Synthesis of a novel series of diphenolic chromone derivatives as inhibitors of NO production in LPS-activated RAW264.7 macrophages

Article abstract of DOI:10.1016/j.bmc.2010.03.020

A novel series of diphenolic chromone derivatives were synthesized and their inhibitory activity on nitric oxide (NO) production and cytotoxicity were evaluated using LPS-activated murine macrophages RAW264.7 assay and MTT method, respectively. Among these compounds, (5,7-dihydroxy-4-oxo-4H-chromen-3-yl) methyl esters (6b, 6c, 6f, 6g, and 6h) showed quite potent inhibitory activities with IC₅₀ values of 2.20, 3.48, 0.35, 0.80, and 0.61 μM, respectively. The MTT results showed that all of the active compounds exhibited no cytotoxicity at the effective concentrations. The preliminary mechanism of the most potent compounds (6b, 6c, 6f, 6g, and 6h) was further examined based on the RT-PCR results and the compounds 6f, 6g, and 6h inhibited NO production by suppressing the expression of iNOS mRNA in a dose dependent manner. Furthermore, a computational analysis of physicochemical parameters revealed that the most of the compounds possessed drug-like properties.

Full text of DOI:10.1016/j.bmc.2010.03.020

Bioorganic & Medicinal Chemistry 18 (2010) 2864–2871
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of a novel series of diphenolic chromone derivatives as inhibitors
of NO production in LPS-activated RAW264.7 macrophages
Guo-Biao Liu a, Jian-Liang Xu b, Mei Geng a, Rui Xu b, Rong-Rong Hui a, Jian-Wei Zhao a, Qiang Xu b,
Hong-Xi Xu c, Jian-Xin Li a,
*
a Key Lab of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China
b State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China
c Chinese Medicine Lab, Hong Kong Jockey Club Institute of Chinese Medicine, Hong Kong, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of diphenolic chromone derivatives were synthesized and their inhibitory activity on nitric
oxide (NO) production and cytotoxicity were evaluated using LPS-activated murine macrophages
RAW264.7 assay and MTT method, respectively. Among these compounds, (5,7-dihydroxy-4-oxo-4H-
chromen-3-yl) methyl esters (6b, 6c, 6f, 6g, and 6h) showed quite potent inhibitory activities with IC50
Received 27 January 2010
Revised 8 March 2010
Accepted 9 March 2010
Available online 12 March 2010
values of 2.20, 3.48, 0.35, 0.80, and 0.61 lM, respectively. The MTT results showed that all of the active
compounds exhibited no cytotoxicity at the effective concentrations. The preliminary mechanism of the
most potent compounds (6b, 6c, 6f, 6g, and 6h) was further examined based on the RT-PCR results and
the compounds 6f, 6g, and 6h inhibited NO production by suppressing the expression of iNOS mRNA in a
dose dependent manner. Furthermore, a computational analysis of physicochemical parameters revealed
that the most of the compounds possessed drug-like properties.
Keywords:
Diphenolic chromone
Derivative
Nitric oxide
iNOS
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Nitric oxide (NO), an endogenous free radical is an important
signaling molecule involved in a wide range of physiological func-
tions, as well as pathophysiological states.1 NO is generated from
L-arginine by a family of nitric oxide synthases (NOSs) including
major of isozymes, endothelial NOS (eNOS), neuronal NOS (nNOS),
and inducible NOS (iNOS).2,3 Both eNOS and nNOS are constitu-
tively expressed and produce NO at a low level. However, iNOS is
often expressed at high levels and is essentially unregulated once
expressed. If activated by many immunological stimuli such as
lipopolysaccharide (LPS), interferon (IFN-c) and a variety of pro-
inflammatory cytokines, iNOS produces a high level of NO to exert
defense against pathogens.4–6 It is well known that NO plays a
major role in anti-inflammatory and immune reactions, however,
an extremely high level of NO induced by iNOS causes inflamma-
tory diseases such as rheumatoid arthritis.7 Therefore, as drug
development targets, inhibitors of NO overproduction and overex-
pression of iNOS might be beneficial for treatment of inflammatory
disorders caused by excessive production of NO.
Figure 1. Some natural and synthetic flavonoids.
expression of iNOS.8–12 Hydroxyl groups might be effective moiety
to improve the potency of NO inhibitory activity for this type of
compounds. Chromone skeleton, very similar with flavonoid struc-
ture, is a class of heterocyclic compounds which possess a variety
of biological effects, such as antioxidant,13,14 anti-inflamma-
tory,15,16 antibacterial,17 antiviral,18 antitumor,19 and HIV-inhibi-
tory20,21 activities. Our previous data revealed that eucryphin, a
Many natural and synthetic flavonoids, such as flavones, isoflav-
ones and azaisoflavones with several hydroxyl groups on their
skeletons (Fig. 1), possess inhibitory effect on NO production and
* Corresponding author. Tel./fax: +86 25 83686419.
E-mail address: lijxnju@nju.edu.cn (J.-X. Li).
chromone rhamnoside possessed
a more potent activity on
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.020

G.-B. Liu et al. / Bioorg. Med. Chem. 18 (2010) 2864–2871
2865
preventing hepatocyte damage caused by inflammatory and
immunological reactions compared with several flavonoids.22
Moreover, it has also been reported that the conjugates of various
pharmacophores with fatty acids, aromatic acids or sulfonic acids
could provide effective bioactivity, and different fatty chain lengths
and different aromatic substituents could also impact the activ-
ity.23–25 These results give us a clue that increasing the number
of hydroxyl groups and different substituents in chromone skele-
ton might improve the NO inhibitory activity. However, chro-
mones, especially multi-hydroxyl chromones together with NO
inhibitory activity were reported rarely. With the aim of exploring
new chemical pharmacophores with anti-inflammatory activity,
we chose to focus on multi-hydroxyl chromone derivatives.
In this paper, a series of diphenolic chromone derivatives were
synthesized. The NO inhibitory activity of the synthesized com-
pounds was evaluated with LPS-activated macrophages assay and
their cytotoxicity was tested using MTT method. The preliminary
mechanism of some compounds with potent activity was exam-
ined by reverse transcription-polymerase chain reaction (RT-PCR)
analysis. A computational calculation on the physicochemical
parameters of all the compounds was also conducted to under-
stand their drug-like properties.
2. Results and discussion
2.1. Chemistry
The syntheses of diphenolic chromone derivatives are outlined
in Scheme 1. Selective protection of two hydroxyl groups of the
commercially available and cheap starting material 2,4,6-trihy-
Scheme 1. Reagents and conditions: (a) BnCl, K2CO3, HMPA, 90 °C, 3 h, 75%; (b) (i) HCOOEt, NaH, ꢀ10 °C, 4 h; (ii) concd HCl, rt, 12 h, 72%; (c) (i) KOH, PhI(OAc)2, MeOH, THF,
0 °C, 5 h; (ii) concd HCl, acetone, rt, 3 h, 62%; (d) POCl3, DMF, 0 °C to rt, 12 h, 70%; (e) basic alumina, i-propanol, 80 °C, 5 h, 68%; (f) NaClO2, NH2SO3H, CH2Cl2, H2O, 0 °C, 1 h,
92%; (g) RCOOH, EDCI, DMAP, CH2Cl2, rt, 6 h; (h) RSO2Cl, Et3N, CH2Cl2, rt, 5 h; (i) oxalyl chloride, CH2Cl2, rt, 6 h; (ii) ROH, Et3N, CH2Cl2, rt, 2 h; (j) Pd/C, cyclohexene, MeOH, THF,
reflux, 6 h, one step yield 85–92% (for 4a, 6a, and 7a); two steps yield 50–83% (for other compounds).

