Synthesis and biological activities of carbonyl cobalt CORMs with selectively inhibiting cyclooxygenase-2

Article abstract of DOI:10.1016/j.jorganchem.2018.08.013

In this paper, three kinds of hybrid carbonyl cobalt CO-releasing molecules (CORMs), RCOOCH₂C₂H[Co₂(CO)₆] (R = NSAIDs-COOH, 1–11; R = celecoxib derivative-COOH, 15), RSO₂(R’) CH₂C₂

Full text of DOI:10.1016/j.jorganchem.2018.08.013

Accepted Manuscript
Synthesis and biological activities of carbonyl cobalt CORMs with selectively inhibiting
cyclooxygenase-2
Jili Li, Jinlong Zhang, Qiuping Zhang, Zhongjie Bai, Quanyi Zhao, Zhen Wang,
Yonglin Chen, Bin Liu
PII:
S0022-328X(18)30450-9
10.1016/j.jorganchem.2018.08.013
JOM 20540
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 4 June 2018
Revised Date: 1 August 2018
Accepted Date: 14 August 2018
Please cite this article as: J. Li, J. Zhang, Q. Zhang, Z. Bai, Q. Zhao, Z. Wang, Y. Chen, B.
Liu, Synthesis and biological activities of carbonyl cobalt CORMs with selectively inhibiting
cyclooxygenase-2, Journal of Organometallic Chemistry (2018), doi: 10.1016/j.jorganchem.2018.08.013.
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ACCEPTED MANUSCRIPT
Synthesis and biological activities of carbonyl cobalt CORMs
with selectively inhibiting cyclooxygenase-2
Jili Li a, Jinlong Zhang a, Qiuping Zhang a, Zhongjie Baia, Quanyi Zhao a
Zhen Wang a, Yonglin Chen b, Bin Liu c
,
(a Institute of Medicinal Chemistry, School of Pharmacy of Lanzhou University; b The First
Affiliated Hospital of Lanzhou University; c School of Stomatology of Lanzhou University,
Lanzhou 730000, China)
* Corresponding author. Tel.: +869318915686; fax: +869318915686.
E-mail address: zhaoqy@lzu.edu.cn

ACCEPTED MANUSCRIPT
Synthesis and biological activities of carbonyl cobalt CORMs
with selectively inhibiting cyclooxygenase-2
Jili Li a, Jinlong Zhang a, Qiuping Zhang a, Zhongjie Baia, Quanyi Zhao a
Zhen Wang a, Yonglin Chen b, Bin Liu c
,
(a Institute of Medicinal Chemistry, School of Pharmacy of Lanzhou University; b The First
Affiliated Hospital of Lanzhou University; c School of Stomatology of Lanzhou University,
Lanzhou 730000, China)
Abstract：
In this paper, three kinds of hybrid carbonyl cobalt CO-releasing molecules
(CORMs), RCOOCH2C2H[Co2(CO)6] (R=NSAIDs-COOH, 1-11; R= celecoxib
derivative-COOH, 15), RSO2(R’) CH2C2H[Co2(CO)6] (R= celecoxib, 12; R=
nimesulide, 13) and ROCH2C2H[Co2(CO)6] (R= ferulic acid etheyl ester, 14), were
synthesized and characterized by IR, NMR and HRMS. The crystal structures of
complexes 9, 11, 14 and 16 were determined by single-crystal X-ray diffraction.
Meanwhile, the anti-tumor activity, protection on oxidative damage of H9c2 cells,
anti-hypertension and myocardial protective effects were evaluated. The results
showed only a part of the complexes had anticancer activity for the five cell lines
compared with 5-FU. Among them, complexes 12 and 13 which modified by selective
COX-2 inhibitors displayed strong antiproliferative activity and selectivity to MCF-7
cell and HT-29 cell lines, and their IC50 values were 33.6-55.8 µM; but compared with
cis-platin (DDP), they showed slightly lower activities. The complex inhibited the
expression of COX-2 in HT-29 cells and MCF-7cells, and complex 12 had stronger
inhibitory effects than the others, which was accordance with its stronger activity
against cell proliferation. Secondly, the complex improved the survival rate of H9c2
cells injured by H2O2. For each tested complex, the survival rate increased obviously
after the cells treated with H2O2 for 1h; but it increased slowly when the cells harmed
for 8h due to the severe cell injury. In addition, the complexes displayed
antihypertensive effects on spontaneously hypertensive rats, accompanying with good
myocardium protection. This indicates these hybrid CORMs reduced myocardial
toxicity of COX-2 inhibitors like, but there still has liver and kidney side-effects when
administrating continuously for 14 days.
* Corresponding author. Tel.: +869318915686; fax: +869318915686.
E-mail address: zhaoqy@lzu.edu.cn

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Key words: carbonyl metal; CO-releasing molecule; cyclooxygenase-2;
antihypertensive; myocardial protective effects
1. Introduction:
Carbon monoxide (CO) has drawn considerable attention because of its
unexpected role in numerous physiological processes. [1] It was found to be beneficial
in therapeutic applications such as systemic pulmonary hypertension, cardiac, renal,
and small bowel graft rejections, as well as intestine disease, hemorrhagic shock, and
[2]
lung injury.
Recently, the researcher found CO was directly related to tumor
[3]
[4]
angiogenesis,
tumor cell energy metabolism
and immune regulation [5], which
indicates CO has a big potential in clinic. But CO is a gas and uncontrollable dose and
un-convenient use. Motterlini first used carbonyl metal CO releasing molecules
(CORMs) as CO substitute, and confirmed CORMs had the same function as
endogenous CO. [1, 6] After that, many carbonyl metal CORMs appeared, involving in
metal Ru, Fe, Mn, Co, Mo, and so on. [7, 8–11]
Among all kinds carbonyl metal complexes, CORMs containing cobalt was
focused on earlier. In 2005, Ott et al found many cobalt carbonyl complexes inhibited
tumor cell proliferation, and their activities were connected with the structures of
[12]
non-CO ligands;
Among these complexes, hexacarbonyl-[2-propinylacetyl
salicylate] dicobalt was more active than cis-platin on the human mammary tumor cell
lines MCF-7 and MDA-MB-231.[13-14] Hexacarbonyl-[ethinylestradiol] dicobalt
[15]
complex was effective to hormone dependent tumors.
The cobalt carbonyl
complexes not only displayed ant-cancer activity, but also had anti-inflammation. In
following years, the researcher found their anti-inflammatory effects owed to
liberating CO to inhibit nitrite, [16] and the effects of proliferative inhibition for cancer
cells were mainly resulting from the CO to inhibit Hsp90 downstream protein activity,
[17]
such as Akt, ER alpha, and cyclin D1;
the complexes also promoted apoptosis,
disturbed cell cycle and interfered NDA synthesis by cobalt fragments [18]
.
Why the CORMs based on acetylsalicylic acid displayed better anti-proliferative
activity to cancer cells, like MCF-7 and MDA-MB-231. With in-depth research on
cancer, many evidences demonstrate inducible cyclooxygenase (COX-2) played an
important role in tumorigenesis, development and metastasis of cancer. The
proliferation of tumor cells accompanies with occurrence of inflammation in tumor
[19-20]
development process.
COX-2 was over-expression in thyroid cancer,
endometrial cancer and colon cancer, [21-22] and it was involved in tumor invasion,
lymph node metastasis and clinical staging in non-small cell lung cancer. The
expression of COX-2 was not only related to tumor size, postoperative pathology and
expression of hypoxia inducible factor [23], but also participated in tumor resistance

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through a variety of mechanisms. Acetylsalicylic acid cobalt carbonyl complexes on
the proliferation of cancer cells inhibition maybe was related to the inhibition of
COX-2 activity.
Considering the COX-2 roles in tumorigenesis and development process, metal
carbonyl CORMs based on COX-2 inhibitor may play the dual effects, on the one
hand, through cyclooxygenase pathway, inhibit tumor cell growth indirectly, reduce or
weaken tumor development and metastasis; on the other hand, ROS and metal
fragments produced by CORMs directly killing tumor cells. This kind of hybrid
CORMs is likely to be a new kind anticancer agent. In addition, non-steriod
anti-inflammatory drugs (NSAIDs), nimesulide and celecoxib are selective COX-2
inhibitors which were withdrawn from market due to myocardiotoxicity. Modified
with CORMs, they will possibly overcome these flaws by CO biological function.
Therefore, on the basis of the above, we synthesized a kind of hybrid CORMs
based on non-steriod anti-inflammatory drugs (NSAIDs), and evaluated biological
function through their anti-tumor activities and regulation of COX-2 expression level;
At the same time, we wonder whether CORMs improve selective COX-2 inhibitors
side-effects through evaluating protective effect on oxidative damage of rat
myocardial H9c2 cells, myocardial protective effects and antihypertensive effect. By
which, we hope to provide a theoretical basis for the development of new drugs.
2. Results and discussion
2.1 Design, syntheses and properties of the complexes
Non-steroidal anti-inflammatory drugs (NSAIDs) are COX inhibitors which
widely applied all over the world; many testing results show they not only have
preventive and anticancer effects.[24-31] but also increase the activity of certain
anticancer drugs when applied in combination[32-39]. Therefore, based on our previous
work about CORMs, the structural fragments of compounds A1-13 (Scheme 1, 2) were
introduced into complexes 1-15 because of their activities, among them, A1 –A11 are
nonselective COXIBs, which are NSAIDs in clinic. A8 and A14 have antithrombotic
activity; A12 and A13 are selective COX-2 inhibitors which regrettably pulled off the
market due to the cardiovascular toxicity. Considering carbonyl cobalt CORMs
protecting cardiovascular effects, the combination of both COXIs and CORMs maybe
overcome their side-effects and even have synergistic action. At the same time, A1-14
can make the corresponding complex target specific organs and tissues, and possess
stronger functions in vivo by primary metabolism.
Complexes 1-11 were prepared through the addition reaction of intermediates
and Co2(CO)8; and M1-11 were synthesized by the reactions of between organic acids
with anti-inflammation and propargyl alcohol; complexes 12 and 13 were synthesized
by replacing H atom of NHSO2 group in nimesulide and celecoxib structures with

