Note: Descriptions are shown in the official language in which they were submitted.
5~6
Ihis invention relates to novel antibacterial agents, the inter-
mediates leading thereto and processes for the preparation of said inter-
mediates. In particular, the invention concerns 4"-deoxy-4"-amino-
erythromycin A antibacterial agents, and process for their preparation.
Erythromycin is an antibiotic formed during the culturing of a
strain of Streptomyces erythreus in a suitable medium as taught in United
States 2,653,899. Erythromycin, which is produced in two forms, A and B, is
represented by the following structure:
~I N (CH3 ) 2
0\~ 0
~ "'~1 110 ~
~ .f ', "~ :
o 2 '~" 0H
~ OCH3
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 des-
cribed, 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.
1~8~
Numerous derivatives of erythromycin have been prepared in an
effort to modify its biological or pharmacodynamic properties.
United States 3,417,077 describes the reaction product of
erythromycin and ethylene carbonate as a very active antibacterial agent.
United States 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 Patent 1,100,504,
Tetrahedron Letters, 1645 ~1967) and Croatica Chemica Ac~a, 39, 273 (1967)]
and some controversy as to its structural identity [Tetrahedron Letters, 157
(1970) and British Patent 1,341,022]. Sulfonamide derivatives of
erythromycylamine are reported in United States 3,983,103 to be useful as
antibacterial agonts. Other derivatives are also reported [Ryden, et al.,
J. M0d. Chem., _, 1059 ~1973) and Massey, et al., J. Med. Chem., 17, 105
~1974)] to have in vitro and in vivo antibacterial activity.
It has now been discovered that certain novel 4"-deoxy-4"-amino-
erythromycin A derivatives are outstanding as antibacterial a~ents. These
are compounds represented by the formulae:
R2O ""~ R40~" ~ ~l0
~o ~
- 'OCH3 O'CH3
III and IV
~l~2~ 6
and pharmaceutically acceptable acid addition salts thereof, wherein Rl and
R4 are each hydrogen or alkanoyl of two to three carbon atoms; R2 is
alkanoyl of two to three carbon atoms; R3 is hydrogen; R2 and R3 when taken
together are -C-; and R3 and R4 when taken together are -C-. :
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 R2 and R3 when taken together are -C-.
A second preferred group of compounds in this class of anti-
bacterial agents are those of Formula I~. ~specially preferred within this
group are those compounds wherein R4 is hydrogen and also wherein R3 and R4
when taken together are -C-.
A class of compounds, useful as intermediates leading to the anti-
bacteriul agents of Yormulae :EII and IV, are the compounds of ~ormulae I and
II
~ R70~ ~J ' ~ ~
~10 ~ R30
~ 0 ~ ~",~
OCH3 '~OCH3
I and II
wherein Rl is hydrogen or alkanoyl of two to three carbon atoms; R2 is
alkanoyl of two to three carbon atoms; Y is N-OH or N-O-CCH3; R3 is hydrogen;
and R2 and R3 when taken together are -C-.
Preerred within this class of intermediates are those compounds
--3--
~BS~
of Formula I. Especially preferred within this grcup of intermediates are
those compounds wherein R1 is hydrogen or acetyl.
A second group of preferred intermediates are those of Formula II.
Especially preferred within this group are those in~:ermediates wherein R1 is
hydrogen and also those wherein R1 is acetyl.
These compounds of Formulae Iand II are the subject of the parent
application, Serial No. 296,037 filed February 2, 1978. According ~o the
parent application, a compound of Formula I or II is prepared by:
(a) reacting a compound selected from the group consisting of:
N~C~l3)2 N(CH3)2
~ o ~u~
ll~ lO~ ~` R20 ~"~ "Q
HO~ ~ R30 ~ ~
O ~, O ~OH
OCH3 3
I' and II'
with one mole each of dimethylsulfoxide and trifluoroacetic anhydride in a
reaction-inert-solvent at about -30 to -65C. followed by contacting the
reaction mixture with at least one mole of triethylamine; or
(b) reacting a compound selected from the group consisting of compounds
of formula I' and II' with one mole each of N-chlorosuccinimide and dimethyl-
sulfide in a reaction-inert-solvent at about O to -25C. followed by contact-
ing the reaction mixture with at least one mole of triethylamine; and where
required subjecting the product to solvolysis.
A preferred feature of process (a) is the oxidation of the com-
s~
pounds of formula I' and II' wherein the reaction-inert-solvent is methylene
chloride.
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 sy~thetic procedure as herein described for the erythromycin A com-
pounds. 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 inven-tion. The erythromycin B amines
are also useful as antibacterial agents.
According to the invention, there is provided a process for pre-
paring 4"-amino epimeric compounds of the formula
2 "" ~ ~ ~ R O ~
2 ~ ~ NH2
OCH3
III and IV
and the pharmaceutically acceptaktle acid addition salts thereof, wherein Rl
and R4 are each selected from the group consisting of hydrogen and alkanoyl
having from two to three carbon atoms; R2 is alkanoyl having from two to
three carbon atoms; R3 is hydrogen, R2 and R3 when taken together are -C-;
and R3 and R4 when taken together are -C-, characterized by reducing com-
pounds of the formula:
N(CH3)2 N~CH~)
'~ \0 '~ u~ '
HO ""~ 110 ~o~ R20 ~
I O ~ ~ ~
wherein Y' is NH, N-OH or N-OCOCH3; and Y" is NH and wherein
(a) when Y" is NH and Y' is NH, N-OH or N-OCOCH3 said reduction is
carried out by catalytic hydrogenation and
(b) when Y' or Y" is NH, generated in situ from the corresponding
ketones of formulae
O - I ~ ~ \ ~ Rl \ ~ 2
~10",~ ` R20'1~
G ~ , U
OCH
I and II
--6--
8~6
wherein Rl is hydrogen or Ac, and Ac is alkanoyl having from two to three
carbon atoms, by condensation of said ketone with ammonium salt of an
alkanoic acid, said reduction is carried out by a hydride reduction~ and if
required,
~ c) when Rl or R4 are hydrogen, converting respectively to alkanoyl
and/or when Rl or R4 are alkanoyl, converting respectively to hydrogen;
(d) forming the pharmaceutically accepta'ble acid addition salts.
