Synthesis and biological study of class I selective HDAC inhibitors with NO releasing activity

Article abstract of DOI:10.1016/j.bioorg.2020.104235

Based on the multi-mechanism antitumor strategy and the regulatory effect of nitric oxide (NO) on histone deacetylases (HDACs), a series of N-acyl-o-phenylenediamine-based HDAC inhibitors equipped with the phenylsulfonylfuroxan module as NO donor was desi

Full text of DOI:10.1016/j.bioorg.2020.104235

BioorganicChemistry104(2020)104235
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and biological study of class I selective HDAC inhibitors with NO
releasing activity
Qin'ge Dinga,1, Chunxi Liub,1, Chunlong Zhaoa, Hang Donga, Qifu Xua, C. James Chouc,
⁎
Yingjie Zhanga,
a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Cheeloo College of Medicine,
Shandong University, 44 West Wenhua Road, Ji’nan, Shandong 250012, PR China
b Department of Pharmacy, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Ji'nan, Shandong 250012, PR China
c Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, Medical University of South Carolina, Charleston, SC 29425, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
Based on the multi-mechanism antitumor strategy and the regulatory effect of nitric oxide (NO) on histone
deacetylases (HDACs), a series of N-acyl-o-phenylenediamine-based HDAC inhibitors equipped with the phe-
nylsulfonylfuroxan module as NO donor was designed, synthesized and biologically evaluated. The in vitro
HDAC inhibitory assays revealed that compared with the clinical class I selective HDAC inhibitor MS275,
compounds 7c, 7d and 7e possessed similar HDAC inhibitory potency and selective profile, which were con-
firmed by the results of western blot analysis. The western blot analysis also showed that NO scavenger N-acetyl
cysteine (NAC) could weaken the intracellular HDAC inhibitory ability of compound 7c, supporting the HDAC
inhibitory effect of NO generated by 7c. It is worth noting that compounds 7c, 7d and 7e exhibited more potent
in vitro antiproliferative activities than MS275 against all four tested solid tumor cell lines. The promising in
vivo antitumor potency of 7c was demonstrated in a HCT116 xenograft model.
Histone deacetylase
Nitric oxide
Hybrid
Anticancer
Phenylsulfonylfuroxan
N-acyl-o-phenylenediamine
1. Introduction
Nitric oxide (NO), an endogenous signal or effector molecule, ac-
counts for various important cellular functions including vasodilation,
Histone deacetylases (HDACs), a family of enzymes, play important
roles in epigenetic regulation and post-translational modification by
removing the acetyl groups from histones and non-histone proteins [1].
Recently, HDACs have been extensively investigated as anticancer tar-
gets. To date, four pan-HDAC inhibitors (vorinostat, belinostat, pano-
binostat, romidepsin, Fig. 1) have received approval in the United
States for the treatment of cutaneous T cell lymphoma, peripheral T cell
lymphoma or multiple myeloma. In addition, a class I selective HDAC
inhibitor chidamide (Fig. 1) has been approved in China for use in
patients with peripheral T cell lymphoma [2]. Despite the success in
treating hematologic malignancies, most HDAC inhibitors showed
limited efficacy as monotherapy against solid tumors. Noteworthily,
HDAC inhibitor-based drug combination or multi-target/multi-me-
chanism agent offers hope to conquer this problem [3–5]. Compared
with drug combination, multi-target/multi-mechanism agents show
advantages in more predictable pharmacokinetic profiles, less drug-
drug interaction and better patient compliance [6–8], despite of the
possible larger molecular weights and more complex synthesis.
neurotransmission, cell migration, immune response and apoptosis [9].
There has been accumulating evidence suggesting that high con-
centrations of NO can exhibit multiple antitumor effects, therefore NO
donors have been researched and developed as potential antitumor
agents [10–12]. Two phase II clinical trials demonstrated that NO donor
nitroglycerin combined with chemotherapy may improve overall re-
sponse, progression-free survival or time to disease progression in pa-
tients with non-small cell lung cancer [13,14]. Moreover, one small
molecular compound named RRx-001 with NO releasing activity is
currently in phase I/II clinical trials for cancer therapy [15,16].
