Disubstituted adamantyl derivatives, pharma- ceutically acceptable salts thereof, and pharmaceutical compositions for inhibiting cancer proliferation containing the same as an active ingredient

Abstract

The present invention relates to a disubstituted adamantyl derivative or a pharmaceutically acceptable salt thereof, and an anticancer pharmaceutical composition and kit containing the same as an active ingredient. The disubstituted adamantyl derivative according to the present invention targets mitochondrial ETC complex I and inhibits cancer cell growth by impairing cancer cell metabolism, and can therefore be usefully used in anticancer pharmaceutical compositions as potent therapeutic agents for cancers that depend on oxidative phosphorylation for ATP production.

Description

[Technical field]

[0001] The present invention relates to a disubstituted adamantyl derivative or a pharma- ceutically acceptable salt thereof, and a pharmaceutical composition and a kit for inhibiting cancer proliferation, each of which contains the derivative as an active ingredient.

[0002] This application claims priority to Korean Patent Application No. 10-2002-0009445, filed on January 23, 2020, the entire contents of which are incorporated herein by reference in their entirety in the specification and drawings. [Background technology]

[0003] Cancer cells increase aerobic glycolysis, glutaminolytic flux, amino acid, and lipid metabolism to grow faster. Competing with neighboring normal cells for limited nutrients, cancer cells consume more glucose. The hypoxic tumor microenvironment of cancer cells also exacerbates cancer cells by inducing the expression of genes involved in metabolism, angiogenesis, metastasis, and resistance to cell death.

[0004] Hypoxia-inducible factor-1 (HIF-1), which consists of HIF-1α and HIF-1β, is a key regulator of tumor hypoxia and plays a key role in metabolic reprogramming of cancer cells. Under hypoxic conditions, HIF-1α increases the expression of glucose transporters (GLUTs) and glycolate enzymes, such as hexokinases (HK) and phosphoglycerate kinase 1 (PGK1), activating glycolysis. Furthermore, HIF-1 increases the expression of pyruvate dehydrogenase kinase isozyme 1 (PDK1), which phosphorylates serine residues and inhibits pyruvate dehydrogenase (PDH) activity. High expression of PDK1 blocks the conversion of pyruvate to acetyl-CoA and suppresses ATP production through the tricarboxylic acid (TCA) cycle and oxidative phosphorylation in mitochondria. In fact, hypoxic cancer cells exhibit these metabolic characteristics through HIF-1-dependent reprogramming. Therefore, inhibition of HIF-1 expression or function can impair cancer metabolism and cause cell death by suppressing the expression of genes involved in the metabolic adaptation of cancer cells.

[0005] Numerous studies have attempted to develop HIF-1 inhibitors. For example, BAY 87-2243, a mitochondrial complex I inhibitor, reduced hypoxia-induced HIF-1α accumulation and showed significant antitumor effects in H460 cancer cell line xenograft models. KCN-1, a benzopyran analog, inhibited HIF-1 activity by preventing the interaction of transcription factor p300 with HIF-1α in glioma cells. The present inventors focused on developing small molecule compounds that target HIF-1α protein in solid tumors (aryloxyacetylamino), and found that LW6, a benzoic acid analog (disclosed in Korean Patent Publication No. 10-2012-0041071), inhibited hypoxia-induced HIF-1α protein accumulation, and its direct target was malate dehydrogenase 2 (MDH2). The clinical candidate IDF-1174 has also been developed as a HIF-1α inhibitor targeting cancer metabolism. Also, moracin O and its benzofuran analogues have been reported as potent inhibitors of HIF-1α protein accumulation. However, no drugs have been approved by the FDA as HIF-1 inhibitors to date.

[0006] Hepatocellular carcinoma (HCC) is a complex heterogeneous tumor type with numerous genetic and epigenetic modifications. Sorafenib has been approved as a first-line treatment for patients with advanced HCC, but the survival benefit is significantly low. Sorafenib is an oral multikinase inhibitor that inhibits tumor growth by suppressing angiogenesis and proliferation by targeting vascular endothelial growth factor (VEGF) receptors and platelet-derived growth factor receptors. Recently, regorafenib and nivolumab have been approved as second-line treatments for patients who do not respond to sorafenib, but effective liver cancer therapies are urgently needed. Summary of the Invention [Problem to be solved by the invention]

[0007] As mentioned above, targeting cancer metabolism has emerged as an important cancer treatment strategy. Therefore, in this invention, we provide the results of synthesis and biological evaluation of disubstituted adamantyl derivatives as inhibitors of HIF-1α, a factor capable of inducing hypoxia.

[0008] More specifically, the present inventors have found that disubstituted adamantyl derivatives, including LW1564 (compound 21-3), inhibit mitochondrial respiration and reduce ATP production by inhibiting electron transport chain (ETC) complex I, and stimulate HIF-1α degradation in HCC cells. These results suggest that disubstituted adamantyl derivatives, including LW1564 (compound 21-3), impair cancer metabolism and suppress cancer cell growth in vitro and in vivo by inhibiting HIF-1α accumulation and fatty acid synthesis. Thus, the present invention provides an anticancer pharmaceutical composition containing the disubstituted adamantyl derivative and its pharma- ceutical acceptable salt as an active ingredient. [Means for solving the problem]

[0009] In order to achieve the above object, the present invention provides a disubstituted adamantyl derivative represented by the following chemical formula 1 (Chemical Formula 1) or a pharma- ceutically acceptable salt thereof.

[0010] [ka]

[0011] The present invention also provides an anti-cancer pharmaceutical composition comprising the disubstituted adamantyl derivative represented by Chemical Formula 1 or a pharma- ceutically acceptable salt thereof as an active ingredient.

[0012] The present invention also provides a method for treating cancer, comprising administering a therapeutically effective amount of a disubstituted adamantyl derivative represented by Chemical Formula 1 (Chemical Formula 1) or a pharma- ceutical acceptable salt thereof to a patient in need of anticancer treatment.

[0013] The present invention also provides a use of the disubstituted adamantyl derivative represented by Chemical Formula 1 or a pharma- ceutical acceptable salt thereof in the manufacture of a preparation for cancer treatment.

[0014] The present invention also provides a use of the disubstituted adamantyl derivative represented by Chemical Formula 1 or a pharma- ceutically acceptable salt thereof for treating cancer.

[0015] In one embodiment of the present invention, the pharmaceutical composition may be an anti-cancer pharmaceutical composition for solid cancer, but is not limited thereto.

[0016] In another embodiment of the present invention, the solid cancer may be selected from the group consisting of breast cancer, cervical cancer, rectal cancer, fibroid cancer, gastric cancer, liver cancer, lung cancer, and pancreatic cancer, but is not limited thereto. Effect of the Invention

[0017] The disubstituted adamantyl derivatives including LW1564 (compound 21-3) according to the present invention significantly inhibited HIF-1α accumulation and inhibited the proliferation of various cancer cell lines including HepG2 and A549. Measurement of oxygen consumption rate (OCR) and ATP production rate showed that LW1564 (compound 21-3) inhibited mitochondrial respiration, thereby increasing intracellular oxygen concentration and stimulating HIF-1α degradation in HepG2 cells. LW1564 (compound 21-3) further increased mitochondrial electron transport chain (ETC) complex I and AMP / ATP ratio, inhibited animal target of rapamycin (mTOR) signaling, increased AMP-activated protein kinase (AMPK) phosphorylation, and significantly decreased total ATP production. As a result, LW1564 (compound 21-3) promoted phosphorylation of acetyl-CoA carboxylase, inhibited lipid synthesis, and significantly inhibited tumor growth in HepG2 mouse xenograft model. In summary, the substituted adamantyl derivatives including LW1564 (compound 21-3) according to the present invention target mitochondrial ETC complex I, impair the metabolism of cancer cells, and inhibit the proliferation of HepG2 cells, and therefore can be usefully used as potent therapeutic agents for cancers that depend on oxidative phosphorylation for ATP production in mitochondria. [Brief description of the drawings]

[0018] [Figure 1a] 1a to 1c show the effect of LW1564 (compound 21-3) on the growth of cancer cells, and FIG. 1a shows the structure of LW1564 (compound 21-3). [Figure 1b] Figures 1a to 1c show the effect of LW1564 (compound 21-3) on the growth of cancer cells, and Figure 1b shows the effect of LW1564 (compound 21-3) on the growth of cancer cells, and Figure 1b shows the proliferation inhibitory efficacy of LW1564 (compound 21-3) on various cancer cell lines. [Figure 1c]Figures 1a to 1c show the effect of LW1564 (compound 21-3) on the growth of cancer cells, and Figure 1c shows the effect of LW1564 (compound 21-3) on the growth of cancer cells, and Figure 1c shows the inhibitory effect of LW1564 (compound 21-3) on the proliferation of HepG2 and A549 cells investigated via an IncuCyte ZOOM system for observing live cell imaging. [Figure 2a] Figures 2a to 2e show the effect of LW1564 (compound 21-3) in suppressing the accumulation of HIF-1α in cancer cells, and Figure 2a shows the inhibitory effect of LW1564 (compound 21-3) on HRE-luciferase activity in HepG2 cells. [Figure 2b] Figures 2a to 2e are graphs showing the effect of LW1564 (compound 21-3) in suppressing the accumulation of HIF-1α in cancer cells, and Figure 2b is a graph showing the effect of LW1564 (compound 21-3) in suppressing the accumulation of HIF-1α in cancer cells, and Figure 2b is a graph showing that LW1564 (compound 21-3) inhibits the accumulation of HIF-1α through proteasome-dependent degradation. [Figure 2c] Figures 2a to 2e show the effect of LW1564 (compound 21-3) in suppressing the accumulation of HIF-1α in cancer cells, and Figure 2c shows the effect of LW1564 (compound 21-3) in suppressing the accumulation of HIF-1α in cancer cells, and Figure 2c shows the mRNA expression level of HIF-1α. [Figure 2d] Figures 2a to 2e show the effect of LW1564 (compound 21-3) in suppressing the accumulation of HIF-1α in cancer cells, and Figure 2d shows the effect of LW1564 (compound 21-3) in suppressing the accumulation of HIF-1α in cancer cells, and Figure 2d shows the effect of LW1564 (compound 21-3) in suppressing HIF-1α accumulation in various cancer cell lines under hypoxic conditions. [Figure 2e]Figures 2a to 2e show the effect of LW1564 (compound 21-3) in suppressing the accumulation of HIF-1α in cancer cells, and Figure 2e shows the effect of LW1564 (compound 21-3) in suppressing the accumulation of HIF-1α in cancer cells, and Figure 2e shows the mRNA expression levels of HIF-1α target genes, GLUT1, PDK1 and VEGFA by qPCR. [Figure 3a] FIG. 3a shows that LW1564 (compound 21-3) inhibits mitochondrial respiration in HepG2 cells, and FIG. 3b shows the effect of LW1564 (compound 21-3) on reducing oxygen consumption in HepG2 cells. [Figure 3b] FIG. 3b shows that LW1564 (compound 21-3) inhibits mitochondrial respiration in HepG2 cells, and FIG. 3b shows the results of measuring the ATP production rate in cancer cells upon treatment with LW1564 (compound 21-3). [Figure 3c] FIG. 3c is a diagram showing that LW1564 (compound 21-3) inhibits mitochondrial respiration in HepG2 cells, and FIG. 3c shows the effect of LW1564 (compound 21-3) on reducing the total amount of intracellular ATP. [Figure 3d] Figure 3d shows that LW1564 (compound 21-3) inhibits mitochondrial respiration in HepG2 cells, and Figure 3c shows the change in intracellular oxygen tension caused by LW1564 (compound 21-3) using MAR, a probe that detects hypoxia. [Figure 4a] Figures 4a to 4d show that LW1564 (compound 21-3) targets complex I in the ETC and inhibits mitochondrial respiration. In Figure 4a, 1 μM rotenone (a complex I activity inhibitor) (Figure 4a) was added to the cells. [Figure 4b]Figures 4a to 4d show that LW1564 (compound 21-3) targets complex I in the ETC and inhibits mitochondrial respiration. Figure 4b shows that LW1564 (compound 21-3) targets complex I in the ETC and inhibits mitochondrial respiration. In Figure 4b, 100 μM LW1564 (compound 21-3) (Figure 4b) was added to cells. [Figure 4c] Figures 4a to 4d show that LW1564 (compound 21-3) targets complex I in the ETC and inhibits mitochondrial respiration. Figure 4c shows that LW1564 (compound 21-3) targets complex I in the ETC and inhibits mitochondrial respiration. Figure 4c shows the effect of LW1564 (compound 21-3) on complex II / III. LW1564 (compound 21-3) was added to cells after the addition of succinate as a substrate for complex II. [Figure 4d] Figures 4a to 4d show that LW1564 (compound 21-3) targets complex I in the ETC and inhibits mitochondrial respiration. Figure 4d shows that LW1564 (compound 21-3) targets complex I in the ETC and inhibits mitochondrial respiration. Figure 4d shows the effect of LW1564 (compound 21-3) on complex IV. LW1564 (compound 21-3) was added to cells after adding TMPD and ascorbate as substrates for complex IV. [Figure 5a] FIG. 5a shows that LW1564 (compound 21-3) activates the AMPK pathway and inhibits fatty acid accumulation in HepG2 cells; FIG. 5a shows the activation of AMPK by LW1564 (compound 21-3). [Figure 5b] FIG. 5b shows that LW1564 (compound 21-3) activates the AMPK pathway and inhibits fatty acid accumulation in HepG2 cells; FIG. 5b shows inhibition of fatty acid accumulation in the presence of LW1564 (compound 21-3). [Figure 5c]FIG. 5c shows that LW1564 (compound 21-3) activates the AMPK pathway and inhibits fatty acid accumulation in HepG2 cells. FIG. 5c shows the effect of LW1564 (compound 21-3) and glucose concentration on cancer cell proliferation. HepG2 cells cultured at different glucose concentrations were treated with LW1564 (compound 21-3), and the graph shows % cell proliferation. [Figure 5d] FIG. 5d shows that LW1564 (compound 21-3) activates the AMPK pathway and inhibits fatty acid accumulation in HepG2 cells, and FIG. 5d shows the combination index (CI) of 2DG (2-deoxy-D-glucose) and LW1564 (compound 21-3) in HepG2 cells. [Figure 6] The tumor growth inhibitory effect of LW1564 (compound 21-3) was shown in a HepG2 xenograft mouse model. The left panel of Figure 6 shows the volume change of the xenograft tumors treated with the control group and LW1564 (compound 21-3) over time; **P≦0.01. The middle panel of Figure 6 shows the weight of the xenograft tumors treated with the control group and LW1564 (compound 21-3); **P≦0.01. The right panel of Figure 6 shows representative images of the tumors shown in the left and middle panels of Figure 6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention will be described in detail below.

