Design, synthesis, and immunosuppressive activity of new deoxybenzoin derivatives

Article abstract of DOI:10.1002/cmdc.201000107

In the search for potential immunosuppressive agents with high efficacy and low toxicity, a series of new deoxybenzoins were synthesized and evaluated for their cytotoxicity and immunosuppressive activity. Among the synthesized compounds, four deoxybenzoi

Full text of DOI:10.1002/cmdc.201000107

DOI: 10.1002/cmdc.201000107
Design, Synthesis, and Immunosuppressive Activity of
New Deoxybenzoin Derivatives
Huan-Qiu Li, Yin Luo, Ran Song, Zi-Lin Li, Tao Yan, and Hai-Liang Zhu*[a]
In the search for potential immunosuppressive agents with
high efficacy and low toxicity, a series of new deoxybenzoins
were synthesized and evaluated for their cytotoxicity and im-
munosuppressive activity. Among the synthesized compounds,
four deoxybenzoin oximes (compounds 31, 32, 37, and 38) ex-
hibited lower cytotoxicity and higher inhibitory activity toward
anti-CD3/anti-CD28 co-stimulated T-cell proliferation than
other compounds. More significantly, compound 31 is >100-
fold less cytotoxic than cyclosporine A (CsA) and is also more
potent (31: SI>684.64, CsA: SI=235.44). The preliminary inhib-
ition mechanism of compound 31 was also identified by flow
cytometry, and this compound exerts immunosuppressive ac-
tivity by inducing apoptosis in activated lymph node cells in a
dose-dependent manner. In addition, the mechanism of apop-
tosis was detected by western blot analysis.
Introduction
Immunosuppressants are an important class of clinical drugs
for an array of medical processes, including transplant rejection
and treatment of autoimmune diseases such as systemic lupus
erythematosus, rheumatoid arthritis, and psoriasis.[1,2] The
knowledge that T-lymphocytes play an integral role in trans-
plant rejection brought cyclosporine A (CsA), tacrolimus
(FK506), and sirolimus (rapamycin) to the fore as therapeutic
immunosuppressants.[3] Although immunosuppressive drugs
have been successfully used for organ transplantation and
treatment of autoimmune diseases in the clinic, their side ef-
fects cannot be neglected.[4–9] Therefore, there is a clinical need
for new therapeutic agents capable of modulating immune re-
sponses with high efficacy and low toxicity.
In this study, 30 novel deoxybenzoin derivatives were syn-
thesized and screened for their immunosuppressive activity.
The immunosuppressive activity of the synthesized com-
pounds was evaluated with a T-cell functional assay and their
cytotoxicity was tested using the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazoliumbromide (MTT) method. Some of the
synthetic compounds exhibited potent immunosuppressive ac-
tivity. The preliminary mechanism of suppression by the most
active compound was further examined by flow cytometry
(FCM) and western blot assays.
Results and Discussion
As the most abundant natural products, isoflavones have
demonstrated potent biological effects such as antitumor,[10]
anti-inflammatory,[11] estrogenic,[12] and immunosuppressive ac-
tivities[13] with low toxicity. Deoxybenzoins, intermediates in
the synthesis of isoflavones, are also found in several plants
and marine plants,[14,15] and their structural similarities with iso-
flavone led us to be interested in studying their biological ac-
tivity, similar to that of isoflavone. The hypothesis is supported
by the fact that deoxybenzoin derivatives exhibit similar bio-
logical effects including antimicrobial,[16] estrogenic,[17] and
FabH inhibitory activity.[18]
Chemistry
A series of novel deoxybenzoins and their Schiff bases were
synthesized by the routes outlined in Scheme 1. Using a
method previously described by Wꢀhꢀlꢀ et al.,[15] we synthe-
sized deoxybenzoins 1–10 by the method detailed in our pre-
vious paper.[24] The synthesis started from phenols and phenyl-
acetic acids. The dihydroxyphenols resorcinol and pyrogallol
reacted with variously substituted phenylacetic acids giving ex-
cellent yields of deoxybenzoins by the Friedel–Crafts reaction
catalyzed by boron trifluoride diethyl etherate. Schiff bases of
deoxybenzoins 11–20 were then obtained in high yields using
para-chloroaniline as the amine and titanium tetrachloride as
the carbonyl activator. Compounds 11–20 were then submit-
Among the deoxybenzoins in previous reports, many com-
pounds with the oxime group displayed potent biological ac-
tivities and low toxicity,[19] some of them have been used as
clinical medical agents and some kinds of oximes were report-
ed to show immunosuppressive properties.[20] Schiff bases also
exhibited antibacterial,[21] antifungal,[22] and antitumor activi-
ty.[23] Based on the previous reports, we focused our interest
on new synthetic deoxybenzoins derivatives with an oxime
group and Schiff base to explore new immunosuppressive
agents with high efficacy and low toxicity.