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propargyl respectively on the alkaline condition, and then esterification; celecoxib has
two H atom on NH2SO2 group, disubstituted product（complex 16）was formed more
easily; Complex 14 was prepared by the reaction of ferulic acid etheyl ester with
propargyl bromide in the presence of potassium carbonate; complex 15 was afforded
by the oxidation of methyl on celecoxib structure with potassium permanganate, and
then esterification. The structures and synthetic routes for complexes 1-15 are shown
in scheme 1and 2.
(CO)3
Co
Co2(CO)8
HOCH2C CH
CH
RCOOCH2C
(OC)3Co
RCOOCH2CCH
RCOOH
EDCI DMAP
A1-11
1-11
F
O
COOH
NH
COOH
CH3
COOH
COOH
CH3
O
H3C
CH3
N
CH3
H3C
O
S
O
CH3
A2
Cl
A4
A3
OH
A1
CH3
OH
O
O
B
O
OH
O
HN
O
N
NH
HO
CH3
Cl
F
O
F3C
A8
A5
A7
A6
CH3
COOH
COOH
NH
COOH
Cl
O
H
N
N
N
Cl
CF3
A11
A9
A10
Scheme 1 The structures and synthetic routes for complexes 1-11

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(CO)3
Co
F3C
F3C
N
N
F3C
N
N
SO2NH2
N
CH
SO2NHCH2 C
N
SO2NH
CH2C CH
Co
(CO)3
b
a
C6H5-p-CH3
A12
C6H5-p-CH3
C6H5-p-CH3
12
(CO)3
Co
CH
SO2CH3
SO2CH3
N
NHSO CH3
2
CH2C CH
N CH2C
O
Co
O
O
(CO)3
a
b
NO2
13
NO2
O
NO2
A13
O
b
O
a
O
O
(CO)3
Co
O
O
O
O
O
O
14
CH2C
F3C
CH
OH
Co
A14
(CO)3
N
F3C
N
F3C
N
N
N
SO2NH2
F3C
b
N
(CO)3
Co
N
SO2NH2
SO2NH2
N
SO2NH2
CH
C6H5-p-COOCH2
C
c
d
Co
C6H5-p-CH3
(CO)3
C6H5-p-COOH
CH
C6H5-p-COOCH2C
15
A12
Reagents and conditions: a, propargyl bromide, K2CO3; b, Co2(CO)8; c, KMnO4; d, propiolic alcohol, EDCI,
DMAP
Scheme 2 The structures and synthetic routes for complexes 12-15
All complexes were characterized by a combination of NMR, ESI-HRMS and IR
spectroscopy. All the complexes are red-black oils or red solids, stable in air for
several days. They are insoluble in water, and easily dissolve in organic solvent. In the
range of 2100–1996 cm-1 appeared several strong terminal carbonyl absorptions in
their IR spectra. The higher absorption bands were attributed to terminal carbonyls
which coordinated to the Co atom. The IR spectra showed corresponding ester
carbonyl absorptions at 1682–1736 cm-1 for all the complexes except complexes 12
13,and 15. In the 1H NMR spectra of the complexes 12 and 15, the chemical shifts of
the parazole ring protons were observed at 6.67–6.79 ppm, signals at 5.26 ppm as
singlet were assigned to hydrogen of OCH2.
In addition, the crystal structures of complexes 9, 11, 14 and 16 were determined
by single-crystal X-ray diffraction, their molecular structures are shown in Fig. 1. The
details of the data collection and structure refinement are listed in Table 1. All four
complexes but 16 have a common tetrahedral framework containing two cobalt atoms,
different side chains make the Co-Co bond lengths changing slightly. For complex 16,
there have two tetrahedral frameworks containing [Co2(CO)6] units to connect
together through –CH2NCH2- group; distinctly, the bond angle of C-N-C (119.2°) is
much bigger than the normal angle of sp3 hybrid N atom, and two fragments of
[Co2(CO)6] are away from each other as far as possible due to steric hindrance; For
celecoxib fragment part, two benzene rings and a parazole ring are not in a plane

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because of the effect of [Co2(CO)6], two benzene rings are on both side of the plane of
parazole ring. Their torsion angles are about 140.
Fig.1 The crystal structures of complexes: A for 11, B for 9, C for 14, D for 16.
Table 1 Details of the data collection and structure refinement
Complex
A
B
C
D
Formula
C22H11Co2F3N2O8
C21H16Co2O10
546.20
C35H18Co4F3N3O14S
1029.30
C26H17Co2NO9
605.27
Formula weight
Crystal system
Dimensions(mm3)
606.19
monoclinic
monoclinic
monoclinic
monoclinic
0.21 × 0.15 × 0.14
0.19 × 0.07 × 0.05
0.25 × 0.24 × 0.17
P21/c
0.25 × 0.07 × 0.04
P21/n
Space group
a(Å)
P21/c
P21/c
24.100(2)
7.9528(5)
13.4035(11)
2547.2(4)
4
11.4258(4)
19.3036(8)
11.4694(4)
2415.88(16))
4
15.3368(6)
12.2990(5)
13.0974(6)
2469.76(17)
4
18.1374(12)
13.2270(9)
13.2270(9)
18.2619(11)
4
b(Å)
c(Å)
Volume(Å3)
Z
Dcalcd(mg/cm3)
1.578
1.357
1224.0
1.667
1.446
1208.0
1.469
1.392
1104.0
1.568
1.619
2048.0
µ
(mm-1)
(000)
-range for data
F
θ
6.618 to 52.038
9637
7.338 to 52.04
10068
6.562 to 52.044
17186
6.94 to 52.04
9299
collection (°)
Reflections
collected
Independent
Reflections
Data/restraints/
Parameters
5015 [Rint=0.0586]
5015/0/344
4620 [Rint=0.0365]
4620/39/361
4850 [Rint= 0.0454]
4850/0/300
8582 [Rint= 0.0484]
8582/36/569
R1 = 0.0546,
wR2 = 0.0948
R1 = 0.0442,
wR2 = 0.0845
R1 = 0.0723,
Final R-indices
R1 = 0.0603,
wR2 = 0.0822
R1 = 0.1400,
R1 = 0.0446,
wR2 = 0.0975
R1 = 0.0742,
[I≥2σ(I)]
Final R indices
R1 = 0.0977,

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[all data]
wR2 = 0.1166
0.42/-0.33
wR2 = 0.1000
0.31/-0.42
wR2 = 0.1093
0.40/-0.39
wR2 = 0.1152
0.45/-0.33
Largest diff.peak
and hole (e Å-3)
The ability of CO-release was evaluated in a myoglobin-based aqueous assay. A
solution of deoxymyoglobin was prepared and subsequently treated with a solution of
the CORM at 37.5 °C. The resulting conversion of deoxy-Mb to CO-Mb was
monitored by UV–vis spectroscopy. To evaluate both the total amount of CO released
and the rate of CO release, all complexes were tested at 10 µ M by following the
changes in absorption. A representative example of the UV spectrum and the
concentration of CO-Mb as a function of time are given in Fig. 2 (for other complexes
see SI-Fig. 1). The half-lives of all the complexes are listed in Table 2. As seen from
the data, the half-lives of complexes 12 and 13 were 25.6 min and 26.8 min
respectively, while complex 15 was 15.8min which shows it was a fast CO-releaser.
Like the complexes reported before [40], the half-lives were also dependent on the
structures of non–CO ligands. For the complexes containing Ar-NH-Ar’ structure
(complexes 2, 6, 7, 9, 10), the T1/2 of the complex having pyridine ring was longer
than the others, the electron withdrawing group at the m-position of Ar ring (CF3, Cl)
made CO releasing slower, for example, the T1/2 of complex 6 was up to 42.6min, in
contrast, complex 7 containing methyl only 26.4min.
Fig. 2 The typical changes in the electronic spectrum of myoglobin as CO releasing from
compounds (myoglobin binds one CO to form carbonyl myoglobin), A, complex 1 (10 µM); B
for 5 (10 µM); C for 10(10 µM); D for 15 (10 µM).
To evaluate the capacity of permeable membrane of complex in vivo, we
measured lgP values using n-octanol/water system. Their lgP values were in the range
of 1.62 and 2.16. The values of complexes 12 and 13 are 1.91 and 2.03 respectively.

