JP2011231053A - Apobec3 expression improver and anti-hiv agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an APOBEC3 expression improver containing as an effective component, a compound for improving expression of APOBEC3 which is a host factor having an anti-HIV activity, and to provide an anti-HIV agent containing the compound as an effective component.SOLUTION: The APOBEC3 expression improver and the anti-HIV agent contain as an effective component, the compound expressed by a general formula (I) [in the formula, Rshows a hydrogen atom, a lower alkoxy group or NRR(Rand Rshow respectively independently a lower alkyl group); n1, n2, m1 and m2 show respectively independently an integer of 1-5; Y1 and Y2 show respectively independently a hydrogen atom or a general formula (II) (in the formula, X shows a lower alkyl group, a halogen atom, a lower halogenoalkyl group, a nitro group, a cyano group or an aldehyde group; p shows an integer of 0-3)], or a physiologically allowable salt thereof.

Description

  The present invention relates to an APOBEC3 expression enhancer and an anti-HIV agent containing a disulfide compound as an active ingredient.

It has been reported that dithiol compounds such as disulfide compounds represented by the following structural formula and reduced forms thereof inhibit the DNA binding of transcription factor HIV-EP1 (Non-patent Document 1). HIV-EP1 is a C ₂ H ₂ type zinc finger protein that binds to a DNAκB site present in the long terminal repeat sequence of HIV provirus, and HIV-1 (human immunodeficiency virus Type-1) by its DNA binding Activates gene expression. Moreover, it has been reported that the said compound inhibits DNA binding of Sp1, which is one of transcription proteins related to HIV-1 gene expression (Non-patent Documents 2 and 3).

  It has also been reported that a compound in which the 4-position of the reduced pyridine ring is changed to an aryl group inhibits the enzyme activity of farnesyl-protein transferase and has anticancer activity (Non-patent Document 4 and Patents). Reference 1). Farnesyl protein transferase is a zinc enzyme involved in intracellular signal transduction, and transfers a farnesyl group to a site called CAAX box at the C-terminal of Ras protein, which is a product of oncogene ras.

  The inhibitory activity of the above compounds is due to the fact that the compounds act on the zinc binding site of the protein.

  On the other hand, APOBEC (apolipoprotein B mRNA editing enzyme catalytic polypeptide) family protein is known as a representative host factor having antiviral activity, and in particular, APOBEC3 protein is obtained by mutating HIV-1 viral cDNA. It is known to inhibit viral replication (Non-patent Document 5). APOBEC3G belonging to this family has been identified in the process of functional analysis of HIV-1 Vif (Virion infectivity factor) protein (Non-patent Document 6), but T cells and macrophages that are target cells for HIV-1 infection Is a host factor having an anti-HIV-1 action and a DNA converting enzyme having an amino acid sequence conserved in cytidine deaminase. APOBEC3G converts cytidine from single-stranded (−) DNA produced during reverse transcription of the HIV-1 genome into uracil (Non-patent Document 7). Due to this mutation, (1) when a double-stranded DNA is formed, a hypermutation from guanine to adenine occurs in the + strand DNA, and an amino acid mutation or a stop codon appears. (2) DNA repair mechanism Subsequent viral replication is inhibited by (UNG: uracil DNA glycosylase), a pathway that causes cleavage and degradation of viral DNA, and (3) a pathway in which U incorporation into single-stranded DNA inhibits the synthesis of + strand DNA itself. (Non-patent Document 8).

JP 2004-196732 A

Journal of Medicinal Chemistry 1996, 39, 503-507 Bioorganic & Medicinal Chemistry 1997, 5, 205-215 Journal of Synthetic Organic Chemistry 1997, 55, 41-48 Bioorganic & Medicinal Chemistry Letters 2003, 13, 1523-1526 Judd F Hultquist & Reuben S Harris, Future Virol. (2009) 4 (6), 605-619 A. M. Sheehy, N. C. Gaddis, J. D. Choi, M. H. Malim, Nature, 418 646 (2002). Q. Yu, R. Konig, S. Pillai, K. Chiles, M. Kearney, S. Palmer, D. Richman, J. M. Coffin, N. R. Landau, Nat. Struct. Mol. Biol., 11, 435 (2004). Takafumi Satoshi, Virus, 55, 267 (2005).

  An object of the present invention is to provide an APOBEC3 expression enhancer containing, as an active ingredient, a compound that improves the expression of APOBEC3, which is a host factor having an anti-HIV-1 action. Another object of the present invention is to provide an anti-HIV agent containing the above compound as an active ingredient.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the disulfide compound represented by the following general formula (I) has the activity of improving the expression of APOBEC3 and reduces the infectious titer of HIV-1 (That is, it has an anti-HIV-1 action). The present invention has been completed based on such findings.

