1,3,4-Oxadiazole-containing histone deacetylase inhibitors: Anticancer activities in cancer cells

Article abstract of DOI:10.1021/jm500303u

We describe 1,3,4-oxadiazole-containing hydroxamates (2) and 2-aminoanilides (3) as histone deacetylase inhibitors. Among them, 2t, 2x, and 3i were the most potent and selective against HDAC1. In U937 leukemia cells, 2t was more potent than SAHA in inducing apoptosis, and 3i displayed cell differentiation with a potency similar to MS-275. In several acute myeloid leukemia (AML) cell lines, as well as in U937 cells in combination with doxorubicin, 3i showed higher antiproliferative effects than SAHA.

Full text of DOI:10.1021/jm500303u

Brief Article
pubs.acs.org/jmc
1,3,4-Oxadiazole-Containing Histone Deacetylase Inhibitors:
Anticancer Activities in Cancer Cells
Sergio Valente,† Daniela Trisciuoglio,‡ Teresa De Luca,‡ Angela Nebbioso,§ Donatella Labella,†
Alessia Lenoci,† Chiara Bigogno,∥ Giulio Dondio,∥ Marco Miceli,§ Gerald Brosch,⊥ Donatella Del Bufalo,‡
Lucia Altucci,§,# and Antonello Mai*,†,∇
†Department of Drug Chemistry and Technologies, Sapienza University of Rome, P. le A. Moro 5, 00185 Rome Italy
‡Regina Elena National Cancer Institute, Via Elio Chianesi, 53, 00144 Rome, Italy
§Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Vico L. De Crecchio 7, 80138 Naples,
Italy
∥Aphad Srl, Via della Resistanza 65, 20090 Buccinasco, Milan, Italy
⊥Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innrain 80/III, 6020 Innsbruck, Austria
#CNR−IGB, Institute of Genetics and Biophysics, via P. Castellino, 80100 Naples, Italy
∇Istituto Pasteur−Fondazione Cenci Bolognetti, Sapienza University of Rome, P. le A. Moro 5 00185, Rome, Italy
S
* Supporting Information
ABSTRACT: We describe 1,3,4-oxadiazole-containing hy-
droxamates (2) and 2-aminoanilides (3) as histone deacetylase
inhibitors. Among them, 2t, 2x, and 3i were the most potent
and selective against HDAC1. In U937 leukemia cells, 2t was
more potent than SAHA in inducing apoptosis, and 3i
displayed cell differentiation with a potency similar to MS-275.
In several acute myeloid leukemia (AML) cell lines, as well as
in U937 cells in combination with doxorubicin, 3i showed
higher antiproliferative effects than SAHA.
A number of HDAC inhibitors are undergoing extensive
clinical evaluation as single agents and in combination with
other chemotherapeutics; among them, vorinostat (SAHA) and
romidepsin (FK-228) have been approved by the U.S. FDA for
the treatment of refractory cutaneous T-cell lymphoma
(CTCL).5,6 In addition, valproic acid (VPA), panobinostat
(LBH589), belinostat (PDX101), givinostat (ITF2357),
resminostat (4SC-201), entinostat (MS-275), mocetinostat
(MGCD0103), 4SC-202, and CHR-3996 (Chart 1) are into
phase I/II/III clinical trials alone or in combination with other
drugs for treatment of hematological and/or solid tumors.4
The pharmacophore model for HDACi includes a cap group
(CAP) interacting with the rim of the catalytic tunnel of the
enzyme, a polar connection unit (CU) linked to a hydrophobic
spacer (HS) which allows the molecule to lie into the tunnel,
and a Zn-binding group (ZBG) able to complex the Zn2+ at the
bottom of the cavity.7 To date, many different CAP, CU, HS,
