KR101624344B1 - Novel 3-Substituted-2-Oxoindoline-Based N-hydroxypropenamides Having Activity of Inhibiting Histone Deacetylase and Antitumor Composition Comprising the Same - Google Patents

Abstract

The present invention relates to novel 3-hydroxyimino-2-oxoindoline-based N-hydroxypropeneamide compounds or 3-methoxyimino-2-oxoindoline-based N-hydroxypropeneamide compounds and their use as active ingredients As an anticancer agent. The hydroxamic acid compound of the present invention has a strong histone deacetylase (HDAC) inhibitory activity and exhibits proliferation inhibitory activity against various cancer cells. The compounds of the present invention can be developed as active ingredients of potent anti-cancer agents.

Description

Substituted 3-substituted-2-oxoindoline-based N-hydroxypropeneamide having histone deacetylase inhibiting activity and an anticancer composition containing the same as an active ingredient. -hydroxypropenamides Having Activity of Inhibiting Histone Deacetylase and Antitumor Composition Comprising the Same}

The present invention relates to a novel 3-substituted-2-oxoindoline-based N-hydroxypropeneamide having histone deacetylase inhibiting activity and an anticancer composition comprising the same as an active ingredient.

Histone deacetylases (HDACs) have been an interesting molecular target for the design and development of anticancer drugs for decades [1-3]. These enzymes remove the acetyl residues from the lysine residues in the tail region of histone proteins, resulting in the suppression of chromosome condensation and transcription [2, 3]. To date, 18 different homologous proteins of HDAC have been identified in eukaryotic cells [4]. They are classified into four classes based on relative sequence similarity. Class I includes four members of HDAC 1, 2, 3, and 8. Class II has six members of HDAC 4, 5, 6, 7, 9, and 10 so far. Class III HDACs are also known as Sirtuins. Sirtuins with seven members (Sirt 1-7) are NAD ⁺ -dependent enzymes. Finally, a Class IV HDAC has only one member (HDAC11). HDAC11 is known to have both characteristics of Class II and Class I HDAC2 [4]. Of the four classes, Class I HDACs are considered to be the most important homologous proteins and the most studied [4]. Suppression of different HDAC homologous proteins in different types of tumor cells results in multiple sequential events associated with cell differentiation, apoptosis and cell cycle arrest [5, 6]. The inhibitory effect of HDAC inhibition on tumor cell growth has been clearly demonstrated in many in vivo pre-clinical models and clinical stages as well as in vitro [7,8]. Thus, recent HDAC inhibition has become an interesting approach in the field of cancer therapy [9]. As a result, hundreds of HDAC inhibitors have been reported by medical chemists until recently. These inhibitors vary from short chain fatty acids (eg, butyrate, phenylbutyrate or valproic acid) to various types of hydroxamic acids or benzamides [10-16]. A number of HDAC inhibitors, such as PXD-01 (Belinostat), LBH-589 (Panobinostat), MS-27-527 (Entinostat) (Figure 1), are in different clinical trial stages and SAHA (vorinostat, Zolinza) Romidepsin (trade name Istodax) (Fig. 1) has been approved for clinical application.

The present inventors have focused on the development of hetero ring analogues of SAHA to develop novel hydroxamic acids having HDAC inhibitors and anticancer activities. As a result of previous research, we have developed hydroxamic acid with various benzothiazole / thiazole series introduced with very strong HDAC inhibitory activity and cytotoxicity against cancer cells [17, 18] (Fig. 2). Among these compounds, the selected compounds exhibited in vivo anti-tumor activity comparable to SAHA in preclinical evaluation in PC-3 prostate cancer cell-transplanted mouse models [18]. Encouraged by this successful outcome, has expanded into a study on 5-substituted phenyl / aryl-1,3,4-thiadiazole-based hydroxamic acids and demonstrates that these compounds are highly potent HDAC inhibitors, (19, 20) (Fig. 2).

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Korean Patent No. 10-1261305 Korean Patent No. 10-1282833 Korean Patent Publication No. 10-2007-0116626

1. Nam NH, Parang K. Curr Drugs Targets 2003; 4: 159-179. 2. Marks PA, Rifkind RA, Richon VM, Breslow R, Miller T, Kelly WK. Nature Rev Cancer 2001; 1: 194-202. 3. Witt O, Deubzer HE, Milde T, Oehme I. HDAC family: Cancer Lett 2009; 277: 8-21. 4. De Ruijter AJM, Gennip AHV, Caron HN, Kemp S, Kuilenburg ABPV. Biochem J 2003; 370: 737-739. 5. Bolden JE, Peart MJ, Johnstone RW. Nat Rev Drug Discov 2006; 5: 769-784. 6. Dokmanovic M, Marks PA. Expert Opin Investi. Drug 2005; 14: 1497-1511. 7. Johnstone RW. Nature Rev Drug Disc 2002; 1: 287-299. 8. Glaser KB. Biochem Pharmacol 2007; 74: 659-871. 9. West AC, Johnstone RW. J Clin Invest 2014; 124: 30-39. 10. Dallavalle S, Cincinelli R, Nannei R, Merlini L, Morini G, Penco S, Pisano C, Vesci L, Barbarino M, Zuco V, De Cesare M, Zunino F. Eur J Med. Chem 2009; 44: 1900-1912. 11. Bracker TU, Sommere, Fichtner I, Faus H, Haendler B, Hess-Stumpp H. Int J Oncol 2009; 35: 909-920. 12. Finnin MS, Donigian JR, Cohen A, Richon VM, Rifkind RA, Marks PA, Breslow R, Pavietich NP. Nature 1999; 401: 188-193. 13. Valente S, Mai A. Expert Opin Ther Pat 2014; 24: 401-415. 14. Li J, Li G, Xu X. Curr Med Chem 2013; 20: 1858-1886. 15. Ververis K, Hionge, Karagiannis TC, Licciardi PV. Biologics 2013; 7: 47-60. 16. Qiu T, Zhou L, Zhu W, Wang T, Wang J, Shu Y, Liu P. Future Oncol 2013; 9: 255-269. 17. Oanh DTK, Hai HV, Hue VTM, Park SH, Kim HJ, Han BW, Kim HS, Hong JT, Han SB, Nam NH. Bioorg Med Chem Lett 2001; 21: 7509-7512. 18. Tung TT, Oanh DT, Dung PT, Hue VT, Park SH, Han BW, Kim Y, Hong JT, Han SB, Nam NH. Med Chem 2013; 9: 1051-1057. 19. Nam NH, Huong TL, Dung DTM, Oanh DTK, Dung PTP, Kim KR, Han BW, Kim YS, Hong JT, Han SB. J Enzyme Inhib Med Chem 2013; published online, doi: 10.3109 / 14756366.2013.832238. 20. Nam NH, Huong TL, Dung DTM, Oanh DTK, Dung PTP, Quyen D, Kim KR, Han BW, Kim YS, Hong JT, Han SB. Eur J Med Chem 2013; 70: 477-486. 21. Skehan P, Storeng R, Scudiero D, Monka, MacMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR. J. Natl Cancer Inst 1990; 82: 1107-1112. 22. You YJ, Kim Y, Nam NH, Bang SC, Ahn BZ. Eur J Med Chem 2004; 39: 189-193. 23. Kim Y, You YJ, Nam NH, Ahn BZ. Bioorg Med Chem. Lett 2002; 12: 3435-3438. 24. Wu L, Smythe AM, Stinson SF, Mullendore LA, Monkse, Scudierode, Paull KD, Koutsoukos AD, Rubinstein LV, Boyd MR, Shoemaker RH. Cancer Res 1992; 52: 3029-3034. 25. Trott O, Olson AJ. AutoDock Vina: J Comput Chem 2010; 31: 455-461. 26. Lauffer BE, Mintzer R, Fong R, Mukund S, Tam C, Zilberley I, Flicke B, Ritschere, Fedorowicz G, Vallero R, Ortwine DF, Gunzner J, Modrusan Z, Neumann L, Koth CM, Lupardus PJ, Kaminker JS, Heise CE, Steiner P. J Biol Chem 2013; 288: 26926-26943. 27. Schuttelkopf AW, van Aalten DM. PRODRG: Biological crystallography 2004; 60: 1355-1363. 28. Pelzel HR, Schlamp CL, Nickells RW. BMC Neuroscience 2010; 11: 62. (Http://www.biomedcentral.com/1471-2202/11/62).

The present inventors have made efforts to develop a novel compound having a histone deacetylase inhibiting activity. As a result, N-hydroxypropene amino compounds based on 3-hydroxyimino-2-oxoindoline and 3-methoxyimino-2-oxoindoline were newly synthesized and their histone deacetylase inhibitory activity And the proliferation inhibitory activity against cancer cells was experimentally confirmed, thereby completing the present invention.