〔式中、Ｒ¹は、水素原子、低級アルコキシ基又はＮＲ²Ｒ³（Ｒ²及びＲ³は、それぞれ独立して低級アルキル基を表す）を表し；ｎ１、ｎ２、ｍ１及びｍ２は、それぞれ独立して、１〜５の整数を表し；Ｙ₁及びＹ₂は、それぞれ独立して、水素原子又は一般式（ＩＩ）：

（式中、Ｘは、低級アルキル基、ハロゲン原子、ハロゲノ低級アルキル基、ニトロ基、シアノ基又はアルデヒド基を表し；ｐは、０〜３の整数を表す）を表す〕
で表される化合物又は生理学的に許容されるその塩を有効成分として含有する、ＡＰＯＢＥＣ３発現向上剤。
〔２〕 一般式（Ｉ）において、Ｒ¹がＮＲ²Ｒ³を表し、Ｒ²及びＲ³がＣ₁−Ｃ₄アルキル基である、〔１〕に記載のＡＰＯＢＥＣ３発現向上剤。
〔３〕 Ｒ²及びＲ³がメチル基である、〔２〕に記載のＡＰＯＢＥＣ３発現向上剤。
〔４〕 一般式（Ｉ）において、Ｙ₁及びＹ₂が一般式（ＩＩ）で表され、Ｘがニトロ基であり、かつｐが１〜２の整数である、〔１〕〜〔３〕のいずれかに記載のＡＰＯＢＥＣ３発現向上剤。
〔５〕 一般式（Ｉ）において、Ｙ₁及びＹ₂が水素原子である、〔１〕〜〔３〕のいずれかに記載のＡＰＯＢＥＣ３発現向上剤。
〔６〕 一般式（Ｉ）において、ｎ１及びｎ２が１であり、かつｍ１及びｍ２が２である、〔１〕〜〔５〕のいずれかに記載のＡＰＯＢＥＣ３発現向上剤。
〔７〕 一般式（Ｉ）で表される化合物が、下記構造式（ＩＩＩ）：

で表される化合物、又は下記構造式（ＩＶ）：

で表される化合物である、〔１〕に記載のＡＰＯＢＥＣ３発現向上剤。
〔８〕 ＡＰＯＢＥＣ３Ｇの発現の向上に用いるための〔１〕〜〔７〕のいずれかに記載のＡＰＯＢＥＣ３発現向上剤。 That is, the aspect of the present invention relates to the following.
[1] General formula (I):

[Wherein, R ¹ represents a hydrogen atom, a lower alkoxy group or NR ² R ³ (R ² and R ³ each independently represents a lower alkyl group); n1, n2, m1 and m2 each represent Each independently represents an integer of 1 to 5; Y ₁ and Y ₂ each independently represent a hydrogen atom or formula (II):

(Wherein X represents a lower alkyl group, a halogen atom, a halogeno lower alkyl group, a nitro group, a cyano group or an aldehyde group; p represents an integer of 0 to 3)]
Or a physiologically acceptable salt thereof as an active ingredient.
[2] The APOBEC3 expression enhancer according to [1], wherein in general formula (I), R ¹ represents NR ² R ³ , and R ² and R ³ are C ₁ -C ₄ alkyl groups.
[3] The APOBEC3 expression enhancer according to [2], wherein R ² and R ³ are methyl groups.
[4] In general formula (I), Y ₁ and Y ₂ are represented by general formula (II), X is a nitro group, and p is an integer of 1 to 2, [1] to [3] The APOBEC3 expression improving agent according to any one of the above.
[5] The APOBEC3 expression enhancer according to any one of [1] to [3], wherein Y ₁ and Y ₂ are hydrogen atoms in the general formula (I).
[6] The APOBEC3 expression enhancer according to any one of [1] to [5], wherein in general formula (I), n1 and n2 are 1, and m1 and m2 are 2.
[7] The compound represented by the general formula (I) is represented by the following structural formula (III):

Or a compound represented by the following structural formula (IV):

The APOBEC3 expression improver according to [1], which is a compound represented by:
[8] The APOBEC3 expression enhancer according to any one of [1] to [7] for use in improving the expression of APOBEC3G.

〔式中、Ｒ¹は、水素原子、低級アルコキシ基又はＮＲ²Ｒ³（Ｒ²及びＲ³は、それぞれ独立して低級アルキル基を表す）を表し；ｎ１、ｎ２、ｍ１及びｍ２は、それぞれ独立して、１〜５の整数を表し；Ｙ₁及びＹ₂は、それぞれ独立して、水素原子又は一般式（ＩＩ）：

（式中、Ｘは、低級アルキル基、ハロゲン原子、ハロゲノ低級アルキル基、ニトロ基、シアノ基又はアルデヒド基を表し；ｐは、０〜３の整数を表す）を表す〕
で表される化合物、その酸化により得られる２量体又は生理学的に許容されるそれらの塩を有効成分として含有する、抗ＨＩＶ剤。
〔１０〕 一般式（Ｉ）において、Ｒ¹がＮＲ²Ｒ³を表し、Ｒ²及びＲ³がＣ₁−Ｃ₄アルキル基である、〔９〕に記載の抗ＨＩＶ剤。
〔１１〕 Ｒ²及びＲ³がメチル基である、〔１０〕に記載の抗ＨＩＶ剤。
〔１２〕 一般式（Ｉ）において、Ｙ₁及びＹ₂が一般式（ＩＩ）で表され、Ｘがニトロ基であり、かつｐが１〜２の整数である、〔９〕〜〔１１〕のいずれかに記載の抗ＨＩＶ剤。
〔１３〕 一般式（Ｉ）において、Ｙ₁及びＹ₂が水素原子である、〔９〕〜〔１２〕のいずれかに記載の抗ＨＩＶ剤。
〔１４〕 一般式（Ｉ）において、ｎ１及びｎ２が１であり、かつｍ１及びｍ２が２である、〔９〕〜〔１３〕のいずれかに記載の抗ＨＩＶ剤。
〔１５〕 一般式（Ｉ）で表される化合物が、下記構造式（ＩＩＩ）：

