DESCRIPTION
RHODACYANINE DERIVATIVE AND PHARMACEUTICAL COMPOSITION FOR
TREATING LEISHMANIASIS
TECHNICAL FIELD
[0001] The present invention relates to a novel rhodacyanine derivative and a
pharmaceutical composition for treating leishmaniasis.
BACKGROUND ART
[0002] Leishmaniasis is a tropical parasitic infectious disease caused by protozoa of the
genus Leishmania parasitizing macrophages of a host such as a human, and is propagated
mainly via sand flies living in the desert. The WHO (World Health Organization) has
designated leishmaniasis as one of the six major tropical diseases. Patients thereof in Africa,
Middle and Near East, Latin America, and Asia account for most of the worldwide patients
(approximately 12 million people / year), and approximately 350 million people are
threatened by infection thereof. Many of the people live in developing countries, and are
hardly able to afford expensive drugs.
[0003] In the current treatment of leishmaniasis, pentavalent antimony formulations, such as
pentostam, are used as first-line drugs. However, pentavalent antimony formulations are
expensive, and side-effects caused by high toxicity thereof are problematic. Further, the
emergence of drug-resistant protozoa has been confirmed in India, which poses a new serious
problem. In a case in which antimony formulations may not be effective, diamidine
compounds such as an isethionate salt of pentamidine
(4,4′-(pentamethylenedioxy)dibenzamidine) are used (see, for example, Japanese
National-Phase Patent Publication (JP-A) No. 9-501653), and macrolide antibiotics such as an
antifungal antibiotic amphotericin B are secondarily used against fungal infection or
mucocutaneous leishmaniasis. However, the efficacy of these diamidine compounds and
amphotericin B is not as high as pentavalent antimony formulations. Further, these drugs are
expensive, and various side-effects thereof have been reported.
[0004] Other known therapeutic drugs for leishmaniasis include antiprotozoal drugs
containing germacrane and guaiane sesquiterpenoid compounds that have been extracted,
purified, and separated from an Asteraceae plant (Elephantopus mollis H.B.K.) (see, for
example, Japanese Patent Application Laid-open (JP-A) No. 2001-226369), and therapeutic
drugs for leishmaniasis of which the active ingredient is a glucopyranose terpenoid derivative
(see, for example, JP-A No. 11-106394). However, these therapeutic drugs have the
3
drawback of low drug efficacy.
[0005] In recent years, it has been disclosed that rhodacyanine dyes of a certain type exhibit
a strong cell-proliferation inhibiting activity against leishmania protozoa although the
cytotoxicity thereof against mammalian cells, which is an index of side-effects, is low, and the
rhodacyanine dyes thus have a high selective toxicity coefficient, and are effective as
anti-leishmania agents (JP-A Nos. 2004-331545 and 2006-104116). However, results
thereof in a leishmania protozoa proliferation inhibiting test in macrophages, and activity
thereof in an experimental system in which a pathological model of infected animals is used,
have not been reported.
SUMMARY OF INVENTION
Technical Problem
[0006] As described above, a compound which has high selective toxicity against parasitic
leishmaniasis, and which is confirmed to have sufficiently high activity and sufficient efficacy
as a pharmaceutical composition for treating leishmaniasis, has not been found.
An object of the present invention is provision of a novel pharmaceutical
composition for treating leishmaniasis which has high selective toxicity against parasitic
leishmaniasis, and which exhibits sufficiently high activity and sufficient efficacy against
leishmaniasis.
Solution to Problem
[0007] According to the invention, the following rhodacyanine derivatives and
pharmaceutical compositions for treating leishmaniasis are provided.
<1> A rhodacyanine derivative represented by the following General Formula (1):
[0008]
S
N
N
S
S
N
O
Y2
R1
R2
R3
Y1
X
􀀀􀀀 􀀀
[0009] In General Formula (1), R1, R2, and R3 each independently represent an alkyl group
4
which may be substituted; Y1 and Y2 each independently represent a hydrogen atom, a
chlorine atom, or a fluorine atom provided that Y1 and Y2 do not represent hydrogen atoms at
the same time; and X represents a counter anion.
<2> The rhodacyanine derivative described in <1>, wherein, in General Formula (1), R1,
R2, or R3 represents an alkyl group having from 1 to 8 carbon atoms, wherein the alkyl group
may be substituted.
<3> The rhodacyanine derivative described in <1> or <2>, wherein, in General Formula
(1), R1, R2, or R3 represents an alkyl group having at least one substituent selected from the
group consisting of a halogen atom, a hydroxyl group, an oxo group, an alkyl group, an
alkoxy group, and a carboxy group.