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Our previous studies show cobalt complexes were unevenly distributed in tissues and
organs; some have a big prone to collect in liver, whereas some easily enter to kidney.
The substituents significantly affected the rate of CO-release, cytotoxicity and their
bio-distribution.
Table 2 Physical-chemical parameters of all the complexes.
Complex T1/2（min） lgP
Complex
T1/2（min） lgP
32.8
21.8
30.5
34.7
38.9
42.6
26.4
16.8
2.16
1.98
1.80
1.96
1.89
1.68
2.10
2.06
30.9
24.8
16.9
25.6
26.8
16.3
15.8
1.85
2.32
2.05
1.91
2.03
2.12
1.62
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
2.2 Anti-tumor activities of complex and effect on COX-2 expression level
Growth inhibitory effect toward the cancer cell line was determined by MTT
method. As shown in Table 3, all the complexes displayed lower activities than
cis-platin on the proliferation of five tumor cells, and most of them had low selectivity
to normal cells W138 and tumor cells. But compared with 5-FU, some displayed
better activity; among them, complexes 4, 12 and 13 had higher selectivity to HT-29
cell lines and MCF-7, and selective index was up to 2.94 at most. Complexes 12 and
13 were significantly more effective against proliferation of all five tumor cells than
others. For HT-29 cell lines, their IC50 values were 37.9µM and 55.8µM respectively,
and 33.6µM and 49.3µM respectively for MCF-7 cell lines. Except the structure
factor of the side chains, this probably closely connected with celecoxib and
nimesulide biological function which were selective COX-2 inhibitors.
Table 3 IC50 values of complex to tumor cells and W138
Complex
IC50(µM)
HT-29 HePG2
136.8±5.8 128.8±5.2 168.9±4.7
Hella
143.6±3.9
129.6±4.5
141.8±6.7
91.8±6.6
A549
MCF-7
W138
126.8±3.5 95.8±4.8
133.4±5.2 83.6±2.1
1
2
3
4
5
6
7
8
9
121.8±4.9 110.8±2.9
146.8±5.1
139.8±6.1
109.8±3.6
128.9±3.9 108.7±1.8 118.8±3.6
98.7±6.3
52.8±3.3
72.3±4.4
65.5±2.4
82.6±4.2
127.7±4.2
141.6±4.9
117.6±5.8
198.8±4.3
128.7±5.1
136.8±5.3 98.6±3.7
118.7±6.1
106.2±4.5 130.8±4.6
152.8±6.1 132.6±2.1 161.8±3.9 121.6±4.1 132.8±5.1
129.8±3.7 109.8±3.4 109.6±4.8 88.9±2.5 168.9±6.8
241±3.6 218.9±4.9 232.5±6.8 178.8±3.9 218.9±4.1
165.6±5.2 139.8±5.3 141.5±4.8 108.3±5.7 136.8±4.9

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132.8±2.9
108.6±6.1
77.9±4.6
71.5±4.8
189.8±4.9
129.8±5.7
86.8±5.4
114.2±4.9
34.7±3.2
159.3±4.7 129.8±3.2 109.8±5.1 118.6±6.1
154.8±6.1
10
109.3±3.8 120.8±1.5 136.8±5.4 102.8±4,4 142.8±4.2
11
59.8±3.9
63.8±4.6
37.9±3.7
55.8±5.9
60.7±3.9
69.8±4.9
33.6±2.4
49.3±2.9
98.8±6.4
92.6±4.8
12
13
209.8±5.1 202.7±4.1 241.8±6.3 238.7±6.9 289.8±3.9
14
90.5±3.6
92.8±5.7
98.7±4.2
29.6±1.9
89.6±3.1
73.2±5.6
89.9±5.2
66.8±6.1
72.8±4.1
66.8±3.6
169.8±5.4
82.5±3.9
15
16
136.2±3.9 124.3±10.3 148.7±5.2 198.6±3.6
30.6±1.2 24.6±2.2 25.5±1.8 50.3±4.2
5-FU
DDP
In order to confirm the important role of COX-2 in anti-proliferation, we
measured the survival rates of HT-29 and MCF-7 cells after treated with precursors
and complexes respectively. Seen from Fig.2-SI, in the range of 6.5-800µM, all
compounds showed better activities against tumor cells, and the survival rates of
HT-29 and MCF-7 cells decreased with the increase of the concentration, this
indicates COX-2 was one of targets of the complex; moreover, complexes 4, 12 and
13 had higher inhibitory rates of proliferation than the corresponding precursors, A4,
A12 and A13, which indicates the complexes may be multi-target anticancer agents. In
contrast, the activities of complexes 12 and 13 were higher than that of 4, this is
because A12 and A13 were selective COX-2 inhibitors and A4 was non-selective
COX-2 inhibitors. This suggests down-regulation of COX-2 level should inhibit the
proliferation of tumor cells.
To figure out the relationship between the up-regulation or down-regulation of
COX-2 expression and the anti-tumor activity of the complex, the effect of
concentration on the expression level of COX-2 in HT-29 cells and MCF-7 cells was
further investigated.
The results showed that all the tested compounds inhibited the expression of
COX-2 in HT-29 cells, and complexes 4, 12 and 13 had stronger inhibitory effects on
COX-2 than the corresponding A4, A12 and A13. Seen from Fig.3, complex 4 displayed
lower activity to inhibit COX-2 expression level at 10uM, but when it was over 50uM,
its activity was very significant. Complex 12 distinctly inhibited the expression of
COX-2 at 10µM, and the down-regulation trend was obvious in a dose-dependent
manner. Complex 13 was similar to 12, but its activity was slightly lower than that of
12. This indicates that the stronger the complex inhibiting COX-2 expression, the
higher the activity of the complex. This was consistent with the conclusion that the
cell survival rate was lower.

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Fig. 3 The effect of compound concentration on COX-2 in HT-29(A) and MCF-7 cells; (B): a
for complex 4 and A4, b for complex 12 and A12, c for complex 13 and A13
For MCF-7 cell lines, all the tested compounds also inhibited the expression of
COX-2 (Fig. 3), and the inhibitory trend with the concentration was similar to HT-29
cells. Distinctly, these compounds inhibited COX-2 level expression in MCF-7 more
strongerly than in HT-29 cells, especially for complex 12.
2.3 Protective effect of complex on oxidative damage of rat myocardial H9c2 cells
Celecoxib was selective COX-2 inhibitors which used clinically as NSAIDs, but it
withdrew from market due to myocardiotoxicity. CORMs had protective effect on
myocardial ischemia-reperfusion injury. [41] Modified with CORMs, they will possibly
improve these side-effects by CO biological function. In order to verify validation of
this idea, we chose H9C2 cells treated with H2O2 as an in vitro model of myocardial
ischemia-reperfusion injury.
To obtain more accurate information about myocardial cell protection, the
toxicities of the tested complexes to H9c2 cells were evaluated at first. The cell
survival rates were greater than 90% after the H9c2 cells treated with the complex at
the set concentration range, which indicates the set concentration was effective and
rational in the protective effect of H9c2 cells. And the cell survival rate was 80.46%,
70.96% and 64.28%, respectively, after treatment with 400µM H2O2 for 1h, 4h and
8h.
Then, the cells were treated with 400µM H2O2 for 1h (or 4h, or 8h), and incubated
with complex (1, or 10 or 12) at different concentrations (10µM, 25µM and 50µM),
the repair effects on H9c2 were detected.
The results showed all three complexes improved the survival rate of H9c2 cells
injured by H2O2 (Fig.4). The survival rate increased most obviously after the cells

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treated with H2O2 for 1h for each tested complex, and it increased least when the cells
harmed for 8h due to the severe cell injury. H9c2 cells treated for 1h, complex 1
increased the survival rate of 13.35%, and for 8h, it increased below 10% (Fig.4); for
complex 12, the survival rate of H9c2 cells was only increased by 3.55% after H2O2
injury for 8h (Fig.4). As a whole, after 1h of injury, complex 1 had the strongest
ability to improve H9c2 cell viability, and complex 12 distinctly improved the cell
survival rate; and for each tested complex, the increasing survival rate of H9c2 cells
was not in a dose-dependent manner. When the tested complex was 50µM, they had a
maximal efficacy to repair damaged H9c2 cells.
Fig. 4 Effect of complex 1, 10 and 12 on the viability rate of H9c2 cell treated with H2O2 for
different time: a for 1h, b for 4h, c for 8h. Each experimental concentration set 6 sets of parallel
experiments, expressed by mean ±SD; * represents testing group and H2O2 group P < 0.05; **
represents testing group and H2O2 group P < 0.01; # represents compared with the blank control (p
< 0.01).
After H9c2 cells were damaged by H2O2, the CO released by the complexes
increased the survival rate of H9c2 cells, and displayed a protection against H9c2
cells injury. To preliminary understand the mechanism of action, the level of SOD and
MDA in H9c2 cells was measured. The results showed, after H9c2 cells treated with
H2O2 for 1h the content of SOD decreased, but it increased significantly after
administration of complex 12; Moreover, with the concentration of the complex 12

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increasing, the content of SOD increased (Fig.5). At the same time, the level of MDA
decreased significantly with the complex increasing (Fig.5). In contrast, the activity of
complex 12 was as strong as that of complex 10, and complex 1 was stronger than
those of complexes 10 and 12. All that suggests the complexes protected H9C2 cells
from antioxidant damage by the way which increase SOD level and low MDA level.
Fig. 5 Effect of complex on SOD and MDA content of H9C2 cells treated with H2O2 for 1h. Six
groups of parallel experiments were set up in each content, and the data were expressed as mean
±SD. ** represents P < 0.01 for testing group and H2O2 group; ## represents P < 0.01 for testing
group and blank group
2.4 Antihypertensive effect of target complex in SHR
It has been shown that CO relaxed the blood vessels and protected the heart,
which was related to the activity of CO regulating the activity of vascular endothelial
factor. [42] CO acted on the peripheral blood vessels and maybe participated in the
regulation of blood pressure. To prove this idea, we selected the spontaneously
hypertensive rats SHR, evaluated the antihypertensive effect of the complex. The
results showed that all the tested complexes had antihypertensive effect on SHR rats.
As shown in Fig. 6, complex 1 which modified by indomethacin decreased the
systolic blood pressure of SHR rats compared with the control group (165mmhg);
When the concentration was 10, 20 and 40 mg/kg, the blood pressure of SHR dropped
to 154.7, 142.6 and 134.6 mmHg; for complex 10 which modified by diclofenac, after
administration of 10, 20 and 40 mg/kg, the contraction pressure of SHR rats decreased
to 147.6, 144.5 and 135.8mmHg. Compared with the control group (165mmhg),
complex 1 almost had the same antihypertensive effect as complex 10. But for
complex 12, it showed stronger activity than those in the same condition, the systolic