and ZBG moieties have been studied, and structurally diverse
HDAC inhibitors have been recently reviewed.8
INTRODUCTION
■
Lysine acetylation and deacetylation are crucial post-transla-
tional modifications in the epigenetic field. These processes are
regulated by two groups of enzymes with opposite activities:
histone acetyltransferases (HATs) and histone deacetylases
(HDACs), leading to gene transcription or silencing,
respectively.1 Apart from histone modification, HATs and
HDACs regulate the post-translational acetylation of many
nonhistone proteins, including transcription factors, chaper-
ones, and signaling molecules, resulting in changes in protein
stability, protein−protein interactions, and protein−DNA
interactions.2
At least 50 nonhistone proteins have been identified as
HDAC substrates, including transcription factors (p53, E2F, c-
Myc, nuclear factor-κB (NF-κB), hypoxia-inducible factor 1 α
(HIF-1α), estrogen receptor α (ERα), and the androgen
receptor (AR), MyoD, chaperones (HSP90), signaling
mediators (Stat3 and Smad7), and DNA repair proteins.2
HDAC inhibitors (HDACi) mediate cancer cell death
through several pathways, and in particular class I HDAC
inhibitors are able to induce apoptosis, differentiation, cell cycle
arrest, inhibition of DNA repair, upregulation or reactivation of
silenced tumor suppressors, downregulation of growth factors,
oxidative stress, autophagy, and control of angiogenesis.3,4
Since 2001, we described various chemically different series
of HDACi [aroylpyrrolylhydroxamates (APHAs) and
(aryloxopropenyl)pyrrolylhydroxamates,9 aroyl/arylamino-,
Received: February 25, 2014
Published: June 27, 2014
© 2014 American Chemical Society
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oxadiazol-yl-benzoates 10a,b, respectively (Scheme 1 and
Scheme S1 in Supporting Information). Heck cross-coupling
reaction carried out onto the iodo-intermediates 6a−v with
palladium acetate, triethylamine, and n-butyl acrylate in N,N-
dimethylacetamide at 110 °C gave the corresponding butyl
esters 7a−v, which were in turn hydrolyzed with 2N potassium
hydroxide in ethanol into the related acids 8a−v (Scheme 1).
The methyl esters 10a,b were hydrolyzed following the same
procedure to furnish the related benzoic acids 11a,b (Scheme
S1 in Supporting Information). Such key compounds 8 and 11
were then converted into the corresponding hydroxamates 2a−
v and 2w,x, respectively, by reaction with ethyl chloroformate/
triethylamine followed by addition of O-(2-methoxy-2-propyl)-
hydroxylamine and final acidic treatment with Amberlyst 15. In
addition, 8h,i,r−v and 11a,b were treated with benzotriazole-1-
yloxytris(dimethylamino)phosphonium hexafluorophosphate
(BOP Reagent, Castro’s Reagent), o-phenylendiamine, and
triethylamine in dry N,N-dimethylformamide to afford the
corresponding 2-aminoanilides 3a−g and 3h,i, respectively
(Scheme 1 and Scheme S1 in Supporting Information).
Chart 1. HDACi Approved by FDA and/or in Clinical Trials
Chemical and physical data for the intermediate compounds
5−8, 10, and 11 are reported in Supporting Information.
Chemical and physical data for the final compounds 2 and 3 are
listed in Tables S4 and S5 in Supporting Information.
