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    • 专利摘要:
    The present inevntion relates to an excellent Vpr functionregulating agent, comprising an aminostilbazole derivativerepresented by the following formula [1] or a salt thereof asan active ingredient: A-B-D-E wherein A represents heteroaryl or an oxide thereof, B representsethenylene, D represents phenylene, and E represents the formula:-N(R)-SO2-G [wherein G represents phenyl, R represents hydrogen,alkyl, or -COR0 (R0 represents alkyl, alkoxy, aryloxy, 5- to6-membered heteroaryl, heteroarylmethyl, aminoalkylene, orcyclic amino alkylene)or the like].
    公开号:EP1382335A1
    申请号:EP02720594
    申请日:2002-04-25
    公开日:2004-01-21
    发明作者:Hideki Tanaka
    申请人:Nippon Shinyaku Co Ltd；
    IPC主号:C07D213-00
    专利说明:
    [0001] The present invention relates to a Vpr function regulatingagent and an anti-HIV agent. ACKGROUND ART
    [0002] In recent years, the prevalence of acquired immunodeficiencysyndrome (AIDS) has been rapidly increased, and there is worldwideneed for establishing methods for its prophylaxis and treatment.AIDS is caused by human immunodeficiency virus (HIV) , a Lentivirusin the family Retroviridae. For HIV, at present, there are knowntwo types of viruses, HIV-1 and HIV-2. HIV mainly infects CD4 (+)T-lymphocytes in humans, anthropoid apes (chimpanzees), and others,and destroys them and causes AIDS.
    [0003] As agents against AIDS are currently used inhibitors ofreverse transcriptase characteristic of HIV [for example,zidovudine (AZT, ZDV), didanosine (ddI), zalcitabine (ddC)] andof viral proteolytic enzymes (for example, saquinavir). Actually,two types of agents having different mechanisms of action arecombined. Although these are intended to kill HIV-1, they displayside effects and may lead to the generation of viruses that areresistant to them by taking them over a long period of time, forexample, and thus it cannot always be said that these agents aresatisfactory ones.
    [0004] The genome of a retrovirus has a major characteristic in thatthe genome is integrated into a chromosome of an infected cellthrough the phenomenon of reverse transcription. Accordingly,it is impossible for agents and others to eradicate the virusthroughout the body, and thus infected individuals necessitateco-existence with the virus in their life.
    [0005] Another characteristic of retroviruses is that they possessgenes encoding for a group of proteins referred to as accessoryproteins (for example, Nef, Vif, Vpr, Tat, Rev), in addition togenes for proteins constituting a virus (for example, Gag, Pol,Env). Among them, Vpr is a small protein consisting of 96 aminoacids and has a variety of functions of, for example, transportingto the nucleus a viral genome-protein complex, designated aspre-integration complex (immature virus); arresting the cell cycleof infected cells at the G2/M period, thereby enhancing theefficiency of transcription of the virus genome within nondividingcells such as macrophages; destroying surrounding cells (bystandercells) in lymph nodes via cell-membrane impairing activities. Infact, it is known that HIV-1 genome is actively replicated whenan infected cell is at the G2/M period, and that an HIV-1 mutantdeficient in Vpr results in extremely reduced production of matureviruses in macrophages.
    [0006] It is also reported that Vpr suppresses the proliferationof infected cells, thereby promoting the destruction of infectedT-cells, as well as inhibiting the establishment of HIV-1 persistent infection (Rogel ME et al., J. Vilol., 69: 882-888,1995). This activity of Vpr suggests a possibility that Vpr isinvolved during the transition of the silent period to AIDS.
    [0007] Vpr has homology in structure as well as in function homologousbetween simian immunodeficiency virus (SIV) and HIV-1 and HIV-2.Actually, it is shown that in monkeys, Vpr is essential for theonset of AIDS causedby SIV (Hoch J. et al., J. Virol., 69: 4807-4813,1995).
    [0008] As described above, Vpr is believed to be involved in theprogression of each pathological stage by exerting its variousfunctions at a particular period in the pathological stages fromthe initial infection to the onset of AIDS. Therefore, compoundsthat regulate Vpr functions display activities of, for example,1) inhibiting HIV-1 infection of macrophages and others, whichwill become reservoirs of HIV in lymphoid tissues such as thymus,and production of mature viruses; 2) inhibiting apoptosis ofHIV-infected CD4 (+) T-cells, thereby causing the host immune systemto induce cytotoxic lymphocytes (CTLs) against infected cells;3) releasing virus producing cells from their arresting at theG2/M period and reducing the ability of producing viruses; 4)interfering with the destruction of bystander cells (for example,dendritic cells, CD8(+) T-cells, NK (natural killer) cells) onthe basis of cell-membrane impairing activities of Vpr, and asa result, such compounds are likely to prevent the reduction ofperipheral un-infected CD4(+) T-cells and to suppress the onset of AIDS and the progression of the AIDS state.
    [0009] In addition, Vpr arresting of the cell cycle is alsoreproducible by extracellular adding of recombinant Vpr, and inthis case, it is reported that addition of the protein at highconcentrations induces cell death (apoptosis) in various cells(Finkel, T.H. et. al., Nature Med., 1: 129-134, 1995). It is saidthat there is on the order of 2-3 nM Vpr in peripheral blood ofactual patients with AIDS, and the effect of this secreted Vpris likely to cause the destruction of bystander cells as describedabove in 4). Furthermore, it would be likely that the de-regulationof the cell cycle by Vpr promotes canceration of normal cells,and in combination with reduced immune states, leads to thegeneration of such AIDS-related tumors as Kaposi's sarcoma.
    [0010] Recently, attention is being focused on accessory proteinsas new targets for creating drugs, with accumulating informationon physiological functions of accessory proteins. At present,however, there is no medicament targeting an accessory proteinsuch as Vpr.
    [0011] With respect to Vpr, the situation has been in confusion:it is already known that when Vpr is overexpressed in various cells,the cell cycle is arrested at the G2/M period, whereas there havebeen reported contradictory results that when weakly expressed,to the contrary, arresting at the G2/M period is not caused, andalso apoptosis does not take place with ease. Recently, however,a cell line that resolves such problems has been established, and screening using these cells has demonstrated that a certainflavonoid (quercetin) inhibits Vpr functions (Shimura M. et. al.,Biochem. Biophys. Res. Commun. 261: 308-316, 1999).
    [0012] Kohyo (National Publication of Translated Version) No. Hei9-511395 (1997) discloses a biologically active peptide fragmentof the Vpr protein of HIV, a pharmaceutical composition comprisingsuch a peptide or its biologically active analogue, an antagonistof the peptide, a pharmaceutical composition comprising theantagonist, and a method for treatment and screening using sucha compound and a compositions.
