JP6844773B2 - Boron-containing dendrimer with intracellular transfer function - Google Patents

Description

The present invention relates to a novel boron-containing dendrimer having an intracellular introduction function and a method for producing the same. More specifically, the present invention relates to a novel boron-containing dendrimer used in Boron Neutron Capture Therapy (BNCT) and a method for producing the same.

Boron neutron capture therapy (BNCT) is a treatment that uses the reaction of neutrons with boron, which has a sensitizing effect, to selectively destroy only tumor cells without causing much damage to normal cells. Specifically, a boron compound is introduced into the tumor cell of the tumor part of the patient, and a low-energy thermal neutron beam or extrathermal neutron beam that is harmless to the human body is irradiated to the boron compound, and a boron ( ¹⁰ B) atom is introduced inside the tumor cell. Causes a nuclear reaction in the tumor and kills the tumor. The fission reaction produces alpha particles and lithium ( ⁷ Li) nuclei ( ¹⁰ B + 1n → ⁷ Li + 4He + 2.4MeV), but their range is less than 10 μm and shorter than the cell diameter, so neutron rays. The effect of is limited to the inside of cells containing ^{10 B.}

Currently, boron compounds are in clinical applications, the following boron phenylalanine (BPA: borono-L-phenylalanine ) and, dodeca Boranchi ol (BSH: disodium mercaptoundecahydrododecaborate, [B 12 H 11 SH] 2- 2Na +) of two Is.

BPA is a boron compound in which one ¹⁰ B atom is bonded to phenylalanine, which is an essential amino acid. BPA crosses the blood-brain barrier and spreads throughout the brain, especially in brain tumors with high amino acid metabolism. BPA is more efficiently taken up by tumor cells compared to normal cells. BPA is, ¹⁸ F has been developed ¹⁸ F-BPA bound, are utilized in the treatment before the test.

(±) -p-boronophenylalanine-conjugated polyamideamine dendrimer (0th generation) to provide boron compounds with high selectivity to tumor cells, high accumulation, high water solubility, and low cytotoxicity to normal cells. ) And (±) -p-boronophenylalanine-linked polyamideamine dendrimer (1st generation) have been reported (see Examples 1 and 2 of Patent Document 1).

^{BSH is a boron compound containing 12 10} B atoms in the molecule and having an icosahedron chemical structure. BSH itself is usually not selectively taken up by tumor cells. However, BSH accumulates due to the EPR (enhanced permeability and retention) effect in brain tumor cells with a broken blood-brain barrier. BSH is highly water soluble and less toxic. BSH is usually not used except for brain tumor cells because it is not naturally taken up into cells and there is no method for imaging pharmacokinetics such as PET (see Non-Patent Document 1).

Japanese Unexamined Patent Publication No. 2006-96870

Kawabata S. et al., "Boron neutron capture therapy for newly diagnosed glioblastoma", J Radiat Res. 2009 Jan; 50 (1): 51-60. Epub 2008 Oct 29 ..

The ratio of BPA to ¹⁰ B per molecule is originally low, 4.7% by weight. A large amount of BPA is required to obtain a therapeutic effect. Furthermore, since BPA is also taken up by normal cells in a small amount, it is necessary to perform neutron irradiation in consideration of the fact that BPA is taken up by normal cells. And since BPA has insufficient uptake of amino acids into cells with low metabolism, for example, cells called tumor stem cells, which have low cell division ability but high tumorigenicity (that is, the source of tumor formation). Insufficient uptake into cells). Low uptake into tumor stem cells and subsequent neutron killing is thought to be the cause of tumor recurrence.

^{The proportion of 10} B of the boron compound of Example 1 described in Patent Document 1 is about 3.5% by weight, and ^{the proportion of 10} B of the boron compound of Example 2 is about 3.1% by weight. These ¹⁰ B proportions need further improvement.
Further, regarding the incorporation of the boron compound of Patent Document 1 Example 2 into Ihara cancer cells, the boron concentration is about 1.1 ppm, and further improvement is required (Patent Document 1 Example 2 and). (See FIG. 2).
Therefore, a new boron compound different from the boron compound of Patent Document 1 is required.

On the other hand, BSH usually does not have the ability to be taken up into tumor cells, but ^{the ratio of 10} B per molecule of the compound is 57.4% by weight, which ^{is higher than that of 10} B, and is highly water-soluble. It is sex and has low toxicity.
Therefore, it is expected to develop a novel boron compound which can be synthesized by an inexpensive and simple method, contains BSH as a basic structure, and has an ability to be taken up into tumor cells. Furthermore, it is expected to clarify the ability of the boron compound to be taken up by tumor cells, cytotoxicity, and the like.

As a result of diligent studies, the present inventors have found a dendrimer containing a core portion, a dendritic branch portion, and a terminal portion, and a divalent aliphatic hydrocarbon group in which at least one oxygen is inserted in the core portion. Certain dendrimers, which contain an amidoamine structure at the dendritic bifurcation and an undecahydrododecaborate structure at the end, are surprisingly relatively easy to synthesize and have low toxicity. Nevertheless, it was found to have a high ability to be taken up by cells and to show a high intracellular boron concentration.

The present invention provides, in one gist, a novel dendrimer containing boron.
A dendrimer that includes a core, a dendritic bifurcation, and an end.
The core contains at least one oxygenated divalent aliphatic hydrocarbon group.
The dendritic bifurcation contains an amidoamine structure and contains
The end contains an undecahydrododecaborate structure.

