Note: Descriptions are shown in the official language in which they were submitted.
l~S6~8
-1- 16112 Y
TITLE OF THE INVENTION
Selective Hydrogenation Products of C-076
Compounds and Derivatives Thereof
BACKGROVND OF THE INVENTION
The term C-076 is used to describe a series
of compounds isolated from the fermentation broth of a
C-076 producting strain of Streptomyces avermitilis.
The morphological characteristics of the culture are
B completely descr~ibe/d6in copending ~A5.application
10 Serial Number 7,~,G01. The C-076 compounds are a
series of macrolides, each of which is substituted
thereon at the 13-position with a 4-(a-L-oleandrosyl)-
a-L-oleandrose group. The l-series of C-076 compounds
also has a 22,23-double bond, as well as several
15 other double bonds. The selective reduction of the
22,23-double bond, without affecting the remaining
double bonds is the subject matter of the instant
application. The C-076 compounds and the instant
derivatives thereof have a very high degree of
20 anthelmintic and antiparasitic activity.
~1156~8
-2- 16112 IA
SUMMARY OF THE INVENTION
The C-076 series of compounds have the
following structure:
Rl
~ ~ ~2
CH3 ~
0~
,~,
wherein R is the 4'-(~-L-oleandrosyl)-a-L-oleandrose
5 group of the structure:
HO - ~ O -
CH3O CH3O
and wherein the broken line indicates a single or a
double bond; Rl is hydroxy and is present only when
said broken line indicates a single bond;
R2 is iso-propyl or sec-butyl; and
R3 is methoxy or hydroxy.
There are eight different C-076 compounds
and they are given the designations Ala, Alb, A2a,
A2b, Bla, Blb, B2a, B2b based upon the structure of
the individual compounds.
~5~8
-3- 16112 IA
In the foregoing structural formula, the
individual C-076 compounds are as set forth below.
Rl R2 R3
Ala Double bond sec-butyl -OCH3
5 Alb Double bond lso-propyl -OCH3
A2a -OH sec-butyl -OCH3
A2b -OH lso-propyl -OCH3
Bla Double bond s -butyl -OH
Blb Double bond iso-propyl -OH
10 B2a -OH sec-butyl -OH
B2b -OH lso-propyl -OH
The C-076 compounds with the 22,23-unsatura-
tions are identified as the "l-series" and it is only
these compounds which are reduced to prepare the
15 instant derivatives. Either before or after the
reduction of the 22,23-double bond further reactions
may be carried out in which one or both of the a-L-
oleandrose moieties are removed, or in which one or
more of the available hydroxy groups are acylated.
Thus, the compounds of the instant invention
have the following structural formula:
3 ~ CH3
-O 1 ~ ~ R
CH3 ~
~ CH3
-
~115698
-4- 16112 IA
wherein
Rl is iso-propyl or sec-butyl;
R2 is methoxy, hydroxy or loweralkanoyloxy;
R3 is hydrogen; loweralkanoyl; a-L-
5 oleandrosyl; 4'-loweralkanoyl-~-L-oleandrosyl; 4'-
(a-L-oleandrosyl)-~-L-oleandrosyl; 4"-loweralkanoyl-
4'-(a-L-oleandrosyl)-a-L-oleandrosyl.
In the instant invention, the term
"loweralkanoyl" is intended to include those alkanoyl
10 groups of from 2 to 6 carbon atoms such as acetyl,
propionyl, butyryl, pivaloyl and the like.
Preferred compounds of the instant
invention are realized in the above structural formula
when:
Rl is lso-propyl or sec-butyl;
R2 is methoxy or hydroxy; and
~ 3 is hydrogen ~-L-oleandrosyl or 4'-(a-L-
oleandrosyl)-~-L-oleandrosyl.
Additional preferred compounds are realized
20 when the "loweralkanoyl" group of R3 is acetyl in the
disaccharide, monosaccharide and aglycone compounds.
As is readily apparent from an analysis of
the structure of the C-076 starting materials, there
are five unsaturations in the l-series of compounds.
