JPH02207086A - Cage-type boron compound, complex thereof with protein and product thereof - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I [M<+> represents alkali metal ion or quaternary ammonium ion; T represents group <10>B12H11 expressed by formula II (. denotes <10>BH; O denotes <10>B); Q represents -(CH2)n- (n is 1-5); R represents halogen or OX (X represents H, alkali metal, imide residue or protecting group)]. USE:Useful for a boronic neutron capture therapy, since boron atoms can be selectively and surely incorporated into tumor cells. PREPARATION:A compound expressed by formula III is reacted with a halide expressed by the formula YQCOOR<1> (Y represents halogen; R<1> represents protecting group) in the presence of a base to form a compound expressed by formula IV, which, as desired, is hydrolyzed to form a compound expressed by formula V, and, as desired, further converted into an acid halogenide, alkali metal salt or succinimide derivative.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a cage-type boron compound suitable for use in boron neutron capture therapy, a complex thereof with a protein, and a method for producing the same.

In recent years, various treatment methods for cancer have been established. However, there is no definitive treatment method for malignant tumors, and patients with malignant tumors have had to be prepared for side effects such as exposure to radiation and undergo radiation therapy.

Neutron capture therapy is attracting attention as a method to solve these problems. In neutron capture therapy, a boron compound or a lithium compound is injected into the affected area, and the affected area is treated with low-energy thermal neutrons supplied from a nuclear reactor. Note that these thermal neutrons are low-energy. Therefore, it has no biological effect and does not damage normal cells.

When a boron compound containing an IOB with a very large thermal neutron absorption cross section is used, the collision of thermal neutrons with the IOB causes α decay as shown in the following formula, and α particles are efficiently generated. Ru.

0B + thermal neutron → F Li + 'He α grain + HHe) is extremely heavy compared to other radiations, so LET (Linear energy transfer)
er) and short range. Therefore, this α particle was generated II! Within tumor cells, its ionizing action reliably kills the tumor cells. Therefore, in boron neutron capture therapy, ′. By selectively incorporating B into tumor cells at high concentrations, only tumor cells can be killed without killing normal cells.

In order to realize a boron neutron capture therapy without such side effects, 1. The above-mentioned boron compound requires the following requirements.

■It must be non-toxic.

■It must be chemically stable enough that it will not be degraded before it reaches the tumor cells.

■Have high affinity specifically for tumor cells.

In recent years, such boron neutron capture therapy has been effective in treating brain tumors and malignant melanoma. Boron compounds conventionally used as neutron capture therapy agents include, for example, +013 concentrated sodium mercaptoundecahydrodododecabore (N a 2'.B).
12HII SH), which has achieved excellent results particularly in the treatment of brain tumor patients. However, B 128
, S H'- are taken up into brain tumor tissues using the so-called blood-brain barrier. The blood-brain barrier is a phenomenon in which water-soluble substances in blood are not taken into normal brain tissue, but are selectively taken into brain tumor tissue. However, B l 2
H+ + S H'- has the disadvantage that it not only has insufficient selectivity for tumor cells using the blood-brain barrier, but also cannot be applied to tumors in other sites.

Recently, as research and discussion on so-called drug delivery systems has become more active, research on the design of so-called missile drugs has also become active in boron neutron capture therapeutic agents. That is, the aim is to bind a peptide such as an antibody that has an affinity for tumor cells to a boron compound containing 16B, and to cause it to be specifically and efficiently taken up by cancer cells.

The prior art of such boron neutron capture therapy agents is
F, ^law et, al,, J, Med, Chem.
, 28, 522 (1985). In this method, B 12H, S H2-
Boron atoms are introduced into proteins such as antibodies using coupling agents such as N-succinimidyl 3-(2-pyridyldithio)propionate, which reacts under mild conditions at room temperature and around pH 7.

