KR102175125B1 - Compound comprising Hsp90 inhibitor targeting mitochondria and pharmaceutical composition for photodynamic therapy comprising the compound - Google Patents

Abstract

The present invention relates to a composition for photodynamic diagnosis, treatment, or cancer treatment comprising a compound obtained by conjugating an indocyanine derivative and a purine derivative, a diastereomer thereof, or a pharmaceutically acceptable salt thereof, as an active ingredient. .

Description

A compound comprising Hsp90 inhibitor targeting mitochondria and pharmaceutical composition for photodynamic therapy comprising the compound}

The present invention relates to a novel photodynamic therapeutic agent having both an HSP90 inhibitory effect and an effect as a photosensitizer and a composition thereof.

Photodynamic therapy (PDT) refers to a technique for treating incurable diseases such as tumors or cancers without surgery by using a photosensitive substance, that is, a photosensitizer. PDT, which is used in cancer treatment, irradiates light to a light-sensitive material, thereby converting oxygen molecules (O ₂ ) into singlet oxygen ( ¹ O _2, singlet oxygen), or by creating new radicals and targeting only tumor cells. It has a killing effect.

Recently, photodynamic therapy is focused on efforts to improve the problems of existing drugs and to search for new materials with excellent photochemical properties. Most of the existing drugs are sensitizers targeting mitochondria, which is the main cause of tumor formation, and sensitizers targeting mitochondria have been found to cause mitochondrial dysfunction and cell death of cancer cells. However, low tumor selectivity of sensitizers during PDT progression and low therapeutic efficacy due to high overexpression of anti-necrosis agents were major problems. This is because heat shock protein (Hsp)-family proteins, such as Hsp90 (90 kDa) and TRAP1 (75 kDa), are overexpressed in cancer cells, thereby inhibiting the activity of proteins targeted by these sensitizers.

Therefore, in PDT, there is a need to develop a technology for enhancing tumor selectivity and enhancing therapeutic efficacy.

One aspect is to provide a compound in which a purine derivative and an indocyanine derivative are conjugated, a diastereomer thereof, or a pharmaceutically acceptable salt thereof.

Another aspect is to provide a use of the compound according to the above aspect for photodynamic diagnosis.

Another aspect is to provide a use of the compound according to the above aspect for photodynamic therapy.

Another aspect is to provide a use of the compound according to the above aspect for treating cancer.

One aspect provides a compound represented by the following formula (I), a diastereomer thereof, or a pharmaceutically acceptable salt thereof.

[Chemical Formula Ⅰ]

From here,

L is N, S, or O;

Z ₁ , Z ₂ and Z ₃ are independently C or N;

Y is -CH ₂ -or -S-;

X is hydrogen or halogen;

,

,

, 또는

이며, R ₁ is

,

,

, or

Is,

From here,

# Is an attachment site with N in Formula I, #'is an attachment site with L,

m is an integer of 1 to 3, n is an integer of 1 to 8; And

₎ 또는 4-술포부틸 (4-sulfobutyl,

)인 것인 화합물이다.R ₂ is independently methyl, n-propyl, 1-pyridyhexyl (1-pyridylhexyl _,

₎ Or 4-sulfobutyl,

) Is a compound.

에 해당하는 부분은 퓨린의 유도체에 해당할 수 있다. 퓨린은 화학식

로 이루어진 헤테로 사이클릭(Heterocyclic) 계열의 유기화합물이다. 헤테로사이클릭은 고리 구조 내에 적어도 하나의 탄소 원자, 및 황(S), 산소(O) 또는 질소(N)와 같은, 적어도 하나의 탄소 이외의 원소를 포함하는 모이어티를 말한다. 이 헤테로사이클릭은 방향족 고리 또는 포화된 및 불포화된 비방향족 고리 중 어느 것일 수 있다. 상기 퓨린의 유도체 부분은 Hsp90과 결합하여 이를 억제하는 기능을 제공할 수 있다. In the above formula I

The part corresponding to may correspond to a derivative of purine. Purine is the chemical formula

It is a heterocyclic organic compound consisting of. Heterocyclic refers to a moiety containing at least one carbon atom in the ring structure and at least one element other than carbon, such as sulfur (S), oxygen (O) or nitrogen (N). This heterocyclic can be either an aromatic ring or a saturated and unsaturated non-aromatic ring. The derivative moiety of the purine may provide a function of inhibiting it by binding to Hsp90.

'Methyl' is an example of an alkyl referring to a saturated monovalent hydrocarbon radical. Alkyl groups can be straight-chain or branched. The number of carbon atoms in the alkyl group may be 1, 2, 3, 4, 5 or 6, or 1, 2, 3, or 4. Depending on the number of carbon atoms, alkyl is methyl (1), ethyl (2), propyl (3) including n-propyl and isopropyl, n-butyl, sec-butyl, isobutyl and tert -butyl. Pentyl (5 pieces), n-hexyl, 3,3-dimethylbutyl and isohexyl including butyl (4), n-pentyl, 1-methylbutyl, isopentyl, neopentyl and tert -pentyl including It may be containing hexyl (6). Substituted alkyl groups may be substituted at any position as long as each compound is sufficiently stable and suitable for the desired purpose, such as use as a pharmaceutical substance. In the above aspect,'n-propyl' is a monovalent hydrocarbon radical having 3 carbon atoms and may be a straight chain.

The term “halogen” includes, for example, but is not limited to fluorine, chlorine, bromine or iodine unless otherwise stated.

