Note: Descriptions are shown in the official language in which they were submitted.
,.a.J~8;367
This invention relates to intermediates Eor novel
antibacterial agents, and processes for the preparation of
said intermediates. In particular, the invention concerns
4"-deoxy-4"-oxo-erythromycin A derivatives as useful inter
mediates leading to the 4"-amino compounds and processes for
the preparation of the 4"-deoxy-4"-oxo-erythromycin A inter-
mediates.
Erythromycin is an antibiotic formed during the
culturing of a strain of Streptomyces erythreus in a sui.table
medium as taught in U.S. 2,653,899. Erythromycin, which is
produced in two forms, A and B, is represented by the following
structure:
1(CH3)2
HO~ HO
12 ' 4
R - ~ 13 3
o " ~ ~ ~/ 6~/
~ "OH
O ~,
OCH3
3~'~
Erythromycin R
A -OH
B -H
The structure reveals that the antibiotic is com-
prised of three main portions: a sugar fragmen-t 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 the erythromycin have
been prepared in an effort to modify its biological or
pharmacodynamic properties.
U.S. 3,417,077 describes the reaction product
of erythromycin and ethylene carbonate as a very active
antibacterial agent. U.S. 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 Acta, 39, 273
(1967)) and some controversy as to its struc-tural identity
(_etrahedron Letters, 157 (1970) and British Patent 1,341,022).
Sulfonamide derivatives of erythromycylamine are reported
in U.S. 3,983,103 to be useful as antibacterial agents.
~;
,
~L~C~6367
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 ln vivo antibacterial activity.
It has now been discovered that certain novel 4"-deoxy-
4"-aminoerythromycin A derivatives are outstanding as antibacter-
ial agents. These compounds are represented by the ~ormulae:
_ R ~(CH3)2
HO ~ ~O" ~
R2 ~ ~ ~ ~ ol ~ R~ ~ 3)2
3 ~ 1 R4q, ,
H ~ ~ O
O NH2
OCH3
III and IV
and a pharmaceutically acceptable acid addition salt thereof,
wherein Rl and R4 are each hydrogen or alkanoyl or two to three
carbon atoms; R2 is alkanoyl of two to three carbon a-toms; 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-.
- 3 -
~636~
A second preferred yroup oE compounds in this class
of antibac-terial agents are those of formula IV. Especially
preferred within this group are -those compounds wherein R4 is
hydrogen and also wherein R3 and R~ when taken together are
O
--C--.
The present invention p:rovides a class of compounds
useful as intermediates leading to the antibacterial agents
of formulae III and IV, represented as follows:
N(CH3)2
0 ~
HO ~"" ' HO
HO , \ / ~
: CH3
and
-- 4
` T~.
'~ ~.,
. .
`- ~ 636~
:` :R ~ N (CH3 ) 2
R201l."~
II
3 ~ ~
OCH3
wherein Rl is hydrogen or Ac, and Ac and R2 are each alkanoyl
: of two to three carbon atoms; R3 is hydrogen; and R2 and R3
when taken together are -C-.
Preferred within this class of intermediates are
those compounds of formula I. Especially preferred within this
group of intermediates are those compounds wherein Rl is hy-
drogen or acetyl.
A second group of preferred intermediates are those
~ 10 of formula II. Especially preferred within this group are those
: intermediates wherein Rl is hydrogen and also those wherein R
is acetyl.
According to -the present invention there is provided
. a process for preparing a compound of formula I or II defined
: above characterised by:
- 5 -
,~
~a) reacting a compound selected from the group consisting
of:
Ac N(CH3)2
AC ~ 2
HO~ /~
OCH 3 o -~
~13
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 dimethylsulfide in a reaction-inert-solvent
at about O to -25C. followed by contacting 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 compounds of formula I' and II' wherein the reaction-inert-
solven-t is methy]ene chloride.
-- 6 --
3~;~
A preferred Eeature of process (b) is the use of
toluene and benzene as the reaction-inert solvent.
Throughout the present invention, the stereochemical
designation of the substituen-ts 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 corres-
ponding to the compounds of formula III and IV of the present
invention. The erythromycin B amines are also useful as
antibacterial agents.
.
~,
.' ,. . ~ .
Detailed Descri~tion of the Invention
In accordance with the processes employed for syn~hesiæing the 4"-
deoxy-4"-amino-erythromycin A derived antibacterial agents of the present ln-
vention, the followin~ ~chema, startin~ with a 2'-alkanoyl-erythromycin A, or
derivative thereoP, are represented as follows:
''~ ,
. . .
_ Ac ~ ~C~3)~ - Ac ~ ~C~3)X
~ H
HO~Il, y ao~ ? O Y HO ~
~ocaU a ~ $~ocXa3
I' I (Y=O)
and
- Ac ~ ~ca~)2 Ac ~ ~ ~ 3
HO ~ ~, ~ HO ~ ~" ~
R20;~ R20~ ~
. OH
II' 3 II ~oc~3
,
--8--
'' -~
~,~
6~
The selective oxidation of I' and II' to I and II, respectively,
(Y=0) is the Eirst of the processes of the present invention and comprises
reacting the compounds I' and II' wieh tri1uoroacetic anhydride and dimethyl-
sulfoxide followed by tbe addition of a tertia~y ami~e such as triethyla~ine.
5 , In practice, the trifluoroacetic anhytride and dimethylsulfoxide are
inltially combined in a reaction-inert-solvent at about -65~C. After ten to
fifteen minutes the alcohols I' and II' are added at such a rate that the tem-
perature is maintained at about -65C. and does no~ rise above -30C. At tem-
; peratures above -30C. the trifluoroacetic anhydride - dimethylsulfoxide com-
plex is no,t stable. The reaction temperature is maintained between -30 and
-65~C. for about fifteen minutes and is then lowered ~o about ~70C. A tertiary
amine is added all at once and the reaction allowed to warm during a ten Co
flfteen minute periot. The reaction mixturs is subsequently treated with water
and worked up.
Regarding the quantities of reactants, for each mole of alcohol sub-
st2ate employed, one mole each of che trifluoroacetic anhydride and dimethyl-
sulfoxide are required. ~xperimentally, 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 certiary amine employed should correspond to
the molar amount of tri~luoroacetic anhydride used.
