KR20180011423A - Diagnostic or therapeutic composition of cancer which is activated by cathepsin B, and near-infrared imaging and phototherapy of tumor using the same - Google Patents

Abstract

The present invention relates to a diagnostic or therapeutic composition of cancer activated by cathepsin B, and near-infrared fluorescence imaging and phototherapy of cancer using the same. A compound comprising cyanine according to the present invention is activated by cathepsin B and shows fluorescence imaging of cancer cells or phototoxicity and strong phototherapeutic effect to cancer cells in cancer cells specifically and selectively, thereby being usefully used as a multifunctional composition capable of performing diagnosis and treatment of cancer at the same time.

Description

[0002] The present invention relates to a composition for diagnosing or treating cancer activated by cathepsin B, a near-infrared fluorescence imaging of cancer using the same, and a radiotherapy for cancer, same}

The present invention relates to a composition for diagnosing or treating cancers activated by cathepsin B, a near-infrared fluorescence imaging of cancer using the same, and a light therapy.

Fluorescent probes are able to provide visual information in real time with high spatial resolution, making it a powerful tool for biologists to study biological processes. In particular, probes with near-infrared (NIR) emission (650-900 nm) are preferred for in vivo imaging.

The near-infrared fluorescence probe has a unique advantage compared with a conventional probe having a short emission wavelength. First, when the near-infrared probe is applied to in-vivo imaging, the near-infrared light emission can avoid interference of the intrinsic fluorescence of the intrinsic biomolecule while exhibiting a high signal-to-noise ratio. Further, since the near-infrared fluorescent probe has a long excitation wavelength, imaging can be performed in deep tissue. Furthermore, near infrared ray excitation and luminescence cause less damage to biological samples than shorter wavelengths of light.

During the past two decades, some near-infrared fluorescent probes have been developed to observe a variety of species in vivo and in vitro. However, such a near-infrared fluorescent probe is a single functional reagent and can be used only for imaging. Therefore, the development of a multifunctional reagent capable of both specific imaging and cancer treatment is of great significance.

Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), is a mild and noninvasive cancer treatment method. With the help of photosensitizers or photothermogens, the treatment modality can cause localized high fever, where light is absorbed to move to active oxygen or induce the death of cancer cells.

The near-IR light-induced PDT or PTT is preferred in short wavelength light due to deep tissue penetration. To date, the most suitable near-infrared photosensitizer molecule is indocyanine green dye (ICG). The indocyanine green dye is a clinically approved near-infrared dye from the Food and Drug Administration (FDA). However, the indocyanine green dye itself is not specific to cancer cells because it is induced by near-infrared laser irradiation and phototherapy accompanied by damage of normal cells or tissues.

Therefore, a phototherapeutic agent which causes specific and efficient damage to target cancer cells more than normal cells is preferable.

Accordingly, the inventors of the present invention have developed a multifunctional composition capable of both imaging and treating cancer, and have found that a compound containing cyanine according to the present invention is activated by cathepsin B, and specifically and selectively, in cancer cells, The present invention provides a multifunctional composition capable of simultaneous diagnosis and treatment of cancer since it exhibits a phototoxic and powerful light therapy effect.

Korean Patent No. 10-1385855

It is an object of the present invention to provide a compound comprising cyanine.

Another object of the present invention is to provide a process for preparing a compound containing the cyanine.

It is still another object of the present invention to provide a composition for diagnosing cancer comprising a compound containing the cyanine.

Another object of the present invention is to provide a composition for treating cancer, which comprises a compound containing the cyanine as an active ingredient.

It is still another object of the present invention to provide a composition for treating near infrared rays of cancer.

It is another object of the present invention to provide a diagnostic method of cancer using the cancer diagnostic composition.

In order to achieve the above object,

The present invention provides a compound comprising cyanine represented by the following Formula 1:

[Chemical Formula 1]

(In the formula 1,

R ¹ are each independently linear or branched C _1-10 alkyl;

n is an integer from 1 to 5;

L is an alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamic acid , Q, glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine Wherein the terminal amino acid of L is 1 to 10, the terminal of L connected to the carbonyl is -NH-, and the terminal connected to the amine is - (C = O) - ;

는 각각 독립적으로 비치환 또는 치환된

,

또는

이고;

Each independently represent an unsubstituted or substituted < RTI ID = 0.0 >

,

or

ego;

는 각각 독립적으로 비치환 또는 치환된

,

또는

이고; 및

Each independently represent an unsubstituted or substituted < RTI ID = 0.0 >

,

or

ego; And

,

및

는 직쇄 또는 측쇄의 C_1-5알킬, 직쇄 또는 측쇄의 C_{1- 5}알콕시 및 할로겐으로 이루어지는 군으로부터 선택되는 1종 이상의 치환기로 하나 이상 치환될 수 있다).The substituted

,

And

May be substituted one or more by one or more substituents selected from the group consisting of linear or branched C _1-5 alkyl, straight or branched C _{1- 5} alkoxy and halogen).

The present invention also relates to a process for producing a compound represented by the formula (1)

Reacting a compound represented by formula (3) with a compound represented by formula (4) to prepare a compound represented by formula (2) (step 1); And

(Step 2) of preparing a compound represented by Formula (1) by a click reaction with a compound represented by Formula (5) prepared in Step 1 above with a compound represented by Formula (5) Lt; RTI ID = 0.0 > of: < / RTI &

[Reaction Scheme 1]

(In the above Reaction Scheme 1,

및

는 상기 화학식 1에서 정의한 바와 같다).R ¹ , n, L

And

Is as defined in the above formula (1)).

Further, the present invention provides a composition for diagnosing cancer comprising the compound represented by Formula 1 above.

The present invention also provides a composition for treating cancer comprising the compound represented by Formula 1 as an active ingredient.

Further, the present invention relates to a composition for treating near infrared rays of cancer comprising, as an active ingredient, a compound represented by the following general formula (8)

[Chemical Formula 8]

(In the above formula (7)

R ¹ are each independently linear or branched C _1-10 alkyl;

R ² is -NH ₂ or -NH ₃ ⁺ ;

는 각각 독립적으로 비치환 또는 치환된

,

또는

이고;

Each independently represent an unsubstituted or substituted < RTI ID = 0.0 >

,

or

ego;

는 각각 독립적으로 비치환 또는 치환된

,

또는

이고; 및

Each independently represent an unsubstituted or substituted < RTI ID = 0.0 >

,

or

ego; And

,

및

는 직쇄 또는 측쇄의 C_1-5알킬, 직쇄 또는 측쇄의 C_{1- 5}알콕시 및 할로겐으로 이루어지는 군으로부터 선택되는 1종 이상의 치환기로 하나 이상 치환될 수 있다).The substituted

,

And

May be substituted one or more by one or more substituents selected from the group consisting of linear or branched C _1-5 alkyl, straight or branched C _{1- 5} alkoxy and halogen).

In addition, the present invention provides a method for diagnosing cancer comprising treating the cancer diagnostic composition with a tissue or cells separated from an animal.

The compound containing cyanine according to the present invention is activated by cathepsin B to exhibit phototoxicity and strong phototherapeutic effect on cancer cells selectively and selectively in cancer cells by fluorescence imaging of cancer cells or cancer cells, And the like.

