Serendipitous discovery of potent human head and neck squamous cell carcinoma anti-cancer molecules: A fortunate failure of a rational molecular design

Article abstract of DOI:10.1016/j.ejmech.2017.09.075

Histone deacetylase inhibitors (HDACis) play an important role as valuable drugs targeted to cancer therapy: several HDACis are currently being tested in clinical trials. Two new potential HDACis 1a and 1d, characterized by the presence of a biphenyl-4-sulfonamide group as a connection unit between the N-{4-[(E)-(2-formylhydrazinylidene)methyl]-3-hydroxyphenyl} and the 2-hydroxy-N-(trifluoroacetyl)benzamide moiety, respectively, as two zinc-binding group (ZBG), have been designed, synthesized and tested for their biological activity. Surprisingly, compounds 1a and 12, this last exclusively obtained in place of 1d, exhibited a very low HDAC inhibitory activity. A serendipitous assay of these two compounds, conducted on three chemoresistant cell lines of head and neck squamous cell carcinoma (HNSCC), showed their antiproliferative activity at low nanomolar concentrations, better than cisplatin. In vitro, biological assays indicated that compounds 1a and 12 are able to increase acetylation of histone H3 and to interfere with the PI3K/Akt/mTOR pathway by inducing the accumulation of PTEN protein.

Full text of DOI:10.1016/j.ejmech.2017.09.075

Accepted Manuscript
Serendipitous discovery of potent human head and neck squamous cell carcinoma
anti-cancer molecules: A fortunate failure of a rational molecular design
Chiara Zagni, Venerando Pistarà, Luciana A. Oliveira, Rogerio M. Castilho, Giovanni
Romeo, Ugo Chiacchio, Antonio Rescifina
PII:
S0223-5234(17)30790-0
10.1016/j.ejmech.2017.09.075
EJMECH 9790
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 July 2017
Revised Date: 1 August 2017
Accepted Date: 29 September 2017
Please cite this article as: C. Zagni, V. Pistarà, L.A. Oliveira, R.M. Castilho, G. Romeo, U. Chiacchio,
A. Rescifina, Serendipitous discovery of potent human head and neck squamous cell carcinoma anti-
cancer molecules: A fortunate failure of a rational molecular design, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.09.075.
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ACCEPTED MANUSCRIPT
Serendipitous discovery of potent human head and neck squamous cell carcinoma anti-cancer
molecules: A fortunate failure of a rational molecular design
a
b
b
Chiara Zagni a, *, Venerando Pistarà , Luciana A. Oliveira , Rogerio M. Castilho , Giovanni
Romeo c, Ugo Chiacchio a, Antonio Rescifina a, *
a
Dipartimento di Scienze del Farmaco, Università degli Studi di Catania, V.le A. Doria, 95125
Catania, Italy.
b
Laboratory of Epithelial Biology, Department of Periodontics and Oral Medicine, University of
Michigan School of Dentistry, Ann Arbor, MI 48109-1078, USA.
c
Dipartimento Farmaco-Chimico, Università di Messina, Viale SS. Annunziata, Messina 98168,
Italy.
Corresponding authors:
Chiara Zagni chiarazagni@gmail.com; Antonio Rescifina arescifina@unict.it
Graphical Abstract
Highlights
•
•
•
•
•
Novel anticancer molecules were designed and synthesized as HDAC inhibitors
Compounds 1a and 12 showed very low inhibitory activity over HDAC
Compounds 1a and 12 exhibited excellent anti-proliferative activity against HNSCCs
Compounds 1a and 12 enhanced PTEN expression
Compounds 1a and 12 were shown to block mTOR signaling
Keywords
HDAC; PTEN; HNSCC; PI3K/Akt/mTOR; Drug design; anti-cancer
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Abstract
Histone deacetylase inhibitors (HDACis) play an important role as valuable drugs targeted to cancer
therapy: several HDACis are currently being tested in clinical trials. Two new potential HDACis 1a
and 1d, characterized by the presence of a biphenyl-4-sulfonamide group as a connection unit
between the N-{4-[(E)-(2-formylhydrazinylidene)methyl]-3-hydroxyphenyl} and the 2-hydroxy-N-
(trifluoroacetyl)benzamide moiety, respectively, as two zinc-binding group (ZBG), have been
designed, synthesized and tested for their biological activity. Surprisingly, compounds 1a and 12,
this last exclusively obtained in place of 1d, exhibited a very low HDAC inhibitory activity. A
serendipitous assay of these two compounds, conducted on three chemoresistant cell lines of head
and neck squamous cell carcinoma (HNSCC), showed their antiproliferative activity at low
nanomolar concentrations, better than cisplatin. In vitro, biological assays indicated that compounds
1a and 12 are able to increase acetylation of histone H3 and to interfere with the PI3K/Akt/mTOR
pathway by inducing the accumulation of PTEN protein.
1. Introduction
The epigenetic control of gene expression is operated through several post-translational
modifications of chromatin such as methylation, acetylation, ubiquitination, phosphorylation,
sumoylation. In particular, acetylation/deacetylation of ε-amino groups of lysine residues of histone
tails plays a relevant role in cancer development [1, 2]. Histone acyl transferases (HATs) transfer an
acetyl group from acetyl CoA to the amino group of the lysine residue to form ε-N-acetyl lysine.
This modification neutralizes the positive charge of the lysine residues of histones, loosening their
interactions with the negatively charged DNA and leading to an “open” chromatin structure that
increases the binding of transcription factors, thus activating gene transcription [3]. Therefore,
increased levels of histone acetylation are associated with enhanced transcriptional activity;
conversely, decreased levels of acetylation are related to the repression of gene expression [4, 5].