,

,

, 또는

일 수 있다. R₁은 합성과정에서 인도시아닌 유도체와 퓨린 부분을 이어주는 부분으로 합성 과정의 변경에 따라 상기와 같이 구성될 수 있다. 상기 부분은 미토콘드리아-관통성 부분에 해당할 수 있다. 상기

부분은 펩티드 결합 (또는 아미드 결합) 링커를 갖는 것으로 양쪽에 알킬기가 치환될 수 있다. 상기 부분은 상기 화합물이 세포 내 미토콘드리아를 관통하여 미토콘드리아 내부의 TRAP1과 결합할 수 있는 효과를 제공한다. 상기 알킬기의 수는 상기에서 정의한 바와 같다. In Formula I, R ₁ is

,

,

, or

Can be R ₁ is a moiety connecting the indocyanine derivative and the purine moiety in the synthesis process, and may be configured as described above according to the change in the synthesis process. This portion may correspond to a mitochondrial-penetrating portion. remind

The moiety has a peptide bond (or amide bond) linker, and an alkyl group may be substituted on both sides. This part provides the effect that the compound can penetrate the mitochondria in the cell and bind to TRAP1 inside the mitochondria. The number of alkyl groups is as defined above.

에 해당하는 부분은 인도시아닌 유도체로서 광역학 치료제에서 광감작제의 효과를 갖을 수 있다. 인도시아닌 유도체 중 잘 알려진 물질에는 IR-780이 있고, 대부분의 유도체는 IR-780 또는 IR-780의 이성질체를 기반으로 유도된 화합물일 수 있다. IR-780은 하기 화학식 A의 화합물이고, 이의 이성질체를 포함할 수 있다.In the above formula I

The moiety corresponding to is an indocyanine derivative and may have the effect of a photosensitizer in photodynamic therapy. Among the indocyanine derivatives, a well-known substance is IR-780, and most of the derivatives may be compounds derived based on the isomer of IR-780 or IR-780. IR-780 is a compound of Formula A below, and may include isomers thereof.

[Formula A]

Another indocyanine derivative may be IR-775, IR-783, and IR-Pyr. IR-775 is a compound of Formula B below, and may include isomers thereof.

[Formula B]

IR-783 is a compound having the structure of Formula C below, and may include isomers thereof.

[Formula C]

IR-Pyr is a compound having the structure of Formula D below and may include isomers thereof. IR-Pyr, a water-soluble indocyanine derivative, may exhibit better light stability than IR-780 in mitochondria. In addition, two 1-pyridylhexyl groups can serve as targets for mitonchondria.

[Formula D]

The term'isomer' refers to molecules with the same molecular formula but different physical/chemical properties. It can be divided into structural isomers and stereoisomers depending on whether the order of chemical bonds is changed. Structural isomers are molecules with different chemical bond order (bonding relationship), and the name of the substituent may be changed in general. Stereoisomers can be broadly classified into optical isomers and diastereomers depending on the presence or absence of optical activity. In addition, diastereomers can be classified into geometric isomers and conformational isomers according to the basis of stereoisomerism.

Isomers of the compounds are geometric isomers (or cis-trans isomers), which may be stereoisomers depending on the direction of functional groups in the molecule. In general, these isomers may contain non-rotating double bonds. Isomers can arise because of the ring structure, which limits the rotation of the bond.

In one aspect, the compound may be a compound represented by Formula II below.

[Chemical Formula Ⅱ]

From here,

L, R _1, and R ₂ are as defined above.

In one aspect, the compound may be a compound represented by Formula III below.

[Chemical Formula Ⅲ]

From here,

L, Z ₁ , Z _2, Z _3, X, Y, R ₁ and Y are as defined above.

In one aspect, the compound may be a compound represented by the following formula (IV).

[Chemical Formula IV]

, 미토콘드리아 관통성 부분인

, 광잠작제 역할을 하는 부분인

, 및 미토콘드리아 표적 부분인 2개의 1-피리딜헥실 부분으로 구성될 수 있다.The compound is a purine derivative moiety capable of inhibiting chaperone such as Hsp90

, The mitochondrial penetrating part

, The part that plays the role of mania

, And mitochondrial target moieties, two 1-pyridylhexyl moieties.

In the above compound, all atoms may have sufficient hydrogen or substituents other than hydrogen to satisfy valency, or may contain a pharmaceutically acceptable counter ion.

Another aspect provides a composition for photodynamic diagnosis using the compound, isomer, or pharmaceutically acceptable salt thereof as an active ingredient.

'Photodynamics' is'photodynamics' or'optical', and a photosensitizer causes a chemical reaction by light and oxygen, resulting in singlet oxygen and free radicals caused by it. This refers to the principle of selectively destroying only the cells of interest. Specifically, if the photosensitizer is selectively absorbed into the cell tissue and then irradiated with a laser having an absorption wavelength sensitive to the photosensitizer, the photosensitizer is excited and the light energy can be delivered to oxygen in the cell tissue. The oxygen in the ground state, which has received energy, generates free radicals with excellent chemical reactivity or oxygen in the radical state, and begins to chemically destroy surrounding cells and blood vessel tissue. As a result, cells undergo apoptosis and necrosis (cytotoxic effect). In addition, thromboxane A2 generated during this process blocks the formation of neovascularization of tumors, causing ischemic necrosis and destroying the desired cells.

