CN1217701C - Near infrared fluorescent contrast agent and fluorescence imaging - Google Patents
Near infrared fluorescent contrast agent and fluorescence imaging Download PDFInfo
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- CN1217701C CN1217701C CN998170437A CN99817043A CN1217701C CN 1217701 C CN1217701 C CN 1217701C CN 998170437 A CN998170437 A CN 998170437A CN 99817043 A CN99817043 A CN 99817043A CN 1217701 C CN1217701 C CN 1217701C
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- chemical compound
- contrast agent
- salt
- molecular formula
- near infrared
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
Abstract
The present invention relates to a near infrared fluorescent contrast agent and a fluorescence imaging method. The contrast agent comprises a compound of which the molecule is provided with three or more sulfonic groups; the method comprises the steps: introducing the near infrared fluorescent contrast agent of the present invention into a living body, exposing the living body to excitation light, and detecting near infrared fluorescence from the contrast agent. The near infrared fluorescent contrast agent of the present invention emits the infrared fluorescence by the excitation of the excitation light, and the infrared fluorescence is superior to transmit and penetrate through biological tissue. Thus, the detection of lesions in the deep part of the living body is made to become possible. In addition, the contrast agent of the present invention is superior in water solubility and low toxicity, and therefore, the contrast agent can be used safely.
Description
Technical field
The fluorescence imaging that the present invention relates to a kind of near infrared fluorescent contrast agent and use described contrast agent.
Background technology
Aspect the treatment disease, most important in the form and function variation that the commitment of disease detects in the live body that is caused by disease.Especially when the treatment cancer, the position of tumor is the important deciding factor of effective therapeutic scheme with size.The known method that is used for this purpose comprises by puncture and carries out biopsy and similar approach, and image-forming diagnose such as x-ray imaging, MRI, ultra sonic imaging and similar approach.Biopsy is effectively to making a definite diagnosis, but it applies very big burden to the experimenter simultaneously, and it is not suitable for the time course variation of following the trail of damage.X-ray imaging and MRI are exposed to the experimenter in radiation and the magnetic wave inevitably.In addition, conventional imaging diagnosis as mentioned above needs complicated operations and long time to be used for measuring and diagnosis.The main equipment that is used for this purpose also makes these methods of utilization in operation become difficult.
The a kind of of image-forming diagnose is fluorescence imaging (Lipspn R.L. etc., J.Natl.CancerInst., 26,1-11 (1961)).This method uses the material of emitting fluorescence when being exposed to the exciting light with specific wavelength as contrast agent.Thereby body is exposed to exogenic exciting light, and the fluorescence that is sent by fluorescent contrast agent in the body can be detected.
Such fluorescent contrast agent can be, for example, cumulative porphyrin compound in tumor, it is used to photodynamic therapy (PDT), as hemoporphyrin.Other example comprises photosensitive sodium and benzoporphyrin (seeing Lipspn R.L. etc., supra, Meng T.S. etc., SPIE, 1641,90-98 (1992), WO 84/04665 etc.).These chemical compounds are used for PDF at first and have phototoxicity, and are needed because this is PDF.Therefore, these are not ideal diagnostic reagent.
Simultaneously, use the retina circulation microangiograph known (United States Patent (USP) 4945239) of known fluorescent dye such as fluorescein, fluorescamin and riboflabin.These fluorescent dyes are at the visible region emitting fluorescence of 400-600nm.In this zone, it is very slow to penetrate living tissue optical transmission, so the detection of deep layer position damage in the body almost is impossible.
In addition, comprise purposes existing file record (the Haglund M.M. etc. of the cyanine compound of indocyanine green (below this paper slightly ICG) as fluorescent contrast agent, Neurosurgery, 35,930 (1994), Li, X. etc., SPIE, 2389,789-797 (1995)), indocyanine green is used to measure liver function and cardiac output.(700-1300nm) shows absorption to cyanine compound in the near infrared region.
Near infrared light shows the high living tissue transmission that penetrates, and can penetrate the skull of the about 10cm of size.Therefore, it attracts much attention on clinical medicine day by day.For example, the light CT technology of working medium optical transmission attracts much attention at clinical field as new technique.This is because near infrared light can penetrate live body, and can be used to monitor oxygen concentration and circulation in the live body.
Cyanine compound is at the near infrared region emitting fluorescence.This regional fluorescence can penetrate living tissue and potentiality as fluorescent contrast agent is provided.Developed many cyanine compounds in recent years, and attempted as fluorescent contrast agent (WO96/17628, WP97/13490 etc.).Yet, have enough water solublity and to live body safety, and have the reagent of distinguishing normal structure and diseased tissue ability (to the selectivity of imaging target position) and also do not exist.
Summary of the invention
Thereby one of purpose of the present invention provides a kind of fluorescent contrast agent.Reagent of the present invention is hypotoxic, and has superior water solublity.In addition, its emission can penetrate the fluorescence of living tissue near infrared region, forms the specific image of tumor and/or blood vessel.
Another object of the present invention provides a kind of fluorescence imaging method that uses described near infrared fluorescent contrast agent.
The present invention is based on three or more sulfonic groups are incorporated into the discovery that causes producing the fluorescent contrast agent with highly-water-soluble in the cyanine dye chemical compound.Also find when using this contrast agent, can set up a kind of fluorescence imaging method.
Thereby, the invention provides following:
(1) a kind of near infrared fluorescent contrast agent, it comprises and has three or more sulfonic chemical compounds in the molecule, and it is represented by molecular formula [I]
R wherein
1And R
2Identical or different, each replaces or unsubstituted alkyl naturally; Z
1And Z
2Each forms naturally and replaces or unsubstituted thick phenyl ring or the required non-metallic atom of thick naphthalene nucleus; R is 0,1 or 2; L
1-L
7Identical or different, each replaces or unsubstituted methine naturally, and condition is when r is 2, simultaneously the L of Chu Xianing
6And L
7Identical or different; And X and Y are identical or different, a group-O-in each following naturally molecular formula ,-S-,-CH=CH-or
R wherein
3And R
4Identical or different, each replaces or unsubstituted alkyl naturally, or the acceptable salt of its pharmacy.
(2) near infrared fluorescent contrast agent in above-mentioned (1) does not have hydroxy-acid group in the molecule.
(3) the near-infrared contrast agent in above-mentioned (1) or (2), wherein, in molecular formula [I], r is 1.
(4) above-mentioned (1) near infrared fluorescent contrast agent in each in (3) wherein has 4 or more a plurality of sulfonic group in the molecule.
(5) above-mentioned (1) near infrared fluorescent contrast agent in each in (4) wherein has 10 or be lower than 10 sulfonic groups in the molecule.
(6) near infrared fluorescent contrast agent in above-mentioned (1) in (4) each wherein has 8 or be lower than 8 sulfonic groups in the molecule.
(7) near infrared fluorescent contrast agent in each in above-mentioned (1) to (6), the acceptable salt of its Chinese materia medica is sodium salt.
(8) near infrared fluorescent contrast agent in each in above-mentioned (1) to (7) is used for tumor imaging and/or angiography.
(9) sodium salt of the chemical compound of molecular formula [II] has three or more sulfonic groups in the molecule
Wherein, R
1, R
2, L
1-L
7, X and Y as above define, and
R
5To R
16Identical or different, each is hydrogen atom, sulfonic group, carboxyl, hydroxyl, alkyl (sulfoalkyl) amino, two (sulfoalkyls) amino, sulphur alkoxyl, (sulfoalkyl) sulfonyl or (sulfoalkyl) amino-sulfonyl naturally, does not comprise the group of following molecular formula
(10) sodium salt in above-mentioned (9), wherein, in molecular formula [II], R
1And R
2Each has the low alkyl group that is replaced by sulfonic group of 1 to 5 carbon atom naturally, and X and Y are identical or different, and each is the following group of molecular formula naturally
R wherein
17And R
18It is unsubstituted low alkyl group with 1 to 5 carbon atom.
