Synthesis, biological evaluation of chrysin derivatives as potential immunosuppressive agents

Article abstract of DOI:10.1016/j.ejmech.2010.10.034

A series of novel chrysin derivatives was firstly synthesized and evaluated on their immunosuppressive activity in the search for potential immunosuppressive agents. Among them, compounds 5c displayed the most potent immunosuppressive inhibitory activity with IC₅₀ of 0.78 μM, which was comparable to that of cyclosporin A (IC₅₀ = 0.06 μM). The preliminary mechanism of compound 5c inhibition effects was also detected by flow cytometry (FCM), and the compound exerted immunosuppressive activity via inducing the apoptosis of activated lymph node cells in a dose dependent manner. Furthermore, the estimated LD₅₀ (in mg/kg) in vivo of compound 5c is 738.2, which indicated that compound 5c was low toxic.

Full text of DOI:10.1016/j.ejmech.2010.10.034

European Journal of Medicinal Chemistry 46 (2011) 393e398
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, biological evaluation of chrysin derivatives as potential
immunosuppressive agents
*
Peng-Cheng Lv, Tian-Tian Cai, Yong Qian, Juan Sun, Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, 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 novel chrysin derivatives was firstly synthesized and evaluated on their immunosuppressive
activity in the search for potential immunosuppressive agents. Among them, compounds 5c displayed
Received 6 March 2010
Received in revised form
15 October 2010
Accepted 31 October 2010
Available online 10 November 2010
the most potent immunosuppressive inhibitory activity with IC50 of 0.78
mM, which was comparable to
that of cyclosporin A (IC50 ¼ 0.06
m
M). The preliminary mechanism of compound 5c inhibition effects
was also detected by flow cytometry (FCM), and the compound exerted immunosuppressive activity via
inducing the apoptosis of activated lymph node cells in a dose dependent manner. Furthermore, the
estimated LD50 (in mg/kg) in vivo of compound 5c is 738.2, which indicated that compound 5c was low
toxic.
Keywords:
Chrysin derivatives
Immunosuppressive agents
Structureeactivity relationship
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
As we know, salicylic acid was identified in willow bark extracts
as an active anti-inflammatory compound over a century ago. It is
Immunosuppressant is an important class of clinical drugs for an
array of medical processes, including transplant rejection and the
treatment of autoimmune diseases such as systemic lupus eryth-
ematosus, rheumatoid arthritis and psoriasis [1e3]. Although
immunosuppressive drugs have been used fororgan transplantation
and treatment of autoimmune diseases in clinic, their side effects
including liver toxicity, nephrotoxicity, infection, cardiovascular
toxicity and others can not be neglected [4e9]. Therefore, there is
a clinical need for new therapeutic agents with anti-inflammatory
activity. A program to produce synthetic compounds is ongoing in
our laboratory to identify new immunosuppressants [10].
Flavonoids are natural polyphenolic phytochemicals that are
ubiquitous in plants and present in the average human diet. Chrysin
(5,7-dihydroxyflavone, 1) is a natural flavonoid found in many plant
extracts, honey, and propolis, which has been reported to have many
different biological activities such as anti-viral [11], anti-cancer [12],
anti-bactericidal [13], anti-inflammatory [14], anti-allergic [15], DNA
cleavage [16], vasodilator [17], anti-mutagenic [18], anti-anxiolytic
[19] and anti-oxidant [20] effects. Recently, Eun Kyung Shin et al.,
reportedthatchrysincouldalleviatethesymptomsofdextransodium
sulfate (DSS)-induced colitis in mice by reducing the production of
inflammatory mediators, which suggesting that chrysin exerts
potentially clinically useful anti-inflammatory effects [21].
reported that some salicylic acid derivatives showed potent anti-
inflammatory activity [22e24]. For example, 5-aminosalicylic acid
(5-ASA) was an active ingredient of agents used for the long term
maintenance therapy to prevent relapses of inflammatory bowel
diseases [25]. Besides, the methyl and some other esters of salicylic
acids and their derivatives were found to have much lower gastric
ulcerogenic activity compared with their corresponding acids
[26e28]. In view of the facts mentioned above, and in continuation
to extend our research on new potential immunosuppressive
agents with high efficacy and low toxicity [10], we turned our
attention to natural product chrysin. Therefore, a series of novel
substituted salicylic acid and chrysin derivatives were firstly
synthesized. Compound 5c displayed potent immunosuppressive
activity by inhibiting CD3/CD28 co-stimulated T cell proliferation,
suggesting that it may have a characteristic to inhibit the T-cell
mediated immune response. Subsequently, the function of
compound 5c on the cell apoptosis in activated mouse T cells was
studied.