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:
Ac ( 3)2 Ac ~ N~CH3)2
0~ ~~ ~'~ '[~
IIQ "" ~ -10 ~ lo
'Oli ~
6C113 OC~13
I' I (Y=O)
and
N(CH ) N(CH3)2
Ac 1 _ ~c ~
~o~ ~lo~ oJ/~
~ ~ ~O R O
R30 ~ ~ ~ ~
OCH3 OCH3
II' II
--7--
~L~Z8~
The selective oxidation of I' and II' to I and II, respectivelyg
(Y-0) 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 tri-
ethylamine.
- In prac~ice, the trifluoroacetic anhydride and dimethylsulfoxide
are initially combined in a reaction-inert-solvent at about -65C. After
ten to fifteen minutes the alcohols I' and II' are added at such a rate that
the temperature is maintained at about -65C. and does not rise above -30C.
At temperatures above -30C. the trifluoroacetic anhydride - dimethyl-
sulfoxide complex is not stable. The reaction temperature is maintained be-
tween -30 and -65C. for about fifteen minutes and is then lowered to about
-70 C. A tertiary amine is added all at once and the reaction allowed to
warm during a ten to flfteen minute per:iod. The reaction mixture is sub-
sequently 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. ExperimentallyJ 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 oxida-
tion process is conducted at -30 to -65C., 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, chloro-
form or tetrahydrofuran. Solvents which meet the above requiremellts but
which have a freezing point above the reac-tion temperature can be employed
~ 2~ 6
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 21-acetyl-4"-deoxy-4"-oxo-0rythromycin A 6,9-hemiketal
11,12-carbonate ester.
The reaction time is not critical and is clependent on reaction
temperature and the inherent reactivity of the starting reagents. At
temperatures of about -30 to -65C., 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 o the requisite alcohol substrate. It is further
suggested, as hereinbefore mentioned, thak the temperature of the reaction
is kept below -30C. This is in accordance with the teaching o:f Omura, et al.,
J. Org. Chem., 41, 957 ~1976).
The second process of the claimed invention, used to prepare inter-
mediates leading to the useful antibacterial agents, is represented by the
following scheme:
N(CH3)2 N(CH3~2
~10~ r~ ~ ~0 ,~ ~ O~o 1
llO ~ ~ HO ~ ~ -
~ ~",""0~1 o ~Xo
OCI13 OCH3
I' I
and
_9_
~21~5~
N(C~13)2 N(CH3)2
R2 ~ ~~`"` R20 " 3' ~1 ` ~
R3~ 1 R30 ~ ~
"~"~ ""`~
OC113 CH3
II' II
The second process represents an oxidation reaction wherein the
4"-hydroxy substituent of I' ancl lI', wherein Ac and R2 are each alkanoy:l of
two to three carbon atoms, R3 is hyclrogen, and R2 and R3 when taken together
are -C-, is oxidized to a 4"-deoxy-4"-oxo-erythromycin A compound.
The process comprises the use of N-chlorosuccinimide and dimethyl-
sulfide as the oxidizing agent. In practice, these two reagents are first
combined together in a reaction-inert-solvent at about 0C. After ten to
twenty minutes the temperature is lowered to O to -25C. and the alcohol sub-
strate I' or II' is added, while maintaining the aforementioned temperature.
After two to four hours reaction time, a tertiary amine, such as triethyl-
amine, 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 dimethyl-
sulfide 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
-10-
35~6
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 to -25C., it is preferred that, in
addition to having the above characteristics~ it should possess a fFeezing
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 to -25C. 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 use~ul intermediate 4"-deoxy-4"-oxo compounds of the formula:
1~285~6 :~
Rl ~ 0 ~ 3)2 ::
R30 ~ ~
1~""0~'""~ ~
OCH3
wherein Rl and R2 are each alkanoyl of two to three carbon atoms ancl R3 is
hydroger1 are prepared by treating a compound of the formula:
Rl ~ ~ 3)2
~/ ~ i o 1
~10
0// ~O \~
O ~Y
~QC~13
I :~
wherein Y is 0, Rl is alkanoyl of two ts three carbon atoms, with an
alkanoic anhydride (R20) and pyridine.
In practice, the ketone I is contacted with an excess of the
anhydride in pyridine as the solvent. It is pre~erred that as much as a
.
four fold excess of the anhydride be employed in ~he 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 inter-
mediate ketones I ~Y = 0) and II is carried out through a solvolysis reac-
tion 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 = O~ and II wherein
Rl is hydrogen.
As previously mentioned, the ketones of Formulae I (Y = O) and II
are useful intermediates leading to the ~"-deoxy-~"-amino-erythromycin A
antibacterial agents of the yresent invention of Formulae III and IV. Pre-
ferred as intermediates in this group are 2'-acetyL-~"-deoxy-~"-oxo-
erythromycin A 6,9-hemiketal 11,12-carbonate ester and ~"-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 FormulaP III and IV from the requisite ketones
I (Y = 0) and II.
Preparation of the ~"-deoxy-~"-amino-erythromycin A compounds of
Formula III is carried out by the conclensation of the ketones II with the
ammonium salt of a lower alkanoic acicl 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
-13-
il2~35~6
hydrolyzed and the product isolated.
