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Description  |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to novel antibacterial agents, the intermediates
leading thereto and processes for the preparation of said intermediates.
In particular, the invention concerns 4"-deoxy-4"-amino-erythromycin A
antibacterial agents, 4"-deoxy-4"-oxo-erythromycin A derivatives as useful
intermediates leading to the 4"-amino compounds and processes for the
preparation of the 4"-deoxy-4"-oxo-erythromycin A intermediates.
2. Description of the Prior Art
Erythromycin is an antibiotic formed during the culturing of a strain of
Streptomyces erythreus in a suitable medium as taught in U.S. Pat. No.
2,653,899. Erythromycin, which is produced in two forms, A and B, is
represented by the following structure:
______________________________________
##STR1##
Erythromycin R
______________________________________
A OH
B H
______________________________________
The structure reveals that the antibiotic is comprised of three main
portions: a sugar fragment known as cladinose, a second sugar moiety
containing a basic amino substituent known as desosamine and a fourteen
membered lactone ring referred to as erythronolide A or B or, as herein
described, the macrolide ring. While the numbering system of the macrolide
ring is in unprimed numbers, that of the desosamine is in primed numbers
and that of cladinose in double-primed numbers.
Numerous derivatives of erythromycin have been prepared in an effort to
modify its biological or pharmacodynamic properties.
U.S. Pat. No. 3,417,077 describes the reaction product of erythromycin and
ethylene carbonate as a very active antibacterial agent. U.S. Pat. No.
3,884,903 discloses 4"-deoxy-4"-oxo-erythromycin A and B derivatives as
being useful as antibiotics.
Erythromycylamine, the 9-amino derivative of erythromycin A, has been the
subject of considerable investigation [British Pat. No. 1,100,504,
Tetrahedron Letters, 1645 (1967) and Croatica Chemica Acta, 39, 273
(1967)] and some controversy as to its structural identity [Tetrahedron
Letters, 157 (1970) and British Pat. No. 1,341,022]. Sulfonamide
derivatives of erythromycylamine are reported in U.S. Pat. No. 3,983,103
to be useful as antibacterial agents. Other derivatives are also reported
[Ryden, et al., J. Med. Chem., 16, 1059 (1973) and Massey, et al., J. Med.
Chem., 17, 105 (1974)] to have in vitro and in vivo antibacterial
activity.
SUMMARY OF THE INVENTION
It has now been discovered that certain novel
4"-deoxy-4"-amino-erythromycin A derivatives are outstanding as
antibacterial agents. These compounds are represented by the formulae:
##STR2##
and a pharmaceutically acceptable acid addition salt thereof, wherein
R.sub.1 and R.sub.4 are each hydrogen or alkanoyl of two to three carbon
atoms; R.sub.2 is alkanoyl of two to three carbon atoms; R.sub.3 is
hydrogen; R.sub.2 and R.sub.3 when taken together are
##STR3##
and R.sub.3 and R.sub.4 when taken together are
##STR4##
A preferred group of compounds within this class of chemotherapeutic agents
are those of Formula III. Especially preferred within this group are those
compounds wherein R.sub.2 and R.sub.3 when taken together are
##STR5##
A second preferred group of compounds in this class of antibacterial agents
are those of Formula IV. Especially preferred within this group are those
compounds wherein R.sub.4 is hydrogen and also wherein R.sub.3 and R.sub.4
when taken together are
##STR6##
A second class of compounds of the present invention, useful as
intermediates leading to the antibacterial agents of Formulae III and IV,
are represented as follows:
##STR7##
wherein R.sub.1 is hydrogen or alkanoyl of two to three carbon atoms;
R.sub.2 is alkanoyl of two to three carbon atoms; Y is N--OH or
##STR8##
R.sub.3 is hydrogen; and R.sub.2 and R.sub.3 when taken together are
##STR9##
Preferred within this class of intermediates are those compounds of Formula
I. Especially preferred within this group of intermediates are those
compounds wherein R.sub.1 is hydrogen or acetyl.
A second group of preferred intermediates are those of Formula II.
Especially preferred within this group are those intermediates wherein
R.sub.1 is hydrogen and also those wherein R.sub.1 is acetyl.
Also within the scope of the present invention are processes for preparing
intermediate compounds of the formulae:
##STR10##
wherein Ac and R.sub.2 are each alkanoyl of two to three carbon atoms;
R.sub.3 is hydrogen; and R.sub.2 and R.sub.3 when taken together are
##STR11##
which comprises reacting a compound selected from the group consisting of
the formulae:
##STR12##
with one mole each of dimethylsulfoxide and trifluoroacetic anhydride in a
reaction-inert-solvent at about -30.degree. to -65.degree. C. followed by
contacting the reaction mixture with at least one mole of triethylamine.
A preferred feature of this process is the oxidation of the compounds of
Formula I' and II' wherein the reaction-inert-solvent is methylene
chloride.
A second process within the scope of the present invention comprises the
preparation of compounds of the formulae:
##STR13##
wherein Ac and R.sub.2 are each alkanoyl of two to three carbon atoms;
R.sub.3 is hydrogen; and R.sub.2 and R.sub.3 when taken together are
##STR14##
which comprises reacting a compound selected from the group consisting of
the formulae:
##STR15##
with one mole each of N-chlorosuccinimide and dimethylsulfide and in a
reaction-inert-solvent at about 0.degree. to -25.degree. C. followed by
contacting the reaction mixture with at least one mole of triethylamine.