25 An object of the instant invention is to reduce the
22,23-double bond while not affecting the remaining
four unsaturations or any other functional group
present on the molecule. It is necessary to select
a specific catalyst for the hydrogenation, one that
30 will selectively hydrogenate the least hindered
from among a series of unsaturations. The
1~15698
-5- 16112IA
preferred catalyst for such a selective hydrogenation
procedure is one having the formula:
[(R4)3P]3RhX
wherein R4 is loweralkyl/ phenyl, or loweralkyl
5 substituted phenyl and X is a halogen.
In the preferred catalyst R4 is phenyl and X
is chlorinel that is the compound tris(triphenyl-
phosphine~-rhodium (I) chloridel which is also known
as Wilkinson's homogeneous catalyst.
The reaction is carried out using a catalytic
amount of the catalyst. The amount of catalyst is not
critical and from 0.05 to 0.5 moles of the catalyst
for each mole of starting material have been
successfully employed. Mslar ratios in the range of
15 0.25 t9 0.40 are preferred.
The hydrogenation is carried out in a
hydrogen atmosphere which may be either at atmospheric
pressure or up to about 4 atmospheres pressure in a
standard laboratory hydrogenation apparatus. A
20 solvent is normally employed to dissolve both the
starting materials and the catalyst. ~referred
solvents are hydrocarbon solvents such as benzenel
toluenel petroleum ether and other alkane hydrocarbons.
The reaction is complete when the calculated amount
25 of hydrogen has been taken up by the reaction. This
will generally require from about 1 to 48 hours.
The reaction may be carried out at from room
temperature to about 75C, however, room temperature
is preferred. The hydrogenation products are isolated
lil5698
-6- 16112 IA
and purified by techniques known to those skilled in
the art.
Other reactions may be carried out on the
C-076 starting materials or on the hydrogenated
5 products to prepar~ the compounds of this invention.
While it is possible to complete all of the other
reactions on the C-076 starting material and have
the hydrogenation step as the final reaction, it is
preferred to carry out the hydrogenation step first.
Because the 22,23-double bond is somewhat susceptible
to nucleophilic addition, reaction conditions for
removing the sugar groups or acylating the hydroxy
groups must be carefully controlled if the 22,23-double
bond is present. If the 22,23-double bond is
15 hydrogenated first, the subsequent sugar removal and
acylation is rendered more facile.
Thus, the additional reactions which may
be carried out to prepare the compounds of this
invention are the selective removal of one or both of
20 the a-L-oleandrosyl moieties or the selective
acylation of the susceptible hydroxy groups.
The reaction conditions which are generally
applicable to the preparation of both the monosaccharide
and aglycone involve dissolving the C-076 compound or
25 the hydrogenated C-076 compound in an aqueous acidic
non-nucleophilic organic solvent, miscible with water,
preferably dioxane, tetrohydrofuran, dimethoxyethane,
dimethyl formamide, b -2-methoxyethyl ether, and
the like, in which the water concentration is from
30 0.1 to 20% by volume. Concentrated acid is added to
the aqueous organic solvent to the extent of 0.01 to
~1~5698
-7_ 16112 IA
10% by volume. The reaction mixture is generally
stirred at about 20-40C, preferably at room
temperature, for from 6 to 24 hours. The lower
concentrations of acid, from about 0.01 to 0.1%
5 will predominately produce the monosaccharide under the
above reaction conditions. Higher acid concentrations,
from about 1 to 10% will predominantly produce the
aglycone under the above reaction conditions.
Intermediate acid concentrations will generally
10 produce mixtures of monosaccharide and aglycone.
The products are isolated, and mixtures are separated
by techniques such as column, thir layer preparative
and high pressure liquid chromatography, and other
known techniques.
The acids which may be employed in the
above process include mineral acids and organic acids
such as sulfuric, hydrohalic, phosphoric, trifluoro-
acetic, trifluoro methane sulfonic and the like.
The hydrohalic acids are preferably hydrochloric or
20 hydrobromic. The preferred acid in the above process
is sulfuric acid.
A further procedure for the preparation of
the monosaccharide or aglycone of the C-076 compounds
or of the hydrogenated C-076 compounds utilizes a
25 different solvent system for the monosaccharide and
the aglycone. The procedure for the preparation of
the monosaccharide uses 1% acid by volume in
isopropanol at from 20-40C, preferably room
temperature, for from 6 to 24 hours. For the
30 preparation of the aglycone, 1% acid, by volume, in
methanol under the foregoing reaction conditions
has been found to be appropriate.