'Photodynamic diagnosis' refers to confirming the existence or characteristic of a pathological condition by utilizing the principle of photodynamics. Diseases subject to photodynamic diagnosis may include tumors, cancer, or superficial skin diseases. In one embodiment, the composition may be a composition for diagnosis of cancer. Cancer means'malignant tumor' or'malignant neoplasm'. In another embodiment, the cancer that can be diagnosed as the composition may be gastric cancer, laryngeal cancer, bladder cancer, lung cancer, esophageal cancer, liver cancer, pancreatic cancer, skin cancer, biliary tract cancer, and cervical cancer. The most commonly adapted cancers may be lung cancer and esophageal cancer. In addition to cancer, it can also be used for diseases that are prone to cancer such as Barrett's esophagus. Photodynamics can be histologically effective in all ranges of cancer, but the treatment method has to transmit light directly to the tumor tissue, and the penetration depth of the laser beam is limited. Therefore, it is applied to tumors with an outer diameter of less than 2cm in case of superficial tumors or solid cancers I can. Therefore, tumors can be seen through bronchoscopy, gastrointestinal endoscope, cystoscope, and colposcope, and cancers with access to optical fibers can be the primary target.

In one embodiment, the composition may be to inhibit the activity by binding to the cell death theory. 'Chaperones' means'molecular Chaperones' or'Chaperones molecular'. Chaperones may be proteins that control protein folding. They non-covalently bind to the exposed surface of the newly synthesized, denatured or misfolded protein, thereby helping the substrate protein to fold into the correct shape without aggregation. In addition, chaperones can be involved in a number of cellular processes such as protein synthesis, protein transfer and DNA replication. Chaperone activity assists the folding of a protein so that it binds to the target protein and exhibits the physiological activity that the protein has naturally, assists the binding of monomers into a multimer, or aggregates the target protein in an inactive form. It means an activity that prevents or assists in the process of targeting to the membrane.

Chafelon may contain heat shock protein (Hsp). Hsp may include Hsp27, Hsp60 (chaperonin), Hsp70, Hsp90, Hsp104, and the like. In one embodiment, the chaperone to which the composition binds may be Hsp90 or Trap1 (TNF receptor-associated protein 1: TRAP1) of cells.

Hsp may be one of the chaperone molecules involved in the folding and transport of proteins in cells. Among them, Hsp90 is a 90 kDa ATP-dependent chaperone molecule that can be involved in the maturation and tertiary structure of various signaling molecules and transcription factors. At this time, the target protein to be bound may be a client protein. Client proteins can contain various types of oncogenes, transcription factors, and steroid receptors. Hsp90 can play a variety of biological functions related to cell growth, differentiation, and survival by regulating the stabilization and activity of various kinases, transcriptional regulators, signaling substances, and enzymes under normal and stress conditions. In addition, Hsp90 can contribute to the evolution of organisms and maintenance of disease states through its role as a buffer for unstable proteins in cells. TRAP1 is one of the homologous proteins of Hsp90, and can be located in organelles, endoplasmic reticulum, and mitochondria. Therefore, in one embodiment, the composition may be a photosensitizer targeting mitochondria of cells.

In one embodiment, the composition may be accumulated in a target cell of interest. In another embodiment, the cells of interest may be cancer cells or tumor cells. In general, photosensitizers can accumulate in cancer cells or tumor cells. Tumor tissues are characterized by an increase in low-density lipoprotein (LDL), macrophages, and acidity. These characteristics are distinguished from normal tissues and may be related to the tumor selectivity of photosensitizers. For example, in the case of lung cancer or brain tumors, the LDL-receptor is overexpressed more than 100 times in normal tissues, so the intravenously injected photosensitizer binds to the LDL in the blood, and when it reaches the tumor tissue, it binds to the LDL-receptor on the surface of the overexpressed cancer cell. Can get into the cell while doing it. However, the composition of one embodiment may improve tumor selectivity and cell accumulation by the following mechanisms different from the above-described mechanisms.

Cancer cells or tumor cells may have activated chaperones such as Hsp, and more than 50 proteins, including Her2/ErbB2, v-Src, Hif-1α, Raf-1, AKT, hTERT, etc., related to the generation of cancer cells It may be a target protein or a client protein of Hsp90. In normal cells, Hsp90 exists in a state that does not bind to the client, but in cancer cells, many activated Hsp90s that bind to oncogenes can be found. Therefore, the composition can selectively bind to and accumulate therein due to the property of binding to chaperones such as Hsp.

On the other hand, during PDT, Hsp90 or TRAP1 overexpressed in cancer cells can act as anti-necrosis agents in cells. Accordingly, the effect of apoptosis or apoptosis, which is a major mechanism of PDT, is limited, and the therapeutic efficacy of PDT may be reduced. In one embodiment, the composition may inhibit the activity of chaperones such as Hsp90 in addition to the binding of Hsp90 or TRAP1 and accumulation of cancer cells, as described above, thus enhancing the apoptosis effect, thereby enhancing the therapeutic efficacy of PDT. .

Accordingly, another aspect provides a pharmaceutical composition for photodynamic therapy using the compound of any one of claims, a diastereomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. Photodynamic therapy refers to a treatment that destroys cells by apoptosis of target cells by using the principle of photodynamics. The composition has a characteristic capable of binding to chaperones, and may be accumulated in cells of interest by infiltrating and binding to the inside of activated cells or cancer cells such as Hsp90 or TRAP1. In addition, when light is irradiated according to the principle of photodynamics, the composition may promote apoptosis or necrosis of cells as described above.

The term “treatment” refers to delaying the onset of symptoms, alleviating the severity, or delaying the symptomatic progression of cancer or other conditions in a subject. In addition, the specific outcome of this treatment goal will vary from individual to individual, and it will be appreciated that some individuals may gain greater or less than the statistical average for a representative population. Therefore, treatment refers to administering a composition to a subject in need with the expectation that a therapeutic benefit will be obtained.

