Design, synthesis and biological evaluation of novel ferrocene-pyrazole derivatives containing nitric oxide donors as COX-2 inhibitors for cancer therapy

Article abstract of DOI:10.1016/j.ejmech.2018.08.048

A series of novel ferrocene-pyrazole derivatives containing nitric oxide donors as COX-2 inhibitors for cancer therapy were designed, synthesized and biologically evaluated. Among them, compound 7l displayed the most potent inhibitory against COX-2 (IC₅₀ = 0.82 μM) and antiproliferative activities against Hela cells (IC₅₀ = 0.34 μM) compared with Celecoxib (IC₅₀ = 0.38 and 7.91 μM). The further mechanistic studies revealed that 7l could induce apoptosis of Hela cells by mitochondrial depolarization and the antiproliferative activities of 7l were positively correlated with the levels of intracellular NO release in Hela cells. Most notably, 7l could dramatically suppress tumor growth in Hela cells xenografted mouse model. In summary, compound 7l may be promising candidates for cancer therapy.

Full text of DOI:10.1016/j.ejmech.2018.08.048

Accepted Manuscript
Design, synthesis and biological evaluation of novel ferrocene-pyrazole derivatives
containing nitric oxide donors as COX-2 inhibitors for cancer therapy
Shen-Zhen Ren, Zhong-Chang Wang, Dan Zhu, Xiao-Hua Zhu, Fa-Qian Shen, Song-
Yu Wu, Jin-Jin Chen, Chen Xu, Hai-Liang Zhu
PII:
S0223-5234(18)30718-9
10.1016/j.ejmech.2018.08.048
EJMECH 10659
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 April 2018
Revised Date: 20 July 2018
Accepted Date: 17 August 2018
Please cite this article as: S.-Z. Ren, Z.-C. Wang, D. Zhu, X.-H. Zhu, F.-Q. Shen, S.-Y. Wu, J.-J. Chen,
C. Xu, H.-L. Zhu, Design, synthesis and biological evaluation of novel ferrocene-pyrazole derivatives
containing nitric oxide donors as COX-2 inhibitors for cancer therapy, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.048.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, Synthesis and Biological evaluation of Novel
Ferrocene-Pyrazole Derivatives Containing Nitric Oxide
Donors as COX-2 Inhibitors for Cancer Therapy
Shen-Zhen Ren†, Zhong-Chang Wang†*, Dan Zhu , Xiao-Hua Zhu , Fa-Qian
Shen, Song-Yu Wu , Jin-Jin Chen, Chen Xu*, Hai-Liang Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210023, PR China
*Corresponding author. Tel. & fax: +86-025-89682572; e-mail: zhuhl@nju.edu.cn
†These two authors equally contributed to this paper.

ACCEPTED MANUSCRIPT
Abstract
A series of novel ferrocene-pyrazole derivatives containing nitric oxide donors as
COX-2 inhibitors for cancer therapy were designed, synthesized and biologically
evaluated. Among them, compound 7l displayed the most potent inhibitory against
COX-2 (IC50 = 0.82 µM) and antiproliferative activities against Hela cells (IC50 = 0.34
µM) compared with Celecoxib (IC50 = 0.38 and 7.91 µM). The further mechanistic
studies revealed that 7l could induce apoptosis of Hela cells by mitochondrial
depolarization and the antiproliferative activities of 7l were positively correlated with
the levels of intracellular NO release in Hela cells. Most notably, 7l could
dramatically suppress tumor growth in Hela cells xenografted mouse model. In
summary, compound 7l may be promising candidates for cancer therapy.
Keywords:
Pyrazole
Ferrocene
COX-2 inhibitor
NO-Releasing
Anticancer

ACCEPTED MANUSCRIPT
1. Introduction
Cyclooxygenase (COX) is a rate-limiting enzymes in the biosynthesis of
prostaglandins (PGs), which are involved in various physiological and
pathophysiological processes such as inflammation, gastrointestinal cytoprotection
and ulceration, as well as tumor angiogenesis[1-3]. In general, there exist mainly two
isoforms: a constitutive form (COX-1) and an inducible form (COX-2), and recently a
third isoform named COX-3 is also discovered[4, 5]. COX-1 is constitutively
expressed in most normal tissues such as the gastrointestinal tract, kidneys, platelets
and other organisms and plays an important role in the synthesis of PGs which are
involved in the regulation of physiological functions[6]. In contrast, COX-2 is
inducible and undetectable in most normal tissues, but is induced by stimuli such as
pro-inflammatory cytokines, growth factors and oncogenes[7]. Moreover, multiple
lines evidences have confirmed that the overexpression of COX-2 in several solid
malignancies is associated with the formation of carcinogens, tumor proliferation,
angiogenesis, invasiveness and prevention of apoptosis[2, 8]. Numerous studies have
demonstrated the inhibition of COX-2 can prevent the formation of tumor and COX-2
inhibitors could act as an chemotherapy agents to diminishing the occurrence and
growth of malignancies. Therefore, the development of novel COX-2 inhibitors seems
be a rational strategy for cancer treatment[9].
At present, a large number of selective COX-2 inhibitors, named coxibs, have been
discovered. As illustrated in Fig. 1, these COX-2 inhibitors (a ~ c) show efficacy in a
great deal of cancer studies, such as antiproliferation, apoptosis, migration, and
invasion[10]. Unfortunately, their clinical use has been limited by numerous side
effects, especially cardiovascular hazard and myocardial infarction, in long term use
and high dosage[11, 12]. Due to increased risk of cardiovascular events, Rofecoxib
and Valdecoxib were withdrawn from the market in 2004 and 2005 respectively.
Recently, a novel class of agents, named COX-inhibiting nitric oxide (NO) donors
(CINODs), have been developed by conjugating a COX inhibiting feature with a NO
donor[13]. Some CINODs, such as Naproxcinod (d, Fig. 1), have underwent clinical
development studies and shown a more favorable clinical profile than the parent

ACCEPTED MANUSCRIPT
Naproxen[14]. As an important signal molecule, NO plays an essential and
far-reaching role in regulating various physiological and pathological process, such as
vasodilation, thrombosis, and immune system[15, 16]. Besides, numerous literature
provide evidence that the high concentration of NO was effective on some malignant
tumors, including breast, ovarian, prostate and pancreatic cancer, by inhibiting the
proliferation and promoting their apoptosis[17, 18]. Considering its specific character
of NO, researchers are increasingly interested in the development of a new class of
coxibs containing NO-releasing moiety.
In general, diarylheterocycle is a known pharmacophore and has been widely
explored for the development of selective COX-2 inhibitors.[19, 20] Additionally,
sulfonamides moiety plays a crucial role in COX-2 selectivity and many COX-2
inhibitors were substituted by this moiety at para-position of one aryl ring, such as
Celecoxib, Valdecoxib and Parecoxib (Fig.1).[21, 22] Among them, Celecoxib
exhibited the most potent COX-2 inhibitory activities and recent study revealed that
this famous COX-2 inhibitor possess promising anticancer properties.[23] Based on
the structure of these coxibs, diarylpyrazole sulfonamides scaffold was extracted and
a series of diarylpyrazole sulfonamides derivatives have been designed by our group.
Meanwhile, we noticed that ferrocene, one of the most promising organometallic
compound, has attracted active interest in medicinal chemistry due to its excellent
biological activities, such as non-toxicity, antimicrobial and anticancer, and several
ferrocene derivatives with excellent biological activities have been reported. [24, 25].
Various studies have revealed that substitution of a phenyl ring of tested biomolecular
with a ferrocene fragment always can result in unexpected biological activity[26-29].
Thus, we substituted a phenyl group of Celecoxib with ferrocene and obtained
compound 2. The result revealed that compound 2 exhibited an equal COX-2
inhibitory activity (IC50 = 0.46 µM) compared with Celecoxib (IC50 = 0.38 µM).
Inspired by the interesting result, we expected that incorporation of the ferrocene
fragment into the diarylpyrazole sulfonamides scaffold might have an attracting
structural result for the development of novel COX-2 inhibitors.
Considerig the side-effects of tNSAIDs, NO donors were introduced into the

ACCEPTED MANUSCRIPT
ferrocene-pyrazole sulfonamide scaffold for purpose of minimizing the side-effects
and enhancing the anticancer activities. Herein, we designed and synthesized a series
of ferrocene-pyrazole sulfonamide hybridizing NO donors with different length to
explore their COX-2 inhibition and anticancer activities.
2. Results and discussion
2.1 Chemistry
In this study, a series of novel ferrocene-pyrazolo sulfonamide derivatives
containing nitric oxide donors were synthesized and their structures were outlined in
Table 1 and Table 2. As depicted in Scheme 1, the target compound 2 was
synthesized
from
5-(1,2,3-trifluoromethyl-2,4-dione)
ferrocene
(1)
and
4-hydrazinylbenzenesulfonamide in ethanol under stirred at 80 ℃ 。 And the
intermediate (1) was obtained by acetylferrocene in THF reacting with ethyl
trifluoroacetate and sodium hydride. The synthesis of compounds 6a ~ 6h was
outlined in Scheme 2. Acetylferrocene and dimethyl oxalate were reacted in benzene
in the presence of t-BuOK to generated intermediate (3), which then reacted with
corresponding monosubstituted hydrazines hydrochlorides in ethanol to produce
intermediate (4a ~ 4h). Meanwhile, these methyl ester (4a ~ 4h) were hydrolyzed by
potassium hydroxide into corresponding pyrazolesulfonamide carboxylic acids (6a ~
6h), which were directly reacted with the nitric acid salts of 3-(nitrooxy)propylamine
(8b) to produce target compounds 6a ~ 6h. Compounds 7a ~ 7q were synthesized
from similar methods shown in Scheme 3. Like Scheme 2, compound 4h was reacted
with compounds 8a ~8h, 9a ~ 9h or 10 ~ 13 in the presence of EDC•HCl, DMAP and
triethylamine through esterification condensation reaction to obtain the corresponding
target compounds 7a ~ 7q. The synthesis of a series of NO-donors was described in
Scheme 4 according to the literature procedure[28, 29]. Corresponding amino alcohol
or diol were reacted with fuming nitric acid or concentrated nitric acid in the presence
of acetic anhydride to produce compounds 8a ~8h, 9a ~ 9h or 10 ~ 13, respectively.
1
1
These synthesized compounds were characterized by H NMR spectroscopy, C
NMR spectroscopy, melting point, ESI-MS and elemental analysis, in accordance
with their depicted structures. In addition, The crystal structures of compounds 2

