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Rapid Discovery of Highly Potent and Selective Inhibitors of Histone Deacetylase8Using Click Chemistry to Generate Candidate Libraries Takayoshi Suzuki,*,?,?Yosuke Ota,§Masaki Ri,∥Masashige Bando,⊥Aogu Gotoh,?,§Yukihiro Itoh,?Hiroki Tsumoto,§Prima R.Tatum,§Tamio Mizukami,#Hidehiko Nakagawa,§Shinsuke Iida,∥

Ryuzo Ueda,∥Katsuhiko Shirahige,⊥and Naoki Miyata*,§

?Graduate School of Medical Science,Kyoto Prefectural University of Medicine,13Taishogun Nishitakatsukasa-Cho,Kita-ku,Kyoto 403-8334,Japan

?PRESTO,Japan Science and Technology Agency(JST),4-1-8Honcho Kawaguchi,Saitama332-0012,Japan

§Graduate School of Pharmaceutical Sciences,Nagoya City University,3-1Tanabe-dori,Mizuho-ku,Nagoya,Aichi467-8603,Japan ∥Graduate School of Medical Sciences,Nagoya City University,1Kawasumi,Mizuho-cho,Mizuho-ku,Nagoya,Aichi467-8601,Japan ⊥Institute of Molecular and Cellular Biosciences,the University of Tokyo,1-1-1Yayoi,Bunkyo-ku,Tokyo113-0032,Japan

#Graduate School of Bio-Science,Nagahama Institute of Bio-Science and Technology,1266Tamura-cho,Nagahama,Shiga,526-0829, Japan

*Supporting Information

ABSTRACT:To?nd HDAC8-selective inhibitors,we designed a library of HDAC inhibitor candidates,each containing group that coordinates with the active-site zinc ion,linked via a triazole moiety to a capping structure that interacts

on the rim of the active site.These compounds were synthesized by using click chemistry.Screening identi?ed

INTRODUCTION

Reversible acetylation/deacetylation of theε-amino groups of lysine residues on histones and nonhistone proteins,catalyzed by histone acetyltransferases and histone deacetylases (HDACs),is involved in various life phenomena such as gene expression and cell cycle regulation.1Thus far,18HDAC family members have been identi?ed and categorized into two groups,namely zinc-dependent enzymes(HDAC1?11)and NAD+-dependent sirtuins(SIRT1?7).2Among them,HDAC8 is expressed tissue speci?cally and is localized in the nucleus3or cytoplasm.4It is responsible for genetic repression in acute myeloid leukemia5and is associated with the actin cytoskeleton in smooth muscle cells.6Furthermore,recent studies have revealed that HDAC8is associated with several disease states. Gallimari et al.reported that siRNA targeting HDAC8showed antitumor e?ects in cell culture.7Balasubramanian et al. reported that inhibition of HDAC8induced apoptosis in T-cell lymphomas.In addition,Oehme et al.suggested that HDAC8is implicated in neuroblastoma tumorigenesis.9 Therefore,HDAC8-selective inhibitors are of great interest, not only as tools for probing the biological functions of this isozyme but also as candidate anticancer agents having few side e?ects.

Although many HDAC inhibitors have been found so far,10 including trichostatin A(TSA,1),11vorinostat(2),12and MS-275(3)13(Chart1),most lack HDAC8selectivity.It has been reported that HDAC8is selectively inhibited by SB-379278A (4),14linkerless hydroxamic acid5,15PCI-34051(6),8 azetidinone7,16and A8B4(8)(Chart2).17Among these compounds,compound6is a representative HDAC8-selective Received:June14,2012

Published:November1,2012

?2012American Chemical https://www.docsj.com/doc/7c13170984.html,/10.1021/jm300837y|J.Med.Chem.2012,55,9562?9575

inhibitor that shows high potency and selectivity for HDAC8 over HDAC1,2,3,6,and10.

Various strategies have been employed to identify HDAC8-selective inhibitors to https://www.docsj.com/doc/7c13170984.html,pound4was identi?ed by the evaluation of a chemical compound collection using an HDAC8 enzyme-based high-throughput screening,14and compounds 5?7were discovered by drug design based on the crystal structure of HDAC8.8,15,16Here,we report the rapid identi?cation of highly potent and selective inhibitors of HDAC8by the use of Cu(I)-catalyzed azide?alkyne cycloaddition,a representative reaction in click chemistry,18 to generate a library of candidates.Although several groups have used click chemistry to?nd HDAC inhibitors,19there has been no previous report on the use of click chemistry for the discovery of isozyme-selective HDAC inhibitors.

Chemistry.The routes used for the synthesis of the compounds prepared for this study are shown in Schemes 1?11.

The preparation of alkyne A1is shown in Scheme 1. Propiolic acid9was reacted with NH2OTHP in the presence of DCC to give the corresponding O-THP hydroxamate10. Deprotection of the THP group of10by p-TsOH a?orded A1. The preparation of alkyne A2(Scheme2)started with5-bromothiophene-2-carboxylic acid11,which was esteri?ed and subjected to Sonogashira cross coupling with trimethylsilylace-tylene to give13.Hydrolysis and EDCI/HOBt-mediated coupling with NH2OTHP a?orded the corresponding hydrox-amate15,which provided A2upon removal of the THP group with https://www.docsj.com/doc/7c13170984.html,pounds A3and A4were prepared from carboxylic acids16a and16b by using the same procedure described for the synthesis of A2(Scheme3).o-Aminoanilides A5?A8were obtained by the condensation of1,2-phenyl-enediamine with an appropriate carboxylic acid in the presence of condensing reagents(Scheme4).

Azides B2?B9,B11,B12,and B14were prepared by azide displacement(NaN3,DMSO)of the corresponding alkyl halides21(Scheme5).Scheme6shows the synthesis of azides B13and B15from alcohols22a and22b via the tosylates23a,b.Azide B10was accessed from carboxylic acid24 by LiAlH4reduction and alcohol-to-azide conversion using DPPA and DBU(Scheme7).Phenethyl azide analogues B16?B34and B36were synthesized by azide displacement of the corresponding alkyl bromides26(Scheme8).Scheme9shows the preparation of azides B35and B37?B43with an ethylene chain from alcohols27via the mesylate,bromide,or tosylates 28.1-Phenylethanone-2-azide B45and(E)-(2-azidovinyl)-benzene B46were prepared by azide displacement of the bromide29and the boronic acid30,respectively(Scheme10). Cu-catalyzed coupling of alkyne A3and azide B1,B2,B37, or B44provided triazoles C31,C32,C142,and C149(Scheme 11).

■RESULTS AND DISCUSSION

Click Chemistry Approach.Fragment-based assembly is a drug discovery approach that enables high-throughput identi-?cation of small-molecular inhibitors using a minimal number of compounds as building blocks.20It allows medicinal chemists to explore n×m possibilities with n+m combinations.Click chemistry is a powerful fragment-based assembly method and was shown to be highly versatile and e?ective for rapid synthesis/assembly of libraries of small molecules directed against a number of enzymes.21As reported before,triazole synthesis by Cu(I)-catalyzed azide?alkyne cycloaddition (CuAAC),22a key reaction in click chemistry,can provide a library of triazole-containing compounds,which are pure enough to be evaluated by means of in vitro assay without puri?cation.

Chart1.Examples of HDAC Inhibitors

Chart2.HDAC8-Selective Inhibitors

Scheme1.Synthesis of Alkyne A1a

a Reagents and conditions:(a)NH

OTHP,DCC,CH2Cl2,0°C to rt, 57%;(b)p-TsOH·H2O,MeOH,rt,70%.

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In general,the pharmacophore for HDAC inhibitors consists of a zinc-binding group (ZBG)that coordinates with the zinc ion in the active site,a capping structure that interacts with residues on the rim of the active site,and a linker connecting the cap part and the ZBG with an appropriate separation.For example,compound 2(Chart 1),a clinically used HDAC

inhibitor,consists of hydroxamic acid (ZBG),anilide (cap),and alkyl chain (linker).Accordingly,we designed a library of candidate HDAC inhibitors in which the ZBG and the cap group are connected by a triazole-containing linker (Figure 1).19a We designed various alkynes with hydroxamic acid (A1?A4)or o -aminoanilide (A5?A8)as the ZBG and azides with various cap structures (B1?B15)as suitable building blocks for click assembly to construct a structurally diverse chemical library that might provide a high hit rate.We then prepared eight alkynes A1?A8and 15alkyl azides B1?B15as shown in Schemes 1?https://www.docsj.com/doc/7c13170984.html,ing the CuAAC reaction,a 120-member HDAC inhibitor library was assembled in microtiter plates.Each of the eight alkynes was mixed with each of the 15azides in DMSO solution,followed by the addition of catalytic amounts of CuSO 4,sodium ascorbate,and tris[(1-benzyl-1H -1,2,3-triazol-4-yl)methyl]amine (TBTA).The disappearance of the starting materials and the quantitative formation of the triazole products were con ?rmed by TLC and LC-MS

Scheme 2.Synthesis of Alkyne A2a

Reagents and conditions:(a)conc H 2SO 4,MeOH,re ?ux,96%;(b)trimethylsilylacetylene,Pd(PPh 3)2Cl 2,CuI,Et 2NH,rt,81%;(c)NaOH,H 2O,MeOH,rt,97%;(d)NH 2OTHP,EDCI,HOBt ·H 2O,DMF,rt,90%;(e)p -TsOH ·H 2O,MeOH,rt,77%.

Scheme 3.Synthesis of Alkynes A3and A4a a

Reagents and conditions:(a)conc H 2SO 4,MeOH,re ?ux,96%for 17a ,99%for 17b ;(b)trimethylsilylacetylene,Pd(PPh 3)2Cl 2,CuI,Et 3N,THF,rt,90%for 18a ,64%for 18b ;(c)2N NaOH,MeOH,rt,99%for 19a ,92%for 19b ;(d)NH 2OTHP,EDCI,HOBt ·H 2O,DMF,rt,93%for 20a ,86%for 20b ;(e)p -TsOH ·H 2O,MeOH,rt,86%for A3,94%for A4.

Scheme 4.Synthesis of Alkynes A5?A8a

Reagents and conditions:(a)1,2-phenylenediamine,DCC,CH 2Cl 2,0°C to rt,44%for A5;(b)1,2-phenylenediamine,EDCI,HOBt ·H 2O,DMF,rt,79%for A6,91%for A7,86%for A8.

Scheme 5.Synthesis of Azides B2?B9,B11,B12,and B14a

Reagents and conditions:(a)NaN 3,DMSO,rt or 80°C,21?92%.

Scheme 6.Synthesis of Azides B13and B15a

Reagents and conditions:(a)TsCl,pyridine,rt,68%for 23a ,84%for 23b ;(b)NaN 3,DMSO,80°C,26%for B13,84%for B15.

Scheme 7.Synthesis of Azide B10a

Reagents and conditions:(a)LiAlH 4,THF,0?80°C,87%;(b)DPPA,DBU,DMF,rt,30%.

Scheme 8.Synthesis of Azides B16?B34and B36a

Reagents and conditions:(a)NaN 3,DMSO,rt or 80°C,62?100%.