    [0013] The compounds of the formula (1) according to the presentinvention are known to have an anti-cancer effect and to be usefulas a remedy for malignant tumors (International PublicationWO95/27699). DISCLOSURE OF THE INVENTION
    [0014] An object of the present invention is to provide an excellentanti-HIV agent having a new mechanism of action, that is, to providean anti-HIV agent that regulates Vpr function to exert an anti-HIVeffect.
    [0015] From the above-described point of view, the present inventorshave carried out searching for new anti-HIV agents that targetVpr. More specifically, since cytobiological changes(abnormalities of cell division machinery) observed whenaminostilbazole derivatives represented by the following general formula (1) (hereinafter sometimes referred to as the compoudsof the present invention) are subjected to action on differentcancer cells are closely similar to cell changes observed in cellsexpressing Vpr, the present inventors have searched about a Vprfunction regulating effect of the compound of the present invention.As a result, we have found that the compounds of the present inventionhave a Vpr function regulating effect, and thus the presentinvention has been completed.
    [0016] The present invention relates to a Vpr function regulatingagent and an anti-HIV agent, comprising a compound representedby the following general formula [1] or a salt thereof as an activeingredient: A-B-D-E [wherein A represents optionally substituted heteroaryl or an oxidethereof, B represents optionally substituted ethenylene, Drepresents optionally substituted phenylene, E represents thefollowing formula:
    [0017] Preferably, the present invention relates to a Vpr functionregulating agent and an anti-HIV agent, comprising the compoundsof present invention or the salt thereof as an active ingredient,wherein A is optionally substituted pyridyl or 1-oxide pyridyl,B is non-substituted ethenylene, D is non-substituted oraminoalkoxy-substituted 1, 2-phenylene, R of -N(R)-SO2-G as E isalkyl which may be substituted with a substituent selected fromthe group consisting of halogen, amino, monoalkylamino,dialkylamino, morpholino, alkoxy, hydroxy, cyano, the formula:-CONR1R2 (wherein R1 and R2 are the same or different and eachrepresents hydrogen, hydroxy, alkyl, alkoxy, cycloalkyl,cycloalkyloxy, aryl, aryloxy, aralkyl, aralkyloxy,cycloalkylalkyl, cycloalkylalkyloxy, or tetrahydrofuranyloxy),and the formula : -SO2NR3R4 (wherein R3 and R4 are the same or differentand each represents hydrogen or alkyl), alkenyl which may be substituted with halogen, or -COR0, R0 is alkyl, cyclic amino alkyl,or dialkylaminoalkyl, and G is 4-alkoxyphenyl.
    [0018] More preferably, the present invention relates to a Vprfunction regulating agent and an anti-HIV agent, comprising thecompounds of present invention or the salt thereof as an activeingredient, wherein A is non-substituted 4-pyridyl or1-oxide-4-pyridyl, B is ethenylene of a non-substitutedtrans form,D is non-substituted or aminoalkoxy-substituted 1,2-phenylene,R of -N(R)-SO2-G as E is hydrogen, hydroxyalkyl, or -COR0, R0 isalkyl or morpholinoalkyl or dialkylaminoalkyl, and G is4-methoxyphenyl.
    [0019] A feature of the present invention resides in the fact thata Vpr function regulating effect and an anti-HIV effect are foundin the compound of the formula [1]. It has never been describedin the document and also has never been known that the above compoundhas these effects.
    [0020] The present invention will be described in detail below.
    [0021] Terms used in the present specification and definitions ofthe respective substituents are as follows.
    [0022] The term "Vpr function regulating agent" as used herein refersto a drug which enhances or relieves a biological phenomenon causedby Vpr itself, or caused by Vpr as a result of binding with moleculesderived from host cells or virus.
    [0023] The term "anti-HIV agent" as used herein refers to a drugwhich reduces the viral load by suppressing the growth of HIV or reducing its pathogenicity. Particularly, it refers to a drugwhich suppresses the destruction of CD4(+) T-cells, otherimmunocompetent cells in the lymphoid tissue and nerve cells causedby a biological activity of HIV, thereby to suppress the onsetof AIDS-related diseases and to relieve the AIDS state.
    [0024] Examples of "heteroaryl" may include 5- to 6-memberedheteroaryl having 1 to 2 nitrogen atoms as an annular atom. Sucha heteroaryl may have 1 to 2 substituents at any positions andexamples of the substituent may include halogen, alkyl, alkoxy,hydroxy, aminoalkyl and the like. Examples of "heteroaryl" asA may include 6-membered heteroaryl, for example, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl,5-pyrimidinyl, 3-pyridazinyl, 4-pyridazinyl and pyrazinyl.Among them, non-substituted 4-pyridyl is preferable. Examplesof the heteroaryl moiety of "heteroaryl" and "heteroarylmethyl"as R0 may include 5- to 6-membered heteroaryl, for example,2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl,5-pyrimidinyl, 3-pyridazinyl, 4-pyridazinyl, 1-imidazolyl,2-imidazolyl and 4-imidazoly. Among them, pyridyl is preferable.
    [0025] "Ethenylene" may have a substituent on the respective atomsand examples of the substituent may include cyano, bromo alkyland the like. Among them, non-substituted ethenylene ispreferable.
    [0026] Examples of "phenylene" may include 1,2-phenylene,1, 3-phenylene and 1,4-phenylene. Such a phenylene group may have 1 to 2 substituents at any positions and examples of the substituentmay include hydroxy, halogen, amino, alkyl, alkoxy, aminoalkyloxyand the like. Non-substituted or aminoalkoxy-substituted1,2-phenylene is preferable.
    [0027] "Phenyl" may have 1 to 2 substituents and examples of thesubstituent may include hydroxy, alkoxy and the like. Among them,alkoxy-substituted phenyl is preferable and 4-methoxyphenyl isparticularly preferable.
    [0028] Examples of "halogen" mayinclude fluorine, chlorine, bromine,iodine and the like.
    [0029] Examples of "alkyl" may include straight-chain orbranched-chain alkyl having 1 to 6 carbon atoms, for example, methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, isopentyl, n-hexyl and isohexyl. Amongthem, alkyl having 1 to 4 carbon atoms is preferable and methylis particularly preferable. Alkyl as R may be substituted witha substituent selected from the group consisting of halogen, amino,monoalkylamino, dialkylamino,morpholino, alkoxy, hydroxy, cyano,the formula: -CONR1R2 (wherein R1 and R2 are the same or differentand each represents hydrogen, hydroxy, alkyl, alkoxy, cycloalkyl,cycloalkyloxy, aryl, aryloxy, aralkyl, aralkyloxy,cycloalkylalkyl, cycloalkylalkyloxy, or tetrahydrofuranyloxy)and the formula: -SO2NR3R4 (wherein R3 and R4 are the same or differentand each represents hydrogen or alkyl).
    [0030] Examples of the alkyl moiety of "hydroxyalkyl", "monoalkylamino", "dialkylamino", "aminoalkyl","cycloalkylalkyl" and "cycloalkylalkyloxy" may include "alkyl"described above.
