Note: Descriptions are shown in the official language in which they were submitted.
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NEAR INFRARED FLUORESCENT CONTRAST AGENT AND
FLUORESCENCE IMAGING
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a near infrared fluorescent contrast
agent and fluorescence imaging using said contrast agent.
In treating diseases, it is vital to detect morphological and functional
changes
caused by the disease in the living body at an early stage of the disease.
Especially when treating cancer, the site and size of tumor are significant
determinant factors of the effective treatment design. Known methods for this
purpose include biopsy by puncture and the like, and imaging diagnoses such
as X-ray imaging, MRI, ultrasound imaging and the like. Biopsy is effective
for
definitive diagnosis, but at the same time it places great burden on test
subjects and is not suitable for tracking time-course changes in lesions. X-
ray
imaging and MRI inevitably expose test subjects to radiation and magnetic
waves. In addition, conventional imaging diagnoses as mentioned above
require complicated operation and a long time for measurement and diagnosis.
A large apparatus used for this purpose also makes it difficult to apply these
methods during operation.
One of the image diagnoses is fluorescence imaging (Lipspn R. L. et al., J.
Natl. Cancer Inst., 26, 1-11 (1961)). This method uses, as a contrast agent, a
substance that emits fluorescence upon exposure to an excitation light having
a specific wavelength. Thus, a body is exposed to an excitation light from
outside the body and the fluorescence emitted from the fluorescent contrast
agent in the body is detected.
Such fluorescent contrast agent may be, for example, a porphyrin compound
that accumulates in tumor and is used for photodynamic therapy (PDT), such
as haematoporphyrin. Other examples include photophrin and benzoporphyrin
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(see Lipspn R. L. et al., supra, Meng T. S. et al., SPIE, 1641, 90-98 (1992),
WO
84/04665 and the like). These compounds are originally used for PDT and
have phototoxicity, since this is what PDT requires. Consequently, these are
not desirable diagnostic agents.
Meanwhile, retinal circulatory microangiography using a known fluorescent
dye, such as fluorescein, fluorescamin and riboflabin, has been known (U.S.
Patent No. 4945239). These fluorescent dyes emit fluorescence in a visible
light region of 400-600 nm. In this region, the light transmission through
living
tissue is very low, so that the detection of lesions in the deep part of a
body is
nearly impossible.
In addition, the use, as fluorescent contrast agent, of cyanine compounds
inclusive of indocyanine green (hereinafter to be abbreviated as ICG), which
are used to determine liver function and cardiac output, has been documented
(Haglund M. M. et al., Neurosurgery, 35, 930 (1994), Li, X. et al., SPIE,
2389,
789-797 (1995)). Cyanine compounds show absorbance in a near infrared
light region (700-1300 nm).
Near infrared light shows high transmission through living tissues and can
pass
through a skull of about 10 cm in size. Because of this, it has been
increasingly attracting attention in clinical medicine. For example, optical
CT
technique using optical transmission of medium has been drawing attention in
the clinical field as a new technology. This is because near infrared light
can
pass through living body and can be used for monitoring oxygen concentration
and circulation in the living body.
Cyanine compounds emit fluorescence in the near infrared region. The
fluorescence in this region can pass through living tissues and offers the
potential for a fluorescent contrast agent. Various cyanine compounds have
been'developed in recent years and tried as fluorescent contrast agents
(W096/17628, WP97/13490 and the like). However, an agent having sufficient
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solubility in water and safety to living body, as well as capability of
distinguishing normal tissues from diseased tissues (selectivity for imaging
target site), is not in existence.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a fluorescent
contrast agent. The inventive agent is low toxic and has a superior solubility
in
water. In addition, it emits fluorescence in a near infrared region that can
pass
through living tissues, and permits specific imaging of tumor and/or blood
vessel.
Another object of the present invention is to provide a method of fluorescence
imaging using said near infrared fluorescent contrast agent.
The present invention is predicated on the finding that introduction of three
or
more sulfonic acid groups into a cyanine dye compound results in the provision
of a fluorescent contrast agent having a high solubility in water. It has been
also found that a method of fluorescence imaging can be established when this
contrast agent is used.
Thus, the present invention provides the following.
(1) A near infrared fluorescent contrast agent comprising a compound having
three or more sulfonic acid groups in a molecule, which is represented by the
formula [I]
X y
~]
/ LLr
CZ Z
aN N
N
R1 R'
wherein R' and R2 are the same or different and each is a substituted or
unsubstituted alkyl; Z' and Z2 are each nonmetallic atoms necessary for
forming a substituted or unsubstituted condensed benzo ring or condensed
SUBSTITUTE SHEET (RULE 26)
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naphtho ring; r is 0, 1 or 2; L' - L' are the same or different and each is a
substituted or unsubstituted methine,
provided that when r is 2, L 6 and L'that occur in duplicate are the same or
different; and
X and Y are the same or different and each is a group of the formula
R3
1
- O - , - S - ~ CH=CH - or - C -
1 4
R
wherein R3 and R4 are the same or different and each is substituted or
unsubstituted alkyl, or a pharmaceutically acceptable salt thereof.
(2) The near infrared fluorescent contrast agent of (1) above, which is free
of a
carboxylic acid group in a molecule.
(3) The near infrared fluorescent contrast agent of (1) or (2) above, wherein,
in
the formula [I], r is 1.
(4) The near infrared fluorescent contrast agent of any of (1) to (3) above,
wherein 4 or more sulfonic acid groups are contained in a molecule.
(5) The near infrared fluorescent contrast agent of any of (1) to (4) above,
wherein 10 or less sulfonic acid groups are contained in a molecule.
(6) The near infrared fluorescent contrast agent of any of (1) to (4) above,
wherein 8 or less sulfonic acid groups are contained in a molecule.
(7) The near infrared fluorescent contrast agent of any of (1) to (6) above,
wherein the pharmaceutically acceptable salt is a sodium salt.
(8) The near infrared fluorescent contrast agent of any of (1) to (7) above,
that
is for tumor imaging and/or angiography.
(9) A sodium salt of a compound of the formula [II] having three or more
sulfonic acid groups in a molecule
SUBSTITUTE SHEET (RULE 26)
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R6 Ri2
Ri l Ri
R7 ZR
3
8 X 1a
R R LIIJ
+~ ~L1=L2-L3=La-L5=L6-L~ 15
N N R
R10 Rl I 2 16
R R
wherein R1, R2, L' - L' , X and Y are as defined above, and
R5 to R's are the same or different and each is a hydrogen atom, a sulfonic
acid
group, a carboxyl group, a hydroxyl group, an alkyl(sulfoalkyl)amino group, a
bis(sulfoalkyl)amino group, a sulfoalkoxy group, a (sulfoalkyl)sulfonyl group
or a
(sulfoalkyl)aminosulfonyl group, exclusive of the groups of the following
formulas
NaO3S
SO3Na
CH3 CH3
CH3 CH3
+
N CH=CH~ CH N
I _ I
(CH2)3SO3 (CH2)3SO3Na
NaO3S SO3Na
I CH3 CH3
CH3 CH3
+
~-CH N
N CH=CH3
I _ I
(CH2)2CHSO3 (CHz)zCHSO3Na
I
CH3 CH3
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NaO3S SO3Na
H3 fC3
CH3 i H3 CH3
CH=CH-CH=C-CH=CH-CH N
N
(CH2)ZCHSO3 (CHz)Z i HSO3Na
I CH3 CH3
NaO3S CH 3 CH3 SO3Na
I I
I ~ CH3 CH3
CH - C~ CH
(CH2)2SO3 ((-H2)2SO3Na
CO2C2H5
NaO3S CH
3 r N CH SO3Na
3
CH3 CH3
CH= CH 6 CH-CH
(CH2)2SO3 (CH2)2SO3Na
iOzC2H5 and
NaO3S N SO3Na
H3 fCH3 CH3 N CH3 Ig
N CH=CH CH-CH N -1 I= I
(CH2)2CHS03 (CH2)2CHSO3Na
I I
CH3 CH3
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(10) The sodium salt of (9) above, wherein, in the formula [11], R' and R2 are
each a lower alkyl having 1 to 5 carbon atoms substituted by a sulfonic acid
R17
1
- C-
I
R1s
group and X and Y are the same or different and each is a group of the formula
wherein R" and R18 are unsubstituted lower alkyl having 1 to 5 carbon atoms.
(11) The sodium salt of (10) above, having the formula
NaO3S SO 3Na NaO3S SO3Na
3 H3
~3 ~3
N CH=-- CH-CH 3 I _ I
((,'H2)4SO3 2)4SO3Na
(12) A sodium salt of a compound of the formula [III-1] having three or more
R21 R25
R22
X' y~ R26
L I=L2-L3=L4-L5=L6-L7
R23
i N Rz7
R24 Rt9 I R 20 R28
[iII- i ]
sulfonic acid groups in a molecule
wherein L'-L' are as defined above, R19 and R20 are lower alkyl having 1 to 5
carbon atoms and substituted by sulfonic acid group, R 21 - R 28 are the same
or
different and each is hydrogen atom, sulfonic acid group, carboxyl group,
hydroxyl group, alkyl(sulfoalkyl)amino group, bis(sulfoalkyl)amino group,
SUBSTITUTE SHEET (RULE 26)
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Rt7
1
- C-
I
R18
sulfoalkoxy group, (sulfoalkyl)sulfonyl group or (sulfoalkyl)aminosulfonyl
group,
and X' and Y' are the same or different and each is a group of the formula
wherein R" and R18 are as defined above, exclusive of the groups of the
following formulas
NaO3S CH3 CH3 S03Na
CH3 CH3
N+ CH~CH-CH N /
I 3 I
(CH2)4SO3 (CH2)4SO3Na
NaO3S CH3 CH3 S03Na
C2H5 CZH5
N CHCH-CH3 N /
~ 1
(CH2)4SO3 (CH2)4SO3Na
NaO3SCH2CH2NHO2S CH3 CH3 S02NHCH2CH2SO3Na
CH3 CH3
N+ CH~CH-CH N
3
(CH2)4SO3 (CH2)4SO3Na
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NaO3S CH CH3 SO3Na
C2H5 CZH5
N CHCH-CH N
1 3 1
(CH2)4SO3 (CH2)4S03K
Na03SCH2CH2O2S CH3 CH3 S02CH2CH2SO3K
CH3 CH3
N+ CH~CH-CH 3 N
(CH2)2S03 (CH2)2S03K
and
NaO3SCH2O2S CH3 CH3 S02CH2CH2SO3K
)aN CH 3 CH3 + CHJCH-CH 3 N
((;H2)2SO3 (CH2)2SO3K
(13) The sodium salt of (12) above, wherein, in the formula [III-1], L4 is
methine
substituted by alkyl having 1 to 4 carbon atoms.
(14) The sodium salt of (12) above, which is a sodium salt of the compound of
the formula [111-2] having three or more sulfonic acid groups in a molecule
R21 R25
R22 A 26
#.I X, Y, R
~3 />- CH=CH CH-CH =<
Z3 R27 R24 R19 I õ20 R28
(III-2] t ~
wherein R19 - R28, X' and Y' are as defined above, Z3 is a non-metallic atom
group necessary for forming a 5- or 6-membered ring and A is hydrogen atom
SUBSTITUTE SHEET (RULE 26)
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or a monovalent group.
(15) The sodium salt of (14) above, having the formula
NaO3S H3 H3 SO3Na
CH3 CH3
~ N CH=CH CH-CH N
I _ I
(CHZ)4S03 (CH2)4SO3Na
(16) The sodium salt of (12) above, having the formula
CH3 CH3
Na03S \ CH3 CH3 CH3 SO3Na
N CH=CH-CH-_C-CH_ -CH-CH N
I I
(CH2)2503 (CH2)ZSO3Na
(17) The sodium salt of any of (9), (10), (12), (13) and (14) above,
comprising 4
or more sulfonic acid groups in a molecule.
(18) The sodium salt of any of (9), (10), (12), (13), (14) and (17) above,
comprising 10 or less sulfonic acid groups in a molecule.
(19) The sodium salt of any of (9), (10), (12), (13), (14) and (17) above,
comprising 8 or less sulfonic acid groups in a molecule.
(20) A near infrared fluorescent contrast agent comprising the sodium salt of
any of (9) to (19) above.
(21) The near infrared fluorescent contrast agent of (20) above, which is for
tumor imaging and/or angiography.
(22) A method of fluorescence imaging comprising introducing the near infrared
fluorescent contrast agent of (1) above into a living body, exposing the body
to
an excitation light, and detecting near infrared fluorescence from the
contrast agent.