2. Results and discussion
2.1. Chemistry
In this study, twenty novel substituted salicylic acid and chrysin
derivatives were synthesized. The synthetic route of them is shown
in Scheme 1. To a stirring solution of chrysin (CR, compound 1) in
* Corresponding author. Tel./fax: þ86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.10.034

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P.-C. Lv et al. / European Journal of Medicinal Chemistry 46 (2011) 393e398
5a R1= H, R2= H, R3= OH, R4= H
5k R1= Br, R2= H, R3= H, R4= H
5l R1= Cl, R2= H, R3= H, R4= H
5m R1= CH3, R2= H, R3= H, R4= H
5n R1= H, R2= Cl, R3= H, R4= Cl
5o R1= H, R2= Br, R3= H, R4= Br
5p R1= H, R2= H, R3= H, R4= F
5q R1= H, R2= H, R3= H, R4= Cl
5r R1= H, R2= H, R3= H, R4= Br
5s R1= H, R2= H, R3= H, R4= CH3
5t R1= H, R2= H, R3= H, R4= OH
5b R1= H, R2= H, R3= CH3, R4= H
5c R1= H, R2= H, R3= Br, R4= H
5d R1= H, R2= H, R3= Cl, R4= H
5e R1= H, R2= H, R3= F, R4= H
5f R1= H, R2= CH3, R3= H, R4= H
5g R1= H, R2= Cl, R3= H, R4= H
5h R1= H, R2= Br, R3= H, R4= H
5i R1= OCH3, R2= H, R3= H, R4= H
5j R1= H, R2= H, R3= H, R4= H
Scheme 1. General synthesis of compounds 5ae5t. Reagents and conditions: i) BrCH2COOEt, K2CO3, acetone; ii) Na2CO3, 5% aq, DMSO; iii) substituted methyl salicylate (4ae4t),
EDC, HOBt, DMAP, DMF, 10e12 h.
acetone, K2CO3 was added and then a mixture of ethyl bromoace-
tate and acetone was added dropwise to give compound 2.
Compound 2 was treated in Na2CO3 solution (DMSO) and after that
most of the volatiles were evaporated. The residue was dissolved in
water and the solution was adjusted to pH 2 by using HCl solution
to give compound 3. Compound 3 was suspended in DMF with
stirring at room temperature, then substituted methyl salicylate
(4ae4t), 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydro-
chloride (EDC.HCl), N-Hydroxybenzotriazole (HOBt) and 4-dime-
thylaminopyridine (DMAP) were added. The reaction solution was
stirred for 12 h at room temperature. Then, compounds 5ae5t were
obtained by subsequent purification with flash chromatography. All
of the synthetic compounds gave satisfactory analytical and spec-
troscopic data, which were in full accordance with their depicted
structures.
CD3/CD28 co-stimulated lymph node cells with cyclosporin A (CsA)
as the control. The pharmacological results of these compounds
were summarized in Table 1. The cytotoxicity of each compound
was expressed as the concentration of compound that reduced cell
viability to 50% (CC50). The immunosuppressive activity of each
compound was expressed as the concentration of the compound
that inhibited CD3/CD28 co-stimulated T cell proliferation to 50%
(IC50) of the control value. The selective index (SI) value was used to
evaluate the bioactivity of the compounds.
Studies were performed by modification of the phenyl ring of
the salicylic acid derivatives to determine how the substituents of
the subunits affected the immunosuppressive activity. As shown in
Table 1, in general, most of the synthesized novel chrysin deriva-
tives exhibited higher immunosuppressive activity than the parent
compound chrysin (IC50: 44.16
to 45.72 M. Among them, compounds with substitutions on the
R1- and R4- position of phenyl ring of the salicylic acid derivatives
(IC50: from 36.78 M to 45.72 M) showed less potent immuno-
suppressive activity than the R2- and R3- position ones (IC50: from
0.78 M to 31.07 M). For the R2- position substituted ones,
replacement of H atom at R2-position by one halogen atom (5g
IC50: 12.36 M and 5h IC50: 17.59 M) resulted in the improving of
their immunosuppressive activity compared to 5f (IC50: 31.07 M).
mM), with IC50 ranging from 0.78 mM
m
2.2. Immunosuppressive activity
m
m
T cells play a pivotal role in immune response. Excessive T-cell
proliferation and activation has been implicated in the pathogen-
esis of a variety of inflammatory diseases.
The new synthetic chrysin derivatives were tested in vitro for
their cytotoxicity on lymph node cells and inhibition activity on
m
m
m
m
m

P.-C. Lv et al. / European Journal of Medicinal Chemistry 46 (2011) 393e398
395
Table 1
Apoptosis is an essential mechanism used to eliminate activated
T cells during the shutdown process of excess immune responses
and maintain proper immune homeostasis, while deficient
apoptosis of activated T cells is associated with a wide variety of
immune disorders. As a representative of these chrysin derivatives,
compound 5c has been under investigations in vitro experiment.
We detected the mechanism of compound 5c inhibition effects by
flow cytometry (FCM) (Fig. 1), and found that the compound could
induce the apoptosis of activated lymph node cells in a dose
dependent manner. As shown in Fig. 1, lymph node cells stimulated
In vitro cytotoxicity on lymph node cells and inhibitory effects of the synthetic
compounds 5ae5t on lymph node cells co-stimulated by CD3/CD28.
a
Compd
CC50
(
m
M)
IC50
(
m
M)
SI b
CR
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
428.27
263.42
306.48
>450
>436
389.26
44.16
29.47
25.68
0.78
3.74
6.82
9.70
8.94
11.93
>576.92
>116.57
50.08
8.04
21.43
18.54
5.34
249.81
264.83
326.18
196.34
264.92
378.63
366.24
298.86
345.26
397.64
318.72
365.84
387.91
297.85
337.69
15.82
31.07
12.36
17.59
36.78
28.54
40.65
38.74
39.08
43.57
45.72
40.27
38.94
41.72
37.16
38.25
0.06
with anti-CD3/anti-CD28 were treated with 1, 2, 4 and 8 mM of
compound 5c for 48 h. The compound increased the percentage of
apoptosis by Annexin V-FITC/PI staining in a dose-dependent
manner. The result indicated that compound 5c induced apoptosis
of anti-CD3/anti-CD28 stimulated lymph node cells.
