Quinoline-3-carbothioamides and related compounds as novel immunomodulating agents

Article abstract of DOI:10.1016/S0960-894X(02)00377-3

A series of quinoline-3-carbothioamides and their analogues was prepared via four synthetic routes and evaluated for their antinephritic and immunomodulating activities. The optimal compound 9g strongly inhibited the T-cell independent antibody production in mice immunized with TNP-LPS and was highly effective in two nephritis models, namely chronic graft-versus-host disease and autoimmune MRL/l mice.

Full text of DOI:10.1016/S0960-894X(02)00377-3

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2427–2430
Quinoline-3-carbothioamides and Related Compounds as Novel
Immunomodulating Agents
Takashi Tojo,a Glen W. Spears,a Kiyoshi Tsuji,a,* Hiroaki Nishimura,a Takashi Ogino,a
Nobuo Seki,c Aiko Sugiyamab and Masaaki Matsuob
aMedicinal Chemistry Research Laboratories, Fujisawa Pharmaceutical Co., Ltd, 2-1-6, Kashima,Yodogawa-ku, Osaka 532-8514, Japan
bResearch Information Management, Fujisawa Pharmaceutical Co., Ltd, 2-1-6, Kashima,Yodogawa-ku, Osaka 532-8514, Japan
cExploratory Research Laboratories, Fujisawa Pharmaceutical Co., Ltd, 5-2-3, Tokodai, Tsukuba, Ibaraki 300-2698, Japan
Received 17 April 2002; accepted 16 May 2002
Abstract—A series of quinoline-3-carbothioamides and their analogues was prepared via four synthetic routes and evaluated for
their antinephritic and immunomodulating activities. The optimal compound 9g strongly inhibited the T-cell independent antibody
production in mice immunized with TNP-LPS and was highly effective in two nephritis models, namely chronic graft-versus-host
disease and autoimmune MRL/l mice. # 2002 Elsevier Science Ltd. All rights reserved.
Typical T-cell immunosuppressants such as cyclosporin A
and FK506 cannot prevent the antibody-mediated rejec-
tion process of xenotransplantation. Leflunomide and the
quinoline-8-carboxamide derivative 1 have been shown to
inhibit antibodies produced by B-cells elicited by T-cell
independent antigens like the trinitrophenyl-lipopoly-
saccharide (TNP-LPS), and this class of B-cell immuno-
suppressants is emphasized to have the substantial
therapeutic potential in antibody-mediated diseases
including xenograft rejection and autoimmune diseases.1
pounds suppress not only proteinuria and histological
changes but also the anti-DNA antibody production in
these models. Recent finding that 2 is also an effective
inhibitor of anti-TNP antibody production in TNP-LPS
immunized mice prompted us to further optimize the
structure of 2. Reported here are the chemical modifi-
cation of 2 and the identification of the quinoline-3-
carbothioamide derivative FR165009 (9g) as the opti-
mal compound.4,5
Chemistry
The quinoline-3-carboxamide derivatives 7a–c were
synthesized as shown in Scheme 1. Isatoic anhydride 3a3
was treated with an appropriate acetate 4 (Z=O or S) in
We have reported on the 2-aminothiazole derivative
FR1150922 and the quinoline-3-carboxamide FR137316
(2)3 as novel antinephritic agents, which are effective in
chronic graft-versus-host disease (GVHD) and auto-
immune W/BF1 mice and MRL/l mice. These com-
Scheme 1. Synthesis of compounds 7a–c. Reagents and conditions:
(a) NaH, DMA, 120 ꢀC; (b) HBr, AcOH, 70 ꢀC; (c) HNR1R2, PCl3,
toluene, 100 ꢀC; (d) HNR1R2, DCC, toluene, 90 ꢀC.
*Corresponding author. Tel.:+81-6-6390-1142; fax:+81-6-6304-5435;
e-mail: kiyoshi_tsuji@po.fujisawa.co.jp
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00377-3