(phenyloxopropenyl)-, and (amidopropenyl)cinnamyl and
pyridinylpropenoic hydroxamates and 2-aminoanilides,10 and
uracil-based hydroxamates and 2-aminoanilides (UBHAs)11]
according to the known pharmacophore model. Among them,
when tested in human leukemia U937 cells, some acylamino-
cinnamyl hydroxamates and 2-aminoanilides 1 (Figure 1)
RESULTS AND DISCUSSION
■
Enzyme Studies. To perform a first preliminary screening,
the hydroxamates 2a−x were tested by determining their IC50
values against maize HD1-B and HD1-A, two homologues of
class I and class II HDACs, and against class I mouse HDAC1
(Tables S8 and S9 in Supporting Information). Afterward, a
series of related 2-aminoanilides 3a−i have been prepared, and
selected compounds 2 and 3a−i were screened against human
recombinant HDAC1 and HDAC4 (% of inhibition at 5 μM)
using histone H3 (HDAC1) or the nonhistone trifluoroacetyl-
lysine12 (HDAC4) as a substrate, respectively (Table S10 in
Supporting Information). From these preliminary screenings, it
emerged that, regardless of bearing an hydroxamate or an
anilide function, (i) the introduction of 1-naphthyl ring at the
1,3,4-oxadiazole C5-position provided the highest inhibitory
potency, (ii) the insertion of one methylene unit (no more)
between the above aryl ring and the C5-position of the
oxadiazole increased the potency of the derivatives, and (iii)
this potency can be further improved by the introduction of an
additional methylene unit between the 1,3,4-oxadiazole C2-
position and the cinnamic/benzoic portion.
Figure 1. Design of 1,3,4-oxadiazole-containing HDAC inhibitors 2
and 3.
displayed higher pro-apoptotic and/or cytodifferentiating
effects than vorinostat, entinostat, and S-HDAC42, used as
reference drugs.10 Prompted by these results, to study the effect
of changes at CU in such compounds, we replaced the amide
group of 1 with the bioisostere 1,3,4-oxadiazole moiety, thus
developing new series of hydroxamates 2 and 2-aminoanilides 3
(Figure 1), which have been tested as HDACi to assess their
capability to induce cell cycle arrest, apoptosis, and/or
cytodifferentiation in human leukemia U937 cells. Afterward,
selected compounds have been tested in a panel of cancer cells
to detect their effects on antiproliferation. Lastly, combination
treatment of selected HDACi with doxorubicin in U937 cells
have been evaluated.
From these first results, we selected the hydroxamates 2h, 2r,
2t, 2w, and 2x as well as the 2-aminoanilides 3a−c, 3e, 3h, and
3i to determine the IC50 values against human HDAC1,
HDAC4, and HDAC6 (Table 1). SAHA was tested as reference
drug.
The hydroxamates 2h, 2r, 2t, and 2x displayed submicro-
molar (or single-digit micromolar, 2x vs HDAC6) inhibition
toward HDAC1 and HDAC6, while they are much less efficient
against HDAC4. The “double methylene space” inserted
between the aryl CAP group and the C5-oxadiazole position
on one hand, and between the C2-oxadiazole and the
cinnamic/benzoic hydroxamate HS-ZBG on the other hand,
conferred to the derivatives 2t and 2x the highest potency
against HDAC1, while the replacement of the cinnamic with
the benzoic moiety at the HS led to less effective HDAC6
inhibition (compare 2t with 2x). In the 2-aminoanilide series,
the change from cinnamic to benzoic linker led to more potent
CHEMISTRY
■
Cyclocondensation of the hydrazides 5 or 9 (5a and 9 are
commercially available, 5b was prepared by reaction between
the methyl-4-iodophenylacetate 4 and hydrazine monohydrate
in methanol at 60 °C) with the appropriate carboxylic acids
provided the iodophenyloxadiazoles 6a−v or the methyl 1,3,4-
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a
Scheme 1
a
(a) NH2NH2·H2O, MeOH, 60 °C, 2 h; (b) RCOOH, POCl3, 110 °C, 2 h; (c) n-butyl acrylate, Pd(OAc)2, (C2H5)3N, DMA, 120 °C, N2, 30 min;
(d) 2N KOH, EtOH, overnight; (e) (1) ClCOOC2H5, (C2H5)3N, dry THF, 0 °C, 30 min, (2) CH3OC(CH3)2ONH2, dry THF, 0 °C, 1 h, (3)
Amberlyst 15, CH3OH, rt, 1.5 h; (f) (1) (C2H5)3N, BOP reagent, dry DMF, N2, rt, (2) o-phenylendiamine, dry DMF, N2, rt, 1 h.
improved the acetyl-H3 levels, and 3e, 3h, and 3i gave a signal
for α-tubulin acetylation, despite their low potency against
HDAC6 (Figure 2C,D). With regard to p21 induction, the
hydroxamates 2r and 2x gave the greatest effect, and the anilide
3i showed higher p21 induction than SAHA used as reference
drug (Figure 2E,F).