A ~
1115698
-8- 16112 IA
When this procedure is employed on the
starting material (the compounds with the 22,23-double
bond) there is a possibility of nucleophilic addition
to the double bond. If such occurs, chromatographic
5 purification will remove the by-product in order to
allow for further reactions.
The acids listed above are appropriate
for this process, and again sulfuric acid is the
preferred acid.
The above described compounds are isolated
from the reaction mixture and mixtures of compounds
are separated using techniques known to those skilled
in this art, and in particular the chromatographic
techniques described above.
The acylated compounds are prepared using
acylation techniques in which the reaction conditions
will vary, depending upon the reactivity of the hydroxy
group being acylated. Where there is more than one
hydroxy group to be acylated, different reaction
20conditions are employed to minimize the formation of
mixtures.
The acylation reagents employed are
generally the halide, preferably the chloride, of the
above loweralkanoyl groups. That is the loweralkanoyl
25halide reagent is generally employed.
In addition, the acylation reagent could be
in the form of the anhydride or of the halo formate.
In the case of reactions carried out with the
halide reagents, it is often advantageous to include
30in the reaction mixture a basic compound capable of
~115698
-9- 16112 IA
reacting with and neutralizing the hydrogen halide
which is liberated during the course of the reaction.
Tertiary amines are preferred such as triethylamine,
pyridine, dimethylamino pyridine, diisopropyl
5 ethylamine and the like. The basic compound is
required in equimolar amounts relative to the
numbered moles of hydrogen halide being liberated,
however excess amounts, even using the basic compound
as a solvent, are not detrimental.
In the case of the Al compounds of C-076, or
of the hydrogenated C-076 Al compounds there is only
a single hydroxy group, 4" hydroxy, which may be
acylated. The formation of the monosaccharide or
the aglycone still leaves only a single hydroxy group
15 which may be acylated, that is the 4' or 13 hydroxy
group.
In the case of the 4", 4' and 13 hydroxy
groups of C-076 Al compounds, the acylating reagent is
dissolved in a suitable solvent, pyridine is preferred,
20 and the acylating reagent added. The reaction is
maintained at from 0C to room temperature for from
4 to 24 hours. The product is isolated using known
techniques.
The Bl compounds have 2 available hydroxy
25 groups: at the 4"(4' or 13) and the 5-positions.
However, the two hydroxy groups have similar
reactivities. When the reaction of the acylating
agent in pyridine is carried out at about room
temperature for from 4 to 24 hours, the diacyl
30 compound is recovered. When the reaction is carried
out at 0C a mixture of the 4"(4' or 13) and 5
monoacyl compounds are recovered. To recover individual
compounds, the mixture is placed on a chromatographic
column or a preparative layer chromatographic plate of
~15698
-10- 16112 IA
alumina or silica gel and the individual compounds
are readily isolated. In addition, techniques such
as high pressure liquid chromatography may be
employed to separate mixtures of acylated compounds.
The acyl compounds thus prepared are isolated
from the reaction mixture using techniques ~nown to
those skilled in this art.
The novel compounds of this invention have
significant parasiticidal activity as anthelmintics,
10 ectoparasiticides, insecticides and acaricides, in
human and animal health and in agriculture.
The disease or group of diseases described
generally as helminthiasis is due to infection of an
animal host with parasitic worms known as helminths.
15 Helminthiasis is a prevalent and serious economic
problem in domesticated animals such as swine, sheep,
horses, cattle, goats, dogs, cats and poultry. Among
the helminths, the group of worms described as nematodes
causes widespread and often times serious infection in
20 various species of animals. The most common genera of
nematodes infecting the animals referred to above are
Haemonchus, Trichostrongylus, Ostertagia, Nematodirus,
Cooperia, Ascaris, Bunostomum, Oesophagostomum,
-
Chabertia, Trichuris, Strongylus, Trichonema,
25 Dictyocaulus, Capillaria, Heterakis, Toxocara,
Ascaridia, Oxyuris, Ancylostoma, Uncinaria,
Toxascaris and Parascaris. Certain of these, such
as Nematodirus, Cooperia, and Oesphagostomum attack
-
primarily the intestinal tract while others, such
30 as Haemonchus and Ostertagia, are more prevalent
~15698
-11- 16112 IA
in the stomach while still others such as
Dictyocaulus are found in the lungs. Still other
parasites may be located in other tissues and organs
of the body such as the heart and blood vessels,
5 subcutaneous and lymphatic tissue and the like.
