JPH04309502A - Polysulfuric ester of cyclodextrin derivative and its production - Google Patents

Abstract

PURPOSE:To provide a polysulfuric ester of cyclodextrin derivative improved in antiretroviral action, especially an action of suppressing the replication if human immuno-defficiency viruses. CONSTITUTION:A cyclodextrin derivative prepared by substituting at least one of the 6-8 D-glucose units constituting cyclodextrin by a unit of the formula [wherein R<1> is a group derived by removing the OH group from the terminal COOH of an amino acid or a peptide, COCH2CH2CO-R<2> (wherein R<2> is a group derived by removing one H atom from the terminal NH2 of an amino acid or a peptide) or a monovalent group derived by removing the terminal NH2 from an amino acid or a peptide] provided that the above NH2 or COOH may be protected is allowed to react with a sulfonating agent and optionally neutralized or salt-exchanged with an alkali metal hydride or the like to obtain the title polysulfuric ester or a salt thereof.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to a cyclodextrin derivative/polysulfate ester having antiretroviral activity and a method for producing the same.

0002

[Prior Art] AIDS (acquired immunodeficiency syndrome) is caused by the human immunodeficiency virus (HIV), a type of retrovirus.
) is a fatal or extremely malignant disease caused by infection, and its prevention and eradication is currently the most important problem for humanity to overcome on a global level. Conventionally, as a compound having an antiretroviral effect, for example, azidothymidine [Igaku no Ayumi; Vol. 142, No. 9, 619-62
2 (1987)] and sulfated polysaccharides (Japanese Unexamined Patent Publication No. 1987-
No. 45223, No. 64-25724), etc. are known.

0003

OBJECTS OF THE INVENTION An object of the present invention is to provide a novel pharmaceutical compound that has excellent antiretroviral activity, particularly excellent antiproliferative activity against HIV, and is free from side effects.

0004

[Means for Solving the Problems] The present invention provides that at least one of the 6 to 8 D-glucose units constituting the cyclodextrin has the general formula

[0005]

[C6]

[However, R1 is (1) a monovalent group obtained by removing the hydroxyl group from the terminal carboxyl group of an amino acid or peptide, (2) a group represented by the formula -COCH2CH2CO-R2 (R
2 represents a monovalent group obtained by removing one hydrogen atom from the terminal amino group of an amino acid or peptide), or 2 represents a monovalent group obtained by removing one hydrogen atom from the terminal amino group of the amino acid or peptide, and The amino group or carboxyl group indicates that the amino group or carboxyl group may be a protected amino group or carboxyl group. The present invention relates to a polysulfate ester of a cyclodextrin derivative (hereinafter referred to as a polysulfate ester compound) substituted with the unit shown in the formula (hereinafter referred to as a polysulfate compound) or a salt thereof. The polysulfate ester compound of the present invention is schematically shown as follows.

[0007]

[Chemical 7]

(However, n is an integer of 6 to 8, m is an integer of 1 or more and n or less, R is all or part -SO3H,
The remainder is a hydrogen atom, and R1 has the same meaning as above. ) The above general formula (I) represents that 6 to 8 structural units are cyclically bonded at the 1st and 4th positions in any order. Furthermore, in the general formula (I), a compound in which n is 6 is a derivative of α-cyclodextrin, and n is 7.
The compound where n is 8 is a derivative of β-cyclodextrin, and the compound where n is 8 is a derivative of γ-cyclodextrin.

[0009] The amino acid used in the present invention is of natural or synthetic origin, and is a compound having at least one amino group and one carboxyl group in one molecule, and is represented by the following formula: .

