ) the separation
was achieved by protection of the amino group as
trifluoroacetamide, as described in Scheme 7; the ami-
noalkyloximes were reacted with trifluoroacetic anhy-
dride in THF and then stirred with 5% aqueous NaH-
CO3 in MeOH overnight (the hydroxy groups were
deprotected by hydrolysis of the trifluoroacetates) to
give, after separation by flash chromatography, 23a (Z
isomer, 22% yield), 23b (E isomer, 40% yield), and 23c
(Z isomer, 25% yield), 23d (E isomer, 41% yield); the
separated isomers were deprotected with K2CO3 in
MeOH/water to give the E (22j and 22l) and Z (22k and
22m ) isomers as pure compounds.
Resu lts a n d Discu ssion
All compounds were tested in vitro for their inhibitory
activity on purified dog kidney Na+,K+-ATPase, mea-
sured by 32P-ATP hydrolysis method (see data in Table
1).19,20 Some compounds showing high inhibitory po-
tency in vitro were investigated in vivo for their ino-
tropic activity and lethal effect by slow intravenous
infusion in anesthetized guinea pigs (the results are
reported in Table 2). Digoxin was chosen as reference
compound because it is the most commonly prescribed
cardiac glycoside in the treatment of congestive heart
failure (CHF).
The following structure-activity relationships (SAR)
are based on the in vitro data shown in Table 1. In the
model described above, the keto or hydroxy functions
in positions C-6 and C-17 are particularly significant.
With regard to the stereochemistry at position C-6, the

3650 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
De Munari et al.
the contrary, differences were found in the correspond-
ing Z derivatives (22k vs 22m ); again, the above-
mentioned hypothesis can explain these results, i.e., an
increased conformational freedom of the chain permits
proper recognition of the receptor in the case of less
potent compounds. The contrasting results of the dif-
ferent chains in position 3 could be due to subtle
adjustments of the ligands at the receptor, depending
on the different substitutions in position 6.
posed by our group, we designed a new series of
compounds starting from a planar steroidal skeleton,
in contrast to the bent skeleton of digitalis-like com-
pounds, substituted in a reversed orientation. By intro-
ducing an aminoalkoxyimino chain in position 3 and oxo
or hydroxy groups at position 6 and 17, we obtained
compounds having inhibitory activity on the Na+,K+-
ATPase comparable with that of digoxin, and with
positive inotropic activity, thus confirming the value of
our model. Most importantly, some of the compounds
also displayed a higher safety index than digoxin. From
preliminary pharmacological data, PST 2744 (22b)
emerged as a very promising novel positive inotropic
compound that may represent an innovative alternative
to digitalis in the treatment of CHF and is a candidate
for Phase I clinical trials.
As reported in our preceding papers,4,8 also in this
series a stronger (about 2 orders of magnitude) inhibi-
tion of Na+,K+-ATPase could be obtained with amino
chains ending with a primary amino group in compari-
son with a tertiary one, as demonstrated by 22b vs 22a
and 22i vs 22h . With the aim to demonstrate the
importance of a basic group at the end of the chain also
in this class of inhibitors, as previously reported for the
other classes in our cited papers, we prepared the
acetamide derivative 22e; the expected reduction of
inhibition was about 2 orders of magnitude in compari-
son with the parent compound 22b.
Exp er im en ta l Section
Gen er a l Deta ils. General experimental details for the
chemistry and pharmacological parts have been reported
elsewhere.8
Ch em istr y. An d r osta n e-3â,6r,17â-tr iol (3) and 5â-a n -
d r osta n e-3â,6â,17â-tr iol (4). To a stirred solution of dehy-
droepiandrosterone (2) (30.0 g, 104 mmol) in THF (450 mL)
at -10 °C, under N2, was added 1 M BH3‚THF complex in THF
(260 mL, 260 mmol). After completing the addition, the
mixture was stirred at room temperature for 3 h. H2O (500
mL) was cautiously added dropwise followed by NaBO3‚4H2O
(31.4 g, 204 mmol). After stirring at room temperature
overnight, the mixture was filtered. The solid was washed with
THF and then discarded. The two liquid phases were separated
and the aqueous layer was saturated with NaCl and extracted
with THF (3 × 200 mL). The combined organic extracts were
dried over NaCl and Na2SO4, filtered, and evaporated to
dryness. The crude product was crystallized from EtOAc/
MeOH (2/1, 10 mL/g) to give a first crop as a white solid (12.5
g). The mother liquors were evaporated and the residue
crystallized from EtOAc/MeOH (2/1, 10 mL/g) to give a second
crop as a white solid (6.4 g). The procedure was repeated to
give a third crop as a white solid (2.1 g; 75% overall yield). 3:
1H NMR (DMSO-d6) δ 0.60 (s, 3H, CH3), 0.72 (s, 3H, CH3),
3.12 (m, 1H, 6-H), 3.26 (m, 1H, 3-H), 3.42 (dt, 1H, 17-H), 4.24
(d, 1H, OH), 4.42 (m, 1H, OH), 4.44 (m, 1H, OH); mp 232-
234 °C. The residue from the final mother liquors was purified
by flash chromatography (EtOAc) to give 4 (4.0 g, 12.5%), a
white solid. 4 1H NMR (acetone-d6) δ 0.61 (s, 3H, CH3), 1.02
(s, 3H, CH3), 3.40 (m, 2H, 6-H, 17-H), 3.81 (m, 1H, 3-H), 4.21
(d, 1H, OH), 4.30 (m, 1H, OH), 4.42 (m, 1H, OH).
An d r osta n e-3,6,17-tr ion e (1a ). J ones oxidation: to a
solution of androstane-3â,6R,17â-triol (3) (21.0 g, 68 mmol)
in acetone (380 mL) was added an excess of J ones reagent (83.5
mL) dropwise, maintaining the temperature below 40 °C. 5
min after completion of the addition, i-PrOH (10 mL) was
added and, after further 10 min, the suspension was filtered
and the filtrate evaporated to dryness. The residue was treated
with H2O (300 mL) and extracted with EtOAc (3 × 100 mL).
The combined organic extracts were washed with H2O (100
mL), 5% aqueous NaHCO3 solution (100 mL), H2O (100 mL),
dried over Na2SO4 and evaporated to dryness to give 1a as a
white solid (15.3 g, 74%); a sample was crystallized from
i-PrOH to give a white solid: mp 195-197 °C. (lit:10b 196-
196.5 °C).
It should be noted that the most potent compound 22b
(PST 2744) showed a higher inhibitory potency than
cassaine, digitoxigenin, and the reference compound
digoxin (see Table 1).
Taken as a whole, the high potencies displayed by
some compounds and SAR evidence reported here for
the inhibition of Na+,K+-ATPase are a validation of our
model based on superposition of cassaine and (trans,
trans, trans) reversed steroids. Furthermore, the intro-
duction in position 3 of the aminoalkyloxime chain
together with the oxygenated functions at positions 6
and 17 onto the flat steroid skeleton, generated a new
series of potent inhibitors of Na+,K+-ATPase. The higher
potencies shown by some E isomers vs the corresponding
Z isomers and by the 6-keto and 6R-hydroxy derivatives
vs the corresponding 6â-hydroxy compounds further
validate our model. Some compounds showing higher
inhibitory potency on the isolated enzyme were inves-
tigated in vivo in the anesthetized guinea pig (Table 2).
