Inhibition of both targets was further confirmed by X-raycrystal structures of Sirt2 and HDAC6 in complex with building blocksof 33. In ovarian cancer cells, 33 evokedenhanced effects on cell viability compared to single or combinationtreatment with the unconjugated Sirt2 and HDAC6 inhibitors. Thus,our dual Sirt2/HDAC6 inhibitors are important new tools to study theconsequences and the therapeutic potential of dual inhibition of tubulindeacetylation.Dysregulation ofboth tubulin deacetylases sirtuin 2(Sirt2) andthe histone deacetylase 6 (HDAC6) has been associated with the pathogenesisof cancer and neurodegeneration, thus making these two enzymes promisingtargets for pharmaceutical intervention. Herein, we report the design,synthesis, and biological characterization of the first-in-class dualSirt2/HDAC6 inhibitors as molecular tools for dual inhibition of tubulindeacetylation. Using biochemical  Despite havingdifferent catalytic mechanisms of lysine deacylation, Sirt2 and HDAC6share several common features, including their substrate spectrum,subcellular localization, and potential as a therapeutic target. Althoughboth deacylases are classified as histone deacetylases, they shareacetylated α-tubulin (α-tubulin K40ac) as their majorsubstrate and are hence frequently referred to as tubulin deacetylases.2 Beside α-tubulin, the cortical actin binding protein (cortactin)and the oncogene K-RAS have been identified as common substrates ofSirt2- and HDAC6-dependent deacetylation,4 therebyalso indicating a certain degree of functional redundancy of bothenzymes. In the case of Sirt2- and HDAC6-mediated tubulin deacetylation,the degree of functional redundancy is dependent on the architectureof hyperacetylated tubulin. Whereas most microtubule structures canbe deacetylated by both enzymes, deacetylation of perinuclear microtubulescan only be achieved by Sirt2.5 A furthercommon feature of Sirt2 and HDAC6 is their subcellular localization.Both enzymes are primarily found in the cytosol, colocalized withmicrotubules, and were even shown to interact with each other by meansof coimmunoprecipitation.2 A dysregulation of bothSirt2 and HDAC6 activity has been associated with the pathogenesisof cancer11 and neurodegeneration,15 thus making these two enzymes promising targets forpharmaceutical intervention. This has prompted intense efforts inthe development of small molecule inhibitors of Sirt2 and HDAC6, whichare extensively reviewed elsewhere.17 A selectionof highly potent and selective inhibitors of Sirt2 (1–8) and HDAC6 (9–14), respectively, is shown in 11) and itssecond generation analogue citarinostat ,entered several clinical trials, primarily for the treatment of multiplemyeloma, but also for other diseases, including malignant melanoma,advanced solid tumors, nonsmall cell lung cancer, lymphoid malignancies,and painful diabetic peripheral neuropathy.17 In most of these clinical trials, the HDAC6 preferential inhibitorwas combined with a second drug.17Both Sirt2 and HDAC6 are protein deacylasesthat cleave off acetylas well as other acyl groups from the ε-amino group of lysinesin their substrate proteins. While Sirt2 features an NAD29 which is especially relevant for cancer-targeted therapies. Additionally,dual-target therapies generally yield a high efficacy,30 thereby enabling a reduction in therapeuticdoses and hence in side effects compared to single-target drug regimens.31 The common practice of combinatorial application of HDAC6 inhibitorsalso triggered the development of dual inhibitors for HDAC6 and asecond target protein. Dual-targeting inhibitors for mTOR/HDAC6,32 LSD1/HDAC6,33 AR/HDAC6,34 and proteasome/HDAC635 were recently reported as novel anticancer drugs (15–18). One thing that has facilitated drug discovery in thisregard is the simple design of HDAC6 inhibitors. As can be seen in 36 Comparedto HDAC6, Sirt2 has been considered less frequently for dual-targetinginhibitor approaches. However, the successful development of a Sirt2affinity probe (19) and a Sirt2-targeted PROTAC  both are highlyselective for their respective target protein,27 (ii) theirbinding modes were previously elucidated via X-ray cocrystallography,35 and (iii) they were already successfullyutilized for the development of heterobivalent ligands or dual-targetedinhibitors, respectively.37 The chemical structures of thereported heterobivalent Sirt2 ligands and the dual HDAC6 inhibitors. For dual Sirt2/HDAC6 inhibitors withlinked Sirt2- and HDAC6-targeted pharmacophores, we incorporated atriazole-based linker unit into the design of these ligands, therebyenabling a conjugation via Cu(I)-catalyzed Huisgen cycloaddition.40 As linker lengths and composition are critical parameters when linkingtwo pharmacophores, we mainly focused on linker variations for structure–activityrelationship studies. In order to also provide a nonselective HDAC/Sirt2inhibitor, we linked suberoylanilide hydroxamic acid , a nonselective inhibitor of Zn2+-dependentHDACs,41 to our SirReal-based pharmacophore(see below).For the design of our dual Sirt2/HDAC6inhibitors, we referred to the SirReals as Sirt2 ligands and hibitors2 provide21 and 22 were synthesized by referring to previouslypublished protocols for the preparation of SirReals  were conjugated via standardsolution-phase Cu(I)-catalyzed Huisgen cycloaddition.39 To this end, we used the previously reported alkynylated SirRealanalogue 35 -catalyzed Huisgen cycloadditionof 34 and 35 gave the triazole 36. Similar to the aforementioned syntheses of the dual Sirt2/HDAC6inhibitors with merged pharmacophores , the final N-hydroxybenzamide of 31 was furnished by hydroxylaminolysis of the ester precursor 36. For the synthesis of the dual Sirt2/HDAC6 inhibitor 32 benzoate withbutyl amine to furnish the secondary amine 40. An amidecoupling between 40 and 5-bromopentanoic acid gave theamide 41. Subsequent conversion of the obtained alkylbromide 41 with sodium azide resulted in the azido-functionalized 42. The triazole 43 was afforded by a conjugationof 42 with the alkynylated SirReal 35 viaclick reaction. Again, the final N-hydroxybenzamide,in this case 33, was obtained by an hydroxylaminolysisof the ethyl benzoate precursor 43.For the synthesis ofdual Sirt2/HDAC6 inhibitors with linked pharmacophores,we utilized both solution-phase and solid-phase chemistry. Compoundswith short alkyl linkers between the Sirt2- and HDAC6-inhibiting pharmacophores-based linkers between the two Sirt2- and HDAC6-targeted pharmacophores,we took advantage of previously published solid-phase-supported protocolsfor the synthesis of hydroxamic acids.44 Pioneeringwork with this respect was reported in 2006 by the group of DavidFairlie.43 First, we immobilized hydroxylamineon commercially available 2-chlorotrityl chloride (2-CTC) resin. Tothis end, we treated the resin with N-hydroxyphthalimideand triethylamine. After deprotection of the phthaloyl (Phth) groupof 47 using hydrazine hydrate, N-Fmoc-protected4-(aminomethyl)benzoic acid for preferential HDAC6 inhibition or N-Fmoc-protected 7-aminoheptanoic acid aiming at nonselectiveHDAC inhibition was coupled to the functionalized resin, to obtain 48 and 50, respectively. Another coupling cyclewith N-Fmoc protected para-aminobenzoicacid provided the respective HDAC inhibitor precursors 49 and 51 -catalyzed Huisgencycloaddition,39 thus showing that the solid-phase-supportedsynthesis of hydroxamic acids can be combined with click chemistry-basedapproaches. Finally, the dual Sirt2/HDAC6 inhibitors 44 and 45 were cleaved from the resin by treatment withtrifluoroacetic acid (TFA). A highly similar procedure was appliedfor the synthesis of the SAHA-derived multitarget Sirt2/HDAC inhibitor 46  evoked both potentSirt2 and HDAC6 inhibition. The fact that the dual Sirt2/HDAC6 inhibitorswith linked pharmacophores exerted similar inhibition of Sirt2 andHDAC6 as compared to the control compounds for sole Sirt2 inhibition(4 and 5) or HDAC6 inhibition (55–57) corroborated the suitability of our designapproach for these dual inhibitors. For 32 and 33, we observed a selective inhibition of the targeted enzymesSirt2 and HDAC6 compared to the off-target deacetylases Sirt1, Sirt3,and HDAC1. The Sirt2/HDAC6 on-target selectivity of 32 and 33 was further confirmed by inhibition tests withHDAC2 and HDAC3. Consistent with the data for HDAC1, 33 also showed weak off-target inhibition of HDAC2 and HDAC3, whichmight be a consequence of its bulky tertiary amide-based cap groupof the HDAC6-targeted pharmacophore. This assumption is supportedby the high HDAC6 selectivity of 57, which is the HDAC6-targetedbuilding block of 33. As expected, the SAHA-derived multitargetSirt2/HDAC inhibitor 46 showed low selectivity for Sirt2and HDAC6. For this compound, we detected a more potent inhibitionof HDAC1 (IC50 = 0.21 μM), HDAC2 (IC50 = 0.34 μM), and HDAC3 (IC50 = 0.14 μM) comparedto Sirt2 inhibition (IC50 = 0.48 μM). To providemore comprehensive data regarding the sirtuin and HDAC selectivityof our lead structure 33, we tested 33 forinhibition of HDAC4–5, HDAC7–10, and Sirt5–6.While for HDAC8 a weak off-target inhibition with an IC50 value of 2.94 ± 0.45 μM was detected, we observed evenweaker activities for the other tested sirtuin and HDAC isotypes,thus further confirming the Sirt2/HDAC6 on-target selectivity of 33.Our setof potential dual Sirt2/HDAC6 inhibitors were first tested on theirpotency and selectivity of target protein inhibition by using previouslyreported biochemical fluorescence-based deacetylation assays 1.44−46 W5, were recently reported to inhibitSirt2-catalyzed demyristoylation.47 Thus,we were interested to see whether our dual Sirt2/HDAC6 inhibitorsalso prevent Sirt2-catalyzed defatty acylation. Using a previouslyreported biochemical in vitro demyristoylation assaythat is based on the small molecule myristoylated substrate ZMML,47 several of our triazole-based dual Sirt2/HDAC6inhibitors were identified as low micromolar inhibitors of Sirt2-catalyzeddemyristoylation  showed the weakest inhibition of Sirt2-mediated demyristoylation.To confirm the inhibition of Sirt2-mediated demyristoylation for ourlead structure 33, we also used a recently publishedbiochemical activity assay that relies on the conversion of a peptide-basedmyristoylated substrate.48 With this assay,which is known to be very sensitive,48 wedetected an IC50 value of 0.88 ± 0.09 μM , thus corroborating the inhibitionof Sirt2-mediated demyristoylation for our dual Sirt2/HDAC6 inhibitor 33.In addition to Sirt2-mediateddeacetylation, severaltriazole-basedSirReals, including oylation 2. Consisoylation 1, the inin vitro activities and selectivityprofiles of the dual Sirt2/HDAC6 inhibitors 32 as wellas 33, we set out to elucidate their binding modes totheir vastly differing targets. As cocrystallization of 32 or 33 with both target proteins as well as the singletarget proteins did not yield suitable crystals for X-ray analyses,we were aiming for cocrystallizing Sirt2 and HDAC6, respectively,with the subunits of our dual inhibitors that target the binding siteof the respective enzyme. We were able to cocrystallize Sirt2 in complexwith the triazole-based SirReal1.2  and provides an excellentbasis to rationalize the Sirt2 binding mode of our dual Sirt2/HDAC6inhibitors, as 5 is highly similar to the Sirt2-targetedsubunit of 32 and 33. The complex crystallizedin space group P21 with one monomer inthe asymmetric unit. Compared to the published structure of Sirt2in complex with a triazole-based SirReal1.1 ,19 no distinct conformational changes were observed for themain chain of the protein (root-mean-square deviation (RMSD) of 0.401Å). The observed binding mode of 5 is highly consistentwith the reported binding mode of 4.19 The dimethyl pyrimidine is tightly anchored in the selectivitypocket by hydrophobic amino acids Tyr139, Phe143, Phe190, and Leu206.A conserved water in the active site forms a hydrogen bond networkwith C=O of 5 and the main chain of Pro94. Thetypical triazole-based SirReal hydrogen bonds are formed with Arg97contributing to the affinity of 5. Additionally, thewell resolved electron density for the normally flexible methoxyethylmoiety protruding from the acyl-lysine channel indicates that thewater mediated hydrogen bond of 3.0 Å distance stabilizes theconformation in the crystal structure. This water mediated hydrogenbond also provides a possible explanation for the slightly improvedpotency of 5 (IC50 = 0.12 μM) comparedto the benzyl analogue 4 (IC50 = 0.16 μM).19Encouraged by the 2 5, see 4. The ob32 and 33, wesubmitted 55 and 57, which represent theHDAC6-targeted subunits of 32 and 33, respectively,for crystallization trials with HDAC6. Crystal structures of catalyticdomain 2 (CD2) of Danio rerio (zebrafish) HDAC6 complexedwith 55 or 57 were determined at resolutionsof 1.87 Å  and 1.77 Å , respectively. As the crystal structuresof human and zebrafish CD2 enzymes are essentially identical,49 the zebrafish HDAC6 CD2 (henceforth simply “HDAC6”)serves as a more readily studied surrogate of the human enzyme. TheHDAC6–55 complex crystallized in space group P21 with one monomer in the asymmetric unit.No significant conformational changes are observed between the structuresof the inhibitor-bound and unliganded (PDB 5EEM) HDAC6 structures, and the RMSD is 0.15Å for the 328 Cα atoms. The hydroxamate N–O– group coordinates to the catalytic Zn2+ ion with a Zn2+–O separation of 2.0 