Environment-sensitive fluorescent inhibitors of histone deacetylase

Article abstract of DOI:10.1016/j.bmcl.2020.127128

Histone deacetylases (HDACs) are proteases that can catalyze the deacetylation of histones to inhibit gene transcription. Since mutations and/or aberrant expression of various HDACs are frequently associated with human diseases, particularly cancers, HDACs are important therapeutic targets for many human tumors. However, there are still relatively few studies on HDAC small molecule fluorescent probes. Herein, we designed and synthesized a class of environment-sensitive fluorescent inhibitors with a switch mechanism to study HDAC activity. In vitro, the enzyme inhibition activity of compound 6b was comparable to the positive control drug SAHA, and it presented suitable imaging in living cells and tumor-tissue slices. This environment-sensitive fluorescent inhibitor provides a new idea for the diagnosis and treatment of HDACs-related diseases.

Full text of DOI:10.1016/j.bmcl.2020.127128

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Environment-Sensitive Fluorescent Inhibitors of Histone Deacetylase
Xin Zhou, Gaopan Dong, Tianjia Song, Guankai Wang, Zhenzhen Li, Xiaojun
Qin, Lupei Du, Minyong Li
PII:
S0960-894X(20)30220-1
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127128
BMCL 127128
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Bioorganic & Medicinal Chemistry Letters
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17 March 2020
19 March 2020
Please cite this article as: Zhou, X., Dong, G., Song, T., Wang, G., Li, Z., Qin, X., Du, L., Li, M., Environment-
Sensitive Fluorescent Inhibitors of Histone Deacetylase, Bioorganic & Medicinal Chemistry Letters (2020), doi:
https://doi.org/10.1016/j.bmcl.2020.127128
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Environment-Sensitive Fluorescent Inhibitors of Histone
Deacetylase
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Xin Zhoua, Gaopan Donga, Tianjia Songa, Guankai Wanga, Zhenzhen Lia, Xiaojun Qina, Lupei Dua, and
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Bioorganic & Medicinal Chemistry Letters
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Environment-Sensitive Fluorescent Inhibitors of Histone Deacetylase
Xin Zhoua, Gaopan Donga, Tianjia Songa, Guankai Wanga, Zhenzhen Lia, Xiaojun Qina, Lupei Dua, and
Minyong Lia, *
aDepartment of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of Pharmacy, Shandong University, Jinan, Shandong 250012, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Histone deacetylases (HDACs) are proteases that can catalyze the deacetylation of histones to
inhibit gene transcription. Since mutations and/or aberrant expression of various HDACs are
frequently associated with human diseases, particularly cancers, HDACs are important
therapeutic targets for many human tumors. However, there are still relatively few studies on
HDAC small molecule fluorescent probes. Herein, we designed and synthesized a class of
environment-sensitive fluorescent inhibitors with a switch mechanism to study HDAC activity.
In vitro, the enzyme inhibition activity of compound 6b was comparable to the positive control
drug SAHA, and it presented suitable imaging in living cells and tumor-tissue slices. This
environment-sensitive fluorescent inhibitor provides a new idea for the diagnosis and treatment
of HDACs-related diseases.
Keywords:
histone deacetylase
HDACs inhibitor
environmental sensitivity
fluorescence inhibitor
cell imaging
2009 Elsevier Ltd. All rights reserved.
The acetylation of histones is essential for epigenetic
regulation, and post-translational modifications of histones
include acetylation, methylation, phosphorylation and
ubiquitination [1, 2]. Allefrey et al. have demonstrated the
relationship between histone acetylation levels and cellular
transcriptional activity in 1964 [3]. Histone acetyltransferases
(HATs) catalyze the acetylation of the ε-NH2 group of histone
lysine residues [4]. In contrast, histone deacetylases (HDACs)
catalyze the deacetylation of histones [5]. The acetylation of
multiforme and cerebral palsy. [11C]-Martinostat is an imaging
probe that is selective for class I HDACs, which firstly showed
gene changes in brain activity in living human brains [16].
Fluorescent probes are mainly used to detect the activity and cell
imaging of HDACs. The existing HDAC fluorescent probes are
mainly based on the mechanism of enzymatic reaction. The
enzymatic reaction deacetylates the probes, leading to release of
the fluorophores [17-19], and aggregation-induced emission [20],
increasing the ability to bind to DNA [21], or stimulating
intramolecular self-crosslinking reactions [22, 23], in order to
achieve the aim of fluorescent switching. In recent years, various
researches on tumor diagnosis and treatment have emerged
increasingly. However, the probes of HDACs are mainly used for
the enzyme activity detection and cell imaging. HDACs are the
important target for malignant tumors, but, due to lack of
researches on the integration of HDAC diagnosis and treatment
of tumors, researches on HDACs should be carried out as soon as
possible.
histones loosens the chromatin structure ，
leading to
transcriptional activation, and the deacetylation of histones
concentrates the chromatin structure， resulting in inhibition of
gene expression[6]. This is a dynamic equilibrium process in
normal cells; however, in many cancer cells, this balance is
broken, and the activity of histone deacetylases is significantly
increased, resulting in decreased activity of tumor suppressor
genes and uncontrolled cell proliferation. Mutations or aberrant
expression of HDACs is often observed in tumor cells. Studies
have shown that lysine in cancer cells is subjected to histone H4
deacetylation, suggesting that HDACs can become an important
therapeutic target for many malignancies [7]. Many studies have
shown that HDACs mediate transcriptional inhibition by
overexpression [8, 9] or abnormal interaction of transcriptional
regulators [10-12]. Moreover, different HDACs have been shown
to have cell-environment-specific effects and to play different
roles in pro- or anti-cancer activities [13].
Most HDAC inhibitors consist of three components: surface
recognition region (Cap structure), zinc ion chelation group
(ZBG structure) and Linker [24]. We designed a series of
environment-sensitive fluorescent inhibitors with
a switch
mechanism (Figure 1. B), and selected SAHA - the most
commonly used hydroxamic acid inhibitor of HDAC - as the
recognition motif. Considering that fluorophores in small volume
can minimally affect the binding affinity of the parent ligand, a
relatively small environment-sensitive fluorophore SBD [25]is
designed to be incorporated into the structure of SAHA (Figure
1. A). In classic design strategies, the fluorophore is not involved
in the binding of the target protein, so that the binding activity of
the parent ligand is somewhat reduced. While in our design
strategy, SAHA was used as lead compound, and the fluorescent
HDAC inhibitor was designed by substituting the aromatic ring
At present, probes for HDACs are mainly divided into radio-
labeled probes and fluorescent probes, in which radio-labeled
probes are mainly used for detecting the biodistribution and
Positron Emission Tomography (PET) imaging of HDAC
inhibitors in vivo. [18F]-FAHA can be used to detect the activity
of class II HDACs in animals [14, 15], and can also be used for
PET imaging in rat breast cancer, human glioblastoma