The parasitic infections known as helminthiases lead
to anemia, malnutrition, weakness, weight loss,
severe damage to the walls of the intestinal tract
and other tissues and organs and, if left untreated,
10 may result in death of the infected host. The
hydrogenated C-076 compounds of this invention have
unexpectedly high activity against these parasites,
and in addition are also active against Dirofilaria
in dogs, Nematospiroides, Syphacia, Aspiculuris
15 in rodents, arthropod ectoparasites of animals and
birds such as ticks, mites, lice, fleas, blowfly,
in sheep Lucilia sp., biting insects and such
migrating diperous larvae as Hypoderma sp. cattle,
Gastrophilus in horses, and Cuterebra sp. in rodents.
The instant compounds are also useful against
parasites which infect humans. The most common genera
of parasites of the gastro-intestinal tract of man are
Ancylostoma, Necator, Ascaris, Strongyloides,
Trichinella, Capillaria, Trichuris, and Enterobius.
25 Other medically important genera of parasites which
are found in the blood or other tissues and organs
outside the gastrointestinal tract are the filiarial
worms such as Wuchereria, Brugia, Onchocerca and
Loa, Dracunculus and extra intestinal stages of the
30 intestinal worms Strongyloides and Trichinella. The
compounds are also of value against arthropods
~15698
-12 16112 IA
parasitizing man, biting insects and other dipterous
pests causing annoyance to man.
The compounds are also active against
household pests such as the cockroach, Blatella sp.,
5 clothes moth, Tineola sp., carpet beetle, Attagenus
sp., and the housefly Musca domestica.
The compounds are also useful against insect
pests of stored grains such as Tribolium sp.,
Tenebrio s and of agricultural plants such as
10 spider mites, (Tetranychus sp.), aphids,
(Acyrthiosiphon sp.); against migratory orthopterans
such as locusts and immature stages of insects living
on plant tissue. The compounds are useful as a
nematocide for the control of soil nematodes and
15 plant parasites such as Meloidogyne spp. which may
be of importance in agriculture.
These compounds may be administered orally in
a unit dosage form such as a capsule, bolus or tablet,
or as a liquid drench where used as an anthelmintic in
20 mammals. The drench is normally a solution, suspension
or dispersion of the active ingredient usually in water
together with a suspending agent such as bentonite and
a wetting agent or like excipient. Generally, the
drenches also contain an antifoaming agent. Drench
25 formulations generally contains from about 0.001 to 0.5%
by weight of the active compound. Preferred drench
formulations may contain from 0.01 to 0.1% by weight.
The capsules and boluses comprise the active ingredient
admixed with a carrier vehicle such as starch, talc,
11i56~8
-13- 16112 IA
magnesium stearate, or di-calcium phosphate.
Where it is desired to administer the C-076
derivatives in a dry, solid unit dosage form, capsules,
boluses or tablets containing the desired amount of
5 active compound usually are employed. These dosage
forms are prepared by intimately and uniformly mixing
the active ingredient with suitable finely divided
diluents, fillers, disintegrating agents and/or binders
such as starch, lactose, talc, magnesium stearate,
10 vegetable gums and the like. Such unit dosage
formulations may be varied widely with respect to
their total weight and content of the antiparasitic
agent depending upon factors such as the type of
host animal to be treated, the severity and type of
15 infection and the weight of the host.
When the active compound is to be administered
via an animal feedstuff, it is intimately dispersed in
the feed or used as a top dressing or in the form of
pellets which may then be added to the finished feed
20or optionally fed separately. Alternatively, the
antiparasitic compounds of our invention may be
administered to animals parenterally, for example, by
intraruminal, intramuscular, intratracheal, or
subcutaneous injection in which event the active
25ingredient is dissolved or dispersed in a liquid carrier
vehicle. For parenteral administration, the active
material is suitably admixed with an acceptable vehicle,
preferably of the vegetable oil variety such as peanut
oil, cotton seed oil and the like. Other parenteral
_ "
11~5698
-14- 16112 IA
vehicles such as organic preparation using solketal,
glycerol formal, and aqueous parenteral formulations
are also used. The active monosaccharide or aglycone
C-076 compound or compounds are dissolved or suspended
5 in the parenteral formulation for administration; such
formulations generally contain from 0.005 to 5% by weight
of the active compound.
