WO2008117935A1

WO2008117935A1 - Nk cell activating molecules, nk cells and pharmaceutical compositons comprising the same

Info

Publication number: WO2008117935A1
Application number: PCT/KR2008/000937
Authority: WO
Inventors: Jong Sun Kim; Jin Sung Tae; Hyung Ran Kim; Young Keun Yang; Kyoung Ho Lee
Original assignee: Industry-Academic Cooperation Foundation, Yonsei University
Priority date: 2007-03-28
Filing date: 2008-02-18
Publication date: 2008-10-02

Abstract

The present invention relates to a compound represented by specific Formula 1; a process for preparing the same; NK cells treated with said compound; a process for treating NK cells with said compound; and a pharmaceutical composition comprising said compound or said NK cells. The present compound of Formula 1 may outstandingly increase expression of natural cytotoxicity receptors on NK cells such as NCR1, NCR2 and NCR3 and death ligands such as FasL and TRAIL. Thus, NK cel ls treated with the present compound have highly increased cytotoxicity against target cells such as cancer cells and host cells infected with viruses. Therefore, the present compound or NK cells treated with the same may be usefully used as a medical use such as anticancer agents and antiviral agents.

Description

[DESCRIPTION] [Invention Title]

NK CELL ACTIVATING MOLECULES, NK CELLS AND PHARMACEUTICAL COMPOSITONS COMPRISING THE SAME

[Technical Field]

The present invention relates to compounds that can activate natural killer cells (referred to "NK cell," below) by increasing expression of natural cytotoxicity receptors (referred to "NCR," below) and death ligands on NK cells; a method for preparing the same; NK cells treated with said compounds; and a pharmaceutical composition comprising said compounds and NK cells.

[Background Art]

The NK cells are effector cells acting in the front of in vivo immune system, such as performing function to remove tumor cells and host cells infected with bacteria, intracellular parasites or viruses even without pre-sensitization by antigens, rejecting anapropriate bone marrow transplant and regulating immune response of T cells. The NK cells exhibit cytolytic potential against target cells (for example, transformed tumor cells and host cells infected with viruses) that fail to express, specifically, major histocompatibility complex (referred to "MHC," below) class I antigens or outstandingly reduce their expression. NK cells are apparently distinguished from other lymphocytes, T cells or B cells, in view of expression aspects of CD markers, cytokine profiles, and the like (Ljunggren et al., In search of the 'missing self: MHC molecules and NK cell recognition, Immunol. Today, 1990; 11: 23744). It is noted that the NK cells show strong cytolytic potential against, especially, hematological malignancy, various solid cancers, and the like to play an important role in in vivo cancer immunity. The mechanisms are relatively well identified, wherein NK cells or cytotoxic T cells show cytotoxicity against target cells. The representative mechanism is one that these cells recognize target cells via activating receptors and secrete cytotoxic materials such as perforin or granzyme A and B to induce their necrosis or apoptosis. The NK cells use a mechanism to induce apoptosis by recognizing death receptors of target cells via death ligands (Fas-L, TNP, TRAIL, and the like) on NK cell surface, being identical to T cells, and perform cytolytic function to directly kill tumor cells or infected cells by using such a mechanism. In addition, the NK cells transfer cell debris of target cells to T cells via macropages or other antigen presenting cells and play a role to strengthen adaptive immunity.

Immunological function of NK cells depends on the balance of stimulatory signals to induce the cytolytic function and inhibitory signals to inhibit it. Specifically, NK cells, which receive the stimulatory signals, attack target cells and remove them and NK cells, which receive the inhibitory signals, keep alive them.

The cytolytic function of NK cells may include ADCC (antibody-dependent cell cytoxicity) and natural killing. ADCC to be induced with antigen-antibody complexes is mediated by Fcγ receptor III (FcγRIII, CD 16), and natural killing is mediated by activating receptors of NK cells. Specific mechanism has some difference in that ADCC proceeds via P13-kinase (phosphatidylinositol-3-kinase), while natural killing proceeds via PKC (protein kinase C). However, ADCC and natural killing both have a common feature that they need activation of PTK (Protein Tyrosine Kinase) and are interrupted with inhibitory signals transferred by inhibitory receptors of NK cells. Thus, since the cytolytic function of NK cells depends on balance of the stimulatory signals and the inhibitory signals, NK cells can distinguish infected cells or tumor cells from normal host cells and remove them.

The target specificity of NK cells is determined by the inhibitory receptors, and the inhibitory receptors of NK cells also recognize MHC of target cells like T cell receptors. While NK cells do not attack cells that MHC class I antigens are normally expressed, they regard cells that MHC class I antigens are not expressed or significantly reduced on expression as abnormal cells and attack the cells. NK cell receptors having this function were identified in NK cells of human and rat, and their primary structure of protein was disclosed by cloning cDNA. Generally, in tumor cells and host cells infected with viruses, expression of MHC class I antigens is significantly reduced. Therefore, the function of inhibitory receptors in such NK cells is necessary to selectively remove transformed tumor cells and host cells infected with viruses.

In case of human, some inhibitory receptors of NK cells are disclosed until now. As inhibitory Killer Ig-like receptors (KIRs) showing specificity to HLA-A, HLA-B and HLA-C and pertaining to immunoglobulin superfamily, the examples may include KJR2DL (p58 KIR) and KIR3DL (p70 KIR), which interact with MHC class I antigens. It was reported that KIR2DL bound to HLA-C, and KIR3DL bound to HLA-A and HLA-B. Although KIR was primarily expressed in NK cells, it was also expressed in some T cells. It was noted that KIR down- regulated immune response of T cells as well as cytolytic function of NK cells. Other examples of NK cell inhibitory receptors may include C-type lectin-like NKG2A and NKG2B, and the like, which comprise complexes with CD94 molecules and show specificity to human leukocyte antigen-E allelic gene. Immunoreceptor tyrosine based inhibitory motifs (ITlMs) are present in intracellular regions of these inhibitory receptors to inhibit activity of NK cells.

Examples of NK cell activating receptors may include CDl 6, involved in ADCC as IgG Fc receptors, NKG2C and NKG2E, showing specificity to HLA-E allelic genes to be coupled to CD94 molecules, activating KIR-like p50 KIR, 2B4 and NKp80 as coreceptors of activating receptors. Since these NK cell activating receptors have short intracellular regions, they should be coupled to adaptor molecules such as FcR γ-chain, CD3 ζ-chain, DAPlO or DAP 12, having immunoreceptor tyrosine-based activation motifs (ITAMs), in order to transfer stimulatory signals. In addition to the above examples, there are included activating receptors such as NCRl(NKp46), NCR2(NKp44), NCR3(NKp30) and NKG2D. NCRs are known to play the highest role in order for NK cells to attack transformed cells or cells infected with viruses (Bottino C, et al. The human natural cytotoxicity receptors (NCR) that induce HLA class I-independentNK cell triggering. Hum. Immunol. 2000; 61:1-6; Pende D, et al. Role of NKG2D in tumor cell lysis mediated by human NK cells: cooperation with natural cytotoxicity receptors and capability of recognizing tumors of nonepithelial origin. Eur. J. Immunol. 2001; 31:1076-86; Moretta A, et al. (2001) Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu. Rev. Immunol. 19, 197-223). NCRs are recently identified among activating receptors of NK cells, and are thought as major activating receptors inducing natural killing of cells in that (1) expression of receptors is mainly to NK cells only, (2) when NCRs are cross-coupled into single clone antibodies, the cytolytic function of target cells by NK cells may be identified via a redirected killing assay, and (3) if receptors are masked by single clone antibodies, NK cell-madiated cytotoxicity is inhibited (Moretta, A. et al., Natural cytotoxicity receptors that trigger human NK-cell- mediated cytolysis. Immunol. Today. 2000; 21(5):228-34; Biron et al., (1999) Natural killer cells in antiviral defence: function and regulation by innate cytokines. Annu. Rev. Immunol. 17, 189-220.; De Maria A, et al. (2001) Identification, molecular cloning and functional characterization of NKp46 and NKp30 natural cytotoxicity receptors in Macaca Fascicularis NK cells. Eur. J. Immunol. 31, 3546-3556). NCRl is expressed in all activated and inactivated NK cells, but not expressed in T cells or other cells. In addition, NCRl is specifically expressed in NK cells of monkey, rat and cattle, as well as human. Analogues are also expressed in such NK cells of mouse, and the like. This implies that the mechanism of activating NK cells via NCRl is preserved between species. In addition, among receptors expressed in human NK cells, NCRl only is the receptor which can recognize ligands of murine cell surface. NCRl is a receptor which belongs to the Ig superfamily having two Ig domains, and its intracellular region comprises 30 amino acids. It does not contain also ITAMs, so that it has the positively charged arginin (Arg) in transmembrane regions and is coupled to CD3ζ- chain to transfer signals. NCRl induces Ca²⁺ mobilization, cytotoxicity and secretion of cytokine in NK cells (Sivori, S. et al. (1997) P46, a novel natural killer cell-specific surface molecule which mediates cell activation. J. Exp. Med. 186, 1129-1136). In addition, it was known that masking of said receptor inhibited lysis against various tumors such as lung cancer, liver caner, breast cancer, melanoma and EBV-transformed cell lines. Gazit et al. disclosed that NCRl is an immune receptor playing a major role in removing influenza viruses in vivo (Gazit R, et al. (2006) Lethal influenza infection in the absence of the natural killer cell receptor gene Ncrl . Nature Immunol. 7, 517-523.). In addition, it was disclosed that NCRl recognized haemagglutinins of influenza viruses as ligands together with NCR2 to activate NK cells (Mandelboim O, et al. (2001) Recognition of haemagglutinins on virus-infected cells by NKp46 activates lysis by NK cells. Nature 409, 1055- 1060.; Arnon TI, et al. (2001) Recognition of viral haemagglutinins by NKp44 but not by NKp30. Eur. J. Immunol. 31, 2680-2689). Vankayalapati et al. disclosed that NCRl on NK cell surface played a major role in killing cells infected with Mycobacterium tuberculosis (Vankayalapati R et al. (2002) The NKp46 receptor contributes to NK cell lysis of mononuclear phagocytes infected with an intracellular bacterium. J. Immunol. 168, 3451-3457).

NCR2, the second NCR, is expressed only in NK cells which are cultured with IL-2 to be activated, but not expressed in lymphocytes or αβ T cells, separated from peripheral blood. Therefore, it may be the specific marker of activated NK cells. Unlike NCRl and NCR3, NCR2 is not found in other species rather than human, and has a characteristic that it is expressed even in some γδ T cells, in addition to activated NK cells. NCR2 belongs to the Ig superfamily having a single Ig domain in extracellular regions and transduces signals by associating it with DAP12 via the positively charged lysine in transmembrane regions, since it does not also have ITAMs in intracellular regions (Cantoni, C. et al (1999) NKP44, a trigering receptor involved in tumor cell lysis by activated human natural killer cells, is a novel member of the immunoglobulin superfamily. J. Exp. Med. 189, 787-796).

NCR3, the third NCR, cooperates with NCRl and NCR2 and plays a role to induce cytotoxicity against various target cells, and is expressed in all NK cells, freshly separated or cultered with IL-2. The extracellular region of NCR3 is the Ig superfamily consisting of a single Ig domain, and NCR3 transduces signals by associating it with CD3ζ-chains, since it does not have ITAMs in the intracellular region. In addition, analogues are present even in murine NK cells. It shows that the mechanism of activating NK cells also preserves between species (Pende, D. et al. Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells. J. Exp. Med. 1999; 190: 1505- 16).

A precise correlation exists between the expression degree of said NCRs and the cytolytic potential of NK cells. That is, NK cells highly expressing NCR show strong cytolytic potential against target cells, whereas NK cells lowly expressing NCR show week cytolytic potential against them (Sivori, S. et al. NKp46 is the major triggering receptor involved in the natural cytotoxicity of fresh of cultured human NK cells: correlation between surface density of NKp46 and natural cytotoxicity against autologous, allogeneic or xenogeneic target cells. Eur. J. Immunol. 1999; 29:1656-66; Moretta L, Moretta A (2004) Unraveling natural killer cell function: triggering and inhibitory human NK receptors. EMBO J. 23, 255-259). In addition, the NK extracellular expression of each NCR is precisely involved in each other and thus there is a characteristic that NCRs are expressed in the same aspects. That is, NK cells highly expressing NCRl highly express NCR3 and being activated, they highly express NCR2 as well. But, NK cells lowly expressing NCRl lowly express NCR3 and being activated, they lowly express NCR2 as well. Regarding expression and regulation of NCRs, researches being conducted until now are as follows. First, CD3ζ-chains, adapter molecules associating NCRl and NCR3, do not affect for these receptors to be expressed in NK cell surfaces, but they are indeed required in expressing NCR2 (Pessino, A. et al. Molecular cloning of NKp46: a novel member of the immunoglobulin superfamily involved in triggering of natural cytotoxicity. J. Exp. Med. 1998; 188: 953-60). And, it is noted that Cortisol, known as human adrenocortical hormone, inhibits the expression of NCRl and NCR2 in NK cells, but prolactin or IL-2, cytokine improving immune response, increases the expression of NCRl and NCR3 (Mavoungou E, Bouyou-Akotet MK, Kremsner PG (2004) Effects of prolactin and Cortisol on natural killer (NK) cell surface expression and function of natural cytotoxicity receptors (NKp46, NKp44 and NKp30). Clinical and Exp. Immunol. 139, 287- 296). Particularly, IL-2 was known as cytokine activating NK cells long ago and has been practiced in a number of animal experiments and clinical trials. Also, it showed from results of animal experiments that prolactin improves anti-cancer function of NK cells (Sun et al., (2003) Recombinant human prolactin improves anti-tumor effects of murine natural killer cells in vitro and in vivo. Neuroimmunomodulation 10, 169-176). In addition, it is reported that TGFβl reduces the expression of NCR3 and NKG2D in NK cell surfaces and consequently decreases cytotoxicity of NK cells. Recently, Mavoungou et al. reported that the cytolytic potential of NK cells was increased by IL-2 or prolactin, but decreased by Cortisol (Mavoungou et al., (2004) Effects of prolactin and Cortisol on natural killer (NK) cell surface expression and function of natural cytotoxicity receptors (NKp46, NKp44 and NKp30) Clinical and Exp. Immunol. 139, 287-296.). From these research results, it may be noted that a hormone such as IL-2, prolactin, TGFβl or Cortisol regulates activity of NK cells through regulating NCR expression, and if the NCR expression increases, cytolytic potential of NK cells increases, conversely, if the NCR decreases, it also decreases. Further, the above fact implies that if the expression of NCR in NK cells increases, the cytolytic potential of NK cells also increases, and thus the above mentioned materials may be usefully used as materials for treating cancer or virus diseases.

In addition to the above fact, most NK cells highly express NCRl, but are also present those that slightly express this. Thus, it is noted that there are differences in the expression of NCRl between NK cells. However, there are insignificant researches about how the NCRs are expressed and regulated in NK cell surfaces rather than the above facts.

Meanwhile, NK cells have also functions that they begin and regulate adaptive immune responses, as well as the cytolytic function of target cells as described above (Raulet DH (2004) Interplay of natural killer cells their receptors with the adaptive immune response. Nature Immunol. 5, 996-1002; Delgi-Esposti MA & Smyth MJ (2005) Close encounters of different kinds: dendritic cells and NK cells take centre stage. Nature Rev. Immunol. 5, 112-124). That is, it is noted that NK cells induce immune responses of T cells through secretion of IFN-γ at the early infection (Martin- Fontecha A, et al. (2004) Induced recruitment of NK cells to lymph nodes provides IFN-g for THl priming. Nature Immunol. 5, 1260-1265), and recently, it is identified that the interaction between NK cells and myeloid cells plays an important role in progressing the following stage of immune responses, and moreover, a research about how NK cells regulate adaptive immune responses is going on. In addition, it is noted that NK cells interact with monocytes such as dendritic cells or macrophages, and consequently, these immune cells are mutually activated to activate the following stage of immune responses. It is noted that the interaction and activation between NK cells and monocytes is mediated by receptors on cell surfaces as well as by aqueous factors. Particularly, it is noted that NCR3 plays an important role. Such results imply that if NK cells are activated, not only natural immune responses but also adaptive immune responses, wherein T cells are involved, may be activated. Therefore, if NK cells are activated by increasing the expression of NCRs on NK cell surfaces, the effect may be exhibited, in which they activate antigen presenting cells such as dendritic cells or macrophages and ultimately activate T cells.

[Disclosure] Technical Problem

The present invention is accomplished by considering the context described above. The present invention has objects to synthesize compounds which can increase the cytolytic potential of NK cells by increasing the expression of NCRs and death ligands on NK cell surfaces and to contrive applications as medical uses such as anticancer agents or antiviral agents by analyzing their action mechanism.

Technical Solution

As means to solve the above objects, the present invention provides a compound represented by Formula 1, a pharmaceutically acceptable salt or solvate thereof; and a process for preparing said compound.

[Formula 1]

As another means to solve the above objects, the present invention provides NK cells treated by the present compound.

As yet another means to solve the above objects, the present invention provides a method of treating NK cells characterized by comprising a step of culturing NK cells in a culture fluid adding the present compound.

As other means to solve the above objects, the present invention provides a pharmaceutical composition comprising the present compound or NK cells treated by the present compound and a pharmaceutically acceptable carrier.

[Description of Drawings]

Fig. 1 is a drawing depicting the mechanism of NK cell activating molecules according to the present invention.

Fig. 2 is a drawing showing results that in Experimental Example 1-1, the degrees of expressing NCR3 were measured by flow cytometry, on treating NK cells with the present compound.

Fig. 3 is a drawing representing the degrees of expressing NCR3 in relative MFI ratios, in Experimental Example 1 - 1 , on treating NK cells with the present compound.

Fig. 4 is a drawing showing results that in Experimental Example 1-2, the degrees of expressing NCR3 were measured by flow cytometry, on treating NK cells with the present compound.

Fig. 5 is a drawing representing the degrees of expressing NCR3 in relative MFI ratios, in Experimental Example 1-2, on treating NK cells with the present compound.

Fig. 6 is a drawing showing results that the degrees of expressing NCRl were measured by flow cytometry, on treating NK cells with the present compound.

Fig. 7 is a drawing representing the degrees of expressing NCRl in relative MFI ratios, on treating NK cells with the present compound. Fig. 8 is a drawing showing results that the degrees of expressing NCR2 in untreated NK cells and NK cells treated with the present compound and PMA were measured by flow cytometry.

Fig. 9 is a drawing representing the results of Fig. 8 in relative MFI ratios.

