Patent Description:
Polycomb Group (PcG) protein was first known to inhibit Hox gene transcription, which plays an important role in the early development of Drosophila, through a mutation experiment using Drosophila. It is known that this function is opposite to that of Trithorax Group (TrxG) proteins found in Drosophila, which promotes specific gene transcription. It was demonstrated that the functions of PcG proteins were well conserved in vertebrates as spinal malformations appeared in the mutants in which the functions of PcG proteins were suppressed. It is known that the gene transcriptional repression by PcG protein is caused by changing the structure of histone that is bound to a specific gene. Methods used for the structural change of histone by these PcG proteins are largely based on methylation and a method of attaching ubiquitin to specific histones. PcG proteins can be referred to as enzyme complexes that play the role of methyltransferase and ubiquitin ligase. In addition, some PcG proteins induce histone deacetylation or induce DNA methylation. Therefore, transcription of a specific gene is suppressed according to the activity of PcG proteins, and a phenotypic change of a specific cell occurs by this function.

PcG proteins form two well-conserved complexes in eukaryotes, and divided into polycomb repressive complex <NUM> (PRC1) and polycomb repressive complex <NUM> (PRC2). There are four types of proteins that make up the complex in PRC1, and these include BMI-<NUM>, CBX2, RING1A/B, and EDR1 (PHC1). There are three types of proteins that make up the complex in PRC2, and these include EED, SUZ12 and EZH2 (ENX2).

Specifically, the EZH2 (enhancer of zeste homolog <NUM>) is a major catalytic subunit of polycomb repressive complex <NUM> (PRC2) that promotes methylation of histone H3 lysine <NUM> (H3K27), and trimethylation of H3K27 (H3K27me3) is a transcriptionally repressive epigenetic mark that regulates gene expression, differentiation and development. In addition to EZH2, dysregulation of other PRC2 components (eg EED and SUZ12) and/or H3K27 trimethylation is associated with a number of cancers.

For example, EZH2 is overexpressed in a wide range of cancers including prostate cancer, breast cancer, myeloma and lymphoma, and it has been known that high EZH2 expression is associated with a bad prognosis of cancer. Hyper-trimethylation of H3K27 catalyzed by PRC2 induces tumorigenesis and progression of cancers including diffused large B cell lymphoma (DLBCL) and malignant rhabdoid tumor (MRT).

Therefore, pharmacological inhibition of EZH2 has been pursued as a targeted therapy to treat these cancers. Indeed, EZH2 inhibitors that effectively inhibit the methyltransferase activity of EZH2 have been demonstrated to exhibit robust antiproliferative activity in DLBCL and MRT cells and animal models, and many EZH2 inhibitors have been reported. Among them, EPZ-<NUM>, GSK126, CPI-<NUM> and PF-<NUM> are in the I/II clinical trial stage to treat lymphoma and several subtypes of MRT.

In addition, EZH2 has been shown to downregulate the tumor/metastasis suppressor RKIP (Raf-<NUM> kinase inhibitor protein), the tumor suppressor KLF2 (Kruppel-like factor), the forkhead box transcription factor FOXC1 (Forkhead box C1), and the tumor suppressor RUNX3 (Runt-related transcription factor <NUM>).

In addition, until now, low-molecular compounds have focused on inhibiting the function of disease-related proteins, but recent research trends are targeting undruggable target proteins for new therapeutic agents and the development of a selective proteolysis method that removes disease-related proteins themselves. This is expected the removal of the "target" and also to be able to overcome the resistance of previously developed drugs. Documents <CIT>, <CIT> and <CIT> describe compounds that can induce the degradation of EZH2.

As part of the development of the selective proteolysis method, PROTAC (Proteolysis-targeting chimaera) technology was devised. PROTAC is a method that can selectively remove target proteins using the ubiquitin-proteasome pathway, and is a low-molecular compound-based drug development platform technology.

Intracellular protein degradation occurs through two pathways by lysosomes and proteasomes. Most (<NUM>%) of cellular proteins are labeled with ubiquitin and then degraded in the cytoplasm and nucleus by the proteasome. This process is called ubiquitin-proteasome system (UPS). A series of enzymes (E1, E2 and E3) are involved in the ubiquitination process in which ubiquitin is labeled to selectively degrade proteins, and the labeled protein is degraded by the <NUM> proteasome, an ATP-dependent protease complex.

In humans, it is estimated that there are <NUM> types of E1, <NUM> types of E2, and <NUM>-<NUM> types of E3. In particular, E3 is divided into HECT, RING-finger, U-Box, and PHD-finger according to the structure and function. Importantly, E3 binds to both E2 and substrate proteins, providing specificity for recognizing substrate proteins to be labeled with ubiquitin. That is, the selection of the target protein to be degraded is determined by the E3 enzyme in the ubiquitination process. At this time, all substrate proteins have a recognition site by a specific E3 enzyme and an ubiquitin linkage site. By E2 complexed with E3 ligase, polyubiquitination is induced on the lysine residue of the target protein, and the target protein is degraded by the proteosome.

PROTAC technology is a bifunctional small molecule composed of E3 ligase binding module-connector-target protein binding module, and by the above mechanism, in vivo degradation of disease-causing target proteins is induced through ubiquitination. The PROTAC-based low-molecular compound used in this way has the advantage that it can be reused.

Accordingly, the present inventors have developed a pharmaceutical composition having the configuration of E3 ligase binding module-connector-EZH2 inhibitor by targeting EZH2 as a target protein.

It is an object of the present invention to provide a compound comprising an EZH2 inhibitor and an E3 ligase binder.

It is another object of the present invention to provide a compound for use in preventing or treating EZH2-related diseases, in particular for use in preventing or treating cancer.

To achieve the above objects, in one aspect of the present invention, the present invention provides a compound represented by formula <NUM>, a stereoisomer thereof, a solvate thereof, a hydrate thereof or a pharmaceutically acceptable salt thereof. <CHM>
wherein in formula <NUM>,
n is an integer of <NUM>,
<CHM>
is
<CHM>
and L<NUM> is any one selected from the group consisting of
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
and
<CHM>
and
<CHM>
is
<CHM>
or
<CHM>.

In another aspect of the present invention, the present invention provides a compound represented by formula <NUM>, a stereoisomer thereof, a solvate thereof, a hydrate thereof or a pharmaceutically acceptable salt thereof for use in the prevention or treatment of cancer.

In another aspect of the present invention, the present invention provides a compound represented by formula <NUM>, a stereoisomer thereof, a solvate thereof, a hydrate thereof or a pharmaceutically acceptable salt thereof for use in the selective degradation of EZH2 protein.

