Efficacy of a New Selective Indole-based Histone Deacetylase 10 Inhibitor in Targeted Anticancer Therapy

Efficacy of a New Selective Indole-based Histone Deacetylase 10 Inhibitor in Targeted Anticancer Therapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Efficacy of a New Selective Indole-based Histone Deacetylase 10 Inhibitor in Targeted Anticancer Therapy Amer H. Tarawneh, Salah A. Al-Trawneh, Talha Z. Yesiloglu, Matthes Zessin, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5950104/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Histone deacetylase (HDAC) inhibitors represent a newer class of anti-cancer agents that play a key role in both epigenetic and non-epigenetic regulation, leading to cancer cell death, apoptosis, and cell cycle arrest.. These inhibitors are being tested in numerous clinical trials against various diseases, including both hematological and solid malignancies. In the present study, we synthesized novel bicyclic hydroxamic acid derivatives and tested them in vitro against class I and IIb HDACs to investigate their inhibitory activity and selectivity. We demonstrate that compound 6 inhibits HDAC10 with high specificity over HDAC6, with no significant impact on class I HDACs. Compound 1 shows the best inhibitory activity against HDAC10, with IC 50 0.41 ± 0.02 nM. Compound 4 revealed a preference toward HDAC6, with an IC50 value of 2.5 ± 0.3 nM. Compounds 2 and 3 demonstrated high selectivity toward class IIb over class I HDACs. Docking and molecular dynamics studies revealed that compound 1 fits well into the active site of HDAC10, forming stable and strong interactions with key residues F204, D94, W205, and E274 in HDAC10. In addition, we tested these compounds against a panel of four human solid tumor cell lines. Furthermore, non-cancerous kidney cell lines (LLC-PK1 and VERO) were employed to determine the anti-cell proliferative activity of these compounds toward noncancerous cells. Physical sciences/Chemistry Physical sciences/Chemistry/Medicinal chemistry Histone deacetylases HDAC10 inhibitors Tumor cell lines Molecular docking Molecular Dynamics Figures Figure 1 Figure 2 Figure 3 Figure 4 1 INTRODUCTION Histones assist in the control of the expression process that converts the coded information in genes into the operational structures in the cell. Histone acetylation (HA) neutralizes the positive charge on the histones by changing amines into amides [ 1 ]. This change decreases the ability of the histone to bind to DNA (chromatin expansion) and permits genetic transcription [ 2 , 3 ]. Histone deacetylases (HDACs) are enzymes that remove the acetyl group from acetylated lysine residues found in both histone and non-histone proteins. This type of removal results in silencing DNA gene expression. Histones are basic proteins present in the chromatin of eukaryotic cell nuclei and play a role in wrapping DNA into structural units called nucleosomes, which are the basic repeating units of chromatin [ 4 , 5 ]. There are 18 subtypes of HDACs within the human genome. These enzymes are categorized into two groups; the first family is zinc-dependent HDACs, which include the following sub-classes: class Ia (HDAC1, HDAC2), class Ib (HDAC3), and class Ic (HDAC8), class IIa (HDAC4, HDAC5, HDAC7, and HDAC9), class IIb (HDAC6 and HDAC10), and class IV (HDAC11). The second group is nicotinamide adenine dinucleotide (NAD + )-dependent HDACs (class III), which are known as sirtuins [ 6 , 7 ]. Many pieces of evidence revealed the crucial role of HDACs in different cellular processes, including autophagy, cell cycle control, and apoptosis [ 8 – 11 ]. Recent data indicate the protective HDAC function against DNA damage, making them validated anti-tumor therapy targets [ 11 ]. Several HDAC inhibitors have been identified, and some (Fig. 1) were approved by FDA. For instance, vorinostat (SAHA) was approved for the treatment of cutaneous T cell lymphoma (CTCL) [ 12 ], velinostat (PXD-101) and romidepsin (FK-228) were approved for the treatment of peripheral T-cell lymphoma (PTCL) [ 13 – 15 ], bipolar disorders and migraine. In addition, panobinostat (LBH-589) was approved for the treatment of multiple myeloma. The inhibitor leads to the growth arrest, differentiation, and apoptosis of many transformed cells, thus eliminating cancer cells [ 16 ]. The catalytic domains of the different zinc-dependent HDACs share a high degree of homology. The lack of specific isoform selectivity of approved HDAC inhibitors leads to potential side effects [ 17 – 19 ]. Both non-selective HDAC inhibitors, vorinostat, and panobinostat modulate multiple pathways and factors in MDA-MB-231, 4T1, and BT-549 cell lines. This modulation includes suppression of growth factor FOXA1 [ 20 ], upregulation of tumor suppressor factors p21 and p27, and downregulation of the survival protein Bcl-2 [ 21 ]. In addition, both can inhibit matrix metalloproteinase (MMP9) activity [ 22 ]. Recently, HDAC10 has been shown to play a unique role in neuroblastoma cells, where inhibition of HDAC10 can cause an accumulation of autolysosomes. This result indicates that HDAC10 may be an effective target for the treatment of neuroblastoma [ 23 ]. Conversely, HDAC10 was found to suppress tumorigenesis in cervical cancer by downregulating the expression of miR-233 and subsequently targeting EPB41L3 expression. [ 24 ]. Investigating the biological relevance of HDAC10 and evaluating its pharmacological role in cancer cell lines highlight the need for selective HDAC10 inhibitors. A few inhibitors that target HDAC10 have so far been identified [ 25 ], such as tubastatin A and tubastatin A derivative (1b), which are selective for HDAC subfamily IIb [ 26 ]. Recently, TH34 was reported as a class I/IIb selective HDAC inhibitor [ 27 ]. Additionally, compounds including 2-(oxazol-2-yl)phenol moiety were considered as HDAC1/6/10 inhibitors [ 28 ]. The importance of a basic moiety in the linker group was also shown in a recently developed series of piperidine-based HDAC10 inhibitors [ 29 , 30 ]. The challenge lies in introducing HDAC10 inhibitors with high selectivity over the class IIb isozyme member HDAC6. Most HDAC inhibitors share common pharmacophoric features, which include a 'cap' group and hydrophobic chain (linker); both groups impact the selectivity. In addition, the zinc-binding group (ZBG) is crucial to the inhibitor's potency [ 31 ]. Herein, we report an effort to optimize indole and quinoline ligands as HDAC10-selective inhibitors. Several modified derivatives that were developed exhibited HDAC10 inhibition and selectivity. 2 RESULTS AND DISCUSSION 2.1 Chemistry The compounds in this study were synthesized as shown in schemes 1 and 2. The starting building block, 1 H -indole-3-carbaldehyde, was treated with 4-methylbenzene-1-sulfonyl chloride in anhydrous N, N' -Dimethylformamide (DMF) using sodium hydride as a base, and subsequently coupled with various amino compounds via a reductive amination reaction using sodium triacetoxyborohydride (STAB) followed by condensation reaction with hydroxylamine and KOH to afford final targeted compounds 2 , 3 , and 6 , Scheme 1. Similarly, indole-3-carboxaldehyde and quinolone were treated in the same manner as indole-3-carboxaldehyde to produce compounds 1 , 4 , 5, and 7 , Scheme 1 and 2. 2.2 Pharmacology/Biology 2.2.1 Synthesis and in vitro testing of novel inhibitors Recently, piperidine-4-acryl hydroxamates ( PZ45 and PZ48 ) were reported as potent HDAC10 inhibitors having high specificity for HDAC10 over HDAC6 and with no significant impact on class I HDACs [ 29 ]. The selectivity of these newly discovered ligands PZ45/48 was also tested in acute myeloid leukemia (AML) cells with the FLT3-ITD oncogene and showed promising activities. In the current work, we extended the structure-activity relationships of HDAC10 inhibitors by synthesizing a series of benzhyldroxamic acid derivatives that bear a bicyclic aromatic moiety as a capping group with a methylene spacer to a basic amine group. All prepared derivatives were tested in vitro against HDAC10 (from zebrafish) and human HDACs 1, 6, and 8 (Table 1 ). Compound 1 , bearing the indole methylene capping group, showed potent inhibition of HDAC10 with an IC 50 of 0.41 ± 0.02 nM (Table 1 ). The tosylatated indole analogue, compound 2 , showed nanomolar HDAC10 inhibition with an IC 50 of 4.5 ± 0.3 nM. Figure 3 , shows that hydrophobic tosyl moiety does not interact with the rim of the HDAC10 binding site and instead remains fully exposed to the solvent. The removal of the methylene spacer between the primary amine and the benzhydroxamic acid moiety in analog 3 and 4 led to a significant reduction in the inhibitory activity (IC 50 of 290 ± 60 nM and 110 ± 10 nM, respectively). The loss of salt bridge interactions with D94 and E274, along with the cation-π interaction with W205 in HDAC10, which were present in the other compounds reported in this study, is evident. As anticipated, only hydrogen bond interactions between the NH-group and D94 are observed (Figure S1 b and Figure S1 c). The second series of compounds comprises of 4-(1-piperazinyl)benzhydroxamic acid derivatives that bear a bicyclic aromatic moiety as a capping group. Compound 5 exhibited potent HDAC10 inhibitory activity (IC 50 = 2.0 ± 0.1 nM); while the methyltosylated indole derivative revealed that compound 6 showed a decrease in the HDAC10 inhibitory activity with an IC 50 of 75 ± 12 nM (Table 2 ). Meanwhile, the replacement of the indole of 5 with aquinoline moiety yielded compound 7 , which was found to be slightly less potent with IC 50 of 11 ± 1 nM. In conclusion of the structure-activity relationship (SAR) analysis, the absence of a strong basic amino group was found to reduce the inhibitory activity against HDAC10. Additionally, modifying the capping group with a hydrophobic tosyl moiety also led to a decrease in inhibitory activity The preliminary structure-activity relationship (SAR) revealed that the additional bicyclic aromatic moiety enhanced the HDAC10 inhibitory activity. We designed and synthesized seven compounds with various substituents at the cap position and linker, the benzhydroxamic acid bearing an indole ring; compound 1 , stood out as the most potent HDAC10 inhibitor with the best enzyme inhibitory with selectivity toward HDAC10. Herein we introduce highly selective ligand 6 for HDAC10 with nanomolar inhibitory activity. 2.2.2 Enzymatic in vitro testing All the synthesized compounds were evaluated in vitro against zebrafish HDAC10 (drHDAC10), and human HDAC 1, 6, and 8 (details see Methods section). DrHDAC10 was chosen as the close homolog of human HDAC10 since it was found to be more stable and easier to express compared to the human HDAC10 [ 29 , 32 ]. It’s worth mentioning that none of the compounds displayed potent inhibitory against class I HDACs. Data in Table 2 indicate that all compounds retained activity on HDAC10 comparable or even higher than the reference HDAC6/10 inhibitor Tubastatin A. We reported that the indole ring significantly affected the potency and selectivity towards HDAC10 [ 29 ]. Compounds 1 bearing a benzhydroxamic acid displays a highly potent inhibitory activity with IC 50 of 0.41 ± 0.02 nM towards HDAC10, but also potent against HDAC6 and moderately potent against HDAC8 with IC 50 value of 37 ± 2 and 350 ± 20 nM, respectively. Table 2 In vitro selectivity of newly synthesized HDAC10 inhibitors. ID hHDAC1 IC 50 [nM] hHDAC6 IC 50 [nM] hHDAC8 IC 50 [nM] 1 4500 ± 200 37 ± 2 350 ± 20 2 22.2%@1 µM 72.8% @10 µM 51 ± 5 65.1%@1 µM 97.3% @10 µM 3 1.3%@1 µM 38.9% @10 µM 53 ± 5 49.2%@1 µM 98.9% @10 µM 4 51.7%@1 µM 91.4% @10 µM 2.5 ± 0.3 210 ± 10 5 48.4%@1 µM 90.6% @10 µM 73 ± 3 74 ± 6 6 7.3%@1 µM 67.2% @10 µM 35.3%@1 µM 95% @10 µM 26.9%@1 µM 87.9% @10 µM 7 32.1%@1 µM 79.4% @10 µM 130 ± 10 300 ± 20 TubastatinA 7.6% @1 µM 29.9%@10 µM 19 ± 1 46.4% @1 µM 84% @10 µM Compounds 5 and 7 showed a preference for HDAC10 (IC 50 2.0 ± 0.1 and 11 ± 1 nM, respectively) compared with HDAC8 and HDAC6. As can be expected, compound 4, which only contains a weakly basic aromatic amine moiety, showed a significant decrease in the HDAC10 inhibitory activity and high preference and potency to HDAC6 with an IC 50 value of 2.5 ± 0.3 nM, which again highlights the importance of the protonated amine group to achieve high HDAC10 potency and selectivity. Meanwhile, compounds 2 and 3 showed a preference for class IIb enzymes (HDAC10 and 6) with no significant inhibition on HDAC1/8. HDAC6 is well documented, it plays a crucial role in microtubule deacetylation and regulation of PDL1 and further targets related to cancer immunotherapy [ 33 , 34 ]. In combination with the observed in vitro potency and selectivity of compounds 2 and 3 , both represent promising hits for further optimization. The lack of specific and potent HDAC10 inhibitors led to limited knowledge about the biological function of HDAC10 [ 29 ]. A more distinguished result showed by compound 6 examined the inhibitory of compound 6 revealed high selectivity with a novel nanomolar inhibitor toward HDAC10 (IC 50 75 ± 12 nM). 2.2.3 Cytotoxic activity All the synthesized compounds (Table 3 ) were screened at three different concentrations for their cytotoxicity towards a panel of four human solid tumor cell lines: melanoma (SK-MEL), epidermal carcinoma (KB), breast carcinoma (BT-549), and ovarian carcinoma (SK-OV-3). Moreover, non-cancer kidney cell lines (LLC-PK1 and VERO) were also included in the study. Table 3 Cytotoxicity of compounds towards a panel of cell lines. ID Cancer cell lines IC 50 µM Kidney cells IC 50 µM SK-MEL KB BT-549 SK-OV-3 LLC-PK1 Vero 1 NC 72.80 ± 2.40 NC 60.95 ± 14.36 NC 62.64 ± 2.39 2 15.12 ± 0.94 20.25 ± 0.32 16.46 ± 0.32 21.36 ± 1.89 16.57 ± 3.93 21.36 ± 1.27 3 8.38 ± 2.11 13.32 ± 0.33 11.37 ± 0.16 12.63 ± 0.98 9.99 ± 0.49 50.52 ± 9.74 4 15.17 ± 1.21 9.72 ± 0.73 14.83 ± 2.65 10.91 ± 2.89 9.38 ± 0.25 3.92 ± 0.24 5 25.97 ± 3.62 12.13 ± 1.41 16.84 ± 3.63 11.42 ± 4.04 13.27 ± 1.41 5.99 ± 0.40 6 8.82 ± 0.70 10.01 ± 0.70 9.71 ± 0.28 8.62 ± 0.42 8.82 ± 1.26 13.18 ± 1.82 7 > 69.03 > 69.03 > 69.03 > 69.03 > 69.07 51.04 ± 1.95 Doxorubicin 2.52 2.30 3.44 2.01 2.34 > 11 NC: No cytotoxicity up to 80 µM. Values are represented as mean ± SD (n = 3). In the first screening, all compounds were tested at a concentration of 80 µM by the MTT assay. The result showed all compounds had cytotoxicity at 80 µM, and hence they were carried out for the second round of screening at a lower concentration. Data showed that all compounds displayed moderate cytotoxicity than the control compound, doxorubicin. Observing the dataset noted that all HDAC10 inhibitors were weakly toxic for LLC-PK1 and VERO kidney cell lines. 2.3 Molecular docking 2.3.1 Molecular modelling studies Docking studies have been conducted to elucidate the binding mode of the synthesized compounds in various HDAC isoforms, including HDAC1 (PDB ID: 5ICN), HDAC6 (PDB ID: 5EDU), HDAC8 (PDB ID: 2V5X) and drHDAC10 (PDB ID: 6UHU). To test the plausibility of the docking results, we compared them with several crystal structures of structurally related hydroxamic acids, including those we recently reported for drHDAC10. Tubastatin A, a known inhibitor of HDAC6 and HDAC10 [ 30 ], along with the newly synthesized inhibitors described here, were subjected to docking studies within the crystal structures of various HDACs. The binding modes were visually analyzed and the docking studies revealed that tubastatin A and the newly synthesized compounds can chelate the zinc ion in a bidentate manner in HDAC1, HDAC8, and HDAC10. On the other hand, in HDAC6, zinc chelation was observed to occur via the hydroxyl oxygen of the hydroxamate moiety in a monodentate fashion that has also been reported for other benzhydroxamic acids [ 35 ]. Docking solutions in all investigated HDAC isoforms (Figs. 2 , 3 , and Supporting information S1: Figures a-d) showed that the phenyl moiety of the linker was embedded in the hydrophobic lysine binding pocket. Furthermore, hydrogen bond interactions with the conserved residues H136/142/140, H137/143/141, and Y307/306/303 were observed in HDAC10, HDAC8, and HDAC1, respectively. In contrast, in HDAC6, the hydroxyl oxygen formed a water-mediated hydrogen bond with the conserved residues H610 and H611. The predicted binding mode of compound 1 in HDAC10 (Fig. 1a, 2 a) provides insights into its significant inhibitory activity against HDAC10. In addition to the previously mentioned hydrogen bond interactions with conserved tyrosine and histidine residues, the capping group exhibits π-π interactions with F204. Moreover, the protonated amine forms electrostatic and salt-bridge interactions with D94 and the gatekeeper residue E274, along with cation-π interactions with W205 in HDAC10. These interactions resemble those observed in previously reported potent HDAC10 inhibitors and polyamine substrates [ 25 , 29 , 30 , 36 ]. HDAC6 belongs to class IIa of HDACs. In comparison to HDAC10, the HDAC6 binding pocket exhibits specific differences, notably involving mutations D94/S568 and E274/L749. These variations in the binding pocket led to the docking results showing only a single hydrogen bond interaction between the protonated nitrogen of compound 1 and S568. This observation could potentially explain the approximately 90-fold selectivity of HDAC10 over HDAC6 (Fig. 2 b). In class I HDAC members (HDAC1 and HDAC8), variations in the gatekeeper residue at the top of the lysine binding tunnel (E274 in HDAC10 is replaced by M274 and L271 in HDAC1 and − 8, respectively) led to the loss of one electrostatic interaction with the protonated nitrogen of 1 as well as the π-π interactions via the capping group, which were observed in HDAC10. The solvent-exposed capping groups together with the loss of electrostatic interaction with the gatekeeper are most likely the reason for the decrease in activity in both isoforms. Furthermore, the narrow binding pocket of HDAC1 due to the E274/L271 variation might lead to steric hindrance for the linker and capping groups of 1 which may explain the higher loss of activity in this isoform (Fig. 2 c and Fig. 2 d). Similar results were observed for compounds 5 and 7 , which bear a piperazine linker instead of the aminomethyl linker of compound 1 . Here, the protonated piperazine- NH was able to undergo two electrostatic interactions with D94 and E274 and cation-π interactions with W205. Additionally, the capping groups showed similar π-π interaction with W205 in the HDAC10 binding pocket as observed for compound 1 (Fig. 3 a and Figure S1 d). Compounds lacking a strong basic amino group including compounds 3 and 4 showed a significant decrease in the HDAC10 inhibitory activity. This can be attributed to the loss of the salt bridge interactions with D94 and E274, and the cation-π interaction with W205 in HDAC10, which were observed with the other compounds reported in this study. As expected, only hydrogen bond interactions between the NH-group and D94 are observed (Figure S1 b and Figure S1 c). These findings further emphasize the significance of the protonated nitrogen and its precise positioning in optimizing the binding interactions with HDAC10. The in vitro data presented in this study demonstrate that substituting the capping group with an additional tosyl moiety results in a decrease in HDAC10 inhibitory activity. Compound 2 exhibits approximately a 10-fold reduction in HDAC10 inhibitory activity compared to compound 1 , while compound 6 shows an almost 38-fold decrease in activity compared to its unsubstituted counterpart, compound 5 . The predicted binding mode of compound 6 (Fig. 3 b) was found to be similar to that of compound 5 (Fig. 3 a). However, an additional hydrogen bond via the sulfonyl group with N207 was observed in compound 6 . Notably, the hydrophobic tosyl moiety does not interact with the rim of the HDAC10 binding site and remains fully exposed to the solvent. This observation could explain the decrease in HDAC10 inhibitory activity compared to compound 5 . 2.3.2 Molecular dynamics (MD) simulations For the validation of the MD protocol, 100 ns MDs were applied to the available humanized drHDAC10 crystal structures (PDB IDs; 7U6B, 7U69, 7U6A, and 7U59, details in the Methods Section). Obtained RMSD plots from the molecular dynamic simulations revealed that the HDAC10-inhibitor structures (Figure S3c, Figure S4c, Figure S5c, and Figure S6c) are stable with an RMSD below 1.5 Å while the Zn ions generally show lower root mean square deviation (RMSD) values (Figure S3d, Figure S4d, Figure S5d and Figure S6d). Meanwhile, the ligand molecules tended to show higher fluctuations (Figure S3a, Figure S4a, Figure S5a, and Figure S6a) which was majorly attributed to the flexibility of the capping groups as observed in the ligand root mean square fluctuations (RMSF) plots (Figure S3b, Figure S4b, Figure S5b and Figure S6b). We further performed molecular dynamics simulation studies on the obtained docking pose of the most active compound 1 in drHDAC10 to examine the stability of the predicted binding mode. The obtained protein-ligand complex was subjected to 100 ns MD simulation protocol three times using AMBER22, and the obtained trajectories were analyzed. RMSD plots showed that the protein structure and the zinc ion in the complex remained stable during the 100 ns simulation time in all three replicas with RMSD values below 1.5 Å (Figure S2a and Figure S2b). Meanwhile, the ligand RMSD plots (Fig. 4 a) demonstrate that the ligand shows significant deviations from the predicted docking pose. Further analyses were performed to assess the stability of the obtained docking pose of compound 1 , and analyze the causes behind the observed fluctuation. Examination of the ligand RMSF plots of compound 1 (Fig. 4 b) demonstrates that the zinc-binding hydroxamate group as well as the linker moiety are stable during the MD simulations with RMSF values < 1 Å. It’s worth noting that the interactions between the hydroxamate group and the zinc ion are maintained during the MD simulations as shown by the unchanged distances between the hydroxamate- O atoms and zinc ion (< 2.5 Å; Fig. 3 c). Meanwhile, the indole-capping group displayed significantly high RMSF values that explains the strong deviations observed in the ligand RMSD plots. Clustering the obtained MD trajectories based on the RMSD of the ligand yielded three clusters with an occupancy > 10%. As can be demonstrated by the inspection of the obtained clusters (Fig. 4 d), the indole capping group can occupy one of two different hydrophobic regions at the rim of the binding pocket: In two clusters (occupancy 55% and 15%, respectively), the capping group is undergoing hydrophobic interactions with I27, A28 and P29. In the third cluster (17% occupancy), the capping group is situated between F204 and W205 (Fig. 4 C). The calculated data show, that the indole-capping group, despite showing strong fluctuations, is well accommodated at the entrance of the lysing binding pocket of HDAC10 where it undergoes hydrophobic interactions with surrounding residues. 3 CONCLUSION In this study, we synthesized novel bicyclic hydroxamic acid derivatives, characterized their inhibitory activity against class I, and class IIb HDACs. Our findings highlight compound 6 as a potent and selective inhibitor of HDAC10, while compound 1 exhibited the best inhibitory activity against HDAC10. Compound 4 displayed a preference towards HDAC6, and compounds 2 and 3 demonstrated high selectivity towards class IIb HDACs over class I HDACs. 