Brief Description of the Drawings Fig. 1 is a schematic diagram of near-infrared imaging and phototherapy of cancer cells induced by cathepsin B using the compound (CyA-P-CyB) prepared in Example 1 according to the present invention.
2A is a graph showing an absorption spectrum (red line) and Cy-N ₃ emission spectrum (black line) of Cy-S-Ph-NH ₂ at λ _ex = 650 nm, Acetate, 1.0 mM EDTA, pH 5.0, and 500 [mu] M GSH) and then incubated with a composition for diagnosis or treatment of cancer comprising the compound of formula (I) according to the present invention at λ _ex = 650 nm CyA-P-CyB with respect to time.
3A to 3 D are photographs showing fluorescence imaging of CyA-P-CyB, a composition for diagnosing or treating cancer, comprising a compound represented by Chemical Formula 1 according to the present invention in a cell, Cells were treated with 150 μM H ₂ O ₂ for 24 hours, 10 μM for camptothecin for 24 hours in FIG. 3C, 30 nM for 30 min for calpain inhibitor I in FIG. 3D, Each of the cells was incubated for 2 hours with 10 μM CyA-P-CyB, a composition for diagnosing or treating cancer comprising the compound represented by Formula 1 according to the present invention. The cells were each washed with DPBS and fluorescent images were obtained by confocal microscopy. The cells were co-stained using LysoTracker (top: CyA-P-CyB treatment (ex 635 nm / em 655-755 nm), central: LysoTracker staining (ex 405 nm / em 430 - 455 nm), bottom: merged into DIC, scale bar: 10 μm).
Fig. 4 is a graph showing the results of cell growth in the presence of CyA-P-CyB, a composition for diagnosing or treating cancer comprising a compound represented by Chemical Formula 1 according to the present invention, FIGS. 4A and 4B are graphs showing cytotoxic effects when laser irradiation is performed or not, FIGS. 4C and 4 are graphs showing cytotoxicity when treated with cathepsin B inhibitor or untreated FIG. 4E is a graph showing a comparison of cytotoxicity between normal cells and cancer cells. FIG. FIG. 4A shows the result of culturing with U87 cells and FIG. 4B with A549 cells with various concentrations of CyA-P-CyB for 16 hours and then removed CyA-P-CyB. The cells were irradiated with a laser (2 W / cm ² , 10 min) at 808 nm and cultured for 24 hours. FIG. 4C shows the result of the pretreatment with 30 nM of the calpain inhibitor I in the U87 cell, FIG. 4D, and A549 cells, and the CyA-P-CyB was removed after culturing with 5 μM CyA-P-CyB for 16 hours. The cells were irradiated with a laser (2 W / cm ² , 10 min) at 808 nm and cultured for 24 hours. Figure 4e shows that the cells were incubated with 5 [mu] M CyA-P-CyB for 16 hours and then removed CyA-P-CyB. The cells were irradiated with a laser (2 W / cm ² , 10 min) at 808 nm and cultured for 24 hours. The results of Figures 4a to 4e represent ± standard deviation values of three independent experiments.
FIG. 5 is a graph showing the near-infrared fluorescence imaging of the composition CyA-P-CyB for the diagnosis or treatment of cancer including the compound represented by the formula 1 according to the present invention and the phototherapeutic effect of CyA-P-CyB in vivo FIG. 5A is a near infrared ray image (tumor is represented by a dotted circle circle) of a tumor-bearing mouse until 48 hours after intravenous injection of PEG-PLA / CyA-P-CyB, FIG. 5B shows a mouse with H640 tumor (Red arrow indicates initiation of intravenous injection of PEG-PLA / CyA-P-CyB, and blue arrow indicates laser irradiation day), and a graph showing the degree of tumor growth over time after intravenous injection of the substance shown in the graph , And Figure 5C is a photograph of a mouse with H640 tumor after different treatments. 5b, mice were treated with 5 mg / kg PEG-PLA / CyA-P-CyB daily for 4 days (total 20 mg / kg) and the results were expressed as ± SEM values (* P <0.05, PEG -PLA treatment and laser irradiation).
Figure 6 shows the toxicity of CyA-P-CyB in vivo; 6A is a graph showing the effect of CyA-P-CyB on cell proliferation and apoptosis; FIG. 6A shows the effect of PEG-PLA or PEG-PLA / CyA-P- CyB (20 mg / FIG. 6B is a graph showing the cell proliferation and cell death of the tumor, and FIG. 6B is a graph showing the results of histological analysis of the organs (lung, liver, heart, kidney, pancreas, spleen and brain) 6c is a graph showing the quantification of the TUNEL fraction. In Figure 6b, cell proliferation is measured by tissue staining with Ki-67 antibody and is shown in brown. The nuclei were contrasted with hematoxylin. Cell death was measured using a TUNEL assay. Green indicates a positive signal. Nuclei were visualized through DAPI staining (blue). The positive signal in Figure 6c is that each slide is counted at 5 frames per slide. The results were expressed as ± SEM (n = 3-6) (** P <0.01, control group (PEG-PLA), laser treated control group (PEG-PLA) or PEG-PLA / CyA- Respectively)
Figure 7 shows the results of incubation with different incubation times in buffer solutions (25 mM acetate, 1.0 mM EDTA, pH 5.0, 500 [mu] M GSH) in the presence of cathepsin B (10 μg / mL) and calpain inhibitor I (100 μM) _and the absorption spectra of CaA-P-CyB at _ex = 650 nm.
Figure 8 is a graph showing the analysis of the photo-thermal effect of Cy-S-Ph-NH ₂ and then a singlet oxygen generation and 808 nm laser radiation, Figure 8a _{Cy-S-Ph-NH 2} (10 at λ _ex = 410 nm FIG. 8B is a graph showing fluorescence spectra of DPBF (10 μM) according to various laser irradiation time periods using an 808 nm laser (0.5 w / cm ² ) in the presence of a laser -Ph-NH ₂ (10 μM) is a graph showing the fluorescence spectra of the DPBF (10 μM) in the presence, Figure 8c is a _{Cy-S-Ph-NH 2} (■) and water (●) 808 nm laser (2 W in / cm &lt; ² &gt;).
FIG. 9 is a graph showing the characteristics of PEG-PLA / CYA-P-CYB nanoparticles through dynamic laser scattering (in FIG. 9, nanoparticle size = 230.9 nm, PDI = 0.461).

Hereinafter, the present invention will be described in detail.

It is to be understood, however, that the following description is provided to assist in understanding the present invention, and the present invention is not limited thereto.

The present invention provides a compound containing cyanine represented by the following formula (1).

[Chemical Formula 1]

(In the formula 1,

R ¹ are each independently linear or branched C _1-10 alkyl;

n is an integer from 1 to 5;

L is an alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamic acid , Q, glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine Wherein the terminal amino acid of L is 1 to 10, the terminal of L connected to the carbonyl is -NH-, and the terminal connected to the amine is - (C = O) - ;

는 각각 독립적으로 비치환 또는 치환된

,

또는

이고;

Each independently represent an unsubstituted or substituted &lt; RTI ID = 0.0 &gt;

,

or

ego;

는 각각 독립적으로 비치환 또는 치환된

,

또는

이고; 및

Each independently represent an unsubstituted or substituted &lt; RTI ID = 0.0 &gt;

,

or

ego; And

,

및

는 직쇄 또는 측쇄의 C_1-5알킬, 직쇄 또는 측쇄의 C_{1- 5}알콕시 및 할로겐으로 이루어지는 군으로부터 선택되는 1종 이상의 치환기로 하나 이상 치환될 수 있다).The substituted

,

And

May be substituted one or more by one or more substituents selected from the group consisting of linear or branched C _1-5 alkyl, straight or branched C _{1- 5} alkoxy and halogen).

Preferably.