(23) The sodium salt of (9) above, which is at least one member selected from
the group consisting of the compounds of the following formulas
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NaO3S SO3Na Na03S SO3Na
CH3 CH3
CH3 CH3
+
N'5~ CH=:eCH-CH 13 N
I I
CH2CH2CHSO3 CH7CH2iHSO3Na
I
CH3 CH3
NaO3S SO3Na
CH3 CH3
CH3 CH3
CH=~: CH-CH 3 N
I _ I
(CH2)4SO3 (CH2)4SO3Na
S03Na
CH3 CH3
CH3 CH3
+
N CH=t-CH-CH 3 N
I _ I
(CH2)4SO3 (CH2)4SO3Na
NaO3S SO3Na NaO3S SO3Na
CH3 CH3
CH3 i CH3 CH3 (
~ N CH=CH-CH=C-CH=CH-CH N
I _ I
(CH2)4SO3 (CHz0O3Na
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NaO3S SO3Na
CH3 CH3
CH3 CH3 CH3
N CH=CH / CH-CH N
I - I
(CHZ)2CHSO3 (CHZ)2CHSO3Na
I I
NaO3S CH3 CH3 SO3Na
I \ CH3 CH3
CH3 C1 CH3
N CH=CH CH-CH N
(CH2)2SO3 (CH2)2SO3Na
NaO3S SO3Na
CH3 CH3
CH3 CH3 CH3
N CH=CH CH-CH N
I (
(CH2)2S03 (CH2)2SO3Na
NaO3S Na0 S 3
SO3Na 3 SO Na
CH3 CH3
CH3 C1 CH3
CH=CH
N CH-CH N
(CHZ)4S03 (CH2)4SO3Na
and
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NaO3S SO3Na Na03S SO3Na
CH3 CH3
CH3 C1 CH3
+,
N CH=CH CH-CH N
I - -r 1= I
(CH2)4SO3 (CH2)4SO3Na
(24) The sodium salt of (12) above, which is at least one member selected
from the group consisting of the compounds of the following formulas
CH3 CH3
CH3 CH3 S03Na
+,
N CH~ CH-CH 3 N
I I
(CH2)2SO3 (CHZ)2SO3Na
CH3 CH3
NaO3S CH3 CH3 CH3 SO3Na
",
N CH=CH-C=CH-CH=CH-CH N
I _ I
(CHZ)4S03 (CH2)4SO3Na
CH3 CH3
NaO3S SO3Na
CH3 i 1 CH3
N CH=CH-CH=CH-C=CH-CH N
( I
(CHZ)4S03- (CHZ)aSO3Na
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NaO3S CH3 CH3 SO3Na
CH3 CH3 ( \
N CH=CH CH-CH N
(CH2)2SO3 (CH2)2SO3Na
CH3 CH3
C~N~ CH3 CH3 CH3 S03Na
CH=CH-CH=C-CH=CH-CH N
_ (
(CH2)4SO3 (CH2)qSO3Na
H3 H3
+
Na0 S SO Na
3 CH3 CH3 3
N CH=CH) 3 C N
I _ I
(CH2)2SO3 (CH2)2SOINa
CzHs \ N CH3 CH3 / C2H5
NaO S CH / N
(CHZ)3S03Na
3( 2)3 CH3 CHiNji::T
+CH=(CH-CH)3 (CH,)3SO3 (CH,)3SO3Na
CH3 CH3
NaO3S SO3Na
CH3 CH3
N CH=i~ CH - CH 3 N
( I
(CH2)3SO3 (CH2);SO3Na
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CH3 ~ N H3 CH3 CH3
NaO3S(CH2)4~ CH3 CH3 (
N
\ CH2)4SO3Na
/
;v CH=== CH- CH 3 N
I I
(CHZ)4SO3 (CH2)4SO3Na
NaO3S-CH2CH2 CN
H3 H3 Na0 CH3 3 S03Na
CH3 CH3
N CH=CH : CH-CH N
I _ I
(CH?)4S03 (CH2)4SO3Na
CH3 CH3
NaO3S ~--. CH3 C1 CH3 SO3Na
I ~ + I
CH=CH-CH=C-CH=CH-CH N
I _ I
(CH2)4SO3 (CH2)4SO3Na
O3Na H3 H3 03Na
CH3 CH3
N11-1 aO3S N CH=:I~ CH -CH 3 N SO3Na
I - I
(CH2)3SO3 (CH7?);SO3Na
SUBSTITUTE SHEET (RULE 26)
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SO3Na CH3 CH3 SO3Na
CH3 CH3
,
N CH=~-- CH-CH 3 N
NaO3S I I SO3Na
(CH2)4SO3 (CH2)4SO3Na
CH3 CH3
NaO3SCH2CH2O~S CH3 CH3 SOzCH2CHZSO3Na
I I
CH=ze CH-C 3 N
I I
(CH2)2SO3 (CH2)2SO3Na
CH3 CH3
NaO3SCH2CHZCHZO C~~'-CH=:(=CH-Ce3 CH3 CH3 OCHzCHZCHzSO3Na
N
I _ I
(CH2)3SO3 (CH2)3SO3Na
CH3 CH3
NaOOC SO3Na
CH3 CH3
N CH=-- CH -CH 3 N /
I _ (
(CH2)3SO3 (CH2)3SO3Na
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CH3 CH3
(NaO3SCH2CHZCH2)ZN I\ CH3 CH3 I\ N(CH2CH,CH1SO3Na)Z
N CH~ CH-CH 3 y ~
I _ I
(CH,)3SO3 (CH,)3SO3Na
NaO3S CH3 CH3 CH3 CH3 SO3Na
CH3 CH3
CH a CH=CH-CH N
I _ I
(CHAS03 (CH2)4SO3Na
NaO3S
NaO3S H3 NaO OC2H5 H3 S03Na
CH3 CH3
N CH=CH CH-CH N
I _ I
(CH2)4S03 (CH2)4SO3Na
N S CH3 CH2CH2SO3Na CH3 SO3Na
CH3 I CH3 I
N CH=CH-CH=C-CH=CH-CH N
I _ I
(CH2)4S03 (CH2)4SO3Na
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SO3Na
I
NaO3S CH3 CH3 SO3Na
CH3 S CH3
N CH=CH CH-CH N
I _ I
(CH2)4SO3 (CHz)4SO3Na
NaO3S CH3 SCH2CH2SO3Na CH3 SO3Na
CH3 CH3 I \
N CH=CH CH-CH N
( _ I
(CH2)4SO3 (CH2)4SO3Na
SO3Na
/ I \
NaO3S CH3 CH3 SO3Na
CH3 O CH3
+,
N CH=CH / CH-CH N
I I
(CH2)4SO3 (CH2)4SO3Na
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SO3Na
I
NaO3S CH3 CH3 SO3Na
)aN CH3 O CH3 CH=CH / CH-CH N
( _ I.
(CH2)4S03 (CH2)4SO3Na
SO3Na
NaO3S CH3 H CH3 SO3Na
\ CH3 N / CH3
+,
/ N CH=CH CH-CH N
I I
(CH2)4SO3 (CH2)4SO3Na
SO3Na
H
Na03S / I
~
N
NaO3S CH3 CH3 SO3Na
CH3 CH3
/ N CH=CH / CH-CH N
I _ I
(CH2)4503 ((-H2)4SO3Na
H CH2CH2SO3Na
NaO3S CH3 N CH3 SO3Na
CH3 CH3 I \
CH=CH CH-CH N
I I
(CHZ)4SO3 (CH2)4SO3Na
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SO3Na
CONH \ /
NC / SO3Na
NaO3S CH3 CH CH3 SO3Na
CH3 CH3
N CH=CH CH-CH N
I I
(CH2)4SO3 (CH2)4SO3Na
CONHCH,CH2SO3Na
NaO3S CH3 NC CH CH3 SO3Na
CH3 CH3
+,
CH=CH CH-CH N
I I
(CH2)4SO3 (CH2)4SO3Na
Na03 CH3 CH3 SO3Na
CH3 i CH3 CH3 N~ CH=CH-CH=C-CH=CH-CH N
I I
(CH2)4S03 (CHz)4SO3Na
and
NaO3S CH3 CH3 S03Na
CH3 H3C
N CHCH-CH N
~ 3 ~
(CH2)2S03 (CH2)2SO3Na
(25) The near infrared fluorescent contrast agent of (1) above, comprising at
SUBSTITUTE SHEET (RULE 26)
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least one compound selected from the group consisting of the compounds of
the following formulas
CH3 CH3
\ CH3 CH3 I SO3Na
I / +,
N CH~ CH - CH 3 N
1 I
(CH2)2SO3 (CH2)2SO3Na
CH3 CH3
NaO3S CH3 CH3 CH3 SO3Na
+ I I
CH=CH-C=CH-CH=CH-CH N
(CHZ)4S03 (CH2)4SO3Na
CH3 CH3
NaO3S CH ci CH3 SO3Na
/ 3 I I
N CH=CH-CH=(.H-C=CH-CH N
I _ I
(CH2)4S03 (CH2)qSO3Na
CH3 CH3
NaO3S SO3Na
I CH3 CH3 c
+
N, CH=CH CH-CH N :
(CHZ)2S03 (CH2)2SO3Na
CH3 CH3
CH3 CH3 CH3 SO3Na
N CH=CH-CH=C-CH=CH-CH N
( _ I
(CH2)4SO3 (CH2)4SO3Na
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CH3 CH3 CH3 CH3
NaO3S SO3Na
~-CH N
N CH=CH3
I I
(CH2)2S03 (CHZ)2SO3Na
C2H5 CH3 CH3 C,H5
N N
NaO3S(CH2)3 ~ ):)7 CH3 CH3 (CH2)3SO3Na
NCH=~:CH-CH 3 N
I _ I
(CH2)3SO3 (CH2)3SO3Na
CH3 CH3
Na0 S SO Na
3 CH3 CH3 3
CH=t- CH - CH 3 N
I_ (
(CHZ)3SO3 (CH2)3SO3Na
CH3 N CH3 CH3 CH3
NaO3S(CH2)4 CH3 CH3 (CH2)4SO3Na
+, H
N C=::CH_CH 3 N
I I
(CH2)4SO3 (CHZ)4SO3Na
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CH3 CH3
NaO3S CH SO3Na
CH3 3 3
N CH=t- CH - CH 3 N
I _ I
(CH2)4SO3 (CH2)4SO3Na
CH3 CH3
Na0 S SO Na
3 C2H5 C2H5 3
N CH=:t- CH CH 3 N
I I
(CH2)4SO3 (CH2)4SO3Na
NaO3S-CHZCHZ CN
NaO3S \ H3 NaO CH3 CH3 S03Na
I CH3 CH3
+/
~ N CH=CH ~ CH-CH N
I _
(CH2)4SO3 (CH2)4SO3Na
CH3 CH3
NaO3S Cl CH SO3Na
I CH3 ~ 3 I
rJ CH=CH-CH=C-CH=CH-CH N
I _ I
(CH2)4SO3 (CH2)4SO3Na
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CH3 CH3
(C2H5)3N H03 C2H5 CH3 SO3H N(C2H5)3
I CH3 ( I
+
N/ CH=CH-CH=C-CH=CH-C N
I _ I
(CHZ)4S03 (CHZ)4S03H N(C2H5)3
SO3Na CH3 CH3 SO3Na
CH3 CH3 I
NaO3S CH=l~ CH-CH 3 N SO3Na
I _ I
(CHZ)3SO3 (CH2)3SO3Na
SO3Na CH3 CH3 SO3Na
CH3 CH3 I
CH=t CH-CH 3 N
NaO3S I I SO3Na
(CH2)4SO3 (CHZ)4SO3Na
H3 CH3
NaO3SCH2CHzOzS CH3 CH3 SO2CHZCH2SO3Na
+/
N CH=t- CH- CH 3 N
(CH,)ZSO3 (CH2)2SO3Na
SUBSTITUTE SHEET (RULE 26)
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CH3 CH3
NaO3SCH2CH2NHO2S I\ CH3 CH3 (\ SO2NHCH2CH2SO3Na
/
N CH=t-CH-C 3 N
I I
(CH2)4SO3 (CH2)4SO3Na
Na03S SO3Na
CH3 H3
CH3 CH3
+/
N CH=--CH-CH 3 N
I _ I
2)3SO3 (CH2)3SO3Na
CH3 CH3
NaO3SCH2CH2NHC \ CH3 CH3 CONHCH2CH2SO3Na
( I
~ N CH=:~:CH- CH 3 N
I _ I
(CH2)4SO3 (CH2)4SO3Na
CH3 CH3
NaO3SCH2CHzCH2 I \+/CH3 CH3 OCH2CH2CH2SO3Na
~ N CH=t-CH -CH 3 N
I - I
(CHZ)3S03 (CH2)3SO3Na
SUBSTITUTE SHEET (RULE 26)
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26
NaO3S SO3Na NaO3S S03Na
CH3 CH3
CH3 CH3
CH=e CH -CH 3 N
I I
CH2CH2CHSO3 CH2CHZCI HSO3Na
I CH3 CH3
NaO3S S03Na
CH3 CH3
CH3 CH3
N~ CH~ CH -CH 3 N
I I
(CH2)4SO3 (CH2)4SO3Na
CH3 CH3
NaOOC SO3Na
CH3 CH3
+~
CH=t- CH-CH 3 N
I _ I
(CH2)3SO3 (CHZ)3SO3Na
SO3Na
I CH3 CH3
I ~ CH3 CH3
/
N CH~ CH-CH 3 N
I _ I
2)4s0-3 (CH2)4SO3Na
SUBSTITUTE SHEET (RULE 26)
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27
CH3 CH3
(NaO3SCH2CH2CH2)2N CH3 CH3 N(CH2CH2CH2SO3Na)2
N CH=e CH-CH 3 N
I _ I
(CH2)3SO3 (CH2)3SO3Na
NaO3S CH3 CH3 CH3 CH3 SO3Na
CH3 CH3
+/
N CH CH=CH-CH N
I _ I
(CH,)4SO3 (CH7)4SO3Na
NaO3S SO3Na NaO3S / SO3Na
3 CH3
CH3 i CH3 CH3
N CH=CH-CH=C-CH=CH-CH N
I _ I
(CHZ)4S03 (CHZ)4SO3Na
NaO3S
N N
1
NaO3S CH3 Na \ OCZHS CH3 S03Na
CH3 CH3
N CH=CH CH-CH N
I _ I
(CH2)4SO3 (CH2)4SO3Na
SUBSTITUTE SHEET (RULE 26)
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28
NaO3S SO3Na
I CH3 CH3
CH3 CH3
N CH=CH~ CH N
I _ I
(CH2)2CHSO3 (CHz)ZCHSO3Na
I I
CH3 CH3
NaO3S SO3Na
CH3 CH3
CH3 i H3 CH3
+
N CH=CH-CH=C-CH=CH-CH N
I _ I
(CH2)2CHSO3 (CHZ), i HSO3Na
I CH3 CH3
NaO3S SO3Na
CH3 CH3
CH3 CH3 CH3
N CH=CH CH-CH N
(CH2)2CHSO3 (CHZ)zCHSO3Na
I I
CH3 CH3
SUBSTITUTE SHEET (RULE 26)
