Novel analogues of Istaroxime, a potent inhibitor of Na+,K+-ATPase: Synthesis, structure-activity relationship and 3D-quantitative structure-activity relationship of derivatives at position 6 on the androstane scaffold

Article abstract of DOI:10.1016/j.bmc.2010.04.095

We report the synthesis and biological properties of novel analogues of Istaroxime acting as positive inotropic compounds through the inhibition of the Na⁺,K⁺-ATPase. We explored the chemical space around the position 6 of the steroidal scaffold by changing the functional groups at that position and maintaining a basic oximic chain in position 3. Some compounds showed inhibitory potencies of the Na⁺,K⁺-ATPase higher than Istaroxime and many of the compounds tested in vivo were safer than digoxin, the classic digitalis compound currently used for the treatment of congestive heart failure as inotropic agent. The 3D-QSAR analyses using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods have been successfully applied to a set of 63 androstane derivatives as Na⁺,K⁺-ATPase inhibitors. The contour plots provide many useful insights into relationships between structural features and inhibitory potency.

Full text of DOI:10.1016/j.bmc.2010.04.095

Bioorganic & Medicinal Chemistry 18 (2010) 4275–4299
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel analogues of Istaroxime, a potent inhibitor of Na+,K+-ATPase: Synthesis,
structure–activity relationship and 3D-quantitative structure–activity
relationship of derivatives at position 6 on the androstane scaffold
Mauro Gobbini a, , , Silvia Armaroli a,à, Leonardo Banfi a, , Alessandra Benicchio a,§, Giulio Carzana a,
Patrizia Ferrari b, Giuseppe Giacalone b, Giuseppe Marazzi a, , Barbara Moro b, Rosella Micheletti b,
Simona Sputore a,–, Marco Torri a, , Maria Pia Zappavigna a, Alberto Cerri a,
,
*
a Department of Medicinal Chemistry, Prassis Istituto di Ricerche Sigma-Tau S.p.A., Via Forlanini 3, 20019 Settimo Milanese (MI), Italy
b Department of Cardiovascular Pharmacology, Prassis Istituto di Ricerche Sigma-Tau S.p.A., Via Forlanini 3, 20019 Settimo Milanese (MI), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the synthesis and biological properties of novel analogues of Istaroxime acting as positive ino-
tropic compounds through the inhibition of the Na+,K+-ATPase. We explored the chemical space around
the position 6 of the steroidal scaffold by changing the functional groups at that position and maintaining
a basic oximic chain in position 3. Some compounds showed inhibitory potencies of the Na+,K+-ATPase
higher than Istaroxime and many of the compounds tested in vivo were safer than digoxin, the classic
digitalis compound currently used for the treatment of congestive heart failure as inotropic agent. The
3D-QSAR analyses using comparative molecular field analysis (CoMFA) and comparative molecular sim-
ilarity indices analysis (CoMSIA) methods have been successfully applied to a set of 63 androstane deriv-
atives as Na+,K+-ATPase inhibitors. The contour plots provide many useful insights into relationships
between structural features and inhibitory potency.
Received 9 March 2010
Revised 27 April 2010
Accepted 28 April 2010
Available online 20 May 2010
Keywords:
Istaroxime
Na+,K+-ATPase
Structure–activity relationship
3D-Quantitative structure–activity
relationship
Ó 2010 Elsevier Ltd. All rights reserved.
Congestive heart failure
Positive inotropic drugs
1. Introduction
with this syndrome and severely limit their normal activities. Ther-
apy of CHF is aimed at improving the pumping performance of the
Heart failure is the pathophysiological state in which the pump-
ing action of the heart is impaired, with a loss of cardiac contractile
function (cardiac dysfunction), to varying degrees whereby the
delivery of blood (cardiac output) becomes inadequate to satisfy
the oxygen and nutritional needs of the body. Decreased cardiac
output leads to excessive fluid accumulation in the body (conges-
tion); the result is congestive heart failure (CHF). Fatigue, breath-
lessness, and peripheral edema are the symptoms in patients
heart and reducing cardiac load by reducing vasoconstriction.
According to treatment guidelines and depending on the stage of
the illness, different classes of drugs are available to limit and con-
trast the decline of cardiac pump efficiency: angiotensin-convert-
ing enzyme inhibitors; angiotensin II receptor blockers; inotropic
agents; diuretics; beta-blockers.1
The digitalis cardiac glycoside digoxin (Chart 1) is the only ino-
tropic drug currently prescribed for chronic treatment of CHF. Car-
diac glycosides improve the pumping performance of the heart via
inhibition of the Na+,K+-ATPase, an ubiquitous membrane protein
that promotes the outward transport of Na+ and the inward trans-
port of K+ against their concentration gradients. Na+,K+-ATPase
inhibition increases intracellular Na+, which is then extruded by
the Na+,Ca2+ exchanger, leading to an increase of intracellular
Ca2+ levels. The availability of higher Ca2+ concentrations increases
cardiac myocytes contractile force (positive inotropic effect).2 De-
spite its proven efficacy,3 a risk related to digoxin therapy is the
proarrhythmogenicity which emerges at a two to three times high-
er serum concentration than the therapeutic with disturbances of
conduction and cardiac arrhythmias.4
* Corresponding author. Present address: Chemistry and Analytical Development,
R&D, Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., Via Pontina Km 30.400,
00040 Pomezia (RM), Italy. Tel.: +39 0691394172; fax: +39 0691393638.
E-mail address: mauro.gobbini@sigma-tau.it (M. Gobbini).
Present address: Chemistry and Analytical Development, R&D, Sigma-Tau
Industrie Farmaceutiche Riunite S.p.A., Via Pontina Km 30.400, 00040 Pomezia
(RM), Italy.
à
Present address: Sigma-Tau Research Switzerland, PO Box 1823, Via Motta 2/a,
CH-6850 Mendrisio, Switzerland.
§
Present address: Jetpharma SA, Via Sottobisio 42 A/C, CH-6828 Balerna,
Switzerland.
–
Present address: Explora Laboratories SA, Department of Research and Develop-
ment, Via Rime 38, CH-6850 Mendrisio, Switzerland.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.095

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M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
Table 1
O
Structure and Na+,K+-ATPase inhibition for compounds bearing the 2-aminoethoxyi-
mino chain (1–33)
O
OH
O
12
17
H
14
9
H
8
5 H
OH
3
H
R
H
O
O
O
H
O
H2N
N
H
H
OH
3
Na+,K+-ATPase inhibition, IC50
(lM)
a
Compounds
R
Digoxin
Istaroxime
Compound A
Compound B
1
2
3
0.31
0.11
0.52
2.1
0.042
0.19
1.1
Oxo
Digoxin
a-OH
b-OH
@CH2
@CF2
O
17
(E) @CHMe
H
9
14
4
5
Spirocyclopropane
b-Me
0.85
6.0
8
5 H
H
3
O
H2N
N
6
7
8
9
a
a
a
-Me
-CH@CH2
-C„CH
0.30
0.53
0.20
0.024
0.017
1.7
43
1.7
64
0.77
0.32
0.18
1.6
0.50
3.0
0.91
0.87
0.71
1.5
3.1
0.33
2.9
16
1.1
6
H
O
(E) @NOH
(E) @NOMe
(E) @NOEt
(E) @NOAllyl
(E) @CHCN
(E) @CHCOOMe
(E) @CHCH2OH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Istaroxime
Chart 1. Structures of digoxin and Istaroxime.
In one of our past works on digitalis-like compounds, the bent
digitalis skeleton was replaced by the flat 5 ,14 -androstane scaf-
a
a
-CH2CH2OH
-CH2OH
a
a
fold properly substituted in position 3 with an aminoalkyloxime
and in position 6 with hydroxy or oxo groups.5 Istaroxime (referred
to as PST 2744; Chart 1), emerged as a very promising positive ino-
tropic compound which is now in phase II clinical trials for the
treatment of CHF.6,7
In the previous paper, the androstane skeleton was used as a
scaffold to study the space around the basic chain in position 3
of our lead compound, Istaroxime; some compounds demonstrated
higher potencies than Istaroxime on the receptor and all tested
compounds resulted less proarrhythmogenic than digoxin.8 In this
paper, we describe new analogues of Istaroxime in which the ef-
fects of substitution at position 6 of the androstane scaffold on
the activity at the Na+,K+-ATPase were studied. We performed a
wide differentiation of the carbonyl group present at position 6
of Istaroxime, with the purpose of obtaining compounds with a
higher potency and better therapeutic index than those of the par-
ent compound.
b-CH2OH
-CH2OMe
a
b-CH2OMe
R-Spirooxirane
S-Spirooxirane
a-OMe
b-OMe
a-OEt
a-ONO2
b-ONO2
a
a
a
a
a
a
-COOH
-CN
-COOMe
-CONH2
-NH–CHO
-NH–COCH3
0.41
0.63
0.16
45
Concentrations able to inhibit 50% of Na+,K+-ATPase enzyme activity; mean of
two or three experiments.
a
.
2. Chemistry
very good stereoselectivity from 60 by reaction with 1 M BH3 THF
complex in THF at 0 °C followed by addition of water, NaOH and
H2O2 (95% yield) and subsequent acidic deprotection of the ethyl-
ene ketals of 64 (85% yield). Etherification of the alcohol 64 with
NaH and CH3I in THF at 0 °C followed by acidic deprotection of
the bis(ethylendioxy) derivative 66 gave 6b-methoxymethyland-
rostane-3,17-dione 67 (76% yield). 6-(Spirocyclopropane)andro-
stane-3,17-dione 68 was obtained in 48% yield from 6-methylene
derivative 60 by reaction with CH2I2 in the presence of Et2Zn in tol-
uene at 60 °C followed by acidic deprotection of the intermediate
bis(ethylendioxy) derivative. Even though a small amount of 6b-
methyl derivative was present in the hydroboration reaction of
60, 6b-methylandrostane-3,17-dione 71 was more easily obtained
by radical deoxygenation of the imidazole-1-carbothioate deriva-
tive 69 (obtained from 64 by reaction with TCDI in CH2Cl2 at
40 °C in the presence of DMAP in 83% yield) with Ph3SnH and AIBN
in toluene (42% yield) and following acidic deprotection of the eth-
ylene ketals of 70.
The oximes 1–58 listed in Tables 1–3, were synthesized from
the appropriate 3,17-diketones and the corresponding O-substi-
tuted hydroxylamine dihydrochlorides in a THF/water solution at
room temperature as mixture of E and Z isomers, in an approxi-
mate 1:1 ratio (Scheme 1, see Section 5).
The required starting 3,17-diketones of Scheme 1 were obtained
as described in Schemes 2–7. As shown in Scheme 2, 6-methylene-
androstane-3,17-dione 61 was obtained by reaction of 3,3:17,17-
bis(ethylendioxy)androstane-6-one5 59 with methyltriphenyl-
phosphonium bromide in THF in the presence of potassium tert-
butoxide followed by deprotection of the ethylene ketals groups
of 60 with PTSA in acetone at room temperature, in 84% overall
yield. From 61, the epimeric mixture of 6-spiro-(20-oxirane) deriv-
atives was obtained by reaction with MCPBA in CH2Cl2 at 0 °C; a
rather difficult separation by flash chromatography gave the pure
(6S)-6-spiro-(20-oxirane)androstane-3,17-dione 62 (30% yield)
and (6R)-6-spiro-(20-oxirane)androstane-3,17-dione 63 (15%). 6b-
Hydroxymethylandrostane-3,17-dione 65 was obtained with a
The compounds obtained from the key intermediate 6
a-carbox-
aldehyde 73 are shown in Scheme 3. The aldehyde 73 was obtained

M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
4277
Table 2
with an efficient method starting from the known 6-keto deriva-
tive 59 (shown in Scheme 2) in 72% overall yield over four steps.
Oxidation of the 6b-hydroxymethylandrostane 64 with IBX9 in
DMSO at room temperature gave the 6b-formylandrostane 72 in
83% yield; isomerization of the axial formyl group of 72 with
K2CO3 in MeOH at room temperature yielded the equatorial epi-
Structure and Na+,K+-ATPase inhibition for compounds bearing the 3-[(3R)-3-
pyrrolidinyloxyimino] chain (34–47)
O
H
H
R
H
meric aldehyde 73 in 94% yield. 6a-Vinylandrostane-3,17-dione
O
N
75 was obtained in 63% yield by reaction of the aldehyde 73 with
methyltriphenylphosphonium bromide in THF at 0 °C in the pres-
ence of potassium tert-butoxide, to give 74, followed by acidic
H
N
H
deprotection of the bis(ethylendioxy) derivative. 6
ethyl)androstane-3,17-dione 77 was prepared from bis(ethylendi-
oxy)-6 -vinyl derivative 74 in 96% yield by oxidative
hydroboration followed by acidic deprotection of the ethylene ke-
tals 76. Acidic deprotection of 3,3:17,17-bis(ethylendioxy)-6
formylandrostane 73 gave 78 in 85% yield; 6 -carboxyandros-
a-(2-Hydroxy-
a
Compound
R
Na+,K+-ATPase inhibition, IC50
(lM)
Compound C
Oxo
@CH2
@CF2
Spirocyclopropane
a
a
0.026
0.046
0.021
0.22
0.34
0.11
0.016
0.018
0.058
0.026
0.32
0.38
0.041
0.14
a
34
35
36
37
38
39
40
41
42
43
44
45
46
47
a-
-Me
-C„CH
a
tane-3,17-dione 79 was obtained by oxidation of the aldehyde
group of 78 with 1 N aqueous KMnO4 in tert-butanol and 5% aque-
(E) @NOH
(E) @NOMe
ous Na2HPO4 solution in 96% yield. 6a-Carbamoylandrostane-3,17-
a
a
a
a
a
a
a
-CH2CH2OH
-CH2OH
-CH2OMe
-ONO2
-NH–CHO
-CONH2
-COOMe
dione 80 and 6 -methoxycarbonylandrostane-3,17-dione 81 were
a
obtained from the acid 79, by reaction with SOCl2 in toluene at
85 °C, followed by treatment of the intermediate with 2 M NH3
solution in THF at 0 °C, (60% yield) or by reaction with DMAP, EDAC
and methanol in CH2Cl2 at 0 °C (70% yield), respectively. 6a-
0.16
Hydroxymethylandrostane-3,17-dione 83 was obtained by reduc-
tion of the aldehyde 73 with NaBH4 followed by acidic deprotec-
tion of the ethylene ketals (73% yield). The intermediate 82 was
reacted with NaH in THF at 0 °C and then with CH3I to give, after
a
Concentrations able to inhibit 50% of Na+,K+-ATPase enzyme activity; mean of
two or three experiments.
deprotection, 6
yield). The ethynyl derivative 87 was prepared from 3,3:17,17-
bis(ethylendioxy)-6 -formylandrostane 73 by reaction with (chlo-
romethyl)triphenylphosphonium chloride in THF and n-butyllith-
ium at ꢀ78 °C followed by heating at 70 °C, and reaction of the
crude product thus obtained with n-butyllithium in THF at
a-methoxymethylandrostane-3,17-dione 85 (74%
Table 3
a
Structure and Na+,K+-ATPase inhibition for compounds bearing the 3-N-methylami-
nopropoxyimino chain (48–58)
O
ꢀ78 °C again; final deprotection gave
3,17-dione 87 in 22% overall yield. Finally, 6
3,17-dione 90 was obtained in 68% yield by the following se-
quence: reaction of the 6 -hydroxymethyl derivative 82 with
6
a
a
-ethynylandrostane-
-methylandrostane-
H
H
H
R
H
N
O
N
a
H
pTSCl in the presence of DABCO in CH2Cl2 at 0 °C to give the corre-
sponding 4-methylbenzenesulfonate 88; reduction of the sulfonate
with NaBH4 in DMSO at 80 °C to the 6a-methylandrostane deriva-
tive 89 and final deprotection of the ethylene ketals.
a
Compounds
R
Na+,K+-ATPase inhibition, IC50
(lM)
Compound D
Oxo
@CH2
Spirocyclopropane
a
0.25
0.57
1.3
0.91
1.2
0.20
0.33
0.69
2.0
48
49
50
51
52
53
54
55
56
57
58
In Scheme 4, the preparations of the exomethylene derivatives
are shown starting from the common intermediate 3,3:17,17-
bis(ethylendioxy)androstane-6-one 59.5 Methyl {3,17-dioxoand-
rostane-6-[(E)-ylidene]}acetate 92 was obtained by reaction of 59
with trimethylphosphonoacetate in DME in the presence of tert-
butyllithium and heating at 110 °C to give the unsaturated ester
91 in 35% yield, followed by deprotection of the latter compound
with PTSA in acetone in 70% yield. 6-[(E)-Ethyliden]androstane-
3,17-dione 94 was obtained in 92% yield by reaction of 59 with
(ethyl)triphenylphosphonium bromide in THF at 0 °C in the pres-
ence of potassium tert-butoxide followed by deprotection of the
ethylene ketals of 93. 6-Difluoromethyleneandrostane-3,17-dione
96 was obtained in 85% yield by reaction of 59 and the anion
generated from diethyl difluoromethylenephosphonate and tert-
butyllithium in DME at ꢀ78 °C; the intermediate 3,3:17,
17-bis(ethylendioxy) derivative 95 was finally deprotected. 3,17-
Dioxoandrostane-6-[(E)-ylidene]acetonitrile 98 was obtained by
reaction of 59 and diethyl cyanomethylphosphonate in the pres-
ence of NaH (60% dispersion in mineral oil) in THF at room temper-
ature to give 97 (71% yield) that in turn was deprotected to afford
98 in 62% overall yield. The nitrile 97 was transformed into the cor-
responding unsaturated aldehyde 99 in 55% yield by reaction with
1 M DIBAH in CH2Cl2 at ꢀ78 °C and following hydrolysis in the
presence of silicagel; the aldehyde 99 was reduced with NaBH4
-Me
-C„CH
a
(E) @NOH
(E) @NOMe
a
a
a
a
a
-CH2OH
-CH2OMe
-NH–CHO
-CONH2
1.1
1.9
4.0
-COOMe
Concentrations able to inhibit 50% of Na+,K+-ATPase enzyme activity; mean of
a
two or three experiments.
O
O
H
H
a
H
R
H
H
R
H
R1O
O
N
H
H
1 - 58
Scheme 1. Reagents and condition: (a) hydroxylamines, THF/water, room
temperature.

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M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
O
O
O
H
H
c
H
H
H
H
O
H
H
O
H
O
O
O
H
H
H
61
62
63
b
R
R
O
O
O
O
a
e
d
H
H
H
H
H
O
H
H
H
O
H
H
H
H
O
R
R
O
O
O
H
O
H
H
H
66 R =
67 R =
64 R =
65 R =
OH
OMe
b
b
O
O
60
59
O
O
f
g
O
R
O
O
h
H
H
H
H
H
S
H
O
H
H
H
O
R
O
O
H
H
H
70 R =
71 R =
O
b
O
O
68
69
N
N
Scheme 2. Reagents and conditions: (a) methyltriphenylphosphonium bromide, potassium tert-butoxide, THF, room temperature; (b) PTSA, acetone, room temperature; (c)
MCPBA, CH2Cl2, 0 °C; (d) BH3.THF complex, THF, 0 °C, then NaOH, H2O2, water, room temperature; (e) NaH, CH3I, THF, 0 °C; (f) CH2I2, Et2Zn, toluene, 60 °C; (g) TCDI, DMAP,
CH2Cl2, 40 °C; (h) Ph3SnH, AIBN, toluene, 110 °C.
O
R
R
O
H
H
f
H
H
e
H
H
H
H
H
H
H
H
O
O
O
R
R
O
74 R =
H
76 R =
H
H
H
COOR1
CHO
O
O
O
O
d
d
78
79 R1 = H
80 R1 = NH2
81 R1 = Me
75 R =
OH
77 R =
g
h
c
d
R
R
O
O
O
O
O
O
a
j
H
H
i
H
H
H
H
b
H
H
H
H
H
H
H
O
H
H
O
O
O
O
R
R
O
H
H
O
H
H
84 R =
85 R =
O
H
82 R =
83 R =
CHO
CHO
d
d
O
O
O
O
HO
OH
MeO
73
72
64
k
R
l
H
H
R
O
O
H
m
H
H
O
R
86 R =
H
H
H
H
H
d
O
O
O
O
R
89 R =
87 R =
H
O
H
88
d
O
O
OTs
90 R =
Scheme 3. Reagents and conditions: (a) IBX, DMSO, room temperature; (b) K2CO3, MeOH, room temperature; (c) methyltriphenylphosphonium bromide, potassium tert-
butoxide, THF, 0 °C; (d) PTSA, acetone, room temperature; (e) BH3.THF complex, THF, 0 °C, then NaOH, H2O2, water, room temperature; (f) 1 N aqueous KMnO4, tert-butanol,
5% aqueous Na2HPO4, room temperature; (g) SOCl2, toluene, 85 °C, then 2 M NH3 THF solution, 0 °C; (h) DMAP, EDAC, MeOH, CH2Cl2, 0 °C; (i) NaBH4, dioxane/H2O 9/1, room
temperature; (j) NaH, CH3I, THF, 0 °C; (k) (chloromethyl)triphenylphosphonium chloride, n-butyllithium, THF, ꢀ78 °C, then heating at 70 °C, then n-butyllithium, ꢀ78 °C; (l)
pTSCl, DABCO, CH2Cl2, 0 °C; (m) NaBH4, DMSO, 80 °C.