4 EXPERIMENTAL 4.1 Chemistry 4.1.1 General Reagents and hydrous or anhydrous organic solvents were purchased from Sigma-Aldrich (Darmstadt, Germany) and Alfa Aesar Chemicals (Tewksbury, MA, USA) and used without further purification. Reaction progressmonitored by thin-layer chromatography (TLC) on pre-coated 0.20mm silica gel GF Uniplates from Macherey-Nagel (Düren, Germany). Plates were visualized with a 254 nm UV lamp and by indicators like ninhydrin, potassium permanganate (KMnO 4 ), dinitrophenylhydrazine (DNP), green girasol, and anisaldehyde. Column chromatography was performed with 63–200 µM of 70–230 mesh silica gel. Nuclear magnetic resonance (NMR) spectra were recorded using a Bruker BB 400 MHz spectrometer. Chemical shifts are reported in ppm (δ) relative to tetramethyl silane (TMS) and coupling constants ( J ) are reported in Hz. Abbreviations for multiplicity are s = singlet d = doublet t = triplet q = quartet dd = doublet of doublets dq = doublet of quartets m = multiplet. 4.1.2 General procedure for the synthesis of HDACs Inhibitor General synthesis methods Preparation of 1-tosyl-1 H -indole-3-carbaldehyde (II) This compound was prepared from 1 H -indole-3-carbaldehyde and p -toluenesulfonyl chloride, according to the literature procedure [ 51 – 53 ]. 1 H -indole-3-carbaldehyde ( I ) (1eq, 3g, 20.7 mmol) was dissolved in (30 ml) anhydrous N , N '-Dimethylformamide (DMF). The solution was stirred under nitrogen for 15 min in ice bath. Sodium hydride (NaH) (1.1 eq, 0.55 g, 22.9 mmol) was then added. After 10 minutes (1.4 eq, 5.52 g, 28.8 mmol) 4-methylbenzenesulfonyl chloride was added slowly to the reaction mixture. The reaction was left to stir overnight at room temperature. The reaction was quenched with distilled water (10 ml), and then (10 ml) saturated solution of sodium bicarbonate (NaHCO 3 ). The product was extracted three time with (25 ml) dichloromethane (DCM). Evaporate the solvent under vacuum, and the product was recrystallized by using 15 mL DCM and 10 mL hexane. The light yellow crystals were soaked with cold methanol. Yield 75%, m.p 148–149°C and R f 0.88 (Hexane:EtOAc, 1:1). 1 H-NMR (400 MHz, CDCl 3 -d 1 , 36°C, TMS): δ 2.36 (s, 3H, Ar-CH 3 ), 7.29(d, J = 8.4Hz, 2H, H-12), 7.36(ddd, J = 7.9, 7.3, 2.0Hz, 1H, H-6), 7.42(ddd, J = 7.3, 8.2, 1.6Hz, 1H, H-7), 7.87(d, J = 8.4Hz, 2H, H-11), 7.97(dt, J = 8.2, 2.0Hz, 1H, H-8), 8.26(s, 1H, H-2), 8.27(dt, J = 7.9, 1.6Hz, 1H, H-5), 10.11(s, 1H, CHO). 13 C-NMR (100MHz, CDCl 3 -d 1 , 36°C, TMS) δ 21.7 (Ar- C H 3 ), 113.3 (C-8), 122.4 (C-3), 122.6 (C-5), 125.1 (C-6), 126.30 (C-4), 126.33 (C-7), 127.3 (C-11), 130.4 (C-12), 134.3 (C-9), 135.2 (C-10), 136.3 (C-2), 146.2 (C-13), 185.4 (- C HO). HRESIMS m / z [M + H] + calcd. for C 16 H 14 NO 3 S: 363.18211, found 300.0700. General procedure for preparation of ((1-tosyl-1 H -indol-3-yl) methyl) amine carboxylate, (quinolin-3-ylmethyl)amine carboxylate or (1 H -indol-3-yl)methyl)amine carboxylate derivatives (Ia-b, IIa-c, and IIIa-b) To a solution of 1-tosyl-1 H -indole-3-carbaldehyde ( II ) (1 eq, 1.0–2.0 mmol), 3-quinolinecaboxaldehyde ( III ) (1 eq, 1.5-2.0 mmol) or 1 H -indole-3-carbaldehyde ( I ) (1.0 eq, 1.5-3.0 mmol) in anhydrous dichloromethane (25 ml) was added the corresponding amine carboxylate (1.1-2.0 eq, 1.1–4.5 mmol) with stirring under nitrogen for 2–4 hours at room temperature. Sodium triacetoxyborohydride (STAB) (1.3-2.0 eq, 1.3–4.5 mmol) was then added to the mixture in the ice bath, and then it was left to be stirred overnight at room temperature. After that, the mixture was then quenched with distilled water (5 ml), followed by the addition of a saturated solution of sodium bicarbonate (5 ml), then it was extracted with DCM (2 x 20 ml), dried over Na 2 SO 4 and concentrated under vacuum. Finally, the crude products were purified by column chromatography using silica gel with the appropriate eluent to give the resulting products ( Ia-b, IIa-c, and IIIa-b ). Preparation of ethyl 4-((((1 H -indol-3-yl)methyl)amino)methyl)benzoate (Ia) This compound was prepared from 1 H -indole-3-carbaldehyde ( I ) (1.0 eq, 0.43 g, 3.0 mmol), Ethyl 4-(aminomethyl)benzoate (1.1 eq, 0.59 g, 3.3 mmol) and (1.5 eq, 0.95 g, 4.5 mmol) of sodium triacetoxyborohydride (STAB) by following the general procedure and reaction conditions as described above. The crude product was purified by column chromatography using silica gel to obtain white crystals (CHCl 3 :MeOH:EtOAc 20:1:1). Yield 42%, m.p = 90–91°C, R f = 0.30 (CHCl 3 :MeOH:EtOAc 20:1:1). 1 H-NMR (400 MHz, CDCl 3 -d 1 , 36°C, TMS) δ 1.44(t, J = 7.1Hz, 3H, -OCH 2 CH 3 ), 3.96(s, 2H, H-CH 2 N-benzylic), 4.04(d, J = 0.8Hz, 2H, H-CH 2 N-pyrrol), 4.42(q, J = 7.1Hz, 2H, -OCH 2 CH 3 ), 7.13(m, 1H, H-2), 7.17(ddd, J = 7.8, 7.0, 1.1Hz, 1H, H-6), 7.24(ddd, J = 8.1, 7.0, 1.1Hz, 1H, H-7), 7.37(dt, J = 8.1, 1.1Hz, 1H, H-8), 7.47(d, J = 8.3Hz, 2H, H-11), 7.69(dt, J = 7.8, 1.1, 1H, H-5), 8.06(d, J = 8.3Hz, 2H, H-12), 8.45(s, 1H, H1-NH).. 13 C-NMR (100MHz, CDCl 3 -d 1 , 36°C, TMS) δ 14.4 (OCH 2 C H 3 ), 44.2 (C- C H 2 N-pyrrol), 52.9 (C- C H 2 N-benzylic), 61.0 (O C H 2 CH 3 ), 111.3 (C-8), 114.4 (C-3), 118.9 (C-5), 119.6 (C-6), 122.2 (C-7), 122.9 (C-2), 127.1 (C-4), 128.1 (C-11), 129.2 (C-13), 129.7 (C-12), 136.5 (C-9), 145.7 (C-10), 166.7 (C = O). HRESIMS m / z [M + Hac-H] + calcd. for C 21 H 23 N 2 O 4 367.16633, found 367.25930. [M + H] + calcd. for C 19 H 21 N 2 O 2 , 309.15975, found 309.15850. Preparation of ethyl 4-(4-((1 H -indol-3-yl)methyl)piperazin-1-yl)benzoate (Ib) This compound was prepared from 1 H -indole-3-carbaldehyde ( I ) (1.0 eq, 0.43 g, 3.0 mmol), ethyl 4-(1-piperazinyl)benzoate (1.2 eq, 0.84 g, 3.6 mmol) and (1.3 eq, 0.83 g, 3.9 mmol) of sodium triacetoxyborohydride (STAB) by following the general procedure and reaction conditions as described above. The crude product was purified by column chromatography using silica gel to obtain white crystals (CHCl 3 :MeOH, 10:1). Yield 73%, m.p = 175–176°C, R f = 0.50 (CHCl 3 : MeOH, 10:1). 1 H-NMR (400 MHz, CDCl 3 -d 1 , 36°C, TMS) δ 1.41(t, J = 7.1Hz, 3H, -OCH 2 CH 3 ), 2.69(dd, J = 6.7, 3.1Hz, 4H, H-10), 3.35(dd, J = 6.7, 2.8Hz, 4H, H-11), 3.82(s, 2H, H-CH 2 N), 4.37(q, J = 7.1Hz, 2H, -OCH 2 CH 3 ), 6.86(d, J = 9.1Hz, 2H, H-13), 7.17(m, 1H, H-2), 7.18(m, 1H, H-6), 7.25(m, 1H, H-7), 7.38(dd, J = 8.0, 1.2Hz, 1H, H-8), 7.80(dd, J = 7.8, 1.2Hz, 1H, H-5), 7.96(d, J = 9.1Hz, 2H, H-14), 8.47(s, 1H, -NH).. 13 C-NMR (100MHz, CDCl 3 -d 1 , 36°C, TMS) δ 14.5(OCH 2 C H 3 ), 47.5 (C-11), 52.7(C-10), 53.6(C- C H 2 N), 60.4 (O C H 2 CH 3 ), 111.2(C-8), 112.0(C-3), 113.6 (C-13), 119.5 (C-5), 119.6(C-6), 119.9(C-15), 122.10(C-7), 124.0(C-2), 128.0(C-4), 131.2(C-14), 136.3(C-9), 154.2(C-12), 166.9(C = O). HRESIMS m / z [M + H] + calcd. for C 22 H 26 N 3 O 2 , 364.20195, found 364.20500. [M-H] + calcd. for C 22 H 24 N 3 O 2 , 362.18740, found 362.18320 . Preparation of ethyl 4-((((1-tosyl-1 H -indol-3-yl)methyl)amino)methyl)benzoate IIa) : This compound was prepared from 1-tosyl-1 H -indole-3-carbaldehyde ( II ) (1.0 eq, 0.45 g, 1.50 mmol), ethyl 4-(aminomethyl)benzoate (1.1 eq, 0.30 g, 1.65 mmol) and (1.5 eq, 0.48 g, 2.25 mmol) of sodium triacetoxyborohydride (STAB) by following the general procedure and reaction conditions as described above. The crude product was purified by column chromatography using silica gel to obtain white crystals (CHCl 3 :MeOH 98:2). Yield 45%, m.p = 110–112°C, R f = 0.54 (CHCl 3 :MeOH, 98:2). 1 H-NMR (400 MHz, CDCl 3 -d 1 , 36°C, TMS) δ 1.42(t, J = 7.1Hz, 3H, -OCH 2 CH 3 ), 2.33(s, 3H, Ar-CH3), 3.89(s, 2H, H-16), 3.92(s, 2H, H-14), 4.40(q, J = 7.1Hz, 2H, -OCH2CH3), 7.21(d, J = 8.2Hz, 2H, H-12), 7.27(ddd, J = 8.2, 7.8, 1.0Hz, 1H, H-7), 7.34(ddd, J = 8.2, 7.3, 1.3Hz, 1H, H-6), 7.43(d, J = 8.2Hz, 2H, H-18), 7.53(s, 1H, H-2), 7.58(dt, J = 7.8, 1.3,, 1H, H-8), 7.78(d, J = 8.2Hz, 2H, H-11), 8.02(m,1H, H-5), 8.04(d, J = 8.2Hz, 2H, H-19). 13 C-NMR (100MHz, CDCl 3 -d 1 , 36°C, TMS) δ 14.4(OCH 2 C H 3 ), 21.6( Ar- C H 3 ), 43.9(C-14), 53.0(C-16), 60.90(O C H 2 CH 3 ), 113.8(C-5), 119.80(C-8), 121.3(C-3), 123.2 (C-7), 123.9(C-2), 124.9(C-6), 126.80(C-11), 128.0(C-18), 129.3(C-20), 129.8(C-19), 129.9(C-12), 130.3(C-4), 135.3(C-10), 135.5 (C-9), 144.9(C-13), 145.3(C-17), 166.6(C = O). HRESIMS m / z [2M + H] + calcd. for C 52 H 53 N 4 O 8 S 2 925.32994, found 925.33900. [M + Na] + calcd for C 26 H 26 NaN 2 O 4 S 485.15055, found 485.15390. Preparation of ethyl 4-(((1-tosyl-1 H -indol-3-yl) methyl) amino) benzoate (IIb) : This compound was prepared from 1-tosyl-1 H -indole-3-carbaldehyde ( II ) (1.0 eq, 0.45 g, 1.50 mmol), Ethyl 4-aminobenzoate (1.2 eq, 0.30 g, 1.8 mmol) and (2 eq, 0.63 g, 2.0 mmol) of sodium triacetoxyborohydride (STAB) by following the general procedure and reaction conditions as described above. The crude product was purified by column chromatography using silica gel to obtain white crystals (CHCl 3 :EtOAc, 4:1). Yield 85%, 154-155C°, R f = 0.85 (CHCl 3 : EtOAc, 4 :1). 1 H-NMR (400 MHz, CDCl 3 -d 1 , 36°C, TMS) δ 1.40(t, J = 7.1Hz, 3H, -OCH 2 CH 3 ), 2.35(s, 3H, Ar-CH 3 ), 4.35(q, J = 7.1Hz, 2H, -OCH 2 CH 3 ), 4.47(d, J = 1.2Hz, 2H, -NCH 2 ), 6.60(d, J = 8.7Hz, 2H, H-15), 7.19(d, J = 8.4Hz, 2H, H-12), 7.26(ddd, J = 8.5, 7.7, 1.0Hz, 1H, H-7), 7.36(ddd, J = 8.5, 7.2, 1.3Hz, 1H, H-6), 7.50(s, 1H, H-2), 7.53(m, 1H, H-8), 7.70(d, J = 8.4Hz, 2H, H-11), 7.89(d, J = 8.7Hz, 2H, H-16), 8.04(m, 1H, H-5). 13 C-NMR (100MHz, CDCl 3 -d 1 , 36°C, TMS) δ 14.5 (OCH 2 C H 3 ), 21.6(Ar- C H 3 ), 39.2(N-CH 2 ), 60.3(O C H 2 CH 3 ), 111.8(C-15), 114.0(C-5), 119.2 (C-17), 119.5 (C-8), 119.7(C-3), 123.4(C-7), 124.3(C-2), 125.1(C-6), 126.8(C-11), 129.6(C-4), 129.9 (C-12), 131.5(C-16), 135.960(C-10), 135.6(C-9), 145.1(C-13), 151.5(C-14), 166.9(C = O). HRESIMS m / z [M + Cl] + calcd for C 25 H 24 ClN 2 O 4 S 483.11458, found 483.10730. Preparation of ethyl 4-(4-((1-tosyl-1 H -indol-3-yl) methyl) piperazin-1-yl) benzoate (IIc) This compound was prepared from 1-tosyl-1 H -indole-3-carbaldehyde ( II ) (1.0 eq, 0.45 g, 1.50 mmol), ethyl 4-(1-piperazinyl)benzoate (1.2 eq, 0.42 g, 1.8 mmol) and (1.3 eq, 0.41 g, 1.95 mmol) of sodium triacetoxyborohydride (STAB) by following the general procedure and reaction conditions as described above. The crude product was purified by column chromatography using silica gel to obtain white crystals (CHCl 3 :EtOAc, 3:1). Yield 78%, m.p = 166–167°C, R f = 0.62 (CHCl 3 :EtOAc, 3:1). 1 H-NMR (400 MHz, CDCl 3 -d 1 , 36°C, TMS) δ 1.39(t, J = 7.1Hz, 3H, -OCH 2 CH 3 ), 2.33(s, 3H, Ar- CH 3 ), 2.60(m, 4H, H-14), 3.31(m, 4H, H-15), 3.69(s, 2H, H-NC H 2 ), 4.36(q, J = 7.1Hz, 2H, -O CH 2 CH 3 ), 6.86(d, J = 9.1Hz, 2H, H-17), 7.21(d, J = 8.4Hz, 2H, H-12), 7.25(m, 1H, H-7), 7.35(ddd, J = 8.4, 7.2, 1.3Hz, 1H, H-6), 7.56(s, 1H, H-2), 7.74(dt, J = 7.8, 1.3Hz, 1H, H-8), 7.80(d, J = 8.4Hz, 2H, H-11), 7.96(d, J = 9.1Hz, 2H, H-18), 8.04(dt, J = 8.4, 1.0Hz, 1H, H-5).. 13 C-NMR (100MHz, CDCl 3 -d1, 36°C, TMS) δ 14.5(OCH 2 C H 3 ), 21.6(Ar- C H 3 ), 47.5(C-15), 52.8(C-14), 53.4(C-N C H 2 ), 60.4(O C H 2 CH 3 ), 113.6(C-17), 113.7(C-5), 119.2(C-19), 120.0(C-3), 120.6 (C-8), 123.2 (C-7), 124.9(C-2), 124.9(C-6), 126.8 (C-11), 129.9 (C-12), 130.8 (C-4), 131.2(C-18), 135.3(C-10), 135.5(C-9), 145.0(C-13), 154.1(C-16), 166.7 (C = O). HRESIMS m / z [M + H] + calcd. for C 29 H 32 N 3 O 4 S 518.21136, found 518.22210. [M + Na] + calcd for C 29 H 31 NaN 3 O 4 S 540.19275, found 540.19350. Preparation of ethyl 4-((quinolin-3-ylmethyl)amino)benzoate (IIIa) This compound was prepared from 3-quinolinecaboxaldehyde ( III ) (1 eq, 0.314 g, 2.0mmol), ethyl 4-aminobenzoate (1.1 eq, 0.36 g, 2.2 mmol) and (1.5 eq, 0.64 g, 3.0 mmol) of sodium triacetoxyborohydride (STAB) by following the general procedure and reaction conditions as described above [ 54 – 56 ]. The crude product was purified by column chromatography using silica gel to obtain white crystals (CHCl 3 :EtOAc, 4:1). Yield 66%, m.p = 135–136°C, R f = 0.38(CHCl 3 :EtOAc, 4:1). 1 H-NMR (400 MHz, CDCl 3 -d 1 , 36°C, TMS) δ 1.35(t, J = 7.1Hz, 3H, -OCH 2 CH 3 ), 4.31(q, J = 7.1Hz, 2H, -O CH 2 CH 3 ), 4.58(d, J = 4.5Hz, 2H, -N CH 2 ), 4.92(s, br, - N H ), 6.64(d, J = 8.8Hz, 2H, H-12), 7.55(ddd, J = 8.1, 6.9, 1.2Hz, 1H, H-7), 7.71(ddd, J = 8.4, 6.9, 1.5Hz, 1H, H-8), 7.76(dd, J = 8.1, 1.5Hz, 1H, H-6), 7.89(d, J = 8.8Hz, 2H, H-13), 8.07(dd, J = 2.2, 1.0Hz, 1H, H-4), 8.11(dt, J = 8.5, 1.0, 1.0Hz, 1H, H-9), 8.90(d, J = 2.2Hz, 1H, H-2). 13 C-NMR (100MHz, CDCl 3 -d 1 , 36°C, TMS) δ 14.5(OCH 2 C H 3 ), 45.4(-N C H 2 ), 60.3(O C H 2 CH 3 ), 111.8(C-12), 119.5 (C-14), 127.1(C-7), 127.7(C-6), 127.9(C-5), 129.1 (C-9), 129.2(C-8), 131.3(C-3), 131.6(C-13), 134.1 (C-4), 147.5(C-10), 150.3(C-2), 151.3(C-11), 166.8(C = O). HRESIMS m / z [M + H] + calcd for C 19 H 19 N 2 O 2 307.14410, found 307.14730. Preparation of ethyl 4-(4-(quinolin-3-ylmethyl)piperazin-1-yl)benzoate (IIIb) This compound was prepared from 3-quinolinecaboxaldehyde ( III ) (1 eq, 0.236 g, 1.50 mmol), ethyl 4-(1-piperazinyl)benzoate (1.2 eq, 0.42 g, 1.8 mmol), and (1.3 eq, 0.42 g, 1.95 mmol) of sodium triacetoxyborohydride (STAB) by following the general procedure and reaction conditions as described above. The crude product was purified by column chromatography using silica gel to obtain white crystals (CHCl 3 :MeOH, 94:6). Yield 63%, m.p = 153–155°C, R f = 0.68 (CHCl 3 :MeOH, 94:6). 1 H-NMR (400 MHz, CDCl 3 -d 1 , 36°C, TMS) δ 1.34(t, J = 7.1Hz, 3H, -OCH 2 CH 3 ), 2.60(t, J = 5.0Hz, 4H, H-11), 3.29(t, J = 5.0Hz, 4H, H-12), 3.69(s, 2H, C H 2 N), 4.30(q, J = 7.1Hz, 2H, -O CH 2 CH 3 ), 6.81(d, J = 8.7Hz, 2H, H-14), 7.52(t, J = 8.3Hz, 1H, H-7), 7.68(ddd, J = 8.4, 6.6, 1.4Hz, 1H, H-8), 7.79(dd, J = 8.3, 1.4Hz, 1H, H-6), 7.91(d, J = 8.7Hz, 2H, H-15), 8.05(d, J = 2.1Hz, 1H, H-4), 8.11(d, J = 8.4Hz, 1H, H-9), 8.92(d, J = 2.1Hz, 1H, H-2). 13 C-NMR (100MHz, CDCl 3 -d 1 , 36°C, TMS) δ 14.5(OCH 2 C H 3 ), 47.5(C-12), 52.8(C-11), 60.3(O C H 2 CH 3 ), 60.4(N C H 2 ), 113.6(C-14), 120.1(C-16), 126.8(C-7), 127.6(C-6), 127.9(C-5), 129.2(C-8), 129.2(C-9), 130.7(C-3), 131.1(C-15), 135.7(C-4), 147.6(C-10), 152.0(C-2), 154.0(C-13), 166.6 (C = O). HRESIMS m / z [M + H] + calcd. for C 23 H 26 N 3 O 2 376.20195, found 376.20380. General procedure for preparation of corresponding hydroxamic acid derivatives of (1–7). To a solution of benzoate derivatives of ( Ia-b, Iia-c and IIIa-b ) (1 eq, 0.27–0.45 mmol) in anhydrous dichloromethane and methanol (1:2, 6 ml) at 0°C, hydroxylamine (50 wt % in water, 7.99–13.37 mmol, 30 eq) was added, followed by the addition of sodium hydroxide (1.08–2.25 mmol, 4.0–5.0 eq). The reaction mixture was allowed to warm to room temperature and stirred for 24 h. Then the solvent was removed under reduced pressure, and the obtained solid was dissolved in water. The pH was adjusted to 7 with a 1N HCl. The resulting precipitate was filtered and dried under a high vacuum, and then the crude products were purified by column chromatography using silica gel with the appropriate eluent or soaking with dichloromethane to give the resulting products ( 1–7 ). Preparation of 4-((((1 H -indol-3-yl)methyl)amino)methyl)-N-hydroxybenzamide (1) This compound was prepared from ethyl 4-((((1 H -indol-3-yl)methyl)amino)methyl)benzoate ( Ia ) (0.10 g, 0.32 mmol, 1 eq), hydroxylamine (50 wt % in water, 0.64 ml, 0.32 g, 9.60 mmol, 30 eq) and sodium hydroxide (0.05 g, 1.28 mmol, 4.0 eq) by following the general procedure and reaction conditions as described above. The crude product was purified by soaking with dichloromethane to afford 4-((((1 H -indol-3-yl)methyl)amino)methyl)- N -hydroxybenzamide (1) as white crystals. Yield 52%, m.p = 158–160°C. 1 H-NMR (400 MHz, DMSO-d 6 , 36°C, TMS) δ 3.80(s, 2H, H-C H 2 N-benzylic), 3.86(d, J = 0.8Hz, 2H, H-C H 2 N-pyrrol), 6.99(s, 1H, H-2), 7.08(m, 1H, H-6), 7.25(m, 1H, H-7), 7.36(m, 1H, H-8), 7.45(m, 2H, H-11), 7.61(m, 1H, H-5), 7.74(m, 2H, H-12), 10.91(s, 1H, -OH). 13 C-NMR (101 MHz, DMSO-d 6 , 36°C, TMS) δ 44.21(C- C H 2 N-pyrrol), 52.42(C- C H 2 N-benzylic), 111.8 (C-8), 113.9(C-3), 118.7(C-2), 119.3(C-5), 121.4(C-6), 124.0(C-7), 127.2(C-12), 127.5(C-4), 128.3(C-11), 131.5(C-13), 136.86(C-9), 144.85(C-10), 164.7(C = O). HRESIMS m / z 294.12507[M-H] + (calc for C 17 H 16 N 3 O 2 , 294.12425), 130.06530 [M-C 8 H 9 N 2 O 2 ] + (calc for C 9 H 8 N, 130.06567). Preparation of N-hydroxy-4-((((1-tosyl-1 H -indol-3-yl)methyl)amino)methyl) benzamide (2) This compound was prepared from ethyl 4-((((1-tosyl-1 H -indol-3-yl)methyl)amino) methyl)benzoate ( IIa ) (0.20g, 0.43mmol, 1eq), hydroxylamine (50 wt % in water, 0.85ml, 0.42g, 12.90mmol, 30eq) and sodium hydroxide (0.086 g, 2.15 mmol, 5.0 eq) by following the general procedure and reaction conditions as described above. The crude product was purified by using silica gel for column chromatography to afford N -hydroxy-4-((((1-tosyl-1 H -indol-3-yl)methyl)amino)methyl)benzamide (2) as light-pink crystals (CH 2 Cl 2 :MeOH 987:13)). Yield 37%, m.p = 151–153°C, R f = 0.49 (CH 2 Cl 2 :MeOH (87:13)). 1 H-NMR (400 MHz, DMSO-d 6 , 36°C, TMS) δ 2.28(s, 3H, Ar- CH 3 ), 3.75(s, 2H, -N CH 2 -16), 3.80(s, 2H, -N CH 2 -14), 7.25(t, J = 7.5Hz, 7.5Hz, 1H, H-7), 7.34(d, J = 7.9Hz, 2H, H-12), 7.35(t, J = 7.4Hz, 1H, H-6), 7.41(d, J = 7.8Hz, 2H, H-18), 7.65(d, J = 7.5Hz, 1H, H-8), 7.67(s, 1H, H-2), 7.75(d, J = 7.8Hz, 2H, H-19), 7.83(d, J = 7.9Hz, 2H, H-11), 7.93(d, J = 8.2Hz, 1H, H-5), 9.04(brs, 1H, -((CO)N H ), 11.17(brs, 1H, -O H ). 13 C-NMR (101 MHz, DMSO-d 6 , 36°C, TMS) δ 21.5( Ar- C H 3 ), 43.5(N- C H 2 -14), 52.4(N- C H 2 -16), 113.7(C-5), 120.8(C-8), 122.5(C-3), 123.7(C-7), 124.5(C-2), 125.2(C-6), 127.1(C-11), 127.3(C-19), 128.3(C-18), 130.6(C-12), 130.6(C-20), 131.6(C-4), 134.6(C-10), 135.2(C-9), 144.5(C-13), 145.8(C-17), 164.6(C = O). HRESIMS m / z [M-H] + calcd. for C 24 H 22 N 3 O 4 S 448.13365, found 448.13420. [2M + H] + calcd for C 48 H 45 N 6 O 8 S 2 897.27403, found 897.27510. Preparation of N-hydroxy-4-(((1-tosyl-1 H -indol-3-yl)methyl)amino)benzamide ( 3 ) This compound was prepared from ethyl 4-(((1-tosyl-1 H -indol-3-yl)methyl)amino)benzoate ( Iib ) (0.20 g, 0.45 mmol, 1.0 eq), hydroxylamine (50 wt % in water, 0.88 ml, 0.44 g, 13.37 mmol, 30 eq) and sodium hydroxide (0.09 g, 2.25 mmol, 5.0 eq) by following the general procedure and reaction conditions as described above. The crude product was purified by using silica gel for column chromatography to afford N-hydroxy-4-(((1-tosyl-1 H -indol-3-yl)methyl)amino)benzamide ( 3 ) as light-pink crystals (CH 2 Cl 2 :MeOH(94:6)). Yield 34%, m.p = 183–184°C, R f = 0.32 (CH 2 Cl 2 :MeOH(94:6)). 1 H-NMR (400 MHz, DMSO-d 6 , 36°C, TMS) δ 2.29(s, 3H, Ar- CH 3 ), 4.43(d, J = 5.6Hz, 2H, -N CH 2 ), 6.65(d, J = 8.4Hz, 2H, H-15), 6.71(t, J = 6.2Hz, 1H, -N H- CH 2 ), 7.26(t, J = 7.5Hz, 1H, H-7), 7.33(d, J = 8.2Hz, 2H, H-12), 7.33(t, J = 7.2Hz, 1H, H-6), 7.53(d, J = 8.3Hz, 2H, H-16), 7.72(d, J = 7.5Hz, 1H, H-8), 7.75(d, J = 8.3Hz, 2H, H-11), 7.77(s, 1H, H-2), 7.91(d, J = 8.2Hz, 1H, H-5), 8.72(brs, 1H, -((CO)N H ), 10.81(brs, 1H, -O H ). 13 C-NMR (101 MHz, DMSO-d 6 , 36°C, TMS) δ 21.46( Ar- C H 3 ), 38.19(N- C H 2 ), 111.85(C-15), 113.76(C-5), 120.00(C-17), 120.87(C-8), 121.16(C-3), 123.79(C-7), 125.12(C-2), 125.37(C-6), 127.04(C-11), 128.63(C-16), 130.26(C-4), 130.64(C-12), 134.44(C-10), 135.24(C-9), 145.82(C-13), 151.27(C-14). HRESIMS m / z [M + Na] + calcd for C 23 H 21 N 3 NaO 4 S 458.11505, found 458.1156. [2M + H] + calcd. for C 46 H 43 N 6 O 8 S 2 871.25839, found 871.26100. Preparation of N -hydroxy-4-((quinolin-3-ylmethyl)amino)benzamide ( 4 ) This compound was prepared from ethyl 4-((quinolin-3-ylmethyl)amino)benzoate ( IIIa ) (0.10 g, 0.32 mmol, 1 eq), hydroxylamine (50 wt % in water, 0.63 ml, 0.32 g, 9.60 mmol, 30 eq) and sodium hydroxide (0.05 g, 1.28 mmol, 4.0 eq) by following the general procedure and reaction conditions as described above. The crude product was purified by soaking with dichloromethane to afford N-hydroxy-4-((quinolin-3-ylmethyl)amino)benzamide ( 4 ) as white crystals. Yield 53%, m.p = 201–203°C. 1 H- NMR (400 MHz, DMSO-d 6 , 36°C, TMS) δ 4.56(d, J = 6.0Hz, 2H, -N CH 2 ), 6.65(d, J = 8.4Hz, 2H, H-12), 6.94(t, J = 6.0Hz, 1H, -N H- CH 2 ), 7.53(d, J = 8.4Hz, 2H, H-13), 7.59(t, J = 7.5Hz, 1H, H-7), 7.73(t, J = 8.4Hz, 1H, H-8), 7.94(d, J = 8.1Hz, 1H, H-6), 8.01(d, J = 8.4Hz, 1H, H-9), 8.25(d, J = 2.2Hz, 1H, H-4), 8.69(brs, 1H, -N H ), 8.93(d, J = 2.2Hz, 1H, H-2), 10.77(s, 1H, -O H ). 13 C-NMR (101 MHz, DMSO-d 6 , 36°C, TMS) δ 44.4(-N C H 2 ), 111.9(C-12), 120.3(C-14), 127.3(C-7), 128.0(C-5), 128.3(C-6), 128.8(C-13), 129.2(C-9), 129.6(C-8), 133.1(C-3), 133.9(C-4), 147.3(C-10), 151.2(C-11), 151.4(C-2), 165.4(C = O). HRESIMS m / z [M-H] + calcd. for C 17 H 14 N 3 O 2 292.10860, found 292.10840. Preparation of 4-(4-((1 H -indol-3-yl)methyl)piperazin-1-yl)- N -hydroxybenzamide (5) This compound was prepared from ethyl 4-(4-((1 H -indol-3-yl)methyl)piperazin-1-yl)benzoate ( Ib ) (0.10 g, 0.27 mmol, 1 eq), hydroxylamine (50 wt % in water, 0.52 ml, 0.26 g, 8.25 mmol, 30 eq) and sodium hydroxide (0.04 g, 1.08 mmol, 4.0 eq) by following the general procedure and reaction conditions as described above. The crude product was purified by soaking with dichloromethane to afford 4-(4-((1 H -indol-3-yl)methyl)piperazin-1-yl)- N -hydroxybenzamide (5) as white crystals. Yield 53%, m.p = 155–157°C. 1 H-NMR (400 MHz, DMSO-d 6 , 36°C, TMS) δ 2.53(m, 4H, H-10), 3.20(m, 4H, H-11), 3.72(s, 2H, H-C H 2 N), 6.87(d, J = 8.9Hz, 2H, H-13), 7.00(t, J = 7.4Hz, 1H, H-6), 7.08(t, J = 7.5Hz, 1H, H-7), 7.27(s, 1H, H-2), 7.37(d, J = 8.0Hz, 1H, H-8), 7.57(d, J = 8.3Hz, 2H, H-14), 7.62(d, J = 7.9Hz, 1H, H-5), 8.81(brs, 1H, -(N H )C = O), 10.93(s, 1H, NH(Ar)), 10.96(s, 1H, OH). 13 C-NMR (101 MHz, DMSO-d 6 , 36°C, TMS) δ 47.7(C-11), 52.7(C-10), 53.6(C- C H 2 N), 110.9(C-3), 111.8(C-8), 114.1(C-13), 118.9(C-6), 119.5(C-5), 121.4(C-7), 122.1(C-15), 125.2(C-2), 128.1(C-4), 128.5(C-14), 136.7 (C-9), 153.2(C-12). HRESIMS m / z [M-H] + calcd for C 20 H 21 N 4 O 2 349.16700, found 349.16776. [M + Cl 35 ] + calcd for C 20 H 22 ClN 4 O 2 385.14368, found 385.14424. Preparation of N -hydroxy-4-(4-((1-tosyl-1 H -indol-3-yl)methyl)piperazin-1-yl)benzamide (6) This compound was prepared from ethyl 4-(4-((1-tosyl-1 H -indol-3-yl)methyl)piperazin-1-yl)benzoate ( IIc ) (0.15 g, 0.29 mmol, 1 eq), hydroxylamine (50 wt % in water, 0.57 ml, 0.287 g, 8.7 mmol, 30 eq) and sodium hydroxide (0.058 g, 1.45 mmol, 5.0 eq) by following the general procedure and reaction conditions as described above. The crude product was purified by using silica gel for column chromatography to afford N -hydroxy-4-(4-((1-tosyl-1 H -indol-3-yl)methyl)piperazin-1-yl)benzamide ( 6 ) as light-pink crystals (CH 2 Cl 2 :MeOH(90:10)). Yield 53%, m.p = 180–182°C, R f = 0.43 (CH 2 Cl 2 :MeOH(90:10)). 1 H-NMR (400 MHz, DMSO-d 6 , 36°C, TMS) δ 2.26(s, 3H, Ar- CH 3 ), 2.49(t, J = 8.2Hz, 4H, H-14), 3.20(t, J = 4.8Hz, 4H, H-15), 3.65(s, 2H, H-C H 2 N), 6.91(d, J = 8.5Hz, 2H, H-17), 7.25(t, J = 7.4Hz, 1H, H-7), 7.33(d, J = 7.9Hz, 2H, H-12), 7.33(t J = 7.6Hz, 1H, H-6), 7.68(d, J = 8.3Hz, 2H, H-18), 7.72(s, 1H, H-2), 7.73(d, J = 8.9Hz, 1H, H-8), 7.84(d, J = 7.8Hz, 2H, H-11), 7.94(d, J = 8.2Hz, 1H, H-5), 8.86(brs, 1H, -N H ), 10.97(brs, 1H, -O H ). 13 C-NMR (101 MHz, DMSO-d 6 , 36°C, TMS) δ 21.4( Ar- C H 3 ), 47.6(C-15), 52.7(C- C H 2 N,14), 113.7(C-5), 114.2(C-17), 119.8(C-19), 121.2(C-8), 122.2(C-3), 123.8(C-7), 125.3(C-2), 125.7(C-6), 127.1(C-11), 128.6(C-18), 130.6(C-12), 131.1(C-4), 135.2(C-10), 135.6(C-9), 145.8 (C-13), 153.2(C-16), 164.8(C = O). HRESIMS m / z . [M + Cl 35 ] + calcd for C 27 H 29 ClN 4 O 4 S 539.15203, found 539.15300. Preparation of N -hydroxy-4-{4-[(quinolin-3-yl)methyl]piperazin-1-yl}benzamide ( 7 ) This compound was prepared from ethyl 4-(4-(quinolin-3-ylmethyl)piperazin-1-yl)benzoate ( IIIb ) (0.10 g, 0.27 mmol, 1 eq), hydroxylamine (50 wt % in water, 0.52 ml, 0.26 g, 7.99 mmol, 30 eq) and sodium hydroxide (0.04 g, 1.08 mmol, 4.0 eq) by following the general procedure and reaction conditions as described above. The crude product was purified by soaking with dichloromethane to afford N -hydroxy-4-{4-[(quinolin-3-yl)methyl]piperazin-1-yl}benzamide ( 7 ) as white crystals. Yield 73%, m.p = 215–216°C. 1 H-NMR (400 MHz, DMSO-d 6 , 36°C, TMS) δ = 2.55(s, 4H, H-11), 3.26(s, 4H, H-12), 3.74(s, 2H, H-C H 2 N), 6.93(d, J = 8.6Hz, 2H, H-14), 7.62(t, J = 7.3Hz, 1H, H-7), 7.64(d, J = 8.3Hz, 2H, H-15), 7.76(t J = 6.6Hz, 1H, H-8), 7.99(d, J = 8.3Hz, 1H, H-6), 8.03(d, J = 8.3Hz, 1H, H-9), 8.26(s, 1H, H-4), 8.84(brs, 1H, -N H ), 8.90(s, 1H, H-2), 10.96(s, 1H, -O H ). 