Synthesis and biological evaluation of nitric oxide-releasing hybrids from gemcitabine and phenylsulfonyl furoxans as anti-tumor agents

Article abstract of DOI:10.1039/c5md00158g

A series of novel hybrids 10a-m were designed and synthesized by coupling phenylsulfonyl furoxans with gemcitabine through various diols or alcohol amine linkers, and their biological activities were evaluated in vitro. Most of the hybrids exhibited good to moderate anti-tumor activities, which are associated with NO release. In particular, hybrid 10e showed excellent anticancer activities which were more potent than or comparable to gemcitabine. However, inhibition of nucleoside transport only significantly decreased the inhibitory rates of gemcitabine against HepG2 cells but not 10e, and the inhibitory rates of 10e were partially reduced by pre-treatment with hemoglobin, demonstrating that the anti-tumor activity of 10e might result from the synergic effect of high levels of NO production and gemcitabine fragment. In addition, compound 10ecould apparently induce cell apoptosis by regulating apoptotic relative proteins. Therefore, our novel findings provide a proof of principle in the design of new furoxan/gemcitabine hybrids for the intervention of human cancers.

Full text of DOI:10.1039/c5md00158g

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Synthesis and biological evaluation of nitric oxide-releasing hybrids
from gemcitabine and phenylsulfonyl furoxans as anti-tumor agents
Xianghua Lia,c,#, Xuemin Wangb,c,#, Chenjun Xub, Junkai Huanga, Chengniu Wangb, Xinyang
Wangb,c, Liqin He*a, Yong Ling∗b,c
Abstract
A series of novel hybrids 10a-m were designed and synthesized by coupling phenylsulfonyl
furoxans with gemcitabine through various diols or alcohol amines linkers, and their biological
activities were evaluated in vitro. Most of hybrids exhibited good to moderate antiꢀtumor activities,
which are associated with NO release. In particular, hybrid 10e showed excellent anticancer
activities which were more potent than or comparable to gemcitabine. However, inhibition of
nucleoside transport only significantly decreased the inhibitory rates of gemcitabine against
HepG2 cells, but not 10e, and the inhibitory rates of 10e were partially reduced by preꢀtreatment
with hemoglobin, demonstrating that the antiꢀtumor activity of 10e might result from the synergic
effect of high levels of NO production and gemcitabine fragment. In addition, compound 10e
could apparently induce cell apoptosis by regulating apoptotic relative proteins. Therefore, our
novel findings provide a proof of principle in the design of new furoxan/gemcitabine hybrids for
the intervention of human cancers.
Keywords: Nitric oxide; Hybrids; Phenylsulfonyl furoxans; Gemcitabine; Antiꢀtumor agents
Introduction
Cancer is one of the most serious diseases threatening human health in the world. Current antiꢀ
tumor agents are still yet to meet human use requirements of high efficiency and low toxicity.
Various nucleoside analogue drugs are widely used for the treatment of cancer.1 Gemcitabine (2′,
2′ꢀdifluorodeoxycytidine, dFdC), a cycleꢀspecific nucleoside analogue, has been approved for the
treatment of various solid tumors,2 such as nonꢀsmallꢀcell lung cancer (NSCLC), pancreatic cancer,
ovary cancer, and breast cancer.3ꢀ7 However, there are some drawbacks of gemcitabine in clinical
use, including poor fatꢀsolubility, poor transmembrane penetration, degraded deaminase
inactivation, short halfꢀlife, low bioavailability, and drug resistance.8 Hence, it is urgent for
researchers to design novel gemcitabine derivatives to further improve its properties.
Based on the literature, several approaches have been applied to the structural modification of
gemcitabine to improve its properties.9,10 It is more common that the N4ꢀposition of the cytidine
ring of gemcitabine be modified to form a hydrolysable amide linkage. For example, the orally
available N4ꢀvalproyl gemcitabine (LY2334737, Figure 1), which is currently in phase I clinical
aAnhui Key Laboratory of Traditional Chinese Medicine, Anhui University of Chinese Medicine, Hefei 230031, China
bSchool of Pharmacy, Nantong University, Nantong, 226001, PR China
cState Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 210009, PR China
∗ Corresponding authors. Tel.: +86 513 85051892 (Y.L.);
E-mail addresses: Lyyy111@sina.com (Y. Ling). Hlq661125@126.com (L. He).