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29
C02C2H5
NaO3S SO3Na
CH3 CH3
CH3 N CH3
N CH=CH CH-CH N
( I - -r r (CHZ)2CHSO3 (CH2)2CHSO3Na
I I
CH3 CH3
NaO3S SO3Na
CH3 CH3
CH3 CH3
i CH=CH ~ CH N
I
(CH2)2SO3 (CHz)zSO3Na
C02C2H5
NaO3S N SO3Na
CH3 CH3
CH3 N CH3
~
N CH=CH CH-CH N
1 -1 r I
(CHZ)2SO3 (CH2)2SO3Na
NaO3S SO3Na
(. \ CH3 CH3
CH3 ci CH3
N CH=CH CH-CH N
(CH2)2SO3 (CH2)2SO3Na
SUBSTITUTE SHEET (RULE 26)
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NaO3S SO3Na
CH3 CH3
CH3 CH3 CH3
N CH=CH / CH-CH N
I - I
(CH,),SO3 (CH2)2S03Na
NaO3S SO3Na Na03S / SO3Na
CH3 CH3 I
CH3 Cl CH3 ~
+
N CH=CH CH-CH N /
(CH1)4SO3 (CH2)4SO3Na
NaO3S SO 3Na NaO3S SO3Na
CH3 CH3
CH3 ci CH3
CH=CH CH-CH N
(CH2)4SO 3 (CH2)4SO3Na
Na0 S CH3 CH2CHzSO3Na CH3 SO Na
3 CH3 I CH3 3
N CH=CH-CH=C-CH=CH-CH N
(CHZ)4S03 (CH2)aSO3Na
SUBSTITUTE SHEET (RULE 26)
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31
SO3Na
I
NaO3S CH3 CH3 SO3Na
CH3 S CH3
N CH=CH CH-CH N
I _ I
(CH2)4SO3 (CHz)4SO3Na
NaO3S CH3 SCH2CH2SO3Na CH3 SO3Na
CH3 CH3 I \
N CH=CH / CH-CH N
I I
(CH2)4SO3 (CH2)4SO3Na
SO3Na
/ I \
NaO3S CH3 CH3 SO3Na
CH3 0 CH3 I\
N CH=CH CH-CH N
I I
(CH2)4S03 (CH2WSO3Na
SO3Na
I
NaO3S CH3 CH3 SO3Na
CH3 0 CH3
N CH=CH / CH-CH N
I _ I
(CH2)4S03 (CH2)4SO3Na
SUBSTITUTE SHEET (RULE 26)
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32
SO3Na
NaO3S CH3 H CH3 S03Na
)::::c CH3 N / CH3 +N CH=CH CH-CH N
I _ I
(CH2)4SO3 (CH2)4SO3Na
SO3Na
H
NaO3S
N
NaO3S CH3 CH3 SO3Na
CH3 CH3
EN,~'CH=CH-[:::::~=CH-CH N
I (
(CH2)4SO3 (CH2)4SO3Na
H CH2CH2SO3Na
NaO3S CH3 N CH3 SO3Na
CH3 CH3
N CH=CH CH-CH N
I I
(CH2)4SO3 (CH2)4SO3Na
SUBSTITUTE SHEET (RULE 26)
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33
SO3Na
CONH
NC / SO3Na
NaO3S CH3 \CH CH3 SO3Na
I CH3 CH3
rJ CH=CH 17 CH-CH N
I _ I
(CH2)4SO3 (CH2)4SO3Na
CONHCH2CH2SO3Na
NaO3S CH3 NC C CH3 SO3Na
CH3 CH3
,
N CH=CH CH-CH N
(CHZ)4SO3 (CHZ)4SO3Na
CH3 CH3
NaO3 SO3Na
CH3 i H3 CH3
TJ CH=CH-CH=C-CH=CH-CH N
I _ I
(CH2)4S03 (CH2)4SO3Na
and
SUBSTITUTE SHEET (RULE 26)
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34
NaO3S CH3 CH3 S03Na
H3 H3C
N+ CH~CH-CH 7N
3 1
(CH2)ZS03 (CH2)2SO3Na
(26) The sodium salt of (14) above, wherein the monovalent group of A is
substituted or unsubstituted alkyl, substituted or unsubstituted aryl,
substituted
or unsubstituted aralkyl, lower alkoxy, optionally substituted substituted
amino,
alkylcarbonyloxy, substituted or unsubstituted alkylthio, substituted or
unsubstituted arylthio, cyano, nitro or halogen atom.
BRIEF DESCRIPTION OF THE DRAWINGS
Figs. 1 to 4 are photographs showing fluorescence imaging at 24 hours after
administration of the compound, wherein administered were A:ICG (5 mg/kg),
B:NK-1967 (5 mg/kg), C:compound (29) (0.5 mg/kg) and D:compound (6) K
salt (5 mg/kg).
Fig. 5 is a photograph showing fluorescence imaging at 24 hours after
administration of the compound, wherein administered was E:compound (31)
(5 mg/kg).
Figs. 6 to 9 are photographs showing fluorescence imaging at 20 seconds and
minutes after administration of the compound (5 mg/kg), wherein
administered were A:ICG (20 seconds later), B:ICG (5 minutes later),
C:compound (29) (20 seconds later) and D:compound (29) (5 minutes later).
Fig. 10 is a graph showing the concentration of the compound in plasma at 0.5,
1, 4 and 24 hours after administration of the compound, wherein the axis of
SUBSTITUTE SHEET (RULE 26)
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ordinates is concentration ( g/ml) of the compound in plasma at each time
point.
Fig. 11 is a chart showing the infrared absorption spectrum of compound (29).
Fig. 12 is a chart showing the infrared absorption spectrum of compound (31).
Fig. 13 is a chart showing the infrared absorption spectrum of compound (6).
Fig. 14 is a chart showing the infrared absorption spectrum of compound (54).
DETAILED DESCRIPTION OF THE INVENTION
The terms used in the present specification are defined in the following.
The near infrared fluorescent contrast agent in the present invention means a
contrast agent that emits fluorescence in a near infrared region.
In the present invention, the sulfonic acid group may mean sulfonate
(-S03 ) when said sulfonic acid group is used to form an inner salt.
In the present invention, preferable X and Y are of the following formula
R3
1
-C-
1 4
R
wherein R3 and R4 are the same or different and each is substituted or
unsubstituted alkyl.
The alkyl of "substituted or unsubstituted alkyl" at R', RZ, R3 and R4 is
preferably linear or branched lower alkyl having 1 to 5 carbon atoms, such as
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,
pentyl,
SUBSTITUTE SHEET (RULE 26)
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36
isopentyl, neopentyl, tert-pentyl, 2-methylpropyl, 1,1-dimethylpropyl and the
like. The substituent may be, for example, sulfonic acid group, carboxyl,
hydroxy and the like. Examples of substituted alkyl include hydroxymethyl, 1-
hydroxyethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 4-
hydroxybutyl, carboxymethyl, carboxyethyl, carboxybutyl, sulfomethyl, 2-
sulfoethyl, 3-sulfopropyl, 4-sulfobutyl and the like. Preferred R' and R2 are
lower alkyl having 1 to 5 carbon atoms that is substituted by sulfonic acid
group (e.g., 2-sulfoethyl, 3-sulfopropyl, 4-sulfobutyl and the like), and R3
and R4
are unsubstituted lower alkyl having 1 to 5 carbon atoms (e.g., methyl, ethyl
and the like).
The unsubstituted lower alkyl having 1 to 5 carbon atoms at R" and R18 is
exemplified by those mentioned above with regard to the alkyl of "substituted
or unsubstituted alkyl" at R1, R2, R3 and R4.
The alkyl group of the lower alkyl having 1 to 5 carbon atoms that is
substituted
by sulfonic acid group at R19 and R20 is exemplified by those mentioned above
with regard to the alkyl of "substituted or unsubstituted alkyl" at R1, Rz, R3
and
R4, and examples of the substituted lower alkyl having 1 to 5 carbon atoms
include 2-sulfoethyl, 3-sulfopropyl and 4-sulfobutyl.
The alkyl moiety of alkyl(sulfoalkyl)amino group, bis(sulfoalkyl)amino group,
sulfoalkoxy group, (sulfoalkyl)sulfonyl group and (sulfoalkyl)aminosulfonyl
group at R21- R28 is preferably a linear or branched lower alkyl having 1 to 5
carbon atoms, which is exemplified by those mentioned above with regard to
the alkyl of "substituted or unsubstituted alkyl" at R', R2, R3 and R4.
In the present invention, the "nonmetallic atoms necessary for forming a
substituted or unsubstituted condensed benzo ring or condensed naphtho ring"
means a bonding group necessary for forming a condensed benzo ring or
condensed naphtho ring, which is a group of the formula
SUBSTITUTE SHEET (RULE 26)
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37
\ \ \ \
or
When the condensed benzo ring or condensed naphtho ring has a substituent,
said bonding group may include a substituent.
Specific examples thereof include carbon atom, nitrogen atom, oxygen atom,
hydrogen atom, sulfur atom, halogen atom (e.g., fluorine atom, chlorine atom,
bromine atom and iodine atom) and the like.
The substituent of the condensed benzo ring and condensed naphtho ring
formed by the nonmetallic atoms at Z' and Z2 is exemplified by sulfonic acid
group, carboxyl, hydroxy, halogen atom (e.g., fluorine atom, chlorine atom,
bromine atom and iodine atom), cyano, substituted amino (e.g.,
dimethylamino, diethylamino, ethyl 4-sulfobutylamino, di-(3-sulfopropyl)amino
and the like), and substituted or unsubstituted alkyl as defined above, which
is
bonded to the ring directly or via a divalent bonding group. Preferable
divalent
bonding group may be, for example, -0-, -NHCO-, -NHSOZ-, -NHCOO-, -
NHCONH-, -COO-, -CO-, SO2-, and the like. The alkyl of substituted or
unsubstituted alkyl that is bonded to the ring directly or via a divalent
connection group is exemplified preferably by methyl, ethyl, propyl and butyl,
and the substituent is preferably exemplified by sulfonic acid group, carboxyl
and hydroxy.
