Note: Descriptions are shown in the official language in which they were submitted.
li338949
-
This Application is a division of Canadian Patent
Application Serial No. 592,219, filed February 27,
1989.
The invention is related to nikkomycin
derivatives, antimycotic compositions containing
nikkomycin derivatives, antimycotic compositions
comprising fungicidally effective amounts of a
nikkomycin derivative and an azole antimycotic, the
preparation of such compositions and the methods of
treating infections of fungi by administering
therapeutically effective amounts of such
compositions.
Compounds inhibitory to the synthesis of fungal
cell wall material (synthase inhibitors) have been
reported recently to have demonstrable effects against
fungi of agricultural importance (U.S. patents
4,315,922 and 4,158,608; see also U.S. patents
4,585,761 and 4,552,954 for descriptions of the
preparation and purification of such compounds). The
agents mentioned in the cited patents, nikkomycins,
together with similar agents known as polyoxins, are
known to act by interfering with the synthesis of
chitin in the cell walls of fungi. Because fungi of
medical importance to humans also have varying amounts
of chitin in their cell walls, experiments have been
conducted to determine if the chitin synthase
inhibitors are capable of inhibiting the growth of
such fungi (Hector and Pappagianis, J. Bacteriol.
154:485 - 498, 1983, and Hector and Braun,
Antimicrobial Agents Chemother, 29:389 - 394, (1986).
In earlier work, certain fungi such as Candida
albicans were reported to be insensitive to chitin
synthase inhibitors ~see Naider et al, Antimicrobial
Agents Chemother, 24:787 - 796, 1983). Subsequently,
C. albicans was found to be more sensitive to
1 338949 -
_ - 2 -
nikkomycins than polyoxins (see Yadan et al, J.
Bacteriol, 160:884 - 888, 1984).
Quite surprisingly, it was now found that
nikkomycin derivatives in combination with
antimycotically active azoles are efficacious in
treating fungal infections in human and non-human
animals, especially by parenteral but also oral
application and administration.
As shown in synergy studies the achieved
antifungal effect is also a synergistic effect based
on combining the nikkomycin derivatives with
antimycotic azoles.
Accordingly, the invention is related to new
nikkomycin derivatives and antimycotic compositions
comprising a fungicidally effective amount of a
nikkomycin derivative and an azole antimycotic.
The nikkomycin derivatives of the invention are
represented by the general formulae (N)
HOOC
R R N I Rn (N)
yw ~0~
HO OH
wherein Rn represents
o
H
o
a) Rw denotes H or -CH2-X, wherein X is H, alkyl,
preferably Cl-C6-alkyl, aryl, preferably C6-Clg-
aryl, aralkyl, preferablY C6-C12-aryl-Cl C6
1 338949
- 2a -
alkyl or any other saturated or unsaturated
organic radical and Rv denotes -CH2-X, wherein X
has the aforementioned meaning:
b) Rw denotes H and
Rv denotes -CH2-CH-X', wherein X' is any
NH2
saturated or unsaturated organic radical,
especially a radical derived from a natural amino
acid;
c) Rw denotes H and
Ry denotes a radical -C-X or -C-CH-X'
O O NH2
wherein X has the meaning mentioned under a) and
b) hereinbefore and
wherein X' has the meaning of the natural amino
acids Gly, Val, Leu Ile, Ser, Asp, Asn, Glu, His,
Gys, Met and Phe or
~CH31
-CH3, -CH2-C~H 3 (D-Configuration on -NH2)
CH3
-C3H7
C6 13~ ~ ~ ~ X , -CH2-0 ~ X",
-CH2-C~X'', -CH2-CH2--~X , ~H243
~3 , ~ , ~N O, -CH2-C-O-CH~,
-CH2-0-CH2~) , -(CH2)2-0-~) CH3
1 338949
- 2b -
wherein
-Xll represents -H,-Cl, -F, -NO2, -NH2, -OCH3, CH3
-Xlll represents -Cl, -F, -NO2, -NH2, -OCH3, -CH3,
-Xllll represents -H, -Cl, -F, -NO2, -NH2, -OCH3, -CH3,
-0-CH2 ~ , -0CH2 ~ , -0-CH2- ~ -F,
-0-CH2 ~ , 0 2 ~ ~I
d) Rw denotes H and
Ry has the meaning of
:) CH 3 OH
-C-CH-CH - CH~H (configuration S,S,S)
I
NRn,RV.
wherein
al) Rn~ denotes H or -CH2-X and Rv, denotes -CH2-
X, wherein X has the meaning mentioned under a)
hereinabove;
bl) Rn~ represents H and Rv, means -CH2-CH-X
NH2
wherein X' has the meaning mentioned under b)
hereinabove;
c') Rn~ denotes H and Rv, represents -C-X and
O
-C-CH-X' wherein X and X' have the meaning
O NH2
given under a) and b) hereinabove
X--
1 338949
- 2c -
or
HOOC
R R ~ ~ Rn
y w l~
~ (N')
HO OH
wherein Rn represents
C~O
H
o
a) Rw denotes H or -CH2-X, wherein X is H, alkyl,
preferably Cl-C6-alkyl, aryl, preferably C6-C18-
aryl, aralkyl, preferably C6-C12-aryl-Cl-C6-alkyl
or any other saturated or unsaturated organic
radical and Ry denotes -CH2-X, wherein X has the
aforementioned meaning:
b) Rw denotes H and
Ry denotes -CH2-CH-X', wherein X' is any
NH2
saturated or unsaturated organic radical,
especially a radical derived from a natural amino
acid;
c) Rw denotes H and
Ry denotes a radical -C-X or -C-CH-X'
O O NH2
wherein X and X' have the meaning mentioned
under a) and b) hereinbefore with the proviso
that X' has not the meaning
1 338949
- 2d -
-CH-CH2 ~ or
-CH-C H -~
OH CH3
d) Rw denotes H and
Ry has the meaning of
O CH3 OH
-C-CH-CH -CH ~ H (configuration S,S,S)
~Rn'Rv~
wherein
a') Rn~ denotes H or -CH2-X and Rv, denotes -CH2-X,
wherein X has the meaning mentioned under a)
hereinabove;
b') Rn~ represents H and Rv, means -CH2-CH-X'
NH2
wherein X' has the meaning mentioned under b)
hereinabovei
c') Rn~ denotes H and Rv, represents -\C-X and
O
-C-CH-X' wherein X and X' have the meaning
1~ ~
O NH2
given under a) and b) hereinabove
X"
~,
1 338949
_ - 3 -
or
HOOC
R R N I Rn
y w ~ (N~)
HO OH
wherein
Rn represents
CH20H
HN ~
~ and
O
a) Rw denotes H or -CH2-X, wherein X is H, alkyl,
preferably Cl-C6-alkyl, aryl; preferably C6-Clg-
aryl, aralkyl, preferably C6-C12-aryl-Cl-C6-alkyl
or any other saturated or unsaturated organic
radical and Ry denotes -CH2-X, wherein X has the
aforementioned meaning;
b) Rw denotes H and
Ry denotes -CH2-CH-X', wherein X' is any saturated
NH2
or unsaturated organic radical, especially a
radical derived from a natural amino acid;
c) Rw denotes H and
R denotes a radical -C-X or -C-CH-X'
Y
O O NH2
wherein X and X' have the meaning mentioned under
a) and b) hereinbefore;
d) Rw denotes H and
Ry has the meaning of
1 338949
_ - 4 -
CH3 OH
O l l
-C-CH-CH -CH-~ ~ H (configuration S,S,S)
I N==~
NRn.RV.
wherein
a') Rn~ and Rv, denote H;
b') Rn~ denotes H or -CH2-X and Rv, denotes -CH2-
X, wherein X has the meaning mentioned under
a) hereinbefore;
c') Rn~ represents H and Rv, means -CH2-CH-X'
NH2
wherein X' has the meaning mentioned under b)
hereinabove;
d') Rn~ denotes H and Rv, represents -C-X and
-C-CH-X' wherein X and X' have the meaning
l\ l
O NH2
given under a) and b) hereinabove.
If X' is an organic radical than preferably a saturated
or unsaturated aliphatic radical, such as Cl-C10-alkyl,
alkenyl or alkinyl or a substituted or unsubstituted
aromatic radical with 6 to 12 C-atoms or a heterocyclic
radical, especially of the group consisting of
,~ Y = N H, O, S
Y = NH, O, S
,~N
J~ N~J
1 338949
_ - 4a -
~r~\ dnd ~ `N~S
A preferred object of the present invention are
antimycotic compositions containing nikkomycin
derivatives and azole antimycotics more specifically
described in GB-PS 1,351,542, CA-PS 225,504, CA-AS
946,391, AU-PS 542,110, AU-PS 551,411, US-PS 4,301,166,
US-PS 4,381,306, US-PS 4,246,274, US-PS 4,238,498, US-PS
4,207,328, US-PS 3,968,229 and DE-OS 3,242,249.
