Structure-activity relationship and mechanistic studies for a series of cinnamyl hydroxamate histone deacetylase inhibitors

Article abstract of DOI:10.1016/j.bmc.2021.116085

Histone deacetylases (HDACs) are a family of enzymes that modulate the acetylation status histones and non-histone proteins. Histone deacetylase inhibitors (HDACis) have emerged as an alternative therapeutic approach for the treatment of several malignanc

Full text of DOI:10.1016/j.bmc.2021.116085

Bioorg. Med. Chem. 35 (2021) 116085
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure-activity relationship and mechanistic studies for a series of
cinnamyl hydroxamate histone deacetylase inhibitors
a
,
1
,
2
b
,
1
c,d
Maurício Temotheo Tavares
, Larissa Costa de Almeida , Thales Kronenberger
,
e
a
Glaucio Monteiro Ferreira , Thain a´ Fujii de Divitiis ,
M oˆ nica Franco Zannini Junqueira Toledo , Neuza Mariko Aymoto Hassimotto ,
Jo a˜ o Agostinho Machado-Neto , Letícia Veras Costa-Lotufo , Roberto Parise-Filho
a
f
b
b
a,*
a
Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of S a˜ o Paulo, S a˜ o Paulo, Brazil
Department of Pharmacology, Institute of Biomedical Sciences, University of S a˜ o Paulo, S a˜ o Paulo, Brazil
b
c
Department of Oncology and Pneumonology, Internal Medicine VIII, University Hospital Tübingen, Otfried-Müller-Straße 10, DE 72076 Tübingen, Germany
School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, 70211 Kuopio, Finland
Laboratory of Molecular Biology Applied to Diagnosis (LBMAD), Department of Pharmacy, Faculty of Pharmaceutical Sciences, University of S a˜ o Paulo, S a˜ o Paulo, SP,
d
e
Brazil
f
Food Research Center-(FoRC-CEPID) and Department of Food Science and Nutrition, Faculty of Pharmaceutical Science, University of S a˜ o Paulo, S a˜ o Paulo, SP, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Histone deacetylases (HDACs) are a family of enzymes that modulate the acetylation status histones and non-
histone proteins. Histone deacetylase inhibitors (HDACis) have emerged as an alternative therapeutic
approach for the treatment of several malignancies. Herein, a series of urea-based cinnamyl hydroxamate de-
rivatives is presented as potential anticancer HDACis. In addition, structure–activity relationship (SAR) studies
have been performed in order to verify the influence of the linker on the biological profile of the compounds. All
tested compounds demonstrated significant antiproliferative effects against solid and hematological human
tumor cell lines. Among them, 11b exhibited nanomolar potency against hematological tumor cells including
Jurkat and Namalwa, with IC50 values of 40 and 200 nM, respectively. Cellular and molecular proliferation
studies, in presence of compounds 11a-d, showed significant cell growth arrest, apoptosis induction, and up to
HDAC6
Selectivity
Hematological malignancies
Cytotoxicity
Acetylated -tubulin
α
4
3-fold selective cytotoxicity for leukemia cells versus non-tumorigenic cells. Moreover, compounds 11a-
d increased acetylated -tubulin expression levels, which is phenotypically consistent with HDAC inhibition, and
α
indirectly induced DNA damage. In vitro enzymatic assays performed for 11b revealed a potent HDAC6 inhibitory
activity (IC50: 8.1 nM) and 402-fold selectivity over HDAC1. Regarding SAR analysis, the distance between the
hydroxamate moiety and the aromatic ring as well as the presence of the double bond in the cinnamyl linker
were the most relevant chemical feature for the antiproliferative activity of the series. Molecular modeling
studies suggest that cinnamyl hydroxamate is the best moiety of the series for binding HDAC6 catalytic pocket
whereas exploration of Ser568 by the urea connecting unity (CU) might be related with the selectivity observed
for the cinnamyl derivatives. In summary, cinnamyl hydroxamate derived compounds with HDAC6 inhibitory
activity exhibited cell growth arrest and increased apoptosis, as well as selectivity to acute lymphoblastic leu-
kemia cells. This study explores interesting compounds to fight against neoplastic hematological cells.
1
. Introduction
were registered by health agencies.1,2 Cancer can be characterized by
uncontrolled cell proliferation, which arises due to the accumulation of
Cancer remains one of the most prevalent causes of death worldwide.
In 2018, >18 million cases were diagnosed and >9.5 million deaths
multiple genetic mutations and epigenetic modifications on targets that
regulate cell proliferation and apoptosis. The genetic aspect results from
*
Corresponding author.
E-mail address: roberto.parise@usp.br (R. Parise-Filho).
These authors contributed equally.
1
2
M.T.T.: Department of Molecular Medicine, Scripps Research, Jupiter, FL 33458, United States.
https://doi.org/10.1016/j.bmc.2021.116085
Received 5 October 2020; Received in revised form 4 February 2021; Accepted 12 February 2021
Available online 23 February 2021
0
968-0896/© 2021 Elsevier Ltd. All rights reserved.

M.T. Tavares et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116085
genotypic modifications that are transferred from a mutated cell to their
descendants. Epigenetic aspects are a result of reversible changes in
chromatin folding, through mechanisms such as the acetylation rate of
date: vorinostat (SAHA, 1), romidepsin (prodrug, 2), panobinostat (3),
belinostat (4), and chidamide (5) (Fig. 1A).2
1–23
A typical HDACi con-
tains a zinc-binding group (ZBG) that coordinates with the active site’s
3
2+
histones and DNA methylation. Those modifications can modulate the
expression level of different genes.4
Zn at the bottom of the catalytic cavity; a linker unity to occupy the
hydrophobic channel of the enzyme; and a capping group (Cap) that can
explore additional interactions all over the surface of the enzyme
(Fig. 1A).24 Recent results suggest that the connecting unit (CU,
Fig. 1A), the region that links Cap and linker, may strongly affect the
The homeostatic balance of histones acetylation, under normal
physiological conditions, is modulated by a dynamic equilibrium be-
tween histone acetyltransferases (HATs) and histone deacetylases
HDACs).5 HATs increase acetylation of histones, resulting in a less
condensed segment of DNA, which is prone to transcription. Meanwhile,
HDACs regulate target genes through deacetylation of -amino groups of
selectivity of the ligand towards HDAC isozymes. Moreover, the
25
(
phenyl hydroxamate moiety (Fig. 1B) has been recognized as a useful
ZBG and also proved to be a suitable hydrophobic linker for the gener-
ε
ation of selective HDAC6is.2
6–35
Furthermore, the cinnamyl hydrox-
lysine residues in histones, thus promoting DNA condensation and,
6
therefore, transcriptional silencing. Moreover, HDACs also modify non-
amate moiety (Fig. 1B) has high potency against all HDAC isozymes and
it is found in the approved HDACis 3 and 4, as well as in compounds
under clinical trials, thus being an attractive scaffold for the design of
histone proteins, which regulate several cell functions like proliferation,
migration and apoptosis independently from the chromatin acetylation
7
–9
36,37
status.
further HDACis.
HDACs overexpression is involved in the regulation of oncogenic
transcription factors and they are involved with several types of solid
and hematological malignancies, such as prostate, gastric, colon, cer-
vical, and breast cancer, as well as B- and T-cell lymphoma, Hodgkin
In general, pan-HDACis are involved in chromosome remodeling,
3
8
cell cycle arrest, apoptosis, and cytotoxicity in cancer cells. In addition
to these effects, there is growing evidence demonstrating that treatment
with pan-HDACis increases differentiation on antigen expression, which
enhances the expression of major histocompatibility complex (MHC)
lymphoma, and chronic lymphocytic leukemia.1
0–18
There are eighteen
known mammalian HDACs, which can be classified into four classes
according to their similarity with yeast deacetylases: class I (HDAC1, 2,
class I and II on the cell surface. This event elevates immunogenicity in
terms of increased expression of CD25, CD40, or CD86.3
9,40
Addition-
3
, and 8), class IIa (HDAC4, 5, 7, and 9), class IIb (HDAC6 and HDAC10),
ally, it has been reported that HDACis can down-regulate the expression
of the immunosuppressive molecule PD-L1 in melanoma cells by
1
9
class III or sirtuins (SIRT 1–7), and class IV (HDAC11). Class I, II, and
IV HDACs require a zinc ion (Zn2 ) as a cofactor for exerting their hy-
drolytic activity and are also referred to as the conventional HDACs. In
contrast, the SIRT 1–7 are dependent on nicotinamide adenine dinu-
+
affecting the recruitment and activation of STAT3.
41,42
Therefore, it is
suggested that HDACis antitumor activity is mediated by two comple-
mentary mechanisms related to inhibition of cell proliferation as well as
+
20
43
cleotide (NAD ) for their catalytic activity.
through improvement of the immune response.
HDACs have been proven to be promising therapeutic targets for
cancer treatment, particularly of hematological malignancies, based on
the successful clinical approval of five HDAC inhibitors (HDACis) to
However, phase I/II clinical trials of pan-HDACi monotherapy (e.g.
1, 3, and quisinostat) exhibited limited efficacy and tolerability while
presenting several side-effects, such as hematological toxicity, fatigue,
Fig. 1. (A) Classical pharmacophore model of HDACis and the approved HDACis. (B) Design of the urea-based cinnamyl hydroxamate HDACis. CU: connecting unit;
ZBG: zinc-binding group.
2