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(Supporting Information Figure S1).The generated triazole compounds C1?C120are shown in Supporting Information Figure S2.The 120triazoles could be directly screened for HDAC-inhibitory activity in the microtiter plates without further puri ?cation.To ?nd HDAC8-selective inhibitors,compounds C1?C120were tested for activity against both HDAC8at 0.3μM and total HDACs from HeLa nuclear extracts at 3μM.We chose total HDACs from HeLa nuclear extracts for primary screening because these extracts are rich in the activity of HDAC1and HDAC2,which are major isozymes of the HDAC family.23In this HDAC8assay,the HDAC8-inhibitory activity of compound 2at 0.3μM in the presence of 10mol %Cu(I)was almost the same as that in the absence of Cu(I)(31.9±0.12%inhibition in the presence of Cu(I),35.0±0.72%inhibition in the absence of Cu(I)),suggesting that Cu(I)does not a ?ect the activity of compounds C1?C120.As shown in Figures 2and 3,two hits emerged from these screens.The HDAC8-inhibitory activity of these two crude compounds C31and C32was comparable to that of compound 6at 0.3μM (Figure 2).Furthermore,at the concentration of 3μM,crude C31and C32showed weak inhibitory activity against total HDACs from HeLa nuclear extract (Figure 3).

To identify HDAC8-selective inhibitors more potent than compound 6,we further designed a C32-based library of compounds,which could be prepared from alkyne A3and azides B16?B46(Schemes 8?10)(Figure 4)using the same click chemistry approach.The CuAAC reaction between alkyne A3and azides B16?B46gave 31triazoles C121?C151,which are structurally related to compound C32(Supporting Information Figure S3).The 31triazole compounds were screened against both HDAC8and total HDACs from HeLa nuclear extracts.Two crude compounds,C142and C149,showed more potent inhibition than C32at the concentration of 0.3μM (Figure 5)while displaying weak inhibition toward HDACs from HeLa nuclear extracts at the concentration of 3μM,indicating high selectivity for HDAC8(Figure 6).

Isozyme https://www.docsj.com/doc/7c13170984.html,pounds C31,C32,C142,and C149were resynthesized and puri ?ed by column chromatog-raphy and recrystallization (Scheme 11).The pure compounds C31,C32,C142,and C149were evaluated for inhibitory e ?ects on HDAC1,HDAC2,HDAC4,HDAC6,and HDAC8as well as total HDACs from nuclear extracts.The results of the enzyme assays are shown in Table 1.In these assays,compound 6inhibited HDAC8with an IC 50of 0.31μM and showed selectivity for HDAC8over HDAC1,HDAC2,HDAC4,and HDAC6.

Compounds C31,C32,C142,and C149all showed potent HDAC8-inhibitory activity.The HDAC8-inhibitory activity of compounds C31and C32was greater than that of compound 2and comparable to that of compound 6(IC 50of 21.5μM,60.31μM,C310.35μM,C320.18μM).Compounds C142and C149,which are derivatives of C32,showed more potent HDAC8-inhibitory activity than compound 6(IC 50of C1420.10μM,C1490.070μM).Furthermore,while compound 2inhibited HDAC1,HDAC2,and HDAC6more potently than HDAC8,compounds C31,C32,C142,and C149inhibited HDAC8in preference to the other isozymes,like compound 6.Thus,C31,C32,C142,and C149are potent and selective inhibitors of HDAC8.

Scheme 9.Synthesis of Azides B35and B37?B43a

Reagents and conditions:(a)MsCl,Et 3N,CH 2Cl 2,rt;(b)TsCl,pyridine,rt;(c)CBr 4,PPh 3,CH 2Cl 2,rt;(d)NaN 3,DMSO,rt or 80°C,16?86%(two steps).

Scheme 10.Synthesis of Azides B45and B46a

Reagents and conditions:(a)NaN 3,DMSO,rt,53%;(b)NaN 3,CuSO 4,MeOH,rt,40%.

Scheme 11.Synthesis of HDAC8-Selective Inhibitors C31,C32,C142,and C149a

Reagents and conditions:(a)CuSO 4·5H 2O,sodium ascorbate,TBTA,MeOH,H 2O,rt,92?100%.

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Molecular Modeling.Although several X-ray crystal structures of HDAC8have been reported,16,23we chose the crystal structure of HDAC8complexed with CRA-A (PDB code 1VKG)for the molecular modeling study on compounds 6and C149with HDAC8.Unlike the other structures,the HDAC8?CRA-A complex structure has a hydrophobic pocket which is thought to be unique to HDAC8.15,23a It is therefore reasonable to assume that HDAC8-selective inhibitors bind to this unique pocket.The lowest energy conformations of 6,a previously reported HDAC8-selective inhibitor,and C149,the most active HDAC8-selective inhibitor in the present series,were obtained when they were docked into a model based on the crystal structure of HDAC8(PDB code 1VKG),using the

software packages Glide 3.5and MacroModel 8.1(Figures 7and 8).

In the simulated HDAC8/compound 6complex,the hydroxamate group of compound 6coordinates in a bidentate fashion to the zinc ion and forms three hydrogen bonds with His 142,His 143,and Tyr 306in the active center of HDAC8(Figure 7).The 4-methoxybenzyl group of compound 6is located in the unique hydrophobic pocket of HDAC8,where the CH of the methoxy group can interact with Tyr 154via CH ?πinteraction.It is also suggested that the indole ring of compound 6orients the hydroxamate group and 4-methox-ybenzyl group into the appropriate geometry.

Inspection of the simulated HDAC8/compound C149complex showed that the hydroxamic acid group coordinates the zinc ion through its CO and OH groups in a similar way to compound 6.It is also suggested that the hydroxamate group of C149forms a hydrogen bond between its CO moiety and His 142and also forms another hydrogen bond between its OH moiety and Tyr 306(Figure 8).Unlike compound 6,it is suggested that C149has a U-shaped conformation in the active site of HDAC8,and most importantly,the phenylthiomethyl group of compound C149binds to a hydrophobic pocket formed by Trp 141,Ile 34,and Pro 35,which is thought to be unique to HDAC8and where the methylthio group can interact with Trp 141and the phenyl group can bind to Pro 35and Tyr 306through hydrophobic interactions.The three nitrogens of the triazole ring of C149do not appear to interact with any amino acid residues of HDAC8,but the triazole ring can interact with the methylene group of Phe 152through CH ?πor hydrophobic interactions (the distance between the CH 2of Phe 152and the triazole is 3.2?).The triazole ring is also considered to be important to ?x the orientation of the zinc-binding hydroxamate and the hydrophobic pocket-binding phenylthiomethyl group appropriately,thereby contributing to the potent HDAC8-inhibitory activity and HDAC8-selectivity of compound https://www.docsj.com/doc/7c13170984.html,parison of the simulated HDAC8/inhibitor complex structures suggests that compound C149can bind to HDAC8more tightly than can compound 6through e ?cient hydrophobic interactions.

In addition,we attempted to carry out a docking study of compound C149to HDAC2(PDB code 3MAX),24another HDAC isozyme.However,compound C149could not be docked in the active site of HDAC2because of a steric clash

Figure 1.Design of alkynes and azides.

Figure 2.HDAC8activity in the presence of 0.3μM C1?C120.

Figure 3.Total HDACs activity in the presence of 3μM C1?C120.

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between the phenylthiomethyl group of C149and the rim of the HDAC2active site.These calculation results are consistent with the experimental fact that compound C149inhibits HDAC8in preference to HDAC2(Table 1).

Cellular Assays.Because HDAC8is a cohesin deacety-lase,25inhibition of HDAC8and that of other HDACs can be assessed by evaluating accumulation of acetylated cohesin and acetylated histone,respectively,using Western blot analysis.Therefore,we examined the e ?ects of compound 6,C142,and C149on accumulation of acetylated cohesin and histone H4(Figure 9).In accordance with the results of enzyme assays,compound 6and compounds C142and C149produced a

dose-dependent increase of cohesin acetylation without a major increase in acetylated H4.These results indicate that compounds C142and C149potently and selectively inhibit HDAC8in preference to other HDACs in cells.

Because it has been reported that inhibition of HDAC8induces apoptosis in T-cell lymphomas 8and HDAC8is implicated in neuroblastoma tumorigenesis,9compounds 6,C32,and C149were tested in cell growth-inhibition assays using human T-cell lymphoma and neuroblastoma cell lines.The results are shown in Table 2.These HDAC8-selective inhibitors showed clear growth-inhibitory e ?ects on both T-cell lymphoma and neuroblastoma cell lines,including Jurkat,HH,MT2,MT4,NB-1,and LA-N-1.In particular,the growth-inhibitory activity against T-cell lymphoma cells of compound C149was greater than that of compound 6.It should be noted that these inhibitors did not a ?ect the growth of healthy donor peripheral blood mononuclear cells (PBMCs)(IC 50values >100μM),suggesting that the cytotoxicity of HDAC8-selective inhibitors is cell-type-speci ?c.These results suggest that HDAC8is involved in the growth of T-cell lymphoma and neuroblastoma cells and that our HDAC8-selective inhibitors may be useful in the treatment of T-cell lymphoma and neuroblastoma.

■

CONCLUSION

In summary,we used click chemistry with the reliable CuAAC reaction initially to prepare a triazole library of 120compounds,among which two potent HDAC8inhibitors C31and C32were identi ?ed.For further structural optimization,we next prepared a 31-member library based on the structure of compound C32.Screening led to the identi ?cation of two highly potent and selective HDAC8inhibitors,C142and C149.A molecular modeling study of the HDAC8/C149complex suggested that the orientation of the hydrophobic pocket-binding phenylthiomethyl group and the zinc-binding hydroxamate,?xed by the triazole ring,is important for both HDAC8-inhibitory activity and https://www.docsj.com/doc/7c13170984.html,-pounds C32and C149also showed in-cell HDAC8selectivity and potent T-cell lymphoma cell growth-inhibitory activity.Thus,we have rapidly identi ?ed a novel series of HDAC8-selective inhibitors by using click chemistry to generate libraries of candidate molecules.It should be possible to obtain even more potent and selective HDAC8inhibitors by means of further structural development.HDAC8-selective inhibitors are thought to have considerable potential both for the develop-Figure 4.Design of a C32-based library of compounds.

Figure 5.HDAC8activity in the presence of 0.3μM C121?C151.

Figure 6.Total HDACs activity in the presence of 3μM C121?C151.

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ment of novel therapeutic agents and as tools for biological research.

■

EXPERIMENTAL SECTION

Chemistry.Melting points were determined using a Yanagimoto micro melting point apparatus or a Bu c hi 545melting point apparatus and were left uncorrected.Proton nuclear magnetic resonance spectra (1H NMR)and carbon nuclear magnetic resonance spectra (13C NMR)were recorded in the indicated solvent on a JEOL JNM-LA500,JEOL JNM-A500,or BRUKER AVANCE600spectrometer.Chemical shifts (δ)are reported in parts per million relative to the internal standard,tetramethylsilane.Elemental analysis was performed with a Yanaco CHN CORDER NT-5analyzer,and all values were within ±0.4%of the calculated values,con ?rming >95%purity.High-resolution mass spectra (HRMS)and fast atom bombardment (FAB)mass spectra were recorded on a JEOL JMS-SX102A mass spectrometer.GC-MS analyses were performed on a Shimadzu GCMS-QP2010.IR spectra were measured on a Shimadzu FTIR-8400S spectrometer.LCMS was performed with a Waters instrument equipped with Unison US-C18(2mm ×5mm/2×150mm,Imtakt

Corporation).Reagents and solvents were purchased from Aldrich,Tokyo Kasei Kogyo,Wako Pure Chemical Industries,and Kanto Kagaku and used without puri ?cation.Flash column chromatography was performed using silica gel 60(particle size 0.046?0.063mm)supplied by Merck.