In a boron neutron capture therapy agent in which a boron atom is introduced into a protein using the above-mentioned coupling agent, disulfide bonds (
B 128 II- is bound to a protein such as an antibody via a S-3 bond (S-3 bond). Therefore, there is a possibility that this disulfide bond is broken and BI28, , SH'- is detached in vivo. The detached B, 2H, and SH'- are not compatible with tumor cells, so they are not taken up by the target tumor cells.
When S H2- is irradiated with thermal neutrons, α particles are generated as described above, which may kill normal cells.

Therefore, the stability of such boron neutron capture therapeutic agents remains problematic.

The purpose of the present invention is to solve the above-mentioned technical problems, to provide a cage-type boron compound, and its peptide, which can be used as a boron neutron capture therapy agent that has excellent chemical stability and can reliably incorporate IIIB into tumor cells. An object of the present invention is to provide a complex with and a method for producing the same.

The present invention is based on the +0131□H1- group represented by - maximum: % formula % () (wherein M+ is an alkali metal ion or quaternary ammonium ion, T is the formula: % formula %), Q is-(CH2
> (n is an integer of 1 to 5), R is halogen or O
The present invention relates to a cage-type boron compound represented by X (X represents a hydrogen atom, an alkali metal, an imide residue, or a protective group, respectively).

Further, the present invention provides a compound represented by -m formula: % formula % () (wherein Mo and T have the same meanings as above) in the presence of a base, -maximum: % formula % () (In the formula, Y is halogen, Q is -(CH2) (n is an integer from 1 to 5), R'
represents a protecting group, respectively), and the general formula: % formula % (
), and if desired, this compound is hydrolyzed to produce a compound represented by the -m formula: % formula % (1), and further, if desired, a compound represented by the formula (
The present invention relates to a method for producing a cage-type boron compound (1), which comprises producing an acid halide (1c) or an alkali metal salt (1d) of the compound (lb).

The raw material compound mercaptoundecahydrododecaborate in the present invention is W, tl, Knowth et, al,
J.

^-,Cbem, Soe,,,ξL-35,,397
3 (1964), and is represented by the formula % (in the formula, Mo and T have the same meanings as above), and is usually an alkali metal or ammonium salt. It is available as . Examples of alkali metals include lithium, sodium, potassium, rubidium, and cesium, and examples of ammoniums include tetramethylammonium and tetraethylammonium. Mercaptoundecahydrododecaborate can be easily converted into any salt by an ion exchange method using a strong acid type cation exchange resin.

The method for producing the cage boron compound (1) of the present invention is shown below.

(Left below) (M”)2T”-SH(II) (M”)2T”-S Q-COOR'(Ia)(M”
>2T2--3-Q-COOH(Ib) (wherein Hal
represents a halogen, A represents an alkali metal, R1 represents a protecting group such as lower alkyl, and M, Q, T and Y have the same meanings as above. ) The first to fourth steps will be explained below.

1L Ci1 The reaction of reacting a raw material (n) with a halide (ff[) to lead to a target substance (1a) is carried out in an appropriate solvent,
In the presence of an alkali hydroxide (e.g., sodium hydroxide, potassium hydroxide, etc.) at 10-50°C, preferably at room temperature (15-30°C) for 5-30 hours, preferably at 10-50°C.
It will be held for 25 hours.

Examples of the halide (III), which is preferably carried out under an inert atmosphere such as nitrogen or argon, include methyl 3-bromopropionate and ethyl 4-bromobutyrate.

The inorganic base that hydrolyzes the ester (1a) obtained above with an inorganic base to lead to the carboxylic acid (1b) is
Examples include alkali hydroxide, alkali carbonate, and alkali hydrogen carbonate, and the method is carried out in a solvent such as water or hydrous alcohol by a conventional method. Note that the number of moles of the inorganic base may be increased to lead to the alkali metal salt (1c). Further, after the carboxylic acid (1b) is purified, it may be similarly reacted with an inorganic base to lead to the alkali metal salt (1c).

