Design, synthesis, and antitumor evaluation of novel histone deacetylase inhibitors equipped with a phenylsulfonylfuroxan module as a nitric oxide donor

Article abstract of DOI:10.1021/acs.jmedchem.5b00317

On the basis of the strategy of creating multifunctional drugs, a novel series of phenylsulfonylfuroxan-based hydroxamates with histone deacetylase (HDAC) inhibitory and nitric oxide (NO) donating activities were designed, synthesized, and evaluated. The

Full text of DOI:10.1021/acs.jmedchem.5b00317

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Article
Design, Synthesis and Antitumor Evaluation of Novel
Histone Deacetylase (HDAC) Inhibitors Equipped with
Phenylsulfonylfuroxan Module as Nitric Oxide (NO) Donor
Wenwen Duan, Jin Li, Elizabeth S. Inks, C. James Chou, Yuping
Jia, Xiaojing Chu, Xiaoyang Li, Wenfang Xu, and Yingjie Zhang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00317 • Publication Date (Web): 23 Apr 2015
Downloaded from http://pubs.acs.org on May 3, 2015
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Design, Synthesis and Antitumor Evaluation of
Novel Histone Deacetylase (HDAC) Inhibitors
Equipped with Phenylsulfonylfuroxan Module as
Nitric Oxide (NO) Donor
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‡
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Wenwen Duan, Jin Li, Elizabeth S. Inks, C. James Chou, Yuping Jia, Xiaojing Chu,
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Xiaoyang Li, Wenfang Xu and Yingjie Zhang
†
Departments of Medicinal Chemistry, School of Pharmacy, Shandong University, Ji'nan,
Shandong, 250012, People's Republic of China
‡
Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy,
Medical University of South Carolina, Charleston, South Carolina 29425, United States
§
Shandong Academy of pharmaceutical Sciences, Ji’nan, Shandong, 250101, People's Republic
of China
‖
Weifang Bochuang International Biological Medicinal Institute, Weifang, Shandong, 261061 ,
People's Republic of China
ABSTRACT: Based on the strategy of “multifunctional drugs,” a novel series of
phenylsulfonylfuroxan-based hydroxamates with histone deacetylase (HDAC) inhibitory and
nitric oxide (NO) donating activities were designed, synthesized, and evaluated. The most potent
NO donor-HDAC inhibitor (HDACI) hybrid 5c exhibited much more potent in vitro
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antiproliferative activity against human erythroleukemia (HEL) cell line than the approved drug
SAHA (Vorinostat), and its antiproliferative activity was diminished by the NO scavenger
hemoglobin in a dose-dependent manner. Further mechanism studies revealed that 5c strongly
induced cellular apoptosis and G1 phase arrest in HEL cells. Animal experiment identified 5c as
an orally active agent with potent antitumor activity in a HEL cell xenograft model. Interestingly,
although compound 5c was remarkably HDAC6 selective in the molecular level, it exhibited
pan-HDAC inhibition in the western blot assay likely due to NO release causing class I HDACs
inhibition in the cellular level.
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
Introduction
The important role of histone deacetylation in gene expression and regulation, especially in
the pathogenesis of cancer has been reported by numerous researchers since the 1960s. Histone
deacetylases (HDACs) are a family of enzymes, which catalyze acetyl-group removal from the
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lysine residue in the histone tails and lead to a transcriptionally repressed chromatin state.
Abnormal HDAC activity has been found to be associated with the aberrant gene expression and
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the development of several kinds of cancer and other human ailments. Accordingly, HDAC
inhibition restores normal gene expression profile resulting in cancer cell cycle arrest, cell
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differentiation, and apoptosis. Thus, HDAC inhibitors (HDACIs, Figure 1),
which block
abnormal HDAC deacetylation have been recently developed and validated as potential
anticancer agents, such as hydroxamic acids, short chain fatty acids, cyclic tetrapeptide, and
benzamides. Among these HDACIs, hydroxamic acids have been the most well-known ones with
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SAHA (6, Figure 1) , PXD-101(7, Figure 1) and LBH-589(8, Figure 1) approved by the US Food
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and Drug Administration (FDA) in October 2006, July 2014 and February 2015 for the cancers
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respectively in the clinic. Many other hydroxamate compounds are in the clinical trials, such as
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SB-939 (9, phase II, Figure 1), and 4SC-201(10, phase II, Figure 1).
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Cellular Nitric Oxide (NO), described in 1980 by Forchgott, participates in the vascular
regulation, nerve transmission delivery, inflammation, and immune response process as an
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important massager molecule in organism.
NO can also inhibit tumor cells proliferation,
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angiogenesis, metastasis, and accelerate tumor cell apoptosis. In addition to the inducible
nitric oxide synthase (iNOS), which can produce a heavy dose of cellular NO by stimulating
factors such as cytokins, chemical NO donor is also an effective way to generate a high
concentration of cellular NO. It was reported that glyceryl trinitrate (GTN) can inhibit the
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proliferation of tumor cells P388 and L-1210 in vitro and in vivo. Sodium Nitroprusside (SNP)
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was reported to exhibit potential cytotoxicity to the leukemia cells of ML, AML, and CMMOL.
Oxadiazole (22, Figure2) was found in 2008 through a high throughput screen to be an important
and potential NO donor, which could produce high levels of NO in vitro and inhibit the growth
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of tumor in vivo.
Phenylsulfonylfuroxan (23, Figure 2), aclassical type of oxadiazole, is
stable in acidic and basic conditions, and its mechanism of NO release in vivo was determined to
be through its reaction with mercapto-compound such as cysteine as described by Feelisch in
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992. It can also release NO to produce activity in variety of tissues and organs through a
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non-enzymatic pathway. Compounds like phenylsulfonylfuroxan coupled with oleanolic acid,
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farnesylthiosalicylic acid, or anilinopyrimidine have displayed synergistic anti-tumor activity.
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Over the past decades, an increasing body of research reported that covalent modifications,
such as S-nitrosylaion or tyrosine-nitration of proteins by NO can dramatically influenced their
cellular functions. Interestingly, many HDAC family members have also been found to be direct
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or indirect targets of NO, and several reports have illustrated that NO-dependent regulation of
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HDAC functions. HDAC family consists of eighteen isoforms belonging to four structurally
and functionally different phylogenetic classes: class I (HDAC 1, 2, 3, and HDAC 8), class II
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class IIa: HDAC 4, HDAC 5, HDAC 7, HDAC 9; class IIb: HDAC 6 and HDAC 10) and class
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IV (HDAC 11) are called “classical HDACs” and are Zn dependent proteases. While the class
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III (SIRT 1~7) are NAD dependent. The enzymatic activity of class I enzyme HDAC 2 and
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HDAC 8 have been reported to be directly inhibited by NO-dependent S-nitrosylation.
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S-nitrosylation of HDAC2 induces its release from chromatin. The nuclear shuttling of class IIa
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HDAC is also induced by NO through sGC-cGMP pathway. Furthermore, NO have been
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shown to allow crosstalk between class III and class I HDACs. In fact, some studies have
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shown that HDACI and NO are synergistic in cardiac hypertrophy and wound healing.
“Multifunctional drugs” through the hybridization strategy is a well-developed approach in
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drug design process. It involves the combination of two complementary or synergistic
pharmacophores directly or via a spacer in order to act on different targets to improve the activity.
Recently, a hybrid (NO-MS275, 25, Figure 3) of NO donor and HDACI MS275 (24, Figure 3)
was reported to promote myogenic differentiation in a more efficient manner than the 1:1
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mixture of the two components, however no antitumor activity of this compound was reported.