9.28
9.31
9.45
7.65
7.92
8.69
7.91
9.39
Our attention in this area is to search for new potential
immunosuppressive agents with high efficacy and low toxicity, so
the acute oral toxicity test of the most potent compound 5c was
examined. It can be seen from Table 2 that the estimated LD50 (in
mg/kg) in vivo of compound 5c is 738.2, which indicated that
compound 5c was low toxic. Furthermore, we also evaluate the
effect of chrysin derivative 5c on three human tumor cell lines:
human hepatocellular liver carcinoma (Hep-G2), human erthro-
myeloblastoid leukemia (K562) and human oral epidermoid
carcinoma (KB), and the normal human liver cell line (L02), and
the in vitro cytotoxicity results were showed in Table 3.
Compound 5c exhibited significant inhibitory activity against
9.28
8.02
8.83
5s
5t
CsA
263.67
a
The compounds tested for immunosuppressive activity were consistent with
the description in the Experimental Section.
b
Selectivity index [SI] is determined as the ratio of the concentration of the
compound that reduced cell viability to 50% (CC50) to the concentration of the
compound needed to inhibit the proliferation to 50% (IC50) of the control value.
K562, KB and Hep-G2 with IC50 of 0.47, 0.79 and 0.61
mM,
respectively. Besides, Compound 5c also displayed little toxicity
against the normal human liver cell line (L02) with IC50 of
274 mM.
For compounds 5n and 5o, introduced two halogen on the R2-
position and R4- position, the immunosuppressive activity was
remarkably decreasing (5n IC50: 43.57 mM and 5o IC50: 45.72 mM)
compared to compounds 5g and 5h. Besides, the SI values of the
compounds differed greatly, ranging from 5.34 to more than 576.92.
Some of the synthesized compounds (5g and 5h), especially 5g
(SI ¼ 21.43) showed good immunosuppressive activity but much
less potent than CsA (SI ¼ 263.67).
3. Conclusions
In summary, a series of novel chrysin derivatives were firstly
synthesized and evaluated for their immunosuppressive activity on
CD3/CD28 co-stimulated T cell proliferation. It is worth noting that
compound 5c displayed the most outstanding immunosuppressive
It is clear that the synthesized compounds with halogen
substitutions in R3- position on the phenyl ring of the salicylic acid
derivatives (compounds 5c, 5d, and 5e) showed higher activity than
others, with IC50 ranging from 0.78
compounds 5c displayed the most potent inhibitory activity with
IC50 of 0.78 M, which was comparable to that of cyclosporin A
(IC50 ¼ 0.06 M). More significantly, compounds 5c exhibited
mM to 6.82 mM. Among them,
effects in vitro with IC50 of 0.78
of cyclosporin A (IC50 ¼ 0.06
m
M, which was comparable to that
mM). Moreover, the preliminary
m
mechanism of compound 5c inhibition effects was also detected by
flow cytometry (FCM), and the compound exerted immunosup-
pressive activity via inducing the apoptosis of activated lymph node
cells in a dose dependent manner. In addition, the acute oral
toxicity test demonstrated that compound 5c was low toxic. All the
results outlined the great potential of compound 5c for further
exploitation as immunosuppressant.
m
reduced cytotoxicity by over 100-fold compared with cyclosporin A
(CsA) and more potent inhibition activity (SI > 576.92) than CsA
(SI ¼ 263.67). Therefore, the IC50 and SI value suggest that
compounds 5c may be a promising lead for the further develop-
ment of novel therapeutic agents.
Fig. 1. Lymph node cells isolated from naive mice were cultured with anti-CD3/anti-CD28 and various concentrations of 5c for 48 h. Cells were stained by Annexin VeFITC/PI and
apoptosis was analyzed by flow cytometry.

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P.-C. Lv et al. / European Journal of Medicinal Chemistry 46 (2011) 393e398
Table 2
Estimated LD50 (in mg/kg) in vivo of compound 5c.
4.2.3. Cell proliferation assay
Cells were incubated in 96-well plate at a density of 5 ꢃ 105 cells
per well and stimulated with anti-CD3/anti-CD28 in the presence of
various concentrations of compounds for 72 h. For proliferation
a
Compound
Estimated LD50 (mg/kg)
738.2
5c
assay, 20
4 h before the end of the incubation. After removing the superna-
tant, 200 L DMSO was added to dissolve the formazan crystals. The
mL MTT (Sigma, MO; 4 mg/mL in PBS) was added per well
a
The LD50 is calculated with AOT425StatPgm based on maximum
likelihood for long term results at 95% confidence interval.
m
absorbance at 540 nm (OD540) was read on an ELISA reader (Tecan,
Austria).