[0020] The present invention provides a disubstituted adamantyl derivative represented by the following chemical formula 1 (Formula 2) or a pharma- ceutically acceptable salt thereof.

[0021] [ka]

[0022] In the above Chemical Formula 1 (Chemical Formula 2), R 1 is -(X)-(CH2)nR3 Or the following formula 3: R 2 is -(Y)-(CH3)m or -(Y)-(CH2)mR 4 and The X is O, NH, or the following formula 4: The Y is NH, the following formula 5 or the following formula 6, The n is 0, 1 or 2, and the m is 0 or 1, R 3 or R 4 are each independently a C1 to C6 linear or branched alkyl group, an unsubstituted or substituted C5 to C 10 or unsubstituted or substituted C5-C9 heteroaryl; wherein the heteroaryl is a 5- or 6-membered heteroaryl containing one or more N or O heteroatoms; The substituted aryl or substituted heteroaryl may be substituted with one or more of halogen, C1-C6 straight or branched alkyl, C1-C6 straight or branched haloalkyl (alkyl halide), hydroxy, or C1-C6 straight or branched alkoxy.

[0023] [ka]

[0024] [ka]

[0025] [ka]

[0026] [ka]

[0027] However, the above R 2In the above, the following formula 7 and formula 8 may be excluded. Preferably, R 1 is -(X)-(CH2)nR 3 Or the following formula 9: R 2 is -(Y)-(CH3)m or -(Y)-(CH2)mR 4 and The X is O, NH, or the following formula 10: The Y is NH, the following formula 11, or the following formula 12, The n is 0, 1 or 2, and the m is 0 or 1, R 3 is methyl, substituted or unsubstituted phenyl, or unsubstituted C5-C9 heteroaryl, where the heteroaryl is a 5- or 6-membered heteroaryl containing a N or O heteroatom, and the substituted phenyl is optionally substituted with one or more of halogen, C1-C6 straight or branched alkyl; C1-C6 straight or branched haloalkyl; hydroxy; or C1-C6 straight or branched alkoxy; R 4 is a substituted phenyl, a substituted or unsubstituted C5-C9 heteroaryl, wherein the heteroaryl is a 5- or 6-membered heteroaryl containing a N or O heteroatom, and the substituted phenyl or heteroaryl may be substituted with a C1-C6 straight or branched chain haloalkyl; However, the above R 2 In the above, the following formula 13 and the following formula 14 may be excluded.

[0028] [ka]

[0029] [ka]

[0030] [ka]

[0031] [ka]

[0032] [ka]

[0033] [ka]

[0034] [ka]

[0035] [ka]

[0036] More preferably, R 1 is represented by Chemical formula 15, R 2 is -(Y)-(CH3)m or -(Y)-(CH2)mR 4 and Y is NH, Formula (16) or Formula (17); The m is 1, R 4 is a substituted phenyl, wherein said substituted phenyl is optionally substituted with a C1-C6 straight or branched chain haloalkyl; However, the above R 2 In the above, formula 18 and formula 19 may be excluded.

[0037] [ka]

[0038] [ka]

[0039] [ka]

[0040] [ka]

[0041] [ka]

[0042] Preferably, The disubstituted adamantyl derivative or a pharma- ceutically acceptable salt thereof may be a disubstituted adamantyl derivative or a pharma- ceutically acceptable salt thereof, characterized by being represented by the following chemical formula 2 (Chemical Formula 20).

[0043] [ka]

[0044] In the above Chemical Formula 2 (Chemical Formula 20), R 1 is -(X)-(CH2)nR 3 Or, X is O, NH or wherein n is 0, 1 or 2; R 3 are each independently a C1 to C6 linear or branched alkyl, an unsubstituted or substituted C5 to C 10 or unsubstituted or substituted C5-C9 heteroaryl; wherein the heteroaryl is a 5- or 6-membered heteroaryl containing one or more N or O heteroatoms; The substituted aryl or substituted heteroaryl may be substituted with one or more of halogen, C1-C6 straight or branched alkyl; C1-C6 straight or branched haloalkyl; hydroxy; or C1-C6 straight or branched alkoxy.

[0045] [ka]

[0046] [ka]

[0047] More preferably, in the chemical formula 2 (Chemical Formula 20), R 1 may be a disubstituted adamantyl derivative, or a pharma- ceutically acceptable salt thereof, characterized in that:

[0048] [ka]

[0049] Preferably, The disubstituted adamantyl derivative or a pharma- ceutically acceptable salt thereof may be a disubstituted adamantyl derivative or a pharma- ceutically acceptable salt thereof, characterized in that it is represented by the following chemical formula 3 (Chemical Formula 24).

[0050] [ka]

[0051] In the above Chemical Formula 3 (Chemical Formula 24), R 2 is -(Y)-(CH3)m or -(Y)-(CH2)mR 4 and Y is NH, formula (25) or formula (26); The m is 0 or 1, R4 are each independently a C1 to C6 linear or branched alkyl, an unsubstituted or substituted C5 to C 10 or unsubstituted or substituted C5-C9 heteroaryl; wherein the heteroaryl is a 5- or 6-membered heteroaryl containing one or more N or O heteroatoms; The substituted aryl or substituted heteroaryl may be substituted with one or more of halogen, C1-C6 straight or branched alkyl; C1-C6 straight or branched haloalkyl; hydroxy; or C1-C6 straight or branched alkoxy. However, the above R 2 In the above, formula 27 and formula 28 may be excluded.

[0052] [ka]

[0053] [ka]

[0054] [ka]

[0055] [ka]

[0056] More preferably, In the above Chemical Formula 3 (Chemical Formula 24), R 2 may be a disubstituted adamantyl derivative, or a pharma- ceutically acceptable salt thereof, characterized in that:

[0057] [ka]

[0058] Preferably, The disubstituted adamantyl derivative or a pharma- ceutically acceptable salt thereof may be a disubstituted adamantyl derivative or a pharma- ceutically acceptable salt thereof, characterized in that it is represented by the following chemical formula 4 (Chemical Formula 30).

[0059] [ka]

[0060] In the above Chemical Formula 4 (Chemical Formula 30), R 1 is -(X)-(CH2)nR 3 Or, X is O, NH or X; wherein n is 0, 1 or 2; R 3 are each independently a C1 to C6 linear or branched alkyl, an unsubstituted or substituted C5 to C 10 or unsubstituted or substituted C5-C9 heteroaryl; wherein the heteroaryl is a 5- or 6-membered heteroaryl containing one or more N or O heteroatoms; The substituted aryl or substituted heteroaryl may be substituted with one or more of halogen, C1-C6 straight or branched alkyl; C1-C6 straight or branched haloalkyl; hydroxy; or C1-C6 straight or branched alkoxy.

[0061] [ka]

[0062] [ka]

[0063] More preferably, In the above Chemical Formula 4 (Chemical Formula 30), R1 may be a disubstituted adamantyl derivative, or a pharma- ceutically acceptable salt thereof, characterized in that:

[0064] [ka]

[0065] Preferably, The disubstituted adamantyl derivative or a pharma- ceutically acceptable salt thereof may be a disubstituted adamantyl derivative or a pharma- ceutically acceptable salt thereof, characterized in that it is represented by the following chemical formula 5 (Chemical Formula 34).

[0066] [ka]

[0067] In the above Chemical Formula 5 (Chemical Formula 34), R 2 is -(Y)-(CH3)m or -(Y)-(CH2)mR 4 and Y is NH, formula (35) or formula (36); The m is 0 or 1, R 4 are each independently a C1 to C6 linear or branched alkyl, an unsubstituted or substituted C5 to C 10 or unsubstituted or substituted C5-C9 heteroaryl; wherein the heteroaryl is a 5- or 6-membered heteroaryl containing one or more N or O heteroatoms; The substituted aryl or substituted heteroaryl may be substituted with one or more of halogen, C1-C6 straight or branched alkyl; C1-C6 straight or branched haloalkyl; hydroxy; or C1-C6 straight or branched alkoxy.

[0068] [ka]

[0069] [ka]

[0070] However, the above R 2 In the above, formula 37 and formula 38 may be excluded.

[0071] [ka]

[0072] [ka]

[0073] More preferably, In the above Chemical Formula 5 (Chemical Formula 34), R 2 may be a disubstituted adamantyl derivative, or a pharma- ceutically acceptable salt thereof, characterized in that:

[0074] [ka]

[0075] Most preferably, The disubstituted adamantyl derivative according to the invention may be any one of the following:

[0076] Methyl-3-(2-(4-(3-((methoxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10a); Methyl-3-(2-(4-(4-((4-methoxybenzyloxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10b); Methyl-3-(2-(4-(4-((3,4-dimethoxybenzyloxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10c); Methyl 3-(2-(4-(2-((3,4-dimethoxyphenethoxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10d); Methyl-3-(2-(4-(3-((furan-2-ylmethoxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10e); Methyl 3-(2-(4-(3-methylcarbamoyl-adamantan-1-yl)phenoxy)acetamido)benzoate (10f); Methyl 3-(2-(4-(3-benzylcarbamoyl-adamantan-1-yl)phenoxy)acetamido)benzoate (10g); Methyl 3-(2-(4-(3-(furan-2-ylmethylcarbamoyl)-adamantan-1-yl)phenoxy)acetamido)benzoate (10h); Methyl 3-(2-(4-(3-(pyridin-2-ylcarbamoyl)-adamantan-1-yl)phenoxy)acetamido)benzoate (10i); Methyl 3-(2-(4-(3-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-adamantan-1-yl)phenoxy)acetamido)benzoate (10j); 3,4-Dimethoxybenzyl 2-(4-(2-oxo-2-(3-(trifluoromethyl)phenylamino)ethoxy)phenyl)adamantane-1-ylcarboxylate (14a); 3,4-Dimethoxybenzyl 2-(4-(2-oxo-2-(quinolin-8-ylamino)ethoxy)phenyl)adamantane-1-ylcarboxylate (14b); 3,4-Dimethoxybenzyl 2-(4-(2-(furan-2-ylmethylamino)-2-oxoethoxy)phenyl)adamantane-1-ylcarboxylate (14c); 3,4-Dimethoxybenzyl 2-(4-(2-(4-methylpiperazin-1-yl)-2-oxoethoxy)phenyl)adamantane-1-ylcarboxylate (14d); N-(Furan-2-ylmethyl)-5-(4-(2-(4-methylpiperazin-1-yl)-2-oxoethoxy)phenyl)adamantan-1-yl-carboxamide (17a); 1-(4-Methylpiperazin-1-yl)-2-(4-(5-(4-(4-(4-(trifluoromethyl)benzyl)piperazine-1-carbonyl)adamantan-1-yl-)phenoxy)ethanone (17b); 2-(4-(5-(4-(4-Methoxybenzyl)piperazine-1-carbonyl)adamantan-1-yl)phenoxy)-1-(4-methylpiperazin-1-yl)ethanone (17c); 2-(4-(3-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)adamantan-1-yl)phenoxy)-1-morpholinoethanone (21a); 2-(4-(3-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)adamantan-1-yl)phenoxy)-1-(4-methylpiperazin-1-yl)ethanone (21b); and 2-(4-(3-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)adamantan-1-yl)phenoxy)-1-(4-(4-(trifluoromethyl)benzyl)piperazin-1-yl)ethanone (21c) (LW1564).

