Double-component diazeniumdiolate derivatives as anti-cancer agents

Article abstract of DOI:10.1016/j.bmc.2020.115405

In this study, we synthesized a series of double-component O²-aryl diazeniumdiolate (DDNO) derivatives, of which each molecule can release up to four nitric oxide molecules. These compounds showed cytotoxic activities to cancer cells, such as human leukemia, breast cancer and lung cancer. Among them, compound 1 (DDNO-1) showed the highest specific activity to human leukemia cells. It induced cell apopotosis and arrest cell cycle of G₂/M phase. The JNK and p38 protein kinases were activated by compound 1 to induce cancer cell apoptosis. Compound 1 also increased pro-apoptotic Bax level, which is a same function compared to a reported NO donor, JS-K. More interestingly, it decreased the level of an anti-apoptotic member Bcl-2, which is an opposite effect compared to JS-K. Compound 1 could be developed as a new anti-cancer agent since it increases the Bax/Bcl-2 ratio to overcome the drug resistance.

Full text of DOI:10.1016/j.bmc.2020.115405

Journal Pre-proofs
Double-component diazeniumdiolate derivatives as anti-cancer agents
Xun Ji, Qi Chen, Viswanath Arutla, Omar Khdour, Qiong-Ying Hu, Shengxi
Chen
PII:
S0968-0896(20)30210-8
https://doi.org/10.1016/j.bmc.2020.115405
BMC 115405
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
27 December 2019
17 February 2020
28 February 2020
Please cite this article as: X. Ji, Q. Chen, V. Arutla, O. Khdour, Q-Y. Hu, S. Chen, Double-component
diazeniumdiolate derivatives as anti-cancer agents, Bioorganic & Medicinal Chemistry (2020), doi: https://
doi.org/10.1016/j.bmc.2020.115405
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Double-component diazeniumdiolate derivatives as anti-cancer agents
Xun Jia1, Qi Chenb1, Viswanath Arutlaa1, Omar Khdoura, Qiong-Yingb,c
Hu*,Shengxi Chen*a
aBiodesign Center for BioEnergetics, Arizona State University, Tempe, Arizona
85287, USA.
bDepartment of Central Laboratory, Taizhou Central Hospital（Taizhou University
Hospital）, Taizhou , Zhejiang, PR China
cSchool of Medcine, Taizhou University , Taizhou , Zhejiang, PR China
1These authors contributed equally to this work
*Corresponding author
Tel.: +1 (480) 965-5969; Fax: +1 (480) 965 0038.
E-mail address: shengxi.chen.1@asu.edu (S. Chen); huqiongying@126.com (Q.
Hu);
Key words: diazeniumdiolate derivatives, nitric oxide, anti-cancer agent, G2/M
phase arrest, Bcl-2 inhibitor, leukemia
1

Abstract
In this study, we synthesized a series of double-component O2-aryl
diazeniumdiolate (DDNO) derivatives, of which each molecule can release up to
four nitric oxide molecules. These compounds showed cytotoxic activities to
cancer cells, such as human leukemia, breast cancer and lung cancer. Among them,
compound 1 (DDNO-1) showed the highest specific activity to human leukemia
cells. It induced cell apopotosis and arrest cell cycle of G2/M phase. The JNK and
p38 protein kinases were activated by compound 1 to induce cancer cell apoptosis.
Compound 1 also increased pro-apoptotic Bax level, which is a same function
compared to a reported NO donor, JS-K. More interestingly, it decreased the level
of an anti-apoptotic member Bcl-2, which is an opposite effect compared to JS-K.
Compound 1 could be developed as a new anti-cancer agent since it increases the
Bax/Bcl-2 ratio to overcome the drug resistance.
2

1. Introduction
Nitric oxide (NO) is a signaling molecule in a number of human cells. It also
involves in many physiological and pathological processes in tumor biology,
regulating tumor blood flow, vascular permeability and angiogenesis, controlling
the growth, migration and invasion of cancer cells.1-5 Interference of NO signaling
might be a promising strategy as a novel cancer treatment. Several studies used NO
to alter blood flow and vascular permeability for increasing the efficiency of drug
delivery to tumors; other studies used NO to induce tumor cell killing or increase
the sensitivity of chemotherapy or radiotherapy.6-10 JS-K, [O2 -(2,4-dinitrophenyl)
1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate] is a NO donor
contains O2-aryl diazeniumdiolates (NONOates) structure (Fig. 1). The 2,4-
dinitrophenyl group of the prodrug JS-K can be removed by a reaction with
glutathione (GSH) to generates NO, which is catalyzed by glutathione-S-
transferases (GSTs).11 GSTs are overexpressed in many kinds of tumors. Especially
in multiple myeloma cells, GSTs are overexpressed about 7-fold comparing to
normal plasma cells. The higher concentration of GSTs in tumor cells catalyzes JS-
K to generate higher intracellular concentration of NO, which results in cytotoxic
activities. JS-K shows a concentration-dependent and high effective
antiproliferative effect to leukemia (HL-60), prostate cancer (PPC-1), multiple
3

