 |
Description  |
|
|
The present invention relates to new steroid derivatives, and more
particularly new androstane 17-carboxylic acid esters substituted at the
3-position by a sugar residue, a process for their preparation, their use
in the production of drugs, as well as the drugs containing them.
French patent application No. 2,464,270 describes 14-amino steroid type
compounds, and in particular hydroxylated derivatives of 14-amino 21-nor
pregnane. In addition, steroid alkaloids of pregnane and androstane
substituted at the 14-position by an amino group are well known, for
example 14.beta.,20.alpha.-pregnanediol is described in A. Astier et al.,
Bull. Soc. Chim., No. 9-10, pp. 1581-1582 (1976); and other 14.beta.-amino
pregnanes and 14.beta.-amino substances are described in A. Astier et al.,
Tetrahedron, Vol. 34, pp. 1481-1486 (1978). However, neither the
pharmaceutical properties of these derivatives nor their use in therapy
has been described in these documents.
French patent application Nos. 2,531,964 and 2,547,586 describe derivatives
of 20-pregnanol, 21-nor 20-pregnanol, 12,20-pregnanediol and 21-nor
12,20-pregnanediol, substituted at the 14-position by an amino group and
at the 3-position by a sugar residue. These derivatives possess positive
inotropic properties enabling consideration of their use as active
ingredients in drugs used in the treatment of congestive heart failure.
East German Pat. No. 138,983 describes cardenolide derivatives,
characterized by the presence of a lactone group at the 17-position, and
an amino group at the 14-position.
In addition, amino derivatives of steroids which are useful in therapy are
known, and for example French patent application Nos. 2,494,697 and
2,494,698 describe 3 (5.alpha.)-amino 17.alpha.,20-pregnanediol, 3
(5.alpha.)-amino 19-nor 20-pregnanol and amino derivatives. These
compounds are thought to possess immunotherapeutic properties enabling
them to be used as drugs for the treatment of auto-immune disorders
resulting from a deficiency in certain lymphocytes.
The work carried out by the applicant has unexpectedly shown that certain
androstane 17-carboxylic acid esters, substituted at the 17-position by an
alkyl ester group and at the 3-position by a sugar residue, possess much
higher and more stable cardiotonic power than known steroid or cardenolide
derivatives, while adverse cardiac reactions are diminished.
An object of the present invention therefore is to provide new androstane
17-carboxylic acid esters substituted at the 3-position by a sugar
residue, possessing cardiotonic properties.
A further object of the present invention is to provide a process for the
preparation of androstane 17-carboxylic acid esters substituted at the
3-position by a sugar residue, from a 14-azido etianic acid ester.
A still further object of the present invention concerns the androstane
17-carboxylic acid esters substituted at the 3-position by a sugar residue
as cardiotonic medication for the treatment of congestive heart failure,
as well as the pharmaceutical compositions containing as an active
ingredient at least one of the new androstane 17-carboxylic acid esters
substituted at the 3-position by a sugar residue, as well as the use of
these derivatives in the production of drugs that can be used in therapy
as cardiotonic agents for the treatment of congestive heart failure.
The new androstane 17-carboxylic acid esters substituted at the 3-position
by a sugar residue according to the present invention can be represented
by the following general formula (I):
##STR5##
wherein R.sub.1 represents a lower alkyl group containing 1 to 4 carbon
atoms, a lower alkyl group from 2 to 4 carbon atoms substituted by an
amino group, or an aralkyl group from 6 to 12 carbon atoms, R.sub.2 and
R.sub.3, which may be the same or different, represent a hydrogen atom or
a lower alkyl group containing 1 to 4 carbon atoms, R.sub.4 represents a
hydrogen atom, a hydroxy group, or an acetoxy group, R.sub.5 represents a
hydroxy group or an acetoxy group, or R.sub.4 and R.sub.5 represent
together a divalent alkylenedioxy group, R.sub.6 represents a hydroxy, a
methoxy group or acetoxy group, R.sub.7 represents a methyl group or a
hydroxymethyl group, R.sub.8 represents a hydrogen atom or a hydroxy group
and
##STR6##
represents either a
##STR7##
group, or a
##STR8##
carbon where the double linkage is at the 4- or 5-position of the steroid
nucleus.
In general formula (I) above, the steroid nucleus can be saturated as shown
in the following formula (Ia), or comprise a 4-5 or 5-6 double linkage,
thus forming a 4- or 5-androstene derivative represented by the following
formulae (Ib) and (Ic).
##STR9##
In the following description, the ords "general formula (I)" means the
three alternatives (Ia), (Ib) and (Ic), unless otherwise indicated.
In general formula (I) above, the lower alkyl group represented by R.sub.1
can be a methyl, ethyl, isopropyl, butyl, etc, and preferably a methyl or
ethyl group. The above alkyl group can be substituted by an amino group,
thus forming for example a 2-aminoethyl or 3-aminopropyl group, or
3-di-(or mono-)methylaminopropyl group. When R.sub.1 represents an aralkyl
group, such group can be for example a phenyl group, a benzyl group, a
tolyl group, or a phenethyl group. R.sub.2 and R.sub.3, which may be the
same or different, preferably represent a hydrogen atom or a methyl group.