Å. Thehydroxamate C=O group accepts a hydrogen bond from the Zn2+-bound water molecule, which also forms hydrogen bonds withthe imidazole side chains of H573 and H574. Additionally, the phenolichydroxyl group of Y745 forms poorly oriented hydrogen bonds with thehydroxamate C=O and NH groups. The phenyl subunit of 55 is sandwiched between F583 and F643, making staggered π-stackinginteractions. The carbonyl of the inhibitor capping group acceptsa hydrogen bond from a nearby water molecule and is positioned 3.6Å away from a molecule of ethylene glycol. The acetamide NH groupof the inhibitor capping group is 3.7 Å from the side chain ofS531 and the acetamide methyl group is oriented toward solvent, althoughelectron density coverage is poor in this region of the electron densitymap  between the structures of inhibitor-bound and unliganded HDAC6(PDB 5EEM).The hydroxamate moiety of 57 coordinates to the catalyticZn2+ ion with bidendate geometry; the hydroxamate N–O– and C=O groups exhibit Zn2+–Oseparations of 2.0 and 2.4 Å, respectively. The Zn2+-bound N–O– group accepts a hydrogen bondfrom H573 and the Zn2+-bound C=O group accepts ahydrogen bond from Y745; the hydroxamate NH group donates a hydrogenbond to H574. The phenyl subunit of 57 is positionedin the aromatic crevice formed by F583 and F643, where it makes staggeredπ-stacking interactions similar to those observed for 55. The peptoid carbonyl group participates in a water-mediatedhydrogen bond network with H614.The HDAC6–in vitro results, we performed docking studies using the solved crystal structuresof HDAC6-57 (PDB 8G20) and Sirt2-5 (PDB 8OWZ). Results from dockingstudies using the monodentate binding mode as observed in the HDAC6-55 cocrystal structure (PDB 8G1Z) are not shown because our docking studiesstrongly supported a bidentate Zn2+-binding for our N-hydroxybenzamide-based dual Sirt2/HDAC6 inhibitors. Thegoodness of the docking setup was first tested on the solved crystalstructures and showed very good agreement between docking solutionsand crystal structures . Dockingof the dual-targeting inhibitors 32 and 33 showed that both the HDAC6 inhibitor part and the Sirt2 inhibitorpart bind to the corresponding target in an analogous way as observedfor the parent compounds in the crystal structures (32 and 33 show the same H-bridges as 5 (conserved water at Pro94 and Arg97). The N-hydroxybenzamideresidue shows van der Waals interactions with Pro99 and His187, whilethe hydroxamate is involved in an H-bridge with Ser263  simulations were performed. First, we tested the MD protocolon the solved crystal structures of Sirt2 and HDAC6. In both cases,stable complexes with low RMSD values were observed in two independent100 ns MD runs . Both protein conformation and ligand interaction exhibited littlevariation during MD simulations. In the case of the 32 and 33–Sirt2 docking complex, MD simulationsshowed stable protein conformations as well as only minor fluctuationsof the Sirt2 inhibitor moiety within the binding pocket . The intramolecular H-bridgeas well as the H-bridges between the carbonyl group of the inhibitorand the conserved water molecule were preserved. The HDAC6 targetinggroup  located at the entranceof the binding pocket showed a higher fluctuation . In the case of the simulated HDAC6–32 and 33 complexes, the protein and zinc coordinationof the inhibitor remained stable. The Sirt2 inhibitor part as wellas the interacting amino acid residues at the surface of the proteinshowed slight rearrangement but resulted in a stabilized complex after10 ns in both simulations . In summary the docking and MD simulations showed that the dual-targetinginhibitors 32 and 33 interact with Sirt2and HDAC6 similarly to the original inhibitors. For both targets,interactions of the additional inhibitor moiety have also been foundbut showed higher flexibility in the MD simulations due to their partialsolvent exposure.To analyze the stability of theobtained dockingsolutions of 50 we first used this method to evaluate the cellulareffects of our synthesized compounds. Based on the previously obtained in vitro results , potentially resultinginimpaired membrane permeability. Because our dual Sirt2/HDAC6 inhibitorswere developed as tools for dual inhibition of tubulin deacetylation,and investigating tubulin acetylation is one of the most commonlyused methods to prove cellular activity for both Sirt2 and HDAC6 inhibitors, results 1, we foc network 7.33, the increase in tubulin hyperacetylationwas especially pronounced. Control experiments with the selectiveSirt2 inhibitors 4 or 5, as well as theHDAC6 selective 57, suggest that dual Sirt2/HDAC6 inhibitionby means of 33 might have a stronger effect on tubulinacetylation compared to single or combination treatment with the unconjugatedSirt2- and HDAC6-targeted inhibitors. As the observed effects of selectiveSirt2 inhibition by single treatment with 4 or 5 were very minor and much less pronounced compared to selectiveHDAC6 inhibition by 57, we utilized a recently publishedNanoBRET assay,47 in order to verify thatthe dual Sirt2/HDAC6 inhibitor 33 really binds to Sirt2on a cellular level. Using this setup for cellular Sirt2 target engagementthat is based on the fluorescent probe SirReal-TAMRA ,51 we detected an IC50 valueof 0.56 μM for 33, which is very similar to theIC50 value of 0.32 μM determined under cell-freeconditions (4 showed a potent interaction with Sirt2 (IC50 = 0.20 μM),whereas the selective HDAC6 inhibitor 57, which was usedas a negative control, showed no Sirt2 binding. To show cellular HDAC6target engagement for 33, we performed a degradationrescue experiment, by applying the recently reported selective HDAC6-targetedPROTAC B4 .52 InMCF-7 breast cancer cells that show an upregulated HDAC6 expression,5333 is able to rescue HDAC6 fromproteasomal degradation induced by 62, thus clearly indicatingcellular HDAC6 target engagement for 33 , despite evokingsimilar in vitro activities, might be a consequenceof conformational effects caused by the additional N-butyl group in 33. The 13C spectra of 33 clearly supports that this compound can exist both as a cis and trans amide bond rotamer, whereasthe 13C spectra of 32 indicates the existenceof only one rotamer . By influencingintramolecular interactions, conformational effects can have a massiveimpact on physicochemical properties, cell permeability, and eventuallycellular effects. This is especially relevant for bifunctional moleculessuch as dual inhibitors or PROTACs, which lie within a chemical spacewhere ADME properties are often complicated.59 Western blot experiments investigating cellular histone H3 acetylation(a marker of reduced HDAC1–3 activity) supported that the effectsof 33 on cancer cell viability are not caused by an off-targetinhibition of class I HDACs . Interestingly, for two of the four tested cancer cell lines , we observed an improved activity of the dual Sirt2/HDAC6inhibitor 33 compared to single or combination treatmentwith Sirt2 and HDAC6 inhibitors. For the cancer cell line W1, theimprovement in activity of the dual Sirt2/HDAC6 inhibitor 33 was statistically significant (p = 0.007), comparedto a combination treatment with the selective Sirt2 inhibitor 4 and the selective HDAC6 inhibitor 57. However,for the interpretation of these data it should be noted that cellviability is primarily impacted by HDAC6 inhibition, the additionaleffects of Sirt2 inhibition are comparably minor, and an off-targetinhibition of other proteins outside the family of lysine deacylasescannot be ruled out.60As both Sirt2 and HDAC6 are involvedin the pathogenesis of cancer,er cells 3. All setylation 7, where in vitro assays, we identified 33 as a potentand selective inhibitor of both target enzymes. In addition to deacetylation,several of our dual Sirt2/HDAC6 inhibitors, including 33, are also able to inhibit Sirt2-catalyzed lysine defatty acylation.Applying a diverse set of cell-based methods, including NanoBRET,PROTAC rescue, and immunofluorescence microscopy, we demonstratedthat 33 is indeed able to bind to and inhibit the twotubulin deacetylases Sirt2 and HDAC6 in a cellular environment. Newcocrystal structures of Sirt2 and HDAC6, respectively, in complexwith the Sirt2- or HDAC6-targeted subunits of 33 enabledthe modeling of the binary Sirt2–33 and HDAC6–33 complexes, thereby indicating that 33 canbind to both target proteins in a similar manner as observed for theunconjugated Sirt2 and HDAC6 inhibitors. In W1 ovarian cancer cells, 33 evoked enhanced effects on cell viability compared to singleor combination treatment with the unconjugated Sirt2 and HDAC6 inhibitors.Thus, our dual Sirt2/HDAC6 inhibitors are highly interesting new toolsto investigate the consequences and the therapeutic potential of dualinhibition of the two tubulin deacetylases Sirt2 and HDAC6.In order to provide a molecular tool fora dual inhibition of thetwo tubulin deacetylases Sirt2 and HDAC6, we either merged or linkedthe pharmacophores of selective Sirt2 and HDAC6 inhibitors. For thesynthesis of these dual Sirt2/HDAC6 inhibitors, we used both solution-phaseand solid-phase chemistry. Our PEG-linker-based dual Sirt2/HDAC6 inhibitorswere synthesized by means of solid-phase support. In the course ofthese syntheses, we showed that hydroxamic acids immobilized on resincan be functionalized with click chemistry-based approaches. In biochemical 35,19 the triazole-based SirReals 4 and ,19 the Sirt2-targeted fluorescent probe SirReal-TAMRA(61),51 and the HDAC6-targetedPROTAC B4 (62) were synthesized as previously reported.52 Solvents were used in pa quality and dried accordingto common procedures, if necessary. Thin-layer chromatography (TLC)for reaction monitoring was performed with alumina plates coated withMerck silica gel 60 F254 (layer thickness: 0.2 mm) or Merck silicagel 60 RP-18 F254 (layer thickness: 0.2 mm) and analyzed under UV-light(254 nm). As an alternative method for reaction monitoring, we usedhigh performance liquid chromatography mass spectrometry (HPLC-MS).HPLC-MS analyses were performed using a Thermo Scientific Dionex UltiMate3000 HPLC system in combination with a DAD detector (220/230/254 nm)and an Agilent ZORBAX ECLIPSE, and XDB-C8 column . Elution was performed at room temperature undergradient conditions. Eluent A was water containing 0.1% (v/v) formicacid; eluent B was methanol. Linear gradient conditions were as follows:0–0.2 min: A = 75%, B = 25%; 0.2–6.0 min: linear increaseto B = 100%; 6.0–8.5 min: B = 100%; 8.5–9.0 min: lineardecrease to A = 75%, B = 25%; 9.0–12.0 min: A = 75%, B = 25%.A flow rate of 0.4 mL·min–1 was maintainedduring the entire elution. Mass detection was performed with a BRUKERamaZon SL mass spectrometer using ESI as ionization source. Flashcolumn chromatography was performed with hand packed Silica Columns60 M  as a stationaryphase on a Biotage SP-1 or Selekt automated flash purification systemwith UV–vis detector. Yields were not optimized. NMR spectrawere recorded using either a Bruker Avance 400 , Bruker Avance 600 , Bruker Avance III HD 400 , Varian/Agilent Mecury-plus-400, or Varian/AgilentMecury-plus-300  instrument.The spectra are referenced against the NMR solvent and are reportedas follows: 1H, chemical shift δ (ppm), multiplicity, integration, coupling constant (J in Hz). 13C: chemical shift δ (ppm),abbreviations: carbons that could not be found in 13C spectra(DEPTQ) but in HMBC or HSQC are additionally marked with a hashtag(#). Signals that are partially overlaid by a solvent signal are markedwith an asterisk (*). The assignment resulted from HMBC and HSQC experiments.High resolution mass spectra were either measured with a timsTOF ProMass Spectrometer from Bruker Daltonics using ESI as ionization source,a Bruker Daltonics micrOTOF coupled to a LC Packings Ultimate HPLCsystem and controlled by micrOTOFControl3.4 and HyStar 3.2-LC/MS,or a Bruker Daltonics ESI-qTOF Impact II coupled to a Dionex UltiMateTM3000 UHPLC system and controlled by micrOTOFControl 4.0 and HyStar3.2-LC/MS. Purity was determined for all tested compounds by HPLCand UV detection and was >95%. HPLC analyses for compounds 21 and 22, 31–33, and 55–57 were performed usingan Agilent1200 series HPLC system employing a diode array detector . If not stated otherwise, the indicatedpurity was determined at a wavelength of 254 nm. For method 1 (M1),we used a ZORBAX ECLIPSE, XDB-C8 column with a flow rate of 0.5 mL·min–1. Elution wasperformed at room temperature under gradient conditions. Eluent Awas water containing 0.1% (v/v) TFA; eluent B was acetonitrile. Lineargradient conditions were as follows: 0–3.0 min: A = 90%, B= 10%; 3.0–18.0 min: linear increase to A = 5%, B = 95%; 18.0–24.0min: A = 5%, B = 95%; 24.0–27.0 min: linear decrease to A =90%, B = 10%; 27.0–30.0 min: A = 90%, B = 10%. For method 2(M2), we used a NUCLEODUR C18 Pyramid column  with a flow rate of 0.5 mL·min–1: Elution was performed at room temperature under gradient conditions.Eluent A was water containing 0.3% (v/v) formic acid; eluent B wasacetonitrile. Linear gradient conditions were as follows: 0–3.0min: A = 90%, B = 10%; 3.0–18.0 min: linear increase to B =100%; 18.0–24.0 min: B = 100%; 24.0–27.0 min: lineardecrease to A = 90%, B = 10%; 27.0–30.0 min: A = 90%, B = 10%.HPLC analyses for compounds 44–46 were performed using method 3 (M3). Here, either a Thermo FisherScientific UltiMateTM 3000 UHPLC system or a Gynkotek Gina 50 HPLCsystem  