Syntheses and anti-cancer activity of CO-releasing molecules with targeting galactose receptors

Article abstract of DOI:10.1039/c8ob01921e

CO-releasing molecules (CORMs) containing cobalt have many bioactivities, but most of them do not dissolve in water and have no selectivity to tissue and organs. On the basis of the specific recognition of galactose or sialic acid by a receptor, a series of CORMs based on carbohydrates were synthesized and evaluated. The test results show that all the complexes displayed anticancer activity. Among them, the effects of the complexes of galactose (1), GalNAc (8) and sialic acid (10) were very distinct. Complex 1 displayed higher activity against HeLa, HePG2, MCF-7 and HT-29 cell proliferation than cis-platin (DDP), and its selectivity was far much better than DDP compared with normal cell W138. Furthermore, the uptakes of complexes 1, 8 and 10 by HePG2, HT-29, A549 and RAW264.7 cell lines were studied. The uptake ratio of each cell line for complex 1 was different, and the order of uptake ratio in the four cell lines was HePG2 > HT-29 > RAW264.7 > A549. The HePG2 cells absorbed complex 1 beyond 60% after incubation for 8 h, while A549 absorbed only 27.8%. For complex 8, the uptake trend was similar to that of complex 1 with it being absorbed by all the four cancer cells, but the uptake rate was lower. However, differently, complex 10 was absorbed heavily by macrophage RAW264.7, followed by HePG2; after 8 h incubation, the uptake ratio of RAW264.7 was over 50%. In addition, the mechanism of action was explored, and the results showed that the complexes inhibited cell cycle arrest at the G2/M phase; complex 1 up-regulated the expression levels of caspase-3 and Bax, and down-regulated the Bcl-2 expression, giving rise to HePG2 cell apoptosis.

Full text of DOI:10.1039/c8ob01921e

Organic &
Biomolecular Chemistry
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Syntheses and anti-cancer activity of CO-releasing
molecules with targeting galactose receptors†
Cite this: Org. Biomol. Chem., 2018,
16, 8115
Jili Li, a Jinlong Zhang,a Qiuping Zhang,a Zhongjie Bai,a Quanyi Zhao,*a Dian He,*a
Zhen Wang,a Yonglin Chenb and Bin Liuc
CO-releasing molecules (CORMs) containing cobalt have many bioactivities, but most of them do not dis-
solve in water and have no selectivity to tissue and organs. On the basis of the specific recognition of
galactose or sialic acid by a receptor, a series of CORMs based on carbohydrates were synthesized and
evaluated. The test results show that all the complexes displayed anticancer activity. Among them, the
effects of the complexes of galactose (1), GalNAc (8) and sialic acid (10) were very distinct. Complex 1 dis-
played higher activity against HeLa, HePG2, MCF-7 and HT-29 cell proliferation than cis-platin (DDP), and
its selectivity was far much better than DDP compared with normal cell W138. Furthermore, the uptakes
of complexes 1, 8 and 10 by HePG2, HT-29, A549 and RAW264.7 cell lines were studied. The uptake ratio
of each cell line for complex 1 was different, and the order of uptake ratio in the four cell lines was HePG2
> HT-29 > RAW264.7 > A549. The HePG2 cells absorbed complex 1 beyond 60% after incubation for 8 h,
while A549 absorbed only 27.8%. For complex 8, the uptake trend was similar to that of complex 1 with it
being absorbed by all the four cancer cells, but the uptake rate was lower. However, differently, complex
10 was absorbed heavily by macrophage RAW264.7, followed by HePG2; after 8 h incubation, the uptake
ratio of RAW264.7 was over 50%. In addition, the mechanism of action was explored, and the results
showed that the complexes inhibited cell cycle arrest at the G2/M phase; complex 1 up-regulated the
expression levels of caspase-3 and Bax, and down-regulated the Bcl-2 expression, giving rise to HePG2
cell apoptosis.
Received 7th August 2018,
Accepted 9th October 2018
DOI: 10.1039/c8ob01921e
rsc.li/obc
[Co2(CO)6] moiety has been studied widely as an antitumor
reagent. Hexacarbonyl-[2-propinyl acetylsalicylate] dicobalt dis-
1. Introduction
CO has potential in clinics because it is involved in neuro- played higher activity against MCF-7 and MDA-MB-231 than
degeneration,1 hypertension,2 inflammation,3–5 oxidative cis-platin,17 and its analogue was effective against hormone
stress,6,7 cell proliferation,8 and apoptosis.9,10 However, the dependent tumors.18 In addition to anti-cancer activity, these
application of CO gas in the clinic remains questionable due complexes also displayed anti-inflammatory activity. The
to its toxicity and lack of quantitative control.11 For this researchers found that their anti-inflammatory effects were
reason, Motterlini proposed the concept of CO releasing mole- due to liberating CO to inhibit nitrite,19 and the effects of pro-
cules (CORMs) ten years ago.12 Over the next few years, a liferative inhibition of cancer cells were mainly resulting from
number of CORMs containing Ru, Fe, Mn, Co, and Mo were the CO to inhibit Hsp90 downstream protein activity, such as
synthesized. The test results showed that most of them exhibi- Akt, ER alpha, and cyclin D1.20 In addition, the complexes also
ted cytoprotective, anti-inflammatory, vasodilatory and other promoted apoptosis, disturbed cell cycle and interfered with
beneficial effects.13–15
DNA synthesis.21
Among these CORMs, the complexes based on cobalt were
Though Co-CORMs have good biological activity, they are
paid more attention than others.16 The complex containing a almost insoluble in water and have no selectivity to tissue and
organs. Recently, T. Tsubomura et al. found that carbohydrate
complexes of palladium and platinum had antitumor activity
aInstitute of Medicinal Chemistry, School of Pharmacy of Lanzhou University,
Lanzhou 730000, China. E-mail: zhaoqy@lzu.edu.cn, hed@lzu.edu.cn;
Fax: +869318915686; Tel: +869318915686
bThe First Affiliated Hospital of Lanzhou University, Lanzhou 730000, China
cSchool of Stomatology of Lanzhou University, Lanzhou 730000, China
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8ob01921e
in vitro and in vivo;22–26 in 2004, D. Schlawe found that iron-
carbonyl complexes had cytotoxicity and triggered apoptotic
effects.27 After that, the CORMs modified with fructose were
found to induce apoptosis at high millimolar concentrations;28
and three new derivatives displayed activity against MCF-7
cells.29,30
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Generally, galactose is specifically recognized and bound by shifts of the protons on the carbon connected with two cobalt
galectin and the asialoglycoprotein receptor (ASGP-R). atoms were observed at 6.08–5.98 ppm as a singlet; and the
Galactose lectin is closely related to the development of chemical shifts of protons on the carbon of the carbohydrate
tumors. The over-expression of galectin-3 in colon cancer cells were observed at 4.79–5.29 ppm. In the 13C NMR spectra of the
was involved in the process of carcinogenesis, development complexes 1–10, the signals at about 90 ppm were assigned to
and metastasis of colon cancer. ASGP-R is a transmembrane C1 of the carbohydrates.
protein of the membrane surface of hepatocytes; when the
Introducing carbohydrates into CORMs increased their
molecules containing the galactose residue bind to the solubility in water. The lgP values of complexes 1–10 were in
ASGP-R, it can be transported to lysosomes. The sialic acid the range of 1.16 and 1.32, which indicated that their polarity
receptor is siglec-1, which has high expression on (tam) tumor- increased greatly. The lgP values of complexes Ac-1–10 were
associated macrophages. Siglec is an endocytosis receptor that between 1.86 and 2.28 (Table 1), and distinctly, they have lower
transports drugs into target cells. Sialic acid (SA) and its polarity than the corresponding unacylated complexes.
derivatives may be used as carriers for drug transport, which Compared with the previously reported complexes,39,40 the
can transport anticancer drugs to tumor-associated macro- carbohydrate structures in the complexes not only help them
phages, and inhibit tumor growth and metastasis.
to dissolve in water, but also decrease metal cobalt toxicity
Therefore, we have synthesized a series of CORMs with (Schemes 1–3).
carbohydrates including galactose and sialic acid, and found
that they not only have good solubility in water, but also target
2.2. CO-releasing tests
Deoxymyoglobin (deoxy-Mb) has
organs or tumor-associated macrophages to give rise to anti-
tumor activity.
a
high affinity for CO,
binding one CO to form carbonyl myoglobin (CO-Mb). The
difference between the absorption maxima of deoxy-Mb and
CO-Mb allows the detection of CO-release from CORMs. The
maximal absorption peak of deoxy-Mb appears at 560 nm
while the two maximal absorption peaks of Mb-CO appear at
540 and 578 nm.