A preferred feature of the claimed process is the use of toluene and
benzene as the reaction-inert solvent.
Throughout the present invention, the stereochemical designation of the
substituents on the sugars and macrolide ring, with the exception of
epimerication at the 4"-position where noted, are those of the naturally
occurring erythromycin A.
Also considered within the purview of the present invention are
erythromycin B derivatives which correspond to those of Formulae I and II.
These erythromycin B compounds are useful intermediates and are prepared
by the same synthetic procedure as herein described for the erythromycin A
compounds. The erythromycin B intermediates are also converted, by the
herein described procedures, to erythromycin B amines corresponding to the
compounds of Formulae III and IV of the present invention. The
erythromycin B amines are also useful as antibacterial agents.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the processes employed for synthesizing the
4"-deoxy-4"-amino-erythromycin A derived antibacterial agents of the
present invention, the following scheme, starting with a
2'-alkanoyl-erythromycin A, or derivative thereof, are represented as
follows:
##STR16##
The selective oxidation of I' and II' to I and II, respectively, (Y.dbd.O)
is the first of the processes of the present invention and comprises
reacting the compounds I' and II' with trifluoroacetic anhydride and
dimethylsulfoxide followed by the addition of a tertiary amine such as
triethylamine.
In practice, the trifluoroacetic anhydride and dimethylsulfoxide are
initially combined in a reaction-inert-solvent at about -65.degree. C.
After ten to fifteen minutes the alcohols I' and II' are added at such a
rate that the temperature is maintained at about -65.degree. C. and does
not rise above -30.degree. C. At temperatures above -30.degree. C. the
trifluoroacetic anhydride - dimethylsulfoxide complex is not stable. The
reaction temperature is maintained below -30.degree. and -65.degree. C.
for about fifteen minutes and is then lowered to about -70.degree. C. A
tertiary amine is added all at once and the reaction allowed to warm
during a ten to fifteen minute period. The reaction mixture is
subsequently treated with water and worked up.
Regarding the quantities of reactants, for each mole of alcohol substrate
employed, one mole each of the trifluoroacetic anhydride and
dimethylsulfoxide are required. Experimentally, it is advantageous to
employ a 1-5 fold excess of the anhydride and dimethylsulfoxide in order
to hasten the completion of the reaction. The tertiary amine employed
should correspond to the molar amount of trifluoroacetic anhydride used.
The reaction-inert-solvent utilized in this process should be one which
appreciably solubilizes the reactants and does not react to any great
extent with either the reactants or the products formed. Since this
oxidation process is conducted at -30.degree. to -65.degree. C., it is
preferred that, in addition to having the above characteristics, said
solvent possess a freezing point below the reaction temperature. Such
solvents or mixtures thereof which meet these criteria are toluene,
methylene chloride, ethyl acetate, chloroform or tetrahydrofuran. Solvents
which meet the above requirements but which have a freezing point above
the reaction temperature can be employed in minor amounts in combination
with one of the preferred solvents. The especially preferred solvent for
this process is methylene chloride.
The preferred compounds prepared by this process are
2'-acetyl-4"-deoxy-4"-oxo-erythromycin A,
11,2'-diacetyl-4"-deoxy-4"-oxo-erythromycin A, 6,9-hemiketal and
2'-acetyl-4"-deoxy-4"-oxo-erythromycin A 6,9-hemiketal 11,12-carbonate
ester.
The reaction time is not critical and is dependent on reaction temperature
and the inherent reactivity of the starting reagents. At temperatures of
about -30.degree. to -65.degree. C., the reaction is complete in fifteen
to thirty minutes.
As to the order of addition of the reagents, it is preferred that the
trifluoroacetic anhydride be combined with the dimethylsulfoxide followed
by the addition of the requisite alcohol substrate. It is further
suggested, as hereinbefore mentioned, that the temperature of the reaction
is kept below -30.degree. C. This is in accordance with the teaching of
Omura, et al., J. Org. Chem., 41, 957 (1976).
The second process of the claimed invention, used to prepare intermediates
leading to the useful antibacterial agents, is represented by the
following scheme:
##STR17##
The second process represents an oxidation reaction wherein the 4"-hydroxy
substituent of I' and II', wherein Ac and R.sub.2 are each alkanoyl of two
to three carbon atoms, R.sub.3 is hydrogen, R.sub.2 and R.sub.3 when taken
together are
##STR18##
is oxidized to a 4"-deoxy-4"-oxo-erythromycin A compound.
The process comprises the use of N-chlorosuccinimide and dimethylsulfide as
the oxidizing agent. In practice, these two reagents are first combined
together in a reaction-inert-solvent at about 0.degree. C. After ten to
twenty minutes the temperature is lowered to 0.degree. to -25.degree. C.
and the alcohol substrate I' or II' is added, while maintaining the
aforementioned temperature. After two to four hours reaction time, a
tertiary amine, such as triethylamine, is added the reaction mixture
hydrolyzed and worked up.
Regarding the quantities of reactants, for each mole of alcohol substrate
employed, one mole each of the N-chlorosuccinimide and dimethylsulfide are
required. Experimentally, it is advantageous to employ a 1-20 fold excess
of the succinimide and sulfide reactants in order to hasten the completion
of the reaction. The tertiary amine employed should correspond to the
molar amount of succinimide used.