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trial, could avoid the fast degradation of gemcitabine and withstand the gastrointestinal
environment with different pH values.11,12 N4ꢀSqualenoyl gemcitabine (SQdFdC) was stable in
plasma and its halfꢀlife of metabolism has been extended in vivo.13 In addition, stearoyl
gemcitabine (C18dFdC), the N4ꢀstearoyl substituted gemcitabine, could prevent the degradation of
gemcitabine by deaminase and therefore improved the bioavailability and halfꢀlife of
gemcitabine.14
Fig. 1. Chemical structures of gemcitabine derivatives.
Nitric oxide (NO) as a signaling and/or effector molecule plays a pivotal role in numerous
physiologic and pathologic processes.15 It is well known that high levels of NO generated from
NOꢀdonors can not only induce apoptosis and inhibit metastasis of tumor cells, but also sensitize
tumor cells to chemotherapy, radiation, and immunotherapy in vitro and in vivo.16,17 Furoxans,
especially phenylsulfonyl furoxans, represent an important class of lipophilic NO donors and have
played an increasingly important role in the development of antiꢀcancer reagents which can
produce high levels of NO and inhibit the growth of tumors in vivo.18ꢀ22 Based on the above
findings, we hypothesized that hybridization of furoxans with gemcitabine may release high levels
of NO to exert synergistic anticancer effects with gemcitabine. According to this hypothesis, a
total of 13 novel hybrids 10a-m were designed and synthesized by coupling phenylsulfonyl
furoxans with the N4ꢀposition of gemcitabine through various diols or alcohol amines linkers.
Their antiꢀtumor effects, membrane transport deficiency, NOꢀreleasing ability and effects on key
regulatory proteins were evaluated in vitro. Herein, we report the synthesis and biological
evaluation of target compounds.
Results and discussion
Chemistry
The synthetic route of 10a-m is presented in Scheme 1. The furoxans were synthesized in a
fourꢀstep sequence. The starting material benzenethiol 1 was converted to 2ꢀ(phenylthio) acetic
acid 2 by treatment with NaOH and chloroacetic acid and Na2CO3 at 80 °C in 89.0 % yield.
Compound 2 was treated with 30 % H2O2 at room temperature to generate 2ꢀ(phenylsulfonyl)
aceticacid 3, which was poured into fuming HNO3 at 90 °C to produce diphenylsulfonyl furoxan 4
in 76.0% yield. Subsequently, compound 4 was converted to various monophenylsulfonylfuroxans
5a-m by linking with different diols or alcohol amines. Then 6a-m were formed by condensation
of succinic anhydride with 5a-m. Gemcitabine 7 was treated with tertꢀbutyldimethylsilyl
chloride (TBDMSCl) and imidazole to give intermediate 8, which was directly reacted with 6a-m
2

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in CH2Cl2 in the presence of Nꢀhydroxysuccinimide/1ꢀethylꢀ3ꢀ(3ꢀdimethyllaminopropyl)
carbodiimide hydrochloride/4ꢀdimethylaminopyridine (NHS/EDCI/DMAP) to generate products
9a-m in yields of 35ꢀ60 %. Finally, compound 9a-m were further deprotected by
tetrabutylammonium fluoride (TBAF) to give target compounds 10a-m. The final products were
1
purified by column chromatography and their structures were characterized by H NMR, 13C
NMR, MS, and elemental analyses.
Scheme 1. Reaction conditions and reagents: a) 30 % NaOH, chloroacetic acid, Na2CO3, reflux, 1h, 89.0 %; b, c) HOAc, 30%
H2O2, rt, 2.5h, 95 % HNO3, 90 °C, 0.5h, 76.0 %; d) diols or alcohol amines, 25 % NaOH or NaH, 0 °C, 0.5 h, 50.0ꢀ70.0 %; e)
Succinic anhydride, CH2Cl2, reflux, 3ꢀ8 h, 85.0ꢀ92.7 %; f) TBDMSCl, imidazole, DMF, rt, 6 h, 87.3 %; g) NHS, EDCI, DMAP, rt,
18 h, 47.8ꢀ59.0 %; h) TBAF, THF, 0 °C, 0.5 h, 66.9ꢀ87.2 %.
