KR20210009919A - Novel Near Infrared Fluorescent Probe Targeting Mitochondria and Composition for Diagnosing and Treating Tumor Containing the Same - Google Patents

Abstract

The present invention relates to a novel near-infrared fluorescent probe targeting mitochondria and a composition for both diagnosis and treatment of tumor containing the same and, more specifically, to a novel near-infrared fluorescent probe (SiR-Mito11) prepared through fluorescence core hydrophobicity control of silicon-rhodamine, that is, NIR phosphor, and a composition for both diagnosis and treatment of tumor containing the same as an active ingredient. SiR-Mito11 which is a near-infrared fluorescent probe according to the present invention can selectively dye only mitochondria of tumor cells, and also SiR-Mito11 accumulated specifically in tumor cells can induce apoptosis by activating apoptosis proteins, thereby being advantageously used as a pharmaceutical composition for both diagnosis and treatment of various tumors including brain tumors.

Description

A novel near-infrared fluorescent probe targeting mitochondria and a composition for diagnosis and treatment of tumors comprising the same.

The present invention relates to a novel near-infrared fluorescent probe targeting mitochondria, and a composition for diagnosis and treatment of tumors including the same, and more particularly, by controlling the hydrophobicity of the fluorescent core of silicon-rhodamine, a NIR fluorescent substance. The present invention relates to a novel near-infrared fluorescent probe (SiR-Mito11) and a composition for both tumor diagnosis and treatment comprising the same as an active ingredient.

Mitochondria are essential organelles for cell survival that produce adenosine triphosphate (ATP) and regulate energy homeostasis and regulate various cellular functions. In particular, mitochondria have a unique double-membrane structure (internal and external mitochondrial membranes), and actively maintain an electronegative potential by adjusting the H+ concentration gradient (DeBerardinis, RJ, et al. , Cell Metab., 7 : 11-20, 2008; Smith, RA, et al. , Trends Pharmacol. Sci., 33:341-352, 2012; Wisnovsky, S., et al. , Cell Chem. Biol., 23:917-927, 2016).

In addition to the bioenergetic function, mitochondria play a variety of roles, including reactive oxygen species (ROS), redox homeostasis, regulation of cellular signals, programmed cell death and production of biosynthetic metabolism. As a result, mitochondria contribute to cell adaptation to a changing environment, thereby affecting cancer development, growth, survival and metastasis. In addition, because the mitochondrial membrane potential (MMP) of cancer cells is more polarized than that of normal cells, mitochondria are considered as potential targets for cancer diagnosis (Hou, XS, et al. , Biomater. Sci., 6: 2786-2797, 2018)

For the intrinsic cell death pathway, mitochondria play a central role in activating the caspase cascade (Mcilwain, DR, et al. , Caspase Functions in Cell Death and Disease, 1-29, 2015). Cellular stress such as DNA damage or ER stress induces the destruction of mitochondrial membrane potential (Ψ _m ) and mitochondrial outer membrane permeabilization (MOMP), which leads to cytochrome C (cytochrome C) from mitochondria to cytosol. c) is released to induce cleavage of caspase and PARP (Ly, JD, et al. , Apoptosis, 8:115-128, 2003; RM, K., et al. , Science, 275: 1132-1136, 1997). Thus, disturbances in mitochondrial function and membrane permeability can directly induce cell death.

Cancer cells usually inhibit mitochondrial cell death by overexpressing BCL-2 protein to prevent MOMP. Paradoxically, tumor cells respond more sensitively to cell death than normal cells due to various factors, such as conditions that induce cell death. In this context, the recent development of a therapy targeting the mitochondrial cell death pathway is considered an effective cancer therapy (Lopez, J, et al. , Br . J. Cancer, 112:957-962, 2015; Schmitt, S., Zischka, H., Dtsch.Zeitschrift fur Onkol., 50:124-130, 2018).

Glioma, a type of malignant brain tumor, is known to be the most aggressive and extremely difficult to treat. Currently, resection, radiation therapy, and some chemotherapy (temozolomide) are possible for brain cancer, but there are many limitations such as tumor sites that cannot be treated, drug resistance or recurrence. For example, temozolomide, an alkylating agent, is the most widely used chemotherapy for glioblastoma for at least 10 years and does not respond to at least 50% of patients by removing DNA alkylated adducts. On the other hand, chemical agents targeting mitochondria are attracting attention as cancer treatments for brain tumors by inhibiting changes in mitochondrial metabolism peculiar to cancer (Guntuku, L., et al. , Curr. Neuropharmacol., 1 4:567-583, 2016)

Accordingly, in the present invention, as a result of efforts to develop a probe for both diagnosis and treatment targeting the mitochondria of tumor cells, a novel near-infrared fluorescent probe, which is a novel near-infrared fluorescent probe, through the hydrophobic regulation of the fluorescent core of silicon-rhodamine, a NIR fluorescent substance. SiR-Mito11 was developed. It was confirmed that SiR-Mito11 has NIR photophysical properties and coordinated hydrophobicity, and specifically stains only mitochondria, and SiR-Mito11 accumulated specifically in tumor cells activates apoptosis proteins to prevent apoptosis. After confirming the induction, the present invention was completed.

An object of the present invention is to provide a near-infrared fluorescent probe targeting mitochondria.

Another object of the present invention is to provide a pharmaceutical composition for diagnosis and treatment of tumors comprising the near-infrared fluorescent probe as an active ingredient.

To achieve the above object,

The present invention provides a near-infrared fluorescent probe (SiR-Mito11) represented by the following formula (1) and targeting mitochondria.

[Formula 1]

According to a preferred embodiment of the present invention, the near-infrared fluorescent probe may be manufactured by controlling the hydrophobicity of a fluorescent core of silicon-rhodamine, which is a near-infrared fluorescent substance.