(11) sodium salt in above-mentioned (10) has following molecular formula
(12) has the sodium salt of three or more sulfonic chemical compounds in the molecule as molecular formula [III-1]
L wherein
1-L
7As above definition, R
19And R
20Be the low alkyl group that is replaced by sulfonic group with 1 to 5 carbon atom, R
21-R
28Identical or different; each is hydrogen atom, sulfonic group, carboxyl, hydroxyl, alkyl (sulfoalkyl) amino, two (sulfoalkyls) amino, sulphur alkoxyl, (sulfoalkyl) sulfonyl or (sulfoalkyl) amino-sulfonyl naturally; and X ' and Y ' are identical or different, a group in each following naturally molecular formula
R wherein
17And R
18As above definition does not comprise following molecular formula
(13) sodium salt in above-mentioned (12), wherein, in molecular formula [III-1], L
4Methine with alkyl replacement of 1 to 4 carbon atom.
(14) sodium salt in above-mentioned (12), it is the sodium salt that has three or more sulfonic chemical compounds as molecular formula [III-2] in the molecule
R wherein
19-R
28, X ' and Y ' as above define Z
3Be to form 5-or the required non-metal atom group of 6-unit ring, and A is hydrogen atom or univalent perssad.
(15) sodium salt in above-mentioned (14) has following molecular formula
(16) sodium salt in above-mentioned (12) has following molecular formula.
(17) sodium salt in each in above-mentioned (9), (10), (12), (13) and (14) comprises 4 or more a plurality of sulfonic group in the molecule.
(18) sodium salt in each in above-mentioned (9), (10), (12), (13), (14) and (17) comprises 10 or be lower than 10 sulfonic groups in the molecule.
(19) sodium salt in each in above-mentioned (9), (10), (12), (13), (14) and (17) comprises 8 or be lower than 8 sulfonic groups in the molecule.
(20) a kind of near-infrared contrast agent that comprises the sodium salt in above-mentioned (9) in (19) each.
(21) the near-infrared contrast agent in above-mentioned (20) is used for tumor imaging and/or angiography.
(22) a kind of fluorescence imaging method comprises the near infrared fluorescent contrast agent in above-mentioned (1) is introduced in the live body, and body is exposed to exciting light, and detects the near-infrared fluorescent from contrast agent.
(23) sodium salt in above-mentioned (9) is a member that is selected from the chemical compound of following molecular formula at least
(24) sodium salt in above-mentioned (12) is a member that is selected from the chemical compound of following molecular formula at least
(25) near infrared fluorescent contrast agent in above-mentioned (1) comprises at least a chemical compound that is selected from the chemical compound of following molecular formula
And
(26) sodium salt in above-mentioned (14), wherein univalent perssad A is replacement or unsubstituted alkyl, replacement or unsubstituted aryl, replacement or unsubstituted aralkyl, lower alkoxy, randomly is amino, alkyl carbonyl oxy (alkylcarbonyloxy), replacement or unsubstituted alkylthio group, replacement or unsubstituted arylthio, cyano group, nitro or the halogen atom that replaces.
Description of drawings
Fig. 1 to 4 gives the fluorescence imaging photo of chemical compound after 24 hours, and that wherein give is A:ICG (5mg/Kg), B:NK-1967 (5mg/Kg), C: chemical compound (29) (0.5mg/Kg) and D: the K salt (5mg/Kg) of chemical compound (6).
Fig. 5 gives the fluorescence imaging photo of chemical compound after 24 hours, and that wherein give is E: chemical compound (31) (5mg/Kg).
Fig. 6 to 9 gives the fluorescence imaging photo of chemical compound (5mg/Kg) after 20 seconds and 5 minutes, and what wherein give is A:ICG (after 20 seconds), B:ICG (after 5 minutes), C: chemical compound (29) (after 20 seconds) and D: chemical compound (29) (after 5 minutes).
Figure 10 is the figure that gives chemical compound compound concentration in blood plasma after 0.5,1,4 and 24 hour, and wherein the longitudinal axis is the compound concentration (μ g/ml) in each time point blood plasma.
Figure 11 is the infrared absorption spectra of chemical compound (29).
Figure 12 is the infrared absorption spectra of chemical compound (31).
Figure 13 is the infrared absorption spectra of chemical compound (6).
Figure 14 is the infrared absorption spectra of chemical compound (54).
The specific embodiment
The used term definition of this description is as follows.
Near infrared fluorescent contrast agent among the present invention refers to launch the contrast agent of near infrared region fluorescence.
In the present invention, when sulfonic group was used to form inner salt, described sulfonic group can refer to sulfonate radical (SO
3 -).In the present invention, preferred X and Y are following molecular formula
R wherein
3And R
4Identical or different, each replaces or unsubstituted alkyl naturally.
At R
1, R
2, R
3And R
4In the alkyl of " replace or unsubstituted alkyl " be preferably the low alkyl group with 1 to 5 carbon atom of straight or branched, as methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group, sec-butyl, the tert-butyl group, amyl group, isopentyl, neopentyl, tertiary pentyl, 2-methyl-propyl, 1,1-dimethyl propyl etc.Substituent group can be, for example sulfonic group, carboxyl, hydroxyl etc.The example of the alkyl that replaces comprises methylol, 1-ethoxy, 2-ethoxy, 2-hydroxypropyl, 3-hydroxypropyl, 4-hydroxyl butyl, carboxymethyl, carboxyethyl, carboxylic butyl, sulphur methyl, 2-sulfoethyl, 3-sulfopropyl, 4-sulphur butyl etc.Preferred R
1And R
2By the low alkyl group with 1 to 5 carbon atom (as 2-sulfoethyl, 3-sulfopropyl, 4-sulphur butyl etc.) that sulfonic group replaces, R
3And R
4It is unsubstituted low alkyl group (as methyl, ethyl etc.) with 1 to 5 carbon atom.
R
17And R
18The not replacement low alkyl group with 1 to 5 carbon atom with above-mentioned those about R
1, R
2, R
3And R
4" replacement or unsubstituted alkyl " alkyl as an example.
R
19And R
20The low alkyl group that is replaced by sulfonic group with 1 to 5 carbon atom alkyl with above-mentioned those about R
1, R
2, R
3And R
4" replace or unsubstituted alkyl " alkyl as an example, the example of low alkyl group with replacement of 1 to 5 carbon atom comprises 2-sulfoethyl, 3-sulfopropyl and 4-sulphur butyl.
R
21-R
28The moieties of alkyl (sulfoalkyl) amino, two (sulfoalkyls) amino, sulphur alkoxyl, (sulfoalkyl) sulfonyl and (sulfoalkyl) amino-sulfonyl preferably have the straight or branched low alkyl group of 1 to 5 carbon atom, with above-mentioned those about R
1, R
2, R
3And R
4The alkyl of " replace or unsubstituted alkyl " be example.
In the present invention, " form and replace or unsubstituted thick phenyl ring or the required non-metallic atom of thick naphthalene nucleus " finger-type becomes thick phenyl ring or the required linking group of thick naphthalene nucleus, and it is the following group of molecular formula
When thick phenyl ring or thick naphthalene nucleus had substituent group, described linking group can have substituent group.
Its specific example comprises carbon atom, nitrogen-atoms, oxygen atom, hydrogen atom, sulphur atom, halogen atom (as fluorine atom, chlorine atom, bromine atoms and iodine atom) etc.
By non-metallic atom at Z
1And Z
2The thick phenyl ring that forms and the substituent group of thick naphthalene nucleus are with the amino (as dimethylamino, lignocaine, ethyl 4-sulphur fourth amino, two-(3-sulfopropyl) amino etc.) of sulfonic group, carboxyl, hydroxyl, halogen atom (as fluorine atom, chlorine atom, bromine atoms and iodine atom), cyano group, replacement, and the replacement or the unsubstituted alkyl direct or that be bonded on the ring by divalent linker of above definition are example.Preferably divalent linker can be, for example-O-,-NHCO-,-NHSO
2-,-NHCOO-,-NHCONH-,-COO-,-CO-,-SO
2-etc.Directly or by divalent linker be bonded to the replacement of ring or the alkyl of unsubstituted alkyl is an example with methyl, ethyl, propyl group and butyl preferably, substituent group is an example with sulfonic group, carboxyl and hydroxyl preferably.