When R.sub.4 and R.sub.5 combine to form a divalent alkylenedioxy group,
this group can comprise a linear or branched alkylene moiety containing 1
to 4 carbon atoms, and for example form a methylenedioxy group, an
ethylenedioxy group or an isopropylidenedioxy group, R.sub.8 preferably
represents a hydrogen atom or a hydroxy group.
The new androstane 17-carboxylic acid esters according to the present
invention contain several asymmetrical carbon atoms in their molecule and
thereby can exist in several stereoisomer forms. The invention of course
concerns the new derivatives represented by the above general formula (I)
in the form of separate isomers or a combination of one or several
isomers.
In particular, the amino group at the 14-position, the ester group at the
17-position and the sugar residue at the 3-position, can have the .alpha.
or .beta. configuration, but they preferably have the .beta.
configuration. In fact, the pharmacological activity of derivatives of
general formula (I) has been found to be generally superior when at least
one of these three substitutes, and preferably the amino group at the
14-position, have the .beta. configuration, and more particularly when the
three substituents have the .beta. configuration.
Among the new androstane 17-carboxylic acid esters represented by general
formula (I) above, the invention preferably relates to methyl
.alpha.-L-rhamnopyranosyloxy-3.beta. amino-14.beta. etianate, methyl
.alpha.-L-rhamnopyranosyloxy-3.beta. methylamino-14.beta. etianate and
methyl .alpha.-L-rhamnopyranosyloxy-3.beta. amino-14.beta. etien-4-ate.
As indicated above, the derivatives of general formula (I) according to the
present invention can be prepared from a 3-hydroxy-14-azido androstane
17-carboxylic acid ester, represented by the following general formula
(II):
##STR10##
wherein R.sub.1 and R.sub.2 are as defined in the general formula (I) of
claim 1, with an activated sugar residue, thus forming a
3-pyranosyloxy-14-azidoandrostane 17-carboxylic acid ester of the
following general formula (III):
##STR11##
wherein R'.sub.4 represents a hydrogen atom or an acetoxy group, and
R'.sub.7 represents a methyl group or a --CH.sub.2 OAc group, an R.sub.8
is as defined in the general formula (I), which is reduced into the
derivative of the general formula (I), followed if necessary by an
aminomethylation.
The derivatives of the general formula (I) wherein R.sub.2 and R.sub.3 each
represent a hydrogen atom, thus comprising an amino groupe at the
14-position, can be transformed into derivatives comprising a mono- or
di-alkylamino group by an aminomethylation reaction according to usual
techniques.
The 3-hydroxy-14-azido androstane 17-carboxylic acid esters of general
formula (II) used as the starting products can easily be obtained from the
methyl 3-acetoxy-14-azido-etianate described in French patent application
No. 2,464,270, by an ordinary deacetylation reaction, for example by
action of an alkaline metal alcoholate such as sodium methanolate, in an
appropriate solvent.
The 3-hydroxy-14-azido androstane 17-carboxylic acid ester of general
formula (II) comprising a double linkage at the 4-5 or 5-6 -position (Z is
.dbd.C-- or --C.dbd.) can be easily prepared from the corresponding ester
comprising a saturated steroid nucleus, according to usual techniques for
double linkage formation, for example as described by J. Fried and J. A.
Edwards Organic Reaction in Steroid Chemistry, Van Nostrand Rheinhold Co.
(1971) and in the following examples.
The sugar derivative used in the coupling reaction with 3-hydroxy-14-azido
androstane 17-carboxylic acid esters is an activated derivative. The
activation can, for example be carried out by transforming the sugar into
a halogenated or acetylated derivative. The halogenated derivative is
preferably a bromide, for example rhamnosyl bromide that is coupled in the
presence of mercury cyanide. In the case of activation by acetylation, it
is possible to use, for example, tri-O-acetyl-digitoxose that is coupled
with methyl 3-hydroxy-14-azido-etianate in the presence of toluene
sulfonic acid.
In general, the sugar, activated and protected as above, can be a
monosaccharide such as a hexose, a 2-deoxyglucose, a 6-deoxyhexose, a
2,6-dideoxyhexose, etc. More particularly, the monosaccharide can be
chosen from among a rhamnose, a glucose, an arabinose, a digitoxose, a
fructose, a galactose; for example, a rhamnopyrannose, a hexopyrranose, a
6-deoxyglucose, a 4,6-dideoxy-glycopyranose, etc, can be used. A rhamnose
is preferably used, and more particularly a rhamnopyranose. Of course, it
is possible to use different existing anomers, and preferably the
.alpha.-L and .beta.-forms.
The hydroxyl functions of the sugar are previously protected in the usual
manner before carrying out the coupling reaction, for example by
acetylation or benzoylation. A technique for the protection of the sugars
usable in the present invention is described in E. Fisher et al., Chem.
Ber. 53, p. 2362 (1920). The activated derivative of the thereby protected
sugar can be for example 2,3,4-(tri-O-acetyl)-1-bromorhamnose that is
coupled in the presence of mercury cyanide, or tri-O-acetyl-digitoxose in
the presence of toluene sulfonic acid. The second case gives rise to a
combination of isomer glycosides that can be separated by chromatography
after deacetylation. When R.sub.8 represents a hydroxyl group, such group
is also preferably protected by the same techniques.
The protective groups of the sugar derivatives can then be eliminated by
means of the usual techniques, after coupling with the 14-azido etianic
acid ester, if so desired. For example, deprotection can be carried out by
heating in an alkaline medium.
