4-(3′α15′β-dihydroxy-5′β-estran- 17′β-yl)furan-2-methyl alcohol: An anti-digoxin agent with a novel mechanism of action

Article abstract of DOI:10.1021/jm0505819

The synthesis and some pharmacological properties of 4-(3′α- 15′β-dihydiOxy-5β-estran-17′β9-yl)furan-2-methyl alcohol (16) have been described. The compound was synthesized by reacting a synthetic 3α-benzyloxy-5β-estr-15-en-17-one with the ethylene acetal

Full text of DOI:10.1021/jm0505819

600
J. Med. Chem. 2006, 49, 600-606
4-(3′r15′â-Dihydroxy-5′â-estran-17′â-yl)furan-2-methyl Alcohol: An Anti-Digoxin Agent with a
Novel Mechanism of Action
Joseph Deutsch,*,†,| Huang G. Jang,† Nura Mansur,† Ohad Ilovich,† Uri Shpolansky,‡ Dana Galili,‡ Tomer Feldman,§
Haim Rosen,§ and David Lichtstein*,‡
Department of Medicinal Chemistry, School of Pharmacy, Department of Physiology, and Department of Parasitology,
The Hebrew UniVersity-Hadassah Medical School, Jerusalem, Israel
ReceiVed June 19, 2005
The synthesis and some pharmacological properties of 4-(3′R-15′â-dihydroxy-5â-estran-17′â-yl)furan-2-
methyl alcohol (16) have been described. The compound was synthesized by reacting a synthetic 3R-
benzyloxy-5â-estr-15-en-17-one with the ethylene acetal of 4-bromo-2-furancarboxyaldehyde, followed by
hydrolysis of the ethylene acetal and reduction of the aldehyde. Despite its resemblance to the structure of
cardiac steroids (CS), 16 does not bind to the CS receptor on Na+,K+-ATPase and does not increase the force
of contraction of heart muscle. However, 16 inhibited the digoxin-induced increase in the force of contraction
and arrhythmias in guinea pig papillary muscle and human atrial appendages. The steroid also inhibited
digoxin-induced alteration in endocytosed membrane traffic, indicating a novel mechanism of action.
Introduction
Cardenolides (i.e. digoxin, ouabain) and bufadienolides (i.e.
bufalin, marinobufogenin) are steroids extracted from plants and
amphibian skin, respectively, which bind to and inhibit the
activity of the plasma membrane Na+,K+-ATPase.1 These
compounds are used in Western and Eastern medicine for the
treatment of cardiac failure and certain types of arrhythmias.2,3
In the past decade, endogenous cardenolides and bufadienolides
(herein collectively termed cardiac steroids, CS) have been
identified in mammals, including humans. Ouabain was identi-
fied in human plasma and adrenal gland,4 digoxin was found
in human urine,5 an ouabain isomer was identified in the bovine
hypothalamus,6 19-norbufalin and its peptide derivative were
identified in cataractous human lenses,7 and dihydropyrone-
substituted bufadienolide was identified in human placenta.8 In
addition, the immunoreactivity of marinobufogenin-like9 and
proscillaridin A-like compounds,10 which also belong to the
bufadienolide family, has been demonstrated in human plasma.
Strong evidence supports the notion that the CS are synthesized
in, and released from, the adrenal gland.11-13 The identification
of endogenous CS raises new questions regarding the physi-
ological roles and pharmacological effects of these compounds.
As part of our study on digoxin-like compounds, we synthesized
and screened estrane derivatives for their effects on cardiac
contractility in guinea pig both in vivo and in vitro. Among the
tested compounds, 4-(3′R,15′â-dihydroxy-5′â-estran-17′â-yl)-
furan-2-methyl alcohol (16, Figure 1a) was found to be of
interest and was chosen for in-depth evaluation.
Figure 1. (a) 4-(3′R-hydroxy-15′â-hydroxy-5â-estran-17′â-yl)furan-
2-methyl alcohol (compound 16). (b) X-ray crystallography of 13.
The most advanced synthesis of bufalin and its analogues was
documented 10 years later.20-23 According to those reports, a
3â-benzyloxy-R,â-unsaturated-17-keto steroid was reacted with
the lithiated ethylene acetal of 4-bromo-2-furancarboxyaldehyde
and the product was further transformed into a bufodienolide
compound in a multistep process. We now describe the synthesis
and in vitro pharmacological characteristics of 4-(3′R,15′â-
dihydroxy-5′â-estran-17′â-yl)furan-2-methyl alcohol, which was
synthesized by a multistep process.
Since 1969, several attempts to synthesize bufadienolides have
been made.14-17 The transformation of digoxin to a bufadieno-
lide compound was also reported.18 In the early 1970s, it was
established that the main prerequisites for a biologically active
cardenolide are the C/D cis ring junction, the 17â-butenolide
moiety, and the 3â,14â-dihydroxy groups. The A/B ring junction
can be either cis or trans without a dramatic change in activity.19
Synthesis and Structural Characterization
The synthesis of 4-(3′R,15′â-dihydroxy-5â-estran-17′â-yl)-
furan-2-methyl alcohol included three major steps: (a) synthesis
of 3R-benzyloxy-5â-estr-15-en-17-one; (b) reaction of the estrane
(norandrostane) moiety with the ethylene acetal of 4-bromo-2-
furancarboxyaldehyde; (c) removal of the benzyl group, hy-
drolysis of the ethylene acetal, and reduction of the aldehyde.