According to another preferred embodiment of the present invention, the near-infrared fluorescent probe may exhibit fluorescence at an excitation wavelength of 651 to 657 nm and an emission wavelength of 662 to 668 nm.

In the present invention, (a) 3-bromo-N,N-dimethylaniline (3-Bromo-N,N-dimethylaniline) and formaldehyde (formaldehyde) reacted to prepare a compound 1 represented by the following formula (A) Step to do;

[Formula A]

(b) synthesizing Compound 2 represented by the following Formula B by lithiating Compound 1, adding dichlorodimethylsilane, and then performing an oxidation reaction with potassium permanganate. ;

[Formula B]

(c) tert - butyl-3-bromo-4-methylbenzoic acid (tert -butyl 3-bromo-4 -methylbenzoate) and tert - butyllithium to by the addition of (tert -Butyllithium) to the compound 2 represented by the chemical formula C Preparing compound 3; And

[Formula C]

(d) reacting the compound 3 with n-octyl amine to prepare a near-infrared fluorescent probe represented by Formula 1 below;

[Formula 1]

It provides a near-infrared fluorescent probe manufacturing method comprising a.

According to a preferred embodiment of the present invention, lithiated in the step (b) Sec Compound 1 may be performed by treating the butyl lithium (Sec -Butyllithium).

According to another preferred embodiment of the invention, the step (c) of tert - butyl 3-bromo-4-methylbenzoic acid (tert -butyl 3-bromo-4 -methylbenzoate) is 3-bromo-4-methyl acid (3-Bromo-4-methylbenzoic acid) and di - tert - by reacting butyl dicarbonate (Di- tert -butyl dicarbonate) may be prepared.

The present invention also provides a pharmaceutical composition for diagnosis and/or treatment of tumors comprising the near-infrared fluorescent probe as an active ingredient.

According to a preferred embodiment of the present invention, the tumor is brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, gastric cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, Ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, anal muscle cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymph adenocarcinoma, bladder cancer, gallbladder cancer, endocrine adenocarcinoma, thyroid cancer, Parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord It may be at least one selected from the group consisting of tumors, brainstem glioma, and pituitary adenoma.

According to another preferred embodiment of the present invention, the near-infrared fluorescent probe may be specifically accumulated in the mitochondria of tumor cells.

According to another preferred embodiment of the present invention, when the near-infrared fluorescent probe is accumulated in the mitochondria of tumor cells, the destruction of mitochondrial membrane potential (MMP) and mitochondrial outer membrane permeabilization (MOMP) is prevented. You can induce.

According to another preferred embodiment of the present invention, when the near-infrared fluorescent probe is accumulated in the mitochondria of tumor cells, it is possible to induce cell death by promoting the release of cytochrome C from the mitochondria to the cytosol. have.

According to another preferred embodiment of the present invention, caspase 3 and PARP cleavage may be induced by the release of cytochrome C.

SiR-Mito11, a near-infrared fluorescent probe of the present invention, can selectively stain only the mitochondria of tumor cells, and SiR-Mito11 accumulated specifically for tumor cells can induce apoptosis by activating apoptosis proteins, It can be usefully used as a pharmaceutical composition for diagnosis and treatment of various tumors including brain tumors.

1 is a schematic diagram of a mechanism of action of apoptosis of the near-infrared fluorescent probe (SiR-Mito11) of the present invention.
2 is a schematic diagram showing the synthesis process of SiR-Mito11.
3 is ¹ H, ¹³ C NMR spectrum data of SiR-Mito11.
4 is data showing (a) absorption and emission spectra (Abs: absorbance, Em: emission) of SiR-Mito11 and (b) chemical and photophysical properties of SiR-Mito11. Here, MW is the molecular weight, Ex/Em is the excitation/emission wavelength, Ф is the quantum yield, and cLogP is the partition coefficient.
5 is a fluorescence imaging experimental data regarding the mitochondrial selectivity of SiR-Mito11, a co-localization test of SiR-Mito11 with Mitotracker Green (top) or Lysotracker Red (bottom) (Co -localization test) was performed. The image on the right shows single cell staining indicated by a white dot box in the merged image, and the image was expressed in three independent experiments and expressed as pseudo-color (Mitotracker or Lysotracker: green, SiR-Mito11: red , Hoechst: blue.Scale bar = 15 μm).
Figure 6 shows the tumor cyto-selective toxicity of SiR-Mito11, the cell viability screening data for a series of SiR-Mito probes in H4 neuroblastoma and HT22 neuronal cells. The red dot circle represents SiR-Mito11, and the cLogP of each compound is indicated by a gradient color according to a color gradation bar.
Figure 7 shows the tumor cyto-selective toxicity of SiR-Mito11, (a) cell viability according to the dose of SiR-Mito11 in HT22 cells and various brain tumor cells, and (b) IC ₅₀ of SiR-Mito11 for various cell types And data expressed by quantifying the selectivity window.
Figure 8 shows the difference in cellular uptake of SiR-Mito11 in normal brain cells and tumor brain cells, (a) difference in cell uptake of SiR-Mito11 in normal brain cells and tumor brain cells using fluorescence imaging, and (b) plate This is the data showing the difference in cellular uptake of SiR-Mito11 in normal brain cells and tumor brain cells using a reader. 8A shows a fluorescence image (left) and quantitative data of fluorescence intensity (right), and the image was expressed in two independent experiments and expressed in pseudocolor (SiR-Mito11: red, Hoechst: blue. Scale bar = 25 μm ). In addition, the graph represents the mean SEM value derived from the data of 20 individual cells (***, p<0.0001 calculated by t-test), and each point represents the intensity of the individual cells.
9 is data showing mitochondrial membrane potential disruption by SiR-Mito11 in normal brain cells and tumor brain cells using fluorescence imaging. Cell merged fluorescence images between red fluorescence (high mitochondrial polarization) and green fluorescence (depolarized mitochondrial region) were visualized pseudochromatic (Scale bar = 25 μm).
10 is data confirming the degree of cytochrome C release according to the tumor cell selective mitochondrial accumulation of SiR-Mito 11.
11 is data confirming the degree of activation of apoptosis proteins according to the accumulation of selective mitochondria in tumor cells of SiR-Mito 11.
12 is data confirming the degree of induction of tumor cell death of SiR-Mito 11 according to the treatment of z-VAD-fmk, which is a pan-caspase inhibitor.