[a] Dr. H.-Q. Li,+ Dr. Y. Luo,+ Dr. R. Song, Z.-L. Li, Dr. T. Yan, Prof. H.-L. Zhu
State Key Laboratory of Pharmaceutical Biotechnology
Nanjing University, Nanjing 210093 (PR China)
Fax: (+86)25-8359 2572
E-mail: zhuhl@nju.edu.cn
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000107.
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H.-L. Zhu et al.
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synthesize oximes for SAR stud-
ies of immunosuppressive activi-
ty. Thus, the treatment of select-
ed deoxybenzoins 1–10 with hy-
droxylamine hydrochloride in
ethanol afforded the oximes 31–
40 with sodium acetate as cata-
lyst and alkaline environment
provider. Based on the results of
single X-ray determination and
1H NMR spectroscopy, the E con-
figuration was determined for
these oximes. The crystal struc-
ture of compound 31 is shown
in Figure 1.
The new synthetic Schiff bases
and amines were tested in vitro
for their cytotoxicity on lymph
Scheme 1. Synthesis of deoxybenzoin derivatives 1–30: a) BF3·OEt2, 808C, 2 h; b) TiCl4, Et3N, p-chloroaniline, THF,
608C, 6 h; c) Zn, HCOOH, THF, room temperature, 17 h.
ted to zinc/formic acid (aqueous) reducing conditions,[25] lead-
ing to the corresponding amines 21–30 with moderate to high
overall yields. All new Schiff bases and amines (Table 1) were
fully characterized by spectroscopic methods and elemental
analysis.
Table 1. Structures of deoxybenzoin derivatives 11–40.[a]
Compd
R1
R2
R3
R4
Compd
R1
R2
R3
R4
Figure 1. Single crystal structure of compound 31.
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
H
OH
OH Br
H
H
H
H
Cl
Cl
Br
Br
OCH3
OCH3
H
H
H
H
Cl
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
OH
OH
H
OH
OH
H
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
Cl
Br
Br
OCH3
OCH3
H
H
H
H
Cl
Cl
Br
Br
OCH3
OCH3
H
Br
F
OH
OH
OH
H
OH Br
H
OH
H
OH
H
OH
OH
OH
H
OH OH
OH
OH OH
OH
OH OH
OH
OH OH
node cells and inhibition activity on anti-CD3/anti-CD28 co-
stimulated lymph node cells with CsA as the control. The phar-
macological results of these compounds are summarized in
Table 2. The cytotoxicity of each compound was expressed as
the concentration of compound that decreases cell viability to
50% (CC50). The immunosuppressive activity of each com-
pound was expressed as the concentration of compound that
inhibits anti-CD3/anti-CD28 co-stimulated T-cell proliferation to
50% (IC50) of the control value. The selectivity index (SI) value
was used to evaluate the bioactivity of compounds. In the ex-
periments testing the bioactivity of each compound, results
were expressed as the mean Æstandard error. Student’s t-test
was used to determine variances between groups where ap-
propriate, although these data are not shown in Table 2.
As shown in Table 2, the SI values of the compounds dif-
fered greatly, ranging from 2.07 to 90.83. Some of the synthe-
sized compounds (11, 18, 21, and 28), especially 11 (SI=
90.83), showed good immunosuppressive activity but much
lower than that elicited by CsA (SI=235.44). The most active
compound, 11, (SI=90.83) had a pyrocatechol and bromine
group, and could be a promising lead for the further develop-
ment of novel therapeutic agents. To compare the Schiff bases,
we also synthesized the corresponding amines 21–30. The ac-
tivity results shown in Table 2 indicated that they were less ef-
fective than the Schiff bases.
