Design, synthesis, and biological evaluation of chalcone oxime derivatives as potential immunosuppressive agents

Article abstract of DOI:10.1016/j.bmcl.2012.03.080

A series of deoxybenzoin oximes were recently reported as potent immunosuppressive agents by our group. In order to continue the original research for potential immunosuppressive agents with high efficacy and low toxicity, we synthesized a series of new c

Full text of DOI:10.1016/j.bmcl.2012.03.080

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3039–3043
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and biological evaluation of chalcone oxime derivatives
as potential immunosuppressive agents
Yin Luo a, , Ran Song b, , Yao Li a, Shuai Zhang a, Zhi-Jun Liu a, Jie Fu c, Hai-Liang Zhu a,
⇑
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
b Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, People’s Republic of China
c State Key Laboratory of Pollution Control & Resource Reuse, Nanjing University, Nanjing 210046, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of deoxybenzoin oximes were recently reported as potent immunosuppressive agents by our
group. In order to continue the original research for potential immunosuppressive agents with high effi-
cacy and low toxicity, we synthesized a series of new chalcone oximes and evaluated them for their cyto-
toxicities and immunosuppressive activities. Among the synthesized compounds, chalcone oximes 25
and 27 exhibited lower cytotoxicities and higher inhibitory activities on anti-CD3/anti-CD28 co-stimu-
lated lymph node cells than other compounds. Specially, compound 27 displayed 200-fold lower cytotox-
Received 27 January 2012
Revised 14 March 2012
Accepted 22 March 2012
Available online 28 March 2012
Keywords:
Chalcone
Oxime
Cytotoxicity
Immunosuppressive activity
icity (CC50 = 2174.39 lM) than cyclosporin A (CC50 = 10.10 lM) and showed SI value (SI = 176.69) close to
cyclosporin A (SI = 154.13). Besides, the preliminary mechanism of inhibition effect of compounds 25 and
27 was also detected by flow cytometry, and the compounds exerted immunosuppressive activities via
inducing the apoptosis of activated lymph node cells in a dose dependent manner. Also, the deep mech-
anism of apoptosis was detected by Western blot analysis.
Ó 2012 Elsevier Ltd. All rights reserved.
Immunosuppressive agents (immunosuppressants) are a class
of chemical or biological substances to reduce tissue damage by
inhibiting cellular and humoral immune response. These agents
can inhibit the body’s abnormal immune response, mainly being
used in organ transplant anti-rejection and treatment for autoim-
mune diseases such as rheumatoid arthritis, rheumatic fever, colla-
gen diseases, lupus erythematosus and autoimmune hemolytic
anemia.1,2
Cyclosporine A (CsA) and tacrolimus (FK506) are important
therapeutic immunosuppressants. They achieve the action process
by inhibiting T-lymphocyte activation, which plays an integral role
in transplant rejection and autoimmune diseases.3 CsA and FK506
both interfere with Ca2+-sensitive T-cell signal transduction path-
ways to prevent the action of transcription factors in the lympho-
kine gene expression process.4–6
Although immunosuppressive drugs have been successfully
used for organ transplantation and treatment of autoimmune dis-
eases in clinic, their side effects cannot be neglected, such as sec-
ondary infection, inducing cancer, kidney toxicity and liver
toxicity. Therefore, there is a clinical need for new therapeutic
agents capable of modulating immune responses with high efficacy
and low toxicity.7–12 The effort of searching for novel classes of po-
tential immunosuppressive compounds has never stopped.
In the search for new potential immunosuppressive agents, we
turned our attention to one kind of products which could be got
from nature: chalcones. Wide range of biological activities of chal-
cones had been reported in the literature. Using the natural chal-
cone structure as a core framework, large quantities of chalcone
derivatives with potent antileishmanial,13 cytotoxic,14 anti-
fungal15 and anti-inflammatory16,17 activities were synthesized.
In this Letter, we also put our interest on new synthetic chalcone
derivatives to explore new immunosuppressive agents with high
efficacy and low toxicity.
As previously reported, many compounds with oxime groups
displayed potent biological activities and low toxicities.18,19 Some
of them had been used as clinical medical agents and some oximes
were reported to show immunosuppressive activities.20 In this Let-
ter, EN3638 (a oxime derivative of salicylic acid) in a certain dose
(50, 100, or 150 mg/kg daily, 250 mg/kg three times a week, or
400 mg/kg twice a week) could suppress both clinical and histo-
logic evidence of experimental allergic encephalomyelitis (EAE)
during the course of therapy of rats.20 Our group also had reported
that a series of deoxybenzoin oximes possessed potential immuno-
suppressive activity.21 In this Letter, the most active compound
was 31 which exhibited immunosuppressive activity with SI
>684.64 even better than CsA (SI = 235.44).
Based on our previous interesting work, we continued our re-
search work: a series of chalcone derivatives including oxime skel-
etons were synthesized. The immunosuppressive activities of the
synthesized compounds were evaluated with lymph node cell
⇑
Corresponding author. Tel./fax: +86 25 83592672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
These two authors equally contributed to this Letter.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.080

