TW201141836A - Histone deacetylase inhibitors - Google Patents

Abstract

This invention relates to novel Histone deacetylases inhibitors. Also disclosed is a method for treating mucositis or cancer with these inhibitors.

Description

201141836 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a novel tissue protein deacetylase inhibitor, and a method for treating mucosal ulcer and cancer. [Prior Art] Since tissue protein deacetylase inhibitors have the potential to treat cancer and inflammation, they are currently receiving extensive attention. Tissue protein deacetylase inhibitors inhibit cancer cell growth while promoting cancer cell differentiation and cells; Lu et al., */· Merf. CAem. 2005, 48, 5530-5535; Kulp et al. , Clin. Cancer Res. 2006, 12, 5199-5206; and Ryan et al., J. Clin. Onclo. 2005, 23, 39 1 2-3922.). At the same time, it has been reported that this deacetylase inhibitor inhibits the expression of pro-inflammatory cytokines, thereby slowing down the inflammatory response, such as mucosal ulcers caused by cancer chemotherapy or radiation therapy (Ref. LM Adcock, British Journal of Pharmacology, 2007, 150(7): 839-83 1; and YL Chung et al., Carcinogenesis 2009 30(8): 1387-1397.). Tissue protein deacetylase inhibitors are currently receiving considerable attention due to their potential to treat cancer and inflammation. SUMMARY OF THE INVENTION The present invention is based on the discovery that certain amine-containing compounds are effective tissue protein deacetylase inhibitors and have an activity of inhibiting mucosal ulcers. Therefore, the present invention relates to specific amine-containing compounds and these compounds Tissue egg 201141836 Application of white acetylase-related diseases. In the first aspect, the invention has the structure of the formula (1):

Formula (1) 虱, alkyl, hydroxy or R is optionally substituted

Wherein 1^, 112, 113, 114, 115, and 尺6 are each independently a hydroalkoxy group; R7 is a hydroxy group, OR, or an alkyl group of 〇C(〇)R, R, the substituent of which is dentate, An aryl group, a heterocyclic aryl group, an amine group, or a carboxylic acid group; R8 is hydrogen, (CH2)mNHC(0)R,) or (CH2)m〇c(〇)R, m is 0, 1, or 2, R' is an optionally substituted alkyl group, the substituent of which is a halogen, an aryl group, a heterocyclic aryl group or a carboxylic acid group. With respect to formula (I) 'where a series of compounds have one or more of the following characteristics: R7 is a substituted OC(〇)R, R is an alkyl group having an NH2 substituent or a benzene substituent; and R, R2 R3, r4, Rs and R6 are each nitrogen, or Ri, R2 and R3 are each hydrogen, I is a hydroxyl group, and Rs and R6 are each an alkyl group. Another aspect of the invention comprises a compound having the structure of formula (II):

Wherein R is hydrogen, hydroxy, amine, alkoxy, arylalkoxy (eg benzyloxy), alkyl thioloxy, or alkoxycarbonyloxy; R 2 is hydrogen, hydroxy , alkoxy, alkoxy, alkoxycarbonyl; r3 is hydroxy or phenyl; X is oxygen 201141836 or NH; Y is phenyl or -CH2; wherein m and η are independently 〇^ 2, 3, 4 or 5 with respect to formula (II) wherein a series of compounds may have one or more of the following characteristics: X is NH or oxy; Y is phenyl or _CH2-; m is 〇, n is 3 R is a hydroxy or alkoxy group; and R3 is a hydroxy group. In a third aspect, the invention comprises a compound having the structure of formula (111): nr2 wherein (X is NH or oxy; R1 is hydroxy, alkyl or aryl; one of r2 and R3 is c(o)〇 R, R is an alkyl group, an alkynyl group, or an aromatic group, and the other is hydrogen or C(〇)〇R, 'R, which is an alkyl group, an alkynyl group, or an aromatic group. With respect to formula (III), wherein a series of compounds have one or more of the following characteristics: X is oxygen or NH, R1 is a benzyl or hydroxy group; and the heart and R3 are each (t-butyloxy)carbonyl (B0C) or (9-fluorenylmethyl)carbonyl (Fm〇c). "Alkyl", unless otherwise indicated, refers to a straight, branched or cyclic saturated hydrocarbon chain containing from 1 to 20 carbons such as, but not limited to, decyl, ethyl, propyl, isopropyl, The butyl, isobutyl 't-butyl, or cyclohexyl" alkyl group may contain one or more heteroatoms (eg, oxygen, gas, sulfur, or selenium) and/or - to multiple double or triple bonds. , aromatic group, refers to (monovalent) monovalent monocyclic, decacyclic bicyclic, tetradecane tricyclic and other aromatic rings, such as benzene ring, Cai ring, onion ring and others., heterocyclic aryl group Refers to one or more heteroatoms (eg, oxygen, I, sulfur or, monovalent aromatic five to eight it single ring or eight to ten - 5 201141836 7US: difficult 5 brothers a 54-- king T four 7G three rings System, such as n bite, biting, squat, benzomethane, pyrimidine, thiophene, quinoline 'carbazole, tetrazole, thiazole and others. i. Derivatives: aryl and heterocyclic aryl Containing substituted and unsubstituted groups. Possible substituents on amine, cycloalkyl, heterocycloalkyl, cycloalkynyl, heterocyclocycloalkynyl, aryl and heterocyclic aromatic groups But not limited to C|_Cl〇 alkyl, C2_C10 alkenyl, c2-c10 alkynyl, c3-c2〇 cycloalkyl, CVCm cycloalkenyl, Ci_c2() heterocycloalkyl, C|_CH Heterocyclenyl, C^-Cm alkoxy, aryl, aryloxy, heterocyclic aryloxy, amine, (:,-(:1()alkylamino, arylamine Base, hydroxyl group, halogen group, divalent oxy group (〇=), divalent sulfur (s=), thiol group, thiol group, Ci_Ci 〇 alkyl thiol group, aryl thiol group, C1_CIG alkyl sulfonic acid Base, arylsulfonic acid group, decylamino group, amine sulfhydryl group, aminothiol group, methionyl group, thiol group, decylamino group, thiourea group, thiocyano group, sulfonamide group, sulfhydryl group, ureido group a cyano group, a nitro group, a decyl group, a thiol group, a decyloxy group, a ureido group, an amine carbaryl group, a carboxylic acid group, and a carboxylate group. On the other hand, an alkyl group, an alkenyl group and an alkynyl group The substituent includes all of the above substituents other than the Ci-Cio^: group. The cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl and heterocyclic aromatic groups may be fused to each other. "Oxygen" refers to -oxy-alkyl groups. Examples include but are not limited to An oxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, an isobutoxy group, a second butoxy group, a third butoxy group. The "amino group" refers to an NH2 'amine group, Aromatic amine group. "Alkylamine" refers to -N(R)-alkyl' wherein R can be hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, hetero a cycloalkenyl group, an aromatic 201141836 aryl group and a heterocyclic aryl group, an 'arylamino group, meaning a nal group', an aromatic group, wherein R may be hydrogen, alkyl, alkenyl, alkyl, cycloalkyl, A cycloaliphatic group, a heterocyclic alkyl group, a heterocyclic group, an aryl group, or a heterocyclic aryl group. The compounds described in the present invention comprise the compound itself, a salt thereof, a melt, and a prodrug. For example, the salt may be a compound formed of an anion and a positively charged group such as an amine group. Suitable anions include chloride, desert, breaking, sulfate, hydrogen sulfate, amine acid, lithate, citrate, sulphate, methionate, triacetate acetate Ions, amphoteric acid ion, grape domain acidate ion, glutarate ion, malate ion, maleate ion, alkanoate ion, fumarate ion, tartrate ion, toluene acid ion , salicylate ion, lactate ion, naphthalene «root ion and acetate ion. The same 'salt may be. The orthoquinone or dihydrogen is formed on the acid compound from a cation and a negatively charged group (e.g., a slow acid ion). Suitable cations include sodium ions, clock ions, town ions, about ions, ammonium (e.g., tetramethylammonium) ions. The above compounds simultaneously contain salts formed by quaternary nitrogen. Examples of prodrugs, including vinegars and other pharmaceutically acceptable derivatives, can provide the above-described active compounds when they are administered at the subject. Exemplary compounds of the invention are listed below: 201141836

