Note: Descriptions are shown in the official language in which they were submitted.
7S7~13
~-1319
Antibiotic preparations having increased effectiveness,
processes for their manufacture and method for increasing
the antibiotic action of antibiotics.
The present invention relates to antibiotic prepara-
tions having increased effectiveness, to processes for their
manufacture and to a method for increasing the antibiotic
effectiveness of antibiotics~
The invention relates especially to antibiotic pre-
parations that contain at least one antibiotic and at least
one muramylpeptide of the formula (I) or a salt thereof
CH20H
/~ O\
7co /~ OH,H ~I~
N-X-R
R 3-CH ~ 1
~D~\ R2
R5 COA1 77
CON-CH-CON-CH-CH2CH-COA2
R~ R~
~ .
' - 2 - ~ ~75~
wherein
X represents carbonyl or carbonyloxy,
Rl represents optionally substituted alkyl
or aryl,
R , R and R represent hydrogen or lower
alkyl,
R represents hydrogen or lower alkyl,
R represents hydrogen, lower alkyl, free or
functionally modified hydroxy-lower alkyl,
free or functionally modified mercapto-
lower alkyl, optionally substituted amino-
lower alkyl, cycloalkyl, cycloalkyl-lower
alkyl, optionally substituted aryl or aralkyl,
nitrogen-containing heterocyclyl or hetero-
cyclyl-lower alkyl, or
R and R together represent also alkylene having 3
or 4 carbon atoms,
R represents hydrogen or optionally esterified or
amidated carboxyl, and
one of the radicals Al and A represents a radical of
the formula
O W
Il
T Y O P O CH (I
I~
IH
z
,
~herein
T represents NH or 0,
Y represents an optionally substituted alkylene
~.7574~
-- 3 --
group that can also be interrupted by one or
two oxycarbonyl and/or iminocarbonyl groups,
W represents hydrogen, and
Z represents a 1,2-dihydroxyethyl or 2-hydroxy-
ethyl group in which at least one hydroxy group
is esterified by an optionally unsaturated
long-chain aliphatic carboxylic acid or
etherified by an optionally unsaturated long-
chain aliphatic alcohol, or
each of W and Z represents a hydroxymethyl group
esterified by an optionally unsaturated long-
chain aliphatic carboxylic acid or etherified
by an optionally unsaturated long-chain
aliphatic alcohol,
and the other of the radicals
A and A represents free or etherified hydroxy, free or
alkylated amino, lower alkylamino or aminocarbonyl-
lower alkylamino,
and to salts thereof.
Alkyl is especially straight-chain or branched alkyl
bonded in any position and having up to 18 carbon atoms,
but is especially lower alkyl.
Suitable substituents of optionally substituted alkyl
groups are especially free or functionally modified hydroxy
or mercapto groups, such as etherified or es~erified hydroxy
or mercapto groups, for example lower alkoxy or lower alkyl-
mercapto groups, or halogen atoms, or free or functionally
modified carboxyl groups, such as lower alkoxycarbonyl
groups or carbamoyl groups. The substituted alkyl radical,
such as a lower alkyl radical, can carry one, two or more
identical or different substituents, especially free hydroxy
groups or halogen atoms.
Aryl radicals are especially monocyclic and bicyclic
aryl radicals, especially phenyl but also naphthyl. They
can optionally be mono-, di- or poly-substituted, for
example by lower alkyl groups, by free, esterified or
~ ~57~8
-- 4 --
etherified hydroxy, for example lower alkoxy or lower
alkylenedioxy, or by halogen atoms, and/or by trifluoro-
methyl groups.
Aralkyl is especially aryl-lower alkyl, wherein aryl
has the meaning given above. Aryl-lower alkyl represents
especially benzyl or phenylethyl, wherein the phenyl nucleus
can be mono-, di- or poly-substituted.
Optionally substituted aralkyl radicals are especially
those radicals that are optionally mono-, di- or poly-
substituted in the aromatic nucleus, for example by lower
alkyl, by free, etherified or esterified hydroxy or mercapto
groups, for example lower alkoxy, lower alkylenedioxy and
lower alkylmercapto groups, or by trifluoromethyl groups,
and/or by halogen atoms.
Cycloalkyl is especially cycloalkyl having 5 or 6
carbon atoms, such as cyclopentyl or cyclohexyl, and cyclo-
alkyl-lower alkyl is especially a radical in which the cyclo-
alkyl moiety has 5 or 6 carbon atoms and the lower alkyl
moiety represents especially methyl or ethyl.
Nitrogen-containing heterocyclyl is especially the
radical of a 5- or 6-membered heterocyclic compound con-
taining one or two nitrogen atoms in the ring. The radical
can be unsaturated or alternatively saturated and can
contain, for example, a condensed-on phenyl radical. Such
radicals may be, for example, pyrrolyl, indolyl, pyridyl
or imidazolyl radicals.
In nitrogen-containing heterocyclyl-lower alkyl the
heterocyclyl radical has the meaning given above and the
lower alkyl radical is especially methyl or ethyl.
The alkylene radical, which can be formed by the
radicals R4 and R , is preferably unsubstituted and
is especially the trimethylene radical.
An optionally esterified or amidated carboxyl group
is especially the carboxyl group itself or a carboxyl group
esterified by a lower alkanol, or alternatively a carbamoyl
group that is unsubstituted at the nitrogen atom or is mono-
_ 5 _ ~ ~7~7~
or disubstituted by alkyl, especially lower alkyl, by aryl,especially phenyl, or by aralkyl, such as benzyl. The
carbamoyl group can, however, also carry an alkylene
radical, such as the tetramethylene or pentamethylene
radical.
