Design, synthesis, and biological evaluation of novel 4-hydro-quinoline-3- carboxamide derivatives as an immunomodulator

Article abstract of DOI:10.1016/j.bmcl.2005.04.040

A series of novel quinoline-3-carboxamide derivatives were synthesized and evaluated for their immunomodulatory activity. The compounds were tested in vitro for effects on spleen lymphocyte proliferation and TNF-α production by macrophage. Three compounds showed immunomodulatory profiles similar to and more potent than those of linomide and FR137316 and were selected for further pharmacological studies in vivo.

Full text of DOI:10.1016/j.bmcl.2005.04.040

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2980–2985
Design, synthesis, and biological evaluation
of novel 4-hydro-quinoline-3-carboxamide
derivatives as an immunomodulator
Jun-Feng He,* Liu-Hong Yun, Ri-Fang Yang, Zhi-Yong Xiao, Jun-Ping Cheng,
Wen-Xia Zhou and Yong-Xiang Zhang
Beijing Institute of Pharmacology and Toxicology, Department of Medicinal Chemistry, 27 Taiping Road, Beijing 100850, China
Received 14 March 2005; revised 11 April 2005; accepted 22 April 2005
Abstract—A series of novel quinoline-3-carboxamide derivatives were synthesized and evaluated for their immunomodulatory activ-
ity. The compounds were tested in vitro for effects on spleen lymphocyte proliferation and TNF-a production by macrophage. Three
compounds showed immunomodulatory profiles similar to and more potent than those of linomide and FR137316 and were selected
for further pharmacological studies in vivo.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
into effector cells.8,9 The SAR study of leflunomide indi-
cated that the active pharmacophore responsible for the
immunosuppressive effects of A-771726 was a b-keto
amide with the enolic hydroxy group fixed in a cis con-
figuration to the amide moiety.10 Interestingly, linomide
and FR137316 have the same enolic hydroxy group fixed
in a cis configuration to the amide moiety. Thus we pre-
Linomide (N-phenylmethyl-1,2-dihydro-4-hydroxyl-1-
methyl-2-oxo-quinoline-3-carboxamide) has been shown
to be effective against various types of autoimmune dis-
eases such as multiple sclerosis, rheumatoid arthritis,
systemic lupus erythematosis, and lupus nephritis.1–3
Recently, linomide and its derivatives FR137316 and
FR165009 have also been reported to have antinephritic
activity.4,5 Although the mechanism of action for lino-
mide is still not completely understood, the results from
several studies have suggested that linomide may exert
its effect via the modulation of antigen presenting cell
(APC) function.6,7
Leflunomide, an isoxazol derivative structurally unre-
lated to other known antirheumatic drugs, is a prodrug
rapidly converted in vivo to its active metabolite (A-
771726), which is a potent noncytotoxic inhibitor of
the dihydroorotate dehydrogenase (DHODH), a key en-
zyme in the de novo synthesis of uridine monophos-
phate (UMP). Activated lymphocytes depend on the
pyrimidine de novo syntheses to fulfill their metabolic
needs for clonal expansion and terminal differentiation
Keywords: Immunomodulator; Quinoline-3-carboxamide; Linomide;
SAR.
*
Corresponding author. Tel.: +86 10 66874611; fax: +86 10
68211656; e-mail: hjf3770@yahoo.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.040