H
F
H
H
H
H
H
H
H
Br
F
F
H
H
H
H
H
H
H
OH
H
OH
OH OH
OH
OH OH
H
OH Br
H
Br
F
H
F
OH
OH
H
[a] See Scheme 1 for scaffold structures.
A series of novel oximes were synthesized by the routes out-
lined in Scheme 2. Our interest in this area was to design and
Scheme 2. Synthesis of deoxybenzoin oximes 31–40: a) NH2OH·HCl, NaOAc,
EtOH, 608C, 4–4.5 h.
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Deoxybenzoin Derivatives
Table 2. In vitro cytotoxicity[a] and inhibitory effects[b] of the synthetic
Table 3. In vitro cytotoxicity[a] and inhibitory effects[b] of the synthetic
Schiff bases and amines.
oximes.
Compd[c]
CC50 [mm]
IC50 [mm]
SI[d]
Compd[c]
CC50 [mm]
>800
IC50 [mm]
SI[d]
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
CsA
201.6Æ22.5
160.4Æ12.4
122.4Æ10.3
113.7Æ8.8
244.6Æ21.5
127.6Æ8.6
247.8Æ28.5
146.8Æ13.7
133.5Æ10.3
130.7Æ11.7
229.1Æ20.5
157.1Æ13.8
123.6Æ7.7
109.6Æ6.5
253.8Æ22.4
126.5Æ8.1
237.1Æ19.5
160.8Æ12.5
130.1Æ10.3
125.1Æ7.6
11.30Æ1.53
2.22Æ0.17
4.91Æ0.38
24.5Æ2.3
38.2Æ3.7
50.1Æ3.5
20.8Æ1.9
5.97Æ0.44
2.03Æ0.18
53.0Æ4.6
59.3Æ4.7
3.01Æ0.21
5.08Æ0.37
30.3Æ2.8
39.1Æ2.8
58.0Æ4.3
35.6Æ3.5
7.97Æ0.68
2.95Æ0.27
58.4Æ5.3
60.3Æ6.9
0.05Æ0.01
90.83
32.68
4.98
2.97
4.88
31
32
33
34
35
36
37
38
39
40
CsA
1.16Æ0.13
2.21Æ0.37
22.8Æ3.5
35.5Æ4.3
41.7Æ5.7
21.3Æ3.1
4.16Æ0.45
1.12Æ0.09
40.6Æ4.8
43.7Æ5.2
0.05Æ0.01
>684.64
74.5
164.6Æ18.1
147.4Æ14.7
127.5Æ13.5
6.47
3.59
>320
>69.41
7.36
69.79
139.12
2.98
6.13
157.4Æ16.2
290.3Æ26.5
155.5Æ14.7
121.1Æ10.1
128.3Æ11.6
11.30Æ1.42
41.51
72.36
2.52
2.20
2.93
235.44
76.09
30.92
4.07
2.80
4.38
[a] On lymph node cells. [b] On lymph node cells co-stimulated by anti-
CD3/anti-CD28. [c] The compounds tested for immunosuppressive activity
are consistent with the description in the Experimental Section. [d] Selec-
tivity index: ratio of the compound concentration that decreases cell via-
bility by 50% (CC50) to the compound concentration that inhibits prolifer-
ation by 50% (IC50), relative to control values.
3.62
29.75
54.52
2.23
2.07
235.44
the acute oral toxicity of the most potent compound, 31, was
examined. The tested animals appeared normal immediately
following administration and did not exhibit any indication of
acute toxicity for 14 days afterward. The body weights in treat-
ed mice were similar to that of the control mice dosed with an
equal volume of vehicle. The result indicated that compound
31 was nontoxic.
[a] On lymph node cells. [b] On lymph node cells co-stimulated by anti-
CD3/anti-CD28. [c] The compounds tested for immunosuppressive activity
are consistent with the description in the Experimental Section. [d] Selec-
tivity index: ratio of the compound concentration that decreases cell via-
bility by 50% (CC50) to the compound concentration that inhibits prolifer-
ation by 50% (IC50), relative to control values.
Cell apoptosis (apoptosis) is an important biological phe-
nomenon in vivo in multicellular organisms. It can be divided
into two continuous processes: commitment and execution.