Even though none of them showed inotropic potencies
(ED80) comparable to digoxin, some displayed safety
ratios (LD/ED80) higher than digoxin, namely, com-
pounds 22b and 22g. Due to its potency and safety,
about six times higher than digoxin, in infused guinea
pig, 22b was chosen to be studied in depth for its
inotropic activity and safety in different experimental
models; results are summarized here and fully reported
elsewhere.22 Intravenous infusion of 22b produced a
positive inotropic effect at doses substantially lower
than those causing cardiac toxicity, without significantly
affecting other hemodynamic parameters. Given iv in
dogs, 22b combined positive inotropic activity with a
favorable hemodynamic profile (no effect on heart rate,
reduction of peripheral resistance), absence of alter-
ations of cardiac rhythm and conduction system, fast/
high reversibility of action and improved safety index.
Preliminary investigations on the pharmacokinetic,
electrophysiological, and receptor interaction properties
of 22b indicate that its profile positively differs from
that of cardiac glycosides.
RuO2/NaBrO3 oxidation: to a solution of NaBrO3 (22.0 g, 146
mmol) in H2O (200 mL), RuO2‚2H2O (0.82 g, 4.87 mmol) and
EtOAc (400 mL) were added. After stirring for 10 min, 3 (10.0
g, 32.5 mmol) was added, while maintaining the temperature
below 30 °C. After 1 h, i-PrOH (40 mL) was added dropwise.
10 min after the addition, the mixture was filtered through a
Celite pad and the filter cake was washed with EtOAc (400
mL). The layers were separated and the aqueous phase was
extracted with EtOAc (3 × 100 mL). The combined organic
extracts were washed with brine (100 mL), 5% aqueous
Con clu sion s
On the basis of the new model derived from super-
position of cassaine and digitoxigenin, previously pro-

Novel Potent Na+,K+-ATPase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3651
1
NaHCO3 solution (50 mL), brine (100 mL), dried over Na2SO4,
and evaporated to dryness to give 1a as a white solid (9.33 g,
95%); a sample was crystallized from i-PrOH to give a white
solid: mp 196-197 °C.
to give 1d as a white solid (0.75 g, 53%). H NMR (CDCl3) δ
0.90 (s, 3H, CH3), 1.04 (d, 3H, 2-CH3), 1.07 (s, 3H, CH3).
6-Meth oxya n d r osta n e-4,6-d ien -3â,17â-d iol (10). To a
solution of CeCl3‚7H2O (3.00 g, 9.4 mmol) and 5 (3.51 g, 9.4
mmol) in MeOH (300 mL) at -5 °C, NaBH4 (0.40 g, 10.6 mmol)
was added in five portions over 15 min. The solution was
stirred at room temperature for 2 h and quenched with AcOH
(2.2 mL, 40 mmol). The mixture was concentrated under
reduced pressure, treated with H2O (100 mL), and extracted
with CHCl3 (3 × 100 mL). The combined organic extracts were
evaporated to dryness to give 10 as a white solid (2.80 g, 93%).
1H NMR (CD3OD) δ 0.81 (s, 3H, CH3), 1.01 (s, 3H, CH3), 3.52
(s, 3H, 6-OCH3), 3.61 (t, 1H, 17-H), 4.20 (ddd, 1H, 3-H), 4.71
(bs, 1H, 7-H), 5.89 (bs, 1H, 4-H).
3â,17â-Dih yd r oxya n d r osta n -4-en -6-on e (11). A solution
of 10 (2.80 g, 8.8 mmol) and 0.5 N HCl (12 mL) in THF (90
mL) was stirred at room temperature for 45 min and then
quenched by addition of saturated Na2HPO4 aqueous solution
(20 mL). The mixture was extracted with EtOAc (3×). The
combined organic extracts were washed with brine, dried over
Na2SO4, and evaporated to dryness to give 11 (2.00 g, 70%), a
white solid. 1H NMR (CD3OD) δ 0.75 (s, 3H, CH3), 1.01 (s, 3H,
CH3), 3.62 (t, 1H, 17-H), 4.19 (m, 1H, 3-H), 6.12 (m, 1H, 4-H).
4r-Meth yl-3â,17â-d ih yd r oxya n d r osta n -6-on e (12). To a
suspension of CuI (31.8 mg, 167 mmol) in Et2O (600 mL) at
-5 °C under N2 was added dropwise a 1.6 M MeLi solution in
Et2O (207 mL, 331 mmol). After stirring of the sample at 0 °C
for 10 min, the solution was cooled at -10 °C, and Me3SiCl
(106 mL, 860 mmol) was added dropwise, followed by a
solution of 11 (3.43 g, 11.1 mmol) in THF (120 mL). After
stirring of the sample at 0 °C for 2 h, the solution was poured
into cold 1 N HCl. EtOAc (100 mL) was added and the mixture
was filtered through Celite. After separation of the organic
layer, the aqueous phase was extracted with EtOAc (3×). The
combined organic extracts were washed with a saturated
aqueous Na2HPO4 solution, dried over Na2SO4, and evaporated
to dryness. The residue was purified by flash chromatography
(CH2Cl2/acetone 90/10). After evaporation of the fractions
containing 12, the residue was crystallized from acetone to give
12 (1.31 g, 36%), a white solid. 1H NMR (CD3OD) δ 0.73 (s,
3H, CH3), 0.76 (s, 3H, CH3), 0.95 (d, 3H, 4-CH3), 2.25 (d, 1H,
5-H), 3.05 (dt, 1H, 3-H), 3.71 (t, 1H, 17-H).
6r-Hyd r oxya n d r osta n e-3,17-d ion e (1b). To a stirred
solution of 3 (15.0 g, 48.6 mmol) in acetone/H2O/pyridine (1485/
300/8 mL), NBS (freshly crystallized from H2O; 14.45 g, 81.05
mmol) was added. After 8 h, NBS (freshly crystallized from
H2O; 14.45 g, 81.05 mmol) was added and after stirring at room
temperature for 24 h, the solution was quenched with conc
HCl (1.5 mL) to pH 3 and, after 10 min, NaOH (small beads,
4.66 g, 0.116 mmol) was added to pH 8. The organic solvent
was evaporated and the aqueous phase was extracted with
EtOAc (4 × 350 mL). The combined organic extracts were dried
over Na2SO4 and evaporated to dryness to give a brown solid.
The crude product was triturated with H2O (2 × 250 mL) and
filtered. After drying under vacuum at 40 °C, 1b (12.58 g, 85%)
was obtained as an off-white solid. 1H NMR (acetone-d6) δ 0.86
(s, 3H, CH3), 1.11 (s, 3H, CH3), 3.48 (m, 1H, 6-H), 3.61 (d, 1H,
OH); a sample was crystallized from EtOAc to give a white
solid: mp 204-206 °C (lit.11 mp 206-207 °C).
6r,17â-Dih yd r oxya n d r osta n -3-on e (1c). To a solution of
3 (3.00 g, 9.72 mmol) in CH2Cl2/acetone/H2O (300/150/6 mL),
activated MnO2 (30.0 g, 345 mmol) was added in three portions
over 8 h. The mixture was stirred at 45 °C overnight. After
cooling the sample to room temperature, the mixture was
filtered through Celite. The filtrate was evaporated and the
residue was purified by flash chromatography (CH2Cl2/n-
hexane/i-PrOH 10/5/1) to give 1c (0.89 g, 30%), a white solid.