1 33~949
Ano~her preferred embodimen~ of ~he present inven-
'ion are an~imyco~ic composi~ions con~aining nikkomycin
derivatives and azole anLimyco~ics described in ~he
following paragraphs.
a) Diazolylalkyl-carbinols of ~he general formula
OH X
I ,B~
R-C---C-N ¦ (I)
I \~
CH2 Y
b~A
~ 11
~ '
in which
A represen~s a ni~rogen a~om or ~he CH group,
B represents a ni~rogen atom or the CH group,
X represen~s hydrogen or alkyl,
r represen~s alkyl, or alkenyl, alkinyl or
optionally substituted benzyl, if X represents
hydrogen, and
R represen~s optionally substituted phenyl or the
grouping
A1k
Rl -C -
Alk2
wherein
Alk1 represents alkyl and
Alk represents alkyl, or
Le A 25 522
1 338949
- 6 -
Alkl and Alk2 together represent a cycloaliphatic
ring, and
Rl represents alkyl, alkenyl or in each case
optionally substituted phenyl, phenylalkyl,
phenoxy, phenylthio, phenoxyalkyl, phenylthioalkyl,
benzyloxy or benzylthio,
and physiologically acceptable acid addition salts
thereof.
The compounds of the formula (I) sometimes have two
asymmetric carbon atoms. In this case, they can exist
in two geometric isomer forms.
The substituted diazolylalkyl-carbinols of the
formula (I) are obtained by a process in which
azolyloxiranes of the formula
X
R----C----C-N ~ (II)
~ I ~
CH2-- Y
in which
B, R, X and Y have the aforementioned meaning, are
reacted with azoles of the formula
,A==1
H-N I (III)
in which
A has the abovementioned meaning,
in the presence of a diluent and, if appropriate, in the
presence of a base.
1 338949
_ - 7 -
Preferably, in formula (I)
A represents a nitrogen atom or the CH group,
B represents a nitrogen atom or the CH group,
X represents hydrogen or straight-chain or branched
alkyl with 1 to 6 carbon atoms,
Y represents straight-chain or branched alkyl with
1 to 6 carbon atoms; or, if X represents hydrogen,
also straight-chain or branched alkenyl or alkinyl
with in each case 3 to 6 carbon atoms or benzyl
which is optionally mono-, di- or tri-substituted
in the phenyl art by identical or different
substituents, substituents which may be mentioned
being; halogen, alkyl with 1 to 4 carbon atoms,
alkoxy and alkylthio with in each case 1 to 4
carbon atoms, halogenoalkyl, halogenoalkoxy and
halogenoalkylthio with in each case 1 or 2 carbon
atoms and 1 to 5 identical or different halogen
atoms, such as, preferably, fluorine and chlorine
atoms, nitro- and cyano; and
R represents phenyl which is optionally mono-, di-
or tri-substituted by identical or different
substituents, preferred substituents which may be
mentioned being; halogen, alkyl with 1 to 4 carbon
atoms, alkoxy and alkylthio with in each case 1 to
4 carbon atoms, halogenoalkyl, halogenoalkoxy and
halogenoalkylthio with in each case 1 or 2 carbon
atoms and 1 to 5 identical or different halogen
atoms, such as, preferably, fluorine and chlorine
atoms, nitro, cyano, hydroxyl, hydroxycarbonyl,
alkoxycarbonyl with 1 to 4 carbon atoms in the
. . - 8 ~ 1 338q49
-
alkyl part, hydroximinoalkyl wi~h 1 to 4 carbon
atoms, alkoximinoalkyl with 1 to 4 carbon atoms in
each alkyl part and phenyl, phenoxy, benzyl and
benzyloxy, each of which is optionally substituted
by halogen andlor alkyl wi~h 1 or 2 carbon atoms;
or
R preferably represents the grouping
Alkl
Rl _f
Alk2
wherein
Alk1 represents s~raight-chain or branched alkyl
wi~h 1 to 4 carbon a~oms; and
Alk2 represents straight-chain or branched alkyl
wi~h 1 ~o 4 carbon atoms; or
Alk1 and Alk2, ~oge~her with the carbon atom to
which ~hey are bonded, represent a 3-membered to
7-membered cycloaliphatic rin~, and
R1 represents straight-chain or branched alkyl with
1 to 6 carbon a~oms, alkenyl wi~h 2 to 4 carbon
a~oms, or phenyl, phenylalkyl with 1 to 4 carbon
atoms in the alkyl part, phenoxy, phenyl~hio,
phenoxyalkyl with 1 to 4 carbon atoms in the alkyl
part, phenyl, ~hioalkyl with 1 to 4 carbon a~oms
in the alkyl part, benzyloxy ~. ben~ylthio, each
of which is optionally mono-, di- or tri-subs~i-
tuted in the phenyl part by identical or diÇferent
Le A 25 522
l 338q49
-
substituents, preferred possible substituents being
the substituents on phenyl already mentioned for R.
Particularly preferred compounds of the formula (I)
are those
in which
A represents a nitrogen atom or the CH group,
B represent a nitrogen atom of the CH group,
X represents hydrogen or straight-chain or branched
alkyl with 1 to 4 carbon atoms,
Y represents straight-chain or branched alkyl with
1 to 4 carbon atoms; or, if X represents hydrogen,
also allyl, methallyl, propargyl, methylpropargyl
or benzyl which is optionally mono- or di-
substituted in the phenyl part by identical or
different substituents, substituents which may be
mentioned being: fluorine, chlorine, bromine,
methyl, isopropyl, tert.-butyl, methoxy,
methylthio, trifluoromethyl, trifluoromethoxy,
trifluoromethylthio, nitro and cyano;
R represents phenyl which is optionally mono- or
di-substituted by identical or different
substituents, substituents which may be mentioned
being: fluorine, chlorine, bromine, methyl,
isopropyl, tert.-butyl, methoxy, methylthio,
trifluoromethyl, trifluoromethoxy,
trifluoromethylthio, nitro, cyano, hydroxyl,
hydroxycarbonyl, methoxycarbonyl, ethoxycarbonyl,
hydroximinomethyl, l-hydroximinoethyl,
methoximinomethyl, l-methoximinoethyl and phenyl,
phenoxy, benzyl and benzyloxy, each of which is
optionally substituted by fluorine, chlorine or
methyl; or
R represents the grouping
y
1 338949
-- 10 --
-
wherein
Alkl represents methyl or ethyl; and
Alk2 represents methyl or ethyl; or
Alkl and Alk2, together with the carbon atom to
which they are bonded, represent cyclobutyl,
cyclopentyl or cyclohexyl; and
Rl represents methyl, ethyl, n-propyl, i-propyl, n-
butyl, neopentyl, or phenyl, benzyl, phenethyl,
phenoxy, phenylthio, phenoxymethyl, phenoxyethyl,
phenylthiomethyl, phenylthioethyl, benzyloxy or
benzylthio, each of which is optionally mono- or
di-substituted in the phenyl part by identical or
different substituents, possible substituents being
the substituents on phenyl which have already been
mentioned for R.
Addition products of acids and those substituted
diazolylalkyl-carbinols of the formula (I) in which the
substituents A, B, X, Y and R have the meanings which
have already been mentioned as preferred for these
substituents are also preferred compounds according to
the invention.
~,
t-338949
-
Preferred acids which can be added on azoles
men~ioned under a), b), c), d) and e) include hydrogen
halide acids, such as, for example, hydrochloric acid
and hydro-bromic acid, in par~icular hydrochloric acid,
and furthermore phosphoric acid, nitric acid, monofunc-
tional and bifunc~ional carboxylic acids and hydroxy-
carboxylic acids, such as, for example acetic acid,
maleic acid, succinic acid, fumaric acid, ~artaric acid,
ci~ric acid, salicyclic acid, sorbic acid and lac~ic
acid, and sulphonic acids, such as o-toluenesulphonic
acid and 1,5-naph~halenedisulphonic acid.
b) SubsLi~u~ed 1,3-diazolyl-2-propanols of ~he general
formula
Alk1 OH X
~X--I
20 R-C C---C-N ~ (I )
Alk2 CH2 Y
~Y
in which
Alk1 represen~s s~raight-chain or branched alkyl
and
Alk2 represen~s s~raight-chain or branched alkyl,
or
3~ Alk1 and Alk2 ~ogeti~ei represen~ a cycloalipha~ic
ring,
X represen~s a ni~rogen atom or ~he CH group,
Y represents a ni~ro,en ato.n or ~he CH group and
Le A 25 522
~ - 12 - 13389~9
R represen~s in each case op~ionally subs~i~u~ed
S pheny~l, phenylalkyl, phenoxy, phenyl~hio, phenoxy-
alkyl, phenyl~hioalkyl, benzyloxy or benzyl~hio,
and physiologically accep~able acid addi~ion sal~s
~hereof.
The subs~i~u~ed 1,3-diazolyl-2-propanols of ~he
formula (I') are ob~ained by a process in which 2-
azolylme~hyl-oxiranes of ~he formula
AIkl
X--I
R-C R C CH2 N I (II')
Alk2 CH2--O \=N
in which
Alk1, Alk2, R and X have ~he abovemen~ioned
: meaning,
are reacted wi~h azoles of the formula
,A--I
H-N I (III')
\9~ .
in which
Y has ~he abovemen~ioned meaning,
in ~he presence of a diluent and, if appropria~e, in ~he
presence of a base.
If appropria~e, an acid can ~hen be added onto ~he
compounds of ~he formula ~ hus ob~ained.
Moreover, ~he compounds of ~he general formula (I')
in which R represen~s in each case op~ionally subs~i-
~u~ed phenyl~hio, phenyl~hioalkyl or benzyl~hio can be
Le A 25 522
- 13 _ 1 33 8 9 4 ~
_
oxidised to the corresponding SO or SO2 derivatives in
the customary manner.
Formula (I') provides a general definition of
substituted l,3-diazolyl-2-propanols according to this
section b) of the invention. Preferably, in this
formula.