Accordingly, it is an object of the present invention to provide novel 3-hydroxyimino-2-oxoindoline-based and 3-methoxyimino-2-oxoindoline-based N-hydroxypropene amino compounds.

Another object of the present invention is to provide an anticancer composition comprising the compound as an active ingredient.

It is still another object of the present invention to provide a method for producing the above compound.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a compound represented by the following general formula (I): or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

Wherein R ₁ is hydrogen or methyl and R ₂ is hydrogen, halogen, C _1-6 alkyl or C _1-6 alkoxy.

According to an embodiment of the present invention, the halogen is fluorine (F), chlorine (Cl) or bromine (Br).

The compounds of the present invention are 3-hydroxyimino-2-oxoindoline-based N-hydroxypropeneamide compounds or 3-methoxyimino-2-oxoindoline-based N-hydroxypropeneamide compounds.

According to another embodiment of the present invention, the compound of the present invention is a compound of any one of the following compounds:

( E ) -N -hydroxy-3- (4 - ((( Z ) -3- (hydroxyimino) -2-oxoindolin-1-yl) methyl) phenyl) propenamide;

(E) -3- (4 - ( ((Z) -5- fluoro-3- (hydroxyimino) -2-oxo-in turned-yl) methyl) phenyl) - N - hydroxy-propenamide ;

( E ) -3- (4 - ((( Z ) -5-chloro-3- (hydroxyimino) -2-oxoindolin-1-yl) methyl) phenyl) -N -hydroxypropenamide;

(E) -3- (4 - ( ((Z) -5- bromo-3- (hydroxyimino) -2-oxo-in turned-yl) methyl) phenyl) - N - hydroxy-propene is mid;

( E ) -N -hydroxy-3- (4 - ((( Z ) -3- (hydroxyimino) -5-methyl-2-oxoindolin-1-yl) methyl) phenyl) propenamide;

( E ) -N -hydroxy-3- (4 - (( Z ) -3- (hydroxyimino) -5-methoxy-2- oxoindolin- ;

( E ) -3- (4 - ((( Z ) -7-chloro-3- (hydroxyimino) -2-oxoindolin-1-yl) methyl) phenyl) -N -hydroxypropenamide;

( E ) -N -hydroxy-3- (4 - ((( Z ) -3- (methoxyimino) -2-oxoindolin-1-yl) methyl) phenyl) propenamide;

( E ) -3- (4 - (( Z ) -5-fluoro-3- (methoxyimino) -2-oxoindolin- 1-yl) methyl) phenyl) -N -hydroxypropenamide ;

( E ) -3- (4 - (( Z ) -5-chloro-3- (methoxyimino) -2-oxoindolin-1-yl) methyl) phenyl) -N -hydroxypropenamide;

(E) -3- (4 - ( ((Z) -5- bromo-3- (methoxy toksiyi mino) -2-oxo-in turned-yl) methyl) phenyl) - N - hydroxy-propenamide ;

( E ) -N -hydroxy-3- (4 - ((( Z ) -3- (methoxyimino) -5-methyl-2-oxoindolin-1-yl) methyl) phenyl) propenamide;

( E ) - N -hydroxy-3- (4 - (( Z ) -3- (methoxyimino) -5-methoxy- ; And

( E ) -3- (4 - (( Z ) -7-chloro-3- (methoxyimino) -2-oxoindolin-1-yl) methyl) phenyl) -N -hydroxypropenamide.

The term " pharmaceutically acceptable salts " as used herein refers to those salts which possess the biological effectiveness and properties of the compounds and which are intended to mean the customary acid addition salts or base addition salts formed from suitable non-toxic organic or inorganic acids, or non-toxic organic or inorganic bases do. Examples of acid addition salts include acid addition salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, Fumaric acid, and the like. Examples of base addition salts include base addition salts derived from ammonium, potassium, sodium, and quaternary ammonium hydroxides, such as tetramethylammonium hydroxide. To obtain improved physical and chemical stability, hygroscopicity, fluidity and solubility of the compound, chemical modification of the pharmaceutical compound (i.e., drug) into a salt is a technique well known to pharmacologists, . Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457, the disclosures of which are incorporated herein by reference.

As used herein, the term " pharmaceutically acceptable " means that, for example, a pharmaceutically acceptable carrier, excipient, etc. is pharmacologically acceptable and substantially non-toxic to a patient to whom a particular compound is administered.

According to another aspect of the present invention, there is provided an anticancer pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

According to one embodiment of the present invention, the compounds of the present invention have an effect of promoting histone acetylation through inhibition of histone deacetylase activity and inhibit the activity of histone deacetylase, The histone is induced to a high acetylated state.

The compound of the present invention exhibits cytotoxic (cell proliferation inhibiting) effect on various cancer cell lines and demonstrates anticancer activity as demonstrated in the following specific example.

Cancer, which is a disease to be treated by the pharmaceutical composition of the present invention, is characterized by aggressive characteristics in which the cells disregard normal growth limits and are divided and grown, invasive characteristics penetrating into surrounding tissues, It is a generic term for diseases caused by cells with metastatic characteristics spreading to other parts.

According to another embodiment of the present invention, the cancer to be treated is a breast cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or ocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer, But are not limited to, cancer, colon cancer, fallopian tube cancer, endometrial cancer, cervical cancer, small bowel cancer, endocrine cancer, thyroid cancer, pituitary cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer or bone cancer. More preferably, the cancer is a colon cancer, a breast cancer, a prostate cancer, a pancreatic cancer, or a lung cancer.

The anticancer pharmaceutical composition of the present invention comprises (i) a pharmaceutical effective amount of a compound represented by the above-described formula (1) or a pharmaceutically acceptable salt thereof; And (ii) a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not.

The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The appropriate dosage of the pharmaceutical composition of the present invention may be determined by various methods depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient It can be prescribed. On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.001-1000 mg / kg (body weight) per day.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, or the like.

The concentration of the active ingredient contained in the composition of the present invention is determined in consideration of the purpose of treatment, the condition of the patient, the period of time required, the severity of disease, and the like, and is not limited to a specific range of concentration.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to another aspect of the present invention, the present invention provides a process for preparing a compound of the present invention as described above, comprising the steps of:

(i) reacting a compound of the formula (a-1) with methyl 4-bromomethylcinnamate under basic conditions to produce a compound of the formula (a-2) And

[Formula (a-1)]

[Formula (a-2)]

(Ii) reacting the resulting compound of formula (a-2) with hydroxylamine to produce a compound of formula (a-3).

[Formula (a-3)]

In the above formulas (a-1) to (a-3), R is hydrogen, halogen, C _1-6 alkyl or C _1-6 alkoxy.

According to another aspect of the present invention, the present invention provides a process for preparing a compound of the present invention as described above, comprising the steps of:

(i ') reacting a compound of formula (a-1) with methoxylamine hydrochloride under basic conditions to produce a compound of formula (a-4);

[Formula (a-1)]

[Formula (a-4)]

(Ii ') reacting the resulting compound of formula (a-4) with methyl 4-bromomethylcinnamate to produce a compound of formula (a-5); And

[Chemical formula (a-5)]

(Iii ') reacting the resulting compound of formula (a-5) with hydroxylamine to produce a compound of formula (a-6).

[Formula (a-6)]

In the above formulas (a-1) to (a-6), R is hydrogen, halogen, C _1-6 alkyl or C _1-6 alkoxy.

The features and advantages of the present invention are summarized as follows:

(i) The present invention relates to novel 3-hydroxyimino-2-oxoindoline based N-hydroxypropeneamide compounds or 3-methoxyimino-2-oxoindoline based N-hydroxypropeneamide compounds and And an anticancer composition comprising the same as an active ingredient.

(Ii) The hydroxamic acid compound of the present invention has a strong histone deacetylase (HDAC) inhibitory activity and exhibits proliferation inhibitory activity against various cancer cells.

(Iii) The compounds of the present invention can be developed as active ingredients of potent anti-cancer agents.