The reaction-inert-solvent utilized in Chis process should be one
which apprec:iably solubilizes the reactants and does not react to any great
extent wich either the reactants or the produccs formed. Since this o~idation
process Ls conducted at ~30 co -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 mixcures thereof which meet these
criteria are toluene, methylene chloride, ethyl acetate, chloroform or
tetrahydrofuran. SoIvents which meet the above requirements but which have
:
_9_
i6~3~7
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"-
5 ' 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 tem-
perature and the inherent reactivity of the starting reagents. At temperatures
of about -30 to -65C., the reaction is complete in fifteen to thirty minuce3.
; As to the order of addition of the reagents, ic i9 preferred that the
trifluoroacetic anhydride be combined with the dimethylsulfoxide followed by the
addition o~ the requisite alcohol substrate. It is further suggested~ as here-
inbefore mentioned, that the-temperature of the reaction is kept below -30C.
15 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 inter-
` mediates leading to the useful antibacterial agents, is represented by the
following scheme:
~,, .
`' :,.
.
~ ,
' :
,.~
-10-
.~
,~
~1~6367
Ac ~ (GY3) z . Ac 2~ 3~ z
ao,.. ,;~ ~
OX ~5~, ~
I ' ,OC~3 'OCH3
and
, ', ' ' ' ' . '
C~3~ 2 ~ 3~ Z
R~OI~ R20 '~
R3 ~AS O~CN3 R30~ ~C~o3
II ' II
-lOa-
~ - ~}
3~
. _ ~
The second process represents an oxidation reaction wherein the
4"-hydroxy substituent of I' and II', wherein Ac ancl R2 are each alkanoyl of
two to three carbon atoms, R3 is hydrogen, 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 agentO 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 0 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 reactionO 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 to -25C., 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 chlorida or tetra-
hydrofuran~ 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 es-
pecially preferred solvent for the claimed process is toluene-benzene.
The preferred compounds prepared by this process are 11,2'-diace-
tyl-~"-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.
- 11 - .
6~6~,~
Reaction time is not crit:ical and i9 dependent on concentration, reac-
tion temperature and the inherent reactivity of the reagents. At a reaction
temperature of O to -25C. the reaction time is about two to four hours.
Regarding the order of addition, as previously mentioned, it is pre-
ferred that the alcohol substrate I' or II' be added to the premixed succinimidederivative and dimethylsulfide.
~ oth 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'l-oxo compounds of the formula:
.
- R ~ ~C~332
R20."~
R30 ~
OCH3
Il
;~ wherein Rl and R2 are each alkanoyl of two to three carbon atoms and R3 is
hydrogen are prepared by trea~ing a compound of the formula:
Rl ~ 5 ~ 3)2
HO~
`: HOI
OCH3
I
-12-
:
6~36~
wherein Y is 0, Rl is alkanoyl of two to ~hree carbon a~o~s, with an alkanoic
anhydride (R20) and pyridine.
In practice, the ketone I is contacted wi~h 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 - 0) and II is carried out through a solvolysis reaction wherein
the 2'-alXanoyl-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 - 0) and rI wherein Rl is
hydrogen.
As previously mentioned, the ketones of Formulae I (Y - 0) and II
are usefuL intermediates leading to the b"-deoxy-4"-amino-erythromycin A anti-
bacterial 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
antibacteria:L agents of Formulae III and IV from the requisite ketones I (Y = 0)
and II.
,
- 13 -
-
3~
Preparation of the 4"-deoxy-4"-amino-erythromycin A compounts 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,
: 5 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 re-
ducing 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 forma~ion of the imine. Such excess amounts
l; appear to have little deleterious effects on the ~uality 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 ~ill vary with concentration, reaction temperature
and the inherent reactivity of the reagents. At room ~emperature, the pre-
ferred 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.
'
: ~L$6~
The preferred ammonium alkanoate, as previously indicated, for this
reaction is ammonium acetate.
In isolating the dejired 4"-deoxy-4"-amino-erythromycin A derivatives
from any non-basic by-products or starting material, advantage i5 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 thac neutral
or non-basic materials are extracted at lower pH's and the product at a p~ 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 solve~t. Additional purification, if necessary, can
be efected 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 ef-
fected by allowing a methanol solu~io~ of said compound to stand overnight at
ambient temperatures.
During the reductive amination of ketones of Formula II wherein R2
and R3 when taken together are -~- and Rl is alkanoyl of two to three carbon
atoms or hydrogen, it is noted eha~ amines related to both For~ulae III and IV
are produced. This is represented by the following scheme:
~,
-15-
3S~7
.. ..
_ Rl ~ ~cg3)2
~OC~
II 3
(R2 + R3
R ~ ~ ~Ç~3~ a R ~ ~ ~ 3
G ~ ~ O
III IV
(R2 + B3 ~ ) (R3 + R4 = -C- )
The amine products III and IV as represented are conveniently separa-
ted by selective crystallization from diethyl ether. Recrystallization of the
miYture of III and IV as represented from acetone-water induces he~iketal ror-
nation in the amine of Formula IV resulting in the isolation of III as the sole
produce.
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 ammonlum alkanoate followed by reduction of the in Sit
generated im:ine with sodium cyanoborohydride.
-16-
~6~6'~'
Compounds of Formula IV, wherein Rl, R3 and R4 are as previously
defined, are also prepared by the reduction of the aforementioned imine using
hydrogen and an appropriate hydroge~nation catalyst. ~xperimentally, the appro-
priate ketone (I) in ~ lower alkanol, such as methanol or isopropanol, is
treated with the ammonium salt of a lower alkanoic acid, such as aceeic acid,
and the hydrogenation catalyst, and the mixture shaken in a hydrogen atmos-
phere until the reaction is essentially complete.
Although one mole of the ammonium alkanoate i9 needed per mole of
ketone, it is preferred that an e~cess, 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 quallty of the product.
The hydrogenation catalyst can be selected from a wide range o f agents;
Raney nickel and 5-10 percent pallad~um-on-charcoal are, hol~ever, ehe 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 reac-
tion time, that an initial pressure of sa 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 tempera-
tur?e, 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 bv filtration of the spent catalys~ and
removal of ~he solvent in vacuo. The residual material is subsequently trea~ed
with water and the product isolated from non-basic materials by e~traction of
the basic product from water at varying pH's previously described .
-16a-
6~3~7
As previously indicated, ~when the lower alkanol solvenc 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 preferrçd 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 - O) to an oxime or oxime derlvative, l.e., Y - N-OH and
N-O-~Ca3, followed by reduction of the oxime or derivative thereof.
