KR20230155310A - Novel N-hydroxypropenamide compound and pharmaceutical composition for preventing or treating cancer comprising the same as an active ingredient - Google Patents

Abstract

The present invention relates to a novel N-hydroxypropenamide compound containing a 2H-benzo[b][1,4]oxazin-3(4H)-one ring and a pharmaceutical for preventing or treating cancer containing the same as an active ingredient. It relates to composition. The compound according to the present invention has the inhibitory activity of histone deacetylase (HDAC) and exhibits anticancer efficacy by exhibiting cytotoxicity in various cancer cells, so it can be used as a powerful anticancer agent.

Description

Novel N-hydroxypropenamide compound and pharmaceutical composition for preventing or treating cancer comprising the same as an active ingredient}

The present invention relates to a novel N-hydroxypropenamide compound and a pharmaceutical composition for preventing or treating cancer containing the same as an active ingredient.

Molecular target-based drugs are the mainstream of anticancer drug research and development today. With advances in cancer pathology, molecules such as protein kinases, farnesyl transferases, telomerases, histone deacetylases, etc. have been identified and validated as attractive targets for anticancer drug design. It has been done.

Histone deacetylase (HDAC) is an enzyme that catalyzes the removal of acetyl groups from histone proteins. Together with histone acetyl transferase (HAT), HDACs play an important role in regulating epigenetic balance. Inhibition of HDAC induces cell cycle arrest, apoptosis, as well as reduction of cell motility and inhibition of angiogenesis. Therefore, HDAC inhibitors are widely known as multi-target anticancer agents. Intensive research by scientists around the world has led to the discovery of short-chain fatty acids (e.g. phenylbutyric acid, valproic acid), cyclic peptides (e.g. depsipeptide), benzamides (e.g. entinostat, gibinostat), and hydroxamic acids (e.g. Hundreds of HDAC inhibitors with different structures (e.g. SAHA) have been discovered and proven to have potent anticancer activity. Of these, four or more, including suberoylanilide hydroxamic acid (SAHA, Zoinza®), belinostat (PXD101), romidepsin (Istodax®), and panobinostat (LBH-589, Farydak®) (Figure 1). The compound has been approved by the U.S. FDA for use in treating various types of cancer. Additionally, chidamide (Epidaza®) was approved by the Chinese FDA for the treatment of relapsed or refractory peripheral T cell lymphoma in 2015.

The structure of HDAC inhibitors has three distinct parts (Figure 2), including a zinc binding group (ZBG) (usually a hydroxamic acid moiety), a linker, and a cap group at the entrance to the enzymatic active binding pocket. In the process of researching new HDAC inhibitors, we have previously used various heterozygous compounds such as 5-aryl-3,4,5-thiadiazole, benzothiazole, 2-oxoindoline and 4-oxoquinazoline, which act as cap groups. Several hydroxamic acids containing cyclic forms were discovered. Among these compounds, N-hydroxypropenamide compounds, especially designed as analogs of LBH-589 (panobinostat) or PXD-101 (belinostat), showed very strong anticancer activity. Inspired by these results, 2H-benzo[b][1,4]oxazin-3(4H)-one containing the 2H-benzo[b][1,4]oxazin-3(4H)-one ring The design of various N-hydroxypropenamide compounds was expanded. The present invention was completed by discovering that 2H-benzo[b][1,4]oxazin-3(4H)-one-containing compounds have various biological activities, especially anticancer activity.

Republic of Korea Patent Publication No. 2009-0094383 Republic of Korea Patent No. 10-1261305 Republic of Korea Patent Publication No. 2007-0043978 Republic of Korea Patent No. 10-1536050

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The present inventors made research efforts to develop a novel N-hydroxypropenamide compound that has a strong inhibitory effect on histone deacetylase (HDAC). As a result, we succeeded in synthesizing a new N-hydroxypropenamide compound with a 2H-benzo[b][1,4]oxazin-3(4H)-one ring, and the synthesized N-hydroxypropenamide The present invention was completed by confirming that the compounds not only inhibit the activity of histone deacetylase but also have anticancer activity against various types of cancer cell lines.

The object of the present invention is to provide novel N-hydroxypropenamide compounds.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer containing a novel N-hydroxypropenamide compound as an active ingredient.

One example of the present invention provides a compound represented by Formula 1 or Formula 2 below and a pharmaceutically acceptable salt, hydrate, solvate, or optical isomer thereof.

[Formula 1]

[Formula 2]

In Formula 1, R is hydrogen, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ alkenyl, substituted or unsubstituted C ₁ -C ₁₀ alkynyl, substituted or unsubstituted It is one of substituted or unsubstituted C ₁ to C ₃₀ cycloalkyl;

In Formula 2, R is one of substituted or unsubstituted C ₅ to C ₃₀ aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted arylalkenyl, or substituted or unsubstituted arylalkynyl;

The substitution involves replacing one or more hydrogens with halogens such as fluorine, chlorine, bromine and iodine; hydroxy; nitro; cyano; oxo (=O); Thioxo (=S); Azido; nitroso; Amino; hydrazino; formyl; alkyl; alkoxy; aryl; It is changed to haloalkyl groups such as trifluoromethyl, tribromomethyl, and trichloromethyl.

Another example of the present invention provides a pharmaceutical composition for preventing or treating cancer containing the above compound and its pharmaceutically acceptable salt, hydrate, solvate, or optical isomer as an active ingredient.

The novel N-hydroxypropenamide compound according to the present invention has inhibitory activity on histone deacetylase (HDAC) and exhibits anticancer efficacy by exhibiting cytotoxicity in various cancer cells, so it is an active ingredient of a powerful anticancer agent. can be developed.

Figure 1 is a diagram showing the chemical structures of various histone deacetylation enzymes.
Figure 2 is a diagram showing the CAP portion, linker portion, and zinc binding group (ZBG) portion of histone deacetylase (belinostat).
Figure 3 is a diagram showing the synthesis mechanism of various N-hydroxypropenamide compounds containing a 2H-benzo[b][1,4]oxazine-3(4H)-one ring.
Figure 4 is a graph analyzing DNA content and cell cycle when cancer cells (SW620) were treated with compound 7f.
Figure 5 is a graph analyzing cells undergoing apoptosis when cancer cells (SW620) were treated with compound 7f.
Figure 6 is a diagram showing the cell morphology when cancer cells (SW620) were treated with compound 7f at 20 times magnification in A) and 40 times magnification in B).