Alkl represents straight-chain or branched alkyl
with 1 to 4 carbon atoms; and
Alk2 represents straight-chain or branched alkyl
with 1 to 4 carbon atoms; or
Alkl and Alk2 together represent a 3-membered to 7-
membered cycloaliphatic ring,
X represents a nitrogen atom or the CH group;
Y represents a nitrogen atom or the CH group; and
R represents phenyl, phenylalkyl with 1 to 4 carbon
atoms in the alkyl part, phenoxy, phenylthio,
phenoxyalkyl with 1 to 4 carbon atoms in the alkyl
part, phenylthioalkyl with 1 to 4 carbon atoms in
the alkyl part, benzyloxy or benzylthio, each of
which is optionally mono-, di- or tri-substituted in
the phenyl part by identical or different
substituents, preferred substituents which may be
mentioned being: halogen, alkyl with 1 to 4 carbon
atoms, alkoxy and alkylthio with in each case 1 to 4
carbon atoms, halogenoalkyl, halogenalkoxy and
halogenoalkylthio with in each case 1 or 2 carbon
atoms and 1 to 5 identical or different halogen
atoms; such as, preferably, fluorine and chlorine
atoms, nitro, cyano, hydroxyl, hydroxycarbonyl,
alkoxycarbonyl with 1 to 4 carbon atoms in the
1 338949
_ - 14 -
alkyl part, alkoximinoalkyl with 1 to 4 carbon atoms
in each alkyl part, and phenyl, phenoxy, benzyl and
benzyloxy, each of which is optionally substituted
by halogen and/or alkyl with 1 or 2 carbon atoms.
Particularly preferred compounds of the formula (I') are
those
in which
Alkl represents methyl or ethyl; and
Alk2 represents methyl or ethyl; or
Alkl and Alk2, together with the carbon atom to
which they are bonded, represent cyclobutyl,
cyclopentyl or cyclohexyl,
X represents a nitrogen atom or the CH group;
Y represents a nitrogen atom or the CH group; and
R represents phenyl, benzyl, phenethyl, phenoxy,
phenylthio, phenoxymethyl, phenoxyethyl,
phenylthiomethyl, phenylthioethyl, benzyloxy or
benzylthio, each of which is optionally mono- or
di-substituted in the phenyl part by identical or
different substituents, substituents which may be
mentioned being: fluorine, chlorine, bromine,
methyl, isopropyl, tert.-butyl, methoxy,
methylthio, trifluoromethyl, trifluoromethoxy,
trifluoromethylthio, nitro, cyano, hydroxyl,
hydroxycarbonyl, methoxycarbonyl, ethoxycarbonyl,
hydroximinoethyl, l-hydroximinoethyl,
methoximinomethyl, l-methoximinoethyl, and phenyl,
phenoxy, benzyl and benzyloxy, each of which is
optionally substituted by fluorine, chlorine or
methyl.
1 338949
- 15 -
Addition products of acids and those substituted
1,3-diazolyl-2-propanols of the formula (I') in which
the substituents Alkl, Alk2, X, Y and R have the
meanings which have already been mentioned as preferred
for these substituents are also preferred compounds
according to the invention.
c) Substituents azolylcyclopropyl-azolylmethyl-
carbinol derivatives of the formula (I")
OR
I \ /',Y--
Ar-C C---N ¦ t I" )
C~2
~ X
in which
Ar represents optionally substituted aryl or
optionally substituted heteroaryl,
R represents hydrogen, alkyl, alkenyl, alkinyl,
trialkylsilyl, optionally substituted phenylalkyl
or the acyl radical,
X represents a nitrogen atom or the CH group, and
Y represents a nitrogen atom or the CH group, and
their acid addition salts have good antimicrobial, in
particular antimycotic, properties when used according
to this invention.
The substituted azolylcyclopropyl-
azolylmethylcarbinol derivatives of the formula (I")
which are to be used according to the invention show a
good spectrum of action in certain areas of indication.
1 338949
- 16 -
The substituted azolylcyclopropyl-azolylmethyl-
carbinol derivatives are generally defined by formula
(I"). In this formula,
Ar preferably represents phenyl which optionally
has one or several, identical or different,
substituents, the substituents which may be
mentioned as being preferred being: halogen,
alkyl, alkoxy and alkylthio each having 1 to 4
carbon atoms; halogenoalkyl, halogenoalkoxy and
halogenoalkylthio each having 1 to 2 carbon atoms
and 1 to 5 identical or different halogen atoms,
such as fluorine and chlorine atoms; as well as
phenyl or phenoxy each of which is optionally
substituted by alkyl having 1 or 2 carbon atoms
and/or halogen; and furthermore represents naphthyl
and a 5- to 6- membered heteroaromatic which
optionally has one or several identical or
different, substituents and nitrogen, oxygen and/or
sulphur as the heteroatoms, the suitable
substituents which are preferred being the
abovementioned phenyl substituents;
R preferably represents hydrogen, straight-chain or
branched alkyl having 1 to 4 carbon atoms,
straight-chain or branched alkenyl and alkinyl each
having 2 to 4 carbon atoms, trialkylsilyl having 1
to 4 carbon atoms in each alkyl moiety, alkyl-
carbonyl having 1 to 4 carbon atoms in the alkyl
moiety, and represents phenylalkyl which optionally
has one or several, identical or different,
substituents, with 1 to 2 carbon atoms in the alkyl
moiety, the suitable substituents which are
t 338949
- 17 -
preferring being the phenyl substituents already
mentioned for Ar; and
X and Y represent the meanings given in the
definition of the invention.
Particularly preferred compounds of the formula
(I") are those in which
Ar represents phenyl which optionally has one to
three, in particular one or two, identical or
different substituents, the substituents which may
be mentioned being: fluorine, chlorine, methyl,
isopropyl, tert.-butyl, methoxy, methylthio,
trifluoromethyl, trifluoromethoxy, trifluoro-
methylthio, and phenyl or phenoxy each of which is
optionally substituted or fluorine, chlorine and/or
methyl; furthermore represents napthyl and
represents furyl, thienyl, pyridinyl or
pyrimidinyl, each of which optionally has one or
two, identical or different, substituents suitable
substituents being the abovementioned phenyl
substituents;
Rl represents hydrogen, methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, allyl, propargyl,
trimethylsilyl, methylcarbonyl, ethylcarbonyl, n-
propylcarbonyl, isopropylcarbonyl, n-butylcarbonyl,
isobutylcarbonyl, and represents benzyl which
optionally has one to three, in particular one or
two, identical or different substituents, suitable
substituents which are preferred being the phenyl
substituents already mentioned for Ar; and
X and Y represent the meanings given in the
definition of the invention.
1 338949
- 18 -
-
Preferred compounds according to this section of
the invention are also addition products of acids and
those substituted azolylcyclopropyl-azolylmethyl-
carbinol derivatives of the formula (I") in which Ar, R,
X and Y have the meanings which have already been
mentioned as preferred for these radicals.
d) Substituted azolylmethyl-cyclopropyl-carbinol
derivatives of the formula (I"')
ORl
\ /
Ar-C C -R2 tI"')
I
CH2
~y
in which
Ar represents optionally substituted aryl or
optionally substituted heteroaryl,
Rl represents hydrogen, alkyl, alkenyl, alkinyl,
trialkylsilyl, optionally substituted phenylalkyl
or the acyl radical,
R2 represents halogen, cyano, thiocyano,
alkylcarbonyloxy, alkylcarbonylthio or the groups
-X-R3 and -NR4R5, as well as hydrogen when Ar
represents optionally substituted heteroaryl,
R3 represents alkyl, cycloalkyl, alkenyl, alkinyl,
hydroxyalkyl, carboxyalkyl, alkoxycarbonylalkyl,
optionally substituted aryl, optionally substituted
aralkyl, or the radical of the formula
1 338949
-- 19 --
-
-CH2-CH2-0
~ ,
R4 and R5 are identical or different and represent
hydrogen or alkyl and, together with the nitrogen
atom to which they are bonded, represent an
optionally substituted cycloaliphatic ring which
optionally contains other heteroatoms,
X represents oxygen, sulphur, the SO or SO2 group,
and
Y represents a nitrogen atom or the CH group, and
their acid addition salts, have good antimycotic
properties.
The substituted azolylmethyl-cyclopropyl-carbinol
derivatives of the formula (I"') which are to be used
according to this section of invention show a good
spectrum of action in certain areas of indication.