;
:
-16b-
~ '
~63~
The oximes of the ketones I (Y - O) are prepared by reacting said ke-
tones with hydroxylamine hydrochloride and barium carbonate in methanol or iso~
propanol at room temperature. In practice, it is preferred that an excess of
hydroxylamine be employed, and as m~ch as a three fold excess provites the de-
sired intermediate in good yields. Employing ambient temperatures and an excessof the hydroxylamine allows for the preparation of the desired oxlme derivative
in a reaction period of one to three hours. The barium carbonate is used ln
molar quantities twice that of the hydroxylamine hydrochloride employed. The
product is isolated by addition of the reaceion 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. Ihe residue is subsequently partitioned be-
tween water at pH 9.0-9.5 and a water-i~miscible solvent.
Prepa~ation of the O-acetyloxime compounds of Formula I (Y = ~-o-~CH
is effected by acetylation of the corresponding oxime. ~xperimentally, one
mole of the oxime is reacted with one mole of acetic anhydride in the presence
of one mole of pyridine or triethylamine. l'he 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 ben-
zene 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 preferrsd oxime and oxime derivatives which are useful interme-
diates 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 oxi~e
and 4"-deoxy-4"-oxo-erythromycin A O-acetyloxime.
Reduction of ehe ketone derivatives tY = N-OH or N~O-CC'~3) ls 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 o~ the spent catalyst followed by removal of
the solvent ~rom the filtrate provides for the isolation of the desired 4"-
deoxy-4"-amino antibacterial agent related to Formula IV. If methanol Ls em-
ployed 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 anti-
bacterial 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"-deoxy-4"-amino-erythromycin .~ 11,12-carbonate ester.
In the utilization of the chemotherapeutic acSivity of those com-
pounds of Formulae III and IV of the present invention which form salts, itis preferred, of course, to use pharmaceutically acceptable salts. Although
water-insolubility, high toxicity, or lack of crystalline nature may make
some particlllar salt species unsuitable or less desirable .or use as such in
a given pharmaceutical application, the water insoluble or toxic salts can be
converted to the corresponding pharmaceutically acceptable bases by decomposi-
tion 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 sul~urous,
phosphoric, acetic, lactic, citric, tartaric, succinic, maleic, gluconic and
aspartic acids.
-18-
'7
.~s previously mentioned, the stereochemistry of the starting materials
leading to the antibacterial agents of the present lnvention is that of ehe
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 opportuniey
for the stereochemistry of the 4"-substituent to change ~rom that of the na-
tural product. AcGordingly, when the compounds I (Y-O) and II are converted
to amlnes by one of the hereinbefore described procedures, it is possible that
two epimeric amines are formed. Experimentally, it is observed that both epi-
meric 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, qaid epimer can be purlfied by repeated recrystalliza-
tion 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 ~nown 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.
-13a-
i36~
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 reactlon. However, it is frequently
advantageous to purify the mixture of epimers by at least one recrystallization
from an appropriate solvent, sub~ectlng it to column or high pressure liquid
chro~atography, solvent partitioning or by trituration in an appropriate sol-
vent. Said purification, whiie 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, e~hibit 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 micro-
organisms, e.~., Staphylococcus aureus and Strepttococcus ~ , and againsc
certain Gram-negative microoganisms such as those of spherical or ellisoidal
shape (cocci). Their activity is readily demonstrated by in vitro tests
agains~ various microorganisms in a brain-heart infusion medium by the usual
two-fold serial dilution technique. Their in vitro activity renders them use-
ful for topical application in the form of ointments, creams and the like; for
sterilization purposes, e.~., sick-room utensils; and as industrial antimicro-
bials, for example, in water treatment, slime control, paint and wood preser-
vation.
'. ' ' '
'.~
--19--
;36~'
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 mi~tures thereof or other pharmaceutically-acceptable
i inert media; that is, media which have no harmful effect on the active ingre-
dient. ~'or such purposes, it will generally be acceptable to employ concen-
trations 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 microoganisms,
e.g., Pasteurella multocida and Neisseria sicca, in vivo via the oral and/or
~,parenteral routes of administration in animals, including man. Their in vivo
activity is more limitqd as regards suscep~ible organisms and is determined by
the usual procedure which comprises infecting mice of substantially uniform
weight with the test organism and subsequently ereating them orally or subcu-
taneously with the test compound. In practice, the mice, e.g., 10, are given
an intraperitoneal inoculation of suitable diluted cultures containing appro~i-
mately 1 to 10 times the LDloo (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 viru-
lence of the test organism. The test compound is administered O.S 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 fav-
ored dosa~e ran~e is from abou~ 5 mg./kv. to about 100 mg./k~. of bodY wei~ht
per day .~nd ~he preferred range from aboue 5 mg./kg. to about 50 mg./kg. of
body weight per day.
-20-
6~
Vehicles suitable for parenteral injec~ion may be either aqueous such as ~ater,
isotonic saline, isotonic dextrose, Ringer's solution, or non-aqueous such as
fatty oils of vegetable origin (cott:on seet, peanut oil, corn, sesame), di-
methylsulfoxide and other non-aqueous vehicles which will not interfere wi~h
therapeutic efficiency of the preparation and are non-toxic in the volume or
proportion used (glycerol, propylene glycol, so~bitol). Additionally, compo-
sitions suitable for extemporaneous preparation of solutions prior to adminis-
tration may advantageously be made. Such compositions may include liquid
diluents, for example, propylene glycol, diethyl carbonate, glycerol, sorbi.tol,
etc.; buff,ering agents, hyaluronidase, local anesthetics and inorganic salts
to afford desirable pharmacological properties. These compounds may also be
combined with various pharmaceutically-acceptable inert carriers incluting
solid diluents, aqueous vehicles, non-toxic organic solvents in the form of
. . .capsules, tablets, lozenges, troches, dry. mixes, suspensions, solutions, elixlrs
. 15 and parenteral solutions or suspensions. In general, the compounds are used in
various dosage forms at concentration levels ranging from about 0.; percent to
about 90 percent by ~eight of the total composition.
The following exa~ples are provided solely for the purpose or illus-
tration and noe to be construed as limitations of this invention, many varia-
cions of whlch are possible ~ chou~ departing erom ~he splric or spo e ~hereoi.
