Boron compounds and methods for producing the same

Abstract

To solve a problem in which, coupling of BSH and an intracellular transport peptide can be performed by once synthesis by modifying the intracellular transport peptide by a maleimide group, in a prior art, however, there is a possibility of generation of chiral carbon on a coupling point when the maleimide group is used and generation of a racemic body.SOLUTION: The invention is configured so that, by coupling BSH and an intracellular transport peptide by a methylene carbonyl linker (-C-C(O)-), generation of a racemic body can be suppressed and a boron compound which can be safely administered to a human body can be acquired.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to a boron compound used in boron neutron capture therapy and a method for producing the same, and also to a drug containing a boron-containing compound. [Background technology]

[0002] Stable isotopes of boron (hereafter referred to as " 10 Also written as "B". When irradiated with neutron beams, a nuclear fission reaction proceeds and alpha particles are emitted. Boron Neutron Capture Therapy (BNCT) is a technique that uses the emitted alpha particles to damage the DNA inside cells and kill cancer cells, and is a new cancer treatment method that combines radiation therapy and chemotherapy.

[0003] BNCT is 10 The effect is enhanced when B is located closer to the DNA, 10 Transporting B into cells is crucial. Therefore, the invention of boron-containing compounds that can enter cells and drugs using them (hereinafter also referred to as "boron preparations") is important.

[0004] On the other hand, BPA (4-Boronophenylalanine) has been used as the boron preparation for BNCT. However, BPA contains one molecule per unit. 10 It contains only B, and inside the cell 10 It is inefficient for transporting B.

[0005] In that respect, 12 molecules in one molecule 10 Boron clusters containing B (hereinafter referred to as BSH: mercaptoundecahydrodecaborate) are produced in large quantities at once. 10 Because it can transport B into cells, it shows promise as a new boron preparation for BNCT. However, 10 Since B cannot enter a cell on its own, it requires a means of transporting it into the cell.

[0006] Patent Document 1 discloses that by attaching a maleimide group to the N-terminus of a cell penetrating peptide (CPP) and covalently linking it to BSH, BSH can be effectively transported into cells.

[0007] Furthermore, Patent Document 2 discloses a compound of BSH with the SH group cyanoethylated and an α-amino acid derivative. Furthermore, Patent Document 3 discloses a compound of BSH with the SH group cyanoethylated and a carbonyl group esterified. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Publication No. 2016-020316 [Patent Document 2] International Publication No. 2010 / 010912 [Patent Document 3] Japanese Patent Publication No. 2008-094730 [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] However, the covalent bond described in Patent Document 1 is formed by the reaction between the thiol group of BSH and the maleimide group modified to CPP, resulting in racemization. Since racemization can have devastating adverse effects on the human body, as seen with thalidomide, a bonding method that avoids racemization is necessary.

[0010] Patent documents 2 and 3 describe a structure that does not produce a racemic mixture when bound to BSH, but they employ a complex process in which the SH group of BSH is cyanoethylated, then reacted with an alkyl halide, and then the cyanoethyl group is removed. Furthermore, patent document 2 assumes that a racemic mixture will be formed as a boron compound. This is because the peptide bound to BSH is not bound to BSH at its N-terminus or C-terminus. [Means for solving the problem]

[0011] The present invention was conceived in view of the above-mentioned problems, and provides a boron compound having a bond between BSH and CPP that does not result in racemization.

[0012] More specifically, the boron compound according to the present invention is: This method is characterized by the linkage of the SH group of mercaptoundecahydrododecaborate (BSH) and the N-terminal NH2 group of the intracellular transport peptide via a methylene carbonyl linker.

[0013] Furthermore, the method for producing boron compounds according to the present invention is: The process involves chloroacetylating the N-terminus of an intracellular transport peptide, The method is characterized by comprising the step of attaching the N-terminus of the chloroacetylated intracellular transport peptide to mercaptoundecahydrodecaborate (BSH). [Effects of the Invention]

[0014] In the boron compound according to the present invention, since BSH and the intracellular transport peptide are linked by a methylene carbonyl linker, racemization does not occur, and the possibility of serious adverse effects on the human body can be eliminated.

