WO2012029609A1 - Compound having silicon-containing substituent introduced thereto, and singlet oxygen-generating agent and cancer therapeutic comprising same - Google Patents

Abstract

A compound represented by any of general formulae (I) to (IV) is to be used as the active ingredient of a singlet oxygen-generating agent. In each of R₁ to R₂₀ in general formula (I), R₁ to R₂₄ in general formula (II), R₁ to R₂₈ in general formula (III) and R₁ to R₁₆ in general formula (IV), at least one is a member selected from substituents represented by general formula (i) and (ii) [in general formulae (i) and (ii), Ra, Rb and Rc independently represent a substituent selected from among hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, phenyl, methoxy, ethoxy and trimethylsilyl; and n is 1 or greater], and at least one is a substituent represented by general formula (a) [in general formula (a), Ra and Rb independently represent a substituent selected from among hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, phenyl, methoxy, ethoxy and trimethylsilyl; and m is 0 or greater].

Description

Compound having silicon-containing substituent introduced therein, singlet oxygen generator and cancer therapeutic agent containing the same

The present invention relates to a novel compound into which a silicon-containing substituent is introduced. The present invention also relates to a singlet oxygen generator and a cancer therapeutic agent comprising the compound.

Cancer is the biggest cause of death among Japanese people, and the development of treatments is strongly desired all over the world. Current main therapies include surgical removal of tumor tissue, long-term radiation therapy or anticancer drug treatment. If the tissue is excised, loss of function or decrease in function becomes a problem, and radiation therapy or anticancer drug treatment is a long-term therapy, and patients suffer from side effects for a long time. Therefore, there is a problem that the quality of life (QOL) of the patient is remarkably lowered.
Photodynamic therapy (PDT) is attracting attention as a new cancer treatment method for solving such problems. In this treatment, a photosensitizing dye, which is a drug, is injected once and irradiated with visible light, so that the tissue can be preserved and the burden on the patient is low. The principle of this therapy is as follows. 1. After waiting several hours after injecting the photosensitizing dye, the photosensitizing dye accumulates in the tumor. 2. When the tumor is selectively irradiated with red light, the accumulated photosensitizing dye is excited and singlet oxygen that is an active oxygen species is generated. 3. Singlet oxygen attacks and kills surrounding cancer cells.

Currently, several photosensitizing dyes have been reported ( Patent Documents 1, 2, Non-Patent Document 1, etc.), and some have been approved and already clinically applied. However, the performance of the photosensitizing dye is not sufficient, Patients are forced to live in a dark room for several weeks after treatment to prevent the side effect of photosensitivity. At the present stage, sufficient effects cannot be obtained for advanced cancer. Therefore, further improvement of the photosensitizing dye is demanded.
The main items that need improvement are: 1. selective accumulation in tumor; 2. Photosensitizing dye uptake efficiency by cells; 3. Light absorption efficiency for red light It is the production efficiency of singlet oxygen. The present inventors have found that the efficiency of singlet oxygen generation is improved by introducing a silicon substituent into porphyrin derivatives, which are typical photosensitizing dyes, and have filed patent applications (patents). Reference 3).

JP 2002-523509 A JP 2004-002438 A No. 2007/023766

The object of the present invention is to further improve the performance of photodynamic therapy, which is currently desired to be improved. Specifically, in addition to the generation efficiency of singlet oxygen, it is an object to improve the selective accumulation in a tumor and the photosensitizing dye uptake efficiency by cells.

The present inventor has intensively studied to solve the above problems. As a result, COO with a substituent containing a silicon compound such as porphyrin analogues and phthalocyanine derivatives ^- by introducing a group, not only the efficiency of generation of singlet oxygen is increased, the uptake and tumor tissues to cells As a result, it has been found that these compounds can be suitably used as singlet oxygen generators useful for cancer treatment and the like, and the present invention has been completed.

That is, the present invention is as follows.
(1) A compound represented by any one of the following general formulas (I) to (IV).
(2) A compound represented by the following (I-1), (II-1) or (III-1).
(3) A singlet oxygen generator containing the compound of (1) or (2).
(4) A cancer therapeutic agent comprising the singlet oxygen generator (3).
(5) A compound represented by any one of formulas (I) to (IV) for cancer treatment.
(6) A method for treating cancer, comprising a step of administering a compound represented by any one of the general formulas (I) to (IV) to a patient.
(7) Use of a compound represented by any one of formulas (I) to (IV) for generating singlet oxygen.
(8) A method for generating singlet oxygen, comprising a step of irradiating a compound represented by any one of the general formulas (I) to (IV) with light.

