JP2010203966A - Near-infrared fluorescent probe for imaging low-oxygen region - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a near-infrared fluorescent probe for non-invasively imaging a low-oxygen region with the low-oxygen region as a target reliably and specifically. <P>SOLUTION: The near-infrared fluorescent probe for imaging a low-oxygen region comprises: a near-infrared fluorescent dye; a low-oxygen binding part that is activated by reductive metabolism at a low-oxygen region and is bound with a nucleophilic biomolecule; and linker bonding. The near-infrared fluorescent dye can be a cyanin-based dye or a BODIPY-based dye. A low-oxygen bonding part can be a unit containing a nitro aromatic ring of benzene, imidazole, and triazole. Linker bonding can be formed by a chain unit including a functional group by one of polyester, triazole, amide, thioamide, ester, thioester, carbamate, amine, sulfide, ether, and alkyl groups, or their combination. A method of imaging a low-oxygen region uses the near-infrared fluorescent probe for imaging a low-oxygen region. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a near-infrared fluorescent probe for non-invasive imaging by specifically visualizing a hypoxic region in a living body with near-infrared fluorescence.

  The hypoxic region present in solid cancer induces angiogenesis, metastasis, and malignant transformation of cancer, and is resistant to chemotherapeutic agents and radiation, so imaging the hypoxic region is an early detection of cancer It is important to select the optimal treatment strategy, and in recent years, cancer stem cells that are attracting attention as the cause of cancer development, metastasis, and recurrence are said to lurk in the hypoxic niche, and imaging the hypoxic region This is considered important in research and development of new tests and treatment methods for cancer stem cells.

  Conventionally, methods for diagnosing the location, size, morphology, etc. of lesions such as cancer include biopsy such as puncture, X-ray contrast imaging, MRI contrast imaging, etc., but these diagnosis methods place a heavy burden on the subject. It can also be said to be a highly invasive diagnostic method that may be exposed to X-rays or electromagnetic radiation. On the other hand, near-infrared light is easy to transmit through living tissue and has high safety. As a non-invasive method for diagnosing a living body, near-infrared fluorescence is used to image a lesion using a near-infrared fluorescent dye. Contrast agents have been proposed (see Patent Document 1 and Patent Document 2).

  On the other hand, there is a proposal of a compound that can be used as a diagnostic imaging probe targeting a hypoxic region of a living body (see Patent Document 3).

JP 2003-261464 A JP-A-2005-120026 JP 2007-77036 A

  However, although the near-infrared fluorescent contrast agents described in Patent Documents 1 and 2 can noninvasively image a lesion, they cannot specifically target a hypoxic region of a living body. In addition, although the diagnostic imaging probe described in Patent Document 3 can use a fluorescent compound as a functional part, it is released in a reducing environment in a low oxygen region by electron transfer of a suppression part, and is nucleophilic in vivo. Since it does not bind to biomolecules, it is difficult to stay in the low oxygen region, and the certainty of imaging in the low oxygen region may be insufficient.

  This invention is made | formed in view of said situation, and makes it a subject to provide the near-infrared fluorescent probe which non-invasively images a hypoxic region by making a hypoxic region target reliably and specifically.

The present invention for solving the above problems is as follows.
A near-infrared fluorescent dye characterized by comprising a near-infrared fluorescent dye, a hypoxic binding site that is activated by reductive metabolism in the hypoxic region and binds to a nucleophilic biomolecule, and a linker bond. The gist is an infrared fluorescent probe. Here, the hypoxic region refers to a region in a living body that is in a hypoxic microenvironment due to a lesion, such as a solid cancer lesion, an ischemic disease such as the brain and heart, or a lesion of retinal macular degeneration. It is done.

  In the above invention, the near-infrared fluorescent dye may be a cyanine dye or a BODIPY dye. The low oxygen binding site may be a unit containing a nitroaromatic ring of benzene, imidazole or triazole. The linker bond may be a chain unit having a functional group of any of polyether, triazole, amide, thioamide, ester, thioester, carbamate, amine, sulfide, ether, alkyl group, or a combination thereof.