R ¹ are each independently C _1-5 alkyl, straight or branched;

n is an integer from 2 to 4;

L is a linker in which at least one amino acid selected from the group consisting of glycine (Gly, G), leucine (Leu, L) and phenylalanine (Phe, F) is linked by a peptide bond, the number of amino acids of L is 1 to 10 , The end connected to the carbonyl of L is -NH- and the end connected to the amine is - (C = O) -;

는 각각 독립적으로 비치환 또는 치환된

또는

이고;

Each independently represent an unsubstituted or substituted &lt; RTI ID = 0.0 &gt;

or

ego;

는 각각 독립적으로 비치환 또는 치환된

또는

이고; 및

Each independently represent an unsubstituted or substituted &lt; RTI ID = 0.0 &gt;

or

ego; And

및

는 직쇄 또는 측쇄의 C_{1- 5}알킬, 직쇄 또는 측쇄의 C_{1- 5}알콕시 및 할로겐으로 이루어지는 군으로부터 선택되는 1종 이상의 치환기로 하나 이상 치환될 수 있다.The substituted

And

May be substituted one or more by one or more substituents selected from the group consisting of C _{1- 5} alkoxy and halogen of C _{1- 5} alkyl, linear or branched linear or branched.

More preferably,

R ¹ are each independently C _1-3 alkyl, straight or branched;

n is 3;

L is a linker in which at least one amino acid selected from the group consisting of glycine (Gly, G), leucine (Leu, L) and phenylalanine (Phe, F) is linked by a peptide bond, the number of amino acids of L is 4, L Is -NH- and the end connected to the amine is - (C = O) -;

는 각각 독립적으로 비치환 또는 치환된

이고;

Each independently represent an unsubstituted or substituted &lt; RTI ID = 0.0 &gt;

ego;

는 각각 독립적으로 비치환 또는 치환된

이고; 및

Each independently represent an unsubstituted or substituted &lt; RTI ID = 0.0 &gt;

ego; And

는 직쇄 또는 측쇄의 C_{1- 5}알킬, 직쇄 또는 측쇄의 C_1-5알콕시 및 할로겐으로 이루어지는 군으로부터 선택되는 1종 이상의 치환기로 하나 이상 치환될 수 있다.The substituted

It may be substituted one or more by one or more substituents selected from the group consisting of C _1-5 alkoxy and halogen, C _{1- 5} alkyl, linear or branched linear or branched.

일 수 있고, 이때, L의 아민 말단은 카보닐과 연결되고, 카보닐 말단은 아민과 연결되며,In the compound represented by the general formula (1), as the most preferable example of the substituent, R ¹ may be a propyl, n may be 3, L is

, Wherein the amine terminus of L is connected to a carbonyl, the carbonyl terminus is connected to an amine,

및

는 각각 독립적으로 비치환 또는 부톡시, 클로로 및 브로모로 이루어지는 군으로부터 선택되는 1종 이상의 치환기로 하나 이상 치환된

일 수 있다.

And

Each independently represent a group selected from the group consisting of unsubstituted or substituted with one or more substituents by one or more substituents selected from the group consisting of butoxy,

Lt; / RTI &gt;

Preferable examples of the compound represented by the formula (1) according to the present invention are those represented by the following compound A.

(A)

The design principle of the compound represented by the formula (1) according to the present invention is based on a peptide linker, which is a protease substrate represented by the following formula (b), with a fluorescent donor represented by the following formula (a) Based on the displayed non-fluorescent acceptor.

(A)

[Formula b]

(C)

(In the above formulas (a), (b) and (c)

및

는 상기 화학식 1에서 정의한 바와 같다).R ¹ , n, L

And

Is as defined in the above formula (1)).

The compound represented by the formula (a) and the compound represented by the formula (c) are not particularly limited as far as they include a chromophore of a cyanine series which can be excited with a wavelength in the infrared region, but the substituent range of the compound represented by the formula .

The present invention also relates to a process for producing a compound represented by the formula (1)

Reacting a compound represented by formula (3) with a compound represented by formula (4) to prepare a compound represented by formula (2) (step 1); And

(Step 2) of preparing a compound represented by Formula (1) by a click reaction with a compound represented by Formula (5) prepared in Step 1 above with a compound represented by Formula (5) Lt; RTI ID = 0.0 &gt; of: &lt; / RTI &

[Reaction Scheme 1]

(In the above Reaction Scheme 1,

및

는 상기 화학식 1에서 정의한 바와 같다).R ¹ , n, L

And

Is as defined in the above formula (1)).

Hereinafter, a method for producing the compound represented by the above formula (1) will be described in detail.

In the process for preparing the compound represented by the formula 1 according to the present invention, the step 1 is a step of reacting the compound represented by the formula 3 with the compound represented by the formula 4 to prepare the compound represented by the formula 2, , A compound represented by the formula (2) can be prepared by reacting a compound represented by the formula (3) in the presence of triposane and a base to form an acid chloride compound and then reacting with an amine represented by the formula (4).

The base includes, but is not limited to, N, N-dimethylaminopyridine (DMAP), pyridine, triethylamine, N, N-diisopropylethylamine (DIPEA), 1,8-diazabicyclo [5.4.0] -7-undecene (DBU), or an inorganic base such as sodium bicarbonate, sodium hydroxide, potassium hydroxide, etc., which may be used alone or in combination, N, N-diisopropylethylamine and N, N-dimethylaminopyridine (DMAP) are preferably used.

Further, as the solvent usable in the above reaction, ether solvents such as tetrahydrofuran, dioxane, dichloromethane and 1,2-dimethoxyethane, aromatic hydrocarbon solvents such as benzene, toluene and xylene, lower alcohols, dimethyl Dimethylformamide (DMF), dimethylsulfoxide (DMSO), acetonitrile, water, chloroform and the like, which can be used singly or in combination.

In the method for preparing the compound represented by the formula 1 according to the present invention, the compound represented by the formula 2 prepared in the step 1 is reacted with the compound represented by the formula 5 by a click reaction to obtain a compound represented by the formula 1 Specifically, the alkane of the compound represented by the formula (2) and the azide of the compound represented by the formula (5) form a triazole through a click reaction in the presence of a metal ion catalyst to produce a compound represented by the formula Is a step for preparing a compound to be displayed.

At this time, the metal ion catalyst is not particularly limited as long as it is a catalyst capable of inducing a click reaction, but preferably a catalyst containing copper ion can be used, and CuCl ₂ can be used as a catalyst containing salicidal copper ion .

Further, as the solvent usable in the above reaction, ether solvents such as tetrahydrofuran, dioxane, dichloromethane and 1,2-dimethoxyethane, aromatic hydrocarbon solvents such as benzene, toluene and xylene, lower alcohols, dimethyl Dimethylformamide (DMF), dimethylsulfoxide (DMSO), acetonitrile, water, chloroform and the like, which can be used singly or in combination.

Each step of the process for preparing the compound represented by the formula (1) according to the present invention may be modified or changed in accordance with the purpose of each stage of the person skilled in the art, and the reaction conditions such as reaction solvent, temperature, Is not particularly limited to the above-mentioned contents. Preferably, the process for preparing the compound represented by the formula (1) according to the present invention can be carried out according to the following embodiments of the present invention.

Further, the present invention provides a composition for diagnosing cancer comprising the compound represented by Formula 1 above.

The present invention also provides a composition for treating cancer comprising the compound represented by Formula 1 as an active ingredient.

Hereinafter, a cancer diagnostic composition or a therapeutic composition comprising the compound represented by Formula 1 according to the present invention will be described in detail.

The composition for diagnosing cancer or the composition for treating cancer comprising the compound represented by the formula (1) according to the present invention comprises a peptide linker which is a protease substrate represented by the following formula (b), a fluorescent donor represented by the following formula donor and a non-fluorescent acceptor represented by the following formula (c).

(A)

[Formula b]

(C)

(In the above formulas (a), (b) and (c)

및

는 상기 화학식 1에서 정의한 바와 같다).R ¹ , n, L

And

Is as defined in the above formula (1)).