The compound of the present invention is 1. 1. Singlet oxygen production efficiency; 2. Uptake efficiency by cells; The three performances of selective accumulation in tumors are improved at the same time, and the cancer treatment effect is remarkably improved. Since the compound of the present invention can be excited even by visible light of 600 to 800 nm, it has a long penetration distance into the human body and has the advantage that singlet oxygen can be generated even in the deep part.

Singlet oxygen spectrum of SiC ₄ and C ₄ . Shows the results of light irradiation after administration C ₄ (left) or SiC ₄ (right) tumor-bearing nude mice (photograph). The SiC ₄ was administered, shows the time course of tumor size after irradiation. It shows the fluorescence images of cancer cells cultured in a medium containing the drug (C _4, SiC ₄ or SiS ₄₎ (photograph). Agent (C _4, SiC ₄ or SiS ₄₎ shows changes in the cell survival rate when subjected to light irradiation in cancer cells cultured in a medium containing. Fluorescence image (photograph) of a tumor-bearing nude mouse intravenously injected with a drug (C ₄ , SiC ₄ or SiS ₄ ). Cancer tissue tumor-bearing nude mice, muscle, shows the time course of concentration of SiC ₄ compound in the blood.

The present invention is described in detail below.
In the present invention, the “singlet oxygen generator” refers to a substance that can generate singlet oxygen when irradiated with light.
The singlet oxygen generator of the present invention contains one or more compounds selected from the group of compounds represented by the following general formulas (I) to (IV).

In the general formula (I), at least one of R ₁ to R ₂₀ (preferably 3 or more, more preferably 3 to 8, particularly preferably 4 to 8) is independent of the following (i) to (ii) And at least one of R ₁ to R ₂₀ (preferably 4 or more, more preferably 4 to 8) is the substituent of the following (a).
In the general formula (II), at least one of R ₁ to R ₂₄ (preferably 3 or more, more preferably 3 to 8, particularly preferably 4 to 8) is independent of the following (i) to (ii) And at least one of R ₁ to R ₂₄ (preferably 4 or more, more preferably 4 to 8) is the substituent of the following (a).
In the general formula (III), at least one of R ₁ to R ₂₈ (preferably 3 or more, more preferably 3 to 8, particularly preferably 4 to 8) is independent of the following (i) to (ii) And at least one of R ₁ to R ₂₈ (preferably 4 or more, more preferably 4 to 8) is the substituent of the following (a).
In the general formula (IV), at least one of R ₁ to R ₁₆ (preferably 3 or more, more preferably 3 to 8, particularly preferably 4 to 8) is independent of the following (i) to (ii) And at least one of R ₁ to R ₁₆ (preferably 4 or more, more preferably 4 to 8) is the substituent of the following (a).
In (i) and (ii), (i) is more preferable.

In the general formulas (i) to (ii), Ra, Rb and Rc are independently a substituent selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, phenyl, methoxy, ethoxy and trimethylsilyl. It is. N is an integer of 1 or more, preferably an integer of 1 to 5.

In the general formula (a), Ra and Rb are each independently a substituent selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, phenyl, methoxy, ethoxy, and trimethylsilyl. M is an integer of 0 or more, preferably an integer of 0 to 5.
Note that COO ⁻ may be coordinated with ions such as Na ⁺ , K ⁺ , Mg ²⁺ , and Ca ^2+, or may be bonded with a hydrogen atom.
In the compounds of the above general formulas (I) to (IV), at least one of R _n (n is an integer of 1 to 20, 1 to 24, 1 to 28, and 1 to 16, respectively) is (i) to ( ii) is a substituent selected from the above, and at least one of them may be the substituent of (a), and other R _n is hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, phenyl, It is a substituent selected from methoxy, ethoxy and trimethylsilyl.

The compound represented by the general formula (I) can be synthesized, for example, as in Synthesis Example 1 described later.
The compound represented by the general formula (II) can be synthesized, for example, as in Synthesis Example 2 described later.
The compound represented by the general formula (III) can be synthesized, for example, as in Synthesis Example 3 described later.

In the compounds represented by the general formulas (I) to (III), the substituents independently selected from (i) to (ii) at the 3-position and 5-position of at least one benzene ring; It is particularly preferable that the above substituent is introduced.

The compound represented by the general formula (IV) can be synthesized, for example, as follows.

As a particularly preferred form of the singlet oxygen generator of the present invention, compounds represented by the following formulas (I-1), (II-1) and (III-1) can be mentioned.