Although the antiparasitic agents of this
invention find their primary use in the treatment and/or
10 prevention of helminthiasis, they are also useful in
the prevention and treatment of diseases caused by other
parasites, for example, arthropod parasites such as
ticks, lice, fleas, mites and other biting insects in
domesticated animals and poultry. They are also
15 effective in treatment of parasitic diseases that
occur in other animals including humans. The optimum
amount to be employed for best results will, of course,
depend upon the particular compound employed, the
species of animal to be treated and the type and
20 severity of parasitic infection or infestation.
Generally good results are obtained with our novel
compounls by the oral administration of from about
0.001 to 10 mg. per kg. of animal body weight, such
total dose being given at one time or in divided doses
25 over a relatively short period of time such as 1-5
days. With the preferred compounds of the invention,
excellent control of such parasites is obtained in
animals by administering from about 0.025 to 0.5 mg.
per kg. of body weight in a single dose. Repeat
30 treatments are given as required to combat re-infections
1~5~8
-15- 16112 IA
and are dependent upon the species of parasite and the
husbandry techniques being employed. The techniques
for administering these materials to animals are known
to those skilled in the veterinary field.
When the compounds described herein are
administered as a component of the feed of the animals,
or dissolved or suspended in the drinking water,
compositions are provided in which the active compound
or compounds are intimately dispersed in an inert
10 carrier or diluent. By inert carrier is meant one that
will not react with the antiparasitic agent and one that
may be administered safely to animals. Preferably, a
carrier for feed administration is one that is, or may
be, an ingredient of the animal ration.
Suitable compositions include feed premixes
or supplements in which the active ingredient is present
in relatively large amounts and which are suitable for
direct feeding to the animal or for addition to the
feed either directly or after an intermediate dilution
20 or blending step. Typical carriers or diluents suitable
for such compositions include, for example, distillers'
dried grains, corn meal, citrus meal, fermentation
residues, ground oyster shells, wheat shorts, molasses
solubles, corn cob meal, edible bean mill feed, soya
25 grits, crushed limestone and the like. The active
hydrogenated C-076 compounds are intimately dispersed
throughout the carrier by methods such as grinding,
stirring, milling or tumbling. Compositions containing
from about 0.005 to 2.0% by weight of the active
30 compound are particularly suitable as feed pxemixes.
Feed supplements, which are fed directly to the
.,.
1115698
-16- 16112 IA
animal, contain from about 0.0002 to 0.3% by weight of
the active compounds.
Such supplements are added to the animal
feed in an amount to give the finished feed the
5 concentration of active compound desired for the
treatment and control of parasitic diseases. Although
the desired concentration of active compound will
vary depending upon the factors previously mentioned
as well as upon the particular C-076 derivative employed,
lO the compounds of this invention are usually fed at
concentrations of between 0.00001 to 0.002% in the
feed in order to achieve the desired antiparasitic
result.
In using the compounds of this invention, the
15 individual hydrogenated C-076 components may be prepared
and used in that form. Alternatively, mixtures of two
or more of the individual hydrogenated C-076 components
may be used, as well as mixtures of the parent C-076
compounds other C-076 compound or other active compounds
20 not related to C-076 and the compounds of this invention.
In the isolation of the C-076 compounds, which
serve as starting materials for the instant processes,
from the fermentation broth, the various C-076 compounds
will be found to have been prepared in unequal amounts.
25 In particular an "a" series compound will be prepared in
a higher proportion than the corresponding "b" series
compound. The weight ratio of "a" series to the
corresponding "b" series is about 75:25 to 99:1. The
29 differences between the "a" series and "b" series is
constant throughout the C-076 compounds and consists of
a sec-butyl group and an iso-propyl group respectively at
1~15698
-17- 16112 IA
the 25 position. This difference, of course, does not
interfere with any of the instant reactions. In
particular may not be necessary to separate the "b"
components from the related "a" component. Separation
5 of these closely related compounds is generally not
practiced since the "b" compound is present only in a
very small percent by weight, and the structural
difference has negligible effect on the reaction
processes and biological activities.