with a Nucleodur 5 μm, C18 100 Å column were used. In the processa flow rate of 1 mL·min–1 and a temperatureof 25 °C were set. Detection was implemented by UV absorptionmeasurement at a wavelength of λ = 254 nm. Elution was performedunder gradient conditions. Eluent A was water containing 0.1% (v/v)TFA; eluent B was acetonitrile containing 0.1% (v/v) TFA. Linear gradientconditions were as follows: 0–5.0 min: A = 95%, B = 5%; 5.0–20.0min: linear increase to A = 5%, B = 95%; 20.0–25.0 min: A =5%, B = 95%.Starting materials were purchased from commercial suppliers  and used without any furtherpurification. The alkynylated 2Cl2 (v/v), treatment for 1 h at ambient temperature) and the compoundswere purified by preparative RP-HPLC. Fractions containing the desiredfinal compounds were collected and lyophilized yielding the dual-or multitarget inhibitors 41–43 with >95% purity in allcases.For all syntheses carried out on solid-phase, 2-chlorotritylchlorideresin  wasused. The manual solid-phase synthesis was carried out in PP-reactorswith PE frit . Syntheses carried out after the modification of the resin anddetermination of the loading followed the standard Fmoc solid-phasemethod. In brief, after resin swelling for 30 min in DMF, the standardprocedure was carried out by repeating the Fmoc-deprotection and amidecoupling. Completion of each coupling step was monitored TNBS testusing a TNBS test kit supplied by TCI. After the last reaction cyclewas performed, the final compounds were cleaved from the resin usingthe standard cleavage cocktail (5% TFA/95% CHA little amount of resin-beads was placedin a 0.5 mL microcentrifuge tube. One drop of picrylsulfonic acid(∼1% in DMF) and two drops of DIPEA (10% in DMF) were addedto the resin beads. The test was evaluated after 5 min reaction timeat room temperature.in vacuo giving the crude products, which were dissolvedin Milli-Q H2O/acetonitrile for analytical purposes viaHPLC. A cleavage on a larger scale was carried out in the same way.For each 40 mg of resin 1.0 mL of cleavage cocktail was used.Fora test cleavage, about2 mg of the dried resin was placed into a tube and treated with thestandard cleavage solution for 1 h. After the reaction period wasfinished, the filtrate was collected and the solvent was removed 300 nm(dibenzofulvene) = 7800 M–1 cm–1). With the concentration of the cleaved dibenzofulvenecalculated by using Lambert–Beer law and the mass of the resin,the loading could be determined. The full synthetic procedures areprovided along with the procedures for the synthesis of the individualcompounds (see below).A small part ofthe different preloaded resin (∼5 mg) was treated with 500μL of the deprotection solution (20% piperidine in DMF) for5 min. The filtrate was collected, and the procedure was repeatedonce. The absorbance of the combined filtrates was measured at a wavelengthof 300 nm and the concentration was determined photometrically  was dissolvedin a mixture of 0.35 mL of dichloromethane and 0.71 mL methanol andcooled on an ice bath to 0 °C. Then 220 μL  of an aqueous 50 wt % hydroxylamine solutionwere added and the mixture was stirred at 0 °C for 10 min before47.3 mg  sodium hydroxide were added. After 30min at 0 °C, the ice bath was removed and stirring of the colorlessclear solution was continued at ambient temperature for 2 h. The reactiondried under reduced pressure and the residue was taken up in 2.5 mLof deionized water. The suspension was cooled in an ice bath and broughtto pH = 8 by the dropwise addition of 1 M HCl, when large amountsof white solid formed. The solid was collected by centrifugation.The precipitate was washed with water (2 × 1 mL) and ice-colddiethyl ether (2 mL) and dried under reduced pressure. For the finalpurification, preparative HPLC (gradient elution from 25% to 50% acetonitrile/water(+ 0.1% TFA)) was used to yield the TFA salt of the title compoundas a colorless solid . 1H NMR : 12.25 , 11.20, 8.86 , 7.65 , 7.58 , 7.43–7.34 , 7.28 , 6.94 ,4.12 , 4.08 , 2.28 . 13C NMR : 168.9 q (pyrimidine C-2),167.0 q , 166.8 q (−NH-CO-CH2-S−), 164.1 q (−CO-NH-OH), 158.3 q , 157.0 q (thiazole C-2), 140.6 q (N-hydroxybenzamide C-3), 134.9 (thiazole C-4), 133.1 q (N-hydroxybenzamide C-1), 131.1 (N-hydroxybenzamideC-4), 130.7 q (thiazole C-5), 128.5 (N-hydroxybenzamideC-2), 127.0 (N-hydroxybenzamide C-5), 124.8 (N-hydroxybenzamide C-6), 116.1 (pyrimidine C-5), 115.3 q, 34.1 (−NH-CO-CH2-S−), 31.7 (thiazole-CH2−), 23.2 (pyrimidine-CH3), 19F NMR : −74.47. HRMS (ESI+): m/z calcd for C19H19N5O3S2Na+: 452.0822 [M+ Na]+; found, 452.0819. LRMS m/z (ESI+): 430 [M + H]+. HPLC retentiontime 14.04 min, 96.0% (M1). (a) 1H signal of and F3C-COOH couldnot be detected.Ethyl 3-thio)acetamido)thiazol-5-yl)methyl)benzoate was dissolvedin a mixture of 0.35 mL of dichloromethane and 0.71 mL of methanoland cooled on an ice bath to 0 °C. Then 220 μL  of an aqueous 50 wt % hydroxylaminesolution were added, and the mixture was stirred at 0 °C for10 min before 47.3 mg  sodium hydroxide wereadded. After 30 min at 0 °C, the ice bath was removed, and stirringof the colorless clear solution was continued at room temperaturefor 2 h. The reaction mixture was dried under reduced pressure, andthe residue was taken up in 2.5 mL of deionized water. The suspensionwas cooled in an ice bath and brought to pH = 8 by the dropwise additionof 1 M HCl, when large amounts of white solid formed. The solid wascollected by filtration. The precipitate was washed with water (2× 1 mL) and ice-cold diethyl ether (2 mL) and dried under reducedpressure. The residue was purified by flash column chromatography(MeOH (+0.5% AcOH)/dichloromethane (+0.5% AcOH): gradient 0–10%).For the final purification, the crude material from column chromatographywas dissolved in 0.5 M NaOH (50 mL). The aqueous layer was washedwith EtOAc (2 × 30 mL). Then, the aqueous layer was acidifiedto pH = 4 with 1 M HCl and extracted with EtOAc (3 × 30 mL).The combined organic layer was washed with brine (50 mL), dried overNa2SO4, filtered, and concentrated under reducedpressure to yield the title compound as a colorless solid . 1H NMR : 12.23 , 11.15 , 9.01 , 7.82–7.58 , 7.34–7.30 , 7.27 , 6.94 ,4.11 , 4.08 , 2.28 . 13C NMR : 169.4 q (pyrimidine C-2), 167.5 q , 167.4 q (−NH-CO-CH2-S−), 164.6 q (−CO-NH-OH), 157.5q (thiazole C-2), 144.0 q (N-hydroxybenzamide C-4),135.5 (thiazole C-4), 131.5 q (thiazole C-5), 131.1 q (N-hydroxybenzamide C-1), 128.8 , 127.7 , 116.6 (pyrimidineC-5), 34.5 (−NH-CO-CH2-S−),32.2 (thiazole-CH2−), 23.7(pyrimidine −CH3). HRMS (ESI+): m/z calcd for C19H19N5O3S2Na+: 452.0822 [M+ Na]+, found: 452.0821. LRMS m/z (ESI+): 430 [M + H]+. HPLC retentiontime 13.89 min, 98.0% (M1).Ethyl 4-thio)acetamido)thiazol-5-yl)methyl)benzoate, CuCl2 ×2H2O , and CaO  in acetone (64 mL). The solution was stirredat room temperature for 2 h, until the nitrogen formation stopped.Acetone was removed by evaporation, and the resulting mixture wasextracted with dichloromethane, filtered, dried over MgSO4, and concentrated in vacuo. The α-chloropropanalintermediate (23) was directly dissolved in ethanol (85mL), and thiourea  was added. The mixturewas refluxed for 24 h. After cooling to room temperature, the reactionmixture was neutralized by the addition of NaHCO3. Theresulting mixture was filtered, and the filtrate was concentratedunder reduced pressure. The crude product was purified by flash columnchromatography (EtOAc/isohexane: gradient 0–100%) to yieldthe title compound as a brown resin . H NMR : 7.86–7.75, 7.55–7.40 , 6.78–6.70 , 4.30 , 3.99 , 1.31 . 13C NMR : 168.2 q (thiazole C-2), 165.7 q (H3C-CH2-O-CO−), 141.3 q (ethyl benzoate C-3),135.8 (thiazole C-4), 133.1 (ethyl benzoate C-4), 130.0 q (ethyl benzoateC-1), 128.9 (ethyl benzoate C-2), 128.7 (ethyl benzoate C-5), 127.1(ethyl benzoate C-6), 124.1 q (thiazole C-5), 60.7 (H3C-CH2-O-CO−), 32.1 (thiazole-CH2−), 14.2 (H3C-CH2-O-CO−). LRMS m/z (ESI+): 263 [M + H]+. Thespectroscopic data are in good agreement with the literature values.61 The attached 1H and 13C NMR spectra contains residual solvent signals (MeOH).A solutionof sodium nitrite in water (32 mL) was added dropwiseto a solution of ethyl 3-aminobenzoate in 18% aqueous HCl (32 mL) at −5 °C. After stirring at0 °C for 30 min, the reaction mixture was carefully neutralizedwith NaHCO3 and subsequently added to an ice-cold suspensionof acrolein , CuCl2·2H2O , and CaO  in acetone (60 mL). The solution was stirred at room temperaturefor 2 h, until the nitrogen formation stopped. Acetone was removedby evaporation and the resulting mixture extracted with dichloromethane,filtered, dried over MgSO4, and concentrated invacuo. The α-chloropropanal intermediate (24) was directly dissolved in ethanol (20 mL), and thiourea  was added. The mixture was refluxed for 2 h.After cooling to room temperature, water (100 mL) was added, and themixture was neutralized with ammonia. The aqueous layer was extractedwith ethyl acetate (3 × 100 mL). Then, the combined organic layerwas extracted with 0.5 M HCl (5 × 100 mL). The combined aqueouslayer was carefully neutralized by the addition of NaHCO3. The neutralized aqueous layer was extracted with EtOAc (3 ×100 mL). The combined organic layer from the last extraction stepwas dried over Na2SO4, filtered, and concentratedunder reduced pressure. The title compound was obtained as a brownresin . 1H NMR : 7.93–7.86 , 7.40–7.31 , 6.76 , 6.73 , 4.29 , 3.98 , 1.30 . 13C NMR : 168.2 q (thiazole C-2), 165.6 q (H3C-CH2-O-CO−), 146.2 q (ethyl benzoate C-4),136.0 (thiazole C-4), 129.3 , 128.5 , 128.0 q (ethyl benzoate C-1), 123.5 q (thiazole C-5), 60.6(H3C-CH2-O-CO−),32.4 (thiazole-CH2−), 14.2(H3C-CH2-O-CO−).LRMS m/z (ESI+): 263[M + H]+. The spectroscopic data are in good agreementwith the literature values.62 The attached 1H and 13C NMR spectra contains residual solventsignals (MeOH).A solutionof sodium nitrite in water (30 mL) was added dropwiseto a solution of ethyl 4-aminobenzoate in 18% aqueous HCl (30 mL) at −5 °C. After stirring at0 °C for 30 min, the reaction mixture was carefully neutralizedwith NaHCO25, 120 mg, 457 μmol, 1 equiv) was dissolved inacetonitrile (4 mL) and N,N-diisopropylethylamine. The mixture was stirredand cooled to 0 °C. Chloroacetyl chloride  was added at 0 °C. After stirring for 2 h at roomtemperature, volatiles were removed under reduced pressure. The red-brown,oily residue was mixed with water (20 mL). The aqueous layer was extractedwith ethyl acetate (3 × 20 mL). The combined organic layer waswashed with 1 M HCl (60 mL), brine (60 mL), dried over Na2SO4, filtered, and dried under reduced pressure. The browncrude product was purified by flash column chromatography (EtOAc/isohexane:gradient 5–80%) to yield the title compound as a tan solid. 1H NMR : 12.38 , 7.86 , 7.83 , 7.56 , 7.47 , 7.32 , 4.34 , 4.30 , 4.19 , 1.31 . 13C NMR : 165.6 q (H3C-CH2-O-CO−), 164.7 q (−NH-CO-CH2-Cl), 156.6 q (thiazole C-2), 140.9 q (ethyl benzoate C-3),135.2 (thiazole C-4), 133.3 (ethyl benzoate C-4), 131.4 q (thiazoleC-5), 130.2 q (ethyl benzoate C-1), 129.0 (ethyl benzoate C-2), 128.9(ethyl benzoate C-5), 127.3 (ethyl benzoate C-6), 60.7 (H3C-CH2-O-CO−), 42.2 (−NH-CO-CH2-Cl), 31.5 (thiazole-CH2−), 14.2 (H3C-CH2-O-CO−). HRMS (ESI+): m/z calcd for C15H16ClN2O3S+: 339.0565 [M + H]+,found: 339.0564. LRMS m/z (ESI+): 339 [M + H]+. The attached 1H and 13C NMR spectra contains residual solvent signals (EtOAc).Ethyl 3-((2-aminothiazol-5-yl)methyl)benzoate was dissolved in acetonitrile(15 mL) and N,N-diisopropylethylamine. The mixture was stirred andcooled to 0 °C. Chloroacetyl chloride  was added at 0 °C. After stirring for 2 h at roomtemperature, volatiles were removed under reduced pressure. The red-brown,oily residue was mixed with water (15 mL). The aqueous layer was extractedwith ethyl acetate (3 × 20 mL). The combined organic layer waswashed with 1 M HCl (20 mL), brine (20 mL), dried over Na2SO4, filtered, and dried under reduced pressure. The browncrude product was purified by flash column chromatography (EtOAc/isohexane:gradient 8–75%) to yield the title compound as a tan solid. 1H NMR : 12.38 , 7.95–7.87 , 7.48–7.37 , 7.32, 4.34 , 4.29 , 4.19 , 1.30 . 13C NMR : 165.5 q (H3C-CH2-O-CO−), 164.7 q (−NH-CO-CH2-Cl), 156.6 q (thiazole C-2), 145.7 q (ethyl benzoate C-4),135.3 (thiazole C-4), 130.8 q (thiazole C-5), 129.5 , 128.7 , 128.2 q (ethyl benzoate C-1),60.6 (H3C-CH2-O-CO−),42.2 (−NH-CO-CH2-Cl), 31.7(thiazole-CH2−), 14.2 (H3C-CH2-O-CO−). HRMS(ESI+): m/z calcd forC15H16ClN2O3S+: 339.0565 [M + H]+, found: 339.0566. LRMS m/z (ESI+): 339 [M + H]+. Theattached 1H and 13C NMR spectra contains residualsolvent signals (EtOAc).Ethyl 4-((2-aminothiazol-5-yl)methyl)benzoate andKI  were added. The mixture was stirredfor 15 min at ambient temperature. Then, ethyl 3-((2-(2-chloroacetamido)thiazol-5-yl)methyl)benzoate was given to the reactionmixture and stirred for 1.5 h. After completion, water (20 mL) wasadded. The aqueous layer was extracted with ethyl acetate (3 ×40 mL). The combined organic layer was dried over Na2SO4, filtered, and dried under reduced pressure. The brown crudeproduct was purified by flash column chromatography (EtOAc/isohexane:gradient 8–80%) to yield the title compound as a tan solid. 1H NMR : 12.25 , 7.84 , 7.81 , 7.53 , 7.45 , 7.29 , 6.93, 4.29 , 4.16 , 4.08 , 2.27 , 1.29 . 