Fig. 10 is a drawing representing expression of NCRs in untreated NK cells and NK cells treated with cytokine in relative MFI ratios.

Figs. 11 and 12 are drawings representing effects that the present compound affects on cytotoxicity of NK cells against cancer cells.

Fig. 13 is a drawing representing effects that the present compound affects on expression of death ligands on NK cells.

Fig. 14 is a drawing representing in vivo anticancer effects in mouse by the present compound.

Figs. 15 and 16 are drawings representing in vivo antimetastatic effects in mouse by the present compound.

[Best Mode]

The present invention relates to a compound represented by Formula 1, a pharmaceutically acceptable salt or solvate thereof. The present compound has an excellent effect to express NCRs and FasL and TRAIL, wherein these are a death ligand, and the like, on NK cells. Since NK cells with increased NCRs and death ligands on their surfaces have the increased cytolytic potential against tumor cells or infected cells, the present compound or NK cells treated with said compound may be effectively applied to medical uses such as treatments of cancers and infectious diseases. In the present specification, the phrase "compound represented by Formula 1" is used in the same meaning as "NK cell activating molecule" or "NKAM." [Formula 1]

wherein, Ri repesents hydrogen, halogen, hydroxy, alkyl, cycloalkyl, alkenyl, alkynyl or alkoxy;

R₂ and R₃ each independently represent hydrogen, halogen, hydroxy, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, amino, cyano, carboxyl, aryl or -R₂₃R₂₄; where R₂₃ represents any one selected from straight or branched alkylene; straight or branched alkenylene; and straight or branchedalkynylene;

R₂₄ represents hydroxy; alkylamino; dialkylamino; amino; cyano; carboxyl; substituted or unsubstituted aryl, heteroaryl or heterocyclic group; thiourea; isothiourea; alkylthio; thiocyanato; sulfamoyl or alkylsulfonyl; and

R₄ to Rn each independently represent hydrogen, alkyl, cycloalkyl or alkoxy.

The term "alkyl" used herein means substituted or unsubstituted straight, branched, cyclic and/or combined alkyl including any carbon-carbon single bond; "alkenyl" means substituted or unsubstituted straight, branched, cyclic and/or combined alkenyl including any carbon-carbon double bond; "alkynyl" means substituted or unsubstituted straight, branched, cyclic and/or combined alkynyl including any carbon-carbon triple bond. In addition, the term "cycloalkyl" used herein means substituted or unsubstituted cycloalkyl which may form a ring and include any unsaturated bond and any heteroatom (for example, O, N or S) in the ring or on the ring; "aryl" means a substituent derived from an aromatic compound, such as substituted or unsubstituted phenyl, biphenyl, napthyl or antracenyl ring system; "heteroaryl" means substituted or unsubstituted, 5 to 12 member heterocyclic or aryl cyclic group containing one or more heteroatoms selected from the group consisting of O, N and S, and includes, but is not limited to, furyl, pyrrolyl, pyrrolidinyl, thienyl, pyridinyl, piperidinyl, indolyl, quinolyl, thiazole, benzthiazole and triazole, and the like.

In addition, from the definitions of substituents in the present compound, alkyl is preferably alkyl having 1 to 12 carbons, more preferably alkyl having 1 to 8 carbons and most preferably alkyl having 1 to 4 carbons; cycloalkyl is preferably cycloalkyl having 3 to 13 carbons, and more preferably cycloalkyl having 3 to 8 carbons; alkoxy is preferably alkoxy having 1 to 12 carbons, more preferably alkoxy having 1 to 8 carbons and most preferably alkoxy having 1 to 4 carbons; alkenyl is preferably alkenyl having 2 to 12 carbons, more preferably alkenyl having 2 to 8 carbons and most preferably alkenyl having 2 to 4 carbons; alkynyl is preferably alkynyl 2 to 12 carbons, more preferably alkynyl having 2 to 8 carbons and most preferably alkynyl having 2 to 4 carbons.

In said compound of Formula 1, preferred examples of Ri may include hydrogen, halogen, hydroxy, alkyl having 1 to 12 carbons, cycloalkyl having 3 to 12 carbons, alkenyl having 2 to 12 carbons, alkynyl having 2 to 12 carbons or alkoxy having 1 to 12 carbons, and are preferably hydrogen or alkyl having 1 to 8 carbons and most preferably hydrogen, methyl or ethyl.

In addition, in said compound of Formula 1, preferred examples of R₂ or R₃ may include hydrogen, hydroxy, alkyl having 1 to 12 carbons, cycloalkyl having 3 to 12 carbons, alkenyl having 2 to 12 carbons, alkynyl having 2 to 12 carbons, alkoxy having 1 to 12 carbons, amino, cyano, carboxyl, phenyl, naphthyl or -R₂₃R₂₄, wherein preferred examples Of R₂₃ may include straight or branched alkylene having 1 to 12 carbons, and preferred examples Of R₂₄ may include hydroxy, alkylamino having 1 to 12 carbons; dialkylamino having 2 to 24 carbons; any one of aryl, heteroaryl or heterocyclic group selected from the group consisting of phenyl, indolyl, pyrrolyl, furanyl, pyridinyl, piperidinyl, pyranyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl and triazolyl; thiourea; or isothiourea, where the aryl, heteroaryl or heterocyclic group may be substituted with one or more selected from the group consisting of hydroxy, amino, cyano, arylalkyl, aryl unsubstituted or substituted with alkyl and alkyl unsubstituted or substituted with carboxyl.

More preferred examples of said R₂ or R₃ may include hydrogen, alkyl having 1 to 8 carbons, cycloalkyl having 3 to 8 carbons, alkenyl having 2 to 8 carbons, alkynyl having 2 to 8 carbons, alkoxy having 1 to 8 carbons or -R₂₃R₂₄, wherein examples OfR₂₃ may include straight or branched alkyleήe having 1 to 8 carbons, and examples Of R₂₄ may include hydroxy, alkylamino having 1 to 8 carbons; dialkylamino having 2 to 16 carbons; any one of aryl, heteroaryl or heterocyclic group selected from the group consisting of phenyl, pyridinyl and triazolyl; thiourea; or isothiourea, where said aryl, heteroaryl or heterocyclic group may be substituted with one or more selected from the group consisting of hydroxy, amino, cyano, arylalkyl having 7 to 12 carbons, aryl unsubstituted or substituted with alkyl having 1 to 4 carbons and, alkyl, having 1 to 12 carbons, unsubstituted or substituted with carboxyl.

Said R₂ or R₃ is, most preferably, hydrogen, alkyl having 1 to 4 carbons, cycloalkyl having 3 to 6 carbons, alkenyl having 2 to 4 carbons, alkynyl having 2 to 4 carbons, alkoxy having 1 to 4 carbons or -R₂₃R₂₄, wherein R₂₃ is straight or branched alkylene having 1 to 4 carbons, and R₂₄ is hydroxy, alkylamino having 1 to 4 carbons; dialkylamino having 2 to 8 carbons; substituted or unsubstituted triazolyl; thiourea or isothiourea, where the substituent of triazolyl is one or more selected from the group consisting of benzyl and, alkyl, having 1 to 4 carbons, unsubstituted or substituted with carboxyl.

In addition, in said compound of Formula 1 , preferred examples OfR₄ to Ri 1 may include hydrogen, alkyl having 1 to 12 carbons, cycloalkyl having 3 to 12 or alkoxy having 1 to 12, and are, more preferably, hydrogen, alkyl having 1 to 8 carbons or alkoxy having 1 to 8 carbons, and most preferably, hydrogen, methyl, ethyl, methoxy or ethoxy.

The present invention also relates to a compound, a pharmaceutically acceptable salt or solvate thereof, characterized in that in said compound of Formula 1,

Ri is hydrogen or alkyl having 1 to 8 carbons,

R₂ and R₃ each independently represent hydrogen, alkyl having 1 to 8 carbons, cycloalkyl having 3 to 8 carbons, alkenyl having 2 to 8 carbons, alkynyl having 2 to 8 carbons, alkoxy having 1 to 8 carbons or -R₂₃R₂₄; wherein R₂₃ represents straight or branched alkylene having 1 to 8 carbons, and R₂₄ represents hydroxy, alkylamino having 1 to 8 carbons; dialkylamino having 2 to 16 carbons; any one of aryl, heteroaryl or heterocyclic group selected from the group consisting of phenyl, pyridinyl and triazolyl; thiourea; or isothiourea; where the aryl, heteroaryl or heterocyclic group may be substited with one or more selected from the group consisting of hydroxy, amino, cyano, arylalkyl having 7 to 12 carbons, aryl unsubstituted or substituted with alkyl having 1 to 4 carbons and, alkyl, having 1 to 12 carbons, unsubstituted or substituted with carboxyl; and

R₄ to Rn are each independently hydrogen, alkyl having 1 to 8 carbons or alkoxy having 1 to 8 carbons.

The present invention also relates to a compound, a pharmaceutically acceptable salt or solvate thereof, characterized in that in said compound of Formula 1 , Ri is hydrogen, methyl or ethyl, R₂ and R₃ each independently represent hydrogen, alkyl having 1 to 4 carbons, cycloalkyl having 3 to 6 carbons, alkenyl having 2 to 4 carbons, alkynyl having 2 to 4 carbons, alkoxy having 1 to 4 carbons or -R23R24; wherein R₂₃ represents straight or branched alkylene having 1 to 4 carbons, and R₂₄ represents hydroxy, alkylamino having 1 to 4 carbons; dialkylamino having 2 to 8 carbons; substituted or unsubstituted triazolyl, thiourea or isothiourea; where the substituent of triazolyl is one or more selected from the group consisting of benzyl and substituted or unsubstituted alkyl having 1 to 4 carbons with carboxyl; and

R₄ to Rn are each independently hydrogen, methyl, ethyl, methoxy or ethoxy.

The present compound of Formula 1 is, more preferably,

3-[l-(3-hydroxypropyl)-5-methoxy-7H-indol-3-yl]-4-(7//-indol-3-yl)pyrrol-2,5-dione;

3-[l-(3-dimethylaminopropyl)-5-methoxy-7H-indol-3-yl]-4-(7H-indol-3-yl)pyrrol-2,5- dione;

3-[l-(2-hydroxyethyl)-5-methoxy-iH-indol-3-yl]-4-(iH-indol-3-yl)-pyrrol-2,5-dione;

3-[l-(2-dime%laminoethyl)-5-methoxy-iH-indol-3-yl]-4-(iH-indol-3-yl)pyrrol-2,5- dione;

3-[l-(3-hydroxypropyl)-5-methoxy-/H-indol-3-yl]-4-(7H-indol-3-yl)-l-methylpyrrol- 2,5-dione;

3-[(l-benzyl-iH-[l,2,3]triazol-4-ylmethyl)-5-methoxy-iH-indol-3-yl]-4-(iH-indol-3-yl)- pyrrol-2,5-dione;

(4.{3-[4.(7_jy-i_ridol-3-yl)-2,5-dioxy-2,5-dihydro-iH-pyrrol-3-yl]-5-methoxy-iH-indol-l- ylmethyl}-[l,2,3]triazDl-l-yl)acetic acid;

3-[l -(2-dimethylaminoethyl)-5-methoxy-7H-indol-3-yl]-4-(7//-indol-3-yl)- 1 - methylpyrrol-2,5-dione;

3-[l-(2-hydroxyethyl)-5-methoxy-iH-indol-3-yl]-4-(7H-indol-3-yl)-l-methylpyrrol-2,5- dione;

2-(2-{3-[4<iH-indol-3-yl)-2,5-dioxo-2,5-dihydro-7H-pyirol-3-yl]-5-methoxyindol-l-yl}- ethyl)isothiourea;

2-(3-{3-[4-(7H-indol-3-yl)-2,5-dioxo-2,5-dihydro-7H-pyrrol-3-yl]-5-methoxyindol-l- yl}propyl)isothiourea;

3-[l-(3-hydroxypropyl)-7H-indol-3-yl]-4-[l-(3-hydroxypropyl)-5-methoxy-7//-indol-3- yl]-pyrrol-2,5-dione;

3-[l-(3-dime1hylanτinopropyl)-7H-indol-3-yl]-4-[l-(3-dimethylaminopropyl)-5-methoxy- 7H-indol-3-yl]-pyrrol-2,5-dione;

2-[3-(3-{4-[l-(3-carbamirnidoylsulfanylpropyl)-iH-indol-3-yl]-2,5-dioxo-2,5-dihydro- 7H-pyrrol-3-yl} -5-methoxyindol- 1 -yl)-propyl]-isothiourea;

3-[l-(4-hydroxybutyl)-5-methoxy-7H-indol-3-yl]-4<iH-indol-3-yl)-pyrrol-2,5-dione;

2-(4-{3-[4-(7H-indol-3-yl)-2,5-dioxo-2,5-dihydro-7H-pyrrol-3-yl]-5-methoxyindol-l- yl}butyl)isothiourea;

3-[l-(4-dimethylan±iobutyl)-5-me1hoxy-7H-indol-3-yl]-4-(77Y-indol-3-yl)pyrrol-2,5- dione;

3,4-bis[l-(3-dimethylaminopropyl)-5-methoxy-77/-indol-3-yl]pyrrol-2,5-dione;

3-[l-(4-hydroxybutyl)-7H-indol-3-yl]4-[l-(4-hydroxybutyl)-5-methoxy-7H-indol-3-yl]- pyrrol-2,5-dione;

2-[4-(3-{4-[l-(4-carbamimidolsulfanylbutyl)-7/7-indol-3-yl]-2,5-dioxo-2,5-dihydro-7/7- pyrrol-3-yl}-5-methoxyindol-l-yl)-butyl]-isothiourea;

3-[l-(4-dime%larninobutyl)-7H-mdol-3-yl]-4-[l-4-dimethylaminopropyl)-5-methoxy- /H-indol-3-yl]-pyrrol-2,5-dione;

3-[l -(4-dimethylaminobutyl)-iH-indol-3-yl]-4-[ 1 -4-dimethylaminopropyl)-5-methoxy- 7H-Indol-3-yl]-pyrrol-2,5-dione;

3-[l-(4-dimethylarninobu1yl)-7H%dol-3-yl]-4-[l-3-dime1hylaminopropyl)-5-methoxy- 7H-indol-3-yl]-pyrrol-2,5-dione;

3-[l-(3-hydroxypropyl)-5-methoxy-7H-indol-3-yl]-4-(l-methyl-7H-indol-3-yl)-pyrrol- 2,5-dione;

3-[l-(3-dimethylaminopropyl)-5-methoxy-7H-indol-3-yl]-4-[l-methyl-i//-indol-3-yl)- pyrrol-2,5-dione;

2-(3-{5-melhoxy-3-[4-(l-methyl-7H-indol-3-yl)-2,5-dioxo-2,5-dihydro-iH-pyrrol-3-yl]- indol-l-yl}-propyl)-isothiourea; or

2-(4-{5-me1hoxy-3-[4-(l-methyl-7H-indol-3-yl)-2,5-dioxo-2,5-dihydro-iH-pyrrol-3-yl]- indol-l-yl}-butyl)-isothiourea.

The present compound of Formula 1 is, most preferably,

3-[l-(3-hydroxypropyl)-5-melhoxy-iH-indol-3-yl]-4-(i/i-indol-3-yl)pyrrol-2,5-dione;

3-[l-(2-dime%larrώioe%l)-5-me1hoxy-i/f-indol-3-yl]-4-(7//-indol-3-yl)pyrrol-2,5- dione;

2-(2-{3-[4-(7H-indol-3-yl)-2,5-dioxo-2,5-dihydro-iH-pyrrol-3-yl]-5-methoxyindol-l- yl}-ethyl)isothiourea;

3-[l-(3-dimethylaniinopropyl)-7H-indol-3-yl]4-[l-(3-dimethylarninopropyl)-5- methoxy-7H-indol-3-yl]-pyrrol-2,5-dione;

2-[3-(3-{4-[l-(3-carbarnirnidolysulfanylpropyl)-7H-indol-3-yl]-2,5-dioxo-2,5-dihydro- iH-pyrrol-3-yl}-5-methoxyindol-l-yl)-propyl]-isothiourea;

2-(4-{344-(iH-indol-3-yl)-2,5-dioxo-2,5-dihydro-7H-pyrrol-3-yl]-5-methoxyindol-l- yl}butyl)isothiourea;

3-[l-(4-dime%laminobutyl)-5-me1hoxy-iH-indol-3-yl]-4-(7H-indol-3-yl)pyrrol-2,5- dione;

3,4-bis[l-(3-dime1hylaminopropyl)-5-methoxy-7//-indol-3-yl]pyrrol-2,5-dione;

3-[l-(3-dimethylaminopropyl)-5-methoxy-iH-indol-3-yl]4-[l-methyl-7/f-indol-3-yl)- pyrrol-2,5-dione;

2-(3-{5-methoxy-3-[4-(l-methyl-iH-indol-3-yl)-2,5-dioxo-2,5-dihydro-iH-pyrrol-3-yl]- indol-l-yl}-propyl)-isothiourea; or

2-(4-{5-methoxy-3-[4-(l-methyl-iH-indol-3-yl)-2,5-dioxo-2,5-dihydro-iH-pyrrol-3-yl]- indol- 1 -yl } -butyl)-isolhiourea.

Said compound of Formula 1 of the present invention may be used by preparing it in a form of a pharmaceutically acceptable salt or a solvate, wherein the inorganic or organic acid or the solvent usually used in this field may be used.

Examples of inorganic or organic acids which may be used in preparing the pharmaceutically acceptable salts of the present compound of Formula 1 may include, but are not limited to, an inorganic acid such as hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid or nitric acid; or an organic acid such as citric acid, acetic acid, latic acid, tararic acid, maleic acid, gluconic acid, succinic acid, formic acid, trifluoroacetic acid, oxalic acid, fumaric acid, methanesulfonic acid, benzenesulfonic acid, paratoluenesulfonic acid or camphorsulfonic acid.

The pharmaceutically acceptable solvents which may be usually used in this field may be used without any limitation, even in preparing said solvate. Example of said solvent may include water or ether, and the like. The present invention also relates to a process for preparing said compound represented by Formula 1.

That is, the present invention relates to a process for preparing a compound of Formula 1 ' characterized by comprising a step of reacting a compound of Formula 2 and a compound of Formula 3.