Another aspect provides a pharmaceutical composition for the treatment of cancer comprising the compound, a diastereomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. The composition may be a photosensitizer for PDT, not only a composition for treating cancer through photodynamic therapy, but also for treating cancer as an Hsp inhibitor. Cancer treatment targets using inhibitors of Hsp90 having various oncogenes as clients may include EGFR, ERBB2, c-MET, PDGFR, IGFR, FGFR3, EML4-ALK fusion protein, and JAK-STAT signaling pathway. In other embodiments, the cancer may be gastric cancer, laryngeal cancer, bladder cancer, lung cancer, esophageal cancer, liver cancer, pancreatic cancer, skin cancer, biliary tract cancer, or cervical cancer.

The compound according to an aspect, a diastereomer thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising the same as an active ingredient have improved tumor selectivity and cell accumulation than conventional PDT therapy, including cancer. There is an effect that can improve treatment efficacy in treatment.

1 is a diagram showing a schematic synthesis method of IR-PU.
Figure 2a is a graph showing a comparison of the singlet oxygen production amount of IR-PU and IR-Pyr according to one embodiment.
Figure 2b is a graph showing data comparing the inhibitory effect of the TRAP1 protein of IR-PU and IR-Pyr, and PU-H71 according to one embodiment.
Figure 2c is a graph showing data comparing the inhibitory effect of the HSP90 protein of IR-PU and IR-Pyr, and PU-H71 according to one embodiment.
3 is an image showing the results of the analysis of the mutual position with the IR-PU using a mito-tracker in order to determine whether the mitochondrial target of the IR-PU according to one embodiment.
4A and 4B are images showing the results of whether IR-PU targets mitochondria through MitoSOX, according to one embodiment.
5A and 5B are images showing a result of inducing apoptosis due to TRAP1 binding of IR-PU and ROS generation by investigating the depolarization change of the mitochondrial membrane according to one embodiment.
6A and 6B are graphs showing the cytotoxicity results of IR-PU against human NCI-H460 tumor cells according to one embodiment.
6C and 6D are graphs showing the results of cytotoxicity of IR-PU against hepatocytes of mice and the HSP inhibitory efficacy of IR-PU compared to the control group using the HSP inhibitor, novobiosin, according to one embodiment.
7A is a graph showing the HSP inhibition and TRAP1 inhibition efficacy of IR-PU according to one embodiment.
7B is a graph showing the results of comparing the cytotoxic effects of IR-PU and IR-Pyr according to one embodiment.
8A and 8B are images showing whether IR-PU targets tumor cells against human NCI-H460 tumor cells implanted in a mouse, according to one embodiment.
8C is a graph showing the tumor cell target and the degree of cell accumulation of IR-PU with respect to human NCI-H460 tumor cells transplanted into a mouse, according to one embodiment.
8D is an image showing whether IR-Pyr targets tumor cells against human NCI-H460 tumor cells transplanted into a mouse, according to one embodiment.
8E and 8F are graphs showing the effect of changes in tumor cell volume and weight of IR-PU on human NCI-H460 tumor cells implanted in a mouse, according to one embodiment.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of IR-PU

The manufacturing process of IR-PU is briefly shown in FIG. 1. Synthetic reagents and materials were obtained from chemical suppliers (Sigma Aldrich and Alfa Aesar). All solvents were used after drying in the standard method before use.

Spectra were recorded on an Agilent 400 MHz spectrometer. The spectra were referenced internally using the resonance of the residual solvent (1Hδ = 3.34 and 13Cδ = 49.86 for CD3OD-d4) for SiMe4. ESI-MS spectra were recorded on Bruker's 1200 series and HCT base systems.

The synthesis of IR-Pyr was described in AP Thomas, L. Palanikumar, MT Jeena, K. Kim, J.-H. Ryu, Chem. Sci. It was synthesized by a method known in 2017, 8 , 8351-8356.

Compound (1) 3-mercapto propanoic acid (3-mercapto propanoic acid) 1g, 9.25 mmol and compound (2) trityl chloride (2) 2.57 g, 9.25 mmol in argon air, dry CH ₂ Cl Dissolved in ₂ . The mixture was stirred at room temperature for 16 hours. The precipitate was washed through CH ₂ Cl ₂ , hexane, and washed again with diethyl ether. Compound (3) was synthesized in a yield of 75% by recrystallization from MeOH/H ₂ O.

¹ H NMR (400 MHz, CDCl ₃ , 298K): δ = 7.35 (d, 6H), 7.18-7.32 (m, 9H), 2.36 (t, 2H), 2.15 (t, 2H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ = 174.21, 144.58, 129.46, 127.79, 126.58, 49.39, 49.18, 48.96, 48.75, 48.54, 33.24, 26.76. ESI-MS: m/z calculated for C ₂₂ H ₂₀ O ₂ S = 348.46; found = 371.55 (M+Na).

A solution (1 g, 2.87 mmol) of compound (3) synthesized by the above method and 0.363 g, 3.1 mmol of N-hydroxysuccinimide were mixed with DCC 0.592 and 2.87 mmol over CH ₂ Cl ₂ , And stirred at room temperature for 16 hours. The solvent was evaporated, and the mixture was purified by a silica gel column to obtain the target compound (5) in 70% yield.