The compound of the present invention comprising an EZH2 inhibitor and an E3 ligase binder can selectively degrade EZH2. Therefore, the compound of the present invention can be effectively used for the treatment of EZH2-related diseases and cancers, particularly, cancers in which EZH2 is overexpressed, and can be usefully used for the selective degradation of EZH2.

The embodiments of this invention can be modified in various other forms, but the scope of the present invention is limited by the claims. It is well understood by those in the art who has the average knowledge on this field that the embodiments of the present invention are given to explain the present invention more precisely. In addition, the "inclusion" of an element throughout the specification does not exclude other elements, but may include other elements, unless specifically stated otherwise.

The compounds as defined in claim <NUM> according to Formula <NUM> (and thus according to the invention) will be described in the context of the following more general Formula <NUM>, which thus also comprising compounds not according to the invention. <CHM>
wherein in formula <NUM>,.

The compound represented by formula <NUM> is intended to be introduced into PROTAC technology, and targets EZH2 as a target protein, and is not limited as long as it is a compound having a residue capable of binding to EZH2. In the present document, an EZH2 inhibitor was introduced.

Specifically, in formula <NUM>, the EZH2 inhibitor can be a low molecular weight compound known as an EZH2 inhibitor or a derivative thereof, and can be a compound selected from the following compound group or a derivative thereof. According to the present invention, a derivative of tazemethostat (epz6438, EZH-<NUM>, E-<NUM>), which is widely known as an EZH2 inhibitor, was introduced.

Further EZH2 inhibitor groups (not according to the invention) in Formula <NUM> may be
<CHM>
<CHM>
<CHM>
<CHM>
and
<CHM>.

In addition, as the EZH2 inhibitor, an EZH1/EZH2 dual inhibitor can be used, and Valemetostat (DS-<NUM>), which is widely known as an EZH1/EZH2 dual inhibitor (not according to the present invention.

In addition, the compound represented by formula <NUM> is intended to be introduced into PROTAC technology. In the present document, an E3 ligase binder was introduced.

In Formula <NUM>, the E3 ligase binder can be any one selected from the group consisting of a β-TRCP binder, a MDM2 binder, a cIAP/XIAP binder, a VHL binder, a HyT binder, an IAP binder, a Bestatin amido binder and a CRBN binder. In one embodiment of the present invention, thalidomide as a CRBN binder, (S,R,S)-AHPC hydrochloride as a VHL binder, and bestatin as an IAP binder were introduced.

In the compound represented by formula <NUM>, an EZH2 inhibitor and an E3 ligase binder are connected by a linker containing -CH<NUM>- and
<CHM>
L<NUM> is a spacer and represents a site where
<CHM>
and an E3 ligase binder are connected. L<NUM> can be absent, or can be a linker consisting of a combination of one or more linkers selected from the group consisting of straight or branched C<NUM>-<NUM> alkylene, straight or branched C<NUM>-<NUM> alkenylene, straight or branched C<NUM>-<NUM> alkynylene, -O-, -S-. - S(=O)- -SO<NUM>-, -NH-, -N=, -C(=S)- and -C(=O)-.

In addition, the spacer can be a linker consisting of a combination of one or more linkers selected from the group consisting of straight or branched C<NUM>-<NUM> alkylene, -O-, -NH-, and -C(=O)-.

In addition, the spacer can be any one selected from the group consisting of
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
and
<CHM>.

In formula <NUM> above,
n can be an integer of <NUM>-<NUM>, can be an integer of <NUM>-<NUM>, and can be <NUM>.

In the first aspect of the present invention, the present invention provides a compound represented by formula <NUM>, a stereoisomer thereof, a solvate thereof, a hydrate thereof or a pharmaceutically acceptable salt thereof. <CHM>
wherein in formula <NUM>,
n is an integer of <NUM>,
<CHM>
is
<CHM>
and L<NUM> is any one selected from the group consisting of
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
and
<CHM>
and
<CHM>
is
<CHM>
or
<CHM>.

Examples of the compound represented by formula <NUM> according to the present invention include the following compounds:.

The present document further describes examples of the compound represented by formula <NUM> (not according to the present invention) including the following compounds:.

The compound represented by formula <NUM> of the present invention can be used as a form of a pharmaceutically acceptable salt, in which the salt is preferably acid addition salt formed by pharmaceutically acceptable free acids. The acid addition salt herein can be obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, and phosphorous acid; non-toxic organic acids such as aliphatic mono/dicarboxylate, phenyl-substituted alkanoate, hydroxy alkanoate, alkandioate, aromatic acids, and aliphatic/aromatic sulfonic acids; or organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, <NUM>-toluenesulfonic acid, tartaric acid, and fumaric acid. The pharmaceutically non-toxic salts are exemplified by sulfate, pyrosulfate, bisulfate, sulphite, bisulphite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutylate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, cabacate, fumarate, maliate, butyne-<NUM>,<NUM>-dioate, hexane-<NUM>,<NUM>-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutylate, citrate, lactate, β-hydroxybutylate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-<NUM>-sulfonate, naphthalene-<NUM>-sulfonate, and mandelate.

The acid addition salt according to the present invention can be prepared by the conventional method known to those in the art. For example, the compound represented by formula <NUM> is dissolved in an organic solvent such as methanol, ethanol, acetone, methylenechloride, and acetonitrile, to which organic acid or inorganic acid is added to induce precipitation. Then, the precipitate is filtered and dried to give the salt. Or the solvent and the excessive acid are distillated under reduced pressure, and dried to give the salt. Or the precipitate is crystallized in an organic solvent to give the same.

A pharmaceutically acceptable metal salt can be prepared by using a base. Alkali metal or alkali earth metal salt is obtained by the following processes: dissolving the compound in excessive alkali metal hydroxide or alkali earth metal hydroxide solution; filtering non-soluble compound salt; evaporating the remaining solution and drying thereof. At this time, the metal salt is preferably prepared in the pharmaceutically suitable form of sodium, potassium, or calcium salt. And the corresponding salt is prepared by the reaction of alkali metal or alkali earth metal salt with proper silver salt (ex; silver nitrate).

In addition, the present invention includes not only the compound represented by formula <NUM> but also a pharmaceutically acceptable salt thereof, and a solvate, an optical isomer, or a hydrate possibly produced from the same.

The term "hydrate" refers to a compound or a salt thereof of the present invention containing a stoichiometric or non-stoichiometric amount of water bound by a non-covalent intermolecular force. The hydrate of the compound represented by formula <NUM> of the present invention can contain a stoichiometric or non-stoichiometric amount of water bonded by a non-covalent intermolecular force. The hydrate can contain <NUM> equivalent or more of water, preferably <NUM> to <NUM> equivalents of water. The hydrate can be prepared by crystallizing the compound represented by formula <NUM>, the isomer thereof, or the pharmaceutically acceptable salt thereof from water or the solvent containing water.