The present invention provides, in one embodiment, a dendrimer in which the core contains a divalent aliphatic hydrocarbon group having 4 to 100 carbon atoms into which oxygen of 1 to 49 is inserted.
The present invention provides, in another aspect, a dendrimer in which the core portion has the structure represented by the formula (2).
[Here, R ¹ is a divalent aliphatic hydrocarbon group having 2 to 20 carbon atoms, and R ¹ may be the same or different from each other. ]
In a preferred embodiment of the present invention, the dendritic bifurcation is represented by the formula (3): -N [R ^2- CONH-R ^2- ] ₂ [where R ² is a divalent fat having 2 to 12 carbon atoms. It is a group hydrocarbon group, and R ² may be the same or different from each other. ] To provide a dendrimer including the structure indicated by.

In another gist, the present invention provides a complex of the above-mentioned dendrimer and carbon nanotube.
The present invention provides, in a preferred gist, a boron preparation comprising the aforementioned dendrimer and / or the aforementioned complex. Boron preparations can be preferably used for BNCT.

Since the dendrimer of the form of the present invention has the above-mentioned characteristics, it can be synthesized by a relatively simple method, has a high uptake ability into cells despite its low toxicity, and exhibits a high intracellular boron concentration. ..

1A and 1B (FIG. 1B is a continuation of FIG. 1A) show a synthetic scheme of a dendrimer 9 having a 1,10-bis (decyloxy) decane in its core. 2A and 2B show scanning electron microscope (SEM) images of an aqueous solution of dendrimer 9. The acceleration voltage is 5.0KV for both. FIG. 2A is 80,000 times, and FIG. 2B is 40,000 times. FIG. 3 shows an ultraviolet-visible near-infrared spectrum of a complex 10 of a dendrimer 9 and a carbon nanotube. FIG. 4 shows the Raman spectrum of the complex 10 of the dendrimer 9 and the carbon nanotube. FIG. 5 shows the fluorescence spectrum of the complex 10 of the dendrimer 9 and the carbon nanotube. FIG. 6 shows an atomic force microscope (AFM) image of a complex 10 of a dendrimer 9 and a carbon nanotube. FIG. 6A shows a range of 3.5 × 3.5 μm, and the fibrous complex 10 was observed. FIG. 6B shows a height profile in the range of 2.8 to 4.3 nm. FIG. 7 shows the results of the cytotoxicity test (WST-1 assay). Dendrimer 9 at concentrations of 1, 10, 50, 100, 250 μM, complex 10 at concentrations of 100, 250 μM, or Control (DMSO) with the malignant brain tumor cell line U87 delta EGFR for 24 hours ( After culturing for 48 hours (left figure) or 48 hours (right figure), cytotoxicity was measured using the WST-1 cell proliferation assay with absorbance (450 nm-690 nm). The data are mean ± standard deviation (n = 5). FIG. 8 shows the results of intracellular boron concentration measurement (ICP). Left figure: With the malignant brain tumor cell line U87 delta EGFR, the intracellular boron concentration was measured after culturing dendrimer 9 at a concentration of 5, 50, 250 μM and complex 10 at a concentration of 250 μM for 24 hours. Right figure: Dendrimer 9 was cultured with U87 delta EGFR at a concentration of 250 μM for 6 hours, 12 hours, and 24 hours, and then the intracellular boron concentration was measured. The data is the mean (n = 6).

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited by these forms.

The present invention is described in one gist.
A dendrimer that includes a core, a dendritic bifurcation, and an end.
The core contains at least one oxygenated divalent aliphatic hydrocarbon group.
The dendritic bifurcation contains an amidoamine structure and contains
The end provides a dendrimer containing an undecahydrododecaborate structure.

The dendrimer of the embodiment of the present invention can be represented by the following formula (1).
In the formula (1), the core portion corresponds to A, dendritic branching unit, two C combined with B and B, i.e., corresponds to BC ₂ structure, end corresponds to D.

The core of the dendrimer contains a divalent aliphatic hydrocarbon group into which at least one oxygen has been inserted (provided that when multiple oxygens are inserted, there are at least two carbon atoms between the oxygens. To do). From the viewpoint of biocompatibility, cell uptake ability, solubility in blood, etc., the chemical structure of the core part is preferably moderately flexible, and the core part is a chain-like 2 in which oxygen is inserted. It preferably contains a valent aliphatic hydrocarbon group.
Since the core part has better biocompatibility, cell uptake ability, solubility in blood, etc., a divalent aliphatic hydrocarbon group containing 4 to 100 carbon atoms into which 1 to 49 oxygens are inserted is used. It is preferably contained, more preferably it contains a divalent aliphatic hydrocarbon group containing 8 to 80 carbon atoms with 1 to 19 oxygen inserted, and 12 to 60 carbon with 1 to 9 oxygen inserted. It is even more preferable to contain a divalent aliphatic hydrocarbon group containing an atom, and even more preferably to contain a divalent aliphatic hydrocarbon group containing 16 to 56 carbon atoms into which 1 to 6 oxygens are inserted. It is particularly preferable to contain a divalent aliphatic hydrocarbon group containing 20 to 50 carbon atoms into which ~ 4 oxygen is inserted.

