Disubstituted adamantyl derivatives, pharmaceutically acceptable salts thereof, and pharmaceutical compositions for inhibiting cancer growth containing the same as an active ingredient

Abstract

The present invention relates to a disubstituted adamantyl derivative or a pharmaceutically acceptable salt thereof, a pharmaceutical composition for anti-cancer containing the same as an active ingredient, and a kit. The disubstituted adamantyl derivative of the present invention inhibits the growth of cancer cells by targeting mitochondrial ETC complex I and disrupting cancer cell metabolism, and thus can be effectively used as a pharmaceutical composition for anti-cancer. This composition is a potent cancer therapeutic agent that depends on oxidative phosphorylation for ATP generation.

Description

Technical Field

[0001] The present invention relates to a disubstituted adamantyl derivative or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition and a kit for inhibiting cancer growth, which contain the same as an active ingredient.

[0002] This application is based on and claims priority to a Korean patent application filed on January 23, 2020, with an application number of 10-2020-0009445. The entire content disclosed in the specification and drawings of this application is incorporated herein by reference. Background Art

[0003] Cancer cells increase aerobic glycolysis, glutaminolytic flux, amino acid, and lipid metabolism to achieve rapid growth. Cancer cells compete with neighboring normal cells for limited nutrients and consume more glucose. In addition, the hypoxic tumor microenvironment of cancer cells malignantly transforms cancer cells by inducing the expression of genes involved in metabolism, angiogenesis, metastasis, and anti-apoptosis.

[0004] Hypoxia-inducible factor-1 (HIF-1), which is increased in expression under hypoxic conditions, consists of HIF-1α and HIF-1β and is a major regulator of tumor hypoxia, playing a key role in the metabolic reprogramming of cancer cells. Under hypoxic conditions, HIF-1α increases the expression of glucose transporters (GLUTs) and glycolytic enzymes such as hexokinases (HKs) and phosphoglycerate kinase 1 (PGK1), resulting in the activation of glycolysis. HIF-1 also increases the expression of pyruvate dehydrogenase kinase isozyme 1 (PDK1), which phosphorylates serine residues and inhibits the activity of pyruvate dehydrogenase (PDH). The high expression of PDK1 prevents the conversion of pyruvate to acetyl-CoA, thereby inhibiting 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, inhibiting HIF-1 expression or function can disrupt cancer metabolism and induce apoptosis by inhibiting the expression of genes involved in cancer cell metabolic adaptation.

[0005] Several studies have attempted to develop HIF-1 inhibitors. For example, the mitochondrial complex I inhibitor BAY 87-2243 can reduce hypoxia-induced HIF-1α accumulation and exhibits significant anti-tumor effects in a xenograft model of the H460 cancer cell line. The benzopyran analog KCN-1 inhibits HIF-1 activity by interfering with the interaction between the transcription factor p300 and HIF-1α in glioma cells. The present inventors focused on developing small molecule compounds that target the HIF-1α protein in solid tumors and found that the (aryloxyacetamido)benzoic acid analog LW6 (disclosed in Korean Patent Publication No. 10-2012-0041071) inhibits hypoxia-induced HIF-1α protein accumulation, and its direct target is malate dehydrogenase 2 (MDH2). The clinical candidate IDF-11774 has also been developed as a HIF-1α inhibitor targeting cancer metabolism. Also, moracin O and its benzofuran analogs have been reported to be potent inhibitors of HIF-1α protein accumulation. However, to date, no drug has been approved by the FDA as a HIF-1 inhibitor.

[0006] Hepatocellular carcinoma (HCC) is a complex heterogeneous tumor type with multiple genetic and epigenetic modifications. Sorafenib has been approved as a first-line treatment for patients with advanced HCC, but the survival effect is very low. Sorafenib is an oral multi-kinase inhibitor that inhibits angiogenesis and proliferation by targeting vascular endothelial growth factor (VEGF) receptors and platelet-derived growth factor receptors, thereby inhibiting tumor proliferation. Recently, regorafenib and nivolumab have been approved as second-line therapeutic agents for patients who are unresponsive to sorafenib, but there is an urgent need for effective liver cancer treatment methods. Summary of the Invention

[0007] Technical Problem

[0008] As described above, targeting cancer metabolism has become an important cancer treatment strategy. Therefore, in the present invention, there are provided the synthesis results of disubstituted adamantyl derivatives as HIF-1α inhibitors and their biological evaluation results, where HIF-1α is a factor that can induce hypoxia.

[0009] More specifically, the present inventors found that a disubstituted adamantyl derivative containing LW1564 (Compound 21-3) inhibits mitochondrial respiration by inhibiting electron transport chain (ETC) Complex I, thereby reducing ATP production and stimulating the degradation of HIF-1α in HCC cells. Such results indicate that a disubstituted adamantyl derivative containing LW1564 (Compound 21-3) disrupts cancer metabolism by inhibiting HIF-1α accumulation and fatty acid synthesis, thereby inhibiting the growth of cancer cells in vivo and in vitro. The present invention provides a pharmaceutical composition for anti-cancer use containing a disubstituted adamantyl derivative and a pharmaceutically acceptable salt thereof as active ingredients.

[0010] Technical solution

[0011] To solve the above-mentioned problems, the present invention provides a disubstituted adamantyl derivative represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[0012] Chemical Formula 1:

[0013] (In the above Chemical Formula 1, R1 and R2 are as defined herein.)

[0014] Furthermore, the present invention provides a pharmaceutical composition for anti-cancer use containing a disubstituted adamantyl derivative represented by the above Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0015] Furthermore, the present invention relates to a method for treating cancer, comprising the step of administering a therapeutically effective amount of a disubstituted adamantyl derivative represented by the above Chemical Formula 1 or a pharmaceutically acceptable salt thereof to a patient in need of anti-cancer treatment.

[0016] Furthermore, the present invention provides the use of a disubstituted adamantyl derivative represented by the above Chemical Formula 1 or a pharmaceutically acceptable salt thereof in the preparation of a preparation for treating cancer.

[0017] Furthermore, the present invention provides the use of a disubstituted adamantyl derivative represented by the above Chemical Formula 1 or a pharmaceutically acceptable salt thereof in the treatment of cancer.

[0018] As an example of the present invention, the above pharmaceutical composition may be a pharmaceutical composition for anti-cancer use against solid cancer, but is not limited thereto.

[0019] As another example of the present invention, the solid cancer may be selected from the group consisting of breast cancer, cervical cancer, rectal cancer, fibrosarcoma, gastric cancer, liver cancer, lung cancer, and pancreatic cancer.

[0020] Technical effect

[0021] The disubstituted adamantyl derivatives containing LW1564 (Compound 21-3) of the present invention significantly inhibit HIF-1α accumulation and inhibit the growth of various cancer cell lines including HepG2 and A549. The measurement results of oxygen consumption rate (OCR) and ATP production rate show that LW1564 (Compound 21-3) increases the intracellular oxygen concentration by inhibiting mitochondrial respiration, thereby stimulating the degradation of HIF-1α in HepG2 cells. LW1564 (Compound 21-3) also inhibits mammalian target of rapamycin (mTOR) signaling by increasing mitochondrial electron transport chain (ETC) Complex I and AMP / ATP ratio, thereby increasing the phosphorylation of AMP-activated protein kinase (AMPK) and significantly reducing the total ATP production. Finally, LW1564 (Compound 21-3) inhibits lipid synthesis by promoting the phosphorylation of acetyl-CoA carboxylase and significantly inhibits tumor growth in a HepG2 mouse xenograft model. In summary, the disubstituted adamantyl derivatives containing LW1564 (Compound 21-3) of the present invention inhibit the proliferation of HepG2 cells by targeting mitochondrial ETC Complex I and disrupting cancer cell metabolism, and thus can be effectively used as a potent cancer therapeutic agent that relies on oxidative phosphorylation for ATP generation in mitochondria. <110> DONGGUK UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> Disubstituted adamantyl derivatives, pharmaceutically acceptable salts thereof, and pharmaceutical compositions for inhibiting cancer growth containing the same as an active ingredient <130> MPCT20-083 <150> KR 10-2020-0009445 <151> 2020-01-23 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial sequence <220> <223> GLUT1 upstream primer <400> 1 tttggctaca acactggagt c 21 <210> 2 <211> 21 <212> DNA <213> Artificial sequence <220> <223> GLUT1 downstream primer <400> 2 catgccccca acagaaaaga t 21 <210> 3 <211> 21 <212> DNA <213> Artificial sequence <220> <223> PDK1 upstream primer <400> 3 caggacagcc aatacaagtg g 21 <210> 4 <211> 21 <212> DNA <213> Artificial sequence <220> <223> PDK1 downstream primer <400> 4 cattacccag cgtgacatga a 21 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <220> <223> VEGFA upstream primer <400> 5 ccttgctgct ctacctccac 20 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220> <223> VEGFA downstream primer <400> 6 atgattctgc cctcctcctt 20 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <220> <223> RPL13A upstream primer <400> 7 cataggaagc tgggagcaag 20 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <223> RPL13A downstream primer <400> 8 gccctccaat cagtcttctg 20 Description of the drawings

[0022] Figures 1a to 1c Shows the effect of LW1564 (Compound 21-3) on cancer cell growth, Figure 1a Shows the structure of LW1564 (Compound 21-3), Figure 1b Shows the proliferation inhibitory effect of LW1564 (Compound 21-3) on multiple cancer cell lines, Figure 1c Is a graph showing the proliferation inhibitory effect of LW1564 (Compound 21-3) on HepG2 and A549 cells using the IncuCyte ZOOM system for live cell imaging.

[0023] Figures 2a to 2e Shows the effect of LW1564 (Compound 21-3) in inhibiting HIF-1α accumulation in cancer cells. Figure 2a Shows the inhibitory effect of LW1564 (Compound 21-3) on HRE-luciferase activity in HepG2 cells. Figure 2b Is a graph showing that LW1564 (Compound 21-3) inhibits HIF-1α accumulation through proteasome-dependent degradation. Figure 2c Shows the mRNA expression level of HIF-1α. Figure 2d Shows the inhibitory effect of LW1564 (Compound 21-3) on HIF-1α accumulation in multiple cancer cell lines under hypoxic conditions. Figure 2e Shows the mRNA expression levels of HIF-1α target genes, GLUT1, PDK1, and VEGFA by qPCR.

[0024] Figures 3a to 3dFigure showing that LW1564 (Compound 21-3) inhibits mitochondrial respiration in HepG2 cells. Figure 3a Figure showing the effect of LW1564 (Compound 21-3) on reducing the oxygen consumption rate in HepG2 cells. Figure 3b Figure showing the measurement results of the rate of ATP production in cancer cells when treated with LW1564 (Compound 21-3). Figure 3c Figure showing the effect of LW1564 (Compound 21-3) on reducing the total amount of ATP in cells. Figure 3d Figure showing the change in intracellular oxygen partial pressure detected by the probe MAR for detecting hypoxia when using LW1564 (Compound 21-3).