The term "solvate" refers to a compound or a salt thereof of the present invention containing a stoichiometric or non-stoichiometric amount of solvent bound by a non-covalent intermolecular force. Preferred solvents therefor include volatile, non-toxic, and/or solvents suitable for administration to human.

The term "isomer" refers to a compound or a salt thereof of the present invention having the same chemical formula or molecular formula, but structurally or sterically different. Such isomers include structural isomers such as tautomers, R or S isomers having an asymmetric carbon center, stereoisomers such as geometric isomers (trans, cis), and optical isomers (enantiomers). Stereoisomers and mixtures thereof are also included in the scope of the present invention.

The compound represented by formula <NUM> of the present invention can be prepared according to the preparation method shown in the following examples, but this is only an example and is not limited thereto. For each preparation step, the method well known to those skilled in the art can be used.

In another aspect of the present invention, the present invention provides a compound represented by formula <NUM>, a stereoisomer thereof, a solvate thereof, a hydrate thereof or a pharmaceutically acceptable salt thereof as an active ingredient for use in the prevention or treatment of cancer.

The cancer can be at least one selected from the group consisting of pseudomyxoma, intrahepatic biliary tract cancer, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testis cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lib cancer, mycosis fungoides, acute myeloid leukemia, acute lymphoid leukemia, basal cell carcinoma, ovarian epithelial cancer, ovarian germ cell cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colorectal cancer, chronic myelogenous leukemia, chronic lymphocytic leukemia, retinoblastoma, choroidal melanoma, ampullar of vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, nasal cavity cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, pediatric leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvic cancer, kidney cancer, heart cancer, duodenal cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye cancer, vulvar cancer, ureteral cancer, urethral cancer, primary site unknown cancer, gastric lymphoma, stomach cancer, gastric carcinoid tumor, gastrointestinal stromal tumor, Wilms cancer, breast cancer, sarcoma, penile cancer, pharyngeal cancer, gestational trophoblastic disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal cancer, rectal cancer, rectal carcinoma, vaginal cancer, spinal cord cancer, acoustic tumor, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleura cancer, blood cancer and thymus cancer.

The compound represented by formula <NUM> of the present invention targets EZH2 as a target protein, and has tazemethostat (epz6438, EZH-<NUM>, E-<NUM>) as the EZH2 inhibitor having a residue capable of binding to EZH2, and thalidomide (cereblon), (S,R,S)-AHPC hydrochloride and bestatin as the E3 ligase binders. As demonstrated in Experimental Example <NUM>, the compound of the present invention can selectively degrade EZH2.

Therefore, the compound represented by formula <NUM> of the present invention can be effectively used for the treatment of EZH2-related diseases and cancers, particularly, cancers in which EZH2 is overexpressed, and can be usefully used for the selective degradation of EZH2.

The compounds for use in preventing or treating cancer of the present invention can be administered alone or in combination with an existing anticancer agent.

In pharmaceutical compositions, the compound represented by formula <NUM> or the pharmaceutically acceptable salt thereof can be administered in various oral and parenteral formulations during clinical administration, more preferably can be a parenteral formulation. In the case of formulation, it is prepared using diluents or excipients such as generally used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration are tablets, pills, powders, granules and capsules. These solid formulations are prepared by mixing one or more compounds with one or more suitable excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Except for the simple excipients, lubricants, for example magnesium stearate, talc, etc, can be used. Liquid formulations for oral administrations are suspensions, solutions, emulsions and syrups, and the above-mentioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to generally used simple diluents such as water and liquid paraffin. Formulations for parenteral administration are sterilized aqueous solutions, water-insoluble excipients, suspensions and emulsions. Water insoluble excipients and suspensions can contain, in addition to the active compound or compounds, propylene glycol, polyethylene glycol, vegetable oil like olive oil, injectable ester like ethylolate, etc..

The pharmaceutical composition comprising the compound represented by formula <NUM> or the pharmaceutically acceptable salt thereof as an active ingredient can be administered by parenterally and the parenteral administration includes subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.

At this time, to prepare the compound represented by formula <NUM> or the pharmaceutically acceptable salt thereof as a formulation for parenteral administration, the compound represented by formula <NUM> or the pharmaceutically acceptable salt thereof is mixed with a stabilizer or a buffering agent in water to produce a solution or suspension, which is then formulated as ampoules or vials. The composition herein can be sterilized and additionally contains preservatives, stabilizers, wettable powders or emulsifiers, salts and/or buffers for the regulation of osmotic pressure, and other therapeutically useful materials, and the composition can be formulated by the conventional mixing, granulating or coating method.

The formulations for oral administration are exemplified by tablets, pills, hard/soft capsules, solutions, suspensions, emulsions, syrups, granules, elixirs, and troches, etc. These formulations can include diluents (for example, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and/or glycine) and lubricants (for example, silica, talc, stearate and its magnesium or calcium salt, and/or polyethylene glycol) in addition to the active ingredient. Tablets can include binding agents such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrolidone, and if necessary disintegrating agents such as starch, agarose, alginic acid or its sodium salt or azeotropic mixtures and/or absorbents, coloring agents, flavours, and sweeteners can be additionally included thereto.

The present document also describes a health functional food comprising a compound represented by formula <NUM>, an isomer thereof, a solvate thereof, a hydrate thereof or a pharmaceutically acceptable salt thereof as an active ingredient for use in the prevention or amelioration of cancer.

Any references to methods of treatment in the subsequent paragraphs of this description are to be interpreted as references to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy (or for diagnosis).

The present document also describes a method for preventing or treating cancer, which comprises a step of administering a pharmaceutical composition or a health functional food comprising a compound represented by formula <NUM>, a stereoisomer thereof, a solvate thereof, a hydrate thereof or a pharmaceutically acceptable salt thereof as an active ingredient to a subject in need.

Hereinafter, the present invention will be described in detail by the following examples and experimental examples.

However, the following examples and experimental examples are only for illustrating the present invention, and the contents of the present invention are not limited thereto.

Examples <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> are examples according to the present invention.

<NUM>-(<NUM>,<NUM>-Dioxopiperidine-<NUM>-yl)-<NUM>-fluoroisoindoline-<NUM>,<NUM>-dione (<NUM>, <NUM> mmol) was dissolved in N-methyl-<NUM>-pyrrolidone, mixed with tert-butyl(<NUM>-aminoethyl)carbamate (<NUM>, <NUM> mmol) and N,N-diisopropylethylamine (<NUM>, <NUM> mmol), and then heated at <NUM> for <NUM> hours. Upon completion of the reaction, the temperature of the reaction mixture was lowered to room temperature, diluted with ethyl acetate, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, green solid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in dichloromethane (<NUM>), trifluoroacetic acid (<NUM>, <NUM> mmol) was added thereto, and the mixture was stirred at room temperature for <NUM> hour. Upon completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a target compound, and the obtained compound was used in the next step without further purification.