で表される化合物、又は下記構造式（ＩＶ）：

で表される化合物である、〔９〕に記載の抗ＨＩＶ剤。 [9] General formula (I):

[Wherein, R ¹ represents a hydrogen atom, a lower alkoxy group or NR ² R ³ (R ² and R ³ each independently represents a lower alkyl group); n1, n2, m1 and m2 each represent Each independently represents an integer of 1 to 5; Y ₁ and Y ₂ each independently represent a hydrogen atom or formula (II):

(Wherein X represents a lower alkyl group, a halogen atom, a halogeno lower alkyl group, a nitro group, a cyano group or an aldehyde group; p represents an integer of 0 to 3)]
The anti-HIV agent which contains the compound represented by these, the dimer obtained by the oxidation, or those physiologically acceptable salts as an active ingredient.
[10] The anti-HIV agent according to [9], wherein in general formula (I), R ¹ represents NR ² R ³ , and R ² and R ³ are C ₁ -C ₄ alkyl groups.
[11] The anti-HIV agent according to [10], wherein R ² and R ³ are methyl groups.
[12] In general formula (I), Y ₁ and Y ₂ are represented by general formula (II), X is a nitro group, and p is an integer of 1 to 2, [9] to [11] The anti-HIV agent according to any one of the above.
[13] The anti-HIV agent according to any one of [9] to [12], wherein in formula (I), Y ₁ and Y ₂ are hydrogen atoms.
[14] The anti-HIV agent according to any one of [9] to [13], wherein in general formula (I), n1 and n2 are 1 and m1 and m2 are 2.
[15] The compound represented by the general formula (I) is represented by the following structural formula (III):

Or a compound represented by the following structural formula (IV):

[9] The anti-HIV agent according to [9].

  The APOBEC3 expression improving agent of the present invention has a remarkable APOBEC3 expression improving activity. The anti-HIV agent of the present invention comprises a disulfide compound having a remarkable APOBEC3 expression enhancing activity as an active ingredient, and has an excellent therapeutic effect against HIV infection and acquired immune deficiency syndrome (AIDS) caused thereby. Compared with conventional anti-HIV infection targeting viral factors, the anti-HIV agent of the present invention targets APOBEC3, which is a host factor, and therefore can cope with the problem of drug resistance due to viral gene mutations. .

FIG. 3 is a diagram showing the results of Western blotting in the APOBEC3G degradation inhibition experiment of Example 1. In FIG. 1, 15 represents compound 15 and 16 represents compound 16. In Example 1, it is a figure which shows the result of the western blotting in the APOBEC3G degradation inhibition experiment including various proteasome inhibitors. In FIG. 2, 15 represents compound 15 and 16 represents compound 16. FIG. 2 is a diagram for explaining an outline of an RT assay in Example 2. FIG. 2 is a diagram for explaining the outline of a MAGI assay in Example 2. In Example 2, it is a graph which shows the HIV-1 infection value about each compound. In FIG. 5, 15 represents compound 15 and 16 represents compound 16.

〔式中、Ｒ¹は、水素原子、低級アルコキシ基又はＮＲ²Ｒ³（Ｒ²及びＲ³は、それぞれ独立して低級アルキル基を表す）を表し；ｎ１、ｎ２、ｍ１及びｍ２は、それぞれ独立して、１〜５の整数を表し；Ｙ₁及びＹ₂は、それぞれ独立して、水素原子又は一般式（ＩＩ）：

（式中、Ｘは、低級アルキル基、ハロゲン原子、ハロゲノ低級アルキル基、ニトロ基、シアノ基又はアルデヒド基を表し；ｐは、０〜３の整数を表す）を表す〕
で表される化合物又は生理学的に許容されるその塩を有効成分として含有する。 The APOBEC3 expression improver of the present invention has the general formula (I):

[Wherein, R ¹ represents a hydrogen atom, a lower alkoxy group or NR ² R ³ (R ² and R ³ each independently represents a lower alkyl group); n1, n2, m1 and m2 each represent Each independently represents an integer of 1 to 5; Y ₁ and Y ₂ each independently represent a hydrogen atom or formula (II):

(Wherein X represents a lower alkyl group, a halogen atom, a halogeno lower alkyl group, a nitro group, a cyano group or an aldehyde group; p represents an integer of 0 to 3)]
Or a physiologically acceptable salt thereof as an active ingredient.

〔式中、Ｒ¹は、水素原子、低級アルコキシ基又はＮＲ²Ｒ³（Ｒ²及びＲ³は、それぞれ独立して低級アルキル基を表す）を表し；ｎ１、ｎ２、ｍ１及びｍ２は、それぞれ独立して、１〜５の整数を表し；Ｙ₁及びＹ₂は、それぞれ独立して、水素原子又は一般式（ＩＩ）：

（式中、Ｘは、低級アルキル基、ハロゲン原子、ハロゲノ低級アルキル基、ニトロ基、シアノ基又はアルデヒド基を表し；ｐは、０〜３の整数を表す）を表す〕
で表される化合物、その酸化により得られる２量体又は生理学的に許容されるそれらの塩を有効成分として含有する。 Further, the anti-HIV agent of the present invention has the general formula (I):

[Wherein, R ¹ represents a hydrogen atom, a lower alkoxy group or NR ² R ³ (R ² and R ³ each independently represents a lower alkyl group); n1, n2, m1 and m2 each represent Each independently represents an integer of 1 to 5; Y ₁ and Y ₂ each independently represent a hydrogen atom or formula (II):

(Wherein X represents a lower alkyl group, a halogen atom, a halogeno lower alkyl group, a nitro group, a cyano group or an aldehyde group; p represents an integer of 0 to 3)]
And a dimer obtained by oxidation thereof or a physiologically acceptable salt thereof as an active ingredient.