[0015] In addition, in the method for producing a boron compound according to the present invention, by simply reacting an intracellular transport peptide with its N-terminus chloroacetylated with BSH, BSH and the intracellular transport peptide can be bound in a method that does not cause racemization, and the production method can be simplified.

Brief Description of Drawings

[0016] [Figure 1] It is a diagram showing the structure of the boron compound according to the present invention. [Figure 2] It is a diagram showing the differences between the prior art and the present invention. [Figure 3] It is a diagram showing the synthesis procedure of the boron compound according to the present invention. [Figure 4] It is a diagram showing the synthesis procedure of the boron compound when CPP is composed of triarginine. [Figure 5] It is a diagram showing the RP-HPLC measurement results of Cl-CH2CO-R3-G-NH2. [Figure 6] It is a diagram showing the MALDI-TofMass measurement results of Cl-CH2CO-R3-G-NH2. [Figure 7] It is a diagram showing the RP-HPLC measurement results of BSH-CH2CO-R3-G-NH2. [Figure 8] It is a diagram showing the MALDI-TofMass measurement results of BSH-CH2CO-R3-G-NH2. [Figure 9] It is a stained photograph when the BSH-CPP (triarginine) conjugate is introduced into cells. [Figure 10] It is a stained photograph when the BSH-CPP (undecararginine) conjugate is introduced into cells. [Figure 11] It is a graph showing the RP-HPLC measurement results for each reaction time. (a) represents the results when the reaction proceeds with time. (b) represents the results when the reaction does not proceed. [Figure 12] It is the result of measuring the reaction rate when the type of peptide is changed. [Figure 13] It is the result of measuring the reaction rate when the length of the peptide is changed.

Best Mode for Carrying Out the Invention

[0017] The boron compound according to the present invention and its production method will be described below with reference to the drawings and examples. The following description illustrates one embodiment and one example of the present invention, and the present invention is not limited to the following description. The following description can be modified without departing from the gist of the present invention.

[0018] The structure of the boron compound according to the present invention is shown in FIG. 1. The boron compound according to the present invention (reference numeral 1) is a mercaptoundecahydrododecaborate (hereinafter referred to as "BSH"; reference numeral 10) in which a thiol group (SH group) is added to a boron cluster containing 12 boron stable isotopes ( 10 B) in one molecule, a methylene carbonyl linker (reference numeral 12), and a cell-penetrating peptide (CPP) (reference numeral 14). The portion other than the N-terminus and C-terminus of CPP is referred to as the "CPP main body". Further, as shown in Example 4 described later, a molecule may be included between the N-terminus of CPP and the methylene carbonyl linker 12.

[0019] The methylene carbonyl linker connects between the thiol group (SH group) of BSH and the N-terminus of CPP, and has the structure shown in the methylene carbonyl linker 12 in FIG. 1. In the formula, it is (-C-C(O)-) ((O) indicates a state of being bonded to carbon by a double bond). This is a structure resulting from introducing a chloroacetyl group to the N-terminus of CPP and the chlorine of the chloroacetyl group leaving and bonding to the sulfur of the thiol group, which will be described in detail later.

[0020] CPP is a peptide with cell membrane permeability used to introduce BSH into cells. Specifically, it is a basic peptide such as Tat protein, oligoarginine, HIV-1Rev(34-50), and FHV Coat. In the boron compound according to the present invention, it is desirable that a cationic group is bonded to the α-carbon (the α-carbon of the N-terminal amino acid of the CPP itself) at the N-terminus. For example, Tat and arginine are desirable in terms of their ability to be transported into cells and their structure. Note that although the C-terminus of CPP is shown as a primary amide (-CONH2), it may also be a carboxyl group.

[0021] Figure 2(a) shows a conceptual diagram of the method for attaching CPP to BSH as described in Patent Document 1. In this method, a maleimide group is attached to the N-terminus of the CPP when attaching CPP to BSH. The double bond of the maleimide group is broken and it binds to the thiol group of BSH, thereby attaching BSH to the CPP with the maleimide group attached. This method allows for the attachment of CPP and BSH in a single synthesis.