Hereinafter, the present invention will be described in detail.

As described above, recently, a method of treating tumors by suppressing changes in mitochondrial metabolism of tumor cells has attracted attention.

In the present invention, as a result of trying to develop a novel near-infrared probe targeting the mitochondria of tumor cells, a novel near-infrared fluorescent probe, SiR-Mito11, was developed, and the SiR-Mito11 not only specifically stains mitochondria, but also It was confirmed that specific accumulation in the mitochondria of tumor cells induces cell death.

1 is a schematic diagram of the mechanism of apoptosis of the near-infrared fluorescent probe (SiR-Mito11) of the present invention. When SiR-Mito11 accumulates in the mitochondria of tumor cells, mitochondrial membrane potential (Ψ _m ) and mitochondrial outer membrane permeability The destruction of membrane permeabilization (MOMP) is induced, and as a result, cytochrome c is released from the mitochondria into the cytosol, leading to the cleavage of caspase and PARP. Therefore, due to the accumulation of tumor cell mitochondrial-specific SiR-Mito11, mitochondrial function and membrane permeability are disturbed, which can directly induce the death of tumor cells.

Accordingly, the present invention relates to a near-infrared fluorescent probe (SiR-Mito11) targeting the mitochondria of tumor cells represented by the following Chemical Formula 1 in a consistent view.

[Formula 1]

In the present invention, the near-infrared fluorescent probe (hereinafter, referred to as "SiR-Mito11") may be manufactured by controlling the hydrophobicity of a fluorescent core of silicon-rhodamine, a near-infrared fluorescent substance. .

In the present invention, the SiR-Mito11 is characterized in that it exhibits fluorescence at an excitation wavelength of 651 ~ 657 nm and an emission wavelength of 662 ~ 668 nm.

In another aspect of the present invention, (a) 3-bromo-N,N-dimethylaniline (3-Bromo-N,N-dimethylaniline) and formaldehyde (formaldehyde) by reacting the compound represented by the formula A Manufacturing a;

[Formula A]

(b) synthesizing Compound 2 represented by the following Formula B by lithiating Compound 1, adding dichlorodimethylsilane, and then performing an oxidation reaction with potassium permanganate. ;

[Formula B]

(c) tert - butyl-3-bromo-4-methylbenzoic acid (tert -butyl 3-bromo-4 -methylbenzoate) and tert - butyllithium to by the addition of (tert -Butyllithium) to the compound 2 represented by the chemical formula C Preparing compound 3; And

[Formula C]

(d) reacting the compound 3 with n-octyl amine to prepare a near-infrared fluorescent probe represented by Formula 1 below;

[Formula 1]

It relates to a method of manufacturing a near-infrared fluorescent probe comprising a.

In the present invention, the lithiated in the step (b) the compound 1 Sec - butyl lithium can be carried out by treating the (Sec -Butyllithium), wherein the (c) step of the tert - butyl 3-bromo-4 methylbenzoic acid (tert -butyl 3-bromo-4 -methylbenzoate) is 3-bromo-4-methylbenzoic acid (3-bromo-4-methylbenzoic acid) and di - tert - butyl dicarbonate (Di- tert -butyl dicarbonate) It can be prepared by reacting.

In a specific embodiment of the present invention, a silicon-rhodamine fluorophore and n-octyl amine are combined to reduce general toxicity of normal cells while maintaining continuous hydrophobicity for mitochondrial targeting properties. SiR-Mito11 was prepared in which an aliphatic chain was introduced instead of a cyclic ring. SiR-Mito11 of the present invention is specifically shown in Figure 2 the manufacturing method.

In another specific embodiment of the present invention, as a result of confirming the near-infrared fluorescence characteristics of SiR-Mito11 prepared in the present invention, as shown in FIG. 4, excitation wavelength 654 nm, emission wavelength 665 nm, quantum yield 0.40 and hydrophobicity It was confirmed that the cLogP value represents 6.55.

In another specific embodiment of the present invention, as a result of confirming the mitochondrial-targeted properties of SiR-Mito11, as shown in FIG. 5, MitoTracker Green and SiR-Mito11, a fluorescent substance that specifically stains only mitochondria Pearson's coefficient with R = 0.94 was confirmed to show a high correlation, whereas LysoTracker Red, a fluorescent substance that specifically stains only lysozyme, had a low correlation with SiR-Mito11 (R = 0.51), that is, it was confirmed that the SiR-Mito11 of the present invention was a mitochondrial-specific near-infrared fluorescent probe.

In another aspect, the present invention relates to a pharmaceutical composition for diagnosis and/or treatment of tumors comprising the near-infrared fluorescent probe (SiR-Mito11) of the present invention as an active ingredient.

In the present invention, the tumor is brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, gastric cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer , Small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer , Soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brain stem glioma And it may be characterized in that at least one selected from the group consisting of pituitary adenoma, preferably may be a brain tumor.

In the present invention, the near-infrared fluorescent probe may be characterized in that it is specifically accumulated in the mitochondria of tumor cells.

In a specific embodiment of the present invention, in order to evaluate the cytotoxic effect and selectivity in brain tumor cells, SiR-Mito11 and the present invention using H4 neuroblastoma cells and HT22 hippocampal neuron cells. It was compared with the toxicity of SiR-Mito1 to SiR-Mito10 (Min, JS, et al. , Bioconjug . Chem ., 30:210-217, 2018) previously prepared by the inventors.