Based on the above mentioned analysis and the “multifunctional drugs” theory, the goal of this
study is to develop novel HDACI containing the NO donor motif (Figure 4) that exhibits
addictive antitumor activity via HDAC inhibition and NO release.
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
Result and discussion
Chemistry
Based on HDACI pharmacophore exemplified by SAHA (Figure 3), a series of novel target
compounds containing phenylsulfonylfuroxan as NO donating module were designed and
synthesized. The target compounds 5a-5o were synthesized from (phenylthio)acetic acid
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purchased from Adamas-beta, China) as shown in Scheme 1. The compounds 2 and 3 were
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synthesized following the method of Zhang’s. One-pot reaction of (phenylthio)acetic acid by
oxidization with 30% H
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adding fuming HNO
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without purification to get compound 2. After nucleophilic substitution by
several corresponding linkers with two hydroxyl groups, we synthesized various
monophenylsulfonylfuroxans (compounds 3a-3n), which were oxidized under Jones agent to get
compounds 4a-4n. The target compounds 5a-5n were obtained from 4a-4n under the conditions
of isobutyl chlorocarbonate, triethylamine, and hydroxylamine hydrochloride. Compound 3j was
also oxidized by PCC to make compound 6, which was reacted with propandioic accid to get
compound 7, and then transformed to the target hydroxamate 5o.
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Biological Results
Inhibiton of Hela Cell Extracts by the Target Compounds.
Based on the fact that all zinc ion dependent HDACs were highly conserved in their active
sites, all target compounds were evaluated for their in vitro HDACs inhibitory activities using
Hela cell extracts (containing primarily HDAC1 and HDAC2) as enzyme source and SAHA as
positive control. The result listed in Table 1 revealed that the IC50 values of the compounds 5a
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0.047 μM), 5c (0.038 μM), 5d (0.054 μM), 5e (0.063 μM) and 5j (0.047 μM) were significantly
lower than that of SAHA. This result revealed that the compounds containing the saturated fatty
linker without any branched chains were more potent compounds than other linkers. When the
linker was substituted with aromatic ring such as benzene, the IC50 values of those without
substitutes were lower than analogs containing substitutes, and the hydroximic acid group was
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best be located in the para-position to the phenylsulfonyllfuroxan. Generally, compounds
containing saturated fatty linkers without any branched chains exhibited much more potency in
HDAC inhibitory activities and the optimal length of the linker should be six to eight carbons.
Our results also showed that compounds 2 and 3c were not active against Hela cell nuclear
extract, validating the crucial role of hydroxamate group in HDAC inhibition.
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In Vitro Antiproliferative Assay.
Antiproliferative activities of target compounds 5a-5o and 3c, 2, SAHA with 2 (1:1) were
tested against HEL (human erythrocyte leukemia cell), HCT-116 (human colorectal carcinoma
cell), Hela (human cervical carcinoma cell), U937 (Human Histiocytic leukemia cell line), 3-AO
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human oophoroma cell), MDA (human breast cancer cell), ES-2 (Ovarian clear cell carcinoma
cell), KG1 (human leukemia cell) cell lines by MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay, using SAHA as
the positive control (Table 2). From the result listed in Table 2, it was found that the inhibitory
activity against tumor cell growth of most of these compounds were better than the clinically
approved drug SAHA, especially 5a, 5b, 5c and 5j, which exhibited higher HDAC enzyme
inhibitory potency and with improved antiproliferative ability against cancerous cells. It should
be noted that NO donors 2 and 3c were also effective at againsting HEL cells proliferation and
combination of SAHA and NO donor 2 with mole ratio of 1:1 exhibited dramatic additive effect.
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In addition, cytotoxicity of 5c against normal primary HUVEC (human umbilical vein
endothelial cells) was tested in our lab, and the IC50 value was 27.1 μM, which revealed our
compounds’ selectivity over non-transformed cells when compared to tumor cells. Encouraged
by these results, compound 5c with the highest Hela nuclear extracts inhibitory potency and the
best antiproliferative activity, was progressed to further in vitro evaluation using the most
sensitive HEL cell line as cellular models.
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NO Generation Measurement.
Regarding the effect of NO generation, the levels of NO generated by the tested compounds
in vitro were detected. HEL cells were respectively exposed to 100 μM of 5a, 5b, 5c, 5j, 2, and
SAHA in the same incubation time (3 hours or 5 hours). The levels of NO released in the cell
lysates were determined using a Griess assay. In Figure 5 we found that the compounds we
designed and synthesized could release NO effectively, in contrast, SAHA hardly released any
NO in HEL. Compound 5c, which exhibited the most potent activity against HDACs and tumor
cells, also released the most NO among these tested compounds.
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In Vitro Antiproliferative Assay with Hemoglobin.
In order to verify that the proliferation of tumor cells were also inhibited by NO generated
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from our compounds, we further examined the anti-proliferation effects of 5c in the presence or
absence of NO scavenger, hemoglobin (Hb). HEL cells were pretreated with the indicated
concentrations of NO scavenger hemoglobin (0, 2.5, 5, 10 and 20 μM) for 1 hour and then
treated by 2 μM of 5c for 24 hours. The results were expressed as the percentage of growth
inhibition relative to the control cells in Figure 6. It was observed that 5c remarkably inhibited
the growth of HEL cells and this inhibitory effect was diminished by pretreatment with
hemoglobin in a dose-dependent manner. These results demonstrated that NO produced by 5c
contributed in part to its inhibition of tumor cell proliferation, which validated our compound
design strategy.
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Induction of Apoptosis in Vitro.
As the fact that high levels of NO act as a tumor cell apoptosis inducer, the ability of 5c as
NO donor to induce tumor cell apoptosis was tested and compared with that of SAHA. HEL cells
were treated with a variable level of 5c, SAHA and 2 for 24 h. The cells were harvested and
stained with 7-aminoactinomycin D (7-AAD) and annexin-V, and the percentage of apoptotic
cells were determined by flow cytometry analysis. Notably, it was found that the ability of 5c to
induce apoptosis was stronger than that of SAHA in HEL cells (Figure 7, A), and the apoptosis
rate was in a dose-dependent manner (Figure 7, B).
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Cell Cycle Arrest Analysis.
The effect of 5c on the various phases of cell cycle progression was tested in HEL cells
Figure 8). In comparison to the control population, the cell cycle data clearly showed 5c arrested
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In Vivo Antitumor Activity.
Compound 5c was further evaluated for its in vivo antitumor efficacy in HEL xenograft
mouse model. Compound 5c (100 mg/kg/day, 120 mg/kg/day) and SAHA (120 mg/kg/day) were
dosed orally by gavage for 21 consecutive days. Tumor growth inhibition (TGI) and relative
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increment ratio (T⁄C) were calculated as described previously at the end of treatment to reveal
the antitumor effects in tumor weight and tumor volume, respectively.
In the HEL xenograft model, compound 5c exhibited dose-dependent antitumor activity, and
at the same dosage of 120 mg/kg/day, compound 5c demonstrated superior antitumor activity
(TGI = 48%, T⁄C = 39%) to that of the reference compound SAHA (TGI = 39%, T/C = 46%).
The tumor growth curve depicted in Figure 9 and the final tumor tissue size visualized in Figure
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higher dose of 5c (120 mg/kg) showed no significant body weight loss (Figure 11) and no
evidence of liver or spleen toxicity.
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In Vitro HDAC Isoform Selectivity of 5c.
In order to explore its HDAC isoform selective profile, 5c was tested for its inhibitory
activity of HDAC1, HDAC2, HDAC3, HDAC4, HDAC6, HDAC8 and HDAC 11 (Table 4).
