JPH10324700A - New anti-hiv conjugate and medicinal composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject conjugate to which (i) a polypeptide having an affinity to HIV surface protein and HIV host target cell surface protein and (ii) a reverse transcriptase inhibitor or an HIV protease inhibitor is linked, and which has low toxicity and high anti-HIV activity. SOLUTION: This conjugate, which is useful as an anti-HIV agent or the like having low toxicity and high activity, is obtained by chemically linking one or more reverse transcriptase inhibitor(s) and/or an HIV protease inhibitor to a peptide having an affinity to HIV surface protein gp120 and/or HIV host target cell surface protein CD4, shown by formula I [A1 is H, or lysine, arginine, or ornithine residue, etc.; A2 is tyrosine or phenylalanine residue, etc.; A3 is lysine or ornithine residue; A4 is lysine or arginine residue, etc.; A5 is OH or NH2 ; Y is a peptide residue shown by formula II (A6 is alanine or valine residue, etc.; A7 is tyrosine or phenylalanine residue, etc.); Cys in positions 3 and 11 can be linked each other by the S-S bond].

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel substance having anti-HIV activity and a pharmaceutical composition containing the substance as an active ingredient. More specifically, a complex in which a reverse transcriptase inhibitor and / or an HIV protease inhibitor are bound to a polypeptide having anti-HIV activity by a chemical bond,
And a pharmaceutical composition containing the substance as an active ingredient, such as an anti-HIV agent.

[0002]

[0002] Human immunodeficiency virus (HIV) is known to cause various clinical symptoms such as acquired immunodeficiency syndrome (AIDS) and AIDS-related syndrome.
As an anti-HIV agent, a drug that inhibits each stage of HIV proliferation is known, but as a drug that is actually approved as a drug or is undergoing clinical trials,
Reverse transcriptase inhibitors (for example, nucleoside 3′-azido-3′-deoxythymidine (hereinafter also referred to as AZT), 2 ′, 3′-deoxyinosine (hereinafter also referred to as ddI), 2 ′, 3 ′ -Deoxycytidine (hereinafter dd)
C)) and HIV protease inhibitors (N '-[1 (S) -benzyl-3- [4a (S), 8
a (S), 3 (S)-(tert-butylcarbamoyl) decahydroisoquinolin-2-yl] -2 (R) -hydroxypropyl] -N "-quinolin-2-ylcarbamoyl) -L-asparaginamide ( The following Ro 31-89
59) is known.

Usually, since these anti-HIV agents need to be administered in large amounts and for a long period of time, side effects (for example, bone marrow disorders) and the emergence of drug-resistant viruses due to amino acid mutations in reverse transcriptase or HIV protease. And other problems. Use of AZT in combination with ddI or ddC for the purpose of solving these problems and enhancing the antiviral effect (for HIV infection / A
IDS, Nihon clinical company, p316-326 (1993)), Ro 31-
Using 8959 in combination with AZT or ddC (J. Infect. Dis., 166 (5), p1143-1146 (1992)) and 3TC with AZT, ddI or Ro 31-89.
59 in combination (see JP-A-6-23464).
1) is known. However, even in the use of these combinations, the antiviral activity is insufficient,
Moreover, the above-mentioned problems have not been sufficiently solved. By the way, a novel polypeptide having an antiviral activity and an anti-HIV agent containing this polypeptide as an active ingredient are known (WO 92/04374, JP-A-5-16329).
8, International Publication WO95 / 10534), it is known that the polypeptide exhibits the same level of anti-HIV activity as AZT, and it can be administered in combination with a reverse transcriptase inhibitor or an HIV protease inhibitor. Excellent anti-HI
It is also known to exhibit V activity (Japanese Unexamined Patent Publication No. 9-2524).
0).

[0004]

SUMMARY OF THE INVENTION Reverse transcriptase inhibitors and H
The side effects due to toxicity such as an IV protease inhibitor cannot be reduced even by administration of a combination of the polypeptide having the anti-HIV activity and a reverse transcriptase inhibitor or an HIV protease inhibitor or the like. And less toxic anti-HIV
Agents are expected.

[0005]

SUMMARY OF THE INVENTION The present inventors have developed an excellent anti-HI
In order to obtain a complex useful as an anti-HIV agent that retains V activity and has fewer side effects, it has an anti-HIV activity
As a result of intensive studies based on a polypeptide having high affinity for the V-surface protein, an anti-HIV active substance such as a reverse transcriptase inhibitor or an HIV protease inhibitor was chemically bound to the amino (N) terminal of the polypeptide. The complex has anti-H
The present inventors have found that they show excellent anti-HIV activity when used as an IV agent and can reduce side effects, and completed the present invention.

That is, the present invention relates to a polypeptide of the following formula (1) having an affinity for an HIV surface protein, comprising one or more anti-HIV active substances (reverse transcriptase inhibitors and / or HIV protease inhibitors) And a pharmaceutical composition containing the complex as an active ingredient.

Embedded image 1 2 3 4 5 6 7 8 9 10 10 11 12 13 A1-A2-Cys-A2-A3-A3-Y-A2-A3-A3-Cys-A4-A5 (1) [Wherein A1 is H- (derived from the amino group of A2) or one basic amino acid selected from lysine, arginine and ornithine, or at least two identical or different amino acid residues selected from these amino acid residues] N-α acyl-substituted amino acid residue in which a hydrogen atom at the N-α position of the amino acid residue of the basic amino acid residue or the peptide residue is substituted with an acyl group or a substituted thiocarbamoyl group. Groups, N-α acyl substituted peptide residues, N-α substituted thiocarbamoylated amino acid residues or N-α substituted thiocarbamoylated peptide residues;

A2 independently represents a tyrosine, phenylalanine or tryptophan residue; A3 independently represents a lysine or arginine residue; A4 represents an amino acid residue having at least one amino acid selected from lysine and arginine; A5 represents a peptide residue;
Indicates OH (A4 from carboxyl group) or -NH ₂ (acid amide derived from the carboxyl group of A4); peptide residue Y is represented by the following formula (a)

Embedded image 1 ′ 2 ′ 3 ′ 4 ′ 5 ′ 6′-A6-A2-A3-Gly-A7-A6- (a) (where A2 and A3 have the same meanings as in the above formula (1)) A6 independently represents an alanine, valine, leucine, isoleucine, serine, cysteine or methionine residue, and A7 represents tyrosine, phenylalanine, tryptophan, alanine, valine, leucine, isoleucine,
Indicates a serine, cysteine or methionine residue;
y represents a glycine residue. However, when both the 1′-position and the 6′-position are cysteine residues, these may be linked by a disulfide bond. ), Or

D-ornithyl-proline, prolyl-D
-Ornithine, prolyl-D-lysine, prolyl-D-
Arginine, D-lysyl-proline, D-arginyl-
Proline, glycyl-lysine, glycyl-arginine,
Ornithyl-glycine, glycyl-ornithine, lysyl-glycine and arginyl-glycine are selected from dipeptides composed of two amino acids, and the constituent amino acids D-lysine, lysine, D-ornithine or The hydrogen atom in the side chain ω-amino group of ornithine indicates a peptide residue which may be substituted with an acyl group; Cys indicates a cysteine residue, and positions 3 and 1
The cysteine residue at position 1 may be linked by a disulfide bond.]

[0009] The complex of the present invention is characterized in that at least the above-mentioned polypeptide and an anti-HIV active substance such as AZT are bound by a chemical bond, whereby H
An anti-HIV active substance is delivered to a CD4-positive cell which is a target of IV infection, the anti-HIV active substance is released in the cell, and the cell exhibits excellent anti-HIV activity. Is highly safe and effective as a pharmaceutical composition such as an anti-HIV agent with less side effects.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. The novel complex of the present invention (hereinafter sometimes referred to as the substance of the present invention) contains the HIV surface protein gp120 and / or
Alternatively, it is a complex in which one or more reverse transcriptase inhibitors and / or HIV protease inhibitors are bound to a polypeptide having an affinity for the HIV target cell surface protein CD4 by a chemical bond. The polypeptide constituting the present invention is not particularly limited as long as it has an affinity for HIV surface protein gp120 and / or HIV host target cell surface protein CD4.
Examples of such a polypeptide include an antibody or a polypeptide represented by the following formula (1).
It also has an affinity for the cell surface protein CD4 of CD4-positive cells (host target cells) that are HIV host targets (Bi
ochem. Biophys. Res.Commun., 219, 555-559 (1996), Bio
Chem. Biophys. Acta., 1298.37-44 (1996)), and a polypeptide represented by the following formula (1), which itself has anti-HIV activity, is preferred.