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T. Tojo et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2427–2430
the presence of sodium hydride in N,N-dimethylacet-
amide to give quinoline-3-carboxylates 5a. The carboxy-
lates were hydrolyzed and condensed with various
Mono-substituted thioamide 19a and thioimidate 20
were prepared as shown in Scheme 4. Quinoline-3-car-
boxylates 5b were hydrolyzed, decarboxylated, and
treated with phenyl isothiocyanate to give 19a,b. Care-
ful methylation of 19b (R3=MeS) afforded 20 in 87%
yield.
6
amines using PCl3 or 1,3-dicyclohexylcarbodiimide7 to
afford 7a–c.
A series of quinoline-3-carbothioamide and sulfonamide
derivatives 9a–s was prepared as depicted in Scheme 2.
Reaction of isatoic anhydrides 3b and an appropriate
acetate or thioacetate 8 by using the same procedure for
the preparation of 5a gave 9a–s.8 Thioamides 8a,b were
obtained by selective amidation of 10.9 Sulfonamide 11
was treated with n-BuLi in THF at ꢁ30 ꢀC, followed by
quenching with (EtO)2CO to afford 8c.
Biological Results and Discussion
Nephritis is a disease that affects millions of people
worldwide, but is poorly treated with current therapies.
The chronic GVHD model is thought to be a good
model for nephritis disease, because its etiology and
symptoms resemble human lupus nephritis.10 In addi-
tion, the production of autoantibodies can be an indi-
cation of the immunomodulating activity of the drug.11
The effects of the compounds on the in vivo T-cell
independent antibody production were also assessed
after immunization of mice with TNP-LPS.12 The test
results,% inhibition of proteinuria in the GVHD model
and% inhibition of anti-TNP antibody in the TNP-LPS
model, are summarized in Tables 1–3.
Preparation of some quinoline-3-carbothioamides 17a–g
is shown in Scheme 3. Heating of a mixture of 12,
chloral hydrate, sodium sulfate, concd HCl and
hydroxylamine hydrochloride in water gave oximes
13, which were treated with sulfuric acid, followed by
methylation and oxidation by m-CPBA to give isatoic
anhydrides 15. Compounds 17a–g were synthesized by
anionic cyclization of 16, which were derived from ring
opening reaction of 15 and subsequent condensation
with the (thiocarbamoyl)acetic acid derivatives.9
Introduction of a sulfur-containing moiety such as MeS
generated potent antinephritic agents in the series of 2-
related compounds.3 It has been reported that replace-
ment of the acetamide to thioacetamide or thiourea
derivatives resulted in enhanced antibacterial activ-
ities.13 There is also a thiocarbamate type of TNF-a
inhibitor.14 Accordingly, we investigated various sulfur-
containing structures as a first step of the optimization
of 2 (Table 1). Incorporation of a MeS moiety on the
N-methyl-N-phenylcarboxamide group (7a,b) and the
sulfonamide analogue (9b) resulted in significant losses
of potency. On the other hand, the thioamide analogues
(7c and 9a) showed good activities. However, bis(thio-
amide) derivative 9c lost the activity. Interestingly, some
activity was maintained in the structurally different
thioimidate 20. The quinoline-3-carbothioamide deriv-
ative 9a displayed the most potent inhibition and thus
was studied further.
Scheme 2. Synthesis of compounds 9a–s. Reagents and conditions: (a)
NaH, DMA, 120 ꢀC; (b) HNR1R2, 180 ꢀC; (c) n-BuLi, (EtO)2CO,
THF, ꢁ30 ꢀC to rt.
Substitutions on the quinoline ring of 9a were optimized
as shown in Table 2. Replacement of the MeS group
with MeO resulted in a similar potency (9e). Halogen
substitution afforded more potent compounds (9g,h) in
the TNP-LPS model. The unsubstitution (9d) and other
Scheme 3. Synthesis of compounds 17a–g. Reagents and conditions:
(a) H2NOH, Cl3CCH(OH)2, Na2SO4, HCl, H2O, reflux; (b) H2SO4,
80 ꢀC; (c) MeI, NaH, DMA, rt; (d) m-CPBA, CH2Cl2, rt; (e) NaOEt,
EtOH, reflux; (f) HOCOCH2C(S)NR1R2, MS, pyridine, tert-BuCOCl,
CH2Cl2, rt; (g) NaOEt, EtOH, rt.
Scheme 4. Synthesis of compounds 19a and 20. Reagents and condi-
tions: (a) NaOH, H2O, reflux; (b) PhNCS, Et3N, DMSO, rt; (c) MeI,
NaH, DMF, ꢁ20 ꢀC.