ΝΗΟΗ OH Ο

化合物 Compound 1 Compound 2 ΒηΟ ΒηΟ

ΝΗΟΗ ΝΗΟΗ

ΝΗΟΗ

ΝΗΟΗ 〇

ΝΗΟΗ ΒηΟ

ΝΗΟΗ

Β ΒηΟ, ΝΗ2

ΝΗΟΗ ΝΗΟΗ ΒηΟ

ΝΗΟΗ The present invention relates to a method for inhibiting tissue proteins by contacting any of the above compounds with cells at a dose effective to have a 201141836 deacetylase (HDAC) activity. In still another aspect, the present invention relates to a method of administering a therapeutically effective dose of any of the above compounds to a mammal, a mucosal ulcer, and a cancer treatment. The present invention also encompasses a box of medicinal compositions of the above-mentioned compounds of the right-recorded, αα^*3, which are used for the treatment of mucosa, scorpion or cancer; Therapeutic use of ulceration or cancer, and the use of the agent for the treatment of the drug, the main + ^ 迂 and / 〇縻 mucosal ulcer or cancer. . Details of one or more specific embodiments of the invention are detailed below. The remaining features, objects, and advantages of the invention will be apparent from the description and appended claims. [Embodiment] The compound of the present invention can be synthesized by a known method. For example, the above compounds 1 to 15 can modify the butyric acid, the lysine and the valine by a conventional chemical conversion method (including the use of a protecting group). Reference can be made to the following documents: R. Larock, Com/?re, ewjive Or Rong flwz.c Transformations, NCY{ Publishers (1989); TW Greene and PGM Wuts, Protective Groups in Organic Synthesis Ed., John Wiley and Sons ( 1999); L. Fieser and M.

ViQSQr, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L.

Vaquette, &d., Encyclopedia of Reagents for Organic 201141836 _M〇hn Wiley and s〇ns (1995) and more recent versions of these documents 0 • Synthetic compounds can be passed through fast column chromatography, high performance liquid chromatography Purification by methods, recrystallization, or other suitable means. The compounds referred to herein contain non-aromatic double bonds and one or more symmetry centers. Such compounds may be racemates, racemates' single mirror phase isomers, individual non-mirrible isomers, mixtures of non-mirror isomers, and cis or trans isomers, All isomers are encompassed by the present invention. The scope of the present invention includes (1) a pharmaceutical composition comprising at least one of the compounds of the present invention and a pharmaceutically acceptable carrier, and (2) a method for treating mucosal ulcers, which is required by the treatment of (4) An effective dose of the compound to a body. "Treatment, as used herein, refers to the administration of an active compound to an individual suffering from mucosal ulcers or cancer' or an individual with an onset of symptoms or a tendency to cure, heal, slow down, toward the discomfort, symptoms or tendency of mucosal ulcers or cancer. Relief, change, shortness, improvement, promotion, influence, or risk reduction. "Effective dose" refers to the amount of effective compound required to achieve the desired therapeutic effect on an individual. The effective dose may vary, as will be appreciated by those skilled in the art, depending on the route of administration, the application of the excipient, and the possible combination with other agents. Mucositis is an inflammation and ulceration of the mucosa of the digestive tract. It is usually a side effect of cancer chemotherapy and radiation therapy, which may occur in the oral cavity or the gastrointestinal tract. Cancer contains solid tumors and blood tumors of different organs. Solid tumor 10 201141836 Contains pancreatic cancer, gallbladder cancer, colon cancer, breast cancer and breast cancer, prostate cancer (including male hormone-dependent and non-dependent prostate cancer), liver cancer, lung cancer (eg non-small) Cell lung cancer (NSCLC), bronchioles and alveolar carcinoma (bac) and lung adenocarcinoma), ovarian cancer (eg, progressive epithelial cancer and primary peritoneal cancer), neck cancer, stomach cancer, esophageal cancer, head and neck cancer ( For example, head and neck squamous cell carcinoma), melanoma, neuroendocrine carcinoma (including metastatic neuroendocrine carcinoma), brain cancer (including neuroglioma, degenerative oligodendroglioma), adult polymorphic malignant glioma, and adult degeneration Astrocytoma), myeloma, and soft tissue sarcoma. Hematological tumors include acute myeloid leukemia (AML); chronic myelogenous leukemia (CML) contains accelerated phase CML and CML acute conversion (CML-BP); acute lymphocytic leukemia (ALL); chronic lymphocytic leukemia (CLL) ;H〇dgkin's disease (HD); non-Hodgkin's lymphoma (NHL) 'containing follicular lymphoma and mantle cell lymphoma), B-cell lymphoma, T-cell lymphoma, Multiple myeloma, Waldenstrom's macroglobulinemia, myelodysplastic syndrome (MDS) 'contains resistant anemia (RA), ring-shaped lateral bud cell-resistant anemia (RARS), excess bud cell-resistant anemia (RAEB), And excessive bud cell refractory anemia and acute transformation (RAEB_T); and myeloproliferative syndromes. The method of the present invention is carried out by orally administering the above-mentioned pharmaceutical composition, or by parenteral administration, inhalation spray, area application, rectal suppository, nasal cavity and infusion, oral injection, vaginal administration, or via implantation. Into the storage tank. "Parenteral administration" refers to the route of administration through the subcutaneous, intradermal, intravenous, muscular, joint, arterial, synovial fluid, sternum, spinal canal, disease 201141836 disease, intracranial, etc. Injection or runoff administration. Sterile injectable compositions, e.g., sterile, aqueous solutions for injection or oils can be prepared from suitable dispersing agents or wet (e.g., butyl ween 80) and suspending agents used in the art. A sterile injectable solution or suspension in a sterile injectable solution, a silkworm I raw material, a gastrointestinal tract, a usable diluent or a solvent, butanediol, and a solvent or solvent containing mannitol, water, and a solution (Ringer's solution) and isotonic water. In addition, sterile, fixed oils are conventionally employed as a solvent or suspension medium (e.g., synthetic mono or diglycerides). Fatty acids, such as oleic acid and its glycerol derivatives, are useful in the preparation of sterile injectables, as are natural pharmaceutically acceptable oils such as eucalyptus oil, cannabis oil, and the like, especially their derivatives of polyoxyethylation. Things. The oil solution and suspension may be obtained by diluting or dispersing a long-chain alcohol or by diluting or dispersing 竣methylcellulose or other dispersing agent. Other commonly used surfactants such as one