<4> The rhodacyanine derivative described in any one of <1> to <3>, wherein, in General
Formula (1), X is selected from the group consisting of a halogen ion, a sulfonate ion, a
sulfamate ion, a sulfate ion, a hydrogensulfate ion, a borate ion, an alkylphosphate ion, a
dialkylphosphate ion, a pyrophosphate ion, a carboxylate ion, a carbonate ion, a
hydrogencarbonate ion, and a hydroxide ion.
<5> The rhodacyanine derivative described in any one of <1> to <4>, wherein, in General
Formula (1), at least one of Y1 or Y2 represents a fluorine atom, R1 and R3 each represent a
methyl group, and R2 represents an ethyl group.
<6> The rhodacyanine derivative described in <1>, wherein the rhodacyanine derivative
is represented by the following Formula (2):
[0010]
S
N
CH3
N
S
S
N
O
F
C2H5
CH3 Cl
􀀀􀀀 􀀀
[0011]
<7> A pharmaceutical composition for treating leishmaniasis, comprising the
rhodacyanine derivative described in any one of <1> to <6> and a pharmaceutically
acceptable carrier.
5
<8> A method of treating leishmaniasis, comprising administering the pharmaceutical
composition for treating leishmaniasis described in <7> to a patient infected with leishmania
or suspected to be infected with leishmania.
<9> A composition for use as a medicament, comprising the rhodacyanine derivative
described in any one of <1> to <6> and a pharmaceutically acceptable carrier.
DESCRIPTION OF EMBODIMENTS
[0012] The rhodacyanine derivative according to the invention is a compound represented
by the following General Formula (1). In General Formula (1), R1, R2, and R3 each
independently represent an alkyl group which may be substituted; Y1 and Y2 each
independently represent a hydrogen atom, a chlorine atom, or a fluorine atom provided that Y1
and Y2 do not represent hydrogen atoms at the same time; and X represents a counter anion.
[0013]
S
N
N
S
S
N
O
Y2
R1
R2
R3
Y1
X
􀀀􀀀 􀀀
[0014] The present inventors have found the above rhodacyanine dye compound, which has
a fluorine atom or a chlorine atom as a substituent, exhibits high efficacy against
leishmaniasis and less side-effects in an experimental system in which a pathology model of
an animal infected with leishmania protozoa is used. Thus, the inventors have completed the
present invention. A pharmaceutical composition for treating leishmaniasis of which a main
component is the above compound has high selective toxicity, and exhibits high activity and
efficacy against leishmaniasis.
The invention is described in detail below.
Hereinafter, a numerical range expressed by "from X to Y" is inclusive of X and Y as
the lower limit value and the higher limit value, respectively.
[0015] The rhodacyanine derivative according to the invention is a compound represented
by General Formula (1), in which R1, R2, and R3 each independently represent an alkyl group
6
which may be substituted, Y1 and Y2 each independently represent a hydrogen atom, a
chlorine atom, or a fluorine atom provided that Y1 and Y2 do not represent hydrogen atoms at
the same time, and X represents a counter anion.
[0016] In the compound represented by General Formula (1), R1, R2, and R3 each
independently represent preferably an alkyl group having from 1 to 8 carbon atoms, wherein
the alkyl group may be substituted. Specific examples of the alkyl group include a methyl
group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl
group, and an octyl group.
[0017] The alkyl group represented by any of R1, R2, or R3 may have one substituent, or two
or more substituents. Examples of substituents include halogen atoms, a hydroxyl group, an
oxo group, alkyl groups, alkoxy groups, and a carboxyl group, and examples of preferable
substituents include halogen atoms, an oxo group, alkyl groups, alkoxy groups, and a carboxyl
group. R1, R2, and R3 each independently represent more preferably an unsubstituted alkyl
group, and still more preferably a methyl group or an ethyl group. In a particularly
preferable example for R1, R2, and R3, R1 and R3 represent methyl groups and R2 represents
an ethyl group.
[0018] In the compound represented by General Formula (1), Y1 and Y2 each independently
represent a hydrogen atom, a fluorine atom, or a chlorine atom. It is more preferable that at
least one of Y1 or Y2 represents a fluorine atom, and it is particularly preferable that Y1
represents a hydrogen atom and Y2 represents a fluorine atom. However, Y1 and Y2 are not
hydrogen atoms at the same time.
[0019] X in General Formula (1) represents a counter anion, which is not particularly limited.