[0022] However, a chiral carbon (symbol 20) appears at the bond site, and mirror image symmetry occurs depending on whether the unbonded hydrogen is on the upper side (symbol 22) or the lower side (symbol 24).

[0023] On the other hand, Figure 2(b) shows the case of the boron compound according to the present invention. A chloroacetyl group (reference numeral 26) is introduced at the N-terminus of CPP. BSH and CPP are bonded by the elimination of chlorine, a halogen, and the hydrogen of the thiol group of BSH. At this time, no chiral carbon appears in the methylene carbonyl linker (reference numeral 12) that links BSH and CPP, and a racemic mixture is not formed in this region. Therefore, it can be safely administered to the human body. Furthermore, this method also allows for the bonding of CPP and BSH in a single synthesis.

[0024] Next, the method for producing the boron compound according to the present invention will be described with reference to Figure 3. In order to chloroacetylate the N-terminus of CPP, first, chloroethylcarbonyloxysuccinimide is prepared by esterifying N-hydroxysuccinimide (hereinafter referred to as "NHS") with chloroacetic acid. This will be referred to below as "chloroacetic acid NHS ester (reference numeral 30)".

[0025] This chloroacetic acid NHS ester is reacted with the N-terminus of CPP to introduce a chloroacetyl group to the N-terminus of CPP (Figure 3(a)). This is called "chloroacetylated CPP (indicated by 32)". Then, the chloroacetylated CPP is bonded to BSH (Figure 3(b)). The chlorine of the chloroacetyl group of CPP and the hydrogen of the thiol group of BSH are eliminated, and BSH and CPP are bonded together to obtain the boron compound according to the present invention. At this time, the link between BSH and CPP is a methylene carbonyl linker (indicated by 12).

[0026] Therefore, it can be said that the methylene carbonyl linker connects the SH group of BSH with the N-terminal NH2 group of CPP.

[0027] The boron compound according to the present invention may be obtained in the form of a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts include salts with inorganic bases, salts with organic bases, salts with inorganic acids, salts with organic acids, and salts with basic or acidic amino acids.

[0028] Suitable examples of salts with inorganic bases include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts; and aluminum salts and ammonium salts.

[0029] Suitable examples of salts with organic bases include, for example, salts with trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine.

[0030] Suitable examples of salts with inorganic acids include, for example, salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid.

[0031] Suitable examples of salts with organic acids include salts with formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid.

[0032] Suitable examples of salts with basic amino acids include salts with arginine, lysine, and ornithine, while suitable examples of salts with acidic amino acids include salts with aspartic acid and glutamic acid. [Examples]

[0033] Examples of the boron compounds according to the present invention are shown below.

[0034] (Example 1) BSH contains 12 molecules 10 This is a boron cluster containing B and one -SH group. The BSH was purchased from Katchem (Prague, Czech Republic).

[0035] The peptide consisting of an oligoarginine chain, known as CPP, was defined as having three arginine (R) molecules linked together, followed by a glycine (G) molecule. This is called CPP (trial arginine). It was synthesized using the peptide solid-phase method according to the following procedure. The synthesis flow is shown in Figure 4. Although CPP (trial arginine) was used here for simplicity, it goes without saying that any amino acid suitable for cell introduction can be linked in place of the glycine.

[0036] <Synthesis of chloroacetylated CPP> Referring to Figure 4(a), R3-G (arginine triple linkage + glycine) was prepared on Fmoc-NH-SAL-PEG resin (Watanabe Chemical Industry, Hiroshima) by solid-phase synthesis of Fmoc peptides. The resin was swollen overnight with dimethylformamide (DMF), washed with DMF, and the Fmoc groups protecting the amino groups on the resin were deprotected (7 min) with 500 μL of 20% piperidine (Wako Pure Chemical Industries, Ltd., Osaka) / DMF solution.

[0037] Four equivalents of 144 μL of Fmoc-Gly-OH (Watanabe Chemical Industry) DMF solution were added to the amino groups on the deprotected resin, and coupling was performed (40 min) with a coupling agent (130 μL of HBTU / DMF solution and 18 μL of N-methylmorpholine (NMM)). HBTU is 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (CAS number 94790-37-1).