  The gist of the present invention is a method for imaging a low oxygen region, characterized by using the above-described near-infrared fluorescent probe for low oxygen region imaging.

  Since the near-infrared fluorescent probe for imaging hypoxic region of the present invention can be visualized and imaged non-invasively and specifically targeting the hypoxic region, important knowledge of lesions such as cancer and ischemic diseases And can be used for early detection of diseases and optimal treatment strategies, and is extremely useful.

  The imaging method of the hypoxic region of the present invention enables early detection of a disease and selection of an optimal treatment strategy without imposing a burden on the subject.

It is a figure which shows typically the near-infrared fluorescent probe for low oxygen area | region imaging. In the figure, “NIR fluorescent dye” represents a near-infrared fluorescent dye. 2 is an excitation-fluorescence spectrum of GPU-167 synthesized in Example 1. 3 is an excitation-fluorescence spectrum of GPU-172 synthesized in Example 2. It is the figure which measured the fluorescence intensity of the near infrared region under hypoxia (hypoxia) and normal oxygen (aerobic) of GPU-172 synthesize | combined in Example 2 using IVIS200 imaging system. It is a graph which shows the result of FIG.

  The near-infrared fluorescent probe for imaging in the hypoxic region of the present invention is an imaging probe that emits fluorescence in the near-infrared region, and is activated by reductive metabolism in the near-infrared fluorescent dye and in the low-oxygen region. It consists of a hypoxic binding site that binds to a biomolecule and a linker bond.

  The near-infrared fluorescent dye is not particularly limited as long as it is excited by excitation light and has a fluorescence of 650 nm to 2500 nm, and examples thereof include a cyanine dye and a BODIPY dye. Fluorescence emitted from near-infrared fluorescent dyes is highly permeable to living tissue and is not affected by autofluorescence in the living body, and the target molecule remains alive in vivo or in vitro with high sensitivity and imaging. it can. Said near-infrared fluorescent pigment | dye is shown by the following general formula group (1). As the near-infrared fluorescent dye, a commercially available dye may be used, or a commercially available dye may be semi-synthesized by a known method.

  The near-infrared fluorescent dye of the general formula group (1) can introduce a functional group into the basic skeleton of the dye in order to adjust the water solubility of the near-infrared fluorescent probe for low oxygen region imaging. A functional group can be introduced at a position indicated by A, B, X, Y, or Z by a known method in synthetic chemistry.

  The hypoxic binding site is a unit containing nitro aromatic rings of benzene, imidazole, and triazole that have become electron deficient due to the nitro group, and is activated by reductive metabolism such as p450 reductase in a hypoxic environment to nucleophile proteins (Takasawa M, Moustafa RR, Baron JC. Applications of nitroimidazole in vivo hypoxia imaging in ischemic stroke. Stroke 2008; 39 (5): 1629-37), showing specificity in the hypoxic region It is. Examples of such compounds include units represented by the following general formula group (2).

A linker bond is a unit interposed between a near-infrared fluorescent dye and a hypoxic binding site.
Such a unit has a functional group based on any one of polyether, triazole, amide, thioamide, ester, thioester, carbamate, amine, sulfide, ether, alkyl group, or a combination thereof shown in the following general formula group (3). Mention may be made of chain units. In the figure, the nitroaromatic ring compound represents a low oxygen binding site (a compound group represented by the general formula group (2) containing Ar), and the NIR chromophore represents a near-infrared fluorescent dye. Moreover, the substituent couple | bonded with the nitro aromatic ring of benzene of the unit shown by said general formula group (2), imidazole, and a triazole is contained in a linker coupling | bonding.

  If the near-infrared fluorescent probe for low-oxygen region imaging of this invention is typically shown, as shown in FIG. 1, a near-infrared fluorescent dye and a nucleophilic biomolecule activated by reductive metabolism in the low-oxygen region It consists of a hypoxic binding site that binds to and a linker bond.

  The near-infrared fluorescent probe for imaging of the hypoxic region of the present invention is activated by reductive metabolism in the hypoxic region and binds to a nucleophilic biomolecule such as a protein and stays in the hypoxic region for a while. It is possible to specifically image the hypoxic region. The near-infrared fluorescent probe for imaging a hypoxic region is excreted outside the body by metabolism after a certain period of time. In addition, the near-infrared fluorescent probe for hypoxic region imaging of this invention can be widely used also for animals other than a human.