Preferable examples thereof include: halogen ions such as a chlorine ion, a bromine ion, and
an iodine ion; sulfonate ions such as aliphatic and aromatic sulfonate ions, such as a
methanesulfonate ion, a trifluoromethanesulfonate ion, a p-toluenesulfonate ion, a
naphthalenesulfonate ion, and a 2-hydroxyethanesulfonate ion; sulfamate ions such as a
cyclohexanesulfamate ion; sulfate ions such as a methylsulfate ion and an ethylsulfate ion; a
hydrogensulfate ion; a borate ion; alkylphosphate ions and dialkylphosphate ions such as a
diethylphosphate ion and a methylhydrogenphosphate ion; pyrophosphate ions such as a
trimethylpyrophosphate ion; carboxylate ions such as an acetate ion, a propionate ion, a
valerate ion, a citrate ion, a maleate ion, a fumarate ion, a lactate ion, a succinate ion, a
tartarate ion, and a benzoate ion, wherein a carboxylate ion having a carboxyl group and a
hydroxyl group is suitable for use; a carbonate ion; a hydrogencarbonate ion; and a hydroxide
ion. Among the above, a halogen anion, a sulfonate anion, or a carboxylate anion is
preferable as the counter anion, and a halogen anion is still more preferable, and a chlorine
7
ion is particularly preferable.
[0020] The compounds represented by General Formula (1) and the compound represented
by Formula (2) can be produced easily from known starting materials according to the
following method disclosed in non-patent documents such as E. B. K Nott, J. Chem. Soc., p.
4762 (1952), ibid p. 949 (1955), Kawakami et al., J. Med. Chem., p. 3151 (1997), and K.
Pudhom, Heterocycles., p. 207 (2009), which are incorporated herein by reference.
[0021]
N
S
S
O
R2
S
N
Y2
R3
H3C
Y1
S
N
R1
N
S
S
O
R2
TsO
PhHN
N-Ph
TsOMe
S
S
O
R2
N
Ph
Ac
N
N
S
O
R2
S
N
Y2
R3
Y1
S
N
R1
N
S
O
R2
SCH3
Y1
S
N
R1
Y1
S
N
R1
CH3
TsO
X
TsO
1)
2) Ac2O/NEt3
-
+ - DMF,Toluene
1)
MeCN,NEt3
2) HX, MeOH
+
Ac2O/NEt3
[0022] Typical examples of rhodacyanine compounds represented by General Formula (1)
according to the invention include, but are not limited to, the compounds shown below.
8
[0023]
S
N
N
S
O
S
N
Cl
S
N
N
S
O
S
N
F
Cl
S
N
N
S
O
S
N
N
S
O
S
N
F
CH3SO3
F
S
N
N
S
O
S
N
Cl
S
N
N
S
O
HOCH2
S
N
F
S
N
N
S
O
COOH
S
N
F
Cl
S
N
N
S
O
S
N
F
Cl
S
N
N
S
O
S
N
F
S
N
F
SO3
Cl
HCO2CHCHCOO
S
N
N
S
O
S
N
Cl
Cl
F
S
N
N
S
O
S
N
Cl
Cl
S
N
N
S
O
S
N
Cl
CH3SO3
A B
C D
F
H
I
K
E
G
J
L
-
F F
Cl
9
[0024] The pharmaceutical composition according to the invention includes the
rhodacyanine derivative represented by General Formula (1) as an active ingredient, and can
be used particularly as a pharmaceutical composition for treating leishmaniasis. The
pharmaceutical composition for treating leishmaniasis according to the invention includes the
rhodacyanine derivative and a pharmaceutically acceptable carrier. The pharmaceutical
composition for treating leishmaniasis according to the invention may be administered
together with a conventional anti-leishmania agent, such as pentostam, amphotericin B, or
miltefosine, or may contain such a known anti-leishmania agent.
[0025] Examples of the pharmaceutically acceptable carrier in the pharmaceutical
composition for treating leishmaniasis according to the invention include physiological saline
and buffer solutions. The pharmaceutical composition for treating leishmaniasis may further
include various known additive components such as diluents, stabilizers, perservatives, and
tonicity agents. Examples of additive components include glucose, saccharose, lactose, ethyl
alcohol, glycerin, mannitol, sorbitol, pentaerythritol, diethyleneglycol, dipropyleneglycol,
polyethyleneglycol 400, other polyethyleneglycols, glycerin trilaurate, mono-, di-, and
tri-glycerides of fatty acids, peptine, starch, alginic acid, xylol, talc, lycopodium, olive oil,
peanut oil, castor oil, corn oil, safflower oil, sesami oil, sunflower oil, gelatin, lecithin, silica,
cellulose, methylcellulose, hydroxyethylcellulose, calcium stearate, calcium laurate,
magnesium oleate, calcium palmitate, cyclodextrins, emulsifying agents (for example, an
ester of a saturated or unsaturated fatty acid having from 2 to 22 carbon atoms and a
monohydric or polyhydric alcohol having from 2 to 20 carbon atoms such as glycol, glycerin,
diethyleneglycol, pentaerythritol, ethyl alcohol, butyl alcohol, or octadecyl alcohol), and
silicones such as methylpolysiloxane.