In the present specification, the “lower alkyl group” is, for example, an alkyl group composed of a linear, branched, cyclic, or combination thereof having 1 to 6 carbon atoms, and more specifically, Methyl group, ethyl group, propyl group, isopropyl group, cyclopropyl group, butyl group, isobutyl group, s-butyl group, t-butyl group, cyclobutyl group, cyclopropylmethyl group, pentyl group, isopentyl group, 2-methylbutyl group , Neopentyl group, 1-ethylpropyl group, hexyl group, isohexyl group, 4-methylpentyl group, 3-methylpentyl group, 2-methylpentyl group, 1-methylpentyl group, 3,3-dimethylbutyl group, 2, 2-dimethylbutyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2,3-dimethylbutyl group Group, 1-ethylbutyl group, and the like 2-ethylbutyl group. Preferably C ₁ -C ₄ alkyl group, more preferably a C ₁ -C ₂ alkyl group, most preferably a methyl group.

The “halogen atom” is a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, preferably a fluorine atom. The “halogeno lower alkyl group” means a group in which one or more halogen atoms are substituted on the “lower alkyl group”. The type, number, and substitution position of the halogen atoms to be substituted are not particularly limited, and when two or more halogen atoms are substituted, the types of halogen atoms may be the same or different. More specifically, as the “halogeno lower alkyl group”, a trifluoromethyl group, a trichloromethyl group, a difluoromethyl group, a dichloromethyl group, a dibromomethyl group, a fluoromethyl group, a 2,2,2-trifluoroethyl group 2,2,2-trichloroethyl group, 2-bromoethyl group, 2-chloroethyl group, 2-fluoroethyl group, 2-iodoethyl group, 3-chloropropyl group, 4-fluorobutyl group, 6-iodohexyl A halogeno C ₁ -C ₆ alkyl group such as a 2,2-dibromoethyl group, preferably a halogeno C ₁ -C ₂ alkyl group, preferably a fluoro C ₁ -C ₂ alkyl group And most preferably a trifluoromethyl group.

The “lower alkoxy group” refers to a group in which the “lower alkyl group” is bonded to an oxygen atom, and more specifically, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, an isobutoxy group, s -Butoxy group, t-butoxy group, pentoxy group, isopentoxy group, 2-methylbutoxy group, 1-ethylpropoxy group, 2-ethylpropoxy group, neopentoxy group, hexyloxy group, 4-methylpentoxy group, 3-methyl Pentoxy group, 2-methylpentoxy group, 3,3-dimethylbutoxy group, 2,2-dimethylbutoxy group, 1,1-dimethylbutoxy group, 1,2-dimethylbutoxy group, 1,3-dimethylbutoxy group , 2,3, etc. and dimethyl butoxy group, preferably a C ₁ -C ₄ alkoxy groups, more preferably An ₁ -C ₂ alkoxy group, most preferably a methoxy group.

Regarding the active ingredient of the APOBEC3 expression enhancer and anti-HIV agent of the present invention, as a preferred embodiment of the general formula (I), R ¹ is NR ² R ³ (R ² and R ³ are each independently C ₁ -C an _alkyl group, more preferably a C ₁ -C ₂ alkyl group, most preferably a methyl group), n1 and n2 are 1 to 3, more preferably 1 to 2, most preferably 1 There, m1 and m2 are 1 to 2, more preferably 2, Y ₁ and Y ₂ is represented by hydrogen atom or formula (II), in the general formula (II) X is a nitro group, p Is preferably 1.

  The most preferred compounds contained as active ingredients of the APOBEC3 expression enhancer and anti-HIV agent of the present invention include compound 15 and compound 1 represented by the following structural formula. Compound 1 can be obtained by reducing compound 15 by using, for example, dithiothreitol (DTT).

  The anti-HIV agent of the present invention may contain an oxidized compound of the compound represented by the general formula (I) or a physiologically acceptable salt thereof as an active ingredient. Examples of such an oxidized compound include compound 16 represented by the following structural formula obtained by oxidizing compound 1 described above.

  The compound of the present invention represented by the general formula (I) and its oxidized compound may exist in the form of a salt such as an acid addition salt or a base addition salt, but any salt is included in the scope of the present invention. Is done. Acid addition salts include mineral acid salts such as hydrochloride or hydrobromide, or organic acid salts such as p-toluenesulfonate, methanesulfonate, oxalate, or tartrate. . As the base addition salt, for example, a metal salt such as sodium salt, potassium salt, magnesium salt, or calcium salt, an ammonium salt, or an organic amine salt such as triethylamine salt or ethanolamine salt can be used. It can also exist as an amino acid salt such as a glycine salt. Furthermore, the compound of the present invention or a salt thereof may exist as a hydrate or a solvate, and these substances are also included in the scope of the present invention. In addition, the compound of the present invention may have one or two or more asymmetric carbons depending on the type of substituent, and may have a stereoisomer such as an optical isomer or a diastereoisomer. Pure forms of stereoisomers, any mixture of stereoisomers, racemates, and the like are all within the scope of the present invention.