13 C-NMR (101 MHz, DMSO-d 6 , 36°C, TMS) δ 47.64(C-12), 52.80(C-11), 59.79(N C H 2 ), 114.21(C-14), 122.37(C-16), 127.17(C-7), 127.97(C-5), 128.38(C-6), 128.54(C-9), 129.16(C-8), 129.64(C-15), 131.44(C-3), 135.80(C-4), 147.44(C-10), 152.47(C-2), 153.15(C-13). HRESIMS m / z [M + 2H] + calcd. for C 21 H 24 N 4 O 2 : 364.18883, found: 364.18853. 4.2 Pharmacological/biological assays The cytotoxic activity The cytotoxic activity of all pure compounds was determined towards a panel of four human solid tumour cell lines: melanoma (SK-MEL), epidermal carcinoma (KB), breast carcinoma (BT-549), and ovarian carcinoma (SK-OV-3). Moreover, non-cancer kidney cell lines (LLC-PK1 and VERO) were also employed to determine if the anti-cell proliferative activity of these compounds was selective for the tested tumour cell lines. All cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were seeded in 96-well plates (10,000 cells/well) and incubated for 24 h. All ligands were dissolved in DMSO, diluted in media, and added to the cells at concentrations of 80, 40, 20, and 10 µM. After incubating for 48 h, cell viability was determined using a tetrazolium dye WST-8, which is converted to a water-soluble formazan product in the presence of 1-methoxy PMS by the activity of cellular enzymes. The colour of the formazan product was measured at 450 nm on a plate reader. Doxorubicin was used as a positive control for the cytotoxicity assay, and DMSO (0.25%) was used as the vehicle control. The IC 50 values were obtained from concentration-response curves. The values are represented as mean ± standard deviation (n = 3). Enzymatic in vitro HDAC inhibitory activity Recombinant human HDAC1, HDAC2, HDAC3/NCOR1, and HDAC6 were purchased from ENZO Life Sciences AG (Lausen, CH). Barinka et al. [ 32 ] produced recombinant drHDAC10 wild type. Recombinant human HDAC8 was produced by Romier et al. [ 37 ]. In vitro testing of the inhibitors in an enzymatic assay was carried out as described in previous publications by us [ 32 , 37 – 40 ]. Inhibitory activity for HDAC1, HDAC2, HDAC3, and HDAC6 was determined using a discontinuous assay with a substrate peptide derived from p53 (Ac-RHKK(Ac)-AMC) in a 384 well-plate. The enzyme at final concentrations (10 nM HDAC1, 3 nM HDAC2 and HDAC3, and 1 nM HDAC6,) and the inhibitors at various concentrations were incubated for 5 min in assay buffer (50 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 1 mM TCEP and 0.2 mg/mL BSA, pH 7.4 adjusted with NaOH). The reaction started with the addition of substrate (20 µM HDAC1-3 and 5 µM HDAC6). Afterwards, the fluorescence was developed with a 0.5 mg/ml trypsin solution (final concentration), and the fluorescence readout was done with an Envision 2104 Multilabel Plate Reader (PerkinElmer, Waltham, USA) with λ Ex = 380 ± 10 nm and λ Em = 430 ± 8 nm. For HDAC8 the fluorogenic peptide derivate Abz-SRGGK(thio-TFA)FFRR-NH2 was applied as described in [ 38 ]. For HDAC10 a spermidine derivative Ac-spermidine-AMC was applied. The HDAC8 assay was performed in the same assay buffer as described above. For HDAC10 the assay buffer was 20 mM HEPES, pH 7.4, and 0.5 mg/ml BSA. The enzyme (1.5 nM HDAC8 or 5 nM HDAC10 final concentration) was incubated for 5 min with various concentrations of the inhibitor. The reaction was started with the addition of 50 µM substrate and the readout was done continuously with an Envision 2104 with λ Ex = 330 ± 75 nm, and λ Em = 430 ± 8 nm. For HDAC1, 2, and 3 a fluorogenic peptide derived from p53 (Ac-RHKK(Acetyl)-AMC) was applied. The measurements were performed in assay buffer (50 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 1 mM TCEP, and 0.2 mg/mL BSA, pH 7.4 adjusted with NaOH) at 37°C. An Envision 2104 Multilabel Plate Reader (PerkinElmer, Waltham, MA), with an excitation wavelength of 380 ± 8 nm and an emission wavelength of 430 ± 8 nm was considered to measure the fluorescence intensity. For HDAC6 the substrate (Abz-SRGGK(thio-TFA)FFRR-NH2) was applied as described before [ 38 ]. The HDAC10 inhibition assay was performed as described before [ 29 ] using Ac-spermidine-AMC as substrate. The idea of this discontinuous assay is the HDAC10 mediated generation of a primary amino function. The released primary amine is reacted wirh 2,3-Naphthalenedicarboxaldehyde (NDA) resulting in an NDA-spermidin-AMC derivative which differences in the AMC fluorescence intensities as compared to Ac-spermidin AMC. The assay was performed in black 96-well plates (PerkinElmer, OptiPlateTM-96 F). The compounds to be tested were incubated for 25 min at 25°C. Before measuring fluorescence (POLARstar plate reader, λex = 330 nm, λem = 390 nm) each well was filled with 200 µL stop solution containing 16mM NDA). (for assay details please refer to [ 29 ]. The enzyme inhibition of HDAC8 was determined by using a homogenous fluorescence assay [ 37 ]. The enzyme was incubated for 90 min at 37°C, with the fluorogenic substrate ZMAL (Z(Ac)Lys-AMC) in a concentration of 10.5 µM and increasing concentrations of inhibitors. Fluorescence intensity was measured at an excitation wavelength of 390 nm and an emission wavelength of 460 nm in a microtiter plate reader (BMG Polarstar). 4.3 Molecular docking Molecular docking Available crystal structures of drHDAC10, hsHDAC1, hsHDAC6 and hsHDAC8 were downloaded (PDB ID: 6UHU, PDB ID: 5ICN, PDB ID: 5EDU, and PDB ID: 2V5X respectively) from the Protein Data Bank (PDB; www.rscb.org ) [ 41 ]. All ligands and all protein-ligand complexes were prepared using similar methods as published before [ 29 , 30 ]. Validation of the molecular docking method was performed by re-docking the ligands co-crystallized in HDAC10 as reported in our previous publication [ 29 ]. For the preparation of the proteins, the wizard implementation of the Schrödinger version 2019.1 was used with the following steps: hydrogen atoms addition, protonation states assignment, and finally, restrained energy minimization using the OPLS force field 2005. The ligand structure was generated using the 2D Sketcher of Schrödinger (version 2019.1). Afterward, the LigPrep tool (Schrödinger version 2019.1) was used for the preparation of the ligands with energy minimization using the OPLS2005 force field [ 42 , 43 ]. 64 conformers for each ligand were generated with the ConfGen tool (Schrödinger version 2019.1). Preparation of the receptor grid for the docking procedure was performed by assigning the co-crystallized ligands as the centroid of the grid box in each PDB crystal structure using the receptor grid preparation module in Schrödinger (version 2019.1). Lastly, docking of all generated conformers was done in the Standard Precision mode using the Glide (Schrödinger-release 2019.1). Molecular Dynamics (MD) Simulations AMBER22 was used to perform GPU-based MD simulations. PDB4Amber command was used for the preparation of the protein structures for further usage within the tLEaP program. Topology and force field parameters of the ligands were assigned with Antechamber [ 44 ] package using the second generation general Amber force field (GAFF2) and the semi-empirical AM1-BCC (Austin Model1 with bond charge correction) as atomic charge method [ 45 , 46 ]. Afterwards, the protein-ligand complexes were created with the AMBER22 tLEaP module. The second-generation general AMBER force field (GAFF2) was used as the ligand force field while force field 14 Stony Brook-ff14SB was used for the protein structures [ 47 , 48 ]. For the catalytic Zn2+, the 12-6-4 LJ-type nonbonded ion model was applied [ 49 ]. After combining the protein and the ligands, complexes were solvated by transferable intermolecular potential 3P-TIP3P water model as an octahedral box around the protein with a 10 Å margin. Then Na + and Cl- ions were added to neutralize the whole system. Parameter/topology files of the entire system were created with tLEaP and the files were used as a starting point for the MD simulations. The solvated systems were first subjected to two energy minimization steps involving 1000 cycles of steepest descent followed by 2000 cycles of conjugate gradient totaling 3000 cycles of minimization. In the first energy minimization step, only the solvent molecules and counter-ions (Na + and Cl-) were minimized while applying constraints with a force constant of 10 kcal*mol-1*Å-2 to the proteins, ligands, and zinc ion. In the second minimization step, the whole system was minimized without constraints. Subsequently, the systems were heated from 0 to 300 K over 100 ps while applying the same constraints on the solute as in minimization step1. Constant volume periodic boundary was set to equilibrate the temperature of the system by Langevin thermostat using a collision frequency of 2 ps-1. Subsequently, a pressure equilibration routine with a constant pressure of 1 bar and at 300 K was performed for 100 ps. Finally, 100 ns free molecular dynamic simulations with the time step of 2 fs were applied utilizing the Particle Mesh Ewald method [ 50 ]. The system temperature was kept at 300 K with a Langevin thermostat using 2 ps-1 collision frequency and the pressure of the system was maintained at 1 bar with the usage of isotropic position scaling and a relaxation time of 2 ps. Implementation of the SHAKE algorithm was done to constrain all bonds containing hydrogens. A total of 1000 frames were written for 100 ns long MDs. Simulations of the prepared crystal structures of humanized drHDAC10 (PDB IDs; 7U6B, 7U69, 7U6A, 7U69) X-ray structures and the docking pose of ligand 1 in drHDAC10 (PDB ID:6UHU) were repeated three times from the minimization steps with non-identical random seeds. The analysis of the MD trajectories was performed using the CPPTRAJ module in AMBER 22. The RMSD of the protein was calculated for the backbone atoms using CPPTRAJ. RMSF of the ligand as well as distances between the hydroxamate group oxygens and the zinc ion were calculated to further examine the stability of the protein-ligand complexes using CPPTRAJ. Additionally, the obtained trajectories were clustered based on the ligand-heavy atoms using the K-means algorithm and pytraj implementation of AMBER. PAINS filter Inhibitors described herein were filtered for pan-assay interference compounds (PAINS). For this purpose, PAINS1, PAINS2, and PAINS3 filters, as implemented in Schroedinger's Canvas program (Schrödinger version 2019.1), were employed. None of the compounds was flagged as PAINS. Declarations CONFLICT OF INTEREST The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution Conceptualization, A.T., W.S., S.R. and S.A.; methodology, A.T., W.S. and S.R.; software, M.S.; validation, W.S., A.T., S.A. and S.R.,.; formal analysis, M.S., T.Y., M.Z., D.R. and C.B..; investigation, A.T,M.S., T.Y., M.Z., D.R. and C.B.; resources, S.R. and W.S.; data curation, A.T and W.S.; writing—original draft preparation, A.T.; writing—review and editing, S.A., M.S. and S.R.,; visualization, W.S and A.T.; supervision, A.T, W.S. and S.R.; project administration, A.T and W.S.; funding acquisition, S.R and W.S. All authors have read and agreed to the published version of the manuscript. ACKNOWLEDGEMENTS We would like to express our appreciation to Dr. Shabana Khan for overseeing the biological assay. Dr. Khan evaluated the cytotoxicity of the synthesized ligands across various cell lines. This work was funded in part by the Deutsche Forschungsgemeinschaft (DFG) SI868/22 − 1, project number 46995445 (to W.S.). Data Availability Data is provided within the manuscript or supplementary information files References J. Fan, J. Baeza, J.M. Denu, Methods Enzymol . 2016 , 574,125. https://doi.org/10.1016/bs.mie.2016.01.007 O. Khan, N.B. La Thangue, Immunol. Cell Biol, 2012 , 90, 85. https://doi.org/10.1038/icb.2011.100 E. Ceccacci, S. Minucci, Br. J. Cancer , 2016 , 114, 605. https://doi.org/10.1038/bjc.2016.36 G. Li, Y. Tian, W.-G. Zhu, Front Cell Dev Biol , 2020 , 8, 576946. https://doi.org/10.3389/fcell.2020.576946 E. San José-Enériz, N. Gimenez-Camino, X. Agirre, F. Prosper, Cancers, 2019 , 11, 1794. https://doi.org/10.3390/cancers11111794 O.H. Krämer, Trends Pharmacol. Sci , 2009 , 30, 647. https://doi.org/10.1016/j.tips.2009.09.007 D.A. Rodrigues, G.À. Ferreira-Silva, A.C.S. Ferreira, R.A. Fernandes, J.K. Kwee, C.M.R. Sant’Anna, M. Ionta, C.A.M. Fraga, J. Med. Chem , 2016, 59, 655. https://doi.org/10.1021/acs.jmedchem.5b01525 Y. Hai, S.A. Shinsky, N.J. Porter, D.W. Christianson, Nat. Commun , 2017 , 8, 15368. https://doi.org/10.1038/ncomms15368 I. Oehme, J.-P. Linke, B.C. Böck, T. Milde, M. Lodrini, B. Hartenstein, I. Wiegand, C. Eckert, W. Roth, M. Kool, S. Kaden, H.-J. Gröne, J.H. Schulte, S. Lindner, A. Hamacher-Brady, N.R. Brady, H.E. Deubzer, O. Witt, Proc. Natl. Acad. Sci. U. S. A , 2013 , 110, E2592. https://doi.org/10.1073/pnas.1300113110 S.A. Shinsky, D.W. Christianson, Biochemistry . 2018 , 57, 3105. https://doi.org/10.1021/acs.biochem.8b00079 D. Zhang, B. Tang, X. Xie, Y.-F. Xiao, S.-M. Yang, J.-W. Zhang, Cancer Biol. Ther , 2015 , 16, 1005. https://doi.org/10.1080/15384047.2015.1046022 T. Kumagai, N. Wakimoto, D. Yin, S. Gery, N. Kawamata, N. Takai, N. Komatsu, A. Chumakov, Y. Imai, H.P. Koeffler, Int. J. Cancer , 2007 , 121, 656. https://doi.org/10.1002/ijc.22558 X. Qian, G. Ara, E. Mills, W.J. LaRochelle, H.S. Lichenstein, M. Jeffers, Int. J. Cancer , 2008 , 122, 1400. https://doi.org/10.1002/ijc.23243 R.L. Piekarz, R. Frye, M. Turner, J.J. Wright, S.L. Allen, M.H. Kirschbaum, J. Zain, H.M. Prince, J.P. Leonard, L.J. Geskin, C. Reeder, D. Joske, W.D. Figg, E.R. Gardner, S.M. Steinberg, E.S. Jaffe, M. Stetler-Stevenson, S. Lade, A.T. Fojo, S.E. Bates, J. Clin. Oncol , 2009 , 27, 5410. https://doi.org/10.1200/JCO.2008.21.6150 M. Rhinn, I. Zapata-Bodalo, A. Klein, J.-L. Plassat, T. Knauer-Meyer, W.M. Keyes, PLoS Biol , 2022 , 20, e3001664. https://doi.org/10.1371/journal.pbio.3001664 L. Ellis, Y. Pan, G.K. Smyth, D.J. George, C. McCormack, R. Williams-Truax, M. Mita, J. Beck, H. Burris, G. Ryan, P. Atadja, D. Butterfoss, M. Dugan, K. Culver, R.W. Johnstone, H.M. Prince, Clin. Cancer Res , 2008 , 14, 4500. https://doi.org/10.1158/1078-0432.CCR-07-4262 N.M. Raghavendra, D. Pingili, S. Kadasi, A. Mettu, S.V.U.M. Prasad, Eur. J. Med. Chem , 2018 , 143, 1277. https://doi.org/10.1016/j.ejmech.2017.10.021 R.R. Ramsay, M.R. Popovic-Nikolic, K. Nikolic, E. Uliassi, M.L. Bolognesi, Clin. Transl. Med , 2018 , 7, 3. https://doi.org/10.1186/s40169-017-0181-2 Y. Li, E. Seto, Cold Spring Harb. Perspect. Med , 2016 , 6. https://doi.org/10.1101/cshperspect.a026831 E. Seto, M. Yoshida, Cold Spring Harb. Perspect. Biol , 2014, 6, a018713. https://doi.org/10.1101/cshperspect.a018713 H.S. Rho, H.S. Baek, J.A. Lee, C.M. Park, D.H. Kim, I.S. Chang, O.S. Lee, Hydroxamic acid derivatives and the preparation method thereof, 7485750, 2009 . https://patentimages.storage.googleapis.com/16/d4/5a/47495080cd5d60/US7485750.pdf (accessed October 18, 2023). Q. Lu, Y.-T. Yang, C.-S. Chen, M. Davis, J.C. Byrd, M.R. Etherton, A. Umar, C.-S. Chen, J. Med. Chem, 2004 , 47, 467. https://doi.org/10.1021/jm0303655 J. Ridinger, E. Koeneke, F.R. Kolbinger, K. Koerholz, S. Mahboobi, L. Hellweg, N. Gunkel, A.K. Miller, H. Peterziel, P. Schmezer, A. Hamacher-Brady, O. Witt, I. Oehme, Sci. Rep , 2018 , 8, 10039. https://doi.org/10.1038/s41598-018-28265-5 Y.Y. Gu, G.N. Zhou, Y. Li, H.Y. He, J.X. Ding, K.Q. Hua, J. Oncol . 2022 , 2022, 8092751. https://doi.org/10.1155/2022/8092751 C.J. Herbst-Gervasoni, R.R. Steimbach, M. Morgen, A.K. Miller, D.W. Christianson, ACS Chem. Biol . 2020 , 15, 2154. https://doi.org/10.1021/acschembio.0c00362 M. Géraldy, M. Morgen, P. Sehr, R.R. Steimbach, D. Moi, J. Ridinger, I. Oehme, O. Witt, M. Malz, M.S. Nogueira, O. Koch, N. Gunkel, A.K. Miller, J. Med. Chem , 2019 , 62, 4426. https://doi.org/10.1021/acs.jmedchem.8b01936 F.R. Kolbinger, E. Koeneke, J. Ridinger, T. Heimburg, M. Müller, T. Bayer, W. Sippl, M. Jung, N. Gunkel, A.K. Miller, F. Westermann, O. Witt, I. Oehme, Arch. Toxicol . 2018 , 92, 2649. https://doi.org/10.1007/s00204-018-2234-8 Y. Li, P.M. Woster, Medchemcomm . 2015 , 6, 613. https://doi.org/10.1039/C4MD00401A P. Zeyen, Y. Zeyn, D. Herp, F. Mahmoudi, T.Z. Yesiloglu, F. Erdmann, M. Schmidt, D. Robaa, C. Romier, J. Ridinger, C.J. Herbst-Gervasoni, D.W. Christianson, I. Oehme, M. Jung, O.H. Krämer, W. Sippl, Eur. J. Med. Chem , 2022 , 234. 114272. https://doi.org/10.1016/j.ejmech.2022.114272 D. Herp, J. Ridinger, D. Robaa, S.A. Shinsky, K. Schmidtkunz, T.Z. Yesiloglu, T. Bayer, R.R. Steimbach, C.J. Herbst-Gervasoni, A. Merz, C. Romier, P. Sehr, N. Gunkel, A.K. Miller, D.W. Christianson, I. Oehme, W. Sippl, M. Jung, Chembiochem , 2022 , 23, e202200180. https://doi.org/10.1002/cbic.202200180 E. Pojani, D. Barlocco, Curr. Med. Chem , 2022 , 29, 2306. DOI: 10.2174/0929867328666210901144658 J. Ptacek, I. Snajdr, J. Schimer, Z. Kutil, J. Mikesova, P. Baranova, B. Havlinova, W. Tueckmantel, P. Majer, A. Kozikowski, C. Barinka, Int. J. Mol. Sc, 2023 , 24. https://doi.org/10.3390/ijms24054720 D.T. Vogl, N. Raje, S. Jagannath, P. Richardson, P. Hari, R. Orlowski, J.G. Supko, D. Tamang, M. Yang, S.S. Jones, C. Wheeler, R.J. Markelewicz, S. Lonial, Ricolinostat, Clin. Cancer Re, 2017 , 23, 3307. https://doi.org/10.1158/1078-0432.CCR-16-2526 T. Li, C. Zhang, S. Hassan, X. Liu, F. Song, K. Chen, W. Zhang, J. Yang, J. Hematol. Oncol , 2018 , 11, 111. https://doi.org/10.1186/s13045-018-0654-9 J.D. Osko, D.W. Christianson, Bioorg. Med. Chem. Lett , 2020 , 30, 127023. https://doi.org/10.1016/j.bmcl.2020.127023 R.R. Steimbach, C.J. Herbst-Gervasoni, S. Lechner, T.M. Stewart, G. Klinke, J. Ridinger, M.N.E. Géraldy, G. Tihanyi, J.R. Foley, U. Uhrig, B. Kuster, G. Poschet, R.A. Casero Jr, G. Médard, I. Oehme, D.W. Christianson, N. Gunkel, A.K. Miller, J. Am. Chem. Soc , 2022 , 144, 18861. https://doi.org/10.1021/jacs.2c05030 T. Heimburg, F.R. Kolbinger, P. Zeyen, E. Ghazy, D. Herp, K. Schmidtkunz, J. Melesina, T.B. Shaik, F. Erdmann, M. Schmidt, C. Romier, D. Robaa, O. Witt, I. Oehme, M. Jung, W. Sippl, J. Med. Chem , 2017, 60, 10188. https://doi.org/10.1021/acs.jmedchem.7b01447 M. Zessin, Z. Kutil, M. Meleshin, Z. Nováková, E. Ghazy, D. Kalbas, M. Marek, C. Romier, W. Sippl, C. Bařinka, M. Schutkowski, Biochemistry , 2019 , 58, 4777. https://doi.org/10.1021/acs.biochem.9b00786 P. Sun, J. Wang, K.S. Khan, W. Yang, B.W.-L. Ng, N. Ilment, M. Zessin, E.F. Bülbül, D. Robaa, F. Erdmann, M. Schmidt, C. Romier, M. Schutkowski, A.S.-L. Cheng, W. Sippl, J. Med. Chem , 2022 , 65, 16313. https://doi.org/10.1021/acs.jmedchem.2c01132 H.S. Ibrahim, M. Abdelsalam, Y. Zeyn, M. Zessin, A.-H.M. Mustafa, M.A. Fischer, P. Zeyen, P. Sun, E.F. Bülbül, A. Vecchio, F. Erdmann, M. Schmidt, D. Robaa, C. Barinka, C. Romier, M. Schutkowski, O.H. Krämer, W. Sippl, Int. J. Mol. Sci , 2021 , 23, 369. https://doi.org/10.3390/ijms23010369 H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne, Nucleic Acids Res , 2000 , 28, 235. https://doi.org/10.1093/nar/28.1.235 C.V. Simoben, E. Ghazy, P. Zeyen, S. Darwish, M. Schmidt, C. Romier, D. Robaa, W. Sippl, Molecules , 2021 , 26. https://doi.org/10.3390/molecules26092584 J.L. Banks, H.S. Beard, Y. Cao, A.E. Cho, W. Damm, R. Farid, A.K. Felts, T.A. Halgren, D.T. Mainz, J.R. Maple, R. Murphy, D.M. Philipp, M.P. Repasky, L.Y. Zhang, B.J. Berne, R.A. Friesner, E. Gallicchio, R.M. Levy, J. Comput. Chem , 2005 , 26, 1752. https://doi.org/10.1002/jcc.20292 J. Wang, W. Wang, P.A. Kollman, D.A. Case, J. Mol. Graph. Model , 2006 , 25, 247. https://doi.org/10.1016/j.jmgm.2005.12.005 A. Jakalian, D.B. Jack, C.I. Bayly, Fast, efficient generation of high‐quality atomic charges. AM1‐BCC model: II. Parameterization and validation, Journal of Computational. ( 2002 ). https://onlinelibrary.wiley.com/doi/abs/10.1002/jcc.10128?casa_token=qpaSyrElG0AAAAAA:13RPtcvphG3Ais-isjxM5xvFOD1ZHoIimPMTpWt5KtqoI2bmsx3btHslXwP3M7_tp6HX_0_u4VXtPZIE. A. Jakalian, B.L. Bush, D.B. Jack, C.I. Bayly, J. Comput. Chem , 2000 , 21, 132. https://doi.org/10.1002/(SICI)1096-987X(20000130)21:23.0.CO;2-P X. He, V.H. Man, W. Yang, T.-S. Lee, J. Wang, J. Chem. Phys , 2020 , 153, 114502. https://doi.org/10.1063/5.0019056 J.A. Maier, C. Martinez, K. Kasavajhala, L. Wickstrom, K.E. Hauser, C. Simmerling, J. Chem. Theory Comput , 2015 , 11, 3696. https://doi.org/10.1021/acs.jctc.5b00255 P. Li, L.F. Song, K.M. Merz Jr, J. Phys. Chem . B, 2015 , 119, 883. https://doi.org/10.1021/jp505875v Toukmaji, C. Sagui, J. Board, T. Darden, Efficient particle-mesh Ewald based approach to fixed and induced dipolar interactions, The Journal of Chemical. (2000). https://pubs.aip.org/aip/jcp/article-abstract/113/24/10913/452430. https://doi.org/10.1063/1.1324708 D.D. Evans, Aust. J. Chem , 1973 , 26, 2555. https://doi.org/10.1071/CH9732555 T.-S. Wu, M.-J. Liou, C.-J. Lee, T.-T. Jong, A.T. McPhail, D.R. McPhail, K.-H. Lee, Tetrahedron Lett , 1989 , 30, 6649. https://doi.org/10.1016/S0040-4039(00)70641-1 H. Xu, Y.-Y. Wang, Bioorg. Med. Chem . Lett, 2010 , 20, 7274. https://doi.org/10.1016/j.bmcl.2010.10.084 M.P. Mertes, A.J. Lin, J. Med. Chem , 1970 , 13, 276. https://doi.org/10.1021/jm00296a026 S.-Y. Cheung, M. Werner, L. Esposito, F. Troisi, V. Cantone, S. Liening, S. König, J. Gerstmeier, A. Koeberle, R. Bilancia, R. Rizza, A. Rossi, F. Roviezzo, V. Temml, D. Schuster, H. Stuppner, M. Schubert-Zsilavecz, O. Werz, T. Hanke, S. Pace, Eur. J. Med. Chem , 2018 , 156, 815. https://doi.org/10.1016/j.ejmech.2018.07.031 F. Gaedcke, R. Knorr, F. Zymalkowski, Arch. Pharm , 1980, 313, 166. https://doi.org/10.1002/ardp.19803130209 Table 1 Table 1 is available in the Supplementary Files section. Schemes Schemes 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files supportingdataHDACMay2.docx Table1ChemicalstructureandIC50valuesforsynthesizedcompoundsagainstHDAC10.docx scheme1.png SCHEME 1 Reagent and condition: (a) DMF, NaH, TsCl, ice bath, 15 min. (b) DCM, STAB, r.t, overnight. (c) DCM/MeOH (1:2), hydroxylamine (50 wt % in H 2 O), NaOH, r.t, 24h scheme2.png Scheme 2 Reagent and condition: (a) DCM, STAB, r.t, overnight. (b) DCM/MeOH (1:2), hydroxylamine (50 wt % in H 2 O), NaOH, r.t, 24h. 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We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5950104","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":412078701,"identity":"925c63bb-4cfa-49ad-a87c-d2b3f8fa6693","order_by":0,"name":"Amer H. Tarawneh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYLADZoYKCEOCgYGNgNoEmJYzBhIkamFsg2vBDfinHT72gfGHXT7/jNyDjwvn/akzOMB88DYPA58cLi0St9OSZzAkJFvOuJGXbDxzm4GEwQG2ZGseBjZjnNbczgHKJTAbABlm0rxgLTxm0kAtiQ04dMjfzv8M1FJvIH87x/w37xyQFv5veLUY3M5hBmo5bABkmDHzNoBtYcOrxfB2mjFDQtpxA8P7b4yleY4ZS848zGZsOccAt1/kbic/ZvhgU20gd+aM4WeeGjl+vuPND2+8qTiGM8TAIAGFxwx28DG8OrCCGtK1jIJRMApGwXAFAIlpSjKLWglxAAAAAElFTkSuQmCC","orcid":"","institution":"Tafila Technical University","correspondingAuthor":true,"prefix":"","firstName":"Amer","middleName":"H.","lastName":"Tarawneh","suffix":""},{"id":412078702,"identity":"a896a8bf-e4ad-4a18-8ef3-47b10f1c1cc0","order_by":1,"name":"Salah A. 