Table 1. IC50 Values of Selected 2 and 3 Derivatives against
Human Recombinant HDAC1, HDAC4, and HDAC6
a
IC50 SD (μM)
compd
HDAC1
HDAC4
HDAC6
2h
2r
0.8 0.02
0.6 0.02
0.2 0.01
9.9 1.6
0.2 0.01
1.0 0.05
2.4 0.5
1.5 0.04
4.1 0.5
0.3 0.02
0.2 0.01
0.3 0.02
>20
15.2 2.1
>20
0.06 0.001
0.03 0.002
0.03 0.001
4.1 0.9
1.2 0.2
>20
Apoptosis Induction and Granulocytic Differentiation
in Human U937 Leukemia Cells. Prompted by results of
enzyme inhibition, we selected 2t, 2x, 3a, 3h, and 3i to
determine their effects on cell cycle, apoptosis induction, and
granulocytic differentiation in human leukemia U937 cells. The
assays were performed at five different doses (0.1, 1, 5, 10, and
25 μM) for each compound for 24 and 48 h. SAHA and MS-
275 (5 μM) were used as reference drugs and positive controls.
In the cell cycle assay (Figure S1 in Supporting Information),
after 48 h both the benzoic hydroxamate 2x and anilide 3i
provided a block of the U937 cells in the S phase similarly to
SAHA, with 3i already acting at 1 μM. Taking the pre-G1 peak
as an index of apoptosis induction, after 48 h of treatment, 2t,
2x, and 3i displayed a clear dose-dependent apoptotic effect in
U937 cells, 2t being the most potent (from 58% up to 74%
when tested from 5 to 25 μM) and more efficient than SAHA
at 5 μM (SAHA, 48%; 2t, 58%) (Figure 3A; for other data at 24
and 48 h see Figure S2 in Supporting Information). The
expression of the surface antigen CD11c in U937 leukemia cells
was determined as a marker of granulocytic differentiation. In
this assay, after 48 h, 2t, 3a, 3h, and 3i induced a dose-
dependent increase of CD11c positive/propidium iodide (PI)
negative cells, with 3i displaying at 5 μM the highest
differentiation effect (45%) similarly to MS-275 (47%) (Figure
3B; for other data at 24 and 48 h, see Figure S3 in Supporting
Information). At higher concentrations, 3i showed low water
2t
2w
2x
>20
7.8 1.9
>20
3a
3b
3c
>20
>20
>20
>20
3e
>20
>20
3h
3i
>20
16.1 2.4
8.9 1.1
0.06 0.003
>20
SAHA
8.8 2.4
a
Values represent the mean of at least three independent experiments.
compounds (3h,i) against HDAC1 respect to the cinnamic
ones, and 3h,i were also the only anilides with little activity
against HDAC6 among those tested.
Functional Assays and p21 Induction in Human U937
Leukemia Cells. As functional tests for HDAC inhibition,
Western blot analyses in human leukemia U937 cells were
performed to determine the acetylation levels of histone H3
and α-tubulin for selected compounds 2 and 3 at 5 μM.
Moreover, the capability of the same compounds to induce p21
expression at 5 μM was assessed. SAHA and MS-275 were used
as reference drugs (Figure 2). All selected hydroxamates
increased both acetyl-histone H3 (with the exception of 2w)
and acetyl-α-tubulin levels, according to their HDAC1/HDAC6
inhibition (Figure 2A,B). Among the 2-aminoanilides, all but 3a
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Figure 2. Effects of selected compounds 2 on acetyl-H3 (A) and acetyl-α-tubulin (B) levels in U937 cells. Effects of selected compounds 3 on acetyl-
H3 (C) and acetyl-α-tubulin (D) levels in U937 cells. Effect of p21 induction by selected compounds 2 (E) and 3 (F). Western blot analyses were
performed with specific antibodies. H4 and ERK proteins were used for equal loading. Blots representative of two independent experiments with
similar results are shown. Acetyl-H3, acetyl-α-tubulin, and p21 levels were quantified by densitometric analyses, and fold increase relative to control
cells is presented.