Although one mole of the ammonium alkanoate is needed per mole of
ke~one, 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 pro-
duct.
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 tempera-
ture 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 deriva-
tives 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 procluct 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. Addi-
tional 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
-14-
~8~
the appropriate 2'-alkanoyl-4"-deoxy-4"-amino-erythromycin A derivative can
be effected by allowing a methanol solution of said compound to stand over-
night at ambient temperatures.
During the reductive amination of ketones of Formula II wherein R2
and R3 when taken together are -C- and Rl is alkanoyl of two to three carbon
atoms or hydrogenJ it is noted that amines related to both Formulae III and
IV are produced. This is represented by the following scheme:
_ l ~ O ~ 3)2
3'/o~ ~ O 1
~ ~ " NH~OCOC
O ~ ~ NaCNB~13
'0"",~
oc~l3
II O
(R2 + R3 = -C-~
1 ~ ~ 2 Rl~O ~ 2
o=< ( ~/ ;~''',1~ i
~/ NH2 ~NH2
"OCH~ OCH3
III O IY O
Il 11
(R + R - -C-) ~R3 + R4 = -C-)
~Z8~
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 syn~hetic pathway to the amine compounds of
Formula IV is the same route as discussed previously and comprises the con- `
densation 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 Rl, R3 and R~ 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 need0d 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 pro-
duct.
The hydrogenation catalyst can be selected from a wide range of
agents; Raney nickel and 5-10 percent palladium-on-charco~l 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.
-16-
~285~
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 aorementioned preferred conditions
the reaction is complete in 12 to 2~ hours.
The product is isolated by filtration of the spent catalyst and
removal of the solvent _ 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 des-
cribed.
~s 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 ~"-deoxy-~"-amino-erythromycln A
antibacterial agents of Formula IV comprises initial conversion of the
ketones of Formula I (Y = O) to an oxime or oxime derivative, i.e., Y = N-OH
and N-O-~CI-13, followed by reduction of the oxime or derivative thereof. `
The oximes of the ketones I (Y = 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 reactionperiod 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
~lZ1~35~6
between water at pH 9.0-9.5 and a water-immiscible solvent.
Preparation of the O-acetyloxime compounds of Formula I (Y =
N-O-CC113) is effected by acetylation of the corresponding oxime. Experiment-
ally, 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 inter-
mediates leading to the 4"-deoxy-4"-amino-erythromycin A derived anti-
bacterial agents include 2'-acetyl-4"-deoxy-4"-oxo-erythromycin A oxime,
2'-acetyl-~"-deoxy-4"-oxo-erythromycin A O-acetyloxime~ 4"-deoxy-4"-oxo-
erythromycill A oxime and 4"-deoxy-4"-oxo-erythromycin A O-acetyloxime.
Reduction of the ketone derivatives ~Y = N-OH or N-O-CC~13) 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 initial 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 isola-
tion of the desired 4"-deoxy-4"-amino antibacterial agent related to Forrnula
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, iso-
propanol 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-41'-
amino-erythromycin A 6,9-hemiketal 11,12 carbonate ester and of 4"-deoxy-4"-
amino-erythromycin A, of 4"-deoxy-4"-amino-erythromycin A 11,12-carbonate
ester.
~21 35~6
In the utilization of the chemotherapeutic activity of those com-
pounds 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 acceptable bases by
decomposition of the salt as described above, or alternately they can be con-
verted to any desired pharmacautically 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 mate-
rials 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 = 0) 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 ob-
served 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 origin-
ally 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.
-19-
~Z~ 6
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 recrystalliza-
tion from an appropriate solvent, subjecting it to column or high pressure
liquid chromatography, solvent partitioning or by trituration in an appropri-
ate solvent. Said purification, while not necessarily separating the
epimers, removes such extraneous materials as starting materials and undesir-
able 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 ~"-deoxy-4"-amino-erythromycin A derivativ0s described
herein exhibit _ vitro activity against a variety o Gram-positive micro-
organisms, e.g., Staphylococcus awreus and Strepttococcus pyogenes, 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-accept-
able 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 pharmaceutic-
ally-acceptable inert media; that is, media which have no harmful effect on
the active ingredient. For such purposes, it will generally be acceptable
-20-
l~Z~35~6
to employ concentrations of active ingredients o~ 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 multocida and Neisseria sicca, in vivo via
the oral and/or parenteral routes of adminis*ration 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 LDloo ~the
lowest concentration oE organisms required to produce 100% dea-ths). Control
tests are simultaneously rull in wh:ich mice receive inoculum Oe lower dilu-
tions as a check on possible variatlon 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.
l~hen used in vivo, these novel compounds can be administered
orally or parenterally, e.g., by subcutaneous or intramuscular injection, Rt
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 solutionJ 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,
s
-21-
~lliLZl~r~6
sorbitol). Additionally, compositions suitable for extemporaneous prepara-
tion of solutions prior to administration may advantageously be made. Such
compositions may include liquid diluents, for example, propylene glycol,
diethyl carbonate, glycerol, sorbital, etc.; buffering agents, hyaluronidase,
local anesthetics and inorganic salts to afford desirable pharmacological
properties. These compounds may also be combined with various pharmaceutic-
ally-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 providos solely for the purpose of
illustrat:ion and not to be construed as l:imitations o this invent:ion, many
variations o 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 -65C. 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 - dimethyl-
sul:Eoxide complex. To the resulting slurry is added dropwise a solution of
1.0 g. of 2'-acetylerythromycin A ethyl acetate, obtained by recrystalliza-
tion of 2'-acetylerythromycin A from ethyl acetate, in 7 ml. of methylene
chloride keeping the temperature at about -65C. The resul~ing mixture is
allowed to stir for 15 min. at about -60C. and is then cooled to -70C.