  The hypoxic region imaging method of the present invention is a method for administering a near-infrared fluorescent probe for hypoxic region imaging to a subject, irradiating the subject with excitation light from an excitation light source, and exciting near-infrared fluorescence excited by the excitation light. Can be detected by detecting with a detector. As the excitation light, visible light or near infrared light can be used by selecting a suitable wavelength according to the type of the near infrared fluorescent probe for low oxygen region imaging. As the excitation light source, a laser light source such as an ion laser, a dye laser, or a semiconductor laser, a halogen light source, a xenon light source, or the like can be used. In this way, hypoxic regions such as cancerous lesions and ischemic lesions can be reliably and specifically targeted and imaged.

  When the near-infrared fluorescent probe for imaging in the low oxygen region of the present invention is a salt, there is no particular limitation as long as it is a pharmacologically acceptable salt. For example, an alkali metal salt such as sodium salt or potassium salt, magnesium salt, calcium An alkaline earth metal salt such as a salt can be used. Moreover, it can be used by being suspended or dissolved in a solvent such as distilled water for injection, physiological saline, Ringer's solution. Furthermore, pharmacologically acceptable additives such as carriers and excipients may be added. The near-infrared fluorescent probe for hypoxic region imaging of the present invention can be administered by injection, spraying, etc. into blood vessels, oral, intraperitoneal, subcutaneous, intradermal and the like. In addition, the dose can be appropriately increased or decreased as long as the site to be diagnosed can be detected, but it is set in consideration of the type of near-infrared fluorescent probe for hypoxic region imaging, the age, sex, weight, application site, etc. of the subject. It is usually 0.1 to 100 mg / kg · body weight, preferably 0.5 to 20 mg / kg · body weight.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited to this.

[Example 1] (Synthesis 1 of near-infrared fluorescent probe for low oxygen region imaging)
1- [4- (aminomethyl) piperidin-1-yl] -3- (2-nitro-1H-imidazol-1-yl) -propan-2-ol (hereinafter referred to as “GPU-174”) was Was synthesized as follows. Compound 1 was synthesized from racemic epichlorohydrin according to the method of Sercel et al. (Sercel AD, Beylin VG, Marlatt ME, Lejab, Showalter HDH, Michel A. Synthesis of the Enantiomers of the Dual Function 2 -Nitroimidazole Radiation Sensitizer RB 6145. J Heterocylic Chem 2006; 43: 1597-604). Compound 2 was prepared from ethanol (10 mL) in the presence of molecular sieves 4A from 1 g (8.76 mmol) 4-aminomethylpiperidine and 1330 mg (13.1 mmol) benzaldehyde according to the method of Diouf et al. And subjected to reaction with compound 1 without purification (Diouf O, Depreux P, Chavatte P, Poupaert JH. Synthesis and preliminary pharmacological results on new naphthalene derivatives as 5-HT4 receptor ligands. Eur. J. Med. Chem. 2000; 35: 699-706). That is, 1407 mg (8.32 mmol) of 2-nitro-1- (2-oxiranylmethyl) -1H-imidazole of Compound 1 was added to the reaction solution containing Compound 2, and the mixture was heated to reflux for 20 hours under a nitrogen atmosphere. After allowing to cool, molecular sieves were removed by centrifugation (3000 rpm, 5 minutes), and the supernatant was distilled off under reduced pressure. To the resulting oily residue 3, 10% hydrochloric acid (20 mL) was added and heated at 40 ° C. for 4 hours. This aqueous phase was washed with chloroform (30 mL × 4), and 10% NaOH aqueous solution was slowly added to the obtained aqueous phase to make it alkaline (pH 9). Subsequently, this aqueous phase was extracted with chloroform (30 mL × 4). The combined organic layers were dried over anhydrous MgSO4 and concentrated under reduced pressure to give 1050 mg of a yellow oily residue. Furthermore, after diluting with a minimum amount of hot ethanol and leaving to stand at 5 ° C. for 12 hours, 978 mg (42%) of GPU-174 (pale yellow crystals) was obtained. A synthesis reaction of GPU-174 is shown in the following formula (4).
1H NMR (CDCl3) δ: 7.29 (s, 1H), 7.12 (s, 1H), 4.70 (dd, J = 14.0, 2.4 Hz, 1H), 4.23 (dd, J = 7.1, 7.1 Hz, 1H), 4.05 (m, 1H), 2.94 (d, J = 11.6 Hz, 1H), 2.79 (d, J = 11.1 Hz, 1H), 2.55 (d, J = 6.3 Hz, 2H), 2.45 (dd, J = 12.3, 4.1 Hz, 1H), 2.26 (m, 2H), 2.00 (td, J = 11.6, 2.4 Hz, 1H), 1.72 (d, J = 12.1 Hz, 2H), 1.31-1.13 (m, 5H); 13C NMR (CDCl3) δ: 144.8, 127.8, 127.4, 65.7, 60.8, 55.1, 53.5, 52.5, 47.8, 38.9, 30.0, 29.8; HRMS (FAB +): calcd.for C ₁₂ H ₂₂ N ₅ O ₃ ([M + H ] +) m / z 284.1723, found 284.1742