1H NMR (acetone-d6) δ 0.75 (s, 3H, CH3), 1.08 (s, 3H, CH3),
3.43 (m, 1H, H-6), 3.51 (d, 1H, 6-OH), 3.58 (m, 2H, 17H and
17-OH); a sample was crystallized from EtOAc to give a white
solid: mp 207-208 °C (lit.12 mp 208-210 °C).
2-Met h ylen e-6-m et h oxy-17â-h yd r oxya n d r ost a n e-4,6-
d ien -3-on e (7). A mixture of 6-methoxy-17â-hydroxyandros-
tane-4,5-dien-3-one16 (5) (4.00 g, 12.6 mmol), paraformaldehyde
(0.48 g, 15.98 mmol), and dimethylamine hydrochloride (1.28
g, 15.7 mmol) in acetonitrile (80 mL) was heated to reflux
under stirring for 7 h. After cooling, the solution was evapo-
rated and the residue was dissolved in an EtOAc/saturated
aqueous Na2HPO4 mixture. The layers were separated, and
the aqueous phase was extracted with EtOAc (3×). The
combined organic extracts were dried over Na2SO4 and evapo-
rated to dryness. The crude product was dissolved in aceto-
nitrile (100 mL) and CH3I (0.40 mL, 6.3 mmol) was added.
After stirring of the sample at room temperature for 2.5 h,
the solution was evaporated to dryness and the yellow crude
product was purified by flash chromatography (n-hexane/CH2-
4r-Meth yla n d r osta n e-3,6,17-tr ion e (1e). A mixture of 12
(1.36 g, 4.2 mmol) and IBX (2.35 g, 8.4 mmol) in acetonitrile
(80 mL) was stirred at reflux for 5 h. After cooling of the
sample to room temperature, the mixture was filtered through
Celite. The solution was evaporated and the crude product was
purified by flash chromatography (n-hexane/EtOAc 6/4) to give
1
1
1e as a white solid (1.02 g, 76%). H NMR (CDCl3) δ 0.91 (s,
Cl2/acetone 6/2/2) to give 7 (3.48 g, 83%), a off-white solid. H
3H, CH3), 1.02 (d, 3H, 4-CH3), 1.02 (s, 3H, CH3), 2.77 (dq, 1H,
4-H).
NMR (CDCl3) δ 0.85 (s, 3H, CH3), 1.05 (s, 3H, CH3), 3.59 (s,
3H, 5-OCH3), 3.71 (t, 1H, 17-H), 5.14 (d, 1H, 7-H), 5.29 (t, 1H),
6.08 (dd, 1H), 6.38 (s, 1H, 4-H).
3,3:17,17-Bis(eth ylen d ioxy)a n d r osta n -6r-ol (13). A solu-
tion of 1b (5.00 g, 16.5 mmol), ethylene glycol (37 mL, 610
mmol) and pTSA (0.219 g, 11.4 mmol) in toluene (530 mL) was
stirred at reflux for 12 h with a Dean-Stark trap. After cooling
to room temperature, the mixture was neutralized with 5%
aqueous NaHCO3 solution. The organic layer was separated
and washed with H2O (2 × 350 mL), dried over Na2SO4 and
evaporated to dryness to give 13 as a white solid (6.40 g, 99%).
1H NMR (DMSO-d6) δ 0.73 (s, 3H, CH3), 0.74 (s, 3H, CH3),
3.11 (m, 1H, 6-H), 3.88-3.70 (m, 8H), 4.25 (d, 1H, OH).
3,3:17,17-Bis(eth ylen d ioxy)a n d r osta n e-6-on e (14). A
solution of 13 (0.75 g, 1.91 mmol) and IBX (0.88 g, 2.87 mmol)
in DMSO (10 mL) was stirred at room temperature for 3 h
and then quenched by addition of H2O (50 mL). After stirring
for 15 min, the mixture was filtered and the cake was washed
with EtOAc. The layers were separated, and the aqueous phase
was extracted with EtOAc (3×). The combined organic extracts
were dried over Na2SO4 and evaporated to dryness to give 14
2-Meth ylen e-17â-h ydr oxyan dr ostan e-4-en -3,6-dion e (8).
A solution of 7 (3.00 g, 9.09 mmol) and 3 N HCl (3 mL) in
dioxane (40 mL) was heated at 80 °C under stirring for 0.5 h.
The brown solution was cooled with an ice bath and then
quenched by careful addition of a saturated aqueous Na2HPO4
solution (50 mL) and brine (20 mL). The mixture was extracted
with Et2O (3×). The combined organic extracts were washed
with brine, dried over Na2SO4 and charcoal, filtered, and
evaporated to dryness to give 8 (1.60 g, 55%), a off-white solid
sufficiently pure to be used in the next step without further
purification. 1H NMR (CDCl3) δ 0.81 (s, 3H, CH3), 1.11 (s, 3H,
CH3), 3.68 (t, 1H, 17-H), 5.38 (m, 1H), 6.11 (m, 1H), 6.42 (bs,
1H, 4-H).
2r-Meth yla n d r osta n e-3,6,17-tr ion e (1d ). A mixture of 8
(1.60 g, 5.0 mmol) and PtO2 (0.15 g) in EtOAc was stirred
under H2 at atm pressure for 4 h. The mixture was filtered
through Celite and the filtrate evaporated to dryness. A
mixture of the residue and IBX (2.80 g, 10.0 mmol) in
acetonitrile (80 mL) was heated at reflux under stirring for 5
h. The mixture was cooled to room temperature and filtered
through Celite. The filtrate was evaporated and the residue
was purified by flash chromatography (n-hexane/EtOAc 6/4)
1
as a white foam (0.70 g, 94%). H NMR (acetone-d6) δ 0.75 (s,
3H, CH3), 0.84 (s, 3H, CH3), 2.55 (m, 1H, 5-H), 3.95-3.75 (m,
8H).
3,3:17,17-Bis(eth ylen d ioxy)a n d r osta n -6â-ol (15). To a
stirred suspension of 14 (1.50 g, 3.82 mmol) in MeOH (20 mL)

3652 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
De Munari et al.
at 0 °C and under N2, NaBH4 (0.22 g, 4.58 mmol) was added.
After 2 h at 0 °C, H2O (50 mL) was added dropwise. The
mixture was extracted with EtOAc (3 × 50 mL). The combined
organic extracts were dried over Na2SO4, filtered, and evapo-
rated to dryness to give 15 as a white solid (1.46 g, 97%). H
NMR (acetone-d6) δ 0.84 (s, 3H, CH3), 1.05 (s, 3H, CH3), 3.33
(d, 1H, 6-OH), 3.70 (m, 1H, 6-H), 3.95-3.75 (m, 8H, OCH2).
3,3:6,6-Bis(eth ylen d ioxy)a n d r osta n e-17r-yl ben zoa te
(20). A solution of 19 (0.48 g, 1.17 mmol), ethylene glycol (2.42
mL, 43.47 mmol), and pTSA (15.3 mg, 0.08 mmol) in toluene
(150 mL) was stirred at reflux for 0.5 h a with a Dean-Stark
apparatus. After cooling of the sample to room temperature,
the mixture was washed with 5% aqueous NaHCO3 solution
(50 mL), H2O (2 × 50 mL), dried over Na2SO4 and evaporated
to dryness to give 20 as a white foam (0.60 g, 100%). 1H NMR
(acetone-d6) δ 0.87 (s, 3H, CH3), 0.99 (s, 3H, CH3), 4.00-3.65
(m, 8H), 5.04 (dd, 1H, 17-H), 8.1-7.45 (m, 5H).