Spans or other similar emulsifiers or accelerators commonly used to make bioavailability of pharmaceutically acceptable solid, liquid or other dosage forms can also be used on the formulation. Orally acceptable dosage forms, dispersions and solutions. Oral and corn house powder. Lubricating agent, in oral capsules, commonly used as a rare oral administration suspension for oral administration of the composition may be any capsule, lozenge, latex, aqueous suspension, tablet used. The usual carrier comprises lactose such as hard Magnesium oleate is a typical addition. The release agent comprises lactose and dried corn starch. In addition to the emulsification or emulsion, the active ingredient can be suspended or dissolved in the oil phase or in a suspension. |Special sweeteners, fragrances, or 12 201141836 stains can be added under certain requirements. Nasal or buccal inhalation of the spray component, j is prepared by methods known in the art of pharmaceutically acceptable dosage forms. The compound J of the present invention is used in the form of an intestinal suppository. The pharmaceutically acceptable carrier must be, acceptable, acceptable, and must be present in a particular dosage form in combination with the active ingredient (preferably to stabilize the active ingredient) without causing harm to the individual at the time of administration. One or more stabilizers (e.g., cyclodextrins) that form a more soluble complex with the compounds of the present invention can be used as a pharmaceutical carrier to deliver the active compound. Other carriers include cerium oxide sol, magnesium stearate, sodium lauryl sulfate, and D&c Yellow No. 10. Suitable in vitro assays can be used to initially assess the effectiveness of the compounds of the invention in inhibiting tissue protein deacetylase. Effective compounds can be further tested for their effectiveness in treating mucosal ulcers or cancer in vitro or in vivo. For example, a compound can be administered in an animal model with mucosal ulcers or cancer (e.g., in a mouse experiment), and his therapeutic effect can be evaluated. The appropriate dosage range and route of administration may be determined based on the results of the experiment. The above list is considered to be sufficient to illustrate the invention. The following examples are for illustrative purposes only and are not limiting as to what is disclosed elsewhere. The published literature cited herein is incorporated herein by reference in its entirety. Example 1: Chemical Synthesis 1. N-Hydroxy-4-Phenylbutyramine (Synthesis of Compound 13 13 201141836

Phenylbutyric acid Compound 1 4-butyric acid (10·0 g, 60.9 mmol) was dissolved in anhydrous tetrahydroanthracene. After South (18 mL), ethyl acetoacetate (7.0 mL, 73_1 mmol) and triethylamine (dichloromethane, 79.2 mmol) were added and stirred at 0 ° C for 15 min. After the solid was filtered, the prepared methanolic hydroxylamine solution (63.0 mL, 6.34 g or 91.4 mmol of hydroxylamine hydrochloride and 5.13 g or 91.4 mmol of hydrogen hydroxide were dehydrated in methanol (30 mL) and mixed for 15 minutes) . The reaction was carried out at room temperature for 3 minutes, and the solvent was evaporated under reduced pressure. <EMI ID=9.1> % (所,2H,

PhCH2-CH2-CH2CONHOH), 2.13 2H

Ph-CH2-CH2CH2CONHOH), 2.64 {t, 2H,

PhCH2CH2-CH

PhCH2CH2-CH2-CONHOH)5 7.15 2H, Ar-H), 7.20 (., 2H

Ar-H), 7.28 (m, 1H, Ar-H), 8.20 (h, iH, NH). 2. Synthesis of 3-dihydroxy-2,2-dimercapto-4_ albendamine (Compound 2)

Compound 2 14 201141836 Zinc powder (〇·85 g, 1.0 mmol) and ethyl acetate (10.0 mL) were added to a Wolff bottle equipped with a distillation apparatus. After the mixture was heated to 40 ° C, it was slowly added dropwise to a-bromoisobutyl bromide (3 45 g, i6.0 mm〇i) and collected at 34 C to obtain a yellow liquid of dimethylpropan-1- Keto-1-one. Dissolve monomethylpropan-1-yl-1-one (〇·〇4 g, 5.0 mmol) with 2-phenylethylidene (〇·60 g' 5.0 mm〇i) in ethyl acetate (3 5 mL) And after the 〇0C was stirred under nitrogen, the zinc (II) (〇.〇3 g, 0.25 mmol) was quickly added, and the reaction mixture was maintained at 〇. (: a short time followed by overnight at room temperature (monitored by TLC). The reaction mixture was treated with aqueous sodium bicarbonate at room temperature. The organic layers were combined, dried and concentrated in vacuo, using dioxane: n-hexane =1: 1 crystallized to give 4-mercaptobenzene-3,3-dimercaptooxycyclobutan-2-one (0.80 g, 84%). 4-Mercaptobenzene-3,3-dimethyl Oxycyclobutane-2-ketone (0.95 g, 5.0 mmol) was dissolved in decyl alcohol (2.0 mL) under a nitrogen atmosphere. (: stirring, then a solution of hydroxylamine hydrochloride dissolved in methanol (40.0 mL). 〇7 g, 1 〇mm〇l) and potassium hydroxide (0_06 g, 10.0 mmol). The reaction mixture was stirred at room temperature and then was taken to EtOAc EtOAc. %). Melting point: 95-96 ° C; !NMR (400 MHz in CDC13,

Bruker AVANCE-400): δ 1.31 {s, 3H, CH3)} 1.32 (s, 3H, CH3), 2.5 5 {dd, \H, J = 10.0Hz, CH2), 2.82 (dd, \U, J = 1.8 Hz, CH2), 3.95 {dd, 1H, J = 2.1Hz), 6.36 (br, 1H, OH), 7.23-7.25 (m, 3H, Ar-H), 7.30-7.33 (m, 2H, Ar- H) 〇3·(R)_Tertibutyl 3-(5-(benzyloxy)°pyridin-2-yl)-1-(4-(4-(hydroxylamine 15 201141836))-4-oxetane Synthesis of thiol-oxypropyl-2-indolecarboxylic acid vinegar (Compound 3)