As optionally functionally modified hydroxy or
mercapto groups special mention may be made of etherified
or esterified hydroxy or mercapto groups, such as lower
alkoxy, lower acyloxy, for example lower alkanoyloxy, or
halogen atoms, lower alkyl~ercapto or lower acylmercapto,
for example lower alkanoylmercapto.
As functionally modified amino-lower alkyl special
mention may be made of mono- or di-lower alkylamino-lower
alkyl or acylated amino-lower alkyl, such as methylamino-
lower alkyl, ethylamino-lower alkyl, dimethylamino-lower
alkyl, diethylamino-lower alkyl and alkanoylamino-lower
alkyl, for example lower alkanoylamino-lower alkyl.
Aminocarbonyl-lower alkylamino is especially l-amino-
carbonyl-lower alkylamino, for example glycylamino, alanyl-
amino, valylamino or isoleucylamino.
The alkylene radical Y contains up to 20 carbon atoms
and is especially a lower alkylene radical, preferably
having 2 or 3 carbon atoms. The alkylene radical Y can,
howéver, also be a lower alkylene radical interrupted by
a radical such as oxycarbonyl or N-R -iminocarbonyl, and
then represents especially a radical of one of the formulae
-Y -COO-Y - (IIIa)
-Y -OOC-Y - ~IIIb)
-Y -CON-Y -
1 2
(IIIc)
] a
7~
or -Y -NOC-Y -
¦ (IIId)
Ra
wherein one of the radicals Y and Y represents an
optionally substituted lower alkylene radical and the other
of the radicals Yl and Y2 represents an optionally sub-
stituted lower alkylene radical that can also be interrupted
by oxycarbonyl or N-R -iminocar~onyl, Yl and Y2
together having more than two carbon atoms, and R
represents hydrogen or lower alkyl. As substituents of
the radicals Y and Y special mention should be made
of free or functionally modified hydroxy or hydroxy-lower
alkyl, free or functionally modified mercapto or mercapto-
lower alkyl, free, mono- or di-lower alkylated or acylated
amino-lower alkyl~ aminocarbonyl, alkyl, cycloalkyl havin~
5 or 6 carbon atoms, aryl or aralkyl, it being possible
for the general terms to have the meanings specified above.
A long-chain aliphatic carboxylic acid i5 especially
one that has from 10 ~o 90 carbon atoms and that may also
have 1 or 2 double bonds and may be straight or branched.
Preferred are those having up to 30 carbon atoms, especially
from 16 to 22 carbon atoms, or natural or synthetic mycolic
acids.
A long-chain aliphatic alcohol is especially an
alkanol that has up to 30 carbon atoms, especially from
10 to 22 carbon atoms, and that may also have one or two
double bonds and may be straight or branched. Preferred
are those alkanols which contain from 12 to 18 carbon atoms
and of which the hydroxy group is terminal.
Those radicals and compounds that are termed "lower"
in connection with the present description and the patent
claims preferably contain up to and including 7 carbon atoms
and especially up to and including 4 carbon atoms.
Hereinbefore and hereinafter the general terms can
have the following meanings:
~7~i7~
-- 7 --
Lower alkyl is, for example, n-propyl, n-butyl,
isobutyl, sec.-butyl or tert.-butyl, also n-pentyl, n-hexyl,
isohexyl or n-heptyl, and especially methyl or ethyl. In
aryl-lower alkyl, cycloalkyl-lower alkyl or heterocyclyl-
lower alkyl radicals the lower alkyl radical is especially
methyl or ethyl, the aryl, cycloalkyl or heterocyclyl
radical having the meaning given above.
Lower alkoxy is, for example, n-propoxy, n-butoxy,
isobutoxy, sec.-butoxy or tert.-butoxy and especially
methoxy or ethoxy.
Lower alkylmercapto is, for example, n-propylmercapto,
n-butylmercapto, isobutylmercapto, sec.-butylmercapto or
tert.~butylmercapto and especially methylmercapto or ethyl-
mercapto.
Lower alkylenedioxy is especially methylenedioxy,
ethylenedioxy or propylenedioxy.
Halogen represents fluorine or bromine but preferably
chlorine.
Lower alkanoyl is especially propionyl or butyryl
but more especially acetyl.
Synthetic mycolic acids are especially -alkyl-~-
hydroxyalkanecarboxylic acids, wherein the alkyl radical
in the ~-position contains from 1 to 20, especially 1 to
14, carbon atoms, and the alkanecarboxylic acid contains
from 20 to 80, especially from 30 to 34, carbon atoms.
They can also contain further hydroxyl groups, and oxo,
methylene or ethylene groups.
Natural mycolic acids are especially those which can
be isolated from living organisms, such as bacteria, for
example Mycobacteria.
The compounds of the formula I can be present in the
form of isomeric mixtures or in the form of pure isomers.
The radical of the formula -CH~R )-(C-O)-, which is linked
to the oxygen atom, if R represents lower alkyl, is pre-
ferably present in optically active form and has especially
the D-form, while the radical of the amino acid of the
~7~;7~
-- 8
formula -N(R4)-CH(R5)-C(=O), if R5 is different from
hydrogen, is also preferably present in optically active
form, especially in the L-form, and the terminal -amino-
glutaric acid radical is preferably present in optically
active form, especially in the D-form. Further, the 1-
hydroxy group can have the ~- or the ~-configuration. The
novel compounds of the formulà I can, however, also be pre-
sent in the form of a mixture of the 1~- and l~-isomers.
In the compounds of the formula I the proton linked
to phosphorus via an oxygen atom can be split off readily
with bases. The compounds of the formula I are customarily
present in the form of a mixture of the free compounds and
their salts. Approximately 40 to 55 % of the muramyl-
peptides of the formula I described in the Examples are
thus present in the form of sodium salts. The invention
relates also to these salts as combination partners.