Figure 1 shows the structure of the HDAC inhibitors developed so far.
Figure 2 shows the structure of benzothiazole-, 5-substituted phenyl-1,3,4-thiadiazole-based hydroxamic acid.
Figure 3 shows the results of measuring the effect of the synthesized compounds on histone acetylation in SW620 cells. Cells were treated with compounds or SAHA at a concentration of 3 μg / mL for 24 h. The levels of acetyl-histone-H3 and -H4 in total cell lysates were determined by Western immunoblot analysis.
Figure 4 shows the steric structure of the SAHA's actual binding pose for HDAC2 and the simulated docking pose of compound 3b (panel A) and compound 3g (panel B). SAHA labeled the carbon, nitrogen and oxygen atoms as pink, blue and red, respectively, as a stick model. Compounds 3b and 3g each have carbon atoms cyan and brown respectively; Nitrogen and oxygen atoms are shown in blue and red, respectively. The crucial regions of the enzymes involved in the interaction were carbon, nitrogen, and oxygen, respectively, in a stick model of gray, blue, and red, respectively. Zn ²⁺ ions are represented by light gray spheres.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Methods

1. Synthetic chemistry of compounds

All the products were homogeneously obtained and confirmed by thin-layer chromatography (TLC) on Whatman 250 μm Silica Gel GF Uniplates and visualization under UV light at 254 nm and 365 nm. The boiling point was measured using a Gallenkamp Melting Point Apparatus (LabMerchant, London, United Kingdom) and not calibrated. Purification using chromatography was performed by open flash silica gel column chromatography using Merck silica gel 60 (240 - 400 mesh). Nuclear magnetic resonance spectrum ( ¹ H NMR) was determined on a Bruker 500 MHz spectrometer using tetramethylsilane as internal standard and dimethylsulfoxide- d ₆ (DMSO- d ₆ ) as solvent unless otherwise specified. Chemical shifts were recorded in parts per million (ppm) downfield from the internal standard tetramethylsilane. Electron ionization (EI), electrospray ionization (ESI) and high resolution mass spectra were performed using PE Biosystems API 200 (Perkin Elmer, Palo Alto, Calif., USA) and Mariner (Azco Biotech, Inc. Oceanside, , USA) mass spectrometer. Reagents and solvents are commercially available from Aldrich or Fluka Chemical Corp. unless otherwise indicated. (Milwaukee, WI, USA) or Merck. The solvent was distilled before use and dried.

2. Synthesis of Compound

( E ) -N-hydroxy-3- (4 - ((Z) -3- (hydroxyimino) -5/7-substituted-2-oxoindolin- Amide (Compound 3a-3g) and ( E ) -N-hydroxy-3- (4 - ((Z) -3- (methoxyimino) -5- Yl) methyl) phenyl) propenamide (Compound 6a-6g) The compounds were synthesized according to the methods described in the following Reactions 1 and 2. Synthesis proceeded as follows.

(One) ( E ) -N-hydroxy-3- (4 - ((Z) -3- (hydroxyimino) -5/7-substituted-2-oxoindolin-1- yl) methyl) phenyl) Synthesis of Compound 3a-3g)

The DMF (3 mL) 1a-1g (1 mmoL) compound in the solution was cooled to -5 ℃, was added _{K 2 CO 3 (165.5 mg,} 1.2 mmoL). The mixture was stirred at room temperature for 1 hour and at -5 ℃ 45 bungan respectively, were added to CH ₃ OH (0.5 mL) and KI (8.3 mg, 0.05 mmoL) . After stirring for 15 min, a solution of ( E ) -methyl 4-bromomethyl cinnamate (255 mg, 1 mmol) in DMF (1 mL) was added and the resulting reaction mixture was stirred at 60 <0> C for 24 h. After complete stirring, the reaction mixture was cooled and acidified to pH ~ 4 with 10% HCl and extracted with DCM (50 mL x 2). After collecting the extracts, DCM was evaporated under reduced pressure to give 2a-2g of the compound as a brown-yellow oil residue and used in the next step without further purification.

The obtained brown-yellow oil (Compound 2a-2g) was dissolved in a methanol / tetrahydrofuran mixture (1/1, 3 mL), respectively, and the resulting solution of each compound thus obtained was cooled to -5 DEG C and hydroxylammonium Chloride (195 mg, 10 mmol) was added. NaOH (0.4 g, 10 mmoL) was dissolved in 2 mL of water, cooled to -5 [deg.] C and then added to the mixture. The mixture was stirred at -5 [deg.] C until the compound 2a-2g was completely reacted. The reaction mixture was neutralized to pH 7 with 15% HCl solution added dropwise to induce precipitation. The precipitate was filtered, washed with water, dried at 60 &lt; 0 &gt; C and recrystallized from ethanol to give compounds 3a-3g.

( E ) - N -Hydroxy-3- (4 - ((( Z ) -3- (hydroxyimino) -2-oxoindolin-1-yl) methyl) phenyl) propenamide (Compound 3a)

Yellow solid; Yield: 74%. mp: 197-198 [deg.] C. _{R f = 0.45 (DCM: MeOH} : AcOH = 90: 5: 1). IR (KBr, cm ^-1 ): 3441 (NH), 3197 (OH), 3049 (CH, aren), 2856 (CH, CH ₂ ), 1714, 1655 1463 (CN). ESI-MS (m / z) : 339 [M + 2H] -. ^{1 H-NMR (500MHz, DMSO} -d 6, ppm): δ 8.01 (1H, d, J = 7.5Hz, H-4 "); 7.52 (2H, d, J = 8.5 Hz, H-2 ', H-6'); 7.40 (1H, d, J = 15.5 Hz, H-3); 7.35 (2H, d, J = 8.5 Hz, H-3 ', H-5'); 7.34 (1H, t, J = 7.5 Hz, H-5 &quot;); 7.07 (1H, t, J = 7.5 Hz, H-6 &quot;); 6.96 (1H, d, J = 8.0 Hz, H-7 &quot;); 6.45 (1H, d, J = 16.0 Hz, H-2); _{4.95 (2H, s, -CH 2} -). ^{13 CNMR (125MHz, DMSO-d} 6, ppm): δ 163.36, 162.69, 143.41, 142.60, 137.79, 137.61, 134.15, 131.92, 127.88, 127.81, 126.91, 122.88, 119.21, 115.41, 109.55, 42.37. Anal. Calcd. _{For C 18 H 15 N 3 O} 4 (337.33): C, 64.09; H, 4.48; N, 12.46. Found: C, 64.11; H, 4.49; N, 12.41.

( E ) -3- (4 - ((( Z ) -5-fluoro-3- (hydroxyimino) -2-oxoindolin-1-yl) methyl) N - hydroxypropenamide (compound 3b)

Yellow solid; Yield: 72%. mp: 198-199 [deg.] C. _{R f = 0.35 (DCM: MeOH} : AcOH = 90: 5: 1). IR (KBr, cm ^-1 ): 3452 (NH), 3224 (OH), 3049 (CH, aren) 2856 (CH, CH ₂ ), 1720, 1662 (C = O), 1615, 1513 , 1474 (CN). ESI-MS (m / z) : 357 [M + 2H] -. ^{1 H-NMR (500 MHz,} DMSO-d 6, ppm): δ 7.78 (1H, dd, J = 8.0Hz, J '= 2.5Hz, H-4 "); 7.52 (2H, d, J = 8.0Hz , H-2 ', H-6'); 7.41 (1H, d, J = 16.0 Hz, H-3); 7.35 (2H, d, J = 8.0 Hz, H-3 ', H-5'); 7.21 (1H, td, J = 9.0 Hz, 3.0 Hz, H-6 "); 6.96 (1H, dd, J = 8.5 Hz, J = 4.0 Hz, H-7 &quot;); 6.44 (1H, d, J = 16.0 Hz, H-2); _{4.95 (2H, s, -CH 2} -). ^{13 CNMR (125 MHz, DMSO-} d 6, ppm): δ 163.35, 162.68, 159.05, 157.15, 143.14, 138.72, 137.81, 137.47, 134.17, 127.89, 127.81, 119.19, 117.91, 117.73, 116.06, 115.98, 113.80, 113.59 , 110.49, 110.42, 42.46. Anal. Calcd. _{For C 18 H 14 FN 3 O} 4 (355.32): C, 58.81; H, 6.58; N, 13.72. Found: C, 58.78; H, 6.55; N, 13.83.