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Yesiloglu","email":"","orcid":"","institution":"Martin-Luther University of Halle-Wittenberg","correspondingAuthor":false,"prefix":"","firstName":"Talha","middleName":"Z.","lastName":"Yesiloglu","suffix":""},{"id":412078704,"identity":"ce91060a-3ebb-441e-a7cd-aaf6d2a957b8","order_by":3,"name":"Matthes Zessin","email":"","orcid":"","institution":"Martin-Luther University of Halle-Wittenberg","correspondingAuthor":false,"prefix":"","firstName":"Matthes","middleName":"","lastName":"Zessin","suffix":""},{"id":412078705,"identity":"ec30359c-cf36-41a7-8f99-ee6672aca4a1","order_by":4,"name":"Dina Robaa","email":"","orcid":"","institution":"Martin-Luther University of Halle-Wittenberg","correspondingAuthor":false,"prefix":"","firstName":"Dina","middleName":"","lastName":"Robaa","suffix":""},{"id":412078706,"identity":"6701319e-4c86-4420-a7b0-e3d3cea9a6de","order_by":5,"name":"Cyril Barinka","email":"","orcid":"","institution":"Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV","correspondingAuthor":false,"prefix":"","firstName":"Cyril","middleName":"","lastName":"Barinka","suffix":""},{"id":412078707,"identity":"d0745a27-da1c-469e-8463-bc7f4e48a307","order_by":6,"name":"Mike Schutkowski","email":"","orcid":"","institution":"Martin-Luther University of Halle-Wittenberg","correspondingAuthor":false,"prefix":"","firstName":"Mike","middleName":"","lastName":"Schutkowski","suffix":""},{"id":412078708,"identity":"a51c382b-3b86-436e-a214-1151b6b1a0ce","order_by":7,"name":"Wolfgang Sippl","email":"","orcid":"","institution":"Martin-Luther University of Halle-Wittenberg","correspondingAuthor":false,"prefix":"","firstName":"Wolfgang","middleName":"","lastName":"Sippl","suffix":""},{"id":412078709,"identity":"230c894f-1285-4be9-9f32-d1e816c71120","order_by":8,"name":"Samir A. Ross","email":"","orcid":"","institution":"University of Mississippi, University","correspondingAuthor":false,"prefix":"","firstName":"Samir","middleName":"A.","lastName":"Ross","suffix":""}],"badges":[],"createdAt":"2025-02-03 10:08:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5950104/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5950104/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-02774-6","type":"published","date":"2025-09-26T15:58:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75699717,"identity":"0a99136a-2473-4bfb-b97a-4b222d5c6827","added_by":"auto","created_at":"2025-02-07 09:09:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":39031,"visible":true,"origin":"","legend":"\u003cp\u003eExample of approved HDAC inhibitors by the Food and Drug Administration (FDA).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5950104/v1/c4baccbce521ba709d643c3d.png"},{"id":75699726,"identity":"fc81b585-e323-4002-9087-3a9154b0d4f4","added_by":"auto","created_at":"2025-02-07 09:09:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":536546,"visible":true,"origin":"","legend":"\u003cp\u003ePredicted binding modes of compound \u003cstrong\u003e1 \u003c/strong\u003ein different HDAC isoforms: \u003cstrong\u003ea)\u003c/strong\u003e \u003cstrong\u003e1 \u003c/strong\u003e(teal sticks) in drHDAC10 (PDB ID 6UHU), \u003cstrong\u003eb)\u003c/strong\u003e \u003cstrong\u003e1\u003c/strong\u003e(violet sticks) in HDAC6 (PDB ID 5EDU), \u003cstrong\u003ec)\u003c/strong\u003e \u003cstrong\u003e1\u003c/strong\u003e (orange sticks) in HDAC8 (PDB ID 2V5X) \u003cstrong\u003ed)\u003c/strong\u003e \u003cstrong\u003e1\u003c/strong\u003e (magenta sticks) in HDAC1 (PDB ID 5ICN). The surface of the proteins is colored according to lipophilicity; green for hydrophobic and magenta for hydrophilic. Side chains of binding site residues are shown as white sticks and the catalytic zinc ion as an orange sphere. H-bond interactions are depicted as blue-dashed lines, salt bridge interactions as magenta-dashed lines, π-π interactions as orange-dashed lines, cation-π interactions as green-dashed lines and zinc ion coordinationby the ligand as yellow-dashed lines.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5950104/v1/6f38a91206404f0bccab1270.png"},{"id":75700170,"identity":"91c6d922-c168-46bd-afc4-43f0f367e4ec","added_by":"auto","created_at":"2025-02-07 09:17:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":463008,"visible":true,"origin":"","legend":"\u003cp\u003ePredicted binding modes of compounds, \u003cstrong\u003ea)\u003c/strong\u003e \u003cstrong\u003e5 \u003c/strong\u003e(peach sticks) and \u003cstrong\u003eb)\u003c/strong\u003e \u003cstrong\u003e6\u003c/strong\u003e (teal sticks) in drHDAC10 (PDB ID 6UHU). The surface of the proteins is colored according to lipophilicity; green for hydrophobic and magenta for hydrophilic. Side chains of binding site residues are shown as white sticks and the catalytic zinc ion as orange spheres. H-bond interactions are depicted as blue-dashed lines, salt bridge interactions as magenta-dashed lines, π-π interactions as orange-dashed lines, cation-π interactions as green-dashed lines, and coordination of the zinc ion by the ligand as yellow-dashed lines.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5950104/v1/52ce6f527e248e79e85fff78.png"},{"id":75699721,"identity":"c6d786f3-1ee1-4031-b30d-3b904f10a6d8","added_by":"auto","created_at":"2025-02-07 09:09:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":200466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea) \u003c/strong\u003eRMSD plots of the compound \u003cstrong\u003e1\u003c/strong\u003e heavy atoms in drHDAC10 (PDB ID 6UHU)-compound \u003cstrong\u003e1\u003c/strong\u003e docked complexes for 3 repeated MD runs each for 100 ns. \u003cstrong\u003eB) \u003c/strong\u003eRMSF plots of compound \u003cstrong\u003e1\u003c/strong\u003eheavy atoms in drHDAC10 (PDB ID 6UHU)-compound \u003cstrong\u003e1\u003c/strong\u003e docked complexes for three repeated MD runs each for 100 ns s\u003cstrong\u003e c)\u003c/strong\u003e The measured distance between both oxygen atoms of the hydroxamate zinc binding moiety and catalytic zinc ion for three repeated MD runs. \u003cstrong\u003eD) \u003c/strong\u003eRepresentative poses of the obtained three clusters with an occupancy \u0026gt; 10%,\u003cstrong\u003e \u003c/strong\u003eteal sticks for the first cluster (occupancy 55%), magenta sticks for the second cluster (occupancy 15%), and yellow sticks for the third cluster (occupancy 17%). Side chains of the relevant binding site residues are shown as white sticks and the catalytic zinc ion as orange spheres.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5950104/v1/e03e1a8f71e7806f793fcd8b.png"},{"id":92430646,"identity":"9057fb79-bd41-411e-a474-fdfc25d6ce0b","added_by":"auto","created_at":"2025-09-29 16:07:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3436440,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5950104/v1/b3c7147e-9ccb-4777-8834-c5e90d960445.pdf"},{"id":75699741,"identity":"509c25aa-0159-4856-8b5e-f5bf178f6bd5","added_by":"auto","created_at":"2025-02-07 09:09:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6465494,"visible":true,"origin":"","legend":"","description":"","filename":"supportingdataHDACMay2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5950104/v1/da69162c36f4ef238fa5981c.docx"},{"id":75699718,"identity":"6e6f69c0-3e5a-451a-aedf-d2a0f7736c89","added_by":"auto","created_at":"2025-02-07 09:09:05","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":94381,"visible":true,"origin":"","legend":"","description":"","filename":"Table1ChemicalstructureandIC50valuesforsynthesizedcompoundsagainstHDAC10.docx","url":"https://assets-eu.researchsquare.com/files/rs-5950104/v1/086a458d2b42c99721d05587.docx"},{"id":75699719,"identity":"da51e1ff-0041-4309-b85a-5f733b4db00f","added_by":"auto","created_at":"2025-02-07 09:09:05","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":35275,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSCHEME 1\u003c/strong\u003e Reagent and condition: (a) DMF, NaH, TsCl, ice bath, 15 min. (b) DCM, STAB, r.t, overnight. (c) DCM/MeOH (1:2), hydroxylamine (50 wt % in H\u003csub\u003e2\u003c/sub\u003eO), NaOH, r.t, 24h\u003c/p\u003e","description":"","filename":"scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-5950104/v1/b7c9816641fcb83c19df19e3.png"},{"id":75699723,"identity":"0fc2962a-c375-4380-ac21-27fc0d506493","added_by":"auto","created_at":"2025-02-07 09:09:05","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":25489,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 2\u003c/strong\u003e Reagent and condition: (a) DCM, STAB, r.t, overnight. (b) DCM/MeOH (1:2), hydroxylamine (50 wt % in H\u003csub\u003e2\u003c/sub\u003eO), NaOH, r.t, 24h.\u003c/p\u003e","description":"","filename":"scheme2.png","url":"https://assets-eu.researchsquare.com/files/rs-5950104/v1/638c912339c8ff53654a79f8.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Efficacy of a New Selective Indole-based Histone Deacetylase 10 Inhibitor in Targeted Anticancer Therapy","fulltext":[{"header":"1 INTRODUCTION","content":"\u003cp\u003eHistones assist in the control of the expression process that converts the coded information in genes into the operational structures in the cell. Histone acetylation (HA) neutralizes the positive charge on the histones by changing amines into amides [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This change decreases the ability of the histone to bind to DNA (chromatin expansion) and permits genetic transcription [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Histone deacetylases (HDACs) are enzymes that remove the acetyl group from acetylated lysine residues found in both histone and non-histone proteins. This type of removal results in silencing DNA gene expression. Histones are basic proteins present in the chromatin of eukaryotic cell nuclei and play a role in wrapping DNA into structural units called nucleosomes, which are the basic repeating units of chromatin [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere are 18 subtypes of HDACs within the human genome. These enzymes are categorized into two groups; the first family is zinc-dependent HDACs, which include the following sub-classes: class Ia (HDAC1, HDAC2), class Ib (HDAC3), and class Ic (HDAC8), class IIa (HDAC4, HDAC5, HDAC7, and HDAC9), class IIb (HDAC6 and HDAC10), and class IV (HDAC11). The second group is nicotinamide adenine dinucleotide (NAD\u003csup\u003e+\u003c/sup\u003e)-dependent HDACs (class III), which are known as sirtuins [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Many pieces of evidence revealed the crucial role of HDACs in different cellular processes, including autophagy, cell cycle control, and apoptosis [\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Recent data indicate the protective HDAC function against DNA damage, making them validated anti-tumor therapy targets [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral HDAC inhibitors have been identified, and some (Fig.\u0026nbsp;1) were approved by FDA. For instance, \u003cb\u003evorinostat\u003c/b\u003e (SAHA) was approved for the treatment of cutaneous T cell lymphoma (CTCL) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], \u003cb\u003evelinostat\u003c/b\u003e (PXD-101) and \u003cb\u003eromidepsin\u003c/b\u003e (FK-228) were approved for the treatment of peripheral T-cell lymphoma (PTCL) [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], bipolar disorders and migraine. In addition, \u003cb\u003epanobinostat\u003c/b\u003e (LBH-589) was approved for the treatment of multiple myeloma. The inhibitor leads to the growth arrest, differentiation, and apoptosis of many transformed cells, thus eliminating cancer cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe catalytic domains of the different zinc-dependent HDACs share a high degree of homology. The lack of specific isoform selectivity of approved HDAC inhibitors leads to potential side effects [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Both non-selective HDAC inhibitors, vorinostat, and panobinostat modulate multiple pathways and factors in MDA-MB-231, 4T1, and BT-549 cell lines. This modulation includes suppression of growth factor FOXA1 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], upregulation of tumor suppressor factors p21 and p27, and downregulation of the survival protein Bcl-2 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In addition, both can inhibit matrix metalloproteinase (MMP9) activity [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, HDAC10 has been shown to play a unique role in neuroblastoma cells, where inhibition of HDAC10 can cause an accumulation of autolysosomes. This result indicates that HDAC10 may be an effective target for the treatment of neuroblastoma [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Conversely, HDAC10 was found to suppress tumorigenesis in cervical cancer by downregulating the expression of miR-233 and subsequently targeting EPB41L3 expression. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInvestigating the biological relevance of HDAC10 and evaluating its pharmacological role in cancer cell lines highlight the need for selective HDAC10 inhibitors. A few inhibitors that target HDAC10 have so far been identified [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], such as tubastatin A and tubastatin A derivative (1b), which are selective for HDAC subfamily IIb [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Recently, TH34 was reported as a class I/IIb selective HDAC inhibitor [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Additionally, compounds including 2-(oxazol-2-yl)phenol moiety were considered as HDAC1/6/10 inhibitors [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The importance of a basic moiety in the linker group was also shown in a recently developed series of piperidine-based HDAC10 inhibitors [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The challenge lies in introducing HDAC10 inhibitors with high selectivity over the class IIb isozyme member HDAC6.\u003c/p\u003e \u003cp\u003eMost HDAC inhibitors share common pharmacophoric features, which include a 'cap' group and hydrophobic chain (linker); both groups impact the selectivity. In addition, the zinc-binding group (ZBG) is crucial to the inhibitor's potency [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Herein, we report an effort to optimize indole and quinoline ligands as HDAC10-selective inhibitors. Several modified derivatives that were developed exhibited HDAC10 inhibition and selectivity.\u003c/p\u003e"},{"header":"2 RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemistry\u003c/h2\u003e \u003cp\u003eThe compounds in this study were synthesized as shown in schemes 1 and 2. The starting building block, 1\u003cem\u003eH\u003c/em\u003e-indole-3-carbaldehyde, was treated with 4-methylbenzene-1-sulfonyl chloride in anhydrous \u003cem\u003eN, N\u0026apos;\u003c/em\u003e-Dimethylformamide (DMF) using sodium hydride as a base, and subsequently coupled with various amino compounds via a reductive amination reaction using sodium triacetoxyborohydride (STAB) followed by condensation reaction with hydroxylamine and KOH to afford final targeted compounds \u003cstrong\u003e2\u003c/strong\u003e, \u003cstrong\u003e3\u003c/strong\u003e, and \u003cstrong\u003e6\u003c/strong\u003e, Scheme 1.\u003c/p\u003e\n\u003cp\u003eSimilarly, indole-3-carboxaldehyde and quinolone were treated in the same manner as indole-3-carboxaldehyde to produce compounds \u003cstrong\u003e1\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;5,\u003c/strong\u003e and \u003cstrong\u003e7\u003c/strong\u003e, Scheme 1 and 2.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Pharmacology/Biology\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Synthesis and \u003cem\u003ein vitro\u003c/em\u003e testing of novel inhibitors\u003c/h2\u003e \u003cp\u003eRecently, piperidine-4-acryl hydroxamates (\u003cb\u003ePZ45\u003c/b\u003e and \u003cb\u003ePZ48\u003c/b\u003e) were reported as potent HDAC10 inhibitors having high specificity for HDAC10 over HDAC6 and with no significant impact on class I HDACs [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The selectivity of these newly discovered ligands \u003cb\u003ePZ45/48\u003c/b\u003e was also tested in acute myeloid leukemia (AML) cells with the FLT3-ITD oncogene and showed promising activities.\u003c/p\u003e \u003cp\u003eIn the current work, we extended the structure-activity relationships of HDAC10 inhibitors by synthesizing a series of benzhyldroxamic acid derivatives that bear a bicyclic aromatic moiety as a capping group with a methylene spacer to a basic amine group. All prepared derivatives were tested in vitro against HDAC10 (from zebrafish) and human HDACs 1, 6, and 8 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Compound \u003cb\u003e1\u003c/b\u003e, bearing the indole methylene capping group, showed potent inhibition of HDAC10 with an IC\u003csub\u003e50\u003c/sub\u003e of 0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 nM (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The tosylatated indole analogue, compound \u003cb\u003e2\u003c/b\u003e, showed nanomolar HDAC10 inhibition with an IC\u003csub\u003e50\u003c/sub\u003e of 4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 nM. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e, shows that hydrophobic tosyl moiety does not interact with the rim of the HDAC10 binding site and instead remains fully exposed to the solvent. The removal of the methylene spacer between the primary amine and the benzhydroxamic acid moiety in analog \u003cb\u003e3\u003c/b\u003e and \u003cb\u003e4\u003c/b\u003e led to a significant reduction in the inhibitory activity (IC\u003csub\u003e50\u003c/sub\u003e of 290\u0026thinsp;\u0026plusmn;\u0026thinsp;60 nM and 110\u0026thinsp;\u0026plusmn;\u0026thinsp;10 nM, respectively). The loss of salt bridge interactions with D94 and E274, along with the cation-π interaction with W205 in HDAC10, which were present in the other compounds reported in this study, is evident. As anticipated, only hydrogen bond interactions between the NH-group and D94 are observed (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb and Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eThe second series of compounds comprises of 4-(1-piperazinyl)benzhydroxamic acid derivatives that bear a bicyclic aromatic moiety as a capping group. Compound \u003cb\u003e5\u003c/b\u003e exhibited potent HDAC10 inhibitory activity (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 nM); while the methyltosylated indole derivative revealed that compound \u003cb\u003e6\u003c/b\u003e showed a decrease in the HDAC10 inhibitory activity with an IC\u003csub\u003e50\u003c/sub\u003e of 75\u0026thinsp;\u0026plusmn;\u0026thinsp;12 nM (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Meanwhile, the replacement of the indole of \u003cb\u003e5\u003c/b\u003e with aquinoline moiety yielded compound \u003cb\u003e7\u003c/b\u003e, which was found to be slightly less potent with IC\u003csub\u003e50\u003c/sub\u003e of 11\u0026thinsp;\u0026plusmn;\u0026thinsp;1 nM.\u003c/p\u003e \u003cp\u003eIn conclusion of the structure-activity relationship (SAR) analysis, the absence of a strong basic amino group was found to reduce the inhibitory activity against HDAC10. Additionally, modifying the capping group with a hydrophobic tosyl moiety also led to a decrease in inhibitory activity\u003c/p\u003e \u003cp\u003eThe preliminary structure-activity relationship (SAR) revealed that the additional bicyclic aromatic moiety enhanced the HDAC10 inhibitory activity. We designed and synthesized seven compounds with various substituents at the cap position and linker, the benzhydroxamic acid bearing an indole ring; compound \u003cb\u003e1\u003c/b\u003e, stood out as the most potent HDAC10 inhibitor with the best enzyme inhibitory with selectivity toward HDAC10. Herein we introduce highly selective ligand \u003cb\u003e6\u003c/b\u003e for HDAC10 with nanomolar inhibitory activity.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Enzymatic \u003cem\u003ein vitro\u003c/em\u003e testing\u003c/h2\u003e \u003cp\u003eAll the synthesized compounds were evaluated in vitro against zebrafish HDAC10 (drHDAC10), and human HDAC 1, 6, and 8 (details see Methods section). DrHDAC10 was chosen as the close homolog of human HDAC10 since it was found to be more stable and easier to express compared to the human HDAC10 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. It\u0026rsquo;s worth mentioning that none of the compounds displayed potent inhibitory against class I HDACs. Data in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e indicate that all compounds retained activity on HDAC10 comparable or even higher than the reference HDAC6/10 inhibitor Tubastatin A. We reported that the indole ring significantly affected the potency and selectivity towards HDAC10 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Compounds \u003cb\u003e1\u003c/b\u003e bearing a benzhydroxamic acid displays a highly potent inhibitory activity with IC\u003csub\u003e50\u003c/sub\u003e of 0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 nM towards HDAC10, but also potent against HDAC6 and moderately potent against HDAC8 with IC\u003csub\u003e50\u003c/sub\u003e value of 37\u0026thinsp;\u0026plusmn;\u0026thinsp;2 and 350\u0026thinsp;\u0026plusmn;\u0026thinsp;20 nM, respectively.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIn vitro selectivity of newly synthesized HDAC10 inhibitors.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehHDAC1 IC\u003csub\u003e50\u003c/sub\u003e [nM]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehHDAC6 IC\u003csub\u003e50\u003c/sub\u003e [nM]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ehHDAC8 IC\u003csub\u003e50\u003c/sub\u003e [nM]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4500\u0026thinsp;\u0026plusmn;\u0026thinsp;200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e350\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.2%@1 \u0026micro;M 72.8% @10 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e51\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e65.1%@1 \u0026micro;M 97.3% @10 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.3%@1 \u0026micro;M 38.9% @10 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49.2%@1 \u0026micro;M 98.9% @10 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51.7%@1 \u0026micro;M 91.4% @10 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e210\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48.4%@1 \u0026micro;M 90.6% @10 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e73\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74\u0026thinsp;\u0026plusmn;\u0026thinsp;6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.3%@1 \u0026micro;M\u003c/p\u003e \u003cp\u003e67.2% @10 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.3%@1 \u0026micro;M\u003c/p\u003e \u003cp\u003e95% @10 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26.9%@1 \u0026micro;M 87.9% @10 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32.1%@1 \u0026micro;M 79.4% @10 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e130\u0026thinsp;\u0026plusmn;\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e300\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTubastatinA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.6% @1 \u0026micro;M\u003c/p\u003e \u003cp\u003e29.9%@10 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e46.4% @1 \u0026micro;M\u003c/p\u003e \u003cp\u003e84% @10 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eCompounds \u003cb\u003e5\u003c/b\u003e and \u003cb\u003e7\u003c/b\u003e showed a preference for HDAC10 (IC\u003csub\u003e50\u003c/sub\u003e 2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 and 11\u0026thinsp;\u0026plusmn;\u0026thinsp;1 nM, respectively) compared with HDAC8 and HDAC6. As can be expected, compound \u003cb\u003e4, which\u003c/b\u003e only contains a weakly basic aromatic amine moiety, showed a significant decrease in the HDAC10 inhibitory activity and high preference and potency to HDAC6 with an IC\u003csub\u003e50\u003c/sub\u003e value of 2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 nM, which again highlights the importance of the protonated amine group to achieve high HDAC10 potency and selectivity.