HDACis selectively alters gene transcription by chromatin remodeling and the structure
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modification of transcription complexes, triggering growth arrest, differentiation, activation of the
extrinsic and/or intrinsic apoptotic pathways, cell death and senescence [6]. Histone deacetylase
(HDAC) inhibitor activation of p21WAF1 involves changes in promoter-associated proteins,
including HDAC1 [7]. Recent studies suggested that acetylation of non-histone proteins may also
play an important role in biological effects of this class of compounds [8]. To date, more than 50
non-histone proteins with regulatory roles in cell proliferation, cell migration, and cell death have
been identified as substrates of HDACis. For this reason, HDACis have recently emerged as an
important target for therapeutic intervention in cancer and potentially other human diseases [9].
Epigenetic changes also play a key role in regulating gene expression through histone
modifications. Inhibition of HDAC constitutes a novel strategy to disrupt the population of cancer
stem cells in head and neck tumors and to create a homogeneous population of cancer cells with
biologically defined signatures and predictable behavior [10].
Figure 1 reports the structure of some HDACis: SAHA (Zolinza®), Romidepsin (Istodax®), and
Belinostat (PXD101) approved by FDA for the treatment of cutaneous T-cell lymphoma (CTCL),
Panobinostat (LBH589, Faridak) licensed for the treatment of multiple myeloma
[https://www.fda.gov], Chidamide [11] approved by the Chinese FDA for the treatment of CTCL
and TSA.
To date, more than 20 inhibitors are under preclinical and clinical investigation as single agents and
in combination therapies against different cancers. The most promising compounds in clinical trials
include MGCD-0103 (Methylgene, phase II) [12] and MS-275 (Syndax, phase II)
[https://clinicaltrials.gov] (Fig. 1).
Co-crystal structures of hydroxamate HDAC inhibitor Trichostatin A (TSA) with histone
deacetylase homologue from the hyperthermophilic bacterium Aquifex aeolicus revealed an active
site consisting of a tubular pocket, a zinc-binding site, and two Asp-His charge-relay systems, all of
which are believed to be involved in the mechanism of HDAC inhibition [13]. All the classical
HDACis (classes I, II, IV) require Zn2+ as a cofactor for their deacetylase activity [14]. HDACis
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contain three key structural elements to their pharmacophore: a zinc-binding group (ZBG), which
coordinates the zinc ion at the bottom of the long narrow active site cavity; a capping group, which
interacts both with the amino acids on the rim of the binding cavity and the protein surface; a linker
domain, whose role is to ensure the correct positioning of the two former groups and interact with
the lipophilic binding tunnel [15].
Fig. 1. Structures of some HDACis in clinical development or clinical use.
Following our recent interest in the research of more potent HDACis, we have designed and studied
in silico four potential HDAC inhibitors. Based on molecular docking results, we decided to
synthesize two of these molecules, the N-{4-[(E)-(2-formylhydrazinylidene)methyl]-3-
hydroxyphenyl}biphenyl-4-sulfonamide (1a) and the 4-[(biphenyl-4-ylsulfonyl)amino]-2-hydroxy-
N-(trifluoroacetyl)benzamide (1d); unfortunately, in place of this last we exclusively obtained the 4-
[(biphenyl-4-ylsulfonyl)amino]-2-hydroxybenzamide (12). Compounds 1a and 12, contrary to in
silico predictions, have shown a not appreciable HDAC activity. However, the synthesized
compounds, serendipitously evaluated for their antiproliferative activity on three human head and
neck squamous cell carcinoma (HNSCCs) cell lines (HN6, HN12, and HN13), exhibited an IC50 in
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the low nanomolar range. To investigate the possible mechanism of action we performed a series of
biological screenings. Considering that in a significant number of HNSCCs tumor progression
results from mutations in genes, such as PTEN and PIK3CA, that causes alterations in cell signaling
cascade PI3K/Akt/mTOR, resulting in aberrant cell growth, migration, and survival [16, 17], we
performed western blot assays that highlighted that compounds 1a and 12 cause a robust
accumulation of the tumor suppressor PTEN, that acts as a negative regulator of the
phosphatidylinositol 3-kinase (PI3K) pathway [18-21].
Despite the not encouraging obtained results on HDAC inhibition, because of compound 1a,
however, present a low activity, we conducted an immunohistochemistry assay in order to verify
the effective acetylation of histones in the cells, on both compounds, for comparison. Surprisingly,
the results indicated that the two molecules promote a dose dependent acetylation of histone H3 in
HNSCCs. These results point out that the acetylation could occur via another mechanism.
2. Results and Discussion
2.1. Molecular drug design
Nowadays, the design of new HDACis is principally focused on changing the capping group and
the linker domain given that the hydroxamic acid functionality is the best ZBG. We, on the search
of new non-hydroxamates HDACis, designed four new ZBGs on the basis of the three-dimensional
structure of the active site of the HDAC8 enzyme. In fact, with the exception of HDAC8, functional
HDACis were found as high-molecular-weight multiprotein complexes, and most purified
recombinant HDACis are enzymatically inactive [22]; therefore, HDAC8 is the best model among
mammalian HDACis, if we look at it from a structural biology perspective. So, looking at the pose
of TSA in the co-crystallized structure with the human HDAC8 (PDB ID: 1T64) and considering
that hydroxamic acid group chelates the zinc ion in a bidentate fashion and forms hydrogen bonds
with Tyr306 and Asp178 (Fig. 2A) we designed, as new ZBGs, a (2-formylhydrazinylidene)methyl
(a), a [2-(trifluoroacetyl)hydrazinylidene]methyl (b), a (2-formylhydrazinyl)carbonyl (c), and a
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(trifluoroacetyl)carbamoyl (d) moieties which could coordinate the zinc ion bi- or tridentately and
could also form hydrogen bonds with Tyr306, Asp178, and, probably, with other amino acids at the
bottom of the channel (Fig. 2B). Contemporarily, we choose as linker a para disubstituted benzene
carrying in ortho to ZBG a hydroxyl functionality that, in principle, could be further involved in the
zinc chelation. Finally, for the CAP we focused on the bifenilsulfonamide basing on an extensive
QSAR modeling study of sulfonamide HDACis [23]. The new four molecules 1a–d have been
represented in Figure 2C.