[0025] Figures 4a to 4d Figure showing that LW1564 (Compound 21-3) inhibits mitochondrial respiration by targeting Complex I in the ETC. In Figure 4a , Figure 4b ,1 μM of rotenone (an inhibitor of Complex I activity) ( Figure 4a ) or 100 μM of LW1564 (Compound 21-3) ( Figure 4b ) was added to the cells. Figure 4c Figure showing the effect of LW1564 (Compound 21-3) on Complex II / III. After adding the Complex II substrate succinate, LW1564 (Compound 21-3) was added to the cells. Figure 4d Figure showing the effect of LW1564 (Compound 21-3) on Complex IV. After adding the Complex IV substrates TMPD and ascorbate, LW1564 (Compound 21-3) was added to the cells.

[0026] Figures 5a to 5d Figure showing that LW1564 (Compound 21-3) inhibits fatty acid accumulation by activating the AMPK pathway in HepG2 cells. Figure 5a Figure showing the activation of AMPK by LW1564 (Compound 21-3). Figure 5b Figure showing the inhibition of fatty acid accumulation in the presence of LW1564 (Compound 21-3). Figure 5c Figure showing the effect of LW1564 (Compound 21-3) and glucose concentration on the proliferation of cancer cells. HepG2 cells cultured at different glucose concentrations were treated with LW1564 (Compound 21-3). The bar graph represents the percentage of cell proliferation. Figure 5d Figure showing the combination index (CI) of 2-DG (2-deoxy-D-glucose) and LW1564 (Compound 21-3) in HepG2 cells.

[0027] Figure 6Shows the tumor proliferation inhibitory effect of LW1564 (Compound 21-3) in a HepG2 xenograft mouse model. Figure 6 The left graph in shows the change in the tumor volume of the xenograft in the control group and the group treated with LW1564 (Compound 21-3) over time; **P≤0.01, Figure 6 The middle graph in shows the weight of the xenograft tumor in the control group and the group treated with LW1564 (Compound 21-3); **P≤0.01, Figure 6 The right graph in is Figure 6 Representative images of the tumors shown in the left graph and the middle graph in.

[0028] Optimal Embodiment

[0029] Hereinafter, the present invention will be described in detail.

[0030] The present invention provides a disubstituted adamantyl derivative represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[0031] Chemical Formula 1:

[0032] In the above Chemical Formula 1,

[0033] R1 is -(X)-(CH2)n-R3 or

[0034] R2 is -(Y)-(CH3)m or -(Y)-(CH2)m-R4,

[0035] The above X is O, NH or

[0036] The above Y is NH,

[0037] The above n is 0, 1 or 2, and the above m is 0 or 1,

[0038] R3 or R4 is each independently a straight-chain or branched-chain alkyl having 1 to 6 carbon atoms, an unsubstituted or substituted aryl having 5 to C 10 aryl, or an unsubstituted or substituted heteroaryl having 5 to 9 carbon atoms,

[0039] wherein, the above heteroaryl is a 5-membered or 6-membered heteroaryl containing one or more N or O heteroatoms,

[0040] The above substituted aryl or substituted heteroaryl can be substituted by one or more halogens, a straight-chain or branched-chain alkyl having 1 to 6 carbon atoms, a straight-chain or branched-chain haloalkyl having 1 to 6 carbon atoms (haloalkane), a hydroxyl group, or a straight-chain or branched-chain alkoxy group having 1 to 6 carbon atoms,

[0041] However, in the above R2, and can be excluded

[0042] Preferably

[0043] the above R1 is -(X)-(CH2)n-R3 or

[0044] R2 is -(Y)-(CH3)m or -(Y)-(CH2)m-R4,

[0045] the above X is O, NH or

[0046] the above Y is NH,

[0047] the above n is 0, 1 or 2, and the above m is 0 or 1,

[0048] the above R3 is methyl, substituted or unsubstituted phenyl, or unsubstituted C5-C9 heteroaryl, wherein the above heteroaryl is a 5- or 6-membered heteroaryl containing N or O heteroatoms, and the above substituted phenyl can be substituted by one or more halogens, C1-C6 straight-chain or branched-chain alkyls, C1-C6 straight-chain or branched-chain haloalkyls, hydroxyl groups, or C1-C6 straight-chain or branched-chain alkoxy groups,

[0049] the above R4 is substituted phenyl, substituted or unsubstituted C5-C9 heteroaryl, wherein the above heteroaryl is a 5- or 6-membered heteroaryl containing N or O heteroatoms, and the above substituted phenyl or heteroaryl can be substituted by C1-C6 straight-chain or branched-chain haloalkyls,

[0050] However, in the above R2, and can be excluded

[0051] More preferably

[0052] the above R1 is

[0053] R2 is -(Y)-(CH3)m or -(Y)-(CH2)m-R4,

[0054] the above Y is NH,

[0055] the above m is 1,

[0056] the above R4 is substituted phenyl, wherein the above substituted phenyl can be substituted by C1-C6 straight-chain or branched-chain haloalkyls,

[0057] However, in the above R2, and can be excluded

[0058] Preferably,

[0059] The disubstituted adamantyl derivative or a pharmaceutically acceptable salt thereof is characterized in that the disubstituted adamantyl derivative or a pharmaceutically acceptable salt thereof can be represented by the following Chemical Formula 2:

[0060] Chemical Formula 2:

[0061] In the above Chemical Formula 2,

[0062] R1 is -(X)-(CH2)n-R3 or

[0063] wherein X is O, NH or

[0064] n is 0, 1 or 2,

[0065] R3 are each independently a straight-chain or branched-chain alkyl having 1 to 6 carbon atoms, an unsubstituted or substituted aryl having 5 to C 10 aryl, or an unsubstituted or substituted heteroaryl having 5 to 9 carbon atoms,

[0066] wherein the heteroaryl is a 5-membered or 6-membered heteroaryl containing one or more N or O heteroatoms,

[0067] The above-mentioned substituted aryl or substituted heteroaryl can be substituted by one or more halogens, a straight-chain or branched-chain alkyl having 1 to 6 carbon atoms, a straight-chain or branched-chain haloalkyl having 1 to 6 carbon atoms, a hydroxyl group, or a straight-chain or branched-chain alkoxy group having 1 to 6 carbon atoms.

[0068] More preferably, in the above Chemical Formula 2,

[0069] The disubstituted adamantyl derivative or a pharmaceutically acceptable salt thereof is characterized in that R1 can be selected from the group consisting of:

[0070]

[0071] Preferably,

[0072] The disubstituted adamantyl derivative or a pharmaceutically acceptable salt thereof is characterized in that the disubstituted adamantyl derivative or a pharmaceutically acceptable salt thereof can be represented by the following Chemical Formula 3:

[0073] Chemical Formula 3:

[0074] In the above Chemical Formula 3,

[0075] R2 is -(Y)-(CH3)m or -(Y)-(CH2)m-R4,

[0076] The above Y is NH,

[0077] The above m is 0 or 1,

[0078] Each R4 is independently a straight-chain or branched-chain alkyl group having 1 to 6 carbon atoms, an unsubstituted or substituted aryl group having 5 to C 10 aryl, or an unsubstituted or substituted heteroaryl group having 5 to 9 carbon atoms,

[0079] wherein the above heteroaryl group is a 5-membered or 6-membered heteroaryl group containing one or more N or O heteroatoms,

[0080] The above substituted aryl group or substituted heteroaryl group can be substituted by one or more halogen atoms, a straight-chain or branched-chain alkyl group having 1 to 6 carbon atoms, a straight-chain or branched-chain haloalkyl group having 1 to 6 carbon atoms, a hydroxyl group, or a straight-chain or branched-chain alkoxy group having 1 to 6 carbon atoms,

[0081] However, in the above R2, and can be excluded.

[0082] More preferably,

[0083] In the above Chemical Formula 3,

[0084] The disubstituted adamantyl derivative or a pharmaceutically acceptable salt thereof is characterized in that the above R2 can be selected from the group consisting of:

[0085]

[0086] Preferably,

[0087] The disubstituted adamantyl derivative or a pharmaceutically acceptable salt thereof is characterized in that the disubstituted adamantyl derivative or a pharmaceutically acceptable salt thereof can be represented by the following Chemical Formula 4:

[0088] Chemical Formula 4:

[0089] In the above Chemical Formula 4,

[0090] R1 is -(X)-(CH2)n-R3 or

[0091] The above X is O, NH or

[0092] The above n is 0, 1 or 2,

[0093] Each R3 is independently a straight-chain or branched-chain alkyl group having 1 to 6 carbon atoms, an unsubstituted or substituted aryl group having 5 to C 10 aryl, or an unsubstituted or substituted heteroaryl group having 5 to 9 carbon atoms,

[0094] Among them, the above-mentioned heteroaryl is a 5- or 6-membered heteroaryl containing more than one N or O heteroatom.

[0095] The above-mentioned substituted aryl or substituted heteroaryl can be substituted by more than one halogen, a straight-chain or branched alkyl group having 1 to 6 carbon atoms, a straight-chain or branched haloalkyl group having 1 to 6 carbon atoms, a hydroxyl group, or a straight-chain or branched alkoxy group having 1 to 6 carbon atoms.

[0096] More preferably,

[0097] In the above Chemical Formula 4,

[0098] The disubstituted adamantyl derivative or a pharmaceutically acceptable salt thereof is characterized in that the above-mentioned R1 can be selected from the group consisting of:

[0099]

[0100] Preferably,

[0101] The disubstituted adamantyl derivative or a pharmaceutically acceptable salt thereof is characterized in that the disubstituted adamantyl derivative or a pharmaceutically acceptable salt thereof can be represented by the following Chemical Formula 5:

[0102] Chemical Formula 5:

[0103] In the above Chemical Formula 5,

[0104] R2 is -(Y)-(CH3)m or -(Y)-(CH2)m-R4,

[0105] The above-mentioned Y is NH,

[0106] The above-mentioned m is 0 or 1,

[0107] Each R4 is independently a straight-chain or branched alkyl group having 1 to 6 carbon atoms, an unsubstituted or substituted aryl group having 5 to C 10 aryl, or an unsubstituted or substituted heteroaryl group having 5 to 9 carbon atoms,

[0108] Among them, the above-mentioned heteroaryl is a 5- or 6-membered heteroaryl containing more than one N or O heteroatom,

[0109] The above-mentioned substituted aryl or substituted heteroaryl can be substituted by more than one halogen, a straight-chain or branched alkyl group having 1 to 6 carbon atoms, a straight-chain or branched haloalkyl group having 1 to 6 carbon atoms, a hydroxyl group, or a straight-chain or branched alkoxy group having 1 to 6 carbon atoms,

[0110] However, in the above R2, and can be excluded.