In this case, the compound represented by the above formula (b) is conjugated between the formula (a) and the formula (c), wherein the peptide linker which is a protease substrate acts as a fluorescent donor and the compound represented by the formula The compound acts as a quencher as a non-fluorescent acceptor.

The composition for diagnosing cancer comprising the compound represented by Formula 1 and the composition for treating cancer according to the present invention,

Is activated through a mechanism including the step of producing a compound represented by formula (6) and a compound represented by formula (7) by decomposing the compound represented by formula (1) with capecin B as shown in the following reaction scheme 2.

[Reaction Scheme 2]

(In the above Reaction Scheme 2,

및

는 상기 화학식 1에서 정의한 바와 같고; 및R ¹ , n, L

And

Is as defined in Formula 1; And

In the compound represented by the general formula (6), the terminal connected to the carbonyl of L is -NH- and the other terminal is -COOH.

In order for the compound represented by Formula 1 according to the present invention to function as a cancer diagnostic or cancer treatment composition, activation should be performed, and cathepsin B must be essential for the activation. Cathepsin B causes the reaction of Scheme 2 to produce the compound of Formula 6 and the compound of Formula 7, and each compound plays a different role.

Specifically, the compound represented by the formula (1) has an effective FRET with the compound represented by the formula (c) and does not show fluorescence. Thus, the peptide linker connecting the formula (a) and the formula (b) is cleaved by cathepsin B to separate the compound represented by the formula (a) as a fluorescent donor and the compound represented by the formula (c) do.

Since the composition for diagnosing or treating cancer comprising the compound represented by Formula 1 is activated by cathepsin B in cells, it can be useful for visualizing the position and activity of cathepsin B.

On the other hand, since cathepsin B is a substance that exists specifically in cancer cells, the cancer diagnosis composition and cancer treatment composition according to the present invention can specifically and selectively diagnose or treat cancer cells only.

The compound represented by the following formula (6) produced by the mechanism shown in the above Reaction formula 2 shows fluorescence upon irradiation with near infrared rays.

[Chemical Formula 6]

(6)

R ¹ are each independently linear or branched C _{1- 10} alkyl;

n is an integer from 1 to 5;

L is an alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamic acid , Q, glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine Wherein the amino acid number of L is 1 to 10, the terminal of L connected to the carbonyl is -NH-, and the other terminal is -COOH;

는 각각 독립적으로 비치환 또는 치환된

,

또는

이고;

Each independently represent an unsubstituted or substituted &lt; RTI ID = 0.0 &gt;

,

or

ego;

는 각각 독립적으로 비치환 또는 치환된

,

또는

이고; 및

Each independently represent an unsubstituted or substituted &lt; RTI ID = 0.0 &gt;

,

or

ego; And

,

및

는 직쇄 또는 측쇄의 C_1-5알킬, 직쇄 또는 측쇄의 C_{1- 5}알콕시 및 할로겐으로 이루어지는 군으로부터 선택되는 1종 이상의 치환기로 하나 이상 치환될 수 있다).The substituted

,

And

May be substituted one or more by one or more substituents selected from the group consisting of linear or branched C _1-5 alkyl, straight or branched C _{1- 5} alkoxy and halogen).

The compound represented by the formula (6) exhibits strong fluorescence in a specific near infrared ray wavelength band.

Further, the present invention provides a composition for treating near infrared rays of cancer, which comprises a compound represented by the following general formula (8) as an active ingredient.

[Chemical Formula 8]

(In the formula (8)

R ¹ are each independently linear or branched C _1-10 alkyl;

R ² is -NH ₂ or -NH ₃ ⁺ ;

는 각각 독립적으로 비치환 또는 치환된

,

또는

이고;

Each independently represent an unsubstituted or substituted &lt; RTI ID = 0.0 &gt;

,

or

ego;

는 각각 독립적으로 비치환 또는 치환된

,

또는

이고; 및

Each independently represent an unsubstituted or substituted &lt; RTI ID = 0.0 &gt;

,

or

ego; And

,

및

는 직쇄 또는 측쇄의 C_1-5알킬, 직쇄 또는 측쇄의 C_{1- 5}알콕시 및 할로겐으로 이루어지는 군으로부터 선택되는 1종 이상의 치환기로 하나 이상 치환될 수 있다).The substituted

,

And

May be substituted one or more by one or more substituents selected from the group consisting of linear or branched C _1-5 alkyl, straight or branched C _{1- 5} alkoxy and halogen).

The compound represented by the formula (8) is a compound produced from the compound represented by the formula (7) generated by decomposing the compound represented by the formula (1) according to the present invention into cathepsin B by the mechanism as shown in the reaction formula (2) Can be used as photodynamic therapy (PDT) and photothermal therapy (PTT) because of its strong phototoxicity.

Accordingly, the composition for diagnosing or treating cancer comprising the compound represented by the formula (1) according to the present invention is a compound represented by the formula (6) which is activated by cathepsin B and exhibits strong fluorescence under irradiation with near infrared rays, , And cathepsin B exhibits specifically higher activity in cancer cells than normal cells. Therefore, the composition for diagnosing or treating cancer, comprising the compound represented by Formula 1 according to the present invention Can be usefully used as a composition for diagnosing or treating cancer.

In addition, the present invention provides a method for diagnosing cancer comprising treating the cancer diagnostic composition with a tissue or cells separated from an animal.

As described above, the cancer diagnostic composition according to the present invention is activated by cathepsin B and exhibits strong fluorescence under irradiation with near infrared rays, and cathepsin B exhibits specifically high activity in cancer cells than normal cells, Therefore, the diagnostic method of cancer of the present invention using the cancer diagnostic composition can also be usefully used for cancer diagnosis.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples, but the present invention is not limited thereto.

< Example  1> A composition for diagnosing or treating cancer according to the present invention CyA -P- CyB )

Step 1: Cy -S- Ph -NH ₂ Manufacturing

(E) -2 - ((E) -2-chloro-3 - ((E) -2- (3,3-dimethyl- 1 -propylindol- 2- ylidene) ethylidene) cyclohex- (IR-780, 320 mg, 0.48 mmol) was added to a solution of 4-aminothiophenol (120 mg, 0.96 mmol) mmol) and DMF (dimethylformamide, 10 mL), and the mixture was stirred at room temperature for 24 hours. Concentration under reduced pressure of DMF in vacuo, the crude (crude) mixture was purified by silica gel column chromatography _{(CH 2 Cl 2 / MeOH 100} : 2) separated through the _{Cy-S-Ph-NH 2} (290 mg, yield 80%) .

^{1 H NMR (300 MHz, CDCl} 3) δ 8.80 (d, J = 15 Hz, 2H), 7.40-7.28 (m, 4H), 7.24 (t, J = 7.5 Hz, 2H), 7.14 (d, J = J = 7.2 Hz, 2H), 7.01 (d, J = 6.6 Hz, 2H), 6.64 (d, J = , 4H), 2.72 (t, J = 6.0 Hz, 4H), 2.04 (m, 2H), 1.92 (m, 4H), 1.57 (s, 12H), 1.07 (t, J = 7.5 Hz, 6H);

¹³ C NMR (75.5 MHz, CDCl ₃ ) δ 172.28, 154.40, 146.52, 145.35, 142.38, 141.13, 134.11, 128.64, 128.19, 125.02, 124.64, 122.20, 116.30, 110.70, 101.40, 49.20, 46.23, 28.06, 26.71, , 11.71;

HRMS (ESI): m / z (M) ⁺ calcd for C42H50N3S, 628.3720; found, 628.3807.