    [0031] Examples of "alkoxy" may include straight-chain orbranched-chain alkoxy having 1 to 6 carbon atoms, for example,methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy,sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, n-hexyloxyand isohexyloxy. Among them, alkoxy having 1 to 4 carbon atomsis preferable and methoxy is particularly preferable.
    [0032] Examples of the alkoxy moiety of "aminoalkoxy" may include"alkoxy" described above.
    [0033] Examples of "aryloxy" may include optionally substitutedaryloxy having 6 to 10 carbon atoms, for example, phenoxy,1-naphthyloxy and 2-naphthyloxy. Among them, phenoxy ispreferable. Examples of the substituent may include alkyl,halogen, hydroxy, alkoxy and the like.
    [0034] Examples of "alkenyl" may include straight-chain orbranched-chain alkenyl having 2 to 6 carbon atoms, for example,vinyl, 1-propenyl, 2-propenyl, isopropenyl, 2-butenyl, 3-butenyl,isobutenyl, metallyl, prenyl, isoprenyl and 1,1-dimethylallyl.Among them, alkenyl having 2 to 4 carbon atoms is preferable."Alkenyl" as R may be substituted with halogen.
    [0035] Examples of "alkynyl" may include straight-chain orbranched-chain alkynyl having 2 to 6 carbon atoms, for example,ethynyl, 1-propynyl, 2-propynyl, 2-butynyl, 3-butynyl and 3-methyl-2-butynyl. Particularly, alkynyl having 2 to 4 carbonatoms is preferable.
    [0036] In akylene represented by "-(CH2)n-", hydrogen atom may besubstituted with one amino or alkyl at any positions.
    [0037] Examples of "cyclic amino" may include 5- to 8-membered cyclicamino, for example, pyrrolidin-1-yl, piperidino,hexamethyleneimino, tetrahydropyridin-1-yl,octahydroazocin-1-yl, piperazin-1-yl, homopiperazin-1-yl,morpholino and thiomorpholino. Such a cyclic amino may have 1to 2 substituents selected from the group consisting of alkyl,alkenyl, alkynyl, aryl, aralkyl and heterocycle group having anitrogen atomat any positions. Among them, 5- to 6-membered cyclicamino is preferable, and piperazin-1-yl substituted with pyridyl,non-substituted pyrrolidin-1-yl, piperidino or morpholino isparticularly preferable.
    [0038] Examples of "aryl" may include aryl having 6 to 10 carbonatoms, for example, phenyl, 1-naphthyl and 2-naphthyl.
    [0039] Examples of "aralkyl" may include aralkyl having 7 to 8 carbonatoms, for example, benzyl and phenethyl.
    [0040] Exampels of the aralkyl moiety of "aralkyloxy" may includethose described above.
    [0041] Examples of "heterocycle having a nitrogen atom" may includecyclic amino and heteroaryl described above. Such a heterocyclemay have 1 to 2 substituents selected from the group consistingof alkyl, amino, hydroxy and oxo.
    [0042] Examples of "cycloalkyl" may include cycloalkyl having 3 to8 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl and cyclooctyl.
    [0043] Examples of the cycloalkyl moiety of "cycloalkyloxy","cycloalkylalkyl" and "cycloalkylalkyloxy" may include thosedescribed above.
    [0044] Examples of "salt" of the compounds of the present inventionmay include pharmacologically acceptable salts, for example, saltswith inorganic acids such as hydrochloric acid, sulfuric acid,nitric acid, phosphoric acid, hydrofluoric acid and hydrobromicacid, and salts with organic acids such as acetic acid, tartaricacid, lactic acid, citric acid, fumaric acid, maleic acid, succinicacid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonicacid, toluenesulfonic acid, naphthalenesulfonic acid andcamphorsulfonic acid.
    [0045] The compounds of the present invention may exist in cis (Zform) and trans (E form) isomers, and each isomer and a mixturethereof are also included in the present invention. Particularly,the E form is preferable.
    [0046] Examples of the compounds of the present invention may includecompounds of the formula [1]. Particularly,(E)-4-[2-{2-[N-acetyl-N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine 1-oxide,(E)-4-[2-(2-[N-acetyl-N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine, (E)-4-[2-{2-[N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine 1-oxide,(E)-4-[2-{2-[N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine,(E)-4-[2-{2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine 1-oxide,(E)-4-[2-{2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine,(E)-4-[2-{3-(2-aminoethyloxy)-2-[N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine, and(E)-4-[2-{3-(2-aminoethyloxy)-2-[N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine 1-oxide are preferable.
    [0047] The compounds of the present invention can be produced bythe method described in International Publication WO95/27699,International Publication WO01/44195 or Japanese UnexaminedPatent Publication No. 2001-261649, or a version thereof.
    [0048] When the compounds of the present invention are administeredas a medicament, they can be administered to a mammal includinghuman as they are or in a mixture with a pharmaceutically acceptablenon-toxic inert carrier, for example, as a pharmaceuticalcomposition containing the compound at a level of 0. 1% to 99.5%,preferably 0.5% to 90%.
    [0049] The carrier that can be used includes solid, semi-solid andliquid diluent, filler and other formulation auxiliaries and atleast one of them is selectivity employed. The pharmaceutical composition is preferably administered in a unit dosage form. Thepharmaceutical composition can be administered into tissue, ororally, intravenously, topically (percutaneously) or rectally.Of course, a dosage form suited for each route of the administrationshould be used. For example, oral or intravenous administrationdue to ambulatory is preferable to prevent the progression of thestage of disease in patients in the primary infection stage andthe asymptomatic stage, while intravenous administration ispreferable for patients in the AIDS state.
    [0050] The dosage of the compound as a Vpr function regulating agentand an anti-HIV agent may preferably be adjusted in considerationof the patient's factors such as age, body weight, etc., natureand severity of disease, etc., as well as the route of administration,a daily dose as an active ingredient in an adult is usually 0.1mg to 100 mg per adult, preferably 0.1 mg to 50 mg per adult whengiven orally, and usually 0.1 mg to 100 mg per adult, preferably0.1 mg to 50 mg per adult when given intravenously. In some cases,a lower dose may be sufficient or a higher dose may be required.Usually, the dose is given once or several times as being dividedinto portions, or given intravenously and continuously over aperiod of 1 to 24 hours a day. When given continuously for a longperiod (administration in the primary infection stage and theasymptomatic stage), the dose and interval are appropriatelycontrolled after periodically examining the number of peripherallymphocytes, the amount of virus and the advent of side effects of patients.
    [0051] Oral administration can be carried out using a solid or liquidunit dosage form, such as a particle, powder, tablet, sugar-coatedtablet, capsule, granule, suspension, liquid, syrup, drop,sublingual tablet or other dosage forms.
