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. 2020 Sep 29;11(11):2268–2276. doi: 10.1021/acsmedchemlett.0c00395

Spiroindoline-Capped Selective HDAC6 Inhibitors: Design, Synthesis, Structural Analysis, and Biological Evaluation

A Prasanth Saraswati †, Nicola Relitti †, Margherita Brindisi †, Jeremy D Osko ‡, Giulia Chemi †, Stefano Federico †, Alessandro Grillo †, Simone Brogi §, Niamh H McCabe ∥, Richard C Turkington ∥, Ola Ibrahim ⊥, Jeffrey O’Sullivan ⊥, Stefania Lamponi †, Magda Ghanim #, Vincent P Kelly #, Daniela Zisterer #, Rebecca Amet #, Patricia Hannon Barroeta #, Francesca Vanni ∇, Cristina Ulivieri ∇, Daniel Herp ○, Federica Sarno ◆, Antonella Di Costanzo ◆, Fulvio Saccoccia , Giovina Ruberti , Manfred Jung ○, Lucia Altucci ◆, Sandra Gemma †, Stefania Butini †, David W Christianson ‡, Giuseppe Campiani †,*
PMCID: PMC7667836  PMID: 33214839

Abstract

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Histone deacetylase inhibitors (HDACi) have emerged as promising therapeutics for the treatment of neurodegeneration, cancer, and rare disorders. Herein, we report the development of a series of spiroindoline-based HDAC6 isoform-selective inhibitors based on the X-ray crystal studies of the hit 6a. We identified compound 6j as the most potent and selective hHDAC6 inhibitor of the series. Biological investigation of compounds 6b, 6h, and 6j demonstrated their antiproliferative activity against several cancer cell lines. Western blotting studies indicated that they were able to increase tubulin acetylation, without significant variation in histone acetylation state, and induced PARP cleavage indicating their apoptotic potential at the molecular level. 6j induced HDAC6-dependent pSTAT3 inhibition.

Keywords: HDAC6, HDAC inhibitors, spiroindoline, cancer therapy, STAT3

Cancer, diabetes, cardiovascular, neurological, and metabolic disorders have an epigenetic etiology. Histone proteins play a crucial role in organizing the DNA into structures called nucleosomes. Histone acetylation and deacetylation represent a prime example of post-translational modifications that function in epigenetic regulation. Histone deacetylases (HDAC) remove acetyl groups from lysine residues and thereby regulate key processes such as gene expression.1 They are clustered in four different classes (I–IV): class I HDAC enzymes consist of isoforms 1, 2, 3, and 8; whereas class II enzymes include the isoforms 4, 5, 6, 7, 9, and 10. Class IV contains only isoform 11, and, similarly to class I and II isoforms, this enzyme is zinc dependent. In contrast, the class III HDACs are NAD+-dependent enzymes called sirtuins (SIRT isoforms 1–7). A common structural feature of HDAC inhibitors (HDACi) is the presence of a zinc binding group (ZBG), a linker moiety and a cap-group portion. All these three moieties can be functionalized to modulate selectivity toward specific HDAC isoforms.2 Many HDACi have been identified as therapeutic tools for the treatment of various pathologies such as cancer, infectious diseases, neurodegenerative disorders, and rare diseases.3−6 To date, four HDACi pan-inhibitors have been approved by the U.S. Food and Drug Administration (FDA) in cancer therapy: vorinostat (1), romidepsin (2), panobinostat (3), and belinostat (4, Figure 1). Their use may lead to unwanted side effects such as thrombocytopenia, neutropenia, diarrhea, nausea, vomiting, and fatigue as the most commonly detected.7

Figure 1.

Figure 1

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Structures of FDA approved HDAC inhibitors: vorinostat (1), romidepsin (2), panobinostat (3), and belinostat (4), spiroindoline inhibitor 5, and novel HDAC6i 6a–j.