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blood pressure of SHR rats were decreased to 135.7, 128.7 and 117.5mmHg. This
effect was also concentration dependent. Meanwhile, the tested complexes also
reduced the diastolic blood pressure, and the trend was consistent with the decrease of
the systolic blood pressure (see in Fig.6). Though the antihypertensive effect of
complex originated from CO released from it, the structure of NSAIDs had
remarkable effect on anti-hypertension, just like the effect of substituent on CO
release rate.
However, compared with nifedipine and captopril, the antihypertensive effects of
the complexes were less at the same concentration. It showed that the regulating effect
of the complexes on blood pressure was limited.
Fig. 6 Effect of the complex on systolic blood pressure and diastolic blood pressure in SHR, A
for complex 1, B for 10, C for 12; D, the anti-hypertension effects of complex 12, captopril
and nifedipine at 10mg/kg
2.5 Myocardial protective effects of complex for SHR
In the process of studying anti-hypertension of the complex, considering the
protection of complex against H9c2 cell injury, we wonder whether the complex is
effective to animal model. By the way, we used optical microscope and TEM to
observe the change of myocardial structure after administrating tested complexes 14
days.
We found that the control (celecoxib) group, myocardial fibers arranged neatly in
SHR, muscle clearance was increased in different degree, and collagen fibers were
found in some of the interstitium. But for the myocardial structures in SHR treated
with complex 1 or 12, myocardial fibers arranged neatly, occasionally found boudoir
disc; the nucleus was located in the center of the muscle fibers, and muscle gap
slightly increased, no obvious interstitial collagen fiber was found. Through TEM, we
can see in Fig.7, in control group, myocardial interstitial vascular swelling, loose

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arrangement of muscle fibers, muscle fiber membrane swelling, myocardial fiber
melting, contraction band formation, myocardial intercalated discs dissociation and
swelling of mitochondria. But in contrast, for complex 1 or 12 group, myofibril basic
was basically regular and no obvious lysis of myofilament; mitochondrial swelling
was not significant and no myocardial intercalated discs dissociation were found.
Distinctly, the complexes protected the myocardium in some degree caused by
celecoxib. We know celecoxib is a selective inhibitor of COX-2, and its toxic effects
for heart and blood vessels may result from inhibiting the COX-2 activities at the
special organs and tissues, because many testing results proved COX-2 mediated
delayed myocardial protection. Therefore, we conclude that this myocardial protective
effect was mediated by CO released from the tested complex.
Fig. 7 Optical microscopy (color) and TEM morphological images of cardiac muscle cell from
tested SHR: Myocardial protective effect of the complex appeared after administrating a certain
dose tested complex successively 14 days. The morphological cells are seen in different states：A
for celecoxib，B for complex 1，C for complex 12. The arrows in A(a), B(a) and C(a) (6,000-fold)
indicate myocardial fiber bundles gap; The arrows in A(b), B(b) and C(b) (10,000-fold) indicate
the swelling of myocardial fibers; A(c), B(c) and C(c) (210,000-fold) indicates dissolution of the
myocardial fibers; A(d), B(d) and C(d) (20,000-fold) arrows indicate myocardial intercalated
discs.

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2.6 Liver and kidney toxicity of complex for SHR
Similarly, through optical microscope (Fig.8), for the SHR treated with complex 1
or 12, we saw the hepatic lobule was complete; congestion of central venous and
congestion of hepatic sinusoid were obvious; liver cell was swelling and cytoplasm
staining was pale. Meanwhile, the the renal glomerulus was intact; the proximal
tubule cells were swelled, the cytoplasm was loose and pale, and some renal tubular
epithelial cells were necrotic. Under TEM, we found liver cell nucleus abnormal,
heterochromatin increase, chromatin margination, lipid droplets increase in the
cytoplasm and hepatic blood sinus Dirichlet space widened.
Fig. 8 Optical microscopy (color) and TEM morphological images of liver and kidney cells from
tested SHR after administrating a certain dose complex successively 14 days: A for and B, the
morphological liver cells treated with complex 1 and complex 12. C and D, the morphological
kidney cells treated with complex 1 and complex 12 respectively. The arrows in A(c, d) and B(c, d)
arrows indicate abnormal nuclear morphology; the arrows in C(c, d) indicate swelling of
glomerular endothelial cells, a large amount of exudation in glomeruli; D(c, d) indicate mesangial
cell proliferation, irregular proximal convoluted tubule villus.
Meanwhile, the glomerular endothelial cells were swelling with stromal
hyperplasia and exudation in the glomerulus; the proximal tubule microvilli exfoliated
loosed and the foot processes were fusion. In contrast, the side-effect on kidney

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caused by complex 12 was more serious. This demonstrates the two complexes which
administrated continuously are still not safe for 14 days.
3. Conclusion
Carbon monoxide (CO) is an important signaling molecule as NO. CORMs are
hoped to deliver controlled amounts of CO directly to tissues or organs. CORMs
containing cobalt are complex. Most of them inhibited tumor cell proliferation, and
their activities were connected with the structures of non-CO ligands. Based on
COX-2 role in tumorigenesis, development and metastasis of cancer, we synthesized
hybrid CORMs containing NSAIDs structure. Compared with cisplatin, all the
complexes displayed low activities against proliferation of five kinds of tumor cells.
But complexes 12 and 13 displayed better activities and selectivity to HT-29 and
MCF-7 cell lines compared with 5-FU. In contrast, complexes 12 and 13 were more
effective against tumor cells; moreover, complex 12 was stronger to down-regulate
COX-2 expression level than complex 4. Possibly, this is because complex 12
contained celecoxib structure fragment, which was a COX-2 selective inhibitor. We
found the complex inhibited the expression of COX-2 in HT-29 cells and MCF-7cells,
in accordance with its activity against tumor cell proliferation. Of course, seen from
the IC50 values of all the complexes, their activities were not very high. However, this
does not stand for these complexes have no value to further investigate, because
whether they inhibitor tumor cell metastasis and tumorigenesis, it needs us to further
investigate. As expected, the complex increased the survival rate of myocardial H9c2
cells after the cells treated with H2O2, which indicates the complexes have protective
effect on oxidative damage of rat myocardial H9c2 cells. But this protective effect
was not obvious when the cells harmed for 8h due to the severe cell injury. In addition,
the compound had concentration dependent antihypertensive effect on SHR rats,
accompanying with good myocardium protection. This indicates these hybrid CORMs
reduced side-effects of COX-2 inhibitors, but there still have liver and kidney
side-effects when administrating continuously for 14 days.
4. Experimental
4.1 Reagents and instruments
Myoglobin (AR, Sigma), BCA protein quantification kit (Sigma), ELISA kit (Sigma).
Captopril and nifedipine nifelat (AR) were purchased from Beijing bailingwei Technology Co.
Hela cell ,HepG2 cell ,MCF-7 cell ,A549 cell ,HT-29 cell and W138 cell lines were purchased
from the cell resources Center for Shanghai Life Science Institute of Chinese Academy of
Sciences (China). Clean grade male spontaneously hypertensive rats (SHR) rats and WKY
(Wistar-Kyoto) rats were provided from the Animal Center of GLP, Lanzhou University.
Laboratory animal qualification: SCXK (GAN) 2009-2004. Rats were fed in cage, kept at

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room temperature (23±2) , relative humidity 60-65%, free eating and drinking water.
Blood press was measured using BP-300A Automatic non-invasive blood pressure
detector (Chengdu, taimeng, China, Software Co. Ltd.).
All reactions were carried under nitrogen atmosphere. Solvents for reactions were
degassed and distilled from the proper drying agents. Column chromatography was carried
out using 200-300 mesh silica gel.
IR spectra were recorded on a Nicolet NEXUS 360 spectrophotometer, and NMR spectra
on a BrukerAM-400 MHz spectrometer. A Lambda 25 UV-Visible spectrophotometer and a
Maxis-4G TOF Mass spectrometer (ESI) were used.
4.2 Synthesis of the complexes
The preparation of compound A1
To the solution of (indomethacin, 357 mg 1.0mmol) in 8mL of acetonitrile, DMAP
(18mg, 0.15mmol) and EDCI (190mg, 1.0mmol) were added with stirring at room
temperature for 0.5h. Then, propargyl alcohol (0.07ml, 1.0mmol) was added to the
mixture solution and stirred for 4h. After filtration, the filtrate was evaporated under
reduced pressure to remove the solvent. The oily residue was dissolved in
trichloromethane; the organic layer was washed with brine and dried with anhydrous
Na2SO4, then filtered; the solvent was removed. The crude product was
chromatographed on a silica gel, and 260 mg white solid was obtained. Yield: 65.8%.
Ethyl acetate/hexane (1:15); Rf =0.28. 1H NMR (CDCl3) δ 7.60 (dd, J = 8.7, 2.0 Hz,
2H), 7.43 – 7.38 (m, 2H), 6.89 (d, J = 2.4 Hz, 1H), 6.80 (d, J = 9.0 Hz, 1H), 6.60 (dd,
J = 9.0, 2.4 Hz, 1H), 4.64 (d, J = 2.4 Hz, 2H), 3.77 (s, 3H), 3.65 (s, 2H), 2.41 (t, J =
2.4 Hz, 1H), 2.32 (s, 3H). ESI-HRMS (m/z): calcd for C23H18ClNNaO4 [M+Na]+
418.0822,found 418.0814.
The preparation of compound A2-A11
The preparation and workup of compounds A2-A11 were similar to A1. Compound
A2: white solid 168 mg. Yield: 60.2%. Ethyl acetate/hexane (1:10); Rf = 0.32. 1H
NMR (CDCl3) δ 9.07 (s, 1H), 7.93 (dd, J = 8.0, 1.6 Hz, 1H), 7.18 (s, 1H), 7.05 (dt, J =
15.0, 7.8 Hz, 2H), 6.96 (d, J = 7.2 Hz, 1H), 6.66 (d, J = 8.6 Hz, 1H), 6.60 (t, J = 7.8
Hz, 1H), 4.85 (d, J = 2.5 Hz, 2H), 2.45 (t, J = 2.5 Hz, 1H), 2.26 (s, 3H), 2.10 (s, 3H).
ESI-HRMS (m/z): calcd. for C18H18NO2 [M+H]+: 280.1338. found 280.1338; calcd.
for C18H17NNaO2 [M+Na]+: 302.1157; found 302.1173.
Compound A3: yellow Oil. Yield: 56.2%. Ethyl acetate/hexane (1:10); Rf =0.32. 1H
NMR (CDCl3) δ 7.77 – 7.70 (m, 2H), 7.68 (t, J = 1.6 Hz, 1H), 7.61 (dt, J = 8.0, 1.6
Hz, 1H), 7.49 (ddt, J = 14.8, 7.7, 1.8 Hz, 2H), 7.40 (dd, J = 15.2, 7.8 Hz, 3H), 4.60
(qd, J = 15.6, 2.4 Hz, 2H), 3.77 (q, J = 7.2 Hz, 1H), 2.37 (t, J = 2.4 Hz, 1H), 1.48 (d, J
= 7.0 Hz, 3H). ES I-HRMS (m/z): calcd. for C19H16NaO3 [M+Na]+: 345.0997; found
315.0986.