Intriguingly, there were research revealing that NO could lead to S-
nitrosylation modification of HDAC2 in neurons, inducing the release of
HDAC2 from chromatin and the subsequent hyperacetylation of his-
tones and gene activation [17,18]. Another research demonstrated that
NO could inhibit the deacetylase activity of HDAC2 and HDAC1 by S-
nitrosylation-dependent and independent mechanism, respectively
[19]. Taken together, it is suggested that NO could negatively regulate
the biological function of many HDACs, at least the class I isoforms of
⁎ Corresponding author.
E-mail address: zhangyingjie@sdu.edu.cn (Y. Zhang).
1 These two authors contributed equally.
https://doi.org/10.1016/j.bioorg.2020.104235
Received 30 June 2020; Received in revised form 4 August 2020; Accepted 12 August 2020
Availableonline26August2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

Q. Ding, et al.
BioorganicChemistry104(2020)104235
Fig. 1. The structures of approved anticancer HDAC inhibitors.
Fig. 2. The structures of reported hybrid molecules of HDAC inhibitor and NO donor. The NO donors are indicated in blue, the functional part of Largazole targeting
HDAC is indicated in purple, and the structure of MS275 is indicated in red. (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
HDAC2 and HDAC1. Therefore, hybrid molecules of HDAC inhibitor
and NO donor is supposed to possess more potent HDAC inhibitory and
antitumor activity than common HDAC inhibitors. In our previous re-
search, a novel series of hydroxamate-based HDAC inhibitors with a
phenylsulfonylfuroxan module as the NO donor was designed and
synthesized, among which compound 1 (Fig. 2) exhibited superior
HDAC inhibitory activity, in vitro and in vivo antitumor potency re-
lative to the approved HDAC inhibitor vorinostat [20]. Lately, two
Largazole analogs as dual HDAC inhibitor and NO donors (compounds
2 and 3, Fig. 2) were developed, which also showed promising in vitro
antitumor potency [21]. In addition, several hybrids of HDAC inhibitor
MS275 and NO donor (compounds 4, 5 and 6, Fig. 2) were developed as
muscle differentiation agents, while no antitumor activity was reported
[22,23].
In the present research, a novel HDAC inhibitor-NO donor hybrid 7c
was designed and synthesized based on the structure of our previously
reported compound 1 (Fig. 3). The main purpose of structural mod-
ification of compound 1 was to get the analogs with improved HDAC
isoform selectivity, given that the N-acyl-o-phenylenediamine group
could generally afford class I selective HDAC inhibitors [2]. Structure-
activity relationship (SAR) study was focused on the linker part of
compound 7c, leading to a series of hybrids of N-acyl-o-phenylenedia-
mine and phenylsulfonylfuroxan (Fig. 3). Among these hybrids, three
analogs 7c, 7d and 7e exhibited not only potent HDAC1/2/3 inhibitory
activities but also significant NO releasing capabilities, which con-
tributed to the remarkable in vitro antiproliferative potency against
Fig. 3. Design strategy of the target compounds.
2

Q. Ding, et al.
BioorganicChemistry104(2020)104235
Scheme 1. Synthesis of compounds 7a-7h. Reagents and conditions: (a) H2O2, CH3COOH; (b) conc. HNO3; (c) HO-R-CH2-OH, 25%NaOH; (d) Jones agent, acetone;
(e) TBTU, TEA, THF; o-phenylenediamine. (f) PCC, DCM, 0℃; (g) propandioic acid, pyrrolidine, pyridine, reflux.
both hematological and solid tumor cell lines. Additionally, the in vivo
antitumor activity of compound 7c in a HCT116 xenograft model was
comparable to, if not better than that of MS275, a class I selective HDAC
inhibitor in clinical trials.
Table 1
Total
HDAC
inhibitory
activities
of
compounds
7a-
7h.
2. Results and discussion
Compd
R
IC50 (μM)a
HeLa nuclear extract
2.1. Chemistry
7a
7b
7c
7d
7e
7f
14.3
8.01
2.16
1.69
2.51
14.9
30.5
2.1
1.54
0.26
0.42
0.27
3.4
The synthesis of target compounds 7a-7h was described in Scheme
1. Oxidization of the starting material 8 by H2O2 led to compound 9.
Without separation and purification, compound 9 was further pro-
gressed to one-pot reaction with fuming HNO3 to get compound 10.
Substitution of 10 with corresponding dihydroxy compounds led to
alcohols 11a-11g, which were oxidized to carboxylic acids 12a-12g,
respectively. The target compounds 7a-7g were obtained by con-
densation of 12a-12g with o-phenylenediamine. The intermediate al-
cohol 11 g could also be oxidized by PCC to get aldehyde 13. Sub-
sequent reaction with propandioic acid and condensation with o-
phenylenediamine afforded the target compound 7h.