M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
4279
R
R
O
O
H
H
O
H
a
91 R =
92 R =
H
H
H
N
H
O
O
H
O
H
O
b
R
R
R
H
H
O
H
OR1
COOMe
H
O
59
93 R =
94 R =
O
H
H
a
O
O
102 R =
103 R =
b
R
R1 = H
c
O
O
O
H
b
O
c
O
R
H
104 R =
105 R =
H
d
R1 = Me
O
O
R
b
b
b
H
O
H
O
H
H
H
F
O
H
O
R
H
95 R =
96 R =
59
O
H
H
CN
O
O
b
106 R =
107 R =
e
R
R1 = Et
H
F
O
O
O
O
110 R =
111 R =
O
O
O
b
H
O
O
b
108 R =
H
H
O
R1 = Allyl
O
O
H
O
H
109 R =
CN
97
H
H
O
Scheme 5. Reagents and conditions: (a) NH2OHꢁHCl or the appropriate O-alkyl
hydroxylamine hydrochloride, Na2HPO4, THF/water, room temperature; (b) PTSA,
acetone, room temperature; (c) toluene-4-sulfonylmethyl isocyanide, potassium
tert-butoxide, DMSO, room temperature.
f
H
CN
98
O
O
H
6b-yl nitrate 121 was obtained starting from 3,3:17,17-bis(ethyl-
R
H
H
O
g
endioxy)androstane-6b-ol5 119. 6
a
-Methoxy derivative 116 was
-ol5 112
O
H
H
obtained from 3,3:17,17-bis(ethylendioxy)androstane-6
a
O
CHO
by reaction with CH3I in the presence of KH in THF at 0 °C (79%
yield) followed by deprotection; the 6b-methoxy derivative 123
was prepared from the epimeric 6b-alcohol 119 by reaction with
CH3I in the presence of NaH in THF at 0 °C in a lower yield (52%),
99
H
H
100 R =
101 R =
O
O
b
R
H
CH2OH
while the 6a-ethoxy derivative 118 was obtained from 112 by
Scheme 4. Reagents and conditions: (a) trimethylphosphonoacetate, tert-butyl-
lithium, DME, 110 °C; (b) PTSA, acetone, room temperature; (c) (ethyl)triphenyl-
phosphonium bromide, potassium tert-butoxide, THF, 0 °C; (d) diethyl
difluoromethylenephosphonate, tert-butyllithium, DME, ꢀ78 °C; (e) diethyl cya-
nomethylphosphonate, NaH (60% dispersion in mineral oil), THF, room tempera-
ture; (f) 1 M DIBAH, CH2Cl2, ꢀ78 °C, then water, THF, silicagel, room temperature;
(g) NaBH4, MeOH, 0 °C.
reaction with EtI and NaH in THF at reflux (49% yield); the depro-
tection of the ethylene ketals gave yields in the 84–95% range.
The preparations of the amides 126 and 128 are shown in
Scheme 7. The key intermediate 3,3:17,17-bis(ethylendioxy)-6a-
aminoandrostane 124 was obtained by reduction of the corre-
sponding 6-(E)-hydroxyiminoandrostane 102 with sodium in n-
propanol at reflux in 53% yield. 6a-Formamidoandrostane-3,17-
dione 126 was obtained in 91% yield by reaction of 124 with formic
acid in CHCl3 and pyridine in the presence of DCC at 0 °C to give
125 followed by deprotection with PTSA in acetone. 6a-Acetami-
doandrostane-3,17-dione 128 was obtained in 90% yield by reac-
tion of 124 with acetic anhydride in pyridine to give 127 which
was then deprotected.
in MeOH at 0 °C to give the allyl alcohol 100 (90% yield); final
deprotection gave 101 in 60% yield.
In Scheme 5, and 6-[(E)-hydroxyimino]androstane-3,17-dione
103 was obtained by reaction of 3,3:17,17-bis(ethylendioxy)andro-
stane-6-one 594 and NH2OHꢁHCl in the presence of Na2HPO4 in
THF/water at room temperature to give the (E)-oxime 102 (93%
yield) that was deprotected with PTSA in acetone at room temper-
ature (70% yield). In the same manner by using the appropriate O-
alkyl hydroxylamine hydrochloride, 6(E)-methoxyiminoandros-
tane-3,17-dione 105 (68%, two steps yield), 6-[(E)-ethoxyimi-
no]androstane-3,17-dione 107 (90%, two steps yield), and 6-[(E)-
allyloxyimino]androstane-3,17-dione 109 (64%, two steps yield),
were obtained. 6a-Cyanoandrostane-3,17-dione 111 was obtained
by reaction of the 6-oxo derivative 59 and toluene-4-sulfonylmeth-
yl isocyanide in the presence of potassium tert-butoxide in DMSO
at room temperature to give 6a-cyano-3,3:17,17-bis(ethylendi-
3. Results and discussion
All target compounds were tested in vitro for their inhibitory
activity on purified dog kidney Na+,K+-ATPase, as measured by
the 32P-ATP hydrolysis method (see data in Tables 1–3).10,11 Some
compounds, showing high inhibitory potency in vitro, were inves-
tigated in vivo for their inotropic activity and lethal effect by slow
intravenous infusion in anesthetized guinea pigs (results reported
in Table 4). Digoxin was chosen as reference compound because it
is the most commonly prescribed cardiac glycoside in the treat-
ment of CHF; Istaroxime is the lead compound in this series of
androstane derivatives and compounds A,5 B,5 C,8 and D8 are re-
ported here for comparison.
oxy)androstane 110 (31% yield) that in turn was deprotected
(75% yield).
In Scheme 6, 3,17-dioxoandrostane-6
tained by esterification of 3,3:17,17-bis(ethylendioxy)androstane-
-ol5 112 with 65% HNO3 in acetic anhydride at 0 °C to give the
a-yl nitrate 114 was ob-
6a
nitrate 113 in 89% yield, which, after deprotection of the ethylene
ketals, gave 114 in 75% yield; analogously, 3,17-dioxoandrostane-
The following structure–activity relationships (SARs) are based
on the in vitro data shown in Tables 1–3. For clarity of discussion,

4280
M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
R
H
O
H
H
113 R =
114 R =
R
O
O
H
b
ONO2
a
O
O
H
R
H
H
O
c
H
O
H
O
O
OH
112
115 R =
H
H
R1 = Me
b
R
H
116 R =
117 R =
O
O
OR1
R1 = Et
O
O
b
118 R =
R
H
H
O
H
120 R =
121 R =
R
O
O
b
H
ONO2
a
O
O
H
R
H
H
O
c
H
O
H
OH
O
H
H
119
122 R =
123 R =
R
O
O
b
H
OMe
Scheme 6. Reagents and conditions: (a) 65% HNO3, acetic anhydride, 0 °C; (b) PTSA, acetone, room temperature; (c) CH3I, NaH, THF, 0 °C or EtI, NaH, THF, reflux.
R
O
O
O
O
H
a
b
H
H
H
H
H
N
H
H
H
O
O
R
H
O
H
O
H
NHCHO
NH2
OH
O
102
124
125 R =
126 R =
O
O
c
d
R
H
H
H
R
H
NHCOCH3
O
127 R =
O
O
c
128 R =
Scheme 7. Reagents and conditions: (a) sodium, n-propanol, reflux; (b) formic acid, DCC, CHCl3 and pyridine, 0 °C; (c) PTSA, acetone, room temperature; (d) acetic anhydride,
pyridine, room temperature.
the different substitutions at position 6 will be treated considering
the three aminoalkyl oxime chains at position 3 separately. The
choice of the three different amines was based on their potencies
and different structures, as reported in a recent study about the
systematic variation of the oximic chain present in Istaroxime.8
The 2-aminoethyl oxime was chosen as reference chain since it is
present in Istaroxime and as example of primary amine with high
potency. The (3R)-3-pyrrolidinyloxyimino chain was used because

M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
4281
Table 4
some cyclic amines on the oximic group at position 3 gave com-
pounds with a higher inhibitory potency than the linear amine
analogues (in particular (E,Z)-3-[(3R)-3-pyrrolidinyloxyimi-
no]androstane-6,17-dione, compound C, was found to be about
four times more potent than Istaroxime) and as example of a sec-
ondary cyclic amine. To complete the series we chose a secondary
linear amine chain on the oximic group in position 3, namely the 3-
N-methylaminopropoxyimino; compound D that carries this chain
is reported with potency about half the value of Istaroxime.
We demonstrated in the preceding papers5,8 that the substitu-
ent at position 6 in Istaroxime plays an important role in the inter-
action with the receptor; referring to the model in which the
rotated E isomer of Istaroxime is superimposed on cassaine12 and
digitoxigenin (Fig. 1a and 1b), the keto group in that position cor-
responds to the keto group at position 7 in cassaine and to the 14b-
hydroxy in digitoxigenin. We have also shown that the reduction of
Inotropic and toxic effects in anesthetized guinea pig
Compound Emaxa% increase EDmax
ED80
Deads/ Lethal
b
c
in dP/dtmax
(
l
mol/kg)
(lmol/kg) treated dose/ED80
Digoxin
Istaroxime 182
6
8
127
0.97
5.7
5.8
9.9
4.3
5.8
17
7.4
18
9.8
27
0.41
1.8
3.1
4.6
0.84
2.4
2.5
1.8
4.3
3.6
2.0
6.7
3.7
1.5
1.9
6.8
1.6
2.2
11
10/10
7/8
0/3
0/3
2/5
0/5
2/3
0/3
1/5
1/5
1/5
0/3
0/3
0/3
0/3
0/3
2/5
0/4
0/4
0/3
0/3
2/2
2/2
3.2
26
—
—
11
—
23
—
9.0
8.0
33
—
—
—
—
—
11
—
—
—
—
203
163
180
183
238
166
167
172
174
244
147
144
183
201
142
109
164
213
158
263
177
9
10
16
17
23
26
32
35
38
39
41
43
45
46
47
50
52
54
56
18
6.0
4.3
5.1
12
6.0
8.8
23
18
15
9.0
2.8
the 6-keto group in Istaroxime (IC50, 0.11
tency and that the decrease is more pronounced for the 6b-hydro-
xy group (compound B, 2.1 M) than for the -hydroxy
(compound A, 0.52 M); an explanation could be that a 6 -hydro-
lM) brings to a lower po-
l
6a
l
a
8.1
7.1
1.5
0.93
xy group places the OH in the same spatial position of the 14b-hy-
droxy in digitoxigenin (Fig. 1d), while the b-hydroxy places the OH
in a spatial position very close to the nonpolar methylene 15 of
digitoxigenin (Fig. 1e). Therefore, it seems that a polar 6-keto or
28
47
a
b
c
Maximal increase in dP/dtmax
Dose inducing maximum positive inotropic effect.
Inotropic potency: dose increasing +dP/dtmax by 80%, calculated from dose–
.
a 6a-hydroxy substituent are of great importance in Istaroxime
while we can assume that there is a space around the 6b position
probably available for nonpolar substituents; the latter space in
digitoxigenin is occupied by the 15 and 16 methylene groups and
response curves.
Figure 1. (a) Structures of rotated E isomer of Istaroxime, cassaine and digitoxigenin. (b) Superposition of rotated E isomer of Istaroxime (light green), cassaine (orange) and
digitoxigenin (dark green). (c) Superposition of rotated E isomer of Istaroxime, cassaine and digitoxigenin with the VDW volumes corresponding to C15 and C16 of
digitoxigenin and C14 of cassaine. (d) Superposition of rotated E isomer of compound A (light green) and digitoxigenin (dark green). (e) Superposition of rotated E isomer of
compound B (light green) and digitoxigenin (dark green).

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M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
in cassaine by the 14a-methyl (see Fig. 1c, in which the cited non-
was substituted with a similar C@N, both in term of geometry
polar groups are surrounded by VDW surface).
To explore the two hypotheses we synthesized derivatives with
nonpolar and polar substituents at position 6.
and electronic distribution; of course the new hydroxyl group
points in a well defined direction (the new oxime is in the E form).
This portion of space in the binding site on the receptor could
accommodate a polar group such as the OH, while the presence
3.1. 3-(2-Aminoethoxyimino) derivatives with nonpolar
substituents at position 6
of a methyl group in 3 negatively impacted on activity (1.1
The introduction of a methyl (10, 0.017 M), ethyl (11, 1.7
and allyl (12, 43
l
l
M).
M),
l
l
M) on the oxime group gave the following infor-
The change of the carbonyl group with an exomethylene in-
mation: a methyl group is well tolerated as the potency is almost
the same as that of the unsubstituted hydroxyl; hence there is no
necessity of a donating hydrogen-bond group, but a receiving
one could be important; the available space fits for a methyl while
an ethyl group and, especially, an allyl group are too bulky (for the
latter the decrease in activity is more than 1700 times when com-
pared with 9). Maintaining a double bond in the substituent, the
creased the inhibitory potency on the pump (1, 0.042
0.11 M for Istaroxime); a difluoroexomethylene (2, 0.19
slightly reduced the activity while the E-ethyliden substituent
sharply reduced it (3, 1.1 M) as well as the b-methyl (5, 6.0 M)
and, to a lesser extent, the spirocyclopropane (4, 0.85 M). -Vinyl
(7, 0.53 M) and -methyl (6, 0.30 M) showed higher potencies
than the b-methyl and the -ethynyl group brought up the activity
at a level similar to that of Istaroxime (8, 0.20 M). An exomethyl-
lM vs
l
lM)
l
l
l
a
l
a
l
a
introduction of a yliden-(E)-acetonitrile (13, 1.7 lM) reduced 70
times the potency in comparison with 9; a very low activity was
l
ene group has some similarities with a ketone, both in term of
shape and in term of electronic distribution; an important differ-
ence is that there is not the possibility to act as a hydrogen-bond
acceptor. In the superposition of the exomethylene derivative 1
and Istaroxime, the new @CH2 group could assume the same posi-
tion of the 6-ketone, but it shows a better interaction with the
receptor notwithstanding its apolarity in comparison with the ke-
tone. This fact could be explained by a slight shift of the position of
the molecule in the receptor (e.g., by rotating around the major
axis of the plane that contains the androstane scaffold; Fig. 2), to
better accommodate the nonpolar substituent in the available non-
polar space described above without losing the other favorable
interactions, the most important being the one due to the basic
chain. The difluoroexomethylene derivative 2 is similar to 1. The
obtained with the introduction of a yliden-(E)-methylacetate (14,
64
lM); a slightly higher activity than that of 13 was shown by
the 2-hydroxy-(E)-ethyliden derivative (15, 0.77
lM). These results
indicate that a polarized double bond in position 6 is important
and the space around the chain is rather limited as the severe fall
of potency of 12 and 14 demonstrates.
Continuing our exploration of the space around the position 6,
we prepared the saturated hydroxy derivatives. The 6
methyl derivative 17, (0.18 M) showed the highest inhibitory po-
tency in this close series; its homologous 6 -(2-hydroxyethyl)
derivative (16, 0.32 M) was slightly more potent than the simple
-hydroxy (compound A, 0.52 M). All these hydroxy derivatives
are more potent than the allylic alcohol 15 (0.77 M); it seems as
though the saturated chains could better fit the space occupied by
the 14b-hydroxy in digitoxigenin. The 6 -methoxymethyl deriva-
tive 19 (0.50 M) decreased the activity: this could be due to the
a-hydroxy-
l
a
l
6a
l
l
reduced potencies of 6b-methyl, 6
a
-methyl and the spirocyclopro-
a
pane derivative (that can be seen as a ‘merge’ of the latter two)
seem to point toward the importance of the presence of an unsat-
urated bond. The same feature is present also in 3, but the extra
methyl group on the double bond is oriented in the opposite direc-
tion with respect to the proposed nonpolar favorable region; this
fact could explain its 25-fold reduced activity compared to that
of 1. The other two compounds carry a double (7) or a triple (8)
bond roughly on the same plane that contains the androstane scaf-
fold; as the more straight alkynyl group gave a higher inhibitory
activity, we can suppose that this zone of the receptor is very selec-
tive in respect to the position of the substituent; again, as for 1, the
molecule could rotate on the major axis of the androstane’s plane,
thus permitting the alkynyl group to reach the nonpolar zone occu-
pied by the D ring of digitoxigenin.
l
increasing space requirement or to the impossibility to act as a
hydrogen-bond donor (moreover, also 14b-OMe digitoxigenin
showed a binding affinity for the Na+,K+-ATPase receptor about
90 times lower than digitoxigenin itself).13 The 6b-epimer of 17
(18, 1.6
for a polar group in the b-face of the androstane scaffold; its O-
methyl ether (20, 3.0 M) further reduced the activity. A similar
decrease in potency was observed for the 6 -methoxy derivative
23 (0.71 M) in comparison to its corresponding hydroxy deriva-
tive compound A; a bulkier ethyl ether (25, 3.1 M) was more det-
lM) confirms the diminished potency already reported
l
a
l
l
rimental. The formal cyclization of the two hydroxymethyl
derivatives to give the corresponding epimeric spirooxiranes gave
practically the same potency (21, 0.91
is no difference in the - or b-face for the two diastereoisomeric
oxiranes, probably because the strained ring reduces the and b-
lM and 22, 0.87 lM); there
a
3.2. 3-(2-Aminoethoxyimino) derivatives with polar
substituents at position 6
a
characteristic of the oxygen; the electronic effect of the oxygen is
negligible since the spirocyclopropane analogue 4 showed a com-
The oxime group at position 6 showed a very interesting incre-
parable potency (0.85
in the -face was confirmed by the two epimeric nitrosyl com-
pounds, 6 -ONO2 (26, 0.33) and 6b-ONO2 (27, 2.9 M).
The introduction of a 6 -carboxylic group gave a very low
inhibitory potency (28, 16 M); the potency increased from the
corresponding -cyano derivative (29, 1.1 M) to the
methoxycarbonyl analogue (30, 0.41 M) and 6 -carboxamide
(31, 0.63 M). As the space occupied by these carboxylic deriva-
lM). A more favorable effect of a polar group
ment of inhibitory activity (9, 0.024
l
M); the double bond C@O
a
a
l
a
l
6a
l
6a-
l
a
l
tives is similar, we can argue that the broad range of activity (about
40 times between 28 and 29) could be explained by the electronic
environment of the substituents, where the negatively charged
carboxylate is hardly recognized by the receptor. The high inhibi-
tion obtained with the carboxamide induced us to prepare the ‘re-
verse’ derivative 6
inhibitory potency; on the other hand the 6
(33, 45 M) fell down of about 280 times.
a
-formamido (32, 0.16
l
a
M) obtaining very high
-acetamido derivative
Figure 2. Superposition of E isomers of Istaroxime (light green) and 1 (gray), the
latter rotated along the major axis of the androstane ring to reach the VDW
volumes. Istaroxime is oriented as in Figure 1.
l

M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
4283
3.3. 3-[(3R)-3-Pyrrolidinyloxyimino]androstane derivatives
higher than the value of the Z isomer: (i) E/Z Istaroxime (0.11
l
M),8
E
isomer of Istaroxime (0.056
(0.63
M);8 (ii) E/Z compound C (0.026
pound C (0.016
Z 3-(2-aminoethoxyimino)androstane-6
isomer (2.0
M)4 and its Z isomer (12.0
l
M),8
Z
l
isomer of Istaroxime
Fourteen 3-[(3R)-3-pyrrolidinyloxyimino]androstane deriva-
tives (see Table 2) were synthesized from the scaffold with the
substituents at position 6 that gave the highest inhibitions when
coupled with the Istaroxime’s oximic chain. Comparing the couples
of compounds with the pyrrolidinyloxy versus aminoethoxy chain,
in 10 of 14 cases the potencies were increased for the cyclic mem-
bers, ranging from 1.5 to about 9 times; in the remaining four cases
there was no change or a very slight decrease. These results are in
agreement with what was observed in the case of Istaroxime and
its cyclic analogue.
l
M),8 E isomer of com-
l
M),8 Z isomer of compound C (0.25 M);8 (iii) E/
l
a
,17b-diol (1.6
l
M),5 its E
l
l
M).5 The inhibitory values
used in the 3D-QSAR analyses are those reported in Tables 1–3, in
their ꢀlog form (pIC50).
Molecules for 3D-QSAR were prepared with the following pro-
cedure. The 3D model of each compound in its E form was con-
structed in MACROMODEL 9.514 and submitted to a cycle of
minimization, conformational analysis and again minimization
with GB/SA continuum model to simulate water solvation (see Sec-
tion 5); a series of conformers for each molecule (all the conform-
ers in an energy range of 3.0 kcal molꢀ1 or the first 30 conformers
for the more flexible molecules) was saved for the subsequent step
of alignment and superposition. The molecules were obviously
aligned on the rigid steroidal skeleton, but the concern regarded
the oxime chain in position 3. The E isomer of compound C was
considered as the driving molecule in this alignment because it is
one of the derivatives of this series with the highest inhibitory po-
tency and has a cyclic amine on the oximic chain, with a smaller
number of conformers in comparison with a linear amino alkyl
chain; the conformer of compound C used was that with the best
superposition with the conformers of Istaroxime (rms). The other
molecules of Tables 1 and 2 were aligned with the respective con-
former that gave the best superposition with the said conformer of
compound C (Fig. 3a). compound D was used as a reference to align
the compounds of Table 3, with a longer aminoalkyl chain. In order
to decide the conformer to be used, compound D was aligned with
cassaine (superimposed with Istaroxime in Fig. 1) to choose its
conformer that placed the amino head closer to that of cassaine
while its alkylene chain was better superposed on the portion
CH–CH2–N of the 3-(R)-pyrrolidinyl ring of compound C (Fig. 3b).
Again, the other molecules of Table 3 were aligned with the respec-
tive conformer that gave the best superposition with compound D.
In SYBYL 8.0 software suite15 the selected conformers were
superposed using the ‘align database’ routine with the E isomer
of compound C as ‘template molecule’ and the androstane scaffold
depleted of the hydrogen atoms, with C@O in position 17, C@N in 3
and without substituents in 6 as ‘common substructure’ (the 63
compounds aligned are reported in Fig. 4); the atom charge for
the aligned molecules were recalculated applying the Gasteiger-
Huckel method and the molecules were imported in the DRAGON
5.016 environment for the calculation of more than 1600 molecular
descriptors.17 Descriptors with constant and near-constant values
and with a pair correlation of 0.95 were discarded; the remaining
494 descriptors were imported in SIMCA-P 11.518 for statistical
analysis. Principal component analysis (PCA) technique was ap-
plied obtaining a matrix in which the descriptors are expressed
in term of scores (t) and loadings (p). Scores t1, t2, etc. are new
3.4. 3-(3-N-Methylaminopropoxyimino) androstane derivatives
As explained above, 11 3-(3-N-methylaminopropoxyimino)
androstane derivatives were synthesized (see Table 3) to complete
the series. A comparison of the couples N-methylaminopropoxy-
versus aminoethoxy-chain shows that in 8 of 11 cases a diminished
potency of more than threefold was obtained, with a maximum of
about 19 times when 53 (0.33
lM) is compared with 10
(0.017 M). These results are in agreement with what was seen
l
in the case of Istaroxime and its N-methylaminopropoxy analogue
(the former was twice as potent as the secondary linear amine),
even though the reduction is sometimes greater than that found
for the parent couple.
3.5. 3D-Quantitative structure–activity relationship
3D-Quantitative structure–activity relationship (3D-QSAR)
studies have been performed on this series of digitalis-like
Na+,K+-ATPase inhibitors to develop models that can bring together
the different classes of molecules, explain and predict the inhibi-
tory activity found on the Na+,K+-ATPase. Comparative molecular
field analysis (CoMFA) and comparative molecular similarity indi-
ces analysis (CoMSIA) were performed on a total of 63 molecules
from which a training set of 44 compounds and a test set of 19
compounds were selected using the chemical descriptors obtained
with the software DRAGON and applying a multivariate analysis on
them with the aid of software SIMCA-P.
We performed the 3D-QSAR on the E isomers of the molecules,
by assigning the experimental data of the E/Z mixture as if they
were the values of the E isomers, based on the consideration that
the experimental inhibitory value of a mixture is closer to the value
hypothetically obtainable with the pure E isomer (the E isomer is
the one with the highest activity in a couple of E and Z isomers,
as predicted by our model and experimentally demonstrated).5,8
This assertion is supported by comparison of the few available
experimental inhibitory data of the correlated E/Z mixture, the
pure E and the Z isomers. In the following examples, both the inhi-
bition value of the E isomer and the one of the mixture are always
Figure 3. (a) Superposition of E isomers of Istaroxime (light green) and compound C (dark green). (b) Superposition of cassaine (orange) and E isomers of compound C (dark
green) and compound D (yellow).