T. Tojo et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2427–2430
2429
substitutions (9f and 17a,b) both resulted in decreased
activities. Substituents on the 7 or 8 position of the quin-
oline ring seemed to diminish the activity (9i and 17c).
which has been under clinical investigation for the
treatment of various autoimmune diseases and cancer.15
In addition, compound 9g exhibited a favorable half-life
(9.3 h) in dog, while 2 showed a half-life of 128 h.
Moreover, 9g was free of the mutagenicity that plagued
both 2 and linomide.
Maintaining the 6-Cl moiety of 9g, we varied the
N-substituents (R1 and R2) of the thioamide part (Table
3). The methoxy, trifluoromethoxy and halogen sub-
stituted phenyl provided a series of highly potent com-
pounds (9j–q). However, the alkylphenyl and thienyl
substitutions exhibited decreased activities (17d–f). As a
surrogate for the methyl in R2, both unsubstitution
(19a) and allyl and 3-methoxypropyl substitutions (9r,s)
resulted in fairly good activities, but the bulky tert-butyl
derivative (17g) was less potent.
The antinephritic and immunomodulating activities of
9g were further evaluated against the spontaneous
Table 3. Optimization of N-substitution of the thioamide part of 9g
One of the most active compounds, 9g, inhibited anti-
TNP antibody dose-dependently from an oral dose of
100 mg/kg down to 0.1 mg/kg in the TNP-LPS model
(Table 4). This potency was comparable with the lead
compound 2, and nearly 10-fold superior to linomide,
Compd
R1
R2
% Inhibition of % Inhibition
proteinuriaa
of anti-TNP
antibodyb
9j
9k
9l
9m
9n
9o
9p
4-MeO–Ph
3-MeO–Ph
4-CF3O–Ph
4-F–Ph
3,4-diF–Ph
4-Cl–Ph
3-Cl–Ph
4-Br–Ph
4-Me–Ph
3-CF3–Ph
2-Thienyl
Ph
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
97**
74**
45**
30**
39**
54**
62**
47**
63**
50**
23*
Table 1. Antinephritic activity of 2-related compounds containing
additional sulfur atom(s)
9q
17d
17e
17f
19a
9r
29
19
38*
Compd
X
Y
Z
% Inhibition of
proteinuriaa
Ph
Ph
Ph
Allyl
MeO(CH2)3
tert-Bu
96**
9s
17g
31**
11
2
CO
CO
CO
CO
CS
SO2
CS
NMePh
NMe(4-MeS–Ph)
NPh(CH2CH2SMe)
NMePh
O
O
O
S
O
O
S
100**
87*
65
96**
99**
53
7a
7b
7c
9a
9b
9c
20
aChronic GVHD, 32 mg/kg po; **p<0.01, *p<0.05 versus control
(Student’s t-test).
bTNP-LPS immunization in mice, 10 mg/kg po; **p<0.01, *p<0.05
versus control (Student’s t-test).
NMePh
NMePh
NMePh
SMe
37
84**
C¼NPh
O
Table 4. Effects of 9g, 2 and linomide on TNP-LPS immunized mice
aChronic GVHD, 32 mg/kg po; **p<0.01, *p<0.05 versus control
(Student’s t-test). See ref 11 for experimental detail.
Compd
Dose (mg/kg po)
% Inhibition of
anti-TNP antibodya
9g
0.01
0.1
1
ꢁ3
Table 2. Optimization of quinoline substitution of 9a
22*
33**
64**
77**
18
50**
67**
13
10
100
0.1
1
10
1
10
2
Compd
R3
R4
% Inhibition of
proteinuriaa
% Inhibition
of anti-TNP
antibodyb
Linomide
41**
49**
100
9a
9d
9e
MeS
H
MeO
M
Cl
H
H
H
e
H
H
7-Cl
H
H
8-Me
99**
85
99*
32**
31*
a**p<0.01, *p<0.05 versus control (Student’s t-test).
9f
H
20
Table 5. Effects of 9g on the autoimmune disease MRL/l mice
9g
9h
9i
17a
17b
17c
100**
98*
ꢁ23
64**
56**
Br
H
Dose (mg/kg po)
% Inhibition of
histological scorea
% Inhibition of
anti-DNA antibodyb
CF3O
1-Pyrrolyl
Cl
19
ꢁ10
17*
0.32
1
3.2
67**
85**
85**
52
73**
83**
aChronic GVHD, 32 mg/kg po; **p<0.01, *p<0.05 versus control
(Student’s t-test).
bTNP-LPS immunization in mice, 10 mg/kg po; **p<0.01, *p<0.05
versus control (Student’s t-test). See ref 12 for experimental detail.
aNephritis in the kidneys **p<0.02 versus control (Mann–Whitney
test).
b**p<0.01 versus control (Cochran–Cox test).