    [0052] A particle is produced by pulverizing the compound of thepresent invention into a suitable particle size. A powder isproduced by pulverizing the compound of the present invention intoa suitable particle size followed by mixing with a pharmaceuticalcarrier, such as an edible carbohydrate including starches ormannitol, which has also been pulverized into a suitable particlesize. Those that may be added if necessary are flavors,preservatives, dispersing agents, colorants, fragrances and thelike.
    [0053] A capsule is produced by filling a particle or powder whichhas been pulverized as described above or a granule obtained asdescribed in the section of a tablet for example in a capsule suchas a gelatin capsule. It is also possible that an additive suchas a lubricant, fluidizing agent, such as colloidal silica, talc,magnesium stearate, calcium stearate or solid polyethylene glycolis mixed with the pulverizedmaterial prior to the filling procedure.The efficacy of the drug after ingestion of capsule may be improvedby adding a disintegrator or solubilizer, such as carboxymethylcellulose, calcium carboxymethyl cellulose, low substitutedhydroxypropyl cellulose, sodium croscarmellose, sodium carboxymethyl starch, calcium carbonate or sodium carbonate, maybe added.
    [0054] The finely pulverized powder may be suspended and dispersedin a vegetable oil, polyethylene glycol, glycerin and surfactant,and then encapsulated in a gelatin sheet, thereby obtaining a softcapsule.
    [0055] A tablet can be produced by preparing a powdery compositionby adding an excipient, granulating or slugging it, adding adisintegrator or lubricant thereto, and then compact into a tablet.
    [0056] The powdery composition can be produced by mixing anappropriately pulverized material with a diluent or base describedabove if necessary together with a binder (for example, sodiumcarboxymethyl cellulose, methyl cellulose, hydroxypropylmethylcellulose, gelatin,polyvinyl pyrrolidone and polyvinylalcohol),a dissolution retardant (for example, paraffin), a resorptionpromoter (for example, quaternary salt), or an adsorbent (forexample, bentonite, kaolin and calcium diphosphate). The powderycomposition can be granulated by wetting with a binder such asa syrup, starch glue, gum arabic, cellulose solution or polymersolution, followed by mixing with stirring, drying and grinding.Instead of the procedure for granulating a powder as describedabove, another procedure may be employed in which a mix is subj ectedfirst to a tablet compacting machine to form a morphologicallyincomplete slug which is then ground. A granule thus obtainedmay contain, as a lubricant, stearic acid, stearates, talc, mineral oil and the like, for the purpose of preventing any adhesion witheach other. The mixture thus lubricated is then compacted intotablets. A plane tablet thus obtained may be film-coated orsugar-coated.
    [0057] The compounds of the present invention may be mixed with afluidized inert carrier and then compacted directly into tabletswithout being subjected to the granulating or slugging processas described above. A transparent or semi-transparent protectivefilm in the form of a shellac sealing film, a film of a sugar orpolymeric material and a glossy film of a wax may also be employed.Other oral dosage forms, such as a solution, syrup and elixir canbe formulated as a unit dosage form whose certain amount containsa certain amount of a medicament. A syrup is produced by dissolvingthe compound of the present invention in an appropriate flavoredaqueous solution, while an elixir is produced by using a non-toxicalcoholic carrier. A suspension is formulated by dispersing thecompound of the present invention in a non-toxic carrier.Additives such as a solubilizing agent, an emulsifier (for exampleethoxylated isostearyl alcohols, polyoxyethylene sorbitolesters), a preservative, a flavor (for example, peppermint oiland saccharin) and the like may be added, if necessary.
    [0058] An oral unit dosage formulation may also be a microcapsuleif desired. Such a formulation may also be coated or embeddedin a polymer, wax or the like to obtain a prolonged activity orsustained release of the active ingredient.
    [0059] In parenteral administration, an injection, a suppositoryand the like can be used. Parenteral administration can beaccomplished by using a liquid unit dosage form, such as a solutionor suspension, for subcutaneous, intramuscular or intravenousadministration. Such a unit dosage form can be produced bysuspending or dissolving predetermined amount of the compound ofthe present invention in a non-toxic liquid carrier such as anaqueous or oily medium compatible with the purpose of the inj ectionfollowed by sterilizing the suspension or solution. It is alsopossible to add a non-toxic salt or salt solution for the purposeof making an injection solution isotonic. It is also possibleto use a stabilizer, preservative, emulsifier and the like.
    [0060] A rectal administration can be accomplished by using asuppository produced by dissolving or suspending the compoundsof the present invention in a water-soluble or water-insolublesolid having a low melting point such as a polyethylene glycol,cocoa butter, semi-synthesized fats and fatty oils (for example,Witepsol (R)), higher esters (for example, myristyl palmitate)as well as a mixture thereof.
    [0061] In the medicament of the present invention, it is possibleto use in combination with other ingredients, for example, reversetranscriptase inhibitors and virus protease inhibitors inherentto HIV. BRIEF DESRIPTION OF THE DRAWINGS
    [0062] Fig. 1 shows in vitro functional antagonism between aflavonoid (quercetin) and the compound B. A is a copy of aphotograph showing the results when microscopic observations weremade of control (untreated) cells; B, cells treated for 72 hourswith the compound B (600 nM) described below; C, cells pretreatedwith 1.0 µM quercetin for 48 hours, followed by treatment withthe compound B (600 nM) for 72 hours. X4 and X20 represent amagnification of 4X and 20X, respectively.
    [0063] Fig. 2 (1) shows electrophoresis views of purified FLAG-fusedVpr protein: (a) a picture of the gel stained with Coomassie Blue,(b) Western blot with an anti-FLAG antibody. Fig. 2 (2) showsdirect binding of the compound and Vpr: lane 1,radioisotope-labeled FLAG-fused Vpr; lane 2, precipitation ofradioisotope-labeled FLAG-fused Vpr with beads not having thecompound immobilized thereto (control); lane 3, precipitation ofradioisotope-labeled FLAG-fused Vpr with beads having the compoundimmobilized thereto; lane 4, radioisotope-labeled FLAG-fused VprΔC; lane 5, precipitation of radioisotope-labeled FLAG-fused VprΔC with beads having the compound immobilized thereto.