-21-
6 3 G~7
EX~MPLE 1
2'-Acetyl-4"-deoxy-4"-oxo-erythromycin A
To 3 ml. oE methylene chloride and 0.328 ml. of dimethylsulfoxide
cooled to about -65C. and maintained under a nitrogen atmosphere is added
5 0.652 ml. of trifluoroacetic anhydride. After about a minute a white slurry
forms indicating the presence of the trifluoroacetic anhydride - dimethylsul-
foxide complex. To the resulting slurry is added dropwise a solution of 1.0 g.
of 2'-acetylerythromycln A - ethyl acetate, obtained by recrystallization of
2'-acetylerythromycin A from ethyl acetate, in 7 ml. of methylene chloride
keeping the temperature at about -65C. The resulting 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 :ixture and the cooling bath is
- removed. After stirring for L5 min. the solution is added to 10 ml. of water
.~ , , . . . . ~ , .and the pH of the aqueous phase adj~sted to 10.` The organic phase is separated,w~shed successively with water (3 x 10 ml.) and brine soLution ~1 x 10) and
dried over sodium sulfate. Removal of the solvene under reduced pressure gives
929 mg. of the crude product. Recrystallization from methylene chloride -
hexane gives 320 mg. of the purified product, m.p. 105-108C.
N~R (~, CDC13): 3.28 (3H?s, 2.21 (6H)s and 2.03 (3H)s.
In a similar manner, starting with 2'-propionylerythromycin ~-ethyl
acetate and following the above procedure gives 2'-propionyl-4'7-deoxy-4"-oxo-
erOythromycin A.
-2~-
6367
:`
E~LE 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 stlr at ambient te~peratures for 20 hrs. The
solvent is removed in vacuo and the residual white foam recrystallized from
methylene chloride - hexane, 3.44 g., m.p. 170.5-172.5~C.
NMR (~, CDC13): 3.36 t3H)s and 2.33 (6H)s.
A product ldentical with the above is isolated when ~'-propionyl-4"-
; deoxy-4"-oxo-erythromycin A is treated with methanol at room temperature.
10EXAMPLE 3
2'-Acetyl-4"-deo~=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 ~1. of acetic anhydride ant ths re-
sulting reaction mi~ture 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.S by the addition of 6~ sodium hydroxide solution. The organic layer is
separaeed, dried over sodium sulfate and concentrated to give 14.5 g. of a
white foam identical, after recrystalli~ation from methylene chloride - hesane,
with the product of Example 1.
' ~
3~
E~AKPLE 4
-
2'-Acet~1-4"-deoxy-4"-oxo-erythromycin A oxime
To 500 ml. of ~ethanol is added 10.8 ~. of 2'-acetyl-4"-teo~-4"-oxo-
erythromycin A, 1.94 g. of hydro~ylamine hydrochloride and 11.0 g. of barium
carbonate, and the resulting suspension allowed to stlr at room temperature for
3.5 hrs. The ~ixtu~e is filtered and the filtrate concentrated under reduced
pressure. The residual foam is taken up i~ ethyl acetate which is subsequently
washed with wa~er at pH 9.5. The organic phase is separated, dried over sodium
sulfate a~d conce~trated in vacuo to give 10.6 g. of the desired product.
NMR (~, CDC13): 3.33 (3H)s, 2.30 (6H)s and 2.06 (3H)s.
EXAMPLE 5
2'-Acetyl-4"-deoxy~4''-oxo-erythromycin A 0-acetyloxime
To a solution of 330 mg. of 2'-acetyl.4"-deoxy-4"-oxo-erythromycin A
oxime in 30 ml. o ethyl acetate is added with stirring 64.2 ~r of acetic an-
hydride, and the reaction allowed to stir overnight at room temperature. Anadditional 15.8 ~1 of acetic anhydride and 23.4 ul of triethylamine are added
ant the stirring continued for 4 hrs. The reaction mixture is added to water
and the pH adiusted to about 9Ø ~he ethyl acetate layer is separated, dried
over sodium sulfate and concentrated under vacuum to give 300 mg. of the de-
2~ sired product:.
NME~ (~, CDC13): 3.38 (3H)s, 2.25 (6H)s, 2.20 (3a)s~ 2.05 (3H)s and
1.56 (3H)s.
In a similar manner by substitut$ng 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.
, _74_
~63~i7
E9~PLE 6
2'-Acetyl-4"-deoxy-4"-amino-erythromycin A
A mlxture of 14.0 g. of 2'-acetyl-4"-deoxy-4"-oxo-erythromycin A
0-acetyloxime and 60 g. of isopropanol washed Raney nickel ln 400 ml. of iso-
propanol is agitated in a hydrogen armosphere at an initial pres~ure of 1000
p.3.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 iso-
propanol and combined with 50 g. of fresh isopropanol washed Raney nickel. The
hydrogenation is continued overnight ac room temperature and an initial hydroge~
pressure of 1000 p.s.i. The catalyst is flltered and the filtrate concentrated
in vacuo to dryness to give 8.1 g. of the desired product.
EXU}~7~ 7
.
Starting with the appropriate 0-acetyloxi~e and employing the pro-
cedure of E~ample 6, the following 4"-a~ino-erythro~ycin A analogs are prepared:
3~2
I H0
~10 ~"~$~NH2
oc~3
Rl
.
H-
o
CH3CH2C-
~1~6~67
EX~h~PLE 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 i9 allowed rO stir at room temperature overnighe. ~he
solYent is removed under reduced pressure and the residual foam treated with a
mi~ture of 50 ~1. of chloroform and S0 ml. of ~ater. The pH of the aqueous
layer is adjusted to 9.5 and the organic layer separated. The chlorofonm layer
is treated with fresh ~ater and the pa ad~usted to 4Ø 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. ~he
extracts at pH 6 and 7 contain the major portion of the product and these are
combined and trested with fresh water at pa 4. The aqueous layer is again ad-
justed through p~ 5, 6 and 7, bei~g extracted at each pN with fresh chloroform.
The chloroform extract at pH 6 is dried over sodium sulfatz and concentrated to
giye 249 mg. of the produ:t as an epimeric ~ixture.
~NR (~, CDCI33: 3.30 ~l~)s, 3.26 (2~3s, 2.30 (6H)s and 1.46 (3H)s.