[Formula 2]

[Formula 3]

[Formula 1']

wherein, Ri and R₄ to Ri i are the same as defined in said Formula 1 , R₂' and R₃' each independently represent hydrogen, halogen, hydroxy, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, amino, cyano, carboxyl, aryl or -R₂₃ ⁵R₂₄'; where Ras'represents any one selected from straight or branched alkylene; straight or branched alkenylene; and straight or branched alkynylene, and R₂₄' represents hydroxy; amino; cyano; carboxyl; substituted or unsubstituted aryl, heteroaryl or heterocyclic group; alkylthio; thiocyanato; sulfamoyl or a protecting group. Here, examples of said protecting group may include alkylsulfonyloxy; tetraalkylsiloxy; ethoxy carbonyl; or acetoxyl, and the like.

The present invention also relates to the process for preparing characterized by further comprising a step of reacting said compound of Formula 1' with a nucleophile. Here, examples of said nucleophile may include, but are not limited to, one or more selected from the group consisting of alkyl amine, dialkyl amine, thiourea and isothiourea.

The present invention also relates to the process for preparing characterized by further comprising a step of reacting said compound of Formula 1 ' with an electrophile. Here, examples of said electrophile may include dialkyl sulfate, preferably dimethyl sulfate.

The process for preparing the present compound of Formula 1 is specifically described below.

In each step of the above described process for preparing the present compound, specific reaction conditions (for example, solvents, reactants, catalysts, added amounts, reaction temperatures and reaction times, etc.) are not particularly restricted. One skilled in synthesis field of organic compounds may use appropriate reaction conditions by easily adapting them depending on the desired compounds.

For example, it is preferred, but not limited to, that in said reaction of compounds of Formulas 2 and 3, a compound of Formula 3 is used in an amount of 1.2 equivalents to 1.5 equivalents relative to a compound of Formula 2 and the reaction is carried out for 12 hours to 15 hours at a temperature of 0 ^°C to 25 ^°C .

In addition, it is preferred, but not limited to, that in a step of reacting said compound of Formula 1' with a nucleophile, the nucleophile is used in an amount of 1.2 equivalents to 1.5 equivalents relative to the compound of Formula 1 ' and the reaction is carried out for 10 hours to 12 hours at a temperature of 80 ^°C to 85 ^°C . Also, it is preferred, but not limited to, that in a step of reacting said compound of Formula 1' with an electrophile, the electrophile is used in an amount of 1.2 equivalents to 1.5 equivalents relative to the compound of Formula 1 ' and the reaction is carried out for 8 hours to 12 hours at a temperature of 60 ^°C to 65 ^°C .

Further, solvents used in each step of said process for preparing the present compound are not also limited, and the solvent may be used, which are usually used in synthesis of organic compounds. Examples of usable solvents may include t-butyllalcohol, dimethylacetamide, dichloromethane, chloroform, diethylether, dioxane, acetonitrile, benzene, toluene, acetone, dimethylformamide (DMF) or tetrahydrofuran (TEIF), and the like, and among said solvents, are, preferably, acetone, dimethylformamide (DMF) or tetrahydrofuran (THF).

The present invention also relates to a process for preparing a compound of Formula 2 used in said process. That is, said compound of Formula 2 may be prepared through a step of reaction of a compound of Formula 4 with oxalyl chloride and sodium methoxide.

[Formula 4]

wherein, R₂' and R₄ to R₇ are the same as defined in the Formula 2. The reaction conditions in preparing said compound of Formula 2 may be also appropriately selected and applied depending on reactants and purpose compounds. For example, it is preferred to use oxalyl chloride and sodium metoxide in an amount of 1.2 equivalents to 1.5 equivalents and 3 equivalents to 4 equivalents, respectively, relative to the compound of Formula 4, and to perform the reaction for 1 to 3 hours at a temperature of -65 ^°C to 25 ^°C , but is not limited to these.

The present invention also relates to a process for preparing a compound of Formula 3 to be used in said process. That is, said compound of Formula 3 may be prepared through a step of reacting a compound of Formula 5 below with a base (for example, potassium hydroxide (KOH), etc.).

[Formula 5]

wherein, R₃' and R₈ to Rn are the same as defined in the Formula 3.

The reaction conditions in preparing said compound of Formula 3 may be also appropriately selected and applied depending on reactants and purpose compounds. For example, it is preferred to use a base in an amount of 7 equivalents to 8 equivalents relative to the compound of Formula 5 and to perform the reaction for 2 to 3 hours at a temperature of 85 ^°C to 90 ^°C , but is not limited to these. Also, the base to be used is not specifically limited. Usual bases in this field can be used without limitation. For example, potassium hydroxide (KOH), and the like, is preferable.

The present invention also relates to NK cells treated with the present compound of Formula 1. The present compound may significantly increase the expression of NCRs inducing natural cytotoxicity on NK cells and death ligands reacting with death receptors of target cells, and thereby increase cytotoxicity of NK cells against tumor cells and host cells infected viruses.

One example of mechanisms that compounds of the present invention increase cytotoxicity of NK cells is specifically described, with reference to Fig. 1 , as follows. As shown in Fig. 1, if NK cells are treated with the present compound of Formula 1, the expression of NCRl, NCR2 and NCR3, and death ligands is significantly increased on their surfaces, and the increased NCRs ultimately increase cytotoxicity of NK cells. This function of the present compound is a unique phenomenon which is not observed by cytokine or PMA, and the like, currently known as activating materials of NK cells. It was identified from substantial examination results of the inventors that various cytokines or PMAs did not almost increase expression of NCRs, and LPSs, activating materials of non-specific immune cells, did not affect on expression of NCRs. NK cells have a function which may effectively kill cancer cells and cells infected with various viruses, and said function is in proportion to expression of NCRs on surfaces. Therefore, the present compound or NK cells treated with this compound can even better kill tumor cells and host cells infected with viruses, and the present compound, which highly increases the expression of NCRs on NK cell surfaces and increases cytotoxicity of NK cells on target cells, may be usefully used as an anticancer agent or an antiviral agent. The present invention also relates to a method of treating NK cells with a compound of the present invention, and specifically to a method of treating NK cells characterized by culturing NK cells identified from host cells in the culture fluid to which the present compound is added. At this time, culture conditions in the used culture fluid are not specifically limited, and those, which are usually used in this field, may be used.

A host includes human, monkey, cattle, sheep, goat, horse, pig, rabbit, dog, cat, rat, mouse or marmot, and the like. Examples of culture fluid to be used may include RPMI, MEM or DMEM, and the like, but are not specifically limited, as long as they are usually used in culturing animal cells such as NK cells. Also, in said culture procedure of NK cells, the culturing temperature is preferably 30 ^°C to 37 ^°C, and the culturing time is preferably 1 to 48 hours, but they are not limited to these. The amount of added compounds in said treating procedure may be appropriately selected depending on the desired use, without specific limitation, but is preferably in a range of 1.0 to 10.0 μM, relative to 10⁵ to 10⁶ cells/ml NK cells.

The present invention also relates to a pharmaceutical composition comprising the present compound of Formula 1 described above, a pharmaceutically acceptable salt or solvate thereof, or NK cells treated with said compound.

As described above, the present compound of Formula 1 increases expression of NCRs and death ligands of NK cells, and NK cells with increased expression of NCRs have the significantly increased cytotoxicity against cancer cells or target cells infected with viruses. Therefore, the present compound or NK cells treated with this may be used as a medical use via various methods. An example of said methods may include, first, a method of obtaining the effect of treatment by administrating a pharmaceutical composition comprising the present compound of Formula 1 directly to a patient in a manner such as oral administration or injection, to activate NK cells in vivo. In addition, it may include, second, a method of using the present compound for activating NK cells on developing cell therapeutics (for example, on developing cell therapeutics by utilizing NK cells, a method which comprises steps of treating NK cells with the present compound in a procedure of culturing NK cells in vitro to activate them, and then transplanting the activated NK cells to a patient).

The pharmaceutical composition of the present invention may be more usefully used as anticancer agents or antiviral agents, and usefully used in treatment of contagious disease, caused by bacteria being parasitic in cells, such as tuberculosis and leprosy.

Particularly, the pharmaceutical composition of the present invention may be usefully utilized in treatment of various diseases related to tumors, for example, various hematological malignancy including leukemia as well as various solid cancers such as lung cancer, liver cancer, gastric cancer, colon cancer, bladder cancer, prostatic cancer, breast cancer, ovarian cancer, cervical cancer, thyroid cancer and melanoma. In addition, NK cells with increased NCRs and death ligands have the increased cytotoxicity against host cells infected with viruses, and thus may be usefully used even in treatment of infectious diseases by viruses or bacteria, for example, viral diseases such as AIDS, avian influenza, influenza and CMV infectious diseases and bacterial diseases such as tuberculosis or leprosy.

Carriers used in said composition according to the present invention include carriers and vehicles usually used in medical fields, and particularly, include, but are not limited to, ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (for example, human serum albumin), buffer materials (for example, various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetative fatty acids), water, salts or electrolytes (for example, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose substrates, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene glycol or lanolin. In addition to the above components, the present composition may further comprise lubricants, wetting agents, emulsifiers, suspensions, or preservatives, and the like.

The pharmaceutical composition of the present invention may be prepared in a dosage form for oral administration or a dosage form for parenteral administration such as injections.

Examples of dosage forms for oral administration may include tablets, troches, lozenges, aqueous or oil suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. In order to formulate dosage forms such as tablets and capsules, binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, vehicles such as dicalcium phosphate, disintegrants such as com starch or sweet potato starch, or lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethyleneglycol wax may be included. Also, in case of capsule dosage forms, liquid carriers such as fatty oils may be included, in addition to the materials mentioned above.

In formulations for oral administration, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsifiers, or lyophilized formulations are included. As non-aqueous solvents or suspending solvents, propylene glycol, polyethylene glycol, vegetative oils such as olive oil or injectable esters such as ethyl oleate, and the like may be used.

In one aspect, the composition according to the present invention may be prepared in aqueous solutions for parenteral administration. Preferably, buffer solutions such as Hank's solution, Ringer's solution or physically buffered saline may be used. In aqueous injection suspensions, substrates, which may increase viscosity of suspensions, such as sodium carboxymethylcellulose, sorbitol or dextran may be added thereto. Other preferred aspect of the present composition may be in a form of formulations, of aqueous or oil suspensions, for sterilized injections. Such suspensions may be formulated by using appropriate dispensing agents or wetting agents (for example, Tween 80) and suspending agents, in accordance with techniques known in this field. Formulations for sterilized injections may be also sterilized injection solutions or suspensions in parenterally acceptable nontoxic diluents or solvents (for example, a solution in 1,3-butanediol). Usable vehicles and solvents include mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterilized non-volatile oils are usually used as a solvent or a suspended medium. For this purpose, any non-volatile oil with less irritability, including synthetic mono- or di-glycerides, may be used.

The effective dose of the pharmaceutical composition according to the present invention is preferably 0.2 mg/kg to 200 mg/kg, and may be administrated once to several times separately per day. When said dose needs to be modified, technicians in this field may appropriately modify said dose, considering physical specificity and body weight of patients, kinds and severity of diseases, characteristic of dosage forms, property of medicine administration and period or interval for administration.

[Example]

The present invention is described in more detail via Examples according to the present invention below, but the scope of the present invention is not restricted to the following Examples.

<Example 1> Preparation of 3-[l-(3-hydroxypropyl)-5-methoxy-i/f-indol-3-yl]-4- (i//-indol-3-yl)pyrroI-2,5-dione (5)

Preparation of [l-O-hydroxypropyD-S-methoxy-iH-indol-S-y^-oxoacetic acid methyl ester (4)

To DMF was added NaH (407 mg, 12.79 mmol), and then 5-methoxy indol (1) (1.00 g, 6.79 mmol) was slowly added thereto at O ^°C, followed by maintaining t he m ixture for 5 minutes. Then, 3-bromopropanol (0.71 ml, 8.15 mmol) was slowly added thereto, followed by maintaining at the same temperature for 10 minutes, increasing the temperature to 70 ^°C and then warming to reflux for 4 hours. The reactant was neutralized with ammonium chloride (5 ml), diluted with water (40 ml) and extracted three times with ethyl acetate (100 ml). Ethyl acetate was removed and the residue was purified by column chromatography (hexane: ethyl acetate = 10:1) to obtain a compound (2) as colorless oil in a yield of 79% (1.10 g, 5.36 mmol).

Said compound (2) (1.10 g, 5.63 mmol) was added to THF (15 ml), and then NaH (214 mg, 5.36 mmol) was slowly added thereto at 0 ^°C . To the mixture was slowly added TBSCl (tert- butyldimethylsilyl chloride) (969 mg, 6.43 mmol), followed by maintaining the mixture at the same temperature for 5 minutes and then at room temperature for 2 hours. The reactant was neutralized with ammonium chloride (6 ml), diluted with water (30 ml) and extracted three times with ethyl acetate (80 ml). Ethyl acetate was removed and the residue was purified by column chromatography (hexane: ethyl acetate = 15:1) to obtain the desired compound (3) as colorless oil in a yield of 93% (1.60 g, 5.00 mmol).

Said compound (3) (520 mg, 1.63 mmol) was added to dichloromethane (20 ml), oxalyl chloride (0.24 ml, 1.96 mmol) was slowly added thereto at 0 ^°C , and the mixture was reacted at the same temperature for 1 hour. Then, the temperature of the reaction vessel was decreased to -65 ^°C , sodium methoxide (100 mg of sodium were added to 2.5 ml of methanol) was slowly added thereto, and the mixture was reacted for 1 hour, with maintaining the same temperature. Then, the mixture was again reacted at room temperature for 1 hour. The reactant was extracted three times with dichloromethane (80 ml), and dichloromethane was removed. The residue was purified by column chromatography (hexane: ethyl acetate = 1:1) to obtain the desired compound (4) as green oil in a yield of 84% (400 mg, 1.37 mmol).

Preparation of 3-[l-(3-hydroxypropyl)-5-methoxy-7H-indol-3-yl]-4-(iH-indol-3- yl)pyrrol-2,5-dione (5)

The mixture of the compound (4) (180 mg, 0.62 mmol) and indol-3-ylacetamide (200 mg, 1.15 mmol) was dissolved in THF (12 ml), and potassium tert-butoxide (3 ml) was slowly added thereto at 0^°C . Said mixture was maintained at the same temperature for 10 minutes and reacted at room temperature for 12 hours. 2M hydrochloric acid (5 ml) was added thereto, and the mixture was further reacted for 1 hour. Then, the reactant was diluted with water (25 ml), and extracted three times with ethyl acetate (50 ml). The residue was purified by column chromatography (hexane: ethyl acetate = 1 :2) to obtain the desired compound (5) as red solid in a yield of 62% (160 mg, 0.85 mmol).

MS (ESI+); 438 [M+Na]⁺

MP; 243 - 245 ^°C ¹H NMR^OOMHZ); DMSCW₆ δ = 11.6O(1H, s), 1O.86(1H, s), 7.83(1H, s), 7.65(1H, s), 7.36(1H, d, J=8.0 Hz), 7.3O(1H, d, J=8.8 Hz), 6.97(1H, t, J=7.2 Hz), 6.88(1H, d, J=4.0 Hz), 6.64(1H, t, J=7.6 Hz), 6.57-6.55(1H, m), 6.09(1H, s), 4.25(2H, t, J=6.8 Hz), 2.98(3H, s), 1.90- 1.83(2H, m)

IR: 3490, 2911, 2850, 1651, 1499, 1440, 1411, 1258, 1090 cm^"1

<ExampIe 2> Preparation of 3-[l-(3-dimethylaminopropyl)-5-methoxy-i/ϊ-indol- 3-yl]-4-(lH-indol-3-yI)pyrrol-2,5-dione (7)

Preparation of 3-(7//-indol-3-yl)-4-5-methoxy-l-[3-(niethanesulfonyloxy)propyl]-.//j^'- indol-3-ylpyrrol-2,5-dione (6)

The compound (5) (70 mg, 0.17 mmol) prepared in Example 1 was dissolved in DMF (4 ml), and triethylamine (0.07 ml, 0.51 mmol) was slowly added thereto at 0 ^°C . After 3 minutes, methanesulfonyl chloride (0.04 ml, 0.34 mmol) was added thereto, and reacted at the same time for 10 minutes and then at room temperature for 6 hours. The reactant was diluted with water (20 ml) and extracted three times with ethyl acetate (50 ml). The residue was purified by column chromatography (hexane: ethyl acetate = 1:3) to obtain the desired compound (6) as red solid in a yield of 94% (80 mg, 0.16 mmol). Preparation of S-ri-G-dimethylaminopropyD-S-methoxy-iH-indol-S-ylM-QH-indol-S- yl)pyrrol-2.5-dione (7)

Said compound (6) (80 mg, 0.168 mmol) and 2M dimethylamine (2.0 ml) were dissolved in DMF (4 ml), and warmed to reflux at 80 ^°C for 13 hours. The reacting mixture was diluted with water (10 ml), and extracted three times with ethyl acetate (30 ml), followed by removing ethyl acetate, and washed three times with hexane. Then, the desired compound (7) as red solid was obtained by recrystallization using ethyl acetate (3 ml), hexane (5 ml) and methanol (1 ml), in ayield of 88% (65 mg, 0.147 mmol).

MS (ESI+); 433 [M+H]⁺

MP; 208^°C ~210^°C

¹H NMR(400 MHz); DMSO-J₆: δ = 11.61(1H, s), 1O.84(1H, s), 7.8O(1H, s), 7.67(1H, s), 7.35(1H, d, J=8.0 Hz), 7.3O(1H, d, J=8.8 Hz), 6.97(1H, t, J=7.2 Hz), 6.84(1H, d, J=4.0 Hz), 6.61(1H, t, J=7.6 Hz), 6.57-6.54(1H, m), 6.11(1H, s), 4.21(2H, t, J=6.4 Hz), 2.30(3H, s), 2.09(6H, s), 1.86-1.81(2H, m)

IR: 3301, 1753, 1708, 1663, 1622, 1532, 1487, 1454, 1434, 1393, 1336, 1091 cm^"1 <Example 3> Synthesis of 3-[l-(2-hydroxyethyl)-5-methoxy-iH-indoI-3-yl]-4-(7H- indol-3-yl)-pyrrol-2,5-dione (11)

The mixture of the compound (10) (450 mg, 1.62 mniol) and indol-3-ylacetamide (380 mg, 2.18 mmol) was dissolved in THF (15 ml), and potassium t-butoxide (4.8 ml) was slowly added thereto at 0 ^°C . The mixture was reacted at the same temperature for 10 minutes, followed by increasing again the temperature to room temperature, and reacted for 12 hours. Then, 2M hydrochloric acid (5 ml) was added thereto and the mixture was reacted for 1 hour to complete the reaction. The reactant was diluted with water (30 ml), and extracted three times with ethyl acetate (50 ml). Ethyl acetate was removed, and the residue was purified by column chromatography (hexane: ethyl acetate = 1 :2) to obtain the desired compound (11) as red solid in a yield of 58% (380 mg, 0.95 mmol).