The reaction-inert-solvent utilized in the claimed process should be one
which appreciably solubilizes the reactants and does not react to any
appreciable extent with either the reactants or the products formed. Since
the reaction is conducted at about 0.degree. to -25.degree. C., it is
preferred that, in addition to having the above characteristics, it should
possess a freezing point below the reaction temperature. Such solvents or
mixtures thereof which meet these criteria are toluene, ethyl acetate,
chloroform, methylene chloride or tetrahydrofuran. Solvents which meet the
above requirements but which have a freezing point above the reaction
temperature can also be employed in minor amounts in combination with one
or more of the preferred solvents. The especially preferred solvent for
the claimed process is toluene-benzene.
The preferred compounds prepared by this process are
11,2'-diacetyl-4"-deoxy-4"-oxo-erythromycin A 6,9-hemiketal,
2'-acetyl-4"-deoxy-4"-oxo-erythromycin A 6,9-hemiketal 11,12-carbonate
ester and 2'-acetyl-4"-deoxy-4"-oxo-erythromycin A.
Reaction time is not critical and is dependent on concentration, reaction
temperature and the inherent reactivity of the reagents. At a reaction
temperature of 0.degree. to -25.degree. C. the reaction time is about two
to four hours.
Regarding the order of addition, as previously mentioned, it is preferred
that the alcohol substrate I' or II' be added to the premixed succinimide
derivative and dimethylsulfide.
Both the herein described processes are viewed as unique because of the
selectivity of the oxidation which takes place exclusively at the
4"-hydroxy substituent, leaving other secondary alcohols in the molecule
unaffected.
The useful intermediate 4"-deoxy-4"-oxo compounds of the formula:
##STR19##
wherein R.sub.1 and R.sub.2 are each alkanoyl of two to three carbon atoms
and R.sub.3 is hydrogen are prepared by treating a compound of the
formula:
##STR20##
wherein Y is O, R.sub.1 is alkanoyl of two to three carbon atoms, with an
alkanoic anhydride (R.sub.2 O) and pyridine.
In practice, the ketone I is contacted with an excess of the anhydride in
pyridine as the solvent. It is preferred that as much as a four fold
excess of the anhydride be employed in the reaction.
The reaction is conveniently carried out at ambient temperatures. At these
reaction temperatures the reaction time is about twelve to twenty-four
hours.
Removal of the alkanoyl moiety at the 2'-position of the intermediate
ketones I (Y.dbd.O) and II is carried out through a solvolysis reaction
wherein the 2'-alkanoyl-4"-deoxy-4"-oxo-erythromycin A related compound is
allowed to stir with an excess of methanol overnight at room temperature.
Removal of the methanol and subsequent purification, where necessary, of
the residual product provides for compounds of Formulae I (Y.dbd.O) and II
wherein R.sub.1 is hydrogen.
As previously mentioned, the ketones of Formulae I (Y.dbd.O) and II are
useful intermediates leading to the 4"-deoxy-4"-amino-erythromycin A
antibacterial agents of the present invention of formulae III and IV.
Preferred as intermediates in this group are
2'-acetyl-4"-deoxy-4"-oxo-erythromycin A 6,9-hemiketal 11,12-carbonate
ester and 4"-deoxy-4"-oxo-erythromycin A 6,9-hemiketal 11,12-carbonate
ester.
Several synthetic pathways can be employed in the preparation of the
antibacterial agents of Formulae III and IV from the requisite ketones I
(Y.dbd.O) and II.
Preparation of the 4"-deoxy-4"-amino-erythromycin A compounds of Formula
III is carried out by the condensation of the ketones II with the ammonium
salt of a lower alkanoic acid and the subsequent reduction of the in situ
generated imine. The term "lower alkanoic" refers, in this instance, to an
acid having two to four carbon atoms.
In practice, a solution of the ketone II in a lower alkanol, such as
methanol or isopropanol, is treated with the ammonium salt of a lower
alkanoic acid, such as acetic acid, and the cooled reaction mixture
treated with the reducing agent sodium cyanoborohydride. The reaction is
allowed to proceed at room temperature for several hours before it is
subsequently hydrolyzed and the product isolated.
Although one mole of the ammonium alkanoate is needed per mole of ketone,
it is preferred that an excess, as great as ten fold, be employed in order
to ensure complete and rapid formation of the imine. Such excess amounts
appear to have little deleterious effects on the quality of the product.
Regarding the amount of reducing agent to be employed per mole of ketone,
it is preferred that about two moles of sodium cyanoborohydride per mole
of ketone be used.
The reaction time will vary with concentration, reaction temperature and
the inherent reactivity of the reagents. At room temperature, the
preferred reaction temperature, the reaction is substantially complete
after two to three hours.
When the lower alkanol solvent is methanol there is, as previously
mentioned, substantial solvolysis of any alkanoyl group at the
2'-position. In order to avoid removal of such a moiety it is preferred
that isopropanol be used as the reaction solvent.
The preferred ammonium alkanoate, as previously indicated, for this
reaction is ammonium acetate.
In isolating the desired 4"-deoxy-4"-amino-erythromycin A derivatives from
any non-basic by-products or starting material, advantage is taken of the
basic nature of the final product. Accordingly, an aqueous solution of the
product is extracted over a range of gradually increasing pH such that
neutral or non-basic materials are extracted at lower pH's and the product
at a pH of greater than 5. The extracting solvents, either ethyl acetate
or diethyl ether, are backwashed with brine and water, dried over sodium
sulfate and the product obtained by removal of the solvent. Additional
purification, if necessary, can be effected by column chromatography on
silica gel according to known procedures.