3040
Y. Luo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3039–3043
functional assay and the cytotoxicities were tested. The prelimin-
ary mechanism of some active compounds was further examined
by using fluorescence activated cell sorter (FACS) and Western blot
assay.
The synthesis of compounds 1–22 was outlined in Scheme 1.
The chalcones were prepared by the Claisen–Schmidt condensa-
tion between ketones and aldehydes in the presence of potassium
hydroxide in ethanol with 80–85% yield.22
The novel oximes were synthesized by the route outlined in
Scheme 1. Our interest in this area was to design and synthesize
chalcone oximes for structure–activity relationship (SAR) studies
in immunosuppressive activities. Thus, the treatment of chalcones
1–22 with hydroxylamine hydrochloride in pyridine afforded the
oximes 23–44 with pyridine as catalyzer and alkaline environment
provider. All newly synthesized oximes (Table 1) were fully charac-
terized by spectroscopic methods and the elemental analysis. All
compounds were recrystallized from ethanol. Crystals of partial
compounds were obtained. The crystal structure of compound 43
was shown in Figure 1. The configuration showed that this com-
pound could be anti isomer. Because all compounds were recrystal-
lized from ethanol, it could suggest that this series of oxime
compounds would exhibit anti configurations.
The synthesized oximes were carried out in vitro tests on the
lymph node cells for cytotoxicities and inhibitory activities on
anti-CD3/anti-CD28 co-stimulated lymph node cells. Pharmacolog-
ical results of these compounds were summarized in Table 2 with
R1
R1
R1
NaOH
R2 CHO
+
NH2OH HCl
R2
R2
CH3CH2OH
O
Pyridine
O
N
OH
23-44
1-22
Scheme 1. Synthetic routes of chalcone oxime derivatives.
Table 1
Chemical structures of compounds 23–34
Compd
R1
R2
Compd
R1
R2
Cl
F
Cl
Cl
23
4-OCH3
34
4-Br
4-Br
24
4-OCH3
35
Cl
F
Br
25
26
27
4-OCH3
4-OCH3
4-OCH3
36
37
38
4-Br
4-Br
4-Br
O
S
O
S
Cl
Cl
Cl
28
4-OCH3
39
4-CH3
Br
O
29
30
31
4-OCH3
4-Cl
40
41
42
4-CH3
4-CH3
4-CH3
Cl
F
4-Cl
S
F
Cl
32
33
4-Cl
4-Cl
43
44
4-CH3
4-F
Cl
Cl
O

