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Description  |
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This invention relates to a novel bis-(meta-amidinophenoxy)-compound and a
pharmacologically acceptable acid addition salt thereof which is
represented by the general formula:
##STR1##
wherein A represents a chain residue selected from the group consisting of
##STR2##
--CH.sub.2 --CH.dbd.CH--CH.sub.2 --, --(CH.sub.2).sub.2 S(CH.sub.2).sub.2
--, and --(CH.sub.2).sub.m --, in which X represents chlorine atom, n is
an integer of 0 to 4, and m is an integer of 4 to 6. As the
pharmacologically acceptable acid addition salt, there may be mentioned
for example, inorganic acid addition salts such as hydrochloride,
hydrobromide, sulfate, bisulfite and the like; and, for example, organic
acid addition salts such as acetate, maleate, fumalate, citrate,
succinate, lactate, tartarate, oxalate, methane sulfonate and the like.
The compound (I) according to this invention has an excellent antifungal,
antibacterial and anti-trichomonal activities, whereby it is effective for
the treatment of the infection of fungi such as Candida, Cryptococcus, and
the like; infection of various bacteria; and Trichomonas.
The compound (I) according to this invention can be prepared by applying
the so called "Pinner amidine synthesis" [J. Am. Chem. Soc. 77, 2341
(1955)], in accordance with the following reaction sequence:
##STR3##
wherein A has the same meanings as defined above, and R represents a lower
alkyl group.
More particularly, the compound (I) is prepared by the step 1 in which
bis-(meta-cyanophenoxy)-compound (II) is reacted with a lower alcohol such
as methanol, ethanol, propanol, isopropanol, butanol and the like in the
presence of an acidic catalyst to yield the corresponding bis-(meta-lower
alkoxycarboimidophenoxy)-compound (III); and followed by the step 2 in
which said intermediate compound (III) is treated with an ammonium
compound to yield the bis-(meta-amidinophenoxy)-compound (I) of this
invention.
In the step 1, the reaction is effected by dissolving the compound (II) in
a solvent, adding a calculated amount of the lower alcohol to said
solution, and further adding the acidic catalyst, followed by allowing to
stand the solution at a room temperature.
As the solvent used in the step 1, there may be exemplified an organic
solvent including a halogenated hydrocarbon solvent such as chloroform,
carbon tetrachloride, dichloromethane, dichloroethane, trichloroethylene
and the like; an aromatic hydrocarbon solvent such as benzene,
nitrobenzene, toluene, xylene and the like; an ether solvent such as
diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, diglyme and
the like. The solvents may be used, by properly selecting from such
solvents, in a form of a sole solvent or a mixed solvent. Alternatively,
an excess of the lower alcohol may be used in place of a solvent. The
acidic catalysts used in this step include dry hydrochloric acid, boron
trifluoride, sulfonic acid and the like. The product of this step is
obtained in a form of the acid addition salt form of the compound (III).
The compound (III) can be provided as the source material for the step 2,
in a form of the acid addition salt per se, or in a freed form. Usually,
the compound (III) is used for the step 2 as it is, without isolation and
purification.
The step 2 is effected by dissolving the compound (III) in the same solvent
as that of the step 1, and subjecting to the reaction with an ammonium
compound. The ammonium compound used may be ammonia per se or an ammonium
salt such as ammonium chloride, ammonium sulfate, ammonium carbonate and
the like. The product of the step 2 is the desired final compound (I) of
this invention.
The compound (II), the starting material for the preparation of the
compound (I) according to this invention is also a novel compound, which
is synthesized according to the following reaction sequence:
##STR4##
wherein A has the same meanings as defined above, Y represents a halogen
atom, methanesulfonyloxy group or tosyloxy group.
Bis-(meta-cyanophenoxy)-compound (II) is produced by reacting
meta-cyanophenol (IV) with the compound (V) in the presence of an alkali.
As the solvent, there may be used an organic solvent including an alcoholic
solvent such as methanol, ethanol, propanol, isopropanol and the like; and
an amidic solvent such as dimethyl acetamide, dimethyl formamide,
hexamethyl phosphoramide and the like. The solvent(s) may be selected from
such solvents, and may be used in a form of a sole solvent or a mixed
solvent.
The compound (I) of this invention has an excellent antifungal,
antibacterial and anti-trichomonal activities. These activities are
clearly proved by the following results of pharmacological tests.