[0111] More preferably,

[0112] In the above chemical formula 5,

[0113] The disubstituted adamantyl derivative or a pharmaceutically acceptable salt thereof is characterized in that the above R2 can be selected from the group consisting of:

[0114]

[0115] Most preferably,

[0116] The disubstituted adamantyl derivative of the present invention can be one or more of the following:

[0117] Methyl-3-(2-(4-(3-((methoxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10a);

[0118] Methyl-3-(2-(4-(4-((4-methoxybenzyloxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10b);

[0119] Methyl-3-(2-(4-(4-((3,4-dimethoxybenzyloxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10c);

[0120] Methyl 3-(2-(4-(2-((3,4-dimethoxyphenethoxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10d);

[0121] Methyl-3-(2-(4-(3-((furan-2-ylmethoxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10e);

[0122] Methyl 3-(2-(4-(3-methylcarbamoyl-adamantan-1-yl)phenoxy)acetamido)benzoate (10f);

[0123] Methyl 3-(2-(4-(3-benzylcarbamoyl-adamantan-1-yl)phenoxy)acetamido)benzoate (10g);

[0124] Methyl 3-(2-(4-(3-(furan-2-ylmethylcarbamoyl)-adamantan-1-yl)phenoxy)acetamido)benzoate (10h);

[0125] Methyl 3-(2-(4-(3-(pyridin-2-ylcarbamoyl)-adamantan-1-yl)phenoxy)acetamido)benzoate (10i);

[0126] Methyl 3-(2-(4-(3-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-adamantan-1-yl)phenoxy)acetamido)benzoate (10j);

[0127] 3,4-Dimethoxybenzyl-2-(4-(2-oxo-2-(3-(trifluoromethyl)phenylamino)ethoxy)phenyl)adamantane-1-ylcarboxylate (14a);

[0128] 3,4-Dimethoxybenzyl-2-(4-(2-oxo-2-(quinolin-8-ylamino)ethoxy)phenyl)adamantane-1-ylcarboxylate (14b);

[0129] 3,4-Dimethoxybenzyl-2-(4-(2-(furan-2-ylmethylamino)-2-oxoethoxy)phenyl)adamantane-1-ylcarboxylate (14c);

[0130] 3,4-Dimethoxybenzyl-2-(4-(2-(4-methylpiperazin-1-yl)-2-oxoethoxy)phenyl)adamantane-1-ylcarboxylate (14d);

[0131] N-(Furan-2-ylmethyl)-5-(4-(2-(4-methylpiperazin-1-yl)-2-oxoethoxy)phenyl)adamantan-1-yl-carboxamide (17a);

[0132] 1-(4-Methylpiperazin-1-yl)-2-(4-(5-(4-(4-(trifluoromethyl)benzyl)piperazine-1-carbonyl)adamantan-1-yl-)phenoxy)ethanone (17b);

[0133] 2-(4-(5-(4-(4-Methoxybenzyl)piperazine-1-carbonyl)adamantan-1-yl)phenoxy)-1-(4-methylpiperazin-1-yl)ethanone (17c);

[0134] 2-(4-(3-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)adamantan-1-yl)phenoxy)-1-morpholinoethanone (21a);

[0135] 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

[0136] 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).

[0137] As used herein, the symbol "Ca-b" or "Ca to Cb" attached to a substituent means that the number of carbon atoms contained in the substituent is from a to b.

[0138] In the present invention, "C1 to C6 straight-chain or branched-chain alkyl" means a straight-chain or branched-chain saturated hydrocarbon group having 1 to 6 carbon atoms. For example, methyl, ethyl, propyl, butyl, 1-methylethyl, diethyl, or dimethyl, etc., but is not limited thereto.

[0139] In the present invention, "C1 to C6 straight-chain or branched-chain alkoxy" means an OR group where R is an alkyl. For example, methoxy, ethoxy, propoxy, butoxy, 1-methylethoxy, 1,1-dimethylethoxy, etc., but is not limited thereto. 1-6 In the present invention, "halogen" is fluorine, bromine, chlorine, or iodine.

[0140] In the present invention, "haloalkyl" means an alkyl substituted with one or more halogen atoms.

[0141]

[0142] ​The disubstituted adamantyl derivative represented by Chemical Formula 1 of the present invention can be used in the form of a pharmaceutically acceptable salt. In the present invention, the term "pharmaceutically acceptable" includes compounds or compositions that meet medical standards and do not pose unreasonable risks of toxicity, irritation, and other problems or complications, and do not cause problems when in contact with tissues of biological objects such as humans. In the present invention, as the salt, an acid addition salt formed from a pharmaceutically acceptable free acid is used. The acid addition salts are formed from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, or phosphorous acid; non-toxic organic acids such as aliphatic monocarboxylates and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids; 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 pharmaceutically non-toxic salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, fluorides, acetates, propionates, decanoates, octanoates, acrylamides, formates, isobutyrates, decanoates, heptanoates, propiolates, oxalates, malonates, succinates, octanedioates, decanedioates, fumarates, maleates, butyne-1,4-dioates, hexane-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, terephthalates, benzenesulfonates, toluenesulfonates, chlorobenzenesulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, β-hydroxybutyrates, glycolates, malates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, or mandelates.

[0143] The acid addition salts of the present invention can be prepared by conventional methods. For example, they can be prepared by dissolving the compound in an excess of an aqueous acid solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. Also, they can be prepared by heating an equimolar amount of the compound and the acid or alcohol in water and then evaporating the mixture to dryness or by filtering off the precipitated salt.

[0144] Furthermore, pharmaceutically acceptable metal salts can be prepared using bases. Salts derived from suitable bases can include, but are not limited to, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium. Alkali metal salts or alkaline earth metal salts can be obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate. At this time, preparing sodium salts, potassium salts, or calcium salts as metal salts is pharmaceutically appropriate, and the corresponding silver salts can be obtained by reacting the alkali metal salts or alkaline earth metal salts with a suitable silver salt (e.g., silver nitrate).

[0145] Furthermore, the present invention not only includes the disubstituted adamantyl derivatives of Formula 1 above and their pharmaceutically acceptable salts, but also includes solvates, hydrates, isomers, etc. that can be prepared therefrom.

[0146] More specifically, the compounds of the present invention may contain asymmetric or chiral centers and exist in the form of stereoisomers. All stereoisomeric forms of the compounds of the present invention, such as diastereoisomers, enantiomers, racemic mixtures, etc., are considered to form part of the present invention. Among them, a mixture of multiple isomers is called a diastereoisomer, and a mixture formed by mixing enantiomers in a ratio of 50:50 is called a racemate.

[0147] In the present invention, "diastereoisomers" refer to stereoisomers having two or more chiral centers, and their molecules are not mirror images of each other. Diastereoisomers have different physical properties, for example, in terms of melting point, boiling point, spectral characteristics, and reactivity. A mixture of diastereoisomers can be separated by high-resolution analysis processes such as electrophoresis and chromatography.

[0148] In the present invention, "enantiomers" refer to two stereoisomers of a compound that are non-superimposable mirror images of each other.

[0149] Furthermore, the present invention provides a method for preparing a disubstituted adamantyl derivative, which is characterized by preparing a compound represented by Formula 1 according to the following reaction formula:

[0150]

[0151] (wherein, R1 and R2 are as defined herein.)

[0152] More specifically, a method for preparing a disubstituted adamantyl derivative is provided, which is characterized by preparing a compound represented by Formula 2 according to the following reaction formula:

[0153]

[0154] (a) AlCl3, bromine, DCM; (b) anisole, AlCl3; (c) BBr3, DCM; (d) benzyl bromide, KHCO3, DMF; (e) ethyl chloroacetate, K2CO3, DMF; (f) LiOH, THF / H2O; (g) methyl 3-aminobenzoate, PyBOP, DMAP, DMF; (h) H2, Pd / C, THF; (i) Synthesis of 10a-d, 10f-g, 10j using EDC, HOBT, DIPEA, DMF; Synthesis of 10e, 10h using ethyl chloroformate, TEA, THF;

[0155] (wherein, R1 is as defined herein.)

[0156] More specifically, a method for preparing a disubstituted adamantyl derivative is provided, which is characterized in that a compound represented by Chemical Formula 3 is prepared according to the following reaction formula:

[0157]

[0158] (a) 3,4-dimethoxybenzyl bromide, K2CO3, DMF; (b) ethyl chloroacetate, K2CO3, DMF; (c) LiOH, THF / H2O; (d) corresponding amine, EDC, HOBT, DIPEA, DMF

[0159] (wherein, R2 is as defined herein.)

[0160] More specifically, a method for preparing a disubstituted adamantyl derivative is provided, which is characterized in that a compound represented by Chemical Formula 4 is prepared according to the following reaction formula:

[0161]

[0162] (a) 1-methylpiperazine, EDC, HOBT, DIPEA, DMF; (b) Pd / C, methanol; (c) corresponding amine, EDC, HOBT, DIPEA, DMF

[0163] (wherein, R1 is as defined herein.)

[0164] More specifically, a method for preparing a disubstituted adamantyl derivative is provided, which is characterized in that a compound represented by Chemical Formula 5 is prepared according to the following reaction formula:

[0165]

[0166] (a) 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, EDC, HOBT, DIPEA, DMF; (b) ethyl chloroacetate, K2CO3, DMF; (c) LiOH, THF / H2O; (d) corresponding amine, EDC, HOBT, DIPEA, DMF

[0167] (wherein, R2 is as defined herein.)

[0168] The method for preparing the disubstituted adamantyl derivative of the present invention is not limited to the above method. Moreover, even in the case of preparing according to the above reaction formula, in addition to the above reagents, the reaction can also be carried out by selectively changing the reagents that do not affect the reaction.

[0169] Furthermore, the present invention provides a pharmaceutical composition for anti-cancer comprising the above disubstituted adamantyl derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

[0170] In the present invention, the above pharmaceutical composition can be a pharmaceutical composition for anti-cancer against solid cancer

[0171] The above solid cancers include, for example, colorectal cancer, lung cancer, liver cancer, gastric cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head and neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, perianal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, prostate cancer, fibrosarcoma, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumors, etc., but are not limited thereto.

[0172] In the present invention, "for anti-cancer" means "preventing or treating cancer", which means inhibiting, delaying the onset of cancer in a biological subject (e.g., human or animal) and further inhibiting or preventing the progression of cancer, reducing the progression rate, improving or alleviating symptoms, etc.

[0173] Moreover, the present invention provides a cancer treatment method comprising the step of administering a therapeutically effective amount of the disubstituted adamantyl derivative represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof to a patient in need of anti-cancer treatment. And, provides the use of the disubstituted adamantyl derivative represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof in the preparation of a preparation for treating cancer. Moreover, provides the use of the disubstituted adamantyl derivative represented by the above chemical formula 1 or a pharmaceutically acceptable salt thereof in the treatment of cancer.

[0174] In the present invention, an "effective amount" refers to an amount of a compound of the present invention that is used for treating or preventing a specific disease or disorder, relieving, attenuating or eliminating the symptoms of a specific disease or disorder, or preventing or delaying the onset of one or more symptoms of a specific disease or disorder described herein. In the present invention, the therapeutically effective amount of a drug refers to inhibiting tumor growth; inhibiting the activation and growth of HIF-1 in cancer cells; inhibiting mitochondrial respiration; relieving or treating the symptoms of complications caused by cancer; delaying and inhibiting (preferably improving) the progression rate of cancer; preventing the deterioration of cancer, etc. The effective amount level may depend on factors such as the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the administration time, the administration route and excretion rate, the duration of treatment, factors including co-administered drugs, and other factors well known in the medical field.