myeloma (MM), hepatoma (Hep 3B), and lung cancer in vitro and in vivo.11-17 The
mechanisms of inducing cell death by JS-K includes arylation of GSH; induction
of protein kinases p38 and JNK (MAPK); and other cellular nucleophiles.15, 16
In this study, we have designed and synthesized a series of analogues of JS-K
and tested their anti-cancer activities. The compound 1 had a higher selectivity to
leukemia cells than to breast cancer and lung cancer. Similar to JS-K, it released
NO via activation by glutathione S-transferase (GST), increased the levels of
protein kinases p38, JNK and Bax to induce the cell apoptosis. However, it
decreased the level of Bcl-2 to promote further apoptosis, which is an opposite
effect compared to JS-K.
2. Results and discussion
JS-K was designed to be activated by GST for NO releasing, which is a part of
the mechanism of its anticancer activity.12 In this study, we postulated that it might
be able to increase the delivery efficiency of nitric oxide by linking two parts of
JS-K together. To synthesize the double-component O2-aryl diazeniumdiolate
derivatives, we tried to couple two molecules of JS-K together via an alkane linker
and obtained four new compounds (1 – 4, Fig. 1). The synthesis of compound 1
was described as shown in Scheme 1. The commercial compound 5 was reacted
with methanesulfonyl chloride following by NaI to generate compound 6 in a yield
4

of 50%; the latter was coupled with compound 7 to obtain compound 8 in a yield
of 66%. The BOC- protection groups of 8 were removed by trifluoroacetic
acid (TFA), and treated with nitric oxide gas to generate the intermediates 9, the
latter was reacted with 2,4-dinitrofluorobenzene to obtain the compounds 1 in a
final yield of 7.9% after three steps of reaction.
As shown in Scheme 2, compound 7 reacted with 1,2-dibromoethane (10a), 1,3-
dibromopropane (10b) or 1,4-bis(bromomethyl)-cyclohexane (10c) to obtain
compounds 11a – c with a yield of 43%, 78% and 75%, respectively. The BOC-
protection groups were removed by TFA followed by treatment of nitric oxide gas
to obtain the intermediates 12a – c, respectively. The intermediates 12a – c were
reacted with 2,4-dinitrofluorobenzene to obtain the compounds 2 – 4 in a final
yield of 10%, 17% and 24%, respectively, after three steps of reaction.
As shown in Table 1, the control compound JS-K had a high inhibition against
all five tested cancer cells including, leukemia cell CCRF-CEM, leukemia Jurket
cell, breast cancer cell BT474, breast cancer cell MDA-MB-231 and lung cancer
cell A549. The new compounds 1 - 4 had comparable activity to JS-K against all
these five tested cancer cells. Among them, the compound 1 showed higher
selectivity than JS-K. It had a similar IC50 to JS-K against leukemia CEM cells
(1.26 μM vs 0.89 μM). However, it showed less efficiency to breast cancer and
5

lung cancer. Its IC50s against other four cancer cells were 1.9 – 8.9 fold higher than
JS-K’s.
To explore the nitric oxide release of these compounds, a GST catalyzed NO
release assay was carried out in the presence of glutathione (GSH) and a NO
quantification reagent, diaminofluorescein-FM diacetate (DAF-FM DA).11 As
shown in Fig. 2, the control compound JS-K released NO faster than these double-
component O2-aryl diazeniumdiolate derivatives in the first hour. However, each
molecule of compounds 1 – 4 released about 2-fold amount of NO when the
incubation time was longer up to 5 h. In other words, each molecule of compounds
1 – 4 released up to four molecules of nitric oxide.
To determine whether apoptosis and cell cycle arrest resulting in inhibition of
cancer cell growth, we used flow cytometry to measure the effect of compound 1
against CEM cells, which had the highest selectivity among four double-
component O2-aryl diazeniumdiolate derivatives. As shown in Fig. 3, the results of
Annexin V-FITC/PI apoptosis detection indicated that the apoptotic population
increased with an incremental incubation periods. Since some suspended cells
showed a slight apoptosis even without any treatment, our measurement indicated
that untreated cells had an around 10% of apoptotic population including early
apoptosis and late apoptosis (Fig. 3, left panel). After 8 h of treatment, a moderate
response of apoptotic cells was observed with a percentage around 38%; and
6