Biological evaluation
All the target compounds 10a-m were evaluated in MTT assay against human hepatocellular
carcinoma cells (HepG2), human colon cancer cell (HCTꢀ116, SWꢀ620), human lung cancer cell
(A549), and human gastric carcinoma cells (SGC7901) in vitro. Gemcitabine was used as a
positive control. The IC50 values of 10a-m against each tumor cell line are presented in Table 1.
All tested compounds exhibited more potent antiꢀtumor activity, especially compounds 10eꢀk
(IC50 = 1.02~9.92 ꢁM) which possessed similar or better inhibition activities for the cancer cell
growth relative to gemcitabine (IC50 = 2.69~7.33 ꢁ M). Notably, the IC50 of 10e (1.02 ꢁM) against
HepG2 cells was eightꢀfold lower than its NO moiety 6e (IC50 = 8.73 ꢁM), nearly threeꢀfold lower
than gemcitabine 7 (IC50 = 2.97 ꢁM), and even lower than the combination of 7 and 6e (IC50
=
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1.85 ꢁM, Figure 2). These results suggest that the antiꢀtumor activity of 10e may result from the
synergetic effects of the NO donor moiety 6e and the gemcitabine moiety by simultaneously
acting on both pathways.
Table 1. The IC50 values of 10a-m against five human cancer cell lines.
In vitro inhibition of human cancer cells proliferation (IC50a, ꢁM)
Compound
HepG2
HCTꢀ116
7.33 ± 0.63
3.75 ± 0.44
9.86 ± 0.92
10.3 ± 0.80
6.89 ± 0.59
3.57 ± 0.41
4.69 ± 0.32
1.19 ± 0.20
11.3 ± 1.10
7.86 ± 0.80
8.78 ± 0.72
>12.5
SWꢀ620
5.62 ± 0.47
5.16 ± 0.63
9.09 ± 0.75
>12.5
5.00 ± 0.61
2.49 ± 0.30
3.98 ± 0.35
1.30 ± 0.16
9.67 ± 1.09
>12.5
A549
2.69 ±0.28
NDb
>12.5
>12.5
9.92 ± 1.05
5.18 ± 0.46
7.82 ± 0.75
3.27 ± 0.39
>12.5
11.5 ± 1.12
>12.5
>12.5
SGC7901
3.58 ± 0.33
ND
11.5 ± 1.02
>12.5
9.26 ± 0.76
6.13 ± 0.71
7.44 ± 0.68
1.36 ± 0.22
10.5 ± 1.00
10.7 ± 1.21
>12.5
9.92 ± 1.02
6.71 ± 0.58
5.10 ± 0.40
5.85 ± 0.63
11.8 ± 1.12
Gemcitabine (7)
2.97 ± 0.32
7.42 ± 0.59
8.73 ± 0.71
>12.5
5.32 ± 0.66
2.44 ± 0.27
3.25 ± 0.43
1.02 ± 0.13
>12.5
7.05 ± 0.64
9.21 ± 0.79
10.5 ± 0.87
6.18 ± 0.59
3.22 ± 0.26
8.39 ± 0.70
10.7 ± 1.01
JSꢀK
6e
10a
10b
10c
10d
10e
10f
10g
10h
10i
>12.5
9.16 ± 0.95
4.97 ± 0.63
4.35 ± 0.51
5.21 ± 0.44
>12.5
10j
10k
10l
4.10 ± 0.46
3.06 ± 0.38
10.8 ± 1.07
>12.5
8.90 ± 0.96
6.04 ± 0.72
10.1 ± 1.05
>12.5
10m
a The inhibitory effects of individual compounds on the proliferation of cancer cell lines were determined by the MTT assay. The
data are the mean values of IC50 from at least three independent experiments. b Not detected.