Triethylamine (1.61 ml.) is added rapidly to the reaction mixture and the
cooling bath is remo~ed. 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
~2~35~6
organic phase is separated, washed successively with water ~3 x 10 ml.) and
brine solution (1 x 10) and dried over sodium sulfate. Removal of the sol-
vent under reduced pressure gives 929 mg. of the crude product. Recrystal-
lization from methylene chloride - hexane gives 320 mg. of the purified pro-
duct, m.p. 105-108C.
MMR (~, CDC13): 3.28 (311)s, 2.21 (6H)s and 2.03 (3H)s.
In a similar manner, starting with 21-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.
Tho solvent is removed in vacuo and the residual white foam recrystallized
from methylene chloride - hexane, 3.44 g., m.p. 170.5-172.5C.
NMR (~, CDC13): 3.36 (3H)s and 2.33 (6H)s.
A ~roduct 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 ace-tic anhydride and the
resul-ting reaction mixture allowed to stir at room temperature for 2 hrs.
The sol-ution is added to 100 ml. of water and the pH of the aqueous phase
raised to 9.5 by the addition of 6N 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.
-23-
~ ~.
~Z~5~6
E~MPLE 4
2'-Acetyl-4"-deoxy-4"-oxo-erythrom _in 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 ll.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 filtra~e concen-
trated under reduced pressure. The residual foam is taken up in ethyl ace-
tate 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 (~, CDC13): 3.33 (3~1)s, 2.30 (6H)s and 2.06 (3~1)s.
XAMPLE 5
2'-Acetyl-4"-deoxy-4"-oxo-ery~hromycin A O-acetyloxime
To a solution of 330 mg. of 2'-acetyl-~"-deoxy-4"-oxo-erythromycin
A oxime in 30 ml. o ethyl acetate is added with stirring 64.2 ~I of acetic
anhydride, and the reaction allowed to stir overnight at room temperature.
An additional 15.~ ~1 of acetic anhydride and 23.4 ~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 9Ø The ethyl acetate layer is
separated, dried over sodium sulfate and concentrated under vacuum to give
300 mg. of the desired product.
M~R ~, CPCl3): 3.38 (3H)s, 2.25 (6~1)s, 2.20 (3H)s, 2.05 (3~1)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
0-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
-24-
Z~35~
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 con-tinued 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 pro-
duct.
EXAMP~E 7
Starting with the appropriate O-acetyloxime and employing the pro-
ceclure of Example 6, the following 4"-amino-erythromycin A analogs are pre-
pared:
~O ~ )2
~10
"~
O y"" ~l2
QC~13
CH3CH2C -
-25-
~:~ZI 3~i~6
EXAMPLE 8
~"-Deoxy-4"-amlno-erythromycin _
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. o~ 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 4Ø
The pH of the acid aqueous layer containing the procluct 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 por~ion of
the product and these are combined and treated with fresh water at pH 4.
The aqueous layer is again adjusted through pl-l 5, 6 and 7, being extracted
at cach pH with fresh chloroform. Tho chloroform extract at pH 6 is dried
over sodium sulfate and concentrated to give 249 mg. of the product as an
epimeric mixture.
NMR ~, CDC13): 3.30 (1H)s, 3.26 (2H)s, 2.30 (6H)s and 1.46 (3H)s.
In a similar manner, ~"-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 ~\
To a stirring solution of 3.0 g. of 4"-deoxy-4"-oxo--erythromycin A
in 30 ml. o m~thanol 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 6Ø The aqueous is extracted at pH
6, 7, 7.5, 8, 9 and 10 using 125 ml. of diethyl ether for each extraction.
The extracts at pH 8~ 9 and lO are combined and washed with 125 ml. of fresh
water. The separated aqueous layer is extracted with ether (1 x 100 ml.) at
-26-
- .
8S~6
pH 7, ethyl acetate (1 x 100 ml.) at pH 7J ether (1 x 100 ml.) at pH 7.5,
ethyl acetate (1 x 100 ml.) at pH 7.5 and ethyl acetate (1 x 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 clried over sodium sulfate. Removal of
the solvent in vacuo gives 30 mg. of an epimeric mixture of the desired pro-
duct 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 chloro-
form layer is separated. The pH of the aqueous phase is subsequently ad-
justed 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 com-
bined, 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 undis-
solved foam to crystallize. The solids are filtered and dried to give 67 mg.
of a single epimer of 4"-de~xy-4"-amino-erythromycin A, m.p. 140-147C.
EXAMPLE 11
11,2'-Diacetyl-4"- _ xy-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 re-
sulting 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
-27-
~lZ8~6
the aqueous is raised to 9.0 and the chloroform separated, dried over sodium
sulfate and concentrated to dryness.
MMR (~ CDC13): 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:
;
N(CH3)2
Rl ~o J~
3~ J~ol
R20 ""'~ ' '1"
HO ~ ~
'~ ~
O ~0 `'
~ ~C~13
-- R2 -
O O
Il 11
C~13C- CH3CH2C-
1l
CH3CH2C- CH3CH2C-
O O
11 11
CH3CH2C- CH3C-
EXAMPLE 13
AcetyI-4"-deoxy-4"-oxo-erythromycin A 6~9-hèmiketal
A solution of 3.0 g. of 11,2'-diacetyl-4"-deoxy-4"-oxo-erythromycin
-28-
~85~6
A 9.6-hemiketal in 50 ml. of methanol is allowed to stir under a nitrogen
atmosphere overnight. lhe solvent is removed ln vacuo to give the desired
product ~3.0 g.) as a yellow foam.