Caspase is considered to be the central executor, whose imple-
mentation depends on the apoptotic function of the enzyme
activity of substrate proteins. Stimulated by a variety of physio-
logical and pathological factors, caspase activates and de-
grades its substrate to cause terminal effect incidents by com-
plex multifactor and multichannel activities. This activation can
cause biochemical changes of characteristic morphology to
complete the process of apoptosis. As a genomic testing and
DNA repair enzyme, poly(ADP-ribose) polymerase (PARP) is the
first enzyme which was identified as being degraded by cas-
pase-3 and other cysteine proteases and is the most character-
istic protease substrate solution.[26] As a representative of these
derivatives, compound 31 was subjected to systematic in vitro
investigation. We detected the mechanism of compound 31
inhibition effects by FCM (Figure 2), and we found that this
compound could induce the apoptosis of activated lymph
node cells in a dose-dependent manner.
As we knew that among the deoxybenzoins, many com-
pounds with oxime group indicated potent biological activities
and low toxicity and to find more active deoxybenzoins show-
ing immunosuppressive activity, the oximes 31–40 were syn-
thesized. The new synthetic oximes were also tested in vitro
for their cytotoxicity on lymph node cells and inhibition activi-
ty on anti-CD3/anti-CD28 co-stimulated lymph node cells with
CsA as the control. The pharmacological results of these com-
pounds were summarized in Table 3.
Structure–activity relationships (SAR) studies of these com-
pounds were performed by modification of the parent com-
pound to determine how the substituents of the subunits
affect the antibacterial activities. In oximes with the same sub-
stituents at the R3 and R4 positions, the compounds (31, 38)
containing pyrocatechol as the subunits exhibited higher in-
hibition activity than those with resorcinol (33, 35, 37, 39) or
pyrogallol (32, 34, 36, 40) as the substituents. In compounds
32 and 34, replacement of a bromine atom (compound 32,
SI=74.5) by a fluoride at the R3 position on deoxybenzoins re-
sulted in little loss of its inhibition activity (compound 34, SI=
3.59). Similarly, 36 (SI=7.36) is not as active as 38 (SI=139.12).
It is also clear that the compounds with a methoxy group (39,
40) showed least activity. Compound 31 (SI>684.64) showed
excellent immunosuppressive activity compared with the other
oximes, even compared with CsA (SI=235.44), and therefore
inspired us to continue the following work.
As shown in Figure 2, lymph node cells stimulated with anti-
CD3/anti-CD28 were treated with compound 31 at 3, 6, 12,
and 24 mm for 48 h. The compound increased the percentage
of apoptosis by Annexin V–FITC/PI staining in a dose-depen-
dent manner. The result indicated that compound 31 induced
apoptosis of anti-CD3/anti-CD28 stimulated lymph node cells.
To examine the status of the caspase-3 protein, we per-
formed western blot analysis by using an anti-(cleaved cas-
pase-3) antibody, which recognizes p17 cleaved caspase-3. The
Our work is focused on finding new potential immunosup-
pressive agents with high efficacy and low toxicity; therefore,
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1119

H.-L. Zhu et al.
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Figure 2. Lymph node cells isolated from naive mice were cultured with anti-CD3/anti-CD28 and various concentrations (0–24 mm, as indicated) of compound
31 for 48 h. Cells were stained by Annexin V–FITC/PI, and apoptosis was analyzed by flow cytometry.
p17 cleavage product appeared in the lysates of lymph node
cells treated with compound 31 at 6, 12, and 24 mm. In addi-
tion, compound 31 at 12 and 24 mm also cleaved PARP to a
band of 89 kDa. Compound 31, at 6 mm, induced a slight cleav-
age of PARP (Figure 3). This clearly indicated that compound
31 activates caspase-3 and PARP to promote apoptosis. Based
on the above results, we can conclude that the compound can
stimulate cells to activate caspase-3. Activated caspase-3 can
cleave the corresponding substrate PARP within the nuclei,
cause PARP to lose its capacity for effecting DNA repair, and
lead to the apoptosis of cells.
volved. All the results outlined the great potential of these
compounds for further exploitation as immunosuppressants.