ACCEPTED MANUSCRIPT
(CCDC: 1834063) and 7l (CCDC:1834102) were determined by X-ray diffraction
analysis and the crystal data presented in Table 3 and Fig. 2. These data are provided
free of charge by The Cambridge Crystallographic Data Centre.
2.2. Biological activity
2.2.1 In vitro COX-2 inhibitory activities
The ability of the synthesized compounds to inhibit COX-1 and COX-2 were
evaluated using human COX-1/COX-2 ELISA kit and Celecoxib was evaluated in
parallel as a standard compound. As summarized in Table 4, the majority of the tested
compounds exhibited excellent COX-2 inhibitory activity. Compound 2, an analog of
Celecoxib, exhibited an equal COX-2 inhibitory activity (IC50 = 0.46 ± 0.12 µM)
compared with Celecoxib (IC50 = 0.38 ± 0.08 µM). This suggested that the
substitution of a phenyl ring of Celecoxib with a ferrocene fragment had no affect on
its COX-2 inhibitory. In addition, it was notable that compound 6h was more potent in
COX-2 inhibition (IC50 = 0.82 ± 0.12 µM) than compounds 6a ~ 6g, which was
consistent with reported conclusion that the benzensulfonamide played a crucial role
in the COX-2 inhibitory activity. After the introduction of different NO donors, all the
obtained hybrid compounds 7a ~ 7q exhibited potent COX-2 inhibitory activity
compared with compound 6h, and among them ,compound 7l exhibited the most
significant selectivity with IC50 values of inhibition COX-1/COX-2 were 0.82 and
38.02 µM. The experiments suggested that these hybrid compounds containing a
ferrocene-pyrazol sulfonamide framework and NO-donors showed good
COX-1/COX-2 selectivity.
2.2.2 In vitro antiproliferative activities
According the COX-2 inhibitory activities, these synthesized compounds were
evaluated by MTT assay for their cytotoxic against four cancer cell lines (A549:
human lungs cell line; HeLa: human cervix cell line; MCF-7: human breast cancer
cell line; B16-F10: murine melanoma cell line) and one non-cancer cell line (293T:
human kidney epithelial cell line). Celecoxib as a reference compound was selected
for comparison. As shown in Table 5, majority of the tested compounds exhibited
excellent growth inhibitory effects against four cancer cell lines, especially against

ACCEPTED MANUSCRIPT
Hela cells, and low cytotoxic effects against non-cancer cell line. Interestingly,
compound 6h displayed more potent antiproliferative activity against four cancer cell
lines compared with compounds 6a ~ 6h, which was consistent with the COX-2
inhibitory activity and suggested that the introduction of sulfonamide group was
crucial to enhance the anti-tumor activity. Moreover, after the introduction of different
NO-donors, we found that the hybrid compounds 7k ~ 7q linking with ester linkage
were more potent antiproliferative activity than those linking with amide linkage,
especially compound 7l, which exhibited the most potent anti-tumor activity against
Hela cells with IC50 of 0.34 µM. It may result from that the NO-donors of compounds
7k ~ 7q were more easily hydrolyzed and the released NO enhanced their
antiliferative activity.
2.2.3 Compound 7l caused cell apoptosis in Hela cells
Considering the antiproliferative and COX-2 inhibitory activity, we evaluated the
effects of compound 7l on the apoptosis of cancer cell using Annexin V/PI double
staining assay though flow cytometry. As shown in Fig. 3A and B, the percentage of
apoptotic cells was significantly elevated from 2.98% to 46.20% after treating Hela
cells with varying does of 0, 1.25, 2.5, 5 and 10 µM compound 7l for 24 h. Meanwhile,
Hela cells were treated with 5 µM compound 7l for different times (0, 12, 24 and 36
h), and the percentage of early and late apoptotic cells was increased from 4.15% to
78.10% (Fig. 3C and D). Taken together, the above results further confirm that
compound 7l could suppress the growth of Hela cells by inducing apoptosis in a dose
and time-dependent manner.
2.2.4 Mitochondrial membranepotential analysis
It is well known that the activation of mitochondrial pathway plays an essential role
in the early event of cells apoptosis, and the loss of mitochondrial membrane potential
(MMP, △Ψm) is a hallmark of mitochondrial pathway activation. JC-1, an indicator of
mitochondrial inner membrane depolarization, was used to monitor the changes of
MMP of Hela cells treated by compound 7l. Our results suggest that, with the
concentration increasing, the percentage of green fluorescence cells was increased in a
dose-dependent manner (Fig. 4). This is consistent with above apoptosis assay.

ACCEPTED MANUSCRIPT
2.2.5 Cellular reactive oxygen species analysis
It is known that the cytotoxic activities of ferrocene were associated with the
production and regulation of reactive oxygen species (ROS) which in turn induce
oxidative DNA damage. Thus, the effect of compound 7l on the ROS levels in Hela
cells was investigated by DCFH-DA stain method. As shown in Fig. 5, when HeLa
cells were treated with compound 7l at concentrations of 2.5, 5 and 10 µM for 12 h,
the levels of reactive oxygen species were increased as the concentration increased. At
a concentration of 10 µM, it caused a 4.6 times higher level of ROS compared with
untreated control cells (Fig. 5B). This results revealed that compound 7l could
significantly increase the level of ROS in HeLa cells.
2.2.6 Compound 7l induces cell-cycle arrest in HeLa cells
To investigate the celluar mechanism underlying the antiproliferative effects of
compound 7l, we further assessed the cytostatic effects of compound 7l on Hela cells
by flow cytometry. Hela cells were treated compound 7l with different concentrations
(0, 1.25, 2.5, 5 and 10 µM) for 24 h, and the results were exhibited in Fig. 6.
Compared with control group, cell cycle distribution underwent significant changes in
all treatment groups, where the percentage of cells in G1 phase was increased from
59.21% to 76.44% and the percentage of cells in G2/M phase was decreased from
18.59% to 11.12%. These changes in cell cycle distribution suggest that compound 7l
could induce cell-cycle arrest in G1 phase with a dose-dependent manner.
2.2.7 Compound 7l reduced the level of PGE2 in Hela cells
We then tested the ability of compound 7l to inhibit the production of PGE2 from
arachidonic acid. Hela cells were treated with different dose of compound 7l,
Celecoxib and LPS for 24 h, and then the cell culture medium was collected to detect
the expression of PGE2 with the PGE2 ELLISA kit. As shown in Fig. 7,
pre-incubation of LPS (1 µg/mL) could significantly elevated the level of PGE2
compared untreated cells. However, the production of PGE2 was seriously reduced
when Hela cells were treated with compound 7l (5 µM) or Celecoxib (5 µM). These
results suggested that compound 7l could participate in COX-2/PGE2 pathway as a
COX-2 inhibitor.

ACCEPTED MANUSCRIPT
2.2.8 In vitro NO released amounts detection
To investigate whether the concentration of NO produced by these compounds was
associated with their cytotoxicities, the levels of NO production in the cell lysates
were determined. Hela cells were treated with 50 µM or 100 µM compounds and the
amounts of nitrite, the principal oxidation product of NO in aqueous solution, were
detected using the Griess assay. As shown in Fig. 8A, variable levels of NO could
release from all of these compounds, and compound 7l could generate the highest
concentration of NO. Interestingly, it was observed that the levels of NO generated by
hybrid compounds 7k ~ 7q linking with ester linkage were more higher than those
linking with amide linkage, which correlated well with their antiproliferative activities
against Hela cells in vitro. Then, NO amounts change curves of compound 7l
producing with time in Hela cells were exhibited in Fig. 8B. The corresponding
concentration/time curves suggested that the level of NO produced by compound 7l
slightly increased with time and maintained steady after 12 h incubation.
In addition, the contribution of NO to the anti-tumor activities of compound 7l was
investigated. After incubation with different dose of an NO scavenger of
carboxy-PTIO (0, 3.125, 6.25, 12.5, 25 and 50 µM) for 1 h, Hela cells were treated
with 5 µM compound 7l, and the cell viability was determined by MTT assay. As
shown in Fig. 8C, compound 7l could remarkably inhibit the proliferation of Hela
cells and the inhibitory effect was reduced after pretreatment with various
concentrations of carboxy-PTIO in a dose-dependent manner. These results suggested
that NO generated by compound 7l could contribute to its cytotoxicity against Hela
cells in vitro.
2.2.9 Compound 7l exhibited antitumor activity in xenografts of Hela cells
To evaluated the antitumor activity of compound 7l, a xenograft model was
established by subcutaneously inoculating Hela cells into the right flank of Balb/C
female nude mice. When the tumor volume was approximately 100 mm3, the mice
were randomly divided into 4 groups (6 mice/group), and intraperitoneally (i.p.)
treated with compound 7l (10 or 20 mg/kg), Celecoxib (10 mg/kg) or vehicle every
second day respectively. The average tumor volume was recorded using micrometer