(a) The synthesis of 3R-hydroxy estrane moieties was carried
out using a modification of a published procedure.20 The starting
material was estrane-4-en-3,17-dione (Scheme 1) which, by
hydrogenation on RhCl3 hydrate, produced a 5â-estrane-3,17-
* To whom correspondence should be addressed. Phone: 972-2-6758695
and 972-2-6758522. Telefax: 972-2-6439736. E-mails: odj@cc.huji.ac.il
and david@md.huji.ac.il.
† Department of Medicinal Chemistry.
‡ Department of Physiology.
§ Department of Parasitology.
| Affiliated with the David R. Bloom Center for Pharmacy.
10.1021/jm0505819 CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/20/2005

Anti-Digoxin Agent
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 601
Scheme 1
dione (compound 2), with cis stereochemistry (80%) at the A/B
ring junction.24,25 Both ketone groups were protected as ethylene
ketal, yielding compound 3. After selective removal of the ketal
at the 3 position by iodine in acetone35 (compound 4), the ketone
was treated with NaBH4 in THF/methanol, selectively producing
the 3R-hydroxy-5â-estran-17-one ethylene ketal (compound 5).
The 3R-hydroxy group was protected as the benzyloxy deriva-
tive by boiling 5 with benzyl bromide in dry toluene in the
presence of NaH. Selective bromination followed by dehydro-
bromination and removal of the ketal group produced the R,â
unsaturated ketone 9 in an overall yield of 13%.
without purification, was subjected to allylic rearrangement in
boiling aqueous acetone in the presence of CaCO3, yielding the
allylic alcohol 12. The purified product was hydrogenated on
Pd/CaCO3 in methanol in the presence of traces of NaOAc,
yielding the crystalline product 13. In addition to the MS and
NMR spectra, the X-ray crystallography pattern of 13 clearly
indicated the ketal structure of 4-(3′R-benzyloxy-5′â-estrane-
15′âhydroxy-17′â-yl)furan-2-carboxyaldehyde ethylene ketal
(Figure 1b). Whereas, the orientation of the 17′â-yl furan
derivative was reported previously,20-22 the â stereochemistry
of the 15′ hydroxy group was observed only by the X-ray
measurement of compound 13. Concerning the mechanism of
formation, we can assume that in an earlier step (in compound
10), the 17′ hydroxyl group can be either R or â or both
conformations. Prior to the occurrence of the allylic rearrange-
(b) Condensation of 9 with the ethylene acetal of 4-bromo-
2-furancarboxyaldehyde26,27 afforded compound 10 (Scheme 1).
The purified alcohol was first acetylated with acetic anhydride
in pyridine to compound 11 (not shown in Scheme 1) which,

602 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Deutsch et al.
Figure 2. Inhibition of digoxin-induced increase in papillary muscle
contractility by 16. Representative records showing changes in force
of contraction of guinea pig papillary muscle induced by digoxin, 16,
and the two drugs together. The experiment was repeated five times
each using different muscle preparations.
Figure 4. Inhibition of digoxin-induced increase in human heart strips
contractility by 16. (a) Representative records showing changes in force
of contraction of human atrial strips, induced by digoxin, 16, and the
two drugs together. The experiment was repeated 10 times each using
different muscle preparations. (b) Effect of increasing 16 concentrations
on digoxin-induced increased force of contraction of human atrial strips.
The columns represent the average ( SE of six experiments (87 ( 16,
57 ( 2.5, and 19 ( 13 for 1 µM digoxin, 1 µM digoxin, and 10 µM
16, and 1 µM digoxin and 20 µM 16, respectively).
Figure 3. Inhibition of digoxin-induced arrhythmias in papillary muscle
by 16. Representative records showing changes in force of contraction
and rate of guinea pig papillary muscle induced by digoxin (4 µM),
16, and the two drugs together. The experiment was repeated five times
each using different muscle preparations.
ment, a SN2 reaction had taken place due to the alkaline
environment, affording mainly the thermodynamically more
stable 17′â hydroxy isomer. Further, the hydroxyl group
migrates to the C-15 retaining the configuration (above the ring
D) to give the 15′â hydroxy product.
(c) Product 13 was hydrolyzed in diluted hydrochloric acid,
yielding the aldehyde 14 and deprotected with Pd(OH)2/C in
the presence of formic acid, producing 15, which was further
reduced with NaBH4 to 4-(3′R,15′â-dihydroxy-5′â-estrane-17â-
yl)furan-2-methyl alcohol (16). 16 was dissolved in ethyl alcohol
and diluted with saline, and the biological activity was measured.