[0175] In the present invention, the above-mentioned "patient" is not limited, and preferably includes mammals including humans.

[0176] The content of the disubstituted adamantyl derivative of the above chemical formula 1 or its pharmaceutically acceptable salt 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 condition of the patient, etc. For example, based on the total weight of the composition, it can be 0.0001% by weight to 99.9% by weight, or 0.001% by weight to 50% by weight, but is not limited thereto. The above content ratio is a value based on the dry amount after removing the solvent.

[0177] The pharmaceutical composition of the present invention may further contain suitable carriers, excipients and diluents commonly used for preparing pharmaceutical compositions. The above excipients may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, humectants, film coating substances and controlled release additives.

[0178] The pharmaceutical composition of the present invention can be formulated into powders, granules, sustained release granules, enteric-coated granules, liquids, eye drops, elixirs, emulsions, suspensions, spirits, lozenges, aromatics, lemonades, tablets, sustained release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained release capsules, enteric-coated capsules, pills, tinctures, soft extracts, dry extracts, liquid extracts, injections, capsules, lavages, plasters, lotions, pastes, sprays, inhalants, patches, sterile injection solutions or aerosols and other external preparations in accordance with conventional methods. The above external preparations can have dosage forms such as creams, gels, patches, sprays, ointments, plasters, lotions, liniments, pastes or cataplasms.

[0179] The carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention can be lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate and mineral oil.

[0180] During formulation, common diluents or excipients are used, such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc.

[0181] As additives for the tablets, powders, granules, capsules, pills, and lozenges of the present invention, excipients can be used, such as corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methyl cellulose, sodium carboxymethyl cellulose, kaolin, urea, colloidal silica, hydroxypropyl starch, hydroxypropyl methyl cellulose, 1928, 2208, 2906, 2910, propylene glycol, casein, calcium lactate, sodium carboxymethyl starch, etc.; binders, such as gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerol, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethyl cellulose, refined shellac, starch paste, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc.; disintegrants, such as hydroxypropyl methyl cellulose, corn starch, agar powder, methyl cellulose, bentonite, hydroxypropyl starch, sodium carboxymethyl cellulose, sodium alginate, calcium carboxymethyl cellulose, calcium citrate, sodium dodecyl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, starch gel, gum arabic, amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, D-sorbitol solution, light anhydrous silicic acid, etc.; lubricants, such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc powder, lycopodium powder, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, polyethylene glycol 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium dodecyl sulfate, magnesium oxide, polyethylene glycol (Macrogol), synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, DL-leucine, light anhydrous silicic acid, etc.

[0182] As additives for the liquid preparations of the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitan fatty acid ester (Tween ester), polyoxyethylene monoalkyl ether, lanolin ether, lanolin ester, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, gliadin, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc. can be used.

[0183] As the syrup of the present invention, a solution of white sugar, other sugars or sweeteners, etc. can be used, and flavoring agents, coloring agents, preservatives, stabilizers, suspending agents, emulsifying agents, thickening agents, etc. can be used as needed.

[0184] As the preservative emulsion of the present invention, purified water can be used, and emulsifying agents, preservatives, stabilizers, flavoring agents, etc. can be used as needed.

[0185] As the suspending agent of the present invention, suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropyl methylcellulose, 1828, 2906, 2910, etc. can be used, and surfactants, preservatives, stabilizers, coloring agents, flavoring agents can be used as needed.

[0186] The injection of the present invention may comprise: solvents such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, glucose injection, glucose + sodium chloride injection, PEG, sodium lactate Ringer's injection, ethanol, propylene glycol, fixed oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, phenyl benzoate; cosolvents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, phenylbutazoline, propylene glycol, Tween, nicotinamide, hexamine, dimethylacetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, gums; isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen (N2), ethylenediaminetetraacetic acid; sulfiting agents such as 0.1% sodium hydrosulfide, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, calcium gluconate; suspending agents such as sodium carboxymethylcellulose, sodium alginate, Tween 80, aluminum monostearate.

[0187] As the suppository of the present invention, matrices can be used, such as cocoa butter, lanolin, semi-synthetic fatty acid esters, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, coconut oil, cocoa butter + cholesterol, lecithin, lanette wax, glyceryl monostearate, Tween or spandex, Imhausen, monoene (propylene glycol monostearate), glycerol, pharmaceutical grade mixed fatty acid glycerides (Adeps solidus), Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydrokote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), masa-MF, masupol, masupol-15, neosuppostal-N, paramount-B, supposiro (OSI, OSIX, A, B, C, D, H, L), suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), suppostal (N, Es), Wecoby (W, R, S, M, Fs), tegester triglyceride substance (TG-95, MA, 57)

[0188] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are formulated by mixing the above-mentioned extracts with at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition, lubricants such as magnesium stearate and talc are used in addition to simple excipients.

[0189] Liquid preparations for oral administration are suspensions, oral liquids, emulsions, syrups, etc., and these liquid preparations can include various types of excipients in addition to simple common diluents such as water and liquid paraffin, such as wetting agents, sweeteners, flavoring agents, preservatives, etc. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used.

[0190] The pharmaceutical composition of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with existing therapeutic agents, and can be administered once or multiple times. For example, the pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiotherapy, hormone therapy, chemotherapy, and biological response modifiers. Considering all of the above factors, it is important to administer the composition in the minimum amount that can achieve the maximum effect without any side effects, and this can be easily determined by those of ordinary skill in the art.

[0191] The pharmaceutical composition of the present invention can be administered to an individual via various routes. All modes of administration can be predicted. For example, it can be administered by oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, perispinal space (intrathecal) injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, intraocular administration, intra-aural administration, intranasal administration, inhalation, via oral or nasal spray, transdermal administration, percutaneous administration, etc.

[0192] The pharmaceutical composition of the present invention is determined according to the type of drug as the active ingredient, as well as various relevant factors such as the disease to be treated, the route of administration, the age, sex, weight of the patient, and the severity of the disease.

[0193] In the present invention, the dosage may vary depending on the condition, age, weight, sex, and weight of the patient, the severity of the disease, the form of the drug, the route of administration, and the time, and can be appropriately selected by those of ordinary skill. For example, in an amount of 0.01 mg / kg / day to 200 mg / kg / day, according to the judgment of a doctor or pharmacist, a predetermined time interval of one day can be divided into several times, preferably 1 to 3 times a day, and administered via oral or parenteral administration routes.

[0194] Hereinafter, the present invention will be described in detail by preparation examples, examples, and experimental examples.

[0195] However, the following preparation examples, examples, and experimental examples only specifically illustrate the present invention, and the content of the present invention is not limited to the preparation examples, examples, and experimental examples.

[0196] Preparation Example

[0197] Preparation Example: Synthesis of Compounds with HIF-1α Inhibitory Activity

[0198] As the HIF-1α inhibitor of the present invention, examples of non-limiting compounds used as the active ingredient of the pharmaceutical composition include the following compounds, their isomers, and their pharmaceutically acceptable salts. The following compounds of the present invention are synthesized by appropriately changing the reactants and / or starting materials of known methods.

[0199] Hereinafter, a specific description will be given.

[0200] To develop novel HIF-1α inhibitors, various disubstituted adamantyl derivatives were prepared by chemical modification at the adamantyl site of LW6. The method for synthesizing the novel compounds is described in Scheme S1-4. Product 2 was synthesized by the bromination reaction of 1-adamantane carboxylic acid 1 with bromine in acetic acid. The intermediate 2 was subjected to Friedel-Crafts alkylation reaction with anisole to obtain the ether product 3 in good yield. Demethylation of product 3 was carried out using boron tribromide (BBr3), and product 4 was chemically selectively protected with benzyl bromide to obtain product 5 under basic conditions. Further alkylation with ethyl chloroacetate produced product 6 in good yield. Product 6 was hydrolyzed with lithium hydroxide (LiOH) to obtain the carboxylic acid product 7, which was then coupled with methyl 3-aminobenzoate to form the amide derivative product 8 in good yield. In the presence of palladium on carbon, the benzyl group of product 8 was removed by catalytic hydrogenation to regenerate the precursor for the synthesis of the target adamantyl derivatives 10a-j. Carboxylic acid compound 9 can react with alcohols and amines to provide esters (10a-e) and amide derivatives (10f-j) respectively. Products 10a-10d, 10f, 10g and 10j were synthesized using hydroxybenzotriazole (HOBt) and EDC·HCl as coupling agents, while the other coupled products 10e, 10h 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 obtain the phenolic ester product 11, which was further alkylated with ethyl chloroacetate to obtain product 12 in good yield. Then, carboxylic acid 13 was obtained by base-mediated hydrolysis of product 12. Finally, the corresponding amides 14a-14d were prepared by coupling of various commercially available amines with EDC·HCl and HOBt (Scheme S2). Similarly, amide derivative 15 was obtained by reacting carboxylic acid intermediate 7 with 1-methylpiperazine in the presence of EDC·HCl and HOBt. The corresponding carboxylic acid product 16 was prepared by catalytic hydrogenation of 15 in the presence of palladium on carbon. Amide derivatives (17a-17c) were prepared by binding with various amines respectively (Scheme S3). Based on the synthetic profile of 14a-14d, as shown in Scheme S4, the ester moiety of the carboxylic acid of product 11 was replaced with an amide to obtain 21a-21c. Product 4 was obtained under amide coupling conditions to obtain intermediate 18, which was then alkylated with ethyl chloroacetate to produce product 19 in good yield. Hydrolysis of product 19 with LiOH provided product 20, which was then coupled with various commercially available amines to obtain the disubstituted adamantyl derivatives 21a-21c (Scheme S4).

[0201] Synthesis of Reactive Disubstituted Adamantyl Derivatives 10a - 10j a

[0202]

[0203] a Reagents and conditions: (a) AlCl3, bromine, DCM; (b) anisole, AlCl3; (c) BBr3, DCM; (d) benzyl bromide, KHCO3, DMF; (e) ethyl chloroacetate, K2CO3, DMF; (f) LiOH, THF / H2O; (g) methyl 3 - aminobenzoate, PyBOP, DMAP, DMF; (h) H2, Pd / C, THF; (i) Synthesis of 10a - 10d, 10f - 10g, 10j using EDC, HOBT, DIPEA, DMF; Synthesis of 10e, 10h using ethyl chloroformate, TEA, THF;

[0204] Synthesis of Reactive Disubstituted Adamantyl Derivatives 14a - 14d a

[0205]

[0206] a Reagents and conditions: (a) 3,4 - dimethoxybenzyl bromide, K2CO3, DMF; (b) ethyl chloroacetate, K2CO3, DMF; (c) LiOH, THF / H2O; (d) corresponding amine, EDC, HOBT, DIPEA, DMF.