Fig. 2. A) Chemical structure of the new designed potential HDACis 1a–d. B) Interactions of TSA in the co-
crystallized structure with the human HDAC8 (PDB ID: 1T64) and proposed tridentate chelation of ZBG A with Zn2+.
The molecular docking simulations of all new compounds showed that compound incorporating the
A and D ZBG could be good candidates as HDACis with a ∆Gbind of –11.63 and –9.84 kcal/mol,
respectively. These energy values are, for 1a, better than that predicted for TSA (∆Gbind = –11.13
kcal/mol) and SAHA (∆Gbind = –10.03 kcal/mol), the two most famous HDACis, chosen as
reference compounds (Table 1). The calculated Ki values for TSA and SAHA have been about 10-
fold underestimated.
Table 1
Predicted Ki for the four designed compounds, TSA, and SAHA, and experimental Ki for the last
two.
Calculated Ki (nM) Experimental Ki (nM)
Compound
1a
∆Gbind (kcal/mol)
–11.63
a
2.9
—
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b
b
c
–8.78
–8.39
–9.84
–10.03
–11.13
363.8
—
—
—
1b
1c
1d
SAHA
TSA
703.0
60.7
44.1
6.9
480d
45d
a IC50 reported in section 2.3.1.
b Not synthetized.
c Not obtained.
d From reference [24].
The 3D and 2D sketch of the docked pose for the best scored compound 1a (Fig. 3) highlight that
this compound chelate the Zn2+ bidentately by the o-OH and the double bonded hydrazinic nitrogen
atom, whereas an extended network of hydrogen bonds establish between carbonyl oxygen and
His142 and Gln263, between NH and Asp178, and between o-OH and Tyr306, confirming the
designed previsions.
Fig. 3. 3D and 2D view of the interactions for 1a inside the binding pocket of HDAC8 (PDB ID: 1T64).
2.2. Chemistry
Basing on the molecular modeling results, and considering the about 10-fold Ki underestimation, we
choose to synthesize only compounds 1a and 1d. The synthetic route towards these two compounds
is shown in Schemes 1 and 2. The first step of the synthesis involved the conversion of p-
aminosalicylic acid 2 into the methyl ester 3 that reacted with diphenyl sulphonyl chloride in order
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to obtain the correspondent sulfonamides 4 (Scheme 1). Compound 4 was subsequently protected at
nitrogen and oxygen atoms by treatment with benzyl bromide providing compound 5. The ester 5
was reduced by lithium aluminum hydride in THF anhydrous to benzyl alcohol 6, successively
oxidized with activated MnO2 to give the aldehyde 7. The reaction of compound 7 with
formylhydrazine gives rise to compound 8 that was deprotected with catalytic hydrogenation to the
target compound 1a.
Scheme 1. Synthetic route to 1a. Reagents and conditions: a) H2SO4, CH3OH, reflux, 4 h; b) diphenylsulfonyl chloride,
Py, THF, r.t., 12 h; c) K2CO3, BnBr, acetone, reflux, 12 h; d) LiAlH4, THF, H2O, –78 °C, 5 h; e) MnO2, CH2Cl2, reflux,
12 h; f) formylhydrazine, EtOH, reflux, 12 h; g) H2, Pd/C, MeOH, r.t., 12 h.
For the synthesis of 1d, the ester 5 was hydrolyzed to acid 9 that was converted into the
corresponding chloride 10 (Scheme 2), which in turn was treated with trifluoromethylacetamide.
Unfortunately, this last step conducted to the amide 11 that was deprotected to afford compound 12.
All attempts to synthesize compound 1d failed. All the reaction carried out on compound 10 with
trifluoroacetamide at different conditions led to the amide derivative 11. This is probably due to a
high reactivity to the hydrolysis process.
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Scheme 2. The tentative synthetic route for 1d. Reagents and conditions: (a) LiOH THF/MeOH, reflux 4 h; b) SOCl2,
reflux 10 h; c) Trifluoroacetamide, CH3CN, overnight; d) H2, Pd/C, MeOH, r.t., 12 h.
Compound 12, docked into HDAC8, gave a predicted Ki of 716.8 nM (∆Gbind = –8.38 kcal/mol). In
fact, the –CONH2 moiety, combined with the hydroxylic one present in ortho to the benzene
scaffold, can be considered as a ZBG group, as suggested by the best obtained docked pose.
2.3. Biology
2.3.1. HDACis inhibition assay and acetylation of histone H3 in HNSCCs
The sulfonamides derivatives 1a and 12 were tested for inhibition of HeLa nuclear HDACis using
TSA as a reference. Surprisingly, compound 1a evaluation revealed a scarce inhibition potency with
an IC50 of 292 µM, so as the unwanted compound 12 lead to 20% of inhibition at 500 µM.
Since the molecular modeling studies showed a certain affinity of 1a with HDAC, we evaluated the
capacity of compound 1a and 12 to influence the acetylation status of histone H3. To this purpose,
the HN13 cells were treated with two different concentrations (1 and 10 nM) of 1a and 12 for 24 h.
The transcribed proteins, analyzed by a Western blot assay as described in material and method,
showed a dose dependent increasing acetylation status of histone H3 (Fig. 4), with the maximum
activity at the IC50 values. Consistently, Western blot analysis revealed that TSA alone, enhanced
acetylation of histone H3 (lys9) in HN13 cells (data not shown). Notably, the compounds showed
acetylation activity at concentration 10-fold lower than the IC50 values. Thus, our data suggest that
1a and 12 might alter acetylation of H3 and cause cell death in HNSCCs through changes in gene
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transcription.