The invention relates generally also to the salts of
compounds of the formula I having any other salt-forming
groups, for example free carboxyl groups, especially phar-
maceutically acceptable non-toxic salts, for example metal
or ammonium salts.
Compounds of the formula I having amino groups, for
example in the radical R , can be present in the form
of internal salts (zwitterions) or in the form of acid addi-
tion salts. Weak and pharmaceutically acceptable acids
are especially suitable for addition. Any antibiotics are
suitable as combination partners. It is possible to use
both individual antibiotics and mixtures of antibiotics.
The latter preferably contain not more than three compounds
having antibiotic action. There are used especially anti-
biotics from the group consisting of ~-lactam antiobiotics,
aminoglycosides, tetracyclines, macrolides, lincomycins,
polyene antibiotics, polypeptide antibiotics, anthra-
cyclines, chloramphenicols and thiamphenicols, cycloserines,
fusidic acids or rifamycins.
~7~
As preferred antibiotics from among the ~-lactams
there may be mentioned the penicillins, cephalosporins,
penems, nocardicins, thienamycins and clavulanic co~pounds.
Penicillin antibiotics are especially amoxycillin,
ampicillin, carbenicillint cloxacillin, cyclacillin,
dicloxacillin, mecillinam, methicillin, penicillin G, peni-
cillin V, pivampicillin, sulbenicillin, azlocillin, ticar-
cillin, m2e610cillin, pivmecillinam or 6-(4-endoazatricyclo-
[~.2.2.0 ]undec-8-enyl)-methyleneaminopenicillanic
acid.
From the group of cephalosporins there may be
mentioned, for example, cefaclor, cefazaflur, cefazolin,
cefadroxil, cefoxitin, cefuroxime, sephacetril, cephalexin,
cephaloglycin, cephaloridins, cephalothin, cefamandole,
cephancn, cephapirin, cefatrizine, cephradine, cefroxadin
(7~-[D-2-amino~2-(1,4-cyclohexadienyl)-acetamido]-3-
methoxy-3-cephem-4-carboxylic acid = CGP 9000), cefsulodin,
cefotaxime, cefotiam, ceftez~le or cefazedon~
Of the nocardicins there may be mentioned, for
example, nocardicin A, and of the thienamycins and clavula-
nic acids there may be mentioned, for example, thienamycin
and clavulanic acid respectively.
Of ~he aminoglycosides special mention should be made
of streptomycins, for example streptomycin and streptomycin
A, neomycins, for example neomycin B, tobramycins, for
example tobramycin or dibekacin, kanamycins, (for example
mixtures of kanamycin-A, B and C), and amicacins, genta-
mycins, (for example mixtures of gentamycin A, C , C
or C ), or sisomycins such as sisomycin oz netilmycin,
also lividomycin, ribocamycin and paromomycin.
As tetracyclines special mention should be made of
tetracycline, doxycycline, chlorotetracycline, oxytetra-
cycline or methacycline.
As macrolides there may be mentioned, for example,
maridomycin, spiramycins, such as spiramycin I, II and III,
erythrornycins, for example erythromycin, oleandomycins,
.
-- 10 --
for example oleandomycin and tetraacetyloleandomycin, and,
as lincomycins, for example, lincomycin and clindamycin.
As polyene antiobiotics special mention should be
made of amphothericin B and the methyl ester thereof, or
nystalin.
As polypeptide antibiotics special mention may be
made, for example, of colistin, gramicidin S, polymyxin B,
virginiamycin, tyrothricin, viomycin or vancomycin.
Suitable rifamycins are especially rifamycin S,
rifamycin SV or rifamycin B or semisynthetic derivatives
thereof, especially rifampicin~
The combination preparations according to the inven-
tion contain the customary amounts of antibiotics per dosage
unit, for example between 50 and 1000 mg, as a rule between
100 and 500 mg. The amount of muramylpeptide depends upon
the intended method of administration. The amount for
orally administrable preparations is higher than that for
injectable preparations. Orally administrable preparations
contain muramylpeptide of the formula I in an amount from
1 mg to half the amount of antibiotics, as a rule between
5 and 50 mg. When using coated tablets that are resistant
to gastric juice the dose can also be less than 1 mg ~down
to 0.01 mg) of muramylpeptide per tablet. Injectable pre-
parations contain between 10 ~9 and 50 mg, preferably
between 100 ~g and 10 mg, of muramylpeptide. These prepara-
tions can additionally contain the customary amounts of
pharmacological carriers, extenders and/or diluents, espe-
cially when they are to be used for oral administration.
Liposomal forms of administration are also suitable espe-
cially for injectable preparations.
Special emphasis is to be given to pharmaceutical or
veterinary preparations, as well as animal feedstuffs or
feedstuffs additives, which contain an effective or less
than effective dose of the specified antibiotics and, in
addition, a muramylpeptide of the formula I.
