JP2016155768A - Porphyrin derivative and photosensitizer containing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a porphyrin derivative capable of being specifically transferring into cancer cells by an oxygen reaction of a membrane protein specifically expressed in the cancer cells, a photosensitizer using characteristic of the porphyrin derivative to being specifically transferred into the cancer cells, a fluorescence visualization agent and MRI contrast medium.SOLUTION: There are provided a porphyrin derivative represented by the general formula (I) and a metal complex where metal ion are inserted into the porphyrin derivative. There is provided a metal complex where metal ions to be inserted are ions of Mn, Zn, Mg, Sn, Pt, Ag and Au and counter ions are Cl, Br, NO, CFSO, hydroxide ion or CHCOO. In the general formula (I), n is a natural number of 0 to 5 and R may be same or different.SELECTED DRAWING: None

Description

  The present invention relates to a porphyrin derivative, a photosensitizer and a fluorescence visualization agent containing the same, a metal complex formed by inserting a metal ion into the porphyrin, and a photosensitizer and an MRI contrast agent containing the metal complex.

  Photodynamic therapy (PDT) is a method in which photosensitizers are administered by intravenous injection, etc., held in cancer tissue, and then irradiated with laser light or LED light of a predetermined wavelength to select only the cancer tissue. It is a method of treating cancer that destroys it.

  More specifically, PDT irradiates a photosensitizer accumulated in cancer tissue with light to excite the photosensitizer to a singlet excited state, and from the singlet excited state to the lowest triplet state by intersystem crossing. By transforming triplet oxygen molecules in the vicinity of the resulting photosensitizer into singlet oxygen (active oxygen) and destroying nearby cells, cancer cells are relatively damaged without relatively damaging the surrounding normal tissue It is a cancer treatment method that destroys and treats cancer tissue.

  Thus, since PDT has little influence on normal tissues, it has attracted attention as a new cancer treatment method following surgery, chemotherapy with anticancer agents, radiation therapy, and the like.

  Well-known porphyrin derivatives such as resafrin, bisdyne, and temoporfin (Foscan) have been used as photosensitizers for PDT, and various derivatives have been studied mainly from porphyrin derivatives. (For example, see Patent Documents 1 to 13 and Non-Patent Document 1.)

  However, conventional porphyrin derivatives are both water-soluble and hydrophobic, do not specifically accumulate only in cancer cells and cancer tissues, and are not rapidly discharged out of the body. Therefore, after the end of PDT, the patient had to be hospitalized for a certain period of time in a dark room so that the photosensitizer remaining in the body did not develop photosensitivity.

  In order to solve these problems, porphyrin derivatives that have been specifically accumulated in cancer tissues and cancer cells have been studied. For example, it is known that a cationic porphyrin having a pyridinium group, a tetramethylammonium group, a guanidinium group, and the like migrates into cells.

  However, these cationic porphyrins cannot be generated by an enzymatic reaction with a membrane protein such as γ-glutar transpeptase that is specifically expressed in cancer cells. Therefore, these cationic porphyrins could not be specifically transferred into cancer cells.

Japanese Patent Laid-Open No. 05-097857 Japanese Patent Laid-Open No. 09-124652 JP 2001-11077 A Japanese Patent Laid-Open No. 2004-2438 JP 2005-507383 A JP 2012-506425 A Japanese Patent No. 3191223 Japanese Patent No. 3718887 Japanese Patent No. 5068920 Japanese Patent No. 5226317 Japanese Examined Patent Publication No. 04-24661 Japanese Patent Publication No. 07-25563 International Publication No. 2002/020536

Yoon et al. Clin. Endosc. 2013, 46, 7-23

  Then, this invention makes it a subject to provide the porphyrin derivative which can be specifically transferred into a cancer cell by the enzyme reaction of the membrane protein specifically expressed in a cancer cell. It is another object of the present invention to provide a photosensitizer, a fluorescence visualization agent, and an MRI contrast agent that utilize the property that the porphyrin derivative specifically migrates into cancer cells.

  As a result of intensive studies, the inventors have paid attention to the fact that the amide group is easily converted into a primary amino group by an enzymatic reaction such as γ-glutar transpeptase. The present inventors have synthesized a water-soluble porphyrin derivative and have found that this porphyrin derivative can be used as a photosensitizer, a fluorescence visualization agent, an MRI contrast agent, and the like.