Buffer Saline (PBS) solution (highly polar) was extremely low at
an almost quenching level; but the quantum yield in the
acetonitrile solution (low polarity) was increased by more than
100 times (Table 1), which preliminarily proved that four
compounds were all environment-sensitive. The fluorescence
intensity of compounds did not change significantly in solutions
at different pH, but the fluorescence intensity increased
significantly with the decrease in the polarity of the solution and
the increase in the viscosity, further confirming that compounds
were environment-sensitive, mainly for the polarity and viscosity
of the environment.
HeLa Cell Extract Inhibition of Compounds. Compounds
6a (204 nM) and 6b (108 nM) had the best in vitro inhibitory
activities, and compound 6b (108 nM) was slightly better than
the positive control drug SAHA (134 nM) (Table 2). The
comparison of compounds 6a and 6b showed that the six-carbon
linker was more conducive to the inhibition of enzyme activity. It
also indicated that our design strategy was successful and the
introduction of the fluorophore did not reduce the activity of the
compounds. The comparison of compounds 11a and 11b with 6a
and 6b indicated that the presence of urea groups reduced the
inhibitory activity. The comparison of compounds 11a and 11b
indicated that the presence of a branched chain at the hydroxamic
acid alpha position greatly reduced the activity of the
compounds. These were all consistent with the study of the
structure-activity relationship of HDAC hydroxamic acid
inhibitors [30-32].
Figure 1 (A) The design strategy of compounds; (B) The structures of four compounds.
moiety of the inhibitor structure with the 4-suifamonyl-7-
aminobenzoxadiazole (SBD) fluorophore. The fluorophores in
the structure of such fluorescent inhibitors are among the
pharmacophores, thereby avoiding the effect of fluorophores on
the binding of probes to target proteins in conventional design
strategies.
Through various analyses of the crystal structure of HDAC
[26-29], it has been found that there is a hydrophobic channel in
the active region of HDAC. Based on the environment-sensitive
fluorophore, the fluorescent probe can detect the hydrophobic
pocket. Therefore, when a compound enters a low-polarity
HDAC active site from a highly polar aqueous environment, the
fluorescence intensity changes significantly, which constitutes of
a fluorescent switch mechanism.
In Vitro HDAC Isoform-Selectivity of Compounds. The
isoform selectivity of compounds 6a and 6b were essentially
identical to the positive control drug SAHA. The result presented
that both compounds 6a and 6b were the broad spectrum HDAC
inhibitors (Table 3).
In Vitro Antiproliferative Assay. The experimental results
showed that the antiproliferative effect of hydroxamic acid
HDAC inhibitors on hematoma was better than on solid tumors.
The most sensitive one among the four cell lines is MOLT4 cell
(Table 4). The anti-tumor proliferative activities of compounds
were inferior to the positive control drug SAHA, but it
represented that the compounds were less cytotoxic and more
beneficial for cell imaging.
Spectroscopic Properties of the Compounds. The results
displayed that compounds possessed excellent fluorescent
properties (Figure S1-S3). The maximum ultraviolet absorption
wavelengths of all compounds were about 440 nm, and the
maximum excitation wavelengths about 440 nm and the
maximum emission wavelengths about 600 nm. The relative
fluorescence quantum yield of the compounds in the Phosphate
Table 1 Photophysical Properties of the Compounds.
Cpd
6a
λmax(PBS)
439 nm
438 nm
438 nm
440 nm
λex(PBS)
440 nm
445 nm
440 nm
440 nm
λem(PBS)
595 nm
590 nm
600 nm
585 nm
Φ(PBS)
0.04%
0.04%
0.05%
0.04%
Φ(MeCN)
8.38%
6b
7.58%
11a
11b
6.10%
6.44%
Table 2 HDAC Inhibition Activity of Compounds
Cpd
6a
IC50
205 ± 19.8 nM
109 ± 9.34 nM
6b