The above-mentioned divalent aliphatic hydrocarbon group preferably contains a linear or branched alkylene group. With the insertion of at least one oxygen, the divalent aliphatic hydrocarbon group is divided into at least a plurality of divalent aliphatic hydrocarbon groups having a lower number of carbon atoms, but they are one kind of more carbon. It may be a divalent aliphatic hydrocarbon group having a small number, or it may be a plurality of types of divalent aliphatic hydrocarbon groups having a smaller number of carbon atoms.

As a divalent aliphatic hydrocarbon group having a smaller number of carbon atoms (without oxygen inserted) in the core part of the dendrimer, for example, an ethylene group, a propylene group, a butylene group, an isobutylene group, a pentylene group, a hexylene group and a heptylene Examples thereof include a group, an octylene group, a nonylene group, a decylene group, an undecylene group, a dodecylene group, a tridecylene group, a tetradecylene group, a pentadecylene group, a hexadecylene group, a heptadecylene group, an octadecylene group, a nonadesilene group, and an eicosilene group.
The divalent aliphatic hydrocarbon group (without oxygen inserted) in the core of the dendrimer is at least one selected from an octylene group, a nonylene group, a decylene group, an undecylene group, a dodecylene group, and a tridecylene group. It is preferable to include it.

The core portion of the dendrimer may have a structure represented by the following formula (2) as the above-mentioned divalent aliphatic hydrocarbon group into which at least one oxygen is inserted.
Here, R ¹ is a divalent aliphatic hydrocarbon group having 2 to 20 carbon atoms, and R ¹ may be the same or different from each other. The ^{number of carbon atoms contained in R 1} is preferably 4 or more, more preferably 6 or more, and 8 or more because biocompatibility, cell uptake ability, solubility in blood, etc. are further improved. It is particularly preferable to have.
Although n is an integer of 1 to 20, the total number of carbon atoms in the carbon chain contained in the formula (2) is 4 because biocompatibility, cell uptake ability, solubility in blood, etc. are further improved. It is preferably ~ 100. The total number of carbon atoms in the carbon chain contained in the formula (2) is preferably 8 to 80, more preferably 12 to 60, further preferably 16 to 56, and 20 to 50. It is particularly preferable to have. n is preferably 1 to 49, more preferably 1 to 19, further preferably 1 to 9, further preferably 1 to 6, and further preferably 1 to 4. More preferred.

R ¹ group according to formula (2) may be a single type of divalent aliphatic hydrocarbon group or may be a more divalent aliphatic hydrocarbon group. Among the formula (2), R ¹ groups need not be a single divalent aliphatic hydrocarbon group.

As R ¹ group according to formula (2), for example, ethylene, propylene, butylene, isobutylene group, pentylene group, hexylene group, heptylene group, octylene group, nonylene group, decylene group, undecylene group, dodecylene group, Examples thereof include a tridecylen group, a tetradecylen group, a pentadecylene group, a hexadecylene group, a heptadecylene group, an octadecylen group, a nonadesilene group, and an eicosilene group.
The R ¹ group preferably contains at least one selected from an octylene group, a nonylene group, a decylenic group, an undecylenic group, a dodecylenic group, and a tridecylenic group.

The dendrimer dendrimer in the form of the present invention contains an amidoamine structure.
It is preferable to include a chain amidoamine structure because the ease of synthesis, biocompatibility, solubility in blood and the like are further improved.
The dendritic bifurcations may be repeatedly joined. By being repeatedly bonded, the number of terminal portions increases to 4, 8, 16, ....
As long as the dendrimer intended by the present invention can be obtained, the number of repetitions is not limited and may not be repeated.
Furthermore, not all (two for the dendrimer of formula 1) need to be repeated, but some (one for the dendrimer of formula 1) are repeated. Is also good.

The tree-shaped branching portion can include, for example, a structure represented by the following formula (3).
(3): -N [R ^2- CONH-R ^2- ] ₂
Here, R ² is a divalent aliphatic hydrocarbon group having 2 to 12 carbon atoms, and R ² may be the same or different from each other. The ^{number of carbon atoms contained in R 2} is preferably 2 to 9 and more preferably 2 to 5 because biocompatibility, cell uptake ability, solubility in blood and the like are further improved. It is particularly preferable that it is ~ 3.

Dendritic branching unit, for _{example, -N [C 2 H 4 -CONH} -C 2 H 4 -] 2, -N [C 3 H 6 -CONH-C 3 H 6 -] 2, -N [C 4 H _{8 -CONH-C 4 H 8 -} ] 2, -N [C 5 H 10 -CONH-C 5 H 10 -] 2, and _{-N [C 6 H 12 -CONH-} C 6 H 12 -] 2, from It preferably contains the structure of choice.
Dendritic _{bifurcation, -N [C 2 H 4 -CONH} -C 2 H 4 -] 2, and _{-N [C 3 H 6 -CONH-} C 3 H 6 -] to include a structure selected from ₂ Is more preferable.

The end contains an undecahydrododecaborate structure [B ₁₂ H ₁₁ ] ^2- as a structure containing boron. Since the terminal part contains an undecahydrododecaborate structure, the dendrimer of the form of the present invention is expected to have a high intracellular boron concentration despite its low toxicity.
Undecahydrododecaborate structure [B ₁₂ H ₁₁ ] ^2- Contains, the dendrimer of the present invention can be obtained, and the end as long as the dendritic bifurcation and the undecahydrododecaborate structure can be connected. The other structure of the part is not particularly limited.