Experimental Section
Chemistry
All chemicals (reagent grade) used were purchased from Sigma Al-
drich (USA) and Sinopharm Chemical Reagent Co. Ltd. (China). TLC
was run on silica gel coated aluminum sheets (silica gel 60 GF254, E.
Merck, Germany) and visualized by UV light (l 254 nm). Melting
points (uncorrected) were determined on a XT4 MP apparatus
(Taike Corp., Beijing, China). ESIMS spectra were recorded on a
1
Mariner System 5304 mass spectrometer, and H NMR spectra were
recorded on Bruker DPX-300, AV-300, or AV-500 spectrometers at
258C with (CH3)4Si and solvent signals allotted as internal stand-
ards. Chemical shifts (d) are reported in ppm. Elemental analyses
were performed on a CHN-O-Rapid instrument. The purity of all
tested compounds was determined by HPLC-DAD obtained on an
LC–MS instrument (Agilent 6210 LC-TOF/MS, HPLC Agilent 1200)
using the following procedure: compounds were dissolved at a
concentration of 0.5 mgmLÀ1 in CH3OH. Then, 1 mL of the sample
was injected into an Agilent Zorbax SB-C18 HPLC column (50ꢂ
4.6 mm, particle size 1.8 mm) and were subjected to chromatogra-
phy using a gradient of H2O/CH3OH from 80:10 to 0:100 for
15 min at a flow rate of 400 mLminÀ1, starting the gradient after
10 min. General preparation procedures and the characterization of
new compounds 11–40 can be found in the Supporting Informa-
tion.
Figure 3. Protein levels of cleaved caspase-3 and cleaved PARP as a function
of concentration of compound 31 (indicated) were examined by western
blotting. Data are representative of three independent experiments.
Conclusions
4-(2-(4-bromophenyl)-1-(4-chlorophenylimino)ethyl)benzene-1,2-
diol (11): Deoxybenzoin 1 (1.84 g, 6 mmol) and p-chloroaniline
(0.76 g, 6 mmol) were dissolved in THF (10 mL), then TiCl4 (1.13 g,
0.006 mol) and Et3N (1.39 mL, 0.01 mol) were added to the mixture
at room temperature over the course of 10 min. The mixture was
stirred at 608C for 6 h and then poured into an ice bath. The re-
sulting mixture was extracted with EtOAc (3ꢂ30 mL), and the or-
ganic layer was washed with aqueous NaHCO3 (4 g, 100 mL), dried
(Na2SO4), and evaporated. The residue was purified by column
chromatography on silica gel, using a mixture of EtOAc and petro-
leum ether (PE). Compound 11 (1.85 g, 4.44 mmol) was obtained
as a yellow solid. Yield: 74%; Rf =0.17 (EtOAc/PE 1:2); mp: 154–
1558C; 1H NMR (300 MHz, [D6]DMSO), 4.42 (s, 2H), 6.67 (d, J=
8.2 Hz, 1H), 6.92 (dd, J=8.2, 2.2 Hz, 2H), 7.21–7.36 (m, 4H), 7.89–
7.96 (m, 4H), 9.13 (s, 1H), 11.07 (s, 1H); 13C NMR (500 MHz,
[D6]DMSO): d=28.8, 113.5, 115.7, 118.1, 119.4, 123.1, 127.4, 130.4,
131.1, 131.6, 133.4, 137.3, 145.2, 146.5, 146.9, 154.6 ppm; ESIMS:
A series of new deoxybenzoins were synthesized and their im-
munosuppressive activity and cytotoxicity were evaluated.
Many deoxybenzoin oximes derivatives displayed good immu-
nosuppressive activity. Compound 31 exhibited significant im-
munosuppressive activity with SI>684.64, better than the ref-
erence compound CsA (SI=235.44). In general, electron-donat-
ing substituents at R2 and R3 were favorable for the immuno-
suppressive activity of the synthesized deoxybenzoins. The cy-
totoxicity assay, using compound 31, revealed potent
immunosuppressive activity and indicated that this molecule
did not exhibit significant cytotoxicity in inactivated mouse
lymph node cells. Furthermore, the acute oral toxicity test
demonstrated that compound 31 was nontoxic. The immuno-
suppression process is mediated by the promotion of apopto-
sis of the lymph node cells, where caspase-3 and PARP are in-
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Deoxybenzoin Derivatives
415.00 ([M+H]+); Anal. calcd for C20H15BrClNO2: C 57.65, H 3.63, Br
19.18, Cl 8.51, N 3.36%, found: C 57.63, H 3.60, Br 19.15, Cl 8.55, N
3.40%.