[0038] Subsequently, the three arginines were condensed using Fmoc-Arg(Pbf)-OH (Watanabe Chemical Industry) by repeatedly performing the deprotection-coupling process on the Fmoc group protecting the N-terminal amino group (indicated as 40 in Figure 4). Therefore, n=3 in Figure 4.

[0039] Next, the amino group of the N-terminal arginine was deprotected, and 10 equivalents of a 0.5M chloromethylcarbonyloxysuccinimide / N-methylpyrrolidone (NMP) solution (which can be called a chloroacetic acid NHS ester / NMP solution (symbol 42)) were added and the reaction was carried out for 40 minutes. The product (Cl-CH2CO-R3-G) was then synthesized.

[0040] After drying the resin using dichloromethane, 500 μL of a cleavage solution (95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIPS), 2.5% distilled water) was added and cleavage from the resin was carried out (90 min) to obtain the target peptide (chloroacetylated CPP (triarginine): Cl-CH2CO-R3-G-NH2) (reference numeral 44). The product from this reaction is a mixture containing chloroacetylated CPP (triarginine) and impurities.

[0041] The impurities in the product were removed by diethyl ether precipitation, isolated and purified by reverse-phase high-performance liquid chromatography (RP-RP-HPLC), and lyophilized to obtain highly pure chloroacetylated CPP (triarginine). The results are shown in Fig. 5. Referring to Fig. 5, the horizontal axis is time (min) and the vertical axis is the detection voltage (mV). The measurement conditions were 0.1% TFA / MeCN, MeCN = 0-50%, the measurement time was 20 min, and the measurement wavelength was 230 nm. As a measurement result, it was 380 mV at 8.84 min.

[0042] Also, the obtained chloroacetylated CPP (triarginine) was confirmed by mass spectrometry (MALDI-Tof Mass). The results are shown in Fig. 6. In Fig. 6, the horizontal axis is m / z and the vertical axis is intensity. (Theoretical value: [M+H] + = 619.32, measured value: 620.69) The yield was 54%.

[0043] <Synthesis of BSH-CH2CO-R3-G-NH2> Referring to Fig. 4(b), the Cl-CH2CO-R3-G-NH2 synthesized by the above operation was dissolved in dimethyl sulfoxide (DMSO) (about 220 μL of DMSO per 1 mg of Cl-CH2CO-R3-G-NH2). Separately, 1.5 equivalents of BSH (reference numeral 10) of Cl-CH2CO-R3-G-NH2 was dissolved in distilled water (the same volume as the above DMSO).

[0044] The two solutions prepared as described above were mixed, and 10 μL of triethylamine (TEA) was added at a time until the pH reached approximately 10. After confirming that the pH was around 10, the mixture was stirred for 30 minutes to allow the reaction to proceed. Subsequently, 100 μL of 0.5% trifluoroacetic acid aqueous solution was added at a time until the pH reached approximately 6, thereby terminating the reaction. This reaction yielded a product containing a boron compound (BSH-CH2CO-R3-G-NH2) in which BSH and chloroacetylated CPP (trialginine) were bonded. This boron compound is also called the BSH-L-CPP (trialginine) conjugate (symbol 46). Here, "L" represents a methylene carbonyl linker.

[0045] This product was isolated and purified by high-performance liquid chromatography (RP-HPLC), then freeze-dried to obtain a high-purity BSH-L-CPP (trialginine) conjugate. The results are shown in Figure 7. Referring to Figure 7, the horizontal axis represents time (minutes), and the vertical axis represents detection voltage (mV). The measurement conditions were 0.1% TFA / MeCN, MeCN = 0-50%, measurement time 20 minutes, and measurement wavelength 230 nm. The measurement result was 209 mV at 11.77 minutes.