similar phenomenon was observed after 24 h of incubation. Whereas, after 48 h
and 72 h treatment, significant apoptosis was induced with an average of more than
55% of apoptotic cells. The above results showed that compound 1 induced
apoptosis in a time-dependent manner. To explore whether compound 1 induced
apoptosis in a dose-dependent manner, we treated CEM cells at 2, 4, 8 and 16 µM
for 72 h, respectively. The data showed that at low concentration of 2 µM, the
apoptotic population was 12% (Fig. 3, right panel). When treated at 4 µM, the
apoptotic population of cells increased to 22%; and at 8 µM, apoptosis was greater
than 55%. When the cells treated at a concentration of 16 µM, the apoptotic
population was more than 95%. These results showed that compound 1 induced
apoptosis of CEM cells in a time- and dose-dependent manner.
As shown in Fig. 4, with the treatment of 8 µM compound 1, the G2/M phase
peak of CEM cells increased (cell accumulation from 28% to 46%) within 24 h.
After 48 h of incubation, around 55% cells went to G2/M phase. Accompanied
with the G2/M phase arrest, S phase peak significantly reduced to 8.2%. The shift
from S phase to G2/M phase became more obvious after 72 h incubation. All the S
phase was disappeared and about 64% cells were arrested in the G2/M phase.
These results indicated that compound 1 mainly arrests cell cycle at G2/M phase.
7

It has been reported that JS-K increased the level of JNK.12 In this study, we
explored whether JNK and other MAPKs family members (p38) were activated by
compound 1 in CEM cells.18 To investigate the cell apoptosis induced by
compound 1, western blot analysis was used to monitor the cell death process at
the molecular level. As shown in Fig. 5, compound 1 increased the levels of JNK
and p38 in CEM cells as early as in a few hours treatment. In addition, compound
1 also increased the levels of phosphorylated JNK and p38 at the same time.
Notably, these results suggest that compound 1 mediated CEM cell apoptosis via
MAPKs pathways.
It was also shown the effects of compound 1 on Bcl-2 family members. Bcl-2
protein is an anti-apoptotic regulator, which prevent apoptotic cell death and
facilitate MYC-induced transformation.19 As shown in Fig. 6, the level of Bcl-2
decreased in a time- and dose-dependent manner in the presence of compound 1.
Comparatively, the level of pro-apoptotic Bax increased in a time- and dose-
dependent manner after compound 1 treatment. Given that apoptotic control
depends on the fine balance between the pro-apoptotic and anti-apoptotic Bcl-2
family members, the increased ratio of Bax/Bcl-2 caused a shift toward apoptosis
in CEM cells. In addition, the increased ratio of Bax/Bcl-2 will facility compound
1 to overcome the chemotherapy resistance for cancer treatment.
8

3. Conclusions
In summary, we have prepared four double-component O2-aryl diazeniumdiolate
derivatives and tested their anti-cancer activities using two leukemia cell lines, two
breast cancer cell lines and one lung cancer line. Among them, compound 1
showed a higher selectivity than JS-K. It activated the JNK and p38 protein kinases
to induce cancer cell apoptosis. In addition, it increased the level of a pro-apoptotic
Bax and decreased the level of an anti-apoptotic member Bcl-2 to promote further
apoptosis. Compound 1 may be developed as a new anti-cancer agent since it
increases the Bax/Bcl-2 ratio to overcome the drug resistance.
Experimental
4.1. General materials and methods
Reagents and solvents for chemical synthesis were purchased from Aldrich
Chemical Co. or Sigma Chemical Co. and were used without further purification.
All reactions involving air- or moisture-sensitive reagents or intermediates were
performed under argon. Analytical TLC was performed using Silicycle silica gel
60 Å F254 plates (0.25 mm), and were (was) visualized by UV irradiation (254
nm). Flash chromatography was performed using Silicycle silica gel (40-60 mesh).
1H and 13C NMR spectra were obtained using a Varian 400 MHz or 500 MHz
NMR spectrometer. Chemical shifts are reported in parts per million (ppm, δ)
9