7g
3.7
7h
27.5
1.56
2.9
MS275
0.17
a
2.2. In vitro HDACs inhibitory assay
Assays were performed in replicate (n ≥ 3), values are shown as
SD.
mean
HeLa cell nuclear extract (mainly contains HDAC1 and 2) was used
to preliminarily evaluate the HDAC inhibitory potency of the target
compounds with the clinical HDAC inhibitor MS275 as the positive
control. The results in Table 1 showed that all newly synthesized hy-
brids exhibited moderate to potent total HDAC inhibitory activities
against HeLa cell nuclear extract, with compounds 7c
Table 2
HDAC isoform selectivity of compounds 7c, 7d, 7e and MS275.
Compd
IC50 (μM)a
Class I
Class IIa
HDAC4
Class IIb
HDAC6
(IC50 = 2.16
(IC50 2.51
(IC50 = 1.56
0.26 μM), 7d (IC50 = 1.69
0.42 μM) and 7e
HDAC1
HDAC2
HDAC3
=
0.27 μM) being comparable to MS275
0.17 μM). For analogs with aliphatic linkers (7a-7f),
7c
0.62
0.47
0.66
0.16
0.14
0.11
0.17
0.03
1.46
1.14
1.20
0.40
0.24
0.18
0.26
0.07
0.62
0.56
0.52
0.61
0.14
0.13
0.14
0.11
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
their HDAC inhibitory activities first increased then decreased with the
7d
linker length, culminating in compound 7d (IC50 = 1.69
0.42 μM).
7e
MS275
Compounds 7g and 7h with aromatic linkers showed much less potent
HDAC inhibitory activities than compounds with aliphatic linkers (7a-
7f). The inferior activities of 7g and 7h could be ascribed to their
aromatic linkers, which might be too rigid to fit in the active site of
HDAC.
a
Assays were performed in replicate (n ≥ 3), values are shown as
SD.
mean
3

Q. Ding, et al.
BioorganicChemistry104(2020)104235
(ac-α-tub). This was in line with the HDAC isoform selective profiles
shown in Table 2. Besides, we performed another set of western blot
analysis to investigate if NO generated by 7c contributed to its in-
tracellular HDAC inhibition. As we can see in Fig. 5B, NO scavenger N-
acetyl cysteine (NAC) could significantly compromise the class I HDAC
inhibitory activity of 7c while showed no influence on MS275, con-
firming the HDAC inhibitory effect of NO as previously reported
[17–19].
2.6. In vitro anti-proliferative assay
Considering their potent HDAC inhibitory and NO donating activ-
ities, compounds 7c, 7d and 7e were evaluated for their anti-
proliferative activities against three hematological tumor cell lines and
four solid tumor cell lines. MS275 was used as the positive control.
According to the results in Table 3, all compounds could attenuate
tumor cell growth effectively. Typically, HDAC inhibitors are more
cytotoxic to hematological cell lines than solid tumor cell lines. This
trend was also observed in MS275 and hybrids 7c, 7d and 7e. For the
three tested hematological tumor cell lines, 7c, 7d and 7e were more
potent against the human erythroleukemia cell line HEL, but less potent
against the human acute lymphoblastic leukemia cell line MOLT-4 and
the human acute T-cell leukemia cell line Jurkat than MS275. On the
whole, the anti-hematological malignancy potency of our hybrid com-
pounds was comparable to that of MS275. It was worth noting that
hybrids 7c, 7d and 7e exhibited more potent activities against all tested
solid tumor cell lines than MS275. Among the four tested solid tumor
cell lines, the human colon cancer cell line HCT116 showed the most
sensitivity to our hybrid compounds, which possessed IC50 values
against HCT116 lower than 1 μM.
Fig. 4. NO production of compounds in HeLa cells. Data are represented as
means
SD obtained from at least three independent experiments. *
p < 0.05 versus the control groups by Student’s two-tailed t test.