MS (ESI+); 424 [M+Na]⁺

MP; 241 - 243 ^°C

¹H NMR(400 MPIz); DMSO^: δ = 11.58(1H, s), 1O.86(1H, s), 7.9O(1H, s), 7.6O(1H, s), 7.37(1H, d, J=8.0 Hz), 7.3O(1H, d, J=8.8 Hz), 7.02-6.97(2H, m), 6.7O(1H, t, J=7.6 Hz), 6.57- 6.54(1H, m), 6.06(1H, s), 4.23(2H, t, J=6.8 Hz), 3.71-3.70(2H, m), 2.94(3H, s)

IR; 3497, 2924, 2856, 1655, 1499, 1440, 1405, 1258, 1095 cm^"1 <Example 4> Synthesis of 3-[l-(2-dimethylaminoethyl)-5-methoxy-7H-indol-3-yl]- 4-(iJΪ-indol-3-yl)pyrrol-2^-dione (13)

The compound (12) (50 mg, 0.104 mmol) and 2M dimethylamine (2.0 ml) were dissolved in DMF (3 ml), and the mixture was warmed to reflux at 80 ^°C for 10 hours. The reactant was diluted with water (10 ml), and extracted three times with ethyl acetate (30 ml). Then, ethyl acetate was removed from the reactant, followed by washing it three times with hexane, and the desired compound (13) as red solid was obtained by recrystallization using ethyl acetate (3 ml), hexane (5 ml) and methanol (1 ml), in a yield of 94% (42 mg, 0.098 mmol).

MS(ESI+);429[M+H]⁺

MP;230-232°C

'HNMR(400MHZ);DMS(W₆: δ= 11.6O(1H,s), 10.85(1H, s),7.93(1H,s),7.65(1H,s), 7.35(1H, d, J=8.0 Hz), 7.3O(1H, d, J=8.8 Hz), 6.97(1H, t, J=7.2 Hz), 6.87(1H, d, J=4.0 Hz), 6.2O(1H,t,J=7.6Hz), 6.57-6.54(1H,m),6.11(1H,s),4.27(2H,t,J=6.4Hz),3.02(3H,s),2.57(2H,t, J=6.4Hz),2.21(6H,s)

IR; 3477, 1757, 1716, 1663, 1438, 1409, 1389, 1336, 1258, 1091, 1054cm-¹ <Example 5> Synthesis of 3-[l-(3-hydroxypropyl)-5-methoxy-7H-indol-3-yl]-4- (/#-indol-3-yl)-l-methylpyrrol-2,5-dione (9)

The compound (5) (50 mg, 0.120 mmol) prepared in Example 1, dimethyl sulfate (0.013 ml) and potassium carbonate (32 mg, 0.232 mmol) were dissolved in acetone (3 ml), and the mixture was warmed to reflux at 60 ^°C for 8 hours. The reactant was diluted with water (10 ml), and extracted three times with ethyl acetate (20 ml), followed by removing ethyl acetate, and the residue was purified by column chromatography (hexane: ethyl acetate = 1 :3) to obtain the desired compound (9) as red solid in a yield of 95% (49 mg, 0.114 mmol).

MS (ESI+); 452 [M+Na]⁺

MP; 115 ~ 117^°C

'HNMR(400 MHZ); CD₃CN : δ = 9.75(1H, s), 7.84(1H, s), 7.71(1H, s), 7.4O(1H, d, J=8.0 Hz), 7.25(1H, d, J=8.8 Hz), 7.01(1H, t, J=7.2 Hz), 6.91(1H, d, J=4.0 Hz), 6.66(1H, t, J=7.6 Hz), 6.62-6.59(1H, m), 6.2O(1H, s), 4.21(2H, t, J=6.4 Hz), 3.50-3.46(2H, m), 3.08(6H, s), 2.01- 1.98(2H, m)

IR; 3452,2929,2864,2803, 1654, 1495, 1434, 1413, 1389, 1254, 1127, 1090, 1054cm^"1 <Example 6> Synthesis of 3-[(lA}mzyl-lH-[l^]iήsaol-4~yϊ)methy\)-5-methoxy- iH-indol-3-yl]-4-(7//-indol-3-yl)-pyrrol-2,5-dione (20)

Synthesis of l-q-benzyl-7H-ri^J11riazol-4-melhylV5-methoxy-iH-indol (IS)

To DMF (16 ml) was added NaH (160 mg, 4.07 mmol), and 5-methoxy-indol (1) (500 mg, 3.40 mmol) was slowly added thereto at 0^°C, and the mixture was reacted at the same temperature for 5 minutes. Then, propargyl bromide (0.35 ml, 4.08 mmol) was slowly added thereto, and tetrabutylammonium iodide (10 mg) was directly added, and the mixture was reacted for 10 minutes and warmed to reflux at 70 ^°C for 8 hours. The reactant was neutralized with ammonium chloride (5 ml), diluted with water (40 ml) and extracted three times with ethyl acetate (100 ml). The residue was purified by column chromatography (hexane: ethyl acetate = 10:1) to obtain the desired compound (17) as colorless oil in a yield of 79% (500 mg, 2.70 mmol).

Said compound (17) (230 mg, 1.24 mmol) and benzyl azide (250 mg, 1.87 mmol) were dissolved in THF (10 ml), and copper sulfoxide (80 mg, 0.32 mmol) and sodium ascorbate (370 mg, 1.87 mmol) were added thereto, and then the mixture was reacted, with slowly adding water (3 ml). The reaction was maintained at room temperature for 8 hours, and the reactant was neutralized with ammonium chloride (5 ml), diluted with water (30 ml) and extracted three times with ethyl acetate (100 ml). The residue was purified by column chromatography (hexane: methylene chloride = 1:1) to obtain the desired compound (18) as colorless oil in a yield of 96% (380 mg, 1.19 mmol). Synthesis of ri-(l-benzyl-7//-ri,2.31triazol-4-ylmethyl)-5-methoxy-i/j-indol-3-yll- oxoacetic acid methyl ester (19)

The compound (18) (700 mg, 2.20 mmol) prepared above was added to dichloromethane (20 ml) and oxalyl chloride (0.23 ml, 2.64 mmol) was slowly added thereto at 0°C, and the mixture was reacted for 1 hour. Then, the temperature of the reaction vessel was decreased to -65 ^°C, and sodium methoxide (100 mg of sodium were added to 2.5 ml of methanol) was slowly added thereto, with maintaining the temperature, and the mixture was reacted for 1 hour. Then, the mixture was again reacted at room temperature for 1 hour to complete the reaction. The reactant was extracted three times with methylene chloride (80 ml). Dichloromethane was removed and the residue was purified by column chromatography (hexane: methylene chloride = 1:1) to obtain the desired compound (19) as yellow solid in a yield of 95% (850 mg, 2.10 mmol).

Synthesis of 3-rd-benzyl-iH-rL2,31tria2ol-4-yl)methyl)-5-methoxy-iH-indol-3-yl14- (7H-indol-3-yl)-pyrrol-2,5-dione (20)

The mixture of the compound (19) (500 mg, 1.24 mmol) prepared above and indol-3- ylacetamide (250 mg, 1.43 mmol) was dissolved in THF (15 ml) and potassium t-butoxide (6.0 ml) was slowly added thereto at 0^°C. The mixture was maintained at the same temperature for 10 minutes and reacted again at room temperature for 13 hours. Then, 2M hydrochloric acid (8 ml) was added thereto and the mixture was further reacted for 1 hour. The reactant was diluted with water (30 ml), and extracted three times with ethyl acetate (50 ml), and the residue was purified by column chromatography(hexane: ethyl acetate = 1:2) to obtain the desired compound (20) as red solid in a yield of 46% (300 mg, 0.56 mmol).

MS (ESI+); 551 |M+Na]⁺

MP; 280 - 282 ^°C

¹H NMR(400 MHz); DMSO- d₆: δ = 11.60(1H, s), 1O.87(1H, s), 8.05(1H, s), 7.95(1H, s), 7.62(1H, s), 7.40-7.25(7H, m), 6.95(1H, t, J=7.6 Hz), 6.88(1H, d, J=8.4 Hz), 6.58-6.54(2H, m), 6.05(1H, s), 5.56(2H, s), 5.50(2H, s), 2.94(3H, s)

IR; 3532, 1720, 1650, 1455, 1123, 1030, 1015 cm^"1

<Example 7> Synthesis of (4-{3-[4-(7//-indol-3-yl)-2,5-dioxy-2,5-dihydro-7H- pyrrol-3-yl]-5-methoxy-iH-indol-l-ylmethyl}-[1^3]triazol-l-yl)acetic acid (23)

Synthesis of [4-(5-methoxyindol-l-ylmethyl)-[l,2,3]triazol-l-yll-acetic acid ethyl ester Oi)

The mixture of the compound (17) (230 mg, 1.24 mmol) and azide acetic acid ethyl ester (200 mg, 1.55 mmol) was dissolved in THF (10 ml) and copper sulfoxide (80 mg, 0.32 mmol) and sodium ascorbate (370 mg, 1.87 mmol) were added thereto, and the mixture was reacted, with slowly adding water (3 ml). The reaction was maintained at room temperature for 8 hours, and the reactant was neutralized with ammonium chloride (5 ml). The reactant was diluted with water (30 ml), extracted three times with ethyl acetate (100 ml), and purified by column chromatography (hexane: methylene chloride = 1 : 1) to obtain the desired compound (21) as colorless oil in a yield of 97% (380 mg, 1.21 mmol).

Synthesis of [l-(l-ethoxycarbonylmethyl-iH-[l,2₁3]triazol-4-ylmethyl)-5-methoxy-iH- indol-3-yll-oxoacetic acid methyl ester (22)

The compound (21) (1.0 g, 3.18 mmol) prepared above was added to dichloromethane (20 ml), and oxalyl chloride (0.27 ml, 3.18 mmol) was slowly added thereto at 0^°C, and the mixture was reacted at the same temperature for 1 hour. Then, the temperature of the reaction vessel was decreased to -65 ^°C and sodium methoxide (100 mg of sodium was added to 2.5 ml of methanol) was slowly added thereto, and the mixture was reacted for 1 hour. The mixture was again reacted at room temperature for 1 hour and the reaction was completed, and the reactant was extracted three times with methylene chloride (80 ml). The residue was purified by column chromatography (hexane: methylene chloride = 1 : 1) to obtain the desired compound (22) as yellow solid in ayield of 86% (1.10 g, 2.75 mmol).

Synthesis of (4-{3-r4-(iH-indol-3-yl)-2,5-dioxy-2,5-dihvdro-iH-pyrrol-3-yl1-5- methoxy-iH-indol-l-ylmethvU-ri^^ltriazol-l-vDacetic acid (23)

The mixture of the compound (22) (500 mg, 1.24 mmol) prepared above and indol-3- ylacetamide (250 mg, 1.43 mmol) was dissolved in TΗF (15 ml) and potassium t-butoxide (6.0 ml) was slowly added thereto at 0^°C. The mixture was maintained at the same temperature for 10 minutes, and reacted at room temperature for 13 hours. Over time, 2M hydrochloric acid (8 ml) was added thereto, and the mixture was further reacted for 1 hour to complete the reaction. The reactant was diluted with water (30 ml) and extracted three times with ethyl acetate (50 ml). The residue was purified by column chromatography (methylene chloride: ethyl acetate = 1 :2) to obtain the desired compound (23) as red solid in a yield of 41% (230 mg, 0.503 mmol).

MS (ESI+); 519 [M+Na]⁺

MP; 180- 182 ^°C

¹H NMR(400 MHz); DMSO-J₆: δ = 11.60(1H, s), 1O.88(1H, s), 8.00(1H, s), 7.61(1H, s), 7.42(1H, d, J=9.2 Hz), 7.35(1H, d, J=8.0 Hz), 6.98(1H, t, J=7.6 Hz), 6.93(1H, d, J=8.0 Hz), 6.65(1H, t, J=7.6 Hz), 6.57-6.55(1H, m), 6.05(1H, s), 5.54(2H, s), 5.24(2H, s), 2.94(3H, s)

IR; 3530, 1716, 1646, 1450, 1189, 1119, 1091, 1049, 1017 cm-¹

<ExampIe 8> Synthesis of 3-[l-(2-dimethylaminoethyl)-5-methoxy-i//-indol-3-yl]- 4-(iH-indol-3-yl)-l-methylpyrrol-2,5-dione (16)

The compound (13) (30 mg, 0.070 mmol), dimethyl sulfate (0.008 ml) and potassium carbonate (20 mg, 0.145 mmol) were dissolved in acetone (3 ml) and the mixture was warmed to reflux at 60 °C for 8 hours. The reactant was diluted with water (10 ml) and extracted three times with ethyl acetate (20 ml), and the residue was purified by column chromatography (hexane: ethyl acetate = 1:3) to obtain the desired compound (16 ) as red solide in a yield of 97% (30 mg, 0.068 mmol).

MS (ESI+); 443 [M+H]⁺ MP; 141 - 143 ^°C

¹H NMR(400 MHz); CD₃OD : δ = 7.72(1H, s), 7.52(1H, s), 7.34(1H, d, J=8.0 Hz), 7.25(1H, d, J=8.8 Hz), 6.97(1H, t, J=7.2 Hz), 6.9O(1H, d, J=4.0 Hz), 6.66(2H, m), 6.21(1H, s), 4.58(2H, t, J=6.4 Hz), 3.59 (2H, t, J=6A Hz), 3.05(12H, s)

IR; 1634, 1462, 1140, 1123, 1091 cm ¹

<Example 9> Synthesis of 3-[l-(2-hydro^ethyl)-5-methoxy-7H-indol-3-yl]-4-(iH- indol-3-yl)-l-methylpyrrol-2,5-dione (15)

The compound (11) (20 mg, 0.050 mmol), dimethyl sulfate (0.005 ml) and potassium carbonate (14 mg, 0.101 mmol) were dissolved in acetone (3 ml), and the mixture was warmed to reflux at 60 ^°C for 8 hours. The reactant was diluted with water (10 ml) and extracted three times with ethyl acetate (20 ml), and the residue was purified by column chromatography (hexane: ethyl acetate = 1:3) to obtain the desired compound (15) as red solid in a yield of 96% (20 mg, 0.048 mmol).

MS (ESI+); 438 [M+Na]⁺

MP; 117 ~ 119^°C

¹H NMR(400 MHz); CD₃CN : δ = 9.75(1H, s), 7.90QH, s), 7.67(1H, s), 7.41(1H, d, J-8.0 Hz), 7.25(1H, d, J=8.8 Hz), 7.04(1H, t, J=7.2 Hz), 6.91(1H, d, J=4.0 Hz), 6.66(1H, t, J=7.6 Hz), 6.62-6.59(1H, m), 6.2O(1H, s), 4.26(2H, t, J=6.4 Hz), 3.85-3.81(2H, m), 3.07(6H, s) IR; 1687, 1622, 1540, 1479, 1442, 1152, 1123, 1095 cm^"1

<Example 10> Synthesis of 2-(2-{3-[4-(iH-indol-3-yl)-2,5-dioxo-2,5-dihydro-iH- pyrrol-3-yl]-5-methoxyindol-l-yl}-ethyl)isothiourea (14)

Synthesis of methanesulfonic acid 2-{3-[4-(7H-indol-3-yl)-2,5-dioxo-2,5-dihvdro-iH- pyrrol-3-yll-5-methoxy-indol-l-yl}ethyl ester (12)

The compound (11) (170 mg, 0.42 mmol) was dissolved in DMF (4 ml) and triethylamine(0.07 ml, 0.51 mmol) w as s lowly added t hereto at O ^°C, and after 3 m inutes, methanesulfonyl chloride (0.04 ml, 0.34 mmol) was added thereto. The mixture was reacted at the same temperature for 10 minutes and again at room temperature for 6 hours. The reactant was diluted with water (20 ml) and extracted three times with ethyl acetate (50 ml), and the residue was purified by column chromatography (hexane: ethyl acetate = 1 :3) to obtain the desired compound (12) as red solid in a yield of 94% (180 mg, 0.37 mmol).

Synthesis of 2-(2-{3-r4-fi//-indol-3-ylV2,5-dioxo-2,5-dihydro-i//-pyrrol-3-yll-5- methoxyindol- 1 -yl 1 -ethvDisothiourea (14)

The compound (12) (120 mg, 0.25 mmol) prepared above and thiourea (38 mg) were dissolved in DMF (3 ml), and the mixture was warmed to reflux at 80 ^°C for 10 hours. The reactant was diluted with water (15 ml) and extracted twice with ethyl acetate (20 ml) to remove materials present in organic layers. Then, water was removed from the reactant, followed by washing it three times with ethyl acetate, and the desired compound (14) as red solid was obtained by recrystallization using ethyl acetate (3 ml), hexane (5 ml) and methanol (1 ml), in a yield of 96% (110 mg, 0.24 mmol).

MS (ESI+); 460 [M+H]⁺

MP; 180 - 182 ^°C

¹H NMR(400 MHz); CD₃OD : δ = 7.8O(1H, s), 7.67(1H, s), 7.34(1H, d, J=8.0 Hz), 7.24(1H, d, J=8.8 Hz), 7.01(1H, t, J=7.2 Hz), 6.93(1H, d, J=4.0 Hz), 6.67-6.60(2H, m), 6.17(1H, s), 4.52(2H, t, J=6.4 Hz), 3.60(2H, t, J=6.8 Hz), 3.01(3H, s)

IR; 1703, 1659, 1528, 1483, 1193, 1140, 1128, 1091, 1061 cm^"1

<Example 11> Synthesis of Z-CS-IS-^-Cliϊ-indol-S-yl^^-dioxo-l^-dihydro-iF- pyrrol-3-yl]-5-methoxyindol-l-yl}propyl)isothiourea (8)

The compound (6) (68 mg, 0.137 mmol) and thiourea (10 mg) were dissolved in DMF (3 ml) and the mixture was warmed to reflux at 80 ^°C for 12 hours. The reactant was diluted with water (15 ml), and extracted twice with ethyl acetate (20 ml) to remove materials present in organic layers. Then, water was removed from the reactants, followed by washing it three times with ethyl acetate, and the desired compound (8) as red solid was obtained by recrystallization using ethyl acetate (3 ml), hexane (5 ml) and methanol (1 ml), in a yield of 92% (60 mg, 0.126 mmol).