[0077] The symbol "Ca-b" or "Ca to Cb" attached to a substituent used in this specification means that the number of carbon atoms contained in the substituent is a to b.

[0078] In the present invention, "C1-C6 straight or branched alkyl" refers to a linear or branched saturated hydrocarbon group having 1 to 6 carbon atoms, including, but not limited to, methyl, ethyl, propyl, butyl, 1-methylethyl, diethyl, or dimethyl.

[0079] In the present invention, "C1-C6 straight or branched alkoxy" refers to an alkoxy group in which R is C 1-6 and OR groups, which are alkyl groups, including, but not limited to, methoxy, ethoxy, propoxy, butoxy, 1-methylethoxy, 1,1-dimethylethoxy, and the like.

[0080] In the present invention, "halogen" is fluorine, bromine, chlorine or iodine.

[0081] As used herein, "haloalkyl" means an alkyl substituted with one or more halogen atoms.

[0082] The disubstituted adamantyl derivatives represented by Chemical Formula 1 (Chemical Formula 1, Chemical Formula 2) of the present invention can be used in the form of a pharma- ceutically acceptable salt, and the term "pharma-ceutically acceptable" in the present invention includes compounds or compositions that are within medical standards and can be used without problems when contacted with tissues of biological subjects similar to humans without undue risk of toxicity, irritation, and other problems or complications. In the present invention, acid addition salts formed with pharma-ceutically acceptable free acids are useful as salts. Acid addition salts can be obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, or phosphorous acid, and non-toxic organic acids such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanolates, hydroxyalkanoates, and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids, and organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, and fumaric acid. Such pharma- ceutically harmless salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyrate-1,4-dioate, hexane-1,6-dioate, benzoate, The term "phenylbenzoate" includes, for example, the phenylsulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, beta-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate salts.

[0083] The acid addition salts according to the invention can be prepared in the usual manner, for example by dissolving the compound in an excess of aqueous acid and precipitating the salt with a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile, or by heating equimolar amounts of the compound and the acid or alcohol in water, followed by evaporating the mixture and drying or suctioning the precipitated salt and filtering it.

[0084] Also, a base can be used to prepare a pharma- ceutically acceptable metal salt. Salts derived from a suitable base can include, but are not limited to, alkali metal salts such as sodium and potassium, alkaline earth metal salts such as magnesium, and ammonium. For example, an alkali metal or alkaline earth metal salt can be obtained by dissolving a compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, it is particularly suitable for pharmaceutical purposes to prepare sodium, potassium, or calcium salts as metal salts, and the corresponding silver salts can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0085] Furthermore, the present invention includes not only the disubstituted adamantyl derivatives of the above formula 1 (Chemical Formula 1, Chemical Formula 2) and their pharma- ceutical acceptable salts, but also all solvates, hydrates, isomers, etc. that can be produced therefrom.

[0086] More specifically, the compounds of the present invention contain asymmetric or chiral centers and can exist in stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention, such as diastereomers, enantiomers, and racemic mixtures, are considered to be part of the present invention, where a mixture of isomers is referred to as an enantiomer, and a 50:50 ratio mixture of enantiomers is referred to as a racemate.

[0087] In the present invention, "diastereomer" refers to a stereoisomer with two or more chiral centers whose molecules are not mirror images of one another. Diastereomers have different physical properties, for example, melting points, boiling points, spectral properties, and reactivity. Mixtures of diastereomers can be separated through high resolution analytical processes such as electrophoresis and chromatography.

[0088] As used herein, "enantiomers" refers to two stereoisomers of a compound which are unique mirror images of one another.

[0089] The present invention also provides a method for producing a disubstituted adamantyl derivative, which comprises producing a compound represented by Chemical Formula 1 (Chemical Formula 1, Chemical Formula 2) according to the following reaction formula (Chemical Formula 40).

[0090] [ka]

[0091] More specifically, the present invention provides a method for producing a disubstituted adamantyl derivative, which comprises producing a compound represented by Chemical Formula 2 (Chemical Formula 20) according to the following reaction formula (Chemical Formula 41).

[0092] [ka]

[0093] More specifically, the present invention provides a method for producing a disubstituted adamantyl derivative, which comprises producing a compound represented by Chemical Formula 3 (Chemical Formula 24) according to the following reaction scheme.

[0094] [ka]

[0095] More specifically, the present invention provides a method for producing a disubstituted adamantyl derivative, which comprises producing a compound represented by Chemical Formula 4 (Chemical Formula 30) according to the following reaction scheme.

[0096] [ka]

[0097] More specifically, the present invention provides a method for producing a disubstituted adamantyl derivative, which comprises producing a compound represented by Chemical Formula 5 (Formula 34) according to the following reaction scheme.

[0098] [ka]

[0099] The method for preparing the disubstituted adamantyl derivative of the present invention is not limited to the above-mentioned method. In addition, in addition to the reagents described in the above reaction scheme, the reaction can be carried out by selectively changing the reagents that do not affect the reaction.

[0100] The present invention also provides an anticancer pharmaceutical composition comprising, as an active ingredient, a disubstituted adamantyl derivative or a pharma- ceutically acceptable salt thereof.

[0101] In the present invention, the pharmaceutical composition may be an anticancer pharmaceutical composition for solid cancer, Examples of the solid cancer include, but are not limited to, colorectal cancer, lung cancer, liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, paraanal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, prostate cancer, fibrosarcoma, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvis cancer, and central nervous system tumors.

[0102] In the present invention, "anticancer use" includes "prevention or treatment of cancer" and means inhibiting or delaying the onset of cancer in a biological subject (e.g., a human or an animal), and further suppressing or stopping the progression of cancer, slowing down the rate of progression, improving or alleviating symptoms, etc.

[0103] The present invention also provides a method for treating cancer, comprising administering a therapeutically effective amount of the disubstituted adamantyl derivative represented by Chemical Formula 1 (Chemical Formula 1 or 2) or a pharma- ceutically acceptable salt thereof to a patient in need of anticancer treatment. The present invention also provides use of the disubstituted adamantyl derivative represented by Chemical Formula 1 (Chemical Formula 1 or 2) or a pharma- ceutically acceptable salt thereof in the manufacture of a preparation for treating cancer. The present invention also provides use of the disubstituted adamantyl derivative represented by Chemical Formula 1 (Chemical Formula 1 or 2) or a pharma- ceutically acceptable salt thereof in treating cancer.

[0104] In the present invention, "effective amount" refers to an amount of the compound of the present invention that treats or prevents a particular disease or condition, or alleviates, reduces or eliminates the symptoms of a particular disease or condition, or prevents or delays the onset of one or more symptoms of a particular disease or condition described herein. In the present invention, an effective amount of a drug treatment refers to tumor growth inhibition; inhibition of HIF-1 activation and proliferation of cancer cells; inhibition of mitochondrial respiration; alleviation or treatment of symptoms of complications caused by cancer; delay and inhibition (preferably improvement) of the rate of cancer progression; prevention of cancer progression, etc. The level of an effective amount can be determined by factors including the type and severity of the patient's disease, the activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, drugs used simultaneously, and other factors well known in the medical field.

[0105] In the present invention, the "patient" is not limited, and is preferably a mammal, including a human. The content of the disubstituted adamantyl derivative of the formula 1 (Chemical Formula 1, Chemical Formula 2) or a pharma- ceutical acceptable salt thereof in the composition of the present invention can be appropriately adjusted according to the symptoms of the disease, the degree of progression of the symptoms, the pathological condition of the patient, etc., and may be, for example, 0.0001 to 99.9% by weight or 0.001 to 50% by weight based on the weight of the total composition, but is not limited thereto. The content is based on the dry weight after removing the solvent.

[0106] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions, such as one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and release control additives.

[0107] The pharmaceutical composition according to the present invention can be formulated and used in the form of external preparations such as powders, granules, or sustained-release granules, enteric-coated granules, liquids, eye drops, elixirs, emulsions, suspensions, cast tablets, troches, aromatic perfumes, lemonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tanning agents, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusion solutions, warning agents, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, etc., according to the usual methods, and the external preparations can have the dosage forms of creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastes, or cataplasms.

[0108] Carriers, excipients and diluents that may be included in pharmaceutical compositions according to the invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0109] When the pharmaceutical preparation is prepared, it is produced using a diluent or excipient such as a commonly used filler, extender, binder, wetting agent, disintegrant, surfactant, etc.

[0110] As additives for tablets, powders, granules, capsules, pills and lozenges according to the present invention, excipients such as corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium monohydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, tablet lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose, 1928, 2208, 2906, 2910, propylene glycol, casein, calcium lactate, primozel; gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, Binders such as dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, tablet shellac, starch powder, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone are used, and disintegrants such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium methylcellulose carboxylate, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelling starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, white sugar, magnesium aluminum silicate, D-sorbitol liquid, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acid, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid may be used.

[0111] Examples of additives that can be used in the liquid preparation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, and sodium carboxymethyl cellulose.

[0112] The syrup preparation according to the present invention may be a solution of sucrose, other sugars or sweeteners, and may contain, if necessary, flavorings, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, etc.

[0113] The emulsion of the present invention may contain purified water, and may contain emulsifiers, preservatives, stabilizers, fragrances, etc., as required.

[0114] As the suspending agent according to the present invention, acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose, 1828, 2906, 2910 and the like can be used, and surfactants, preservatives, stabilizers, coloring agents and fragrances can be used as necessary.

[0115] Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, benzene benzoate and other solvents; sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, twines, nicotinamide, hexamine, dimethylacetamide and other solubilizing agents; weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), ), buffering agents such as organic compounds, proteins, albumin, peptones, and gums; isotonicity agents such as sodium chloride; stabilizers such as sodium bisulfite (Na2SO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; sulfating agents such as sodium disulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and acetone sodium bisulfite; soothing agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium CMC, sodium alginate, Tween 80, and aluminum monostearate.

[0116] Suppositories according to the invention include, but are not limited to, cocoa butter, lanolin, Witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, coconut oil, cocoa butter + cholesterol, lecithin, lanet wax, glycerol monostearate, Tween or Span, Imhausen, mono (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, Hexalite Base 95, Cotomar, Hydrokote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrokote 25 Bases such as Hydrocote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Masa-MF, Maspol, Maspol-15, Neospostal-en, Paramound-B, Sposilo (OSI, OSIX, A, B, C, D, H, L), Suppository Base Type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Spostal (N, Es), Wecoby (W, R, S, M, Fs), and Tezestar triglyceride base (TG-95, MA, 57) can be used.

[0117] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and are prepared by mixing at least one of the extracts with excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stylate talc are also used.

[0118] Liquid preparations for oral administration include suspensions, liquid preparations for internal use, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0119] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered in a single or multiple doses. For example, the pharmaceutical composition of the present invention can be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using biological response modifiers. Taking all of the above factors into consideration, it is important to administer an amount that provides maximum effect with the minimum amount without side effects, which can be easily determined by a person of ordinary skill in the art to which the present invention pertains.

[0120] The pharmaceutical composition of the present invention can be administered to an individual by various routes, all manners of administration can be envisaged, for example, oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intradural) injection, sublingual administration, buccal administration, rectal insertion, vaginal insertion, ocular administration, aural administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, etc.

[0121] The pharmaceutical composition of the present invention is determined by the type of drug as the active ingredient along with several related factors such as the disease to be treated, the administration route, the patient's age, sex, weight and the severity of the disease.

[0122] In the present invention, the dosage may vary depending on the condition, age, body weight, sex and body weight of the patient, the severity of the disease, the drug form, the administration route and time, and may be appropriately selected by a person of ordinary skill in the art. For example, the dosage may be 0.01 to 200 mg / kg / day, which may be administered orally or parenterally at regular intervals several times a day, preferably once to three times a day, according to the judgment of a physician or pharmacist.