2.3. In vitro HDACs isoform selectivity assay
Considering their potent total HDAC inhibitory activities, com-
pounds 7c, 7d and 7e were further tested in HDAC isoform selectivity
assay. Their IC50 values against three class I isoforms (HDAC1, HDAC2
and HDAC3), one class IIa isoform (HDAC4) and one class IIb isoform
(HDAC6) were determined and compared with MS275 (Table 2). As
expected, all N-acyl-o-phenylenediamine-based compounds were class I
selective but showed no significant preference among different class I
isoforms. In general, compared with MS275, the HDAC1 and HDAC2
inhibitory activities of 7c, 7d, 7e were slightly less potent, while their
HDAC3 inhibitory activities were quite similar.
2.7. In vivo antitumor evaluation
2.4. In vitro NO release assay
Finally, a HCT116 xenograft model in nude mice was established to
evaluate the in vivo antitumor potency of the representative compound
7c with MS275 as the positive control. On the basis of our previous
research, oral administration of MS275 in the dosage of 50 mg/kg/d
was effective but toxic in xenograft models [25]. Therefore, in the
present research the dosage of MS275 was reduced to 30 mg/kg/d. To
better compare the in vivo antitumor potency of 7c and MS275, the
dosage of 7c was also set as 30 mg/kg/d. After 15 consecutive days of
treatment, tumor growth inhibition (TGI) and relative increment ratio
(T/C) were calculated to evaluate the antitumor effects in the aspects of
tumor weight and tumor volume, respectively. As shown in Table 4,
compound 7c exhibited potent in vivo antitumor efficacy, which was
comparable to, if not better than that of MS275 at the same dosage. The
tumor growth curve was presented in Fig. 6. Moreover, mice treated by
7c showed no significant body weight loss (Fig. 7), suggesting the ac-
ceptable toxicity of 7c.
The NO releasing capabilities of compounds 7c, 7d and 7e were
characterized in HeLa cells, which were incubated with different
compounds (10 μM) for 1 h or 5 h. After incubation, the intracellular
levels of NO were measured using the Griess method, which measures
the nitrate/nitrite levels to quantify the NO released [24]. As shown in
Fig. 4, compounds 7c, 7d and 7e bearing the phenylsulfonylfuroxan
moiety could effectively release NO in a time dependent manner, re-
presented as fold increase based on the control group with endogenous
nitrate/nitrite. In contrast, MS275 displayed no NO releasing cap-
ability, which was in line with the literature results [22,23].
2.5. Western blot analysis
With confirmed HDAC inhibitory activity and NO donating cap-
ability, one representative compound 7c was progressed to western blot
analysis to investigate their intracellular target engagement. The results
in Fig. 5A showed that both MS275 and 7c could dramatically increase
the intracellular levels of the class I HDAC substrate acetyl-histone H4
(ac-HH4) without influencing the HDAC6 substrate acetyl-α-tubulin
3. Conclusion
In summary, a novel series of N-acyl-o-phenylenediamine-based
Fig. 5. (A) HeLa cells were treated with compounds (2 μM) or DMSO for 5 h. (B) HeLa cells were preincubated with 10 μM of NAC, then were treated with 2 μM of
MS275 or 7c for 5 h. The levels of ac-α-tub and ac-HH4 were determined by immunoblotting. β-Actin was used as a loading control.
4

Q. Ding, et al.
BioorganicChemistry104(2020)104235
Table 3
In vitro anti-proliferative activity of compounds 7c, 7d, 7e and MS275.
Cpd
IC50 (μM)a
Hematological tumor cell lines
Solid tumor cell lines
HEL
MOLT-4
Jurkat
HeLa
PC-3
HCT116
A549
7c
0.32
0.41
0.42
0.68
0.06
0.05
0.05
0.07
0.59
0.54
0.72
0.46
0.09
0.02
0.15
0.02
0.54
0.77
0.47
0.25
0.05
0.12
0.07
0.03
1.23
1.50
1.56
3.36
0.06
0.11
0.11
0.22
2.69
2.50
1.92
9.67
0.15
0.10
0.09
1.62
0.93
0.77
0.79
1.64
0.08
0.06
0.09
0.22
1.03
0.98
1.55
2.05
0.09
0.06
0.03
0.14
7d
7e
MS275
a
Assay were performed in replicate (n ≥ 3), values are shown as mean
SD.
Table 4
In vivo antitumor efficacy of compounds 7c and MS275 in the HCT116 xeno-
graft mode.
Compd
Administration
T/C (%)a
TGI (%)a
7c
30 mg/kg/d, PO
30 mg/kg/d, PO
57
64
36
31
MS275
a
Compared with the control group, all treated groups showed statistically
significant (P < 0.05) T/C and TGI by Student’s two-tailed t test.