1
6â-Hyd r oxya n d r osta n e-3,17-d ion e (1f). 1 N HCl (2.1 mL)
was added to a solution of 15 (1.46 g, 3.71 mmol) in dioxane
(47.7 mL). After stirring at room temperature for 2 h, the
solution was quenched by careful addition of 5% aqueous
NaHCO3 solution, and dioxane was evaporated. The aqueous
phase was extracted with EtOAc (2 × 100 mL). The combined
organic extracts were washed with H2O (2 × 100 mL), dried
over Na2SO4, filtered, and evaporated. The residue was puri-
fied by flash chromatography (cyclohexane/EtOAc 1/1) to give
3,3:6,6-Bis(eth ylen d ioxy)a n d r osta n e-17r-ol (21). To a
solution of 20 (0.59 g, 1.18 mmol) in dry THF (40 mL) at -5
°C under N2, 1.6 M MeLi in Et2O (1.9 mL, 3.04 mmol) was
added dropwise. The solution was stirred at -5 °C for 1.5 h
and then quenched by careful addition of brine. The mixture
was extracted with THF (3 × 50 mL). The combined organic
extracts were washed with brine, dried over Na2SO4, and
evaporated to dryness. The residue was purified by flash
chromatography (n-hexane/EtOAc 1/1) to give 21 (0.38 mg,
1
1f (0.68 g, 60%), a white solid. H NMR (DMSO-d6) δ 0.81 (s,
3H, CH3), 1.13 (s, 3H, CH3), 3.57 (m, 1H, 6-H), 4.47 (d, 1H,
OH).
1
Testoster on e 17r-ben zoa te (16). To a solution of test-
osterone (6.00 g, 20.8 mmol) in dry toluene (168 mL) under
N2, benzoic acid (3.04 g, 25 mmol), triphenylphosphine (6.54
g, 25 mmol) and DIAD (5.05 g, 25 mmol) were added. The
solution was heated at 80 °C under stirring for 1 h. After
cooling of the sample, the solution was evaporated to dryness
and the crude yellow oil was purified by flash chromatography
(n-hexane/acetone 95/5) to give 16 (5.88 g, 72%), as a white
solid. 1H NMR (DMSO-d6) δ 0.82 (s, 3H, CH3), 1.15 (s, 3H,
CH3), 4.94 (d, 1H, 17-H), 5.63 (bs, 1H, 4-H), 8.00-7.45 (m, 5H);
mp 133-135 °C (lit.18 mp 135-137 °C).
83%), a white foam. H NMR (acetone-d6) δ 0.67 (s, 3H, CH3),
0.97 (s, 3H, CH3), 3.38 (d, 1H, OH), 3.95-3.60 (m, 9H, OCH2
and 17-H).
17r-Hyd r oxya n d r osta n e-3,6-d ion e (1i). A solution of 21
(380 mg, 0.97 mmol) and pTSA‚H2O (0.92 g, 4.81 mmol) in
acetone (38 mL) was stirred at room temperature for 1.5 h.
The solution was neutralized by addition of 5% aqueous
NaHCO3, and acetone was evaporated. The aqueous phase was
extracted with CH2Cl2 (3 × 50 mL). The combined organic
extracts were washed with H2O, dried over Na2SO4, and
evaporated to dryness. The residue was purified by flash
chromatography (n-hexane/EtOAc 1/1) to give 21 as a white
solid (167 mg, 57%). 1H NMR (acetone-d6) δ 0.71 (s, 3H, CH3),
0.99 (s, 3H, CH3), 3.53 (d, 1H, OH), 3.71 (dd, 1H, 17-H).
3,3-Eth ylen d ioxya n d r ost-5-en -17r-yl ben zoa te (17). A
solution of 16 (1.71 g, 4.35 mmol), ethylene glycol (8.9 mL,
161 mmol), and pTSA (58 mg, 3.0 mmol) in toluene (140 mL)
was stirred at reflux for 12 h with a Dean-Stark trap. After
cooling to room temperature, the mixture was neutralized with
5% aqueous NaHCO3 solution. The organic layer was sepa-
rated, washed with H2O (2 × 100 mL), dried over Na2SO4, and
evaporated to dryness. The residue was triturated with Et2O/
n-hexane 3/7 (10 mL), filtered, and washed with n-hexane to
Gen er a l P r oced u r e for Com p ou n d s 22a -d , 22f-i, and
22n -q. To a stirred solution of the appropriate ketone (1
equiv) in THF (0.15 M), a solution of the appropriate amino-
alkoxyamine dihydrochloride (1 equiv) in H2O (0.30 M) was
rapidly added dropwise. After 1.5 h, NaCl (7.5 equiv) was
added and the mixture was stirred for 10 min. The phases were
separated and the aqueous phase was extracted with THF
(2×). The combined organic extracts were dried over Na2SO4,
filtered, and evaporated to give an oily residue. The crude
product was dissolved in CH2Cl2 (0.15 M) and washed with a
saturated aqueous solution of NaCl (3×). The organic layer
was dried again over Na2SO4 and evaporated to dryness.
1
give 17 as a white foam (1.23 g, 66%). H NMR (acetone-d6) δ
0.87 (s, 3H, CH3), 1.06 (s, 3H, CH3), 3.95-3.80 (m, 4H), 5.03
(dd, 1H, 17-H), 5.29 (m, 1H, 6-H), 8.10-7.45 (m, 5H).
3,3-Eth ylen d ioxy-5r,6r-ep oxya n d r osta n -17r-yl ben zo-
a te (18a ) and 3,3-eth ylen d ioxy-5â,6â-ep oxya n d r osta n -
17r-yl ben zoa te (18b). To a stirred solution of 17 (1.22 g,
2.81 mmol) in CH2Cl2 (7.4 mL) cooled at 0 °C, a solution of
mCPBA (0.77 mg, 4.4 mmol) in CH2Cl2 (13.6 mL) was added
dropwise. After 0.5 h at 0 °C and 0.5 h at room temperature,
a 10% aqueous solution of Na2SO3 was added. The mixture
was neutralized by addition of 5% NaHCO3 solution and
extracted with CH2Cl2 (3 × 100 mL). The combined organic
extracts were washed with H2O, dried over Na2SO4, and
evaporated to dryness to give 18a and 18b as a white foam
(1/1; 1.24 g, 97%).
Compounds 18a and 18b were separated by flash chroma-
tography (cyclohexane/EtOAc 30/70) on a small sample. 18a ,
a white solid: 1H NMR (acetone-d6) δ 0.81 (s, 3H, CH3), 1.12
(s, 3H, CH3), 2.72 (d, 1H, 6-H), 3.93-3.77 (m, 4H), 4.99 (dd,
1H, 17-H), 8.1-7.45 (m, 5H). 18b, a white solid: 1H NMR
(acetone-d6) δ 0.83 (s, 3H, CH3), 0.99 (s, 3H, CH3), 3.00 (d, 1H,
6-H), 3.95-3.80 (m, 4H), 5.01 (dd, 1H, 17-H), 8.1-7.4 (m, 5H).
3,6-Dioxoa n d r osta n -17r-yl ben zoa te (19). To a suspen-
sion of 18a and 18b (1/1; 1.24 g, 2.73 mmol) in MeOH (290
mL) was added 4% aqueous H2SO4 (5 mL) and the mixture
was stirred at reflux for 1 h. The solution was concentrated
in vacuo and H2O (100 mL) and CH2Cl2 (150 mL) were added.