As previously mentioned, solvolysis of the 2'-alkanoyl group from the
appropriate 2'-alkanoyl-4"-deoxy-4"-amino-erythromycin A derivative can be
effected by allowing a methanol solution of said compound to stand
overnight at ambient temperatures.
During the reductive amination of ketones of Formula II wherein R.sub.2 and
R.sub.3 when taken together are
##STR21##
and R.sub.1 is alkanoyl of two to three carbon atoms or hydrogen, it is
noted that amines related to both Formulae III and IV are produced. This
is represented by the following scheme:
##STR22##
The amine products III and IV as represented are conveniently separated by
selective crystallization from diethyl ether. Recrystallization of the
mixture of III and IV as represented from acetone-water induces hemiketal
formation in the amine of Formula IV resulting in the isolation of III as
the sole product.
The first direct synthetic pathway to the amine compounds of Formula IV is
the same route as discussed previously and comprises the condensation of
the ketone I with an ammonium alkanoate followed by reduction of the in
situ generated imine with sodium cyanoborohydride.
Compounds of Formula IV, wherein R.sub.1, R.sub.3 and R.sub.4 are as
previously defined, are also prepared by the reduction of the
aforementioned imine using hydrogen and an appropriate hydrogenation
catalyst. Experimentally, the appropriate ketone (I) in a lower alkanol,
such as methanol or isopropanol, is treated with the ammonium salt of a
lower alkanoic acid, such as acetic acid, and the hydrogenation catalyst,
and the mixture shaken in a hydrogen atmosphere until the reaction is
essentially complete.
Although one mole of the ammonium alkanoate is needed per mole of ketone,
it is preferred that an excess, as great as ten fold, be employed in order
to insure complete and rapid formation of the imine. Such excess amounts
appear to have little deleterious effects on the quality of the product.
The hydrogenation catalyst can be selected from a wide range of agents;
Raney nickel and 5-10 percent palladium-on-charcoal are, however, the
preferred catalysts. These may be used in varying amounts depending on how
fast the reaction is to be completed. Amounts from 10-200 percent of the
weight of I can be employed effectively.
The pressure of the hydrogen gas in the hydrogenation vessel also
influences the rate of reaction. It is preferred, for the convenience of
reaction time, that an initial pressure of 50 p.s.i, be employed. It is
also preferred, for convenience, that the reduction be carried out at
ambient temperatures.
Reaction time is dependent on a number of factors including temperature,
pressure, concentration of the reactants and the inherent reactivity of
the reagents. Under the aforementioned preferred conditions the reaction
is complete in 12 to 24 hours.
The product is isolated by filtration of the spent catalyst and removal of
the solvent in vacuo. The residual material is subsequently treated with
water and the product isolated from non-basic materials by extraction of
the basic product from water at varying pH's previously described.
As previously indicated, when the lower alkanol solvent is methanol there
is substantial solvolysis of any alkanoyl group at the 2'-position. In
order to avoid removal of such a moiety it is preferred that isopropanol
be used as the reaction solvent.
The second synthetic route to the 4"-deoxy-4"-amino-erythromycin A
antibacterial agents of Formula IV comprises initial conversion of the
ketones of Formula I (Y.dbd.O) to an oxime or oxime derivative, i.e.,
Y.dbd.N--OH and
##STR23##
followed by reduction of the oxime or derivative thereof.
The oximes of the ketones I (Y.dbd.O) are prepared by reacting said ketones
with hydroxylamine hydrochloride and barium carbonate in methanol or
isopropanol at room temperature. In practice, it is preferred that an
excess of hydroxylamine be employed, and as much as a three fold excess
provides the desired intermediate in good yields. Employing ambient
temperatures and an excess of the hydroxylamine allows for the preparation
of the desired oxime derivative in a reaction period of one to three
hours. The barium carbonate is used in molar quantities twice that of the
hydroxylamine hydrochloride employed. The product is isolated by addition
of the reaction mixture to water followed by basification to pH 9.5 and
extraction with a water-immiscible solvent such as ethyl acetate.
Alternately, the reaction mixture can be filtered and the filtrate
concentrated in vacuo to dryness. The residue is subsequently partitioned
between water at pH 9.0-9.5 and a water-immiscible solvent.
Preparation of the O-acetyloxime compounds of Formula I
##STR24##
is effected by acetylation of the corresponding oxime. Experimentally, one
mole of the oxime is reacted with one mole of acetic anhydride in the
presence of one mole of pyridine or triethylamine. The use of an excess of
the anhydride and pyridine aid in the completion of the reaction and an
excess of 30-40% is preferred. The reaction is best conducted in an
aprotic solvent such as benzene or ethyl acetate at room temperature
overnight. On completion of the reaction, water is added, the pH adjusted
to 9.0 and the product separated in the solvent layer.
The preferred oxime and oxime derivatives which are useful intermediates
leading to the 4"-deoxy-4"-amino-erythromycin A derived antibacterial
agents include 2'-acetyl-4"-deoxy-4"-oxo-erythromycin A oxime,
2'-acetyl-4"-deoxy-4"-oxo-erythromycin A O-acetyloxime,
4"-deoxy-4"-oxo-erythromycin A oxime and 4"-deoxy-4"-oxo-erythromycin A
O-acetyloxime.