（式中、nは0〜5の自然数であり、Rは同一又は互いに異なっていてもよい。） That is, the invention described in claim 1 is a porphyrin derivative represented by the following general formula (I).

(In the formula, n is a natural number of 0 to 5, and R may be the same or different from each other.)

  The invention according to claim 2 is the porphyrin derivative according to claim 1, wherein n is 3.

  The invention according to claim 3 is a photosensitizer comprising the porphyrin derivative according to claim 1 or 2 as an active ingredient.

  The invention according to claim 4 is a fluorescence visualization agent comprising the porphyrin derivative according to claim 1 or 2 as an active ingredient.

（式中、nは0〜5の自然数であり、Rは同一又は互いに異なっていてもよい。また、Mは、マンガン、亜鉛、マグネシウム、スズ、パラジウム、銀、金の内の何れかである。さらに、Xは塩素イオン、臭素イオン、硝酸イオン、トリフルオロメタンスルホン酸イオン、水酸化物イオン、酢酸イオンのうちの何れかである。） The invention according to claim 5 is a metal complex represented by the following general formula (II).

(In the formula, n is a natural number of 0 to 5, and R may be the same or different from each other. M is any one of manganese, zinc, magnesium, tin, palladium, silver, and gold. Furthermore, X is any one of chlorine ion, bromine ion, nitrate ion, trifluoromethanesulfonate ion, hydroxide ion and acetate ion.)

  The invention according to claim 6 is the metal complex according to claim 5, wherein n is 3.

  Furthermore, the invention according to claim 7 is a photosensitizer containing the metal complex according to claim 5 or 6 as an active ingredient.

  In addition, the invention according to claim 8 is an MRI contrast agent comprising the metal complex according to claim 5 or 6 as an active ingredient.

  The porphyrin derivative of the present invention is more efficiently and specifically transferred into cancer cells than conventional cationic porphyrin derivatives. Therefore, if the photosensitizer containing the porphyrin derivative of the present invention is used, it can be accumulated at a high concentration in a cancer tissue even if the dose is small, and a high therapeutic effect by a photodynamic therapy method and a high fluorescence visualization effect of the cancer tissue are obtained. I can expect.

  In addition, the metal complex of the present invention in which manganese ions are inserted into the porphyrin derivative of the present invention also migrates into cancer cells specifically with high efficiency. Therefore, even if a photosensitizer containing the metal complex of the present invention is used, a high therapeutic effect by photodynamic therapy can be expected. Moreover, if the MRI contrast agent containing the metal complex of this invention is used, even if there is little dosage, it can accumulate | store in cancer tissue at high concentration, and cancer tissue can be detected more clearly.

FIG. 1 is a diagram showing an example of a synthesis route of a porphyrin derivative of the present invention. FIG. 2 is a graph showing the relationship between the concentration of the porphyrin derivative of the present invention and the cancer cell killing effect.

1. Porphyrin Derivative The porphyrin derivative of the present invention is represented by the following general formula (I). Note that n is a natural number of 0 to 5, and 3 is preferable because it is easy to synthesize and has excellent water solubility. The four Rs may be the same or different from each other, but are preferably the same because they are easily synthesized.

  The porphyrin derivative of the present invention can be synthesized, for example, by combining the following chemical reactions (A) and (B). In addition, the porphyrin derivative of this invention is not limited to what was manufactured by the following synthetic methods.

(A) Synthesis of Carboxylic Acid Amide As shown in the following chemical formula (III), carboxylic acid amide c is synthesized by amide bonding of carboxylic acid a which is a porphyrin derivative and primary amine b having an azide group. .

(B) Reduction of azide group As shown in the following chemical formula (IV), the azide group at the terminal of the carboxylic acid amide c is reduced to synthesize an amine d.

2. Photosensitizer and fluorescence visualization agent The photosensitizer and fluorescence visualization agent of the present invention contain the porphyrin derivative of the present invention as an active ingredient and can be administered to humans or other animals.

  In addition, the dosage form of the photosensitizer and the fluorescence visualization agent of the present invention is not particularly limited, and may be appropriately selected as necessary. Specifically, it is generally used as a parenteral preparation such as an injection or an infusion, but can be used as an oral preparation such as a tablet, capsule, granule, fine granule or powder. The concentration of the porphyrin derivative in the photosensitizer and the fluorescence visualization agent and the dose of the contrast agent to the patient can be freely selected according to the patient's age, weight, and degree of disease.