The substituent of methine at L' - L' is exemplified by substituted or
unsubstituted alkyl (defined above), halogen atom (defined above), substituted
or unsubstituted aryl, lower alkoxy and the like. The aryl of "substituted or
unsubstituted aryl" is exemplified by phenyl, naphthyl and the like,
preferably
phenyl. Examples of the substituent include halogen atom (defined above,
preferably chlorine atom) and the like. The substituted aryl includes, for
SUBSTITUTE SHEET (RULE 26)
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38
example, 4-chlorophenyl and the like. The lower alkoxy is preferably linear or
branched alkoxy having 1 to 6 carbon atoms, which is specifically methoxy,
ethoxy, propoxy, butoxy, tert-butoxy, pentyloxy and the like, preferably
methoxy
and ethoxy. In addition, the substituents of methine at L' - L' may be bonded
each other to form a ring containing three methine groups, and this ring may
further form a condensed ring with a ring containing different methine group.
The ring containing three methine groups that is formed by the bonding of the
substituents of methine at L' - L' is exemplified by 4,4-dimethylcyclohexene
ring and the like.
The conjugated methine chain consisting of the groups of L' - L', and having a
ring is preferably the group of the formula (a):
A
= CH - CH CH = CH - (a)
e3
z
wherein Z3 denotes nonmetallic atoms necessary to form a 5- or 6-membered
ring and A is hydrogen atom or a monovalent group.
The "nonmetallic atoms necessary to form a 5- or 6-membered ring" is
exemplified by those mentioned above.
In the formula (a) and [111-2] to be mentioned later, - or 6-membered ring at
Z3
is exemplified by cyclopentene ring, cyclohexene ring, 4,4-
dimethylcyclohexene ring and the like, with particular preference given to
cyclopentehe ring.
The monovalent group represented by A includes, for example, substituted or
unsubstituted alkyl (defined above), substituted or unsubstituted aryl
(defined
above), substituted or unsubstituted aralkyl, lower alkoxy (defined above),
substituted amino which is optionally substituted, alkylcarbonyloxy (e.g.,
SUBSTITUTE SHEET (RULE 26)
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39
acetoxy), substituted or unsubstituted alkylthio, substituted or unsubstituted
arylthio, cyano, nitro, halogen atom (defined above), and the like. As used
herein, araikyl of the "substituted or unsubstituted aralkyl" is exemplified
by
benzyl, 2-phenylethyl, 1-phenylethyl, 3-phenylpropyl and the like, and the
substituent may be sulfonic acid group, carboxyl, hydroxy, substituted or
unsubstituted alkyl (defined above), alkoxy (defined above), halogen atom
(defined above), and the like. The substituted amino of the "substituted amino
which is optionally substituted" includes, for example, alkylamino (e.g.,
methylamino, ethylamino and the like), dialkylamino (dimethylamino,
diethylamino and the like), diphenylamino, methylphenylamino, cyclic amino
(e.g., morpholino, imidazolidino, ethoxycarbonylpiperadino and the like) and
the like. The substituent with regard to the optional substitution of the
"substituted amino which is optionally substituted" includes sulfonic acid
group,
carboxyl and the like. The alkylthio of the "substituted or unsubstituted
alkylthio" may be, for example, methylthio, ethylthio and the like. Examples
of
the substituent include sulfonic acid group, carboxyl and the like. The
arylthio
of the "substituted or unsubstituted arylthio" is exemplified by phenylthio,
naphthylthio and the like. Examples of the substituent include sulfonic acid
group, carboxyl and the like.
The monovalent group represented by A is preferably fluorine atom, chlorine
atom, dialkylamino (preferably having 6 or less carbon atoms, and optionally
forming a ring) or morpholino. This group particularly preferably has a
sulfonic
acid group.
In the formula [I], r is preferably 1.
The pharmaceutically acceptable salt may be any as long as it forms a
nontoxic salt with the compound of the formula [I]. Examples thereof include
alkali metal salts such as sodium salt, potassium salt; salt of alkaline earth
metal such as magnesium salt, calcium salt and the like; organic ammonium
salt such as ammonium salt, triethyl ammonium salt, tributyl ammonium salt,
pyridinium salt and the like; salt of amino acid such as lysine salt, arginine
salt
SUBSTITUTE SHEET (RULE 26)
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and the like. Particulariy preferred is sodium salt causing less toxicity in
the
living body.
The fluorescent contrast agent to be used in a living body should be
particularly water soluble. In the present invention, the near infrared
fluorescent contrast agent has a noticeably improved water solubility by the
introduction of 3 or more sulfonic acid groups into the above-mentioned
compound. For superior water solubility, the number of the sulfonic acid
groups is preferably 4 or more. For easy synthesis, the number of the sulfonic
acid groups is not more than 10, preferably not more than 8. The improvement
in the water solubility can be determined by measuring partition coefficient
of
each compound, which for example, may be measured in a two-phase system
of butanol/water. More specifically, introduction of 3 or more sulfonic acid
groups results in a partition coefficient log Po/w of n-butanol/water of not
more
than -1.00.
The sulfonic acid groups are particularly preferably introduced into the
positions of R1, R2, Z' and/or Z2 of the formula [I] and R', R2, R5, R', R"
and/or
R13 of the formula [11].
In addition, these sulfonic acid groups are preferably introduced into L4 of
the
conjugated methine chain at the position A of the above-mentioned formula (a)
via a divalent group such as alkylene.
Of the sodium salts of compounds of the formula [II] having three or more
sulfonic acid groups in a molecule, preferred is a sodium salt of a compound
wherein R' and R2 are lower alkyl having 1 to 5 carbon atoms which is
substituted by sulfonic acid group, and X and Y are the same or different and
each is a group of the formula
R' 7
1
- C-
(
RIs
SUBSTITUTE SHEET (RULE 26)
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41
wherein R" and R'$ are the same or different and each is unsubstituted lower
alkyl having 1 to 5 carbon atoms alkyl, said salt having three or more
sulfonic
acid groups in a molecule, with particular preference given to a compound of
the formula
NaO3S SO3Na NaO3S SO3Na
CH3 H3
CH3 CH3
I~i CH~ CH -CH 3 N
I _ I
(CH2)4SO3 (CH2)4SO3Na
Of the compounds of the formula [I] having three or more sulfonic acid
groups in a molecule and pharmaceutically acceptable salts thereof, preferred
is a sodium salt of a compound of the formula [III-1]
R2 1 R25
R22
y R26
L1=L2-L3=L~-L5=L6-L7
----~-1<
23
N N R27
R "'~: Y I I
R2a R 19 R20 R28
wherein Ll - L' are as defined above, R19 and R20 are lower alkyl having 1 to
5
carbon atoms which is substituted by sulfonic acid group, and R21 to R28 are
the same or different and each is hydrogen atom, sulfonic acid group, carboxyl
group, hydroxyl group, alkyl(sulfoalkyl)amino group, bis(sulfoalkyl)amino
group, sulfoalkoxy group, (sulfoalkyl)sulfonyl group or (sulfoalkyl)amino-
sulfonyl group, and X' and Y' are the same or different and each is a group of
R17
1
- C-
I
R18
SUBSTITUTE SHEET (RULE 26)
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42
the formula
wherein R" and R18 are as defined above, said salt having three or more
sulfonic acid groups in a molecule, with particular preference given to the
compound of the following formula.
CH3 CH3
SO3Na
NaO3S CH3 CH3 CH3
N
CH=CH CH=C-CH=CH-CH N
I I
(CHZ)ZS03 (CHZ)ZSO3Na
Of the sodium salts of the compounds of the formula [III-1] having
three or more sulfonic acid groups in a molecule, preferred is a sodium salt
of a
compound of the formula [111-2]
R21 R25
R22 `q 26
/ X~ Y, \ R
CH=CH CH-CH ~ I
~3 \ N
Z3 ~ 7
R24 R19 R20 R28
(III-2J
wherein R19 - R28, X' and Y' are as defined above, Z3 is nonmetallic atoms
necessary to form a 5- or 6-membered ring and A is hydrogen atom or a
monovalent group, said salt having three or more sulfonic acid groups in a
molecule, with particular preference given to the compound of the following
formula
NaO3S CH3 CH3 SO3Na
CH3 CH3
N CH=CH CH-CH N
I _ I
(CH2)4SO3 (CH2)4SO3Na
The compounds contained in the inventive near infrared fluorescent
SUBSTITUTE SHEET (RULE 26)
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43
contrast agent may be any as long as it has formula [I] or [II], as well as 3
or
more, preferably 4 or more, sulfonic acid groups in a molecule. These
compounds can be synthesized according to a known production method of
cyanine dye compounds disclosed in The Cyanine Dyes and Related
Compounds, F.M. Hamer, John Wiley and Sons, New York, 1964, Cytometry,
10, 3-10 (1989), Cytometry, 11, 418-430 (1990), Cytometry, 12 723-730
(1990), Bioconjugate Chem. 4, 105-111 (1993), Anal. Biochem., 217, 197-204
(1994), Tetrahedron, 45, 4845-4866 (1989), EP-A-0591820A1, EP-A-
0580145A1, and the like. Alternatively, they can be semisynthesized from a
commercially available cyanine dye compound by a known method. To be
specific, they can be synthesized by reacting a dianyl compound and a
heterocyclic quaternary salt.
The compound of the formula [I] of the present invention can be synthesized
by, for example, the following method.
(i) when r=0
(a)L'=L5, X=Y, R'=R2 and Z'=Z2
A hetero ring quaternary salt compound (2 moles) of the formula
[IV-1]
X
L1H2
[IV-1
ol 1 ]
N
R1
wherein L', X, Z' and R' are as defined above, and a dianyl compound (1
mole) of the formula [V-1]
NH-L 2=L3-L4=N / \ = HCl [V-1 ]
wherein L2, L3 and L4 are as defined above, are reacted in the presence of a
base and a solvent to give a compound of the formula [VI-1]
SUBSTITUTE SHEET (RULE 26)
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44
X X
t
I x L1=L'-L3=L4-Ll I Z1 [VI-I
Z]
+
+
N N
I I
R Rl
wherein L', LZ, L3, L4, R1, Z' and X are as defined above, and this compound
[VI-1] (1 mole) and a necessary molar amount of the compound of the formula
[VII]
T' - Na [VII]
wherein T' is an organic acid residue, are reacted to give a sodium salt of
the
compound of the above-mentioned formula [VI-1].
(b) L'#L5 or X#Y or R'*R2 or Z'#Z2
A hetero ring quaternary salt compound (1 mole) of the above-
mentioned formula [IV-1] and the above-mentioned dianyl compound (1 mole)
of the formula [V-1] are reacted in the presence of a base and a solvent to
give
a compound of the formula [VIII-1]
X
~ I
Z + L1=L2-L3=L4 - N = HC1 [VIII-I
]
+ N H
I
R
wherein L', L2, L3, L4, R1, Z' and X are as defined above, and this compound
[VIII-1] (1 mole) and a hetero ring quaternary salt compound (1 mole) of the
formula [XI-1 ]
Y
Z2
L 5 Hz [XI- I ]
N
R2
SUBSTITUTE SHEET (RULE 26)
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wherein L5, Y, Z2 and R2 are as defined above, are reacted to give a compound
of the formula [X-1]
X y
t
ZI ~ L 1=L'-L3=L4-LS I Z2 [X-1 ]
+
+
N N
R R2
wherein L', L2, L3, L4, L5, R1, R2, Z', Z2, X and Y are as defined above, and
this
compound of the formula [X-1] (1 mole) and a necessary molar amount of the
above-mentioned compound of the formula [VII] are reacted to give a sodium
salt of the compound of the above-mentioned formula [X-1].
(ii) when r=1
(a) L'=L', X=Y, R'=R2 and Z'=Z2
A hetero ring quaternary salt compound (2 moles) of the formula [IV-1]
X
Z1
I x L t H2 [IV-1 ]
+
N
R1
wherein L', X, Z' and R' are as defined above, and a dianyl compound (1
mole) of the formula [V-2]
NH-L2=L3-L4=L5-L6=N / \ - HCl [V-2]
wherein L2, L3, L4, L5 and L 6 are as defined above, are reacted in the
presence
of a base and a solvent to give a compound of the formula [VI-2]
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X
t
Z I ~ L1=L2-L3=L4-L5=L6-L1 I Zl [VI-2]
N N
I I R1 t
R
wherein L', L2, L3, L4, L5, L6, R', Z' and X are as defined above, and this
compound [VI-2] (1 mole) and a necessary molar amount of the compound of
the formula [VII]
T' - Na [VII]
wherein T' is as defined above, are reacted to give a sodium salt of the
compound of the above-mentioned formula [VI-2].