The singlet oxygen generator of the present invention is a compound selected from the above general formulas (I) to (IV) as it is or pharmacologically according to known means generally used in the production of pharmaceutical preparations. It can be mixed with an acceptable carrier and administered orally or parenterally (eg, topical, intravenous administration, etc.) as a pharmaceutical preparation such as an injection. The compounds of general formulas (I) to (IV) are easy to accumulate in cancer tissue and may be administered alone, but they reach the target tissue by a drug delivery system (DDS) method using liposomes. It may be administered in a form that is easy to do.
As the compounds of the general formulas (I) to (IV), those in which a metal such as Zn ²⁺ , Cu ²⁺ (II), Ca ²⁺ , Mg ²⁺ is bonded to the center of the ring can also be used.

The content of the compounds of the general formulas (I) to (IV) in the preparation is about 0.01 to about 100% by weight of the whole preparation. The dose of the compounds of the general formulas (I) to (IV) varies depending on the administration subject, target organ, symptom, administration method, etc., and is not particularly limited, but is generally about 0.1 to 1000 mg per administration. , Preferably about 1.0 to 100 mg.

Examples of pharmacologically acceptable carriers include excipients, lubricants, binders and disintegrants in solid formulations, or solvents, solubilizers, suspending agents, isotonic agents, buffers in liquid formulations. And soothing agents. If necessary, additives such as conventional preservatives, antioxidants, colorants, sweeteners, adsorbents, wetting agents and the like can be used in appropriate amounts.

In order to generate singlet oxygen and use it for photodynamic therapy, light of an appropriate wavelength is irradiated after administration of the general formulas (I) to (IV).
The irradiation time is appropriately selected depending on the age and sex of the patient, the type and degree of the disease, the distance between the light source and the affected part, and the like. The wavelength is not particularly limited as long as it is a wavelength capable of generating singlet oxygen, but 600 to 800 nm is preferable when performing photodynamic therapy. Irradiation may be performed from outside the body, or may be performed by inserting an optical fiber or the like in the vicinity of the target tissue. Also included are embodiments in which bone marrow is removed from a leukemia patient, treated with compounds of general formulas (I)-(IV) in vitro, and the treated bone marrow is returned to the patient.

The cells can be destroyed by generating singlet oxygen. Therefore, the disease to which the singlet oxygen generator of the present invention can be applied is not particularly limited as long as it can be treated by generating singlet oxygen and destroying cells. Examples include diseases, transplant rejection, autoimmune diseases and T cell-mediated immune allergy, bacterial infections, viral infections, age-related macular degeneration, acne and the like. Of these, cancer is particularly preferred. The type of cancer is not particularly limited, but lung cancer, malignant lymphoma (eg, reticulosarcoma, lymphosarcoma, Hodgkin's disease, etc.), digestive cancer (eg, gastric cancer, gallbladder / bile duct cancer, pancreatic cancer, liver cancer, colon cancer, rectum) Cancer), breast cancer, ovarian cancer, disseminated multiple myeloma, bladder cancer, leukemia (for example, acute leukemia including acute transformation of chronic myeloid leukemia), kidney cancer, prostate cancer and the like.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to the following examples.

Synthesis example 1
<Synthesis of 5,10,15,20-tetrakis (3-carboxyl-5-trimethylsilylphenyl) porphyrin sodium salt>
5,10,15,20-Tetrakis (3-carboxyl-5-trimethylsilylphenyl) porphyrin sodium salt (hereinafter sometimes referred to as SiC ₄ ) was synthesized by the following procedure.
1,3,5-Tribromobenzene was lithiated with an equal amount of n-butyllithium in dry diethyl ether at -78 ° C. and then reacted with chlorotrimethylsilane to give 95% yield in 3,5- Dibromotrimethylsilylbenzene was obtained.
This was then lithiated with an equal amount of n-butyllithium in dry diethyl ether at −78 ° C. and then reacted with DMF to give 3-bromo-5-trimethylsilylbenzaldehyde in 45% yield. .
This was dissolved in dry toluene and reacted with ethylene glycol in the presence of p-toluenesulfonic acid to obtain 3-bromo-5-trimethylsilylbenzaldehyde ethylene acetal in 90% yield.
Next, this was reacted with magnesium in dry THF and then with carbon dioxide at 0 ° C. to obtain 3-carboxy-5-trimethylsilylbenzaldehyde ethylene acetal in a yield of 40%. This was treated with hydrochloric acid to obtain 3-carboxy-5-trimethylsilylbenzaldehyde.
Next, this was reacted with ethyl iodide in DMF in the presence of potassium carbonate to obtain 3-carboxy-5-trimethylsilylbenzaldehyde ethyl ester.
This was reacted with pyrrole and boron fluoride ether complex in chloroform, and then treated with DDQ to obtain 5,10,15,20-tetrakis (3-carboxyl-5-trimethylsilylphenyl) porphyrin ethyl ester.
Finally, this was treated with sodium hydroxide to obtain 5,10,15,20-tetrakis (3-carboxyl-5-trimethylsilylphenyl) porphyrin sodium salt.