In a si~ilar mannar, 4"-deoxy-4"-amino-erythromycin A is prepared by
the methanol solvolysis of 2'-propionyl-4"-deoxy-4"-amino-erythromycin A.
-26-
i3L~t;~36'~
EX~LE 9
4"-Deoxy-4"-amino-erythromycin A
To a stirri~g solution of 3.0 g. of 4"-deoxy-4"-oxo-erythromycin A
i~ 30 ~1. of methanol u~der a nitrogen atmosphere is added 3.16 ~. of dry
ammonium acetate. After 5 ~in. 188 mg. of sodium cyanoborohydride is washed
into the reactlon mixture with 5 ml. of methanol and the reactlon allowed to
stlr at room température overnight. The light yellow solution is poured into
300 ml. of water and the pH adjusted to 6Ø The aqueous is extracted at p~ 6,
7, 7.5, 8, 9 and 10 using 125 ml. of diethyl ether for each extraction. The
extracts at pa 8, 9 and 10 are combined and washed with 125 ml. of fresh water.
The separated aqueous layer is extracted with ether (1 x 100 ml.) at p~ 7,
ethyl acetate (l x 100 ml.) at pH 7, ether (1 x 100 ml.) at pa 7.5, ethyl
.
acetate (1 x 100 ml.) at pH 7.5 and ethyl acetate (1 x 100 ml.j at pH 8, 9 and
10. The ethyl acetate extracts at pH 9 and lO 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 a~ ivory
colored foam.
':~
:;
,
.
,
~ -27-
~;636~
EXAMPLE 10
4"-~eox~-4"-amino~ ry~hromycin A (single eplmer)
A solution of 10.0 g. of the epimeric mixture of 2'-acetyl-4"-deoxg-
4"-amino-erythromycin A in 150 ml. of meehanol ls allowed to stir at room tem-
perature under nitrogen for 72 hrs. The solvent is removed in vacuo and the
residue ia dissolved i~ a stirring mixture of 150 ml. of water and 200 ml. of
chloroform~ The aqueous layer is discarded and 150 ~1. of fresh water is added.
The pH of the aqueous layer is adjusted to 5 and the chloroform layer is sepa-
rated. The p~ of ehe aqueous phase is subsequently adjusted to 5.5, 6, 7, 8
and 9, bein8 extracted after each adjusc~ent with 100 ml. of fresh chloroform.
The chloroform extracts from p~ 6, 7 and 8 are combined, 3uccessively with water
and a saturated brine solution and dried aver sodium sulfate. Removal of the
solvent under reduced pressure gives~2.9 g. of an epi~eric mlxture of 4"-deoxy-
4"-amino-erythromycin A. A 1.9 g. sample of the mixture is triturated with
- 15 diethyl ether causing some of the u~dissolved 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. 140-147C.
, .
.
.
-23-
6~
EXAMPLE 11
11,2'-Diacetyl-4"-deoxy-4"-oxo-erythromycin A 6,9-hemike~al
A solution of 10 g. of 2'-acetyl-4"-deoxy-4"-oxo-erythro~ycin A in
250 ml. of pyridine is treated with 40 ml. of acetic anhydride and the resultlngreaction 3ixture allowed to stand at room temperature for 10 days. The bulk
of the solvent is removed in vacuo and the remaining concentr~te added to a
; mixture of lSO ml. of watar 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.
10~MR (~, CDC13): 3.33 (3H)s, 2.26 (6H)s, 2.10 t3a)s, 2.03 (3H)s and
1.55 (3a)s.
EX~MPLE 12
Starting with the appropriace 4"-deoxy-4"-oxo-erythro~ycin A and
r.equisite alkanoic anhydride and employing the procedure of Exa~ple 11, the
following compounds ~re synthesized:
'
R ~ ~ ( ~ 3~2
R20~"",~
; HO ~
OCH3
Rl R2
O O
CH3C- CH3CH2C-
O O
,. ..
CH3CH2C- CH3CH2C-
O ~
,. -
3CH2C_ CH3C-
-29-
~f Si~6~
~XA~PLE 13
ll-Acetyl-4"-deoxy-4"-oxo-erythrom~cin A 6,9-hemiXetal
A solution of 3.0 g. of 11,2'-diacetyl-4"-deox~-4"-oxo-erythromycin A
9,6-hemiketal in 50 ml. of methanol iq allowed to stir u~der a nitrogan atmos-
phere overnight. The solvent is removed in vacuo to give the desired product
(3.0 g.) a~ a yellow foam.
NMR (~, CDC13): 3.35 (3~)s, 2.31 (6H)s, 2.13 (3H) and 1.55 (3~)9.
In a similar ~anner, the compounds of Exa~ple 12 are converted by
the procedure of Example 13 to 11-acetYl-4"-deox~-4"-oxo-er~thromYcin A 6,9-
h0~i~eeal and 11-propionyl-4"-deoxy-4"-oxo-erythromycin A 6,9-hemik tal.
EXAMPLE 14
ll-Acetyl-4"-deoxy-4"-amino-er2thromycin A 6,9-hemiketal
To a s~irrirg solution of 4.4 g. of 11-acetyl-4"-deoxy-4"-oxo-
erythromycln A 6,9-hemiketal and 4.38 g. of ammonium acetate in 75 ml. of
methanol is added 305 mg. of 85X 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
fr2sh chloroform and the p~ 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~ 6 and 7 are co~bined, washed with a saturated brine solution and dried
over sodium sulfate. Removal of the solvent provides 1.72 g. of the desired
-30-
~36~36~
product as a white foam. The protuct is dissolved in a minimal amount of
diethyl ether and is subsequently treated with hexane to turbidity. The crys-
talline producc which forms is filtered and dried, 1.33 g., m.p. 204.5-206.5C.
~NR (~, CDC13): 3.31 ~2H)s, 3.28 (lH)s, 2.31 (6H)s, 2.11 (3H)s and
1.5 (3H)s.
~XAXPLE 15
The procedure of Example 14 is repeated~ starting with the appropria~e
4"-deoxy-4"-oxo-erythromycin A and substituting isopropa~ol for msthanol as
the reac~io~ solvent to giYe the following co~pounts:
.
R ~C~3)2
= ~ 2
OCH3
~ 10 Rl 2
- O O
,. ..
CH3C- cH3c-
O O
" . .
CH3C CH3C~2C-
O O
:
Ca3CH2C- CH3CH2C-
O ' O
,. ..