L
1-L
7The substituent group of methine to replace or unsubstituted alkyl (as above definition), halogen atom (as above definition), replacement or unsubstituted aryl, lower alkoxy etc. are example.The aryl that " replaces or unsubstituted aryl " is an example with phenyl, naphthyl etc., is preferably phenyl.Substituent example comprises halogen atom (as above definition is preferably the chlorine atom) etc.The aryl that replaces comprises, as the 4-chlorphenyl etc.Low alkyl group is preferably the straight or branched alkoxyl with 1 to 6 carbon atom, specifically is methoxyl group, ethyoxyl, propoxyl group, butoxy, tert-butoxy, amoxy etc., is preferably methoxyl group and ethyoxyl.In addition, L
1-L
7The substituent group of methine can be interconnected to form ring with three methines, this ring can further form condensed ring with the ring with other methine.Pass through L
1-L
7The substituent group of methine connect and the ring that forms with three methines with 4,4-dimethyl cyclohexene etc. is an example.
By L
1-L
7The conjugation methine chain with ring that group is formed is preferably the group of molecular formula (a):
Z wherein
3Expression forms 5-or the required non-metallic atom of 6-unit ring, and A is hydrogen atom or univalent perssad.
" forming the required non-metallic atom of 5-or 6-unit ring " with above-mentioned those atoms is example.
Molecular formula (a) and back with the molecular formula of mentioning [III-2] in, Z
35-or 6-unit ring with cyclopentenes ring, cyclohexene ring, 4,4-dimethyl cyclohexene ring etc. is an example, particularly preferably is the cyclopentenes ring.
The univalent perssad of A representative comprises, as replacing or amino, alkyl carbonyl oxy (as acetoxyl group), replacement or unsubstituted alkylthio group, replacement or the unsubstituted arylthio of unsubstituted alkyl (as above definition), replacement or unsubstituted aryl (as above definition), replacement or unsubstituted aralkyl, lower alkoxy (as above definition), the replacement that randomly replaced, cyano group, nitro, halogen atom (as above definition) etc.As used herein, the aralkyl that " replaces or unsubstituted aralkyl " is an example with benzyl, 2-phenethyl, 1-phenethyl, 3-phenylpropyl etc., and substituent group can be sulfonic group, carboxyl, hydroxyl, replacement or unsubstituted alkyl (as above definition), alkoxyl (as above definition), halogen atom (as above definition) etc.The amino of the replacement that the quilt of " amino of replacement " randomly replaces comprises, for example, alkylamino (as methylamino, ethylamino etc.), dialkylamino (dimethylamino, lignocaine etc.), diphenyl amino, aminomethyl phenyl amino, cyclic amino (as morpholino, imidazolidino, ethoxycarbonylpiperadino etc.) etc.Substituent group about the optional replacement of " amino of the replacement that is randomly replaced " comprises sulfonic group, carboxyl etc.The alkylthio group of " replace or unsubstituted alkylthio group " can be, for example, and methyl mercapto, ethylmercapto group etc.Substituent example comprises sulfonic group, carboxyl etc.The arylthio that " replaces or unsubstituted arylthio " is an example with thiophenyl, naphthalene sulfenyl etc.Substituent example comprises sulfonic group, carboxyl etc.
The univalent perssad of A representative is preferably fluorine atom, chlorine atom, dialkyl amido (preferably have 6 or be lower than 6 carbon atoms, and randomly ring formation) or morpholino.This group particularly preferably has sulfonic group.
In molecular formula [I], r is preferably 1.
The acceptable salt of pharmacy can be any nontoxic salts with the compound formation of molecular formula [I].Its example comprises alkali metal salt such as sodium salt, potassium salt, alkali salt such as magnesium salt, calcium salt etc., organic ammonium salt such as ammonium salt, triethyl ammonium salt, tributyl ammonium salt, pyridiniujm etc., amino acid salts such as lysinate, arginine salt etc.Particularly preferably be the lower sodium salt of toxicity in vivo.
The fluorescent contrast agent that is used for live body should be water miscible especially.In the present invention, because of in above-claimed cpd, introducing the water solublity that 3 or more a plurality of sulfonic group have near infrared fluorescent contrast agent to significantly improve.For better water-solubility is arranged, sulfonic number is preferably 4 or more.For synthesizing for simplicity, sulfonic number is no more than 10, preferably is no more than 8.Water miscible raising can be determined by the partition coefficient of measuring each chemical compound, for example, can measure in the two-phase system of butanol/water.More particularly, 3 or more a plurality of sulfonic introducing cause the partition coefficient log Po/w of n-butanol/water to be no more than-1.00.
Sulfonic group especially preferably is incorporated into the R of molecular formula [I]
1, R
2, Z
1And/or Z
2The position and the R of molecular formula [II]
1, R
2, R
5, R
7, R
11And/or R
13The position on.
In addition, these sulfonic groups preferably are incorporated into the L of the conjugation methine chain of above-mentioned molecular formula (a) at position A by divalent group such as alkylidene
4On.
In molecule, have in the sodium salt of chemical compound of 3 or more a plurality of sulfonic molecular formula [II], preferably the sodium salt of following chemical compound, wherein R
1And R
2Be the low alkyl group that is replaced by sulfonic group with 1 to 5 carbon atom, X and Y are identical or different, and each is the group of following molecular formula naturally
R wherein
17And R
18Identical or different, each has the unsubstituted alkyl of 1 to 5 carbon atom naturally, has 3 or more a plurality of sulfonic group in the described molecules of salt, particularly preferably is the following chemical compound of molecular formula
In molecule, have in the chemical compound and the acceptable salt of pharmacy thereof of 3 or more a plurality of sulfonic molecular formula [I], preferably the sodium salt of the chemical compound of molecular formula [III-1]
L wherein
1-L
7As above definition, R
19And R
20By the low alkyl group that sulfonic group replaces, R with 1 to 5 carbon atom
21To R
28Identical or different; each is hydrogen atom, sulfonic group, carboxyl, hydroxyl, alkyl (sulfoalkyl) amino, two (sulfoalkyls) amino, sulphur alkoxyl, (sulfoalkyl) sulfonyl or (sulfoalkyl) amino-sulfonyl naturally; and X ' and Y ' are identical or different, and each is the group of following molecular formula naturally
R wherein
17And R
18As above definition has 3 or more a plurality of sulfonic salt in the described molecule, particularly preferably is the chemical compound of following molecular formula
In molecule, have in the sodium salt of chemical compound of 3 or more a plurality of sulfonic molecular formula [III-1], preferably the sodium salt of the chemical compound of molecular formula [III-2]
R wherein
19-R
28, X ' and Y ' as above define Z
3Be to form 5-or the required non-metallic atom of 6-unit ring, A is hydrogen atom or univalent perssad, has 3 or more a plurality of sulfonic salt in the described molecule, particularly preferably is the chemical compound of following molecular formula
The chemical compound that is contained in the near infrared fluorescent contrast agent of the present invention can be any have molecular formula [I] or [II], also has 3 or more a plurality of in the molecule, is preferably 4 or more a plurality of sulfonic chemical compound.These chemical compounds can synthesize according to the production method of known cyanine dye chemical compound, and these methods are at " cyanine dye and related compound " (The Cyanine Dyesand Related Compounds), F.M.Hamer, John Wiley and Sons, New York, 1964, " cytometry " (Cytometry), 10,3-10 (1989), " cytometry ", 11,418-430 (1990), " cytometry ", 12,723-730 (1990), " bioconjugate chemistry " (Bioconjugate Chem.), 4,105-111 (1993), " bioanalysis chemistry " (Anal.Biochem.), 217,197-204 (1994), " tetrahedron " are (Tetrahedron), 45,4845-4866 (1989), EP-A-0591820A1 is disclosed among the EP-A-0580145A1 etc.Perhaps, they can be semi-synthetic from commercial cyanine dye by known method.Specifically, they can be by synthesizing dianyl chemical compound and the reaction of heterocycle quaternary salt.
The chemical compound of molecular formula of the present invention [I] can synthesize by the following method.