[0046] Furthermore, the obtained BSH-L-CPP (trialginine) conjugate (indicated as 46: see Figure 4) was confirmed by mass spectrometry (MALDI-Tof Mass). The results are shown in Figure 8. In Figure 8, the horizontal axis is m / z and the vertical axis is intensity. (Theoretical value: [M+H]) + (=748.44, measured value: 749.53) The yield was 33%. As a result, the yield of the operation from peptide synthesis to reaction with BSH was 17% relative to the resin.

[0047] <Cell introduction> To confirm whether the obtained BSH-L-CPP (trialginine) conjugates could enter cells, cultured U87ΔEGFR cells were inoculated with 100 μM of BSH-L-CPP (trialginine) conjugates and incubated for 24 hours.

[0048] After removing the cell culture medium and washing, measurements were taken using a confocal laser microscope. The results are shown in Figure 9. The scale bars in the photographs each represent 20 μm.

[0049] Figure 9(a) is a nuclear staining image using Hoechst. The image has been converted to a black and white photograph so that the stained areas appear white. The same process was used for the following photographs (including the photograph in Figure 10). Figure 9(b) is a stained image using BSH antibody. The white areas indicate the presence of BSH. Figure 9(c) is a cytoplasmic staining image using F-actin, and Figure 9(d) is a superimposed image of these two. The areas where BSH is present, which appear white in the photograph in Figure 9(b), have diffused into the region including the cytoplasm in Figure 9(c) and the cell nucleus in Figure 9(a). This indicates that BSH can be effectively transported into the cell by the BSH-L-CPP conjugate (trialginine).

[0050] Figure 10 shows the results when undecaarginine was used instead of trialginine in the BSH-L-CPP (trialginine) conjugate. In other words, it is the stained image when the BSH-L-CPP (undecaarginine) conjugate was used. The scale bar in the image represents 20 μm.

[0051] Similar to Figure 9, the region where BSH is present (Figure 10(b)) diffuses into the cytoplasm (Figure 10(c)) and the cell nucleus (Figure 10(a)). Therefore, it was found that BSH can be successfully transported into the cell even when using the BSH-L-CPP (undekaarginine) conjugate. Thus, it is considered that the arginine conjugates constituting CPP can consist of at least 3 to 11 (also referred to as 3 to 11) molecules.

[0052] (Example 2) Similar to Example 1, CPP was prepared by substituting triarginine (R3), which constitutes CPP, with trilysine (K3), trihistidine (H3), trityrosine (Y3), triaspartic acid (D3), triglutamic acid (E3), tricerine (S3), trithreonine (T3), triasparagine (N3), triglutamine (Q3), triglycine (G3), trialanine (A3), trivaline (V3), trileucine (L), triisoleucine (I), triphenylalanine (F), and tryptophan (W).

[0053] These are called CPP (triarginine), CPP (trilysine), CPP (histidine), CPP (trityrosine), CPP (triaspartic acid), CPP (triglutamic acid), CPP (tricerine), CPP (trithreonine), CPP (triasparagine), CPP (triglutamine), CPP (triglycine), CPP (trialanine), CPP (trivaline), CPP (trileucine), CPP (triisoleucine), CPP (triphenylalanine), and CPP (tritryptophan). We then attempted to synthesize BSH from chloroacetylated CPP.

[0054] <Reactivity of chloroacetyl groups and BSH due to adjacent amino acids> S between chloroacetyl group and BSH N In the two reactions, to evaluate the differences in reactivity depending on the type of chloroacetyl group and the adjacent amino acid (the N-terminal amino acid of the CPP body), reaction tracking by HPLC and identification by MALDI-Tof-Mass were performed. Representative examples are shown in Figures 11(a) and 11(b).

[0055] Referring to Figure 11, in both Figures 11(a) and (b), the horizontal axis represents time (minutes) and the vertical axis represents output voltage (mV). The horizontal axis can be considered as the time from the start of the reaction. For substances of the same mass, peaks can be observed at the same time. In Figure 11(a), at 0 minutes (dashed line), a peak was observed around 8 minutes 50 seconds. After 30 minutes (solid line), the peak shifted to around 10 minutes 20 seconds, and even after 1 day (dotted line), the peak around 10 minutes 20 seconds was maintained. From these results, it can be concluded that the reaction with amino acids proceeded.