3
Biological Activity
Figure 5. Displacement of H-digoxin by digoxin, ouabain, bufalin,
and 16 from guinea pig crude synaptosomal fraction. Crude synaptosom-
al fraction preparation and binding assays are described in the Biological
Methods. A 200 µL volume (60 µg of protein) was incubated with
3H-digoxin in the presence of increasing concentrations of digoxin (open
triangles), ouabain (open circles), bufalin (open squares), or 16 (closed
circles). Following 1 h incubation, the membranes were passed through
Whatman GF/B filters, which were then washed twice. The radioactivity
retained on the filters was measured by â-counter liquid scintillation.
The experiments resulted in IC50 of 10, 20, and 30 nM for digoxin,
ouabain, and bufalin, respectively, and undefined value for 16.
The effect of 16 on guinea pig papillary muscle contractility
was determined using conventional techniques.28 Preliminary
experiments demonstrated that in this preparation at short time
(minutes) periods, 0.5-4 µM digoxin elicited significant
increase in contractility and arrhythmias at the high concentra-
tion. Compound 16, on the other hand, up to 20 µM, did not
significantly affect muscle contractility and rhythm (data not
shown). Thus, these concentration ranges were chosen in the
following experiments. The addition of digoxin (1 µM) to the
muscle bathing solution elicited a 60% increase in the force of
contraction within 10 min. Compound 16 (10 µM) slightly
reduced the force of contraction. However, when digoxin and
16 (at the above concentrations) were added simultaneously, a
complete blockade of digoxin-induced increase in contraction
was apparent (Figure 2). An anti-digoxin effect of 16 was also
observed by measuring digoxin-induced arrhythmias (Figure 3).
Papillary muscle exposed to 4 µM digoxin developed severe
arrhythmias within several min. Digoxin-induced arrhythmias
had an average time for onset of 7 ( 1 min (mean ( SD) and
were sustained for more than 20 min. The addition of 16 (1
µM) 1 min after the onset of arrhythmias lowered arrhythmias
irregularity, and the rhythm returned to the normal stimulatory
rate after within 10 ( 3 min (Figure 3).
An anti-digoxin effect of 16 was also demonstrated on human
atrial appendages. Human atrial appendages are routinely
discarded during open heart surgery and were used in the
following experiments. The addition of digoxin (500 nM)
increased the force of contraction of the human atrial strip by
∼60% (Figure 4). However, in the presence of 16 (1 µM), the
same concentration of digoxin elicited an increase of only ∼20%
in the force of contraction (Figure 4). The effect of different
concentrations of 16 on the digoxin-induced increase in the force
of contraction of the human atrial appendages is shown in Figure

Anti-Digoxin Agent
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 603
Figure 6. Inhibition of digoxin-induced transferrin accumulation in NT2 cells by 16. NT2 cells were grown in DMEM-F12 on glass coverslips for
24 h. The DMEM-F12 was then replaced with drug-free medium (A-C) or medium containing 20 nM digoxin (D-F), 20 nM 16 (G-I), or 20 nM
digoxin, and 20 nM 16 (J-L). Following 20 min incubation, the medium was removed and the cells were incubated in medium containing transferrin
Alexa Fluor, as described in Materials and Methods. Following 4 h incubation, the cells were observed under a fluorescence microscope. Phase
contrast images (A, D, G, and J), fluorescent images (B, E, H, and K), and the merged images of the two (C, F, I, and L) are shown. The green
color corresponds to transferrin signals. Scale bar, 20 µm.
5. Clearly, the effect of 16 is dose-dependent, and an almost
complete block of the digoxin-induced effect was obtained at a
ratio of 20:1 (16:digoxin).
We recently demonstrated that the bufadienolide bufalin in-
duces the accumulation of vesicles in the perinuclear region of
human cells.31 On the basis of the effect of CS on the accumu-
It is generally accepted that the mechanism of action of CS-
lation of FM1-43 in colocalization with specific Rab proteins,
induced increase in the force of contraction of heat muscle is
these vesicles were identified as late endosomes, and we sug-
mediated by the inhibition of the plasma membrane Na+,K+-
gested that endogenous CS-like compounds are involved in the
ATPase.1,2 Although alternative mechanisms were recently
regulation of endocytosed membrane traffic.31 Thus, it can be
suggested,29 it is believed that this inhibition results in a local
postulated that CS exert their action on heart contractility and
increase in intracellular Na+ and a consequent reduction in Na+/
rhythm, partially by their effect on intracellular membrane
Ca2+ exchange and increased intracellular Ca2+ and contractil-
traffic. This possibility is supported by recent findings that CS
ity.30 Thus, a plausible mechanism for the inhibition of the
effects are inhibited by endocytosis inhibitors.32,33 The effect
digoxin-induced effect by 16 is the inhibition by this steroid of
digoxin binding to its receptor on Na+,K+-ATPase. Binding
experiments, however, do not support this notion. The displace-
of digoxin on the accumulation of transferrin in human neuronal
NT2 cells is shown in Figure 6. In the absence of digoxin, no
accumulation of transferrin was apparent (Figur 6a-c). Like
bufalin,31 digoxin induced a 20-fold increase in the accumulation
of transferrin (Figure 6d-f), indicating reduced recycling of
the plasma membrane. Interestingly, the new steroid 16 com-
pletely blocked the digoxin-induced transferrin accumulation.