[0026] The pharmaceutically effective amount and administration method or means of the
pharmaceutical composition for treating leishmaniasis according to the invention vary
depending on the type of leishmania protozoa, the parasitic site, the seriousness of the disease,
the therapeutic method, the age, weight, sex, and health condition of the patient, and the
genetic and racial background of the patient. The dose of the pharmaceutical composition
for treating leishmaniasis according to the invention, in terms of active ingredient amount, is
generally from 1 to 2,000 mg/day/70 kg body weight, and is more generally from 50 to 500
mg/day/70 kg body weight. Examples of preferable dosage forms of the pharmaceutical
composition for treating leishmaniasis according to the invention include a liquid formulation
in which the rhodacyanine derivative according to the invention and optionally the additional
components described above are dissolved in a 5% by mass glucose aqueous solution, a gel
formulation, and an ointment. Examples of preferable administration methods for the
10
pharmaceutical composition for treating leishmaniasis according to the invention include
intravenous administration, intraperitoneal administration, subcutaneous injection, oral dosage,
and application to the skin.
[0027] The method of treating leishmaniasis according to the invention includes
administering, to a patient infected with leishmania or suspected to be infected with
leishmania, the pharmaceutical composition for treating leishmaniasis containing the
rhodacyanine derivative represented by General Formula (1), preferably represented by
Formula (2), and a pharmaceutically acceptable carrier.
The pharmaceutical composition for treating leishmaniasis according to the invention
has high selective toxicity against parasitic leishmaniasis. Examples of the leishmaniasis
include cutaneous leishmaniasis, visceral leishmaniasis, and mucocutaneous leishmaniasis.
The pharmaceutical composition for treating leishmania infection according to the invention
has particularly high activity against visceral leishmaniasis. Therefore, it is particularly
preferable that the pharmaceutical composition for treating leishmaniasis according to the
invention is used for treating visceral leishmaniasis.
[0028] Examples of the present invention are described below. However, the technical
scope of the invention is not limited to the examples.
EXAMPLES
[0029] Synthesis Example 1
Synthesis of 2-((3-ethyl-5-(2-(3-methylbenzo[d]thiazole-2(3H)-ylidene)ethylidene)-
4-oxothiazolidine-2-ylidene)methyl-5-fluoro-3-methylbenzo[d]thiazole-3-ium chloride
(Compound A)
Under an argon atmosphere, a mixture of 0.5 g (1.5 mmol) of
1-(3-ethyl-5-(2-(3-methylbenzo[d]thiazole-2(3H)-ylidene)ethylidene))-2-thioxo-4-oxothiazoli
dine, 0.84 g (4.5 mmol) of methyl p-toluenesulfonate, and 1.5 mL of DMF was stirred in
toluene at 115 °C for 6 hours. After the mixture was cooled to ambient temperature, a liquid
obtained by adding 0.51 g (1.5 mmol) of 5-fluoro-3-methylbenzo[d]thiazole-3-ium
p-toluenesulfonate to 50 mL acetonitrile was added to the mixture. The resultant mixture
was stirred for 12 hours at 75 °C. The precipitate formed was collected and washed with