  When the photosensitizer and fluorescence visualization agent of the present invention are produced as parenteral preparations such as injections and infusions, well-known diluents such as distilled water for injection, physiological saline diluents, aqueous glucose solutions, etc. It can be manufactured by a method. In addition, you may add a disinfectant, antiseptic | preservative, and a stabilizer as needed. In addition, from the viewpoint of stability, this parenteral preparation can be frozen after filling into a vial or the like, the water removed by a normal freeze-drying treatment, and re-prepared from a freeze-dried product immediately before use. Furthermore, an isotonic agent, stabilizer, preservative, and soothing agent may be added as necessary.

  When producing the photosensitizer and fluorescent visualization agent of the present invention as an oral preparation such as a tablet, together with known excipients, binders, disintegrants, surfactants, lubricants, fluidity promoters, etc. It can be produced by a known production method. The photosensitizer and fluorescence visualization agent of the present invention can also be administered orally as suspensions, emulsions, syrups and elixirs. In this case, a flavoring agent, a flavoring agent, a colorant and the like may be contained.

  The photosensitizer and fluorescence visualization agent of the present invention may be administered in the body by encapsulating the known DDS technique, for example, the contrast agent of the present invention in a carrier such as a liposome. In this case, if a carrier that specifically recognizes cells at the target site is used, the photosensitizer and fluorescence visualization agent of the present invention can be efficiently transported to the target site.

3. Metal Complex The porphyrin complex of the present invention is obtained by inserting manganese ions into the porphyrin derivative of the present invention and represented by the following chemical formula (II). Note that n is a natural number of 0 to 5, and 3 is preferable because it is easy to synthesize and has excellent water solubility. The four Rs may be the same or different from each other, but are preferably the same because they are easily synthesized.

  Further, M is any one of manganese, zinc, magnesium, tin, palladium, silver, and gold, and manganese is preferable because it has a high MRI contrast effect. In addition, X is any one of chlorine ion, bromine ion, nitrate ion, trifluoromethanesulfonic acid ion, hydroxide ion, and acetate ion, and chlorine ion is preferable because it is easy to purify.

  The metal complex of the present invention can be produced using a known method in which the porphyrin derivative of the present invention is coordinated to a metal ion such as manganese. Specifically, a metal ion solution in which a salt containing a metal ion serving as a central metal is dissolved in a polar organic solvent such as methanol or acetonitrile, and a porphyrin derivative solution in which the porphyrin derivative of the present invention is dissolved in a polar organic solvent are prepared. If the metal ion solution and the porphyrin derivative solution are mixed and stirred, crystals of the complex can be precipitated. If this crystal is washed with a polar organic solvent, a crystal with higher purity can be obtained.

4. Photosensitizer and MRI contrast agent The other photosensitizer and MRI contrast agent of the present invention contain the metal complex of the present invention as an active ingredient and can be administered to humans or other animals. it can. The dosage form, dosage, etc. of the MRI contrast agent can be arbitrarily set and changed in the same manner as the photosensitizer and fluorescence visualization agent of the present invention.

  Hereinafter, the present invention will be described in more detail based on examples. The claims of the present invention are not limited in any way by the following examples.

1. Synthesis of Porphyrin Derivative The porphyrin derivative TAEP according to the present invention was synthesized along the synthetic route of FIG. In addition, in order to make it easy to understand, in the following description, the same symbol as FIG. 1 was used about the same compound.

(1) Synthesis of carboxylic acid amide (compound c) Tetrakis (4-carboxyphenyl) porphyrin (compound a, CAS # 14609-54-2, 100 mg, 0.125 mmol) was placed in a two-necked eggplant flask and dichloromethane under nitrogen. (10 mL) was added. While stirring the reaction solution, oxalyl chloride (0.25 mL, 3 mmol) and N, N-dimethylformamide (1 μL) were added, and the mixture was stirred at room temperature for 19 hours under a light-shielded condition under nitrogen. The reaction solution was concentrated with a rotary evaporator connected to an alkali trap and a liquid nitrogen trap, and washed with dichloromethane (5 mL). After repeating this operation twice, dichloromethane (10 mL) was added to the reaction solution under nitrogen (hereinafter abbreviated as reaction solution 1).