Cells and reagents
Lymph node cells isolated from BALB/c mice were incubated in
RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented
with 10% fetal bovine serum, under a humidified atmosphere of
5.0% CO2 at 378C. Anti-mouse antibodies against CD3 (clone 145-
2C11) and CD28 (clone 37.51), were purchased from BD Pharmin-
gen (Becton Dickinson, San Diego, CA, USA). The Annexin V–FITC/
PI Kit was purchased from Jingmei Biotech (Nanjing, China). Anti-
(cleaved PARP) antibody was purchased from Cell Signaling Tech-
nology (Beverly, MA, USA). Anti-(cleaved caspase-3) antibody and
anti-b-actin antibody were purchased from Santa Cruz Biotech-
nology (Santa Cruz, CA, USA). All other chemicals were obtained
from Sigma Chemical Co. (St. Louis, MO, USA).
4-(2-(4-bromophenyl)-1-(4-chlorophenylamino)ethyl)benzene-
1,2-diol (21): The resulting Schiff base 11 (3.75 g, 9 mmol) was dis-
solved in THF (20 mL), 70% HCOOH(aq) (17 mL) was added, and Zn
powder (1.74 g, 26.7 mmol) was added portion-wise over 30 min
after cooling the solution with an ice bath. The reaction mixture
was stirred for 17–19 h at room temperature, filtered on sand, and
washed with EtOAc (3ꢂ30 mL). The filtrate was neutralized with a
concentrated ammonia solution to pH 8 and then extracted with
EtOAc (3ꢂ30 mL). The organic phase was washed with H2O
(50 mL), dried over anhydrous Na2SO4, and evaporated in vacuo to
give a residue that was purified by column chromatography on
silica gel using a mixture of EtOAc and PE as the eluent. Com-
pound 21 (2.52 g, 6.03 mmol) was obtained as a yellow solid.
Yield: 67%; Rf =0.15 (EtOAc/PE 1:3); mp: 112–1138C; 1H NMR
(300 MHz, [D6]DMSO), 1.52 (s, 1H), 4.29 (s, 2H), 6.62 (d, J=8.1 Hz,
1H), 6.82 (dd, J=8.2, 2.1 Hz, 2H), 7.17–7.34 (m, 4H), 7.86–7.94 (m,
4H), 9.08 (s, 1H), 11.13 (s, 1H); 13C NMR (500 MHz, [D6]DMSO): d=
36.7, 65.5, 112.4, 117.4, 119.2, 121.4, 114.1, 126.6, 127.4, 130.4,
131.1, 132.6, 135.3, 142.1, 145.5, 149.2 ppm; ESIMS: 417.01
([M+H]+); Anal. calcd for C20H17BrClNO2: C 57.37, H 4.09, Br 19.08,
Cl 8.47, N 3.35%, found: C 57.33, H 4.05, Br 19.10, Cl 8.49, N 3.38%.
Cell proliferation assay
Cells were incubated in a 96-well plate at a density of 5ꢂ105 cells
per well and stimulated with anti-CD3/anti-CD28 in the presence
of various concentrations of compounds for 72 h. For the prolifera-
tion assay, 20 mL MTT (Sigma, 4 mgmLÀ1 in PBS) was added per
well 4 h before the end of incubation. After removing the superna-
tant, 200 mL DMSO was added to dissolve the formazan crystals.
The absorbance at l 540 nm (OD540) was read on an ELISA reader
(Tecan, Austria).