MMR (~, CDC13): 3.35 ~3H)s, 2.31 (6H)s, 2.13 ~3H) and 1.~5 (3H)s.
In a similar manner, the compounds of Example 12 are conver~ed by
the procedure of Example 13 to 11-acetyl-4"-deoxy-4"-oxo-erythromycin A 6,9-
hemiketal and ll-propionyl-4"-deoxy-4"-oxo-erythromycin A 6,9-hemiketal.
XAMPLE 14
ll-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. o chloro:Eorm. The pH of the aqueous
layer is adjusted to 9.8 ancl 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 p~l 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. 204.5-206.5C.
MMR ~, CDC13): 3.31 (2H)s, 3.28 (lH)s, 2.31 (6H)s, 2.11 ~3H)s
and 1.5 ~3H)s.
-29-
~L~Z~ 6
EXAMPLE 15
__
The procedure of Example 14 is repeated9 starting with the
appropriate 4"-deoxy-4"-oxo-erythromycin A and substituting isopropanol for
methanol as the reaction solvent to give the following compounds:
N(C 3)2
- I ~ O ~ `
~ ~\ /\ 1
R20 ~
~10
t""` ~ " ""
~r ~,
~", 2
OCl13
Rl R2
O O
Il 11
CH3C- C~13C-
O O
Il 11
C~13C- CH3CH2C-
O O
Il 11
C~13C~12C- C~13C~12C-
O
Il ~1
CH3CHzC-CH3C-
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 (United States 3,417,077) in 150 ml. of benzene is added 1.8
ml. of acetic anhydride, and the reaction mixture allowed to stir at room
-30-
5~6
temperature for 1.5 hrs. The solution is poured into 200 ml~ of water and
th0 aqueous phase basiied to pH 9Ø The benzene layer is separated, dried
over sodium sulfate and concentrated in vacuo to 15.3 g. of a white foam.
On trituration with 50 ml. of diethyl ether the foarn crystallizes. Filtra-
tion and drying of the product gives 12.6 g. of pure product, m.p. 224.5-
228.5C.
N~R ~, CDC13): 3.36 (3H)s, 2.30 (6H)s, 2.06 (3H)s and 1.61 (3H)s.
In a similar manner, by substituting an equivalent amount of pro-
pionic anhydride for acetic anhydride in the procedure of Example 16, 2'-
propionylerythromycin A 6,9-hemiketal 11,12-carbonate ester is prepared.
EXA~IPLE 17
2'-Acetyl-4"-deoxy-4"-oxo-erythromycin A
6,9-hemiketal 11,12-carbonate ester
To a suspension o 6.19 g. of N-chlorosuccinimide in 150 ml. of
toluene and 50 ml. of benzene cooled to -5C. is added 4.46 ml. oE dimet~lyl-
sulfide. After stirring for 20 min. the resulting suspension is cooled to
-25C. and 12.4 g. of 2'-acetylerythromycin A 6,9-hemiketal 11,12-carbonate
ester, partially dissolved in 80 ml. of toluene, is added dropwise. The
temperature, which is maintained between -19 to -25C. during the addition,
is kept at -25C. for 2 hrs. At the end of this period 6.79 ml. of triethyl-
amine is added all at once. The cooling bath is removed and the temperature
al:Lowed to rise to -10C. The reaction mixture is then poured into water
and the aqueous phase adjusted rom 8.4 to 9Ø The organic layer is
separated, dried over sodium sulfate and concentrated under vacuum to a
white foam (14.0 g.). Trituration of the residue with diethyl ether causes
the foam to crystallize. Filtration and drying of the product gives 11.3 g.
of crystalline material, m.p. 212-213.5C.
NMR ~, CDC13): 5.26 (lH)t, 3.36 (3H)s, 2.30 ~6H)s, 2.13 (3H)s,
1.63 (3H)s and 1.50 (3H)s.
Similarly, 2'-propionyl-4"-deoxy-4"-oxo-erythromycin A 6,9-hemi-
ketal 11,12-carbonate ester is prepared by the procedure of Example 17 by
-31-
`
1~2~35~
the replacement of the 2'-acetyl ester with an equivalent amount of
2'-propionyl-erythromycin A 6,9-hemiketal 11,12-carbonate ester.
EXAMPL~ 18
4"-Deoxy-4"-oxo-erythromycin A 6,9-hemiketal 11,12-carbonate ester
.
Forty-two and nine-tenths grams of 27-ace~yl-4"-deoxy-4"-oxo-
erythromycin A 6,9-hemiketal 11,12-carbonate ester is added to 800 ml. of
methanol and the resulting solution allowed to stir at room temperature for
72 hrs. On removal of the solvent in vacuo there remains 41 g. of the pro-
duct as a white foam. The residual material is dissolved in about 100 ml.
of acetone followed by the careful addition of water to the precipitation
point. Ihe resulting crystalline solid is allowed to stir for 40 min., and
this then filtered and dried to give 34.2 g. of desired product~ m.p. 186.5-
188C.
NMR (~, CDC13): 5.66 ~lH)t, 3.35 (3H)s, 2.35 (611)x, 1.65 ~311)s
and 1.51 (3H)s.
In a similar manner the same product is obtained when an equiva-
lent amount of 2'-propionyl-4"-deoxy-4"-oxo-erythromycin A 6,9-hemiketal
11,12-carbonate ester is employed in the above procedure in place of the
2'-acetyl ester.