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M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
Figure 4. The 63 molecules of training and test sets superposed as for the 3D-QSAR analyses.
Figure 5. (a) PCA score plot of first component (t1) versus second component (t2). The dots represented the molecules are colored according to Table 1 (black), Table 2 (red)
and Table 3 (blue). (b) PCA 3D score plot of first component (t1), second component (t2) and third component (t3); dots colored as previously. (c) PCA score plot of first
component (t1) versus second component (t2). The dots are colored according to training (black) and test (red) sets.
variables summarizing the X variables (the descriptors); they are
orthogonal, that is, completely independent of each other. The
score t1 (first component) explain the largest variation of the X
space, followed by t2, etc. Hence, the scatters plot of t1 versus t2

M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
4285
displays how the molecules are situated with respect to each other
in the chemical space. Molecules that are close to each other are
similar; molecules far away from each other are dissimilar. By
means of PCA the scores t1 and t2 were able to separate to some
extent the three classes of compounds, that is, the compounds
belonging to the three tables (Fig. 5a), and even a better and almost
complete separation among the three classes was obtained in the
3D graphic in which also the t3 component was added (Fig. 5b).
This partition among the different classes of compounds indicates
that the molecular descriptors obtained with DRAGON and elabo-
rated with SIMCA-P were able to extract the ‘chemical nature’ of
ing set and of the test set are reported in Figure 6 and, in tabulated
form, in Supplementary data. Referring to the results for the test
set, 16 compounds of 19 are predicted within a range less than
one order of magnitude (and 9 of these 16 are less than 0.5) giving
an adequate result taking into consideration the compelled
assumption of assigning the experimental data of the E/Z mixture
to the E isomers, thus probably underestimating the ‘real’ inhibi-
tory potencies of the E isomers. The three compounds with an error
in the prediction greater than one (in the oval in Fig. 6) are all pre-
dicted more potent than the experimental value and all belong to
the first class of compounds (Table 1; bearing the 2-aminoethoxyi-
mino chain); the substituents in position 6 present in these three
derivatives, for the low activity that they convey to the com-
pounds, are not represented in the other two classes and for that
reason they are not well characterized in the 3D-QSAR model.
The CoMFA steric and electrostatic contour plots for all 63 mol-
ecules are displayed in Figure 7. Significant green contours sur-
the molecules. Only one molecule (Table 2, 44, 6a-ONO2) is a weak
outlier, being outside the Hotelling T2 circle describing the 95%
confidence region of the scores’ graph. With the aid of this graph
we manually selected a test set of 19 compounds (about 30% of
the entire dataset), taking care to choose representative com-
pounds in term of position in the X space, activity on Na+,K+-ATP-
ase (expressed as pIC50) and presence of the three different oximic
chains; the remaining 44 compounds constituted the training set
(Fig. 5c). The training set was used in the CoMFA and CoMSIA anal-
yses to build a predictive model of the inhibition of the Na+,K+-ATP-
ase; the test set was used to validate the model.
3D-QSAR analyses were performed with the tools included in
SYBYL 8.0. In the CoMFA method, a relationship is seeked among
the biological activities of a set of compounds and their electro-
static and steric properties.19,20 The molecules under study are
superposed in their putative bioactive conformations and then
placed in a box of a regularly spaced grid of 2.0 Å. The lattice
was extended to 4 Å beyond the van der Waals volume of each
molecule in the X, Y, and Z directions. A sp3 carbon atom of radius
1.52 Å and charge +1.0 was used as a probe to calculate both the
steric and electrostatic fields at each node of the grid. A 3D-QSAR
model has been derived from partial least-squares (PLS)21 linear
regression that permits to correlate the inhibitory activities
(pIC50; the values span over more than three orders of magnitude)
with CoMFA values. The optimum number (N) of PLS components,
corresponding to the smallest standard error of prediction (SEP),
was determined by the leave-one-out (LOO) cross-validation pro-
cedure, applying a column filter of 2.0 kcal molꢀ1 so that only the
steric and electrostatic energies with values greater than
2.0 kcal molꢀ1 were considered in the PLS analysis. The optimal N
obtained was used in a non-cross-validated PLS analysis to get
the model parameters such as correlation coefficient (r2), SEE,
and F value. Good statistical results were obtained from the train-
ing set of 44 molecules as reported in Table 5. From this analysis
the predictions of the Na+,K+-ATPase inhibitory values of the train-
Figure 6. CoMFA predicted Na+,K+-ATPase inhibition of training (cyan) and test
(red) sets. In addition to the line of ideal correlation, dotted lines are given,
indicating deviations of 1 logarithmic unit. Outliers are labeled by their compound
numbers.
Table 5
CoMFA and CoMSIA statistical results
Statistical parameters
CoMFA
SE
CoMSIA
SEHDA
SEDA
SEHA
SEHD
SEA
0.52
0.49
0.82
0.30
28.61
6
SED
0.57
0.46
0.85
0.27
SEH
0.58
0.45
0.86
0.26
45.30
5
SE
0.57
0.45
0.78
0.32
34.84
4
q2
SEP
r2
SEE
F
0.68
0.39
0.93
0.18
98.20
5
0.43
0.53
0.85
0.28
33.62
6
0.44
0.53
0.83
0.29
30.20
6
0.53
0.48
0.84
0.29
33.19
6
0.55
0.48
0.85
0.27
35.63
6
34.9
6
N
Contributions
S
E
H
D
A
0.64
0.36
0.09
0.25
0.12
0.36
0.18
0.09
0.29
0.15
0.36
0.21
0.10
0.31
0.15
0.44
0.20
0.46
0.13
0.35
0.22
0.48
0.30
0.33
0.67
0.40
0.22
0.52
0.28
0.34
a
q2, Leave-one-out (LOO) cross-validated correlation coefficient; SEP, standard error of prediction; r2, non-cross-validated correlation coefficient; SEE, standard error of
estimate; F, F-test value; N, optimum number of components; S, steric field; E, electrostatic field; H, hydrophobic field; D, hydrogen-bond donor field; A, hydrogen-bond
acceptor field.

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M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
Figure 7. CoMFA standard deviation coefficient steric and electrostatic contour plots; all the 63 molecules of training and test sets are displayed for reference. Sterically
favored areas (contribution level 80%) are represented by green polyhedral. Sterically disfavored areas (contribution level 20%) are represented by yellow polyhedral. Positive
charge favored areas (contribution level 80%) are represented by blue polyhedral. Negative charge favored areas (contribution level 20%) are represented by red polyhedral.
rounding the heterocyclic rings of the oximic chain and the space
under the substituent in position 6 (i.e., toward the -face of the
culations on the aligned molecules of the training set and
validated with the 19 molecules of the test set. Five different sim-
ilarity fields are calculated: steric, electrostatic, hydrogen-bond do-
nor, hydrogen-bond acceptor, and hydrophobic. Maintaining the
steric and electrostatic fields, we systematically explored the com-
bination with the other three fields: the PLS statistical values ob-
tained for each model are reported in Table 5. Compared to
CoMFA, no one of the CoMSIA’s models performed better, conse-
quently we do not use these results to predict the inhibitory activ-
ities of the molecules, but simply to obtain a representation of the
fields around the molecules, especially for those not present in
CoMFA. The CoMSIA’s steric and electrostatic contours were found
to be comparable to the corresponding CoMFA contours even if in
reverse percentage; since these contours were discussed above, the
following section will deal with the CoMSIA H-bond donor and
acceptor contours as well as the CoMSIA hydrophobic contours of
the three different models: SEA, SED, and SEH (Fig. 8). In SEA and
SED models the accepting and donating contours describe regions
of space where hydrogen-bond donors or acceptors on the putative
Na+,K+-ATPase receptor will enhance binding affinity.23 These
fields are created using extension points from acceptors and do-
nors from the ligands into space where supposed corresponding
groups would lie on the receptor: the acceptor field contains infor-
mation about where hydrogen-bond donating groups should be on
the receptor; the donor field describes where hydrogen-bond
acceptor groups should be located on the receptor. These results
can be seen as an indication of an existing correlation between
the activities of the molecules of the training set and the features
accepting or donating H bonds present on the compounds. In Fig-
ure 8a the contour maps of hydrogen-bond acceptor field in the
CoMSIA model SEA are shown; the magenta contour, acceptor fa-
vored, encloses areas where acceptor hydrogen-bonds on the li-
a
steroid scaffold), represent favored steric areas to increase inhibi-
tion of the Na+,K+-ATPase; the small green area located in front
of the position 6 is less significant. The green area around the het-
erocyclic rings supports the evidence that most of the compounds
of Table 2 with this feature showed enhanced Na+,K+-ATPase inhib-
itory potency than the corresponding compounds with a linear oxi-
mic chain. Examples of compounds that locate the substituents in
6
in the green area below that position are 42
(
a
-CH2OH,
M).
0.026 M), 17 ( -CH2OH, 0.18 M), and 47 ( -COOMe, 0.16 l
l
a
l
a
A yellow contour above the substituent in position 6 (i.e., toward
the b-face of the steroid scaffold) suggests that bulkier substituents
in 6b or spanning towards the b-face, are close to the sterically
unfavorable yellow region and therefore are detrimental for the
inhibitory activity as demonstrated by compounds like 12 (NOAl-
lyl, 43
exhibit lower inhibitory activities than the corresponding ana-
logues 10 (@NOMe, 0.017 M), 19 ( -CH2OMe, 0.50 M), and 26
-ONO2, 0.33 M) which have groups at position 6 that do not
lM), 20 (b-CH2OMe, 3.0 lM), and 27 (b-ONO2, 2.9 lM) that
l
a
l
(a
l
span in the yellow region. Taking into account that electrostatic
characteristics report for about one third of the CoMFA model, sig-
nificant blue contours can be found above the position 6 and on the
axis of the 6a position of the androstane scaffold and represent re-
gions where positively charged substituents favor the Na+,K+-ATP-
ase inhibitory activity. The blue region above the position 6
receives the alkyl part of the 6-substituents of some compounds
(20, b-CH2OMe, 3.0
the blue region below the position 6 accepts compounds like 38 (
C„CH, 0.11 M) and 47 ( -COOMe, 0.16 M). A single small red
lM; 5, b-Me, 6.0 lM; 11, @NOEt, 1.7 lM) while
a
-
l
a
l
contour, where negatively charged substituents favor activity, is
located above position 6 close to the disfavored steric yellow re-
gion; the red region accounts for only a little contribution on the
activity; in fact compounds that put substituents with a partial
negative charge in this region showed relatively low potencies be-
cause they are not able to counteract the negative steric effect
gands are expected to enhance activity; it lies primarily in the 6
region reflecting the high potency of compounds having this kind
of substituents as for those of 30 and 47 ( -COOMe; 0.41 and
0.16 M, respectively), 19 and 43 ( -CH2OMe; 0.50 and 0.32 M,
respectively). The two red contours, acceptor disfavored, are lo-
cated in the 6b-face and on the axis of the 4 position; in the 6b
a
a
l
a
l
pointed out by the yellow region: 24 (b-OMe, 1.5
ONO2, 2.9 M); and 12 (@NOAllyl, 43 M).
lM); 27 (b-
l
l
a
CoMSIA22 is an extension of the CoMFA method and both are
based on the assumption that changes in binding affinities of li-
gands are related to changes in molecular properties, represented
by fields. They differ only in the implementation of the fields; in
CoMSIA Gaussian-type distance dependence was applied to mea-
sure the relative attenuation of the field position of each atom in
the grid leading to a smoother sampling of the fields around the
molecules when compared to CoMFA. CoMSIA analysis was per-
formed with the same lattice box as that used for the CoMFA cal-
region hydrogen-bond acceptors on ligands are expected to reduce
activity as demonstrated by relatively low potency of compounds
like 20 (b-CH2OMe, 3.0
red region close to 4 probably accounts for the very high potency
of compounds having a 6-oxo substituent (Istaroxime, 0.11 M and
compound C, 0.026 M) from which an acceptor extension point in
lM) or 27 (b-ONO2, 2.9 lM). The smaller
a
l
l
this direction originates; therefore lack of this feature reduces the
potency. In Figure 8b the contour maps of hydrogen-bond donor
field in the CoMSIA model SED are shown; the cyan contours, do-

M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
4287
Figure 8. (a) CoMSIA standard deviation coefficient hydrogen-bond acceptor contour plots; all the 63 molecules of training and test sets are displayed for reference. Favored
areas (contribution level 80%) are represented by magenta polyhedral. Disfavored areas (contribution level 20%) are represented by red polyhedral. (b) CoMSIA standard
deviation coefficient hydrogen-bond donor contour plots; all the 63 molecules of training and test sets are displayed for reference. Favored areas (contribution level 80%) are
represented by cyan polyhedral. Disfavored areas (contribution level 20%) are represented by purple polyhedral. (c) CoMSIA standard deviation coefficient hydrophobic
contour plots; all the 63 molecules of training and test sets are displayed for reference. Favored areas (contribution level 80%) are represented by violet polyhedral. Disfavored
areas (contribution level 20%) are represented by gray polyhedral.
nor favored (H-bond acceptor on the receptor), cover four regions:
the two close to the oximic chains explain the high activity of mol-
ecules with a 2-aminoethoxyimino or (3R)-3-pyrrolidinyloxyimino
chains (9, 10, 39, and 40), that in these regions project the donor
extension points originated from the amine groups; the small re-
philes favored), are located in the 6a region and in the proximity
of the pyrrolidinyl ring on the oximic chain: the former is ex-
plained by the hydrophilic effect of substituents like those of com-
pounds with high potency as for 16 and 41 (
0.058 M, respectively), 17 and 42 ( -CH2OH; 0.18 and 0.026
respectively), 26 and 44 ( -ONO2; 0.33 and 0.38 M, respectively);
a-CH2CH2OH; 0.32 and
l
a
lM,
gion near position 4 is expression of the 6
present in 17, 42, and 54; the region in 6 position receives the do-
nor extension points of compounds like 16 and 41 (6 -CH2CH2OH),
17 and 42 (6 -CH2OH). The single small purple contour, donor dis-
a
-CH2OH substituents
a
l
a
the latter is entirely due to the compounds of Table 3 present in the
training set, that, bearing the 3-N-methylaminopropoxyimino
chain, do not have a nitrogen atom in this ‘gray region’ and there-
fore account for a hydrophobic environment negatively correlated
to the potency.
a
a
favored, is hardly explained, but probably can be attributable to the
compound of the 2-aminoethoxyimino class in the training set
with very low activity and that has a donor extension point in
the vicinity, namely 12. In Figure 8c the contour maps of hydro-
phobic fields in the CoMSIA model SEH are shown. The violet con-
tour, hydrophobes favored (or hydrophiles disfavored), lies on the
axis of a double bonded substituent in position 6 and can be ratio-
nalized by contribute of high potency compounds like 2 (@CF2,
3.6. In vivo activity in guinea pigs
Some compounds showing high inhibitory potency on the
Na+,K+-ATPase were investigated in vivo in the anesthetized guinea
pig (Table 4). Even though none of the tested compounds showed
inotropic potencies (ED80) comparable to digoxin, all displayed
safety ratios higher than digoxin, and even higher than Istaroxime,
0.19
0.22
l
l
M), 34 (@CH2, 0.046
M). The gray contours, hydrophobes disfavored (or hydro-
lM), and 36 (spirocyclopropane,

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M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
as for 32, 54, and 56. Further, many compounds showed not deter-
minable lethal dose/ED80 ratios, since no animals died (although a
small number of animals was used). The reported ability of low di-
goxin concentrations to stimulate Ca2+ release from the sarcoplas-
mic reticulum24 may partly account for its proarrhythmic
properties. The contribution of other mechanisms, such as Sarco/
Endoplasmic Reticulum Calcium ATPase Isoform 2a (SERCA2a) in-
volved in the lower proarrhythmogenicity of Istaroxime,25 was
not investigated on these compounds.
It was not possible to find particular substituents conferring
higher safety to the compounds: only a trend might be attributable
to the 6a-NHCHO group, even though with a high variability (com-
pounds 32, 45, and 56, with lethal dose/ED80 ratios 33, 11, and 47,
respectively).
5% aqueous NaHCO3 and acetone was evaporated. The aqueous
suspension was extracted with CH2Cl2 (3 ꢂ 50 mL). The combined
organic extracts were washed with H2O, dried over Na2SO4 and
evaporated to dryness.
5.3. Target compounds 1–58
5.3.1. (E,Z)-3-(2-Aminoethoxyimino)-6-methyleneandrostane-
17-one hydrochloride (1)
Prepared in 90% yield from 6-methyleneandrostane-3,17-dione
61 and 2-aminoethoxyamine dihydrochloride.26 1H NMR
(300 MHz, DMSO-d6, ppm from TMS; the specific E and Z signal
attributions were not assigned): d 8.08 (br b, 3H, NH3þ), 4.83 (br
s, 0.5H, C@CH2), 4.80 (br s, 0.5H, C@CH2), 4.52 (br s, 0.5H, C@CH2),
4.49 (br s, 0.5H, C@CH2), 4.09 (m, 2H, CH2–O), 0.77 (s, 3H, CH3),
0.75 (s, 3H, CH3); white solid. Anal. (C22H34N2O2ꢁHCl) C, H, Cl, N.
4. Conclusions
The compounds reported in this paper confirm the interest in
the androstane skeleton, with a proper basic substituent at posi-
tion 3 as a pharmacophore for positive inotropic activity, and dif-
ferent groups at position 6. Such compounds have the desirable
advantage of a higher safety as compared to digoxin, the classic
digitalis compound currently used for the treatment of congestive
heart failure as inotropic agent.
The 3D-QSAR analysis using CoMFA and CoMSIA methods have
been successfully applied to a set of 63 androstane derivatives as
Na+,K+-ATPase inhibitors. The contour plots provide many useful in-
sights into relationships between structural features and inhibitory
activity. These features could be used to expand the class exploring
different positions of the androstane scaffold, such as 5 and 7.
5.3.2. (E,Z)-3-(2-Aminoethoxyimino)-6-difluoromethylene-
androstane-17-one hydrochloride (2)
Prepared in 61% yield from 6-difluoromethyleneandrostane-
3,17-dione 96 and 2-aminoethoxyamine dihydrochloride. 1H
NMR (300 MHz, DMSO-d6, ppm from TMS): d 7.62 (br b, 3H,
NH3þ), 4.07 (m, 2H, CH2–O), 3.25 (m, 0.5H, H-4eq Z isomer), 3.07
(m, 0.5H, H-2eq E isomer), 3.01 (m, 2H, CH2–N), 0.89 (s, 3H, CH3),
0.78 (s, 3H, CH3); white solid. Anal. (C22H32F2N2O2ꢁHCl) C, H, Cl, N.
5.3.3. (E,Z)-3-(2-Aminoethoxyimino)-6-[(E)-ethyliden]
androstane-17-one hydrochloride (3)
Prepared in 71% yield from 6-[(E)-ethyliden]androstane-3,17-
dione 94 and 2-aminoethoxyamine dihydrochloride. 1H NMR
(300 MHz, DMSO-d6, ppm from TMS; the specific E and Z signal
attributions were not assigned): d 8.01 (br b, 3H, NH3þ), 5.01 (q,
0.5H, C@CH), 4.97 (q, 0.5H, C@CH), 4.08 (m, 2H, CH2–O), 2.69 (m,
1H, H-7eq), 0.77 (s, 3H, CH3), 0.72 (s, 3H, CH3); white solid. Anal.
(C23H36N2O2ꢁHCl) C, H, Cl, N.
5. Experimental section
5.1. General details
General experimental details regarding chemistry are reported
elsewhere.26 Compound purity was assessed by 1H NMR and ele-
mental analysis (reported as Supplementary data).
5.3.4. (E,Z)-3-(2-Aminoethoxyimino)-6b-methylandrostane-17-
one hydrochloride (4)
Prepared in 64% yield from 6b-methylandrostane-3,17-dione 71
and 2-aminoethoxyamine dihydrochloride. 1H NMR (300 MHz,
DMSO-d6, ppm from TMS): d 7.89 (br b, 3H, NH3þ), 4.06 (m, 2H,
CH2–O), 3.07 (m, 0.5H, H-2eq E isomer), 3.02 (m, 2H, CH2–N),
2.81 (m, 0.5H, H-4eq Z isomer), 0.96 (s, 1.5H, CH3), 0.95 (s, 1.5H,
CH3), 0.91 (d, 1.5H, CH3), 0.90 (d, 1.5H, CH3), 0.81 (s, 3H, CH3);
white solid. Anal. (C23H36N2O2ꢁHCl) C, H, Cl, N.
5.2. Chemistry
5.2.1. General procedure for the reaction of Scheme 1
To a stirred solution of the appropriate ketone (1 equiv) in THF
(0.15 M), a solution of the appropriate hydroxylamine dihydro-
chloride (1 equiv) in H2O (0.30 M) was rapidly added dropwise.
After 1.5 h, NaCl (7.5 equiv) was added and the mixture stirred
for 10 min. The phases were separated and the aqueous phase ex-
tracted with THF (2 ꢂ 50 mL). The combined organic extracts were
dried over Na2SO4, filtered and evaporated to dryness.
5.3.5. (E,Z)-3-(2-Aminoethoxyimino)-6-(spirocyclopropane)
androstane-17-one hydrochloride (5)
Prepared in 92% yield from 6-(spirocyclopropane)androstane-
3,17-dione 68 and 2-aminoethoxyamine dihydrochloride. 1H
NMR (300 MHz, DMSO-d6, ppm from TMS): d 7.88 (br b, 3H,
NH3þ) 4.06 (m, 2H, CH2–O), 3.06 (m, 0.5H, H-4eq Z isomer), 3.00
(m, 2H, CH2–N), 2.70 (m, 0.5H, H-2eq E isomer), 0.96 (s, 3H, CH3),
0.79 (s, 3H, CH3), 0.57–0.16 (m, 4H, cyclopropane); white solid.
Anal. (C22H36N2O2ꢁHCl) C, H, Cl, N.
5.2.2. Fumarate salts
The crude product was purified by flash chromatography (SiO2,
CHCl3/MeOH/26% NH4OH 90/10/1). To the concentrated fractions
containing the pure compound a stoichiometric amount of fumaric
acid in MeOH was added. After addition of a 1/1 mixture of EtOAc/
Et2O, the precipitate was filtered.
5.3.6. (E,Z)-3-(2-Aminoethoxyimino)-6a-methylandrostane-17-
5.2.3. Hydrochloride salts
one hydrochloride (6)
The crude product was triturated with EtOAc/Et2O and filtered.
Prepared in 83% yield from 6a-methylandrostane-3,17-dione
90 and 2-aminoethoxyamine dihydrochloride. 1H NMR (300 MHz,
DMSO-d6, ppm from TMS): d 7.83 (br b, 3H, NH3þ), 4.07 (m, 2H,
CH2–O), 3.16 (m, 0.5H, H-4eq Z isomer), 3.06 (m, 0.5H, H-2eq E iso-
mer), 3.00 (m, 2H, CH2–N), 0.89 (s, 1.5H, CH3), 0.87 (s, 1.5H, CH3),
0.84 (d, 1.5H, CH3), 0.81 (d, 1.5H, CH3), 0.78 (s, 3H, CH3); white so-
lid. Anal. (C22H36N2O2ꢁHCl) C, H, Cl, N.
5.2.4. General procedure for the hydrolysis of the 3,3:17,17-
bis(ethylendioxy) groups to give the 3,17-Diketo derivatives
A solution of 3,3:17,17-bis(ethylendioxy) derivative (1.0 mmol)
and PTSAꢁH2O (4.8 mmol) in acetone (40 mL) was stirred at room
temperature for 3 h. The solution was neutralized by addition of