2. Results and discussion
2.1. Syntheses and characterization
All complexes 1–7 were synthesized using sugar as the starting
material. Sugar mainly included galactose, methyl glucuro-
nate, mannose, etc. Complexes 1–7 were prepared by the reac-
tion of carbohydrates A1–7 and acetic anhydride to form acetyl-
ated sugars, and then reacted with thiourea in the presence of
The CO-release ability was therefore evaluated in a myoglo-
bin-based aqueous assay. A solution of deoxy-Mb was prepared
and subsequently treated with an aqueous solution of CORMs
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 40 μM concentration by following the
changes in absorption. The UV spectra of some complexes and
the concentrations of CO-Mb as a function of time are given in
Fig. 1. The half-lives of some complexes are listed in Table 1.
As seen from the data, all complexes 1–10 were effective relea-
sers with half-lives varying between 30.6 min and 48.1 min,
and most of them were about 40 min. They are slower CO
releasers, and their half-lives (t1/2) are longer than CORMs con-
taining ruthenium.41 After acylation of the complexes, acylated
complexes released CO faster than the corresponding com-
boron trifluoride to form active intermediates, which reacted
31–34
with propargyl bromide to afford B1–7
;
B1–7 was deacety-
lated using sodium methoxide to afford the deacetylated pro-
ducts, and then reacted with octacarbonyl dichromate to
obtain the target molecules 1–7. For complexes 8–9, the start-
ing material was amino sugar; the intermediates B8–9 were
obtained in the presence of ytterbium trifluorosulfonate.35,36
Sialic acid was treated by the reaction of esterification, then
acetylation, glycosylation and deacetylation, and finally reacted
with cobalt carbonyl to afford complex 10.37,38
The complexes were either a red-black oil or red solid, and
stable in air for several days. They are soluble in water, and
easily soluble in polar organic solvents, including CH3OH,
DMF and DMSO. Complexes Ac-1–10 easily dissolve in organic
solvents, but do not dissolve in water. All the complexes
showed a number of strong terminal carbonyl absorption
bands at 2079–1849 cm−1 in their IR spectra. The higher
absorption bands were attributed to terminal carbonyls co-
ordinated to the Co atom. The IR spectra showed the corres-
ponding ester carbonyl absorptions at 1755–1735 cm−1 for
complexes Ac-1–9; for the spectra of complexes 8, Ac-8, 9 and
Ac-9, there was another strong carbonyl absorption band of
the acetamido group at 1680–1700 cm−1. Because of the para-
Table 1 Physical–chemicalparameters of all the complexes
Complex
T1/2 (min)
lgP
Complex
T1/2 (min)
lg P
1
Ac-1
2
Ac-2
3
Ac-3
4
Ac-4
5
Ac-5
42.8
44.1
47.9
49.6
32.6
36.8
44.2
46.4
48.1
46.2
1.16
1.86
1.19
1.92
1.23
2.10
1.21
1.82
1.20
1.98
6
Ac-6
7
Ac-7
8
Ac-8
9
Ac-9
10
Ac-10
40.6
33.8
30.6
34.9
38.6
42.6
39.6
44.8
38.2
40.6
1.23
2.06
1.25
2.12
1.26
1.98
1.22
2.08
1.32
2.28
1
magnetism of cobalt atoms, the peaks in the H NMR spectra
became wider, and some characteristic signals were superim-
posed. In the 1H NMR spectra of complexes 1–10, the chemical
Ac appoints the acetylated product of the complex.
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Scheme 1 Syntheses and structures of complexes 1–7.
plexes except for complexes 5 and 6. This is possibly because complexes displayed cell proliferative inhibition activities to
acylation of the hydroxyls in complexes 1–10 decreased disturb some degree, and their IC50 values were in the 5.69–149.02 μM
of the hydroxyl group to CO releasing. In addition, the half- range. All complexes 1–10 displayed higher activities than their
lives of CO release were connected with the structures of the corresponding acetylated products. Among complexes 1–10,
carbohydrate.
complexes 1, 8 and 10 displayed higher activities than the
2.2.3 Anti-tumor activities of the complexes. In order to others. Complex 1 displayed higher activities against HeLa,
obtain more information about anticancer activity, we chose HePG2, MCF-7 and HT-29 cell proliferation than DDP. Its
HeLa, A549, HePG2, MCF-7 and HT-29 as test cancer lines; all efficacy (IC50 5.69 µM) was about as high as 2.8 times DDP
the complexes were evaluated for the growth inhibitory effect (15.87 µM) against HePG2 cell proliferation, and 2.3 times
by the MTT method. Cis-platin (DDP) was used as a positive (21.96 µM) against HT-29 cells. Meanwhile, complex 1 dis-
control in the process of testing. As shown in Table 2, all the played better activity against MCF-7 cell proliferation than
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Scheme 2 Syntheses and structures of complexes 8–9.
Scheme 3 Synthesis and structure of complex 10.
DDP. For normal cell W138, the selectivity of complex 1 was activity against the proliferation of HePG2 cell lines. This may
far better than that of DDP. Complex 8 had a similar inhibitory be related to the over-expression of the galactose receptor on
effect to complex 1 for all the cancer cells, and its efficacy was the surface of HePG2 cells. Complex 10 displayed the highest
lower than that of complex 1 for all the cell lines but HeLa activity against A549 cells among the tested cell lines, and its
cells. This demonstrates that the structure of the complex was IC50 value was 9.74 µM. It suggests that complex 10 has selecti-
connected with its effect. In all, complexes 1 and 8 had good vity to A549 cells to some degree.
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Fig. 1 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 2 (10 μM); c for 8 (10 μM); d for 10 (10 μM).
Table 2 IC50 values of complexes to the tumor cells and W138a
IC50 (µM)
Complex
HeLa
A549
HePG2
5.69 0.21
MCF-7
HT-29
9.62 0.15
W138
1
Ac-1
2
Ac-2
3
Ac-3
4
Ac-4
5
Ac-5
6
Ac-6
7
Ac-7
8
Ac-8
9
Ac-9
10
Ac-10
DDP
16.26 0.18
19.82 0.22
20.52 0.12
35.21 0.16
42.84 0.27
52.73 0.31
82.49 0.29
119.74 0.19
56.73 0.21
149.02 0.26
28.43 0.23
90.03 0.29
54.74 0.11
117.34 0.36
13.84 0.16
63.59 0.29
21.38 0.19
84.74 0.33
10.23 0.20
18.67 0.33
18.74 0.28
14.17 0.14
21.16 0.34
17.09 0.22
29.46 0.41
29.68 0.18
62.51 0.41
78.67 0.31
101.23 0.5
49.63 0.32
110.81 0.4
42.51 0.26
126.75 0.6
74.07 0.41
128.65 0.6
14.43 0.14
56.49 0.28
18.74 0.27
76.25 0.39
9.74 0.18
14.92 0.17
32.27 0.29
14.53 0.42
20.17 0.36
62.35 0.37
48.92 0.38
64.29 0.27
92.41 0.48
71.28 0.28
128.54 0.6
37.68 0.20
111.54 0.3
62.13 0.19
94.87 0.39
16.41 0.22
71.52 0.37
26.32 0.47
57.68 0.51
11.32 0.17
22.36 0.33
18.24 0.33
36.74 0.22
30.75 0.28
24.68 0.27
35.25 0.29
69.27 0.30
78.79 0.26
80.56 0.21
129.67 0.4
77.34 0.41
145.87 0.7
55.36 0.19
131.36 0.5
80.08 0.29
123.58 0.4
19.56 0.14
88.91 0.35
28.94 0.23
85.38 0.39
20.15 0.24
38.19 0.41
22.19 0.33
26.63 0.33
16.82 0.32
24.52 0.33
34.52 0.31
70.24 0.42
58.63 0.19
113.81 0.6
67.47 0.37
118.06 0.5
36.24 0.29
89.28 0.33
49.35 0.23
85.23 0.45
7.69 0.19
52.16 0.32
22.02 0.41
59.07 0.45
11.03 0.15
33.74 0.27
15.87 0.19
20.54 0.32
21.16 0.29
27.46 0.44
58.27 0.42
67.62 0.51
59.81 0.33
139.02 0.6
69.46 0.30
131.27 0.6
24.78 0.28
118.26 0.4
56.32 0.31
90.85 0.41
12.36 0.24
85.48 0.41
20.28 0.25
73.24 0.49
17.14 0.21
30.25 0.41
21.96 0.17
26.73 0.22
13.63 0.20
a Values represent mean SEM from at least three independent experiments.
