WO1998043995A1 - Novel anti-hiv complexes and medicinal compositions - Google Patents

Abstract

Medicinal compositions comprising complexes in which one or more reverse transcriptase inhibitors and/or HIV protease inhibitors are chemically bonded to polypeptides having affinity for HIV surface protein gp120 and/or HIV host target cell surface protein CD4 and which thus can lead substances with anti-HIV activity on the target cells due to the affinity for the CD4-positive cell surface protein and HIV surface protein, thus achieving high anti-HIV activity.

Description

 Specification

 New anti-HIV conjugates and pharmaceutical compositions

 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 a Z or HIV protease inhibitor are bound to a polypeptide having an anti-HIV activity by a chemical bond, an anti-HIV agent containing the substance as an active ingredient, and the like. And a pharmaceutical composition. Background art

 Human immunodeficiency virus (HIV) is known to cause various clinical symptoms, such as acquired immunodeficiency syndrome (AIDS) and AIDS-related syndrome.

 Drugs that inhibit each stage of HIV growth are known as anti-HIV drugs, but drugs that are actually licensed or undergoing clinical trials include reverse transcriptase inhibitors ( For example, nucleoside 3′-azido-3, -deoxythymidine (hereinafter also referred to as AZT), 2,, 3,1-dexoxyinosine (hereinafter also referred to as ddl), 2 ′, 3′-deoxycytidine (hereinafter ddC) Etc.) and HIV protease inhibitors (N, 1 [1 (S) 1 benzyl-3--3- [4 a (S), 8 a (S), 3 (S)-(tert-butyl carbamoyl) ) Decahydroisoquinoline 1-2-yl] 1-2 (R) -hydroxypropyl] 1N "-quinoline-2-ylcarbamoyl) 1-L-asparagineamide (hereinafter also referred to as Ro 31-8959) ) Etc. are known.

 In general, these anti-HIV drugs require large doses and long-term administration, so that side effects (such as bone marrow damage) and the development of drug-resistant viruses due to reverse transcriptase or amino acid mutations in HIV protease are required. There were problems such as appearance.

Use of AZT in combination with ddI or ddC for the purpose of solving these problems and enhancing the antiviral effect (HIV infectious disease · AIDS, Nihon Gakusha, p316-326 (1993)), Ro 31—8959 and 82 Use in combination (J. Infect. Dis ,, 166 (5), pll43-1146 (1992)) and use in combination with 3 TC and AZT, dd I or Ro 31-8959 (Kaihei 6_2 3 4 6 4 1) is known.

Even when used in combination, the antiviral activity is insufficient, and the above-mentioned problems are not 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 (International Publication WO92 / 043734, JP-A-5-1633). The polypeptide is known to exhibit the same level of anti-HIV activity as AZT, such as reverse transcriptase inhibitors and HIV proteases. It is also known that when administered in combination with a monose inhibitor, it exhibits excellent anti-HIV activity (Japanese Patent Application Laid-Open No. 9-252440).

 Toxic side effects such as reverse transcriptase inhibitors and HIV protease inhibitors can also be caused by administration of a combination of a polypeptide having anti-HIV activity and a reverse transcriptase inhibitor or HIV protease inhibitor, etc. It cannot be reduced and remains a problem to be solved, and anti-HIV drugs with lower toxicity are expected. Disclosure of the invention

 In order to obtain a useful complex as an anti-HIV agent having excellent anti-HIV activity and less side effects, the present inventors have developed a polypeptide based on a polypeptide having anti-HIV activity and having high affinity for HIV surface protein. As a result of intensive studies, a complex in which an anti-HIV active substance such as a reverse transcriptase inhibitor or an HIV protease inhibitor has been chemically bonded to the amino (N) terminus of the polypeptide has been used as an anti-HIV agent. Excellent anti-H

The present inventors have found that it is possible to exhibit IV activity and reduce side effects, and thus completed the present invention.

That is, the present invention relates to a polypeptide having an affinity for an HIV surface protein, preferably a polypeptide of the following formula (1), and one or more anti-HIV active substances (a reverse transcriptase inhibitor and a Z or HIV protein). And a pharmaceutical composition containing the complex as an active ingredient. I 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3

A 1 -A 2 -C y s -A 2 -A 3 -A 3 -Y-A 2 -A 3 -A 3 -C y s -A 4-A 5

 (1)

[Wherein, 81 is ^ 1— (derived from the amino group of A2), or one basic amino acid selected from lysine, arginine and ordinine, or the same or selected from these amino acid residues. A peptide residue having at least two different amino acid residues, or a 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 an acyl group or substitution A Ν-α-substituted amino acid residue, a ァ -substituted peptide residue, a 置換 -α-substituted rubamoylated amino acid residue or a Ν-substituted thiocarbamoylated peptide residue substituted with a thiocarbamoyl group. Show;

 A 2 independently represents a tyrosine, phenylalanine or tributophan residue; A 3 independently represents a lysine or arginine residue;

 A 4 represents an amino acid residue or a peptide residue having at least one amino acid selected from lysine and arginine;

8 represents —011 (derived from the carboxyl group of A4) or 1 NH ₂ (derived from the acid amide of the carboxyl group of A4); Y represents a peptide residue represented by the following formula (a):

 1 2 * 3, 4 '5 * 6'

 -A 6 -A 2-A 3 -G 1 y -A 7 -A 6-(a)

 (In the formula, A 2 and A 3 have the same meanings as in the above formula (1).

 A 6 independently represents an alanine, nocrine, leucine, isoleucine, serine, cysteine or methionine residue;

 A7 represents a tyrosine, phenylalanine, tryptophan, alanine, palin, oral isin, isoleucine, serine, cysteine or methionine residue;

G 1 y represents a glycine residue.

However, when both the Γ-position and the 6-position are cysteine residues, these may be linked by a disulfide bond. ), Or

D-Ordityl-proline, Prolyl-D-Orditin, Prolyl-D-lysine, Prolyl-D-arginine, D-Rigid-loop phosphorus, D-Arginyl-proli And glycyl-lysine, glycyl-arginine, ornithyl-glycine, glycylornithine, lysyl-glycine, and arginyl-glycine. A hydrogen atom of a side chain ω-amino group of a certain D-lysine, lysine, D-orudin or ordinin indicates a peptide residue which may be substituted with an acyl group;

 Cys indicates a cysteine residue, and the cysteine residues at positions 3 and 11 may be linked by a disulfide bond.]

 The conjugate 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 CD4 positive cells, which are targets for HIV infection in vivo, are tested. It transports an HIV active substance and releases an anti-HIV active substance in the cells to exhibit excellent anti-HIV activity.A composition containing this novel complex as an active ingredient is safe. It is effective as a pharmaceutical composition such as an anti-HIV drug which is high and has few side effects.

 (Embodiment of 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) is a polypeptide which has affinity for HIV surface proteins gp120 and Z or HIV host target cell surface protein CD4, 1 or A complex in which two or more reverse transcriptase inhibitors and / or HIV protease inhibitors are bound by a chemical bond.

 The polypeptide constituting the present invention is not particularly limited as long as it is a polypeptide having an affinity for the HIV surface proteins gp120 and Z or the HIV host target cell surface protein CD4. Examples of suitable polypeptides include antibodies and polypeptides represented by the following formula (1). The protein also has affinity for the cell surface protein CD4 of CD4 positive cells (host target cells), which are host targets of HIV. (Biochem. Biophys. Res. Commun., 219, 555-559 (1996), Biochem. Biophys. Acta., 1298.37-44 (1996)), which is itself represented by the following formula (1) having anti-HIV activity. Polypeptides are preferred.