Step 2: Cy -N ₃ Manufacturing

(E) -2 - ((E) -2-chloro-3 - ((E) -2- (3,3-dimethyl- 1 -propylindol- 2- ylidene) ethylidene) cyclohex- (IR-780, 150 mg, 0.22 mmol) was added to a solution of 3-azidopropane-1-amine 42 mg, 0.42 mmol) and DMF (10 mL), and the mixture was stirred at 90 ^° C for 3 hours. The DMF was concentrated under reduced pressure in vacuo and the crude mixture was separated through silica gel column chromatography (CH ₂ Cl ₂ / MeOH 100: 2) to give Cy-N ₃ (90 mg, yield 56%).

^{1 H NMR (300 MHz, CDCl} 3) δ 8.226 (br, 1H), 7.77 (d, J = 12.9 Hz, 2H), 7.30-7.25 (m, 4H), 7.09 (t, J = 7.2 Hz, 2H) 2H), 6.88 (d, J = 7.2 Hz, 2H), 5.65 (d, J = 12.9 Hz, 2H), 4.03-3.97 (m, 2H), 3.97-3.80 J = 7.5 Hz, 2H), 3.51 (t, J = 6.3 Hz, 2H), 2.33-2.25 (m, 2H), 1.86-1.82 , 6H);

¹³ C NMR (75.5 MHz, CDCl ₃ ) δ 169.15, 167.47, 140.18, 138.20, 128.00, 122.65, 122.10, 119.99, 108.37, 94.18, 53.47, 49.46, 47.84, 30.60, 29.16, 25.30, 21.56, 20.07, 11.76;

HRMS (ESI): m / z (M) ⁺ calcd for C39H51N6, 603.4170; found, 603.4317.

Step 3: Alkane - Peptide - PABC - CyB ( Alkyne -Peptide- PABC - CyB )

Anhydrous DIPEA (diisopropylethylamine, 85%) was added to Cy-S-Ph-NH ₂ (100 mg, 0.13 mmol) obtained in the above step 1 and anhydrous CHCl ₃ (10 mL) mg, 0.65 mmol) was added dropwise. The solution was stirred at room temperature for 8 hours. Unreacted phosgene gas was removed by argon purge. Alkyne-peptide-PABC (74 mg, 0.13 mmol, purchased from DgPeptides Co., Ltd., Hangzhou, China) and DMAP the CHCl ₃ solution of dimethylaminopyridine, 32 mg, 0.26 mmol) was added. The reaction mixture was stirred overnight. After evaporation of the solvent, the residue was separated through silica gel column chromatography (CH ₂ Cl ₂ / MeOH 100: 7) to give an alkane-peptide-PABC-CyB (85 mg, yield 53%).

^{1 H NMR (300 MHz, CDCl} 3) δ 8.69 (d, J = 14.1 Hz, 2H), 8.55 (s, 1H), 8.47-8.43 (t, J = 5.1 Hz, 1H), 7.61 (m, 2H) , 7.50 (d, J = 9 Hz, 2H), 7.33-6.99 (m, 2H), 6.05 (d, J = 14.1 Hz, 2H), 4.91 , 3.96-3.89 (m, 6H), 3.78-3.41 (m, 2H), 3.18-2.95 (m, 2H), 2.62 (t, J = 6 Hz, 4H) ), 2.28-2.08 (m, 2H), 1.97-1.93 (m, 2H), 1.84-1.77 (m, 5H), 1.68-1.60 , 1H), 0.98 (t, J = 7.2 Hz, 6H), 0.83 (d, J = 6.9 Hz, 6H);

¹³ C NMR (75.5 MHz, CDCl ₃ ) δ 173.68, 173.38, 172.71, 172.24, 168.22, 153.55, 146.66, 142.08, 141.06, 138.22, 135.70, 133.41, 129.59, 129.15, 128.87, 128.72, 127.20, 126.97, 125.46, , 120.45, 110.62, 100.97, 82.99, 69.09, 56.05, 52.98, 49.35, 45.99, 43.67, 39.46, 36.05, 34.49, 28.01, 26.39, 24.49, 23.28, 20.95, 20.84, 14.21, 11.68;

HRMS (ESI): m / z (M) ⁺ calcd for C74H87N8O7S ⁺ , 1231.6413, found, 1231.6424; ^{m / z (M) 2+ calcd} for C74H88N8O7S 2 +, 616.3243, found, 616.3276.

Step 4: CyA -P- CyB  Produce

To a solution of MeOH-H ₂ O (5 mL, 1: 4, v / v) containing sodium ascorbate (4 mg, 0.02 mmol) and CuCl ₂ (1 mg, 0.007 mmol) (40 mg, 0.03 mmol) and Cy 2-N ₃ (24 mg, 0.03 mmol) prepared in Step 2 in CH ₂ Cl ₂ (10 mL) were added thereto. After the reaction solution was stirred for 36 hours, the water layer was divided into two portions. The organic layer was dried over Na ₂ SO ₄ and the solvent was evaporated in vacuo. The residue was separated through silica gel column chromatography (CH ₂ Cl ₂ / MeOH 100: 5) to give CyA-P-CyB (25 mg, yield 45%).

^{1 H NMR (300 MHz, CDCl} 3) δ 8.73-8.69 (m, 3H), 8.2 (t, J = 4.8 Hz, 1H), 7.84-7.65 (m, 8H), 7.56 (d, J = 7.8 Hz, 2H), 7.46 (d, J = 9 Hz, 2H), 7.36-7.07 (m, 21H), 7.02 (t, J = 7.2 Hz, 2H) (d, J = 15 Hz, 2H), 5.63 (d, J = 12.9 Hz, 2H), 4.97 (s, 2H), 4.51-4.29 (m, 4H), 4.03-3.95 2H), 2.91 (t, J = 5.1 Hz, 2H), 2.69 (t, J = 6.6 Hz, 4H), 2.53 (m, (M, 2H), 1.90-1.75 (m, 14H), 1.63 (s, 12H), 1.48 -0.98 (m, 12H), 0.80-0.75 (m, 6H);

¹³ C NMR (75.5 MHz, CDCl ₃ ) 隆 175.09, 173.37, 172.92, 172.58, 171.95, 168.95, 168.29, 167.75, 162.55, 153.61, 146.59, 146.51, 143.09, 142.15, 141.08, 140.14, 137.95, 137.13, 133.60, 129.41 , 128.90, 128.69, 128.61, 128.05, 126.96, 125.33, 122.85, 122.34, 122.13, 120.49, 120.19,110.71,110.35,108.57,107.74,101.16,94.56,65.30, 53.46, 49.30, 47.85, 47.56, 46.08, 44.86, 36.51 , 31.92, 31.44, 29.70, 29.65, 29.36, 29.15, 29.10, 28.01, 26.49, 25.48, 24.58, 23.24, 23.26, 21.44, 21.01, 20.86, 20.11, 14.13, 11.76, 11.69, 11.11;

MS (ESI): m / z (M) 2+ calcd for C113H138N14O7S 2 +, 918.0; found, 918.0.

< Experimental Example  1> Car taping  Activation of compositions for diagnosis or treatment of cancer by B

As shown in the following Reaction Scheme 3, the following experiment was conducted to confirm that the composition for diagnosis or treatment containing the compound represented by Formula 1 according to the present invention is activated by cathepsin B, 2A and 2B.

[Reaction Scheme 3]

Specifically, cathepsin B was first activated for 60 minutes at room temperature in DL-diterothreitol (DTT, 60 mM) / EDTA (30 mM, 5 μL) Therapeutic composition (CyA-P-CyB) was added to the solution of DMSO in DMSO to an initial concentration of 1 mM and incubated at various time intervals in reaction buffer (25 mM acetate, 1.0 mM EDTA, pH 5.0, and 500 μM GSH) Lt; / RTI &gt; The final concentrations of CyA-P-CyB and cathepsin B were 10 μM and 10 μg / mL, respectively. The enzymatic reaction was observed while measuring with a fluorescence spectrometer at an excitation wavelength of 650 nm.