M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
4289
5.3.7. (E,Z)-3-(2-Aminoethoxyimino)-6
one hydrocloride (7)
a
-vinylandrostane-17-
1H NMR (300 MHz, DMSO-d6, ppm from TMS): d 8.07 (br b, 3H,
NH3þ), 5.26 (br s, 1H, C@CH-CN), 4.08 (m, 2H, CH2–O), 3.11 (m,
0.5H, H-2eq E isomer), 3.07 (m, 0.5H, H-4eq Z isomer), 3.02 (m,
2H, CH2–N), 2.82 (m, 1H, H-7eq), 0.79 (s, 3H, CH3), 0.75 (s, 3H,
CH3); white solid. Anal. (C23H33N3O2ꢁHCl) C, H, Cl, N.
Prepared in 90% yield from 6a-vinylandrostane-3,17-dione 75
and 2-aminoethoxyamine dihydrochloride. 1H NMR (300 MHz,
DMSO-d6, ppm from TMS): d 7.95 (br b, 3H, NH3þ), 5.51 (m, 1H,
C@CH), 4.98 (m, 2H, C@CH2), 4.05 (m, 2H, CH2–O), 3.06 (m, 0.5H,
H-2eq E isomer), 3.01 (m, 2H, CH2–N), 2.97 (m, 0.5H, H-4eq Z iso-
mer), 0.91 (s, 1.5H, CH3), 0.90 (s, 1.5H, CH3), 0.78 (s, 3H, CH3); white
solid. Anal. (C22H36N2O2ꢁHCl) C, H, Cl, N.
5.3.14. Methyl {3-{(E,Z)-[2-aminoethoxyimino]}-17-
oxoandrostane-6-[(E)-ylidene]}acetate hydrochloride (14)
Prepared in 87% yield from methyl {3,17-dioxoandrostane-6-
[(E)-ylidene]}acetate 92 and 2-aminoethoxyamine dihydrochlo-
ride. 1H NMR (300 MHz, DMSO-d6, ppm from TMS; the specific E
and Z signal attributions were not assigned): d 8.09 (br b, 3H,
NH3þ), 5.45 (s, 0.5H, C@CH), 5.41 (s, 0.5H, C@CH), 4.10 (m, 2H,
CH2–O), 3.94 (m, 1H, H-7eq), 3.62 (s, 3H, CH3–O), 0.77 (s, 3H,
CH3), 0.75 (s, 3H, CH3); white solid. Anal. (C24H36N2O4ꢁHCl) C, H,
Cl, N.
5.3.8. (E,Z)-3-(2-Aminoethoxyimino)-6a-ethynylandrostane-17-
one hydrochloride (8)
Prepared in 76% yield from 6a-ethynylandrostane-3,17-dione
87 and 2-aminoethoxyamine dihydrochloride. 1H NMR (300 MHz,
DMSO-d6, ppm from TMS; the specific E and Z signal attributions
were not assigned): d 7.90 (br b, 3H, NH3þ), 4.06 (m, 2H, CH2–O),
2.98 (d, 0.5H, C„CH), 2.97 (d, 0.5H, C„CH), 0.88 (s, 1.5H, CH3),
0.87 (s, 1.5H, CH3), 0.77 (s, 3H, CH3); white solid. Anal.
(C23H34N2O2ꢁHCl) C, H, Cl, N.
5.3.15. (E,Z)-3-(2-Aminoethoxyimino)-6-{(E)-[2-
hydroxyethylidene]}androstane-17-one hydrochloride (15)
Prepared in 70% yield from 6-[(E)-2-hydroxyethylidene]andro-
stane-3,17-dione 101 and 2-aminoethoxyamine dihydrochloride.
1H NMR (300 MHz, DMSO-d6, ppm from TMS): d 7.75 (br b, 3H,
NH3þ), 5.08 (br t, 0.5H, C@CH), 5.05 (br t, 0.5H, C@CH), 4.56 (t,
0.5H, OH), 4.53 (t, 0.5H, OH), 4.12–3.92 (m, 4H, CH2–O, CH2–O),
3.09 (m, 0.5H, H-2eq E isomer), 3.05 (m, 2H, CH2–N), 3.02 (m,
0.5H, H-4eq Z isomer), 2.65 (m, 1H, H-7eq), 0.77 (s, 3H, CH3),
0.75 (s, 3H, CH3); white solid. Anal. (C23H36N2O3ꢁHCl) C, H, Cl, N.
5.3.9. (E,Z)-3-(2-Aminoethoxyimino)-6-[(E)-hydroxyimino]
androstane-17-one hydrochloride (9)
Prepared in 70% yield from 6-[(E)-hydroxyimino]androstane-
3,17-dione 103 and 2-aminoethoxyamine dihydrochloride. 1H
NMR (300 MHz, DMSO-d6, ppm from TMS): d 10.58 (s, 0.5H, N–
OH), 10.51 (s, 0.5H, N–OH), 7.98 (m, 3H, NH3þ), 4.08 (m, 2H,
CH2–O), 3.29 (m, 1H, H-7eq), 3.13 (m, 0.5H, H-4eq Z isomer),
3.10 (m, 0.5H, H-2eq E isomer), 3.02 (m, 2H, CH2–N), 0.79 (s, 6H,
CH3, CH3); white solid. Anal. (C21H33N3O3ꢁHCl) C, H, Cl, N.
5.3.16. (E,Z)-3-(2-Aminoethoxyimino)-6a-(2-hydroxyethyl)
androstane-17-one hydrochloride (16)
5.3.10. (E,Z)-3-(2-Aminoethoxyimino)-6-[(E)-methoxyimino]
androstane-17-one hydrochloride (10)
Prepared in 85% yield from 6a-(2-hydroxyethyl)androstane-
3,17-dione 77 and 2-aminoethoxyamine dihydrochloride. 1H
NMR (300 MHz, DMSO-d6, ppm from TMS): d 7.95 (br b, 3H,
NH3þ), 4.35 (br, 1H, OH), 4.08 (m, 2H, CH2–ON), 3.42 (m, 2H,
CH2–O), 3.22 (m, 0.5H, H-4eq Z isomer), 3.06 (m, 0.5H, H-2eq E iso-
mer), 3.02 (m, 2H, CH2–N), 0.88 (s, 1.5H, CH3), 0.87 (s, 1.5H, CH3),
0.78 (s, 3H, CH3); white solid. Anal. (C23H38N2O3ꢁHCl) C, H, Cl, N.
Prepared in 60% yield from 6-[(E)-methoxyimino]androstane-
3,17-dione 105 and 2-aminoethoxyamine dihydrochloride. 1H
NMR (300 MHz, DMSO-d6, ppm from TMS): d 8.03 (br b, 3H, NH3þ),
4.09 (m, 2H, CH2–O), 3.75 (s, 1.5H, CH3–O), 3.73 (s, 1.5H, CH3–O),
3.19 (m, 1H, H-7eq), 3.11 (m, 0.5H, H-4eq Z isomer), 3.08 (m, 0.5H,
H-2eq E isomer), 3.02 (m, 2H, CH2–N), 0.78 (s, 3H, CH3), 0.77 (s, 3H,
CH3); white solid. Anal. (C22H35N3O3ꢁHCl) C, H, Cl, N.
5.3.17. (E,Z)-3-(2-Aminoethoxyimino)-6a-hydroxymethy-
landrostane-17-one hydrochloride (17)
5.3.11. (E,Z)-3-(2-Aminoethoxyimino)-6-[(E)-ethoxyimino]
androstane-17-one hydrochloride (11)
Prepared in 60% yield from 6a-hydroxymethylandrostane-3,17-
dione 83 and 2-aminoethoxyamine dihydrochloride. 1H NMR
(300 MHz, DMSO-d6, ppm from TMS): d 7.73 (br b, 3H, NH3þ),
4.37 (t, 1H, OH), 4.06 (m, 2H, CH2–ON), 3.37 (m, 2H, CH2–O), 3.16
(m, 0.5H, H-4eq Z isomer), 3.06 (m, 0.5H, H-2eq E isomer), 3.02
(m, 2H, CH2–N), 0.89 (s, 1.5H, CH3), 0.87 (s, 1.5H, CH3), 0.78 (s,
3H, CH3); white solid. Anal. (C22H36N2O3ꢁHCl) C, H, Cl, N.
Prepared in 80% yield from 6-[(E)-ethoxyimino]androstane-
3,17-dione 107 and 2-aminoethoxyamine dihydrochloride. 1H
NMR (300 MHz, DMSO, ppm from TMS): d 7.85 (br b, 3H, NH3þ),
4.07 (m, 2H, CH2–O), 4.00 (q, 1H, CH2–O), 3.98 (q, 1H, CH2–O),
3.20 (m, 1H, H-7eq), 3.11 (m, 0.5H, H-4eq Z isomer), 3.08 (m,
0.5H, H-2eq E isomer), 3.03 (m, 2H, CH2–N), 1.17 (t, 1.5H, CH3),
1.16 (t, 1.5H, CH3), 0.78 (s, 6H, CH3, CH3); white solid. Anal.
(C23H37N3O3ꢁHCl) C, H, Cl, N.
5.3.18. (E,Z)-3-(2-Aminoethoxyimino)-6b-hydroxymethy-
landrostane-17-one hydrochloride (18)
Prepared in 85% yield from 6b-hydroxymethylandrostane-3,17-
dione 65 and 2-aminoethoxyamine dihydrochloride. 1H NMR
(300 MHz, DMSO-d6, ppm from TMS): d 8.14 (br b, 3H, NH3þ),
4.42 (t, 0.5H, OH), 4.40 (t, 0.5H, OH), 4.08 (m, 2H, CH2–ON), 3.35
(m, 2H, CH2–O), 3.05 (m, 0.5H, H-2eq E isomer), 3.00 (m, 2H,
CH2–N), 2.91 (m, 0.5H, H-4eq Z isomer), 0.84 (s, 1.5H, CH3), 0.82
(s, 1.5H, CH3), 0.80 (s, 3H, CH3); white solid. Anal. (C22H36N2O3ꢁHCl)
C, H, Cl, N.
5.3.12. (E,Z)-3-(2-Aminoethoxyimino)-6-[(E)-allyloxyimino]
androstane-17-one fumarate (12)
Prepared in 75% yield from 6-[(E)-allyloxyimino]androstane-
3,17-dione 109 and 2-aminoethoxyamine dihydrochloride. 1H
NMR (300 MHz, DMSO, ppm from TMS): d 9.01 (br b, 4H, COOH,
NH3þ), 6.40 (s, 2H, CH@CH), 5.93 (m, 1H, C@CH), 5.18 (m, 2H,
C@CH2), 4.49 (m, 2H, C@C–CH2–O), 4.05 (m, 2H, CH2–O), 3.22 (m,
1H, H-7eq), 3.09 (m, 0.5H, H-4eq Z isomer), 3.07 (m, 0.5H, H-2eq
E isomer), 2.98 (m, 2H, CH2–N), 0.78 (s, 6H, CH3, CH3); white solid.
Anal. (C24H37N3O3ꢁC4H4O4) C, H, N.
5.3.19. (E,Z)-3-(2-Aminoethoxyimino)-6a-methoxymethy-
landrostane-17-one hydrochloride (19)
Prepared in 33% yield from 6a-methoxymethylandrostane-
5.3.13. (E,Z)-3-(2-Aminoethoxyimino)-17-oxoandrostane-6-
[(E)-ylidene]acetonitrile hydrochloride (13)
Prepared in 61% yield from 3,17-dioxoandrostane-6-[(E)-yli-
dene]acetonitrile 98 and 2-aminoethoxyamine dihydrochloride.
3,17-dione 85 and 2-aminoethoxyamine dihydrochloride. 1H
NMR (300 MHz, DMSO-d6, ppm from TMS): d 7.82 (br b, 3H,
NH3þ), 4.06 (m, 2H, CH2–ON), 3.22 (m, 2H, CH2–O), 3.20 (s, 3H,
CH3–O), 3.11 (m, 0.5H, H-4eq Z isomer), 3.05 (m, 0.5H, H-2eq E

4290
M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
isomer), 3.02 (m, 2H, CH2–N), 0.89 (s, 3H, CH3), 0.78 (s, 3H, CH3);
5.3.26. (E,Z)-3-(2-Aminoethoxyimino)-17-oxoandrostane-6a-yl
white solid. Anal. (C23H38N2O3ꢁHCl) C, H, Cl, N.
nitrate fumarate (26)
Prepared in 33% yield from 3,17-dioxoandrostane-6a-yl nitrate
114 and 2-aminoethoxyamine dihydrochloride. 1H NMR (300 MHz,
DMSO-d6, ppm from TMS): d 8.76 (br b, 4H, NH3þ, COOH), 6.41 (s,
2H, CH@CH), 4.98 (m, 1H, CH–O), 4.04 (m, 2H, CH2–ON), 3.16 (m,
0.5H, H-4eq Z isomer), 3.06 (m, 0.5H, H-2eq E isomer), 2.98 (m,
2H, CH2–N), 0.98 (s, 1.5H, CH3), 0.97 (s, 1.5H, CH3), 0.80 (s, 3H,
CH3); white solid. Anal. (C21H33N3O5ꢁC4H4O4) C, H, N.
5.3.20. (E,Z)-3-(2-Aminoethoxyimino)-6b-methoxymethy-
landrostane-17-one hydrochloride (20)
Prepared in 60% yield from 6b-methoxymethylandrostane-3,17-
dione 67 and 2-aminoethoxyamine dihydrochloride. 1H NMR
(300 MHz, DMSO-d6, ppm from TMS): d 8.06 (br b, 3H, NH3þ),
4.07 (m, 2H, CH2–ON), 3.35 (m, 2H, CH2–O), 3.23 (s, 1.5H, CH3),
3.22 (s, 3H, CH3), 3.07 (m, 0.5H, H-2eq E isomer), 3.02 (m, 2H,
CH2–N), 2.92 (m, 0.5H, H-4eq Z isomer), 0.86 (s, 1.5H, CH3), 0.85
(s, 1.5H, CH3), 0.81 (s, 3H, CH3); white solid. Anal. (C23H38N2O3ꢁHCl)
C, H, Cl, N.
5.3.27. (E,Z)-3-(2-Aminoethoxyimino)-17-oxoandrostane-6b-yl
nitrate fumarate (27)
Prepared in 60% yield from 3,17-dioxoandrostane-6b-yl nitrate
121 and 2-aminoethoxyamine dihydrochloride. 1H NMR
þ
(300 MHz, DMSO-d6, ppm from TMS): d 8.41 (br b, 4H, NH3
,
5.3.21. (E,Z)-3-(2-Aminoethoxyimino)-(6R)-6-(spiro-20-oxirane)
androstane-17-one hydrochloride (21)
COOH), 6.40 (s, 2H, CH@CH), 5.23 (m, 0.5H, CH–O), 5.19 (m, 0.5H,
CH–O), 4.03 (m, 2H, CH2–O), 3.07 (m, 0.5H, H-2eq E isomer), 3.05
(m, 0.5H, H-4eq Z isomer), 2.96 (m, 2H, CH2–N), 1.00 (s, 1.5H,
CH3), 0.99 (s, 1.5H, CH3), 0.80 (s, 3H, CH3); white solid. Anal.
(C21H33N3O5ꢁC4H4O4) C, H, N.
Prepared in 50% yield from (6R)-6-spiro-(20-oxirane)andro-
stane-3,17-one 63 and 2-aminoethoxyamine dihydrochloride. 1H
NMR (300 MHz, DMSO-d6, ppm from TMS): d 7.75 (br b, 3H,
NH3þ), 4.06 (m, 2H, CH2–ON), 3.07 (m, 0.5H, H-2eq E isomer),
2.99 (m, 2H, CH2–N), 2.83 (m, 0.5H, H-4eq Z isomer), 2.75 (d,
0.5H, CH2–O), 2.72 (d, 0.5H, CH2–O), 2.30 (d, 0.5H, CH2–O), 2.27
(d, 0.5H, CH2–O), 0.96 (s, 1.5H, CH3), 0.94 (s, 1.5H, CH3), 0.80 (s,
3H, CH3); white solid. Anal. (C22H34N2O3ꢁHCl) C, H, Cl, N.
5.3.28. (E,Z)-3-(2-Aminoethoxyimino)-6a-carboxyandrostane-
17-one hydrochloride (28)
Prepared in 80% yield from 6a-carboxyandrostane-3,17-dione 79
and 2-aminoethoxyamine dihydrochloride. 1H NMR (300 MHz,
DMSO-d6, ppm from TMS): d 8.24 (br b, 4H, NH3þ, COOH), 4.07 (m,
2H, CH2–O), 3.07 (m, 0.5H, H-2eq E isomer), 3.01 (m, 2H, CH2–N),
2.93 (m, 0.5H, H-4eq Z isomer), 0.90 (s, 1.5H, CH3), 0.89 (s, 1.5H,
CH3), 0.78 (s, 3H, CH3); white solid. Anal. (C22H34N2O4ꢁHCl) C, H, Cl, N.
5.3.22. (E,Z)-3-(2-Aminoethoxyimino)-(6S)-6-(spiro-20-oxirane)
androstane-17-one hydrochloride (22)
Prepared in 40% yield from (6S)-6-spiro-(20-oxirane)andro-
stane-3,17-one 62 and 2-aminoethoxyamine dihydrochloride. 1H
NMR (300 MHz, DMSO-d6, ppm from TMS): d 7.80 (br b, 3H,
NH3þ), 4.06 (m, 2H, CH2–ON), 3.07 (m, 0.5H, H-2eq E isomer),
3.01 (m, 2H, CH2–N), 2.90 (m, 0.5H, H-4eq Z isomer), 2.76 (d, 1H,
CH2–O), 2.57 (d, 1H, CH2–O), 0.92 (s, 3H, CH3), 0.78 (s, 3H, CH3);
white solid. Anal. (C22H34N2O3ꢁHCl) C, H, Cl, N.
5.3.29. (E,Z)-3-(2-Aminoethoxyimino)-6a-cyanoandrostane-17-
one fumarate (29)
Prepared in 65% yield from 6a-cyanoandrostane-3,17-dione
111 and 2-aminoethoxyamine dihydrochloride. 1H NMR
þ
(300 MHz, DMSO-d6, ppm from TMS): d 9.07 (br b, 4H, NH3
,
COOH), 6.40 (s, 2H, CH@CH), 4.07 (m, 2H, CH2–ON), 3.25 (m,
0.5H, H-4eq Z isomer), 3.07 (m, 0.5H, H-2eq E isomer), 2.99 (m,
2H, CH2–N), 2.77 (m, 1H, CH-CN), 0.88 (s, 1.5H, CH3), 0.87 (s,
1.5H, CH3), 0.77 (s, 3H, CH3); white solid. Anal. (C22H33N3O2ꢁ-
C4H4O4) C, H, N.
5.3.23. (E,Z)-3-(2-Aminoethoxyimino)-6a-methoxyandrostane-
17-one hydrochloride (23)
Prepared in 62% yield from 6a-methoxyandrostane-3,17-dione
116 and 2-aminoethoxyamine dihydrochloride. 1H NMR
(300 MHz, DMSO-d6, ppm from TMS): d 7.98 (br b, 2H, NH3þ),
4.07 (m, 2H, CH2–O), 3.40 (dd, 1H, CH–O), 3.24 (s, 1.5H, CH3–O),
3.23 (s, 1.5H, CH3–O), 3.05 (m, 0.5H, H-4eq Z isomer), 2.97 (m,
0.5H, H-2eq E isomer), 0.89 (s, 1.5H, CH3), 0.88 (m, 1.5H, CH3),
0.78 (m, 3H, CH3); white foam. Anal. (C22H36N2O3ꢁHCl) C, H, Cl, N.
5.3.30. (E,Z)-3-(2-Aminoethoxyimino)-6a-methoxycarbony-
landrostane-17-one hydrochloride (30)
Prepared in 62% yield from 6a-methoxycarbonylandrostane-
3,17-dione 81 and 2-aminoethoxyamine dihydrochloride. 1H
NMR (300 MHz, DMSO-d6, ppm from TMS): d 7.75 (br b, 3H,
NH3þ), 4.06 (m, 2H, CH2–ON), 3.60 (s, 3H, CH3–O), 3.07 (m, 0.5H,
H-2eq E isomer), 3.01 (m, 2H, CH2–N), 2.79 (m, 0.5H, H-4eq Z iso-
mer), 0.91 (s, 1.5H, CH3), 0.90 (s, 1.5H, CH3), 0.78 (s, 3H, CH3); white
solid. Anal. (C23H36N2O4ꢁHCl) C, H, Cl, N.
5.3.24. (E,Z)-3-(2-Aminoethoxyimino)-6b-methoxyandrostane-
17-one hydrochloride (24)
Prepared in 34% yield from 6b-methoxyandrostane-3,17-dione
123 and 2-aminoethoxyamine dihydrochloride. 1H NMR
(300 MHz, DMSO-d6, ppm from TMS): d 7.98 (br b, 3H, NH3þ),
4.08 (m, 2H, CH2–O), 3.23 (m, 1H, CH–O), 3.21 (s, 1.5H, CH3),
3.20 (s, 1.5H, CH3), 3.06 (m, 0.5H, H-4eq Z isomer), 3.02 (m, 2H,
CH2–N), 2.88 (m, 0.5H, H-2eq E isomer), 0.97 (s, 1.5H, CH3), 0.96
(s, 1.5H, CH3), 0.79 (s, 3H, CH3); white solid. Anal. (C22H36N2O3ꢁHCl)
C, H, Cl, N.
5.3.31. (E,Z)-3-(2-Aminoethoxyimino)-6a-carbamoyland-
rostane-17-one fumarate (31)
Prepared in 40% yield from 6a-carbamoylandrostane-3,17-
dione 80 and 2-aminoethoxyamine dihydrochloride. 1H NMR
þ
(300 MHz, DMSO-d6, ppm from TMS): d 8.00 (br b, 4H, NH3
,
COOH), 7.38 (s, 0.5H, CONH2), 7.32 (s, 0.5H, CONH2), 6.80 (s,
0.5H, CONH2), 6.78 (s, 0.5H, CONH2), 6.40 (s, 2H, CH@CH), 4.05
(m, 2H, CH2–ON), 3.06 (m, 0.5H, H-2eq E isomer), 2.99 (m, 2H,
CH2–N), 2.91 (m, 0.5H, H-4eq Z isomer), 0.89 (s, 3H, CH3), 0.78 (s,
3H, CH3); white solid. Anal. (C22H33N3O2ꢁ1.5C4H4O4) C, H, N.
5.3.25. (E,Z)-3-(2-Aminoethoxyimino)-6a-ethoxyandrostane-
17-one fumarate (25)
Prepared in 88% yield from 6a-ethoxyandrostane-3,17-dione
118 and 2-aminoethoxyamine dihydrochloride. 1H NMR
(300 MHz, DMSO-d6, ppm from TMS; the specific E and Z signal
attributions were not assigned): d 8.50 (m, 3H, NH3þ), 4.02 (m,
2H, CH2–O), 1.08 (t, 1.5H, CH3), 1.07 (t, 1.5H, CH3), 0.88 (s, 1.5H,
CH3), 0.87 (s, 1.5H, CH3), 0.77 (s, 3H, CH3); white solid. Anal.
(C23H38N2O3ꢁC4H4O4) C, H, N.
5.3.32. (E,Z)-3-(2-Aminoethoxyimino)-6a-formamidoand-
rostane-17-one fumarate (32)
Prepared in 59% yield from 6
a-formamidoandrostane-3,17-
dione 126 and 2-aminoethoxyamine dihydrochloride. 1H NMR

