Bioorganic & Medicinal Chemistry Letters 30 (2020) 127537

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier.com/locate/bmcl

Design, synthesis and evaluation of novel indirubin-based N- T

hydroxybenzamides, N-hydroxypropenamides and N-hydroxyheptanamides
as histone deacetylase inhibitors and antitumor agents
Duong Tien Anha, Pham-The Haia, Do Thi Mai Dunga, Phan Thi Phuong Dunga,
Le-Thi-Thu Huonga, Eun Jae Parkb, Hye Won Junb, Jong Soon Kangc, Joo-Hee Kwonc,
Truong Thanh Tungd,e, Sang-Bae Hanb, , Nguyen-Hai Nama,
⁎ ⁎

a
Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hanoi, Viet Nam
b
College of Pharmacy, Chungbuk National University, 194-31, Osongsaengmyung-1, Heungdeok, Cheongju, Chungbuk 28160, Republic of Korea
c
Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju, Chungbuk, Republic of Korea
d
Faculty of Pharmacy, PHENIKAA University, Hanoi 12116, Viet Nam
e
PHENIKAA Institute for Advanced Study (PIAS), PHENIKAA University, Hanoi 12116, Viet Nam

ARTICLE INFO ABSTRACT

Keywords: Several novel indirubin-based N-hydroxybenzamides, N-hydropropenamides and N-hydroxyheptanamides (4a-

Histone deacetylase (HDAC) inhibitors h, 7a-h, 10a-h) were designed using a fragment-based approach with structural features extracted from several
N-hydroxybenzamide previously reported HDAC inhibitors, such as SAHA (vorinostat), MGCD0103 (mocetinostat), nexturastat A and
Hydroxamic acids PXD-101 (belinostat). The biological results reveal that our compounds showed excellent cytotoxicity toward
N-hydroxypropenamide
three common human cancer cell lines (SW620, PC-3 and NCI-H23) with IC50 values ranging from 0.09 to
Docking simulation
ADMET profiling
0.007 µM. The cytotoxicity of the compounds was equipotent or even up to 10-times more potent than adria­
mycin and up to 205-times more potent than SAHA. Among the series of N-hydroxypropenamides, compounds
10a-d were the most potent HDAC inhibitors as well as cytotoxicity toward the cell lines tested. In addition, the
strong inhibitory activites toward HDAC of our compounds were observed with IC50 values of below-micromolar
range. Especially, compound 4a inhibited HDAC6 with an IC50 value of 29-fold lower than that against HDAC2
isoform. Representative compounds 4a and 7a were found to significantly arrest SW620 cells at G0/G1 phase.
Compounds 7a and 10a were found to strongly induce apoptosis in SW620 cells. Docking studies revealed some
important features affecting the selectivity against HDAC6 isoform. The results clearly demonstrate the potential
of the indirubin-hydroxamic acid hybrids and these compounds should be very promising for further develop­
ment.

Histone deacetylases (EC 3.5.1.98, HDAC) are the family of enzymes which resulted in hundreds of structurally diverse and potent HDAC
which play a crucial role in removal of the acetyl groups from the lysine inhibitors. These include principally hydroxamic acids (e.g. sub­
termini of histone proteins.1 To date, 18 HDAC isoforms have been eroylanilide hydroxamic acid, SAHA, Zolinza®), cyclic peptides (e.g.
described in human and based on their homology to yeast HDACs, these depsipeptide), short-chain fatty acids (e.g. valproic acid), and benza­
isoforms are divided into four main classes. Class I with four members mides.4–9
(HDACs 1-3, 8), class II with six members (HDACs 4-7, 9, 10) and class Hitherto, five HDAC inhibitors have been approved for use clinically
IV with only one member (HDAC 11) are characterized as zinc-depen­ to treat several types of cancer. These include suberoylanilide hydro­
dent enzymes, while class III (known as sirtuins 1-7) are NAD+-de­ xamic acid (SAHA, Zolinza®), romidepsin (Istodax®), belinostat
pendent enzyme.2 Because of their very critical role in tumor cell (PXD101), panobinostat (LBH-589 Farydak®) (approved by the U.S.
biology, HDACs have become one of the most important targets for FDAC in 2006, 2009, 2014, and 2015, respectively), and chidamide
anticancer drug design and development currently.3 Medicinal chemists (Epidaza®) (approved in 2015 by the Chinese FDA). Dozens of other
worldwide in the past decade have spent extensive research efforts HDAC inhibitors, such as entinostat (MS-27-527), mocetinostat