13C NMR : 168.9 q (pyrimidine C-2), 167.0 q , 166.9q (−NH-CO-CH2-S−),165.6 q (H3C-CH2-O-CO−),157.1 q (thiazole C-2), 141.0 q (ethyl benzoate C-3), 135.0 (thiazoleC-4), 133.2 (ethyl benzoate C-4), 130.7 q (thiazole C-5), 130.2 q(ethyl benzoate C-1), 129.0 (ethyl benzoate C-5), 128.8 (ethyl benzoateC-2), 127.3 (ethyl benzoate C-6), 116.1 (pyrimidine C-5), 60.7 (H3C-CH2-O-CO−), 34.0(−NH-CO-CH2-S-), 31.5 (thiazole-CH2−), 23.2 (pyrimidine-CH3), 14.1 (H3C-CH2-O-CO−).HRMS (ESI+): m/z calcdfor C21H22N4O3S2Na +: 465.1026 [M + Na]+, found: 465.1025.LRMS m/z (ESI+): 443[M + H]+. The attached 1H and 13CNMR spectra contains residual solvent signals (EtOAc).4,6-Dimethyl-2-methylsulfanyl-pyrimidine was dissolved in dimethyl sulfoxide . Na2CO3 , and KI  were added. The mixture was stirred for 15 min atroom temperature. Then, ethyl 4-((2-(2-chloroacetamido)thiazol-5-yl)methyl)benzoate was given to the reaction mixtureand stirred for 2 h. After completion, water (20 mL) was added. Theaqueous layer was extracted with ethyl acetate (3 × 40 mL). Thecombined organic layer was dried over Na2SO4, filtered, and dried under reduced pressure. The brown crude productwas purified by flash column chromatography (EtOAc/isohexane: gradient8–80%) to yield the title compound as a tan solid . 1H NMR : 12.26 , 7.93–7.86 , 7.43–7.36 , 7.28 , 6.93 , 4.28 , 4.16 , 4.08 , 2.27 , 1.29. 13C NMR : 168.9 q (pyrimidine C-2), 167.0 q , 166.8q (−NH-CO-CH2-S−),165.5 q (H3C-CH2-O-CO−),157.1 q (thiazole C-2), 145.8 q (ethyl benzoate C-4), 135.1 (thiazoleC-4), 130.1 q (thiazole C-5), 129.4 , 128.7, 128.1 q (ethyl benzoate C-1), 116.1 (pyrimidineC-5), 60.6 (H3C-CH2-O-CO−),34.0 (−NH-CO-CH2-S−),31.7 (thiazole-CH2−), 23.2(pyrimidine-CH3), 14.1 (H3C-CH2-O-CO−). HRMS (ESI+): m/z calcd for C21H22N4O3S2Na+: 465.1026 [M + Na]+, found: 465.1024. LRMS m/z (ESI+): 443 [M + H]+.4,6-Dimethyl-2-methylsulfanyl-pyrimidine was dissolved in dimethyl sulfoxide . NaH-1,2,3-triazol-1-yl)methyl)benzoate  was dissolved in a mixture of 0.71 mLof dichloromethane and 0.71 mL of methanol and cooled in an ice bathto 0 °C. Then 69.4 μL  of an aqueous 50 wt % hydroxylamine solution were added, andthe mixture was stirred at 0 °C for 10 min before 14.9 mg  sodium hydroxide were added. After 30 min at 0 °C,the ice bath was removed, and stirring of the colorless clear solutionwas continued at ambient temperature for 3 h. The reaction mixturewas dried under reduced pressure and the residue was taken up in 2mL of deionized water. The suspension was cooled in an ice bath andbrought to pH = 8 by the dropwise addition of 1 M HCl, when largeamounts of white solid formed. The solid was collected by centrifugation,washed with ice-cold diethyl ether (2 mL), and dried under reducedpressure. For the final purification preparative HPLC (gradient elutionfrom 25% to 50% acetonitrile/water (+0.1% TFA)) was used to yieldthe TFA salt of the title compound as a colorless solid . 1H NMRa : 12.23 , 11.22 , 9.10 , 8.29 , 7.81–7.63 , 7.40–7.30 , 7.25 , 7.22, 6.92, 6.91–6.79 ,5.65 , 5.10 , 4.08, 4.03 , 2.27 . 13C NMR : 168.9 q (pyrimidine C-2), 167.0 q , 166.8 q (−NH-CO-CH2-S−), 163.7 q (−CO-NH-OH), 158.1q , 158.1 q (phenyl C-3), 156.9 q (thiazoleC-2), 143.0 q (triazole C-4), 141.9 q (phenyl C-1), 138.9 q (N-hydroxybenzamide C-4), 134.7 (thiazole C-4), 132.6 q (N-hydroxybenzamide C-1), 131.0 q (thiazole C-5), 129.6 (phenylC-5), 127.9 , 127.3 , 124.8 (triazole C-5), 120.9 (phenylC-6), 116.1 (pyrimidine C-5), 116.0 ,115.0 (phenyl C-2), 112.4 (phenyl C-4), 61.0 (triazole-CH2-O−), 52.4 (N-hydroxybenzamide-CH2-triazole), 34.1 (−NH-CO-CH2-S-), 31.9 (thiazole-CH2-phenyl),23.2 (pyrimidine −CH3). 19F NMR : −74.0.HRMS (ESI+): m/z calcdfor C29H29N8O4S2+: 617.1748 [M + H]+, found: 616.1745. LRMS m/z (ESI+): 617 [M + H]+. HPLC retention time 15.33 min, >99.5% (M1). a)1thio)acetamido)thiazol-5-yl)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)pentanamido)methyl)benzoate  was dissolved in amixture of 0.78 mL of dichloromethane and 0.78 mL of methanol andcooled in an ice bath to 0 °C. Then 76 μL  of an aqueous 50 wt % hydroxylamine solutionwere added, and the mixture was stirred at 0 °C for 10 min, before16.4 mg  sodium hydroxide were added. After30 min at 0 °C, the ice bath was removed and stirring of thecolorless clear solution was continued at ambient temperature for3 h. The reaction mixture was dried under reduced pressure, and theresidue was taken up in 2 mL of deionized water. The suspension wascooled in an ice bath and brought to pH = 8 by the dropwise additionof 1 M HCl, then large amounts of white solid formed. The solid wascollected by centrifugation, washed with ice-cold diethyl ether (2mL), and dried under reduced pressure. For the final purification,preparative HPLC (gradient elution from 25% to 50% acetonitrile/water(+0.1% TFA)) was used to yield the TFA salt of the title compoundas a colorless solid . 1H NMRa : 12.23 ,11.18 , 8.40 4-triazole),8.20 , 7.72–7.62 , 7.34–7.17 , 6.94 , 6.91–6.79 , 5.09 , 4.36 3-CH2-triazole), 4.284-triazole), 4.08 , 4.03 , 2.27 , 2.19 3-triazole),1.87–1.72 2-CH2-CH2-triazole), 1.55–1.42 2-triazole). 13C NMR : 171.8 q (−CH2-NH-CO-(CH2)4-triazole), 168.9 q (pyrimidine C-2),167.0 q , 166.9 q (−NH-CO-CH2-S−), 164.0 q (−CO-NH-OH), 158.3 q , 158.2 q (phenyl C-3), 157.0 q (thiazoleC-2), 143.0 q (N-hydroxybenzamide C-4), 142.6 q (triazoleC-4), 141.9 q (phenyl C-1), 134.8 (thiazole C-4), 131.2 q (N-hydroxybenzamide C-1), 131.1 q (thiazole C-5), 129.7 (phenylC-5), 127.0 , 126.9 , 124.4 (triazole C-5), 120.9 (phenylC-6), 116.1 (pyrimidine C-5), 115.3 ,115.0 (phenyl C-2), 112.5 (phenyl C-4), 61.1 (triazole-CH2-O−), 49.1 (−CH2-NH-CO-(CH2)3-CH2-triazole), 41.8 (−CH2-NH-CO-(CH2)4-triazole), 34.5 (−CH2-NH-CO-CH2-(CH2)3-triazole), 34.1 (−NH-CO-CH2-S−), 31.9 (thiazole-CH2-phenyl), 29.4 (−CH2-NH-CO-(CH2)2-CH2-CH2-triazole), 23.3 (pyrimidine −CH3), 22.2(−CH2-NH-CO-CH2-CH2-(CH2)2-triazole). 19F NMR :−74.6. HRMS (ESI+): m/z calcd for C34H38N9O5S2+: 716.2432 [M + H]+, found: 716.2431.LRMS m/z (ESI+): 716[M + H]+. HPLC retention time 15.03 min, >99.5% (M1).Theattached 1H and 13C NMR spectra contains residualsolvent signals . a)1thio)acetamido)thiazol-5-yl)methyl)phenoxy)methyl)-1N-Butyl-5-thio)acetamido)thiazol-5-yl)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)pentanamido)methyl)-N-hydroxybenzamide . Ethyl 4-thio)acetamido)thiazol-5-yl)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)pentanamido)methyl)benzoate  was dissolved in a mixtureof 0.78 mL of dichloromethane and 0.78 mL of methanol and cooled inan ice bath to 0 °C. Then 71 μL  of an aqueous 50 wt % hydroxylamine solution wereadded, and the mixture was stirred at 0 °C for 10 min, before15.2 mg  sodium hydroxide were added. After30 min at 0 °C, the ice bath was removed and stirring of thecolorless clear solution was continued at ambient temperature for3 h. The reaction mixture was dried under reduced pressure, and theresidue was taken up in 2 mL of deionized water. The suspension wascooled in an ice bath and brought to pH = 8 by the dropwise additionof 1 M HCl, when large amounts of white solid formed. The solid wascollected by centrifugation, washed with ice-cold diethyl ether (2mL), and dried under reduced pressure. For the final purification,preparative HPLC (gradient elution from 35% to 56% acetonitrile/water(+0.1% TFA)) was used to yield the TFA salt of the title compoundas a colorless solid . 1H NMRa : 12.19 ,11.16 , 8.20, 8.16, 7.77–7.64 , 7.30–7.18 , 6.93, 6.91–6.87 , 6.85–6.79, 5.10, 5.08 , 4.59–4.52-CO-(CH2)4−), 4.38, 4.31 -CO-(CH2)3-CH2-triazole), 4.08 , 4.03, 3.24–3.17 -CO-(CH2)4−), 2.42, 2.30 -CO-CH2-(CH2)3−),2.28 , 1.89–1.74 -CO-(CH2)2-CH2-CH2−), 1.57–1.35 -CO-CH2-CH2-(CH2)2−), 1.28–1.15 2-CH2-CH3)-CO-(CH2)4−), 0.84, 0.83 3-CH3)-CO-(CH2)4−). 13CNMRb : 171.71, 171.70 q (−CH2-N(C4H9)-CO-(CH2)4−),168.9 q (pyrimidine C-2), 167.0 q , 166.8 q (−NH-CO-CH2-S−), 164.0, 163.9 q (−CO-NH-OH), 158.2 q , 158.2q (phenyl C-3), 156.9 q (thiazole C-2), 142.58, 142.55 q (triazoleC-4), 141.8 q (phenyl C-1), 141.8, 141.3 q (N-hydroxybenzamideC-4), 134.7 (thiazole C-4), 131.7, 131.4 q (N-hydroxybenzamideC-1), 131.0 q (thiazole C-5), 129.6 (phenyl C-5), 127.3, 126.3 , 127.2, 126.9 , 124.32, 124.28 (triazole C-5), 120.9 (phenylC-6), 116.1 (pyrimidine C-5), 115.5 ,114.9 (phenyl C-2), 112.5 (phenyl C-4), 61.1 (triazole-CH2-O−), 50.0, 47.5 (−CH2-N(C4H9)-CO-(CH2)4−), 49.3, 49.2 (−CH2-N(C4H9)-CO-(CH2)3-CH2−), 46.8, 45.2 (−CH2-N(CH2-C3H7)-CO-(CH2)4−), 34.1 (−NH-CO-CH2-S−), 31.9 (thiazole-CH2-phenyl), 31.6, 31.2 (−CH2-N(C4H9)-CO-CH2-(CH2)3−), 30.3, 29.3 (−CH2-N(CH2-CH2-C2H5)-CO-(CH2)4−), 29.4,29.2 (−CH2-N(C4H9)-CO-CH2-CH2-(CH2)2−), 23.2 (pyrimidine-CH3), 21.9, 21.8 (−CH2-N(C4H9)-CO-(CH2)2-CH2-CH2−), 19.6,19.4 (−CH2-N((CH2)2-CH2-CH3)-CO-(CH2)4−), 13.7, 13.6 (−CH2-N((CH2)3-CH3)-CO-(CH2)4−). 19F NMR : −74.3. HRMS (ESI+): m/z calcd for C38H46N9O5S2+: 772.3058 [M + H]+, found: 772.3066. LRMS m/z (ESI+) 772 .HPLC retention time 16.53 min, >99.5% (M1). a)Rotamerswere observed.4- to yield the title compound as a colorless liquid . 1H NMR : 8.01–7.93 , 7.54–7.47 , 4.58, 4.31 , 1.32 . 13C NMR : 165.4 q (H3C-CH2-O-CO−), 141.1q , 129.5 , 129.5 q (ethylbenzoate C-1), 128.5 , 60.8 (H3C-CH2-O-CO-), 53.0 (−CH2-N3), 14.2 (H3C-CH2-O-CO−). LRMS m/z (ESI+): 206 [M + H]+. The spectroscopic dataare in good agreement with the literature values.63Ethyl 4-(bromomethyl)benzoate was dissolved in 15 mL of dimethylformamide (DMF), followedby the addition of sodium azide . Thereaction mixture was stirred at ambient temperature for 16 h. Water(50 mL) was added to the reaction mixture, followed by the extractionof the aqueous layer with EtOAc (3 × 50 mL). The combined organiclayer was washed with brine (50 mL), dried over NaN-(5-(3-(prop-2-yn-1-yloxy)benzyl)thiazol-2-yl)acetamide and ethyl 4-(azidomethyl)benzoate were dissolved in a water/tert-BuOH mixture . Tris(benzyltriazolylmethyl)amine was dissolved in DMF (800 μL)and added to the mixture. An aqueous CuSO4 solution  and an aqueous solution of sodium ascorbate  were added in that order. The resulting reactionmixture was stirred for 12 h at ambient temperature under nitrogenatmosphere. After completion, water (30 mL) was added to the reactionmixture. The aqueous layer was extracted with EtOAc (3 × 30 mL).The combined organic layer was washed with brine (30 mL), dried overNa2SO4, and concentrated under reduced pressure.The residue was purified by flash column chromatography (EtOAc/isohexane:gradient 10–100%) to yield the title compound as a colorlesssolid . 1H NMR : 12.22 , 8.29 ,8.02–7.89 , 7.45–7.34 , 7.27–7.18 , 6.92 , 6.91–6.80 , 5.70 , 5.11 , 4.30, 4.08, 4.02 , 2.27 , 1.30 . 13C NMR : 168.9 q (pyrimidine C-2), 167.0 q , 166.8q (−NH-CO-CH2-S−),165.3 q (H3C-CH2-O-CO−),158.1 q (phenyl C-3), 156.9 q (thiazole C-2), 143.1 q (triazole C-4),141.9 q (phenyl C-1), 141.2 q , 134.8 (thiazoleC-4), 131.1 q (thiazole C-5), 129.7 (phenyl C-5), 129.6 , 128.1 , 124.9(triazole C-5), 120.9 (phenyl C-6), 116.1 (pyrimidine C-5), 115.0(phenyl C-2), 112.5 (phenyl C-4), 61.0 (triazole-CH2-O−), 60.9 (H3C-CH2-O-CO−), 52.4 (ethyl benzoate-CH2-triazole), 34.1 (−NH-CO-CH2-S−), 31.9 (thiazole-CH2-phenyl), 23.3 (pyrimidine-CH3), 14.2 (H3C-CH2-O-CO−). HRMS(ESI+): m/z calcd forC31H32N7O4S2+: 630.1952 [M + H]+, found: 630.1950. LRMS m/z (ESI+): 630 [M + H]+.2-thio)-34, 1.4 g, 6.82 mmol, 1 equiv) was dissolved in a mixtureof 23 mLtetrahydrofuran (THF) and 4.4 mL water. Triphenylphospine  was added to the mixture followed by stirringat ambient temperature for 1 h and further at 75 °C for 1 h.The reaction mixture was ice-cooled, and water was added (30 mL).The mixture was adjusted to pH 2 by adding 1 M HCl and was subsequentlywashed with TBME. Then, the aqueous layer was neutralized with saturatedaqueous sodium bicarbonate, followed by the extraction with EtOAc(3 × 30 mL). The combined organic layer was washed with brine(50 mL), dried over Na2SO4, filtered, and concentratedunder reduced pressure. The title compound was obtained as a pale-yellowoil  and was used for subsequent reactions without anyfurther purification. 1H NMR : 7.94–7.83 , 7.51–7.43 , 4.29 , 3.78 ,1.86 , 1.30 . 13C NMR : 165.8 q (H3C-CH2-O-CO−), 150.1 q , 129.0 , 127.8 q (ethyl benzoate C-1),127.2 , 60.5 (H3C-CH2-O-CO−), 45.4 (−CH2-NH2), 14.2 (H3C-CH2-O-CO−). LRMS m/z (ESI+): 180 [M + H]+. The spectroscopic dataare in good agreement with the literature values.64 The attached 1H and 13C NMR spectracontains residual solvent signals (EtOAc).Ethyl 4-(azidomethyl)benzoate(O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium-hexafluorphosphat  dissolved in DMF (2 mL), N,N-diisopropylethylamine ,and ethyl 4-(aminomethyl)benzoate  dissolved in DMF (2 mL) were added under nitrogen atmosphereand ice-cooling in this order. After 5 min of stirring at 0 °C,further DIPEA  was added under nitrogen atmosphereand ice-cooling. The reaction mixture was allowed to warm up to ambienttemperature and was stirred for further 16 h. Then, the reaction mixturewas partitioned between water (50 mL) and EtOAc (50 mL). The organiclayer was washed with 1 M HCl (2 × 30 mL), saturated sodium bicarbonatesolution (50 mL), water (50 mL), brine (50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure.The residue was purified by flash column chromatography (EtOAc/isohexane:gradient 10–80%) to yield the title compound as a pale-redsolid . 1H NMR : 8.44 4-N3), 7.97–7.86 , 7.41–7.33 , 4.38–4.234-N3, H3C-CH2-O-CO−),3.33* 3-CH2-N3), 2.19 3-N3), 1.67–1.42 , 1.30 . 