<NUM>-Bromo-N-((<NUM>,<NUM>-dimethyl-<NUM>-oxo-<NUM>,<NUM>-dihydropyridine-<NUM>-yl)methyl)-<NUM>-(ethyl(tetrahydro-<NUM>-pyran-<NUM>-yl)amino)-<NUM>-methylbenzamide (<NUM>, <NUM> mmol) was dissolved in <NUM>,<NUM>-dioxane (<NUM>), to which (<NUM>-((<NUM>-(tert-butoxycarbonyl)piperazine-<NUM>-yl)methyl)phenyl)boronic acid (<NUM>, <NUM> mmol), tetrakis(triphenylphosphine)palladium(<NUM>) (<NUM>, <NUM> mmol), and <NUM> sodium carbonate aqueous solution (<NUM>, <NUM> mmol) were added, followed by heating at <NUM> for <NUM> hour. Upon completion of the reaction, the temperature of the reaction mixture was lowered to room temperature, diluted with dichloromethane, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, yellow solid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in dichloromethane (<NUM>), trifluoroacetic acid (<NUM>, <NUM> mmol) was added thereto, and the mixture was stirred at room temperature for <NUM> hour. Upon completion of the reaction, the mixture was diluted with dichloromethane and neutralized by addition of a sodium hydrogen carbonate aqueous solution. After washing with water and brine, the residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, yellow solid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in toluene (<NUM>), to which triethylamine (<NUM>, <NUM> mmol) and ethyl <NUM>-bromoacetate (<NUM>, <NUM> mmol) were added at <NUM>, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, yellow solid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in tetrahydrofuran/methanol (<NUM>:<NUM>, <NUM>), to which lithium hydroxide (<NUM>, <NUM> mmol) dissolved in water (<NUM>) was added, followed by stirring at room temperature for <NUM> hour. Upon completion of the reaction, 1N hydrochloric acid solution was added until the pH of the reaction mixture reached <NUM>, the mixture was diluted with dichloromethane, and washed several times with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to give a target compound. The obtained compound was used in the next step without further purification.

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in N,N-dimethylformamide (<NUM>), to which HATU (<NUM>, <NUM> mmol), N,N-diisopropylethylamine (<NUM>, <NUM> mmol) and the compound prepared in step <NUM> (<NUM>, <NUM> mmol) were added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was purified by Prep HPLC, and neutralized by adding sodium hydrogen carbonate aqueous solution to give a target compound (<NUM>, <NUM>%, yellow solid).

The compound (<NUM>, <NUM> mmol) prepared in [Example <NUM>-Step <NUM>] was dissolved in N,N-dimethylformamide (<NUM>), to which potassium carbonate (<NUM>, <NUM> mmol) and <NUM>-(<NUM>-bromopentyl)isoindoline-<NUM>,<NUM>-dione (<NUM>, <NUM> mmol) were added, followed by heating at <NUM> for <NUM> hour. Upon completion of the reaction, the temperature of the reaction mixture was lowered to room temperature, the mixture was diluted with ethyl acetate, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, yellow liquid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in ethanol (<NUM>), to which hydrazinyl hydrate (<NUM>, <NUM> mmol) was added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was concentrated under reduced pressure and purified by MPLC to give a target compound (<NUM>, <NUM>%, yellow solid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in N-methyl-<NUM>-pyrrolidone (<NUM>), to which <NUM>-(<NUM>,<NUM>-dioxopiperidine-<NUM>-yl)-<NUM>-fluoroisoindoline-<NUM>,<NUM>-dione (<NUM>, <NUM> mmol) and N,N-diisopropylethylamine (<NUM>, <NUM> mmol) were added, followed by heating at <NUM> for <NUM> hour. Upon completion of the reaction, the temperature of the reaction mixture was lowered to room temperature, the mixture was diluted with ethyl acetate, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, purified by Prep HPLC, and neutralized by addition of a sodium hydrogen carbonate aqueous solution to give a target compound (<NUM>, <NUM>%, yellow solid).

The compound (<NUM>, <NUM> mmol) prepared in [Example <NUM>-Step <NUM>] was dissolved in N,N-dimethylformamide (<NUM>), to which tert-butyl-<NUM>-(<NUM>-(<NUM>-(<NUM>-aminoethoxy)ethoxy)ethoxy)propanoate (<NUM>, <NUM> mmol), N,N-diisopropylethylamine (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with ethylacetate, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a target compound, and the obtained compound was used in the next step without further purification.

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in dichloromethane (<NUM>), to which trifluoroacetic acid (<NUM>, <NUM> mmol) was added, followed by stirring at room temperature for <NUM> hour. Upon completion of the reaction, the reaction mixture was concentrated under reduced pressure, diluted with dichloromethane, and neutralized by addition of a sodium hydrogen carbonate aqueous solution to give a target compound. The obtained compound was used in the next step without further purification.

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in N,N-dimethylformamide (<NUM>), to which the compound prepared in [Example <NUM>-Step <NUM>] (<NUM>, <NUM> mmol), N,N-diisopropylethylamine (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was purified by Prep HPLC, and neutralized by adding a sodium hydrogen carbonate aqueous solution to give a target compound (<NUM>, <NUM>%, yellow solid).

<NUM>-(<NUM>,<NUM>-Dioxopiperidine-<NUM>-yl)-<NUM>-hydroxyisoindoline-<NUM>,<NUM>-dione (<NUM>, <NUM> mmol) was dissolved in N,N-dimethylformamide (<NUM>), to which tert-butyl(<NUM>-bromoethyl)carbamate (<NUM>, <NUM> mmol) and potassium carbonate (<NUM>, <NUM> mmol) were added, followed by heating at <NUM> for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, yellow solid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in dichloromethane (<NUM>), to which trifluoroacetic acid (<NUM>, <NUM> mmol) was added, followed by stirring at room temperature for <NUM> hour. Upon completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a target compound, and the obtained compound was used in the next step without further purification.

The compound (<NUM>, <NUM> mmol) prepared in [Example <NUM>-Step <NUM>] was dissolved in N,N-dimethylformamide (<NUM>), to which the compound prepared in step <NUM> (<NUM>, <NUM> mmol), N,N-diisopropylethylamine (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was purified by Prep HPLC, and neutralized by adding a sodium hydrogen carbonate aqueous solution to give a target compound (<NUM>, <NUM>%, yellow solid).