M.T. Tavares et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116085
nausea, and clinical abnormalities.41 The broad inhibition of pan-
HDACis can lead to toxicity as a result of unwanted off-target effects,
thus impairing their clinical usage. Therefore, there is an emerging in-
terest in the investigation of the molecular mechanisms related to the
antitumor effects of HDACis.44 Herein, we have examined a series of
urea-based HDACi in which the cinnamyl hydroxamate linker-ZBG
moiety has been changed (Fig. 1B). For instance, we have designed a
series of cinnamyl hydroxamate inhibitors as well as their phenyl and di-
hydrocinnamyl analogues (Fig. 1B). Moreover, the urea group has been
selected as CU of the series as an attempt for exploring additional in-
teractions at the rim of the HDAC catalytic cavity (Fig. 1B). These efforts
have been made in order to assess the influence of these changes on the
cytotoxic activity of the compounds against solid and hematological
tumor cells, thus focusing on improved compounds with drug-like
properties and clinical potential. Mechanistic studies of cell death,
enzymatic inhibiton, as well as in silico SAR studies also have been made.
Finally, we describe the development of novel compounds with potent
HDAC inhibition and cytotoxic profile against tumorigenic cells.
2.2. Cell cytotoxic screening assay
The cytotoxic effects of compounds 8a-d, 11a-d, and 14a-d were
tested against solid (MCF-7, breast cancer; and HCT-116, colon carci-
noma) and hematological (Jurkat, T-cell leukemia; and Namalwa, Bur-
kitt’s lymphoma) tumor cells. Vorinostat (SAHA, 1) and doxorubicin
were included in the experiments as positive controls. The results were
expressed as half-maximal inhibitory concentration (IC50
) values
(Table 1). Most analogues presented cytotoxic activities against HCT-
116, Jurkat and Namalwa cell lines, but the cells of hematological
origin were found to be more sensitive to the influence of the compounds
than those from solid tumors. Regarding the activity against Jurkat,
compound 11b (0.04
potent analogues, remarkably 11b was 2.5-fold more active than 1
(0.10 M). For Namalwa cells, compounds 8a, 8b, 11a-d, 14a, and 14b
displayed potent cell growth inhibition with IC50 ranging from 0.20
to 0.87 M. The modest activity was obtained against HCT-116 (IC50
range 11.9 M to 31.0 M), except for 11a and 14d that displayed
moderate activity (IC50: 6.5 M and 6.4 M, respectively). These values
μM) and 11d (0.09
μ
M) emerged as the most
μ
μM
μ
μ
μ
μ
μ
2
. Results and discussion
are comparable to that observed for 1, but they were around 13 times
higher than the values obtained for doxorubicin. On the other hand,
investigations of the cytotoxic activity against MCF-7 indicated that all
compounds displayed no significant activity at tested concentrations
2
.1. Chemistry
Three different linkers attached to the hydroxamate ZBG, and four
(IC50 > 50
μ
M). Moreover, our findings indicated that 1 did not exert
para-substituted phenyl ureas (Scheme 1) were synthesized in order to
evaluate potency and cytotoxic activity. We first carried out catalytic
hydrogenation of compound 9 with Pd/C in ethanol, providing 12.
Subsequently, intermediates 7a-d, 10a-d and 13a-d were obtained via
addition of appropriate 4-aminophenyl derivative 6, 9 and 12 with
different phenyl isocyanates in dichloromethane with 20–95% yield.
The ester intermediates were converted into their corresponding
hydroxamic acids 8a-d, 11a-d, and 14a-d by treatment with aqueous
hydroxylamine (50% w/v) and NaOH with 70–95% yield.
antiproliferative effects over MCF-7 cells, which is in contrast with
4
5
previous results reported by Gryder and coworkers (IC50 = 4.4
Replacement of the cinnamyl linker (e.g. 11a, Jurkat IC50 = 0.16
by phenyl ring (e.g. 8a, Jurkat IC50 = 2.50 M), led to reduction of the
cytotoxic activity. Moreover, the cinnamyl replacement (e.g. 11c, Jurkat
M)
μ
M).
μ
M)
μ
IC50 = 0.11 μM) by di-hydrocinnamyl (e.g. 14c, Jurkat IC50 = 2.80 μ
also led to impairments on cytotoxicity. These results suggest that the
length and the low flexibility of the linker are relevant structural fea-
tures that favored the activity. Moreover, it was not possible to observe
significant differences in cytotoxicity among the para-substituted
Scheme 1. Synthesis of analogues 8a-d, 11a-d, and 14a-d. Reagents and conditions: (i) phenyl isocyanate, DCM, r.t., 16 h; (ii) NH
THF/MeOH (1:1), 0 ◦C – r.t., 2 h; (iii) 10% Pd/C, H
, EtOH, r.t., 18 h.
2 2
OH (50 wt% in H O), NaOH,
2
3

M.T. Tavares et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116085
Table 1
Cytotoxicity for compounds 8a-d, 11a-d, and 14a-d against solid and hematological tumor cells.
IC50 (µM) ± ±SEM€
Solid tumors
Hematological tumors
Jurkat
Non-tumorigenic
HS-5
¥
§
Compound
HCT-116
MCF-7
Namalwa
SI
TS
£
8
8
8
8
1
1
1
1
1
1
1
1
a
14.71 ± 2.82
>50
>50
2.50 ± 0.76
1.06 ± 0.45
8.03 ± 3.07
12.65 ± 3.86
0.16 ± 0.04
0.04 ± 0.01
0.11 ± 0.05
0.09 ± 0.03
1.59 ± 0.72
0.41 ± 0.23
2.80 ± 0.86
0.81 ± 0.32
0.05 ± 0.01
0.10 ± 0.03
0.53 ± 0.14
0.87 ± 0.46
4.79 ± 1.93
13.25 ± 5.87
0.30 ± 0.09
0.20 ± 0.05
0.46 ± 0.19
0.58 ± 0.22
0.78 ± 0.18
0.37 ± 0.13
1.99 ± 0.83
2.50 ± 0.98
0.09 ± 0.03
0.11 ± 0.04
n.d.
–
–
–
–
–
–
–
–
b
>50
n.d.
c
22.28 ± 5.27
28.40 ± 6.97
6.49 ± 1.16
29.86 ± 8.44
11.86 ± 3.86
12.57 ± 2.44
16.22 ± 3.66
17.92 ± 7.12
31.03 ± 6.82
6.37 ± 3.17
0.50 ± 0.20
4.20 ± 1.60
>50
n.d.
d
>50
n.d.
1a
1b
1c
1d
4a
4b
4c
4d
>50
1.0 ± 0.35
1.7 ± 0.40
3.0 ± 1.25
3.0 ± 1.38
n.d.
6/3
43/9
27/7
33/5
–
41/22
747/149
108/26
140/22
–
>50
>50
>50
>50
>50
n.d.
–
–
>50
n.d.
–
–
>50
n.d.
–
–
doxorubicin
1.90 ± 0.70
>50
0.15 ± 0.07
7.50 ± 1.70
3/2
75/68
10/6
42/38
vorinostat (1)
€
£
¥
Data are expressed as means ± SEM obtained from two independent experiments. n.d.: not determined. SI: selectivity index, determined by the ratio between IC50
§
non-tumor cells:tumor cells - HS-5:Jurkat (left) and HS-5:Namalwa (right). TS: tumor specificity, determined by the ratio between IC50 tumor cells 1:tumor cells 2 – HCT-
16:Jurkat (left) and HCT–116:Namalwa (right).
1
phenyl-ureide moiety. Thus, results suggest that both electron-donor/
acceptor and/or lipophilic/hydrophilic substituents do not substan-
tially influence either tumor cell penetration or the activity of the
compounds over HDACs.
for hematological tumor cells when compared to solid tumors. Com-
pounds 11a-d were more specific for hematological tumors and pre-
sented TS values ranging from 41 to 747 (Table 1). Particularly,
compound 11b showed not only a remarkable TS but also the highest
value (747), followed by 11d and 11c. The TS values of 11a-d are
prominent in comparison to the positive controls doxorubicin and 1
(Table 1).
Although it was not possible to evaluate the influence of para sub-
stituents on cytotoxicity, compounds 8b, 11b and 14b, bearing a chlo-
rine at the phenyl-ureide moiety, showed the highest activity of each
subseries against Jurkat and Namalwa cells. Interestingly, chlorine,
more than other halogens, is frequently found in approved drugs. Over
time, it has been empirically found that the introduction of a chlorine in
a biologically active molecule can substantially improve the biological
activity.46 For instance, the increase in the lipophilicity of a molecule,
when introducing a chlorine substituent, can lead to a greater partition
of a compound in the lipophilic phase of a cell membrane or through the
lipophilic domains of a protein.47 Indeed, compounds 8b, 11b and 14b
were the most lipophilic compounds, among others, as corroborated by
their clogP values: 2.28, 2.62 and 2.51, respectively. To evaluate the
influence of each linker moiety on potency and isoform selectivity, we
tested the potency of 8b, 11b, 14b along with trichostatin A (TSA)
against human HDAC1 and HDAC6 (see Section 2.4). Noteworthy,
compounds 11a-d have also shown the most potent cytotoxic activity
against Jurkat cells, with IC50 in the nanomolar range (from 40 to 160
nM, Table 1). Consequently, the cellular mechanisms of cytotoxicity of
2.3. Cytotoxicity kinetics, cell proliferation, apoptosis, tumor selectivity,
and molecular analysis
Regarding the cellular mechanisms induced by 11a-d, their cyto-
toxicity kinetics was accessed by the MTT assay after 24, 48 and 72 h-
treatments. Compounds 11a-d presented time-dependent cytotoxicity,
with positive relationship tendency observed between cell growth in-
hibition and exposure time of compounds (Figs. 2 and S2). The time-
dependent cytotoxicity is also observed in 1 treatment, otherwise, this
phenomenon is not apparent in doxorubicin treatment. Based on this
data, 48 h-time was chosen to perform proliferation and cell death
assays.
The cytotoxic potential of 11a-d was then confirmed by flow
cytometry, where the proliferation status was assessed by targeting Ki-
67, a protein highly expressed in proliferating cells.50 It was observed
that 11a-d can reduce both Jurkat and Namalwa proliferation in com-
parison to 1 and doxorubicin (Fig. 3). Additionally, compounds 11a-
d displayed apoptotic induction by increasing annexin V-positive cells
(Fig. 4).
1
1a-d have been further investigated (Section 2.3).
The selectivity index (SI)48 was calculated aiming to evaluate the
selectivity of 11a-d in relation to the non-tumorigenic cells and the
hematological cell lines (Table 1). Consequently, high SI values indicate
that a compound is more cytotoxic to the latter than to the former
These data indicate that cinnamyl hydroxamate derived compounds
have potential antileukemia effects, by reducing cell proliferation and
inducing apoptosis. Noteworthy, 11a-d were more potent than 1, a pan-
HDACi approved for the treatment of hematological malignancies.
Modulation of different types of HDACs can lead to diverse effects,
including repression of tumor suppressor genes expression as well as
regulation of oncogenic cell signaling pathways.21 In this context, for a
better phenotipically understand of the molecular mechanisms involved
in 11a-d activity, we further evaluated proteins related with two distinct
apoptotic mechanisms. For instance, mechanisms of inhibition mediated
(
Fig. S1). In the present work, HS-5, bone marrow/stroma cells, were
used as non-tumorigenic cells for SI calculation. Compounds 11a-
d showed remarkably interesting SI ranging from 6.3 to 42.5 for Jurkat
cells, and 3.3 to 8.5 for Namalwa cells, respectively, when compared to
doxorubicin SI ranging from 1.6 for Namalwa and 3.0 for Jurkat.
However, the positive control 1 remains the most selective compound
among the others, with SI ranging from 68.2 for Namalwa and 75 for
Jurkat. These results suggest that compounds be quite selective for
tumor cells. Compounds 11b and 11d were the most selective and, in
this context, 11b stands out for its high selectivity for Jurkat cells.
Noteworthy, the SI of 11b for Namalwa cells also showed the highest
observed selectivity.49
by HDACs (e.g. acetyl-
serine and phosphotyrosine), were considered along with DNA damage
-Tub and
α
-tubulin, ac-
α
-Tub) and kinases (e.g. phospho-
(γH2AX). Treatment with 11a-d increases the levels of ac-
α
Tumor specificity (TS), herein defined as the ratio between IC50 for
HCT-116 cells and IC50 for hematological tumors, was calculated in
order to assess the ability of the compounds on targeting a specific type
of tumor. High TS values may indicate, for instance, higher cytotoxicity
γH2AX, while phosphoserine and phosphotyrosine expression remains
unchanged for both Jurkat and Namalwa cells (Fig. 5).
The increased levels of ac-
α
-Tub point out the HDAC inhibition by
compounds 11a-d. Worthy of note, HDAC6 isozyme is the unique
4