Fig. 4. A) Immunofluorescence images of Ac.H3 (lys9), DAPI staining and merge in untreated or treated HN13 cells
with 1a, 12, and TSA as a positive control. B) Western blot for Ac. H3 (lys9) in HN13 cells in the presence or absence
of 1a and 12 at different concentrations. C) Quantification of acetyl H3 levels; **p < 0.01, ****p < 0.001.
2.3.2. Cell viability assay
Aiming at a preliminary screen for cellular activity, we tested the effect of compounds 1a and 12 on
the viability of three human head and neck squamous cell carcinoma (HNSCC) cell lines: HN6,
HN12, and HN13.
Initially, the half-maximal inhibitory concentration (IC50) was determined using the CellTiter 96TM
Aqueous non-radioactive cell proliferation kit (Promega) according to manufacturer instructions.
Interestingly, both compounds showed strong antiproliferative activities over all tested cell lines
with IC50 in the range between 4.73 and 9.85 nM (Table 2, Fig. 5).
Table 2
In vitro efficacy assay of compounds 1a and 12 on selected HNSCC cell lines and on non-cancer cells expressed as
IC50a (nM) at 24 h
Compound
HN6
HN12
HN13
184B5
4.94 ±0.12
4.73 ±0.34
5.03 ±0.08
4.99 ±0.19
9.85 ±0.42
9.66 ±0.34
1840 ±125
1955 ±98
1a
12
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Cisplatin
16660b
30000c
41650b
25540d
a Mean of three independent triplicate experiments ± standard error.
b From reference [16].
c Value at 48 h. From reference [25].
d GI50 value at 48 h. From reference [26].
Moreover, the tested compounds showed better growth inhibition than cisplatin (>3300-fold), an
established and effective treatment for HNSCC [27], at which these cell lines are particularly
resistant. Finally, to determine if the newly synthesized compounds have differential cytotoxic
effects on cancer and non-cancer cells, their cytotoxicity was also evaluated using 184B5 non-
cancer immortalized breast epithelial cell lines. We found that the antiproliferative activity of
compounds 1a and 12 was not pronounced against the noncancer cell lines (Table 2) with a >186-
fold and >200-fold selectivity, respectively, respect to the more resistant HN13 cancer cell lines.
Fig. 5. Percentage of cell viability after 24 h of treatment with 1a (A) and 12 (C). The concentration is expressed in
nanomolar. B) Representation of HNSCC receiving vehicle, 1a or 12 after 24 h.
2.3.3. Evaluation of PTEN
PTEN (phosphatase and tensin homolog deleted on chromosome ten) is one of the most frequently
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mutated tumor suppressors in human cancer. PTEN functions through the regulation of the cell
cycle, induction of tumor apoptosis, and inhibition of tumor cell growth, invasion and metastasis.
Genetic analysis of PTEN in HNSCC has demonstrated alterations in PTEN, suggesting that PTEN
may play a role in HNSCC tumorigenesis [28]. The PI3K/Akt/mTOR pathway has been shown to
play an important role in cancer [29]. Therefore, molecules that increase PTEN expression are
promising antitumoral drugs. On these bases, we further decided to evaluate if compounds 1a and
12 would have any effect over the reactivation of tumor suppressor genes. Towards this goal, we
decided to explore the activation status of phosphatase and tensin homolog (PTEN) at the protein
level. Using two different concentrations of compounds 1a and 12 (1.0 and 10.0 nM) we treated
HNSCC cell lines for 24 h and compared them to vehicle treated controls. Interestingly, we
observed that compounds 1a and 12 induced the expression of PTEN in a dose dependent manner
(Fig. 6), similar to that observed with histone H3. Moreover, administration of compounds 1a and
12 resulted in a dose dependent reduction in the levels of pS6, a protein used to access the
activations status of the PI3K/Akt/mTOR pathway. Our results are exciting once that they
demonstrate the molecular mechanism by which compounds 1a and 12 reduced cellular viability of
HNSCC cancer cells.
Fig. 6. A) Western blot of HNSCC cell line treated with compounds 1a and 12 (1.0 and 10.0nM) for 24 h. Note the
accumulation of PTEN upon and reduced activation of the PI3K/Akt/mTOR pathway evident by reduced accumulation
of pS6, a marker for an active mTOR pathway. GAPDH served as a loading control. B) Quantification of PTEN levels;
**p < 0.01, ****p < 0.001.
3. Conclusions
Two new molecules, N-{4-[(E)-(2-formylhydrazinylidene)methyl]-3-hydroxyphenyl}biphenyl-4-
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sulfonamide (1a) and 4-[(biphenyl-4-ylsulfonyl)amino]-2-hydroxy-N-(trifluoroacetyl)benzamide
(1d) have been designed and synthesized; unfortunately, in place of 1d we exclusively obtained the
4-[(biphenyl-4-ylsulfonyl)amino]-2-hydroxybenzamide (12). Compounds 1a and 12 were assayed,
with negative results, as HDACis. Fortunately, these compounds were evaluated as antitumoral
agents, exhibiting potent antiproliferative activities against HNSCCs cell lines with an IC50 in the
low nanomolar range (4.73–9.85 nM). Both compounds are also able to increase the acetylation
status of histone H3, a classical marker of HDAC inhibition. Finally, these two compounds interfere
with PI3K/Akt/mTOR pathway by increasing PTEN expression and downregulate mTOR, as shown
by western blot results.
With these results, we can speculate that the antitumoral activity exhibited by 1a and 12 is due to a
combined effect over histone H3 acetylation and PI3K/Akt/mTOR pathway.
Considering the significance of PTEN as a tumor suppressor gene and that germline mutations in
PTEN are associated with several autosomal dominant hamartoma syndromes [20] including
Cowden’s disease, Bannayan-Riley-Ruvalcaba syndrome, and Lhermitte-Duclos syndrome, the
importance of the results obtained with these two compounds deserve to be further deepened.