The substituted azolylmethyl-cyclopropyl-carbinol
derivatives according to the invention are generally
defined by formula (I"'). In this formula,
Ar preferably represents phenyl which optionally
has one or several, identical or different,
substituents, the substituents which may be
mentioned as being preferred being: halogen,
alkyl, alkoxy and alkylthio each having 1 to 4
carbon atoms; halogenoalkyl, halogenoalkoxy and
halogenoalkylthio each having 1 to 2 carbon atoms
and 1 to 5 identical or different halogen atoms,
such as fluorine and chlorine atoms; as well as
phenyl or phenoxy each of which is optionally
substituted by alkyl having 1 or 2 carbon atoms
and/or halogen; and
y
1 338949
- 20 -
furthermore represents naphthyl and a 5- to 6-
membered heteroaromatic which optionally has one
several, identical or different, substituents and
nitrogen, oxygen and/or sulphur as the heteroatoms,
the suitable substituents which are preferred being
the above mentioned phenyl substituents;
Rl preferably represents hydrogen, straight-chain
or branched alkyl having 1 to 4 carbon atoms,
straight-chain or branched alkenyl and alkinyl,
each having 2 to 4 carbon atoms, trialkylsilyl
having 1 to 4 carbon atoms in each alkyl part,
alkylcarbonyl having 1 to 4 carbon atoms in the
alkyl part, and phenylalkyl which has one or two
carbon atoms in the alkyl part and is optionally
monosubstituted or polysubstituted by identical or
different substituents, preferred substituents
being the phenyl substituents already mentioned for
Ar,
R2 preferably represents fluorine, chlorine,
bromine, cyano, thiocyano, alkylcarbonyloxy having
1 to 4 carbon atoms in the alkyl part, alkyl-
carbonylthio having 1 to 4 carbon atoms in the
alkyl part, or the groupings -X-R3 and -NR4R5,
wherein
R3 preferably represents straight-chain or branched
alkyl having 1 to 18 carbon atoms, cycloalkyl
having 3 to 8 carbon atoms, straight-chain or
branched alkenyl having 2 to 18 carbon atoms,
straight-chain or branched alkinyl having 2 to 18
carbon atoms, hydroxyalkyl having 1 to 18 carbon
1 338949
- 21 -
atoms, alkylthioalkyl having 1 to 6 carbon atoms in
the alkylthio part and 1 to 6 carbon atoms in the
alkyl part, carboxyalkyl having 1 to 18 carbon
atoms in the alkyl part, alkoxycarbonylalkyl having
1 to 6 carbon atoms in the alkoxy part and 1 to 6
carbon atoms in the alkyl part, and phenyl or,
phenylalkyl having 1 to 2 carbon atoms in the alkyl
part, each of which is optionally monosubstituted
or polysubstituted by identical or different
substituents, preferred substituents in each case
being the phenyl substituents mentioned as being
preferred for Ar, or
R3 represents the radical of the formula
-CH2-CH2-0~
R4 and R5 independently or one another preferably
represent hydrogen or straight-chain or branched
alkyl having 1 to 4 carbon atoms, or
R4 and R5, together with the nitrogen atom to which
they are bonded, preferably represent a 5-membered
or 6-membered ring which is optionally substituted
by alkyl having 1 to 4 carbon atoms or
alkylcarbonyl having 1 to 4 carbon atoms in the
alkyl part, and can contain oxygen, sulphur and/or
nitrogen as further heteroatoms, and
X preferably represents hydrogen when Ar represents
an optionally substituted 5-membered or 6-membered
heteroaromatic, and
Y preferably represents nitrogen or a CH group.
X
1.338q49
- 22 -
Particularly preferred compounds of the formula (I"')
are those in which
Ar represents phenyl which is optionally
monosubstituted or trisubstituted, in particular
monosubstituted or disubstituted, by identical or
different substituents, the following being
mentioned as substituents: fluorine, chlorine,
methyl, isopropyl, tert.-butyl, methoxy,
methylthio, trifluoromethyl, trifluoromethoxy,
trifluoromethylthio, and phenyl or phenoxy, each of
which is optionally substituted by fluorine,
chlorine and/or methyl; and furthermore represents
naphthyl, and represents furyl, thienyl, pyridinyl
or pyrimidinyl, each of which is optionally
monosubstituted or disubstituted by identical or
different substituents, suitable substituents being
the above mentioned phenyl substituents;
R2 represents hydrogen, methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, allyl, propargyl,
trimethylsilyl, methylcarbonyl, ethylcarbonyl, n-
propyl-carbonyl, isopropylcarbonyl, n-
butylcarbonyl, isobutylcarbonyl, and benzyl which
is optionally monosubstituted or disubstituted, in
particular monosubstituted or disubstituted, by
identical or different substituents, preferred
substituents being the phenyl substituents already
mentioned as being preferred for Ar,
R2 represents fluorine, chlorine, bromine, cyano,
thiocyano, methylcarbonyloxy, ethylcarbonyloxy, n-
propylcarbonyloxy, isopropylcarbonyloxy, n-butyl-
1 338949
- 23 -
carbonyloxy, isobutylcarbonyloxy, methylcarbonyl-
thio, ethylcarbonylthio, n-propylcarbonylthio,
isopropylcarbonylthio, n-butylcarbonylthio,
isobutylcarbonylthio or the groupings -X-R3 or
-NR4R5
wherein
R3 represents straight-chain or branched alkyl
having 1 to 12 carbon atoms, cycloalkyl having 5 to
7 carbon atoms, straight-chain or branched alkenyl
having 2 to 12 carbon atoms, straight-chain or
branched alkinyl having 2 to 12 carbon atoms,
hydroxyalkyl having 1 to 12 carbon atoms, alkyl-
thioalkyl having 1 to 4 carbon atoms in the alkyl-
thio part and 1 to 4 carbon atoms in the alkyl
part; carboxyalkyl having 1 to 12 carbon atoms in
the alkyl part, alkoxycarbonylalkyl having 1 to 4
carbon atoms in the alkoxy part and 1 to 4 carbon
atoms in the alkyl part, and phenyl or benzyl, each
of which is optionally monosubstituted to
trisubstituted, in particular monosubstituted to
trisubstituted, in particular monosubstituted or
disubstituted, by identical or different
substituents already mentioned above for Ar as
being particularly preferred, or
R3 represents the radical of the formula
2 2 2
R4 and R5 independently of one another represent
hydrogen, methyl, ethyl, n-propyl, isopropyl, n-
butyl or isobutyl, or
1 338949
- 24 -
-
R4 and R5 together with the nitrogen atom to which
they are bonded, represent piperidinyl, piperazinyl
or morpholinyl each of which is optionally
substituted by methyl, ethyl, methylcarbonyl or
ethylcarbonyl, and
X represents oxygen, sulphur, an SO group or an SO2
group, and furthermore
R2 also represents hydrogen when Ar represents one
of the above mentioned optionally substituted
heteroaromatics, and
Y represents nitrogen or a CH group,
other preferred compounds according to the invention are
addition products of acids and those substituted
azolylmethyl-cyclopropyl-carbinol derivatives of the
formula (I"') in which Ar, Rl, R2 and Y have the
meanings which have already been mentioned as being
preferred for these radicals,
The substituted azolylmethyl-cyclopropyl-carbinol
derivatives which are to be used according to this
section of the invention and their acid addition salts
have not yet been described. They can be obtained in a
generally known manner.
e) Hydroxyethyl-azole derivatives of the general
formula
OH CH3
Rl-l C-C~=Z (I"")
CH2 CH3
l~X
,~
.
1 338949
- 25 -
in which
Rl represents alkyl or the grouping Ar-Y,
Ar represents optionally substituted aryl,
Y represents a direct bond or the groupings -CH2-,
CH2-CHz-, -OCH2-, -SCH2-, -CH=CH- or -C=C-,
X represents a nitrogen atom or the CH group,
Z represents oxygen or the NOR2 group and
R2 represents hydrogen, alkyl, alkenyl, alkinyl,
optionally substituted aralkyl or optionally
substituted cycloalkylalkyl,
and acid addition salts thereof, have good antimycotic
properties for the purpose of the present invention.
The compounds of the formula (I"") have an
asymmetric carbon atom and can therefore be obtained in
the two optical isomer forms.
The hydroxyethyl-azole derivatives of the formula
(I"") to be used according to this section of the
invention have a good action spectrum in certain fields
of indication.
Formula (I"") provides a general definition of the
hydroxyethyl-azole derivatives according to the
invention. Preferably, in this formula
Rl represents straight-chain or branched alkyl with
1 to 6 carbon atoms or the grouping Ar-Yi
Ar represents naphthyl, or phenyl which is
optionally monosubstituted or polysubstituted by
identical or different substituents, preferred
substituents which may be mentioned being:
halogen, alkyl with 1 to 4 carbon atoms, alkoxy and
alkylthio with in each case 1 or 2 carbon atoms,
y
1 338949
- 26 -
nitro, halogenoalkyl, halogenoalkoxy and
halogenoalkylthio with in each case 1 or 2 carbon
atoms and 1 to 5 identical or different halogen
atoms, such as, preferably, fluorine and chlorine
atoms, the -CH=NOR2 radical, and phenyl, phenoxy,
benzyl and benzyloxy, each of which is optionally
substituted by halogen and/or alkyl with 1 or 2
carbon atoms;
X represents a nitrogen atom or the CH group;
Y represents a direct bond or the groupings -CH2-,
CH2CH2-, -OCH2-, -SCH2-, -CH=CH- or -C-C-;
Z represents oxygen or the NOR2 group; and
R2 represents hydrogen, straight-chain or branched
alkyl with 1 to 6 carbon atoms, alkenyl or alkinyl
with in each case 2 to 6 carbon atoms, or
phenylalkyl which has 1 or 2 carbon atoms in the
alkyl part and is optionally monosubstituted or
polysubstituted by identical or different
substituents, possible substituents on the phenyl
being the substituents on phenyl which have already
been mentioned in the case of Ar; or represents
cycloalkylmethyl which has 5 or 6 carbon atoms in
the cycloalkyl part and is optionally mono-, di- or
trisubstituted by identical or different alkyl
radicals with 1 to 3 carbon atoms.