13C NMR : 172.0 q (−CH2-NH-CO-(CH2)4-N3), 165.6 q (H3C-CH2-O-CO−), 145.4 q , 129.2 , 128.4 q (ethyl benzoate C-1), 127.3 , 60.7 (H3C-CH2-O-CO−), 50.4 (−CH2-NH-CO-(CH2)3-CH2-N3), 41.8 (−CH2-NH-CO-(CH2)4-N3), 34.7 (−CH2-NH-CO-CH2-(CH2)3-N3), 27.9 (−CH2-NH-CO-(CH2)2-CH2-CH2-N3), 22.5 (−CH2-NH-CO-CH2-CH2-(CH2)2-N3), 14.2 (H3C-CH2-O-CO−). HRMS (ESI+): m/z calcd for C15H21N4O3+: 305.1608 [M + H]+, found: 305.1614.LRMS m/z (ESI+): 305[M + H]+. The attached 1H and 13CNMR spectra contains residual solvent signals (EtOAc).5-Azidopentanoic acid was dissolvedin DMF (1 mL). N-(5-(3-(prop-2-yn-1-yloxy)benzyl)thiazol-2-yl)acetamide and ethyl 4-((5-azidopentanamido)methyl)benzoate were dissolved in a water/tert-BuOH mixture . Tris(benzyltriazolylmethyl)amine was dissolved in DMF (800 μL)and added to the mixture. An aqueous CuSO4 solution  and an aqueous solution of sodium ascorbate  were added in that order. The resulting reactionmixture was stirred for 12 h at room temperature under nitrogen atmosphere.After completion, water (30 mL) was added to the reaction mixture.The aqueous layer was extracted with EtOAc (3 × 30 mL). The combinedorganic layer was washed with brine (30 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure.The residue was purified by flash column chromatography (MeOH/dichloromethane:gradient 0–8%) to yield the title compound as a colorless solid. 1H NMR : 12.22 , 8.44 4-triazole), 8.19 , 7.93–7.87, 7.41–7.32 , 7.28–7.17 , 6.93 , 6.92–6.81 , 5.09, 4.44–4.24 3-CH2-triazole, -CH2-NH-CO-(CH2)4-triazole, H3C-CH2-O-CO−), 4.08 , 4.03, 2.27 , 2.20 3-triazole), 1.89–1.73 2-CH2-CH2-triazole),1.55–1.44 2-triazole), 1.30 . 13CNMR :171.9 q (−CH2-NH-CO-(CH2)4-triazole), 168.9 q (pyrimidine C-2), 167.0 q, 166.8 q (−NH-CO-CH2-S−), 165.6 q (H3C-CH2=O-CO−), 158.2 q (phenyl C-3), 156.9 q (thiazoleC-2), 145.3 q , 142.6 q (triazole C-4), 141.9q (phenyl C-1), 134.8 (thiazole C-4), 131.1 q (thiazole C-5), 129.7(phenyl C-5), 129.2 , 128.4 q (ethyl benzoateC-1), 127.3 , 124.4 (triazole C-5), 120.9 (phenylC-6), 116.1 (pyrimidine C-5), 115.0 (phenyl C-2), 112.4 (phenyl C-4),61.1 (triazole-CH2-O−), 60.7(H3C-CH2-O-CO−),49.1 (−CH2-NH-CO-(CH2)3-CH2-triazole), 41.8 (−CH2-NH-CO-(CH2)4-triazole), 34.5(−CH2-NH-CO-CH2-(CH2)3-triazole), 34.1 (−NH-CO-CH2-S−), 31.9 (thiazole-CH2-phenyl), 29.4 (−CH2-NH-CO-(CH2)2-CH2-CH2-triazole), 23.3 (pyrimidine-CH3), 22.2 (−CH2-NH-CO-CH2-CH2-(CH2)2-triazole), 14.2 (H3C-CH2-O-CO−). HRMS (ESI+): m/z calcd for C36H41N8O5S2+: 729.2636 [M + H]+, found: 729.2633. LRMS m/z (ESI+): 729 [M + H]+.2-thio)-2SO4, filtered, and concentrated under reduced pressure. The title compoundwas obtained as a tan oil and was used for subsequent reactions withoutfurther purification . 1H NMR : 7.92–7.86 , 7.49–7.42 ,4.29 ,3.74 3-CH3), 2.45 2-CH3), 2.15 2-CH3), 1.46–1.242-CH3, H3C-CH2-O-CO−),0.85 3-CH3). 13C NMR : 165.7 q (H3C-CH2-O-CO−), 147.1 q , 128.9 , 128.1 q (ethyl benzoate C-1), 127.9 , 60.5 (H3C-CH2-O-CO−), 52.7 (−NH-CH2-(CH2)2-CH3), 48.4 (−CH2-NH-(CH2)3-CH3), 31.7 (−NH-CH2-CH2-CH2-CH3), 20.0 (−NH-CH2-CH2-CH2-CH3), 14.2 (H3C-CH2-O-CO−), 13.9(−NH-CH2-CH2-CH2-CH3); HRMS (ESI+): m/z calcd for C14H22NO2+: 236.1645 [M + H]+, found: 236.1645. LRMS m/z (ESI+): 236 [M + H]+.Butylamine was added to a solution of ethyl4-(bromomethyl)benzoate  in THF (20 mL). After 3h of stirring at ambient temperature, the reaction mixture was partitionedbetween chloroform (20 mL) and a saturated sodium bicarbonate solution(20 mL). The organic layer was dried over NaH-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumtetrafluoroborate  and DIPEA  were dissolved in DMF (1.5 mL). This mixture was addeddropwise at 0 °C to the flask containing the solution of 5-bromopentanoic.After addition, the mixture was stirred for 15 min at 0 °C. Then,ethyl 4-((butylamino)methyl)benzoate  dissolved in 1.5 mL of DMF was added to the reaction mixtureat 0 °C. After 2 h of stirring at ambient temperature, TLC indicatedcompletion of the reaction . Then, 30 mL of water were addedto the reaction mixture. The aqueous layer was extracted with EtOAc(3 × 30 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure.The residue was purified by flash column chromatography (EtOAc/isohexane:gradient 4–30%) to yield 84 mg of ethyl 4-((5-bromo-N-butylpentanamido)methyl)benzoate (41) asa colorless liquid (LRMS m/z (ESI+): 398 ([M(79Br + H]+), 400 ([M(81Br + H]+). The obtained intermediate 4-((5-bromo-N-butylpentanamido)methyl)benzoate  and NaN3  were dissolvedin DMSO (2.5 mL) and stirred overnight at 45 °C. After additionof water (20 mL), the aqueous layer was extracted with EtOAc (3 ×20 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residuewas purified by flash column chromatography (EtOAc/isohexane: gradient4–32%) to yield the title compound as a colorless solid . 1H NMRa : 7.98–7.88 , 7.36–7.30 ,4.65, 4.57 -CO-(CH2)4-N3), 4.30, 4.29 , 3.37–3.18*-CO-(CH2)3-CH2-N3), 2.44, 2.27 -CO-CH2-(CH2)3-N3),1.67–1.14 2-CH3)-CO-CH2-(CH2)2-CH2-N3)), 0.85, 0.84 3-CH3)-CO-(CH2)4-N3). 13C NMRa : 171.9 q (−CH2-N(C4H9)-CO-(CH2)4-N3), 165.6, 165.5 q (H3C-CH2-O-CO−), 144.3, 143.8 q , 129.6,129.2 , 128.8, 128.5 q (ethyl benzoate C-1),127.5, 126.7 , 60.7, 60.7 (H3C-CH2-O-CO−), 50.54, 50.45 (−CH2-N(C4H9)-CO-(CH2)3-CH2-N3), 50.1, 47.7(−CH2-N(C4H9)-CO-(CH2)4-N3), 47.0, 45.4 (−CH2-N(CH2-C3H7)-CO-(CH2)4-N3), 31.7, 31.4 (−CH2-N(C4H9)-CO-CH2-(CH2)3-N3), 30.4, 29.3 (−CH2-N(CH2-CH2-C2H5)-CO-(CH2)4-N3), 28.0,27.8 (−CH2-N(C4H9)-CO-CH2-CH2-(CH2)2-N3), 22.2, 22.0 (−CH2-N(C4H9)-CO-(CH2)2-CH2-CH2-N3), 19.6, 19.5(−CH2-N((CH2)2-CH2-CH3)-CO-(CH2)4-N3), 14.2 (H3C-CH2-O-CO−), 13.8, 13.7 (−CH2-N((CH2)3-CH3)-CO-(CH2)4-N3). HRMS (ESI+): m/z calcd for C19H29N4O3+: 361.2234 [M+ H]+, found: 361.2239; LRMS m/z (ESI+) 361 . a) wasdissolved in 4 mL of a mixture of dichloromethane and DMF (1:1 (v/v)).The solution was cooled to 0 °C. 2-(1N-(5-(3-(prop-2-yn-1-yloxy)benzyl)thiazol-2-yl)acetamide and ethyl 4-((5-azido-N-butylpentanamido)methyl)benzoate  were dissolved in a water/tert-BuOH mixture . Tris(benzyltriazolylmethyl)amine was dissolved in DMF (800 μL)and added to the mixture. An aqueous CuSO4 solution  and an aqueous solution of sodium ascorbate  were added in that order. The resulting reactionmixture was stirred overnight at ambient temperature under nitrogenatmosphere. After completion, water (30 mL) was added to the reactionmixture. The aqueous layer was extracted with EtOAc (3 × 30 mL).The combined organic layer was washed with brine (30 mL), dried overNa2SO4, filtered, and concentrated under reducedpressure. The residue was purified by flash column chromatography(MeOH/dichloromethane: gradient 0.4–5%) to yield the titlecompound as a colorless solid . 1H NMRa :12.22 , 8.21, 8.16 , 7.98–7.87, 7.38–7.17 , 6.95–6.78 , 5.09, 5.07 , 4.63,4.56 -CO-(CH2)4−), 4.41–4.24 -CO-(CH2)3-CH2-triazole,H3C-CH2-O-CO−), 4.08 , 4.03 , 3.27–3.15-CO-(CH2)4−), 2.47–2.19 -CO-CH2-(CH2)3–, pyrimidine-CH3), 1.97–1.66 -CO-(CH2)2-CH2-CH2−),1.56–1.35 -CO-CH2-CH2-(CH2)2−), 1.31,1.30 , 1.25–1.11 2-CH2-CH3)-CO-(CH2)4−), 0.83, 0.82 3-CH3)-CO-(CH2)4−). 13C NMRa :171.8 q (−CH2-N(C4H9)-CO-(CH2)4−), 168.9 q (pyrimidineC-2), 167.0 q , 166.8 q (−NH-CO-CH2-S−), 165.54, 165.46 q (H3C-CH2-O-CO−), 158.2 q (phenyl C-3),156.9 q (thiazole C-2), 144.2, 143.7 q , 142.6,142.5 q (triazole C-4), 141.8 q (phenyl C-1), 134.8 (thiazole C-4),131.0 q (thiazole C-5), 129.6 (phenyl C-5), 129.5, 129.2 , 128.8, 128.5 q (ethyl benzoate C-1), 127.5, 126.6 , 124.3, 124.3 (triazole C-5), 120.9 (phenyl C-6), 116.1 (pyrimidineC-5), 114.9 (phenyl C-2), 112.4 (phenyl C-4), 61.1 (triazole-CH2-O−), 60.7, 60.5 (H3C-CH2-O-CO−), 50.1, 47.7 (−CH2-N(C4H9)-CO-(CH2)4−), 49.3, 49.2 (−CH2-N(C4H9)-CO-(CH2)3-CH2−), 46.9, 45.3 (−CH2-N(CH2-C3H7)-CO-(CH2)4−), 34.0 (−NH-CO-CH2-S−), 31.9 (thiazole-CH2-phenyl), 31.6, 31.2 (−CH2-N(C4H9)-CO-CH2-(CH2)3−), 30.3, 29.2 (−CH2-N(CH2-CH2-C2H5)-CO-(CH2)4−), 29.3,29.2 (−CH2-N(C4H9)-CO-CH2-CH2-(CH2)2−), 23.2 (pyrimidine-CH3), 21.88, 21.7 (−CH2-N(C4H9)-CO-(CH2)2-CH2-CH2−), 19.6,19.4 (−CH2-N((CH2)2-CH2-CH3)-CO-(CH2)4−), 14.2 (H3C-CH2-O-CO−), 13.7, 13.6 (−CH2-N((CH2)3-CH3)-CO-(CH2)4−). HRMS (ESI+): m/z calcd for C40H49N8O5S2+: 785.3262 [M + H]+, found: 785.3271. LRMS m/z (ESI+): 785 [M + H]+. a)thio)-49  in DMF for 30min, Fmoc deprotection was performed by treatment with 20% piperidinein DMF . Afterward, the resin was washed withDMF (5 × 2 mL), CH2Cl2 (5 × 2 mL),and DMF (5 × 2 mL). For the subsequent amide coupling reaction,a solution of N3-PEG4-COOH , HATU , and DIPEA  in DMF (500 μL) was agitatedfor 5 min and then added to the resin. The reaction was shaken for5 h at ambient temperature followed by washing with DMF (5 ×2 mL), CH2Cl2 (5 × 2 mL), and DMF (5 ×2 mL). The obtained azido-functionalized preferential HDAC6i precursorresin 52 was directly used for the subsequent reaction.For the Huisgen cycloaddition, a solution of SirReal-alkyne 35  in DMF (1.00 mL), TBTA, 0.1 M aqueous CuSO4·5H2O in DMF (0.50 mL), 0.2 M aqueous ascorbic acid  in DMF (0.50 mL), and t-BuOH(990 μL) were added in that sequence to the resin. The reactionwas shaken for 18 h at ambient temperature. After washing with DMF(5 × 2 mL) and CH2Cl2 (5 × 2 mL),the resin was dried under reduced pressure followed by the cleavageof the crude product from the resin by treatment with cleavage solution for 1 hat ambient temperature. The filtrates were concentrated under reducedpressure, and the crude final product was purified by preparativeHPLC (gradient elution from 5% to 95% acetonitrile (+0.1% TFA)/water(+0.1% TFA)). Lyophilization of the respective fractions yielded thetitle compound  as a TFA salt. 1H NMRa : 12.20 , 11.16 , 10.15 ,8.95 ,8.15 , 7.95–7.82 , 7.75–7.61 , 7.40–7.32 , 7.26–7.19 , 6.93 , 6.91–6.86 , 6.83 , 5.09 , 4.56–4.41 , 4.08 , 4.03 ,3.82–3.76* ,3.68* , 3.51–3.37 , 2.56 , 2.28. 13C NMR : 169.7 q(−NH-CO-CH2-CH2-O−), 168.9 q (pyrimidine C-2), 167.0 q ,166.8 q (−NH-CO-CH2-S−),165.8 q (N-hydroxybenzamide-CH2-NH-CO−), 164.1 q (−CO-NH-OH), 158.2 q (phenyl C-3), 156.9 q (thiazole C-2), 143.1 q (N-hydroxybenzamide C-4), 142.5 q (triazole C-4), 141.9 q(4-acylamidobenzamide C-4), 141.8 q (phenyl C-1), 134.7 (thiazoleC-4), 131.3 q (N-hydroxybenzamide C-1), 131.1 q (thiazoleC-5), 129.6 (phenyl C-5), 128.5 q (4-acylamidobenzamide C-1), 128.1, 127.1 , 126.9 , 124.9 (triazoleC-5), 120.9 (phenyl C-6), 118.2 , 116.1(pyrimidine C-5), 114.9 (phenyl C-2), 112.4 (phenyl C-4), 69.7 (−O-CH2-CH2-O−), 69.7 (−O-CH2-CH2-O−), 69.7 (−O-CH2-CH2-O−), 69.6 (−O-CH2-CH2-O−), 69.6 (−O-CH2-CH2-O−), 69.5 (−O–CH2-CH2-O−), 68.6 (−O-CH2-CH2-triazole−), 66.5(−NH-CO-CH2-CH2-O−), 61.0 (−triazole-CH2-O−), 49.4 (−O-CH2-CH2-triazole−), 42.4 (N-hydroxybenzamide-CH2-NH-CO−), 37.2 (−NH-CO-CH2-CH2-O−), 34.1 (−NH-CO-CH2-S−), 31.9 (thiazole-CH2-phenyl), 23.2 (pyrimidine −CH3). 19F NMR : −74.9. HRMS (ESI+): m/z calcd for C47H55N10O10S2+: 983.3539 [M+ H]+, found: 983.3536. HPLC retention time 16.04 min,97.4% (M3). a)113C signalsof F3C-COOHcould not be detected.After swellingof the resin 49  in DMF for 30min, Fmoc deprotection was performed by treatment with 20% piperidinein DMF . Afterward, the resin was washed withDMF (5 × 2 mL), CH2Cl2 (5 × 2 mL),and DMF (5 × 2 mL). For the subsequent amide coupling reaction,a solution of N3-PEG10-COOH , HATU , and DIPEA  in DMF (500 μL) was agitatedfor 5 min and then added to the resin. The reaction was shaken for5 h at ambient temperature followed by washing with DMF (5 ×2 mL), CH2Cl2 (5 × 2 mL), and DMF (5 ×2 mL). The obtained azido-functionalized preferential HDAC6i precursorresin 53 was directly used for the subsequent reaction.For the Huisgen cycloaddition solutions of SirReal-alkyne 35  in DMF (1.00 mL), TBTA , 0.1 M aqueous CuSO4·5H2O  in DMF (0.50mL), 0.2 M aqueous ascorbic acid  in DMF (0.50 mL), and tert-BuOH (990 μL)were added in that sequence to the resin. The reaction was shakenfor 18 h at ambient temperature. After washing with DMF (5 ×2 mL) and CH2Cl2 (5 × 2 mL), the resinwas dried under reduced pressure followed by the cleavage of the crudeproduct from the resin by treatment with cleavage solution  for 1 h at ambienttemperature. The filtrates were concentrated under reduced pressure,and the crude final product was purified by preparative HPLC (gradientelution from 5% to 95% acetonitrile (+0.1% TFA)/water (+0.1% TFA)).Lyophilization of the respective fractions yielded the title compound as a TFA salt. 1H NMRa :12.22 , 11.18 , 10.18 , 8.97,8.17 , 7.92–7.78 , 7.78–7.60 , 7.44–7.31 , 7.28–7.15 , 6.94 , 6.92–6.87 , 6.83 , 5.09 , 4.64–4.35 , 4.08 , 4.03 ,3.90–3.75* ,3.70* , 3.59–3.32 , 2.58 , 2.28. 13C NMRb :169.6 q (−NH-CO-CH2-CH2-O−), 168.9 q (pyrimidine C-2), 167.0 q , 166.8 q (−NH-CO-CH2-S−), 165.7 q (N-hydroxybenzamide-CH2-NH-CO−), 164.1 q (−CO-NH-OH), 158.2 q (phenyl C-3), 158.1 q ,156.9 q (thiazole C-2), 143.1 q (N-hydroxybenzamideC-4), 142.5 q (triazole C-4), 141.9 q (4-acylamidobenzamide C-4),141.8 q (phenyl C-1), 134.7 (thiazole C-4), 131.2 q (N-hydroxybenzamide C-1), 131.0 q (thiazole C-5), 129.6 (phenyl C-5),128.5 q (4-acylamidobenzamide C-1), 128.1 ,127.0 , 126.9 , 124.8 (triazole C-5), 120.9 (phenylC-6), 118.2 , 116.1 (pyrimidine C-5),114.9 (phenyl C-2), 112.4 (phenyl C-4), 69.73 (7 × −O-CH2-CH2-O−),69.67 (−O-CH2-CH2-O−), 69.65 (−O-CH2-CH2-O−), 69.6 (−O-CH2-CH2-O−), 69.5 (−O-CH2-CH2-O−), 68.6 (−O-CH2-CH2-triazole−), 66.5(−NH-CO-CH2-CH2-O−), 61.0 (−triazole-CH2-O−), 49.4 (−O-CH2-CH2-triazole−), 42.3 (N-hydroxybenzamide-CH2-NH-CO−), 37.2 (−NH-CO-CH2-CH2-O−), 34.0 (−NH-CO-CH2-S−), 31.9 (thiazole-CH2-phenyl), 23.2 (pyrimidine −CH3). 