acetonitrile and ethyl acetate to give 0.64 g of a tosylate as a dark-green solid. The yield was
65.8%. The analytical values of the obtained solid are as described below:
1H NMR (400MHz, DMSO-d6) δ: 8.20 (dd, J=8.8 and 5.2Hz, 1H), 7.82 (d, J=7.7Hz, 1H),
7.71(dd, J=9.9 and 2.1Hz, 1H), 7.57(d, J=13.2Hz, 1H), 7.49 (d, J=8.0Hz, 2H), 7.42-7.18 (m,
4H), 7.11 (d, J=8.0Hz, 2H), 6.69 (s, 1H), 5.90 (d, J=13.2Hz, 1H), 4.15 (q, J=7.1Hz, 1H), 3.96
11
(s, 3H), 3.65 (s, 3H), 2.29 (s, 3H), 1.29 (t, J=7.1Hz, 3H);
MS(ESI+) m/z: 482.1 [M+]
[0030] 0.5 g (0.76 mmol) of the above tosylate was stirred in 50 mL methanol at 80ºC for 30
minutes. Then, about 2.3 mL concentrated hydrochloric acid was slowly added to the
mixture. After the mixture was stirred for 30 minutes, the precipitate was filtered and
washed with methanol to give 0.39 g of
2-((3-ethyl-5-(2-(3-methylbenzo[d]thiazole-2(3H)-ylidene)ethylidene)-4-oxothiazolidine-2-yli
dene)methyl-5-fluoro-3-methylbenzo[d]thiazole-3-ium chloride (Compound A) as a
dark-green solid. The yield was 99%. The physical property values and analytical values
of this compound are as described below:
Mp: 274.5-275.6 ºC;
UV-vis (H2O): λ(nm)(log ε/L mol-1cm-1): 528(4.54), 356(4.23);
IR ν (neat, cm-1): 2972, 1685, 1525, 1471, 1376, 1362, 1314, 1276, 1198, 1055, 1033, 941,
891, 819, 747;
1H NMR (400MHz, DMSO-d6) δ: 8.27 (dd, J=8.9 and 5.2Hz, 1H), 7.83 (d, J=7.7Hz, 1H),
7.75 (dd, J=9.9 and 2.3Hz, 1H), 7.59 (d, J=13.2Hz, 1H), 7.43-7.31 (m, 3H), 7.28 (t, J=6.0Hz,
1H), 6.72 (s, 1H), 5.88 (d, J=13.2Hz, 1H), 4.17 (q, J=7.1Hz, 2H), 3.98 (s, 3H), 3.68 (s, 3H),
1.29 (t, J=7.1Hz, 3H);
13C NMR (125MHz, DMSO-d6) δ: 164.0, 163.4, 163.0, 161.5, 157.3, 141.6, 134.6, 127.5,
125.0, 124.2, 123.8, 122.6, 121.5, 113.6, 113.4, 112.4, 102.3, 102.1, 101.7, 90.9, 86.5, 34.8,
32.9, 12.4;
MS (ESI+) m/z: 482.1 [M+]
Anal. calcd. for C24H21ClFN3OS3·2H2O: C, 51.97; H, 5.04; N, 7.55. Found: C, 52.02; H,
4.55; N, 7.58.
[0031] Synthesis Example 2
Synthesis of 2-((3-ethyl-5-(2-(3-methylbenzo[d]thiazol-2(3H)-ylidene)ethylidene)-
4-oxothiazolidin-2-ylidene)methyl)-5-fluoro-3-methylbenzo[d]thiazol-3-ium maleate
(Compound C)
After dissolving Compound A, which was obtained in Synthesis Example 1, in
methanol, the anion thereof was replaced with maleic acid according to an ordinary method to
give 2-((3-ethyl-5-(2-(3-methylbenzo[d]thiazol-2(3H)-ylidene)ethylidene)-
4-oxothiazolidin-2-ylidene)methyl)-5-fluoro-3-methylbenzo[d]thiazol-3-ium maleate
(Compound C) as a dark-green solid. The physical property values and analytical values of
this compound are as described below:
12
Mp: 260.1-260.8 ºC;
UV-vis (H2O):  (nm) (log /L mol-1cm-1): 526 (4.68), 355 (4.37);
IR  (neat, cm-1): 2972, 2937, 2866, 1684, 1526, 1472, 1376, 1360, 1346, 1314, 1275, 1198,
1055, 1032, 1013, 940, 891, 818, 747;
1H NMR (400 MHz, DMSO-d6) δ : 8.28 (dd, J = 8.8 and 5.1 Hz, 1H), 7.99 (dd, J = 9.9 and
1.9 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.75 (d, J = 13.2 Hz, 1H), 7.59 (d, J = 8.2 Hz, 1H), 7.49
(dd, J = 17.0 and 8.3 Hz, 2H), 7.33 (t, J = 7.6 Hz, 1H), 6.78 (s, 1H), 6.03 (d, J = 13.2 Hz, 3H),
4.19 (q, J = 7.1 Hz, 2H), 4.06 (s, 3H), 3.78 (s, 3H), 1.27 (t, J = 7.1 Hz, 3H);
MS (ESI+) m/z: 482.3 [M+].