As indicated above, a reduction is carried out after the coupling reaction
with the sugar derivative in order to obtain the derivative of general
formula (I). The reduction of methyl 3-pyranosyloxy-14-azido-etianate of
general formula (III) is carried out by action of sodium borohydride in
the presence of tellure in an alcohol, followed if necessary by
deacetylation.
According to an alternative, the reduction of methyl
3-pyranosyloxy-14-azido-etianate of general formula (III) is carried out
by hydrogenation reaction in the presence of Raney nickel, preceded if
necessary by deacetylation.
The following examples provide a more detailed illustration of the
invention without limiting the scope there of.
EXAMPLE 1
Methyl 3.beta.-(.alpha.-L-rhamnopyranosyloxy)-14.beta.-amino-etianate
A solution is prepared containing 20 g of methyl
3.beta.-hydroxy-14.beta.-azido-etianate and 37.6 g of
2,3,4-(tri-O-acetyl)-1-bromo rhamnose in 1.5 liters of acetonitrile
extemporaneously distilled on P.sub.2 O.sub.5. 26.7 g of mercury cyanide
is added. The solution is shaken at room temperature for about one hour.
The reactive medium is then poured into 650 ml of a saturated solution of
sodium hydrogenocarbonate. This solution is shaken for 50 min., then 500
ml of toluene is added. The aqueous phase is extracted three times with
toluene, washed with water, then with water saturated with sodium
chloride.
The toluene phases are collected, dried, concentrated until dry. After
recrystallization in methanol, 26.2 g (76% yield) of white crystals of
methyl 3.beta.-(tri-O-acetyl
.alpha.-L-rhamnopyranosyloxy)-14.beta.-azido-etianate are obtained.
The methyl 3.beta.-hydroxy-14.beta.-azido-etianate used as initial product
is obtained from a corresponding acetoxylated derivative, by heating in
the presence of sodium methanolate, extraction and crystallization in
hexane.
In a one liter balloon-flask, a mixture of 8.7 g of sodium borohydride and
12.3 g of ground tellure, in 313 ml of absolute ethanol extemporaneously
distilled on magnesium and degasified with nitrogen, is brought to reflux
for about 6 hours under argon.
After cooling at room temperature, 25 g of methyl 3.beta.-(tri-O-acetyl
.alpha.-L-rhamnopyranosyloxy)-14.beta.-azido-etianate obtained as
indicated above, is progressively added, under inert atmosphere, and kept
at room temperature while being shaken for about 20 hours.
The reactive mixture is then shaken in air for 30 minutes, filtered on
celite, rinsed with absolute ethanol, and concentrated until dry. The
white crystals obtained are picked up with 330 ml of ethyl acetate, then
an acid-base extraction is carried out (extraction by 5% sulfamic acid,
washing with ethyl acetate, alkalinization, shaking for one hour,
extraction with methylene chloride, then washing with water).
After separation of the neutral substances (2.9 g), 14.3 g of bases in the
form of white crystals are obtained (raw yield: 75%).
The obtained product, purified by chromatography on Merck silica (solvent:
CH.sub.2 Cl.sub.2 /MeOH/NH.sub.4 OH 85/15/1.5 Rf=0.32), then
recrystallization in ethanol, is methyl
3.beta.-(.alpha.-L-rhamnopyranosyloxy)-14.beta.-amino-etianate.
Melting point: 253.degree./254.degree. C. (solvent EtOH/H.sub.2 O).
IR spectrum (Nujol): .nu.=3370, 3320, 1735, 1605 cm.sup.-1.
NMR spectrum: (CDCl.sub.3 +CD.sub.3 OD 3.5/1). .delta.=0.91 (s, Me-19) 0.93
(s, Me-18) 1.27 (d, Me-6', J.sub.5'-6' =6 Hz) 3.68 (s, --COOCH.sub.3) 4.80
(H-1') ppm.
EXAMPLE 2
Methyl 3.beta.-(.alpha.-L-rhamnopyranosyloxy)-14.beta.-methylamino etianate
0.16 g of the derivative obtained as indicated in example 1 is dissolved in
3.5 ml of acetonitrile and 0.03 ml of a 35% aqueous solution of
formaldehyde and 22 mg of sodium borocyanohydride are added. The reaction
is allowed to develop for 15 minutes.
155 mg of the methylation product is obtained after neutralization with
acetic acid and evaporation of the solvent, recovery of the 2N potash and
extraction of the base. The product is separated by chromatography on
silica.
Chromatography (T.L.C.): Rf=0.3.
Eluent: CH.sub.2 Cl.sub.2 /MeOH/NH.sub.4 OH (80/20/2).
IR spectrum (Nujol): .nu.=3700-2300 (max. 3360), 1725, 1695 cm.sup.-1.
EXAMPLE 3
Methyl 3.beta.-(.alpha.-L-rhamnopyranosyloxy)-14.beta.-dimethylamino
etianate
This derivative is formed simultaneously with the derivative in example 2
above. It is isolated by chromatography on column and crystallization in
ethanol.
Chromatography (T.L.C.): Rf=0.4.
Eluent: CH.sub.2 Cl.sub.2 /MeOH/NH.sub.4 OH (85/15/1.5).
IR spectrum (Nujol): .nu.=3620-2300 (max. 3550, 3380), 2780, 1735, 1705
cm.sup.-1.