( E ) -3- (4 - ((( Z ) -5-chloro-3- (hydroxyimino) -2-oxoindolin-1-yl) methyl) N -Hydroxypropenamide (Compound 3c) &lt; RTI ID = 0.0 &gt;

Yellow solid; Yield: 69%. mp: 196-198 [deg.] C. _{R f = 0.38 (DCM: MeOH} : AcOH = 90: 5: 1). IR (KBr, cm ^-1 ): 3279 (OH), 3069 (CH, aren), 2924, 2847 (CH, CH ₂ ), 1716, 1656 (CN). ESI-MS ( m / z): 370.4 [MH] ^&lt;&quot;&gt;. ^{1 H-NMR (500 MHz,} DMSO-d 6, ppm): δ 13.80 (1H, s, OH); 10.75 (1H, s, NH); 9.03 (1H, s, OH); 7.97 (1H, d, J = 2.0 Hz, H-4 &quot;); 7.52 (2H, d, J = 8.0 Hz, H-2 ', H-6'); 7.43-7.40 (2H, m, H-6 &quot;,H-3); 7.35 (2H, d, J = 8.0 Hz, H-3 ', H-5'); 6.99 (1H, d, J = 8.5 Hz, H-7 &quot;); 6.43 (1H, d, J = 15.5 Hz, H-2); 4.96 (2H, s, H-7'a, H-7'b). ^{13 CNMR (125 MHz, DMSO-} d 6, ppm): δ 162.90, 162.67, 142.68, 141.39, 137.79, 137.21, 134.17, 131.40, 127.85, 127.75, 126.67, 126.24, 119.17, 116.51, 110.10, 42.46. Anal. Calcd. For C ₁₈ H ₁₄ ClN ₃ O ₄ (371.78): C, 58.15; H, 3.80; N, 11.30. Found: C, 58.19; H, 3.76; N, 11.26.

( E ) -3- (4 - ((( Z ) -5-bromo-3- (hydroxyimino) -2-oxoindolin-1-yl) methyl) N -Hydroxypropeneimide (compound 3d)

Yellow solid; Yield: 67%. mp: 205-207 [deg.] C. _{R f = 0.47 (DCM: MeOH} : AcOH = 90: 5: 1). IR (KBr, cm ^-1 ): 3431 (NH), 3290 (OH), 3089 (CH, aren), 2925 (CH, CH ₂ ), 1715, 1656 ), 1433 (CN). ESI-MS ( m / z): 415.3 [MH] ^&lt;&quot;&gt;. ^{1 H-NMR (500 MHz,} DMSO-d 6, ppm): δ 13.85 (1H, s, OH); 10.74 (1H, s, NH); 9.02 (1H, s, OH); 8.10 (1H, d, J = 2.0 Hz, H-4 &quot;); 7.56 (1H, dd, J = 8.5 Hz, J = 2.0 Hz, H-6 &quot;); 7.52 (2H, d, J = 8.0 Hz, H-2 ', H-6'); 7.42 (1H, d, J = 15.5 Hz, H-3); 7.35 (2H, d, J = 8.0 Hz, H-3 ', H-5'); 6.95 (1H, d, J = 8.5 Hz, H-7 "), 6.43 (1H, d, J = 15.5 Hz, H-2); 4.96 (2H, s, H-7'a, H-7'b). ^{13 CNMR (125 MHz, DMSO-} d 6, ppm): δ 162.76, 162.64, 142.54, 141.77, 137.78, 137.16, 134.21, 134.14, 128.92, 127.82, 127.72, 119.16, 116.92, 114.30, 111.59, 42.41. Anal. Calcd. For C ₁₈ H ₁₄ BrN ₃ O ₄ (416.23): C, 51.94; H, 3.39; N, 10.10. Found: C, 51.98; H, 3.42; N, 10.14.

( E ) - N -Hydroxy-3- (4 - ((( Z Yl) methyl) phenyl) propenamide (Compound 3e) was reacted with 3-amino-2-

Yellow solid; Yield: 71%. mp: 195-196 [deg.] C. _{R f = 0.46 (DCM: MeOH} : AcOH = 90: 5: 1). IR (KBr, cm ^-1 ): 3226 (OH), 3049 (CH, aren), 2921 (CH, CH ₂ ), 1711, 1660 (C = O), 1616, 1478 ). ESI-MS ( m / z): 349.9 [MH] ^- . ^{1 H-NMR (500MHz, DMSO} -d 6, ppm): δ 13.50 (1H, s, OH); 10.76 (1H, s, NH); 9.03 (1H, s, OH); 7.85 (1H, s, H-4 &quot;); 7.54 (2H, d, 8.0 Hz, H-2 ', H-6'); 7.41 (1H, d, J = 15.5 Hz, H-3); 7.34 (2H, d, J = 8.0 Hz, H-3 ', H-5'); 7.16 (1H, d, J = 7.5 Hz, H-7 &quot;); 6.85 (1H, d, J = 8.0 Hz, H-6 &quot;); 6.41 (1H, d, J = 16.0 Hz, H-2); 4.92 (2H, s, H-7'a, H-7'b); _{2.25 (3H, s, -CH 3} ). ¹³ CNMR (125 MHz, DMSO-d ₆ , ppm) : δ 163.27, 162.74, 143.58, 140.45, 137.89, 137.67, 134.08, 132.19, 131.94, 127.86, 127.76, 127.52, 119.11, 115.40, 109.35, 42.35, 20.52. Anal. Calcd. _{For C 19 H 17 N 3 O} 4 (351.36): C, 64.95; H, 4.88; N, 11.96. Found: C, 64.99; H, 4.51; N, 11.92.

( E ) - N -Hydroxy-3- (4 - ((( Z Yl) methyl) phenyl) propenamide (Compound 3f) was reacted with 4-

Yellow solid; Yield: 75%. mp: 201-202 [deg.] C. _{R f = 0.38 (DCM: MeOH} : AcOH = 90: 5: 1). IR (KBr, cm ^-1 ): 3212 (OH), 3054 (CH, aren), 2884 (CH, CH ₂ ), 1709, 1660 (C = O), 1625, 1514, 1480 (CN). ESI-MS ( m / z): 368.3 [M + H] &lt; ⁺ &gt;. ^{1 H-NMR (500MHz, DMSO} -d 6, ppm): δ 13.61 (1H, s, OH); 7.69 (1H, d, J = 2.0 Hz, H-4 &quot;); 7.52 (2H, d, J = 8.0 Hz, H-2 ', H-6'); 7.41 (1H, d, J = 15.5 Hz, H-3); 7.34 (2H, d, J = 8.0 Hz, H-3 ', H-5'); 6.94 (1H, dd, J = 8.5, J = 2.5 Hz, H-6 &quot;); 6.87 (1H, d, J = 8.5 Hz, H-7 &quot;); 6.44 (1H, d, J = 15.5 Hz, H-2); 4.92 (2H, s, H- 7'a, H-7'b), 3.74 (4H, s, -OCH 3). ^{13 CNMR (125MHz, DMSO-d} 6, ppm): δ 163.15, 162.70, 155.33, 143.70, 137.83, 137.67, 136.28, 134.12, 127.87, 127.80, 119.17, 116.90, 115.95, 113.28, 110.20, 55.68, 42.41. Anal. Calcd. _{For C 19 H 17 N 3 O} 5 (367.36): C, 62.12; H, 4.66; N, 11.44. Found: C, 62.17; H, 4.62; N, 11.47.

( E ) -3- (4 - ((( Z ) -7-chloro-3- (hydroxyimino) -2-oxoindolin-1-yl) methyl) N - hydroxypropenamide (compound 3g)

Yellow solid; Yield: 72%. mp: 190-192 [deg.] C. _{R f = 0.44 (DCM: MeOH} : AcOH = 90: 5: 1). IR (KBr, cm ^-1 ): 3214 (OH), 3054 (CH, aren), 2855 (CH, CH ₂ ), 1717, 1659 (C = O), 1602, 1514, 1470 (CN). ESI-MS (m / z) : 370.6 [M-2H] -. ^{1 H-NMR (500MHz, DMSO} -d 6, ppm): δ 13.90 (1H, s, OH); 10.74 (1H, s, NH); 9.01 (1H, s, OH); 8.10 (1H, dd, J = 8.5Hz, J '= 1.0Hz, H-4 "); 7.52 (2H, d, J = 8.0 Hz, H-2 ', H-6'); 7.42 (1H, d, J = 15.5 Hz, H-3); 7.38 (1H, dd, J = 8.5Hz, J '= 1.0Hz, H-6 "); 7.24 (2H, d, J = 8.0 Hz, H-3 ', H-5'); 7.12 (1H, t, J = 8.0 Hz, H-5 "), 6.42 (1H, d, J = 15.5 Hz, H-2) ). ^{13 CNMR (125MHz, DMSO-d} 6, ppm): δ 163.90, 162.70, 142.11, 138.96, 138.43, 137.88, 133.80, 133.67, 127.76, 126.50, 125.95, 124.39, 118.91, 118.26, 114.58, 40.01. Anal. Calcd. For C ₁₈ H ₁₄ ClN ₃ O ₄ (371.78): C, 58.15; H, 3.80; N, 11.30. Found: C, 58.19; H, 3.86; N, 11.25.