Table 3
4.2.4. Cytotoxicity test
In vitro cytotoxicity (IC50 value,
mM) on three human tumor cell lines human
Cells were incubated in 96-well plate at a density of 5 ꢃ 105 cells
hepatocellular liver carcinoma (Hep-G2), human erthromyeloblastoid leukemia
(K562) and human oral epidermoid carcinoma (KB), and the normal human liver cell
line (L02) of compound 5c.
per well with various concentrations of compounds for 48 h. For
cytotoxicity assay, 20
added per well 4 h before the end of the incubation. MTT formazan
production was dissolved by 200 L DMSO replacing the medium.
mL of MTT (Sigma, MO; 4 mg/mL in PBS) was
Compound
K562
KB
Hep-G2
L02
m
5c
0.47 ꢁ 0.06
38 ꢁ 7
0.79 ꢁ 0.18
2.16 ꢁ 0.22
0.61 ꢁ 0.13
2.78 ꢁ 0.31
274 ꢁ 26
56 ꢁ 9
The absorbance was read on an ELISA reader (Tecan, Austria) at
540 nm (OD540).
5-fluorouracil
4.2.5. Apoptosis assay
4. Experimental Section
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 VeFITC (fluorescein iso-
thiocyanate) and propidium iodide (PI). Then samples were
analyzed by FACSCalibur flow cytometer (Becton Dickinson, San
Jose, CA).
4.1. General
Separation of the compounds by column chromatography was
carried out with silica gel 60 (200e300 mesh ASTM, E. Merck). The
quantity of silica gel used was 50e100 times the weight charged on
the column. Then, the eluates were monitored using TLC. Melting
points (uncorrected) were determined on an XT4 MP apparatus
(Taike Corp., Beijing, China). ESI mass spectra were obtained on
a Mariner System 5304 mass spectrometer, and 1H NMR spectra
were recorded on a Bruker PX500 or DPX300 spectrometer at 25 ꢀC
with TMS and solvent signals allotted as internal standards.
4.2.6. Acute oral Toxicity of compound 5c
Healthy nulliparous and non-pregnant BALB/C female mice, 12
weeks of age at the initiation of the experiment were used. All
animals were fastening prior to dosing by withholding food (not
water) for 3e4 h and 1e2 h after dosing. After that period access to
food and water was ad libitum. Animals were kept in conventional
circumstances: light/dark rhythms 12/12 h, temperature 22 ꢀC, and
humidity 55%. All experiments were performed according to the
OECD 425 Guideline for the testing of chemicalsdAcute oral
Toxicity: Up-and-down procedure [29]. Altogether 35 animals were
used in experiments to determine LD50 for compound 5c. Animals
are dosed, one at a time, at 24 h intervals. The first animal receives
a dose at the level of the best estimate of the LD50. Depending on
the outcome for the previous animal, the dose for the next animal is
adjusted up or down. If an animal survives, the dose for the next
animal is increased; if it dies, the dose for the next animal is
decreased. After reaching the reversal of the initial outcome, that
is, the point where an increasing (or decreasing) dose pattern is
reversed by giving a smaller (or a higher) dose, four additional
animals are dosed following the same UDP.
Chemical shifts were reported in ppm (d). Elemental analyses were
performed on a CHN-O-Rapid instrument and were within ꢁ0.4% of
the theoretical values.
4.2. Biological assay
4.2.1. Animals
Specific-pathogen-free female BALB/c mice, 6e8 week-old, were
obtained from Experimental Animal Center of Yangzhou University
(Yangzhou, China). They were maintained in plastic cages at
22 ꢁ 2 ꢀC and kept on a 12 h lightedark cycle with free access to
pellet food and water. Animal welfare and experimental procedures
were carried out strictly in accordance with the guide for the care
and use of laboratory animals (National Research Council of USA,
1996). All efforts were made to minimize animals’ suffering and to
reduce the number of animals used.
4.2.7. Statistical analysis
Statistical analyses were conducted by the ‘Acute Oral Toxicity
(Guideline 425) Statistical Program’ (AOT425StatPgm) which is
developed by Westat for the US EPA and designed to be used with
the acute oral toxicity testing procedure presented in OECD
Guideline for the testing of chemicals, Section 4: Health Effects Test
No. 425, Acute Oral Toxicity: Up-and-Down Procedure [29].
4.2.2. Reagents and materials
T cells were incubated in RPMI 1640 medium supplemented
with 100 U mLꢂ1 of penicillin, 100 gm Lꢂ1 of streptomycin and 10%
m
fetal calf serumunder a humidified 5% (v/v) CO2 atmosphere at
37 ꢀC. Chrysin (purity>98%, obtained from the Shanxi Huike co.,
Shanxi, China) is dissolved at a concentration of 0.05 mol/L in 100%
DMSO as a stock solution, stored at ꢂ20 ꢀC, and diluted with
medium before each experiment. The final DMSO concentration did
not exceed 0.1% throughout the study (all the control groups are
composed of 0.1% DMSO). Other drugs and reagents used in this
study are as follows: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium bromide (MTT, Sigma Chemical Co., St. Louis, MO);
Cyclosporin A (CsA, Sigma Chemical Co., St. Louis, MO); Conca-
navalin A (Con A, Sigma Chemical Co., St. Louis, MO); Annexin V-
FITC/PI Kit (Jingmei Biotech, Nanjing, China).