111 [By reacting the carboxylic acid (1b) obtained in the above second step with a halogenating agent such as thionyl chloride or thionyl bromide,
It may lead to acid halide (1d).

This reaction is carried out in the presence of a basic deoxidizing agent such as triethylamino or pyridine at 10 to 80°C according to a conventional method.

Strategy m Add N, N to the carboxylic acid (1b) obtained in the second step above.
"-Dicyclohexylcarbodiimide (DCC>), N-hydroxysuccinimide is reacted in the presence of a condensing agent such as polyphosphoric acid to lead to a succinimide compound (1e).

This reaction was carried out in accordance with the conventional method for peptide synthesis, using 1.
It is carried out by heating to 0 to 100°C, preferably 50 to 60°C. Examples of the solvent include dimethyl sulfoxide, dimethylformamide, hexamethylphosphoric triamide, and chloroform.

The method for producing the complex (1f) of the cage-type boron compound (1) and protein of the present invention is shown below.

Complex with protein (1f) The succinimide compound (1e) obtained in the fifth step is reacted with a protein such as mouse IgG to form a complex with the target protein (1f). lead.

However, in the present invention, proteins include synthesized polypeptides and amide conjugates of natural proteins and synthetic polypeptides. This reaction is carried out in a suitable solvent at a temperature of 10 to 30°C, preferably 15 to 25°C, according to a conventional method. Examples of the solvent include dimethyl sulfoxide, dimethylformamide, chloroform, dioxane, and hexamethylphosphoric triamide.

As described above, in the present invention, a carboxylic acid is introduced into the compound (■) via an S-C bond that is extremely stable in vivo, and a monoclonal antibody that has an affinity for, for example, tumor cells is produced using the carboxylic acid group. The amino groups of lysine residues of proteins such as peptides bond to form a complex. Therefore, such a cage-type boron compound-protein complex can be reliably and selectively attached to tumor cells because the protein has affinity with tumor cells and the S-C bond is stable. be included.

As mentioned above, antibodies containing 10B atoms are expected to be specifically absorbed into cancer cells as so-called missile drugs, and are expected to become useful boron neutron capture therapy agents. In particular, by selecting proteins such as monoclonal antibodies, it is possible to selectively incorporate 1013 into tumor cells in areas other than brain tumors, making it possible to widely apply boron neutron capture therapy to cancer treatment in all areas. .

Further, in the sixth step, using polylysine, the amino group thereof is reacted with the active ester (1e), and an appropriate amount of the cage-type boron compound is bonded to obtain a polylysine-cage-type boron compound complex. By binding this to a monoclonal antibody, a large number of cage-type boron compounds can be bound to the monoclonal antibody without extremely reducing antibody activity, and it can be used more effectively in boron neutron therapy.

Examples of R of the present invention are shown below by way of examples.

Example 1 Cesium Monomer Hydrodote Borate Hydrate (CS 2”B 12HIIs H-H20
: 2.3g of “B concentration 95% or more), 26ml of distilled water
Dissolved in cation exchange resin column (Amberlite I
R-120. Sodium type; diameter 24mm, height 100
mm, volume 50 ml) to pre-convert into the sodium salt. The aqueous solution containing the sodium salt is concentrated to dryness using a rotary evaporator, a small amount of benzene is added, and azeotropic dehydration is repeated 2 to 3 times.
After drying under reduced pressure at 70°C for about 1 hour, sodium monomercaptoundecahydrododecaborate anhydride (N
a 210B +28 Its H)1, 1 g is obtained.