Inhibition experiments were carried out by adding 100 [mu] M calpain inhibitor I under the same conditions as above, and the results are shown in Fig.

2A is a graph showing an absorption spectrum (red line) and Cy-N ₃ emission spectrum (black line) of Cy-S-Ph-NH ₂ at λ _ex = 650 nm, Acetate, 1.0 mM EDTA, pH 5.0, and 500 [mu] M GSH) and then incubated with a composition for diagnosis or treatment of cancer comprising the compound of formula (I) according to the present invention at λ _ex = 650 nm CyA-P-CyB with respect to time.

Figure 7 shows the results of incubation with different incubation times in buffer solutions (25 mM acetate, 1.0 mM EDTA, pH 5.0, 500 [mu] M GSH) in the presence of cathepsin B (10 μg / mL) and calpain inhibitor I (100 μM) _and the absorption spectra of CaA-P-CyB at _ex = 650 nm.

As shown in FIG. 2A, it was found that spectral overlapping occurred between the absorption of Cy-S-Ph-NH ₂ prepared in step 1 of Example 1 and the emission of Cy-N ₃ produced in step 2 number and, which, indicates that the Cy-S-Ph-NH ₂ the CyB moiety effective receptor for CyA donors included in, CyB is fluorescent light emission is not limited, denotes a photon carrying a small role in the FRET system.

As shown in FIG. 2B, the intensity of fluorescence increases at a wavelength of 720 nm as time elapses. This indicates that CyA emission is occurring. CyA-P- Indicates that CyB is activated.

Further, as shown in Fig. 7, it can be seen that when cathepsin B is inhibited, no increase in fluorescence is observed, indicating that cathepsin B activates CyA-P-CyB.

< Experimental Example  2> 808 nm  By laser irradiation Cy -S- Ph - NH ₂ from Sunshine  Oxygen production

DPBF (10 μM) fluorescence labeled with fluorescence at a laser irradiation time of 0 (t = 0) in an aqueous solution containing Cy-S-Ph-NH ₂ (10 μM) before irradiation with an 808 nm laser under 410 nm excitation Were measured. The results are shown in Fig.

While irradiating the solution up to 5 minutes, and near-infrared light ^{(808nm, 0.5 W / cm 2} ), it was measured for 10 s, 30 s, 1 min , 2 min, and the emission spectra (430 nm-600 nm) at 5 min .

The control group was experimented under the same conditions except that no laser irradiation was applied.

Figure 8 is a graph showing the analysis of the photo-thermal effect of Cy-S-Ph-NH ₂ and then a singlet oxygen generation and 808 nm laser radiation, Figure 8a _{Cy-S-Ph-NH 2} (10 at λ _ex = 410 nm FIG. 8B is a graph showing fluorescence spectra of DPBF (10 μM) according to various laser irradiation time periods using an 808 nm laser (0.5 w / cm ² ) in the presence of a laser a graph showing the fluorescence spectra of the DPBF (10 μM) under -Ph-NH ₂ (10 μM) there.

As shown in FIG. 8A, after the solution containing Cy-S-Ph-NH ₂ and DPBF was irradiated with the 808 nm laser, it was found that the fluorescence of DPBF gradually decreased at 480 nm as the laser irradiation time increased there was. In contrast, as shown in FIG. 8B, no change was observed when the laser was not irradiated. This is because the generation of singlet oxygen in the Cy-S-Ph-NH ₂ indicates that solvent is caused by irradiation of the near infrared laser.

< Experimental Example  3> 808 nm  After laser irradiation, Cy -S- Ph -NH ₂ of Light heat  Effect measurement

To measure the photothermal effect of Cy-S-Ph-NH ₂ in aqueous solution, Cy-S-Ph-NH ₂ (10 M) was irradiated with 808 nm laser (2 W / cm ² ). The negative control irradiated the same amount of water with the same laser. The solution temperature was measured with an infrared imaging device (FLIR E5) at 0 s, 30 s, 1 min, 1.5 min, 2.0 min, 2.5 min, 3.0 min, 3.5 min, 4.0 min, 4.5 min and 5 min. The results are shown in FIG. 8C.

8C is a graph showing a temperature change with time when an 808 nm laser (2 W / cm ² ) is irradiated onto Cy-S-Ph-NH ₂ () and water ().

Further, as shown in Fig. 8C,

After 5 minutes of laser irradiation, no noticeable temperature change was detected in the control water, whereas the temperature of the Cy-S-Ph-NH ₂ solution increased by 6 ° C. This indicates that when near infrared rays are irradiated, Cy-S-Ph-NH ₂ absorbs light and can transfer it to heat, indicating that light energy can be converted into heat energy.

< Experimental Example  4> PEG- By PLA CyA -P- CyB  Encapsulation CyA -P- CyB Encapsulation Gt; PEG-PLA &lt; / RTI &gt; nanoparticles)

In order to increase the solubility in water and delay the clearance in blood of a composition for diagnosing or treating cancer comprising the compound represented by Chemical Formula 1 according to the present invention, a micelle structure is formed with PEG-PLA, In order to prepare nanoparticles encapsulating the compound represented by Formula 1 according to the present invention, the following experiment was conducted.

CyA-P-CyB (5 mg ) and _{PEG 5000 -PLA 3000 (45 mg,} Daigang Biomaterial Co., Ltd, Jinan, purchased from China) (weight ratio of the CyA-P-CyB and PEG ₅₀₀₀ -PLA ₃₀₀₀ 1: 9) was dissolved completely in CH ₂ Cl ₂ (0.5 mL) with stirring and dried for 2 hours by rotary evaporation to obtain a thin film. Thereafter, deionized water (10 mL) was added dropwise to form a micellar solution. The size of the PEG-PLA nanoparticles encapsulating CyA-P-CyB was measured using a dynamic laser scattering spectrometer and the results are shown in FIG.

FIG. 9 is a graph showing characteristics of PEG-PLA / CYA-P-CYB nanoparticles through dynamic laser scattering.

As shown in FIG. 9, the size of the PEG-PLA nanoparticles encapsulating CyA-P-CyB according to the present invention was 230.9 nm and the polydispersion index (PDI) was 0.461.

< Experimental Example 5 > In vivo CyA -P- CyB Near infrared  Fluorescence analysis

In order to analyze the near infrared fluorescence of the composition for diagnosing cancer comprising the compound represented by the formula (1) according to the present invention, the following experiment was conducted and the results are shown in FIG.

All animal procedures were approved by the Institutional Animal Care Committee at Yonsei University. H640 lung cancer cells (1.0 x 10 ⁷ cells per mouse) were injected subcutaneously into male BALB / c nude mice. When the volume of the cancer reached approximately 100 mm ³ , the PEG-PLA / CyA-P-CyB capsules (2 mg / kg) prepared in Experimental Example 4 were intravenously injected into the mice.

Near infrared images were captured using an animal optical imaging system (IVIS, Caliper Life Sciences) at 0, 1, 2, 4, 8, 11, 24, 32 and 48 hours after PEG-PLA / CyA- Respectively.

FIG. 5A shows near-infrared imaging (tumor is represented by a dotted circle) of a gingival mouse until 48 hours after intravenous injection of PEG-PLA / CyA-P-CyB.

As shown in FIG. 5A, after 11 hours of intravenous injection of PEG-PLA / CyA-P-CyB, it was found that the fluorescence signal reaches the point where the tumor is located. Indicating that CyA-P-CyB can accumulate in the tumor. After 24 hours of intravenous injection of PEG-PLA / CyA-P-CyB, the fluorescence signal gradually decreased. This indicates that the optimum time for laser irradiation is 24 hours after CyA-P-CyB injection. In contrast, when treated with PEG-PLA alone, significant signifi- cant signals did not appear.

