JPH10203967A - Simple herpes virus type i protease inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an enzyme activity inhibitor against serine protease derived from simple herpes virus type I (HSV-1 protease) and further to prepare an anti-herpes virus agent including the HSV-1 protease inhibitor as an active ingredient. SOLUTION: This HSV-1 protease inhibitor includes a compound selected from a group consisting of 1,4-dihydronaphthalene, 5-amino-8-hydroxyquinoline, juglone, purpurin, 4-methylcatechol, coumestrol, fluoroglycinolcarbaldehyde and 3-(2,4,5-trihydroxyphenyl)alanine, or a pharmaceutically acceptable salt thereof as an active ingredient. The anti-herpes virus agent includes a compound selected from a group consisting of 1,4-dihydronaphthalene, 5-amino-8- hydroxyquinoline and juglone, or a pharmaceutically acceptable salt thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an inhibitor of an enzyme activity of a serine protease derived from herpes simplex virus type 1. The present invention also relates to a pharmaceutical composition comprising the substance that inhibits the enzyme activity of serine protease as an active ingredient, that is, an anti-herpes virus agent.

[0002]

2. Description of the Related Art Among herpes viruses, herpes simplex virus type 1 (HSV-1) infects many adults and latently infects the trigeminal ganglion. In general, they do not show any serious symptoms, but with various additional factors (accumulation of fatigue, ultraviolet irradiation, trauma, drug use, etc.)
Activity is activated, keratitis, Kaposi varicella-like rash,
Causes symptoms such as cold sores. That is, when any defect or disorder occurs in the immune system of a latently infected person, it causes unpleasant symptoms, and it frequently occurs in combination with other diseases.

[0003] As the anti-herpes virus agent, Acyclovir (ACV) which is a nucleic acid antiviral agent is used.
Has been used in chemotherapy to utilize its thymidine kinase inhibitory activity and the selective inhibitory effect of the viral gene replication process by DNA polymerase. Although nucleic acid-based antiviral agents are extremely effective as symptomatic treatments, virus strains exhibiting resistance to these nucleic acid-based antiviral agents are gradually appearing. The emergence of this resistant strain is a phenomenon frequently reported for nucleic acid-based antiviral agents that utilize an inhibitory effect on DNA polymerase and the like, and has become an incentive and motivation for the development of non-nucleic acid-based antiviral agents.

One direction of non-nucleic acid antiviral agents inhibits the maturation of proteins such as core proteins forming capsids confining these viral genes and enzyme proteins specific to the virus, or proteins forming the envelope of the virus outer shell. Then, there is the development of a drug that inhibits the production of virus particles. Specifically, the virus-specific protein group includes:
The translation of viral genes into polypeptide chains, followed by cleavage into individual proteins, involves the development of viral-derived cleavage enzymes; protease inhibitors that are involved in the process.

[0005] Herpes simplex virus type 1 (HSV-1)
Also produce a protease derived from the virus (HSV-1 protease), and this HSV-1
-1 protease is essential. That is, the HSV-1
By inhibiting the enzymatic activity of the protease, the process of producing virus particles in the host cell can be interrupted. Unless virus particles having infectivity are newly generated, it is possible to prevent the onset of various diseases caused by the proliferation of the virus. In addition, when various diseases develop from latent infection, by inhibiting the enzyme activity of HSV-1 protease to weaken the infection itself,
The target symptom can be cured. It is desired to propose a compound which inhibits the enzyme activity of HSV-1 protease and which is suitable for such a therapeutic purpose.
A new proposal for a compound that inhibits the enzyme activity with high specificity for one protease is expected.

[0006]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide a herpes simplex virus type 1 serine protease (HSV-1 protease) using a specific known compound. )) Is to provide an HSV-1 protease inhibitor containing the compound as an active ingredient as a new means for inhibiting the enzyme activity, that is, as a new use of a specific known compound. In addition, this H
An object of the present invention is to provide a pharmaceutical composition containing a compound having SV-1 protease inhibitory activity as an active ingredient, that is, an anti-herpes virus agent.

[0007]

Means for Solving the Problems The inventors of the present invention have studied various compounds to determine whether or not they have HSV-1 protease inhibitory ability. Showed excellent HSV-1 protease inhibitory ability. From the plurality of compounds exhibiting the HSV-1 protease inhibitory ability, those compounds whose inhibitory ability is not significantly impaired even in an environment in which a compound having a weak reducing ability coexists are selected. Further examination was also made as a selection condition that it was in a range suitable for a medicine. As a result, the inventors have found several compounds that also satisfy these conditions, and have completed the present invention.