    [0064] Fig. 3 (1) shows the inhibition of cytotoxic activity of Vprby the compound, which is indicated by detecting (PI staining)dead cells on a flow cytometer. The compound concentration is0 nM in (a) , 0.1 nM in (b) , and 1. 0 nM in (c). The ordinate axisrepresents the number of counts and the abscissa axis representsthe intensity of fluorescence. Fig. 3 (2) shows copies of photographs showing the results when microscopic observations weremade of the state in which the compound removes the inhibitionof cell proliferation induced by purified FLAG-fused Vpr. Thecompound concentration is 0.1 nM in (a), 1.0 nM in (b) , and 10. 0nM in (c). BEST MODE FOR CARRYING OUT THE INVENTION
    [0065] The present invention will now be described in more detailwith reference to Production Examplesof typicalstarting materials(Reference Examples) , Production Examples of the compounds of thepresent invention (Examples), Pharmaceutical PreparationExamples and Test Examples, but the present invention is not limitedthereto. The specific rotation was measured at 20°C. In the TestExamples,(E)-4-[2-{2-[N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine 1-oxide (hereinafter referred to as a compound A) and(E)-4-[2-{3-(2-aminoethyloxy)-2-[N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine (hereinafter referred to as acompound B) were used. In the Formulation Examples, the compoundA and(E)-4-{2-[2-{N-(4-methoxybenzenesulfonyl)-N-[4-(2-pyridyl)piperadino]acetylamino}phenyl]ethenyl}pyridine 1-oxidedihydrochloride (hereinafter referred to as a compound 2) wereused. Reference Example 1Production of(E)-2-(2-tert-butoxycarbonylaminoethoxy)-6-[2-(4-pyridyl)ethenyl]aniline(Step 1) Syntehsis of 3-hydroxy-2-nitrobenzaldehyde
    [0066] 3.62 g of 3-methoxy-2-nitrobenzaldehyde was dissolved in 80ml of methylene chloride and a boron tribromide-methylene chloridesolution (15. 03 g of boron tribromide, 40 ml of methylene chloride)was added dropwise under ice cooling, followed by stirring at 0°Cfor one hour. The reaction solution was poured into ice, extractedwith chloroform, dried and then concentrated to obtain 3.32 g ofthe desired compound. (Step 2) Production of(E)-2-acetoxy-6-[2-(4-pyridyl)ethenyl]nitrobenzene
    [0067] To 3.17 g of the compound obtained in the step 1, 1.94 g of4-picoline and 4.79 g of acetic anhydride were added, and thenthe mixture was refluxed with stirring for 12 hours. The reactionsolutionwaspouredintoice, neutralizedwithpotassiumcarbonate,extracted with chloroform and then dried over magnesium sulfate.After the solvent was distilled off under reduced pressure, theresidue was subjected to silica gel column chromatography (CHCl3)to obtain 4.78 g of the desired compound. (Step 3) Production of (E)-2-hydroxy-6-[2-(4-pyridyl)ethenyl]nitrobenzene
    [0068] 4.54 g of the compound obtained in the step 2 was dissolvedin 160 ml of methanol and 4.42 of potassium carbonate was added, followed by stirring at room temperature for 2 hours. After thereaction solution was concentrated, the residue was combined withiced water and neutralized with 2 N-hydrochloric acid and thenthe deposited crystal was collected by filtration to obtain 3.47g of the desired compound. The resulting compound was used asa material without purification. (Step 4) Production of(E)-2-(2-tert-butoxycarbonylaminoethoxy)-6-[2-(4-pyridyl]ethenyl] nitrobenzene
    [0069] 2.18 g of the compound obtained in the step 3 was dissolvedin 9 ml of DMSO and 0. 54 g of 60% sodium hydride was added underan argon gas flow, followed by stirring at room temperature forone hour. 4.63 g of 2-bromoethyl-tert-butoxycarbonylamine wasadded, followed by stirring with heating at 120°C for 3 hours.The reaction solution was poured into iced water, combined withethyl acetate, washed with water (3 times) , washed with saturatedbrine and then dried over magnesium sulfate. After the solventwas distilled off under reduced pressure, the residue was subjectedto silica gel column chromatography (CHCl3:MeOH = 50:1) to obtain1.90 g of the desired compound. (Step 5) Production of(E)-2-(2-tert-butoxycarbonylaminoethoxy)-6-[2-(4-pyridyl)ethenyl]aniline
    [0070] 1.90 g of the compound obtained in the step 4 was dissolvedin 50 ml of 70% hydrous methanol and 4.90 g of reduced iron and 0.30 g of calcium chloride were added, and then the mixture wasrefluxed with stirring for 4 hours. After the reaction solutionwas filtered through celite, the filtrate was concentrated andthe residue was subjected to silica gel column chromatography(CHCl3:MeOH = 50:1) to obtain 1.45 g of the desired compound. Example 1(E)-4-[2-{2-[N-phenoxycarbonyl-N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine
    [0071] 1.00 g of(E)-4-[2-{2-[N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine was suspended in 40 ml of chloroform and, after adding1.82 g of phenyl chlorocarbonate, 1.20 g of triethylamine was slowlyadded under ice cooling. Then, the mixture was stirred at roomtemperature for 5 minutes. The solvent was distilled off underreduced pressure and the desired product was purified by silicagel column (carrier:Wako Gel C200, developing solvent chloroform)to obtain the desired compound. The desired compound was treatedwith ethanol to obtain 0.71 g of a white granular crystal. Elemental analysis (for C27H22N2O5S) Calcd. (%) H, 4.55; C, 66.65; N, 5.75 Found (%) H, 4.51; C, 66.44; N, 5.67
    [0072] In the same manner, the following desired compounds weresynthesized. Example 2(E)-4-[2-{2-[N-methoxycarbonyl-N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine
    [0073] Elemental analysis (for C22H20N2O5S)Calcd. (%)H,4.75;C, 62.25;N, 6.60Found (%)H, 4.83;C, 61.97;N, 6.51 Example 3(E)-4-[2-{2-[N-ethoxycarbonyl-N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine
    [0074] Elemental analysis (for C23H22N2O5S·1/5H2O)Calcd. (%)H, 5.11;C, 62.49;N, 6.34Found (%)H, 5.09;C, 62.39;N, 6.34 Example 4(E)-4-[2-{2-[N-n-propoxycarbonyl-N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine hydrochloride
    [0075] Elemental analysis (for C24H24N2O5S·HCl·1/2H2O)Calcd. (%)H, 5.26;C, 57.88;N, 5.63Found (%)H, 5.23;C, 58.12;N, 5.72 Example 5(E)-4-[2-{2-[N-n-butoxycarbonyl-N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine hydrochloride
    [0076] Elemental analysis (for C25H26N2O5S·HCl·1/2H2O)Calcd. (%)H, 5.51;C, 58.64;N, 5.47Found (%)H, 5.47;C, 58.44;N, 5.49 Example 6(E)-4-[2-{3-(2-aminoethoxy)-2-[N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine dihydrochloride(Step 1) Production of (E)-4-[2-{3-(2-tert-butoxycarbonylaminoethoxy)-2-[N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine
    [0077] 1.42 g of(E)-2-(2-tert-butoxycarbonylaminoethoxy)-6-[2-(4-pyridyl)ethenyl]aniline obtained in the step5 of Reference Example 3 wasdissolved in 14 ml of pyridine and 0.99 g of4-methoxybenzenesulfonyl chloride was added, followed by stirringat room temperature overnight. The reaction solution wasconcentrated and the residue was combined with iced water,extracted with chloroform and then dried over magnesium sulfate.After the solvent was distilled off under reduced pressure, theresidue was subjected to silica gel column chromatography (CHCl3:MeOH = 30:1) to obtain 2.19 g of the desired compound. (Step 2) Production of (E)-4-[2-{3-(2-tert-butoxycarbonylaminoethoxy)-2-[N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine 1-oxide
    [0078] 0.56 g of the compound obtained in the step 1 was dissolvedin 5 ml of chloroform and m-chloroperbenzoic acid was added,followed by stirring at room temperature for one hour. The reactionsolution was washed with water three times and then dried overmagnesium sulfate. After the solvent was distilled off underreduced pressure, the residue was subjected to silica gel columnchromatography (CHCl3:MeOH = 20:1) to obtain 0.46 g of the desiredcompound. (Step 3) 2.10 g of the compound obtained in the step 2 was dissolvedin 10 ml of methylene chloride and 10. 5 ml of trifluoroacetic acidwas added dropwise under ice cooling, followed by stirring at roomtemperature for 2 hours. The reaction solution was combined withiced water, made weak basic by the addition of potassium carbonate,extracted with chloroform and then dried over magnesium sulfate.After the solvent was distilled off under reduced pressure, theresidue was subjected to silica gel column chromatography(CHCl3:MeOH= 20:1) to obtain 1.33 g of a free form.