<NUM>-(<NUM>,<NUM>-Dioxopiperidine-<NUM>-yl)-<NUM>-hydroxyisoindoline-<NUM>,<NUM>-dione (<NUM>, <NUM> mmol) was dissolved in N,N-dimethylformamide (<NUM>), to which (((<NUM>-bromopentyl)oxy)methyl)benzene (<NUM>, <NUM> mmol) and potassium carbonate (<NUM>, <NUM> mmol) were added, followed by heating at <NUM> for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, yellow solid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in tetrahydrofuran (<NUM>) and methanol (<NUM>), to which Pd(OH)<NUM>/C (<NUM>, <NUM> mmol) and two drops of concentrated hydrochloric acid were added, followed by stirring at room temperature for <NUM> hour under a hydrogen atmosphere. Upon completion of the reaction, the reaction mixture was filtered through Celite. The separated filtrate was concentrated under reduced pressure to give a target compound, and the obtained compound was used in the next step without further purification.

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in dichloromethane (<NUM>), to which Dess-MartinPeriodinane (<NUM>, <NUM> mmol) was added at <NUM>, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and a sodium hydrogen carbonate aqueous solution. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, yellow solid).

The compound (<NUM>, <NUM> mmol) prepared in [Example <NUM>-Step <NUM>] was dissolved in methanol (<NUM>), to which the compound prepared in step <NUM> (<NUM>, <NUM> mmol), two drops of acetic acid and NaBH<NUM>CN (<NUM>, <NUM> mmol) were added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, purified by Prep HPLC, and neutralized by adding a sodium hydrogen carbonate aqueous solution to give a target compound (<NUM>, <NUM>%, yellow solid).

The compound (<NUM>, <NUM> mmol) prepared in [Example <NUM>-Step <NUM>] was dissolved in dichloromethane (<NUM>), to which triethylamine (<NUM>, <NUM> mmol) and <NUM>-bromoacetylbromide (<NUM>, <NUM> mmol) were added at <NUM>, followed by stirring for <NUM> hour. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and purified by MPLC to give a target compound (<NUM>, <NUM>%).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in N,N-dimethylformamide (<NUM>), to which the compound (<NUM>, <NUM> mmol) prepared in [Example <NUM>-Step <NUM>] and triethylamine (<NUM>, <NUM> mmol) were added, followed by stirring at room temperature for <NUM> hour. Upon completion of the reaction, the reaction mixture was purified by Prep HPLC, and neutralized by adding a sodium hydrogen carbonate aqueous solution to give a target compound (<NUM>, <NUM>%, yellow solid).

Tert-butyl(<NUM>-(<NUM>-hydroxypropoxy)ethyl)carbamate (<NUM>, <NUM> mmol) was dissolved in dichloromethane (<NUM>), to which carbon tetrabromide (<NUM>, <NUM> mmol) and triphenylphosphine (<NUM>, <NUM> mmol) were added, followed by stirring for <NUM> minutes and further stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was concentrated under reduced pressure and purified by MPLC to give a target compound (<NUM>, <NUM>%).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in N,N-dimethylformamide (<NUM>), to which sodium azide (<NUM>, <NUM> mmol) was added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%).

<NUM>-(<NUM>,<NUM>-Dioxopiperidine-<NUM>-yl)-<NUM>-fluoroisoindoline-<NUM>,<NUM>-dione (<NUM>, <NUM> mmol) was dissolved in N-methyl-<NUM>-pyrrolidone (<NUM>), to which the compound prepared in step <NUM> (<NUM>, <NUM> mmol) and N,N-diisopropylethylamine (<NUM>, <NUM> mmol) were added, followed by stirring at <NUM> for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in methanol (<NUM>), to which Pd/C (<NUM>, <NUM> mmol) was added, followed by stirring at room temperature for <NUM> hours under a hydrogen atmosphere. Upon completion of the reaction, the reaction mixture was filtered through Celite. The separated filtrate was concentrated under reduced pressure to give a target compound, and the obtained compound was used in the next step without further purification.

<NUM>-(Benzyloxy)ethanol (<NUM>, <NUM> mmol) was dissolved in N,N-dimethylformamide (<NUM>), to which sodium hydride (<NUM>, <NUM> mmol) was added at <NUM>. Then, <NUM>-(<NUM>-bromohexyl)isoindoline-<NUM>,<NUM>-dione (<NUM>, <NUM> mmol) was added to the reaction mixture while stirring at <NUM>, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with ethyl acetate and an aqueous ammonium chloride solution and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, colorless liquid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in a <NUM>:<NUM> solution (<NUM>) of ethyl acetate and methanol, followed by stirring under palladium and hydrogen for <NUM> hours. Upon completion of the reaction, the reaction mixture was filtered and concentrated to give a target compound (<NUM>, <NUM>%, colorless liquid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in dichloromethane (<NUM>), to which carbon tetrabromide (<NUM>, <NUM> mmol) and triphenylphosphine (<NUM>, <NUM> mmol) were added, followed by stirring at room temperature for <NUM> hours. Then, the reaction mixture was diluted with hexane, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, colorless liquid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in N,N-dimethylformamide (<NUM>) , to which the compound (<NUM>, <NUM> mmol) prepared in [Example <NUM> - step <NUM>] and potassium carbonate (<NUM>, <NUM> mmol) were added, followed by stirring at <NUM> for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with ethyl acetate, and washed with water. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, yellow liquid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in ethanol (<NUM>), to which hydrazinyl hydrate (<NUM>, <NUM> mmol) was added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was concentrated under reduced pressure and purified by MPLC to give a target compound (<NUM>, <NUM>%, yellow liquid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in N,N-dimethylformamide (<NUM>), to which <NUM>-(<NUM>,<NUM>-dioxopiperidine-<NUM>-yl)-<NUM>-fluoroisoindoline-<NUM>,<NUM>-dione (<NUM>, <NUM> mmol) and N,N-diisopropylethylamine (<NUM>, <NUM> mmol) were added, followed by stirring at <NUM> for <NUM> hours. Upon completion of the reaction, the temperature of the reaction mixture was lowered to room temperature, diluted with ethyl acetate, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, purified by Prep HPLC, and neutralized by adding a sodium hydrogen carbonate aqueous solution to give a target compound (<NUM>, <NUM>%, yellow solid).

<NUM>-(<NUM>,<NUM>-Dioxopiperidine-<NUM>-yl)-<NUM>-fluoroisoindoline-<NUM>,<NUM>-dione (<NUM>, <NUM> mmol) was dissolved in N-methyl-<NUM>-pyrrolidone (<NUM>), to which <NUM>-aminohexan-<NUM>-ol (<NUM>, <NUM> mmol) and N,N-diisopropylethylamine (<NUM>, <NUM> mmol) were added, followed by stirring at <NUM> for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in dichloromethane (<NUM>), to which Dess-MartinPeriodinane (<NUM>, <NUM> mmol) was added at <NUM>, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%).