[0056] On the other hand, in the case of Figure 11(b), the peak positions at 0 minutes (dashed line), 30 minutes (solid line), and 1 day (dotted line) remained almost unchanged, indicating that the reaction did not proceed.

[0057] Based on these results, the amino acids were grouped into those that reacted and those that did not. The results are shown in Table 1.

[0058] [Table 1]

[0059] Referring to Table 1, the three CPPs that have a basic group in the side chain of the amino acid adjacent to the chloroacetyl group—CPP(triarginine), CPP(trilysine), and CPP(histidine)—reacted with BSH. However, CPP(trilysine) and CPP(histidine) did not show as significant reactivity as arginine. Furthermore, the CPPs that do not have a basic group in the side chain of the amino acid adjacent to the chloroacetyl group—CPP(trityrosine), CPP(triaspartic acid), CPP(triglutamic acid), CPP(tricerine), CPP(trithreonine), CPP(triasparagine), CPP(triglutamine), CPP(triglycine), CPP(trialanine), CPP(trivaline), CPP(trileucine), CPP(triisoleucine), CPP(triphenylalanine), and CPP(tritryptophan)—did not react with BSH.

[0060] From the above results, it was found that the common characteristic of the amino acids that reacted was that they were basic amino acids. In other words, the reaction between BSH and CPP proceeds when a basic amino acid is used as the amino acid adjacent to the chloroacetyl group (the N-terminal amino acid of the CPP compound).

[0061] (Example 3) For the reaction between BSH and CPP to proceed, a basic amino acid must be used as the amino acid adjacent to the chloroacetyl group. This is suggested to depend on the electrostatic interaction between BSH and CPP containing a basic amino acid. Therefore, we evaluated the effects of reducing the number of arginine molecules and changing the charge.

[0062] In addition to the trial arginine (denoted as "RRR") from Example 1, CPPs consisting of arginine-arginine-alanine (denoted as "RRA") and arginine-alanine-alanine (denoted as "RAA") were synthesized, and after chloroacetylation, a reaction with BSH was attempted. The reaction rate of each peptide was calculated from the HPLC results and plotted in the graph in Figure 12.

[0063] Referring to Figure 12, the horizontal axis represents reaction time (minutes), and the vertical axis represents reaction rate (%). RRR is arginine-arginine-arginine (trialginine), RRA is arginine-arginine-alanine, and RAA is arginine-alanine-alanine. When CPP was converted to RRR, the reaction with BSH showed a 100% reaction rate in 30 minutes. On the other hand, when CPP was converted to RAA, the reaction rate decreased with increasing alanine content. The increase in alanine can be considered to be due to a decrease in charge within CPP.

[0064] In other words, as shown in Figure 12, it was confirmed that the reaction rate with BSH decreased with decreasing charge within the CPP molecule. On the other hand, even with only one arginine molecule, a reaction rate of 50% or more could be achieved, making it practically usable. That is, in order to rapidly advance the reaction between BSH and CPP, at least one arginine molecule is required within the CPP molecule. Although examples using arginine were shown here, as shown in Table 1, it is thought that the reaction between BSH and CPP can also be rapidly advanced by using lysine or histidine, which can react with BSH in a similar way to arginine.

[0065] (Example 4) To evaluate the effect of increasing the distance between the charge-holding site and the reaction site on the reaction, Cl-CH2CO-C1-R3-Sp6-NH2, Cl-CH2CO-C2-R3-Sp6-NH2, Cl-CH2CO-C4-R3-Sp6-NH2, Cl-CH2CO-C7-R3-Sp6-NH2, and Cl-CH2CO-C11-R3-Sp6-NH2, which incorporate arbitrary hydrocarbon chains between the arginine and chloroacetyl groups, were synthesized in the same manner as in Example 1, and their reaction with BSH was attempted. The reaction site refers to the reaction site between the hydrogen of the thiol group of BSH and the chlorine of the chloroacetyl group.