Particularly preferred compounds of the formula
(I"") are those
in which
Rl represents straight-chain alkyl with 1 to 6
carbon atoms or the grouping Ar-Y-;
Ar represents naphthyl, or represents phenyl which
1 338949
- 27 -
is optionally mono-, di- or trisubstituted by
identical or different substituents, substituents
which may be mentioned being: fluorine, chlorine,
methyl, trifluoromethyl, trifluoromethoxy,
trifluoromethylthio, methoximinomethyl,
ethoximinomethyl and allyloximinomethyl, and
phenyl, phenoxy, benzyl and benzyloxy, each of
which is optionally substituted by chlorine and/or
methyl;
X represents a nitrogen atom or the CH group;
Y represents a direct bond or the groupings -CH2-,
-CH2CH2-, -OCH2-, -SCH2-, -CH=CH- or -C-C-; and
Z represents oxygen or the NOR2 group,
wherein
R2 represents hydrogen, methyl, ethyl, n-propyl, n-
butyl, allyl or propargyl, or represents benzyl
which is optionally mono-, di- or trisubstituted by
identical or different substituents from the group
comprising fluorine, chlorine, methyl,
trifluoromethyl and trifluoromethoxy, or represents
cyclohexylmethyl which is optionally substituted by
methyl or ethyl,
Another preferred embodiment of the invention are
compositions comprising a fungicidally effective amount
of a nikkomycin derivative selected from the group
consisting of nikkomycins Zt~ Xt, B, Bx, C and Cx (see
US-PS 4,585,761, US-PS 4,315,922, US-PS 4,158,608 and
US-PS 4,046,881) and an antimycotically active and
optionally orally applicable azole.
Moreover, antimycotic compositions are preferred
comprising an azole antimycotic selected from the group
1 338949
- 28 -
consisting of ketoconazole, itraconazole, fluconazole,
clotrimazole, miconazole, bifonazole, 1-(4-chlorphenyl)-
2-methyl-2-methoximo-methyl-1-(1,2,4-triazol-1-yl-
methyl)-l-propanol, l-(4-chlorphenyl)-3,3-dimethyl-2-
(1,2,4-triazol-1-yl-methyl)-2-butanol and 4-
(fluorphenyl)-(l-methylsulfinyl-l-cyclopropyl)-(1,2,4-
triazol-l-yl-methyl)-methanol.
The antimycotic compositions according to the
invention contain a fungicidally effective amount of a
nikkomycin derivative and an azole antimycotic in a
ratio of 1:1 to 30:1, preferably 3:1 to 10:1.
Preferred are also antimycotic compositions
according to the invention comprising a fungicidally
effective amount of a nikkomycin derivative and an azole
antimycotic selected from the group consisting of
ketoconazole, itraconazole, fluconazole, clotrimazole,
miconazole, bifonazole, 1-(4-chlorphenyl)-2-methyl-2-
methoximo-methyl-1-(1,2,4-triazol-1-yl-methyl-1-
propanol, l-(4-chlorphenyl)-3,3-dimethyl-2-(1,2,4-
triazol-l-yl-methyl)-2-butanol and 4-(fluorphenyl)-(1-
methylsulfinyl-l-cyclopropyl)-(1,2,4-triazol-1-yl-
methyl)-methanol and, moreover, the antimycotic
compositions comprising a fungicidally effective amount
of nikkomycin Z and 1(4-chlorphenyl)-2-methyl-2-
methoximo-methyl l-(1,2,3-triazol-1-yl-methyl-1-
propanol, l-(4-chlorphenyl)-3,3-dimethyl-2-(1,2,4-
triazol-l-yl-methyl)-2-butanol and/or 4-(fluorphenyl)-
(1-methylsulfinyl-1-cyclopropyl)-(1,2,4-triazol-1-yl-
methyl)-methanol.
A further embodiment of the invention are nikko-
mycins in combination with antimycotically active and
- 29 - 1338949
op~ionally orally applicable azoles for use in a meLhod
for ~herapeutical ~rea~men~ of ~he human or non-human animal
body infected with fungi.
The inven~ion is also rela~ed ~o ~he use of nikko-
~ycin deriva~ives in combinaLion wi~h an~imyco-ically
ac~ive and op~ionally orally applicable azoles for ~he
prepara~ion of an~imyco~ically acLive pharmaceu~ical
composi~ions and ~o a process for ~he prepara~ion of
an~imyco~ically ac~ive pharmaceu~ical composi~ions
comprising mixing a combina~ion of a fungicidally
effec~ive amoun~ of a nikkomycin deriva~ive and an azole
deriva~ive wi~h a solid or liquid diluen~ and/or carrier
or o~her auxiliaries useful for ~he prepara~ion of phar-
maceu~ical composi~ions, said nikkomycin deriva~ive and
azole deriva~ive pref~ra~ly being presen~ in a ra~io of
~ o 30:1.
Finally, ~he inven~ion is also rela~ed ~o a me~hod
of ~rea~ing human or non-human animals infec~ed wi~h
fungi, ~he me~hod comprising adminic~ering ~o ~he human
or non-human animal ~herapeu~ically effec~ive amoun~s
of a nikkomycin derivaLive and an azole an~imyco~ic.
More specifically ~he inven~ion is rela~ed ~o a
me~hod comprising adminis~ering to Lhe human or non-
human animal of from 1 mg/kg ~o 1000 mglkg, preferably
10 mg/kg ~o 100 mg/kg of ~ody weigh~ per day of a nikko-
mycin deriva~ive and an azole an~imyco~ic of ~he above
idenLified !ype. The me~hod comprising adminis~ering ~he
nikkomycin and the azole in a ra~io of 1:1 ~o 30:1 is
also preferred.
Prefelably ~he compos'i~ions according ~o ~he inven-
t.ion are orally administ.ered, but may be administered parenterally
or via the inhalation of an aerosilized preparation.
_ - 30 - 1338949
Experimental part for nikkomycin derivatives and
analogues
N-alkylation of nikkomycin Z with aldehydes and ketones
(Table 1, General Procedure)
10 mmol of nikkomycin Z are dissolved in 100 ml of
an H2O/methanol mixture and 50-100 mmol of the aldehyde
or ketone dissolved in 20 ml of methanol are added.
After stirring for 30 min. 10-15 mmol of NaBH3CN are
added to the reaction mixture.
The mixture is stirred overnight (TLC control)
until the reaction is complete. The excess NaBH3CN is
eliminated by adding glacial acetic acid. The mixture
is concentrated to dryness by evaporation and triturated
with ether. The powdery product is chromatographed on
LH-20 (ethanol/H2O = 1:1). In many cases the mono- and
dialkylation products are produced concomitantly in the
reaction with aldehydes. They are separated from each
other by chromatography.
N-Alkylation of nikkomycin Z with a-amino aldehydes
(Table 2)
The procedure is analogous to the N-alkylation of
nikkomycin Cz with a-amino aldehydes (Table 6).
10 mmol of nikkomycin Z and 10 mmol of N-BOC-D/L-a-
aminooctanal are dissolved (partly suspended) in 150 mlof methanol and 15 mmol of NaBH3CN are added. By adding
acetic acid the pH value is adjusted to pH 6-7.
1 338949
- 31 -
The solution is filtered free of unreacted
nikkomycin Z, concentrated by evaporating and
chromatographed on LH-20 (ethanol/H2O = 1:1).
The BOC protecting group is cleaved off in 30 ml of
TFA/CH2C12 (1:1) at 0C over a period of 2 hours. The
product is chromatographed once again on LH-20
(H2O/acetol = 99.9:0.1).
Yield after chromatography: 33%.
N-acylation of nikkomycin Z with a-amino acid reactive
esters (Table 3, examPle).
L-phenylalanine-nikkomYcin Z
10 mmol of nikkomycin Z (= 4.95 g) are dissolved in
50 ml of a DMF/H2O mixture and 1.38 ml of triethylamine
are added while cooling with ice (protection of the
carboxyl group by a salt).
Then 11 mmol (= 6.26 g) of N-BOC-L-phenylalanine-
ONP-ester in 5 ml of DMF and 1.38 g of hydroxy-
benztriazole are added. TLC control on cellulose using
n-propanol/H2O/acetic acid = 60:40:0.5 or 80:20:0.5.
The mixture is stirred overnight. After the
reaction is complete the DMF/H2O mixture is stripped off
under a high vaccum and the sample is dried (under a
high vacuum). It is taken up in water and extracted
first with ether and then with ethyl acetate in order to
substantially remove residual DMF, hydroxybenztriazole
and nitrophenol (pH kept at between 6-7). Then the
aqueous phase is concentrated by evaporation and dried
under a high vacuum.
1 338949
- 32 -
To cleave the BOC protecting group from the
tripeptide the product is dissolved in CH2C12/TFA = 1:1
while cooling with ice and the solution is allowed to
reach room temperature (TLC control). When the cleavage
is complete the mixture is concentrated by evaporation,
the remaining TFA is removed azeotropically with toluene
and the product is dried under a high vacuum.
The product is chromatographed on LH-20 with
ethanol/H2O/ethanol/H2O/acetic acid = 50:50:0.1 (twice).
1.65 g of a lH-NMR-pure tripeptide are obtained (yield:
25.7%).
Identification: lH-NMR.
N-acylation of nikkomycin Z with reactive esters (Table
4, example)
The procedure is similar to the N-acylation of
nikkomycin Z with a-amino acid reactive esters.
10 mmol of nikkomycin Z are dissolved in 50 ml of a
DMF/H2O mixture and 1.38 ml of triethylamine are added
while cooling with ice. Then 11 mmol of palmitic acid
ONP ester and 1.38 g of hydroxybenztriazole are added.
The yield following chromatography on LH-20 (mobile
solvent: ethanol) is 41 %.