The mixture was stirred for 15 min. The layers were separated
and the aqueous phase was extracted three times with CH2-
Cl2. The combined organic extracts were washed with a 5%
aqueous NaHCO3 solution, dried over Na2SO4 and evaporated.
The crude product was purified by flash chromatography (n-
hexane/EtOAc 7/3) to give 19 as a white solid (0.67 g, 60%).
1H NMR (acetone-d6) δ 0.91 (s, 3H, CH3), 1.01 (s, 3H, CH3),
5.06 (dd, 1H, 17-H), 8.1-7.4 (m, 5H).
(E,Z)-3-[2-(N,N-Dim et h yla m in o)et h oxyim in o]a n d r os-
t a n e-6,17-d ion e (22a ). Prepared in 65% yield from 1a
and 2-(N,N-dimethylamino)ethoxyamine dihydrochloride. The
crude product was purified by flash chromatography (CHCl3/
MeOH/26% NH4OH 90/10/1) and crystallized from MeOH/
1
EtOAc. H NMR (DMSO-d6) δ 0.88 (s, 3H, CH3), 0.93 (s, 3H,
CH3), 2.30 (s, 6-H, N(CH3)2), 2.62 (m, 2H, NCH2), 4.13 (m, 2H,
OCH2); white solid: mp 141-145 °C. Anal. (C23H36N2O2) C,
H, N.
(E,Z)-3-(2-Am in oeth oxyim in o)an dr ostan e-6,17-dion e h y-
d r och lor id e (22b). Prepared in 53% yield from 1a and
2-aminoethoxyamine dihydrochloride. The crude product was
crystallized from MeOH/EtOAc. 1H NMR (DMSO-d6) δ 0.78
(s, 3H, CH3), 0.79 (s, 3H, CH3), 3.00 (t, 2H, NCH2), 3.08 (m,
1H, 4-H eq. Z isomer and 2-H eq. E isomer), 4.09 (t, 2H, OCH2),
8.12 (bb, 3H, NH3+); white solid: mp 220-225 °C (dec). Anal.
(C21H32N2O3‚HCl) C, H, N, Cl.
(E)-2r-Meth yl-3-(2-am in oeth oxyim in o)an dr ostan e-6,17-
d ion e fu m a r a te (22c). Prepared in 55% yield from 1d and
2-aminoethoxyamine dihydrochloride. The crude product was
purified by flash chromatography (CHCl3/MeOH/26% NH4OH
90/10/1). To the concentrated fractions, a stoichiometric amount
of fumaric acid in MeOH was added. After addition of a 1/1
mixture of EtOAc/Et2O, the precipitate was filtered to give 22c.
1H NMR (DMSO-d6) δ 0.76 (s, 3H, CH3), 0.81 (s, 3H, CH3),
0.99 (d, 3H, CH3 eq), 2.98 (m, 2H, NCH2), 3.10 (m, 1H, 4-H
eq), 4.07 (m, 2H, OCH2); white solid: mp 140-147 °C dec.
Anal. (C22H34N2O3‚C4H4O4‚1.5 H2O) C, H, N, H2O.

Novel Potent Na+,K+-ATPase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3653
(E)-4r-Meth yl-3-(2-am in oeth oxyim in o)an dr ostan e-6,17-
d ion e h yd r och lor id e (22d ). Prepared in 60% yield from 1e
and 2-aminoethoxyamine dihydrochloride. The crude product
was crystallized from MeOH/EtOAc. 1H NMR (DMSO-d6) δ
0.77 (s, 3H, CH3), 0.78 (s, 3H, CH3), 0.98 (d, 3H, CH3 eq), 2.60
(dq, 1H, 4-H ax), 3.03 (m, 2H, NCH2), 3.12 (m, 1H, 2-H eq),
4.12 (m, 2H, OCH2), 8.12 (bb, 3H, NH3+); white solid: mp 226-
229 °C dec. Anal. (C22H34N2O3‚HCl) C, H, N, Cl.
(E,Z)-3-(2-Aceta m id oeth oxyim in o)a n d r osta n e-6,17-d i-
on e (22e). To a stirred solution of 22b (1.14 g, 2.9 mmol) in
CH2Cl2 (11.5 mL) at room temperature, Ac2O (0.54 mL, 5.7
mmol) and pyridine (0.46 mL, 5.7 mmol) were added. The
solution was stirred at room temperature for 4 h and evapo-
rated. The residue was dissolved with EtOAc (100 mL), washed
with 1 N HCl (1 × 20 mL) and brine (4 × 20 mL). The organic
solution was dried over Na2SO4, filtered, and evaporated to
dryness. The residue was purified by flash chromatography
(cyclohexane/CH2Cl2/acetone 2/4/4) to give 22e (0.80 g, 69%).
1H NMR (DMSO-d6) δ 0.79 (s, 3H, CH3), 0.81 (s, 3H, CH3),
1.76 (s, 1.5H, COCH3), 1.77 (s, 1.5H, COCH3) 2.96 (m, 0.5H,
4-H eq Z isomer), 3.03 (m, 0.5H, 2-H eq E isomer), 3.22 (m,
2H, NCH2), 3.88 (m, 2H, OCH2), 7.88 (bb, 1H, NH); white
solid: mp 101-103 °C. Anal. (C23H34N2O4‚0.5 H2O) C, H, N,
H2O.
(E ,Z)-3-(3-Am in op r op oxyim in o)a n d r ost a n e -6,17-d i-
on e h yd r och lor id e (22f). Prepared in 73% yield from 1a and
3-aminopropoxyamine dihydrochloride. The crude product was
crystallized from MeOH/EtOAc. 1H NMR (DMSO-d6) δ 0.74
(s, 3H, CH3), 0.75 (s, 3H, CH3), 2.81 (m, 2H, NCH2), 2.95 (m,
0.5H, 4-H eq Z isomer), 3.00 (m, 0.5H, 2-H eq E isomer), 3.99
(m, 2H, OCH2), 7.85 (bb, 3H, NH3+); white solid: mp 83.5-
139.5 °C. Anal. (C22H34N2O3‚HCl) C, H, N, Cl.
(E,Z)-3-(2-Am in oeth oxyim in o)-6r-h yd r oxya n d r osta n e-
17-on e h yd r och lor id e (22g). Prepared in 70% yield from 1b
and 2-aminoethoxyamine dihydrochloride. The crude product
was crystallized from MeOH/EtOAc. 1H NMR (DMSO-d6) δ
0.77 (s, 3H, CH3), 0.86 (s, 1.5H, CH3), 0.87 (s, 1.5H, CH3), 3.02
(t, 2H, NCH2), 3.06 (m, 0.5H, 2-H eq. E isomer), 3.25 (m, 1H,
6-H), 3.45 (m, 0.5H, 4-H eq. Z isomer), 4.08 (t, 2H, OCH2), 4.52
(bb, 1H, OH), 7.99 (bb, 3H, NH3+); white solid: mp 152-155
°C. Anal. (C21H34N2O3‚HCl‚H2O) C, H, N, Cl, H2O.
(E,Z)-3-[2-(N,N-Dim et h yla m in o)et h oxyim in o]a n d r os-
ta n e-6r,17â-d iol (22h ). Prepared in 80% yield from 1c and
2-(N,N-dimethylamino)ethoxyamine dihydrochloride. The crude
product was purified by flash chromatography (CHCl3/MeOH/
26% NH4OH 90/10/1) to give 22h . 1H NMR (CD3OD) δ 0.75 (s,
3H, CH3), 0.94 (s, 3H, CH3), 2.30 (s, 6H, N(CH3)2), 2.64 (m,
2H, NCH2), 3.58 (m, 1H, 17-H), 4.11 (m, 2H, OCH2); white
solid: mp 102-106 °C. Anal. (C23H40N2O3‚H2O) C, H, N, H2O.