Fig. 7. Body weight curve of nude mice. Data are expressed as the mean
SD.
4. Experimental section
4.1. Chemistry
Unless specified otherwise, all starting materials, reagents and sol-
vents were commercially available. 1H NMR and 13C NMR spectra were
obtained using a Bruker DRX spectrometer at 400 and 100 MHz re-
spectively. Chemical shifts were reported in parts per million (ppm).
Multiplicity of 1H NMR signals was reported as singlet (s), doublet (d),
triplet (t), quartert (q) and multiplet (m). ESI-MS data was recorded on
an API 4000 spectrometer. High resolution mass spectra were con-
ducted by Shandong Analysis and Test Center in Ji’nan. Melting points
were determined on an electrothermal melting point apparatus and
were uncorrected.
Fig. 6. Growth curve of implanted HCT116 xenograft in nude mice. Data are
expressed as the mean
SD. * p < 0.05 versus the control groups by
Student’s two-tailed t test.
HDAC inhibitors equipped with the phenylsulfonylfuroxan module was
designed, synthesized and biologically evaluated. Among these analogs,
three compounds (7c, 7d and 7e) with aliphatic linkers of suitable
length (4–6 methylene groups) showed remarkable inhibition and se-
lectivity towards class I HDACs. The NO donating activities of these
three analogs were validated using the Griess method. The intracellular
class I HDAC inhibitory potency and selectivity of one representative
compound 7c were validated by western blot analysis, which also re-
vealed that NO generated by 7c also contributed to its intracellular
HDAC inhibition. The dual biological activities endowed compounds
7c, 7d and 7e with considerable in vitro antiproliferative potencies,
especially against the solid tumor cell lines. The results of xenograft
experiment presented the favorable in vivo antitumor potency and toxic
profile of 7c, which supported its further research and development as a
novel antitumor agent.
3,4-bis(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (10), 4-(4-hydro-
xybutoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (11a), 4-((5-
hydroxypentyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (11b),
4-((6-hydroxyhexyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide
(11c), 4-((7-hydroxyheptyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-
oxide (11d), 4-((8-hydroxyoctyl)oxy)-3-(phenylsulfonyl)-1,2,5-ox-
adiazole 2-oxide (11e), 4-((9-hydroxynonyl)oxy)-3-(phenylsulfonyl)-
1,2,5-oxadiazole 2-oxide (11f), 4-(4-(2-hydroxyethyl)phenoxy)-3-(phe-
nylsulfonyl)-1,2,5-oxadiazole 2-oxide (11g), 4-(3-carboxypropoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12a), 4-(4-carboxybutoxy)-
3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12b), 4-((5-carbox-
ypentyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12c), 4-((6-
carboxyhexyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (12d),
4-((7-carboxyheptyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide
(12e), 4-((8-carboxyoctyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-
oxide (12f), 4-(4-(carboxymethyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-
5

Q. Ding, et al.
BioorganicChemistry104(2020)104235
oxadiazole 2-oxide (12g), 4-(4-formylphenoxy)-3-(phenylsulfonyl)-
1,2,5-oxadiazole 2-oxide (13), (E)-4-(4-(2-carboxyvinyl)phenoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (14) were obtained as pre-
viously described [20].
137.70, 136.61, 130.50, 128.74, 126.14, 125.72, 124.06, 116.64,
116.36, 110.90, 71.93, 36.22, 29.21, 29.09, 28.92, 28.31, 25.77, 25.47.
HRMS (AP-ESI) m/z calcd for C22H27N4O6S [M + H]+: 475.1651;
found: 475.1633.