Synthesis of compound 3 (1) 4-(4-nitrophenyl)butyric acid methyl ester and 4-(2-nitrophenyl)butyric acid decanoate 4-(p-nitrophenyl)butyric acid and 4-(ortho-nitrite A mixture of butyric acid (3.22 g, 15.38 mmol) was dissolved in a saturated aqueous solution of methanol (6 mL) and heated to reflux for 24 hours (monitored by TLC). The solvent was removed and then di-methane (5 mL) was added to dissolve the residue. The product was washed with a saturated aqueous solution of sodium carbonate. The methylene chloride solution was separated and then evaporated to dryness <RTI ID=0.0> Preparation of methyl butyrate a mixture of decyl 4-(4-nitrophenyl)butanoate and decyl 4-(2-nitrophenyl)butanoate (2.24 g, 10.03 mmol) with 0.2 g of 1 〇 % pd/c (0.2 g) was dissolved in methanol (40 mL) and stirred vigorously under a hydrogen atmosphere. The mixture was stirred at room temperature for 4 hours (monitored by TLC) and then removed by C e ite transition. The solvent 'residual' was purified by chromatography to give the title compound (〇.8〇g, 4 l %1 νMR (400 MHz in CDCl3, Bruker AVANCE-400); § 1.89 (m, 2H CH2), 2.31 ( t, 2H, CH2-CH2~CO〇Me), 2 .53 (t, 2H, CH2) 16 201141836 3.58 (br, 2H, NH2), 3.65 (s, 3H, OMe), 6.61 (d, 2H, J = 3.5Hz, Ar-H), 6.95 (ί/, 2H, = 3.4 Hz) (iii) (R)-4-(4-(3-(5-(Benzyloxy) phthalate-2-yl)-2-(t-butoxycarbonylamino) propyl hydrazine Preparation of Amino)Phenyl) Butyrate Butyl Ester Methyl 4-(4-phenylamine)butyrate Butyrate (1.24 g, 6.42 mmol) ' 3-(5-(Benzyloxypyridin-2-yl)) 2-(t-butoxycarbonylamino)propionic acid (2.17 g, 5.84 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.12 g, 5.84 Methyl acetate was dissolved in 25 mL of dichlorohydrazine and allowed to stand at room temperature for 2 hours (monitored by TLC). The mixture was washed with water, dried over anhydrous magnesium sulfate and purified by chromatography (EA:Hex = 1:1). The title compound (2.37 g, 74%). m.p.: 166-167°C; (400 MHz in CDC13, Bruker AVANCE-400): δ 1.31 (s, 9H, Boc), 1.93 (m, 2H, CH2), 2.30 (t, 2H, CH2-CH2-COOMe), 2.59 (t, 2H, CH2), 3.22 (m 2H, CH2), 3.66 (sy 3H, OMe), 4.43 (t 1H CH) 5.13 (s 2H OBn), 6.97 (d, 2H, J= 7.9Hz, Ar-H), 7.09 (d, 1H, J= 8.0Hz, Pyr-H), 7.20-7.48 (m, 9H Ar-H), 8.26 (br, 1H, NH), 9.91 (br, 1H, NH). (lv) (R)-4-(4-(3-(5-(benzyloxy))pyridin-2-yl)-2(t-butoxycarbonylamino)propanamide) phenyl)butyric acid Synthesis of 4-(4-(3-(5-(benzyloxy)&quot;bipyridin-2-yl)-2-(t-butoxycarbonylamino)propanamide) phenyl)butyrate 0.59 g, 1.07 mmol) was dissolved in 10 mL of tetrahydrofuran/water (1:1) with lithium hydroxide (〇_26 g, 10.7 mmol). The drops were acidified to pH = 2 and extracted with dichloromethane. The methane methane was separated and the residue was dried with EtOAc EtOAc EtOAc EtOAc EtOAc (EtOAc) Boc), 1.74 (m, 2H, CH2), 2A7(t, 2H, CH2-CH2-COOMe, /= 7.34Hz), 2.52 (t, 2H, CH2, J = 7.55Hz), 2.92-3.07 (m, 2H, CH2), 4.44 (m, 1H CH), 5.13 (s, 2H OBn), 7.09 (d, 1H, J = 8.47Hz, Ar-H), 7.21 (d, 1H, Ar-H, J = 8.52 Hz), 7.31-7.48 (m, 9H Ar-H), 8.26 (br, 1H, NH), 9.91 {br, 1H, OH). (v) (R)-Tertiary Butyl 3-(5-( Benzyloxy)&quot;bipyridin-2-yl)-1-(4-(4-(hydroxyamino)-4-butylbutyl)anilino)-1-oxopropyl-2-ylamine citrate ( Synthesis of Compound 3) (Compound 3) Preparation of 4-(4-(3-(5-(Benzyloxy)&lt;*bipyridin-2-yl)-2(t-butoxycarbonylamino)propanoid Amine)Phenyl)butyric acid (0.43 g, 0.80 mmol) was dissolved in anhydrous tetrahydromethane, then ethyl chloroformate (1 mL, 0.88 mmol) and triethylamine (0.15 mL, 0.96 mmol). ). Stir at 〇 ° C for 30 minutes. After the insoluble matter was removed by filtration, the filtrate was added to a hydroxylamine methanol solution. The hydroxylamine sterol solution was prepared in situ by adding a solution of hydroxylamine hydrochloride (0.11 g, 1.60 mmol) in methanol (1.1 mL) at 0 〇c to the stirred solution of KO(0.09 g, 1.60 mmol) of methanol ( 〇·5 mL) solution. After stirring for 15 minutes, the precipitate was removed and the filtrate was stirred at room temperature for 3 min (monitored by TLC). The solvent was evaporated in vacuo. (0.3 g, 74% Bu Melting Point: &gt; 300 ° C; NMR (400 MHz in CDC13s Bruker AVANCE-400): δ 1.23 (s, 9H, Boc), 1.75 (m, 2H, CH2), 1.93 (t , 2H, CH2-CH2-CONHOH, J= 7.0Hz), 2.51 (t, 2H, CH2, /= 6.5Hz)s 2.95 (m, 2H, CH), 4.45 (m, 1H, CH), 5.14 (5 , 2H, OBn), 6.97 201141836 (d, 1H, J= 8.0Hz, Ar-H), 7.09 (d, 2H, J= 8.5Hz), 7.21 (d, 1H, J = 8.5Hz, Ar-H) , 7.32-7.47 (m, 8H, Ar-H), 8.26 (br, 1H, NH), 8.62 (br, 1H, NH), 9.91 (br, 1H, NH), 10.32 (br, 1H, OH). 4. Ternyl-1-(4-(4-(hydroxylamine)-4-oxobutyl)aniline) 3-(5-hydroxyxanthrin-2-yl)-1-oxopropan-2-yl Preparation of urethane carboxylic acid (compound 4)

BnO

NHOH

NHOH will be a second butyl 3-(5-(&gt;oxy).biter-2-yl)-1-(4-(4-(amino)-4 oxobutyl)amino)- 1-oxopropyl-2-ylamine phthalic acid vinegar (oi g, 0.19 mm〇i) and 1 〇% Pd/C dissolved in 2 mL of sterol in 1 〇g, vigorously stirred under hydrogen for 4 hours at room temperature (monitored by TLC). Then filter with Celite. The solvent was evaporated, and the residue was crystalljjjjjjjj Melting point: 166-167 ° C; NMR (400 MHz in CDC13, Bruker AVANCE-400): δ 1.31 (s, 9H, Boc), 1.75 (m, 2H, CH2), 1.93 (t, 1H, CH2, /= 7.10 Hz), 2.52 (t, 2H, CH2, J = 7.10Hz), 2.94 (m, 2H, CH2), 4.41 (m, 1H, CH), 6.94 (d, 1H, Ar-H, J = 7.99Hz ), 7.05 (m, 1H, Ar-H), 7.08 (d, 2H, Ar-H, /= 7.88Hz), 7.21 (m, 1H, Ar-H), 7.46 (d, 2H, Ar-H, J = 8.15 Hz), 8.03 (br, 1H, NH), 9.88 (br, 1H, NH), 10.32 (br, 1H, OH). 19 201141836 5.4-(4-(2-Amino- 3-(5) -Benzyloxy-2-° ratio bite) Synthesis of n-propionamine)phenyl)-N-gas-based butyrate (compound 5)