These findings support the hypothesis that 16 antagonism of
the digoxin-induced effect on cardiac muscle is mediated by
3
ment of H-digoxin by digoxin, ouabain, bufalin, and 16 from
rat brain synaptosomal fraction is shown in Figure 6. Whereas
digoxin, ouabain, and bufalin displaced 3H-digoxin in a dose-de-
pendent manner (IC50 10 and 20 nM, and 30 nM, respectively),
16 had only a marginal effect. Thus, it can be concluded that 16
does not interact with the CS binding site on Na+,K+-ATPase,
and its digoxin antagonism is mediated by a different pathway.

604 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Deutsch et al.
was dissolved in EtOAc (100 mL), washed twice with brine, and
dried over Na2SO4, and the solvents were removed under reduced
pressure, affording 6.5 g (98.5% yield) of the title compound as an
the inhibition of digoxin-induced changes in intracellular
membrane traffic.
CS are used for the treatment of cardiac failure and certain
types of arrhythmias,2,3 despite the frequent toxic side effects
of these drugs.2 In severe cases of CS toxicity, treatment with
specific digoxin antibodies is recommended.34 The discovery
that 16 is an antagonist of CS action raises the possibility of
developing such compounds and their derivatives for the
treatment of CS toxicity.
1
oil. MS: m/z (M+‚) 362. H NMR: δ 3.81 (m, 4H,OCH2CH2O),
0.87 (s, 3H, CH3[18]).
3-Oxo-5â-estrane 17-Ethylene Ketal (compound 4). Diketal
3 (300 mg, 1, 5 mmol) was dissolved in acetone (15 mL) and cooled
in an ice bath to 10 °C, and iodine (90 mg, 0.16 mmol) was then
added. The solution was stirred at 10 °C for 20 min. The reaction
was terminated by the addition of 10% aqueous Na2S2O3 until the
color of the iodine disappeared. Most of the acetone was then
removed under vacuum, and the residue was extracted with
dichloromethane (50 mL) and washed with brine. The organic layer
was separated, dried over Na2SO4, and filtered. The solvent was
removed, affording an oily product (300 mg, 99% yield) that was
reduced with NaBH4 without additional purification. MS: m/z (M+‚
) 318. IR 3737 cm-1. 1H NMR: δ 3.62 (m, 1H, CHOH), 3.81 (m,
4H, OCH2CH2O), 0.87 (s, 3H, CH3[18]).
Materials and Methods
General Procedures. Solvents were purchased from Biolab
(Jerusalem, Israel), Aldrich Chemical Co. (Milwaukee, WI), and
Frutarom (Haifa, Israel). Chemical reagents RhCl3‚H2O, Aliquat-
336, Pd (OH)2/10%C, n-butyllithium, pyridinium tribromide, eth-
ylene glycol, and potassium tert-butoxide were purchased from
Aldrich Chemical Co. Estrane-4-ene-3,17-dione was purchased from
Taizhou Xingye Chemical Co., Ltd, Shanghai 201507, P.R. China.
4-Bromo-2-furaldehyde was synthesized as described previously.12,13
Electron impact mass spectra were obtained using a Thermo-
Quest Trace MS (San Jose, CA) mass spectrometer, operating at
70 eV and with the ion source heated to 200 °C. The samples were
inserted by direct inlet with external heating of up to 220 °C or by
a GC inlet provided with a capillary column Phenomenex (Torrance,
CA) ZB 5, 5% phenyl-95% dimethyl polysiloxane (25m × 0.25
mm and 0.25 µm film thickness). Separation was performed in a
splitless mode with a temperature program. The starting temperature
was 80 °C which, after 6 min, was increased to 260 °C, 6 °C /min,
maintained for 10 min, and then decreased to the starting temper-
3-Hydroxy-5â-estrane 17-Ethylene Ketal (compound 5). Com-
pound 4 (3.18 g, 10.0 mmol) was dissolved in a 20% solution of
methanol in THF (50 mL), NaBH4 (1.0 g, 26.0 mmol) was slowly
added, and the solution was stirred overnight at room temperature.
The reaction was terminated by the addition of water (20 mL) and
stirred for 10 min. Removal of the organic solvent at reduced pres-
sure produced a white residue that was dissolved in diethyl ether,
washed with water, and dried over Na2SO4, affording an oily pro-
1
duct (3.0 g, 93.7% yield). MS: m/z (M+‚) 320. H NMR: δ 3.62
(m, 1H, CHOH), 3.81 (m, 4H, OCH2CH2O), 0.87 (s, 3H, CH3[18]).
3r-Benzyloxy-5â-estran-17-one Ethylene Ketal (6). A solution
of compound 5 (6.0 g, 18.7 mmol) in dry toluene (80 mL), NaH
powder (1.5 g, 60 mmol), and benzyl bromide (3.2 g, 18.7 mmol)
was boiled under nitrogen for 6 h. After cooling, the toluene solution
was poured slowly over ice. The organic phase was separated,
washed with water, and dried over Na2SO4. Removal of the solvent
resulted in an oily product. Flash chromatography on silica gel with
diethyl ether:hexane (1:9) produced compound 6 (6.2 g, 78% yield)
1
ature. The H NMR (300 MHz) measurements were performed
using a Varian VXR-300S (Palo Alto, CA) spectrometer in CDCl3.