Reduction of the ketone derivatives (Y.dbd.N--OH or
##STR25##
is carried out by catalytic hydrogenation wherein a solution of the oxime
or derivative thereof in a lower alkanol, such as isopropanol, and a Raney
nickel catalyst is shaken in a hydrogen atmosphere at an intial pressure
of 1000 p.s.i. at room temperature overnight. Filtration of the spent
catalyst followed by removal of the solvent from the filtrate provides for
the isolation of the desired 4"-deoxy-4"-amino antibacterial agent related
to Formula IV. If methanol is employed as the solvent in this reduction,
solvolysis of a 2'-alkanoyl moiety is probable. In order to avoid this
side-reaction, isopropanol is employed.
Preferred among these 4"-deoxy-4"-amino-erythromycin A derived
antibacterial agents of Formulae III and IV are both epimers of
4"-deoxy-4"-amino-erythromycin A 6,9-hemiketal 11,12-carbonate ester and
of 4"-deoxy-4"-amino-erythromycin A, of 4"-amino-erythromycin A
11,12-carbonate ester.
In the utilization of the chemotherapeutic activity of those compounds of
Formulae III and IV of the present invention which form salts, it is
preferred, of course, to use pharmaceutically acceptable salts. Although
water-insolubility, high toxicity, or lack of crystalline nature may make
some particular salt species unsuitable or less desirable for use as such
in a given pharmaceutical application, the water insoluble or toxic salts
can be converted to the corresponding pharmaceutically bases by
decomposition of the salt as described above, or alternately they can be
converted to any desired pharmaceutically acceptable acid addition salt.
Examples of acids which provide pharmaceutically acceptable anions are
hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, or sulfurous,
phosphoric, acetic, lactic, citric, tartaric, succinic, maleic, gluconic
and aspartic acids.
As previously mentioned, the stereochemistry of the starting materials
leading to the antibacterial agents of the present invention is that of
the natural material. The oxidation of the 4"-hydroxyl group to a ketone
and the subsequent conversion of said ketone to the 4"-amines presents an
opportunity for the stereochemistry of the 4"-substituent to change from
that of the natural product. Accordingly, when the compounds I (Y.dbd.O)
and II are converted to amines by one of the hereinbefore described
procedures, it is possible that two epimeric amines are formed.
Experimentally, it is observed that both epimeric amines are present in
the final product in varying ratios depending on the choice of synthetic
method. If the isolated product consists predominantly of one of the
epimers, said epimer can be purified by repeated recrystallization from a
suitable solvent to a constant melting point. The other epimer, the one
present in smaller amounts in the originally isolated solid material, is
the predominant product in the mother liquor. It can be recovered
therefrom by methods known to those skilled in the art, as for example,
the evaporation of the mother liquor and repeated recrystallization of the
residue to a product of constant melting point.
Although said mixture of epimers can be separated by methods known to those
skilled in the art, for practical reasons it is advantageous to use said
mixture as it is isolated from the reaction. However, it is frequently
advantageous to purify the mixture of epimers by at least one
recrystallization from an appropriate solvent, subjecting it to column or
high pressure liquid chromatography, solvent partitioning or by
trituration in an appropriate solvent. Said purification, while not
necessarily separating the epimers, removes such extraneous materials as
starting materials and undesirable by-products.
The absolute stereochemical assignment for the epimers has not been
completed. Both epimers of a given compound, however, exhibit the same
type of activity, e.g., as antibacterial agents.
The novel 4"-deoxy-4"-amino-erythromycin A derivatives described herein
exhibit in vitro activity against a variety of Gram-positive
microorganisms, e.g., Staphylococcus aureus and Strepttococcus pyrogenes,
and against certain Gram-negative microorganisms such as those of
spherical or ellisoidal shape (cocci). Their activity is readily
demonstrated by in vitro tests against various microorganisms in a
brain-heart infusion medium by the usual two-fold serial dilution
technique. Their in vitro activity renders them useful for topical
application in the form of ointments, creams and the like; for
sterilization purposes, e.g., sick-room utensils; and as industrial
antimicrobials, for example, in water treatment, slime control, paint and
wood preservation.
For in vitro use, e.g., for topical application, it will often be
convenient to compound the selected product with a
pharmaceutically-acceptable carrier such as vegetable or mineral oil or an
emollient cream. Similarly, they may be dissolved or dispersed in liquid
carriers or solvents, such as water, alcohol, glycols or mixtures thereof
or other pharmaceutically-acceptable inert media; that is, media which
have no harmful effect on the active ingredient. For such purposes, it
will generally be acceptable to employ concentrations of active
ingredients of from about 0.01 percent to about 10 percent by weight based
on total composition.
Additionally, many compounds of this invention and their acid addition
salts are active versus Gram-positive and certain Gram-negative
microorganisms, e.g., Pasteurella multocide and Neisseria sicca, in vivo
via the oral and/or parenteral routes of administration in animals,
including man. Their in vivo activity is more limited as regards
susceptible organisms and is determined by the usual procedure which
comprises infecting mice of substantially uniform weight with the test
organism and subsequently treating them orally or subcutaneously with the
test compound. In practice, the mice, e.g., 10, are given an
intraperitoneal inoculation of suitable diluted cultures containing
approximately 1 to 10 times the LD.sub.100 (the lowest concentration of
organisms required to product 100% deaths). Control tests are
simultaneously run in which mice receive inoculum of lower dilutions as a
check on possible variation in virulence of the test organism. The test
compound is administered 0.5 hour post-inoculation, and is repeated 4, 24
and 48 hours later. Surviving mice are held for four days after the last
treatment and the number of survivors is noted.