In particular it has been found that the
starting materials for the compounds of this
invention are very often prepared in a ratio of about
80% C-076 Bla or Ala and 20% C-076 Blb or Alb. Thus
the preferred composition of this invention is one
lS which contains about 80% of the "a" component and
20% of the "b" component.
The C-076 compounds of this invention are
also useful in combatting agricultural pests that
inflict damage upon crops while they are growing or
20 while in storage. The compounds are applied using
known techniques as sprays, dusts, emulsions and the
like, to the growing or stored crops to effect
protection from such agricultural pests.
The following examples are provided in order
25 that this invention might be more fully understood;
they are not to be construed as limitative of the
invention.
The hydrogenated C-076 derivatives prepared
in the following examples are generally isolated as
30 amorphous solids and not as crystalline solids. They
are thus characterized analytically using techniques
such as mass spectrometry, nuclear magnetic resonance,
and the like. Being amorphous, the compounds are not
characterized by sharp melting points, however, the
35 chromatographic and analytical methods employed
indicate that the compounds are pure.
lllS698
-18- 16112 IA
EXAM2LE 1
22,23-Dihydro C-076 Ala
51.0 Mg. of C-076 Ala and 14.4 mg. of tris
(triphenylphosphine) rhodium (I) chloride are combined
5 in 3.5 ml. of benzene and hydrogenated for 20 hours at
room temperature under atmospheric pressure. The
crude reaction mixture is chromatographed on a
preparative layer chromatography plate eluting twice
with 10% tetrahydrofuran in chloroform. The product
10 is removed from the support using ethyl acetate
which is evaporated to dryness and the residue analyzed
with 300 MXz nuclear magnetic resonance and mass
spectroscopy indicating the preparation of 22,23-
dihydro C-0 76 Ala.
EXA~LE 2
?2,23-Dihydro C-076 Bla
The solution of 87. 3 mg. of C-076 Bla in 6 ml.
of benzene containing 25 mg. of tris (triphenylphos-
phine rhodium (I) chloride is hydrogenated for 4 hours
20 at room temperature under 1 atmosphere of hydrogen
pressure. Preparative layer chromatography on silica
gel eluting with 20~ tetrahydrofuran in chloroform
recovers starting material. The sample is rehydro-
genated following the above conditions for 19 hours.
25 Preparative layer chromatography recovers 55 mg.
of 22,23-dihydro C-076 Bla which is identified by mass
spectrometry and 300 MHz nuclear magnetic resonance.
11~5~i~8
-19- 16112 IA
EXAM2LE 3
22,23-Dihydro C-076 Bla
A solution of 1.007 g. of C-076 Bla, 314 mg.
of tris (triphenylphosphine) rhodium (I) chloride and
5 33 ml. of benzene is hydrogenated for 21 hours at
room temperature under 1 atmosphere of hydrogen
pressure. The solvent is removed in vacuo and the
residue dissolved in a l:l mixture of methylene
chloride and ethyl acetate and filtered. The filtrate
10 is placed on a column of 60 g. of silica gel eluting
with a 1:1 mixture of methylene chlorid and ethyl
acetate taking 10 ml. fractions. Fractions 14-65
are combined and evaporated to dryness affording
1.118 g. of a solid material which is indicated by
15 high pressure liquid chromatography to be a 60/40
mixture of the hydrogenated product and starting
material. The mixture is rehydrogenated in 55 ml.
of benzene adding 310 mg. of tris (triphenylphosphine)
rhodium (I) chloride and stirring for 21 hours at
20 room temperature under 1 atmosphere of hydrogen
pressure. The solvent is removed in vacuo and the
residue chromatographed on 80 g. of silica gel using
40:60 mixture of ethyl acetate and methylene chloride
as eluant. 10 Ml. fractions are taken and the product
25 appears in fractions 26-80. These fractions are
combined and evaporated to dryness in vacuo affording
a yellow oil. The oil is dissolved in benzene and
lyophilized affording a pale yellow powder which is
identified as 22,23-dihydro C-076 Bla by mass
30 spectrometry and 300 MHz nuclear magnetic resonance.