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Compound A4: pale yellow solid 288.8mg; Yield: 73.1%. Ethyl acetate/hexane
1
(1:20);Rf=0.43. H NMR (400 MHz, Chloroform-d) δ 7.76 – 7.51 (m, 4H), 7.17 (s,
1H), 7.10 (s, 1H), 6.81 (dd, J = 8.9, 2.4 Hz, 1H), 6.50 (td, J = 8.8, 2.5 Hz, 1H), 4.64 (d,
J = 2.5 Hz, 2H), 3.55 (s, 2H), 2.74 (s, 3H), 2.42 (t, J = 2.5 Hz, 1H), 2.14 (s, 3H).
ESI-HRMS (m/z): calcd. for C22H20FO3S [M+H]+: 395.1117; found 395.1125,
C22H20FO3SNa [M+Na]+: 417.0937; found 417.0945.
Compound A5: white solid 138.2 mg; Yield: 48.94%. Ethyl acetate/hexane
(1:4);Rf=0.28.1H NMR (CDCl3) δ 7.46 (dt, J = 8.0, 1.6 Hz, 2H), 7.39 – 7.33 (m, 2H),
7.32 – 7.27 (m, 2H), 7.10 – 7.03 (m, 2H), 4.70 – 4.53 (m, 2H), 3.73 (q, J = 7.2 Hz,
1H), 2.39 (t, J = 2.4 Hz, 1H), 1.48 (d, J = 7.2 Hz, 3H). ESI-HRMS (m/z): calcd. for
C18H15FNaO2 [M+Na]+: 305. 0954; found 305.0944.
Compound A6: white solid 126.9mg ； Yield: 42.30%. Ethyl acetate/hexane
(1:10);Rf=0.361H NMR (400 MHz, Chloroform-d) δ 9.74 (s, 1H), 8.37 – 8.13 (m, 2H),
7.77 (dd, J = 7.5, 2.0 Hz, 1H), 7.12 – 7.07 (m, 2H), 6.67 (dd, J = 7.8, 4.7 Hz, 1H),
4.88 (d, J=2.5Hz,2H), 2.49(t, J=2.4Hz, 1H), 2.32(s,3H). ESI-HRMS(m/z): calcd.for
C16H14ClN2O2[M+H]+:301.0744;found301.0740,C16H13ClN2NaO2[M+Na]+: 323.0563;
found 323.0560.
Compound A7: white solid 150.2mg；Yield: 46.93%. Ethyl acetate/hexane (1:6);
Rf=0.30.1H NMR (CDCl3) δ 9.43 (s, 1H), 7.96 (dd, J = 8.1, 1.7 Hz, 1H), 7.41 (s, 1H),
7.38 – 7.28 (m, 3H), 7.24 (d, J = 7.3 Hz, 1H), 7.22 – 7.17 (m, 1H), 6.84 – 6.61
(m,1H),4.84(d,J=2.5Hz,2H),2.46(t,J=2.5Hz,1H).ESI-HRMS(m/z):calcd.forC17H13F3N
O2[M+H]+:320.0986;found320.0999.
Compound A8: white solid 120.6 mg； Yield: 42.17%. Ethyl acetate/hexane
(1:10);Rf=0.32.1H NMR (CDCl3) δ 8.42 (s, 1H), 8.09 (dt, J = 7.8, 1.6 Hz, 1H), 7.96 –
7.91 (m, 1H), 7.40 (d, J = 7.6 Hz, 1H), 4.86 (d, J = 2.4 Hz, 2H), 2.44 (t, J = 2.4 Hz,
1H), 1.29 (s, 12H). ESI-HRMS (m/z): calcd. for C16H19BNaO4 [M+Na]+: 309.1274;
found 309.1265.
Compound A9: white solid 120.6 mg；Yield: 42.17%. Ethyl acetate/hexane
(1:3);Rf=0.36.1H NMR (CDCl3) δ 10.18 (s, 1H), 8.58 – 8.19 (m, 2H), 8.01 (s, 1H),
7.81 (s, 1H), 7.38 (t, J = 7.8 Hz, 1H), 7.24 (s, 1H), 6.76 (d, J = 4.6 Hz, 1H), 4.88 (d, J
= 2.4 Hz, 2H), 2.50 (t, J = 2.4 Hz, 1H). ESI-HRMS (m/z): calcd. ForC16H12F3N2O2
[M+H]+: 321.0861，found 321.0891.
Compound A10: white solid 136.6 mg. Yield: 40.8%. Ethyl acetate/hexane
(1:5);Rf=0.43.1H NMR (CDCl3) δ 7.27 (d, J = 8.0 Hz, 2H), 7.19 (d, J = 2.8 Hz, 1H),
7.07 (td, J = 7.6, 1.6 Hz, 1H), 6.94 – 6.87 (m, 2H), 6.49 (dd, J = 8.0, 1.2 Hz, 1H), 4.68
(d,J=2.5Hz,2H),3.80(s,2H),3.42(s,1H),2.42(t,J=2.5Hz,1H).ESI-HRMS(m/z):calcd.For
C17H14Cl2NO2[M+H]+:334.0462;found334.0475.
Compound A11: white solid 112.4 mg. Yield: 35.3%. Ethyl acetate/hexane (1:2);
Rf=0.31. 1H NMR (CDCl3) δ 7.83 (d, J = 7.6 Hz, 1H), 7.75 (d, J = 8.2 Hz, 2H), 7.51

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(t, J = 7.4 Hz, 1H), 7.43 (d, J = 8.0 Hz, 2H), 7.30 (s, 2H), 4.77 (s, 2H), 4.68 – 4.49 (m,
2H), 3.70 (q, J = 7.2 Hz, 1H), 2.38 (t, J = 2.5 Hz, 1H), 1.45 (d, J = 7.2 Hz, 3H).
ESI-HRMS (m/z): C20H17NNaO3 [M+Na]+: 342.1156; found 342.1177.
The preparation of compound A12-A13
Compound A12: To the solution of (celecoxib, 1013 mg 3.0mmol) in 20mL of
acetone, anhydrous potassium carbonate (167 mg, 1.2mmol) were added with stirring
at room temperature for 0.5h. Then 3-bromopropyne (0.08 ml, 1.0mmol) was added
and stirred for 24h at room temperature. After filtration, the filtrate was evaporated
under reduced pressure to remove the solvent. The crude product was
chromatographed on a silica gel (ethyl acetate: petroleum ether10:1→3:1), and 298.0
mg white solid was obtained. Yield:23.8 %.1H NMR (400 MHz, Chloroform-d) δ 7.81
(d, J = 8.7 Hz, 2H), 7.41 (dd, J = 8.9, 2.2 Hz, 2H), 7.19 (s, 1H), 7.11 (s, 2H), 7.03 (d,
J = 8.2 Hz, 2H), 6.67 (s, 1H), 3.80 (dd, J = 6.1, 2.6 Hz, 2H), 2.31 (s, 3H), 2.02 (t, J =
2.5 Hz, 1H).ESI-HRMS (m/z): calcd. for C20H16F3N3NaO2S [M+Na]+:442.0813;
found 442.0820.
Compound A13: To the solution of (nimesulide, 308 mg 1.0mmol) in 10mL of
acetone, anhydrous potassium carbonate (167mg, 1.2mmol) were added with stirring
at room temperature for 0.5h. To the reaction 3-bromopropyne (0.08ml, 1.0mmol)
was added and stirred for 24h at room temperature. After filtration, the filtrate was
evaporated under reduced pressure to remove the solvent. The crude product was
chromatographed on a silica gel (thyl acetate: petroleum ether 5:1, Ethyl
acetate/hexane (1:5);Rf=0.35), and 280.0 mg white solid was obtained. Yield: 80.6 %.
1H NMR (400 MHz, DMSO-d6) δ 8.02 (dd, J = 8.7, 2.6 Hz, 1H), 7.82 (d, J = 8.7 Hz,
1H), 7.56 – 7.50 (m, 2H), 7.48 (d, J = 2.6 Hz, 1H), 7.33 (s, 1H), 7.28 – 7.24 (m, 2H),
4.58 (d, J = 2.5 Hz, 2H), 3.39 (t, J = 2.4 Hz, 1H), 3.24 (s, 3H).ESI-HRMS (m/z):
calcd. for C16H15N2O5S [M+H]+: 347.0702; found 347.0727, C16H14N2NaO5S
[M+Na]+: 369.0521; found 369.0544.
The preparation of compound A14
To the solution of (ferulic acid ethyl ester 223 mg 1.0mmol) in 10mL of DMF,
anhydrous potassium carbonate (207mg, 1.5mmol) were added with stirring at room
temperature for 0.5h. Then 3-bromopropyne (0.08ml, 1.0mmol) was added and
stirred for 24h at room temperature. After filtration, the filtrate was evaporated under
reduced pressure to remove the solvent. The crude product was chromatographed on a
silica gel (thyl acetate: petroleum ether 5:1 Ethyl acetate/hexane (1:5); Rf=0.36). And
white powder 114 mg was obtained; Yield: 43.6%,1H NMR (400 MHz, Chloroform-d)
δ 7.56 (d, J = 15.8 Hz, 1H), 7.05 – 6.94 (m, 3H), 6.26 (d, J = 15.9 Hz, 1H), 4.73 (d, J
= 2.4 Hz, 2H), 4.19 (q, J = 7.1 Hz, 2H), 3.84 (s, 3H), 2.47 (t, J = 2.3 Hz, 1H), 1.27 (t,
J = 7.1 Hz, 3H). ESI-HRMS (m/z): calcd. For C15H17O4[M+H]+: 261.1127; found
261.1115, C15H16NaO4 [M+Na]+: 283.0946; found 280.0934.