  Separately, 11-azido-3,6,9-trioxaundecan-1-amine (compound b, CAS # 134179-38-7, 318 mg, 1.25 mmol) was weighed into a two-necked eggplant flask under nitrogen. After adding dichloromethane (5 mL), N, N-diisopropylethylamine (0.5 mL) was added (hereinafter abbreviated as reaction solution 2). Thereafter, the reaction solution 2 was added to the reaction solution 1 under nitrogen, and the mixture was stirred at room temperature for 22 hours under light-shielding under nitrogen.

The obtained reaction solution was diluted with dichloromethane (100 mL), and then the organic layer was washed with 1N hydrochloric acid, 1N aqueous sodium hydroxide solution and saturated brine in this order. The organic layer was dehydrated with sodium sulfate and filtered, and then concentrated on a rotary evaporator and dried in vacuo. The vacuum dried product was purified by flash chromatography, and then concentrated and vacuum dried by a rotary evaporator to obtain a dark purple powder (yield: 142.4 mg, yield: 72%). This compound was identified from the measurement results of nuclear magnetic resonance spectroscopy ( ¹ H NMR) and Fourier transform infrared spectrophotometer (FT-IR). The results are shown below.

¹ HNMR (500MHz, CDCl ₃ ): δ [ppm] = 8.83 (s, 8H, pyrrolic-β-CH), 8.29 (d, J = 8.3 Hz, 8H, phenylic CH), 8.21 (d, J = 8.3 Hz 8H, phenylic CH), 7.16 (t, J = 5.2 Hz, 4H, amido), 3.86 (t, J = 4.8 Hz, 8H, N ₃ CH ₂ CH ₂ ), 3.83 (d, J = 4.1 Hz, 8H, CONHCH ₂ ), 3.73 (m, 32H, PEG), 3.66 (t, J = 5.2 Hz, 8H, CONHCH ₂ CH ₂ ), 3.35 (t, J = 4.8 Hz, 8H, N ₃ CH ₂ ), -2.81 ( s, 2H, pyrrolic-NH).
FTIR (neat): 2099 cm ^-1 (ν _as N ₃ ), 1100 cm ^-1 (ν _as COC).

(2) Reduction of azide group (synthesis of compound d)
Triphenylphosphine (99 mg, 0.38 mmol) was weighed into a two-necked eggplant flask and dissolved in tetrahydrofuran (2 mL). This triphenylphosphine solution was added to a Schlenk tube in which compound c (60 mg, 0.038 mmol) was measured, and stirred at room temperature for 18 hours under nitrogen. Water (0.2 mL) was added to the reaction solution, and the mixture was again stirred at room temperature under nitrogen for 23 hours.

The obtained reaction liquid was concentrated with a rotary evaporator and vacuum-dried. The vacuum dried product was diluted with 0.5 M hydrochloric acid (50 mL) and washed 3 times with ethyl acetate. The aqueous layer was washed with 1N aqueous sodium hydroxide solution and saturated brine in this order, and extracted with chloroform. The chloroform layer was dehydrated with sodium sulfate and filtered, and the filtrate was concentrated on a rotary evaporator and dried in vacuo to obtain a dark purple gel material (compound d) (yield: 62.4 mg, quant.). This compound was identified from the measurement results of nuclear magnetic resonance spectroscopy ( ¹ H NMR) and mass spectrometry. The results are shown below.

¹ H NMR (500MHz, CDCl ₃ ): δ [ppm] = 8.82 (s, 8H, pyrrolic-β-CH), 8.27 (d, J = 8.7 Hz, 8H, phenylic CH), 8.24 (d, J = 8.0 Hz 8H, phenylic CH), 7.88 (t, J = 5.1 Hz, 4H, amido), 3.64-3.85 (m, 48H, PEG), 3.50 (t, J = 5.1 Hz, 8H, NH ₂ CH ₂ CH ₂ ) , 2.83 (t, J = 5.1 Hz, 8H, NH ₂ CH ₂₎ , 1.61 (s, 8H, NH ₂ ), -2.84 (s, 2H, pyrrolic-NH).
m / z (ESI): Calcd. for C ₈₀ H ₁₀₄ N ₁₂ O ₁₆ [M + 2H] ²⁺ 496.92; found 496.92, Calcd. for C ₈₀ H ₁₀₅ N ₁₂ O ₁₆ [M + 3H] ³⁺ 744.37; found 744.37.