19F-NMR : −74.1. HRMS (ESI+): m/z calcd for C59H79N10O16S2+: 1247.5111[M + H]+, found: 1247.5105; HPLC retention time 16.07 min,95.0% (M3). The attached 1H and 13C NMR spectracontains residual solvent signals (DMSO). a)13C signal of F3C-COOH could not be detected.After swellingof the resin 51  in DMF for 30 min, Fmoc deprotection was performed bytreatment with 20% piperidine in DMF . Afterward,the resin was washed with DMF (5 × 2 mL), CH2Cl2 (5 × 2 mL), and DMF (5 × 2 mL). For the subsequentamide coupling reaction, a solution of N3-PEG4-COOH , HATU , and DIPEA  in DMF(500 μL) was agitated for 5 min and then added to the resin.The reaction was shaken for 5 h at ambient temperature followed bywashing with DMF (5 × 2 mL), CH2Cl2 (5× 2 mL), and DMF (5 × 2 mL). The obtained azido-functionalizednonselective HDACi precursor resin 54 was directly usedfor the subsequent reaction. For the Huisgen cycloaddition a solutionof SirReal-alkyne 35 in DMF (1.00 mL), TBTA , 0.1M aqueous CuSO4·5H2O  in DMF (0.50 mL), 0.2 M aqueous ascorbic acid in DMF (0.50 mL), andand tert-BuOH (990 μL) were added in that sequenceto the resin. The reaction was shaken for 18 h at ambient temperature.After washing with DMF (5 × 2 mL) and CH2Cl2 (5 × 2 mL), the resin was dried under reduced pressure followedby the cleavage of the crude product from the resin by treatment withcleavage solution  for 1 h at ambient temperature. The filtrates were concentratedunder reduced pressure, and the crude final product was purified bypreparative HPLC (gradient elution from 5% to 95% acetonitrile (+0.1%TFA)/water (+0.1% TFA)). Lyophilization of the respective fractionsyielded the title compound  as a TFAsalt. 1H NMRa : 12.20 , 10.33 , 10.12 ,8.29 6-NH-CO-phenyl−),8.15 , 7.83–7.74 , 7.68–7.59 , 7.28–7.18, 6.95–6.86 , 6.83 , 5.09 , 4.51 , 4.08 , 4.03, 3.79 ,3.67 , 3.54–3.37 , 3.22 5-CH2-NH-CO−),2.56 ,2.28 , 1.94 5−),1.56–1.41 2-CH2-CH2−),1.35–1.18 2-CH2-CH2-(CH2)2−). 13C NMRb : 169.6 q (−NH-CO-CH2-CH2-O−), 169.1 q (−CO-NH-OH), 168.9 q (pyrimidine C-2), 167.0 q , 166.8q (−NH-CO-CH2-S−),165.5 q (−(CH2)6-NH-CO-phenyl−), 158.4 q , 158.2 q (phenylC-3), 156.9 q (thiazole C-2), 142.5 q (triazole C-4), 141.9 q (phenylC-1), 141.6 q (4-acylamidobenzamide C-4), 134.7 (thiazole C-4), 131.1q (thiazole C-5), 129.6 (phenyl C-5), 129.1 q (4-acylamidobenzamideC-1), 128.0 , 124.9 (triazole C-5), 120.9(phenyl C-6), 118.1 , 116.1 (pyrimidineC-5), 114.9 (phenyl C-2), 112.4 (phenyl C-4), 69.7 (−O-CH2-CH2-O−), 69.71 (−O-CH2-CH2–O−), 69.66(−O-CH2-CH2-O−),69.64 (−O-CH2-CH2-O−), 69.57 (−O-CH2-CH2-O−), 69.5 (−O-CH2-CH2-O−), 68.6 (−O-CH2-CH2-triazole−), 66.5(−NH-CO-CH2-CH2-O−), 61.0 (−triazole-CH2-O−), 49.4 (−O-CH2-CH2-triazole−), 37.2 (−NH-CO-CH2-CH2-O−), 34.1 (−NH-CO-CH2-S−), 32.2 (HO-NH-CO-CH2-(CH2)5−),31.9 (thiazole-CH2-phenyl), 29.1(HO-NH-CO-(CH2)4-CH2-CH2−), 28.4 (HO-NH-CO-(CH2)2-CH2-(CH2)3−), 26.3 (HO-NH-CO-(CH2)3-CH2-(CH2)2−),25.1 (HO-NH-CO-CH2-CH2-(CH2)4−), 23.2 (pyrimidine −CH3). 19F-NMR : 75.1. HRMS (ESI+): m/z calcd for C46H60N10O10S2Na+: 999.3828 [M + H]+, found: 999.3835. HPLC retention time 16.10 min, 95.2% (M3). a)1135-CH2–  could not be detected.After swelling of the resin 47  for 30 min in DMF, the resin was washed withMeOH (3 × 5 mL). The phthaloyl protecting group was removed bytreatment with 5% hydrazine monohydrate in MeOH for 15 min (2 ×5 mL/g resin). Afterward, the resin was washed with DMF (3 ×5 mL), MeOH (3 × 5 mL), CH2Cl2 (3 ×5 mL), and DMF (3 × 5 mL). For the subsequent amide couplingreaction, a solution of Fmoc-4-aminomethylbenzoic acid , HATU , HOBt·H2O , and DIPEA  in DMF (3 mL) was agitated for 5 min and thenadded to the resin. The amide coupling was performed for 20 h at ambienttemperature. Afterward, the resin was washed with DMF (3 × 5mL) and CH2Cl2 (3 × 5 mL). Completion ofthe reaction was monitored via TNBS test. The preloaded resin 48 was dried under reduced pressure and stored at 4 °C.Determined loading: 0.69 mmol/g.After swelling of themodified resin 48  in DMF for 30 min, Fmoc deprotection was performedby treatment with 20% piperidine in DMF. Afterward, the resin waswashed with DMF (3 × 5 mL), CH2Cl2 (3 ×5 mL), and DMF (3 × 5 mL). For the subsequent amide couplingreaction, a solution of Fmoc-aminobenzoic acid , HATU , and DIPEA  in DMF (1.5 mL) was agitated for5 min and then added to the resin. After shaking the reaction mixturefor 20 h, the resin was washed with DMF (3 × 5 mL) and CH2Cl2 (3 × 5 mL). Completion of the reactionwas monitored via TNBS test. The resin was dried under reduced pressureand stored at 4 °C.After swelling of the preloaded resin 47  for 30 min in DMF, the resin was washed withMeOH (3 × 5 mL). The phthaloyl protecting group was removed bytreatment with 5% hydrazine monohydrate in MeOH for 15 min (2 ×3 mL). Afterward, the resin was washed with DMF (3 × 5 mL), MeOH(3 × 5 mL), CH2Cl2 (3 × 5 mL), andDMF (3 × 5 mL). For the subsequent amide coupling reaction asolution of Fmoc-7-aminoheptanoic acid ,HATU , HOBt·H2O , and DIPEA  in DMF (2 mL) was agitated for 5 min and then added to theresin. The amide coupling was performed for 20 h at ambient temperature.Afterward, the resin was washed with DMF (3 × 5 mL) and CH2Cl2 (3 × 5 mL). Completion of the reactionwas monitored via TNBS test. Upon completion of the reaction and washing,the resin was dried under reduced pressure and a loading between 0.77mmol/g and 0.97 mmol/g was determined for different batches.After swelling of themodified resin 50  in DMF for 30 min, Fmoc-deprotection was performedby treatment with 20% piperidine in DMF. Afterward, the resin waswashed with DMF (3 × 5 mL), CH2Cl2 (3 ×5 mL), and DMF (3 × 5 mL). For subsequent amide coupling reaction,a solution of Fmoc-4-aminobenzoic acid ,HATU , and DIPEA  in DMF (1.5 mL) was agitated for 5 min and thenadded to the resin. After 20 h reaction time at room temperature,the resin was washed with DMF (3 × 5 mL) and CH2Cl2 (3 × 5 mL). Completion of the reaction was monitoredvia TNBS test. The resin was dried under reduced pressure and storedat 4 °C.After swelling of theresin 58, 163 mg, 0.737 mmol, 1 equiv) was dissolved in a mixtureof 14.1mL of dichloromethane and 14.1 mL of methanol and cooled in an icebath to 0 °C. Then 1.354 mL  of an aqueous 50 wt % hydroxylamine solution were added,and the mixture was stirred at 0 °C for 10 min before 294.6 mg sodium hydroxide were added. After 30 min at0 °C, the ice bath was removed and stirring of the colorlessclear solution was continued at room temperature for 2 h. The reactionmixture was freed of all volatiles under reduced pressure, and theresidue was taken up in 40 mL of deionized water. The suspension wascooled in an ice bath and brought to pH = 6 by the dropwise additionof 1 M HCl. The mixture was freed of all volatiles under reduced pressure.The residue was purified by flash column chromatography (MeOH (+1%AcOH)/dichloromethane (+1% AcOH): gradient 0–10%). For thefinal purification, normal phase preparative thin layer chromatographywas used. As an eluent we used a mixture of dichloromethane, methanol,and AcOH (89/10/1). The title compound was obtained as a colorlesssolid . 1H NMR : 11.21 , 9.02 , 8.44 , 7.80–7.64, 7.37–7.24, 4.28 , 1.88 . 13C NMR : 169.3 q (−CH2-NH-CO-CH3), 164.1 q (−CO-NH-OH), 143.0 q (N-hydroxybenzamide C-4), 131.3q (N-hydroxybenzamide C-1), 127.1 , 126.9 , 41.8 (−CH2-NH-CO-CH3), 22.6 (−CH2-NH-CO-CH3). HRMS (ESI+): m/z calcd for C10H13N2O3+: 209.0921 [M + H]+, found: 209.0920.LRMS m/z (ESI+): 439[2M + Na]+. HPLC retention time 9.11 min, 99.2% (M2).Ethyl 4-(acetamidomethyl)benzoate  was dissolved in a mixture of 13.4mL of dichloromethane and 13.4 mL of methanol and cooled in an icebath to 0 °C. Then 1.291 mL  of an aqueous 50 wt % hydroxylamine solution were added,and the mixture was stirred at 0 °C for 10 min before 281.0 mg sodium hydroxide were added. After 30 min at0 °C, the ice bath was removed and stirring of the colorlessclear solution was continued at room temperature for 2 h. The reactionmixture was freed of all volatiles under reduced pressure, and theresidue was taken up in 40 mL of deionized water. The solution wascooled in an ice bath and brought to pH = 8 by the dropwise additionof 1 M HCl, when large amounts of white solid formed. The precipitatewas collected by filtration, washed with water (2 × 1 mL) andice-cold diethyl ether (2 × 2 mL), and dried under reduced pressure.For the final purification preparative HPLC (gradient elution from10% to 35% acetonitrile/water (+0.1% TFA)) was used to yield the titlecompound as a pale-red solid . 1H NMR : 11.16 , 9.84–8.21 , 7.94–7.87, 7.75–7.68 , 7.54 , 7.48 , 7.42–7.35 , 4.51 . 13C NMR : 169.3 q(−CH2-NH-CO-C6H5), 164.1 q (−CO-NH-OH),142.9 q (N-hydroxybenzamide C-4), 134.2 q (benzamideC-1), 131.29 (benzamide C-4), 131.26 q (N-hydroxybenzamideC-1), 128.3 , 127.2 , 127.0 , 126.9 , and 42.4 (−CH2-NH-CO-C6H5). HRMS (ESI+): m/z calcd for C15H15N2O3+: 271.1077 [M + H]+, found: 271.1080.LRMS m/z (ESI+): 541[2M+H]+. HPLC retention time 12.10 min, >99.5% (M1).Ethyl 4-(benzamidomethyl)benzoate (N-butylpentanamido)methyl)benzoate  was dissolved in a mixture of 5 mL dichloromethaneand 5 mL methanol and cooled in an ice bath to 0 °C. 698 μL of an aqueous 50 wt% hydroxylamine solution were added, and the mixture was stirred at0 °C for 10 min before 149.5 mg  of sodiumhydroxide were added. After 30 min at 0 °C, the ice bath wasremoved and stirring of the colorless clear solution was continuedat room temperature for 3 h. The reaction mixture was freed of allvolatiles under reduced pressure and the residue was taken up in 8mL of deionized water. The suspension was cooled in an ice bath andbrought to pH = 8 by the dropwise addition of 1 M HCl, when largeamounts of white solid formed. The solid was collected by centrifugation,washed with ice-cold water (5 mL), and dried under reduced pressure.The residue was purified by reversed phase flash column chromatography(acetonitrile/water (1% TFA (v/v)): gradient 3–40%) to yieldthe title compound as a colorless solid . 1H NMRa : 11.20, 11.17 , 8.97 , 7.77–7.66 , 7.29–7.21 , 4.60, 4.53 -CO-C4H9), 3.24, 3.21 -CO-C4H9), 2.38, 2.23 -CO-CH2-C3H7), 1.60–1.152-CH3)-CO-CH2-(CH2)2-CH3), 0.93–0.77 3-CH3)-CO-(CH2)3-CH3). 13CNMRa : 172.2, 172.1 q (−CH2-N(C4H9)-CO-C4H9), 164.1,163.9 q (−CO-NH-OH), 141.9, 141.5 q(N-hydroxybenzamide C-4), 131.6, 131.4 q (N-hydroxybenzamide C-1), 127.3, 126.3 , 127.2, 127.0 , 50.1, 47.5 (−CH2-N(C4H9)-CO-C4H9), 46.9, 45.3 (−CH2-N(CH2-C3H7)-CO-C4H9), 32.1, 31.7 (−CH2-N(C4H9)-CO-CH2-C3H7), 30.4, 29.3 (−CH2-N(CH2-CH2-C2H5)-CO-C4H9), 27.2, 27.1 (−CH2-N(C4H9)-CO-CH2-CH2-C2H5), 22.0, 21.8 (−CH2-N(C4H9)-CO-(CH2)2-CH2-CH3), 19.6, 19.5 (−CH2-N((CH2)2-CH2-CH3)-CO-C4H9), 13.9, 13.8(−CH2-N(C4H9)-CO-(CH2)3-CH3), 13.8, and 13.7(−CH2-N((CH2)3-CH3)-CO-C4H9). HRMS(ESI+): m/z calcd forC17H27N2O3+: 307.2016 [M + H]+, found: 307.2019; LRMS m/z (ESI+): 307 [M + H]+. HPLCretention time 15.56 min, 99.3% (M1). a) and DIPEA  were dissolved in 7 mL of dichloromethaneand cooled in an ice bath to 0 °C. Acetyl chloride  was added, and the mixture was stirredat 0 °C for 10 min. Then, the ice bath was removed and stirringwas continued at room temperature for 1 h. After complete conversion,50 mL of dichloromethane were added and the reaction mixture was washedaqueous HCl , saturated NaHCO3 solution(50 mL), and brine (50 mL). The organic layer was dried over Na2SO4, filtered, and dried under reduced pressure.The residue was purified by flash column chromatography (EtOAc/isohexane:gradient 12–100%) to yield the title compound as a colorlesssolid . 1H NMR : 8.45 , 8.00–7.80 , 7.47–7.28, 4.39–4.24 , 1.90 , 1.32 . 13C NMR : 169.3 q (−CH2-NH-CO-CH3), 165.6 q (H3C–CH2-O-CO−), 145.3 q , 129.2 , 128.4 q (ethyl benzoate C-1), 127.3 , 60.7 (H3C-CH2-O-CO−), 41.9 (−CH2-NH-CO–CH3), 22.6 (−CH2-NH-CO-CH3), 14.2 (H3C-CH2-O-CO−). HRMS (ESI+): m/z calcd for C12H16NO3+: 222.1125 [M + H]+, found: 222.1126.LRMS m/z (ESI+): 443[2M + H]+. The spectroscopic data are in good agreementwith the literature values.65Ethyl 4-(aminomethyl)benzoate and DIPEA were dissolved in 7 mL of dichloromethane and cooled in an ice bathto 0 °C. Benzoyl chloride  was added, and the mixture was stirred at 0 °C for10 min. Then, the ice bath was removed and stirring was continuedat room temperature for 1 h. After complete conversion, 50 mL of dichloromethanewere added and the reaction mixture was washed aqueous HCl , saturated NaHCO3 solution (50 mL), andbrine (50 mL). The organic layer was dried over Na2SO4, filtered, and dried under reduced pressure. The residuewas purified by flash column chromatography (EtOAc/isohexane: gradient3–45%) to yield the title compound as a colorless solid . 1H NMR : 9.14 , 7.96–7.87 , 7.59–7.52 , 7.51–7.42, 4.55 , 4.30 ,1.31 . 13C NMR : 166.3 q (−CH2-NH-CO-C6H5), 165.6 q (H3C-CH2-O-CO−), 145.3 q ,134.1 q (benzamide C-1), 131.4 (benzamide C-4), 129.2 , 128.4 , 127.33 , 127.27 , 60.7 (H3C-CH2-O-CO−), 42.5 (−CH2-NH-CO-C6H5), and14.2 (H3C-CH2-O-CO−).HRMS (ESI+): m/z calcdfor C17H18NO3+: 284.1281[M + H]+, found: 284.1279. LRMS m/z (ESI+): 567 [2M + H]+.Ethyl 4-(aminomethyl)benzoate  was dissolved in 3 mL of dichloromethane.The solution was cooled to 0 °C. Pentanoyl chloride  was added. Then, pyridine was added in a dropwise manner. The reaction mixture was allowed towarm up to ambient temperature and was stirred for further 4 h. Aftercompletion of the reaction, the reaction mixture was partitioned betweendichloromethane (20 mL) and a saturated aqueous solution of sodiumbicarbonate (20 mL). The organic layer was separated, and the aqueouslayer was extracted with dichloromethane (2 × 20 mL). The combinedorganic layer was dried over Na2SO4, filtered,and concentrated under reduced pressure. The residue was purifiedby flash column chromatography (EtOAc/isohexane: gradient 3–27%)to yield the title compound as a colorless liquid . 