4.3. Chemistry
4.3.1. General procedure for the preparation of compound 2
A solution of compound 1 (2.84 g, 11.2 mmol), ethyl bromoa-
cetate (3.8 g, 22.7 mmol), and powdered potassium carbonate (3 g,
22 mmol) in acetone (50 mL) was refluxed for 48 h (the reaction
was monitored by TLC). After cooling, removal of the solvent under
reduced pressure gave a crude product, which was filtered off and
purified by washing with water (50 mL), NaOH (1 M, 30 mL), and

P.-C. Lv et al. / European Journal of Medicinal Chemistry 46 (2011) 393e398
397
water again (30 mL) to give the product 2 as a yellow powder. Yield:
86%. M.p.: 277e279 ꢀC 1H NMR (300 MHz, d6-DMSO): 1.25 (t,
J ¼ 6.9 Hz, 3H); 4.19 (m, 2H); 4.96 (s, 2H); 6.43 (m,1H); 6.85 (m, 1H),
7.01 (s, 1H), 7.60 (m, 3H), 8.10 (m, 2H), 12.81 (s, 1H). MS (ESI): 340.1
(C19H17O6, [M þ H]þ). Anal. Calcd for C19H16O6: C, 67.05; H, 4.74%;
Found: C, 67.18; H, 4.91%.
(s, 2H); 6.55e6.56 (d, J ¼ 2.19 Hz, 1H); 6.98e6.99 (d, J ¼ 2.40 Hz,
1H); 7.08 (s, 1H); 7.42e7.44 (d, J ¼ 5.58 Hz, 1H); 7.59e7.64 (m, 3H);
7.92e7.95 (dd, J1 ¼ 2.57, J2 ¼ 8.64 Hz, 1H); 7.96e7.97 (d, J ¼ 2.73 Hz,
1H); 8.10e8.12 (m, 2H); 12.83 (s, 1H). MS (ESI): 481.0 (C25H18ClO8,
[M þ H]þ). Anal. Calcd for C25H17ClO8: C, 62.45; H, 3.56%; Found: C,
62.73; H, 3.35%.
4.3.2. General procedure for the preparation of compound 3
A solution of sodium carbonate (5% aqueous, 8 mL, excess) was
added to a solution of compound 2 (0.98 g, 2.82 mmol) in DMSO
(50 mL). The mixture was refluxed for 48 h (the reaction was
monitored by TLC). After cooling, removal of the solvent under
reduced pressure, and acidification with HCl (1 M) gave a crude
product, which was filtered off and purified by washing with water
(50 mL) to give the product 3 as a yellow powder. Yield: 80%. M.p.:
296e298 ꢀC; 1H NMR (500 MHz, d6-DMSO): 2.51 (s, 3H); 4.85 (s,
2H); 6.41 (m, 1H), 6.82 (m, 1H); 7.04 (s, 1H); 7.61 (m, 3H), 8.11 (m,
2H),12.80 (s,1H),13.20 (s,1H). MS (ESI): 312.1 (C17H13O6, [M þ H]þ).
Anal. Calcd for C17H12O6: C, 65.39; H, 3.87%; Found: C, 65.17; H,
3.73%.
4.3.3.5. Methyl 5-fluoro-2-(2-(5-hydroxy-4-oxo-2-phenyl-4H-chro-
men-7-yloxy)acetoxy)benzoate (5e). White powder. Yield: 75%.
M.p.: 207e209 ꢀC 1H NMR (300 MHz, d6-DMSO): 3.84 (s, 3H); 5.27
(s, 2H); 6.54e6.56 (d, J ¼ 2.17 Hz, 1H); 6.94e6.96 (d, J ¼ 2.41 Hz,
1H); 7.07 (s, 1H); 7.36e7.38 (d, J ¼ 5.17 Hz, 1H); 7.59e7.64 (m, 3H);
7.91e7.94 (dd, J1 ¼ 2.64, J2 ¼ 8.73 Hz, 1H); 8.08e8.09 (d, J ¼ 2.73 Hz,
1H); 8.08e8.10 (m, 2H); 12.81 (s, 1H). MS (ESI): 465.0 (C25H18FO8,
[M þ H]þ). Anal. Calcd for C25H17FO8: C, 64.66; H, 3.69%; Found: C,
64.37; H, 3.46%.
4.3.3.6. Methyl 2-(2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)
acetoxy)-4-methylbenzoate (5f). White powder. Yield: 70%. M.p.:
183e184 ꢀC 1H NMR (300 MHz, d6-DMSO): 2.39 (s, 3H); 3.83 (s,
3H); 5.27 (s, 2H); 6.54e6.55 (d, J ¼ 1.97 Hz, 1H); 6.98e6.99 (d,
J ¼ 2.43 Hz, 1H); 7.06 (s, 1H); 7.17 (s, 1H); 7.25e7.27 (d, J ¼ 7.74 Hz,
1H); 7.59e7.62 (m, 3H); 7.87e7.89 (d, J ¼ 4.74 Hz, 1H); 8.09e8.11
(m, 2H); 12.82 (s, 1H). MS (ESI): 461.1 (C26H21O8, [M þ H]þ). Anal.
Calcd for C26H20O8: C, 67.82; H, 4.38%; Found: C, 67.63; H, 4.52%.