Other structures at the end for connecting to the dendritic bifurcation include, for example, -NHCO-C ₃ H _6- NC ₄ O ₂ H ₃ -S-, -CO-S-, and -CONH-C ₃ H. ₆ -S- and the like can be exemplified, and it is preferable that the other structure at the terminal portion includes a structure selected from these.

As long as the desired dendrimer containing boron can be obtained, the production method thereof is not particularly limited. The boron-containing dendrimer of the embodiment of the present invention can be produced, for example, as follows, as can be seen from Examples described later and FIGS. 1A and 1B.
_{For example, a diamine compound 5 [NH 2-} C ₁₀ H ₂₀ O-C ₁₀ H ₂₀ O-C] having amino groups at both ends of a divalent aliphatic hydrocarbon group having 30 carbon atoms in which two oxygen atoms are inserted. ₁₀ H _20- NH ₂ ] is prepared. Methyl acrylate was reacted with this to form a tetraester compound 6 [(CH ₃ OCO-C ₂ H ₄ ) ₂ N-C ₁₀ H ₂₀ O-C ₁₀ H ₂₀ O-C ₁₀ H _20- N (C _2). H _4- COO-CH ₃ ) ₂ ] is obtained. Next, reacted with diethylamine, tetraamine compound _{7 [(NH 2 -C 2 H} 4 -NHCO-C 2 H 4) 2 N-C 10 H 20 O-C 10 H 20 O-C 10 H 20 -N (C ₂ H _4- CONH-C ₂ H _4- NH ₂ ) ₂ ] is obtained. Then, an N-succinimidyl group is attached to the amino group, and the N-succinimidyl group _{is reacted with mercaptoundecahydrododecaborate (Na 2} ¹⁰ B ₁₂ H ₁₁ SH) to obtain a dendrimer 9 containing boron. it can.

By repeating the reaction with diethylamine and the subsequent reaction with methyl acrylate, the dendritic branch can be repeatedly introduced. For example, it is possible to obtain a dendrimer containing boron _{in which the BC 2} structure in the formula (1) is repeated. The number of end portions D can be increased as the number of repetitions of this dendritic branch portion increases.
^{Further, the bond between [10} B ₁₂ H ₁₁ ] contained in the terminal portion and the dendritic branch portion is not particularly limited as long as the boron-containing dendrimer in the form of the present invention can be obtained.

The dendrimer of the form of the present invention can form a complex with carbon nanotubes. That is, the present invention provides a composite of a boron-containing dendrimer and carbon nanotubes in the form of the present invention.
In the present specification, the carbon nanotube means a substance in which a six-membered ring network (graphene sheet) made of carbon is formed into a single-layer or multi-layer coaxial tubular, and a composite of a dendrimer and a carbon nanotube, which is the object of the present invention. As long as it can be obtained, there are no particular restrictions.

The method for producing the composite is not particularly limited as long as the composite of the dendrimer and the carbon nanotubes which is the object of the present invention can be obtained, but usually, the dendrimer and the carbon nanotubes are added to a suitable solvent. It can be produced by appropriately stirring (heating if necessary).
The formation of the complex can be confirmed by an ultraviolet-visible near-infrared spectrum, a Raman spectrum, a fluorescence spectrum, an image obtained by an atomic force microscope, and the like.
The composite preferably covers at least a part of the carbon nanotubes with a dendrimer containing boron, and more preferably covers at least a part of the outside of the carbon nanotubes with a dendrimer containing boron.

The present invention can provide a boron preparation containing a boron-containing dendrimer in the form of the present invention and / or a complex of the dendrimer and a carbon nanotube.
The boron preparation containing a dendrimer containing boron in the form of the present invention can be synthesized by a relatively simple method, has a high ability to be taken up by cells despite its low toxicity, and exhibits a high intracellular boron concentration. It can be used preferably.
The boron preparation containing the complex of the form of the present invention can be synthesized by a relatively simple method, has a high uptake ability into cells despite its low toxicity, and exhibits a high intracellular boron concentration, which is preferable. Can be used. Further, it can be more preferably used as a fluorescent probe that absorbs near-infrared light and emits fluorescence.

Boron preparations of the form of the invention are commonly used as suitable pharmaceutically acceptable excipients, carriers, solvents, gel-forming agents, antioxidants, diluents, tonicity agents, pH stabilizers and the like. Additives can be included. Those skilled in the art can appropriately select these additives.
Such boron preparations can be prepared in various dosage forms such as solutions, suspensions, injections, capsules, gels, ointments, creams, emulsions and the like. In other words, it should be used as an injection together with saline, isotonic solutions, and additives for injections used in general clinical practice, and it should be encapsulated as an internal medicine or mixed with a gelling agent or a dermatological agent to be introduced transdermally. It is also possible to.

The boron preparation in the form of the present invention can generally be administered intravenously or transarterically. Furthermore, administration by oral medicine, administration by intramuscular injection, percutaneous administration and the like can also be performed.
The boron preparation in the form of the present invention is preferably administered in a therapeutically effective amount at a concentration and amount that can be accumulated in the tumor cells.

Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples, but these Examples are only one aspect of the present invention, and the present invention is not limited to these Examples.