11a
11b
7.58 ±1.05 μM
> 100 μM
SAHA
135 ± 4.54 nM
aAssays were performed in replicate (n ≥ 2); data are shown as mean ± SD.
Table 3 In Vitro Inhibition of HDACs Isoforms of Compounds.
IC50 of HDAC1
(nM)a
IC50 of HDAC2
(nM)a
IC50 of HDAC3
(nM)a
IC50 of HDAC6
(nM)a
IC50 of HDAC7
IC50 of HDAC8
Cpd
6a
(μM)a
(μM)a
112±29.0
30.0±1.00
69.0±8.00
357±22.0
304±12.0
178±20.0
82.0±4.00
37.0±1.00
56.0±4.00
41.0±8.00
11.0±2.00
25.0±1.00
24.7±1.40
12.7±2.10
1.10±0.10
2.00±0.10
6b
15.7±2.30
8.20±1.30
SAHA
aAssays were performed in replicate (n ≥ 2); data are shown as mean ± SD.
Table 4 In Vitro Antiproliferative Activity of Compounds.
IC50 (μM)a
K562
Cpd
MOLT4
3.55±0.16
PC-3
A549
>100
6a
6b
15.2±1.38
11.0±1.29
1.51±0.04
31.2±1.48
52.6±7.32
60.8±5.67
7.84±0.38
0.38±0.01
>100
SAHA
7.06±0.09
aAssays were performed in replicate (n ≥ 2); data are shown as mean ± SD.
Protein-Binding Assay. We can see (Figure 2) that the
fluorescence intensity increased with the increase in the enzyme
concentration until saturation, while the concentration of
compound 6b was kept constant. These results indicated that
compound 6b exhibited its environmental sensitivity at the
enzyme level, and had a fluorescent switch function, which might
be used for inhibitors screening and determination of binding
kinetic parameters.
Flow-Cytometry
Analysis.
To
future
explore
the
antiproliferative mechanism, the MOLT4 cells were treated with
compounds 6a, 6b and SAHA, respectively, and then were
Figure 3 Results of MOLT-4 cells apoptosis experiments of 5 μM 6a, 6b and
SAHA by flow cytometry analysis. (A) Control; (B) SAHA; (C) 6a; (D) 6b.
stained with Annexin V-PE. The results indicated that, as the
antitumor drugs, compounds 6a, 6b and SAHA could induce
apoptosis of tumor cells. At the same concentration (5 μM),
compounds 6a and 6b induced apoptosis (23.30%, and 20.02%,
respectively), and the apoptosis rate was lower than that induced
by SAHA (29.58 %) (Figure 3). The results of apoptosis
experiments performed by flow cytometry were consistent with
the anti-tumor proliferation activity test in vitro, indicating that
Figure 2 The fluorescence intensity saturation curve of 6b (1 nM) as the enzyme
concentration changes.

compounds 6a and 6b were less toxic and more suitable for cell
imaging.
Fluorescence Imaging. Both PC-3 and MOLT4 cells with
high expression of HDACs were well imaged, while HEK-293
cells with low expression of HDACs were almost unstained
(Figure 4), indicating that compound 6b performed well in cell
imaging and had selectivity for cells with high expression of
HDACs. To explore the distribution of HDACs in cells, we
studied the co-localization of compound 6b with cell membrane
dye DID and nuclear dye Hoechst 33342 under confocal
microscopy. The results of co-localization imaging showed that
the main staining site of compound 6b was cytoplasm, which was
consistent with the reported results, but compound had a high co-
staining rate of 62.1% with the membrane dye and almost no co-
staining with the nuclear dye (Figure 5). It is well known that
HDACs were expressed mainly in the nucleus and cytoplasm,
and were hardly expressed in cell membrane. Therefore, we
speculated that compound 6b had selectivity for HDACs,
However, since the membrane permeability of compound 6b was
poor, it was accumulated on the surface of the cell membrane and
was difficult to pass through the nuclear membrane. Moreover,
due to the off-on mechanism, compound 6b was near-
fluorescence quenching in a highly polar aqueous solution, and
the cell imaging background was very low, which could
eliminate the repetitive washing operations, allowing for greater
convenience and cost-efficiency.
Figure 5 (A) Co-staining results of 5 μM 6b and 2.5 μM cell membrane dye DID for
MOLT-4 cells; (B) Co-staining results of 5 μM 6b and 0.25 μM nuclear dye Hoechst
33342 for MOLT-4 cells.
Hematoxylin-Eosin (HE) and Tissue Section Staining. To
investigate the ability of 6b for imaging in mouse tumor tissue
sections, we established a subcutaneous PC-3-xenograft mouse
model where the tumor was paraffin-embedded when reaching
about 1 cm in diameter. Hematoxylin-Eosin (HE) staining was
performed using the sections provided by Qilu Hospital of
Shandong University. The results showed that the tumor sections
were in significant pathological state compared to normal tissues
(Figure 6).
was much stronger than that of normal tissue sections, and the
fluorescence intensity of tumor tissue sections was significantly
reduced when co-incubated with inhibitor SAHA (see Figure 7),
indicating that compound 6b could selectively label HDACs and
had potential value in clinical cancer diagnosis.
To confirm whether our probes could selectively label
HDACs and located tumor sites, these tissue sections were
subjected to fluorescence imaging measurements. The
experimental results showed that, under the same experimental
conditions, the fluorescence intensity of tumor tissue sections
Figure 6 Hematoxylin−eosin staining of (A) tumor tissue and (B) normal tissue of
male nude mice.
Figure 4 (A) Fluorescence imaging after incubation of 5 μM 6b with PC-3 cells (A1:
bright field, A2: green channel, A3: merged image.); (B) Fluorescence imaging after
incubation with 5 μM 6b and MOLT4 cells (B1: bright field, B2: green channel, B3:
merged image.); (C) Fluorescence imaging after incubation of 5 μM 6b with HEK293
cells (C1: bright field, C2: green channel, C3: merged image.); Objective lens: 63×. Scale
bar = 67 μm.
Figure 7 (A) Fluorescence imaging of tumor tissues incubated with 10 μM 6b. (B)
Fluorescence imaging of tumor tissues incubated with 10 μM 6b and 100 μM
SAHA. (C)Fluorescence imaging of normal tissues incubated with 10 μM 6b.
S6