That is, the herpes simplex virus type 1 protease inhibitor of the present invention comprises 1,4-dihydroxynaphthalene (1,4-naphthalenediol), 5-amino-8-
Hydroxyquinoline, juglone (5-hydroxy-
1,4-naphthoquinone), purpurin (1,2,4-trihydroxyanthraquinone), 4-methylcatechol (4-methyl-1,2-benzenediol), cumestrol (7 ', 6-dihydroxycoumarino (3 ',
4 ', 3,2) coumarone), phloroglucinol carbaldehyde (2,4,6-trihydroxybenzaldehyde) and 3- (2,4,5-trihydroxyphenyl)
A protease inhibitor comprising, as an active ingredient, a compound selected from the group consisting of alanine (2-amino-3- (2,4,5-trihydroxyphenyl) propionic acid) or a pharmaceutically acceptable salt thereof. . Preferably,
1,4-dihydroxynaphthalene (1,4-naphthalenediol), 5-amino-8-hydroxyquinoline and juglone (5-hydroxy-1,4-naphthoquinone)
And a pharmaceutically acceptable salt thereof selected from the group consisting of:

The anti-herpes virus agent of the present invention comprises:
1,4-dihydroxynaphthalene (1,4-naphthalenediol), 5-amino-8-hydroxyquinoline and juglone (5-hydroxy-1,4-naphthoquinone)
And a pharmaceutical composition containing a compound selected from the group consisting of or a pharmaceutically acceptable salt thereof.

The pharmaceutically acceptable salts include, for example, inorganic acid salts such as hydrochloride and organic acid salts such as acetate. Further, as the 3- (2,4,5-trihydroxyphenyl) alanine, any of a DL form, a D form and an L form may be used.

[0011]

DETAILED DESCRIPTION OF THE INVENTION HS used in the present invention
All compounds exhibiting V-1 protease inhibitory ability are known compounds, and all are commercially available as chemical reagents. For example, it is described in a compound catalog of a reagent sales company such as Merck; Merck Index and the like.

The molecular structures of these compounds are shown below. 1,4-dihydroxynaphthalene (1,4-naphthalene diol): molecular weight = 160.17

[0013]

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5-amino-8-hydroxyquinoline: molecular weight = 233.1 (as dihydrochloride)

[0015]

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This compound is commercially available as a dihydrochloride. Juglone (5-hydroxy-1,4-naphthoquinone): molecular weight = 174.16

[0017]

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Purpurin (1,2,4-trihydroxyanthraquinone): molecular weight = 256.21

[0019]

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The purpurin is obtained from western madder (Rub)
ia tinctorum L. ) And Akane (Rubia Ak)
ane N.) is contained as a glycoside in the roots. 4-methylcatechol (4-methyl-1,2-benzenediol): molecular weight = 124.14

[0021]

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Coumetrol (7 ', 6-dihydroxycoumarino (3', 4 ', 3,2) coumarone): molecular weight =
268.15

[0023]

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Phloroglucinol carbaldehyde (2,
4,6-trihydroxybenzaldehyde): molecular weight =
154.12

[0025]

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3- (2,4,5-trihydroxyphenyl) -DL-alanine (2-amino-3- (2,4,5-
Trihydroxyphenyl) propionic acid): molecular weight = 21
6.21

[0027]

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These compounds all have in common that they have a hydroxyl group on the aromatic ring, have a molecular weight in the range of less than 300, and have water solubility.

As shown in the following test examples, the above eight compounds were used in an in vitro test system to obtain recombinant HSV-1.
When the inhibitory ability was evaluated using protease,
In each case, the enzyme activity was 50
% Or less. In particular, 1,4-dihydroxynaphthalene, 5-amino-8-hydroxyquinoline, and juglone are all superior in their inhibitory ability, and 0.1% bovine serum albumin (BSA) is contained in the system.
Under the condition of the presence of 0.5 mM dithiothreitol (DTT), or the condition of the presence of 0.5 mM dithiothreitol (DTT). In addition, as shown in the example of 1,4-dihydroxynaphthalene, various proteases produced by humans and present in the body do not inhibit their enzymatic activities, and thus are caused by the inhibition of these in vivo proteases. It has specificity suitable for pharmaceutical use without unwanted side effects. That is, it has no inhibitory ability against various human serine proteases and specifically inhibits HSV-1 protease, so that it can be applied as an anti-herpes virus agent. Note that HSV-
One protease has the same partial structure of the enzyme active site, for example, a protease derived from human cytomegalovirus (HCMV), or a serine derived from various viruses constituting the Herpesviridae family to which HSV-1 and HCMV belong. It also inhibits the enzyme activity of protease. Therefore, in general, it can also be used as an anti-herpes virus agent for treating diseases caused by these herpes viruses.