Meanwhile, 2.6 g of pulverized potassium hydroxide is dissolved in 140 ml of dry dimethyl sulfoxide under a dry nitrogen gas atmosphere. To this, 10 ml of a solution of 1.1 g of the aforementioned sodium monomercaptoundecahydrododecaborate anhydride in dry dimethyl sulfoxide was added, and then 5 ml of a solution of 8.5 g of methyl 3-bromopropionate in dry dimethyl sulfoxide was added dropwise over about 30 minutes. do. At this time, the reaction temperature rises slightly and the reaction progresses, but
Adjust the dropping rate to maintain approximately 30°C. After the dropwise addition, stirring was continued for 24 hours to complete the reaction.

Then, the reaction solution was concentrated under reduced pressure to remove the solvent.

Continue to add distilled water and add 1.7me (10゜1m
mo1) A cesium chloride aqueous solution was added. The resulting precipitate was collected and recrystallized sequentially from acetonitrile-ether and methanol-ether, yielding 1.9 g (yield 69%) of cesium carbomethoxyethylthioundecahydrododecaborate hydrate (Cs 2.6B l
2 H, S (CH2) 2 COOCH3・H2
O) was obtained.

Elemental analysis: C4Hx. ” B + x OS CS x
Calculated value (%): C, 8,99: H, 3,77,
"B, 22.56S, 6.00 Actual value (%): C, 9,13; H, 3,72,"B, 2
2.80S, 5.87 IR (Nujol): 3560.3360.248
0 (v-->.

1730 (v-,.'), 1595, 1228, 121
0°1185.1150, 1078.1005.982
940.872,832,740 cm-'PMR(
020), τ: 6.36 (OCH3).

7.35 (CH2CH2) ppm Example 2 Cesium carbomethoxyethylthioundecahydrododecaborate-hydrate (CS2CH2) obtained in Example 1
” B l 2 H+ + S (CH2) 2 C
OOCHs ・H2O) 67.7mg was dissolved in methanol 0.
7mj! and suspended in 4N aqueous sodium hydroxide solution.
7 ml was added and stirred at room temperature for about 30 minutes.
Approximately 2 ml of N aqueous hydrochloric acid solution is added, and the mixture is concentrated to dryness to distill off excess hydrochloric acid. The carboxylic acid obtained here has an I R: 2
485 (L/all) +1715 (c-o) Cm
showed that.

1.5 ml' of dry dimethyl sulfoxide was added to the solid residue of the above carboxylic acid, and N-hydroxysuccinimide (
NHS) 49 mg dry dimethyl sulfoxide solution 0.
5 ml, and 0.5 ml of a solution of 75 mg of N,N''-dicyclohexylcarbodiimide in dry dimethyl sulfoxide.
j! was added and reacted at 60°C for 2 hours with stirring.

This reaction yielded an active ester of a boron compound carboxylic acid.

This active ester was added to mouse IgG (Inmun) at room temperature.
ogIobulinll:: Jackson
Immunoresearch Laboratory
After the completion of the reaction, the solvent was distilled off at 40-45°C under reduced pressure, and 20 mN of purified water was added to dissolve the water-insoluble material. removed. Water soluble bone 2.
Concentrate to 5 ml and use a Sephadex G-25 column (trade name: manufactured by Pharmacia; volume 9.1 mt', height 5 cm).
m), the desired fractions were collected and thoroughly dialyzed against purified water. Furthermore, the dialysis membrane is a fractionated molecule! i
35005spectra/Por3 (Spectru
(manufactured by mMedical Industries) was used. When this was freeze-dried, an IgG complex into which 4.3 mg of 108 had been introduced was obtained.

As a result of elemental analysis, IOB was 56.4 mg/g.

Example 3 A reaction was carried out using 4.3 g of ethyl 3-bromopropionate and 1.1 g of tetramethylammonium chloride instead of methyl 3-bromopropionate as a reaction reagent. The reaction product is separated and purified as a tetramethylammonium salt. In this way, tetramethylammonium carboethoxyethylthioundecahydrododecaborate (2-), i.e. ((CH3) 4N) 210B, tHIls (CH
2) 2COOC21-1゜1.5g yield 70.8%
Obtained with.