2. Cancer cell killing effect Using the porphyrin derivative of the present invention as a photosensitizer, the killing effect of cancer cells by photodynamic therapy was examined. Specifically, the following procedure was used. A conventional porphyrin derivative, Mesotetra (4-N-methylpyridyl) porphine tetraiodide (hereinafter abbreviated as TMPyP, CAS # 36674-90-5) was used as an experimental control.

(1) Cancer cell culture
A cell culture medium (D-MEM, 1 × 10 ⁵ cells / mL, 100 μL / well) containing HeLa cells (RIKEN RCB007) was placed in a 96-well microplate and cultured in a cell culture incubator for 24 hours. A total of four similar plates were prepared.

(2) Preparation of photosensitizer solution The porphyrin derivative TAEP manufactured in Example 1 and a TMPyP DMSO solution (TAEP: 2.18 mM, TMPyP: 1.23 mM) were prepared. These DMSO solutions of porphyrin derivatives were diluted to 10 μM using D-MEM (without serum). Furthermore, these were serially diluted using D-MEM (without serum) to prepare 10 μM, 8 μM, 6 μM, 5 μM, 4 μM, 3 μM, 2.5 μM, 2 μM, and 1 μM porphyrin derivative-diluted media.

(3) Photodynamic treatment The 96-well microplate was taken out from the incubator, and all the medium was removed on a clean bench. Using a 12-pipetman, add porphyrin derivative-diluted medium (100 μL / well) to each well of a 96-well microplate, and include two 96-well microplates containing TAEP-diluted medium and TMPyP-diluted medium Two 96-well microplates were prepared. The prepared four 96-well microplates were cultured in an incubator for 24 hours.

  Select one from a 96-well microplate containing each porphyrin diluted solution, remove all medium from the selected 96-well microplate with a clean bench, wash twice with D-PBS (120 μL / well), and D-MEM ( Serum free, 100 μL / well) was added.

Two unwashed 96-well microplates and two washed 96-well microplates were transferred to a dark room. Using a xenon light source (manufactured by Asahi Spectroscope, MAX-303, λ = 375 to 700 nm), a 96-well microplate was irradiated with 400 ± 20 mW / m ² of light for 30 minutes and then cultured in an incubator for 20 hours. .

(4) Measurement of survival rate
The viability of cancer cells surviving in each well of a 96-well microplate was examined according to the MTT assay. First, an MTT solution (2.5 mg / mL, 20 μL / well) was added to each well, followed by culturing in an incubator for 3 hours, and all the medium was removed in a clean bench. Next, after adding a formazan solution (100 μL / well) to each well and pipetting, the absorbance at 570 nm was measured with a microplate reader (manufactured by Molecular Devices, OptiMax Tunable Microplate Reader). From the measured absorbance, the survival rate of the cancer cells was calculated. The result is shown in FIG.

(5) Summary As shown in FIG. 2, TAEP is highly cancerous killed by light irradiation even after washing cancer cells (TAEP + Wash in FIG. 2) and before washing cells (TAEP in FIG. 2). It showed an effect (cancer cells died nearly 100% at 1 μM or less) and was found to have high cell migration. In addition, in TMPyP, which is an experimental control, comparing cancer cells before washing (TMPyP in FIG. 2) and after washing (TMPyP + Wash in FIG. 2), IC ₅₀ = 1 μM to IC ₅₀ = 5 μM. The effect is decreasing.

Claims (8)

Porphyrin derivatives represented by the following general formula (I).

(In the formula, n is a natural number of 0 to 5, and R may be the same or different from each other.)   The porphyrin derivative according to claim 1, wherein n is 3.   The photosensitizer which contains the porphyrin derivative in any one of Claim 1 or Claim 2 as an active ingredient.   A fluorescence visualization agent comprising the porphyrin derivative according to claim 1 as an active ingredient. A metal complex represented by the following general formula (II).

(In the formula, n is a natural number of 0 to 5, and R may be the same or different from each other. M is any one of manganese, zinc, magnesium, tin, palladium, silver, and gold. Furthermore, X is any one of chlorine ion, bromine ion, nitrate ion, trifluoromethanesulfonate ion, hydroxide ion and acetate ion.)   6. The metal complex according to claim 5, wherein n is 3.   The photosensitizer which contains the metal complex of Claim 5 or Claim 6 as an active ingredient.   An MRI contrast agent comprising the metal complex according to claim 5 or 6 as an active ingredient.

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