EXAMPLE 19
4"-Deoxy-4"-amino-erythromycin A 6,9-hemiketal 11,12-carbonate ester
To 189 g. of 4"-deoxy-4"-oxo-erythromycin A 6,9-hemiketal 11,12-
carbonate ester in 1200 ml. of methanol at room temperature is added with
stirring 193 g. of ammonium acetate. After 5 min. the resulting solu~ion is
cooled to about -5C. and is subsequently treated with 13.4 g. of 85% sodium
cyanoborohydride in 200 ml. of methanol over a 45 min. addition period. The
cooling bath is removed and the reaction mixture allowed to stir at room
temperature overnight. The reaction mixture is reduced in volume to 800 ml.
in vacuo and added to a stirring mixture of 1800 ml. of water and 900 ml. of
chloroform. The pH is adjusted from 6~2 to 4.3 with 6N hydrochloric acid
-32-
~lZBSQ6
and the chloroform layer separated. The chloroform is combined with 1 1. of
water and the pH adjusted to 9.5. The organic phase is separated, dried
over sodium sulfate and concentrated under reduced pressure to give 174 g. of
a white foam. The residual material is dissolved in a mixture of 1 1. of
water and 500 ml. of ethyl acetate and the pll adjusted to 5.5. The ethyl
acetate layer is separated and the aqueous layer adjusted to pH 5.7 and 9.5
successively, being extracted after each pH adjustment with 500 ml. of fresh
ethyl acetate. The ethyl acetate extract at pH 9.5 is dried over sodium
sulfate and concentrated in vacuo to dryness, 130 g. One-hundred and twenty
grams of the residual foam is dissolved in a mixture of 1 1. of water and
1 1. of methylene chloride the pH of the aqueous layer is adjusted to 4.4,
4.9 and 9.4 successively, being extracted after each adjustment with 1 1. of
fresh methylene chloride. The methylene chloride extract at pH 9.4 is dried
over sodium sulfate and concentrated under reduced pressure to give 32 g. of
the product as a white foam. Crystallization from 250 ml. of acetone-water
(1:1, v:v) gives 28.5 g. of the crystalline epimers.
NMR 100 Mz (f~t, CDC13): 5.20 (lH)m, 3.37 (1.5H)s, 3.34 (l.SH~s, ~ -
2.36 (6H)s, 1.66 (3H)s and 1.41 (3H)s.
EXAMPL~ 20
Separation of the ~pimers of 4"-Deoxy-4"-amino-erythromycin A
6,9-hemiketal 11,12-carbonate ester
On to a high-pressure-lif~uid-chromatography column (1/2" x 9 cm.)
packed with Gf 254 silica gel impregnated with formamide and eluted with
chloroform is applied 200 mg. A pressure of 240 p.s.i. is applied with a
rate of 4.76 cc. per min. and a fraction size of 10 ml. is employed. Frac-
tions 14 thru 21 and 24 thru 36 are collected.
Fractions 14 thru 21 are combined and concentrated to about 50 ml.
Water (50 ml.) is added and the pH adjusted to 9Ø The chloroform layer is
separated, dried over sodium sulfate and concentrated to give 106 mg. of a
white foam. Trituration with diethyl ether causes the foam to crystallize.
-33-
~3 2~5Q6
After stirring at room temperature for one hour the crystalline product is
filtered and dried, 31.7 mg., m.p. 194-196C.
NhIR 100 Mz (~, CDC13~: 5.24 (llI)d, S.00 ~lII)t, 3.40 (311)s,
2.40 (6H)s, 1.66 (3H)s and 1.40 (3H)s.
Fractions 24 thru 36 are combined and worked up as above to give
47.1 mg. of product as a white Eoam, which is identical to the material from
Example 25.
EXAMPLE 21
To a suspension of ll.l g. of 2'-acetyl-47'-deoxy-4"-oxo-
erythromycin A 6,9-hemiketal 11,12-carbonate ester in 300 ml. of isopropanol
at room temperature is added with stirring 10.7 g. of a~lonium acetate.
Afl:er 5 min., 747 mg. of sodium cyanoborohydride in 130 ml. of isopropanol
is added over a period of 30 Illin. and the resulting reaction ~Ilixture is
aLlowed to stir at room temperature o~ernight. The pale yellow solution is
poured into 1100 ml. of water to which is then added 400 ml. of diethyl
ether. The pH is adjusted to 4.5 and the ether layer is separated, The
aqueous layer is basified to pH 9.5 and extracted (2 x 500 ml.) with chloro-
form. The chloroform extracts are combined, dried over sodium sulfate and
concentrated to give 7.5 g. of a yellow foam. Recrystallization of the
residual material from diethyl ether gives 1.69 g. which is retained along
with the mother liquors.
The mother liquor is treated with 75 ml. of water, and the pH ad-
justed to 5Ø The ether layer is replaced with 75 ml. of fresh ether and
the pH adjusted to 5.4. The ether is replaced with ethyl acetate and the pH
raised to 10. The basified aqueous layer is extracted (2 x 75 ml.) with
ethyl acetate and the first ethyl acetate extrac* dried over sodium sulfate
and concentrated to dryness. The residual foam (1.96 g.) is added ~o a mix-
ture of 75 ml. of water and 50 ml. of diethyl ether and the pi-l adjusted to
5.05. Th~ ether is separated and the aqueous layer adjusted successively to
pH 5.4, 6.0, 7.05 and 8.0, being extracted after each pH adjustment with
-34-
~lZ85!~6
50 ml. of fresh diethyl ether. Ihe pll is finally adjusted to 9.7 and the
aqueous layer extracted with 50 ml. of ethyl acetate. The ether extract
carried out at pH 6.0 is combined with 75 ml. of water and the pH adjusted
to 9.7. The ether layer is separatedJ dried and concentrated in vacuo to
give 460 mg. of a white foam.