[0207] Synthesis of Reactive Disubstituted Adamantyl Derivatives 17a - 17c a

[0208]

[0209] a Reagents and conditions: (a) 1 - methylpiperazine, EDC, HOBT, DIPEA, DMF; (b) Pd / C, methanol; (c) corresponding amine, EDC, HOBT, DIPEA, DMF.

[0210] Synthesis of Reactive Disubstituted Adamantyl Derivatives 21a - 21c a

[0211]

[0212] 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 amine, EDC, HOBT, DIPEA, DMF. Example

[0213] Example 1. Synthesis of Compounds 2 to 9

[0214] The synthetic procedures of Compounds 2 to 9 used in the above reaction formula are as described below.

[0215] Compound 2: 3-Bromoadamantane-1-carboxylic acid (2):

[0216] AlCl3 (4.80 g, 36.10 mmol) was added to a two-necked round flask equipped with a reflux condenser and argon. The flask was stirred in a low-temperature reactor. Bromine (17.10 mL, 33.29 mmol) was added at -5 °C and stirred for 15 minutes. Then, 1-adamantane carboxylic acid (5.00 g, 27.70 mmol) was added to the flask, stirred at -5 °C for 1 hour and at room temperature for 48 hours. The reaction solution was diluted with chloroform. Excess bromine was treated with sodium pyrosulphite until the color completely changed. The organic layer was dried over anhydrous MgSO4, filtered and concentrated. The residue was recrystallized from hexane to obtain a white solid (2) (6.67 g, yield: 93%). 1 1H-NMR (400 MHz, DMSO-d6) δ (s, 1H), 2.36 (s, 2H), 2.26 - 2.13 (m, 6H), 1.79 (d, J = 2.8 Hz, 4H), 1.68 - 1.60 (m, 2H).

[0217] Compound 3: 3-(4-Methoxyphenyl)-adamantane-1-carboxylic acid (3):

[0218] 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. After the reaction mixture was stirred at room temperature for 24 h, ice and concentrated HCl (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 above concentrate was recrystallized from hexane to give a white solid (3) (5.20 g, yield: 94%). 1 1H-NMR (400 MHz, 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).

[0219] Compound 4: 3-(4-Hydroxyphenyl)-adamantane-1-carboxylic acid (4):

[0220] At -10 °C, a solution of 1.0 M BBr3 (17.40 mL, 17.45 mmol) diluted in DCM was added to a solution of compound 3 (2.00 g, 6.98 mmol) in DCM under argon. 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 a white solid (4) (1.80 g, yield: 94%). 1 1H-NMR (400 MHz, DMSO-d6) δ 1H), 9.11 (s, 1H), 7.13 (d, J = 9.2 Hz, 2H), 6.69 (d, J = 8.8 Hz, 2H), 2.12 (s, 2H), 1.83 - 1.75 (m, 10H), 1.65 (s, 2H).

[0221] Compound 5: 3-(4-Hydroxyphenyl)-adamantane-1-carboxylic acid benzyl ester (5):

[0222] Benzyl bromide (1.12 g, 6.61 mmol) was added to a mixture of compound 4 (1.50 g, 5.50 mmol) and KHCO3 (0.66 g, 6.61 mmol) in 20 mL of DMF. After heating the reaction mixture at 40 °C for 4 h, 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 above concentrate was purified by silica gel column chromatography to obtain a white solid (5) (1.75 g, yield: 87%). 1 1H-NMR (400 MHz, CDCl3) δ 7.35 δ J = J = 10H, 1.69 (s, 2H).

[0223] Compound 6: 3-(4-Ethoxycarbonylmethoxyphenyl)-adamantane-1-carboxylic acid benzyl ester (6):

[0224] 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. After stirring the reaction mixture overnight at room temperature, 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 above concentrate was purified by silica gel column chromatography to obtain a white solid (6) (1.05 g, yield: 85%). 1 1H-NMR (400 MHz, DMSO-d6) 7.39 - 7.31 (m, 5H), 7.26 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.8 Hz, 2H), 5.00 (s, 2H), 4.72 (s, 2H), 4.16 (q, J = 7.1 Hz, 2H), 2.15 (s, 2H), 1.90 - 1.79 (m, 10H), 1.67 (s, 2H), 1.21 (t, J = 7.0 Hz, 3H).

[0225] Compound 7: 3-(4-Carboxymethoxyphenyl)-adamantane-1-carboxylic acid benzyl ester (7):

[0226] 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 the mixture was stirred at room temperature for 90 minutes. Then, 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. After obtaining the above concentrated solution, it was purified by silica gel column chromatography to obtain a white solid (7) (0.60 g, yield: 92%). 1 1H-NMR (400 MHz, DMSO-d6) 12.93 (s, 1H), 7.37 - 7.31 (m, 5H), 7.26 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.8 Hz, 2H), 5.09 (s, 2H), 4.61 (s, 2H), 2.15 (s, 2H), 1.98 - 1.75 (m, 10H), 1.67 (s, 2H).

[0227] Compound 8: 3-{4-[(3-Methoxycarbonylphenylcarbamoyl)-methoxy]-phenyl}-adamantane-1-carboxylic acid benzyl ester (8):

[0228] A mixture of compound 7 (0.73 g, 1.74 mmol), methyl 3-aminobenzoate (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 overnight at room temperature. 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 above concentrated solution was purified by silica gel column chromatography to obtain a white crystal (8) (0.68 g, 72.00%). 1 1H-NMR (400 MHz, DMSO-d6) δ (s, 1H), 8.33 (s, 1H), 7.90 (d, J = 8.1 Hz, 1H), 7.68 (d, J = 8.1 Hz, 1H), 7.48 (t, J = 8.1 Hz, 1H), 7.40 - 7.33 (m, 5H), 7.30 (d, J = 8.7 Hz, 2H), 6.94 (d, J = 8.7 Hz, 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).

[0229] Compound 9: 3-{4-[(3-Methoxycarbonylphenylcarbamoyl)-methoxy]-phenyl}-adamantane-1-carboxylic acid (9):

[0230] 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 under H2 at room temperature for 2 hours and filtered through a Celite pad. The solution was concentrated under reduced pressure to give 0.78 g of a white solid (9). 1 1H-NMR (400 MHz, DMSO-d6) δ 1H), 10.31 (s, 1H), 8.33 (s, 1H), 7.90 (d, J = 8.1 Hz, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.48 (t, J = 8.1 Hz, 1H), 7.30 (d, J = 8.7 Hz, 2H), 6.94 (d, J = 8.7 Hz, 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).

[0231] Example 2. Synthesis of Compounds 10-1 to 10-10

[0232] According to the above reaction scheme S1, starting from compound 1 and using compounds 2, 3, 4, 5, 6, 7, 8 and 9 as intermediates, compounds 10-1 to 10-10 were synthesized.

[0233] Compound 10-1: Methyl-3-(2-(4-(3-((methoxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10a):

[0234] Compound 9 (0.31 g, 0.66 mmol), methanol (0.03 ml, 0.99 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (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 then stirred overnight. After completion of the stirring, the reaction was terminated by adding water and extracted with EA. The extracted organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure. The concentrated reactant was purified by silica gel column chromatography (DCM / EA) to obtain white solid 10a (0.37 g, 78%). 1 1H-NMR (400 MHz, CDCl3) δ 8.41 (s, 1H), 8.07 (s, 1H), 8.01 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.44 (t, J = 8.0 Hz, 1H), 7.34 (d, J = 8.8 Hz, 2H), 6.96 (d, J = 8.8 Hz, 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).

[0235] Compound 10-2: Methyl-3-(2-(4-(4-((4-methoxybenzyloxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10b):

[0236] Compound 9 (0.31 g, 0.66 mmol), 4-methoxybenzyl alcohol (0.12 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. After completion of the stirring, the reaction was terminated by adding water and extracted with EA. The extracted organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure. The concentrated reactant was purified by silica gel column chromatography (DCM / EA) to obtain the target compound 10b as a white solid (0.21 g, 54%). 1H-NMR (400 MHz, CDCl3) δ 8.49 (s, 1H), 8.10 (s, 1H), 7.99 (d, J = 7.6 Hz, 1H), 7.81 (d, J = 7.2 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 7.31 - 7.25 (m, 4H), 6.90 (dd, J = 8.8, 23.2 Hz, 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).

[0237] Compound 10 - 3: Methyl - 3-(2-(4-(4-((3,4-dimethoxybenzyloxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10c):

[0238] 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. After completion of stirring, the reaction was terminated by adding water and extracted with EA. The extracted organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure. The concentrated reactant was purified by silica gel column chromatography (DCM / EA) to give the target compound 10c as a white solid (0.25 g, 63%). 1 H-NMR (400 MHz, CDCl3) δ 8.38 (s, 1H), 8.07 (s, 1H), 8.01 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.45 (t, J = 7.8 Hz, 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).

[0239] Compound 10-4: Methyl 3-(2-(4-(2-((3,4-dimethoxyphenethoxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10d):

[0240] 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. After completion of stirring, the reaction was terminated by adding water and extracted with EA. The extracted organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure. The concentrated reactant was purified by silica gel column chromatography (DCM / EA) to obtain the target compound 10d as a white solid (0.36 g, 58%). 1 1H-NMR (400 MHz, CDCl3) δ 8.44 (s, 1H), 8.09 (s, 1H), 8.01 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.31 (d, J = 8.8 Hz, 1H), 6.95 (d, J = 8.8 Hz, 2H), 6.81 - 6.74 (m, 3H), 4.60 (s, 2H), 4.26 (t, J = 6.8 Hz, 2H), 3.91 (s, 3H), 3.85 - 3.83 (m, 6H), 2.88 (t, J = {7.0} Hz, 2H), 2.22 (s, 2H), 1.97 - 1.80 (m, 10H), 1.72 (s, 2H).

[0241] Compound 10-5: Methyl 3-(2-(4-(3-((furan-2-ylmethoxy)carbonyl)adamantane-1-yl)phenoxy)acetamido)benzoate (10e):

[0242] Note: In the 1H-NMR data, the value in the brackets for the coupling constant of 2.88 (t, J = {7.0} Hz) might be a specific value provided in the original text that was not clearly visible in the given input. Here it is kept as it is with the curly braces for clarity. If there was a clear error in the input, it should be corrected before translation.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 into the column. After the reaction mixture was stirred at room temperature for 30 minutes, water was poured in to terminate the reaction. The reactants were extracted with EA, and the extracted organic layer was dried over MgSO4 and concentrated under reduced pressure. The concentrated reactants were purified by silica gel column chromatography (DCM / methanol) to obtain the target compound 10e (49.0 mg, 70%) as a white solid. 1 1H-NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 8.10 (s, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 7.6 Hz, 1H), 7.43 - 7.39 (m, 2H), 7.30 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 6.36 (dd, J = 2.2, 11.4 Hz, 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).