7~7~1~
The pharmaceutical preparations of the present inven-
tion are preferably tablets or gelatin capsules which con-
tain the active substances together with diluents, for
example lactose, dextrose, saccharose, mannitol, sorbitol;
cellulose and/or glucosej and lubricants, for example
siliceous earth, talc, stearic acid or salts thereof, such
as magnesium or calcium stearate, and/or polyethylene
glycol. Tablets also contain binders, for example magnesium
aluminium silicate, starches, such as corn, wheat, rice
or arrowroot starch, gelatin, tragacanth, methylcellulose,
sodium carboxymethylcellulose and/or polyvinylpyrrolidone,
and, if desired, disintegrators, for example starches, agar,
alginic acid or a salt thereof, such as sodium alginate,
and/or effervescent mixtures, or adsorbents, colourants,
flavourings and sweeteners. Injectable preparations are
preferably isotonic aqueous solutions or suspensions. Sup-
positories, ointments or creams are especially fatty
emulsions or suspensions. The pharmaceutical preparations
can be sterilised and/or contain adjuncts, for example
preservatives, stabilisers, wetting agents and~or
emulsifiers, solubilisers, salts for regulating the osmotic
pressure and/or buffers. The present pharmaceutical
preparations which, if desired, can contain further
pharmacologically valuable substances, are manufactured
in a manner known per se, for example by means of
conventional mixing, granulating or coating methods, and
contain rom approximately 0.1 ~ to approximately 75 %,
especially from approximately 1 % to approximately 50 %,
of the specified active substances.
The orally administrable preparations of the present
invention can also be provided with a coating that is
resistant to gastric juice.
The high antibiotic action of the novel preparations
and of the novel method can be demonstrated by "in vivo"
tests on various species of animals, especially mammals
such as mice. For this purpose, animals are infected with
- 12 - ~7~7~
a lethal or sub-lethal dose of a pathogenic micro-organism
and then the specified novel preparation or the individual
doses of muramylpeptide and antibiotic are administered.
The action is determined as ED50, that is to say, the
dose at which 50 % o~ the animals survive.
Surprisingly, it has now been found that infection
with pathogenic germs, especially of the less easily con-
trollable gram-negative bacteria, such as, for example,
strains of Aerobacter, Brucella, Escherichia, Klebsiella,
_
Malleomyces, Neisseria, Pasteurella, Proteus, Pseudomonas,
Shigella and Vibro, but a~so of gram-positive bacteria such
as actinomycetes/ clostridia, corynebacteria, diplococci,
mycobacteria or staphylococci, or of fungi such as Candida
albicans, Cryptococcus neoformans_ Plastomyces dermatitides
or Hystoplasma capsulatum, are inhibited and combated to
an increased extent.
The invention relates also to a method for increasing
the antibiotic activity of antibiotics. This method is
characterised in that at least one antibiotic, especially
an antibiotic as described in the combination preparations
according to the invention, is administered together with
at least one of the above-mentioned muramylpeptides of the
formula I.
In the method of the present invention, an effective
or less than effective dose of the antibiotic is used depen-
ding on the nature of the latter, for example from approxi-
mately 10 to approximately 1000 mg, especially from approxi-
mately 50 to approximately 500 mg, per individual dose.
The dosage of muramylpeptides of the formula I depends
on the method of administration and corresponds to the
dosage specified for the pharmaceutical preparations. The
muramylpeptide derivative can be administered up to 24 hours
before or after, but preferably approximately simultaneously
with, the antibiotic.
The antibiotics are administered in the customary
manner, such as subcutaneously, intravenously or orally,
- 13 - ~ ~757~
while the muramylpeptides are usually administered subcu-
taneously, especially when they are administered separately
from the antibiotics.
Especially suitable as combination partners are
muramylpeptides of the formula I
CH2H
O
OH,H (I~
~-X-R1
R3-CH 2
OAl l7
CON- H-CON-CH-CH CH-COA
¦ (L~ ¦ (D~ 2 2
R R6
wherein
~ X represents carbonyl,
Rl represents optionally substituted alkyl having
up to 18 carbon atoms or aryl having up to 30
carbon atoms,
R , R , R and R represent hydrogen or lower alkyl,
R represents hydrogen; lower alkyl optionally
substituted by hydroxy, lower alkoxy, mercapto,
lower alkylmercapto, amino, lower alkylamino or
by halogen; cycloalkyl or cycloalkyl-lower
alkyl, wherein the cycloalkyl radical contains
from 4 to ~ carbon atoms; optionally substituted
phenyl or phenyl-lower alkyl; heterocyclyl or
heterocyclyl-lower alkyl, each having 5 or 6
ring members and containing one or two a~a
atoms, or
R and R together represent also alkylene having 3 or 4
carbon atoms,
R represents hydrogen, and
- 14 - ~ ~7~7~8
: one of the radicals Al and A represents a radical of
the formula
O W
T Y O P O IH (II~
I
OH Z
wherein
T represents NH or O,
Y represents optionally substituted alkylene
that has up to 20 carbon atoms and can also
be interrupted by carbonyloxy or
carbonylimino,
W represents hydrogen, and
Z represents a 1,2-dihydroxyethyl or 2-hydroxy-
ethyl group in which at least one hydroxy
group is esterified by an optionally
unsaturated long-chain aliphatic carboxylic
acid having up to 30 carbon atoms or by a
mycolic acid, or is etherified by an option-
unsaturated long-chain aliphatic alcohol
having up to 30 carbon atoms, or
each of W and Z represents a hydroxymethyl group
esterified by an optionally unsaturated long-
- chain aliphatic carboxylic acid having up to
30 carbon atoms or by a mycolic acid or
etherified by an optionally unsaturated long-
chain aliphatic alcohol having up to 30
carbon atoms, and
- 15 - ~ ~7~7~
the other of the radicals A and A represents free or
etherified hydroxy, amino, lower alkylamino or amino-
carbonyl-lower alkylamino,
and salts thereof.