[0123] The present invention will be described in detail below with reference to Production Examples, Examples and Experimental Examples.

[0124] However, the following Production Examples, Examples and Experimental Examples are intended to specifically illustrate the present invention, and the contents of the present invention are not limited to these Production Examples, Examples and Experimental Examples.

[0125] [Production Example] Manufacturing example: Synthesis of compounds with HIF-1α inhibitory activity Non-limiting examples of compounds that are HIF-1α inhibitors according to the present invention and that can be used as active ingredients in pharmaceutical compositions include the following compounds, their isomers, and pharma- ceutically acceptable salts thereof. By appropriately modifying reactants and / or starting materials of known methods, the following compounds according to the present invention were synthesized.

[0126] The following is a detailed explanation:

[0127] To develop novel HIF-1α inhibitors, chemical modifications were performed at the adamantyl moiety of LW6 to produce a variety of disubstituted adamantyl derivatives. The synthetic methods of the novel compounds are illustrated in Scheme S1-4. Bromination of 1-adamantanecarboxylic acid 1 with bromine in acetic acid gave product 2, which was then subjected to Friedel-Crafts alkylation with anisole to give ether product 3 in good yield. Demethylation of product 3 with boron tribromide (BBr3) and subsequent chemo-selective protection of product 4 with benzyl bromide gave product 5 under basic conditions, which was further alkylated with ethyl chloroacetate to give product 6 in good yield. Hydrolysis of product 6 with lithium hydroxide (LiOH) gave carboxylic acid product 7, which was then coupled with methyl 3-aminobenzoate to give amide derivative product 8 in good yield. The benzyl group of product 8 was removed by catalytic hydrogenation in the presence of palladium on carbon to regenerate the precursor for the synthesis of the target adamantyl derivatives 10a-j. Carboxylic acid compound 9 could be reacted with alcohols and amines to provide esters (10a-e) and amide derivatives (10f-j), respectively. Products 10a-d, f, g, and j were synthesized using coupling agents hydroxybenzotriazole (HOBt) and EDC.HCl, whereas other coupled products 10e, h were synthesized using ethyl chloroformate and triethylamine (TEA) (Scheme S1). As shown in Scheme S2, product 4 was treated with 3,4-dimethoxybenzyl bromide in the presence of potassium carbonate (K2CO3) to generate the phenolic ester product 11, which was further alkylated with ethyl chloroacetate to give product 12 in good yield. Carboxylic acids 13 were then obtained through base-mediated hydrolysis of products 12. Finally, the corresponding amides 14a-d were prepared through EDC.HCl and HOBt mediated coupling with various commercially available amines (Scheme S2).Similarly, the carboxylic acid intermediate 7 was reacted with 1-methylpiperazine in the presence of EDC.HCl and HOBt to give the amide derivative 15. Catalytic hydrogenation of 15 in the presence of palladium on carbon produced the corresponding carboxylic acid product 16. Coupling with various amines produced the respective amide derivatives (17a-c) (Scheme S3). Based on the synthesis profile of 14a-d, the ester moiety of the carboxylic acid in product 11 was replaced with amide to give 21a-c, as shown in Scheme S4. Coupling of product 4 under amide coupling conditions produced intermediate 18, which was then alkylated with ethyl chloroacetate to give product 19 in good yield. Hydrolysis of product 19 with LiOH provided product 20, which was then coupled with various commercially available amines to give disubstituted adamantyl derivatives 21a-c (Scheme S4).

[0128] [ka]

[0129] a Reagents and conditions: (a) AlCl3,Bromine,DCM;(b)Anisole,AlC3;(c)BBr3,DCM;(d) Benzyl bromide,KHCO3,DMF; (e) Ethyl chloroacetate,K2CO3,DMF;(f)LiOH,THF / H2O; (g) Methyl 3-amino benzoate,PyBOP,DMAP,DMF;(h) H2,Pd / C,THF; (i) EDC,HOBT,DIPEA,DMF for10a-d,10f-g,10j; ethyl chloroformate,TEA,THF for 10e,h;

[0130] [ka]

[0131] aReagents and conditions: (a) 3,4-Dimethoxybenzyl bromide,K2CO3,DMF; (b) ethyl chloroacetate,K2CO3,DMF;(c)LiOH,THF / H2O;(d)correspondingamine,EDC,HOBT,DIPEA,DMF.

[0132] [ka]

[0133] a Reagents and conditions: (a) 1-Methyl piperazine,EDC,HOBT,DIPEA,DMF; (b) Pd / C,methanol; (c) corresponding amines,EDC,HOBT,DIPEA,DMF.

[0134] [ka]

[0135] a Reagents and conditions: (a) 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline,EDC,HOBT,DIPEA,DMF; (b) Ethyl chloroacetate,K2CO3,DMF;(c)LiOH,THF / H2O;(d)corresponding amines,EDC,HOBT,DIPEA,DMF.

[0136] [Example] Example 1. Synthesis of Compounds 2 to 9 The synthesis steps of compounds 2 to 9 used in the above reaction scheme are as follows.

[0137] Compound 2: 3-Bromoadamantane-1-carboxylic acid (2): AlCl3 (4.80 g, 36.10 mmol) was placed in a two-necked round flask equipped with a reflux condenser and argon. The flask was stirred in a cold reactor. Bromine (17.10 mL, 33.29 mmol) was added at -5°C and stirred for 15 min. 1-Adamantanecarboxylic acid (5.00 g, 27.70 mmol) was then added to the flask and stirred at -5°C for 1 h and room temperature for 48 h. The reaction was diluted with chloroform. Excess bromine was treated with sodium pyrosulphite until complete discoloration occurred. The organic layer was dried over anhydrous MgSO4, filtered, and concentrated. The residue was recrystallized from hexane to give (2) as a white solid (6.67 g, 93% yield). 1 H-NMR (400MHz, DMSO-d6) δ(s,1H), 2.36(s,2H), 2.26-2.13(m,6H), 1.79(d,J=2.8Hz,4H), 1.68-1.60(m,2H).

[0138] Compound 3: 3-(4-Methoxyphenyl)-adamantane-1-carboxylic acid (3): Aluminum chloride (5.14 g, 38.58 mmol) was suspended in 52 mL of anisole, and compound 2 (5.00 g, 19.29 mmol) was added at -10°C. The reaction mixture was stirred at room temperature for 24 h, and then ice and conc. HCl were added dropwise. The mixture was extracted with EA and separated. The organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure. The concentrate was recrystallized from hexane to give (3) as a white solid (5.20 g, 94% yield). 1 H-NMR (400MHz, DMSO-d6) δ(s,1H), 7.27-7.24(m,2H), 6.88-6.84(m,2H), 3.71(s,3H), 2.13(s,2H), 1.90-1.77(m,10H), 1.66(s,2H).

[0139] Compound 4: 3-(4-Hydroxyphenyl)-adamantane-1-carboxylic acid (4): To a solution of compound 3 (2.00 g, 6.98 mmol) in DCM was added a solution of 1.0 M BBr3 (17.40 mL, 17.45 mmol) diluted in DCM under argon at -10 °C. The reaction solution was stirred at room temperature until the starting material disappeared (monitored by TLC). The organic layer was dried over anhydrous MgSO4 and concentrated to give (4) as a white solid (1.80 g, 94% yield). 1 H-NMR(400MHz,DMSO-d6)δ1H), 9.11(s,1H), 7.13(d,J=9.2Hz,2H), 6.69(d,J=8.8Hz,2H), 2.12(s,2H), 1.83-1.75(m,10H), 1.65(s,2H).

[0140] Compound 5: 3-(4-Hydroxyphenyl)-adamantane-1-carboxylic acid benzyl ester (5): Benzyl bromide (1.12 g, 6.61 mol) was added to a mixture of compound 4 (1.50 g, 5.50 mmol) and KHCO3 (0.66 g, 6.61 mol) in 20 mL of DMF. The reaction mixture was heated at 40° C. for 4 h, and then saturated NaHCO3 was added dropwise. The mixture was extracted with EA and separated. The organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure. The concentrate was purified by column chromatography on silica gel to give (5) as a white solid (1.75 g, 87% yield). 1 H-NMR (400MHz, CDCl3) δ7.35δJ=J=10H,1.69(s,2H).

[0141] Compound 6: 3-(4-Ethoxycarbonylmethoxyphenyl)-adamantane-1-carboxylic acid benzyl ester (6): Ethyl chloroacetate (0.67 g, 5.50 mmol) was added to a mixture of compound 5 (1.0 g, 2.75 mmol) and potassium carbonate (1.14 g, 8.27 mmol) in 10 mL of DMF. The reaction mixture was stirred at room temperature overnight, and then saturated NaHCO3 was added dropwise. The mixture was extracted with EA and dried over anhydrous MgSO4. The mixture was filtered and concentrated under reduced pressure. The concentrate was purified by column chromatography on silica gel to give (6) as a white solid (1.05 g, 85% yield). 1 H-NMR(400MHz,DMSO-d6)7.39-7.31(m,5H), 7.26(d,J=8.4Hz,2H), 6.84(d,J=8.8Hz,2H), 5.00(s,2H), 4 .72(s,2H), 4.16(q,J=7.1Hz,2H), 2.15(s,2H), 1.90-1.79(m,10H), 1.67(s,2H), 1.21(t,J=7.0Hz,3H).

[0142] Compound 7: 3-(4-carboxymethoxyphenyl)-adamantane-1-carboxylic acid benzyl ester (7): Compound 6 (0.70 g, 1.56 mmol) was dissolved in 7 mL of THF / H2O (1:1). Lithium hydroxide (0.26 g, 6.24 mmol) was added and stirred at room temperature for 90 min, after which 10% HCl was added dropwise. The mixture was extracted with EA and separated. The organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure to obtain the concentrate, which was then purified by column chromatography on silica gel to obtain (7) as a white solid (0.60 g, 92% yield). 1 H-NMR(400MHz,DMSO-d6)12.93(s,1H), 7.37-7.31(m,5H), 7.26(d,J=8.4Hz,2H), 6.84(d ,J=8.8Hz,2H), 5.09(s,2H), 4.61(s,2H), 2.15(s,2H), 1.98-1.75(m,10H), 1.67(s,2H).

[0143] Compound (8): 3-{4-[(3-Methoxycarbonylphenylcarbamoyl)-methoxy]-phenyl}-adamantane-1-carboxylic acid benzyl ester (8): A mixture of compound 7 (0.73 g, 1.74 mmol), 3-aminobenzoic acid methyl ester (0.52 g, 3.47 mmol), PyBOP (1.80 g, 3.47 mmol) and DMAP (0.42 g, 3.47 mmol) was dissolved in 20 mL of DMF solvent. The reaction solution was stirred at room temperature overnight, and then 10% HCl was added dropwise. The mixture was extracted with EA and separated. The organic layer was dried over anhydrous MgSO4 and concentrated. The concentrate was purified by column chromatography on silica gel to obtain (8) as white crystals (0.68 g, 72.00%). 1 H-NMR(400MHz,DMSO-d6)δ(s,1H), 8.33(s,1H), 7.90(d,J=8.1Hz,1H), 7.68(d,J=8.1Hz,1H), 7.48(t,J=8.1Hz,1H), 7.40-7.33(m,5H), 7.30(d,J =8.7Hz,2H), 6.94(d,J=8.7Hz,2H), 5.09(s,2H), 4.69(s,2H), 3.86(s,3H), 2.16(br,2H), 1.92(s,2H), 1.86(s,4H), 1.81(br,4H), 1.68(br,2H).

[0144] Compound 9: 3-{4-[(3-Methoxycarbonylphenylcarbamoyl)-methoxy]-phenyl}-adamantane-1-carboxylic acid (9): Compound 8 (0.62 g, 1.12 mmol) was dissolved in 30 mL of THF and 10% Pd / C was added. The reaction mixture was stirred at room temperature under H2 for 2 h and filtered through a Celite pad. The solution was concentrated under reduced pressure to give (9) as a white solid (0.78 g). 1 H-NMR(400MHz,DMSO-d6)δ1H), 10.31(s,1H), 8.33(s,1H), 7.90(d,J=8.1Hz,1H), 7.67(d,J=8.1Hz,1H), 7.48(t,J=8.1Hz,1H), 7.30(d,J=8.7Hz,2H), 6.94(d,J=8.7Hz,2H), 4.69(s,2H), 3.85(s,3H), 2.14(br,2H), 1.87(s,2H), 1.80(br,8H), 1.66(br,2H).

[0145] Example 2. Synthesis of Compounds 10-1 to 10-10 According to the above-mentioned reaction formula S1, compounds 10-1 to 10-10 were synthesized using compound 1 as a starting material, via compounds 2, 3, 4, 5, 6, 7, 8 and 9 as intermediates.