Accordingly, significant research efforts are currently focused on the development of isoform-selective HDACi.2,6 HDAC6 represents a unique member of the HDAC family due to two main factors: (a) it contains two distinct catalytic domains and is primarily found in the cytoplasm (unlike HDAC1, -2, and -3, which are nuclear localized isoforms and HDAC8, displaying both nuclear and cytoplasmic distribution; interestingly, the cytosolic enzyme HDAC10, which is closely related to HDAC6, is ineffective as a lysine deacetylase);8 (b) it predominantly acts on non-histone substrates, such as α-tubulin, Hsp90, and cortactin.2 In spite of a couple of recent reports stating the challenges of using HDAC6 inhibitors in cancer,9,10 ample evidence outweighs their utility as anticancer agents.6,11 In particular, the involvement of HDAC6 in cancer cell migration and metastasis11 prompted us to develop novel anticancer agents as selective HDAC6 inhibitors. Further, the contribution of non-histone proteins such as α-tubulin and Hsp90 in HDAC6-mediated tumorigenesis makes selective HDAC6 inhibition a unique therapeutic strategy for cancer chemotherapy, with respect to the use of classical pan-HDAC inhibitors.6

In an effort to develop potent and selective HDAC6i, our group recently identified spiroindoline-capped HDACi (5, Figure 1) which exhibited significant anticancer potential against several cancer cell lines.12 We have herein investigated the impact of a strategical overturning of the linker and the ZBG moieties from the indoline nitrogen (compound 5) to the piperidine nitrogen (compounds 6a–j) in order to improve biological properties. The prototype 6a (Table 1) demonstrated a promising hHDAC6 IC50 value of 264.4 nM with a selectivity index of 85 over hHDAC1 and of 7 over hHDAC8. To gain a deeper understanding of the binding mode of 6a and to proceed to the design of analogues, a 2.09 Å resolution X-ray crystal structure of the complex between 6a and catalytic domain 2 of HDAC6 from Danio rerio (zebrafish) was determined. The active site structure of zebrafish HDAC6 (zfHDAC6) is essentially identical to that of hHDAC6, and zfHDAC6 yields crystals of much better quality compared with crystals of hHDAC6.13 Subsequently, molecular modeling approaches were exploited to analyze the binding mode and the structural requirements to design novel “reversed” spiroindolines with an improved HDAC6 inhibitory profile and selectivity index. This was achieved following two main strategies: (i) synthesizing derivatives with bulkier cap-groups and (ii) modulating the outdistancing between the cap-group and the ZBG, through the insertion of amide, urea, and carbamate functionalities in the linker portion. The resulting compounds (6b–j) were tested for their ability to inhibit the HDAC1, -6, and -8 isoforms. In addition, the best performing compounds were further evaluated for their effects on cell cycle progression and apoptosis in various cancer cell lines.

Table 1. Inhibitory Activity of Compounds 6a–j and Reference Compounds (5 and Tubastatin A) against hHDAC1, as IC50 (μM), and hHDAC6, as IC50 (nM)a.

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a

Each value is the mean of at least three determinations; compounds were assayed at eight concentrations; results are expressed with SD.

For the synthesis of compounds 6a–j, five key steps were employed to obtain the desired products which include (i) an interrupted Fischer indolization, starting from suitable arylhydrazines and N-Boc-piperidine-4-carboxaldehyde providing 3,3-disubstituted indolenines, (ii) reduction of the imine bond of the indolenines to get the respective indolines, (iii) appropriate substitution at the N-1 position of the indoline, (iv) insertion at the piperidine nitrogen with suitable linkers, and (v) conversion of the ester into hydroxamic acid (Scheme 1). See the Supporting Information, section 1, for more details.

Scheme 1. Synthesis of the Final Compounds 6a–j.

