Synthesis and evaluation of a novel series of quinoline derivatives with immunosuppressive activity

Article abstract of DOI:10.1016/j.bmc.2009.06.043

A series of quinoline derivatives were synthesized and their immunosuppressive activity and cytotoxicity were evaluated with a T-cell functional assay and MTT method, respectively. Most of 5,7-dimethoxyquinolin-4-yl ortho-substituted benzoate derivatives

Full text of DOI:10.1016/j.bmc.2009.06.043

Bioorganic & Medicinal Chemistry 17 (2009) 5433–5441
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of a novel series of quinoline derivatives
with immunosuppressive activity
Guo-Biao Liu a, Jian-Liang Xu b, Cui-Cui He a, Gong Chen b, 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 series of quinoline derivatives were synthesized and their immunosuppressive activity and cytotoxicity
were evaluated with a T-cell functional assay and MTT method, respectively. Most of 5,7-dimethoxyquin-
olin-4-yl ortho-substituted benzoate derivatives (18, 22, 24, 28, 31 and 37) showed a quite stronger
inhibitory activity compared to other analogs. Among the synthesized compounds, 5,7-dimethoxyquin-
olin-4-yl 2,6-dichlorobenzoate (22) and 5,7-dimethoxyquinolin-4-yl 4-methylbenzenesulfonate (40)
Received 20 May 2009
Revised 18 June 2009
Accepted 19 June 2009
Available online 26 June 2009
exhibited a potent inhibitory activity without significant cytotoxicity at 10 lM concentration. The preli-
Keywords:
Quinoline derivatives
Synthesis
minary mechanism of the active compounds 22 and 40 was further clarified based on the fluorescence
activated cell sorter (FACS) assay, and the compounds exerted immunosuppressive activity via inhibiting
the T cell activation in a dose dependent manner.
Immunosuppressive activity
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
and natural products, is a class of heterocyclic compounds which
possess a variety of biological activities, such as antimalarial,16
Immunosuppressant is an important class of clinical drugs used
in prevention of the transplant rejection and treatment of autoim-
mune diseases such as systemic lupus erythematosus, rheumatoid
arthritis and psoriasis. T-lymphocyte plays an integral role in
transplant rejection and autoimmune diseases. Inhibition of T-
lymphocyte activation has made cyclosporine A (CsA), tacrolimus
(FK506), and sirolimus (rapamycin) become therapeutic immuno-
suppressants.1 CsA and FK506 both interfere with a Ca2+-sensitive
T-cell signal transduction pathway, thereby preventing the activa-
tion of specific transcription factors involved in lymphokine gene
expression.2–4 Rapamycin suppresses T-cell activation mainly
through inhibition of proliferation induced by growth-promoting
lymphokines.1 Although immunosuppressive drugs have been suc-
cessfully used for organ transplantation and treatment of autoim-
mune diseases in clinic, their side effects including liver toxicity,
nephrotoxicity, malignancy, infection, cardiovascular toxicity and
others can not be neglected.5–11 Many compounds derived from
traditional Chinese medicines, such as artemisinin,12,13 bakuchiol14
and their analogs exhibit immunosuppressive activity by inhibiting
T-cell activation and proliferation. The effort of searching for novel
classes of potential immunosuppressive compounds with high effi-
cacy and low toxicity has never stopped.
antiinflammatory,17,18 antibacterial,19 antiviral,20 antitubercular,21
and antitumor.22 However, the effect of quinoline and its deriva-
tives on immunosuppressive activity have not been studied suffi-
ciently. Considering about the structural similarity of quinoline
and chromone, we put our interest on quinoline derivatives in or-
der to explore new chemical pharmacophores which might exhibit
the immunosuppressive activity.
It has been reported that conjugates of fatty acids and pharma-
cophores could provide effective bioactivity, and different chain
length greatly impacted the activity.23 Therefore fatty acids might
be effective moieties to improve the potency of the immunosup-
pressive activity. The same bioactivity improvements could also
be observed on the glycosides and benzoic acid derivatives of nat-
ural products.24–26 In the present study, a series of quinoline deriv-
atives including fatty acid esters, glycosides and different
substituted benzoic acid esters were synthesized. The immunosup-
pressive activity of the synthesized compounds was evaluated
with T-cell functional assay and their cytotoxicity was tested using
MTT method. The preliminary mechanism of some active com-
pounds was further examined in a fluorescence activated cell sor-
ter (FACS) assay.
We have previously reported that a natural product, eucryphin
with chromone skeleton possesses an immunological hepatocyte
protective activity.15 Quinoline skeleton, found in many synthetic
2. Results and discussion
2.1. Chemistry
The syntheses of target compounds 4–9 are outlined in Scheme
1. Protection of the starting material 3,5-dimethoxyaniline (1) with
* Corresponding author. Tel./fax: +86 25 83686419.
E-mail address: lijxnju@nju.edu.cn (J.-X. Li).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.043

5434
G.-B. Liu et al. / Bioorg. Med. Chem. 17 (2009) 5433–5441
Scheme 1. Reagents and conditions: (a) Ac2O, Et3N, CH2Cl2, 0 °C, 1 h, 90%; (b) AcCl, SnCl4, ClCH2CH2Cl, 0 °C, 4 h, 60%; (c) (i) HCOOEt, NaH, rt, 20 min; (ii) concd HCl, 0 °C, 3 h,
92%; (d) BBr3, CH2Cl2, ꢀ78 °C to rt, 12 h, 45–50%; (e) tBuOK, tBuOH, reflux, 2 h, 95%; (f) R-PhCOCl, Et3N, CH2Cl2, rt, 81–84%.
acetic anhydride afforded 2 in a satisfactory yield (90%). Compound
3 was prepared via a Friedel–Crafts reaction of compound 2 with
acetyl chloride in the presence of anhydrous stannic chloride.27
Compound 3 was then reacted with sodium hydride in ethyl for-
mate at rt for 20 min, subsequently acidified using concentrated
hydrochloric acid to provide 4 in 92% yield. This reaction might in-
volve two steps, the hydrolysis of acetamide and intermolecular
cyclization of o-aminoacetophenone.28 An intramolecular cycliza-
tion of compound 3 in tertiary butyl alcohol in the presence of
potassium t-butoxide afforded target compound 5 in 95% yield.29
Demethylations of 4 and 5 using BBr3 at ꢀ78 °C to rt afforded 6
and 7 in moderate yields. Compound 5 was then reacted with cor-
responding acyl chlorides to get 8 and 9, respectively.
densing agent and DMAP as a catalyst provided corresponding
fatty acid esters of 10–13.
D
-Sugar bromides 14a–16a were prepared through benzoyla-
tion (BzCl in pyridine) and bromination (HBr in HOAc).32,33
A
phase-transfer-catalyzed method was applied for glycosidation
due to its mild conditions and high yields.34,35 The present glycos-
idations of 4 were performed by the reaction of acceptor 4 with do-
nors (14a–16a) under the phase-transfer-catalyzed conditions
(NaOH, Bu4NBr, CH2Cl2–H2O, reflux). Subsequent removal of the
protecting groups (benzoyl) using sodium methoxide (cat.) in a
mixed solution of MeOH and CH2Cl2 gave the target glycosides
14–16.36
For the stereochemistry of the glycosidic bonds, bromides 14a–
In order to understand the influences on activity, modifications
at 4-OH of quinolines were conducted and compounds 10–40 were
prepared. The synthesis routes are depicted in Schemes 2 and 3.
DDC/DMAP-mediated esterification was commonly used in or-
ganic synthesis for years.30,31 As shown in Scheme 2, the reactions
of 4 with different chain length of fatty acids using DCC as a con-
16a were a-configuration which were identical with the reported
data.32,37 To target glycosides, all of the glycosidic bonds were
b-configuration (14–16: H-10, 5.80–5.89 ppm, d, J = 6.0–7.5 Hz).
Esterifications of 4 with 23 different substituted benzoic acids
using EDCI and DMAP were outlined in Scheme 3. In the reactions,
two condensing agents EDCI and DCC were tested. Although the
Scheme 2. Reagents and conditions: (a) RCOOH, DCC, DMAP, CH2Cl2, rt, 3 h, 70–75%; (b) (i) sugar donor, NaOH, TBAB, CH2Cl2/H2O, reflux, 18 h; (ii) MeONa, CH2Cl2/MeOH, rt,
3 h, two steps 31–38%.

G.-B. Liu et al. / Bioorg. Med. Chem. 17 (2009) 5433–5441
5435
compared with the same modifiers of 4 (compounds 17 and 28)
at lower concentration, which will discuss late.