Throughout this specification, unless otherwise specified, “include” or “contains” refers to the inclusion of a certain component (or component) without particular limitation, excluding the addition of other components (or components). cannot be interpreted as

As used herein, the term “treatment” means, for an individual suffering from a disease or at risk of developing a disease, improving the condition of the individual, delaying the progression of the disease, delaying the onset of symptoms, or slowing the progression of symptoms, etc. It means any form of treatment or prevention that provides effects including. Accordingly, the term “treatment” includes prophylactic treatment of an individual, preventing the occurrence of symptoms. Additionally, the terms “treatment” and “prevention” are not intended to imply cure or complete elimination of symptoms.

As used herein, the term “pharmaceutically acceptable” refers to a composition that is physiologically acceptable when administered to a subject, such as a human subject, and typically does not produce an allergic reaction or similar adverse effect. For example, this may mean approved by a state or federal regulatory agency for use in humans, or listed in the Korean Pharmacopoeia, United States Pharmacopoeia, or other generally accepted pharmacopoeia.

As used herein, the terms “cancer,” “neoplasm,” and “tumor” refer to cells that exhibit autonomous, uncontrolled growth, resulting in an abnormal growth phenotype characterized by significant loss of control over cell proliferation. It is used in this institution for this purpose. Cells of interest for detection, analysis, and/or treatment in the context of the present invention include cancer cells (e.g., cancer cells from an individual with cancer), malignant cancer cells, pre-metastatic cancer cells, metastatic cancer cells, and non-metastatic cancer cells. -Contains metastatic cancer cells. Cancer of almost all tissues is known.

As used herein, the term “cancer cell” refers to a cancer cell (e.g., one that can be isolated from an individual with cancer, e.g., any cancer for which the individual can be treated) or derived from a cancer cell, e.g. For example, it refers to any cell that is a clone of a cancer cell. For example, the cancer cells can be derived from an established cancer cell line, can be a primary cell isolated from an individual with cancer, can be progeny cells from a primary cell isolated from an individual with cancer, etc. In some embodiments, the term may also refer to a portion of a cancer cell, such as an intracellular portion, a cell membrane portion, or a cell lysate of a cancer cell. Many types of cancer are known to those skilled in the art, including solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, and myeloma, and circulatory cancers such as leukemias.

One embodiment of the present invention provides a compound represented by Formula 1 or Formula 2 below and a pharmaceutically acceptable salt, hydrate, solvate, or optical isomer thereof.

[Formula 1]

[Formula 2]

In Formula 1, R is hydrogen, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ alkenyl, substituted or unsubstituted C ₁ -C ₁₀ alkynyl, substituted or unsubstituted It is one of substituted or unsubstituted C ₁ to C ₃₀ cycloalkyl;

In Formula 2, R is one of substituted or unsubstituted C ₅ to C ₃₀ aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted arylalkenyl, or substituted or unsubstituted arylalkynyl;

The substitution involves replacing one or more hydrogens with halogens such as fluorine, chlorine, bromine and iodine; hydroxy; nitro; cyano; oxo (=O); Thioxo (=S); Azido; nitroso; Amino; hydrazino; formyl; alkyl; alkoxy; aryl; It may be a haloalkyl group such as trifluoromethyl, tribromomethyl, trichloromethyl, etc., preferably fluorine, chlorine, bromine, hydroxy, or alkyl.

In the above compounds, when halogen is present, the halogen is fluorine, chlorine, bromine or iodine; If an alkyl group is present, the alkyl group is methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl or substituted thereof;

When present, the cycloalkyl group may be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, cycloheptyl, perhydronaphthyl, adamantyl, bridged cyclic groups or spirobicyclic groups. are spirobicyclic groups;

If an aryl group is present, the aryl group is phenyl, naphthyl, anthracenyl, indanyl or biphenyl;

If a hydroxyalkyl group is present, the hydroxyalkyl group may be hydroxymethyl or hydroxyethyl.

Specifically, in Formula 1, R is ethyl, 2-chloroethyl, 2-hydroxyethyl, n-propyl, n-butyl, , , or It may be, and in Formula 2, R is , , , , , or It can be.

For example, the compound represented by Formula 1 or Formula 2 may be any of the following compounds:

(E)-3-(4-ethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacrylamide;

(E)-3-(4-(chloroethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacrylamide ;

(E)-N-hydroxy-3-(4-(hydroxymethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)acrylic amides;

(E)-N-hydroxy-3-(3-oxo-4-propyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)acrylamide;

(E)-3-(4-Butyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacrylamide;

(E)-3-(4-(cyclopropylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacryl amides;

(E)-3-(4-(Cyclobutylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacryl amides;

(E)-3-(4-(cyclopentylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacryl amides;

(E)-3-(4-(cyclohexylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacryl amides;

(E)-3-(4-benzyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacrylamide;

(E)-3-(4-(2-fluorobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydride Roxyacrylamide;

(E)-3-(4-(3-fluorobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydride Roxyacrylamide;

(E)-3-(4-(4-fluorobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydride Roxyacrylamide;

(E)-3-(4-(4-chlorobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxy acrylamide;

(E)-3-(4-(4-bromobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydride Roxyacrylamide; and

(E)-N-hydroxy-3-(4-(4-methylbenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl) It may be acrylamide.

Another embodiment of the present invention provides a pharmaceutical composition for preventing or treating cancer containing the above compound and its pharmaceutically acceptable salt, hydrate, solvate, or optical isomer as an active ingredient.

The pharmaceutical composition may include a peptide compound, a stereoisomer, an optical isomer, a hydrate, a solvate, or a pharmaceutically acceptable salt thereof as an active ingredient. The pharmaceutical composition may further include known active ingredients with anticancer activity. The pharmaceutical composition may additionally include a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and the lubricant may be magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, disintegrant, binder, lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium monohydrogen phosphate anhydride, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The pharmaceutical composition may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions according to conventional methods. When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

In the pharmaceutical composition, the solid preparation for oral administration may be a tablet, pill, powder, granule, or capsule. The solid preparation may further include excipients. Excipients may be, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. Additionally, the solid preparation may further include a lubricant such as magnesium stearate or talc. In the pharmaceutical composition, the oral liquid preparation may be a suspension, oral solution, emulsion, or syrup. The liquid formulation may contain water or liquid paraffin. The liquid formulation may contain excipients such as wetting agents, sweeteners, flavoring agents, or preservatives. In the pharmaceutical composition, preparations for parenteral administration may be sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried products, and suppositories. Non-aqueous solvents or suspensions may contain vegetable oil or ester. The vegetable oil may be, for example, propylene glycol, polyethylene glycol, or olive oil. The ester may be, for example, ethyl oleate. The base of the suppository may be witepsol, macrogol, tween 61, cacao, laurel, or glycerogelatin. The preferred dosage of the pharmaceutical composition varies depending on the individual's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. However, the compound, its isomer, derivative, solvate, or pharmaceutically acceptable salt may be used in an amount of, for example, about 0.0001 mg/kg to about 100 mg/kg, or about 0.001 mg/kg to about 100 mg/kg. The amount can be divided and administered 1 to 24 times per day, 1 to 7 times per 2 days to 1 week, or 1 to 24 times per month to 12 months. In the pharmaceutical composition, the compound, its isomer, derivative, solvate, or pharmaceutically acceptable salt is present in an amount of about 0.0001% to about 10% by weight, or about 0.001% to about 1% by weight, based on the total weight of the composition. may be included.