Scheme 1

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Reagents and conditions: (A) (a) Phenylhydrazine or (1,1-biphenyl)-2-ylhydrazine·HCl, AcOH, 80 °C, 2 h; (b) H2, Pd/C, MeOH, 25 °C, 3 h; (c) paraformaldehyde or benzaldehyde, NaBH3CN, MeOH, 25 °C, 8 h; (d) benzoyl chloride, TEA, DCM, 25 °C, 1 h; (e) 1 N HCl in MeOH, 25 °C, 15 min; (f) methyl 4-formyl benzoate, NaBH3CN, MeOH, 25 °C, 8 h; (g) NH2OH (50 wt % in H2O), 4 M KOH in MeOH, DCM/MeOH, 25 °C, 2 h; (B) (h) NMO, MeCN, 25 °C, 12 h; (i) 10a, NaBH3CN, MeOH, 25 °C, 8 h; (C) (j) 2-(4-(methoxycarbonyl)phenyl)acetic acid, EDCI, HOBt, DIPEA, DCM, 0 to 25 °C, 24 h; (k) methyl 4-(isocyanatomethyl)benzoate, TEA, dry THF, 45 °C, 2 h; (l) methyl 4-(hydroxymethyl)benzoate, CDI, DCM, 0 to 25 °C, 6 h.

The 2.09 Å resolution crystal structure of the zfHDAC6 CD2-6a complex revealed that the inhibitor hydroxamate group coordinates to the catalytic Zn2+ with bidentate geometry (Figure 2). The Zn2+-bound hydroxamate C=O group accepts a hydrogen bond from Y745, the Zn2+-bound hydroxamate N–O– group accepts a hydrogen bond from H573, and the hydroxamate NH group donates a hydrogen bond to H574. This constellation of intermolecular interactions with catalytically relevant residues accounts for the high affinity generally retrieved for the hydroxamate-based inhibitors in the HDAC6 active site.

Figure 2.

Figure 2

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Stereoview of a Polder omit map of the HDAC6-6a complex for which the atomic coordinates of 6a were omitted from the structure factor calculation (PDB 6V7A; contoured at 5.0 σ). Atoms are color-coded as follows: C = light blue (HDAC6 catalytic domain 2), light gray (symmetry mate), or wheat (inhibitor), N = blue, O = red, Zn2+ = gray sphere, and solvent = small red spheres. Metal coordination and hydrogen bond interactions are indicated by solid and dashed black lines, respectively.

The para-substituted phenyl linker makes favorable offset π–π interactions in the aromatic crevice defined by F583 and F643. The piperidine ring adopts a chair conformation, and the piperidine nitrogen forms a hydrogen bond with a water molecule that in turn hydrogen bonds with the backbone carbonyl of R798. The spiroindoline group is oriented toward the L2 pocket at the mouth of the active site. There, the indoline nitrogen hydrogen bonds with a water molecule that in turn is linked to N645 and a second water molecule; a third water molecule completes a hydrogen bond network between the indoline nitrogen and Zn2+ ligand H614. Although the inhibitor makes no direct enzyme–inhibitor hydrogen bonds apart from those made with the hydroxamate moiety, it is interesting that three water molecules comprise a “wet” hydrogen bonded interface in such a high-affinity enzyme–inhibitor pair.

It is relatively rare to see inhibitor capping groups bind in the L2 pocket, since most tend to bind in the L1 pocket on the opposite side of the active site.14−18 It appears that the chair conformation of the piperidine ring combined with the molecular structure of the novel spiro-fused indoline moiety yields a structure and a conformation that is ideal for binding within the L2 pocket.

A computational investigation (Figure S1) highlighted that 6a accommodates in a similar fashion in both zfHDAC6 and hHDAC6 enzymes, with only slight changes. Three main differences could be observed, involving the residues D567, T678, and M682 in hHDAC6 which are replaced by N530, A641, and N645 in zfHDAC6. The presence of M682 in hHDAC6 contributed to a slightly different orientation of the cap-group that is more solvent exposed with respect to the crystal structure and the docked pose within zfHDAC6. This study confirms that zfHDAC6 could represent a valuable structural model for translating the results of potential inhibitors to hHDAC6.