M.T. Tavares et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116085
Fig. 2. Compounds 11a-d present time-dependent cytotoxicity. Dose-response curves for compounds 11a-d treatments after 24 h (black line), 48 h (blue line) and
2 h (gray line) in Jurkat cells (n = 3) and their respective values of half inhibitory concentration (IC50). Values are expressed in terms of the mean and standard error
of the mean (mean ± SEM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
7
member of the HDAC family able to deacetylate non-histone proteins
Biology Corp, Malvern, PA). As illustrated in Table 2, all analogues
presented a strong total inhibition of HDACs in nuclear extracts (>90%),
which is in accordance with our previous mechanistic studies and
cytotoxic assays. Subsequent analysis against HDAC1 and HDAC6 iso-
zymes showed that 8b and 11b are potent HDAC6 inhibitors (IC50: 20.7
and 8.1 nM, respectively). The HDAC6 selectivity of 8b (64-fold) was
expected since it is structurally related to 15. Noteworthy, the absence of
the n-butyl chain in the proximal nitrogen of the urea CU, and
replacement of the 3–dimethylamino substituent by para-chloro caused
a 86-fold improvement on potency of 8b compared to 15. Moreover,
insertion of a methylene unity between the phenyl linker and the urea
CU may impair both HDAC6 potency and selectivity of the series. As
presented on Table 2, 8b has 6.7–fold potency against HDAC6 compared
to 16 (IC50: 139 nM) and ~34-fold selectivity, thus endorsing the in-
fluence of linker’s length on potency and selectivity of these compounds.
The cinnamyl hydroxamate moiety is known by its pan-HDAC inhi-
bition, that is found in two FDA-approved HDACis (3 and 4). These
drugs are potent inhibitors of both HDAC1 and HDAC6 isoforms
such as
α
-tubulin, cortactin, and HSP90, and its inhibition leads to
1–53
hyperacetylation of
α
-tubulin.5
Selective inhibitors of HDAC6, such
as ricolinostat, nexturastat A and its analogues, increase the level of
acetylated
–tubulin with no significant effects over the acetylation
status of histones.
α
2
5,54,55
Nonetheless, the increased levels of ac- -Tub
α
observed for compounds 11a-d does not exclude the possibility of in-
hibition of other HDAC isozymes, which was further investigated and is
discussed on Section 2.4 of this manuscript. HDAC inhibition causes
5
6
DNA damage, which explains the increased levels of γH2AX. The
constant levels of phosphoserine and phosphotyrosine, markers of ki-
nase function,57 suggest a specific mechanism of HDAC inhibition for
compounds 11a-d. Interestingly, HDACs are overexpressed in acute
myeloid leukemia (e.g. Namalwa) and acute lymphoblastic leukemia (e.
g. Jurkat), which makes them interesting targets for tackling hemato-
logical cancers.7
,53,58
2
.4. In vitro HDAC potency assessment
5
9
(
Table 2) and have low SI values (0.3 and 1.2, respectively). Intrigu-
ingly, 11b presented a slight improvement on potency compared to 3
and 4, and 402-fold selectivity over HDAC1. From our knowledge, this is
the higher HDAC6 selectivity index reported to date for a cinnamyl
To evaluate the influence of each linker on potency and selectivity,
we screened the inhibitory activity of 8b, 11b, 14b against HeLa nuclear
extract, human HDAC1, and HDAC6 in vitro (performed by Reaction
5

M.T. Tavares et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116085
Fig. 3. Compounds 11a-d reduce cell proliferation. Ki-67 mean fluorescence intensity (M.F.I.) was determined by flow cytometry after incubation of Jurkat and
Namalwa cells treated with vorinostat and doxorubicin IC50, and compounds 11a-d IC50 for 48 h; histogram traces are illustrated. The bar graphs represent the Ki-67
M.F.I normalized to the respective untreated control cells, and the results are shown as mean ± SEM of at least three independent experiments. *p < 0.05, **p < 0.01
and ***p < 0.001, ANOVA test and Bonferroni post-test.
2
+
hydroxamate-derived HDACi. As discussed above, the linker length
might be affecting both potency and selectivity of these compounds. In
this sense, 11b is the only compound that has a cinnamyl linker directly
attached to the urea CU at para position, which may confer a unique
steric feature for the compound, thus allowing an improved interaction
with the catalytic pocket of HDAC6. Regarding flexibility of the linker,
HDAC6, all compounds showed a monodentate coordination with Zn
…
2+
ꢀ … 2+
Zn ) during the whole simulation. On the other
(CO Zn or N
–
O
hand, the coordination geometry transitioned from the bidentate
…
2+… ꢀ
O
(CO Zn
–
N) towards monodentate upon HDAC1 simulations.
These findings may suggest that, in short timescale simulations, the
structural feature of each linker (Fig. 1B) could be affecting the coor-
dination fashion depending on the HDAC isozyme. Previous studies re-
ported that HDACis may present both monodentate and bidentate
1
4b had 13–fold reduction on potency compared to 11b (IC50: 105 nM
and 8.1 nM, respectively), though 3.5–fold improvement on potency
over HDAC1, which may be related to the submicromolar activity of 14b
on cellular assays (Table 1), as well as with its high inhibition rate (98%)
over HeLa nuclear extracts.
2
+
coordination with Zn , switching one to another during the interac-
tion.61 Even though changes in the coordination geometry do not
necessarily reflect on gain or loss of affinity neither potency of
HDACis,6
2–65
they may be exerting influence over the HDAC6 selectivity
of 8b, 11b and 14b.
2
.5. Docking and molecular dynamics simulations
Simulations with 8b, 11b and 14b, presented interactions with res-
idues such as Glu779 and Tyr782 at the bottom of the cavity, besides a
stable interaction with Gly619. Compounds 11b and 14b displayed
more often interactions with Glu779 in comparison to 8b, due to its
The biological results suggest the compounds synthesized herein are
HDAC6 inhibitors with certain activity also over HDAC1. In this sense,
we employed molecular dynamics (MD) simulations on docking poses of
′
shorter linker. Interestingly, the interaction with HDAC1 s Gly149 is less
8
b, 11b and 14b, as well as known substrates and inhibitors over the
frequent (Fig. S3), which would endorse the higher affinity of the
compounds towards HDAC6.
HDAC1 and HDAC6 isoforms. The in silico studies aimed to assess the
differences of interaction that the cinnamyl hydroxamate,
di–hydrocinnamyl hydroxamate and phenyl hydroxamate groups could
cause with the biological target. In addition, we sought to correlate
potential differences on interactions with the inhibitory profile observed
in vitro for each scaffold (Section 2.4).
It is worthy of note that 11b, among the other compounds, had the
…
…
highest hydrogen bond frequency with Ser568 (CO HO and NH O,
Fig 6B/E). Ser568 is located at the rim of the catalytic pocket of HDAC6
59,66
and is an essential residue responsible for substrate recognition.
Among other interactions, the hydrogen bond with Ser568 is likely
responsible for the HDAC6 potency and selectivity presented by
As it can be seen in Figs. 6 and S3, compounds presented different
coordination geometry depending on the HDAC isozyme. Regarding
6