We, for the first time, designed, synthesized and evaluated
the environment-sensitive fluorescent inhibitors of HDACs,
which exhibited good optical properties and environmental
sensitivity. In vitro inhibitory activity and isoform selectivity of
the compounds were similar to those of the positive control drug
SAHA. The anti-tumor cell proliferation activity in vitro of the
compounds was slightly lower than that of SAHA, which made
the compounds more suitable for cell imaging. The staining with
compound 6b was good for cells with high expression of HDACs
(MOLT4 and PC-3), while the cells with low expression of
HDAC (HEK293) were almost unstained, indicating that 6b had
good selectivity. Furthermore, 6b was successfully applied to the
detection of tumor tissue sections. The imaging results of tumor
tissue and normal tissue showed significant differences, and the
addition of inhibitors significantly reduced the imaging effect of
tumor tissue, indicating that 6b could selectively identify HDACs
and tumor sites. Therefore, we believe that this compound may
be used in the pathology and physiology studies of HDACs, and
provide a new idea for the integration of clinical diagnosis and
treatment of HDAC-related tumors.
Protein-Binding Assay. In this experiment, we incubated 6b
with different concentrations of Hela cell extract and measured
the changes in fluorescence intensity at 450/590 nm with a
microplate reader (POLARstar Omega), to verify whether 6b
could reflect its environment-sensitivity at the enzyme
level.More experimental details were provided in the Supporting
Information.
In Vitro Antiproliferative Assay. The cytotoxicity of 6a and
6b was assayed on solid tumors (PC-3 and A549) and
hematological tumor (K562 and MOLT4) cell lines by the Cell
Counting Kit-8 (CCK-8) method [34]. In brief, solid tumor cell
lines (5×103 / well) and hematological tumor cell lines (1×104 /
well) were cultured for 12 h, and then different concentrations of
compounds and SAHA were added, respectively. Then, they
were incubated for another 24 h. Finally, the absorbance was
measured at 450 nm two hours after CCK8 mixture was added
and IC50 value was calculated by GraphPad Prism 5.
Flow-Cytometry Analysis. The apoptosis rate of MOLT4 cell
line induced by the compounds was detected by Annexin V-PE
apoptosis assay kits. 2×105 cells/well were added to the 6-well
plate and incubated for 12 h. Compounds or SAHA (final
concentration 5 μM) was then added to the wells and incubated
for additional 24 h. Then the cells were collected and washed
with 1X PBS for three times and gently re-suspended and
counted. 1×105 re-suspended cells were centrifugated (6 min,
1200 rmp) and the supernatant was removed. Then 195 μL
Annexin V-PE binding solution and 5 μL Annexin V-PE were
added sequentially and mixed gently. After incubated for about
20 min in dark, the tubes were analyzed by a Beckman flow
cytometer.
Supplementary Material
Materials and Instruments. All reagents were used in this
work without further purification unless otherwise specified.
Boc-Lys (acetyl)-AMC and HeLa Cell Extract were all purchased
from Bachem AG, Switzerland. The melting points were
determined on an electrothermal melting-point apparatus. 1H
NMR and 13C NMR of the compounds were obtained on Bruker
400 or 600 MHz NMR spectrometer with TMS as the internal
standard. High-resolution mass spectra were performed by
Shandong Analysis and Test Center. HPLC examination was
performed on an Agilent 1260 HPLC system. Absorption spectra
were performed by a PUXI TU-1901 spectrophotometer, and
fluorescence spectra were performed by a PerkinElmer EnSight
microplate reader.
Cell Staining and Fluorescence Imaging. Compound 6b with
low cytotoxicity was selected for the cell fluorescence imaging.
And we selected PC-3 and MOLT4 cells with high expression of
HDACs and HEK293 cells with normal expression of HDACs in
the
experiments,
with
reference
to
Fluorescence-Spectroscopy Test. We determined the
ultraviolet absorption, fluorescence quantum yield and
fluorescence spectra in 10 μM solutions of PBS Buffer (pH 7.4),
and studied the environmental sensitivity of the compounds in
solvents of different polarities, viscosities and pH. The
fluorescent properties of compounds were tested by a PUXI TU-
1901 spectrophotometer and a PerkinElmer EnSight microplate
reader. More details can be found in the Supporting Information.
https://www.proteinatlas.org/search/HDAC. PC-3 and HEK293
cells (1×104/mL, 1 mL, DMEM medium + 10% FBS), as well as
MOLT4 cells (2×104/mL, 1 mL, RPMI-1640 medium + 10%
FBS) were inoculated into confocal culture dishes and incubated
for 12 h, respectively. Then the medium was aspirated, and a
solution of 6b (5 μM) diluted with serum-free medium was
added, followed by incubation for 30 minutes. Subsequently,
these cells were imaged by Zeiss Axio Observer A1 fluorescence
microscope (objective lens: 63×). Using the same procedure, 6b
(5 μM) was co-stained with the commercial dyes DID (2.5 μM)
and Hoechst 33342 (0.25 μM). The fluorescence imaging was
captured by a Zeiss LSM780 confocal fluorescence microscope.
Fluorescence Staining of Mouse Tumor and Normal Tissue
Sections. PC-3 (2×107) cells were seeded under the armpit of the
male nude mice. About one month later, the tumor tissues and
normal tissues (skin and thigh muscle tissues) of the nude mice
were made into paraffin sections for immunostaining experiment.
After baking, dewaxing, hydration, and antigen retrieval, the
sections were incubated with 6b (10 μM) or 6b (10 μM) and 100
μM SAHA in Krebs solution overnight at 4 °C. On the next day,
it was washed once with Krebs solution, and a drop of anti-
fluorescence quencher was added. Subsequently, the imaging was
performed with the Olympus VS120 fluorescence upright
microscope (40×).
HeLa Cell Extract Inhibition of the Compounds. The
experimental detail was conducted with reference [33]. The Hela
cell extract was used as the enzyme source and the fluorescence
analysis method with small error and high sensitivity was
adopted. Firstly, the HDACs enzymes, compounds and the
fluorescent substrate Boc-Lys (acetyl)-AMC were incubated
together, so that the histone deacetylase catalyzed the substrate to
deacetylate and form Boc-Lys-AMC; and then Boc-Lys-AMC
was treated with trypsin to form the fluorescent group AMC (4-
amino-7-methylcoumarin), and the fluorescence intensity of the
substance was measured at 390/460 nm (excitation
wavelength/emission wavelength) with a microplate reader.
Finally, the IC50 value was calculated by GraphPad Prism 5
software.
In Vitro HDAC Isoform-Selectivity of the Compounds. We
selected HDAC1, HDAC2, HDAC3, HDAC6, HDAC7 and
HDAC8 for in vitro inhibition of enzyme activity to further
confirm the HDAC isoform selectivity of 6a and 6b. More
experimental details were provided in the Supporting
Information.
Conflicts of interest
There are no conflicts to declare
S7