Reagents, measuring devices, etc. used in Examples and Comparative Examples are described.
All solvents and reagents used were purchased from Kanto Chemical Co., Inc., Tokyo Chemical Industry Co., Ltd. or Wako Junyaku Co., Ltd. Unless otherwise specified, it was used as it was without purification.
High performance liquid chromatography (HPLC) for preparative use is model LC-918V (product name) manufactured by Japan Analytical Industry Co., Ltd., and the column is manufactured by Japan Analytical Industry Co., Ltd. JAIGEL GS-320 (product name) (eluent: CH ₃ OH) was used.
The nuclear magnetic resonance spectrum (NMR) was measured using JEOL AL300 (product name) manufactured by JEOL Ltd. TMS was used as an internal standard.
The ultraviolet-visible near-infrared absorption spectrum was measured using SHIMADZU UV-3150 (product name) manufactured by Shimadzu Corporation.
The infrared absorption spectrum (IR) was measured using the SHIMADZU IR Affinity-1 (product name) manufactured by Shimadzu Corporation using the KBr method.
The Raman spectrum was measured using a JASCO NRS-3100 (product name) manufactured by JASCO Corporation and a laser beam of 488 nm.

The dendrimer and the complex 10 of the dendrimer and the carbon nanotube were produced, for example, using 1,10-decanediol 1 as a starting material as shown in FIGS. 1A and 1B. Hereinafter, a detailed description will be given.
<Synthesis of compound (diol) 2>
Sodium hydride (0.432 g, 18.0 mmol), 1,10-decanediol 1 (2.613 g, 15.0 mmol) and DMF (50 ml) are added to a 500 ml three-necked flask, and the solution is mixed at 80 ° C. for 3 The mixture was heated and stirred for an hour. Then, a solution of 1,10-dibromodecane (0.750 g, 2.50 mmol) of DMF (40 ml) was added dropwise to the DMF solution while heating and stirring at 80 ° C. Further, the mixture was heated and stirred at 80 ° C. for 24 hours under an argon atmosphere.
After adding water to the reaction mixture, the solvent was distilled off to obtain a residue. The residue was suction filtered with chloroform, and chloroform was distilled off with an evaporator to obtain a solid. The solid was purified using column chromatography (silica gel, eluent: chloroform) and compound 2 (11,22-dioxadotriacontane-1,32-diol: 11,22-dioxadotriacontane-1,32-diol). ) (0.220 g, 0.452 mmol, yield 18%) was obtained as colorless and transparent crystals.
Compound 2: mp 81 --82 ° C; ^{1 1} H NMR (300 MHz, CDCl ₃ ) δ 1.29 (s, 36H), 1.56 (t, J = 6.5 Hz, 12H), 3.39 (t, J = 6.6 Hz, 8H) , 3.64 (t, J = 6.0 Hz, 4H); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 16.39 (t), 16.46 (t), 22.29 (t), 24.11 (t), 46.34 (t), 46.77 (t), 51.09 (t), 61.50 (t), 61.59 (t); Anal. Calcd for C ₃₀ H ₆₂ O ₄ ; C, 74.02; H, 12.84; N, 0. Found: C, 74.11; H, 13.32; N, 0.

<Synthesis of compound (dichloride) 3>
In a 200 ml eggplant flask, add the above-mentioned compound 2 (0.100 g, 0.205 mmol), thionyl chloride (0.596 ml), catalytic amounts of DMF (0.128 ml) and chloroform (20 ml), and bring the solution to room temperature. Stirred for 1 day. Thionyl chloride and DMF were distilled off to obtain a residue. The residue is then purified by column chromatography (silica gel, eluent: chloroform) and compound 3 (11,22-dioxa-1,32-dichlorodotriacontane: 11,22-dioxa-1,32-dichlorodotriacontane). (0.876 g, 0.167 mmol, yield 81%) was obtained as a colorless and transparent solid.
Compound 3: mp 47-48 ° C; ^{1 1} H NMR (300 MHz, CDCl ₃ ) δ 1.29 (s, 32H), 1.42 (t, J = 7.2 Hz, 4H), 1.56 (t, J = 6.6 Hz, 8H) , 1.74 (m, 4H), 3.39 (t, J = 6.8 Hz, 8H), 3.53 (t, J = 6.8 Hz, 4H); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 26.2 (t), 26.9 ( t), 28.8 (t), 29.36 (t), 29.42 (t), 29.46 (t), 29.53 (t), 29.7 (t), 32.6 (t), 45.2 (t), 70.92 (t), 70.95 ( t); Anal. Calcd for C ₃₀ H ₆₀ Cl ₂ O ₂ ; C, 68.80; H, 11.55; N, 0. Found: C, 68.75; H, 11.86; N, 0.