M.T. Tavares et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116085
Fig. 4. Compounds 11a-d induce apoptosis at IC50 concentration. Apoptosis was detected by flow cytometry in Jurkat (A) and Namalwa (C) cells treated with
vorinostat and doxorubicin IC50, and compounds 11a-d IC50 and half of IC50 for 48 h using annexin V-FITC/7AAD staining method. Representative point graphs are
shown for each condition. The upper and lower right quadrants (Q2 plus Q3) cumulatively contain the apoptotic population (annexin V-positive cells). Annexin V
positive Jurkat (B) and Namalwa (D) cells percentages in doxorubicin and vorinostat IC50, and compounds IC50 and half of the IC50 treatments obtained by the 7AAD
and annexin V-FITC labelled flow cytometry assay. Values expressed by the mean and standard error of the mean (n = 3). The results are presented as the mean ± SD
of three independent experiments. *p < 0.01, **p < 0.001, ***p < 0.0001, ANOVA test and Bonferroni post-test, all pairs were analyzed and statistically significant
differences are indicated.
7

M.T. Tavares et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116085
Fig. 5. Compounds 11a-d induce DNA damage and increase acetyl-
α
-tubulin (ac-
α
-Tub). Immunoblotting analysis for ac-
α
-Tub, phosphorylated histone H2AX
(
γH2AX), phosphoserine (pSer), phosphotyrosine (pTyr),
α-tubulin (
α
-Tub), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of Jurkat (A) and Namalwa (B)
cells after incubation with DMSO (0.05%, control), vorinostat (1), and compounds 11a-d IC50 for 48 h. Pixel quantification of ac-
mean ± SEM), ANOVA, one way, followed by Tukey’s multiple comparison test. *p < 0.01, **p < 0,001, ***p < 0.0001.
α-Tub, γH2AX, pSer and pTyr (n = 3;
ricolinostat.62 Consequently, these preliminary findings highlight the
role of the urea moiety as the CU of the cinnamyl hydroxamate de-
rivatives 11a-d, which presented the most potent activities of the series
in the cellular assays. Moreover, the length of the linker may be related
to the increased potency of 11b when compared to 8b, since the phenyl
replacement by the cinnamyl moiety allows a deeper exploration of the
narrow catalytic tunnel, besides it looks to have the ideal interatomic
distance for a simultaneous hydrogen bonding interaction with Ser568 –
through the urea – and coordination with the zinc ion at the bottom of
the cavity. On the other hand, the decrease in 14b’s activity, compared
to 11b, may be related to potential entropic impairments, resulted from
the hydrogenation of the cinnamyl double bond. Conversion to the di-
8

M.T. Tavares et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116085
Table 2
a
HDAC1 and HDAC6 enzymatic evaluation of analogues 8b, 11b, and 14b.
Structure
IC50 (nM)
b
c
Compound
Cap –±CU –±Linker –±ZBG
Inhibition rate
HDAC1
HDAC6
SI
8
1
1
b
92%
1,320
20.7
64
402
9
1b
4b
93%
98%
3,260
941
8.1
105
1.5
d
Trichostatin (TSA)
n.a.
3.9
2.6
e
Panobinostat (3)
n.a.
3.0
11.0
0.3
e
Belinostat (4)
n.a.
n.a.
18.0
15.0
1.2
f
1
1
5
>30,000
1,790
>17
g
6
n.a.
265
139
1.9
a
IC50 values are the mean of two experiments obtained from curve-fitting of a 10-point enzymatic assay starting from 100 M with 3-fold serial dilution against HDAC1
μ
b
c
and HDAC6 (Reaction Biology Corp, Malvern, PA). Total HDAC activity determined in a single dose duplicate at 100 µM against HeLa nuclear extract. SI: HDAC6
selectivity over HDAC1. n.a.: not available. IC50 values were extracted from Ref. . IC50 values were extracted from Ref. . IC50 values were extracted from Ref.
d
e
59
f
60
g
25
.
hydrocinnamyl linker can impair the suitable interaction with Ser568 as
well as the fit of 14b into the cavity, thus resulting in a reduced potency
of this compound.
generation of potent HDAC inhibitors with improved selectivity over the
HDAC6 isoform.
4
. Experimental section
3
. Conclusions
4
.1. Chemistry
Herein, a series of cinnamyl hydroxamates, di-hydrocinnamyl
hydroxamates and phenyl hydroxamates derivatives with HDAC inhib-
itory activity have been synthesized and evaluated in a panel of solid
and hematological cancer cells. Compounds 11a-d presented selective
nanomolar potency against hematological Jurkat and Namalwa cells.
The solvents were purified according to standard procedures.67 The
reagents and solvents were purchased from Synth®, Merck®, Sigma-
Aldrich-Merck®, and Oakwood Chemicals®. All air-sensitive and/or
water-sensitive reactions were carried out with dry solvents under
anhydrous conditions and nitrogen atmosphere. The reactions were
monitored by TLC on Merck silica gel (60 F 254) by using UV light (λ =
254 nm) as a visualizing agent and ninhydrin, vanillin or molybdate
staining solution. Sigma-Aldrich silica gel (particle size 0.040–0.063
nm) was used for flash chromatography (performed manually or using
The increased expression of ac- -Tub, DNA fragmentation and the
α
absence of effects on tyrosine and serine kinases pointed out HDAC as
the biological target of these compounds. By evaluating the inhibitory
activities of 8b, 11b, and 14b at HDAC1/6 isoforms, we found that: (a)
the cinnamyl moiety generated the most potent and HDAC6–selective
analogue of the series (11b); (b) the length and flexibility of the linker
strongly affect the selectivity of the compounds; (c) disubstitution of the
proximal nitrogen from the urea CU may impair HDAC6 potency. In
silico studies suggest that the cinnamyl linker would have the ideal
length among the series for interaction with the HDAC6 catalytic cavity.
Moreover, the urea CU was able to establish an additional hydrogen
bond interaction with Ser568 at the rim of HDAC6 binding cavity. The
cinnamyl-derived compound 11b was the analogue who better explored
the interaction with Ser568, which could be related to its high HDAC6
potency. Taken together, these results highlight the potential of urea-
1
13
Isolera Prime® - Biotage™). H and C NMR spectra were obtained on a
300/75 MHz Bruker spectrometer, using the solvent residual peak as the
6
internal reference (chemical shifts: DMSO‑d , 2.50/39.52). HRMS
spectra were measured with a Bruker Daltonics micrOTOF QII/ESI-TOF.
Analytical HPLC was carried out on an Shimadzu™ Proeminence® in-
strument under the following conditions: column, C-18 Gemini® (5
μ
m,
150 × 4.6 mm), mobile phase, 5–100% H
O/CH CN containing 0.1%
2 3
TFA at a flow rate of 1.0 mL/min for 25 min, UV detection at 254 nm.
The purities of all tested compounds were > 95%, as determined by
analytical HPLC. The isolated compounds (final products) were
analyzed using a high liquid chromatograph (Prominence liquid
based cinnamyl hydroxamates as
a privileged scaffold for the
9