[0146] Compound 10-1: Methyl-3-(2-(4-(3-((methoxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10a): Compound 9 (0.31 g, 0.66 mmol), methyl alcohol (0.03 ml, 0.99 mmol), 1-(3-dimethylaminopropyl)-3-hydrate (0.13 g, 0.99 mmol), HOBt (0.13 g, 0.99 mmol) and DIPEA (0.17 ml, 0.99 mmol) were dissolved in dimethylformamide at room temperature and stirred overnight. When stirring was completed, water was added to quench the reaction, followed by extraction with EA. The extracted organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure. The concentrated reaction was purified by column chromatography on silica gel (DCM / EA) to give 10a as a white solid (0.37 g, 78%). 1H-NMR(400MHz,CDCl3)8.41(s,1H), 8.07(s,1H), 8.01(d,J=8.4Hz,1H), 7.83(d,J=8.0Hz,1H), 7.44(t,J=8.0Hz,1H), 7.34(d,J=8. 8Hz,2H), 6.96(d,J=8.8Hz,2H), 4.61(s,2H), 3.92(s,3H), 3.67(s,3H), 2.23(s,2H), 2.01(s,2H), 1.92-1.86(m,8H), 1.73(s,2H).

[0147] Compound 10-2: Methyl-3-(2-(4-(4-((4-methoxybenzyloxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10b): Compound 9 (0.31 g, 0.66 mmol), 4-methoxybenzoyl alcohol (0.12 ml, 0.99 mmol), EDC.HCI (0.19 g, 0.99 mmol), HOBt (0.13 g, 0.99 mmol) and DIPEA (0.17 ml, 0.99 mmol) were dissolved in dimethylformamide at room temperature and then stirred overnight. When stirring was completed, water was added to quench the reaction, followed by extraction with EA. The extracted organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure. The concentrated reaction was purified by column chromatography on silica gel (DCM / EA) to give the target compound 10b as a white solid (0.21 g, 54%). 1 H-NMR(400MHz,CDCl3)8.49(s,1H), 8.10(s,1H), 7.99(d,J=7.6Hz,1H), 7.81(d,J=7.2Hz,1H), 7.41(t,J=7.8Hz,1H), 7.31-7.25(m,4 H), 6.90(dd,J=8.8,23.2Hz,4H), 5.22(s,2H), 4.58(s,2H), 3.89(s,3H), 3.78(s,3H), 2.21(s,2H), 2.03-1.85(m,10H), 1.71(s,2H).

[0148] Compound 10-3: 3-(2-(4-(4-((3,4-dimethoxybenzyloxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10c): Compound 9 (0.31 g, 0.66 mmol), 3,4-dimethoxybenzyl alcohol (0.14 ml, 0.99 mmol), EDC.HCl (0.19 g, 0.99 mmol), HOBt (0.13 g, 0.99 mmol) and DIPEA (0.17 ml, 0.99 mmol) were dissolved in dimethylformamide at room temperature and then stirred overnight. Upon completion of stirring, water was added to quench the reaction, followed by extraction with EA. The extracted organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure. The concentrated reaction was purified by column chromatography on silica gel (DCM / EA) to give the target compound 10c as a white solid (0.25 g, 63%). 1 H-NMR(400MHz,CDCl3)8.38(s,1H), 8.07(s,1H), 8.01(d,J=8.4Hz,1H), 7.83(d,J=8.0Hz,1H), 7.45(t,J=7.8Hz,1H), 7.33(d,J=8.8 Hz,2H), 6.96-6.83(m,5H), 5.06(s,2H), 4.61(s,2H), 3.93(s,3H), 3.88-3.87(m,6H), 2.23(s,2H), 2.03-1.73(m,10H), 1.57(s,2H).

[0149] Compound 10-4: Methyl 3-(2-(4-(2-((3,4-dimethoxyphenethoxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10d): Compound 9 (0.31 g, 0.66 mmol), 2-(3,4-dimethoxyphenyl)ethanol (0.18 g, 0.99 mmol), EDC.HCl (0.19 g, 0.99 mmol), HOBt (0.13 g, 0.99 mmol) and DIPEA (0.17 ml, 0.99 mmol) were dissolved in DMF at room temperature and then stirred overnight. When stirring was completed, water was added to quench the reaction and then extracted with EA. The extracted organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure. The concentrated reaction was purified by column chromatography on silica gel (DCM / EA) to give the target compound 10d as a white solid (0.36 g, 58%). 1 H-NMR(400MHz, CDCl3)8.44(s,1H), 8.09(s,1H), 8.01(d,J=8.4Hz,1H), 7.82(d ,J=8.0Hz,1H), 7.43(t,J=8.0Hz,1H), 7.31(d,J=8.8Hz,1H), 6.95(d,J=8.8Hz,2 H), 6.81-6.74(m,3H), 4.60(s,2H), 4.26(t,J=6.8Hz,2H), 3.91(s,3H), 3.85-3. 83(m,6H), 2.88(t,J=7.0Hz,2H), 2.22(s,2H), 1.97-1.80(m,10H), 1.72(s,2H).

[0150] Compound 10-5: Methyl-3-(2-(4-(3-((furan-2-ylmethoxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10e): Compound 9 (60.0 mg, 0.13 mmol) was dissolved in tetrahydrofuran (THF) and cooled to -10°C. Then, 2-(bromomethyl)furan (0.02 ml, 0.17 mmol), ethyl chloroformate (0.02 ml, 15 mmol) and triethylamine (0.02 ml, 0.15 mmol) were loaded onto the column. The reaction mixture was stirred at room temperature for 30 minutes, and then water was poured in to terminate the reaction. The reaction solution was extracted with EA, and the extracted organic layer was dried over MgSO4 and concentrated under reduced pressure. The concentrated reaction product was purified by silica gel column chromatography (DCM / methanol) to obtain the target compound 10e as a white solid (49.0 mg, 70%). 1 H-NMR(400MHz,CDCl3)8.51(s,1H), 8.10(s,1H), 7.99(d,J=8.0Hz,1H), 7.81(d,J=7.6Hz,1H), 7.43-7.39(m,2H), 7.30(d,J=8.8Hz,2H), 6 .93(d,J=8.4Hz,2H), 6.36(dd,J=2.2,11.4Hz,2H), 5.05(s,2H), 4.59(s,2H), 3.90(s,3H), 2.21(s,2H), 1.99-1.84(m,10H), 1.70(s,2H).

[0151] Compound 10-6: Methyl 3-(2-(4-(3-methylcarbamoyl-adamantan-1-yl)phenoxy)acetamido)benzoate (10f): EDC.HCl (80 mg, 0.42 mmol), HOBt (56 mg, 0.42 mmol) and DIPEA (0.15 mL, 0.87 mmol) were added into a solution of compound 9 (160 mg, 0.35 mmol) and methylamine hydrochloride (0.02 g, 0.35 mmol) in DMF (5.0 mL). After stirring the reaction mixture at room temperature overnight, the resulting mixture was diluted with EtOAc and washed with water and brine. The organic layer was separated, dried over anhydrous MgSO4, filtered and concentrated in vacuum. The resulting residue was purified by silica gel column chromatography (hexane:EA=6:4) to give 10f as a white solid (110 mg, 66.1% yield). 1 H-NMR (CDCl3,400MHz) 8.40(s,1H), 8.07(s,1H), 8.00(dd,J=8.2,1.4Hz,1H), 7.83(d,J=7.8Hz,1H), 7.44(t,J=8.0Hz,1H), 7.34(d,J=8.4H) z,2H), 6.95(d,J=9.2Hz,2H), 5.67(s,1H), 4.61(s,2H), 3.92(s,3H), 2.81(d,J=4.4Hz,3H), 2.26(s,2H), 1.97-1.87(m,10H), 1.73(s,2H).

[0152] Compound 10-7: Methyl 3-(2-(4-(3-benzylcarbamoyl-adamantan-1-yl)phenoxy)acetamido)benzoate (10g): EDC.HCl (80 mg, 0.42 mmol), HOBt (56 mg, 0.42 mmol) and DIPEA (0.15 mL, 0.87 mmol) were added to a solution of compound 9 (160 mg, 0.35 mmol) and benzylamine (37 mg, 0.35 mmol) in DMF (5.0 mL). After stirring the reaction mixture at room temperature overnight, the resulting mixture was diluted with EtOAc and washed with water and brine. The organic layer was separated, dried over anhydrous MgSO4, filtered and concentrated in vacuum. The resulting residue was purified by silica gel column chromatography (hexane:EA=6:4) to give 10g as a white solid (110 mg, 56.9% yield). 1 H-NMR(400MHz,CDCl3)8.38(s,1H), 8.07(s,1H), 8.01(d,J=8.4Hz,1H), 7.83(d,J=8.0Hz,1H), 7.45(t,J=8.0Hz,1H), 7.33(t,J=7.6Hz) ,4H), 7.29-7.26(m,5H), 5.89(s,1H), 4.61(s,2H), 4.45(d,J=5.2Hz,2H), 3.92(s,3H), 2.27(s,2H), 2.04-1.90(m,10H), 1.74(s,2H).

[0153] Compound 10-8: Methyl 3-(2-(4-(3-(furan-2-ylmethylcarbamoyl)-adamantan-1-yl)phenoxy)acetamido)benzoate (10h): Triethylamine (0.022 mL, 0.155 mmol) was added to a solution of compound 9 (60 mg, 0.129 mmol) in 1 mL of THF and the mixture was cooled to -10°C. Ethyl chloroformate (0.016 mL, 0.168 mmol) was then added and stirred at -10°C for 30 min. Furfurylamine (0.015 mL, 0.155 mmol) was then added to the solution and allowed to warm to room temperature. The reaction solution was stirred at room temperature for an additional 30 min and the solvent was evaporated under reduced pressure. The residue was purified by preparative TLC to give 10h as a pale yellow solid (49 mg, 70% yield).1 H-NMR(400MHz,CDCl3)δ(s,1H), 8.07(s,1H), 8.01(dd,J=1.2-8.4Hz,1H), 7.83(d, J=8.0Hz,1H), 7.45(t,J=8.0Hz,1H), 7.34(d,J=8.8Hz,3H), 6.96(d,J=9.2Hz,2H), 6.32(t,J=2.4Hz,1H), 6.21(d,J=3.2Hz,1H), 4.61(s,2H), 4.44(d,J=5.2Hz,2H), 3 .92(s,3H), 2.26(s,2H), 1.98(s,2H), 1.90-1.88(m,8H), 1.73(s,2H), 1.55(s,2H).

[0154] Compound 10-9: 3-2-(4-(3-(pyridin-2-ylcarbamoyl)-adamantan-1-yl)phenoxy)acetamido)benzoate (10i): DMF (0.5 mL) was added to a mixture of compound 9 (50 mg, 0.11 mmol), 2-aminopyridine (20 mg, 0.21 mmol), PyBOP (110 mg, 0.21 mmol) and DMAP (26 mg, 0.216 mmol). The reaction solution was then stirred at room temperature overnight, after which 10% HCl was added dropwise. The mixture was extracted with EA, and the combined organic layer was dried over anhydrous MgSO4. The filtrate was concentrated under reduced pressure, and the residue was purified by preparative TLC (n-hexane:EA=1:1) to give 10i as a yellow solid (30 mg, 52% yield). 1H-NMR(300MHz,DMSO-d6)δ1H), 9.78(s,1H), 8.33(d,J=2.1Hz,1H), 8.31(s,1H), 8.06 (d,J=8.7Hz,1H), 7.90(d,J=7.8Hz,1H), 7.56(t,J=7.5Hz,1H), 7.67(d,J=7.2Hz,1H), 7.48(t,J=8.1Hz,1H), 7.36(d,J=9Hz,2H), 7.09(t,J=7.5Hz,1H), 6.96(d,J=9Hz,2H), 4.69(s,2H), 3.85(s,3H), 2.18(br,2H), 2.04(s,2H), 1.94(s,3H), 1.89-1.69(m,7H).