Compound 3 χχα

Compound 5 will be a second butyl 3-(5-(anthoxyl ratio.din-2-yl)-1-(4-(4-(amino)-4-oxobutyl)anilino)-1- Oxypropyl-2-ylamine decanoate (〇1g, 〇19 mmol) dissolved in DCM/TFA (v/v = 1:1) (30 mL), stirred at room temperature for 4 hours (monitored by TLC) The solvent was removed in vacuo to give the title compound <RTI ID=0.0>(</RTI> </RTI> <RTIgt; </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt; , 2H, CH2&gt; 7= 7.37Hz), 2.52 (t, 2H, CH2, J = 7.55Hz), 3.16 (m, 2H, CH2), 4.30 (m, 1H, CH), 5.16 (s, 2H, OBn ), 7.13 (d, 2H, Ar-H, J = 8.06Hz), 7.24 (d, 1H, Ar-H, J = 8.47Hz), 7.33-8.46 (m, 9H, Ar-H), 8.22 (br , 2H, NH2), 8.30 (m, 1H, NH), 8.64 (br, 1H, NH), 10.33 (br, 1H, OH), 10.36 (br, 1H, TFA). 6. 4-(4-( Preparation of 2-amino-3-(5-benzyloxy-2-pyridyl)-n-propylamine) phenyl) hydroxy-butyryl ester (Compound 6)

The third butyl-1-(4-(4-(amino)-4-oxybutyl)aniline)-3-(5-°°°201141836 bit-2-yl)-1-oxo- 2-2 The amide (oi g, 〇 19 mmol) was dissolved in 1 mL of di-methane and 1 mL of TFA and stirred at room temperature for 4 hours (monitored by TLC). The solvent was evaporated, and the residue was purified mjjjjjjj Melting point: 230 〇c decomposition; (400 MHz in CDC13, Bruker AVANCE-400): δ 1.77 (m, 2H, CH2), 2.20 (/, 2H, CH2, J = 7.01 Hz), 2.51 (^, 2H, CH2 , J = 8.27Hz), 3.43(m, 2H, CH2), 4.48(m, 1H, CH), 7.13(J, 1H, Ar-H, J = 8.19Hz), 7.26-7.32(m, 4H, Ar -H), 7.5l (d, 1H, Ar-H, /= 8.07Hz), 7.61(m, 1H, Ar-H), S.39(br, 1H, NH), %.65(br, 2H , NH2), I0.44(br, 1H, OH), ll.〇4(^r, 1H, TFA). 7. Third butyl-3-(5-(benzyloxy)"pyridin-2- Preparation of 1-(4-(hydroxylamine)benzylamino)-1-oxopropyl-2-amine decanoic acid g (Compound 7)

(1) Preparation of 4-((3-(5-(benzyloxypyridin-2-yl)-2-(t-butoxycarbonylamino)propionamide) methyl benzoate oxime ester will 4-Aminomethyl)benzoic acid oxime ester (2 g, 1 〇mm〇1), 3-(5-(benzyloxy)-pyridin-2-yl)-2-(t-butoxycarbonylamino)propionic acid (1〇4 g, ^ mm〇i ), with 21 201141836 1-ethyl-3-(3-dioguanylpropyl) carbodiimide hydrochloride (2 g, 1〇4 mmol) dissolved in 50 The methane methane was stirred at room temperature for 2 hours (monitored by TLC). The reaction mixture was extracted with water and then dried over anhydrous magnesium sulfate. The residue was purified by chromatography (EA:Hex = 1:1) ,49%)e nmr

(400 MHz in CDC13, Bruker AVANCE-400): δ 1.31 (5, 9H)

Boc), 2.90(m, 2H, CH2), 3.81(^, 3H, OMe) 4.30(m, 2H, CH2), 4.33(m, 1H,CH), 5.12(j, 2H, OBn), 6.97-7.09 (ί/, 1H, J = 3.5Hz, Ar-H), 7.15 (i/, lH, t/=8.53Hz, Ar-H), 7.24~7.46(w, 8H, Ar-H), T.S4 (d, 2H, Ar-H, J = 8.24 Hz), 8.24 (6^, 1H, NH), SA3(br, 1H, NH). (ii) 4-((3-(5-(benzyloxy)) Synthesis of "bipyridin-2-yl)-2-(t-butoxycarbonylamino)propanamide) methyl)benzoic acid 4-((3) in 50 mL of tetrahydrofuran/water (1:1) -(5-(benzyloxy)pyridin-2-yl)-2-(t-butoxyamino)propanamide)methyl)benzoic acid (1.3 g, 2.58 mmol) and lithium hydroxide (0.6 g , 25 mmol) The mixture was heated at 60 ° C for 4 hours. The drops were acidified to pH = 2 and extracted with di-methane. The dioxane was evaporated and dried <RTI ID=0.0> NMR (400 MHz in CDC13, Bruker AVANCE-400): δ 1.31 (^, 9H, Boc), 2.97 (m, 2H, CH2), 4.27 (m, 2H, CH2), 4.33 (m, 1H, CH), 5.13(^, 2H, OBn), 6.96(^, 1H, J = 8.32Hz, Ar-H), 7.l5-7.18(m, 3H, Ar-H), 7.3l~7.39(m, 4H, Ar -H), 7A4(d, 2H, Ar-H, J = 7.01Hz), 7.80(ii, 2H, Ar-H, J = 7.67Hz), S.24(br, 1H, NH), S.43 (br, 1H, NH). (iii) Tert-butyl-3-(5-(benzyloxy)pyrene-But-2-yl)-1-(4-(amino-branched) 22 201141836 Benzylamine Synthesis of 1-yloxypropyl-2-amine decanoate (Compound 7) 4-((3-(5-(Benzyloxy)-2-yl)-2-) A solution of tributoxyamino) acrylamide) mercapto) benzoic acid (0.65 g, 1.25 mmol) in anhydrous tetrahydrogenate was added to ethyl benzoate (0.15 mL, 1.5 mmol) and triethylamine ( 0.21 mL, 1_88 mmol), stirred for 30 minutes at 〇 ° C. The solid was removed by filtration, and the filtrate was added to a hydroxylamine methanol solution in the form of a hydroxylamine methanol solution. Hydroxylamine hydrochloride (0.14 g, 2.5 mmol) at 0 ° C. A solution of methanol (20 mL) was added to a solution of .KOH (0.11 g, 2.5 mmol) in methanol (1 mL). After stirring for 15 minutes, The precipitate was removed and the filtrate was stirred at room temperature for 30 min (mdqqqqqqqqqqqqqqqQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQQ NMR (400 MHz in CDC13, Bruker AVANCE-400): δ 30 (5, 9H, Boc), 2.91 (m, 2H, CH2), 4.26 (m, 2H, CH2), 4.32 ( m, 1H, CH), 5.12(^, 2H, OBn), 6.95{d, 1H, Ar-H, J= 8.24Hz), 7.14-7.21 (m, 3H, Ar-H), 7.30-7.44(m , 6H, Ar-H), 7.65(d, 1H, Ar-H, /= 8.38Hz), 7.8(^, 1H, Ar-H, J = 8.38Hz), S.24(br, 1H, NH) , 8.45(6r, 1H, NH), 9AS(br, 1H, NH), 10.39(6r, 1H, OH). 8.4-((2-Amino-3-(5-(benzyloxypyridin-2-) Synthesis of propylamine fluorenyl)-n-hydroxybenzoguanamine trifluoroacetate (compound 5) 23 201141836