referenced to the residual 1H of the solvent (CDCl3, δ 7.26). 13C NMR spectra were
referenced to the residual 13C resonance of the solvent (CDCl3, δ 77.16). Splitting
patterns are designated as follows: s, singlet; d, doublet; q, quartet; m, multiplet.
High resolution mass spectra were obtained at the Arizona State University CLAS
High Resolution Mass Spectrometry Facility. Fluorescence spectral measurements
were made using a Hitachi F2000 fluorescence spectrophotometer.
Diaminofluorescein-FM diacetate (DAF-FM DA) and L-glutathione reduced
were purchased from Sigma Chemicals (St. Louis, MO). Glutathione S-
Transferase was purchased from GenScript (Piscataway, NJ). BT474 and MDA-
MB-231 breast cancer cells, A549 lung cancer cells, DU145 prostate cancer cells,
CCRF-CEM and Jurkat E6-1 leukemia cells were cultured at 37 oC in a 5% CO2
atmosphere and grown in Gibco® RPMI 1640 medium supplemented with 10%
fetal bovine serum (FBS) and 1% antibotic-antimycotic mix antibiotic supplement
before use. Bax, p38, p-JNK and JNK antibodies were purchased from Bioss Inc
(Boston, USA). Phospho-p38 and β-actin antibodies were obtained from Santa
Cruz biotechnology (Santa Cruz, USA) unless otherwise indicated.
4.2. Synthesis and characterization of double-component diazeniumdiolate
derivatives.
10

4.2.1. tert-Butyl 4-(iodomethyl)piperidine-1-carboxylate (6)
To an ice-cooled stirred solution of 400 mg (1.86 mmol) of compound 5 in 5 mL
of CH2Cl2 was added 0.52 mL (375 mg, 3.72 mmol) of triethylamine and 0.17 mL
(256 mg, 2.23 mmol) of methanesulfonyl chloride. The reaction mixture was
allowed to warm to ambient temperature and stirred for 2 h. The reaction mixture
was diluted with 35 mL of CH2Cl2, washed successively with 20 mL of water, 20
mL of satd. aq. NaHCO3, 20 mL of brine, dried (MgSO4) and concentrated under
reduced pressure. The resulting crude product was dissolved in 2 mL of acetonitrile
and 1.39 g (9.30 mmol) of NaI was added. The reaction mixture was stirred at r.t.
for 12 h, and poured into 50 mL of water, extracted with two 30-mL portions of
ethyl acetate. The combined organic extract was washed with 20 mL of brine, dried
(MgSO4) and concentrated under diminished pressure. The residue was purified by
flash chromatography on a silica gel column (15 × 2 cm). Elution with 2:3 hexane–
ethyl acetate afforded 6 as a colorless oil; yield 302 mg (50%); silica gel TLC Rf
0.31 (2:3 hexane–ethyl acetate); 1H NMR (400 MHz, CDCl3) δ 1.11-1.19 (m, 2H),
1.45 (s, 9H), 1.59-1.62 (m, 1H), 1.82 (d, 2H, J = 13.2 Hz), 2.66 (brs, 2H), 3.12 (d,
2H, J = 6.8 Hz) and 4.10 (brs, 2H); 13C NMR (100 MHz, CDCl3) δ 13.5, 28.4,
32.5, 38.4, 43.5, 79.3 and 154.5; HRMS (ESI) m/z: [M + H]+ Calcd for
C11H21INO2, 326.0617; Found 326.0615.
11

4.2.2. tert-Butyl 4-((1-(tert-Butoxycarbonyl)piperidin-4-yl)methyl)piperazine-
1-carboxylate (8)20
To a stirred solution of 250 mg (0.77 mmol) of 6 in 2 mL of anhydrous DMF was
added 319 mg (2.31 mmol) of K2CO3 followed by 0.14 mg (0.77 mmol) of 1-(tert-
Butoxycarbonyl)piperazine (7) at rt. After 15 h, the reaction mixture was poured
into 50 mL of water and extracted with three 30-mL portions of ethyl acetate. The
combined organic extract was washed with 40 mL of brine, dried (MgSO4) and
concentrated under diminished pressure. The residue was purified by flash
chromatography on a silica gel column (15 × 2 cm). Elution with 1:1 hexane–
EtOAc afforded 8 as a colorless oil; yield 194 mg (66%); silica gel TLC Rf 0.36
(1:1 hexane–EtOAc); 1H NMR (500 MHz, CDCl3) δ 1.06 (qd, 2H, J = 12.4, 4.3
Hz), 1.45 (d, 18H, J = 2.3 Hz), 1.62 (ddd, 1H, J = 11.2, 7.4, 3.7 Hz), 1.67 – 1.78
(m, 2H), 2.15 (d, 2H, J = 7.2 Hz), 2.32 (t, 4H, J = 5.0 Hz), 2.67 (t, 2H, J = 12.6
Hz), 3.39 (t, 4H, J = 5.1 Hz), 4.07 (s, 2H); 13C NMR (126 MHz, CDCl3) δ 28.58,
28.62, 30.85, 33.71, 43.61, 44.07, 64.69, 79.36, 79.66, 154.92, 155.04. HRMS
(ESI) m/z: [M + H]+ Calcd for C20H38N3O4, 384.2862; Found 384.2859.
4.2.3. Compound 1
To a solution of 152 mg (0.40 mmol) of 8 in 500 µL of CH2Cl2 was added 500 µL
of CF3COOH.21,22 The reaction mixture was stirred overnight and then
12