⁎
Corresponding authors.
E-mail addresses: [email protected] (S.-B. Han), [email protected] (N.-H. Nam).

https://doi.org/10.1016/j.bmcl.2020.127537
Received 6 July 2020; Received in revised form 8 August 2020; Accepted 2 September 2020
Available online 08 September 2020
0960-894X/ © 2020 Elsevier Ltd. All rights reserved.
D.T. Anh, et al. Bioorganic & Medicinal Chemistry Letters 30 (2020) 127537

Fig. 1. Rational design of indirubin-based N-hydroxybenzamides/N-hydroxypropenamides and N-hydroxyheptanamides as HDAC inhibitors.

(MGCD0103), and givinostat (ITF2357) are also currently undergoing reaction occurred under alkaline conditions, and methanol was found
different phases of clinical trials for several types of cancer.10–14 as an optimum solvent. Overall, compounds 4a-g were obtained with
Previously, we have reported several series of heterocycle-con­ acceptable yields under our reaction conditions (57–67%).
taining hydroxamic acids and N-hydroxypropenamides as analogues of A series of indirubin-based N-hydroxypropenamides (7a-g) were
SAHA or belinostat/panobinostat,15–19 which incorporated different synthesized by a similar synthetic pathway described for 4a-g, except
heterocyclic systems, especially 2-oxoindoline one (Fig. 1). In addition, that methyl (E)-4-bromomethylcinnamate was used instead of methyl
based on the structure of nexturastat A and panobinostat, we also 4-bromomethylbenzoate (Scheme 1). Likewise, seven indirubin-based
synthesized and evaluated some series of 2-oxoindoline-based N-hy­ N-hydroxyheptanamides (10a-g) were obtained from isatin derivatives
droxybenzamides/N-hydroxypropenamides (Fig. 1).20–22 As a result, via three-step reactions (Scheme 1). The procedures were exactly the
the above analogues displayed very potent HDAC inhibitory activities same as described for compounds 4a-g with the modification that me­
and cytotoxicity against several human cancer cell lines.20,22 Further­ thyl 7-bromoheptanoate was used in place of methyl 4-bromo­
more, in the in vivo screening in nude mice bearing PC-3 cancer cells, methylbenzoate.
the excellent antitumor activities were observed.17,23 In this report, we Characterization of our synthesized compounds were determined
extended our investigation into novel series of N-hydroxybenzamides, straightforwardly based on analysis of IR, MS, 1H and 13C NMR spec­
N-hydroxypropenamides and N-hydroxyheptanamides incorporating 2- troscopic data. A singlet peak at 4.48–5.54 ppm presents two-proton for
oxoindoline system (novel 2-oxoindolin-based hydroxamic acids) methylene moiety in the structures of 4a-g and 7a-g. In addition to
(Fig. 1). In designing these compounds, we have adopted a hybridiza­ methylene moiety, trans-olefinic protons were recorded as two doublets
tion approach, in which 2-oxoindoline moiety has been expanded to at around 6.4 and 7.4 ppm with the coupling constant (J) of approxi­
include structural features of indirubins (Fig. 1). The indirubins, as mately 15.5–16.0 Hz. The 2E,3Z configuration in the indirubin skeleton
represented by indirubin 3′-oxime, are known as anticancer agents.24 It has been well established.25 Full NMR characterization (1H,13C) and MS
was expected that the indirubins, acting as cap groups, would create can be found in Supporting Information.
more favorable interactions with the amino acid chain at the CAP The synthesized compouds were screened toward three common
binding region of HDAC active binding site, thus increasing the affinity human cancer cell lines, including SW620, PC-3 and NCI-H23 (colon
of the compounds towards HDACs. cancer, prostate cancer and lung cancer, respectively). The biological
The target indirubin-based N-hydroxybenzamides (4a-g) were syn­ sceening methodology was described previously using a colorimetric
thesized via a three-step parthway, as depicted in Scheme 1. Firstly, method26 with slight modifications.27–29 The IC50 values were calcu­
isatins (1a-g) reacted with methyl 4-bromobenzoate via nucleophilic lated as averages from three independent experiments using a Probits
substitution mechanism under basic conditions (K2CO3) and KI (cata­ method 30. SAHA and adriamycin (ADR) were used as a positive con­
lytic amount) in DMF to give N-alkyl isatin derivatives. The yields of trol. The overall sceening results were summarized in Table 1. Gen­
this step were generally from very good to excellent (85–95%). In the erally, our synthesized compounds exhibited very potent cytotoxicity
second step, the indirubin coumpounds were synthesized from the in­ against all three human cancer cell lines tested with IC50 values of
termediates 2 and 3-indoxylacetate in presence of K2CO3 in DMF in 0.09 μM or lower as shown in Table 1. Seven compounds, including 4e,
good yields (~75%) were obtained. In the final step, the target hy­ 7a-c, 7e, 10b, and 10e, even showed IC50 values lower than 0.01 μM in
droxamic acids were obtained by the nucleophilic acyl substitution of NCI-H23 cell line. The IC50 values of adriamycin, a strongly cytotoxic
hydroxylamine hydrochloride with the corresponding esters 3. This anticancer drug currently used widely in clinical setting, were 0.09 μM