化合物 Compound 7 Compound 8 tert-butyl-3-(5-(benzyloxy)pyridin-2-yl)-1-(4-(hydroxylamine benzyl)benzylamino)-1-oxopropyl 2-Amino phthalate (0.26 g, 0.5 mmol) was dissolved in TFA / DCM (v/v = 1:1) (30 mL) and stirred at room temperature for 4 hours (monitored by TLC). The DCM was removed from the solution three times to obtain 0.021 g of HPW101x048 (Sl/^oi^^lfc.lsi-lSjoC^NMRKOOMHzinCDClhBruker AVANCE-400): δ 3.12 (m, 2H, CH2), 4.25 (m, 2H, CH2), 4.40 ( m, 1H, CH), 5.15(5, 1H, OBn), 7.21(m, 3H, Ar-H), 7.40(w, 7H, Ar-H), 1.61{d, 1H, Ar-H, /= 8.21Hz), 7.84(i/, 1H, Ar-H, /= 7.96Hz), S.26(br, 2H, NH2), 8.27(5, 1H, NH), S.92(br, 1H, NH ), 11.19(6r, 1H, OH). 9·Secondyl 1-(4-(aminocarbamoyl)benzylamino)_3·(5_ via n to bit_2)-1-oxyl Preparation of propyl-2-ylamine decanoate (Compound 9)

ΝΗΟΗ ο Compound 9 NHOH pd/c Me〇H ( Hz Ο Compound 7 tert-butyl-3-(5-(oxygen)-deno-2-yl) small (4_(aminoguanidino) amide a mixture of 1-oxypropyl-2-amine phthalic acid vinegar (0.26 g, 0.5 mm 〇1) and 26 mg of 10% Pd/C dissolved in 2 mL of methanol under hydrogen and at room temperature 24 201141836 The mixture was stirred for 4 hours, then filtered with EtOAc (EtOAc). δ 1.28(ί, 9Η, Boc), 3.1 5 (m, 2 Η, C Η 2), 4.2 8 (/, 2 Η, C Η 2, / = 5 · 1 2 Η ζ), 4.3 6 (w, 1 Η, CH), 7.05-7.25(m, 4Η, Ar-H), 7.64-7.84(w, 3Η, Ar-H), S.23(br, 1H, NH), 8.51(fer, 1H, NH ), 10.18(0r, 1H, NH), UA6(br, 1H, OH). 10.4-((2-Amino-3-(5-hydroxypyridin-2-yl)-n-propylamine)methyl)· Synthesis of ν-hydroxyphenylguanamine (Compound 10)

The third butyl 1-(4-(hydroxylaminomethyl)benzylamino)-3-(5-hydroxy)pyridin-2-yl)- oxapropyl-2-ylamine citrate (0.10) g, 0.19 mmol) was stirred at room temperature for 4 hours with 1 mL of TFA in hexanes (25 mL). The solution was volatilized (monitored by TLC). The residue was purified by chromatography to give EtOAc (EtOAc: EtOAc (EtOAc: EtOAc) ), 4.36 (m, 1H, CH), 7.08-7.28 (m, 4H, Ar-H), 62-7.70 (m, 2H, Ar-H), 7.82-7.88 (m, 1H, Ar-H), 8.96(w, 2H, NH2), 9A3(br, 1H, NH)S 9.91(br, 1H, NH), 11.15(6r, 1H,OH)· 25 201141836 11. (R)-4-(4-( Hydroxylamine)-4-oxobutane)phenyl_3_(5.(benzyloxy)pyridin-2-yl)-2-(2nd-butoxy-amino)propyl]Preparation of (Compound η)

Bn0NT^ NHBoc BnO.

DCC/DMAP/CH2CI2/r.t.

EtOH βη〇Τ^ι rBocVx^X, Compound 11 (i) Preparation of 4-(4-hydroxyphenyl)butyric acid 4-(4-methoxyphenyl)butyric acid (0.97 g, 5 mm〇1) ) mixed with desertified argon (5 〇 mL) and glacial acetic acid (80 mL). The mixture was heated to reflux for 1 hour (monitored by TLC) and poured into ice to mix for 1 hour and then extracted with dcm. The solvent was removed in vacuo to give the title compound (0.83 g, 92%). NMR ¢400 MHz in CDC13, Bruker AVANCE-400): δ 1.72(m, 2H, CH2), 2.16(ί, 2H, CH2j J = 7.4Hz), 2.45(ί, 2H, CH2, J = 7.6Hz), 6.65 (d, 2H, Ar-H, J = 8.3 Hz), 6.95 (^/, 2H, Ar-H, J = 8.3 Hz). (11) 4-(4-Hydroxyphenyl)_N-(tetrahydrogen) Preparation of -21^pyran-2-yloxy)butanamine 4-(4-pyridyl)butyric acid (〇.9〇吕, 5 111111〇1), 〇-(tetrahydro-211-〇 Bis-2-yl)hydroxylamine (0-59 g, 5 mmol) and 1-ethyl-3-(3-diamidinopropyl) 1⁄4 diimine hydrochloride (1.15 g, 6 mmol) dissolved in 50 The mL methylene chloride was stirred at room temperature for 2 hours (monitored by TLC). The mixture was extracted with water and dried with EtOAc EtOAc EtOAc EtOAc (EtOAc) 400): δ 1.50 (m, 3H, OTHP), 1.71 (m, 26 201141836 3H, OTHP), 1.73 (w, 2H, CH2), 1.96 (m, 2H, CH2), 2.44 (m, 2H, CH2) , 3.48(ί, 1H, / = 5.0Hz, OTHP), 3.91(ί, 1H, 7 = 10.0Hz, OTHP), 4.80(5, 1H, OTHP), 6.65{d, 2H, /= 5.0Hz, Ar -H), 6.95(ί/, 2H, J= 10.0Hz, Ar-H), 9.10(5, 1H, NH), 10.87(^, 1H, OH). (iii) (2R)-4-(4 -oxy-4-(tetra-argon-2H-11 than 2-yloxyamine)butyl)phenyl-3-(5-(oxygen)° ratio bit-2-yl)-2-(third Synthesis of butoxy-alginyl) propyl vinegar 3-(5-(benzyloxy)pyridin-2-yl)-2-(t-butoxycarbonylamino)propanoic acid (0.19 g, 0.5 mmol), 4- (4-Hydroxyphenyl)-N-(tetrahydro-2H-pyran-2-yloxy)butanamine (0.14 g, 0.5 mmol), with DMAP (0.06 g, 0.5 mmol) in DCM (10 mL) ) Stir at 0 ° C for 10 minutes. A solution of DCC (0.12 g, 0.6 mmol) in DCM (5 mL) was added dropwise to the above solution and allowed to stand at room temperature for 1 hour (monitored by TLC). The white precipitate was removed and the filtrate was evaporated under vacuum. The solid was removed again by the addition of ethyl acetate. The solution was volatilized in vacuo and the residue was purified by chromatography (EA:Hex = 1:5) to give the title compound. (iv) (R)-4-(4-(transamino)-4-oxobutyl)phenyl-3-(5-(oxygen) η than bite_2_yl)-2-(third Oxycarbonylamino)propionate (2R)-4-(4-oxo-4-(tetrahydro-2Η-°pyran-2-yloxyamine)butyl)phenyl-3-(5- (Benzyloxy)pyridin-2-yl)-2-(t-butoxycarbonylamino)propyl ester (〇32匕0.5 mmol) and Dowex 50wX8 (0.5 mmol) in ethanol (25 mL) heated to reflux for 1 hour . The precipitate was removed, and the filtrate was evaporated in vacuo to give purified crystals. 27 201141836 Preparation of benzyl 3-(5-(benzyloxypyridin-2-yl)-2-(third duck (compound 1 5 ))