M.T. Tavares et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116085
Fig. 6. Potential binding mode of 8b, 11b, and 14b in the HDAC6 catalytic cavity. (A) Frequency of hydrogen interactions observed along with the production phase
of molecular dynamics simulation. Residues highlighted with (W) have also water-mediated hydrogen bonds. (B) Frequency of hydrogen interactions observed
between the urea CU and Ser568. Representative snapshots from the molecular dynamics simulation (200 ns per system), highlighting the residues involved in
interactions with (C) trichostatin A (TSA, dark gray); (D) 8b (red); (E) 11b (orange); and (F) 14b (yellow). Interactions are represented by yellow dashed lines.
HDAC6 residues are colored according to the atom types of the interacting amino acid residues (protein’s carbon, light gray; nitrogen, blue; oxygen, red, zinc, light
purple sphere). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
chromatography, Shimadzu, Japan) coupled to an accurate Q-TOF mass
spectrometer, Compact model (Bruker Daltonics GmbH, Bremen, Ger-
many), and electrospray ionization interface. The isolated compounds
4.1.1. General method A (7a-d, 10a-d, 13a-d)
To a solution of 4-aminophenyl derivative 6, 9, or 12 (5 mmol, 1 eq.)
in DCM (10 mL), the appropriate phenyl isocyanate (1 eq.) was added.
The mixture was kept under constant stirring at room temperature and
were dissolved in DMSO and separated using an Kinetex 1.7 m EVO
μ
C18 100 Å (100 × 2.1 mm; Phenomenex Ltd.; California, USA) and 0.1%
formic acid in water and 0.1% formic acid in acetonitrile as the mobile
phase at a flow rate of 0.4 mL/min. The gradiente program was as
follow: 10% B at the beginning, 100% B at 5 min, 25% B at 7 min, and a
2
N atmosphere for 16 h. The resulting suspension was filtered in vacuo,
and washed with DCM (3 × 30 mL) to afford the intermediates 7a-d,
10a-d, and 13a-d.
5
min post-run at 10% B. The injection volume was 20
μ
L and the col-
4.1.1.1. Methyl 4-(3-phenylureido)benzoate (7a). The intermediate was
prepared following the general method A from methyl 4-aminobenzoate
(6) and phenyl isocyanate. The product was isolated as a white solid
◦
umn temperature was maintained at 40 C. The Q-TOF/MS instrument
was operated in positive mode using the following parameters: ion gas
◦
(0.422 g, 31%). 1H NMR (300 MHz, DMSO‑d ) δ 9.08 (s, 1H), 8.79 (s,
source (N
2
) temperature 200 C; nebulizer pressure 45 psi; and capillary
6
voltage of 2,800 V. The mass spectometer was operated in MS scan
mode. The sodium formate was used as internal mass calibration.
1H), 7.91 (d, J = 6.9 Hz, 2H), 7.61 (d, J = 7.0 Hz, 2H), 7.49 (d, J = 7.4
Hz, 2H), 7.31 (t, J = 6.9 Hz, 2H), 7.01 (t, J = 7.0 Hz, 1H), 3.83 (s, 3H).
1
0

M.T. Tavares et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116085
13C NMR (75 MHz, DMSO‑d
) δ 165.9, 152.1, 144.4, 139.3, 130.4 (2C),
7.76–7.60 (m, 5H), 7.59–7.50 (m, 2H), 6.49 (d, J = 16.0 Hz, 1H), 4.18
6
1
3
1
28.8 (2C), 122.4, 122.2, 118.4 (2C), 117.3 (2C), 51.7.
(q, J = 7.0 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H). C NMR (75 MHz,
6
DMSO‑d ) δ 166.4, 151.7, 146.1, 144.0, 141.2, 129.3 (2C), 128.1, 125.0
4
.1.1.2. Methyl 4-(3-(4-chlorophenyl)ureido)benzoate (7b). The inter-
(2C), 118.4 (2C), 117.9, 117.6 (2C), 115.9, 59.8, 14.2.
mediate was prepared following the general method A from methyl 4-
aminobenzoate (6) and 4-chlorophenyl isocyanate. The product was
4.1.1.9. Ethyl 3-(4-(3-phenylureido)phenyl)propanoate (13a). The in-
termediate was prepared following the general method A from ethyl 3-
(4-aminophenyl)propanoate (12) and phenyl isocyanate. The product
was isolated as a white solid (0.12 g, quant.). 1H NMR (300 MHz,
1
isolated as a white solid (0.845 g, 55%). H NMR (300 MHz, DMSO‑d
6
) δ
9
.11 (s, 1H), 8.92 (s, 1H), 7.90 (d, J = 8.1 Hz, 2H), 7.59 (d, J = 8.1 Hz,
1
3
2
H), 7.50 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 3.83 (s, 3H).
C
NMR (75 MHz, DMSO‑d
6
) δ 165.9, 152.1, 144.2, 138.3, 130.4 (2C),
DMSO‑d
6
) δ 8.68 (s, 1H), 8.63 (s, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.37 (d, J
1
28.6 (2C), 125.8, 122.6, 120.0 (2C), 117.4 (2C), 51.7.
= 7.5 Hz, 2H), 7.28 (t, J = 7.3 Hz, 2H), 7.14 (d, J = 7.7 Hz, 2H), 6.97 (t,
J = 6.9 Hz, 1H), 4.06 (q, J = 6.9 Hz, 2H), 2.80 (t, J = 7.2 Hz, 2H), 2.60 (t,
1
3
4
.1.1.3. Methyl 4-(3-(4-methoxyphenyl)ureido)benzoate (7c). The inter-
J = 7.3 Hz, 2H), 1.17 (t, J = 7.0 Hz, 3H). C NMR (75 MHz, DMSO‑d
6
) δ
mediate was prepared following the general method A from methyl 4-
166.4, 152.2, 144.2, 141.9, 139.4, 129.3 (2C), 128.8 (2C), 127.5, 122.0,
aminobenzoate (6) and 4-methoxiphenyl isocyanate. The product was
118.3 (2C), 117.9 (2C), 115.5, 59.8, 14.2.
1
isolated as a white solid (0.308 g, 20%). H NMR (300 MHz, DMSO‑d
6
) δ
8
2
.99 (s, 1H), 8.59 (s, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.58 (d, J = 8.6 Hz,
H), 7.37 (d, J = 8.7 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 3.82 (s, 3H), 3.73
) δ 168.3, 154.4, 152.8, 137.9,
4.1.1.10. Ethyl 3-(4-(3-(4-chlorophenyl)ureido)phenyl)propanoate (13b
). The intermediate was prepared following the general method A from
ethyl 3-(4-aminophenyl)propanoate (12) and 4-chlorophenyl isocya-
(
s, 3H). 13C NMR (75 MHz, DMSO‑d
6
1
1
34.1, 132.9, 128.4 (2C), 119.9 (2C), 118.2 (2C), 113.9 (2C), 55.1, 51.7.
nate. The product was isolated as a white solid (0.326 g, 94%). H NMR
(
300 MHz, DMSO‑d
6
) δ 8.64 (s, 1H), 8.59 (s, 1H), 7.49 (d, J = 7.7 Hz,
4
.1.1.4. Methyl 4-(3-(4-nitrophenyl)ureido)benzoate (7d). The interme-
2H), 7.40 (d, J = 6.9 Hz, 2H), 7.17 (d, J = 7.1 Hz, 2H), 6.99 (d, J = 7.4
Hz, 2H), 4.09 (q, J = 7.0 Hz, 2H), 2.84 (t, J = 7.2 Hz, 2H), 2.62 (t, J =
diate was prepared following the general method A from methyl 4-ami-
1
3
nobenzoate (6) and 4-nitrophenyl isocyanate. The product was isolated
7.3 Hz, 2H), 1.20 (t, J = 7.1 Hz, 3H). C NMR (75 MHz, DMSO‑d
6
) δ
1
as a yellow solid (1.109 g, 70%). H NMR (300 MHz, DMSO‑d
6
) δ 9.50 (s,
172.2, 152.6, 139.8, 137.8, 133.9, 128.8 (2C), 128.5 (2C), 121.7, 118.3
1
H), 9.29 (s, 1H), 8.20 (d, J = 8.6 Hz, 2H), 7.92 (d, J = 8.1 Hz, 2H), 7.71
(2C), 118.2 (2C), 59.8, 35.3, 29.7, 14.1.
13
(
d, J = 8.7 Hz, 2H), 7.62 (d, J = 8.1 Hz, 2H), 3.83 (s, 3H). C NMR (75
MHz, DMSO‑d ) δ 165.8, 151.7, 145.9, 143.6, 141.3, 130.4 (2C), 125.1
2C), 123.1, 117.9 (2C), 117.7 (2C), 51.8.
6
4.1.1.11. Ethyl 3-(4-(3-(4-methoxyphenyl)ureido)phenyl)propanoate (13
c). The intermediate was prepared following the general method A from
(
ethyl 3-(4-aminophenyl)propanoate (12) and 4-methoxyphenyl isocya-
1
4
.1.1.5. Ethyl (E)-3-(4-(3-phenylureido)phenyl)acrylate (10a). The in-
nate. The product was isolated as a white solid (0.341 g, quant.).
NMR (300 MHz, DMSO‑d
H
termediate was prepared following the general method A from ethyl 4-
6
) δ 8.51 (s, 1H), 8.45 (s, 1H), 7.40–7.32 (m,
aminocinnamate (9) and phenyl isocyanate. The product was isolated as
4H), 7.12 (d, J = 8.3 Hz, 2H), 6.87 (d, J = 8.9 Hz, 2H), 4.05 (q, J = 7.1
Hz, 2H), 3.72 (s, 3H), 2.79 (t, J = 7.5 Hz, 2H), 2.57 (t, J = 7.4 Hz, 2H),
1
a white solid (1.296 g, 83%). H NMR (300 MHz, DMSO‑d
6
) δ 8.92 (s,
1
3
1
H), 8.74 (s, 1H), 7.71–7.43 (m, 7H), 7.30 (t, J = 7.7 Hz, 2H), 7.01 (t, J
1.17 (t, J = 7.1 Hz, 3H). C NMR (75 MHz, DMSO‑d
6
) δ 172.2, 154.4,
=
7.2 Hz, 1H), 6.49 (d, J = 15.9 Hz, 1H), 4.19 (q, J = 6.9 Hz, 2H), 1.26 (t,
152.8, 138.0, 133.6, 132.8, 128.5 (2C), 119.9 (2C), 118.2 (2C), 114.0
1
3
J = 7.0 Hz, 3H). C NMR (75 MHz, DMSO‑d
6
) δ 166.4, 152.2, 144.2,
(2C), 59.7, 55.2, 35.3, 29.7, 14.1.
1
41.9, 139.4, 129.3 (2C), 128.8 (2C), 127.5, 122.0, 118.3 (2C), 117.9
(
2C), 115.5, 59.8, 14.2.
4.1.1.12. Ethyl 3-(4-(3-(4-nitrophenyl)ureido)phenyl)propanoate (13d).
The intermediate was prepared following the general method A from
ethyl 3-(4-aminophenyl)propanoate (12) and 4-nitrophenyl isocyanate.
4
.1.1.6. Ethyl (E)-3-(4-(3-(4-chlorophenyl)ureido)phenyl)acrylate (10b
1
)
. The intermediate was prepared following the general method A from
The product was isolated as a yellow solid (0.553 g, 90%). H NMR (300
ethyl 4-aminocinnamate (9) and 4-chlorophenyl isocyanate. The prod-
MHz, DMSO‑d
6
) δ 9.46 (s, 1H), 9.13 (s, 1H), 8.20 (d, J = 9.0 Hz, 2H),
uct was isolated as a white solid (1.223 g, 71%). 1H NMR (300 MHz,
7.76–7.67 (m, 4H), 7.55 (d, J = 8.6 Hz, 2H), 4.18 (q, J = 7.1 Hz, 2H),
2.84 (t, J = 7.3 Hz, 2H), 2.63 (t, J = 7.3 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H).
DMSO‑d
6
) δ 8.95 (s, 1H), 8.88 (s, 1H), 7.70–7.45 (m, 7H), 7.34 (d, J =
1
3
8
.8 Hz, 2H), 6.49 (d, J = 16.0 Hz, 1H), 4.18 (q, J = 7.0 Hz, 2H), 1.26 (t, J
6
C NMR (75 MHz, DMSO‑d ) δ 166.4, 151.8, 146.2, 141.1, 140.3, 129.0
=
7.1 Hz, 3H). 13C NMR (75 MHz, DMSO‑d
6
) δ 166.4, 152.1, 144.1,
(2C), 128.3, 125.1 (2C), 118.6 (2C), 117.6 (2C), 59.8, 34.1, 30.2, 14.2.
1
41.7, 138.4, 129.3 (2C), 128.6 (2C), 127.6, 125.6, 119.9 (2C), 118.1
(
2C), 115.6, 59.8, 14.2.
4.1.2. General method B (8a-d, 11a-d, 14a-d)
In a round bottom flask, 0.243 g of sodium hydroxide (NaOH, 6.08
mmol, 8 eq.) was dissolved in 1.059 mL of aqueous hydroxylamine so-
4
.1.1.7. Ethyl (E)-3-(4-(3-(4-methoxyphenyl)ureido)phenyl)acrylate (10c
◦
lution (50% p/v, 38 mmol, 50 eq.) at 0 C. Then, a solution containing
)
. The intermediate was prepared following the general method A from
the intermediate (7a-d, 10a-d, 13a-d, 0.76 mmol, 1 eq.) in tetrahy-
drofuran (THF) and methanol (1:1, 6 mL) was added dropwise. The
mixture was kept under constant stirring at room temperature for 2 h.
The resulting solution was neutralized (pH = 7.0) with 2 N HCl and
poured into 20 mL of ice-cold water. The suspension was filtered under
vacuum, washed with ice-cold water (3 × 30 mL) and dried in a vacuum
pump to afford the final product.
ethyl 4-aminocinnamate (9) and 4-methoxyphenyl isocyanate. The
1
product was isolated as a white solid (1.619 g, 95%). H NMR (300 MHz,
DMSO‑d
6
) δ 8.85 (s, 1H), 8.55 (s, 1H), 7.71–7.46 (m, 5H), 7.38 (d, J =
8
.9 Hz, 2H), 6.89 (d, J = 8.9 Hz, 2H), 6.48 (d, J = 16.0 Hz, 1H), 4.19 (q,
1
3
J = 7.1 Hz, 2H), 3.73 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H). C NMR (75 MHz,
DMSO‑d ) δ 166.4, 154.7, 152.4, 144.2, 142.2, 132.4, 129.3 (2C), 127.2,
20.2 (2C), 117.8 (2C), 115.3, 114.0 (2C), 59.7, 55.1, 14.2.
6
1
4
.1.2.1. N-hydroxy-4-(3-phenylureido)bezamide (8a). The final product
4
.1.1.8. Ethyl (E)-3-(4-(3-(4-nitrophenyl)ureido)phenyl)acrylate (10d).
was prepared following the general method B from 7a. The product was
The intermediate was prepared following the general method A from
ethyl 4-aminocinnamate (9) and 4-nitrophenyl isocyanate. The product
was isolated as a yellow solid (1.599 g, 90%). 1H NMR (300 MHz,
1
isolated as a white solid (0.270 g, quant.). H NMR (300 MHz, DMSO‑d
δ 11.07 (s, 1H), 8.90 (s, 2H), 8.74 (s, 1H), 7.72 (d, J = 8.6 Hz, 2H), 7.53
d, J = 8.6 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 7.30 (t, J = 7.8 Hz, 2H),
6
)
(
DMSO‑d
6
) δ 9.46 (s, 1H), 9.13 (s, 1H), 8.19 (d, J = 9.2 Hz, 2H),
1
1