The term “cancer” refers to a mass or tumor composed of undifferentiated cells that proliferates without limit within the tissue. The pharmaceutical composition can be used for any cancer, including pancreatic cancer, colon cancer, lung cancer, breast cancer, stomach cancer, liver cancer, colon cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, brain cancer, laryngeal cancer, prostate cancer, bladder cancer, esophagus cancer, and thyroid cancer. , it may be bladder, kidney, or rectal cancer.

The above cancers include, for example, intrahepatic bile duct cancer, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, mycosis fungoides, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, and basal cell cancer. , ovarian epithelial cancer, ovarian germ cell tumor, male breast cancer, brain tumor, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colon cancer, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, Non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain tumor, pediatric lymphoma, pediatric leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, neuroblastoma, pyeloureteral cancer, kidney cancer, malignant soft tissue tumor, malignant bone tumor, malignant Lymphoma, malignant mesothelioma, malignant melanoma, eye tumor, vulvar cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, stomach cancer, gastric carcinoid tumor, gastrointestinal stromal tumor, Wilm's tumor, breast cancer, sarcoma, penile cancer, pharyngeal cancer, gestational villi. Diseases, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic brain tumor, rectal cancer, rectal carcinoid, vaginal cancer, spinal tumor, acoustic neurula, pancreatic cancer, salivary gland cancer, tonsil cancer, squamous cell carcinoma, pulmonary adenocarcinoma, lung cancer, lung It may be squamous cell cancer, skin cancer, anal cancer, laryngeal cancer, or a combination thereof, which cancers include, for example, breast cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreas cancer, colon cancer, skin cancer, head and neck cancer, skin or eye cancer. Melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, It may be bronchial cancer, or bone marrow cancer.

The compound may be a pharmaceutical composition for the prevention or treatment of cancer, characterized in that it has the activity of promoting histone acetylation through inhibition of histone deacetylase. Specifically, the compounds of the present invention can induce intracellular histones into a hyperacetylated state by inhibiting the activity of histone deacetylation enzymes.

Hereinafter, the present invention will be described in more detail through experimental examples and examples. However, these experimental examples and examples are for illustrative purposes only and the scope of the present invention is not limited to these experimental examples and examples.

In addition, unless there is a special definition in this specification, all scientific and technical terms used in this specification may have the same meaning as commonly understood by a person skilled in the art in the technical field to which the present invention pertains.

chemical substance

Thin layer chromatography was performed using Whatman® 250 J mSilica Gel GF Uniplates and visualized under UV light at 254 and 365 nm wavelengths to confirm the progress of the reaction and perform a preliminary assessment of the homogeneity of the compounds. . The melting point was measured using the Gallenkamp Melting Point Apparatus (LabMerchant, London, United Kingdom) and was not separately corrected. Purification of the compound was performed using a crystallization method using Merck silica gel 60 (240 to 400 mesh) as a stationary phase and/or open silica gel column flash chromatography. Nuclear magnetic resonance spectra ( ^1H NMR) were measured on a Bruker 500 MHz spectrometer using DMSO- d6 as _the solvent unless otherwise indicated. Tetramethylsilane was used as an internal standard, and the chemical shift was measured in parts per million (ppm) downfield from tetramethylsilane. Mass spectra with various ionization modes, including electron ionization (EI) and electrospray ionization (ESI), were collected using PE Biosystems API2000 (Perkin Elmer, Palo Alto, CA, USA) and Mariner® (Azco Biotech, Inc. Oceanside, CA, USA), respectively. ) was measured through mass spectrometry. Elemental (C, H, N) analysis was performed on a Perkin Elmer model 2400 elemental analyzer. All reagents and solvents were purchased from Aldrich or Fluka Chemical Corp. (Milwaukee, WI, USA) or Merck, unless otherwise specified. Solvents were used as purchased unless otherwise indicated.

<Example>

Example 1. Preparation of N-hydroxypropenamide compounds containing alkyl substituents

K 2 CO 3 (342.5 mg, 2.5 mmol) in a solution _of 6-bromo-2H _- benzo[b][1,4]oxazin-3(4H)-one (Compound 1, 2 mmol) in DMF (10 mL). ) was added. The resulting mixture was heated at 80° C. with stirring for 30 minutes, then KI (16.6 mg, 0.1 mmol) was added. After stirring for another 15 minutes, alkyl bromide (2.0 mmol) diluted with DMF (1 mL) was added dropwise to the mixture. The mixture was stirred again at 60°C until the reaction was complete (6 hours, confirmed by thin layer chromatography). The resulting mixture was poured into ice water (10 mL). The brown precipitate thus obtained was washed with water and dried under vacuum for 24 hours at 40° C. The compound was used in the next step without further purification.

In the next step, dried triethylamine (0.5 mL) and ethyl acrylate (0.5 mL) were added to the DMF (10 mL) solution containing the intermediate (Compound 2, 1.5 mmol). After stirring for an additional 15 minutes, a solution of triphenylphosphine (105 mg, 0.5 mL, 0.4 mmol) and palladium diacetate (0.2 mmol, 45 mg) was added. The mixture was heated at 110° C. for 8 hours. After the reaction was complete, the mixture was cooled to room temperature and then poured into ice water. Insoluble material was filtered off. The solid was collected and dried under vacuum at 40°C for 24 hours. The product was then purified by column chromatography (silica gel; DCM/methanol = 100/5) to obtain intermediate esters (compounds 3a-3i) in a yield of 65-79%.