2.2.4 Cellular uptake of the complex. In order to further for more details).42–45 The results are shown in Fig. 2. We can see
demonstrate that some complexes had selective proliferation that the uptake of target molecules by three kinds of tumor cells
inhibitory activity toward cancer cells, the uptakes of com- and macrophage RAW264.7 gradually increased with the pro-
plexes 1, 8 and 10 by HePG2, HT-29, A549 and RAW264.7 cells longation of time. The uptake of the same cell line to different
were studied respectively. For this purpose, the cells were exposed complexes was different, and for the same complex, the uptake
to 1, 8 or 10 over a period of 2 h, 4 h and 8 h, and then the metal of different cell lines varies greatly.
levels of the cells were measured by using inductively coupled
For complex 1, after 8 h of incubation, the uptake of every
plasma mass spectrometry (ICP-MS, see the Experimental section cell line was more distinct than the control, but HePG2 cells
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Fig. 2 The uptake of the complex with the time, (a) for complex 1, (b) for 8 and (c) for 10.
Fig. 3 Effects of the complexes on the HePG2 cell cycle: a and b are for complex 1 with 10 µM and 20 µM; c and d for complex 8; e and f for
complex 10; h for treatment with 10 μM; i with 20 μM.
absorbed it far much more than the other cell lines. The of the cancer cell, and entered into the cell by endocytosis.
uptake ratios for HePG2 for 2 h, 4 h and 8 h were about 3.7%, However, under the same conditions, the uptake of complex 8
23.5% and 60.3%, respectively. The uptake ratio of each cancer was less than that of complex 1 for every cell line. This
cell line was different, and the order of uptake ratio was suggests that the amino group takes the place of the hydroxyl
HePG2 > HT-29 > RAW264.7 > A549. After 8 h, the uptake ratio group at the 2-position in galactose attenuating the binding
of HePG2 was more than 60%, while that of A549 was only ability to the galactose receptor. Complex 10 was absorbed
27.8%. For complex 8, the uptakes of the four cells were heavily by macrophage RAW264.7, this is because complex 10
similar to those of complex 1, but the uptake rates were lower has a sialic acid unit, which is selectively recognized by the
than those of complex 1. Unexpectedly, complex 10 was sialic acid receptor. Macrophages are connected with some
absorbed heavily by macrophage RAW264.7, followed by immune diseases, so complex 10 possibly has a controlling
HePG2; and the uptake ratio of the other two kinds of cells effect on immune diseases.
was about as much as each other. After 8 h incubation, the
uptake ratio of RAW264.7 for complex 10 was more than 50%.
2.2.5 Effects of complex on cell cycle and apoptosis. To
investigate whether the complexes have anti-mitotic effects on
The structure of the complex has a great influence on its HePG2 cells, cell cycle analysis was performed by flow cytome-
bio-distribution and activity. Complexes 1 and 8 were ingested try. The treatment of HePG2 cells with 10 μM and 20 μM com-
in large quantities by HePG2, this is because both of them plexes for 24 h resulted in significant alterations in cell cycle
contain galactose and amino-galactose structural units, which phases. The results are shown in Fig. 3. For every tested
are recognized by the galactose receptor on the cell membrane complex, there was a significant increase in the percentage of
of the HePG2 cells. Possibly, it was through the receptor selec- the cells at the pre-G2/M phase with the increase in the con-
tive recognition that the complexes aggregated to the surface centration. When HePG2 cells were treated with complex 1 at
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Fig. 4 Effects of the complexes on HePG2 cell apoptosis: a and b are for complex 1 with 10 µM and 20 µM; c and d for complex 8; e and f for
complex 10.
20 μM, the percentage accounted for 52.15%, while that of the
control was 4.40%; under the same condition, it was 48.98%
with complex 8 and 25.59% with complex 10. Hence, we con-
clude that the complexes inhibited the cell proliferation
through cell cycle arrest at the G2/M phase.
The complexes arrested the cell cycle at the G2/M phase, so
they almost surely induce apoptosis in HePG2 cells. In order to
obtain more information about apoptosis, we further per-
formed apoptosis detection in a dose-dependent manner
(Fig. 4). We first treated HePG2 cells with varying concentrations
(10 and 20 μM) of the complexes. Then AnnexinV-FITC/PI assay
was performed with the cells. As a result, HePG2 cells showed
considerable sensitivity to the tested complexes, and their
effects were in a dose-dependent manner. When the complexes
were 20 μM, the proportion of apoptotic cells was up to 83.20%
Fig. 5 The nucleus of HePG2 cells by DAPI and Mito-Tracker Green
for complex 1; under the same conditions, it was 70.6% for
fluorescence staining: a (control), b, c, d are for complex 1, complex 8
and complex 10 with 10 μM; e, f, g are for complex 1, complex 8 and
complex 8 and 41.9% for complex 10. Thus, it can be concluded
that the complexes significantly induced apoptosis of HePG2
cells in a dose dependent manner.
complex 10 with 20 μM.
2.2.6 Mechanism of action of the complex. How did the
complexes work? In order to understand more intuitively the
effect of complexes on organelles, we made a morphological
observation through fluorescence staining. After treating with the
control or the complexes for 24 h, the HePG2 cells were washed
with phosphate buffered saline (PBS) and immobilized with 3.7%
paraformaldehyde PBS solution at room temperature for 10 min.
The immobilized cells were washed with PBS and stained with
4′,6-diamidino-2-phenylindole (DAPI) and Mito-Tracker Green for
20 minutes at room temperature. Then, the cells were washed
with PBS and observed using a fluorescence microscope.
Under the fluorescence microscope, the fluorescence of the
cells in the control group was more diffuse and uniform, the
nucleus was relatively complete and regular, the nucleus was
ellipsoid, and the nucleolus was obvious (Fig. 5a). The mito-
Fig. 6 The mitochondria of HePG2 cells by DAPI and Mito-Tracker
Green fluorescence staining: a (control), b, c, and d are for complex 1,
complex 8 and complex 10 with 10 μM; e, f and g are for complex 1,
chondria were short rod or ball shaped, and the shape was complex 8 and complex 10 with 20 μM.
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Fig. 7 Effects of complex 1 on the protein levels in human HePG2 cells. HePG2 cells were exposed to 10 or 20 μM complex 1 for 24 h. The cell
extract protein isolated from complex 1 treated HePG2 cells was subjected to Tris-glycine gel electrophoresis and immunoblotted with the anti-
bodies against caspase-3, Bcl-2 or Bax. β-Actin was used as a loading control. Western blot data presented are representative of those obtained
from three separate experiments.
regular. The central depression was the location of the tive administrations, the cobalt did not accumulate, and was
nucleus, and it was obvious (Fig. 6a). But when the cells were excreted with the urine. In view of these aspects, the CORMs
exposed to the test complex for some time, the cell nucleus based on cobalt have potential to be a medicine. The struc-
and mitochondria were obviously damaged. Most nuclei were ture-activity relationship study suggests that the complex bio-
densified or edge-stained, or densified in mass; some nucleoli distribution is related to side chain substituents, and the
disappeared and nucleolysis occurred in some nuclei (Fig. 5b–g). alkyne substituents significantly affect the rate of CO-release,
Mitochondria showed pyknosis, smaller matrices, deeper cytotoxicity and cell viability. However, it is a pity that, at
matrices, irregular morphology, less obvious nucleus position, present, most CORMs obtained have a poor solubility and no
and an increased number of apoptotic cells. When the concen- selectivity to tissue and organs.
tration of the complex was 10 µM, the damage to the nucleus
For this purpose, a series of CORMs based on carbo-
and mitochondria was slight; but when the concentration of hydrates were synthesized. As a result, introducing galactose
the complex was 20 µM, the damage degree of the nucleus and and sialic acid into CORMs, not only increased their solubility,
mitochondria became more serious.