The compounds under test:
(i) The compound for control: Nystatin
(ii) The compound according to this invention:
1,4-Bis-(m,m'-amidinophenoxymethyl)-cyclohexane-dihydrochloride
(hereinafter refer to compound A of this invention)
PHARMACOLOGICAL TEST 1
Antifungal Test
Procedure of the test:
Respective agar plates were prepared by dissolving compounds under test in
aseptic water to make a two fold dilution series, taking each one
milliliter of said diluted solutions and 9 ml of Sabouraud medium into a
petri dish, and sufficiently admixing the same.
On the other hand, an inoculum was prepared by cultivating an examined
fungus (microorganism) on the Sabouraud slant medium and suspending two
loopful of the incubated fungus in 10 ml of sterilized physiological
saline water.
One loopful of the inoculum was streaked on the abovementioned agar plate.
After the agar plate was incubated at a defined temperature for a
predetermined period of time to determine the minimal inhibitory
concentration (M.I.C.).
RESULTS OF THE TEST
(1) antifungal activity
Incubation conditions
Incubation temperature: 27.degree. C.
Incubation time: 3 days (72 hours) and 7 days (168 hours)
Table 1
______________________________________
Minimal inhibitory concentration against various fungi
M.I.C. (.mu.g/ml)
Compound A according
to this invention
3 days 7 days
Microorganism incubation incubation
______________________________________
Candida albicans
AHU 3656 3.13 6.25
Candida albicans NAKAGAWA
IFM 4017 3.13 3.13
Candida albicans
Yu-1200 IFM 4020 < 1.56 3.13
Candida albicans
IFM 4080 3.13 6.25
Candida tropicalis
AHU 3071 3.13 3.13
Candida utilis AHU 3053
3.13 3.13
Candida guilliermondii
AHU 3654 25 25
Cryptococcus neoformans
3.13 3.13
Saccharomyces cerevisiae
< 1.56 < 1.56
(2) Anticandida activity
Incubation conditions
Incubation temperature
: 37.degree. C.
Incubation time : 3 days (72 hours)
______________________________________
Table 2
______________________________________
Minimal inhibitory concentration against Candida alibicans
M.I.C. (.mu.g/ml)
Compound A
according
Microorganism Nystatin to this invention
______________________________________
Candida albicans
AHU 3656 6.25 3.13
Candida albicans Yu-1200
IFM 4020 6.25 0.8
Candida albicans NAKAGAWA
IFM 4017 6.25 1.56
(3) Effect of incubation temperature on the antifungal
activity
Incubation conditions:
Incubation temperature:
27.degree. C.
Incubation time 3 days (72 hours)
Incubation time 7 days (168 hours)
Incubation temperature:
37.degree. C.
Incubation time 2 days (48 hours)
Incubation time 3 days (72 hours)
______________________________________
Table 3
______________________________________
Minimal inhibitory concentration of Compound A of
this invention
M.I.C. (.mu.g/ml)
Incubation
conditions 27.degree. C.
37.degree. C.
Microorganism 3 days 7 days 2 days
3 days
______________________________________
Candida albicans
AHU 3656 3.13 6.25 3.13 3.13
Candida albicans NAKAGAWA
IFM 4017 3.13 3.13 0.8 1.56
Candida alibicans Yu-1200
IFM 4020 1.56 3.13 0.8 0.8
Candida albicans
IFM 4080 3.13 6.25 0.8 3.13
______________________________________
In viewpoint of the result of the pharmacological test 1, it is apparent
that the compound A of this invention has an excellent antifungal activity
against the yeast-like fungi such as Candida, Cryptococcus, and the
activity is not influenced by the incubation temperature. In addition, it
is recognized that the compound A of this invention has a more excellent
anti-candida activity than that of the nystatin.
PHARMACOLOGICAL TEST 2
Antifungal test when a serum is added.
Procedure of the test:
Minimal inhibitory concentration (M.I.C.) was determined by preparing the
agar plate medium in the same manner of the preceding pharmacological test
1, except for the addition of the horse serum. The concentration of the
component in the medium was finally corrected, so that it may amount to
the concentration of the component when the horse serum was not added.
RESULTS OF THE TEST
Incubation conditions
Incubation temperature: 27.degree. C.