ACCEPTED MANUSCRIPT
caliper every other day. As shown in Fig. 9A, the tumor volume in vehicle control
group was rapidly development, whereas the treated groups showed a suppressed
growth of tumor volume. Among them, compound 7l (20 mg/kg) group exhibited the
smallest tumor volume (0.149 cm3 ± 0.045). After 15 days of treatment, the tumors
were removed and the average weight was measured. All treated groups showed a
dramatic reduced in the tumor weight, and the average weight of control group tumors
was more 4-fold to the compound 7l (20 mg/kg) group (Fig. 9C). During the whole
treatment period, no dramatic weight changes were observed in compound 7l groups
and control group (Fig 9B), suggesting that compound 7l had minimal toxicity.
However, severe weight loss took place in Celecoxib group and one of the mice
suddenly died on the 11th day (Fig. 9D). All of our results showed that compound 7l
exhibits more safe and more efficient in antitumor therapy compared with Celecoxib.
3. Conclusion
The over-expression of COX-2 was clearly associated with carcinogenesis in
various types of cancer and the concept of COX-2 as a possible target is a promising
treatment strategy. Thus, we designed a series of novel ferrocene-pyrazolo
sulfonamide derivatives containing nitric oxide donors as potential inhibitors of
COX-2. Most of the synthesized compounds exhibited potent bioactivities, and
compound 7l displayed the most potent inhibitory activities against COX-2 and
antiproliferative activities against Hela cells with IC50 value of 0.34 µM. In further
mechanistic studies, the representative compound 7l could induce apoptosis of Hela
cells by mitochondrial depolarization and arrest cell cycle in G1 phase in a
dose-dependent manner. The Gressis assay revealed that most of the tested
compounds could release moderate amounts of NO in vitro especially compound 7l,
and the level of NO correlated well with their antiproliferative activities. Finally, the
in vivo antitumor activity of compound 7l was validated in Hela cells xenograft mouse
model. In summary, our studies of these well designed compounds may be great
helpful for the development of novel antitumor candidates in the future.
4. Experimental section
4.1 Materials and measurements

ACCEPTED MANUSCRIPT
All chemicals (reagent grade) used were purchased from Nanjing Chemical
Reagent Co. Ltd. (Nanjing, China) and used in this study without further purification.
1
All the H NMR spectra 13C NMR spectra were recorded on Bruker DPX400 or
Bruker DPX600 model spectrometer in DMSO-d6, and chemical shifts (δ) are
reported as parts per million (ppm). Melting points were determined on a X4 MP
apparatus (Jingsong Corp, Shanghai, China). Thin layer chromatography (TLC) was
performed on the glass backed silica gel sheets (Silica Gel 60 GF254) and visualized
in UV light (254 nm and 365 nm). Elemental analyses were performed on a
CHN-O-Rapid instrument and were within 0.4% of the theoretical values.
The COX-1 (human) Inhibitor Screening Assay Kit (catalog No.701070) and
COX-2 (human) Inhibitor Screening Assay Kit (catalog No.701080) were purchased
from
Cayman
Chemical
(MI,
USA).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) were purchased from
Beyotime Institute of Biotechnology (Haimen, China). Annexcin V-FITC cell
apoptosis assay kit (catalog No.BA11100) was purchased from BIO-BOX (Nanjing,
China). PGE2 enzyme immunoassay (EIA) kit-monoclonal (catalog No.514010) were
purchased from Cayman Chemical.
4.2 General procedure for the synthesis of 5-(1,2,3-trifluoromethyl-2,4-dione)
ferrocene (1)
Acetylferrocene (1.82 g, 8 mmol) was added to the suspension of sodium hydride
(0.38 g, 16 mmol) in dry THF (50 mL) at 0 ℃. Then ethyl trifluoroacetate (2.27 g, 16
mmol) was added and the mixture was stirred at room temperature for 6 h. The
reaction mixture was evaporated and the remains were poured into 100 mL ice water,
acidified with dilute hydrochloric acid (1 M) to pH 6 and extracted with EtOAc (100
mL × 2). The organic layer was washed with water and brine, dried over anhydrous
sodium sulfate and concentrated under vacuum. The crude product was purified by
silica gel column chromatography (VEtOAc / VPE = 1:4) to obtain a deep red solid (1),
yield 87.3%.
4.3 General procedure for the synthesis of compound 2

ACCEPTED MANUSCRIPT
A solution of compound 1 (0.32 g, 1 mmol) and 4-hydrazinylbenzenesulfonamide
(0.45 g, 2 mmol) in ethanol (20 mL× 2) was stirred at 80 ℃ for 12 h. After cooling
to room temperature, the precipitate was isolated by filtration and washed with cold
ethanol (10 mL ). The crude product was purified by silica gel column
chromatography (VEtOAc / VPE = 1:2) to obtain a yellow bolid (2).
Yellow powder, yield 44%; mp: 217.6 – 218.4 ℃; 1H NMR (400 MHz, DMSO-d6)
δ: 7.98 (d, J = 8.5 Hz, 2H), 7.67 (d, J = 8.5 Hz, 2H), 7.60 (s, 2H), 7.23 (s, 1H), 4.41 –
13
4.31 (m, 2H), 4.30 – 4.21 (m, 2H), 4.14 (s, 5H). C NMR (150 MHz, DMSO-d6) δ:
145.18, 145.05, 141.86, 127.45, 127.19, 105.39, 72.87, 70.33, 69.72, 69.00. MS(ESI):
476.33 [M + H]+. Anal. Calcd for C20H16F3FeN3O2S: C, 50.54; H, 3.39; N, 8.84%;
Found: C, 50.61; H, 3.34; N, 8.72.
4.4 General procedure for the synthesis of compound 3
Acetylferrocene (6.84 g, 30 mmol) was added to the suspension of 5.04 g of
t-BuOK (45 mmol) in benzene (100 mL) at 0 ℃. Then dimethyl oxalate (5.40 g, 45
mmol) was added dropwise over 10 min, and the mixture was heated at 80 ℃ for 6 h.
After cooling to room temperature, the solid was filtered off and suspended in
dichloromethane (30 mL), washed with an aqueous solution of hydrochloric acid (1 M,
50 mL × 2). The organic layer was dried over anhydrous sodium sulfate and
concentrated to give a deep purple solid (3).
Red powder, yield 61%; mp: 173.2 – 174.5 ℃; 1H NMR (400 MHz, DMSO-d6) δ:
15.06 (s, 1H), 6.64 (s, 1H), 5.04 (t, J = 1.8 Hz, 2H), 4.79 (t, J = 1.8 Hz, 2H), 4.27 (s,
5H), 3.86 (s, 3H). MS(ESI): 315.73 [M + H]+. Anal. Calcd for C15H14FeO4: C, 57.36;
H, 4.49%; Found: C, 57.61; H, 4.54.
4.5 General procedure for the synthesis of compounds 4a ~ 4h
2 g of methyl ester of ferrocenoylpyruvic acid (3) was dissolved in 20 mL ethanol,
and then corresponding monosubstituted hydrazines hydrochlorides (12.8 mmol) were
added under vigorous stirring. The mixture was heated while refluxing for 6 h. After
cooling to room temperature, the precipitated was isolated by filtration, and washed
with cold ethonl (10 mL). The crud product was purified by recrystallization from
ethanol to give a yellow crystal 4a ~ 4h.

ACCEPTED MANUSCRIPT
1
(4h) Yellow powder, yield 77%; mp: 198.2 – 199.4 ℃; H NMR (400 MHz,
DMSO-d6) δ: 7.97 (d, J = 8.6 Hz, 2H), 7.65 (d, J = 8.6 Hz, 2H), 7.58 (s, 2H), 7.20 (s,
1H), 4.33 (t, J = 1.9 Hz, 2H), 4.26 (t, J = 1.9 Hz, 2H), 4.12 (s, 5H), 3.87 (s, 3H).
MS(ESI): 466.13 [M + H]+. Anal. Calcd for C21H19FeN3O4S: C, 54.21; H, 4.12; N,
9.03%; Found: C, 53.91; H, 4.13; N, 9.12.
4.6 General procedure for the synthesis of compounds 5a ~ 5h
To a solution of compound 4 (6.4 mmol) in methanol (20 mL), potassium
hydroxide (20 mL, 1M) was added and the mixture was refluxing for 3 h under
vigorous stirring. After cooling to room temperature, the resulting mixture was poured
into ice water (100 mL) and acidified with dilute hydrochloric acid (1M) to pH 3 ~ 4.
The precipitate was isolated by flitration and washed with water (10 mL). The
obtained product dried under vacuum to get a yellow solid (5a ~ 5h).
1
(5h) Yellow powder, yield 90%; mp: 201.4 – 205.6 ℃; H NMR (400 MHz,
DMSO-d6) δ: 13.05 (s, 1H), 7.96 (d, J = 8.5 Hz, 2H), 7.67 – 7.55 (m, 4H), 7.13 (s,
1H), 4.32 (t, J = 1.9 Hz, 2H), 4.25 (t, J = 1.9 Hz, 2H), 4.12 (s, 5H). MS(ESI): 452.43
[M + H]+. Anal. Calcd for C20H17FeN3O4S: C, 53.23; H, 3.80; N, 9.31%; Found: C,
53.41; H, 3.73; N, 9.32.
4.7 General procedure for the synthesis of compounds 6a ~ 6h
The nitric acid salts of 3-(nitrooxy)propylamine (8b) (0.3 g, 1.5 mmol),
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC•HCl) (0.29 g,
1.5 mmol), 4-(dimethylamino)pyridine (DMAP) (0.18 g, 1.5 mmol), and
triethylamine (0.60 ml, 4 mmol) were added to the solution compound 5 (1 mmol) in
dichloromethane (20 mL), and the mixture was stirred at room temperature for 8 h.
Then, the reaction mixture was partitioned between dilute hydrochloric acid (30 mL,
1M) and dichloromethane (50 mL × 2). The organic layer was washed with water and
brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The
crude product was purified by silica gel column chromatography (VEtOAc /VPE = 1:1) to
give a yellow solid (6a ~ 6h).
1
(6a) Yellow powder, yield 52%; mp: 237.4 – 238.6 ℃; H NMR (600 MHz,
DMSO-d6) δ: 8.52 (t, J = 5.8 Hz, 1H), 7.57 (dd, J = 5.1, 1.9 Hz, 3H), 7.51 – 7.42 (m,

