Novel 2-oxoindoline-based hydroxamic acids: Synthesis, cytotoxicity, and inhibition of histone deacetylation

Article abstract of DOI:10.1016/j.tetlet.2015.09.147

In a continuation of a research program to discover novel small molecules targeting histone deacetylases, a series of 2′-oxospiro[1,3]dioxolane/dithiolane-2,3′-indoline-based hydroxamic acids have been designed and synthesized. These 2-oxoindoline-based hydroxamic acids displayed potent cytotoxicity against three human cancer cell lines, including SW620 (colon cancer), PC-3 (prostate cancer) and AsPC-1 (pancreatic cancer), with IC₅₀ values as low as 0.05-0.07 μM, 74-fold lower than that of SAHA (1.64-3.70 μM). Additionally, compounds in this series exhibited good inhibition against histone-H3 and histone-H4 deacetylation, as evaluated by Western blot assay. These compounds also strongly inhibited HDAC2 with IC₅₀ values as low as 0.03 μM. Docking studies performed using Autodock Vina showed all compounds bound to HDAC2 with relatively higher affinities (from -7.7 to -8.0 kcal/mol) compared to SAHA (-7.4 kcal/mol).

Full text of DOI:10.1016/j.tetlet.2015.09.147

Accepted Manuscript
Novel 2-oxoindoline-based hydroxamic acids: Synthesis, cytotoxicity and in-
hibition of histone deacetylation
Tran Thi Lan Huong, Do Thi Mai Dung, Phan Thi Phuong Dung, Phung Thanh
Huong, Tran Khac Vu, Hyunggu Hahn, Byung Woo Han, Jisung Kim, Minji
Pyo, Sang-Bae Han, Nguyen-Hai Nam
PII:
S0040-4039(15)30188-X
DOI:
http://dx.doi.org/10.1016/j.tetlet.2015.09.147
TETL 46819
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 January 2015
18 August 2015
29 September 2015
Please cite this article as: Huong, T.T.L., Dung, D.T.M., Dung, P.T.P., Huong, P.T., Vu, T.K., Hahn, H., Han, B.W.,
Kim, J., Pyo, M., Han, S-B., Nam, N-H., Novel 2-oxoindoline-based hydroxamic acids: Synthesis, cytotoxicity and
inhibition of histone deacetylation, Tetrahedron Letters (2015), doi: http://dx.doi.org/10.1016/j.tetlet.2015.09.147
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Novel 2-oxoindoline-based hydroxamic acids: synthesis, cytotoxicity and inhibition of histone
deacetylation
Tran Thi Lan Huong,a Do Thi Mai Dung,a Phan Thi Phuong Dung,*a
Phung Thanh Huong,a Tran Khac Vu,b Hyunggu Hahn,c Byung Woo
Han,c Jisung Kim,d Minji Pyo,d Sang-Bae Han,*d Nguyen-Hai Nam*a

1
Tetrahedron Letters
Novel 2-oxoindoline-based hydroxamic acids: synthesis, cytotoxicity and
inhibition of histone deacetylation
Tran Thi Lan Huong,a Do Thi Mai Dung,a Phan Thi Phuong Dung,*a Phung Thanh Huong,a Tran Khac Vu,b
Hyunggu Hahn,c Byung Woo Han,c Jisung Kim,d Minji Pyo,d Sang-Bae Han,*d Nguyen-Hai Nam*a
aHanoi University of Pharmacy, 13-15 Le Thanh Tong, Hanoi, Vietnam
bSchool of Chemical Engineering, Hanoi University of Science and Technology, N0 1 Dai Co Viet, Hai Ba Trung, Hanoi, Vietnam
cResearch Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Korea
dCollege of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 361-763, Korea
ARTICLE INFO
ABSTRACT
In a continuation of a research program to discover novel small molecules targeting histone
deacetylases, a series of 2´-oxospiro[1,3]dioxolane/dithiolane-2,3´-indoline-based hydroxamic
acids have been designed and synthesized. These 2-oxoindoline-based hydroxamic acids
displayed potent cytotoxicity against three human cancer cell lines, including SW620 (colon
cancer), PC-3 (prostate cancer) and AsPC-1 (pancreatic cancer), with IC50 values as low as 0.05-
0.07M, 74-fold lower than that of SAHA (1.64-3.70 M). Additionally, compounds in this
series exhibited good inhibition against histone-H3 and histone-H4 deacetylation, as evaluated
by Western Blot assay. These compounds also strongly inhibited HDAC2 with IC50 values as
low as 0.03 M. Docking studies performed using Autodock Vina showed all compounds bound
to HDAC2 with relatively higher affinities (from -7.7 to -8.0 kcal/mol) compared to SAHA (-7.4
kcal/mol).