(i) when r=0
(a)L
1＝L
5，X＝Y，R
1＝R
2，Z
1＝Z
2
The heterocycle quaternary salt of molecular formula [IV-1] (2 moles)
L wherein
1, X, Z
1And R
1As above definition is with the dianyl chemical compound (1 mole) of molecular formula [V-1]
L wherein
2, L
3And L
4As above definition is reacted in the presence of alkali and solvent, generates the chemical compound of molecular formula [VI-1]
L wherein
1, L
2, L
3, L
4, R
1, Z
1As above define this chemical compound [VI-1] (1 mole) and need the chemical compound of the molecular formula [VII] of mole with X
T
1-Na [VII]
T wherein
1Be organic acid residue, reaction generates the sodium salt of the chemical compound of above-mentioned molecular formula [VI-1].(b) L
1≠ L
5Or X ≠ Y or R
1≠ R
2Or Z
1≠ Z
2
The dianyl chemical compound (1 mole) of heterocycle quaternary salt compounds of above-mentioned molecular formula [IV-1] (1 mole) and above-mentioned molecular formula [V-1] reacts the chemical compound that generates molecular formula [VIII-1] in the presence of alkali and solvent
L wherein
1, L
2, L
3, L
4, R
1, Z
1As above define the heterocycle quaternary salt compounds (1 mole) of this chemical compound [VIII-1] (1 mole) and molecular formula [XI-1] with X
L wherein
5, Y, Z
2And R
2As above definition, reaction generates the chemical compound of molecular formula [X-1]
L wherein
1, L
2, L
3, L
4, L
5, R
1, R
2, Z
1, Z
2, X and Y as above define, the chemical compound of this molecular formula [X-1] (1 mole) and need the chemical compound reaction of the above-mentioned molecular formula [VII] of mole to generate the sodium salt of the chemical compound of above-mentioned molecular formula [X-1].
(ii) when r=1
(a) L
1=L
7, X=Y, R
1=R
2And Z
1=Z
2
The heterocycle quaternary salt compounds of molecular formula [IV-1] (2 moles)
L wherein
1, X, Z
1And R
1As above definition, and the dianyl chemical compound of molecular formula [V-2] (1 mole)
L wherein
2, L
3, L
4, L
5And L
6As above definition, reaction generates the chemical compound of molecular formula [VI-2] in the presence of alkali and solvent
L wherein
1, L
2, L
3, L
4, L
5, L
6, R
1, Z
1As above define the chemical compound of this molecular formula [VI-2] (1 mole) and need the chemical compound of the molecular formula [VII] of mole with X
T
1-Na [VII]
Wherein T1 as above defines, and reaction generates the sodium salt of the chemical compound of above-mentioned molecular formula [VI-2].
(b) L
1≠ L
7Or X ≠ Y or R
1≠ R
2Or Z
1≠ Z
2
The dianyl chemical compound (1 mole) of heterocycle quaternary salt compounds of above-mentioned molecular formula [IV-1] (1 mole) and above-mentioned molecular formula [V-2] reacts the chemical compound that generates molecular formula [VIII-2] in the presence of alkali and solvent
L wherein
1, L
2, L
3, L
4, L
5, L
6, R
1, Z
1As above define the heterocycle quaternary salt compounds (1 mole) of this chemical compound [VIII-2] (1 mole) and molecular formula [IX-2] with X
L wherein
7, Y, Z
2And R
2As above definition, reaction generates the chemical compound of molecular formula [X-2]
L wherein
1, L
2, L
3, L
4, L
5, L
6, L
7, R
1, R
2, Z
1, Z
2, X and Y as above define, the chemical compound of this molecular formula [X-2] (1 mole) and need the chemical compound reaction of the above-mentioned molecular formula [VII] of mole generates the sodium salt of the chemical compound of above-mentioned molecular formula [X-2].
(iii) when r=2
When r is 2, L
6And L
7Overlapping in molecular formula [I].For fear of this point, eclipsed L
6And L
7Be called as L
8And L
9Express with clear.
(a) L
1=L
9, X=Y, R
1=R
2And Z
1=Z
2
The heterocycle quaternary salt compounds of molecular formula [IV-1] (2 moles)
L wherein
1, X, Z
1And R
1As above definition, and the dianyl chemical compound of molecular formula [V-3] (1 mole)
L wherein
2, L
3, L
4, L
5, L
6And L
7As above definition, L
8The inferior first justice base that is randomly replaced reacts in the presence of alkali and solvent, generates the chemical compound of molecular formula [VI-3]
L wherein
1, L
2, L
3, L
4, L
5, L
6, L
7, L
8, R
1, Z
1As above define this chemical compound [VI-3] (1 mole) and need the chemical compound of the molecular formula [VII] of mole with X
T
1-Na [VII]
Wherein T1 as above defines, and reaction generates the sodium salt of the chemical compound of above-mentioned molecular formula [VI-3].
(b) L
1≠ L
9Or X ≠ Y or R
1≠ R
2Or Z
1≠ Z
2
The dianyl chemical compound (1 mole) of heterocycle quaternary salt compounds of above-mentioned molecular formula [IV-1] (1 mole) and above-mentioned molecular formula [V-3] reacts in the presence of alkali and solvent, generates the chemical compound of molecular formula [VIII-3]
L wherein
1, L
2, L
3, L
4, L
5, L
6, L
7, L
8, R
1, Z
1As above define the heterocycle quaternary salt compounds (1 mole) of this chemical compound [VIII-3] (1 mole) and molecular formula [IX-3] with X
Wherein Y, Z
2And R
2As above definition, L
9The methine that is randomly replaced, reaction generates the chemical compound of molecular formula [X-3]
L wherein
1, L
2, L
3, L
4, L
5, L
6, L
7, L
8, L
9, R
1, R
2, Z
1, Z
2, X and Y as above define.The chemical compound of this molecular formula [X-3] (1 mole) and need the chemical compound reaction of the above-mentioned molecular formula [VII] of mole generates the sodium salt of the chemical compound of above-mentioned molecular formula [X-3].
The chemical compound that needs the molecular formula [VII] of mole is the amount that the amount of the sodium that contains in the molecule that is no less than with the target sodium salt of the chemical compound of molecular formula [I] equates.
L
8And L
9The substituent group of methine of replacement with above-mentioned L
1To L
7The substituent group of methine be example.
In above-mentioned synthetic method (i), (ii) and (iii); the reaction of chemical compound [IV-1] and [V-1]; the reaction of chemical compound [VIII-1] and [XI-1]; the reaction of chemical compound [IV-1] and [V-2]; the reaction of chemical compound [VIII-2] and [IX-2], the reaction of chemical compound [IV-1] and [V-3], and chemical compound [VIII-3] and [IX-3] be reflected at-20 ℃ to-80 ℃; preferably between-10 ℃ to-40 ℃ temperature, preferably in the presence of acylating reagent such as acetic anhydride, carry out.
In above-mentioned synthetic method (i), (ii) and (iii), the reaction of chemical compound [IV-1] and [VII], the reaction of chemical compound [X-1] and [VII], the reaction of chemical compound [VI-2] and [VII], the reaction of chemical compound [X-2] and [VII], the reaction of chemical compound [VI-3] and [VII], and being reflected between 0 ℃ to 40 ℃ temperature of chemical compound [X-3] and [VII] are preferably carried out in the presence of solvent such as second alcohol and water.
In above-mentioned synthetic method (i), (ii) and (iii), used alkali can be, for example, and triethylamine, tri-n-butylamine, pyridine, diazabicyclo hendecene (diazabicycloundecene), Feldalat NM etc.; Solvent for use can be, for example, and amide compound such as N,N-dimethylacetamide, N-Methyl pyrrolidone and N, N-diethylformamide, or alcohols such as methanol; Organic acid residue can be, for example, and CH
3COO etc.
Production about the acceptable salt of various pharmacy of the chemical compound of aforementioned molecular formula [I], the ammonium salt and the potassium salt of the chemical compound of molecular formula [I] can pass through, for example use the chemical compound of molecular formula [VII], wherein sodium atom becomes ammonium group or potassium atom, the chemical compound of the molecular formula [VII] of replace above-mentioned synthetic method (i), using in (ii) and (iii) and obtaining; The different cationic salts of the chemical compound of aforementioned molecular formula [I] can obtain by making spent ion exchange resin convert described ammonium salt and potassium salt to different cationic salts on demand.
The chemical compound that is used for above-mentioned molecular formula of the present invention [I] that comprises the chemical compound of molecular formula [II] is an example with following molecule specifically, and the present invention is not limited to them.
Be contained in above-claimed cpd in the near infrared fluorescent contrast agent of the present invention at 700-1300nm, particularly the near infrared region of about 700-900nm has and absorbs and fluorescence, and molar absorption coefficient is not less than 100000.