[0011]

Embedded image 1 2 3 4 5 6 7 8 9 10 10 11 12 13 A1-A2-Cys-A2-A3-A3-Y-A2-A3-A3-Cys-A4-A5 (1) [Wherein, A1 has at least two identical or different amino acid residues selected from H- (derived from A2 amino group) or one basic amino acid selected from lysine, arginine and ornithine, or these amino acid residues] A peptide residue, or an N-α acyl-substituted amino acid residue in which the hydrogen atom at the N-α position of the amino terminal amino acid residue of the basic amino acid residue or the peptide residue is substituted with an acyl group or a substituted thiocarbamoyl group A, N-α acyl-substituted peptide residue, N-α-substituted thiocarbamoylated amino acid residue or N-α-substituted thiocarbamoylated peptide residue; A2 is independently tyrosine, A3 independently represents a lysine or arginine residue; A4 represents an amino acid residue or a peptide residue having one or at least two amino acids selected from lysine and arginine; A5 Is -OH (from the carboxyl group of A4) or-
NH ₂ (from the acid amide of the carboxyl group of A4);

Y is a peptide residue represented by the following formula (a):

Embedded image 1 ′ 2 ′ 3 ′ 4 ′ 5 ′ 6′-A6-A2-A3-Gly-A7-A6- (a) (where A2 and A3 have the same meanings as in the above formula (1)) A6 independently represents alanine, valine, leucine, isoleucine, serine, cysteine or methionine residue; A7 represents tyrosine, phenylalanine, tryptophan, alanine, valine, leucine, isoleucine,
Indicates a serine, cysteine or methionine residue;
y represents a glycine residue. However, when both the 1′-position and the 6′-position are cysteine residues, these may be linked by a disulfide bond. ), Or

D-ornithyl-proline, prolyl-D
-Ornithine, prolyl-D-lysine, prolyl-D-
Arginine, D-lysyl-proline, D-arginyl-
Proline, glycyl-lysine, glycyl-arginine,
Ornithyl-glycine, glycyl-ornithine, lysyl-glycine and arginyl-glycine are selected from dipeptides composed of two amino acids, and the constituent amino acids D-lysine, lysine, D-ornithine or The hydrogen atom in the side chain ω-amino group of ornithine indicates a peptide residue which may be substituted with an acyl group; Cys indicates a cysteine residue, and positions 3 and 1
The cysteine residue at position 1 may be linked by a disulfide bond].

Since the polypeptide represented by the formula (1) is a polypeptide synthesized based on a horseshoe crab-derived tachyplesin family polypeptide, a horseshoe crab-derived tachyplesin I, tachyplesin II, tachyplesin III, polyphemsin I or polyphemsin is used. II (metabolism, 26, p429-439 (1
989)) can be used as an alternative to the above polypeptides.

As used herein, having anti-HIV activity means having the ability to exhibit effects such as suppressing HIV infection, suppressing HIV proliferation, and reducing HIV. The polypeptide represented by the above formula (1) (hereinafter referred to as a polypeptide chain for convenience) is synthesized by a known polypeptide synthesis method, in particular, a liquid phase synthesis method or a solid phase synthesis method (New Chemistry Laboratory Course 1 Protein VI). , Tokyo Chemical Dojin, p3-29 (1992)). Alternatively, DNAs encoding the amino acid sequences of these polypeptides can be synthesized by introducing them into host cells by gene recombination techniques and expressing them. That is, for example, in the case of the solid phase synthesis method, for example, the carboxyl group of the N-protected amino acid corresponding to the carboxyl terminal amino acid residue of the polypeptide chain is directly or, optionally, via an intervening substance, and is appropriately converted to an amino group or hydroxyl group. After binding to an insoluble resin having a functional group, the protected amino acids from position 11 to position 1 of the amino acid sequence represented by the above formula (1) are sequentially bonded according to a solid phase synthesis method to obtain a protected group-protected peptide resin. .

As the insoluble resin, carboxyl (C)
It can be covalently bonded directly to the carboxyl group of the terminal N-protected amino acid or optionally through an intervening agent,
There is no particular limitation as long as it can be desorbed thereafter. Examples of such an insoluble resin include a resin that releases an amino acid or a peptide acid after desorption of the insoluble resin, an alkoxy-type resin (such as Alko-resin),
Or an amide-type resin that releases an amino acid amide or a peptide amide after elimination (Rink amideresire
n etc.). Specifically, resins that release amino acids or peptide acids include chloromethyl resin, oxymethyl resin, PAM (4- (hydroxymethyl) phenylacetamidomethyl) resin, and Alko-resin.
As p-alkoxybenzyl alcohol (Wan
g) Resin, hydroxymethylphenoxyacetic acid (HMP
A) Resins, dialkoxybenzyl alcohol-type resins, trialkoxydiphenylmethyl alcohol-type resins, and the like, and amide-type resins include benzhydrylamine (BHA) resin and p-methylbenzhydrylamine (M
BHA) resin, trialkoxybenzhydrylamine type resin, PAL (peptide amide link)
er) Resin and Rink amide resin (4-
(2 ′, 4′-dimethoxyphenyl-aminomethyl) -phenoxy resin and the like).

The protected amino acid is an amino acid in which a functional group is protected with a protecting group by a known method, and various protected amino acids are commercially available. The protecting group is preferably selected from those known per se according to the conditions for peptide synthesis. The binding of the protected amino acid can be carried out according to a usual condensation method, but includes a DCC (dicyclohexylcarbodiimide) method, a DCC-HOBt (1-hydroxybenzotriazole) method, a DIPCI (N, N-diisopropylcarbodiimide) -HOBt method, and a BOP method. (Benzotriazole-N-hydroxytrisdimethylaminophosphonium hexafluorophosphide) -HOBt method,
HBTU (2- (1H) -benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate) -HOBt method, symmetric anhydride method and the like are preferable. These condensation reactions are usually performed in an organic solvent such as dichloromethane, dimethylformamide (DMF), N-methylpyrrolidone (NMP) or a mixture thereof.

In formula (1), an N-α-acyl amino acid residue or N-α-acyl peptide residue in which the hydrogen atom at the N-α position of the amino-terminal amino acid residue in A1 is substituted with an acyl group is selected. In the case of using the above-mentioned protecting group-protected peptide resin or the peptide released from the protecting group-protected polypeptide resin, and the corresponding acid anhydride of the acyl group or the corresponding carboxylic acid, using a condensing agent The N-terminal free amino group of the peptide resin or peptide is acylated to obtain an N-acylated peptide resin. Further, an N-α-substituted thiocarbamoylated amino acid residue or N-α-substituted thiocarbamoylated peptide in which the hydrogen atom at the amino terminal N-α position in A1 in the formula (1) is substituted with a substituted thiocarbamoyl group. When a residue is selected, the substance of the present invention can be obtained by reacting a substituted isothiocyanate compound with a peptide protected from the desired protected group-protected peptide resin or the protected group-protected polypeptide resin under slightly alkaline conditions. N-terminal N-α-substituted thiocarbamoylated polypeptides can be obtained. In this case, by appropriately selecting an insoluble resin, the carboxyl terminus of the amino acid residue at position 13 is free (in the formula (1), A5 is -O
H) or it can be converted to an acid amide (A5 in formula (1) corresponds to —NH ₂ ). The polypeptide having the protecting group and bound to the insoluble resin is hereinafter also referred to as a protecting group-protected polypeptide resin.