concentrated under diminished pressure to obtain the deprotected amine, which
was used in next step without further purification.
To a stirred solution of obtained amine in 15 mL of 1:3 methanol-diethyl ether was
added 1.19 mL (1.19 mmol) of 1M NaOMe solution in Parr bottle at room
temperature. The reaction mixture was charged with 60 psi of nitric oxide and
stirred at room temperature for 24 h. The white crystalline solid of 9 was collected
by filtration and washed with diethyl ether and dried under vacuum.
To an ice-cooled solution of the above obtained solid 9 in 1 mL of aqueous 5%
NaHCO3 solution was added a solution of 47.0 mg (0.25 mmol) of 2,4-
dinitrofluorobenzene in 1 mL of 1,4-dioxane. The reaction mixture was allowed to
ambient temperatures and stirred for overnight. The reaction mixture was diluted
with 5 mL of water and extracted with two 10 mL portions of ethyl acetate. The
organic phase was dried (MgSO4) and concentrated under diminished pressure.
The residue was purified by chromatography on a silica gel column (10 × 1 cm).
Elution with 15:1 ethyl acetate/methanol gave desired product 1 as a yellow foam;
yield 7.2 mg (7.9%); silica gel TLC Rf 0.19 (2:1 ethyl acetate/hexane); 1H NMR
(400 MHz, CDCl3) δ 1.36-1.45 (m, 1H), 1.56 (brs, 3H), 1.78 (brs, 1H), 1.91 (d, 2H,
J = 12.0 Hz), 2.33 (d, 2H, J = 4.0 Hz), 2.67 (s, 3H), 3.09 (t, 2H, J = 12.0 Hz), 3.47
(d, 2H, J = 16.0 Hz), 3.67 (s, 3H), 7.09 (d, 1H, J = 8.0 Hz), 7.66 (d, 1H, J = 8.0
13

Hz), 8.23 (dd, 1H, J1 = 8.0 Hz, J2 = 4.0 Hz), 8.45 (dd, 1H, J1 = 8.0 Hz, J2 = 4.0
Hz), 8.68 (d, 1H, J = 4.0 Hz) and 8.88 (d, 1H, J = 4.0 Hz); 13C NMR (100 MHz,
CDCl3) δ 29.0, 30.3, 50.7, 51.1, 51.9, 63.3, 117.8, 119.4, 122.3, 124.0, 128.3,
129.2, 137.4, 137.9, 141.6, 142.4, 149.7, and 154.1; HRMS (ESI) m/z: [M + H]+
Calcd for C22H26N11O12 636.1762; Found 636.1757.
4.2.4. Di-tert-butyl 4,4'-(ethane-1,2-diyl)bis(piperazine-1-carboxylate) (11a)
To a stirred solution of 200 mg (1.07 mmol) of 1-(tert-Butoxycarbonyl)piperazine
(7) in 5 mL of dry DMF were added 47.0 µL (101 mg, 0.54 mmol) of 1,2-
dibromoethane (10a) and 592 mg (4.28 mmol) of K2CO3 at room temperature.
After 24 h, reaction mixture was diluted with 25 mL of water and extracted with
two 50 mL portions of ethyl acetate. The organic phase was dried (MgSO4) and
concentrated under diminished pressure. The residue was purified by
chromatography on a silica gel column (10 × 2 cm). Elution with 19:1 ethyl
acetate/methanol gave desired product 11a as a colorless oil; yield 183 mg (43%);
silica gel TLC Rf 0.27 (9:1 ethyl acetate/methanol); 1H NMR (500 MHz, CDCl3) δ
1.45 (s, 18H), 2.42 (t, 8H, J = 5.1 Hz), 2.52 (s, 4H), 3.42 (t, 8H, J = 5.1 Hz); 13C
NMR (126 MHz, CDCl3) δ 28.57, 43.33, 44.07, 53.59, 56.07, 79.76, 154.85.
HRMS (ESI) m/z: [M + H]+ Calcd for C20H39N4O4 399.2971; Found 399.2969.
14