Table 2. Antiproliferative IC50 Values of 2t and 3i in a Panel
a
of Cancer Cell Lines
IC50 SD (μM)
cell line
M14
cancer type
2t
3i
melanoma
melanoma
lung
45
8
43.3 2.9
17.4 3.9
23.6 1.4
24.7 0.2
6.7 1.5
SANTO
A549
24 6.4
29.5 9.2
21.3 5.8
5.1 0.03
33.6 6.9
18.4 6.7
50 13.9
7.8 0.9
6.0 2.4
4.1 0.2
2.6 0.2
2.2 0.1
HCT116
SW620
HCC937
MB468
U2OS
colon
colon
breast
29.8 1.9
19.6 5.7
breast
osteosarcoma
leukemia
leukemia
leukemia
leukemia
leukemia
20.4
4
U937
1.8 1.2
2.8 1.9
2.0 0.2
1.8 0.9
1.6 0.6
HL60
HEL
KG1
MOLM13
a
Values represent the mean of at least three separate experiments.
most responsive cancer cell lines were the colorectal
adenocarcinoma SW620 and all the tested human acute
myeloid leukemia (AML) cell lines (U937, HL60, HEL, KG1,
and MOLM13), whose proliferation was inhibited by 3i and 2t
at single-digit micromolar concentrations. When compared to
SAHA, 3i displayed higher cell growth arrest against all the
tested AML U937, HL60, HEL, KG1, and MOLM13 cells
(SAHA IC50 values: 3.0, 2.9, 2.3, 4.4, and 2.9 μM, respectively).
Because of their high antiproliferative effects in leukemia, we
tested 2t, 3i, or SAHA (each at 2.5 μM) in combination with
doxorubicin (0.2 μM) in U937 for 24 h (Figure 4). In this
assay, all the combinations between doxorubicin and HDACi
displayed increased cell growth arrest with respect to the
treatment with the single agent. Interestingly, the use of
doxorubicin plus 3i was more effective than doxorubicin plus
SAHA (75% vs 69% inhibition of proliferation), thus
confirming the higher potency of 3i respect to SAHA.
Figure 3. Dose-dependent apoptosis induction (A) and cytodiffer-
entiation (B) in human leukemia U937 cells treated with selected 2
and 3 derivatives.
solubility and high apoptosis so that the differentiation effect
was not detectable.
Antiproliferative Activities of 2t and 3i in Cancer
Cells. Combination Treatment with Doxorubicin. The
hydroxamate 2t and the 2-aminoanilide 3i were tested in a
panel of different cancer cell lines (lung, colon, liver, and breast
cancer, melanoma, osteosarcoma, and leukemias) to determine
their antiproliferative activities (Table 2).
In general, the anilide 3i showed similar or lower IC50 values
than the hydroxamate 2t along the various cancer cell lines. The
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Journal of Medicinal Chemistry
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reference tetramethylsilane (Me4Si). EIMS spectra were recorded with
a Fisons Trio 1000 spectrometer; only molecular ions (M+) and base
1
peaks are given. All compounds were routinely checked by TLC, H
NMR, and 13C NMR spectra. TLC was performed on aluminum-
backed silica gel plates (Merck DC, Alufolien Kieselgel 60 F254) with
spots visualized by UV light. All solvents were reagent grade and, when
necessary, were purified and dried by standard methods. Concen-
tration of solutions after reactions and extractions involved the use of a
rotary evaporator operating at reduced pressure of ca. 20 Torr.