When used in vivo, these novel compounds can be administered orally or
parenterally, e.g., by subcutaneous or intramuscular injection, at a
dosage of from about 1 mg./kg. to about 200 mg./kg. of body weight per
day. The favored dosage range is from about 5 mg./kg. to about 100 mg./kg.
of body weight per day and the preferred range from about 5 mg./kg. to
about 50 mg./kg. of body weight per day. Vehicles suitable for parenteral
injection may be either aqueous such as water, isotonic saline, isotonic
dextrose, Ringer's solution, or non-aqueous such as fatty oils of
vegetable origin (cotton seed, peanut oil, corn, sesame),
dimethylsulfoxide and other non-aqueous vehicles which will not interfere
with therapeutic efficiency of the preparation and are non-toxic in the
volume or proportion used (glycerol, propylene glycol, sorbitol).
Additionally, compositions suitable for extemporaneous preparation of
solutions prior to administration may advantageously be made. Such
compositions may include liquid diluents, for example, propylene glycol,
diethyl carbonate, glycerol, sorbitol, etc.; buffering agents,
hyaluronidase, local anesthetics and inorganic salts to afford desirable
pharmacological properties. These compounds may also be combined with
various pharmaceutically-acceptable inert carriers including solid
diluents, aqueous vehicles, non-toxic organic solvents in the form of
capsules, tablets, lozenges, troches, dry mixes, suspensions, solutions,
elixirs and parenteral solutions or suspensions. In general, the compounds
are used in various dosage forms at concentration levels ranging from
about 0.5 percent to about 90 percent by weight of the total composition.
The following examples are provided solely for the purpose of illustration
and not to be construed as limitations of this invention, many variations
of which are possible without departing from the spirit or scope thereof.
EXAMPLE 1
2'-Acetyl-4"-deoxy-4"-oxo-erythromycin A
To 3 ml. of methylene chloride and 0.328 ml. of dimethylsulfoxide cooled to
about -65.degree. C. and maintained under a nitrogen atmosphere is added
0.652 ml. of trifluoroacetic anhydride. After about a minute a white
slurry forms indicating the presence of the trifluoroacetic
anhydride-dimethylsulfoxide complex. To the resulting slurry is added
dropwise a solution of 1.0 g. of 2'-acetylerythromycin A.ethyl acetate,
obtained by recrystallization of 2'-acetylerythromycin A from ethyl
acetate, in 7 ml. of methylene chloride keeping the temperature at about
-65.degree. C. The resulting mixture is allowed to stir for 15 min. at
about -60.degree. C. and is then cooled to -70.degree. C. Triethylamine
(1.61 ml.) is added rapidly to the reaction mixture and the cooling bath
is removed. After stirring for 15 min. the solution is added to 10 ml. of
water and the pH of the aqueous phase adjusted to 10. The organic phase is
separated, washed successively with water (3.times.10 ml.) and brine
solution (1.times.10) and dried over sodium sulfate. Removal of the
solvent under reduced pressure gives 929 mg. of the crude product.
Recrystallization from methylene chloride-hexane gives 320 mg. of the
purified product, m.p. 105.degree.-108.degree. C.
NMR (.delta., CDCl.sub.3): 3.28 (3H)s, 2.21 (6H)s and 2.03 (3H)s.
In a similar manner, starting with 2'-propionylerythromycin A.ethyl acetate
and following the above procedure gives
2'-propionyl-4"-deoxy-4"-oxo-erythromycin A.
EXAMPLE 2
4"-Deoxy-4"-oxo-erythromycin A
A solution of 4.0 g. of 2'-acetyl-4"-deoxy-4"-oxo-erythromycin A in 75 ml.
of methanol is allowed to stir at ambient temperatures for 20 hrs. The
solvent is removed in vacuo and the residual white foam recrystallized
from methylene chloride-hexane, 3.44 g., m.p. 170.5.degree.-172.5.degree.
C.
NMR (.delta., CDCl.sub.3): 3.36 (3H)s and 2.33 (6H)s.
A product identical with the above is isolated when
2'-propionyl-4"-deoxy-4"-oxo-erythromycin A is treated with methanol at
room temperature.
EXAMPLE 3
2'-Acetyl-4"-deoxy-4"-oxo-erythromycin A
To a stirring solution of 13.7 g. of 4"-deoxy-4"-oxo-erythromycin A in 100
ml. of ethyl acetate is added 2.3 ml. of acetic anhydride and the
resulting reaction mixture allowed to stir at room temperature for 2 hrs.
The solution is added to 100 ml. of water and the pH of the aqueous phase
raised to 9.5 by the addition of 6 N sodium hydroxide solution. The
organic layer is separated, dried over sodium sulfate and concentrated to
give 14.5 g. of a white foam identical, after recrystallization from
methylene chloride-hexane, with the product of Example 1.
EXAMPLE 4
2'-Acetyl-4"-deoxy-4"-oxo-erythromycin A oxime
To 500 ml. of methanol is added 10.8 g. of
2'-acetyl-4"-deoxy-4"-oxo-erythromycin A, 1.94 g. of hydroxylamine
hydrochloride and 11.0 g. of barium carbonate, and the resulting
suspension allowed to stir at room temperature for 3.5 hrs. The mixture is
filtered and the filtrate concentrated under reduced pressure. The
residual foam is taken up in ethyl acetate which is subsequently washed
with water at pH 9.5. The organic phase is separated, dried over sodium
sulfate and concentrated in vacuo to give 10.6 g. of the desired product.
