D.T. Anh, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127537
Table 2
Inhibition of HDAC2 and HDAC6 isoforms by compounds 4a-g.
1
HDAC6 inhibition (IC50,1 μM)
HDAC2 inhibition (IC50,1 μM)
HDAC6 inhibition (IC50,1 μM)
Cpd. code
R
HDAC2 inhibition (IC50
,
μM)
Cpd. Code
R
4
4
4
4
a
b
c
H
0.205 ± 0.038
0.194 ± 0.017
0.187 ± 0.015
0.088 ± 0.001
0.007 ± 0.001
0.043 ± 0.001
0.016 ± 0.000
0.049 ± 0.000
4e
4f
5-Br
0.075 ± 0.006
0.021 ± 0.002
0.031 ± 0.005
0.033 ± 0.003
0.024 ± 0.001
0.020 ± 0.002
0.030 ± 0.002
0.030 ± 0.001
5-F
5-Cl
7-Cl
5-CH
3
4g
5-OCH
3
2
d
SAHA
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
4
4
4
4
4
4
4
a
b
c
d
e
f
−19.822
−17.371
−20.137
−20.772
−20.238
−23.119
−21.747
−20.884
−7.578
−6.246
−7.367
−6.918
−9.065
−9.676
−8.725
−8.287
2.19
2.35
2.35
2.18
2.16
2.18
2.16
2.18
2.35
2.39
2.19
2.20
2.30
2.23
2.32
2.29
−22.762
−18.542
−21.569
−16.393
−18.421
−20.142
−18.497
−20.231
−17.547
−11.598
−10.110
−11.121
−10.502
−10.796
−10.523
−10.987
−10.664
−11.064
2.09
2.13
2.07
2.09
2.08
2.19
2.08
2.07
2.13
2.06
2.10
2.06
2.07
2.05
2.03
2.03
2.07
1.96
g
SAHA
TSA
*
*
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
with N-hydroxybenzamide, N-hydroxypropenamide or N-hydro-
xyheptannamide scaffolds has strongly enhanced the cytotoxicity of the
indirubin-3′-oximes and furnished powerful cytotoxic agents. All com-
pounds in three series 4a-g, 7a-g and 10a-g have logP values in the
range of 2.20–3.17, as calculated by KowWin program v1.67, thus fa-
vorable and promising for further development as orally active antic-
ancer agents.
In this series, both electron-widrawing substituents (-F, -Cl, -Br) or
electrong releasing substituents were favorable for HDAC inhibition.
Considering in more detail, it could be noted that, the electron-wi-
drawing substituents (-F, -Cl, -Br) more strongly enhanced the HDAC
inhibitory activity, as manifested by the IC50 values of compounds 10b-
e (0.003–0.014 μM) vs. compounds 10f, g (0.018–0.025 μM). All
compounds in series 10a-g were more potent than SAHA in term of
HDAC inhibition, which correlated well with their stronger cytotoxicity
in comparison to SAHA. However, in series 7a-g, and also in series 4a-
g, some compounds were less potent as HDAC inhibitors in comparison
to SAHA, but exhibited much stronger cytotoxicity than SAHA in all
three human cancer cell lines assayed. It is likely that these compounds
might act on other targets of the indirubin-3′-oxime cores (e.g. cyclin-
dependent kinases, CDKs) and this possibility remains to be in-
vestigated further. On the other hand, compounds bearing N-hydro-
xybenzamide scaffolds have recently been demonstrated as more se-
The addition of the hydroxamic acid scaffolds to the indirubin-3′-
oxime cores resulted in compounds 4a-g, 7a-g and 10a-g with cyto-
toxicity thousand-fold more potent than the original indirubin-3′-
oximes. Thus, compounds 4a-g, 7a-g and 10a-g would be expected to
act more prominently as HDAC inhibitors. We therefore evaluated the
compounds for their inhibition of HDAC using a Fluorogenic HDAC
Assay Kit (abcam, MA, USA) with SAHA as a positive control. The re-
sults of inhibition of HDAC are also presented in Table 1. As the results
demonstrate, all compounds in three series 4a-g, 7a-g and 10a-g were
potent HDAC inhibitors with IC50 values ranging from 0.003 to
3
1
lective inhibitors of HDAC6 isoform. Meanwhile, a Fluorogenic HDAC
Assay Kit using an HDAC extract which is a rich source of HDAC ac-
tivity, but contains predominantly class-I isoforms (HDAC1, 2, 3, and
0
5
.604 μM. Within the N-hydroxybenzamides 4a-g, compound 4b with
3
2
-fluoro substituent (IC50, 0.218 μM) was slightly less potent than the
8). Considering this information, it is possible that the current N-
hydroxybenzamides 4a-g might possess better inhibitory effects againts
HDAC6 isoform. We therefore decided to evaluate compounds 4a-g for
unsubstituted one (4a, IC50, 0.195 μM). All other compounds in the
series were more potent than 4a. Chloro substituted at position 7 (4d,
IC50, 0.071 μM) seemed to better enhanced the HDAC inhibition than 5-
chlorosubstitution (4c, IC50, 0.174 μM). Bulkier halogen (e.g. Br) and
their inhibitory effects against HDAC6 isoform. HDAC2,
a re-
presentative of Class-I HDACs was included. The results are summar-
ized in Table 2. It is very interesting to note that 5 compounds, in-
cluding 4a-e, showed more potent inhibitory activity against HDAC6,
as compared to HDAC2. Especially, compound 4a inhibited HDAC6
with an IC50 value of 0.007 μM, 29-fold lower than its IC50 value against
HDAC2 isoform (0.205 μM). Compounds 4b and 4c exhibited 4.5- and
11.7-fold more potent inhibition towards HDAC6 in comparison to
HDAC2 (IC50 values of 0.043 and 0.016 μM vs. 0.194 and 0.187 μM,
respectively). Thus, from this study, compound 4a showed potentials as
a lead compound for further development of HDAC6 potent inhibitors
in the future.
electron-releasing substituents (–CH
3
, –OCH ) were more favorable for
3
HDAC inhibition ((4e-g, IC50, 0.039, 0.005, and 0.025 μM, respec-
tively). For N-hydroxypropenamides (7a-g), a very clear structure-ac-
tivity relationship was observed. Electron-withdrawing substituents (-F,
-
Cl, and -Br) reduced the HDAC inhibition, while electron-releasing
substituents (–CH
activity of the compounds. In this series, it seemed that bulkier sub-
stituents (-Br, –OCH ) were more favorable for bioactity. It was inter-
esting to note that compounds 4f, 4g and compounds 7f, 7g bearing 5-
3
, –OCH ) significantly enhanced the HDAC inhibitory
3
3
CH
3
and 5-OCH substituents were the most potent HDAC inhibitors
3
among the compounds in two series 4a-g and 7a-g.
In addition to cytotoxicity and HDAC inhibition, three re-
presentative compounds, including 4a, 7a and 10a, were selected to
investigate for their effects on cell cycle and apoptosis using flow
Seven compounds in series 10a-g were the most potent HDAC in-
hibitors among three series with IC50 values from 0.003 to 0.022 μM).
4