Since all of the above-mentioned compounds having HSV-1 protease inhibitory activity are highly water-soluble, they can achieve a blood concentration at which a therapeutic effect is observed even in the body, and the pharmaceutical composition, that is, the efficacy of the anti-herpes virus agent It can be used as a component. In consideration of the fact that the blood concentration decreases over time due to metabolism, excretion, etc. in the body, a compound showing protease inhibitory activity even at a lower concentration is, of course, more preferable as an anti-herpes virus agent. is there. In consideration of this point, the aforementioned 1,4-dihydroxynaphthalene and 5-amino-8-
Hydroxyquinoline and juglone are more suitable as active ingredients of anti-herpes virus agents. 1,4
-Dihydroxynaphthalene and juglone are more preferred.

The anti-herpesvirus agent of the present invention comprises 1,4
-A compound selected from the group consisting of -dihydroxynaphthalene, 5-amino-8-hydroxyquinoline and juglone or a pharmaceutically acceptable salt thereof as an active ingredient, and its dosage form depends on the administration method. Is appropriately selected. Since both are highly water-soluble, they can be made into liquid preparations such as injections and drops for intravascular administration,
Alternatively, various dosage forms for oral administration can be used. In addition, diseases caused by herpes virus, for example, Kaposi varicella-like rash, cold sores, etc., often show local symptoms, and in these, various external preparations that can act directly on the affected area You can also.

An anti-herpes virus agent,
The dose of the compound depends on the purpose of administration and the route of administration.
In addition, it is appropriately determined in consideration of the patient's age, gender, weight, and the like. For example, in the case of intravenous administration or oral administration, at least the blood concentration indicates that the compound has HSV.
Usually, the dose and the administration interval are usually determined appropriately so as to exceed the concentration required to reduce the enzyme activity of -1 protease to half. In general, 10-100 mg / kg of the above compound is administered to an adult once or several times a day. Hereinafter, the present invention will be described in detail with reference to specific examples.

[0033]

EXAMPLES The following test examples show that all the compounds used in the HSV-1 protease inhibitor of the present invention can achieve sufficient inhibition at low concentrations.

Test Method In this example, recombinant HV-1 protease was used as recombinant H
Using SV-1 protease and a synthetic peptide having a specific amino acid sequence cleaved by natural HSV-1 protease as a substrate, the ability to inhibit enzyme activity was evaluated.

The recombinant HSV-1 protease used was a fusion protein in which the HSV-1 protease was linked to the C-terminal of glutathione-S-transferase (GST). Glutathione constituting the N-terminal side of this fusion protein
S-transferase (GST) is a commercially available glutathione-S-transferase expression vector-p.
It has an amino acid sequence translated based on a known nucleobase sequence encoded by GEX-4T-1 (Pharmacia Biotech). On the other hand, the HSV-1 protease region constituting the C-terminal of the fusion protein is derived from the HSV-1 (F) strain, as shown below. The H
SV-1 (F) plasmid with a U _L 26 gene strains
pRB4057 (F. Liu and B. Roizman, J. Virology, 65 ,
p.206-212 (1991), F. Liu and B. Roizman, J. Virol
ogy, 65 , p. 5149-5156 (1991)) and the translation region thereof are identical.

The recombinant HSV-1 protease is constructed by constructing a circular plasmid vector containing the DNA encoding the recombinant HSV-1 protease according to the following procedure, and introducing the circular plasmid vector into the plasmid. A transformed microorganism having the ability to produce the recombinant HSV-1 protease produced by the transformation is prepared, the transformed microorganism is cultured, and the recombinant HSV-1 protease is isolated and collected from the culture. Can be prepared. In addition,
The preparation of the recombinant HSV-1 protease and its enzymatic activity have already been published by the present inventors (the 8th Annual Meeting of the Protein Engineering Society of Japan, lecture number o-08, and Japanese Patent Application No. 8-108). 120703).