As shown in Table 1 and Figure 6, in the present invention, it was confirmed that the increase in the hydrophobicity of the probe (SiR-Mito4: Bz, SiR-Mito5: Phenylethyl, SiR-Mito6: Cyclohexylmethyl) decreases H4 cell viability (33.0, respectively, 28.6, 4.8%). In particular, a probe having a cycloalkane substitution agent (SiR-Mito6: Cyclohexylmethyl, SiR-Mito8: Cyclooctyl, SiR-Mito9: Cyclohexylethyl) generally exhibited high cytotoxicity to both HT22 and H4 cells. In addition to the cycloalkane moiety, a long chain alkyl chain (SiR-Mito10: 2-Methylheptyl) having a branched aliphatic substituent also showed cytotoxic activity in both cell lines. In addition, the probe having a long n-alkyl chain substituent (SiR-Mito2: Ethyl, SiR-Mito3: n-butyl) showed only marginal selectivity for H4 than in HT22.

On the other hand, when treated with SiR-Mito11 of the present invention on normal cells and brain tumor cells, HT22 cells showed a cell viability of 90% or more, whereas H4 cells were confirmed that most of the cells were killed by SiR-Mito11. In normal cells, the IC ₅₀ value of SiR-Mito11 was 9.1, and the IC ₅₀ value was 0.3 to 5.8 in brain tumor cells, which means that SiR-Mito11 has 1.5 to 30.3 times more selectivity in tumor cells than in normal cells. do.

That is, in the present invention, it was confirmed that the side chain of the SiR fluorophore plays an important role for tumor cell-specific toxicity, among which n-octyl amine chains. It was confirmed that SiR-Mito11 acts specifically for tumor cells. Considering the safety of normal neuronal cells and a wide range of dosages for imaging, in the present invention, it was determined that SiR-Mito11, which is less toxic, would be useful in the application of brain tumor diagnosis and treatment.

As shown in FIG. 7, in order to confirm the anticancer effect of SiR-Mito11 on a wide range of brain tumor cells, various types of malignant brain tumors such as neuroblastoma, astrocytoma, glioblastoma, and metastatic cancer The glioma cell line of the morphology was added and examined. SiR-Mito11 showed strong toxicity with an IC ₅₀ of 0.3 ~ 5.8 μM, and the selectivity value for normal neuronal cells was confirmed to be 1.5 ~ 30.3 times.

In the present invention, in order to rationalize the tumor cytotoxicity of SiR-Mito11, metabolic energy consumption of tumor cells increases, cell uptake of SiR-Mito11 increases compared to normal neuronal cells, and ultimately direct activation of apoptosis is induced. Hypothesized. Figure 8a shows the difference in cellular uptake of SiR-Mito11 in normal brain cells and tumor brain cells. Fluorescence imaging for cellular uptake of SiR-Mito11 shows that the accumulation of SiR-Mito11 is significantly increased in H4 cells compared to HT22 cells. Confirmed. In addition, for the quantitative evaluation at the single cell level, the average fluorescence intensity of SiR-Mito11 was measured around ~10 ⁴ cells in both HT22 and H4 cells, and as shown in FIG.8B, the cellular uptake level of SiR-Mito11 was HT22 cells. It was confirmed that it was clearly elevated in H4 cells when compared with.

That is, the SiR-Mito11 of the present invention not only enables specific imaging of tumor cells, but also induces death of tumor cells, and thus can be usefully used as a composition for both tumor diagnosis and treatment.

In the present invention, when the near-infrared fluorescent probe is accumulated in the mitochondria of tumor cells,

(1) induction of disruption of mitochondrial membrane potential (MMP) and mitochondrial outer membrane permeabilization (MOMP);

(2) promoting cytochrome C release from mitochondria to cytosol; And

(3) It may be characterized in that caspase 3 and PARP cleavage is induced by the cytochrome C release, thereby inducing tumor cell-specific cell death.

Mitochondrial membrane potential (MMP) disruption caused by intrinsic death signals leads to membrane permeabilization and apoptosis. Therefore, MMP is one of the important indicators for distinguishing between unhealthy cells and healthy cells through disturbance of mitochondrial function.

In a specific embodiment of the present invention, the effect of SiR-Mito11 on MMP was analyzed, and as a result, as shown in FIG. 9, a shift in fluorescence emission from red to green was detected in both HT22 and H4 cells. It means that the number of unhealthy cells increases by the destruction of MMP in the cell. In particular, it was observed that H4 cells had a greater decrease in MMP than HT22 cells due to SiR-Mito11 treatment? It was confirmed that H4 cells were very sensitive to MMP change and apoptosis by SiR-Mito11.

In the present invention, for further mechanistic studies, the SiR-Mito11-induced apoptosis pathway was observed in H4 and HT22 cells. As shown in FIG. 10, in the present invention, cytochrome C release was observed in H4 cells by treatment with SiR-Mito11, but not in HT22 cells. In addition, as shown in FIG. 11, it was confirmed that apoptosis was clearly activated in H4 cells by SiR-Mito11 treatment through Western blot analysis of cleaved caspase 3 and PARP, whereas HT22 cells It was confirmed that it was not observed in.

In another specific embodiment of the present invention, in order to confirm whether the tumor cell selective toxicity of SiR-Mito11 is caused by caspase-dependent cell death, a pan-caspase inhibitor z-VAD-fmk was used. After treatment to suppress cell death, cell viability was confirmed. As a result, as shown in FIG. 12, it was confirmed that apoptosis was inhibited in H4 cells treated with z-VAD-fmk even with SiR-Mito11 treatment, whereas no change was observed in HT22 cells.

That is, it was confirmed that SiR-Mito11 promotes cytochrome C release and induces caspase-dependent apoptosis in tumor cells.In the present invention, the uptake of SiR-Mito11 in glial cells is increased due to high energy demand and mitochondrial metabolism. , It was confirmed that this induces cell death.