(b) L'#L' or X#Y or R':#R2 or Z':#Z2
A hetero ring quaternary salt compound (1 mole) of the above-
mentioned formula [IV-1] and the above-mentioned dianyl compound (1 mole)
of the formula [V-2] are reacted in the presence of a base and a solvent to
give
a compound of the formula [VIII-2]
X
1 I
Z / L1=LZ-L3=L4-L5=L6 N ~ ~ = HC1
+ H
N
I [VIII-2]
R1
wherein L', L2, L3, L4, L5, Ls, R1, Z' and X are as defined above, and this
compound [VIII-2] (1 mole) and a hetero ring quaternary salt compound (1
mole) of the formula [IX-2]
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Z
L 7 H2 [IX-2]
N
2
wherein L', Y, Z2 and R2 are as defined above, are reacted to give a compound
of the formula [X-2]
X
t I
Z + L1=L2-L3=L4-L5=L6-L7 Z2 [X-2]
a N N
R1 R-
wherein L', L2, L3, L4, L5, Ls, L', R', R2, Z', Z2, X and Y are as defined
above,
and this compound of the formula [X-2] (1 mole) and a necessary molar
amount of the above-mentioned compound of the formula [VII] are reacted to
give a sodium salt of the compound of the above-mentioned formula [X-2].
(iii) when r=2
When r is 2, L 6 and L' overlap in the formula [I]. To avoid this, the
overlapping L 6 and L' are referred to as L8 and L9 for clarification.
(a) L'=L9, X=Y, R'=R2 and Z'=Z2
A hetero ring quaternary salt compound (2 moles) of the formula
[IV-1]
X
1
+ L H2 [IV-1
]
a/\-
N
R1
wherein L', X, Z' and R' are as defined above, and a dianyl compound (1
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mole) of the formula [V-3]
/ \ NH-L2=L'-L4=L5-L6=L7-Lg=N / \ = HC1 [V-3]
wherein L2, L3, L4, L5, Ls and L' are as defined above and L 8 is an
optionally
substituted methine group, are reacted in the presence of a base and a solvent
to give a compound of the formula [VI-3]
X
a+/_ L1=L'-L3=L4-L5=L6-L7=Ls-Li [VI-3]
Z
N N
I
R1 R1
wherein L', L2, L3, L4, L5, Ls, L', L8, R1, Z' and X are as defined above, and
this
compound [VI-3] (1 mole) and a necessary molar amount of the compound of
the formula [VII]
T' - Na [VII]
wherein T' is as defined above, are reacted to give a sodium salt of the
compound of the above-mentioned formula [VI-3].
(b) L1#L9 or X#Y or R':#R2 or Z':#Z2
A hetero ring quaternary salt compound (1 mole) of the above-
mentioned formula [IV-1] and the above-mentioned dianyl compound (1 mole)
of the formula [V-3] are reacted in the presence of a base and a solvent to
give
a compound of the formula [VIII-3]
X
Zl I / L1=L2-L3=L4-L5=L6-L7=Lg- N O = HCI
+
N H
I 1 [VIII-3]
R
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wherein L', LZ, L3, L4, L5, L6, L', L8, R', Z' and X are as defined above, and
this
compound [VIII-3] (1 mole) and a hetero ring quaternary salt compound (1
mole) of the formula [IX-3]
Z
1 L 9 H2 [IX-3]
+
N
I
R2
wherein Y, Z2 and R2 are as defined above and L9 is an optionally substituted
methine group, are reacted to give a compound of the formula [X-3]
X Y
I / LI=L2-L3=L4-L5=L6-L'=L8-L9 Z2
Z1~=K I
+
N N N C
~1 I
R R'" [X-3]
wherein L', LZ, L3, L4, L5, L6, L', L8, L9, R1, R 2, Z', Z2X and Y are as
defined
above, and this compound of the formula [X-3] (1 mole) and a necessary molar
amount of the above-mentioned compound of the formula [VII] are reacted to
give a sodium salt of the compound of the above-mentioned formula [X-3].
The necessary molar amount of the compound of the formula [VII] is not less
than the amount equivalent to the amount of sodium contained in one molecule
of the objective sodium salt of the compound of the formula [I].
The substituent of the substituted methine group at L8 and L9 is exemplified
by
those mentioned with regard to the substituent of the above-mentioned
methine groups at L'to L'.
In the synthetic methods of the above-mentioned (i), (ii) and (iii), the
reaction of
SUBSTITUTE SHEET (RULE 26)
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the compounds [IV-1] and [V-1], that of the compounds [VIII-1] and [XI-1],
that
of the compounds [IV-1 ] and [V-2], that of the compounds [VII I-2] and [IX-
2],
that of the compounds [IV-1] and [V-3] and that of the compounds [VIII-3] and
[IX-3] proceed at a temperature of -20 C - 80 C, preferably -10 C - 40 C,
preferably in the presence of an acylating agent such as acetic anhydride.
In the synthetic methods of the above-mentioned (i), (ii) and (iii), the
reaction of
the compounds [IV-1] and [VII], that of the compounds [X-1] and [VII], that of
the compounds [VI-2] and [VII], that of the compounds [X-2] and [VII], that of
the compounds [VI-3] and [VII] and that of the compounds [X-3] and [VII]
proceed at a temperature of preferably 0 C - 40 C, preferably in the presence
of a solvent such as alcohol and water.
In the synthetic methods of the above-mentioned (i), (ii) and (iii), the base
to be
used may be, for example, triethylamine, tributylamine, pyridine,
diazabicycloundecene, sodium methoxide and the like; the solvent to be used
may be, for example, an amide compound such as N,N-dimethylacetamide, N-
methylpyrrolidone and N,N-diethylformamide or alcohols such as methanol;
and the organic acid residue may be, for example, CH3COO and the like.
With regard to the production of various pharmaceutically acceptable salts of
the compounds of the aforementioned formula [I], ammonium salt and
potassium salt of the compounds of the formula [I] can be obtained by, for
example, substituting the compound of the formula [VII] used in the above-
mentioned synthetic methods (i), (ii) and (iii) with a compound of the formula
[VII] wherein the sodium atom has been changed to ammonium group or
potassium atom; and different cationic salts of the compounds of the
aforementioned formula [I] can be obtained by converting said ammonium salt
and potassium salt to different cationic salts by the use of ion exchange
resins
as necessary.
The compound of the above-mentioned formula [I] inclusive of the compound
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of the formula [II] to be used in the present invention are specifically
exemplified in the following, to which the present invention is not limited.
(1) CH3 CH3
I\ CH3 CH3 S03Na
N CH=f CH-C 3 N
I I
(CHZ)2SO3 (CH2)2SO3Na
(2) f.{3 CH3
NaO3S CI{ SO3Na
I \ CH3 i H3
/ +~
N CH=CH-C=CH-CH=CH-C N
~ (CH2)4SO3Na
(CH2)aS03
CH3 CH3
(3) NaO3S I\ CH3 il CH3 I\ SO3Na
+ CH=CH-CH=CH-C=CH-CH N
N
I
(CH-)4S03 (CH-))4SO3Na
(4) CH3 CH3
NaO3S S03Na
)[:) CH3 CH3
N CH=CH / CH-C N
I (
(CH-7)2S63 (CH2)2SO3Na
CH3 CH3
(5) I\ CH3 CH3 CH3 SO3Na
N CH=CH-CH=C-CH=CH-CH N
I I
(CH,)4SO3 (CHZ)4SO3Na
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(6) NaO3S CH3 CH3 SO3Na
CH; CH3 I ~
N CH=CH CH-CH i
_ (CH,)4S03 (CH,)4SO3Na
H3 CH3
(7) Na0 S SO Na
3 CH3 CH3 I 3
~--CH
CH=CH 3
(CH,)2S03 (CH2)zSO3Na
C2H5 \ N H3 CH3 / C?H5
g
( ) Na03S(CH2)3/ CH3 CH3 \ N \
(CHz)3SO3Na
N CH=t- CH- CH 3 N
- I
(CHZ)3SO3 (CH2)3SO3Na
CH3 CH3
NaO3S SO3Na
(9) I CH3 CH3
+/
N CH ==~= CH -CH 3
I
(CH2)3SO3 (CH2)3SO3Na
CH3\ CH3 CH3 / CH3
N
NaO3S(CH2)4/ CH3 CH3 N~(CH2)4SO3Na
+/ I
N CH CH- CH
3 N
- (
(CH2)4SO3 (CHZ)aSO3Na
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(11)
CH3 CH3
NaO3S CH3 CH3 S03Na
+~ ~ I
CH ==E CH- C
(CH2)4SO3 (CH2)4SO3Na
(12)
CH3 CH3
NaO3S SO3Na
C2H5 C25
CH=E CH- C 3
( _
(CH2)4SOs (CH2)4SO3Na
(13)
0
Na03S-CHzCHz~ CN
CH3 N CH3
Na03S CH3 \ I S03Na
3 H 3C
Na CH
+
N CHCH ~ CH-CH N
I I
(CH2)4503 (CH 2)dSO3Na
(14)
CH3 CH3
NaO3S CH3 Cl CH3 / SO3Na
I
CH=CH-CH=C-CH=CH-CH
(CH2)4503 ((-H2)4SO3Na
(15)
CH3 CH3
(C2H5)3NHO3S I CH3 C2H5 CH3 SO3HN(C2H5)3
I ~
CH=CH- CH=C- CH=CH- CH
(CH2)4 SO3 ((-H2)4 SO3HN(C2Hs)3
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(16) SO3Na H3 CH3 SO3Na
CH3 CH3
+/
NaO3S N CH =~: CH - CH N SO3Na
( - I
(CH2)3SO3 (CH2)3SO3Na
SO3Na H3 CH3 SO3Na
(17)
CH3 CH3 I
+/
N CH==e= CH -CH 3 N
NaO3S I I SO3Na
(CH2)4SO3 (CHz)4SOINa
(18) H3 CH3
NaO3SCH2CH2O2S \ CH3 CH3 SO2CH,CH,SO3Na
~ N j CH==(=CH-CH 3 N
I _ I
(CH,)2S03 (CH2)2SO3Na
(19)
H3 CH3
NaO3SCH2CH2NHO2S \ CH3 CH3 SO2NHCH2CH2SO3Na
~
~ N CH=t- CH-CH 3 N
I I
(CH2)4SO3 (CH2)4SO3Na
NaO3S SO3Na
I
(20) H3 CH3
- - 7
CH3 CH3
CH=~-- CH-CH 3 N
I _ I
(CH,)3SO3 (CH,)3SO3Na
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(21)
CH3 CH3
NaO3SCH2CH2NHCO CH3 CH3 CONHCH2CH2SO3Na
CH===6-CFt- C 3
(CH2)4SO3 (CH2)4SO3Na
(22)
CH3 CH3
Na03SCH2CH2CH2O OCH2CH2CH2SO3Na
CH3 CH3
+-
CH~ CH- C 3 I
(CH2)3SO3 (CH2)3SO3Na
(23)
NaO3S SO3Na ~H3S SO3Na
CH3
CH3 CH3
+ CH ==E CH- C 3
I CH2CH2CHSO3Na
CH2CH2CHSO3 I
I CH3
CH3
(24)
SO3Na
NaO3S CH3 CH3 ~
CH3 CH3
I / +~ \
CH ==ECH- C 3 I
I (CH2)4SO3Na
(CH2)4SO3
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(25) H3 CH3
NaOOC SO3Na
CH3 CH3
N CH=:(-- CH-CH t3N
I _ I
(CHI-)3SO3 (CH2)3SO3Na
H3 CH3 SO3Na
(26)
CH3 CH3
+~
N CH~ CH-CH 3 N
I _ I
(CH,)4S03 (CHz)4SO3Na (27) H CH3
(NaO3SCH2CHZCH2)ZN CH3 CH3 N(CHZCHZCHZSO3Na)2
+~
N CH=(= CH-CH 3 N
I _ I
(CH,)3S03 (CH,)3SO3Na
(28)
Na03S H3 CH3 CH3 CH3 SO3Na
CH3 CH3
N CH a CH=CH-CH N
I (
(CH,)4SO; (CHZ)4SO3Na
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(29)
NaO3S SO3Na NaO3S SO3Na
CH3 CH3
CH3 CH3
N CH~ CH - CH 3
I N
CHZ)aSO3- I(CH ( 2)4SO3 Na
(30)
NaO3S SO3Na NaO3S SO3Na
CH3 CH3
CH3 CH3 CH3
N ~ CH=CH-CH=C-CH=CH-C N
(CH2)4SO3- (CH,)4SOzNa
(31)
CH3 CH3
NaO3S SO Na
I \ CH3 CH3 CH3 3
CH=CH-CH=C-CH=CH-CHO~
I I
(CH2)ZSO3- (CHz)ZSO3Na
(32) NaO3S
N N
K
NaO3S CH3 Na0 \ OCZH5 CH3 SO3Na
CH3 CH3
CH=CH
rJT / CH-CH
N
I I
(CH,),,S03 (CH,)4SO3Na
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(33)
NaO3S SO3Na
I CH3
CH3
CH3 CH3
N CH=CH ~ CH N
_ I
(CH2)2CHSO3 (CH2)2CHSO3Na
1 1
(34) CH3 CH3
NaO3S SO3Na
CH3 CH3
CH3 i CH3 CH3
N CH=CH-CH=C-CH=CH-CH N
I _ (
(CH2)2CHSO3 (CH2)2 HSO3Na
1 (35) CH3 CH3
NaO3S SO3Na
I \ CH3 CH3
CH3 CH3 CH3 I~
+
CH=CH CH-CH N
(CH2)2CHSO3 (CHZ)zCHSO3Na
CH3 CH3
(36)
COzC2H5
NaO3S N SO3Na
CH3 CH3
CH3 N CH3
+
N~ CH=CH CH-CH N
(CHZ)2CHSO3 - (CH,)2CHSO3Na
I
CH3 CH3
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(37)
NaO3S SO3Na
CH3 CH3
CH3 CH3
+
N -:-" CH=CH --3
CH N
I
(CH2)ZS03
(CHZ)ZSO3Na
(38)
CrO2C2H5
Na03S S03Na
CH3 CH3
CJ N
CH3 N CH3
+/
CH=CH CH-CH N
1 -1 r 1
(39) (CH2)2SO3 (CH2)2SO3Na
NaO3S SO3Na
I \ CH3 CH3
I~ CH3 Cl CH3
~ N CH=CH CH-CH N
(40) (CH2)2S03 (CH2)2SO3Na
NaO3S SO3Na
CH3 CH3
CH3 CH3 CH3
+/
N CH=CH CH-CH N
(CH2)2S03 (CH2)2SO3Na
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(41)
NaO3S SO Na
SO3Na NaO3S 3
CH3 CH3
CH3 C1 CH3
/
N CH=CH / CH-CH N
(CHZ)4SO3 (CH2)4SO3Na
(42)
NaO3S SO3Na NaO3S SO3Na