\u003c/p\u003e \u003cp\u003eMeanwhile, compounds \u003cb\u003e2\u003c/b\u003e and \u003cb\u003e3\u003c/b\u003e showed a preference for class IIb enzymes (HDAC10 and 6) with no significant inhibition on HDAC1/8. HDAC6 is well documented, it plays a crucial role in microtubule deacetylation and regulation of PDL1 and further targets related to cancer immunotherapy [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn combination with the observed \u003cem\u003ein vitro\u003c/em\u003e potency and selectivity of compounds \u003cb\u003e2\u003c/b\u003e and \u003cb\u003e3\u003c/b\u003e, both represent promising hits for further optimization. The lack of specific and potent HDAC10 inhibitors led to limited knowledge about the biological function of HDAC10 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A more distinguished result showed by compound \u003cb\u003e6\u003c/b\u003e examined the inhibitory of compound \u003cb\u003e6\u003c/b\u003e revealed high selectivity with a novel nanomolar inhibitor toward HDAC10 (IC\u003csub\u003e50\u003c/sub\u003e 75\u0026thinsp;\u0026plusmn;\u0026thinsp;12 nM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Cytotoxic activity\u003c/h2\u003e \u003cp\u003eAll the synthesized compounds (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) were screened at three different concentrations for their cytotoxicity towards a panel of four human solid tumor cell lines: melanoma (SK-MEL), epidermal carcinoma (KB), breast carcinoma (BT-549), and ovarian carcinoma (SK-OV-3). Moreover, non-cancer kidney cell lines (LLC-PK1 and VERO) were also included in the study.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCytotoxicity of compounds towards a panel of cell lines.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eCancer cell lines IC\u003csub\u003e50\u003c/sub\u003e \u0026micro;M\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eKidney cells IC\u003csub\u003e50\u003c/sub\u003e \u0026micro;M\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSK-MEL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBT-549\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSK-OV-3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLLC-PK1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eVero\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72.80\u0026thinsp;\u0026plusmn;\u0026thinsp;2.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e60.95\u0026thinsp;\u0026plusmn;\u0026thinsp;14.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e62.64\u0026thinsp;\u0026plusmn;\u0026thinsp;2.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.57\u0026thinsp;\u0026plusmn;\u0026thinsp;3.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.38\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e13.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e50.52\u0026thinsp;\u0026plusmn;\u0026thinsp;9.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.91\u0026thinsp;\u0026plusmn;\u0026thinsp;2.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25.97\u0026thinsp;\u0026plusmn;\u0026thinsp;3.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.84\u0026thinsp;\u0026plusmn;\u0026thinsp;3.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.42\u0026thinsp;\u0026plusmn;\u0026thinsp;4.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.27\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;69.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;69.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;69.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;69.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;69.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e51.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDoxorubicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eNC: No cytotoxicity up to 80 \u0026micro;M. Values are represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003cp\u003eIn the first screening, all compounds were tested at a concentration of 80 \u0026micro;M by the MTT assay. The result showed all compounds had cytotoxicity at 80 \u0026micro;M, and hence they were carried out for the second round of screening at a lower concentration. Data showed that all compounds displayed moderate cytotoxicity than the control compound, doxorubicin. Observing the dataset noted that all HDAC10 inhibitors were weakly toxic for LLC-PK1 and VERO kidney cell lines.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Molecular docking\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Molecular modelling studies\u003c/h2\u003e \u003cp\u003eDocking studies have been conducted to elucidate the binding mode of the synthesized compounds in various HDAC isoforms, including HDAC1 (PDB ID: 5ICN), HDAC6 (PDB ID: 5EDU), HDAC8 (PDB ID: 2V5X) and drHDAC10 (PDB ID: 6UHU). To test the plausibility of the docking results, we compared them with several crystal structures of structurally related hydroxamic acids, including those we recently reported for drHDAC10.\u003c/p\u003e \u003cp\u003eTubastatin A, a known inhibitor of HDAC6 and HDAC10 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], along with the newly synthesized inhibitors described here, were subjected to docking studies within the crystal structures of various HDACs. The binding modes were visually analyzed and the docking studies revealed that tubastatin A and the newly synthesized compounds can chelate the zinc ion in a bidentate manner in HDAC1, HDAC8, and HDAC10. On the other hand, in HDAC6, zinc chelation was observed to occur via the hydroxyl oxygen of the hydroxamate moiety in a monodentate fashion that has also been reported for other benzhydroxamic acids [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Docking solutions in all investigated HDAC isoforms (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e, and Supporting information S1: Figures a-d) showed that the phenyl moiety of the linker was embedded in the hydrophobic lysine binding pocket. Furthermore, hydrogen bond interactions with the conserved residues H136/142/140, H137/143/141, and Y307/306/303 were observed in HDAC10, HDAC8, and HDAC1, respectively. In contrast, in HDAC6, the hydroxyl oxygen formed a water-mediated hydrogen bond with the conserved residues H610 and H611.\u003c/p\u003e \u003cp\u003eThe predicted binding mode of compound \u003cb\u003e1\u003c/b\u003e in HDAC10 (Fig.\u0026nbsp;1a, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) provides insights into its significant inhibitory activity against HDAC10. In addition to the previously mentioned hydrogen bond interactions with conserved tyrosine and histidine residues, the capping group exhibits π-π interactions with F204. Moreover, the protonated amine forms electrostatic and salt-bridge interactions with D94 and the gatekeeper residue E274, along with cation-π interactions with W205 in HDAC10. These interactions resemble those observed in previously reported potent HDAC10 inhibitors and polyamine substrates [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. HDAC6 belongs to class IIa of HDACs. In comparison to HDAC10, the HDAC6 binding pocket exhibits specific differences, notably involving mutations D94/S568 and E274/L749. These variations in the binding pocket led to the docking results showing only a single hydrogen bond interaction between the protonated nitrogen of compound \u003cb\u003e1\u003c/b\u003e and S568. This observation could potentially explain the approximately 90-fold selectivity of HDAC10 over HDAC6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In class I HDAC members (HDAC1 and HDAC8), variations in the gatekeeper residue at the top of the lysine binding tunnel (E274 in HDAC10 is replaced by M274 and L271 in HDAC1 and \u0026minus;\u0026thinsp;8, respectively) led to the loss of one electrostatic interaction with the protonated nitrogen of \u003cb\u003e1\u003c/b\u003e as well as the π-π interactions via the capping group, which were observed in HDAC10. The solvent-exposed capping groups together with the loss of electrostatic interaction with the gatekeeper are most likely the reason for the decrease in activity in both isoforms. Furthermore, the narrow binding pocket of HDAC1 due to the E274/L271 variation might lead to steric hindrance for the linker and capping groups of \u003cb\u003e1\u003c/b\u003e which may explain the higher loss of activity in this isoform (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eSimilar results were observed for compounds \u003cb\u003e5\u003c/b\u003e and \u003cb\u003e7\u003c/b\u003e, which bear a piperazine linker instead of the aminomethyl linker of compound \u003cb\u003e1\u003c/b\u003e. Here, the protonated piperazine-\u003cem\u003eNH\u003c/em\u003e was able to undergo two electrostatic interactions with D94 and E274 and cation-π interactions with W205. Additionally, the capping groups showed similar π-π interaction with W205 in the HDAC10 binding pocket as observed for compound \u003cb\u003e1\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eCompounds lacking a strong basic amino group including compounds \u003cb\u003e3\u003c/b\u003e and \u003cb\u003e4\u003c/b\u003e showed a significant decrease in the HDAC10 inhibitory activity. This can be attributed to the loss of the salt bridge interactions with D94 and E274, and the cation-π interaction with W205 in HDAC10, which were observed with the other compounds reported in this study. As expected, only hydrogen bond interactions between the NH-group and D94 are observed (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb and Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec). These findings further emphasize the significance of the protonated nitrogen and its precise positioning in optimizing the binding interactions with HDAC10.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe in vitro data presented in this study demonstrate that substituting the capping group with an additional tosyl moiety results in a decrease in HDAC10 inhibitory activity. Compound \u003cb\u003e2\u003c/b\u003e exhibits approximately a 10-fold reduction in HDAC10 inhibitory activity compared to compound \u003cb\u003e1\u003c/b\u003e, while compound \u003cb\u003e6\u003c/b\u003e shows an almost 38-fold decrease in activity compared to its unsubstituted counterpart, compound \u003cb\u003e5\u003c/b\u003e. The predicted binding mode of compound \u003cb\u003e6\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) was found to be similar to that of compound \u003cb\u003e5\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). However, an additional hydrogen bond via the sulfonyl group with N207 was observed in compound \u003cb\u003e6\u003c/b\u003e. Notably, the hydrophobic tosyl moiety does not interact with the rim of the HDAC10 binding site and remains fully exposed to the solvent. This observation could explain the decrease in HDAC10 inhibitory activity compared to compound \u003cb\u003e5\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Molecular dynamics (MD) simulations\u003c/h2\u003e \u003cp\u003eFor the validation of the MD protocol, 100 ns MDs were applied to the available humanized drHDAC10 crystal structures (PDB IDs; 7U6B, 7U69, 7U6A, and 7U59, details in the Methods Section). Obtained RMSD plots from the molecular dynamic simulations revealed that the HDAC10-inhibitor structures (Figure S3c, Figure S4c, Figure S5c, and Figure S6c) are stable with an RMSD below 1.5 \u0026Aring; while the Zn ions generally show lower root mean square deviation (RMSD) values (Figure S3d, Figure S4d, Figure S5d and Figure S6d). Meanwhile, the ligand molecules tended to show higher fluctuations (Figure S3a, Figure S4a, Figure S5a, and Figure S6a) which was majorly attributed to the flexibility of the capping groups as observed in the ligand root mean square fluctuations (RMSF) plots (Figure S3b, Figure S4b, Figure S5b and Figure S6b).\u003c/p\u003e \u003cp\u003eWe further performed molecular dynamics simulation studies on the obtained docking pose of the most active compound \u003cb\u003e1\u003c/b\u003e in drHDAC10 to examine the stability of the predicted binding mode. The obtained protein-ligand complex was subjected to 100 ns MD simulation protocol three times using AMBER22, and the obtained trajectories were analyzed. RMSD plots showed that the protein structure and the zinc ion in the complex remained stable during the 100 ns simulation time in all three replicas with RMSD values below 1.5 \u0026Aring; (Figure S2a and Figure S2b). Meanwhile, the ligand RMSD plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) demonstrate that the ligand shows significant deviations from the predicted docking pose. Further analyses were performed to assess the stability of the obtained docking pose of compound \u003cb\u003e1\u003c/b\u003e, and analyze the causes behind the observed fluctuation. Examination of the ligand RMSF plots of compound \u003cb\u003e1\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) demonstrates that the zinc-binding hydroxamate group as well as the linker moiety are stable during the MD simulations with RMSF values\u0026thinsp;\u0026lt;\u0026thinsp;1 \u0026Aring;. It\u0026rsquo;s worth noting that the interactions between the hydroxamate group and the zinc ion are maintained during the MD simulations as shown by the unchanged distances between the hydroxamate-\u003cem\u003eO\u003c/em\u003e atoms and zinc ion (\u0026lt;\u0026thinsp;2.5 \u0026Aring;; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Meanwhile, the indole-capping group displayed significantly high RMSF values that explains the strong deviations observed in the ligand RMSD plots. Clustering the obtained MD trajectories based on the RMSD of the ligand yielded three clusters with an occupancy\u0026thinsp;\u0026gt;\u0026thinsp;10%. As can be demonstrated by the inspection of the obtained clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), the indole capping group can occupy one of two different hydrophobic regions at the rim of the binding pocket: In two clusters (occupancy 55% and 15%, respectively), the capping group is undergoing hydrophobic interactions with I27, A28 and P29. In the third cluster (17% occupancy), the capping group is situated between F204 and W205 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The calculated data show, that the indole-capping group, despite showing strong fluctuations, is well accommodated at the entrance of the lysing binding pocket of HDAC10 where it undergoes hydrophobic interactions with surrounding residues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3 CONCLUSION","content":"\u003cp\u003eIn this study, we synthesized novel bicyclic hydroxamic acid derivatives, characterized their inhibitory activity against class I, and class IIb HDACs. Our findings highlight compound \u003cb\u003e6\u003c/b\u003e as a potent and selective inhibitor of HDAC10, while compound \u003cb\u003e1\u003c/b\u003e exhibited the best inhibitory activity against HDAC10. Compound \u003cb\u003e4\u003c/b\u003e displayed a preference towards HDAC6, and compounds \u003cb\u003e2\u003c/b\u003e and \u003cb\u003e3\u003c/b\u003e demonstrated high selectivity towards class IIb HDACs over class I HDACs.\u003c/p\u003e"},{"header":"4 EXPERIMENTAL","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Chemistry\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e4.1.1 General\u003c/h2\u003e \u003cp\u003eReagents and hydrous or anhydrous organic solvents were purchased from Sigma-Aldrich (Darmstadt, Germany) and Alfa Aesar Chemicals (Tewksbury, MA, USA) and used without further purification. Reaction progressmonitored by thin-layer chromatography (TLC) on pre-coated 0.20mm silica gel GF Uniplates from Macherey-Nagel (D\u0026uuml;ren, Germany). Plates were visualized with a 254 nm UV lamp and by indicators like ninhydrin, potassium permanganate (KMnO\u003csub\u003e4\u003c/sub\u003e), dinitrophenylhydrazine (DNP), green girasol, and anisaldehyde. Column chromatography was performed with 63\u0026ndash;200 \u0026micro;M of 70\u0026ndash;230 mesh silica gel. Nuclear magnetic resonance (NMR) spectra were recorded using a Bruker BB 400 MHz spectrometer. Chemical shifts are reported in ppm (δ) relative to tetramethyl silane (TMS) and coupling constants (\u003cem\u003eJ\u003c/em\u003e) are reported in Hz. Abbreviations for multiplicity are s\u0026thinsp;=\u0026thinsp;singlet d\u0026thinsp;=\u0026thinsp;doublet t\u0026thinsp;=\u0026thinsp;triplet q\u0026thinsp;=\u0026thinsp;quartet dd\u0026thinsp;=\u0026thinsp;doublet of doublets dq\u0026thinsp;=\u0026thinsp;doublet of quartets m\u0026thinsp;=\u0026thinsp;multiplet.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e4.1.2 General procedure for the synthesis of HDACs Inhibitor\u003c/h2\u003e \u003cp\u003e \u003cb\u003eGeneral synthesis methods\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of 1-tosyl-1\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e-indole-3-carbaldehyde (II)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis compound was prepared from 1\u003cem\u003eH\u003c/em\u003e-indole-3-carbaldehyde and \u003cem\u003ep\u003c/em\u003e-toluenesulfonyl chloride, according to the literature procedure [\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e1\u003cem\u003eH\u003c/em\u003e-indole-3-carbaldehyde (\u003cb\u003eI\u003c/b\u003e) (1eq, 3g, 20.7 mmol) was dissolved in (30 ml) anhydrous \u003cem\u003eN\u003c/em\u003e, \u003cem\u003eN\u003c/em\u003e'-Dimethylformamide (DMF). The solution was stirred under nitrogen for 15 min in ice bath. Sodium hydride (NaH) (1.1 eq, 0.55 g, 22.9 mmol) was then added. After 10 minutes (1.4 eq, 5.52 g, 28.8 mmol) 4-methylbenzenesulfonyl chloride was added slowly to the reaction mixture. The reaction was left to stir overnight at room temperature. The reaction was quenched with distilled water (10 ml), and then (10 ml) saturated solution of sodium bicarbonate (NaHCO\u003csub\u003e3\u003c/sub\u003e). The product was extracted three time with (25 ml) dichloromethane (DCM). Evaporate the solvent under vacuum, and the product was recrystallized by using 15 mL DCM and 10 mL hexane. The light yellow crystals were soaked with cold methanol. Yield 75%, m.p 148\u0026ndash;149\u0026deg;C and R\u003csub\u003ef\u003c/sub\u003e 0.88 (Hexane:EtOAc, 1:1). \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e-d\u003csub\u003e1\u003c/sub\u003e, 36\u0026deg;C, TMS): δ 2.36 (s, 3H, Ar-CH\u003csub\u003e3\u003c/sub\u003e), 7.29(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4Hz, 2H, H-12), 7.36(ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 7.3, 2.0Hz, 1H, H-6), 7.42(ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3, 8.2, 1.6Hz, 1H, H-7), 7.87(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4Hz, 2H, H-11), 7.97(dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2, 2.0Hz, 1H, H-8), 8.26(s, 1H, H-2), 8.27(dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 1.6Hz, 1H, H-5), 10.11(s, 1H, CHO).\u003csup\u003e13\u003c/sup\u003eC-NMR (100MHz, CDCl\u003csub\u003e3\u003c/sub\u003e-d\u003csub\u003e1\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 21.7 (Ar-\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e3\u003c/sub\u003e), 113.3 (C-8), 122.4 (C-3), 122.6 (C-5), 125.1 (C-6), 126.30 (C-4), 126.33 (C-7), 127.3 (C-11), 130.4 (C-12), 134.3 (C-9), 135.2 (C-10), 136.3 (C-2), 146.2 (C-13), 185.4 (-\u003cem\u003eC\u003c/em\u003eHO). HRESIMS \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e calcd. for C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003eS: 363.18211, found 300.0700.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneral procedure for preparation of ((1-tosyl-1\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e-indol-3-yl) methyl) amine carboxylate, (quinolin-3-ylmethyl)amine carboxylate or (1\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e-indol-3-yl)methyl)amine carboxylate derivatives (Ia-b, IIa-c, and IIIa-b)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo a solution of 1-tosyl-1\u003cem\u003eH\u003c/em\u003e-indole-3-carbaldehyde (\u003cb\u003eII\u003c/b\u003e) (1 eq, 1.0\u0026ndash;2.0 mmol), 3-quinolinecaboxaldehyde (\u003cb\u003eIII\u003c/b\u003e) (1 eq, 1.5-2.0 mmol) or 1\u003cem\u003eH\u003c/em\u003e-indole-3-carbaldehyde (\u003cb\u003eI\u003c/b\u003e) (1.0 eq, 1.5-3.0 mmol) in anhydrous dichloromethane (25 ml) was added the corresponding amine carboxylate (1.1-2.0 eq, 1.1\u0026ndash;4.5 mmol) with stirring under nitrogen for 2\u0026ndash;4 hours at room temperature. Sodium triacetoxyborohydride (STAB) (1.3-2.0 eq, 1.3\u0026ndash;4.5 mmol) was then added to the mixture in the ice bath, and then it was left to be stirred overnight at room temperature. After that, the mixture was then quenched with distilled water (5 ml), followed by the addition of a saturated solution of sodium bicarbonate (5 ml), then it was extracted with DCM (2 x 20 ml), dried over Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and concentrated under vacuum. Finally, the crude products were purified by column chromatography using silica gel with the appropriate eluent to give the resulting products (\u003cb\u003eIa-b, IIa-c, and IIIa-b\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of ethyl 4-((((1\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e-indol-3-yl)methyl)amino)methyl)benzoate (Ia)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis compound was prepared from 1\u003cem\u003eH\u003c/em\u003e-indole-3-carbaldehyde (\u003cb\u003eI\u003c/b\u003e) (1.0 eq, 0.43 g, 3.0 mmol), Ethyl 4-(aminomethyl)benzoate (1.1 eq, 0.59 g, 3.3 mmol) and (1.5 eq, 0.95 g, 4.5 mmol) of sodium triacetoxyborohydride (STAB) by following the general procedure and reaction conditions as described above. The crude product was purified by column chromatography using silica gel to obtain white crystals (CHCl\u003csub\u003e3\u003c/sub\u003e:MeOH:EtOAc 20:1:1). Yield 42%, m.p\u0026thinsp;=\u0026thinsp;90\u0026ndash;91\u0026deg;C, R\u003csub\u003ef\u003c/sub\u003e = 0.30 (CHCl\u003csub\u003e3\u003c/sub\u003e:MeOH:EtOAc 20:1:1). \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e-d\u003csub\u003e1\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 1.44(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1Hz, 3H, -OCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 3.96(s, 2H, H-CH\u003csub\u003e2\u003c/sub\u003eN-benzylic), 4.04(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.8Hz, 2H, H-CH\u003csub\u003e2\u003c/sub\u003eN-pyrrol), 4.42(q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1Hz, 2H, -OCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 7.13(m, 1H, H-2), 7.17(ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 7.0, 1.1Hz, 1H, H-6), 7.24(ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1, 7.0, 1.1Hz, 1H, H-7), 7.37(dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1, 1.1Hz, 1H, H-8), 7.47(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3Hz, 2H, H-11), 7.69(dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 1.1, 1H, H-5), 8.06(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3Hz, 2H, H-12), 8.45(s, 1H, H1-NH).. \u003csup\u003e13\u003c/sup\u003eC-NMR (100MHz, CDCl\u003csub\u003e3\u003c/sub\u003e-d\u003csub\u003e1\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 14.4 (OCH\u003csub\u003e2\u003c/sub\u003e\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e3\u003c/sub\u003e), 44.2 (C-\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003eN-pyrrol), 52.9 (C-\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003eN-benzylic), 61.0 (O\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 111.3 (C-8), 114.4 (C-3), 118.9 (C-5), 119.6 (C-6), 122.2 (C-7), 122.9 (C-2), 127.1 (C-4), 128.1 (C-11), 129.2 (C-13), 129.7 (C-12), 136.5 (C-9), 145.7 (C-10), 166.7 (C\u0026thinsp;=\u0026thinsp;O). HRESIMS \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e [M\u0026thinsp;+\u0026thinsp;Hac-H]\u003csup\u003e+\u003c/sup\u003e calcd. for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e367.16633, found 367.25930. [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e calcd. for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 309.15975, found 309.15850.