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G.-B. Liu et al. / Bioorg. Med. Chem. 18 (2010) 2864–2871
Table 1
droxyacetophenone (1) was carried out via reaction with benzyl
chloride in hexamethylphosphotriamide (HMPA) to give 2 in good
yield (75%) following recrystallization from CH2Cl2/MeOH.26 Con-
densation of compound 2 with ethyl formate in the presence of so-
dium hydride, followed by dehydration of the resulting chromanol
utilizing concentrated hydrochloric acid furnished 3 in an overall
yield of 72%.27 3-Hydroxychromone can be prepared through the
oxidation of an appropriate chromone derivative using an oxidiz-
ing agent, such as m-chloroperoxybenzoic acid (MCPBA),28 NbCl5,29
or PhI(OAc)2.30 In recent years, the use of hypervalent iodine com-
pounds in synthetic organic chemistry has gained attention be-
cause of their high selectivity, therefore, a hypervalent iodine
reagent PhI(OAc)2 was used in the present reaction. Mild oxidation
in basic solution followed by acidification provided target com-
pound 3-hydroxychromone (4). Elucidation of the structure of 4
was facilitated by the presence of a characteristic singlet associated
with the 2-H, as opposed to a doublet in compound 3.
Compound 5 was prepared via Vilsmeier–Haack formylation of
compound (2) using POCl3/DMF as formylation reagents in a mod-
erate yield (70%).31 Compound 5 was a key intermediate and could
undergo both reduction and oxidation. Reduction of 5 was carried
out in a suspension of basic alumina and isopropanol to get 6 in
68% yield. This reaction is the reverse process of Oppenauer oxida-
tion, and increasing of the amount of isopropanol could be benefi-
cial for the reduction yield.32 Oxidation of 5 using sodium chlorite
in the presence of aminosulfonic acid provided 7 in a satisfactory
yield (92%) after a recrystallization in MeOH.33 The structures of
compounds 5, 6, and 7 could be easily determined by their charac-
teristic proton signals in 1H NMR spectra, and the singlet peak of
CHO at 10.38 ppm for 5, the two hydrogen atoms of CH2OH at
4.50 ppm for 6 and the broad peak of COOH at 14.26 ppm for 7
were clearly displayed. The protective groups (Bn) of 4, 6, and 7
were removed by catalytic hydrogenolysis using Pd/C as a catalyst,
cyclohexene as a hydrogen donor and THF as an auxiliary solvent
to yield 4a, 6a, and 7a, respectively.
In order to understand the influences on activity, esterifications
of the hydroxyl and carboxyl of chromone were conducted and the
corresponding esters of 4a, 6a, and 7a were prepared, respectively.
EDC/DMAP-mediated esterification is commonly used in organic
synthesis due to its highly reactive performance and the easy re-
moval of the condensing byproduct of EDC.34,35 Thus, as shown
in Scheme 1, esterifications of compounds 4 or 6 with different
fatty acids or aromatic acids using 1-ethyl-3-(3-dimethyllamino-
propyl)carbodiimide hydrochloride (EDCI) as a condensing agent
and 4-dimethylaminopyridine (DMAP) as a catalyst afforded corre-
sponding esters. Compound 4 was also reacted with substituted
sulfonyl chlorides to yield the sulfonyl esters. While compound 7
was first converted to acyl chloride using oxalyl chloride which
was reacted with different alcohols to yield the corresponding es-
ters. These esters of 4, 6, and 7 were deprotected by catalytic
hydrogenolysis to obtain the final products 4b–4j, 6b–6d, 6f–6h,
and 7b–7f, respectively.
Inhibitory activities on the NO production in LPS-activated RAW264.7 macrophages
and cytotoxicity of diphenolic chromone derivatives
a
Compd
Activity IC50
(
Cytotoxicity IC50
M)
(
l
M)
l
g/mL)
(l
4a
4b
4c
4d
4e
4f
4g
4h
4i
>100
60.01
60.36
62.25
>100
86.56
47.20
16.97
19.22
53.71
>100
2.20
3.48
29.16
0.35
0.80
0.61
>19.40
14.16
16.78
20.80
>46.03
25.79
15.48
5.29
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
67.38
80.21
81.58
54.69
51.76
55.61
>100
>100
>100
>100
>100
>100
6.69
4j
14.61
>20.80
0.55
1.02
10.15
0.11
6a
6b
6c
6d
6f
6g
6h
7a
7b
7c
7d
7e
7f
0.27
0.20
>100
>100
>100
58.80
24.91
>100
>22.20
>25.00
>27.81
16.35
8.32
>47.43
a
Cytotoxicity IC50: the concentration of inhibition of 50% cell growth.
The fatty acids esters (4b–4d) of 4a showed similar inhibitory
activity, with IC50 60.01, 60.36, and 62.25
lM, respectively, but
compound 4e showed no effect on NO production. This result sug-
gested that the appropriate length of the fatty chains almost did
not affect the inhibitory activity to some extent (4b–4d), but the
extremely long chain length might decrease the activity (4e). Most
of the esters of 4a with aromatic acids showed a better activity
than fatty acids derivatives, and the activity of substituted aro-
matic acids modifiers (4g and 4h, with IC50 47.20 and 16.97
respectively) were higher than that of non-substituted aromatic
acid one (4f, IC50 86.56 M). The aromatic sulfonic acid modifier
4i (IC50 19.22 M) exhibited better activity than fatty sulfonic acid
lM,
l
l
modifier 4j (IC50 53.71 lM), which was consistent with the result
above.
The esters of 6a exhibited quite potent inhibitory activity com-
pared with the derivatives of 4a. Interestingly, unlike the case of 4a
derivatives, the length of fatty acids was closely related to the
inhibitory activity for the esters of 6a. As the chain length in-
creased, the activity of fatty acids modifiers decreased obviously
(6b > 6c > 6d, with IC50 2.20, 3.48, and 29.16 lM, respectively).
While the aromatic acids modifiers (6f–6h) of 6a displayed very
strong inhibitory effect on NO production. In contrast to 4a deriv-
atives, the non-substituted aromatic acid modifier (6f, IC50
0.35
l
M) showed better inhibitory effect than the substituted aro-
matic acids ones (6g and 6h, with IC50 0.80 and 0.61
lM). From all
2.2. Biological activity
of the derivatives, the three compounds (6f, 6g, and 6h) displayed
much more potent activity than others.