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Acknowledgements
The present work was supported by grants from the Shandong
Natural Science Foundation (No. ZR2018ZC0233), the Taishan
Scholar Program at Shandong Province, and the Key Research
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Environment-Sensitive
Fluorescent Inhibitors of Histone
Deacetylase
Xin Zhou,† Gaopan Dong,† Tianjia Song,†
Guankai Wang,† Zhenzhen Li,†Xiaojun Qin†,
Lupei Du,† and Minyong Li*,†
† Department of Medicinal Chemistry, Key
Laboratory of Chemical Biology (MOE), School of
Pharmacy, Shandong University, Jinan, Shandong
250012, China
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([(18)F]fluoroacetamido) -1-hexanoicanilide for PET imaging of histone
deacetylase in the baboon brain. Nucl Med Biol. 2009, 36:247-58.
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deacetylases in the brain using PET/MRI with (18)F-FAHA. Neuroimage.
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through human histone deacetylase PET imaging. Sci Transl Med. 2016,
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Assay Well Suited for High-Throughput Activity Screening. Chem Biol.
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histone deacetylases. Nat Chem Biol. 2010, 6:238.
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with a transesterification switch for detection of histone deacetylase activity.
J Am Chem Soc. 2012, 134:14310-3.
Table of Contents
1. Synthesis...............................................................
2. Fluorescence-Spectroscopy Test..........................
3. In Vitro HDAC Isoform-Selectivity of
Compounds...............................................................
4. Protein-Binding Assay .........................................
5. Characterization of Compounds...........................
[20] Koushik D, Yuichiro H, Reisuke B. A fluorescent probe for detection of
histone deacetylase activity based on aggregation-induced emission. Chem
Commun (Camb). 2012, 48:11534-6.
[21] Masafumi M, Tetsuaki M, Kazuya K. Development of a fluorogenic
probe based on a DNA staining dye for continuous monitoring of the histone
deacetylase reaction. Anal Chem. 2014, 86:7925-30.
[22] Rooker DR, Buccella D. Real-time detection of histone deacetylase
activity with a small molecule fluorescent and spectrophotometric probe.
Chem Sci. 2015, 6:6456-61.
[23] Liu X, Xiang M, Tong Z, et al. Activatable Fluorescence Probe via Self-
Immolative Intramolecular Cyclization for Histone Deacetylase Imaging in
Live Cells and Tissues. Anal Chem. 2018:acs.analchem.8b00709.
[24] Miller TA, Witter DJ, Belvedere S. Histone deacetylase inhibitors. J
Med Chem. 2003, 46:5097-116.
[25] Zhuang YD, Chiang PY, Wang CW. Environment-sensitive fluorescent
turn-on probes targeting hydrophobic ligand-binding domains for selective
protein detection. Angew Chem Int Ed Engl. 2013, 52:8124-8.
S8

Compound 2 (520 mg, 2.05 mmol) was
dissolved in 10 mL of acetonitrile. The mixture of
dimethylamine hydrochloride (135.34 mg, 2.05
mmol), triethylamine (462 μL, 3.32 mmol) and 20
mL MeCN was added dropwise with stirring in an
ice bath for 30 min, and allowed to react at room
temperature for 3 h. The acetonitrile was
concentrated in vacuo. The residue was dissolved
with ethyl acetate and filtrated, and the filtrate was
concentrated in vacuo. The crude product was
purified by column chromatography on silica gel
(petroleum: ethyl acetate = 3:1) to obtain
compound 3 as a white solid.
1. Synthesis
Scheme S1. Synthetic Route of SBD fluorophore
4-Chloro-7-chlorosulfonyl-2,1,3-benzoxadiazole
(2)
The 4-chloro-2,1,3-benzooxadiazole solid was
placed in a 100 mL round bottom flask, and
chlorosulfonic acid (38.8 mL, 60.0 mmol) was
placed in a constant pressure dropping funnel,
which was then slowly added dropwise to the 4-
chloro-2,1,3-benzoxadiazole (4.0 g, 25.88 mmol)
solid and stirred evenly under ice bath. After the
addition, the mixture was stirred at room
temperature for 30 min and transferred to the oil
bath at 120 °C. After reacting for 6 h, the mixture
was cooled to room temperature, and the reaction
solution was slowly added to crushed ice water
while stirring. When the crushed ice had been
completely melted, the solution was filtered, and
the precipitate was washed three times with water
to obtain an off-white solid. The crude product was
purified by column chromatography on silica gel
(petroleum: ethyl acetate = 3:1) to obtain
compound 2 as an off-white solid.
A white solid, yield: 222.3 mg, 41.3%, mp:
138-140 °C.
Scheme S2. Synthetic Route of 6a and 6b
Methyl 6-(7-(N,N-dimethylsulfamoyl)-2,1,3-
benzoxadiazole-4-aminohexane) (5a)
Methyl 6-aminohexanoate hydrochloride
(138.1 mg, 0.76mmol) was dissolved in 10 mL
MeCN in a 50 mL round bottom flask under ice
bath, and then triethylamine (212.8μL, 1.52 mmol)
was added to free the amino groups. Five minutes
later, 4-chloro-7-N, N-dimethylsulfonyl-2, 1, 3-
benzoxadiazole (100 mg, 0.38 mmol) was added to
the reaction solution, and the round bottom flask
An off-white solid, yield: 5.57 g, 85.0%, mp:
85- 86 ° C.
5-chloro-7-N,N-dimethylsulfonyl-2,1,3-
benzoxadiazole (3)
was transferred to oil bath at 60℃ to react for 8 h.
The reaction solution was then cooled and 1 M HCl
S9