EXAMPLE 4
Ethyl 3.beta.-(.alpha.-L-rhamnopyranosyloxy)-14.beta.-amino etianate
0.3 g of the derivative obtained as indicated in example 1 is allowed to
react in 3.5 ml of ethanol containing an equivalent amount of
extemporaneously prepared sodium ethanolate.
The reaction is allowed to develop for 24 hours. 0.2 g of pure product
consisting of methyl 3.beta.-(.alpha.-L-rhamnopyranosyloxy)-14.beta.-amino
etianate is obtained after extraction and washing according to the
technique described in example 1, then trituration in isopropyl ether and
crystallization in ethyl ether.
Chromatography (T.L.C.): Rf=0.25.
Eluent: CH.sub.2 Cl.sub.2 /MeOH/NH.sub.4 OH (85/15/1.5).
IR spectrum (Nujol): .nu.=3250-3600, 1730, 1700, 1660, 1515, 1040
cm.sup.-1.
EXAMPLE 5
Methyl
3.beta.-(O-methyl-4'.alpha.-L-rhamnopyranosyloxy)14.beta.-amino-5.beta.,17
.alpha.(H) etianate
The procedure is similar to that indicated in example 1 from methyl
3.beta.-hydroxy-14.beta.-azido etianate but the
2,3,4-tri-O-acetyl)-1-bromo-rhamnose is replaced with
2,3-(di-O-acetyl)-4-methoxy-1-bromo-rhamnose in 1.5 liters of distilled
acetonitrile.
Methyl
3.beta.-(O-methyl-4'.alpha.-L-rhamnopyranosyloxy)-14.beta.-azido-5.beta.,1
7.alpha.(H) etianate is thereby obtained. 70 mg of the product is put in
suspension in 1.5 ml of a mixture consisting of 40 mg of tellurium, 28 mg
of sodium borocyanohydride and 1.5 ml of absolute ethanol. The reactive
mixture is kept at room temperature with constant stirring for about 21
hours under argon atmosphere.
The excess reagent is destroyed by stirring for 15 minutes in open air. The
reactive mixture is then filtered on celite by rinsing with a mixture of
methylene chloride and ethanol. 57 mg of base (yield: 85%) are obtained
after evaporation of the filtrate until dry, extraction by toluene,
washing with a 2% aqueous solution of sulfamic acid, alkalinization and
extraction with methylene chloride.
Purification is carried out by chromatography on silica gel column under
pressure. The CH.sub.2 Cl.sub.2 /EtOH/N.sub.4 OH mixture (89.1/9/0.9) is
used as eluant. 47 mg of methyl
3.beta.-(O-14.beta.-amino-5.beta.,17.alpha.(H) etianate (yield: 71%) is
obtained after crystallization in isopropanol.
Melting point: F=106.degree.-108.degree. C. (isopropanol solvent).
IR spectrum (Nujol): .nu.=3700-3100 (max. 3360), 1725, 600, 1195, 1170,
1110, 1045 cm.sup.-1.
NMR spectrum: (CDCl.sub.3 /CD.sub.3 OD). .delta.=0.90 (s, Me 19), 0.94 (s,
Me 19) 1.26 (d, J=6, Me 6'), 2.51 (m, H 17), 3.02 (t, J=9.5, H 4'), 3.54
(s, C-OMe), 3.65 (s, COOMe), 3.4 to 4.1 (m, H 2', 3', 5' and H 3), 4.75
(s, H 1') ppm.
EXAMPLE 6
Methyl
3.beta.-desoxy-6'.alpha.-L-tallo-hexopyranosyloxy)-14.beta.-amino-5.beta.,
17.alpha.(H) etianate
1.8 g of methyl 3.beta.-(tri-O-acetyl-.alpha.-L-rhamnopyranosyloxy)
14.beta.-azido etianate, obtained as indicated in example 1, are added to
8.4 ml of 1N sodium methylate under nitrogen atmosphere. 2.5 ml of
methanol is then added and the solution is allowed to react for about 1
hour at room temperature.
1.4 g of methyl 3.beta.-(.alpha.-L-rhamnopyranosyloxy)-14.beta.-azido
etianate is obtained after neutralization with icy acetic acid and
extraction with methylene chloride. 0.5 g are removed and dissolved in 4
ml of dimethyl formamide and then 5 ml of dimethoxy-2,2 propane and 15 ml
of tosylic acid are added. The solution is left to react for 2 hours at
room temperature. 0.4 g of methyl 3.beta.-(O-isopropylidene-2',3'
.alpha.-L-rhamnopyranosyloxy)-14.beta.-azido-5.beta.,17.alpha.(H) etianate
(raw yield: 96%) are obtained after evaporation of the solvent and
extraction with toluene.
The thereby obtained derivative is purified by chromatography on silica
column under pressure by eluting with the CH.sub.2 Cl.sub.2 /EtOH (98/2)
mixture (Rf=0.65).
IR spectrum (Nujol): .nu.=3650-3250 (max. 3470), 2110, 1740 cm.sup.-1.
0.1 ml of dry dimethylsulfoxide is added to a solution consisting of 0.1 ml
of oxalyl chloride in 2.1 ml of methylene chloride cooled to -60.degree.
C. The solution is stirred for 5 minutes at -60.degree. C. and then for 5
minutes at -40.degree. C. 100 mg of methyl 3.beta.-(O-isopropylidene-2',3'
.alpha.-L-rhamnopyranosyloxy)-14.beta.-azido-5.beta.,17.alpha.(H)
etianate, obtained as indicated above, dissolved in 0.7 ml of methylene
chloride, is added drop by drop.