CH3 CH3
CH3 ci CH3
N CH=CH CH-CH N
I _ I
(CH2)4SO 3 (CH2)4SO3Na
(43)
Na0 S CH3 CHZCH2SO3Na CH3 SO Na
3 CH3 CH3 3
N CH=CH-CH=C-CH=CH-CH N
I I
(CH2)4SO3 (CHz)4SO3Na
(44) SO3Na
I
NaO3S CH3 CH3 S03Na
)C)~~N CH3 S CH3 CH=CH CH-CH N
I I
(CHZ)4S03 (CH2)4SO3Na
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(45)
NaO3S CH3 SCH2CH2SO3Na CH3 SO3Na
CH3 CH3
,
N CH=CH CH-CH N
I I
(CHZ)4SO3 (CH2)4SO3Na
(46) SO3Na
/ I \
NaO3S CH' CH, SO3Na
CH3 O CH;
+
CH=CH CH-CH N
I I
(CH2)4SO3 (CH2)4SO3Na
(47)
SO3Na
I
NaO3S CH3 CH3 SO3Na
CH3 O CH3
N CH=CH / CH-CH N
I _ I
(CH2)4SO3 (CH2)4SO3Na
(48)
SO3Na
NaO3S CH3 H CH3 SO3Na
CH3 N CH3 CH=CH
)aN CH-CH NI
I (
(CHZ)qSO; (CH2)4SO3Na
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(49) SO3Na
H
NaO3S
N/
NaO3S CH3 CH3 SO3Na
I ~ CH3 CH3
N CH=CH L CH-CH N
I _ I
(CH2)4SO3 (CH2)4SO3Na
(50) H CH,CH,S03Na
NaO3S CH3 N CH3 SO3Na
CH3 CH3
,
N CH=CH CH-CH N
I _ I
(CH,)4SO3 (CH,)4SO3Na
SO3Na
(51) -
CONH \ /
NC SO3Na
NaO3S CH3 \ CH CH3 SO3Na
CH3 CH3 I ~
N CH=CH CH-CH N
I
(CH2)4SO3 (CH2)4SO3Na
(52)
CONHCH2CH2SO3Na
NaO3S CH3 NC C CH3 SO3Na
CH3 CH3 I
N CH=CH CH-CH N
I I
(CH2)4SO3 (CH2)4SO3Na
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(53)
CH3 CH3
Na03 SO3Na
CH3 CH3 CH3
CH=CH-CH=C-CH=CH-CH N
(CH2)4S03 (CH,)4SO3Na
(54)
NaO3S CH3 CH3 S03Na
CH3 H3C
N CHtCH-CH N
3
(CHZ)2S03 (CH2)2SO3Na
The above-mentioned compound to be contained in the near infrared
fluorescent contrast agent of the present invention shows absorbance and
fluorescence in the near infrared light region of 700 - 1300 nm, particularly
about 700 - 900 nm, and has a molar absorption coefficient of not less than
100,000.
The near infrared fluorescent contrast agent of the present invention is
subject
to no particular limitation as long as it contains a compound of the formula
[I] or
formula [II] and/or a pharmaceutically acceptable salt thereof, and has 3 or
more, preferably 4 or more, sulfonic acid groups in a molecule. This
compound or a salt thereof alone or in combination may be contained in said
contrast agent.
To be specific, said contrast agent includes said compound or said compound
suspended or dissolved in a solvent, such as injectable distilled water,
physiological saline, Ringer solution and the like. Where necessary,
pharmacologically acceptable additives such as carrier, excipient and the like
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may be added. These additives contain substances such as pharmacologically
acceptable electrolyte, buffer, detergent and a substance for adjusting
osmotic
pressure and improving stability and solubility (e.g., cyclodextrin, liposome
and
the like). Various additives generally used in the pertinent fields may be
used.
The near infrared fluorescent contrast agent of the present invention is
preferably produced through a sterilization process when it is intended for
pharmaceutical use.
Said contrast agent can be administered to a living body by injecting,
spraying
or coating, intravascularly (venous, arterial), orally, intraperitoneally,
percutaneously, subcutaneously, intracystically or intrabronchially.
Preferably,
the agent is administered into blood vessels in the form of an aqueous agent,
emulsion or suspension.
The dose of the near infrared fluorescent contrast agent of the present-
invention is not particularly limited insofar as the dose enables detection of
the
site to be ultimately diagnosed. It is appropriately adjusted depending on the
kind of compound to be used that emits near infrared fluorescence, age, body
weight and target organ of administration subjects, and the like. Typically,
the
dose is 0.1 - 100 mg/kg body weight, preferably 0.5 - 20 mg/kg body weight,
in the amount of said compound.
The contrast agent of the present invention can be appropriately used for
various animals other than human. The administration form, route and dose
are suitably determined depending on the body weight and conditions of the
target animals.
In the present invention, moreover, the above-mentioned compound of the
formula [I], particularly preferably [II], having 3 or more, preferably 4 or
more,
sulfonic acid groups in a molecule tends to be noticeably accumulated in tumor
tissues. Utilizing this characteristic, a tumor tissue can be specifically
imaged
using the inventive fluorescent contrast agent. In addition, a series of said
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compounds can reside in blood vessel for a long time and they are expected to
serve well as angiography contrast agents.
The fluorescence imaging method of the present invention is characterized by
the use of the inventive near infrared fluorescent contrast agent. This method
is practiced following known methods, and each parameter, such as excitation
wavelength and fluorescence wavelength to be detected, is appropriately
determined to achieve optimal imaging and evaluation, depending on the kind
of near infrared fluorescent contrast agent to be administered and
administration targets. The time spent from administration of the inventive
near infrared fluorescent contrast agent to determination target to the
initiation
of determination by the inventive fluorescence imaging method varies
depending on the kind of the near infrared fluorescent contrast agent to be
used and administration targets. For example, when the agent contains a
compound of the formula [I] for tumor imaging, the lapse time will be about 4-
120 hours after administration. In the case of compound of formula [II], the
lapse time will be about 24-120 hours after administration. When the lapse
time is too short, the fluorescence is so intense that the target site and
other
site cannot be clearly divided. When it is too long, said contrast agent may
be
cleared from the body. When imaging of blood vessel is desired, the
compound of the formula [I] or formula [II] is detected immediately after
administration or in about 30 minutes thereafter.
The method typically includes the following steps.
That is, a near infrared fluorescent contrast agent of the present
invention is administered to a detection target and the detection target is
exposed to an excitation light from an excitation light source. Then,
fluorescence from the near infrared fluorescent contrast agent, which has been
caused by said excitation light, is detected with a fluorescence detector.
The wavelength for excitation varies depending on the near infrared
fluorescent contrast agent to be used. It is free of limitation as long as
said
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compound efficiently emits fluorescence in the near infrared region.
Preferably, a near infrared light having superior biotransmission capability
is
used.
The wavelength of the near infrared fluorescence to be detected also varies
depending on the contrast agent to be used. In general terms, an excitation
light having a wavelength of 600-1000 nm, preferably 700-850 nm, is used and
near infrared fluorescence in a region at a wavelength of 700-1000 nm,
preferably 750-900 nm, is detected. In this case, the excitation light source
may be a conventional excitation light source, such as various lasers (e.g.,
ion
laser, dye laser and semiconductor laser), halogen light source, xenon light
source and the like. Where necessary, various optical filters may be used to
obtain optimal excitation wavelength. Likewise, fluorescence may be detected
using various optical filters to pick up only fluorescence from said near
infrared
fluorescent contrast agent.
The detected fluorescence is data-processed as fluorescence information and
used to generate fluorescence images that can be recorded. The fluorescence
images are generated by irradiating a wide area including the target tissue,
detecting fluorescence with a CCD camera and image-processing the obtained
fluorescence information. Alternatively, an optical CT device may be used, an
endoscope may be used, or a fundus camera may be used.
The fluorescence imaging method of the present invention enables visualizing
systemic diseases, tumors, blood vessels and the like without damaging a
living body.
The present invention is explained in more detail by way of Examples and
Experimental Examples, to which the present invention is not limited. The
compound numbers in the following Examples and Experimental Examples
correspond to those of the compounds explained by structural formulas.
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The compound wherein a symbol designating "potassium salt", "calcium salt"
or "pyridinium salt" is indicated after the compound number (e.g. compound
(29) K salt) means a compound that is the same as the compound expressed
by the compound number (sodium salt) except that the counter ion is
potassium salt, calcium salt or pyridinium salt instead of sodium salt. For
example, "compound (31)K salt" means a compound that is the same as the
compound (31) except that the counter ion is potassium instead of sodium;
"compound (31)Ca salt" means a compound that is the same as compound
(31) except that the counter ion is calcium instead of sodium; and "compound
(31)pyridinium" salt means a compound that is the same as compound (31)
except that the counter ion is pyridinium instead of sodium.
The synthetic method of the compound to be contained in the near infrared
fluorescent contrast agent of the present invention as an active ingredient is
explained in Examples.
The following synthetic methods mostly consist of reactions of heterocyclic
quaternary salt compound shown in Table 1 and dianyl compounds shown in
Tables 2 and 3.
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Table 1 Heterocyclic quaternary salt compound
Structural formula [Registration No.
Symbol Source/Synthetic method
in Chemical Abstract (CA)]
In the same manner as Q2,
HO3S SO3H corresponding indolenin derivative
CH3 is reacted with butanesulton.
Q1 CH3
N+ CH3
(CH2)4S03
(138913-76-5)
JP-A 63-55544
HO3S EP 251282
I CH3
CH3
Q2 N CH3
I
CH2CH2CHSO3
(113995-56-5) CH3
JP-A 2-233658
HO3S CH3 CH3 CA 114:122053
N+ CH3
Q3 I
(CH2)4S03
(76588-81-3)
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In the same manner as Q3,
CH3 corresponding indolenin derivative
HO3S
CH3 is reacted with 2-
N+ CH bromoethanesulfonic acid.