4.3.3. General procedure for the preparation of compounds 5ae5t
A solution of compound 3, substituted methyl salicylate (4ae4t),
1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride
(EDC.HCl), N-Hydroxybenzotriazole (HOBt) and 4-dimethylamino-
pyridine (DMAP) in DMF (50 mL) was stirred for 12 h at room
temperature. Then, compounds 5ae5t were obtained by subse-
quent purification with flash chromatography (acetate: petroleum
ether ¼ 1:5e1:2).
4.3.3.7. Methyl 4-chloro-2-(2-(5-hydroxy-4-oxo-2-phenyl-4H-chro-
men-7-yloxy)acetoxy)benzoate (5g). White powder. Yield: 71%.
M.p.: 171e174 ꢀC 1H NMR (300 MHz, d6-DMSO): 3.82 (s, 3H); 5.26
(s, 2H); 6.53e6.54 (d, J ¼ 2.14 Hz,1H); 6.97e6.98 (d, J ¼ 2.41 Hz,1H);
7.06 (s, 1H); 7.18 (s, 1H); 7.26e7.28 (d, J ¼ 7.81 Hz, 1H); 7.58e7.63
(m, 3H); 7.88e7.89 (d, J ¼ 4.71 Hz, 1H); 8.09e8.13 (m, 2H); 12.81 (s,
1H). MS (ESI): 481.0 (C25H18ClO8, [M þ H]þ). Anal. Calcd for
C25H17ClO8: C, 62.45; H, 3.56%; Found: C, 62.73; H, 3.71%.
4.3.3.1. Methyl 5-hydroxy-2-(2-(5-hydroxy-4-oxo-2-phenyl-4H-chro-
men-7-yloxy)acetoxy)benzoate (5a). White powder. Yield: 74%. M.p.:
187e188 ꢀC 1H NMR (300 MHz, d6-DMSO): 3.86 (s, 3H); 5.31 (s, 2H);
6.53e6.56 (d, J ¼ 2.17 Hz,1H); 6.94e6.97 (d, J ¼ 2.43 Hz,1H); 7.08 (s,
1H); 7.41e7.43 (d, J ¼ 5.67 Hz, 1H); 7.57e7.62 (m, 3H); 7.91e7.94
(dd, J1 ¼ 2.47, J2 ¼ 8.56 Hz, 1H); 7.94e7.96 (d, J ¼ 2.61 Hz, 1H);
8.11e8.13 (m, 2H); 12.82 (s, 1H). MS (ESI): 463.1 (C25H19O9,
[M þ H]þ). Anal. Calcd for C25H18O9: C, 64.94; H, 3.92%; Found: C,
64.76; H, 3.74%.
4.3.3.8. Methyl 4-bromo-2-(2-(5-hydroxy-4-oxo-2-phenyl-4H-chro-
men-7-yloxy)acetoxy)benzoate (5h). White powder. Yield: 74%.
M.p.: 176e178 ꢀC 1H NMR (300 MHz, d6-DMSO): 3.84 (s, 3H); 5.27
(s, 2H); 6.52e6.54 (d, J ¼ 2.19 Hz, 1H); 6.96e6.97 (d, J ¼ 2.43 Hz,
1H); 7.06 (s, 1H); 7.16 (s, 1H); 7.27e7.29 (d, J ¼ 7.83 Hz, 1H);
7.56e7.61 (m, 3H); 7.86e7.88 (d, J ¼ 4.74 Hz, 1H); 8.08e8.12 (m,
2H); 12.83 (s, 1H). MS (ESI): 525.0 (C25H18BrO8, [M þ H]þ). Anal.
Calcd for C25H17BrO8: C, 57.16; H, 3.26%; Found: C, 57.43; H, 3.47%.
4.3.3.2. Methyl
2-(2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetoxy)-5-methylbenzoate (5b). White powder. Yield: 71%.
M.p.: 196e197 ꢀC 1H NMR (300 MHz, d6-DMSO): 2.38 (s, 3H); 3.85
(s, 3H); 5.31 (s, 2H); 6.53e6.55 (d, J ¼ 2.06 Hz, 1H); 6.96e6.97 (d,
J ¼ 2.43 Hz, 1H); 7.07 (s, 1H); 7.41e7.43 (d, J ¼ 5.61 Hz, 1H);
7.57e7.62 (m, 3H); 7.93e7.96 (dd, J1 ¼ 2.41, J2 ¼ 8.61 Hz, 1H);
7.95e7.96 (d, J ¼ 2.73 Hz, 1H); 8.09e8.11 (m, 2H); 12.81 (s, 1H). MS
(ESI): 461.1 (C26H21O8, [M þ H]þ). Anal. Calcd for C26H20O8: C, 67.84;
H, 4.38%; Found: C, 67.62; H, 4.59%.
4.3.3.9. Methyl
2-(2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetoxy)-3-methoxybenzoate (5i). White powder. Yield: 73%.
M.p.: 180e182 ꢀC 1H NMR (300 MHz, d6-DMSO): 3.87 (s, 3H); 3.89
(s, 3H); 5.30 (s, 2H); 6.92e6.93 (d, J ¼ 2.40 Hz, 1H); 7.07 (s, 1H);
7.37e7.51 (m, 3H); 7.58e7.64 (m, 3H); 8.09e8.12 (m, 2H); 12.84 (s,
1H). MS (ESI): 477.1 (C26H21O9, [M þ H]þ). Anal. Calcd for C26H20O9:
C, 65.55; H, 4.23%; Found: C, 65.72; H, 4.43%.