Organic solutions were dried over anhydrous sodium sulfate.
Elemental analysis has been used to determine purity of the described
compounds, that is, >95%. Analytical results are within 0.40% of the
theoretical values. All chemicals were purchased from Aldrich Chimica,
Milan (Italy), or from Alfa Aesar, Karlsruhe (Germany), and were of
the highest purity.
General Procedure for the Synthesis of the N-Hydroxy-3-(4-
(5-aryl/arylalkyl-1,3,4-oxadiazol-2-yl)phenyl)acrylamides and
-benzamides (2a−x). Example: N-Hydroxy-4-((5-(naphthalen-
1-ylmethyl)-1,3,4-oxadiazol-2-yl)methyl)benzamide (2x). Trie-
thylamine (1.13 mmol, 0.16 mL) and ethyl chloroformate (1.04 mmol,
0.10 mL) were added to a solution of 4-((5-(naphthalen-1-ylmethyl)-
1,3,4-oxadiazol-2-yl)methyl)benzoic acid 11b (0.87 mmol, 0.3 g) in
dry tetrahydrofuran (10 mL) at 0 °C, and the resulting mixture was
stirred at this temperature for 15 min. After this time, O-(2-methoxy-2-
propyl)hydroxylamine (2.61 mmol, 0.19 mL) was added and the
mixture was stirred for 1 h. The solvent was evaporated, the residue
was eluted with methanol (10 mL), and the ionic resin Amberlyst 15
(25 mg of resin per 0.25 mmol of acid) was added. After 2 h, the resin
was filtered off, the product concentrated in vacuo, washed with
diethyl ether (3 × 10 mL), and recrystallized by benzene/acetonitrile.
1H NMR (DMSO-d6) δ 4.22 (s, 2H, −CH2-naphthalene), 7.34−7.36
Figure 4. Antiproliferative effect of combination treatment between
doxorubicin (0.2 μM) and SAHA, 2t, or 3i (each at 2.5 μM), in U937
cells for 24 h.
CONCLUSIONS
■
On the basis of structural motifs of the aroylaminocinnamyl
hydroxamates and 2-aminoanilides 1 previously described by us
and displaying high pro-apoptotic and/or cytodifferentiating
effects in human leukemia U937 cells,10 we planned to replace
the amide group (CU) of such compounds with its bioisoster
1,3,4-oxadiazole group and to synthesize a new series of
hydroxamates 2 and 2-aminoanilides 3 to be tested as HDACi
and anticancer agents.
Among all the prepared hydroxamates 2a−x and 2-
aminoanilides 3a−i, we selected compounds 2h,r,t,w,x and
3a−c,e,h,i to determine their IC50 values against human
HDAC1, -4, and -6. In general, the hydroxamates 2 were
active across 2 out of 3 isoforms where the anilides 3 displayed
high inhibition only against HDAC1. In Western blot
experiments, the hydroxamates 2 increased both histone H3
and α-tubulin acetylation, while among the anilides, only 3e,
3h, and 3i showed effects on both the targets. About p21
induction, 2r, 2x, and 3i gave a signal at 5 μM, 3i being more
effective than SAHA. When tested in U937 cells for 48 h as pro-
apoptotic and/or cytodifferentiation agents, 2t, 2x, and 3i
showed a dose-dependent apoptotic effect, 2t being more
potent than SAHA at 5 μM, and 2t, 3a, 3h, and 3i induced a
dose-dependent differentiation determined as increased per-
centage of CD11c positive/PI negative cells respect to the
control. In this assay, at 5 μM, 3i displayed the same potency as
MS-275, one of the most efficient HDACi in cytodifferentia-
tion. When tested against a panel of cancer cell lines to assess
their antiproliferative potential, 3i and 2t displayed single-digit
micromolar activity against SW620 colon adenocarcinoma and
five AML cell lines (U937, HL60, HEL, KG1, and MOLM13),
3i always being more potent than SAHA in leukemias. In
combination treatment with doxorubicin, 3i was more effective
than SAHA in inhibiting U937 cell proliferation.