12.(R)-4-(hydroxylamine-butoxycarbonylamino)propionic acid

(1) 4 (Methyl)_N-(tetrahydro-211_〇 〇_2_yloxy) benzamide synthesis 4-(methyl) benzoic acid (〇75 g, 5 _〇1), 〇 (tetrazine 2h "pyran-2-yl" via a base amine (0.59g, 5mm 〇im丨 ethyl 3 (3-didecylaminopropyl) carbon-imine hydrochloride compound (115 g, 6 Ment) Dissolved in 50 mL of di-methane and stirred at room temperature for 2 hours (monitored by tlc). The mixture was extracted with water and dried with dry sulphuric acid to remove water and the residue was purified by chromatography (Me 〇H: DCM = 1:50). The title compound (0.88 g, 7〇%) was obtained. Inmr^4〇〇MHz in DMSO, Bruker AVANCE-400): δ \.52{m, 3H, OTHP), 1.72 (m, 3H, OTHP), 3.51 ( m, 1H, OTHP), 4.04(m, 1H, OTHP), 4.53(j, 2H, CH2), 4.98(5, 1H, OTHP), 5.28(^, 1H, NH), 7.38(^/, 2H, J =10.0 Hz, Ar-H), 1.12{d, 2H, J = 5.0 Hz, Ar-H), 11.55 (5, 1H, OH). (ii) (2R)-4-(tetrahydro-2H- °pyran-2-yloxyaminecarboxamidobenzyl 3-(5-(benzyloxy)-pyridin-2-yl)-2-(t-butoxycarbonylamino)propionate 3-( 5-(Benzyloxy)acridin-2-yl)-2-(t-butoxycarbonylamino)propionic acid (0.19 g, 0.5 mmol), 4-(hydroxyindenyl)-N-(tetrahydro-2H -pyran-2- Oxygen) 28 201141836 Benzamide (0.13 g, 0.5 mmol) and DMAP (0.06 g, 0.5 mmol) were added to DCM (10 mL) and stirred at 0 ° C for 10 min. DCC (0.12 g, 0·6 mmol) was Dissolved in DCM (5 mL), the solution was slowly added to the above solution and stirred at room temperature to room temperature for 1 hour (monitored by TLC). The white precipitate was removed by filtration, and the filtrate was evaporated in vacuo. (MeOH '.DCM = 1:50) gave the title compound (0·18 g, 60%). NMR (400 MHz in DMSO, Bruker AVANCE-400): δ 1.32 (5, 9H, Boc), 1.59 (m, 3H, OTHP), 1.70(m, 3H, OTHP), 3.0(m, 2H, CH2), 3.50(ί/, 1H, J =10.0Hz, OTHP), 4.04(ftr, 1H, OTHP), 4.44(m , 1H, CH), 4.98 (~ 1H, OTHP), 5.12 (1 4H, OBn and C(0)OCH2), 5.54 (1 1H, NH), 5.55 (^, 1H, NH), 7A6(d, 1H , /= 10.0Hz, Ar-H), 7.28-7.39(m, 7H, Ar-H), 7.44{d, 2H, J = 5.0Hz, Ar-H), 7.72(^, 2H, J = 5.0Hz , Ar-H), 8.24 (^, 1H, Ar-H). (iii) (R)-4-(hydroxylamine decyl)benzyl 3-(5-(benzyloxy). (2R)-4-(tetrahydro-2H-.0-0-2-oxoamine oxime)benzyl benzylidene-2-yl)-2((butoxycarbonylamino)propionate 3-(5-(oxygen) ° ratio -2-yl)-2-(t-butoxyamino)propionic acid vinegar (〇.3〇g, 0.5 mmol) and Dowex 50wX8 (0.5 mmol) Heated in ethanol (2.5 mL) for 1 hour (monitored by TLC). The sediment was removed and the liquid was evaporated under vacuum. The residue was purified by chromatography (MeOH: DCM = 1:25) to give the title compound 15. Example 2: Biological activity 1 · Tissue protein deacetylase (HDAC) inhibitory activity assay 29 201141836 Activity assay of tissue protein deacetylase (HDAC) for the use of tissue proteins extracted from HeLa cells to deacetylase and used The Fluor de LysTM fluorescent color rendering system (Biomol, Enzo Life Science; PA, USA) was tried and adjusted as described above (PΠum e/α/., 2007). Briefly, HeLa extract was added to a solution with or without a test substance (DMSO, 0.2 〇 / 〇 v / v) and preheated at 30 ° C for 1 Torr. Then add Fluor de LysTM substrate (125 μΜ) and heat to 37. (: Select for 45 minutes. Finally, add the developing reagent (95 μιη, ι X) to the above mixture and leave it for another 10 minutes. Use the microplate fluorescence analyzer (Thermo Varioskan Flash) to the extent of the formation of the acetamidine group. , Thermofisher; MA, USA) to monitor 'to determine the fluorescence intensity of the Fluor de LysTM substrate (stimulus wave is 3 60 nm 'the emission wavelength is 46 〇. The experimental results are expressed as the percent inhibition of the deacetylation reaction formation interval. The marketed HDAC inhibitor SAHA (1〇μΜ) is used as a positive control group. See Bieiiauskas et al., 5zo〇rg· Λ/ec?. CAew. Ze&quot;., 2007, 17: 2216-2219.

Compounds 1-8 were tested and their test results showed that their jjdAC inhibitory activity was lower than the concentration of 1 〇-5 Μ. The inhibitory strength of compounds 3, 4 and 7 and SAHA for HDAC increased with increasing concentration, and its IC5g appears in the lower list. Table 1: Inhibition of HDAC inhibitory activity IC5 〇 compound 1 3 4 7 SAHA IC50 1.73χχ10'5Μ 3.98x1 〇·7 Μ 4.13χ10'6Μ 1,32χ10·6Μ 3.98χ1〇·7Μ 30 201141836 2. In vitro wound healing test compound The wound healing activities of 1 and 3 are Valster et al., Methods, 2005, 37, 208-215 and Rodriguez et al., "Methods in Molecular Biology" in Cell Migration: Developmental Methods and Protocols, vol. 294, Ed by Guan Et al., Humana

The methods provided in Press Inc. were performed ex vivo. First, IMR90 cells were cultured in a 6-well culture dish and grown in a serum-containing environment containing 10% FBS. When the cells are full, the culture solution (MEM containing 10% FBS) is replaced with a serum-free medium (MEM without FBS) for starvation for 24 hours. Next, the wound was scraped off with a pipette tip of ριοοο, and Compound 1 (〇, 0.1, 0.5, and 1 mM) or Compound 3 (0, 〇.〇5, 0.5, and 1 μΜ) was added for 24 hours. The experimental results were observed and photographed by a phase difference microscope. Images were captured at regular intervals from the intersection of the scar and marking lines over a 24-hour period and analyzed by a digital imaging system. Use Image-Pro Plus® (Media Cybernetics, Silver