ACCEPTED MANUSCRIPT
2H), 7.04 (s, 1H), 4.64 (t, J = 5.1 Hz, 2H), 4.26 (s, 2H), 4.18 (s, 2H), 4.09 (s, 5H),
3.61 (q, J = 5.4 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) δ: 162.13, 146.86, 144.11,
140.13, 129.83, 129.61, 127.43, 106.27, 73.76, 72.85, 70.11, 69.31, 68.39, 36.50.
MS(ESI): 461.25 [M + H]+. Anal. Calcd for C22H20FeN4O4: C, 57.41; H, 4.38; N,
12.17%; Found: C, 57.51; H, 4.53; N, 12.15.
1
(6b) Yellow powder, yield 50%; mp: 223.6 – 224.5 ℃; H NMR (600 MHz,
DMSO-d6) δ: 8.49 (s, 1H), 7.36 (q, J = 9.2, 8.6 Hz, 4H), 7.01 (s, 1H), 4.77 – 4.56 (m,
2H), 4.26 (s, 2H), 4.18 (s, 2H), 4.08 (s, 5H), 3.60 (s, 2H), 2.42 (s, 3H). 13C NMR (150
MHz, DMSO-d6) δ: 162.19, 161.91, 146.67, 144.19, 142.95, 139.73, 139.42, 137.66,
129.98, 127.16, 108.43, 106.10, 72.85, 70.08, 69.29, 68.34, 36.49, 21.27. MS(ESI):
474.11 [M + H]+. Anal. Calcd for C23H22FeN4O4: C, 58.24; H, 4.68; N, 11.81%;
Found: C, 58.31; H, 4.55; N, 11.77.
1
(6c) Yellow powder, yield 48%; mp: 247.5 – 249.1 ℃; H NMR (600 MHz,
DMSO-d6) δ: 8.49 (t, J = 5.7 Hz, 1H), 7.40 (d, J = 8.4 Hz, 2H), 7.11 (d, J = 8.5 Hz,
2H), 7.00 (s, 1H), 4.63 (t, J = 4.5 Hz, 2H), 4.25 (s, 2H), 4.18 (s, 2H), 4.07 (s, 5H),
13
3.84 (s, 3H), 3.60 (q, J = 5.4 Hz, 2H). C NMR (150 MHz, DMSO-d6) δ: 162.19,
160.13, 146.58, 144.30, 133.04, 128.91, 114.61, 105.71, 73.76, 72.86, 70.06, 69.31,
68.17, 56.00, 36.47. MS(ESI): 491.61 [M + H]+. Anal. Calcd for C23H22FeN4O5: C,
56.34; H, 4.52; N, 11.43%; Found: C, 56.51; H, 4.33; N, 11.39.
1
(6d) Yellow powder, yield 59%; mp: 237.2 – 239.1 ℃; H NMR (600 MHz,
DMSO-d6) δ: 8.53 (t, J = 5.3 Hz, 1H), 7.56 (dd, J = 8.6, 4.7 Hz, 2H), 7.43 (t, J = 8.5
Hz, 2H), 7.03 (s, 1H), 4.64 (t, J = 4.5 Hz, 2H), 4.28 (s, 2H), 4.19 (s, 2H), 4.10 (s, 5H),
13
3.60 (q, J = 5.3 Hz, 2H). C NMR (150 MHz, DMSO-d6) δ: 162.06, 161.68, 146.93,
144.37, 136.52, 129.82, 116.64, 116.49, 106.22, 73.58, 72.84, 70.16, 70.11, 69.41,
68.34, 36.49. MS(ESI): 479.03 [M + H]+. Anal. Calcd for C22H19FFeN4O4: C, 55.25;
H, 4.00; N, 11.71%; Found: C, 55.31; H, 4.11; N, 11.62.
1
(6e) Yellow powder, yield 63%; mp: 248.6 – 249.2 ℃; H NMR (600 MHz,
DMSO-d6) δ: 8.54 (t, J = 5.8 Hz, 1H), 7.64 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz,
2H), 7.05 (s, 1H), 4.64 (t, J = 5.1 Hz, 2H), 4.29 (s, 2H), 4.21 (s, 2H), 4.11 (s, 5H),
13
3.61 (q, J = 5.4 Hz, 2H). C NMR (150 MHz, DMSO-d6) δ: 162.01, 147.17, 144.21,

ACCEPTED MANUSCRIPT
138.88, 134.17, 129.66, 129.03, 106.77, 73.64, 72.83, 70.16, 69.44, 68.58, 36.53.
MS(ESI): 495.06 [M + H]+. Anal. Calcd for C22H19ClFeN4O4: C, 53.41; H, 3.87; N,
11.33%; Found: C, 53.38; H, 4.02; N, 11.27.
1
(6f) Yellow powder, yield 54%; mp: 223.1 – 224.6 ℃; H NMR (600 MHz,
DMSO-d6) δ: 8.54 (t, J = 5.7 Hz, 1H), 7.77 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.2 Hz,
2H), 7.05 (s, 1H), 4.64 (t, J = 5.1 Hz, 2H), 4.30 (s, 2H), 4.22 (s, 2H), 4.11 (s, 5H),
13
3.61 (q, J = 5.4 Hz, 2H). C NMR (150 MHz, DMSO-d6) δ: 162.00, 147.20, 144.15,
139.28, 132.60, 129.24, 122.65, 106.85, 73.65, 72.83, 70.17, 69.45, 68.63, 36.53.
MS(ESI): 540.11 [M + H]+. Anal. Calcd for C22H19BrFeN4O4: C, 49.01; H, 3.55; N,
10.39%; Found: C, 49.22; H, 3.47; N, 10.49.
1
(6g) Yellow powder, yield 57%; mp: 231.0– 232.6 ℃; H NMR (600 MHz,
DMSO-d6) δ: 8.63 (t, J = 5.7 Hz, 1H), 8.37 (d, J = 8.5 Hz, 2H), 7.71 (d, J = 8.6 Hz,
2H), 7.13 (s, 1H), 4.66 (t, J = 5.0 Hz, 2H), 4.32 (d, J = 22.1 Hz, 4H), 4.15 (s, 5H),
13
3.63 (q, J = 5.3 Hz, 2H). C NMR (150 MHz, DMSO-d6) δ: 161.80, 147.95, 147.37,
144.73, 144.32, 127.48, 125.01, 108.27, 73.72, 72.79, 70.35, 69.59, 69.20, 36.60.
MS(ESI): 506.27 [M + H]+. Anal. Calcd for C22H19FeN5O6: C, 52.30; H, 3.79; N,
13.86%; Found: C, 52.36; H, 3.55; N, 13.84.
1
(6h) Yellow powder, yield 69%; mp: 241.0 – 242.3 ℃; H NMR (400 MHz,
DMSO-d6) δ: 8.58 (s, 1H), 7.96 (d, J = 8.0 Hz, 2H), 7.66 (d, J = 7.9 Hz, 2H), 7.57 (s,
2H), 7.10 (s, 1H), 4.65 (s, 2H), 4.32 (s, 2H), 4.25 (s, 2H), 4.13 (s, 5H), 3.74 – 3.50 (m,
2H). 13C NMR (150 MHz, DMSO-d6) δ: 170.79, 161.93, 147.44, 144.77, 144.13,
142.34, 127.40, 127.26, 127.03, 107.42, 73.71, 72.80, 70.22, 69.47, 68.87, 60.21,
36.56, 21.23, 14.55. MS(ESI): 540.21 [M + H]+. Anal. Calcd for C22H21FeN5O6S: C,
48.99; H, 3.92; N, 12.99%; Found: C, 49.11; H, 3.78; N, 12.84.
4.8 General procedure for the synthesis of compounds 7a ~ 7q
A
solution
of
compound
4h
(0.45
g,
1
mmol),
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC•HCl) (0.29 g,
1.5 mmol), 4-(dimethylamino)pyridine (DMAP) (0.18 g, 1.5 mmol), triethylamine
(0.60 ml, 4 mmol) and corresponding NO-donors (1.5 mmol) in dichloromethane (20
mL) was stirred at room temperature for 8 h. Then, the reaction mixture was