As described above, fatty acid modification and glycosylation
might improve the activity. Unfortunately, in the present studies,
the fatty acids derivatives (10–13) and the glycosides (14–16) of
4 did not display ideal improvements of the inhibitory activity,
although those compounds showed almost no cytotoxicity. To en-
hance the inhibitory activity of quinoline derivatives, different
substituted benzoic acids were further introduced to the 4-hydroxy
of quinolines, which resulted in compounds 17–39. Fortunately,
the most of 5,7-dimethoxyquinolin-4-yl substituted benzoate
derivatives showed much stronger inhibitory activity than fatty
acid derivatives and glycosides. As shown in Table 1, compounds
9 and 28 were obtained by modifying compounds 5 and 4 with
ortho-iodobenzoic acid, the later showed much more potent inhib-
itory activity than 9, which seemed that the activity of skeleton 4
derivatives is better than that of 5. The interesting thing is the loca-
tions of substituent at benzoic acid were closely related to the
immunosuppressive activity. The ortho located substituent deriva-
tives (18, 22, 24, 28, 31, and 37) had more potent activity than
other derivatives. Furthermore, the inhibitory activity of com-
pounds modified with ortho-halogen-substituted benzoic acids
was increased in the following order: F < Cl < Br < I. Fortunately,
compound 40 displayed very good inhibitory activity and without
cytotoxicity at three concentrations (10, 30 and 100
lM), which
indicated that the para-toluene sulfonyl may be a potentially active
functional group of immunosuppressor.
For proving these results obtained by MTT assay, several active
compounds (18, 22, 24, 28, 31, 37 and 40) were assessed their ef-
fect on T cell proliferation induced by ConA in terms of uptake of
[3H]thymidine which is more sensitive and accurate than MTT as-
say. As shown in Table 2, the results were similar and consistent
with the data got from MTT assay as described above. It is note-
worthy to indicate that compound 22 got an inhibition rate of
35% at 1 lM.
To further understand the immunosuppressive mechanism of
these derivatives, the fluorescence activated cell sorter (FACS) assay
wasappliedtoassesstheeffectof some activecompounds(18, 22, 24,
28, 31, 37 and 40) on T cell activation induced by ConA. As shown in
Table 3, compared to control, the percentage of activated T cells was
increased from 38.39 to 93.84 when stimulated with ConA. CsA, a
clinically available immunosuppressor, inhibited the activation to
control level. Both 22 and 40 inhibited the T cell activation in a dose
dependent manner. The other quinoline derivatives (18, 24, 28, 31
and 37) showed weak effects which indicated that they did not sup-
press T cell proliferation through inhibiting cell activation.
Scheme 3. Reagents and conditions: (a) R-PhCOOH, EDCI, DMAP, CH2Cl2, rt, 3–10 h,
60–90%; (b) p-TsCl, Et3N, CH2Cl2, reflux, 8 h, 83%.
yield rates of two agents were the almost same, removal of con-
densing byproduct of DCC (DCHU) was quite difficult, while the
EDCI’s byproduct could be easily scoured off by diluted hydrochlo-
ric acid. Therefore, EDCI was used in the reaction. As expected,
after removal of EDCI’s byproduct and DMAP with diluted hydro-
chloric acid, only a simple crystallization process of the reaction
product in methanol provided each pure target compound (17–
39) without any chromatography purifications. Compound 40
was obtained in good yield by refluxing 4 with excess p-toluene
sulfonyl chloride in the presence of triethyl amine.
3. Conclusion
2.2. Biological activity
We have synthesized a series of quinoline derivatives with
inhibitory effect on ConA-induced T cell proliferation. Quinoline
with methylated-hydroxyl groups displayed a better immunosup-
pressive activity than unmethylated one. Fatty acid derivatives and
glycosides of 5,7-dimethoxyquinolin-4-yl did not show any benefit
on the improvement of the activity, while, benzoic acid derivatives
positively impacted the activity. ortho-Substituted benzoate deriv-
atives exhibited a close correlation with the stronger activity and
The inhibitory activity on ConA-induced T cell proliferation of
all the synthesized compounds was tested and their cytotoxicity
on murine spleen cells was evaluated with MTT assay. The results
of these compounds are summarized in Table 1.
As can been seen from Table 1, compounds 6 and 7 showed a
weak inhibitory activity on ConA-induced T cell proliferation only
at 100
exhibited a moderate activity at the concentration of 100 and
30 M. This result revealed that the methylation of hydroxyl
groups might elevate the activity. While, the activity between
compound 6 and its methylated product (4) showed no difference.
Further modification of 5 with substituted benzoic acids (com-
pounds 8 and 9) did not exhibit any advancement of the activity
lM. However, the compound 5, a methylated derivative of 7
no significant cytotoxicity at 10 lM was observed. Especially, com-
pound 22 and 40 exerted a strongest immunosuppressive activity
by inhibiting the T cell activation in a dose dependent manner.
The present results revealed that the quinoline derivatives as a
new class of immunosuppressive leads deserve further studies.
The in vivo activity and further detailed immunosuppressive
mechanism of 22 and 40 are in process in our lab.
l

5436
G.-B. Liu et al. / Bioorg. Med. Chem. 17 (2009) 5433–5441
Table 1
Effect of quinoline derivatives 4–40 on murine T lymphocyte proliferation induced by ConA (5 lg/mL) measured with MTT method
Compound
Inhibition rate (%)
30 ( M)
Cytotoxicity (growth inhibition in %)
30 ( M) 100 (lM)
10 (l
M)
l
100 (l
M)
10 (l
M)
l
4
5
6
7
8
0.1 2.6
15.9 2.8*
ꢀ0.9 1.5
7.5 4.9
3.7 2.9
35.4 2.4**
1.7 1.9
12.3 6.9
6.1 5.8
6.5 0.7
1.8 9.2
9.1 7.8
ꢀ7.6 4.9
ꢀ3.4 5.1
8.3 4.1
4.5 3.7
6.4 5.9
4.1 6.0
7.6 7.1
6.2 4.8
6.9 6.5
0.0 5.1
2.9 6.9
3.1 4.4
3.4 1.9
16.5 9.0
10.9 6.3
3.4 2.7
9.8 5.2
1.7 11.6
8.6 9.4
5.5 4.5
14.3 10.9
3.0 10.0
6.0 6.0
10.7 3.9
3.3 5.1
6.0 14.0
3.4 7.0
6.6 5.3
7.7 15.3
9.4 12.2
10.3 3.5
8.2 7.5
6.2 17.2
7.8 3.6
14.7 8.5
10.9 7.4
9.9 1.5
ꢀ0.2 7.1
18.2 7.6
11.2 5.9
10.5 14.0
2.0 6.9
8.8 8.8
9.1 3.8
4.2 5.6
9.1 7.1
4.6 4.0
6.6 5.4
9.4 8.4
0.3 11.9
20.8 7.5
52.7 3.7**
10.7 8.4
22.5 9.6
1.7 3.6
7.5 4.1
23.6 4.7*
8.6 6.6
11.2 13.4
13.7 8.7
37.6 3.5**
11.3 6.0
9.4 9.0
11.6 12.6
7.2 3.9
9.5 4.3
17.0 9.3
11.2 3.3*
7.7 6.9
57.2 2.2**
18.0 6.4*
26.7 2.9**
81.5 8.0**
91.5 2.2**
22.7 6.8*
13.9 2.3**
31.9 4.7**
22.2 2.5**
20.4 3.0**
17.8 3.3*
41.0 1.2**
35.4 1.0**
89.8 1.8**
90.8 2.1**
86.3 2.8**
79.2 5.6**
94.3 0.5**
7.9 2.4*
4.4 3.0
5.7 4.4
34.8 14.9
54.0 6.9**
9.7 2.8*
9
19.9 3.0**
2.2 2.3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
4.1 3.7
4.4 2.2
4.4 3.3
3.6 2.4
5.3 4.6
2.0 6.1
8.5 1.9*
6.0 3.9
4.2 2.3
11.2 3.9*
14.9 5.7*
13.1 1.4**
73.7 5.7**
42.0 3.1**
47.4 11.4*
62.3 10.6**
94.6 0.2**
5.1 2.6
10.3 4.9
6.2 1.1*
46.1 4.7**
20.6 7.8*
30.1 3.9**
35.3 5.2**
94.0 0.2**
3.9 2.6
39.8 5.0**
12.2 12.3
21.9 5.5*
22.7 5.6*
83.4 1.4**
7.2 3.4
51.2 16.0*
16.9 5.7*
9.2 0.6**
6.4 4.1
85.7 4.1**
48.8 13.4*
17.5 0.4**
10.0 9.4
90.6 2.6**
50.5 11.3*
47.2 5.8**
75.2 3.8**
42.0 3.6**
34.1 2.0**
5.1 4.3
93.3 4.1**
91.0 3.1**
48.2 11.2*
12.1 1.8
29.2 6.4*
21.0 7.1*
20.7 7.9*
6.3 4.9
7.3 10.0
9.4 7.7
63.0 10.9**
24.7 5.7*
26.4 7.8*
38.6 12.6*
18.8 6.1*
18.4 0.4**
6.3 4.6
93.0 4.4**
85.2 2.9**
91.3 4.5**
90.5 3.7**
82.8 5.7**
64.0 9.9**
5.6 5.0
26.5 11.7
5.6 12.0
6.6 5.3
54.7 12.4*
11.6 16.7
23.4 10.8
33.4 0.2**
12.2 11.8
9.5 15.7
13.4 7.4
23.6 20.8
19.5 18.1
64.0 5.7**
47.9 13.5*
13.4 10.2
13.2 16.8
32.0 5.2**
8.1 10.0
11.1 16.9
9.5 7.8
15.7 9.9
9.3 6.7
27.8 8.7*
14.3 4.4*
91.4 0.7**
35.2 17.5
11.0 4.7
94.7 0.2**
86.4 5.4**
91.4 2.5**
31.0 5.8*
92.2 2.5**
85.3 8.2**
17.7 3.2*
94.2 0.3**
2.2 18.5
12.8 16.3
53.9 7.6**
17.6 7.2
11.3 5.6
5.4 15.2
8.70 12.4
70.3 4.6**
11.3 2.9*
7.8 3.8
82.3 5.9**
CsA (1 lM)
Data are expressed as mean S.D., n = 3. Significant differences compared with control group (0.0%).