DMF (10 mL) containing the above intermediate esters (Compounds 3a-3i) was dissolved in methanol (10 mL). Then, hydroxylamine, HCl (685 mg, 10 mmol) was added, and aqueous NaOH solution (1 mL, 400 mg) was added dropwise. Afterwards, the mixture was stirred at 0 °C for 1-2 hours until the reaction was complete. When the reaction was completed as confirmed by TLC, the resulting mixture was poured into ice water and neutralized with 5% HCl solution until pH reached 7. The precipitate was filtered, dried, and recrystallized from methanol to obtain the following desired compounds (compounds 4a-4i) (Figure 3):

Compound 4a: (E)-3-(4-ethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacrylamide

Yield: 55%. mp: 167-168℃. Rf = 0.40 (DCM/MeOH/AcOH = 90:5:1). IR (KBr, cm ^-1 ) : 3202 (OH); 2922 (CH, aren); 2851 (CH, CH ₂ ); 1668 (C=O); 1603 (C=C). ¹ H-NMR (500 MHz, DMSO- d ₆ ): δ 7.49 (1H, d, J = 16.00 Hz, H-3); 7.42 (1H, s, H-5'); 7.25 (1H, d, J = 7.50 Hz, H-7'); 7.06 (1H, d, J = 10.50 Hz, H-8'); 6.44 (1H, d, J = 15.50 Hz, H-2); 4.68 (2H, s, H-2'a, H-2'b); 3.99-3.98 (2H, m, H-1”a, H-1”b); 1.18 (3H, t, J = 6.50 Hz, -CH ₃ ). ¹³ C-NMR (125 MHz, DMSO- d ₆ ): δ 163.75, 163.32, 146.28, 138.27, 134.97, 134.89, 130.90, 130.32, 128.82, 122.77, 118.54, 117.54, 1 15.19, 67.47, 21.86, 14.41. ESI-MS m/z: 261.0866 [MH] ^- .

Compound 4b: (E)-3-(4-(chloroethyl)-3-oxo-3,4-dihydro-2H- benzo[b][1,4]oxazin-6-yl)-N-hydride Roxyacrylamide

Yield: 52%. mp: 172-173℃. Rf = 0.44 (DCM/MeOH/AcOH = 90:5:1). IR (KBr, cm ^-1 ) : 3204 (OH); 2955, 2920 (CH, aren); 2851 (CH, CH ₂ ); 1682 (C=O); 1597 (C=C). ¹ H-NMR (500 MHz, DMSO- d ₆ ): δ 7.50 (1H, d, J = 15.50 Hz, H-3); 7.40 (1H, s, H-5'); 7.25 (1H, d, J = 7.50 Hz, H-7'); 7.04 (1H, d, J = 8.50 Hz, H-8'); 6.40 (1H, d, J = 15.50 Hz, H-2); 4.67 (2H, s, H-2'a, H-2'b); 3.95 (2H, t, J = 6.75 Hz, H-2”a, H-2”b); 3.68 (2H, t, J = 5.25 Hz, H-1”a, H-1”b). ¹³ C-NMR (125 MHz, DMSO- d ₆ ): δ 164.39, 144.76, 133.62, 132.80, 132.52, 132.50, 132.00, 131.92, 131.38, 129.28, 129.18, 126.28, 1 19.10, 118.74, 114.42, 67.51, 58.25, 44.02 . ESI-MS m/z: 297.0646 ( ³⁵ Cl), 299.0626 ( ³⁷ Cl) [M+H] ⁺ .

Compound 4c: (E)-N-Hydroxy-3-(4-(hydroxymethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6- 1) Acrylamide

Yield: 59%. mp: 169-170℃. Rf = 0.37 (DCM/MeOH/AcOH = 90:5:1). IR (KBr, cm ^-1 ) : 3383 (NH); 3217(OH); 2953, 2918 (CH, aren); 2851 (CH, CH ₂ ); 1668 (C=O); 1599 (C=C). 1H-NMR (500 MHz, DMSO- d ₆ ): δ 7.50 (1H, d, J = 15.50 Hz, H-3); 7.40 (1H, s, H-5'); 7.25 (1H, d, J = 7.50 Hz, H-7'); 7.04 (1H, d, J = 8.50 Hz, H-8'); 6.40 (1H, d, J = 15.50 Hz, H-2); 5.05 (1H, s, OH); 4.67 (2H, s, H-2'a, H-2'b), 3.95-3.94 (2H, m, H-1”a, H-1”b); 3.70-3.69 (2H, m, H-2”a, H-2”b). ¹³ C-NMR (125 MHz, DMSO- d ₆ ): δ 164.58, 146.14, 135.88, 132.37, 132.00, 131.92, 130.25, 129.33, 129.27, 129.13, 128.84, 123.07, 1 18.78, 117.51, 115.53, 67.51, 43.22, 31.15 . ESI-MS m/z: 279.0946 [M+H] ⁺ .

Compound 4d: (E)-N-hydroxy-3-(3-oxo-4-propyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)acrylamide

Yield: 61%. mp: 172-173℃. Rf = 0.41 (DCM/MeOH/AcOH = 90:5:1). IR (KBr, cm ^-1 ) : 3188 (OH); 2963, 2932 (CH, aren); 2876 (CH, CH ₂ ); 1665 (C=O); 1605 (C=C). ¹ H-NMR (500 MHz, DMSO- d ₆ ): δ 7.50 (1H, d, J = 15.50 Hz, H-3); 7.41 (1H, s, H-5'); 7.25 (1H, d, J = 7.50 Hz, H-7'); 7.03 (1H, d, J = 8.00 Hz, H-8'); 6.42 (1H, d, J = 15.00 Hz, H-2); 4.68 (2H, s, H-2'a, H-2'b), 3.91-3.89 (2H, m, H-1”a, H-1”b); 1.61-1.59 (2H, m, H-2”a, H-2”b); 0.93-0.90 (2H, m, H-3”a, H-3”b). ¹³ C-NMR (125 MHz, DMSO- d ₆ ): δ 164.06, 163.33, 146.30, 138.27, 130.29, 129.39, 129.02, 127.92, 122.61, 118.45, 117.57, 115.61, 6 7.46, 42.06, 20.37, 11.44. ESI-MS m/z: 275.1041 [MH] ^- .

Compound 4e: (E)-3-(4-butyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacrylamide

Yield: 61%. mp: 168-169℃. Rf = 0.41 (DCM/MeOH/AcOH = 90:5:1). IR (KBr, cm ^-1 ) : 3175 (OH); 2920 (CH, aren); 2851 (CH, CH ₂ ); 1676, 1659 (C=O); 1601 (C=C). ¹ H-NMR (500 MHz, DMSO- d ₆ ): δ 10.72 (1H, s, NH); 9.02 (1H, s, OH); 7.50 (1H, d, J = 15.50 Hz, H-3); 7.40 (1H, s, H-5'); 7.25 (1H, d, J = 7.50 Hz, H-7'); 7.03 (1H, d, J = 8.50 Hz, H-8'); 6.40 (1H, d, J = 15.50 Hz, H-2); 4.67 (2H, s, H-2'a, H-2'b), 3.95 (2H, t, J = 6.75 Hz, H-1”a, H-1”b); 1.57-1.54 (2H, m, H-2”a, H-2”b); 1.37-1.32 (2H, m, H-3”a, H-3”b); 0.94-0.91 (2H, m, H-4”a, H-4”b). ¹³ C-NMR (125 MHz, DMSO- d ₆ ): δ 164.02, 163.40, 146.35, 138.42, 130.24, 129.00, 122.62, 118.40, 117.58, 115.59, 67.47, 29.17, 19. 89, 14.15. ESI-MS m/z: 289.1198 [MH] ^- .