0010

[Chemical formula 8]

[However, A represents a group represented by the formula -CH(Rbo)- or an alkylene group, where Rbo is a guanidino group, a carboxyl group, a carbamoyl group, an imidazolyl group, a mercapto group, an amino group, a lower alkylthio group. ,
A lower alkyl group which may have a substituent selected from a phenyl group, a hydroxyl group, an indolyl group and a hydroxyphenyl group; or a phenyl group which may have a hydroxy group, and Rao represents a hydrogen atom; or Rb
It represents that it is bonded to o at the terminal to form a trimethylene group. Among these amino acids, naturally occurring amino acids include, for example, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, which are component amino acids of biological proteins. Methionine, phenylalanine, proline, serine,
Examples include threonine, tryptophan, tyrosine, and valine. Furthermore, naturally occurring amino acids and synthetically occurring amino acids other than the above-mentioned amino acids include, for example, phenylglycine, hydroxyphenylglycine, ω-aminoalkylcarboxylic acids (such as β-alanine, aminopentylcarboxylic acid, aminodecylcarboxylic acid, etc.). ) and other amino acids. Moreover, the peptide as used in the present invention refers to a peptide consisting of 2 to 3 amino acids as described above, and includes, for example, phenylalanylglycine, aspartoylbis(phenylalanine), and the like.

[0012] Among these amino acids or peptides, asparagine, aspartic acid, glycine, methionine, phenylalanine, proline, serine, tryptophan, hydroxyphenylglycine, ω-aminoalkylcarboxylic acid, phenylalanylglycine, and aspartoid are preferred. Rubis (phenylalanine), and more preferred are aspartic acid, methionine, phenylalanine, serine, tryptophan, and hydroxyphenylglycine.

00
13] Furthermore, the amino group or carboxyl group of the above amino acid or peptide may be a protected amino group or carboxyl group, and as the protecting group, those normally used in this field can be suitably used. . Examples of the protected amino group include lower alkanoyl group, benzoyl group, halogenobenzoyl group, lower alkylbenzoyl group, lower alkoxybenzoyl group, lower alkoxycarbonyl group, benzyloxycarbonyl group, lower alkoxyphenylcarbamoyl lower alkyl group, etc. A protected amino group is mentioned, and a protected carboxyl group includes, for example, a lower alkyl esterified, amidated, lower alkyl amidated, or lower alkoxyphenylamidated carboxyl group.

As a preferable example of the polysulfate ester compound of the present invention, in the general formula [I], R1 is

[
0015

[Chemical formula 9]

[However, P represents 0 or 1, A1 represents a group represented by the formula -CH(Rb1)- or an alkylene group, where Rb1 represents an optionally protected carboxyl group, carbamoyl group, lower represents a lower alkyl group which may have a substituent selected from an alkylthio group, a phenyl group, a hydroxyl group and an indolyl group; or a phenyl group which may have a hydroxy group; Ra1 represents a hydrogen atom; or It represents that it is bonded with Rb1 at the terminal to form a trimethylene group, and Rc represents a hydrogen atom or a protective group. ], or

■Formula -COCH2CH2CO-R21 [However, R21 is a group obtained by removing a hydrogen atom from the terminal amino group of aspartoylbis (phenylalanine) whose carboxyl group may be protected, or an α-amino group and a carboxyl group represents a group obtained by removing a hydrogen atom from an optionally protected ω-amino group of lysine. ], or (ii) a group obtained by removing an amino group from aspartic acid whose carboxyl group may be protected.

[0018] Furthermore, the polysulfate ester compound of the present invention (
I) has 9 to 23 sulfate groups (-S
O3H) is preferred. Compound of the present invention (I
), the amino acid unit of the group represented by R1 is in the L-form or the D-form, and the present invention includes any of these isomers or mixtures thereof. The polysulfate compound (I) of the present invention can be suitably used either in its free form or in its pharmacologically acceptable salt form. Examples of such salts include alkali metal salts such as sodium salts, potassium salts, and lithium salts; alkaline earth metal salts such as calcium salts, magnesium salts, and barium salts; trimethylamine salts, triethylamine salts, pyridine salts, glycine ethyl ester salts, Examples include organic amine salts such as ethanolamine salts and basic amino acid salts.