*
p <0.05.
p <0.01.
**
1H and 13C NMR spectra were taken on a Bruker DPX-300 spec-
trometer, using TMS as an internal standard (chemical shifts in
d). ESI-HR-MS were obtained on Esquire 4000 mass spectrometer.
TLC was carried out on precoated Kieselgel F254 plates
(0.25 mm), and spots were detected under a UV lamp (254 nm).
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.
4.1.2. N-(3,5-Dimethoxyphenyl)acetamide (2)
To a solution of 1 (15.3 g, 0.10 mol) in CH2Cl2 (250 mL) at 0 °C
was added triethylamine (16.8 mL, 0.12 mol) and then acetic
Table 2
Table 3
Effect of some selected quinoline derivatives on murine T lymphocyte proliferation
induced by ConA (5
l
g/mL) measured in terms of uptake of [3H] thymidine
Effect of some selected quinoline derivatives on murine T cell activation induced by
ConA (5 lg/mL) measured with FACS assay
Compound
Inhibition rate (%)
Compound
Activated T cell% (CD4+CD25+/CD4+)
3 ( M) 10 ( M)
1 (l
M)
10 (
lM)
30 (lM)
1 (
l
M)
l
l
30 (lM)
18
22
24
28
31
37
40
4.3 4.1
34.7 2.6**
4.8 7.8
8.2 6.0
10.2 5.6
2.2 3.0
37.9 1.4**
97.8 0.6**
42.0 1.7**
47.5 1.1**
21.1 1.2**
55.4 1.3**
87.7 1.0**
95.0 0.4**
52.0 0.8**
99.2 0.2**
85.4 1.8**
87.1 1.2**
64.6 1.1**
94.6 0.1**
98.9 0.5**
18
22
24
28
31
37
40
95.63
92.96
94.92
93.10
94.79
93.81
85.74
92.80
88.80
93.23
91.87
94.01
92.54
81.66
93.10
59.64
93.63
90.04
92.75
85.45
68.39
90.86
19.41
86.21
83.23
86.19
75.65
13.70
12.9 2.8
CsA (1 lM)
Alone
ConA
CsA (1 lM)
38.39
93.84
35.14
Data are expressed as mean S.D., n = 3. Significant differences compared with
control group (0.0%).
**
p <0.01.

G.-B. Liu et al. / Bioorg. Med. Chem. 17 (2009) 5433–5441
5437
anhydride (11.4 mL, 0.12 mol) was added dropwise. The reaction
mixture was stirred for 1 h, quenched by the addition of water
(30 mL), and then the pH was adjusted to 7 by adding saturated
NaHCO3. The aqueous layer was extracted with CH2Cl2
(3 ꢁ 200 mL). The combined organic layer was washed with di-
luted HCl, saturated NaHCO3, and brine, and dried over anhydrous
Na2SO4. The solvent was removed in vacuo to give 2. Yield: 90%; 1H
NMR (CDCl3, 300 MHz) d: 2.15 (3H, s), 3.76 (6H, s), 6.22 (1H, s),
6.74 (2H, s), 7.34 (1H, br).
4.1.6.2. 5,7-Dihydroxy-2-methylquinolin-4(1H)-one (7). Yield: 50%;
Carbonization before mp; 1H NMR (CF3COOD, 300 MHz) d: 3.07 (3H,
s), 6.82 (1H, s), 6.86 (2H, s); 13C NMR (CF3COOD, 75 MHz) d: 23.96,
107.48, 108.66, 112.41, 119.91, 159.45, 160.94, 161.52, 162.10,
162.68; HRMS-ESI (m/z): calcd for C16H10NO3 [M+H]+: 192.0661, found:
192.0654.
4.1.7. General procedure for compounds 8 and 9
To a solution of 5 (219 mg, 1 mmol) in CH2Cl2 (10 mL) at rt was
added Et3N (0.28 mL, 2 mmol) and corresponding substituted ben-
zoyl chloride (1.2 mmol). The reaction solution was stirred for 3 h,
quenched by the addition of water (10 mL), and then the aqueous
layer was extracted with CH2Cl2 (3 ꢁ 20 mL). The organic layer was
washed with diluted hydrochloric acid, saturated NaHCO3, and
brine, and dried over anhydrous Na2SO4. After evaporation of the
solvent in vacuo, the crude product was recrystallized in MeOH
to yield 8 and 9.
4.1.3. N-(2-Acetyl-3,5-dimethoxyphenyl)acetamide (3)
To a solution of 2 (12.4 g 63.5 mmol) in anhydrous ClCH2CH2Cl
(250 mL) at 0 °C was added anhydrous SnCl4 (15 mL 128.4 mmol)
dropwise and acetyl chloride (10 mL 140.1 mmol) was added drop-
wise. The solution was stirred at rt for 4 h and poured into 100 mL
ice water. The aqueous layer was extracted with CH2Cl2
(3 ꢁ 200 mL). The combined organic layer was washed with satu-
rated NaHCO3, brine, and then dried over anhydrous Na2SO4. After
removal of solvent in vacuo, chromatography of the crude product
on silica gel, eluting with PE/EtOAc (4/1) afforded 3. Yield: 60%; 1H
NMR (CDCl3, 300 MHz) d: 2.17 (3H, s), 2.56 (3H, s), 3.85 (6H, s),
6.17 (1H, s), 7.95 (1H, s), 11.73 (1H, br).
4.1.7.1. 5,7-Dimethoxy-2-methylquinolin-4-yl benzoate (8). Yield:
84%; mp 183–185 °C; 1H NMR (CDCl3, 300 MHz) d: 2.85 (3H, s), 3.90
(3H, s), 3.91 (3H, s), 6.50 (1H, d, J = 2.2 Hz), 6.87 (1H, s), 6.98 (1H, d,
J = 2.2 Hz), 7.51 (2H, t, J = 7.5 Hz), 7.64 (1H, t, J = 7.5 Hz), 8.25 (2H, d,
J = 7.5 Hz); 13C NMR (CDCl3, 75 MHz) d: 24.67, 55.68, 55.71, 98.71,
100.54, 114.48, 115.68, 128.68, 129.38, 130.57, 133.94, 150.62,
150.72, 157.29, 158.83, 161.27, 165.10; HRMS-ESI (m/z): calcd for
C19H18NO4 [M+H]+: 324.1236, found: 324.1233.
4.1.4. 5,7-Dimethoxyquinolin-4(1H)-one (4)
To a solution of 3 (3.0 g, 12.6 mmol) in ethyl formate (40 mL) was
added NaH (3.1 g, 60% in mineral oil, 77 mmol). After a short induc-
tion period, an exothermal reaction was carried out and the suspen-
sion began to reflux. The reaction solution was stirred at rt for
20 min, then cooled to 0 °C. The reaction was quenched by the addi-
tion of water (1 mL), subsequently concentrated HCl (9 mL) was
added, and the suspension was stirred at 0 °C for 4 h. After adjusting
the pH to 7 by 5 M NaOH solution, the resulting solid was filtered,
washed with water, dioxane, ethyl acetate, and then dried to give
4. Yield: 92%; mp 233–235 °C; 1H NMR (DMSO-d6, 300 MHz) d:
3.72 (3H, s), 3.78 (3H, s), 5.74 (1H, d, J = 7.0 Hz), 6.26 (1H, s), 6.42
(1H, s), 7.55 (1H, d, J = 7.0 Hz), 11.22 (1H, br); 13C NMR (DMSO-d6,
75 MHz) d: 55.68, 56.05, 91.87, 94.79, 111.44, 111.87, 137.38,
144.68, 161.35, 162.21, 176.75; HRMS-ESI (m/z): calcd for
C11H12NO3 [M+H]+: 206.0817, found: 206.0810.