Specific examples of the polypeptide chains (protective groups are not described) constituting the protective group-protected polypeptide resin include those shown in Table 1 below.

[0020]

[Table 1]

[0021]

[Table 2]

[0022]

[Table 3]

[0023]

[Table 4]

[0024]

[Table 5]

Similarly, in Table 1, a polypeptide chain having a carboxyl group at the carboxy terminus (A in formula (1)
5 corresponds to -OH). In the description of the polypeptide in the present specification, each symbol represents an amino acid residue or a substituted amino acid residue in internationally recognized three-letter display, and each symbol represents the following amino acid residue or substituted amino acid residue. In addition, when there is no "D-" before an amino acid indication, it means that they are all "L-" forms. Arg: arginine, Trp: tryptophan, Cy
s: cysteine, Tyr: tyrosine, Lys: lysine,
Gly: glycine, Phe: phenylalanine, Il
e: isoleucine, Ser: serine, Leu: leucine, Met: methionine, Val: valine, Ala: alanine, Pro: proline, Orn: ornithine, DO
rn: D-ornithine, DAArg: D-arginine, D
Lys: D-lysine, Ac-Arg: N-α-acetylarginine, Oct-Arg: N-α-octanoylarginine, Laur-Arg: N-α-laurylarginine, Myr-Arg: N-α-myristylarginine , Palm-Arg: N-α-palmitoyl arginine, FTC-Arg: N-α-fluorescein thiocarbaminated arginine, PTC-Arg: N-α-phenylthiocarbaminated arginine, Nicot-Arg:
N-α-nicotinyl arginine, ε-N-Ac-DLy
s: ε-N-ω-aminoacetyl-D-lysine, ε-N
-But-DLys: ε-N-ω-aminobutyryl-D
-Lysine, Parm-Orn: N-α-palmitoyl ornithine Examples of the protective group for the side chain functional group of the amino acid include the following. Bu ^t: tert-butyl, Boc: t-butoxycarbonyl, Fmoc: 2,2,5,7,8-pentamethyl chroman-6-sulfonyl

As a method for obtaining only a polypeptide chain from the protecting group-protected polypeptide resin, a known method and a method for removing a protecting group and an insoluble resin and forming a disulfide bond in one step described later can be mentioned. That is, α-
Examples of the reagent for removing an amino-protecting group include TFA (trifluoroacetic acid) / dichloromethane, HCl / dioxane,
Piperidine / DMF or piperidine / NMP is used, and is appropriately selected depending on the type of the protecting group. The protecting group-protected polypeptide resin includes hydrogen fluoride, TFMSA (trifluoromethanesulfonic acid), TMSOTf (trimethylsilyl triflate), TMSBr (trimethylsilyl bromide), and TMSCl (trimethylsilyl chloride).
Alternatively, by treating with TFA or the like, the resin and the protecting group can be simultaneously eliminated. The above elimination reagent,
It is appropriately selected depending on the synthesis strategy (Boc method or Fmoc method), the insoluble resin to be used, and the type of protective group.

Further, the obtained polypeptide chain has a 3
A disulfide bond (-SS-) can be formed via a mercapto group between the cysteines at the 1-position and the 11-position, or optionally between the 1'-position and the 6'-position. These disulfide bonds can be formed by a method known per se, for example, mild air oxidation. Typically, an oxidizing agent such as atmospheric oxygen or ferricyanate (eg, potassium ferricyanide) is used. The protecting group and the polypeptide thus formed by removing the resin and forming a disulfide bond can be further isolated and purified by a known method, but the method by reversed-phase high performance liquid chromatography is most effective. is there.

In the substance of the present invention, a reverse transcriptase inhibitor or an HIV protease inhibitor which is an anti-HIV active substance is chemically bound to the above-mentioned protecting group-protected polypeptide resin. Reverse transcriptase inhibitors are substances that inhibit the activity of HIV reverse transcriptase, and are classified into nucleoside-based inhibitors and non-nucleoside-based inhibitors. The nucleoside-based inhibitor is preferably a nucleoside or an analog thereof composed of a pyrimidine base, a purine base, an imidazole base or a triazole base, and a furanose having at least one hydroxyl group or an acyclo-form thereof. For example, AZT (CAS REGISTRY NUMBE
RS: 30516-87-1: zidovudine), ddI
(CAS REGISTRY NUMBERS: 69655-05-6: didanosine (did
anosine)), ddC (CAS REGISTRY NUMBERS: 7481-89-
2: zalcitabine), 2 ′, 3′-didehydro-2 ′, 3′-dideoxythymidine (CAS REGISTRY)
NUMBERS: 3056-17-5: d4T: stavudin
e)) 3′-thia-2 ′, 3′-dideoxycytidine (CAS REGISTRY NUMBERS: 134678-17-4: 3TC: lamivudine), 2′-β-fluoro-ddC,
3'-Fluorothymidine (CAS REGISTRY NUMBERS: 2552
6-93-6: FLT), 9- (2-phosphonyl-methoxyethyl) -adenine (CAS REGISTRY NUMBERS: 106941-25-
7: PMEA), 6-Cl-ddI, 6-Cl-ddC
And the like.

Examples of the non-nucleoside inhibitors include tetrahydro-imidazo-benzo-diazepin-one or -thione (TIBO) derivatives (specifically, (+)-S-4,5,6,7 -Tetrahydro-5-
Methyl-6- (3-methyl-2-butenyl) imidazo [4,5,1-jk] [1,4] benzodiazepine-2
(1H) -Thion) (CAS REGISTRY NUMBERS: 167206-2)
9-3: R82913), hydroxyethoxy-methylphenylthiothymine (HEPT) derivative, nevirapine (Ne
virapine) (CAS REGISTRY NUMBERS: 129618-40-2),
And pyridinone derivatives. Of these, nucleoside-based reverse transcriptase inhibitors are preferred, and nucleoside-based reverse transcriptase inhibitors are preferred, considering the ease of binding to the polypeptide chain and the mechanism by which DNA synthesis effectively inhibits DNA synthesis. Among the HIV transcriptase inhibitors, AZT, ddI, ddC, d4T or 3TC, which are preferably already administered to humans in clinical settings,
More preferably, AZT is chemically synthesizing with the polypeptide chain to particularly synergistically enhance the antiviral activity when the substance of the present invention is obtained. These nucleoside reverse transcriptase inhibitors and the like are capable of HIV
When NA is synthesized, it is incorporated into DNA, resulting in D
It is preferably a non-natural nucleoside or nucleoside analog to inhibit the synthesis of NA. The nucleoside analog refers to a non-nucleoside compound having a steric structure similar to that of a nucleoside. In addition, as these reverse transcriptase inhibitors, commercially available ones or those prepared according to known synthesis methods can be used.

The HIV protease inhibitor is preferably a substance which inhibits the activity of an HIV protease, and which is a substrate transition state mimic compound of the protease. The substrate transition state mimic is a substance that can bind to a substrate binding site of an enzyme, and refers to a substance having a three-dimensional structure similar to that of a substrate in an enzyme-substrate complex. For example, Ro 31-8959 (CAS REGISTRY NUMBERS: 127
779-20-8: saquinavir), A-77003
(CAS REGISTRY NUMBERS: 134878-17-4), A-809
87 (CAS REGISTRY NUMBERS: 144141-97-9), KNI
-93 (CAS REGISTRY NUMBERS: 138258-64-7), KN
I-102 (CAS REGISTRY NUMBERS: 139694-65-8),
KNI-174, KNI-227 (CAS REGISTRY NUMBE
RS: 147384-69-8), KNI-272 (CAS REGISTRY NU)
MBERS: 147318-81-8), L-735527 (CAS REGIST)
RY NUMBERS: 150378-17-9: Indinavir
r)), SC-52151 (CAS REGISTRY NUMBERS: 1432
24-34-4: Telinavir), VX-478,
ABT-538 (CAS REGISTRY NUMBERS: 155213-67-
5: ritonavir), DMP-323 (CAS RE
GISTRY NUMBERS: 151867-81-1), U-96988 (CAS
REGISTRY NUMBERS: 149394-65-0). More preferably, Ro 31- having high antiviral activity
8959, L-735527 and KN-272 are preferred, but not particularly limited. As these HIV protease inhibitors, commercially available ones or those prepared according to known synthesis methods can be used. Ro 31-
For 8959, for example, J. Med. Chem. 36, p2300-2310
(1993).