[0243] Compound 10-6: Methyl 3-(2-(4-(3-methylcarbamoyl-adamantan-1-yl)phenoxy)acetamido)benzoate (10f):

[0244] 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 methylamine hydrochloride (0.02 g, 0.35 mmol) in DMF (5.0 mL). After the reaction mixture was stirred 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 vacuo. The resulting residue was purified by silica gel column chromatography (hexane:EA = 6:4) to obtain 10f (110 mg, yield: 66.1%) as a white solid. 1H-NMR (CDCl3, 400 MHz): δ 8.40 (s, 1H), 8.07 (s, 1H), 8.00 (dd, J = 8.2, 1.4 Hz, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.44 (t, J = 8.0 Hz, 1H), 7.34 (d, J = 8.4 Hz, 2H), 6.95 (d, J = 9.2 Hz, 2H), 5.67 (s, 1H), 4.61 (s, 2H), 3.92 (s, 3H), 2.81 (d, J = 4.4 Hz, 3H), 2.26 (s, 2H), 1.97 - 1.87 (m, 10H), 1.73 (s, 2H).

[0245] Compound 10 - 7: Methyl 3-(2-(4-(3-benzylcarbamoyl-adamantan-1-yl)phenoxy)acetamido)benzoate (10 g):

[0246] 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). The reaction mixture was stirred overnight at room temperature, and 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 vacuo. The resulting residue was purified by silica gel column chromatography (hexane:EA = 6:4) to give 10 g (110 mg, yield: 56.9%) of a white solid. 1 H-NMR (400 MHz, CDCl3): δ 8.38 (s, 1H), 8.07 (s, 1H), 8.01 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H), 7.33 (t, J = 7.6 Hz, 4H), 7.29 - 7.26 (m, 5H), 5.89 (s, 1H), 4.61 (s, 2H), 4.45 (d, J = 5.2 Hz, 2H), 3.92 (s, 3H), 2.27 (s, 2H), 2.04 - 1.90 (m, 10H), 1.74 (s, 2H).

[0247] Compound 10-8: Methyl 3-(2-(4-(3-(furan-2-ylmethylcarbamoyl)-adamantan-1-yl)phenoxy)acetamido)benzoate (10h):

[0248] 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. Then, ethyl chloroformate (0.016 mL, 0.168 mmol) was added and the mixture was stirred at -10 °C for 30 minutes. After that, furfurylamine (0.015 mL, 0.155 mmol) was added to the solution and the temperature was raised to room temperature. The reaction solution was further stirred at room temperature for 30 minutes, and the solvent was evaporated under reduced pressure. The residue was purified by preparative TLC to give yellow solid 10h (49 mg, yield: 70%). 1 1H-NMR (400 MHz, CDCl3) δ (s, 1H), 8.07 (s, 1H), 8.01 (dd, J = 1.2 - 8.4 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H), 7.34 (d, J = 8.8 Hz, 3H), 6.96 (d, J = 9.2 Hz, 2H), 6.32 (t, J = 2.4 Hz, 1H), 6.21 (d, J = 3.2 Hz, 1H), 4.61 (s, 2H), 4.44 (d, J = 5.2 Hz, 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).

[0249] Compound 10-9: Methyl 3-(2-(4-(3-(pyridin-2-ylcarbamoyl)-adamantan-1-yl)phenoxy)acetamido)benzoate (10i):

[0250] 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). Then, the reaction solution was stirred overnight at room temperature, and 10% HCl was added dropwise. The mixture was extracted with EA, and the combined organic layers were 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 obtain yellow solid 10i (30 mg, yield: 52%). 1 1H-NMR (300 MHz, DMSO-d6) δ 1H), 9.78 (s, 1H), 8.33 (d, J = 2.1 Hz, 1H), 8.31 (s, 1H), 8.06 (d, J = 8.7 Hz, 1H), 7.90 (d, J = 7.8 Hz, 1H), 7.56 (t, J = 7.5 Hz, 1H), 7.67 (d, J = 7.2 Hz, 1H), 7.48 (t, J = 8.1 Hz, 1H), 7.36 (d, J = 9 Hz, 2H), 7.09 (t, J = 7.5 Hz, 1H), 6.96 (d, J = 9 Hz, 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).

[0251] Compound 10-10: Methyl 3-(2-(4-(3-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-adamantan-1-yl)phenoxy)acetamido)benzoate (10j):

[0252] 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 methyl 3-aminobenzoate (53 mg, 0.35 mmol) in DMF (5.0 mL). After the reaction mixture was stirred overnight at room temperature, 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 vacuo. The resulting residue was purified by silica gel column chromatography (hexane:EA = 6:4) to give white solid 10j (107 mg, yield: 48%). 1 1H-NMR (CDCl3, 400 MHz) δ 8.39 (s, 1H), 8.07 (s, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 7.6 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.36 (d, J = 8.8 Hz, 1H), 6.97 (d, J = 8.4 Hz, 2H), 6.60 (d, J = 5.2 Hz, 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).

[0253] Example 3. Synthesis of Compounds 14-1 to 14-4

[0254] According to the above reaction scheme S2, using compound 4 as the starting material and compounds 11, 12, and 13 as intermediates, compounds 14-1 to 14-4 were synthesized.

[0255] Compound 14-1: 3,4-Dimethoxybenzyl-2-(4-(2-oxo-2-(3-(trifluoromethyl)phenylamino)ethoxy)phenyl)adamantane-1-ylcarboxylate (14a):

[0256] 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 the reaction mixture was stirred overnight at room temperature, 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 vacuo. The resulting residue was purified by silica gel column chromatography (hexane:EA = 6:4) to give white solid 14a (64 mg, yield: 54%). 1 1H-NMR (CDCl3, 400 MHz) δ 8.39 (s, 1H), 7.87 (s, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.48 (t, J = 7.8 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 8.8 Hz, 2H), 6.95 (d, J = 9.2 Hz, 2H), 6.91 (dd, J = 8.4, 2.0 Hz, 1H), 6.85 (s, 1H), 6.84 (d, J = 10.8 Hz, 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).

[0257] Compound 14-2: 3,4-Dimethoxybenzyl-2-(4-(2-oxo-2-(quinolin-8-ylamino)ethoxy)phenyl)adamantane-1-ylcarboxylate (14b):

[0258] 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 the reaction mixture was stirred overnight at room temperature, 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 vacuo. The resulting residue was purified by silica gel column chromatography (hexane:EA = 6:4) to give white solid 14b (79 mg, yield: 61%). 1 1H-NMR (400 MHz, CDCl3) δ 10.97 (s, 1H), 8.86 (dd, J = 1.4 - 4.2 Hz, 1H), 7.56 (d, J = 1.6 Hz, 1H), 7.55 (s, 1H), 7.47 (q, J = 4.1 Hz, 1H), 7.33 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H), 6.91 (d, J = 10.0 Hz, 1H), 6.84 (d, J = 8.0 Hz, 2H), 5.05 (s, 2H), 4.73 (s, 2H), 3.88 (s, 6H), 2.23 (s, 2H), 2.04 - 1.87 (m, 10H), 1.72 (s, 2H); 13C-NMR (100 MHz, CDCl3) δ 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.

[0259] Compound 14-3: 3,4-Dimethoxybenzyl-2-(4-(2-(furan-2-ylmethylamino)-2-oxoethoxy)phenyl)adamantane-1-ylcarboxylate (14c):

[0260] 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-ylmethylamine (20 mg, 0.20 mmol) in DMF (5.0 mL). The reaction mixture was stirred at room temperature overnight, then diluted with EtOAc and washed with water and brine. The organic layer was separated, dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (hexane:EA = 6:4) to afford compound 14c (57 mg, 54% yield) as a white solid. 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).

[0261] Compound 14-4: 3,4-Dimethoxybenzyl-2-(4-(2-(4-methylpiperazin-1-yl)-2-oxoethoxy)phenyl)adamantane-1-ylcarboxylate (14d):

[0262] 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). The reaction mixture was stirred at room temperature overnight, then diluted with EtOAc and washed with water and brine. The organic layer was separated, dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (hexane:EA = 6:4) to afford 14d (50 mg, 47% yield) as a white solid. 11H-NMR (400 MHz, CDCl3) δ 7.26 (d, J = 8.8 Hz, 2H), 6.89 - 6.83 (m, 5H), 5.05 (s, 2H), 4.65 (s, 2H), 3.87 (s, 6H), 3.61 (d, J = 23.2 Hz, 4H), 2.39 (s, 4H), 2.29 (s, 3H), 2.21 (s, 2H), 2.01 - 1.85 (m, 10H), 1.71 (s, 2H).

[0263] Example 4. Synthesis of Compounds 17-1 to 17-3

[0264] According to the above reaction formula S3, using compound 7 as the starting material and compounds 15 and 16 as intermediates, compounds 17-1 to 17-3 were synthesized.

[0265] Compound 17-1: N-(Furan-2-ylmethyl)-5-(4-(2-(4-methylpiperazin-1-yl)-2-oxoethoxy)phenyl)adamantan-1-yl-carboxamide (17a):

[0266] 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-ylmethylamine (25 mg, 0.26 mmol) in DMF (3.0 mL). The reaction mixture was stirred overnight at room temperature, and 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 vacuo. The resulting residue was purified by silica gel column chromatography (DCM:MeOH = 1:9) to give white solid 17a (0.30 mg, yield: 29%). 11H-NMR (CDCl3, 400 MHz): δ 7.33 (s, 1H), 7.29 (s, 1H), 7.25 (s, 1H), 6.87 (d, J = 3.7 Hz, 2H), 6.31 - 6.29 (m, 1H), 6.19 (d, J = 3.6 Hz, 1H), 5.96 (s, 1H), 4.64 (s, 2H), 4.42 (d, J = 5.2 Hz, 2H), 3.63 (t, J = 4.8 Hz, 2H), 3.57 (t, J = 5.0 Hz, 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).

[0267] Compound 17 - 2: 1-(4-Methylpiperazin-1-yl)-2-(4-(5-(4-(4-(trifluoromethyl)benzyl)piperazine-1-carbonyl)adamantan-1-yl-)phenoxy)ethanone (17b):

[0268] 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). The reaction mixture was stirred overnight at room temperature, and 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 vacuo. The resulting residue was purified by silica gel column chromatography (DCM:MeOH = 1:9) to give white solid 17b (25 mg, yield: 19%). 1 1H-NMR (CDCl3, 400 MHz): δ 7.57 (d, J = 8.0 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.8 Hz, 2H), 4.67 (s, 2H), 3.70 - 3.54 (m, 10H), 2.43 - 2.37 (m, J = 3.3 Hz, 8H), 2.29 (s, 3H), 2.23 (s, 2H), 2.06 - 2.00 (m, 6H), 1.85 (s, 4H), 1.71 (s, 2H).

[0269] Compound 17-3: 2-(4-(5-(4-(4-Methoxybenzyl)piperazine-1-carbonyl)adamantan-1-yl)phenoxy)-1-(4-methylpiperazin-1-yl)ethanone (17c):

[0270] 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 the reaction mixture was stirred overnight at room temperature, 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 vacuo. The resulting residue was purified by silica gel column chromatography (DCM:MeOH = 1:9) to give white solid 17c (37 mg, yield: 30%). 1 1H-NMR (CDCl3, 400 MHz) δ 7.26 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 8.8 Hz, 2H), 6.90 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.8 Hz, 2H), 4.66 (s, 2H), 3.80 (s, 3H), 3.70 - 3.66 (m, 6H), 3.61 (t, J = 4.8 Hz, 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).