Spri, ng, MD) software to determine the location of migrated cells at the edge of the scar. The distance the cells migrate can be calculated by subtracting the starting width from the width of the flaw at each time interval. The experimental results show that Compounds 1 and 3 promote wound healing. 3. COX-2 protein expression in vitro This experiment used Western blotting to detect intracellular COX-2 protein expression. First, IMR90 cells were scraped with a pipette tip of P1000, and compound 3 was added for treatment for 24 hours. The cells were lysed and extracted with 1 2011 cell lysis buffer to extract proteins. Next, protein quantification was carried out by Bio-Rad Quantitative Reagent (Bio-Rad Laboratory, CA), subjected to 10% SDS-PAGE electrophoresis at 2 〇盹 protein amount, and disrupted to PVDF 骐. The antibody used to identify COX-2 in this experiment was purchased from Epitomics (Burlingame'CA), and the antibody recognizing β-actin was purchased from Sigma (St Louis, MO). Except for the β-actin antibody, which is a mouse monoclonal antibody, all antibodies are rabbit polyclonal antibodies. The secondary antibodies are anti-rabbit IgG and anti-m〇use IgG (anti-m〇use IgG) (anti-rabbit peroxide-related antibodies). All antibodies are TBS-T with 5% BSA. The dilution of the solution 'primary antibody is hl〇〇〇, and the dilution of the secondary antibody is 1:5000. Finally, a cold light color reaction was performed with an ECL detection reagent group (Millipore), and exposure was performed with an X-ray film to detect an immunoreactive protein. The experimental results show that Compound 3 inhibits the expression of COX-2 protein at a concentration of 0.05-1.0 μΜ. 4. Protein expression of in vitro acetylated histone protein-3 (Acetyl-H3) This experiment used western blotting to detect the expression of acetylated histone protein-3. First, IMR-90 cells were treated with Compound 1-4 and the protein extract was collected using the methods described in Sections 2 and 3 above and subjected to Western blotting. The acetylated histone protein-3 protein is mainly detected by primary antibodies and secondary antibodies. The acetylated histone protein-3 protein antibody used in this experiment was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and the β-actin 32 201141836 antibody was purchased from ICN (Costa Mesa, CA). Anti-rabbit immunoglobulin and anti-mouse immunoglobulin (antibody related to horseradish peroxide) were purchased from Amersham Biociences (Piscataway, NJ) and all antibodies were diluted to 1 with TBS-T solution containing 5% milk. :1000 use. Finally, a cold light color reaction was performed with an ECL detection reagent group (Millipore), and an X-ray film was used to expose the immunoreactive protein. The results of the experiment showed that Compound 1-4 increased the expression of the acetylated tissue protein-3 protein of IMR-90 cells. 5. Anti-mucosal activity live animal experiments 20 young Syrian golden hamsters aged 7 to 8 weeks and weighing 90 to 130 grams were selected and numbered separately and randomly divided into 5 groups (4 in each group). The right cheek intima of each of the 5 groups was wound with a 6 mm diameter wound on the second day using a skin perforator. Four groups were randomly selected from these five groups for intraperitoneal injection of 5-FU 20 mg/kg (20 mg/kg) from the second day to the second day, twice a day. The remaining group was left untreated for comparison. Three groups were administered to the four groups given 5-FU, and subcutaneous injection of Compound 1 was given from day 0 to day 5, 36, 72 and 144 mg/kg per kg or subcutaneously administered compound 3' per kg 0.5 , 5.0 and 50.0 mg (mg/kg), once daily. Macroscopic and microscopic studies on the 0.5th, 1st, 2nd, 3rd, 4th, 5th and 6th day after the wound was made. Hematological parameters, serum biochemical values, degree of inflammatory cell infiltration, size of hemorrhagic area, and ulceration Records of abscesses, etc. · After day 6 'All hamsters were anesthetized with isoflurane (2-chloro-2-di 33 201141836 fluoromethoxy-1,1,1-trifluoroethane) for macroscopic observation, Histological analysis and Western blot analysis. The results showed that both Compound-1 and Compound 3 reduced the wound area of 5-U-induced membranous inflammation, and its inhibition was concentration-dependent. 34

Claims (1)

201141836 VII. Patent application scope: 1 · A compound with the following structure, Ri R2 RsRe N-R7 Η wherein RbUhU5, and independently independently hydrogen, affiliation, oxime or alkoxy; I is hydroxy, OR, or OC(0)R, and the aryl is optionally substituted alkyl' Is a halogen, an aryl group, a heterocyclic aryl group, an amine group, or a carboxylic acid group; R8 is hydrogen, (CH2)mNHC(0)R,) or (Cl^UOC^COR, m is 0, 1, or 2 And R' is an optionally substituted alkyl group, the substituent of which is a halogen, an aryl group, a heterocyclic aryl group or a carboxylic acid group. 2. A compound according to the item of claim 2, wherein r7 is a hydroxyl group. The compound of claim 2, wherein Ru R2, r3, R4, R5 and each are hydrogen. 4. The compound of claim 1 is a hydroxyl group. 5. A compound of the formula wherein R, R2 and R3 are each hydrogen, and Rs and R6 are each an alkyl group. 6. A compound according to claim 1, wherein R7 is 35 201141836 0C(0)R. 7. A compound according to claim 6 wherein R is an alkyl group having a NH2 or a phenyl substituent. 8. A compound according to the scope of the patent application, wherein the compound is from the following New Zealand group consisting of the formula ^ selected. 9. A compound having the formula: Wherein R1 is hydrogen, hydroxy, amino, alkoxy, arylalkoxy, alkylcarbonyloxy, or alkoxycarbonyloxy; R2 is hydrogen, hydroxy, alkoxy, alkyl, and oxygen R3 is a thiol group or a phenyl group; X is oxygen or Nh; Y is phenyl or -CH2-; wherein m and η are each independently 〇, 丨, 2, 3, 4 or 5. 10. The compound of claim 9, wherein X is νη. 11. The compound of claim 10, wherein γ is phenylene group 0 36 201141836 12. The compound of claim 11 wherein m is 0 and η is 3. A compound according to claim 12, wherein R3 is a hydroxyl group. 14. A compound according to claim 10, wherein hydrazine is -CH2-. 1 5. The compound of claim 9 wherein R3 is hydroxy. 16. The compound of claim 9, wherein X is oxygen. 17. The compound of claim 9, wherein R in the ear is hydroxy or alkoxy. 18. The compound of claim 9, wherein R is benzyloxy. 19. A compound as claimed in claim 9 wherein the compound is selected from the group consisting of: 37 201141836 20. Treatment of the desired compound. 21. Treatment of the desired compound. 22. What is needed. The invention relates to a method for treating mucosal ulcer, comprising the method of administering an effective dose of the compound 'oral mucosal ulcer> as described in the scope of the patent application, comprising administering an effective dose to the body. The method for treating a cancer/alpha cancer according to claim 9 of the invention, comprising administering the therapeutic dose to a body, as described in claim 1 of claim 1 2011 2011. The method comprises the compound according to claim 9 in the effective dosage required for administering the treatment to a body. 0 39 201141836 IV. Designated representative figure: (1) The representative representative figure of the case is: ( ). (2) A brief description of the symbol of the representative figure: (none) 5. If there is a chemical formula in this case, please disclose the chemical formula that best shows the characteristics of the invention: 2