[0155] Compound 10-10: 3-(2-(4-(3-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-adamantan-1-yl)phenoxy)acetamido)benzoate (10j): EDC.HCl (80 mg, 0.42 mmol), HOBt (56 mg, 0.42 mmol) and DIPEA (0.15 mL, 0.87 mmol) were added to a solution of compound 20 (100 mg, 0.35 mmol) and 3-aminobenzoic acid methyl ester (53 mg, 0.35 mmol) in DMF (5.0 mL). After stirring the reaction mixture at room temperature overnight, the resulting mixture was diluted with EtOAc and washed with water and brine. The organic layer was separated, dried over anhydrous MgSO4, filtered and concentrated in vacuum. The resulting residue was purified by silica gel column chromatography (hexane:EA=6:4) to give a white solid as 10j (107 mg, 48% yield). 1H-NMR(CDCl3,400MHz)8.39(s,1H), 8.07(s,1H), 8.02(d,J=8.0Hz,1H), 7.83 (d,J=7.6Hz,1H), 7.45(t,J=7.8Hz,1H), 7.36(d,J=8.8Hz,1H), 6.97(d,J=8.4 Hz,2H), 6.60(d,J=5.2Hz,2H), 4.71(s,2H), 4.61(s,2H), 3.92(s,5H), 3.85(s ,6H), 2.81(s,2H), 2.28(s,2H), 2.15-2.07(m,6H), 1.91(s,4H), 1.77(s,4H).

[0156] Example 3. Synthesis of Compounds 14-1 to 14-4 According to the above-mentioned reaction formula S2, compounds 14-1 to 14-4 were synthesized using compound 4 as a starting material and compounds 11, 12, and 13 as intermediates.

[0157] Compound 14-1: Compound 3,4-Dimethoxybenzyl 2-(4-(2-oxo-2-(3-(trifluoromethyl)phenylamino)ethoxy)phenyl)adamantane-1-ylcarboxylae (14a): EDC.HCl (42 mg, 0.22 mmol), HOBt (29 mg, 0.22 mmol) and DIPEA (0.080 mL, 0.47 mmol) were added to a solution of compound 13 (90 mg, 0.19 mmol) and 3-(trifluoromethyl)aniline (33 mg, 0.20 mmol) in DMF (5.0 mL). After stirring the reaction mixture at room temperature overnight, the resulting mixture was diluted with EtOAc and washed with water and brine. The organic layer was separated, dried over anhydrous MgSO4, filtered and concentrated in vacuum. The resulting residue was purified by silica gel column chromatography (hexane:EA=6:4) to give 14a as a white solid (64 mg, 54% yield). 1H-NMR (CDCl3,400MHz)8.39(s,1H), 7.87(s,1H), 7.82(d,J=8.4Hz,1H), 7.48(t,J= 7.8Hz,1H), 7.41(d,J=8.0Hz,1H), 7.33(d,J=8.8Hz,2H), 6.95(d,J=9.2Hz,2H), 6. 91(dd,J=8.4,2.0Hz,1H), 6.85(s,1H), 6.84(d,J=10.8Hz,1H), 5.05(s,2H), 4.61( s,2H), 3.88(s,3H), 3.87(s,3.87), 2.23(s,2H), 2.02-1.86(m,10H), 1.72(s,2H).

[0158] Compound 14-2: 3,4-Dimethoxybenzyl 2-(4-(2-oxo-2-(quinolin-8-ylamino)ethoxy)phenyl)adamantane-1-ylcarboxylate (14b): EDC.HCl (42 mg, 0.22 mmol), HOBt (29 mg, 0.22 mmol) and DIPEA (0.080 mL, 0.47 mmol) were added to a solution of compound 13 (90 mg, 0.19 mmol) and quinolin-8-amine (29 mg, 0.20 mmol) in DMF (5.0 mL). After stirring the reaction mixture at room temperature overnight, the resulting mixture was diluted with EtOAc and washed with water and brine. The organic layer was separated, dried over anhydrous MgSO4, filtered and concentrated in vacuum. The resulting residue was purified by silica gel column chromatography (hexane:EA=6:4) to give 14b as a white solid (79 mg, 61% yield). 1H-NMR(400MHz,CDCl3)10.97(s,1H), 8.86(dd,J=1.4-4.2Hz,1H), 7.56(d,J=1.6Hz,1H), 7.55(s,1H), 7.47(q,J=4.1Hz,1H), 7.33(d,J=8.4Hz,2H), 7.0 8(d,J=8.4Hz,2H), 6.91(d,J=10.0Hz,1H), 6.84(d,J=8.0Hz,2H), 5.05(s,2 H), 4.73(s,2H), 3.88(s,6H), 2.23(s,2H), 2.04-1.87(m,10H), 1.72(s,2H); 13 C-NMR (100MHz, CDCl3)177.1, 167.0, 155.5, 148.9, 148.8, 143.9, 143.9, 138.8, 136.2, 133.8, 128.9, 128.0, 127.2, 126 .2, 122.2, 121.7, 120.7, 116.8, 114.9, 111.3, 111.0, 68.4, 66.0, 55.9, 55.8, 44.3, 42.2, 41.9, 38.1, 36.0, 35.6, 28.7.

[0159] Compound 14-3: 3,4-Dimethoxybenzyl 2-(4-(2-(furan-2-ylmethylamino)-2-oxoethoxy)phenyl)adamantane-1-ylcarboxylate (14c): EDC.HCl (42 mg, 0.22 mmol), HOBt (29 mg, 0.22 mmol) and DIPEA (0.080 mL, 0.47 mmol) were added to compound 13 (90 mg, 0.19 mmol) and furan-2-ylmethanamine (20 mg, 0.20 mmol) in DMF (5.0 mL). After stirring the reaction mixture at room temperature overnight, the resulting mixture was diluted with EtOAc and washed with water and brine. The organic layer was separated, dried over anhydrous MgSO4, filtered and concentrated in vacuum. The resulting residue was purified by silica gel column chromatography (hexane:EA=6:4) to give 14c as a white solid (57 mg, 54% yield).1 H-NMR(400MHz,CDCl3)7.35(s,1H), 7.29(d,J=1.6Hz,2H), 6.91-6.82(m,5H), 6.32(q,J=1.6Hz,1H), 6.22(d,J=3.2Hz,1H), 5.05(s,2H), 4 .53(d,J=6.0Hz,2H), 4.49(s,2H), 3.87(d,J=2.4Hz,6H), 2.22(s,2H), 2.00(s,2H), 1.97-1.88(m,4H), 1.85(d,J=1.6Hz,2H), 1.71(s,2H).

[0160] Compound 14-4: 3,4-Dimethoxybenzyl 2-(4-(2-(4-methylpiperazin-1-yl)-2-oxoethoxy)phenyl)adamantane-1-ylcarboxylate (14d): EDC.HCl (42 mg, 0.22 mmol), HOBt (29 mg, 0.22 mmol) and DIPEA (0.080 mL, 0.47 mmol) were added to a solution of compound 13 (90 mg, 0.19 mmol) and 1-methylpiperazine (20 mg, 0.20 mmol) in DMF (5.0 mL). After stirring the reaction mixture at room temperature overnight, the resulting mixture was diluted with EtOAc and washed with water and brine. The organic layer was separated, dried over anhydrous MgSO4, filtered and concentrated in vacuum. The resulting residue was purified by silica gel column chromatography (hexane:EA=6:4) to give 14d as a white solid (50 mg, 47% yield). 1 H-NMR(400MHz,CDCl3)7.26(d,J=8.8Hz,2H), 6.89-6.83(m,5H), 5.05(s,2H), 4.65(s,2H), 3.87(s,6) H), 3.61(d,J=23.2Hz,4H), 2.39(s,4H), 2.29(s,3H), 2.21(s,2H), 2.01-1.85(m,10H), 1.71(s,2H).

[0161] Example 4. Synthesis of Compounds 17-1 to 17-3 According to the above-mentioned reaction formula S3, compounds 17-1 to 17-3 were synthesized using compound 7 as a starting material and compounds 15 and 16 as intermediates.

[0162] Compound 17-1: N-(Furan-2-ylmethyl)-5-(4-(2-(4-methylpiperazin-1-yl)-2-oxoethoxy)phenyl)adamantan-1-yl-carboxamide (17a): EDC.HCl (50 mg, 0.26 mmol), HOBt (35 mg, 0.26 mmol) and DIPEA (0.90 mL, 0.52 mmol) were added to a solution of compound 16 (90 mg, 0.21 mmol) and furan-2-ylmethanamine (25 mg, 0.26 mmol) in DMF (3.0 mL). After stirring the reaction mixture at room temperature overnight, the resulting mixture was diluted with EtOAc and washed with water and brine. The organic layer was separated, dried over anhydrous MgSO4, filtered and concentrated in vacuum. The resulting residue was purified by silica gel column chromatography (DCM:MeOH=1:9) to give 17a as a white solid (0.30 mg, 29% yield). 1 H-NMR(CDCl3,400MHz)7.33(s,1H), 7.29(s,1H), 7.25(s,1H), 6.87(d,J=3.7H z,2H), 6.31-6.29(m,1H), 6.19(d,J=3.6Hz,1H), 5.96(s,1H), 4.64(s,2H), 4. 42(d,J=5.2Hz,2H), 3.63(t,J=4.8Hz,2H), 3.57(t,J=5.0Hz,2H), 2.38-2.36( m,4H), 2.28(s,3H), 2.23(s,2H), 1.95(s,2H), 1.88-1.85(m,8H), 1.71(s,2H).

[0163] Compound 17-2: 1-(4-Methylpiperazin-1-yl)-2-(4-(5-(4-(4-(4-(trifluoromethyl)benzyl))piperazine-1-carbonyl)adamantan-1-yl-)phenoxy)ethanone (17b): EDC.HCl (50 mg, 0.26 mmol), HOBt (35 mg, 0.26 mmol) and DIPEA (0.90 mL, 0.52 mmol) were added to a solution of compound 16 (90 mg, 0.21 mmol) and 1-(4-(trifluoromethyl)benzyl)piperazine (49 mg, 0.35 mmol) in DMF (3.0 mL). After stirring the reaction mixture at room temperature overnight, the resulting mixture was diluted with EtOAc and washed with water and brine. The organic layer was separated, dried over anhydrous MgSO4, filtered and concentrated in vacuum. The resulting residue was purified by silica gel column chromatography (DCM:MeOH=1:9) to give 17b as a white solid (25 mg, 19% yield). 1 H-NMR (CDCl3,400MHz) 7.57(d,J=8.0Hz,2H), 7.45(d,J=8.4Hz,2H), 7.26(d,J=8.8Hz,2H), 4.67(s,2H), 3.70-3. 54(m,10H), 2.43-2.37(m,J=3.3Hz,8H), 2.29(s,3H), 2.23(s,2H), 2.06-2.00(m,6H), 1.85(s,4H), 1.71(s,2H).

[0164] Compound 17-3: 2-(4-(5-(4-(4-Methoxybenzyl)piperazine-1-carbonyl)adamantan-1-yl)phenoxy)-1-(4-methylpiperazin-1-yl)ethanone (17c): EDC.HCl (50 mg, 0.26 mmol), HOBt (35 mg, 0.26 mmol) and DIPEA (0.90 mL, 0.52 mmol) were added to a solution of compound 16 (90 mg, 0.21 mmol) and 1-(4-methoxybenzyl)piperazine (53 mg, 0.26 mmol) in DMF (3.0 mL). After stirring the reaction mixture at room temperature overnight, the resulting mixture was diluted with EtOAc and washed with water and brine. The organic layer was separated, dried over anhydrous MgSO4, filtered and concentrated in vacuum. The resulting residue was purified by silica gel column chromatography (DCM:MeOH=1:9) to give 17c as a white solid (37 mg, 30% yield). 1 H-NMR (CDCl3,400MHz) 7.26(d,J=8.0Hz,2H), 7.23(d,J=8.8Hz,2H), 6.90(d,J=8.4Hz,2H), 6.86(d,J=8.8Hz,2H), 4.66(s,2H), 3.80(s,3H), 3.70-3.66(m,6H), 3.61(t,J=4.8Hz,2H), 3.46(s,2H), 2.42(s,8H), 2 .31(s,3H), 2.23(s,2H), 2.06-1.99(m,6H), 1.85(s,4H), 1.71(s,2H).

[0165] Example 5. Synthesis of Compounds 19 and 20 The synthesis steps of compounds 19 and 20 used in the above reaction scheme are as described below.

[0166] Compound 19: {4-[3-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinoline-2-carbonyl)-adamantan-1-yl]-phenoxy}-acetic acid ethyl ester (19): Compound 15 (2.26 g, 6.30 mmol) was dissolved in acetonitrile, and triethylamine (2.25 g, 0.02 mol) and 50% PPAA (4.81 g, 7.57 mmol) were added at room temperature. The mixture was stirred for 30 min, and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride (1.74 g, 7.57 mmol) was added. The reaction solution was then stirred overnight at room temperature and evaporated under reduced pressure. The reaction mixture was purified by column chromatography on silica gel to give compound 19 as a white solid (2.21 g, 66% yield). 1 H-NMR(400MHz,DMSO-d6)δ(d,J=8.7Hz,2H), 6.84(d,J=9Hz,2H), 6.83(s,1H), 6.70(s,1H), 4.73(s,2H), 4.64(s,2H), 4.16(q,J=6.3Hz,2H), 3.8 1(q,J=5.7Hz,2H), 3.71(s,3H), 3.70(s,3H), 2.69(q,J=5.1Hz,2H), 2.1 7(br,2H), 1.95-1.86(m,7H), 1.78-1.67(m,5H), 1.21(t,J=7.5Hz,3H).