Propynoic Acid Hydroxyamide (A1).Step 1:Preparation of Propynoic Acid Tetrahydropyran-2-yloxyamide (10).To a solution of propynoic acid (9,300mg,4.28mmol)in CH 2Cl 2(6mL)and NH 2OTHP (552mg,4.71mmol)was added dropwise a solution of DCC (972mg,4.71mmol)in CH 2Cl 2(9mL)at 0°C.The reaction mixture was stirred for 3h at room temperature.Filtration,concentration in vacuo,and puri ?cation of the residue by silica gel ?ash chromatography (AcOEt/n -hexane =2/3)gave 416mg (57%)of 10as a pale-yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):8.76(1H,s),5.01(1H,s),3.96(1H,t,J =10.2Hz),3.96?3.65(1H,m),2.89(1H,s),1.94?1.78(3H,m),1.71?1.61(3H,m).

Step 2:Preparation of Propynoic Acid Hydroxyamide (A1).To a solution of 10(416mg,2.46mmol)obtained above in MeOH (21mL)was added TsOH ·H 2O (47mg,0.246mmol).The reaction mixture was stirred for 30h at room temperature.After removal of the solvent,the residue was puri ?ed by silica gel ?ash

column

Table 1.HDAC-Inhibitory Activity of Compounds 2,6,C31,C32,C142,and C149a

6>100>100>1000.31>1009.3C313741650.35307.9C3223>100760.18>100 3.2C14244>100>1000.10>100 1.1C149

>100

0.070

2.4

Values are means of at least three experiments.

Figure 7.The lowest energy conformation of 6(ball-and-stick representation)in the HDAC8catalytic https://www.docsj.com/doc/7c13170984.html,pound 6was docked into a model based on the crystal structure of HDAC8(PDB code 1VKG),using the software packages Glide 3.5and MacroModel 8.1.(top left)Surface view of 6docked in the HDAC8catalytic pocket.(top right)View of the catalytic center of HDAC8complexed with 6.Residues within 3?of the zinc ion are displayed.(bottom)Schematic representation of 6binding with HDAC8.

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chromatography (AcOEt/n -hexane =3/1)to give 147mg (70%)of A1as a white solid.The solid was recrystallized from AcOEt/n -hexane to give 70mg of A1as colorless crystals;mp 69?71°C.1H NMR (DMSO-d 6,500MHz,δ;ppm):11.2(1H,broad s),9.28(1H,s),4.18(1H,s).13C NMR (DMSO-d 6,150MHz,δ;ppm):149.15,77.32,76.03.HRMS (EI)Calcd.for C 3H 3O 2N 85.0160,Found 85.0159.5-Ethynylthiophene-2-carboxylic Acid Hydroxyamide (A2).Step 1:Preparation of 5-Bromothiophene-2-carboxylic Acid Methyl Ester (12).To a solution of 5-bromo-2-thiophenecarboxylic acid (11,1.00g,4.83mmol)in MeOH (30mL)was added concentrated H 2SO 4(1.00mL).The reaction mixture was re ?uxed for 30h.After removal of the solvent,the residue was poured into water and extracted with AcOEt.The organic layer was separated,washed with brine,and dried over Na 2SO 4.Filtration,concentration in vacuo,and puri ?cation of the residue by silica gel ?ash column chromatography (AcOEt/n -hexane =1/10)gave 1.03g (96%)of 12as a white solid.1H NMR (CDCl 3,500MHz,δ;ppm):7.55(1H,d,J =4.0Hz),7.07(1H,d,J =4.0Hz),3.88(3H,s).

Step 2:Preparation of 5-Trimethylsilanylethynylthiophene-2-carboxylic Acid Methyl Ester (13).To a solution of 12(1.03g,4.66mmol)obtained above,PdCl 2(PPh 3)2(32.7mg,46.6μmol),and CuI (13.3mg,69.9μmol)in diethylamine (15.5mL)was added trimethylsilylacetylene (687mg,6.99mmol).The mixture was stirred for 18h at room temperature.After removal of the solvent,the residue was diluted with Et 2O and the Et 2O solution was washed with 1N aqueous HCl,saturated NaHCO 3,and brine,and then dried over Na 2SO 4.Filtration,concentration in vacuo,and puri ?cation of the residue by silica gel ?ash column chromatography (AcOEt/n -hexane =1/20)gave 895mg (81%)of 13as a brown solid.1H NMR (CDCl 3,500MHz,δ;ppm):7.63(1H,d,J =4.0Hz),7.16(1H,d,J =4.0Hz),3.88(3H,s),0.257(9H,s).

Step 3:Preparation of 5-Ethynylthiophene-2-carboxylic Acid (14).To a solution of 13(895mg,3.75mmol)obtained above in MeOH (10mL)was added 2N aqueous NaOH (3.75mL,7.50mmol)at 0°C.The reaction mixture was stirred for 5h at room temperature.The mixture was acidi ?ed with 2N aqueous HCl and

Figure 8.The lowest energy conformation of C149(ball-and-stick representation)in the HDAC8catalytic https://www.docsj.com/doc/7c13170984.html,pound C149was docked into a model based on the crystal structure of HDAC8(PDB code 1VKG),using the software packages Glide 3.5and MacroModel 8.1.(top left)Surface view of C149docked in the HDAC8catalytic pocket.(top right)View of the catalytic center of HDAC8complexed with C149.Residues within 3?of the zinc ion are displayed.(bottom)Schematic representation of C149binding with HDAC8.

Figure 9.Western blot detection of acetylated structural maintenance of chromosome 3(SMC3),a subunit of cohesin,and H4levels in HeLa cells after 4h treatment with compounds 6,C142,and C149.Values of Ac SMC3/SMC3ratio determined by optical density measurement of the blots are shown below the uppermost photograph.

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concentrated in vacuo.The residue was diluted with AcOEt and the AcOEt solution was washed brine and dried over Na 2SO 4.Filtration and concentration in vacuo gave 552mg (97%)of 14as a brown solid.1

H NMR (DMSO-d 6,500MHz,δ;ppm):7.64(1H,d,J =4.0Hz),7.40(1H,d,J =4.0Hz),4.80(1H,s).

Step 4:5-Ethynylthiophene-2-carboxylic Acid Tetrahydropyran-2-yloxyamide (15).To a solution of 14(150mg,0.986mmol),EDCI (226mg,1.18mmol),and HOBt ·H 2O (159mg,1.18mmol)in DMF (3mL)was added NH 2OTHP (173mg,1.48mmol),and the mixture was stirred at room temperature for 24h.The reaction mixture was poured into water and extracted with AcOEt.The organic layer was separated,washed with saturated NaHCO 3and brine,and dried over Na 2SO 4.Filtration,concentration in vacuo,and puri ?cation of the residue by silica gel ?ash column chromatography (AcOEt/n -hexane =1/30)gave 224mg (90%)of 15as a yellow solid.1H NMR (DMSO-d 6,500MHz,δ;ppm):11.9(1H,broad s),7.62(1H,d,J =4.0Hz),7.39(1H,d,J =4.0Hz),4.96(1H,s),4.77(1H,s),4.05?4.01(1H,m),3.55?3.53(1H,m),1.73?1.71(3H,m),1.55?1.53(3H,m).Step 5:Preparation of 5-Ethynylthiophene-2-carboxylic Acid Hydroxyamide (A2).To a solution of 15(224mg,0.891mmol)obtained above in MeOH (10mL)was added TsOH ·H 2O (16.9mg,0.0891mmol).The reaction mixture was stirred for 12h at room temperature.After removal of the solvent,the residue was puri ?ed by silica gel ?ash column chromatography (AcOEt/n -hexane =1/1)to give 147mg (77%)of A2as a brown solid;mp 146?147°C.1H NMR (DMSO-d 6,500MHz,δ;ppm):11.4(1H,broad s),9.25(1H,broad s),7.53(1H,d,J =4.0Hz),7.36(1H,d,J =4.0Hz),4.74(1H,s).13C NMR (CDCl 3,125MHz,δ;ppm):158.32,138.73,133.83,127.36,124.66,86.93,76.20.MS (EI)m /z 167(M +);Anal.Calcd for C 7H 5NO 2S ·1/5H 2O:C,49.23;H,3.19;N,8.20.Found:C,49.10;H,3.18;N,8.07.

Compounds A3and A4were prepared from 16a and 16b ,respectively,using the same procedure described for A2.

3-Ethynyl-N-hydroxybenzamide (A3).Yield 68%;colorless crys-tals;mp 165?166°C.1H NMR (DMSO-d 6,500MHz,δ;ppm):11.3(1H,broad s),9.14(1H,broad s),7.82(1H,s),7.78(1H,d,J =7.9Hz),7.62(1H,d,J =7.6Hz),7.49(1H,t,J =7.8Hz),4.28(1H,s).13

C NMR (DMSO-d 6,150MHz,δ;ppm):163.07,134.07,133.08,129.81,128.89,127.35,121.75,82.65,81.40.MS (EI)m /z :161(M +).Anal.(C 9H 7NO 2)C,H,N.

4-Ethynyl-N-hydroxybenzamide (A4).Yield 47%;colorless crys-tals;mp 166?167°C.1H NMR (DMSO-d 6,500MHz,δ;ppm):11.3(1H,broad s),9.12(1H,broad s),7.75(2H,d,J =8.2Hz),7.55(2H,d,J =8.2Hz),4.37(1H,s).13C NMR (DMSO-d 6,150MHz,δ;ppm):163.24,132.77,131.61,127.04,124.25,82.72,82.66.MS (EI)m /z 161(M +).Anal.(C 9H 7NO 2·1/10H 2O)C,H,N.

Propynoic Acid 2-Aminophenylamide (A5).Compound A5was prepared as colorless crystals in 44%yield from 8and 1,2-phenylenediamine,using the same procedure described for A1(step 1);mp 127?128°C.1H NMR (DMSO-d 6,500MHz,δ;ppm):9.97(1H,s),7.15(1H,d,J =7.9Hz),6.93(1H,t,J =7.6Hz),6.73(1H,d,

J =7.9Hz),6.55(1H,t,J =7.6Hz),4.91(2H,s),4.32(1H,s).13C NMR (DMSO-d 6,125MHz,δ;ppm):149.94,141.94,126.59,125.52,121.65,116.01,115.96,78.43,76.61.MS (EI)m /z 160(M +).Anal.(C 9H 8N 2O)C,H,N.

Compounds A6?A8were prepared from 1,2-phenylenediamine and the corresponding carboxylic acids 14and 189a ,b using the same procedure described for A2(step 4).