The in vitro inhibitory profile of the newly developed compounds 6a–j (Table 1) was evaluated against hHDAC1 and hHDAC6. SAR studies were performed by taking into consideration the data obtained from in vitro, X-ray, and computational studies. To get a better understanding of the behavior of the compounds in the binding sites of hHDAC1 and hHDAC6, we performed docking studies based on a previously reported protocol (Figures S2–S9).3,12 It was observed that the hindrance imposed by a bulkier cap-group allowed the compound to be better accommodated into the HDAC6 enzyme with respect to the HDAC1 isoform.

Based on these studies, limited contacts were established by 6a within the HDAC1 binding site (Figure 3A) compared to those established within HDAC6 binding site (Figure 3B). 6a was able to coordinate Zn2+ in HDAC1 by its hydroxamic moiety through polar contacts with the backbone of G149 and the side chain of Y303. In addition, we observed only a π–π stacking with H141 and some hydrophobic interactions with Y204, F205, and L271. On the contrary, the docking output of 6a into HDAC6 showed an increased number of contacts. The hydroxamic acid moiety coordinated with Zn2+ and established supplementary H-bonds with the side chain of Y782 and H610 and with the backbone of G619. The benzyl linker was able to establish a double π–π stacking with F620 and H651. We also noted relevant hydrophobic interactions with F679, F680, M682, and L749. This pattern of interaction perfectly supported the selectivity of 6a toward HDAC6 over HDAC1 (IC50 HDAC1 = 22.4 μM; IC50 HDAC6 = 264.4 nM).

Figure 3.

Figure 3

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Docked poses of 6a into HDAC1 (A) and HDAC6 (B). Compound 6a is represented by purple sticks, the residues in the active sites are represented by lines, and the protein is represented as a cartoon. Zn2+ is represented by a gray sphere. H-bonds are shown as black dotted lines, and the red solid lines represent the metal coordination bonds.

The docking studies of analogues 6b–g are reported in the Supporting Information, and poses are shown in Figures S1–S8.

In order to investigate the role of the linker portion, compounds 6h–j were synthesized. 6h, in addition to the contacts found for 6a, was able to produce a π–π stacking with F205 of HDAC1 through the benzyl functionality. In HDAC6, compound 6h showed the same interactions of 6a and the crucial π–π stacking with F680 (IC50 HDAC1 = 10.2 μM; IC50 HDAC6 = 227 nM). Compound 6i demonstrated that the urea functionality determines an improvement in inhibitory potency against both isoforms. Therefore, 6i established two further interactions beside those described for 6h, namely, (i) a H-bond with the side chain of H178 and (ii) a cation−π stacking with K200. With respect to the HDAC6 enzyme, 6i interacts with the same residues described for 6h, displaying an additional H-bond between the side chain of S568 and the urea NH (IC50 HDAC1 = 3.6 μM; IC50 HDAC6 = 110 nM).

The carbamic functionality of 6j determined an improvement in potency and selectivity toward HDAC6 over HDAC1 (Figure 4). The hydroxamic acid moiety, in addition to the metal coordination bond with the Zn2+, established a series of H-bonds with the side chain of Y782 and with the backbone of G619 of HDAC6. Moreover, its benzyl linker established a triple π–π stacking with F620, F680, and H651. Also, relevant hydrophobic interactions with T678, F679, M682, and L749 were observed. Notably, the phenyl group of the indole established a cation−π stacking with R673 (IC50hHDAC1 = 6.8 μM; IC50hHDAC6 = 48 nM; IC50hHDAC8 = 3.9 μM).

Figure 4.