¹ H NMR data of 5,10,15,20-tetrakis (3-carboxyl-5-trimethylsilylphenyl) porphyrin is shown below.
δH (300 MHz, CD ₃ CN) 9.40 (8H, s, pyrrole β-H), 9.35 (4H, s, ph-H), 9.19 (4H, s, ph-H), 9.16 (4H, s, ph -H), 0.99 (36H, s, Me) and -2.29 (2H, s, NH).

Synthesis example 2
<Synthesis of 5,10,15,20-tetrakis (3-carboxyl-5-trimethylsilylphenyl) chlorine sodium salt>
5,10,15,20-Tetrakis (3-carboxyl-5-trimethylsilylphenyl) porphyrin ethyl ester is treated with p-toluenesulfonyl hydrazide and potassium carbonate in dry pyridine, followed by treatment with chloranil. 15,20-Tetrakis (3-carboxyl-5-trimethylsilylphenyl) chlorin ethyl ester was obtained. This was treated with sodium hydroxide to obtain 5,10,15,20-tetrakis (3-carboxyl-5-trimethylsilylphenyl) chlorin sodium salt.

Synthesis example 3
<Synthesis of 5,10,15,20-tetrakis (3-carboxyl-5-trimethylsilylphenyl) bacteriochlorin sodium salt>
5,10,15,20-Tetrakis (3-carboxyl-5-trimethylsilylphenyl) porphyrin ethyl ester is treated with p-toluenesulfonyl hydrazide and potassium carbonate in dry pyridine to give 5,10,15,20-tetrakis ( 3-carboxyl-5-trimethylsilylphenyl) bacteriochlorin ethyl ester was obtained. This was treated with sodium hydroxide to obtain 5,10,15,20-tetrakis (3-carboxyl-5-trimethylsilylphenyl) bacteriochlorin sodium salt.

In the following examples using the compound C ₄ below for comparison.

Example 1
<Generation of singlet oxygen>
Each compound of C ₄ and SiC ₄ was dissolved in ethanol at a concentration of 0.1, and then irradiated with 355 nm light using YAG LASER (Lotis TII LS-2137U). For the measurement of singlet oxygen, a self-made infrared emission measuring device using a near-infrared photomultiplier tube (Hamamatsu Photonics R5509-42) was used. The measurement was performed 17 times, and the average value is shown in FIG.
As a result, the phosphorescence spectrum of singlet oxygen sensitized by the compound was measured at the position of λmax = 1270 nm. The quantum yield Φ for singlet oxygen generation of SiC ₄ was 0.72, 1.29 times that of C ₄ . As a result, it was found that the singlet oxygen generation efficiency was increased by introducing a substituent containing Si into C ₄ .
In addition, when argon gas (Ar) was injected into the ethanol solution, almost no phosphorescence was generated, which confirmed that the observed spectrum was due to singlet oxygen.

Example 2
<Therapeutic effects of cancer in animals bearing cancer>
Silicon substituents developed using nude mice inoculated with cancer cells (SCC7: Shaojuan Zhang, Masahiro Hosaka, Toshitada Yoshihara, Kazuya Negishi, Yasuhiko Iida, Seiji Tobita, and Toshiyuki Takeuchi, Cancer Research, 2010, 70, 4490.) 4 hours after intravenous injection of the therapeutic agent SiC ₄ (100 nmol) (400-800 nm, 28 mW / cm ² , 30 min), the tumor site disappeared after 6 days, It became a scab (Figure 2). In compound C ₄ having no silicon substituent, such discoloration was hardly observed. The time course of tumor size after treatment is shown in FIG. Tumor size increases over C ₄ In time, the SiC ₄ tumors disappeared. As described above, the performance as a therapeutic agent was greatly improved by the introduction of the silicon substituent.