[0167] Compound 20: {4-[3-(6,7-Dimethoxy-3,4-dihydro-1H-isoquinoline-2-carbonyl)-adamantan-1-yl]-phenoxy}-acetic acid (20): Compound 19 (2.21 g, 4.14 mmol) was dissolved in a solution of THF and HO, lithium hydroxide (0.26 g, 6.21 mmol) was added at room temperature, and the mixture was stirred for 1 h, followed by dropwise addition of 10% HCl. The filtrate was evaporated under reduced pressure, and the solid was washed with n-hexane to give product 20 as a white solid (2.07 g). 1H-NMR(400MHz,DMSO-d6)7.29(d,J=8.7Hz,2H), 6.83(d,J=8.1Hz,2H), 6.81(s,1H), 6.70(s,1H), 4.64(s,2H), 4.62(s,2H), 3.81(q,J=5.1Hz,2H), 3.71(s,3H), 3.70(s,3H), 2.69(q,J=5.7Hz,2H), 2.17(br,2H), 1.99-1.88(m,8H), 1.78-1.66(m,4H).

[0168] Example 6. Synthesis of Compounds 21-1 to 21-3 According to the above-mentioned reaction formula S1, compounds 21-1 to 21-3 were synthesized using compound 7 as a starting material and compounds 15 and 16 as intermediates.

[0169] Compound 21-1: 2-(4-(3-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)adamantan-1-yl)phenoxy)-1-morpholinoethanone (21a): EDC.HCl (191 mg, 0.35 mmol), HOBt (135 mg, 0.35 mmol) and DIPEA (0.129 mL, 0.60 mmol) were added to a solution of compound 20 (150 mg, 0.29 mmol) and morpholine (313 mg, 0.35 mmol) in DMF (5.0 mL). After stirring the reaction mixture at room temperature overnight, the resulting mixture was diluted with EtOAc and washed with water and brine. The organic layer was separated, dried over anhydrous MgSO4, filtered and concentrated in vacuum. The resulting residue was purified by silica gel column chromatography (DCM:MeOH=1:9) to give 21a as a white solid (81 mg, 48% yield). 1H-NMR(400MHz,CDCl3)δ(d,J=2.4Hz,2H), 7.28(d,J=3.2Hz,2H), 6.60(d,J=6.4Hz,2H), 4.71(s,2H), 4.67(s,2H), 3.92-3.88( m,2H), 3.85(s,4H), 3.67-3.60(m,9H), 2.80(t,J=6.4Hz,2H), 2.26(s,2H), 2.13-2.02(m,6H), 1.89-1.86(m,4H), 1.75(s,2H).

[0170] Compound 21-2: 2-(4-(3-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)adamantan-1-yl)phenoxy)-1-(4-methylpiperazin-1-yl)ethanone (21b): EDC.HCl (191 mg, 0.35 mmol), HOBt (135 mg, 0.35 mmol) and DIPEA (0.129 mL, 0.60 mmol) were added to compound 20 (150 mg, 0.29 mmol) and 1-methylpiperazine (100 mg, 0.35 mmol) in DMF (5.0 mL). After stirring the reaction mixture at room temperature overnight, the resulting mixture was diluted with EtOAc and washed with water and brine. The organic layer was separated, dried over anhydrous MgSO4, filtered and concentrated in vacuum. The resulting residue was purified by silica gel column chromatography (DCM:MeOH=1:9) to give 21b as a white solid (80 mg, 45% yield). 1H-NMR(400MHz,CDCl3)δ(d,J=6.8Hz,2H), 6.90(d,J=8.8Hz,2H), 6.60(d,J=6.2Hz,2H), 4.71(s,2H), 4.66(s,2H), 3.89(s,2H), 3.8 5(s,6H), 3.62(d,J=22.4Hz,4H), 2.80(s,2H), 2.40(s,4H), 2.29(s,3H), 2.26(s,2H), 2.13-2.02(m,6H), 1.89(s,4H), 1.75(s,2H).

[0171] Compound 21-3: 2-(4-(3-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)adamantan-1-yl)phenoxy)-1-(4-(4-(trifluoromethyl)benzyl)piperazin-1-yl)ethanone (21c) (LW1564): EDC.HCl (191 mg, 0.35 mmol), HOBt (135 mg, 0.35 mmol) and DIPEA (0.129 mL, 0.60 mmol) were added to a solution of compound 20 (150 mg, 0.29 mmol) and 1-(4-(trifluoromethyl)benzyl)piperazine (71 mg, 0.29 mmol) in DMF (5.0 mL). After stirring the reaction mixture at room temperature overnight, the resulting mixture was diluted with EtOAc and washed with water and brine. The organic layer was separated, dried over anhydrous MgSO4, filtered and concentrated in vacuum. The resulting residue was purified by silica gel column chromatography (DCM:MeOH=1:9) to give LW1564 (21c) as a white solid (0.123 mg, 57% yield). 1H-NMR(400MHz,CDCl3)δ(d,J=8.0Hz,2H)7.44(d,J=8.0Hz,2H), 7.29(d,J=8.8Hz,2H),6. 89(d,J=8.4Hz,2H), 6.60(d,J=6.0Hz,2H), 4.71(s,2H), 4.66(s,2H), 3.90(t,J=5.8Hz,2H) ), 3.85(d,J=2.0Hz,6H), 3.64(s,2H), 3.59(t,J=4.8Hz,2H), 3.55(s,2H), 2.80(t,J=5.4H) z,2H), 2.44(s,4H)), 2.26(s,2H), 2.13-2.05(m,6H), 1.89(d,J=2.4Hz,4H), 1.76(s,2H).

[0172] [Experimental Example] Screening of HIF-1 inhibitors by HRE-luciferase assay HCT116 cells stably expressing an HRE-dependent luciferase reporter were treated with the disubstituted adamantyl derivatives prepared in the above examples under hypoxic conditions for 12 hours before measuring luciferase activity.

[0173] Luciferase activity was examined in HCT-116 cells treated with disubstituted adamantyl derivatives using the HRE-luciferase assay. The HRE-luciferase reporter assay was performed using the Luciferase Assay System (Promega, Madison, WI, USA) according to the manufacturer's guidelines as previously described (Naik, R. et al. Synthesis and Structure-Activity Relationship of (E) Phenoxyacrylic Amide Derivatives as Hypoxia-Inducible Factor (HIF) 1α Inhibitors. J. Med. Chem. 55, 10564-1057 (2012)). Luciferase activity was measured using a Victor X light luminescence reader (PerkinElmer, Boston, MA, USA).

[0174] As a result, the activities of the disubstituted adamantyl derivatives are shown in Tables 1 and 2 below.

[0175] [Table 1]

[0176] [Table 2]

[0177] LW1564 (compound 21-3) inhibits HIF-1 activation and proliferation in cancer cells Among the newly synthesized compounds, compound 21c (LW1564 (compound 21-3), Figure 1a) was evaluated for its effect on the inhibition of proliferation of various cancer cells. Human breast cancer cells (MCF7), human cervical cancer cells (HeLa), human colorectal cancer cells (HCT116, HCT15, LoVo, SW480, SW620, and WiDr), human fibrosarcoma (HT1080), human gastric cancer cells (NCI-N87, NUGC3, and SNU484), human liver cancer cells (HepG2, HT17, Huh7, SHJ1, and SK-Hep1), human lung cancer cells (A549 and H1299), human pancreatic cancer cells (AsPC1 and MIA-PaCa2), and normal lung fibroblast cells (CCD-34Lu and WI-38) were cultured using the American Type Culture Collection (ASC). Cells were purchased from the Cell Line Collection (Manassas, VA, USA) or the KRIBB Cell Line Bank (Daejeon, Korea). For cell culture, cells were cultured in DMEM medium (Gibco, Grand Island, NY, USA) supplemented with 5% (v / v) fetal bovine serum (WelGENE, Daegu, Korea) and 100 U / ml penicillin and 100 μg / ml streptomycin (Gibco). Cells were maintained at 37 °C in a humidified incubator with 5% CO2 and 1% O2 in a hypoxic and multi-gas incubator (Sanyo, Osaka, Japan). 2、The atmosphere was adjusted to 94% N2 and 5% CO2. Cell viability was determined by methylene blue colorimetric assay as described in "Chou, TC Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol. Rev. 58, 621-681 (2006)". After treatment with LW1564 (compound 21-3), cells were fixed with 4% formaldehyde (Sigma-Aldrich) and washed three times with PBS. After staining with methylene blue (Sigma-Aldrich), cells were washed with water. The dye was eluted with 0.1% HCl (v / v) and quantified at 600 nm on a Molecular Devices EMax (Molecular Devices, CA, USA). We found that LW1564 (compound 21-3) inhibited the proliferation of various cancer cells (GI 50 =0.4-4.6 μM), normal cells, CCD-34Lu and WI-38 (GI 50 We confirmed that LW1564 (compound 21-3) had no effect on cell growth (>20 μM) (Fig. 1b). The effect of LW1564 (compound 21-3) on cell proliferation was then investigated using a live cell analysis system, IncuCyte ZOOM (Essen Bioscience, Ann Arbor, MI, USA). LW1564 (compound 21-3) induced a dose-dependent inhibition of proliferation of HepG2 and A549 cells (Fig. 1c).

[0178] LW1564 (compound 21-3) suppresses HIF-1α accumulation in cancer cells In the presence of oxygen, proline residues in the oxygen-dependent degradation (ODD) domain of HIF-1α are hydroxylated by prolyl hydroxylase (PHD), which binds to the Von Hippel-Lindau syndrome (VHL) associated VCB-Cul2 E3 ligase that induces protein degradation, promoting HIF-1α degradation. Under hypoxic conditions, proline hydroxylation does not occur, and the amount of HIF-1α protein increases. HIF-1α activity was examined using the HRE-luciferase assay in HepG2 cells previously treated with LW1564 (compound 21-3). HRE-luciferase reporter assays were performed using a luciferase assay system (Promega, Madison, WI, USA) according to the manufacturer's guidelines as previously described (Naik, R. et al. Synthesis and Structure-Activity Relationship of (E) Phenoxyacrylic Amide Derivatives as Hypoxia-Inducible Factor (HIF) 1α Inhibitors. J. Med. Chem. 55, 10564-1057 (2012)). Luciferase activity was measured using a Victor X light luminescence reader (PerkinElmer, Boston, MA, USA). LW1564 (compound 21-3) had an IC of 1.2 μM in HepG2 cells. 50 We also found that LW1564 (compound 21-3) inhibited HIF-1α accumulation in a concentration-dependent manner under hypoxic conditions (Fig. 2b). Indeed, Western blot experiments confirmed that LW1564 increased HIF-1α accumulation by inhibiting the activity of prolyl hydroxylase, since LW1564 (compound 21-3) had no effect on HIF-1α protein abundance in the presence of cobalt chloride (Fig. 2b).

[0179] In addition, the amount of HIF-1α mRNA was investigated in LW1564-treated cells by qPCR. Total RNA was isolated using TRIzol reagent (Invitrogen) and analyzed by TOPscript TM cDNA was synthesized using DryMIX (Enzynomics, Seoul, Korea). The HIF1A primer sequences were Fwd 5'-CTG ACC CTG CAC TCA ATC AAG-3' and Rev 5'-TGG GAC TAT TAG GCT CAG GTG-3. By observing that HIF-1α mRNA levels were unchanged, we confirmed that LW1564 (compound 21-3) inhibited hypoxia-mediated accumulation of HIF-1α through proteasomal degradation rather than transcriptional processes (Fig. 2c). We next investigated whether LW1564 (compound 21-3) had a similar effect on HIF-1 degradation in other cancer cell lines. LW1564 (compound 21-3) significantly reduced hypoxia-induced HIF-1α protein accumulation in various cancer cell lines, including A549, WiDr, MIA-CaCa2, and HCT-116 (Fig. 2d).