(2) ( E ) -N-hydroxy-3- (4 - ((Z) -3- (methoxyimino) -5/7-substituted-2-oxoindolin-1- yl) methyl) phenyl) Synthesis of Compound 6a-6g)

A mixture of 1a-1 g (1 mmol) and methoxylamine hydrochloride (501 mg, 6 mmol) in pyridine anhydride (5 mL) was refluxed for 3 h. The reaction mixture was cooled to ambient temperature and poured into a cooled HCl 5M (30 mL) solution. The precipitate (compounds 4a-4g) was washed with water, dried at 40 &lt; 0 &gt; C under vacuum for 24 hours, and used in the next step without further purification. The compound 4a-4g obtained above was dissolved in DMF (3 mL). The resulting solution was cooled to -5 ℃ and added to _{K 2 CO 3 (165.5 mg,} 1.2 mmoL). The mixture was stirred at -5 ° C for 1 hour and at room temperature for 45 minutes, respectively, and CH ₃ OH (0.5 mL) and KI (8.3 mg, 0.05 mmol) were added. After stirring for 15 min, a solution of ( E ) -methyl 4-bromomethyl cinnamate (255 mg, 1 mmol) in DMF (1 mL) was added and the resulting reaction mixture was stirred at 60 <0> C for 24 h. After complete stirring, the reaction mixture was cooled and acidified to pH ~ 4 with 10% HCl and extracted with DCM (50 mL x 2). After collecting the extracts, the DCM was evaporated under reduced pressure to give 5a-5g as a brown-yellow oil residue.

The resulting brown-yellow oil (Compound 5a-5g) was dissolved in a methanol / tetrahydrofuran mixture (1/1, 3 mL), respectively, and the resulting solution of each compound thus obtained was cooled to -5 占 폚 and hydroxylammonium Chloride (195 mg, 10 mmoL). NaOH (0.4 g, 10 mmoL) was dissolved in 2 mL of water, cooled to -5 [deg.] C and then added to the mixture. The mixture was stirred at -5 [deg.] C until 5a-5g of the compound was completely reacted. The reaction mixture was neutralized to pH 7 with 15% HCl solution added dropwise to induce precipitation. The precipitate was filtered, washed with water, dried at 60 &lt; 0 &gt; C and recrystallized from ethanol to give compounds 6a-6g.

( E ) - N -Hydroxy-3- (4 - ((( Z Yl) methyl) phenyl) propenamide (Compound 6a) was reacted with 4-

Pale yellow solid; Yield: 76%. mp: 207-208 [deg.] C. _{R f = 0.43 (DCM: MeOH} : AcOH = 90: 5: 1). IR (KBr, cm ^-1 ): 3313 (NH), 3220 (OH), 3038 (CH, aren), 2938 (CH, CH ₂ ), 1718, 1648 (C = O), 1622, 1607, = C), 1437 (CN). ESI-MS (m / z) : 349.4 [M-2H] -. ^{1 H-NMR (500MHz, DMSO} -d 6, ppm): δ 10.76 (1H, s, NH), 7.89 (1H, d, J = 7.5Hz, H-4 "), 7.52 (2H, d, J = 8.0Hz, H-2 ', H -6'), 7.44-7.35 (4H, m, H-3, H-3 ', H-5', H-5 "), 7.07 (1H, t, J = 8.5Hz, H-6 "), 6.98 (1H, d, J = 8.0Hz, H-7"), 6.44 (1H, d, J = 16.0Hz, H-2), 4.95 (2H, s, H- 7'a, H-7'b), 4.23 (3H, s, -NOCH 3), 13 CNMR (125MHz, DMSO-d 6, ppm): δ 162.63, 162.41, 143.25, 143.16, 137.75, 137.31, 134.13, 132.85, 127.81, 127.75, 127.37, 122.95, 119.16, 115.01, 109.79, 64.53, 42.40. Anal. Calcd. _{For C 19 H 17 N 3 O} 4 (351.36): C, 64.95; H, 4.88; N, 11.96. Found: C, 64.91; H, 4.92; N, 11.91.

( E ) -3- (4 - ((( Z ) -5-fluoro-3- (methoxyimino) -2-oxoindolin-1-yl) methyl) N - hydroxypropenamide (Compound 6b)

Pale yellow solid; Yield: 67%. _{^{mp: 194-195 o C. R f =}} 0.41 (DCM: MeOH: AcOH = 90: 5: 1). IR (KBr, cm ^-1 ): 3426 (NH), 3199 (OH), 2946 (CH, CH ₂ ), 1715 (C = O), 1614, 1601, 1513, 1476 ). ESI-MS ( m / z): 368.0 [MH] ^&lt;&quot;&gt;. ^{1 H-NMR (500MHz, DMSO} -d 6, ppm): δ 10.76 (1H, s, NH), 9.03 (1H, s, OH), 7.69 (1H, d, J = 6.5Hz, H-4 ") , 7.52 (2H, d, J = 7.0 Hz, H-2 ', H-6'), 7.42 (1H, d, J = 15.5 Hz, H- H-3 ', H-5 '), 7.27 (1H, t, J = 7.5Hz, H-6 "), 6.99 (1H, s, H-7"), 6.44 (1H, d, J = 15.5Hz , H-2), 4.95 ( 2H, s, H-7'a, H-7'b), 4.25 (3H, s, -NOCH 3). ¹³ CNMR (125 MHz, DMSO-d ₆ , ppm) :? 162.66, 162.29,158.99,157.09,142.87,139.59,137.75,137.11,134.17,127.83,127.76,119.20,118.99,115.59,115.51,114.55,114.34,110.87, 110.81, 64.78, 42.53. Anal. Calcd. _{For C 19 H 16 FN 3 O} 4 (369.35): C, 61.79; H, 4.37; N, 11.38. Found: C, 61.83; H, 4.41; N, 11.34.

( E ) -3- (4 - ((( Z ) -5-chloro-3- (methoxyimino) -2-oxoindolin-1-yl) methyl) N - hydroxypropenamide (Compound 6c)

Yellow solid; Yield: 70%. mp: 202-203 [deg.] C. _{R f = 0.37 (DCM: MeOH} : AcOH = 90: 5: 1). IR (KBr, cm ^-1 ): 3426 (NH), 3224 (OH), 2942 (CH, CH ₂ ), 1722, 1661 (C = O), 1608, 1467 (C = C), 1444 (CN). ESI-MS (m / z) : 386.4 [M + H] -. ^{1 H-NMR (500MHz, DMSO} -d 6, ppm): δ 10.75 (1H, s, NH), 9.02 (1H, s, OH), 7.87 (1H, d, J = 2.0Hz, H-4 ") , 7.42 (1H, dd, J = 8.5 Hz, J = 2.0 Hz, H-6 "), 7.52 (2H, d, J = 8.0 Hz, H- d, J = 16.0Hz, H- 3), 7.35 (2H, d, J = 8.0Hz, H-3 ', H-5'), 7.00 (1H, d, J = 8.0Hz, H-7 ") , 6.43 (1H, d, J = 15.5Hz, H-2), 4.95 (2H, s, H-7'a, H-7'b), 4.26 (3H, s, -NOCH 3). ^{13 CNMR (125MHz, DMSO-d} 6, ppm): δ 162.62, 162.08, 142.41, 142.02, 137.72, 136.97, 134.17, 132.26, 127.81, 127.72, 126.79, 126.64, 119.20, 116.21, 111.34, 64.85, 42.52. Anal. Calcd. For C ₁₉ H ₁₆ ClN ₃ O ₄ (385.80): C, 59.15; H, 4.18; N, 10.89. Found: C, 59.19; H, 4.21; N, 10.83.

( E ) -3- (4 - ((( Z ) -5-bromo-3- (methoxyimino) -2-oxoindolin-1-yl) methyl) N - hydroxypropenamide (Compound 6d)

Yellow solid; Yield: 65%. mp: 190-191 [deg.] C. _{R f = 0.42 (DCM: MeOH} : AcOH = 90: 5: 1). IR (KBr, cm ^-1 ): 3435 (NH), 2942 (CH, CH ₂ ), 1723, 1627 (C = O), 1604, 1516, 1466 (C = C), 1437 (CN). ESI-MS ( m / z): 429.9 [MH] ^{-. 1 H-NMR (500MHz, DMSO} -d 6, ppm): δ 8.00 (1H, s, H-4 "), 7.65-7.51 (4H, m, H-6", H-2 ', H-6' M, H-3 ', H-5'), 6.96 (1H, d, J = 8.0 Hz, H-7), 6.50-6.41 H-2), 4.98-4.95 (2H , m, H-7'a, H-7'b), 4.26 (3H, s, -NOCH 3). ^{13 CNMR (125MHz, DMSO-d} 6, ppm): δ 167.43, 161.98, 143.27, 142.40, 142.38, 142.27, 137.78, 137.71, 135.08, 133.56, 129.32, 128.51, 127.79, 127.69, 127.64, 119.28, 119.19, 116.64, 114.43, 111.81, 64.86, 42.49. Anal. Calcd. _{For C 19 H 16 BrN 3 O} 4 (430.25): C, 53.04; H, 3.75; N, 9.77. Found: C, 53.11; H, 3.79; N, 9.72.