The procedure to synthesize compounds 11b and 11c is same to 11a. And the
procedure to synthesize compounds 2 – 4 is same to compound 1.
4.2.5. Compound 2
Pale yellow foam; yield 8.0 mg (10%); silica gel TLC Rf 0.19 (9:1 ethyl
acetate/methanol); 1H NMR (400 MHz, CDCl3) δ 2.50 (brs, 2H), 2.65 (brs, 4H),
2.76 (brs, 6H), 3.70 (brs, 4H), 3.86 (t, 2H, J = 4.0 Hz), 4.29 (t, 2H, J = 4.0 Hz),
7.66 (d, 2H, J = 8.0 Hz), 8.46 (dd, 2H, J1 = 8.0 Hz, J2 = 4.0 Hz), 8.87 (d, 2H, J =
4.0 Hz); 13C NMR (100 MHz, CDCl3) δ 52.1, 53.6, 55.3, 117.7, 122.2, 129.2,
137.3, 142.3 and 153.9; HRMS (ESI) m/z: [M + H]+ Calcd for C22H27N12O12
651.1871; Found 651.1868.
4.2.6. Di-tert-butyl 4,4'-(propane-1,3-diyl)bis(piperazine-1-carboxylate) (11b)
Colorless oil; yield 521 mg (78%); silica gel TLC Rf 0.27 (9:1 ethyl
acetate/methanol); 1H NMR (500 MHz, CDCl3) δ 1.44 (s, 18H), 1.79 (q, 2H, J =
7.5 Hz), 2.45 (t, 12H, J = 5.5 Hz), 3.47 (t, 8H, J = 5.0 Hz). 13C NMR (126 MHz,
CDCl3) δ 28.53, 43.12, 43.81, 53.03, 56.49, 79.96, 154.73. HRMS (ESI) m/z: [M +
H]+ Calcd for C21H41N4O4 413.3128; Found 413.3127.
4.2.7. Compound 3
15

Pale yellow foam; yield 28 mg (17%); silica gel TLC Rf 0.19 (9:1 ethyl
acetate/methanol); 1H NMR (400 MHz, CDCl3) δ 1.74 (brs, 2H), 2.48 (brs, 6H),
2.70 (brs, 6H), 3.69-3.87 (m, 8H), 7.66 (d, 2H, J = 8.0 Hz), 8.45 (dd, 2H, J1 = 8.0
Hz, J2 = 4.0 Hz) and 8.88 (d, 2H, J = 4.0 Hz); 13C NMR (100 MHz, CDCl3) δ 31.3,
36.5, 52.7, 56.5, 117.5, 122.2, 129.3, 137.4, 142.1 and 153.9; HRMS (ESI) m/z: [M
+ H]+ Calcd for C23H29N12O12 665.2028; Found 665.2023.
4.2.8. Di-tert-butyl 4,4'-(cyclohexane-1,4-diylbis(methylene))bis(piperazine-1-
carboxylate) (11c)
Colorless oil; yield 317 mg (75%); silica gel TLC Rf 0.22 (1:1 hexane–EtOAc); 1H
NMR (400 MHz, CDCl3) δ 1.29-1.36 (m, 4H), 1.45 (s, 18H), 1.47-1.51 (m, 4H),
1.69 (m, 2H), 2.20 (d, 4H, J = 7.6 Hz), 2.31 (t, 8H, J = 4.0 Hz) and 3.40 (t, 8H, J =
2.6 Hz); 13C NMR (100 MHz, CDCl3) δ 27.1, 28.4, 31.4, 32.3, 53.4, 62.5, 79.2 and
154.6; HRMS (ESI) m/z: [M + H]+ Calcd for C26H49N4O4 481.3754; Found
481.3751.
4.2.9. Compound 4
Pale yellow foam; yield 8.4 mg (24%); silica gel TLC Rf 0.13 (19:1 ethyl
acetate/methanol); 1H NMR (400 MHz, CDCl3) δ 1.29-1.35 (m, 4H), 1.48-1.54 (m,
4H), 1.69 (m, 2H), 2.19 (d, 4H, J = 6.8 Hz), 2.31 (t, 8H, J = 4.4 Hz), 3.44 (t, 8H, J
= 2.6 Hz), 7.64 (d, 2H, J = 8.0 Hz), 8.40 (dd, 2H, J = 9.2 Hz and 2.4 Hz) and 8.43
16