[0037] [Construction of circular plasmid vector comprising DNA encoding a recombinant HSV-1 protease, and the preparation of transformed microorganisms introduced with plasmid vectors] First, a plasmid pRB4057 with the U _L 26 gene As a template, a DNA encoding the HSV-1 protease region, specifically, a peptide chain consisting of 247 amino acid residues of Met ¹ to Ala ²⁴⁷ is amplified and prepared by PCR. At this time, a restriction enzyme EcoR I cleavage site is located 5 'to the start codon ATG (Met), and Ser ^248, which is originally present immediately after GCG encoding Ala ²⁴⁷ , is encoded.
Introduce the stop codon TGA instead of AGC. At the same time, a Hind III cleavage site was introduced into the non-translated region downstream of the 3 ′ side. That is, already with reference to the nucleotide sequence of the U _L 26 gene has been elucidated, and synthesized primer added these artificial nucleotide sequences, the amplification by the PCR method using the same.

The obtained DNA fragment was digested with the restriction enzyme EcoR I.
Digest with Hind III to form cut ends. On the other hand, the cloning site E of the commercially available plasmid pMT1
The DNA fragment is ligated and inserted between the coRI and HindIII sites to construct the plasmid pMTHP. This plasmid pMTHP is once introduced into Escherichia coli JM109 for transformation. This recombinant bacterium is cultured to prepare a large amount of plasmid pMTHP. Bacteria are disrupted, and plasmid pMTHP is isolated and collected.

After digestion of the plasmid pMTHP with the restriction enzyme HindIII, the protruding end of HindIII in the cut part is blunt-ended using a large DNA polymerase I fragment derived from Escherichia coli. Then, it is cut with the restriction enzyme EcoR I and placed on the 5 'side of the start codon ATG (Met) with the restriction enzyme EcoR
I The cleavage site is a DNA fragment having blunt ends downstream of the termination codon TAG. The resulting DNA fragment is
Commercially available GST expression vector pGEX-4T-1 (Pharmacia Biot
ech) (EcoR I-Sma I) to construct the plasmid pGSTThHP. That is, pGEX-4T
-1 tac promoter (Ptac) and G immediately following it
A translation region encoding the fusion protein, in which the ST gene and the HSV-1 protease gene are linked, is formed. The link between the GST gene and the HSV-1 protease gene is the same as the previously reported GST and HSV-1 protease gene.
Expression plasmid for fusion protein of 1 protease pGST247
(CLDiIanni et al., J. Biological Chemistry, 268
(34), p.25449-25454 (1993)).

The plasmid pGSTThHP was transformed into the host E. coli E.
coli BL21 (DE3) pLysS strain for transformation. Using the marker gene Amp ^r present in plasmid PGSTThHP, screened transformants showing ampicillin resistance, in addition, whether to produce a fusion protein of GST and HSV-1 protease, goat anti-GST antibody The plasmid was confirmed by Western blotting using
Screen recombinant bacteria harboring pGSTThHP. In this example,
Retains plasmid pGSTThHP and contains GST and HSV-
A recombinant strain showing the ability to produce a fusion protein of one protease was used. Specifically, E. coli BL21 (DE3) /pLysS.pGS
A name for identification of TThHP was attached, and the accession number FERM P-
The strain deposited as 15610 was used.

[Preparation of Recombinant HSV-1 Protease of GST-HSV-1 Protease Fusion Protein Type] Culture production of fusion protein by recombinant bacteria and isolation of active recombinant HSV-1 protease from culture Purification is performed according to the following procedure.

Recombinant E. coli BL21 (DE3) /pLysS.pG
STThHP (FERM P-15610) was added to ampicillin 1
Using 2 L of 2 × YT as a culture medium, shake culture is performed in a 5 L Erlenmeyer flask at 30 ° C. for 6 hours. Then, GST-H
In order to induce the expression of the SV-1 protease fusion protein, isopropyl-β-D-thiogalactopyranoside (IPTG) is added to the medium at a final concentration of 0.1 mM. Subsequently, shaking culture is performed at 30 ° C. for 14 hours, and the obtained culture is centrifuged to collect the bacteria.

The collected cells were mixed with a glutathione Sepharose 4B column equilibration buffer solution (50 mM Tris-HCl, 15 mM).
0 mM NaCl, 1 mM EDTA, pH 8.0, buffer solution A
) And disrupt the cells by ultrasonication. Then, the insoluble fraction was separated by centrifugation,
A supernatant containing the soluble protein is obtained. The GST-HSV-1 protease fusion protein dissolved in the supernatant is isolated and purified by the following means.