ACCEPTED MANUSCRIPT
partitioned between dilute hydrochloric acid (30 mL, 1M) and dichloromethane (50
mL × 2). The organic layer was washed with water and brine, dried over anhydrous
sodium sulfate, and concentrated under vacuum. The crude product was purified by
silica gel column chromatography (VEtOAc /VPE = 1:1) to give a yellow solid (7a ~ 7q).
1
(7a) Yellow powder, yield 66%; mp: 251.2 – 252.7 ℃; H NMR (400 MHz,
DMSO-d6) δ: 8.53 (t, J = 5.9 Hz, 1H), 7.96 (d, J = 8.5 Hz, 2H), 7.65 (d, J = 8.5 Hz,
2H), 7.58 (s, 2H), 7.08 (s, 1H), 4.56 (t, J = 6.2 Hz, 2H), 4.32 (s, 2H), 4.25 (s, 2H),
13
4.13 (s, 5H), 3.37 – 3.28 (m, 2H), 1.91 (p, J = 6.3 Hz, 2H). C NMR (150 MHz,
DMSO-d6) δ: 161.71, 147.81, 144.71, 143.99, 142.38, 127.35, 127.03, 107.46, 73.84,
72.22, 70.23, 69.46, 68.91, 60.23, 35.49, 26.99, 21.24, 14.56. MS(ESI): 554.18 [M +
H]+. Anal. Calcd for C23H23FeN5O6S: C, 49.92; H, 4.19; N, 12.66%; Found: C, 49.88;
H, 4.19; N, 12.48.
1
(7b) Yellow powder, yield 72%; mp: 229.3 – 230.3 ℃; H NMR (400 MHz,
DMSO-d6) δ: 7.95 (d, J = 8.5 Hz, 2H), 7.61 (d, J = 8.5 Hz, 2H), 7.57 (s, 2H), 7.09 (s,
1H), 4.50 (t, J = 8.7 Hz, 1H), 4.33 (s, 3H), 4.28 (s, 1H), 4.24 (s, 1H), 4.12 (s, 5H),
3.92 (t, J = 7.9 Hz, 1H), 1.27 (d, J = 6.6 Hz, 3H). MS(ESI): 554.33 [M + H]+. Anal.
Calcd for C23H23FeN5O6S: C, 49.92; H, 4.19; N, 12.66%; Found: C, 49.88; H, 4.21; N,
12.73.
1
(7c) Yellow powder, yield 61%; mp:245.8 – 247.2 ℃; H NMR (600 MHz,
DMSO-d6) δ: 8.58 (s, 1H), 7.96 (d, J = 7.0 Hz, 2H), 7.65 (d, J = 7.1 Hz, 2H), 7.55 (s,
2H), 7.09 (s, 1H), 5.33 (s, 1H), 4.32 (s, 2H), 4.26 (s, 2H), 4.13 (s, 5H), 3.63 (d, J =
12.4 Hz, 1H), 3.53 – 3.46 (m, 1H), 1.36 – 1.26 (m, 3H). MS(ESI): 554.28 [M + H]+.
Anal. Calcd for C23H23FeN5O6S: C, 49.92; H, 4.19; N, 12.66%; Found: C, 49.87; H,
4.32; N, 12.75.
1
(7d) Yellow powder, yield 52%; mp: 232.5 – 234.4 ℃; H NMR (600 MHz,
DMSO-d6) δ: 7.96 (d, J = 8.5 Hz, 2H), 7.61 (d, J = 8.5 Hz, 2H), 7.57 (s, 2H), 7.08 (s,
1H), 4.36 – 4.29 (m, 2H), 4.28 – 4.22 (m, 2H), 4.12 (s, 5H), 4.09 (s, 2H), 1.31 (s, 6H).
13C NMR (150 MHz, DMSO-d6) δ: 156.69, 144.66, 143.80, 142.44, 142.21, 127.21,
127.06, 108.15, 78.65, 73.61, 70.24, 69.48, 68.87, 67.89, 28.70. MS(ESI): 568.16 [M
+ H]+. Anal. Calcd for C24H25FeN5O6S: C, 50.80; H, 4.44; N, 12.34%; Found: C,

ACCEPTED MANUSCRIPT
50.77; H, 4.39; N, 12.31.
1
(7e) Yellow powder, yield 50%; mp: 251.2 – 253.4 ℃; H NMR (600 MHz,
DMSO-d6) δ: 7.95 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.5 Hz, 2H), 7.56 (s, 2H), 7.09 (s,
1H), 4.42 (t, J = 8.6 Hz, 1H), 4.37 – 4.27 (m, 3H), 4.22 (m, 1H), 4.18 – 4.00 (m, 7H),
0.98 (d, J = 6.7 Hz, 3H), 0.90 (d, J = 6.7 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ:
158.05, 144.62, 143.76, 142.40, 142.14, 127.15, 127.05, 108.23, 73.65, 72.42, 70.24,
70.04, 69.49, 69.02, 68.81, 32.71, 19.19, 18.72. MS(ESI): 582.38 [M + H]+. Anal.
Calcd for C25H27FeN5O6S: C, 51.64; H, 4.68; N, 12.05%; Found: C, 52.71; H, 4.72; N,
12.12.
1
(7f) Yellow powder, yield 61%; mp: 221.2 – 223.3 ℃; H NMR (600 MHz,
DMSO-d6) δ: 8.25 (d, J = 8.6 Hz, 1H), 7.96 (d, J = 8.5 Hz, 2H), 7.67 (d, J = 8.5 Hz,
2H), 7.55 (s, 2H), 7.08 (s, 1H), 4.66 (q, J = 8.4 Hz, 1H), 4.47 (t, J = 7.1 Hz, 2H), 4.32
(s, 2H), 4.28 – 4.20 (m, 2H), 4.14 (s, 5H), 1.71 – 1.51 (m, 2H), 1.34 – 1.25 (m, 1H),
13
0.90 (t, J = 6.8 Hz, 6H). C NMR (150 MHz, DMSO-d6) δ: 161.61, 147.50, 144.82,
144.15, 142.36, 127.56, 127.00, 107.53, 75.73, 73.78, 70.21, 69.46, 68.87, 44.59,
24.58, 23.56, 22.00. MS(ESI): 596.21 [M + H]+. Anal. Calcd for C26H29FeN5O6S: C,
52.45; H, 4.91; N, 11.76%; Found: C, 52.32; H, 4.95; N, 12.01.
1
(7g) Yellow powder, yield 47%; mp: 241.2 – 243.4 ℃; H NMR (400 MHz,
DMSO-d6) δ: 8.74 (t, J = 5.8 Hz, 1H), 7.96 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 8.3 Hz,
2H), 7.57 (s, 2H), 7.11 (s, 1H), 5.58 (s, 1H), 4.96 (dd, J = 12.5, 2.3 Hz, 1H), 4.73 (dd,
J = 12.6, 6.7 Hz, 1H), 4.33 (s, 2H), 4.26 (d, J = 4.1 Hz, 2H), 4.13 (s, 4H), 3.75 (m,
1H), 3.64 (m, 1H). 13C NMR (150 MHz, DMSO-d6) δ: 162.33, 147.23, 144.81, 144.23,
142.32, 127.42, 127.07, 107.58, 79.17, 73.70, 70.93, 70.24, 69.51, 68.94, 37.74.
MS(ESI): 615.82 [M + H]+. Anal. Calcd for C23H22FeN6O9S: C, 44.97; H, 3.61; N,
13.69%; Found: C, 44.99; H, 3.52; N, 13.68.
1
(7h) Yellow powder, yield 49%; mp: 251.2 – 253.6 ℃; H NMR (600 MHz,
DMSO-d6) δ: 8.65 (d, J = 8.3 Hz, 1H), 7.98 (d, J = 8.5 Hz, 2H), 7.69 (d, J = 8.5 Hz,
2H), 7.56 (s, 2H), 7.11 (s, 1H), 4.79 (m, 2H), 4.67 (m, 2H), 4.32 (q, J = 2.2 Hz, 2H),
4.25 (t, J = 1.9 Hz, 2H), 4.13 (s, 5H), 1.25 (d, J = 14.2 Hz, 1H). MS(ESI): 615.08 [M
+ H]+. Anal. Calcd for C23H22FeN6O9S: C, 44.97; H, 3.61; N, 13.68%; Found: C,

ACCEPTED MANUSCRIPT
44.73; H, 3.62; N, 13.83.
1
(7i) Yellow powder, yield 52%; mp: 223.7 – 225.3 ℃; H NMR (600 MHz,
DMSO-d6) δ: 7.96 (d, J = 8.1 Hz, 2H), 7.63 (d, J = 8.3 Hz, 2H), 7.56 (d, J = 12.6 Hz,
2H), 7.10 (s, 1H), 4.65 (s, 2H), 4.33 (s, 2H), 4.26 (s, 2H), 4.14 (s, 2H), 4.12 (s, 5H),
1.39 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ: 144.77, 144.04, 142.36, 141.55,
127.27, 127.08, 108.35, 77.48, 74.58, 73.47, 70.25, 69.70, 69.53, 68.88, 24.24.
MS(ESI): 629.09 [M + H]+. Anal. Calcd for C24H24FeN6O9S: C, 45.87; H, 3.85; N,
13.37%; Found: C, 45.72; H, 3.93; N, 13.35.
1
(7j) Yellow powder, yield 61%; mp: 206.2– 208.1 ℃; H NMR (400 MHz,
DMSO-d6) δ: 7.98 (d, J = 8.5 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.59 (s, 2H), 7.21 (s,
1H), 4.90 (d, J = 11.9 Hz, 2H), 4.76 (d, J = 11.9 Hz, 2H), 4.35 (s, 2H), 4.27 (s, 2H),
4.13 (s, 5H), 2.84 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ: 169.49, 146.65, 144.88,
144.48, 142.15, 127.11, 108.83, 73.32, 72.56, 70.30, 69.61, 69.03. MS(ESI): 690.11
[M + H]+. Anal. Calcd for C24H23FeN7O12S: C, 41.81; H, 3.36; N, 14.22%; Found: C,
41.74; H, 3.42; N, 14.36.
1
(7k) Yellow powder, yield 60%; mp: 218.2 – 219.7 ℃; H NMR (400 MHz,
DMSO-d6) δ: 7.99 (d, J = 8.5 Hz, 2H), 7.66 (d, J = 8.4 Hz, 2H), 7.60 (s, 2H), 7.21 (s,
1H), 4.95 – 4.81 (m, 2H), 4.69 – 4.56 (m, 2H), 4.33 (s, 2H), 4.26 (s, 2H), 4.12 (s, 5H).
13C NMR (150 MHz, DMSO-d6) δ: 161.78, 145.11, 144.51, 143.57, 142.22, 127.46,
127.16, 109.37, 73.19, 72.05, 70.26, 69.62, 68.84, 61.14. MS(ESI): 541.27 [M + H]+.
Anal. Calcd for C22H20FeN4O7S: C, 48.90; H, 3.73; N, 10.37%; Found: C, 48.92; H,
3.66; N, 10.44.
1
(7l) Yellow powder, yield 55%; mp: 220.5 – 222.4 ℃; H NMR (600 MHz,
DMSO-d6) δ: 7.98 (d, J = 8.5 Hz, 2H), 7.65 (d, J = 8.5 Hz, 2H), 7.58 (s, 2H), 7.19 (s,
1H), 4.69 (t, J = 6.3 Hz, 2H), 4.39 (t, J = 6.3 Hz, 2H), 4.33 (s, 2H), 4.25 (s, 2H), 4.13
13
(d, J = 5.2 Hz, 5H), 2.16 (p, J = 6.2 Hz, 2H). C NMR (150 MHz, DMSO-d6) δ:
161.92, 145.03, 144.34, 143.97, 142.25, 127.40, 127.14, 127.06, 126.69, 109.31,
73.30, 71.45, 70.26, 69.58, 69.40, 69.15, 68.85, 61.64, 26.31. MS(ESI): 555.11 [M +
H]+. Anal. Calcd for C23H22FeN4O7S: C, 49.83; H, 4.00; N, 10.11%; Found: C, 49.79;
H, 4.12; N, 10.17.