3 2 C~3C-
-3~-
EXAMPLE 16
2'-Acety~erythro~ycin A 6,9-hemiketal 11,12-carbonate este_
To a solution of 13.2 g. of erythromycin A 6,9-hemikeeal 11,12-
carbonate ester (U.S. 3,417,077) in 150 mL. of benzene i9 added 1.8 ml. o-f
acetic anhydride, and the reaction mixture allowed to stir at room eemperature
for 1.5 hrs. The solutio~ is poured into 200 ml. of water and the aqueous
phase basified 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 ehe foam crystallizes. Filtration and drying of
10 the product gives 12.6 g. of pura product, m.p. 224.5-228.5C.
~R (~, CDC13): 3.36 (3H)s, 2.30 (6a)B7 2.06 (3~)s and 1.61 (3H)s.
In a simi}ar manner, by substituting an equivalent amount of pro-
pionic anhydride for acetlc anhydride in the procedure of Example 16, 2'-
.. . . . . . .
propionylerythromycin A 6,9-hemiketal 11712-carbonaté ester is prepared.
: .
E~AMPLE 17
:, :
2'-Acetyl-4"-deoxy-4"-oxo-erythromycin A
6,9-hemiketal 11,12-carbonate ester _
To a suspension of 6.19 g. of N-chlorosuccinimide in 150 ml. of
toluene and 50 ~1. of benzene cooled to -5C. i9 adted 4.46 ml. of dimethyl-
ZO sulfide. After stirring for 20 min. the resul~ing suspension is cooled to-25C. and 12.4 g. of 2'-acetylerythromycin A 6,9-hemiketal 11,12-carbonate
ester, partially dissolved ln 80 ml. of toluene, lS added dropwise. The
temperature, which is ~aintained 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 temperatureallowed to rise to -10C. The reaction mixture is then poured into water and
: :~
-32-
~1~6367
the aqueous phase ad~usted from 8.4 to 9Ø The organic layer is separated,
dried over sodium sulfate and concentrated under vacuum to a white foam (14.0g.).
Trituration of the residue with diethyl ether causes the foam to crystallize.
Filtration and drying of the product gives 11.3 g. of crystalline macerial,
m.p. 212-213.SC.
N~R (~, CDC13): 5.26 (lH)~, 3.36 (3H)s, 2.30 (6H)s, 2.13 (3H)s,
1.63 t3H)s and 1.50 (3H)~.
Similarly, 2'-propionyl-4"-deoxy-4"-oxo-erythromycin A 6,9-hemiketal
11,12-carbona e ester is prepared by the procedure of Exa~pla 17 by the re-
placement of the 2'-acetyl ester with an equivalent amount of 2'-propionyl-
erythromycin A 6,9-hemiketal 11,12-carbonate ester.
E~AMPLE 13
4"-Deoxy-4"-oxo-erythro~ycin A
6,9-hemiketal 11.12-carbonate ester
Forty-two and nlne-cenths gra~s of 2'-acetyl-4"-deoxy-4"-oxo-
erythro~ycin 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 product
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.
The resulting crystalline solid is allowed to stir for 40 ~in., and this then
filtered and dried to give 34.2 g. of desired product, m.p. 186.5-188C.
~R (~, CDC13): 5.66 (lH)t, 3.35 (3H)s, 2.3S (6H)s, 1.65 (3H)s and
l.Sl (3H)s.
-33-
-
63~;~
In a similar manner the same product is obtainsd when an equivalent
a~ount 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 ~ 6,9-hemiketal
_ 11,12-carbonate ester
To 189 g. of 4"-deoxy-4"-oxo-erythromycin A 6,9-hemi~etal 11,12-
car~onate ester in 1200 mL. of methanol at room temperature is added with
stirring 193 8- of ar~onium acetate. After 5 min. the resulting solution is
cooled to about -5C. and is subsequently treated with 13.4 g. of 85% sodium
cyanoborohydride i~ 200 ~l. of methanol over a 45 min. addition period. The
cooling bath is removed and the reaction mixture alIowed to stir at room tem-
perature overnight. The reaction mixture is reduced in ~oLume to 800 mL. in
vacuo and added to a stirring mixture of 1800 ml. of water and 900 ml. of
chloroform. The p~ i5 adjusted from 6.2 to 4.3 with 6N hydrochloric acid and
the chloroform layer separated. The chlorofor~ 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 pr2ssure to give 174 g. of a white foam.
The residùal material is dissolved in a mixture of 1 1. of water and 500 ml.
of ethyl acetate and the pH adjusted to 5.5. The ethyl acetate 13yer is
separaced and the aqueous layer adjusted to pH 5.7 and 9.5 successi~ely, being
extracted after each pH adjustment with 500 ml. of fresh ethyl acetate. The
ethyl acetat:e extract at pH 9.5 is dried over sodium sulfate and concentrated
in vacuo to dryness, 130 g. One-hundred and twenty grams or the residual
foam is dis~;olved in a mixture of l 1. of water and 1 1. of methylene chloride.:
~6~6~
the pH of the aqueous layer is adjusted to 4.4, 4.9 and 9.4 successively, being
extracted after each atjustment with 1 1. of fresh methylene chloride. The
methylene chloride extract at pH 9.4 is dried over sodium sulfate and concen-
tratsd under reduced pressure to give 32 g. of the product as a white Eoam.
Crystallization from 250 ml. of aceeone-water (1:1, v:v) give~ 28.5 g. of the
crystalline epimers.
N~R 100 ~ t~, CDC13): 5.20 tlH)m, 3.37 (1.5H)s, 3.34 (1.5H)s,
2.36 (6H)s, 1.66 t3H)s and 1.41 (3H)s.
EXA~PLE 20
Separation of the Epimers of 4"-Deo~y-4"-amino-erytbro~ycin A
6,9-hemiketal 11,12-carbonate Ester
On to a high-pressure-liquid-chromatography colu~n (l/2" x 9 cm.)
packed w~th G~ 254 siLica gel i~pregnated 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. Fractions 14
thru 21 and 24 thru 36 are collected.
Fractions 14 thru 21 are combined and concentrated to about 50 ml.
Water (50 ~1.) is added and the pH adjusted to 9Ø The chloroform layer i9
separated, dried over sodium sulfate and concentrated to give 106 mg. of a
white foam. Trituration with diethyl ether causes the Eoam to crystallize.