(15mL) was added, followed by three extractions
with DCM. The organic layer was washed three
times with distilled water, three times with
saturated NaCI solution, and dried over anhydrous
Na2SO4 overnight, then filtered, and the filtrate was
concentrated in vacuo. The crude product was
purified by column chromatography on silica gel
(petroleum: ethyl acetate = 3:1) to obtain
compound 5 as a yellow solid.
the reaction mixture was dissolved and extracted
three times with ethyl acetate, washed three times
with distilled water, washed three times with
saturated NaCl solution, and dried over anhydrous
Na2SO4, then filtrated, and the the filtrate was
concentrated in vacuo. The crude product was
purified by column chromatography on silica gel
(dichloromethane: methanol = 10:1) to obtain
compound 4 as an orange solid.
A yellow solid, yield: 93 mg, 66.1%, mp: 90-
91℃. 1H NMR (400 MHz, MeOD) δ 7.87 (d, J =
8.1 Hz, 1H), 6.27 (d, J = 8.2 Hz, 1H), 3.64 (s, 3H),
3.44 (t, J = 7.1 Hz, 2H), 2.79 (s, 6H), 2.36 (t, J =
7.4 Hz, 2H), 1.76 (dt, J = 15.2, 7.6 Hz, 2H), 1.68
(dd, J = 15.2, 7.5 Hz, 2H), 1.54 -1.41 (m, 2H).
Methyl 7-(7-(N,N-dimethylsulfamoyl)-2,1,3-
benzoxadiazole-4-aminoheptanoate) (5b)
The procedure was the same as 5a
An orange solid, yield: 18.3 mg, 22.9 %, mp:
108-110 ℃. 1H NMR (600 MHz, MeOD) δ 7.87 (d,
J=7.8 Hz, 1H), 6.27 (d, J=7.6 Hz, 1H), 3.44 (t, 2H),
2.79 (s, 6H), 2.12 (s, 2H), 1.77 (t, J=6.1 Hz, 2H),
13
1.70 (t, J=6.2 Hz, 2H), 1.48 (t, J=5.9 Hz, 2H). C
NMR (151 MHz, MeOD) δ 171.36, 146.54, 144.41,
141.80, 140.27, 105.96, 98.02, 42.80, 36.80, 32.21,
27.59, 26.16, 25.01. HRMS (AP-ESI)m/z calcd for
C14H21N5O5S[M + Na]+ 394.1263, found 394.1149.
7-(7-(N,N-dimethylsulfamoyl)-2,1,3-
An orange solid, yield: 120 mg, 82.2%, mp:
79-80 ℃. 1H NMR (400 MHz, MeOD) δ7.87 (d, J
= 8.1 Hz, 1H), 6.26 (d, J = 8.2 Hz, 1H), 3.64 (s,
3H), 3.43 (t, J = 7.1 Hz, 2H), 2.79 (s, 6H), 2.33 (t, J
= 7.4 Hz, 2H), 1.80-1.70 (m, 2H), 1.68-1.59 (m,
2H), 1.52-1.35 (m, 4H).
benzoxadiazole-4-amino)-N-Hydroxyheptamide
(6b)
The procedure was the same as 6a.
An orange solid, yield: 58.9 mg, 58.7%, mp:
125-127℃. 1H NMR (400 MHz, MeOD) δ 7.87 (d,
J = 8.1 Hz, 1H), 6.26 (d, J = 8.2 Hz, 1H), 3.43 (t, J
= 7.1 Hz, 2H), 2.10 (t, J = 7.3 Hz, 2H), 1.83-1.70
(m, 2H), 1.69-1.59 (m, 2H), 1.53-1.37 (m, 4H). 13C
NMR (101 MHz, MeOD) δ 171.29, 146.54, 144.41,
141.83, 140.28, 105.86, 97.98, 42.93, 36.80, 32.29,
28.42, 27.76, 26.36, 25.27. HRMS (AP-ESI) m/z
calcd for C15H23N5O5S [M + Na]+ 408.1420, found
408.1307.
6-(7-(N,N-dimethylsulfamoyl)-2,1,3-
benzoxadiazole-4-amino)-N-
Hydroxyhexanamide (6a)
Compound 6 (90 mg, 0.24 mmol) was
dissolved in 10 mL saturated NH2OK-MeOH
solution, and allowed to react at room temperature
for 30 min. The reaction solution was concentrated
under reduced pressure and the residue was
dissolved in 20 mL of distilled water. The pH was
adjusted to 2 with 4 M HCl under ice bath. A large
amount of yellow solid was precipitated, and then
S10