[0032] Synthesis Example 3
Synthesis of 2-((3-ethyl-5-(2-(3-methylbenzo[d]thiazol-2(3H)-ylidene)ethylidene)-
4-oxothiazolidin-2-ylidene)methyl)-5-fluoro-3-methylbenzo[d]thiazol-3-ium
methanesulfonate (Compound D)
After dissolving Compound A, which was obtained in Synthesis Example 1, in
methanol, the anion thereof was replaced with methanesulfonic acid according to an ordinary
method to give 2-((3-ethyl-5-(2-(3-methylbenzo[d]thiazol-2(3H)-
ylidene)ethylidene)-4-oxothiazolidin-2-ylidene)methyl)-5-fluoro-3-methylbenzo[d]thiazol-3-
ium mesylate (Compound D) as a dark-green solid. The physical property values and
analytical values of this compound are as described below:
Mp >300 oC;
UV-vis (H2O):  (nm) (log /L mol-1cm-1): 524 (4.83), 355 (4.52);
IR (neat, cm-1): 2971, 2937, 1682, 1525, 1470, 1376, 1358, 1317, 1276, 1197, 1057, 1033,
939, 891, 819, 747;
1H NMR (400 MHz, DMSO-d6) δ: 8.29 (dd, J = 8.8 and 5.2 Hz, 1H), 8.01 (dd, J = 9.9 and 2.0
Hz, 1H), 7.90 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 13.2 Hz, 1H), 7.61 (d, J = 8.3 Hz, 1H), 7.49 (m,
2H), 7.34 (t, J = 7.8 Hz, 1H), 6.77 (s, 1H), 6.05 (d, J = 13.2 Hz, 1H), 4.19 (q, J = 7.1 Hz, 2H),
4.05 (s, 3H), 3.77 (s, 3H), 2.29 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H);
MS (ESI+) m/z: 483.2 [M+].
[0033] Example 1
(1) Culture of Leishmania Protozoa
In this test, Leishmania donovani (L. Don, MHOM/ET/67/L82 strain) was used.
The protozoa were maintained in the Syrian Golden hamster, and amastigotes were collected
from the spleen of an infected hamster. A SM medium supplemented with 10%
heat-inactivated fetal bovine serum was used in the experiment, and the pH was adjusted to
5.4. The protozoa were grown at 37 ºC under an atmosphere having a CO2 concentration of
13
5%.
[0034]
(2) Leishmania Protozoa Proliferation Inhibition Test
The activity with respect to inhibition of the proliferation of leishmania protozoa was
studied for Compounds A, C, and D according to the invention for test, and for MKT-077,
Compound X, and miltefosine (positive compound, existing drug) as comparative compounds.
The structures of the compounds are as shown below. Each compound was dissolved in
DMSO to give a test solution having a predetermined concentration.
[0035]
S
N
N
S
S
N
O
F
Cl
S
N
N
S
O
S
N
F
CH3SO3
HCO2CHCHCOO
A C
D
S
N
N
S
O
S
N
F
Cl2
MKT-077
S
N
N
S
O
N
S
N
N
S
S
N
O Cl
X
-
[0036] Each of the compounds to be used in the test was dissolved in DMSO to give a test
solution having a concentration of 10 mg/mL. Assays were performed in 96-well culture
plates, with each well containing 100 μL of the culture medium containing 105 amastigotes
with or without the test compound at a predetermined serial dilution. The test was
conducted twice.
[0037] After incubating the culture plates for 72 hours in an incubator, proliferation
inhibiting activity was assayed. The assay was conducted in the following manner. 10 μL
of an Alamar Blue aqueous solution (12.5 mg resazurin dissolved in 100 mL of distilled
water) was added to each well, and the plates were incubated for another 2 hours. Each
culture plate was mounted on a microplate fluorometer, and irradiated with light at an
14
excitation wavelength of 536 nm. The intensity of fluorescence at 588 nm was measured,
and the residual ratios of leishmania protozoa in the group added with the test solution and in
the control were calculated.
The 50% proliferation inhibitory concentration (IC50) values were determined based
on the residual ratios of protozoa obtained above, and the results are shown in Table 1.
[0038]
(3) Rat L6 Cell Proliferation Inhibition Test
Rat-derived L6 cells (rat skeletal myoblast cells) were used. The cells were
cultured at 37 ºC at a CO2 concentration of 5%, using, as a culture medium, a RPMI 1640
medium supplemented with 1% L-glutamine (200 mM) and 10% fetal bovine serum (FBS).
Each of the compounds to be tested was dissolved in DMSO to give a test solution
having a concentration of 10 mg/mL.
96-well culture plates of which each well contained 4 × 104 cells in 100 μL of the
culture medium were prepared. After 24 hours, a 3-fold dilution series was prepared
according to an ordinary method by adding a medium containing the compound to be tested,
and the test was conducted.
After incubating the culture plates for 72 hours in an incubator, the plates were
inspected with respect to proliferation activity. The inspection was conducted as follows.
10 μL of the Alamar Blue aqueous solution as described above was added to each well, and
the plates were incubated for another 2 hours. Then, the culture plates were mounted on a
microplate fluorometer, and irradiated with light at an excitation wavelength of 536 nm.
The intensity of fluorescence at 588 nm was measured, and the residual ratios of L6 cells in
the group added with the test solution and in the control were calculated.
The 50% proliferation inhibitory concentration (IC50) values were determined based
on the residual ratios of cells obtained above, and the results are shown in Table 1. In Table
1, "ND" represents that the test was not conducted.