[0271] Example 5. Synthesis of Compounds 19 and 20

[0272] The synthetic procedures of compound 19 and compound 20 used in the above reaction formula are as described below.

[0273] Compound 19: Ethyl {4-[3-(6,7-dimethoxy-3,4-dihydro-1H-isoquinoline-2-carbonyl)-adamantan-1-yl]-phenoxy}-acetate (19):

[0274] Dissolve compound 15 (2.26 g, 6.30 mmol) in acetonitrile, add triethylamine (2.25 g, 0.02 mol) and 50% PPAA (4.81 g, 7.57 mmol) at room temperature. Stir the mixture for 30 minutes and add 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride (1.74 g, 7.57 mmol). Then, stir the reaction solution overnight at room temperature and evaporate it under reduced pressure. Purify the reaction mixture by silica gel column chromatography to obtain compound 19 as a white solid (2.21 g, yield: 66%). 1 1H-NMR (400 MHz, DMSO-d6) δ (d, J = 8.7 Hz, 2H), 6.84 (d, J = 9 Hz, 2H), 6.83 (s, 1H), 6.70 (s, 1H), 4.73 (s, 2H), 4.64 (s, 2H), 4.16 (q, J = 6.3 Hz, 2H), 3.81 (q, J = 5.7 Hz, 2H), 3.71 (s, 3H), 3.70 (s, 3H)), 2.69 (q, J = 5.1 Hz, 2H), 2.17 (br, 2H), 1.95 - 1.86 (m, 7H), 1.78~1.67 (m, 5H), 1.21 (t, J = 7.5 Hz, 3H).

[0275] Compound 20: {4-[3-(6,7-dimethoxy-3,4-dihydro-1H-isoquinoline-2-carbonyl)-adamantan-1-yl]-phenoxy}-acetic acid (20):

[0276] Dissolve compound 19 (2.21 g, 4.14 mmol) in a solution of THF and H2O, add lithium hydroxide (0.26 g, 6.21 mmol) at room temperature and stir for 1 hour, then add 10% HCl dropwise. Evaporate the filtrate under reduced pressure and wash the solid with n-hexane to obtain product 20 as a white solid (2.07 g). 11H-NMR (400 MHz, DMSO-d6) δ 7.29 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.1 Hz, 2H), 6.81 (s, 1H), 6.70 (s, 1H), 4.64 (s, 2H), 4.62 (s, 2H), 3.81 (q, J = 5.1 Hz, 2H), 3.71 (s, 3H), 3.70 (s, 3H), 2.69 (q, J = 5.7 Hz, 2H), 2.17 (br, 2H), 1.99 - 1.88 (m, 8H), 1.78 - 1.66 (m, 4H).

[0277] Example 6. Synthesis of Compounds 21-1 to 21-3

[0278] According to the above reaction formula S1, using compound 7 as the starting material and compounds 15 and 16 as intermediates, compounds 21-1 to 21-3 were synthesized.

[0279] Compound 21-1: 2-(4-(3-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)adamantan-1-yl)phenoxy)-1-morpholinoethanone (21a):

[0280] 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). The reaction mixture was stirred overnight at room temperature, and 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 vacuo. The resulting residue was purified by silica gel column chromatography (DCM:MeOH = 1:9) to give white solid 21a (81 mg, yield: 48%). 11H-NMR (400 MHz, CDCl3) δ (d, J = 2.4 Hz, 2H), 7.28 (d, J = 3.2 Hz, 2H), 6.60 (d, J = 6.4 Hz, 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 = 6.4 Hz, 2H), 2.26 (s, 2H), 2.13 - 2.02 (m, 6H), 1.89 - 1.86 (m, 4H), 1.75 (s, 2H).

[0281] 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):

[0282] 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 the reaction mixture was stirred overnight at room temperature, 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 vacuo. The resulting residue was purified by silica gel column chromatography (DCM:MeOH = 1:9) to give white solid 21b (80 mg, yield: 45%). 1 1H-NMR (400 MHz, CDCl3) δ (d, J = 6.8 Hz, 2H), 6.90 (d, J = 8.8 Hz, 2H), 6.60 (d, J = 6.2 Hz, 2H), 4.71 (s, 2H), 4.66 (s, 2H), 3.89 (s, 2H), 3.85 (s, 6H), 3.62 (d, J = 22.4 Hz, 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).

[0283] 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):

[0284] 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 the reaction mixture was stirred overnight at room temperature, 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 vacuo. The resulting residue was purified by silica gel column chromatography (DCM:MeOH = 1:9) to afford white solid LW1564 (21c) (0.123 mg, yield: 57%). 1 1H-NMR (400 MHz, CDCl3) δ (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.29 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.4 Hz, 2H), 6.60 (d, J = 6.0 Hz, 2H), 4.71 (s, 2H), 4.66 (s, 2H), 3.90 (t, J = 5.8 Hz, 2H), 3.85 (d, J = 2.0 Hz, 6H), 3.64 (s, 2H), 3.59 (t, J = 4.8 Hz, 2H), 3.55 (s, 2H), 2.80 (t, J = 5.4 Hz, 2H), 2.44 (s, 4H)), 2.26 (s, 2H), 2.13 - 2.05 (m, 6H), 1.89 (d, J = 2.4 Hz, 4H), 1.76 (s, 2H).

[0285] Experimental Example

[0286] Screening for HIF-1 Inhibitors by HRE-Luciferase Assay

[0287] Prior to measuring luciferase activity, under hypoxic conditions, HCT116 cells stably expressing the HRE-dependent luciferase reporter gene were treated with the disubstituted adamantyl derivatives prepared in the above-described examples for 12 hours.

[0288] Luciferase activity was studied in HCT116 cells treated with the disubstituted adamantyl derivatives by HRE-luciferase assay. The luciferase assay system (Promega, Madison, WI, USA) was used and the HRE-luciferase reporter gene assay was performed according to the manufacturer's instructions by the method described previously (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).

[0289] As a result, the activities exhibited by the disubstituted adamantyl derivatives are shown in Table 1 below.

[0290] Table 1

[0291]

[0292] LW1564 (Compound 21-3) Inhibits the Activation and Proliferation of HIF-1 in Cancer Cells

[0293] Among the newly synthesized adamantyl compounds, compound 21c (LW1564 (compound 21-3)) was evaluated. Figure 1a)How it affects the proliferation inhibition 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 cells (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 fibroblasts (CCD-34Lu and WI-38) were purchased from the American Type Culture Collection (Manassas, VA, USA) or the KRIBB Cell Line Bank (Daejeon). To culture the cells, the cells were cultured in DMEM medium (Gibco, Grand Island, NY, USA) supplemented with 5% (v / v) fetal bovine serum (WelGENE, Daegu, Korea), 100 U / ml penicillin, and 100 μg / ml streptomycin (Gibco). The cells were maintained at 37 °C in a humidified incubator with 5% CO2, and the hypoxia and multi-gas incubator (Sanyo, Osaka, Japan) was adjusted to 1% O2, 94% N2, and 5% CO2. The cell viability was determined by the methylene blue colorimetric assay 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)". After treatment with LW1564 (Compound 21-3), the cells were fixed with 4% formaldehyde (Sigma-Aldrich) and washed three times with PBS. After staining with methylene blue (Sigma-Aldrich), the cells were washed with water. The dye was eluted with 0.1% HCl (v / v), and quantification was performed at 600 nm on a Molecular Devices EMax (Molecular Devices, CA, USA). It was found that LW1564 (Compound 21-3) inhibited the proliferation of various cancer cells (GI 50 = 0.4 - 4.6 μM), and it was confirmed that it did not affect CCD-34Lu and WI-38 (GI 50Growth at a concentration greater than 20 μM Figure 1b ) Subsequently, an IncuCyte ZOOM live cell imaging system (Essen Bioscience, Ann Arbor, MI, USA) was used to study the effect of LW1564 (Compound 21-3) on cell proliferation. LW1564 (Compound 21-3) inhibited the proliferation of HepG2 and A549 cells in a dose-dependent manner Figure 1c ).

[0294] LW1564 (Compound 21-3) Inhibits HIF-1α Accumulation in Cancer Cells

[0295] When proline residues in the oxygen-dependent degradation (ODD) domain of HIF-1α are hydroxylated by prolyl hydroxylase (PHD) in the presence of oxygen, von Hippel-Lindau syndrome (VHL), which binds to the VCB-Cul2 E3 ligase that induces proteolysis, promotes HIF-1α degradation. Under hypoxic conditions, proline is not hydroxylated, so the amount of HIF-1α protein increases. First, HIF-1α activity was studied in HepG2 cells treated with LW1564 (Compound 21-3) by HRE-luciferase assay. A luciferase assay system (Promega, Madison, WI, USA) was used, and the HRE-luciferase reporter gene assay was performed according to the manufacturer's instructions and the previously described method (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) inhibited HIF-1α activity in HepG2 cells, with an IC 50 of 1.2 μM Figure 2a ). Moreover, it was found that LW1564 (Compound 21-3) inhibited HIF-1α accumulation in a concentration-dependent manner under hypoxic conditions Figure 2b)。Obviously, LW1564 (Compound 21-3) does not affect the amount of HIF-1α protein in the presence of cobalt chloride. Therefore, it was confirmed by Western blot experiments that LW1564 increases the accumulation of HIF-1α by inhibiting the activity of prolyl hydroxylase ( Figure 2b )。

[0296] Furthermore, the amount of HIF-1α mRNA in cells treated with LW1564 was investigated by qPCR. Total RNA was isolated using TRIzol reagent (Invitrogen), and cDNA was synthesized using TOPscript TM DryMIX (Enzynomics, Seoul, Korea). The primer sequences for HIF1A were Fwd 5'-CTG ACC CTG CAC TCA ATC AAG-3' and Rev 5'-TGG GAC TAT TAG GCT CAG GTG-3. No change in the amount of HIF-1α mRNA was observed, confirming that LW1564 (Compound 21-3) inhibits hypoxia-mediated HIF-1α accumulation during proteasomal degradation rather than transcription ( Figure 2c )。Then, it was investigated whether LW1564 (Compound 21-3) has a similar effect on HIF-1 degradation in other cancer cell lines. LW1564 (Compound 21-3) significantly reduced the accumulation of HIF-1α protein increased under hypoxic conditions in various cancer cell lines including A549, WiDr, MIA-CaCa2, and HCT116 ( Figure 2d )。

[0297] Next, the mRNA levels of HIF-1 target genes GLUT1, PDK1, and VEGF were measured. For GLUT1, the primer sequences were Fwd 5'-TTT GGC TAC AAC ACT GGA GTC-3' (Sequence 1) and Rev 5'-CAT GCC CCC AAC AGA AAAGAT-3' (Sequence 2); for PDK1, the primer sequences were Fwd 5'-CAG GAC AGC CAA TAC AAG TGG-3' (Sequence 3) and Rev 5'-CAT TAC CCA GCG TGA CAT GAA-3' (Sequence 4); for VEGFA, the primer sequences were Fwd 5'-CCTTGC TGC TCT ACC TCC AC-3' (Sequence 5) and Rev 5'-ATG ATT CTG CCC TCC TCC TT-3' (Sequence 6); for RPL13A, the primer sequences were Fwd 5'-CAT AGG AAG CTG GGA GCA AG-3' (Sequence 7) and Rev 5'-GCC CTC CAA TCA GTC TTC TG-3' (Sequence 8). LW1564 (Compound 21-3) decreased the amount of GLUT1, PDK1, and VEGF mRNAs that increased under hypoxic conditions in HepG2 cells ( Figure 2e ). In summary, LW1564 (Compound 21-3) inhibited HIF-1α accumulation through stimulated proteasomal degradation, thereby reducing the mRNA expression of target genes.