The mixture is allowed to react under constant stirring for one hour at
-40.degree. C. The reaction is then stopped by adding 0.6 ml of
N,N-diisopropylethylamine.
The mixture is allowed to return to room temperature. The solvent is
evaporated under vacuum in order to obtain the methyl 3.beta.-(deoxy-6'
O-isopropylidene-2',3'
.alpha.-L-lyxohexopyranulos-4'-yloxy)-14.beta.-azido-5.beta.,17.alpha.(H)
etianate.
6.7 g of sodium borohydride is added to a solution cooled to 0.degree. C.
of 100 mg of the derivative obtained as indicated above and 66 mg of
CeCl.sub.3 7H.sub.2 O in 0.5 ml of methanol. The resulting suspension is
kept at room temperature for one hour while stirring. After extraction by
methylene chloride and washing with water saturated with sodium chloride,
the residue is purified by chromatography on silica column under pressure
by eluting with the CH.sub.2 Cl.sub.2 /EtOH (98/2) mixture (Rf=0.46).
The methyl 3.beta.-(desoxy-6' O-isopropylidene-2',3'
.alpha.-L-tallo-hexopyranosyloxy)-14.beta.-azido-5.beta.,17.alpha.(H)
etianate obtained (70 mg) is hydrolyzed in solution in 1.4 ml of
chloroform by 0.16 ml of trifluoroacetic acid in the presence of about 1%
water. After extraction by the chloroform, 70 mg of crystallizable dry
residue in methanol is obtained, consisting of the methyl
3.beta.-(deoxy-6'
.alpha.-L-tallo-hexopyranosyloxy)-14.beta.-azido-5.beta.,17.alpha.(H)
etianate.
The thereby obtained derivative is reduced with a mixture of sodium
borohydride (26 mg), ground tellurium (37 mg) and absolute ethanol (2 ml)
according to the method described in example 1, by allowing the reaction
to continue overnight.
The methyl 3.beta.-(deoxy-6' .alpha.-L-tallo
hexopyranosyloxy)-14.beta.-amino-5.beta.,17.alpha.(H) etianate (isomer of
the derivative of example 1) is obtained after filtration, rinsing and
extraction as indicated in example 1.
Melting point: F=210.degree. C. (isopropanol solvent).
IR spectrum (Nujol): .nu.=3600-3050 (3480, 3370, 3100), 1725, 1600 1105,
100, 1050, 1025, 975 cm.sup.-1.
NMR spectrum: (CDCl.sub.3 /CD.sub.3 OD 4/1). .delta.=0.92 (s, Me-19), 0.94
(s, Me-18) 1.25 (d, J=6, Me 6'), 2.55 (m, H 17), 3.67 (s, OCH.sub.3) 4.08
(s, H 1') 7.44 (CHCl.sub.3) ppm.
EXAMPLE 7
Methyl 3.beta.-(O-isopropylidene-2',3'
.alpha.-L-rhamnopyranosyloxy)-14.beta.-amino-5.beta.,17.alpha.(H) etianate
150 mg of ground tellurium and 103 mg of sodium borohydride are added to
5.4 ml of absolute ethanol deoxygenized by argon. The solution is heated
for 2 hours under ethanol reflux, under argon. 270 mg of methyl
3.beta.-(O-isopropylidene-2',3'
.alpha.-L-rhamnopyranosyloxy)-14.beta.-azido-5.beta.,17.alpha.(H)
etianate, obtained as indicated in example 6, in solution in 2 ml of
deoxygenized absolute ethanol is added after cooling to room temperature.
The reactive medium is continuously stirred for 24 hours. The reaction is
then stopped by exposing to open air for 15 minutes in order to destroy
the excess reagent.
The solution is filtered on a celite column and eluted with the CH.sub.2
Cl.sub.2 /EtOH (9/1) mixture. The filtrate is then evaporated until dry.
The residue is picked up by toluene and the bases are the extracted with a
2% aqueous solution of sulfamic acid. 189 mg of base and 42 mg of a
neutral product are obtained after alkalinization with sodium carbonate
and extraction with methylene chloride.
By saturating the aqueous phase with sodium chloride, 26 mg of very polar
product that is also present in the first basic extract is obtained.
The main basic fraction is purified by chromatography on 5.7 g on silica
column under 500 mB of pressure by eluting with the CH.sub.2 Cl.sub.2
/EtOH/NH.sub.4 OH (94.5/5/0.5) mixture.
150 mg of methyl 3.beta.-(O-isopropylidene-2',3'
.alpha.-L-rhamnopyranosyloxy)-14.beta.-amino-5.beta.,17.alpha.(H) etianate
(yield: 58%) is thereby obtained in the form of colorless crystals that
are soluble in isopropyl ether.
Melting point: F=179.degree. C. (isopropyl ether solvent).
IR spectrum (Nujol): .nu.=3700-3050 (max. 3350, 3300, 3170), 1735, 1600,
1170, 1075, 1555 cm.sup.-1.
NMR spectrum: (CDCl.sub.3). .delta.=0.91 (s, Me-19) 0.94 (s, Me-18) 1.25
(d, J=6, Me 6'), 1.36 and 1.51 (2s, CH.sub.3 --C--CH.sub.3) 2.51 (m, H
17), 3.64 (s, OCH.sub.3) 3.2 to B 4.2 (m, H 2', 3', 4',5', H 3) 4.99 (s,
H1') ppm.