Figure 2. Comparison of anticancer activity of hybrid 10e with that of 7, 6e, and an equimolar mixture of 7 and 6e.
Given the strong growth inhibitory activity of 10e in vitro, the selectivity profile of 10e was
investigated on the inhibitory effects of HepG2 and LO2 cells using gemcitabine as a control. The
treatment with increased dose of 10e had no significant effect on the survival of nonꢀtumor LO2
cells while the same treatment induced the majority of HepG2 cell death (Figure 3), suggesting
10e may possess selective antiproliferation activity against tumor cells.
Nucleoside transporters have been thought to have an important role in membrane transport
systems of gemcitabine. To study the role of membrane transport, the sensitivity to gemcitabine
and 10e in the presence of nucleoside transport inhibitor dipyridamole (Dipy) was determined. As
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shown in Figure 4, cell inhibitory rates were determined for gemcitabine and 10e with different
concentrations in HepG2 cells with or without 4 ꢁg/mL dipyridamole. It was found that
dipyridamole sharply decreased sensitivity to gemcitabine in HepG2 cells, whereas the sensitivity
to 10e remained almost unchanged. These results suggested that the furoxan/gemcitabine hybrid
10e may not be dependent on the nucleoside transporter and may independently transport over the
cell membrane.
90
10e in HepG2
80
10e in LO2
Gem in HepG2
Gem in LO2
70
60
50
40
30
20
10
0
0
0.5
1.0
2.0
5.0
Concentrations (ꢁM)
Figure 3. Inhibitory effects of 10e on the proliferation of HepG2 and LO2 cells. Cells were incubated with the indicated
concentrations of 10e for 48 h. Cell proliferation was assessed using the MTT assay. Data are means ± SD of the inhibition (%)
from three independent experiments.
gemcitabine
100
gemcitabine + Dipy
10e
80
60
40
20
0
10e + Dipy
50
20
5.0
2.0
0.50
Concentrations (ꢁM)
Figure 4. The effect of the nucleoside transport inhibitor dipyridamole on sensitivity to gemcitabine and 10e was studied in
HepG2 cells. HepG2 cells were treated with different concentrations (50, 20, 5.0, 2.0, and 0.50 ꢁM) of gemcitabine and 10e with
or without dipyridamole (4 ꢁg/mL) for 48 h. Data are expressed as means ± SD from three separate experiments.
The correction between NO production and anti-tumor activities. Furthermore, we tested
whether the strong inhibition of active compounds on the proliferation of tumor cells could be
associated with high levels of NO production in HepG2, HCTꢀ116, and SWꢀ620 cells. These cells
were exposed to each compound (100 ꢁM) for varying durations (30ꢀ300 min). The levels of
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nitrite/nitrate produced in the lysates of these cells were characterized in the Griess assay (Figure
5A). As expected, treatment with gemcitabine resulted in little nitrite/nitrate similar to control
group in any tested cells, while treatment with hybrids (10b-e, 10j, and 10k) with high
cytotoxicities produced significantly variable levels of nitrate/nitrite. In particular, 10e exhibited
the highest levels of nitrate/nitrite in these cells. These results indicate that the amounts of NO
released intracellularly are closely associated with their in vitro antiproliferative activity. The NO
release behaviors of 10e along with JSꢀK (a known NO donor) were further examined using a NOꢀ
sensitive fluorophore, 4ꢀaminoꢀ5ꢀ(methylamino)ꢀ2′,7′ꢀdifluorofluorescein diacetate (DAFꢀFM
DA).27 It was observed that 10e showed significant fluorescence in a doseꢀdependent manner and
produced greater amounts of NO in tumor cells than JSꢀK (Figure 5B).