After stirring at room temperature for one hour the crystalline product is
fiLtered and dried, 31.7 mg., m.p. 194-196C.
N~E~ I00 Mz (~, CDC13): 5.24 (lH)d, 5.00 (lH)t, 3.40 (3H)s,
2.40 (6H)s; 1.66 (3H)s and 1.40 (3H)s.
-
-35-
~636~
Fractions 24 thru 36 are combi~Led and worked up as above to give
47.1 mg. of product as a white foam, which is identical to the material from
Example 25.
E~A~PLE 21
To a suspensio~ of 11.1 8- Of 2'-acetyl-4"-deoxy-4"-oxo-erythromycin
A 6,9-hemiketal 11,12-carbonate ester in 300 ml. of isopropanol at room tem-
peraeure is added with stirring io.7 g. of ammonium acetate. After S min.,
747 mg. of sodium cyanoborohydride in 130 ml. of isopropanol is added over a
period of 30 ~iIL. and the resulting reaction ~lxture is allowed to stir at
room temperature overnight. The pa~e yellow solution is poured into 1100 ml.
of water to which is then added 400 ~1. of diethyl ether. The pH is adj U8 ted
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 chloroform. The chloroform extracts
are combined, dried over sodium sulfate and concentrated to give 7.5 g. of a
yellow foam. Recrystallization of ths 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 pa raised
to 10. The basified aqueous layer is extracted (2 x 75 ml.) with eehyl
acetate and the first ethyl acetate e~tract dried over sodium sulfate and con-
centrated to dryness. The residual foam (1.96 g.) is added to a mixture of
75 ml. of water and 50 ml. of diethyl ether and the pa adjusted to 5.05. The
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 50 ml. of fresh
diethyl ether. The pH is finally adjusted to 9.7 and the aqueous layer extracted
-36-
~6~
with 50 ml. of ethyl acetate. The ether extract carried out at pH 6.0 is com-
bined ~ith 75 ml. of water ant the pH adjusted to 9.7. The ether layer is
separated, dried and concen~rated in vacuo to give 460 mg. of a white oam.
N~R 100 MZ ~J CDC13): 5.20 (lH)t, 3.43 (2El)g, 3.40 (lH)s, 2.38 (6H)s,
2.16 (3H)s, 1.70 (3H)s and 1.54 (3H).
The ~R data indicates the product to be the epimer30f 2'-acetyl-4"-
deoxy-4"-ami~a-erythromycin A 6,9-hemiketal 11,12-carbonate e_ter.
~he 1.69 g. indicated above is dissolved in a mixture of 75 ml. of
water and 75 ml. of diethyl ether and the pH adiusted to 4.7. The ether is
separated a~d the aqueous layer further extracted ~ith ~resh ether (75 ml.)
at pH 5.05 and 5.4, and with ethyl acetate (2 x 75 ~1.) at pa 9.7. The com-
bined ethyl acetate extracts are dried over sodium s~lfate and concentrated
under reduced pressure to give 1.26 g. of a ~hite foa~. Crystalli~ation o~
this residual material gives 411 ~g. of oroduct, m.p. i93-196C. (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 ~hich precipitate are filtered and dried, 182 mg., m.p.
198~202C. (dec.) to give additional product.
~R 100 Mz (~, CDCl3): 5.10 (lH~t, 3.34 (2H)s, 3.30 (lH)s, 2.30 (6H)s,
2.08 (3H)s, 1.62 (3H)s and 1.48 (3H)s.
The N~R data indicates the produce to be the epimers o~ 2'-acetyl-
4"-deoxy-4"-amino-er~thromycin A 11jl2-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 eseer,
there is obtained 2'-propionyl-4"-deoxy 4"-amino-erythromvcin A 6,9-hemiketal
11,12-carbonate ester and 2'-propionyl-4"-deoxy-4"-amino-erythromycin A 11,12-
carbonate ester.
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~636'7
EXAMPLE 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
overnlght at room temperature. The reaction solution i3 poured into 100 ml.
of water followed by the additio~ of 50 ml. o~ ethyl acetate. The pH is ad-
~usted to 9.5 and the orga~ic phase separated. The extraction is repeated
again with 50 ml. of fresh ethyl acetate. The combined ethyl acetate ex~ract~
are dried over sodium sul~ate and concentrated to give 392 mg. of a white foam.
Trlturation with dlethyl ether and scratching with a glass rod af~ects cry-
stallization. After standing at room temperature for 30 min., the crystall~ie
solids are filtered and drisd, 123 mg., and the mother Iiquor is retained. The
product is identical by N~R to material prepared in Example 24.
NMR 100 Mz (~, CDC13~: 3.26 (3H)s, 2.32 (6H)s, 1.61 (3H)s and
: 1.44 (3H~ s .
~he ~MR data indicates tha~ the crystalline product is a single
epimer of 4"-deoxy-4"-amino-erythromvcin A 11,12-carbonace ester.
Tke retained mother liquor i9 coDcentrated in vacuo to give 244 mg.
of a white foam.
The product is identical with materlal from Example 19.
Fhe NMR data indicaces that this product is a mixture of ehe epimers
:~ :
~ ~ o 4"-deoa~-4"-amino-erythromycin A 6,9-hemiketal 11,12-carbonate ester and
i identical wlth the product of Example 19.
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EXA PLE 23
In a manner similar to the procedure of E~ample 22, methanolysis oE
2'-propionyl-4"-deoxy-4"-amino-erythromycin A 6,9-hemiketal 11,12-carbonate
ester gives 4"-deoxy-4"-amino-er~thromycin A _1,12-carbonate ester and 4"-
deoxy-4"-amino-erythrom~cin A 629-hemiketal 11,12-carbonate ester.
EXAMP~E 24
Eight grams of the epimeric mixture of 4"-deoxy-h"-amino-erythromycin
A 11,12-carbonate ester from the non-crystalline product of Example 19 is dis-
solved in 50 ml. of diethyl ether. The product is induced to crystalliæe by
scratching wieh a glass rod. After 20 min. stirring,the crystalline product is
filtered and dxied, 1.91 g., m.p. 198.5-200C.