[0298] LW1564 (Compound 21-3) Reduces Oxygen Consumption by Inhibiting Mitochondrial Respiration

[0299] Compounds that inhibit mitochondrial respiration promote the degradation of HIF-1α by increasing the oxygen content of cells. Therefore, the oxygen consumption rate (OCR) was measured 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) Recovered after culturing with 2 μM of LW1564 (Compound 21-3) for 6 hours, and then, the OCR was measured at 37 °C for 5 minutes using a thermoregulated controlled circulating system. For epidemiological studies, digitonin-permeabilised HepG2 cells (2×10 7 ) were read on a detection device 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). Then, the following substrates that provide electrons for the components in the ETC were 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.

[0300] LW1564 (Compound 21-3) significantly inhibits OCR ( Figure 3a ) at concentrations that inhibit HIF-1 accumulation, indicating inhibition of mitochondrial respiration. Then, the effect of mitochondrial respiration on ATP production by oxidative phosphorylation was evaluated by comparing the amounts of ATP generated by glycolysis and mitochondrial respiration in the presence of LW1564 (Compound 21-3). The ATP content was determined by the method 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).". After inoculating HepG2 cells and culturing them with LW1564 (Compound 21-3) for 6 hours, the ATP content was measured using an ATP assay kit (Promega Corporation, Madison, Wisconsin, USA) according to the manufacturer's instructions. Under normoxic conditions, HepG2 cells obtain 47% of their ATP through mitochondrial respiration and 53% through glycolysis. As expected, in cells treated with LW1564 (Compound 21-3), mitochondrial-derived ATP production was significantly reduced (16%), while glycolysis-derived ATP production increased ( Figure 3b)。This compensatory upregulation of degradation in response to oxidative phosphorylation inhibition occurs via AMPK activation induced by a high AMP / ATP ratio. LW1564 (Compound 21-3) slightly reduces the total ATP production amount within a short time ( Figure 3b ), but significantly reduces the total intracellular ATP level over a long period of time ( Figure 3c ). These data indicate that LW1564 (Compound 21-3) reduces ATP production by inhibiting mitochondrial oxidative phosphorylation.

[0301] The reduction of mitochondrial respiration means an increase in the amount of intracellular oxygen. Therefore, the amount of intracellular oxygen increase was evaluated using MAR, an azo-based hypoxia probe that produces fluorescence under hypoxic conditions. Under hypoxic conditions, HepG2 cells were cultured with LW1564 (Compound 21-3) for 6 hours, stained with 500 nM MAR according to the manufacturer's instructions (Chou, T.C. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol. Rev. 58, 621-681 (2006).), and analyzed using the IncuCyte system (Essen BioScience, Michigan, USA). Under hypoxic conditions, LW1564 (Compound 21-3) significantly reduced the fluorescence in HepG2 cells, indicating an increase in the amount of intracellular oxygen due to reduced mitochondrial respiration ( Figure 3d ).

[0302] LW1564 (Compound 21-3) Inhibits the Activity of Complex I in the Mitochondrial Electron Transport Chain

[0303] To clarify the mechanism of action of LW1564 (Compound 21-3) in reducing mitochondrial respiration in the ETC, mitochondrial respiration analysis was performed. To analyze the coupled respiration process, experiments were conducted by applying specific substrates and inhibitors to each complex ( Figure 4a ). For electron transfer of Complex I, malate / pyruvate was added to HepG2 cells, and rotenone, a well-known inhibitor of Complex I, was used as a positive control group for reducing oxygen consumption ( Figure 4a ). LW1564 (Compound 21-3) reduced the oxygen consumption caused by Complex I, which was overcome by directly supplying electrons to Complex III by adding succinate ( Figure 4b)。These results indicate that LW1564 (Compound 21-3) is an inhibitor of mitochondrial electron transport chain complex I. Then, it was evaluated whether LW1564 (Compound 21-3) affects other ETC complexes. The present inventors administered succinate or cytochrome as an electron source for ETC complex II to cells and administered ascorbate / TMPD as an electron source for complex IV, and treated the cells with LW1564 (Compound 21-3). It was 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 ). Therefore, these results indicate that LW1564 (Compound 21-3) inhibits mitochondrial respiration only by inhibiting mitochondrial ETC complex I.

[0304] LW1564 (Compound 21-3) Activates the AMPK Signaling and Inhibits Lipid Synthesis

[0305] A significant decrease in the total ATP content was observed in cells treated with LW1564 (Compound 21-3) ( Figure 3c ). Since these results indicate an increase in the AMP / ATP ratio, AMPK activation and downstream signaling phenomena in HepG2 cells were investigated. It was confirmed by Western blot analysis that LW1564 (Compound 21-3) promoted the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC), a downstream protein thereof, in HepG2 cells ( Figure 5a)。The AMPK activation induced by LW1564 (Compound 21-3) inhibits the phosphorylation of mTOR in a concentration-dependent manner and inhibits the expression and / or activation of downstream proteins such as 4EBP1, cyclin D1, and sterol regulatory element-binding protein 1 (SREBP-1), thus explaining this result. The increase in the amount of ACC phosphorylation and the decrease in the amount of SREBP-1 indicate that LW1564 (Compound 21-3) can inhibit lipid synthesis. To explore this possibility, the effect of LW1564 (Compound 21-3) on lipid synthesis was evaluated using Nile red, a fluorescent probe that binds to hydrophobic molecules such as lipid droplets. Nile red staining was performed according to the method 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).". After treating HepG2 cells with LW1564 (Compound 21-3) for 24 hours, they were stained with 200 nM Nile red and 1 μM calcein AM for 10 minutes. Data were collected and analyzed using the IncuCyte system (Essen BioScience). To normalize, cell viability was measured using calcein AM, a cell-permeant fluorescent dye. It was confirmed by the results that LW1564 (Compound 21-3) reduces lipid accumulation in a concentration-dependent manner ( Figure 5b ). In summary, LW1564 (Compound 21-3) promotes the anti-cancer effect in HepG2 cells by regulating the AMPK / mTOR signaling pathway and lipid synthesis.

[0306] LW1564 (Compound 21-3) Inhibits Glucose-Dependent Cancer Metabolism

[0307] Glycolytic function is activated in many cancer cells. Therefore, it was investigated whether LW1564 (Compound 21-3) affects cancer cell proliferation according to glucose concentration. LW1564 (Compound 21-3) showed a stronger inhibitory effect on cell proliferation in cells cultured in a medium with a low glucose concentration (3 mM) than in a medium with a high glucose concentration (25 mM) ( Figure 5c)。Then, to investigate the combined effect of LW1564 (Compound 21-3) and 2-deoxy-D-glucose (2DG) on the proliferation of HepG2 cells, combination index (CI) and isobologram analyses were performed. 2-deoxy-D-glucose is both a glucose analog and a competitive glycolysis inhibitor. The combination index (CI) value quantitatively represents the relationship between the two drugs through GI50 analysis indicating the concentration inhibiting cell proliferation. Synergistic, additive, and antagonistic effects are defined as CI < 1, CI = 1, and CI > 1, respectively. The analysis results showed that LW1564 (Compound 21-3) and 2-DG had a synergistic effect in inhibiting the proliferation of HepG2 cells (0.4 ≤ CI ≤ 0.92)( Figure 5d ). Therefore, it was demonstrated that the combination therapy of LW1564 (Compound 21-3) and glycolysis inhibitors could exert better anti-cancer efficacy in liver cancer than LW1564 (Compound 21-3) alone.

[0308] LW1564 (Compound 21-3) Inhibits Tumor Growth in a HepG2 Xenograft Mouse Model

[0309] The present 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 investigated the efficacy of LW1564 (Compound 21-3) on tumor proliferation in a HepG2 xenograft mouse model. All animal experiments were approved and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology (Daejeon, Korea). HepG2 cells (1×10 7 ) were subcutaneously inoculated into the right side of female nude mice (6 weeks old) specific pathogen-free. When the tumor volume reached 100 - 150 mm 3 , the mice were randomly assigned to a control group (vehicle) and an LW1564 (Compound 21-3) treatment group (n = 6). LW1564 (Compound 21-3) (10 mg / kg) was intraperitoneally administered daily for 2 weeks. Tumor size was measured using a microcalliper, and tumor volume was calculated using the standard volume (length × width 2 × 0.5). Significant differences between the two groups were determined by Student's t-test.

[0310] Compared with the control group, the tumor size decreased by 67% in mice intraperitoneally injected with LW1564 (Compound 21-3) (10 mg / kg)( Figure 6(left figure in) and, upon confirmation, the weight of the tumors removed from the mice administered with LW1564 (Compound 21-3) ( Figure 6 (middle figure in) and the size ( Figure 6 (right figure in) decreased. These results indicate that LW1564 (Compound 21-3) can be developed into a potent therapeutic agent for patients with liver cancer.

[0311] Table 2

[0312] g g Mg mg Mmol mmol mL ml h h min min Z all (together) E relative (opposite) Fig. Figure Conc. Concentration Dil. Dilution Sat. Saturated Aq. Aqueous Solution ℃ Celsius Temperature ％ Percentage R.T / r.t Room Temperature Expt. Experiment Temp. Temperature o ortho m meta p para Psi pound per square inch Cat. Catalyst Atm. under the atmosphere of... et al etc. (and others) TLC Thin Layer Chromatography Calcd. Calculated

[0313] Table 3

[0314]

[0315] Industrial Applicability

[0316] The disubstituted adamantyl derivatives of the present invention are expected to be effectively used as potent cancer therapeutic agents that rely on oxidative phosphorylation for ATP generation in mitochondria.

Claims

1. A disubstituted adamantyl derivative or a pharmaceutically acceptable salt thereof, characterized in that it is represented by the following Chemical Formula 5: Chemical formula 5: , wherein R2 is: 。 2. A pharmaceutical composition for anti-cancer, characterized in that, comprising the disubstituted adamantyl derivative or a pharmaceutically acceptable salt thereof described in Claim 1 as an active ingredient.

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