The inhibitory effect of the synthesized diphenolic chromone
derivatives on nitric oxide production was evaluated using LPS-
activated murine macrophages-like RAW264.7 cell culture sys-
tems. The cytotoxicity of these compounds was checked using
MTT assay. The results are summarized in Table 1.
As can been seen from Table 1, compounds 4a, 6a, and 7a did
not show obvious inhibitory activity, while most of the esters of
4a, 6a, and 7a exhibited a great improvement of the activity when
compared to the corresponding parent compounds. This result re-
vealed that the esterification of diphenolic chromone was benefi-
cial in elevating activity.
Among the esters of 7a, only 7d and 7e exhibited moderate
inhibitory activity, while other compounds were inactive, suggest-
ing that the carboxyl group is inimical to inhibitory activity.
The results cited above revealed that the 3-CH2OH group of
chromone skeleton, an appropriate length of fatty chain and aro-
matic substituents play a critical role in the enhancement of activ-
ity and might be effective moieties to improve the potency of
inhibitory activity.
It is worthy to mention that the molecular weight of this series
of diphenolic chromone derivatives was low (about 300). As can be
seen in Table 1, when expressed in the unit of
lg/mL, the IC50

G.-B. Liu et al. / Bioorg. Med. Chem. 18 (2010) 2864–2871
2867
values of the compounds decreased to about 1/3 of the former val-
ues. The active compounds 6f, 6g, and 6h displayed noteworthy
IC50 values of 0.11, 0.27, and 0.20 lg/mL, respectively.
From the MTT results, the cytotoxicity IC50 (the concentration of
inhibition of 50% cell growth) values of all the synthesized com-
pounds were above 50 lM, some compounds even to 100 lM,
The electronic properties such as HOMO, LUMO, and GAP en-
ergy values of all the compounds were very similar and no correla-
tion with the activity values was observed. However, the most
active compounds (6f, 6g, and 6h) showed higher dipole values
than the rest of the compounds, suggesting that dipole might be
an important factor concerned with activity.
which were much larger than those of inhibitory activity IC50. It
could be concluded that all the compounds displayed no obvious
cytotoxicity at the effective concentrations.
‘Lipinski rule of 5’ is very important in drug design and bioavail-
ability prediction. Molecules violating more than one of these rules
may have problems with bioavailability.36 As shown in Table 2, all
parameters of the compounds were consistent with the ‘rule of 5’ ex-
cept the miLog P values of 4e and 7f, which possessed extremely
long chain alkyl substituent. It was reported that a molecule with
ten or fewer rotatable bonds and PSA less than 140 Å2 might have
a high probability of good oral bioavailability.37 As can be seen in Ta-
ble 2, all the compounds fulfilled both of the criteria except com-
pounds 4e and 7f. From the calculation results and the biological
activity above, it could be concluded that extremely long alkyl chain
is not suitable for improving the activity, and miLog P at the range of
2.7–2.9 and a big dipole value might relate with a potent activity.
Because the high level of NO is mainly generated by iNOS, iNOS
expression is one of the key steps during the process of LPS-activated
NO production. To further study the mechanism of the active com-
pounds, the RT-PCR experiment was applied to assess the effect of
the active compounds on mRNA expression of iNOS. As can be seen
in Figure 2, the aromatic acids derivatives (6f, 6g, and 6h) of 6a
strongly inhibited the expression of iNOS mRNA in a dose dependent
manner. However, the two derivatives (6b and 6c) did not suppress
the expression of iNOS, suggesting that the two compounds might
inhibit NO production through other mechanisms.
2.3. In silico study
3. Conclusion
In order to understand the drug-like properties of the com-
We have synthesized a series of diphenolic chromone derivatives
with inhibitory effect on overproduction of NO and overexpression
of iNOS in LPS-activated RAW264.7 macrophages. Esterification of
diphenolic chromone could be beneficial to elevate the activity.
Most of the esters of 6a, including fatty acids esters (6b and 6c)
and aromatic acids esters (6f, 6g, and 6h), showed quite potent
inhibitory activity and no cytotoxicity at effective concentrations.
The most active compounds (6f, 6g, and 6h) inhibited NO production
by suppressing the expression of iNOS in a dose dependent manner.
The physicochemical parameters suggested that most of the com-
pounds possessed drug-like properties. The present results revealed
that the diphenolic chromone derivatives as a new class of anti-
inflammatory leads warrant further studies. Further design and syn-
thesis of related compounds, and the in vivo activity together with
detailed mechanism of 6f, 6g, and 6h are in progress in our lab.
pounds,
a preliminary assessment of their geometrical and
electronic structures and physicochemical parameters was con-
ducted. A density functional theory calculation was performed at
*
DFT:B3LYP/6-31G level for the determination of geometrical and
electronic structures including EHOMO, ELUMO, GAP, and dipole.
The final RMS gradient less than 0.01 kcal/mol was achieved for
all fully optimized molecules, which was performed using
Gaussian 03 software. The physicochemical parameters including
miLog P, Mw, HBD, HBA, TPSA, and Rotatable bonds were
calculated using molinspiration server (http://www.molinspira
tion.com/cgi-bin/properties). All the calculated results are summa-
rized in Table 2.
4. Experimental
4.1. Synthesis
4.1.1. General
All reagents and solvents were commercially available and used
without further purification. Melting points were determined on a
Taike X-4 digital micromelting point apparatus and uncorrected.
1H and 13C NMR spectra were taken on a Bruker DPX-300 spectrom-
eter, using TMS as an internal standard (chemical shifts in d). ESI-MS
were obtained on ThermoFisher LCQ Fleet mass spectrometer. ESI-
HR-MS were obtained on Esquire 4000 mass spectrometer.
4.1.2. 1-(2,4-Bis(benzyloxy)-6-hydroxyphenyl)ethanone (2)
To a solution of 1 (60 g, 0.36 mol) in HMPA (300 mL) was added
K2CO3 (148 g, 1.07 mol) and BnCl (86.3 mL, 0.75 mol), and the sus-
pension was stirred at 90 °C for 3 h. The solid was filtered, and the
filtrate was poured into ice-water. The pH of the solution was ad-
justed to 2 by adding diluted hydrochloric acid and the resulting
yellow solid was filtered and recrystallized in CH2Cl2/MeOH to give
2. Yield: 75%; 1H NMR (CDCl3, 300 MHz) d: 2.56 (3H, s), 5.06 (4H, s),
6.11 (1H, s), 6.17 (1H, s), 7.41 (10H, m), 14.04 (1H, s); HRMS-ESI
(m/z): calcd for C22H21O4 [M+H]+: 349.1440 found: 349.1446.
4.1.3. 5,7-Bis(benzyloxy)-4H-chromen-4-one (3)
To a solution of 2 (8.0 g, 23.0 mmol) in HCOOEt (70 mL) at
ꢀ10 °C was added NaH (8.0 g, 60%, 200 mmol) in batches, and
Figure 2. The RT-PCR analysis of iNOS mRNA for compounds 6b–6c, 6f–6h.