ACCEPTED MANUSCRIPT
1
(7m) Yellow powder, yield 47%; mp: 231.4 – 233.5 ℃; H NMR (600 MHz,
DMSO-d6) δ: 7.98 (d, J = 8.5 Hz, 2H), 7.65 (d, J = 8.5 Hz, 2H), 7.59 (d, J = 6.7 Hz,
2H), 7.17 (s, 1H), 4.61 (t, J = 5.8 Hz, 2H), 4.37 – 4.31 (m, 4H), 4.26 – 4.21 (m, 2H),
4.13 (d, J = 7.5 Hz, 5H), 1.84 – 1.80 (m, 3H). MS(ESI): 569.82 [M + H]+. Anal.
Calcd for C24H24FeN4O7S: C, 50.72; H, 4.26; N, 9.86%; Found: C, 50.62; H, 4.19; N,
9.87.
1
(7n) Yellow powder, yield 53%; mp: 214.7 – 216.2 ℃; H NMR (600 MHz,
DMSO-d6) δ: 7.98 (d, J = 8.0 Hz, 2H), 7.65 (d, J = 8.0 Hz, 2H), 7.58 (s, 2H), 7.17 (s,
1H), 4.55 (t, J = 6.2 Hz, 2H), 4.32 (d, J = 13.3 Hz, 4H), 4.25 (s, 2H), 4.12 (s, 5H),
1.88 – 1.68 (m, 4H), 1.52 – 1.42 (m, 2H). MS(ESI): 583.19 [M + H]+. Anal. Calcd for
C25H26FeN4O7S: C, 51.56; H, 4.50; N, 9.62%; Found: C, 51.59; H, 4.69; N, 9.63.
1
(7o) Yellow powder, yield 59%; mp: 226.5 – 228.3 ℃; H NMR (600 MHz,
DMSO-d6) δ: 8.04 – 7.91 (m, 2H), 7.69 – 7.62 (m, 2H), 7.58 (s, 2H), 7.17 (s, 1H),
5.40 – 5.13 (m, 1H), 4.66 (d, J = 4.6 Hz, 1H), 4.47 – 4.36 (m, 1H), 4.33 (s, 2H), 4.28
– 4.21 (m, 2H), 4.13 (s, 5H), 2.21 – 2.05 (m, 2H), 1.39 (dd, J = 24.6, 6.2 Hz, 3H). 13C
NMR (150 MHz, DMSO-d6) δ: 161.88, 161.59, 145.04, 144.98, 144.30, 144.22,
143.95, 142.28, 142.22, 127.47, 127.32, 127.13, 109.36, 109.23, 79.91, 73.34, 70.97,
70.24, 69.58, 68.92, 68.87, 68.81, 68.76, 61.13, 40.26, 32.95, 32.77, 20.30, 18.64.
MS(ESI): 569.17 [M + H]+. Anal. Calcd for C24H24FeN4O7S: C, 50.72; H, 4.26; N,
9.86%; Found: C, 50.73; H, 4.15; N, 9.73.
1
(7p) Yellow powder, yield 56%; mp: 261.2 – 262.8 ℃; H NMR (600 MHz,
DMSO-d6) δ: 7.98 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 8.4 Hz, 2H), 7.58 (s, 2H), 7.21 (s,
1H), 4.50 (s, 2H), 4.34 (s, 2H), 4.26 (s, 2H), 4.14 (d, J = 20.4 Hz, 7H), 1.08 (s, 6H).
13C NMR (150 MHz, DMSO-d6) δ: 161.77, 145.03, 144.37, 143.87, 142.27, 127.41,
127.14, 109.36, 78.40, 73.35, 70.25, 69.57, 69.40, 68.88, 35.24, 21.74. MS(ESI):
583.48 [M + H]+. Anal. Calcd for C25H26FeN4O7S: C, 51.56; H, 4.50; N, 9.62%;
Found: C, 51.53; H, 4.52; N, 9.57.
1
(7q) Yellow powder, yield 61%; mp: 233.7 – 235.6 ℃; H NMR (600 MHz,
DMSO-d6) δ: 7.98 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 8.3 Hz, 2H), 7.58 (s, 2H), 7.18 (d,
J = 7.5 Hz, 1H), 5.49 (m, 1H), 4.69 (m, 1H), 4.43 (dd, J = 12.6, 5.9 Hz, 1H), 4.33 (s,

ACCEPTED MANUSCRIPT
2H), 4.25 (d, J = 21.0 Hz, 2H), 4.13 (s, 5H), 1.40 (m, 3H). MS(ESI): 555.67 [M + H]+.
Anal. Calcd for C23H22FeN4O7S: C, 49.83; H, 4.00; N, 10.07%; Found: C, 50.01; H,
4.02; N, 10.15.
4.9 General procedure for the synthesis of compounds 8a ~ 8h
Compounds 8a ~ 8h and 10 ~ 13 were obtained according to the literature
procedure[30]. Briefly, corresponding amino alcohol (40 mmol) was added, dropwise,
to an ice-cooled solution of fuming nitric acid (5.4 mL, 90%) in acetic anhydride (30
mL). For the synthesis of compounds 10 ~ 12, double the amount of fuming nitric
acid and acetic anhydride was used. And triple the amount of fuming nitric acid and
acetic anhydride was used for the synthesis of compound 13. The mixture was stirred
in an ice-bath for 1 h and then at room temperature for another 1 h. Then, excessive
Et2O (200 mL) was poured into the reaction mixture with stirring until the product
precipitated. The solid was isolated by flitration and washed with Et2O (10 mL). The
white product was dried under vacuum to give the target compound.
1
(8b) White powder, yield 81%; mp: 63.2 – 65.1 ℃; H NMR (400 MHz,
DMSO-d6) δ: 7.80 (s, 3H), 4.59 (t, J = 6.1 Hz, 2H), 2.91 (q, J = 6.1 Hz, 2H), 2.12 –
1.82 (m, 2H). MS(ESI): 184.03 [M + H]+. Anal. Calcd for C3H9N3O6: C, 19.68; H,
4.95; N, 22.95%; Found: C, 19.62; H, 4.88; N, 22.91.
4.10 General procedure for the synthesis of compounds 9a ~ 9h
Compounds 9a ~ 9h were obtained according to the literature procedure[31].
Briefly, concentrated nitric acid (1.6 mL, 32 mmol), glacial acetic acid (5.9 mL), and
acetic anhydride (6.2 mL) were consecutively added to an ice-cold solution of the
appropriate diol (20 mmol) in 50 mL ethyl acetate under vigorous stirring for 1 h. The
reaction mixture was stirred at room temperature overnight, and the solution was
washed with a 10% aqueous solution of sodium bicarbonate. The organic layer was
dried over anhydrous sodium sulfate, filtered, and concentrated under reduced
pressure to give a oily product.
4.11 Cell culture
A549 (human lungs cell line), HeLa (human cervix cell line), MCF-7 (human
breast cancer cell line), B16-F10 (murine melanoma cell line) and 293T (human

ACCEPTED MANUSCRIPT
kidney epithelial cell line) were preserved in the State Key Laboratory of
Pharmaceutical Biotechnology of Nanjing University. Cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS,
BI), 100 U/mL penicillin and 100 mg/mL streptomycin (Hyclone), and incubated at
37 ℃ in a humidified atmosphere containing 5% CO2.
4.12 In vitro COX inhibition assay
The ability of the synthesized compounds to inhibit COX-1 and COX-2 was
determined by COX-1/COX-2 ELISA Kit (catalog number 701170 and 701180,
Cayman Chemical). COX-1 or COX-2 enzyme was pre-incubated with test
compounds at 0 µM, 1 µM, 10 µM and 100 µM in the supplied reaction buffer (0.1 M
Tris–HCl, pH 8.0, 5 mM EDTA, 2 mM phenol and 1 µM heme) for 10 min at 37 ℃.
The reactions were initiated by adding arachidonic acid to a final concentration of 100
µM and incubated 2 min at 37 ˚C for. Afterwards, 1 M HCl was added to the reaction
mixtures to stop the reaction, followed by one tenth the volume of saturated stannous
chloride (50 mg/mL). The reaction mixtures were incubated for 5 min at room
temperature, and the amount of PGF2α produced from PGH2 by reduction with
stannous chloride was measured using enzyme immunoassay at 450 nm.
4.13 Anti-proliferative assay
The antiproliferation activities of the synthesized compounds against Hela, A549,
MCF-7, F10 and 293T cell lines were evaluated using a standard MTT-based
colorimetric assay with some modification. Briefly, the cells were seeded at a density
of 5 × 104 cells/well into 96-well plates and then allowed to adhere for 12 h at 37 ℃,
5% CO2 atmosphere. Then the cells were treated with the synthesized compounds at
final concentrations of 0, 0.1, 1, 10 and 100 µM with Celecoxib as positive reference.
After 48 h incubation, cell viability was assessed by the conventional
3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) reduction assay
carried out strictly according to the manufacturer’s instructions (Sigma). The
absorbance (OD 570 nm) was read on an ELISA reader (ELx800, BioTek, USA) with
reference wavelength of 630 nm. The IC50 was calculated according to the
dose-dependent curves and expressed as mean ± S.D. of three independent