EXAMPLE 8
Methyl 3.beta.-O-(dideoxy-2',6'
.alpha.-D-ribohexopyranosyloxy)-14.beta.-amino-5.beta. etianate
A solution of 1.6 g of methyl 3.beta.-hydroxy-14.beta.-azido etianate and
2.3 g of tri-O-acetyl digitoxose in the presence of 1.9 g of dry p-toluene
sulfonic acid and in 24 ml of benzene is kept while stirring for 2 hours
at room temperature.
A residue is obtained after adding an aqueous saturated solution of sodium
bicarbonate and extraction by benzene. The residue is purified by
chromatography on silica column under pressure, by eluting with the
AcOEt/hexane (25/75) mixture. A mixture of acetylated glycosides is
obtained that is subject to a desacetylation reaction with 5.3 ml of
sodium methylate. The products are then separated by chromatography on
column.
The separated .alpha.-D-digitoxoside (T.L.C.: eluant CH.sub.2 Cl.sub.2
/MeOH 95/5, Rf=0.57) is used as is in the following reduction stage.
The reduction is carried out according to the technique described in
example 1, with the mixture of sodium borohydride, ground tellurium and
ethanol.
The methyl 3.beta.-O-(dideoxy-2',6'
.alpha.-D-ribo-hexopyranosyloxy)-14.beta.-amino-5.beta.-etianate crystals
are obtained after extraction as indicated in example 1 and chromatography
on silica column, under pressure, by eluting with the CHCl.sub.3
/EtOH/NH.sub.4 OH (94.5/5/0.5) mixture and crystallization in the ethyl
acetate-isopropyl ether mixture.
Melting point: F=178.degree.-182.degree. C. (ethyl acetate-isoproyl ether
solvent).
IR spectrum (Nujol): .nu.=3250, 3470, 1730 cm.sup.-1.
NMR spectrum: (CDCl.sub.3 /CD.sub.3 OD 4/1). .delta.=0.94 (s, Me-18 and
Me-19) 1.31 (d, J=6, Me 6) 2.52 (m, H-17), 3.11 (dd, J.sub.3'4' =3,
J.sub.4'5' =10, H4') 3.66 (s, OCH.sub.3), 3.95 (H-3, H-3') 4.95 (s, H 1')
7.37 (CHCl.sub.3) ppm.
EXAMPLE 9
Methyl 3.beta.-O-(diesoxy-2',6'
.beta.-D-ribohexopyranoxyloxy)-14.beta.-amino-5.beta.-etianate
The procedure is indicated in example 8. The .beta.-D-digitoxoside, an
isomer of .alpha.-D-digitoxoside in example 8, is separated by
chromatography.
The thereby obtained .beta.-D-digitoxoside is reduced according to the
technique indicated in example 8 with the mixture of sodium borohydride,
ground tellurium and ethanol.
The methyl 3.beta.-O-(dideoxy-2',6'
.beta.-D-ribo-hexopyranosyloxy)-14.beta.-amino-5.beta.-etianate is
obtained after extraction of the bases as indicated in example 1,
chromatography on silica column under pressure, by eluting with the
CHCl.sub.3 /EtOH/NH.sub.4 OH (92.3/7/0.7) mixture and crystallization in
the ethyl acetate-isopropyl ether mixture.
Melting point: F=162.degree.-167.degree. C. (ethyl acetate-isopropyl ether
solvent).
IR spectrum (Nujol): .nu. 3600-3040 (3350), 1725, 1595, 1070 cm.sup.-1.
NMR spectrum: (CDCl.sub.3 /CD.sub.3 OD 4/1). .delta.=0.95 and 0.96 2 (s,
Me-18 and Me-19) 1.27 (d, J=6, Me 6'), 2.54 (m, H-17), 3.21 (dd,
J.sub.3'4' =3, J.sub.4'5' =10 H-4'), 3.70 (s, OCH.sub.3), 4.05 (H-3,
H-3'), 4.90 (dd, J.sub.1'2'a =10, J,.sub.1'2'e =2, H 1') 7.52 (CDCl.sub.3)
ppm.
EXAMPLE 10
Methyl
O-3.beta.-(dideoxy-2',6'.beta.-D-arabino-hyxopyranosyloxy)-14.beta.-amino-
5.beta.,17.alpha.(H)-etianate
Tri-O-acetyl digitoxoside is coupled with methyl
3.beta.-hydroxy-14.beta.-azido-etianate as indicated in the technique
described in example 8. The solution is then saponified by sodium
methylate and put through chromatography on silica column under pressure
in accordance with the usual technique.
Methyl O-3.beta.-(dideoxy-2',6'
.beta.-D-arabino-hexopyranosyloxy)-14.beta.-azido-5.beta.,17.alpha.(H)
etianate is obtained after extraction and chromatography on silica column
under pressure, by eluting with the CH.sub.2 Cl.sub.2 /EtOH/NH.sub.4 OH
(89/10/1) mixture.
Melting point: F=210.degree. C. (solvent:isopropanol-isopropyl solvent).
IR spectrum (CHCl.sub.3 film):=3700-3100 (3360, 3220), 1700, 1625, 1215
cm.sup.-1.