4.1.7.2. 5,7-Dimethoxy-2-methylquinolin-4-yl 2-iodobenzoate
(9). Yield: 81%; mp 145–146 °C; 1H NMR (CDCl3, 300 MHz) d: 2.86
(3H, s), 3.90 (3H, s), 3.91 (3H, s), 6.50 (1H, d, J = 2.2 Hz), 6.91 (1H, s),
6.96 (1H, d, J = 2.2 Hz), 7.22 (1H, td, J = 7.8, 1.5 Hz), 7.48 (1H, t,
J = 7.8 Hz), 8.08 (1H, d, J = 7.8 Hz), 8.20 (1H, dd, J = 7.8, 1.5 Hz);
13C NMR (CDCl3, 75 MHz) d: 24.71, 55.70, 55.73, 95.29, 98.78,
100.54, 114.28, 115.75, 128.19, 132.32, 133.19, 133.67, 142.04,
150.57, 150.85, 156.90, 158.81, 161.29, 164.30; HRMS-ESI (m/z):
calcd for C19H17INO4 [M+H]+: 450.0202, found: 450.0205.
4.1.8. General procedure for compounds (10–13)
To a solution of 4 (205 mg, 1.0 mmol) in CH2Cl2 (10 mL) at 0 °C
was added corresponding fatty acid (1.2 mmol), DCC (247 mg,
1.2 mmol) and a few crystals of DMAP. The suspension was stirred
for 3 h. The resulting solid was filtered and the filtrate was washed
with diluted HCl, saturated NaHCO3, and brine, dried over anhy-
drous Na2SO4 and concentrated in vacuo. Purification through sil-
ica gel chromatography eluting with PE/EtOAc afforded
corresponding compounds 10–13.
4.1.5. 5,7-Dimethoxy-2-methylquinolin-4(1H)-one (5)
To a solution of 3 (1.0 g, 4.2 mmol) in tBuOH (30 mL) was added
tBuOK (2.3 g, 21 mmol), and the suspension was refluxed for 2 h.
The pH of the suspension was adjusted to 7 by adding diluted
hydrochloric acid. The resulting solid was filtered, washed with
water and dried to provide 5. Yield: 95%; mp 263–265 °C; 1H
NMR (DMSO-d6, 300 MHz) d: 2.49 (3H, s), 3.78 (3H, s), 3.82
(3H, s), 6.02 (1H, s), 6.30 (1H, s), 6.44 (1H, s), 11.38 (1H, br); 13C
NMR (CF3COOD, 75 MHz) d: 24.24, 54.83, 55.08, 107.74, 108.71,
112.46, 119.97, 159.00, 160.73, 161.30, 161.88, 162.45; HRMS-ESI
(m/z): calcd for C12H14NO3 [M+H]+: 220.0974, found: 220.0969.
4.1.8.1. 5,7-Dimethoxyquinolin-4-yl acetate (10). Yield 70%;
mp 225–227 °C; 1H NMR (CDCl3, 300 MHz) d: 2.36 (3H, s), 3.89
(3H, s), 3.91 (3H, s), 6.51 (1H, d, J = 2.1 Hz), 6.86 (1H, d,
J = 4.8 Hz), 7.08 (1H, d, J = 2.1 Hz), 8.73 (1H, d, J = 4.8 Hz); 13C
NMR (CDCl3, 75 MHz) d: 21.59, 55.62, 55.92, 92.30, 96.32,
109.92, 110.82, 139.46, 144.98, 160.24, 163.09, 176.01, 177.30;
HRMS-ESI (m/z): calcd for C13H14NO4 [M+H]+: 248.0923, found:
248.0924.
4.1.6. General procedure for compounds 6 and 7
To a solution of 4 or 5 (1.0 mmol) in anhydrous CH2Cl2 (10 mL)
was added BBr3 solution (2.2 mL, 1 M in CH2Cl2, 2.2 mmol) drop-
wise at ꢀ78 °C, and the mixture was stirred at rt for 24 h. Water
(1 mL) was added to quench the reaction, and the solvent was
evaporated in vacuo. Chromatography of the crude product on sil-
ica gel, eluting with CH2Cl2/MeOH afforded 6 or 7.
4.1.8.2. 5,7-Dimethoxyquinolin-4-yl pentanoate (11). Yield
72%; mp 103–105 °C; 1H NMR (CDCl3, 300 MHz) d: 0.98 (3H, t,
J = 7.5 Hz), 1.47 (2H, sextet, J = 7.5 Hz), 1.77 (2H, quintet,
J = 7.5 Hz), 2.64 (2H, t, J = 7.5 Hz), 3.86 (3H, s), 3.91 (3H, s), 6.51
(1H, d, J = 1.8 Hz), 6.84 (1H, d, J = 4.5 Hz), 7.06 (1H, d, J = 1.8 Hz),
8.72 (1H, d, J = 4.5 Hz). 13C NMR (CDCl3, 75 MHz) d: 13.83, 22.35,
27.15, 34.39, 55.51, 55.78, 92.09, 96.04, 110.23, 111.09, 139.05,
144.89, 160.39, 162.98, 177.94, 178.77; HRMS-ESI (m/z): calcd for
C16H20NO4 [M+H]+: 290.1392, found: 290.1386.
4.1.6.1. 5,7-Dihydroxyquinolin-4(1H)-one (6). Yield: 45%; Car-
bonization before mp; 1H NMR (CD3OD, 300 MHz) d: 6.03 (1H, d,
J = 7.2 Hz), 6.11 (1H, d, J = 1.6 Hz), 6.25 (1H, d, J = 1.6 Hz), 7.70
(1H, d, J = 7.2 Hz); 13C NMR (CD3OD, 75 MHz) d: 91.51, 97.73,
106.11, 107.91, 139.63, 142.55, 162.32, 162.82, 181.90; HRMS-ESI
(m/z): calcd for C9H8NO3 [M+H]+: 178.0504, found: 178.0498.

5438
G.-B. Liu et al. / Bioorg. Med. Chem. 17 (2009) 5433–5441
4.1.8.3. 5,7-Dimethoxyquinolin-4-yl nonanoate (12). Yield 72%;
mp 73–76 °C; 1H NMR (CDCl3, 300 MHz) d: 0.87 (3H, t, J = 6.3 Hz),
1.26 (10H, m), 1.64 (2H, m), 2.35 (2H, t, J = 7.5 Hz), 3.73 (3H, s),
3.77 (3H, s), 6.26 (1H, s), 6.30 (1H, d, J = 6.9 Hz), 6.65 (1H, s), 7.72
(1H, d, J = 6.9 Hz). 13C NMR (CDCl3, 75 MHz) d: 14.22, 22.76,
25.07, 29.26, 29.29, 29.38, 31.93, 34.60, 55.58, 55.90, 92.53,
96.12, 110.12, 110.97, 139.54, 145.23, 160.28, 162.88, 177.22,
179.15; HRMS-ESI (m/z): calcd for C20H28NO4 [M+H]+: 346.2018,
found: 346.2006.
4.1.10. General procedure for compounds (17–39)
To a solution of 4 (205 mg, 1.0 mmol) in CH2Cl2 (10 mL) at rt
was added substituted benzoic acid (1.2 mmol), EDCI (288 mg,
1.5 mmol) and a few crystals of DMAP. The solution was stirred
for 6 h. Water (10 mL) was added, and the aqueous layer was ex-
tracted with CH2Cl2 (3 ꢁ 20 mL). The organic layer was washed
with diluted hydrochloric acid, saturated NaHCO3, and brine, and
dried over anhydrous Na2SO4. After evaporation of the solvent in
vacuo, the crude product was recrystallized in MeOH to yield
17–39.
4.1.8.4. 5,7-Dimethoxyquinolin-4-yl oleate (13). Yield 75%; oil;
1H NMR (CDCl3, 300 MHz) d: 0.85 (3H, m), 1.25–1.34 (20H, m),
1.77 (2H, t, J = 7.2 Hz), 2.01 (4H, m), 2.63 (2H, t, J = 7.5 Hz), 3.86
(3H, s), 3.92 (3H, s), 5.34 (2H, s), 6.51 (1H, d, J = 1.8 Hz), 6.84 (1H,
d, J = 4.8 Hz), 7.06 (1H, d, J = 1.8 Hz), 8.72 (1H, d, J = 4.8 Hz). 13C
NMR (CDCl3, 75 MHz) d: 14.20, 24.66, 24.57, 27.15, 27.21, 29.10,
29.22, 29.31, 29.50, 29.68, 29.74, 31.76, 34.16, 55.57, 56.11,
99.71, 100.56, 110.94, 112.64, 122.66, 122.96, 151.35, 153.18,
154.86, 155.83, 161.15, 171.66; HRMS-ESI (m/z): calcd for
C29H44NO4 [M+H]+: 470.3270 found:470.3265.