0.976 G. of product is obtained.
11~56~8
-20- 16112 IA
BXAMPLE 4
22,23-Dihydro C-076 Ala Monosaccharide
ll.2 Mg. of 22,23-dihydro C-076 Ala is
dissolved in 1.1 ml. of 1% sulfuric acid in isopro-
5 panol and stirred for 20 hours at room temperature.
The reaction mixture is diluted with chloroform to
a volume of about 5.0 ml. and washed with saturated
aqueous sodium bicarbonate solution and sodium
chloride solution. The organic layer is dried over
10 sodium sulfate and evaporated to dryness in vacuo
affording an oil. The oil is placed on a silica gel
preparative layer chromatography plate and eluted
with 5% tetrahydrofuran in chloroform. The product
is removed from the plate and lyophilized from
15 benzene affording 5.2 mg. of a white powder which is
identified by 300 MHz nuclear magnetic resonance and
mass spectrometry as 22,23-dihydro C-076 Ala
monosaccharide.
EXAMPLE 5
20 22,23-Dihydro C-076 Ala Aglycone
10.1 Mg. of 22,23-dihydro C-076 Ala is
stirred for 20 hours in 1.1 ml. of 1% sulfuric acid
in methanol at room temperature. The reaction mixture
is treated as in Example 4 affording an oil which is
25 purified by preparative layer chromatography on silica
gel eluting with 5~ tetrahydrofuran in chloroform.
The product is removed from the chromatography plate
and lyophilized from benzene affording 4.2 mg. of a
white powder which 300 MHz nuclear magnetic resonance
30 and mass spectrometry indicate to be 22,23-dihydro
C-076 Ala aglycone.
-, ~ . .....
1115~98
-21- 16112 IA
EXAMPLE 6
22,23-Dihydro C-076 Bla Monosaccharide
395 Mg. of 22,23-dihydro C-076 Bla is added
to a stirred solution of 50 ml. of 1~ sulfuric acid
5 in isopropanol and the solution is stirred for 14
hours at room temperature. The reaction mixture is
treated as in Example 4 affording 0.404 g. of a foam
after lyophilization from benzene. The foam is
chromatographed on 6 preparative layer silica gel
10 chromatography plates eluting twice with 4~ tetra-
hydrofuran in chloroform. The monosaccharide with
a Rf 0.15 is collected and washed from the silica
gel with a total of 650 ml. of ethyl acetate. The
combined washings are evaporated to dryness and the
15 residue lyophilized from benzene to afford 0.2038 g.
of 22,23-dihydro C-076 Bla monosaccharide which high
pressure liquid chromatography indicates to be
essentially pure.
EXAMPLE 7
2022,23-Dihydro C-076 Bla Aglycone
9.7 Mg. of 22,23-dihydro C-076 Bla is
stirred overnight in 1 ml. of a 1% sulfuric acid in
methanol solution. The reaction mixture is treated as
in Example 4 and the solid material treated with
2spreparative layer chromatography on silica gel eluting
with 10% tetrahydrofuran in chloroform. The oil
recovered from the chromatography plate is lyophilized
from benzene affording 4.7 mg. of a white powder
which 300 MHz nuclear magnetic resonance and mass
30spectrometry indicate to be 22,23-dihydro C-076 Bla
aglycone.