J.-F. He et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2980–2985
2981
Scheme 1. Reagents and conditions: (a) toluene, 105 ꢁC; (b) diphenyl oxide, 250 ꢁC; (c) R2R3NH, diphenyl oxide, 210 ꢁC.
sume that this structural element of the enolic hydroxy
group is necessary for the immunomodulatory activity
of these amide compounds. In addition, the immunomod-
ulatory SAR studies of 4-hydroxyl-2-oxo-quinoline-
3-carboxamide derivatives have been published,11–13
and the action of 2-oxo group at the quinoline ring is
not clear. In this letter, we report the synthesis and
biological evaluation of novel 4-hydroxyl-quinoline-3-
carboxamides(I) whose scaffolds are free of 2-oxo group.
modulatory activity. Although it is not valid at the 6-
and 8-position, the activity could be preserved when
two methyl groups were simultaneously introduced at
the 6- and 8-position. For example, the immunomodula-
tory activity of compounds 6, 7, 12, and 19 was higher
than that of compounds 5, 9, and 11. Tables 1 and 2
show that the activities of compounds 6 and 7 substi-
tuted with electron-donating substituents (MeS, MeO)
were remarkably higher than those of compounds 8
and 10 substituted with electron-withdrawing substitu-
ents (CF3, F), indicating that the class of substituents
is an important factor to immunomodulatory activity.
It is surprising to find that the introduction of carboxy
group at 6-position of quinoline ring strongly enhanced
the inhibition of spleen lymphocyte proliferation reac-
tion but there was no evident effect on TNF-a produc-
tion by macrophage compared to linomide and
FR137316, which supposes that compounds 15 and 16
might have different immunomodulatory activities from
those of linomide and FR137316. We can also see in the
table that the activities were maintained for most of the
compounds with R2 and R3 substituted by methyl and
aromatic groups, except for compound 18 in which R2
and R3 were hydrogen and 2-hydroxyethyl group, hav-
ing a high immunomodulatory activity; it means the
substituents at the N atom of amide (R2, R3) also in-
fluence the immunomodulatory activity.
2. Chemistry
Compounds were synthesized as outlined in Scheme 1.
The syntheses of the target compounds required access
to a number of different substituted 4-hydroyl-3-quino-
linecarboxylic ester derivatives. In detail, both the con-
densation of the substituted aniline with diethyl
ethoxymethylenemalonate (EMME) in toluene at reflux-
ing temperature and the subsequent thermal cyclization
of anilinomethylenemalonate in diphenyl ether at about
250 ꢁC gave good yields.14,15 The target compounds
could be prepared by condensation of the 4-hydroyl-3-
quinolinecarboxylic ester with the appropriate amine
in diphenyl ether at about 210 ꢁC while the formed
ethanol is distilled off. The products were purified by
chromatography on silica gel column (dichlorometh-
ane–methanol, 20:1) or by recrystallization from ethanol.
4. Conclusion
In this paper, we have described the synthesis and
immunomodulatory activity of a series of novel quino-
line-3-carboxamide derivatives. The SAR of those com-
pounds demonstrated that the 2-oxo group of the
quinoline ring in linomide and FR137316 might be
unimportant for their immunomodulatory activities;
the type and position of quinoline ring substitution
played an important role in sustaining the activity, and
furthermore the N-carboxamide substitution has an evi-
dent effect. Mostly, introduction of electron-donating
substituents at the 7-position of quinoline ring is advan-
tageous to sustain the activity. The introduction of car-
boxy group into quinoline ring may change the
immunomodulatory type. Compounds 6, 18, and 1917
showed immunomodulatory profiles similar to and
more potent than those of linomide and FR137316
and were selected for further pharmacological and
3. Results and discussion
Linomide and FR137316 were inhibitors of T-cell prolif-
eration induced by ConA and TNF-a secreted by
macrophage in vitro. Thus, the synthesized quinoline-
3-carboxamide derivatives were tested for their effects
on spleen lymphocyte proliferation and TNF-a produc-
tion by macrophage in preliminary screens.16 The test
results rate % inhibition of spleen lymphocyte and
TNF-a are summarized in Tables 1 and 2.
It can be seen from Tables 1 and 2 that the immuno-
modulatory activity was either preserved or enhanced
with the introduction of substituents at the 7-position,
compared to linomide and FR137316; this means that
the substituents position is related with the immuno-