NMR 100 Mz ~3, CDC13): 3.26 (3H)s, 2.30 (6H)s, 1.61 (3H)s and
. 1.45 (3H~s
The NMR data indicates that the crystalline product is a single epimer
15 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 epimeF of Example 24 is dissolved in 20 ml. of acetone
`~ ~ and heated at steam bath temperatures until the boiling point is reached. Water
t25 ml.) is added and the resulting solution allowed to stir at roo~ ~empera-
ture. After one hour of stirring; the precipitate which for~s is filtered and
dried to give 581 mg., m.p. 147-149C.
N~ 100 ~z (~, CDC13): 5.12 (lH)d, 3.30 (3H)s, 2.30 (6H)s,
1.62 (3H)s and 1.36 (3H)s.
The NMR data indicates the product to be a single epimer of 4"-deoxy-
4"-amino-erythromycin A 6,9-hemiketal 11,12-carbonate ester and identical to
the epimer in fractions 24-36 of Example 2D.
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636"7
EX~h~PLE 26
4"-3eo-~y-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 concen-
trated to dryness in vacuo. The residue i9 partitioned between water-chloroform
at a pH of 5.5. The aqueous layer is separated, the pH 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 ~l. of diethyl ether a~ room temperature for
~0 minutes. The resulting solids are filtered and dried to give 9.45 g. of a
single epimer indlsti~guishable from that in E~ample.10.
The diethyl ether filtrate is concentrated to dryness to give 6.89 g.
; 15 of product consisting of the other epimer plus some impurities.
- 40
~1~63~
EXA~ 27
4"-~eoxy-4"-Amino-Erythromycin A
Two grams of 4"-deoxy-4"-oxo-erythromycin A, 3.1 g. of a~monium
acetate and 2.0 g. of Raney nickel in 50 ~1. of rnethanol is shaken at room
temperature in a hydrogen atmosphere at an initlal pressure of 50 p.s.i. over-
night. An additional 3.16 ~. of ammonium acetate and 2.0 g. of Raney nickel
are added and the hydrogenation continued for an additional 5 hours. The solids
are filtered and the filtrate concentrated to dryness in vacuo. The residue
is added with stirring to a mixture of water-chloroform, and the pa adjusted
from 6.4 co 5.5. The aqueous phase is separated, ehe pH adjustet to 9.6 and
fresh chloroform added. The chlorofor~ extract is separated, dried over sodium
sulfate and concentrated under reduced pressure to give 1.02 g. of the product
as a yellow foam. The predominant isomer has the opposite configuration at
: . ' ' '
4" al3 the compo~D~ o~ ~xa:lple 10.
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- 41 -
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EXAMPLE 28
2'-Ace~yl-4"-deoxy-4"-amino-er7thromys~L_~
To a sol~tion of 4.5 g. oE 2'-acetyl-4"-deoxy-4"-oxo-erythromycin 3
(U.S. #3,884,903) in 45 ml. of isopropanol under a nitrogen at~osphere is
S added with stirring 4.66 g. of dry a~monium acetate. After lO min. 376 mg. of
sodium cyanoborohydride is washed into che reaction mixture with 10 ml. of
isopropanol and the reaction allowed to stir at room temperature overnighe.
The lighe yellow solution is poured into 400 ml. of water and the pH adjusted
to 6Ø The aqueous is extracted at pH 6, 7, 7.5, 8, 9 and 10 using 250 ml.
of diethyl ether for each extraction. The extracts at pH 8, 9 and 10 are co~-
bined and washed with 250 ml. of fresh water. The separated aqueous layer is
extracted with ether (l x 100 ml.) at pH 7, ethyl acetate (l x 100 ml.) at pH 7,
ether (1 x 100 m~.) a~ pH ?.5, ethyl acetate (1 x lOO 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 dried
over sodium sulfate. Removal o the solvent in vacuo gives an epimeric mix-
ture of the desired product as a crea~ colored foam.
In a similar manner, 4"-deoxy-4"-ami~o-erythromycin G is prepared
from 4"-deoxy-4"-oxo-erythromycin B.
~,, 7
116367
EXUhMPLE 29
4"-Deoxy-4"-amino-erythromycin B
A solution of 4.34 g. of 2'-acetyl-4"-deoxy-4"-amino-erythromycin 3
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 wlth a
mixture of 100 ml. of chloroform and 100 ml. of water. The pH of the aqueous
layer is adiusted to 9.5 and the organic layer separaced. The chlorofor~ layer
i9 treated ~ith fresh water and the pH adjusted to 4Ø The pH of the acid
aqueous layer containing the product is gradually adiusted to 5, 6, 7, 8 and 9
by the addition of base, being extracted at each pH with fresh chloroform. The
extracts ac 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 ad-
justed 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 co
l; give the product as an epimeric mixture. -
.
_ 43
~636~
EX~.PLE 30
4"-Deoxy-4"-amino-erythromycin A 6,9-hemi~etal
11,12-carbonate ester aspartate
To a solution of 1.0 g. of 4"-deoxy-4"-amino-erythromycin A 6,9-
hemiketal ll,l2-carbonate ester i~ 6 ml. of acetone ~armed to 40C. is added
20 ml. of ~ater followed by 175 mg. of L-a~partic acid. The mixture i9 heated
to reflux for 1.5 hours and is then filtered while hot. The filtraee is con-
centrated by removal of the acetone and is subsequently freeze-dried to give
1.1 g. of the desired product as a white solid.
EXA~PLE 31
4"-Deoxy-4"-amino-erythromycin A 6t9-hemiketal
11,12-carbonate ester dihydrochloride
' . .
To 7.58 g. o~ 4"-deoxy-4"-amino-erythromycin A 6,9-hemiketaL 11,12-
carbonate ester in 50 ml. of dry ethyl acetate i5 added ~0 Ql. of a lN ethyl
acetate solution of hydrogen chloride, and the resulting solution concentrated
to dryness under reduced pressure. The residual material is triturated with
ether and filtered to give the desired salt.
By a similar procedure the amine compounds of the present invention
:re converted to thelr dl-acld additlon slltt.
.
.
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i36~
EXAMP'LE 32
4"-Deoxy-4"-amino-eryehromycin A 6,9-hemiketal
11,12-carbonate ester hydrochloride
The procedure of Example 60 is repeaced with the exception that 10 ml.
of a 1~ ethyl acetate solution of hydrogen chloride is added. The solution is
concentrated to dryness in vacuo and the residual mono-hydrochloride salt is
triturated with ether and filtered.
By a similar procedure the amine compounds oc che present invention
are converted to their ~ono-acid addition salts.
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