2868
G.-B. Liu et al. / Bioorg. Med. Chem. 18 (2010) 2864–2871
Table 2
Calculated physicochemical properties of diphenolic chromone derivatives
Compd
EHOMO (eV)
ELUMO (eV)
GAPa (eV)
Dipole (debye)
Lipinski rule of 5
TPSAe (Å2)
Rotatable bonds
miLog Pb
Mw
HBDc
HBAd
4a
4b
4c
4d
4e
4f
4g
4h
4i
ꢀ0.215
ꢀ0.225
ꢀ0.224
ꢀ0.224
ꢀ0.222
ꢀ0.228
ꢀ0.220
ꢀ0.227
ꢀ0.235
ꢀ0.234
ꢀ0.231
ꢀ0.231
ꢀ0.231
ꢀ0.231
ꢀ0.232
ꢀ0.222
ꢀ0.233
-0.233
ꢀ0.035
ꢀ0.040
ꢀ0.039
ꢀ0.039
ꢀ0.039
ꢀ0.042
ꢀ0.041
ꢀ0.040
ꢀ0.050
ꢀ0.047
ꢀ0.045
ꢀ0.043
ꢀ0.043
ꢀ0.042
ꢀ0.043
ꢀ0.041
ꢀ0.044
ꢀ0.051
ꢀ0.047
ꢀ0.046
ꢀ0.047
ꢀ0.046
ꢀ0.046
0.180
0.185
0.185
0.185
0.183
0.186
0.179
0.187
0.186
0.187
0.186
0.188
0.188
0.188
0.190
0.181
0.189
0.182
0.182
0.182
0.175
0.182
0.182
5.139
3.148
2.996
2.948
2.968
3.729
1.787
3.625
3.655
5.637
5.863
4.372
4.028
3.982
6.026
6.910
5.723
4.992
4.117
4.018
5.929
3.979
3.977
0.732
0.504
2.237
4.258
8.590
2.820
2.876
2.882
2.365
0.383
0.289
0.993
2.418
4.439
2.717
2.774
2.779
0.524
1.160
2.222
1.969
4.242
8.830
194.142
236.179
278.260
334.368
460.611
298.250
328.276
312.277
348.332
272.234
208.169
250.206
292.287
348.395
312.277
342.303
326.304
222.152
250.206
278.260
278.260
334.368
474.638
3
2
2
2
2
2
2
2
2
2
3
2
2
2
2
2
2
3
2
2
2
2
2
5
6
6
6
6
6
7
6
7
7
5
6
6
6
6
7
6
6
6
6
6
6
6
90.895
96.972
96.972
96.972
96.972
96.972
106.206
96.972
114.043
114.043
90.895
96.972
96.972
96.972
96.972
106.206
96.972
107.966
96.972
96.972
96.972
96.972
96.972
0
2
5
9
18
3
4
3
3
2
1
3
6
10
4
5
4
1
3
4j
6a
6b
6c
6d
6f
6g
6h
7a
7b
7c
7d
7e
7f
ꢀ0.229
ꢀ0.229
ꢀ0.221
ꢀ0.228
ꢀ0.228
5
3
9
19
a
b
c
GAP = ELUMO ꢀ EHOMO
.
miLog P: the log P value calculated using molinspiration server.
HBD: hydrogen bond donor (expressed as the sum of OH and NH).
HBA: hydrogen bond acceptor (expressed as the sum of O and N atoms).
d
e
TPSA: topological polar surface area (defined as a sum of surfaces of polar atoms in a molecule).
the suspension was stirred for 4 h. The reaction was quenched by
the addition of methanol. Subsequently concentrated hydrochloric
acid (20 mL) was added, and the suspension was stirred overnight.
Water (50 mL) was added, and the aqueous layer was extracted
with CH2Cl2 (3 ꢁ 100 mL), the combined organic layer was washed
with brine and dried over anhydrous Na2SO4. Solvent was evapo-
rated in vacuo and the residue was recrystallized in methanol to
provide 3. Yield: 72%; 1H NMR (CDCl3, 300 MHz) d: 5.09 (2H, s),
5.21 (2H, s), 6.19 (1H, d, J = 6.0 Hz), 6.49 (1H, d, J = 2.4 Hz), 6.52
(1H, d, J = 2.4 Hz), 7.30–7.42 (8H, m), 7.60 (1H, d, J = 6.0 Hz), 7.61
(2H, d, J = 7.8 Hz); ESI-MS (m/z): 381 [M+Na]+; HRMS-ESI (m/z):
calcd for C23H19O4 [M+H]+: 359.1283 found: 359.1288.
layer was washed with brine, dried over anhydrous Na2SO4 and
concentrated in vacuo. The residue was purified through chroma-
tography eluting with CH2Cl2/EtOAc (25/1) to give 5. Yield: 70%;
1H NMR (CDCl3, 300 MHz) d: 5.11 (2H, s), 5.22 (2H, s), 6.56 (1H,
d, J = 2.1 Hz), 6.58 (1H, d, J = 2.1 Hz), 7.42 (8H, m), 7.59 (2H, d,
J = 7.5 Hz), 8.32 (1H, s), 10.38 (1H, s); ESI-MS (m/z): 387 [M+H]+;
HRMS-ESI (m/z): calcd for C24H19O5 [M+H]+: 387.1232 found:
387.1237.
4.1.6. 5,7-Bis(benzyloxy)-3-(hydroxymethyl)-4H-chromen-4-one
(6)
To a solution of 5 (3.0 g, 7.8 mmol) in isopropanol (150 mL) un-
der N2 atmosphere was added basic Al2O3 (30.0 g, 294 mmol). The
suspension was stirred at 80 °C for 4 h. The solid was filtered,
washed with hot isopropanol (3 ꢁ 50 mL) and the filtrate was con-
centrated in vacuo to afford 6. Yield: 68%; 1H NMR (CDCl3,
300 MHz) d: 4.50 (2H, s), 5.07 (2H, s), 5.21 (2H, s), 6.49 (1H, d,
J = 2.1 Hz), 6.51 (1H, d, J = 2.1 Hz), 7.41 (8H, m), 7.58 (2H, d,
J = 7.5 Hz), 7.70 (1H, s); ESI-MS (m/z): 389 [M+H]+, 411 [M+Na]+;
HRMS-ESI (m/z): calcd for C24H21O5 [M+H]+: 389.1389 found:
389.1396.
4.1.4. 5,7-Bis(benzyloxy)-3-hydroxy-4H-chromen-4-one (4)
To a solution of KOH (2.4 g, 42.8 mmol) in anhydrous methanol
(60 mL) was added a solution of 3 (3.2 g, 8.9 mmol) in anhydrous
THF (25 mL) dropwise and the solution was stirred for 1 h.