5-Ethynylthiophene-2-carboxylic Acid 2-Aminophenylamide (A6).Yield 79%;colorless crystals;mp 143°C.1H NMR (DMSO-d 6,500MHz,δ;ppm):9.88(1H,broad s),7.98(1H,d,J =4.0Hz),7.51(1H,d,J =4.0Hz),7.17(1H,d,J =7.9Hz),7.07?7.04(1H,m),6.86?6.84(1H,m),6.68?6.64(1H,m),5.01(2H,s),4.83(1H,s).13

C NMR (CDCl 3,125MHz,δ;ppm):159.04,143.46,141.22,133.93,128.97,127.03,126.98,125.45,122.13,116.17,116.01,87.04,76.42.MS (EI)m /z 242(M +).Anal.(C 7H 5NO 2S ·1/4H 2O)C,H,N.

N-2-Aminophenyl-3-ethynylbenzamide (A7).Yield 91%;colorless crystals;mp 194?195°C.1H NMR (DMSO-d 6,500MHz,δ;ppm):9.73(1H,broad s),8.09(1H,broad s),7.99(1H,d,J =7.9Hz),7.67(1H,d,J =7.6Hz),7.53(1H,t,J =7.8Hz),7.15(1H,d,J =7.9Hz),6.97(1H,m),6.78(1H,dd,J =1.2,7.9Hz),6.59(1H,t,J =7.3Hz),4.93(2H,broad s),4.30(1H,s).13C NMR (DMSO-d 6,150MHz,δ;ppm):164.47,143.28,135.09,134.35,130.88,128.85,128.38,126.86,126.69,122.96,121.76,116.20,116.06,82.92,81.43.MS (EI)m /z :236(M +).Anal.(C 15H 12N 2O ·1/3H 2O)C,H,N.

N-2-Aminophenyl-4-ethynylbenzamide (A8).Yield 86%;colorless crystals;mp 181?183°C.1H NMR (DMSO-d 6,500MHz,δ;ppm):9.73(1H,broad s),7.99(2H,d,J =7.9Hz),7.61(2H,d,J =8.5Hz),7.15(1H,d,J =7.3Hz),6.99?6.96(1H,m),6.78?6.77(1H,m),6.61?6.58(1H,m),4.92(2H,s),4.41(1H,s).13C NMR (DMSO-d 6,125MHz,δ;ppm):164.41,143.13,134.62,131.47,127.96,126.67,126.52,124.45,122.90,116.06,115.95,82.82,82.79.MS (EI)m /z 236(M +).Anal.(C 15H 12N2O ·1/5H 2O)C,H,N.

General Procedure for the Synthesis of Azides B2?B9,B11,B12,B14,B16?B34,B36,and B45.To a 0.5M solution of NaN 3(1.1equiv)in DMSO was added an appropriate alkyl halide (1.0equiv),and the mixture was stirred at room temperature or 80°C and periodically monitored by TLC.When the reaction was completed,the mixture was quenched with water and stirred until it cooled to room temperature and then extracted with AcOEt or Et 2O.The organic layer was separated,washed with water and brine,and dried over Na 2SO 4.Filtration,concentration in vacuo,and puri ?cation of the residue by silica gel ?ash column chromatography gave the corresponding alkyl azide.

Phenethyl Azide (B2).26Yield 76%;a pale-yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.33?7.21(5H,m),3.50(2H,t,J =7.3Hz),2.89(2H,t,J =7.31Hz).13C NMR (CDCl 3,150MHz,δ;ppm):137.99,128.72,128.62,126.76,52.44,35.33.FTIR (neat,cm ?1)2090.MS (EI)m /z 119(M +?28).

3-Phenylpropyl Azide (B3).26Yield 55%;a pale-yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.31?7.17(5H,m),3.28(2H,t,J =6.9Hz),2.69(2H,t,J =7.6Hz),1.91(2H,m).13C NMR (CDCl 3,150MHz,δ;ppm):140.85,128.51,128.45,126.14,50.64,32.76,30.43.FTIR (neat,cm ?1)2090.MS (EI)m /z 133(M +?28).

4-Phenylbutyl Azide (B4).27Yield 58%;a pale-yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.30?7.17(5H,m),3.28(2H,t,J =6.9Hz),2.65(2H,t,J =7.5Hz),1.74?1.68(2H,m),1.66?1.60(2H,m).13

C NMR (CDCl 3,125MHz,δ;ppm):141.80,128.37,128,35,125.89,51.34,35.35,28.42.FTIR (neat,cm ?1)2090.

5-Phenylpentyl Azide (B5).Yield 75%;a pale-yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.29?7.16(5H,m),3.25(2H,t,J =7.0Hz),2.65(2H,t,J =7.6Hz),1.68?1.59(4H,m),1.44?1.38(2H,m).13

C NMR (CDCl 3,125MHz,δ;ppm):142.26,128.34,128.29,125.73,51.34,35.74,30.94,28.72,26.34.FTIR (neat,cm ?1)2090.MS (EI)m /z 133(M +?28).

4-Methoxybenzyl Azide (B6).28Yield 60%;a pale-yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.24(2H,d,J =8.8Hz),6.91(2H,d,J =8.5Hz),4.26(2H,s),3.81(2H,s).13C NMR (CDCl 3,150MHz,δ;ppm):159.60,129.72,127.36,114.16,55.26,54.36.FTIR (neat,cm ?1)2090.MS (EI)m /z 163(M +).HRMS (EI)calcd for C 8H 9ON 3,163.0758;found,

163.0761.

Table 2.Growth Inhibition of Various Cancer Cells by Compounds 6,C32,and C149a

HH >1007923MT2151911MT4253015neuroblastoma cell

NB-1143912LA-N-1 3.927 6.4healthy PBMC cell

PB-O 97>100>100PB-N

>100

Values are means of at least three experiments.

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3-Phenylbenzyl Azide (B7).Yield 78%;colorless crystals.1H NMR (CDCl 3,500MHz,δ;ppm):7.59?7.52(4H,m),7.37?7.34(1H,m),7.46?7.42(3H,m),7.29(1H,d,J =7.6Hz),4.39(2H,s).13C NMR (CDCl 3,125MHz,δ;ppm):141.95,140.66,135.94,129.28,128.84,127.56,127.20,127.14,127.00,126.98,54.87.FTIR (neat,cm ?1)2094.MS (EI)m /z 209(M +).HRMS (EI)calcd for C 13H 11N 3,209.0957;found,209.0960.

4-Phenylbenzyl Azide (B8).Yield 21%;a pale-yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.62?7.59(4H,m),7.45(2H,t,J =7.6Hz),7.40?7.35(3H,m),4.39(2H,s).13C NMR (CDCl 3,125MHz,δ;ppm):141.31,140.54,134.37,128.84,128.67,127.58,127.52,127.13,54.57.FTIR (neat,cm ?1)2090.MS (EI)m /z 209(M +).HRMS (EI)calcd for C 13H 11N 3,209.0952;found,209.0953.

3-Phenoxybenzyl Azide (B9).Yield 86%;a pale-yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.37?7.32(3H,m),7.12(1H,t,J =7.5Hz),7.05?7.01(3H,m),6.96?6.89(2H,m),4.30(2H,s).13C NMR (CDCl 3,125MHz,δ;ppm):157.79,156.78,137.29,130.19,129.85,123,60,122.77,119.15,118.48,118.27,54.42.FTIR (neat,cm ?1)2090.MS (EI)m /z 225(M +).HRMS calcd for C 13H 11ON 3,225.0901;found,225.0902.

1-Azidomethylnaphthalene (B11).Yield 91%;a pale-yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):8.01(1H,d,J =8.2Hz),7.88?7.83(2H,m),7.58?7.50(2H,m),7.46?7.42(2H,m),4.74(2H,s).13

C NMR (CDCl 3,125MHz,δ;ppm):133.87,131.32,130.93,129.39,128.77,127.23,126.69,126.12,125.17,123.442,52.97.FTIR (neat,cm ?1)2094.MS (EI)m /z 183(M +).HRMS (EI)calcd for C 11H 9N 3,183.0804;found,183.0802.

2-Azidomethylnaphthalene (B12).29Yield 83%;colorless crystals.1

H NMR (CDCl 3,500MHz,δ;ppm):7.88?7.84(3H,m),7.78(1H,s),7.53?7.49(2H,m),7.43(1H,dd,J =1.5,8.2Hz),4.51(2H,s).13

C NMR (CDCl 3,125MHz,δ;ppm):133.28,133.11,132.83,128.79,127.96,127.77,127.19,126.48,126.35,125,86,55.05.FTIR (neat,cm ?1)2090.MS (EI)m /z 183(M +).HRMS (EI)calcd for C 11H 9N 3,183.0803;found.183.0803.

Azidomethylcyclohexane (B14).Yield 92%;a pale-yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):3.11(2H,d,J =6.7Hz),1.78?1.67(5H,m),1.59?1.51(1H,m),1.29?1.11(3H,m),1.01?0.92(2H,m).13C NMR (CDCl 3,125MHz,δ;ppm):58.08,38.09,30.67,26.27,25.76.FTIR (neat,cm ?1)2094.

2-Methylphenethyl Azide (B16).Yield 95%;a colorless oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.16(4H,s),3.47(2H,t,J =7.6Hz),2.91(2H,t,J =7.6Hz),2.34(3H,s).13C NMR (CDCl 3,125MHz,δ;ppm):136.20,136.07,130.48,129.34,126.96,126.25,51.44,32.62,19.31.FTIR (neat,cm ?1)2090.MS (EI)m /z 133(M +?28).3-Methylphenethyl Azide (B17).Yield 81%;a colorless oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.21(1H,t,J =7.5Hz),7.07?7.01(3H,m),3.49(2H,t,J =7.3Hz),2.86(2H,t,J =7.3Hz),2.34(3H,s).13C NMR (CDCl 3,125MHz,δ;ppm):138.28,137.94,129.55,128.54,127.52,125.73,52.50,35.27,21.38.FTIR (neat,cm ?1)2090.MS (EI)m /z 133(M +?28).

4-Methylphenethyl Azide (B18).Yield 85%;a colorless oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.13(2H,d,J =8.2Hz),7.11(2H,d,J =8.2Hz),3.48(2H,t,J =7.3Hz),2.86(2H,t,J =7.3Hz),2.33(3H,s).13C NMR (CDCl 3,125MHz,δ;ppm):136.36,134.92,129.33,128.62,52.59,34.92,21.05.FTIR (neat,cm ?1)2090;.HRMS (EI)calcd for C 9H 11N 3,161.0950;found,161.0947.

2-Methoxyphenethyl Azide (B19).Yield 72%;a pale-yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.24(1H,dt,J =1.8,5.9Hz),7.16(2H,dd,J =1.5,5.8Hz),6.91(1H,dt,J =1.2,6.2Hz),6.86(1H,d,J =8.2Hz),3.83(3H,s),3.47(2H,t,J =7.6Hz),2.92(2H,t,J =7.6Hz).13C NMR (CDCl 3,125MHz,δ;ppm):157.57,130.61,128.17,126.23,120.56,110.35,55.24,50.97,30.34.FTIR (neat,cm ?1)2090.HRMS (EI)calcd for C 9H 11ON 3,177.0901;found,177.0903.