¹ H NMR (400 MHz, CDCl ₃ , 298K): δ = 7.44 (dd, 6H), 7.29 (t, 6H), 7.22-7.26 (m, 3H), 2.78 (s, 4H), 2.55 (t, 2H ), 2.40 (t, 2H); ¹³ C NMR (100 MHz, CDCl ₃ ): δ = 168.85, 167.04, 144.31, 129.50, 128.04, 126.82, 49.29, 33.81, 30.50, 26.09, 25.57, 25.53, 24.89. ESI-MS: m/z calculated for C ₂₆ H ₂₃ NO ₄ S = 445.53; found = 449.69).

0.5 g and 1.12 mmol of compound (5) and 0.575 g and 1.12 mmol of compound (6) were dissolved in dry DMF, and 0.170 g and 1.68 mmol of TEA were slowly added. The mixture was stirred at room temperature for 16 hours. The solvent was removed in vacuo, and the mixture was purified by silica gel column chromatography to obtain the target compound (7) in a yield of 80%.

¹ H NMR (400 MHz, CD ₃ OD, 298K): δ = 8.22 (s, 1H), 7.47 (s, 1H), 7.37-7.39 (m, 6H), 7.28 (s, 1H), 7.26 (t, 3H), 7.24 (t, 2H), 7.23 (t, 1H), 7.21 (t, 1H), 7.19 (t, 1H), 6.08 (s, 2H), 3.17 (t, 2H), 2.4 (t, 2H ), 2.21 (t, 2H), 1.86 (t, 2H), 1.48-1.54 (m, 2H), 1.38-1.45 (m, 4H); ¹³ C NMR (100 MHz, CD ₃ OD): δ = 176.31, 155.72, 154.49, 154.37, 153.44, 152.80, 148.69, 133.27, 132.38, 131.45, 130.31, 128.19, 123.31, 118.65, 106.87, 99.53, 70.24, 47.79 42.69, 38.42, 32.92, 32.71, 31.71, 29.85, 29.83. ESI-MS: m/z calculated for C ₄₀ H ₃₉ IN ₆ O ₃ S ₂ =842.82; found = 844.25.

The solution of compound 7 (0.20 g, 0.24 mmol) was stirred at room temperature with TFA 0.067 g, 0.59 mmol, triethylsilane 0.110 g, 0.95 mmol on CH ₂ Cl ₂ until exhaustion of the starting material. The reaction mixture was diluted with CH ₂ Cl ₂ and washed with 5% NaHCO ₃ . It was dried over anhydrous Na ₂ SO ₄ and the solvent was evaporated. The mixture was purified on a silica gel column using an ethyl acetate/hexane eluent to give compound (8) in a yield of 70%.

1H NMR (400 MHz, CD3OD, 298K): δ = 8.22 (s, 1H), 7.38 (s, 1H), 7.16 (s, 1H), 5.99 (s, 2H), 4.22 (t, 2H), 3.07 ( t, 2H), 2.61-2.64 (m, 2H), 2.34-2.37 (m, 2H), 1.76-1.79 (m, 2H), 1.39-1.41 (m, 2H), 1.29-1.32 (m, 6H); 13C NMR (100 MHz, CD3OD): δ = 172.30, 151.29, 150.67, 150.38, 149.53, 144.7, 124.51, 119.36, 118.7, 114.54, 102.91, 95.30, 39.31, 38.75, 28.97, 28.78, 25.97, 25.84, 19.77; ESI-MS: m/z calculated for C ₂₁ H ₂₅ IN ₆ O ₃ S ₂ =600.50; found = 601.39.

The solution of compound (8) (0.10 g, 0.16 mmol) and IR-Pyr already prepared 0.169 g and 0.16 mmol were stirred at room temperature for 16 hours, with 0.025 g and 0.25 mmol of TEA over DMF. The solvent was evaporated and the mixture was purified by reverse phase HPLC (C18 column using methanol/water). The desired compound IR-PU was prepared in a yield of 65%.

¹ H NMR (400 MHz, CD ₃ OD, 298K): δ = 8.99 (dd, 4H), 8.88 (s, 1H), 8.40 (d, 1H), 8.58 (t, 2H), 8.28 (d, 1H) , 8.08-8.12 (m, 4H), 7.47-7.50 (dd, 2H), 7.42 (d, 1H), 7.40 (d, 1H), 7.38 (d, 1H), 7.25-7.29 (m, 3H) , 7.22 (d, 1H), 6.28 (s, 1H), 6.24 (d, 1H), 6.07 (d, 2H), 4.61-4.66 (m, 4H), 4.26 (t, 2H), 4.15 (t, 4H) ), 3.14 (t, 2H), 3.05 (t, 2H), 2.65 (t, 4H), 2.52 (t, 2H), 2.65 (t, 4H), 2.52 (t, 2H), 2.00-2.06 (m, 4H), 1.85 (t, 6H), 1.74 (d, 11H), 1.47-1.55 (m, 10H), 1.38 (t, 4H), 1.28 (d, 1H); ¹³ C NMR (100 MHz, CD ₃ OD): δ = 174.68, 173.61, 162.72, 162.35, 159.00, 147.73, 146.75, 145.94, 144.96, 143.24, 135.52, 130.69, 130.36, 127.16, 126.53, 124.34, 121.57, 120. 119.81, 116.93, 112.83, 105.17, 97.75, 63.81, 51.34, 46.02, 48.88, 41.19, 38.09, 35.62, 33.15, 31.17, 31.01, 29.31, 29.09, 28.37, 28.24, 28.10, 28.05, 27.88, 25.06, 22.98 ESI-MS: m/z calculated for C ₇₃ H ₈₈ IN ₁₀ O ₃ S ₂ ³⁺ = 447.85; found = 448.69.