pale-yellow oily material was obtained, with the
yield of 54.0%, which was stored sealed in a
refrigerator at 4℃, and was to be used in the next
step without purification.
Methyl 2-isocyanato-4-methylpentanoate (9b)
The procedure was the same as 9a
A pale-yellow oily material was obtained, with
the yield of 488.6 mg, 95%.
Scheme S3. Synthetic Route of 11a and 11b
4-((5-aminopentyl)amino)-7-(N,N-
Methyl((5-((7-(N,N-dimethylsulfamoyl)-2,1,3-
benzoxadiazole-4-
dimethylsulfamoyl)-2,1,3-benzoxadiazole (7)
yl)amino)pentyl)carbamoyl)glycinate (10a)
Compound 9a (85.2 mg, 0.74 mmol) was
dissolved in DCM (3 mL), and slowly added
dropwise to a solution of compound 7 (121.2 mg,
0.37 mmol) in DCM (5 mL) under ice bath, and
stirred for 1 h. The reaction mixture was washed
three times with 1 M HCl and saturated NaCl
solution, respectively, and dried over anhydrous
Na2SO4. The crude product was purified by silica
gel column (dichloromethane : methanol = 10:1)
and dried, resulting in 124.5 mg of yellow solid
Compound
3
(200 mg, 0.76 mmol),
acetonitrile (14 mL) and 1,5-pentanediamine (0.71
mL, 6.08 mmol) were sequentially added to a 50
mL round bottom flask, and reacted at 60 ° C for 6
h. After cooling to room temperature, 20 mL of
saturated NaHCO3 solution was added, and
extracted three times with DCM. The organic phase
was washed three times with distilled water and
saturated NaCl solution, respectively, and dried
over anhydrous MgSO4. Following filtration and
concentration of the filtrate under reduced pressure,
173.8 mg of orange solid was obtained with the
yield 77.2%. The crude product was to be used in
the next step without purification.
1
(mp: 100-101℃), with the yield 76.1%. H NMR
(600 MHz, DMSO) δ 8.41 (t, J = 5.5 Hz, 1H), 7.81
(dd, J = 8.2, 2.3 Hz, 1H), 6.31 (dd, J = 8.2, 3.9 Hz,
1H), 6.16 (dd, J = 11.5, 5.8 Hz, 2H), 3.76 (d, J = 6.0
Hz, 2H), 3.61 (s, J = 8.2 Hz, 3H), 2.99 (td, J = 12.8,
6.6 Hz, 2H), 2.69 (s, J = 2.0 Hz, 6H), 1.67 (dt, J =
14.4, 7.3 Hz, 2H), 1.46-1.39 (m, 2H), 1.39-1.30 (m,
2H).
Methyl 2-isocyanatoacetate (9a)
Glycine methyl ester hydrochloride (376.6 mg,
3 mmol) was dissolved in a mixture of
dichloromethane and saturated NaHCO3 (12 mL /
12 mL) in ice bath, subsquently triphos (296.7 mg,
1 mmol) was added and the mixture was stirred
vigorously for 15 min. The aqueous phase was
extracted three times with DCM. The organic
phases were pooled and dried over anhydrous
MgSO4. Following filtration and concentration of
the filtrate under reduced pressure, 186.5 mg of
methyl((5-((7-(N,N-dimethylsulfamoyl)-2,1,3-
benzoxadiazole-4-
yl)amino)pentyl)carbamoyl)leucinate (10b)
The procedure was the same as 10a.
A yellow solid, yield: 220.0 mg, 59.1%, mp:
1
98-99℃. H NMR (600 MHz, DMSO) δ 8.40 (t,
S11