Preferred combination partners are muramylpeptides
of the formula I wherein X represents carbonyl, R
represents lower alkyl optionally substituted by hydroxy,
lower alkoxy or by halogen; or phenyl optionally substituted
by hydroxy, lower alkoxy, lower alkyl or by halogen, R ,
R and R represent hydrogen or methyl, R represents
hydrogen, methyl or ethyl, R represents hydrogen: lower
alkyl having from 1 to 7 carbon atoms optionally substituted
by hydroxy, lower alkoxy, mercapto, lower alkylmercapto
or by halogen; 4-aminobutyl; cycloalkyl or cycloalkyl-lower
alkyl wherein the cycloalkyl radical contains from 4 to
6 carbon atoms and the lower alkyl radical contains from
1 to 3 carbon atoms; phenyl or phenyl-lower alkyl having
from 1 to 3 carbon atoms in the lower alkyl radical and
optionally substituted by hydroxy, lower alkoxy or by
halogen; 4-imidazolylmethyl or 3-indolylmethyl, or R
and R5 together represent also alkylene having 3 or 4
carbon atoms, R represents hydrogen and one of the
radicals A and A represents a radical of the formula
O W
Il I
- T - Y - O - P - O - CH (II~
.
- OH Z
wherein T represents NH or O, Y represents optionàlly sub-
stituted lower alkylene or a radical of the formulae
~L~.7~7~3
- 16 -
~Y - COO - y (IIIa)
or
-Yl - CONH - Y (IIIe)
in which each of Y and Y independently of the other
represents lower alkylene that has from 1 to 7 carbon atoms
and is optionally substituted by hydroxy, lower alkoxy,
mercapto, methylthio, phenyl, 4-imidazolyl or by 3-indolyl,
W represents hydrogen and Z represents a 1,2-dihydroxyethyl
or 2-hydroxyethyl group in which at least one hydroxy group
is esterified by an aliphatic carboxylic acid having from
14 to 24 carbon atoms and optionally containing one or two
unsaturated bonds, or by a natural or synthetic mycolic
acid or etherified by an aliphatic alcohol having from 10
to 20 carbon atoms and optionally containing one or two
unsaturated bonds, or each of W and Z represPnts a hydroxy-
methyl group esterified by an aliphatic carboxylic acid
having from 16 to 22 carbon atoms and optionally containing
one or two unsaturated bonds, or by a natural or synthetic
mycolic acid or etherified by an aliphatic alcohol having
from 12 to 18 carbon atoms and optionally containing one
or two unsaturated bonds, and the other of the radicals
A and A is hydroxy, lower alkoxy, amino, lower alkyl-
amino or aminocarbonyl-lower alkylamino, and salts thereof.
Especially preferred combination partners are muramyl-
peptides of the formula I wherein X represents carbonyl,
R represents lower alkyl or phenyl, R , R and R
represent hydrogen, R and R represent hydrogen or
methyl, R5 represents hydrogen, lower alkyl having from
1 to 7 carbon atoms optionally substituted by phenyl, or
R and R together represent also trimethylene, wherein
A represents hydroxy, lower alkoxy, amino, lower alkyl-
amino or aminocarbonyl-lower alkylamino and A2 represents
~ ~.7~7~
- 17 -
- a radical of the formula
O W
- T - Y - O - P - O - 1H (II~
1H
.
wherein T represents NH or O, Y represents ethylene or a
radical of the formulae
Y - COO - Y or Y -CONH-Y
1 2 1 2
(IIIa) (IIIe)
wherein each of Yl and Y2 independently of the other
represents unsubstituted lower alkylene, W is hydrogen and
Z is a 1,2-dihydroxyethyl or 2-hydroxyethyl group in which
at least one hydroxy group is esterified by an alkanecarb-
oxylic acid having from 16 to 20 carbon atoms and optionally
containing one or two unsaturated bonds, or etherified by
an alkanol having from 12 to 18 carbon atoms and optionally
containing one or two unsaturated bonds, or each of W and
Z represents a hydroxymethyl group esterified by an alkane-
carboxylic acid having from 16 to 22 carbon atoms and
optionally containing one or two unsaturated bonds, or
etherified by an alkanol having from 12 to 18 carbon atoms
and optionaliy containing one or two unsaturated bonds,
and those compounds in which the meanings for A and A2
are interchanged and salts thereof.
Most especially preferred as combination partners
are muramylpeptides of the formula I wherein X represents
carbonyl, R represents lower alkyl or phenyl, R , R ,
R and R represent hydrogen, R represents hydrogen
- 18 -
or methyl, R represents lower alkyl having from 1 to
3 carbon atoms, A represents amino and A represents
a radical of the formula
Il (IV~
2 2 P CH2
¦ I d
~IO CH-O-R
CH2-0-Rd
.wherein R represents the acyl radical of an alkanecarb-
oxylic acid having from 16 to 20 carbon atoms and option-
ally containing one or two unsaturated bonds, and salts
thereof.
The novel phosphorylmuramylpeptides of the formula
I can be prepared according to various processe~ that are
known per se. They are prepared, for example, by conden-
sing, in a manner known per se, a compound of the formula V
CH20Rl o
~ \¦OR H (Vl
N - X ~ R
R3 - CH 2
COI - ~H - CON - CH - CH2CH - COA2
R4 R6
wherein X, R , R , R ~ R , R , R and R have
1 2 3 4 5 6 7
the meanings given above, R , R and R represent
hydrogen or a protecting group that can readily be split
off and one of the radicals A and A represents an
activated hydroxy group and the other of these radicals
is etherified hydroxy, amino, lower alkylamino or
aminocarbonyl-lower alkylamino, with a compound of the
formula (VI),
O W
Il I
H - T - Y - O - P ~ O - CH (VI~
I I
OH Z
wherein T, Y, W and Z have the meanings specified for the
end products, and splitting off the protecting groups that
are present.