<Synthesis of compound (diphthalimidyl) 4>
In a 200 ml eggplant flask, potassium phthalimide (0.213 g, 115 mmol), the above-mentioned compound 3 (0.100 g, 0.191 mmol) and DMF (16.3 ml) were added, and the mixture was heated and stirred at 80 ° C. for 2 days. DMF was distilled off, and the obtained residue was suction-filtered with chloroform, and chloroform was distilled off with an evaporator to obtain a residue. The resulting residue was purified by column chromatography (silica gel, eluent: hexane: chloroform = 1: 5) and compound 4 (1,32-diphtalimidyl-11,22-dioxadotriacontane: 1,32-diphthalimidyl-11, 22-Dioxadotriacontane) (0.111 g, 0.150 mmol, yield 78%) was obtained as a colorless and transparent solid.
Compound 4: mp 82 ^{--83 ° C; 1 1} H NMR (300 MHz, CDCl ₃ ) δ1.27 (s, 36H), 1.55 (t, J = 6.6 Hz, 8H), 1.66 (t, J = 7.4 Hz, 4H) ), 3.38 (t, J = 6.6 Hz, 8H), 3.67 (t, J = 7.4Hz, 4H), 7.71 (AA'BB', J = 8.4, 2.8 Hz, 8H), 7.84 (AA'BB', J = 8.4, 2.8 Hz, 8H); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 26.2 (t), 26.9 (t), 28.6 (t), 29.2 (t), 29.4 (t), 29.49 (t) , 29.54 (t), 29.8 (t), 38.1 (t), 71.0 (t), 123.1 (d), 132.2 (d), 133.2 (d), 168.5 (q); Anal. Calcd for C ₄₆ H ₈₈ N ₂ O ₆ ; C, 74.16; H, 9.20; N, 3.76. Found: C, 73.75; H, 9.46; N, 3.68.

<Synthesis of compound (diamine) 5>
In a 200 ml eggplant flask, add the above-mentioned compound 4 (0.111 g, 0.150 mmol), benzene (6 ml), ethanol (6 ml), and hydrazine monohydrate (0.6 ml, 0.012 mol) to 80 ° C. The mixture was heated and refluxed for 3 hours. The solvent was distilled off, the mixture was extracted with chloroform, and washed with saturated sodium chloride solution to obtain an organic layer. After drying the organic layer with magnesium sulfate, chloroform is distilled off with an evaporator, and the mixture is vacuum dried to compound 5 (11,22-dioxa-1,32-dotriacontanediamine: 11,22-dioxa-1,32-dotriacontane). Diamine) (0.051 g, 0.105 mmol, yield 70.5%) was obtained as a colorless transparent solid.
Compound 5: ^{1 1} H NMR (300 MHz, CDCl ₃ ) δ 1.28 (s, 32H), 1.47 (m, 4H), 1.56 (t, J = 6.5 Hz, 8H), 2.67 (t, J = 6.9 Hz 4H) , 3.39 (t, J = 6.7 Hz, 8H); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 26.2, 26.9, 29.5, 29.6, 29.8, 70.98; IR (KBr): 3327 (-NH ₂ ), 2930 ( CH), 2855 (CH), 1114 (COC) cm ^-1 .

<Synthesis of compound (tetraester) 6>
Methyl acrylate (0.189 ml, 2.11 mmol) was added dropwise to a solution of compound 5 (0.0511 g, 0.105 mmol) in methanol (7.5 ml) in a 100 ml eggplant flask, and then the mixture was added dropwise at 45 ° C. for 3 days. It was heated and stirred. The solvent was evaporated on an evaporator to give a residue. The obtained residue was purified by HPLC (eluent; methanol) to obtain Compound 6 (0.0612 g, 0.0738 mmol, yield 70%) as a colorless and transparent oily substance.
Compound 6: ^{1 1} H NMR (300 MHz, CDCl ₃ ) δ 1.27 (d, J = 6.6 Hz, 36H), 1.40 (t, J = 6.8 Hz, 4H), 1.56 (t, J = 6.5 Hz, 8H), 2.37-2.46 (m, 12H), 2.76 (t, J = 7.2 Hz, 8H), 3.39 (t, J = 6.8 Hz, 8H), 3.67 (s, 12H); ¹³ C NMR (75 MHz, CDCl ₃ ) δ 26.1 (t), 27.1 (t), 27.3 (t), 29.45 (t), 29.50 (t), 29.6 (t), 29.7 (t), 32.5 (t), 49.2 (t), 51.5 (q) , 53.8 (t), 70.9 (t), 173.1 (s); Anal. Calcd for C ₄₆ H ₈₈ N ₂ O ₁₀ ; C, 66.63; H, 10.70; N, 3.38. Found: C, 66.02; H, 10.58 N, 3.28.

<Synthesis of compound (tetraamine) 7>
In a 200 ml eggplant flask, a methanol dispersion (50 ml) of the above-mentioned compound 6 (0.151 g, 0.182 mmol) was added dropwise to an excess amount of ethylenediamine (9.69 ml, 0.145 mol) while cooling in an ice bath. Then, the mixture was stirred at room temperature for 3 days. Methanol and ethylenediamine were distilled off under reduced pressure to obtain a residue. The residue was dissolved in methanol, diethyl ether was added, and centrifugation was performed at 3800 rpm for 20 minutes to obtain a precipitate. The precipitate was dried to give compound 7 as a clear, colorless solid. Compound 7 was used as it was in the next reaction without purification.