Incubation period: 3 days (72 hours) 7 days (168 hours)
Table 4
______________________________________
Minimal inhibitory concentration of the compound A of
this invention when the serum was added (.mu.g/ml)
M.I.C. (.mu.g/ml) of serum added
0 % 10 % 30 %
3 7 3 7 3 7
Microorganism
days days days days days days
______________________________________
Candida albicans
AHU 3656 3.13 6.25 25 25 25 25
Candida albicans
NAKAGAWA IFM
3.13 3.13 25 25 6.25 6.25
4017
Candida albicans
Yu-1200 IFM <1.56 3.13 6.25 6.25 25 25
4020
Candida albicans
IFM 4080 3.13 6.25 12.5 12.5 12.5 12.5
Candida tropicalis
AHU 3071 3.13 3.13 6.25 6.25 12.5 12.5
Candida utilis
AHU 3053 3.13 3.13 3.13 6.25 <0.8 <0.8
Candida guillier-
mondii
AHU 3654 25 25 25 25 50 50
Candida Krusei
AHU 3993 >100 >100 200 >200 100 100
Cryptococcus neofor-
3.13 3.13 6.25 12.5 12.5 12.5
mans
______________________________________
From the results of the pharmacological test 2, it is noted that the
antifungal activity of the compound A of this invention, when 10% and 30%
by volume of the horse serum was added to the agar plate medium for test,
is nearly same as that when the horse serum was not added; or slight
difference to the extent of 1- 2 dilution step(s). It is therefore shown
that the antifungal activity of the compound A of this invention is hardly
affected by the addition of the horse serum.
PHARMACOLOGICAL TEST 3
Antibacterial test
Procedure of the test:
A compound under test was dissolved in aseptic water to prepare a two fold
dilution series. Each one milliliter of the solution was taken in a petri
dish. Nine ml of infusion agar medium (Difco) was added to the solution,
thereby preparing the agar plate for test. A pre-culture of each test
strain in Tripticase soy broth medium (BBL) at 37.degree. C. for 18 hours
was streaked on the above-mentioned agar plate, and the said agar plate
was incubated at 37.degree. C. for 18 hours to determine the minimal
inhibitory concentration (M.I.C.).
RESULTS OF THE TEST
Table 5
______________________________________
Minimal inhibitory concentration of the compound A of
this invention
Microorganism M.I.C. (.mu.g/ml)
______________________________________
Staphylococcus aureus
209-P JO-1 3.13
Staphylococcus aureus 6.25
13-6
Streptococcus hemolyticus
6.25
Y-73-5
Escherichia coli
NIHJ JC-1 100
Escherichia coli
E-15 200
Salmonella typhimurium
1406 200
Klebsiella pneumonial
NO-1 200
Proteus mirabilis
OM-1 >200
Pseudomonas aeruginosa >200
______________________________________
The compound A of this invention showed a strong antibacterial activity in
that minimal inhibitory concentration against Staphylococcus aureus 209-P,
Staphylococcus anreus 13-6 and Streptococcus pyogenes amounts to 3.13-
6.25 .mu.g/ml. The compound A also showed, though it is rather weak, the
antibacterial activity in that minimal inhibitory concentration against
gram-negative bacteria such as Escherichia coli NIHJ and Escherichia coli
E-15 amounts to 100- 200 .mu.g/ml or above 200 .mu.g/ml.
PHARMACOLOGICAL TEST 4
Anti-trichomonal test
Procedure of the test:
The compound under test was dissolved in aseptic water, and filtered
aseptically to prepare a two fold dilution series. Each 2.6 ml ASAMI
medium containing 20% of the horse serum were respectively taken into
small test tubes. Onto the medium, there were incubated 0.3 ml of the two
fold dilution solution of the compounds and 0.1 ml of the suspension of
the ASAMI medium of Trichomonas vaginalis which was cultured at 37.degree.
C. for 3 days respectively. Incubation was carried out at 37.degree. C.
for 3 days to determine the minimal inhibitory concentration.
RESULT OF THE TEST
It was recognized that the compound A of this invention exhibits the
minimal inhibitory concentration of 200 .mu.g/ml and has anti-trichomonal
activity.
In view point of the results of the pharmacological tests 1- 4, it was
found that the compound A of this invention has excellent antifungal,
antibacterial and anti-trichomonal activities. It is seen that the
antifungal activity of the compound A is not affected by the incubation
temperature and addition of serum, and particularly, the compound (A) is
effective against Candida albicans, Cryptococcus neoformans and the like.
The compound according to this invention represented by the compound (A)
of this invention has excellent antifungal, antibacterial and
anti-trichomonal activities, hereinbefore mentioned. It is therefore
concluded that the compound according to this invention is effective to
treatment of candidiasis such as digestive tract-candidiasis, cutaneous
candidiasis, vaginal candidiasis, cryptococcosis, trichomoniasis and mixed
infection of those with various bacteria.
Following Examples will serve to illustrate the preparation of the
compounds of the present invention.