but also improved their activity and selectivity to target mole-
To further elucidate the mechanism of apoptosis induced cules. Among them, complexes 1, 8 and 10 displayed higher
by the complexes, the expression levels of three apoptosis-regu- activities than others. Complex 1 displayed higher activities
lating genes in the bcl-2 protein family and caspase family against HeLa, HePG2, MCF-7 and HT-29 cell proliferation than
were measured. Among them, the bcl-2 gene and bax gene are DDP. Its activity against HePG2 cells was about as high as 2.8
two important regulatory genes which have opposite functions times DDP. Moreover, compared with normal cell W138, its
in the process of apoptosis regulation. Bcl-2 inhibits apoptosis, selectivity was far better than that of DDP. Importantly, the
while bax promotes apoptosis. Caspase-3 is a key protease in uptake ratios of complexes 1, 8 and 10 by the HePG2, HT-29,
the process of apoptosis. The results showed that, compared A549 and RAW264.7 cell lines were high. The uptake ratio of
with the control group, the expression of caspase-3 and Bax in HePG2 cells for complex 1 was more than 60% after 8 h.
the cells treated with the complex up-regulated obviously, but Complex 10 was absorbed selectively by macrophage RAW264.7,
the Bcl-2 down-regulated (Fig. 7). When the complex concen- and its uptake ratio was more than 50% after 8 h. Possibly,
tration was 20 µM, the change in protein expression levels was complex 1 aggregated on the surface of the cancer cell through
obviously higher than that of the control group. The galactose receptor selective recognition, and then entered
expression amount of caspase-3 (the death executive protein) into the HePG2 cells. Complex 10 was absorbed heavily by
was 2 times higher than that of the control; and bax (apopto- macrophage RAW264.7 because it has a sialic acid unit, which
sis-promoting protein) increased by 80% of the control, but is selectively recognized by the sialic acid receptor. This kind
the expression of bcl-2 (anti-apoptotic protein) decreased by of compound may have a controlling effect on immune
about 40% of the control. This indicated that complex 1 diseases.
induced HePG2 cell apoptosis by affecting the expression of
the apoptotic gene.
As for the mechanism of action, the complexes inhibited
the cell cycle arrest at the G2/M phase; meanwhile, they up-
regulated the expression of caspase-3 and Bax, and down-regu-
lated Bcl-2. Of course, it also involved reducing reactive oxygen
species (ROS) and lowering the mitochondrial membrane
potential, and so on, like other Co-CORMs reported before.21
The complex containing galactose or sialic acid showed
3. Conclusion
CORMs containing cobalt have many bioactivities, such as
anti-inflammatory and antitumor cell proliferation. The com- better solubility and higher selectivity to target cells, but to
plexes displayed a lower toxicity in animal tests. Though the determine whether they can be used as candidates in clinics or
complexes damaged the liver and kidney after several consecu- not, there is a lot of further research to do.
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ESI-HRMS (m/z): calcd for C17H22NaO9S [M + Na]+ 425.0882,
found 425.0851.
4. Experimental
4.1. Reagents and instruments
Then B1 (402.1 mg, 1 mmol) was dissolved in 2 mL of 0.5 M
Myoglobin (AR, Sigma), monosaccharides (Sigma), thiourea NaOMe in MeOH. The pH was 12 and 13. The mixture was
(Sigma), ytterbium(III) trifluoromethanesulfonate (Sigma), stirred at ambient temperature for 15 min, and TLC analyses
Annexin V-FITC (BD Bio-Pharmingen), PI (BD Bio- on the mixture (petroleum ether/AcOEt 1/1 v/v) showed com-
Pharmingen), and deionized water were used. All reactions plete conversion of the substrate. Then Amberlite® IR120 H+
were carried out under a nitrogen atmosphere. Solvents for was added until pH 7. The resin was filtered off and the
reactions were degassed. Column chromatography was carried solvent was removed in vacuo, and the crude product was
out using 200–300 mesh silica gel; HeLa cells, HepG2 cells, obtained. The crude product was dissolved in 20 mL of dry
MCF-7 cells, A549 cells, HT-29 cells and W138 cell lines were THF, and then 456 mg (1.2 mmol) of dicobaltoctacarbonyl
purchased from the cell resources (Center for Shanghai Life was added. The reaction mixture was stirred for 12 h at room
Science Institute of Chinese Academy of Sciences, China). IR temperature. The solvent was removed in vacuo, the residue
spectra were recorded on a Nicolet NEXUS 360 spectrophoto- was extracted with a small amount of CH2Cl2, and the extract
meter, and NMR spectra on a Bruker AM-400 MHz spectro- was subjected to silica gel column chromatography. Ethyl
meter; a Lambda 25 UV-visible spectrophotometer and a acetate/hexane (1 : 10) eluted the main red band, from which
Maxis-4G TOF mass spectrometer (ESI) were used; BD 353.6 mg (68.2%) of brown-red oil was obtained. Yield:
FACSverse™ flow cytometry was used for cell cycle arrest and 75.8%, IR (KBr disk, cm−1): 2098vs, 2055vs, 2024vs (Co–CO);
1
apoptosis.
[α]2D3:7 −89.8° (c = 1.0, CH3OH), H NMR (CDCl3-d) δ 6.08 (s,
1H, CH), 5.32 (m, 2H, SCH2), 4.71–3.83 (m, 7H, C1–5H and
CH2 of sugar), 3.41–2.573 (m, 4H, 4OH); 13C NMR (DMSO-d6)
4.2. Synthesis of the complexes
4.2.1. Complex 1. A1 galactose (180.2 mg, 1 mmol) was dis- δ 200.1 (Co–C), 85.6, 79.6, 79.2, 78.9, 75.3, 70.4, 68.7, 60.8,
solved in pyridine (2 mL); acetic anhydride (2 mL) was added 33.4(CH3); ESI-HRMS (m/z): calcd for C15H16Co2O12
S
S
S
to the mixture solution. The mixture was stirred at ambient [M + H2O]+ 537.9026, found 538.9066; C15H14Co2NaO11
temperature for several hours. TLC analyses (petroleum ether/ [M
+
Na]+ 542.8819, found 542.8818; C15H14Co2KO11
AcOEt 1/1 v/v) showed complete conversion of the substrate, [M + K]+ 558.8558, found 558.8566. Elemental anal., calcd
and the mixture was diluted with ethyl acetate. Then ice was for C15H14Co2O11S: C, 34.63; H, 2.71%, found C, 34.43, H,
added to the solution. The organic layer was washed with 0.5N 2.81%.
HCl aq and NaHCO3-saturated aqueous solutions, repeatedly.
Complex Ac-1: 402.1 mg (1.0 mmol) B1 was dissolved in
The organic layer was dried with anhydrous MgSO4, filtered 20 mL of dry THF, and then 456 mg (1.2 mmol) of dicobalt-
and the solvent was removed. The product was purified using octacarbonyl was added. The reaction mixture was stirred for
column chromatography with silica gel (petroleum ether/ 12 h at room temperature. The solvent was removed in vacuo,
AcOEt 3/1 v/v). Peracetylated galactose (358.2 mg) was obtained the residue was extracted with a small amount of CH2Cl2, and
1
as a white solid. Yield: 65.8%. H NMR (CDCl3) δ 5.69 (d, J = the extract was subjected to silica gel column chromatography.
8.3 Hz, 1H), 5.41 (d, J = 3.3 Hz, 1H), 5.11–5.06 (m, 1H), 4.13 (t, Ethyl acetate/hexane (1 : 10) eluted the main red band, from
J = 6.7 Hz, 2H), 4.04 (t, J = 6.6 Hz, 1H), 2.15 (s, 3H), 2.11 (s, which 590.8 mg (86.1%) of complex Ac-1 as a brown-red oil
3H), 2.03 (s, 6H), 1.99 (s, 3H). ESI-HRMS (m/z): calcd for was obtained. IR (KBr disk, cm−1): 2099vs, 2054vs, 2018vs,
C16H22NaO11 [M + Na]+ 413.1060, found 413.1012; C16H22KO11 1753vs, (COO); [α]D21:5 −79.1° (c = 1.0, CH3OH), 1H NMR
[M + K]+ 429.0799, found 429.0746.