M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
4291
(300 MHz, DMSO-d6, ppm from TMS): d 8.20 (m, 4H, NH3þ, COOH),
5.3.39. (E,Z)-3-[(3R)-3-Pyrrolidinyloxyimino]-6-[(E)-hydrox-
8.10–7.60 (m, 2H, NHCH–O), 6.44 (s, 2H, CH@CH), 4.05 (m, 2H,
CH2–ON), 3.72 (m, 1H, CH–N), 3.16 (m, 0.5H, H-4eq Z isomer),
3.06 (m, 0.5H, H-2eq E isomer), 2.97 (m, 2H, CH2–N), 0.93 (s,
1.5H, CH3), 0.92 (s, 1.5H, CH3), 0.78 (s, 3H, CH3); white solid. Anal.
(C22H35N3O3ꢁC4H4O4) C, H, N.
yimino]androstane-17-one hydrochloride (39)
Prepared in 77% yield from 6-[(E)-hydroxyimino]androstane-
3,17-dione 103 and 3-(R)-pyrrolidinyloxyamine dihydrochloride.
1H NMR (300 MHz, DMSO-d6, ppm from TMS): d 10.56 (s, 0.5H,
N–OH), 10.52 (s, 0.5H, N–OH), 9.25 (br b, 2H, NH2þ), 4.74 (m, 1H,
CH–O), 0.78 (s, 6H, CH3, CH3); white solid. Anal. (C23H35N3O3ꢁHCl)
C, H, Cl, N.
5.3.33. (E,Z)-3-(2-Aminoethoxyimino)-6a-acetamidoand-
rostane-17-one hydrochloride (33)
Prepared in 84% yield from 6
a
-acetamidoandrostane-3,17-
5.3.40. (E,Z)-3-[(3R)-3-Pyrrolidinyloxyimino]-6-[(E)-methoxy-
imino]androstane-17-one hydrochloride (40)
dione 128 and 2-aminoethoxyamine dihydrochloride. 1H NMR
(300 MHz, DMSO-d6, ppm from TMS): d 7.87 (br b, 3H, NH3þ),
7.83 (d, 0.5H, NH-CO), 7.67 (d, 0.5H, NH-CO), 4.07 (m, 2H, CH2–
ON), 3.65 (m, 1H, CH–N), 3.15 (m, 0.5H, H-4eq Z isomer), 3.07
(m, 0.5H, H-2eq E isomer), 3.03 (m, 2H, CH2–N), 1.81 (s, 1.5H,
CH3–CO), 1.79 (s, 1.5H, CH3–CO), 0.93 (s, 1.5H, CH3), 0.91 (s,
1.5H, CH3), 0.78 (s, 3H, CH3); white solid. Anal. (C23H37N3O3ꢁHCl)
C, H, Cl, N.
Prepared in 70% yield from 6-[(E)-methoxyimino]androstane-
3,17-dione 105 and 3-(R)-pyrrolidinyloxyamine dihydrochloride.
1H NMR (300 MHz, DMSO-d6, ppm from TMS; the specific E and
Z signal attributions were not assigned): d 9.05 (br b, 2H, NH2þ),
4.74 (br s, 1H, CH–O), 3.75 (s, 1.5H, CH3–O), 3.73 (s, 1.5H, CH3–
O), 0.81 (s, 3H, CH3), 0.76 (s, 3H, CH3); white solid. Anal.
(C24H37N3O3ꢁHCl) C, H, Cl, N.
5.3.34. (E,Z)-3-[(3R)-3-Pyrrolidinyloxyimino]-6-methyleneand-
rostane-17-one hydrochloride (34)
5.3.41. (E,Z)-3-[(3R)-3-Pyrrolidinyloxyimino]-6a-(2-hydroxy-
ethyl)androstane-17-one hydrochloride (41)
Prepared in 75% yield from 6-methyleneandrostane-3,17-dione
61 and 3-(R)-pyrrolidinyloxyamine dihydrochloride.8 1H NMR
(300 MHz, DMSO-d6, ppm from TMS): d 9.01 (br b, 2H, NH2þ),
4.82 (m, 1H, C@CH), 4.74 (m, 1H, CH–O), 4.50 (m, 1H, C@CH),
0.77 (s, 3H, CH3), 0.76 (s, 3H, CH3); white solid. Anal.
(C24H36N2O2ꢁHCl) C, H, Cl, N.
Prepared in 78% yield from 6a-(2-hydroxyethyl)androstane-
3,17-dione 77 and 3-(R)-pyrrolidinyloxyamine dihydrochloride.
1H NMR (300 MHz, DMSO-d6, ppm from TMS; the specific E and
Z signal attributions were not assigned): d 8.95 (br b, 2H, NH2þ),
4,74 (m, 1H, CH–O), 4.34 (t, 1H, OH), 0.89 (s, 1.5H, CH3), 0.88 (s,
1.5H, CH3), 0.76 (s, 3H, CH3); white solid. Anal. (C25H40N2O3ꢁHCl)
C, H, Cl, N.
5.3.35. (E,Z)-3-[(3R)-3-Pyrrolidinyloxyimino]-6-difluorometh-
yleneandrostane-17-one hydrochloride (35)
5.3.42. (E,Z)-3-[(3R)-3-Pyrrolidinyloxyimino]-6a-hydroxymeth-
Prepared in 71% yield from 6-difluoromethyleneandrostane-
3,17-dione 96 and 3-(R)-pyrrolidinyloxyamine dihydrochloride.
1H NMR (300 MHz, DMSO-d6, ppm from TMS): d 9.10 (br b, 2H,
NH2þ), 4.70 (m, 1H, CH–O), 0.89 (s, 3H, CH3), 0.78 (s, 3H, CH3);
white solid. Anal. (C24H34F2N2O2ꢁHCl) C, H, Cl, N.
ylandrostane-17-one hydrochloride (42)
Prepared in 57% yield from 6a-hydroxymethylandrostane-3,17-
dione 83 and 3-(R)-pyrrolidinyloxyamine dihydrochloride. 1H
NMR (300 MHz, DMSO-d6, ppm from TMS; the specific E and Z signal
attributions were not assigned): d 9.23 (br b, 2H, NH2þ), 4.72 (m, 1H,
CH–O), 4.37 (t, 1H, OH), 0.88 (s, 1.5H, CH3), 0.86 (s, 1.5H, CH3), 0.86 (s,
3H, CH3); white solid. Anal. (C24H38N2O3ꢁHCl) C, H, Cl, N.
5.3.36. (E,Z)-3-[(3R)-3-Pyrrolidinyloxyimino]-6-(spirocyclopro-
pane)androstane-17-one hydrochloride (36)
Prepared in 91% yield from 6-(spirocyclopropane)androstane-
3,17-dione 68 and 3-(R)-pyrrolidinyloxyamine dihydrochloride.
1H NMR (300 MHz, DMSO-d6, ppm from TMS): d 9.02 (br b, 2H,
NH2þ), 4.72 (m, 1H, CH–O), 2.98 (m, 0.5H, H-2eq E isomer), 2.63
(m, 0.5H, H-4eq Z isomer), 0.96 (s, 1.5H, CH3), 0.95 (s, 1.5H, CH3),
0.79 (s, 3H, CH3), 0.55-(-0.15) (m, 4H, cyclopropane); white solid.
Anal. (C25H38N2O2ꢁHCl) C, H, Cl, N.
5.3.43. (E,Z)-3-[(3R)-3-Pyrrolidinyloxyimino]-6
methoxymethylandrostane-17-one fumarate (43)
Prepared in 60% yield from 6 -methoxymethylandrostane-
3,17-dione 85 and 3-(R)-pyrrolidinyloxyamine dihydrochloride.
1H NMR (300 MHz, DMSO-d6, ppm from TMS; the specific E and
Z signal attributions were not assigned): 1H NMR (300 MHz,
DMSO-d6, ppm from TMS): d 9.00 (br b, 3H, NH2þ, COOH), 6.40
(s, 2H, CH@CH), 4.71 (m, 1H, CH–O), 3.22 (s, 1.5H, CH3–O), 3.21
(s, 1.5H, CH3–O), 0.88 (s, 3H, CH3), 0.78 (s, 3H, CH3); white solid.
Anal. (C25H40N2O3ꢁC4H4O4) C, H, N.
a-
a
5.3.37. (E,Z)-3-[(3R)-3-Pyrrolidinyloxyimino]-6a-methyland-
rostane-17-one fumarate (37)
Prepared in 84% yield from 6a-methylandrostane-3,17-dione
90 and 3-(R)-pyrrolidinyloxyamine dihydrochloride. 1H NMR
5.3.44. (E,Z)-3-[(3R)-3-Pyrrolidinyloxyimino]-17-oxoand-
þ
(300 MHz, DMSO-d6, ppm from TMS): d 8.50 (br b, 3H, NH2
,
rostane-6a-yl nitrate hydrochloride (44)
COOH), 6.41 (m, 2H, CH@CH), 4.73 (m, 0.5H, CH–O), 4.70 (m,
0.5H, CH–O), 3.12 (m, 0.5H, H-4eq Z isomer), 2.98 (m, 0.5H, H-
2eq E isomer), 0.88 (s, 1.5H, CH3), 0.87 (s, 1.5H, CH3), 0.84 (d,
1.5H, CH3), 0.81 (s, 1.5H, CH3), 0.78 (s, 3H, CH3); white solid. Anal.
(C24H38N2O2ꢁC4H4O4) C, H, N.
Prepared in 41% yield from 3,17-dioxoandrostane-6a-yl nitrate
114 and 3-(R)-pyrrolidinyloxyamine dihydrochloride. 1H NMR
(300 MHz, DMSO-d6, ppm from TMS; the specific E and Z signal
attributions were not assigned): d 8.96 (br b, 2H, NH2þ), 4.99 (m,
1H, CH–ONO2), 4.74 (m, 1H, CH–O), 0.99 (s, 1.5H, CH3), 0.98 (s,
1.5H, CH3), 0.80 (s, 3H, CH3); white solid. Anal. (C23H35N3O5ꢁHCl)
C, H, Cl, N.
5.3.38. (E,Z)-3-[(3R)-3-Pyrrolidinyloxyimino]-6a-ethynyland-
rostane-17-one hydrochloride (38)
Prepared in 90% yield from 6a-ethynylandrostane-3,17-dione
5.3.45. (E,Z)-3-[(3R)-3-Pyrrolidinyloxyimino]-6a-formamido-
87 and 3-(R)-pyrrolidinyloxyamine dihydrochloride. 1H NMR
(300 MHz, DMSO-d6, ppm from TMS; the specific E and Z signal
attributions were not assigned): d 8.98 (br b, 2H, NH2þ), 4.75 (m,
1H, CH–O), 2.98 (d, 0.5H, C„CH), 2.94 (m, 0.5H, C„CH), 0.88 (s,
1.5H, CH3), 0.87 (s, 1.5H, CH3), 0.79 (s, 3H, CH3); white solid. Anal.
(C25H36N2O2ꢁHCl) C, H, Cl, N.
androstane-17-one hydrochloride (45)
Prepared in 70% yield from 6a-formamidoandrostane-3,17-
dione 126 and 3-(R)-pyrrolidinyloxyamine dihydrochloride. 1H
NMR (300 MHz, DMSO-d6, ppm from TMS; the specific E and Z sig-
nal attributions were not assigned): d 9.38 (br b, 3H, NH2þ), 8.42–
7.50 (m, 2H, NH-CH–O), 4.76 (m, 0.5H, CH–O), 4.71 (m, 0.5H, CH–