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D.T. Anh, et al. Bioorganic & Medicinal Chemistry Letters 30 (2020) 127537

Scheme 1. Synthesis of indirubin-based N-hydroxybenzamides (4a-g), N-hydroxpropenamides (7a-g), and N-hydroxyheptanamides (10a-g); a, R = H; b, R = 5-F; c,
R = 5-Cl; d, R = 7-Cl; e, R = 5-Br; f, R = 5-CH3; g, R = 5-OCH3.

Table 1
Inhibition of HDAC activity in Hela cell extract and cytotoxicity of the synthesized compounds against several human cancer cell lines.

HDAC (Hela extract) Inhibition (IC50,1 μM) Cytotoxicity (IC50,1 μM)/Cell lines2

Cpd. code R SW620 PC3 NCI-H23

4a H 0.195 ± 0.041 0.07 ± 0.03 0.06 ± 0.02 0.07 ± 0.03

4b 5-F 0.218 ± 0.019 0.02 ± 0.01 0.05 ± 0.001 0.03 ± 0.001
4c 5-Cl 0.174 ± 0.022 0.06 ± 0.03 0.05 ± 0.001 0.05 ± 0.001
4d 7-Cl 0.071 ± 0.001 0.09 ± 0.001 0.06 ± 0.001 0.06 ± 0.001
4e 5-Br 0.039 ± 0.004 0.07 ± 0.02 0.05 ± 0.01 0.007 ± 0.000
4f 5-CH3 0.005 ± 0.001 0.03 ± 0.001 0.09 ± 0.001 0.01 ± 0.001
4g 5-OCH3 0.025 ± 0.003 0.04 ± 0.01 0.03 ± 0.01 0.09 ± 0.001
7a H 0.095 ± 0.024 0.05 ± 0.01 0.05 ± 0.001 0.009 ± 0.002
7b 5-F 0.422 ± 0.074 0.05 ± 0.01 0.05 ± 0.001 0.008 ± 0.001
7c 5-Cl 0.318 ± 0.064 0.08 ± 0.01 0.06 ± 0.04 0.007 ± 0.001
7d 7-Cl 0.604 ± 0.050 0.06 ± 0.01 0.09 ± 0.01 0.05 ± 0.01
7e 5-Br 0.183 ± 0.003 0.06 ± 0.01 0.09 ± 0.001 0.009 ± 0.001
7f 5-CH3 0.020 ± 0.008 0.05 ± 0.01 0.06 ± 0.01 0.01 ± 0.001
7g 5-OCH3 0.005 ± 0.000 0.04 ± 0.001 0.03 ± 0.001 0.05 ± 0.01
10a H 0.022 ± 0.000 0.09 ± 0.001 0.05 ± 0.02 0.09 ± 0.001
10b 5-F 0.014 ± 0.006 0.08 ± 0.01 0.012 ± 0.001 0.008 ± 0.001
10c 5-Cl 0.003 ± 0.000 0.06 ± 0.02 0.09 ± 0.001 0.03 ± 0.01
10d 7-Cl 0.007 ± 0.000 0.09 ± 0.001 0.03 ± 0.001 0.05 ± 0.01
10e 5-Br 0.004 ± 0.000 0.09 ± 0.001 0.03 ± 0.001 0.009 ± 0.001
10f 5-CH3 0.018 ± 0.005 0.05 ± 0.001 0.08 ± 0.02 0.010 ± 0.001
10g 5-OCH3 0.021 ± 0.001 0.03 ± 0.01 0.05 ± 0.01 0.07 ± 0.02
Indirubin-3′-oxime # 13.5 ± 2.18 15.8 ± 2.81 11.25 ± 2.11
SAHA3 0.025 ± 0.002 1.12 ± 0.10 1.82 ± 0.09 1.44 ± 0.17
ADR4 # 0.09 ± 0.001 0.09 ± 0.001 0.09 ± 0.001