Moreover, also considering that HNSCC is the most frequent malignant tumor of the head and neck
region and it is the sixth leading cancer by incidence worldwide, that the five-year survival rate of
patients with HNSCC is about 40-50% [30], and that, despite advances in treatment, the mortality
rate of patients treated with platinum-based chemotherapy has not changed markedly over the last
few decades, due to chemoresistance [31], we have engaged further studies to better identify the
antitumoral activity of these new compounds.
4. Experimental Section
4.1. In silico studies
4.1.1. Preparation of protein and ligands
The fully optimized crystal structure of the HDAC8/TSA complex (PDB ID: 1T64) was retrieved
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from the PDB_REDO databank. The PDB_REDO procedure combines re-refinement and
rebuilding within a unique decision-making framework to improve structures, using a variety of
existing and custom-built software modules. This approach allows to choose an optimal refinement
protocol (e.g. anisotropic, isotropic or overall B-factor refinement, TLS model) and to optimize the
geometry versus data-refinement weights. Next, it proceeds to rebuild side chains and peptide
planes before a final optimization round [32].
From the refined structure were retained only the A chain, one TSA molecule, and Zn2+ atom. Only
for docking purpose, the 3D structure of the TSA was extracted from the PDB files whereas all
other ligands were built using Gabedit (2.4.8) software [33] and all geometries were fully
optimized, in the same software, with the semi-empirical PM6 [34] Hamiltonian implemented in
MOPAC2016 (17.130W) [35].
4.1.2 Docking protocol
Macromolecule and ligands were prepared with Vega ZZ (3.1.1) [36] assigning Gasteiger charges
to the protein and AM1BCC ones to the ligand. Fine docking was performed by applying the
Lamarckian genetic algorithm (LGA) implemented in AutoDock 4.2.6 [37] in combination with a
specialized potential describing the interactions of zinc-coordinating ligands, named AutoDock4Zn
[38]. The ligand-centered maps were generated by the program AutoGrid (4.2.6) with a spacing of
0.375 Å and dimensions that encompass all atoms extending 5 Å from the surface of the ligand. All
of the parameters were inserted at their default settings. In the docking tab, the macromolecule and
ligand are selected, and GA parameters are set as ga_runs = 100, ga_pop_size = 150, ga_num_evals
= 20000000, ga_num_generations = 27000, ga_elitism = 1, ga_mutation_rate = 0.02,
ga_crossover_rate = 0.8, ga_crossover_mode = two points, ga_cauchy_alpha = 0.0, ga_cauchy_beta
= 1.0, number of generations for picking worst individual = 10.
Because no water molecule is directly involved in complex stabilization they were not considered in
the docking process. All protein amino acidic residues were kept rigid whereas all single bonds of
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ligands were treated as full flexible.
4.2. Chemistry. General
Solvents and reagents were used as received from commercial sources. Melting points were
determined with a Kofler apparatus and are reported uncorrected. Elemental analyses were
performed with a Perkin–Elmer elemental analyzer. NMR spectra (1H NMR recorded at 500 MHz,
13C NMR recorded at 125 MHz) were obtained on Varian Instruments and are referenced in ppm
relative to TMS or the solvent signal. Thin-layer chromatographic separations were performed on
Merck silica gel 60-F254 pre coated aluminum plates. Flash chromatography was accomplished on
Merck silica gel (200–400 mesh).
Analyses indicated by the symbols of the elements or functions were within 0.4% of the theoretical
values.
All reagents were purchased from Aldrich Co. All solvents were dried according to literature
methods.
4.2.1. Synthesis of methyl 4-([1,1'-biphenyl]-4-ylsulfonamido)-2-hydroxybenzoate (4)
To a solution of biphenyl-4-sulfonyl chloride (10.58 g, 41.86 mmol) in dry THF (100 mL) at 0 °C,
compound 3 (7.00 g, 41.86 mmol) in dry THF (50 mL) and dry pyridine (16.94 mL, 5 eq.) was
added dropwise. The reaction mixture was allowed to warm to room temperature, stirred overnight
and the solvent was evaporated under reduced pressure. The residue was purified by flash
chromatography (ethyl acetate/cyclohexane 30:70) to afford the desired product 4 (88% yield) as
1
yellow sticky solid. H NMR spectrum (CD3COCD3, 500 MHz): δ = 3.87 (s, 3H, CH3), 6.80–6.87
13
(m, 2H, CH), 7.38–7.99 (m, 10H, CH), 10.81 (s, 1H, OH). C NMR (CDCl3, 125 MHz): 52.67,
106.63, 108.71, 110.73, 128.07, 128.51, 128.61, 129.42, 129.93, 132.11, 145.73, 146.59. HRMS
(ESI) calcd. for C20H17NO5S [M+H]+, 383.0827; found, 383.0825.
4.2.2. Synthesis of methyl 4-(N-benzyl-[1,1'-biphenyl]-4-ylsulfonamido)-2-(benzyloxy)benzoate (5)
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Compound 4 (2.87 g, 7.48 mmol) was dissolved in acetone (100 mL) and mixed with K2CO3 (5.17
g, 37.42 mmol, 5 eq.). The mixture was stirred at room temperature for 15 minutes. Then benzyl
bromide (4.45 mL, 5 eq) was added and the reaction was heated under reflux for 12 h and
neutralized with 1 M solution of HCl. After concentration under reduced pressure, the residue was
purified through flash column chromatography (ethyl acetate/cyclohexane 20:80) to give the desired
product 5 (93% yield) as light grey solid foam. 1H NMR spectrum (CDCl3, 500 MHz): δ = 3.85 (s,
13
3H, CH3), 4.73 (s, 2H, CH2), 4.98 (s, 2H, CH2), 6.60-7.70 (m, 22H, CH). C NMR (CDCl3, 125
MHz): 52.02, 54.31, 70.62, 114.87, 119.56, 119.74, 126.74, 127.27, 127.37, 127.50, 127.81,
128.16, 128.41, 128.46, 128.51, 128.62, 129.09, 131.98, 135.25, 136.21, 136.66, 139.01, 143.46,
145.84, 158.22, 165.99. HRMS (ESI) calcd. for C34H29NO5S [M+H]+, 563.1766; found, 563.1762.