( E ) - N -Hydroxy-3- (4 - ((( Z Yl) methyl) phenyl) propenamide (Compound 6e) was reacted with 2-

Pale yellow solid; Yield: 68%. mp: 193-194 [deg.] C. _{R f = 0.44 (DCM: MeOH} : AcOH = 90: 5: 1). IR (KBr, cm ^-1 ): 3428 (NH), 3296 (OH), 2938, 2856 (CH, CH ₂ ), 1709, 1669 (C = O), 1616, 1594, 1516, 1480 , 1441 (CN). ESI-MS ( m / z): 364.1 [MH] ^&lt;&quot;&gt;. ^{1 H-NMR (500MHz, DMSO} -d 6, ppm): δ 10.74 (1H, s, NH), 9.01 (1H, s, OH), 7.74 (1H, s, H-4 "), 7.51 (2H, d, J = 8.0Hz, H- 2 ', H-6'), 7.41 (1H, d, J = 16.0Hz, H-3), 7.43 (2H, d, J = 8.0Hz, H-3 ', H-5 '), 7.20 ( 1H, d, J = 7.5Hz, H-7 "), 6.87 (1H, d, J = 8.0Hz, H-6"), 6.42 (1H, d, J = 16.0Hz , H-2), 4.93 ( 2H, s, H-7'a, H-7'b), 4.23 (3H, s, -NOCH 3), 2.26 (3H, s, -CH 3). ^{13 CNMR (125MHz, DMSO-d} 6, ppm): δ 162.61, 162.39, 143.28, 141.03, 137.75, 137.38, 134.07, 133.05, 132.07, 127.83, 127.78, 127.70, 119.11, 115.05, 109.58, 64.48, 42.37, 20.40. Anal. Calcd. _{For C 20 H 19 N 3 O} 4 (365.38): C, 65.74; H, 5.24; N, 11.50. Found: C, 65.77; H, 5.21; N, 11.54.

( E ) - N -Hydroxy-3- (4 - ((( Z (Methoxyimino) -5-methoxy-2-oxoindolin-1-yl) methyl) phenyl) propenamide (Compound 6f)

Pale yellow solid; Yield: 71%. mp: 201-202 [deg.] C. _{R f = 0.42 (DCM: MeOH} : AcOH = 90: 5: 1). IR (KBr, cm ^-1 ): 3413 (NH), 3214 (OH), 3018 (CH, aren), 2942 (CH, CH ₂ ), 1699 (C = O), 1617, 1595, 1478 ). ESI-MS (m / z) : 379.4 [M-2H] -. ^{1 H-NMR (500MHz, DMSO} -d 6, ppm): δ 10.73 (1H, s, NH), 9.01 (1H, s, OH), 7.52 (2H, d, J = 8.0Hz, H-2 ', H-6 '), 7.49 ( 1H, d, J = 2.5Hz, H-4 "), 7.41 (1H, d, J = 16.0Hz, H-3), 7.34 (2H, d, J = 8.0Hz, H-3 ', H-5 '), 6.99 (1H, dd, J = 8.0Hz, J '= 2.5Hz, H-6 "), 6.90 (1H, d, J = 8.5Hz, H-7") , 6.42 (1H, d, J = 16.0Hz, H-2), 4.92 (2H, s, H-7'a, H-7'b), 4.24 (3H, s, = NOCH 3), 3.72 (3H , s, -OCH _3). ^{13 CNMR (125MHz, DMSO-d} 6, ppm): δ 162.63, 162.25, 155.32, 143.41, 137.76, 137.40, 136.82, 134.09, 127.79, 127.73, 119.12, 117.61, 115.57, 113.80, 110.40, 64.60, 55.69, 42.42. Anal. Calcd. _{For C 20 H 19 N 3 O} 5 (381.38): C, 62.99; H, 5.02; N, 11.02. Found: C, 63.06; H, 5.10; N, 10.91.

( E ) -3- (4 - ((( Z ) -7-chloro-3- (methoxyimino) -2-oxoindolin-1-yl) methyl) N - hydroxypropenamide (compound 6g)

Pale yellow solid; Yield: 75%. mp: 203-204 [deg.] C. _{R f = 0.39 (DCM: MeOH} : AcOH = 90: 5: 1). IR (KBr, cm ^-1 ): 3307 (NH), 3204 (OH), 3018 (CH, aren), 2938 (CH, CH ₂ ), 1722, 1662 (C = O), 1624, 1601, 1514, 1470 (C = C), 1442 (CN). ESI-MS (m / z) : 383.3 [M-2H] -. ^{1 H-NMR (500MHz, DMSO} -d 6, ppm): δ 10.74 (1H, s, NH), 9.01 (1H, s, OH), 7.98 (1H, d, J = 7.5Hz, H-4 ") , 7.51 (2H, d, J = 8.0Hz, H-2 ', H-6'), 7.44-7.41 (2H, m, H-3, H-6 "), 7.25 (2H, d, J = 8.0 Hz, H-3 ', H -5'), 7.12 (1H, t, J = 8.0Hz, H-5 "), 6.42 (1H, d, J = 16.0Hz, H-2), 5.28 (2H, s, H-7'a, H- 7'b), 4.27 (3H, s, -NOCH 3). ^{13 CNMR (125MHz, DMSO-d} 6, ppm): δ 163.12, 162.69, 141.91, 139.06, 138.74, 137.85, 134.68, 133.70, 127.76, 126.50, 126.44, 124.49, 118.94, 117.98, 114.79, 64.95, 44.14. Anal. Calcd. For C ₁₉ H ₁₆ ClN ₃ O ₄ (385.80): C, 59.15; H, 4.18; N, 10.89. Found: C, 59.18; H, 4.21; N, 10.84.

3. Cytotoxicity analysis of cancer cell lines for evaluation of antitumor activity

Human cancer cell lines from SW620 (colorectal cancer cells), PC3 (prostate cancer cells), and AsPC-1 (pancreatic cancer cell) cell lines were purchased from a cancer cell bank in the Korea Research Institute of Bioscience and Biotechnology (KRIBB). The medium, serum, and other reagents used for cell culture were purchased from GIBCO Co. Ltd. (Grand Island, New York, USA). Cells play them in a 96-well plate at a concentration of 9 x 10 ³ cells / well plated and treated with samples for 48 hours and incubated overnight. Compounds to be tested were dissolved in dimethylsulfoxide (DMSO) and used. Cytotoxicity was measured by a method [20, 22-23] in which a slight modification was made to the contents described in the known document [21]. IC ₅₀ values were calculated according to the Probits method [24]. The values measured for each compound were expressed as the average value of three independent measurement results.

4. Protein analysis for evaluation of HDAC inhibitory activity (Western blotting analysis)

Cells were lysed in RIPA buffer (50 mM TrisCl pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% SDS, and 1 mM phenylmethylsulfonyl fluoride) . Protein concentrations in the lysates were determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories Inc., Hercules, Calif., USA). Samples were separated on SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were incubated with blocking buffer (TBS containing 0.2% Tween-20 and 3% skim milk) and then detected using primary antibodies against acetyl histone-H3, -H4, and GAPDH. After washing, the membranes were detected using horseradish peroxidase-conjugated secondary antibodies. Detection was performed using an enhanced chemiluminescent protein (ECL) detection system (Amersham Biosciences, Little Chalfont, UK).

5. HDAC2 enzyme analysis

The HDAC2 enzyme was purchased from BPS Bioscience (San Diego, Calif., USA), and HDAC enzyme activity was analyzed using a fluorescent HDAC assay kit (BPS Bioscience) according to the manufacturer's instructions. Briefly, HDAC2 enzymes were incubated with various concentrations of KBH-A42 or vehicle in the presence of HDAC fluorescent substrate for 30 min at 37 &lt; 0 &gt; C. The HDAC assay developer, which produces fluorophore in the reaction mixture, was added and fluorescence was measured at 360 nm excitation wavelength and 460 nm emission wavelength using VICTOR3 (PerkinElmer, Waltham, Mass., USA). The measured activity was subtracted from the vehicle-treated control enzyme activity and IC ₅₀ values were determined using GraphPad Prism (GraphPad Software, San Diego, Calif., USA).