The route of administration of the pharmaceutical composition of the present invention may be administered through any general route as long as it can reach the target tissue. Parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, or intradermal administration, but is not limited thereto. At this time, in order to formulate a formulation for parenteral administration, the probe of the present invention may be mixed with water with a stabilizer or buffer to prepare a solution or suspension, which may be prepared in an ampoule or vial unit dosage form. The composition may be sterilized and/or contain adjuvants such as preservatives, stabilizers, hydrating agents or emulsification accelerators, salts and/or buffers for controlling osmotic pressure, and other therapeutically useful substances, which are conventional methods of mixing, granulating It can be formulated according to the method of painting or coating.

In addition, the dosage of the pharmaceutical composition of the present invention to the human body may vary depending on the patient's age, weight, sex, dosage form, health condition and degree of disease, and based on an adult patient with a body weight of 60 kg, It is generally 0.001 to 1,000 mg/day, preferably 0.01 to 500 mg/day, and may be dividedly administered once a day or several times a day at regular time intervals according to the judgment of a doctor or pharmacist.

Hereinafter, the present invention will be described in more detail through examples.

These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

SiR-Mito11 synthesis

All intermediates for synthesizing SiR-Mito11 were prepared according to previously known methods (Sung, J., et al. , Bioconjug. Chem., 30:210-217, 2018), and all reagents used in the experiment were commercially available. It was purchased from and used without further purification. The specific synthesis process is shown in FIG. 2.

Compound 1

3-Bromo-N,N-dimethylaniline (3-Bromo-N,N-dimethylaniline; 400 mg, 2.00 mmol) and formaldehyde (880 μL) were added to 20 mL of acetic acid (AcOH) Then, the mixture was stirred at 85° C. for 1 hour. After completion of the reaction, the reaction mixture was cooled to room temperature, and acetic acid was removed by evaporation, and NaHCO ₃ (sat) and 1N NaOH were treated to the crude product. Then, the organics were extracted three times with DCM, then the combined organic layers were dried over Na ₂ SO ₄ and concentrated in vacuo.

The reaction mixture was purified by silica gel column chromatography (EA: Hex = 1: 100 → 1:10), and compound 1 (4,4'-methylenebis(3-bromo- N , N- dimethylaniline) represented by the following formula A; 204 mg, 49.5%) was obtained as a white solid.

[Formula A]

Compound 2

A hexane solution (1.0 mL) containing 1.3 M sec-BuLi was slowly added dropwise to a THF (20 mL) solution containing Compound 1 (200 mg, 0.485 mmol), and the resulting reaction mixture It was stirred at -78 °C for 30 minutes. Then, THF (3.5 mL) containing Si ₂ MeCl ₂ (106 μL, 0.873 mmol) was added dropwise, and the resulting reaction mixture was warmed to room temperature and then stirred overnight. 12 mL of 1N HCl solution was added to the cloudy solution, and the resulting blue reaction mixture was basified with NaHCO ₃ (sat) and concentrated under vacuum. The resulting green oil was diluted with acetone (3 mL) and stirred at -20 °C. KMnO ₄ was added in small portions (6 x 30 mg) for 30 minutes to the reaction mixture, followed by stirring at the same temperature for 2 hours.

The resulting purple suspension was filtered through a Celite pad, then washed with acetone and the yellow filtrate was evaporated. Then, the reaction mixture was purified by silica gel column chromatography (Hex: DCM = 1: 4) to obtain compound 2 (35 mg, 22.3%) represented by the following formula B as a white solid.

[Formula B]

Compound 3

First, 3-Bromo-4-methylbenzoic acid (1.40 g, 6.51 mmol), di-tert-butyl dicarbonate (3.62 g, 16.6 mmol) and DMAP (180 mg, 1.47 mmol) was added to THF (10 mL) and then refluxed overnight. After 1 day, the reaction mixture was cooled to ambient temperature and concentrated in vacuo, the resulting white solid was basified with NaHCO ₃ (sat) and the organic material was extracted 3 times with EA. The combined organic layers were dried over Na ₂ SO ₄ and concentrated in vacuo.

The reaction mixture was purified by silica gel column chromatography (EA: Hex = 1: 100 to 1: 10) to to give the tert represented by the formula D - butyl 3-bromo-4-methylbenzoic acid (tert -butyl 3-bromo- 4-methylbenzoate; 923 mg, 52.1%) was obtained as a clear oil.

[Formula D]

Then, a solution of THF (5 mL) containing tert -butyl 3-bromo-4-methylbenzoic acid (200 mg, 0.741 mmol) prepared above was stirred at -78°C, and then tert -butyllithium ( tert Pentane containing -butyllithium ( tert- BuLi); 450 μL, 0.765 mmol) was slowly added dropwise, and the reaction mixture was stirred at the same temperature for 1 hour. Then, THF (3 mL) containing Compound 2 (50 mg, 0.154 mmol) was added dropwise to the reaction mixture, and the resulting reaction mixture was warmed to room temperature and then stirred for 2 hours.

0.1N HCl (aq.) was added to the reaction mixture, and the resulting dark blue solution was evaporated with NaHCO ₃ (sat) and extracted three times with DCM. The combined organic layers were dried over Na ₂ SO ₄ and concentrated in vacuo. The resulting blue solid was used in the next reaction without further purification, and the blue solid was added to 2 mL MeCN and 8 mL 6N HCl (aq.), followed by stirring at 40° C. for 1 hour. After cooling to room temperature, the reaction mixture was basified with 0.1N NaOH to adjust the pH to 2-3, and then extracted three times with DCM. The combined organic layers were dried over Na ₂ SO ₄ and concentrated in vacuo.