As long as near infrared fluorescent contrast agent of the present invention contains chemical compound and/or the acceptable salt of its pharmacy of molecular formula [I] or [II], and have 3 or more a plurality of in the molecule, be preferably 4 or more a plurality of sulfonic group, it is not subjected to special restriction.This compound or its salt can be contained in the described contrast agent alone or with combining form.
Specifically, described contrast agent comprises described chemical compound or at solvent, as suspend in distilled water for injection, normal saline, the Ringer's mixture etc. or dissolved as described in chemical compound.When needs, can add pharmacy acceptable additive such as carrier, excipient etc.These additives contain just like the acceptable electrolyte of pharmacology, buffer agent, detergent and regulate osmotic pressure and the material of raising stability and deliquescent material (for example cyclodextrin, liposome etc.).Can use various additives commonly used in association area.When near infrared fluorescent contrast agent of the present invention was medicinal the design, it was preferably by aseptic method preparation.
Described contrast agent can be by in (intravenous, intra-arterial) in injection, spraying or coating, the blood vessel, oral, intraperitoneal, percutaneous, subcutaneous, the capsule or give in the trachea.Preferably, this agent is administered in the blood vessel with aqueous pharmaceutical, Emulsion or suspensoid.
As long as dosage makes the position of wanting last diagnostic can be detected, the dosage of near infrared fluorescent contrast agent of the present invention has no particular limits.It depend on the chemical compound of employed emission near-infrared fluorescent kind, administration object age, body weight and target organ etc. and suitably regulated.Usually, dosage is preferably the 0.5-20mg/kg body weight in the scope of 0.1-100mg/kg body weight, in the amount of described chemical compound.
Except the people, contrast agent of the present invention can suitably be used for various animals.Form of medication, approach and dosage depend on the body weight of target animal and symptom and are appropriately determin.
In addition, in the present invention, have 3 or more a plurality of in the above-mentioned molecule, be preferably 4 or more a plurality of sulfonic molecular formula [I], especially preferably the chemical compound of molecular formula [II] tends to accumulate in significantly tumor tissues.Utilize this characteristic, use fluorescent contrast agent of the present invention, tumor tissues can be by imaging specifically.In addition, a series of described chemical compounds can be present in the blood vessel for a long time, and their expections can be used as the contrast agent of angiography well.
The feature of fluorescence imaging method of the present invention is the use of near infrared fluorescent contrast agent of the present invention.This method is implemented with known method, and each parameter as excitation wavelength and the wavelength of fluorescence that detected, is to depend on the kind of the near infrared fluorescent contrast agent that will give and administration target and be appropriately determin to realize best image and evaluation.Depend on the kind of employed near infrared fluorescent contrast agent and administration target and change to the time cost that begins to measure from measuring target near infrared fluorescent contrast agent of the present invention with fluorescence imaging method of the present invention.For example, when this agent contained the chemical compound of the molecular formula [1] that tumor imaging uses, be after the administration about 4-120 hour blanking time.In the chemical compound situation of molecular formula [II], be after the administration about 24-120 hour blanking time.When blanking time too in short-term, to such an extent as to the too strong target site of fluorescence and other position can not clearly be distinguished.When blanking time was oversize, described contrast agent may be eliminated in body.When needs carry out blood vessel imaging, the chemical compound of molecular formula [I] or [II] after the administration immediately or detected after about 30 minutes thereafter.
This method generally includes following steps.
That is, detect target near infrared fluorescent contrast agent of the present invention and also will detect target exposure in exciting light from excitation source.Then, the fluorescence that is caused by described exciting light from the near-infrared contrast agent detects with fluorescence detector.
Excitation wavelength depends on employed near infrared fluorescent contrast agent and changes.As long as described chemical compound is emitting fluorescence effectively in the near infrared region, it without limits.Preferably, use near infrared light with better biological transmittability.
The wavelength of the near-infrared fluorescent that is detected also depends on employed contrast agent and changes.Generally, use to have 600-1000nm, be preferably the exciting light of 700-850nm wavelength, the detection wavelength is 700-1000nm, is preferably the near-infrared fluorescent in 750-900nm district.In this case, excitation source can be conventional excitation source, as various laser (as ion laser, dye laser, semiconductor laser), halogen light source, xenon light source etc.When needs, can use various optical filters to obtain maximum excitation wavelength.Equally, can use various optical filters to measure fluorescence, only to obtain fluorescence from described near infrared fluorescent contrast agent.
Detected fluorescence carries out date processing and is used to produce the fluoroscopic image that can be recorded as fluorescence information.Fluoroscopic image comprises the big zone of target tissue by irradiation, carries out image processing and produces with CCD phase machine testing fluorescence and to the fluorescence information that obtains.Perhaps, can use optical ct equipment, can use endoscope, perhaps can use fundus camera.
Fluorescence imaging method of the present invention makes people can estimate general disease, tumor, blood vessel etc. and does not damage live body.
The present invention explains in more detail by the mode of embodiment and experimental example, but the invention is not restricted to these embodiment.Chemical compound sequence number in following examples and experimental example is consistent with the chemical compound sequence number that structural formula is represented.Wherein be meant except counter ion counterionsl gegenions it is potassium salt, calcium salt or pyridiniujm rather than the sodium salt, the identical chemical compound of representing with this chemical compound sequence number of chemical compound (sodium salt) at the chemical compound that the symbol of indicating " potassium salt ", " calcium salt " or " pyridiniujm " is arranged after the chemical compound sequence number (as chemical compound (29) K salt).For example, " chemical compound (31) K salt " is meant except counter ion counterionsl gegenions are potassium rather than sodium, with the identical chemical compound of chemical compound (31); " chemical compound (31) Ca salt " is meant except counter ion counterionsl gegenions are calcium rather than sodium, with the identical chemical compound of chemical compound (31); " chemical compound (31) pyridiniujm " is meant except counter ion counterionsl gegenions are pyridine rather than sodium, with the identical chemical compound of chemical compound (31).
Obtain obtaining in an embodiment explaining as the synthetic method of the chemical compound of the active component of near infrared fluorescent contrast agent of the present invention.
Following synthetic method mainly is made up of the reaction of the dianyl chemical compound of listing in the heterocycle quaternary salt compounds of listing in the table 1 and table 2 and 3.
Table 1 heterocycle quaternary salt compounds
Table 2 Dianyl chemical compound-1
Table 3 Dianyl chemical compound-2
Embodiment
In following examples, for simplicity, chemical compound is with the symbolic representation (as A1, Q1 etc.) of table 1 in 3
Embodiment 1: chemical compound (29) synthetic
In heterocycle quaternary salt compounds Q1 (5g), add methanol (100ml), N, dinethylformamide (25ml), triethylamine (5.6ml), dianyl compd A 1 (1.83g) and acetic anhydride (3ml), mixture at room temperature stirred 4 hours.Add triethylamine (2.2ml) and acetic anhydride (2ml), mixture at room temperature stirred 3 hours.The filtering insoluble matter adds the solution of sodium acetate (2g) in methanol (15ml) in filtrate, then at room temperature stirred 1 hour.Filtration is collected the crystal of generation and is washed with small amount of methanol.Add the coarse crystal (3.5g) that entry (20ml) dissolving obtains.Add sodium acetate (1g), add methanol (30ml) then, then stirred 1 hour.Filter and collect the crystal that generates,, obtain 3g chemical compound (29) with small amount of methanol washing and dry.The chemical compound that obtains (29) shows yellow in flame test.
Maximum absorption wavelength (H
2O): 780nm
Molar absorption coefficient (H
2O): 243000
Maximum emission wavelength (H
2O): 802nm
Use Fourier Tranform infrared spectrometer (VALOR-III, JASCO makes), measure the infrared absorption spectroscopy of the chemical compound (29) that obtains with pellet technique.Detect following peak.Spectrum as shown in figure 11.
IR(νmax(KBr))：1414，1086，1037，995，889cm
-1
Embodiment 2: chemical compound (34) synthetic
Add methanol (20ml) in heterocycle quaternary salt compounds Q2 (2.13g), mixture is chilled to 10 ℃.To wherein adding dianyl compd A 2 (0.75g), triethylamine (4ml) and acetic anhydride (2ml), mixture stirred 20 minutes.Add acetic anhydride (2ml), mixture stirred 4 hours down at 10 ℃.The filtering insoluble matter adds the solution of sodium acetate (2g) in small amount of methanol in filtrate.Filtration is collected the crystal of generation and is washed with small amount of methanol.Add the coarse crystal that water (7ml) dissolving obtains.Add methanol (7ml) precipitation crystal.Filter and collect the crystal that generates,, obtain 1.2g chemical compound (34) with small amount of methanol washing and dry.The chemical compound that obtains (34) shows yellow in flame test.