IR spectra were recorded with a Perkin-Elmer FT-2000 or an
Analect FT-IR spectrometer (Analect Instruments FX-6160, Irvine,
CA). Liquid samples were recorded as a film between two NaCl
plates. Solid samples were pressed into KBr pellets. The melting
point was determined using an electrothermal apparatus (Electro-
thermal, England) and was uncorrected. Elemental analyses were
performed by the Microanalytical Laboratory at The Hebrew
University, Jerusalem, Israel. Crystallographic structure analysis was
done on an ENRAF-NONIUS CAD-4 computer-controlled diffrac-
tometer, and all crystallographic computing was done on a
VAX9000 computer at the Hebrew University of Jerusalem. The
HPLC measurement was performed with a Hewlett-Packard (HP)
1050 Series chromatograph provided with a HP 104OM detection
system and an Agilent ChemStation (Waldbron, Germany). The
detector was set to 225 nm and the chromatography of samples
was performed on a Luna C-18, 5 µm column (250 × 4.6 mm)
(Phenomenex, Torrance, CA) with an isocratic 60% acetonitrile/
water system with a flow of 0.8 mL/min. One milligram of sample
was dissolved in 1.0 mL ethyl alcohol in an autosample vial, and
10 µL was injected into chromatograph for HPLC analysis.
5â-Estrane-3,17-dione (2). A solution of estran-4-ene-3,17-dione
(5 g, 18.38 mmol) in dichloromethane (90.0 mL) was mixed with
the catalyst previously prepared from RhCl3‚H2O (40 mg) and
Aliquat-336 (200 mg) in water (2 mL) and dichloromethane (10.0
mL). The catalyst solution was added to the steroid solution and
hydrogenated overnight at 50 psi. The product was filtered, washed
twice with water (40 mL), and dried over Na2SO4, yielding 80%
of the cis isomer. Recrystallization from ethyl acetate produced
compound 2 (3.0 g, 60% yield) as a white solid. Crystallization
from ethyl acetate produced a white solid, mp 182-184 °C. MS:
m/z (M+‚) 274. IR 1737, 1705 cm-1. 1H NMR: δ 2.58 (t, 1H, J )
16 Hz, 4-Hax), 0.88 (s, 3H, CH3[18]). Elemental Analysis: C18H26O2
(MW 274), Calcd C (78.8%), H (9.48%), Found C (78.64%), H
(9.21%).
1
as a white solid. MS: m/z (M+‚) 410. H NMR: δ 7.35 (m, 5H,
Ph), 4.50 (s, 2H, CH2Ph), 3.89 (m, 4H, OCH2CH2O), 3.38 (m,1H,
H-C(3)), 0.83 (s, 3H, CH3[18]).
3r-Benzyloxy-16-bromo-5â-estran-17-one Ethylene Ketal (7).
Pyridinium tribromide (9 g, 25.2 mmol) was added slowly to a
stirred solution of compound 6 (9 g, 21.9 mmol) in dry THF (45
mL). After 20 h at 4 °C, the mixture was poured into a cold solution
of 5% NaHCO3 (30 mL) and 10% Na2S2O3 (30 mL). The organic
phase was extracted with hexane (2 × 100 mL), washed with brine,
and dried over Na2SO4, and the solvent was removed under reduced
pressure, yielding 9 g (75.3% yield) of the title compound as an
oil. The crude oil was subjected to dehydrobromination without
additional purification. MS: m/z (M+‚) 488, 490.
3r-Benzyloxy-5â-estr-15-en-17-one Ethylene Ketal (8). The
dry compound 7 (12 g, 24.4 mmol) was dissolved in dry benzene
(100 mL) and dry DMSO (50 mL), and potassium tert-butoxide
(8.0 g) was slowly added. The mixture was stirred and heated to
60 °C under nitrogen. After 20 h, the product was cooled and brine
(50 mL) was added. The mixture was extracted with benzene and
washed with brine. Solvent removal afforded compound 8 (5.6 g,
57% yield) as an oily compound, from which the protecting group
was removed without additional purification. MS: m/z (M+‚) 408.
1H NMR: δ 7.34 (m, 5H, Ph), 6.19 (d, 1H, H-C[16]) 5.68 (d ×
d, 1H, H-C[15]), 4.59(m, 2H CH2Ph), 3.93 (m, 4H, OCH2CH2O),
3.39 (m, 1H, H-C[3]) 0.93 (s, 3H, CH3[18′]).
3r-Benzyloxy-5â-estr-15-en-17-one (9). Pyridinium p-toluene-
sulfonate (1 g, 4 mmol) was added to a solution of compound 8
(5.6 g, 13.7 mmol) in acetone:water 10:2 (120 mL). The mixture
was stirred overnight, and deprotection was monitored by TLC.