The reaction mixture was purified by silica gel column chromatography (MeOH: DCM = 0: 100 → 1: 5) to obtain compound 4 (45 mg, 73.4%) represented by the following formula C as a dark blue solid.

[Formula C]

SiR-Mito11

DMF (0.5 mL, 0.09 mM) solution containing compound 3 (20 mg, 1.0 eq), TSTU (15 mg, 1.1 eq) and DIPEA (30 μl, 3.8 eq) was stirred for 10 minutes at room temperature and under argon atmosphere. I did. After the reaction, octylamine (15 μl, 2.0 eq) was added to the reaction mixture, followed by stirring at room temperature overnight. The reaction mixture was purified using Biotage MPLC and 18C reverse phase column chromatography, and finally SiR-Mito11[N-(7-(dimethylamino)-), a dark blue solid product (6.1 mg, 24.4%) represented by the following Formula 1 5,5-dimethyl-10-(2-methyl-5-(octylcarbamoyl)phenyl)dibenzo[b,e]silin-3(5H)-ylidene)-N-methylmethanaminium] was obtained.

[Formula 1]

The results of analyzing the product by NMR are as follows (Fig. 3).

1H NMR (600 MHz, Methanol- d 4) δ7.94 (dd, J = 7.8 Hz, 1H), 7.60 (d, J = 1.8 Hz, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.38 (d, J = 3.0 Hz, 2H), 7.06 (d, J = 9.6 Hz, 2H), 6.79 (dd, J = 9.6 Hz, 2H), 3.35 (s, 12H), 2.10 (s, 3H), 1.34 (m, 2H), 1.31 (m, 12H), 0.88 (m, 3H), 0.62 (s, 6H); 13C NMR (150 MHz, Methanol- d 4) δ169.3, 168.9, 155.8, 149.5, 142.1, 141.0, 140.4, 133.4, 131.6, 129.0, 128.7, 128.4, 122.3, 155.4, 41.1 40.9, 33.0, 30.5, 30.4, 30.4, 28.1, 23.7, 19.5, 14.4, -1.2; LRMS (ESI) m/z calcd. for C35H48N3OSi+ [M] 554.36; found: 554.30.; HRMS (ESI) m/a calcd. 554.3561; found 554.3654.

Confirmation of chemical and photophysical properties of SiR-Mito11

In the present invention, in order to confirm the chemical and photophysical properties of SiR-Mito11 synthesized in Example 1, the absorption and emission spectrum of SiR-Mito11, quantum yield, and cLogP value indicating hydrophobicity were measured. To this end, a PBS 1X solution containing SiR-Mito11 (10 μM) is put in a quartz cuvette, and a UV-Vis spectrometer (JASCO V-670), a fluorescence spectrometer (JASCO FP-8200), and an absolute luminous efficiency measuring device (QE-2000) Photophysical properties were measured using, and cLogP was predicted using the Chemdraw program.

As a result, as shown in FIG. 4, it was confirmed that SiR-Mito11 exhibited an excitation wavelength of 654 nm, an emission wavelength of 665 nm, a quantum yield of 0.40, and a cLogP value indicating hydrophobicity of 6.55.

Identification of mitochondrial targeting properties of SiR-Mito11

In the present invention, it was attempted to confirm the mitochondrial targeting properties of SiR-Mito11.

H4 neuroblastoma cells were purchased and used from ATCC, and prepared by culturing in DMEM (Dulbecco's modified eagle media, High glucose) medium containing 10% FBS and 1% penicillin at 5% CO ₂ and 37 ℃. I did.

Reagents for cell culture, including medium, serum and penicillin, were purchased from Hyclone and Corning, respectively. All culture vessels used in the cell-based assay were purchased and used from COSTAR and SPL.

Then, H4 cells were inoculated into a confocal dish and incubated overnight, and then 2.5 μM SiR-Mito11, 500 nM MitoTracker Green (Invitrogen) or 1 μM LysoTracker Red (Invitro) Rosen) and Hoechst (Hoechst; Thermo Fisher Scientific) in DMEM medium containing 1 hour. Then, the cells were washed with PBS and observed with an inverted-fluorescence microscope (DMi8, LEICA) in the state containing the medium.

Images are Hoechst's DAPI channel (ex/em = 325 ~ 375 nm/435 ~ 485 nm), Mitotracker Green's FITC channel (460 ~ 500 nm/512 ~ 542 nm), Lysotracker Red's Rhodamine channel (541 ~ 551 nm). /565 ~ 605 nm), was obtained using a Cy5 channel (545 ~ 625 nm/663 ~ 738 nm) of SiR-Mito11.

Pearson's coefficient was measured as an image containing 80-100 cells using ImageJ FIJI. Quantitative analysis of fluorescence intensity was performed using ImageJ FIJI.

As shown in FIG. 5, it was confirmed that the Pearson's coefficient with the phosphors MitoTracker Green and SiR-Mito11, which are fluorescent substances that specifically stain only mitochondria, showed a high correlation with R = 0.94, whereas lysozyme It was confirmed that LysoTracker Red, a fluorescent substance that stains only specifically, shows a low correlation (R = 0.51) with SiR-Mito11, that is, the SiR-Mito11 of the present invention is a mitochondrial-specific near-infrared fluorescent probe. It was confirmed that it was.

Identification of SiR-Mito11's tumor cell-selective toxicity characteristics

In the present invention, the tumor cytotoxicity of SiR-Mito11 was confirmed. H4 cells and HT22 hippocampal neuron cells were used in the experiment, and HT22 cells were sold and used at the NN center of KIST. In addition, SiR-Mito1 to SiR-Mito10 (Sung, J., et al. , Bioconjug. Chem., 30:210-217, 2018) produced by the present inventors previously and SiR-Mito11 of the present invention were compared and analyzed. I did.

H4 cells and HT22 cells were inoculated into a 384-well plate and incubated overnight, and then DMSO was added at a concentration of 5 μM as each SiR-Mito probe or control, followed by incubation for 48 hours.