4.2.2. Synthesis
of N-benzyl-N-(3-(benzyloxy)-4-(hydroxymethyl)phenyl)-[1,1'-biphenyl]-4-
sulfonamide (6)
A solution of ester 5 (1.75 g, 3.10 mmol) in THF (60 mL) at –78 °C was treated with LiAlH4 (0.35
g, 9.31 mmol, 3 eq.). The reaction mixture was stirred at –78 °C until completion (generally 6 h) as
determined by TLC. The cooling bath was then removed, and the reaction mixture was quenched
with a 1 M solution of HCl followed by extraction of the aqueous layer with ethyl acetate. The
combined organic layers were washed with water and brine, dried over Na2SO4, and concentrated in
vacuum. The combined organic layers were washed with water and brine, dried with Na2SO4, and
concentrated in vacuum. Chromatography on silica gel with ethyl acetate/cyclohexane 30:70 as the
1
eluent afforded the targeted alcohol 6 (yield 71%) as light yellow sticky solid. H NMR spectrum
(CDCl3, 500 MHz): δ = 4.62 (s, 2H, CH2), 4.72 (s, 2H, CH2), 4.89 (s, 2H, CH2), 6.52-7.74 (m, 22H,
13
CH). C NMR (CDCl3, 125 MHz): 54.88, 61.43, 70.17, 113.65, 120.36, 127.28, 127.40, 127.67,
128.14, 128.27, 128.37, 128.41, 128.55, 128.65, 129.08, 129.29, 135.75, 136.21, 137.07, 139.15,
139.36, 145.60, 156.33. HRMS (ESI) calcd. for C33H28N2O4S [M+H]+, 536.1896; found, 536.1900.
4.2.3. Synthesis of N-benzyl-N-(3-(benzyloxy)-4-formylphenyl)-[1,1'-biphenyl]-4-sulfonamide (7)
16

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The alcohol 6 (0.87 g, 1.63 mmol) was stirred with anhydrous CH2Cl2 (50 mL) and heated slowly
until reflux. Activated MnO2 (1.42 g, 16.0 mmol, 10 eq.) was added in small portions over 15 min.
Reflux was maintained for 10h after which the reaction was monitored by TLC. The reaction
mixture was cooled to room temperature and filtered through celite using CH2Cl2 for washings. The
yellow filtrate was concentrated under vacuum a purified by flash column chromatography (ethyl
1
acetate/cyclohexane 10:90) to raise the aldehyde 7 (63% yield) as yellow solid foam. H NMR
spectrum (CDCl3, 500 MHz): δ = 4.77 (s, 2H, CH2), 5.03 (s, 2H, CH2), 6.62-7.70 (m, 22H, CH),
13
10.40 (s, 1H, CHO). C NMR (CDCl3, 125 MHz): 54.14, 70.69, 114.48, 119.32, 124.04, 127.30,
127.35, 127.60, 127.89, 128.12, 128.38, 128.55, 128.73, 129.13, 135.18, 135.58, 136.60, 138.98,
145.69, 146.03, 160.93, 188.77. Anal. HRMS (ESI) calcd. for C33H28N2O4S [M+H]+, 534.1739;
found, 534.1735.
4.2.4. Synthesis of (N-benzyl-N-(3-(benzyloxy)-4-((2-formylhydrazono)methyl)phenyl)-[1,1'-
biphenyl]-4-sulfonamide (8)
Aldehyde 7 (0.20 g, 0.37 mmol) was heated in boiling EtOH (20 mL) until complete dissolution. N-
formyl hydrazine (0.07 g, 1.12 mmol, 3 eq.) was added portionwise and the reaction maintained
under reflux for 12 h. The mixture was then extracted with ethyl acetate, washed with brine, dried
over Na2SO4, and concentrated in vacuum. Chromatography on silica gel, using a gradient of ethyl
acetate/cyclohexane and 0.1% of triethylamine as eluent, gave product 8 (79% yield) as light yellow
1
sticky solid. H NMR (CDCl3, 500 MHz): δ = 4.75 (s, 2H, CH), 4.93 (s, 2H, CH), 6.58-8.15 (m,
23H, CH), 8.72 (d, 1H, CHO), 9.28 (bs, 1H, NH). 13C NMR (CDCl3, 125 MHz): 61.44, 70.17,
113.66, 120.37, 127.29, 127.41, 127.67, 127.49, 128.15, 128.28, 128.38, 128.42, 128.55, 128.66,
129.01, 129.30, 135.76, 136.22, 137.08, 139.16, 139.35, 145.61, 156.34. HRMS (ESI) calcd. for
C33H28N2O4S [M+H]+, 576.1957; found, 576.1951.