The acetone was removed under reduced pressure, and the product
was extracted with dichloromethane. The dichloromethane solution
was washed with brine and dried over Na2SO4. Solvent removal
produced compound 9 (4.0 g, 80% yield) as an oil. Flash
chromatography on silica gel with diethyl ether:hexane (3:7) and
crystallization afforded a white solid (3.4 g, 68%), mp 92-94 °C
5â-Estrane-3,17 Ethylene Diketal (compound 3). Compound
2 (5 g, 18.2 mmol) was dissolved in dry benzene (100 mL), mixed
with ethylene glycol (25 mL, 280 mmol) and pyridinium p-
toluenesulfonate (1 g, 3.9 mmol), and refluxed, using a Dean-
Stark separator overnight or until no starting material was detected
by TLC. The benzene was removed at low pressure, the residue
(from diethyl ether). MS: m/z (M+‚) 364. IR 1707 cm-1 1H
.

Anti-Digoxin Agent
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 605
NMR: δ 7.52 (d, 1H, H-C[16]), 7.34 (m, 5H, Ph), 6.02 (d × d,
1H, H-C[15]), 4.56 (m, 2H CH2Ph), 3.4 (m, 1H, H-C[3]) 1.06
(s, 3H, CH3[18′]). Anal. C25H32O2 (MW 364), Calcd C 82.42%, H
8.79%; Found C 82.10%, H 8.80%.
14 (120 mg, 66% yield) as an oily product. MS: m/z (M+‚) 464.
1H NMR: δ 7.34 (m, 5H, Ph), 7.20 (s, 1H, H-C[5]), 6.38 (s, 1H,
H-C[3]), 5.85 (s, 1H, H-C-C[2]), 4.56 (s, 2H, CH2Ph), 4.38 (m,
1H, H-C[15′]), 3.40 (m, 1H, H-C[3′]) 0.78 (s, 3H, CH3 [18′]).
4-(3′a,15′â-Dihydroxy-5′â-estran-17′â-yl)furan-2-carboxyal-
dehyde (15). A solution of compound 14 (300 mg, 0.65 mmol) in
10% formic acid in ethanol (10 mL) and 10% Pd(OH)2/C (300 mg)
was boiled for 60 min. The solution was filtered, and water (5 mL)
was added. The product was extracted with dichloromethane,
washed with water, and dried over Na2SO4. Removal of the solvent
afforded compound 15 (220 mg, 91% yield) as a yellow solid.
MS: m/z (M+‚) 372.
Ethylene Acetal of 4-(3′a-Benzyloxy-17′-hydroxy-5′â-estr-15′-
en-17′-yl)furan-2-carboxyaldehyde (10). A 4.0 mL volume (10
mmol) of 2.5 M n-butyllithium was slowly added to an ethylene
acetal of 4-bromo-2-furancarboxyaldehyde (2.5 g, 11.4 mmol)
solution in freshly distilled dry THF (10 mL), and the solution was
cooled to -70 °C under N2. After 30 min at -70 °C, compound 9
(2.0 g, 5.5 mmol) dissolved in dry THF (20 mL) was added through
a syringe. After 1 h at -70 °C, the mixture was allowed to warm
to -10 °C and citric acid (100 mM, 10 mL) was slowly added.
The product was extracted with diethyl ether, washed with 100 mM
citric acid (30 mL) and then with water, and dried over Na2SO4.
Solvent removal and purification by flash chromatography on silica
gel with diethyl ether:hexane (7:3) afforded compound 10 (2.3 g,
4-(3′a15′â-dihydroxy-5′â-estran-17′â-yl)furan-2-methanol (16).
NaBH4 (200 mg) was slowly added to a solution of compound 15
(200 mg, 0.53 mmol) in THF (15 mL) and methanol (3 mL). After
the exothermic reaction was completed, the solution was stirred
for 1 h. Water (10 mL) was added, and the solution was stirred for
15 min. The organic solvents were removed under low pressure,
and the remaining solution was extracted with diethyl ether, washed
with water, and dried Na2SO4. Removal of the solvent afforded
compound 16 (170 mg, 85% yield) as a white solid. HPLC measure-
ment showed a large peak with a λmax at 220 nm with more than
90% compound 16. MS: m/z (M+‚) 374. 1H NMR: δ 7.20 (s, 1H,
H-C[5]), 6.20 (s, 1H, H-C[3]), 4.58 (s, 2H, CH2-O), 4.38 (m,
1Ht, H-C[15′]), 3.63 (m, 1H, H-C[3′]) 0.78 (s, 3H, CH3 [18′]).13C
NMR: δ 153.96 (C2), 139.55 (C5), 125.69 (C4), 109.51 (C3), 71.91
(C3′), 71.04 (C6), 59.54 (C15′), 57.93 (C14′), 15.85 (C18′)
Biological Methods. Measurements of Heart Muscle Con-
tractility. All experiments were carried out in accordance with the
guidelines of The Hebrew University Ethics Committee. Guinea
pigs were sacrificed by cervical dislocation. The hearts were
immediately excised in Krebs-Henseleit bicarbonate buffer (com-
position in mM: 118.4 NaCl, 4.7 KCl, 25 NaHCO3, 1.2 KH2PO4,
2 CaCl2, 1.2 MgSO4, and 5.5 glucose, pH 7.4). Papillary muscles
were excised from the left and right ventricles, secured with silk
thread to a polypropylene tissue holder, and mounted vertically in
a 12 mL bath. The nutrient solution was aerated with 95% O2/5%
CO2 and maintained at 36 °C. The papillary muscles were driven
by a pair of platinum electrodes (filed stimulation) with a rectangular
current pulse (1 Hz, 0.5 ms, about 1.2× threshold voltage) generated
by an electronic stimulator (Master-8, A.M. P.I and a custom-made
isolated current amplifier). The developed tension was measured
isometrically with a force-displacement transducer (FSG-01, Ex-
perimetria) connected to a bridge amplifier (Lablinc, Coulbourn
Instruments). The data were displayed and recorded on a PC based
PowerLab/16sp system.