First, the supernatant to be collected is buffered A
The GST-HSV-1 protease fusion protein dissolved in the supernatant is specifically adsorbed to the column by loading onto the glutathione Sepharose 4B column equilibrated in the above. The column is washed with the buffer solution A to remove unadsorbed components. After that, GST-H adsorbed on the column was added with buffer solution A to which 20 mM reduced glutathione was added.
Elute the SV-1 protease. The fraction containing GST-HSV-1 protease eluted from this column is once concentrated with an ultrafiltration membrane. This concentrated solution is mixed with 0.2 M NaC
l, Sephacryl S equilibrated with 0.1 M sodium phosphate, pH 7.2 (hereinafter referred to as buffer solution B)
Load on a -300 HR column and perform gel filtration chromatography. The fraction containing the GST-HSV-1 protease that has been subjected to the gel filtration is concentrated again using an ultrafiltration membrane. This was added to 50 mM Tris-HCl, 100 mM NaCl, pH 7.5
(Hereinafter referred to as buffer solution C), and loaded onto a Sephadex G-25 column equilibrated with the buffer solution C to obtain a purified GST-HSV-1 protease solution. Finally, the purified GST-HSV
-1 Protease solution is concentrated by an ultrafiltration membrane, and glycerol is added to a final concentration of 50%.
Store at -20 ° C. The concentration of the fusion protein contained in the GST-HSV-1 protease solution was measured using commercially available BCST.
Calculate based on the quantification result of the fusion protein using A Protein Assay Reagent (manufactured by PIERCE).

[Synthetic Substrate] HSV-1 protease is
Between ^{Ser 248, Ala 610 - - U} L 26 gene translation consisting 635 amino acid residues is the product peptide chain Ala ²⁴⁷ Ser
It has been reported to selectively disconnect both during ⁶¹¹ . In addition, it has been confirmed that protease activity is also observed when a synthetic peptide consisting of a partial amino acid sequence containing these cleavage sites is used as a substrate (CL DiIanni).
et al., J. Biological Chemistry, 268 (34), p.25449-2
5454 (1993)). With reference to this report, Ala ^247-
Corresponds to the cleavage site between the Ser ^248, created a synthetic substrate below.

[0046]

Embedded image H-Thr-Tyr-Leu-Gln-Ala-Ser-Glu-Lys-Phe-Lys-
Met-Trp-Gly-NH _{2 When} this synthetic substrate is cleaved with HSV-1 protease, the following two types of peptide fragments are obtained.

[0047]

Embedded image H-Thr-Tyr-Leu-Gln-Ala-OH H-Ser-Glu-Ly
s-Phe-Lys-Met-Trp-Gly-NH ₂

[Measurement of Recombinant HSV-1 Protease Activity In Vitro Using Synthetic Substrate] The above-mentioned synthetic substrate was previously prepared in a buffer solution (10% glycerol, 25 mM
Tris-HCl, pH 7.5, 50 mM NaCl) to prepare a synthetic substrate solution dissolved at a predetermined concentration. On the other hand, the aforementioned recombinant HSV
-1 protease; prepared from a GST-HSV-1 protease solution into a recombinant HSV-1 protease solution having a protein concentration of 9.4 pmol / μl, 50% glycerol, 25 mM Tris-HCl, pH 7.5, and 50 mM NaCl. did. A test substance for evaluating enzyme inhibition was used by dissolving a predetermined amount in dimethyl sulfoxide (DMSO) in advance. As an assay buffer, 8.8% glycerol, 25.7 mM Tris
The composition used was -HCl, pH7.5, 51.3 mM NaCl.

First, 2.7 μl of the above-mentioned recombinant HSV-1 protease solution and 2 μl of a test substance DMSO solution are added to 75.3 μl of an assay buffer solution, and incubated at 30 ° C. for 30 minutes. Next, 20 μl of the synthetic substrate solution is added, and the mixture is incubated at 30 ° C. for 1 hour to cause an enzyme reaction. The enzymatic reaction is stopped by adding 20 μl of 6% trifluoroacetic acid (TFA). Under these reaction conditions, the final concentration of the recombinant HSV-1 protease is 250 nM. The test substance DMSO solution 2
The same operation as described above was performed using 2 μl of DMSO instead of μl, to obtain a reference value for enzyme inhibition of 0%.

After the termination of the reaction, the synthetic substrate remaining in the reaction solution and the fragments of the synthetic substrate cleaved by the enzymatic reaction are analyzed by reversed-phase HPLC. The conditions for reverse phase HPLC are
Using a reverse phase _C18 column (inner diameter 4.6 mm × 7.5 cm, Tosoh), elution was performed using 0.1% TFA (4%) with 20% acetonitrile.
Minutes).