The compound (<NUM>, <NUM> mmol) prepared in [Example <NUM>-Step <NUM>] was dissolved in N,N-dimethylformamide (<NUM>), to which tert-butyl(<NUM>-oxoethyl)carbamate (<NUM>, <NUM> mmol) and NaBH<NUM>CN (<NUM>, <NUM> mmol) were added, and acetic acid (<NUM>, <NUM> mmol) was added at <NUM>. Then, the mixture was stirred at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and a sodium hydrogen carbonate aqueous solution. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in dichloroethane (<NUM>), to which the compound prepared in step <NUM> (<NUM>, <NUM> mmol), NaBH<NUM>CN (<NUM>, <NUM> mmol) and two drops of acetic acid were added at <NUM>, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and a sodium hydrogen carbonate aqueous solution. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, purified by Prep HPLC, and neutralized by adding a sodium hydrogen carbonate aqueous solution to give a target compound (<NUM>, <NUM>%, yellow solid).

<NUM>-Azidopropane-<NUM>-amine (<NUM>, <NUM> mmol) was dissolved in N,N-dimethylformamide (<NUM>), to which <NUM>-(<NUM>,<NUM>-dioxopiperidine-<NUM>-yl)-<NUM>-fluoroisoindoline-<NUM>,<NUM>-dione (<NUM>, <NUM> mmol) and N,N-diisopropylethylamine (<NUM>, <NUM> mmol) were added, followed by heating at <NUM> for <NUM> hours. Upon completion of the reaction, the temperature of the reaction mixture was lowered to room temperature, diluted with ethyl acetate, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, yellow solid).

The compound (<NUM>, <NUM> mmol) prepared in [Example <NUM> - Step <NUM>] was dissolved in N,N-dimethylformamide (<NUM>), to which potassium carbonate (<NUM>, <NUM> mmol) and <NUM>-bromoprop-<NUM>-yn (<NUM>, <NUM> mmol) were added, followed by stirring at <NUM> for <NUM> hours. Upon completion of the reaction, the temperature of the reaction mixture was lowered to room temperature, diluted with ethyl acetate, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, white solid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) and the compound prepared in step <NUM> (<NUM>, <NUM> mmol) were dissolved in tetrahydrofuran (<NUM>), to which copper sulfate (<NUM>, <NUM> mmol), sodium ascorbate (<NUM>, <NUM> mmol) and water (<NUM>) were added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with ethyl acetate, and washed with water. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, purified by Prep HPLC, and neutralized by adding a sodium hydrogen carbonate aqueous solution to give a target compound (<NUM>, <NUM>%, yellow solid).

The compound (<NUM>, <NUM> mmol) prepared in [Example <NUM> - Step <NUM>] was dissolved in N,N-dimethylformamide (<NUM>), to which propiolic acid (<NUM>, <NUM> mmol), N,N-diisopropylethylamine (<NUM>, <NUM> mmol)) and HATU (<NUM>, <NUM> mmol) were added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with ethyl acetate, and washed with water. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, white solid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) and the compound prepared in [Example <NUM> - Step <NUM>] (<NUM>, <NUM> mmol) were dissolved in tetrahydrofuran (<NUM>), to which copper sulfate (<NUM>, <NUM> mmol), sodium ascorbate (<NUM>, <NUM> mmol) and water (<NUM>) were added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with ethyl acetate, and washed with water. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, purified by Prep HPLC, and neutralized by adding a sodium hydrogen carbonate aqueous solution to give a target compound (<NUM>, <NUM>%, yellow solid).

Propane-<NUM>,<NUM>-diol (<NUM>, <NUM> mmol) was dissolved in N,N-dimethylformamide (<NUM>), to which NaH (<NUM>, <NUM> mmol) was added at <NUM>, followed by stirring for <NUM> minutes. Then, (((<NUM>-bromopentyl)oxy)methyl)benzene (<NUM>, <NUM> mmol) was added to the mixture, and the mixture was stirred at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in toluene (<NUM>), to which tert-butyl bromoacetate (<NUM>, <NUM> mmol) and tetrabutylammonium bromide (<NUM>, <NUM> mmol) were added, and <NUM>% sodium hydroxide aqueous solution (<NUM>) was added at <NUM>, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in ethyl acetate (<NUM>) and methanol (<NUM>), to which Pd/C (<NUM>, <NUM> mmol) was added, followed by stirring at room temperature for <NUM> hours under a hydrogen atmosphere. Upon completion of the reaction, the reaction mixture was filtered through Celite. The separated filtrate was concentrated under reduced pressure to give a target compound, and the obtained compound was used in the next step without further purification.

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in dichloromethane (<NUM>), to which carbon tetrabromide (<NUM>, <NUM> mmol) and triphenylphosphine (<NUM>, <NUM> mmol) were added at <NUM>, followed by stirring for <NUM> minutes. Then, the reaction mixture was further stirred at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was concentrated under reduced pressure, and purified by MPLC to give a target compound (<NUM>, <NUM>%).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in ethyl acetate (<NUM>), to which Pd/C (<NUM>, <NUM> mmol) was added, followed by stirring for <NUM> hours under a hydrogen atmosphere. Upon completion of the reaction, the reaction mixture was filtered through Celite. The separated filtrate was concentrated under reduced pressure to give a target compound, and the obtained compound was used in the next step without further purification.

The compound (<NUM>, <NUM> mmol) prepared in [Example <NUM>-Step <NUM>] was dissolved in N,N-dimethylformamide (<NUM>), to which the compound prepared in step <NUM> (<NUM>, <NUM> mmol), N,N-diisopropylethylamine (<NUM>, <NUM> mmol), HOBT (<NUM>, <NUM> mmol) and EDCI (<NUM>, <NUM> mmol) were added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in dichloromethane (<NUM>), to which <NUM>,<NUM>,<NUM>-trifluoroacetic acid (<NUM>, <NUM> mmol) was added, followed by stirring at room temperature for <NUM> hour. Upon completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a target compound, and the obtained compound was used in the next step without further purification.

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in N,N-dimethylformamide (<NUM>), to which <NUM>-(<NUM>-amino-<NUM>-oxoisoindoline-<NUM>-yl)piperidine-<NUM>,<NUM>-dione (<NUM>, <NUM> mmol), propylphosphonic anhydride solution in DMF (<NUM>, <NUM> mmol) and pyridine (<NUM>, <NUM> mmol) were added, followed by stirring at <NUM> for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, yellow solid).