3-Methoxyphenethyl Azide (B20).Yield 89%;a colorless oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.24(1H,t,J =7.9Hz),6.82?6.76(3H,m),3.81(3H,s),3.50(2H,t,J =7.3Hz),2.87(2H,t,J =7.3Hz).13C NMR (CDCl 3,125MHz,δ;ppm):159.83,139.61,129.66,121.07,114.62,112.05,55.20,52.38,35.40.FTIR (neat,cm ?1)2090.HRMS (EI)calcd for C 9H 11ON 3,177.0909;found,177.0910.

4-Methoxyphenethyl Azide (B21).Yield 100%;a pale-yellow oil.1

H NMR (CDCl 3,500MHz,δ;ppm):7.14(2H,d,J =8.8Hz),6.86(2H,d,J =8.5Hz),3.80(3H,s),3.46(2H,t,J =7.2Hz),2.84(2H,t,J =7.2Hz).13C NMR (CDCl 3,125MHz,δ;ppm):158.50,130.06,129.73,114.09,55.28,52.73,34.50.FTIR (neat,cm ?1)2090.HRMS (EI)calcd for C 9H 11ON 3,177.0904;found,177.0903.

2-Chlorophenethyl Azide (B22).Yield 90%;a colorless oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.37(1H,d,J =7.0Hz),7.26?7.21(3H,m),3.53(2H,t,J =7.3Hz),3.03(2H,t,J =7.3Hz).13C NMR (CDCl 3,125MHz,δ;ppm):135.57,134.07,131.11,129.71,128.39,127.02,50.63,33.29.FTIR (neat,cm ?1)2090.HRMS (EI)calcd for C 8H 8N 3Cl,181.0405;found,181.0404.

3-Chlorophenethyl Azide (B23).Yield 80%;a pale-yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.25?7.22(3H,m),7.11(1H,d,J =6.7Hz),3.51(2H,t,J =7.2Hz),2.87(2H,t,J =7.2Hz).13C NMR (CDCl 3,125MHz,δ;ppm):140.06,134.43,129.90,128.92,127.04,126.97,52.13,35.03.FTIR (neat,cm ?1)2090.MS (EI)m /z 153(M +?28).

4-Chlorophenethyl Azide (B24).Yield 92%;a colorless oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.29(2H,d,J =8.2Hz),7.15(2H,d,J =8.2Hz),3.49(2H,t,J =7.2Hz),2.86(2H,t,J =7.2Hz).13C NMR (CDCl 3,125MHz,δ;ppm):136.52,132.66,130.11,128.79,52.27,34.72.FTIR (neat,cm ?1)2090.HRMS (EI)calcd for C 8H 8N 3Cl,181.0406;found,181.0404.

2-Fluorophenethyl Azide (B25).Yield 82%;a colorless oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.26?7.22(2H,m),7.11?7.03(2H,m),3.52(2H,t,J =7.0Hz),2.94(2H,t,J =7.0Hz).13C NMR (CDCl 3,150MHz,δ;ppm):161.24,131.15,128.69,124.90,124.23,115.45,51.08,29.00.FTIR (neat,cm ?1)2090.MS (EI)m /z 137(M +?28).

3-Fluorophenethyl Azide (B26).Yield 95%;a colorless oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.31?7.26(1H,m),7.01?6.93(3H,m),3.52(2H,t,J =7.3Hz),2.89(2H,t,J =7.3Hz).13C NMR (CDCl 3,125MHz,δ;ppm):162.96,140.54,130.12,124.42,115.69,113.75,52.13,35.09.FTIR (neat,cm ?1)2090.MS (EI)m /z 137(M +?28).

4-Fluorophenethyl Azide (B27).Yield 95%;a colorless oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.19?7.17(2H,m),7.01(2H,t,J =8.7Hz),3.49(2H,t,J =7.3Hz),2.86(2H,t,J =7.3Hz).13C NMR (CDCl 3,125MHz,δ;ppm):161.83,133.72,130.22,115.49,52.51,34.58.FTIR (neat,cm ?1)2090.HRMS (EI)calcd for C 8H 8N 3Cl,165.0702;found,165.0701.

2-(Tri ?uoromethyl)phenethyl Azide (B28).Yield 88%;a colorless oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.66(1H,d,J =7.6Hz),7.51(1H,t,J =7.6Hz),7.39?7.35(2H,m),3.51(2H,t,J =7.3Hz),3.08(2H,t,J =7.3Hz).13C NMR (CDCl 3,125MHz,δ;ppm):131.93,131.73,128.99,127.00,126.29,125.56,123.38,52.08,32.30.FTIR (neat,cm ?1)2094.MS (EI)m /z 187(M +?28).

3-(Tri ?uoromethyl)phenethyl Azide (B29).Yield 87%;a colorless oil.1H NMR (CDCl 3,600MHz,δ;ppm):7.52(1H,d,J =7.8Hz),7.48(1H,s),7.46?7.41(2H,m).13C NMR (CDCl 3,125MHz,δ;ppm):139.01,132.22,129.11,125.52,123.74,52.11,35.16.FTIR (neat,cm ?1)2094.MS (EI)m /z 153(M +?28).

4-(Tri ?uoromethyl)phenethyl Azide (B30).Yield 90%;a pale-yellow oil.1H NMR (CDCl 3,600MHz,δ;ppm):7.58(2H,d,J =8.1Hz),7.34(2H,d,J =8.0Hz),3.55(2H,t,J =7.1Hz),2.95(2H,t,J =7.1Hz).13C NMR (CDCl 3,150MHz,δ;ppm):142.16,129.13,125.60,125.08,123.27,52.02,35.16.FTIR (neat,cm ?1)2094.MS (EI)m /z 153(M +?28).

2-Nitrophenethyl Azide (B31).Yield 91%;a yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.99(1H,d,J =8.2Hz),7.58(1H,t,J =7.5Hz),7.45?7.41(2H,m),3.64(2H,t,J =6.9Hz),3.18(2H,t,J =6.9Hz).13C NMR (CDCl 3,125MHz,δ;ppm):149.34,133.28,133.13,132.86,128.14,125.14,51.48,33.00.FTIR (neat,cm ?1)2090,1519,1342.MS (EI)m /z 164(M +?28).

3-Nitrophenethyl Azide (B32).Yield 62%;a yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):8.13?8.11(2H,m),7.58(1H,d J =7.6Hz),7.51(1H,t J =7.8Hz),3.60(2H,t,J =6.9Hz),3.00(2H,t,J =6.9Hz).13C NMR (CDCl 3,125MHz,δ;ppm):148.47,140.15,

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135.09,129.58,123.70,122.01,51.86,34.98.FTIR (neat,cm ?1)2090,1523,1346.MS (EI)m /z 164(M +?28).

4-Nitrophenethyl Azide (B33).Yield 93%;a yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):8.19(2H,d,J =8.5Hz),7.40(2H,d,J =8.5Hz),3.59(2H,t,J =6.9Hz),3.00(2H,t,J =6.9Hz).13C NMR (CDCl 3,125MHz,δ;ppm):147.05,145.77,129.70,123.89,51.72,35.19.FTIR (neat,cm ?1)2090,1516,1342.MS (EI)m /z 164(M +?28).

1-(2-Azidoethyl)naphthalene (B34).Yield 91%;a yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):8.00(1H,d,J =8.5Hz),7.89?7.87(1H,m),7.77(1H,d,J =8.2Hz),7.56?7.38(4H,m),3.64(2H,t,J =7.6Hz),3.38(2H,t,J =7.6Hz).13C NMR (CDCl 3,150MHz,δ;ppm):133.93,133.86,131.70,129.01,127.68,127.00,126.27,125.72,125.55,123.14,51.74,32.49.FTIR (neat,cm ?1)2090.HRMS (EI)calcd for C 12H 11N 3,197.0953;found,197.0954.

3-(2-Azidoethyl)indole (B36).Yield 92%;a pale-yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):8.01(1H,s),7.59(1H,dd,J =1.2,7.9Hz),7.37(1H,dt,J =0.9,8.2Hz),7.21(1H,m),7.14(1H,m),7.07(1H,d,J =2.4Hz),3.57(2H,t,J =7.2Hz),3.07(2H,dt,J =0.9,7.0Hz).13C NMR (CDCl 3,150MHz,δ;ppm):136.25,127.13,122.24,122.21,119.56,118.52,112.37,111.27,51.66,25.09.FTIR (neat,cm ?1)2090.HRMS (EI)calcd for C 10H 10N 4,186.0906;found,186.0904.

2-Azidoacetophenone (B45).Yield 53%;a yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.93?7.90(2H,m),7.65?7.61(1H,m),7.52?7.49(2H,m),4.57(2H,s).13C NMR (CDCl 3,150MHz,δ;ppm):193.19,134.41,134.15,129.00,127.95,54.90.FTIR (neat,cm ?1)2098,1693.MS (EI)m /z 133(M +?28).

Azidomethylcyclopentane (B13).Step 1:Preparation of Cyclo-pentylmethyl Tosylate (23a ).To a solution of cyclopentanemethanol (22a ,500mg,4.99mmol)in pyridine (5mL)was added TsCl (1.43g,7.49mmol)at 0°C.The reaction mixture was stirred for 36h at room temperature.After removal of the solvent,the residue was diluted with AcOEt.The AcOEt solution was washed with saturated NaHCO 3and brine and dried over Na 2SO 4.Filtration,concentration in vacuo,and puri ?cation of the residue by silica gel ?ash column chromatography (AcOEt/n -hexane =1/50)gave 864mg (68%)of 23a as a colorless oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.79(2H,d,J =8.2Hz),7.35(2H,d,J =7.9Hz),3.89(2H,d,J =7.3Hz),2.50(3H,s),2.24?2.15(1H,m),1.74?1.68(2H,m),1.57?1.49(4H,m),1.22?1.15(2H,m).

Step 2:Preparation of Azidomethylcyclopentane (B13).To a 0.5M solution of NaN 3in DMSO (26.6mL,13.3mmmol)was added 23a (1.13g,13.3mmmol)obtained above,and the mixture was stirred at 80°C for 5h.The reaction mixture was quenched with water and stirred until it cooled to room temperature,then extracted with AcOEt.The AcOEt layer was separated,washed with water and brine,and dried over Na 2SO 4.Filtration and concentration in vacuo gave 147mg (26%)of B13as a yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):3.20(2H,d,J =7.3Hz),2.19?2.10(1H,m),1.83?1.77(2H,m),1.64?1.55(4H,m),1.29?1.21(2H,m).13C NMR (CDCl 3,125MHz,δ;ppm):56.51,39.64,30.33,25.28;FTIR (neat,cm ?1)2098.Azides B15and B41?B43were prepared from the corresponding alcohols using the same procedure described for B13.

1-Azidomethyladamantane (B15).30Yield 45%;a colorless oil.1H NMR (CDCl 3,500MHz,δ;ppm):2.95(2H,s),1.99(3H,s),1.72(3H,d,J =12Hz),1.64(3H,d,J =12Hz),1.52(6H,d,J =2.4Hz).13

C NMR (CDCl 3,125MHz,δ;ppm):64.37,40.09,36.85,34.78,28.21.FTIR (neat,cm ?1)2094.MS (EI)m /z 191(M +).HRMS (EI)calcd for C 11H 17N 3,191.1423;found,191.1424.