In the substance of the present invention, the above-mentioned polypeptide chain and the above-mentioned anti-HIV active substance are chemically bonded. However, the bond is not particularly limited as long as the bond is a chemically formed bond. Include an ester bond, an amide bond, an ether bond, and a disulfide bond. Of these, the ester bond is formed by transporting the bound anti-HIV active substance into a target cell in a living body, followed by intracellular esterase or the like. And can release the anti-HIV active substance near the action point of the anti-HIV active substance. The ester bond is preferably, but not limited to, a bond having such a stability that it is not easily cleaved during transportation.

In the substance of the present invention, the site of the polypeptide chain that binds to the anti-HIV substance is not particularly limited, but the polypeptide chain itself and the HIV surface protein gp120
In addition, an amino-terminal site capable of maintaining the affinity of the CD4 positive cell which is the host target cell with the cell surface protein CD4 is preferable. Specifically, the α-amino group or ω-amino group of the amino acid at the amino terminal, or the MAP system developed by Tam for the production of antibodies (multiple a)
ntigenic peptide system) (JPTam, Proc.Natl.Acad.Sc
iUSA, 85,5409-5413 (1988); JPTam, "Peptides: synt
hesis, structures, and applications "(B. Gutte, ed.), p.
p.456-500, Academic Press, Inc., New York (1995)) dendritic structure using radially-branched lysine (dendrime
r) is prepared at the amino terminus of the polypeptide chain by the method of Tam described above, and a plurality of anti-HIV active substances are bonded to a plurality of amino groups present at the terminus and side chains to form one molecule per polypeptide chain. It is also possible to bind more than one anti-HIV active substance. The substance used for forming the dendritic structure is most preferably the lysine described above, but is a substance containing two or more amino groups even if it is a basic amino acid such as ornithine or an amino acid prepared by synthesis in addition to lysine. It is possible and preferable to use it.

The binding site of the anti-HIV active substance that binds to the amino group of the polypeptide chain should be appropriately selected depending on the type of the active substance to be bound, and a carboxyl group or a hydroxyl group is present in the substance. In such a case, a chemical bond formed through these functional groups is preferable, and a chemical bond through a hydroxyl group is particularly preferable. Even if the position of the functional group is a site having a central role in anti-HIV activity, a chemical bond is cleaved in the target cell,
The position in the molecule of the functional group serving as a binding site is not particularly limited. For example, when the substance of the present invention is obtained by binding an anti-HIV active substance to a polypeptide chain via a carboxyl group in the substance, the amino group at the amino terminal of the protecting group-protected polypeptide resin and the anti-HIV active substance It is also possible to form an amide bond between the carboxyl groups and directly bond the same, or to form a chemical bond that can be easily cleaved in vivo through an appropriate spacer substance.

When the substance of the present invention is obtained by binding an anti-HIV active substance to a polypeptide chain via a hydroxyl group in the substance, the functional group at the amino terminal of the protecting group-protected polypeptide resin and the anti-HIV It can be attached via a multifunctional spacer having a functional group that can be chemically attached to both hydroxyl groups of the active substance. Such a polyfunctional spacer has one carboxyl group capable of binding to the amino group at the amino terminal of the polypeptide chain used, and forms an ester bond with the hydroxyl group in the anti-HIV active substance. It is preferable to have a carboxyl group different from the above, specifically, an aromatic compound or an aliphatic compound having two or more carboxyl groups. Group compounds are preferred.

That is, when a dicarboxylic acid is used as a polyfunctional spacer, an amide bond is formed between one carboxyl group and the amino group at the amino terminal of the polypeptide chain, and the other carboxyl group and the anti-HIV activity are used. By forming an ester bond with a hydroxyl group in the substance, the polypeptide chain and the anti-HIV active substance can be bound. Examples of the multifunctional spacer include simple synthesis, easy cleavage by an enzyme in a living body, and anti-HIV of the substance of the present invention.
In consideration of the activity and the like, an aliphatic dicarboxylic acid having 4 to 12 carbon atoms is preferable, and an aliphatic dicarboxylic acid having 4 to 8 carbon atoms is more preferable. Among the dicarboxylic acids, succinic acid or glutaric acid is particularly preferred from the viewpoint of stability and easy handling. In addition, dicarboxylic acid is a polypeptide chain and anti-HIV.
It may have another functional group as long as it does not prevent the binding of the active substance.

The protecting group-protected polypeptide resin and AZ
As a method of bonding T with a dicarboxylic acid (for example, succinic acid or glutaric acid) as a polyfunctional spacer, first, a conjugate of AZT and a polyfunctional spacer is formed. That is, for example, AZT is reacted with succinic anhydride or glutaric anhydride in the presence of dimethylaminopyridine, and after a conventional treatment, a conjugate of AZT in which succinic acid or glutaric acid is ester-bonded to AZT and a polyfunctional spacer is obtained. Next, the AZT-polyfunctional spacer conjugate is linked to the α-amino or ω-amino group of the amino acid at the N-terminal of the protecting group-protected polypeptide resin, or to a dendrimer-like structure in which the amino acid is dendrimer-bonded to the amino acid. Can be condensed and bound to a plurality of amino groups present at the terminal and side chains of the compound by a known method, for example, the DIPCI-HOBt method (this substance is also used as a protecting group-protected polypeptide resin-spacer-AZT complex). Described).

The protecting group-protected polypeptide resin-spacer-AZT complex obtained as described above can be obtained, for example, by removing the insoluble resin and the protecting group and forming a disulfide bond in one step by the following method. Thus, the substance of the present invention can be obtained. That is, for example, 10% is added to the protecting group-protected polypeptide resin-spacer-AZT complex.
Below, preferably less than 5%, more preferably 0.5 to 5%
1.5% (v / v) anisole, 1 equivalent or more, preferably 5 to 15 equivalents, more preferably 8 to 12 equivalents of TM
SCl (trimethylsilyl chloride) and TFA are added for 5 minutes or more, preferably for 30 to 120 minutes, at 0 to 80 ° C,
Preferably 10 to 40 ° C, more preferably 15 to 30 ° C
The reaction is performed under the following conditions, and then preferably at most 10 ° C., more preferably at least 100 equivalents under ice-cooling, and preferably at least 200
By adding ~ 400 equivalents of DMSO and reacting for 20 minutes or more, preferably 30 to 120 minutes, more preferably 60 to 100 minutes, the deprotection group, resin removal and formation of disulfide bond are performed in one step. A system for removing the protecting group and the resin and forming a disulfide bond is described below as TMS.
Described as Cl-DMSO / TFA system. When preparing the substance of the present invention in which a plurality of anti-HIV active substances are bound, it is preferable to bind not more than 32 anti-HIV active substances per polypeptide chain in consideration of the molecular size of the substance of the present invention, More preferably, the number is 16 or less. The substance of the present invention thus obtained can be isolated and purified by a known method, but the method by reversed-phase high performance liquid chromatography is most effective.

The active ingredient of the pharmaceutical composition of the present invention is the above-mentioned substance of the present invention or a pharmacologically acceptable salt thereof. Pharmaceutically acceptable salts include, for example, inorganic acids (hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, etc.), organic carboxylic acids (acetic acid, propionic acid, maleic acid, succinic acid, malic acid, citric acid, citric acid). Acid, tartaric acid, salicylic acid, etc.), acidic sugars (glucuronic acid, galacturonic acid, gluconic acid, ascorbic acid, etc.), or salts with organic sulfonic acids (methanesulfonic acid, p-toluenesulfonic acid, etc.), and the like. , Sodium salt, potassium salt, calcium salt,
A magnesium salt and the like are also included, but are not particularly limited. While it is possible to administer the substance of the present invention as it is, it may also be a pharmaceutical composition to which a pharmacologically acceptable carrier selected according to the administration method and administration form of the drug is further added. The pharmaceutical composition is orally or parenterally administered according to the treatment method or the like, and is injected, injectable, externally applied, suppository (for example, powder, Granules, injectable or oral liquids, tablets, pessaries, ointments, creams, aerosols, etc.). When the pharmaceutical composition of the present invention is directly administered to a living body, for example, as an injection, 10 m of a human body weight per day is 1 kg as a drug component.
g to 5 g can be administered.