Compound 4f: (E)-3-(4-(cyclopropylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N- Hydroxyacrylamide

Yield: 64%. mp: 173-174℃. Rf = 0.41 (DCM/MeOH/AcOH = 90:5:1). IR (KBr, cm ^-1 ) : 3202 (OH); 2918 (CH, aren); 2851 (CH, CH ₂ ); 1680 (C=O); 1601 (C=C). 1H-NMR (500 MHz, DMSO- d ₆ ): δ 7.47 (1H, s, H-5'); 7.44 (1H, d, J = 16.50 Hz, H-3); 7.22 (1H, d, J = 7.00 Hz, H-7'); 7.01 (1H, d, J = 7.50 Hz, H-8'); 6.42 (1H, d, J = 15.50 Hz, H-2); 4.66 (2H, s, H-2'a, H-2'b), 3.86 (2H, d, J = 6.00 Hz, CH ₂ ); 1.14-1.12 (1H, m, H-1”); 0.55-0.46 (4H, m, H-2”a, H-2”b, H-3”a, H-3”b). ¹³ C-NMR (125 MHz, DMSO-d6): δ 164.17, 146.19, 131.99, 130.38, 129.25, 129.19, 122.92, 118.68, 117.54, 115.52, 67.44, 44.46, 31.1 5. ESI-MS m/z: 287.1042 [MH] ^- .

Compound 4g: (E)-3-(4-(cyclobutylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N- Hydroxyacrylamide

Yield: 61%. mp: 180-181℃. Rf = 0.42 (DCM/MeOH/AcOH = 90:5:1). IR (KBr, cm ^-1 ) : 3202 (OH); 2974, 2934 (CH, aren); 2855 (CH, CH ₂ ); 1672 (C=O); 1603 (C=C). ¹ H-NMR (500 MHz, DMSO- d ₆ ): δ 7.46 (1H, d, J = 16.00 Hz, H-3); 7.42 (1H, s, H-5'); 7.24 (1H, d, J = 8.00 Hz, H-7'); 7.03 (1H, d, J = 8.50 Hz, H-8'); 6.54 (1H, d, J = 16.00 Hz, H-2); 4.68 (2H, s, H-2'a, H-2'b), 4.05 (2H, d, J = 7.00 Hz, CH ₂ ); 2.66-2.63 (1H, m, H-1”); 1.96-1.74 (6H, m, H-2”a, H-2”b, H-3”a, H-3”b, H-4”a, H-4”b). ¹³ C-NMR (125 MHz, DMSO- d ₆ ): δ 164.40, 146.31, 138.73, 131.92, 129.20, 122.72, 118.42, 117.57, 115.91, 67.44, 44.86, 33.67, 33.6 2, 26.18, 18.34. ESI-MS m/z: 301.1198 [MH] ^- .

Compound 4h: (E)-3-(4-(cyclopentylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N- Hydroxyacrylamide

Yield: 64%. mp: 187-188℃. Rf = 0.44 (DCM/MeOH/AcOH = 90:5:1). IR (KBr, cm ^-1 ) : 3202 (OH); 2918 (CH, aren); 2853 (CH, CH ₂ ); 1672 (C=O); 1603, 1589 (C=C). ¹ H-NMR (500 MHz, DMSO- d ₆ ): δ 10.72 (1H, s, NH); 9.01 (1H, s, OH); 7.50 (1H, d, J = 15.50 Hz, H-3); 7.46 (1H, s, H-5'); 7.25 (1H, d, J = 8.00 Hz, H-7'); 7.04 (1H, d, J = 8.00 Hz, H-8'); 6.40 (1H, d, J = 16.00 Hz, H-2); 4.68 (2H, s, H-2'a, H-2'b); 3.94 (2H, d, J = 8.00 Hz, CH ₂ ); 2.26-2.23 (1H, m, H-1”); 1.63-1.24 (8H, m, H-2”a, H-2”b, H-3”a, H-3”b, H-4”a, H-4”b, H-5”a, H-5”b). 13C-NMR (125 MHz, DMSO- d ₆ ): δ 164.40, 146.46, 138.42, 131.17, 129.08, 122.60, 118.40, 117.64, 115.99, 67.48, 55.37, 44.26, 37.8 3, 30.08, 24.83. ESI-MS m/z: 317.1508 [M+H] ⁺ .

Compound 4i: (E)-3-(4-(cyclohexylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N- Hydroxyacrylamide

Yield: 66%. mp: 193-194℃. Rf = 0.45 (DCM/MeOH/AcOH = 90:5:1). IR (KBr, cm ^-1 ) : 3200 (OH); 2922 (CH, aren); 2851 (CH, CH ₂ ); 1670 (C=O); 1603 (C=C). 1H-NMR (500 MHz, DMSO- d ₆ ): δ 10.73 (1H, s, NH); 9.02 (1H, s, OH); 7.51 (1H, d, J = 16.00 Hz, H-3); 7.43 (1H, s, H-5'); 7.25 (1H, d, J = 8.00 Hz, H-7'); 7.04 (1H, d, J = 8.50 Hz, H-8'); 6.39 (1H, d, J = 16.00 Hz, H-2); 4.68 (2H, s, H-2'a, H-2'b); 3.84 (2H, d, J = 7.00 Hz, CH ₂ ), 1.65-1.14 (11H, m, H-1”, H-2”a, H-2”b, H-3”a, H-3” b, H-4”a, H-4”b, H-5”a, H-5”b, H-6”a, H-6”b). ¹³ C-NMR (125 MHz, DMSO- d ₆ ): δ 164.37, 163.42, 146.38, 138.49, 130.17, 129.39, 129.32, 122.38, 118.37, 117.65, 116.21, 67.46, 55 .36, 45.78, 35.53, 30.45, 26.36, 25.70 . ESI-MS m/z: 329.1520 [MH] ^- .

Example 2. Preparation of N-hydroxypropenamide compound containing benzyl

The target compounds (compounds 7a-g) were synthesized using the same method as in Example 1, except that a benzyl bromide derivative was used instead of alkyl bromide (FIG. 3).