NMR (.delta., CDCl.sub.3): 3.33 (3H)s, 2.30 (6H)s and 2.06 (3H)s.
EXAMPLE 5
2'-Acetyl-4"-deoxy-4"-oxo-erythromycin A O-acetyloxime
To a solution of 330 mg. of 2'-acetyl-4"-deoxy-4"-oxo-erythromycin A oxime
in 30 ml. of ethyl acetate is added with stirring 64.2 .mu.l of acetic
anhydride, and the reaction allowed to stir overnight at room temperature.
An additional 15.8 .mu.l of acetic anhydride and 23.4 .mu.l of
triethylamine are added and the stirring continued for 4 hrs. The reaction
mixture is added to water and the pH adjusted to about 9.0. The ethyl
acetate layer is separated, dried over sodium sulfate and concentrated
under vacuum to give 300 mg. of the desired product.
NMR (.delta., CDCl.sub.3): 3.38 (3H)s, 2.25 (6H)s, 2.20 (3H)s, 2.05 (3H)s
and 1.56 (3H)s.
In a similar manner by substituting
2'-propionyl-4"-deoxy-4"-oxo-erythromycin A oxime and
4"-deoxy-4"-oxo-erythromycin A oxime for
2'-acetyl-4"-deoxy-4"-oxo-erythromycin A oxime in the above procedure, the
respective O-acetyl derivatives are prepared.
EXAMPLE 6
2'-Acetyl-4"-deoxy-4"-amino-erythromycin A
A mixture of 14.0 g. of 2'-acetyl-4"-deoxy-4"-oxo-erythromycin A
O-acetyloxime and 60 g. of isopropanol washed Raney nickel in 400 ml. of
isopropanol is agitated in a hydrogen atmosphere at an initial pressure of
1000 p.s.i. overnight at room temperature. The catalyst is filtered and
the filtrate concentrated to a white foam. The residue is redissolved in
400 ml. of isopropanol and combined with 50 g. of fresh isopropanol washed
Raney nickel. The hydrogenation is continued overnight at room temperature
and an initial hydrogen pressure of 1000 p.s.i. The catalyst is filtered
and the filtrate concentrated in vacuo to dryness to give 8.1 g. of the
desired product.
EXAMPLE 7
Starting with the appropriate O-acetyloxime and employing the procedure of
Example 6, the following 4"-amino-erythromycin A analogs are prepared:
______________________________________
##STR26##
R.sub.1
______________________________________
##STR27##
______________________________________
example 8
4"-deoxy-4"-amino-erythromycin A
A solution of 2.17 g. of 2'-acetyl-4"-deoxy-4"-amino-erythromycin A in 50
ml. of methanol is allowed to stir at room temperature overnight. The
solvent is removed under reduced pressure and the residual foam treated
with a mixture of 50 ml. of chloroform and 50 ml. of water. The pH of the
aqueous layer is adjusted to 9.5 and the organic layer separated. The
chloroform layer is treated with fresh water and the pH adjusted to 4.0.
The pH of the acid aqueous layer containing the product is gradually
adjusted to 5, 6, 7, 8 and 9 by the addition of base, being extracted at
each pH with fresh chloroform. The extracts at pH 6 and 7 contain the
major portion of the product and these are combined and treated with fresh
water at pH 4. The aqueous layer is again adjusted through pH 5, 6 and 7,
being extracted at each pH with fresh chloroform. The chloroform extract
at pH 6 is dried over sodium sulfate and concentrated to give 249 mg. of
the product as an epimeric mixture.
NMR (.delta., CDCl.sub.3): 3.30 (1H)s, 3.26 (2H)s, 2.30 (6H)s and 1.46
(3H)s.
In a similar manner, 4"-deoxy-4"-amino-erythromycin A is prepared by the
methanol solvolysis of 2'-propionyl-4"-deoxy-4"-amino-erythromycin A.
EXAMPLE 9
4"-Deoxy-4"-amino-erythromycin A
To a stirring solution of 3.0 g. of 4"-deoxy-4"-oxo-erythromycin A in 30
ml. of methanol under a nitrogen atmosphere is added 3.16 g. of dry
ammonium acetate. After 5 min. 188 mg. of sodium cyanoborohydride is
washed into the reaction mixture with 5 ml. of methanol and the reaction
allowed to stir at room temperature overnight. The light yellow solution
is poured into 300 ml. of water and the pH adjusted to 6.0. The aqueous is
extracted at pH6, 7, 7.5, 8, 9 and 10 using 125 ml. of diethyl ether for
each extraction. The extracts at pH 8, 9 and 10 are combined and washed
with 125 ml. of fresh water. The separated aqueous layer is extracted with
ether (1.times.100 ml.) at pH 7, ethyl acetate (1.times.100 ml.) at pH 7,
ether (1.times.100 ml.) at pH 7.5, ethyl acetate (1.times.100 ml.) at pH
7.5 and ethyl acetate (1.times.100 ml.) at pH 8, 9 and 10. The ethyl
acetate extracts at pH 9 and 10 are combined, washed with a saturated
brine solution and dried over sodium sulfate. Removal of the solvent in
vacuo gives 30 mg. of an epimeric mixture of the desired product as an
ivory colored foam.