(E,Z)-3-(2-Am in oeth oxyim in o)a n d r osta n e-6r,17â-d iol
oxa la te (22i). Prepared in 85% yield from 1c and 2-amino-
ethoxyamine dihydrochloride. The crude product was purified
by flash chromatography (CHCl3/MeOH/26% NH4OH 90/10/
1). To the concentrated fractions a stoichiometric amount of
oxalic acid in MeOH was added. After addition of a 1/1 mixture
of EtOAc/Et2O, the precipitate was filtered to give 22i. 1H NMR
(DMSO-d6) δ 0.61 (s, 3H, CH3), 0.83 (s, 1.5H, CH3), 0.84 (s,
1.5H, CH3), 3.04 (t, 2H, NCH2), 3.07 (m, 0.5H, 2-H eq. E
isomer), 3.20 (m, 1H, 6-H), 3.42 (m, 1.5H, 4-H eq. Z isomer
and 17-H), 4.06 (t, 2H, OCH2), 7.71 (bb, 3H, NH3+); white
solid: mp 151-156 °C. Anal. (C21H36N2O3‚C2H2O4‚H2O) C, H,
N, H2O.
(Z)-3-(2-Tr iflu or oa ceta m id oeth oxyim in o)a n d r osta n e-
6r,17â-d iol (23a ) and (E)-3-(2-tr iflu or oa ceta m id oeth oxy-
im in o)a n d r osta n e-6r,17â-d iol (23b). To a suspension of
(E,Z) 3-(2-aminoethoxyimino)androstane-6R,17â-diol hydro-
chloride (22i) (1.10 g, 2.77 mmol) in THF (40 mL) at 0 °C,
(CF3CO)2O (0.86 mL, 6.18 mmol) was added dropwise. After
stirring at room temperature overnight, the mixture was
diluted with EtOAc (100 mL) and washed with a 5% aqueous
NaHCO3 solution (2 × 120 mL), H2O (100 mL), dried over Na2-
SO4, filtered, and evaporated to dryness. To the crude product,
MeOH (150 mL) and 5% aqueous NaHCO3 solution (40 mL)
were added. After stirring at 35 °C overnight, MeOH was
evaporated and the residue was extracted with EtOAc (2 ×
100 mL). The combined organic extracts were washed with
H2O (2 × 100 mL), dried over Na2SO4, filtered, and evaporated
to dryness. The residue was purified by flash chromatography
(CH2Cl2/EtOAc/acetone 7/2/1) to give 23a as a white foam (0.28
g, 22%) and 23b as a white foam (0.50 g, 40%). 23a : 1H NMR
(CDCl3) δ 0.73 (s, 3H, CH3), 0.92 (s, 3H, CH3), 3.48 (m, 1H,
6-H), 3.55 (m, 1H, 4-H eq.), 3.61 (t, 2H, NCH2), 3.64 (m, 1H,
17-H), 4.14 (m, 2H, OCH2), 7.40 (bb, 1H, CONH). 23b: 1H NMR
(CDCl3) δ 0.73 (s, 3H, CH3), 0.92 (s, 3H, CH3), 3.12 (m, 1H,
2-H eq), 3.48 (m, 1H, 6-H), 3.61 (m, 2H, NCH2), 3.64 (m, 1H,
17-H), 4.14 (m, 2H, OCH2), 7.30 (bb, 1H, CONH).
(E )-3-(2-Am in oe t h oxyim in o)a n d r ost a n e -6r,17â-d iol
h em ifu m a r a te (22j). To a solution of 23b (0.50 g, 1.08 mmol)
in MeOH (6.6 mL) and H2O (4.0 mL), K2CO3 (0.75 g, 5.42
mmol) was added. After stirring of the sample at room
temperature overnight, the solution was diluted with CH2Cl2
(50 mL) and 1 N NaOH (5 mL). The layers were separated
and the aqueous phase was extracted with CH2Cl2 (2 × 50 mL).
The combined organic extracts were dried over Na2SO4,
filtered, and evaporated to dryness. The residue was dissolved
in MeOH (4 mL) and fumaric acid (126 mg, 1.08 mmol) and
Et2O (2 mL) were added. The precipitate was filtered to give
22j (0.415 g, 80%). 1H NMR (DMSO-d6) δ 0.61 (s, 3H, CH3),
0.83 (s, 3H, CH3), 3.08 (m, 3H, NCH2 and 2-H eq.), 3.21 (m,
1H, 6-H), 3.42 (m, 1H, 17-H), 4.08 (t, 2H, OCH2), 7.78 (bb, 3H,
NH3+); white solid: mp 110-180 °C. Anal. (C21H36N2O3‚0.5
C4H4O4‚1.5 H2O) C, H, N, H2O.
(Z)-3-(2-Am in oe t h oxyim in o)a n d r ost a n e -6r,17â-d iol
h em ifu m a r a te (22k ). Prepared in 75% yield from 23a by the
procedure described above for the preparation of 22j. 1H NMR
(DMSO-d6) δ 0.61 (s, 3H, CH3), 0.84 (s, 3H, CH3), 2.89 (m, 2H,
NCH2), 3.21 (m, 1H, 6-H), 3.44 (m, 2H, 17-H and 4-H eq), 3.99
(t, 2H, OCH2); white solid: mp 180-183 °C. Anal. (C21H36N2O3‚
0.5 C4H4O4‚H2O) C, H, N, H2O.
(Z)-3-(3-Tr iflu or oacetam idopr opoxyim in o)an dr ostan e-
6r,17â-d iol (23c) and (E)-3-(3-tr iflu or oa ceta m id op r op oxy-
im in o)a n d r osta n e-6r,17â-d iol (23d ). Prepared from 1c and
3-aminopropoxyamine dihydrochloride by the general proce-
dure described above to give (E,Z)-3-(3-aminopropoxyimino)-
androstane-6R,17â-diol. Compounds 23c and 23d were pre-
pared from (E,Z)-3-(3-aminopropoxyimino)androstane-6R,17â-
diol by the general procedure described above for the preparation
of 23a and 23b. 23c (0.40 g, 25%), a white foam: 1H NMR
(MeOD) δ 0.74 (s, 3H, CH3), 0.98 (s, 3H, CH3), 3.35 (t, 2H,
NCH2), 3.37 (m, 1H, 6-H), 3.57 (m, 1 H, 4-H eq.), 3.58 (m, 1H,
17-H), 4.02 (m, 2H, OCH2). 23d (0.66 g, 41%), a white foam:
1H NMR (MeOD) δ 0.74 (s, 3H, CH3), 0.98 (s, 3H, CH3), 3.18
(m, 1H, 2-H eq), 3.35 (t, 2H, NCH2), 3.37 (m, 1H, 6-H), 3.58
(m, 1H, 17-H), 4.02 (m, 2H, OCH2).