Cell viability was analyzed using Cell Titer Glo (PROMEGA), and luminescent signal was measured using Flexstation 3. Cell viability and IC ₅₀ were specified 48 hours after treatment with each individual probe (5 μM) and normalized to DMSO control. Selectivity (selectivity window) was calculated by the IC ₅₀ value for the in HT22 IC ₅₀ values in the H4.

The IC ₅₀ values, cLogP and selectivity window values in HT22 cells and H4 cells of SiR-Mito1 to SiR-Mito10 and SiR-Mito11 are shown in Table 1 below.

As shown in Table 1 and Figure 6, in the present invention, it was confirmed that the increase in the hydrophobicity of the probe (SiR-Mito4: Bz, SiR-Mito5: Phenylethyl, SiR-Mito6: Cyclohexylmethyl) decreases H4 cell viability (33.0, respectively, 28.6, 4.8%). In particular, a probe having a cycloalkane substitution agent (SiR-Mito6: Cyclohexylmethyl, SiR-Mito8: Cyclooctyl, SiR-Mito9: Cyclohexylethyl) generally exhibited high cytotoxicity to both HT22 and H4 cells. In addition to the cycloalkane moiety, a long chain alkyl chain (SiR-Mito10: 2-Methylheptyl) having a branched aliphatic substituent also showed cytotoxic activity in both cell lines. In addition, probes having a long n-alkyl chain substituent (SiR-Mito2: Ethyl, SiR-Mito3: n-butyl) were more toxic to H4 than HT22, but did not show high toxicity to H4.

On the other hand, when treated with the SiR-Mito11 of the present invention to normal cells and brain tumor cells, HT22 cells showed a cell viability of 90% or more, whereas H4 cells were confirmed that most of the cells were killed by SiR-Mito11. In normal cells, the IC ₅₀ value of SiR-Mito11 was 9.1, and the IC ₅₀ value was 0.3 to 5.8 in brain tumor cells, which means that SiR-Mito11 has 1.5 to 30.3 times more selectivity in tumor cells than in normal cells. .

Confirmation of toxicity of SiR-Mito11 against various tumor cells

To confirm the anticancer effect of SiR-Mito11 on various tumor cell lines, various types of glioma cell lines such as malignant brain tumors such as neuroblastoma, astrocytoma, glioblastoma, and metastatic cancer were added. And examined.

HT22, H4, N2a, C6 and SK-N-MC cells were cultured in DMEM (Dulbecco's modified eagle media, High glucose) medium containing 10% FBS and 1% penicillin. A172 and U87-MG cells were cultured in RPMI 1640 medium containing L-glutamine, 10% FBS and 1% penicillin. All cells were cultured in 5% CO ₂ , 37°C.

SiR-Mito11 was added to the cells, added at a concentration of 5 μM, and cultured for 48 hours, and then cell viability and selectivity window values were measured in the same manner as in Example 4.

As a result, as shown in FIG. 7, it was confirmed that SiR-Mito11 exhibited strong toxicity with IC ₅₀ 0.3 ~ 5.8 μM for various tumor cells, and the selectivity value for normal neuronal cells was 1.5 ~ 30.3 times or more. .

Confirmation of absorption of SiR-Mito11 in tumor cell mitochondria

In the present invention, in order to confirm that the absorption of SiR-Mito11 is higher in tumor cells than in normal cells, SiR-Mito11 (5 μM) was added to HT22 cells and H4 cells and cultured for 1 hour, and then in the same manner as in Example 3. The fluorescence image was observed.

As a result of measuring the quantitative data of fluorescence images and fluorescence intensity of different cell types, as shown in Fig.8a, the fluorescence imaging of the cellular uptake of SiR-Mito11 showed that the accumulation of SiR-Mito11 was significantly higher in H4 cells than in HT22 cells. It was confirmed to increase.

In addition, for the quantitative evaluation at the single cell level, the average fluorescence intensity of SiR-Mito11 was measured around the cells including the range of HT22 cells 2 to 4 x 10 ⁴ and H4 cells 0.6 to 1.8 x 10 ⁴ , as shown in FIG. As shown, it was confirmed that the cellular uptake level of SiR-Mito11 was clearly elevated in H4 cells when compared to HT22 cells.

Comparison of difference in mitochondrial membrane potential according to SiR-Mito11 absorption

In the present invention, changes in mitochondrial membrane potential (MMP) according to SiR-Mito11 absorption were observed.

Membrane potential analysis was performed according to the manual using a JC-1 Mitochondrial membrane potential assay (Abcam), and HT22 cells and H4 cells were cultured in the same manner as in Example 4, and then , After treatment with JC-1 for 6 hours in the presence or absence of SiR-Mito11 (5 μM), fluorescence of JC-1 was observed with a fluorescence microscopy (ZOE, Bio-rad). Green (ex/em = 463 ~ 497 nm/496 ~ 540 nm) and red (ex/em = 536 ~ 586 nm/554 ~ 676 nm) were observed, and red fluorescence (high mitochondrial polarization) and green fluorescence (depolarization Fluorescence images that merged cells between the mitochondrial regions) were visualized with pseudochromatic.

As a result, as shown in FIG. 9, a shift in fluorescence emission from red to green was detected in both HT22 and H4 cells, which means that unhealthy cells increase by destruction of MMP in both cells. In particular, it was observed that H4 cells had a greater decrease in MMP than HT22 cells due to SiR-Mito11 treatment? It was confirmed that H4 cells were very sensitive to MMP change and apoptosis by SiR-Mito11.

Confirmation of the pathway for inducing tumor cell death by SiR-Mito11

8-1: Confirmation of the degree of cytochrome C release, caspase 3 and PARP activity by SiR-Mito11

In the present invention, in order to confirm the tumor cell death induction pathway by SiR-Mito11, the SiR-Mito11-induced apoptosis pathway in H4 and HT22 cells was observed.