Figure 4

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Docked poses of 6j into HDAC1 (A) and HDAC6 (B). Compound 6j is represented by orange sticks, the residues in the active sites are represented by lines and the protein is represented as cartoon. Zn2+ is represented by a gray sphere. H-bonds are shown as black dotted lines, and the red solid lines represent the metal coordination bonds.

6a, 6i, and 6j were tested on the hHDAC8 isoform (Table 2), and their selective profile was confirmed toward the HDAC6 enzyme.

Table 2. Inhibitory Activity of 6a, 6i, and 6j, as IC50 (μM), against hHDAC8a.

compd 6a 6i 6j TubA12
IC50 (μM) 1.91 ± 0.33 2.48 ± 0.67 3.19 ± 1.51 0.695
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a

Each value is the mean of at least three determinations; compounds were assayed at eight concentrations; results are expressed with SD.

We also investigated the potential affinity toward HDAC10 of the new compounds. We used the crystallized structure of the mentioned enzyme from Danio rerio due to the high identity with the human enzyme, especially in the binding site (sequence identity >44%; sequence similarity >65%) (PDB ID 6WBQ).19 The output of this calculation is presented in Figures S10 and S11. In general, we observed that our molecules are poor binders of zfHDAC10 enzyme, indicating that HDAC10 is not a preferred target for this series of derivatives, confirming HDAC6 as the main target (see Supporting Information section 4.4).

In a range of malignancies, HDAC6 has been found to be overexpressed and shown to correlate with increased tumor aggressiveness including oral squamous cell carcinoma20 and esophageal squamous cell carcinoma.21 Therefore, the new molecules were tested against leukemic, multiple myeloma, oral, and esophageal cancer cells to evaluate their antiproliferative activity and mechanism of action. Cell cycle distribution and propidium iodide (PI) analysis studies were performed on U937 and NB4 cell lines, with selected compounds 6b, 6h, and 6j. Specifically, in U937 cells, 6b exhibited cell death and a significant S phase reduction at a concentration of 10 μM (Figure S15A). 6j (10 μM) only after 48 h of treatment induces an increase of the pre-G1 phase, without significant cell cycle variation (Figure S15B). Interestingly, in the NB4 cell line, both 6b and 6h displayed a similar phenotypic effect in terms of cell death at the two time intervals 24 and 48 h (Figure S16A, B), whereas 6j shows this only at 48 h of treatment (Figure S16C). Western blot analyses on NB4 total cell extracts using compounds 6b, 6h, and 6j were also performed. Induction of acetylated tubulin was observed without a significant variation in histone acetylation state (Figure S16D–F), which was detected only after treatment with 6h and 6j at higher concentration, thus confirming selective HDAC6 inhibition. Moreover, cleavage of PARP at 24 h by all molecules indicated apoptosis at a molecular level at both 24 and 48 h at 10 μM concentration (Figure S16D–F). 6a, 6i, and 6j were preliminarily screened against the multiple myeloma (U266) cell line. All three compounds reduced the viability of U266 cells with 6j exhibiting the greatest potency (IC50 = 20.25 μM, Figure S17 and Table 5 SI). Flow cytometric analysis of these compounds in annexin V/PI stained U266 cells showed an induction of apoptotic cell death, with compound 6j exhibiting the highest potency (Figure S18).

STAT3 represents an important signal transducer and transcription factor displaying a key role in the tumorigenic process. This has been confirmed by the fact that 70% of cancers express activated STAT3.22,23 Recent reports highlighting the important crosstalk between HDAC6 and STAT3 demonstrated that HDAC6 inhibition leads to a decrease of pSTAT3 and reduce the expression of STAT3-targeted genes.24 The enhanced survival of leukemic cells in chronic lymphocytic leukemia and in multiple myeloma has been associated with the constitutive activation of the JAK/STAT3 signaling pathway.22,23 Therefore, we proceeded to test STAT3 inhibition using the HDAC6 inhibitors 6a and 6j at 5 and 10 μM concentrations in the human chronic lymphocytic leukemia cell line (MEC1)23 and at 25 μM against multiple myeloma cells (U266).22 Both compounds showed a marked decrease in the levels of pSTAT3 in both cell lines. Specifically, 6j demonstrated the most potent activity with a dose-dependent effect (Figure 5).