The compound (<NUM>, <NUM> mmol) prepared in [Example <NUM>-Step <NUM>] was dissolved in N,N-dimethylformamide (<NUM>), to which tert-butyl4-(<NUM>-aminoethyl)piperazine-<NUM>-carboxylate (<NUM>, <NUM> mmol), N,N-diisopropylethylamine (<NUM>, <NUM> mmol), HOBT (<NUM>, <NUM> mmol) and EDCI (<NUM>, <NUM> mmol) were added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in N,N-dimethylformamide (<NUM>), to which potassium carbonate (<NUM>, <NUM> mmol) and <NUM>-bromoethylamine hydrobromide (<NUM>, <NUM> mmol) were added, followed by stirring at <NUM> for <NUM> hour. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in N-methyl-<NUM>-pyrrolidone (<NUM>), to which <NUM>-(<NUM>,<NUM>-dioxopiperidine-<NUM>-yl)-<NUM>-fluoroisoindoline-<NUM>,<NUM>-dione (<NUM>, <NUM> mmol) and N,N-diisopropylethylamine (<NUM>µl, <NUM> mmol) were added, followed by stirring at <NUM> for <NUM> hours. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, purified by Prep HPLC, and neutralized by adding a sodium hydrogen carbonate aqueous solution to give a target compound (<NUM>, <NUM>%, yellow solid).

<NUM>-Azido-N-(<NUM>-(<NUM>,<NUM>-dioxopiperidine-<NUM>-yl)-<NUM>,<NUM>-dioxoisoindoline-<NUM>-yl)acetamide (<NUM>, <NUM> mmol) and <NUM>-hexyn-<NUM>-ol (<NUM>, <NUM> mmol) were dissolved in tetrahydrofuran (<NUM>), to which copper sulfate (<NUM>, <NUM> mmol), sodium ascorbate (<NUM>, <NUM> mmol) and water (<NUM>) were added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with ethyl acetate, and washed with water. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, yellow solid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in dichloromethane (<NUM>), to which carbon tetrabromide (<NUM>, <NUM> mmol) and triphenylphosphine (<NUM>, <NUM> mmol) were added, followed by stirring at room temperature for <NUM> hours. Then, the reaction mixture was diluted with hexane, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, yellow solid).

The compound (<NUM>, <NUM> mmol) prepared in [Example <NUM> - Step <NUM>] was dissolved in N,N-dimethylformamide (<NUM>), to which the compound (<NUM>, <NUM> mmol) prepared in step <NUM> and potassium carbonate (<NUM>, <NUM> mmol) were added, followed by stirring at <NUM> for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with ethyl acetate, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, purified by Prep HPLC, and neutralized by adding a sodium hydrogen carbonate aqueous solution to give a target compound (<NUM>, <NUM>%, yellow solid).

T-butyl <NUM>,<NUM>-diazaspiro[<NUM>]heptane-<NUM>-carboxylate (<NUM>, <NUM> mmol) and <NUM>-(<NUM>-bromobutyl)isoindoline-<NUM>,<NUM>-dione (<NUM>, <NUM> mmol) were dissolved in N,N-dimethylformamide (<NUM>), to which potassium carbonate (<NUM>, <NUM> mmol) was added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with ethyl acetate, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, white solid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in ethanol (<NUM>), to which hydrazinyl hydrate (<NUM>, <NUM> mmol) was added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was concentrated under reduced pressure, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, yellow liquid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in N-methyl-<NUM>-pyrrolidone (<NUM>), to which <NUM>-(<NUM>,<NUM>-dioxopiperidine-<NUM>-yl)-<NUM>-fluoroisoindoline-<NUM>,<NUM>-dione (<NUM>, <NUM> mmol) and N,N-diisopropylethylamine (<NUM>, <NUM> mmol) were added, followed by stirring at <NUM> for <NUM> hours. Upon completion of the reaction, the temperature of the reaction mixture was lowered to room temperature, diluted with ethyl acetate, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, yellow solid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in dichloromethane (<NUM>), to which <NUM>,<NUM>,<NUM>-trifluoroacetic acid (<NUM>) was added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction was completed, the reaction mixture was concentrated under reduced pressure to give a target compound, and the obtained compound was used in the next step without further purification.

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) and the compound (<NUM>, <NUM> mmol) prepared in [Example <NUM> - Step <NUM>] were dissolved in N,N-dimethylformamide (<NUM>), to which EDC (<NUM>, <NUM> mmol), HOBT (<NUM>, <NUM> mmol) and N,N-diisopropylethylamine (<NUM>, <NUM> mmol) were added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with ethyl acetate, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, purified by Prep HPLC, and neutralized by adding a sodium hydrogen carbonate aqueous solution to give a target compound (<NUM>, <NUM>%, yellow solid).

The compound (<NUM>, <NUM> mmol) prepared in [Example <NUM>-Step <NUM>] was dissolved in dichloromethane (<NUM>), to which <NUM>,<NUM>,<NUM>-trifluoroacetic acid (<NUM>, <NUM> mmol) was added, followed by stirring at room temperature for <NUM> hour. Upon completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a target compound, and the obtained compound was used in the next step without further purification.

(<NUM>,4R)-<NUM>-((S)-<NUM>-amino-<NUM>,<NUM>-dimethylbutanoyl)-<NUM>-hydroxy-N-(<NUM>-(<NUM>-methylthiazole-<NUM>-yl)benzyl)pyrrolidine-<NUM>-carboxamide (<NUM>, <NUM> mmol) was dissolved in N,N-dimethylformamide (<NUM>), to which the compound prepared in step <NUM> (<NUM>, <NUM> mmol), N,N-diisopropylethylamine (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in tetrahydrofuran (<NUM>) and water (<NUM>), to which triphenylphosphine (<NUM>, <NUM> mmol) was added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%).

The compound (<NUM>, <NUM> mmol) prepared in [Example <NUM>-Step <NUM>] was dissolved in N,N-dimethylformamide (<NUM>), to which the compound prepared in step <NUM> (<NUM>, <NUM> mmol), N,N-diisopropylethylamine (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, and washed with water and brine. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, white solid).

<NUM>-Azidopropanoic acid (<NUM>, <NUM> mmol) and (<NUM>, 4R)-<NUM>-((S)-<NUM>-amino-<NUM>,<NUM>-dimethylbutanoyl)-<NUM>-hydroxy-N-(<NUM>-<NUM>-methylthiazol-<NUM>-yl)benzyl)pyrrolidine-<NUM>-carboxamide (<NUM>, <NUM> mmol) were dissolved in N,N-dimethylformamide (<NUM>), to which N,N-diisopropylethylamine (<NUM>, <NUM> mmol), EDC (<NUM>, <NUM> mmol) and HOBT (<NUM>, <NUM> mmol) were added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with ethyl acetate, and washed with water. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, white solid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) and the compound (<NUM>, <NUM> mmol) prepared in [Example <NUM> - Step <NUM>] were dissolved in tetrahydrofuran (<NUM>), to which copper sulfate (<NUM>, <NUM> mmol), sodium ascorbate (<NUM>, <NUM> mmol) and water (<NUM>) were added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with ethyl acetate, and washed with water. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, purified by Prep HPLC, and neutralized by adding a sodium hydrogen carbonate aqueous solution to give a target compound (<NUM>, <NUM>%, white solid).