MS (ESI+); 474 [M+H]⁺

MP; 230 - 232 ^°C

¹H NMR(400 MHz); CD₃OD : δ = 7.74QH, s), 7.7O(1H, s), 7.35(1H, d, J=8.0 Hz), 7.24(1H, d, J=8.8 Hz), 7.01(1H, t, J=7.2 Hz), 6.88QH, d, J=AJd Hz), 6.64-6.60(2H, m), 6.28(1H, s), 4.32(2H, t, J=6.4 Hz), 3.08(3H, s), 3.02(2H, t, J=6.8 Hz), 2.22-2.19(2H, m)

IR: 1654, 1622, 1479, 1148, 1095, 1119 cm^'1

<Example 12> Synthesis of 3-[l-(3-hydroxypropyl)-lH-indol-3-yl]-4-[l-(3- hydroxypropyl)-5-methoxy-2i^:/-indol-3-yl]-pyrrol-2,5-dione (31)

The mixture of the compound (4) (500 mg, 1.44 mmol) prepared in Example 1 and 2-[l- (3-hydroxypropyl)-7H-indol-3-yl]-acetarnide (800 mg, 3.45 mmol) was dissolved in TΗF (20 ml) and potassium t-butoxide (10.0 ml) was slowly added thereto at 0^°C. Then, the mixture was maintained at the same temperature for 10 minutes, and then reacted at room temperature for 12 hours. 2M hydrochloric acid (5 ml) was added thereto, and the mixture was reacted for 1 hour to complete the reaction. The reactant was diluted with water (30 ml) and extracted three times with ethyl acetate (50 ml), and the residue was purified by column chromatography (hexane: ethyl acetate = 1:3) to obtain the desired compound (31) as red solid in a yield of 44% (300 mg, 0.64 mmol).

MS (ESI+); 496 [M+Na]⁺

MP; 190 - 192 ^"C

¹H NMR(400 MHz); CD₃OD : δ = 7.76(1H, s), 7.56(1H, s), 7.35(1H, d, J=8.0 Hz), 7.21(1H, d, J=8.8 Hz), 7.03(1H, t, J=7.2 Hz), 6.97(1H, d, J=8.0 Hz), 6.68(1H, t, J=7.2 Hz), 6.11(1H, s), 4.25-4.19(4H, m), 3.54-3.47(4H, m), 2.00-1.89(4H, m)

IR; 3351, 1708, 1659, 1618, 1532, 1471, 1140, 1123, 1095 cm^"1

<ExampIe 13> Synthesis of 3-[l-(3-dimethylaminopropyl)-7H-indol-3-yl]-4-[l-(3- dimethylaminopropyl)-5-methoxy-7//-indol-3-yl]-pyrrol-2,5-dione (33)

Synthesis of methanesulfonic acid 3-(3-{4-ri-(methanesulfonyloxy-propyl)--/H-indol-3- yll-2,5-dioxo-2,5-dihydro-i//-pyrrol-3-yl}-5-methoxy-indol-l-yl)propyl ester (32)

The compound (31) (250 mg, 0.53 mmol) prepared in Example 12 was dissolved in DMF (6.0 ml), triethylamine (0.30 ml, 2.12 mmol) was slowly added thereto at 0^°C, and after 3 minutes, methanesulfonyl chloride (0.16 ml, 2.12 mmol) was added thereto. The mixture was maintained at the same temperature for 10 minutes, and further reacted at room temperature for 6 hours. The reactant was diluted with water (20 ml) and extracted three times with ethyl acetate (50 ml). Ethyl acetate was removed from the reactant and the residue was purified by column chromatography (hexane: ethyl acetate = 1 :3) to obtain the desired compound (32) as red solid in a yield of 81% (270 mg, 0.43 mmol).

Synthesis of 3-ri-(3-dimethylaminopropyl)-7//-indol-3-yll-4-ri-(3- dimethylammopropyl)-5-methoxy-7/j-indol-3-yll-pyrrol-2,5-dione (33)

The compound (32) (150 mg, 0.238 mmol) prepared above and 2M dimethylamine (1.5 ml) were dissolved in DMF (4.0 ml) and the mixture was warmed to reflux at 80 ^°C for 13 hours. The reactant was diluted with water (10 ml) and extracted three times with ethyl acetate (30 ml), followed by removing ethyl acetate and washing it three times with hexane. Then, the desired compound (33) as red solid was obtained by recrystallization using ethyl acetate (3 ml), hexane (5 ml) and metiianol (1 ml), in a yield of 88% (110 mg, 0.208 mmol).

MS (ESl+); 550 [M+Na]⁺

MP; 182 - 184 ^°C

'H NMR(400 MHZ); OMSOd₆: δ = 1O.87(1H, s), 7.82(1H, s), 7.63(1H, s), 7.44(1H, d, J=8.0 Hz), 7.31(1H, d, J=8.8 Hz), 7.04(1H, t, J=7.6 Hz), 6.93(1H, d, J=8.0 Hz), 6.68(1H, t, J=7.6 Hz), 6.59-6.56(1H, m), 6.06(1H, s), 4.21-4.17(4H, m), 2.94(3H, s), 2.09-205(16H, m), 1.85- 1.74(4H, m)

IR; 3399, 1659, 1054, 1033, 1013 cm^'1

<Example 14> Synthesis of 2-[3-(3-{4-[l-(3-carbamimidolysulfanylpropyl)-lH- indol-3-yl]-2,5-dioxo-2,5-dihydro-lH-pyrrol-3-yl}-5-methoxyindol-l-yl)-propyl]-isothiourea (34)

The compound (32) (100 mg, 0.159 mmol) prepared in Example 13 and thiourea (48 mg) were dissolved in DMF (3.5 ml), and the mixture was warmed to reflux at 80 ^°C for 13 hours. The reactant was diluted with water (15 ml), and extracted twice with ethyl acetate (20 ml) to remove materials present in organic layers. Then, water was removed from the reactant, followed by washing it three times with ethyl acetate, and the desired compound (34) as red solid was obtained by recrystallization using ethyl acetate (3 ml), hexane (5 ml) and methanol (1 ml), in a yield of 91% (85 mg, 0.144 mmol).

MS (ESI+); 590 [M+H]⁺

MP; 120 - 122 ^°C

¹H NMR(400 MHz); CD₃OD : δ = 7.82(1H, s), 7.57(1H, s), 7.45(1H, d, J=8.4 Hz), 7.31(1H, d, J=8.8 Hz), 7.12-7.08(2H, m), 6.75(1H, t, J-7.2 Hz), 6.68-6.65(1H, m), 6.12(1H, s), 4.38-4.31(4H, m), 3.10-2.98(7H, s), 2.26-2.23(2H, m), 2.16-2.13(2H, m)

IR; 3211, 1761, 1712, 1650, 1532, 1487, 1442, 1397, 1344, 1189, 1054cm^"1

<Example 15> Synthesis of3-[l-(4-hydroxybutyl)-5-methoxy-i//-indol-3-yl]-4-(iH- indol-3-yl)-pyrrol-2,5-dione(27)

Synthesis of acetic acid 4-(5-methoxyindol-l-yl)-butyl ester (24)

To DMF (30 ml) was added NaH (600 mg, 15.29 mmol), and then 5-methoxy indol (1.5 g, 10.19 mmol) was slowly added thereto at 0^°C , and the mixture was reacted for 5 minutes, with maintaining the temperature. To the reactant was slowly added 4-bromobutyl acetate (1.90 ml, 13.25 mmol) and tetrabutylammonium iodide (10 mg) was directly added thereto, and title mixture was reacted for 10 minutes, and then further reacted at room temperature for 2 hours. The reactant was neutralized with ammonium chloride (6.0 ml), diluted with water (40 ml) and extracted three times with ethyl acetate (100 ml). Ethyl acetate was removed from the reactant and the residue was purified by column chromatography (hexane: ethyl acetate = 10:1) to obtain the desired compound (24) as green oil in a yield of 93% (2.5 g, 9.59 mmol).

Synthesis of ri-^-acetoxybutvD-S-methoxy-./H-indol-S-vli-oxoacetic acid methyl ester

05}

The compound (24) (2.50 g, 9.57 mmol) prepared above was added to dichloromethane (20 ml) and oxalyl chloride (1.0 ml, 11.48 mmol) was slowly added thereto at 0 ^°C , and the mixture was reacted for 1 hour, with maintaining the temperature. Then, the temperature of the reaction vessel was decreased to -65 ^°C and sodium methoxide (150 mg of sodium was added to 4.0 ml of methanol) was slowly added thereto to react for 1 hour and then at room temperature for further 1 hour. The reactant was extracted three times with methylene chloride (80 ml), and dichloromethane was removed from the reactant. The residue was purified by column chromatography (hexane: ethyl acetate = 1 :1) to obtain the desired compound (25) as yellow oil in a yield of 54% (1.80 g, 5.18 mmol). In the above reaction, the compound (26) as yellow oil was further formed in a yield of 41% (1.20 g, 3.93 mmol).

Synthesis of 3-ri-(4-hvdroxybutyl)-5-methoxy-iH-indol-3-yll-4-(iH-indol-3-yl)-pyrrol-

2.5-dione (27)

The mixture of the compound (25) (500 mg, 1.44 mmol) prepared above and indol-3- ylacetamide (300 mg, 1.72 mmol) was dissolved in THF (15 ml) and potassium t-butoxide (4.3 ml) was slowly added thereto at 0^°C. The mixture was maintained at the same temperature for 10 minutes and reacted at room temperature for 12 hours. Then, 2M hydrochloric acid (5 ml) was added thereto, and the mixture was reacted for further 1 hour. The reactant was diluted with water (30 ml) and extracted three times with ethyl acetate (50 ml), and the residue was purified by column chromatography (hexane: ethyl acetate = 1:3) to obtain the desired compound (27) as red solid in a yield of 74% (460 mg, 1.07 mmol).

MS (ESI+); 452 [M+Na]⁺

MP; 220 - 222 ^°C

¹H NMR(400 MHz); DMSO-J₆: δ - 11.61(1H, s), 1O.87(1H, s), 7.85(1H, s), 7.68(1H, s), 7.37(1H, d, J=4.0 Hz), 7.3O(1H, d, J=8.8 Hz), 6.98(1H, t, J=7.6 Hz), 6.9O(1H, d, J=8.0 Hz), 6.65(1H, t, 7.6 Hz), 6.12-6.16(1H, m), 4.20(2H, t, J=6.4 Hz), 3.40(2H, s), 1.77(2H, t, J=6.4 Hz), 1.38-1.39(2H, m)

IR; 3412, 1712, 1659, 1532, 1487, 1340, 1205, 1054, 1021^', 1013 cm^"1

<Example 16> Synthesis of 2-(4-{3-[4-(iH-indol-3-yl)-2,5-dioxo-2^-dihydro-iH- pyrrol-3-yl]-5-methoxyindol-l-yl}butyl)isothiourea (30)

Synthesis of methanesulfonic acid 4-{3-[4-(7ij-indol-3-yl)-2,5-dioxo-2,5-dihydro-7//- pyrrol-3-yll-5-methoxy-indol-l-vU-butyl ester (28)

The compound (27) (800 mg, 1.86 mmol) prepared in Example 15 was dissolved in DMF (10 ml), triethylamine (0.50 ml, 3.72 mmol) was slowly added thereto at 0^°C, and after 3 minutes, methanesulfonyl chloride (0.29 ml, 3.72 mmol) was added thereto. The mixture was reacted at the same temperature for 10 minutes, and then at room temperature for further 6 hours. The reactant was diluted with water (20 ml) and extracted three times with ethyl acetate (50 ml). Then, ethyl acetate was removed from the reactant and the residue was purified by column chromatography (hexane: ethyl acetate = 1 :3) to obtain the desired compound (28) as red solid in a yield of 95% (900 mg, 1.77 mmol).

Synthesis of 2-(4-{3-r4-(iH-indol-3-yl)-2,5-dioxo-2,5-dihvdro-iH-pyrrol-3-yll-5- methoxyindoΗ-yl}buM)isothiourea(30)

The compound (28) (500 mg, 1.03 mmol) prepared above and thiourea (140 mg) were dissolved in DMF (6.0 ml), and the mixture was warmed to reflux at 80 ^°C for 10 hours. The reactant was diluted with water (15 ml) and extracted twice with ethyl acetate (20 ml) to remove materials present in organic layers. Then, water was removed from the reactant, followed by washing it three times with ethyl acetate, and the desired compound (30) as red solid was obtained by recrystallization using ethyl acetate (3 ml), hexane (5 ml) and methanol (1 ml), in a yield of 92% (460 mg, 0.94 mmol). MS (ESI+); 488 [M+H]⁺

MP; 182- 184 ^°C

¹H NMR(400 MHz); CD₃OD : δ = 7.78(1H, s), 7.68(1H, s), 7.35(1H, d, J=8.0 Hz), 7.21(1H, d, J=8.8 Hz), 6.99(1H, t, J=7.6 Hz), 6.89(1H, d, J=4.0 Hz), 6.64-6.58(2H, m), 6.2O(1H, s), 4.20(2H, t, J=6.8 Hz), 3.10(2H, t, J=7.2 Hz), 3.04(3H, s), 1.95-1.93(2H, m), 1.67-1.63(2H, m)

IR; 3387, 3285,3179, 1699, 1650, 1605, 1528, 1475, 1417, 1336, 1193, 1091, 1058cm^"1

<Example 17> Synthesis of3-[l-(4-dimethylaminobutyl)-5-methoxy-iH-indol-3-yl]- 4-(iH-indol-3-yl)pyrrol-2,5-dione(29)

The compound (28) (400 mg, 0.79 mmol) prepared in Example 16 and 2M dimethylamine (0.8 ml) were dissolved in DMF (6 ml), and the mixture was warmed to reflux at 80 ^°C for 12 hours. The reactant was diluted with water (10 ml) and extracted three times with ethyl acetate (30 ml), followed by removing ethyl acetate and washing it three times with hexane. Then, the desired compound (29) as red solid was obtained by recrystallization using ethyl acetate (3 ml), hexane (5 ml) and methanol (1 ml), in a yield of 94% (340 mg, 0.74 mmol).

MS (ESI+); 457 [M+H]⁺

MP; 100 - 102 ^°C ¹H NMR(400 MHz); CD₃OD : δ = 7.78(1H, s), 7.63(1H, s), 7.33(1H, d, J=4.4 Hz), 7.18(1H, d, J=8.8 Hz), 6.99(1H, t, J=7.6 Hz), 6.94(1H, d, J=4.0 Hz), 6.63(1H, t, J=7.6 Hz), 6.59- 6.6O(1H, m), 6.18(1H, s), 4.15-4.12(2H, m), 3.01(3H, s), 2.28(2H₅1, J=7.2 Hz), 1.80-1.76(2H, m), 1.47-1.44(2H, m)

IR; 3305, 2946, 1761, 1699, 1659, 1614, 1528, 1479, 1446, 1332, 1134, 1123, 1086 cm^"1

<Example 18> Synthesis of 3,4-bis[l-(3-dimethylaminopropyl)-5-methoxy-7H- indol-3-yl]pyrroI-2,5-dione (40)

Synthesis of 2-ri-(3-hvdroxypropyl)-5-methoxy-i//-indol-3-yl]acetamide (37)

36 37

In DMF (20 ml) was dissolved NaH (130 mg, 3.22 mmol), 5-methoxyindol-3- ylacetonitrile (500 mg, 2.685 mmol) (35) was slowly added thereto at 0^°C, and the mixture was reacted for 5 minutes. Then, 3-bromopropanol (0.35 ml, 4.03 mmol) was slowly added thereto, and the mixture was reacted at the same temperature for 10 minutes and warmed to reflux at 70 ^°C for 4 hours. The reactant was neutralized with ammonium chloride (5 ml), diluted with water (40 ml) and extracted three times with ethyl acetate (100 ml). Ethyl acetate was removed from the reactant and the residue was purified by column chromatography (hexane: ethyl acetate = 1:1) to obtain the desired compound (36) as colorless oil in a yield of 76% (500 mg, 2.046 mmol).

The mixture of the prepared compound (36) (500 mg, 2.046 mmol) and potassium hydroxide (800 mg, 14.26 mmol) was dissolved in tert-butylalcohol (7.0 ml) and warmed to reflux at 85 °C for 2 hours. Then, 2M hydrochloric acid (6.0 ml) was added thereto, and the reactant was diluted water (20 ml) and extracted three times with ethyl acetate (50 ml). Ethyl acetate was removed from the reactant and the residue was purified by column chromatography (hexane: ethyl acetate = 1 :3) to obtain the desired compound (37) as oil in a yield of 84% (450 mg, 1.716 mmol).

Synthesis of 3,4-bis[l-(3-hydroxypropyl)-5-methoxy-7//-indol-3-yllpyrrol-2,5-dione (38)

to RT

The mixture of the prepared compound (37) (500 mg, 1.91 mmol) and the compound (4) (400 mg, 1.37 mmol) prepared in Example 1 was dissolved in THF (20 ml), and potassium t- butoxide (4.1 ml) was slowly added thereto at 0^°C. The mixture was maintained at the same temperature for 10 minutes and reacted at room temperature for 12 hours. Then, 2M hydrochloric acid (5 ml) was added thereto and the mixture was reacted for 1 hour. The reactant was diluted with water (30 ml) and extracted three times with ethyl acetate (50 ml), and the residue was purified by column chromatography (hexane: ethyl acetate = 1:3) to obtain the desired compound (38) as red solid in a yield of 42% (290 mg, 0.576 mmol).

Synthesis of methanesulfonic acid 3-(3-{4-[l-(3-methanesulfonyloxy-propyl)-5- methoxy-7H-indol-3-yll-2,5-dioxo-2,5-dihvdro-iH-pyrrol-3-vU-5-methoxy-indol-l-yl)-propyl ester (39)

The prepared compound (38) (160 mg, 0.318 mmol) was dissolved in DMF (6.0 ml) and triethylamine (0.17 ml, 1.272 mmol) was slowly added thereto at 0^°C, and after 3 minutes, methanesulfonyl chloride (0.10 ml, 1.272 mmol) was added thereto. Then, the mixture was maintained at the same temperature for 10 minutes and reacted at room temperature for further 6 hours. The reactant was diluted with water (20 ml) and extracted three times with ethyl acetate (50 ml). Ethyl acetate was removed from the reactant, and the residue was purified by column chromatography (hexane: ethyl acetate = 1 :3) to obtain the desired compound (39) as red solid in a yield of 85% (200 mg, 0.270 mmol).