Example 3
<Evaluation of uptake into cancer cells>
FIG. 4 shows fluorescence images of cancer cells (U251) cultured for 12 hours in a medium containing 15 μM SiC ₄ or C ₄ . Strong fluorescence was observed with SiC ₄ , but almost no fluorescence was observed with C ₄ . Since the fluorescence quantum yield (0.054) of SiC ₄ is almost the same as that of C ₄ (0.046), it was found that introduction of silicon substituents significantly improved the uptake efficiency by cells. In addition, SiC ₄ shows stronger fluorescence than the following compound (SiS ₄ ) described in the reissued 2007/023766 publication. Fluorescence quantum yield of SiS ₄ is 0.059, since a SiC ₄ equivalent quantum yield, towards the SiC ₄ is present compounds than SiS ₄ was found to be easily taken into the cancer cells.

Example 4
<Effects on cultured cancer cells>
The activity of drugs was evaluated at the level of cultured cells using cancer cells (U251: Masanori Aihara, Ken-ichi Sugawara, Seiji Torii, Masahiro Hosaka, Hideyuki Kurihara, Nobuhito Saito and Toshiyuki Takeuchi, Laboratory Investigation (2004) 84, 1581). However, SiC ₄ with a silicon substituent introduced (15 μM) was able to kill almost 100% of the cancer cells even with a slight light irradiation dose (8 J / cm ² ), but with C ₄ without a silicon substituent, Even if light irradiation of the same degree was performed, the cancer cells could hardly be killed (FIG. 5). From this result, it can be seen that the introduction of the silicon substituent improves both the singlet oxygen generation efficiency and the cellular uptake efficiency, and increases the drug efficiency. In addition, when comparing the activity of SiC ₄ against cultured cancer cells with that of SiS ₄ , it was also found that SiC ₄ has an activity approximately 6 times that of SiS ₄ .

Example 5
<Evaluation of tumor accumulation in cancer-bearing animals>
Furthermore, in order to clarify the tumor accumulation property of the drug by the silicon substituent, the localization of the drug was evaluated using a tumor bearing nude mouse (FIG. 6). In FIG. 6, the tumor site is surrounded by a circle. SiS ₄ (100 nmol) was intravenously injected from the tail of the mouse, and the fluorescence image after 2 hours is shown in the lower part of FIG. SiS ₄ remained in the tail and was found not transported throughout the body. Furthermore, 6 hours after the administration, the mouse died and was toxic. Therefore, it became clear that SiS ₄ cannot be used for photodynamic therapy. In contrast, SiC ₄ and C ₄ spread throughout the body when injected intravenously, are not toxic, and have solved the problems of SiS ₄ . The fluorescence images 24 hours after intravenous injection of C ₄ and SiC ₄ are shown in FIG. In C ₄ , almost no fluorescence due to the drug was observed from the tumor part, but in SiC ₄ , strong fluorescence was observed from the tumor. From this, it was found that the tumor accumulation was improved by the introduction of the silicon substituent.

Moreover, in FIG. 7, the accumulation property of SiC ₄ in each tissue in the cancer-bearing mouse was quantitatively evaluated. As a result, assuming that the concentration in the tumor was 1 at 12 hours after administration, the concentration in the muscle was 0.25, the concentration in the blood was 0.46, and it was found that SiC ₄ was selectively collected in the tumor.

From the above results, the problem of SiS ₄ described in Table 2007/023766 was solved by changing the sulfonic acid group to a carboxyl group. Furthermore, it was found that by introducing a silicon substituent into the drug, the generation efficiency of singlet oxygen, the uptake efficiency by cells, and the tumor accumulation were improved, and the therapeutic effect of cancer was improved.

The compound introduced with a silicon-containing substituent of the present invention can be suitably used for photodynamic therapy used for phototherapy of cancer.

Claims (4)

1. A compound represented by any one of the following general formulas (I) to (IV).
   

   

   R ₁ to R ₂₀ in the general formula (I), R ₁ to R _{24 in} the general formula (II), R ₁ to R ₂₈ in the general formula (III), and R ₁ to R ₁₆ in the general formula (IV). In the formula, at least one is a substituent selected from the following (i) or (ii), and at least one is a substituent (a) below.
   

   

   In general formula (i) or (ii), Ra, Rb, and Rc are each independently a substituent selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, phenyl, methoxy, ethoxy, and trimethylsilyl. is there. N is an integer of 1 or more.
   

   

   In the general formula (a), Ra and Rb are each independently a substituent selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, phenyl, methoxy, ethoxy, and trimethylsilyl. M is an integer of 0 or more.
2. The compound of Claim 1 which is the following compound.
   

   
3. A singlet oxygen generator comprising the compound according to claim 1 or 2.
4. A cancer therapeutic agent comprising the singlet oxygen generator according to claim 3.

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