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
Histone deacetylase (HDAC) inhibitors
Hydroxamic acids
Isatin
Heterocycle
2015 Elsevier Ltd. All rights reserved.
Target-based drug design and discovery has become
mainstream in anticancer drug discovery and development. With
the advances of the molecular biology, many molecular targets
for anticancer drugs have been identified.1 Among these, histone
deacetylases (HDACs) are a group of targets which currently
attract a great deal of interest from medicinal chemists
worldwide.2,3
Lastly, class IV has only one member (HDAC11) and displays
properties of both class I and class II HDACs.4
It has been widely demonstrated that inhibition of HDACs
results in cell differentiation, apoptosis and cell-cycle arrest in a
number of cancer cell lines. The effects have also been
demonstrated in in vivo preclinical models.5-12 HDAC inhibitors
have, therefore, become a promising class of anticancer agents.8-
11
HDACs are enzymes that catalyze the removal of acetyl
groups from specific lysine residues in the histone tails. This
removal results in chromatin condensation and transcriptional
repression.2,3 Besides their involvement in the regulation of gene
expression and chromatin structure, HDACs have also been
demonstrated to play an important role in cell-cycle progression
and carcinogenic process.2,3 To date, 18 mammalian HDAC
enzymes have been identified. These HDACs are categorized
into four classes based on their structural and functional
characteristics.4 Class I containing four members include HDAC
1, 2, 3, and 8. Class II contains six members, including HDACs
4, 5, 6, 7, 9 and 10 while class III HDACs, also known as
Sirtuins, are NAD+-dependent enzymes and have seven members
(Sirt1-7).
Extensive effort over the past decades has resulted in the
identification of many potent HDAC inhibitors, including SAHA
(suberoylanilide hydroxamic acid, Vorinostat), PXD-01
(Belinostat), LBH-589 (Panobinostat), MS-27-527 (Entinostat),
and romidepsin (Figure 1).13-15 Of these, two inhibitors including
SAHA (trade name, Zolinza®) and romidepsin (tradename,
Istodax®) have been approved by the FDA in 2006 and 2009,
respectively, to treat cutaneous T-cell lymphoma.
As part of our research program to further explore novel
hydroxamic acids as potential inhibitors of HDACs and
anticancer agents, we have directed our efforts on heterocyclic
analogues of SAHA. Previously, we have reported a series of
benzothiazole-based hydroxamic acids with very potent HDAC
inhibitory activity as well as cytotoxicity (Figure 2).16 From this
series,
N1-(6-chlorobenzo[d]thiazol-2-yl)-N8-
*Corresponding authors: Tel.: +84-4-39330531; Fax: +84-4-39332332;
Emails:
namnh@hup.edu.vn
(N.H.
Nam
&
P.T.P.
Dung);
hydroxyoctanediamide (HUP00752), which possessed the most
potent HDAC inhibition and cytotoxicity, was selected for
further in vivo evaluation. It was shown that in the PC-3 prostate
shan@chungbuk.ac.kr (S.B. Han)

2
Tetrahedron
cancer cells xenografted mice model, this compound exhibited
have also investigated a series of isatin-based hydroxamic acids
and found these compounds exhibited potent HDAC inhibition as
well as strong cytotoxicity against several human cancer cell
lines.19 In continuity of our research program, we have designed,
synthesized and evaluated a novel series of 2-oxoindoline-based
hydroxamic acids. The current paper reports the results of this
study.
in vivo antitumor efficacy equipotent to SAHA.17 Currently,
HUP00752 is being further developed as a promising anticancer
agent. Encouraged by this success, we expanded our research
with a series of 5-substitutedphenyl/aryl-1,3,4-thiadiazole-based
hydroxamic acids (Figure 2) which are also very potent as HDAC
inhibitors, possessing strong cytotoxicity.18 Additionally, we
Figure 1. Structures of selected HDAC inhibitors
Figure 2. Structures of benzothiazole- and 5-substituted phenyl-1,3,4-thiadiazole-based hydroxamic acids
The synthesis of hydroxamic acids 3a-h is illustrated in
Scheme 1. In the first step, isatin and its 5- or 7-substituted
derivatives were reacted with an excess of ethylene glycol in the
presence of catalytic p-toluenesulfonic acid (p-TsOH) in
refluxing dry toluene to give the dioxolane intermediates 2a-h.