From the results we found that at the molecular level, compound 5c exhibited remarkable
selectivity for HDAC6 over other isoforms.
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Western Blot Analysis.
In order to investigate its HDAC isoform selectivity in cellular environment, compound 5c
was further evaluated in western blot assays to determine acetylated protein levels (Figure 12).
The results in Figure 12A showed that at its very low concentration of 12.5 nM, compound 5c
still could dramatically increase cellular level of ac-tubulin, the substrate of HDAC6, which was
in line with its highly potent HDAC6 inhibitory activity (IC50 of 7 nM). However, 5c could also
effectively increase cellular levels of AcHH3 and AcHH4, which were the main nuclear
substrates of HDAC1 and HDAC2. This is not very surprising because 5c could release NO
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effectively and some research showed that NO not only could inhibit the enzyme activity and
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chromatin binding of HDAC2 by S-nitrosylation, but also could minimally inhibit HDAC1 by
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an S-nitrosylation-independent mechanism, Moreover, our results also showed that 5c could
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decrease the cellular level of HDAC2 at higher concentrations (1 μM and 10 μM, Figure 12B),
which could also be attributed to release NO because the NO scavenger PTIO could recover the
cellular level of HDAC2 (Figure 12C). Additional research to elucidate the mechanism on how
NO affecting cellular levels of HDAC2 is warranted.
(
Figure 12 should be listed here)
 CONCLUSION
In the present research, a novel series of compounds capable to simultaneously release NO
and inhibit HDACs were designed and synthesized for the treatment of cancer based on the
“
multifunctional drugs” approach. All synthesized compounds were evaluated for their HDAC
inhibitory potency against Hela cell nuclear extracts and for their antiproliferative effects
against several tumor cell lines. Several representative compounds were evaluated in HEL cell
line and displayed potent NO releasing activity with compound 5c being the most potent one.
Pretreatment of HEL cells with NO scavenger hemoglobin could moderately reduce the
antiproliferative activity of 5c demonstrated the additive effect between NO release and HDAC
inhibition. Further mechanistic studies revealed that 5c induced a much stronger apoptotic effect
and G1 phase arrest in HEL cells than SAHA, which was in line with their abilities to inhibit
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HDAC and to generate NO. Because of its very promising in vitro activity, compound 5c was
progressed to a HEL xenograft model and exhibited greater oral antitumor potency than SAHA
in a dose-dependent manner. It is worth noting that compound 5c was a remarkably HDAC6
selective inhibitor in the molecular level, which could be used as a design template for HDAC6
selective inhibitors. Taken together, compound 5c, a very potent HDAC inhibitor with NO
releasing activity was discovered, which deserves further research and development as a
promising therapeutic agent for hematologic malignancies.
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
EXPERIMENTAL SECTION
Chemistry Materials and Methods. All commercially available starting materials, reagents,
and solvents were used without further purification unless otherwise stated. All reactions were
monitored by TLC with 0.25 mm silica gel plates (60GF-254). UV light, iodine stain, and ferric
chloride were used to visualize the spots. Silica gel or C18 silica gel was used for column
1
13
chromatography purification. H NMR and C spectra were recorded on a Bruker DRX
spectrometer at 600 MHz, δ in parts per million and J in hertz, using TMS as an internal standard.
High-resolution mass spectra were conducted by Shandong Analysis and Test Center in Ji’nan,
China. ESI-MS spectra were recorded on an API 4000 spectrometer. Melting points were
determined uncorrected on an electrothermal melting point apparatus. All tested compounds
are >95% pure by HPLC analysis, performed on a Agilent 1100 HPLC instrument using an 5 μm
ODS HYPERSIL column (4.6 mm×250 mm) according to the following methods. All target
compounds were eluted with 35% acetonitrile/65% water (containing 0.1% acetic acid) over 20
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min, with detection at 254 nm and a flow rate of 1.0 mL/min.
Synthesis of compound 2 (3,4-bis(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide):
To a solution of (phenylthio)acetic acid (10.0 g，55.0 mmol ) in glacial acetic acid (16 mL),
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aqueous H
2
2
O (30%, 13.6 mL) was added at 0℃, after 1.0 h of stirring at room temperature, the
mixture was continued stirred at 80℃ for 3 h after additional of fuming nitric acid (24 mL)
controlling the inner temperature lower than 20℃. The reaction was checked by TLC and the
mixture was cooled after it reacted completely, and then the compound 2 was collected by
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filtration and dry under vacuum as a white solid (Yield: 56%). H NMR (400 MHz, CDCl ) δ
3
8.20 (t, J = 7.4 Hz, 4H), 7.83 (dd, J = 14.3, 7.3 Hz, 2H), 7.69 (dt, J = 16.1, 8.2 Hz,4H).
General procedure for the preparation of compounds (3a-3o)
4
-(4-hydroxybutoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (3a): To a solution of
compound 2 (1.1 g, 2.7 mmol) in 10 mL THF 1,4-Butanediol was added (1.2 mL, 11.4 mmol) at
approximately 5℃, followed by dropwise addition of 2.5 N aqueous NaOH (1 mL) followed by
3
0 min stirring. The solution was concentrated under vacuum at room temperature. The residue
was extracted with EtOAc three times (3×50 mL) and washed twice with water and brine. The
combined organic layers were dried by anhydrous Na SO , and evaporated under vacuum to give
a residue that was purified by silica-gel column chromatography tresulting in a white solid
2
4
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31
product. (Yield:71%). H NMR (400 MHz, CDCl
3
) δ 8.05 (d, J = 7.7 Hz, 2H), 7.76 (t, J = 7.3
Hz, 1H), 7.62 (t, J = 7.6 Hz, 2H), 4.47 (t, J = 6.1 Hz, 2H), 3.76 (t, J = 6.1 Hz, 2H), 2.07 – 1.91
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(
m, 2H), 1.82 – 1.71 (m, 2H), 1.62 (s, 1H).
4-((5-hydroxypentyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (3b): white solid. (Yield:
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59
60
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6
7
6%). H NMR (600 MHz, d
6
-DMSO) δ 8.02 (d, J = 7.7 Hz, 2H), 7.91 (t, J = 7.4 Hz, 1H),
.76 (t, J = 7.5 Hz, 2H), 4.39 (t, J = 6.2 Hz, 2H), 3.42 (t, J = 6.2 Hz, 2H), 1.79 – 1.72 (m, 2H),
1.50 – 1.44 (m, 2H), 1.42 – 1.37 (m, 2H).
4
7
7
1
-((6-hydroxyhexyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (3c): white solid. (Yield:
1
32
3%). H NMR (600 MHz, d -DMSO) δ 8.02 (d, J = 7.7 Hz, 2H), 7.91 (t, J = 7.3 Hz, 1H),
6
.76 (t, J = 7.8 Hz, 2H), 4.38 (t, J = 6.3 Hz, 2H), 3.98 (t, J = 6.7 Hz, 2H), 1.79 – 1.71 (m, 2H),
.58 – 1.52 (m, 2H), 1.35 (d, J = 3.1 Hz, 2H), 1.29 (d, J = 3.1 Hz, 2H).