The polysulfate compound (I) of the present invention or a pharmacologically acceptable salt thereof can be administered orally or parenterally (for example, intravenously, intramuscularly, subcutaneously), and can be administered orally or parenterally (for example, intravenously, intramuscularly, subcutaneously). For example, tablets, granules, capsules, powders,
It can be used in appropriate pharmaceutical preparations such as injections, suppositories, vaginal preparations, and creams. The dosage of the polysulfate ester compound of the present invention or its pharmacologically acceptable salt varies depending on the age, weight, condition, and type of disease of the patient, but is usually about 0.1 to 200 mg/kg per day. It is suitable, and particularly preferably about 0.5 to 50 mg/kg.

In the polysulfate compound (I) of the present invention, at least one of the 6 to 8 D-glucose units constituting the cyclodextrin has the general formula

[0021]

[Chemical formula 10]

(However, the symbols have the same meanings as above.) It can be produced by reacting a cyclodextrin derivative substituted with the unit shown in () with a sulfonating agent. The above reaction is schematically illustrated as follows.

[0023]

[Chemical formula 11]

(However, the symbols have the same meanings as above.) Examples of the sulfonating agent include sulfur trioxide complex (for example, sulfur trioxide-pyridine complex, sulfur trioxide-trialkylamine complex, sulfur oxide-dioxane complex,
Sulfur trioxide-N,N-dimethylformamide complex, etc.), sulfuric anhydride, concentrated sulfuric acid, chlorosulfuric acid, etc. can all be suitably used. Further, the amount of the sulfonating agent used is preferably in excess of the cyclodextrin derivative (II). For example, when using a sulfur trioxide-pyridine complex or a sulfur trioxide-trialkylamine complex as a sulfonating agent, the amount is 1 to 10 equivalents, especially 2 to 5 equivalents, based on the hydroxyl group of the cyclodextrin derivative (II). It is preferable to use it in moderation.

The reaction between the cyclodextrin derivative (II) and the sulfonating agent can be carried out in a suitable solvent or without a solvent. Examples of the reaction solvent include pyridine, N
, N-dimethylformamide, formamide, hexamethylenephosphoryltriamide, chloroform, benzene, toluene, xylene, water, and mixtures thereof, etc. can be suitably used. This reaction can be carried out under cooling or under heating (-30°C to 140°C) depending on the sulfonating agent used, especially under heating (50°C to 100°C) when using a sulfur trioxide complex. It is desirable to carry out the In addition, in the above reaction, if a compound having an amino group substituted with a trityl group is used as the raw material cyclodextrin derivative (II), detritylation occurs simultaneously with sulfuric acid esterification by the above treatment, and the trityl group is removed. The removed object (I) is obtained.

After the reaction is completed, the target product is isolated and
Can be purified. For example, the crude product obtained from the reaction completion solution is neutralized or salt-exchanged with alkali metal hydroxide, etc., and then subjected to a column packed with cross-linked dextran gel, etc., or purified with an anion exchange resin, and then hydroxylated. By treating with an alkali metal or the like, the target product can be obtained as an alkali metal salt.

The cyclodextrin derivative (II), which is the raw material compound of the present invention, is a new compound, among which R
1 is ■ a monovalent group obtained by removing the hydroxyl group from the terminal carboxyl group of an amino acid or peptide, or ■ formula -COCH2
Compound (II-a), which is a group represented by CH2CO-R2 (R2 has the same meaning as above), is a mono- or poly(6-amino-6-deoxy)-α, β or γ-cyclodextrin and an amino acid. , a peptide, or a compound represented by the general formula HOOCCH2CH2COR2 (however, the symbols have the same meanings as above). In addition, compounds in which R1 is a monovalent group excluding the terminal amino group of an amino acid or peptide include mono- or poly(6-iodo-6-deoxy)-α,
It can be produced by reacting β- or γ-cyclodextrin with an amino acid or a peptide. The present invention will be described below with reference to Examples, but the present invention is not limited thereto.