  The compounds included in the above general formula (I) can be synthesized by employing the methods disclosed in Non-Patent Documents 1 to 4 and Patent Document 1 described above, or by combining these methods with known conventional techniques. .

  The APOBEC3 expression enhancer of the present invention can be used for improving the expression of a gene (protein) belonging to APOBEC3. Here, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3DE, APOBEC3F, APOBEC3G, and APOBEC3H are known as belonging to APOBEC3. Of these, the APOBEC3 expression enhancer of the present invention is preferably used for improving the expression of APOBEC3G.

  The APOBEC3 expression enhancer and anti-HIV agent of the present invention are one of substances selected from the group consisting of the compounds represented by the above general formula (I) and salts thereof, and hydrates and solvates thereof. Contains seeds or two or more active ingredients. The anti-HIV agent of the present invention is selected from the group consisting of a compound obtained by oxidizing the compound represented by the general formula (I) and a salt thereof, and a hydrate and a solvate thereof. One type or two or more types of substances may be included as active ingredients. As the APOBEC3 expression enhancer and anti-HIV agent of the present invention, the above substances themselves may be administered, but preferably administered as an oral or parenteral pharmaceutical composition that can be produced by methods well known to those skilled in the art. can do. Examples of the pharmaceutical composition suitable for oral administration include tablets, capsules, powders, fine granules, granules, liquids, and syrups. The pharmaceutical composition suitable for parenteral administration includes Examples include injections, instillations, suppositories, inhalants, eye drops, nasal drops, ointments, creams, patches, transdermal absorbents, transmucosal absorbents, and the like.

  Examples of the additive for preparation used in the production of the above pharmaceutical composition include excipients, disintegrants or disintegration aids, binders, lubricants, coating agents, dyes, diluents, bases, and solubilizers. Or a solubilizing agent, an isotonic agent, a pH adjuster, a stabilizer, a propellant, an adhesive, and the like, which can be appropriately selected by those skilled in the art depending on the form of the pharmaceutical composition. It may be used in combination of two or more. The above-mentioned pharmaceutical composition can be used as a pharmaceutical composition in the form of a so-called mixture by further blending one or more active ingredients of other pharmaceuticals. The pharmaceutical composition can be prepared in any form for oral administration or parenteral administration.

  The administration route of the APOBEC3 expression enhancer and anti-HIV agent of the present invention is not particularly limited, and may be oral administration or parenteral administration (for example, rectal administration, subcutaneous administration, intramuscular administration, nasal administration, intravenous administration, etc.). Oral administration is preferred.

  The doses of the APOBEC3 expression improver and anti-HIV agent of the present invention can be appropriately set according to the age, weight, symptoms, etc. of the administration subject or patient. For example, the amount of the active ingredient compound is 0.1 μg. ˜100 mg / kg / day, preferably 1 μg to 10 mg / kg / day.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not particularly limited by the following examples.

[Example 1]
<APOBEC3G degradation inhibition experiment>
It was investigated whether the compound of the present invention inhibits the degradation of APOBEC3G in cells. Briefly, an expression vector of APOBEC3G (APOBEC3G-myc) to which 293T cells have been added and VIF (Vif-FLAG) to which a FLAG tag has been added, and Vif-deficient pNL- of HIV-1 infectious clone pNL4-3 Nd was introduced. Six hours after the start of introduction, compound 15 (4- (Dimethylamino) -2,6-bis [[N- [2-[(2-nitrophenyl) dithio] ethyl] amino] methyl] pyridine), compound 16 (4- ( Dimethylamino) -2,6-bis [oxidized form of [[(2-mercaptoethyl) amino] methyl] pyridine) and / or DTT was added, and the mixture was further allowed to stand for 36 hours to collect virus solution and cells. The collected cells were lysed and Western blotting was performed to measure the expression levels of APOBEC3G and Vif. The synthesis method of compound 16 and the procedure of APOBEC3G degradation inhibition experiment are described below.

(Synthesis of Compound 16)
Compound 15 (109.0 mg, 0.181 mmol) was dissolved in 5.8 ml of CHCl ₃ , dithiothreitol (83.6 mg, 0.542 mmol) was added, and the mixture was stirred at room temperature for 3 hr. Purified water was added, the pH was adjusted to 1 with 1M aqueous HCl, and the mixture was washed with CH ₂ Cl ₂ (30 ml × 5 times). The aqueous layer was neutralized with sat. NaHCO ₃ aqueous solution and extracted with CH ₂ Cl ₂ (30 ml × 4 times). MgSO ₄ was added, dried and filtered. The mixture was stirred at room temperature for 12 hours under an O ₂ atmosphere. MgSO ₄ was added, dried and filtered. Concentration under reduced pressure gave Compound 16 as amorphous.
Yield 35.0 mg (0.117 mmol)
Yield 65%
¹ H NMR (C DCl ₃ ) δ: 2.59 (4H, t, J = 6.9Hz, S-CH ₂ CH ₂ -N × 2), 2.82 (4H, t, J = 6.9Hz, S-CH ₂ CH ₂ -N × 2), 2.95 (6H, s, N (CH ₃ ) ₂ ), 3.76 (4H, s, -CH ₂ -N- × 2), 6.27 (2H, s, pyridine)
¹³ C NMR (CDCl ₃ ) δ: 39.2 (N (CH ₃ ) ₂ ), 41.5 (S-CH ₂ CH ₂ -N), 47.7 (S-CH ₂ CH ₂ -N), 54.9 (-CH ₂ -N -), 104.5 (pyridine), 155.6 (pyridine), 159.5 (pyridine)
FAB-MS (m / z): 299, 597 (M + H) ⁺