EXAMPLE 1
Preparation of 1,4-bis-(m,m'-amidinophenoxymethyl)-cyclohexane
dihydrochloride
5 Grams of 1,4-bis-(m,m'-cyanophenoxymethyl)-cyclohexane are thoroughly
pulverized, which are then added to a mixed solvent consisting of 50 ml of
dried chloroform and 5 ml of absolute ethanol. Dried gaseous hydrogen
chloride is passed under the ice-cooling to the mixture, until the gas is
saturated. The vessel for the mixture is sealed, followed by allowing to
stand for a week at a room temperature. The reaction mixture is
concentrated under a reduced pressure without heating, so that the
hydrochloric acid is completely removed. Thereafter, the residue is
dissolved in 80 ml of methanol. The solution is saturated with dried
gaseous ammonia at a room temperature, is heated under reflux for one
hour, and is concentrated to form a crystalline mass, which is then
recrystallized from 90% ethanol-water to obtain the desired compound as
white needles.
Yield: 4 g.
Melting Point: 290.degree. - 292.degree. C.
Elementary analysis of the compound having a presumed formula C.sub.22
H.sub.28 N.sub.4 O.sub.2.2HCl gave:
______________________________________
C H N
______________________________________
Calculated (%)
58.32 6.67 12.36
Found (%) 58.02 6.92 12.32
______________________________________
The starting material, 1,4-bis-(m,m'-cyanophenoxymethyl)-cyclohexane used
with this example is produced by the following steps:
4.8 Grams of meta-cyanophenol and 6 g. of 1,4-bis-(methanesulfonyloxy
methyl)-cyclohexane are added to 50 ml of methanol containing 2.65 g. of
sodium ethylate. To the solution are further added 50 ml of dimethyl
formamide, and the whole is heated under reflux for two hours. The
reaction solution is poured into water, and the resulting crystalline
substance is filtered and washed with water. After drying, the substance
is recrystallized from chloroform-methanol, to obtain white granule.
Yield: 5 g.
Melting Point: 182.degree. - 183.degree. C.
Elementary analysis of the compound having a presumed formula: C.sub.22
H.sub.22 N.sub.2 O.sub.2 gave:
______________________________________
C H N
______________________________________
Calculated (%)
76.27 6.40 8.09
Found (%) 75.91 6.41 8.10
______________________________________
EXAMPLE 2
Preparation of bis-(m,m'-amidinophenoxy)-para-xylene dihydrochloride
5 Grams of bis-(m,m'-cyanophenoxy)-para-xylene are pulverized, which are
then added to a mixed solvent consisting of 50 ml of dried chloroform and
5 ml of absolute ethanol. Dried gaseous hydrogen chloride is passed under
the ice-cooling to the mixture, until the gas is saturated. The vessel for
the mixture is sealed, and allowed to stand for a week at a room
temperature. The reaction mixture is concentrated under a reduced pressure
without heating, so that the hydrochloric acid is thoroughly removed. The
residue is then dissolved in 80 ml of methanol. The solution is saturated
with dried gaseous ammonia at a room temperature, is heated under reflux
for one hour, and is concentrated to form a crystalline mass, which is
then recrystallized from ethanol-water to obtain the desired compound as
white needles.
Yield: 2.5 g.
Melting Point: 298.degree. - 300.degree. C.
Elementary analysis of the compound having a presumed formula C.sub.22
H.sub.22 N.sub.4 O.sub.2.2HCl gave:
______________________________________
C H N
______________________________________
Calculated (%)
59.06 5.41 12.52
Found (%) 58.99 5.43 12.29
______________________________________
The starting material, bis-(m,m'-cyanophenoxy)-para-xylene used with this
example is produced by the following steps:
13.2 Grams of meta-cyanophenol are dissolved in 200 ml of ethanol
containing 7.5 g. of sodium ethylate. To the solution are added 8.7 g. of
.alpha.,.alpha.'-dichloro-para-xylene, and the whole is heated under
reflux with stirring for two hours. After cooling, the reaction solution
is poured into water, the resulting crystalline substance is filtered and
washed with water. After drying, the substance is recrystallized from
chloroform-methanol, to obtain white powder.
Yield: 14.5 g.
Melting Point: 174.degree. - 175.degree. C.
Elementary analysis of the compound having a presumed formula C.sub.22
H.sub.16 N.sub.2 O.sub.2 gave:
______________________________________
C H N
______________________________________
Calculated (%)
77.63 4.74 8.23
Found (%) 77.85 4.81 8.15
______________________________________
EXAMPLE 3
Preparation of
bis-(m,m'-amidinophenoxy)-2,3,4,6-tetrachloro-meta-xylene-dihydrochloride-
monohydrate
The procedure in Example 1 is repeated, except for the use of 5 g. of
bis-(m,m'-cyanophenoxy)-2,3,4,6-tetrachloro-meta-xylene having the melting
point of 229.degree. C. The resulting crystalline mass is recrystallized
from ethanol-water, to obtain the desired compound as white powdery
substance.