On the other hand, a standard product of the above-mentioned C-terminal side cleavage product of the synthetic substrate was separately synthesized in advance, and the synthetic substrate and the C-terminal side cleavage product were dissolved in a buffer solution having the same composition as the reaction solution to obtain a standard sample. The analysis is also performed by reverse phase HPLC. Quantification was performed by comparison with this standard spectrum. The enzyme reaction rate is calculated based on the peak intensity of the C-terminal cleavage product. The inhibitory activity was defined as the difference between the reference value of the enzyme activity and the measured residual value of the enzyme activity.

Table 1 shows the inhibitory activity when each of the test substances was used in a final concentration of 100 μM when the final concentration of the synthetic substrate was 500 μM in the reaction solution. In the following, 5-
As amino-8-hydroxyquinoline, 5-amino-8-hydroxyquinoline dihydrochloride was used.

[0053]

[Table 1] Test substance inhibitory activity (%) 1,4-dihydroxynaphthalene 100 5-amino-8-hydroxyquinoline 75 juglone 100 purpurin 75 4-methylcatechol 55 cumestrol 90 phloroglucinol carbaldehyde 483- (2, (4-5-trihedraloxyphenyl) -DL-alanine 73

In addition, a 0.1% final concentration of BSA was added to the reaction solution.
(Bovine serum albumin) or DTT at a final concentration of 0.5 mM was added, and the inhibitory activity in the presence of these coexisting substances was evaluated under the same conditions. That is, these coexisting substances were added to the assay buffer solution, and the other conditions were the same. Table 2 shows the results. The addition of BSA corresponds to an environment in blood, and the addition of DTT corresponds to a weak reducing condition. In particular, the case where the inhibitory activity is significantly reduced with the addition of BSA is considered to reflect that the effective amount of the test substance participating in enzyme inhibition decreases because the serum albumin component contained in the plasma undergoes capture and the like. .

[0055]

Inhibitory activity (%) Test substance 0.1% BSA 0.5 mM DTT 1,4-dihydroxynaphthalene 100 55 5-amino-8-hydroxyquinoline 67 44 Juglone 97 66 Purpurine 4 73 4-Methylcatechol 51-38 Cumestrol 11 90 phloroglucinol carbaldehyde 3 28 3- (2,4,5-trihedraloxyphenyl) -DL-alanine 6 63

From the above results, it can be seen that all of the above eight compounds are excellent in the inhibitory activity against HSV-1 protease. Among them, 1,4-dihydroxynaphthalene, 5-amino-8-hydroxyquinoline dihydrochloride,
The three juglone species also have high inhibitory activity and are only slightly affected by the presence of serum albumin,
It is determined to be preferable for pharmaceutical use. As an example, FIGS. 1 to 3 show the results obtained by measuring the dependency of the inhibitory activity on the addition concentration of these three compounds while changing the addition amounts of the three compounds. When the 50% inhibitory concentration IC ₅₀ of 1,4-dihydroxynaphthalene was calculated from the results in FIG.
The C ₅₀ value is estimated at 6.4 μM. From the results in FIG. 2, the IC ₅₀ value of 5-amino-8-hydroxyquinoline dihydrochloride is estimated to be 33 μM, and from the results in FIG. 3, the IC ₅₀ value of juglone is estimated to be 4.6 μM.

For the purpose of examining the mechanism by which these inhibitors inhibit the enzymatic activity of HSV-1 protease, the concentration of the inhibitor was varied in various ways.
The dependence on the synthetic substrate concentration was determined. The enzyme concentration was the same as the measurement in Table 1 described above. As an example,
The dependence of 1,4-dihydroxynaphthalene on the addition concentration and the concentration of the synthetic substrate was described by Lineweaver-Bur
The result of k plot is shown in FIG. The same result, Dixon plot
The result is shown in FIG. From the plot of FIG.
Inhibition constants K _i value of dihydroxynaphthalene is estimated to be about 3 [mu] M.

The zinc ion, which is presumed to inhibit the reaction with the substrate by coordinating to the enzyme active site with respect to the HSV-1 protease, also shows the dependence on the concentration of the inhibitor and the concentration of the synthetic substrate. Was measured. In this case, zinc sulfate (ZnSO ₄ ), which is an inhibitor for a protease derived from Moller Accenfeld, was used as a zinc ion source. The result of Lineweaver-Burk plot is
FIG. 6 is a Dixon plot of FIG. Incidentally, from the results shown in FIG. 8, the IC ₅₀ value of zinc sulfate was 1
3.8 μM, from the results shown in FIG. 7, the inhibition constant of zinc sulfate
K _i values are estimated to be about 6μM.