40% yield) as a white solid. MS: m/z (M+‚) 504, IR: 3550 cm-1
.
1H NMR: δ 7.33 (m, 5H, Ph), 7.18 (s, 1H, H-C[5]), 6.42 (s, 1H,
H-C[3]), 6.06 (d, 1H, H-C[16′]), 5.89 (s, 1H, H-C-C[2]), 5.68
(d × d, 1H, H-C[15′]), 4.54 (s, 2H CH2Ph), 4.08 (m,4H, OCH2-
CH2O), 3.36 (m, 1H, H-C[3′]) 1.02 (s, 3H, CH3 [18′]).
Ethylene Acetal of 4-(3′a-Benzyloxy-17′-acetyl-5′â-estr-15′-
en-17′-yl)furan-2-carboxyaldehyde (11). Compound 10 (2.3 g, 4.5
mmol) was dissolved in a mixture of pyridine (20 mL), acetic
anhydride (10 mL), and dimethylaminopyridine (100 mg) and stirred
overnight at room temperature. The reaction was terminated by the
addition of 0.5 M HCl (30 mL). The product was extracted with
diethyl ether, washed with 5% NaHCO3, and dried over Na2SO4.
Solvent removal afforded compound 11 (2.4 g, 96% yield) as an
oily product. MS: m/z [M - 60]‚+ 486. 1H NMR: δ 7.32 (m, 5H,
Ph), 7.19 (s, 1H, H-C[5]), 6.39 (d, 1H, H-C[15′]), 6.28 (s, 1H,
H-C[3]) 6.10 (d, 1H, H-C[16′]) 5.87 (s, 1H, H-C-C[2]), 5.68
(d × d, 1H, H-C[15′]), 4.52 (s, 2H, CH2Ph), 4.08 (m,4H, OCH2-
CH2O), 3.36 (m, 1H, H-C[3′]), 2.02 (s, 3H, OCOCH3), 1.02 (s,
3H, CH3 [18′]).
Ethylene Acetal of 4-(3′a-Benzyloxy-15′â-hydroxy-5′â-estr-
16′-en-17′-yl)furan-2-carboxyaldehyde (12). Compound 11 (1.8
g, 3.3 mmol) was dissolved in acetone (75 mL) and water (15 mL),
and CaCO3 (1 g) was added. The mixture was stirred and boiled
for 3 days and the reaction monitored by TLC. After filtration, the
acetone was removed under reduced pressure and the product
dissolved in diethyl ether, washed with water, and dried over Na2-
SO4. Solvent removal and flash chromatography on silica gel with
diethyl ether:hexane (3:7) afforded compound 12 (0.9 g, 54% yield)
1
as a yellow solid. MS: m/z (M+‚) 504. IR 3550 cm-1. H NMR-
(CDCl3) δ 7.52 (s, 1H, H-C[5]), 7.32 (m, 5H, Ph), 6.57 (s, 1H,
H-C[3]), 5.94 (d, 1H, H-C[16′]), 5.90 (s, 1H, H-C-C[2]), 4.57
(m, 1H, H-C[15′] and s, 2H, CH2Ph), 4.08 (m,4H, OCH2CH2O),
3.40 (m, 1H, H-C[3′]) 1.28 (s, 3H, CH3[18′]).
Human atrial appendages are routinely discarded in the course
of open heart surgery. The tissue was transferred within minutes
to the laboratory, dissected into isolated trabecula (5-10 mm long
and 2-6 mm in diameter), and mounted on the same system as
described above. The trabecula was driven at 0.5 Hz field
stimulation, and the force of contraction was followed as described
for the papillary muscle preparation.
Membrane Preparation. A crude synaptosomal membrane
preparation was prepared from guinea pig brain by homogenizing
whole brain (minus cerebellum) in 10 volumes (w/v) of ice-cold
0.32 M sucrose, using a Teflon-glass homogenizer (Zivan, Haifa,
Israel). The homogenate was centrifuged at 1000g for 10 min at 4
°C. The supernatant was decanted and centrifuged at 28500g for
10 min at 4 °C. The pellet was re-suspended in the sucrose solution
and recentrifuged under the same conditions. The resulting pellet
was resuspended in 50 mM Tris buffer, pH 7.4, and dispersed, using
the glass-glass homogenizer. The preparation was diluted to a final
protein concentration of 5-10 mg/mL and was stored at -70 °C
in aliquots of 1 mL until used.