[0039] The anti-HIV activity of the pharmaceutical composition of the present invention is determined by transporting an anti-HIV active substance into a target cell by a polypeptide chain having at least an affinity for an HIV surface protein, and a polypeptide chain on the target cell. An anti-HIV agent having a different mechanism of action of a polypeptide chain and an anti-HIV active substance by breaking a chemical bond between the polypeptide and the anti-HIV active substance;
That is, it is thought to be caused by a combination of a polypeptide which is thought to inhibit the steps of adsorption, fusion and invasion of HIV to CD4-positive cells and an anti-HIV agent which inhibits the enzyme activity of HIV reverse transcriptase or HIV protease. Can be That is, a therapeutic effect equivalent to that of the multidrug combination therapy is expected by using the substance of the present invention alone. In addition, the reason why the toxicity is reduced as compared with the conventional anti-HIV agent is that an anti-HIV active substance such as AZT, which has cytotoxicity itself, is chemically bound to the target cell and accurately delivered to the target cell. It is considered that this is because the effective concentration is suppressed and the toxicity to normal cells is suppressed.

[0040]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these without departing from the gist thereof. Test Example 1 TMSCl-DMSO-TFA-based de-side chain protection capability ^{Fmoc-Asp (Bu t) -OH} , Fmoc-Glu
^{(Bu t) -OH, Fmoc-} His (Boc) -O
^{H, Fmoc-Ser (Bu t} ) -OH, Fmoc-T
^{hr (Bu t) -OH, Fmoc} -Lys (Boc) -
OH, Fmoc-Arg (Pmc) -OH
MSCl (42 μl), DMSO (762 μl), and TFA (5.2 ml) were added and treated at 4 ° C.
After 0, 30, 60 and 90 minutes, sample 100 μl each and dilute to 1 ml with H ₂ O-MeCN (1: 1),
A part of the solution is injected into HPLC to obtain F
The moc amino acids and the side chain-protected Fmoc amino acids were quantified. In the following, HPLC analysis was performed using Cosmosil
The measurement was performed at a flow rate of 1 ml / min using a 5C18-AR (4.6 × 150 mm) column. Elution is 0.1%
(V / v) TFA-H 2 O and 0.1% (v / v) TFA
Performed with a linear gradient of -MeCN (acetonitrile). For separation by HPLC, Cosmosil C18-
AR (2.0 × 25 cm) was used at a flow rate of 7 ml / min. As a result, it was found that any of the side chain protecting groups could be quantitatively removed by this cleavage reagent system within 60 minutes.

Test Example 2 Amino Acid Release Ability from TMSCl-DMSO-TFA-Based Resin H-Leu-Al in which Leu is bonded to an alkoxyl-type resin that releases amino acids or peptide acids by cleavage treatment
ko resin (0.60 mmol / g, 2 μmo
1) Or Arg (Pmc) is applied to an amide type resin which releases amino acid amide or peptide amide by cleavage treatment.
-Arg (Pmc) -Rink amide bound to
e resin (0.32 mmol / g, 2 μmol)
Boc-Gly-OH (3 μmol) as an internal standard
, TMSCl (7 μl, 750 eq) and TFA
(870 μl), and the mixture was stirred at room temperature for 1 hour.
SO (127 μl, 750 eq) was added, and the mixture was further stirred for 1 hour. 10 minutes after 30, 60, 90, 120 minutes
Each 0 μl was sampled and diluted to 1 ml with distilled water. Regenerated amino acids were quantified using an amino acid analyzer. As a result, the regeneration rate of amino acids after 2 hours of treatment was Le
u: 100%, Arg: 87%. This indicates that Alko resin and Ri were used in this cleavage reagent system.
It was found that amino acids bound to nk amide resin could be quantitatively cleaved and released.

Preparation Example 1 Preparation of a protecting group-protected polypeptide resin having a polypeptide chain of the following formula (A)

[0043]

Embedded image (In the formula, Arg, Tyr, Cys, Lys, DLys and Pro indicate the amino acid residues described above, and the solid line between Cys at positions 3 and 11 indicates a disulfide bond.) The polypeptide chain of formula (A) The preparation of the protecting group-protected polypeptide resin was manually performed using an Fmoc-type solid phase synthesis method. As a resin for peptide synthesis, Fmoc-Ar
g (Pmc) -Alko resin (0.52 mmol / g in terms of Arg) was used. The mixture was treated with 20% piperidine / DMF for 15 minutes to remove the Fmoc group. For the condensation of Fmoc amino acids, DIPCI-HOB
t / DMF was used. The Fmoc amino acid, the condensing reagent and the like were used in 2.5 equivalents to the resin, respectively. Less than,
The substance synthesized in this manner is referred to as a protecting group-protected polypeptide resin 1 for convenience. Further, a protecting group-protected polypeptide resin that produces an amide of a polypeptide chain in which the 13-position of the formula (A) is NH ₂ is used as a peptide synthesis resin as Fmoc-Arg (Pmc) -Rink ami.
de resin (0.34 mmol / Arg conversion /
g) was synthesized by the same Fmoc-type solid phase synthesis method as described above.

Further, a protecting group-protected polypeptide resin having a polypeptide chain represented by the following formula (B) is used as a resin for peptide synthesis. Fmoc-PAL (peptide) which releases amino acid amide or peptide amide by cleavage treatment
Amide linker) resin (0.38 mmol / g in terms of amino group) using the same Fmo
It was synthesized by the c-type solid phase synthesis method. The substance synthesized in this manner is conveniently converted to a protecting group-protected polypeptide resin 2.
It is described.

[0045]

Embedded image (In the formula, Arg, Trp, Tyr, Cys, Gly and Lys represent the amino acid residues described above, and the solid lines connecting Cys between the 3-position and the 11-position, and between the 7-position indicate a disulfide bond.) Each amino acid condensation reaction in the phase synthesis was performed according to the operating conditions shown in Table 2.

[0046]

[Table 6]

Preparation Example 2 Preparation of 3'-azido-3'-deoxythymidine-5'-monosuccinate (AZT succinate) 3'-azido-3'-deoxythymidine (AZT) (5
34.8 mg, 2 mmol) was dissolved in THF (10 ml), and succinic anhydride (200.1 mg, 2 mmol) was added at 4 ° C.
l) then dimethylaminopyridine (48.8 mg,
0.4 mmol) and stirred for 48 hours. Furthermore,
Succinic anhydride (400.2 mg, 4 mmol) was added and the mixture was stirred for 24 hours. The reaction solution was treated with AcOEt (ethyl acetate:
50 ml), and the organic layer was washed with saturated saline, and then a saturated aqueous solution of NaHCO ₃ was added to carry out liquid separation. The aqueous layer was adjusted to pH 4 by adding citric acid, and AcOEt (50 ml) was added.
× 3), the organic layer was washed with a saturated saline solution, dried over anhydrous MgSO ₄ , MgSO ₄ was filtered, and the filtrate was distilled off under reduced pressure to obtain the desired AZT succinic ester crystals (5).
67.8 mg, yield 77.3%). ¹ H NMR (270 MHz, CDCl ₃ ) δ 1.86
(D, 3H), 2.6-2.9 (m, 6H), 4.11-
4.13 (m, 1H), 4.23-4.29 (dd, 1
H) 4.34-4.39 (m, 1H), 4.82-4.8
8 (dd, 1H), 5.88-5.93 (q, 1H), 7.
45 (d, 1H), 10.68 (s, 1H)