Maximum absorption wavelength (H
2O): 794nm
Molar absorption coefficient (H
2O): 176000
Maximum emission wavelength (H
2O): 812nn
Embodiment 3: chemical compound (6) synthetic
Add methanol (50ml), triethylamine (7ml), dianyl compound A-13 (3.1g) and acetic anhydride (3.9ml) in heterocycle quaternary salt compounds Q3 (9.5g), mixture at room temperature stirred 7 hours.The filtering insoluble matter adds the solution of sodium acetate (5g) in small amount of methanol in filtrate.Mixture is placed and is spent the night.Filtration is collected the crystal of generation and is washed with small amount of methanol.Add water (30ml) dissolving crystal.Add sodium acetate (2g), add methanol (30ml) then.Filter and collect the crystal that generates,, obtain chemical compound (6) with small amount of methanol washing and dry.
Embodiment 4: chemical compound (45) synthetic
Add methanol (50ml), triethylamine (4ml), dianyl compd A 4 (1.7g) and acetic anhydride (2ml) in heterocycle quaternary salt compounds Q3 (4.8g), mixture at room temperature stirred 3 hours.The filtering insoluble matter adds the solution of sodium acetate (4g) in small amount of methanol in filtrate.Filtration is collected the crystal of generation and is washed with small amount of methanol.Add water (10ml) dissolving crystal.Add methanol (10ml) then.The crystal that filter to collect generates with small amount of methanol washing and air-dry, obtains the 1.6g chemical compound, remove substituent group on the methine chain to be-Cl rather than-SCH
2CH
2SO
3Outside the Na, this chemical compound is identical with chemical compound (45).
Repeat above step, obtain the described chemical compound of 4.2g.To wherein adding entry (30ml), triethylamine (1.2ml) and 2-ethane thiol sodium sulfonate (0.8g), mixture at room temperature stirred 4 hours.The filtering insoluble matter adds the solution of sodium acetate (2g) in low amounts of water in filtrate.Filter and collect the crystal that generates,, obtain 2.3g chemical compound (45) with methanol (20ml) washing and air-dry.The chemical compound that obtains (45) shows yellow in flame test.
Maximum absorption wavelength (H
2O): 815nm
Molar absorption coefficient (H
2O): 196000
Maximum emission wavelength (H
2O): 827nm
Embodiment 5: chemical compound (2) synthetic
Add methanol (25ml), triethylamine (2.8ml), dianyl compound A-45 (1.5g) and acetic anhydride (2.4ml) in heterocycle quaternary salt compounds Q3 (4.7g), mixture at room temperature stirred 1 hour.To wherein adding triethylamine (3.5ml) and acetic anhydride (1.5ml) in addition, mixture at room temperature stirred 3.5 hours.The filtering insoluble matter adds the solution of sodium acetate (3g) in small amount of methanol in filtrate.Mixture at room temperature stirred 1 hour.Filtration is collected the crystal of generation and is washed with small amount of methanol.Add entry (15ml) dissolving crystal.Add methanol (15ml) then.Filter and collect the crystal that generates,, obtain chemical compound (2) with small amount of methanol washing and dry.
Embodiment 6: chemical compound (43) synthetic
Add methanol (25ml), triethylamine (3.5ml), dianyl compd A 6 (1.95g) and acetic anhydride (2.4ml) in heterocycle quaternary salt compounds Q3 (3.75g), mixture at room temperature stirred 1 hour.The filtering insoluble matter adds the solution of sodium acetate (3.9g) in small amount of methanol in filtrate.Mixture at room temperature stirred 1 hour.Filtration is collected the crystal of generation and is washed with small amount of methanol.Add entry (10ml) dissolving crystal.Add sodium acetate (2g), add methanol (10ml) then.Filter and collect the crystal that generates,, obtain 1.8g chemical compound (43) with small amount of methanol washing and dry.The chemical compound that obtains (43) shows yellow in flame test.
Maximum absorption wavelength (H
2O): 773nm
Molar absorption coefficient (H
2O): 204000
Maximum emission wavelength (H
2O): 789nm
Embodiment 7: chemical compound (4) synthetic
Add methanol (20ml), triethylamine (3.5ml), dianyl compd A 7 (1.2g) and acetic anhydride (1.9ml) in heterocycle quaternary salt compounds Q3 (3.5g), mixture at room temperature stirred 10 hours, placed then and spent the night.Mixture stirred 5 hours at 50 ℃ under heating.Add water (2ml) and filtering insoluble matter.In filtrate, add the solution of sodium acetate (5g) in low amounts of water.Mixture at room temperature stirred 30 minutes.Filter and collect the crystal that generates,, obtain chemical compound (4) with small amount of methanol washing and dry.
Embodiment 8: chemical compound (31) synthetic
Add methanol (35ml), triethylamine (3.5ml) and acetic anhydride (2ml) in heterocycle quaternary salt compounds Q4 (3.5g), add dianyl compd A 2 (1.8g) under stirring in batches, mixture at room temperature stirred 1 hour in addition.Add acetic anhydride (2ml), mixture at room temperature stirred 5 hours.The filtering insoluble matter adds the solution of sodium acetate (4g) in small amount of methanol in filtrate.Filtration is collected the crystal of generation and is washed with small amount of methanol.Add entry (10ml) dissolving crystal.Add methanol (10ml), mixture at room temperature stirred 2 hours.Filter and collect the crystal that generates,, obtain 1.3g chemical compound (31) with small amount of methanol washing and dry.The chemical compound that obtains (31) shows yellow in flame test.
Maximum absorption wavelength (H
2O): 755nm
Molar absorption coefficient (H
2O): 228000
Maximum emission wavelength (H
2O): 774nm
Use Fourier Tranform infrared spectrometer (VALOR-III, JASCO makes), measure the infrared absorption spectroscopy of the chemical compound (31) that obtains with pellet technique.Detect following peak.Spectrum as shown in figure 12.
IR(νmax(KBr))：1518，1183，1149，1111，995cm
-1
Embodiment 9: chemical compound (41) synthetic
Add methanol (120ml), triethylamine (13.6ml), dianyl compound A-28 (4.4g) and acetic anhydride (2.4ml) in heterocycle quaternary salt compounds Q1 (12g), mixture stirred 30 minutes.Add acetic anhydride (2.4ml), mixture stirred 1.5 hours, added acetic anhydride (2.4ml) then, and mixture at room temperature stirred 6 hours.Add heterocycle quaternary salt compounds Q1 (1g), triethylamine (3ml) and acetic anhydride (3ml) in addition, mixture at room temperature stirred 2 hours.Mixture is placed and is spent the night.Add sodium acetate (5g), filtration is collected the crystal of generation and is washed with small amount of methanol.Add the coarse crystal that water (200ml) dissolving obtains.The filtering insoluble matter also adds sodium acetate (10g) in filtrate.Filtration is collected the crystal of generation and is washed with small amount of methanol.In crystal, add entry (200ml) and triethylamine (10ml), add the solution of sodium acetate (10g) in methanol (100ml) to generate crystal.Repeat this step twice.Filter and collect the crystal that generates,, obtain 9.7g chemical compound (41) with small amount of methanol washing and dry.The chemical compound that obtains (41) shows yellow in flame test.
Maximum absorption wavelength (H
2O): 811nm
Molar absorption coefficient (H
2O): 230000
Maximum emission wavelength (H
2O): 822nm
Embodiment 10: chemical compound (3) synthetic
According to embodiment 5, use heterocycle quaternary salt compounds Q3 and corresponding dianyl chemical compound, obtain chemical compound (3).
Embodiment 11
Except using potassium acetate (2g) rather than sodium acetate (2g), use with embodiment 1 in the synthetic same way as of chemical compound (29) synthetic with the identical chemical compound of chemical compound (29), except its counter ion counterionsl gegenions are potassium rather than sodium.Hereinafter, this chemical compound is called chemical compound (29) K salt.Chemical compound (29) the K salt that obtains shows purple in flame test.