NMR lO0 Mz (~, CDC13): 5.20 (lH)t, 3.43 (2H)s, 3.40 (lH)s,
2.38 (6H)s, 2.16 (3H)s, 1.70 (3H3s and
1.54 (3H). ;
The NMR data indicates the product to be the epimers of 2'-acetyl- `
4"-deoxy-4"-amino-erythromycin A 6,9-hemiketal 11,12-carbonate ester.
-- - :
The 1.69 g. indicated abov0 is dissolved in a mixture of 75 ml. of
water and 75 ml. of diethyl ether and the pH adjusted to 4.7. The ether is
separated and the aqueous layer further extracted with fresh e~her ~75 ml.)
at pH 5.05 and 5.4, and with ethyl acetate ~2 x 75 ml.) at pH 9.7. The com- `
bined ethyl acetate extracts are dried over sodium sulfate and concentrated ~`
under reduced pressure to give 1.26 g. of a white foam. Crystalliza~ion of
this residual material gives 411 mg. of product, m.p. 193-196C. (dec.).
The mother liquor is concentrated to dryness, and the residue dissolved in
hot ethyl acetate. The solution is allowed to stand overnight at room
temperature. The crystalline solids which precipitate are filtered and
dried, 182 mg., m.p. 198-202C. ~dec.) to give additional product.
NMR lO0 Mz (~, CDCl3): 5.10 ~lH)t, 3.34 ~2H)s~ 3.30 ~lH)s,
2.30 ~6H)s, 2.08 t3l-l)s, 1.62 ~3H)s and
1.48 ~3H)s.
The NMR data indicates the product to be the epimers of 2'-acetyl-
4"-deoxy-4"-amino-erythromycin A 11,12-carbonate ester.
-. :
In a similar manner, when Example 21 is repeated, starting with
2'-propionyl-4"-deoxy-4"-oxo-erythromycin A 6,9-hemiketal 11,12-carbonate
ester, there is obtained 2'-Eropionyl-4ll-deoxy-4l~-amino-erythromycin A
6,9-hemiketal 11,12-carbonate Pster and _ propionyl-4"-deoxy-4"-amino-
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- ~2~S~
erythromycin A 11,12-carbonate ester.
E~PLE 22
A solution of 400 mg. of 2'-acetyl-4"-deoxy-4"-amino-erythromycin
A 6,9-hemiketal 11,12-carbonate ester in 20 ml. of methanol is allowed to
stir overnight at room temperature. The reaction solution is poured into
100 ml. of water followed by the addition of 50 ml. of ethyl acetate. The
p}l is adjusted to 9.5 and the organic phase separated. The extraction is
repeated again with 50 ml. of fresh ethyl acetate. The combined ethyl ace-
tate extracts are dried over sodium sulfate and concentrated to give 392 mg.
of a white foam. Trituration with diethyl ether and scratching with a glass
rod affects crystallization. After standing at room temperature for 30 min.,
the crystalline solids are filtered and dried, :123 mg., and the mother
liquor is retained. The product is identical by NMR to material prepared in
Example 24.
NMR 100 Mz (~, CDC13): 3.26 (3H)s, 2.32 (611)s, 1.61 (3H)s and
1.44 (3H)s.
The NMR data indicates that the crystalline product is a single
epimer of 4''-deoxy-4''-amino-erythromycin A 11,12-carbonate ester.
The retained mother liquor is concentrated in vacuo to give 244 mg.
of a white foam.
The product is identical with material from Example 19.
The NMR data indicates that this product is a mixture of the
epimers of 4"-deoxy-4"-amino-erythromycin A 6,9-hemiketal 11,12-carbonate
ester and identical with the product of Example 19.
EXAMP~E 23
In a manner similar to the procedure of Example 22, methanolysis
of 2'-propionyl-4"-deoxy-4"-amino-erythromycin A 6,9-hemiketal 11,12-carbon-
ate ester gives 4"-deoxy-4"-amino-erythromycin A 11,12-carbonate ester and
~ .
4"-deoxy-4"-amino-erythromycin A 6,9-hemiketal 11,12-carbonate ester.
.,
~2~3S~6
~XAMPLE 24
Eight grams of the epimeric mixture of 4"-deoxy-4"-amino-
erythromycin A 11,12-carbonate ester from the non-crystalline product of
Example 19 is dissolved in 50 ml. of diethyl ether. The product is induced
to crystallize by scratching with a glass rod. After 20 min. stirring, the
crystalline product is filtered and dried, l.91 g., m.p. 198.5-200C.
NMR 100 Mz (~, CDC13~: 3.26 (3H)s, 2.30 (6H)s, 1.51 (3H)s and
1.45 ~3~l)s.
The NMR data indicates that the crystalline product is a single
epimer of 4"-deoxy-4"-amino-erythromycin A 11,12-carbonate ester and
identical with the ketone product from Example 22.
EXAMPLE 25
One gram of the epimer of Example 24 is dissolved in 20 ml. of
acetone and heated at steam bath temperatures until the boiling point is
reached. Water (25 ml.) is added and the resulting solution allowed to stir
at room temperature. After one hour of stirring, the precipitate which
forms is filtered and dried to give 581 mg., m.p. 147-149C.
MMR 100 Mz (~, CDC13): 5.12 (lH)d5 3.30 (3H)s, 2.30 (6H)s,
1.62 (3H~s and 1.36 (3H)s.
The MMR data indlcates the product to be a single epimer of 4"-
deox~4"-amino-erythromycin A 6,9-hemiketal 11,12-carbonate ester and
identical to the epimer in fractions 24-36 of Example 20.