4.1.10.1. 5,7-Dimethoxyquinolin-4-yl benzoate (17). Yield 78%;
mp 169–171 °C; 1H NMR (CDCl3, 300 MHz) d: 3.55 (3H, s), 3.93
(3H, s), 6.47 (1H, d, J = 2.1 Hz), 7.00 (1H, d, J = 4.8 Hz), 7.10 (1H,
d, J = 2.1 Hz), 7.54 (2H, t, J = 7.5 Hz), 7.67 (1H, t, 7.5 Hz), 8.23 (2H,
d, J = 7.5 Hz), 8.79 (1H, d, J = 4.8 Hz); 13C NMR (CDCl3, 75 MHz) d:
55.58, 55.81, 99.59, 100.51, 110.81, 112.80, 128.58, 129.86,
130.29, 133.55, 151.45, 153.24, 155.19, 155.89, 161.25, 164.95;
HRMS-ESI (m/z): calcd for C18H16NO4 [M+H]+: 310.1079, found:
310.1072.
4.1.9. General procedure for compounds (14–16)
4.1.10.2. 5,7-Dimethoxyquinolin-4-yl 2-chlorobenzoate (18). Yield:
80%; mp 155–157 °C; 1H NMR (CDCl3, 300 MHz) d: 3.71 (3H, s), 3.96
(3H, s), 6.53 (1H, d, J = 1.8 Hz), 7.04 (1H, d, J = 4.8 Hz), 7.12 (1H, d,
J = 1.8 Hz), 7.47 (1H, m), 7.56 (2H, m), 8.26 (1H, d, J = 7.2 Hz), 8.82
(1H, d, J = 4.8 Hz). 13C NMR (CDCl3, 75 MHz) d: 55.61, 55.94, 99.71,
100.65, 110.74, 112.71, 126.71, 128.41, 131.57, 132.56, 133.43,
135.10, 151.52, 153.29, 154.63, 155.76, 161.27, 162.91; HRMS-ESI (m/
z): calcd for C18H15ClNO4 [M+H]+: 344.0690, found: 344.0682.
To a solution of 4 (205 mg, 1.0 mmol) in CH2Cl2 (20 mL) and
water (20 mL) was added sugar donor (1.5 mmol), NaOH
(320 mg, 8.0 mmol) and TBAB (386 mg, 1.2 mmol). The suspension
was stirred at 50 °C for 18 h. Water (10 mL) was added and the
aqueous layer was extracted with CH2Cl2 (3 ꢁ 20 mL). The organic
layer was washed with diluted acid, saturated NaHCO3, and brine,
dried over anhydrous Na2SO4, and concentrated in vacuo. Purifica-
tion through silica gel chromatography eluting with PE/EtOAc
afforded corresponding product. The product was dissolved in a
mixed solution of CH2Cl2 (3 mL) and MeOH (3 mL), and MeONa
(catalytic amount) was added. The mixture was stirred for 3 h.
The reaction was quenched by the addition of water (2 mL), and
the pH was adjusted to 7. The resulting solid was filtered, washed
with a little water and dried to obtain 14–16.
4.1.10.3. 5,7-Dimethoxyquinolin-4-yl 3-chlorobenzoate (19). Yield:
85%; mp 149–151 °C; 1H NMR (CDCl3, 300 MHz) d: 3.57 (3H, s), 3.92
(3H, s), 6.48 (1H, d, J = 2.1 Hz), 7.00 (1H, d, J = 4.8 Hz), 7.11 (1H, d,
J = 2.1 Hz), 7.48 (1H, t, J = 8.0 Hz), 7.64 (1H, d, J = 8.0 Hz), 8.11 (1H, d,
J = 8.0 Hz), 8.22 (1H, s), 8.79 (1H, d, J = 4.8 Hz); 13C NMR (CDCl3,
75 MHz) d: 55.77, 56.05, 99.88, 100.72, 110.68, 112.76, 128.55,
130.10, 130.44, 131.40, 133.77, 134.92, 151.60, 153.38, 154.96,
155.87, 161.46, 163.88; HRMS-ESI (m/z): calcd for C18H14ClNO4Na
[M+Na]+: 366.0509, found: 366.0505.
4.1.9.1. 5,7-Dimethoxyquinolin-4-yl b-D-glucopyranosyl ether
(14). Yield 31%; mp 188–190 °C; 1H NMR (C5D5N, 300 MHz) d:
3.63 (3H, s), 3.76 (3H, s), 4.21 (1H, s), 4.40–4.58 (5H, m), 5.86
(1H, d, J = 6.8 Hz), 6.65 (1H, s), 7.30 (1H, d, J = 4.8 Hz), 7.35
(1H, s), 8.86 (1H, d, J = 4.8 Hz); 13C NMR (C5D5N, 75 MHz) d:
56.92, 57.70, 63.75, 72.52, 76.53, 80.20, 80.74, 100.89, 102.71,
102.84, 105.20, 111.03, 153.67, 155.71, 160.08, 162.81, 164.12;
HRMS-ESI (m/z): calcd for C17H22NO8 [M+H]+: 368.1345, found:
368.1344.
4.1.10.4. 5,7-Dimethoxyquinolin-4-yl 4-chlorobenzoate (20). Yield:
87%; mp 139–140 °C; 1H NMR (CDCl3, 300 MHz) d: 3.56 (3H, s), 3.93
(3H, s), 6.49 (1H, d, J = 2.1 Hz), 7.00 (1H, d, J = 4.9 Hz), 7.11 (1H, d,
J = 2.1 Hz), 7.53 (2H, d, J = 8.5 Hz), 8.17 (2H, d, J = 8.5 Hz), 8.79 (1H, d,
J = 4.9 Hz); 13C NMR (DMSO-d6, 75 MHz) d: 55.77, 56.01, 99.85,
100.67, 110.78, 112.83, 128.09, 129.14, 131.82, 140.27, 151.54,
153.32, 155.10, 155.89, 161.46, 164.21; HRMS-ESI (m/z): calcd for
C18H14ClNO4Na [M+Na]+: 366.0509, found: 366.0508.
4.1.9.2. 5,7-Dimethoxyquinolin-4-yl b-
(15).
D-galactopyranosyl ether
Yield 33%; mp 214–216 °C; 1H NMR (C5D5N, 300 MHz) d:
3.55 (3H, s), 3.78 (3H, s), 4.36–4.55 (4H, m), 4.68 (1H, d, J = 3.0 Hz),
4.96 (1H, dd, J = 9.3, 7.8 Hz), 5.80 (1H, d, J = 7.5 Hz), 6.64 (1H, d,
J = 2.4 Hz), 7.35 (1H, d, J = 5.4 Hz), 7.36 (1H, d, J = 2.4 Hz), 8.86
(1H, d, J = 5.4 Hz). 13C NMR (C5D5N, 75 MHz) d: 56.46, 57.29,
63.66, 71.22, 73.50, 76.55, 79.17, 100.54, 102.42, 103.40, 104.91,
109.08, 153.38, 155.27, 159.82, 162.48, 163.97; HRMS-ESI (m/z):
calcd for C17H22NO8 [M+H]+: 368.1345, found: 368.1345.
4.1.10.5. 5,7-Dimethoxyquinolin-4-yl
2,4-dichlorobenzoate
(21). Yield: 81%; mp 174–175 °C; 1H NMR (CDCl3, 300 MHz) d:
3.67 (3H, s), 3.92 (3H, s), 6.51 (1H, d, J = 2.1 Hz), 6.99 (1H, d,
J = 4.8 Hz), 7.10 (1H, d, J = 2.1 Hz), 7.42 (1H, dd, J = 8.5, 1.8 Hz),
7.58 (1H, d, J = 1.8 Hz), 8.19 (1H, d, J = 8.5 Hz), 8.79 (1H, d,
J = 4.8 Hz); 13C NMR (CDCl3, 75 MHz) d: 55.78, 56.12, 99.94,
100.83, 110.69, 112.77, 126.83, 127.34, 131.64, 133.73, 136.42,
139.50, 151.65, 153.40, 154.55, 155.75, 161.43, 162.22; HRMS-ESI
(m/z): calcd for C18H14Cl2NO4 [M+H]+: 378.0300, found: 378.0296.