 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3

A 1 -A 2 -C ys -A 2 -A 3 -A 3 -YA 2 -A 3-A 3 -C ys -A 4-A 5 (1)

[Wherein, A UiH— (derived from the amino group of A2) or one basic amino acid selected from lysine, arginine and orditin, or the same or different amino acids selected from these amino acid residues A peptide residue having at least two residues, or a hydrogen atom at the N-la position of the amino terminal amino acid residue of the basic amino acid residue or the peptide residue is an acyl group or a substituted thiocarno <moyl group Represents a α-α-acyl-substituted amino acid residue, a Ν-α-acyl-substituted peptide residue, a Ν-substituted thiamine-substituted rubamoylated amino acid residue or a Ν-substituted thiocarbamoylated peptide residue;

 A 2 independently represents a tyrosine, phenylalanine or tribubutane residue; A 3 independently represents a lysine or arginine residue;

 A 4 represents an amino acid residue or a peptide residue having one or at least two amino acids selected from lysine and arginine;

85 represents —011 (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)

 1, 2 * 3 * 4 '5' 6 '

 -A 6 -A 2-A 3 -G 1 y-A 7 -A 6-(a;

 (Where A 2 and A 3 have the same meanings as in the above formula (1).

 A 6 independently represents an alanine, nocrine, leucine, isoleucine, serine, cysteine or methionine residue;

 A 7 represents a tyrosine, phenylalanine, tryptophan, alanine, palin, oral isin, isoleucine, serine, cysteine or methionine residue;

G 1 y represents a glycine residue.

However, when both the Γ-position and the 6-position are cysteine residues, these may be linked by a disulfide bond. ), Or

D-ornithyl-loop phosphorus, prolyl-D-ornithine, prolyl-D_lysine, prolyl-D-arginine, D-lysylloop-mouth phosphorus, D-arginyl-prolin, glycyrrhizin, glycyl-arginine, ornityl-glycine , Grishi It is selected from dipeptides composed of two amino acids represented by ruorutin, lysyl-glycine and arginyl-glycine, and the constituent amino acids are D-lysine, lysine, and D-orutin Or a hydrogen atom of a side chain ω-amino group of ordinine represents a peptide residue which may be substituted with an acyl group;

Cys represents a cysteine residue, and the cysteine residues at positions 3 and 11 may be linked by a disulfide bond.] In the formula (1), the number indicating the position of the amino acid roughly indicates the amino acid sequence, but does not necessarily correspond to the amino acid residue in a one-to-one correspondence. are those attached to, H from the N-terminus, one OH and from the C-terminus - are NH ₂ and one digit when the amino acid residue inside the other amino acid sequences present one is used, If the amino acid residue is a peptide residue according to the definition, the number of its constituent amino acid residues is used.

 Since the polypeptide represented by the formula 8 (1) is a polypeptide synthesized based on a horseshoe crab-derived tachyplesin family polypeptide, a horseshoe crab-derived tachyplesin I, a tachyplesin II, a tachyplesin III, a polyhumsine I or Polyphencin II (metabolism, 26, p429-439 (1989)) can be used as an alternative to the above polypeptides.

 Having an anti-HIV activity in the present specification means having the ability to exert 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 a polypeptide synthesis method known per se, in particular, a liquid phase synthesis method or a solid phase synthesis method (New Protein 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, the carboxyl group of the N-protected amino acid corresponding to the carboxyl terminal amino acid residue of the polypeptide chain, for example, directly or optionally via an intervening substance such as an amino group or a hydroxyl group Has a functional group After binding to the insoluble resin, the protected amino acids from position 11 to position 1 of the amino acid sequence represented by the above formula (1) are sequentially bonded in accordance with the solid phase synthesis method to obtain a protecting group-protected peptide resin. .

 The insoluble resin is not particularly limited as long as it can be covalently bonded directly to the carboxyl group of the N-protected amino acid at the carboxyl (C) terminal, or optionally through an intervening substance, and can be subsequently removed. Is not done. Examples of such an insoluble resin include a resin that releases an amino acid or a eptidic acid after elimination of the insoluble resin, an alkoxy resin (A1 ko-resin or the like), an amino acid amide after elimination, An amide-type resin (Rinkami de resin or the like) that releases a peptide amide may be used. Specifically, resins that release amino acids or eptidic acids include chloromethyl resin, oxymethyl resin, PAM (4- (hydroxymethyl) phenylacetamidomethyl) resin and Alko-resin. Examples of the amide resin include p-alkoxybenzyl alcohol (Wang) resin, hydroxymethyl phenoxy acetic acid (HMPA) resin, dialkoxy benzyl alcohol type resin, and trialkoxy diphenyl methyl alcohol type resin. Are benzhydrylamine (BHA) resin, p_methylbenzhydrylamine (MBHA) resin, trialkoxybenzhydrylamine type resin, PAL (pepti de ami de 1 i nker resin and R i n k ami de resin) (4- (2,4, -dimethoxyphenylaminomethyl) monophenyl resin, etc.).

A 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 peptide synthesis conditions. The coupling of the protected amino acids can be carried out according to a conventional condensation method. However, the DCC (dicyclohexylcarbodiimide) method, the DCC-HOBt (1-hydroxybenzotriazole) method, the DIPCI (N, N-diisopropyl carpoimide) -HOB t method, BOP (benzotriazolyl-N-hydroxytrisdimethylaminophosphonium hexafluorophosphoride) -HOB t method, HBTU (2- (1H) -benzotria 1-yl) 1,1,1,3,3-tetramethylperoniumhexafluoro (Phosphate) -HOBt method, symmetric anhydride method, etc. are preferred. 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), select a α-α-acylamino acid residue or a Ν-hyacyl peptide residue in which the hydrogen atom at the N _ position of the amino-terminal amino acid residue in A1 is replaced with an acyl group. In this case, condensation is carried out with the target protecting group-protected peptide resin or the peptide released from the protecting group-protected polypeptide resin, and the acid anhydride of the corresponding acyl group or the corresponding carboxylic acid. Using an agent, the Ν-terminal amino group of the peptide resin or peptide is acylated to obtain a ァ -acylated peptide resin. Also, a Ν-α-substituted thiocarbamoylated amino acid residue or 一 -monosubstituted thiocarbamoyl in which the hydrogen atom at the ァ -α-position of the amino terminal in A 1 in the above formula (1) is substituted with a substituted thiocarbamoyl group. When a peptide residue is selected, the desired protecting group-protected peptide resin or the peptide released from the protecting group-protected polypeptide resin and the substituted isothiocyanate compound are slightly diluted. By reacting under the conditions, a {terminal} -monosubstituted thiocarbamoylated polypeptide of the substance of the present invention can be obtained. In this case, by appropriately selecting the insoluble resin, the carboxyl terminus of the amino acid residue at position 13 can be free (A 5 in formula (1) corresponds to -Ο), or the acid amide (formula (In (1), A 5 is equivalent to —NH ₂ ). The polypeptide having the protecting group and bonded to the insoluble resin is hereinafter also referred to as a protecting group-protected polypeptide resin.

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

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1 1 1 1

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 1 1 2 3 4 5 6 7 7 7 7 7 7 8 9 10 1 1 12 12 13