3
Q4 CI
H2CH2SO3
(183272-36-8)
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Table 2 Dianyl compound-1
Structural formula [Registration No. in
Symbol Source/Synthetic method
Chemical Abstract (CA)]
Reagent commercially
available from Aldrich and
NHCH=CH-CH=CH-CH=N ~ \ others
Al (1497-49-0) = HCI
JP-A 8-295658
CH3 CA 126:90721
NHCH=CH-C=CH-CH=N / \
A2
(1979-58-4) = HCI
Zh.Org.Khim., 13(6) 1189-92
/ (1977)
A3 CA 87:102034
= HCI
(53019-66-2)
Zh.Org.Khim., 13(6) 1189-92
ci (1977)
O NHCH &CH=N / \
- CA 87:102034
A4
= HCI
(63856-99-5)
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Table 3 Dianyl compound-2
Structural formula [Registration No. in
Symbol Source/Synthetic method
Chemical Abstract (CA)]
Nukleofil' nye Reacts.
~ H3 Karbonil' nykn Soedin (1982),
NHCH=C-CH=CH-CH=N / \ 52-53
A5 -
(77146-76-0) = HCI CA 101:130179
Ger Offem,
CH2CH2SO3H DE 2928184
/ \ NHCH=CH-C=CH-CH=N / \ CA 94:176696
A6 - -
(125577-71-1)
= HCI
Zh.Org.Khim., 18(10) 2176-9
(1982)
A7 O CA 98:73808
NHCH CH=N / \
= HCI
(56709-94-5)
Zh.Org.Khim., 13(6) 1189-92
ci (1977)
O-NHCH 1: CH=N / \
A8 - CA 87:102034
(63857-00-1) = HCI
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Examples
In the following Examples, the compounds are referred to with the symbols
(e.g., Al, Q1 and the like) used in Tables I to 3 for the convenience's sake.
Example 1: Synthesis of compound (29)
To hetenocydic quatemary salt compound Q1 (5 g) were added methanol (100
ml), N,N-dimethylformamide (25 mi), triethylamine (5.6 ml), dianyl compound
Al (1.83 g) and acetic anhydride (3 ml), and the mixture was stirred at room
temperature for 4 hours. Triethyl amine (2.2 ml) and acetic anhydride (2 ml)
were added, and the mixture was stirred at room temperature for 3 hours. The
insoluble matter was filtered off, and a solution of sodium acetate (2 g) in
methanol (15 ml) was added to the filtrate, which was followed by stining at
room temperature for 1 hour. The resui6ng crystals were collected by
filtration
and washed with a small amount of methanol. To the obtained crude crystals
(3.5 g) was added water (20 ml) for dissolution: Sodium acetate (1 g) was
added, and then methanol (30 mi) was added, which was followed by stirring
for 1 hour. The resulting crystals wbre collected by filtrafion, washed with a
small amount of methanol and dried to give 3 g of compound (29). The
obtained compound (29) showed yellow in a flame test
Maximum wavelength of absorbance (H20) : 780 nm
Molar absorption coefficient (Hz0) : 243,000
Maximum wavelength of fluorescence emission (HZO) : 802 nm
The infrared absorption spectrum was measured for the obtained
compound (29) by potassium bromide tablet method using a Fourier transform
TM
infrared spectrometer (VALOR-111, manufactured by JASCO). The following
peaks were detected. The spectrum is shown in Fig. 11.
IR (Omax(KBr)) : 1414, 1086, 1037, 995, 889 cm'
Example 2: Synthesis of compound (34)
To heterocydic quatemary salt compound Q2 (2.13 g) was added methanol
(20 ml) and the mixture was cooled to 10 C. Thereto were added dianyl
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compound A2 (0.75 g), triethylamine (4 ml) and acetic anhydride (2 ml), and
the mixture was stirred for 20 minutes. Acetic anhydride (2 ml) was added,
and the mixture was stirred at 10 C for 4 hours. The insoluble matter was
filtered off, and a solution of sodium acetate (2 g) in a small amount of
methanol was added to the filtrate. The resulting crystals were collected by
filtration and washed with a small amount of methanol. To the obtained crude
crystals was added water (7ml) for dissolution. Methanol (7 ml) was added to
precipitate crystals. The resulting crystals were collected by filtration,
washed
with a small amount of methanol and dried to give 1.2 g of compound (34).
The obtained compound (34) showed yellow in a flame test.
Maximum wavelength of absorbance (H20) : 794 nm
Molar absorption coefficient (H20) : 176,000
Maximum wavelength of fluorescence emission (H20) : 812 nm
Example 3: Synthesis of compound (6)
To heterocyclic quaternary salt compound Q3 (9.5 g) are added methanol (50
ml), triethylamine (7 ml), dianyl compound A3 (3.1 g) and acetic anhydride
(3.9
ml), and the mixture is stirred at room temperature for 7 hours. The insoluble
matter is filtered off, and a solution of sodium acetate (5 g) in a small
amount of
methanol is added to the filtrate. The mixture is stood overnight. The
resulting
crystals are collected by filtration and washed with a small amount of
methanol.
To the crystals is added water (30 ml) for dissolution. Sodium acetate (2 g)
is
added, and then methanol (30 ml) is added. The resulting crystals are
collected by filtration, washed with a small amount of methanol and dried to
give compound (6).
Example 4: Synthesis of compound (45)
To heterocyclic quaternary salt compound Q3 (4.8 g) were added methanol (50
ml), triethylamine (4 ml), dianyl compound A4 (1.7 g) and acetic anhydride (2
ml), and the mixture was stirred at room temperature for 3 hours. The
insoluble matter was filtered off, and a solution of sodium acetate (4 g) in a
small amount of methanol was added to the filtrate. The resulting crystals
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were collected by filtration and washed with a small amount of methanol. To
the crystals was added water (10 ml) for dissolution. Then methanol (10 ml)
was added. The resulting crystals were collected by filtration, washed with a
small amount of methanol and air dried to give 1.6 g of a compound that is the
same as compound (45) except that the substituent on the methine chain is
-CI instead of -SCH2CH2SO3 Na.
The above step was repeated to give 4.2 g of said compound. Thereto were
added water (30 ml), triethylamine (1.2 ml) and sodium 2-
mercaptoethanesulfonate (0.8 g), and the mixture was stirred at room
temperature for 4 hours. The insoluble matter was filtered off, and a solution
of
sodium acetate (2 g) in a small amount of water was added to the filtrate. The
resulting crystals were collected by filtration, washed with methanol (20 ml)
and
air dried to give 2.3 g of compound (45). The obtained compound (45) showed
yellow in a flame test.
Maximum wavelength of absorbance (HZO) : 815 nm
Molar absorption coefficient (H20) : 196,000
Maximum wavelength of fluorescence emission (H20): 827 nm
Example 5: Synthesis of compound (2)
To heterocyclic quaternary salt compound Q3 (4.7 g) are added methanol (25
ml), triethylamine (2.8 ml), dianyl compound A5 (1.5 g) and acetic anhydride
(2.4 ml), and the mixture is stirred at room temperature for 1 hour. Thereto
are
further added triethyl amine (3.5 ml) and acetic anhydride (1.5 ml), and the
mixture is stirred at room temperature for 3.5 hours. The insoluble matter is
filtered off, and a solution of sodium acetate (3 g) in a small amount of
methanol is added to the filtrate. The mixture is stirred at room temperature
for
1 hour. The resulting crystals are collected by filtration and washed with a
small amount of methanol. To the crystals is added water (15 ml) for
dissolution. Then methanol (15 ml) is added. The resulting crystals are
collected by filtration, washed with a small amount of methanol and dried to
give compound (2).
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Example 6 : Synthesis of compound (43)
To heterocyclic quaternary salt compound Q3 (3.75 g) were added methanol
(25 ml), triethylamine (3.5 ml), dianyl compound A6 (1.95 g) and acetic
anhydride (2.4 ml), and the mixture was stirred at room temperature for 1
hour.
The insoluble matter was filtered off, and a solution of sodium acetate (3.9
g) in
a small amount of methanol was added to the filtrate. The mixture was stirred
at room temperature for 1 hour. The resulting crystals were collected by
filtration and washed with a small amount of methanol. To the crystals was
added water (10 ml) for dissolution. Sodium acetate (2 g) was added, and then
methanol (10 ml) was added. The resulting crystals were collected by
filtration,
washed with a small amount of methanol and dried to give 1.8 g of compound
(43). The obtained compound (43) showed yellow in a flame test.
Maximum wavelength of absorbance (H20) : 773 nm
Molar absorption coefficient (H20) : 204,000
Maximum wavelength of fluorescence emission (H20) : 789 nm
Example 7 : Synthesis of compound (4)
To heterocyclic quaternary salt compound Q3 (3.5 g) are added methanol (20
mi), triethylamine (3.5 ml), dianyl compound A7 (1.2 g) and acetic anhydride
(1.9 ml), and the mixture is stirred at room temperature for 10 hours, and
then
stood overnight. The mixture is stirred under heating at 50 C for 5 hours.
Water (2 ml) is added and the insoluble matter is filtered off. A solution of
sodium acetate (5 g) in a small amount of water is added to the filtrate. The
mixture is stirred at room temperature for 30 minutes. The resulting crystals
are collected by filtration and washed with a small amount of methanol and
dried to give compound (4).
Example 8 : Synthesis of compound (31)
To heterocyclic quaternary salt compound Q4 (3.5 g) were added methanol (35
ml), triethylamine (3.5 ml) and acetic anhydride (2 ml), and dianyl comound A2
(1.8 g) was added portionwise with stirring. The mixture was further stirred
for
1 hour. Acetic anhydride (2 ml) was added, and the mixture was stirred at
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room temperature for 5 hours. The insoluble matter was filtered off, and a
solution of sodium acetate (4 g) in a small amount of methanol was added to
the filtrate. The resulting crystals were collected by filtration and washed
with a
small amount of methanol. To the crystals was added water (10 mi) for
dissolution. Then methanol (10 ml) was added, and the mixture was stirred at
room temperature for 2 hours. The resulting crystals were collected by
filtration, washed with a small amount of methanol and dried to give 1.3 g of
compound (31). The obtained compound (31) showed yellow in a flame test.
Maximum wave(ength of absorbance (H20) : 755 nm
Molar absorption coefficient (H20) : 228,000
Maximum wavelength of fluorescence emission (H20) : 774 nm
The infrared absorption spectrum was measured for the obtained compound
(31) by potassium bromide tablet method using a Fourier transform infrared
rM
spectrometer (VALOR-{II, manufactured by JASCO). The foNowing peaks were
detected. The spectrum is shown in Fig. 12.
IR (vmax(KBr)) : 1518, 1183, 1149, 1111, 995 cm'
Example 9: Synthesis of compound (41)
To heterocyclic quatemary satt compound Q1 (12 g) were added methanol
(120 ml), triethylamine (13.6 mi), dianyl compound A8 (4.4 g) and acetic
anhydride (2.4 mi), and the mixture was stirred for 30 minutes. Acetic
anhydride (2.4 ml) was added and the mixture was stirred for 1.5 hours, and
then acetic anhydride (2.4 mi) was added and the mixture was stirred at room
temperature for 6 hours. Heterocyclic quatemary salt compound Q1 (1 g),
triethyl amine (3 ml) and acetic anhydride (3 ml) were further added and the
mixture was stirred at room temperature for 2 hours. The mixture was stood
overnight. Sodium acetate (5 g) was added and the resulting crystals were
collected by filtration and washed with a small amount of methanol. To the
obtained crude crystals was added water (200 mi). The insoluble matter was
filtered off, and sodium acetate (10 g) was added to the filtrate. The
resulting
crystals were collected by filtration and washed with a small amount of
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methanol. To the crystals were added water (200 ml) and triethylamine (10
ml), and a solution of sodium acetate (10 g) in methanol (100 ml) was added to
give crystals. This step was repeated twice. The resulting crystals were
collected by filtration, washed with a small amount of methanol and dried to
give 9.7 g of compound (41). The obtained compound (41) showed yellow in a
flame test.
Maximum wavelength of absorbance (H20) : 811 nm
Molar absorption coefficient (H20) : 230,000
Maximum wavelength of fluorescence emission (HZO) : 822 nm
Example 10 : Synthesis of compound (3)
According to Example 5, heterocyclic quaternary salt compound Q3 and the
corresponding dianyl compound are used to give compound (3).