Maximum absorption wavelength (H
2O): 780nm
Molar absorption coefficient (H
2O): 254000
Maximum emission wavelength (H
2O): 800nm
Other above-mentioned chemical compound uses the mode identical with this embodiment to handle, and obtains having the chemical compound of potassium counter ion counterionsl gegenions rather than sodium.
These chemical compounds with potassium counter ion counterionsl gegenions distinguish with above-claimed cpd by additional " K salt " is next after the respective compound sequence number.
Embodiment 12
Mode with identical with embodiment 11 obtains chemical compound (6) K salt.The chemical compound that obtains (6) K salt shows purple in flame test.
Maximum absorption wavelength (H
2O): 788nm
Molar absorption coefficient (H
2O): 226000
Maximum emission wavelength (H
2O): 806nm
Embodiment 13
Mode with identical with embodiment 11 obtains chemical compound (2) K salt.The chemical compound that obtains (2) K salt shows purple in flame test.
Maximum absorption wavelength (H
2O): 743nm
Molar absorption coefficient (H
2O): 266000
Maximum emission wavelength (H
2O): 762nm
Embodiment 14
Mode with identical with embodiment 11 obtains chemical compound (4) K salt.The chemical compound that obtains (4) K salt shows purple in flame test.
Maximum absorption wavelength (H
2O): 753nm
Molar absorption coefficient (H
2O): 212000
Maximum emission wavelength (H
2O): 767nm
Embodiment 15
Mode with identical with embodiment 11 obtains chemical compound (3) K salt.The chemical compound that obtains (3) K salt shows purple in flame test.
Maximum absorption wavelength (H
2O): 751nm
Molar absorption coefficient (H
2O): 241000
Maximum emission wavelength (H
2O): 767nm
Embodiment 16
Chemical compound (6) K salt (50mg) is dissolved in the low amounts of water, and the potassium of chemical compound (6) K salt is changed into proton by ion exchange resin.Use the saturated methanol of sodium acetate with the precipitation crystal to wherein adding.Repeat twice of this process.Filter and collect the crystal that generates,, obtain chemical compound (6) (32mg) with small amount of methanol washing and dry.The chemical compound that obtains (6) shows yellow in flame test.
Use Fourier Tranform infrared spectrometer (VALOR-III, JASCO makes), measure the infrared absorption spectroscopy of the chemical compound (6) that obtains with pellet technique.Detect following peak.Spectrum as shown in figure 13.
IR(νmax(KBr))：1395，1372，1188，1102，1020cm
-1
Embodiment 17: chemical compound (54) synthetic
Add methanol (20ml), triethylamine (3.5ml) and acetic anhydride (2ml) in heterocycle quaternary salt compounds Q4 (3.5g), add dianyl compd A 1 (1.4g) under stirring in batches, mixture stirred 20 minutes in addition.Add acetic anhydride (1ml), mixture at room temperature stirred 1.5 hours.The filtering insoluble matter adds the solution of sodium acetate (4g) in small amount of methanol in filtrate.Filtration is collected the crystal of generation and is washed with small amount of methanol.Crystal is dissolved in the low amounts of water.Solution dilutes with methanol (10ml) then, and mixture at room temperature stirred 1 hour.Filter and collect the crystal that generates,, obtain 1.5g chemical compound (54) with small amount of methanol washing and dry.The chemical compound that obtains (54) shows yellow in flame test.
Maximum absorption wavelength (H
2O): 743nm
Molar absorption coefficient (H
2O): 244000
Maximum emission wavelength (H
2O): 766nm
Use Fourier Tranform infrared spectrometer (VALOR-III, JASCO makes), measure the infrared absorption spectroscopy of the chemical compound (54) that obtains with pellet technique.Detect following peak.Spectrum as shown in figure 14.
IR(νmax(KBr))：1511，1421，1099，1004，926cm
-1
Experimental example 1
Chemical compound (29), chemical compound (43), chemical compound (45), chemical compound (31), chemical compound (3) K salt, chemical compound (11) [NK-3261 have been measured, from Nippon Kankoh-ShikisoKenkyusho CO., LTD.], the n-butanol/water partition coefficient (log Po/w) of chemical compound (6) K salt, chemical compound (2) K salt, chemical compound (4) K salt, chemical compound (34) and chemical compound (54).
Chemical compound in contrast, used is only have in the molecule 2 sulfonic NK-1967 (Nippon Kankoh-Shikiso Kenkyusho CO., LTD.) and ICG (TokyoKasei Kogyo).The results are shown in table 4.
Table 4
Experimental example 2: fluorescence imaging test (1)
(5 weeks are big, Clea Japan, Inc.) left lung to the BALB/c nude mice with the tumor tissues fragment subcutaneous transplantation of mouse junction cancer (colon 26 cancers).After 10 days when tumor long during to about 8 centimetres of diameter, this nude mice is used for test.
Titanium sapphire laser is used as the fluorescence excitation light source.Use ring light guide device (Sumita Optical Glass Co.), test mice is exposed in the laser equably, and wherein the peptizaiton of illumination is in 10%.The output of illumination power is adjusted at the mouse skin near surface and is about 40 μ W/cm
2Fluorescence excitation under the maximum excitation wavelength of each chemical compound passes through short wavelength's edge filter (IR84, IR86 from the fluorescent emission of mice, IR88, FujiPhoto Film CO. LTD) detects and photograph with CCD camera (C4880, Hamamatsu Photonics K.K.).Select edge filter to be suitable for the excitation wavelength of chemical compound.Open-assembly time depends on the fluorescence intensity of each chemical compound and regulates.
Used test compounds is chemical compound of the present invention (29), chemical compound (31) and chemical compound (6) K salt, and only has 2 sulfonic NK-1967 and ICG in the molecule of chemical compound in contrast.Each test compounds (0.5mg/ml) is dissolved in the distilled water and through the tail vein gives mice.The dosage of chemical compound (31), chemical compound (6) K salt, NK-1967 and ICG is 5.0mg/Kg, and the dosage of chemical compound (29) is 0.5mg/Kg.Give chemical compound after 24 hours, mice is used etherization, and the fluoroscopic image of the whole health of mice is taken.The result is shown in Fig. 1 to 5.
Compare with having 2 sulfonic control compounds (NK-1967 and ICG), have benzo three carbocyanine structures and 6 sulfonic chemical compounds (29) and both and all have three carbocyanine structures and 4 sulfonic chemical compounds (6) K salt and chemical compound (31) and obviously produce clearly tumor imaging with three carbocyanine structures with benzo three carbocyanine structures.Especially, chemical compound (29) is also effective significantly even also can clearly depict tumor when low dosage.
Experimental example 3: fluorescence imaging test (2)
Nude mice is used in this test.Sucking continuously under the anesthesia with sevoflurane, chemical compound of the present invention (29) and control compound ICG are injected into from the tail vein with the dosage of 5.0mg/Kg.Simultaneously, fluoroscopic image is taken in beginning off and on.In order to take fluoroscopic image, be exposed to excitation laser and extract fluorescence by optical filter, wherein open-assembly time was 1 second.Give chemical compound after 20 seconds, blood vessel is by suitably imaging.Take fluoroscopic image up to administration after 5 minutes.Fig. 6 to 9 has shown the fluoroscopic image of the whole health of mice administration after 20 seconds and 5 minutes.
ICG can not optionally show blood vessel after 5 minutes, and chemical compound (29) can make the time of blood vessel imaging be longer than ICG.
Experimental example 4: the stop in blood vessel
With with experimental example 2 in identical mode, the tumor tissues fragment is transplanted to CDF
1Mice (female, 5 weeks are big, Japan SLC, and Inc.), when tumor is long when arriving about 1 centimetre of diameter, mice is accepted test after about 2 weeks.
The chemical compound of test is to have benzo three carbocyanine structures and 6 sulfonic chemical compounds (29) K salt and chemical compound (41) K salt, have three carbocyanine structures and 4-5 sulfonic chemical compound (6) K salt, chemical compound (4) K salt, chemical compound (45) K salt, chemical compound (31), chemical compound (31) K salt, chemical compound (3) K salt, chemical compound (2) K salt, chemical compound (43) K salt and chemical compound (11), and control compound ICG and NK-1967.Each test compounds is dissolved in (0.5mg/ml) use in the distilled water.The solution of each chemical compound that obtains gives (5.0mg/Kg) from mouse tail vein.Get blood and centrifugally go out blood plasma from mice after 0.5,1,4 and 24 hour in administration.