Y. Luo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3039–3043
3041
Figure 1. Crystal structure diagram of compound 43. H atoms are shown as small spheres of arbitrary radii.
compounds were more effective when substituents were both
OCH3. This result was similar with previous study that esterifica-
tion could improve activity.23 For comparison, we also evaluated
the corresponding chalcones (compound 3 and 5) of compound
25 and 27 for their activities. As shown in Table 2, the chacones
showed good immunosuppressive activities but not better than
the corresponding oximes.
Table 2
In vitro cytotoxicitiesa and inhibitory effectsb of the synthetic oximes
Compoundc
CC50
(
lM)
sd
IC50
Avg
(l
M)
SId
a
b
Avg
sd
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
3
290.57
13.47
737.17
10.60
2174.39
25.01
28.60
14.08
11.8
11.49
24.29
24.74
<5
15.83
2.19
38.83
6.91
334.94
1.26
5.83
1.70
11.48
10.95
13.93
8.49
12.31
11.18
11.81
9.15
9.59
10.64
5.85
14.72
<5
0.78
0.34
0.22
0.76
1.87
0.43
0.09
0.24
0.74
1.73
0.16
0.03
25.31
1.23
52.93
1.25
176.69
2.24
2.42
1.54
1.23
1.08
4.16
1.68
—
1.53
>4.75
1.38
1.84
2.94
18.82
1.17
2.32
1.36
3.73
75.98
154.13
Table 1 summarized the substituents (R1 and R2) of all chalcone
oximes. When R1 groups (such as methoxy groups) were un-
changed, if R2 groups were changed (such as compounds 23–29),
the facts listed below were clear: (1) the SI value order was 27
(R2 = 4-methoxyphenyl, SI = 176.69) > 25 (R2 = 4-bromophenyl,
SI = 52.93) > 23 (R2 = 4-chlorophenyl, SI = 25.31) > 29 (R2 = phenyl,
SI = 2.42) > 28 (R2 = 2,4-dichlorophenyl, SI = 2.23) > 26 (R2 = 2-thio-
phene, SI = 1.25) > 24 (R2 = 4-fluorophenyl, SI = 1.23). The SI value
order of halogen substituents was bromine > chlorine > fluorine,
which conformed to our previously reported results: bromine pos-
sessed weakest electron withdrawing capability,21 so the com-
pound with bromine substituent showed more inhibitory activity
(SI = 52.93). (2) The activity of thiophene substituted compound
26 (SI = 1.25) to be less than the phenyl substituted compound’s
activity (compound 29, SI = 2.42) showed that aromatic properties
of compounds had effect on activities. The sulfur 3p orbital and
carbon 2p orbital conjugated in thiophene ring. The track was
not matched very well and conjugation was not very good. (3) In
addition, the activity of the compound with two same substituents
in two different positions such as 28 (SI = 2.23) was significantly
weaker than 23 (SI = 25.31), which should be attributed to the in-
crease amount of substituents which could improve the electron
withdrawing capability.
1.24
1.65
0.63
1.09
20.26
23.73
15.35
21.31
34.17
117.36
13.21
14.74
14.83
93.67
1789.42
10.10
3.13
1.61
0.45
0.91
2.56
1.92
1.20
0.45
0.31
5.03
215.25
1.55
13.26
<5
11.15
11.59
11.61
6.24
11.25
6.34
10.88
25.08
23.55
0.07
0.77
0.03
0.24
0.12
0.26
0.64
0.47
0.09
0.97
1.83
0.01
5
CsA
a
b
c
On lymph node cells.
On lymph node cells co-stimulated by anti-CD3/anti-CD28.
The compounds tested for immunosuppressive activity are consistent with the
description in the Experimental Section.
d
Selectivity index: ratio of the compound concentration that decreases cell
When R2 groups were remained and R1 groups were changed,
such as compound 23, 30, 34, 39 and 44. Their activity was not very
different from each other. The order was: 23 (R1 = 4-methoxyl,
SI = 2.23) > 39 (R1 = 4- methyl, SI = 1.84) > 34 (R1 = 4-bromine,
SI = 1.68) > 30 (R1 = 4-chlorine, SI = 1.53) > 44 (R1 = 4-fluorine,
SI = 1.36). ThisorderwassimilarwiththeorderwhenR1 groupswere
remained and R2 groups were changed. Methoxyl and methyl groups
were both electron donating groups, however, because the former
group’s electron donating capability was weaker than the latter,
the latter inhibited stronger activity. The activity could be greatly
improved when R1 and R2 were both electron donating groups.
In general, the most potential compound was 27 (SI = 176.68)
owning two methoxy groups compared with the other oximes,
even close to CsA (SI = 154.13). Through SAR analysis, it could be
seen that electron withdrawing ability and the aromatic property
viability by 50% (CC50) to the compound concentration that inhibits proliferation by
50% (IC50), relative to control values.
CsA as control. The cytotoxicity of each compound, which was the
concentration when cell survival rate was 50%, was expressed as
CC50. Immunosuppressive activity of each compound, which was
the concentration when cell survival rate of anti-CD3/anti-CD28
co-stimulation of lymph node cell survival rate was 50%, was ex-
pressed as IC50. Selectivity index (SI = CC50/IC50) was used to assess
the compounds’ biological activities in the previous papers.21,23
As shown in Table 2, the SI values of the compounds varied from
1.17 to 176.68. Some compounds (25 and 27),25,26 especially 27
(SI = 176.69) showed good immunosuppressive activity, and 27
was even comparable to the control CsA (SI = 154.13). Clearly, the