PhI(OAc)2 (4.0 g, 12.4 mmol) was added in batches, and the sus-
pension was stirred for 5 h. The reaction mixture was concentrated
in vacuo, and water (50 mL) was added. The aqueous layer was ex-
tracted with CH2Cl2 (3 ꢁ 100 mL). The combined organic layer was
dried over anhydrous MgSO4 and concentrated in vacuo. The resi-
due was dissolved in acetone (5 mL), and concentrated hydrochlo-
ric acid (2 mL) was added. The suspension was stirred for 3 h at rt,
and the resulting solid was filtered, washed with cold acetone and
dried to get 4. Yield: 62%; 1H NMR (CDCl3, 300 MHz) d: 5.10 (2H, s),
5.23 (2H, s), 6.49 (1H, d, J = 2.4 Hz), 6.52 (1H, d, J = 2.4 Hz), 7.32–
7.42 (8H, m), 7.60 (2H, d, J = 7.5 Hz), 7.78 (1H, s); ESI-MS (m/z):
397 [M+Na]+; HRMS-ESI (m/z): calcd for C23H19O5 [M+H]+:
375.1232 found: 375.1239.
4.1.7. 5,7-Bis(benzyloxy)-4-oxo-4H-chromene-3-carboxylic acid
(7)
To a solution of 5 (4.0 g, 10.4 mmol) in CH2Cl2 (160 mL) at 0 °C
was added an aqueous solution (120 mL) of NH2SO3H (5.0 g,
51.5 mmol). An aqueous solution (80 mL) of NaClO2 (4.7 g, 80%,
41.6 mmol) was added dropwise. The suspension was stirred
for 30 min. The aqueous layer was extracted with CH2Cl2 (3 ꢁ
200 mL). The organic layer was washed with brine, dried over anhy-
drous Na2SO4 and concentrated in vacuo. The residue was recrystal-
lized in MeOH to afford 7. Yield: 92%; 1H NMR (CDCl3, 300 MHz) d:
5.14 (2H, s), 5.24 (2H, s), 6.61 (1H, d, J = 2.1 Hz), 6.65 (1H, d,
J = 2.1 Hz), 7.42 (8H, m), 7.56 (2H, d, J = 7.2 Hz), 8.78 (1H, s), 14.26
(1H, br); ESI-MS (m/z): 403 [M+H]+, 425 [M+Na]+; HRMS-ESI (m/z):
calcd for C24H19O6 [M+H]+: 403.1181 found: 403.1186.
4.1.5. 5,7-Bis(benzyloxy)-4-oxo-4H-chromene-3-carbaldehyde (5)
To a solution of 2 (15.0 g, 43 mmol) in DMF (100 mL) was added
POCl3 (19.5 mL, 213 mmol) dropwise at 0 °C and the solution was
stirred at rt for 12 h. The reaction mixture was poured into ice-
water, and resulting solid was dissolved in CH2Cl2. The organic

G.-B. Liu et al. / Bioorg. Med. Chem. 18 (2010) 2864–2871
2869
4.1.8. General procedure for compounds 4a, 6a, and 7a
To a solution of 4 (6 or 7, 0.5 mmol) in a mixed solution of
MeOH (5 mL), THF (5 mL) and cyclohexene (2 mL) was added Pd/
C (212 mg, 10%, 0.2 mmol). The suspension was refluxed for 8 h.
Pd/C was filtered, and the filtrate was concentrated in vacuo. The
crude product was purified through chromatography to give 4a,
6a, and 7a.
106.07, 134.97, 148.51, 157.66, 162.17, 163.06, 172.16, 175.62;
HRMS-ESI (m/z): calcd for C18H23O6 [M+H]+: 335.1495 found:
335.1491.
4.1.9.4. 5,7-Dihydroxy-4-oxo-4H-chromen-3-yl stearate (4e).
Yield 72%; mp 127–128 °C; 1H NMR (CDCl3, 300 MHz) d: 0.88
(3H, t, J = 6.0 Hz), 1.26 (28H, m), 1.75 (2H, m), 2.63 (2H, t,
J = 7.2 Hz), 6.24 (1H, s), 6.28 (1H, s), 7.84 (1H, s), 11.95 (1H, s);
13C NMR (CDCl3, 75 MHz) d: 14.26, 22.84, 24.87, 29.16, 29.85,
32.07, 33.82, 94.72, 99.96, 106.24, 135.13, 148.69, 157.80, 162.32,
163.20, 172.42, 175.77; HRMS-ESI (m/z): calcd for C27H41O6
[M+H]+: 461.2903 found: 461.2904.
4.1.8.1. 3,5,7-Trihydroxy-4H-chromen-4-one (4a). Yield: 85%;
mp 258–260 °C; 1H NMR (C5D5N, 300 MHz) d: 6.66 (1H, d,
J = 2.1 Hz), 6.73 (1H, d, J = 2.1 Hz), 8.17 (1H, s), 13.24 (1H, br); 13C
NMR (C5D5N, 75 MHz) d: 96.09, 101.04, 107.16, 142.61, 143.69,
160.09, 164.41, 167.17, 179.63; HRMS-ESI (m/z): calcd for C9H7O5
[M+H]+: 195.0293 found: 195.0287.
4.1.9.5. 5,7-Dihydroxy-4-oxo-4H-chromen-3-yl benzoate (4f).
Yield 68%; mp 191–193 °C; 1H NMR (CD3COCD3, 300 MHz) d:
6.33 (1H, d, J = 2.1 Hz), 6.49 (1H, d, J = 2.1 Hz), 7.60 (2H, t,
J = 7.5 Hz), 7.75 (1H, t, J = 7.5 Hz), 8.16 (2H, d, J = 7.5 Hz), 8.45
(1H, s), 12.17 (1H, s); 13C NMR (CD3COCD3, 75 MHz) d: 95.21,
100.13, 106.00, 129.29, 129.74, 130.94, 135.00, 136.18, 150.55,
158.94, 163.02, 163.26, 164.53, 165.58, 176.39; HRMS-ESI (m/z):
calcd for C16H11O6 [M+H]+: 299.0556 found: 299.0563.
4.1.8.2. 5,7-Dihydroxy-3-(hydroxymethyl)-4H-chromen-4-one
(6a). Yield: 88%; mp 208–210 °C; 1H NMR (CD3OD, 300 MHz) d:
4.46 (2H, s), 6.19 (1H, d, J = 2.0 Hz), 6.31 (1H, d, J = 2.0 Hz), 7.99
(1H, s); 13C NMR (CD3OD, 75 MHz) d: 56.64, 94.91, 100.08,
105.95, 123.36, 155.36, 159.90, 163.50, 166.33, 182.60; HRMS-ESI
(m/z): calcd for C10H9O5 [M+H]+: 209.0450 found: 209.0449.
4.1.8.3. 5,7-Dihydroxy-4-oxo-4H-chromene-3-carboxylic acid
(7a). Yield: 92%; mp 293–294 °C; 1H NMR (DMSO-d6, 300 MHz)
d: 6.27 (1H, d, J = 2.1 Hz), 6.45 (1H, d, J = 2.1 Hz), 8.88 (1H, s),
11.15 (1H, br), 12.35 (1H, br); 13C NMR (DMSO-d6, 75 MHz) d:
94.69, 100.05, 104.36, 113.43, 157.51, 161.89, 163.29, 163.49,
165.10, 178.96; HRMS-ESI (m/z): calcd for C10H7O6 [M+H]+:
223.0243 found: 223.0240.