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The preparation of compound A15
To 0.51 g (1.32 mmol) Celecoxib, 0.42 g (2.65 mmol) potassium permanganate in
10 mL water 0.52 mL 2M sodium hydroxide were added and heated to reflux for 6 h.
The cooled, brown suspension was filtrated and washed with water. The filtrate was
acidified with 2M hydrochloric acid and the white voluminous preticipate was
collected and dried in vacuum affording 0.36 g (66%) carboxylic acid B15.1H NMR
(400 MHz, Chloroform-d) δ 8.05 – 7.97 (m, 2H), 7.85 (d, J = 8.2 Hz, 2H), 7.37 (d, J =
8.1 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 6.79 (s, 1H), 4.94 – 4.74 (m, 2H), 2.46 (t, J =
2.3 Hz, 1H). ESI-HRMS (m/z): calcd. for C17H13F3N3O4S [M+H]+: 412.0579; found
412.0658, C17H12F3N3KO4S [M+Na]+: 450.0138; found 450.0138. To the solution of
B15, 411.80mg 1.0mmol) in 20mL of DMF, DMAP (18mg, 0.15mmol) and EDCI
(190mg, 1.0mmol) were added with stirring at room temperature for 0.5h. To the
reaction propargyl alcohol (0.07ml, 1.0mmol) was added and stirred for 4h at room
temperature. After filtration, the filtrate was evaporated under reduced pressure to
remove the solvent. The oily residue thus obtained was dissolved in trichloromethane;
the organic layer was washed with brine, with NaCl 5%, and then dried on anhydrous
Na2SO4, filtered and the solvent evaporated. The crude product was chromatographed
on a silica gel, and 220.6 mg white solid was obtained. Yield: 49.1%. Ethyl
1
acetate/hexane (1:3); Rf=0.32. H NMR (400 MHz, Chloroform-d) δ 8.01 (d, J = 8.2
Hz, 2H), 7.87 (d, J = 8.6 Hz, 2H), 7.39 (d, J = 8.5 Hz, 2H), 7.27 (d, J = 8.2 Hz, 2H),
7.19 (s, 2H), 6.79 (s, 1H), 4.87 (d, J = 2.4 Hz, 2H), 2.47 (t, J = 2.4 Hz, 1H).
ESI-HRMS (m/z): calcd. for C20H14F3N3O4S [M+H]+: 450.0735; found 450.0748.
2. The preparation of complexes 1-15
The preparation of complex 1: 395 mg (1.0 mmol) of compound A1 was dissolved
in 20 mL of dry THF, and then 456 mg (1.2 mmol) of dicobaltoctacarbonyl was added.
The reaction mixture was stirred for 12 h at room temperature. The solvent was
removed in vacuo, the residue was extracted by a small amount of CH2Cl2, and the
extract was subjected to silica gel column chromatography. Ethyl acetate/hexane (1:5)
eluted the main red band, from which 410 mg (60.29%) of complex 1 as a brown-red
oil was obtained.IR (KBr disk, cm-1): 2100vs, 2063vs, 2059vs, 2033vs (Co-CO),
1762s, (COO). 1H NMR (400 MHz, Chloroform-d) δ 7.59 (d, J = 8.0 Hz, 2H), 7.40 (d,
J = 8.1 Hz, 2H), 6.89 (s, 1H), 6.79 (d, J = 9.0 Hz, 1H), 6.60 (d, J
=9.0Hz,1H),5.99(s,1H),5.26(s,2H),3.76(s,3H),3.69(s,2H),2.31(s,3H).13CNMR(101M
Hz,CDCl3)δ198.1,169.5167,3,155.1,138.2,135.2,132.9,130.2,129.8,129.6,128.1,113.9
,111.0,110.7,100.2,87.5,70.9,64.4,54.6,28.9,12.3.ESI-HRMS(m/z):calcd.forC28H18Cl
Co2NO10Na[M+Na]+:703.9181; found 703.9175.
The procedure and workup of complexes 2-15 are similar to the process of
complex 1.

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Complex 2: 298.2mg was obtained as red powder. Yield: 52.8%. Ethyl
acetate/hexane (1:5);Rf=0.43.IR (KBr disk, cm-1): 2099vs, 2053vs, 2031vs, 2007vs
(Co-CO), 1686s, (COO). 1H NMR (400 MHz, Chloroform-d) δ 9.21 (s, 1H), 8.00 –
7.95 (m, 1H), 7.21 – 7.15 (m, 1H), 7.05 (dd, J = 15.3, 8.0 Hz, 2H), 6.96 (d, J = 7.3 Hz,
1H),6.66(d,J=8.5Hz,1H),6.58(t,J=7.6Hz,1H),6.06(s,1H),5.44(s,2H),2.26(s,3H),2.10(s,
3H).13CNMR(101MHz,CDCl3)δ198.1,167.3,148.9,137.6,137.2,133.5,131.7,130.4,12
5.9,124.9,122.4,115.0,112.61,109.0,87.9,76.1,71.1,64.0,19.6,12.9.ESI-HRMS(m/z):ca
lcd.forC24H17NCo2NaO8[M+Na]+: 587.9516; found 587.9506.
Complex 3:301.6 mg was obtained as red powder. Yield: 52.1%. Ethyl
acetate/hexane (1:6); Rf=0.36.IR (KBr disk, cm-1): 2094vs, 2050vs, 2032vs, 2016vs,
2002vs (Co-CO), 1735s, (COO). 1H NMR (400 MHz, Chloroform-d) δ 7.72 (d, J =
9.3 Hz, 3H), 7.65 – 7.58 (m, 1H), 7.56 – 7.46 (m, 2H), 7.42 (t, J = 8.6 Hz, 2H), 7.19
(s, 1H), 5.95 (s, 1H), 5.39 – 5.01 (m, 2H), 3.88 – 3.70 (m, 1H), 1.60 – 1.45 (m, 3H).
13C NMR (101 MHz, CDCl3) δ199.4, 196.5, 173.6, 140.6, 138.0, 137.5, 132.5,
131.6,130.5, 129.2, 129.1,128.6, 128.3, 77.2, 71.97, 65.72, 45.32, 18.48.ES I-HRMS
(m/z): calcd. for C25H16NaCo2O9 [M+Na]+: 600.9356; found 600.9348.
Complex 4: 396.6 mg was obtained as red powder. Yield: 58.4%. Ethyl
acetate/hexane (1:5);Rf=0.36.IR (KBr disk, cm-1): 2096vs, 2063vs, 2034vs, 2010vs
1
(Co-CO), 1720s (COO). H NMR (400 MHz, Chloroform-d) δ 7.76 – 7.48 (m, 4H),
7.21 (s, 1H), 7.14 (s, 1H), 6.82 (dd, J = 8.9, 2.4 Hz, 1H), 6.50 (td, J = 8.8, 2.5 Hz, 1H),
5.96(s,1H),5.26(s,2H)3.56(s,2H),2.78(s,3H),2.16(s,3H).13CNMR(101MHz,Chlorofor
m-d)δ197.9,170.8,162.1,147.2,145.1,141.9,136.3,130.1,129.4,127.7,125.5,123.5,112.
2,71.7,65.2,54.7.ESI-HRMS (m/z): calcd. for C29H20Fco2O9S [M+H]+:680.9476;
found 680.9558.
Complex 5: 302.6 mg was obtained as red powder. Yield: 53.4%. Ethyl
acetate/hexane (1:4); Rf=0.44 IR (KBr disk, cm-1): 2098vs, 2051vs, 2037vs, 2026vs
(Co-CO), 1736s (COO). 1H NMR (400 MHz, Chloroform-d) δ 7.46 (d, J = 7.5 Hz,
2H), 7.37 (t, J = 7.7 Hz, 2H), 7.32 (s, 1H), 7.19 (s, 1H), 7.08 (t, J = 11.0 Hz, 2H), 5.96
(s, 1H), 5.48 – 5.01 (m, 2H), 3.76 (q, J = 7.4, 7.0 Hz, 1H), 1.51 (d, J = 6.2 Hz,
3H).13CNMR(101MHz,Chloroform-d)δ205.9,172.7,159.9,140.4,134.5,129.9,129.8,12
7.9,127.4,126.6,122.6,114.4,114.2,76.2,70.9,64.7,43.9.ESI-HRMS(m/z):calcd.forC24
H15FCo2NaO8
[M+Na]+:
590.9313;
found
590.9308.
calcd.
for
C24H15Co2FKO8[M+K]+: 606.9052; found 606.9057.
Complex 6: 302.1mg was obtained as red powder. Yield: 51.6%. Ethyl
acetate/hexane (1:5); Rf=0.44. IR (KBr disk, cm-1): 2098vs, 2060vs, 2034vs, 2021vs,
1998vs(Co-CO),1690s(COO).1HNMR(400MHz,Chloroform-d)δ9.90(s,1H),8.27(s,2H
),7.83(s,1H),7.09(s,2H),6.64(s,1H),6.08(s,1H),5.48(s,2H),2.33(s,3H).13CNMR(101M
Hz,Chloroform-d)δ198.0,166.3,155.7,152.7,139.1,138.2,133.8,127.9,125.6,124.0,121.
1,112.4,105.4,87.1,71.1,64.8,14.0.ESI-HRMS(m/z):calcd.forC22H14ClCo2N2O8