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J.-F. He et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2980–2985
Table 1. 4-Hydroxyl-quinoline-3-carboxamide and its effect on spleen lymphocyte proliferation
Compound
R1
R2
R3
%Inhibition of T-cell
proliferation induced by ConA
%Inhibition of B-cell proliferation
reaction induced by LPS
Dose (lg/mL)
Dose (lg/mL)
1
10
70.6a
100
78.5a
1
10
100
Linomide
FR137316
58.1a
52.0a
À24.7a
À7.55
11.4
0.00
13.6
24.6
30.3
62.7a
37.8a
14.1
75.3b
À27.6a
24.9
79.6b
41.2b
À9.33
81.7b
76.0b
53.7b
93.6b
96.3b
17.1
3
7-Cl
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
À4.33
17.7
À26.7
4
8.46
32.4
5
8-MeS
7-MeS
7-MeO
7-CF3
6-MeS
7-F
52.4a
11.8
À2.46
36.5a
17.0
24.9
6.41
71.7a
83.5
6
26.2b
36.5a
54.7b
31.1
7
29.3
41.3a
39.8a
57.5b
64.9b
65.6b
À126.7a
8
4.11
9
À209b
À46.8a
À59.3
1.84
À155a
À21.5
À20.0
12.9
À47.8
4.00
À114a
10
11
12
12.5
6-CH3
6-CH3, 8-CH3
À1.70
47.1a
13
H
H
28.6b
38.8b
À20.9
À1.94
4.33
28.0
14
6-Cl
H
12.7
61.5b
98.9b
17.8
52.9b
87.0a
15
16
6-CO2H
6-CO2H
H
H
Ph
71.3b
87.6b
57.5a
84.8b
39.8
82.5b
78.5b
80.6b
94.3b
48.5a
94.7b
73.4b
17
H
H
45.8a
61.0b
99.1b
38.0
75.8b
88.5b
18
7-Cl
H
42.5a
49.8a
93.8b
21.3
29.1
53.3a
19
20
21
6-CH3, 8-CH3
7-Cl
7-Cl
Et
H
Ph
Ph
31.9b
16.7a
42.1a
35.5b
32.6a
49.5a
64.2b
22.3
51.2b
4.49
À2.65
À3.57
36.0
12.3
18.1
87.5b
27.1
12.6
n-C3H7
n-C3H7
22
23
24
25
26
6-F
H
À30.7
32.6
32.9
29.4
87.4b
98.5b
96.3a
98.2b
99.9b
21.3
17.8
70.5b
91.6b
7-MeO
7-F
H
H
H
H
48.8b
72.3b
20.8
37.2
6-MeS
8-MeS
À181.6
32.0b
À42.6
69.1b
À4.33
À9.69
87.7b

J.-F. He et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2980–2985
2983
Table 1 (continued)
Compound
R1
R2
R3
%Inhibition of T-cell
proliferation induced by ConA
%Inhibition of B-cell proliferation
reaction induced by LPS
Dose (lg/mL)
Dose (lg/mL)
1
10
100
1
10
100
27
28
7-MeS
6-NO2
H
H
30.3a
41.5a
38.5b
100b
53.2b
33.7b
99.9b
99.9b
73.6b
94.9b
99.7b
99.6b
92.8b
87.2b
29
30
31
32
33
34
35
7-Cl
H
H
H
H
H
H
H
14.0
H
À14.4a
À27.5
À19.6b
0.95
À6.94a
À11.2
31.6b
6-CH3
6-Br
6-CH3, 8-CH3
7-MeO
7-F
38.5b
À40.7
À24.6
À15.3
À9.00
a P < 0.05 versus control (StudentÕs t-test).
b P < 0.01.
Table 2. 4-Hydroxyl-quinoline-3-carboxamide and its effect on TNF-a production by macrophage
Compound
R1
R2
R3
%Inhibition of TNF-a secreted by macrophage
Dose (lg/mL)
1
10
18.2
11.2
5.61
100
Linomide
FR137316
9.95
14.7
18.6
7.59
2.08
14.6
12.1
27.3
29.7
3
4
5
6
7
8
7-Cl
H
CH3
CH3
CH3
CH3
CH3
CH3
Ph
Ph
Ph
Ph
Ph
Ph
25.6
À17.4
À22.5
22.2
À1.33
À9.52
52.7
8-MeS
7-MeS
7-MeO
7-CF3
35.7
À5.45
35.7
À3.57
À10.9
13
H
H
31.2
5.61
27.5
(continued on next page)