3042
Y. Luo et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3039–3043
Figure 2. Lymph node cells isolated from naïve mice were cultured with anti-CD3/anti-CD28 and various concentrations of 25 and 27 for 24 h. Cells were stained by Annexin
V–FITC /PI and apoptosis was analyzed by flow cytometry.
might be important factors to determine the activity of these com-
pounds. This might provide direction to search for more active and
promising treatment agent in the future.
In our previous study, we knew that synthesized oximes posed
their immunosuppressive effect through cell apoptosis. Caspase 3
and poly(ADP-ribose) in polymerse (PARP) beared important parts
in this process. Caspase was considered to be the central executor
of apoptosis process to activate and degrade its substrate to cause
terminal effect incidents by the complex multi-factor and multi-
channel interaction. This activation could cause biochemical
changes of characteristic morphology to complete the process of
apoptosis. Also, PARP was a genomic testing and DNA repair en-
zyme, degraded by the caspase-3 and other cysteine protease
and the most characteristic protease substrate solution.24
As the most active agents, compounds 25 and 27 had been un-
der systematic investigations in vitro experiments. We detected
the mechanism of proliferation inhibition by FACS, and found that
these compounds could induce the apoptosis of activated lymph
node cells in a dose-dependent manner. As shown in Figure 2,
lymph node cells co-stimulated with anti-CD3/anti-CD28 were
Figure 3. Protein levels of cleaved caspase-3 and PARP examined by Western
blotting. Data were representative of three independent experiments.
activities of the synthesized oximes. The immunosuppression pro-
cess was achieved through the promotion of apoptosis of the lymph
node cells, by increasing the cleaved caspase-3 and the cleaved
PARP. All the results outlined the potential of compound 27 for fur-
ther exploitation as immunosuppressant.
treated with 10, 20 and 40 lg/mL of 25 or 27 for 24 h. The com-
pound increased the percentage of apoptosis detected by Annexin
V-FITC/PI staining in a dose-dependent manner. The result indi-
cated that compound 25 and 27 induced apoptosis of anti-CD3/
anti-CD28 stimulated lymph node cells and compound 27 dis-
played more outstanding activity.
Acknowledgment
To examine the status of the caspase 3 protein, we performed
Western blot analysis by using an anti-cleaved caspase-3 antibody,
which recognized p17 cleaved caspase-3. The p17 cleavage product
of the lymph node cells treated with 25 or 27 appeared a signifi-
cant increase in a dose-dependent manner (Fig. 3). This result con-
firmed the promotion function of compounds 25 and 27 on the cell
apoptosis.
In conclusion, a series of new chalcone oximes were synthesized
and their immunosuppressive activities and cytotoxicities were
evaluated. Most chalcone oximes derivatives displayed potent
immunosuppressive activities. Compound 27 exhibited significant
immunosuppressive activity with SI = 176.69, even comparable to
CsA (SI = 154.13). In general, electron donating substituents at both
of the two rings were favorable for the immunosuppressive
This work was supported by the National Natural Science Foun-
dation of China (NSFC J1103512).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.
2012.03.080.
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