When the result of 1,4-dihydroxynaphthalene shown above is compared with the result of this zinc ion (zinc sulfate), the results converge to one point on the vertical axis in the Lineweaver-Burk plot. It can be seen that dihydroxynaphthalene and the like are also mechanisms that inhibit the reaction with the substrate by coordinating the inhibitor with the enzyme active site similarly to the zinc ion.

In addition, 1,4-dihydroxynaphthalene having the highest inhibitory ability among the above compounds is HSV-
It was verified that the compound exhibited an excellent inhibitory activity against one protease, but did not have any inhibitory activity on other human-specific proteases. Specifically, the ability to inhibit the enzymatic activity against six types of trypsin, chymotrypsin, elastase, plasmin, thrombin, and factor Xa was measured. FIG. 9 shows the results obtained by adding 1,4-dihydroxynaphthalene to the enzyme reaction system of these proteases and measuring the enzyme activity. Note that the relative activity in FIG. 9 indicates% relative to the control (when the amount of addition was set to zero). This figure 9
As shown in Table 2, when the added concentration was 100 μM, the enzyme activity tended to be slightly suppressed, but it was within the range of measurement error, and it was judged that there was no inhibition. That is, as represented by the results of 1,4-dihydroxynaphthalene, HSV
-1 protease specifically inhibits the enzyme activity.

The above-described specificity can be explained as follows in association with the mechanism of its inhibition.
First, considering the enzyme active site of HSV-1 protease, it can be estimated that it is substantially the same as the enzyme active site of human-cytomegalovirus (HCMV) -derived protease which has been found to be an enzyme very similar to this. That is, both, Herpesviridae family (Herpesviridae family)
And the amino acid sequence homology is extremely high.
In particular, the partial amino acid sequence containing three amino acids essential for its activity, determined from the results of X-ray structural analysis of the HCMV protease and the like; Ser ¹²⁹ , His ⁶¹ , and His ¹⁴⁸ , has the HSV-1 Extremely high homology between protease and HCMV protease (Ping Chen et al.
al., Cell, 86 , p.835-843 (1996) and the like). In addition, two Arg ¹⁵⁶ and Arg ¹⁵⁷ characteristic of the active site are similarly retained.

Results of three-dimensional structural analysis of HCMV protease (Nature, 383 , p.272-275, p.275-279, p.279-
282 (1996), etc.) can be compared with each other.
Ser ¹³² , His ⁶³ , His ¹⁵⁷ corresponding to the amino acid at the position,
It is located at a position close to the hydrophobic region showing protease activity. Also in this HCMV protease, Zn ion inhibits the enzymatic activity, and the results of crystal structure analysis show that HCMV protease His ⁶³ and His ¹⁵⁷
Have been confirmed to exist as coordinated zinc (see Nature, 383 , p.272-275 (1996), etc.). The proton transfer between His ⁶³ and His ¹⁵⁷ is
Since it is indispensable for the enzymatic reaction of the protease, coordination with zinc makes the above-mentioned proton transfer impossible, resulting in loss of enzymatic activity. In addition, the presence of zinc in this region also has the effect of preventing the peptide chain of the substrate from reaching the active site, and it can be inferred that two mechanisms are responsible for the high inhibitory activity. In other serine proteases; trypsin, chymotrypsin, etc., three amino acids essential for enzyme activity are Ser, His, Asp
It is known that when His becomes Asp, inhibition of enzyme activity by zinc ions is no longer found.

As with this HCMV protease, HS
Ser ¹²⁹ , His ⁶¹ , His ¹⁴⁸ are other serine proteases; three amino acids constituting the active site of trypsin, chymotrypsin, etc .; Ser, His, Asp , But Asp has been changed to His ¹⁴⁸ . The specificity of the above-mentioned inhibitor is that the conventional serine proteases; trypsin, chymotrypsin, etc., were difficult to form a hydrogen bond between the inhibitor and the enzyme due to this conversion.
It is understood that the conversion to His reflects the possibility of hydrogen bond formation. Zn ions also coordinate to these two Hiss, and it is said that the substrate inhibits the process of approaching this active site, and the mechanism of enzyme inhibition in 1,4-dihydroxynaphthalene also has essential similarities. .

Compounds such as 1,4-dihydroxynaphthalene utilize the above two groups by utilizing a substituent on the aromatic ring.
Form a hydrogen bond with His, preventing proton transfer between His ⁶¹ and His ¹⁴⁸ . In addition, it can be inferred that the hydrophobic nature of the aromatic ring also has a stabilizing effect by forming a hydrophobic intermolecular bond in the enzyme active center. For this reason, it is considered that it becomes difficult to interact with the substrate as if it were a lid. By these two actions, the inhibitory activity becomes excellent.