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T. Tojo et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2427–2430
autoimmune disease MRL/l mice.16 Compound 9g
strongly suppressed the development of glomerulone-
phritis in the kidneys17 as shown in Table 5. In addition,
the production of autoantibodies, that has been shown
to play an important role in the development of lupus
nephritis,18 was also reduced.
6. Matsuo, M.; Tsuji, K.; Nakamura, K.; Spears, G.W.
WO92–18483, 1992; Chem. Abstr. 1993, 118, 212903b.
7. Matsuo, M.; Tsuji, K.; Spears, G.W.; Tojo, T. Japanese
Patent JP07224040, 1995; Chem. Abstr. 1996, 124, 29618x.
8. Matsuo, M.; Tsuji, K.; Spears, G.W.; Ogino, T.; Nishi-
mura, H.; Tojo, T. WO95–24395, 1995; Chem. Abstr. 1996,
124, 117102e.
9. Spears, G. W.; Tsuji, K.; Tojo, T.; Nishimura, H.; Ogino,
T. Synth. Commun. 2000, 30, 565.
10. Bruijn, J. A.; Hogendoorn, P. C. W.; Corver, W. E.; van
den Broek, L. J. C. M.; Hoedemaeker, P. J.; Fleuren, G. J.
Clin. Exp. Immunol. 1990, 79, 115.
In conclusion, it has been demonstrated that the quino-
line-3-carbothioamide derivatives are potent immuno-
modulating agents and have potential for treating
various kind of autoimmune diseases and nephritis. Our
recent results have suggested that 9g and related com-
pounds inhibit autoimmune responses and potentiate
normal immune responses by augmentation of NKT
cells,19 which play a crucial role in controlling the
development of autoimmune diseases.20
11. Six-week old female (C57BL/6 ꢂ DBA/2)F1 and DBA/2
mice were used. Chronic GVHD was induced in (C57BL/6 ꢂ
DBA/2)F1 mice with two injections of DBA/2 spleen cells
given 5 days apart. Each injection contained 5 ꢂ 107 cells.
From 3 days after the second cell injection, drug was admi-
nistered orally once a day for 8 weeks. To assess the renal
disease, proteinuria were measured after the last drug admin-
istration. The concentration of serum albumin in the urine was
determined by the single radial immunodiffusion method using
rabbit anti-mouse serum albumin antiserum. Ten mice were
used per group. The activity was expressed as a% inhibition of
proteinuria. As an indication of autoimmune disease, 4 weeks
after the last cell injection anti-DNA antibodies were mea-
sured by ELISA.
Acknowledgements
The authors are grateful to Drs. Kazuo Sakane,
Hachiro Senoh and Takashi Fujii for their continuing
guidance and encouragement.
12. Female (C57BL/6 ꢂ DBA/2) F1 mice (6 weeks old) were
immunized intravenously with TNP-LPS (10 mg/mice) on day
0. Mice were bled on day 4 after immunization, and anti-TNP
IgMlevels in each serum were determined by ELISA. To test
the inhibitory activity of the compound, mice were randomly
divided after immunization (7 mice/group) and were treated
orally with the compound on day 0 and day 1. The activity of
the compound was expressed as% inhibition of anti-TNP IgM
level.
13. Tokuyama, R.; Takahashi, Y.; Tomita, Y.; Suzuki, T.;
Yoshida, T.; Iwasaki, N.; Kado, N.; Okezaki, E.; Nagata, O.
Chem. Pharm. Bull. 2001, 49, 347.
14. Fischer, R; Mueller, U; Handke, G; Petesch, N; Schmeck,
C. German Patent DE10034628, 2002.
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1630, 1595, 1575, 1495 cmꢁ1 1H NMR (DMSO-d6) d 3.42
;
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11.17 (1H, s); MS m/z 359 (M+H)+. Anal. calcd for
C18H15ClN2O2S: C,60.24; H,4.21; N,7.81. Found: C,60.64;
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