NMR spectrum: (CDCl.sub.3 /CD.sub.3 OH 4/1). .delta.=0.93 2 (s, Me-19),
1.01 (s, Me-18) 1.29 (d, J=6, Me 6'), 3.72 (s, OCH.sub.3), 4.03 (H-3) 4.56
(dd, J=9, H-1') 7.35 (CHCl.sub.3) ppm.
EXAMPLE 11
Methyl
O-3.beta.-(dideoxy-2',6'.alpha.-D-arabino-hexopyranosyloxy)-14.beta.-amino
-5.beta.,17.alpha.(H)-etianate
The method followed is indicated in example 10. However, the intermediate
used is methyl
O-3.beta.-(dideoxy-2',6'.beta.-D-arabino-hexopyranosyloxy)14.beta.-azido-5
.beta.,17.alpha.(H)-etianate, a .beta.-D isomer of the .alpha.-D glycoside
of example 10, isolated by chromatography from the same reactive mixture.
Reduction in the same conditions indicated in example 10, after
chromatography on silica column under pressure, by eluting with the
CH.sub.2 Cl.sub.2 /EtOH/NH.sub.4 OH (89/10/1) mixture and crystallization
in the CH.sub.2 Cl.sub.2 /EtOh mixture produces crystals of methyl
O-3.beta.-(dideoxy-2',6'.alpha.-D-arabino-hexpy-ranosyloxy)-14.beta.-amino
-5.beta.,17(H) etianate.
Melting point: F=270.degree. C. (solvent: methylene chloride-isopropanol).
IR spectrum (Nujol): .nu.=3500-3150 (3350, 3270), 1730, 1605 cm.sup.-1.
EXAMPLE 12
Methyl 3.beta.-(.alpha.-L-rhamnopyranosyloxy)-14.beta.-amino-4-etianate
5 g of methyl 3.beta.-hydroxy-14.beta.-azido-etianate is treated with 5 ml
of Jones reagent in 100 ml of acetone. The reagent is added drop by drop
to the solution cooled on an ice-bath. The oxidation reaction is allowed
to develop for 20 minutes. 5 g of ketone is obtained after extraction and
is crystallized in ethyl acetate (F=189.degree.-191.degree. C.).
The ketone obtained as indicated above is bromated by pouring, one drop at
a time, 1.5 g of bromide in solution in 36 ml of dimethyl formamide in a
solution of 75 ml of dimethyl formamide containing 2.2 g of ketone, in the
presence of 0.07 g of TsOH. The solution is allowed to stand for 6 hours
at room temperature. The mixture of two isomers is obtained after
extraction and is separated by chromatography on silica column (ethyl
acetate/hexane 22/78) in order to yield 1.5 g of methyl
3-oxo-4.beta.-bromo-14.beta.-azido-5.beta.-etianate (the more polar
fraction) and 0.5 g of methyl
3-oxo-2.beta.-bromo-14.beta.-azido-5.beta.-etianate (the less polar
fraction).
0.8 g of methyl 3-oxo-4.beta.-bromo-14.beta.-azido-5.beta.-etianate in 20
ml of dimethyl formamide is allowed to react with 0.7 g of lithium
chloride by heating for 2 hours at 110.degree. C. under nitrogen. 0.5 g of
methyl 3-oxo-14.beta.-azido-4-etianate is obtained after extraction and
chromatography on silica (ethyl acetate/hexane 28/72) (T.L.C.: ethyl
acetate/hexane 1/1, Rf=0.3).
The ketone obtained as indicated above is reduced by allowing 0.5 g of
CeCl.sub.3 and then 50 mg of NaBH.sub.4 react with 0.5 g of ketone in 10
ml of methanol. N hydrochloric acid (pH=6) is used to acidify. Ethyl
acetate is used to extract in order to obtain 0.5 g of methyl
3.beta.-hydroxy-14.beta.-azido-4-etianate.
Thin layer chromatography: CH.sub.2 Cl.sub.2 /MeOH 97/3, Rf=0.35.
Ir spectrum (Nujol): .nu.=3300-3600 (3220, 3320,) 2100, 1715, 1665, 1285,
1205, 1180 cm.sup.-1.
The thereby obtained methyl 3.beta.-hydroxy-14.beta.-azido-4-etianate,
analogous to the starting product in example 1 comprising a double bond in
the 4-position, is treated as indicated in example 1 with
2,3,4-(tri-O-acetyl)-1-bromo-rhamnose and mercury cyanide in order to form
the methyl 3.beta.-(tri-O-acetyl
.alpha.-L-rhamnopyranosyloxy)-14.beta.-azido-4-etianate.
The methyl 3.beta.-(tri-O-acetyl
.alpha.-L-rhamnopyranosyloxy)-14.beta.-azido-etianate is desacetlyated by
the action of sodium methanolate in methanol at room temperature and then
neutralized by dilute hydrochloric acid and extracted by CH.sub.2
Cl.sub.2.
The 14-azido derivative described above is reduced to the corresponding
14-amino derivative by the action of sodium borohydride in the presence of
ground tellurium, in absolute ethanol, according to the method described
in example 1.
Methyl 3.beta.-(.alpha.-L-rhamnopyranosyloxy)-14.beta.-amino-etianate is
obtained after chromatography on silica (CH.sub.2 Cl.sub.2 /MeOH/NH.sub.4
OH 85/12/06). It crystallizes in absolute ethanol.