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[M+H]+: 586.9163; found 586.9133. calcd. for C22H13ClCo2N2NaO8 [M+Na]+:
608.8922; found 608.8957.
Complex 7: 336.6mg was obtained as red powder. Yield: 54.2%. Ethyl
acetate/hexane (1:5); Rf=0.40. IR (KBr disk, cm-1): 2097vs, 2045, 2012 vs, (Co-CO),
1682s (COO). 1H NMR (400 MHz, Chloroform-d) δ 9.52 (s, 1H), 8.01 (d, J = 8.0 Hz,
1H), 7.44 – 7.19 (m, 6H), 6.74 (t, J = 7.6 Hz, 1H), 6.07 (s, 1H), 5.44
(s,2H).13CNMR(101MHz,Chloroform-d)δ205.9,167.0,146.1,140.4,133,6,131.0,130.7,
128.9,123.4,118.7,117.3,113.2,76.2,71.2,64.4.ESI-HRMS(m/z):calcd.ForC23H12F3Nco
2NaO8 [M+Na]+: 627.9077; found 627.9067.
Complex 8: 268.6mg was obtained as red powder. Yield: 47.2%. Ethyl
acetate/hexane (1:5); Rf=0.35.IR (KBr disk, cm-1): 2098vs, 2056vs, 2034vs, 2019vs
(Co-CO), 1715s (COO). 1H NMR (400 MHz, Chloroform-d) δ： 8.49 (s, 1H), 8.11 (s,
1H), 7.92 (s, 1H), 7.38 (s, 1H), 6.05 (s, 1H), 5.46 (s, 2H), 1.27 (s, 12H). 13C NMR
(101MHz,Chloroform-d)δ：198.0, 165.4, 138.3, 135.2, 131.2, 126.8, 76.1, 71.0,
64.4,23.8.ESI-HRMS(m/z):calcd.for C22H19Bco2NaO10 [M+Na]+: 594.9633; found
594.9664.
Complex 9: 316.2mg was obtained as red powder. Yield: 52.6%. Ethyl
acetate/hexane (1:5);Rf=0.38. IR (KBr disk, cm-1): 2098vs, 2061vs, 2038vs, 2021vs
(Co-CO), 1691s (COO). 1H NMR (400 MHz, Chloroform-d) δ 10.31 (s, 1H), 8.41 (s,
1H), 8.30 (d, J = 7.8 Hz, 1H), 8.00 (s, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.33 (t, J = 7.9
Hz, 1H), 7.21 (d, J = 7.4 Hz, 1H), 6.73 (dd, J = 8.1, 3.1 Hz, 1H), 6.09 (s, 1H),
5.49(s,2H).13CNMR(101MHz,Chloroform-d)δ206.9,167.2,155.9,153.5,140.2,129.2,1
23.7,119.2,117.3,114.1,76.96,72.2,66.0.ESI-HRMS(m/z):calcd.forC22H11Co2F3N2NaO
8 [M+Na]+:628.9029;found 628.9008.
Complex 10: 320.6mg was obtained as red powder. Yield: 51.8%. Ethyl
acetate/hexane (1:5);Rf=0.33 IR (KBr, cm-1): 2097vs, 2068vs, 2021vs, 2007, 1996
(Co-CO), 1728s (COO). 1H NMR (400 MHz, Chloroform-d) δ 7.27 (s, 1H), 7.19 (s,
1H),7.14(d,J=7.5Hz,1H),7.06(t,J=7.9Hz,1H),6.90(dt,J=14.0,7.7Hz,3H),6.49(d,J=8.0
Hz,1H),5.98(s,1H),5.28(s,2H),3.82(s,2H).13CNMR(101MHz,Chloroformd)δ198.2,171
.1,141.1,130.0,128.4,127.9,127.1,123.0,122.9,121.0,117.3,76.20,72.4,,64.8,37.27.
ESI-HRMS (m/z): C23H13Cl2Co2NNaO8 [M+Na]+: 641.8580; found 641.8542.
Complex 11: 326.1mg was obtained as red powder.Yield:53.9%. Ethyl
acetate/hexane (1:5);Rf=0.44.IR (KBr, cm-1): 2099vs, 2056vs, 2033vs, 2006 (Co-CO),
1728s (COO). 1H NMR (400 MHz, Chloroform-d) δ 7.88 – 7.31 (m, 8H), 5.95 (s, 1H),
5.31(d,J=14.2Hz,1H),5.07(d,J=14.4Hz,1H),4.78(s,2H),3.74(s,1H),1.49(s,3H).13CNM
R(101MHz,Chloroform-d)δ199,2,174.2,167.5,140.1,138.6,136.3,133.2,132.1,128.4,1
28.3,124.2,122.6,119.7, 88.5 , 71.9 , 65.5 , 50.7 , 44.9 , 18.4.ESI-HRMS (m/z): calcd.
for C26H18NCo2O9 [M+H]+: 605.9646.found 605.9785.
Complex 12: 306.1mg was obtained as red powder. Yield: 50.9%. Ethyl

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1
acetate/hexane (1:5);Rf=0.46.IR (KBr, cm-1): 2098vs, 2057vs, 2025vs (Co-CO) H
NMR (400 MHz, Chloroform-d) δ 7.81 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.3 Hz, 2H),
7.19(s,1H),7.10(s,2H),7.04(d,J=7.9Hz,2H),6.67(s,1H),5.88(s,1H),4.26(d,J=6.5Hz,2H)
,2.32(s,3H).13CNMR(101MHz,Chloroform-d)δ197.4,141.8,138.8,138.4,128.8,127.7,1
27.1,124.7,124.6,105.5,76.19,71.52,45.0,20.3.ESI-HRMS:calcd.forC26H17F3Co2N3O8
S[M+H]+705.9352,found705.9355, calcd. for C26H16F3Co2N3NaO8S[M+Na]+
727.9172, found 727.9169.
Complex 13: 455.2.1mg was obtained as red powder.Yield:61.9%. Ethyl
1
acetate/hexane (1:6);Rf=0.42. IR (KBr, cm-1): 2096vs, 2058vs, 2021vs (Co-CO). H
NMR (400 MHz, Chloroform-d) δ 7.98 (dd, J = 8.6, 2.4 Hz, 1H), 7.73 (d, J = 8.7 Hz,
1H), 7.65 (d, J = 2.4 Hz, 1H), 7.48 (t, J = 7.8 Hz, 2H), 7.26 (s, 1H), 7.06 (d, J = 7.9
Hz,2H),5.82(s,1H),5.22(s,2H),3.05(s,3H).13CNMR(101MHz,Chloroform-d)δ207.0,15
3.8,148.3,134.7,133.7,130.9,126.3,119.9,117.8,112.3,77.3,72.9,52.6,40.0.ESI-HRMS:
calcd.forC22H14Co2N2NaO11S[ M+Na+ ]+654.8880, found 654.8926.
Complex 14: 325.1mg was obtained as red powder.Yield:60.9%. Ethyl
acetate/hexane (1:8); Rf=0.36. IR (KBr, cm-1): 2099vs, 2056vs, 2034vs, 2014vs
(Co-CO). 1H NMR (400 MHz, Chloroform-d) δ 7.56 (d, J = 15.9 Hz, 1H), 7.02 (d, J =
10.8 Hz, 2H), 6.85 (d, J = 8.3 Hz, 1H), 6.26 (d, J = 15.9 Hz, 1H), 5.98 (s, 1H), 5.24 (s,
2H),4.19(q,J=7.2Hz,2H),3.78(s,3H),1.27(t,J=7.2Hz,3H).13CNMR(101MHz,Chlorofor
m-d)δ198.1,166.2,149.1,148.5,143.4,127.4,121.0,115.3,112.7,109.5,87.5,71.3,68.2,59.
4,54.7,13.3.ESI-HRMS(m/z):calcd.forC21H17Co2O10[M+H]+:
546.9486;
found
546.9461.
Complex 15: 455.2.1mg was obtained as red powder. Yield: 61.9%. Ethyl
acetate/hexane (1:10); Rf=0.38.IR (KBr, cm-1): 2098vs, 2062vs, 2035vs, 2023
1
(Co-CO), 1712s (COO). H NMR (400 MHz, Chloroform-d) δ 8.02 (d, J = 8.4 Hz,
2H),7.88(d,J=8.8Hz,2H),7.42(d,J=8.6Hz,2H),7.30(d,J=8.4Hz,2H),7.19(s,2H),6.79(s,1
H),5.96(s,1H),5.26(s,2H).13CNMR(101MHz,Chloroform-d)δ198.3,168.3,155.1,149.2,
145.5,143.1,131.3,129.2,127.4,120.3,87.7,64.2,54.6.ESI-HRMS:calcd.forC26H15F3Co2
N3O10S[M+H]+735.9004,found735.9267,calcd.forC26H14F3Co2N3NaO10S[M+Na]+757
.8914, found 757.9084.
4.3 Crystallographic analysis
Crystallographic analysis Diffraction data were collected at 291 K on a Bruker
Smart Apex II diffractometer with Mo-Ka radiation (k = 0.71070 A ° ) using a
SMART CCD camera. Diffractometer control, data collection and initial unit cell
determination were performed using ‘‘SMART.’’ Frame integration and unit cell
refinement were carried out with the ‘‘SAINT’’ software package. Absorption
corrections were applied with SADABS (v 2.10 Sheldrick). Structureswere solved by
direct methods using SHELXS-97 and refined by full-matrix least-squares using