4
-((7-hydroxyheptyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (3d): white solid. (Yield:
1
6
6%). H NMR (600 MHz, CDCl
3
) δ 8.05 (d, J = 7.8 Hz, 2H), 7.74 (t, J = 7.5 Hz, 1H), 7.61 (t,
J = 7.7 Hz, 2H), 4.42 (t, J = 6.5 Hz, 2H), 3.66 (t, J = 6.6 Hz, 2H), 1.91 – 1.84 (m, 2H), 1.59 (dd,
1
3
J = 13.0, 6.5 Hz, 2H), 1.50 – 1.46 (m, 2H), 1.42 (dd, J = 6.5, 3.5 Hz, 4H). C NMR (151 MHz,
CDCl
3
) δ 159.2, 138.4, 135.7, 129.8, 129.3, 128.7, 128.0, 110.6, 71.7, 63.0, 32.7, 29.0, 28.4, 25.7,
+
25.7. ESI-MS [M+H] m/z: 357.1.
4-((8-hydroxyoctyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (3e): white solid. (Yield:
1
7
1%). H NMR (600 MHz, CDCl
3
) δ 8.04 (d, J = 7.8 Hz, 2H), 7.75 (t, J = 7.5 Hz, 1H), 7.61 (t, J
=
7.6 Hz, 2H), 4.40 (t, J = 6.5 Hz, 2H), 3.62 (s, 2H), 1.88 – 1.82 (m, 2H), 1.65 – 1.49 (m, 8H),
1
3
1.44 (dd, J = 13.6, 6.4 Hz, 2H). C NMR (151 MHz, CDCl ) δ 159.1, 138.1, 135.6, 129.6, 129.2,
3
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28.5, 127.8, 110.5, 71.6, 62.9, 32.7, 29.2, 29.0, 28.4, 25.6, 25.6, 25.5. ESI-MS [M+H] m/z:
371.1.
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-(2-(2-hydroxyethoxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (3f): white solid.
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(
Yield: 56%). H NMR (400 MHz, CDCl
3
)
δ 8.06 (d, J = 7.7 Hz, 2H), 7.75 (t, J = 7.5 Hz, 1H),
7.61 (t, J = 7.8 Hz, 2H), 4.62 – 4.51 (m, 2H), 3.97 – 3.89 (m, 2H), 3.81 – 3.74 (m, 2H), 3.72 –
3
.67 (m, 2H).
4
-(3-hydroxy-2,2-dimethylpropoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (3g): white
1
solid. (Yield: 80%). H NMR (400 MHz, CDCl
3
) δ 8.04 (d, J = 8.0 Hz, 2H), 7.75 (t, J = 7.1 Hz,
13
1H), 7.61 (t, J = 7.7 Hz, 2H), 4.23 (s, 2H), 3.55 (s, 2H), 1.05 (s, 6H). C NMR (400 MHz,
+
CDCl
3
) δ 153.5, 137.4, 132.6, 132.1, 127.6, 76.7, 69.6, 37.1, 19.4. ESI-MS [M+H] m/z: 329.1.
(
E)-4-((4-hydroxybut-2-en-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (3h): white
1
solid. (Yield: 70%). H NMR (600 MHz, CDCl ) δ 8.12 – 8.02 (m, 2H), 7.96 – 7.87 (m, 1H),
3
7.81 – 7.69 (m, 2H), 5.98 – 5.77 (m, 2H), 4.68 (dd, J = 11.5, 1.0 Hz, 2H), 4.23 – 4.11 (m, 2H).
13
C NMR (400 MHz, CDCl ) δ 155.5, 137.4, 132.6, 132.1, 131.7, 128.1, 127.6, 69.6, 63.3.
3
+
ESI-MS [M+H] m/z: 313.0.
4
-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (3i):
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white solid. (Yield:58%). H NMR (600 MHz, CDCl ) δ 8.03 (d, J = 7.6 Hz, 2H), 7.90 (d, J =
3
7
2
.5 Hz, 1H), 7.76 (t, J = 7.9 Hz, 2H), 4.53 – 4.50 (m, 2H), 3.81 – 3.79 (m, 2H), 3.64 – 3.61 (m,
H), 3.57 – 3.54 (m, 2H), 3.49 (d, J = 5.3 Hz, 2H), 3.43 (d, J = 5.0 Hz, 2H).
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-(4-(hydroxymethyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (3j): white solid.
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(Yield: 68%). H NMR (600 MHz, d
H), 7.78 (t, J = 7.6 Hz, 2H), 7.43 (d, J = 8.3 Hz, 2H), 7.37 (d, J = 8.3 Hz, 2H), 5.27 (t, J = 5.7
Hz, 1H).
6
-DMSO) δ 8.06 (d, J = 7.9 Hz, 2H), 7.93 (t, J = 7.3 Hz,
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-(3-(hydroxymethyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (3k): white solid.
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(
Yield: 74%). H NMR (600 MHz, d -DMSO) δ 8.05 (d, J = 7.7 Hz, 2H), 7.91 (t, J = 7.5 Hz,
6
1
2
H), 7.77 (t, J = 7.8 Hz, 2H), 7.43 (t, J = 7.9 Hz, 1H), 7.35 (s, 1H), 7.27 (dd, J = 11.4, 9.1 Hz,
H), 5.27 (t, J = 5.5 Hz, 1H), 4.54 (d, J = 5.1 Hz, 2H).
4-(4-(hydroxymethyl)-2-methoxyphenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (3l):
1
white solid. (Yield: 67%). H NMR (600 MHz, CDCl
3
) δ 8.09 (d, J = 8.2 Hz, 2H), 7.95 (t, J =
7
.4 Hz, 1H), 7.81 (t, J = 7.7 Hz, 2H), 7.36 (d, J = 8.2 Hz, 1H), 7.17 (s, 1H), 6.98 (d, J = 8.2 Hz,
1
3
1H), 5.31 (d, J = 5.7 Hz, 1H), 4.53 (d, J = 5.8 Hz, 2H), 3.72 (s, 3H). C NMR (400 MHz, CDCl )
3
+
δ 155.3, 150.9, 137.4, 136.6, 132.6, 132.1, 128.2, 127.6, 120.7, 63.2, 39.4. ESI-MS [M+H] m/z:
3
79.1.
4
-(4-(2-hydroxyethyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (3m): colorless oil.
1
(Yield: 61%). H NMR (600 MHz, CDCl
3
) δ 8.06 (d, J = 8.1 Hz, 2H), 7.92 (t, J = 7.4 Hz, 1H),
7
.77 (t, J = 7.6 Hz, 2H), 7.32 (q, J = 8.6 Hz, 4H), 4.66 (t, J = 5.1 Hz, 1H), 3.64 (dd, J = 12.4, 6.3
13
Hz, 2H), 2.76 (t, J = 6.8 Hz, 2H). C NMR (400 MHz, CDCl ) δ 154.3, 137.4, 132.6, 132.1,
3
+
127.6, 72.6, 70.4, 70.0, 66.6, 61.0. ESI-MS [M+H] m/z: 363.1.
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-((2-(hydroxymethyl)-2-methylpentyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (3n):
1
colorless oil. (Yield: 63%). H NMR (600 MHz, CDCl
3
) δ 8.08 – 8.01 (m, 2H), 7.95 – 7.86 (m,
1
1
H), 7.76 – 7.68 (m, 2H), 4.27 (dd, J = 172.0, 24.7 Hz, 2H), 3.42 (dd, J = 127.1, 24.7 Hz, 2H),
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1
3
.49 (s, 1H), 1.45 – 1.33 (m, 4H), 0.95 (d, J = 8.4 Hz, 3H), 0.91 – 0.83 (m, 3H). C NMR (400
MHz, CDCl
3
) δ 153.5, 137.4, 132.6, 132.1, 127.6, 73.7, 69.9, 38.8, 34.1, 20.3 17.1, 14.7.
+
ESI-MS [M+H] m/z: 357.1.