4-(4-((2-aminophenyl)amino)-4-oxobutoxy)-3-(phenylsulfonyl)-
1,2,5-oxadiazole 2-oxide (7a). At 0 °C, to a solution of compound 12a
(0.46 g, 1.4 mmol) in dried THF (30 mL), were added TBTU (0.53 g,
1.6 mmol) and TEA (0.28 mL, 2.0 mmol). After stirring for 40 min, o-
phenylenediamine (0.17 g, 1.6 mmol) was added. The mixture was
stirred for an additional 4 h at room temperature, then the solvent was
removed under vacuum, with the residues being taken up in EtOAc. The
organic portion was washed by water and brine, dried with anhydrous
Na2SO4. The solvent was evaporated to get the crude product, which
was purified by silica chromatography column (DCM/MeOH = 20:1) to
obtain 7a (0.29 g, 39% yield), white solid. Mp: 150 − 152℃. 1H NMR
(400 MHz, DMSO‑d6) δ 9.20 (s, 1H), 8.05 (d, J = 8.6 Hz, 2H), 7.89 (t,
J = 4.0 Hz, 1H), 7.75 (t, J = 8.6 Hz, 2H), 7.17 (d, J = 9.0 Hz, 1H),
6.91 (t, J = 8.5 Hz, 1H), 6.73 (d, J = 9.1 Hz, 1H), 6.60 – 6.52 (m, 1H),
4.48 (t, J = 7.3 Hz, 2H), 2.08 (q, J = 7.5 Hz, 2H). 13C NMR (101 MHz,
DMSO‑d6) δ 170.66, 159.40, 142.50, 137.61, 136.62, 130.50, 128.87,
126.34, 125.99, 123.80, 122.99, 116.63, 116.32, 111.06, 71.46, 31.90,
24.60. HRMS (AP-ESI) m/z calcd for C18H19N4O6S [M + H]+:
419.1025; found: 419.0989.
4-((9-((2-aminophenyl)amino)-9-oxononyl)oxy)-3-(phenylsulfonyl)-
1,2,5-oxadiazole 2-oxide (7f). White solid. 33% yield. Mp: 130-132℃.
1H NMR (400 MHz, DMSO‑d6) δ 9.09 (s, 1H), 8.01 (d, J = 7.9 Hz, 2H),
7.89 (t, J = 7.4 Hz, 1H), 7.75 (t, J = 7.6 Hz, 2H), 7.15 (d, J = 7.9 Hz,
1H), 6.88 (t, J = 7.6 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 6.53 (t,
J = 7.6 Hz, 1H), 4.85 (s, 2H), 4.38 (t, J = 6.3 Hz, 2H), 2.32 (t,
J = 7.3 Hz, 2H), 1.78–1.71 (m, 2H), 1.65–1.55 (m, 2H), 1.33 (s, 8H).
13C NMR (101 MHz, DMSO‑d6) δ 171.63, 159.36, 142.31, 137.72,
136.60, 130.49, 128.74, 126.14, 125.72, 124.08, 116.66, 116.38,
110.90, 71.95, 36.24, 29.21, 29.09, 28.92, 28.32, 25.77, 25.48. HRMS
(AP-ESI) m/z calcd for C23H29N4O6S [M + H]+: 489.1808; found:
489.1800.
4-(4-((2-aminophenyl)carbamoyl)phenoxy)-3-(phenylsulfonyl)-
1,2,5-oxadiazole 2-oxide (7g). White solid. 30% yield. Mp: 180-182℃.
1H NMR (400 MHz, DMSO‑d6) δ 9.73 (s, 1H), 8.08 (dd, J = 16.5,
8.1 Hz, 4H), 7.93 (t, J = 7.4 Hz, 1H), 7.78 (t, J = 7.7 Hz, 2H), 7.58 (d,
J = 8.4 Hz, 2H), 7.17 (d, J = 7.9 Hz, 1H), 6.98 (t, J = 7.6 Hz, 1H),
6.78 (d, J = 8.0 Hz, 1H), 6.60 (t, J = 7.6 Hz, 1H), 4.95 (s, 2H). 13C
NMR (101 MHz, DMSO) δ 164.75, 158.53, 155.36, 143.80, 137.29,
136.78, 133.22, 130.52, 130.47, 129.10, 127.36, 127.14, 123.44,
119.73, 116.61, 116.47, 111.89. HRMS (AP-ESI) m/z calcd for
Compounds 7b-7h were prepared in a similar method as described
for compound 7a.