1
The concentration (μM) of compounds that produces a 50% reduction in enzyme activity or cell growth, the numbers represent the averaged results from
triplicate experiments; 2Cell lines: SW620, colon cancer; PC3, prostate cancer; NCI-H23, lung cancer; 3SAHA, suberoylanilide acid, a positive control; 4ADR,
adriamycin, a positive control.

in all three cancer cell lines. Thus, in term of cytotoxicity, it was clear Meanwhile, against NCI-H23 cells the compounds (IC50 values of
that all compounds from three series 4a-g, 7a-g and 10a-g were equi­ 0.007–0.09 μM) were found to be about 15-fold to 205-fold as potent as
potent or even more potent than adriamycin towards these human SAHA (IC50 value of 1.44 μM). The IC50 values for indirubin-3′-oxime
cancer cell lines. Against SW620 cells, the compounds (IC50 values of against SW620, PC-3 and NCI-H23 cell lines were 13.5 ± 2.18,
0.09–0.02 μM) were found to be approximately 12-fold to 56-fold more 15.8 ± 2.81, and 11.25 ± 2.11 μM, respectively. Other 5/-7-sub­
potent than SAHA (IC50 value of 1.12 μM). Against PC3 cells, the stitutedindirubin-3′-oxime derivatives all showed weak cytotoxicity in
compounds (IC50 values of 0.012–0.09 μM) were found to be from 20- the above three cancer cell lines with IC50 values of > 10 μM (data not
to approximately 152-fold as potent as SAHA (IC50 value of 1.82 μM). shown). These results clearly demonstrate that hydridization of the

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D.T. Anh, et al. Bioorganic & Medicinal Chemistry Letters 30 (2020) 127537

Table 2
Inhibition of HDAC2 and HDAC6 isoforms by compounds 4a-g.
Cpd. code R HDAC2 inhibition (IC50,1 μM) HDAC6 inhibition (IC50,1 μM) Cpd. Code R HDAC2 inhibition (IC50,1 μM) HDAC6 inhibition (IC50,1 μM)

4a H 0.205 ± 0.038 0.007 ± 0.001 4e 5-Br 0.075 ± 0.006 0.024 ± 0.001

4b 5-F 0.194 ± 0.017 0.043 ± 0.001 4f 5-CH3 0.021 ± 0.002 0.020 ± 0.002
4c 5-Cl 0.187 ± 0.015 0.016 ± 0.000 4g 5-OCH3 0.031 ± 0.005 0.030 ± 0.002
4d 7-Cl 0.088 ± 0.001 0.049 ± 0.000 SAHA2 0.033 ± 0.003 0.030 ± 0.001

1
The concentration (μM) of compounds that produces a 50% reduction in enzyme activity. 2SAHA, suberoylanilide acid, a positive control.