General procedure for the preparation of compounds (4a-4n)
4
-(3-carboxypropoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (4a): To a solution of 3(680
mg，2.2 mmol) in 10 mL acetone, Jones reagent (1.1 mL) was added at 0-5℃.This mixture was
stirred at r.t. for 10 h. The precipitate was filtered off and acetone was removed in vacuo, and
then EtOAc (3×50 mL) was added,the organic portion was washed with water and brine, and
dried by anhydrous Na
2
4
SO . The crude product was obtained by removing EtOAc and purified
1
by silica chromatography column (P:E=3:1) to get the product. white solid. (Yield: 81%). H
NMR (400 MHz, CDCl
2H), 4.50 (t, J = 6.0 Hz, 2H), 2.60 (t, J = 7.0 Hz, 2H), 2.26 – 2.19 (m, 2H), 2.17 (s, 1H). C
NMR (400 MHz, CDCl ) δ 177.2, 154.3, 137.4, 132.6, 132.1, 127.6, 68.0, 31.8, 24.3. ESI-MS
3
) δ 8.05 (d, J = 7.6 Hz, 2H), 7.76 (t, J = 7.4 Hz, 1H), 7.62 (t, J = 7.8 Hz,
13
3
+
[
M+H] m/z: 329.0.
4
-(4-carboxybutoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (4b): white solid. (Yield:
1
80%). H NMR (600 MHz, CDCl
3
) δ 12.09 (s, 1H), 8.02 (d, J = 8.0 Hz, 2H), 7.90 (t, J = 7.4 Hz,
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H), 7.75 (t, J = 7.6 Hz, 2H), 4.40 (t, J = 6.0 Hz, 2H), 2.30 (t, J = 7.3 Hz, 2H), 1.82 – 1.74 (m,
1
3
2H), 1.62 (t, J = 6.4 Hz, 2H). C NMR (400 MHz, CDCl ) δ 177.2, 154.3, 137.4, 132.6, 132.1,
3
+
1
27.6, 69.3, 34.5, 28.8, 22.1. ESI-MS [M+H] m/z: 343.0.
10
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-((5-carboxypentyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (4c): white solid. (Yield:
1
73%). H NMR (600 MHz, d
6
-DMSO) δ 12.04 (s, 1H), 8.04 – 8.00 (m, 2H), 7.91 (t, J = 7.4 Hz,
1
2
1
H), 7.76 (t, J = 7.9 Hz, 2H), 4.38 (t, J = 6.2 Hz, 2H), 2.24 (t, J = 7.3 Hz, 2H), 1.79 – 1.71 (m,
13
H), 1.58 – 1.54 (m, 2H), 1.38 (dd, J = 15.3, 8.0 Hz, 2H). C NMR (400 MHz, CDCl
3
) δ 177.2,
+
54.3, 137.4, 132.6, 132.1, 127.6, 69.3, 34.5, 29.3, 25.9, 24.9. ESI-MS [M+H] m/z: 357.1.
4-((6-carboxyhexyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (4d): white solid. (Yield:
1
8
1
3%). H NMR (600 MHz, CDCl
3
) δ 11.58 (s, 1H), 8.07 (d, J = 8.0 Hz, 2H), 7.78 (t, J = 7.5 Hz,
H), 7.64 (t, J = 7.8 Hz, 2H), 4.43 (t, J = 6.4 Hz, 2H), 2.38 (d, J = 7.4 Hz, 2H), 1.94 – 1.85 (m,
1
3
2H), 1.69 (dd, J = 14.8, 7.3 Hz, 4H), 1.48 – 1.43 (m, 2H). C NMR (400 MHz, CDCl
3
) δ 180.0,
1
59.0, 138.1, 135.6, 129.7, 129.2, 128.5, 127.8, 110.5, 71.4, 33.8, 28.5, 28.2, 25.3, 24.2. ESI-MS
+
[
M+H] m/z: 371.1.
4
-((7-carboxyheptyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (4e): white solid. (Yield:
1
79%). H NMR (600 MHz, CDCl
3
) δ 8.06 (d, J = 7.9 Hz, 2H), 7.77 (t, J = 7.4 Hz, 1H), 7.62 (d, J
=
7.8 Hz, 2H), 4.42 (t, J = 6.5 Hz, 2H), 2.37 – 2.34 (m, 2H), 1.91 – 1.84 (m, 2H), 1.68 – 1.58 (m,
1
3
8
H). C NMR (151 MHz, CDCl
3
) δ 179.6, 159.0, 138.2, 135.5, 129.6, 129.2, 128.5, 127.8, 71.6,
+
33.9, 28.8, 28.7, 28.3, 25.4, 24.5. ESI-MS [M+H] m/z: 385.1.
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6
4
-(2-(carboxymethoxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (4f): white solid.
1
(Yield: 85%). H NMR (600 MHz, CDCl
3
) δ 8.07 (s, 2H), 7.74 (s, 1H), 7.61 (s, 2H), 4.61 (s,
) δ 173.8, 154.3, 137.4, 132.6, 132.1,
1
3
2
1
H), 4.28 (s, 2H), 4.02 (s, 2H). C NMR (400 MHz, CDCl
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
27.6, 69.8, 67.9, 66.6. ESI-MS [M+H] m/z:345.0.
4
-(2-carboxy-2-methylpropoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (4g): white solid.
1
(
Yield: 79%). H NMR (600 MHz, CDCl
3
) δ 8.12 – 7.95 (m, 2H), 7.79 – 7.70 (m, 1H), 7.60 (t, J
13
=
7.8 Hz, 2H), 4.43 (s, 2H), 1.41 (s, 6H). C NMR (400 MHz, CDCl
3
) δ 182.7, 153.5, 137.4,
+
1
32.6, 132.1, 127.6, 77.0, 43.3, 23.4. ESI-MS [M+H] m/z:343.0.
(E)-4-((3-carboxyallyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (4h): white solid.
1
(
Yield: 88%). H NMR (600 MHz, CDCl
3
) δ 12.80 (s, 1H), 8.04 (d, J = 8.1 Hz, 2H), 7.90 (t, J =
7
.4 Hz, 1H), 7.75 (t, J = 7.6 Hz, 2H), 6.54 – 6.47 (m, 1H), 5.97 (d, J = 11.6 Hz, 1H), 5.48 (dd, J
1
3
= 4.7, 2.2 Hz, 2H). C NMR (400 MHz, CDCl ) δ 171.8, 155.5, 149.5, 137.4, 132.6, 132.1,
3
+
1
27.6, 119.7, 68.3. ESI-MS [M+H] m/z:327.0.
4
-(2-(2-(carboxymethoxy)ethoxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (4i):
1
white solid. (Yield: 82%). H NMR (600 MHz, CDCl
3
) δ 8.10 – 8.00 (m, 2H), 7.98 – 7.88 (m,
1H), 7.78 – 7.70 (m, 2H), 4.33 (s, 2H), 4.31 (t, J = 7.8 Hz, 2H), 3.77 (t, J = 7.8 Hz, 2H), 3.52 (s,
1
3
4
6
H). C NMR (400 MHz, CDCl ) δ 173.8, 154.3, 137.4, 132.6, 132.1, 127.6, 70.4, 70.0, 69.7,
3
+
7.9, 66.6. ESI-MS [M+H] m/z:389.1.