ACCEPTED MANUSCRIPT
experiments.
4.14 Cell apoptosis assay
Hela cells were seeded in six-well plates (2×105 cells/well) and treated with
compound 7l at final concentrations of 0, 1.25, 2.5, 5 and 10 µM for 12, 24 or 36 h to
induce cell apoptosis. After incubation, the cells were harvested by trypsinization and
washed twice with ice-cold PBS. Subsequently, the cells were resuspended in 500 µL
of binding buffer and then Annexin-V/FITC (5 µL) and PI (5 µL) were added to the
medium for another 10 min incubation in dark. The stained cells were analyzed by a
flow cytometer (Becton Dickinson, USA). Statistical analysis was done using Flowjo
7.6.1 software.
4.15 Mitochondrial membrane potential evaluation
Mitochondrial membrane potential (MMP,
△Ψm) was determined using JC-1 dye
(5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-imidacarbocyanine). Briefly, Hela cells were
seeded in six-well plates (2×105 cells/well) and grown for 12 h. Then, the cells were
treated with compound 7l at the indicated concentration (0, 1.25, 2.5, 5 and 10 µM)
for 12 h. After incubation, the cells were harvested by centrifugation and incubated
with JC-1 solution for 30 min, and then washed twice with PBS and resuspended in
medium. The stained cells were analyzed by a flow cytometer (Becton Dickinson,
USA). CCCP (carbonyl cyanide 3-chlorophenylhydrazone) was used as a positive
control.
4.16 Cellular reactive oxygen species analysis
HeLa cells in logarithmic growth phase were collected and seeded into 6-well
plates at a density of 5×105/mL. After 24 hours of culture, they were treated with
different concentrations of the test compounds for 12 h. Rosup, a mixture which was
offered in the kit and could increase the level of ROS, was used as positive contrast.
After 12 hours of culture, the cells were collected and re-suspended in 500 µL of
DCFH-DA. The cells were incubated in a 37°C incubator for 30 minutes. The stained
cells were washed three times with DMEM. 200 µL of PBS was added to resuspend in
a flow tube, placed on ice. Reactive oxygen detection was monitored on flow
cytometer (Becton Dickinson, USA). Statistical analysis was done using Flowjo 7.6.1

ACCEPTED MANUSCRIPT
software.
4.17 Cell cycle assay
Hela cells were seeded in six-well plates (2×105 cells/well) and incubation at 37 ℃
for 12h. Then, the cells were treated with compound 7l at final concentrations of 0,
1.25, 2.5, 5 and 10 µM for 24 h. After incubation, the cells were harvested by
trypsinization and washed twice with ice-cold PBS. Subsequently, the cells were fixed
in ice-cold 70% ethanol for a least 24 h. After being centrifuged, the cells treated with
RNase A (0.1 mg/mL) at 37 ℃ for 30 min, and stained with PI (5 µg/mL) for 30 min.
Cell-cycle distribution was analyzed by a flow cytometer (Becton Dickinson, USA).
Statistical analysis was done using Flowjo 7.6.1 software.
4.18 The levels of PGE2 detection
Prostaglandin E2 (PGE2) levels were determined by the colorimetric using a PGE2
Enzyme Immunoassay kit. Briefly, approximately 2×105 cells of Hela cells were
seeded in six-well plates and grown for 12 h. The replaced the culture media with
fresh media containing LPS (1 µg/mL) to induce PGE2 expression. Subsequently, the
cells were treated with compound 7l (5 µM) or Celecoxib (5 µM) for another 24 h.
The media were collected by centrifugation and the PGE2 levels were determined
using a PGE2 Enzyme Immunoassay kit (Catalog NO.514010, Cayman chemical)
according to the manufacturer’s instructions.
4.19 NO released amounts detection
The levels of intracellular NO generated by synthesized compounds were
determined using the nitrite colorimetric assay kit (Beyotime, China) according to the
manufacturer’s instructions. Briefly, Hela cells were seeded in 12-well plates (1×105
cells) and incubated for 12 h. Then, the cells were treated with 50 or 100 µM of each
compound for 6 h in the presence of 5 mM L-cysteine at pH 7.4. Subsequently, the
cells were harvested and their cell lysates were mixed with Griess reagent for 10 min
at 37 ℃. Then, the samples were measured at 540 nm using a Microplate Reader and
the nitrite levels were calculated according to the standard curve. Each compound was
measured in triplicate.
4.20 In vivo xenograft model

ACCEPTED MANUSCRIPT
In this experiment, animal welfare and experimental procedures were followed in
accordance with the Guide for Care and Use of Laboratory Animals (National
Institutes of Health, the United States) and the related ethical regulations of Nanjing
University.
Female BALB/c nude mice (5 weeks, 18-20 g) were housed in air-conditioned
rooms and fed a standard rodent and water. Hela cells grown in logarithmic phase
were harvested and resuspended in ice-cold PBS. Then 100 µL of the cell suspension
(2×105 cells) was subcutaneously injected into the right flank of each mouse. When
the tumor volume reached approximately 100 mm3, the mice were randomized into 4
groups and then treated intraperitoneally (i.p.) with 100 µL of either vehicle or
compound 7l and Celecoxib for every second days. Tumor volume and body weights
were measured every second days, and the volume was calculated according to the
following formula: tumor volume (mm3) = 0.5 × length × width2. Tumor tissue were
excised and weight on day 15.
Acknowledgements
The work was financed by Public Science and Technology Research Fund Project
of Ocean (No. 201505023).
References：
[1] W.L. Smith, R. Langenbach, Why there are two cyclooxygenase isozymes, J. Clin. Invest. 107
(2001) 1491-1495.
[2] N. Ghosh, R. Chaki, V. Mandal, S.C. Mandal, COX-2 as a target for cancer chemotherapy,
Pharmacol. Rep. 62 (2010) 233-244.
[3] A.K. Tewari, V.P. Singh, P. Yadav, G. Gupta, A. Singh, R.K. Goel, P. Shinde, C.G. Mohan,
Synthesis, biological evaluation and molecular modeling study of pyrazole derivatives as selective
COX-2 inhibitors and anti-inflammatory agents, Bioorg. Chem. 56 (2014) 8-15.
[4] K.Y. Shamsudin, H. Gutiérrezdeterán, L. Boukharta, Å. J, Toward an optimal docking and free
energy calculation scheme in ligand design with application to COX-1 inhibitors, J. Chem. Inf.
Model. 54 (2014) 1488-1499.
[5] N.V. Chandrasekharan, H. Dai, K.L.T. Roos, N.K. Evanson, J. Tomsik, T.S. Elton, D.L.
Simmons, COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other
analgesic/antipyretic drugs: Cloning, structure, and expression, Proc. Natl. Acad. Sci. U.S.A. 99
(2002) 13926-13931.

ACCEPTED MANUSCRIPT
[6] A.S. Girgis, S.R. Tala, P.V. Oliferenko, A.A. Oliferenko, A.R. Katritzky, Computer-assisted
rational design, synthesis, and bioassay of non-steroidal anti-inflammatory agents, Eur. J. Med.
Chem. 50 (2012) 1-8.
[7] E. Yıldırım, O. Sağıroğlu, F.S. Kılıç, K. Erol, Effects of Nabumetone and Dipyrone on
Experimentally Induced Gastric Ulcers in Rats, Inflammation. 36 (2013) 476-481.
[8] B. Sever, M.D. Altıntop, G. Kuş, M. Özkurt, A. Özdemir, Z.A. Kaplancıklı, Indomethacin
based new triazolothiadiazine derivatives: Synthesis, evaluation of their anticancer effects on T98
human glioma cell line related to COX-2 inhibition and docking studies, Eur. J. Med. Chem. 113
(2016) 179-186.
[9] P.M. De, S. Baeza, M.T. Escudero, T. Dierssensotos, I. Gómezacebo, M. Pollán, J. Llorca,
Effect of COX-2 inhibitors and other non-steroidal inflammatory drugs on breast cancer risk: a
meta-analysis, Breast Cancer Res. Treat. 149 (2015) 525-536.
[10] K.A.M. Abouzid, N.A. Khalil, E.M. Ahmed, H.A.A. El-Latif, M.E. El-Araby, Structure-based
molecular design, synthesis, and in vivo anti-inflammatory activity of pyridazinone derivatives as
nonclassic COX-2 inhibitors, Med. Chem. Res. 19 (2010) 629-642.
[11] J. Grover, V. Kumar, V. Singh, K. Bairwa, M.E. Sobhia, S.M. Jachak, Synthesis, biological
evaluation, molecular docking and theoretical evaluation of ADMET properties of nepodin and
chrysophanol derivatives as potential cyclooxygenase (COX-1, COX-2) inhibitors, Eur. J. Med.
Chem. 80 (2014) 47-56.
[12] B.P. Bandgar, R.J. Sarangdhar, S. Viswakarma, F.A. Ahamed, Synthesis and biological
evaluation of orally active prodrugs of indomethacin, J. Med. Chem. 54 (2011) 1191-1201.
[13] M. Anzini, C.A. Di, S. Valenti, S. Brogi, M. Rovini, G. Giuliani, A. Cappelli, S. Vomero, L.
Chiasserini, A. Sega, Novel analgesic/anti-inflammatory agents: 1,5-diarylpyrrole nitrooxyalkyl
ethers and related compounds as cyclooxygenase-2 inhibiting nitric oxide donors, J. Med. Chem.
56 (2013) 3191-3206.
[14] M. Biava, G.C. Porretta, G. Poce, C. Battilocchio, S. Alfonso, M. Rovini, S. Valenti, G.
Giorgi, V. Calderone, A. Martelli, Novel Analgesic/Anti-Inflammatory Agents: Diarylpyrrole
Acetic Esters Endowed with Nitric Oxide Releasing Properties, J. Med. Chem. 54 (2011)
7759-7771.
[15] M. Ghasemi, A. Fatemi, Pathologic role of glial nitric oxide in adult and pediatric
neuroinflammatory diseases, Neurosci. Biobehav. Rev. 45 (2014) 168-182.
[16] P.G. Wang, M. Xian, X. Tang, X. Wu, Z. Wen, T. Cai, A.J. Janczuk, Nitric oxide donors:
chemical activities and biological applications, Chem. Rev. 102 (2002) 1091-1134.
[17] D. Hirst, T. Robson, Targeting nitric oxide for cancer therapy, J. Pharm. Pharmacol., 59 (2007)
3-13.
[18] Y. Ai, F. Kang, Z. Huang, X. Xue, Y. Lai, S. Peng, J. Tian, Y. Zhang, Synthesis of CDDO–
Amino Acid–Nitric Oxide Donor Trihybrids as Potential Antitumor Agents against Both
Drug-Sensitive and Drug-Resistant Colon Cancer, J. Med. Chem. 58 (2015) 2452-2464.
[19] J. Mozziconacci, E. Arnoult, P. Bernard, C. Marot, L. Morinallory, Optimization and
Validation of a Docking-Scoring Protocol; Application to Virtual Screening for COX-2 Inhibitors,
J. Med. Chem. 48 (2005) 1055-1068.
[20] A. Pacelli, J. Greenman, C. Cawthorne, G. Smith, Imaging COX-2 expression in cancer using
PET/SPECT radioligands: current status and future directions, J. Labelled. Comp. Radiopharm. 57
(2014) 317-322.