(CDCl3) δ 6.03 (s, 1H, CH), 5.23 (s, 2H, SCH2), 5.03–3.69 (m,
To a solution of peracetylated galactose (1.0 mmol) in anhy- 7H, C1–5H and CH2 of sugar), 1.96 (br, 12H, COCH3); 13C
drous acetonitrile was added thiourea (1.1 mmol) and boron NMR (CDCl3) δ 198.3 (Co–C), 169.5 (CO), 169.1 (CO), 168.4
trifluoride diethyl etherate (2.1 mmol). The reaction mixture (CO), 90.6, 81.9, 75.2, 72.8, 72.6, 68.7, 67.2, 61.1, 32.4 (SCH2),
was refluxed until the starting material was consumed comple- 19.6 (CH3); ESI-HRMS (m/z): calcd for C23H22Co2NaO15S [M +
tely, and then left to cool to rt. Propargyl bromide (1.1 mmol) Na]+ 710.9241, found 710.9229; C23H22Co2KO15S [M + K]+
and triethylamine (4.5 mmol) were added and the solution 726.8980, found 726.8968. Elemental anal., calcd for
was stirred at rt overnight. The reaction mixture was concen-
C23H22Co2O15S: C, 40.13; H, 3.22%, found C, 40.03, H,
trated and the residue was diluted in CH2Cl2 before washing 3.34%.
with aqueous 5% HCl and brine, and then filtered and evapor-
The procedure and workup of complexes 2–7 were similar
ated. The residue was purified by phase chromatography (pet- to the process of complex 1 and the procedure and workup of
roleum ether/AcOEt 5/1 v/v). B1 was obtained as a yellow oil. complexes Ac-2–Ac-7 were similar to the process of complex
1H NMR (CDCl3-d) δ 5.38 (d, J = 2.7 Hz, 1H), 5.18 (t, J = Ac-1.
10.0 Hz, 1H), 5.04 (dd, J = 10.0, 3.3 Hz, 1H), 4.70 (d, J = 10.0
4.2.2. Complex 2. Yield: 77.8%; IR (KBr disk, cm−1):
Hz, 1H), 4.07 (td, J = 11.1, 5.8 Hz, 2H), 3.98–3.89 (m, 1H), 3.51 2099vs, 2051vs, 2024vs (Co–CO); [α]2D2:5 −68.9° (c = 1.0,
(dd, J = 16.5, 2.5 Hz, 1H SCH2), 3.26 (dd, J = 16.5, 2.6 Hz, 1H, CH3OH), 1H NMR (CDCl3) δ 6.06 (s, 1H, CH), 5.30 (m, 2H,
SCH2), 2.25 (t, J = 2.5 Hz, 1H, CH), 2.09 (s, 3H, COCH3), 1.98 (s, SCH2), 4.52–3.66 (m, 7H, C1–5H and CH2 of sugar), 3.36–2.51
3H, COCH3), 1.97 (s, 3H, COCH3), 1.92 (s, 3H, COCH3). (m, br, 4H, 4OH). 13C NMR (DMSO-d6) δ 199.1 (Co–C), 86.6,
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78.6, 78.2, 77.9, 74.3, 70.4, 68.7, 61.8, 33.6; ESI-HRMS (m/z):
4.2.5. Complex 5. Yield: 80.1%; IR (KBr disk, cm−1):
calcd for C15H15Co2O11S [M + H]+ 520.8999, found 520.9671; 2092vs, 2058vs, 2034vs (Co–CO); [α]D23:5 127.3° (c
=
1.0,
C15H16Co2O12
C15H14Co2NaO11S [M
S [M +
H2O]+ 537.9026, found 538.9262; CH3OH), 1H NMR (CDCl3) δ 6.05 (s, 1H, CH), 5.31 (m, 2H,
+
Na]+ 542.8819, found 542.8818; SCH2), 4.90–3.56 (m, 7H, C2–4H and CH2 of sugar), 3.42–3.06
C15H14Co2KO11S [M + K]+ 558.8558, found 558.8555. Elemental (m, 4H, 4OH). 13C NMR (DMSO-d6) δ 199.9 (Co–C), 85.1, 76.4,
anal., calcd for C15H14Co2O11S, C, 34.63; H, 2.71%, found C, 72.9, 68.3, 65.3, 61.2, 53.6, 32.6. ESI-HRMS (m/z): calcd for
34.80, H, 2.89%.
C15H14Co2NaO11S [M +
Na]+ 542.8819, found 542.8849.
Complex Ac-2: Yield: 67.8%; IR (KBr disk, cm−1): 2091vs, Elemental anal., calcd for C15H14Co2O11S: C, 34.63; H, 2.71%,
2053vs, 2034vs, 1851vs, (COO); [α]D20:5 −48.3° (c = 1.0, CH3OH), found C, 34.44, H, 2.86%.
1H NMR (CDCl3) δ 6.02 (s, 1H, CH), 5.24 (s, 2H, SCH2),
Complex Ac-5: Yield: 79.6%; IR (KBr disk, cm−1): 2095vs,
5.06–3.68 (m, 7H, C1-5H and CH2 of sugar), 1.98 (s, 12H, 2048vs, 2019vs, 1749vs (COO); [α]2D0:5 97.3° (c = 1.0, CH3OH),
COCH3). 13C NMR (CDCl3) δ 199.3 (Co–C), 169.8 (CO), 169.3 1H NMR (CDCl3) δ 6.02 (s, 1H, CH), 5.56 (m, 2H, SCH2),
(CO), 168.0 (CO), 85.6, 81.9, 75.2, 72.8, 72.6, 68.7, 66.2, 60.1, 4.92–4.07 (m, 7H, C2–4H and CH2 of sugar), 2.02 (s, 12H,
32.8 (SCH2), 19.9 (CH3); ESI-HRMS (m/z): calcd for COCH3). 13C NMR (CDCl3) δ 199.1, 170.4, 170.0, 168.9, 80.8,
C23H22Co2NaO15S [M
Elemental anal., calcd for C23H22Co2O15S: C, 40.13; H, 3.22%, ESI-HRMS (m/z): calcd for C23H22Co2NaO15S [M
found C, 40.21, H, 3.39%. 710.9241, found 710.9237. Elemental anal., calcd for
+
Na]+ 710.9241, found 710.9231. 79.8, 79.2, 78.0, 77.2, 76.8, 73.6, 65.0, 62.7, 32.5, 20.7;
Na]+
+
4.2.3. Complex 3. Yield: 68.1%; IR (KBr disk, cm−1): C23H22Co2O15S: C, 40.13; H, 3.22%, found C, 40.24, H, 3.37%.
2099vs, 2054vs, 2045vs (Co–CO); [α]2D2:7 −100.2° (c = 1.0,
4.2.6. Complex 6. Yield: 77.2%; IR (KBr disk, cm−1):
1
CH3OH), H NMR (CDCl3) δ6.05 (s, 1H, CH), 5.46–5.26 (m, 2H, 2097vs, 2054vs, 2033vs, (Co–CO), [α]2D0:6 −186.8° (c = 1.0,
SCH2), 5.26–3.88 (m, 5H, C1-5H of sugar), 3.72 (s, 3H, OCH3), CH3OH), 1H NMR (CDCl3) δ 6.10 (s, 1H, CH), 5.29 (s, 2H,
3.25–2.23 (m, 3H, 3OH). 13C NMR (DMSO-d6) δ 199.3 (Co–C), SCH2), 4.88–3.94 (m, 5H, C1–5H of sugar), 3.86–2.86 (m, 4H,
170.3, 94.5, 85.1, 76.4, 72.9, 68.3, 53.6, 32.6; ESI-HRMS (m/z): 4OH), 1.25 (s, 3H, CH3). 13C NMR (CDCl3) δ 198.3 (Co–C), 90.8,
calcd for C15H14Co2NaO11S [M
+
Na]+ 570.8768, found 83.2, 76.2, 72.6, 72.1, 71.2, 68.2, 33.6(SCH2), 16.5(CH3); CORM:
570.8766; C16H14Co2KO12S [M + K]+ 586.8507, found 586.8512. ESI-HRMS (m/z): calcd for C15H14Co2KO10S [M + K]+ 542.8609,
Elemental anal., calcd for C16H14Co2O12S: C, 35.06; H, 2.57%, found 542.8914. Elemental anal., calcd for C15H14Co2O10S: C,
found C, 35.18, H, 2.71%.
35.73; H, 2.80%, found C, 35.54, H, 2.93%.