In the present invention, lower alkyl groups and lower alkoxy groups refer to those having 1 to 6 carbon atoms, especially those having 1 to 4 carbon atoms, and lower alkanoyl groups refer to those having 2 to 7 carbon atoms, especially those having 1 to 4 carbon atoms. It refers to those with 2 to 5 carbon atoms, and the alkyl group and alkylene group have 1 to 1 carbon atoms.
5, particularly those having 1 to 10 carbon atoms.

[0029]

[Example] Example 1 70 ml of pyridine was added to 0.70 g of mono[6-(Nα-benzyloxycarbonyltryptophyl)amino-6-deoxy]-β-cyclodextrin, and then 10
Sulfur trioxide-pyridine complex at 0°C 4.59
g and stirred at the same temperature for 6 hours. After the reaction, remove the supernatant, wash the residue with methanol, and add 10ml of water to the residue.
1 and 2.05 g of sodium acetate, stirred for 30 minutes, and concentrated under reduced pressure. After washing the residue, it was dissolved in water, purified using a Sephadex G-10 column (manufactured by Pharmacia), and then lyophilized to obtain mono[6-(Nα-benzyloxycarbonyltryptophyl)amino-6-
1.25 g of the sodium salt of [deoxy]-β-cyclodextrin polysulfate is obtained as a pale yellow powder. Yield: 179% (wt% of target product relative to raw material compound,
(Same below) IRνMaxKBrcm-1: 3500, 1710, 1
650, 1550, 1460, 1250, 1165, 1
050,1005,950,820 1H-NMR (D2O) δ: 6.8-7.6 (m), 7
．． 7-8.0 (brd) Number of sulfate ester groups in the molecule calculated from elemental analysis value: 17

Examples 2 to 35 The corresponding starting compounds and sulfur trioxide-pyridine complexes are treated in the same manner as in Example 1 to obtain the compounds listed in Table A below.

[0031]

[Table 1]

[0032]

[Table 2]

[0033]

[Table 3]

[0034]

[Table 4]

[0035]

[Table 5]

[0036]

[Table 6]

[0037]

[Table 7]

Example 36 By treating bis[6-(11-tritylaminoundecanoyl)amino-6-deoxy]-β-cyclodextrin and sulfur trioxide-pyridine complex in the same manner as in Example 1, The sodium salt of bis[6-(11-aminoundecanoyl)amino-6-deoxy]-β-cyclodextrin polysulfate is obtained as a colorless powder. Yield: 144% IRνMaxKBrcm-1:1640,1540,1
240,1165,1040,1000,945,82
0,740 1H-NMR (D2O) δ: 0.9-1.8 (m), 2
．． 0-2.5 (m), 3.02 (t) Number of sulfate ester groups in the molecule calculated from elemental analysis values: 16

Reference Example 1 2.268 g of mono(6-amino-6-deoxy)-β-cyclodextrin, 0.368 g of 1-hydroxybenztriazole, N-benzyloxycarbonyl-L-
Tryptophan 0.81g, anhydrous magnesium sulfate 1.
A mixture of 0 g and 10 ml of N,N-dimethylformamide was stirred at room temperature for 1 hour, cooled on ice, 0.494 g of N,N'-dicyclohexylcarbodiimide was added, and the mixture was stirred at room temperature for 1 day. Add 0.2 ml of acetic acid and 1 ml of water to the reaction mixture.
After stirring for 1 hour, concentrate under reduced pressure, add 50 ml of ethanol to the residue, and remove the soluble portion. 100 ml of a 1:1 mixture of methanol and water was added to the residue, the soluble portion was distilled off under reduced pressure, and the resulting precipitated crystals were recrystallized from water-containing methanol to obtain mono[6-(Nα-benzyloxycarbonyltryptophyl)]. )amino-6-deoxy]-β-
1.65 g of cyclodextrin are obtained as colorless crystals. IRνMaxKBrcm-1:3400,1710,1
660, 1540, 1250, 1155, 1080, 1
030,950

Reference Examples 2 to 34 Mono- or poly(6-amino-6-deoxy)-β-cyclodextrin and the corresponding raw material compounds are treated in the same manner as in Reference Example 1 to obtain the compounds listed in Table B below. .