First, HT22 cells and H4 cells were treated with SiR-Mito11 at different concentrations for 6 hours, and then the degree of cytochrome C release, caspase signaling markers caspase 3 and PARP were analyzed using Western blot.

After harvesting the cells, the cells were lysed using RIPA buffer containing protease & phosphatase inhibitor cocktail (THERMO) and Benzonase Nuclease (SIGMA). After centrifuging the lysate, a supernatant was obtained, and the protein was quantified using a protein assay kit (THERMO).

The cell lysate obtained by quantifying the protein was analyzed using SDS-PAGE using an acrylamide gel. After SDS-PAGE, the protein was transferred to a PVDF membrane, blocked with 5% BSA, and washed with TBST.

The membrane was treated with each of the primary antibodies (anti-caspase3, anti-PARP, anti-cytochrome c, anti-GAPDH, 1:1000 dilution) and reacted at 4° C. overnight, followed by washing with TBST. Thereafter, the HRP-conjugated secondary antibody was treated and reacted for 1 hour, washed with TBST, treated with ECL solution (THERMO), and then used for chemiluminescent images with ChemiDOC (Bio-rad). It was observed using.

In the case of cytochrome C, the degree of release was quantitatively analyzed using densitometry. As shown in FIG. 10, in the present invention, cytochrome C release was observed in H4 cells by treatment with SiR-Mito11, but not in HT22 cells.

In addition, as shown in FIG. 11, it was confirmed that apoptosis was clearly activated in H4 cells by SiR-Mito11 treatment through Western blot analysis of cleaved caspase 3 and PARP, whereas HT22 cells It was confirmed that it was not observed in.

8-2: Confirmation of caspase-dependent cell death by SiR-Mito11

In the present invention, it was attempted to confirm whether the tumor cytotoxicity of SiR-Mito11 was caused by caspase-dependent cell death.

First, H4 cells and HT22 cells were cultured in the same manner as in Example 4, and then z-VAD-fmk (R&D SYSTEMS), which is a pan-caspase inhibitor, was added at 40 μM and for 1 hour. After pretreatment, SiR-Mito11 or DMSO (control) was treated, respectively, and incubated for 24 hours. The cell survival rate was measured in the same manner as in Example 4.

As a result, as shown in FIG. 12, it was confirmed that apoptosis was inhibited in H4 cells treated with z-VAD-fmk even with SiR-Mito11 treatment, whereas no change was observed in HT22 cells.

That is, it was confirmed that SiR-Mito11 promotes cytochrome C release and induces caspase-dependent apoptosis in tumor cells.In the present invention, the uptake of SiR-Mito11 in glial cells is increased due to high energy demand and mitochondrial metabolism. , It was confirmed that this induces cell death.

Claims (10)

A near-infrared fluorescent probe represented by the following formula (1) and targeting mitochondria.
[Formula 1]

The near-infrared fluorescence probe of claim 1, wherein the near-infrared fluorescence probe exhibits fluorescence at an excitation wavelength of 651 to 657 nm and an emission wavelength of 662 to 668 nm.
(a) reacting 3-Bromo-N,N-dimethylaniline and formaldehyde to prepare compound 1 represented by the following formula A;
[Formula A]

(b) synthesizing Compound 2 represented by the following Formula B by lithiating Compound 1, adding dichlorodimethylsilane, and then performing an oxidation reaction with potassium permanganate. ;
[Formula B]

(c) tert - butyl-3-bromo-4-methylbenzoic acid (tert -butyl 3-bromo-4 -methylbenzoate) and tert - butyllithium to by the addition of (tert -Butyllithium) to the compound 2 represented by the chemical formula C Preparing compound 3; And
[Formula C]

(d) reacting the compound 3 with n-octyl amine to prepare a near-infrared fluorescent probe represented by Formula 1 below;
[Formula 1]

Near-infrared fluorescent probe manufacturing method comprising a.
The method of claim 3, wherein the lithiated in the step (b) the compound 1 Sec-method produced a near infrared fluorescent probe, characterized in that for performing the treatment of butyl lithium (Sec -Butyllithium).
The method of claim 3, wherein the step (c) of tert - butyl 3-bromo-4-methylbenzoic acid (tert -butyl 3-bromo-4 -methylbenzoate) is 3-bromo-4-methylbenzoic acid (3-Bromo -4-methylbenzoic acid) and di - tert - butyl dicarbonate (method of producing near-infrared fluorescent probes characterized in that produced by reacting Di- tert -butyl dicarbonate).
A pharmaceutical composition for diagnosis or treatment of a tumor comprising the near-infrared fluorescent probe of claim 1 or 2 as an active ingredient.
The method of claim 6, wherein the tumor is brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, gastric cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon Cancer, small intestine cancer, rectal cancer, fallopian tube carcinoma, anal muscle cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, minor Renal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem nerve A pharmaceutical composition for diagnosis or treatment of tumors, characterized in that at least one selected from the group consisting of gliomas and pituitary adenomas.
The pharmaceutical composition for diagnosis or treatment of a tumor according to claim 6, wherein the near-infrared fluorescent probe is specifically accumulated in the mitochondria of tumor cells.
The tumor according to claim 8, wherein when the near-infrared fluorescent probe accumulates in the mitochondria of tumor cells, destruction of mitochondrial membrane potential (MMP) and mitochondrial outer membrane permeabilization (MOMP) is induced. A pharmaceutical composition for diagnosis or treatment.
According to claim 8, When the near-infrared fluorescent probe accumulates in the mitochondria of tumor cells, cytochrome C release is promoted from the mitochondria, and caspase 3 and PARP cleavage is induced by cytochrome C release. A pharmaceutical composition for diagnosis or treatment of tumors, characterized in that the tumor cell death is induced.

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