    [0079] The free form was dissolved in 20 ml of methanol and an excessamount of a 20% hydrochloric acid-ether solution was added underice cooling, followed by stirring for one hour. The reactionsolution was concentrated and treated with ethanol to obtain 1. 30g of the desired compound (pale yellow crystal). Elemental analysis (for C22H23N3O4S·2HCl ·H2O) Calcd. (%) C, 51.17; H, 5.27; N, 8.14 Found (%) C, 51.04; H, 4.99; N, 8.02 Example 7(E)-4-[2-{3-(2-aminoethoxy)-2-[N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine 1-oxide dihydrochloride
    [0080] 2.38 g of the compound obtained in the step 2 of Example 6was dissolved in 23 ml of methylene chloride and 23 ml oftrifluoroacetic acid was added dropwise underice cooling, followedby stirring at room temperature for 2 hours. The reaction solutionwas combined with iced water, made weak basic by the addition of potassium carbonate, extracted with chloroform and then dried overmagnesium sulfate. After the solvent was distilled off underreduced pressure, the residue was subjected to silica gel columnchromatography (CHCl3: MeOH: 28%NH3 (aq.) = 90:10:1) to obtain 1.48g of a free form.
    [0081] The free form was dissolved in 50 ml of methanol and an excessamount of a 20% hydrochloric acid -ether solution was added underice cooling, followed by stirring for one hour. The reactionsolution was concentrated and treated with ethanol to obtain 1. 50g of the desired compound (pale yellow crystal). Elemental analysis (for C22H23N3O5S·2HCl ·0.3H2O) Calcd. (%) C, 50.83; H, 4.96; N, 8.08 Found (%) C, 50.86; H, 4.88; N, 7.99 Formulation Example 1 (180 mg per tablet for internal use) Compound A 10 mg Lactose 100 mg Corn starch 55 mg Low substituted hydroxypropyl cellulose 9 mg Polyvinyl alcohol (partially saponified product) 5 mg Magnesium stearate 1 mg
    [0082] The above ingredients are weighed in accordance with theformulation. The ingredients, excluding polyvinyl alcohol andmagnesium stearate, are uniformly mixed and tabletting granulesare produced by a wet granulation method using an aqueous polyvinyl alcohol solution as a binder. Aftermixing the tabletting granuleswith magnesium stearate, the mixture is subjected to a tabletcompacting machine to mold into tablets each 8 mm in diameterweighing 180 mg per tablet, thereby obtaining a tablet for internaluse. Formulation Example 2 (220 mg per hard capsule) Compound A 10 mg Lactose 187 mg Microcrystalline cellulose 20 mg Magnesium stearate 3 mg
    [0083] The above ingredients are weighed in accordance with theformulation and, after mixing uniformly, 220 mg of the mixtureis filled into a #2 capsule using a capsule filling machine, therebyobtaining a hard capsule. Formulation Example 3 (1 g per granule) Compound A 10 mg Lactose 880 mg Low substituted hydroxypropyl cellulose 70 mg Hydroxypropyl cellulose 40 mg
    [0084] The above ingredients are weighed in accordance with theformulation and, after mixing and kneading uniformly, the kneadedmixture is subj ected to a granulating machine to mold into granuleseach 0.7 mm in diameter, thereby obtaining a granule. Formulation Example 4 (1 ml per injection) Compound 2 10 mg Mannite 50 mg Water for injection q.s. (Compound 2 is (E)-4-{2-[2-{N-(4-methoxybenzenesulfonyl)-N-[4-(2-pyridyl)piperadino]acetylamino}phenyl]ethenyl}pyridinel-oxidedihydrochloride)Preparation Method
    [0085] After the compounds of the present invention and mannite aredissolved in water for injection, the resulting solution isaseptically filtered through a membrane filter (pore size: 0.22µm). After filling into a vial, the vial is subjected tofreeze-drying to obtain an injection to be dissolved before using. Formulation Example 5 (1 ml per injection) Compound 2 10 mg Maltose 100 mg Water for injection q.s. Preparation Method
    [0086] After the compounds of the present invention and maltose aredissolved in water for injection, the resulting solution isaseptically filtered through a membrane filter (pore size: 0.22µm). After filling into a vial, the vial is subjected tofreeze-drying to obtain an injection to be dissolved before using. Test Example 1In vitro functional antagonism between flavonoid (quercetin) andcompound B
    [0087] Three types of cells were employed (HeLa, ECV304, and HBL-100,purchased from ATCC) , and for each type, 1 x 105 cells were seededin a 10 cm dish and cultured in D-MEM/F-12 medium (Gibco BRL) for18-20 hours. On the next day, the medium was changed to the samemedium containing 1.0 µM quercetin (Sigma; hereinafterabbreviated to QCT), followed by culturing for an additional 2days. The compound A was then added at a concentration of 600nM, and 72 hours later observations under a microscope were madeof cell morphologies. As a representative example, Fig. 1 showsthe results obtained when HeLa cells were employed. Similarresults were obtained when the other types of cells were employed.