4.1.9.6. 5,7-Dihydroxy-4-oxo-4H-chromen-3-yl 4-methoxyben-
zoate (4g). Yield 60%; mp 196–197 °C; 1H NMR (CD3COCD3,
300 MHz) d: 3.92 (3H, s), 6.32 (1H, d, J = 1.8 Hz), 6.48 (1H, d,
J = 1.8 Hz), 7.11 (2H, d, J = 9.0 Hz), 8.11 (2H, d, J = 9.0 Hz), 8.40
(1H, s), 12.21 (1H, br); 13C NMR (CD3COCD3, 75 MHz) d: 56.08,
95.15, 100.07, 106.04, 115.00, 121.34, 133.17, 136.23 150.51,
158.94, 163.04, 164.16, 165.34, 165.54, 176.59; HRMS-ESI (m/z):
calcd for C17H13O7 [M+H]+: 329.0661 found: 329.0659.
4.1.9. General procedure for compounds 4b–4h, 6b–6d, and 6f–
6h
4.1.9.7. 5,7-Dihydroxy-4-oxo-4H-chromen-3-yl 2-methylbenzo-
ate (4h). Yield: 64%; mp 190–192 °C; 1H NMR (CD3OD, 300 MHz)
d: 2.63 (3H, s), 6.26 (1H, d, J = 2.0 Hz), 6.40 (1H, d, J = 2.0 Hz), 7.35–
7.38 (2H, m), 7.53 (1H, td, J = 8.1, 1.5 Hz), 8.12 (1H, dd, J = 7.2,
1.5 Hz), 8.29 (1H, s); 13C NMR (CD3OD, 75 MHz) d: 22.71, 96.25,
101.31, 127.97, 129.71, 133.27, 133.81, 135.17, 137.47, 143.39,
151.79, 160.56, 164.33, 167.00, 167.39, 178.70; HRMS-ESI (m/z):
calcd for C17H12O6Na [M+Na]+: 335.0532 found: 335.0536.
To a solution of 4 or 6 (1.0 mmol) in CH2Cl2 (15 mL) was added
RCOOH (1.0 mmol), EDCI (230 mg, 1.2 mmol) and a few crystals of
DMAP. The solution was stirred at rt for 6 h. Diluted hydrochloric
acid (10 mL) was added, and the aqueous layer was extracted with
CH2Cl2 (3 ꢁ 20 mL). The organic layer was washed with diluted
hydrochloric acid, saturated NaHCO3, and brine, dried over anhy-
drous Na2SO4 and concentrated in vacuo. The residue was purified
through chromatography eluting with PE/EtOAc to give the corre-
sponding compound which was deprotected using the same proce-
dure as described for the preparation of 4a to give 4b–4h, 6b–6d,
and 6f–6h.
4.1.9.8. (5,7-Dihydroxy-4-oxo-4H-chromen-3-yl)methyl acetate
(6b). Yield: 66%; mp 189–191 °C; 1H NMR (C5D5N, 300 MHz) d:
2.04 (3H, s), 5.16 (2H, s), 6.63 (1H, d, J = 1.8 Hz), 6.72 (1H, d,
J = 1.8 Hz), 8.22 (1H, s), 13.18 (1H, br); 13C NMR (C5D5N, 75 MHz)
d: 21.11, 58.08, 95.39, 100.76, 105.74, 118.46, 156.98, 159.11,
163.52, 166.77, 171.26, 181.44; HRMS-ESI (m/z): calcd for
C12H11O6 [M+H]+: 251.0555 found: 251.0555.
4.1.9.1. 5,7-Dihydroxy-4-oxo-4H-chromen-3-yl acetate (4b). Yield:
68%; mp 296–297 °C; 1H NMR (CD3COCD3, 300 MHz) d: 2.28 (3H,
s), 6.30 (1H, d, J = 2.0 Hz), 6.44 (1H, d, J = 2.0 Hz), 8.25 (1H, s),
12.18 (1H, br); 13C NMR (CD3COCD3, 75 MHz) d: 19.17, 94.22,
99.19, 105.04, 135.09, 149.41, 157.99, 164.75, 167.79, 175.58;
HRMS-ESI (m/z): calcd for C11H9O6 [M+H]+: 237.0399 found:
237.0401.
4.1.9.9. (5,7-Dihydroxy-4-oxo-4H-chromen-3-yl)methyl pent-
anoate (6c). Yield: 63%; oil; 1H NMR (C5D5N, 300 MHz) d: 0.77
(3H, t, J = 7.4 Hz), 1.24 (2H, sxt, J = 7.4 Hz), 1.58 (2H, quint,
J = 7.4 Hz), 2.36 (2H, t, J = 7.4 Hz), 5.20 (2H, s), 6.64 (1H, d,
J = 2.1 Hz), 6.73 (1H, d, J = 2.1 Hz), 8.27 (1H, s), 13.20 (1H, br); 13C
NMR (C5D5N, 75 MHz) d: 14.17, 22.74, 27.53, 34.31, 57.99, 95.36,
100.72, 105.53, 118.53, 156.98, 159.14, 163.48, 166.73, 173.87,
181.34; HRMS-ESI (m/z): calcd for C15H17O6 [M+H]+: 293.1025
found: 293.1014.
4.1.9.2. 5,7-Dihydroxy-4-oxo-4H-chromen-3-yl pentanoate (4c).
Yield: 50%; mp 174–177 °C; 1H NMR (CD3COCD3, 300 MHz) d: 0.91
(3H, t, J = 8.0 Hz), 1.45 (2H, m), 1.66 (2H, m), 2.59 (2H, t, J = 6.0 Hz),
6.30 (1H, d, J = 2.0 Hz), 6.44 (1H, d, J = 2.0 Hz), 8.26 (1H, s), 12.20
(1H, s); 13C NMR (CD3COCD3, 75 MHz) d: 13.09, 21.77, 26.71,
32.68, 94.21, 99.13, 105.17, 149.36, 157.99, 162.13, 162.38,
164.51, 170.52; HRMS-ESI (m/z): calcd for C14H15O6 [M+H]+:
279.0869 found: 279.0863.
4.1.9.10. (5,7-Dihydroxy-4-oxo-4H-chromen-3-yl)methyl non-
anoate (6d). Yield: 69%; oil; 1H NMR (C5D5N, 300 MHz) d: 0.83
(3H, t, J = 6.8 Hz), 1.15–1.24 (10H, m), 1.64 (2H, quint, J = 7.5 Hz),
2.40 (2H, t, J = 7.5 Hz), 5.23 (2H, s), 6.64 (1H, d, J = 2.1 Hz), 6.73
(1H, d, J = 2.1 Hz), 8.29 (1H, s), 13.22 (1H, br); 13C NMR (C5D5N,
75 MHz) d: 14.61, 23.24, 25.60, 29.66, 29.73, 29.84, 32.34, 34.66,
57.99, 95.37, 100.74, 105.74, 118.57, 157.03, 159.11, 163.52,
4.1.9.3. 5,7-Dihydroxy-4-oxo-4H-chromen-3-yl nonanoate (4d).