M.T. Tavares et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116085
18
6
1
.99 (t, J = 7.3 Hz, 1H). 13C NMR (75 MHz, DMSO‑d
6
) δ 164.1, 152.3,
(2C), 116.5, 114.0 (2C), 55.2. HRMS calc. for C17
H
N
3
O
4
: [M + H]+,
42.4, 139.4, 128.8 (2C), 127.8 (2C), 125.8, 122.1, 118.3 (2C), 117.3
m/z 328.1297. Found 328.1318. Analytical HPLC retention time: 8.9
(
2C). HRMS calc. for C14
H
14
N
3
O
3
: [M + H]+, m/z 272.1035. Found
min; purity: 99.8%.
2
72.1059. Analytical HPLC retention time: 9.4 min; purity: 98.2%.
4
.1.2.8. (E)-N-hydroxy-3-(4-(3-(4-nitrophenyl)ureido)phenyl)acrylamide
4
.1.2.2. 4-(3-(4-chlorophenyl)ureido)-N-hydroxybenzamide
(8b). The
(11d). The final product was prepared following the general method B
1
final product was prepared following the general method B from 7b. The
from 10d. The product was isolated as a yellow solid (0.324 g, 95%). H
1
product was isolated as a white solid (0.215 g, 70%). H NMR (300 MHz,
6
NMR (300 MHz, DMSO‑d ) δ 10.71 (br s, 1H), 9.51 (s, 1H), 9.14 (s, 1H),
DMSO‑d
6
) δ 11.06 (s, 1H), 8.95 (s, 1H), 8.90 (s, 2H), 7.71 (d, J = 8.3 Hz,
9.03 (br s, 1H), 8.19 (d, J = 9.0 Hz, 2H), 7.71 (d, J = 9.0 Hz, 2H),
1
3
13
2
H), 7.57 – 7.46 (m, 4H), 7.35 (d, J = 8.6 Hz, 2H). C NMR (75 MHz,
7.65–7.39 (m, 5H), 6.39 (d, J = 15.7 Hz, 1H). C NMR (75 MHz,
DMSO‑d
6
) δ 164.1, 152.2, 142.2, 138.4, 128.6 (2C), 127.8 (2C), 125.9,
13ClN
6
DMSO‑d ) δ 163.1, 151.8, 146.2, 141.1, 140.2, 129.0, 128.3 (2C), 125.1
(2C), 118.6 (2C), 117.9, 117.5 (2C), 117.0. HRMS calc. for C16H N O :
15 4 5
1
25.6, 119.9 (2C), 117.4 (2C). HRMS calc. for C14
H
3
O
3
: [M + H]+,
m/z 306.0645. Found 306.0649. Analytical HPLC retention time: 12.3
[M + H]+, m/z 343.1042. Found 343.1065. Analytical HPLC retention
min; purity: 97.3%.
time: 13.8 min; purity: 99.8%.
4
.1.2.3. N-hydroxy-4-(3–4(-methoxyphenyl)ureido)benzamide (8c). The
4.1.2.9. N-hydroxy-3-(4-(3-phenylureido)phenyl)propanamide (14a).
final product was prepared following the general method B from 7c. The
The final product was prepared following the general method B from
1
1
product was isolated as a white solid (0.233 g, 85%). H NMR (300 MHz,
13a. The product was isolated as a white solid (0.220 g, 73%). H NMR
DMSO‑d
6
) δ 11.04 (s, 1H), 8.88 (s, 1H), 8.82 (s, 1H), 8.54 (s, 1H), 7.70
(300 MHz, DMSO‑d
6
) δ 9.48 (br s, 1H), 8.84–8.82 (m, 2H), 7.50 (d, J =
(
d, J = 8.6 Hz, 2H), 7.51 (d, J = 8.7 Hz, 2H), 7.37 (d, J = 8.9 Hz, 2H),
7.9 Hz, 2H), 7.40 (d, J = 8.1 Hz, 2H), 7.31 (t, J = 7.7 Hz, 2H), 7.14 (d, J
= 8.0 Hz, 2H), 6.99 (t, J = 7.3 Hz, 1H), 3.39 (br s, 1H), 2.80 (t, J = 7.6
1
3
6
.88 (d, J = 8.9 Hz, 2H), 3.73 (s, 3H). C NMR (75 MHz, DMSO‑d
64.2, 154.6, 152.5, 142.6, 132.4, 127.8 (2C), 125.5, 120.2 (2C), 117.1
: [M + H]+, m/z
02.1140. Found 302.1155. Analytical HPLC retention time: 12.4 min;
6
) δ
1
3
1
Hz, 2H), 2.28 (t, J = 7.6 Hz, 2H). C NMR (75 MHz, DMSO‑d
6
) δ 168.3,
(
2C), 114.0 (2C), 55.2. HRMS calc. for C15
H
16
N
3
O
4
152.7, 139.9, 137.8, 134.3, 128.7 (2C), 128.4 (2C), 121.6, 118.3 (2C),
+
3
118.1 (2C), 34.1, 30.2. HRMS calc. for C16
H
18
N
3
O
3
: [M + H] , m/z
purity: 97.2%.
300.1348. Found 300.1369. Analytical HPLC retention time: 9.6 min;
purity: 96.5%.
4
.1.2.4. N-hydroxy-4-(3-(4-nitrophenyl)ureido)benzamide
(8d). The
final product was prepared following the general method B from 7d. The
product was isolated as a yellow solid (0.269 g, 85%). 1H NMR (300
4.1.2.10. 3-(4-(3-(4-chlorophenyl)ureido)phenyl)-N-hydrox-
ypropanamide (14b). The final product was prepared following the
MHz, DMSO‑d
6
) δ 11.10 (s, 1H), 9.49 (s, 1H), 9.15 (s, 1H), 8.93 (s, 1H),
general method B from 13b. The product was isolated as a white solid
1
8
2
1
.20 (d, J = 9.0 Hz, 2H), 7.73 (t, J = 9.4 Hz, 4H), 7.55 (d, J = 8.5 Hz,
(0.189 g, 86%). H NMR (300 MHz, DMSO‑d
6
) δ 11.23 (br s, 1H), 9.97
1
3
H). C NMR (75 MHz, DMSO‑d
28.3 (2C), 127.0, 125.6 (2C), 118.2 (2C), 118.1 (2C). HRMS calc. for
: [M + H]+, m/z 317.0885. Found 317.0880. Analytical
HPLC retention time: 11.4 min; purity: 99.7%.
6
) δ 164.5, 152.3, 146.6, 142.2, 141.7,
(br s, 1H), 9.14 (s, 1H), 9.09 (s, 1H), 7.49 (d, J = 7.7 Hz, 2H), 7.40 (d, J
= 6.9 Hz, 2H), 7.17 (d, J = 7.1 Hz, 2H), 6.99 (d, J = 7.4 Hz, 2H), 2.84 (t,
1
3
C
14
H
13
4
N O
5
J = 7.2 Hz, 2H), 2.62 (t, J = 7.3 Hz, 2H). C NMR (75 MHz, DMSO‑d
6
) δ
168.3, 152.7, 139.9, 137.8, 134.3, 128.7 (2C), 128.4 (2C), 121.6, 118.3
+
(
2C), 118.1 (2C), 34.1, 30.3. HRMS calc. for C16
H
17ClN
3
O
3
: [M + H] ,
4
.1.2.5. (E)-N-hydroxy-3-(4-(3-phenylureido)phenyl)acrylamide (11a).
m/z 334.0958. Found 334.0958. Analytical HPLC retention time: 11.6
The final product was prepared following the general method B from
min; purity: 97%.
1
1
0a. The product was isolated as a white solid (0.296 g, quant.). H NMR
(
300 MHz, DMSO‑d ) δ 10.68 (s, 1H), 8.97 (br s, 1H), 8.87 (s, 1H), 8.71
6
4.1.2.11. N-hydroxy-3-(4-(3-(4-methoxyphenyl)ureido)phenyl)prop-
(
s, 1H), 7.57–7.42 (m, 7H), 7.29 (t, J = 7.9 Hz, 2H), 6.99 (t, J = 7.3 Hz,
anamide (14c). The final product was prepared following the general
1
3
1
H), 6.36 (d, J = 15.8 Hz, 1H). C NMR (75 MHz, DMSO‑d
52.3, 141.0, 139.5, 138.1, 129.1, 128.8 (2C), 128.3 (2C), 122.0, 118.3
: [M + H]+, m/z
98.1191. Found 298.1188. Analytical HPLC retention time: 9.9 min;
6
) δ 163.2,
method B from 13c. The product was isolated as a white solid (0.216 g,
1
1
90%). H NMR (300 MHz, DMSO‑d
6
) δ 10.36 (br s, 1H), 9.81 (br s, 1H),
(
2C), 118.1 (2C), 116.6. HRMS calc. for C16
H
16
N
3
O
3
8.62 (s, 1H), 8.57 (s, 1H), 7.46–7.30 (m, 4H), 7.10 (d, J = 7.2 Hz, 2H),
2
6.87 (d, J = 8.0 Hz, 2H), 3.73 (s, 3H), 2.77 (t, J = 6.2 Hz, 2H), 2.26 (t, J
1
3
purity: 96.1%.
= 6.4 Hz, 2H). C NMR (75 MHz, DMSO‑d
6
) δ 168.3, 154.4, 152.8,
1
37.9, 134.1, 132.9, 128.4 (2C), 119.9 (2C), 118.2 (2C), 113.9 (2C),
+
4
.1.2.6. (E)-3-(4-(3-(4-chlorophenyl)ureido)phenyl)-N-hydrox-
55.1, 34.1, 30.2. HRMS calc. for C17
H
20
N
3
O
4
: [M + H] , m/z 330.1453.
yacrylamide (11b). The final product was prepared following the gen-
Found 330.1480. Analytical HPLC retention time: 10.4 min; purity:
99.8%.
eral method B from 10b. The product was isolated as a yellow solid
1
(
(
0.331 g, quant.). H NMR (300 MHz, DMSO‑d
6
) δ 11.27 (br s, 1H), 9.97
br s, 1H), 9.87 (s, 1H), 9.71 (s, 1H), 7.62–7.26 (m, 9H), 6.37 (d, J =
4.1.2.12. N-hydroxy-3-(4-(3-(4-nitrophenyl)ureido)phenyl)propanamide
1
1
5.7 Hz, 1H). 13C NMR (75 MHz, DMSO‑d
39.0, 138.0, 128.5 (2C), 128.2 (2C), 125.2, 119.8 (2C), 119.6, 118.2
15ClN
: [M + H]+, m/z 332.0801.
Found 332.0814. Analytical HPLC retention time: 12.3 min; purity:
6
) δ 163.2, 152.5, 141.2,
(14d). The final product was prepared following the general method B
1
from 13d. The product was isolated as a yellow solid (0.250 g, 85%). H
(
2C), 116.7. HRMS calc. for C16
H
3
O
3
6
NMR (300 MHz, DMSO‑d ) δ 11.28 (br s, 1H), 9.51 (s, 1H), 9.14 (s, 1H),
9.03 (br s, 1H), 8.19 (d, J = 9.0 Hz, 2H), 7.71 (d, J = 9.0 Hz, 2H), 7.17 (d,
9
9.3%.
J = 7.1 Hz, 2H), 6.99 (d, J = 7.3 Hz, 2H), 2.84 (t, J = 6.2 Hz, 2H), 2.62 (t,
1
3
J = 6.4 Hz, 2H). C NMR (75 MHz, DMSO‑d
6
) δ 163.4, 151.8, 146.2,
4
.1.2.7. (E)-N-hydroxi-3-(4-(3-(4-methoxyphenyl)ureido)phenyl)acryl-
141.1, 140.2, 129.0, 128.3 (2C), 125.1 (2C), 118.6 (2C), 117.6 (2C),
+
amide (11c). The final product was prepared following the general
34.1, 30.2. HRMS calc. for C16
H
17
N
4
O
5
: [M + H] , m/z 345.1198. Found
method B from 10c. The product was isolated as a white solid (0.327 g,
345.1160. Analytical HPLC retention time: 11.3 min; purity: 99.3%.
1
quant.). H NMR (300 MHz, DMSO‑d
6
) δ 10.67 (s, 1H), 9.00 (s, 1H), 8.82
(
(
s, 1H), 8.55 (s, 1H), 7.61–7.26 (m, 7H), 6.89 (d, J = 8.8 Hz, 2H), 6.36
4.1.3. General method C for the synthesis of ethyl 3-(4-aminophenyl)
propanoate (12)
1
3
d, J = 15.8 Hz, 1H), 3.73 (s, 3H). C NMR (75 MHz, DMSO‑d
6
) δ 163.2,
1
54.6, 152.5, 141.2, 138.2, 132.5, 128.3 (2C), 128.1, 120.2 (2C), 118.0
In a solution of ethyl 4-aminocinnamate (9, 0.382 g, 2 mmol, 1 eq.)
1
2