l~lS6~8
-22- 16112 IA
EXAMPLE 8
22,23-Dihydro C-076 Bla Aglycone
0.486 G. of 22,Z3-dihydro C-076 Bla is
added to a stirred solution of S0 ml. of 1~ sulfuric
5 acid in methanol and the reaction mixture stirred
for 13 hours at room temperature. The reaction
mixture is diluted with 250 ml. of methylene chloride
and washed with 50 ml. of saturated aqueous potassium
bicarbonate and 50 ml. of water. The aqueous layer
10 is washed twice with 20 ml. portions of methylene
chloride and the combined organic phases are dried
with saturated brine and sodium sulfate and
evaporated to dryness in vacuo affording 0.480 g. of
a pale yellow foam. The foam is dissolved in 4 ml. of
15 methylene chloride and placed on 4 preparative layer
chromatography silica gel plates and eluted 4 times
with 4~ tetrahydrofuran and chloroform. The product is
recovered from the silica gel plates affording an oily
residue which is lyophilized from benzene affording
20 255.8 mg. of a white solid. Traces of methyl
oleandroside are indicated to be present in the solid
material. The white solid is then lyophilized again
from benzene ar,d placed under high vacuum for 20 hours
to remove the impurity affording 22,23-dihydro C-076
25 Bla aglycone.
~15698
-23- 16112 IA
EXAM2LE 9
4"-0-acetyl-22,23-Dihydro C-076 Ala
6.8 Mg. of 22,23-dihydro C-076 Ala is
dissolved in 40 drops of anhydrous pyridine, chilled
5 to 0C and treated with 20 drops of acetic anhydride.
The reaction mixture is allowed to warm to room
temperature and stirred overnight. The reaction
mixture is diluted with 5 ml. of ether and 6 ml. of
water and the layers separated. The aqueous phase
10 is washed twice with ether and the organic layers
combined and back washed 3 times with water. The
ether layer is dried over magnesium sulfate and
evaporated to dryness in vacuo affording an oil. The
oil is chromatographed on silica gel preparative
15 layer chromatography plates eluting with 5% tetra-
hydrofuran in chloroform. The product is recovered
from the plates and lyophilized from benzene affording
6.1 mg. of 4"-O-acetyl-22,23-dihydro C-076 Ala as
determined by mass spectrometry at 300 MHz nuclear
20 magnetic resonance.
EXAMPLE 10
4"-O-acetyl-22,23-Dihydro C-076 Bla and 4",5-di-O-acetyl
22,23-Dihydro C-076 Bla
18.6 Mg. of 22,23-dihydro C-076 Bla is
25 dissolved in 63 drops (about 1 ml.) of dry pyridine
and treated with 9 drops of acetic anhydride at 0C.
The reaction is stirred under nitrogen for 6 hours at
0C. The mixture is then quenched with 5 ml. of
water and extracted 3 times with 3 ml. portions of
30 ether. The combined ether extracts are then washed
3 times with 3 ml. portions of water and dried
~1'15~98
-24- 16112IA
over magnesium sulfate and evaporated to dryness
in vacuo. The oil is chromatographed on preparative
layer silica gel chromatography plates eluting twice
with 5% tetrahydrofuran in chloroform affording
5 5.8 mg. of 4"-O-acetyl-22,23-dihydro C-076 Bla and
5.8 mg. of 4",5-di-O-acetyl-22,23-dihydro C-076 Bla
after lyophilization from benzene. The structures
are confirmed by 300 I~Hz nuclear magnetic resonance
and mass spectrometry.
EXAMPLE 11
22,23-Dihydro C-076 Bla
39 G. of C-076 Bla is dissolved in 1540 ml.
of toluene and introduced into a 4 liter stirred
autoclave. To this is added 3.9 g. of tris(triphenyl-
15 phosphine)rhodium(I) chloride (Wilkinson's catalyst).A hydrogenation pressure of 40 psi. and a temperature
of 40C is maintained with stirring for 4 1/2 hours.
At the end of this period liquid chromatographic
analysis indicates 98% yield of dihydro C-076 Bla with
20 1.5% of tetrahydro C-076 Bla. The toluene is removed
by evaporation in vacuo and the dark red gum is
dissolved in ethanol at a rate of 4 ml. of ethanol per
gram of product. Formamide at a rate of 10 ml. per
gram of product is added and the solution heated on
25 the steam bath to 40-50 while added water at a rate
of 2 ml. per gram of product. After crystallization
commences the heat is removed and the solution allowed
to cool slowly with stirring overnight. The solid is
filtered off and washed with a mixture 3 parts water
30 and 1 part ethanol and dried in vacuo overnight. The
solids are dissolved in 150 ml. of ethanol and warmed
to 35-40C on the steam bath. Water, 150 ml. is added
slowly with stirring. When solution is complete at
35C the heat is removed and the solution allowed to
35 cool slowly overnight. The crystals are removed by
~115698
-25-- 16112IA
filtration and washed with 50% aqueous ethanol and dried
in vacuo overnight affording 32 . 55 g . of 22, 23-
dihydro C-076 Bla with a m.p. of 155-157C.