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SHELXL-97 [43]. All nonhydrogen atoms were refined anisotropically. Hydrogen
atoms attached to the nitrogen of amino groups were located in the electron difference
map, and remaining hydrogen atoms were placed using a ‘‘riding model’’ andincluded
inthe refinement at calculated positions. CCDC 1820688 (9), 1820689(11),
1820690(14) and 1820687 (16) contain the supplementary crystallographic data for
this paper.
4.4. Lipophilicity (log P o/w)
The lipophilicity of complex was determined using the flaskshaking method
where n-octanol and 0.1 M PBS were used as the organic and aqueous phase
respectively. n-Octanol was presaturated with 0.1 M PBS by swirling at 45 rpm for 24
h. The tested complex was dissolved in the isolated organic phase at a concentration
of 50 uM. An equal volume of aqueous PBS was added, and the mixture was swirled
for 8 h at 45 rpm at 37 . The solution was then centrifuged, and the amounts of
complex in both layers were determined by ICP-AES.
4.5 CO-release tests with myoglobin assay
The release of CO from the metal carbonyl complexes was studied
spectrophotometrically by measuring the conversion of deoxy-myoglobin (deoxy-Mb)
to carbonyl myoglobin (Mb-CO). The amount of Mb-CO formed was quantified by
measuring the absorbance at 540 nm. A stock solution of myoglobin (lyophilised
horse heart) (60 µM final concentration) was prepared fresh by dissolving the protein
in phosphate buffered saline (PBS, 0.1M, pH=7.4). Sodium dithionite (0.1%) was
added to convert the myoglobin stock to deoxy-Mb. A 2 ml quantity of this solution
was assayed to obtain a deoxy-Mb spectrum and then bubbled with CO to get an
Mb-CO spectrum. Each complex was dissolved in water and added to deoxy-Mb in
the cuvette, mixed using a pipette and then overlaid with 500 µL light mineral oil to
prevent CO escaping or the myoglobin being oxygenated. This is the standard
procedure; other experiments have been undertaken using different concentrations of
myoglobin.
In these experiments, the absorption peak of deoxy-Mb at 560 nm was replaced by
the two peaks of Mb-CO at 540 and 578 nm. The concentration of myoglobin in the
stock solution was calculated from the absorption maximum of the Mb-CO solution at
540 nm.
4.6 Cytotoxicity assays
Hela cell, HepG2 cell, MCF-7 cell, A549 cell, HT-29 cell and W138 cells line
were purchased from cell resources Center for Shanghai Life Science Institute of
Chinese Academy of Sciences (China). The cells were cultivated at 37 °C, 10% CO2,
100% humidity in RPMI1640 medium, enriched with glucose and supplemented with

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10 % fetal bovine serum, non-essential amino acids, antibiotics (penicillin/
streptomycin) and antifungals.Typically, 8 µmol of the complex was dissolved in 1.0
mL of culture medium with vigorous stirring. Growth inhibitory effect toward the cell
line was determined by means of MTT colorimetric assay. The cells (100 µL,
1×105cells mL−1) were seeded into 96-well plates and left to adhere for 24h. The
media was removed from the wells and replaced with fresh media containing the
complexes with different concentrations (6.5, 12.5, 25, 50, 100, 200, 400 and 800
µmol L−1), respectively. The cells were then incubated for another 24 h before the
incubation media were replaced with the complete medium and MTT (10 µL, 5 mg
mL−1 in phosphate buffer solution, PBS) was added to each well of the plate. The cells
were further incubated for 4 h before the media were replaced with DMSO (100 µL).
Absorbance at 490 nm for each well of the plates was recorded with a microplate
reader. In the MTT assay, DMSO (100 µL) in a well was used as blank and cells in the
well without the addition of any complexes were taken as a control (100 % in cell
viability).
4.7 Bicinchoninic acid (BCA) protein assay
In order to exclude the influence of the total amount of protein on the determination
of the following cytokine levels, protein quantification of each protein was carried out
using BCA protein Assay. Briefly, samples were removed and placed into a 96-well
plate (20µL per well). BCA reagent A and reagent B were formulated as working
solutions, which was added to each well (200µL per well) and left to react for 0.5 h in
37℃. And then the plate was shaken for 10 min and the absorbance read at 562 nm on
a microplate reader. The concentration of protein in each sample was calculated from
a standard curve generated with standard solution.
4.8 Determination of COX-2 levels
The level COX-2 in each sample was determined using a commercially available
kit from Shanghai MLBIO Biotechnology Co.Ltd. The assay was performed
according to the manufacturers’ instructions. Briefly, cell were collected from 6-well
plate immediately after lysed by RIPA Lysis Buffer and spun at 12,000×g for 10 min
to remove any particulates. Each protein solution was measured by BCA Protein
Assay Kit. The protein solution for concentration of 0.1mg ml-1 was added to a
96-well plate precoated with affinity-purified polyclonal antibodies specific for the
mouse of our cytokines. An enzyme-linked polyclonal antibody specific for the mouse
of our cytokines was added to the wells and left to react for 0.5 h followed by a final
wash to remove any unbound antibody-enzyme reagent. The intensity of the color
detected at 450 nm was measured after addition of chromogen solution A, B and stop
solution and was proportional to the amount of four cytokines produced. The COX-2

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levels which in each sample was calculated from a standard curve generated with
standard solution.
4.9 Myocardial protective effects of the complexes
The rat cardiac H9c2 cells were purchased from the Cell Bank of the Chinese
Academy of Sciences (Shanghai,China) and cultured in DMEM supplemented with
10% FBS,100 U•mL-1 of penicillin, and 100 µg•mL-1 of streptomycin at 37 °C in a
humidified atmosphere of 5% CO2 .Cardiomyocyte survival rate .The H9c2 cells were
cultured in 96 - well plates at a density of 5 × 104 cells /mL and cultured for 8 h at
37 ℃ and 5 % CO2 incubator for 8 hours. The tests were divided into blank group,
H2O2 group, complex（10µM、25µM、50µM）-H2O2 group. H2O2 was 400µM, and
the control group only added cells without drugs. The cells survival rate was detected
after 24h. After cells were treated with H2O2 for 1h, 4h and 8h, respectively, the
culture medium was discarded and the H2O2 group was supplemented with fresh
medium. Then the cell viability was measured after 24 hours of incubation.
Assays for enzyme activities The H9c2 cells were cultured in 6-well plates
overnight and then treated with H2O2 (400µM) for another 1h. The activities of SOD
and the MDA content were measured using commercial kits following the
manufacturer’s instruction (NanJing JianCheng Bioengineering Institute, Nanjing,
China).
4.10 Antihypertensive effect in SHR
A total of 40 male clean spontaneously hypertensive rats (SHR) were divided into
normal control group, captopril group (10 mg kg-1), nifedipine nifelat group (10 mg.
kg-1) and compound group (10, 20, 40 mg. kg-1) by randomized block design. Male
WKY rats were used as the normal control group. All the drugs and compounds were
administered by intraperitoneal injection as a 2mL/kg. The solvent was the mixed
solution of 0.5% sodium carboxymethyl cellulose and DMSO (v/v 3:1). The control
group and WKY group were injected mixed solution without containing compound or
drug. Tail blood pressure and heart rate were measured before administration. After
administration, blood pressure and heart rate were measured every 10min, lasting for
3 h. The arterial systolic pressure, diastolic arterial pressure (DBP) and heart rate were
recorded before and after administration.
Method of measurement: In a brief, in a quiet state of consciousness, the rats were
fixed to a special fixture. The rat tail was covered until the roots by pressure cuff,
which strapped pulse transducer to contact closely to the ventral tail artery. When the
rat pulse wave stabilized, it began to measure. The pressure cuff was inflated and
pressurized until the blocking pressure was 250 mmHg. (The pulse wave disappeared
when the pressure reached the systolic pressure level.) After the pressure maintains 6

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seconds, it slowly decompresses. When the first pulse waves appear, the
corresponding blood pressure is the arterial systolic pressure, the peak of the
corresponding curve is the arterial diastolic pressure, and the system automatically
displays the heart rate. Each group was continuously measured 6 times. All data are
expressed as the mean ± standard error (SE); n refers to number of animals studied in
each group. Significance was accepted at P < 0.05.
Conflict of Interest
The authors confirm that this article content has no conflict of interest.
Acknowledgements
This work was financially supported by the National Natural Science Foundations
of China (no. 21171079) and the Recruitment Program of Global Experts (1000
Talents Plan).
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