Human cervical carcinoma cells (HeLa cells) and human lung cancer cells (NCI-H460 cells) for 37 ℃, 10% in the wet state of 5% CO ₂ fetal bovine serum (fetal bovine serum: FBS) ( Life Technologies) and 1% penicillin / Incubated in DMEM medium or RPMI medium supplemented with streptomycin (Life Technologies). To isolate primary hepatocytes from 8-week-old BALB/c mice, the mice were anesthetized, the liver was perfused with collagenase solution, dissected, and the clump was destroyed by pipetting, and a 100-μm cell filter (BD Biosciences) Filtered through. Cells were washed several times by repeatedly centrifuging and resuspending in M199/EBSS medium (Hyclone). Cell debris and non-parenchymal cells were separated from hepatocytes by gradient centrifugation using Percoll (Sigma). After repeated washing, the cell pellet was resuspended and cultured in M199/EBSS medium supplemented with 10% FBS at 37° C. and 5% CO ₂ .

Experimental Example 1. 1O of IR-PU ₂ (Singlet oxygen) generation comparison

Of IR-PU light-induced singlet oxygen ⁽¹ O ₂₎ generated and HSP90 / TRAP1 in order to investigate the inhibitory effect, singlet oxygen sensor green (singlet oxygen sensor green: SOSG) is the primary standard for PDT ¹ using The ability to produce O ₂ was investigated.

IR-PU in PBS was mixed with an equimolar solution of SOSG to measure the change in fluorescence intensity. The electron absorption spectrum and steady state fluorescence spectrum were recorded on a spectrophotometer (JASCO V-670) and a fluorescence spectrophotometer (Hitachi F-7000), respectively. Before and after irradiation with laser light (808 nm, 200 mW) at different time intervals, SOSG was observed at 530 nm after excitation at 504 nm. The enhanced emission intensity of SOSG contains a single increased product oxygen. The results are shown in Figure 2A.

As shown in Figure 2a, IR-PU showed a higher generation of ¹ O ₂ compared to IR-Pyr. At the same time, irradiation with only SOSG without IR-PU and fluorescence measurement without irradiation did not show fluorescence intensity, and confirmed the generation of photoexcitation of ¹ O ₂ . These results indicate that IR-PU has more triple populations to generate ¹⁰ O ₂ at light excitation than IR-Pyr.

Experimental Example 2. Comparison of inhibitory efficacy of HSP90 and TRAP1

The PU-H71 portion of IR-PU is a competitive inhibitor for the ATP binding pocket of Hsp90 and TRAP1. In order to examine the binding affinity of IR-PU to HSP90 and TRAP1, fluorescence polarization was measured using a PU-H71-FITC3 probe. The results are shown in Figures 2b and 2c.

As shown in Figures 2b and 2c, the data show that IR-PU has a higher binding affinity for TRAP1 and a somewhat lower affinity for HSP90 compared to PU-H71 (a known HSP inhibitor), whereas the control compound IR -Pyr showed no affinity compared to IR-PU. These results suggest that IR-PU has high affinity for both HSP90 and TRAP1.

Experimental Example 3. Analysis of the Mutual Location of Mito-Trecker and IR-PU in HeLa Cell Line

In order to determine whether IR-PU is a mitochondrial target, a mutual position analysis of mito-tracker and IR-PU was performed.

A (HeLa cells) Human cervical cancer cells in 37 ℃, the wet state of 5% CO ₂ 10% fetal bovine serum (fetal bovine serum: FBS) supplemented with (Life Technologies) and 1% penicillin / streptomycin (Life Technologies) It was cultured in DMEM medium or RPMI medium.

HeLa cells were seeded on one well cover glass (Lab Tek II, Thermo Scientific) at a seeding density of 2×10 ⁵ cells/well. After 24 hours, the cells were treated with 2.5 μM of IR-PU for 4 hours and replaced with a fresh medium, followed by treatment with Mito-tracker green. Cell absorption was periodically monitored using a multiphoton microscope (Carl Zeiss LSX 780 NLO) connected to a CO ₂ incubator, and the excitation of the incubator was 720 nm, the emission was 488 nm excitation between 725-758 nm, and the emission was It was set up with cross-location analysis with Mito-Tracker Green FM set between 500-550 nm. The Pearson coefficient for mitochondrial localization was calculated as 0.73, which is higher compared to the widely used indocyanine dye IR-780 (Pearson coefficient 0.61). The results are shown in FIG. 3.

As shown in FIG. 3, whether the IR-PU targets mitochondria was confirmed by confocal microscopy analysis in HeLa cell lines compared to mito-Trecker Green. Intense red fluorescence after irradiation indicates the production of 10 2 of IR-PU. As shown in Figure 3, it can be seen that the IR-PU injected into the cells was accumulated in the mitochondria.

Experimental Example 4. Analysis of MitoSox ROS occurrence

In order to investigate the effect of generating reactive oxygen species on cells as a photodynamic therapy of IR-PU, the generation of ROS through MitoSox was analyzed. HeLa cells were sprinkled on a Lab Tek II chamber cover glass so as to have a density of 90% in DMEM media supplemented with 10% FBS, 100 μgmL ^-1 streptomycin, and 100 μgmL ^-1 penicillin, and cultured at 37°C and 5% CO ₂ . After incubation with 2.5 μM IR-PU for 4 hours, the cell culture medium was replaced with a medium containing 5 μM MitoSox reagent (MitoSox, M36008) working solution to cover adherent cells according to the manufacturer's protocol. Thereafter, the cells were cultured at 37° C. for 10 minutes, protected from light, and irradiated (200 mWcm ^-2) . Then, the cells were analyzed with an FV1000 laser confocal scanning microscope. The results are shown in Figs. 4a and 4b.