4-((5-((2-aminophenyl)amino)-5-oxopentyl)oxy)-3-(phe-
C
21H17N4O6S [M + H]+: 453.0869; found: 453.0861.
nylsulfonyl)-1,2,5-oxadiazole 2-oxide (7b). White solid. 40% yield. Mp:
136-138℃. 1H NMR (400 MHz, DMSO‑d6) δ 9.16 (s, 1H), 8.07–7.98 (m,
2H), 7.93–7.84 (m, 1H), 7.78–7.70 (m, 2H), 7.18 (dd, J = 7.8, 1.6 Hz,
1H), 6.91 (td, J = 7.6, 1.6 Hz, 1H), 6.73 (dd, J = 8.0, 1.5 Hz, 1H), 6.55
(td, J = 7.5, 1.5 Hz, 1H), 4.89 (s, 2H), 4.43 (t, J = 6.1 Hz, 2H), 2.40 (t,
J = 7.2 Hz, 2H), 1.86–1.78 (m, 2H), 1.76–1.68 (m, 2H). 13C NMR
(101 MHz, DMSO‑d6) δ 171.28, 159.36, 142.41, 137.67, 136.61,
130.53, 128.81, 126.24, 125.78, 123.93, 116.64, 116.36, 110.93,
71.66, 35.48, 27.95, 21.89. HRMS (AP-ESI) m/z calcd for C19H21N4O6S
[M + H]+: 433.1182; found: 433.1178.
(E)-4-(4-(3-((2-aminophenyl)amino)-3-oxoprop-1-en-1-yl)phe-
noxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (7h). White solid.
47% yield. Mp: 168-170℃. 1H NMR (400 MHz, DMSO‑d6) δ 9.41 (s,
1H), 8.05 (d, J = 7.8 Hz, 2H), 7.93 (t, J = 7.4 Hz, 1H), 7.84–7.71 (m,
4H), 7.64–7.45 (m, 3H), 7.35 (d, J = 7.9 Hz, 1H), 6.92 (q, J = 6.9 Hz,
2H), 6.76 (d, J = 8.0 Hz, 1H), 6.59 (t, J = 7.6 Hz, 1H), 4.95 (s, 2H). 13
C
NMR (101 MHz, DMSO) δ 163.78, 158.63, 153.85, 142.10, 138.67,
137.34, 136.76, 133.75, 130.52, 129.85, 129.05, 126.32, 125.23,
123.88, 123.52, 120.63, 116.75, 116.47, 111.81. HRMS (AP-ESI) m/z
calcd for C23H19N4O6S [M + H]+: 479.1025; found: 479.1004.
4-((6-((2-aminophenyl)amino)-6-oxohexyl)oxy)-3-(phenylsulfonyl)-
1,2,5-oxadiazole 2-oxide (7c). White solid. 35% yield. Mp: 129-131℃.
1H NMR (400 MHz, DMSO‑d6) δ 9.36 (s, 1H), 8.01 (d, J = 7.9 Hz, 2H),
7.89 (t, J = 7.4 Hz, 1H), 7.74 (t, J = 7.7 Hz, 2H), 7.21 (d, J = 7.9 Hz,
1H), 6.97 (t, J = 7.5 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.69 (t,
J = 7.5 Hz, 1H), 4.43–4.39 (m, 2H), 2.37 (t, J = 7.5 Hz, 2H), 1.85–1.74
(m, 2H), 1.70–1.62 (m, 2H), 1.46–1.39 (m, 2H). 13C NMR (101 MHz,
DMSO) δ 171.75, 159.36, 137.65, 136.61, 130.52, 128.77, 126.32,
125.93, 125.88, 119.59, 118.33, 110.92, 71.83, 36.01, 28.17, 25.23,
25.15. HRMS (AP-ESI) m/z calcd for C20H23N4O6S [M + H]+:
447.1338; found: 447.1328 .
4.2. In vitro HDACs inhibition fluorescence assay
10 μL of enzyme solution (HeLa cell nuclear extract, HDAC1,
HDAC2, HDAC3 or HDAC6) was mixed with different concentrations of
tested compound (50 μL). The mixture was incubated at 37 °C for 5
mins, followed by adding 40 μL fluorogenic substrate (Boc-Lys(acetyl)-
AMC for HeLa cell nuclear extract, HDAC1, HDAC2, HDAC3 and
HDAC6, Boc-Lys(trifluoroacetyl)-AMC for HDAC4). After incubation at
37 °C for 30 mins, the mixture was quenched by addition of 100 μL of
developer containing trypsin and Trichostatin A. Over another in-
cubation at 37 °C for 20 min, fluorescence intensity was measured using
a microplate reader at excitation and emission wavelengths of 390 and
460 nm, respectively. The inhibition ratios were calculated from the
fluorescence intensity readout of tested wells relative to those of control
wells, and the IC50 values were calculated using Prism non-linear curve
fitting method.