Synthesis of 3,4-bisri-(3-dime1hylaminopropyl)-5-methoxy-i//-indol-3-yllpyrrol-2,5- dione (40)

The prepared compound (39) (40 mg, 0.054 mmol) and 2M dimethylamine (0.2 ml) were dissolved in DMF (3.0 ml), and the mixture was warmed to reflux at 80 ^°C for 12 hours, and the reactant was diluted with water (10 ml) and extracted three times with ethyl acetate (30 ml). Ethyl acetate was removed from the reactant, followed by washing it three times with hexane, and the desired compound (40) as red solid was obtained by recrystallization using ethyl acetate (3 ml), hexane (5 ml) and methanol (1 ml), in a yield of 99% (110 mg, 0.0538 mmol).

MS (ESI+); 558 [M+H]⁺

MP; 100 - 102 ^°C

¹H NMR(400 MHz);CD₃OD : δ = 7.68(1H, s), 7.22(1H, d, J=8.4 Hz), 6.65-6.62(1H, m), 6.31(1H, s), 4.12(2H, t,J=7.2 Hz), 3.30(3H, s), 2.26-2.22(2H, m), 2.17(6H, s), 1.92-1.88(2H, m) IR; 1708, 1622, 1487, 1454, 1193, 1144, 1127, 1091, 1054, 1025 cm^"1

<ExampIe 19> Synthesis of 3-[l-(4-hydroxybuiyl)-l//-indol-3-yl]-4-[l-(4- hydroxybutyl)-5-methoxy-iH-indol-3-yl]-pyrrol-2,5-dione (41)

The mixture of the compound (24) (800 mg, 2.30 mmol) prepared in Example 15 and indol-3-ylacetamide-N-butylacetate (620 mg, 2.15 mmol) was dissolved in THF (20 ml), and potassium t-butoxide (9.2 ml) was slowly added thereto at 0 ^°C . Then, the mixture was maintained at the same temperature for 10 minutes and reacted at room temperature for 13 hours. Then, 2M hydrochloric acid (5 ml) was added thereto, and the mixture was reacted for further 1 hour. The reactant was diluted with water (20 ml) and extracted three times with ethyl acetate (70 ml), and the residue was purified by column chromatography (hexane: ethyl acetate = 1 :4) to obtain the desired compound (41) as red solid in a yield of 61% (702 mg, 1.40 mmol).

MS (ESl+); 524 |M+Na]⁺

MP; 102 - 104 ^°C

¹H NMR (400 MHz); CD₃OD :δ= 7.24 (s, IH), 7.51 (s, IH), 7.31 (d, J= 8.0 Hz, IH), 7.16 (d, J= 8.8 Hz, IH), 7.02-6.96 (m, 2H), 6.65 (t, J= 8.0 Hz, IH), 6.56-6.54 (m, IH), 4.13-4.07 (m, 4H), 3.51-3.44 (m, 4H), 1.85-1.74 (m, 4H), 1.48-1.30 (m, 4H)

IR; 3305, 1699, 1650, 1622, 1471, 1197, 1144, 1119, 1086 cm^'1

<ExampIe 20> Synthesis of 2-[4-(3-{4-[l-(4-carbamimidobulfanylbutyl)-/H-indol-3- yl]-2^-dioxo-2,5-dihydro-/H-pyrrol-3-yl}-5-methoxyindol-l-yl)-butyl]-isothiourea (43)

The compound (41) (240 mg, 0.38 mmol) prepared in Example 19 and thiourea (117 mg) were dissolve in DMF (4.0 ml), and the mixture was warmed to reflux at 80 ^°C for 12 hours. Then, the reactant was diluted with water (15 ml), and extracted twice with ethyl acetate (20 ml) to remove materials present in organic layers. Then, water was removed from the reactant, followed by washing it three times with ethyl acetate, and the desired compound (43) as red solid was obtained by recrystallization using ethyl acetate (3 ml), hexane (5 ml) and methanol (1 ml), in a yield of 80% (210 mg, 0.34 mmol).

MS (ESI+); 618 [M+H]⁺

MP; 101 ~ 103 ^°C

¹H NMR (400 MHz); CD₃OD :δ= 7.76 (s, IH), 7.51 (s, IH), 7.36 (d, J= 8.4 Hz, IH), 7.22 (d, J= 8.8 Hz, IH), 7.05 (t, J= 7.2 Hz, IH), 6.99 (d, J= 8.0 Hz, IH), 6.69 (t, J= 7.2 Hz, IH), 6.62-6.59 (m, IH), 6.08 (s, IH), 4.16-4.09 (m, 4H), 3.09-3.01 (m, 4H), 2.95 (s, 3H), 1.90-1.79 (m, 4H), 1.64- 1.56 (m, 4H)

IR; 3305, 3187, 1699, 1642, 1524, 1393, 1340, 1193, 1050 cm^"1

<Example 21> Synthesis of 3-[l-(4-dimethylaminobutyl)-7//-indol-3-yI]-4-[l-(4- dimethylaminobutyl)-5-methoxy-/i/-indol-3-yl]-pviτol-2,5-dione (44)

The compound (41) (200 mg, 0.32 mmol) prepared in Example 19 and 2M dimethylamine (1.28 ml) were dissolved in DMF (6.0 ml), and the mixture was warmed to reflux at 80 ^°C for 12 hours. The reactant was diluted with water (10 ml) and extracted three times with ethyl acetate (30 ml), followed by removing ethyl acetate and washing it three times with hexane. Then, the desired compound (44) as red solid was obtained by recrystallization using ethyl acetate (3 ml), hexane (5 ml) and methanol (1 ml), in ayield of 80% (150 mg, 0.27 mmol).

MS (ESI+); 556 [M+H]⁺

MP; 78 - 80 ^°C

¹H NMR (400 MHz); CD₃OD :δ= 7.77 (s, IH), 7.54 (s, IH), 7.33 (d, J= 8.0 Hz, IH), 7.18 (d, J= 8.8 Hz, IH), 7.03-7.00 (m, 2H), 6.67 (t, J= 7.2 Hz, IH), 6.60-6.57 (m, IH), 6.12 (s, IH) 4.11-4.05 (m, 4H), 2.95 (s, 3H), 2.33-2.15 (m, 16H), 1.78-1.66 (m, 4H), 1.46-1.40 (m, 4H)

IR; 2933, 2766, 1708, 1618, 1528, 1462, 1332, 1135, 1119, 1091 cm^"1

<Example 22> Synthesis of 3-[l-(4-hydroxybutyl)-7//-indol-3-yl]-4-[l-(3- hydroxypropyl)-5-methoxy-/H^r-indol-3-yl]-pyrroI-2,5-dione (45)

The mixture of the compound (4) (300 mg, 1.03 mmol) prepared in Example 1 and indol- 3-ylacetamide-N-butylacetate (450 mg, 1.29 mmol) was dissolved in THF (20 ml), and potassium t-butoxide (6.0 ml) was slowly added thereto at 0^°C . The mixture was maintained at the same temperature for 10 minutes and reacted at room temperature for 12 hours. Then, 2M hydrochloric acid (8.0 ml) was added thereto, and the mixture was reacted for 1 hour to complete the reaction. The reactant was diluted with water (30 ml) and extracted ihree times with ethyl acetate (50 ml), and the residue was purified by column chromatography (hexane: ethyl acetate = 1 :4) to obtain the desired compound (45) as red solid in a yield of 44% (210 mg, 0.43 mmol).

MS (ESI+); 510 [M+Na]⁺

MP; 75 - 77 ^°C

¹H NMR (400 MHz); CD₃OD :δ= 7.77 (s, IH), 7.55 (s, IH), 7.35 (d, J= 8.4 Hz, IH), 7.23 (d, J= 8.8 Hz, IH), 7.06-6.99 (m, 2H), 6.68 (t, J= 7.2 Hz, IH), 6.59 (dd, J= 2.4, 8.8 Hz, IH), 6.12 (d, J= 2.4 Hz, IH), 4.25 (t, J= 6.8 Hz, 2H), 4.12 (t, J= 7.2 Hz, 2H), 3.55-3.48 (m, 4H), 2.97 (s, 3H), 2.02-1.98 (m, 2H), 1.82-1.78 (m, 2H), 1.47-1.44 (m, 2H)

IR; 3318, 1740, 1732, 1720, 1695, 1630, 1536, 1495, 1462, 1385, 1193, 1119, 1095, 1054 cm^"1

<Example 23> Synthesis of 3-[l-(4-dimethylaminobutyl)-7H-indol-3-yl]-4-[l-(3- dimethylaminopropyl)-5-methoxy-///-indol-3-yl]-pyrroI-2,5-dione (47)

Synthesis of methanesulfonic acid 3-(3-{4-[l-(4-methanesulfonyloxybutyl)-7//-indol-3- yl1-2,5-dioxo-2,5-dihydro-7H-pyrrol-3-vU -5-methoxy-indol- 1 -vD-propyl ester (46)

The compound (45) (210 mg, 0.43 mmol) prepared in Example 22 was dissolved in DMF (10 ml). Then, at 0^°C, triethylamine (0.24 ml, 1.72 mmol) was slowly added thereto and after 3 minutes, methanesulfonyl chloride (0.13 ml, 1.72 mmol) was added. The mixture was maintained at the same temperature for 10 minutes and reacted at room temperature for further 10 hours. Then, the reactant was diluted with water (10 ml) and extracted three times with ethyl acetate (30 ml). Ethyl acetate was removed from the reactant and the residue was purified by column chromatography (hexane: ethyl acetate = 1:3) to obtain the desired compound (46) as red solid in a yield of 70% (200 mg, 0.31 mmol).

Synthesis of 3-[l-(4-dimethylarnMobutyl)-7H-indol-3-yl1-4-[l-(3-dimethylaminopropyl)- 5-methoxy-i//-indol-3-yll-pyrrol-2,5-dione (47)

The compound (46) (60 mg, 0.09 mmol) prepared above and 2M dimethylamine (1.0 ml) were dissolved in DMF (5.0 ml) and the mixture was warmed to reflux at 80 °C for 8 hours. The reactant was diluted with water (10 ml) and extracted three times with ethyl acetate (30 ml), followed by removing ethyl acetate and washing it three times with hexane. Then, the desired compound (47) as red solid was obtained by recrystallization using ethyl acetate (3 ml), hexane (5 ml) and methanol (1 ml), in a yield of 99% (50 mg, 0.09 mmol).

MS (ESI+); 542 [M+H]⁺

MP; 73 - 75 ^°C

¹H NMR (400 MHz); CD₃OD :δ= 7.78 (s, IH), 7.60 (s, IH), 7.37 (d, J= 8.4 Hz, IH), 7.24 (d, J= 8.8 Hz, IH), 7.05 (t, J= 7.2 Hz, 2H), 7.00 (d, J= 12 Hz, IH), 6.70 (t, J= 7.2 Hz, IH), 6.60 (dd, J= 2.4, 8.8 Hz, IH), 6.15 (d, J= 2.4 Hz, IH), 4.20 (t, J= 6.8 Hz, 2H), 4.14 (t, J= 7.2 Hz, 2H), 2.84 (s, 3H), 2.29-2.25 (m, 4H), 2.21 (s, 6H), 2.18 (s, 6H), 1.99-1.95 (m, 2H), 1.76-1.71 (m ,2H), 1.48-1.46 (m, 2H)

IR; 3318, 2946, 1704, 1626, 1540, 1471, 1393, 1221, 1140, 1123, 1095 cm^"1

<Example 24> Synthesis of 3-[l-(3-hydroxypropyl)-5-methoxy-lH-indol-3-yI]-4-(l- methyl-i//-indol-3-yl)-pyiτol-2,5-d[ione (51)

The mixture of the compound (4) (500 mg, 1.50 mmol) prepared in Example 1 and 2-(l- methyl-lH-indol-3-yl)-acetamide (50) (300 mg, 1.59 mmol) was dissolved in THF (20 ml) and potassium t-butoxide (7.0 ml) was slowly added thereto at 0 ^°C . Then, the mixture was maintained at the same temperature for 10 minutes and reacted at room temperature for 12 hours. 2M hydrochloric acid (8.0 ml) was added thereto, and the mixture was reacted for 1 hour to complete the reaction. The reactant was diluted with water (30 ml) and extracted three times with ethyl acetate (50 ml), and the residue was purified by column chromatography (hexane: ethyl acetate = 1 : 4) to obtain the desired compound (51) as red solid in a yield of 53% (340 mg, 0.79 mmol).

MS (ESI+); 452 [M+Na]⁺

MP; 222 -224 ⁰C

¹H NMR (400 MHz); DMSO-^ :δ= 10.87 (s, IH), 7.82 (s, IH), 7.74 (s, IH), 7.40 (d, J= 8.4 Hz, IH), 7.29 (d, J= 8.8 Hz, IH), 7.03 (t, J= 7.2 Hz, IH), 6.77 (d, J= 8.0 Hz, IH), 6.63 (1, J= 7.2 Hz, IH), 6.56 (dd, J= 2.4, 8.8 Hz, IH), 6.12 (d, J= 2.4 Hz, IH), 4.61 (t, J= 2.4 Hz, IH), 4.24 (t, J= 12 Hz, 2H), 3.82 (s, 3H), 3.36-3.35 (m, 2H), 3.00 (s, 3H), 1.88-1.84 (m, 2H)

IR; 3383, 2251, 2124, 1752, 1703, 1642, 1532, 1462, 1336, 1217, 1131, 1095, 1050, 1029, 1005 cm^"1

<Example 25> Synthesis of 3-[l-(3-dimethylaminopropyl)-5-methoxy-2H-indol-3- yI]-4-(l-methyl-lH-indol-3-yl)-pyrrol-2,5-dione (53)

Synthesis of methanesulfonic acid 3-r5-methoxy-3-r4-(l-methyl-7H-indol-3-yl^')-2,5- dihydro-i//-pyrrol-3-yl]-indol-propyl ester (52)

The compound (51) (340 mg, 0.79 mmol) prepared in Example 24 was dissolved in DMF (10 ml) and triethylamine (0.22 ml, 1.58 mmol) was slowly added thereto at 0°C . After 3 minutes, methanesulfonyl chloride (0.12 ml, 1.58 mmol) was added thereto, and the mixture was reacted at the same temperature for 10 minutes and at room temperature for further 10 hours. The reactant was diluted with water (10 ml) and extracted three times with ethyl acetate (30 ml). Then, ethyl acetate was removed from the reactant and the residue was purified by column chromatography (hexane: ethyl acetate = 1 :3) to obtain the desired compound (52) as red solid in a yield of 80% (300 mg, 0.59 mmol).

Synthesis of 3-[l-(3-dimethylaminopropyl)-5-methoxy-./H-indol-3-yll-4-[l-methyl-iH- indol-3-yl)-pyrrol-2,5-dione (53)

The compound (52) (120 mg, 0.24 mmol) prepared above and 2M dimethylamine (0.24 ml) were dissolved in DMF (5.0 ml) and the mixture was warmed to reflux at 80 ^°C for 12 hours. The reactant was diluted with water (10 ml) and extracted three times with ethyl acetate (30 ml), followed by removing ethyl acetate and washing it three times with hexane. Then, the desired compound (53) as red solid was obtained by recrystallization using ethyl acetate (3 ml), hexane (5 ml) and methanol (1 ml), in a yield of 93% (100 mg, 0.22 mmol).

MS (ESI+); 457 [M+H]⁺

MP; 180 - 182 ^°C ¹H NMR (400 MHz); DMSO-c/₆:δ= 10.83 (bs, IH), 7.78 (d, J= 13.6 Hz, IH), 7.40 (d, J= 8.0 Hz, IH), 7.29 (d, J= 8.8 Hz, IH), 7.02 (t, J= 7.2 Hz, IH), 6.75 (d, J= 8.0 Hz, IH), 6.61 (t, J= 8.8 Hz, IH), 6.56 (dd, J= 2.4, 8.8 Hz, IH), 6.14 (d, J= 2.4 Hz₅ IH), 4.21 (t, J= 6.4 Hz, 2H), 3.82 (s, 3H), 3.02 (s, 3H), 2.09-2.05 (m, 8H), 1.84-1.80 (m, 2H)

IR; 2917, 1708, 1622, 1532, 1462, 1328, 1221, 1152, 1123, 1091, 1054 cm^"1

<Example 26> Synthesis of 2-(3-{5-methoxy-3-[4-(l-methyI-iH-indol-3-yl)-2,5- dioxo-2,5-dihydro-7H-pyrrol-3-yl]-indol-l-yl}-propyl)-isothiourea (54)

The compound (52) (40 mg, 0.08 mmol) prepared in Example 25 and thiourea (12 mg) were dissolved in DMF (3.0 ml) and the mixture was warmed to reflux at 80 ^°C for 12 hours. The reactant was diluted with water (15 ml), and extracted twice with ethyl acetate (20 ml) to remove materials present in organic layers. Then, water was removed from the reactant, followed by washing it three times with ethyl acetate and the desired compound (54) as red solid was obtained by recrystallizationusing ethyl acetate (3 ml), hexane (5 ml) and methanol (1 ml), in a yield of 91% (35 mg, 0.07 mmol).

MS (ESI+); 488 [MH-H]⁺

MP; 103 - 105 ^°C

¹H NMR (400 MHz); CD₃OD :δ= 7.77 (s, IH), 7.68 (s, IH), 7.36 (d, J= 8.4 Hz, IH), 7.26 (d, J= 8.8 Hz, IH), 7.07 (t, J= 8.0 Hz, IH), 6.86 (d, J= 8.0 Hz, IH), 6.67-6.62 (m, 2H), 6.25 (d, J= 2.4 Hz, IH), 4.35 (t, J= 6.4 Hz, 2H), 3.84 (s, 3H), 3.08 (s, 3H), 3.04 (t, J= 8.0 Hz, 2H), 2.27-2.21 (m, 2H)

IR; 3318, 1753, 1704, 1650, 1618, 1532, 1454, 1348, 1197, 1119, 1058, 1095 cm^"1

<Example 27> Synthesis of 2-(4-{5-methoxy-3-[4-(l-methyI-7H-indol-3-yl)-2,5- dioxo-2,5-dihydro-lH^r-pyrroI-3-yl]-indol-l-yl}-bufyl)-isothiourea (56)

Synthesis of 3-[l-(4-hvdroxybuM)-5-methoxy-i//-indol-3-yll-4-(l-methyl-7//-indol-3- yl)-pyrrol-2,5-dione (55)

The mixture of the compound (24) (225 mg, 0.65 mmol) prepared in Example 15 and 2- (l-methyl-7H-indol-3-yl)-acetamide (50) (250 mg, 1.32 mmol) was dissolved in THF (20 ml) and potassium t-butoxide (4.0 ml) was slowly added thereto at 0 ^°C . The mixture was maintained at the same temperature for 10 minutes and reacted at room temperature for 12 hours. Then, 2M hydrochloric acid (8.0 ml) was added thereto, and the mixture was reacted for 1 hour to complete the reaction. The reactant was diluted water (30 ml) and extracted three times with ethyl acetate (50 ml), and the residue was purified by column chromatography (hexane: ethyl acetate = 1 :3) to obtain the desired compound (55) as red solid in a yield of 52% (150 mg, 0.34 mmol). Synthesis of methanesulfonic acid 4-{5-methoxy-3-r4-(l-methyl-7H-indol-3-yl)-2,5- dioxo-2,5-dihvdro-iH-pyrrol-3-yll-indol-l-yl|-butyl ester (56)

The compound (55) (150 mg, 0.34 mmol) prepared above was dissolved in DMF (10 ml) and triethylamine (0.14 ml, 0.70 mmol) was slowly added thereto at 0^°C, and after 3 minutes, methanesulfonyl chloride (0.04 ml, 0.50 mmol) was added thereto. The mixture was maintained at the same temperature for 10 minutes and reacted at room temperature for 8 hours. Then, the reactant was diluted with water (10 ml) and extracted three times with ethyl acetate (30 ml). Then, ethyl acetate was removed from the reactant, and the residue was purified by column chromatography (hexane: ethyl acetate = 1 :3) to obtain the desired compound (56) as red solid in a yield of 96% (170 mg, 0.33 mmol).