(d, 2H, benzene protons), 7.40−7.45 (m, 2H, naphthalene protons),
7.53−7.54 (d, 2H, benzene protons), 7.76−7.78 (m, 2H, naphthalene
protons), 7.86−7.88 (d, 1H, naphthalene proton), 7.92−7.94 (d, 1H,
naphthalene proton), 8.13 (d, 1H, naphthalene proton), 9.56 (bs, 1H,
CONHOH), 10.95 (bs, 1H, CONHOH). 13C NMR (DMSO-d6) δ
29.2, 30.9, 124.2, 124.3, 125.6, 125.8, 126.5, 126.9, 127.4 (2C), 128.6,
129.2 (2C), 131.2, 132.6, 133.5, 134.0, 139.7, 163.0, 166.4 (2C). MS
(EI): m/z [M]+: 359.1270.
General Procedure for the Synthesis of the N-(2-Amino-
phenyl)-3-(4-(5-aryl/arylalkyl-1,3,4-oxadiazol-2-yl)phenyl)-
acrylamides and -Benzamides (3a−i) Example: N-(2-Amino-
phenyl)-4-((5-(naphthalen-1-ylmethyl)-1,3,4-oxadiazol-2-yl)-
methyl)benzamide (3i). Triethylamine (2.32 mmol, 0.32 mL) and
benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluoro-
phosphate (BOP Reagent) (0.70 mmol, 0.31 g) were added to a
solution of 4-((5-(naphthalen-1-ylmethyl)-1,3,4-oxadiazol-2-yl)-
methyl)benzoic acid 11b (0.58 mmol, 0.2 g) in anhydrous N,N-
dimethylformamide (5 mL) under nitrogen atmosphere. The resulting
mixture was stirred for 30 min at room temperature, afterward, 1,2-
phenylenediamine (0.58 mmol, 0.06 g) was added under nitrogen
atmosphere and the mixture was stirred overnight. The reaction was
quenched with water (50 mL) and extracted with ethyl acetate (3 × 30
mL). The combined organic layers were washed with sodium chloride
solution (3 × 20 mL), dried with sodium sulfate, and the residue
obtained upon evaporation of solvent was purified by column
chromatography (SiO2 eluting with ethyl acetate/n-hexane 1/1) to
1
give pure 3i. H NMR (DMSO-d6, 400 MHz, δ; ppm) δ 4.27 (s, 2H,
From the data presented, it appears that specific HDAC1
inhibition well correlates with induction of apoptosis, cell
differentiation, and cell growth arrest. The anticancer properties
showed by 3i, higher than SAHA in both enzyme (HDAC1)
and cellular assays, will be further studied in other cancer
contexts.
CH2-benzene), 4.67 (s, 2H, −CH2-naphthalene), 4.88 (s, 2H, NH2),
6.59−6.61 (t, 1H, naphthalene proton), 6.76−6.78 (d, 1H,
naphthalene proton), 6.96−6.99 (t, 1H, naphthalene proton), 7.14−
7.16 (d, 1H, benzene proton), 7.36−7.39 (d, 2H, benzene protons),
7.48−7.52 (d, 2H, naphthalene protons), 7.54−7.57 (m, 2H, benzene
proton), 7.87- 7.96 (m, 4H, benzene protons and naphthalene
protons), 8.06−8.08 (d, 1H, benzene proton), 9.37 (bs, 1H,
CONHPh). 13C NMR (DMSO-d6) δ 29.2, 30.9, 116.5
̧ 119.0, 122.4,
EXPERIMENTAL SECTION
■
122.9, 124.2, 124.3, 125.2, 125.6, 125.8, 126.5, 126.9, 127.4 (2C),
128.6, 129.2 (2C), 131.2, 132.6, 133.5, 134.0, 139.7, 141.8, 164.8,
166.4 (2C) ppm. MS (EI): m/z [M]+: 434.1734.