M.T. Tavares et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116085
in ethanol (30 mL), 0.240 g of 10% palladium on charcoal (10% Pd/C)
IC50 concentration, as well as the vorinostat and doxorubicin controls,
◦
◦
was added at 0 C. Then, the reaction was kept under constant stirring at
for 48 h, fixed with 70% ethanol and then stored at ꢀ 20 C. Ki-67
room temperature under H
2
atmosphere for 16 h. The product was
staining was performed following the manufacturer’s instructions (BD
Bioscience) and the mean of fluorescence intensity (M.F.I.) was obtained
by flow cytometry using the FACSCalibur, as previously described. IgG
isotype was used as a negative control for each condition.
filtered through a small pad of Celite®, and concentrated to aford the
1
product as a yellow liquid (0.386 g, quant.). H NMR (300 MHz, DMSO-
d
6
) δ 10.19 (s, 2H), 7.31 (d, J = 8.6 Hz, 2H), 7.27 (d, J = 8.6 Hz, 2H),
.04 (q, J = 7.1 Hz, 2H), 2.86 (t, J = 7.4 Hz, 2H), 2.62 (t, J = 7.4 Hz, 2H),
4
1
1
1
3
.15 (t, J = 7.1 Hz, 3H). C NMR (75 MHz, DMSO- d
6
) δ 172.0, 139.6,
4.6. Western blot
30.9, 129.3 (2C), 122.6 (2C), 59.8, 34.9, 29.7, 14.0.
Equal amounts of protein were used as total extracts, followed by
SDS-PAGE,69 western blot analysis with the indicated antibodies and
imaging using the SuperSignal™ West Dura Extended Duration Sub-
strate System (Thermo Fisher Scientific, San Jose, CA, USA) and G: BOX
Chemi XRQ (Syngene, Cambridge, UK). Antibodies against phosphory-
4
.2. Cell culture
The HCT-116 (colon carcinoma, ATCC® 247™), MCF-7 (mammary
gland adenocarcinoma, ATCC® HTB-22™), Jurkat (acute T-cell leuke-
mia, ATCC® TIB-152™), Namalwa (Burkitt’s lymphoma, ATCC® CCRL-
1
1
lated histone H2AX (γH2AX, #9718), acetyl-
##5335), phosphoserine (pSer, #2981), phosphotyrosine (pTyr,
#9411), -tubulin ( -Tub, #2144) and glyceraldehyde 3-phosphate
α
-tubulin (ac- -Tub,
α
432™) cancer cells, and the HS-5 (bone marrow/stroma, ATCC® CRL-
1882™) non-tumorigenic cells were obtained from American Tissue
α
α
and Cell Collection (ATCC). Cell culture conditions were developed
using the Roswell Park Memorial Institute (RPMI) medium (Thermo
Fisher Scientific, San Jose, CA, USA) supplemented with 10% fetal
bovine serum (FBS) (Thermo Fisher Scientific, San Jose, CA, USA) and
dehydrogenase (GAPDH, #5174) were obtained from Cell Signaling
Technology (Danvers, MA, USA). Antibody against p21 (sc-6246) was
obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Band
intensities were determined using UN-SCAN-IT gel 6.1 software (Silk
Scientific; Orem, UT, USA).
1
% penicillin–streptomycin (Thermo Fisher Scientific, San Jose, CA,
◦
USA). Cells were kept in an incubator with 5% CO
2
at 37 C. Doxorubicin
and vorinostat (SAHA) were obtained from Sigma-Aldrich (Sigma-
Aldrich, St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) (Synth, Dia-
dema, SP, Brazil) was used as a vehicle for the dilution of the tested
compounds.
4.7. In vitro HDAC inhibition assays
HDAC inhibition assays were performed by Reaction Biology Corp.
(Malvern, PA) using isolated human, recombinant full-length HDAC1
and HDAC6 from a baculovirus expression system in Sf9 cells. The
acetylated fluorogenic peptide, RHKK(Ac)AMC, derived from residues
4
.3. MTT assay
3
79–382 of p53 was used as substrate. The reaction buffer was made up
The cytotoxicity of the compounds was measured by methylthiazol
of 50 mM Tris-HCl pH 8.0, 127 mM NaCl, 2.7 mM KCl, 1 mM MgCl , 1
2
tetrazolium (MTT) assay (Thermo Fisher Scientific, San Jose, CA,
mg/mL BSA, and a final concentration of 1% DMSO. Compounds were
delivered in DMSO and delivered to enzyme mixture with preincubation
of 5–10 min followed by substrate addition and incubation for 2 h at
6
8
4
USA). HCT-116, MCF-7, and HS-5 (1 × 10 cells/well), and Jurkat and
4
Namalwa (2 × 10 cells/well) were cultured in a 96-well plate in the
◦
RPMI medium, containing 10% FBS and 1% penicillin–streptomycin
30 C. Trichostatin A (TSA) and a developer were added to quench the
with increasing concentrations of doxorubicin (0.0032–10
μ
M), vor-
M) for 24,
8 and 72 h. DMSO 1% was used as a negative control. After treatment,
reaction and generate fluorescence, respectively. Dose ꢀ response
inostat (0.0032–50
μ
M) and tested compounds (0.0032–50
μ
curves were generated starting at 100 M compound with 3-fold serial
μ
4
dilutions to generate a 10-dose plot. IC50 values were then generated
from the resulting plots, and the values expressed are the average of
duplicate trials.
the culture media of HCT-116, MCF-7, and HS-5 adherent cells were
replaced with fresh media containing MTT solution (0.5 mg/mL) and
incubated for additional 3 h. Then, the MTT solution was removed, and
◦
the plates were dried at 35 C for 30 min. The formazan product was
4.8. Molecular modeling – Docking and molecular dynamics simulations
solubilized by 150
μ
L of DMSO, and the absorbance was measured at
5
40 nm using a microplate reader. For non-adherent cells (Jurkat and
We aimed to suggest potential targets for our compounds based on
the similarity against databases of reported target-inhibitor pairs. In this
Namalwa), 10 L/well of MTT solution was added (0.5 mg/mL final
μ
7
0
concentration) after treatments and incubated for additional 3 h. After
MTT incubation, 100 L of 0.1 N HCl in isopropanol solution was added
sense, we used the function TargetHunter from the server cBligand,
μ
with standard settings, where compounds are compared in terms of 2D
similarity against the ChEmbl and PubChem databases. Among the
resulting list, we chose the top hits by score, whose template compounds
maintained the hydroxyamate moiety which resulted in HDAC1 and
HDAC6 as suggested molecular targets.
per well. IC50 values and their 95% confidence intervals were calculated
using sigmoidal nonlinear regression analysis performed with GraphPad
Prism 5 (GraphPad Software, Inc., San Diego, CA, USA).
4
.4. Apoptosis assay
The crystallographic structures of HDAC1 and HDAC6 were obtained
from the Protein Data Bank (PDB) with the access code 5ICN (resolution:
5
71
72
Jurkat and Namalwa cells were seeded in 24-well plates (2 × 10
3.30 Å) and 5EDU (resolution: 2.79 Å), respectively. Despite the low
resolution of those complexes, both systems were chosen due to the
interesting co-crystallized peptide substrate and/or inhibitor contained
cells/well) and treated with compounds’ IC50 and half of IC50, vorinostat
IC50 and doxorubicin IC50 for 48 h. Cells were then washed twice with
′
ice-cold PBS and resuspended in binding buffer containing 1
μ
g/mL
in the active site. HDAC1 s chains B (catalytic domain) and C (substrate
7
AAD and 1 g/mL APC-labeled annexin V (BD Biosciences, San Jose,
μ
peptide) were maintained, by removing the metastasis-associated pro-
tein (Chain A, MTA1). HDAC6 structure was edited to keep only the CD2
catalytic domain (Uniprot Q9UBN7 residues 479–835), by removing the
maltose purification domain. The protein structure was then prepared
by adding hydrogen atoms and fixing missing sidechains using the
Protein Preparation Wizard (PrepWiz),73 missing loops were generated
using Prime and sulfate/crystallization buffer molecules such as potas-
sium ions were removed. Crystallographic waters were removed during
the docking runs.
CA, USA). All specimens were acquired by flow cytometry (FACSCali-
bur; Becton Dickinson, Lincoln Park, NJ, USA) after incubation for 15
min at room temperature in a light-protected area and analyzed using
FlowJo software (Treestar, Inc., San Carlos, CA, USA).
4
.5. Assessment of cell proliferation by Ki-67 staining
Jurkat and Namalwa cells were treated with our compounds at the
1
3