(2-Azidoethyl)cyclopentane (B41).Yield 53%;a pale-yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):3.27(2H,t,J =7.3Hz),1.87?1.78(3H,m),1.64?1.62(4H,m),1.60?1.49(2H,m).13C NMR (CDCl 3,150MHz,δ;ppm):50.83,37.41,34.92,32.48,25.05.FTIR (neat,cm ?1)2090.

(2-Azidoethyl)cyclohexane (B42).Yield 55%;a pale-yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):3.29(2H,t,J =7.2Hz),1.72?1.61(5H,m),1.50(2H,q,J =7.1Hz),1.38?1.37(1H,m),1.28?1.11(3H,m),0.951(2H,m).13C NMR (CDCl 3,125MHz,δ;ppm):49.20,36.05,35.00,33.02,26.44,26.14.FTIR (neat,cm ?1)2090.

(2-Azidoethyl)-1-adamantane (B43).Yield 79%;a colorless oil.1H NMR (CDCl 3,500MHz,δ;ppm):3.27(2H,t,J =7.9Hz),1.96(3H,s),1.72?1.62(6H,m),1.51(6H,d,J =2.1Hz),1.40(2H,t,J =7.9Hz).13C NMR (CDCl 3,125MHz,δ;ppm):46.61,42.42,42.34,36.98,31.80,30.93,28.55.FTIR (neat,cm ?1)2090.

4-Phenoxybenzyl Azide (B10).Step 1:4-Phenoxyphenylmethanol (25).A solution of 4-phenoxybenzoic acid (24,2.00g,9.34mmol)in THF (45mL)was added dropwise to a suspension of LiAlH 4(1.06g,28.0mmol)in THF (65.0mL)with cooling by an ice-bath.The mixture was heated at re ?ux for 7h.After cooling of the reaction mixture,water (1.00mL),15%aqueous NaOH (1.00mL),and water (3.00mL)were successively added,and the slurry was ?ltered.The solid was washed with THF,the combined ?ltrates were concentrated in vacuo,and the residue was puri ?ed by silica gel ?ash column chromatography (AcOEt/n -hexane =1/3)to give 1.63g (87%)of 25as a white solid.1H NMR (CDCl 3,500MHz,δ;ppm):7.35?7.31(4H,m),7.10(1H,t,J =7.5Hz),7.02?7.00(4H,m),4.67(2H,d,J =5.8Hz).

Step 2:Preparation of 4-Phenoxybenzyl Azide (B10).To a mixture of 25(300mg,1.50mmol)obtained above and DPPA (495mg,1.80mmol)in dry DMF (2.7mL)was added neat DBU (274mg,1.80mmol)at 0°C under Ar.The reaction mixture was stirred at room temperature for 24h and then poured into water and extracted with AcOEt.The AcOEt layer was separated,washed with water,2N aqueous HCl,and brine,and dried over Na 2SO 4.Filtration,concentration in vacuo,and puri ?cation of the residue by silica gel ?ash column chromatography (AcOEt/n -hexane =1/50)gave 103mg (30%)of B10as a colorless oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.37?7.31(2H,m),7.10(1H,t,J =7.5Hz),7.02?7.00(4H,m),2H (d,J =5.8Hz).13C NMR (CDCl 3,125MHz,δ;ppm):157.49,156.81,130.01,129.81,123.57,119.15,118.89,54.30.FTIR (neat,cm ?1)2090.MS (EI)m /z 225(M +).HRMS (EI)calcd for C 13H 11ON 3,225.0903;found,225.0900.

2-(2-Azidoethyl)naphthalene (B35).To a solution of 2-naphtha-leneethanol (27a ,1.00g,5.81mmol)in CH 2Cl 2(4mL)were added triethylamine (705mg,6.97mmol)and MsCl (798mg,6.97mmol)at 0°C.The mixture was stirred for 3h at room temperature and then poured into saturated aqueous NaHCO 3and extracted with CHCl 3.The organic layer was washed with water and brine and dried over Na 2SO 4.Filtration and concentration in vacuo gave the crude mesylate.To a 0.5M solution of NaN 3in DMSO (13.9mL,6.97mmmol)was added the mesylate,and the mixture was stirred at 80°C for 2h and then poured into water and extracted with AcOEt.The AcOEt layer was washed with water and brine and dried over Na 2SO 4.Filtration,concentration in vacuo,and puri ?cation of the residue by silica gel ?ash column chromatography (AcOEt/n -hexane =1/50)gave 906mg (79%,2steps)of B35as a white solid.The solid was recrystallized from AcOEt/n -hexane to give 319mg of B35as a white solid.1H NMR (CDCl 3,500MHz,δ;ppm):7.83?7.79(3H,m),7.68(1H,s),7.49?7.43(2H,m),7.35(1H,dd,J =1.8,8.2Hz),3.60(2H,t,J =7.3Hz),3.06(2H,t,J =7.3Hz).13C NMR (CDCl 3,125MHz,δ;ppm):135.49,133.58,132.38,128.33,127.69,127.55,127.31,126.98,126.19,125.65,52.41,35.52.FTIR (neat,cm ?1)2075.HRMS (EI)calcd for C 12H 11N 3,197.0950;found,197.0952.

Azides B37,B39,and B40were prepared from the corresponding alcohols using the same procedure described for B35.

3-(2-Azidoethyl)thiophene (B37).Yield 84%;a colorless oil.1H NMR (CDCl 3,500MHz,δ;ppm):7.29(1H,m),7.06(1H,d,J =1.8Hz),6.98(1H,d,J =4.3Hz),3.51(2H,t,J =7.0Hz),2.93(2H,t,J =7.0Hz).13C NMR (CDCl 3,150MHz,δ;ppm):138.26,127.97,125.95,121.79,51.79,29.81.FTIR (neat,cm ?1)2086.MS (EI)m /z 125(M +?28).

5-(2-Azidoethyl)-4-methylthiazole (B39).Yield 86%;a colorless oil.1H NMR (CDCl 3,500MHz,δ;ppm):8.62(1H,s),3.51(2H,t,J =6.9Hz),3.04(2H,t,J =6.9Hz),2.43(3H,s).13C NMR (CDCl 3,125MHz,δ;ppm):150.06,150.02,127.01,52.06,26.27,14.95.FTIR (neat,cm ?1)2090.HRMS (EI)calcd for C 6H 8N 4S,168.0473;found,198.0475.

4-(2-Azidoethyl)morpholine (B40).Yield 50%;a yellow oil.1H NMR (CDCl 3,500MHz,δ;ppm):3.73(4H,t,J =4.7Hz),3.35(2H,

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t,J=5.9Hz),2.60(2H,t,J=5.9Hz),2.51(4H,t,J=4.6Hz).13C NMR(CDCl3,125MHz,δ;ppm):66.91,57.63,53.62,47.94.FTIR (neat,cm?1)2094.

2-(2-Azidoethyl)pyridine(B38).To a solution of2-pyridineethanol (27c,1.10g,8.93mmol)and CBr4(3.26g,9.83mmol)in CH2Cl2(10 mL)was added a solution of PPh3(2.58g,9.83mmol)in CH2Cl2(5 mL)at0°C,and the mixture was stirred for1h at room temperature and then concentrated in vacuo.Puri?cation of the residue by silica gel ?ash column chromatography(AcOEt/n-hexane=1/4)gave596mg (36%)of the bromide as a colorless oil.To a0.5M solution of NaN3 in DMSO(7.68mL,3.84mmmol)was added the bromide(596mg, 3.20mmol),and the mixture was stirred at room temperature for2h. The reaction mixture was poured into water and extracted with AcOEt. The AcOEt layer was washed with water and brine and dried over Na2SO4.Filtration,concentration in vacuo,and puri?cation of the residue by silica gel?ash column chromatography(AcOEt/n-hexane= 1/3)gave207mg(44%)of B38as a pale-yellow oil.1H NMR (CDCl3,500MHz,δ;ppm):8.57(1H,d,J=4.3Hz),7.64(1H,dt,J= 1.5,7.6Hz),7.21?7.16(2H,m),3.72(2H,t,J=7.0Hz),3.06(2H,t, J=7.0Hz).13C NMR(CDCl3,150MHz,δ;ppm):158.04,149.60, 136.55,123.57,121.84,50.70,37.52.FTIR(neat,cm?1)2090.MS (EI)m/z119(M+?28).

(E)-(2-Azidovinyl)benzene(B46).To a solution of CuSO4(118mg,

0.737mmol)and NaN3(575mg,8.84mmol)in MeOH(20mL)was added(E)-2-phenylvinylboronic acid(30,1.09g,7.37mmol),and the mixture was stirred for30h at room temperature.After removal of the solvent,the residue was diluted with AcOEt.The AcOEt solution was washed water and brine and dried over Na2SO4.Filtration, concentration in vacuo,and puri?cation of the residue by silica gel ?ash column chromatography(AcOEt/n-hexane=1/10)gave425mg (40%)of B46as a yellow oil.1H NMR(CDCl3,500MHz,δ;ppm): 7.32?7.20(5H,m),6.61(1H,d,J=14Hz),6.27(1H,d,J=14Hz).

13C NMR(CDCl

3,125MHz,δ;ppm):135.02,128.76,127.39,126.68,

125.82,119.80.FTIR(neat,cm?1)2090,1635.HRMS(EI)calcd for C8H7N3,145.0644;found,145.0645.

Construction of a120-Member Triazole Library(C1?C120) and a31-Member Triazole Library(C121?C151).To a solution of an alkyne(25mM,20μL),an azide(30mM,20μL),and TBTA(5 mM,10μL)in DMSO was added an aqueous solution of CuSO4·5H2O(2mM,25μL)on a96-well plate.To the resulting mixture was added an aqueous solution of sodium ascorbate(10mM, 25μL),and the mixture was shaken for1?3days at room temperature.The reaction was monitored by TLC and LCMS. When the reaction was completed,DMSO(150μL)was added to the mixture,in which the concentration of the triazole is assumed to be2 mM.The crude triazoles were diluted to a desired concentration for enzyme assays.

3-(1-Benzyl-1H-[1,2,3]triazol-4-yl)-1-carboxylic Acid Hydroxya-mide(C31).To a solution of A3(43.2mg,0.268mmol),B1(51.8 mg,0.322mmol),and TBTA(14.2mg,10mol%)in MeOH(5mL) was added a solution of CuSO4·5H2O(6.69mg,10mol%)and sodium ascorbate(26.5mg,50mol%)in water(5mL).The reaction mixture was stirred for24h at room temperature and then poured into water and extracted with AcOEt.The AcOEt layer was separated, washed with water and brine,and dried over Na2SO4.Filtration, concentration in vacuo,and puri?cation of the residue by silica gel ?ash column chromatography(AcOEt/n-hexane=2/1)gave82.2mg (q.y.)of C31as a pale-yellow solid.The solid was recrystallized from MeOH to give40.9mg of C31as colorless crystals;mp154?155°C.

1H NMR(DMSO-d

6,600MHz,δ;ppm):11.3(1H,broad s),9.08

(1H,s)8.69(1H,s),8.23(1H,t,J=1.6Hz),7.98(1H,d,J=7.7Hz), 7.69(1H,dt,J=1.4,7.9Hz),7.52(1H,t,J=7.7Hz),7.42?7.34(5H, m),5.67(2H,s).13C NMR(DMSO-d6,125MHz,δ;ppm):163.93, 146.07,135.89,133.45,130.80,128.99,128.79,128.18,127.93,127.63, 126.18,123.73,121.90,53.06.MS(FAB)m/z295(MH+).Anal. (C16H14N4O2·H2O)C,H,N.