(d, 2H, J = 2.8 Hz); 13C NMR (100 MHz, CDCl3) δ 26.7, 31.8, 32.1, 53.3, 62.1,
117.2, 122.3, 129.3, 136.7, 141.5 and 154.3; HRMS (ESI) m/z: [M + H]+ Calcd for
C28H37N12O12 733.2654; Found 733.2651.
4.3. Nitric oxide release assay
Glutathione S-Transferase catalyzed nitric oxide release was determined with a
fluorescence-based assay. A double-component diazeniumdiolate derivative (1
μM) was dissolved in 100 µL of 100 mM phosphate-buffered saline (PBS), pH 7.4,
containing 1 unit of Glutathione S-Transferase, 50 μM of L-glutathione reduced
(GSH) and 5 μM of DAF-FM DA. The reaction mixture was incubated at rt in the
dark. The fluorescence intensity was measured to determine the levels of released
NO at desired time points with 2.5 nm band width, 495 nm excitation and 515 nm
emission wavelength. Fluorescence spectral was measured setting same conditions
in all experiments using a Hitachi F2000 fluorescence spectrophotometer.23-25
4.4. Cell culture
Exponentially growing cells were harvested and plated in 96-well plates at a
concentration of 1 × 104 cells/well. After incubation at 37 oC for 24 h, the cells
were treated with analogues of JS-K at different concentrations for an additional
24, 48 or 72 h. Then 20 µL of MTT (5 mg/mL) or MTS was added to each well
and the plates were incubated at 37 oC for 4 h. The supernatant was discarded for
MTT assay, and 100 µL of DMSO was added to each well. For the MTS assay,
this step is not necessary. The absorbance was recorded at 490 nm after 15 min.
Inhibition of cell growth was obtained by the following formula: Inhibition of cell
17

growth (%) = (ODnegative control – ODtreatment) × 100% / (ODnegative control
–
ODbackground).26,27 Data are reported as the mean of three independent experiments,
each run in quintuplicate.
4.5. Annexin V-FITC apoptosis detection
Phosphatidylser (PS) translocated from the inner to the outer leaflet of the plasma
membrane during cell apoptosis. FITC Annexin V apoptosis detection kit (BD,
USA) was used to detect PS by staining with fluorescent-conjugated Annexin V
which has a high affinity to PS.28 The experiment was carried out in two groups
according to different administration methods. Cells (2 × 106 cells/10cm cell
culture dish) were treated with 8 μM 1 for 8, 24, 48 or 72 h in one group. Cells
with the same concentration in another group were cultivated for 72 h with 2, 4, 8
or 16 μM 1. After twice wash-centrifuge steps, cells were resuspended in 1 ×
binding buffer at a concentration of 1 × 106 cells/mL. Cells (1 × 105 cells) were
incubated for 15 min at rt in the dark after stained with 5 L of FITC Annexin V
and analyzed by BD FACS Aria II after adding 5 L of propidium iodide (PI) and
400 L of 1 × binding buffer. Cytographs were performed using FlowJo software.
4.6. Cell cycle analysis29
CEM cells were treated with 1 for indicated concentration and time as described
above in cell apoptosis assay. After washing twice, cells were fixed with ice-cold
70% ethanol slowly and permeabilized at -20 oC overnight. Cells were resuspended
in 1× PBS at a concentration of 1×106 cells/mL. Then, cells (1×105 cells) were
incubated with 400 L of PI/RNase staining buffer (BD, USA) for 20 min at rt in
the dark. At last, cells were analyzed using BD FACS Aria II.
4.7. Western blotting
18

CEM cells were cultured with the indicated concentrations of 1 for the
determinate times. After twice wash-centrifuge steps, cells were lysed using lysis
buffer (2.1 μg/mL aprotinin, 0.5 μg/mL leupeptin, 4.9 mM MgCl2, 1 mM
orthovanadate, 1% Triton X-100, and 1 mM PMSF). Proteins in cell lysate were
loaded on a denaturing SDS-PAGE gel and transferred to PVDF membrane
(Millipore, Billerica, MA).30 After blocking with 5% fat-free milk, the membranes
were washed by PBS containing 0.1% Tween 20 and immunoblotted with primary
antibodies, such as Bcl-2, Bax, phospho(Thr183/Tyr185)-JNK, JNK, phospho-p38
and p-38 followed by respective horseradish peroxidase conjugated secondary
antibodies. Signals were visualized with enhanced ECL chemiluminescence
detection reagents (Millipore, Billerica, MA) and visualized on X-ray film (Fuji
Photo Film, Tokyo, Japan). β-actin was used as reference to determine the protein
relative level.
19