4
-(4-carboxyphenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (4j): white solid. (Yield:
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1
7
8%). H NMR (600 MHz, CDCl
3
) δ 13.11 (s, 1H), 8.05 (t, J = 8.0 Hz, 4H), 7.92 (t, J = 7.5 Hz,
13
1H), 7.77 (t, J = 7.9 Hz, 2H), 7.56 (d, J = 8.8 Hz, 2H). C NMR (400 MHz, CDCl
3
) δ 168.8,
+
1
57.7, 155.3, 137.4, 132.6, 132.1, 129.9, 127.6, 126.7, 120.3. ESI-MS [M+H] m/z:363.0.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
-(3-carboxyphenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (4k): white solid. (Yield:
1
81%). H NMR (600 MHz, CDCl
3
) δ 13.34 (s, 1H), 8.07 (d, J = 7.8 Hz, 2H), 7.99 (s, 1H), 7.92 (t,
13
J = 7.4 Hz, 2H), 7.77 (t, J = 7.8 Hz, 2H), 7.73 – 7.69 (m, 1H), 7.64 (t, J = 7.9 Hz, 1H). C NMR
(
400 MHz, CDCl
3
) δ 168.01, 155.3, 152.2, 137.4, 132.6, 132.1, 130.0, 128.5, 127.6, 126.4, 126.3,
+
1
22.3. ESI-MS [M+H] m/z:363.0.
4-(4-carboxy-2-methoxyphenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (4l): white solid.
1
(
Yield: 73%). H NMR (600 MHz, CDCl
3
) δ 13.20 (s, 1H), 8.08 (d, J = 7.6 Hz, 2H), 7.95 (t, J =
7
.5 Hz, 1H), 7.81 (t, J = 7.9 Hz, 2H), 7.69 (d, J = 1.4 Hz, 1H), 7.64 (dd, J = 8.4, 1.6 Hz, 1H),
1
3
7.57 (d, J = 8.3 Hz, 1H), 3.75 (s, 3H). C NMR (400 MHz, CDCl
3
) δ 168.1, 156.2, 149.0, 146.8,
+
1
37.4, 132.6, 132.1, 127.6, 125.1, 122.2, 121.9, 117.6, 56.8. ESI-MS [M+H] m/z:393.0.
4
-(4-(carboxymethyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (4m): colorless oil.
1
(
Yield: 88%). H NMR (600 MHz, CDCl
3
) δ 12.33 (s, 1H), 8.04 (d, J = 7.8 Hz, 2H), 7.92 (t, J =
1
3
7.4 Hz, 1H), 7.77 (t, J = 7.7 Hz, 2H), 7.37 (q, J = 8.8 Hz, 4H), 3.64 (s, 2H). C NMR (400 MHz,
CDCl
3
) δ 174.9, 155.3, 147.0, 137.4, 132.6, 132.1, 131.1, 127.6, 126.6, 118.9, 44.4. ESI-MS
+
[
M+H] m/z:376.0.
4
-((2-carboxy-2-methylpentyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (4n): colorless
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2
2
2
2
2
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4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
oil. (Yield: 86%). H NMR (600 MHz, CDCl
3
) δ 8.07 (d, J = 8.9 Hz, 2H), 7.93 (t, J = 7.5 Hz,
1H), 7.75 (t, J = 7.4 Hz, 2H), 4.85 (dd, J = 283.1, 12.4 Hz, 2H), 1.67 (dt, J = 98.5, 7.7 Hz, 2H),
13
1
1
.46 – 1.36 (m, 2H), 0.97 (s, 3H), 0.89 (t, J = 6.6 Hz, 3H). C NMR (400 MHz, CDCl
3
) δ 179.7,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
53.5, 137.4, 132.6, 132.1, 127.6, 73.5, 44.8, 40.1, 26.4, 17.2, 14.7. ESI-MS [M+H] m/z: 370.1.
The preparation of compound 6 (4-(4-formylphenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole
-oxide): To a stirred suspension of PCC (648 mg, 3 mmol) in DCM at 0℃, compound 3j (600
2
mg, 1.8 mmol) was added, and the solution was stirred at 0℃ for 5 h. The solution was
concentrated under vacuum at room temperature. The residue was extracted with EtOAc three
times (3×50 mL) and washed twice with water and brine. The combined organic layers were
dried by anhydrous Na
2
SO
4
, and evaporated under vacuum to give a residue that was purified by
silica-gel column chromatography (P:E=3:1) to get the product as a white solid. (Yield: 68%).
1
19
H NMR (600 MHz, d
6
-DMSO) δ 9.98 (s, 1H), 7.95 (d, J = 8.5 Hz, 2H), 7.91 (d, J = 7.7 Hz,
2
H), 7.85 (t, J = 7.5 Hz, 1H), 7.70 (t, J = 7.9 Hz, 2H), 7.29 (d, J = 8.5 Hz, 2H).
The
preparation
of
compound
7
((E)-4-(4-(2-carboxyvinyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide): To
a
solution of propandioic acid (710 mg, 2.4 mmol) in pyridine, Pyrrolidine (0.03 mL) was added
and the mixture was stirred at room temperature for 20 minutes. After that, the compound 6 (750
mg, 2.4 mmol)was added and the reaction mixture was heated to 100℃ in reflux for 3h.
Pyridine was removed and the residue was extracted with EtOAc (3×50 mL) and washed twice
with water and brine. The organic portion was dried by anhydrous Na
2
SO . The crude product
4
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was obtained by removing EtOAc and purified by silica chromatography column (P:E=3:1) to
1
get the product as white solid. (Yield: 80%). H NMR (600 MHz, d
6
-DMSO) δ 12.47 (s, 1H),
8
2
.05 (d, J = 7.8 Hz, 2H), 7.93 (t, J = 7.4 Hz, 1H), 7.83 (d, J = 8.7 Hz, 2H), 7.77 (t, J = 7.8 Hz,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
H), 7.63 (d, J = 16.0 Hz, 1H), 7.48 (d, J = 8.7 Hz, 2H), 6.57 (d, J = 16.0 Hz, 1H). C NMR
(400 MHz, d
6
-DMSO) δ 170.5, 155.3, 153.2, 143.7, 137.4, 132.6, 132.1, 131.9, 130.9, 127.6,
+
1
21.7, 115.0. ESI-MS [M+H] m/z:389.0.
General procedure for the preparation of compounds (5a-o)
4
-(4-(hydroxyamino)-4-oxobutoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (5a): To a
solution of compound 4a (1.2 g, 3.8 mmol) in dried THF, Isobutyl chlorocarbonate (0.9 mL, 3.9
mmol) was added by drop at 0℃, after stirring 0.5h, triethylamine (0.9 mL, 2.5 mmol) was
added dropwise at 0℃. The mixture was further stirred for additional 1 h at room temperature
and solid precipitates were filtered resulting in the first filtrate. Potassium hydroxide (319.2 mg,
5
.7 mmol) and hydroxylamine hydrochloride (396.1 mg, 5.7 mmol) were dissolved in anhydrous
menthol completely and the residue was filtered to get the second filtrate. The first filtrate was
added into the second filtrate and the mixture was stirred for additional 4 h at room temperature
under nitrogen atmosphere. Ferric trichloride was the color-producing reagent. THF and menthol
were removed after the pH of the reacted solution was adjusted to 3.0 with 2N HCl. And then the
residue was extracted with EtOAc.The organic portion was dried by anhydrous Na
2
SO . The
4
crude product was obtained by removing EtOAc and purified by silica chromatography column
1
(
P:E=3:1) to get the product. white solid. (Yield: 73%). M.p. 139-141℃. H NMR (600 MHz,
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
d
6
-DMSO) δ 10.51 (s, 1H), 8.80 (s, 1H), 8.03 (d, J = 8.2 Hz, 2H), 7.89 (t, J = 7.4 Hz, 1H), 7.74 (t,
1
3
J = 7.5 Hz, 2H), 4.40 (t, J = 6.2 Hz, 2H), 2.13 (t, J = 7.3 Hz, 2H), 2.02 – 1.97 (m, 2H). C NMR
(
400 MHz, d
6
-DMSO) δ 168.4, 159.1, 137.3, 136.4, 130.2, 128.6, 128.5, 110.7, 71.0, 28.3, 24.4.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
HRMS (ESI) m/z calcd for C12
H
14
N
3
O
7
S [M+H] 344.0547, found 344.0548.