Compound 7a: (E)-3-(4-benzyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacrylamide

Yield: 65%. mp: 188-189℃. Rf = 0.41 (DCM/MeOH/AcOH = 90:5:1). IR (KBr, cm ^-1 ) : 3233 (OH); 2988 (CH, aren); 2853 (CH, CH ₂ ); 1647 (C=O); 1614, 1593 (C=C). ¹ H-NMR (500 MHz, DMSO- d ₆ ): δ 10.72 (1H, s, NH); 8.99 (1H, s, OH); 7.37-7.21 (8H, m, H-3, H-5', H-7', H-2”, H-3”, H-4”, H-5”, H-6”); 7.04 (1H, d, J = 8.50 Hz, H-8'); 6.27 (1H, d, J = 15.50 Hz, H-2); 5.21 (2H, s, CH2); 4.84 (2H, s, H-2'a, H-2'b). ¹³ C-NMR (125 MHz, DMSO- d ₆ ): δ 164.53, 163.26, 146.37, 138.17, 136.77, 129.91, 129.17, 129.01, 127.75, 127.33, 123.14, 118.37, 1 17.53, 115.80, 67.52, 43.80. ESI-MS m/z: 323.1032 [MH] ^- .

Compound 7b: (E)-3-(4-(2-fluorobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)- N-Hydroxyacrylamide

Yield: 68%. mp: 195-196℃. Rf = 0.44 (DCM/MeOH/AcOH = 90:5:1). IR (KBr, cm ^-1 ) : 3240 (OH); 2918 (CH, aren); 2851 (CH, CH ₂ ); 1649 (C=O); 1618, 1589 (C=C). ¹ H-NMR (500 MHz, DMSO- d ₆ ): δ 10.73 (1H, s, NH); 9.00 (1H, s, OH); 7.36-7.21 (6H, m , H-3, H-5', H-7', H-3”, H-4”, H-6”); 7.15 (1H, t, J = 7.25 Hz, H-5”); 7.06 (1H, d, J = 8.50 Hz, H-8'), 6.28 (1H, d, J = 15.50 Hz, H-2); 5.24 (2H, s, CH ₂ ); 4.84 (2H, s, H-2'a, H-2'b). ¹³ C-NMR (125 MHz, DMSO- d ₆ ): δ 164.61, 163.24, 161.51, 159.56, 146.43, 138.14, 130.08, 129.88, 129.82, 129.40, 129.05, 128.89, 1 25.13, 125.11, 123.45, 123.40, 123.34, 118.44 , 117.62, 116.06, 115.90, 115.37, 67.58. ESI-MS m/z: 341.0937 [MH] ^- .

Compound 7c: (E)-3-(4-(3-fluorobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-yl)- N-Hydroxyacrylamide

Yield: 66%. mp: 197-198℃. Rf = 0.44 (DCM/MeOH/AcOH = 90:5:1). IR (KBr, cm ^-1 ) : 3235 (OH); 2920 (CH, aren); 2851 (CH, CH ₂ ); 1649 (C=O); 1612, 1589 (C=C). ¹ H-NMR (500 MHz, DMSO- d ₆ ): δ 10.71 (1H, s, NH); 8.99 (1H, s, OH); 7.43-7.05 (8H, m, H-3, H-5', H-7', H-8', H-2”, H-4”, H-5”, H-6”); 6.28 (1H, d, J = 16.00 Hz, H-2); 5.22 (2H, s, CH2); 4.86 (2H, s, H-2'a, H-2'b). ¹³ C-NMR (125 MHz, DMSO- d ₆ ): δ 164.62, 163.82, 163.28, 161.88, 146.33, 139.81, 139.76, 138.15, 131.20, 131.13, 129.98, 128.95, 1 23.26, 123.24, 123.07, 118.42, 117.58, 115.80 , 114.70, 114.53, 114.18, 114.00, 67.51, 43.49. ESI-MS m/z: 341.0943 [MH] ^- .

Compound 7d: (E)-3-(4-(4-fluorobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-yl)- N-Hydroxyacrylamide

Yield: 69%. mp: 194-195℃. Rf = 0.44 (DCM/MeOH/AcOH = 90:5:1). IR (KBr, cm ^-1 ) : 3300 (NH); 3183(OH); 2992 (CH, aren); 2762 (CH, CH ₂ ); 1643 (C=O); 1599, 1558 (C=C). ¹ H-NMR (500 MHz, DMSO- d ₆ ): δ 10.71 (1H, s, NH); 8.99 (1H, s, OH); 7.41-7.16 (7H, m, H-3, H-5', H-7', H-2”, H-3”, H-5”, H-6”); 7.04 (1H, d, J = 8.00 Hz, H-8'); 6.28 (1H, d, J = 16.00 Hz, H-2); 5.19 (2H, s, CH ₂ ); 4.84 (2H, s, H-2'a, H-2'b). 13C-NMR (125 MHz, DMSO- d ₆ ): δ 164.55, 163.28, 162.78, 160.85, 146.35, 138.18, 132.98, 132.95, 129.96, 129.51, 129.44, 129.40, 1 28.88, 123.13, 118.41, 117.55, 116.05, 115.88, 115.79, 67.51, 43.13. ESI-MS m/z: 341.0945 [MH] ^- .

Compound 7e: (E)-3-(4-(4-chlorobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N -Hydroxyacrylamide

Yield: 71%. mp: 210-211℃. Rf = 0.46 (DCM/MeOH/AcOH = 90:5:1). IR (KBr, cm ^-1 ) : 3300 (NH); 3183(OH); 2992 (CH, aren); 1643 (C=O); 1599, 1558 (C=C). ¹ H-NMR (500 MHz, DMSO- d ₆ ): δ 10.71 (1H, s, NH); 8.99 (1H, s, OH); 7.43-7.22 (7H, m, H-3, H-5', H-7', H-2”, H-3”, H-5”, H-6”); 7.05 (1H, d, J = 8.50 Hz, H-8'); 6.28 (1H, d, J = 15.50 Hz, H-2); 5.20 (2H, s, CH ₂ ); 4.84 (2H, s, H-2'a, H-2'b). ¹³ C-NMR (125 MHz, DMSO- d ₆ ): δ 164.56, 163.26, 146.33, 138.13, 135.88, 132.36, 130.00, 129.31, 129.13, 128.87, 123.15, 118.44, 1 17.57, 115.75, 67.51, 43.23. ESI-MS m/z: 357.0652 ( ³⁵ Cl), 359.0632 ( ³⁷ Cl) [MH] ^- .

Compound 7f: (E)-3-(4-(4-bromobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)- N-Hydroxyacrylamide

Yield: 73%. mp: 223-224℃. Rf = 0.47 (DCM/MeOH/AcOH = 90:5:1). IR (KBr, cm ^-1 ) : 3296 (NH); 3183(OH); 2992 (CH, aren); 1643 (C=O); 1599 (C=C). ¹ H-NMR (500 MHz, DMSO- d ₆ ): δ 10.72 (1H, s, NH); 9.01 (1H, s, OH); 7.55-7.22 (7H, m, H-3, H-5', H-7', H-2”, H-3”, H-5”, H-6”); 7.04 (1H, d, J = 8.50 Hz, H-8'); 6.28 (1H, d, J = 15.50 Hz, H-2); 5.18 (2H, s, CH ₂ ); 4.84 (2H, s, H-2'a, H-2'b). ¹³ C-NMR (125 MHz, DMSO- d ₆ ): δ 164.57, 163.28, 146.33, 138.17, 136.29, 132.04, 129.98, 129.65, 129.40, 128.85, 123.20, 120.85, 1 18.42, 117.58, 115.72, 67.50, 43.28. ESI-MS m/z: 401.0139 ( ⁷⁹ Br), 403.1024 ( ⁸¹ Br) [MH] ^- .