Preparation 3 Preparation of 3'-azido-3'-deoxythymidine-5'-monoglutarate (AZT glutarate) Reaction conditions and reactions similar to those of Preparation 2 of AZT succinate AZT (534.8m)
g, 2 mmol) and glutaric anhydride (228.2 mg,
2 mmol), and after the same treatment, the desired AZT
Glutaric acid ester crystals were obtained (yield: 80.0%). ¹ H NMR (270 MHz, CDCl ₃ ) δ 1.91
(S, 3H), 1.96-2.05 (m, 2H), 2.3
6-2.57 (m, 6H), 3.98-4.08 (m, 1
H), 4.24-4.33 (m, 2H), 4.51-4.5
5 (dt, 1H), 6.05-6.09 (t, 1H), 7.
39 (s, 1H), 10.27 (s, 1H)

Preparation Example 4 Preparation of Polypeptide from Protecting Group-Protected Polypeptide Resin To protecting group-protected polypeptide resin 1 (50 mg, 9.6 mmol) prepared in Preparation Example 1 above, anisole (0.
15ml), TMSCl (0.105ml, 10eq)
And TFA (13 ml) were added, and the mixture was stirred at room temperature for 1 hour. Then, at 4 ° C., DMSO (1.9 ml, 300 e
q) was added and the mixture was stirred for 1 hour and 30 minutes. After the reaction, cold ether (50 ml) was added to precipitate the polypeptide, and the polypeptide was obtained by centrifugation. The crude polypeptide was washed with ether (50 ml × 3) and air-dried. After purification by HPLC and freeze-drying, the desired polypeptide of the above formula (A) was obtained (6.2 mg, yield 33%). The following ion spray mass spectrum (IS
-MS) was measured using a triple stage quadrupole mass spectrometer APIIE (Perkin-Elmer Scie).
x company). IS-MS (reconstructed) m / z: F
ound 1974.0 (1973.2 Calc. for C 86 H 140 N 32 P 18 S 2),
[α] ²⁶ _D = -24.5 ° (c = 0.2, H ₂ O), acetonitrile concentration gradient of 10 to 40% for 30 minutes, Wat at a flow rate of 1 ml / min
ers μBondasphere 5 μC18-10
Analytical H by 0Å (3.9 × 150mm, manufactured by Nippon Millipore)
The retention time in the PLC was 14.2 minutes, and the amino acid analysis of the hydrolyzate with 6N HCl (the numerical value in parentheses is a theoretical value). Cys not determined (1), Tyr3.00 (3), Lys and D-Lys2.86 ( 3), Arg 5.11 (5),
Pro 0.95 (1). By the same reaction operation as above,
Polypeptide amide of the above formula (A) (protecting group-protected polypeptide resin (Rink amide resin is used))
(Yield 56% (8.4 mg)). IS-MS
(Reconstructed) m / z: Found 1972.5 (1972.1 Calc. For
C ₈₆ H ₁₄₁ N ₃₃ P ₁₇ S ₂ ), [α] ²⁶ _D = -18.8 ° (c = 0.2, H ₂ O), 1
Acetonitrile concentration gradient of 0 to 40% for 30 minutes, flow rate
Waters μBondasphere at 1ml / min
Retention time by analytical HPLC using 5 μC18-100Å (3.9 × 150 mm, manufactured by Nippon Millipore) was 14.8 minutes, 6N
In the amino acid analysis of the hydrolyzate by HCl (the values in parentheses are theoretical values), Cys not determined (1), Tyr3.0
0 (3), Lys and D-Lys2.80 (3), Ar
g 5.13 (5), Pro 0.88 (1). Similarly, a polypeptide amide of the above formula (B) (43% yield from protecting group-protected polypeptide 2) was obtained. Hereinafter, for convenience, the substance obtained by the present method is referred to as a synthetic polypeptide, and the synthetic polypeptide (T
131) was synthesized using a synthetic polypeptide 1 and a synthetic polypeptide (T22) prepared in the same manner as described above using a protecting group-protected polypeptide resin 2 having a polypeptide chain of the above formula (B). Describe.

Preparation Example 5 Preparation of Substance of the Present Invention (Polypeptide-AZT Succinate Complex) The α-amino group of the N-terminal amino acid of the protecting group-protected polypeptide resin 1 or 2 prepared in Preparation Example 1 was prepared. AZT succinate prepared in Preparation Example 2 (2.5
The carboxyl group of eq) was condensed by the DIPCI-HOBt method. The protecting group-protected polypeptide-AZT succinate complex resin thus obtained (50 mg)
Is subjected to deprotection, deresining, and disulfide bond formation in the same manner as in the preparation method of the synthetic polypeptide of Preparation Example 4, and isolated and purified in the same manner as in the preparation of the target polypeptide-AZT succinate complex of the present invention. I got Hereinafter, for convenience, the substance of the present invention according to the present preparation method is referred to as substance 1 of the present invention, and substance 1 of the present invention (AZT-Su
c-T131) is referred to as substance 11 of the present invention, and substance 1 of the present invention (AZT-Suc-T22) having the polypeptide chain of formula (B) is referred to as substance 12 of the present invention. Inventive substance 11: yield 29.99% (5.5 mg), I
S-MS (reconstructed) m / z: Found 2323.23 (2322.1 C
alc. for C ₁₀₀ H ₁₅₅ N ₃₇ O ₂₄ S ₂ , [α] ¹⁸ _D = -16.7 ° (c = 0.1,1
N AcOH), Waters μBonda at 10-50% acetonitrile gradient over 30 minutes, flow rate 1 ml / min.
The retention time in analytical HPLC using sphere 5 μC18-100− (3.9 × 150 mm, manufactured by Nippon Millipore) was 1
The amino acid analysis of the hydrolyzate with 6N HCl for 7.5 minutes (the numerical values in parentheses are theoretical values) shows that Cys has not been determined (1), Tyr3.00 (3), Lys and D-Lys.
3.00 (3), Arg 5.37 (5), Pro1.2
4 (1). Inventive substance 12: 2.76% yield, IS-MS (reconst
ructed) m / z: Found 2834.47 (2836.3 Calc. for C ₁₂₃ H
₁₇₉ N ₄₃ O ₂₈ S ₄ , [α] ²⁰ _D = 47.8 ° (c = 0.01,1N AcOH)

Preparation Example 6 Preparation of Substance of the Present Invention (Polypeptide-AZT Glutaric Acid Ester Complex) The α-amino group of the N-terminal amino acid of the protecting group-protected polypeptide resin 1 or 2 prepared in Preparation Example 1 was AZT glutaric acid ester prepared in Preparation Example 3 (2.5
The carboxyl group of eq) was condensed by the DIPCI-HOBt method. Preparation Example 4 of Protecting Group-Protected Polypeptide Resin-AZT Glutaric Ester Complex thus Obtained
In the same manner as in the preparation of the synthetic polypeptide of the above, the substance of the present invention of the target polypeptide-AZT glutaric acid ester complex was obtained. Hereinafter, the substance of the present invention according to the present preparation method is referred to as substance 2 of the present invention for convenience, and substance 2 of the present invention (AZT-Glu-T131) having a polypeptide chain of the above formula (A) is referred to as substance 21 of the present invention, The substance 2 of the present invention (AZT-Glu-T22) having the polypeptide chain of (2) is referred to as substance 22 of the present invention. Inventive substance 21: yield 6.27% (2.3 mg), IS
−MS (reconstructed) m / z: Found 2337.73 (2336.2 Cal
c. for C ₁₀₁ H ₁₅₇ N ₃₇ O ₂₄ S ₂ , [α] ²⁰ _D = -38.2 ° (c = 0.3,1N
AcOH), Waters μ Bondas at 10-50% acetonitrile gradient over 30 minutes, flow rate 1 ml / min.
The retention time in analytical HPLC using a 5 μC18-100− (3.9 × 150 mm, manufactured by Nippon Millipore) was 1
Amino acid analysis of the hydrolyzate with 6N HCl for 7.4 minutes (the values in parentheses are theoretical values) showed that Cys was not determined (1), Tyr2.92 (3), Lys and D-Lys
3.00 (3), Arg 5.08 (5), Pro1.2
2 (1). Inventive substance 22: yield: 8.81%, IS-MS (reconst
ructed) m / z: Found 2849.97 (2850.3 Calc. for C ₁₂₁ H
₁₈₁ N ₄₃ O ₂₈ S ₄ , [α] ²⁰ _D = 21.4 ° (c = 0.1,1N AcOH)