Melting point: F=240.degree.-246.degree. C.
NMR spectrum: (CDCl.sub.3 +CD.sub.3 O.sub.4 4/1). .delta.=1.02 (s,
Me-18+Me-19), 1.26 (d, J=6, Me-6'), 2.6 (m, H-17) 3.69 (s, MeO), 4.86 (s,
H-1'), 5.26 (s, H-4) ppm.
EXAMPLE 13
Methyl
3.beta.-(.alpha.-L-rhamnopyranosyloxy)-14.beta.-amino-5.alpha.-etianate
37 mg of the ester of example 12 are treated in the presence of 20 mg of
PtO.sub.2 according to Adams' reaction. The mixture in suspension in 6 ml
of ethyl acetate and 0.6 ml of acetic acid is shaken for 48 hours.
28 mg of product is obtained after extraction according to the usual
technique, from the basic fraction. The product is purified by
crystallization in isopropyl ether, to yield 16 mg of
.alpha.-rhanmopyranosyloxy)-14.beta.-amino-5.alpha.-etianate.
Melting point: F=206.degree. C.
NMR spectrum: (CHCl.sub.3 /MeOH 4/1). .delta.=0.80-0.98 (s, Me-18+Me-19),
1.26 (d, J=6, Me-6'), 2.6 (m, H-17), 3.71 (s, OMe), 4.85 (s, H-1') ppm.
The pharmacological and clinical studies carried out on the new 14-amino
etianic acid derivatives according to the present invention have shown
that they possess interesting properties enabling their application as
active ingredients in drugs used in human and veterinary therapy.
The derivatives according to the invention in particular have a major
inotropic effect that is much greater than that of digoxin, a well-known
reference drug used in the treatment of congestive heart failure. In
addition, their activity is more stable and their cardiac toxicity is
lower.
The inotropic activity, demonstrating the cardiotonic power, was verified
on the stimulated left auricle in the Guinea-pig, by measuring the force
of contraction, according to the technique described in J. Ghysel-Burton
et al., Brit. J. Pharmacol., 66, 2, pp. 175-184 (1979).
By way of example, the derivative of example 1 according to the present
invention develops a very hight positive maximum inotropic effect (130% at
a concentration of 10.sup.-5 M), while the inotropic effect of digoxin, in
the same conditions, is much lower (70% at a concentration of 10.sup.-6
M). In addition, the positive inotropic effect of the derivative complying
with the invention is stable for about 60 minutes, whereas digoxin rapidly
induces a negative inotropism. This phenomenon indicates lower cell
toxicity for the derivative of the invention when compared with digoxin.
Similarly, the well-known contacturant effect of digitalis glycosides, and
particularly digoxin (refer to Handbook of Cardiac Glycosides, Ed. K.
Greef), is much lower in the case of derivatives of the invention.
The positive inotropic effect of the derivatives complying with the present
invention described in examples 1 to 11 above is indicated in the
following table. The inotropic effect is given as a percentage of the
increase in the force of contraction of the stimulated left auricle in the
Guinea-pig, at a concentration of 5.10.sup.-6 M.
______________________________________
Ex-
ample
1 2 3 4 5 6 7 8 9 10 11 12 13
______________________________________
Inotropic
97 36 17 27 29 34 37 11 35 29 16
95 41
effect
______________________________________
The positive inotropic effect in the dog anaesthetized with sodium
pentobarbital (in vivo tests) is confirmed by the measurement of the
hemodynamics parameters, according to the method described in R. E.
Patterson et al., Cardiology, 57, pp. 277-294 (1972). The product in
example 1, after intravenous perfusion of 3 to 4.10.sup.-9 moles per
kg.min. for 20 minutes is found to be about 20 to 40%. If the perfusion is
continued for several hours at the rate of 2.2.10.sup.-9 moles per
kg.min., the cardiotonic activity develops and is maintained at about
60-70%. In the case of digoxin, for a similar inotropic effect, when the
perfusion is maintained, heart rhythm troubles appear as of hour 1, then
death as of hour 3.
Comparative tests were carried out on 10 dogs for the following therapeutic
indexes: A--reversible arrhytmia dose/efficacious dose; B--toxic
dose/efficacious dose; C--lethal dose/efficacious dose. The reversible
arrhythmia dose is the dose where spontaneously resolving arrhythmia
begins; the toxic dose is the dose where the arrhythmia is sustained; the
lethal dose is the dose inducing death (no blood pressure and cardiac
output); the efficacious dose is the dose where there is a 30% increase in
the force of cardiac contraction as compared with the control level.
The results are provided in the following table:
______________________________________
Therapeutic Index
Substance A B C
______________________________________
Digoxin 1.5 2.0 3.2
Example 1 3.1 5.8 14.0
______________________________________
These results show that the efficacy of the derivatives according to the
present invention is superior to that of digoxin and the toxicity is
lower.
The androstane 17-carboxylic acid esters according to the present invention
can be used as active ingredients in drugs, that can be administered in
the usual forms, the active ingredient being diluted in a suitably chosen
pharmaceutically acceptable excipient, for example in tablet, capsule or
injectable solution form.
The doses administered are comparable to those of the known cardiotonic
drugs derived from the cardenolids, and are determined by the physician in
the usual manner according to the means of administration and the
condition of patient.
* * * * *
|
|
 |
|
|