(APOBEC3G degradation inhibition experiment)
293T cells (JS Lebkowski, S. Clancy, MP Calos, Nature, 317, 169 (1985)), an epithelial cell derived from human fetal kidney, are suspended in a D-MEM medium containing 10% FCS and seeded in a petri dish for cell culture. Then, static culture was performed in a CO ₂ incubator at 37 ° C. and 5% CO ₂ . Cells that reached confluence were detached from the dish by trypsin-EDTA method, and D-MEM containing 10% FCS was added and pipetted. The cell suspension was seeded in a petri dish for cell culture to which 10% FCS-containing D-MEM medium had been added in advance, and the cells that had been allowed to stand for 48 hours were used for transfection experiments.

  HIV-1 infectious clone pNL4-3 [A. Adachi, HE Gendelman, S. Koenig, T. Folks, R. Willey, A. Rabson, MA Martin, J. Virol., 59, 284 (1986)] Vif deficient pNL-Nd [A. Adachi, N. Ono, H. Sakai, K. Ogawa, R. Shibata, T. Kiyomatsu, H. Masuike, S. Ueda, Arch. Virol., 117, 45 (1991) And pNL-ASCF-fWT [B. Khamsri, M. Fujita, K. Kamada, A. Piroozmand, T. Yamashita, T. Uchiyama, A. Adachi, Int. J. Mol as a vector for expressing Vif-FLAG. Med., 18, 679, (2006)]. As these mock, pUC or pNL-A1 Vif (−) (provided by Dr. Strebel Klaus, NIH, USA) was used. PcDNA-APO3G (provided by Dr. Strebel Klaus, NIH, USA) was used as a vector for expressing APOBEC3G-myc, and pcDNA 3.1 (-) was used as the mock thereof.

Gene transfer was carried out by the calcium phosphate method, and was followed in a standard manner. First, a total of 220 μL of gene expression plasmid was used with ultrapure water. To this mixed solution, 25 μL of an aqueous CaCl ₂ solution (2.5 M) was added and added dropwise to a mixed solution of 250 μL of 2 × Hebs (42 mM HEPES, 290 mM NaCl, pH 7.1) and 5 μL of Na ₂ HPO ₄ (70 mM). After incubating at room temperature for 30 minutes, it was added to 293T cells in a subconfluent state using a cell culture petri dish (6.0 cm). After culturing at 37 ° C. for 6 hours, it was replaced with D-MEM. Cells were collected 48 hours after the start of gene transfer. Table 1 shows the compositions of the gene expression plasmids.

  As described above, 293T cells were transfected with various plasmid vectors, and when the supernatant was replaced with D-MEM 6 hours after gene introduction, DTT (15 μM), compound 15 (final concentration was 5 μM), Compound 16 (final concentration 5 μM), compound 15 (final concentration 5 μM) and a mixture of DTT (15 μM), compound 16 (final concentration 5 μM) and DTT (15 μM) were added, respectively. 48 hours after gene transfer, the cells were detached with a scraper, transferred to a 15 mL tube, and washed with PBS (−). PBS (-) 150 μL and 2 × sampling buffer 150 μL are added to the cells, heated with a heat block (100 ° C., 1 hour), centrifuged (14,000 rpm, 1 minute, rt), and the supernatant sample for Western blotting It was.

  Next, the collected cells were analyzed by a usual western blotting method. The sample for Western blotting was subjected to SDS-PAGE and then transferred to a PVDF membrane (100 V, 4 ° C., 3 hours). The transferred PVDF membrane was masked by shaking for 1 hour in PBST containing 5% skim milk (PBS (-) containing 0.1% Tween-20). Antibody reaction was performed for 1 hour using a primary antibody (anti-myc or anti-FLAG) on the PVDF membrane. After washing with PBST, an antibody reaction was carried out for 1 hour using a secondary antibody (anti-mouse IgG). After completion of the reaction, the PVDF membrane was washed with PBST, reacted with the substrate, and detected using FUJIFILM LAS-3000 (Fuji Film). The result is shown in FIG.

  As a result, expression of APOBEC3G was observed in the absence of Vif as expected, but the expression level decreased in the presence of Vif. Surprisingly, in the presence of Compound 15 and Compound 15 + DTT added, the expression level of APOBEC3G was significantly increased. Since its expression level is higher than in the absence of Vif, it is suggested that APOBEC3G is degraded by factors other than Vif in this experimental system, and that it is inhibited by the addition of Compound 15 and Compound 15 + DTT. It was done.

  Next, in order to examine whether this degradation is proteasome degradation, the activity was compared with MG132 and Lactacystin, which are proteasome inhibitors, in the same manner as in the experiment described above. At the same time, the same experiment was conducted for Bis (2-nitrophenyl) disulfide. The result of the Western blotting is shown in FIG.

  As a result, the expression level of APOBEC3G was also increased in MG132 and Lactacystin as in Compound 15, indicating that this degradation was proteasome degradation and that proteasome degradation was inhibited by Compound 15, MG132, or Lactacystin. Bis (2-nitrophenyl) disulfide did not increase the expression level of APOBEC3G.