[0041]

[Table 8]

[0042]

[Table 9]

[0043]

[Table 10]

[0044]

[Table 11]

[0045]

[Table 12]

Reference Example 35 (1) To a mixture of 26.3 g of mono(6-O-mesitylenesulfonyl)-β-cyclodextrin and 180 ml of N,N-dimethylformamide, 60 ml of sodium iodide was added.
g and stirred at 100°C for 2 hours. After cooling the reaction mixture on ice, the precipitate was filtered off, and the filtrate was added to 1 liter of acetone. By filtering the precipitated crystals and recrystallizing them from 50 ml of water, mono(6-deoxy-6-iodo)-β-
18.5 g of cyclodextrin was obtained. m. p. 190-193°C (decomposition) (2) 2.49 g of mono(6-deoxy-6-iodo)-β-cyclodextrin, 2.06 g of N,N'-bis(4-methoxyphenyl)-L-aspartic acid diamide
A mixture of 12 ml of N,N-dimethylformamide and N,N-dimethylformamide is stirred at 80° C. for 4 days under a nitrogen stream. After adding 200 ml of water to the reaction mixture, it was neutralized by adding sodium bicarbonate, washed, concentrated, purified, and freeze-dried to obtain mono[6-[1,2-bis(4-methoxyphenylamino)]. 0.72 g of carbonyl)ethyl]amino-6-deoxy]-β-cyclodextrin is obtained. m. p. 240℃ (decomposition)

Reference Example 36 Bis[6-(11
-tritylaminoundecanoyl)amino-6-deoxy]-β-cyclodextrin is obtained. m. p. 213℃ (decomposition)

[0048]

[Action] Experimental example HIV-1 proliferation inhibitory action (Principle) Human T-cell leukemia virus type I [human
T-cell Leukemia virus, HTLV
-I] HIV-1 in MT-4 cells, a persistently infected cell line
It is known that when infected with MT-4, HIV-1 rapidly proliferates, and MT-4 cells die due to cytotoxicity in 5 to 6 days. Therefore, the HIV-1 proliferation inhibitory effect of the sample can be investigated using the number of viable MT-4 cells infected with HIV-1 as an index.

(Method) MT-4 cells were infected with HIV-1 (TA
LL-1/LAV-1 culture supernatant) at 0.001 T
CID50 (median tissue
After infecting the cells for 1 hour at 37°C, the cells were infected with RPMI containing various concentrations of the specimen.
-1640 medium [FCS (fetal calf se
rum: 1×105ce containing 10% fetal bovine serum)
It was suspended at a concentration of 1/ml. Pour this cell suspension into a 24-well flat bottom culture plate at an amount of 1 ml/well.
The cells were cultured for 5 days at 37°C in the presence of 5% carbon dioxide. After culturing, the number of living cells in the cell suspension was counted by trypan blue staining. The HIV-1 proliferation inhibitory effect of the specimen is M
It was determined as the concentration of the specimen that inhibited 100% of the infectivity and cytopathic effects of HIV-1 on T-4 cells. (Results) The results are as shown in Table C below.

[0050]

[Table 13]

[0051]

[Table 14]

[0052]

[Effects of the Invention] As mentioned above, the polysulfate ester compound of the present invention has an excellent antiretroviral effect, especially an excellent antiproliferation effect against HIV, and is also characterized by low toxicity and high safety as a medicine. be. Also,
The polysulfate ester compound of the present invention also has weak side effects such as anticoagulant effects, which are known from sulfated polysaccharides.