4.3.3.3. Methyl 5-bromo-2-(2-(5-hydroxy-4-oxo-2-phenyl-4H-chro-
men-7-yloxy)acetoxy)benzoate (5c). White powder. Yield: 76%.
M.p.: 202e203 ꢀC 1H NMR (300 MHz, d6-DMSO): 3.86 (s, 3H); 5.28
(s, 2H); 6.54e6.55 (d, J ¼ 2.09 Hz, 1H); 6.96e6.97 (d, J ¼ 2.43 Hz,
1H); 7.06 (s, 1H); 7.34e7.36 (d, J ¼ 5.13 Hz, 1H); 7.57e7.63 (m, 3H);
7.92e7.95 (dd, J1 ¼ 2.55, J2 ¼ 8.61 Hz, 1H); 8.07e8.08 (d, J ¼ 2.71 Hz,
1H); 8.09e8.11 (m, 2H); 12.80 (s, 1H). 13C NMR (d6-DMSO): 54.6,
65.2, 93.5, 98.7, 104.6, 106.2, 114.8, 117.4, 122.6, 126.1, 127.7, 127.9,
128.8, 129.1, 129.8, 139.2, 148.5, 156.7, 160.6, 162.3, 164.7, 171.4,
184.9, 191.4. MS (ESI): 525.0 (C25H18BrO8, [M þ H]þ). Anal. Calcd for
C25H17BrO8: C, 57.16; H, 3.26%; Found: C, 57.41; H, 3.38%.
4.3.3.10. Methyl
2-(2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetoxy)benzoate (5j). White powder. Yield: 76%. M.p.:
174e175 ꢀC 1H NMR (300 MHz, d6-DMSO): 3.89 (s, 3H); 5.30 (s, 2H);
6.56e6.57 (d, J ¼ 2.07 Hz,1H); 6.99e7.00 (d, J ¼ 2.41 Hz,1H); 7.07 (s,
1H); 7.35e7.37 (d, J ¼ 7.95 Hz,1H); 7.45e7.48 (m,1H); 7.58e7.63 (m,
3H); 7.72e7.75 (m, 1H); 7.98e8.01 (dd, J1 ¼ 2.43, J2 ¼ 8.67 Hz, 1H);
8.10e8.12 (m, 2H); 12.83 (s, 1H). MS (ESI): 447.1 (C25H20O8,
[M þ H]þ). Anal. Calcd for C25H18O8: C, 67.26; H, 4.06%; Found: C,
67.47; H, 4.31%.
4.3.3.4. Methyl 5-chloro-2-(2-(5-hydroxy-4-oxo-2-phenyl-4H-chro-
men-7-yloxy)acetoxy)benzoate (5d). White powder. Yield: 73%.
M.p.: 204e205 ꢀC 1H NMR (300 MHz, d6-DMSO): 3.87 (s, 3H); 5.30
4.3.3.11. 3-Bromo-2-(2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetoxy)benzoic acid (5k). White powder. Yield: 70%. M.p.:
163e165 ꢀC 1H NMR (300 MHz, d6-DMSO): 3.88 (s, 3H); 5.31 (s, 2H);

398
P.-C. Lv et al. / European Journal of Medicinal Chemistry 46 (2011) 393e398
6.91e6.93 (d, J ¼ 2.42 Hz, 1H); 7.09 (s, 1H); 7.45e7.53 (m, 3H);
7.59e7.66 (m, 3H); 8.02e8.11 (m, 2H); 12.83 (s, 1H). MS (ESI): 511.1
(C24H16BrO8, [M þ H]þ). Anal. Calcd for C24H15BrO8: C, 56.38; H,
2.96%; Found: C, 56.41; H, 2.89%.
(C24H16BrO8, [M þ H]þ). Anal. Calcd for C24H15BrO8: C, 56.38; H,
2.96%; Found: C, 56.42; H, 3.04%.
4.3.3.19. 2-(2-(5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)
acetoxy)-6-methylbenzoic acid (5s). White powder. Yield: 74%.
M.p.: 179e181 ꢀC 1H NMR (300 MHz, d6-DMSO): 2.37 (s, 3H); 3.87
(s, 3H); 5.34 (s, 2H); 6.73e6.82 (d, J ¼ 2.54 Hz, 1H); 7.17 (s, 1H);
7.22e7.34 (m, 3H); 7.56e7.67 (m, 3H); 8.23e8.36 (m, 2H); 12.87 (s,
1H). MS (ESI): 447.1 (C25H19O8, [M þ H]þ). Anal. Calcd for C25H18O8:
C, 67.26; H, 4.06%; Found: C, 67.32; H, 4.12%.
4.3.3.12. 3-Chloro-2-(2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetoxy)benzoic acid (5l). White powder. Yield: 73%. M.p.:
168e169 ꢀC 1H NMR (300 MHz, d6-DMSO): 3.87 (s, 3H); 5.30 (s,
2H); 6.94e6.99 (d, J ¼ 2.47 Hz, 1H); 7.07 (s, 1H); 7.51e7.58 (m, 3H);
7.63e7.69 (m, 3H); 8.06e8.17 (m, 2H); 12.86 (s, 1H). MS (ESI): 467.1
(C24H16ClO8, [M þ H]þ). Anal. Calcd for C24H15ClO8: C, 61.75; H,
3.24%; Found: C, 61.69; H, 3.29%.