  In addition, the in vitro proteasome inhibitory activity of the compounds was determined using A. Asai, T. Tsujita, SV Sharma, Y. Yamashita, S. Akinaga, M. Funakoshi, H. Kobayashi, T. Mizukami, Biochem. Pharmacol., 67, 227 (2004). The results are shown in Table 2. As a result, it was found that compounds 15, 16, 16 + DTT showed proteasome degradation inhibitory activity, and among them, compound 15 had the highest proteasome degradation inhibitory activity.

  Further, when the expression level of Vif was examined, no increase in the expression was observed in the presence of Compound 15, but the expression was strongly increased in the presence of MG132. It has already been reported that Vif undergoes proteasome degradation and is inhibited by MG132 (M. Fujita, H. Akari, A. Sakurai, A. Yoshida, T. Chiba, K. Tanaka, K. Strebel , A. Adachi, Microbes and Infection, 6, 791 (2004)). However, it was suggested here that compound 15 does not inhibit proteasomal degradation of Vif but selectively inhibits proteasomal degradation of APOBEC3G.

(Example 2)
<HIV-1 infection inhibition experiment>
Next, it was examined whether compounds 15 and 16 actually suppressed HIV-1 infection in cells expressing APOBEC3G. The infectivity titer corrected by the amount of HIV-1 released (result of RT assay described below) was used as the index.

(1) RT assay (measurement of HIV-1 release)
This RT assay is based on the method described in RL Willey, PH Smith, LA Lasky, TS Thedore, PL Earl, B. Moss, DJ Capon, MA Mavtia, J. Virol., 62, 139 (1988). .

The outline of the RT assay procedure is as follows: APOBEC3G expression vector and HIV-1 infectious clone pNL4-3 are introduced into 293T cells, and after 15 hours from the start of introduction, compounds 15, 16 and / or DTT and TPEN are added for an additional 42 hours. After stationary culture, the virus solution was recovered. The virus solution was dissolved to remove the reverse transcriptase, and PolyA, Oligo (dT) _12-18 primer and [α ³² P] -TTP were added. The amount of reverse transcriptase (RT) was calculated by isolating oligonucleotides and measuring β-rays, and was used as the amount of HIV-1 released. An outline of the RT assay method is shown in FIG. 3, and the procedure will be described in detail below.

  The plasmids used for gene transfer below are the above-described HIV-1 infectious clone pNL4-3 and its Vif-deficient pNL-Nd. PUC was used as these mock. PcDNA-APO3G (provided by Dr. Strebel Klaus, NIH, USA) was used as an expression vector for APOBEC3G.

Gene transfer was carried out by the calcium phosphate method, and was followed in a standard manner. First, a total of 3.3 μg of gene expression plasmid was used, and the total volume was adjusted to 73.3 μL with purified water. To this mixed solution was added 8.3 μL of an aqueous CaCl ₂ solution (2.5 M), and mixed with 83.3 μL of 2 × Hebs (42 mM HEPES, 290 mM NaCl, pH 7.1) and 1.7 μL of Na ₂ HPO ₄ (70 mM). It was dripped. After incubating at room temperature for 30 minutes, it was added to 293T cells in a subconfluent state using a cell culture petri dish (3.5 cm). After culturing at 37 ° C. for 6 hours, it was replaced with D-MEM. Cells were collected 48 hours after the start of gene transfer. Table 3 shows the composition of the plasmid for gene expression.

6 hours after gene transfer into 293T cells using various plasmid vectors, when the supernatant was replaced with D-MEM, DTT (15 μM), compound 15 (final concentration 5 μM), compound 16 (final concentration 5 μM), A mixture of Compound 15 (final concentration 5 μM) and DTT (15 μM), Compound 16 (final concentration 5 μM) and DTT (15 μM), and TPEN (final concentration 5 μM) were added, respectively. After 48 hours from gene transfer, the supernatant was collected and centrifuged (3,000 rpm, 10 minutes, rt), and the supernatant was obtained as a virus solution. RT cocktail (50 mM Tris-HCl, pH 7.8, 75 mM KCl, 2.0 mM DTT, 5.0 mM MgCl ₂ , 0.5 μg / mL Poly A, 6.3 μg / ml Oligo d (T) _12-18 , 0.05% NP40 25 μL, [α ³² P] -TTP (appropriate amount (Day 1: 0.025 μL)) was added, and the mixture was incubated at 37 ° C. for 3 hours. This was soaked in 10 μL of chromatography paper, washed with 2 × SSC (300 mM NaCl, 30.0 mM Sodium citrate), dried, and β-rays were detected with a scintillation counter. The obtained value was defined as the amount of HIV-1 released.

(2) MAGI assay (measurement of HIV-1 infectivity titer)
This MAGI assay is based on the method described in J. Kimpton, M. Emerman, J. Virol., 66, 2232 (1992).

  The outline of the MAGI assay procedure was as follows. The virus solution used in the RT assay was added to MAGI-CXCR4 that had been cultured in advance for infection. In MAGI-CXCR4, CD4, CXCR4, and LTR-β-gal are introduced. When HIV-1 is infected, the transcriptional activation protein Tat of HIV-1 is expressed, which binds to TAR in the LTR region and promotes transcription of the β-gal gene downstream thereof. The resulting β-galactosidase was added with the substrate X-Gal, and the blue-stained cells were counted to determine the HIV-1 infectivity. An outline of the MAGI assay is shown in FIG. The procedure will be described in detail below.