Complex Ac-3: Yield: 80.4%; IR (KBr disk, cm−1): 2091vs,
Complex Ac-6: Yield: 83.9%; IR (KBr disk, cm−1): 2099vs,
2055vs, 2047vs, 1750vs (COO); [α]2D3:2 −153.6° (c = 1.0, CH3OH), 2051vs, (Co–CO), 1750vs, (COO); [α]2D1:5 −236.5° (c = 1.0,
1H NMR (CDCl3) δ6.04 (s, 1H, CH), 5.13 (br, 2H, SCH2), CH3OH); 1H NMR (CDCl3) δ5.92 (s, 1H, CH), 5.22 (m, 2H,
4.60–4.05 (m, 5H, C1-5H of sugar), 3.69 (s, 3H, OCH3), 1.96 (s, SCH2), 4.91–3.88 (m, 7H, C1–5H and CH2 of sugar), 2.09 (s, 3H,
9H, COCH3). 13C NMR (CDCl3) δ199.2, 169.9, 169.3, 166.7, COCH3), 1.99 (s, 3H, COCH3), 1.92 (br, 6H, COCH3); 13C NMR
91.8, 83.3, 76.4, 73.0, 69.4, 52.9, 33.6, 20.5; ESI-HRMS (m/z): (CDCl3) δ 199.2, 170.4, 170.2, 169.6, 85.3, 82.8, 73.6, 72.4, 70.4,
calcd for C22H20Co2NaO15S [M
696.9056. Elemental anal., calcd for C22H20Co2O15S: C, 39.19; C23H22Co2KO15S [M + K]+ 726.8980, found 726.8968. Elemental
H, 2.99%, found C, 39.04, H, 3.08%. anal., calcd for C21H20Co2O13S: C, 40.02; H, 3.20%, found C,
4.2.4. Complex 4. Yield: 83.1%; IR (KBr disk, cm−1): 40.11, H, 3.29%.
2093vs, 2057vs, 2022vs (Co–CO); [α]2D3:4 340.8° (c 4.2.7. Complex 7. Yield: 77.6%; IR (KBr disk, cm−1):
1.0,
+
Na]+ 696.9085, found 67.2, 33.3, 20.8, 16.9; ESI-HRMS (m/z): calcd for
=
CH3OH), 1H NMR (CDCl3) δ6.85 (s, 1H, CH), 5.18 (m, 2H, 2095vs, 2053vs, 2030vs, (Co–CO); [α]2D3:1 −40.9° (c = 1.0,
SCH2), 4.85–3.88 (m, 7H, C1-5H of sugar and CH2), 3.67–3.34 CH3OH), 1H NMR (CDCl3) δ 6.07 (s, 1H, CH), 5.22 (s, 2H,
(m, 4H, 4OH). 13C NMR (DMSO-d6) δ 200.3 (Co–C), 94.5, 85.1, SCH2), 4.40–3.86 (m, 5H, C1–5H and CH2 of sugar), 3.57–3.06
75.4, 71.9, 67.3, 49.06, 33.4; ESI-HRMS (m/z): calcd for (br, 3H, 3OH), 1.22 (s, 3H, CH3); 13C NMR (CDCl3) δ 199.3
C15H14Co2NaO11S [M
C15H14Co2KO11S [M + K]+ 558.8558, found 558.8560. Elemental 16.8 (CH3); ESI-HRMS (m/z): calcd for C15H14Co2NaO10
anal., calcd for C15H14Co2O11S, C, 34.63; H, 2.71%, found C, [M + Na]+ 526.8869, found 526.8862. Elemental anal., calcd
34.82, H, 2.88%. for C15H14Co2O10S: C, 35.73; H, 2.80%, found C, 35.57, H,
+
Na]+ 542.8819, found 542.8842. (Co–C), 89.8, 81.2, 72.2, 71.6, 70.1, 69.2, 67.2, 33.9 (SCH2),
S
Complex Ac-4: Yield: 85.0%; IR (KBr disk, cm−1): 2097vs, 2.95%.
2049vs, 2024vs, 1753vs (COO); [α]2D2:6 178.1° (c = 1.0, CH3OH),
Complex Ac-7: Yield: 85.1%; IR (KBr disk, cm−1): 2093vs,
1H NMR (CDCl3) δ 6.08 (br, 1H, CH), 5.39 (m, 2H, SCH2), 20516vs, 2017vs, 1755vs (COO); [α]D23:2 −79.1° (c = 1.0, CH3OH),
4.87–3.95 (m, 7H, C1-5H and CH2 of sugar), 2.12 (br, 3H, 1H NMR (CDCl3) δ 6.03 (s, 1H, CH), 5.12 (m, 2H, SCH2),
COCH3), 2.02 (br, 3H, COCH3), 1.98 (br, 3H, COCH3), 1.93 (s, 4.90–3.79 (m, 5H, C1-5H and CH2 of sugar), 1.97 (s, 9H, COCH3),
3H, COCH3). 13C NMR (DMSO-d6) δ 198.2 (Co–C), 170.1, 169.6, 1.18 (s, 3H, CH3). 5.12 (d, J = 88.3 Hz, 3H), 4.50 (s, 1H), 4.12 (s,
169.0, 168.7 (CvO), 85.6, 79.6, 79.2, 78.9, 75.3, 70.4, 68.7, 60.8, 1H), 4.06 (s, 1H), 3.79 (s, 1H), 1.97 (s, 9H), 1.18 (s, 3H). 13C
33.4; ESI-HRMS (m/z): calcd for C23H22Co2KO15S [M + K]+ NMR (CDCl3) δ 199.4, 170.6, 170.2, 169.7, 92.3, 82.8, 73.6, 72.3,
726.8980, found 726.8906. Elemental anal., calcd for 70.4, 67.2, 33.2, 20.6, 16.3; ESI-HRMS (m/z): calcd for
C23H22Co2O15S: C, 40.13; H, 3.22%, found C, 40.24, H, 3.37%.
C21H20Co2NaO13S [M +
Na]+ 652.9186, found 652.9179.
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Elemental anal., calcd for C21H20Co2O13S: C, 40.02; H, 3.20%, was stirred for 12 h at room temperature. The solvent was
found C, 40.17; H, 3.31%. removed in vacuo, the residue was extracted with a small
4.2.8. Complex 8. A8 (2.00 g, 9.28 mmol) was dissolved in amount of CH2Cl2, and the extract was subjected to silica gel
anhydrous pyridine (20 mL), and acetic anhydride (10.5 mL, column chromatography. Ethyl acetate/hexane (1 : 10) eluted
111.3 mmol) was added. The mixture was stirred at room the main red band, and complex Ac-8 as a brown-red oil was
temperature until the disappearance of the starting material, obtained (86.1%). IR (KBr disk, cm−1): 2099vs, 2052vs, 2021vs,
and poured into ice-cold water. A white solid precipitate was 1747vs (COO), 1687s (CON); [α]2D3:877.1 (c = 1.0, CH3OH), 1H
collected by filtration, washed with ice-cold water and co-evap- NMR (CDCl3) δ5.99 (s, 1H, CH), 5.51 (s, 2H, OCH2), 4.90–3.63
orated with toluene (3 × 20 mL) to remove residual water to (m, 8H, C1–5H of sugar, CH2, NH), 2.33–1.64 (m, 12H, COCH3).
yield 2.96 g (82%) of powdery solid (peracetylated aminogalac- 13C NMR (CDCl3) δ 199.5, 198.3, 198.2 (Co–C), 169.6, 169.2,
1
tose). H NMR (CDCl3) δ 5.70 (d, J = 8.4 Hz, 1 H), 5.40 (d, J = 168.5 (CvO), 98.29, 71.08, 70.93, 70.88, 67.94, 67.75, 61.09,
9.0 Hz, 1 H), 5.30 (d, J = 2.5 Hz, 1 H), 5.04 (dd, J = 3.0, 11.0 Hz, 54.38, 22.24 (CH3), 19.66(CH3); ESI-HRMS (m/z): calcd for
1 H), 4.44–4.360 (m, 1 H), 4.20–4.08 (m, 2 H), 4.07–4.02 (m, C23H23Co2KNO15 [M
+
K]+ 709.9369, found 709.9757.
1H), 2.16 (s, 3 H), 2.14 (s, 3 H), 2.00 (s, 3 H), 1.98 (s, 3 H), 1.94 Elemental anal., calcd for C23H23Co2NO15: C, 41.15; H, 3.45%,
(s, 3H). ESI-HRMS (m/z): calcd for C16H25NO11 [M + H2O]+ found C, 41.01; H, 3.56%.
407.1428, found 407.1458; C16H23NNaO10 [M + Na]+ 412.1220,
found 412.1229.
The procedure and workup of complex 9 were similar to the
process of complex 8 and the procedure and workup of com-
Then propargyl alcohol (29.9 μL, 0.514 mmol) and Yb(OTf)3 plexes Ac-9 were similar to the process of complex Ac-8.