4.2.5. Synthesis of N-{4-[(E)-(2-formylhydrazinylidene)methyl]-3-hydroxyphenyl}biphenyl-4-
sulfonamide (1a)
17

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Pd/C (10%, 0.2 equiv) was added to a solution of 8 (0.5 g, 0.87 mmol) in ethyl acetate (10 mL). The
mixture was refluxed under hydrogen at atmospheric pressure. After consumption of the required
volume of hydrogen, the mixture was filtered on Celite, and the filtrate was concentrated under
reduced pressure. The solid was purified by column chromatography (ethyl acetate/cyclohexane
1
40:60) (81% yeld) as pale yellow solid; m.p. 243–244 °C. H NMR (CD3COCD3, 500 MHz): δ =
6.84–8.18 (m, 13 H), 8.36 (s, 1H, syn-CHO), 8.74 (s, 1H, anti-CHO), 9.42 (bs, 1H, NH), 10.34 (bs,
NH), 10.67 (bs, 1H, CHO), 11.42 (bs, 1H, OH). 13C NMR (CD3COCD3, 125 MHz): 107.65, 111.23,
111.63, 114.89, 115.26, 128.06, 128.39, 128.41, 128.60, 129.38, 129.40, 129.94, 132.38, 132.92,
139.57, 139.85, 141.72, 142.03, 146.33, 147.85, 150.63, 157.15, 159.17, 164.40. HRMS (ESI)
calcd. for C20H18N3O4S [M+H]+, 396.1018; found, 396.1021.
4.2.6. Synthesis of 4-[benzyl(biphenyl-4-ylsulfonyl)amino]-2-(benzyloxy)benzoic acid (9)
To a solution of ester 5 (2.46 g, 4.36 mmol) in a THF/H2O mixture (1:1, 50 mL) LiOH (0.52 g,
21.82 mmol, 5 eq.) was added. The reaction proceed under reflux for 24 h. The reaction mixture
was netraulized with a 6M solution of HCl and extracted with EtOAc. The combined organic layer
was collected, dried over anhydrous Na2SO4 and concentrated under reduced pressure to give a
white residue, which was purified by silica gel chromatography (EtOAc: Hexane, 30:70) to give 9
as off white solid foam in 76% yield. 1H NMR (CDCl3, 500 MHz): δ = 4.82 (d, 2H, CH2), 4.99 (d,
2H, CH2), 6.79-7.94 (m, 22H, CH), 12.68 (s, 1H, OH). 13C NMR (CDCl3, 125 MHz): 54.03, 72.17,
114.83, 117.29, 119.91, 127.25, 127.62, 127.81, 127.92, 128.04, 128.34, 128.56, 128.67, 129.00,
129.01, 133.69, 134.19, 134.97, 136.35, 139.35, 144.72, 146.09, 157.39, 165.12. HRMS (ESI)
calcd. for C33H28NO5S [M+H]+, 550.1688; found, 550.1691.
4.2.7. Synthesis of 4-[benzyl(biphenyl-4-ylsulfonyl)amino]-2-(benzyloxy)benzoyl chloride (10)
To the acid 9 (0.85 g, 1.55 mmol) SOCl2 (8 mL) was added. The reaction was refluxed for 4 h after
which the excess thionyl chloride was removed under vacuum and the product was used without
further purification.
4.2.8. Synthesis of 4-[benzyl(biphenyl-4-ylsulfonyl)amino]-2-(benzyloxy)benzamide (11)
18

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To chloride 10 (0.60 g, 1,06 mmol), in CH3CN (30 mL), trifluoracetamide (0.18 g, 1.59 mmol, 1,5
eq.) was added. The reaction was conducted for 12 h. Finally, the product was purified by flash
1
chromatography (EtOAc/Hexane, 40:60) to give 11 (64% yield) as white sticky solid. H NMR
(CDCl3, 500 MHz): δ = 4.77 (s, 2H, CH2), 5.03 (s, 2H, CH2), 5.63 (bs, 2H, NH2) 6.61-8.05 (m, 22
H, CH). 13C NMR (CDCl3, 125 MHz): 52.02, 72.39, 114.86, 117.08, 119.95, 126.33, 127.28,
127.65, 127.95, 128.06, 128.24, 128.37, 128.60, 128.72, 129.12, 133.88, 133.97, 135.00, 136.35,
138.88, 144.88, 146.13, 157.27, 164.73. HRMS (ESI) calcd. for C33H28N2O4S [M+H]+, 548.1770;
found, 548.1775.
4.2.9. Synthesis of 4-[(biphenyl-4-ylsulfonyl)amino]-2-hydroxybenzamide (12)
Pd/C (0.04 g, 0.338 mmol) was added to a solution of 11 (0.2 g, 0.36 mmol) in ethyl acetate (10
mL). The mixture was refluxed under hydrogen at atmospheric pressure. After consumption of the
required volume of hydrogen, the mixture was filtered on Celite, and the filtrate was concentrated
under reduced pressure. The solid was purified by column chromatography (ethyl
1
acetate/cyclohexane 40:60) to give 12 (80% yield) as white solid; m.p. 285–286 °C. H NMR
(CD3COCD3, 500 MHz): δ = 6.78–7.89 (m, 12 H), 7.22 (bs, 2H, NH2), 9.45 (bs, 1H, NH), 13.11 (s,
1H, OH). 13C NMR (CD3COCD3, 125 MHz): 107.67, 109.99, 110.62, 110.89, 128.08, 128.47,
128.59, 129.41, 129.60, 129.63, 129.94, 139.54, 139.85, 144.30, 146.43, 164.32, 173.38. HRMS
(ESI) calcd. for C19H17N2O4S [M+H] +, 369.0909; found, 369.0904.
4.3. Biological Evaluation
4.3.1. Measurement of HDAC Activity against HeLa Cell Nuclear Extract
HDAC inhibition was measured by a HDAC fluorometric drug screening kit procured from
Biovision (catalog number K340-100). The kit was used according to the manufacturer’s
guidelines. The candidate compounds, assay buffer, and HDAC fluorometric substrate, which
comprises an acetylated lysine side chain, were added to HeLa nuclear extracts in a 96-well plate
and incubated at 37 °C for 60 min. The reaction was stopped by adding lysine developer, and the
19

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mixture was incubated for another 30 min at 37 °C. An additional positive control included
incubation with double-distilled water, and the inhibitor control consisted of incubation with
Trichostatin A (TSA) at 20 µM. HDAC activities were quantified by a fluorescence plate reader
with excitation at 370 nm and emission at 450 nm. The data analysis was performed using
GraphPad Prism 6.0.