Compounds 2a-h were then reacted with ethyl 7-
bromoheptanoate in DMF under alkaline conditions (K2CO3)
with catalytic KI to furnish the heptanoates 3a-h as brown-
yellow oils in good overall yields (75-95%). Nucleophilic acyl
substitution of heptanoates 3a-h by hydroxylamine under basic
conditions gave the final hydroxamic acids 4a-h in good yields.
In a similar manner, two dithiolane compounds 7a and 7f were
synthesized using ethylene dithiol instead of ethylene glycol
(Scheme 1). The structures of the products were unambiguously
1
determined using spectroscopic methods, including IR, MS, H
NMR and 13C NMR, as well as elemental analysis (See ESI).
Scheme 1. Synthesis of 2´-oxospiro[1,3]dioxolane/dithiolane-2,3´-indoline-based hydroxamic acids. Reagents and conditions: a) ethylene
glycol/ethylene dithiol, p-TsOH, toluene, ∆, 24 h; b) ethyl 7-bromoheptanoate, K2CO3, KI, DMF, 60 oC, 24 h; c) hydroxylamine hydrochloride,
NaOH, MeOH/THF, -5 oC, 30-60 min.

3
Because the compounds in this study were designed as
analogues of SAHA, we expected them to be at least cytotoxic as
SAHA. Therefore, all synthesized compounds were evaluated for
their cytotoxicity. Initially the compounds were screened for cell
growth inhibition against the SW620 (human colon cancer) cell
line at a fixed concentration of 30 µM using the SRB method20
with some modifications.21,22 It was found that at this
concentration, all ten compounds completely inhibited the growth
of SW620 cells. The compounds, therefore, were further
evaluated at 5 concentrations (e.g. 30, 10, 3, 1, 0.3 µM) with
SW620 and two further human cancer cell lines, including PC-3
(prostate cancer) and AsPC-1 (pancreas cancer). The IC50 values
of each compound were calculated23 (Table 1). In this assay,
SAHA was employed as a positive control. The data shown in
Table 1 clearly indicated that most compounds in this series
displayed potent cytotoxicity in all three human cancer cell lines
tested, with IC50 values ranging from 0.05 to 2.90 µM. It was
found that compound 4g was the most potent with IC50 values of
0.05, 0.07 and 0.07 µM in the SW620, PC-3, and AsPC-1 cell
lines, respectively. This compound was up to 74-fold more potent
than SAHA in terms of cytotoxicity. The least cytotoxic
compound in these series, 4a, was still comparably potent (IC50:
2.69, 2.90, 1.60 µM) to SAHA (IC50: 3.70, 1.64, 2.24 µM) in the
three cell lines tested. The introduction of substituents such as
halogens (-F, -Cl, -Br), -NO2, -CH3, or OCH3, either at position
5´ or 7´ on the indoline moiety, in all cases, resulted in
enhancement of cytotoxicity. At least within the 4a-f series,
substitution at position 7´ (compound 4h, IC50, 0.07, 0.09, 0.09
µM) seemed to be more favorable than substitution at position 5´
(compound 4c, IC50: 1.04, 0.53, 0.49 µM). Using the current
results, it was not possible to the electronic effect of the
substituents on the compounds’ cytotoxicity. The 2´-
oxospiro[1,3]dithiolane-2,3´-indoline derivatives (7a, 7f) showed
better cytotoxicity than the two corresponding dioxolane
compounds (4a, 4f). These results suggest that a bulkier 1,3-
dioxolane moiety at position 3´ of the indoline part was tolerated
and favorable for the cytotoxicity of the compounds, despite the
fact that compounds 7a and 7f showed much higher logP values
(Table 1) compared to others (4a-f, SAHA).