Table 3
Binding energies estimated for all compounds docked into HDAC2 and HDAC6 enzymes.
Cpd. code HDAC2 HDAC6

E_Score1** E_score2** Distance to Zn2+ * E_Score1 E_score2 Distance to Zn2+

eOH ]O eOH ]O

4a −19.822 −7.578 2.19 2.35 −22.762 −11.598 2.09 2.06

4b −17.371 −6.246 2.35 2.39 −18.542 −10.110 2.13 2.10
4c −20.137 −7.367 2.35 2.19 −21.569 −11.121 2.07 2.06
4d −20.772 −6.918 2.18 2.20 −16.393 −10.502 2.09 2.07
4e −20.238 −9.065 2.16 2.30 −18.421 −10.796 2.08 2.05
4f −23.119 −9.676 2.18 2.23 −20.142 −10.523 2.19 2.03
4g −21.747 −8.725 2.16 2.32 −18.497 −10.987 2.08 2.03
SAHA −20.884 −8.287 2.18 2.29 −20.231 −10.664 2.07 2.07
TSA −17.547 −11.064 2.13 1.96

* The docking score (kcal/mol) calculated from the London (with refinement) and affinity scoring function from MOE software.
** Distances (Å) from oxygen atoms (eO and ]O) of hydroxamate group to zinc ion.

indirubin-3′-oxime or 5/-7-substituted-indirubin-3′-oxime derivatives In this series, both electron-widrawing substituents (-F, -Cl, -Br) or
with N-hydroxybenzamide, N-hydroxypropenamide or N-hydro­ electrong releasing substituents were favorable for HDAC inhibition.
xyheptannamide scaffolds has strongly enhanced the cytotoxicity of the Considering in more detail, it could be noted that, the electron-wi­
indirubin-3′-oximes and furnished powerful cytotoxic agents. All com­ drawing substituents (-F, -Cl, -Br) more strongly enhanced the HDAC
pounds in three series 4a-g, 7a-g and 10a-g have logP values in the inhibitory activity, as manifested by the IC50 values of compounds 10b-
range of 2.20–3.17, as calculated by KowWin program v1.67, thus fa­ e (0.003–0.014 μM) vs. compounds 10f, g (0.018–0.025 μM). All
vorable and promising for further development as orally active antic­ compounds in series 10a-g were more potent than SAHA in term of
ancer agents. HDAC inhibition, which correlated well with their stronger cytotoxicity
The addition of the hydroxamic acid scaffolds to the indirubin-3′- in comparison to SAHA. However, in series 7a-g, and also in series 4a-
oxime cores resulted in compounds 4a-g, 7a-g and 10a-g with cyto­ g, some compounds were less potent as HDAC inhibitors in comparison
toxicity thousand-fold more potent than the original indirubin-3′- to SAHA, but exhibited much stronger cytotoxicity than SAHA in all
oximes. Thus, compounds 4a-g, 7a-g and 10a-g would be expected to three human cancer cell lines assayed. It is likely that these compounds
act more prominently as HDAC inhibitors. We therefore evaluated the might act on other targets of the indirubin-3′-oxime cores (e.g. cyclin-
compounds for their inhibition of HDAC using a Fluorogenic HDAC dependent kinases, CDKs) and this possibility remains to be in­
Assay Kit (abcam, MA, USA) with SAHA as a positive control. The re­ vestigated further. On the other hand, compounds bearing N-hydro­