Compounds C32,C142,and C149were prepared from A3and the corresponding azides using the same procedure described for C31. 3-(1-Phenethyl-1H-[1,2,3]triazol-4-yl)-1-carboxylic Acid Hydrox-yamide(C32).Yield92%;colorless crystals;mp168?170°C.1H NMR(DMSO-d6,600MHz,δ;ppm):11.3(1H,broad s),9.08(1H, s)8.59(1H,s),8.20(1H,d,J=1.6Hz),7.93(1H,d,J=7.7Hz),7.68 (1H,dt,J=1.2,6.5Hz),7.52(1H,t,J=7.7Hz),7.30?7.20(5H,m), 4.68(2H,t,J=7.3Hz),3.23(2H,t,J=7.3Hz).13C NMR(DMSO-d6,125MHz,δ;ppm):163.98,145.53,137.56,133.49,130.96,128.99, 128.65,128.41,127.54,126.58,126.03,123.68,121.90,50.65,35.49. MS(FAB)m/z309(MH+).Anal.(C17H16N4O2·H2O)C,H,N.

N-Hydroxy-3-[1-(2-thiophen-3-yl-ethyl)-1H-[1,2,3]triazol-4-yl]-benzamide(C142).Yield100%;colorless crystals;mp164?166°C. 1H NMR(DMSO-d

,500MHz,δ;ppm):11.3(1H,broad s),9.09 (1H,s)8.59(1H,s),8.21(1H,s),7.94(1H,d,J=7.6Hz),7.68(1H, d,J=7.9Hz),7.52(1H,t,J=7.6Hz),7.48?7.46(1H,m),7.24(1H, s),7.00(1H,d,J=4.8Hz),4.67(2H,t,J=7.3Hz),3.25(2H,t,J= 7.3Hz).13C NMR(DMSO-d6,150MHz,δ;ppm):163.88,145.47, 137.68,133.40,130.89,128.93,128.11,127.48,126.12,125.95,123.62, 122.01,121.59,49.94,30.08.MS(FAB)m/z315(MH+).Anal. (C15H14N4O2S)C,H,N.

N-Hydroxy-3-(1-phenylsulfanylmethyl-1H-[1,2,3]triazol-4-yl)-benzamide(C149).Yield92%;colorless crystals;mp160?161°C.1H NMR(DMSO-d6,500MHz,δ;ppm):11.3(1H,broad s),9.09(1H, s)8.63(1H,s),8.21(1H,t,J=1.5Hz),7.96(1H,d,J=7.9Hz),7.70 (1H,dt,J=1.2,7.6Hz),7.52(1H,t,J=7.9Hz),7.45?7.43(2H,m), 7.37?7.30(3H,m),6.01(2H,s).13C NMR(DMSO-d6,125MHz,δ; ppm):163.87,146.10,133.47,132.27,130.66,130.52,129.31,129.06, 127.79,127.70,126.35,123.71,121.37,52.02.MS(FAB)m/z327 (MH+).Anal.(C16H14N4O2S)C,H,N.

Biology.Enzyme Assays.The HDAC activity assay was performed using an HDACs/HDAC8deacetylase?uorometric assay kit(CY-1150/CY-1158,Cyclex Company Limited),HDAC1/HDAC6?uo-rescent activity drug discovery kit(AK-511/AK-516,BIOMOL Research Laboratories),?uorescent SIRT1activity assay/drug discovery kit(AK-555,BIOMOL Research Laboratories),or ?uorogenic HDAC class2αassay kit(BPS Bioscience Incorporated) with HDACs(CY-1150,Cyclex Company Limited),HDAC1(SE-456, BIOMOL Research Laboratories),HDAC2(SE-500,BIOMOL Research Laboratories),HDAC4(BPS Bioscience Incorporated), HDAC6(SE-508,BIOMOL Research Laboratories),and HDAC8 (CY-1158,Cyclex Company Limited),according to the supplier’s instructions.The?uorescence of the wells was measured on a ?uorometric reader with excitation set at360nm and emission detection set at460nm,and the%inhibition was calculated from the ?uorescence readings of inhibited wells relative to those of control wells.The concentration of a compound that results in50%inhibition (IC50)was determined by plotting log[Inh]versus the logit function of %inhibition.IC50values were determined by regression analysis of the concentration/inhibition data.For determination of IC50values, various concentrations(0.0001,0.001,0.01,0.1,1.0,10,and100μM)of inhibitors were used.

Cell Growth Inhibition Assay.The cells were plated at the initial density of2×105cells/well(50μL/well)in96-well plates in medium culture and exposed to inhibitors for72h at37°C in5%CO2 incubator.A solution of3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme-thoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt(MTS)was then added(20μL/well),and incubation was continued for2h.The solubilized dye was quanti?ed by colorimetric reading at490nm using a reference wavelength of650nm.The absorbance values of control wells(C)and test wells(T)were measured.The absorbance of the test wells(T0)was also measured at time0(addition of compounds). Using these measurements,cell growth inhibition(percentage of growth)by a test inhibitor at each concentration used was calculated as:%growth=100[(T?T0)/(C?T0)],when T>T0and%growth =100[(T?T0)/T],when T

Western Blot Analysis.The cohesin-or H4-acetylating activities of the test compounds were assayed according to the method reported in ref24.

Molecular Modeling.The X-ray structure of HDAC8(PDB code 1VKG)was used as the target structure for docking.Protein preparation,receptor grid generation,and ligand docking were

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performed using the software https://www.docsj.com/doc/7c13170984.html,pounds C149and6were docked into the ligand binding site of HDAC8.The standard precision mode of Glide was used to determine favorable binding poses,which allowed the ligand conformation to be?exibly explored while holding the protein as a rigid structure during docking.Then,the predicted complex structure was fully energy-minimized,with both the protein and the ligand allowed to move,using Macromodel8.1software.The conformation of compounds C149and6in the ligand binding site was minimized by MM calculation based on the OPLS-AA force?eld with the following parameter set:solvent,water;method,LBFGS;max no. of iterations,10000;converge,gradient;convergence threshold,0.05.■ASSOCIATED CONTENT

*Supporting Information

Results of LCMS analysis of representative compounds,the 120-member triazole library for HDAC inhibitors obtained by the combination of alkynes A1?A8and azides B1?B15,the 31-member C32-based library for HDAC8-selective inhibitors obtained by the combination of alkyne A3and azides B16?B46,and results of elemental analysis of A3?A8,C31,C32, C142,and C149are reported.This material is available free of

charge via the Internet at https://www.docsj.com/doc/7c13170984.html,.

■AUTHOR INFORMATION

Corresponding Author

*Phone/Fax:+81-52-836-3407.E-mail:suzukit@koto.kpu-m. ac.jp(T.S.);miyata-n@phar.nagoya-cu.ac.jp(N.M.).

Notes

The authors declare no competing?nancial interest.■ACKNOWLEDGMENTS

We thank Mie Tsuchida for her technical support.This work was supported in part by JST PRESTO program(T.S.),a Grant-in-Aid for Scienti?c Research from the JapanSociety for the Promotion of Science(T.S.),Foundation NAGASE Science Technology Development(T.S.),and the Shorai Foundation

for Science and Technology(T.S.).

■ABBREVIATIONS USED

HDAC,histone deacetylase;SIRT,sirtuin;TSA,trichostatin A; CuAAC,Cu(I)-catalyzed azide?alkyne cycloaddition;ZBG, zinc-binding group;TBTA,tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine;SMC,structural maintenance of chromo-

some;PBMC,peripheral blood mononuclear cell

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(22)(a)Rostovtsev,V.V.;Green,L.G.;Fokin,V.V.;Sharpless,K.B.

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外文文献: Designing Against Fire Of Buliding John Lynch ABSTRACT: This paper considers the design of buildings for fire safety. It is found that fire and the associ- ated effects on buildings is significantly different to other forms of loading such as gravity live loads, wind and earthquakes and their respective effects on the building structure. Fire events are derived from the human activities within buildings or from the malfunction of mechanical and electrical equipment provided within buildings to achieve a serviceable environment. It is therefore possible to directly influence the rate of fire starts within buildings by changing human behaviour, improved maintenance and improved design of mechanical and electrical systems. Furthermore, should a fire develops, it is possible to directly influence the resulting fire severity by the incorporation of fire safety systems such as sprinklers and to provide measures within the building to enable safer egress from the building. The ability to influence the rate of fire starts and the resulting fire severity is unique to the consideration of fire within buildings since other loads such as wind and earthquakes are directly a function of nature. The possible approaches for designing a building for fire safety are presented using an example of a multi-storey building constructed over a railway line. The design of both the transfer structure supporting the building over the railway and the levels above the transfer structure are considered in the context of current regulatory requirements. The principles and assumptions associ- ated with various approaches are discussed. 1 INTRODUCTION Other papers presented in this series consider the design of buildings for gravity loads, wind and earthquakes.The design of buildings against such load effects is to a large extent covered by engineering based standards referenced by the building regulations. This is not the case, to nearly the same extent, in the

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New technique of the computer network Abstract The 21 century is an ages of the information economy, being the computer network technique of representative techniques this ages, will be at very fast speed develop soon in continuously creatively, and will go deep into the people's work, life and study. Therefore, control this technique and then seem to be more to deliver the importance. Now I mainly introduce the new technique of a few networks in actuality live of application. keywords Internet Network System Digital Certificates Grid Storage 1. Foreword Internet turns 36, still a work in progress Thirty-six years after computer scientists at UCLA linked two bulky computers using a 15-foot gray cable, testing a new way for exchanging data over networks, what would ultimately become the Internet remains a work in progress. University researchers are experimenting with ways to increase its capacity and speed. Programmers are trying to imbue Web pages with intelligence. And work is underway to re-engineer the network to reduce Spam (junk mail) and security troubles. All the while threats loom: Critics warn that commercial, legal and political pressures could hinder the types of innovations that made the Internet what it is today. Stephen Crocker and Vinton Cerf were among the graduate students who joined UCLA professor Len Klein rock in an engineering lab on Sept. 2, 1969, as bits of meaningless test data flowed silently between the two computers. By January, three other "nodes" joined the fledgling network.