Compound 7g: (E)-N-hydroxy-3-(4-(4-methylbenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6 -1) Acrylamide

Yield: 65%. mp: 188-189℃. Rf = 0.43 (DCM/MeOH/AcOH = 90:5:1). IR (KBr, cm ^-1 ) : 3291 (NH); 3221 (OH); 2959, 2918 (CH, aren); 2851 (CH, CH ₂ ); 1649 (C=O); 1601 (C=C). ¹ H-NMR (500 MHz, DMSO- d ₆ ): δ 10.73 (1H, s, NH); 9.00 (1H, s, OH); 7.34-7.14 (7H, m, H-3, H-5', H-7', H-2”, H-3”, H-5”, H-6”); 7.03 (1H, d, J = 8.50 Hz, H-8'); 6.28 (1H, d, J = 16.00 Hz, H-2); 5.16 (2H, s, CH ₂ ); 4.83 (2H, s, H-2’a, H-2’b), 2.26 (3H, s, 4”-CH3). ¹³ C-NMR (125 MHz, DMSO- d ₆ ): δ 164.48, 163.26, 146.38, 138.20, 136.92, 133.69, 129.87, 129.73, 128.96, 127.63, 123.10, 118.36, 1 17.49, 115.84, 67.52, 43.50, 21.12. ESI-MS m/z: 337.1210 [MH] ^- .

<Experimental example>

Experimental Example 1. Cytotoxicity analysis

The compounds prepared in Examples 1 and 2 were treated with three human cancer cell lines, including SW620 (colon cancer), PC3 (prostate cancer), and MDA-MB-231 (breast cancer), and cytotoxicity on cancer cell lines was evaluated. Cell lines were purchased from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Cancer Cell Bank. Media, serum, and other reagents used for cell culture in this assay were purchased from GIBCO Co. Ltd. (Grand Island, New York, USA). Each cell was cultured in DMEM (Dulbecco's Modified Eagle Medium) until confluence, and each cell was trypsinized and suspended at 3 x 10 ⁴ cells/mL. On day 0, 180 μL of the cell suspension was inoculated into each well of a 96 well plate. The plate was then incubated in a 5% CO ₂ incubator at 37°C for 24 hours. Compounds were initially dissolved in dimethyl sulfoxide (DMSO) and diluted to appropriate concentrations with culture medium. Then, 20 μL of the prepared sample of each compound was added to each well of the 96-well plate, and the plate was further cultured for 48 hours. The cytotoxicity of the compounds was measured by a modified colorimetric assay. IC ₅₀ values were calculated using the Probits method and are the average of three independent measurements (SD ≤ 10%). The results are shown in Table 1 below.

compound RCH ₂ - or
MW LogP Cell growth rate/cell line SW620 PC3 MDA-MB-231 4a ethyl 262.10 -0.40 92.70 ± 4.33 94.87 ± 3.52 62.76 ± 5.73 4b 2-chloroethyl 297.06 -0.15 89.18 ± 1.52 95.17 ± 2.26 92.65 ± 4.98 4c 2-Hydroxyethyl 278.09 -1.86 71.19 ± 4.17 71.75 ± 4.11 61.87 ± 3.71 4d n-profile 276.11 0.09 99.65 ± 5.94 104.43 ± 3.00 64.00 ± 3.42 4e n-butyl 290.13 0.58 93.06 ± 1.24 106.83 ± 2.11 74.20 ± 3.11 4f 288.11 0.40 93.41 ± 3.54 101.34 ± 2.24 89.97 ± 6.25 4g 302.13 0.89 87.63 ± 3.33 95.34 ± 4.55 86.07 ± 6.33 4h 316.14 1.38 74.79 ± 2.82 92.79 ± 4.16 73.75 ± 3.40 4i 330.16 1.87 55.28 ± 2.81 36.64 ± 5.59 38.70 ± 3.34 7a 324.11 0.82 52.11 ± 3.84 52.95 ± 2.51 50.46 ± 5.38 7b 342.10 1.02 45.44 ± 2.15 44.74 ± 3.94 49.41 ± 1.88 7c 342.10 1.02 44.38 ± 2.14 37.22 ± 4.23 44.81 ± 3.42 7d 342.10 1.02 47.39 ± 3.25 39.51 ± 4.92 45.25 ± 4.92 7e 359.07 1.46 6.14 ± 2.27 14.63 ± 1.63 37.83 ± 2.60 7f 403.02 1.71 0.92 ± 1.35 5.52 ± 2.08 36.32 ± 6.90 7g 338.13 1.36 43.60 ± 3.12 35.16 ± 4.87 36.40 ± 5.03

As can be seen from Table 1, it was confirmed that compounds 4a to 4i and 7a to 7g according to the present invention can significantly inhibit the growth of SW620, PC3 and MDA-MB-231 cell lines.

Experimental Example 2. HDAC enzyme activity analysis

HDAC enzyme (HeLa cell nuclear extract) was purchased from Enzo Life Sciences Inc. (Farmingdale, New York, USA). HDAC enzyme assay was performed using the Fluorogenic HDAC Assay Kit (Enzo Life Sciences Inc.) according to the manufacturer's instructions. HDAC enzyme was incubated with vehicle or various concentrations of the analyzed sample or SAHA for 30 min at 37°C in the presence of HDAC fluorescent substrate. HDAC analysis developer was added and fluorescence was measured using VICTOR (PerkinElmer, Waltham, MA, USA) with excitation set at 360 nm and emission set at 460 nm. The measured activity value was subtracted from the vehicle-treated control enzyme activity, and the IC ₅₀ value was calculated using GraphPad Prism (GraphPad Software, San Diego, CA, USA).