    [0088] As is obvious from Fig. 1, in the cell group receiving onlythe treatment with the present compound (Fig. 1, B) , the cell cycleis arrested at the G2/M period, many cells which became globularare visible, and the number of multinucleate cells having pluralnuclei within single cells are apparently increased, also for cellsadhered onto culturing dishes, compared with the control group(Fig. 1, A). On the other hand, in the cell group subjected tothe pre-treatment with quercetin, a Vpr inhibitor, effects of thecompound as described above disappear almost completely (Fig. 1,C). These results reveal that the present compound results infunctional antagonism with quercetin. Test Example 2In vitro direct binding of Vpr and compound
    [0089] 1) Preparation of a Vpr gene fused to N-terminal FLAG (SEQ ID NO:1)A Vpr gene fused to N-terminal FLAG (SEQ ID NO:1) was obtainedby PCR (polymerase chain reaction) using as a template pBT-1(purchased from ATCC) into which the HIV genome without env hadbeen incorporated. Primers utilized were synthetic DNAs havingthe following sequences (SEQ ID NOs:1 and 2).N-terminal
    [0090] The DNA fragment of interest (300 bp) was recovered from PCRreaction products by agarose gel electrophoresis, and then clonedinto the pGEM-T vector (Promega). This fragment was base analyzedon both plus and minus strands and was verified to correspond tothe intended fused Vpr. The vector into which the Vpr gene fusedto FLAG has been incorporated is hereinafter referred to aspGEM-T/Vpr-wt. Furthermore, using this pGEM-T/Vpr-wt as atemplate, a second Vpr gene fused to FLAG in which a C-terminalregion of Vpr (a. a. 73-96) was deleted was obtained by PCR utilizingprimers shown below (SEQ ID Nos:4 and 5).
    [0091] This fused gene was cloned into the pGEM-T vector as describedabove, and the resulting vector is hereinafter referred to aspGEM-T/Vpr ΔC. 2) Preparation of agarose beads having compound immobilized thereto
    [0092] One milligram of the compound B was dissolved in 1 ml of 50%DMSO (dimethyl sulfoxide), and to this NHS(N-hydroxysuccimide)-activated Sepharose 4 FF(Amersham-Pharmacia Biotech) of a bed volume of 2 ml was addedand subjected to reaction at room temperature for 2 hours withshaking. After the reaction, a 2M Tris solution (pH 8. 0) was addedand allowed to stand at 4°C overnight, followed by washing, anda 50% suspension in PBS was prepared for experiments. 3) Preparation of FLAG-fused Vpr
    [0093] Preparation of FLAG-fused Vpr was performed by transcriptionand translation from the T7 promoter employing the TNT-coupledwheat germ extract in vitro transcription/translation system(wheat germ extract transcription/translation system) (Promega).Specifically, 1.0 µg of pGEM-T/Vpr-wt (or pGEM-T/Vpr ΔC) digestedwith a restriction enzyme PstI was transcribed and translated withT7 RNA polymerase in 25 µl of the wheat germ extract in the presenceof 100 µCi of 35S-methionine. Whether these products were Vprwas analyzed by carrying out western blotting according to typicalprocedures employing an anti-FLAG antibody after samples resultingfrom transcription and translation in the absence of the radiolabelwere denatured with SDS (sodium dodecylsulfate). 4) Affinity Precipitation
    [0094] Observations of direct binding of the compound B and theFLAG-fused Vpr (or FLAG-fused Vpr ΔC) were made by mixing Sepharosebeads having the compound B immobilized thereto and the FLAG-fusedVpr (or FLAG-fused Vpr ΔC), followed by collecting the beads andidentifying co-precipitation with Vpr.
    [0095] 10 µl of a reaction solution containing the radiolabeledFLAG-fused Vpr (or FLAG-fused Vpr ΔC), 10 µl (bed volume) ofSepharose beads having the compound B immobilized thereto, and10 µl of PBS were mixed and the mixture was gently stirred atroom temperature for 2 hours or at 4°C overnight. The Sepharosebeads were then washed 3 times or more with PBS containing 0.1%Brij (Sigma), and SDS denature was carried out, followed byacrylamide gel electrophoresis. After drying the gel, analysiswas performed on a BAS 2000 Image Analyzer (Fuji Photo Film Co.,Ltd.). The results are shown in Fig. 2.
    [0096] As shown in Fig. 2 (2) , there were observed bands of moleculeswhich seemed to be FLAG-fused Vpr and FLAG-fused Vpr ΔC producedin the wheat germ extract transcription/translation system (lanes1 and 4). Both bands displayed the mobility corresponding withthe predicted molecular weight. Also, the results from westernblotting employing an anti-FLAG antibody revealed that themolecules corresponding to these bands have a FLAG epitope. Fromthe results of affinity precipitation, FLAG-fused Vpr was observedto bind to the compound-immobilized beads (lane 3), whereas FLAG-fused Vpr ΔC having the C-terminal deleted was not observedto bind to the beads (lane 5). From these results, it has turnedout that the compound B binds directly to the C-terminal regionwhich is believed to be important to Vpr effects of arresting thecell cycle and cytotoxicity. Test Example 3Inhibition of Vpr cytotoxic activity by compound1) Mass purification of FLAG-fused Vpr
    [0097] An E. coli strain DH5α transformed with pGEM-T/Vpr-wt wascultured overnight at 37°C in 60 ml of LB medium containing 0.5%glucose as pre-culture. On the next day, 30 ml aliquots of thisculture were added to one-liter LB mediums containing 0.5% glucose,which in turn were cultured at 37°C, and to which, at the timeof reaching an OD600 of 0.6, IPTG (isopropyl-β-D-thiogalactopyranoside) was added at 0.1 mM and culturing wascontinued for an additional two hours. Cells were then harvestedby centrifugation. Cells were suspended in a lysis solution [10mM Tris-HCl (pH 7.5), 2% CHAPS(3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid),2% n-octyl-β-D-glucoside (both from Dojindo), 1M NaCl, 10 µMPMSF (phenylmethylsulfonyl fluoride)] and then dispersed with anelectric homogenizer for 5 minutes and by 10 strokes in a Teflonhomogenizer, followed by further processing with aultrasonificator on ice for 5 minutes. Supernatant was collectedby low-speed centrifugation (8000 g, 20 minutes), and this supernatant was subjected further to high-speed centrifugation(50000 g, 20 minutes) to obtain its supernatant. The supernatantwas passed through a filter having a pore size of 2 µm and thendiluted 4 times or more in 10 mM Tris (pH 7.5), 10 µM PMSF, towhich 2 ml (bed volume) of anti-FLAG-antibody immobilized agarosebeads was added, followed by mixing overnight at 4°C with stirring.On the next day, the beads were collected, washed 3 times withTBST, and then suspended in 1 ml TBST containing 100 µM of FLAGpeptide (Sigma) and stirred overnight to elute FLAG-fused Vpr fromthe beads. The eluate was dialyzed to reduce CHAPS and theconcentration of n-octyl-β-D-glucoside to 0.05%, and thenconcentrated about 5 times with a Centricon (Amicon).
    [0098] The obtained protein run together with lysozyme having almostthe same molecular weight ( a molecular weight of 19 kDa; Gibco-BRL),and its amount was calculated by comparing the staining intensityof the bands stained with Coomassie Blue. 2) Suppression effects of the compound on cytotoxic activity ofFLAG-fused Vpr
    [0099] Fig. 2 (1) shows an electrophoresis image of purified FLAG-fusedVpr (stained with Coomassie Blue). Western blotting confirmedthat an anti-FLAG antibody recognizes this molecule.