Synthesis of 2-C4- (5-methoxy-3-r4-(l -methyl-iH^r-indol-3-ylV2,5-dioxo-2.5-dihydro-7H- Pyrrol-3-yll-indol-l-vU-butyl)-isothiourea(57)

The compound (56) (34 mg, 0.07 mmol) prepared above and thiourea (10 mg) were dissolved in DMF (3.0 ml) and warmed to reflux at 80 ^°C for 7 hours. The reactant was diluted with water (15 ml), and extracted twice with ethyl acetate (20 ml) to remove materials present in organic layers. Then, water was removed from the reactant, followed by washing it three times with ethyl acetate, and the desired compound as red solid was obtained by recrystallization using ethyl acetate (3 ml), hexane (5 ml) and methanol (1 ml), in a yield of 92% (30 mg, 0.06 mmol).

MS (ESl+); 502 [M+H]⁺

MP; 82 - 84 ⁰C

¹H NMR (400 MHz); DMS(W₆ :δ= 7.78 (s, IH), 7.63 (s, IH), 7.34 (d, J= 8.4 Hz, IH), 7.23 (d, J= 8.8 Hz, IH), 7.06 (t, J= 8.0 Hz, IH), 6.86 (d, J= 8.0 Hz, IH), 6.66-6.59 (m, 2H), 6.18 (d, J= 2.4 Hz, IH), 4.23 (t, J= 6.4 Hz, 2H), 3.82 (s, 3H), 3.30 (t, J= 8.0 Hz, 2H), 3.29 (s, 3H), 2.00-1.95 (m, 2H), 1.71-1.66 (m, 2H)

IR; 3326, 3199, 1753, 1699, 1654, 1609, 1528, 1446, 1336, 1193, 1152, 1127, 1095, 1062 cm

<Experimental Example 1> Analysis of effects that the present compound affects on expression of NCR3 Expermental Example 1-1

In order to identify effects that the present compounds affect the expression of NCR3 (NKp30) on NK cell surfaces, experiments were carried out, using immunofluorescent staining and flow cytometry. Specifically, compounds (Examples 2, 4, 10, 13, 14, 16, 17 and 18) randomly selected from the prepared examples each were added by 5 μM to culture NK cells for 24 hours. The completed NK cells in culture were washed twice with PBS, and reacted with 10 μi of PE-conjugated anti-NCR3 antibody (Beckman Coulter) at 4 ^°C for 30 minutes, in 90 μi, of PBS comprising 0.1% BSA. Then, the reactant was washed twice with PBS to measure fluorescence intensity by FACStar (Becton Dickinson) and analyze measurements using WinMDI 2.8 program, and then the results were represented in Figs. 2 and 3.

Fig. 2 shows results that the expression of NCR3 on surfaces of NK cells treated with the present compound is measured using the flow cytometry, wherein the bold black line represents the isotype control, the green line the basically expressed NCR3 and the filled portion with gray the expressed NCR3 after treating with the present compound. As could be seen from Fig. 2, a very small quantity of NCR3 was expressed in NK cells fresh isolated or cultured for 24 hours (green line), but a significantly large quantity of NCR3 was expressed on surfaces of NK cells treated with the compounds prepared in Examples, compared with non-treatment case (portion filled with gray). Results analysing the results of Fig. 2 by relative mean fluorescence intensity ratio (MFI) are the same as Table 1 below, which is represented in Fig. 3.

[Table 1]

As could be seen from Table 1 above and Fig. 3, the expression of NCR3 in NK cells treated with compounds of Examples 2, 13, 16 and 18 was observed about 6 times than that of untreated case. In addition, in case of compounds of Examples 4, 10, 14 and 17, the expression of NCR3 was observed about 3 times or more.

Experimental Example 1-2

Compounds (Examples 2, 20, 21, 23 and 25 to 27) selected from the prepared examples each were added by 5 μM to culture NK cells, whose donor was different from that used in Experimental Example 1-1, for 24 hours. The completed NK cells in culture were washed twice with PBS, and reacted with 10 μl of PE-conjugated anti-NCR3 antibody (Beckman Coulter) at 4°C for 30 minutes, in 90 μJt of PBS comprising 0.1% BSA. Then, the reactant was washed twice with PBS to measure fluorescence intensity by FACStar (Becton Dickinson) and analyze measurements using WinMDI 2.8 program, and then the results were represented in Fig.4. Fig.4 shows results that the expression of NCR3 on surfaces of NK cells treated with the present compound is measured using the flow cytometry, wherein the bold black line represents the isotype control, the green line the basically expressed NCR3 and the filled portion with gray the expressed NCR3 after treating with the present compound. Results analysing the results of Fig. 4 by relative mean fluorescence intensity ratio (MFI) are the same as Table 2 below, which is represented in Fig. 5.

[Table 2]

As could be known from Table 2 above and Fig. 5, the present compounds increased outstandingly the expression of NCR3 on surfaces of NK cells. That is, the expression of NCR3 in NK cells treated with compounds of Examples 2 and 25 to 27 was observed about 8 times or more than that of untreated case. Specifically, in case of Examples 26 and 27, the expression of NCR3 was observed about 10 times or more. Experimental Example 2> Analysis of effects that the present compound affects on expression of NCRl

In order to identify effects that the present compounds affect the expression of NCRl on NK cell surfaces, the experiment was carried out by the same method as that of Experimental Example 1-1, using compounds (Examples 2, 4, 5, 6, 10, 11, 13, 14, 16, 17 and 18) randomly selected from the prepared examples, and the results were represented in Figs. 6 and 7.

NCRl is a receptor expressed in both activated and inactivated NK cells. As shown in Fig. 6, it was observed that a significant quantity of NCRl was expressed in all NK cells fresh isolated or cultured for 24 hours (green line). As the case of NCR3, it was identified that much larger quantity of NCRl was expressed on surfaces of NK cells treated with the compound of Example 2, compared with non-treatment case (portion filled with gray). Results analyzing quantatively the results of Fig. 6 by comparing relative MFIs are the same as Table 3 below and Fig. 7.

[Table 3]

As could be seen from the results of Table 2 and Fig. 7, it could be identified that the expression of NCRl was highly increased even in cases treated with compounds of Examples 2, 13 and 16 (about twice or more), and a large quantity of NCRl was also expressed in other cases, compared with the untreated case.

<Experimental Example 3> Analysis of effects that the present compound affects on expression of NCR2 In order to identify effects that the present compounds affect the expression of NCR2 on NK cell surfaces, the experiment was carried out by the similar method to that of Experimental Example 1, using the compound of Example 2. In addition, NK cells were treated with PMA currently known as activating materials of NK cells, and their results were compared and represented in Figs. 8 and 9, together with the untreated case. As could be seen from Fig. 8, NCR2 was not almost expressed in NK cells untreated or treated with PMA, while the expression of NCR2 was very strongly induced in NK cells treated with the compound of Example 2. Results analyzing quantatively each relative MFI are the same as Table 4 below and Fig. 9.

[Table 4]

As shown in Table 4 above and Fig. 9, it was identified that NK cells treated with the present compound of Example 2 expressed about 30 times or more of NCR2 than cases untreated or treated with PMA.

As could be seen from the results of Experimental Examples 1 to 3 above, the present compound outstandingly increased the extracellular expression of NCRl, 2 and 3. On reviewing relative MFI ratios analyzing quantatively the degree of increasing each NCR expression, it can be noted that the expression quantity after treatment of the present compound has a little difference depending on the NCR types. This difference is developed, because NCR2 is not almost expressed in normal NK cells, but NCR3 is very slightly expressed therein. Although the direct comparison is difficult due to the difference of expression according to such NCR types, it can be identified that comparing with the quantity of NCRl, 2 and 3 expressed in NK cells treated with the present compound, all NCRs are strongly expressed.

<Experemental Example 4> Analysis of effects that cytokines affect on expression of NCRs

In order to analyze effects that cytokines affect on expression of NCRs in NK cells, an experiment was carried out, similar to Experimental Example 1 , using immunofluorescent staining and flow cytometry. The cytokines used in the experiment were IL-2 (1 ng/ml), IL-15 (1 ng/ml), IL-8 (10 ng/ml), IL-12 (10 ng/ml), IL-18 (10 ng/ml), IFN-αl (10 ng/ml) and IFN-α2b (10 ng/ml). When G average value of NCR expression in the untreated NK cells as simply cultured was estimated as 1, the expression quantities of NCRs on surfaces of NK cells after treating them with each cytokine for 24 hours were represented as relative MFIs in Fig. 10.

As could be seen from Fig. 10, in cytokines used in the experiment, only IL-12 increased the expression of NCR3 more or less (Fig. 10 (A)), and IL- 15 and IFN-α2 slightly increased the expression of NCRl (Fig. 10 (B)), but the degrees of both were insignificant. In addition, in case of NK cells treated with other cytokines rather than the above cytokines, all expression quantities of NCRl and NCR3 had no difference with that of the untreated NK cells (Fig. 10).

As described above, it was known that cytokines such as IL-2, IL-12 and IL- 15 played an important role in differentiation and development of NK cells. Therefore, effects that cytokines such as IL-2, IL-8, IL-12, IL-15, IL-18, IFNaI and IFN-a2b affect on expression of NCRs on surfaces of NK cells were investigated. But, as a result, it was identified that all these cytokines did not affect highly on expression of NCRs (Fig. 10). That is, some of cytokines (IL-12, IL-15 and IFN-α2, and the like) slightly increased the expression of NCRl and NCR3, which were hard to understood them as meaningful results. It could be seen from such results that the present compound activated NK cells in a manner different from action of cytokines on differentiation, development, growth and proliferation of NK cells.

Experimental Example 5> Analysis of effects that the present compound affects on cytotoxicity of NK cells against cancer cells

Experimental Example 5-1 (PMA and Compound of Example 2)

Effects that NK cells treated with 100 ng/ml of PMA and 5μM of the compound of Example 2 affected on apoptosis of hepatocellular carcinoma were identified through JAM test. Specifically, target cells were seeded in 96-well plate by 1 x 10⁴ cells/well, and cultured for HepG2 and Hep3B to be attached to the bottom of plate. The cell culture fluid was removed and ³[H]- thymidine was uptaked to 20 μCi/well. The remaining [H]-thymidine was removed and NK cell and target cells were cocultured in a ratio of 3:1. After 2 hours, cells attached to the bottom were taken off from the bottom using 0.125% trypsin/0.5 mM EDTA (Gibco BRL). Target cells were absorbed on glass fiber filter (Wallac Oy), followed by well washing it, and then, meltilex (Wallac) was melted to cover the filter, and the quantity of radiation was measured by β-counter (Wallac).

Then, using the following equation (1), the quantity to induce apoptosis by NK cells was measured.

Equation (1)

% DNA fragmentation=((l - experimental value)/negative control value) x 100

Then, the cytotoxicity of NK cells treated with the compound of Example 2 against cancer cells. HepG2 and Hep3B, hepatoma cell lines, were used as target cells. NK cells were treated with the compound of Example 2, and then mixed with hepatoma cell lines to culture them, and the quantity of killed hepatoma cell lines was measured. In addition, apoptosis of hepatoma cell lines by NK cells was measured using JAM test, and represented in Fig. 11. As shown in Fig. 11 , it was identified that NK cells untreated or treated with PMA killed HepG2 or Hep3B hepatoma cell lines within about 20% for 2 hours. But, it was identified that NK cells treated with the compound of Example 2 killed them up to 40% to show about twice or more of cytotoxicity.

Experimental Example 5-2 (Compounds of Examples 2 and 27)

Effects that the compounds of Examples 2 and 27 affected on the cytotoxicity of NK cells against cancer cells were measured by a similar method to Experimental Example 5-1 except for using HepG2 and Hep3B, hepatoma cell lines, and HeLa, cervical cancer cell line, as target cells, using NK cells provided by a donor different from that of Experimental Example 5-1 and treating them with the compounds of Examples 2 and 27 for 4 hours, and then the results were represented in Fig. 12.

As could be seen from Fig. 12, it could be noted that NK cells treated with the present compounds of Examples 2 and 27 had excellent cytotoxicity against cancer cell lines over the untreated NK cells. In addition, the cytotoxicity of NK cells treated with Example 27 was more excellent than that of NK cells treated with Example 2. As shown in Experimental Example 1 above, such a result is because the compound of Example 27 increases the expression of NCRs on surfaces of NK cells better than the compound of Example 2, together with natural cytolytic potential of NK cells.

Experimental Example 6> Analysis of effects that the present compound affects on excellular expression of death ligands on NK cells

Effects that the present compound affects on the expression of FasL and TRAIL, death ligands, on surfaces of NK cells were measured using immunofluorescent scanning and flow cytometry. Specifically, NK cells were cultured with 5 μM of the comound of Example 2 for 24 hours. Then, said cultured NK cells were washed twice with PBS, and 10 jΛ, of PE-conjugated anti-FasL antibody or PE-conjugated anti-TRAIL antibody was added to 90 jύ, of PBS comprising 0.1% BSA to react them at 4^°C for 30 minutes. Then, the reactant was washed twice with PBS, and fluorescence intensity was measured by FACScalibur (Becton Dickinson), and then the results were analized using WinMDI 2.8 programm. NK cells were treated with the compound of Example 2 as above, and after 24 hours, the quantities of FasL and TRAIL expressed on surfaces of NK cells were analized by flow cytometry. Comparing said analyzing results through relative MFI, the results were represented in Fig. 13. As could be seen from Fig. 13, it could be noted that NK cells treated with the present compound further expressed about 30 times or more of FasL and about 40 times or more of TRAIL over the untreated NK cells.

Death ligands of NK cells as above have functions inducing apoptosis by recognizing death receptors of target cells such as cancer cells. Therefore, NK cells treated with the present compound show to have higher cytotoxicity against cancer cells and the like by increasing the expression of death ligands in addition to increase of expression of NCRs.

Experimental Example 7> Analysis of anticancer effects of the present compound on mouse

In order to identify anticancer effects of the present compound on mouse, differences of survival times were investigated between a control group and a group (treated group) injected by the compound of Example 2 in tumor mouse models. C57BL/6 mice of 8 week old, in which 1 x 10⁶ of B16BL6 cells, malignant melanoma cell line, were subcutaneously injected on a portion of the back, were used as tumor mouse models. 4.4 βg (2 nmole) of the present compound of Example 2 was dissolved in PBS, and this solution gave treatment thereto in a total of 5 times, each at two or three day intervals, by once at 2 days before injection of cancer cells, once at the day and three times after their injection (treated group). As a control group, one was used, which injected only PBS 5 times at the same period as above. After injecting cancer cells, the survival rates were observed over two months, and the results were represented in Fig. 14. In both the control group and the treated group, tumor began to grow after about 2 weeks and the outstanding difference in tumor size was not observed between two groups. However, in case of the control group, all were killed before 35 days after injecting cancer cells, whereas, in case of the treated group, about 1/3 were killed at the similar period to the control group, about 1/3 were further lived for 1 week, and about 1/3 were lived for the experiment period (63 days) without death. It implies from this result that the present compound plays a role to extend the survival time in the tumor mouse models and has excellent in vivo anticancer effect.

Experimental Example S>In vivo antimetastasis effect of the present compound in mouse The compound of Example 2

C57BL/6 mice of 8 week old, in which 1 x 10⁵ of B16BL6 cells, malignant melanoma cell line, were intravenously injected at the tail, were used as mouse metastasis models. 4.4μg (2 nmole) or 22μg (5 nmole) of the compound of Example 2 dissolved in PBS gave treatment the treated group in a total of 5 times, each at two to three day intervals, by once at 2 days before injection of cancer cells, once at the day and three times after their injection. PBS was injected 5 times to the control group at the same period as the treated group. After injecting cancer cells, mice in the control group and the treated group were simultaneously caught at the 18 ¹ day and numbers of melanoma caused in lung were compared. In each group the statistics for melanoma spots were in except for each one case of the most case and the least case. As a result, as shown in Fig. 15, the melanoma spots spread to the lung in the control group amounted to average 60, wheras the treated groups treated with 4.4 μg (2 nmole) or 22 μg (5 nmole) of the compound of Example 2 showed 34.9 and 26.3 of spots, respectively. Therefore, it could be known that the metastasis was effectively prohibited.

The compound of Example 27

C57BL/6 mice of 8 week old, in which 1 x 10⁵ of B16BL6 cells, malignant melanoma cell line, were intravenously injected at the tail, were used as mouse metastasis models. 5μg (2 nmole) or 25μg (5 nmole) of the compound of Example 27 dissolved in PBS gave treatment the treated group in a total of 5 times, each at two to three day intervals, by once at 2 days before injection of cancer cells, once at the day and three times after their injection. PBS was injected 5 times to the control group at the same period as the treated group. After injecting cancer cells, mice in the control group and the treated group were simultaneously caught at the 21^st day and numbers of melanoma caused in lung were compared. In each group the statistics for melanoma spots were in except for each one case of the most case and the least case. As a result, as shown in Fig. 16 (A), the melanoma spots spread to the lung in the control group amounted to average 78, wheras the treated groups treated with 5 μg (2 nmole) or 25 μg (5 nmole) of the compound of Example 27 showed average 33.6 and 32.6 of spots, respectively. Therefore, it could be known that the metastasis was effectively prohibited. In Fig. 16 (B), the above results are arranged and represented as percent about antimetastasis effects of the compounds of Examples 2 and 27.