SAHA was used as a positive control. The results are shown in Table 2 below.

compound RCH ₂ -
or

MW
LogP HDAC inhibition
(IC ₅₀ ,mM) Cytotoxicity (IC ₅₀ , mM)/cell line SW620 PC3 MDA-MB-231 4a ethyl 262.10 -0.40 > 10 > 10 > 10 > 10 4b 2-chloroethyl 297.06 -0.15 > 10 > 10 > 10 > 10 4c 2-Hydroxyethyl 278.09 -1.86 9.185±0.366 > 10 > 10 > 10 4d n-profile 276.11 0.09 > 10 > 10 > 10 > 10 4e n-butyl 290.13 0.58 > 10 > 10 > 10 > 10 4f 288.11 0.40 > 10 > 10 > 10 > 10 4g 302.13 0.89 > 10 > 10 > 10 > 10 4h 316.14 1.38 > 10 > 10 > 10 > 10 4i 330.16 1.87 7.117±0.309 9.470±0.410 7.941±0.850 6.625±0.467 7a 324.11 0.82 2.699±0.187 > 10 > 10 > 10 7b 342.10 1.02 6.363±0.275 > 10 > 10 > 10 7c 342.10 1.02 5.178±0.608 8.773±0.113 8.736±1.228 8.351±0.756 7d 342.10 1.02 4.473±0.181 8.257±0.693 9.713±1.697 9.809±1.143 7e 359.07 1.46 1.498±0.020 3.616±0.282 5.475±0.126 5.137±0.951 7f 403.02 1.71 1.794±0.159 1.918±0.080 3.910±0.272 4.584±0.651 7g 338.13 1.36 4.598±0.273 8.669±0.411 8.231±1.019 7.654±0.625 SAHA 264.32 1.44 0.128±0.019 0.691±0.010 0.716±0.095 0.403±0.006 ADR 543.53 1.85 # 0.128±0.005 0.123±0.005 0.068±0.001

As can be seen in Table 2, compounds 4a to 4i and 7a to 7g according to the present invention all showed excellent HDAC inhibitory activity, and among them, 7e and 7f were confirmed to show significantly excellent HDAC inhibitory activity.

Experimental Example 3. Cell cycle, apoptosis and cell shape analysis

The results of Experimental Examples 1 and 2 showed that compound 7f was the most cytotoxic compound in three human cancer cell lines. Due to this surprising cytotoxicity, we selected this compound to analyze its effects on cell cycle and apoptosis.

In the experimental procedure, SW620 cells (human colon cancer) (2x10 ⁵ cells) were treated with compound 7f (5 μM) or SAHA (5 μM) for 24 hours. Harvested cells were stained with propidium iodide (PI) in the presence of RNase and then analyzed for DNA content, cell cycle, and apoptosis (Figures 4 and 5). UN: untreated, VH: vehicle (DMSO. 0.05%). Cell morphology was photographed using a Biostation with a 20X lens (A) or a 40X lens (B) (Figure 6).

As a result, compound 7f caused SW620 cells to stay in the G2/M phase, while SAHA stopped SW620 cells in the S and G2/M phases. In addition, it was confirmed that compound 7f increased late-stage apoptosis to some extent in SW620 cells. In terms of cell morphology, it was confirmed that compound 7f flattens SW620 cells in a manner almost similar to SAHA.

Claims (7)

A compound represented by Formula 1 or Formula 2 below and its pharmaceutically acceptable salt, hydrate, solvate, or optical isomer.
[Formula 1]

[Formula 2]

In Formula 1, R is hydrogen, substituted or unsubstituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₁ -C ₁₀ alkenyl, substituted or unsubstituted C ₁ -C ₁₀ alkynyl, substituted or unsubstituted It is one of substituted or unsubstituted C ₁ to C ₃₀ cycloalkyl;
In Formula 2, R is one of substituted or unsubstituted C ₅ to C ₃₀ aryl, substituted or unsubstituted arylalkyl, substituted or unsubstituted arylalkenyl, or substituted or unsubstituted arylalkynyl;
The substitution involves replacing one or more hydrogens with halogens such as fluorine, chlorine, bromine and iodine; hydroxy; nitro; cyano; oxo (=O); Thioxo (=S); Azido; nitroso; Amino; hydrazino; formyl; alkyl; alkoxy; aryl; It is changed to haloalkyl groups such as trifluoromethyl, tribromomethyl, and trichloromethyl.
According to paragraph 1,
If halogen is present, the halogen is fluorine, chlorine, bromine or iodine; If an alkyl group is present, the alkyl group is methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl or substituted thereof;
When present, the cycloalkyl group may be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, cycloheptyl, perhydronaphthyl, adamantyl, bridged cyclic groups or spirobicyclic groups. are spirobicyclic groups;
If an aryl group is present, the aryl group is phenyl, naphthyl, anthracenyl, indanyl or biphenyl;
A compound and a pharmaceutically acceptable salt, hydrate, solvate, or optical isomer thereof, wherein, if a hydroxyalkyl group is present, the hydroxyalkyl group is hydroxymethyl or hydroxyethyl.
According to paragraph 1,
R in Formula 1 is ethyl, 2-chloroethyl, 2-hydroxyethyl, n-propyl, n-butyl, , , or ego,
R in Formula 2 is , , , , , or A compound and a pharmaceutically acceptable salt, hydrate, solvate, or optical isomer thereof.
According to paragraph 1,
The compound represented by Formula 1 or Formula 2 is a compound characterized in that it is any one of the following compounds and a pharmaceutically acceptable salt, hydrate, solvate, or optical isomer thereof:
(E)-3-(4-ethyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacrylamide;
(E)-3-(4-(chloroethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacrylamide ;
(E)-N-hydroxy-3-(4-(hydroxymethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)acrylic amides;
(E)-N-hydroxy-3-(3-oxo-4-propyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)acrylamide;
(E)-3-(4-Butyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacrylamide;
(E)-3-(4-(cyclopropylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacryl amides;
(E)-3-(4-(Cyclobutylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacryl amides;
(E)-3-(4-(cyclopentylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacryl amides;
(E)-3-(4-(cyclohexylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacryl amides;
(E)-3-(4-benzyl-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxyacrylamide;
(E)-3-(4-(2-fluorobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydride Roxyacrylamide;
(E)-3-(4-(3-fluorobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydride Roxyacrylamide;
(E)-3-(4-(4-fluorobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydride Roxyacrylamide;
(E)-3-(4-(4-chlorobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxy acrylamide;
(E)-3-(4-(4-bromobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydride Roxyacrylamide; and
(E)-N-hydroxy-3-(4-(4-methylbenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl) Acrylamide.
A pharmaceutical composition for the prevention or treatment of cancer comprising the compound according to any one of claims 1 to 4 and a pharmaceutically acceptable salt, hydrate, solvate, or optical isomer thereof as an active ingredient. According to clause 5,
The above cancers include breast cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, colon cancer, skin cancer, head and neck cancer, skin or eye melanoma, uterine sarcoma, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube cancer, and endometrium. A pharmaceutical composition for the prevention or treatment of cancer, characterized in that it is cancer, cervical cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer, or bone marrow cancer.
According to clause 5,
The compound is a pharmaceutical composition for preventing or treating cancer, characterized in that it has the activity of promoting histone acetylation through inhibition of histone deacetylase.