1H NMRa : 7.98–7.88 ,7.35–7.29 , 4.65, 4.57 -CO-C4H9), 4.31, 4.30 , 3.26, 3.22 -CO-C4H9), 2.38,2.22 -CO-CH2-C3H7), 1.60–1.12 2-CH3)-CO-CH2-(CH2)2-CH3), 0.93–0.74 3-CH3)-CO-(CH2)3-CH3). 13CNMRa : 172.2, 172.1 q (−CH2-N(C4H9)-CO-C4H9), 165.54,165.47 q (H3C-CH2-O-CO−), 144.3, 143.9 q , 129.5, 129.2 , 128.7, 128.5 q (ethyl benzoate C-1), 127.5, 126.6, 60.7, 60.6 (H3C-CH2-O-CO−), 50.1, 47.6 (−CH2-N(C4H9)-CO-C4H9), 47.0, 45.4 (−CH2-N(CH2-C3H7)-CO-C4H9), 32.0, 31.6 (−CH2-N(C4H9)-CO-CH2-C3H7), 30.4, 29.3 (−CH2-N(CH2-CH2-C2H5)-CO-C4H9), 27.1, 27.0 (−CH2-N(C4H9)-CO-CH2–CH2-C2H5), 21.9, 21.8(−CH2-N(C4H9)-CO-(CH2)2-CH2-CH3), 19.6, 19.4 (−CH2-N((CH2)2-CH2-CH3)-CO-C4H9), 14.2 (H3C-CH2-O-CO−), 13.8, 13.7 (−CH2-N(C4H9)-CO-(CH2)3-CH3).13.7, 13.6 (−CH2-N((CH2)3-CH3)-CO-C4H9). HRMS (ESI+): m/z calcd for C19H30NO3+: 320.2220 [M + H]+, found: 320.2220.LRMS m/z (ESI+): 320[M + H]+. a)(Rotamers wereobserved.Ethyl 4-((butylamino)methyl)benzoate  cells were transformed with the expression vectorspET30S-hSirt1134–747, pET30S-hSirt256–356, or pET15dt-Sirt3118–395. Cells were grown toan OD600 of 0.6 at 37 °C in 2XYT medium (supplementedwith 50 μg mL–1 kanamycin). Overexpressionof sirtuins was induced by IPTG  and afterfurther cultivation at 20 °C for 12 h cells were harvested bycentrifugation . Cells were resuspendedin lysis buffer  glycerol) and cell lysis was achieved by ultrasonication at 70% amplitude for 10 min . After centrifugation with 100 000g for1 h the supernatant was loaded on a Strep-Tactin Superflow cartridge. After elution with lysisbuffer containing d-desthiobiotin , the proteins were further purified by size-exclusion chromatography equilibrated with Tris/HClbuffer  and concentrated. Purity and identityof the target proteins were verified by SDS–PAGE and proteinconcentration was determined by BCA-assay using BSA as a standard.Recombinant humanSirt145 In black 96-well plates , the respective sirtuin was mixed with 5 μL of ZMAL,3 μL of inhibitor in DMSO in varying concentrations or DMSOas a control ) and filled up to55 μL with assay buffer . Substrate conversion was adjustedto 15–30% for the DMSO control and a blank control with noenzyme and a 100% conversion control without enzyme but with AMC instead of ZMAL were performed as well. The enzymatic reaction wasstarted by addition of 5 μL NAD+ , and plates were incubated at 37 °Cand 140 rpm for 4 h. Addition of 60 μL of a trypsin containingstop solution  DMSO, 5.5 U μL–1 trypsin, 8 μM nicotinamide, pH 8.0) stoppedthe catalytic reaction. Further incubation at 37 °C and 140 rpmfor 20 min led to release of the fluorophore AMC. Fluorescence intensitywas measured using a microplate reader . All experiments were performed in duplicate series in atleast two independent experiments. Inhibition was calculated in %in relation to the DMSO control after blank subtraction. IC50 values were determined using OriginPro 9G  or GraphPad7.0 by a nonlinear regression to fit the dose response curve. Allexperiments were performed at least in duplicates.Inhibitionof Sirt1, Sirt2, and Sirt3 activities was determined using a trypsin-basedfluorescence assay, previously described by Heltweg et al.Invitro deacetylation assays for HDAC6 and HDAC1–3 wereperformed as previously reported.44 Allreactions were performed in OptiPlate-96 black microplates (PerkinElmer)with duplicate series in at least two independent experiments. Anassay end volume of 50 μL was set for each experiment, and allreactions were carried out in assay buffer  withappropriate concentrations of substrate and inhibitors obtained bydilution from 10 mM stock solutions. Control wells without enzymeand control wells with vorinostat as reference were included in eachplate. After addition of 5 μL of test compound or control inassay buffer, 35 μL of the fluorogenic substrate ZMAL (21.43μM in assay buffer) and 10 μL of the enzyme solution,the plates were incubated at 37 °C for 90 min. Human recombinantHDAC1 , HDAC2 , HDAC3/NcoR2 ,or HDAC6  was applied. Afterward,50 μL of 0.4 mg/mL trypsin in trypsin buffer  was added, followed by further incubation at37 °C for 30 min. Plates were analyzed using a Fluoroskan Ascentmicroplate reader (Thermo Scientific), and fluorescence was measuredwith an excitation wavelength of 355 nm and an emission wavelengthof 460 nm.50 values given in 47 In brief, the assaywas performed in a similar manner as described for sirtuin deacetylationactivity. However, instead of ZMAL, a myristoylated substrate (ZMML)was used, and the Tris buffer was exchanged for a HEPES buffer (25mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 137mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.015% Triton X-100, pH8.0). AMC was used again as 100% conversion control. All experimentswere performed in duplicate series in at least two independent experiments.OriginPro 9.0 and GraphPad 7.0 were used for the analysis of results.The discontinuous Sirt2 demyristoylation assay thatwas used to determine the IC33, we used a continuous Sirt2 demyristoylationassay as reported by Zessin et al.48 Inbrief, this assay was performed in a black 384-well fluorescence platein a total volume of 40 μL of Sirt assay buffer . Dilutionseries of the inhibitor was done in DMSO. A total of 2 μL ofthe inhibitor dilutions were incubated with 14 μL of the peptidesubstrate, referred to as F4 in Zessin et al.48  and 14 μL of Sirt2 solution  for 5 min at room temperature. In addition tothe inhibitor-containing samples, a sample without inhibitors wasmeasured as a positive control, and a sample without enzymes was measuredas a negative control. The DMSO concentration was set to 5% (v/v).The reaction was started by the addition of 10 μL of NAD+ . The product formationwas monitored by the change in fluorescence intensity, and the reactionrate represented the activity. Fluorescence readout was performedwith a BMG Fluostar Omega plate reader at following wavelengths: Excitationwavelength: λEx = 485 ± 10 nm, emission wavelength:λEm = 530 ± 10 nm. Inhibition was calculatedusing the following equation:xc, slopeof compound; xpos, mean slope of positivecontrol; xneg, mean slope of negativecontrol. Data fitting was carried out by GraphPad 9.0.2 using nonlinearfit ([inhibitor] vs response–variable slope (four parameters)).Experiments were performed in triplicate.As a second method to confirm the inhibition of Sirt2-mediateddemyristoylation by 67 In brief, the reactions of Sirt5 and Sirt6 were performed in a totalvolume of 120 μL in Sirt buffer, containing 20 mm TRIS (pH 7.8),150 mM NaCl, 5 mM MgCl2, with 0.2 mg/mL BSA. The inhibitorswere dissolved in DMSO, and the final DMSO concentration was 5% (v/v).The inhibitors (20–500 μM) were preincubated togetherwith the master mix for 5 min at 37 °C. The master mix consistedof NAD+ (1 mM) and the respective substrate (Sirt5: Bz-GVLK(Succ)EYGV-NH2 final conc = 5 μM and Sirt6: Ac-EALPKK(Myr)Y(NO2)GG-NH2 final conc = 20 μM). The reactionswere started by the addition of enzyme . After shaking at 37 °C for 5 min for Sirt5 or after 1 hfor Sirt6, 100 μL of the reaction was added to 20 μL ofstop solution consisting of 1% (v/v) trifluoroacetic acid (TFA) and10% (v/v) ACN in dd. H2O. The reaction solution in a volumeof 100 μL was injected into the RP-HPLC system, and compoundswere separated using linear gradients.These discontinuousHPLC assays for Sirt5 and Sirt6, respectively,were performed as previously described.p-nitrotyrosine (λ = 360 nm) was usedto quantify product and substrate peak areas. The reactions were performedonce in duplicates. The conversion in the noninhibited reaction wasassigned to 100%, and the conversion in a reaction without enzymewas defined as 0%. The normalized conversions were plotted as a functionof the logarithm inhibitor concentration and the IC50 valueswere calculated using nonlinear regression for normalized dose–responsecurves with GraphPad Prism8.The solvents used werewater (solvent A) and ACN (solvent B), both containing 0.1% TFA. Theseparations were performed on a 3.0 mm × 50 mm reversed phasecolumn  with a flow rateof 0.6 mL/min. The mixtures of the Sirt5 reactions were separatedusing a linear gradient from 10% to 60% solvent B within 6 min. Theabsorbance at 220 nm was used to quantify product and substrate peakareas. The mixtures of the Sirt6 reactions were separated using alinear gradient from 5% to 95% solvent B within 6 min. The absorbanceof the 68 In brief, all reactions were performed in 384-well plates  with a total volume of 40 μL per wellin HDAC buffer (20 mM HEPES buffer (pH 7.4), 140 mM NaCl, 10 mM KCl,1 mM TCEP, and 0.2 mg/mL BSA). Inhibitors were dissolved in DMSO andthe final DMSO concentration was 3% (v/v). DMSO only was used forboth negative and positive control. The inhibitors (6–20000nM) were preincubated with the enzyme  for 5 min at room temperature whileshaking. The reaction was started by adding the substrate Abz-SRGGK(STFA)FRR-NH2 (STFA = thiotrifluoroacetyl residue = fluorescence quencher).The change in florescence was monitored over 30 min every 30 s. Then20 mM HEPES buffer (pH 7.4) containing 0.5 mg/mL BSA was used forthe HDAC10 measurements. The inhibitors and the enzyme (5 nM) wereprepared as described above. The reaction was also started by theaddition of the substrate . The change in florescence was monitoredover 60 min every 50 s. All measurements were performed on an EnVision2104 Multilabel reader  with a 330 ±75 nm excitation filter and a 430 ± 8 nm emission filter. Allmeasurements were performed in triplicates with n = 2. The fluorescence intensity was plotted as a function of time.The initial slope of these plots was set as activity. The activityin the noninhibited reaction was assigned to 100%, and the activityin a reaction without enzyme was defined as 0%. The normalized activitywas plotted as a function of the logarithm inhibitor concentrationand the IC50 values were calculated using nonlinear regressionfor normalized dose–response curves with GraphPad Prism8.These continuous fluorescence-basedassays for monitoring the enzymatic activity of HDAC4, 5, 7, and 9were performed as previously described.69 In brief, inhibition of HDAC8 was measured in 1/2 AREAPLATE-96 F microplates (PerkinElmer) with a total assay volume of 30 μL.HDAC8 enzyme was obtained as described before.71 The conversion rate of the enzyme was adjusted to 10–20%.22.5 μL of enzyme in HDAC buffer  was mixed with 2.5 μL of inhibitorin DMSO and 5 μL of Z-l-Lys(ε-trifluoroacetyl)-AMC. The plate was incubatedat 37 °C for 90 min. The reaction was stopped by adding 30 μLof freshly prepared stop solution to each well, composed of trichostatinA (2.75 μM), trypsin from bovine pancreas . After incubation for 20min at 37 °C, fluorescence intensity was measured at an excitationwavelength of 390 nm and an emission wavelength of 460 nm in a microtiterplate reader (BMG Fluostar). IC50 values were determinedby using 10 different compound concentrations. All experiments wereperformed in triplicate. Calculations were performed using standardmethods and the GraphPad Prism software package (version 9.0.2).This assay was performed as previously described.47 In brief, PC-3M-luc cells (25 000 cells perwell) were plated in Ibidi 8-well slides and incubated overnight at 37 °C, 5% CO2. Next, cellswere treated with 20 μM of inhibitor. After 5 h, the mediumwas removed and the cells washed with PBS and fixed with 4% PFA for8–10 min at ambient temperature. Cells were rinsed three timeswith PBS and lysed with extraction buffer for 3–5 min at ambient temperature. After another washing stepwith PBS, blocking buffer  was added forat least 10 min before incubating with monoclonal acetylated α-tubulinantibody  in blocking bufferovernight at 4 °C. The cells were rinsed three times with blockingbuffer and incubated with goat antimouse IgG H&L Alexa Fluor 647, diluted 1:2000 in blocking buffer, for 30 minin the dark. The cells were rinsed two times with blocking bufferand once with PBS and DAPI in mounting medium  diluted 1:50 inPBS was added and it was incubated for 10 min in the dark. Confocalmicroscopy was performed with a Leica SP8 confocal microscope equippedwith a 40×/1.40 oil objective (Leica Microsystems) keeping thelaser settings of the images constant to allow direct comparison ofsignal intensities between images.The detection of acetylation levels of α-tubulinby immunofluorescence was performed as previously described.47 In brief, HEK293T cells were plated in 6-wellplates  at a density of 8 ×105 cells per well and incubated 2–4 h at 37 °Cand 5% CO2 before transfection. The fusion protein plasmidswere transfected using Fugene HD transfection reagent (Promega) accordingto the manufacturer’s protocol. First, 2 μg of fusionprotein DNA were dissolved in 100 μL of medium without serumand phenol red to obtain a concentration of 0.02 μg of DNA perμL. Next, Fugene reagent was added to form DNA:Fugene complexesin a ratio of 1:3 and the mix was shortly vortexed and incubated for15 min at ambient temperature. The mix was added dropwise to the HEK293Tcells followed by incubation for 20–24 h at 37 °C and5% CO2. Cells were trypsinized, resuspended