EXAMPLE 10
4"-Deoxy-4"-amino-erythromycin A (single epimer)
A solution of 10.0 g. of the epimeric mixture of
2'-acetyl-4"-deoxy-4"-amino-erythromycin A in 150 ml. of methanol is
allowed to stir at room temperature under nitrogen for 72 hrs. The solvent
is removed in vacuo and the residue is dissolved in a stirring mixture of
150 ml. of water and 200 ml. of chloroform. The aqueous layer is discarded
and 150 ml. of fresh water is added. The pH of the aqueous layer is
adjusted to 5 and the chloroform layer is separated. The pH of the aqueous
phase is subsequently adjusted to 5.5, 6, 7, 8 and 9, being extracted
after each adjustment with 100 ml. of fresh chloroform. The chloroform
extracts from pH 6, 7 and 8 are combined, successively with water and a
saturated brine solution and dried over sodium sulfate. Removal of the
solvent under reduced pressure gives 2.9 g. of an epimeric mixture of
4"-deoxy-4"-amino-erythromycin A. A 1.9 g. sample of the mixture is
triturated with diethyl ether causing some of the undissolved foam to
crystallize. The solids are filtered and dried to give 67 mg. of a single
epimer of 4"-deoxy-4"-amino-erythromycin A, m.p. 140.degree.-147.degree.
C.
EXAMPLE 11
11,2'-Diacetyl-4"-deoxy-4"-oxo-erythromycin A 6,9-hemiketal
A solution of 10 g. of 2'-acetyl-4"-deoxy-4"-oxo-erythromycin A in 250 ml.
of pyridine is treated with 40 ml. of acetic anhydride and the resulting
reaction mixture allowed to stand at room temperature for 10 days. The
bulk of the solvent is removed in vacuo and the remaining concentrate
added to a mixture of 150 ml. of water and 100 ml. of chloroform. The pH
of the aqueous is raised to 9.0 and the chloroform separated, dried over
sodium sulfate and concentrated to dryness.
NMR (.delta., CDCl.sub.3): 3.33 (3H)s, 2.26 (6H)s, 2.10 (3H)s, 2.03 (3H)s
and 1.55 (3H)s.
EXAMPLE 12
Starting with the appropriate 4"-deoxy-4"-oxo-erythromycin A and requisite
alkanoic anhydride and employing the procedure of Example 11, the
following compounds are synthesized:
______________________________________
##STR28##
R.sub.1 R.sub.2
______________________________________
##STR29##
##STR30##
##STR31##
##STR32##
##STR33##
##STR34##
______________________________________
example 13
11-acetyl-4"-deoxy-4"-oxo-erythromycin A 6,9-hemiketal
A solution of 3.0 g. of 11,2'-diacetyl-4"-deoxy-4"-oxo-erythromycin A
9,6-hemiketal in 50 ml. of methanol is allowed to stir under a nitrogen
atmosphere overnight. The solvent is removed in vacuo to give the desired
product (3.0 g.) as a yellow foam.
NMR (.delta., CDCl.sub.3): 3.35 (3H)s, 2.31 (6H)s, 2.13 (3H) and 1.55
(3H)s.
In a similar manner, the compounds of Example 12 are converted by the
procedure of Example 13 to 11-acetyl-4"-deoxy-4"-oxo-erythromycin A
6,9-hemiketal and 11-propionyl-4"-deoxy-4"-oxo-erythromycin A
6,9-hemiketal.
EXAMPLE 14
11-Acetyl-4"-deoxy-4"-amino-erythromycin A 6,9-hemiketal
To a stirring solution of 4.4 g. of 11-acetyl-4"-deoxy-4"-oxo-erythromycin
A 6,9-hemiketal and 4.38 g. of ammonium acetate in 75 ml. of methanol is
added 305 mg. of 85% sodium cyanoborohydride. After stirring at room
temperature overnight, the reaction mixture is poured into 300 ml. of
water to which is then added 250 ml. of chloroform. The pH of the aqueous
layer is adjusted to 9.8 and the chloroform layer separated. The aqueous
layer is extracted with chloroform again, and the chloroform extracts are
combined, dried over sodium sulfate and concentrated to a white foam. The
residual foam is dissolved in a stirring mixture of 125 ml. of water and
125 ml. of fresh chloroform and the pH adjusted to 4.9. The chloroform is
separated and discarded, and the aqueous layer adjusted to pH 5, 6, 7 and
8, being extracted after each adjustment with fresh chloroform. The
extracts from the aqueous at pH 6 and 7 are combined, washed with a
saturated brine solution and dried over sodium sulfate. Removal of the
solvent provides 1.72 g. of the desired product as a white foam. The
product is dissolved in a minimal amount of diethyl ether and is
subsequently treated with hexane to turbidity. The crystalline product
which forms is filtered and dried, 1.33 g., m.p.
204.5.degree.-206.5.degree. C.
NMR (.delta., CDCl.sub.3): 3.31 (2H)s, 3.28 (1H)s, 2.31 (6H)s, 2.11 (3H)s
and 1.5 (3H)s.
EXAMPLE 15
The procedure of Example 14 is repeated, starting with the appropriate
4"-deoxy-4"-oxo-erythromycin A and substituting isopropanol for methanol
as the reaction solvent to give the following compounds:
______________________________________
##STR35##
R.sub.1 R.sub.2
______________________________________
##STR36##
##STR37##
##STR38##
##STR39##
##STR40##
##STR41##
##STR42##
##STR43##
______________________________________
example 16
2'-acetylerythromycin A 6,9-hemiketal 11,12-carbonate ester
To a solution of 13.2 g. of erythromycin A 6,9-hemiketal 11,12-carbonate
ester (U.S. Pat. No. 3,417,077) in 150 ml. of benzene is added 1.8 ml. of
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