The activated carboxylic acid group COA or COA
can be, for example, an acid anhydride, for example with
a carbonic acid lower alkyl ester, such as carbonic acid
ethyl ester or carbonic acid isobutyl ester, an acid azide,
an acid amide, such as an imidazolide, isoxazolide or an
activated ester. As activated esters special mention may
be made of: cyanomethyl ester, carboxymethyl ester,
p-nitrophenyl ester, methoxyethylthio ester, acetyl-
aminoethylthio ester, p-nitrophenyl ester, 2,4,5-
trichlorophenyl ester, N-hydroxysuccinimide ester, N-
hydroxyphthalimide ester, ~-hydroxyquinoline ester, N-
hydroxypiperidine ester. Active esters can also be obtained
optionally with a carbodiimide with the addition of N-
hydroxysuccinimide or a l-hydroxybenzotriazole or 3-hydroxy
4-oxo-3,4-dihydrobenzo[d]-1,2,3-triazine that is
unsubstituted or substituted, for example by halogen, methyl
or methoxy.
Preferred active esters are those with N-hydroxy-
succinimide or the C-substitution products thereof, such
as N-hydroxymethylsuccinimide or N-hydroxydimethylsuccin-
imide, or activation by reacting with a carbodiimide, such
~7~7~8
- 20 -
as carbodiimide itself or l-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide is preferred.
The starting materials used for this purpose are known
or may be prepared in a manner known per se.
If in the novel compounds of the formula I T
represents O, this reaction may also be carried out in such
a manner that the free acid is esterified with the alcohol
in the presence of an agent that splits off water, such
as a carbodiimide, for example dicyclohexylcarbodiimide,
and an amine, such as pyridine and dimethylaminopyridine,
or a trialkylamine, for example trimethylamlne. It is also
possible, however, to react the carboxylic acid, for example
in the form of a salt, such as a sodium or potassium salt,
with a reactive ester of the alcohol, for example an ester
with a strong inorganic or organic acid, such as a hydro-
halic acid, for example hydrochloric, hydrobromic or
hydriodic acid, or an organic sulphonic acid, such as p-
toluenesulphonic acid or methanesulphonic or ethanesul-
phonic acid. Furthermore, it is also possible to react
the alcohol, optionally in the form of a salt, for example
a sodium or potassium salt, with an activated carboxylic
acid.
Protecting groups that can readily be split off are
those known from peptide or sugar chemistry. For carboxy
groups special mention should be made of tertiary-butyl,
benzyl or benzhydryl, and for hydroxy groups special mention
should be made of acyl radicals, for example lower alkanoyl
radicals, such as acetyl, aroyl radicals, such as benzoyl,
and more especially radicals that are derived from carbonic
acid, such as benzyloxycarbonyl or lower alkoxycarbonyl,
or alkyl, especially tert-butyl, benzyl optionally substi-
tuted by nitro, lower alkoxy or by halogen, or tetrahydro-
pyranyl or triphenylmethyl optionally substituted by halo-
gen or by lower alkoxy, such as methoxy, or optionally
substituted alkylidene radicals that bond the oxygen atoms
in the 4-and 6-position of the glucose moiety. Such
~ ~ 7~i7~8
- 21 -
alkylidene radicals are especially a lower alkylidene
radical, more especially the ethylidene, isopropylidene
or propylidene radical, or alternatively an optionally
substituted benzylidene radical, preferably substituted
in the p-position.
These protecting groups can be split off in a manner
known per se. It is thus possible to remove them by acid
hydrolysis, in the case of benzyl or benzylidene radicals
also by hydrogenolysis, for example with hydrogen in the
presence of a noble metal catalyst, such as a palladium or
platinum catalyst.
The following Examples serve to illustrate the
invention. Temperatures are given in degrees Centigrade.
- 22 - ~ ~7~
Example 1 : Manufacture of 1000 capsules each containing
260 mg of active ingredients:
Composition
rifampicin 250 g
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-
alanyl-2-(1',2'-dipalmitoyl-sn-glycero-3'-
hydroxyphosphoryloxy)-ethylamide in admixture
with from 40 to 55 % of the sodium salt10 g
.. . .
t~lc 36 g
wheat starch 24 g
magnesium stearate 16 9
~. .
lactose 4 g
34~ g
Preparation
The pulverulent substances are forced through a sieve
having a mesh width of 0.6 mm and mixed ~horoughly. Gelatin
capsules are each filled with 340 mg of this mixture using a
capsule filling machine.
Example 2 : Manufacture of 1000 capsules each containing
105 mg of active substances:
Composition
rifampicin ~ 100 9
N-acetyldesmethylmuramyl-L-alanyl-D-
isoglutaminyl-2-(1',2'-dipalmitoyl-sn-
glycero-3'-hydroxyphosphoryloxy)-ethylamide
- 23 _ ~ 7~8
in admixture with from 40 to 55 % of the
sodium salt 5 9
ethylcellulose 3 9
stearic acid 3 g
111 9
Preparation
The ethylcellulose and the stearic acid are dissolved
in 120 ml of methylene chloride, the antibiotic is added and
the composition is forced through a sieve of 0.6 mm mesh
width at a temperature of approximately 40 C, the
methylene chloride evaporating off. Gelatin capsules having
a capacity of 0.5 ml are filled with 156 mg of the resulting
granulate using a capsule filling machine.
Example 3 Manufacture of an animal feedstuff containing
0.005 % of active substances.