4-((7-((2-aminophenyl)amino)-7-oxoheptyl)oxy)-3-(phe-
nylsulfonyl)-1,2,5-oxadiazole 2-oxide (7d). White solid. 42% yield. Mp:
137-139℃. 1H NMR (400 MHz, DMSO‑d6) δ 9.18 (s, 1H), 8.02 (d,
J = 7.8 Hz, 2H), 7.90 (t, J = 7.4 Hz, 1H), 7.75 (t, J = 7.6 Hz, 2H), 7.17
(d, J = 7.8 Hz, 1H), 6.91 (t, J = 7.6 Hz, 1H), 6.75 (d, J = 8.0 Hz, 1H),
6.57 (t, J = 7.6 Hz, 1H), 4.39 (t, J = 6.3 Hz, 2H), 2.34 (t, J = 7.5 Hz,
2H), 1.80–1.73 (m, 2H), 1.66–1.58 (m, 2H), 1.38 (d, J = 7.0 Hz, 4H).
13C NMR (101 MHz, DMSO‑d6) δ 171.65, 159.34, 141.41, 137.67,
136.60, 130.50, 128.76, 126.19, 125.77, 124.48, 117.34, 116.84,
110.90, 71.87, 36.13, 28.62, 28.21, 25.64, 25.30. HRMS (AP-ESI) m/z
calcd for C21H25N4O6S [M + H]+: 461.1495; found: 461.1489
4-((8-((2-aminophenyl)amino)-8-oxooctyl)oxy)-3-(phenylsulfonyl)-
1,2,5-oxadiazole 2-oxide (7e). White solid. 47% yield. Mp: 127-129℃.
1H NMR (400 MHz, DMSO‑d6) δ 9.10 (s, 1H), 8.05–7.97 (m, 2H),
7.93–7.83 (m, 1H), 7.78–7.69 (m, 2H), 7.15 (dd, J = 7.9, 1.5 Hz, 1H),
6.89 (td, J = 7.6, 1.6 Hz, 1H), 6.71 (dd, J = 8.0, 1.5 Hz, 1H), 6.53 (td,
J = 7.5, 1.5 Hz, 1H), 4.82 (s, 2H), 4.39 (t, J = 6.3 Hz, 2H), 2.33 (t,
J = 7.4 Hz, 2H), 1.75 (t, J = 6.6 Hz, 2H), 1.65–1.57 (m, 2H), 1.38–1.33
(m, 6H). 13C NMR (101 MHz, DMSO‑d6) δ 171.62, 159.35, 142.32,
4.3. In vitro NO measurement
The in vitro NO measurement was tested using the Griess method
[24]. The level of NO generated by individual compound in the cells
was presented as that of nitrite and determined by the colorimetric
assay using the colorimetric assay kit (purchased from Beyotime,
China), according to the manufacturer’s instruction. Briefly, HeLa cells
(5 × 105/well) were treated with a 10 μM of tested compounds for 1 h
or 5 h. Subsequently, the cells were harvested, and their cell lysates
were prepared and then mixed with Griess for 10 min at 37℃, followed
by measurement at 540 nm by an enzyme-linked immunosorbent assay
plate reader. The cells treated with DMSO were used as negative
6

Q. Ding, et al.
BioorganicChemistry104(2020)104235
controls for the background level of nitrite production, while sodium
nitrite at different concentrations was prepared for the standard curve.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
4.4. Western blot analysis
For regular western blot analysis, the HeLa cells were treated with
MS275 (2 μM), 7c (2 μM) or DMSO for 5 h. For western blot analysis to
investigate if NO generated by 7c contributed to its intracellular HDAC
inhibition, the HeLa cells were preincubated with 10 μM of NAC, then
were treated with 2 μM of MS275 or 7c for 5 h. After treatment, the
cells were washed twice with cold PBS and lysed in ice-cold RIPA
buffer. Lysates were cleared by centrifugation. Protein concentrations
were determined using the BCA assay. Equal amounts of cell extracts
were then resolved by SDS-PAGE, transferred to nitrocellulose mem-
branes and probed with ac-histone H4 antibody, ac-α-tubulin antibody
and β-actin antibody, respectively. Blots were detected using an en-
hanced chemiluminescence system.
Acknowledgement
This work was supported by Natural Science Foundation of
Shandong Province (Grant No. ZR2018QH007), Key Research and
Development Program of Shandong Province (2017CXGC1401), Young
Scholars Program of Shandong University (YSPSDU, 2016WLJH33).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104235.
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