(E)-3-(2-Am in op r op oxyim in o)a n d r ost a n e-6r,17â-d iol
h em ifu m a r a te (22l). Prepared in 82% yield from 23d by the
procedure described above for the preparation of 22j. 1H NMR
(DMSO-d6) δ 0.61 (s, 3H, CH3), 0.84 (s, 3H, CH3), 2.75 (m, 2H,
NCH2), 2.98 (m, 1H, 2-H eq), 3.21 (m, 1H, 6-H), 3.41 (m, 1H,
17-H), 3.98 (m, 2H, OCH2), 7.78 (bb, 3H, NH3+); white solid:
mp 147-181 °C. Anal. (C22H38N2O3‚0.5 C4H4O4‚0.5 H2O) C, H,
N, H2O.
(Z)-3-(2-Am in op r op oxyim in o)a n d r ost a n e-6r,17â-d iol
h em ifu m a r a te (22m ). Prepared in 80% yield from 23c by the
procedure described above for the preparation of 22j. 1H NMR
(DMSO-d6) δ 0.61 (s, 3H, CH3), 0.84 (s, 3H, CH3), 2.75 (m, 2H,
NCH2), 3.21 (m, 1H, 6-H), 3.40 (m, 2H, 17-H and 4-H eq), 3.98
(m, 2H, OCH2), 7.80 (bb, 3H, NH3+); white solid: mp 125-
174 °C. Anal. (C22H38N2O3‚0.5 C4H4O4‚H2O) C, H, N, H2O.
(E,Z)-3-(2-Am in oeth oxyim in o)-6â-h yd r oxya n d r osta n -
17-on e h yd r och lor id e (22n ). Prepared in 86% yield from 1f
and 2-aminoethoxyamine dihydrochloride. The product was
crystallized from MeOH/EtOAc. 1H NMR (DMSO-d6) δ 0.80
(s, 3H, CH3), 1.02 (s, 1.5H, CH3), 1.04 (s, 1.5H, CH3), 2.82 (dd,
0.5H, 4-H eq. Z isomer), 3.03 (t, 2H, NCH2), 3.06 (m, 0.5H,
2-H eq. E isomer), 3.65 (m, 1H, 6-H), 4.07 (t, 2H, OCH2), 4.45
(bb, 1H, OH), 7.95 (bb, 3H, NH3+); white solid: mp 160-170
°C dec. Anal. (C21H34N2O3‚HCl‚H2O) C, H, N, H2O.

3654 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
De Munari et al.
(7) Cerri, A.; Almirante, N.; Barassi, P.; Benicchio, A.; De Munari,
S.; Marazzi, G.; Molinari, I.; Serra, F.; Melloni, P. Synthesis and
Inotropic Activity of 1-(O-Aminoalkyloximes) of Perhydroindene
Derivatives as Simplified Digitalis-like Compounds Acting on
the Na+,K+-ATPase. J . Med. Chem. 2002, 45, 189-207.
(8) Cerri, A.; Almirante, N.; Barassi, P.; Benicchio, A.; Fedrizzi, G.;
Ferrari, P.; Micheletti, R.; Quadri, L.; Ragg, E.; Rossi, R.;
Santagostino, M.; Schiavone, A.; Serra, F.; Zappavigna, M. P.;
Melloni, P. 17â-O-Aminoalkyloximes of 5â-Androstane-3â,14â-
diol with Digitalis-Like Activity: Synthesis, Cardiotonic Activity,
Structure-Activity Relationships and Molecular Modeling of the
Na+,K+-ATPase Receptor. J . Med. Chem. 2000, 43, 2332-2349.
(9) Rosenkranz, G.; Velasco, M.; Sondheimer, F. Cycloethylene
Ketals of Androstane-3,6,17-trione. Synthesis of Androstan-3-
one-6â,17â-diol. J . Am. Chem. Soc. 1954, 76, 5024-5026.
(10) (a) Amendolla, C.; Rosenkranz, G.; Sondheimer, F. Synthesis of
Steroidal Hormone Analogues Hydroxylated at C(6). J . Chem.
Soc. 1954, 1226-1233. (b) Numazawa, M.; Osawa, Y. Synthesis
and Some Reactions of 6-Bromoandrogens: Potential Affinity
Ligand and Inactivator of Estrogen Synthetase. Steroids 1979,
34, 347-360.
(11) Hammerschmidt, F. J .; Spiteller, G. Zur Lokalisierung Funk-
tioneller Gruppen mit Hilfe der Massenspektrometrie-VIII.
6-Hydroxyandrostan-3,17-dione und 6,17â-Dihydroxyandrostan-
3-one. Tetrahedron 1973, 29, 2465-2472.
(12) Chambers, V. E. M.; Denny, W. A.; Evans, J . M.; J ones, E. R.
H.; Kasal, A.; Meakins, G. M.; Pragnell, J . Microbiological
Hydroxylation of Steroids. Part VIII. The Pattern of Monohy-
droxylation of Diketones and Keto-alcohols Derived from 5R-
Androstane with Cultures of the Fungus, Rhizopus nigricans.
J . Chem. Soc., Perkin Trans. 1 1973, 1500-1511.
(13) Nicholson, S. H.; Turner, A. B. Marine Steroids. Part III. On
the Structures of Marthasterone Glucoside, from the Starfish
Marthasterais glacialis. J . Chem. Soc., Perkin Trans. 1 1976,
1357-1360.
(14) Giddings, S.; Mills, A. Optimization of a Simple System for the
Oxidation of Octan-2-ol with Sodium Bromate, Mediated by
Ruthenium Tetraoxide Generated in Situ. J . Org. Chem. 1988,
53, 1103-1107.
(15) (a) Mohamadi, F.; Richards, N. G. J .; Guida, W. C.; Liskamp,
R.; Lipton, M.; Caufield, C.; Chang, G.; Hendrikson, T.; Still,
W. C. MacroModel - an Integrated Software System for Model-
ing Organic and Bioorganic Molecules Using Molecular Mechan-
ics. J . Comput. Chem. 1990, 11, 440-467. (b) For the lowest
energy conformations of each pair of isomers, the following
differences were found: 0.01 kJ /mol between (E)-22b and (Z)-
22b (no substitutions at C-2 or C-4); 12.14 kJ /mol between (E)-
22c and (Z)-22c (2R-Me derivatives) with the former as the lower
energy isomer (note that E and Z for 22c have the opposite
position in the space in comparison with 22b and 22d because
the substituted C-2 has a higher priority than C-4), and this
difference is equivalent to a distribution of about 8/1000 for the
higher energy isomer (Z) at room temperature; 15.80 kJ /mol
between (E)-22d and (Z)-22d (4R-Me derivatives) with the
former as the most favored isomer (Z/E distribution ratio of
about 2/1000 at room temperature).
(E,Z)-3-(2-Am in oet h oxyim in o)a n d r ost a n e-6â,17â-d iol
fu m a r a te (22o). Prepared in 75% yield from 1g and 2-amino-
ethoxyamine dihydrochloride. The crude product was purified
by flash chromatography (CHCl3/MeOH/26% NH4OH 90/10/
1). To the concentrated fractions, the stoichiometric amount
of fumaric acid in MeOH was added, followed by a 1/1 mixture
of EtOAc/Et2O. The precipitate formed was filtered to give 22o.
1H NMR (DMSO-d6) δ 0.64 (s, 3H, CH3), 1.00 (s, 1.5H, CH3),
1.01 (s, 1.5H, CH3), 2.79 (dd, 0.5H, 4-H eq. Z isomer), 3.03 (t,
2H, NCH2), 3.06 (m, 0.5H, 2-H eq. E isomer), 3.42 (t, 1H, 17-
H), 3.59 (m, 1H, 6-H), 4.05 (t, 2H, OCH2), 4.40 (bb, 2H, OH),
6.58 (s, 2H fumaric ac.), 8.21 (bb, 3H, NH3+); white solid: mp
148-156 °C dec. Anal. (C21H36N2O3‚C4H4O4‚2 H2O) C, H, N,
H2O.