(33 ys-ys-Trp-C ys ■ T yr ™ ys-Lys-Cys ■ Tyr. Ys 'Gly T yr-Cys' T yr-ys-ys-Cys-ys- ys-shi ys-NH (34 Arg-Arg-Trp-Cys-Tyr-Lys-Arg-Cys-Γ yr ■ shi ys-GlyTyr ~ Cys-Tyr-shiys-Ai 'g-Cys-Arg-H (3δArg-Ar-Trp-Cys ■ Tyϊ- -rg-Lys-Cys-Tyr- ys-GlyPhe-Cys ■ lyr-Arg -Ly s-Cy s- Arg -NH (36 Arg-Af -Trp-Cys-Tyf-A rg -shis -C ys -T yr -Arg- Gly.T yr -C ys -Ty r-Arg -L ys -C ys -A rg -NH (37 A rg -T rp -C ys -T yr -A rg -L ys -C ys -Ty r ■ Ar g- Gly.Γ yr C ys' T yr- Arg-shis-Cys-Arg-Nil (38 Arg-Arg-Tr-Cys-Tyr-Arg-Lys-Cys-Phe-Arg-GlyPhe-Cys-Tyr-Arg -Lys-Cys-Arg-NH: (39 A rg-Tri)-Cys T yr Λ rg Ly s-ys. Phe-Arg-Gly Plie-Cys-T yr-Arg-Lys- C ys ■ Arg-N II. (40 Λ rg-Λ rg-T rp-Cys-T yr-Lys ■ Λ rg-Cys-T y I · Arg- G ly P! Ie-Cys- T yr -shi ys-A rg- Cys-Ar-Nil: (41 rg-Trp-Cys-Tyr- ^ rg ys-Cys-Tyr-Arg-GlyPhe-Cys-Tyr-Arg-Lys -Cys-Arg-NH: (42 Λr-g-Λr-g-Trp-Cys-Tyr-ΛrgLys-Cys · Pe-shiys-Gly-PIIe-C ys-Ty r- Arg-L ys-Cys-Arg-NH. (43 Arg- T rp-Cys-T yr-Λ rg L ys-Cys-Pe-Shi ys-G! y Pe- Cys-Tyr-Arg-ys-Cys-Arg-NH: (44 Λ rg-Aig-Tr-Cys-Tyr-Arg-Lys-Cys-Phe- ys -Gly P he-Cys Tyr-Arg- Lys-Cys Arg ■ A rg-NH; (45 Λ rg ~ Λ fg-T rp ■ Cys-T yr-Ly s-Ar r-g- C ys-Phe- ys-Gly- Phe-Cy s- T yr-ys-Ar g-Cy s- Arg-NH: (46 r '-T rp-Cy-T yr- ys-Λ rg-C ys ■ T yr ■ Λ rg-Gly Ί 'yr-Cys-T yr-shi ys-Λ rg-C ys A rg-Nl (47 Λ rg-A r-T r-C ys-T yr-Λ rg ■ Λ rg-Cys-Tyr-ys-Gly-T yr-Cys-T yr-Ar g-Λ rg-Cys ■ Ar g-NH: (48 Λ rg-T rp-C ys ■ Tyr- Λ r -shi ys-C ys-T yr ■ ys-Gly T yr-Cys-T y Λ rg-Lys-Cys Arg-NH (49 T rp · Cys-ΐ yr-A rg-shi ys-Cys-T yr-ys-Gly T yr--Cys-T yr-Arg-ys-Cys-Ai'g-NH (50 T rp ■ Cys-T yr -Ar g-Lys-Cys ■ Tyr Gly P he-Cys-Tyr-Ar g- ys-Cys • Arg-N II

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 1 1 2 3 4 5 6 7 7 7 8 9 10 11 12 12 13 (69) Ac-Arg-Arg-Trp-Cys-Tyr-Arg-Lys-D ys -Pro -Tyr-Arg-ys- Cys Arg -NH

(70) Oct-Arg-Arg-Trp-Cys-TyrArg-Shys-D-ysys-Pro-Tyr-Arg-Shys-Cys-Arg-NH

(71) aur-Arg-Arg-'Γι.ρ-Cys-Tyr-Arg-Lys-D y s -Pro-Tyr—Arg—shis—Cys—Arg—NH

(72) My r-Arg-Arg-Trp Cys-Tyr Arg-Lys-D ys-Pro-Tyr-Arg-ys-Cys-Arg-Nil:

(73) Parm-Arg- Arg-Trp Cys- Tyr-Arg-ys-D-y s -Pro-Tyr-Arg-ys-Cys-Arg-NH:

(74) FTC-Arg-Arg-Trp-Cys-Tyr-Arg-Lys-D-ys-Pro.ryr-Arg-sh ys-CysArg-NH:

(75) PTC -8 rg-Arg-Trp- Cys- Tyr- Arg-Lys -DLy s -Pro -Tyr-Arg-Lys-Cys-Arg-H:

(76) Nicot Arg- Arg-Trp-Cys-Tyr- Arg -Lys-D Lys -Pro • ryr-Arg-Lys-Cys Arg -NH:

s -Pro •Tyr - Arg - Lys - Cys - Arg -NH: (77) Λ rg-Λ rg-Ί ri)-Cys T yr-A rg-shi y:

s -Pro • Tyr-Arg-Lys-Cys-Arg -NH:

(78) Arg Arg-Trp-Cys-Tyr-Arg Lys-D Lys-Pro ryr-Arg-ys-Cys-Arg-Nil:

 N-But ^,

 (79) Parm-Arg Arg-Trp-Cys Tyr-Arg-ys-D-ys-Pro-Tyr- Arg-Lys-Cys-Arg-NH:

(80) Parm Or n-Arg-"rp Cys Tyr-Arg-Lys-D ys Pro-Tyr-Arg-ys-Cys-Arg-NH:

Similarly, in Table 1, a polypeptide chain having a carboxyl group at the carboxy terminus (A 5 in formula (1) corresponds to 1 OH) can also be mentioned.

 In the description of polypeptides 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 label, it means that all are "L".

 A rg: Arginine, Trp: Tryptophan, Cys: Cystine, Tyr: Tyrosine, Lys: Lysine, G1y: Glycine, Phe: Phenylalanine, I1e: Isoloicin, Ser: Serine, Leu: Leucine , Met: methionine, Va 1 phosphorus, A 1 a: alanine, Pro: proline, 0 rn: ornithine, DO rn D—ornitin, DA rg D—arginine, DL ys D—lysine, A c— A rg N—Hi-acetyl arginine, Oct—Arg: N—α—Octanoyl arginine, Laur— Arg: N—α—Lauryl arginine, Μ yr—Arg: N—α—mi Listinolearginine, Parm—ArgN-«-lumitoylarginine, FTC—Argg—α-fluoresceinthiocarbaminated arginine, PTC—ArgΝ_α—phenylthiocarbaminated arginine , Ni co t— Ar g: N— α—nicotinyl argini , Ε—N—Ac—DL ys ε—N—ω—aminoacetyl-D-lysine, ε—N—But—DLy s ε—N—ω—aminobutyryl D—lysine, P arm—0 r η Ν — Α-palmi toyl ordinine

 Examples of the amino acid used as a protecting group for the side chain functional group of the amino acid include the following.

 B u ': tertiary butyl, B oc: tertiary butyloxy, Fmo c 2 2,5,7 8-pentamethylchroman-1-6-sulfo

 Examples of a method for obtaining only a polypeptide chain from the protective group-protected polypeptide resin include a known method and a method for removing a protective group and an insoluble resin and forming a disulfide bond in one step described below.

That is, as a reagent for removing the α-amino group protecting group, TFA (trifluoroacetic acid) / dichloromethane, HC 1Z dioxane, piperidine / DMF or piperidine Zin ZNMP or the like is used, and is appropriately selected depending on the type of the protecting group.

 Protected group-protected polypeptide resins include hydrogen fluoride, TFMSA (trifluoromethanesulfonic acid), TMSOTf (trimethylsilino retriflate), TMSB r (trimethylsilyl bromide), TMSC1 (trimethylsilino chloride) ) Or by treating with TFA, etc., the resin and the protecting group can be simultaneously eliminated. The above elimination reagent is appropriately selected depending on the synthesis strategy (Boc method or Fmoc method), the insoluble resin to be used, and the type of protecting group.

 Further, the obtained polypeptide chain forms a disulfide bond (1-S—S—) via a mercapto group between the cysteines at positions 3 and 11 or, optionally, at positions と and 6 ,. Can be. These disulfide bonds can be formed by a method known per se, for example, mild air oxidation or the like. Usually, an oxidizing agent such as oxygen perfluorophosphate in the atmosphere (for example, potassium perfluoride) is used.

 Thus, the protecting group and the resin are eliminated, and the polypeptide formed by forming a disulfide bond can be further isolated and purified by a known method. Most effective.

In the substance of the present invention, a reverse transcriptase inhibitor and a NO or HIV protease inhibitor, which are anti-HIV active substances, are chemically bound to the 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 includes a nucleoside comprising 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. Its analogs are preferred, for example, AZT (CAS REGISTRY NUMBERS: 30516-87-1: zidovudine), dd I (CAS REGISTRY NUMBERS: 69655-05-6: didanosine), d dC (CAS REGISTRY NUMBERS: 7481-89- 2: zalci tabin e), 2 ', 3'—didehydro 1', 3, 1-dideoxythymidine (CAS REGISTRY NUMBERS: 3056-17 -5: d 4 T: stavudine), 3'-thia-2 ', 3' didoxycytidine (CAS REGISTRY NUMBERS: 134678-17-4: 3 TC: l 5 Lamivudine), 2 '-3-Fluoro dd C, 3' Fluorotymidine (CAS REGISTRY NUMBERS: 25526-93-6: FLT). 9-1 (2-phosphonyl-methoxyxetil) 1 adenine (CAS REGISTRY) NUMBERS: 106941-25-7: PME A), 6—CI—ddl, 6—C 1—dd C and the like.