Figure 5.

Figure 5

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(A) Immunoblot analysis of pSTAT3 in MEC-1 cells treated with 6a or 6j (5 or 10 μM) for 30 h. Actin was used as a loading control. The histogram shows the quantification by densiometric analysis of the levels of pSTAT3 relative to actin (n = 2). Data are presented as mean value ± SD. One way ANOVA; *p < 0.05. (B) Immunoblot analysis of pSTAT3 in U266 cells treated with 6j (25 μM) for 4, 8, 16, 24, and 48 h. Actin was used as a loading control. The blot is representative of three independent experiments with similar results.

Our compounds were also screened against KYSE520 (esophageal squamous cell carcinoma), OE33 (esophageal adenocarcinoma), Ca9-22 (gingival squamous cell carcinoma), and TR-146 (buccal mucosa squamous cell carcinoma) cell lines. 6b demonstrated the highest activity against KYSE520 (IC50 = 12.76 μM), OE33 (IC50 = 5.56 μM), Ca9-22 (IC50 = 19.00 μM), and TR-146 (IC50 = 18.00 μM, Supporting Information, section 6) cell lines. Flow cytometric analysis established that 6b was able to trigger apoptosis after 48 h of treatment in the KYSE520 cell line.

Furthermore, cytotoxicity assays were performed on compounds 6a and 6b to establish the effect on mouse fibroblasts NIH3T3. Compound 6b showed a TC50 of 40 μM being slightly less toxic than 6a and 6j (TC50 of 24–27 μM, Tables 6 and 7 in the Supporting Information). Potential mutagenicity caused by the use of hydroxamic-based compounds remains a major concern affecting their druglike profile.25 To asses this property, the Ames test was carried out. Compounds 6a and 6b showed no mutagenic effect on the TA98 strain (with or without S9 activation), while low mutagenicity on the TA100 strain (above 8 μM for compound 6b or above 24– 40 μM for compounds 6a and 6j as shown in the Supporting Information, section 9.1) was detected. This effect has been reported also for FDA approved HDACi (1-4), and it is mostly ascribable to the Lossen rearrangement of the hydroxamate group, generating reactive isocyanates, which can trigger mutagenicity by damaging the DNA.26

In summary we have developed a new series of HDAC6 selective inhibitors rationally designed on the basis of the crystallographic study of compound 6a in complex with ZfHDAC6. Compound 6j resulted the best inhibitor of this series (IC50 = 48.5 nM, selectivity index of 140 over HDAC1 and 66 over HDAC8). Notably, the selectivity of these compounds toward HDAC6 over HDAC8 is in general higher respect to the previously published spiroindoline-based HDAC6 inhibitors. Cell-based studies on compounds 6a, 6b, 6h, and 6j uncovered their antiproliferative activity against several cancer cell lines. We believe that the low of activity in cell-based assays for compound 6j could be ascribable to the weak stability of the carbamate moiety during the study, which determines the decomposition of the molecule. Oral and esophageal cancers are usually refractory to most of the current therapeutic treatments; notably, our HDAC6 selective inhibitors displayed promising effect in various oral and esophageal cancer cell lines (KYSE520, OE33, Ca9-22, TR-146, and U266B, Table 3). For compounds 6h, 6b, and 6j, PARP cleavage was quantified in NB4 cell extracts, thus indicating its proapoptotic potential at the molecular level. The increased levels of tubulin acetylation without a significant variation in histone acetylation status confirmed the selectivity of 6h, 6b, and 6j for the HDAC6 isoform. Moreover, 6j was able to inhibit the phosphorylation of STAT3 in MEC1 and U266 cell lines. The studies herein discussed may pave the way for the structure-based design of novel HDAC6i as anticancer agents endowed with promising potentialities in the treatment of esophageal and oral cancers.