Next, to clarify whether the compounds exhibited cytotoxicity
through inhibition of HDAC in a manner similar to SAHA, the
synthesized hydroxamic acids (4a-h, 7a, 7f) were evaluated for
inhibitory effect of histone-H3 and histone-H4 deacetylation at
concentrations of 3 M using the Western Blot assay. Since
deacetylation of histone proteins is generally affected by
HDACs, the inhibitory effects of the compounds against HDACs
could be postulated through their ability to inhibit the level of
histone deacetylation in cells. As shown in Figure 3, overall, it
was found that the compounds exhibited significant inhibition of
histone deacetylation in SW620 cells. The levels of acetyl-
histones-H4 in cells treated with compounds 4a-h, and 7a, 7f had
almost comparable intensity as that in cells treated with SAHA,
suggesting these compounds were equally effective in the
inhibition of histone-H4 deacetylation as SAHA. The levels of
acetyl-histones-H3 in cells treated with compounds 4a, 4f and 4g
also appeared to be similar to that in cells treated with SAHA.
Meanwhile, in cells treated with 4b-e, 4h and 7a, 7f, the levels of
acetyl-histones-H3 were somewhat less intense than that in cells
treated with SAHA. Nevertheless, the above results showed a
relatively good correlation between the inhibition of histones-H3
and H4 deacetylation and cytotoxicity. Since histones-H3 and H4
deacetylation is regulated principally by HDAC2 and HDAC3,24
it is reasonable to extrapolate that these compounds exert HDAC
inhibitory effects. To further confirm this, we performed an
additional evaluation of the HDAC inhibitory effects of the
compounds using purified HDAC2 enzymes. The results in Table
1 clearly demonstrate that all compounds exhibited comparable
inhibitory effects to SAHA against HDAC2 enzymes with some
compounds, e.g. 4b-e, even showing 2- to 6-fold greater effect
than SAHA in terms of HDAC2 activity inhibition. It was,
however, difficult to correlate the HDAC2 inhibitory activity and
cytotoxicity. Thus, the possibility that the compounds, especially
compounds 4b-e, 4h and 7a, 7f, might exert cytotoxicity through
inhibition of other HDAC subtypes, e.g. HDAC6 or HDAC8,
should not be excluded. A more comprehensive evaluation of
HDAC inhibitory effects of these compounds, therefore, needs to
be further performed to deduce a complete correlation between
HDAC inhibition and cytotoxicity.
Table 1: HDAC inhibition and cytotoxicity of the compounds synthesized against several cancer cell lines
Comps
Molecular
formula
Molecular
weight
LogP1
HDAC2
inhibition
(IC50,2 M)
Cytotoxicity (IC50,2 M)/Cell lines3
R
SW620
PC-3
AsPC-1
-H
5´-F
C17H22N2O5
C17H21FN2O5
C17H21ClN2O5
C17H21BrN2O5
C17H21N3O7
334.37
352.36
368.81
413.26
379.36
348.39
364.39
352.36
366.50
396.52
1.15
1.35
1.79
2.04
1.37
1.70
1.23
1.79
3.95
4.50
2.68
1.48
1.04
0.50
1.11
1.48
0.05
0.07
0.84
0.84
2.90
1.86
0.53
0.34
0.86
0.92
0.07
0.09
0.89
0.39
1.60
1.01
0.49
0.22
0.74
0.68
0.07
0.09
0.86
0.36
4a
4b
4c
4d
4e
4f
4g
4h
7a
7f
0.16
0.06
0.03
0.03
0.08
0.16
0.12
0.15
0.32
0.26
5´-Cl
5´-Br
5´-NO2
5´-CH3
5´-OCH3
7´-Cl
C18H24N2O5
C18H24N2O6
C17H21FN2O5
C17H22N2O3S2
C18H24N2O4S2
-H
5´-CH3

4
Tetrahedron
SAHA4
264.32
0.20
0.053
1.44
1.44
2.24
1Estimated by KOWWIN Software v1.67; 2Concentration (M) that produces a 50% reduction in cell growth or enzyme activity, the numbers represent the
averaged results from triplicate experiments with deviation of less than 10%.; 3Cell lines: SW620, colon cancer; PC-3, prostate cancer; AsPC-1, pancreatic
cancer; 4SAHA, suberoylanilide acid (positive control).