4.1.9.3. 5,7-Dimethoxyquinolin-4-yl b-D-xylopyranosyl ether
(16). Yield 38%; mp 118–120 °C; 1H NMR (C5D5N, 300 MHz) d:
3.72 (3H, s), 3.78 (3H, s), 3.99 (1H, dd, J = 11.2, 7.8 Hz), 4.31–4.50
(4H, m), 5.89 (1H, d, J = 6.0 Hz), 6.67 (1H, d, J = 2.1 Hz), 7.26 (1H,
d, J = 5.4 Hz), 7.39 (1H, d, J = 2.1 Hz), 8.87 (1H, d, J = 5.4 Hz); 13C
NMR (C5D5N, 75 MHz) d: 56.91, 57.64, 67.96, 72.04, 75.21, 78.27,
100.91, 102.65, 102.91, 105.14, 110.91, 153.55, 155.60, 159.72,
162.83, 163.58; HRMS-ESI (m/z): calcd for C16H20NO7 [M+H]+:
338.1240, found: 338.1242.
4.1.10.6. 5,7-Dimethoxyquinolin-4-yl 2,6-dichlorobenzoate (22). Yield:
60%; mp 173–174 °C; 1H NMR (CDCl3, 300 MHz) d: 3.85 (3H, s), 3.92
(3H, s), 6.53 (1H, d, J = 2.1 Hz), 7.08 (1H, d, J = 2.1 Hz), 7.21 (1H, d,
J = 5.0 Hz), 7.32–7.43 (3H, m), 8.80 (1H, d, J = 5.0 Hz); 13C NMR (CDCl3,
75 MHz) d: 55.74, 56.15, 99.80, 100.39, 110.70, 112.53, 128.62, 131.84,
131.94, 132.97, 151.31, 153.66, 154.54, 156.45, 161.56, 162.45; HRMS-ESI
(m/z): calcd for C18H14Cl2NO4 [M+H]+: 378.0300, found: 378.0305.

G.-B. Liu et al. / Bioorg. Med. Chem. 17 (2009) 5433–5441
5439
4.1.10.7. 5,7-Dimethoxyquinolin-4-yl 3,5-dichlorobenzoate (23). Yield:
88%; mp 232–233 °C; 1H NMR (CDCl3, 300 MHz) d: 3.61 (3H, s), 3.94 (3H,
s), 6.51 (1H, d, J = 2.1 Hz), 7.00 (1H, d, J = 4.9 Hz), 7.12 (1H, d, J = 2.1 Hz),
7.66 (1H, t, J = 1.8 Hz), 8.11 (2H, d, J = 1.8 Hz), 8.80 (1H, d, J = 4.9 Hz); 13C
NMR (CDCl3, 75 MHz) d: 55.83, 56.18, 100.06, 100.78, 110.47, 112.61,
128.78, 132.50, 133.61, 135.75, 151.58, 153.38, 154.68, 155.76, 161.55,
162.77; HRMS-ESI (m/z): calcd for C18H14Cl2NO4 [M+H]+: 378.0300, found:
378.0303.
4.1.10.14. 5,7-Dimethoxyquinolin-4-yl 4-iodobenzoate (30). Yield:
90%; mp 185–186 °C; 1H NMR (CDCl3, 300 MHz) d: 3.56 (3H, s), 3.93
(3H, s), 6.48 (1H, d, J = 2.1 Hz), 6.99 (1H, d, J = 5.0 Hz), 7.10 (1H, d,
J = 2.1 Hz), 7.90 (2H, d, J = 8.9 Hz), 7.94 (2H, d, J = 8.9 Hz), 8.78 (1H, d,
J = 5.0 Hz); 13C NMR (CDCl3, 75 MHz) d: 55.76, 56.02, 99.83, 100.64,
101.77, 110.74, 112.80, 129.09, 131.78, 138.13, 151.53, 153.30,
155.06, 155.86, 161.44, 164.61; HRMS-ESI (m/z): calcd for C18H15INO4
[M+H]+: 436.0046, found: 436.0045.
4.1.10.8. 5,7-Dimethoxyquinolin-4-yl 2-bromobenzoate (24). Yield:
90%; mp 155–156 °C; 1H NMR (CDCl3, 300 MHz) d: 3.70 (3H, s), 3.93
(3H, s), 6.52 (1H, d, J = 1.9 Hz), 7.02 (1H, d, J = 5.0 Hz), 7.11 (1H, d,
J = 1.9 Hz), 7.42–7.52 (2H, m), 7.78 (1H, d, J = 7.8 Hz), 8.23 (1H, dd,
J = 7.4, 1.8 Hz), 8.79 (1H, d, J = 5.0 Hz); 13C NMR (CDCl3, 75 MHz) d:
55.77, 56.15, 99.88, 100.71, 110.86, 112.83, 123.28, 127.44, 130.29,
132.77, 133.62, 135.12, 151.58, 153.34, 154.83, 155.88, 161.44,
163.47; HRMS-ESI (m/z): calcd for C18H15BrNO4 [M+H]+: 388.0184,
found: 388.0187.
4.1.10.15. 5,7-Dimethoxyquinolin-4-yl 2-nitrobenzoate (31). Yield:
80%; mp 170–172 °C; 1H NMR (CDCl3, 300 MHz) d: 3.76 (3H, s), 3.93
(3H, s), 6.53 (1H, d, J = 1.8 Hz), 7.10–7.11 (2H, m), 7.71–7.81 (2H, m),
7.97 (1H, d, J = 7.9 Hz), 8.04 (1H, d, J = 7.2 Hz), 8.81 (1H, d, J = 5.0 Hz);
13C NMR (CDCl3, 75 MHz) d: 55.81, 56.30, 100.18, 100.76, 110.71,
112.39, 124.33, 126.40, 130.56, 132.80, 132.90, 151.69, 153.31,
154.23, 155.82, 161.49, 162.99; HRMS-ESI (m/z): calcd for C18H15N2O6
[M+H]+: 355.0930, found: 355.0925.
4.1.10.16. 5,7-Dimethoxyquinolin-4-yl 3-nitrobenzoate (32). Yield:
90%; mp 210–211 °C; 1H NMR (CDCl3, 300 MHz) d: 3.58 (3H, s), 3.94
(3H, s), 6.51 (1H, d, J = 2.1 Hz), 7.04 (1H, d, J = 4.9 Hz), 7.12 (1H, d,
J = 2.1 Hz), 7.77 (1H, t, J = 8.0 Hz), 8.51–8.57 (2H, m), 8.81 (1H, d,
J = 4.9 Hz), 9.09 (1H, s); 13C NMR (CDCl3, 75 MHz) d: 55.79, 56.13,
100.07, 100.84, 110.34, 112.59, 125.35, 128.13, 130.08, 131.49, 135.98,
148.58, 151.58, 153.42, 154.56, 155.72, 161.51, 162.96; HRMS-ESI (m/
z): calcd for C18H15N2O6 [M+H]+: 355.0930, found: 355.0937.
4.1.10.9. 5,7-Dimethoxyquinolin-4-yl 3-bromobenzoate (25). Yield:
88%; mp 165–166 °C; 1H NMR (CDCl3, 300 MHz) d: 3.58 (3H, s), 3.93
(3H, s), 6.49 (1H, d, J = 2.1 Hz), 7.00 (1H, d, J = 4.8 Hz), 7.11 (1H, d,
J = 2.1 Hz), 7.43 (1H, t, J = 8.0 Hz), 7.79 (1H, d, J = 8.0 Hz), 8.16 (1H, d,
J = 8.0 Hz), 8.39 (1H, s), 8.79 (1H, d, J = 4.8 Hz); 13C NMR (CDCl3,
75 MHz) d: 55.76, 56.05, 99.87, 100.71, 110.69, 112.73, 122.78,
128.98, 130.32, 131.57, 133.35, 136.66, 151.57, 153.36, 154.94,
155.86, 161.44, 163.72; HRMS-ESI (m/z): calcd for C18H15BrNO4
[M+H]+: 388.0184, found: 388.0179.
4.1.10.17. 5,7-Dimethoxyquinolin-4-yl 4-nitrobenzoate (33). Yield:
90%; mp 220–221 °C; 1H NMR (DMSO-d6, 300 MHz) d: 3.53 (3H, s),
3.91 (3H, s), 6.68 (1H, d, J = 2.0 Hz), 7.11 (1H, d, J = 2.0 Hz), 7.26 (1H,
d, J = 4.7 Hz), 8.38 (2H, d, J = 8.7 Hz), 8.46 (2H, d, J = 8.7 Hz), 8.84 (1H,
d, J = 4.7 Hz); 13C NMR (DMSO-d6, 75 MHz) d: 55.64, 56.28, 99.86,
100.63, 109.62, 112.82, 124.18, 131.36, 134.22, 150.64, 151.92,
152.64, 153.92, 155.28, 160.99, 163.06; HRMS-ESI (m/z): calcd for
C18H15N2O6 [M+H]+: 355.0930, found: 355.0932.