Premix
rifampicin or chlorotetracycline 30 g
N-benzoyldesmethylmuramyl-L-alanyl-D-
isoglutaminyl-2-(1',2'-dipalmitoyl-sn-
glycero-3l-hydroxyphosphoryloxy)-
ethylamide in admixture with from 40 to
55 ~ of the sodium salt 10 g
sugar powder 50 g
soybean feed (extracted with solvents~275 g
365 9
~L~7~7~3
- 24 -
Additives
corn meal 500.0 kg
soybean flour, 44 % protein 300.0 kg
alfalfa flour 13.5 kg
dicalcium phosphate 18.0 kg
calcium carbonate (ground) 4.5 kg
salt 2.3 kg
fish meal, 60 ~ protein 18.0 kg
stabilised fat 27.0 kg
dry whey residue 18.0 kg
manganese sulfate 0.2 kg
zinc oxide 1.3 kg
d,l-methionine 0.7 kg
vitamin premix 4.5 kg
908.0 kg
The vitamin premix contains in 4.5 kg:
16,000,000 I.U. of vit. A, l,000,000 I.U. of vit. D ,
5,000 I.U. of vit. E acetate, 6 g of vit. K , 6 mg of
vit. B , 3 g of riboflavin, 30 g of niacin, 5 g of
calcium pantothenate and lO0 g of ethoxyquin (1,2-dihydro-6-
ethoxy-2,2,4-trimethylquinoline) and corn meal to make up
4.5 kg.
Method of preparation
The active substances and sugar are thoroughly mixed,
the mixture is forced through a sieve of 0.6 mm mesh width
and then mixed with the soybean flour. The premix is then t
added to the animal feedstuff in an amount corresponding
to the desired final concentration and the whole is then
homogenised in a horizontal drum mixer.
Example 4 4
Laboratory white mice are infected i.p. with 2 x lO
cfu (colony forming units) of Klebsiella pneumoniae, 0.1 x
~7~
- 25 -
4 4
cfu of Pasteurella multocida or 40 x 10 cfu of
Salm nella typhimurium in salt solution so that 90 to 100 %
of the untreated control animals die within 4B hours.
Immediately after infection and 3 hours after infection,
groups of 10 mice are treated with cefroxadin, both alone
(without muramylpeptide) and in combination with 10 mg/kg
of N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanyl-2-
(1',2'-dipalmitoyl-sn-glycero-3'-hydroxyphosphoryloxy)-
ethylamide, these being administered twice subcutaneously
in the form of aqueous solutions or suspensions. A count
of the surviving animals was made 4 and 10 days after infec-
tion. The ED50 values [mg/kg] for the antibiotic were
lower ~y a multiple when administered in combination with
the muramylpeptide.
Example 5
In a manner analogous to that described in Examples 1
and 2 combination preparations are obtained which in
addition to the adjuncts and carriers contain the following
active ingredients tfrom 40 to 55 % of muramylpeptides
in the form of sodium salts) in the specified amounts per
capsule:
a) 500 my of cephalexin and 5 mg of N-acetylmuramyl-L-
alanyl-D-isoglutaminyl-2-(1~,2'-dihexadecyl-rac-glycero-
3'-hydroxyphosphoryloxy)-ethylamide,
b) 750 mg sf ampicillin and 40 m~ of N-benzoylnormuramyl-
( ~methylalanyl)-D-isoglutaminyl-2-(1',2'-dioleoyl-sn-
glycero-2'-hydroxyphosphoryloxy)-ethylamide,
c) 100 mg of doxycycline and 15 mg of N-acetylmuramyl-
L-alanyl-D-glutamyl-2-(1'-hexadecyl-rac-glycero-3'-hydroxy-
phosphoryloxy)-ethylamide,
d) 300 mg of methacycline and 15 mg of N-benzoyldes-
methylmuramyl-L-alanyl-D-glutamyl-r-methyl ester--2-(1',2'-
-
- 26 - ~ ~7~7~
distearoyl-sn-glycero-3'-hydroxyphosphoryloxy)-ethylamide,
e) 250 mg of erythromycin estolate and 30 mg of N-
propionyldesmethylmuramyl-L--aminobutyryl-D-isoglutaminyl-
Y-oxymethylcarbonyl-2-(l~3~-di[~3uR)-hydroxy-(2ns)-
palmitoylaminooctadecyloxy)-2'-hydroxyphosphoryloxy]-
ethylamide.
xample 6 : Manufacture of a sterile dry substance for
injection (lyophilisation).
500 mg of cefsulodin and 10 mg of N-acetylmuramyl-
N-methyl-L-alanyl-N,N'-dimethylglutaminyl-2-(1',2'-
dipalmitoyl-sn-~lycero-3'-hydroxyphosphoryloxy)-ethylamide
in admixture with from 40 to 55 ~ of the sodium salt are
dissolved, while stirring, in 5 ml of water. The solution
is sterile-filtered and, under aseptic conditions, is
introduced into a sterile ampoule (phial) and lyophi-
lised. The dry substance can be used for parenteral admini-
stration after dissolving in water or physiological
solutions.
Example 7 : Manufacture of a sterile dry substance for
injection (powder filling).
500 mg of sterile cefsulodin and 15 mg of sterile
N-acetyldesmethylmuramyl-L-alanyl-D-isoglutaminyl-2-(1'-
palmitoyl-2'-oleoyl-sn-glycero-3'-hydroxyphosphoryloxy)-
ethylamide (in admixture with from 40 to 55 % of the sodium
salt) are, under asep`tic conditions, homogeneously mixed and
introduced into an ampoule. The dry substance can be
used for parental administration after dissolving in water
or physiological solutions.