1H), 7.82 (d, J = 8.1 Hz, 1H), 6.31 (d, J = 8.1 Hz,
1H), 6.16 (d, J = 8.2 Hz, 1H), 5.93 (t, J = 4.9 Hz,
1H), 4.17 (dd, J = 15.2, 7.6 Hz, 1H), 3.60 (s, 3H),
3.36 (s, 2H), 3.00 (d, J = 6.0 Hz, 2H), 2.70 (s, 6H),
1.71-1.64 (m, 2H), 1.61 (dt, J = 13.1, 6.6 Hz, 1H),
1.43 (m, J = 15.3, 8.0 Hz, 4H), 1.36 (d, J = 6.4 Hz,
2H), 0.86 (dd, J = 17.3, 6.5 Hz, 6H).
29.61, 27.60, 24.49, 23.93, 21.88, 20.96. HRMS
(AP-ESI) m/z calcd for C17H27N7O6S [M + Na]+
522.2213, found 522.2097.
2. Fluorescence-Spectroscopy Test.
The compounds were dissolved in DMSO to
obtain 10 mM concentrated solutions. The
concentrated solutions were diluted in organic
solvents of different polarities, mixed solutions of
glycerol and water with different viscosities, and
Britton-Robinson buffer solutions at different pH
values to acquire 5 µM solutions, and in PBS
(PH=7.4) to acquire 5 µM, 10 µM, 20 µM, 40 µM
and 80 µM solutions. The fluorescent properties of
the compounds were determined by PUXI TU-1901
and PerkinElmer EnSight. Moreover, the quantum
yields in PBS solution (pH=7.4) and acetonitrile
were calculated in comparison with 500 nM sodium
fluorescein. The calculation formula of the
fluorescence quantum yield is as follows:
ΦX= ΦS (AS /AX) (FX/FS ) (ƞX/ƞS )2
2-(3-(5-((7-(N,N-dimethylsulfamoyl)-2,1,3-
benzoxadiazole-4-yl)amino)pentyl)ureido)-N-
hydroxyacetamide (11a)
The procedure was the same as 6a.
A yellow solid, yield: 62 mg, 56.4%, mp: 93-
94℃. 1H NMR (600 MHz, MeOD) δ 7.87 (d, J =
8.1 Hz, 1H), 6.27 (d, J = 8.2 Hz, 1H), 3.73 (s, 2H),
3.44 (t, J = 7.0 Hz, 2H), 3.14 (t, J = 6.8 Hz, 2H),
2.79 (s, 6H), 1.80-1.74 (m, 2H), 1.56 (dt, J = 14.2,
6.9 Hz, 2H), 1.51-1.45 (m, 2H). 13C NMR (151
MHz, MeOD) δ 168.61, 159.54, 146.54, 144.41,
141.83, 140.29, 105.88, 98.02, 42.93, 40.71, 39.48,
36.80, 29.56, 27.60, 23.90. HRMS (AP-ESI) m/z
calcd for C16H25N7O6S [M + Na]+ 466.1587, found
466.1477.
Where, S represents the standard; X represents
the test; Φ is the quantum yield,; F is the integrated
area; A is the absorbance; and η is the refractive
index .
2-(3-(5-((7-(N,N-dimethylsulfamoyl)2,1,3-
benzoxadiazole-4-yl)amino)pentyl)ureido)-N-
hydroxy-4-methylpentanamide (11b)
The procedure was the same as 6a.
A yellow solid, yield: 128 mg, 58%, mp: 95-
1
96℃. H NMR (600 MHz, MeOD) δ 7.87 (d, J =
8.1 Hz, 1H), 6.27 (d, J = 8.2 Hz, 1H), 4.16 (t, J =
7.4 Hz, 1H), 3.43 (t, J = 7.0 Hz, 2H), 3.13 (t, J =
6.4 Hz, 2H), 2.79 (s, 6H), 1.78 (m, 2H), 1.65(m,
2H), 1.52-1.43 (m, 3H), 0.93 (dd, J = 13.0, 6.6 Hz,
6H). 13C NMR (151 MHz, MeOD) δ 171.02,
158.95, 146.55, 144.41, 141.83, 140.29, 105.87,
98.03, 56.93, 49.92, 42.96, 41.71, 39.35, 36.81,
S12

Figure S1. Ultraviolet absorption spectrum of the
compounds.
Figure S3. Excitation and emission spectra of
compounds in solvents of different polarities
3. In Vitro HDAC Isoform-Selectivity of
Compounds.
All of the enzymatic reactions were conducted
at 37 ℃ for 30 minutes. The 50 μL reaction mixture
contained 25 mM Tris, pH 8.0, 1 mM MgCl2, 0.1
mg/ml BSA, 1 37mM NaCl, 2.7 mM KCl, Hela cell
extract and the substrate. The compounds were
diluted in 10% DMSO and 5 μL of the dilution was
added to 50 μL reaction solution, to achieve the
final concentration of DMSO of 1% among all of
reaction solutions. The assay was performed by
Figure S2. (A): Excitation (A1) and emission (A2)
spectra of 6b in different concentrations of PBS
solution; (B): Excitation (B1) and emission (B2)
spectra of 6b (5 μM) in mixes solution of different
viscosities (glycerol:H2O); (C): Excitation (C1)
and emission (C2) spectra of 6b (5 μM) in solvents
at different pH values.
S13

quantitating the fluorescent product in solution
following an enzyme reaction. Fluorescence was
then analyzed with an excitation wavelength of
350-360 nm and an emission wavelength of 450-
460 nm on a SpectraMax M5 microtiter plate
reader. The IC50 values were calculated by
nonlinear regression with normalized dose-response
fit using Prism GraphPad software.
4. Protein-Binding Assay
Firstly, the DMSO stock solution (10 mM) of
compound 6b was diluted to 10μM with HDACs
Buffer (as same as used in the in vitro inhibitory
activity assay). Then Hela cell extract was prepared
to obtain a six-concentration gradient with HDACs
Buffer, ie., 3.12 ng/μL, 6.26 ng/μL, 12.5 ng/μL, 25
ng/μL, 50 ng/μL, and 100 ng/μL. A total of 100 μL
solution was added to each well, including 10 μL
6b dilution and 90 μL enzyme solution; and three
replicate wells were set for each concentration. In
the negative control group, 10 μL 6b dilution and
90 μL HDAC Buffer were added, and in the blank
control group was, 100 μL HDAC Buffer was
added. The solutions were incubated at 37 ℃ for 30
minutes on a shaker, and the fluorescence intensity
was measured at 450/590 nm (excitation
1H NMR of 6a
wavelength/emission wavelength) by a microplate
reader. Finally, the saturation curve was fitted and
the Kd value was calculated by GraphPad Prism 5.
5. Characterization of Compounds
13C NMR of 6a
S14

13C NMR of 6b
ESI-HRMS of 6a
1H NMR of 6b
ESI-HRMS of 6b
S15

1H NMR of 11a
13C NMR of 11a
ESI-HRMS of 11a
S16

1H NMR of 11b
ESI-HRMS of 11b
HPLC assessment of purity.
13C NMR of 11b
9
S17

☐The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests:
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Environment-Sensitive Fluorescent Inhibitors of Histone
Deacetylase
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Xin Zhoua, Gaopan Donga, Tianjia Songa, Guankai Wanga, Zhenzhen Lia, Xiaojun Qina, Lupei Dua, and
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S18