Figure 5. (A) Human cancer cells were treated with each compound at 100 ꢁM and the contents of nitrate/nitrite in the cell
lysates were determined by Griess assay through the duration of 30–300 min. The individual values were determined by
measuring absorbance at 540 nm, and calculated according to the standard curve. Nitrate/nitrite production in these cells was
observed at other experimental time points (data not shown). (B) DAFꢀFM DA (Beyotime) was used as a fluorescent indicator of
intracellular NO. When cells grown in a 96ꢀwell plate reached 80% confluence, they were washed with PBS. After being loaded
with 5 ꢁM DAFꢀFM DA at 37 °C for 20 min, the cells were rinsed three times with PBS and incubated with test compounds for
24 h. NO production was measured with the flow cytometer with excitation and emission wavelengths of 495 and 515 nm,
respectively. (C) HepG2 cells were pretreated with, or without, the indicated concentrations of hemoglobin for 1 h and treated
with 2 or 6 ꢁM of 10e for 48 h. Data are expressed as mean% of inhibition on the growth of HepG2 cells. The cells treated with
different concentrations of hemoglobin alone did not affect their growth (data not shown).
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To further determine the role of NO in cytotoxicity of these hybrids against cancer cells, we
preꢀtreated HepG2 cells with various concentrations of hemoglobin, a wellꢀknown NO scavenger,
followed by treatment with different concentrations of hybrid compound 10e to detect their
inhibitory activities and NO production (Figure 5C). As expected, 10e displayed a strong antiꢀ
proliferation of HepG2 cells without preꢀtreatment with hemoglobin. In contrast, preꢀtreatment
with different concentrations of hemoglobin dramatically reduced the cytotoxicity of 10e against
the HepG2 cells. Furthermore, the inhibitory effects of different concentrations of hemoglobin
were doseꢀdependent and negatively correlated with the inhibition of the proliferation of tumor
cells. Notably, treatment with 10 ꢁM of hemoglobin reduced the cytotoxicity of 10e by near 50%.
Therefore, the high concentrations of NO produced by NO donor moieties and the bioactivity of
gemcitabine synergistically contribute to the antiꢀtumor effects of these new hybrids in vitro.
Flow cytometry assay of cell apoptosis. To test whether the higher potency of compound 10e
is due to cell apoptosis, HepG2 cells were incubated with vehicle alone, different concentrations
of 10e, or gemcitabine, respectively, for 48 h and the percentages of apoptotic cells were
determined by FITCꢀAnnexin V/PI staining and flow cytometry. The effect of 10e on cell
apoptosis is presented in Figure 6. In the untreated group, the frequency of HepG2 cells apoptosis
was not noticable. In contrast, the percentage of the cell apoptosis showed an upward trend in 10eꢀ
treated HepG2 cells when the dose increased. More importantly, treatment with 2.0 ꢁM of 10e
induced 78.3% of HepG2 cells apoptosis, which was superior to that with 2.0 ꢁM of gemcitabine.
These results demonstrated that incubation with 10e induced HepG2 cell apoptosis in a doseꢀ
dependent manner.
Figure 6. Compound 10e induced HepG2 cells apoptosis in vitro. HepG2 cells were incubated with the indicated concentrations
of 10e and gemcitabine for 48 h, and the cells were stained with FITCꢀAnnexin V/PI, followed by flow cytometry analysis. (A)
Flow cytometry analysis. (B) Quantitative analysis of apoptotic cells. Data are expressed as means ± SD of the percentages of
apoptotic cells from three independent experiments.
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Western blot assay. To gain insight on the underlying molecular mechanism with
furoxan/gemcitabine hybrid 10e on cell apoptosis, we conducted western blot analysis to examine
the expression of apoptosis proteins in HepG2 cells. It is well known that Bclꢀ2 and Bax are antiꢀ
apoptotic or proꢀapoptotic regulator proteins, respectively, and caspaseꢀ3 is the execution factor of
apoptosis. In addition, Parpꢀ1 is one of several known cellular substrates for caspaseꢀ3 and
cleavage of Parpꢀ1 by caspaseꢀ3 is considered to be a hallmark of apoptosis.23 As shown in Figure
7, the protein level of Bclꢀ2 was dramatically reduced and the expression of Bax proteins was
significantly increased in 10e-treated cells in a doseꢀdependent manner. Moreover, the level of
activated caspaseꢀ3 was greatly enhanced, and the decreased expression of Parpꢀ1 in 10e-treated
cells was also found which was greater than that treated with 2.0 ꢁM of gemcitabine. These
changes indicated that the 10e could significantly induce cell apoptosis through the regulation of
apoptosis proteins.