Figure 3. Effect on histone acetylation in SW620 cells. Cells were treated with compounds (3 M) for 24 hrs. Levels of acetylated histone-H3
and -H4 in total cell lysates were determined by Western immunoblot analysis.
To gain insight into the main interaction between these
compounds and HDAC, we performed docking experiments
using the active site of HDAC. Since histone-H3 and histone-H4
deacetylation is principally regulated by HDAC2 and HDAC324
and the crystal structure of HDAC2 in complex with SAHA
(PDB ID: 4LXZ) has been reported,25 we decided to select the
structure of HDAC2 in complex with SAHA as a docking
template. In this study, we used AutoDock Vina25 and executed
control docking experiments with SAHA to the crystal structure
of HDAC2 after SAHA was removed from the complex
structures, as described previously.16-19 It was found from
docking experiments, that all of the compounds were located in
the active site (Figure 4) of the enzyme and showed higher
binding affinities (stabilization energy from -7.6 to -8.0 kcal/mol)
than that of SAHA (stabilization energy, (-7.4 kcal/mol) (r.m.s.d
distance from the original SAHA in the crystal structure :
0.609/2.056 Å). Overall, the binding affinities of individual
compounds in both series 4 and 7 did not vary significantly in
these docking studies. It seemed that changing the functional
group at position 5´ or 7´ on the indoline moiety was not enough
to affect calculation of the binding affinities using the program
employed. On the binding mode, it was found that all
compounds, except 4e and 4h, displayed very similar binding
modes to that of compound 4a (Figure 4). Compound 4e was
found to have a flipped orientation due to steric clash caused by
the 5´-NO2 functional group with Tyr209 and Leu276,
meanwhile, the head group of compound 4h was found to be
rotated outward compared to compound 4a, probably due to the
presence of a chlorine substituent at position 7´ instead of
position 5´ on the indoline system. The docking results indicate
that within compound series 4 and 7, changing the substituents
has little effect on the binding affinities but are significantly
influenced by the way the head group is orientated in the
hydrophobic environment at the surface around the binding site
of HDAC2.
In conclusion, we have reported
a
series of 2´-
two 2´-
oxospiro[1,3]dioxolane-2,3´-indoline
and
oxospiro[1,3]dithiolane-2,3´-indoline-based hydroxamic acids
with potent cytotoxicity in tested human cancer cell lines and
significant inhibitory effects against histone deacetylation in
SW630 cells. From the results obtained it is clearly indicated that
2´-oxospiro[1,3]dioxolane-2,3´-indoline as well as 2´-
oxospiro[1,3]dithiolane-2,3´-indoline systems can replace a
phenyl ring as a cap group to create novel antitumor agents.
Additionally, diverse substituents could be introduced at the 2´-
oxospiro[1,3]dioxolane-2,3´-indoline moiety at both 5´ and 7´
positions, while still retaining bioactivity. These findings should
encourage further exploration of the 2-oxoindoline moiety to
produce more potent HDAC inhibitors with potential anticancer
activity. Further evaluation of the inhibitory effects of these
compounds against different HDAC subtypes and more in-depth
docking studies are currently being carried out to delineate a
quantitative correlation between the enzyme inhibition, enzyme
affinities and their cytotoxicity.
Acknowledgements
We acknowledge the principal financial supports from the
National Foundation for Science and Technology of Vietnam
(NAFOSTED, Grant number 104.01-2013.16). The biological
study was supported by the Medical Research Center program
(MRC, Grant number 2010-0029480) and the docking study was
supported by the Global Core Research Center (GCRC, Grant
number NRF-2011-0030001) from the National Research
Foundation (NRF) of Korea.
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Figure 4. Stereoview of the actual binding pose of SAHA and simulated docking poses of compounds 4a, 4e and 4h to HDAC2.
SAHA is represented as a stick model in grey color. Compounds 4a, 4e and 4h are shown as a stick model with carbon colored in
bright green, blue and yellow; nitrogen, and oxygen atoms colored in dark blue and red, respectively. Important parts of the
enzyme for interaction were shown as a stick model with carbon, nitrogen, and oxygen colored as grey, blue and red,
respectively. Zn2+ ion is shown as a cyan sphere.