The above-mentioned mechanism is effective when the phenolic hydroxyl group present in the compound such as 1,4-dihydroxynaphthalene does not dissociate with the acid. When the phenolic hydroxyl group is ionized by dissociation with the acid, the inhibitory ability is remarkably reduced. A decrease in inhibitory activity in the presence of DTT would reflect this effect. On the other hand, when the hydrophobicity associated with the aromatic ring is increased, hydrophobic intermolecular bonding with serum albumin or the like is likely to occur, so that the protein is captured by a protein other than the target HSV-1 protease. This makes it impossible to make full use of the high bonding strength. Considering the results in Table 2 in consideration of these points as well, it can be seen that 1,4-dihydroxynaphthalene, 5-amino-8-hydroxyquinoline, and juglone have both hydrophobic and hydrophilic properties appropriately. However, it is more suitable in an actual place.

[0066]

EFFECTS OF THE INVENTION The herpes simplex virus type 1 protease inhibitor of the present invention is suitable for a pharmaceutical composition because of its high inhibitory ability, high water solubility and relatively low molecular weight. . That is, having an aromatic ring showing hydrophobicity,
Moreover, since it is rich in water solubility, it has the advantage that the drug can be absorbed into the body even in various administration forms, for example, like various vitamins. In particular, since it has both hydrophobic and hydrophilic properties in addition to its low molecular weight, it can be easily transferred into a host cell infected with a virus, and can exert its inhibitory activity. Because of these advantages, the anti-herpes virus agent of the present invention can exhibit a high therapeutic effect regardless of the affected area.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing the concentration of 1,4-dihydroxynaphthalene added and the enzyme inhibitory ability against HSV-1 protease.

FIG. 2 is a graph showing the addition concentration of 5-amino-8-hydroxyquinoline dihydrochloride and the enzyme inhibitory ability against HSV-1 protease.

FIG. 3 is a graph showing the addition concentration of juglone and the enzyme inhibitory ability against HSV-1 protease.

FIG. 4. HSV-1 of 1,4-dihydroxynaphthalene
In the enzyme inhibitory ability to protease, the 1,4-
Lineweaver-Burk plot showing dependence on the concentration of dihydroxynaphthalene added and the concentration of substrate peptide.

FIG. 5: HSV-1 of 1,4-dihydroxynaphthalene
In the enzyme inhibitory ability to protease, the 1,4-
A Dixon plot showing the dependence on the concentration of dihydroxynaphthalene added and the concentration of the substrate peptide.

FIG. 6 is a Lineweaver-Burk plot showing the dependence of zinc sulfate on the enzyme inhibitory activity against HSV-1 protease on the concentration of zinc sulfate added and the concentration of substrate peptide.
FIG.

FIG. 7 is a Dixon plot showing the dependence of zinc sulfate on enzyme inhibition of HSV-1 protease with the concentration of zinc sulfate added and the concentration of substrate peptide.

FIG. 8 is a graph showing the addition concentration of zinc sulfate and the enzyme inhibitory ability against HSV-1 protease.

FIG. 9 shows whether 1,4-dihydroxynaphthalene has the ability to inhibit enzyme activity against various serine proteases.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI A61K 31/47 AED A61K 31/47 AED // A61K 35/78 35/78 C C07D 493/04 106 C07D 493/04 106A

Claims (3)

[Claims] (1) 1,4-dihydroxynaphthalene, 5-
A compound selected from the group consisting of amino-8-hydroxyquinoline, juglone, purpurin, 4-methylcatechol, cumestrol, phloroglucinol carbaldehyde, and 3- (2,4,5-trihydroxyphenyl) alanine, or a pharmaceutical thereof A herpes simplex virus type 1 protease inhibitor comprising a chemically acceptable salt as an active ingredient. (2) 1,4-dihydroxynaphthalene, 5-
2. A compound selected from the group consisting of amino-8-hydroxyquinoline and juglone or a pharmaceutically acceptable salt thereof as an active ingredient.
A herpes simplex virus type 1 protease inhibitor as described. 3. A compound selected from the group consisting of 1,4-dihydroxynaphthalene, 5-amino-8-hydroxyquinoline and juglone, or a pharmaceutically acceptable compound thereof, as an active ingredient exhibiting herpes simplex virus type 1 protease inhibitory activity. An anti-herpes virus agent comprising a salt to be used.