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of ethyl 4-(4-((1\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e-indol-3-yl)methyl)piperazin-1-yl)benzoate (Ib)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis compound was prepared from 1\u003cem\u003eH\u003c/em\u003e-indole-3-carbaldehyde (\u003cb\u003eI\u003c/b\u003e) (1.0 eq, 0.43 g, 3.0 mmol), ethyl 4-(1-piperazinyl)benzoate (1.2 eq, 0.84 g, 3.6 mmol) and (1.3 eq, 0.83 g, 3.9 mmol) of sodium triacetoxyborohydride (STAB) by following the general procedure and reaction conditions as described above. The crude product was purified by column chromatography using silica gel to obtain white crystals (CHCl\u003csub\u003e3\u003c/sub\u003e:MeOH, 10:1). Yield 73%, m.p\u0026thinsp;=\u0026thinsp;175\u0026ndash;176\u0026deg;C, R\u003csub\u003ef\u003c/sub\u003e = 0.50 (CHCl\u003csub\u003e3\u003c/sub\u003e: MeOH, 10:1). \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e-d\u003csub\u003e1\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 1.41(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1Hz, 3H, -OCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 2.69(dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.7, 3.1Hz, 4H, H-10), 3.35(dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.7, 2.8Hz, 4H, H-11), 3.82(s, 2H, H-CH\u003csub\u003e2\u003c/sub\u003eN), 4.37(q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1Hz, 2H, -OCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 6.86(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.1Hz, 2H, H-13), 7.17(m, 1H, H-2), 7.18(m, 1H, H-6), 7.25(m, 1H, H-7), 7.38(dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0, 1.2Hz, 1H, H-8), 7.80(dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 1.2Hz, 1H, H-5), 7.96(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.1Hz, 2H, H-14), 8.47(s, 1H, -NH).. \u003csup\u003e13\u003c/sup\u003eC-NMR (100MHz, CDCl\u003csub\u003e3\u003c/sub\u003e-d\u003csub\u003e1\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 14.5(OCH\u003csub\u003e2\u003c/sub\u003e\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e3\u003c/sub\u003e), 47.5 (C-11), 52.7(C-10), 53.6(C-\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003eN), 60.4 (O\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 111.2(C-8), 112.0(C-3), 113.6 (C-13), 119.5 (C-5), 119.6(C-6), 119.9(C-15), 122.10(C-7), 124.0(C-2), 128.0(C-4), 131.2(C-14), 136.3(C-9), 154.2(C-12), 166.9(C\u0026thinsp;=\u0026thinsp;O). HRESIMS \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e calcd. for C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 364.20195, found 364.20500. [M-H]\u003csup\u003e+\u003c/sup\u003e calcd. for C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 362.18740, found 362.18320 .\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of ethyl 4-((((1-tosyl-1\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e-indol-3-yl)methyl)amino)methyl)benzoate IIa)\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eThis compound was prepared from 1-tosyl-1\u003cem\u003eH\u003c/em\u003e-indole-3-carbaldehyde (\u003cb\u003eII\u003c/b\u003e) (1.0 eq, 0.45 g, 1.50 mmol), ethyl 4-(aminomethyl)benzoate (1.1 eq, 0.30 g, 1.65 mmol) and (1.5 eq, 0.48 g, 2.25 mmol) of sodium triacetoxyborohydride (STAB) by following the general procedure and reaction conditions as described above. The crude product was purified by column chromatography using silica gel to obtain white crystals (CHCl\u003csub\u003e3\u003c/sub\u003e:MeOH 98:2). Yield 45%, m.p\u0026thinsp;=\u0026thinsp;110\u0026ndash;112\u0026deg;C, R\u003csub\u003ef\u003c/sub\u003e = 0.54 (CHCl\u003csub\u003e3\u003c/sub\u003e:MeOH, 98:2). \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e-d\u003csub\u003e1\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 1.42(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1Hz, 3H, -OCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 2.33(s, 3H, Ar-CH3), 3.89(s, 2H, H-16), 3.92(s, 2H, H-14), 4.40(q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1Hz, 2H, -OCH2CH3), 7.21(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2Hz, 2H, H-12), 7.27(ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2, 7.8, 1.0Hz, 1H, H-7), 7.34(ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2, 7.3, 1.3Hz, 1H, H-6), 7.43(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2Hz, 2H, H-18), 7.53(s, 1H, H-2), 7.58(dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 1.3,, 1H, H-8), 7.78(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2Hz, 2H, H-11), 8.02(m,1H, H-5), 8.04(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2Hz, 2H, H-19).\u003csup\u003e13\u003c/sup\u003eC-NMR (100MHz, CDCl\u003csub\u003e3\u003c/sub\u003e-d\u003csub\u003e1\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 14.4(OCH\u003csub\u003e2\u003c/sub\u003e\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e3\u003c/sub\u003e), 21.6( Ar-\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e3\u003c/sub\u003e), 43.9(C-14), 53.0(C-16), 60.90(O\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 113.8(C-5), 119.80(C-8), 121.3(C-3), 123.2 (C-7), 123.9(C-2), 124.9(C-6), 126.80(C-11), 128.0(C-18), 129.3(C-20), 129.8(C-19), 129.9(C-12), 130.3(C-4), 135.3(C-10), 135.5 (C-9), 144.9(C-13), 145.3(C-17), 166.6(C\u0026thinsp;=\u0026thinsp;O). HRESIMS \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e [2M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e calcd. for C\u003csub\u003e52\u003c/sub\u003eH\u003csub\u003e53\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e 925.32994, found 925.33900. [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e calcd for C\u003csub\u003e26\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eNaN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eS 485.15055, found 485.15390.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of ethyl 4-(((1-tosyl-1\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e-indol-3-yl) methyl) amino) benzoate (IIb)\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eThis compound was prepared from 1-tosyl-1\u003cem\u003eH\u003c/em\u003e-indole-3-carbaldehyde (\u003cb\u003eII\u003c/b\u003e) (1.0 eq, 0.45 g, 1.50 mmol), Ethyl 4-aminobenzoate (1.2 eq, 0.30 g, 1.8 mmol) and (2 eq, 0.63 g, 2.0 mmol) of sodium triacetoxyborohydride (STAB) by following the general procedure and reaction conditions as described above. The crude product was purified by column chromatography using silica gel to obtain white crystals (CHCl\u003csub\u003e3\u003c/sub\u003e:EtOAc, 4:1). Yield 85%, 154-155C\u0026deg;, R\u003csub\u003ef\u003c/sub\u003e = 0.85 (CHCl\u003csub\u003e3\u003c/sub\u003e: EtOAc, 4 :1). \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e-d\u003csub\u003e1\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 1.40(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1Hz, 3H, -OCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 2.35(s, 3H, Ar-CH\u003csub\u003e3\u003c/sub\u003e), 4.35(q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1Hz, 2H, -OCH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 4.47(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.2Hz, 2H, -NCH\u003csub\u003e2\u003c/sub\u003e), 6.60(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.7Hz, 2H, H-15), 7.19(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4Hz, 2H, H-12), 7.26(ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5, 7.7, 1.0Hz, 1H, H-7), 7.36(ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5, 7.2, 1.3Hz, 1H, H-6), 7.50(s, 1H, H-2), 7.53(m, 1H, H-8), 7.70(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4Hz, 2H, H-11), 7.89(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.7Hz, 2H, H-16), 8.04(m, 1H, H-5). \u003csup\u003e13\u003c/sup\u003eC-NMR (100MHz, CDCl\u003csub\u003e3\u003c/sub\u003e-d\u003csub\u003e1\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 14.5 (OCH\u003csub\u003e2\u003c/sub\u003e\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e3\u003c/sub\u003e), 21.6(Ar-\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e3\u003c/sub\u003e), 39.2(N-CH\u003csub\u003e2\u003c/sub\u003e), 60.3(O\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 111.8(C-15), 114.0(C-5), 119.2 (C-17), 119.5 (C-8), 119.7(C-3), 123.4(C-7), 124.3(C-2), 125.1(C-6), 126.8(C-11), 129.6(C-4), 129.9 (C-12), 131.5(C-16), 135.960(C-10), 135.6(C-9), 145.1(C-13), 151.5(C-14), 166.9(C\u0026thinsp;=\u0026thinsp;O). HRESIMS \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e [M\u0026thinsp;+\u0026thinsp;Cl]\u003csup\u003e+\u003c/sup\u003e calcd for C\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eClN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eS 483.11458, found 483.10730.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of ethyl 4-(4-((1-tosyl-1\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e-indol-3-yl) methyl) piperazin-1-yl) benzoate (IIc)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis compound was prepared from 1-tosyl-1\u003cem\u003eH\u003c/em\u003e-indole-3-carbaldehyde (\u003cb\u003eII\u003c/b\u003e) (1.0 eq, 0.45 g, 1.50 mmol), ethyl 4-(1-piperazinyl)benzoate (1.2 eq, 0.42 g, 1.8 mmol) and (1.3 eq, 0.41 g, 1.95 mmol) of sodium triacetoxyborohydride (STAB) by following the general procedure and reaction conditions as described above. The crude product was purified by column chromatography using silica gel to obtain white crystals (CHCl\u003csub\u003e3\u003c/sub\u003e:EtOAc, 3:1). Yield 78%, m.p\u0026thinsp;=\u0026thinsp;166\u0026ndash;167\u0026deg;C, R\u003csub\u003ef\u003c/sub\u003e = 0.62 (CHCl\u003csub\u003e3\u003c/sub\u003e:EtOAc, 3:1). \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e-d\u003csub\u003e1\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 1.39(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1Hz, 3H, -OCH\u003csub\u003e2\u003c/sub\u003e\u003cem\u003eCH\u003c/em\u003e\u003csub\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sub\u003e), 2.33(s, 3H, Ar-\u003cem\u003eCH\u003c/em\u003e\u003csub\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sub\u003e), 2.60(m, 4H, H-14), 3.31(m, 4H, H-15), 3.69(s, 2H, H-NC\u003cem\u003eH\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e), 4.36(q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1Hz, 2H, -O\u003cem\u003eCH\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 6.86(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.1Hz, 2H, H-17), 7.21(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4Hz, 2H, H-12), 7.25(m, 1H, H-7), 7.35(ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4, 7.2, 1.3Hz, 1H, H-6), 7.56(s, 1H, H-2), 7.74(dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 1.3Hz, 1H, H-8), 7.80(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4Hz, 2H, H-11), 7.96(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.1Hz, 2H, H-18), 8.04(dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4, 1.0Hz, 1H, H-5).. \u003csup\u003e13\u003c/sup\u003eC-NMR (100MHz, CDCl\u003csub\u003e3\u003c/sub\u003e-d1, 36\u0026deg;C, TMS) δ 14.5(OCH\u003csub\u003e2\u003c/sub\u003e\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e3\u003c/sub\u003e), 21.6(Ar-\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e3\u003c/sub\u003e), 47.5(C-15), 52.8(C-14), 53.4(C-N\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003e), 60.4(O\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 113.6(C-17), 113.7(C-5), 119.2(C-19), 120.0(C-3), 120.6 (C-8), 123.2 (C-7), 124.9(C-2), 124.9(C-6), 126.8 (C-11), 129.9 (C-12), 130.8 (C-4), 131.2(C-18), 135.3(C-10), 135.5(C-9), 145.0(C-13), 154.1(C-16), 166.7 (C\u0026thinsp;=\u0026thinsp;O). HRESIMS \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e calcd. for C\u003csub\u003e29\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eS 518.21136, found 518.22210. [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e calcd for C\u003csub\u003e29\u003c/sub\u003eH\u003csub\u003e31\u003c/sub\u003eNaN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eS 540.19275, found 540.19350.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of ethyl 4-((quinolin-3-ylmethyl)amino)benzoate (IIIa)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis compound was prepared from 3-quinolinecaboxaldehyde (\u003cb\u003eIII\u003c/b\u003e) (1 eq, 0.314 g, 2.0mmol), ethyl 4-aminobenzoate (1.1 eq, 0.36 g, 2.2 mmol) and (1.5 eq, 0.64 g, 3.0 mmol) of sodium triacetoxyborohydride (STAB) by following the general procedure and reaction conditions as described above [\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The crude product was purified by column chromatography using silica gel to obtain white crystals (CHCl\u003csub\u003e3\u003c/sub\u003e:EtOAc, 4:1). Yield 66%, m.p\u0026thinsp;=\u0026thinsp;135\u0026ndash;136\u0026deg;C, R\u003csub\u003ef\u003c/sub\u003e = 0.38(CHCl\u003csub\u003e3\u003c/sub\u003e:EtOAc, 4:1). \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e-d\u003csub\u003e1\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 1.35(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1Hz, 3H, -OCH\u003csub\u003e2\u003c/sub\u003e\u003cem\u003eCH\u003c/em\u003e\u003csub\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sub\u003e), 4.31(q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1Hz, 2H, -O\u003cem\u003eCH\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 4.58(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.5Hz, 2H, -N\u003cem\u003eCH\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e), 4.92(s, br, \u003cem\u003e-\u003c/em\u003eN\u003cem\u003eH\u003c/em\u003e), 6.64(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8Hz, 2H, H-12), 7.55(ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1, 6.9, 1.2Hz, 1H, H-7), 7.71(ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4, 6.9, 1.5Hz, 1H, H-8), 7.76(dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1, 1.5Hz, 1H, H-6), 7.89(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8Hz, 2H, H-13), 8.07(dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.2, 1.0Hz, 1H, H-4), 8.11(dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5, 1.0, 1.0Hz, 1H, H-9), 8.90(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.2Hz, 1H, H-2). \u003csup\u003e13\u003c/sup\u003eC-NMR (100MHz, CDCl\u003csub\u003e3\u003c/sub\u003e-d\u003csub\u003e1\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 14.5(OCH\u003csub\u003e2\u003c/sub\u003e\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e3\u003c/sub\u003e), 45.4(-N\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003e), 60.3(O\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 111.8(C-12), 119.5 (C-14), 127.1(C-7), 127.7(C-6), 127.9(C-5), 129.1 (C-9), 129.2(C-8), 131.3(C-3), 131.6(C-13), 134.1 (C-4), 147.5(C-10), 150.3(C-2), 151.3(C-11), 166.8(C\u0026thinsp;=\u0026thinsp;O). HRESIMS \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e calcd for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 307.14410, found 307.14730.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of ethyl 4-(4-(quinolin-3-ylmethyl)piperazin-1-yl)benzoate (IIIb)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis compound was prepared from 3-quinolinecaboxaldehyde (\u003cb\u003eIII\u003c/b\u003e) (1 eq, 0.236 g, 1.50 mmol), ethyl 4-(1-piperazinyl)benzoate (1.2 eq, 0.42 g, 1.8 mmol), and (1.3 eq, 0.42 g, 1.95 mmol) of sodium triacetoxyborohydride (STAB) by following the general procedure and reaction conditions as described above. The crude product was purified by column chromatography using silica gel to obtain white crystals (CHCl\u003csub\u003e3\u003c/sub\u003e:MeOH, 94:6). Yield 63%, m.p\u0026thinsp;=\u0026thinsp;153\u0026ndash;155\u0026deg;C, R\u003csub\u003ef\u003c/sub\u003e = 0.68 (CHCl\u003csub\u003e3\u003c/sub\u003e:MeOH, 94:6). \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e-d\u003csub\u003e1\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 1.34(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1Hz, 3H, -OCH\u003csub\u003e2\u003c/sub\u003e\u003cem\u003eCH\u003c/em\u003e\u003csub\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sub\u003e), 2.60(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.0Hz, 4H, H-11), 3.29(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.0Hz, 4H, H-12), 3.69(s, 2H, C\u003cem\u003eH\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003eN), 4.30(q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1Hz, 2H, -O\u003cem\u003eCH\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 6.81(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.7Hz, 2H, H-14), 7.52(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3Hz, 1H, H-7), 7.68(ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4, 6.6, 1.4Hz, 1H, H-8), 7.79(dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3, 1.4Hz, 1H, H-6), 7.91(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.7Hz, 2H, H-15), 8.05(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.1Hz, 1H, H-4), 8.11(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4Hz, 1H, H-9), 8.92(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.1Hz, 1H, H-2). \u003csup\u003e13\u003c/sup\u003eC-NMR (100MHz, CDCl\u003csub\u003e3\u003c/sub\u003e-d\u003csub\u003e1\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 14.5(OCH\u003csub\u003e2\u003c/sub\u003e\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e3\u003c/sub\u003e), 47.5(C-12), 52.8(C-11), 60.3(O\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 60.4(N\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003e), 113.6(C-14), 120.1(C-16), 126.8(C-7), 127.6(C-6), 127.9(C-5), 129.2(C-8), 129.2(C-9), 130.7(C-3), 131.1(C-15), 135.7(C-4), 147.6(C-10), 152.0(C-2), 154.0(C-13), 166.6 (C\u0026thinsp;=\u0026thinsp;O). HRESIMS \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e calcd. for C\u003csub\u003e23\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 376.20195, found 376.20380.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneral procedure for preparation of corresponding hydroxamic acid derivatives of (1\u0026ndash;7).\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo a solution of benzoate derivatives of (\u003cb\u003eIa-b, Iia-c and IIIa-b\u003c/b\u003e) (1 eq, 0.27\u0026ndash;0.45 mmol) in anhydrous dichloromethane and methanol (1:2, 6 ml) at 0\u0026deg;C, hydroxylamine (50 wt % in water, 7.99\u0026ndash;13.37 mmol, 30 eq) was added, followed by the addition of sodium hydroxide (1.08\u0026ndash;2.25 mmol, 4.0\u0026ndash;5.0 eq). The reaction mixture was allowed to warm to room temperature and stirred for 24 h. Then the solvent was removed under reduced pressure, and the obtained solid was dissolved in water. The pH was adjusted to 7 with a 1N HCl. The resulting precipitate was filtered and dried under a high vacuum, and then the crude products were purified by column chromatography using silica gel with the appropriate eluent or soaking with dichloromethane to give the resulting products (\u003cb\u003e1\u0026ndash;7\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of 4-((((1\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e-indol-3-yl)methyl)amino)methyl)-N-hydroxybenzamide (1)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis compound was prepared from ethyl 4-((((1\u003cem\u003eH\u003c/em\u003e-indol-3-yl)methyl)amino)methyl)benzoate (\u003cb\u003eIa\u003c/b\u003e) (0.10 g, 0.32 mmol, 1 eq), hydroxylamine (50 wt % in water, 0.64 ml, 0.32 g, 9.60 mmol, 30 eq) and sodium hydroxide (0.05 g, 1.28 mmol, 4.0 eq) by following the general procedure and reaction conditions as described above. The crude product was purified by soaking with dichloromethane to afford 4-((((1\u003cem\u003eH\u003c/em\u003e-indol-3-yl)methyl)amino)methyl)-\u003cem\u003eN\u003c/em\u003e-hydroxybenzamide \u003cb\u003e(1)\u003c/b\u003e as white crystals. Yield 52%, m.p\u0026thinsp;=\u0026thinsp;158\u0026ndash;160\u0026deg;C. \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 3.80(s, 2H, H-C\u003cem\u003eH\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003eN-benzylic), 3.86(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.8Hz, 2H, H-C\u003cem\u003eH\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003eN-pyrrol), 6.99(s, 1H, H-2), 7.08(m, 1H, H-6), 7.25(m, 1H, H-7), 7.36(m, 1H, H-8), 7.45(m, 2H, H-11), 7.61(m, 1H, H-5), 7.74(m, 2H, H-12), 10.91(s, 1H, -OH). \u003csup\u003e13\u003c/sup\u003eC-NMR (101 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 44.21(C-\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003eN-pyrrol), 52.42(C-\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003eN-benzylic), 111.8 (C-8), 113.9(C-3), 118.7(C-2), 119.3(C-5), 121.4(C-6), 124.0(C-7), 127.2(C-12), 127.5(C-4), 128.3(C-11), 131.5(C-13), 136.86(C-9), 144.85(C-10), 164.7(C\u0026thinsp;=\u0026thinsp;O). HRESIMS \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e 294.12507[M-H]\u003csup\u003e+\u003c/sup\u003e (calc for C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 294.12425), 130.06530 [M-C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e9\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e (calc for C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eN, 130.06567).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of N-hydroxy-4-((((1-tosyl-1\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e-indol-3-yl)methyl)amino)methyl) benzamide (2)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis compound was prepared from ethyl 4-((((1-tosyl-1\u003cem\u003eH\u003c/em\u003e-indol-3-yl)methyl)amino) methyl)benzoate (\u003cb\u003eIIa\u003c/b\u003e) (0.20g, 0.43mmol, 1eq), hydroxylamine (50 wt % in water, 0.85ml, 0.42g, 12.90mmol, 30eq) and sodium hydroxide (0.086 g, 2.15 mmol, 5.0 eq) by following the general procedure and reaction conditions as described above. The crude product was purified by using silica gel for column chromatography to afford \u003cem\u003eN\u003c/em\u003e-hydroxy-4-((((1-tosyl-1\u003cem\u003eH\u003c/em\u003e-indol-3-yl)methyl)amino)methyl)benzamide \u003cb\u003e(2)\u003c/b\u003e as light-pink crystals (CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e:MeOH 987:13)). Yield 37%, m.p\u0026thinsp;=\u0026thinsp;151\u0026ndash;153\u0026deg;C, R\u003csub\u003ef\u003c/sub\u003e = 0.49 (CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e:MeOH (87:13)). \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 2.28(s, 3H, Ar-\u003cem\u003eCH\u003c/em\u003e\u003csub\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sub\u003e), 3.75(s, 2H, -N\u003cem\u003eCH\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e-16), 3.80(s, 2H, -N\u003cem\u003eCH\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e-14), 7.25(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5Hz, 7.5Hz, 1H, H-7), 7.34(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9Hz, 2H, H-12), 7.35(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4Hz, 1H, H-6), 7.41(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8Hz, 2H, H-18), 7.65(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5Hz, 1H, H-8), 7.67(s, 1H, H-2), 7.75(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8Hz, 2H, H-19), 7.83(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9Hz, 2H, H-11), 7.93(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2Hz, 1H, H-5), 9.04(brs, 1H, -((CO)N\u003cem\u003eH\u003c/em\u003e), 11.17(brs, 1H, -O\u003cem\u003eH\u003c/em\u003e). \u003csup\u003e13\u003c/sup\u003eC-NMR (101 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 21.5( Ar-\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e3\u003c/sub\u003e), 43.5(N-\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003e-14), 52.4(N-\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003e-16), 113.7(C-5), 120.8(C-8), 122.5(C-3), 123.7(C-7), 124.5(C-2), 125.2(C-6), 127.1(C-11), 127.3(C-19), 128.3(C-18), 130.6(C-12), 130.6(C-20), 131.6(C-4), 134.6(C-10), 135.2(C-9), 144.5(C-13), 145.8(C-17), 164.6(C\u0026thinsp;=\u0026thinsp;O). HRESIMS \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e [M-H]\u003csup\u003e+\u003c/sup\u003e calcd. for C\u003csub\u003e24\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eS 448.13365, found 448.13420. [2M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e calcd for C\u003csub\u003e48\u003c/sub\u003eH\u003csub\u003e45\u003c/sub\u003eN\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e 897.27403, found 897.27510.