Figure 7. Effect of compound 10e on the expression of apoptosisꢀrelated proteins in HepG2 cells. (A) The expression of Bax,
Bcl2, caspase 3, Parpꢀ1, and βꢀactin were examined by western blot analysis. HepG2 cells were incubated with or without 10e, or
gemcitabine at the indicated concentrations for 48 h and the levels of protein expression were detected using specific antibodies.
Data shown are representative images of each protein for three separate experiments. (B) Quantitative analysis: the relative levels
of each protein compared to the control βꢀactin were determined by densimetric scanning. Data are expressed as means ± SD
from three separate experiments.
Analysis of SAR revealed that most of 10a-m exhibited remarkable antiꢀtumor activities against
five human cancer cells. It was discovered that the length of the linker and linker type have
significant influence on the inhibitory activities against the cancer cell growth. For example,
compounds 10a-h with diol linker chain showed relatively higher antiproliferative activities
compared to compounds 10i-m with alcohol amine chain. As for the length of the linker, hybrids
(10b-e, 10j, and 10k) with the linker comprised of three or four carbon units displayed
considerably greater growth inhibitory effects than the other hybrids. However, some compounds
with aryl or ether bond chain were less effective than those with the aliphatic linker chain. In sharp
contrast, among tested compounds, the hybrid 10e with 1,4ꢀbutyl acetylene glycol group had the
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strongest inhibitory activity, which may result from the synergic effects of the NO donor moiety
and gemcitabine moiety.
Conclusion
In summary, a series of novel nitric oxide (NO)ꢀreleasing hybrids 10a-m from gemcitabine and
phenylsulfonyl furoxans were designed and synthesized. Most hybrids possessed potent
antiproliferative activities against five human cancer cell lines in vitro. In particular, compound
10e not only exhibited the strongest antiꢀtumor activity, but also produced high levels of NO.
Furthermore, inhibition of nucleoside transport significantly reduced the inhibitory activity of
gemcitabine, but not 10e, suggesting the independence of a nucleoside transporter. More
importantly, treatment with hemoglobin partially reduced cytotoxicities of 10e in HepG2 cells.
These results suggested that the high concentration of NO release by NO donor moieties of 10e
and the bioactivity of gemcitabine segment may synergistically contribute to the antiꢀtumor
activity of 10e in vitro. In addition, 10e induced cancer cells apoptosis by reducing the levels of
Bclꢀ2 and Parpꢀ1 and upꢀregulating the expression of Bax and caspaseꢀ3. Our findings suggest that
10e may hold promise as therapeutic agents for the intervention of human cancers.
Acknowledgments
We gratefully acknowledge the financial support by the Natural Science Foundation of China
(Grant No. 81302628), the Project of “Jiangsu Six Peaks of Talent” (2014-SWYYꢀ044), and China
Pharmaceutical University for the Open Project Program of State Key Laboratory of Natural
Medicines (SKLNMKF201415).
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Page 11 of 11
MedChemComm
View Article Online
DOI: 10.1039/C5MD00158G
Synthesis and biological evaluation of nitric oxide-releasing hybrids from
gemcitabine and phenylsulfonyl furoxans as anti-tumor agents
Xianghua Lia,c,#, Xuemin Wangb,c,#, Chenjun Xub, Junkai Huanga, Chengniu Wangb, Xinyang Wangb,c, Liqin
He*a, Yong Ling∗b,c
aAnhui Key Laboratory of Traditional Chinese Medicine, Anhui University of Chinese Medicine, Hefei
230031, China
bSchool of Pharmacy, Nantong University, Nantong, 226001, PR China
cState Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 210009, PR China
Graph Abstract
Novel furoxan/gemcitabine hybrids displayed significant antitumor activities, in particular 10 e, which could
be independent to the nucleoside transporter, release high levels of NO, and induce cell apoptosis by
regulating apoptotic related proteins in tumor cells in vitro.
∗ Corresponding authors. Tel.: +86 513 85051892 (Y.L.);
E-mail addresses: Lyyy111@sina.com (Y. Ling). Hlq661125@126.com (L. He).