(47.8 mg, 0.231 mmol) were added to a solution of peracety-
4.2.9. Complex 9. Yield: 65.1%; IR (KBr disk, cm−1):
lated aminogalactose (100 mg, 0.257 mmol) in dry DCE 2096vs, 2059vs, 2033vs (Co–CO), 1683s (CON). [α]2D3:7 −73.8°
1
(6 mL). The solution was stirred for 24 h at 70 °C. The solution (c = 1.0, CH3OH), H NMR (CDCl3) δ 5.82 (s, 1H, CH), 5.10 (s,
was diluted with DCM and washed with water. The organic 2H, OCH2), 4.80–3.86 (m, 8H, C1-5H of sugar, CH2, NH),
layer was dried with sodium sulfate and concentrated in vacuo 3.70–3.35 (m, 3H, 3OH), 2.02 (s, 3H, CH3). 13C NMR (CDCl3)
1
to furnish B8 (86 mg, 87%). H NMR (CDCl3) δ 5.48 (d, J = 8.8 δ200.1 (Co–C), 169.9, 81.0, 72.9, 71.0, 70.4, 69.5, 67.3, 35.6
Hz, 1H), 5.20 (t, J = 10.0 Hz, 1H), 5.06 (t, J = 9.8 Hz, 1H), 4.84 (SCH2), 19.5; ESI-HRMS (m/z): calcd for C17H17Co2NNaO12
(d, J = 8.4 Hz, 1H), 4.38 (s, 2H), 4.27 (dd, J = 12.2, 4.6 Hz, 1H), [M + Na]+ 567.9312, found 567.3191. Elemental anal., calcd
4.16 (d, J = 12.0 Hz, 1H), 3.90 (dd, J = 18.6, 9.2 Hz, 1H), 3.70 (d, for C17H17Co2NO12: C, 37.45; H, 3.14%, found C, 37.55;
J = 9.8 Hz, 1H), 2.43 (d, J = 1.3 Hz, 1H), 2.11–1.96 (m, 12H). H, 3.33%.
ESI-HRMS (m/z): calcd for C17H25NO10 [M + H2O]+ 403.1478,
Complex Ac-9: Yield: 75.1%; IR (KBr disk, cm−1): 2091vs,
found 403.1408; C17H23NNaO9 [M + Na]+ 408.1271, found 2055vs, 2047vs, 1750vs (COO), 1684s (CON); [α]2D3:9 −117.3° (c =
1
408.1287.
1.0, CH3OH), H NMR (CDCl3) δ 6.02 (br, 1H, CH), 5.60 (s, 2H,
Then B8 was dissolved in 2 mL of 0.5 M NaOMe in MeOH. OCH2), 4.78–3.58 (m, 8H, C1-5H of sugar, CH2, NH), 2.36–1.68
The pH was between 12 and 13. The mixture was stirred at (m, 12H, COCH3). 13C NMR (CDCl3) δ 201.6, 198.3, 198.2 (Co–
ambient temperature for 15 min, and TLC analyses (petroleum C), 169.6, 169.1, 169.5 (CvO), 98.3, 71.2, 70.9, 70.8, 67.8, 67.6,
ether/AcOEt 1/1 v/v) showed complete conversion of the sub- 61.1, 54.4, 22.2 (CH3), 19.6(CH3); ESI-HRMS (m/z): calcd for
strate. Then Amberlite® IR120 H+ was added until pH 7 was C23H23Co2KNO15 [M
+
K]+ 709.9369, found 709.9357.
reached; the resin was filtered off and the solvent was removed Elemental anal., calcd for C23H23Co2NO15: C, 41.15; H, 3.45%,
in vacuo, the crude product was obtained, then the crude found C, 41.00; H, 3.66%.
product was dissolved in 20 mL of dry THF, and then dicobalt-
4.2.10. Complex 10. N-Acetylneuraminic acid (5.07 g,
octacarbonyl was added. The reaction mixture was stirred for 16.4 mmol) dissolved in dry MeOH (100 mL) with a strong
12 h at room temperature. The solvent was removed in vacuo, cation-exchange resin (Dowex 50W-X2) (H+) (5.08 g) was stirred
the residue was extracted with a small amount of CH2Cl2, and at rt under N2 gas. After the reaction mixture had been stirred
the extract was subjected to silica gel column chromatography. for 48 h, the cation-exchange resin was removed and the
Ethyl acetate/hexane (1 : 6) eluted the main red band, and MeOH solution was concentrated. To a solution of the residue
complex 8 was obtained (yield 68.2%), IR (KBr disk, cm−1): in dry pyridine (100 mL) was added Ac2O (125 mL,
2094vs, 2054vs, 2019vs (Co–CO), 1686vs (CON); [α]2D3:3 57.2° (c = 1.32 mmol), and then stirred at rt in the dark for 20 h. The
1
1.0, CH3OH); H NMR (CDCl3) δ 5.86 (s, 1H, CH), 5.04 (s, 2H, solution was concentrated to remove pyridine and acetic acid.
OCH2), 4.82–3.94 (m, 8H, C1–5H of sugar, CH2, NH), 3.51–2.26 The crude product was purified by column chromatography on
(m, 3H, 3OH), 1.84 (s, 3H, CH3). 13C NMR (DMSO-d6) δ 200.1 silica gel (CHCl3–isopropanol 50 : 1 → 3 : 1) to afford penta-O-
(Co–C), 85.6, 79.6, 79.2, 78.9, 75.3, 70.4, 68.7, 60.8, 33.4(CH3); acetyl-N-acetylneuraminic acid methyl ester (72%). 1H NMR
ESI-HRMS (m/z): calcd for C17H17Co2NNaO12 [M + Na]+ (CDCl3): δ 6.22 (d, J = 8.9 Hz, 1H), 5.35 (d, J = 4.4 Hz, 1H), 5.18
567.9312, found 567.9626. Elemental anal., calcd for (s, 1H), 5.00 (s, 1H), 4.46 (d, J = 14.6 Hz, 1H), 4.34 (dd, J = 12.4,
C
17H17Co2NO12: C, 37.45; H, 3.14%, found C, 37.57; H, 2.5 Hz, 0H), 4.12 (d, J = 5.7 Hz, 2H), 3.71 (d, J = 13.7 Hz, 3H),
3.31%.
2.48 (dd, J = 13.4, 4.9 Hz, 1H), 2.11–1.93 (m, 15H), 1.82 (s, 3H).
Complex Ac-8: B8 was dissolved in 20 mL of dry THF, and ESI-HRMS (m/z): calcd for C22H31NNaO14 [M + Na]+ 556.1642,
then dicobaltoctacarbonyl was added. The reaction mixture found 556.1612.
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Then propargyl alcohol (29.9 μL, 0.514 mmol) and Yb(OTf)3 4.3. Lipophilicity (log P o/w)
(47.8 mg, 0.231 mmol) were added to a solution of penta-O-
acetyl-N-acetylneuraminic acid methyl ester (136 mg,
0.257 mmol) in dry DCE (6 mL). The solution was stirred for
24 h at 70 °C. The solution was diluted with DCM and
washed with water. The organic layer was dried with sodium
sulfate and concentrated in vacuo to furnish B10 (85 mg,
The lipophilicity of the complex was determined using the
flask shaking method where n-octanol and 0.1 M PBS were
used as the organic and aqueous phases respectively.
n-Octanol was presaturated with 0.1 M PBS by swirling at
45 rpm for 24 h. The test complex was dissolved in the iso-
lated organic phase at a concentration of 50 μM. An equal
volume of aqueous PBS was added, and the mixture was
swirled for 8 h at 45 rpm at 37 °C. The solution was then
centrifuged, and the amounts of complex in both layers
were determined by ICP-AES, and then log P was calculated.
1
82%). H NMR (CDCl3) δ 5.40 (s, 1H), 5.32 (s, 1H), 5.24–5.18
(m, 1H), 4.86 (s, 1H), 4.39 (s, 1H), 4.28 (s, 1H), 4.15 (s, 1H),
4.11–4.02 (m, 3H), 3.76 (s, 3H), 2.58 (dd, J = 12.8, 4.6 Hz,
1H), 2.40 (t, J = 2.4 Hz, 1H), 2.18–1.80 (m, 15H), 2.06–1.90
(m, 1H).
Then B10 was dissolved in 2 mL of 0.5 M NaOMe in MeOH.
The pH was between 12 and 13. The mixture was stirred at
ambient temperature for 15 min, and TLC analyses on the
mixture (petroleum ether/AcOEt 1/1 v/v) showed complete con-
version of the substrate. Then Amberlite® IR120 H+ was added
until pH 7 was reached. The resin was filtered off and the
solvent was removed in vacuo, and the crude product was
obtained. The crude product was dissolved in 20 mL of dry
THF, and then dicobaltoctacarbonyl was added. The reaction
mixture was stirred for 12 h at room temperature. The solvent
was removed in vacuo, the residue was extracted with a small
amount of CH2Cl2, and the extract was subjected to silica gel
column chromatography. Ethyl acetate/hexane (1 : 4) eluted the
main red band, and complex 10 was obtained (yield 68.2%).