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of N-hydroxy-4-(((1-tosyl-1\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e-indol-3-yl)methyl)amino)benzamide\u003c/b\u003e (\u003cb\u003e3\u003c/b\u003e)\u003c/p\u003e \u003cp\u003eThis compound was prepared from ethyl 4-(((1-tosyl-1\u003cem\u003eH\u003c/em\u003e-indol-3-yl)methyl)amino)benzoate (\u003cb\u003eIib\u003c/b\u003e) (0.20 g, 0.45 mmol, 1.0 eq), hydroxylamine (50 wt % in water, 0.88 ml, 0.44 g, 13.37 mmol, 30 eq) and sodium hydroxide (0.09 g, 2.25 mmol, 5.0 eq) by following the general procedure and reaction conditions as described above. The crude product was purified by using silica gel for column chromatography to afford N-hydroxy-4-(((1-tosyl-1\u003cem\u003eH\u003c/em\u003e-indol-3-yl)methyl)amino)benzamide (\u003cb\u003e3\u003c/b\u003e) as light-pink crystals (CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e:MeOH(94:6)). Yield 34%, m.p\u0026thinsp;=\u0026thinsp;183\u0026ndash;184\u0026deg;C, R\u003csub\u003ef\u003c/sub\u003e = 0.32 (CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e:MeOH(94:6)). \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 2.29(s, 3H, Ar-\u003cem\u003eCH\u003c/em\u003e\u003csub\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sub\u003e), 4.43(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.6Hz, 2H, -N\u003cem\u003eCH\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e), 6.65(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4Hz, 2H, H-15), 6.71(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.2Hz, 1H, -N\u003cem\u003eH-\u003c/em\u003eCH\u003csub\u003e2\u003c/sub\u003e), 7.26(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5Hz, 1H, H-7), 7.33(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2Hz, 2H, H-12), 7.33(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2Hz, 1H, H-6), 7.53(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3Hz, 2H, H-16), 7.72(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5Hz, 1H, H-8), 7.75(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3Hz, 2H, H-11), 7.77(s, 1H, H-2), 7.91(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2Hz, 1H, H-5), 8.72(brs, 1H, -((CO)N\u003cem\u003eH\u003c/em\u003e), 10.81(brs, 1H, -O\u003cem\u003eH\u003c/em\u003e). \u003csup\u003e13\u003c/sup\u003eC-NMR (101 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 21.46( Ar-\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e3\u003c/sub\u003e), 38.19(N-\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003e), 111.85(C-15), 113.76(C-5), 120.00(C-17), 120.87(C-8), 121.16(C-3), 123.79(C-7), 125.12(C-2), 125.37(C-6), 127.04(C-11), 128.63(C-16), 130.26(C-4), 130.64(C-12), 134.44(C-10), 135.24(C-9), 145.82(C-13), 151.27(C-14). HRESIMS \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e [M\u0026thinsp;+\u0026thinsp;Na]\u003csup\u003e+\u003c/sup\u003e calcd for C\u003csub\u003e23\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eNaO\u003csub\u003e4\u003c/sub\u003eS 458.11505, found 458.1156. [2M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e calcd. for C\u003csub\u003e46\u003c/sub\u003eH\u003csub\u003e43\u003c/sub\u003eN\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003e 871.25839, found 871.26100.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of\u003c/b\u003e \u003cb\u003eN\u003c/b\u003e\u003cb\u003e-hydroxy-4-((quinolin-3-ylmethyl)amino)benzamide\u003c/b\u003e (\u003cb\u003e4\u003c/b\u003e)\u003c/p\u003e \u003cp\u003eThis compound was prepared from ethyl 4-((quinolin-3-ylmethyl)amino)benzoate (\u003cb\u003eIIIa\u003c/b\u003e) (0.10 g, 0.32 mmol, 1 eq), hydroxylamine (50 wt % in water, 0.63 ml, 0.32 g, 9.60 mmol, 30 eq) and sodium hydroxide (0.05 g, 1.28 mmol, 4.0 eq) by following the general procedure and reaction conditions as described above. The crude product was purified by soaking with dichloromethane to afford N-hydroxy-4-((quinolin-3-ylmethyl)amino)benzamide (\u003cb\u003e4\u003c/b\u003e) as white crystals. Yield 53%, m.p\u0026thinsp;=\u0026thinsp;201\u0026ndash;203\u0026deg;C. \u003csup\u003e1\u003c/sup\u003eH- NMR (400 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 4.56(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0Hz, 2H, -N\u003cem\u003eCH\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e), 6.65(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4Hz, 2H, H-12), 6.94(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0Hz, 1H, -N\u003cem\u003eH-\u003c/em\u003eCH\u003csub\u003e2\u003c/sub\u003e), 7.53(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4Hz, 2H, H-13), 7.59(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5Hz, 1H, H-7), 7.73(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4Hz, 1H, H-8), 7.94(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1Hz, 1H, H-6), 8.01(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4Hz, 1H, H-9), 8.25(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.2Hz, 1H, H-4), 8.69(brs, 1H, -N\u003cem\u003eH\u003c/em\u003e), 8.93(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.2Hz, 1H, H-2), 10.77(s, 1H, -O\u003cem\u003eH\u003c/em\u003e). \u003csup\u003e13\u003c/sup\u003eC-NMR (101 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 44.4(-N\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003e), 111.9(C-12), 120.3(C-14), 127.3(C-7), 128.0(C-5), 128.3(C-6), 128.8(C-13), 129.2(C-9), 129.6(C-8), 133.1(C-3), 133.9(C-4), 147.3(C-10), 151.2(C-11), 151.4(C-2), 165.4(C\u0026thinsp;=\u0026thinsp;O). HRESIMS \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e [M-H]\u003csup\u003e+\u003c/sup\u003e calcd. for C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 292.10860, found 292.10840.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of 4-(4-((1\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e-indol-3-yl)methyl)piperazin-1-yl)-\u003c/b\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-hydroxybenzamide (5)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis compound was prepared from ethyl 4-(4-((1\u003cem\u003eH\u003c/em\u003e-indol-3-yl)methyl)piperazin-1-yl)benzoate (\u003cb\u003eIb\u003c/b\u003e) (0.10 g, 0.27 mmol, 1 eq), hydroxylamine (50 wt % in water, 0.52 ml, 0.26 g, 8.25 mmol, 30 eq) and sodium hydroxide (0.04 g, 1.08 mmol, 4.0 eq) by following the general procedure and reaction conditions as described above. The crude product was purified by soaking with dichloromethane to afford 4-(4-((1\u003cem\u003eH\u003c/em\u003e-indol-3-yl)methyl)piperazin-1-yl)-\u003cem\u003eN\u003c/em\u003e-hydroxybenzamide \u003cb\u003e(5)\u003c/b\u003e as white crystals. Yield 53%, m.p\u0026thinsp;=\u0026thinsp;155\u0026ndash;157\u0026deg;C. \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 2.53(m, 4H, H-10), 3.20(m, 4H, H-11), 3.72(s, 2H, H-C\u003cem\u003eH\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003eN), 6.87(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.9Hz, 2H, H-13), 7.00(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4Hz, 1H, H-6), 7.08(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5Hz, 1H, H-7), 7.27(s, 1H, H-2), 7.37(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0Hz, 1H, H-8), 7.57(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3Hz, 2H, H-14), 7.62(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9Hz, 1H, H-5), 8.81(brs, 1H, -(N\u003cem\u003eH\u003c/em\u003e)C\u0026thinsp;=\u0026thinsp;O), 10.93(s, 1H, NH(Ar)), 10.96(s, 1H, OH). \u003csup\u003e13\u003c/sup\u003eC-NMR (101 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 47.7(C-11), 52.7(C-10), 53.6(C-\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003eN), 110.9(C-3), 111.8(C-8), 114.1(C-13), 118.9(C-6), 119.5(C-5), 121.4(C-7), 122.1(C-15), 125.2(C-2), 128.1(C-4), 128.5(C-14), 136.7 (C-9), 153.2(C-12). HRESIMS \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e [M-H]\u003csup\u003e+\u003c/sup\u003e calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 349.16700, found 349.16776. [M\u0026thinsp;+\u0026thinsp;Cl\u003csup\u003e35\u003c/sup\u003e]\u003csup\u003e+\u003c/sup\u003e calcd for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eClN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 385.14368, found 385.14424.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of\u003c/b\u003e \u003cb\u003eN\u003c/b\u003e\u003cb\u003e-hydroxy-4-(4-((1-tosyl-1\u003c/b\u003e\u003cb\u003eH\u003c/b\u003e\u003cb\u003e-indol-3-yl)methyl)piperazin-1-yl)benzamide (6)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis compound was prepared from ethyl 4-(4-((1-tosyl-1\u003cem\u003eH\u003c/em\u003e-indol-3-yl)methyl)piperazin-1-yl)benzoate (\u003cb\u003eIIc\u003c/b\u003e) (0.15 g, 0.29 mmol, 1 eq), hydroxylamine (50 wt % in water, 0.57 ml, 0.287 g, 8.7 mmol, 30 eq) and sodium hydroxide (0.058 g, 1.45 mmol, 5.0 eq) by following the general procedure and reaction conditions as described above. The crude product was purified by using silica gel for column chromatography to afford \u003cem\u003eN\u003c/em\u003e-hydroxy-4-(4-((1-tosyl-1\u003cem\u003eH\u003c/em\u003e-indol-3-yl)methyl)piperazin-1-yl)benzamide (\u003cb\u003e6\u003c/b\u003e) as light-pink crystals (CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e:MeOH(90:10)). Yield 53%, m.p\u0026thinsp;=\u0026thinsp;180\u0026ndash;182\u0026deg;C, R\u003csub\u003ef\u003c/sub\u003e = 0.43 (CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e:MeOH(90:10)). \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 2.26(s, 3H, Ar-\u003cem\u003eCH\u003c/em\u003e\u003csub\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sub\u003e), 2.49(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2Hz, 4H, H-14), 3.20(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.8Hz, 4H, H-15), 3.65(s, 2H, H-C\u003cem\u003eH\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003eN), 6.91(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5Hz, 2H, H-17), 7.25(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4Hz, 1H, H-7), 7.33(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9Hz, 2H, H-12), 7.33(t \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6Hz, 1H, H-6), 7.68(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3Hz, 2H, H-18), 7.72(s, 1H, H-2), 7.73(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.9Hz, 1H, H-8), 7.84(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8Hz, 2H, H-11), 7.94(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2Hz, 1H, H-5), 8.86(brs, 1H, -N\u003cem\u003eH\u003c/em\u003e), 10.97(brs, 1H, -O\u003cem\u003eH\u003c/em\u003e). \u003csup\u003e13\u003c/sup\u003eC-NMR (101 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 21.4( Ar-\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e3\u003c/sub\u003e), 47.6(C-15), 52.7(C-\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003eN,14), 113.7(C-5), 114.2(C-17), 119.8(C-19), 121.2(C-8), 122.2(C-3), 123.8(C-7), 125.3(C-2), 125.7(C-6), 127.1(C-11), 128.6(C-18), 130.6(C-12), 131.1(C-4), 135.2(C-10), 135.6(C-9), 145.8 (C-13), 153.2(C-16), 164.8(C\u0026thinsp;=\u0026thinsp;O). HRESIMS \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e. [M\u0026thinsp;+\u0026thinsp;Cl\u003csup\u003e35\u003c/sup\u003e]\u003csup\u003e+\u003c/sup\u003e calcd for C\u003csub\u003e27\u003c/sub\u003eH\u003csub\u003e29\u003c/sub\u003eClN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eS 539.15203, found 539.15300.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of\u003c/b\u003e \u003cb\u003eN\u003c/b\u003e\u003cb\u003e-hydroxy-4-{4-[(quinolin-3-yl)methyl]piperazin-1-yl}benzamide\u003c/b\u003e (\u003cb\u003e7\u003c/b\u003e)\u003c/p\u003e \u003cp\u003eThis compound was prepared from ethyl 4-(4-(quinolin-3-ylmethyl)piperazin-1-yl)benzoate (\u003cb\u003eIIIb\u003c/b\u003e) (0.10 g, 0.27 mmol, 1 eq), hydroxylamine (50 wt % in water, 0.52 ml, 0.26 g, 7.99 mmol, 30 eq) and sodium hydroxide (0.04 g, 1.08 mmol, 4.0 eq) by following the general procedure and reaction conditions as described above. The crude product was purified by soaking with dichloromethane to afford \u003cem\u003eN\u003c/em\u003e-hydroxy-4-{4-[(quinolin-3-yl)methyl]piperazin-1-yl}benzamide (\u003cb\u003e7\u003c/b\u003e) as white crystals. Yield 73%, m.p\u0026thinsp;=\u0026thinsp;215\u0026ndash;216\u0026deg;C. \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e, 36\u0026deg;C, TMS) δ\u0026thinsp;=\u0026thinsp;2.55(s, 4H, H-11), 3.26(s, 4H, H-12), 3.74(s, 2H, H-C\u003cem\u003eH\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003eN), 6.93(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.6Hz, 2H, H-14), 7.62(t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3Hz, 1H, H-7), 7.64(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3Hz, 2H, H-15), 7.76(t \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.6Hz, 1H, H-8), 7.99(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3Hz, 1H, H-6), 8.03(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3Hz, 1H, H-9), 8.26(s, 1H, H-4), 8.84(brs, 1H, -N\u003cem\u003eH\u003c/em\u003e), 8.90(s, 1H, H-2), 10.96(s, 1H, -O\u003cem\u003eH\u003c/em\u003e). \u003csup\u003e13\u003c/sup\u003eC-NMR (101 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e, 36\u0026deg;C, TMS) δ 47.64(C-12), 52.80(C-11), 59.79(N\u003cem\u003eC\u003c/em\u003eH\u003csub\u003e2\u003c/sub\u003e), 114.21(C-14), 122.37(C-16), 127.17(C-7), 127.97(C-5), 128.38(C-6), 128.54(C-9), 129.16(C-8), 129.64(C-15), 131.44(C-3), 135.80(C-4), 147.44(C-10), 152.47(C-2), 153.15(C-13). HRESIMS \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e [M\u0026thinsp;+\u0026thinsp;2H]\u003csup\u003e+\u003c/sup\u003e calcd. for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e: 364.18883, found: 364.18853.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Pharmacological/biological assays\u003c/h2\u003e \u003cp\u003e \u003cb\u003eThe cytotoxic activity\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe cytotoxic activity of all pure compounds was determined towards a panel of four human solid tumour cell lines: melanoma (SK-MEL), epidermal carcinoma (KB), breast carcinoma (BT-549), and ovarian carcinoma (SK-OV-3). Moreover, non-cancer kidney cell lines (LLC-PK1 and VERO) were also employed to determine if the anti-cell proliferative activity of these compounds was selective for the tested tumour cell lines. All cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were seeded in 96-well plates (10,000 cells/well) and incubated for 24 h. All ligands were dissolved in DMSO, diluted in media, and added to the cells at concentrations of 80, 40, 20, and 10 \u0026micro;M. After incubating for 48 h, cell viability was determined using a tetrazolium dye WST-8, which is converted to a water-soluble formazan product in the presence of 1-methoxy PMS by the activity of cellular enzymes. The colour of the formazan product was measured at 450 nm on a plate reader. Doxorubicin was used as a positive control for the cytotoxicity assay, and DMSO (0.25%) was used as the vehicle control. The IC\u003csub\u003e50\u003c/sub\u003e values were obtained from concentration-response curves. The values are represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEnzymatic\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eHDAC inhibitory activity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRecombinant human HDAC1, HDAC2, HDAC3/NCOR1, and HDAC6 were purchased from ENZO Life Sciences AG (Lausen, CH). Barinka et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] produced recombinant drHDAC10 wild type. Recombinant human HDAC8 was produced by Romier et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In vitro testing of the inhibitors in an enzymatic assay was carried out as described in previous publications by us [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Inhibitory activity for HDAC1, HDAC2, HDAC3, and HDAC6 was determined using a discontinuous assay with a substrate peptide derived from p53 (Ac-RHKK(Ac)-AMC) in a 384 well-plate. The enzyme at final concentrations (10 nM HDAC1, 3 nM HDAC2 and HDAC3, and 1 nM HDAC6,) and the inhibitors at various concentrations were incubated for 5 min in assay buffer (50 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 1 mM TCEP and 0.2 mg/mL BSA, pH 7.4 adjusted with NaOH). The reaction started with the addition of substrate (20 \u0026micro;M HDAC1-3 and 5 \u0026micro;M HDAC6). Afterwards, the fluorescence was developed with a 0.5 mg/ml trypsin solution (final concentration), and the fluorescence readout was done with an Envision 2104 Multilabel Plate Reader (PerkinElmer, Waltham, USA) with λ\u003csub\u003eEx\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;380\u0026thinsp;\u0026plusmn;\u0026thinsp;10 nm and λ\u003csub\u003eEm\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;430\u0026thinsp;\u0026plusmn;\u0026thinsp;8 nm.\u003c/p\u003e \u003cp\u003eFor HDAC8 the fluorogenic peptide derivate Abz-SRGGK(thio-TFA)FFRR-NH2 was applied as described in [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. For HDAC10 a spermidine derivative Ac-spermidine-AMC was applied. The HDAC8 assay was performed in the same assay buffer as described above. For HDAC10 the assay buffer was 20 mM HEPES, pH 7.4, and 0.5 mg/ml BSA. The enzyme (1.5 nM HDAC8 or 5 nM HDAC10 final concentration) was incubated for 5 min with various concentrations of the inhibitor. The reaction was started with the addition of 50 \u0026micro;M substrate and the readout was done continuously with an Envision 2104 with λ\u003csub\u003eEx\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;330\u0026thinsp;\u0026plusmn;\u0026thinsp;75 nm, and λ\u003csub\u003eEm\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;430\u0026thinsp;\u0026plusmn;\u0026thinsp;8 nm.\u003c/p\u003e \u003cp\u003eFor HDAC1, 2, and 3 a fluorogenic peptide derived from p53 (Ac-RHKK(Acetyl)-AMC) was applied. The measurements were performed in assay buffer (50 mM HEPES, 150 mM NaCl, 5 mM MgCl2, 1 mM TCEP, and 0.2 mg/mL BSA, pH 7.4 adjusted with NaOH) at 37\u0026deg;C. An Envision 2104 Multilabel Plate Reader (PerkinElmer, Waltham, MA), with an excitation wavelength of 380\u0026thinsp;\u0026plusmn;\u0026thinsp;8 nm and an emission wavelength of 430\u0026thinsp;\u0026plusmn;\u0026thinsp;8 nm was considered to measure the fluorescence intensity. For HDAC6 the substrate (Abz-SRGGK(thio-TFA)FFRR-NH2) was applied as described before [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The HDAC10 inhibition assay was performed as described before [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] using Ac-spermidine-AMC as substrate. The idea of this discontinuous assay is the HDAC10 mediated generation of a primary amino function. The released primary amine is reacted wirh 2,3-Naphthalenedicarboxaldehyde (NDA) resulting in an NDA-spermidin-AMC derivative which differences in the AMC fluorescence intensities as compared to Ac-spermidin AMC. The assay was performed in black 96-well plates (PerkinElmer, OptiPlateTM-96 F). The compounds to be tested were incubated for 25 min at 25\u0026deg;C. Before measuring fluorescence (POLARstar plate reader, λex\u0026thinsp;=\u0026thinsp;330 nm, λem\u0026thinsp;=\u0026thinsp;390 nm) each well was filled with 200 \u0026micro;L stop solution containing 16mM NDA). (for assay details please refer to [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The enzyme inhibition of HDAC8 was determined by using a homogenous fluorescence assay [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The enzyme was incubated for 90 min at 37\u0026deg;C, with the fluorogenic substrate ZMAL (Z(Ac)Lys-AMC) in a concentration of 10.5 \u0026micro;M and increasing concentrations of inhibitors. Fluorescence intensity was measured at an excitation wavelength of 390 nm and an emission wavelength of 460 nm in a microtiter plate reader (BMG Polarstar).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Molecular docking\u003c/h2\u003e \u003cp\u003e \u003cb\u003eMolecular docking\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAvailable crystal structures of drHDAC10, hsHDAC1, hsHDAC6 and hsHDAC8 were downloaded (PDB ID: 6UHU, PDB ID: 5ICN, PDB ID: 5EDU, and PDB ID: 2V5X respectively) from the Protein Data Bank (PDB;\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.rscb.org\u003c/span\u003e\u003cspan address=\"http://www.rscb.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. All ligands and all protein-ligand complexes were prepared using similar methods as published before [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Validation of the molecular docking method was performed by re-docking the ligands co-crystallized in HDAC10 as reported in our previous publication [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. For the preparation of the proteins, the wizard implementation of the Schr\u0026ouml;dinger version 2019.1 was used with the following steps: hydrogen atoms addition, protonation states assignment, and finally, restrained energy minimization using the OPLS force field 2005. The ligand structure was generated using the 2D Sketcher of Schr\u0026ouml;dinger (version 2019.1). Afterward, the LigPrep tool (Schr\u0026ouml;dinger version 2019.1) was used for the preparation of the ligands with energy minimization using the OPLS2005 force field [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. 64 conformers for each ligand were generated with the ConfGen tool (Schr\u0026ouml;dinger version 2019.1). Preparation of the receptor grid for the docking procedure was performed by assigning the co-crystallized ligands as the centroid of the grid box in each PDB crystal structure using the receptor grid preparation module in Schr\u0026ouml;dinger (version 2019.1). Lastly, docking of all generated conformers was done in the Standard Precision mode using the Glide (Schr\u0026ouml;dinger-release 2019.1).