sults of inhibition of HDAC are also presented in Table 1. As the results xybenzamide scaffolds have recently been demonstrated as more se­
demonstrate, all compounds in three series 4a-g, 7a-g and 10a-g were lective inhibitors of HDAC6 isoform.31 Meanwhile, a Fluorogenic HDAC
potent HDAC inhibitors with IC50 values ranging from 0.003 to Assay Kit using an HDAC extract which is a rich source of HDAC ac­
0.604 μM. Within the N-hydroxybenzamides 4a-g, compound 4b with tivity, but contains predominantly class-I isoforms (HDAC1, 2, 3, and
5-fluoro substituent (IC50, 0.218 μM) was slightly less potent than the 8).32 Considering this information, it is possible that the current N-
unsubstituted one (4a, IC50, 0.195 μM). All other compounds in the hydroxybenzamides 4a-g might possess better inhibitory effects againts
series were more potent than 4a. Chloro substituted at position 7 (4d, HDAC6 isoform. We therefore decided to evaluate compounds 4a-g for
IC50, 0.071 μM) seemed to better enhanced the HDAC inhibition than 5- their inhibitory effects against HDAC6 isoform. HDAC2, a re­
chlorosubstitution (4c, IC50, 0.174 μM). Bulkier halogen (e.g. Br) and presentative of Class-I HDACs was included. The results are summar­
electron-releasing substituents (–CH3, –OCH3) were more favorable for ized in Table 2. It is very interesting to note that 5 compounds, in­
HDAC inhibition ((4e-g, IC50, 0.039, 0.005, and 0.025 μM, respec­ cluding 4a-e, showed more potent inhibitory activity against HDAC6,
tively). For N-hydroxypropenamides (7a-g), a very clear structure-ac­ as compared to HDAC2. Especially, compound 4a inhibited HDAC6
tivity relationship was observed. Electron-withdrawing substituents (-F, with an IC50 value of 0.007 μM, 29-fold lower than its IC50 value against
-Cl, and -Br) reduced the HDAC inhibition, while electron-releasing HDAC2 isoform (0.205 μM). Compounds 4b and 4c exhibited 4.5- and
substituents (–CH3, –OCH3) significantly enhanced the HDAC inhibitory 11.7-fold more potent inhibition towards HDAC6 in comparison to
activity of the compounds. In this series, it seemed that bulkier sub­ HDAC2 (IC50 values of 0.043 and 0.016 μM vs. 0.194 and 0.187 μM,
stituents (-Br, –OCH3) were more favorable for bioactity. It was inter­ respectively). Thus, from this study, compound 4a showed potentials as
esting to note that compounds 4f, 4g and compounds 7f, 7g bearing 5- a lead compound for further development of HDAC6 potent inhibitors
CH3 and 5-OCH3 substituents were the most potent HDAC inhibitors in the future.
among the compounds in two series 4a-g and 7a-g. In addition to cytotoxicity and HDAC inhibition, three re­
Seven compounds in series 10a-g were the most potent HDAC in­ presentative compounds, including 4a, 7a and 10a, were selected to
hibitors among three series with IC50 values from 0.003 to 0.022 μM). investigate for their effects on cell cycle and apoptosis using flow