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M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
O), 3.72 (m, 1H, CH–N), 0.93 (s, 1.5H, CH3), 0.92 (s, 1.5H, CH3), 0.78
0.87 (s, 3H, CH3), 0.77 (s, 3H, CH3); white solid. Anal.
(s, 3H, CH3); white solid. Anal. (C23H37N3O3ꢁHCl) C, H, Cl, N.
(C25H38N2O2ꢁHCl) C, H, Cl, N.
5.3.46. (E,Z)-3-[(3R)-3-Pyrrolidinyloxyimino]-6
landrostane-17-one hydrochloride (46)
a-carbamoy-
5.3.52. (E,Z)-3-(3-N-Methylaminopropoxyimino)-6-[(E)-hydrox-
yimino]androstane-17-one hydrochloride (52)
Prepared in 65% yield from 6
a
-carbamoylandrostane-3,17-
Prepared in 72% yield from 6-[(E)-hydroxyimino]androstane-
3,17-dione 103 and 3-N-methylaminopropoxyamine dihydrochlo-
ride. 1H NMR (300 MHz, DMSO-d6, ppm from TMS; the specific E
and Z signal attributions were not assigned): d 10.57 (s, 0.5H, N–
OH), 10.53 (s, 0.5H, N–OH), 8.70 (br b, 2H, NH2þ), 3.96 (m, 2H,
CH2–O), 2.51 (s, 1.5H, CH3–N), 2.50 (s, 1.5H, CH3–N), 0.77 (s, 6H,
CH3, CH3); white solid. Anal. (C23H37N3O3ꢁHCl) C, H, Cl, N.
dione 80 and 3-(R)-pyrrolidinyloxyamine dihydrochloride. 1H
NMR (300 MHz, DMSO-d6, ppm from TMS; the specific E and Z sig-
nal attributions were not assigned): d 8.80 (br b, 2H, NH2þ), 7.38
(br b, 0.5H, NH-CO), 7.31 (br b, 0.5H, NH-CO), 6.92 (br b, 0.5H,
NH-CO), 6.78 (br b, 0.5H, NH-CO), 4.62 (m, 1H, CH–O), 0.89 (s,
3H, CH3), 0.78 (s, 3H, CH3); white solid. Anal. (C23H37N3O3ꢁHCl) C,
H, Cl, N.
5.3.53. (E,Z)-3-(3-N-Methylaminopropoxyimino)-6-[(E)-meth-
oxyimino]androstane-17-one hydrochloride (53)
5.3.47. (E,Z)-3-[(3R)-3-Pyrrolidinyloxyimino]-6
carbonylandrostane-17-one hydrochloride (47)
Prepared in 74% yield from 6 -methoxycarbonylandrostane-
3,17-dione 81 and 3-(R)-pyrrolidinyloxyamine dihydrochloride.
1H NMR (300 MHz, DMSO-d6, ppm from TMS): d 8.88 (br b, 2H,
NH2þ), 4.72 (m, 1H, CH–O), 3.61 (s, 1.5H, CH3–O), 3.60 (s, 1.5H,
CH3–O), 2.99 (m, 0.5H, H-2eq E isomer), 2.74 (m, 0.5H, H-4eq Z iso-
mer), 0.91 (s, 1.5H, CH3), 0.90 (s, 1,5H, CH3), 0.78 (s, 3H, CH3); white
solid. Anal. (C25H38N2O4ꢁHCl) C, H, Cl, N.
a-methoxy-
Prepared in 60% yield from 6-[(E)-methoxyimino]androstane-
3,17-dione 105 and 3-N-methylaminopropoxyamine dihydrochlo-
ride. 1H NMR (300 MHz, DMSO-d6, ppm from TMS): d 8.60 (br b,
2H, NH2þ), 3.97 (m, 2H, CH2–O), 3.75 (s, 1.5H, CH3–O), 3.73 (s,
1.5H, CH3–O), 3.19 (dd, 1H, H-7eq), 3.05 (m, 0.5H, H-4eq Z isomer),
2.99 (m, 0.5H, H-2eq E isomer), 2.90 (m, 2H, CH2–N), 2.52 (s, 3H,
CH3–N), 0.78 (s, 1.5H, CH3), 0.78 (s, 1.5H, CH3), 0.77 (s, 3H, CH3);
white solid. Anal. (C24H39N3O3ꢁHCl) C, H, Cl, N.
a
5.3.48. (E,Z)-3-(3-N-Methylaminopropoxyimino)-6-methylene-
androstane-17-one fumarate (48)
5.3.54. (E,Z)-3-(3-N-Methylaminopropoxyimino)-6a-hydrox-
ymethylandrostane-17-one hydrochloride (54)
Prepared in 70% yield from 6-methyleneandrostane-3,17-dione
Prepared in 67% yield from 6a-hydroxymethylandrostane-3,17-
61 and 3-N-methylaminopropoxyamine dihydrochloride.7 1H NMR
dione 83 and 3-N-methylaminopropoxyamine dihydrochloride. 1H
NMR (300 MHz, DMSO-d6, ppm from TMS): d 8.64 (br b, 2H, NH2þ),
4.36 (t, 1H, OH), 3.96 (m, 2H, CH2–ON), 3.33 (m, 2H, CH2–O), 3.16
(m, 0.5H, H-4eq Z isomer), 2.97 (m, 0.5H, H-2eq E isomer), 2.89 (m,
2H, CH2–N), 2.51 (s, 3H, CH3-N), 0.88 (s, 1.5H, CH3), 0.87 (s, 1.5H,
CH3), 0.78 (s, 3H, CH3); white solid. Anal. (C24H40N2O3ꢁHCl) C, H,
Cl, N.
þ
(300 MHz, DMSO-d6, ppm from TMS): d 9.10 (br b, 3H, NH2
,
COOH), 6.41 (s, 2H, CH@CH), 4.82 (m, 0.5H, C@CH), 4.80 (m,
0.5H, C@CH), 4.52 (m, 0.5H, C@CH), 4.49 (m, 0.5H, C@CH), 3.96
(t, 2H, CH2–O), 3.01 (m, 0.5H, H-2eq E isomer), 2.96 (m, 0.5H, H-
4eq Z isomer), 2.80 (m, 2H, CH2–N), 2.46 (s, 1.5H, CH3–N), 2.45
(s, 1.5H, CH3–N), 0.77 (s, 3H, CH3), 0.75 (s, 3H, CH3); white solid.
Anal. (C25H40N2O3ꢁC4H4O4) C, H, N.
5.3.55. (E,Z)-3-(3-N-Methylaminopropoxyimino)-6a-meth-
5.3.49. (E,Z)-3-(3-N-Methylaminopropoxyimino)-6-(spirocy-
clopropane)androstane-17-one hydrochloride (49)
oxymethylandrostane-17-one fumarate (55)
Prepared in 67% yield from 6a-methoxymethylandrostane-
Prepared in 93% yield from 6-(spirocyclopropane)androstane-
3,17-dione 68 and 3-N-methylaminopropoxyamine dihydrochlo-
ride. 1H NMR (300 MHz, DMSO-d6, ppm from TMS): d 8.55 (br b,
2H, NH2þ), 3.95 (m, 2H, CH2–O), 2.96 (m, 0.5H, H-2eq E isomer),
2.88 (m, 2H, CH2–N), 2.62 (m, 0.5H, H-4eq Z isomer), 2.52 (s, 3H,
CH3–N), 0.96 (s, 1.5H, CH3), 0.95 (s, 1.5H, CH3), 0.79 (s, 3H, CH3),
0.60–0.05 (m, 4H, cyclopropane); white solid. Anal.
(C25H40N2O2ꢁHCl) C, H, Cl, N.
3,17-dione 85 and 3-N-methylaminopropoxyamine dihydrochlo-
ride. 1H NMR (300 MHz, DMSO-d6, ppm from TMS): d 10.00 (br b,
3H, NH2þ, COOH); 6.44 (s, 2H, CH@CH), 3.95 (t, 2H, CH2–O), 3.21
(s, 1.5H, CH3–O), 3.20 (s, 1.5H, CH3–O), 3.08 (m, 0.5H, H-4eq Z iso-
mer), 2.96 (m, 0.5H, H-2eq E isomer), 2.85 (t, 2H, CH2–N), 2.50 (s,
1.5H, CH3-N), 2.48 (s, 1.5H, CH3-N), 0.87 (s, 3H, CH3), 0.77 (s, 3H,
CH3); white solid. Anal. (C25H42N2O3ꢁHCl) C, H, Cl, N.
5.3.56. (E,Z)-3-(3-N-Methylaminopropoxyimino)-6a-formam-
5.3.50. (E,Z)-3-(3-N-Methylaminopropoxyimino)-6
a-methy-
idoandrostane-17-one hydrochloride (56)
landrostane-17-one hydrochloride (50)
Prepared in 70% yield from 6a-formamidoandrostane-3,17-
Prepared in 79% yield from 6
a
-methylandrostane-3,17-dione
dione 126 and 3-N-methylaminopropoxyamine dihydrochloride.
1H NMR (300 MHz, DMSO-d6, ppm from TMS): d 8.57 (br b, 2H,
NH2þ), 8.06–7.57 (m, 2H, NH-CH–O), 3.96 (m, 2H, CH2–O), 3.72
(m, 1H, CH–N), 3.07 (m, 0.5H, H-4eq Z isomer), 2.97 (m, 0.5H, H-
2eq E isomer), 2.88 (m, 2H, CH2–N), 2.52 (s, 3H, CH3-N), 0.93 (s,
1.5H, CH3), 0.92 (s, 1.5H, CH3), 0.78 (s, 3H, CH3); white solid. Anal.
(C24H39N3O3ꢁHCl) C, H, Cl, N.
90 and 3-N-methylaminopropoxyamine dihydrochloride. 1H NMR
(300 MHz, DMSO-d6, ppm from TMS): d 8.54 (br b, 2H, NH2þ),
3.95 (t, 2H, CH2–O), 2.96 (m, 0.5H, H-2eq E isomer), 2.88 (t, 2H,
CH2–N), 2.61 (m, 0.5H, H-4eq Z isomer), 2.52 (s, 3H, CH3–N), 0.95
(s, 1.5H, CH3), 0.94 (s, 1.5H, CH3), 0.79 (s, 3H, CH3); white solid.
Anal. (C24H40N2O2ꢁHCl) C, H, Cl, N.
5.3.51. (E,Z)-3-(3-N-Methylaminopropoxyimino)-6
a-ethynyl-
5.3.57. (E,Z)-3-(3-N-Methylaminopropoxyimino)-6a-carba-
androstane-17-one hydrochloride (51)
moylandrostane-17-one hydrochloride (57)
Prepared in 70% yield from 6
a-ethynylandrostane-3,17-dione
Prepared in 84% yield from 6a-carbamoylandrostane-3,17-
87 and 3-N-methylaminopropoxyamine dihydrochloride. 1H NMR
(300 MHz, DMSO-d6, ppm from TMS): d 8.90 (br b, 2H, NH2þ),
3.98 (m, 2H, CH2–O), 3.44 (m, 0.5H, H-4eq Z isomer), 3.00 (m,
0.5H, H-2eq E isomer), 3.01 (d, 0.5H, C„CH), 2.97 (d, 0.5H, C„CH),
2.86 (m, 2H, CH2–N), 2.49 (s, 1.5H, CH3–N), 2.48 (s, 1.5H, CH3–N),
dione 80 and 3-N-methylaminopropoxyamine dihydrochloride.
1H NMR (300 MHz, DMSO-d6, ppm from TMS; the specific E and
Z signal attributions were not assigned): 8.53 (br b, 2H, NH2þ),
7.36 (br b, 0.5H, NH2-CO), 7.32 (br b 0.5H, NH2-CO), 6.79 (br b,
1H, NH2-CO), 3.95 (m, 2H, CH2–O), 2.54 (s, 1.5H, CH3-N), 2.51 (s,

M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
4293
1.5H, CH3-N), 0.89 (s, 3H, CH3), 0.78 (s, 3H, CH3); white solid. Anal.
(C24H39N3O3ꢁHCl) C, H, Cl, N.
CH2CH2–O, O–CH2CH2–O), 3.52 (m, 2H, CH2–O), 3.36 (t, 1H, OH),
0.84 (s, 3H, CH3), 0.81 (s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 64 was hydrolyzed
according to the general method to give 65 in 85% yield. 1H NMR
(300 MHz, acetone-d6, ppm from TMS): d 3.71–3.47 (m, 3H, CH2–
OH), 1.08 (s, 3H, CH3), 0.89 (s, 3H, CH3).
5.3.58. (E,Z)-3-(3-N-Methylaminopropoxyimino)-6
methoxycarbonylandrostane-17-one hydrochloride (58)
Prepared in 65% yield from 6 -methoxycarbonylandrostane-
a-
a
3,17-dione 81 and 3-N-methylaminopropoxyamine dihydrochlo-
ride. 1H NMR (300 MHz, DMSO-d6, ppm from TMS): d 8.36 (br b,
2H, NH2þ), 3.95 (m, 2H, CH2–O), 3.61 (s, 1.5H, CH3–O), 3.60 (s,
1.5H, CH3–O), 2.98 (m, 0.5H, H-2eq E isomer), 2.87 (m, 2H, CH2–
N), 2.77 (m, 0.5H, H-4eq Z isomer), 2.53 (s, 1.5H, CH3-N), 2.52 (s,
1.5H, CH3-N), 0.91 (s, 3H, CH3), 0.78 (s, 3H, CH3); white solid. Anal.
(C25H40N2O4ꢁHCl) C, H, Cl, N.
5.4.4. 6b-Methoxymethylandrostane-3,17-dione (67)
To a stirred solution of 3,3:17,17-bis(ethylendioxy)-6b-hydrox-
ymethylandrostane 64 (0.80 g, 1.9 mmol) in dry THF (11 mL) at
0 °C, under N2, NaH (60% dispersion, 96 mg, 2.4 mmol) was added.
After stirring the mixture at 0 °C for 1 h, CH3I (144 lL, 0.33 g,
1.7 mmol) was added. After stirring overnight at room tempera-
ture, H2O (10 mL) was added. The mixture extracted with EtOAc
(2 ꢂ 20 mL). The combined organic extracts were dried over
Na2SO4, filtered and evaporated to dryness. The residue was puri-
fied by flash chromatography (SiO2, n-hexane/acetone 90/10) to
5.4. 3,17-Diketo intermediates
5.4.1. 6-Methyleneandrostane-3,17-dione (61)
To a stirred suspension of methyltriphenylphosphonium bro-
mide (9.50 g, 26.6 mmol) in dry THF (77 mL) cooled at 0 °C under
N2, potassium tert-butoxide (2.91 g, 25.9 mmol) was added. After
stirring for 10 min, a solution of 3,3:17,17-bis(ethylendioxy)andro-
stane-6-one 59 (2.60 g, 6.6 mmol) in dry THF (77 mL) was added
dropwise at room temperature over 0.5 h. After 0.5 h at room tem-
perature, the mixture was quenched by addition of 5% NaH2PO4
aqueous solution and extracted with Et2O (2 ꢂ 60 mL). The com-
bined organic extracts were washed with 5% NaH2PO4 aqueous solu-
tion, brine, driedover Na2SO4 and evaporatedtodryness. Theresidue
was purified by flash chromatography (SiO2, cyclohexane/EtOAc 85/
15) to give 3,3:17,17-bis(ethylendioxy)-6-methyleneandrostane 60
(2.66 g, 97%). 1H NMR (300 MHz, acetone-d6, ppm from TMS): d 4.68
(m, 1H, C@CH2), 4.36 (m, 1H, C@CH2), 3.88–3.71 (m, 8H, O–CH2CH2–
O, O–CH2CH2–O), 0.74 (s, 3H, CH3), 0.62 (s, 3H, CH3).
give
3,3:17,17-bis(ethylendioxy)-6b-methoxymethylandrostane
66 (0.70 g, 84%). 1H NMR (300 MHz, acetone-d6, ppm from TMS):
d 3.80 (m, 8H, O–CH2CH2–O, O–CH2CH2–O), 3.32 (m, 2H, CH2–O),
3.24 (s, 3H, CH3–O), 0.84 (s, 3H, CH3), 0.83 (s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 66 was hydrolyzed
according to the general method to give 67 in 90% yield. The crude
was purified by flash chromatography (SiO2, n-hexane/CH2Cl2/ace-
tone 70/10/20). 1H NMR (300 MHz, acetone-d6, ppm from TMS): d
3.45 (m, 2H, CH2–O), 3.27 (s, 3H, CH3–O), 1.10 (s, 3H, CH3), 0.89 (s,
3H, CH3).
5.4.5. 6-(Spirocyclopropane)androstane-3,17-dione (68)
To a stirred solution of 3,3:17,17-bis(ethylendioxy)-6-methyl-
eneandrostane 60 (200 mg, 0.51 mmol) in dry toluene (10 mL) un-
der N2, 1 M Et2Zn in n-hexane (2.5 mL) was added. After heating at
60 °C, CH2I2 (0.42 mL, 1.4 g, 5.21 mmol) was added in portions over
15 min. After 26 h the mixture was cooled and quenched by careful
addition of 1 N HCl. The suspension was extracted with Et2O. The
combined organic extracts were washed with 5% aqueous NaHCO3
solution, brine, dried over Na2SO4 and evaporated to dryness. The
crude product was dissolved in acetone (20 mL) and PTSAꢁH2O
(39 mg, 0.20 mmol) was added and the solution stirred at room
temperature for 1 h. The solution was neutralized by addition of
5% aqueous NaHCO3 and acetone was evaporated. The aqueous
suspension was extracted with EtOAc. The combined organic ex-
tracts were washed with H2O, dried over Na2SO4, and evaporated
to dryness. The residue was purified by flash chromatography
(SiO2, n-hexane/CH2Cl2/EtOAc 90/5/5) to give the title compound
68 (78 mg, 48%). 1H NMR (300 MHz, acetone-d6, ppm from TMS):
d 1.17 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.60-(-0.15) (m, 4H,
cyclopropane).
The 3,3:17,17-bis(ethylendioxy) derivative 60 (1.05 g, 2.7 mmol)
was hydrolyzed according to the general method to give 61 in 87%
yield. 1H NMR (300 MHz, acetone-d6, ppm from TMS): d 4.85 (m,
1H, C@CH2), 4.50 (m, 1H, C@CH2), 0.92 (s, 3H, CH3), 0.86 (s, 3H, CH3).
5.4.2. (6S)-6-Spiro-(20-oxirane)androstane-3,17-dione (62) and
(6R)-6-spiro-(20-oxirane)androstane-3,17-dione (63)
To a solution of 6-methyleneandrostane-3,17-dione 61 (0.76 g,
2.5 mmol) in CH2Cl2 (44 mL) stirred at 0 °C, MCPBA (0.933 mg,
5.4 mmol) was added in three portions over 0.5 h. After stirring
at room temperature for 3 h the mixture was washed with 5%
aqueous NaHCO3 solution, 40% aqueous NaHSO3 solution, 5% aque-
ous Na2HPO4 solution and brine. The organic layer was dried over
Na2SO4 and evaporated to dryness. The residue was purified by
flash chromatography (SiO2, toluene/acetone 95/5) to give the title
compounds 62 (30%) and 63 (15%). 1H NMR (300 MHz, acetone-d6,
ppm from TMS): 62: d 2.79 (d, 1H, C–CH2–O), 2.59 (d, 1H, C–CH2–
O), 1.16 (s, 3H, CH3), 0.88 (s, 3H, CH3); 63: d 1.17 (s, 3H, CH3), 0.90
(s, 3H, CH3).
5.4.6. 6b-Methylandrostane-3,17-dione (71)
To a stirred solution of 3,3:17,17-bis(ethylendioxy)-6b-hydrox-
ymethylandrostane 64 (90 mg, 0.22 mmol) and DMAP (5 mg,
0.041 mmol) in CH2Cl2 (3 mL) under N2, TCDI (78 mg, 0.44 mmol)
was added. After stirring 2 h at 40 °C H2O was added and the mix-
ture was extracted with CH2Cl2 (2ꢂ). The combined organic ex-
tracts were washed with brine, dried over Na2SO4, filtered and
evaporated to dryness. The mixture was purified by flash chroma-
tography (SiO2, n-hexane/CH2Cl2/acetone 70/15/15) to give O-
[3,3:17,17-bis(ethylendioxy)androstane-6b-ylmethyl] imidazole-
1-carbothioate 69 (95 mg, 83%). 1H NMR (300 MHz, DMSO-d6,
ppm from TMS): d 8.43 (dd, 1H, imidazole), 7.76 (dd, 1H, imidaz-
ole), 7.08 (dd, 1H, imidazole), 4.82 (dd, 1H, CH2–O), 4.68 (dd, 1H,
CH2–O), 3.80 (m, 8H, O–CH2CH2–O, O–CH2CH2–O), 0.84 (s, 3H,
CH3), 0.79 (s, 3H, CH3).
5.4.3. 6b-Hydroxymethylandrostane-3,17-dione (65)
To a stirred solution of 3,3:17,17-bis(ethylendioxy)-6-methy-
lene-androstane 60 (2.89 g, 7.4 mmol) in dry THF (29 mL) at 0 °C
.
under N2, 1 M BH3 THF complex in THF (5.21 mL, 5.21 mmol) was
added. After completing the addition, the mixture was stirred at
0 °C for 3 h. H2O (2.3 mL) was cautiously added dropwise followed
by 3 N NaOH (3 mL) and 9.8 M H2O2 (0.91 mL). After stirring at
room temperature overnight, H2O (20 mL) was added. The mixture
was extracted with EtOAc (2 ꢂ 20 mL). The combined organic ex-
tracts were washed with brine, dried over Na2SO4, filtered and
evaporated to dryness. The residue was purified by flash chroma-
tography (SiO2, n-hexane/EtOAc 45/55) to give 3,3:17,17-bis(ethyl-
endioxy)-6b-hydroxymethylandrostane 64 (2.86 g, 95%). 1H NMR
(300 MHz, acetone-d6, ppm from TMS): d 3.94–3.75 (m, 8H, O–
To a stirred solution of Ph3SnH (193 mg, 0.55 mmol) in dry tol-
uene (2 mL) under Ar, AIBN (5 mg, 0.030 mmol) was added. After

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M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
stirring 20 min at 90 °C, a solution of 69 (95 mg, 0.18 mmol) in dry
toluene (2 mL) was added dropwise. After stirring for 2 h at 110 °C,
the mixture was evaporated to dryness and the residue purified by
flash chromatography (SiO2, n-hexane/EtOAc 98/2) to give
3,3:17,17-bis(ethylendioxy)-6b-methylandrostane 70 (30 mg,
42%). 1H NMR (300 MHz, acetone-d6, ppm from TMS): d 3.80 (m,
8H, O–CH2CH2–O, O–CH2CH2–O), 0.92 (s, 3H, CH3), 0.88 (d, 3H,
CH3), 0.85 (s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 70 was hydrolyzed
according to the general method to give 71 in 94% yield. 1H NMR
(300 MHz, acetone-d6, ppm from TMS): d 1.18 (s, 3H, CH3), 0.98
(d, 3H, CH3), 0.90 (s, 3H, CH3).
5.4.9. 6
Carbamoylandrostane-3,17-dione (80)
-Formylandrostane-3,17-dione 78 was prepared in 85% yield
from 3,3:17,17-bis(ethylendioxy)-6 -formylandrostane 73 by the
Wittig reaction according to the procedure described above for
the preparation of 6-methyleneandrostane-3,17-dione 61. The
combined organic extracts were washed with H2O, dried over
a-Carboxyandrostane-3,17-dione (79) and 6a-
6a
a
Na2SO4 and evaporated to dryness to give 6a-formylandrostane-
3,17-dione 78. 1H NMR (300 MHz, acetone-d6, ppm from TMS): d
9.50 (d, 1H, CH–O), 1.16 (s, 3H, CH3), 0.88 (s, 3H, CH3).
To a stirred suspension of 6a-formylandrostane-3,17-dione 78
(1.77 g, 5.6 mmol) in t-ButOH (35 mL) and 5% aqueous Na2HPO4
solution (21.5 mL), 1 N aqueous KMnO4 (35 mL) was added. After
five minutes at room temperature, the mixture was quenched by
addition of 40% aqueous NaHSO3 solution. The suspension was fil-
tered, washed with H2O and the filtrate was freeze-dried. The res-
idue was taken up with H2O (50 mL) and extracted with EtOAc
(4 ꢂ 70 mL). The combined organic extracts were dried over
5.4.7. 6a-Vinylandrostane-3,17-dione (75)
To a solution of 3,3:17,17-bis(ethylendioxy)-6b-hydroxymethy-
landrostane 64 (0.63 g, 1.5 mmol) in DMSO (6 mL), IBX (0.87 g,
3.1 mmol) was added and stirred at room temperature for 1 h.
The mixture was quenched by addition of H2O (30 mL) and Et2O
(30 mL). After stirring for 15 min, the mixture was filtered and
the cake was washed with Et2O. The layers were separated and
the aqueous phase was extracted with Et2O (3ꢂ). The combined or-
ganic extracts were washed with brine, dried over Na2SO4 and
evaporated to dryness. The residue was purified by flash chroma-
tography (SiO2, n-hexane/EtOAc 75/35) to give 3,3:17,17-bis(ethyl-
endioxy)-6b-formylandrostane 72 (0.52 g, 83%). 1H NMR
(300 MHz, acetone-d6, ppm from TMS): d 9.92 (d, 1H, CH–O),
3.80 (m, 8H, O–CH2CH2–O, O–CH2CH2–O), 0.81 (s, 3H, CH3), 0.77
(s, 3H, CH3).
Na2SO4 and evaporated to dryness to give 6a-carboxyandrostane-
3,17-dione 79 (1.80 g, 96%). 1H NMR (300 MHz, acetone-d6, ppm
from TMS): d 11.99 (br b, 1H, COOH), 1.01 (s, 3H, CH3), 0.79 (s,
3H, CH3).
To a stirred suspension of 6a-carboxyandrostane-3,17-dione 79
(1.20 g, 3.9 mmol) in dry toluene (12 mL), SOCl2 (1.2 mL, 1.9 g,
118.97 mmol) was added. After stirring 5.5 h at 85 °C the solution
was cooled at 0 °C and 2 M NH3 solution in THF (6 mL) was added.
After stirring overnight at room temperature, the mixture was
evaporated to dryness. The residue was treated with CH2Cl2 and
H2O and extracted with CH2Cl2. The combined organic extracts
were washed with 10% K2CO3 solution, brine, dried over Na2SO4
and evaporated to dryness. The residue was purified by flash chro-
matography (SiO2, n-hexane/acetone 50/50) to give the title com-
pound 80 (720 mg, 60%). 1H NMR (300 MHz, DMSO-d6, ppm from
TMS): d 7.27 (br s, 1H, NH-CO), 6.78 (br s, 1H, NH-CO), 1.00 (s,
3H, CH3), 0.80 (s, 3H, CH3).
A mixture of 3,3:17,17-bis(ethylendioxy)-6b-formylandrostane
72 (0.61 g, 1.5 mmol), K2CO3 (0.90 g, 6.5 mmol) in MeOH (57 mL)
was stirred overnight at room temperature. After evaporation,
the residue was treated with H2O (20 mL) and extracted with
EtOAc (3 ꢂ 30 mL). The combined organic extracts were washed
with brine (3 ꢂ 20 mL), dried over Na2SO4 and evaporated to dry-
ness to give 3,3:17,17-bis(ethylendioxy)-6a-formylandrostane 73
(0.57 g, 94%). 1H NMR (300 MHz, acetone-d6, ppm from TMS): d
9.41 (d, 1H, CH–O), 3.80 (m, 8H, O–CH2CH2–O, O–CH2CH2–O),
0.90 (s, 3H, CH3), 0.84 (s, 3H, CH3).
5.4.10. 6
To a stirred solution of 6
(680 mg, 2.0 mmol) in CH2Cl2 (30 mL) at 0 °C, MeOH (160
a
-Methoxycarbonylandrostane-3,17-dione (81)
-carboxyandrostane-3,17-dione 79
L),
a
3,3:17,17-Bis(ethylendioxy)-6
pared in 70% yield from 3,3:17,17-bis(ethylendioxy)-6
a
-vinylandrostane 74 was pre-
-formy-
l
a
DMAP (20 mg, 0.16 mmol), and EDAC (800 mg, 4.1 mmol) were
added. After stirring overnight at room temperature, H2O was
added and the mixture was extracted with CH2Cl2 (2ꢂ). The com-
bined organic extracts were washed with H2O, brine, dried over
Na2SO4, filtered and evaporated to dryness. The residue was puri-
fied by flash chromatography (SiO2, n-hexane/EtOAc 60/40) to give
the title compound 81 (500 mg, 70%). 1H NMR (300 MHz, DMSO-d6,
ppm from TMS): d 3.59 (s, 3H, CH3–O), 1.02 (s, 3H, CH3), 0.79 (s, 3H,
CH3).
landrostane 73 according to the procedure described above for
the preparation of 3,3:17,17-bis(ethylendioxy)-6-methyleneand-
rostane 60. The crude was purified by flash chromatography
(SiO2, n-hexane/EtOAc 88/12). 1H NMR (300 MHz, acetone-d6,
ppm from TMS): d 5.47 (m, 1H, C@CH), 4.91 (m, 2H, C@CH2),
3.80 (m, 8H, O–CH2CH2–O, O–CH2CH2–O), 0.88 (s, 3H, CH3), 0.83
(s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 74 was hydrolyzed
according to the general method to give 75 in 92% yield. 1H NMR
(300 MHz, acetone-d6, ppm from TMS): d 5.51 (m, 1H, C@CH),
4.97 (m, 2H, C@CH2), 1.14 (s, 3H, CH3), 0.98 (s, 3H, CH3).
5.4.11. 6
a
-Hydroxymethylandrostane-3,17-dione (83)
To
a
stirred suspension of 3,3:17,17-bis(ethylendioxy)-6a-
formylandrostane 73 (0.52 g, 1.3 mmol) in dioxane/H2O 9/1
(25 mL), NaBH4 (49.0 mg, 1.3 mmol) was added and the mixture
was stirred overnight at room temperature. To the solution NaCl
was added and the layers were separated. The aqueous phase
was extracted with EtOAc (3ꢂ). The combined organic extracts
were washed with brine, dried over Na2SO4 and evaporated to dry-
5.4.8. 6
3,3:17,17-Bis(ethylendioxy)-6
76 was prepared in 96% yield from 3,3:17,17-bis(ethylendioxy)-
-vinylandrostane 74 by the oxidative hydroboration according
to the procedure described above for the preparation of
3,3:17,17-bis(ethylendioxy)-6b-hydroxymethylandrostane 64.
The crude was purified by flash chromatography (SiO2, n-hexane/
acetone 80/20). 1H NMR (300 MHz, DMSO-d6, ppm from TMS): d
4.25 (t, 1H, OH), 3.80 (m, 8H, O–CH2CH2–O, O–CH2CH2–O), 3.35
(m, 2H, CH2–O), 0.75 (s, 3H, CH3), 0.74 (s, 3H, CH3).
a
-(2-Hydroxyethyl)androstane-3,17-dione (77)
a
-(2-hydroxyethyl)androstane
6a
ness to give 3,3:17,17-bis(ethylendioxy)-6a-hydroxymethyland-
rostane 82 (0.45 g, 86%) 1H NMR (300 MHz, acetone-d6, ppm
from TMS): d 3.80 (m, 8H, O–CH2CH2–O, O–CH2CH2–O), 3.57–
3.25 (m, 3H, CH2–OH), 0.86 (s, 3H, CH3), 0.83 (s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 82 was hydrolyzed
according to the general method to give 83 in 85% yield. 1H NMR
(300 MHz, acetone-d6, ppm from TMS): d 3.50 (m, 3H, CH2–OH),
1.11 (s, 3H, CH3), 0.88 (s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 76 was hydrolyzed
according to the general method to give 77 in quantitative yield.
1H NMR (300 MHz, DMSO-d6, ppm from TMS): d 4.32 (t, 1H, OH),
3.39 (m, 2H, CH2–O), 0.98 (s, 3H, CH3), 0.79 (s, 3H, CH3).