Yield 54%; mp 119–121 °C; 1H NMR (CDCl3, 300 MHz) d: 0.88 (3H,
m), 1.28 (10H, m), 1.75 (2H, m), 2.64 (2H, t, J = 6.5 Hz), 6.26 (1H, s),
6.30 (1H, s), 7.85 (1H, s), 12.00 (1H, s); 13C NMR (CDCl3, 75 MHz) d:
14.12, 22.65, 24.71, 29.00, 29.09, 29.71, 31.79, 33.65, 94.57, 99.80,

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G.-B. Liu et al. / Bioorg. Med. Chem. 18 (2010) 2864–2871
166.78, 173.93, 181.45; HRMS-ESI (m/z): calcd for C19H25O6
acid chloride in CH2Cl2 (10 mL) was added corresponding ROH
(1.5 mmol) and Et3N (0.22 mL, 1.5 mmol). The reaction mixture
was stirred at rt for 2 h, and then concentrated and chromato-
graphed to yield the compound which was deprotected using the
same procedure as described for the preparation of 4a to give the
final product 7b–7f.
[M+H]+: 349.1651 found: 349.1637.
4.1.9.11. (5,7-Dihydroxy-4-oxo-4H-chromen-3-yl)methyl benzo-
ate (6f). Yield: 64%; mp 208–209 °C; 1H NMR (C5D5N, 300 MHz) d:
5.43 (2H, s), 6.66 (1H, d, J = 1.9 Hz), 6.74 (1H, d, J = 1.9 Hz), 7.40
(2H, t, J = 7.3 Hz), 7.52 (1H, t, J = 7.3 Hz), 8.21 (2H, d, J = 7.3 Hz),
8.38 (1H, s), 13.21 (1H, br); 13C NMR (C5D5N, 75 MHz) d: 58.81,
95.44, 100.78, 105.72, 118.38, 129.24, 130.40, 130.88, 133.88,
157.30, 159.28, 163.54, 166.77, 166.88, 181.46; HRMS-ESI (m/z):
calcd for C17H13O6 [M+H]+: 313.0712 found: 313.0708.
4.1.11.1. Ethyl 5,7-dihydroxy-4-oxo-4H-chromene-3-carboxyl-
ate (7b). Yield: 72%; mp 224–226 °C; 1H NMR (C5D5N, 300 MHz)
d: 1.24 (3H, t, J = 7.1 Hz), 4.31 (2H, q, J = 7.1 Hz), 6.30 (1H, d,
J = 2.1 Hz), 6.70 (1H, d, J = 2.1 Hz), 8.81 (1H, s), 13.41 (1H, br); 13C
NMR (C5D5N, 75 MHz) d: 15.54, 62.47, 96.61, 102.26, 106.81,
116.31, 159.18, 163.94, 164.90, 167.71, 179.97; HRMS-ESI (m/z):
calcd for C12H11O6 [M+H]+: 251.0556 found: 251.0544.
4.1.9.12. (5,7-Dihydroxy-4-oxo-4H-chromen-3-yl)methyl 4-meth-
oxybenzoate (6g). Yield: 64%; mp 186–188 °C; 1H NMR (C5D5N,
300 MHz) d: 3.68 (3H, s), 5.43 (2H, s), 6.66 (1H, d, J = 2.2 Hz),
6.74 (1H, d, J = 2.2 Hz), 7.01 (2H, d, J = 9.1 Hz), 8.23 (2H, d,
J = 9.1 Hz), 8.36 (1H, s), 13.23 (1H, br); 13C NMR (C5D5N, 75 MHz)
d: 55.81, 58.48, 95.40, 100.74, 105.72, 114.62, 118.57, 123.15,
132.52, 157.21, 159.17, 163.53, 164.37, 166.65, 166.72, 181.40;
HRMS-ESI (m/z): calcd for C18H15O7 [M+H]+: 343.0818 found:
343.0812.
4.1.11.2. Butyl 5,7-dihydroxy-4-oxo-4H-chromene-3-carboxyl-
ate (7c). Yield: 68%; mp 204–206 °C; 1H NMR (C5D5N, 300 MHz) d:
0.85 (3H, t, J = 7.4 Hz), 1.40 (2H, sxt, J = 7.4 Hz), 1.64 (2H, quint,
J = 6.7 Hz), 4.42 (2H, t, J = 6.7 Hz), 6.64 (1H, d, J = 2.1 Hz), 6.70
(1H, d, J = 2.1 Hz), 8.82 (1H, s), 13.40 (1H, br); 13C NMR (C5D5N,
75 MHz) d: 14.18, 19.79, 31.27, 65.43, 95.75, 101.41, 105.94,
115.61, 158.34, 162.97, 163.00, 164.06, 166.86, 179.11; HRMS-
ESI (m/z): calcd for C14H14O6Na [M+Na]+: 301.0688 found:
301.0685.
4.1.9.13. (5,7-Dihydroxy-4-oxo-4H-chromen-3-yl)methyl 2-meth-
ylbenzoate (6h). Yield: 67%; mp 212–214 °C; 1H NMR (C5D5N,
300 MHz) d: 2.66 (3H, s), 5.40 (2H, s), 6.66 (1H, d, J = 2.0 Hz),
6.73 (1H, d, J = 2.0 Hz), 7.20–7.26 (2H, m), 7.39 (1H, t, J = 7.5 Hz),
8.11 (1H, d, J = 7.5 Hz), 8.36 (1H, s), 13.20 (1H, br); 13C NMR
(C5D5N, 75 MHz) d: 22.12, 58.65, 95.48, 100.82, 105.70, 118.54,
126.59, 131.51, 132.48, 132.93, 141.15, 157.24, 159.32, 163.59,
166.80, 167.83, 174.84, 181.46; HRMS-ESI (m/z): calcd for
C18H15O6 [M+H]+: 327.0868 found: 327.0865.
4.1.11.3. tert-Butyl 5,7-dihydroxy-4-oxo-4H-chromene-3-car-
boxylate (7d). Yield: 65%; mp 252–254 °C; 1H NMR (C5D5N,
300 MHz) d: 1.59 (9H, s), 6.63 (1H, d, J = 1.9 Hz), 6.69 (1H, d,
J = 1.9 Hz), 8.71 (1H, s), 13.44 (1H, br); 13C NMR (C5D5N, 75 MHz)
d: 28.51, 82.23, 95.65, 101.27, 105.94, 116.92, 158.36, 162.39,
164.22, 166.75, 179.38; HRMS-ESI (m/z): calcd for C14H14O6Na
[M+Na]+: 301.0688 found: 301.0686.
4.1.10. General procedure for compounds 4i and 4j
To a solution of 4 (374 mg, 1.0 mmol) in CH2Cl2 (10 mL) was
added RSO2Cl (1.5 mmol) and Et3N (0.42 mL, 3.0 mmol). The solu-
tion was stirred at rt for 4 h. CH2Cl2 (20 mL) was added, and the or-
ganic phase was washed with diluted hydrochloric acid, saturated
NaHCO3 and brine, and dried over anhydrous Na2SO4. The solvent
was evaporated in vacuo and the residue was purified through
chromatography eluting with PE/EtOAc to give the compound
which was deprotected using the same procedure as described
for the preparation of 4a to give 4i and 4j.