Ethylene Acetal of 4-(3′a-Benzyloxy-15′â-hydroxy-5′â-estran-
17′â-yl)furan-2-carboxyaldehyde (13). Compound 12 (0.5 g, 1.0
mmol) was dissolved in THF (9 mL), methanol (20 mL), and 5%
NaOAc (0.6 mL), mixed with 10% Pd/CaCO3 (150 mg), and
hydrogenated at 50 psi for 6 h.. The solution was filtered and the
solvent evaporated under reduced pressure. The product was
dissolved in dichloromethane, washed with water, and dried over
Na2SO4. Removal of the solvent afforded compound 13 (0.5 g, 99%
yield) as a white solid. Crystallization afforded a white solid, mp
147-149 °C (from hexane:diethyl ether). X-ray diffraction mea-
surements of compound 13 (Figure 1b) showed the 3′R-benzyloxy-
15′â-hydroxy--5′â-estran-17′â-yl configuration. MS: m/z (M+‚)
1
506. IR 3550 cm-1. H NMR: δ 7.34 (m, 5H, Ph), 7.20 (s, 1H,
H-C[5]), 6.38 (s, 1H, H-C[3]), 5.85 (s, 1H, H-C-C[2]), 4.56
(s, 2H, CH2Ph), 4.38 (m, 1H, H-C[15′]), 4.08 (m,4H, OCH2CH2O),
3.40 (m, 1H, H-C[3′]) 0.78 (s, 3H, CH3 [18′]).
3H-Digoxin Binding to Synaptosomal Fraction. Two hundred
microliters of synaptosomes (60 µg of protein) was incubated for
1 h at 37 °C with 300 µL of a solution containing final
concentrations of 30 mM Tris-HCl buffer, pH 7.4, 0.4 mM EDTA,
80 mM NaCl, 4 mM MgSO4, 2 mM ATP (Tris salt, vanadium-
free), 40 nM 3H-digoxin, and varying concentrations of nonradioac-
tive digoxin, ouabain, bufalin, or 16. The reactions were terminated
4(3′a-Benzyloxy-15′â-hydroxy-5′â-estran-17′â-yl)furan-2-car-
boxyaldehyde (14). A solution of 13 (200 mg, 0.39 mmol) in THF
(14 mL) and 1 N HCl (7 mL) was stirred for 3 h. The solution was
diluted with water and extracted twice with diethyl ether. The
organic solution was washed with 5% NaHCO3 and water and dried
over anhydrous Na2SO4. Removal of the solvent afforded compound

606 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2
Deutsch et al.
(10) Sich, B.; Kirch, U.; Tepel, M.; Zidek, W.; Schoner, W. Pulse pressure
correlates in humans with a Proscillaridin A immunoreactive
compound. Hypertension 1996, 7, 1073-1078.
by the addition of 3 mL of ice-cold 50 mM Tris-HCl, pH 7.4,
followed by passage over Whatman GF/B filters (Whatman
International Ltd., Maidstone, U.K.). The filters were washed twice
with 3 mL of Tris buffer, dried, and counted in a liquid scintillation
counter. Specific binding was calculated by subtracting the binding
observed in the presence of 100 µM unlabeled digoxin from that
observed in the absence of unlabeled digoxin and representing 93%
of the total binding under control conditions.
Cell Culture. Human neuronal precursor cells (NT2)) were
obtained from Stratagene Cloning Systems (La Jolla, CA). The cells
were grown in T25 tissue culture flasks in DMEM-F12 supple-
mented with 2 mM glutamine, 10% fetal bovine serum, 100 U/mL
penicillin, and 100 µg/mL streptomycin, in an atmosphere of 5%
CO2 at 37 °C, as described previously.31 The cells were grown to
confluence and then split 1:4-1:5 by trypsinization and replated.
In the fluorescence microscopy studies, cells were grown on glass
coverslips, which were mounted with neutral contact glue on a 1.4
× 2.4-cm aperture in the center of 6-cm Petri dishes, 24-48 h
before the experiments.
Fluorescence Microscopy. Conventional fluorescence micros-
copy was performed as previously described.31 Images were
acquired with a cooled SensiCAM charge-coupled device camera
and analyzed using the IP Plus 4.1 version (Signal Analytics, San
Diego, CA) software. Unlabeled cells were used to determine
autofluorescence. The image background was corrected as fol-
lows: Two to three regions were selected from cell-free areas in
each field and the average intensity of these regions was considered
the background value for that field. This value was then subtracted
from each pixel in the field. An integrated fluorescence power
reading for each image was recorded after background correction.
Images were saved in TIFF format and transferred to Adobe
Photoshop version 5.5 software for printing.
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Acknowledgment. This study was supported in part by The
Nophar program of the Chief Scientist Office, Ministry of
Industry, Trade & Labor, State of Israel.
Note Added after ASAP Publication. In the version of this
paper posted December 20, 2005, an incorrect compound
number (16) was present in part b of the Figure 1 caption. The
compound number was changed to 13, and the paper was
reposted December 23, 2005.
Supporting Information Available: Detailed X-ray data is
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