Fluorimetric Human Recombinant HDAC1, -4, and -6
Assays. The HDAC fluorescent activity assay for HDAC1, -4, and
Chemistry. Melting points were determined on a Buchi 530
melting point apparatus and are uncorrected. 1H NMR and 13C NMR
spectra were recorded at 400 MHz on a Bruker AC 400 spectrometer;
chemical shifts are reported in δ (ppm) units relative to the internal
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Journal of Medicinal Chemistry
Brief Article
-6 was based on the Fluor de Lys Substrate and Developer
combination (BioMol) and has been carried out according to
supplier’s instructions. First, the inhibitors and purified recombinant
HDAC1, HDAC4, or HDAC6 enzymes were preincubated at room
temperature for 15 min before substrate addition, the Fluor de Lys
Substrate, which comprises an acetylated lysine side chain. For
HDAC4 assay, the HDAC4-selective, nonhistone substrate reported
by Lahm et al.12 was used. Full length HDAC1 and HDAC4 fused with
a C-terminal His tag were expressed using baculovirus systems.
Deacetylation sensitized the substrates that, in the second step, treated
with the developer produced a fluorophore. Fluorescence was
quantified with a TECAN Infinite M200 station.
Cell Cultures. Human acute myeloid leukemia (AML) cell lines
(HL-60, HEL 92.1.7, MOLM-13, KG1, U-937)13 were cultured in
RPMI with 10% fetal calf serum, 100 U/mL penicillin, 100 μg/mL
streptomycin, and 250 ng/mL amphotericin-B, 10 mM HEPES, and 2
mM glutamine. AML cells were kept at the constant concentration of
200 000 cells per milliliter of culture medium. HCT116 and SW620
(colon carcinoma cancer), M14 and SANTO (melanoma), H1299 and
A549 (nonsmall-cell lung carcinoma), HCC937 and MB468 (breast
carcinoma), and U2OS (osteosarcoma) were cultured in RPMI
(Invitrogen) with 10% fetal calf serum (Hyclone), 100 U/mL
penicillin.
Western Blot Analysis for α-Tubulin and Histone H3
Acetylation in U937 Cells. For quantification of histone H3
acetylation, 5 mg of total histone extracts were separated on a 15%
polyacrylamide gel and blotted as described.14 Western blots were
shown for acetylated histone H3 (Upstate) antibodies, and histone H4
(Abcam) antibodies was used to normalize for equal loading. For
determination of α-tubulin acetylation, 25 mg of total protein extracts
were separated on a 10% polyacrylamide gel and blotted as
described.15 Western blots were shown for acetylated α-tubulin
antibodies (Sigma), and total ERKs (Santa Cruz) were used to
normalize for equal loading.
AUTHOR INFORMATION
Corresponding Author
*Phone: +39 06 49913392. Fax: +39 06 49693268. E-mail:
antonello.mai@uniroma1.it.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by FIRB RBFR10ZJQT, Italian
Association for Cancer Research (MFAG no. 11502), Progetto
Ateneo Sapienza 2013, Progetto IIT-Sapienza, RF-2010-
2318330, and FP7 projects BLUEPRINT/282510 and A-
ParaDDisE/602080.
ABBREVIATIONS USED
■
AML, acute myeloid leukemia; AR, androgen receptor; CD11c-
PE, phycoerythrine-conjugated CD11c; CTCL, cutaneous T-
cell lymphoma; ERα, estrogen receptor α; FACS, fluorescence
activated cell sorting; HAT, histone acetyltransferase; HDAC,
histone deacetylase; HIF-1α, hypoxia-inducible factor 1 alpha;
HSP90, heat shock protein 90; NF-κB, nuclear factor-κB; PBS,
phosphate buffered saline; PI, propidium iodide; SAHA,
suberoylanilide hydroxamic acid; Smad7, mothers against
decapentaplegic homologue 7; Stat3, signal transducer and
activator of transcription 3
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