[Industrial Applicability]

The present compound of Formula 1 increased the expression of NCRs and death ligands of NK cells. Such NK cells with increased NCRs and death ligands increase cytotoxicity against target cells, and thus may be usefully used as a medical use such as anticancer agents or antiviral agents. The present invention has superiority different from conventional arts in that, first, the inventors found out primarily that organic compounds with specific formulas increase cytotoxicity of NK cells, rather than conventional arts to be subject to hormones or cytokines as materials regulating activity of NK cells, and that, second, they identified primarily that specific compounds characteristically increase the expression of NCRs on surfaces of NK cells and thus cytotoxicity of NK cells. Third, the present compounds may significantly increase the expression of NCR2 and NCR3 as well as NCRl up to 30 times over current IL-2 or prolactin increasing the expression of NCRs about 1.5 to 2 times. Fourth, the present compounds also increase the expression of death ligands as well as NCRs and thus outstandingly increase cytotoxicity of NK cells against target cells. Therefore, it is excellent to apply the present compound as a medical use.

Claims

[CLAIMS][Claim 1]A compound represented by Formula 1 :

[Formula 1]

wherein, Rj repesents hydrogen, halogen, hydroxy, alkyl, cycloalkyl, alkenyl, alkynyl or alkoxy;

R₂ and R₃ represent independently hydrogen, halogen, hydroxy, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, amino, cyano, carboxyl, aryl or -R₂₃R₂₄; wherein R₂₃ represents any one selected from straight or branched alkylene; straight or branched alkenylene; and straight or branched alkynylene;

R₄ to Rn represent independently hydrogen, alkyl, cycloalkyl or alkoxy. [Claim 2]

The compound according to claim 1, wherein R₁ is hydrogen, halogen, hydroxy, alkyl having 1 to 12 carbons, cycloalkyl having 3 to 12 carbons, alkenyl having 2 to 12 carbons, alkynyl having 2 to 12 carbons or alkoxy having 1 to 12 carbons. [Claim 3]

The compound according to claim 1, wherein, R₂ and R₃ represent independently hydrogen, hydroxy, alkyl having 1 to 12 carbons, cycloalkyl having 3 to 12 carbons, alkenyl having 2 to 12 carbons, alkynyl having 2 to 12 carbons, alkoxy having 1 to 12 carbons, amino, cyano, carboxyl, phenyl, naphthyl or -R₂₃R₂₄, wherein, R₂₃ represents straight or branched alkylene having 1 to 12 carbons, and

R₂₄ represents hydroxy, alkylamino having 1 to 12 carbons; dialkylamino having 2 to 24 carbons; any one of aryl, heteroaryl or heterocyclic group selected from the group consisting of phenyl, indolyl, pyrrolyl, furanyl, pyridinyl, piperidinyl, pyranyl, pyridazinyl, pyrimidinyl, pyrazinyl, piperazinyl and triazolyl; thiourea; or isothiourea, wherein, the aryl, the heteroaryl or the heterocyclic group may be substituted with one or more selected from the group consisting of hydroxy; amino; cyano; arylalkyl; aryl unsubstituted or substituted with alkyl; and alkyl unsubsituted or substituted with carboxyl. [Claim 4]

The compound according to claim 3, wherein, R₂ and R₃ represent independently hydrogen, alkyl having 1 to 8 carbons, cycloalkyl having 3 to 8 carbons, alkenyl having 2 to 8 carbons, alkynyl having 2 to 8 carbons, alkoxy having 1 to 8 carbons or -R₂₃R₂₄, wherein, R₂₃ reperesents straight or branched alkylene having 1 to 8 carbons, and

R₂₄ represents hydroxy, alkylamino having 1 to 8 carbons; dialkylamino having 2 to 16 carbons; any one of aryl, heteroaryl or heterocyclic group selected from the group consisting of phenyl, pyridinyl and triazolyl; thiourea; or isothiourea, wherein, the aryl, the heteroaryl or the heterocyclic group may be substituted with one or more selected from the group consisting of hydroxy, amino, cyano, arylalkyl having 7 to 12 carbons, aryl unsubstituted or substituted with alkyl having 1 to 4 carbons, and alkyl, having 1 to 4 carbons, unsubstituted or substituted with carboxyl. [Claim 5]

The compound according to claim 4, wherein, R₂ and R₃ represent independently hydrogen, alkyl having 1 to 4 carbons, cycloalkyl having 3 to 6 carbons, alkenyl having 2 to 4 carbons, alkynyl having 2 to 4 carbons, alkoxy having 1 to 4 carbons or -R₂3R2₄, wherein, R₂₃ represents straight or branched alkylene having 1 to 4 carbons, and

R₂₄ represents hydroxy, alkylamino having 1 to 4 carbons; dialkylamino having 2 to 8 carbons; substituted or unsubstituted triazolyl; thiourea or isothiourea, where the substituent of triazolyl is one or more selected from the group consisting of benzyl, and aUkyl, having 1 to 4 carbons, unsubstituted or substituted with carboxyl. [Claim 6]

The compound according to claim 1, wherein, R₄ to Rn represents independently hydrogen, alkyl having 1 to 8 carbons or alkoxy having 1 to 8 carbons. [Claim 7]

The compound according to claim 1, wherein, Rj represent hydrogen or alkyl having 1 to 8 carbons,

R₂ and R₃ represent independently hydrogen, alkyl having 1 to 8 carbons, cycloalkyl having 3 to 8 carbons, alkenyl having 2 to 8 carbons, alkynyl having 2 to 8 carbons, alkoxy having 1 to 8 carbons or -R₂₃R₂₄; wherein, R₂₃ represents straight or branched alkylene having 1 to 8 carbons, and R₂₄ represents hydroxy, alkylamino having 1 to 8 carbons; dialkylamino having 2 to 16 carbons; any one of aryl, heteroaryl or heterocyclic group selected from the group consisting of phenyl, pyridinyl and triazolyl; thiourea; or isothiourea; wherein, the aryl, the heteroaryl or the heterocyclic group may be substited with one or more selected from the group consisting of hydroxy, amino, cyano, arylalkyl having 7 to 12 carbons, aryl unsubstituted or substituted with alkyl having 1 to 4 carbons, and alkyl, having 1 to 12 carbons, unsubstituted or substituted with carboxyl; and

R₄ to R₁₁ represent independently hydrogen, alkyl having 1 to 8 carbons or alkoxy having 1 to 8 carbons. [Claim 8]

The compound according to claim 7, wherein, R₁ represents hydrogen, methyl or ethyl,

R₂ and R₃ represent independently hydrogen, alkyl having 1 to 4 carbons, cycloalkyl having 3 to 6 carbons, alkenyl having 2 to 4 carbons, alkynyl having 2 to 4 carbons, alkoxy having 1 to 4 carbons or -R₂₃R₂₄; wherein, R₂₃ represents straight or branched alkylene having 1 to 4 carbons, and R₂₄ represents hydroxy, alkylamino having 1 to 4 carbons; dialkylamino having 2 to 8 carbons; substituted or unsubstituted triazolyl, thiourea or isothiourea; where the substituent of triazolyl is one or more selected from the group consisting of benzyl and alkyl, having 1 to 4 carbons, unsubstituted or substituted with carboxyl; and R₄ to Rn represent independently hydrogen, methyl, ethyl, methoxy or ethoxy. [Claim 9] The compound according to claim 1, wherein, the compound of Formula 1 is

3-[l-(3-hydroxypropyl)-5-methoxy-iH-indol-3-yl]-4-(iH-indol-3-yl)pyrrol-2,5-dione;

3-[l-(3-dimethylaminopropyl)-5-me1hoxy-iH-indol-3-yl]-4-(7H-indol-3-yl)pyrrol-2,5- dione;

3-[l-(2-dimethylaminoethyl)-5-methoxy-7H-indol-3-yl]-4-(7H-indol-3-yl)pyrrol-2,5-dione;

3-[l-(3-hydroxypropyl)-5-methoxy-7H-indol-3-yl]-4-(7H-indol-3-yl)-l-methylpyrrol-2,5- dione;

3-[(l-berizyl-iH-[l,2,3]triaz»l-4-ylme%l)-5-methoxy-iH-indol-3-yl]-4-(7//-indol-3-yl)- pyrrol-2,5-dione;

(4.{3.[4.(iH.indol-3-yl)-2,5-ώoxy-2,5-d%dro-7H-pyrrol-3-yl]-5-merhoxy-7H-indol-l- ylmethyl}-[l,2,3]triazol-l-yl)acetic acid;

3-[l-(2-dimelhylaminoethyl)-5-me1hoxy-7H-indol-3-yl]-4-(7H-indol-3-yl)-l-methylpyrrol- 2,5-dione;

3-[l-(2-hydroxyethyl)-5-methoxy-i//-indol-3-yl]-4-(7H-indol-3-yl)-l-methylpyrrol-2,5- dione;

2-(2-{3-[4-(7H-indol-3-yl)-2,5-dioxo-2,5-dihydro-7H-pyrrol-3-yl]-5-methoxyindol-l-yl}- ethyl)isothiourea;

2.(3-{3-[4-(7H-mdol-3-yl)-2,5-ώoxo-2,5-dihydro-iH-pyrrol-3-yl]-5-methoxyindol-l- yl}propyl)isothiourea;

3-[l-(3-hydroxypropyl)-iH-indol-3-yl]-4-[l-(3-hydroxypropyl)-5-methoxy-iH-indol-3- yl]-pyrrol-2,5-dione;

3-[l-(3-dime%larriinopropyl)-iH-indol-3-yl]4-[l-(3-dimethylarninopropyl)-5-methoxy- 7H-indol-3-yl]-pyrrol-2,5-dione;

2-[3-(3-{4-[l-(3-carbamimidoylsdfanylpropyl)-i/f-mdol-3-yl]-2,5-dioxo-2,5-dihydro-i/f- pyrrol-3-yl}-5-methoxyindol-l-yl)-propyl]-isothiourea;

3-[l-(4-hydroxybutyl)-5-methoxy-iH-indol-3-yl]-4-(/H-indol-3-yl)-pyiτol-2,5-dione;

2-(4-{3-[4-(7H-indol-3-yl)-2,5-dioxo-2,5-dihydro-iH-pyrrol-3-yl]-5-methoxyindol-l- yl}butyl)isothiourea;

3-[l-(4-dimethylaminobutyl)-5-methoxy-7H-indol-3-yl]-4-(7/J-indol-3-yl)pyrrol-2,5- dione;

3,4-bis[l-(3-dimethylaminopropyl)-5-methoxy-7//-indol-3-yl]pyrrol-2,5-dione;

3-[l-(4-hydroxybutyl)-7H-indol-3-yl]-4-[l-(4-hydroxybutyl)-5-methoxy-iH-indol-3-yl]- pyrrol-2,5-dione;

2-[4-(3-{4-[l-(4-carbamimidolsulfanylbu1yl)-iH-indol-3-yl}-2,5-dioxo-2,5-dihydro-7H- pyrrol-3-yl]-5-niethoxyindol- 1 -yl)-butyl]-isothiourea;

3-[l-(4-dime%laminobu1yl)-iH-indol-3-yl]-4-[14-dime%laminopropyl)-5-me1hoxy-iH- indol-3-yl]-pyrrol-2,5-dione;

3-[l-(4-dimethylarninobutyl)-7H-indol-3-yl]-4-[l-4-dimethylairunopropyl)-5-methoxy-7H- indol-3-yl]-pyrrol-2,5-dione;

3-[l-(4-dime1hylaminobutyl)-iH-indol-3-yl]-4-[l-3-dimethylaminopropyl)-5-methoxy-7H- indol-3-yl]-pyrrol-2,5-dione;

3-[l-(3-hydroxypropyl)-5-me1hoxy-iH-indol-3-yl]-4-(l-methyl-7H-indol-3-yl)-pyiτol-2,5- dione;

3-[l-(3-dime%laminopropyl)-5-melhoxy-7H-indol-3-yl]4-[l-methyl-///-indol-3-yl)- pyrrol-2,5-dione;

2-(3-{5-methoxy-3-[4-(l-methyl-i//-indol-3-yl)-2,5-dioxo-2,5-dihydro-i//-pyrrol-3-yl]- indol-l-yl}-propyl)-isothiourea; or

2-(4-{5-methoxy-3-[4-(l-methyl-7H-indol-3-yl)-2,5-dioxo-2,5-dihydro-7H-pyrrol-3-yl]- indol- 1 -yl } -butyl)-isothiourea. [Claim 10]

The compound according to claim 1, wherein, the compound of Formula 1 is

3-[l-(3-hydroxypropyl)-5-methoxy-7H-indol-3-yl]-4-(iH-indol-3-yl)pyrrol-2,5-dione;

3-[l-(2-dimethylaminoethyl)-5-methoxy-iH-indol-3-yl]-4-(7H-indol-3-yl)pyrrol-2,5-dione;

2-(2-{3-[4-(iH-indol-3-yl)-2,5-ώoxo-2,5-dihydro-iH-pyrrol-3-yl]-5-methoxyindol-l-yl}- ethyl)isothiourea;

3-[l-(3-dimethylaminopropyl)-iH-indol-3-yl]4-[l-(3-dimethylaminopropyl)-5-methoxy- i//-indol-3-yl]-pyrrol-2,5-dione;

2-[3-(3-{4-[l-(3-carbarrώrudolysulfany^^ pyrrol-3-yl}-5-methoxyindol-l-yl)-propyl]-isothiourea;

2-(4-{3-[4-(iH-indol-3-yl)-2,5-dioxo-2,5-dihydro-iH-pyrrol-3-yl]-5-methoxyindol-l- yl}butyl)isothiourea;

3-[l-(4-dime%laminobu1yl)-5-methoxy-7H-indol-3-yl]-4-(7/f-indol-3-yl)pyrrol-2,5- dione;

3,4-bis[l-(3-dime1hylaminopropyl)-5-methoxy-i//-indol-3-yl]pyrrol-2,5-dione;

341-(3-dime%laminopropyl)-5-methoxy-iH-indol-3-yl]-4-[l-methyl-iH-indol-3-yl)- pyrrol-2,5-dione;

2-(3-{5-methoxy-3-[4-(l-methyl-7H-indol-3-yl)-2,5-dioxo-2,5-dihydro-/H-pyrrol-3-yl]- indol-l-yl}-propyl)-isothiourea; or

2-(4-{5-methoxy-3-[4-(l-methyl-7H-indol-3-yl)-2,5-dioxo-2,5-dihydro-;H-pyrrol-3-yl]- indol- 1 -yl } -butyl)-isothiourea. [Claim 11]

A process for preparing a compound of Formula 1', comprising a step of reacting a compound of Formula 2 and a compound of Formula 3 :

[Formula 2]

[Formula 3]

[Formula 1']

R1

wherein, Rj and R₄ to Ri _\ are the same as defined in Claim 1 ,

R₂' and R₃' represent independently hydrogen, halogen, hydroxy, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, amino, cyano, carboxyl, aryl or -R₂₃ ⁵R₂₄'; where R₂₃'represents any one selected from straight or branched alkylene; straight or branched alkenylene; and straight or branched alkynylene, and R₂₄' represents hydroxy; amino; cyano; carboxyl; substituted or unsubstituted aryl, heteroaryl or heterocyclic group; alkylthio; thiocyanato; sulfamoyl or a protecting group. [Claim 12]

The process according to Claim 11, further comprising a step of reacting a compound of Formula 1 ' with a necleophile. [Claim 13]

The process according to Claim 11, further comprising a step of reacting a compound of Formula 1 ' with an electrophile. [Claim 14]

The process according to Claim 11 , wherein, said compound of Formula 2 is prepared by reacting a compound of Formula 4 with oxalyl chloride and sodium methoxide:

[Formula 4]

wherein, R₂' and R₄ to R₇ are the same as defined in Claim 11. [Claim 15]

The process according to Claim 11 , wherein, said compound of Formula 3 is prepared by reacting a compound of Formula 5 with a base:

[Formula 5]

wherein, R₃' and Rg to Rn are the same as defined in Claim 11. [Claim 16]

NK cells treated with the compound according to any one of Claims 1 to 10. [Claim 17]

A process for treating NK cells identified from a host, comprising a step of culturing NK cells in a culture fluid comprising the compound according to any one of Claims 1 to 10. [Claim 18]

The process according to Claim 17, wherein, the host is human, monkey, cattle, sheep, goat, horse, pig, rabbit, dog, cat, rat, mouse or marmot. [Claim 19]

The process according to Claim 17, wherein, the culture fluid is RPM, MEM or DMEM. [Claim 20]

The process according to Claim 17, wherein, the culturing temperature is 30 to 37 ^°C , and the culturing time is 1 to 48 hours. [Claim 21]

The process according to Claim 17, wherein, characterized in that the culture fluid comprises the compound in an amount of 1 to 10 μM, relative to 10⁵ to 10⁶ cells/ml NK cells. [Claim 22]

A pharmaceutical composition for treating a disease, comprising the compound according to any one of Claims 1 to 10, a pharmaceutically acceptable salt or solvate thereof; and a pharmaceutically acceptable carrier. [Claim 23]

The pharmaceutical composition according to Claim 22, wherein, the disease is cancer; or an infectious disease by viruses or bacteria [Claim 24]

The pharmaceutical composition according to Claim 23, wherein, the cancer is lung cancer, liver cancer, gastric cancer, colon cancer, bladder cancer, prostatic cancer, breast cancer, ovarian cancer, cervical cancer, thyroid cancer, melanoma or hematological malignancy. [Claim 25]

The pharmaceutical composition according to Claim 23, wherein, the infectious disease by viruses or bacteria is AIDS, avian influenza, influenza, CMV infectious disease, tuberculosis or leprosy. [Claim 26]

A pharmaceutical composition for treating a disease, comprising the NK cells according to Claim 16, and a pharmaceutically acceptable carrier. [Claim 27] The pharmaceutical composition according to Claim 26, wherein, the disease is cancer; or an infectious disease by viruses or bacteria. [Claim 28]

The pharmaceutical composition according to Claim 27, wherein, the cancer is lung cancer, liver cancer, gastric cancer, colon cancer, bladder cancer, prostatic cancer, breast cancer, ovarian cancer, cervical cancer, thyroid cancer, melanoma or hematological malignancy. [Claim 29]

The pharmaceutical composition according to Claim 27, wherein, the infectious disease by viruses or bacteria is AIDS, avian influenza, influenza, CMV infectious disease, tuberculosis or leprosy.