 Examples of the non-nucleoside inhibitors include, for example, tetrahydroimidazo-benzodiazepine one-one or one-thione (T IBO) derivative (specifically,

(+)-S-4,5,6,7-tetrahydro-5-methyl-6- (3-methyl-12-butenyl) imidazo [4,5,1-jk] [1,4] benzodiazepine-1 2

(1 H) -thione) (CAS REGISTRY NUMBERS: 167206-29-3: R 829 13), hydroxyethoxyquin-methylphenylthiothymine (HEPT) derivative, nevirapine (Nevirapine) (CAS REGISTRY NUMBERS: 129618-40-2) ) And pyridinone derivatives.

 Of these, considering the ease of binding to the above polypeptide chains and the mechanism of effectively inhibiting DNA synthesis by being incorporated into DNA, nucleoside reverse transcriptase inhibitors are Preferably, among the nucleoside-based HIV transcriptase inhibitors, it is preferably AZT, ddl, ddC, 14 or 3 fingers already administered to humans in the clinic, more preferably the polypeptide chain and AZT whose antiviral activity is particularly synergistically enhanced when chemically combined with the substance of the present invention. These nucleoside reverse transcriptase inhibitors are incorporated into DNA when HIV synthesizes DNA from RNA by reverse transcription.As a result, unnatural nucleosides or nucleoside analogs are inhibited to inhibit DNA synthesis. Nigs are preferred. The nucleoside analog refers to a non-nucleoside compound having a steric structure similar to that of a nucleoside. In addition, these reverse transcriptase inhibitors can be commercially available or those prepared according to known synthesis methods.

Further, as the HIV protease inhibitor, an inhibitor which is a substance which inhibits the activity of HIV protease and which is a mimic compound of the substrate transition state of the protease is preferable. The substrate transition state mimic refers to a substance that can bind to the substrate binding site of the enzyme and has a similar tertiary structure to the substrate in the enzyme-substrate complex. example For example, Ro 3 1—8 9 5 9 (CAS REGISTRY NUMBERS: 127779-20-8: sa quinavir), A—7703 (CAS REGISTRY NUMBERS: 134878-17-4), A—8 0 9 8 7 (CAS REGISTRY NUMBERS: 144141-97-9), KN I—93 (CAS REGISTR Y NUMBERS: 138258-64-7), KN I—102 (CAS REGISTRY NUMBERS: 139694-6 5- 8), KN I- 1 7 4, KN I-2 2 7 (CAS REGISTRY NUMBERS: 147384-69-8) .KN I-2 7 2 (CAS REGISTRY NUMBERS: 147318-81-8), L- 7 3 5 5 2 7 (CAS REGISTRY NUMBERS: 150378-17-9: indinavir), SC-5 2 1 5 1 (CAS REGISTRY NUMBERS: 143224-34-4: Tel inavir). VX- 4 7 8, ABT- 5 3 8 (CAS REGISTRY NUMBERS: 155213-67-5: ritonavir), DMP—32 3 (CAS REGISTRY NUMBERS: 151867-81-1), U- 9 6 9 8 8 ( CAS REGISTRY NUMBERS: 149394-65-0). More preferably, Ro31-8959, L1-735527 and KN-272 having high antiviral activity are preferred, but there is no particular limitation. As these HIV protease inhibitors, commercially available ones or those prepared according to known synthetic methods can be used. As for Ro 31 -8959, for example, the preparation method described in J. Med. Chem. 36, P2300-2310 (1993) can be mentioned.

 The substance of the present invention is not particularly limited as long as the force at which the polypeptide chain and the anti-HIV active substance are chemically bonded is not particularly limited as long as the bond is a chemically formed bond. , An ester bond, an amide bond, an ether bond, a disulfide bond and the like. Among 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. The anti-HIV active substance can be released near the action point of the anti-HIV active substance. The ester bond is a bond having such a stability that the ester bond is not easily cleaved during transportation, and is not limited to a preferable force.

In the substance of the present invention, the position of the polypeptide chain that binds to the anti-HIV active substance is not particularly limited, but the polypeptide chain itself, HIV surface protein gp120, and CD4 positive which is a host target cell An amino terminal site capable of maintaining affinity with cell surface protein CD4 of the cell is preferred. Specifically, the α-amino group or ω-amino group of the amino acid at the amino terminal, or Tam was used to prepare the antibody. MAP system (multiple antigen peptide peptide system) (JP Tam ^ Proc. Natl. Acad. Sci. USA, 85, 5409-5413 (1988); JP Tam 'Peptides: synthesi s, structures, and applications "(B. Gut tered.) _N pp. 456-500 ^ Academic Press, Inc. New York (1995)). Dendrimer using radially branched lysine. Is formed at the amino terminus of the polypeptide chain by the method of Tam described above, and a plurality of anti-HIV active substances are bound to a plurality of amino groups present at the terminus and side chains to obtain one molecule of the polypeptide chain. The substance used for forming the dendritic structure is most preferably the above-described lysine, but may be prepared by a basic amino acid such as ornithine or by synthesis in addition to lysine. Even amino acids and the like can be used as long as they are substances containing two or more amino groups. .

 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, but when a carboxyl group or a hydroxyl group is present in the substance. Is preferably a chemical bond formed through these functional groups, particularly preferably a hydroxyl group. Further, 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, so that the functional group in the molecule serving as the binding site is The position 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 is reacted with the amino group. It is possible to form an amide bond between the carboxyl groups of the HIV active substance and to make the bond directly, and to form a chemical bond that is easily cleaved in vivo through an appropriate spacer substance. It is also possible to form.

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 activity It can be attached through a multifunctional spacer having a functional group that can be chemically attached to both hydroxyl groups of the substance. Such a multifunctional supplier has one carboxyl group capable of binding to an amino group at the amino terminal of the polypeptide chain to be used, and further contains water in the anti-HIV active substance. It is preferable to have a carboxyl group different from the above which can form an ester bond with an acid group, and specific examples include an aromatic compound having two or more carboxyl groups or an aliphatic compound. Aliphatic compounds are preferred because they form a chain structure.

 That is, when a dicarboxylic acid is used as a polyfunctional spacer, an amide bond is formed between one of the carboxyl groups and the amino group at the amino end of the polypeptide chain, and the other carboxyl group is formed. By forming an ester bond with the hydroxyl group in the anti-HIV active substance, a polypeptide chain and the anti-HIV active substance can be bound. Considering the simplicity of synthesis, the ease of enzymatic cleavage in vivo, and the anti-HIV activity of the substance of the present invention, an aliphatic dicarboxylic acid having 4 to 12 carbon atoms is a multifunctional spacer. Preferably, an aliphatic dicarboxylic acid having 4 to 8 carbon atoms is more preferable. Succinic acid or glutaric acid is particularly preferred from the viewpoint of stability and ease of handling. The dicarboxylic acid may have another functional group as long as it does not prevent the binding between the polypeptide chain and the anti-HIV active substance.

 As a method of bonding the protecting group-protected polypeptide resin and AZT to a dicarboxylic acid (for example, succinic acid or glutaric acid) as a polyfunctional spacer, first, AZT and a polyfunctional spacer are combined. To form a conjugate. That is, for example, AZT is reacted with succinic anhydride or glucuric anhydride in the presence of dimethylaminopyridine, and after a conventional treatment, the binding of AZT, in which succinic acid or glutaric acid is ester-bonded to AZT, to a multifunctional spacer. Get the body. Then, the AZT-polyfunctional spacer conjugate is added to the α-amino group or ω-amino group of the amino acid at the N-terminal of the protecting group-protected polypeptide resin, or dendrimer-like to the amino acid. Can be condensed and bound to a plurality of amino groups present at the terminal and side chain of the dendritic structure bonded to DNA by a known method, for example, the DIPCI-HOBt method or the like. Peptide resin is also referred to as AZT complex).