M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
4295
5.4.12. 6
a
-Methoxymethylandrostane-3,17-dione (85)
added in portions over 15 min. After stirring for 3 h at 80 °C, the
mixture was quenched at room temperature by careful addition
of H2O (200 mL). The suspension was extracted with Et2O. The
combined organic extracts were washed with brine, dried over
Na2SO4 and evaporated to dryness. The mixture was purified by
flash chromatography (SiO2, n-hexane/EtOAc 90/10) to give
To a stirred solution of 3,3:17,17-bis(ethylendioxy)-6
ymethylandrostane 82 (14, 0.80 g, 1,9 mmol) in dry THF (11 mL) at
0 °C, under N2, NaH (60% dispersion, 96 mg, 2,4 mmol) was added.
After stirring the mixture at 0 °C for 1 h, CH3I (144 lL, 0.33 g,
1.7 mmol) was added. After stirring overnight at room tempera-
ture, H2O (10 mL) was added and the mixture extracted with EtOAc
(2 ꢂ 20 mL). The combined organic extracts were dried over
Na2SO4, filtered and evaporated to dryness. The residue was puri-
fied by flash chromatography (SiO2, n-hexane/acetone 90/10) to
a
-hydrox-
3,3:17,17-bis(ethylendioxy)-6a-methylandrostane 89 (0.70 g,
90%). 1H NMR (300 MHz, acetone-d6, ppm from TMS): d 3.80 (m,
8H, O–CH2CH2–O, O–CH2CH2–O), 0.85 (s, 3H, CH3), 0.83 (s, 3H,
CH3), 0.79 (d, 3H, CH3).
give 3,3:17,17-bis(ethylendioxy)-6
a-methoxymethylandrostane
The 3,3:17,17-bis(ethylendioxy) derivative 89 was hydrolyzed
according to the general method to give 90 in 94% yield. 1H NMR
(300 MHz, acetone-d6, ppm from TMS): d 1.18 (s, 3H, CH3), 0.98
(d, 3H, CH3), 0.90 (s, 3H, CH3).
84 (0.70 g, 84%). 1H NMR (300 MHz, acetone-d6, ppm from TMS):
d 3.80 (m, 8H, O–CH2CH2–O, O–CH2CH2–O), 3.25 (dd, 1H, CH2–O),
3.23 (s, 3H, CH3–O), 3.14 (dd, 1H, CH2–O), 0.85 (s, 3H, CH3), 0.82
(s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 84 was hydrolyzed
according to the general method to give 85 in 88% yield. 1H NMR
(300 MHz, acetone-d6, ppm from TMS): d 3.25 (s, 3H, CH3–O),
3.24 (m, 2H, CH2–O), 1.11 (s, 3H, CH3), 0.87 (s, 3H, CH3).
5.4.15. Methyl {3,17-dioxoandrostane-6-[(E)-ylidene]}acetate
(92)
To a stirred solution of trimethylphosphonoacetate (5.17 mL,
5.8 g, 31.9 mmol) in DME (5.75 mL) at 0 °C under N2 a 1.5 M solution
of tert-butyllithium in n-pentane (18.5 mL) was added dropwise.
After stirring 15 min at the same temperature a solution of
3,3:17,17-bis(ethylendioxy)androstane-6-one 59 (1.00 g, 2.5 mmol)
in DME (15 mL) was added dropwise. The mixture was heated at
110 °C for 8 h and, after cooling, quenched by addition of H2O and ex-
tracted with EtOAc (3 ꢂ 50 mL). The combined organic extracts were
dried over Na2SO4 and evaporated to dryness. The residue was puri-
fiedbyflash chromatography(SiO2, n-hexane/CH2Cl2/acetone80/10/
10) to give {3,3:17,17-bis(ethylendioxy)androstane-6-[(E)-yli-
dene]}acetic acid methyl ester 91 (400 mg, 35%). 1H NMR
(300 MHz, acetone-d6, ppm from TMS: d 5.34 (s, 1H, C@CH), 4.05–
3.75 (m, 9H, O–CH2CH2–O, O–CH2CH2–O, H-7eq), 3.62 (s, 3H, CH3–
O), 0.82 (s, 3H, CH3), 0.72 (s, 3H, CH3).
5.4.13. 6a-Ethynylandrostane-3,17-dione (87)
To a stirred solution of (chloromethyl)triphenylphosphonium
chloride (1.20 g, 3.4 mmol) in dry THF (20 mL) at ꢀ78 °C under ar-
gon, 1.6 M n-butyllithium in n-hexane (1.5 mL) was added drop-
wise. After 30 min at room temperature, a solution of 3,3:17,17-
bis(ethylendioxy)-6a-formylandrostane 73 (0.28 g, 0.7 mmol) in
dry THF (7 mL) was added dropwise. The mixture was heated at
70 °C for 1 h and then cooled to room temperature. The mixture
was quenched by addition of brine and extracted with EtOAc
(3 ꢂ 50 mL). The combined organic extracts were dried over
Na2SO4, and evaporated to dryness. The crude product was dis-
solved in dry THF (20 mL) and stirred at ꢀ78 °C. To the resulting
solution 1.6 M n-butyllithium in n-hexane (2.24 mL) under argon
was added dropwise. After 1 h at room temperature the mixture
was quenched by addition of brine and extracted with Et2O
(3 ꢂ 50 mL). The combined organic extracts were dried over
Na2SO4, and evaporated to dryness to give 3,3:17,17-bis(ethylendi-
The 3,3:17,17-bis(ethylendioxy) derivative 91 was hydrolyzed
according to the general method to give 92 in 70% yield. The resi-
due was purified by flash chromatography (n-hexane/CH2Cl2/ace-
tone 75/15/15). 1H NMR (300 MHz, acetone-d6, ppm from TMS: d
5.42 (br s, 1H, C@CH), 4.16 (dd, 1H, H-7eq), 3.66 (s, 3H, CH3–O),
0.99 (s, 3H, CH3), 0.87 (s, 3H, CH3).
oxy)-6
be used in the next step without further purification. 1H NMR
(300 MHz, acetone-d6, ppm from TMS): 3.85 (m, 8H, O–
a-ethynylandrostane 86 (160 mg, 46%), sufficiently pure to
d
5.4.16. 6-[(E)-Ethyliden]androstane-3,17-dione (94)
CH2CH2–O, O–CH2CH2–O), 2.46 (d, 1H, C„CH), 0.82 (s, 3H, CH3),
0.86 (s, 3H, CH3).
3,3:17,17-Bis(ethylendioxy)-6-[(E)-ethyliden]androstane
93
was prepared in 96% yield from 3,3:17,17-bis(ethylendioxy)andro-
stane-6-one 59 and (ethyl)triphenylphosphonium bromide by the
Wittig reaction according to the procedure described above for
the preparation of 3,3:17,17-bis(ethylendioxy)-6-methyleneand-
rostane 60. The mixture was purified by flash chromatography
(SiO2, cyclohexane/EtOAc 85/15). 1H NMR (300 MHz, acetone-d6,
ppm from TMS): d 4.91 (m, 1H, C@CH), 3.80 (m, 8H, O–CH2CH2–
O, O–CH2CH2–O), 2.69 (m, 1H, H-7eq), 0.81 (s, 3H, CH3), 0.66 (s,
3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 93 was hydrolyzed
according to the general method to give 94 in 96% yield. 1H NMR
(300 MHz, acetone-d6, ppm from TMS): d 4.99 (m, 1H, C@CH),
2.86 (dd, 1H, H-7eq), 0.93 (s, 3H, CH3), 0.86 (s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 86 was hydrolyzed
according to the general method to give 87 in 46% yield. The resi-
due was purified by flash chromatography (SiO2, cyclohexane/
CH2Cl2/acetone 80/10/10). 1H NMR (300 MHz, acetone-d6, ppm
from TMS): d 2.56 (d, 1H, C„CH), 1.12 (s, 3H, CH3), 0.87 (s, 3H,
CH3).
5.4.14. 6
To a stirred solution of DABCO (0.55 g, 4.9 mmol) and 3,3:17,17-
bis(ethylendioxy)-6 -hydroxymethylandrostane 82 (1.00 g,
a-Methylandrostane-3,17-dione (90)
a
2.4 mmol) in dry CH2Cl2 (20 mL), under N2 at 0 °C, pTSCl (0.703 g,
3.4 mmol) was added. After stirring 2 h at room temperature, the
mixture was filtered and the cake was washed with CH2Cl2. The or-
ganic layer was washed with brine, dried over Na2SO4, filtered and
evaporated to dryness. The crude product was triturated with n-
hexane/EtOAc (60/40) and filtered. After drying under vacuum at
5.4.17. 6-Difluoromethyleneandrostane-3,17-dione (96)
To a stirred solution of diethyl difluoromethylene phosphonate
(0.67
lL) in DME (5.75 mL) in n-pentane (1.1 mL) at ꢀ78 °C, 1.5 M
40 °C, 3,3:17,17-bis(ethylendioxy)-6
a-[4-methyl(benzenesulfonyl-
pentane solution of tert-butyllithium (2.75 mL) was added drop-
wise under argon. After 15 min at the same temperature, a solution
of 3,3:17,17-bis(ethylendioxy)androstane-6-one 59 (0.50 g,
1.3 mmol) in DME (4.5 mL) and n-pentane (1.25 mL) was added
dropwise. The mixture was stirred at ꢀ78 °C for further 30 min
and warmed up to room temperature. n-Pentane was distilled off
and after heating at 80 °C for 4 h the mixture was quenched with
H2O and extracted with CH2Cl2 (3 ꢂ 50 mL). The combined organic
oxy)methyl]androstane 88 (1.11 g, 80%) was obtained. 1H NMR
(300 MHz, acetone-d6, ppm from TMS): d 4.00–3.74 (m, 10H, O–
CH2CH2–O, O–CH2CH2–O, CH2–O), 0.82 (s, 3H, CH3), 0.80 (s, 3H,
CH3).
To a stirred solution of NaBH4 (0.15 g, 3.9 mmol) in dry DMSO
(90 mL), under N2, 3,3:17,17-bis(ethylendioxy)-6a-[4-methyl(ben-
zenesulfonyloxy)methyl]androstane 88 (1.11 g, 1.9 mmol) was

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M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
extracts were dried over Na2SO4 and evaporated to dryness. The
residue was purified by flash chromatography (SiO2, cyclohex-
ane/Et2O 70/30) to give 3,3:17,17-bis(ethylendioxy)-6-difluorom-
ethyleneandrostane 95 (0.47 g, 85%). 1H NMR (300 MHz, acetone-
d6, ppm from TMS): d 3.85 (m, 8H, O–CH2CH2–O, O–CH2CH2–O),
0.83 (s, 3H, CH3), 0.84 (s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 95 was hydrolyzed
according to the general method to give 96 in 99% yield. 1H NMR
(300 MHz, acetone-d6, ppm from TMS): d 1.12 (s, 3H, CH3), 0.88
(s, 3H, CH3).
5.4.20. 6-[(E)-Hydroxyimino]androstane-3,17-dione (103)
To a stirred solution of 3,3:17,17-bis(ethylendioxy)androstane-
6-one 59 (1.10 g, 2.8 mmol) in THF (22 mL)
a solution of
.
NH2OHꢁHCl (0.33 g, 4.7 mmol), Na2HPO4 12H2O (1.71 g, 4.7 mmol)
in H2O (7.2 mL) was added. After stirring overnight at room tem-
perature, NaCl was added and the mixture was extracted with
EtOAc (2ꢂ). The combined organic extracts were washed with
brine, dried over Na2SO4, filtered and evaporated to dryness to give
3,3:17,17-bis(ethylendioxy)-6-[(E)-hydroxyimino]androstane 102
(1.08 g, 93%). 1H NMR (300 MHz, DMSO-d6, ppm from TMS): d
10.34 (s, 1H, N–OH), 3.80 (m, 8H, O–CH2CH2–O, O–CH2CH2–O),
3.16 (dd, 1H, H-7eq), 0.74 (s, 3H, CH3), 0.64 (s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 102 was hydrolyzed
according to the general method to give 103 in 70% yield. The res-
idue was purified by flash chromatography (SiO2, n-hexane/ace-
tone 70/30). 1H NMR (300 MHz, DMSO-d6, ppm from TMS): d
10.61 (s, 1H, N–OH), 3.29 (dd, 1H, H-7eq), 0.88 (s, 3H, CH3), 0.79
(s, 3H, CH3).
5.4.18. 3,17-Dioxoandrostane-6-[(E)-ylidene]acetonitrile (98)
To a stirred suspension of NaH (60% dispersion in mineral oil,
204 mg, 5.1 mmol) in THF (6 mL) at room temperature, diethyl
cyanomethylphosphonate (895 lL, 980 mg, 5.5 mmol) was added.
After stirring for 0.5 h 3,3:17,17-bis(ethylendioxy)androstane-6-
one 59 (200 mg, 0.5 mmol) was added to the yellow mixture. After
stirring at reflux for 2 h the mixture was quenched by addition of
brine and extracted with Et2O (3 ꢂ 50 mL). The combined organic
extracts were dried over Na2SO4, and evaporated to dryness. The
residue was purified by flash chromatography (SiO2, n-hexane/
CH2Cl2/acetone 70/20/20) to give 3,3:17,17-bis(ethylendi-
oxy)androstane-6-[(E)-ylidene]acetonitrile 97 (150 mg, 71%). 1H
NMR (300 MHz, acetone-d6, ppm from TMS): d 5.03 (t, 1H, C@CH),
3.85 (m, 8H, O–CH2CH2–O, O–CH2CH2–O), 2.90 (dd, 1H, H-7eq),
0.84 (s, 3H, CH3), 0.73 (s, 3H, CH3).
5.4.21. 6-[(E)-Methoxyimino]androstane-3,17-dione (105)
3,3:17,17-Bis(ethylendioxy)-6-[(E)-methoxyimino]androstane
104 was prepared from 3,3:17,17-bis(ethylendioxy)androstane-6-
one 59 (1.00 g, 2.5 mmol) and NH2OCH3ꢁHCl by the procedure de-
scribed above for the preparation of 3,3:17,17-bis(ethylendioxy)-
6-[(E)-hydroxyimino]androstane 103. The combined organic ex-
tracts were washed with brine, dried over Na2SO4, filtered and
evaporated to dryness to give 3,3:17,17-bis(ethylendioxy)-6-[(E)-
methoxyimino]androstane 104 (1.04 g, 97%). 1H NMR (300 MHz,
acetone-d6, ppm from TMS): d 3.80 (m, 8H, O–CH2CH2–O, O–
CH2CH2–O), 3.73 (s, 3H, CH3–O), 3.22 (dd, 1H, H-7eq), 0.82 (s, 3H,
CH3), 0.75 (s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 97 was hydrolyzed
according to the general method to give 98 in 87% yield. 1H NMR
(300 MHz, acetone-d6, ppm from TMS): d 5.15 (s, 1H, C@CH),
3.04 (dd, 1H, H-7eq), 1.01 (s, 3H, CH3), 0.90 (s, 3H, CH3).
5.4.19. 6-[(E)-2-Hydroxyethylidene]androstane-3,17-dione
(101)
The 3,3:17,17-bis(ethylendioxy) derivative 104 was hydrolyzed
according to the general method to give 105 in 70% yield. 1H NMR
(300 MHz, acetone-d6, ppm from TMS): d 3.78 (s, 3H, CH3–O), 3.37
(dd, 1H, H-7eq), 1.01 (s, 3H, CH3), 0.98 (s, 3H, CH3).
To a stirred solution of 3,3:17,17-bis(ethylendioxy)androstane-
6-[(E)-ylidene]acetonitrile 97 (214 mg, 0.5 mmol) in dry CH2Cl2
(10 mL) at ꢀ78 °C under N2, 1 M DIBAH in CH2Cl2 (1.56 mL) was
added dropwise. The mixture was stirred at ꢀ78 °C for 1 h and then
quenched by careful addition of a 2 M solution of isopropyl alcohol
5.4.22. 6-[(E)-Ethoxyimino]androstane-3,17-dione (107)
3,3:17,17-Bis(ethylendioxy)-6-[(E)-ethoxyimino]androstane
106 was prepared in 90% yield starting from 3,3:17,17-bis(ethylen-
dioxy)androstane-6-one 59 and NH2OCH2CH3ꢁHCl by the proce-
dure described above for the preparation of 3,3:17,17-
bis(ethylendioxy)-6-[(E)-hydroxyimino]androstane 103. 1H NMR
(300 MHz, acetone-d6, ppm from TMS): d 3.99 (q, 2H, CH2–O),
3.80 (m, 8H, O–CH2CH2–O, O–CH2CH2–O), 3.25 (dd, 1H, H-7eq),
1.17 (t, 3H, CH3), 0.82 (s, 3H, CH3), 0.75 (s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 106 was hydrolyzed
according to the general method to give 107 in quantitative yield.
1H NMR (300 MHz, acetone-d6, ppm from TMS): d 4.03 (q, 2H, CH2–
O), 3.40 (dd, 1H, H-7eq), 1.20 (t, 3H, CH3), 1.01 (s, 3H, CH3), 0.87 (s,
3H, CH3).
in toluene (0.80 mL). After 1 h, H2O (70 lL) and THF (2.8 mL) were
added and after an additional hour SiO2 (0.76 g) and Na2SO4
(1.52 g) were added. The mixture was stirred for 1 h, filtered
through a Celite pad and the filter cake washed with EtOAc. The fil-
trate was dried over Na2SO4, evaporated to give 3,3:17,17-bis(eth-
ylendioxy)androstane-6-[(E)-ylidene]acetaldehyde 99 (0.18 g,
55%). 1H NMR (300 MHz, acetone-d6, ppm from TMS): d 9.98 (dd,
1H, CH–O), 5.46 (d, 1H, C@CH), 3.80 (m, 8H, O–CH2CH2–O, O–
CH2CH2–O), 3.35 (dd, 1H, H-7eq), 0.75 (s, 3H, CH3), 0.64 (s, 3H,
CH3).
To a stirred suspension of 3,3:17,17-bis(ethylendioxy)andro-
stane-6-[(E)-ylidene]acetaldehyde 99 (110 mg, 0.26 mmol) in
MeOH (2.5 mL) at 0 °C, NaBH4 (5 mg, 0.13 mmol) was added and
the mixture was stirred for 1 h. Acetone (100
l
L) was added and
5.4.23. 6-[(E)-Allyloxyimino]androstane-3,17-dione (109)
Using the same reaction conditions described for the prepara-
tion of 103 and starting from 3,3:17,17-bis(ethylendioxy)andro-
stane-6-one 59 (250 mg, 0.6 mmol) and O-allylhydroxylamine
hydrochloride (140 mg, 1.27 mmol), 3,3:17,17-bis(ethylen-dioxy)-
6-[(E)-allyloxyimino]androstane 108 was obtained (260 mg, 91%).
1H NMR (300 MHz, acetone-d6, ppm from TMS): d 5.97 (m, 1H,
C@CH), 5.22 (m, 1H, C@CH2), 5.11 (m, 1H, C@CH2), 4.47 (m, 2H,
CH2–O), 3.80 (m, 8H, O–CH2CH2–O, O–CH2CH2–O), 3.28 (dd, 1H,
H-7eq), 0.82 (s, 3H, CH3), 0.75 (s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 108 was hydrolyzed
according to the general method to give 109 in 70% yield. 1H NMR
(300 MHz, acetone-d6, ppm from TMS): d 5.99 (m, 1H, C@CH), 5.25
(m, 1H, C@CH2), 5.14 (m, 1H, C@CH2), 4.52 (m, 2H, CH2–O), 1.01 (s,
3H, CH3), 0.88 (s, 3H, CH3).
the mixture evaporated. The residue was treated with H2O and ex-
tracted with EtOAc. The combined organic extracts were washed
with brine, dried over Na2SO4 and evaporated to dryness to give
3,3:17,17-bis(ethylendioxy)-6-[(E)-2-hydroxyethylidene]andro-
stane 100 (100 mg, 90%). 1H NMR (300 MHz, acetone-d6, ppm from
TMS): d 5.07 (m, 1H, C@CH), 4.11 (m, 2H, CH2–O), 3.80 (m, 8H, O–
CH2CH2–O, O–CH2CH2–O), 3.41 (t, 1H, OH), 2.67 (m, 1H, H-7eq),
0.80 (s, 3H, CH3), 0.69 (s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 100 was hydrolyzed
according to the general method to give 101 in 60% yield. The
crude was purified by flash chromatography (SiO2, n-hexane/ace-
tone 70/30). 1H NMR (300 MHz, acetone-d6, ppm from TMS: d
5.14 (m, 1H, C@CH), 4.17 (m, 2H, CH2–O), 3.53 (t, 1H, OH), 2.84
(dd, 1H, H-7eq), 0.97 (s, 3H, CH3), 0.85 (s, 3H, CH3).