4
-((5-(hydroxyamino)-5-oxopentyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (5b):
1
white solid. (Yield: 68%). M.p. 119-121℃. H NMR (600 MHz, d
6
-DMSO) δ 10.39 (s, 1H), 8.74
(
s, 1H), 8.02 (d, J = 7.9 Hz, 2H), 7.90 (t, J = 7.3 Hz, 1H), 7.76 (t, J = 7.8 Hz, 2H), 4.38 (t, J = 6.1
13
Hz, 2H), 1.79 – 1.71 (m, 2H), 1.66 – 1.58 (m, 3H), 1.53 (t, J = 6.8 Hz, 2H). C NMR (400 MHz,
d
6
-DMSO) δ 169.2, 159.3, 137.6, 136.6, 130.6, 128.8, 110.9, 71.5,32.1, 27.9, 21. 8. HRMS (ESI)
+
m/z calcd for C13
H
16
N
3
O
7
S [M+H] 358.0703, found 358.0705.
4
-((6-(hydroxyamino)-6-oxohexyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (5c): white
1
solid. (Yield: 62%). M.p. 135-137℃. H NMR (600 MHz, d
6
-DMSO) δ 10.37 (s, 1H), 8.70 (s,
H), 8.04 – 7.99 (m, 2H), 7.90 (t, J = 7.5 Hz, 1H), 7.76 (t, J = 7.9 Hz, 2H), 4.37 (t, J = 6.3 Hz,
H), 1.97 (t, J = 7.4 Hz, 2H), 1.78 – 1.69 (m, 2H), 1.57 – 1.51 (m, 2H), 1.32 (dt, J = 15.3, 7.7 Hz,
1
2
13
2H). C NMR (400 MHz, d
6
-DMSO) δ 169.4, 159.3, 137.6, 136.6, 130.5, 128.8, 110.9, 71.8,
+
3
3
2.6, 28.1, 25.2, 25.1. HRMS (ESI) m/z calcd for C14
72.0866.
H
18
N
3
O
7
S [M+H] 372.0860, found
4
-((7-(hydroxyamino)-7-oxoheptyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (5d):
1
white solid. (Yield: 70%). H NMR (600 MHz, DMSO) δ 10.35 (s, 1H), 8.62 (s, 1H), 8.01 (d, J =
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.4 Hz, 2H), 7.91 (s, 1H), 7.76 (t, J = 7.9 Hz, 2H), 4.38 (t, J = 6.3 Hz, 2H), 1.97 (t, J = 7.4 Hz,
1
3
2H), 1.78 – 1.69 (m, 2H), 1.54 – 1.49 (m, 2H), 1.36 – 1.25 (m, 4H). C NMR (151 MHz,
d
6
-DMSO) δ 169.6, 159.3, 136.6, 130.5, 130.2, 128.7, 127.9, 110.9, 71.9, 32.7, 28.5, 28.2, 25.5,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
2
5.2. HRMS (ESI) m/z calcd for C15
H
20
N
3
O
7
S [M+H] 386.1016, found 386.1032.
4
-((8-(hydroxyamino)-8-oxooctyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (5e): white
1
solid. (Yield: 64%). H NMR (600 MHz, DMSO) δ 10.34 (s, 1H), 8.66 (s, 1H), 8.01 (d, J = 7.5
Hz, 2H), 7.91 (t, J = 7.4 Hz, 1H), 7.76 (t, J = 7.9 Hz, 2H), 4.38 (t, J = 6.3 Hz, 2H), 1.96 (t, J =
7
.3 Hz, 2H), 1.73 (dd, J = 13.5, 6.4 Hz, 2H), 1.51 (dt, J = 14.5, 7.4 Hz, 2H), 1.35 – 1.21 (m, 6H).
1
3
C NMR (151 MHz, DMSO) δ 169.6, 159.3, 137.8, 136.6, 130.5, 128.7, 110.9, 72.0, 32.7, 28.9,
+
2
4
8.7, 28.3, 25.5, 25.4. HRMS (ESI) m/z calcd for C16
00.1172.
H
22
N
3
O
7
S [M+H] 400.1173, found
4-(2-(2-(hydroxyamino)-2-oxoethoxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (5f):
1
white solid. (Yield: 66%). M.p. 143-145℃. H NMR (600 MHz, d
6
-DMSO) δ 10.54 (s, 1H),
8
.89 (s, 1H), 8.03 (d, J = 7.5 Hz, 2H), 7.90 (t, J = 7.5 Hz, 1H), 7.75 (t, J = 7.9 Hz, 2H), 4.58 –
1
3
4.49 (m, 2H), 3.97 (s, 2H), 3.86 – 3.80 (m, 2H). C NMR (400 MHz, d
6
-DMSO) δ 171.0, 158.8,
1
37.2, 136.0, 129.9, 128.2, 110.4, 72.1, 70.6, 68.9, 68.2, 42.8. HRMS (ESI) m/z calcd for
+
C
12
H
14
N
3
O S [M+H] 360.0496, found 360.0497.
8
4
-(3-(hydroxyamino)-2,2-dimethyl-3-oxopropoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide
1
(5g): white solid. (Yield: 71%). M.p. 128-130℃. H NMR (600 MHz, d
6
-DMSO) δ 10.61 (s,
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7
8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
H), 8.81 (s, 1H), 7.99 (d, J = 7.5 Hz, 2H), 7.90 (t, J = 6.8 Hz, 1H), 7.76 (t, J = 7.4 Hz, 2H), 4.38
13
(s, 2H), 1.22 (s, 6H). C NMR (400 MHz, d
6
-DMSO) δ 171.4, 159.3, 137.9, 136.5, 130.6, 130.6,
+
1
28.62, 110.7, 76.99, 41.7, 22.2. HRMS (ESI) m/z calcd for C13
H
16
N
3
O
7
S [M+H] 358.0704,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
found 358.0703.
(
E)-4-((4-(hydroxyamino)-4-oxobut-2-en-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole
1
2
-oxide (5h): white solid. (Yield: 76%). M.p. 144-146℃. H NMR (600 MHz, d
6
-DMSO) δ
8
.10 (d, J = 7.5 Hz, 2H), 7.86 (t, J = 7.5 Hz, 1H), 7.73 (t, J = 7.9 Hz, 2H), 6.96 (dt, J = 15.3, 4.3
1
3
Hz, 1H), 6.27 (d, J = 15.5 Hz, 1H), 5.15 (d, J = 3.1 Hz, 2H). C NMR (400 MHz, d
6
-DMSO) δ
1
61.2, 158.6, 139.1, 136.0, 129.9, 128.2, 120.4, 76.7, 69.0. HRMS (ESI) m/z calcd for
+
C
12
H
12
N
3
O S [M+H] 342.0390, found 342.0393.