Claims (9)

[Claims] Claim 1: 6 to 8 constituting cyclodextrin
At least one of the D-glucose units has the general formula:
A group represented by CH2CH2CO-R2 (R2 represents a monovalent group obtained by removing one hydrogen atom from the terminal amino group of an amino acid or peptide), or a monovalent group obtained by removing one hydrogen atom from the terminal amino group of an amino acid or peptide. and represents that the amino group or carboxyl group of the above amino acid or peptide may be a protected amino group or carboxyl group. ] A polysulfate ester of a cyclodextrin derivative or a salt thereof substituted with a unit represented by the following. 2. The amino acid or peptide has the general formula: , imidazolyl group, mercapto group, amino group, lower alkylthio group, phenyl group,
Represents a lower alkyl group which may have a substituent selected from a hydroxyl group, an indolyl group and a hydroxyphenyl group; or a phenyl group which may have a hydroxy group, and Rao represents a hydrogen atom or Rbo and Indicates that the terminus is bonded to form a trimethylene group. The compound according to claim 1, which is an amino acid represented by the following formula or a peptide consisting of 2 to 3 such amino acids. 3. The amino acid or peptide is aspartic acid, asparagine, methionine, phenylalanine, glycine, proline, serine, tryptophan, hydroxyphenylglycine, ω-aminoalkylcarboxylic acid, phenylalanylglycine, aspartoylbis(phenylalanine). A compound according to claim 1. 4. The compound according to claim 1, wherein the amino acid or peptide is aspartic acid, methionine, phenylalanine, serine, tryptophan, or hydroxyphenylglycine. 5. R1 is a compound of the formula (3) [wherein p represents 0 or 1, and A1 is a compound of the formula -CH(Rb
1) represents a group represented by - or an alkylene group, where R
b1 is a lower alkyl group that may have a substituent selected from an optionally protected carboxyl group, a carbamoyl group, a lower alkylthio group, a phenyl group, a hydroxyl group, and an indolyl group; or a hydroxy group; Ra1 represents a hydrogen atom or is bonded with Rb1 at the terminal to form a trimethylene group, and Rc represents a hydrogen atom or a protective group. ], or the formula -COCH2CH2CO-R
21 [However, R21 is a group obtained by removing a hydrogen atom from the terminal amino group of aspartoylbis(phenylalanine), which may have a protected carboxyl group, or lysine, whose α-amino group and carboxyl group may be protected. ω
-Represents a group obtained by removing a hydrogen atom from an amino group. 2. The compound according to claim 1, which is a group represented by the following formula, or (2) a group obtained by removing an amino group from aspartic acid whose carboxyl group may be protected. 6. The compound according to claim 1, having 9 to 23 sulfate groups. Claim 7: 6 to 8 constituting cyclodextrin
At least one of the D-glucose units has the general formula:
A group represented by CH2CH2CO-R2 (R2 represents a monovalent group obtained by removing one hydrogen atom from the terminal amino group of an amino acid or peptide), or a monovalent group obtained by removing one hydrogen atom from the terminal amino group of an amino acid or peptide. and represents that the amino group or carboxyl group of the above amino acid or peptide may be a protected amino group or carboxyl group. ] A method for producing a polysulfate ester of a cyclodextrin derivative or a salt thereof, which comprises reacting a cyclodextrin derivative substituted with the unit represented by the above with a sulfonating agent, and optionally converting the product into a salt thereof. . 8. The method according to claim 7, wherein the sulfonating agent is sulfur trioxide complex, anhydrous sulfuric acid, concentrated sulfuric acid or chlorosulfuric acid. Claim 9: 6 to 8 constituting cyclodextrin
At least one of the D-glucose units has the general formula:
A group represented by CH2CH2CO-R2 (R2 represents a monovalent group obtained by removing one hydrogen atom from the terminal amino group of an amino acid or peptide), or a monovalent group obtained by removing one hydrogen atom from the terminal amino group of an amino acid or peptide. and represents that the amino group or carboxyl group of the above amino acid or peptide may be a protected amino group or carboxyl group. ] Cyclodextrin derivatives substituted with the unit shown.