M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
4297
5.4.24. 6a-Cyanoandrostane-3,17-dione (111)
5.4.27. 6a-Ethoxyandrostane-3,17-dione (118)
To a solution of toluene-4-sulfonylmethyl isocyanide (2.23 g,
11.4 mmol) in anhydrous DMSO (13 mL), stirred under N2, potas-
sium tert-butoxide (3.55 g, 31.5 mmol) was added. After stirring
for 5 min, anhydrous MeOH (0.40 mL) was added dropwise, fol-
lowed after 10 min by 3,3:17,17-bis(ethylendioxy)androstane-6-
one 59 (3.27 g, 8.4 mmol). After 72 h at room temperature, the
reaction was quenched by addition of H2O and the mixture was
neutralized by addition of 1 N HCl and extracted with EtOAc
(3 ꢂ 50 mL). The combined organic extracts were washed with
H2O, 5% NaHCO3 solution, dried over Na2SO4, and evaporated to
dryness. The residue was purified by flash chromatography (SiO2,
To a solution of 3,3:17,17-bis(ethylendioxy)androstane-6a-ol
112 (1.40 g, 3.56 mmol) in dry THF (35 mL) at 0 °C, 60% NaH in
oil (0.45 mg, 11.0 mmol) was added. After 30 min C2H5I (0.86 mL,
10.8 mmol) was added and the mixture was heated at reflux for
5 h. After cooling the reaction was quenched with water and ex-
tracted with EtOAc. The organic extracts were dried over Na2SO4,
and evaporated to dryness. The crude was purified by flash chro-
matography (SiO2, cyclohexane/acetone 95/5) to give 6
a
-ethoxy-
3,3:17,17-bis(ethylendioxy)androstane 117 as
a white foam
(0.78 g, 52%). 1H NMR (300 MHz, acetone-d6, ppm from TMS): d
3.85 (m, 8H, O–CH2CH2–O, O–CH2CH2–O), 3.60–3.20 (m, 2H,
CH2–O), 3.01 (ddd, 1H, CH–O), 1.08 (t, 3H, CH3), 0.85 (s, 3H, CH3),
0.82 (s, 3H, CH3).
n-hexane/EtOAc 70/30) to give 6a-cyano-3,3:17,17-bis(ethylendi-
oxy)androstane 110 (1.05 g, 31%). 1H NMR (300 MHz, acetone-d6,
ppm from TMS): d 3.80 (m, 8H, O–CH2CH2–O, O–CH2CH2–O), 2.60
(m, 1H, CH-CN), 0.89 (s, 3H, CH3), 0.82 (s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 117 was hydrolyzed
according to the general method. The crude was purified by flash
The 3,3:17,17-bis(ethylendioxy) derivative 110 was hydrolyzed
according to the general method to give 111 in 75% yield. 1H NMR
(300 MHz, acetone-d6, ppm from TMS): d 2.82 (ddd, 1H, CH-CN),
1.16 (s, 3H, CH3), 0.87 (s, 3H, CH3).
chromatography (SiO2, cyclohexane/acetone 80/20) to give 6a-eth-
oxyandrostane-3,17-dione 118 as a white solid (0.50 g, 84%). 1H
NMR (300 MHz, acetone-d6, ppm from TMS): d 3.70–3.30 (m, 2H,
CH2–O), 3.20 (m, 1H, CH–O), 1.12 (s, 3H, CH3), 1.11 (t, 3H, CH3),
0.87 (s, 3H, CH3).
5.4.25. 3,17-Dioxoandrostane-6a-yl nitrate (114)
5.4.28. 3,17-Dioxoandrostane-6b-yl nitrate (121)
To a solution of acetic anhydride (2.53 mL, 2.7 g, 26.4 mmol)
3,3:17,17-Bis(ethylendioxy)androstane-6b-yl nitrate 120 was
prepared in 50% yield from 3,3:17,17-bis(ethylendioxy)andro-
stane-6b-ol5 119 following the procedure described above for the
and 65% HNO3 (0.592 mL) cooled at 0 °C, 3,3:17,17-bis(ethylendi-
oxy)androstane-6a
-ol5 112 (2.5 g, 6.4 mmol) was added in one
portion. After 2 h the mixture was quenched by careful addition
of ice and 5% aqueous NaHCO3 solution and was extracted with
CH2Cl2 (3 ꢂ 50 mL). The combined organic extracts were washed
with H2O, dried over Na2SO4, and evaporated to dryness to give
preparation of 3,3:17,17-bis(ethylendioxy)androstane-6a-yl ni-
trate 113. 1H NMR (300 MHz, acetone-d6, ppm from TMS): d 5.16
(m, 1H, CH–ON), 3.80 (m, 8H, O–CH2CH2–O, O–CH2CH2–O), 1.00
(s, 3H, CH3), 0.85 (s, 3H, CH3).
3,3:17,17-bis(ethylendioxy)androstane-6a-yl nitrate 113 as a
The 3,3:17,17-bis(ethylendioxy) derivative 120 was hydrolyzed
according to the general method to give 121 in 75% yield. The
crude product was purified by flash chromatography (SiO2, cyclo-
hexane/acetone/CH2Cl2 70/15/15) to give II-ab. 1H NMR
(300 MHz, acetone-d6, ppm from TMS): d 5.24 (ddd, 1H), 2.72
(dd, 1H, CH–ON), 1.25 (s, 3H, CH3), 0.90 (s, 3H, CH3).
white solid (2.50 g, 89%). 1H NMR (300 MHz, acetone-d6, ppm from
TMS): d 4.94 (m, 1H, CH–ON), 3.80 (m, 8H, O–CH2CH2–O, O–
CH2CH2–O), 0.98 (s, 3H, CH3), 0.85 (s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 113 was hydrolyzed
according to the general method to give 114 in 75% yield. 1H NMR
(300 MHz, acetone-d6, ppm from TMS): d 5.09 (ddd, 1H, CH–ON),
1.25 (s, 3H, CH3), 0.90 (s, 3H, CH3).
5.4.29. 6b-Methoxyandrostane-3,17-dione (123)
To a solution of 3,3:17,17-bis(ethylendioxy)androstane-6b-ol
119 (1.10 g, 2.8 mmol) in dry THF (16 mL) at 0 °C, 60% NaH in oil
(0.45 mg, 3.3 mmol) was added. After 30 min CH3I (0.29 lL,
5.4.26. 6a-Methoxyandrostane-3,17-dione (116)
20% KH in oil (0.59 mg, 3.5 mmol) were washed with dry Et2O
(2 ꢂ 5 mL) in the reaction flask under nitrogen. The remaining so-
lid was suspended in dry THF (5 mL) and 3,3:17,17-bis(ethylendi-
4.5 mmol) was added. After stirring at room temperature for
4 days, the reaction was quenched with water and extracted with
EtOAc. The organic extracts were dried over Na2SO4, and evapo-
rated to dryness. The crude was purified by flash chromatography
(SiO2, cyclohexane/acetone 80/20) to give 6b-methoxy-3,3:17,17-
bis(ethylendioxy)androstane 122 as a white foam (0.56 g, 49%).
1H NMR (300 MHz, acetone-d6, ppm from TMS): d 3.85 (m, 8H,
O–CH2CH2–O, O–CH2CH2–O), 3.21 (s, 3H, CH3–O), 3.16 (m, 1H,
CH–O), 0.95 (s, 3H, CH3), 0.84 (s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 122 was hydrolyzed
according to the general method to give 123 as a white foam in 95%
yield. 1H NMR (300 MHz, acetone-d6, ppm from TMS): d 3.27 (s, 3H,
CH3–O), 3.24 (m, 1H, CH–O), 1.18 (s, 3H, CH3), 0.88 (s, 3H, CH3).
oxy)androstane-6
added dropwise. After 30 min CH3I (220
a
-ol5 112 (1.15 g, 2.9 mmol) in THF (11 mL) was
lL, 3.5 mmol) was added
in one portion. After 1 h the mixture was quenched with water
and extracted with EtOAc. The organic extracts were dried over
Na2SO4, and evaporated to dryness. The crude was purified by
flash chromatography (SiO2, cyclohexane/acetone 80/20) to give
6a
-methoxy-3,3:17,17-bis(ethylendioxy)androstane 115 as
a
white solid (0.93 g, 79%). 1H NMR (300 MHz, acetone-d6, ppm
from TMS): d 3.78 (m, 8H, O–CH2CH2–O, O–CH2CH2–O), 3.18 (s,
3H, CH3–O), 2.85 (m, 1H, CH–O), 0.76 (s, 3H, CH3), 0.75 (s, 3H,
CH3).
A solution of 3,3:17,17-bis(ethylendioxy) derivative 115 (0.80 g,
1.96 mmol) and PTSAꢁH2O (1.86 g) in acetone (8 mL) was stirred at
room temperature for 1 h. The solution was added with water (7 L)
and after 1 h acetone was evaporated. The aqueous suspension was
extracted with CH2Cl2 (3ꢂ). The combined organic extracts were
dried over Na2SO4 and evaporated to dryness. The crude was puri-
fied by flash chromatography (SiO2, cyclohexane/acetone 80/20) to
5.4.30. 6
a
-Formamidoandrostane-3,17-dione (126)
stirred solution of 3,3:17,17-bis(ethylendioxy)-6-(E)-
To
a
hydroxyiminoandrostane 102 (0.88 g, 2.1 mmol) in n-PrOH
(26 mL), Na (2.0 g) was added in small pieces over 20 min. The
mixture was stirred at reflux for 2 h. After cooling to room temper-
ature, the mixture was quenched by careful addition of MeOH. To
the solution H2O was added carefully and the organic solvent was
evaporated. The mixture was extracted with CH2Cl2 (3 ꢂ 50 mL).
The combined organic extracts were washed with brine, dried over
Na2SO4, filtered and evaporated to dryness. The mixture was puri-
give
6a-methoxyandrostane-3,17-dione 116 as a white solid
(0.54 g, 86%). 1H NMR (300 MHz, acetone-d6, ppm from TMS): d
3.22 (s, 3H, CH3–O), 3.02 (ddd, 1H, CH–O), 0.99 (s, 3H, CH3), 0.79
(s, 3H, CH3).

4298
M. Gobbini et al. / Bioorg. Med. Chem. 18 (2010) 4275–4299
fied by flash chromatography (SiO2, CHCl3/MeOH/26% NH4OH 90/
5.6.2. Inotropic and Toxic Effects in Anesthetized Guinea pigs
Male guinea pigs weighing 400-450 g were anesthetized with
urethane (1.5 g/kg ip). Body temperature was maintained at 37°C
bya homeothermic blanketsystem(HarwardApparatus). Amicrotip
pressure transducer (Millar SPR-407, Houston TX) was introduced
into the left ventricle through the right carotid artery to measure
ventricular pressure (LVP). Recordings were fed to a Gould (RS
3800) polygraph and to an AST 486 computer and analyzed by IDAS
software (Mangoni, Pisa, Italy). A polythene cannula (PE50) was in-
serted into a jugular vein for drug infusion and the trachea was intu-
bated to facilitate spontaneous respiration. After a stabilization
period, the test substance was injected at the rate of 0.16 mL/min
by means of a Harvard P22 pump (Harward Apparatus) until the ani-
mal died or up to a maximum of 90 min. The doses inducing the max-
imum inotropic effect and death were determined. The compounds
were dissolved in DMSO and the resulting solution diluted with
physiological solution to obtain final solutions containing 1% DMSO.
The starting concentration of the infused solution was based on the
EC50 found in the electrically driven guinea pig atria. If no effect was
found, the concentration was raised until satisfactory inotropic
effect was found or death occurred.
10/1) to give 3,3:17,17-bis(ethylendioxy)-6a-aminoandrostane
124 (0.45 g, 53%). 1H NMR (300 MHz, DMSO-d6, ppm from TMS):
d 3.80 (m, 8H, O–CH2CH2–O, O–CH2CH2–O), 2.29 (m, 1H, CH–N),
0.75 (s, 3H, CH3), 0.74 (s, 3H, CH3).
A 2 M solution of formic acid in CHCl3 (0.67 mL) was added
dropwise to a solution of DCC (106 mg, 0.5 mmol) in CHCl3 at
0 °C. The mixture was stirred for further 5 min and then added to
an ice-cooled solution of 3,3:17,17-bis(ethylendioxy)-6a-amino-
androstane 124 (100 mg, 0.2 mmol) in pyridine (0.70 mL) over
30 min. The mixture was then stirred in an ice bath for 4 h. Evap-
oration of the solvent was followed by addition of Et2O. The precip-
itate was removed by filtration and washed with Et2O. The
combined organic extracts were evaporated to dryness to give
3,3:17,17-bis(ethylendioxy)-6
a
-formamidoandrostane
125
(100 mg, 95%). 1H NMR (300 MHz, DMSO-d6, ppm from TMS): d
7.98–7.43 (m, 2H, NH–CHO), 3.89–3.00 (m, 9H, O–CH2CH2–O, O–
CH2CH2–O, CH–N), 0.81 (s, 3H, CH3), 0.77 (s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 125 was hydrolyzed
according to the general method to give 126 in 96% yield. 1H NMR
(300 MHz, DMSO-d6, ppm from TMS): d 8.02–7.56 (m, 2H, NH–
CHO), 3.74 (m, 1H, CH–N), 1.04 (s, 3H, CH3), 0.80 (s, 3H, CH3).
Supplementary data
5.4.31. 6
To a stirred solution of 3,3:17,17-bis(ethylendioxy)-6
androstane 124 (100 mg, 0.2 mmol) in dry pyridine (0.5 mL) at
0 °C, under N2, (CH3CO)2O (48 L, 51.8 mg, 0.5 mmol) was added
a-Acetamidoandrostane-3,17-dione (128)
a
-amino-
Supplementary data (elemental analysis results for target com-
pounds and Table of CoMFA predictions of the Na+,K+-ATPase
inhibitory values of the training set and of the test set) associated
with this article can be found, in the online version, at doi:10.1016/
j.bmc.2010.04.095.
l
dropwise. The mixture was stirred at room temperature for 1.5 h
and the solution was evaporated to dryness. The residue was taken
up with H2O and extracted with EtOAc (2 ꢂ 50 mL). The combined
organic extracts were washed with brine, dried over Na2SO4, fil-
tered and evaporated to dryness to give 3,3:17,17-bis(ethylendi-
References and notes
oxy)-6a
-acetamidoandrostane 127 (103 mg, 94%). 1H NMR
1. The Task Force for the Diagnosis and Treatment of Acute and Chronic Heart
Failure 2008 of the European Society of Cardiology Eur. Heart J. 2008, 2008,
2388; (b) Jessup, M.; Abraham, W. T.; Casey, D. E.; Feldman, A. M.; Francis, G. S.;
Ganiats, T. G.; Konstam, M. A.; Mancini, D. M.; Rahko, P. S.; Silver, M. A.;
Stevenson, L. W.; Yancy, C. W. Circulation 2009, 119, 1977.
(300 MHz, DMSO-d6, ppm from TMS): d 7.55 (d, 1H, NH), 3.80
(m, 8H, O–CH2CH2–O, O–CH2CH2–O), 3.53 (m, 1H, CH–N), 1.75 (s,
3H, CH3–CO), 0.80 (s, 3H, CH3), 0.75 (s, 3H, CH3).
The 3,3:17,17-bis(ethylendioxy) derivative 128 was hydrolyzed
according to the general method to give 127 in 96% yield. 1H NMR
(300 MHz, DMSO-d6, ppm from TMS): d 7.61 (d, 1H, NH), 3.67 (m,
1H, CH–N), 1.78 (s, 3H, CH3–CO), 1.04 (s, 3H, CH3), 0.80 (s, 3H, CH3).
2. Gobbini, M.; Cerri, A. Curr. Med. Chem. 2005, 12, 2343. and references cited
therein.
3. (a) The Digitalis Investigation Group N. Engl. J. Med. 1997, 336, 525; (b) Ahmed,
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K. F.; Gheorghiade, M. Eur. Heart J. 2006, 27, 178; (c) Ahmed, A.; Rich, M. W.;
Fleg, J. L.; Zile, M. R.; Young, J. B.; Kitzman, D. W.; Love, T. E.; Aronow, W. S.;
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5.5. Conformational energy calculations
4. Ooi, H.; Colucci, W. S. Cardiac Glycosides. In The Pharmacological Basis of
Therapeutics; Hardman, J. G., Limbird, L. E., Goodman Gilman, A., Eds., 10th ed.;
McGraw-Hill: New York, 2001; pp 916–921.
The conformational analyses of the target compounds 1–58 in
their E isomer form were performed using the OPLS2005 all-atom
force field as implemented in the MACROMODEL 9.5 program; 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. The Monte Carlo multiple minimum method
was used in the conformational search (OPLS2005, 5000 steps, tor-
sion rotations allowed on the amino chain and, where appropriate,
on the substituent in position 6 of the androstane scaffold). All con-
formations within 50 kJ/mol of the identified lowest energy con-
former were minimized again (Multiple Minimization routine in
the program) using OPLS2005 in conjunction with the GB/SA con-
tinuum model to simulate water solvation.
5. De Munari, S.; Cerri, A.; Gobbini, M.; Almirante, N.; Banfi, L.; Carzana, G.;
Ferrari, P.; Marazzi, M.; Micheletti, R.; Schiavone, A.; Sputore, S.; Torri, M.;
Zappavigna, M. P.; Melloni, P. J. Med. Chem. 2003, 46, 3644.
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A.; Zhang, D.; Cowart, D.; Valentini, G.; Carminati, P.; Gheorghiade, M. Am. J.
Cardiol. 2007, 99, 47A.
7. (a) Gheorghiade, M.; Blair, J. E. A.; Filippatos, G. S.; Macarie, C.; Ruzyllo, W.;
Korewicki, J.; Bubenek-Turconi, S. I.; Ceracchi, M.; Bianchetti, M.; Carminati, P.;
Kremastinos, D.; Valentini, G.; Hani, N.; Sabbah, H. N. J. Am. Coll. Cardiol. 2008,
51, 2276; (b) Khan, H.; Metra, M.; Blair, J. E. A.; Vogel, M.; Harinstein, M. E.;
Filippatos, G. S.; Sabbah, H. N.; Porchet, H.; Valentini, G.; Gheorghiade, M. Heart
Fail. Rev. 2009, 14, 277; (c) Shah, S. J.; Blair, J. E. A.; Filippatos, G. S.; Macarie, C.;
Ruzyllo, W.; Korewicki, J.; Bubenek-Turconi, S. I.; Ceracchi, M.; Bianchetti, M.;
Carminati, P.; Kremastinos, D.; Grzybowski, J.; Valentini, G.; Sabbah, H. N.;
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