The cyanine dye 4 was synthesized according to Mujumdar et al. (Mujumdar RB, Ernst LA, Mujumdar SR, Lewis CJ, Waggoner AS. Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjugate Chem 1993; 4: 105-11). In a nitrogen atmosphere, 300 mg (0.34 mmol) of cyanine dye 4 and 553.4 mg (2.16 mmol) of N, N'-disuccineimidylcarbonate were added to 6.3 mL of anhydrous N, N- Dissolved in a mixed solution of dimethylformamide (N, N-dimethylformamide): anhydrous pyridine (20: 1) and heated at 60 ° C. for 2 hours. After cooling, the reaction mixture was poured into 50 mL of anhydrous ethyl acetate, and the resulting suspension was centrifuged (3000 rpm, 5 minutes) to remove the supernatant. The precipitate was dried under reduced pressure to obtain an active ester. Under a nitrogen atmosphere, this active ester and 102 mg (0.32 mmol) of GPU-174 synthesized above are dissolved in 6 mL of anhydrous N, N-dimethylformamide and 146.7 mg (1.44 mmol, 202 μL) Of triethylamine was added and stirred at room temperature for 1 hour. The reaction solution was poured into 50 mL of cold chloroform to obtain a suspension. This was centrifuged (3000 rpm, 5 minutes), and the supernatant was removed. The precipitate was purified by amine-coated silica gel column chromatography (chloroform / 66% methanol) to obtain a dark green solid (140 mg). The crude product was purified by reversed-phase HPLC to obtain GPU-167 (4.2 mg, 0.9%), a near-infrared fluorescent probe for low oxygen region imaging. The synthesis reaction is shown in the following formula (5).
HRMS (ESI, positive): calcd. For C ₆₃ H ₈₇ N ₁₂ O ₁₄ S ₂ ([M + H] ⁺ ) m / z 1299.5906, found 1299.5897

[Example 2] (Synthesis 2 of near-infrared fluorescent probe for low oxygen region imaging)
The cyanine dye 5 was synthesized according to the method of Narayanan et al. (Narayanan N, Patonay G. A New Method for the Synthesis of Heptamethine Cyanine Dyes: Synthesis of New Near-Infrared Fluorescent Labels. J. Org. Chem. 1995; 60: 2391-5). 50 mg (0.077 mmol) cyanine dye 5 and 87 mg (0.31 mmol) GPU-174 were dissolved in 6.3 mL anhydrous N, N-dimethylformamide and heated at 85 ° C. for 12 hours. . After cooling, 202 mg of the residue obtained by distilling off the solvent under reduced pressure was separated by ODS column chromatography (water / 50% methanol) to obtain a blue solid (28 mg). Furthermore, it was purified by semi-preparative reverse-phase HPLC to obtain GPU-172 (7.6 mg, 12%), a near-infrared fluorescent probe for imaging in a low oxygen region. The synthesis reaction is shown in the following formula (6).
HRMS (ESI, positive): calcd. For C ₅₀ H ₆₈ N ₇ O ₉ S ₂ ([M + H] ⁺ ) m / z 974.4520, found 974.4523