Preparation Example 7 Preparation of Dendrimer-Type Substance of the Present Invention The same amino acid condensation method (DIPCI-HOBt) was further applied to the α-amino group of the N-terminal amino acid of the protecting group-protected polypeptide resin 1 or 2 prepared in Preparation Example 1 above. Fmoc-Lys (Fmo-Lys)
c) was coupled. Furthermore, the removal of Fmoc group operation and Fmoc
The introduction of -Lys (Fmoc) was repeated twice to prepare a Lys-introduced protecting group-protected polypeptide resin having a dendritic structure in which seven Lys were introduced into the amino terminus of the polypeptide. Hereinafter, the resin prepared from the protecting group-protected polypeptide 1 obtained in Preparation Example 1 was designated as Lys-introduced protecting group-protected polypeptide resin 1, and the protecting group-protected polypeptide 2
The resin prepared from the above is referred to as Lys-introduced protecting group-protected polypeptide resin 2. The Fmoc group of the Lys-introduced protecting group-protected polypeptide resin 1 was removed, and the AZT succinate (2.5 eq) prepared in Preparation Example 2 in the same manner as in Preparation Example 5 was condensed by the DIPCI-HOBt method. The Lys-introduced protecting group-protected polypeptide resin-AZT succinate complex thus obtained was subjected to deprotection, deresining, and disulfide bond formation in the same manner as in the preparation method of synthetic polypeptide 1 of Preparation Example 4. A dendrime into which eight target AZTs have been introduced by the same isolation and purification method.
An r-type substance of the invention was obtained. Hereinafter, the substance of the present invention according to the present preparation method is referred to as a dendrimer-type substance of the present invention 1 for convenience. Dendrimer type substance of the present invention 1: 13.18% yield
(7.3 mg), IS-MS (reconstructed) m / z: Fou
nd 5665.47 (5664.5 Calc. for C ₂₄₀ H ₃₄₆ N ₈₆ O ₇₃ S ₂ , [α]
¹⁸ _D = 22.6 ° (c = 0.1,1N AcOH), acetonitrile gradient of 10-50% for 30 minutes, Wate at a flow rate 1 ml / min
rs μBondasphere 5 μC18-100
Analytical HPL by 、 (3.9 × 150mm, manufactured by Nippon Millipore)
The retention time at C was 26.5 minutes, and amino acid analysis of the hydrolyzate with 6N HCl (numerical values in parentheses were theoretical values) showed that Cys had not been determined (1), Tyr 2.87 (3), Ly
s and D-Lys10.00 (10), Arg5.42
(5), Pro 1.26 (1).

In the same manner as described above, the AZT glutaric acid ester prepared by the preparation method 3 is bonded to the Lys-introduced protecting group-protected polypeptide resin 1, and the same deprotection, deresinization, disulfide bond formation, isolation, and purification are performed. To obtain the desired substance of the present invention. Hereinafter, the substance of the present invention is referred to as a dendrimer-type substance 2 of the present invention. dendrimer type substance 2 of the present invention: yield 0.72%,
IS-MS (reconstructed) m / z: Found 5775.22 (5780.
3 Calc. For C ₂₄₈ H ₃₆₂ N ₈₆ O ₇₃ S ₂ , [α] ²⁰ _D = -12.3 ゜ (c = 0.
05,1N AcOH) The AZT succinate prepared in Preparation Example 2 was bound to the Lys-introduced protecting group-protected polypeptide resin 2 in the same manner as described above. The resulting Lys-introduced protecting group protected polypeptide-AZT succinate complex resin (100 m
g), m-cresol (300 μl), 1,2-ethanedithiol (300 μl), thioanisole (300 μl)
l), TFA (4.8 ml), and distilled water (300 μl) were added, and the mixture was stirred at room temperature for 2 hours, deprotected and de-resined, followed by the procedure for preparing synthetic polypeptide 1 or 2 of Preparation Example 4. The treatment operation and air drying were performed, and the obtained crude peptide derivative was dissolved in 1NAcOH (3 ml) and diluted to 40 ml with ultrapure water. DMSO (10 ml) was added to this solution at 4 ° C., and the mixture was allowed to stand at room temperature for 24 hours to form a disulfide bond. After further isolation and purification using HPLC and lyophilization, the dendri with 8 target AZTs introduced
A mer-type substance of the present invention was obtained. Hereinafter, for convenience, the substance of the present invention according to the present preparation method is referred to as dendrimer type substance of the present invention 3
It is described. However, since two disulfide isomers were obtained, they are represented as isomers 1 and 2, respectively.

The dendrimer type substance 3 of the present invention 3: two disulfide isomers were obtained. Isomer 1: yield 2.62%, IS-MS (reconstructe
d) m / z: Found 6178.22 (6180.7 Calc. for C ₂₆₃ H ₃₇₀ N ₉₂ O
₇₇ S ₄ , [α] ²⁰ _D = 3.1 ゜ (c = 0.1,1N AcOH) Isomer 2: Yield 4.31%, [α] ²⁰ _D = -15.4 ° (c = 0.1,1N
AcOH) In the same manner as described above, the AZT glutaric acid ester prepared in Preparation Example 3 is bonded to the Lys-introduced protecting group-protected polypeptide resin 2, and the same deprotection, deresining, disulfide bond formation, isolation, and purification are performed. The intended substance of the present invention was obtained. Hereinafter, the substance of the present invention is described as a dendrimer-type substance 4 of the present invention. dendrimer type substance 4 of the present invention: yield 6.20%,
IS-MS (reconstructed) m / z: Found 6292.97 (6292.
9 Calc / for C ₂₇₁ H ₃₈₆ N ₉₂ O ₇₇ S ₄ , [α] ²⁰ _D = 10.3 ° (c = 0.
2,1N AcOH)

Example 1 Measurement of Affinity of the Substance of the Present Invention for gp120 and CD4 The substance of the present invention 12 (AZT-Suc-T22) obtained in Preparation Examples 5 and 6
BIAcor ^™ biosensor (Pharmacia Bios), which measures the affinity of AZT-Glu-T22 and gp120 and CD4 for gp120 and CD4 using surface plasmon resonance
Biochem. Biophys. Res. Commun., 21
9,555-559 (1996), Biochem. Biopys. Acta., 1298, 37-44.
The measurement and analysis were carried out according to the method of (1996) (FIG. 1). As a result, the affinity constant (Kaff) of the substance 12 of the present invention for gp120 and CD4 was 2.20 × 10 ⁷ M and 3.44 × 10 ⁶ M, respectively. Similarly, Kaf of substance 22 of the present invention with respect to gp120 and CD4 was 6.05 × 10 ⁶ M and 1.07 × 10 ⁷ M, respectively. These affinity constants correspond to those of the synthetic polypeptide 2 obtained in Preparation Example 4.
Kaff against gp120 and CD4 (5.99 × 10 ⁶ M and 9.90 × 10 ⁶ M, respectively: Biochem. Bio
phys. Res. Commun., 219, 555-559 (1996), Biochem. Bio.
pys. Acta., 1298, 37-44 (1996)), and it was revealed that the substances 12 and 22 of the present invention show high affinity for gp120 and CD4.