Example 11
In the same manner as in the synthesis of compound (29) in Example 1 except
that potassium acetate (2 g) was used instead of sodium acetate (2 g), a
compound that is the same as compound (29) except that the counter ion was
potassium instead of sodium was obtained. Hereinafter this compound is
referred to as compound (29) K salt. The obtained compound (29) K salt
showed purple in a flame test.
Maximum wavelength of absorbance (H20) : 780 nm
Molar absorption coefficient (H20) : 254,000
Maximum wavelength of fluorescence emission (HZO) : 800 nm
Other aforementioned compounds are treated in the same manner as
in this Example to give compounds having potassium counter ion instead of
sodium.
These compounds having potassium counter ion are distinguished
from the above compounds by attaching "K salt" after the corresponding
compound numbers.
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Example 12
In the same manner as in Example 11, compound (6) K salt was obtained. The
obtained compound (6) K salt showed purple in a flame test.
Maximum wavelength of absorbance (H20) : 788 nm
Molar absorption coefficient (H20) : 226,000
Maximum wavelength of fluorescence emission (H20) : 806 nm
Example 13
In the same manner as in Example 11, compound (2) K salt was obtained. The
obtained compound (2) K salt showed purple in a flame test.
Maximum wavelength of absorbance (H20) : 743 nm
Molar absorption coefficient (H20) : 266,000
Maximum wavelength of fluorescence emission (H20) : 762 nm
Example 14
In the same manner as in Example 11, compound (4) K salt was obtained. The
obtained compound (4) K salt showed purple in a flame test.
Maximum wavelength of absorbance (H20) : 753 nm
Molar absorption coefficient (H20) : 212,000
Maximum wavelength of fluorescence emission (H20) : 767 nm
Example 15
In the same manner as in Example 11, compound (3) K salt was obtained. The
obtained compound (3) K salt showed purple in a flame test.
Maximum wavelength of absorbance (H20) : 751 nm
Molar absorption coefficient (H20) : 241,000
Maximum wavelength of fluorescence emission (H20) : 767 nm
Example 16
The compound (6) K salt (50 mg) was dissolved in a small amount of water
and passed through an ion exchange resin to convert potassium of the
compound (6) K salt to proton. Thereto was added methanol saturated with
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sodium acetate to allow precipitatiorr of crystals. This procedure was
repeated
twice. The resulting crystals were collected by filtration, washed with a
small
amount of methanol and dried to give (32 mg) of compound (6). The obtained
compound (6) showed yellow in a flame test.
The infrared absorption spectrum was measured for the obtained compound
(6) by potassium bromide tablet method using a Fourier transfonn infrared
TM
spectrometer (VALOR-III, manufactured by JASCO). The following peaks were
detected. The spectrum is shown in Fig. 13.
IR (vmax(KBr)) : 1395, 1372, 1188, 1102, 1020 crn'
Example 17: Synthesis of compound (54)
To heterocyclic quatemary saft compound Q4 (3.5 g) were added methanol (20
ml), trlethylamine (3.5 mi) and acetic anhydride (2 mi), and dianyl comound Al
(1.4 g) was added portionwise with stirring. The mixture was further stirred
for
20 minutes. Acetic anhydride (1 ml) was added, and the mixture was stirred at
room temperature for 1.5 hours. The insoluble matter was filtered off, and a
solution of sodium acetate (4 g) in a small amount of methanol was added to
the filtrate. The resulting crystals were collected by fiitration and washed
with a
small amount of methanol. The crystals were dissolved in a small amount of
water. Then the solution was diluted with methanol (10 ml), and the mixture
was stirred at room temperature for 1 hour. The resulting crystals were
collected by filtration, washed with a small amount of methanol and dried to
give 1.5 g of compound (54). The obtained compound (54) showed yellow in a
flame test.
Maximum wavelength of absorbance (HZO) : 743 nm
Molar absorption coefficient (H2O) : 244,000
Maximum wavelength of fluorescence emission (HZO) : 766 nm
The infrared absorption spectrum was measured for the obtained compound
(54) by potassium bromide-tablet method using a Fourier transform infrared
TM .
spectrometer (VALOR-I{I, manufactured by JASCO). The following peaks were
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detected. The spectrum is shown in Fig. 14.
IR (vmax(KBr)) : 1511, 1421, 1099, 1004, 926 cm"'
Experimental Example 1
The partition coefficient (log Po/w) of n-butanol/water was determined with
respect to compound (29), compound (43), compound (45), compound (31),
compound (3) K salt, compound (11) [available from Nippon Kankoh-Shikiso
Kenkyusho CO., LTD. as NK-3261], compound (6) K salt, compound (2) K salt,
compound (4) K salt, compound (34) and compound (54).
As a control compound, used were NK-1967 (Nippon Kankoh-Shikiso
Kenkyusho CO., LTD.) and ICG (Tokyo Kasei Kogyo) having only 2 sulfonic
acid groups in a molecule. The results are shown in Table 4.
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Table 4
Compound Number of sulfonic log Po/w
acid group (butanol/water)
Compound (29) 6 -2.00 or less
------------------------------------------------------------ ------------------
---------- ----------------------------
Compound (43) 5 -2.00 or less
------------------------------------------------------------ ------------------
---------- ----------------------------
Compound (45) 5 -2.00 or less
------------------------------------------------------------ ------------------
----------- ----------------------------
Compound (31) 4 -2.00 or less
------------------------------------------------------------ ------------------
---------- ----------------------------
Compound (3) K salt 4 -2.00 or less
------------------------------------------------------------ ------------------
---------- ----------------------------
Compound (11) (NK-3261) 4 -2.00 or less
------------------------------------------------------------ ------------------
---------- ----------------------------
Compound (6) K salt 4 -2.00 or less
-------------------------- --------------------------------- ------------------
---------- ----------------------------
Compound (2) K salt 4 -2.00 or less
--------- -------------------------------------------------- ------------------
---------- ----------------------------
Compound (4) K salt 4 -1.51
--------- -------------------------------------------------- ------------------
---------- ----------------------------
Compound (34) 4 -1.49
--------- -------------------------------------------------- ------------------
---------- ----------------------------
Compound (54) 4 -2.00 or less
NK-1967
CH3 CH3 CH3 CH3
JjI_CH=CH)-CH='JIJ + \2 0.34
N
I I
(CH2)3SO3 (CHz)3SO3Na
------------------------------------------------------------ ------------------
---------- ----------------------------
ICG
/
CH3 CH3 CH3 CH3 I
2 1.41
CH=CH~CH- I \
N+ N /
I I
(CHZ)4S03 (CHZ)4SO3Na
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Experimental Example 2: Fluorescence imaging test (1)
Tumor tissue pieces of mouse colon carcinoma (colon 26 carcinoma) were
subcutaneously grafted to the left breast of BALB/c nude mice (5 weeks old,
Clea Japan, Inc.). Ten days later when the tumor grew to a diameter of about
8 mm, the mice were subjected to the test.
As a fluorescence excitation light source, a titanium sapphire laser was used.
The test mice were uniformly exposed to the laser light using a ring type
light
guide (Sumita Optical Glass Co.) wherein dispersion of irradiation was within
10%. The irradiation power output was adjusted so that it was about 40
W/cm2 near skin surface of the mice. The fluorescence was excited at the
maximum excitation wavelength of each compound and fluorescence emission
from the mice was detected and photographed through a short wavelength
cutoff filter (IR84, IR86, IR88, Fuji Photo Film CO., LTD.) with a CCD camera
(C4880, Hamamatsu Photonics K.K.). The cutoff filter was selected to fit the
excitation wavelength of the compound. The exposure time was adjusted
depending on the fluorescence intensity of each compound.
The test compounds used were compound (29), compound (31) and
compound (6) K salt of the present invention, and NK-1967 and ICG having
only 2 sulfonic acid groups in a molecule as control compounds. Each test
compound (0.5 mg/mI) was dissolved in distilled water and administered to the
mice via a tail vein. The dose was 5.0 mg/kg for compound (31), compound
(6) K salt, NK-1 967 and ICG, and 0.5 mg/kg for compound (29). At 24 hours
after the administration of the compouds, the mice were anesthetized with
diethyl ether and fluorescent light images of the entire body of the mice was
photographed. The results are shown in Figs. 1 to 5.
The compound (29) having a benzotricarbocyanine structure and six sulfonic
acid groups, as well as compound (6) K salt and compound (31) both having a
tricarbocyanine structure and four sulfonic acid groups obviously generated
clearer images of tumor as compared to control compounds (NK-1967 having
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benzotricarbocyanine structure and ICG having tricarbocyanine structure)
having two sulfonic acid groups. In particular, compound (29) could clearly
depict the tumor even at a low dose and was noticeably effective.
Experimental Example 3: Fluorescence imaging test (2)
Nude mice were used for the test. compound (29) of the present invention and
control compound ICG were intravenously injected from the tail vein at a dose
of 5.0 mg/kg each under sevoflurane continuous inhalation anesthesia. At the
same time, intermittent photographing of the fluorescence images was
initiated. For photographing of the fluorescence images, exposure to
excitation
laser beam and extraction of fluorescence through a filter were done, wherein
exposure time was one second. At 20 seconds after the administration of the
compounds, blood vessel was suitably imaged. The fluorescence images were
photographed until 5 minutes after the administration. Figs. 6 to 9 show
fluorescence images of the entire body of the mice at 20 seconds and 5
minutes after the administration.
ICG failed to contrastively show blood vessel in 5 minutes, whereas compound
(29) could image the blood vessel for longer time than ICG.
Experimental Example 4: Residence in blood vessel
In the same manner as in Experimental Example 2, tumor tissue pieces were
grafted to CDF, mice (female, 5 weeks old, Japan SLC, Inc.), and about 2
weeks later when the tumor grew to a diameter of about 1 cm, the mice were
subjected to the test.
The test compounds were compound (29) K salt and compound (41) K salt
having a benzotricarbocyanine structure and 6 sulfonic acid groups; compound
(6) K salt, compound (4) K salt, compound (45) K salt, compound (31),
compound (31) K salt, compound (3) K salt, compound (2) K salt, compound
(43) K salt and compound (11) having a tricarbocyanine structure and 4-5
sulfonic acid groups; and control compounds ICG and NK-1967. Each test
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compound was dissolved in distilled water (0.5 mg/ml) and used. The obtained
each compound solution was administered from the tail vein of the mice (5.0
mg/kg). Blood was taken from the mice at 0.5, 1, 4 and 24 hours after the
administration of the compounds and centrifuged to give plasma.
The fluorescence intensity of the plasma was measured by a
spectrofluorescence meter (RF 5300 PC, SHIMADZU CORPORATION). A
calibration curve of each compound was drawn and compound concentration
in plasma was calculated. The results are shown in Fig. 10.
The compounds of the present invention remained in plasma at high
concentration for a long time.
Experimental Example 5: Acute toxicity
Reduction of toxicity by the introduction of sulfonic acid group and reduction
thereof by conversion into sodium salt were studied.
The test compounds were those listed in Table 5.
Each test compound was dissolved in distilled water to give a compound
solution. This solution was intravenously injected to the conscious mice from
the tail vein. The mice were monitored for 3 days after the administration,
and
acute toxicity [LD50 (mg/kg body weight)] was estimated. The results are
shown in Table 5.
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Table 5
Number of sulfonic Compound LD50 (mg/kg
acid group body weight)
compound (11)K salt 350
compound (11) 1980
compound (31)K salt 350
compound (31) >3550
compound (31)Ca salt 2000
compound (31)
pyridinium salt 1000-2000
Three or more
compound (45)K salt 550
compound (45) 1100-1220
compound (43)K salt 300-350
compound (43) 1630
compound (41)K salt 470
compound (41) >1010
compound (29)K salt 470
compound (29) >1010
compound (54) >5000
compound (6)K salt 350
compound (3)K salt 530
compound (4)K salt 450
compound (2)K salt 610
ICG 70
Two or less
NK1967 20
An increase in the number of sulfonic acid group in a molecule or
conversion to a sodium salt resulted in striking reduction of acute toxicity.
SUBSTITUTE SHEET (RULE 26)
CA 02394539 2002-05-24
WO 01/43781 PCT/EP99/09959
86
The near infrared fluorescent contrast agent of the present invention is
excited by an excitation light and emits near infrared fluorescence. This
infrared fluorescence is superior in transmission through biological tissues.
Thus, detection of lesions in the deep part of a living body has been made
possible. In addition, the inventive contrast agent is superior in water
solubility
and low toxic, and therefore, it can be used safely.
SUBSTITUTE SHEET (RULE 26)