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J.-F. He et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2980–2985
Table 2 (continued)
Compound
R1
R2
R3
%Inhibition of TNF-a secreted by
macrophage
Dose (lg/mL)
1
10
100
14
6-Cl
H
0.78
32.1
À5.35
15
17
6-CO2H
H
H
H
Ph
15.2
15.5
12.1
À27.1
1.82
30.4
18
7-Cl
H
À10.9
6.06
77.7
19
20
21
6-CH3, 8-CH3
7-Cl
7-Cl
Et
H
n-C3H7
Ph
Ph
46.3
10.5
14.7
29.7
10.4
18.4
47.6
23.1
n-C3H7
À4.03
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We wish to acknowledge the support granted by the
National Science Foundation of China (30371677).
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cells were passed through a 200 gauge nylon sieve to get a
single cell suspension. Red blood cells were lysed with
0.16 M NH4Cl–Tris buffer. Then cells were washed twice
and resuspended in RPMI-1640 medium containing 10%
fetal bovine serum. The cells were seeded in a 96-well
microtiter plate (Nunc) at 2.5 · 106/mL and were stimu-
lated with ConA at 0.5 lg/mL or with LPS at 10 lg/mL or
left nonstimulated. The final concentration of the drug
was 1, 10, and 100 lg/mL, respectively. The final volume
per well was 200 lL and all treatment regimens were run in
triplicates. The cells were cultured at 37 ꢁC for 72 h and
during the final 16 h of culture 0.5 lCi of [3H]thymidine
was added to each well. The cells were harvested onto
glass–fiber filters, processed and counted in a b-counter
(Perkin–Elmer, USA). The results are expressed as the
mean SD of counts per minute (cpm). The activity was
expressed as a %inhibition of T cell or B cell proliferation.
The %inhibition = (the mean of control well À the mean
of administered drug well)/the mean of control well. (ii)
TNF-a production by macrophage cultures. RAW264.7
cells were grown in RPMI 1640 medium, supplemented
with 50 lg/mL gentamycin, 2 mM glutamine, and 10%
fetal calf serum (FCS; Hyclone). Cells (2 · 105/mL) were
seeded into each well in 96-well plates (Nunc, Roskilde,
Denmark) for collection of supernatants, and incubated in
37 ꢁC in 5% CO2 and 95% humidity. The experiments were
started when the cells were growing confluently (after
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J.-F. He et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2980–2985
2985
1
approx. 3 days).To stimulate the macrophages, lipopoly-
saccharide (LPS; Sigma–Aldrich) was added at 10 lg/mL.
The final concentration of drug was 1, 10, and 100 lg/mL,
respectively. After 24 h incubation, supernatants were
collected to measure the TNF-a level. The level of TNF-a
in the supernatants was measured with commercial ELISA
kits according to the instructions of the producer and the
results were expressed as mean SD pg/mL. The activity
was expressed as a %inhibition of TNF-a production. The
%inhibition = (the mean of control well À the mean of
administered drug well)/the mean of control well.
66.26; H, 5.03; N, 8.70. H NMR (DMSO-d6, d): 11.71 (s,
1H), 7.93 (s, 1H), 7.85 (d, 1H, J = 8.7 Hz), 7.20–7.30 (m,
5H), 7.07–7.14 (m, 2H), 3.30 (s, 3H), 2.50 (s, 3H).
Compound 18: mp 200 ꢁC (dec). MS m/z 267 (M+1,
ESI). Anal. Calcd for C12H11ClN2O3: C, 54.05; H, 4.16; N,
10.50. Found: C, 53.67; H, 4.20; N, 10.37. 1H NMR
(DMSO-d6, d): 10.37 (br, 1H), 8.77 (s, 1H), 8.20 (d, 1H,
J = 8.7 Hz), 7.64 (s, 1H), 7.35 (d, 1H, J = 8.7 Hz), 3.50 (m,
2H), 3.37 (q, 2H, J = 5.7Hz), 2.78 (t, 1H, J = 5.6 Hz).
Compound 19: mp 216–218 ꢁC. MS m/z 321 (M+1, ESI).
Anal. Calcd for C20H20N2O2: C, 74.98; H, 6.29; N, 8.74.
Found: C, 74.62, H, 6.28; N, 8.62. 1HNMR (DMSO-d6, d):
11.07 (s, 1H), 7.79 (d, 1H, J = 6.2 Hz), 7.62 (s, 1H), 7.18–
7.29 (m, 5H), 7.08–7.11 (m, 1H), 3.79 (q, 2H, J = 7.0 Hz),
2.38 (s, 3H), 2.31 (s, 3H), 1.07 (t, 3H, J = 7.0 Hz).
17. All new compounds reported herein showed satisfactory
spectral data (1H NMR, MS). Compound 6: mp 238–
240 ꢁC. MS m/z 325(M+1, ESI). Anal. Calcd for
C18H16N2O2S2: C, 66.65; H, 4.97; N, 8.64. Found: C