The compound (<NUM>, <NUM> mmol) prepared in [Example <NUM> - Step <NUM>] was dissolved in N,N-dimethylformamide (<NUM>), to which N,N-diisopropylethylamine (<NUM>, <NUM> mmol) and HATU (<NUM>, <NUM> mmol) were added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with ethyl acetate, and washed with water. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by MPLC to give a target compound (<NUM>, <NUM>%, white solid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in dichloromethane (<NUM>), to which trifluoroacetic acid (<NUM>) was added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was concentrated under reduced pressure to give a target compound, and the obtained compound was used in the next step without further purification.

<NUM>-(<NUM>-Amino-<NUM>-hydroxy-<NUM>-phenylbutanamido)-<NUM>-methylpentanoic acid (<NUM>, <NUM> mmol) was dissolved in dichloromethane (<NUM>), to which sodium carbonate (<NUM>, <NUM> mmol) and di-t-butyl dicarbonate (<NUM>, <NUM> mmol) were added, followed by stirring at room temperature for <NUM> hour. Upon completion of the reaction, the reaction mixture was washed with water. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then used in the next step without further purification.

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) and the compound prepared in step <NUM> (<NUM>, <NUM> mmol) were dissolved in N,N-dimethylformamide (<NUM>), to which N,N-diisopropylethylamine (<NUM>, <NUM> mmol), EDC (<NUM>, <NUM> mmol) and HOBT (<NUM>, <NUM> mmol) were added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with ethyl acetate, and washed with water. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and then purified by Prep HPLC to give a target compound (<NUM>, <NUM>%, white solid).

The compound prepared in step <NUM> (<NUM>, <NUM> mmol) was dissolved in dichloromethane (<NUM>), to which trifluoroacetic acid (<NUM>) was added, followed by stirring at room temperature for <NUM> hours. Upon completion of the reaction, the reaction mixture was diluted with dichloromethane, neutralized by adding a sodium hydrogen carbonate aqueous solution, and washed with water. The residue of the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to give a target compound (<NUM>, <NUM>%, white solid).

Chemical structures, compound names, analysis data, yields, and purification methods of the compounds prepared in Examples <NUM>-<NUM> are summarized and shown in Table <NUM> below.

In order to confirm the proteolytic effect of the compound represented by formula <NUM> or formula <NUM>, the following experiment was performed, and specifically, the proteolytic effect of the compounds of Examples <NUM>-<NUM> on colorectal cancer cells was evaluated.

Particularly, <NUM> × <NUM><NUM> LS174T cells were placed in each <NUM>-well plate, and after <NUM> hours of stabilization, each well was treated with the example compound at the final concentrations of <NUM>, <NUM>, <NUM>, and <NUM>. One well was treated with DMSO at the same percentage. After <NUM> hours of the treatment, the cells were collected and lysed using RIPA buffer (<NUM> Tris, pH <NUM>, <NUM> NaCl, <NUM>% Triton X-<NUM>, <NUM>% SDS, <NUM> EDTA, <NUM>% deoxycholate, and protease inhibitor cocktail), followed by sonication (<NUM> sec on /<NUM> sec off, <NUM> cycles) to prepare a cell lysate. Western blotting was performed after the protein quantification through cell lysate BCA using the cell lysate, and the results are shown in table <NUM>.

In Table <NUM>, the grade is the percent value of the protein degradation for each section when treated with <NUM>, and the values for each grade are as follows.

A:><NUM>%, B: <NUM>~<NUM>%, C: <NUM>~<NUM>%, D: <<NUM>%.

As shown in table <NUM>, the compounds of the present invention were excellent in the effect of degrading EZH2 protein when treated to colorectal cancer cells. In particular, it was found that the compounds of Examples <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> exhibit excellent effects.

In addition, as shown in <FIG>, the novel compound according to the present invention is a Degraducer compound that induces degradation of EZH2, the target protein, and significantly induces degradation of the target protein through UPS (Ubiquitin Proteasome System). Therefore, it can be seen that the pharmaceutical composition for preventing or treating EZH2-related diseases or conditions containing the compound as an active ingredient has a preventive or ameliorating effect on the diseases.

In order to evaluate the EZH2 proteolytic activity of the compound according to the present invention, the following experiment was performed.

Specifically, the CRISPR/Cas9 technique was used to construct a stable cell line of HiBiT-EZH2. After the transfection of EZH2 targeted CRISPR/Cas9 vector and single-stranded oligo-deoxynucleotide (ssODN) containing HiBiT nucleotide sequence into HEK293T cells, cell stabilization was performed for <NUM>-<NUM> days. After harvesting the cells, the cell solution was prepared at a concentration of <NUM> cell/<NUM> ul, and <NUM> ul of the cell solution was dispensed into each well of a <NUM>-well plate and seeded so that a single cell could proliferate in each well. After proliferating the cells for <NUM>-<NUM> weeks, some of the proliferated cells from one cell were recovered and gDNA was extracted. To confirm that the HiBiT sequence was correctly inserted into the EZH2 target, deep sequencing was performed to select the cells containing the HiBiT sequence, and after further verification through Sanger sequencing, the final EZH2-HiBiT HEK293T cells were obtained.

<NUM> × <NUM><NUM> of the obtained cells were placed in a <NUM>-well white plate, and after <NUM> hours of stabilization, each well was treated with the example compound at a final concentration of <NUM>. The control group was treated with the same concentration of DMSO which was used as the solvent of the example compound. After <NUM> hours of the treatment, an assay was performed using Nano-Glo HiBiT Lytic Detection System (N3040, Promega). After removing the medium, <NUM>µL of lysis buffer, <NUM>µL of LgBiT protein and <NUM>µL of substrate buffer were added to each well. Then, the plate was reacted in a shaking incubator at <NUM> rpm for <NUM> minutes, and then the mixture of each well was pipetted to mix well. Thereafter, the plate was reacted at room temperature for <NUM> minutes while light was blocked, and luminescence was measured. The results are shown in table <NUM> and <FIG>.

Claim 1:
A compound represented by formula <NUM>, a stereoisomer thereof, a solvate thereof, a hydrate thereof or a pharmaceutically acceptable salt thereof:
<CHM>
wherein, in formula <NUM>,
n is an integer of <NUM>;

<CHM>
is
<CHM>
and

<CHM>
is
<CHM>
or
<CHM>
and
L<NUM> is any one selected from the group consisting of
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
and
<CHM>