Example 2 Antiviral activity against human immunodeficiency virus (HIV) Synthetic polypeptides 1 (T131) and 2 obtained in Preparation Example 4 above
(T22), substance 11 of the present invention (AZT-Suc
-T131), the antiviral activity against HIV of the dendrimer type substance 1 of the present invention and AZT obtained in Preparation Example 7 was tested and evaluated according to the following method. That is, in a 96-well microtiter plate, HIV-infected MT-4 cells (2.5 × 10 ⁴ cells / well,
Multiplicity of infection (MOI): 0.001) is added immediately after infection. After culturing at 37 ° C. for 5 days in a CO ₂ incubator, the number of surviving cells was determined by the MTT method (Pauwel et. Al., J. Viro
l. Methods 20, 309-321 (1988)). The antiviral activity is represented by a concentration that inhibits cell death due to HIV infection by 50% (EC ₅₀ : 50% effective concentration).
On the other hand, in order to know the cytotoxicity of the test substance on MT-4 cells, virus-uninfected cells were cultured with various concentrations of the test substance in the same manner as described above. Cytotoxicity is 50% cytotoxic concentration (CC ₅₀ : 50% cytotoxic conc)
entration). The approximate ratio (CC ₅₀ / EC ₅₀ ) between CC ₅₀ and EC ₅₀ was expressed as a selection coefficient (SI). Table 3 shows the results of the anti-HIV activity measurement. The anti-HIV activity of the substance 11 of the present invention was twice as high as the anti-HIV activity of AZT (EC ₅₀ value comparison), and the selectivity was twice or more, indicating a clear enhancement of the anti-HIV activity. It also has higher anti-HIV activity than synthetic polypeptide 1, which is a known anti-HIV active polypeptide. Furthermore, the dendrimer type substance 1 of the present invention showed the highest anti-HIV activity.

[0057]

[Table 7]

Example 3 HIV Infection Inhibitory Activity Synthetic polypeptides 1 and 2 obtained in Preparation Example 4 above, substances 11, 22 and den of the present invention obtained in Preparation Examples 4, 5, and 6 above
The HIV infection inhibitory activity of the drimer type substance 1 of the present invention and AZT was tested and evaluated according to the following method. That is, using T cell line-directed HIV-1 (NL4-3) and macrophage-directed HIV-1 (JR-CSF),
PHA (Phaseolus vulgaris agglutinin) activated PB
HI to MC (peripheral blood mononuclear cells)
The V-1 infection inhibitory activity was measured by a p24 antigen expression suppression test by ELISA using an antibody against HIV-1 p24 antigen. That is, various concentrations of the test substance
In addition to the MT-4 cells immediately after the infection with IV-1, the cells were cultured at 37 ° C. for 3 days (4 or 7 days in some cases) in a CO ₂ incubator, and then HIV-derived p2 in the culture supernatant was removed.
ELISA kit (Cellular Pr)
products (manufactured by E. products). Table 4 shows the results.
Macrophage-directed HIV in which the synthetic polypeptides 1 and 2 obtained in Preparation Example 4 do not originally show any anti-HIV activity
All of the substances of the present invention exhibited anti-HIV activity against JR-CSF which is -1 as well as AZT, and it was found that the substance was also effective against HIV-1 directed to macrophages.

[0059]

[Table 8]

[0060]

According to the present invention, a pharmaceutical composition having a high anti-HIV activity by effectively inducing an anti-HIV active substance on target cells by affinity for CD4 positive cell surface protein and HIV surface protein is provided. can do.

[Brief description of the drawings]

FIG. 1 shows substances 12 of the present invention (AZT-Suc-T22) and 2
FIG. 2 is a BIAcore analysis diagram showing the affinity of AZ120-Glu-T22 for gp120 and CD4.

Claims (13)

[Claims] 1. The HIV surface protein gp120 and / or
Alternatively, a complex characterized in that one or more reverse transcriptase inhibitors and / or HIV protease inhibitors are bound to a polypeptide having affinity for the HIV host target cell surface protein CD4 by a chemical bond. body. 2. The HIV surface protein gp120 and / or
2. The complex according to claim 1, wherein the polypeptide having affinity for the HIV host target cell surface protein CD4 is a polypeptide represented by the following formula (1). Embedded image 1 2 3 4 5 6 7 8 9 9 10 11 11 12 13 A1-A2-Cys-A2-A3-A3-Y-A2-A3-A3-Cys-A4-A5 (1) Wherein A1 is H- (derived from the amino group of A2) or one basic amino acid residue selected from lysine, arginine and ornithine, or at least two identical or different amino acid residues selected from these amino acid residues. N-α acyl substitution in which a hydrogen atom at the N-α position of the amino acid residue of the basic amino acid residue or peptide residue is substituted with an acyl group or a substituted thiocarbamoyl group Indicates an amino acid residue, an N-α acyl substituted peptide residue, an N-α substituted thiocarbamoylated amino acid residue or an N-α substituted thiocarbamoylated peptide residue; A2 is independently tyrosine; Represents a phenylalanine or tryptophan residue; A3 independently represents a lysine or arginine residue; A4 represents an amino acid residue or a peptide residue having at least one amino acid selected from lysine and arginine;
Is -OH (from the carboxyl group of A4) or -NH
₂ (derived from the acid amide of the carboxyl group of A4);
Is a peptide residue represented by the following formula (a): 1 ′ 2 ′ 3 ′ 4 ′ 5 ′ 6′-A6-A2-A3-Gly-A7-A6- (a) (where A2 and A3 has the same meaning as in the above formula (1), A6 independently represents an alanine, valine, leucine, isoleucine, serine, cysteine or methionine residue; A7 represents tyrosine, phenylalanine, tryptophan, alanine, valine, leucine, Gly represents a glycine residue, provided that both 1 ′ and 6 ′ are cysteine residues, which may be linked by a disulfide bond; ) Or D-ornithyl-
Proline, prolyl-D-ornithine, prolyl-D-
Lysine, prolyl-D-arginine, D-lysyl-proline, D-arginyl-proline, glycyl-lysine,
Glycyl-arginine, ornithyl-glycine, glycyl-ornithine, lysyl-glycine and arginyl-glycine are selected from dipeptides composed of two amino acids represented by, and the constituent amino acids D-lysine, lysine, The hydrogen atom of D-ornithine or the ω-amino group in the side chain of ornithine represents a peptide residue which may be substituted with an acyl group; Cys represents a cysteine residue; May be linked by a bond]. 3. The method according to claim 1, wherein the chemical bond is at least one of the α- and ω-amino groups of the amino-terminal amino acid of the polypeptide represented by the formula (1) and a reverse transcriptase inhibitor and / or HI.
The complex according to claim 2, wherein the complex is a bond with a V protease inhibitor via a multifunctional spacer. 4. The conjugate according to claim 3, wherein the multifunctional spacer is a substance capable of binding to both an amino group and a hydroxyl group. 5. The conjugate according to claim 3, wherein the multifunctional spacer is a compound having at least two carboxyl groups. 6. The conjugate according to claim 3, wherein the polyfunctional spacer is an aliphatic dicarboxylic acid. 7. The conjugate according to any one of claims 1 to 6, wherein the reverse transcriptase inhibitor is a nucleoside reverse transcriptase inhibitor. 8. The complex according to claim 7, wherein the nucleoside reverse transcriptase inhibitor is a non-natural nucleoside or a nucleoside analog. 9. The nucleoside reverse transcriptase inhibitor comprises a base selected from the group consisting of pyrimidine bases, purine bases, imidazole bases and triazole bases, and a nucleoside comprising furanose having at least one hydroxyl group or an acyclo-form thereof. The complex according to claim 7, which is an analog thereof. 10. The nucleoside reverse transcriptase inhibitor,
8. A compound according to claim 7, which is 3'-azido-3'-deoxythymidine.
The conjugate of claim 1. 11. The method of claim 11, wherein the HIV protease inhibitor is HIV.
3. The complex according to claim 1, which is a mimic compound of a substrate transition state of a protease. 12. The method according to claim 12, wherein the HIV protease inhibitor is Ro.
31-8959, A-77003, KNI-93, KN
I-102, KNI-174, KNI-227, KNI
-272, L-735527, SC-52151, VX
-478, ABT-538, DMP-323 and U-9
The complex according to any one of claims 1 to 6, which is one or more substances selected from the group consisting of 6988. 13. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof as an active ingredient.