Example 8 : In a manner analogous to that described in
Examples 6 and 7 sterile dry substance mixtures for
injection are obtained that contain the following amounts
of effective constituents (muramylpeptides in admixture
~7~;7~3
- 27 -
with from 40 to 55 % of their sodium salt):
a) 1000 mg of oxacillin (sodium salt) and 20 mg of N-
acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanyl-2-t3'-
palmitoyl-rac-glycero-l'-hydroxyphosphoryloxy)-ethylamide,
b) 500 mg of cefazolin and 10 mg of N-acetyldesmethyl-
muramyl-L-prolyl-D-glutaminylglycyl-2-(1',2'-dimyristoyl-
sn-glycero-3'-hydroxyphosphoryloxy)-ethylamide,
c~ 80 mg of gentamycin and 1 mg of N-4-(methylbenzoyl)-
desmethylmuramyl-L-valyl-D-glutaminyl-2-(1',2'-dilauroyl-
sn-glycero~3'~hydroxyphosphoryloxy)-ethylamide,
d) 200 mg of doxycycline and 0O2 mg of N-propionyldes-
methylmuramyl-L-seryl-D-isoglutaminyl-2-(1',2'-dilinoloyl-
sn-glycero-3'-hydroxyphosphoryloxy)-ethylamide,
e) 50 mg of amphotericin B, 41 mg of sodium desoxycholate
and 0.05 mg of N-acetylmuramyl-L-lysyl-D-isoglutaminyl-2-
(1',2'-distearoyl-sn-glycerohydroxyphosphoryloxy)-ethylamide
for the manufacture of the stock solution,
f) 500 mg of vancomycin and 0.01 mg of N-4-methoxybenzoyl-
normuramyl-L-threonyl-D-isoglutaminyl-2-(1',2'-dioleoyl-sn-
glycero-3'-hydroxyphosphoryloxy)-ethylamide.
The muramylpeptides of the formula I serving as the
starting materials for the manufacture of the combination
preparations according to the invention are manufactured,
for example, in a manner analogous to that described in thet
following Examples.
The compounds of the formula I cannot be characterised
by a melting point nor is spectroscopic data, such as NMR
and IR spectra, suitable for accurate characterisation.
The data provided by R values is also unsuitable
for precise characterisation because of the dominant nature
of the lipid constituents.
;7~
- 28 -
Since, however, the structure of the starting
materials is known accurately (cf. German Offenlegungs-
scheift 26 55 500; the phospholipid constituent used in
each case is commercially available) and since the linking
of phospholipid and muramylpeptide is clear, the sequence
of the units in the end product and the structure of the
end product is thus also clearly determined.
Example 9
A solution of 2 mmol of N-acetylmuramyl-L-alanyl-D-
isoglutamine-N-hydroxysuccinimide ester in 6.5 ml of
dimethylacetamide is added dropwise to a solution of 1.4
mmol of 1,2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine
and 2.5 mmol of triethylamine in 25 ml of a mixture of
chloroform, methanol and water in the ratio 65:25:4. After
stirring for 18 hours at 20 C, the solution is concen-
trated under reduced pressure to approximately 15 ml pro-
ducing an emulsion. This emulsion is diluted with 200 ml
of water and freeze-dried. The residue is suspended in
30 ml of water and dialysed first of all against water,
then against a 0.1 molar solution of sodium phosphate
buffer p~ 7 and subsequently extensively against water.
The internal dialysate, which contains the desired product
and residues of dicyclohexylurea, is diluted with water
to make up 150 ml and is centrifuged for one hour at
30 000 g. The supernatant liquid, which contains a mixture
of pure N-acetylmuramyl-L-alanyl-D-isoglutaminyl-2-(1',2'-
dipalmitoyl-sn-glycero-3'-phosphoryl)-ethylamide with from
40 to 55 mole % of its sodium salt, is freeze-dried. In
a thin-layer chromatogram on silica gel the compound
exhibits the following R values: 0.31 (in chloroform/
methanol/water, 65:25:4) and 0.64 (in chloroform/methanol/
acetic acid/water, 25:15:4:2).
The novel compound is characterised analytically in
that the units N-acetylmuraminic acid, palmitic acid, phos-
phate, L-alanine and D-glutaminic acid are determined
~.~ 7~i7~3
- 29 -
quantitatively: N-acetylmuraminic acid is determined spec-
trophometrically with the aid of the Morgan Elson reaction
according to the modification of J. M. Ghuysen et al [in
"Methods in Enzymologya 8, 629 (1966)].
Phosphate is determined quantitatively according to
Lowry et al [J. Biol. Chem. 207, 1 ll954)3.
Palmitic acid and the amino acids are determined by
gas chromatography in a total hydrolysate (6N HCl, 24 hours,
110 C) or quantitatively with the aid of an amino acid
analyser using pentadecanoic acid or norleucine as internal
standards.
The molar ratios found, calculated on phosphate, are
as follows:
PO ~': N-acetylmuraminic acid:L-alanine:D-glutaminic
acid:palmitic acid = 1:~.92:0.91:0.95:2.18.
The N-acetylmuramyl-L-alanyl-D-isoglutamine-N-
hydroxysuccinimide ester, which is used as starting
material, may be prepared, for example, as follows:
2 mmol of N acetylmuramyl-L-alanyl-D-isoglutamine,
2.2 mmol of N-hydroxysuccinimide and 2.2 mmol of dicyclo-
hexylcarbodiimide are dissolved in 6 5 ml of dimethylacet-
amide and stirred for 18 hours at 20 C. The precipitated
dicyclohexylurea is separated off and the solution is used
directly for the condensation with the phospholipid.~
The 1,2-dipalmitoyl-sn-glycero-3-phosphoryle hanol-
amine, which is used as starting material, is a commercially
available synthetic preparation.
Example 10
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanyl-
2-(1',2'-dipalmitoyl-sn-glycero-3'-phosphoryl)-ethylamide
is obtained in a manner analogous to that of Example 1 using
1,2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine and
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-N-
hydroxysuccinimide ester as starting materials. R value
_ 30 _ ~ ~757~
in a thin-layer chromatogram on silica gel: 0.3 (in the
system chloroform/methanol/water, 65:25:4).