[0066] C1, C2, C3, C4, and C11 are straight-chain hydrocarbon chains with 1, 2, 3, 4, and 11 carbon atoms, respectively. These straight-chain hydrocarbons are inserted between the CPP and chloroacetyl groups. The reaction rates of each peptide were calculated from the HPLC results and plotted in the graph shown.

[0067] Referring to Figure 13, the horizontal axis represents reaction time (minutes), and the vertical axis represents reaction rate (%). The reaction rate decreased as the length of the hydrocarbon chain increased. From the results in Figure 13, it was confirmed that the reaction rate with BSH decreased with increasing distance between the charge-holding site CPP and the chloroacetyl group.

[0068] Furthermore, even with a linear carbon chain of 7 carbon atoms, the reaction rate is over 20%, which is within a practically usable range. Therefore, in order to rapidly proceed the reaction between BSH and chloroacetylated CPP, the distance between CPP and the chloroacetyl group, which is the reaction site, must be kept within seven carbon atoms. In other words, the N-terminus of CPP may have a carbon chain with 7 or fewer carbon atoms. It can also be said that a carbon chain with 7 or fewer carbon atoms can be placed between CPP and the methylene carbonyl linker.

[0069] Furthermore, carbon chains with 7 or fewer carbon atoms can be considered as the seven carbon atoms in the arginine linkage, and by replacing the entire carbon chain with arginine, it is possible to attach conjugates of up to 10 arginine atoms. Although examples using arginine are shown here, as shown in Table 1, it is thought that the reaction between BSH and CPP can also be rapidly advanced by using lysine or histidine, which can react with BSH in a similar way to arginine. [Industrial applicability]

[0070] The boron compound according to the present invention can be suitably used as a drug for boron neutron capture therapy. [Explanation of Symbols]

[0071] 1. Boron compounds 10 BSH 12 Methylene Carbonyl Linker 14 CPP 20 Chiral carbon 22 When the hydrogen formed by the bond is on top 24. When the hydrogen formed by the bond is at the bottom 26 Chloroacetyl group 30. NHS chloroacetate 32 Chloroacetylated CPP 40 CPP (Trialginine) 42. Chloroacetate NHS ester / NMP solution 44. Chloroacetylated CPP (Trialginine) (Cl-CH2CO-R3-G-NH2) 46 BSH-L-CPP (Trialginine) Conjugate

Claims

1. The SH group of mercaptoundecahydrododecaborate (BSH) and the N-terminal NH of the intracellular transport peptide ２ A boron compound characterized by having groups linked by a methylene carbonyl linker.

2. The boron compound according to claim 1, characterized in that a basic group is attached to the α-carbon to which the N-terminus of the intracellular transport peptide is bound.

3. The boron compound according to claim 1 or 2, characterized in that the intracellular transport peptide comprises a linkage of at least one of arginine, lysine, and histidine, from one to ten such linkages.

4. The SH group of mercaptoundecahydrodecaborate (BSH) is linked to a methylene carbonyl linker, The aforementioned methylene carbonyl linker is linked to a straight-chain hydrocarbon having 7 or fewer carbon atoms. A boron compound characterized by the linkage of the linear hydrocarbon to the NH2 group of the α-carbon of the N-terminal amino acid of an intracellular transport peptide.

5. A step of chloroacetylating the NH2 group of the α-carbon at the N-terminus of an intracellular transport peptide, A method for producing a boron compound, characterized by comprising the step of attaching the N-terminus of a chloroacetylated intracellular transport peptide to mercaptoundecahydrodecaborate (BSH).

6. The method for producing a boron compound according to claim 5, characterized in that a basic group is attached to the α-carbon to which the N-terminus of the intracellular transport peptide is bound.

7. A method for producing a boron compound according to claim 5 or 6, characterized in that the intracellular transport peptide contains a linkage of 3 to 11 arginine molecules.

8. A step of chloroacetylating the terminal NH2 group of an intracellular transport peptide in which a linear hydrocarbon having 7 or fewer carbon atoms and terminal NH2 groups is bonded to the NH2 group of the α-carbon at the N-terminus, A method for producing a boron compound, characterized by comprising the step of attaching the chloroacetylated terminal NH2 group to mercaptoundecahydrodecaborate (BSH).

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