<Synthesis of compound (Tetra-N-succinimidyl) 8>
Compound 7 (0.113 g, 0.12 mmol) above was dissolved in methanol (2 ml) and N-succinimidyl 4-maleimidebutyrate (0.202 g, 0.72 mmol), triethylamine (0.067 mL, 4.8 mmol) and chloroform (5 mL). Was added. After stirring at room temperature for 12 hours, the solvent was distilled off and the obtained residue was suction-filtered with methanol to obtain a filtrate. The filtrate was purified by HPLC to give compound 8 as a clear, colorless oil (0.062 g, 0.039 mmol, 32% yield).
Compound 7: 1H NMR (300 MHz, CDCl ₃ ) δ 1.27 (36H, d, J = 6.3 Hz), 1.45 (4H, br), 1.55 (8H, t, J = 6.3 Hz), 1.92 (8H, quin, J = 6.8 Hz), 2.19 (8H, t, J = 7.2 Hz), 2.35 (8H, t, J = 5.9 Hz), 2.43 (8H, t, J = 7.4 Hz), 2.70 (8H, t, J = 5.9 Hz), 3.30-3.40 (28H, m), 3.56 (8H, t, J = 6.8 Hz), 6.71 (8H, s). IR (KBr): 3302, 3089, 2928, 2855, 1709, 1693,1552 cm ^-1 .

<Synthesis of Dendrimer 9>
Compound 8 (0.033 g, 0.021 mmol) described above was dissolved in methanol (3 ml) and Na ₂ ¹⁰ B ₁₂ H ₁₁ SH (0.017 g, 0.084 mmol) was added. After stirring at room temperature for 3 hours, the solvent was concentrated to give dendrimer 9 as a clear, colorless oil (0.050 g, 0.020 mmol).
Dendrimer 9: ^{1 1} H NMR (300 MHz, CD ₃ OD) δ 0.5-1.8 (11 Η, m), 1.24 (36H, s), 1.55 (12H, br), 1.90 (8H, br), 2.26 (8H, br) ), 2.58 (12H, br), 3.04 (8H, br), 3.17 (8H, br), 3.42 (28H, t, J = 6.5 Hz), 3.51 (8H, t, J = 6.3 Hz), 3.89 (4H) , br). IR (KBr): 3373, 2927, 2854, 2498, 1694, 1643, 1549 cm ^-1
The boron content of dendrimer 9 was 19.3% by weight.

<Observation of dendrimer 9 using scanning microscope (SEM)>
The above-mentioned dendrimer 9 aqueous solution (10, 100 μM) was placed on a cover glass (MATHUNAMI) attached to an SEM sample table (Nissin EM Co., Ltd. PK29T Type-KM) and air-dried for 1 hour. The sample is osmium-coated with an osmium coater (vacuum device HPC-1S type hollow cathode plasma CVD), and the shape of the above-mentioned dendrimer 9 is formed using a scanning electron microscope (S-4800 type (product name) manufactured by Hitachi, Ltd.). Observed. The acceleration voltage was 5.0 KV, and the magnifications were 80,000 times and 40,000 times.

Scanning electron microscope (SEM) images are shown in FIGS. 2A and 2B. FIG. 2A is 80,000 times and FIG. 2B is 40,000 times. When the particle size of each was measured from the SEM image, it was 129.37 ± 46.86 nm. In the SEM image, the shape of the dendrimer 9 showed a shape slightly close to an ellipse. Moreover, the size of each particle appeared to be almost uniform.

<Synthesis of complex 10 of dendrimer 9 and carbon nanotubes>
The above-mentioned dendrimer 9 (0.002 g, 0.82 μmol) was dissolved in 5 ml of ion-exchanged water to prepare an aqueous solution of dendrimer 9. Next, 1 mg of carbon nanotube (HiPco (trade name) tube manufactured by Nano Integris) was placed in a screw cap test tube, and then the above-mentioned dendrimer 9 aqueous solution (5 ml) was added. Then, ultrasonic irradiation was performed for 4 hours, and centrifugation was performed at 3000 G for 30 minutes to obtain a black transparent supernatant solution of the (supramolecular) complex 10 of dendrimer 9 and carbon nanotubes.

The ultraviolet-visible-near-infrared spectrum, Raman spectrum, fluorescence spectrum, and atomic force microscope (AFM) image of the complex 10 are shown in FIGS. 3 to 6, respectively.
According to the ultraviolet-visible-near-infrared spectrum, an absorption band in which the light absorption of carbon nanotubes and the light absorption of dendrimer 9 are added was observed. This indicates that the carbon nanotube and the dendrimer 9 are compounded.
According to the Raman spectrum, no new peak was observed and no new chemical bond formation was observed.
According to the fluorescence spectrum, carbon nanotubes were dispersed and present, and a characteristic peak was observed indicating that a complex in which the dendrimer was physically adsorbed was formed around one carbon nanotube.
According to the atomic force microscope (AFM) image, the fibrous structure of the complex 10 was observed (left figure), and the height profile showed that the thickness was about 1.5 nm. As the AFM, SPA 400 manufactured by Seiko Instruments Inc. was used.

<Cell proliferation assay (Water-Soluble Tetrazolium: WST-1 assay)>
The toxicity of dendrimer 9 and complex 10 described above was evaluated using the WST-1 assay. U87ΔEGFR cells were cultured in 96 well plates (BD) for 24 hours (1 × 103 cells / well). The culture was replaced with the same amount of culture containing dendrimer 9 or complex 10 (final concentrations 1, 10, 50, 100, 250 μM). As Control, Dulbecco's Modified Eagle Medium (DMEM, Wako Pure Chemical Industries) containing 10% (v / v) fetal bovine serum (GE Healthcare, Fairfield CA, USA), 100 units / mL penicillin-100 μg / mL streptomycin (Wako Pure Chemical Industries, Ltd.) Pharmaceutical industry) was used. After 24, 48, 72 hours, Cell Proliferation Reagent WST-1 (Roche, Basel, Switzerland) was added at 10 μL / well. After incubation at 37 ° C for 1 hour, the absorbance (A450 nm-690 nm) of each sample was measured with a microplate reader (Vient XS, DS Pharma Biomedical, Osaka).