7
4
-(2-(2-(2-(hydroxyamino)-2-oxoethoxy)ethoxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole
1
2-oxide (5i): white solid. (Yield: 70%). M.p. 139-141℃. H NMR (600 MHz, DMSO) δ 10.40
(
s, 1H), 8.77 (s, 1H), 8.03 (d, J = 7.8 Hz, 2H), 7.91 (t, J = 7.4 Hz, 1H), 7.76 (t, J = 7.3 Hz, 2H),
.54 – 4.50 (m, 2H), 3.89 (s, 2H), 3.84 – 3.80 (m, 2H), 3.65 (d, J = 3.0 Hz, 2H), 3.61 (d, J = 3.2
4
1
3
Hz, 2H). C NMR (400 MHz, d
6
-DMSO) δ 165.5, 158.8, 137.2, 136.0, 129.9, 128.1, 70.8, 70.2,
+
6
9.62, 68.9, 67.7. HRMS (ESI) m/z calcd for C17
H
14
N
3
O
7
S [M+H] 404.0547, found 404.0549.
4
-(4-(hydroxycarbamoyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (5j): white
1
solid. (Yield: 78%). M.p. 171-172℃. H NMR (600 MHz, d
6
-DMSO) δ 11.29 (s, 1H), 9.10 (s,
1H), 8.04 (d, J = 7.3 Hz, 2H), 7.93 (t, J = 6.8 Hz, 1H), 7.87 (d, J = 7.9 Hz, 2H), 7.77 (t, J = 7.4
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7
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9
1
3
Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H). C NMR (400 MHz, d -DMSO) δ 163.6, 158.5, 155.1, 137.3,
6
136.8, 131.4, 130.5, 129.5, 129.1, 119.9, 111.8. HRMS (ESI) m/z calcd for C15
H
12
N
3
7
O S
+
[
M+H] 378.0390, found 378.0439.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
-(3-(hydroxycarbamoyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (5k): white
1
solid. (Yield: 64%). M.p. 177-179℃. H NMR (600 MHz, d
6
-DMSO) δ 11.31 (s, 1H), 9.16 (s,
1
H), 8.04 (d, J = 7.6 Hz, 2H), 7.92 (d, J = 7.4 Hz, 1H), 7.78 – 7.72 (m, 4H), 7.62 – 7.57 (m, 2H).
13
C NMR (400 MHz, d
6
-DMSO) δ 167.3, 157.6, 144.0, 135.4, 129.8, 119.0, 118.9, 117.3, 114.4.
+
HRMS (ESI) m/z calcd for C15
H
12
N
3
O
7
S [M+H] 378.0390, found 378.0378.
4-(4-(hydroxycarbamoyl)-2-methoxyphenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (5l):
1
white solid. (Yield: 72%). M.p. 198-200℃. H NMR (600 MHz, d
6
-DMSO) δ 11.26 (s, 1H),
9
.07 (s, 1H), 8.09 (d, J = 7.8 Hz, 2H), 7.95 (t, J = 7.4 Hz, 1H), 7.81 (t, J = 7.6 Hz, 2H), 7.57 (s,
13
1H), 7.52 (d, J = 8.2 Hz, 1H), 7.44 (d, J = 8.5 Hz, 1H), 3.79 (s, 3H). C NMR (400 MHz,
d
6
-DMSO) δ 163.0, 158.4, 149.4, 142.8, 137.0, 136.4, 130.2, 128.4, 121.6, 119.8, 112.1, 110.8,
+
5
6.3. HRMS (ESI) m/z calcd for C16
H
14
N
3
O
8
S [M+H] 408.0496, found 408.0500.
4
-(4-(2-(hydroxyamino)-2-oxoethyl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole
2-oxide
1
(5m): white solid. (Yield: 73%). H NMR (600 MHz, d
6
-DMSO) δ 10.62 (s, 1H), 8.79 (s, 1H),
8
.04 (d, J = 7.8 Hz, 2H), 7.92 (t, J = 7.4 Hz, 1H), 7.77 (t, J = 7.6 Hz, 2H), 7.35 (dd, J = 17.3, 8.6
1
3
Hz, 4H), 3.34 (s, 2H). C NMR (400 MHz, d -DMSO) δ 158.3, 136.9, 136.1, 131.3, 130.4,
6
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
1
29.9, 129.5, 128.5, 128.3, 119.3, 29.9.HRMS (ESI) m/z calcd for C16
H
14
N
3
O S [M+H]
7
392.0547, found 392.0550.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
-((2-(hydroxycarbamoyl)-2-methylpentyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole
2-oxide
1
(
5n): white solid. (Yield: 54%). H NMR (600 MHz, DMSO-d
6
) δ 10.63 (s, 1H), 8.79 (s, 1H),
8.04 (d, J = 7.5 Hz, 2H), 7.92 (t, J = 7.5 Hz, 1H), 7.77 (t, J = 7.7 Hz, 2H), 7.35 (dd, J = 17.9, 8.4
1
3
Hz, 4H), 3.34 (s, 2H). C NMR (400 MHz, d
6
-DMSO) 177.2, 172.4, 131.3, 129.96, 129.5, 128.5,
+
6
3
7.0, 64.9, 47.2, 36.3, 35.7, 19.8, 17.7, 13.7. HRMS (ESI) m/z calcd for C15
86.1016, found 386.1017.
H
20
N
3
7
O S [M+H]
(E)-4-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)phenoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazol
1
e 2-oxide (5o): white solid. (Yield: 69%). M.p. 153-155℃. H NMR (600 MHz, DMSO) δ
1
0.79 (s, 1H), 9.08 (s, 1H), 7.91 (d, J = 8.5 Hz, 1H), 7.76 (dd, J = 25.3, 7.9 Hz, 2H), 7.63 (d, J =
8.4 Hz, 2H), 7.47 (d, J = 15.9 Hz, 1H), 7.34 (dd, J = 15.9, 8.4 Hz, 2H), 7.29 (d, J = 8.5 Hz, 2H),
1
3
6
.44 (d, J = 15.8 Hz, 1H). C NMR (400 MHz, d
6
-DMSO) δ 163.3, 158.7, 138.3, 129.0, 125.8,
+
115.6, 115.4. HRMS (ESI) m/z calcd for C14
H N
18 3
O
9
S [M+H] 404.0758, found 404.0755.
In Vitro Hela Extracts Inhibition Fluorescence Assay.
3
1
In vitro HDACs inhibition assays were conducted as previously described in our group. In
brief, 10 μL of HELA extracts were mixed with various concentrations of 50 μL tested
compounds (0.039, 0.39, 1.56, 6.25, 25, 100 μM) and SAHA as the positive control. Five
minutes later, fluorogenic substrate Boc-Lys (acetyl)-AMC (40 μL) was added, and the mixture
2
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was incubated at 37℃ for 30 minutes and then stopped by addition of 100 μL of developer
containing trypsin and TSA. After incubation at 37℃ for 20 minutes, fluorescence intensity was
measured using a microplate reader at excitation and emission wavelengths of 390 nm and 460
nm, respectively. The inhibition ratios were calculated from the fluorescence intensity readings
of tested wells relative to those of control wells, and the IC50 values were calculated using a
regression analysis of the concentration/inhibition data.
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60
In Vitro Antiproliferative Assay.
In
vitro
antiproliferative
assays
were
determined
by
the
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method as previously
3
1
described. Briefly, all cell lines were maintained in RPMI1640 medium containing 10% FBS at
3
7℃ in 5% CO humidified incubator. Cells were passaged the day before dosing into a 96-well
2
cell plate and allowed to grow for a minimum of 4h prior to addition of compounds. After
compounds addition, the plates were incubated for an additional 48h, and then 0.5% MTT
solution was added to each well. After further incubation for 4h, formazan formed from MTT
was extracted by adding 200 μL of DMSO for 15 min. Absorbance was then determined using an
ELISA reader at 490 nm and 630 nm, and the IC50 values were calculated according to the
inhibition ratios.
In Vitro Nitrite Measurement.
33
The nitrite measurement in vitro was tested using the reported method. The levels of NO
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