The protecting group-protected polypeptide resin spacer-AZT complex obtained as described above can be prepared in a single step, for example, by the following method to remove the insoluble resin and the protecting group and form a disulfide bond. By performing, the substance of the present invention can be obtained. That is, for example, 10% or less, preferably less than 5%, and more preferably 0.5 to 1.5% (v / v) of aniso is added to the protecting group-protected polypeptide resin-spacer AZT complex. 1 liter, 1 equivalent or more, preferably 5 to 15 equivalents, more preferably 8 to 12 equivalents of TMSC1 (trimethylsilyl chloride) and TFA are added for 5 minutes or more, preferably 30 to 12 The reaction is carried out for 0 minutes at 0 to 80 ° C, preferably at 10 to 40 ° C, more preferably at 15 to 30 ° C, followed by preferably 10 ° C or less, more preferably ice. 100 equivalents or more, preferably 200 to 400 equivalents of DMSO is added under cooling, and 20 minutes or more, preferably 30 to 120 minutes, more preferably 60 to 100 minutes. By reacting, the deprotection group, the resin removal, and the formation of the disulfide bond are performed in one step. The system for removing the protecting group and the resin and forming a disulfide bond is hereinafter referred to as TMSC1-1DMSO / TFA system. When preparing the substance of the present invention in which a plurality of anti-HIV active substances are bound, it is necessary 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. And more preferably 16 or less.

The thus-obtained substance of the present invention 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, Salts with citric acid, tartaric acid, salicylic acid, etc., acidic sugars (eg, glucuronic acid, galacturonic acid, dalconic acid, ascorbic acid, etc.), or organic sulfonic acids (eg, methanesulfonic acid, p-toluenesulfonic acid, etc.). In addition, sodium salts, potassium salts, calcium salts, magnesium salts and the like can be mentioned, but there is no particular limitation. Although the substance of the present invention can be administered as it is, it may be a pharmaceutical composition further containing a pharmacologically acceptable carrier selected according to the administration method and administration form of the drug. This pharmaceutical composition is administered orally or parenterally according to the treatment method and the like, and is injected, injectable, externally applied, suppository (for example, powder) using an appropriate drug carrier according to the administration method. , Granules, liquid for injection or oral use, tablets, pessaries, ointments, creams, aerosols, etc.) It can be a formulation.

 When the pharmaceutical composition of the present invention is directly administered to a living body, for example, as an injection, 10 mg to 5 g per 1 kg of human body weight per day can be administered as a drug component.

 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 affinity for at least an HIV surface protein, and a polypeptide chain on the target cell. By breaking the chemical bond with the anti-HIV active substance, the anti-HIV agent with a different mechanism of action, the polypeptide chain and the anti-HIV active substance, ie, the steps of adsorption, fusion and entry of HIV to CD4-positive cells It is thought to be caused by the combination of a polypeptide that is thought to suppress the activity and an anti-HIV agent that inhibits the enzyme activity of HIV reverse transcriptase or HIV protease. That is, a therapeutic effect equivalent to that of the multidrug combination therapy is expected by using the substance of the present invention alone. The reason for the lower toxicity compared to conventional anti-HIV agents is that an anti-HIV active substance such as AZT, which has cytotoxicity on its own, is chemically bonded and accurately delivered to target cells. It is thought that this is to suppress the effective concentration of the drug and suppress toxicity to normal cells. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a BIAcore analysis diagram showing the affinity of the substances 12 (AZT-Suc-T22) and 22 (AZT-Glu-T22) of the present invention for gp120 and CD4. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto unless it exceeds the gist.

 Test example 1

 TMS C 1— DMSO— Deprotection ability of TFA system

Fmo c — As p (But) — OH, Fmo c — G lu (B u ') — OH, Fm oc — H is (Bo c) _0H, Fmo c — Ser (Bu ^l ) — 0H, Fmo c- Th r (B u ') -OH, Fmo c— Lys (Bo c) one 0H, Fmo c— A rg (Pmc) — To each 6 mg of OH, add TMSC 1 (42〃1), DMSO (762〃1), and TFA (5.2 ml), and treat at 4 ° C to 5, 1 0, 3 0, 6 0, 9 and sampled by 1 0 0 beta 1 after 0 minutes _{Η 2 0- Me CN (1:} 1) with diluted lml, by injection a part of the solution to the H PLC Fmoc amino acids, side chain protected Fmoc amino acids were quantified.

 In the following, HPLC analysis was performed using a Cosmosi15C18-AR (4.6 x 150 mm) column at a flow rate of 1 m1 Zmin. Elution is 0.1%

_{(v / v) TFA- H 2} 0 and 0. 1% (v / v) TFA- was achieved using a linear gradient of Me CN (Asetonito Lil). Cosmo si 1 CI 8 -AR (2.0 × 25 cm) was used for fractionation with HP LC at a flow rate of 7 ml 1 Zmin.

 As a result, it was found that any side-chain protecting groups could be quantitatively removed within 60 minutes by this cleavage reagent system.

 Test example 2

 TMS C 1 DMSO—Amino acid release ability from TFA resin

 H—Leu—A1 koresin (0.60 mmo1 / g, 2〃mo1) in which Leu is bound to an alkoxyl-type resin that releases amino acids or eptidic acid by cleavage, or amino acids by cleavage H-Arg (Pmc) with Arg (Pmc) bound to an amide-type resin that releases amide or peptide amide An internal standard for Rink am ideresin (0.32 mmo1 / g, 2 no1) Boc—Gly—OH (3 no 1), TMS C 1 (7 ^ 1, 750 eq) and TFA (870 (1) were added, and the mixture was stirred at room temperature for 1 hour. DMSO (127 / l, 750 eq) was added, and the mixture was further stirred for 1 hour. After 30 minutes, 60 minutes, 90 minutes, and 120 minutes, samples were sampled at 1001 each 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 the amino acid after treatment for 2 hours was 100% for Leu and 87% for Arg. From this result, it was revealed that the amino acid bound to A1koresin and Rinkamiderensin can be quantitatively cleaved and released by this cleavage reagent system.

 Preparation Example 1

Preparation of Protecting Group-Protected Polypeptide Resin Having Polypeptide Chain of the Following Formula (A) ¹ 1 2 3 4 5 6 7 7 8 9 10 11 12 13

Arg-Arg-Tyr-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Lys-Cys-Arg-OH

• · · · (A)

(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.)

 Preparation of the protecting group-protected polypeptide resin having the polypeptide chain of the formula (A) was performed manually using the Fmoc-type solid phase synthesis method. As a resin for peptide synthesis, Fmoc-Arg (Pmc) -A1 koresin (0.52 mmo1 / g in terms of Arg) was used. The cells were treated with 20% piperidine ZDMF for 15 minutes to remove the Fmoc group. DIPCI-HOBt / DMF was used for the condensation of Fmoc amino acids. Fmoc amino acids, condensing reagents, and the like were each used in 2.5 equivalents to the resin. Hereinafter, the substance synthesized in this manner is referred to as a protecting group-protected polypeptide resin 1 for convenience.