The results of the cell proliferation assay (cytotoxicity) are shown in FIG. Evaluation was made 24 hours (left figure) and 48 hours (right figure) after administration of dendrimer 9 and complex 10. None of them showed a significant difference from the control (Cont).
Therefore, no cytotoxicity was observed by administration of dendrimer 9 and complex 10.

^{<Measurement of 10} B concentration of tumor cells>
The malignant brain tumor cell line U87ΔEGFR was cultured in a φ35 mm-dish (BD) for 24 hours (1 × 105 cells / dish). The culture was replaced with the same amount of culture containing the above-mentioned dendrimer 9 or complex 10 (final concentration 5, 50, 250 μM). After the set time (6, 12, 24 hours) had elapsed, the culture medium was removed and the cells were washed once with PBS. Trypsin (0.25w / v% trypsin-1 mmol / l EDTA · 4Na solution, Wako Pure Chemical Industries, Ltd.) was added at 300 μL / well and incubated at 37 ° C. for 2 minutes. After 700 μL / well of PBS was added and recovered, 400 μL / well of PBS was added and washed and recovered. Centrifuge at 1500 rpm for 10 minutes with a micro-cooled centrifuge (Kubota Seisakusho Co., Ltd.), and the supernatant was aspirated. 61% (v / v) nitric acid (HNO ₃ , boron determination grade, Wako Pure Chemical Industries, Ltd.) was added in an amount of 300 μL, and the mixture was dissolved at room temperature for 30 minutes. 700 μL of water was added and heated at 100 ° C. for 2 hours. ^{The amount of 10} B contained in the obtained sample, that is, the intracellular ^{concentration of 10} B was measured by an inductively coupled plasma emission spectrophotometer (ICP-AES, VISTA-PRO, Seiko Instruments Inc., Chiba).

The ^{results of intracellular 10} B concentration measurement are shown in FIG. The intracellular introduction effect was confirmed 24 hours after the administration of dendrimer 9. This intracellular boron concentration ( ¹⁰ B) was 316.3338 (ng / 106 cells), (ppm) in the 5 μM group, 596.1537 (ng / 106 cells), (ppm) in the 50 μM group, and 2182.93 (ng / 106 cells) in the 250 μM group. ), (ppm), showing dependence on the concentration of dendrimer 9.
Furthermore, the 250 μM group of dendrimer 9 was 770.3263 (ng / 106 cells), (ppm) 6 hours after administration, 1229.552 (ng / 106 cells), (ppm) 12 hours after administration, and 2182.93 (2182.93 cells) 24 hours after administration. ng / 106 cells), (ppm), showing time dependence.
In addition, the 250 μM group of complex 10 showed a higher intracellular introduction effect than the 250 μM group of dendrimer 9. For example, the 250 μM group of complex 10 showed a high intracellular introduction effect of 6411.329 (ng / 106 cells), (ppm) 24 hours after administration.
Considering that the boron concentration in the cell uptake test shown in Experimental Example 2 and FIG. 2 of Patent Document 1 is about 1 ppm, the intracellular introduction of the dendrimer 9 and the complex 10 according to the present invention. The effect was shown to be significantly higher.

The present invention provides a novel boron-containing dendrimer having an intracellular introduction function and a complex containing the dendrimer and carbon nanotubes. Boron preparations containing such dendrimers and / or complexes can be preferably used in boron neutron capture therapy (BNCT).

Claims (6)

A dendrimer that includes a core, a dendritic bifurcation, and an end.
The core part is a divalent aliphatic hydrocarbon group in which at least one oxygen is inserted,
The dendritic bifurcation contains an amidoamine structure and contains
The distal end, only contains the down decahydro dodecaborate structure,
A dendrimer represented by the following formula.
[Here, the core portion corresponds to A, dendritic branching unit, two C combined with B and B, i.e., corresponds to BC ₂ structure, end corresponds to D. ]
The tree-shaped branch has a structure shown by the following formula.
[Here, R ² is a divalent aliphatic hydrocarbon group having 2 to 12 carbon atoms, and R ² may be the same or different from each other. ]
The end contains other structures for connecting the dendritic bifurcation to the undecahydrododecaborate structure, and the other structures are:
Is selected from. The dendrimer according to claim 1, wherein the core portion contains a divalent aliphatic hydrocarbon group containing 4 to 100 carbon atoms into which 1 to 49 oxygens are inserted. The dendrimer according to claim 1, wherein the core portion contains at least one oxygenated divalent aliphatic hydrocarbon group having a structure represented by the following formula.
[Here, R ¹ is a divalent aliphatic hydrocarbon group having 2 to 20 carbon atoms, and R ¹ may be the same or different from each other. ] The dendritic branch is
The dendrimer according to claim 1, which is a structure selected from. The complex of the dendrimer and carbon nanotubes according to any one of claims 1 to 4. A boron preparation comprising the dendrimer according to any one of claims 1 to 4 and / or the complex according to claim 5.