in medium withoutserum and phenol red, and adjusted to a concentration of 2 ×105 cells per mL. All compounds were prepared as concentratedstock solutions dissolved in DMSO. To determine affinities of theinhibitors, a final tracer concentration of 2 μM was used. Asa fluorescent Sirt2 tracer, we used SirReal-TAMRA with a reported Kd value of 0.25 μM.47 Serially diluted inhibitors and tracer were added to thecell suspension, and 100 μL of the mixture were seeded in 96-wellwhite, sterile nonbinding surface plates . Plates were incubated at 37 °C and 5% CO2 for 2 h. For BRET measurements, 25 μL of 1:100 diluted NanoBRETNanoGlo Substrate  was added to the wellsaccording to the manufacturer’s protocol and incubated for2–3 min at ambient temperature. For all measurements, the 2102EnVisionTM Multilabel reader (PerkinElmer) was used, equipped with460 nm filter (donor) and 615 nm (acceptor) filter. The BRET ratioswere calculated as difference between the acceptor/donor channel emissionratio of the sample and the acceptor/donor emission ratio of the control:NanoBRET experimentswere performed as previously described.50 valueswere calculated with a four-parameter logistic fit. Data analysiswas performed using GraphPad Prism 7.0.The obtained NanoBRET ratios of thesamples were normalized to the DMSO control. IC2. Compound or vehiclewere added to a final concentration of 0.5% DMSO. After 72 h of incubationtime, 20 μL of a mixture (20:1) consisting of MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-2H-tetrazolium) and PMS (phenazine methosulfate) were addedto each well. Absorbance was measured after another 2–4 h witha BMG LABTECH POLARstar OPTIMA plate reader . Experiments were performed in triplicates, and EC50 values were calculated using the Prism . EC50 was defined as the concentration that ledto 50% viable cells. Cell viability assays with W1 and MCF-7 cellswere performed by applying an MTT assay using 3--2,5-diphenyltetrazoliumbromide . Cells wereseeded at densities of 104 cells/well for W1 cells or 2× 103 cells/well for MCF-7 cells. Tests were performedin 96-well plates . Afterincubation overnight, cells were supplemented with half logarithmicdilution series of the indicated compounds and DPBS  as control. Then 72 h after addition of cytostatictreatment, the MTT solution  was added intothe wells for 1 h at 37 °C and 5% CO2 until formazancrystals were formed. The supernatant was removed, and the cells weredissolved in 200 μL of DMSO. Absorption was analyzed at 570nm, with background subtraction at 690 nm, using a plate reader . Cell viability was then determinedby nonlinear regression and a four-parameter logistic equation withvariable hill slope. All experiments were performed in triplicate(n = 4), and inflection points of sigmoidal curveswere used to calculate EC50 values Prism . EC50 was defined as the concentrationthat led to 50% viable cells. The significance of the difference ofthe effects of 33 and 4 + 57 was determined by using an unpaired, two-tailed t test with Welch’s correction, implemented in GraphPad Prism8.4.0 . Statistical significance was accepted if p < 0.05. Asterisks indicate *p <0.05, **p < 0.01.Cell viability assays of HGC27and PC-3M-luc cells were performed by using the Celltiter 96 AqueousNonradioactive Proliferation Assay (Promega). Cells were seeded insterile 96-well plates at a density of 2000 cells per well and incubatedovernight at 37 °C and 5% CO72 In brief, MCF-7 were centrifuged and lysed with cell extractionbuffer (Thermo Fisher), supplemented with 0.1 mM PMSF, Halt ProteaseInhibitor (Thermo Fisher), and Natriumorthovanadat (Thermo Fisher).Precast gels with a polymerization degree of 4–15% were used.Proteins were transferred to Trans-Blot Turbo-PVDF membrane (Bio-Rad).The membrane was blocked with skimmed milk powder in Tris-bufferedsaline-Tween 20 (with 0.2% Tween 20) for 60 min, followed by threewashing cycles of 10 min using Tris-buffered saline-Tween 20. Afterward,membranes were incubated with primary antibodies for ac.-Histon H3 and GAPDH  fora total of 60 min at room temperature and then incubated at 4 °Covernight. Membranes were rinsed again three times before applyingthe secondary antirabbit IgG HRP-conjugated mAbs  or antimouse IgG HRP-conjugated mAbs  for 90 min. Primary antibodies werediluted 1:500 (ac.-Histon H3) or 1:20.000 (GAPDH), respectively, andthe secondary antibodies were diluted 1:20 000. For HDAC6 rescueexperiments, precast gels with a polymerization degree of 10% wereused , and the primary antibody for HDAC6 was diluted 1:1000. After rinsing of the secondary antibody, membraneswere detected using ClarityECL Western Blotting Substrate (Bio-Rad).For quantitative determination, the StainFree technique was employed(Bio-Rad), as well as normalization, against the housekeeping proteinGAPDH, which allows the imaging of whole lysates in SDS-PAGE beforeblotting and normalization against the total protein. Pixel densityanalysis was performed with the IMAGE LAB software (Bio-Rad).Western blots on histone H3 hyperacetylationin MCF-7 cells were performed according to a previously publishedprotocol.56–356 was expressed and purifiedas described previously with minor modifications summarized hereafter.18 Sirt256–356 was overexpressedin 2xYT medium using strain E. coli BL21 Star (DE3) at 20 °Covernight. Overexpression was induced with IPTG  atan OD600 of 0.6–0.8. The cleavage of the His10-Tag via TEV protease was performed in lysis buffer  glycerol, 0.5 mM Tris(2-carboxyethyl)phosphine(TCEP), pH 8.0) at 4 °C for 36 h. Sirt256–356 was applied on a HisTrap HP column  again toobtain pure fractions of Sirt256–356 with cleavedHis10-Tag in the flowthrough. For the last purificationstep a Superdex S75 26/600 gel filtration column  was used. Crystallization assays were set up withthe Oryx Nano pipetting robot  using the vapor diffusion sitting drop method  at 20 °C. Sirt256–356 (13.2 mg/mL) was incubated with 1.8 mM compound 5  final DMSO concentration)on ice for 1 h prior to crystallization. To remove precipitates, thesolution was centrifuged 10 min at 4 °C. Crystals of the Sirt2–5 complex formed after 30 days in wells with a 1:1 ratio of0.3 μL protein solution to 0.3 μL reservoir solution containing25% (w/v) PEG 3,350 in 0.1 M Bis-Tris at pH 6.5. The crystals werecryoprotected with a mixture of 10% (v/v) 2R,3R-(−)-butanediol and reservoir solution, mountedon a nylon loop, and flash-cooled in liquid nitrogen.For cocrystallization experiments withSirt2, human Sirt2Danio rerio  encoded in the His6-MBP-TEV-HDAC6-pET28a-(+) vector washeterologously expressed in Escherichia coli BL21(DE3)cells. Protein was purified as described.73 The HDAC6–55 and HDAC6–57 complexes were crystallized via sitting drop vapor diffusion at13 °C. Briefly, the protein solution was mixed with 2 mM inhibitor and equilibrated on ice at 4 °Cfor 1 h. The enzyme–inhibitor solution was spin-filtered witha 0.22 μm centrifuge filter prior to crystallization. Usinga mosquito crystallization robot (SPT Labtech), a 300 nL drop of enzyme–inhibitorsolution was mixed with a 150 nL drop of precipitant solution fromthe Additive Screen (Hampton Research)  and 25 nL of seeding solution preparedfrom crystals of wild-type HDAC6. The resulting 475 nL sitting dropwas equilibrated against 80 μL of precipitant solution in thecrystallization well reservoir. Precipitant solutions used for crystallizationof HDAC6 complexes contained the following additives: HDAC6–55 complex, 0.2 M NDSB-211 (Hampton Research); HDAC6–57 complex, 3% (v/v) ethanol. Crystals appeared after 1 dayand grew to full size after 3 days. Prior to harvesting, crystalswere soaked in a cryoprotectant solution containing mother liquoralong with 25% (v/v) ethylene glycol and flash-cooled in liquid nitrogen.For co-crystallizationexperiments with HDAC6, the catalytic domain 2 of HDAC6 from 5 complex, X-ray diffraction data were collectedon ID30A-3 (MASSIF-3) beamline74 at theEuropean Synchrotron Radiation Facility  usingan Eiger X 4M detector. The data set was processed using autoPROC75 and scaled using Aimless.76 The structure was solved by molecular replacement withPhaser77 using the Sirt2–3–AcLysH3 complex (PDB 4RMH)18 as a searchmodel. The model was built and refined in iterative steps with COOT78 and either REFMAC79 or Phenix.refine.80 Restraints for 5 were generated with the Grade Web Server . All residues except for Met299, Ile300, Met301,Gly302, and Leu303 of the Sirt2-specific insertion are defined inthe electron density. Amino acids Gly102–Tyr114 exhibit poorelectron density and higher B-factors compared tothe rest of the protein. The electron density of 5 iswell refined. Final structures were validated using MolProbity.81 All data collection and refinement statisticsare reported in the Supporting Information (SI), For theSirt2–55 complex,X-ray diffraction data were collected on NE-CAT beamline 24-ID-C atthe Advanced Photon Source, Argonne National Laboratory. Data wereprocessed using Rapid Automated Processing of Data with X-ray detectorsoftware (XDS).82 For the HDAC6–57 complex, X-ray diffraction data were collected on the NSLS-IIFMX beamline at Brookhaven National Laboratory.83 Data were processed and analyzed using autoPROC.75 The initial electron density map of each complexwas phased by molecular replacement using Phaser77 with the atomic coordinates of unliganded HDAC6 (PDB 5EEM)49 as a search model. Iterative model building and refinementcycles were performed using Coot78 andPhenix,80 respectively. In the final stagesof refinement, inhibitor and solvent molecules were added into well-definedelectron density. Final structures were validated using MolProbity.81 All data collection and refinement statisticsare reported in SI, For the HDAC6–5, PDB 8OWZ, HDAC6 cocrystallized with 55, PDB 8G1Z and 57, PDB 8G20). Protein preparation and ligand docking were performed using theSchrödinger software version 2021.3v.84 Protein structure preparation for docking was done using the ProteinPreparation Wizard within Schrödinger software using defaultsetting. This included addition of missing hydrogen atoms and aminoacid side chains, removal of solvent molecules and metal ions, optimizationof protonation states and minimization applying the OPLS Force Fieldimplemented in Schrödinger version 2021.3v. Docking was doneusing Glide in standard precision (SP) mode. The grid box includingthe information on active site coordinates of the proteins was definedwith 20 Å radius around the ligand. Ten docking poses were employedfor each ligand. The other settings were kept as default. The dockingsetup was previously used for Sirt2/HDAC inhibitor docking and couldbe validated by the structures of cocrystallized inhibitors.86 Redocking procedure was alsoable to reproduce the binding mode of 5 in Sirt2 withRMSD value of 0.227 Å and that of 55/57 in HDAC6 with RMSD values of 0.548 and 1.448 Å, respectively. RMDS value of 57 was slightlyhigher due to the flexibility of the two n-butylchains in the capping group.The 3D structures of HDAC6 and Sirt2were taken from the crystal structures solved in this study  atomic charge model were used.89 Using the AMBER22 tLEaP module the protein–ligandcomplexes were generated. The AMBER force field (GAFF2) was used asligand force field, while force field 14 Stony Brook-ff14SB was usedfor the protein structures.90 For the catalyticZn2+ ion the 12–6–4 LJ-type nonbonded ionmodel was applied.91 The final complexeswere solvated by TIP3P water model as octahedral box around the protein(10 Å margin). Na+ and Cl– ionswere added for neutralization of the whole system. Parameter/topologyfiles of the entire system were saved to use as starting point ofthe MD simulations. The solvated and neutralized systems were subjectedto two energy minimization steps involving 1000 cycles of steepestdescent followed by 2000 cycles of conjugate gradient minimization.In the first minimization solvent molecules and counterions (Na+ and Cl–) were minimized using a force constantof 10 kcal·mol–1·Å–2 for the protein, ligands, and zinc ion. In the second minimizationstep, the whole system was minimized. Afterward, the entire systemwas heated over 100 ps from 0 to 300 K. Constant volume periodic boundarywas set to equilibrate the temperature of the system by Langevin thermostatusing a collision frequency of 2 ps–1. Thereafter,a pressure equilibration routine at 300 K was performed for 100 pswith a constant pressure of 1 bar. Ultimately, free MD simulationutilizing the Particle Mesh Ewald method with the time step of 2 fswas applied for 100 ns. During these 100 ns, the system temperaturewas kept at 300 K and pressure of the system was maintained at 1 barimplementation of the SHAKE algorithm was done to constrain all bondcontaining hydrogens. As resul0,t 1000 frames were written for each100 ns long simulation. Simulations of the crystal structures as wellas the docking poses were repeated two times with nonidentical randomseeds. Figures were generated using Pymol implemented in the Schrödingersoftware suite.GPU-based MD simulationswere performed using the AMBER22 program.http://zinc15.docking.org/patterns/ home/. None of the final compounds  was flagged asPAINS. Intermediates 34, 38, and 42 were flagged due their azide group but were only used for synthesisand were not evaluated in biological assays.For the identificationof potentialpan-assay-interferers (PAINS), all compounds were reviewed using