In addition, a protecting group-protected polypeptide resin that generates an amide form of a polypeptide chain in which the 13-position of the above formula (A) is NH ₂ is used as a resin for peptide synthesis, as Fmoc-Arg (Pmc). — Synthesized by the Fmoc-type solid-phase synthesis method as described above, using Rink ami de resin (0.34 mmo 1 / g in terms of Arg). Furthermore, a protecting group-protected polypeptide resin having a polypeptide chain represented by the following formula (B) is used as a resin for synthesizing a peptide, which releases an amino acid amide or a peptide amide by a cleavage treatment. Fmoc-PAL (peptideamid) The compound was synthesized by the Fmoc-type solid phase synthesis method as described above using delinker) resin (0.38 mmo 1 / g in terms of amino group). The substance synthesized in this manner is described as a protecting group-protected polypeptide resin 2 for convenience. 1 1 2 3 4 5 6 7 7 7 7 7 7 8

Arg-Arg-Trp-Cys-Tyr-Arg-Lys-Cys-Tyr-Lys-Gly-Tyr-Cys-Tyr

9 10 11 12 13

(B)

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

Table 2

Preparation Example 2

 Preparation of 3,1-azido-3, -deoxythymidine-15,1-monosuccinate (AZT succinate)

3,1-Azido-1,3-deoxythymidine (AZT) (534.8 mg, 2 mmol) was dissolved in THF (10 ml). At C, succinic anhydride (200.lmg, 2mmo1) and dimethylaminopyridine (48.8mg, 0.4mmo1) were added, and the mixture was stirred for 48 hours. Further, succinic anhydride (40.2 mg, 4 mmo 1) was added, and the mixture was stirred for 24 hours. The reaction solution A COE t (acetate Echiru: 50 ml) and extracted with, and the organic layer was washed with saturated brine and liquid separation added a saturated NaHCO ₃ solution. Adding Kuen acid aqueous layer was adjusted to PH 4, and extracted with AcOE t (5 0 mlx 3) , and the organic layer was washed with saturated brine, dried over anhydrous MgSO _4, and MgSO ⁴ was filtered and the filtrate Was distilled off under reduced pressure to obtain the desired AZT succinate ester crystals (5 67.8 mg, yield 77.3%).

Ή-NMR (27 0 MHz, CDC 1 3) (51. 86 (d, 3H), 2.6 -2 9 (m, 6H), 4. 1 1 -. 4. 1 3 (m, 1 H), 4.23 -4.29 (dd, 1 H), 4.34-4.39 (m, 1 H), 4.8 2-4.88 (dd, 1 H), 5.8 8—5.93 (q, l H), 7.45 ( d, 1 H), 10.6.8 (s, 1 H)

 Preparation Example 3

 Preparation of 3,1 azido 3'-deoxythymidine-1 5'-monoglutarate (AZT glutarate)

 AZT (534.8 mg, 2 mmo 1) was reacted with glutaric anhydride (228.2 mg, 2 mmo 1) under the same reaction conditions and the same reaction procedure as in the preparation of AZT succinate in Preparation Example 2. After the same treatment, the desired crystals of AZT glutaric acid ester were obtained (yield: 80.0%).

Ή-NMR (2 7 0 MHz , CD C 1 3) 51.9 1 (s, 3H), 1.96- 2.0 5 (m, 2H), 2. 3 6 - 2. 57 (m, 6H), 3.9 8- 4.08 (m, 1 H), 4.24-4.33 (m, 2 H), 4.5 1—4.55 (dt, 1 H), 6.05-6.09 (t, lH), 7.39 (s, 1H), 10.27 (s, 1H) Preparation Example 4

 Preparation of Polypeptides from Protecting Group-Protected Polypeptide Resins

To the protecting group-protected polypeptide resin 1 (50 mg, 9.6 mmo 1) prepared in Preparation Example 1 above, anisol (0.15 m 1), TMS C 1 (0.15 m K 1) 0 eq) and TFA (13 ml) were added, and the mixture was stirred at room temperature for 1 hour. Next, DMSO (1.9 ml, 300 eQ) was added at 4 ° C., 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 using HPLC and lyophilization, the desired polypeptide of the above formula (A) was obtained (6.28, yield 33%). The following measurements of the ion spray mass spectrum (IS-MS) were performed using a triple stage quadrupole mass spectrometer type ΑΡΙΠΕ (Perkin-E1mer S ciex). IS -MS (reconstructed) m / z: Found 1974.0 (1973.2 Calc. For C ₈₆ H, ₄ oN ₃₂ Pi a S ₂ ), [] ²⁶ _D = -24.5 ° (c = 0.2, H ₂₀ ), 30 to 40% acetonitrile concentration gradient of 30 to 40%, Waters Bondasphere 5 nC at a flow rate of lml / min 1 8-100 A (3.9 x 150 mm; manufactured by Nippon Millipore) Retention time on an analytical HP line is 14.2 minutes, Amino acid analysis of hydrolyzate by 6 N HC1 (values in force) Is the theoretical value) Cys undecided (l), Tyr3.00 (3), Lys and D-Lys2.86 (3), Arg5.11 (5), Pro O. 95 (1).

By the same reaction procedure as above, the yield from the polypeptide amide form of the above formula (A) (protecting group-protected polypeptide resin (using Rink Amide resin) 56% (8.4 mg)) I got IS- MS (reconstructed) m / z : Found 1972.5 (. 19 72.1 Calc for C 86 H, 4, N 33 P. 7 S 2), [a] 26 D = -18.8 ° (c = 0.2, H 2 0 ), 10-4

0% acetonitrile gradient for 30 minutes, flow rate at lml / min

Bondasphere 5 C18—100 people (3.9 x 50 mm, manufactured by Nippon Millipore) has a retention time of 14.8 minutes in analytical HPLC, and amino acid analysis of hydrolyzate with 6N HCl (in Nikko) Cy s not determined (theoretical values are theoretical) (1), Tyr

3.00 (3), Lys and D—Lys 2.80 (3). Arg 5.13 (5), ProO. 88 (1).

 Similarly, the polypeptide amide of the above formula (B) (protecting group-protected polypeptide)

(Yield from 2% 43%).

 Hereinafter, for convenience, the substance obtained by this method is referred to as a synthetic polypeptide, and the synthetic polypeptide (T13 31) having the polypeptide chain of the above formula (A) is referred to as synthetic polypeptide 1, the above formula (B) The synthetic polypeptide (T22) prepared by the same method as described above using the protecting group-protected polypeptide resin 2 having the above polypeptide chain will be referred to as synthetic polypeptide 2.

 Preparation Example 5

Preparation of substance of the present invention (polypeptide-AZT succinate complex) The α-amino group of amino acid at the N-terminal of the protecting group-protected polypeptide resin 1 or 2 prepared in Preparation Example 1 above The carboxyl group of the succinate (2.5 eq) prepared in 2 was condensed by DIPC I-HOBt method. The protecting group-protected polypeptide AZT succinate complex tree thus obtained Fat (5 Omg) is subjected to deprotection, deresining, and disulfide bond formation in the same manner as in the preparation of the synthetic polypeptide in Example 4, and the same polypeptide and AZT succinic acid are obtained by the same isolation and purification methods. An ester complex of the substance of the present invention was obtained. Hereinafter, for convenience, the substance of the present invention according to the present preparation method will be referred to as substance 1 of the present invention, and substance 1 of the present invention (AZT-Suc-T131) having the polypeptide chain of the above formula (A) will be referred to as substance 11 of the present invention. The substance 1 of the present invention (AZT-Sue-T22) having the polypeptide chain of (B) is referred to as the substance 12 of the present invention.

Inventive substance 11: Yield 29.99% (5.5 mg), IS-MS (reconstructed) m / z: Found 2323.23 (2322.1 Calc. For C, ₀ oH, ₅₅ N ₃₇ 0 ₂₄ S ₂ , [ «] ' ⁸ _D = — 16.7 ° (c = 0.1, IN AcOH), 30 to 50% acetonitrile gradient from 10 to 50%, Waters / Bon dasphere 5 / C 18-1 at lml / min flow rate Analytical HP LC retention time with 00A (3.9X150 thigh, manufactured by Nippon Millipore) is 17.5 min, amino acid analysis of hydrolyzate with 6 N HC1 (values in force are theoretical values) Then, Cys undecided (l), Tyr3.00 (3), Lys and D-Lys3.00

(3), Arg 5.37 (5), Prol. 24 (1).

Inventive substance 12: Yield 2.76%, I S-MS (reconstructed) m / z: Found 28 34.47 (2836.3 Calc. For Ci ₂₃ Hi ₇₉ N ₄₃ 0 ₂₈ S ₄ , [a] ² % = 47.8 ° (c = 0.01, IN AcOH) Preparation Example 6

 Preparation of the 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 above was added to Preparation Example 3 above. The carboxyl group of the prepared glutaric acid ester (2.5 eq) was condensed by DIPCI-HOBt method. The protecting group-protected polypeptide resin-AZT glutaric acid ester complex thus obtained was prepared in the same manner as in the preparation of the synthetic polypeptide of Preparation Example 4 to obtain the desired polypeptide AZT glutaric acid ester complex. A body of the substance of the invention was obtained.