4.3.3.20. 2-Hydroxy-6-(2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-
7-yloxy)acetoxy)benzoic acid (5t). White powder. Yield: 71%. M.p.:
210e211 ꢀC 1H NMR (300 MHz, d6-DMSO): 3.86 (s, 3H); 5.33 (s, 2H);
6.83e6.92 (d, J ¼ 2.74 Hz, 1H); 7.16 (s, 1H); 7.47e7.53 (m, 3H);
7.61e7.73 (m, 3H); 8.23e8.37 (m, 2H); 12.87 (s, 1H). MS (ESI): 449.1
(C24H17O9, [M þ H]þ). Anal. Calcd for C24H16O9: C, 64.29; H, 3.60%;
Found: C, 64.34; H, 3.71%.
4.3.3.13. 2-(2-(5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)
acetoxy)-3-methylbenzoic acid (5m). White powder. Yield: 71%.
M.p.: 171e173 ꢀC 1H NMR (300 MHz, d6-DMSO): 2.36 (s, 3H); 3.84
(s, 3H); 5.32 (s, 2H); 6.87e6.95 (d, J ¼ 2.41 Hz, 1H); 7.13 (s, 1H);
7.39e7.46 (m, 3H); 7.51e7.62 (m, 3H); 8.14e8.21 (m, 2H); 12.81 (s,
1H). MS (ESI): 447.1 (C25H19O8, [M þ H]þ). Anal. Calcd for C25H18O8:
C, 67.26; H, 4.06%; Found: C, 67.19; H, 4.12%.
Acknowledgements
4.3.3.14. 2,4-Dichloro-6-(2-(5-hydroxy-4-oxo-2-phenyl-4H-chro-
men-7-yloxy)acetoxy)benzoic acid (5n). White powder. Yield: 68%.
M.p.: 192e193 ꢀC 1H NMR (300 MHz, d6-DMSO): 3.81 (s, 3H); 5.28
(s, 2H); 6.56e6.64 (d, J ¼ 2.34 Hz, 1H); 6.84e6.89 (d, J ¼ 2.47 Hz,
1H); 7.08 (s, 1H); 7.13 (s, 1H); 7.27e7.34 (d, J ¼ 7.69 Hz, 1H);
7.54e7.61 (m, 2H); 7.86e7.92 (d, J ¼ 4.76 Hz, 1H); 8.09e8.14 (m,
2H); 12.84 (s, 1H). MS (ESI): 501.1 (C24H15Cl2O8, [M þ H]þ). Anal.
Calcd for C24H14Cl2O8: C, 57.51; H, 2.82%; Found: C, 57.63; H, 2.89%.
This work was supported by the Jiangsu National Science
Foundation (No. BK2009239).
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4.3.3.15. 2,4-Dibromo-6-(2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-
7-yloxy)acetoxy)benzoic acid (5o). White powder. Yield: 73%. M.p.:
207e209 ꢀC 1H NMR (300 MHz, d6-DMSO): 3.83 (s, 3H); 5.26 (s, 2H);
6.52e6.60 (d, J ¼ 2.31 Hz, 1H); 6.78e6.84 (d, J ¼ 2.56 Hz, 1H); 7.11 (s,
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4.3.3.16. 2-Fluoro-6-(2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetoxy)benzoic acid (5p). White powder. Yield: 70%. M.p.:
185e187 ꢀC 1H NMR (300 MHz, d6-DMSO): 3.86 (s, 3H); 5.33 (s, 2H);
6.86e6.91 (d, J ¼ 2.47 Hz, 1H); 7.11 (s, 1H); 7.47e7.56 (m, 3H);
7.65e7.73 (m, 2H); 8.09e8.17 (m, 2H); 12.81 (s, 1H). MS (ESI): 451.1
(C24H16FO8, [M þ H]þ). Anal. Calcd for C24H15FO8: C, 64.00; H,
3.36%; Found: C, 64.08; H, 3.41%.
4.3.3.17. 2-Chloro-6-(2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetoxy)benzoic acid (5q). White powder. Yield: 72%. M.p.:
190e192 ꢀC 1H NMR (300 MHz, d6-DMSO): 3.87 (s, 3H); 5.30 (s,
2H); 6.74e6.87 (d, J ¼ 2.56 Hz, 1H); 7.13 (s, 1H); 7.55e7.62 (m, 3H);
7.69e7.76 (m, 2H); 8.17e8.24 (m, 2H); 12.85 (s, 1H). MS (ESI): 467.1
(C24H16ClO8, [M þ H]þ). Anal. Calcd for C24H15ClO8: C, 61.75; H,
3.24%; Found: C, 61.82; H, 3.31%.
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4.3.3.18. 2-Bromo-6-(2-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)acetoxy)benzoic acid (5r). White powder. Yield: 69%. M.p.:
183e184 ꢀC 1H NMR (300 MHz, d6-DMSO): 3.86 (s, 3H); 5.32 (s,
2H); 6.79e6.86 (d, J ¼ 2.43 Hz, 1H); 7.11 (s, 1H); 7.46e7.56 (m, 3H);
7.64e7.77 (m, 2H); 8.21e8.34 (m, 2H); 12.87(s, 1H). MS (ESI): 511.1