[0039]
(4) Determination of Drug Efficacy against Leishmania
The selective toxicity coefficient, which is used as an index of the selectivity against
leishmania protozoa, was calculated according to the following equation, and the drug
efficacy was evaluated. The selective toxicity coefficient of each compound is shown in
Table 1.
Selective toxicity coefficient = (IC50 value of the test compound against rat L6 cells)
/ (IC50 value of the test compound against leishmania protozoa)
15
[0040]
Table 1
50% Proliferation Inhibitory
Concentration (μM)
Compound
IC50
L. Don
IC50
L-6 Cells
Selective Toxicity
Coefficient
Compound A 0.011 >173 > 15,000
Compound C 0.025 71.7 2,870
Compound D 0.020 84.5 4,220
MKT-077 (Comparative
Compound)
0.25 115 450
Compound X (Comparative
Compound)
0.052 125 2,400
Miltefosine (Comparative
Compound)
0.43 ND ―
[0041] Compounds A, C, and D according to the invention exhibited far higher protozoa
inhibitory activity and far higher selective toxicity coefficient than MKT-077, and higher
activity and higher selective toxicity coefficient than Compound X having hydrogen atoms as
Y1 and Y2 in Formula (1). Further, Compounds A, C, and D exhibited more than 10 times
higher activity than miltefosine, which is an existing drug, and only a weak toxicity toward
normal cells. That is, Compounds A, C, and D are assessed as effective anti-leishmania drug
with less side-effects. Therefore, it is found that the introduction of a fluorine atom is
important.
[0042] Example 2
Leishmania Protozoa Proliferation Inhibition Test in Macrophages
The inhibitory activity on the proliferation of leishmania protozoa in macrophages
was examined for Compounds A and B according to the invention and comparative
compounds MKT-077, Compound X, Compound Y, Compound Z, and miltefosine (positive
compound, existing drug). The structures of the compounds are as shown below.
16
[0043]
S
N
N
S
S
N
O
F
Cl
S
N
N
S
O
S
N
F
F
A B
Cl2
MKT-077
S
N
N
S
O
N
S
N
N
S
S
N
O Cl
X
Cl
O
O
S
N
N
S
O
S
N
Y
O
O
S
N
N
S
O
S
N OH
Z
[0044] Peritoneal macrophages from NMRI mice are collected 1 day after stimulation of
macrophage production with an intraperitoneal injection of 2 mL of a 2% potato starch
suspension. The present test was conducted at 37 ºC under an atmosphere having a CO2
concentration of 5%. Each of the compounds to be used in the test was dissolved in DMSO
to give a test solution having a predetermined concentration.
100 μL of a murine macrophage suspension (4 × 105/mL) in a RPMI 1640 medium
containing bicarbonate and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)
and supplemented with 10% heat-inactivated fetal bovine serum (RPMI/FBS medium) was
added to wells of 96-well culture plates. After 24 hours, 100 μL of a suspension containing
amastigotes (1.2 × 106/mL) was further added to each well, giving a suspension having a
amastigotes/macrophages ratio of 3/1. The amastigotes were prepared in the same manner
as above, and suspended in the RPMI/FBS medium. After 24 hours, the medium containing
free amastigotes was removed, the cells were washed once with medium, and fresh media
containing the test compound at serial dilutions (four 3-fold dilutions for each compound)
were added to the wells. The growth of the protozoa in samples treated with the
test-compound-containing media was compared with that of the control. Specifically, after 4
days of incubation, the samples were fixed with methanol, and thereafter stained with a 10%
17
Giemsa solution. The IC50 values were determined based on the number of infected
macrophages.
The IC50 value of each compound against leishmania protozoa is shown in Table 2.
[0045]
Table 2
IC50 L.Don (μM)
Compound A 0.353
Compound B 0.452
X (Comparative compound) Could not be tested as the macrophages died
MKT-077
(Comparative compound)
Could not be tested as the macrophages died
Compound Y
(Comparative compound)
Could not be tested as the macrophages died
Compound Z
(Comparative compound)
Could not be tested as the macrophages died
Miltefosine
(Comparative compound)
0.811
[0046] As shown in Table 2, the compound according to the invention has a high ability to
inhibit the proliferation of leishmania protozoa in macrophages, as compared to conventional
anti-leishmania drugs as comparative compounds.
MKT-077 and Compounds Y and Z were all unable to be tested since the
macrophages were killed by strong toxic effects exerted by these compounds in the present
macrophage test. It is unexpected and surprising finding that such a harsh toxic effect
against macrophages is reduced by introduction of a halogen atom, particularly a fluorine
atom, according to the invention, and anti-leishmania activity is manifested.