< Experimental Example  6> In vivo CyA -P- CyB  Measurement of light treatment effect

In order to measure the phototherapeutic effect of the composition for treating cancer comprising the compound represented by the formula (I) according to the present invention as an active ingredient, the following experiment was conducted and the results are shown in FIGS. 5B and 5C.

Specifically, H640 lung cancer cells (1.0 x 10 ⁷ cells per mouse) were injected subcutaneously into male BALB / c nude mice. When the volume of cancer reached 100-200 mm ³ in almost hayeoteul, every four days the experiment Example 4 PEG-PLA / CyA-P- CyB capsule (5 mg / kg, n = 3-6) prepared in the experimental group mice Intravenous injection. Control mice received the same amount of PEG-PLA (n = 3). After 24 hours of PEG-PLA / CyA-P-CyB injection, the mice were irradiated with 808 nm laser (1 W / cm ² ) for 5 minutes. Tumor diameters were measured using a Vernier caliper. The volume (V) of the tumor was calculated by the following equation (1).

[Equation 1]

x (W x H x D)/6V =

x (W x H x D) / 6

In Equation (1), W is the diameter of the smallest tumor, H is the diameter of the largest tumor, and D is the thickness of the tumor.

The relative tumor volume was calculated as V / V ₀ (V ₀ being the initial volume of the tumor before treatment).

The data are expressed as ± SEM values. Various groups were compared using ANOVA. Statistical significance was set at P <0.05.

FIG. 5B is a graph showing the degree of tumor growth over time after intravenous injection of the substance shown in the graph into a mouse having H640 tumor (the red arrow indicates the start of intravenous injection of PEG-PLA / CyA-P-CyB, Shows the day of laser irradiation), and Figure 5C is a photograph of a mouse with H640 tumor after different treatments.

As shown in FIG. 5B, after 24 hours of injection, the mouse was irradiated with a near-infrared laser. PEG-PLA / CyA-P-CyB injected and laser irradiated mice were treated with PEG-PLA alone, PEG-PLA injection and laser irradiation, It was confirmed that the mice were significantly smaller than the mice not irradiated.

As shown in FIG. 5C, the tumor volume of the mice subjected to the PEG-PLA / CyA-P-CyB injection and laser irradiation was found to be 1/3 of the tumor volume of the mice of the other three groups.

These results demonstrate that CyA-P-CyB exhibits significant phototherapeutic efficacy in tumor growth inhibition.

< Experimental Example  7> In vivo CyA -P- CyB  Toxicity assessment

In order to evaluate the in vivo toxicity of the composition for treating cancer comprising the compound represented by the formula (1) according to the present invention as an active ingredient, the following experiment was conducted and the results are shown in FIGS. 6A to 6C.

Specifically, the same amount of PEG-PLA (n = 3) or PEG-PLA / CyA-P-CyB (20 mg / kg, n = 3-6) was intravenously injected to male BALB / c nude mice. Male BALB / c nude mice (n = 3-6) were injected via the tail vein daily for 4 days with PEG-PLA / CyA-P-CyB (50 mg / kg, 200 microliters per mouse). BALB / c nude mice (n = 3) for use as a control injected the same amount of PEG-PLA. After 14 days, the mice were euthanized and the lung, liver, heart, kidney, pancreatic spleen and brain were harvested and fixed in formalin, and cell proliferation and apoptosis were assessed by H & E staining and immunohistochemistry, Respectively.

For cell proliferation analysis, fractions from paraffin-fixed tissues were paraffin-removed, rehydrated, and sections were re-deparaffinized and antigen-retrieved from the tissues. After endogenous peroxidase and protein were blocked, the tissues were incubated with anti-Ki-67 antibody followed by secondary rabbit IgG (Dako, Denmark) and detected with DAKO Envision + System-HRP DAB (Dako, Denmark). The fractions were counterstained with hematoxylin (Sigma-Aldrich, USA). Cell death was performed using a TUNEL assay kit (Life Technologies, USA). Cell nuclei were visualized by DAPI staining.

Figure 6 shows the toxicity of CyA-P-CyB in vivo; 6A is a graph showing the effect of CyA-P-CyB on cell proliferation and apoptosis; FIG. 6A shows the effect of PEG-PLA or PEG-PLA / CyA-P- CyB (20 mg / FIG. 6B is a graph showing the cell proliferation and cell death of the tumor, and FIG. 6B is a graph showing the results of histological analysis of the organs (lung, liver, heart, kidney, pancreas, spleen and brain) 6c is a graph showing the quantification of the TUNEL fraction. In Figure 6b, cell proliferation is measured by tissue staining with Ki-67 antibody and is shown in brown. The nuclei were contrasted with hematoxylin. Cell death was measured using a TUNEL assay. Green indicates a positive signal. Nuclei were visualized through DAPI staining (blue). The positive signal in Figure 6c is that each slide is counted at 5 frames per slide. The results were expressed as ± SEM (n = 3-6) (** P <0.01, control group (PEG-PLA), laser treated control group (PEG-PLA) or PEG-PLA / CyA- Respectively)

As shown in FIG. 6A, the toxicity of CyA-P-CyB was evaluated in PEG-PLA / CyA-P-CyB or PEG-PLA tail vein injection, 20 mg / kg) mice did not die and survived for 16 days. (Lung, liver, heart, kidney, pancreas, spleen and brain) from mice 14 days old after injection of PEG-PLA or PEG-PLA / CyA-P- CyB , And toxicity was assessed using H & E staining. There were no apparent lesions (necrosis, edema, inflammatory infiltrates, or hyperplasia) in the 7 organs. These pathological results indicate that CyA-P-CyB does not have cytotoxic effects in mice.

As shown in FIG. 6B, PEG-PLA was injected to perform laser irradiation or non-performed mouse and PEG-PLA / CyA-P-CyB were injected, and laser irradiation or untreated tumor lumps were used for cell proliferation and cell proliferation Cell proliferation was measured by anti-Ki-67 staining. Cell proliferation was significantly reduced in mice treated with PEG-PLA / CyA-P-CyB injection and laser treatment Respectively.

As shown in FIG. 6C, apoptosis analysis performed using TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining showed that apoptosis was decreased in PEG-PLA / CyA-P- , Whereas in the other groups, the number of killed cells was significantly increased.

< Experimental Example  8> CyA -P- CyB  Neon Imaging  analysis

In order to analyze fluorescence imaging of a composition for diagnosing cancer comprising the compound represented by Formula 1 according to the present invention, the following experiment was conducted and the results are shown in FIGS.

Specifically, Figure 3a is a HeLa cell, Figure 3b is a 150 μM H ₂ O ₂ 24 times that are going through the pre-treatment, Figure 3c is 10 μM kaemto camptothecin (camptothecin) for 24 hours, Figure 3d 30 nM knife Payne inhibitor I (calpain inhibitor I) was treated for 30 minutes. Then, each cell was incubated for 2 hours with 10 μM CyA-P-CyB, a composition for diagnosing or treating cancer comprising the compound represented by Chemical Formula 1 according to the present invention. The cells were each washed with DPBS and fluorescent images were obtained by confocal microscopy. The cells were co-stained using LysoTracker (top: CyA- P- CyB treatment (ex 635 nm / em. 655-755 nm), central: LysoTracker staining (ex 405 nm / em 430 - 455 nm), bottom: merged into DIC, scale bar: 10 μm).

3A to 3D are photographs showing fluorescence imaging of a composition CyA-P-CyB for the diagnosis or treatment of cancer comprising the compound represented by Chemical Formula 1 according to the present invention in a cell.