N-acylation of nikkomycin Cz with a-amino acid reactive
esters ~Table 5, examPle)
p-MethoxY-D/L PhenYlqlYcine C7,
10 mmol of nikkomycin Cz (2.87 g) are dissolved in
* - Trade-mark
V
i
1 338949
_ - 33 -
20 ml of a DMF/H2O mixture and 1.38 mol of triethylamine
are added while cooling with ice (protection of the
carbonyl group by a salt). Then 10 mmol (4.02 g) of N-
BOC-D/L-phenylglycine ONP ester is 5 mol of DMF and 1.35
g of hydroxybenztriazole are added.
Partially precipitated components dissolve on
stirring the mixture overnight.
TLC control on cellulose using n-
propanol/H2O/acetic acid = 60:40:0.5 or 80:20:0.5.
When the reaction is complete (pH kept at between
6-7) the DMF/H2O mixture is stripped off and the sample
dried under a high vacuum. The product is worked up and
TFA cleaved off following the same procedure as for the
N-acylation of Nikkomycin Z with a-amino acid reactive
esters.
The product is chromatographed on LH-20 using
ethanol/H2O/acetic acid = 50:50:0.1.
Yield: Fractions 72-85 1.1 g (slightly contaminated)
86-105 2.5 g
= 79 % total yield,
Identification: lH-NMR
N-Alkylation of NikkomYcin C7-with a-aminoaldehYdes
(Table 6, examPle) N-BOC-homo Phenylalaninal
2.93 g of N-BOC homo phenylalanine methyl ester (10
mmol) are dissolved in 25 ml of absolute toluene under
nitrogen and the solution is cooled to -80C. 20 ml of
-20% strength DIBA in toluene are added dropwise and the
temperature maintained. Since the reaction was not
complete after 1.5 h a further 3 ml of 20 % strength
DIBA were added.
X~
_ ~ 34 ~ 1 3 3 8 94 9
Workinq uP
After 1 ml of methanol has been added the reaction
mixture is added to 10 ml of a saturated potassium
sodium tartrate solution of 60 ml of H2O (OC). After
stirring a gelatinous precipitate forms. It is filtered
off and washed with ether. The product is extracted
from the aqueous phase quantitatively, using ether. The
combined ether and toluene phases are dried over Na2SO4
and concentrated by evaporation. An oil is obtained
which is separated by means of a small silica gel column
using pentane/ethyl acetate (85/15).
Yield: 60.8%.
TLC/silica gel: Rf = 0.78 (ester)
Rf = 0.68 (aldehyde)
CHC13/methanol (9/1);
spray reagent: DNP/ethanol
Identification: lH-NMR (ppm)
9.42 (lH, CHO); 7.25 (5H, m, Aryl); 5.2 (lH,br, BOCNH);
4.18 (lH,br.m, NH-CH-CHO); 2.7 (2H,br.m, -CH2-);
2.0 (2H,br.m, -CH2-); 1.43 (9H, s, (CH3)3-C)
CouPlinq of nikkomycin C~ with N-BOC-homo Phenylalanina
2 g of nikkomycin Cz (7 mmol) are dissolved in 70
ml of methanol at room temperature and 2.2g of N-BOC-
homo phenylalaninal in 30 ml of methanol are added
dropwise. After stirring for 15 min. 441 mg of NaBH3CN
are added. After stirring overnight the reaction was
~ 35 ~ 1338949
comple~e (TLC con~rol). The excess NaBH3CN is decomposed
with ace~ic acid. Af~er concen~ra~ing ~he mix~ure by
evapora~i~n ~he residue is ~ri~uraLed wi~h e~her.
Af~er cleaving off ~he BOC group wi~h CH2Cl2lTFA
a~ 0C~RT (TLC con~rol) ~he produc~ is chroma~ographed
*
on Sephadex LH-20 using e~hanol/H20/ace~ic acid
(50/S0lO.1). The coupling produc~ is ob~ained in an 80
'~. yield.
TLC/silica gel: propanol/H20/ace~ic acid (80l20l0.5).
Iden~ifica~ion: lH-NMR
Table 1: 1H shif~s of ~he dias~ereomer mix~ure in D20
H-a~oms (ppm) H-a~oms (ppm)
aroma~, H 7.16-7.34 2H on C-l 3.318
-olef.H 7.606;7.598 lH on C-2 3.598
(broadened)
-olef.H 5.823;5.813 2H on C-3 2.015
H on C-1' 5.756 (broadened) 2H on C-4 2.723
H on C-2' 4.266:4.251
H on C-3' 4.523,4.498
H on C-4' 3.965;3.911
H on C-5' 4.330;4.311
The syn~hesis in ~he case where R = CH2 phenyl was
~2 carried lou~ by ~he samQ procedure.
* trade-mark
Le A 25 522
1 338949
_ - 36 -
N-alkylation of nikkomycin Cz with aldehydes (Table 7,
example)
The N-alkylation of nikkomycin Cz is similar to the
N-alkylation of nikkomycin Z with a-amino aldehydes.
10 mmol of nikkomycin Cz are dissolved in 200 mol
of an H2O/methanol mixture and 20 mmol of
phosphonoaldehyde disodium salt (Mw: 157) are added.
After stirring the mixture for 30 min 1 g of NaBH3CN is
added. The pH value of the reaction mixture is kept at
pH 5-6 using acetic acid.
The mixture is stirred overnight (TLC control).
When the reaction is complete the excess NaBH3CN is
eliminated by adding glacial acetic acid. The mixture
is concentrated to dryness by evaporation. The powdery
product is chromatographed on LH-20 (ethanol/H2O = 1:1).
Yield: 77~ (monoalkylation product).
Reduction of nikkomycin X to nikkomycin X alcohol
(Table 8)
100 mmol of nikkomycin X are dissolved in 30 ml of
acetic acid/H2O (1:1). The solution is hydrogenated (6
hours, RT, 3 bar) with 1 g of freshly reduced PO2 and
worked up. It is then chromatographed on LH-20 with
water and 0.1% acetic acid.
Yield after chromatography: 45%.
Nikkomycin derivatives according to the invention
are specified in the following tables:
1 338949
- 37 ~
Table 1: N-Alkylation of nikkomycin Z with aldehydes
and ketons
HN
HO ~ ¦
OH NRl ~
R2 HO OH
Rl R2 Physical Data
Confirmed by
-CH3 H lH-NMR data
-CH3 CH3
-C2H5 -C2H5
C3H7 C3H7
CH3
25 -CH< H
CH3
-C4H9 -C4H9
CH3
-CH2-CH< H
CH3
CH3
35 -CH< H
CH2-CH3
- 38 - t338949
Table 1: N-Alkyla~ion of nikkomycin Z wi~h
al~eh~des and ketons (con~inua~ion)
R1 R2 Physical Da~a.
CH2-CH3 Confirmed by
10 -CH< H NMR data
CH2 - CH2
CH3
-CH( H
CH2-CH2-cH3
-tCHz)6~CH3 H
-CH2-CH< H
C4H9
CH20H
-CH~ H
CH20H
25 -CH2-CH=CH2 H
-CH2-CH=CH-CH3 H
t
H H
-CH2-(C=c)2-cH3 H
. H H H H
-CH2-(C-C)2-CH3 -CH2-(C=C)2-CH3
i
-(CH2)11-NH2 H
Le A 25 5Z2
_ ` _ 39 _ l 338 94 9
Table 1: N-Alkyla~ion of nikkomycin Z wi~h
aldehydes and keLons ~con~inua~ion)
R1 R2 Physical Da~a
10 -CH2 ~ H Confirmed by
-CH2 ~ 3 H
H0
-CH2-CH2 ~ H3 H
-CH2-CH2 ~ -CHz-CH2 { 3~
CH3 CH3
-CH2-CH=CH ~ 3 -CH2-CH=CH { 3
-(CH2)3 { 3 H
-(CH2)3 ~ 3 -'CH2)3 { 3
Le A 25 522
' ~ _ 40 - 1338949
'.
Table 1' N-Alkvla~ion of nikkomycin Z wi~h
aldeh~des and ke~ons (con~inua~ion)
R1 R2 Physical Da~a
Confirmed by
~ data
-CH2-P-O~Na t H
OH
O O
1 5
-(CH2)2-P-(oc2Hs)2 -(CH2)-P-(OC2H5)2
, t
Le A 25 S22
" - 41 - 1338949
Table 2: N-Alkyla~ion of nikkomycin Z w;~h ~-amino
aldehYdes
O
HN
HO HOOC ~
~ ' .
10H ,
OH ~
HO OH
HN-CH2-CH-Rz
NH2
Rz Configura~ion PHysical Da~a
- of ~-NH2
. Confir~.ed by
-H - lH-NMR da~a
-CH3 L
ICH3
-C-CH3 D/L
CH3
-C6Hl3 DIL
-(CH2)4-NH2 L
-CH2 ~ D/L
F
-CH2 ~ F D/L
Le A 25 522
~ 42 - 1 338949
Ta~le 3: N-Acyla~ion of nikkomvcin Z wi~h ~-amino
acid reac~ive es~ers
HN
~O~Q~C I I
OH ~
HO OH
HN-CH-CH-Rz
O NH2
Rz Configura~ion Physical Da~a
of ~-NH2
Confirmed by
20 -H _ 1H-NMR da~a
-CH3 L
CH3
-CH( L
H CH~
-CH2-C< L
CH3
H CH3
_~( L
C~2-CH3
35 -cH2-cH2-s-cH3 L
-CH2-COOH L
Le A 25 522
.