EXAMPLE 26
4"-Deoxy-4"-amino-erythromycin A
Twenty grams of 4"-deoxy-4"-oxo-erythromycin A, 31.6 g. of
ammonium acetate and 10 g. of 10% palladium-on-charcoal in 200 ml. of
methanol is shaken at ambient temperatures in a hydrogen atmosphere at an
initial pressure of 50 p,s.i. overnight. The spent catalyst is filtered and
the filtrate concentrated to dryness in vacuo. The residue is partitioned
between water-chloroform at a pH of 5.5. The aqueous layer is separated,
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5~ Ei
the pll adjusted to 9.6 and chloroform added. The organic layer is separated,
dried over sodium sulfate and concentrated under reduced pressure to dryness,
The residual white foam ~19 g.) is triturated with 150 ml. of diethyl ether
at room temperature for 30 minutes. The resulting solids are filtered and
dried to give 9.45 g. of a single epimer indistinguishable from that in
Example 10.
The diethyl ether filtrate is concentrated to dryness to give 6.89
g. of product consisting of the other epimer plus some impurities.
EXAMPLE 27
4"-Deoxy-4"-amino-erythromycin A
Two grams of 4"-deoxy-4"-oxo-erythromycin A, 3.1 g. of ammonium
acetate and 2.0 g. of Raney nickel in 50 ml. of methanol is shaken at room
temperature in a hydrogen atmosphere at an initial pressure o:E 50 p.s.i.
overnight. An additional 3.16 g. of ammonium acetate and 2.0 g. of Raney
nickel are added and the hydrogenation continued for an additional 5 hours.
The solids are fi.ltered and the filtrate concentrated to dryness in vacuo.
The residue is added with stirring to a mixture of water-chloroform, and the
pH adjusted from 6.4 to 5.5. The aqueous phase is separated, the pH ad-
justed to 9.6 and fresh chloroform added. The chloroform extract is
separated, dried over sodium sulfate and concentrated under reduced pressure
~o give 1.02 g. of the product as a yellow foam. The predominant isomer has
the opposite configuration at 4" as the compound of Example 10.
EXAMPLE 28
2'-Acetyl-4"-deoxy-4"-amino-erythromycin B
To a solution of 4.5 g. of 2'-acetyl-4"-deoxy-4"-oxo-erythromycin
B (United States #3,884,903~ in 45 ml. of isopropanol under a nitrogen
atmosphere is added with stirring 4.66 g. of dry ammonium acetate. After 10
min. 376 mg. of sodium cyanoborohydride is washed into the reaction mixture
with 10 ml. of isopropanol and the reaction allowed to stir at room tempera-
ture overnight. The light yellow solution is poured into 400 ml. of water
.
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~85~6
and the pll adjusted to 6Ø The aqueous is extracted at pH 6, ~ 7.5J 8, 9
and 10 using 250 ml. of diethyl e-ther for each extraction. The extracts at
pH 8, 9 and 10 are combined and washed with 250 ml. of fresh water. The
separated aqueous layer is extracted with ether (1 x 100 ml.) at pH 7, ethyl
acetate (1 x 100 ml.) at pH 7, ether (1 x lOQ ml.) at pH 7.5, ethyl acetate
(1 x 100 ml.) at pH 7.5 and ethyl acetate (1 x 100 ml.) at pH 8, 9 and 10.
The ethyl acetate extracts at pH 9 and 10 are combined, washed with a satur-
ated brine solution and dried over sodium sulfate. Removal of the solvent
in vacuo gives an epimeric mixture of the desired product as a cream colored
_ _ .
foam.
In a similar manner, 4"-deoxy-4"-amino-erythromycin B is prepared
from 4"-deoxy-4"-oxo-erythromycin B.
EXAMPLE 29
4"-Deoxy-4"-amino-erythromycin B
A solution of 4.34 g. of 2'-acetyl-4"-deoxy-4"-amino-erythromycin
B in 100 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 100 ml. of chloroform and 100 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 4Ø
The pH of the acid aqueous layer containing the product is gradually ad-
justed 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 por-
tion 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 the product as an
epimeric mixture.
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~lz~cj~6
EXAMPLE 30
4"-Deoxy-4"-amino-erythromycin A 6,9-hemiketal
11,12-carbonate ester aspartate
To a solution of 1.0 g. of 4"-deoxy-4"-amino-erythromycin A 6,9-
hemiketal 11,12-carbonate ester in 6 ml. of acetone warmed to 40C. is added
20 ml. of water followed by 175 mg. of L-aspartie acid. The mixture is
heated to reflux for 1.5 hours and is then filtered while hot. The filtrate
is concentrated by removal of the acetone and is subsequently free7e-dried
to give 1.1 g. of the desired product as a white solid.
EXAMPLE 3I
4"-Deoxy-4"-amino-erythromycin A 6,9-hemiketal
11,12-carbonate ester dihydrochIoride _
To 7.58 g. of 4"-deoxy-4"-amino-erythromycin A 6,9-hemiketal 11,12-
carbonate ester in 50 ml. of dry ethyl acetate is added 20 ml. of a lN ethyl
acetate solution of hydrogen chloride, and the resulting solution concen-
trated to dryness under reduced pressure. The residual material is tritur-
ated with ether and filtered to give the desired salt.
By a similar procedure the amine compounds of the present invention
are converted to their di-acid addition salts.
EXAMPLE 32
4"-Deoxy-4"-amino-erythromycin A 6,9-hemiketal
11,12-carbonate ester hydrochloride ,
-
The procedure of Example 60 is repeated with the exception that 10
ml. of a lN ethyl acetate solution of hydrogen chloride is added. The
solution is concentrated to dryness in vacuo and the residual mono-hydro-
chloride salt is triturated with ether and filtered.
By a similar procedure the amine compounds of the present invention
are converted to their mono-acid addition salts.
40-