6. Docking Research

A docking study was performed using the AutoDock Vina program [25] (The Scripps Research Institute, CA, USA). The initial structures of HDAC2 [26] that form a complex with SAHA were obtained from the Protein Data Bank (PDB) (PDB ID: 4LXZ) and the coordinates for the compounds were GlycoBioChem PRODRG2 Server (http://davapc1.bioch.dundee.ac .uk / prodrg /) [27]. The grid map for the docking study was located at the center of the SAHA binding site, and after removing the SAHA from the composite structure, it was 26 x 26 x 22 points at intervals of 1.0 [17-20]. The AutoDock Vina program is run with eight-way multithreading, and the other parameters are set as defaults in the AutoDock Vina program.

Ⅱ. Results and discussion

1. Synthesis of Compound

The synthesis of N-hydroxypropenamide (3a-3g) is illustrated in Reaction Scheme 1 below. In the first step, isatin and its 5-or 7-substituted derivatives are reacted with an equivalent amount of methyl 4-bromomethyl cinnamate in dimethyl formamide (DMF) with a catalytic amount of KI under alkaline conditions (K ₂ CO ₃ ) To give the cinnamate intermediate 2a-2h as a brown-yellow oil in good overall yield (85-96%). Subsequently, the final propeneamide was produced in high yield by nucleophilic acyl substitution of cinnamate 2a-2h with hydroxylamine under basic conditions.

The 3-methoxyimino-2-oxoindoline N-hydroxypropenamide (6a-6g) was also first treated with isatin 1a-1g in anhydrous pyridine under refluxing conditions using methoxylamine hydrochloride to give 3- 2-oxoindole derivative 4a-4g (see Reaction Scheme 2 below).

The structure of the final products was clearly confirmed using spectroscopic methods such as IR, MS, ¹ H NMR and ¹³ C NMR. Hydroxylamine reacted on both the ester and the 3-oxo group on the indolin side of the ester compound 2a-2g to give 3-hydroxyimino-2-oxoindoline N-hydroxypropenamide (3a-3g) . This was evident from the mass spectroscopic data of compounds 3a-3g and the downfield shift of H-4 '(delta 8.01-7.78 ppm) in ¹ H NMR spectrum. The down-field shift of H-4 indicated the presence of the hydroxyimino group at position 3 of the indole ring [20]. In addition, in the ¹ H NMR spectrum of compounds 3a-3g, generally measured in DMSO- d ₆ , three broad singlets appeared near δ 9.02, 10.75, and 13.50-13.80 ppm, -NH (hydroxamic acid moiety), and -OH (3-hydroxyimino moiety on indole ring). In the ¹³ C NMR spectrum of the compound 3a-3g, no peak corresponding to the 3-oxo carbon of the indole ring [20] which should appear near δ 184.00-188.00 ppm was found. These evidences further confirmed the presence of 3-hydroxyimino group on the indoline moiety. The simultaneous reaction of the hydroxyl group with the ester group and the 3-oxo group of indole cyclic has been observed under the same previous conditions [20]. Efforts to reduce the molar amount of hydroxylamine. HCl to maintain the 3-oxo group intact were unsuccessful. When used in a 1: 1 molar equivalent to the corresponding ester intermediate, a mixture of many products was observed with unreacted starting material (esters), making it impossible to separate the target product.

[Reaction 1]

Reaction 1 is a synthetic reaction scheme of 3-oxime-2-oxoindoline-based N-hydroxypropenamide. The reagents and conditions are as follows: a) Methyl 4-bromomethyl cinnamate, K ₂ CO ₃ , KI, DMF, 60 ° C, 24 hrs; b) NH ₂ OH.HCl, NaOH, MeOH / THF, -5 ° C, 30-60 min.

[Reaction 2]

Reaction 2 is a synthetic reaction scheme of 3-methoxycin-2-oxoindoline-based N-hydroxypropenamide. The reagents and conditions are as follows: a) NH ₂ OCH ₃ .HCl, pyridine, DMF; b) Methyl 4-bromomethyl cinnamate, K ₂ CO ₃ , KI, DMF, 60 ° C, 24 hrs; c) NH ₂ OH.HCl, NaOH, MeOH / THF, -5 ° C, 30-60 min.

2. Measurement of biological activity

The inhibitory activity of histone-H3 and histone-H4 was evaluated using Western blot analysis for all of the synthesized hydroxamic acids (compounds 3a-3g, 6a-6g). Since histone deacetylation is generally determined by HDACs in general, the HDAC inhibitory effect of compounds can be assumed to be the ability to inhibit histone deacetylation levels. As shown in Figure 3, in the presence of the known HDAC inhibitor SAHA, the levels of acetyl-histone H3 and acetyl-histone H4 were strongly elevated, suggesting that HDAC is strongly inhibited and the acetyl groups on histone H3 and histone H4 It can not be removed. Similarly, the levels of acetyl-histone H3 and acetyl-histone H4 clearly appear in the presence of compounds 3a, 3b, 3d-f, 6a-c, 6d and 6e, Implying the fact. Acetyl-histone H3 and acetone-histone H4 were still observed in the presence of compound 3c, but at a much lower level. Thus, compound 3c possessed activity but was weaker than the above compounds. In contrast, the levels of acetyl-histone H3 and acetyl-histone H4 were scarcely observed in the presence of 3 g of compound and 6 g of compound, indicating that HDACs still have activity and deacetylated histone-H3 and histone- . These results suggest that substitution with chlorine and possibly other substituents at position 7 is not desirable for biological activity, while at position 5 it is tolerable for various substituents.

In contrast, Compound 3c showed strong anticancer activity with an IC ₅₀ value of 0.086 μM in HDAC2 assay, but weak activity in Western blot analysis. Since histone-H3 and histone-H4 are substrates of HDAC2 and HDAC3 [28], HDAC3 did not appear to be still inhibited by compound 3c. A similar explanation may be applied to compounds 3g, 6d, and 6g.

Next, anti-proliferative activity against cancer cells was evaluated using SRB (sulforhodamine B) cell proliferation assay on the synthesized compounds. First, compounds were screened for cell growth inhibitory activity at 30 [mu] M against SW620 (human colon cancer) cell line. All compounds 3a-3g and 6a-6g completely inhibited the growth of SW620 cells at this concentration. Thus, the compounds were also tested for inhibition of cell growth on SW620 cell lines and other human cancer cell lines PC-3 (proliferative adenocarcinoma) and AsPC-1 (pancreatic cancer) cell lines at five additional concentrations (30, 10, 3, 1, . The IC ₅₀ values (concentration causing 50% inhibition of cell proliferation) of each compound were measured and summarized in Table 1 below. SAHA was used as a positive control.

The data in Table 1 show that all compounds except compound 6f exhibited equivalent or superior anticancer activity compared to SAHA in terms of cytotoxicity against the analyzed 3 week cancer cell lines. Also, in all three cancer cell lines tested, compounds 3a and 3b exhibited equivalent cancer cell cytotoxicity indicating that methylation at oxime in compound 3a was tolerable for cytotoxicity. Substitution of electron withdrawing groups (-F, -Cl, -Br) (compounds 3b-3d, 6b-6d) at position 5 on the indole site appeared to increase cytotoxicity, while substitution at position 7 3g and 6g) appeared less desirable in biological activity. Of the electron donor groups (-CH ₃ , -OCH ₃ ), only the methyl group slightly increased cytotoxicity to the cancer cells (compounds 3e, 6e vs. compounds 3a, 6a), while the methoxy group did not Compound 3f). The simultaneous presence of a 5-methoxy substituent and methoxime on the indole ring further reduced cytotoxicity (compound 6f). These results imply that the biological activity of these compounds may be very sensitive to the bulkiness of the substituents on the indole moiety.

Relevant good correlations were found within the series compounds 3a-3h with regard to the association between cytotoxicity against cancer cells and inhibitory effects of compounds on HDAC2 or histone-H3 and histone-H4 deacetylation. Compounds 3a-3f, which exhibited activity in Western blotting assays and exhibited an excellent inhibitory activity against HDAC2 activity, were tested against (3b-3e) for all 3 cancer cell lines for 3 weeks or against cancer cells for at least 2 weeks in 3 cancer cells Compounds 3a and 3f) exhibited significantly superior cancer cell cytotoxicity. On the other hand, 3 g of compounds that were inactive in Western blot analysis and showed the highest IC ₅₀ values for HDAC 2 were the compounds showing the weakest cancer cell cytotoxicity in compound series 3a-3g. In compound series 6a-6g, a similar correlation was observed except for compound 6d. Compound 6d did not show activity in Western Blot analysis and showed a relatively high IC ₅₀ value for HDAC2 (1.320 [mu] M). However, in an analysis of the three week cancer cell line, the compound showed potent cancer cell cytotoxicity. Other types of HDAC enzymes are affected by compound 6d and still lead to cell growth inhibition as a whole. Of course, other than HDAC inhibition, other cytotoxic mechanisms by compound 6d can not be excluded. Therefore, these compounds have a complete correlation between HDAC inhibition and cancer cell cytotoxicity, and additional experimentation is required for the remaining HDAC subtypes. Nonetheless, the 3-substituted 2-oxoindole-based N-hydroxypropene amides of the present invention are promising as potent HDAC inhibitors and cytotoxic agents.