4.1.10.10. 5,7-Dimethoxyquinolin-4-yl 4-bromobenzoate(26). Yield:
86%; mp 165–166 °C; 1H NMR (CDCl3, 300 MHz) d: 3.54 (3H, s), 3.91 (3H,
s), 6.47 (1H, d, J = 2.2 Hz), 6.98 (1H, d, J = 4.9 Hz), 7.09 (1H, d, J = 2.2 Hz),
7.67 (2H, d, J = 8.5 Hz), 8.08 (2H, d, J = 8.5 Hz), 8.78 (1H, d, J = 4.9 Hz);
13C NMR (CDCl3, 75 MHz) d: 55.72, 55.98, 99.79, 100.68, 110.70, 112.79,
128.53, 128.94, 131.59, 131.89, 132.10, 151.58, 153.35, 154.99, 155.83,
161.39, 164.33; HRMS-ESI (m/z): calcd for C18H15BrNO4 [M+H]+:
388.0184, found: 388.0187.
4.1.10.18. 5,7-Dimethoxyquinolin-4-yl
3,5-dinitrobenzoate
(34). Yield: 81%; mp 251–253 °C; 1H NMR (CDCl3–CF3COOD,
300 MHz) d: 3.84 (3H, s), 4.06 (3H, s), 6.87 (1H, s), 7.21 (1H, s),
7.62 (1H, d, J = 6.4 Hz), 8.94 (1H, d, J = 6.4 Hz), 9.43 (3H, s); 13C
NMR (CF3COOD, 75 MHz) d: 55.80, 56.14, 102.93, 108.67, 112.43,
112.65, 119.93, 123.85, 130.13, 143.86, 148.97, 160.90, 161.47,
162.06; HRMS-ESI (m/z): calcd for C18H14N3O8 [M+H]+: 400.0781,
found: 400.0782.
4.1.10.11. 5,7-Dimethoxyquinolin-4-yl 3,5-dibromobenzoate (27).
Yield: 86%; mp 224–225 °C; 1H NMR (CDCl3, 300 MHz) d: 3.62 (3H, s), 3.94
(3H, s), 6.51 (1H, d, J = 2.0 Hz), 7.00 (1H, d, J = 4.9 Hz), 7.12 (1H, d, J = 2.0 Hz),
7.96 (1H, t, J = 1.5 Hz), 8.31 (2H, d, J = 1.5 Hz), 8.79 (1H, d, J = 4.9 Hz); 13C
NMR (CDCl3, 75 MHz) d: 55.84, 56.20, 110.07, 100.75, 110.47, 112.61,
123.43, 132.11, 132.86, 139.08, 151.54, 153.34, 154.70, 155.76, 161.58,
162.52; HRMS-ESI (m/z): calcd for C18H14Br2NO4 [M+H]+: 465.9290, found:
465.9289.
4.1.10.19. 5,7-Dimethoxyquinolin-4-yl methyl terephthalate
(35). Yield: 82%; mp 167–168 °C; 1H NMR (CDCl3, 300 MHz) d:
3.53 (3H, s), 3.92 (3H, s), 3.97 (3H, s), 6.48 (1H, d, J = 2.2 Hz), 7.01
(1H, d, J = 5.0 Hz), 7.10 (1H, d, J = 2.2 Hz), 8.20 (2H, d, J = 8.4 Hz),
8.30 (2H, d, J = 8.4 Hz), 8.79 (1H, d, J = 5.0 Hz); 13C NMR (CDCl3,
75 MHz) d: 52.71, 55.77, 55.98, 99.88, 100.70, 110.68, 112.77,
129.89, 130.41, 133.39, 134.64, 151.58, 153.35, 155.03, 155.84,
161.47, 164.29, 166.32; HRMS-ESI (m/z): calcd for C20H18NO6
[M+H]+: 368.1134, found: 368.1131.
4.1.10.12. 5,7-Dimethoxyquinolin-4-yl 2-iodobenzoate (28). Yield:
82%; mp 151–152 °C; 1H NMR (CDCl3, 300 MHz) d: 3.68 (3H, s), 3.93
(3H, s), 6.51 (1H, d, J = 2.0 Hz), 7.03 (1H, d, J = 5.0 Hz), 7.11 (1H, d,
J = 2.0 Hz), 7.26 (1H, m), 7.53 (1H, t, J = 7.5 Hz), 8.12 (1H, d, J = 8.0 Hz),
8.26 (1H, dd, J = 7.5, 1.2 Hz), 8.79 (1H, d, J = 5.0 Hz); 13C NMR (CDCl3,
75 MHz) d: 55.76, 56.19, 95.62, 99.86, 100.70, 110.82, 112.82, 128.19,
132.46, 132.87, 133.66, 142.20, 151.58, 153.33, 154.88, 155.85,
161.41, 163.89; HRMS-ESI (m/z): calcd for C18H15INO4 [M+H]+:
436.0046, found: 436.0042.
4.1.10.20. 5,7-Dimethoxyquinolin-4-yl 2-fluorobenzoate (36). Yield:
80%; mp 143–145 °C; 1H NMR (CDCl3, 300 MHz) d: 3.62 (3H, s), 3.92
(3H, s), 6.49 (1H, d, J = 2.0 Hz), 7.00 (1H, d, J = 4.8 Hz), 7.10 (1H, d,
J = 2.0 Hz), 7.20–7.32 (2H, m), 7.58–7.65 (1H, m), 8.14–8.19 (1H, m),
8.78 (1H, d, J = 4.8 Hz); 13C NMR (CDCl3, 75 MHz) d: 55.73, 56.01,
99.83, 100.71, 110.83, 112.83, 117.20, 117.50, 124.26, 132.81, 135.35,
151.60, 153.35, 154.79, 155.89, 160.78, 161.41, 164.25; HRMS-ESI (m/
z): calcd for C18H15FNO4 [M+H]+: 328.0985, found: 328.0981.
4.1.10.13. 5,7-Dimethoxyquinolin-4-yl 3-iodobenzoate (29). Yield:
85%; mp 176–178 °C; 1H NMR (CDCl3, 300 MHz) d: 3.56 (3H, s), 3.90
(3H, s), 6.46 (1H, d, J = 2.1 Hz), 6.97 (1H, d, J = 4.8 Hz), 7.08 (1H, d,
J = 2.1 Hz), 7.26 (1H, m), 7.96 (1H, d, J = 7.9 Hz), 8.16 (1H, d,
J = 7.9 Hz), 8.56 (1H, s), 8.76 (1H, d, J = 4.8 Hz); 13C NMR (CDCl3,
75 MHz) d: 55.78, 56.08, 94.05, 99.87, 100.69, 110.68, 112.76, 129.58,
130.43, 131.51, 139.22, 142.54, 151.56, 153.35, 154.96, 155.87,
161.45, 163.57; HRMS-ESI (m/z): calcd for C18H15INO4 [M+H]+:
436.0046, found: 436.0042.
4.1.10.21. 5,7-Dimethoxyquinolin-4-yl 2-methylbenzoate (37). Yield:
85%; mp 138–139 °C; 1HNMR(CDCl3, 300 MHz) d: 2.67, (3H, s) 3.61, (3H, s)

5440
G.-B. Liu et al. / Bioorg. Med. Chem. 17 (2009) 5433–5441
3.92 (3H, s), 6.49 (1H, d, J = 2.2 Hz), 6.98 (1H, d, J = 4.8 Hz), 7.10 (1H, d,
J = 2.2 Hz), 7.32–7.38 (2H, m), 7.47–7.52 (1H, m), 8.26 (1H, d, J = 7.2 Hz),
8.78 (1H, d, J = 4.8 Hz); 13C NMR (CDCl3, 75 MHz) d: 21.84, 55.70, 55.89,
99.67, 100.68, 111.09, 113.04, 126.04, 128.26, 131.75, 132.03, 132.90,
141.71, 151.59, 153.35, 155.28, 155.97, 161.33, 165.30; HRMS-ESI (m/z):
calcd for C19H18NO4 [M+H]+: 324.1236, found: 324.1230.
mals’ (National Research Council, 1996) and the related ethical reg-
ulations of our university. All efforts were made to minimize the
animals’ suffering and to reduce the number of animals used.
4.2.3. Preparation of spleen cell suspensions
BALB/c mice were sacrificed and their spleens were removed
aseptically. A single cell suspension was prepared after cell debris,
and clumps were removed. Erythrocytes were depleted with Tris–
buffered ammonium chloride (0.155 M NH4Cl, 16.5 mM Tris, pH
7.2). Lymphocytes were washed three times with RPMI-1640 con-
taining 10% FBS and were resuspended in RPMI-1640 medium sup-
plemented with 10% FBS, penicillin (100 U/mL), and streptomycin
(100 U/mL).