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMolecular Dynamics (MD) Simulations\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAMBER22 was used to perform GPU-based MD simulations. PDB4Amber command was used for the preparation of the protein structures for further usage within the tLEaP program. Topology and force field parameters of the ligands were assigned with Antechamber [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] package using the second generation general Amber force field (GAFF2) and the semi-empirical AM1-BCC (Austin Model1 with bond charge correction) as atomic charge method [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Afterwards, the protein-ligand complexes were created with the AMBER22 tLEaP module. The second-generation general AMBER force field (GAFF2) was used as the ligand force field while force field 14 Stony Brook-ff14SB was used for the protein structures [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. For the catalytic Zn2+, the 12-6-4 LJ-type nonbonded ion model was applied [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. After combining the protein and the ligands, complexes were solvated by transferable intermolecular potential 3P-TIP3P water model as an octahedral box around the protein with a 10 \u0026Aring; margin. Then Na\u0026thinsp;+\u0026thinsp;and Cl- ions were added to neutralize the whole system. Parameter/topology files of the entire system were created with tLEaP and the files were used as a starting point for the MD simulations. The solvated systems were first subjected to two energy minimization steps involving 1000 cycles of steepest descent followed by 2000 cycles of conjugate gradient totaling 3000 cycles of minimization. In the first energy minimization step, only the solvent molecules and counter-ions (Na\u0026thinsp;+\u0026thinsp;and Cl-) were minimized while applying constraints with a force constant of 10 kcal*mol-1*\u0026Aring;-2 to the proteins, ligands, and zinc ion. In the second minimization step, the whole system was minimized without constraints. Subsequently, the systems were heated from 0 to 300 K over 100 ps while applying the same constraints on the solute as in minimization step1. Constant volume periodic boundary was set to equilibrate the temperature of the system by Langevin thermostat using a collision frequency of 2 ps-1. Subsequently, a pressure equilibration routine with a constant pressure of 1 bar and at 300 K was performed for 100 ps. Finally, 100 ns free molecular dynamic simulations with the time step of 2 fs were applied utilizing the Particle Mesh Ewald method [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The system temperature was kept at 300 K with a Langevin thermostat using 2 ps-1 collision frequency and the pressure of the system was maintained at 1 bar with the usage of isotropic position scaling and a relaxation time of 2 ps. Implementation of the SHAKE algorithm was done to constrain all bonds containing hydrogens. A total of 1000 frames were written for 100 ns long MDs. Simulations of the prepared crystal structures of humanized drHDAC10 (PDB IDs; 7U6B, 7U69, 7U6A, 7U69) X-ray structures and the docking pose of ligand 1 in drHDAC10 (PDB ID:6UHU) were repeated three times from the minimization steps with non-identical random seeds. The analysis of the MD trajectories was performed using the CPPTRAJ module in AMBER 22. The RMSD of the protein was calculated for the backbone atoms using CPPTRAJ. RMSF of the ligand as well as distances between the hydroxamate group oxygens and the zinc ion were calculated to further examine the stability of the protein-ligand complexes using CPPTRAJ. Additionally, the obtained trajectories were clustered based on the ligand-heavy atoms using the K-means algorithm and pytraj implementation of AMBER.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePAINS filter\u003c/b\u003e \u003c/p\u003e \u003cp\u003eInhibitors described herein were filtered for pan-assay interference compounds (PAINS). For this purpose, PAINS1, PAINS2, and PAINS3 filters, as implemented in Schroedinger's Canvas program (Schr\u0026ouml;dinger version 2019.1), were employed. None of the compounds was flagged as PAINS.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, A.T., W.S., S.R. and S.A.; methodology, A.T., W.S. and S.R.; software, M.S.; validation, W.S., A.T., S.A. and S.R.,.; formal analysis, M.S., T.Y., M.Z., D.R. and C.B..; investigation, A.T,M.S., T.Y., M.Z., D.R. and C.B.; resources, S.R. and W.S.; data curation, A.T and W.S.; writing\u0026mdash;original draft preparation, A.T.; writing\u0026mdash;review and editing, S.A., M.S. and S.R.,; visualization, W.S and A.T.; supervision, A.T, W.S. and S.R.; project administration, A.T and W.S.; funding acquisition, S.R and W.S. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eWe would like to express our appreciation to Dr. Shabana Khan for overseeing the biological assay. Dr. Khan evaluated the cytotoxicity of the synthesized ligands across various cell lines. This work was funded in part by the Deutsche Forschungsgemeinschaft (DFG) SI868/22\u0026thinsp;\u0026minus;\u0026thinsp;1, project number 46995445 (to W.S.).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript or supplementary information files\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJ. Fan, J. Baeza, J.M. Denu, \u003cem\u003eMethods Enzymol\u003c/em\u003e. \u003cstrong\u003e2016\u003c/strong\u003e, 574,125. https://doi.org/10.1016/bs.mie.2016.01.007\u003c/li\u003e\n\u003cli\u003eO. Khan, N.B. La Thangue, \u003cem\u003eImmunol. \u003c/em\u003eCell Biol, \u003cstrong\u003e2012\u003c/strong\u003e, 90, 85. https://doi.org/10.1038/icb.2011.100 \u003c/li\u003e\n\u003cli\u003eE. Ceccacci, S. Minucci, \u003cem\u003eBr. J. Cancer\u003c/em\u003e, \u003cstrong\u003e2016\u003c/strong\u003e, 114, 605. https://doi.org/10.1038/bjc.2016.36 \u003c/li\u003e\n\u003cli\u003eG. Li, Y. Tian, W.-G. Zhu, \u003cem\u003eFront Cell Dev Biol\u003c/em\u003e, \u003cstrong\u003e2020\u003c/strong\u003e, 8, 576946. https://doi.org/10.3389/fcell.2020.576946 \u003c/li\u003e\n\u003cli\u003eE. San Jos\u0026eacute;-En\u0026eacute;riz, N. Gimenez-Camino, X. Agirre, F. Prosper, \u003cem\u003eCancers, \u003c/em\u003e\u003cstrong\u003e2019\u003c/strong\u003e, 11, 1794. https://doi.org/10.3390/cancers11111794 \u003c/li\u003e\n\u003cli\u003eO.H. Kr\u0026auml;mer, \u003cem\u003eTrends Pharmacol. Sci\u003c/em\u003e, \u003cstrong\u003e2009\u003c/strong\u003e, 30, 647. https://doi.org/10.1016/j.tips.2009.09.007 \u003c/li\u003e\n\u003cli\u003eD.A. Rodrigues, G.\u0026Agrave;. Ferreira-Silva, A.C.S. Ferreira, R.A. Fernandes, J.K. Kwee, C.M.R. Sant\u0026rsquo;Anna, M. Ionta, C.A.M. Fraga, \u003cem\u003eJ. Med. Chem\u003c/em\u003e, 2016, 59, 655. https://doi.org/10.1021/acs.jmedchem.5b01525 \u003c/li\u003e\n\u003cli\u003eY. Hai, S.A. Shinsky, N.J. Porter, D.W. Christianson, \u003cem\u003eNat. Commun\u003c/em\u003e, \u003cstrong\u003e2017\u003c/strong\u003e, 8, 15368. https://doi.org/10.1038/ncomms15368 \u003c/li\u003e\n\u003cli\u003eI. Oehme, J.-P. Linke, B.C. B\u0026ouml;ck, T. Milde, M. Lodrini, B. Hartenstein, I. Wiegand, C. Eckert, W. Roth, M. Kool, S. Kaden, H.-J. Gr\u0026ouml;ne, J.H. Schulte, S. Lindner, A. Hamacher-Brady, N.R. Brady, H.E. Deubzer, O. Witt, \u003cem\u003eProc. \u003c/em\u003e\u003cem\u003eNatl. Acad. Sci. U. S. A\u003c/em\u003e, \u003cstrong\u003e2013\u003c/strong\u003e, 110, E2592. https://doi.org/10.1073/pnas.1300113110 \u003c/li\u003e\n\u003cli\u003eS.A. Shinsky, D.W. Christianson, \u003cem\u003eBiochemistry\u003c/em\u003e. \u003cstrong\u003e2018\u003c/strong\u003e, 57, 3105. https://doi.org/10.1021/acs.biochem.8b00079 \u003c/li\u003e\n\u003cli\u003eD. Zhang, B. Tang, X. Xie, Y.-F. Xiao, S.-M. Yang, J.-W. Zhang, \u003cem\u003eCancer Biol. Ther\u003c/em\u003e, \u003cstrong\u003e2015\u003c/strong\u003e, 16, 1005. https://doi.org/10.1080/15384047.2015.1046022 \u003c/li\u003e\n\u003cli\u003eT. Kumagai, N. Wakimoto, D. Yin, S. Gery, N. Kawamata, N. Takai, N. Komatsu, A. Chumakov, Y. Imai, H.P. Koeffler, \u003cem\u003eInt. J. Cancer\u003c/em\u003e, \u003cstrong\u003e2007\u003c/strong\u003e, 121, 656. https://doi.org/10.1002/ijc.22558 \u003c/li\u003e\n\u003cli\u003eX. Qian, G. Ara, E. Mills, W.J. LaRochelle, H.S. Lichenstein, M. Jeffers, \u003cem\u003eInt. J. Cancer\u003c/em\u003e, \u003cstrong\u003e2008\u003c/strong\u003e, 122, 1400. https://doi.org/10.1002/ijc.23243 \u003c/li\u003e\n\u003cli\u003eR.L. Piekarz, R. Frye, M. Turner, J.J. Wright, S.L. Allen, M.H. Kirschbaum, J. Zain, H.M. Prince, J.P. Leonard, L.J. Geskin, C. Reeder, D. Joske, W.D. Figg, E.R. Gardner, S.M. Steinberg, E.S. Jaffe, M. Stetler-Stevenson, S. Lade, A.T. Fojo, S.E. Bates, \u003cem\u003eJ. Clin. \u003c/em\u003e\u003cem\u003eOncol\u003c/em\u003e, \u003cstrong\u003e2009\u003c/strong\u003e, 27, 5410. https://doi.org/10.1200/JCO.2008.21.6150 \u003c/li\u003e\n\u003cli\u003eM. Rhinn, I. Zapata-Bodalo, A. Klein, J.-L. Plassat, T. Knauer-Meyer, W.M. Keyes, \u003cem\u003ePLoS Biol\u003c/em\u003e, \u003cstrong\u003e2022\u003c/strong\u003e, 20, e3001664. https://doi.org/10.1371/journal.pbio.3001664 \u003c/li\u003e\n\u003cli\u003eL. Ellis, Y. Pan, G.K. Smyth, D.J. George, C. McCormack, R. Williams-Truax, M. Mita, J. Beck, H. Burris, G. Ryan, P. Atadja, D. Butterfoss, M. Dugan, K. Culver, R.W. Johnstone, H.M. Prince, \u003cem\u003eClin. \u003c/em\u003e\u003cem\u003eCancer Res\u003c/em\u003e, \u003cstrong\u003e2008\u003c/strong\u003e, 14, 4500. https://doi.org/10.1158/1078-0432.CCR-07-4262 \u003c/li\u003e\n\u003cli\u003eN.M. Raghavendra, D. Pingili, S. Kadasi, A. Mettu, S.V.U.M. Prasad, \u003cem\u003eEur. J. Med. Chem\u003c/em\u003e, \u003cstrong\u003e2018\u003c/strong\u003e, 143, 1277. https://doi.org/10.1016/j.ejmech.2017.10.021 \u003c/li\u003e\n\u003cli\u003eR.R. Ramsay, M.R. Popovic-Nikolic, K. Nikolic, E. Uliassi, M.L. Bolognesi, \u003cem\u003eClin. Transl. Med\u003c/em\u003e, \u003cstrong\u003e2018\u003c/strong\u003e, 7, 3. https://doi.org/10.1186/s40169-017-0181-2 \u003c/li\u003e\n\u003cli\u003eY. Li, E. Seto, \u003cem\u003eCold Spring Harb. Perspect. Med\u003c/em\u003e, \u003cstrong\u003e2016\u003c/strong\u003e, 6. https://doi.org/10.1101/cshperspect.a026831\u003c/li\u003e\n\u003cli\u003eE. Seto, M. Yoshida, \u003cem\u003eCold Spring Harb. Perspect. Biol\u003c/em\u003e, 2014, 6, a018713. https://doi.org/10.1101/cshperspect.a018713 \u003c/li\u003e\n\u003cli\u003eH.S. Rho, H.S. Baek, J.A. Lee, C.M. Park, D.H. Kim, I.S. Chang, O.S. Lee, Hydroxamic acid derivatives and the preparation method thereof, 7485750, \u003cstrong\u003e2009\u003c/strong\u003e. https://patentimages.storage.googleapis.com/16/d4/5a/47495080cd5d60/US7485750.pdf (accessed October 18, 2023).\u003c/li\u003e\n\u003cli\u003eQ. Lu, Y.-T. Yang, C.-S. Chen, M. Davis, J.C. Byrd, M.R. Etherton, A. Umar, C.-S. Chen, \u003cem\u003eJ. Med. Chem, \u003cstrong\u003e2004\u003c/strong\u003e, \u003c/em\u003e47, 467. https://doi.org/10.1021/jm0303655 \u003c/li\u003e\n\u003cli\u003eJ. Ridinger, E. Koeneke, F.R. Kolbinger, K. Koerholz, S. Mahboobi, L. Hellweg, N. Gunkel, A.K. Miller, H. Peterziel, P. Schmezer, A. Hamacher-Brady, O. Witt, I. Oehme, \u003cem\u003eSci. \u003c/em\u003e\u003cem\u003eRep\u003c/em\u003e, \u003cstrong\u003e2018\u003c/strong\u003e, 8, 10039. https://doi.org/10.1038/s41598-018-28265-5 \u003c/li\u003e\n\u003cli\u003eY.Y. Gu, G.N. Zhou, Y. Li, H.Y. He, J.X. Ding, K.Q. Hua, \u003cem\u003eJ. Oncol\u003c/em\u003e. \u003cstrong\u003e2022\u003c/strong\u003e, 2022, 8092751. https://doi.org/10.1155/2022/8092751 \u003c/li\u003e\n\u003cli\u003eC.J. Herbst-Gervasoni, R.R. Steimbach, M. Morgen, A.K. Miller, D.W. Christianson, \u003cem\u003eACS Chem. Biol\u003c/em\u003e. \u003cstrong\u003e2020\u003c/strong\u003e, 15, 2154. https://doi.org/10.1021/acschembio.0c00362 \u003c/li\u003e\n\u003cli\u003eM. G\u0026eacute;raldy, M. Morgen, P. Sehr, R.R. Steimbach, D. Moi, J. Ridinger, I. Oehme, O. Witt, M. Malz, M.S. Nogueira, O. Koch, N. Gunkel, A.K. Miller, \u003cem\u003eJ. Med. \u003c/em\u003e\u003cem\u003eChem\u003c/em\u003e, \u003cstrong\u003e2019\u003c/strong\u003e, 62, 4426. https://doi.org/10.1021/acs.jmedchem.8b01936 \u003c/li\u003e\n\u003cli\u003eF.R. Kolbinger, E. Koeneke, J. Ridinger, T. Heimburg, M. M\u0026uuml;ller, T. Bayer, W. Sippl, M. Jung, N. Gunkel, A.K. Miller, F. Westermann, O. Witt, I. Oehme, \u003cem\u003eArch. \u003c/em\u003e\u003cem\u003eToxicol\u003c/em\u003e. \u003cstrong\u003e2018\u003c/strong\u003e, 92, 2649. https://doi.org/10.1007/s00204-018-2234-8 \u003c/li\u003e\n\u003cli\u003eY. Li, P.M. Woster, \u003cem\u003eMedchemcomm\u003c/em\u003e. \u003cstrong\u003e2015\u003c/strong\u003e, 6, 613. https://doi.org/10.1039/C4MD00401A \u003c/li\u003e\n\u003cli\u003eP. Zeyen, Y. Zeyn, D. Herp, F. Mahmoudi, T.Z. Yesiloglu, F. Erdmann, M. Schmidt, D. Robaa, C. Romier, J. Ridinger, C.J. Herbst-Gervasoni, D.W. Christianson, I. Oehme, M. Jung, O.H. Kr\u0026auml;mer, W. Sippl, \u003cem\u003eEur. J. Med. Chem\u003c/em\u003e, \u003cstrong\u003e2022\u003c/strong\u003e, 234. 114272. https://doi.org/10.1016/j.ejmech.2022.114272 \u003c/li\u003e\n\u003cli\u003eD. Herp, J. Ridinger, D. Robaa, S.A. Shinsky, K. Schmidtkunz, T.Z. Yesiloglu, T. Bayer, R.R. Steimbach, C.J. Herbst-Gervasoni, A. Merz, C. Romier, P. Sehr, N. Gunkel, A.K. Miller, D.W. Christianson, I. Oehme, W. Sippl, M. Jung, \u003cem\u003eChembiochem\u003c/em\u003e, \u003cstrong\u003e2022\u003c/strong\u003e, 23, e202200180. https://doi.org/10.1002/cbic.202200180 \u003c/li\u003e\n\u003cli\u003eE. Pojani, D. Barlocco, \u003cem\u003eCurr. Med. Chem\u003c/em\u003e, \u003cstrong\u003e2022\u003c/strong\u003e, 29, 2306. DOI: 10.2174/0929867328666210901144658\u003c/li\u003e\n\u003cli\u003eJ. Ptacek, I. Snajdr, J. Schimer, Z. Kutil, J. Mikesova, P. Baranova, B. Havlinova, W. Tueckmantel, P. Majer, A. Kozikowski, C. Barinka, \u003cem\u003eInt. J. Mol. Sc, \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e, 24. https://doi.org/10.3390/ijms24054720 \u003c/li\u003e\n\u003cli\u003eD.T. Vogl, N. Raje, S. Jagannath, P. Richardson, P. Hari, R. Orlowski, J.G. Supko, D. Tamang, M. Yang, S.S. Jones, C. Wheeler, R.J. Markelewicz, S. Lonial, Ricolinostat, \u003cem\u003eClin. Cancer Re, \u003c/em\u003e\u003cstrong\u003e2017\u003c/strong\u003e, 23, 3307. https://doi.org/10.1158/1078-0432.CCR-16-2526 \u003c/li\u003e\n\u003cli\u003eT. Li, C. Zhang, S. Hassan, X. Liu, F. Song, K. Chen, W. Zhang, J. Yang, \u003cem\u003eJ. Hematol. \u003c/em\u003e\u003cem\u003eOncol\u003c/em\u003e, \u003cstrong\u003e2018\u003c/strong\u003e, 11, 111. https://doi.org/10.1186/s13045-018-0654-9 \u003c/li\u003e\n\u003cli\u003eJ.D. Osko, D.W. Christianson, \u003cem\u003eBioorg. \u003c/em\u003e\u003cem\u003eMed. Chem. Lett\u003c/em\u003e, \u003cstrong\u003e2020\u003c/strong\u003e, 30, 127023. https://doi.org/10.1016/j.bmcl.2020.127023 \u003c/li\u003e\n\u003cli\u003eR.R. Steimbach, C.J. Herbst-Gervasoni, S. Lechner, T.M. Stewart, G. Klinke, J. Ridinger, M.N.E. G\u0026eacute;raldy, G. Tihanyi, J.R. Foley, U. Uhrig, B. Kuster, G. Poschet, R.A. Casero Jr, G. M\u0026eacute;dard, I. Oehme, D.W. Christianson, N. Gunkel, A.K. Miller, \u003cem\u003eJ. Am. Chem. Soc\u003c/em\u003e, \u003cstrong\u003e2022\u003c/strong\u003e, 144, 18861. https://doi.org/10.1021/jacs.2c05030 \u003c/li\u003e\n\u003cli\u003eT. Heimburg, F.R. Kolbinger, P. Zeyen, E. Ghazy, D. Herp, K. Schmidtkunz, J. Melesina, T.B. Shaik, F. Erdmann, M. Schmidt, C. Romier, D. Robaa, O. Witt, I. Oehme, M. Jung, W. Sippl, \u003cem\u003eJ. Med. Chem\u003c/em\u003e, 2017, 60, 10188. https://doi.org/10.1021/acs.jmedchem.7b01447 \u003c/li\u003e\n\u003cli\u003eM. Zessin, Z. Kutil, M. Meleshin, Z. Nov\u0026aacute;kov\u0026aacute;, E. Ghazy, D. Kalbas, M. Marek, C. Romier, W. Sippl, C. Bařinka, M. Schutkowski, \u003cem\u003eBiochemistry\u003c/em\u003e, \u003cstrong\u003e2019\u003c/strong\u003e, 58, 4777. https://doi.org/10.1021/acs.biochem.9b00786 \u003c/li\u003e\n\u003cli\u003eP. Sun, J. Wang, K.S. Khan, W. Yang, B.W.-L. Ng, N. Ilment, M. Zessin, E.F. B\u0026uuml;lb\u0026uuml;l, D. Robaa, F. Erdmann, M. Schmidt, C. Romier, M. Schutkowski, A.S.-L. Cheng, W. Sippl, \u003cem\u003eJ. Med. Chem\u003c/em\u003e, \u003cstrong\u003e2022\u003c/strong\u003e, 65, 16313. https://doi.org/10.1021/acs.jmedchem.2c01132 \u003c/li\u003e\n\u003cli\u003eH.S. Ibrahim, M. Abdelsalam, Y. Zeyn, M. Zessin, A.-H.M. Mustafa, M.A. Fischer, P. Zeyen, P. Sun, E.F. B\u0026uuml;lb\u0026uuml;l, A. Vecchio, F. Erdmann, M. Schmidt, D. Robaa, C. Barinka, C. Romier, M. Schutkowski, O.H. Kr\u0026auml;mer, W. Sippl, \u003cem\u003eInt. J. Mol. Sci\u003c/em\u003e, \u003cstrong\u003e2021\u003c/strong\u003e, 23, 369. https://doi.org/10.3390/ijms23010369 \u003c/li\u003e\n\u003cli\u003eH.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne, \u003cem\u003eNucleic Acids Res\u003c/em\u003e, \u003cstrong\u003e2000\u003c/strong\u003e, 28, 235. https://doi.org/10.1093/nar/28.1.235 \u003c/li\u003e\n\u003cli\u003eC.V. Simoben, E. Ghazy, P. Zeyen, S. Darwish, M. Schmidt, C. Romier, D. Robaa, W. Sippl, \u003cem\u003eMolecules\u003c/em\u003e, \u003cstrong\u003e2021\u003c/strong\u003e, 26. https://doi.org/10.3390/molecules26092584 \u003c/li\u003e\n\u003cli\u003eJ.L. Banks, H.S. Beard, Y. Cao, A.E. Cho, W. Damm, R. Farid, A.K. Felts, T.A. Halgren, D.T. Mainz, J.R. Maple, R. Murphy, D.M. Philipp, M.P. Repasky, L.Y. Zhang, B.J. Berne, R.A. Friesner, E. Gallicchio, R.M. Levy, \u003cem\u003eJ. Comput. \u003c/em\u003e\u003cem\u003eChem\u003c/em\u003e, \u003cstrong\u003e2005\u003c/strong\u003e, 26, 1752. https://doi.org/10.1002/jcc.20292 \u003c/li\u003e\n\u003cli\u003eJ. Wang, W. Wang, P.A. Kollman, D.A. Case, \u003cem\u003eJ. Mol. \u003c/em\u003e\u003cem\u003eGraph. Model\u003c/em\u003e, \u003cstrong\u003e2006\u003c/strong\u003e, 25, 247. https://doi.org/10.1016/j.jmgm.2005.12.005 \u003c/li\u003e\n\u003cli\u003eA. Jakalian, D.B. Jack, C.I. Bayly, Fast, efficient generation of high‐quality atomic charges. AM1‐BCC model: II. Parameterization and validation, Journal of Computational. (\u003cstrong\u003e2002\u003c/strong\u003e). https://onlinelibrary.wiley.com/doi/abs/10.1002/jcc.10128?casa_token=qpaSyrElG0AAAAAA:13RPtcvphG3Ais-isjxM5xvFOD1ZHoIimPMTpWt5KtqoI2bmsx3btHslXwP3M7_tp6HX_0_u4VXtPZIE. \u003c/li\u003e\n\u003cli\u003eA. Jakalian, B.L. Bush, D.B. Jack, C.I. Bayly, \u003cem\u003eJ. Comput. Chem\u003c/em\u003e, \u003cstrong\u003e2000\u003c/strong\u003e, 21, 132. https://doi.org/10.1002/(SICI)1096-987X(20000130)21:2\u0026lt;132::AID-JCC5\u0026gt;3.0.CO;2-P \u003c/li\u003e\n\u003cli\u003eX. He, V.H. Man, W. Yang, T.-S. Lee, J. Wang, \u003cem\u003eJ. Chem. Phys\u003c/em\u003e, \u003cstrong\u003e2020\u003c/strong\u003e, 153, 114502. https://doi.org/10.1063/5.0019056 \u003c/li\u003e\n\u003cli\u003eJ.A. Maier, C. Martinez, K. Kasavajhala, L. Wickstrom, K.E. Hauser, C. Simmerling, \u003cem\u003eJ. Chem. \u003c/em\u003e\u003cem\u003eTheory Comput\u003c/em\u003e, \u003cstrong\u003e2015\u003c/strong\u003e, 11, 3696. https://doi.org/10.1021/acs.jctc.5b00255 \u003c/li\u003e\n\u003cli\u003eP. Li, L.F. Song, K.M. Merz Jr, \u003cem\u003eJ. Phys. Chem\u003c/em\u003e. B, \u003cstrong\u003e2015\u003c/strong\u003e, 119, 883. https://doi.org/10.1021/jp505875v \u003c/li\u003e\n\u003cli\u003eToukmaji, C. Sagui, J. Board, T. Darden, Efficient particle-mesh Ewald based approach to fixed and induced dipolar interactions, The Journal of Chemical. (2000). https://pubs.aip.org/aip/jcp/article-abstract/113/24/10913/452430. https://doi.org/10.1063/1.1324708\u003c/li\u003e\n\u003cli\u003eD.D. Evans, \u003cem\u003eAust. J. Chem\u003c/em\u003e, \u003cstrong\u003e1973\u003c/strong\u003e, 26, 2555. https://doi.org/10.1071/CH9732555 \u003c/li\u003e\n\u003cli\u003eT.-S. Wu, M.-J. Liou, C.-J. Lee, T.-T. Jong, A.T. McPhail, D.R. McPhail, K.-H. Lee, \u003cem\u003eTetrahedron Lett\u003c/em\u003e, \u003cstrong\u003e1989\u003c/strong\u003e, 30, 6649. https://doi.org/10.1016/S0040-4039(00)70641-1 \u003c/li\u003e\n\u003cli\u003eH. Xu, Y.-Y. Wang, \u003cem\u003eBioorg. Med. Chem\u003c/em\u003e. Lett, \u003cstrong\u003e2010\u003c/strong\u003e, 20, 7274. https://doi.org/10.1016/j.bmcl.2010.10.084 \u003c/li\u003e\n\u003cli\u003eM.P. Mertes, A.J. Lin, \u003cem\u003eJ. Med. Chem\u003c/em\u003e, \u003cstrong\u003e1970\u003c/strong\u003e, 13, 276. https://doi.org/10.1021/jm00296a026 \u003c/li\u003e\n\u003cli\u003eS.-Y. Cheung, M. Werner, L. Esposito, F. Troisi, V. Cantone, S. Liening, S. K\u0026ouml;nig, J. Gerstmeier, A. Koeberle, R. Bilancia, R. Rizza, A. Rossi, F. Roviezzo, V. Temml, D. Schuster, H. Stuppner, M. Schubert-Zsilavecz, O. Werz, T. Hanke, S. Pace, \u003cem\u003eEur. J. Med. Chem\u003c/em\u003e, \u003cstrong\u003e2018\u003c/strong\u003e, 156, 815. https://doi.org/10.1016/j.ejmech.2018.07.031 \u003c/li\u003e\n\u003cli\u003eF. Gaedcke, R. Knorr, F. Zymalkowski, \u003cem\u003eArch. \u003c/em\u003e\u003cem\u003ePharm\u003c/em\u003e, 1980, 313, 166. https://doi.org/10.1002/ardp.19803130209\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"},{"header":"Schemes","content":"\u003cp\u003eSchemes 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Histone deacetylases, HDAC10 inhibitors, Tumor cell lines, Molecular docking, Molecular Dynamics","lastPublishedDoi":"10.21203/rs.3.rs-5950104/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5950104/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHistone deacetylase (HDAC) inhibitors represent a newer class of anti-cancer agents that play a key role in both epigenetic and non-epigenetic regulation, leading to cancer cell death, apoptosis, and cell cycle arrest.. These inhibitors are being tested in numerous clinical trials against various diseases, including both hematological and solid malignancies. In the present study, we synthesized novel bicyclic hydroxamic acid derivatives and tested them in vitro against class I and IIb HDACs to investigate their inhibitory activity and selectivity. We demonstrate that compound \u003cb\u003e6\u003c/b\u003e inhibits HDAC10 with high specificity over HDAC6, with no significant impact on class I HDACs. Compound \u003cb\u003e1\u003c/b\u003e shows the best inhibitory activity against HDAC10, with IC\u003csub\u003e50\u003c/sub\u003e 0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 nM. Compound \u003cb\u003e4\u003c/b\u003e revealed a preference toward HDAC6, with an IC50 value of 2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 nM. Compounds \u003cb\u003e2\u003c/b\u003e and \u003cb\u003e3\u003c/b\u003e demonstrated high selectivity toward class IIb over class I HDACs. Docking and molecular dynamics studies revealed that compound \u003cb\u003e1\u003c/b\u003e fits well into the active site of HDAC10, forming stable and strong interactions with key residues F204, D94, W205, and E274 in HDAC10. In addition, we tested these compounds against a panel of four human solid tumor cell lines. Furthermore, non-cancerous kidney cell lines (LLC-PK1 and VERO) were employed to determine the anti-cell proliferative activity of these compounds toward noncancerous cells.\u003c/p\u003e","manuscriptTitle":"Efficacy of a New Selective Indole-based Histone Deacetylase 10 Inhibitor in Targeted Anticancer Therapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-07 09:08:59","doi":"10.21203/rs.3.rs-5950104/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-25T04:26:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-22T02:41:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"302842814005162614521320765916824574722","date":"2025-02-14T13:30:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-14T07:18:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"127899658012923796405994611844941750547","date":"2025-02-14T05:37:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-02-13T17:01:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-13T16:52:07+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-02-06T12:15:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-05T09:36:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-02-03T09:56:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"254db877-23a2-450f-8649-7469d79c94b8","owner":[],"postedDate":"February 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":43942090,"name":"Physical sciences/Chemistry"},{"id":43942091,"name":"Physical sciences/Chemistry/Medicinal chemistry"}],"tags":[],"updatedAt":"2025-09-29T16:04:25+00:00","versionOfRecord":{"articleIdentity":"rs-5950104","link":"https://doi.org/10.1038/s41598-025-02774-6","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-09-26 15:58:15","publishedOnDateReadable":"September 26th, 2025"},"versionCreatedAt":"2025-02-07 09:08:59","video":"","vorDoi":"10.1038/s41598-025-02774-6","vorDoiUrl":"https://doi.org/10.1038/s41598-025-02774-6","workflowStages":[]},"version":"v1","identity":"rs-5950104","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5950104","identity":"rs-5950104","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}