[α]D22:7 −34.3°(c = 1.0, CH3OH), IR (KBr disk, cm−1): 2097vs,
2052vs, 2033vs, 2024vs (Co–CO); 1H NMR (CDCl3) δ 6.01 (br,
1H, CH), 5.36 (br 2H, OCH2), 4.79–3.88 (m, 10H, C2–5H of
sugar, CH2, CH, NH), 3.75 (br, 3H, OCH3), 3.65–2.89 (m, 4H,
4OH), 2.09 (s, 3H, CH3); 13C NMR (CDCl3) δ 198.5 (Co–C), 198.2
(Co–C), 169. 6 (CO), 169.3 (CO), 97.9, 88.6, 71.5, 71.5, 70.5,
67.6, 67.5, 65.0, 61.4, 51.8, 48.3, 36.3, 22.2; ESI-HRMS (m/z):
calcd for C21H23Co2KNO15 [M + K]+ 685.9369, found 685.9314.
Elemental anal., calcd for C21H23Co2NO15: C, 38.97; H, 3.58%,
found C, 39.05; H, 3.66%.
4.4. 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 concen-
tration) was prepared fresh by dissolving the protein in phos-
phate buffered saline (PBS, 0.1 M, pH = 7.4). Sodium dithio-
nite (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
obtain 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 from escaping or the myoglobin from being oxy-
genated. 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.
Complex Ac-10: B10 was dissolved in 20 mL of dry THF, and
then dicobaltoctacarbonyl was added. The reaction mixture
was stirred for 12 h at room temperature. The solvent was
4.5. Cytotoxicity assays
removed in vacuo, the residue was extracted with a small HeLa cells, HepG2 cells, MCF-7 cells, A549 cells, HT-29 cells
amount of CH2Cl2, and the extract was subjected to silica gel and W138 cell lines were purchased from cell resources
column chromatography. Ethyl acetate/hexane (1 : 6) eluted (Center for Shanghai Life Science Institute of Chinese
the main red band, complex Ac-10, as a brown-red oil was Academy of Sciences, China). The cells were cultivated at
obtained (86.1%). [α]2D0:7 −10.6°(c = 1.0, CH3OH); IR (KBr disk, 37 °C, 10% CO2, 100% humidity in RPMI1640 medium,
1
cm−1): 2098vs, 2056vs, 2034vs, 1745vs (COO); H NMR (CDCl3) enriched with glucose and supplemented with 10% fetal
δ 6.02 (s, 1H, CH), 5.35 (br, 2H, OCH2), 4.85–3.98 (br, 10H, bovine serum, non-essential amino acids, antibiotics (penicil-
C2–5H of sugar, CH2, CH, NH), 3.75 (s, 3H, OCH3), 2.09–1.81 lin/streptomycin) and antifungals. Typically, 8 μmol of the
(m, 15H, COCH3 and CNCH3). 13C NMR (CDCl3) δ 198.5 (Co–C), complex was dissolved in 1.0 mL of culture medium with vig-
198.2 (Co–C), 169.8 (CO), 169.7 (CO), 169, 6 (CO), 169.3 (CO), orous stirring. The growth inhibitory effect toward the cell line
169.2 (CO), 166.1 (CO), 97.9, 88.6, 71.5, 71.5, 70.5, 67.6, 67.5, was determined by means of the MTT colorimetric assay. The
65.0, 61.4, 51.8, 48.3, 36.3, 22.2, 20.0, 19.8, 19.8, 19.7; cells (100 µL, 1 × 105 cells per mL) were seeded into 96-well
ESI-HRMS (m/z): calcd for C29H32Co2NO19 [M + H]+ 816.0233, plates and left to adhere for 24 h. The media were removed
found 816.0679; C29H33Co2NO20 [M + H2O]+ 833.0260, found from the wells and replaced with fresh media containing the
833.0952; C29H31Co2NNaO19 [M
838.0509. Elemental anal., calcd for C29H31Co2NO19: C, 42.72; 100, 200 and 400 µmol L−1), respectively. The cells were then
+
Na]+ 838.0052, found complexes of different concentrations (3.25, 6.5, 12.5, 25, 50,
H, 3.83%, found C, 42.58; H, 3.99%.
incubated for another 24 h before the incubation media were
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ðfind Co in cell lysateÞ ꢀ ðvolume of cell lysateÞ
Uptake ratio ð%Þ ¼
ðfind Co in cell lysateÞ ꢀ ðvolume of cell lysateÞ þ ðfind Co in mediumÞ ꢀ ðvolume of mediumÞ
replaced with the complete medium and MTT (10 µL, NP-40, 2 mM EDTA, 10 mM NaCl, 10 μg ml−1 aprotinin, 10
5 mg mL−1 in phosphate buffered saline, PBS) was added to μg ml−1 leupeptin, 1 mM DTT, 0.1% SDS and 1 mM phenyl
each well of the plate. The cells were further incubated for 4 h methyl sulfonyl fluoride). Total proteins were obtained by cen-
before the media were replaced with DMSO (100 µL). trifugation (12 000g for 20 min at 4 °C). The protein concen-
Absorbance at 490 nm for each well of the plates was recorded trations were determined by using the Bradford method. For
with a microplate reader. In the MTT assay, DMSO (100 µL) in western blot analysis, equal amounts of proteins (30 μg) were
a well was used as a blank and the cells in the well without the separated on 10% or 12% SDS-PADE gels and transferred to
addition of any complexes were taken as a control (100% cell polyvinylidine difluoride (PVDF) membranes (Millipore
viability).
Corporation, USA). The blot was blocked in blocking buffer
(5% non-fat dry milk in TBST) for 2 h at room temperature,
and then incubated with a dilute solution (1 : 500–1 : 1000) of
caspase-3, Bax or bcl-2 antibodies (BioLegend, USA) in block-
ing buffer overnight at 4 °C. The blot was then incubated with
an appropriate secondary antibody (ZSGB-BIO, Beijing, China)
(1 : 5000–1 : 10 000 dilution), and β-actin (ZSGB-BIO, Beijing,
China) was used as a loading control. The protein bands were
visualized using the Gel Imaging System (ChemDoc-It610,
UVP, USA).
4.6. Cellular uptake
Cancer cells were grown until at least 70% confluency in
75 cm2 cell-culture flasks. Stock solutions of the compounds
in DMSO were prepared and diluted in cell-culture medium to
a final concentration of 100 μM immediately before use (final
DMSO concentration: 0.1% v/v). The cell-culture medium of
the flasks was replaced with the medium that contained the
metal compound (10 mL) and the flasks were incubated at
37 °C/5% CO2 for 2 h, 4 h and 8 h. After the desired incubation
period the uptake was stopped by removing the cell-culture
medium. The pellet cells were washed with sterilized 10 mM Conflicts of interest
PBS (pH 7.4) and centrifuged to obtain cell pellets. The pellets
The authors confirm that this article content has no conflicts
of interest.
were lysed with 10% Tween 20 (50 μL). The concentration of
Co atoms in the cell lysate was measured by ICP-MS. The per-
centage of uptake was calculated from the amount of Co atoms
in culture medium and in the cell lysates using the following
equation.
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).
4.7. Flow cytometry assays
The HePG2 cell lines were cultured at 37 °C, 5% CO2, 100%
humidity in RPMI 1640 medium supplemented with 10% fetal
bovine serum. The cells were cultured in 6-well plates for 24 h.
The media were removed from the wells and treated with
various concentrations of complexes 1, 8 or 10 for 24 h. The
control cells were treated with vehicle. Next, the cells were
detached using 0.25% trypsin-EDTA (0.5 ml) and then resus-
pended in media (4 ml) prior to centrifugation (1000 rpm for
5 min). Cell pellets were washed twice using PBS (2 ml). For
cell-cycle analysis, the cells were fixed in cold 70% ethanol,
RNase treated, and stained with propidium iodide (PI) in the
dark for 30 min at 37 °C. The cells were analyzed for their DNA
content by BD FACSverse™ flow cytometry. For apoptosis ana-
lysis, the cells were stained with Annexin V-FITC (BD Bio-
Pharmingen) and PI in the dark for 15 min. The percentages
of apoptotic cells were determined by BD FACSverse™ flow
cytometry for 1 h.
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