(E)-2-(4-bromophenyl)-1-(3,4-dihydroxyphenyl)ethanone oxime
(31):
A solution of deoxybenzoin 1 (0.28 g, 0.90 mmol) and
NH2OH·HCl (0.38 g, 5.4 mmol) in EtOH (18 mL) was held at reflux
for 4–4.5 h at 608C, and then NaOAc (0.074 g, 0.90 mol) was
added. The reaction mixture was allowed to cool to room tempera-
ture and evaporated to dryness in vacuo, and the residue was par-
titioned between H2O (10 mL) and CH2Cl2 (10 mL). The organic ex-
tract was washed with H2O (10 mL), and the aqueous layer was ex-
tracted with CH2Cl2 (10 mL). The combined organic extracts dried
over anhydrous Na2SO4 and evaporated in vacuo to give a residue
that was purified by column chromatography on silica gel using a
mixture of EtOAc and PE as eluent. Compound 31 (0.24 g,
0.73 mmol) was obtained as a yellow crystalline solid. Yield: 81%;
Rf =0.13 (EtOAc/PE 1:3); mp: 144–1458C; 1H NMR (300 MHz,
[D6]DMSO), 3.99 (s, 2H), 6.67 (d, J=8.2 Hz, 1H), 6.92 (dd, J=8.2,
2.2 Hz, 2H), 7.10(d, J=2.2 Hz, 1H), 7.15 (d, J=8.4 Hz, 1H), 7.44 (d,
J=8.4 Hz, 1H), 8.96 (s, 1H), 9.09 (s, 1H), 11.10 (s, 1H); 13C NMR
(500 MHz, [D6]DMSO): d=30.4, 113.5, 115.7, 118.1, 119.4, 127.4,
131.1, 131.6, 137.6, 145.5, 146.9, 154.6 ppm; ESIMS: 321.00([M+H]+
); Anal. calcd for C14H12BrNO3: C 52.20, H 3.75, Br, 24.80, N 4.35%,
found: C 52.11, H 3.70, Br 24.89, N 4.38%.
Cytotoxicity test
Cells were incubated in a 96-well plate at a density of 5ꢂ105 cells
per well with various concentrations of compounds for 48 h. For
the cytotoxicity assay, 20 mL of MTT (Sigma, 4 mgmLÀ1 in PBS) was
added per well 4 h before the end of the incubation. MTT forma-
zan production was terminated by replacing the medium with
200 mL DMSO. The absorbance (OD540) was read on an ELISA
reader (Tecan, Austria).
Apoptosis assay
Lymph node cells were stimulated with anti-CD3/anti-CD28 in the
presence of various concentrations of compounds for 48 h and
then stained with both Annexin V–FITC (fluorescein isothiocyanate)
and propidium iodide (PI). Samples were then analyzed by a FACS-
Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA).
Western blot analysis
After incubation, cells were washed with PBS and lysed using lysis
buffer (30 mm Tris, pH 7.5, 150 mm NaCl, 1 mm phenylmethylsul-
fonyl fluoride, 1 mm Na3VO4, 1% Nonidet P-40, 10% glycerol, and
phosphatase and protease inhibitors). After centrifugation at
10,000 g for 10 min, the protein content of the supernatant was
determined by a BCATM protein assay kit (Pierce, Rockford, IL,
USA). The protein lysates were separated by 10% SDS-PAGE and
subsequently electrotransferred onto a polyvinylidene difluoride
membrane (Millipore, Bedford, MA, USA). The membrane was
blocked with 5% nonfat milk for 2 h at room temperature. The
blocked membrane was probed with the indicated primary anti-
bodies overnight at 48C, and then incubated with a horse radish
peroxidase (HRP)-coupled secondary antibody. Detection was per-
formed using a LumiGLO chemiluminescent substrate system (KPL,
Guildford, UK).
Biological assays
Animals
Six- to eight-week-old female BALB/c mice were purchased from
the Experimental Animal Center of Jiangsu Province (Jiangsu,
China). Mice were maintained with free access to pellet food and
water in plastic cages at 21Æ28C and kept on a 12 h light/dark
cycle. Animal welfare and experimental procedures were carried
out in strict accordance with the Guide for the Care and Use of
Laboratory Animals (Ministry of Science and Technology of China,
2006) and the related ethical regulations of Nanjing University. All
efforts were made to minimize animal’s suffering and to minimize
the number of animals used.
ChemMedChem 2010, 5, 1117 – 1122
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1121

H.-L. Zhu et al.
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Acknowledgements
This work was supported by the National Natural Science Foun-
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Keywords: cytotoxicity
immunosuppression · oximes
·
deoxybenzoins
·
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Published online on May 5, 2010
1122
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ChemMedChem 2010, 5, 1117 – 1122