ACCEPTED MANUSCRIPT
[21] S. Ovais, S. Yaseen, R. Bashir, P. Rathore, M. Samim, S. Singh, V. Nair, K. Javed, Synthesis
and anti-inflammatory activity of celecoxib like compounds, J. Enzyme. Inhib. Med. Chem, 28
(2013) 1105-1112.
[22] M.M. Ghorab, F.A. Ragab, M.M. Hamed, Design, synthesis and anticancer evaluation of
novel tetrahydroquinoline derivatives containing sulfonamide moiety, Eur. J. Med. Chem. 44
(2009) 4211-4217.
[23] M. Kaneko, S. Kaneko, K. Suzuki, Prolonged low-dose administration of the
cyclooxygenase-2 inhibitor celecoxib enhances the antitumor activity of irinotecan against
neuroblastoma xenografts, Cancer Sci. 100 (2010) 2193-2201.
[24] D. Plazuk, S. Top, A. Vessières, M.A. Plamont, M. Huché, J. Zakrzewski, A. Makal, K.
Woźniak, G. Jaouen, Organometallic cyclic polyphenols derived from 1,2-(alpha-keto tri or tetra
methylene) ferrocene show strong antiproliferative activity on hormone-independent breast cancer
cells, Dalton. Trans. 39 (2010) 7444-7450.
[25] C. Biot, G. Glorian, L.A.M. And, J.S. Brocard, O. Domarle, G. Blampain, A. Pascal Millet,
A.J. Georges, H.A. And, D. Dive, Synthesis and Antimalarial Activity in Vitro and in Vivo of a
New Ferrocene−Chloroquine Analogue, J. Med. Chem. 40 (1997) 3715-3718.
[26] D.R. van Staveren, N. Metzlernolte, Bioorganometallic chemistry of ferrocene, Chem. Rev.
104(2004) 5931-5986.
[27] A. Burmudžija, Z. Ratković, J. Muškinja, N. Janković, B. Ranković, M. Kosanić, S. Đorđević,
Ferrocenyl based pyrazoline derivatives with vanillic core: synthesis and investigation of their
biological properties, Rsc Adv. 6 (2016) 91420-91430.
[28] K. Kowalski, P. Hikisz, Ł. Szczupak, B. Therrien, A. Kocevachyła, Ferrocenyl and dicobalt
hexacarbonyl chromones--new organometallics inducing oxidative stress and arresting human
cancer cells in G2/M phase, Eur. J. Med. Chem. 81 (2014) 289-300.
[29] J.K. Muenzner, B. Biersack, A. Albrecht, T. Rehm, U. Lacher, W. Milius, A. Casini, J.J.
Zhang, I. Ott, V. Brabec, Ferrocenyl‐Coupled N‐Heterocyclic Carbene Complexes of Gold(I):
A Successful Approach to Multinuclear Anticancer Drugs, Chem. Eur. J. 22 (2016) 18953 –
18962.
[30] M. Calas, M. Ouattara, G. Piquet, Z. Ziora, Y. Bordat, M.L. Ancelin, R. Escale, H. Vial,
Potent Antimalarial Activity of 2-Aminopyridinium Salts, Amidines, and Guanidines, J. Med.
Chem. 50 (2007) 6307-6315.
[31] G.N. Ziakas, E.A. Rekka, A.M. Gavalas, P.T. Eleftheriou, K.C. Tsiakitzis, P.N. Kourounakis,
Nitric oxide releasing derivatives of tolfenamic acid with anti-inflammatory activity and safe
gastrointestinal profile, Bioorg. Med. Chem. 13 (2005) 6485-6492.

ACCEPTED MANUSCRIPT
Figure Captions
Table 1. Structures of compounds 6a ~ 6h.
Table 2. Structures of compounds 7a ~ 7q.
Table 3. Crystal data for compounds 2 and 7l.
Table 4. In vitro COX-1/COX-2 inhibitory activity of the designed compounds.
Table 5. Antiproliferative activities of selected compounds against four cancer cell lines and
cytotoxicity towards non-cancer cells.
Fig. 1. Structures of Reference Compounds and design strategy of the target compounds.
Fig. 2. Crystal structure diagrams of compounds 2 (CCDC: 1834063) and 7l (CCDC:1834102).
Fig. 3. Compound 7l induced apoptosis in Hela cells. (A, B) Effect of treatment of 7l (0, 1.25, 2.5,
5 or 10 µM) for 24 h on the apoptosis of Hela cells was determined; (C,D) Effect of treatment of
7l (5 µM) for 12, 24 or 36 h on the apoptosis of Hela cells was determined; The sum of early and
late apoptotic cells ratio (%) were quantitated by flow cytometer analysis of Annexin V-FITC/PI.
Data are shown as mean ± SD of three independent experiments; *p < 0.05, **p < 0.01.
Fig. 4. MMP levels were detected by JC-1 dye. Hela cells treated with 0, 1.25, 2.5, 5 and 10 µM 7l
for 12 h were collected and analyzed. CCCP was used as a positive control.
Fig. 5. Intracellular ROS accumulation in HeLa cells treated with compound 7l were analyzed. (A)
Flow cytometry analyses of Hela cells incubated with 0, 2.5, 5, 10 µM 71 and Rosup for 12 h; (B)
Mean fluorescence intensity (MFI) quantification by flow cytometry analyses. Rosup was set as
positive control. Data is shown as mean ± SD of three independent experiments.
Fig. 6. Effect of compound 7l on cell-cycle progression of HeLa cells was determined by flow
cytometry analysis. Hela cells treated with 0, 1.25, 2.5, 5 and 10 µM 7l for 24 h were collected and
analyzed. The percentage of cells in G1 graph are showed in the dot plots. (G1 phase, green; S
phase, yellow and G2/M phase, blue) ; *p < 0.05.
Fig. 7. The effects of 7l (5 µM), Celecoxib (5 µM), LPS (1 µg/ml) on PGE2 secretion in HeLa cells.
The levels of PGE2 in culture media was determined by ELISA assay. Data are shown as mean ±
SD of three independent experiments; *p < 0.05, **p < 0.01.
Fig. 8. The levels of NO generated by tested compounds and cytotoxicities against Hela cells. (A)
Hela cells were treated with 50 µM or 100 µM compounds for 6 h and the levels of NO (present as
nitrite) in cell lysates were determined by Griess assay; (B) Time course of NO release of 7l at 100
µM; (C) Effects of NO scavenging, carboxy-PTIO (PTIO) on the antiproliferative effect of 7l..
Data are shown as mean value ± SD of three independent experiments; *p < 0.05, **p < 0.01.
Fig. 9. Antitumor activity of compound 7l in xenografts of Hela cells. (A) Tumor volume after
treatment with 7l (10 or 20 mg/kg), Celecoxib (10 mg/kg) or vehicle. Data were measured every
other day by using a Vernier caliper and calculated as 0.5 × length × width2 (mm3); (B) Changes in
relative body weights of each group. The weight was monitored and recorded every two days; (C)
Weight of the excised tumors from each group; (D) Photograph of the excised tumors from each
group after treatment. The red dotted line represents the death of a mice on the 15th day; *p < 0.05,
**p < 0.01.
Scheme 1
Scheme 2
Scheme 3
Scheme 4

ACCEPTED MANUSCRIPT
Table 1. Structures of compounds 6a ~ 6h.
Compounds
R1
Compounds
R1
H
CH3
OCH3
F
Cl
Br
6a
6b
6c
6d
6e
6f
NO2
6g
6h
SO2NH2
Table 2. Structures of compounds 7a ~ 7q.
Compounds
R2
Compounds
R2
7j
7k
7l
7a
7b
7c