    [0100] Test 1: HeLa cells were cultured in 6 cm dishes. At aconfluency (saturation) of about 30%, 1 µM of FLAG-fused Vpr anddifferent concentrations of the compound were added, and culturingwas done for 48 hours. After culturing, PI (propidium iodide) was added into the medium for staining, and cells were treatedwith trypsin and collected. Cells were analyzed on a flow cytometerand the percentage of PI-stained dead cells was determined. Asshown in Fig. 3 (1), it has turned out that the compound reducesthe number of dead cells in a dose dependent manner.
    [0101] Test 2: Jurkat T-cells were plated at a density of 2 x 105cells/ml in 24-well plates, into which FLAG-fused Vpr was addedat a concentration of 1 µM. Different concentrations of thecompound were further added, and culturing was carried out at 37°Cfor 48 hours, followed by counting the number of cells. Thisexperiment was repeated three times and the average percentageof inhibiting the growth was calculated.
    [0102] As shown in Fig. 3 (2), the presence of FLAG-fused Vpr inhibitedthe growth by 30%, whereas the coexistence with 10 nM of the compoundresulted in reducing the suppression of growth to 13%.
    [0103] From the results of the above-described Test Examples 1 to3, the present compound inhibits the cytotoxic activity of Vprat a concentration (10 nM) at which it does not display a significantanti-cancer activity. It is known that the present compound haslow toxicity and high safety. INDUSTRIAL APPLICABILITY
    [0104] The present compound is a superior agent for regulating Vprfunctions, and inhibits the destruction of various cells basedon HIV infection, and therefore can be employed as an agent for preventing the onset of and treating AIDS. Although it is reportedthat extracellular addition of Vpr promotes the production ofviruses from persistent infection cells, it is also possible thatthe present compound suppresses this and retains persistentinfection. Molecules functionally equivalent to Vpr have not beenfully identified at present, but the present compound also couldfind applications as a treating agent for human adult T-cellleukemia, an infectious disease caused by another human pathogenicretrovirus HTLV-1.
    权利要求:
    Claims (15)
    [1] A Vpr function regulating agent comprising anaminostilbazole derivative represented by the following generalformula [1] or a salt thereof as an active ingredient: A-B-D-E {wherein A represents optionally substituted heteroaryl or an oxidethereof, B represents optionally substituted ethenylene, Drepresents optionally substituted phenylene, E represents thefollowing formula:
    [2] The Vpr function regulating agent according to claim 1,wherein A is optionally substituted pyridyl or 1-oxide pyridyl,B is non-substituted ethenylene, D is non-substituted oraminoalkoxy-substituted 1,2-phenylene, R of -N(R)-SO2-G as E isalkyl which may be substituted with a substituent selected fromthe group consisting of halogen, amino, monoalkylamino,dialkylamino, morpholino, alkoxy, hydroxy, cyano, the formula:- CONR1R2 (wherein R1 and R2 are the same or different and eachrepresents hydrogen, hydroxy, alkyl, alkoxy, cycloalkyl,cycloalkyloxy, aryl, aryloxy, aralkyl, aralkyloxy,cycloalkylalkyl, cycloalkylalkyloxy, or tetrahydrofuranyloxy),and the formula: -SO2NR3R4 (wherein R3 and R4 are the same or differentand each represents hydrogen or alkyl), alkenyl which may besubstituted with halogen, or -COR0, R0 is alkyl, cyclic amino alkyl,or dialkylaminoalkyl, and G is 4-alkoxyphenyl.
    [3] The Vpr function regulating agent according to claim 1,wherein A is non-substituted 4-pyridyl or 1-oxide-4-pyridyl, Bis ethenylene of a non-substituted trans form, D is non-substitutedor aminoalkoxy-substituted 1, 2-phenylene, R of -N(R)-SO2-G as Eis hydrogen, hydroxyalkyl, or -COR0, R0 is alkyl or morpholinoalkyl or dialkylaminoalkyl, and G is 4-methoxyphenyl.
    [4] The Vpr function regulating agent according to claim 1,wherein the compound represented by the formula [I] is a compoundselected from the group consisting of(E)-4-[2-{2-[N-acetyl-N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine 1-oxide,(E)-4-[2-{2-[N-acetyl-N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine,(E)-4-[2-{2-[N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine 1-oxide,(E)-4-[2-{2-[N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine,(E)-4-[2-{2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine 1-oxide,(E)-4-[2-{2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine,(E)-4-[2-{3-(2-aminoethyloxy)-2-[N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine, and(E)-4-[2-{3-(2-aminoethyloxy)-2-[N-(4-methoxybenzenesulfonyl)amino]phenyl)ethenyl]pyridine 1-oxide.
    [5] A Vpr function regulating agent comprising the compoundof any of claims 1 to 4, or a salt thereof, in an amount effectivefor regulation of Vpr function.
    [6] A method for regulation of Vpr function, which comprisesusing the compound of any of claims 1 to 4, or a salt thereof,in an amount effective for regulation of Vpr function.
    [7] Use for production of a Vpr function regulating agentcomprising the compound of any of claims 1 to 4, or a salt thereof,as an active ingredient.
    [8] An anti-HIV agent comprising the compound representedby the general formula [1] of claim 1, or a salt thereof, as anactive ingredient.
    [9] An anti-HIV agent comprising the compound of claim 2,or a salt thereof, as an active ingredient.
    [10] An anti-HIV agent comprising the compound of claim 3,or a salt thereof, as an active ingredient.
    [11] The anti-HIV agent according to claim 8, wherein thecompound represented by the formula [I] is a compound selectedfrom the group consisting of(E)-4-[2-{2-[N-acetyl-N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine 1-oxide,(E)-4-[2-{2-[N-acetyl-N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine, (E)-4-[2-{2-[N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine 1-oxide,(E)-4-[2-{2-[N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine,(E)-4-[2-{2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine 1-oxide,(E)-4-[2-{2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine,(E)-4-[2-{2-[N-(4-methoxybenzenesulfonyl)-N-morpholinoacetylamino]phenyl}ethenyl]pyridine 1-oxide,(E)-4-[2-{3-(2-aminoethyloxy)-2-[N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine, and(E)-4-[2-{3-(2-aminoethyloxy)-2-[N-(4-methoxybenzenesulfonyl)amino]phenyl}ethenyl]pyridine 1-oxide, or a salt thereof.
    [12] An anti-HIV agent comprising the compound of any of claims8 to 11, or a salt thereof, in an amount effective for prophylaxisor treatment of a mammal infected with HIV or a mammal with AIDS.
    [13] A method for treatment of AIDS, which comprisesadministering an anti-HIV agent comprising the compound of anyof claims 8 to 11, or a salt thereof, in an amount effective forprophylaxis or treatment of a mammal infected with HIV or a mammalwith AIDS.
    [14] The anti-HIV agent according to claim 12, wherein themammal is human.
    [15] Use for production of an anti-HIV agnet comprising thecompound of any of claims 8 to 11, or a salt thereof, as an activeingredient.
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