10kV小区供配电英文文献及中文翻译

在广州甚至广东的住宅小区电气设计中,一般都会涉及到小区的高低压供配电系统的设计.如10kV高压配电系统图,低压配电系统图等等图纸一大堆.然而在真正实施过程中,供电部门(尤其是供电公司指定的所谓电力设计小公司)根本将这些图纸作为一回事,按其电脑里原有的电子档图纸将数据稍作改动以及断路器按其所好换个厂家名称便美其名曰设计(可笑不?),拿出来的图纸根本无法满足电气设计的设计意图,致使严重存在以下问题:(也不知道是职业道德问题还是根本一窍不通) 1.跟原设计的电气系统货不对板,存在与低压开关柜后出线回路严重冲突,对实际施工造成严重阻碍,经常要求设计单位改动原有电气系统图才能满足它的要求(垄断的没话说). 2.对消防负荷和非消防负荷的供电(主要在高层建筑里)应严格分回路(从母线段)都不清楚,将消防负荷和非消防负荷按一个回路出线(尤其是将电梯和消防电梯,地下室的动力合在一起等等,有的甚至将楼顶消防风机和梯间照明合在一个回路,以一个表计量). 3.系统接地保护接地型式由原设计的TN-S系统竟曲解成"TN-S-C-S"系统(室内的还需要做TN-C,好玩吧?),严格的按照所谓的"三相四线制"再做重复接地来实施,导致后续施工中存在重复浪费资源以及安全隐患等等问题.. ............................(违反建筑电气设计规范等等问题实在不好意思一一例举,给那帮人留点混饭吃的面子算了) 总之吧,在通过图纸审查后的电气设计图纸在这帮人的眼里根本不知何物,经常是完工后的高低压供配电系统已是面目全非了,能有百分之五十的保留已经是谢天谢地了. 所以.我觉得:住宅建筑电气设计,让供电部门走!大不了留点位置,让他供几个必需回路的电,爱怎么折腾让他自个怎么折腾去.. Guangzhou, Guangdong, even in the electrical design of residential quarters, generally involving high-low cell power supply system design. 10kV power distribution systems, such as maps, drawings, etc. low-voltage distribution system map a lot. But in the real implementation of the process, the power sector (especially the so-called power supply design company appointed a small company) did these drawings for one thing, according to computer drawings of the original electronic file data to make a little change, and circuit breakers by their the name of another manufacturer will be sounding good design (ridiculously?), drawing out the design simply can not meet the electrical design intent, resulting in a serious following problems: (do not know or not know nothing about ethical issues) 1. With the original design of the electrical system not meeting board, the existence and low voltage switchgear circuit after qualifying serious conflicts seriously hinder the actual construction, often require changes to the original design unit plans to meet its electrical system requirements (monopoly impress ). 2. On the fire load and fire load of non-supply (mainly in high-rise building in) should be strictly sub-loop (from the bus segment) are not clear, the fire load and fire load of non-qualifying press of a circuit (especially the elevator and fire elevator, basement, etc.

3英文文献及翻译格式示例

哈尔滨工业大学毕业设计（论文）英文原文（原文也可以直接将PDF版打印） ASSESSING CREDIT OR DETERMINING QUANTITY? THE EVOLVING ROLE OF RATING AGENCIES Lynnette D. Purda* This version: April 21, 2011 Abstract Over the past ten years, credit rating agencies have come under intense criticism from both practitioners and academics, first for their failure to identify problems resulting in bankruptcies at Enron and Worldcom and second for providing overly optimistic ratings for structured finance products. While many investors question the value of rating agencies in light of these criticisms, they have proven remarkably resilient. This paper provides a brief background on how rating agencies secured competitive advantages in evaluating credit quality. It then reviews the empirical evidence on the information content of ratings given these advantages. I argue that the information content of ratings stems from two intertwined sources: 1) information related to credit quality and 2) information related to the firm’s ability to access debt. Based on this evidence, I suggest that the dominant role for ratings today is as a benchmark for financial contracting. In this way, ratings remain influential in establishing the supply and demand of debt securities. 译文评级机构的发展的作用评估信用还是决定数量？本文：2011.4.21 摘要在过去的十年，信用评级机构一直处于来自实践者和学者的激烈的批评中，首先他们未能发现问题，导致安然和世通破产；其次对结构性金融产品提供过于乐观的评级。虽然许多投资者因为这些批评对评级机构的价值提出了质疑，但他们仍然被证明是相当有活力的。这篇文章首先在评估机构如何在信用评级质量中获得竞争优势提供一个简单地背景介绍，然后考虑到这些优势回顾了一些信息内容方面的评级经验证据。个人认为信息内容的评级来自两种交织在一起（错综复杂）的来源：1）和信贷质量相关的信息；2)和公司获取债务资本能力相关的信息。以此为据，我建议当前评级的主导作用是作为基准的金融收缩。以这种方式，在建立债券的供应和需求方面评级仍然是有效的。 - -1

土木工程外文文献翻译

专业资料学院：专业：土木工程姓名：学号：外文出处：Structural Systems to resist (用外文写) Lateral loads 附件：1.外文资料翻译译文；2.外文原文。

附件1：外文资料翻译译文抗侧向荷载的结构体系常用的结构体系若已测出荷载量达数千万磅重，那么在高层建筑设计中就没有多少可以进行极其复杂的构思余地了。确实，较好的高层建筑普遍具有构思简单、表现明晰的特点。这并不是说没有进行宏观构思的余地。实际上，正是因为有了这种宏观的构思，新奇的高层建筑体系才得以发展，可能更重要的是：几年以前才出现的一些新概念在今天的技术中已经变得平常了。如果忽略一些与建筑材料密切相关的概念不谈，高层建筑里最为常用的结构体系便可分为如下几类： 1.抗弯矩框架。 2.支撑框架，包括偏心支撑框架。 3.剪力墙，包括钢板剪力墙。 4.筒中框架。 5.筒中筒结构。 6.核心交互结构。 7. 框格体系或束筒体系。特别是由于最近趋向于更复杂的建筑形式，同时也需要增加刚度以抵抗几力和地震力，大多数高层建筑都具有由框架、支撑构架、剪力墙和相关体系相结合而构成的体系。而且，就较高的建筑物而言，大多数都是由交互式构件组成三维陈列。将这些构件结合起来的方法正是高层建筑设计方法的本质。其结合方式需要在考虑环境、功能和费用后再发展，以便提供促使建筑发展达到新高度的有效结构。这并

不是说富于想象力的结构设计就能够创造出伟大建筑。正相反，有许多例优美的建筑仅得到结构工程师适当的支持就被创造出来了，然而，如果没有天赋甚厚的建筑师的创造力的指导，那么，得以发展的就只能是好的结构，并非是伟大的建筑。无论如何，要想创造出高层建筑真正非凡的设计，两者都需要最好的。虽然在文献中通常可以见到有关这七种体系的全面性讨论，但是在这里还值得进一步讨论。设计方法的本质贯穿于整个讨论。设计方法的本质贯穿于整个讨论中。抗弯矩框架抗弯矩框架也许是低，中高度的建筑中常用的体系，它具有线性水平构件和垂直构件在接头处基本刚接之特点。这种框架用作独立的体系，或者和其他体系结合起来使用，以便提供所需要水平荷载抵抗力。对于较高的高层建筑，可能会发现该本系不宜作为独立体系，这是因为在侧向力的作用下难以调动足够的刚度。我们可以利用STRESS，STRUDL 或者其他大量合适的计算机程序进行结构分析。所谓的门架法分析或悬臂法分析在当今的技术中无一席之地，由于柱梁节点固有柔性，并且由于初步设计应该力求突出体系的弱点，所以在初析中使用框架的中心距尺寸设计是司空惯的。当然，在设计的后期阶段，实际地评价结点的变形很有必要。支撑框架支撑框架实际上刚度比抗弯矩框架强，在高层建筑中也得到更广泛的应用。这种体系以其结点处铰接或则接的线性水平构件、垂直构件和斜撑构件而具特色，它通常与其他体系共同用于较高的建筑，并且作为一种独立的体系用在低、中高度的建筑中。

变电站_外文翻译_外文文献_英文文献_变电站的综合概述

英文翻译 A comprehensive overview of substations Along with the economic development and the modern industry developments of quick rising, the design of the power supply system become more and more completely and system. Because the quickly increase electricity of factories, it also increases seriously to the dependable index of the economic condition, power supply in quantity. Therefore they need the higher and more perfect request to the power supply. Whether Design reasonable, not only affect directly the base investment and circulate the expenses with have the metal depletion in colour metal, but also will reflect the dependable in power supply and the safe in many facts. In a word, it is close with the economic performance and the safety of the people. The substation is an importance part of the electric power system, it is consisted of the electric appliances equipments and the Transmission and the Distribution. It obtains the electric power from the electric power system, through its function of transformation and assign, transport and safety. Then transport the power to every place with safe, dependable, and economical. As an important part of power’s transport and control, the transformer substation must change the mode of the traditional design and control, then can adapt to the modern electric power system, the development of modern industry and the of trend of the society life. Electric power industry is one of the foundations of national industry and national economic development to industry, it is a coal, oil, natural gas, hydropower, nuclear power, wind power and other energy conversion into electrical energy of the secondary energy industry, it for the other departments of the national economy fast and stable development of the provision of adequate power, and its level of development is a reflection of the country's economic development an important indicator of the level. As the power in the industry and the importance of the national economy, electricity transmission and distribution of electric energy used in these areas is an indispensable component.。Therefore, power transmission and distribution is critical. Substation is to enable superior power plant power plants or power after adjustments to the lower load of books is an important part of power transmission. Operation of its functions, the capacity of a direct impact on the size of the lower load power, thereby affecting the industrial production and power consumption.Substation system if a link failure, the system will protect the part of action. May result in power outages and so on, to the production and living a great disadvantage. Therefore, the substation in the electric power system for the protection of electricity reliability,

英文论文及中文翻译

International Journal of Minerals, Metallurgy and Materials Volume 17, Number 4, August 2010, Page 500 DOI: 10.1007/s12613-010-0348-y Corresponding author: Zhuan Li E-mail: li_zhuan@https://www.docsj.com/doc/7c13170984.html, ? University of Science and Technology Beijing and Springer-Verlag Berlin Heidelberg 2010 Preparation and properties of C/C-SiC brake composites fabricated by warm compacted-in situ reaction Zhuan Li, Peng Xiao, and Xiang Xiong State Key Laboratory of Powder Metallurgy, Central South University, Changsha 410083, China (Received: 12 August 2009; revised: 28 August 2009; accepted: 2 September 2009) Abstract: Carbon fibre reinforced carbon and silicon carbide dual matrix composites (C/C-SiC) were fabricated by the warm compacted-in situ reaction. The microstructure, mechanical properties, tribological properties, and wear mechanism of C/C-SiC composites at different brake speeds were investigated. The results indicate that the composites are composed of 58wt% C, 37wt% SiC, and 5wt% Si. The density and open porosity are 2.0 g·cm–3 and 10%, respectively. The C/C-SiC brake composites exhibit good mechanical properties. The flexural strength can reach up to 160 MPa, and the impact strength can reach 2.5 kJ·m–2. The C/C-SiC brake composites show excellent tribological performances. The friction coefficient is between 0.57 and 0.67 at the brake speeds from 8 to 24 m·s?1. The brake is stable, and the wear rate is less than 2.02×10?6 cm3·J?1. These results show that the C/C-SiC brake composites are the promising candidates for advanced brake and clutch systems. Keywords: C/C-SiC; ceramic matrix composites; tribological properties; microstructure [This work was financially supported by the National High-Tech Research and Development Program of China (No.2006AA03Z560) and the Graduate Degree Thesis Innovation Foundation of Central South University (No.2008yb019).] 温压-原位反应法制备C / C-SiC刹车复合材料的工艺和性能李专，肖鹏，熊翔粉末冶金国家重点实验室，中南大学，湖南长沙410083，中国（收稿日期：2009年8月12日修订：2009年8月28日;接受日期：2009年9月2日）摘要：采用温压?原位反应法制备炭纤维增强炭和碳化硅双基体(C/C-SiC)复合材