4.1.11.4. Octyl 5,7-dihydroxy-4-oxo-4H-chromene-3-carboxyl-
ate (7e). Yield: 76%; mp 158–160 °C; 1H NMR (C5D5N, 300 MHz)
d: 0.85 (3H, t, J = 6.6 Hz), 1.21 (8H, m), 1.39 (2H, quint, J = 7.7 Hz),
1.71 (2H, quint, J = 6.9 Hz), 4.36 (2H, t, J = 6.9 Hz), 6.64 (1H, d,
J = 1.9 Hz), 6.70 (1H, d, J = 1.9 Hz), 8.85 (1H, s), 13.40 (1H, br); 13C
NMR (C5D5N, 75 MHz) d: 14.64, 23.26, 26.59, 29.32, 29.80, 29.83,
32.37, 65.80, 95.73, 101.38, 105.94, 115.64, 158.35, 162.99,
163.34, 164.06, 166.82, 179.17; HRMS-ESI (m/z): calcd for
C18H23O6 [M+H]+: 335.1495 found: 335.1490.
4.1.10.1. 5,7-Dihydroxy-4-oxo-4H-chromen-3-yl 4-methylben-
zenesulfonate (4i). Yield: 83%; mp 232–234 °C; 1H NMR (CD3OD,
300 MHz) d: 2.46 (3H, s), 6.20 (1H, d, J = 2.1 Hz), 6.35 (1H, d,
J = 2.1 Hz), 7.43 (2H, d, J = 8.2 Hz), 7.86 (2H, d, J = 8.2 Hz), 8.19
(1H, s); 13C NMR (CD3OD, 75 MHz) d: 22.54, 96.35, 101.48,
111.91, 117.60, 130.88, 131.87, 134.24, 135.98, 147.30, 148.64,
153.98, 159.82, 164.34, 167.55; HRMS-ESI (m/z): calcd for
C16H13O7S [M+H]+: 349.0382 found: 349.0384.
4.1.11.5. Octadecyl 5,7-dihydroxy-4-oxo-4H-chromene-3-car-
boxylate (7f). Yield: 70%; mp 144–146 °C; 1H NMR (C5D5N,
300 MHz) d: 0.87 (3H, t, J = 6.6 Hz), 1.28–1.43 (30H, m), 1.74 (2H,
quint, J = 6.8 Hz), 4.37 (2H, t, J = 6.8 Hz), 6.50 (1H, d, J = 2.2 Hz),
6.71 (1H, d, J = 2.2 Hz), 8.88 (1H, s), 13.43 (1H, br); 13C NMR
(C5D5N, 75 MHz) d: 15.58, 24.24, 27.51, 30.21, 30.81, 30.91,
31.10, 31.18, 31.22, 31.25, 31.29, 33.41, 66.70, 96.61, 102.26,
106.81, 116.47, 159.22, 163.94, 164.35, 164.94, 167.74, 180.06;
HRMS-ESI (m/z): calcd for C28H42O6Na [M+Na]+: 497.2879 found:
497.2875.
4.1.10.2. 5,7-Dihydroxy-4-oxo-4H-chromen-3-yl methanesulfo-
nate (4j). Yield: 80%; mp 226–228 °C; 1H NMR (CD3OD, 300 MHz)
d: 3.44 (3H, s), 6.26 (1H, d, J = 2.2 Hz), 6.39 (1H, d, J = 2.2 Hz), 8.37
(1H, s); 13C NMR (CD3OD, 75 MHz) d: 39.43, 95.63, 100.73, 106.55,
135.75, 153.56, 159.59, 163.59, 166.79, 170.01, 177.39; HRMS-ESI
(m/z): calcd for C10H9O7S [M+H]+: 273.0069 found: 273.0057.
4.2. Biological assay
4.2.1. Materials
Murine macrophages-like RAW264.7 cell line was obtained
from Institute of Biochemistry and Cell Biology (Shanghai, China).
Stock solutions of compounds were prepared with 100% dimethyl-
sulfoxide (DMSO, Sigma) and diluted with RPMI 1640 medium
containing 10% fetal bovine serum (FBS, Life Technologies Inc.,
Grand Island, NY). MTT (3-(4,5-dimethylthylthiazol-2-yl)-2,5-di-
phenyl-tetrazolium bromide) and lipopolysaccharide (LPS, Esche-
richia coli) were purchased from Sigma (St. Louis, Mo). Griess
reagent was obtained from Promega (Madison, USA). Trizol reagent
4.1.11. General procedure for compounds 7b–7f
To a solution of 7 (301 mg, 0.75 mmol) in CH2Cl2 (10 mL) was
added oxalyl chloride (1 mL), and the solution was stirred at rt
for 6 h. Then the mixture was concentrated in vacuo, and cyclohex-
ane (3 ꢁ 20 mL) was added to the residue and the solution was
concentrated in vacuo (this procedure was repeated three times)
to eliminate the excess oxalyl chloride. To the solution of the above

G.-B. Liu et al. / Bioorg. Med. Chem. 18 (2010) 2864–2871
2871
was purchased from Invitrogen (Carlsbad, USA). M-MLV reverse
Acknowledgments
transcriptase was obtained from Toyobo (Osaka, Japan).
This work was supported by National Natural Science Founda-
tion of China (90913023), National Natural Science Fund for Crea-
tive Research Groups of China (20821063), and National Science
and Technology Major Project of China (2009ZX09102-129 and
2009ZX09303-001).
4.2.2. Cell culture
RAW264.7 cells were cultured in RPMI 1640 medium supple-
mented with penicillin (100 U/mL), streptomycin (100 lg/mL),
and 10% FBS at 37 °C in a humidified 5% CO2 and 95% air. Cell con-
centration was adjusted to 4 ꢁ 105 cells/mL, and 200
lL of cell sus-
pension was seeded in each well of a 96-well flat-bottomed plate.
Supplementary data
After a 2 h incubation, medium was replaced with a fresh 2% FBS-
RPMI 1640 medium containing 2
g/mL LPS plus the test compounds at various concentrations
(0.01–100 M) and cells was then incubated for 24 h. Cells incu-
lg/mL of LPS plus 0.1% DMSO or
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.03.020.
2
l
l
bated in fresh 2% FBS-RPMI 1640 medium containing 0.1% DMSO
were used as control. The levels of NO in the culture supernatant
were analyzed. When determining the mRNA expressions of iNOS
in RAW264.7 cells, cells were treated with LPS and compounds for
8 h.
References and notes
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To determine iNOS mRNA expression levels in RAW264.7 cells,
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according to manufacturer’s instructions. The sense primer of
iNOS was 50-GGAGCGAGTTGTGGATTGTC-30, and the antisense pri-
mer was 50-CTCTGCCTATCCGTCTCGTC-30. The sense primer of
b-Actin was 50-TGCTGTCCCTGTATGCCTCT-30, and the antisense pri-
mer was 50-TTTGATGTCACGCACGATTT-30. The PCR reaction was
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