Elemental analysis: Calculated value (%) as C13144q”B+zNzO□S: C, 37,85, H, 10,75°N,
6.79; S, 7.77 Actual value (%): C, 37,24; H, 10,33; N,
5.78. S, 7.66 I R (Nujol): 3025.2480 (νs
J, 1730 (c-o).

1485, 1418.1332.1301.1362.
1188°1064, 1030.992.930.85
2.775゜735 cm- Example 4 Tetramethylammonium monomer borate (1), i.e. ((CH3)4N)
2B, □H, 1S H is thoroughly dried by azeotropic dehydration several times from dry benzene, and 644 mg thereof is dissolved in 15 rnl of dry dimethylformamide. Under a stream of dry nitrogen, 160 mg of sodium hydride (NaH: 60% mineral oil dispersion) was added dropwise to a 3 ml suspension of dry dimethylformamide with stirring over a period of about 10 minutes. As the temperature rose to 40 to 50°C, the temperature of the solution rose to 40 to 50°C. , hydrogen gas is violently generated and the reaction progresses. After the generation of hydrogen gas has finished and the reaction has ended, bring the temperature to room temperature and add 4 ml of dry dimethylformamide.
334 mg of B r CH2COOCtH5 dissolved in
was gradually added and allowed to react at room temperature for about 2 hours.

After the reaction is completed, the brown solid is removed by filtration, and the P solution is concentrated to dryness. After this, 10 ml of purified water was added to dissolve it, and 440 mg of tetramethylammonium chloride (CH2>4NCI) dissolved in 2 to 3 ml of produced water was added. , < (CH3), N)2″B
, □H,S CH2COOCx 110 mg of H5 are obtained with a yield of 12.5%.

IR (Nujol): 3030.2480b'm-
), 1722 (ce-o) 1488, 1296.12
70.1200.1170°1145, 1052.96
5.950.842.721 In this Example 4, the reaction yield was relatively low, but this was because the product, carboxylic acid ethyl ester, was allowed to coexist with a fairly strong alkali in the separation and purification process of the reaction product. It is presumed that this is due to the hydrolysis of carboxylic acid salts. When the water-soluble portion obtained during the purification process is neutralized with sulfuric acid, washed with ethanol, and recrystallized with water-ethanol, ((CH)) 4N > 2B12H1l, which has weak hygroscopicity as a by-product, is produced.
450 mg of SCH2COONa are obtained with a yield of 55.9%.

IR (Nujol): 3595, 3350, 3030° 2480 (ν, engineering), 1578 (ν.,.), 14
90°1292.1220,1135,1052.97
0952.842,721 cm Example 5 The active ester obtained in the same manner as in Example 2 was heated at room temperature.
28.1 mg of polylysine hydrochloride (molecular weight 15,000
．． Dissolve peptide research cleavage) in 0.5 ml of water and add 0.5 ml of water.
After neutralization with 2 ml of 0.01N sodium hydroxide,
Freeze dry. This is dissolved in 2 ml' of dry dimethylformamide and reacted with the above active ester overnight at room temperature. When treated in the same manner as in Example 2 and freeze-dried, the result is 10.0
2 mg of polylysine-10B complex is obtained.

As a result of elemental analysis, +013 was 90.8 mg/g.

Example 6 The stability of various boron compounds in rat liver extracts was compared. As a control compound, CS 2”B1201
1S S (CH2)2 C0OH was used, and the compound obtained in Example 2 was used as the compound of the present invention.

Table 1 p) (7,2 P B S (phosphate-b
0.2 m of a solution containing the boron compound in the amount shown in Table 1 in 1 mi
l of rat liver extract (Wistar rat, male, 105
00
Follow-up measurements of C) were performed. The HPLC conditions are as follows.