Yield: 2.2 g.
Melting Point: 289.degree. - 291.degree. C.
Elementary analysis of the compound having a presumed formula C.sub.22
H.sub.18 N.sub.4 O.sub.2.2HCl.H.sub.2 O gave:
______________________________________
C H N
______________________________________
Calculated (%)
43.81 3.68 9.29
Found (%) 44.01 3.81 9.23
______________________________________
EXAMPLE 4
Preparation of 1,4-bis-(m,m'-amidinophenoxy)-2-butene.dimethane sulfonate
The procedure in Example 1 is repeated, except for the use of 5 g. of
bis-(m,m'-cyanophenoxy)-2-butene having the melting point of 156.degree. -
157.degree. C., to obtain
bis-(m,m'-amidinophenoxy)-2-butene.hydrochloride.
The resulting compound is dissolved in water. To the solution is added
sodium carbonate to adjust its alkalinity to pH 9. The resulting
crystalline mass is filtered, and is suspended in ethanol. To the
suspension is added ethanol containing methane sulfonic acid, so as to
dissolve all the crystalline mass. After concentration, the mass is
recrystallized from ethanol-water, to obtain the desired compound as white
granule.
Yield: 2.2 g.
Melting Point: 204.degree. -205.degree. C.
Elementary analysis of the compound having a presumed formula C.sub.18
H.sub.20 N.sub.4 O.sub.2.2CH.sub.3 SO.sub.3 H gave:
______________________________________
C H N
______________________________________
Calculated (%)
46.50 5.46 10.84
Found (%) 46.16 5.39 10.70
______________________________________
EXAMPLE 5
Preparation of bis-(m,m'-amidinophenoxy)-1,4-butane.dimethane sulfonate
The procedure in Example 1 is repeated, except for the use of 4 g. of
bis-(m,m'-cyanophenoxy)-1,4-butane, to obtain
bis-(m,m'-amidinophenoxy)-1,4-butane-dihydrochloride.
The resulting compound is dissolved in water. To the solution is added 10%
solution of sodium carbonate, so that its alkalinity may amount to pH 9.
The resulting crystalline mass is filtered, and suspended in 30 ml of
ethanol. To the suspension is added ethanol containing methane sulfonic
acid, until the solution shows slight acidity. The resulting crystalline
substance is filtered, and is recrystallized from ethanol, to obtain the
desired compound as colourless granules.
Yield: 1.5 g.
Melting Point: 217.5.degree. - 218.degree. C.
Elementary analysis of the compound having a presumed formula C.sub.18
H.sub.22 N.sub.4 O.sub.2.2CH.sub.3 SO.sub.3 H gave:
______________________________________
C H N
______________________________________
Calculated (%)
46.32 5.83 10.80
Found (%) 46.58 5.88 10.77
______________________________________
The following Table 6 illustrates Examples 6 - 10 wherein data are given in
the compound of this invention.
##STR5##
Table 6
__________________________________________________________________________
Elementary Analysis
Ex. Molecular Formula
Calculated (%)
No.
A Melting Point (.degree.C)
Found (%)
C H N
__________________________________________________________________________
C.sub.22 H.sub.22 N.sub.4 O.sub.2.2HCl.2H.sub.2 O 149 -
150.degree.C.
54.66 55.01
5.84 5.84
11.59 11.73
7
##STR6## C.sub.22 H.sub.18 N.sub.4 O.sub.2 Cl.sub.4.2HCl.H.sub.2
O >300.degree.C.
43.81 44.20
6.68 6.63
9.29 9.11
8 (CH.sub.2).sub.2 S(CH.sub.2).sub.2
C.sub.18 H.sub.18 N.sub.4 S.2HCl.2H.sub.2 O
46.25
6.39
11.99
90.degree.C. 46.34
6.03
12.04
9 (CH.sub.2).sub.5
C.sub.19 H.sub.24 N.sub.4 O.sub.2.2HCl.H.sub.2 O
52.91
6.54
12.98
122 - 123.degree.C.
53.36
6.47
13.22
10 (CH.sub.2).sub.6
C.sub.20 H.sub.26 N.sub.4 O.sub.2.2HCl
56.21
6.60
13.11
250 - 251.degree.C.
56.44
6.58
13.30
__________________________________________________________________________
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