In FIG. 4A, intense red fluorescence after irradiation indicates the generation of 10 ₂ . The red signal of the control without laser irradiation is presumed to be a signal indicating the production of active oxygen due to HSP90/TRAP1 inhibition of the PU-H71 residue.

Experimental Example 5. TMRM depolarization analysis

In order to investigate the degree of depolarization of the mitochondrial membrane, the degree of occurrence of ROS in IR-PU was measured. After preparing a HeLa cell line in the same manner as in Example 3, it was incubated with IR-PU (2.5 μM) for 2 hours. The medium was replaced and used as an index for measuring mitochondrial membrane depolarization. TMRM (Tetramethylrhodamine methyl ester perchlorate) was added and incubated for another 30 minutes. The medium was replaced again and analyzed using an FV1000 laser confocal scanning microscope. And it irradiated for 2 minutes with a laser (808 nm). The results are shown in Figures 5a, 5b, and 5c.

5A and 5B, the confocal image after irradiation showed a decrease in the fluorescence intensity of the dye compared to the initial intensity, which indicates the depolarization of the mitochondrial membrane due to the binding of TRAP1 and purine groups in the mitochondria. Looking at the graph of FIG. 5C, IR-PU showed higher cell accumulation than IR-pyr in NCI-H460 cells.

Experimental Example 6. Cell viability analysis (MTT Assay)

In order to measure the cytotoxicity of IR-PU, cell viability was investigated. Human lung cancer cells (NCI-H460 cells) were added to DMEM medium or RPMI medium supplemented with 10% fetal bovine serum (Life Technologies) and 1% penicillin/streptomycin (Life Technologies) at 37° C. and 5% CO ₂ Cultured. To isolate primary hepatocytes from 8-week-old BALB/c mice, the mice were anesthetized, the liver was perfused with collagenase solution, dissected, and the clump was destroyed by pipetting, and a 100-μm cell filter (BD Biosciences) Filtered through. Cells were washed several times by repeatedly centrifuging and resuspending in M199/EBSS medium (Hyclone). Cell debris and non-parenchymal cells were separated from hepatocytes by gradient centrifugation using Percoll (Sigma). After repeated washing, the cell pellet was resuspended and cultured in M199/EBSS medium supplemented with 10% FBS at 37° C. and 5% CO ₂ .

The prepared cells were cultured in a 96-well plate overnight (5Х10 ³ cells /well), and treated in the dark for 12 hours with IR-PU, and then the medium was replaced with a new medium. The cells were irradiated with a laser (808 nm, 200mWcm ^-2 ) for 2 minutes and cultured for an additional 12 hours, and then cytotoxicity of the cells was confirmed using a cell viability assay (MTT assay). To determine cell viability, cells were treated with 3(4,5-dimethyl-thiazol-2-yl) 2,5 diphenyltetrazolium bromide (3(4,5-dimethyl-thyzoyl-2-yl)2,5 diphenyltetrazolium bromide: MTT), and the crystallized formazan was quantified by measuring absorbance (595 nm) with a micro plate reader (SYNERGY NEO, BioTek Instruments, Inc.). The absorbance data was compared with the absorbance data of the control cells and expressed as a survival rate (%). The control experiment was performed when there was no laser irradiation, when IR-Pyr was used, and when there was no laser irradiation while using IR-Pyr. The results are shown in Figure 6.

By the above analysis method, the results for NCI-H460 cells are shown in Figs. 6A and 6B. In the case of hepatocytes, a sample pretreated with novobiosin (competitive binding agent for HSP90) was tested to confirm the change in the effect of reducing the viability of IR-PU. The results are shown in Figs. 6c and 6d.

6A and 6B, in the NCI-H460 cell line, IR-PU showed concentration-dependent phototoxicity after irradiation. The phototoxicity of IR-PU was significantly higher than that of IR-Pyr. In addition, as the cell accumulation of the drug caused by binding to HSP90 increased, additional toxicity was induced.

Referring to FIG. 6C, the normal hepatocytes of mice were inhibited from binding of IR-PU with HSP90 due to the binding of novobiosin and HSP90. As a result, the accumulation of IR-PU was decreased, and the result of phototoxicity reduction of IR-PU was confirmed. 6D shows that PU-H71 has a lower cytotoxic effect than IR-PU, and thus indirectly demonstrates the synergistic effect of HSP inhibition of IR-PU and PDT.

Experimental Example 7. Apoptosis induction, Western blot and FACS analysis

Western blot and FACS analysis were performed to confirm the effect of IR-PU inducing cell death according to HSP90 inhibition. Human lung cancer cells NCI-H460 prepared in the same manner as in Example 6 were cultured overnight in a 6-well plate (4Х105 cells/well) and treated with a drug for 12 hours. The cells were harvested 12 hours after laser irradiation (200mWcm-2) for 2 minutes. And Western blot analysis was performed. The results are shown in Figure 7a. Caspase 3 and PARP1, which direct cell death with IR-PU, were cleaved during PDT, which was further confirmed by FACS analysis. For FACS analysis, DNA content (propidium iodide) was identified and exogenous phosphatidyl serine (Annexin V) was used using an apoptosis kit (Molecular Probe). Labeled cells were quantified using the FACS Calibur system (BD Biosciences). Data was processed using FlowJo software (TreeStar). The results of the FACS analysis are shown in Figure 7b.