In Table 1 superscript 1 was calculated with ChemDraw 9.0 software; Superscript 2 is the concentration of compounds that reduce enzyme activity or cell growth by 50% ([mu] M), and the number is the average result of triplicate experiments with an error of less than 10%; Superscript 3 is SW620, respectively; colon cancer; PC3, prostate cancer; AsPC-1, a cell line of pancreatic cancer; Superscript 4 is a suberoylanilide acid (SAHA) as a positive control.

3. Docking Study Results

In order to study the mechanism for the interaction between the synthesized compounds and HDAC, docking experiments were performed using HDAC active sites. Because histone-H3 and histone-H4 deacetylation is mainly regulated by HDAC2 and HDAC3 [28], the structure of HDAC2 complexed with SAHA as a docking template was selected. The crystal structure of HDAC2 as a complex with SAHA has been reported by Lauffer and co-workers [26]. Control docking experiments were performed with SAHA on the crystal structure of HDAC2 using the AutoDock Vina program [25] after SAHA was removed from the composite structure [17-20], as is known in the prior art. As a result of the docking experiments, it was confirmed that all the synthesized compounds were located in the active sites with higher binding affinity than SAHA. The stabilization energies of these compounds were -8.3 kcal / mol to -9.4 kcal / mol, Had a low value. For example, the expected stabilization energies for the coupled mode for HDAC2 were calculated to be -9.0 kcal / mol and -8.6 kcal / mol for compounds 3b and 6b, respectively, whereas the stabilization energy for SAHA was -7.4 kcal / mol (the rmsd distance from the original SAHA in the crystal structure is 0.609 / 2.056). As a result of docking experiments, zinc ions (gray spheres) were coordinated by Asp181, His183 and Asp269, three residues of HDAC2. All of the synthesized compounds combined with zinc ions in a manner similar to that of SAHA. It was confirmed that, in all the compounds, benzene connecting the indoline and the N-hydroxypropenamide moiety was tightly sandwiched between the Phe155 and Phe210 of the enzyme (see FIG. 4), and the interaction by this intercalating structure Lt; RTI ID = 0.0 &gt; HDAC2. &Lt; / RTI &gt; However, in these docking experiments, the indolin moiety appeared to interact negatively with the enzyme. Thus, the binding affinities of the compounds having different substituents were almost unchanged. For example, the predicted stabilization energies of 3g and 6g of the binding mode for HDAC2 were -8.9 kacl / mol and -8.4 kcal / mol, respectively. These values were almost similar to those of compounds 6b and 6g. Thus, the combined difference stabilization energy is difficult to explain the 5-fold difference in the compound 3b and compounds 3g of the IC ₅₀ value for the IC 3 times the ₅₀ value difference or compound 6b and Compound 6g in HDAC2 enzyme inhibition assay (Table 1). Nonetheless, the binding stabilization energies of the compound 6g were much lower than those of SAHA (-8.4 kcal / mol vs. -7/4 kcal / mol, respectively), which is comparable to that of SAHA (IC ₅₀ , 1.840 μM) for HDAC2 By comparison, a relatively high inhibition of this compound (IC ₅₀ , 1.399 [mu] M) could be accounted for.

Ⅲ. conclusion

The present inventors have synthesized two series of compounds of 3-substituted-2-oxoindoline-based N-hydroxypropenamide with strong HDAC inhibitory effect and strong cancer cell cytotoxicity. As is clear from the results obtained from the above experimental results, various substituents were introduced into both the 5-position and the 7-position of 3-hydroxyimino-2-oxoindoline or 3-methoxyimino-2-oxoindoline, The biological activity remained intact.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

A compound represented by the following formula (1):
[Chemical Formula 1]

Wherein R ₁ is hydrogen or methyl, and R ₂ is hydrogen, halogen, C _1-6 alkyl, or C _1-6 alkoxy.
The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the halogen is fluorine (F), chlorine (Cl) or bromine (Br).
The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is any one of the following compounds:
( E ) -N -hydroxy-3- (4 - ((( Z ) -3- (hydroxyimino) -2-oxoindolin-1-yl) methyl) phenyl) propenamide;
(E) -3- (4 - ( ((Z) -5- fluoro-3- (hydroxyimino) -2-oxo-in turned-yl) methyl) phenyl) - N - hydroxy-propenamide ;
( E ) -3- (4 - ((( Z ) -5-chloro-3- (hydroxyimino) -2-oxoindolin-1-yl) methyl) phenyl) -N -hydroxypropenamide;
(E) -3- (4 - ( ((Z) -5- bromo-3- (hydroxyimino) -2-oxo-in turned-yl) methyl) phenyl) - N - hydroxy-propene is mid;
( E ) -N -hydroxy-3- (4 - ((( Z ) -3- (hydroxyimino) -5-methyl-2-oxoindolin-1-yl) methyl) phenyl) propenamide;
( E ) -N -hydroxy-3- (4 - (( Z ) -3- (hydroxyimino) -5-methoxy-2- oxoindolin- ;
( E ) -3- (4 - ((( Z ) -7-chloro-3- (hydroxyimino) -2-oxoindolin-1-yl) methyl) phenyl) -N -hydroxypropenamide;
( E ) -N -hydroxy-3- (4 - ((( Z ) -3- (methoxyimino) -2-oxoindolin-1-yl) methyl) phenyl) propenamide;
( E ) -3- (4 - (( Z ) -5-fluoro-3- (methoxyimino) -2-oxoindolin- 1-yl) methyl) phenyl) -N -hydroxypropenamide ;
( E ) -3- (4 - (( Z ) -5-chloro-3- (methoxyimino) -2-oxoindolin-1-yl) methyl) phenyl) -N -hydroxypropenamide;
(E) -3- (4 - ( ((Z) -5- bromo-3- (methoxy toksiyi mino) -2-oxo-in turned-yl) methyl) phenyl) - N - hydroxy-propenamide ;
( E ) -N -hydroxy-3- (4 - ((( Z ) -3- (methoxyimino) -5-methyl-2-oxoindolin-1-yl) methyl) phenyl) propenamide;
( E ) - N -hydroxy-3- (4 - (( Z ) -3- (methoxyimino) -5-methoxy- ; And
( E ) -3- (4 - (( Z ) -7-chloro-3- (methoxyimino) -2-oxoindolin-1-yl) methyl) phenyl) -N -hydroxypropenamide.
An anticancer pharmaceutical composition comprising the compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof as an active ingredient.
The method of claim 4, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or ocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer, Wherein the cancer is cancer, endometrial cancer, cervical cancer, small bowel cancer, endocrine cancer, thyroid cancer, pituitary cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer or bone cancer.
5. The anticancer pharmaceutical composition according to claim 4, wherein the compound or a pharmaceutically acceptable salt thereof has an activity of promoting histone acetylation through inhibition of histone deacetylase.
Process for preparing 3-hydroxyimino-2-oxoindoline-based N -hydroxypropeneamide-based compounds of the compounds of claims 1 to 3, comprising the steps of:
(i) reacting a compound of the formula (a-1) with methyl 4-bromomethylcinnamate under basic conditions to produce a compound of the formula (a-2) And
[Formula (a-1)]

[Formula (a-2)]

(Ii) reacting the resulting compound of formula (a-2) with hydroxylamine to produce a compound of formula (a-3).
[Formula (a-3)]

In the above formulas (a-1) to (a-3), R is hydrogen, halogen, C _1-6 alkyl or C _1-6 alkoxy.
Process for preparing 3-methoxyimino-2-oxoindoline-based N -hydroxypropeneamide-based compound of any one of claims 1 to 3, comprising the steps of:
(i ') reacting a compound of formula (a-1) with methoxylamine hydrochloride under basic conditions to produce a compound of formula (a-4);
[Formula (a-1)]

[Formula (a-4)]

(Ii ') reacting the resulting compound of formula (a-4) with methyl 4-bromomethylcinnamate to produce a compound of formula (a-5); And
[Chemical formula (a-5)]

(Iii ') reacting the resulting compound of formula (a-5) with hydroxylamine to produce a compound of formula (a-6).
[Formula (a-6)]

In the above formulas (a-1) to (a-6), R is hydrogen, halogen, C _1-6 alkyl or C _1-6 alkoxy.

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