MAGI-CXCR4 cells in which CD4, CXCR4, and LTR-β-gal genes were introduced into HeLa cells, which are human cervical cancer-derived cells, were treated with 10% FCS, 0.2 mg / mL G418, 0.1 mg / mL Hydromycin B, 1 μg. The suspension was suspended in / ml Puromycin-containing D-MEM (hereinafter referred to as D-MEM for MAGI-CXCR4), seeded in a cell culture dish, and statically cultured in a CO ₂ incubator at 37 ° C. and 5% CO ₂ . Cells that reached confluence were detached from the dish by trypsin-EDTA method, and D-MEM for MAGI-CXCR4 was added and pipetted. Count the number of cells with a calculation plate, transfer only the required amount to a 15 mL tube, and after centrifugation, add the D-MEM containing 10% FCS and pipet so that the number of cells is 2 × 10 ⁴ cells / 300 μL. . A cell suspension was seeded in a 96-well plate in an amount of 300 μL, and the cells that were allowed to stand for 48 hours were used for the MAGI assay.

The virus solution used in the RT assay was diluted with D-MEM to 1/10, 1/100, or 1/1000, and DEAE-dextran was added to a final concentration of 20 μg / mL. The supernatant of MAGI cells cultured in a 96-well plate was removed, and 100 μL of the prepared virus solution was added. After incubation at 37 ° C. for 12 hours, 100 μL of D-MEM was added. After further static culture for 36 hours, the supernatant was removed, the fixative was added, incubated for 5 minutes, washed with PBS (−), and then stained (4.0 mM K ₃ [Fe (CN) ₆ ], 4.0 mM). K ₄ [Fe (CN) ₆ ], 2.0 mM MgCl ₂ , 0.4 mg / mL X-Gal) was added and incubated at 37 ° C. for 50 minutes. After washing with PBS (−), the number of cells stained blue was counted, and the obtained value was used as the infectious titer of HIV-1.

  Representative results (corrected by RT activity) are shown in FIG. From the results obtained, it was found that Compound 15, Compound 16, and the reduced products thereof by DTT have the effect of reducing the infectious titer of HIV-1.

  The APOBEC3 expression enhancer and anti-HIV agent of the present invention have high industrial applicability in the fields of medicine, medicine and the like.

Claims (15)

Formula (I):

[Wherein, R ¹ represents a hydrogen atom, a lower alkoxy group or NR ² R ³ (R ² and R ³ each independently represents a lower alkyl group); n1, n2, m1 and m2 each represent Each independently represents an integer of 1 to 5; Y ₁ and Y ₂ each independently represent a hydrogen atom or formula (II):

(Wherein X represents a lower alkyl group, a halogen atom, a halogeno lower alkyl group, a nitro group, a cyano group or an aldehyde group; p represents an integer of 0 to 3)]
Or a physiologically acceptable salt thereof as an active ingredient. The APOBEC3 expression improving agent according to claim 1, wherein in general formula (I), R ¹ represents NR ² R ³ , and R ² and R ³ are C ₁ -C ₄ alkyl groups. The APOBEC3 expression enhancer according to claim ^2, wherein R ² and R ³ are methyl groups. The general formula (I), wherein Y ₁ and Y ₂ are represented by the general formula (II), X is a nitro group, and p is an integer of 1 to 2. APOBEC3 expression improving agent as described in 2. above. The APOBEC3 expression improving agent according to any one of claims 1 to 3, wherein in formula (I), Y ₁ and Y ₂ are hydrogen atoms. The APOBEC3 expression enhancer according to any one of claims 1 to 5, wherein, in the general formula (I), n1 and n2 are 1, and m1 and m2 are 2. The compound represented by the general formula (I) is represented by the following structural formula (III):

Or a compound represented by the following structural formula (IV):

The APOBEC3 expression improving agent of Claim 1 which is a compound represented by these. The APOBEC3 expression improving agent according to any one of claims 1 to 7, which is used for improving the expression of APOBEC3G. Formula (I):

[Wherein, R ¹ represents a hydrogen atom, a lower alkoxy group or NR ² R ³ (R ² and R ³ each independently represents a lower alkyl group); n1, n2, m1 and m2 each represent Each independently represents an integer of 1 to 5; Y ₁ and Y ₂ each independently represent a hydrogen atom or formula (II):

(Wherein X represents a lower alkyl group, a halogen atom, a halogeno lower alkyl group, a nitro group, a cyano group or an aldehyde group; p represents an integer of 0 to 3)]
The anti-HIV agent which contains the compound represented by these, the dimer obtained by the oxidation, or those physiologically acceptable salts as an active ingredient. The anti-HIV agent according to claim 9, wherein, in the general formula (I), R ¹ represents NR ² R ³ , and R ² and R ³ are C ₁ -C ₄ alkyl groups. The anti-HIV agent according to claim 10, wherein R ² and R ³ are methyl groups. 12. The general formula (I), wherein Y ₁ and Y ₂ are represented by the general formula (II), X is a nitro group, and p is an integer of 1 to 12. The anti-HIV agent described in 1. The anti-HIV agent according to any one of claims 9 to 11, wherein in formula (I), Y ₁ and Y ₂ are hydrogen atoms. The anti-HIV agent according to any one of claims 9 to 13, wherein, in the general formula (I), n1 and n2 are 1, and m1 and m2 are 2. The compound represented by the general formula (I) is represented by the following structural formula (III):

Or a compound represented by the following structural formula (IV):

The anti-HIV agent of Claim 9 which is a compound represented by these.

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