[Example 3] (Excitation-fluorescence spectrum of GPU-167 and GPU-172)
FIG. 2 shows an excitation-fluorescence spectrum in GPU-167, DMSO solvent, 2.307 × 10 ⁻⁶ M. FIG. 2 shows the result of measuring the fluorescence wavelength and intensity by selecting 765 nm as the excitation light from the result of the ultraviolet-visible absorption spectrum. The left vertical axis indicates the excitation light intensity and the right vertical axis indicates the fluorescence intensity. From Figure 2, the fluorescence wavelength of the GPU-167 is, lambda] ex _max = 695 nm, was found to be λEm _max = 801nm.

FIG. 3 shows an excitation-fluorescence spectrum in GPU-172, solvent methanol, and 6.833 × 10 ⁻⁶ M. In FIG. 3, 640 nm was selected as the excitation light from the results of the ultraviolet-visible absorption spectrum, and the fluorescence wavelength and intensity were measured. From FIG. 3, it was found that the fluorescence wavelengths of GPU-172 were λEx _max = 632 nm and λEm _max = 763 nm.

  Table 1 below summarizes the excitation-fluorescence spectrum data of GPU-167 and GPU-172.

[Example 4] (Examination of hypoxia selectivity using cells)
Using cells, we examined the hypoxia selectivity of GPU-172. Using cells cultured under normal oxygen (aerobic) or cells cultured under hypoxia (hypoxia), GPU-172 incorporated into the cells was compared and evaluated by measuring fluorescence intensity. That is, a certain number of human fetal kidney cells (HEK293) were seeded on chamber slides under normoxic or anaerobic culture devices. The cells were cultured at 37 ° C. for 24 hours under normal oxygen or hypoxia (oxygen concentration 0.02%). Then, GPU-172 was added to each and cultured for 2 hours. In the case of hypoxia, the cells were cultured while maintaining a low oxygen state. After removing the medium, the wells were washed several times with PBS. Using the IVIS 200 imaging system (Xenogen), the fluorescence intensity in the near infrared region was measured and compared.

The concentration of GPU-172 was adjusted to 0.1-100 μg / mL (7.7 × 10 ⁻⁸ M-7.7 × 10 ⁻⁵ M) and added.
Moreover, 640 nm was selected as excitation light, and the obtained fluorescence intensity was measured and compared by a 20 nm band pass. The results are shown in FIG. 4 and FIG.

  4 and 5, the strongest fluorescence intensity was obtained at 760 nm, and concentration dependence was observed. When the GPU-172 concentration was 100 μg / mL, the strongest fluorescence intensity was obtained in hypoxia, but strong fluorescence was observed in aerobic, and hypoxia recognition was low. The result was too high. The ratio of hypoxia to aerobic, H / A was the maximum at 10 μg / mL.

  The present invention can be used in the field of medicine, particularly in the field of diagnostic agents.

Claims (5)

  A near-infrared fluorescent dye characterized by comprising a near-infrared fluorescent dye, a hypoxic binding site that is activated by reductive metabolism in the hypoxic region and binds to a nucleophilic biomolecule, and a linker bond. Infrared fluorescent probe.   The near-infrared fluorescent probe for low oxygen region imaging according to claim 1, wherein the near-infrared fluorescent dye is a cyanine dye or a BODIPY dye.   The near-infrared fluorescent probe for low-oxygen region imaging according to claim 1 or 2, wherein the low-oxygen binding site is a unit containing a nitro aromatic ring of benzene, imidazole, or triazole.   The linker bond is a chain unit having a functional group of any one of polyether, triazole, amide, thioamide, ester, thioester, carbamate, amine, sulfide, ether, alkyl group, or a combination thereof. The near-infrared fluorescent probe for low oxygen region imaging in any one of Claims 1-3.   An imaging method for a hypoxic region, wherein the near-infrared fluorescent probe for hypoxic region imaging according to any one of claims 1 to 4 is used.

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