NO2
O
N+
O
N
O
N
O
N
N
N
N
O2N
O2N
NO2
1
O
N
O
N
N
N
O2N
NO2
O2N
NO2
N
N
N
O
2
N
O
O
O
N
O
N
O
N
N
N
N
N
N
O2N
NO2
O2N
NO2
3
NO2
O
N
O2N
NO2
N
O
N
N
N
N
N
N
O
O2N
O
4
O
O
N
N
N
O
N
O2N
NO2
O
JS-K
Fig. 1. Structures of compounds 1 – 4 and JS-K.
20

350
300
250
200
150
100
50
JSK
1
2
3
4
0
0
100
200
300
400
Time (min)
Fig. 2. NO release from 1 – 4 and JS-K in an aqueous solution.
21

Fig. 3. Determination of cell death by flow cytometry of Annexin V-FITC/PI
staining in CEM cells treated with compound 1 at indicated periods. The CEM
cells treated with compound 1 at a concentration of 8 µM for 0 – 72 hours (left
panel); or at a concentration of 0 – 16 µM for 72 hours (right panel).
22

Fig. 4. Determination of cell cycle blockade by flow cytometry of PI staining in
CEM cells treated with compound 1 at indicated periods. The CEM cells treated
with compound 1 at a concentration of 8 µM for 24, 48 and 72 hours.
23

Fig. 5. Western blot analysis of compound 1 induced MAPKs pathway in CEM
cells. CEM cells were exposed to 8 µM compound 1 for 0 – 6 hours, and then
whole-cell lysates were subjected to Western blotting with p-JNK and p-p38
antibodies, respectively. Reblotting with JNK and p38 antibodies, respectively.
24

Fig. 6. Western blot analysis of compound 1 induced apoptotic pathway. CEM
cells were treated with compound 1 at 8 µM for 0 – 72 h (left panel), and at 2 – 8
µM for 72 h. The level of Bcl-2 and Bax were determined with Bcl-2 and Bax
antibodies, respectively.
25

Table 1. Half maximal inhibitory concentration (IC50) of compounds 1 – 4 against
different cancer cells after 72 h.
Cell line
Half maximal inhibitory concentration (IC50, μM)
1
2
3
4
JS-K
CCRF-CEM
Jurkat E6-1
BT474
1.26 ± 0.08
1.94 ± 0.08
7.06 ± 1.03
17.86 ± 1.01
11.3 ± 1.61
2.64 ± 0.37
3.71 ± 0.14
10.7 ± 0.97
17.6 ± 0.68
17.1 ± 1.02
3.73 ± 0.15
4.06 ± 0.18
10.8 ± 0.18
> 20
2.54 ± 0.14
3.01 ± 0.22
6.01 ± 0.21
0.89 ± 0.05
0.92 ± 0.07
3.66 ± 0.41
MDA-MB-231
A549
16.4 ± 0.53 7.93 ± 0.47
18.5 ± 0.15 2.11 ± 0.15
18.8 ± 0.21
26

Scheme 1. Synthesis of compound 1.
Boc
Boc
b
OH
I
a
N
N
c
N
N
N
Boc
Boc
Boc
N
5
6
8
NH
7
O
N
NO2
O
N
NaO
N
ONa
O
N
O
d
N
N
N
N
N
N
N
N
O
O
N
N
O2N
O2N
NO2
9
1
a: (1), CH3SO2Cl, NEt3, CH2Cl2; (2) NaI, CH3CN. b: K2CO3, DMF. c: (1) CF3COOH, CH2Cl2; (2) NO, 60 psi,
NaOMe, MeOH/Et2O. d: 2,4-dinitrofluorobenzene, NaHCO3, dioxane/H2O.
27

Scheme 2. Synthesis of compounds 2 – 4.
NH
Boc
Boc
a
b
Br
X
Br
+
N
N
N
Boc
N
X
N
7
10a - c a: X = 0
b: X = -CH2-
11a - c
c: X =
O
N
O
N
O
N
O
N
O
O
c
NaO
ONa
N
N
N
N
N
N
N
N
N
X
N
N
X
N
O2N
NO2
O2N
NO2
2: X = 0
3: X = -CH2-
12a - c
4: X =
a: K2CO3, DMF. b: (1) CF3COOH, CH2Cl2; (2) NO, 60 psi, NaOMe, MeOH/Et2O. c: 2,4-dinitrofluorobenzene,
NaHCO3, dioxane/H2O.
28

Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was performed with the support of the Bill & Melinda Gates Foundation
through the Grant Challenges Explorations initiative (Grant No. OPP1108171); the
Experiment Animal Science and Technology Project of Zhejiang Province (Grant
No. 2016C37143) and Taizhou science and technology plan project (Grant No.
15yw07).
29