 Hereinafter, for convenience, the substance of the present invention according to the present preparation method is referred to as substance 2 of the present invention, and substance 2 of the present invention (AZT-G1U-T131) having the 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-G1U-T22) having the polypeptide chain of (B) is referred to as substance 22 of the present invention.

Present substance 21: Yield 6.27% (2.3 mg), IS-MS (reconstructed ) m / z: Found 2337.73 (2336.2 Calc. for C, ₀ .H, ₅ τΝ ₃ 7O24S2, [«] ²⁰ _D = -38.2 ° (c = 0.3, IN AcOH), 10 to 50% of 30 Acetonitrile concentration gradient of 1 min per minute, Waters Bondasphere 5 C18—100 (3.9 X 150 mm manufactured by Nippon Millipore) at a flow rate of lml / min, retention time by analytical HPLC of 17.4 minutes, Amino acid analysis of the hydrolyzate with 6 N HC1 (the values in force are theoretical values) indicate that Cys has not been determined (l), Tyr 2.92 (3), Lys and D-Lys 3.00 (3), Arg 5.08 (5), and Pro1.22 (1).

The present invention material 2 2: Yield 8. 8 1%, I S- MS (reconstructed) m / z:. Found 28 49.97 (2850.3 Calc for C, 2, H, 8, N 4 .0 28 S 4, [ α] ² % = 21.4 ° (c = 0.1, IN AcOH) Preparation Example 7

 Preparation of De n d r i mer type substance of the present invention

 The protecting amino acid is further protected on 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 using a similar amino acid condensation method (DIPCI-HOBt method, etc.). Fmo c — Lys (Fmo c) was bound. Furthermore, the removal of Fmoc group and the introduction of Fmoc-Lys (Fmoc) were repeated twice, and Lys having a dendritic structure in which seven Lys were introduced into the amino terminal of the polypeptide. An introduced protecting group-protected polypeptide resin was prepared. Hereinafter, the resin prepared from the protecting group-protected polypeptide 1 obtained in Preparation Example 1 is referred to as Lys-introduced protecting group-protected polypeptide resin 1, and the resin prepared from the protecting group-protected polypeptide 2 is referred to as Lys Described as introduced protection group-protected polypeptide resin 2.

 The F moc 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 was prepared in the same manner as in Preparation Example 5 by DI PC I- Condensed by the HOBt method. The Lys-introduced protecting group-protected polypeptide resin-AZT succinate complex thus obtained was subjected to deprotection, deresinization and disulfide bond formation in the same manner as in the preparation of synthetic polypeptide 1 in Preparation Example 4. By the same isolation and purification methods, a dendrimer-type substance of the present invention into which eight target AZTs were introduced was obtained. Hereinafter, the substance of the present invention according to the present preparation method will be referred to as “dendrimer type substance 1 of the present invention” for convenience.

dendrimer-type substance of the present invention 1: Yield 13.18% (7.3 mg), IS— MS (reconstructed) m / z: . Found 5665.47 (5664.5 Calc for C 24 .H 346 N 86 0 73 S 2, [] 18 D two 22.6 ° (c = 0.1,1N AcOH)ヽ1 0-50% of the 30 Acetonitrile concentration per minute min., Water retention rate at lml / min 5 〃C 18-100 people (3.9xl50mm, manufactured by Nippon Millipore) retention time in HPLC by 26.5 minutes, In the amino acid analysis of the hydrolyzate by 6N HC1 (the values in kazuko are theoretical values), Cys undetermined (1), Tyr2.87 (3), Lys and DLys100.0 (1). 0) .A rg 5.42 (5), Pro l. 26 (1) o

 In the same manner as described above, the AZT glutaric acid ester prepared in Preparation method 3 is bonded to the Lys-introduced protecting group-protected polypeptide resin 1, and the same deprotection, deresinization, disulfide bond formation, isolation, Purification was performed to obtain the target substance of the present invention. Hereinafter, the substance of the present invention is referred to as “dendrimer type present substance 2”.

dendri me r-type substance of the invention 2: Yield 0. 7 2%, I S- MS (reconstruc ted) m / z:. Found 5775.22 (5780.3 Calc for C 248 H 362 N 86 0 73 S 2, [α] ² % =-12.3 ° (c = 0.05, IN AcOH)

 In the same manner as described above, the AZT succinate prepared in Preparation Example 2 was bonded to the Lys-introduced protecting group-protected polypeptide resin 2. The obtained Lys-introduced protecting group-protected polypeptide-AZT succinate complex resin (10 Omg) was added to m-cresol (3001), 1,2-ethanedithiol (300/1), Add thioanisole (30031), TFA (4.8 ml) and distilled water (300〃1) and stir at room temperature for 2 hours to deprotect and deresin. After treating and air-drying according to the procedure for preparing synthetic polypeptide 1 or 2, the crude peptide derivative obtained was 1 N

It was dissolved in AcOH (3 ml) and diluted to 40 ml with ultrapure water. To this solution was added DMSO (10 ml) at 4 ° C, and the mixture was allowed to stand at room temperature for 24 hours to form a disulfide bond. Furthermore, isolation and purification were carried out using HPLC, and after lyophilization, a dendrimer-type substance of the present invention into which eight target AZTs had been introduced was obtained. Hereinafter, the substance of the present invention according to the present preparation method is referred to as dendrimer-type substance 3 of the present invention for convenience. However, since two disulfide isomers were obtained, they are represented as isomers 1 and 2, respectively. Dendrimer-type substance 3 of the present invention 3: two kinds of disulfide isomers were obtained.

Isomer 1: Yield 2. 6 2% IS- MS (reconstructed ) m / z: Found 6178.22 (6 180.7 Calc for C 2 s 3 H 37 .N 92 0 77 S 4, [] 2> =..! 3.Γ (c = 0.1, IN AcOH)

Isomer 2: Yield 4.31%, [α] ² % =-15.4. (c = 0.1, IN AcOH)

 In the same manner as described above, the AZT glutaric acid ester prepared in Preparation Example 3 was bonded to the Lys-introduced protecting group-protected polypeptide resin 2, and the same deprotection, resin removal, disulfide bond formation, isolation, Purification was performed to obtain the target substance of the present invention. Hereinafter, the substance of the present invention is referred to as “dendrimer type present substance 4”.

dendri me r-type substance of the invention 4: Yield 6. 2 0%, I S- MS (reconstruc ted) m / z:. Found 6292.97 (6292.9 Calc for C 271 H 386 N 92 0 "S 4, [ shed] ^2. _D- 2 10.3 ° (c = 0.2, IN AcOH)

 Example 1

 Measurement of the affinity of the substance of the present invention for gp120 and CD4

The affinity of substances 12 (AZT-Sue-T22) and 22 (AZT-Glu-T22) of the present invention obtained in Preparation Examples 5 and 6 to gp120 and CD4 was measured using surface plasmon resonance. Biosensors, BIA c0 r ™ biosensor (Pharmacia BiosensorAB ^), Biochem. Biophys. Res. Commun., 219, 555-559 (1996), Biochem. Biopys. Act a., 1298, 37 Measurements and analyzes were performed according to the method of -44 (1996) (Fig. 1). As a result, affinity constant for gp l 2 0 and CD4 of the onset bright material 1 2 (K aff) were respectively 2 and ^{20 X 1 0 7 M 3. 44} X 1 0 6 M. Similarly Ka ff against gp 1 20 and CD 4 of the present invention material 22 were respectively 6.0 5 1 0 ⁶ and ^{1. 0 7 X 1 0 7 M} . These affinity constants, K aff (respectively for gp 1 20 and CD 4 synthetic polypeptide 2 obtained in Preparation Example 4 5 and ^{9 9 x 1 0 6 M 9} 9 0 x 1 0 6 M:. Biochem. Biophys. Res. Commun., 219, 555-559 (1996), Biochem. Biopys. Acta., 1298, 37-44 (1996)). It was revealed that it shows high affinity for CD4.