(RF 5300 PC SHIMADZUCORPORATION) measure the fluorescence intensity of blood plasma by spectrofluorophotometer.Make the calibration trace of each chemical compound, calculate compound concentrations in the blood plasma.The result as shown in figure 10.
Be retained in the blood plasma to chemical compound long time high concentration of the present invention.
Experimental example 5: acute toxicity
Studied by introducing sulfonic group and changing into sodium salt and reduced toxicity.
Test compounds is listed in the table 5.
Each chemical compound is dissolved in the solution that obtains chemical compound in the distilled water.This solution is injected into sentient mice from the tail cava vein.After the administration, monitored 3 days of mice, assessment acute toxicity [LD
50(mg/Kg body weight)].The results are shown in table 5.
Table 5
| The sulfonic group number | Chemical compound | LD 50(mg/Kg body weight) |
| 3 or more a plurality of | Chemical compound (11) K salt | 350 |
| Chemical compound (11) | 1980 | |
| Chemical compound (31) K salt | 350 | |
| Chemical compound (31) | ＞3550 | |
| Chemical compound (31) Ca salt | 2000 | |
| Chemical compound (31) pyridiniujm | 1000-2000 | |
| Chemical compound (45) K salt | 550 | |
| Chemical compound (45) | 1100-1220 | |
| Chemical compound (43) K salt | 300-350 | |
| Chemical compound (43) | 1630 | |
| Chemical compound (41) K salt | 470 | |
| Chemical compound (41) | ＞1010 | |
| Chemical compound (29) K salt | 470 | |
| Chemical compound (29) | ＞1010 | |
| Chemical compound (54) | ＞5000 | |
| Chemical compound (6) K salt | 350 | |
| Chemical compound (3) K salt | 530 | |
| Chemical compound (4) K salt | 450 | |
| Chemical compound (2) K salt | 610 | |
| ICG | 70 | |
| 2 or be lower than 2 | NK1967 | 20 |
The increase of sulfonic group number or be converted into sodium salt and cause acute toxicity obviously to descend in the molecule.
Be excited optical excitation and launch near-infrared fluorescent of near infrared fluorescent contrast agent of the present invention.This near-infrared fluorescent penetrate aspect the biological tissue comparatively superior.Thereby, make the damage of measuring the live body further portion become possibility.In addition, contrast agent of the present invention is comparatively superior aspect water solublity and hypotoxicity, thereby it can be used safely.
Claims (1)
1, near infrared fluorescent contrast agent, it comprises the following chemical compound of molecular formula:
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|---|---|---|---|
| PCT/EP1999/009959 WO2001043781A1 (en) | 1999-12-15 | 1999-12-15 | Near infrared fluorescent contrast agent and fluorescence imaging |
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| SK (1) | SK8142002A3 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7682602B2 (en) * | 2003-12-19 | 2010-03-23 | Konica Minolta Medical & Graphic, Inc. | Near-infrared fluorescent contrast medium |
| JP2005220045A (en) * | 2004-02-04 | 2005-08-18 | Konica Minolta Medical & Graphic Inc | Fluorescent contrast agent |
| JP5074044B2 (en) * | 2007-01-18 | 2012-11-14 | オリンパス株式会社 | Fluorescence observation apparatus and method of operating fluorescence observation apparatus |
| US8344158B2 (en) * | 2007-08-15 | 2013-01-01 | Washington University | Fluorescent polymethine cyanine dyes |
| US20090214436A1 (en) | 2008-02-18 | 2009-08-27 | Washington University | Dichromic fluorescent compounds |
| EP2470887B1 (en) | 2009-08-28 | 2020-03-11 | VisEn Medical, Inc. | Systems for tomographic imaging in diffuse media using a hybrid inversion technique |
| JP2011046663A (en) | 2009-08-28 | 2011-03-10 | Fujifilm Corp | Near infrared fluorescent imaging agent |
| AU2010298388B2 (en) | 2009-09-22 | 2015-05-28 | Visen Medical, Inc. | Systems and methods for virtual index-matching of diffusive media |
| CA2787129A1 (en) * | 2010-01-14 | 2011-07-21 | The Regents Of The University Of California | Devices, systems and methods to detect and reduce or prevent entry of inflammatory mediators into milk ducts |
| GB201010878D0 (en) | 2010-06-29 | 2010-08-11 | Ge Healthcare As | Dye compositiion and dye syntheses |
| US10230943B2 (en) | 2012-01-23 | 2019-03-12 | Washington University | Goggle imaging systems and methods |
| EP2906106B1 (en) | 2012-10-15 | 2023-06-14 | VisEn Medical, Inc. | Systems, methods, and apparatus for imaging of diffuse media featuring cross-modality weighting of fluorescent and bioluminescent sources |
| CN105228628B (en) | 2013-03-15 | 2019-10-18 | 普渡研究基金会 | Synthesis and composition with the amino acid linker of the compound conjugation for cancer target to be imaged |
| EP3089653A1 (en) | 2013-12-31 | 2016-11-09 | Memorial Sloan Kettering Cancer Center | Systems, methods, and apparatus for multichannel imaging of fluorescent sources in real time |
| JP6751715B2 (en) | 2014-12-15 | 2020-09-09 | メモリアル スローン ケタリング キャンサー センター | Cyclic peptide with enhanced neural binding selectivity, nanoparticles bound to the cyclic peptide, and use of the cyclic peptide for real-time in vivo neural tissue imaging. |
| WO2016179350A1 (en) | 2015-05-06 | 2016-11-10 | Washington University | Compounds having rd targeting motifs and methods of use thereof |
| PL3166606T3 (en) * | 2015-06-03 | 2018-10-31 | Surgimab S.A.S. | Fluorescent conjugates |
| CN105222828B (en) * | 2015-09-30 | 2017-05-24 | 东南大学 | Synchronization measurement device for wall-adherent jet speed field and concentration field and method |
| KR20180093961A (en) | 2015-12-15 | 2018-08-22 | 메모리얼 슬로안-케터링 캔서 센터 | System distinctions, for example imaging systems and methods for intraoperative visualization |
| US11660354B2 (en) | 2016-11-30 | 2023-05-30 | Memorial Sloan Kettering Cancer Center | Inhibitor-functionalized ultrasmall nanoparticles and methods thereof |
| US11712482B2 (en) | 2019-12-13 | 2023-08-01 | Washington University | Near infrared fluorescent dyes, formulations and related methods |
| EP4015004A1 (en) | 2020-12-18 | 2022-06-22 | Phi Pharma SA | Proteoglycan specific branched peptides |
| CN114459862A (en) * | 2022-01-13 | 2022-05-10 | 华腾实业（深圳）股份有限公司 | High-identification fungus fluorescent staining solution |
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| DE69132750T2 (en) * | 1990-05-15 | 2002-06-20 | Hyperion Inc | FLUORESCENT PORPHYRIN AND FLUORESCENT PHTHALOCYANIN-POLYETHYLENE GLYCOL, POLYOL AND SACCHARID DERIVATIVES AS FLUORESCENT PROBE |
| EP0800831B9 (en) * | 1995-01-30 | 2004-11-10 | Daiichi Pure Chemicals Co., Ltd. | Diagnostic marker |
| JP2000095758A (en) * | 1998-09-18 | 2000-04-04 | Schering Ag | Near-infrared, fluorescent contrast medium, and its production |
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| HUP0204024A2 (en) | 2003-04-28 |
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| AU2097500A (en) | 2001-06-25 |
| MXPA02005806A (en) | 2010-10-26 |
| EE200200321A (en) | 2003-10-15 |
| NO20022837D0 (en) | 2002-06-14 |
| WO2001043781A1 (en) | 2001-06-21 |
| SK8142002A3 (en) | 2002-12-03 |
| BR9917587A (en) | 2002-08-06 |
| YU44402A (en) | 2005-03-15 |
| CN1384760A (en) | 2002-12-11 |
| BG106822A (en) | 2003-01-31 |
| CA2394539C (en) | 2009-10-27 |
| IL149798A0 (en) | 2002-11-10 |
| NO20022837L (en) | 2002-06-14 |
| KR20020082207A (en) | 2002-10-30 |
| TR200201567T2 (en) | 2002-11-21 |
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