[0047] Example 3
Anti-leishmania Activity Test Using Mice Infected with Leishmania
Compound A was used as the test compound in this test. Further, a
pentostam-sensitive L. donovani (strain MHOM/ET/67/HU3) was used in this test, and the
protozoa were maintained in Syrian Golden hamsters, and amastigotes were isolated from
spleen cells of the infected hamster. Then, an inoculum containing 7.5 × 107 amastigotes/mL
in RPMI 1640 was prepared. Female BALB/c mice (20 g) were infected with the
amastigotes by intravenous injection at the tail vein on day 0. On day 7 after infection, one
mouse was sacrificed, liver smears were methanol-fixed and Giemsa-stained, thereby
confirming infection. The test compound was dissolved in an aqueous solution containing
18
10% ethanol and 5% glucose, and administered to the mice once a day for 5 days from day 7
to day 11. 3 days after the completion of the administration of the drug, livers were removed
and weighed, and smears were methanol-fixed and then Giemsa-stained. The number of
amastigotes (average value) per 500 liver cells was determined by counting, and the infection
inhibition rate was determined based on the comparison thereof with mice that had not been
treated with the drug. The results are shown in Table 3.
Infection inhibition rate (%) = [1-(b-a)/(c-a)] × 100
a: Initial infection rate
b: Infection rate in the mice treated with the test solution
c: Infection rate in the control mice
[0048]
Table 3
Dose (Intravenous Injection)
(mg/kg/day)
Infection Inhibition Rate
(%)
Compound A 4.10 × 5days 97.1
Compound A 1.30 × 5days 94.9
Compound A 0.20 × 5 days 16.1
[0049] As shown in Table 3, it was found that the compound according to the invention
exhibits high infection-inhibition rate when administered by intravenous injection. Due to
the easiness of synthesis thereof, the compound according to the invention is assessed as an
inexpensive and effective anti-leishmania drug, and is effective as a single drug or a combined
formulation with one or more other anti-leishmania drugs.
Therefore, it is confirmed that the pharmaceutical composition for treating
leishmaniasis containing the rhodacyanine derivative according to the invention as an active
ingredient has high selective toxicity and exhibits high activity and efficacy against
leishmaniasis.
[0050] The disclosure of Japanese Patent Application No. 2009-251292 filed on October 13,
2009 is herein incorporated by reference.
All publications, patent applications, and technical standards mentioned in this
specification are herein incorporated by reference to the same extent as if each individual
publication, patent application, or technical standard was specifically and individually
indicated to be incorporated by reference.
19
CLAIMS
1. A rhodacyanine derivative represented by the following General Formula (1):
S
N
N
S
S
N
O
Y2
R1
R2
R3
Y1
X
􀀀􀀀 􀀀
wherein, in General Formula (1), R1, R2, and R3 each independently represent an
alkyl group which may be substituted; Y1 and Y2 each independently represent a hydrogen
atom, a chlorine atom, or a fluorine atom provided that Y1 and Y2 do not represent hydrogen
atoms at the same time; and X represents a counter anion.
2. The rhodacyanine derivative according to claim 1, wherein, in General Formula
(1), R1, R2, or R3 represents an alkyl group having from 1 to 8 carbon atoms, wherein the
alkyl group may be substituted.
3. The rhodacyanine derivative according to claim 1 or 2, wherein, in General
Formula (1), R1, R2, or R3 represents an alkyl group having at least one substituent selected
from the group consisting of a halogen atom, a hydroxyl group, an oxo group, an alkyl group,
an alkoxy group, and a carboxy group.
4. The rhodacyanine derivative according to any one of claims 1 to 3, wherein, in
General Formula (1), X is selected from the group consisting of a halogen ion, a sulfonate ion,
a sulfamate ion, a sulfate ion, a hydrogensulfate ion, a borate ion, an alkylphosphate ion, a
dialkylphosphate ion, a pyrophosphate ion, a carboxylate ion, a carbonate ion, a
hydrogencarbonate ion, and a hydroxide ion.
20
5. The rhodacyanine derivative according to any one of claims 1 to 4, wherein, in
General Formula (1), at least one of Y1 or Y2 represents a fluorine atom, R1 and R3 each
represent a methyl group, and R2 represents an ethyl group.
6. The rhodacyanine derivative according to claim 1, wherein the rhodacyanine
derivative is represented by the following Formula (2):
S
N
CH3
N
S
S
N
O
F
C2H5
CH3 Cl
􀀀􀀀 􀀀 .
7. A pharmaceutical composition for treating leishmaniasis, comprising the
rhodacyanine derivative of any one of claims 1 to 6 and a pharmaceutically acceptable carrier.
8. A composition for use as a medicament, comprising the rhodacyanine derivative
according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.