 Example 2

Antiviral activity against human immunodeficiency virus (HIV) Synthetic polypeptides 1 (T131) and 2 (T22) obtained in Preparation Example 4 above, substance 11 (AZT-Sue-T131) of the present invention obtained in Preparation Example 5 above, dendrimer type book obtained in Preparation Example 7 above. The antiviral activity against HIV of Invention Substance 1 and AZT was tested and evaluated according to the following method. That is, HIV-infected MT-4 cells (2.5 × 10 ⁴ cells, Zwell, multiplicity of infection (MOI): 0.001) are added to a 96-well microtiter plate immediately after infection, together with various concentrations of the test substance. Among C0 ₂ incubator one, they were cultured for 5 days at 37 ° C, the number of viable cells the MTT method (Pauwel et. Al., J. Virol. M ethods 20, 309-321 (1988)) was measured in. Antiviral activity is expressed as the concentration that inhibits cell death by 50% by HIV infection (E Cso: 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 the test substance at various concentrations in the same manner as described above. Cytotoxicity is expressed as 50% cytotoxic concentration (C Cso: 50¾ cytotoxic concentration) by the test substance. Also CC ₅ . And EC ₅ . The approximate ratio (CC _5; ZEC ₅ ) was expressed as the 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 _5. Comparison of values), the selection coefficient was more than twice, and the anti-HIV activity was clearly enhanced. 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. Table 3 Measurement of anti-HIV activity by MTT method

CC ₅₀ (^ M) ECso (nM) SI Synthetic polypeptide 1> 80 17> 4615 Synthetic polypeptide 2 27 8.4 3208 Substance of the present invention 11 6.8 1.1 5713 dendrimer type substance of the present invention 1 1.7 0.18 9694

AZT 5.6 2.5 2299 Example 3

 HIV infection inhibitory activity

HIV infection inhibitory activity of synthetic polypeptides 1 and 2 obtained in Preparation Example 4 above, substances 1 to 22 of the present invention obtained in Preparation Examples 4, 5, and 6, and dendrimer-type substance 1 of the present invention, and AZ-cho. Was tested and evaluated according to the following methods. That is, PHA (Phaseolus vulgaris agglutinin) activated P BMC (perip heral blood mononuclear cells) using T cell line tropic HIV-1 (NL 4-3) and macrophage tropic HIV-1 (JR-CSF) ) Was measured by a p24 antigen expression suppression test by an ELISA method using an antibody against the HIV-1 p24 antigen. That addition of the test substance of various concentrations MT- 4 cells immediately after HIV- 1 infection, C0 in ₂ incubator one, 37 ° C for 3 days (possibly four days one had seven days) After culturing, the culture The amount of HIV-derived p24 antigen in the supernatant was measured using an ELISA kit (manufactured by Cellular Products). The results are shown in Table 4. In contrast to JR-CSF, which is a macrophage-directed HIV-1 in which synthetic polypeptides 1 and 2 obtained in Preparation Example 4 above originally show no anti-HIV activity, the present invention shows that all of the present substances are anti-HIV as well as AZT. It showed activity and was also effective against macrophage-tropic HIV-1.

 Table 4 Measurement of HIV infection inhibitory activity by p24 antigen expression suppression test

ECso (nM)

 NL4-3 JF-CSF

 (T cell directivity) (? D D-fa, A directivity)

 Synthetic polypeptide 1 1790> 4000

 Synthetic polypeptide 2 63> 3000

 Substance of the present invention 11 168 7.4

 Substance of the present invention 22 11.4 4.2

 dendrimer type substance of the present invention 1 88 7.3

AZT 65 4.¾ Industrial applicability

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

Claims

The scope of the claims 1. One or more reverse transcriptase inhibitors and / or HIV protease inhibitors are applied to polypeptides having an affinity for HIV surface protein gp120 and / or HIV host target cell surface protein CD4. A complex, wherein the agents are bound by a chemical bond.  2. The polypeptide having affinity for HIV surface protein gp120 and / or HIV host target cell surface protein CD4 is a polypeptide represented by the following formula (1): The composite of claim 1.  1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 A 1 -A 2 -C y s -A 2 -A 3 -A 3 -Y -A 2 -A 3 -A 3 -C y s -A 4 -A 5  (1) [Wherein, 1 is 11- (derived from the amino group of A2) or one basic amino acid residue selected from lysine, arginine and orditin, or the same or a selected from these amino acid residues; A peptide residue having at least two different amino acid residues, or a hydrogen atom at the N-α-position of the amino terminal amino acid residue of the basic amino acid residue or the peptide residue having a hydrogen atom or a substituted thiocarbamoyl group Represents a Ν-α-substituted amino acid residue, a Ν-α-substituted peptide residue, a Ν-substituted thiosulfonate amino acid residue or a Ν-substituted thiocarbamoylated peptide residue. ;  A 2 independently represents a tyrosine, phenylalanine or tryptophan residue; A 3 independently represents a lysine or arginine residue;  A 4 represents an amino acid residue or a peptide residue having at least one amino acid selected from lysine and arginine; 85 represents −1 to 1 (from the carboxyl group of A4) or 1 NH ₂ (from the acid amide of the carboxyl group of A4); and Y represents a peptide residue represented by the following formula (a).  1, 2, 3, 4, 5, 6,, -A 6-A 2-A 3-G 1 yA 7-A 6-( _a ) (Where A2 and A3 have the same meanings as in the above formula (1), A 6 independently represents an alanine, valine, leucine, isoleucine, serine, cysteine or methionine residue;  A7 represents a tyrosine, phenylalanine, tryptophan, alanine, phosphine, oral isine, isoleucine, serine, cysteine or methionine residue; G 1 y represents a glycine residue. However, when both the Γ and ， positions are cysteine residues, they may be linked by a disulfide bond. ), Or  D-ornichiloop mouth phosphorus, prolyl-D-ornithin, prolyl-D-lysine, prolyl-D-arginine, D-lysiloop-mouth phosphorus, D-arginyl-proline, glycyl-lysine, glycyl-arginine, ornithyl-glycine Glycylorutin, lysyl-daricin and arginyl-daricin, which are selected from dipeptides composed of two amino acids, and the constituent amino acids of which are D-lysine, lysine, The hydrogen atom of D-orditin or the side chain of orditin ω-amino group represents a peptide residue which may be substituted with an acyl group; Cys represents a cysteine residue; The cysteine residue at position 1 may be linked by a disulfide bond.]  3. The chemical bond between at least one of the amino acid terminal amino acid elongation and the ω-amino group of the polypeptide represented by the above formula (1), a reverse transcriptase inhibitor and a Ζ or HIV protease inhibitor, 3. The conjugate according to claim 2, wherein the conjugate is a bond via a multifunctional spacer.  4. The composite 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 composite according to claim 3, wherein the polyfunctional spacer is a compound having at least two carboxyl groups.  6. The conjugate according to any one of claims 3 to 5, wherein the multifunctional spacer is an aliphatic dicarboxylic acid.  7. The complex according to any one of claims 1 to 6, wherein the reverse transcriptase inhibitor is a nucleoside reverse transcriptase inhibitor. 8. When the nucleoside reverse transcriptase inhibitor is a non-natural nucleoside or nucleoside 8. The composite according to claim 7, which is a door analog.  9. When the nucleoside reverse transcriptase inhibitor is a base selected from the group consisting of pyrimidine bases, purine bases, imidazole bases and triazole bases, and a furanose having at least one hydroxyl group or its acyclo isomer. 8. The complex according to claim 7, which is a nucleoside or an analog thereof.  10. The conjugate according to claim 7, wherein the nucleoside reverse transcriptase inhibitor is 3, -azido 3'-deokymthymidine.  3. The conjugate according to claim 1, wherein the HIV protease inhibitor is a substrate transition state mimic compound of HIV protease.  1 2. HIV protease inhibitors are Ro 31 1-89 59, A-77 003, KNI-93, KNI-102, KNI-174, KNI-227, KNI -2 7 2, L 1 7 3 5 527, SC-5 2 1 5 1, VX-478, ABT-538, DMP-323, and U-969 88 8 Or the complex according to any one of claims 1 to 6, which is two or more substances.  1 3. 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.