As a result of the Western blot shown in FIG. 7A, IR-PU strongly inhibited Hsp90, which is associated with a decreased level of HSP90 client protein (chk1) and a heat shock reaction (rise of Hsp70). In addition, there was a slight decrease in the TRAP1 client protein Sorcin in the IR-PU treated samples. Control experiments using IR-Pyr showed no signs of HSP90 and TRAP1 inhibition as expected.

As a result of the FACS analysis shown in FIG. 7B, after irradiation with an 808 nm laser for 2 minutes, the late cell death population increased from 4% to 21%. It was much higher than IR-Pyr under similar conditions. Treatment with a caspase inhibitor (Z-VAD) reduced the apoptosis population to 5%, confirming the apoptosis pathway by PDT of IR-PU.

Experimental Example 8. Tumor xenograft animal model and photodynamic treatment

In order to confirm the tumor treatment effect of IR-PU through an animal model, the following experiment was performed. NCI-H460 cells were injected subcutaneously into both sides of 9-week-old BALB/c nu/nu male mice. Nude mice with NCI-H460 (tumor volume: 200 mm ³ ) cells were intraperitoneally injected with DMSO and IR-PU (containing 20% Chremophor in PBS, concentration of 0.5 mg/mL) at 10 mg/kg daily. Tumor volume volume = (tumor length) × (width of the tumor) was calculated by a ^2/2. Mice were divided into three groups: DMSO, IR-PU and laser-irradiated IR-PU. For PDT treatment, a group of mice irradiated with laser was administered IR-PU for 24 hours and then irradiated for 2 minutes using a NIR laser (808 nm, 200 mWcm ^-2 ). This injection pattern was repeated until the 9th day. An in vivo fluorescence imaging system (Bruker Xtreme model) with excitation at 830 nm and emission at 760 nm was used to regularly image with a standard X-ray background. The results are shown in FIG. 8.

As shown in FIGS. 8A, 8B, and 8C, accumulation of IR-PU in the tumor site of the mouse and IR-PU fluorescence signal in the tumor site appeared. In the case of the mice treated with IR-PU, a signal was significantly higher than that of the mice treated with IR-pyr shown in Fig. 8d. This indicates the excellent cancer targeting ability of IR-PU due to its binding with HSP.

In addition, as shown in Fig. 8e, compared to the control DMSO group, the tumor volume of the PDT-treated mouse group with IR-PU decreased. This indicates that tumor growth was weakened by IR-PU. Figure 8f shows the weight of the tumor after euthanizing all mice and obtaining the tumor, the tumor weight of the PDT-treated mouse group was much lighter than that of the control group (DMSO). These results show the superior tumor specificity of IR-PU.

Claims (17)

A compound represented by the following formula (I), a diastereomer thereof, or a pharmaceutically acceptable salt thereof:
[Chemical Formula Ⅰ]

From here,
L is NH, S or O;
Z ₁ , Z ₂ and Z ₃ are independently C or N;
Y is -CH ₂ -or -S-;
X is hydrogen or halogen;
R ₁ is

,

,

, or

Is,
From here,
# Is an attachment site with N in Formula I, #'is an attachment site with L,
m is an integer of 1 to 3, n is an integer of 1 to 8; And
R ₂ is independently, alkyl, n- propyl, a 1-pyridyl-hexyl _{(1-pyridylhexyl)} or 4-sulfo-butyl being the (4-sulfobutyl) compound. The method according to claim 1, wherein the compound is a compound represented by the following formula (II), a diastereomer thereof, or a pharmaceutically acceptable salt thereof:
[Chemical Formula Ⅱ]

From here,
L, R _1, and R ₂ are as defined above. The method according to claim 1, wherein the compound is a compound represented by the following formula (III), a diastereomer thereof, or a pharmaceutically acceptable salt thereof:
[Chemical Formula Ⅲ]

From here,
L, Z ₁ , Z _2, Z _3, X, R _1, and Y are as defined above. The method according to claim 1, wherein the compound is a compound represented by the following formula (IV), a diastereomer thereof, or a pharmaceutically acceptable salt thereof:
[Chemical Formula IV]

A pharmaceutical composition for diagnosis of cancer comprising the compound of any one of claims 1 to 4, a diastereomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. The composition of claim 5, wherein the composition binds to chaperones of cells. The composition of claim 6, wherein the chaperone is a heat shock protein 90 (Hsp90) or a trap 1 (TNF receptor-associated protein 1: TRAP1). The composition of claim 5, wherein the composition accumulates in target cells. A pharmaceutical composition for photodynamic treatment of cancer comprising the compound of any one of claims 1 to 4, a diastereomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. The composition of claim 9, wherein the composition inhibits activity by binding to chaperones of cells. The composition of claim 10, wherein the chaperone is Hsp90 or TRAP1. The composition of claim 9, wherein the composition accumulates in target cells. A pharmaceutical composition for the treatment of cancer comprising the compound of any one of claims 1 to 4, a diastereomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. The composition of claim 13, wherein the composition inhibits activity by binding to chaperones of cells. The composition of claim 14, wherein the chaperone is Hsp90 or TRAP1. The pharmaceutical composition of claim 13, wherein the composition accumulates in target cells. The composition of claim 13, wherein the cancer is gastric cancer, laryngeal cancer, bladder cancer, lung cancer, esophageal cancer, liver cancer, pancreatic cancer, skin cancer, biliary tract cancer, or cervical cancer.

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