4.3.2. Cell Lines and Cell Culture
For cell culture experiments, human head and neck squamous cell carcinoma (HNSCCs) were
obtained from University of Michigan (Ann Arbor, Michigan) All cancer cell lines were grown at
37 °C under humidified air supplemented with 5% CO2 in DMEM containing 10% fetal calf serum,
120 µg/mL streptomycin. The cells were grown to 70–80% confluence before using them for the
appropriate assays.
4.3.3. MTT Cell Viability Assay
The rate of cell survival under the action of test substances was evaluated by an improved MTT
assay as previously described.[39] The assay is based on the ability of viable cells to metabolize
yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Applichem,) to violet
formazan that be detected spectrophotometrically. In brief, HN6, HN12, and HN13 cell lines were
seeded in 96-well plates (Corning, USA). After 24 h, cells were exposed to increased concentrations
of the test compounds. Incubation was ended after additional 24 h, and cell survival was determined
by addition of MTT solution (5 mg/mL in phosphate buffered saline). The formazan precipitate was
dissolved in DMSO (VWR, USA). Absorbance was measured at 590 nm in an FLUOstar
microplate reader (BMG LabTech).
4.3.4. Western Blot
HN13 cells were treated with compounds 1a and 12 and lysed with lysis buffer containing 0.2 M
20

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Tris pH 7.5, SDS 20%, 14.3 M 2-betamercaptoetanol and protease inhibitors at 4 °C, and the
supernatant was obtained after centrifuged at 16000 rpm, 4 °C for 10 min. A BCA protein assay
reagent kit (Beyotime Institute of Biotechnology) was used to quantify the protein concentration.
Identical amounts of protein from each sample (30 µg) were applied for SDS-PAGE and then
transferred to the polyvinylidene difluoride membrane (Millipore, USA). The membranes were
blocked for 1h at room temperature with 5% milk and incubated with PTEN (Cell Signaling
Technology, 9559), Antiacetyl-Histon H3 (Cell Signaling, USA) and GAPDH (EMD Millipore,
CB1001) primary antibodies. Membranes were then incubated with the IgG-HRP conjugated
secondary antibody, and immunoblots were developed using a chemiluminescent-HRP substrate.
4.3.5. Immunofluorescence
HN13 were seeded onto glass coverslips, incubated at 37 °C overnight and treated with compounds
1a and 12 for 24 h. The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.5%
Triton X-100, washed with PBS and blocked with 3% bovine serum albumin (BSA)/PBS. Samples
were incubated in anti-AcH3 primary antibody (Cell Signaling, USA) for 2 h, followed by
incubation with TRITC-conjugated anti-rabbit (Santa Cruz, USA) for 1 h. The samples were then
stained with Hoechst 33342 (Invitrogen, H357C) diluted to 1:ꢀ5000 in H2O. Coverslips were
mounted in Fluoroshield (Sigma, USA) mounting medium. Images were taken using a QImaging
ExiAqua monochrome digital camera attached to a Nikon Eclipse 80i Microscope (Nikon, Melville,
NY) and visualized with QCapturePro software.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/
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Biomol. Screen., 9 (2004) 506-515.
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The changes have been marked in yellow
Please, arrange the paragraph 2.1 as follow:
2.1. Molecular drug design
Nowadays, the design of new HDACis is principally focused on changing the capping group and
the linker domain given that the hydroxamic acid functionality is the best ZBG. We, on the search
of new non-hydroxamates HDACis, designed four new ZBGs on the basis of the three-dimensional
structure of the active site of the HDAC8 enzyme. In fact, with the exception of HDAC8, functional
HDACis were found as high-molecular-weight multiprotein complexes, and most purified
recombinant HDACis are enzymatically inactive [22]; therefore, HDAC8 is the best model among
mammalian HDACis, if we look at it from a structural biology perspective. So, looking at the pose
of TSA in the co-crystallized structure with the human HDAC8 (PDB ID: 1T64) and considering
that hydroxamic acid group chelates the zinc ion in a bidentate fashion and forms hydrogen bonds
with Tyr306 and Asp178 (Fig. 2B) we designed, as new ZBGs, a (2-formylhydrazinylidene)methyl
(a), a [2-(trifluoroacetyl)hydrazinylidene]methyl (b), a (2-formylhydrazinyl)carbonyl (c), and a
(trifluoroacetyl)carbamoyl (d) moieties (Fig. 2A) which could coordinate the zinc ion bi- or
tridentately and could also form hydrogen bonds with Tyr306, Asp178, and, probably, with other
amino acids at the bottom of the channel (Fig. 2B). Contemporarily, we choose as linker a para
disubstituted benzene carrying in ortho to ZBG a hydroxyl functionality that, in principle, could be
further involved in the zinc chelation. Finally, for the CAP we focused on the bifenilsulfonamide
basing on an extensive
QSAR modeling study of sulfonamide HDACis [23]. The new four molecules 1a–d have been
represented in Figure 2A.
Fig. 2. A) Chemical structure of the new designed potential HDACis 1a–d. B) Interactions of TSA in the co-crystallized
structure with the human HDAC8 (PDB ID: 1T64) and proposed tridentate chelation of the ZBG of 1a with Zn2+.

ACCEPTED MANUSCRIPT
In paragraph 2.3.1. lines 7 and 9 please substitute:
“The transcribed proteins, analyzed by a Western blot assay as described in material and method
…”
with
“The transcribed proteins, analyzed by an immunofluorescence assay as described in material and
method …”
and
“Consistently, Western blot analysis revealed that TSA alone, …”
with
“Consistently, the immunofluorescence analysis revealed that TSA alone, …”