- 43 - 1 338949
Ta~le 3: N-Acyla~ion of nikkomYcin Z wi~h ~-amino
a~i' reac~ive es~ers (con~inua~ion)
Rz Configura~ion Physical Da~a
of ~-NH2
0 Confirmed by
Il ~ data
-~CH2)2-C-NH2 L
-(CH2)3-NH2 L
-~CH2)4-NH2 L
-~CH2)4-NH2xCF3COOH L
20 -~CH2)3-N-Il_NH2 L
-CH2 ~ L
-CH2 ~ F DIL
30 -CH2 ~ DIL
F
-CH2 ~ H L
Le A 25 522
_ _ 44 1338949
Table 4: N-Acvlderiva~ives of nikkomvcin Z wiLh reactive
S esters
O
HO HOOC ~
10 ~ ,
OH
N~-C-Rz ~-~
O HO OH
15 Rz Ph~sical Data
Conf1rmed by
-~CHz)l4-CH3 lH-NMR da~a
-(cH=cH)2-cH3
-(CH2)10-NH2
~0
Le A 25 522
_ 45 _ 1 3389 49
.
Table 5: N-Acvla~ion of nikkomycin Cz wi~h K-amino
acid reac~lve es~ers
O
HOOC
o 1l
Rz-CH-C-N
I H O
NH
HO OH
15 Rz Configura~ion Physical Da~a
of K-NH2
Confirmed by
-CH2-CH2-CH3 D lH-NMR da~a
20 -CH2-CH2-S-CH3 L
-(CH2)3-CH3 . D
25 ~ D/L
~ NH2 D/L
.
3G
~ OCH3 D/L
35 ~ D/L
Le A 25 522
1 338949
- 46 -
Table 5: N-Acvla~ion of nikkomycin Cz wi~h a-amino acid
reac~ive es~ers (con~inua~ion)
,' ~
Rz Configura~ion Physical Da~a
of a-NH2
DIL Confirmed ~y
NMR data
~ \
N O D/L
-CH2 ~ D
-CH2 ~ F D/L
-CH2 ~ D
-CH2 ~ -CH2 ~ L
3~ .
Le A 25 522
1 338q49
r
Table 5: ~-AcylaLion of nikkomycin Cz wi~h ~-amino-
5 acld reac~ive es~ers ~con~inua~ion)
Rz Configura~ion Physical Da~a
of ~-NH2
~ Confirmed bv
10 -CH2-CH2-~ ~ DIL 'H-N~R da~a
CH2-C ~ L
CH2 ~ D
-CH2- ~ L
-(CH2)2- ~ DIL
CH3
-C-CH2 ~ D/L
CH3
addi~ionally:
N~_~S~ o H~
N---~~ ~ R HHOOC ~I
N-OCH3 HO OH
Le A 25 522
1 338949
- 4a -
Table 6: N-Alkvla~ion of nikkomycin Cz wi~h ~-ami~o-
aldehydes -
HN~
HOOC
R DIL
Z--N'/
I H ~
NH2
HO OH
15 ~z Configura~ion Physical Da~a
of ~-NH2
Confir~d by
C6H13 D/L lH-N~R da~a
20 -CH3-S-CH2-cH2 L
F ~
DIL
~ ~ .
"~
F ~ D/L
~ DIL
H ~
I II D/L
~,
Le A 25 522
1 338949
- 49 -
Table 6: N-Alkvla~ion of nikkomvcin Cz wi~h -amino-
aldehydes
Rz Configura~ion Physical Da~a
of ~-NH2
CH x) Confirmed by
3 ~ data
DIL
OH
x) D/L
OH
~ L
Le A 25 522
' ~ 50 ~ t338949
Table 6: N-Alkvla~ion of nikkomycin Cz wi~h a-amino-
S aldehvdes (conLinua~ion)
Rz Configura~ion Physical Da~a
of -NH2
10 ~ H3C CH3 x) DIL Confirmed by
NMR data
OH
CH30 ~ D/L
OH
(R,R-configuraLion)
HO ~ CH3 x~
x CF3COOH D/L
OH
HO ~ CH3
~y
OH
x) mix~ures of dias~ereoisomers
Le A 25 522
- 51 -
- 1 338949
Table 7: N-Alkvla~ion of nikkomycin Cz
O
HN
HOOC
R
z~
H
~`
HO OH
15 Rz Phvsical Da~a
Confirmed by
-CH2-(CH2)l0-NH2 lH-NMR da~a
-(CH2)2-P-(OC2H5)2
(dialkylat.ion
o
-CH2-P-O~Na+
OH
Le A 25 522
- 52 - 13 389 ~9
Table 8: Deriva~ives wi~h subs~i~u~ion in ~he nucleoside
por~ion
HO HOOC
~ CH3 0
~ )~
OH NH2 ~
HO OH
R Physical Da~a
Confirmed by
Nikkomvcin Z 1H-NMR da~a
o
HN ~
~ :~.
O I '
Nikkomycin X
HN___T~CHO
0~
HN CH20H
I IJ
~
HN ~CH20H
! 11
.
x TFA
Le A 25 522
1338949
- 53 -
~
The composi~ions accordinq ~o ~he inven~ion can be
used accordin~ to ~he invention and display an~imicro-
bial ac~i~ns, in par~icular powerful an~imyco~ic
ac~ions. They possess a very broad an~imyco~ic ac~ion
spec~rum, especially aqains~ derma~ophy~es and yeasts
as well as biphasicfun~i, for example a~ains~
varie~ies of Candida, such as Candida albicans, varie-
~ies of Epidermophy~on, such as Epidermophy~on flocco-
sum, varie~ies of Asper~illus, such as Asper~illus niger
and Asper~illus fumi~a~us, varie~ies of Trichophy~on,
such as Trichophy~on men~agrophy~es, varie~ies of ~icro-
sporon, such as Microsporon felineum and varie~ies of
Torulopsis, such as Torulopsis qlabra~a. The lis~in~ of
~hese micro-or~anisms in no way implies a limi~a~ion of
~he ~erms which can be comba~ed bu~ is only of iilus~ra-
~ive charac~er. Such fun~i include also Coccidioides
immi~is, Hist.oplasma capsulat.um, Blastomyces derma~i~idis
-and Paracoccidioides brasiliensis.
~ xamples which may be men~ioned of fields of
indica~ion in human medicine are: derma~omycoses and
sys~emic mycoses caused by Trichophy~on men~a~rophy~es
and other varie~ieS of Trichophy~on, varie~ies of
~licrosporon, Epidermophyton floccosum, veasts and
biphasicfun~i as well as moulds and varie~ies of
Candida.
~xamples which may be men~ioned of field of indi-
ca~ion in veLerinary medicine are: all derma~omycoses
and sys~emic mycoses, especially ~hose caused by ~heabovemen~ioned pa~ho~ens.
The presen~ inven~i_A i~C ' uaes pharmaceu~ical
formula~ions which, in addi~ion ~o non-Loxic, inert
Le A 25 522
1 338949
- 54 -
pharmaceutically suitable excipients, contain one or
more active compounds according to the invention or
which consist of one or more active compounds
according to the invention, as well as processes for
the preparation of these formulations.
The present invention also includes pharma-
ceutical formulations in dosage units. This means that
the formulations are in the form of individual parts,
for example, tablets, dragees, capsules, pills,
suppositories and ampoules, of which the content of
active compound corresponds to a fraction or a
multiple of an individual dose. The dosage units can
contain, for example, 1, 2, 3 or 4 individual doses or
1/2, 1/3 or 1/4 of an individual dose. An individual
dose preferably contains the amount of active compound
which is given in one administration and which usually
corresponds to a whole, a half, a third or a quarter
of a daily dose.
By non-toxic, inert pharmaceutically suitable
excipients there are to be understood solid, semi-
solid or liquid diluents, fillers and formulation
auxiliaries of every kind.
Tablets, dragees, capsules, pills, granules,
suppositories, solutions, suspensions and emulsions,
pastes, ointments, gels, creams, lotions, powders and
sprays may be mentioned as preferred pharmaceutical
formulations.
Tablets, dragees, capsules, pills and granules
can contain the active compound or compounds along-
side the customary excipients, such as (a) fillers andextenders, for example starches, lactose, sucrose,
glucose, mannitol and silica, (b) binders, for example
carboxymethylcellulose, alginates, gelatine and
polyvinylpyrrolidone, (c) humectants, for example
glycerol, (d) disintegrating agents, for example
agar-agar, calcium carbonate and sodium bicarbonate,
1 338949
_ - 55 -
(d) solution retarders, for example paraffin, and (f)
resorption accelerators, for example cetyl alcohol and
glycerol monostearate, (h) adsorbents, for example
kaolin and bentonite, and (i) lubricants, for example
talc, calcium stearate and magnesium stearate and
solid polyethylene glycols, or mixtures of the
substances listed under (a) to (i).
The tablets, dragees, capsules, pills and
granules can be provided with the customary coatings
and shells, optionally containing opacifying agents,
and can also be of such composition that they release
the active compound or compounds only, or prefer-
entially, in a certain part of the intestinal tracts,
optionally in a delayed manner, examples of embedding
compositions which can be used being polymeric
substances and waxes.
The active compound or compounds, optionally
together with one or more of the above-mentioned
excipients can also be in a micro-encapsulated form.
Suppositories can contain, in addition to the
active compound or compounds, the customary water-
soluble or water-insoluble excipients, for example
polyethylene glycols, fats, for example cacao fat, and
higher esters (for example C14-alcohol with C16-fatty
acid), or mixtures of these substances.