M.T. Tavares et al.
Bioorganic & Medicinal Chemistry 35 (2021) 116085
The three-dimensional (3D) structures of compounds 8b, 11b and
numbers: 2013/19311-6, 2017/00689-0, 2015/17177-6) for financial
support. The authors wish to acknowledge the CSC – IT Center for Sci-
ence, Finland, for the generous computational resources.
1
4b were generated in their neutral forms and then prepared using
7
4
LigPrep, with standard options, in order to generate charges for
docking. Molecular docking was performed in a grid encompassing
residues around 12 Å from the centroid of the co-crystallized Zn2 , using
the default settings of the InducedFit software (available in Maestro
v.2019.4),75 with at least ten poses selected for further visual inspection.
Redocking: protein complexes with the original co-crystallized li-
gands and the docked ligands were submitted for molecular dynamics
+
Appendix A. Supplementary material
Additional data collection and refinement statistics, and H and 13C
NMR spectra of the compounds are available in the Supporting Infor-
mation free of charge at DOI: . Data referring the molecular modeling is
available at 10.5281/zenodo.4064775. Supplementary data to this
article can be found online at https://doi.org/10.1016/j.bmc.2021.11
6085.
1
(
MD) simulations in order to evaluate both the ligand stability and the
effects of ligand-binding upon the protein. The entire protocol was
previously described elsewhere,76 with details about the minimization
protocol and interaction evaluation. MD simulations were carried out
using Desmond,77 with the OPLS3e force-field. The simulated system
encompassed the protein–ligand complexes, a predefined water model
78
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