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D.T. Anh, et al. Bioorganic & Medicinal Chemistry Letters 30 (2020) 127537

ligand (the RMSD value of 0.563 Å). The main interactions between
TSA and residues in the active site of HDAC6 were conserved, including
H-bonds with His610, His651 and Tyr782, and stacking interactions
with Pro501, Phe620 and Phe680. On the other hands, SAHA was re­
docked into the active sites of HDAC2 and HDAC6 enzymes. As the
results, the redocked and co-crystal poses of SAHA in HDAC2 were
appropriately superimposed (RMSD of 0.823 Å). All the key interactions
with Asp104, His145, and Tyr308 (H-bonds), His33 and Pro34 (hy­
drophobic interactions) were conserved. In addition, the docking results
of SAHA against HDAC6 highlighted the importance of multiple H-
bonding interactions with residues at the base of the pocket, especially
His610, His611, His651 and Tyr782 (Fig. S6). These results were con­
sistent with those reported previously.34
After validating the docking method, all the compounds 4a-g were
docked into HDAC2 and HDAC6 isoforms. In general, these compounds
were well accomodated in the pockets of the two enzymes. The free
binding energies dG were computed after energy minimization using
the London (E_score1) and affinity GBVI/WSA (E_score2) scoring
functions.21 Interestingly these functions matched well with the ex­
perimental assessments. As can be seen in Table 3, 4f exhibited the
highest energy with HDAC2, meanwhile 4a and 4c showed the best
scores in complex with HDAC6; all of them showed higher dG values
than reference compounds SAHA and TSA. On the basis of in vitro tests
in HDAC6, the correlation between log(E_score1) and IC50 (R2) was
~80%, higher than that of function between E_score2 and logIC50
(R2 ~ 73%), suggesting a suitable rank-order relationship between
experimental and computational estimations.
As can be seen in Fig. 2, 4a exhibited similar interactions to TSA in
the HDAC6 pocket. However, 4a docked into HDAC2 did not show H-
bond interactions with the key residues at the base of the pocket, such
as His145, His146, Asp181, His183 and Asp269. On the other hands,
oxoindoline with 5-halogen substituents lacked of doble stacking in­
teractions with Leu749 while forming two additional van der Waals
interactions with Ser568. Enhancement of the hydrophobic interactions
with residues at the entrance of the pocket, including His500, Pro501
and Ser568 from 4b to 4e could be observed. These interactions could
make positive effect on selectivity towards HDAC6 but this effect de­
creases from fluorine to bromine analogues (4b, 4c, 4e). In contrast to
4a, 4f showed better interactions with HDAC2 compared to HDAC6.
This compound regenerated a complex H-bonding network from the
hydroxamate group towards residues His146, Gly154 and Tyr308 of
HDAC2. At last, the indirubin capping groups showed multiple pi
stacking interactions with hydrophobic residues at the entrance of
HDAC pockets, such as Pro34, Leu276 (HDAC2), and Pro501, Leu740
(HDAC6). Given the importance of bulky branched cap groups for im­
proving protein surface interaction as well as HDAC isoform-selectivity
(e.g. HDAC6),35 it is emphasized the roles of indirubins as promising
capping groups in designing potent, selective HDAC inhibitors.
Fig. 2. Docking poses of compounds 4a, 4c and 4f inside the active site of More than 40% potential therapeutic agents fail to be an effective
HDAC2 and HDAC6. clinical candidate because of their unfavorable absorption, distribution,
metabolism, elimination and toxic (ADMET) factors.36 We therefore
evaluated accomplishment of Lipinski’s Ro5. The results (shown in
cytometry technique. It was found that compounds 7a and 10a sigini­
Table S1, Supporting Information) demonstrate that these compounds
ficantly induced early apoptosis of SW620 cells at a level similar to that
fulfilled Ro5 criteria and possessed satisfactory ADMET profile. In
observed with SAHA. Compound 4a appeared to significantly arrest
comparison to SAHA (ZolinzaTM), compounds 4f and 7g show favorable
cells at G0/G1 phase meanwhile compound 7a was found to sig­
physicochemical and pharmacokinetic profiles similar to the reference
nificantly arrest cells at both G0/G1 and S phases (See Figs. S1–S5,
compound. In our opinion, these results served as a good starting point
Supporting Information).
for further structural optimization for new anticancer agents.
Based on the experimental evaluations, a comparative docking
In conclusion, we have reported three series of indirubin-based N-
study was performed for compounds of series 4a-g against HDAC6 and
hydroxybenzamides, N-hydroxypropenamides, and N-hydro­
HDAC2 (PDB ID: 4LXZ and 5EDU, respectively), a preferentially ex­
xyheptannamides with excellent HDAC inhibitory activities and potent
pressed Class-I isoform in Hela nuclear extract,33 in order to figure out
cytotoxicity towards three human cancer cell lines, including SW620,
which chemical features could be responsible for HDAC selectivity. To
PC-3 and NCI-H23 (colon cancer, prostate cancer and lung cancer, re­
validate the docking procedures applied, redocking simulations were
spectively). Most compounds displayed cytotoxicity of 20- to more than
performed with the native ligands. As the results, after redocking in the
205-fold stronger than SAHA. In term of cytotoxicity our synthesized
binding site of HDAC6, TSA was highly overlapped with the co-crystal
compounds were also equipotent or even more potent than that of

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interests or personal relationships that could have appeared to influ­ hydroxypropenamides as histone deacetylase inhibitors and antitumor agents. Med
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We acknowledge the principal financial supports from the National
corporating 2-oxoindoline with unexpected potent histone deacetylase inhibitory
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