Ointments, pastes, creams and gels can contain,
in addition to the active compound or compounds, the
customary excipients, for example animal and vegetable
fats, waxes, paraffins, starches, tragacanth,
cellulose derivatives, polyethylene glycols,
silicones, bentonites, silica, talc and zinc oxide, or
mixtures of these substances.
Powders and sprays can contain, in addition to
the active compound or compounds, the customary
excipients, for example lactose, talc, silica,
i ~ i
- 56 - 1 338949
aluminium hydroxide, calcium silicate and polyamide
powders or mixtures of these substances. Sprays can
additionally contain the customary propellants, for
example chlorofluorohydrocarbons.
Solutions and emulsions can contain, in addition
to the active compound or compounds, the customary
excipients, such as solvents, solubilizing agents and
emulsifiers, for example water, ethyl, alcohol,
isopropyl alcohol, ethyl carbonate, ethyl acetate,
benzyl alcohol, benzyl benzoate, propylene glycol,
1,3-butylene glycol, dimethylformamide, oils,
especially cottonseed oil, groundnut oil, maize germ
oil, olive oil, castor oil and sesame oil, glycerol-
formal, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitane, or
mixtures of these substances.
For parenteral administration, the solutions and
emulsions can also be in a sterile form which is
isotonic with blood.
Suspensions can contain, in addition to the
active compound or compounds, the customary
excipients, such as liquid diluents, for example
water, ethyl alcohol or propylene glycol, suspending
agents, for example ethoxylated isostearyl alcohols,
polyoxy-ethylene sorbitol esters and sorbitane esters,
micro-crystalline cellulose, aluminium metahydroxide,
bentonite, agar-agar and tragacanth, or mixtures of
these substances.
The formulation forms mentioned can also contain
colorants, preservatives and additives which improve
the odour and flavour, for example peppermint oil and
eucalyptus oil, and sweeteners, for example saccharin.
The therapeutically active compounds should
preferably be present in the abovementioned pharma-
1 338949
- 57 -
ceutical formulations in a concentration of about 0.1
to 99.5, preferably of about 0.5 to 95% by weight of
the total mixture.
The abovementioned pharmaceutical formulations
can also contain other pharmaceutical active com-
pounds in addition to the active compounds according
to the invention.
The abovementioned pharmaceutical formulations
are prepared in the customary manner according to
known methods, for example by mixing the active
compound or compounds with the excipient or
excipients.
The present invention also includes the use of
the active compounds according to the invention, and
of pharmaceutical formulations which contain one or
more active compounds according to the invention, in
human and veterinary medicine, for the prevention,
alleviation and/or cure of the abovementioned
diseases.
The active compounds or the pharmaceutical
formulations can be administered locally, orally,
parenterally, intraperitoneally and/or rectally,
preferably parenterally, and in particular
intravenously, and aerosilized.
In general, it has proved advantageous both in
human medicine and in veterinary medicine, to
administer the active compound or compounds according
to the invention in total amounts of about 10 to 300,
preferably 50 to 200 mg/kg of body weight every 24
hours, optionally in the form of several individual
administrations, in order to achieve the desired
results.
However, it can be necessary to deviate from the
dosages mentioned, and in particular to do so as a
function of the species and the body weight of the
X'~
1 338949
_ - 58 -
subject to be treated, the nature and severity of the
disease, the nature of the formulation and of the
administration of the medicament and the time or
interval over which the administration takes place.
Thus it can in some cases suffice to manage with less
than the abovementioned amount of active compound,
whilst in other cases the abovementioned amount of
active compound must be exceeded. The particular
optimum required and the type of administration of the
active compounds can easily be determined by anyone
skilled in the art on the basis of his expert
knowledge.
The following formulae show compounds according
to the invention. The figures in parenthesis stand for
their efficacy in C. albicans infected mice. CFW-l
mice were infected intraveneously with 6 x 105 cfu of
Candida albicans. 100 mg/kg of active substance were
administered orally 2 times daily during 3 days
starting with the day of infection. Data were
determined after 12 to 14 days when 80-90% of the
control animals were dead.
Efficacy is defined as follows: 2 means that
compound show activity, 3 means good activity and 4
represents very good efficacy.
X
i
- 59 _
1 1 33894-~
HN
HOOC
~ - C H 3 0 ~
1) ~, (4
~H NH 2 `>
HO OH
o
- HOOC
CH 3 O~H - C - N-- ~ 2
NH2 `~
HO OH
HN
HOOC
3 ) ~NH ~
OH HN-CH3 `X
HO OH
;, J~
HN
'Jnr'~
4 ) ~ Cl H 3 11 ! ~
H / \
OH HN-CH2~ 0
HO OH
Le A 25 5 22
- 60 -
~ 1 338949
- HN
HOOC ¦
~ ~ CH3 0 ~
5) ~ ' (2)
OH NH '~
10 CIH2 HO OH
01~3
H~/
HOOC I ¦
OH~,~ CH3 0 ~
6 ) 1~,~ ( 2 )
- OH NH
C\ HO OH
CH20H CH20H
2 5 - HOOC 0~3
HO ~ CH ~ O
.~~
3 0 OH NH ~ ~
f = ~ HO OH
HC -NH2
CH3
Le A 25 5 22
-- 6 1
~1, 1 338949
HOOC I I i
OH ~,~ CH3 O ~
~/ O
OH NH ~,
C=O HO OH
HC -NH2
CH2~
1 5 O
HN~
HOOC I ¦ l
2)
CH ~ HO OH
CH2C~
CH3 CH3
HOOC
OH~,~ CH3 O
";~, 1 0 ) ~ ( 2 )
OH N ~
C4H9 C4H9 HO OH
Le A 25 522
- 62 -
1 338949
HN~
HO~ CH O ~
11) ~-- O (3) .
7 ~
C=O HO OH
HC - NH2
CH
/ \
CH3 CH3
~5
HN~
HOOC l ¦
HO ~,~ CH3 0~
12) ~ , O ~ (3)
OH NH ~
CH HO OH
CH3 CH3
COOH o
HOOC - CHz - CH2 ~ H ^~H HNl
OH~ O = C o~J
3 0 1 3 ) 1~ ~ O ( 2 )
OH NH 2 ~ ~
HO OH
Le A 25 5 22
- - 63 -
-
- 1 338949
HNJ~,
1 5 OH~OOCC~
14 ) OH 7H ~ (2)
C=O HO OH
Hf -NH2
CH2
C H
CH3 CH3
HO oOCO~
OH NH2 ~ <~
R,S HO OH
Le A 25 S 22
~ - 64 -
~ 1 338949
HN
HOOC
30~C CH3 0 ~
16) ~ ~ CH2-N- (2)
OH NH2 ` ~
HO OH
o
HN ~
HOOC l 1¦
17) ~ CH2-N (3/4)
OH NH2 ~
HO OH
H HOOC ~ 'CH20H
18)
OH NH2 \ /
r-~
HO OH
x CF3COOH
o
HN
HOOC I IJ
OH ~ oD~
CH2HO OH
¦ CH3
NH -CH - C-CH3
CH3
Le A 25 522
- 65 -
~ 1 338949
HN
HOOC
OH ~ CH3 0 ~
20); ~ - ~ O ~ (3/4)
OH NH ~
C=O HO OH
HC-(CH2)4-NH2
NH2
HOOC ¦
OH~,~
Z0 OH NH ~
C=O HO OH
CH-NH2
(CH2)3-NH2
11
HN~
HOOC I ¦
' ~ (3/4)
On NH ~
C=O HO OH
NH2-CH
(CH2)3-N-lCl-NH2
- O
Le A 25 52Z
1 338949
- 66
O
HN~
HOOC
OH~ o~/
OH NH ~
CH2 HO OH
HO - P - ON a
O
HN~,
HOOC
20 )
- OH NH
1 ~
- C=O HO OH
( CH2 ) 4 -NH2 x CF3COOH
Le A 25 5 Z2
t 338949
- - 67 -
-
To test the ability of nikkomycin derivatives and azoles to act
synergistically, checkerboard in vitro assay was performed using the
yeast Candida albicans.
Materials and Methods
Candida albicans strain B311 was grown overnight in glucose-yeast
extract broth, washed, and adjusted to the desired density.
The tests were performed in 96-well microtiter plates using yeast
nitrogen broth with dextrose and asparagine as the growth medium. The
eest materials were dissolved, diluted serlally, then transferred to
the appropriate wells of the microtiter plates so that the final concentrations
ranged from 0.02 - 1 millimolar for the nikkomycins, and 0.06 - 64 micrograms
for the azoles. All wells were inoculated with lO,000 organisms per
milliliter (final concentration) and the plates incubated at 30C for
48 hours. The plates were then examined and the degree of growth for
each well noted. Endpoints were determined for each row as being the
lowest concentration showing the complete inhibition of growth. Data
were plotted as isobolograms showing the endpoints for the single compounds,
as well as the endpoints for the various combinations.
Results
The data for the assays are presented in Figures 1-4. As can be
seen, for the azole R3783, the addition of nikkomycin derivatives results
in a dramatic lowering of the endpoints in comparison to the endpoints
for the single agents. As an example, Figure l shows the endpoints for
R3783 alone to be 64 micrograms/milliliter and l.0 millimolar for the
nikkomycins. Surprisingly, with the concentrations of tne .ombinations
employed for this example, no growth was detected in the wells containing
at least 0.125 m~l nikkomycin R 5124 and 0.125 ~g/~i of the azole R3783.
Thus, the endpoint for the combination was reduced a minimum of 600-fold
with respect to the azole endpoint, clear evidence of synergy for these
two classes of drugs.