[0180] Next, we measured the mRNA levels of the HIF-1 target genes GLUT1, PDK1 and VEGF. The primer sequences are, for GLUT1, Fwd 5'-TTT GGC TAC AAC ACT GGA GTC-3' (SEQ ID NO:1) and Rev 5'-CAT GCC CCC AAC AGA AAA GAT-3' (SEQ ID NO:2); for PDK1, Fwd 5'-CAG GAC AGC CAA TAC AAG TGG-3' (SEQ ID NO:3) and Rev 5'-CAT TAC CCA GCG TGA CAT GAA-3' (SEQ ID NO:4); for VEGFA, Fwd 5'-CCT TGC TGC TCT ACC TCC AC-3' (SEQ ID NO:5) and Rev 5'-ATG ATT CTG CCC TCC TCC TT-3' (SEQ ID NO:6); and for RPL13A, Fwd 5'-CAT AGG AAG CTG GGA GCA AG-3' (SEQ ID NO:7) and Rev 5'-GCC CTC CAA TCA GTC TTC TG-3' (SEQ ID NO:8). LW1564 (compound 21-3) reduced the mRNA levels of GLUT1, PDK1, and VEGF, which were increased under hypoxic conditions in HepG2 cells (Figure 2e). In summary, we confirmed that LW1564 (compound 21-3) inhibited HIF-1α accumulation and reduced the mRNA expression of target genes through stimulated proteasomal degradation.

[0181] LWR1564 (compound 21-3) inhibits mitochondrial respiration and reduces oxygen consumption. Compounds that inhibit mitochondrial respiration promote the degradation of HIF-1α by increasing the oxygen content of cells. Therefore, we measured the oxygen consumption rate (OCR) to investigate whether LW1564 (compound 21-3) affects mitochondrial respiration. Oxygen consumption was measured using an Oxytherm Clark-type electrode system (Hansatech, Norfolk, UK) (Chou, T. C. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol. Rev. 58, 621-681 (2006)). HepG2 cells (2 × 10 7 ) were incubated with 2 μM LW1564 (compound 21-3) for 6 h, harvested, and the OCR was measured over 5 min at 37 °C in a thermoregulated controlled circulating system. For kinetic studies, digitonin-permeabilised HepG2 cells (2 × 10 7 The mixture was subjected to a detection apparatus containing 2 mL of respiratory buffer (0.25 M sucrose, 2 mM KH2PO4, 5 mM MgCl2, 1 mM EDTA, 1 mM ADP, 20 mM MOPS, pH 7.4). The following substrates, which provide electrons to components within the ETC, were then added: 5 mM sodium pyruvate, 5 mM sodium malate, 5 mM sodium succinate, 5 mM L-ascorbic acid, and 0.2 mM N,N,N,N-tetramethyl-p-phenylenediamine (TMPD). Complex I, III, and IV inhibitors (rotenone, antimycin A, and KCN, respectively) were added to a final concentration of 1 μM.

[0182] LW1564 (compound 21-3) significantly inhibited OCR at concentrations that inhibited HIF-1 accumulation (Figure 3a), indicating suppression of mitochondrial respiration. Next, the effect of mitochondrial respiration on ATP production by oxidative phosphorylation was evaluated by comparing the amount of ATP generated by glycolysis and mitochondrial respiration in the presence of LW1564 (compound 21-3). ATP content was determined as described in "Chou, T C Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol. Rev. 58, 621-681 (2006)." HepG2 cells were seeded and incubated with LW1564 (compound 21-3) for 6 hours, and then treated with ENLITEN according to the manufacturer's guidelines. ATP content was measured using an ATP assay kit (Promega, Madison, WI, USA). Under normoxic conditions, HepG2 cells obtained 47% of ATP from mitochondrial respiration and 53% from glycolysis. As expected, mitochondria-derived ATP production was significantly decreased (16%) and glycolysis-derived ATP production was increased in cells treated with LW1564 (compound 21-3) (Figure 3b). Compensatory upregulation of such degradation in response to inhibition of oxidative phosphorylation occurred through activation of AMPK, which was induced by a high AMP / ATP ratio. LW1564 (compound 21-3) slightly decreased total ATP production for a short period of time (Figure 3b), but significantly decreased total intracellular ATP levels over a long period of time (Figure 3c). These data indicate that LW1564 (compound 21-3) decreases ATP production by inhibiting mitochondrial oxidative phosphorylation.

[0183] Since a decrease in mitochondrial respiration means an increase in the amount of oxygen in the cells, the presence or absence of an increase in the amount of oxygen in the cells was evaluated using an azo-based hypoxia probe (MAR) that generates fluorescence under hypoxic conditions. Under hypoxic conditions, HepG2 cells were incubated with LW1564 (compound 21-3) for 6 hours, stained with 500 nM MAR according to the manufacturer's guidelines (Chou, T. C. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol. Rev. 58, 621-681 (2006)). Then, the cells were analyzed using an IncuCyte ZOOM. The results were analyzed using a ELISA system (Essen BioScience, MI, USA). Under hypoxic conditions, LW1564 (compound 21-3) significantly reduced fluorescence in HepG2 cells, indicating that the amount of oxygen in the cells was increased due to a decrease in mitochondrial respiration (Figure 3d).

[0184] LW1564 (compound 21-3) inhibits the activity of complex I in the mitochondrial electron transport chain. To determine the mechanism of action by which LW1564 (compound 21-3) reduces mitochondrial respiration in the ETC, mitochondrial respiration analysis was performed. Experiments were performed by administering specific substrates and inhibitors to analyze the respiratory chain process for each complex (Figure 4a). HepG2 cells were treated with malate / pyruvate for electron transport of complex I, and rotenone, a known complex I inhibitor, was used as a positive control to reduce oxygen consumption (Figure 4a). LW1564 (compound 21-3) reduced oxygen consumption by complex I, which was overcome by adding succinate and providing electrons directly to complex III (Figure 4b). This result indicated that LW1564 (compound 21-3) is an inhibitor of mitochondrial electron transport complex I. We then evaluated whether LW1564 (compound 21-3) affects other ETC complexes. We treated cells with LW1564 (compound 21-3) that had been administered succinate as an electron source for ETC complex II or ascorbate / TMPD as an electron source for cytochrome c and complex IV. We confirmed that LW1564 (compound 21-3) did not affect the oxygen consumption rate in the presence of succinate (Figure 4c) or ascorbate / TMPD (Figure 4d). Thus, these results suggest that LW1564 (compound 21-3) inhibits mitochondrial respiration by inhibiting only mitochondrial ETC complex I.

[0185] LW1564 (compound 21-3) activates AMPK signaling and inhibits lipid synthesis A significant decrease in total ATP content was observed in LW1564 (compound 21-3) treated cells (Figure 3c). Since these results indicate an increase in the AMP / ATP ratio, AMPK activation and downstream signaling events in HepG2 cells were examined. Western blot analysis confirmed that LW1564 (compound 21-3) promoted the phosphorylation of AMPK and its downstream protein, acetyl-CoA carboxylase (ACC), in HepG2 cells (Figure 5a). This result was interpreted as indicating that activation of AMPK by LW1564 (compound 21-3) inhibited the phosphorylation of mTOR in a concentration-dependent manner and inhibited the expression and / or activation of downstream proteins of mTOR, such as 4EBP1, cyclin D1, and sterol regulatory element-binding protein 1 (SREBP-1). The increase in ACC phosphorylation and decrease in SREBP-1 levels suggested that LW1564 (compound 21-3) could inhibit lipid synthesis. To explore this possibility, we evaluated the effect of LW1564 (compound 21-3) on lipid synthesis using Nile red, a fluorescent probe that binds to hydrophobic molecules such as lipid droplets. Nile red staining was performed as described in 1. Huang, D. et al. HIF-1-mediated suppression of acyl-CoA dehydrogenases and fatty acid oxidation is critical for cancer progression. Cell Rep. 8, 1930-1942 (2014) . HepG2 cells were treated with LW1564 (compound 21-3) for 24 h and then stained with 200 nM Nile red and 1 μM calcein AM for 10 min. Data were collected and analyzed with an IncuCyte ZOOM system (Essen BioScience).For normalization, cell viability was measured using calcein AM, a cell-permeant fluorescent dye. The results confirmed that LW1564 (compound 21-3) reduced lipid accumulation in a concentration-dependent manner (Figure 5b). In summary, we show that LW1564 (compound 21-3) promotes anticancer effects in HepG2 cells by regulating the AMPK / mTOR signaling pathway and lipid synthesis.

[0186] LW1564 (compound 21-3) inhibits glucose-dependent cancer metabolism Glycolysis is activated in many cancer cells. Therefore, we investigated whether LW1564 (compound 21-3) affects cancer cell proliferation depending on the glucose concentration. LW1564 (compound 21-3) showed a stronger cell proliferation inhibitory effect in cells in a low glucose concentration medium (3 mM) than in cells in a high glucose concentration medium (25 mM) (Figure 5c). Next, to investigate the combined effect of LW1564 (compound 21-3) and 2-deoxy-D-glucose (2DG), a glucose analogue but also a competitive glycolysis inhibitor, on the proliferation of HepG2 cells, we performed combination index (CI) and isobologram analysis. The combination index (isobologram) value is the GI, which indicates the cell proliferation inhibitory concentration. 50 Through the analysis, the interrelationship between the two drugs is quantitatively shown, and the synergistic, additive and antagonistic effects are defined as CI<1, CI=1 and CI>. The analysis showed that LW1564 (compound 21-3) and 2-DG had a synergistic effect (0.4≦CI≦0.92) in inhibiting HepG2 cell proliferation (Figure 5d). Therefore, it is suggested that the combination therapy of LW1564 (compound 21-3) with glycolysis inhibitors can exert a better anticancer efficacy against liver cancer than LW1564 (compound 21-3) alone.

[0187] LW1564 (compound 21-3) inhibits tumor growth in a HepG2 xenograft mouse model The inventors found that LW1564 (compound 21-3) inhibited the growth of HepG2 cells but did not affect the growth of normal cells (Figure 1), and therefore investigated the efficacy of LW1564 (compound 21-3) on tumor growth in a HepG2 xenograft mouse model. All animal experiments were approved and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Korea Institute of Bioscience and Biotechnology (Daejeon, Korea). Specific pathogen-free female nude mice (6 weeks old) were inoculated with HepG2 cells (1 × 10 7 ) was inoculated subcutaneously on the right side. 3 Upon reaching a tumor size of 100 mm, mice were randomly assigned to vehicle and LW1564 (compound 21-3) treatment groups (n=6). LW1564 (compound 21-3) (10 mg / kg) was administered intraperitoneally daily for 2 weeks. Tumor size was measured using a microcalliper and standard volume: length x width. 2 Tumor volumes were calculated using a factorial covariate analysis (Fcos) of 0.5 × 0.5. Significant differences between both groups were determined by Student's t-test.

[0188] LW1564 (compound 21-3) showed a 67% reduction in tumor size in mice that received an intraperitoneal injection of LW1564 (compound 21-3) (10 mg / kg) compared to the control group (left panel of Figure 6). We also confirmed that the weight (middle panel of Figure 6) and size (right panel of Figure 6) of tumors excised from mice administered LW1564 (compound 21-3) were reduced. These results suggest that LW1564 (compound 21-3) could be developed as a potent therapeutic agent for liver cancer patients.

[0189] [Table 3]

[0190] [Table 4] [Industrial Applicability]

[0191] The disubstituted adamantyl derivatives according to the present invention are expected to be useful as potent therapeutic agents for cancers dependent on oxidative phosphorylation for ATP production in mitochondria.

Claims

1. A disubstituted adamantyl derivative represented by the following chemical formula 1 (Chemical Formula 1) or a pharma- ceutical acceptable salt thereof: 【Chemistry 1】 In the above Chemical Formula 1, R ２ is -(Y)-(CH ２ ) m-R ４ and The Y is 【Chemistry 2】 and The m is 1, R ４ is the substituted C ５ ~C １０ is an aryl of wherein the substituted aryl is C １ ~C ６ or a pharma- ceutically acceptable salt thereof.

2. The R ２ is represented by the following formula 3: 【Chemistry 3】 The disubstituted adamantyl derivative according to claim 1 or a pharma- ceutically acceptable salt thereof.

3. The disubstituted adamantyl derivative is the compound: 2-(4-(3-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)adamantan-1-yl)phenoxy)-1-(4-(4-(trifluoromethyl)benzyl)piperazin-1-yl)ethanone) (21c) (LW1564) The disubstituted adamantyl derivative according to claim 1 or a pharma- ceutically acceptable salt thereof.

4. 4. An anticancer pharmaceutical composition comprising the disubstituted adamantyl derivative or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 3 as an active ingredient.

5. The pharmaceutical composition according to claim 4, which is an anticancer pharmaceutical composition for solid cancers.

6. The pharmaceutical composition according to claim 5, wherein the solid cancer is selected from the group consisting of breast cancer, cervical cancer, rectal cancer, fibrosarcoma, gastric cancer, liver cancer, lung cancer and pancreatic cancer.

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