4.1.10.22.
5,7-Dimethoxyquinolin-4-yl 2-methoxybenzoate
(38). Yield: 83%; mp 158–160 °C; 1H NMR (CDCl3, 300 MHz) d:
3.66 (3H, s), 3.92 (3H, s), 3.93 (3H, s), 6.48 (1H, d, J = 2.1 Hz), 7.00
(1H, d, J = 4.9 Hz), 7.04–7.11 (3H, m), 7.55–7.60 (1H, m), 8.16
(1H, dd, J = 7.6, 1.8 Hz), 8.76 (1H, d, J = 4.9 Hz); 13C NMR (CDCl3,
75 MHz) d: 55.68, 55.95, 56.10, 99.55, 100.57, 111.21, 112.25,
113.17, 118.54, 120.25, 132.88, 134.65, 151.55, 153.30, 155.28,
156.10, 160.39, 161.27, 163.42; HRMS-ESI (m/z): calcd for
C19H18NO5 [M+H]+: 340.1185, found: 340.1189.
4.2.4. MTT assay
Fresh spleen cells were obtained from BALB/c mice (male,
6–8 weeks old). 5 ꢁ 105 spleen cells were cultured in triplicate in
4.1.10.23. 5,7-Dimethoxyquinolin-4-yl
4-methoxybenzoate
(39). Yield 86%; mp 178–181 °C; 1H NMR (CDCl3, 300 MHz) d:
3.55 (3H, s), 3.90 (3H, s), 3.91 (3H, s), 6.46 (1H, d, J = 2.1 Hz), 6.98
(1H, d, J = 4.8 Hz), 7.01 (2H, d, J = 8.7 Hz), 7.08 (1H, d, J = 2.1 Hz),
8.18 (2H, d, J = 8.7 Hz), 8.77 (1H, d, J = 4.8 Hz). 13C NMR (CDCl3,
75 MHz) d: 55.49, 55.55, 55.81, 99.48, 100.51, 110.96, 112.92,
113.83, 121.82, 132.41, 151.46, 153.27, 155.30, 155.98, 161.17,
163.83, 164.63; HRMS-ESI (m/z): calcd for C19H18NO5 [M+H]+:
340.1185, found: 340.1180.
96-well flat plates with 200 lL RPMI-1640 media containing 10%
FBS, 100 U/mL penicillin, and 100 U/mL streptomycin in a humidi-
fied, 37 °C, 5% CO2-containing incubator for 48 h in the presence or
absence of various concentrations of compounds. The cells with
media alone were used as control. 20
was added 4 h before the end of culture. Then 90
l
L MTT (5 mg/mL) reagent
L of lysis buffer
l
(10% SDS, 50% DMF, pH 7.2) was added to each well for 6 h and the
absorbance value at 570 nm was collected by microplate reader.
The percentage of cell growth inhibition was determined using
the following formula:
4.1.11. 5,7-Dimethoxyquinolin-4-yl 4-methylbenzenesulfonate
(40)
Cytotoxicity ð%Þ
To a solution of 4 (205 mg, 1 mmol) in CH2Cl2 (10 mL) at rt was
added Et3N (0.56 mL, 4 mmol) and p-toluene sulfonyl chloride
(380 mg, 2 mmol), and the solution was refluxed for 5 h. The reac-
tion was quenched by the addition of water (10 mL), and the aque-
ous layer was extracted with CH2Cl2 (3 ꢁ 20 mL). The organic layer
was washed with diluted hydrochloric acid, saturated NaHCO3, and
brine, and dried over anhydrous Na2SO4. After evaporation of the
solvent in vacuo, the crude product was recrystallized in MeOH
to yield 40. Yield: 83%; mp 148–149 °C; 1H NMR (CDCl3,
300 MHz) d: 2.40 (3H, s), 3.76 (3H, s), 3.88 (3H, s), 6.45 (1H, d,
J = 2.2 Hz), 6.80 (1H, d, J = 5.0 Hz), 6.99 (1H, d, J = 2.2 Hz), 7.27
(2H, d, J = 8.2 Hz), 7.72 (2H, d, J = 8.2 Hz), 8.63 (1H, d, J = 5.0 Hz);
13C NMR (CDCl3, 75 MHz) d: 21.79, 55.70, 55.90, 100.06, 100.30,
110.80, 112.06, 128.49, 129.76, 133.48, 145.49, 151.13, 153.20,
153.63, 156.20, 161.62; HRMS-ESI (m/z): calcd for C18H18NO5S
[M+H]+: 360.0906, found: 360.0910.
¼ ð1 ꢀ ½Compounds ðOD570Þ ꢀ Background ðOD570Þꢂ
=½Control ðOD570Þ ꢀ Background ðOD570ÞꢂÞ ꢁ 100
The compounds were dissolved in dimethylsulfoxide (DMSO)
followed by dilution with culture medium to desired concentra-
tions, and DMSO final concentration was 0.1%. DMSO at 0.1% was
added into control group and showed no effects on cells.
4.2.5. ConA-induced T cell proliferation assay
Fresh spleen cells were obtained from BALB/c mice (male,
6–8 weeks). 5 ꢁ 105 Spleen cells were cultured at the same condi-
tions as those mentioned above. The cultures were unstimulated or
stimulated with 5 lg/mL ConA to induce T cell proliferative re-
sponse. The compounds were added to cultures with desired con-
centrations to test their bio-activities. The cells stimulated with
ConA were used as control. Proliferation was assessed by MTT as-
say as above. In some experiments, proliferation was assessed in
terms of uptake of [3H]thymidine during 8 h of pulsing with
20 kBq [3H]thymidine for each well, and then cells will be har-
vested onto glass fiber filters by a Basic 96 harvester. The incorpo-
rated radioactivity was counted by a liquid scintillation counter.
4.2. Biological assays
4.2.1. Materials
Stock solutions of compounds were prepared with dimethyl-
sulfoxide (DMSO, Sigma) and diluted with RPMI-1640 medium
containing 10% fetal bovine serum (FBS). MTT (3-(4,5-dim-
ethylthylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide), Conca-
navalin A (ConA) was purchased from Sigma (St Louis, Mo).
Cyclosporin A (CsA) was obtained from Sandoz Ltd (Basel, Switzer-
land). [3H]Thymidine (1 mCi/mL) was purchased from Nanjing
Medical University. CD25-FITC and CD4-APC antibody were pur-
chased from BD Pharmingen (San Diego, CA).
Inhibition rate ð%Þ ¼ ð1 ꢀ ½3Hꢂ Compound=½3Hꢂ ControlÞ ꢁ 100
4.2.6. FACS assay of mitogen-induced T cell activation
Fresh spleen cells were obtained from BALB/c mice (male,
6–8 weeks old). 5 ꢁ 105 spleen cells were cultured at the same
conditions as those mentioned above. The cultures were unstimu-
lated or stimulated with 5 lg/mL ConA to induce T cell activation
4.2.2. Animals
response. Cells were incubated in the presence or absence of vari-
ous concentrations of compounds. Twenty four hours later cells
were collected and washed with cold PBS twice and stained with
CD25-FITC and CD4-APC antibody in 4 °C for 30 min. After washed
Female BALB/c mice (6–8 weeks old, 18–22 g) were supplied by
the Laboratory Animal Center of Yangzhou University (Jiangsu, Chi-
na). They were maintained with free access to pellet food and water
in plastic cages at 21 2 °C and kept on a 12-h light–dark cycle. Ani-
mal welfare and experimental procedures were carried out strictly
in accordance with the ‘Guide for the Care and Use of Laboratory Ani-
once with cold PBS, cells were resuspended in 400
lL and applied
for FACS assay.

G.-B. Liu et al. / Bioorg. Med. Chem. 17 (2009) 5433–5441
5441
14. Chen, H.; Du, X.; Tang, W.; Zhou, Y.; Zuo, J.; Feng, H.; Li, Y. Bioorg. Med. Chem.
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4.2.7. Statistics
All experiments were repeated 3–5 times with the similar out-
come. The p-values between two experimental groups were tested
by two-tailed Student’s t-test, and p-values of 0.05 or less were
considered to be statistically significant.
17. Baba, A.; Kawamura, N.; Makino, H.; Ohta, Y.; Taketomi, S.; Sohda, T. J. Med.
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Acknowledgment
18. Green, N.; Hu, Y. H.; Janz, K.; Li, H. Q.; Kaila, N.; Guler, S.; Thomason, J.; Joseph-
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This work was supported by National Natural Science Fund for
Creative Research Groups of China (20521503) and National Natu-
ral Science Foundation of China (30730107 and 30672472).
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Supplementary data
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