(E,Z)-3-(2-Am in oeth oxyim in o)-17â-h yd r oxya n d r osta n -
6-on e fu m a r a te (22p ). Prepared in 55% yield from 1h and
2-aminoethoxyamine dihydrochloride. The crude product was
purified by flash chromatography (CHCl3/MeOH/26% NH4OH
90/10/1). To the concentrated fractions, the stoichiometric
amount of fumaric acid in MeOH was added, followed by a
1/1 mixture of EtOAc/Et2O. The precipitate formed was filtered
1
to give 22p . H NMR (DMSO-d6) δ 0.62 (s, 3H, CH3), 0.74 (s,
3H, CH3), 2.42 (dd, 0.5H, 5-H, Z isomer), 2.47 (dd, 0.5H, 5-H,
E isomer), 2.99 (m, 2H, NCH2), 3.02 (m, 0.5H, 4-H eq. Z
isomer), 3.08 (m, 0.5H, 2-H eq. E isomer), 3.46 (t, 1H, 17-H),
4.06 (t, 2H, OCH2); white solid: mp 110-170 °C. Anal.
(C21H34N2O3‚C4H4O4‚H2O) C, H, N, H2O.
(E,Z)-3-(2-Am in oeth oxyim in o)-17r-h ydr oxyan dr ostan e-
6-on e h yd r och lor id e (22q). Prepared in 62% yield from 1i
and 2-aminoethoxyamine dihydrochloride. 1H NMR (DMSO-
d6) δ 0.58 (s, 3H, CH3), 0.75 (s, 3H, CH3), 3.01 (t, 2H, NCH2),
3.53 (dd, 1H, 17-H), 4.09 (t, 2H, OCH2), 4.35 (d, 1H, OH), 8.01
(bb, 3H, NH3+); off-white solid: mp 170-172 °C dec. Anal.
(C21H34N2O3‚HCl‚H2O) C, H, N, H2O.
Con for m a tion a l En er gy Ca lcu la tion s of E a n d Z iso-
m er s. The calculations of E and Z isomers of 22b, 22c, and
22d were performed using the AMBER* all atom force field
as implemented in the program MacroModel 7.2; PR conjugate
gradient was used in all the minimization steps, with the
derivative convergence set to 0.05 (kJ /mol)/Å, with a maximum
of 5000 iterations. Monte Carlo multiple minimum method was
used in the conformational search (AMBER*, 5000 steps,
torsion rotations allowed on the amino chain). All conforma-
tions within 50 kJ /mol of the identified lowest energy con-
former were minimized again (Multiple Minimization routine
in the program) using AMBER* in conjunction with the GB/
SA continuum model to simulate water solvation.
(16) Sollman, P. B.; Dodson, R. M. Alkoxylation of Steroids with
Cupric Bromide: Alcohol. J . Org. Chem. 1961, 26, 4180-4181.
(17) Frigerio, M.; Santagostino, M.; Sputore, S.; Palmisano, G.
Oxidation of Alcohols with o-Iodoxybenzoic Acid (IBX) in
DMSO: A New Insight into on Old Hypervalent Iodine Reagent.
J . Org. Chem. 1995, 60, 7272-7276.
Refer en ces
(1) Hoffman, B. F.; Bigger, J . T. Digitalis and Allied Cardiac
Glycosides. In The Pharmacological Basis of Therapeutics, 8th
ed.; Goodman Gilman, A., Nies, A. S., Rall, T. W., Taylor, P.,
Eds.; Pergamon Press: New York, 1990; pp 814-839.
(2) a) Thomas, R.; Gray, P.; Andrews, J . Digitalis: its Mode of
Action, Receptor and Structure Activity Relationships. In Ad-
vances in Drug Research; Academic Press: New York, 1990; Vol.
19, pp 311-562. (b) Repke, K. R. H.; Sweadner, K. J .; Weiland,
J .; Megges, R.; Scho¨n, R. In Search of Ideal Inotropic Steroids:
Recent Progress. Prog. Drug Res. 1996, 47, 9-52.
(3) De Munari, S.; Barassi, P.; Cerri, A.; Fedrizzi, G.; Gobbini, M.;
Mabilia, M.; Melloni, P. A New Approach to the Design of Novel
Inhibitors of Na+,K+-ATPase: 17R-Substituted Seco-D 5â-An-
drostane as Cassaine Analogues. J . Med. Chem. 1998, 41, 3033-
3040.
(4) Gobbini, M.; Barassi, P.; Cerri, A.; De Munari, S.; Fedrizzi, G.;
Santagostino, M.; Schiavone, A.; Torri, M.; Melloni, P. 17R-O-
Aminoalkyloxime Derivatives of 3â,14â-Dihydroxy-5â-andros-
tane and 3â-Hydroxy-14-oxo-seco-D-5â-androstane as Inhibitors
of the Digitalis Receptor on Na+,K+-ATPase. J . Med. Chem.
2001, 44, 3821-3830.
(5) Repke, K. R. H.; Wieland, J .; Menkes, K. H. Chemical Models
for the Chemical Nature of Endogenous Digitalis. J . Enzyme
Inhib. 1991, 5, 25-32.
(18) Loibner, H.; Zbiral, E. Nucleophilic Substitution Reactions of
Hydroxysteroids Using Triphenylphosphane/Diethylazodicar-
boxylate. Helv. Chim. Acta 1977, 60, 417-425.
(19) J ørghensen, P. L. Purification and Characterization of Na+,K+-
ATPase. III. Purification from the Outer Medulla of Mammalian
Kidney after Selective Removal of Membrane Components by
Sodium Dodecylsulphate. Biochim. Biophys. Acta 1974, 356, 36-
52.
(20) Noel, F.; Godfraind, T. Heterogeneity of Ouabain Specific
Binding Sites and Sodium-Potassium ATPase Inhibition in
Microsomes from Rat Heart. Biochem. Pharmacol. 1984, 33, 47-
53.
(21) Scho¨nfeld, W.; Weiland, J .; Lindig, C.; Masnyk, M.; Kabat, M.
M.; Kurek, A.; Wicha, J .; Repke, K. R. H. The lead structure in
cardiac glycosides is 5â,14â-androstane-3â,14-diol. Naunyn-
Schmiedeberg’s Arch. Pharmacol. 1985, 329, 414-426.
(22) Micheletti, R.; Mattera, G. G.; Rocchetti, M.; Schiavone, A.; Loi,
M. F.; Zaza, A.; Gagnol, R. J . P.; De Munari, S.; Melloni, P.;
Carminati, P.; Bianchi, G.; Ferrari, P. Pharmacological Profile
of the Novel Inotropic Agent (EZ)-3-((2-Aminoethoxy)imino)-
androstane-6,17-dione Hydrochloride (PST2744). J . Pharmacol.
Exp. Ther. 2002, 303, 592-600.
(6) Sevillano, L. G.; Melero, C. P.; Caballero, E.; Tome´, F.; Lelievre,
L. G.; Geering, K.; Crambert, G.; Carron, R.; Medarde, M.; San
Feliciano, A. Inotropic Activity of Hydroindene Amidinohydra-
zones. J . Med. Chem. 2002, 45, 127-136.
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