Table 3. Antiproliferative Activities of Compounds 6a–b and 6h–j against KYSE520, OE33, Ca9-22, TR-146, and U266B Cell Lines after 48–72 h of Drug Treatment.

IC50 (μM)a
compd KYSE520 OE33 Ca9-22 TR-146 U266B
6a 197 17.73 >1000 >1000  
6b 12.76 5.56 19 18  
6c     >1000 >1000  
6h 33.81 13.82 >1000 54  
6i     55 >1000 59.38
6j   37.83 >1000 133 20.25
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a

Each value is the mean of at least three determinations.

Acknowledgments

Support from the European Union’s Horizon 2020 (EU) Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement No. 721906-TRACT is acknowledged. Cost Action EPICHEMBIO CM1406 (G.C, M.B.) and the synchrotron beamline staff at the Advanced Photon Source (APS) for assistance, especially David Neau are also gratefully acknowledged. We thank the Northeastern Collaborative Access Team (NE-CAT) funded by the National Institute of General Medical Sciences (NIGMS) from the NIH (P30 GM124165), and US National Institutes of Health grant GM49758. The Pilatus 6M detector on beamline 24-ID-C is funded by a NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source (APS), a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. N.R. Tuscany strategic project POR-FSE 2014-2020, ‘Medicina di Precisione e Malattie Rare’(MePreMaRe), (ACE-ESCC). G.R. acknowledges the CNR-CNCCS “Rare, Neglected and Poverty Related Diseases - Schistodiscovery Project” (DSB.AD011.001.003). L.A. acknowledges MIUR20152TE5PK; VALERE: Vanvitelli per la Ricerca; Campania Regional Government Technology Platform Lotta alle Patologie Oncologiche: iCURE and Campania Regional Government FASE2: IDEAL. MIUR, Proof of Concept POC01_00043.

Glossary

Abbreviations

HDAC

histone deacetylase

HDACi

HDAC inhibitors

ZBG

zing binding group

Hsp90

heat shock protein 90

SAR

structure–activity relationship

KYSE520

esophageal squamous cell carcinoma

OE33

esophageal adenocarcinoma

Ca9-22

gingival squamous cell carcinoma

TR-146

buccal mucosa squamous cell carcinoma

PI

propidium iodide

U937

monocytic leukemia

NB4

acute promyelocytic leukemia

PARP

poly(ADP-ribose) polymerase

STAT3

signal transducer and activator of transcription 3

JAK

Janus kinase

MEC1

human chronic lymphocytic leukemia

U266

multiple myeloma cells

NIH3T3

mouse fibroblast

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00395.

  • Supplementary figures, details of the synthetic chemistry, in silico studies, and biological assays, plot of 1H and 13C NMR spectra (PDF)

Author Present Address

● M.B.: University of Napoli Federico II, Department of Pharmacy, via Montesano 49, I-80131 Napoli, Italy.

Author Present Address

□ G.C.: Wellcome Centre for Anti-Infectives Research, Drug Discovery Unit, Division of Biological Chemistry and Drug Discovery, University of Dundee, DD1 5EH Dundee, United Kingdom.

Author Present Address

■ A.G.: Promidis S.r.L., Via Olgettina, 60 Milano, Torre San Michele 1, Ospedale San Raffaele, Italy.

Author Contributions

+ A.P.S., N.R., and M.B. contributed equally to this work.

The authors declare no competing financial interest.

Notes

The atomic coordinates and crystallographic structure factors of the HDAC6 complex with inhibitor 6a has been deposited in the Protein Data Bank (www.rcsb.org) with accession code 6V7A. Authors will release the atomic coordinates and experimental data upon article publication.

Supplementary Material

ml0c00395_si_001.pdf (4.9MB, pdf)

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