As shown in Table 3a, when the HeLa cells were pretreated with CyA-P-CyB (10 μM) for 2 hours, near-infrared fluorescence was observed in LysoTracker lysosomes showing blue fluorescence, indicating that CyA-P- Indicating that CyB can image cathepsin B in lysosomes.

As shown in FIGS. 3b and 3c, cells were treated with 150 μM H ₂ O ₂ (active oxygen, ROS) or 10 μM of camptothecin (CPT) to activate the DNA tomo isomerase I inhibitor causing DNA damage , It was confirmed that fluorescence diffused in the cytoplasm was increased. This indicates that LMP (lysosome membrane permeabilization) was induced by H ₂ O ₂ or CPT and catechin, indicating that the cathethy was transferred from the lysosomal lumen to the cytoplasm.

As shown in FIG. 3D, near-infrared fluorescence was not observed in HeLa cells pretreated with Calpain Inhibitor I before culturing with CyA-P-CyB, and CyA-P-CyB was cleaved by catenin B, Indicating that light emission occurs.

< Experimental Example  9> CyA -P- CyB  Analysis of effect on cell growth

In order to analyze the effect of the compound represented by the formula (1) according to the present invention on the cell growth of a composition for treating cancer comprising the active ingredient, the following experiment was carried out and the results are shown in FIGS. 4A to 4E .

Specifically, in the presence of CyA-P-CyB, a composition for diagnosing or treating cancer comprising a compound represented by Chemical Formula 1 according to the present invention, MTT measurement results are shown in a graph.

FIG. 4A shows the result of culturing with U87 cells and FIG. 4B with A549 cells with various concentrations of CyA-P-CyB for 16 hours and then removed CyA-P-CyB. The cells were irradiated with a laser (2 W / cm ² , 10 min) at 808 nm and cultured for 24 hours.

FIG. 4C shows the result of the pretreatment with 30 nM of the calpain inhibitor I in the U87 cell, FIG. 4D, and A549 cells, and the CyA-P-CyB was removed after culturing with 5 μM CyA-P-CyB for 16 hours. The cells were irradiated with a laser (2 W / cm ² , 10 min) at 808 nm and cultured for 24 hours. Figure 4e shows that the cells were incubated with 5 [mu] M CyA-P-CyB for 16 hours and then removed CyA-P-CyB. The cells were irradiated with a laser (2 W / cm ² , 10 min) at 808 nm and cultured for 24 hours. The results of Figures 4a to 4e represent ± standard deviation values of three independent experiments.

FIGS. 4A and 4B are graphs showing cytotoxic effects when laser irradiation is performed or not, FIGS. 4C and 4 are graphs showing cytotoxicity when a cathepsin B inhibitor is treated or untreated, FIG. FIG. 2 is a graph showing a comparison of cytotoxicity between normal cells and cancer cells. FIG.

As shown in Figs. 4a to 4e, CYA-P-CYB showed phototoxicity in tumor cells by confirming that the cell viability of tumor cells was decreased in a concentration-dependent manner using MTT assay Could know. In addition, when the cathepsin B inhibitor was used, it was confirmed that the phototoxicity of CYA-P-CYB was dependent on the activity of cathepsin B, as the degree of decrease in cell viability was reduced.

Accordingly, the composition for diagnosing or treating cancer comprising the compound represented by Formula 1 according to the present invention is activated by cathepsin B to specifically and selectively and selectively detect cancerous cells by fluorescent imaging of cancer cells or phototoxicity and strong phototherapy Therefore, it can be usefully used as a multifunctional composition capable of simultaneously diagnosing or treating cancer.

Claims (10)

A compound comprising cyanine represented by the following formula (1):
[Chemical Formula 1]

(In the formula 1,
R ¹ are each independently linear or branched C _{1- 10} alkyl;
n is an integer from 1 to 5;
L is an alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamic acid , Q, glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine Wherein the terminal amino acid of L is 1 to 10, the terminal of L connected to the carbonyl is -NH-, and the terminal connected to the amine is - (C = O) - ;

Each independently represent an unsubstituted or substituted &lt; RTI ID = 0.0 &gt;

,

or

ego;

Each independently represent an unsubstituted or substituted &lt; RTI ID = 0.0 &gt;

,

or

ego; And
The substituted

,

And

May be substituted one or more by one or more substituents selected from the group consisting of linear or branched C _1-5 alkyl, straight or branched C _{1- 5} alkoxy and halogen).
The method according to claim 1,
R ¹ are each independently linear or branched C _{1- 5} alkyl;
n is an integer from 2 to 4;
L is a linker in which at least one amino acid selected from the group consisting of glycine (Gly, G), leucine (Leu, L) and phenylalanine (Phe, F) is linked by a peptide bond and the number of amino acids of L is 1 to 10 , The end connected to the carbonyl of L is -NH- and the end connected to the amine is - (C = O) -;

Each independently represent an unsubstituted or substituted &lt; RTI ID = 0.0 &gt;

or

ego;

Each independently represent an unsubstituted or substituted &lt; RTI ID = 0.0 &gt;

or

ego; And
The substituted

And

Compounds, characterized in that one or more of which may be substituted by one or more substituents selected from the group consisting of C _{1- 5} alkoxy and halogen of C _{1- 5} alkyl, linear or branched linear or branched.
The method according to claim 1,
R ¹ are each independently linear or branched C _{1- 3} alkyl;
n is 3;
L is a linker in which at least one amino acid selected from the group consisting of glycine (Gly, G), leucine (Leu, L) and phenylalanine (Phe, F) is linked by a peptide bond, the number of amino acids of L is 4, L Is -NH- and the end connected to the amine is - (C = O) -;

Each independently represent an unsubstituted or substituted &lt; RTI ID = 0.0 &gt;

ego;

Each independently represent an unsubstituted or substituted &lt; RTI ID = 0.0 &gt;

ego; And
The substituted

Compounds, characterized in that one or more of which may be substituted by one or more substituents selected from the group consisting of C _1-5 alkoxy and halogen, C _{1- 5} alkyl, linear or branched linear or branched.
As shown in Scheme 1 below,
Reacting a compound represented by formula (3) with a compound represented by formula (4) to prepare a compound represented by formula (2) (step 1); And
(Step 2) of preparing a compound represented by the formula (1) by a click reaction with the compound represented by the formula (5) prepared in the step (1) &Lt; / RTI &gt;:&lt;
[Reaction Scheme 1]

(In the above Reaction Scheme 1,
R ¹ , n, L

And

Are as defined in formula (1) of claim 1).
A composition for diagnosing cancer comprising a compound represented by the general formula (1) of claim 1.
6. The method of claim 5,
Wherein said composition is activated by cathepsin B.
A composition for treating cancer, comprising a compound represented by the general formula (1) of claim 1 as an active ingredient.
8. The method of claim 7,
Wherein the composition is activated by cathepsin B.
A composition for treating near infrared rays of cancer comprising, as an active ingredient, a compound represented by the following formula (8):
[Chemical Formula 8]

(In the formula (8)
R ¹ are each independently linear or branched C _{1- 10} alkyl;
R ² is -NH ₂ or -NH ₃ ⁺ ;

Each independently represent an unsubstituted or substituted &lt; RTI ID = 0.0 &gt;

,

or

ego;

Each independently represent an unsubstituted or substituted &lt; RTI ID = 0.0 &gt;

,

or

ego; And
The substituted

,

And

May be substituted one or more by one or more substituents selected from the group consisting of linear or branched C _1-5 alkyl, straight or branched C _{1- 5} alkoxy and halogen).
A method for diagnosing cancer, comprising the step of treating the cancer diagnostic composition of claim 5 with a tissue or cell isolated from an animal subject.

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