Column: Nucleosil+oC+a 4φX
300 mrn mobile phase: 5mM PI CA
:CH*01l=55:45 Flow rate: 1m 11min Detector: Ultraviolet absorption photometer (254nm) For the control compound, the peak identified as 16B12H11SH2- was observed at the HPLC retention time of 8°5 to 8.6 min. It was observed that the intensity increased over time, and the number of peaks and the width of the peaks in PLC also became wider. This is due to the cleavage of disulfide bonds (SS bonds), [SH”- is generated,
This indicates that other decomposition and recombination are occurring. The amount of "B,,H9H2-" produced and the remaining amount of the starting boron compound estimated by this production are shown in Table 2, and the relationship between the relative concentration of the starting boron compound and the elapsed time is shown by line 11 in the drawing. has been done.

Table 2 Table 3 On the other hand, in the I-I PLC measurement results of the compounds of the present invention, no peak corresponding to '812Hz S H''- appeared even after 120 minutes or more, and other peaks did not appear. There was no appearance of a peak or increase in intensity.

(Margin below) Therefore, no decomposition of the compound of the present invention is observed.

The HPLC results of the compound of the present invention are shown in Table 9, and the relationship between the relative concentration of the compound of the present invention and elapsed time is shown in line 12 of the drawing.
It is indicated by. In the drawing, "○" indicates the HPL of the control compound.
The C measurement value and "x" indicate the HPLC measurement value of the compound of the present invention.

To 11 11 As described above, in the present invention, a boron compound that is bound to a protein and is extremely stable in vivo can be obtained.

Therefore, for example, if a peptide with specific affinity for tumor cells, such as a monoclonal antibody, is conjugated with a boron compound and used for boron neutron supplementation therapy,
Boron atoms can be selectively and reliably incorporated into tumor cells, greatly improving the effectiveness of neutron capture therapy.

[Brief explanation of the drawing]

The drawing is a graph for explaining the stability of the boron compound of the present invention and the boron compound of the conventional example. Agent Patent Attorney Keiichi Nishikyo

Claims (9)

[Claims] (1) General formula: (M^+)_2T^2^--S-Q-CO-R…( I
) (In the formula, M^+ is an alkali metal ion or quaternary ammonium ion, T is a formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (● is ^1^0BH, ○ is ^1^0B) ^1^0B_1_2H_1_1- group, Q
represents -(CH_2)_n- (n is an integer of 1 to 5), and R represents a halogen or OX (X is a hydrogen atom, an alkali metal, an imide residue, or a protective group), respectively. ) A cage-type boron compound represented by (2) A complex in which the cage-type boron compound according to claim 1 is bound to a protein. (3) The complex according to claim 2, wherein the bond with the protein is an amide bond between an amino group of the protein and a carboxy group of the cage boron compound. (4) The amino group is ω- of a lysine residue of a protein.
The complex according to claim 3, which is an amino group. (5) The complex according to claim 2, wherein the protein is IgG. (6) The complex according to claim 5, wherein the IgG is mouse IgG. (7) The complex according to claim 2, wherein the protein is polylysine. (8) The complex according to claim 2, wherein the protein is an amide conjugate of IgG and polylysine. (9) In the presence of a base, the general formula: (M^+)_2T^2^--SH...(II) (wherein M^+ is an alkali metal ion or quaternary ammonium ion, T is the formula: ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (● represents ^1^0BH, ○ represents ^1^0B) The general formula: YQCOOR^ 1...(III) (In the formula, Y is halogen, Q is -(CH_2)_n- (n is an integer from 1 to 5), R^
1 represents a protecting group, respectively. ) is reacted with a halide represented by the general formula: (M^+
)_2T^2^--S-Q-COOR^1...(I a)
A compound represented by is produced, and if desired, this compound is hydrolyzed to form the general formula: (M^+)_2T^2^--S-Q-COOH...( I
b) to produce a compound represented by b), and optionally further,
Acid halide (Ic) or alkali metal salt (Id) or succinimide (I
e) A method for producing a cage-type boron compound and an alkali metal salt thereof.