Synthesis and bioactivities of novel pyridazine derivatives: Inhibitors of interleukin-1 beta (IL-1β) production

Article abstract of DOI:10.1016/S0960-894X(01)00432-2

New pyridazine derivatives were prepared, and their abilities to inhibit IL-1β production were evaluated. Some compounds showed potent inhibitory activity against IL-1β production in HL-60 cells stimulated with lipopolysaccharide (LPS). The synthesis and structure-activity relationships of these compounds are described.

Full text of DOI:10.1016/S0960-894X(01)00432-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2369–2372
Synthesis and Bioactivities of Novel Pyridazine Derivatives:
Inhibitors of Interleukin-1 Beta (IL-1ꢀ) Production
Takayuki Matsuda,* Taro Aoki, Tadaaki Ohgiya, Tomoyuki Koshi,
Masao Ohkuchi and Hiromichi Shigyo
Tokyo Research Laboratories, Kowa Company Ltd., 2-17-43, Noguchi-Cho, Higashimurayama, Tokyo 189-0022, Japan
Received 16 May 2001;accepted 21 June 2001
Abstract—New pyridazine derivatives were prepared, and their abilities to inhibit IL-1b production were evaluated. Some com-
pounds showed potent inhibitory activity against IL-1b production in HL-60 cells stimulated with lipopolysaccharide (LPS). The
synthesis and structure–activity relationships of these compounds are described. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
Compound 6 was synthesized as shown in Scheme 2.
Dimethoxybenzil 3 was condensed with glycinamide to
provide pyrazinone 4 followed by chlorination with
phosphorus oxychloride for conversion to chloropyr-
azine 5.10 Compound 5 was treated with tetra-
kis(triphenylphosphine)palladium in the presence of
potassium cyanide to produce cyanopyrazine 6.11
Interleukin-1b (IL-1b) plays a pivotal role in the patho-
genesis of inflammatory diseases.1,2 Recently, an endog-
enous IL-1 receptor antagonist (IL-1Ra) was isolated,3
and has been shown to be effective in animal models of
septic shock,4 endotoxin shock,5 and rheumatoid
arthritis (RA).6 Moreover, Gabay7 reported that IL-1b
played a major role in synovitics and in the mechanisms
that lead to the progressive joint destruction in RA.
Inhibition of IL-1b thus offers an attractive target for
the design of antirheumatic agents.
Preparation of cyanopyrimidine 12 is shown in Scheme
3. Although the synthesis of 2-cyano-4,5-diphenylpyr-
imidine was reported previously by Yamanaka et al.,12
the main product was the 6-cyano isomer, and not the 2-
cyano isomer. 2-Cyanopyrimidine 12 was obtained in
good yield by a procedure similar to that described
above for the synthesis of pyrazine 6. Desoxyanisoin 7
was treated with ethyl formate under basic conditions to
produce formylate 8.13À15 Condensation of 8 with N-
methylurea afforded pyrimidinone 9 and intermediate
10.13À16 The intermediate 10 was also converted to 9 by
dehydration using p-TsOH.15 The pyrimidinone 9 was
treated with phosphorus oxychloride and phosphorus
Screening of our in-house compounds on inhibition of
IL-1b production allowed us to identify 3,4-bis(4-
methoxyphenyl)-6-cyanopyridazine 1 as a potent inhib-
itor. However, compound 1 showed acute toxicity in
mice. To reduce its toxicity, pyrimidine, pyrazine and
other analogues of compound 1 were synthesized. In
this paper, we describe synthesis and the structure–
activity relationships on 3,4-bis(4-methoxyphenyl)-pyr-
idazine analogues.
Chemistry and Biology
Cyanopyridazine derivative 1 was synthesized from
pyridazine-N-oxide 28 according to the method described
in the literature (Scheme 1).9
*Corresponding author. Tel.: +81-42-391-6211;fax: +81-42-395-
0312;e-mail: tamatuda@kowa.co.jp
Scheme 1. Reagents and conditions: (a) KCN, BnCl, H2O, under N2,
0 ꢀC, 69%.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00432-2

2370
T. Matsuda et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2369–2372
Scheme 2. Reagents and conditions: (a) H2NCOCH2NH2HCl, NaOH, EtOH, 80 ꢀC, 85%;(b) POCl , benzene, reflux, 27%;(c) KCN, Pd(PPh ) ,
3
3 4
DMF, 150 ꢀC, 81%.
Scheme 3. Reagents and conditions: (a) NaOEt, HCO2Et, EtOH, 0 ꢀC, 47%;(b) H 2NCONHMe, p-TsOH, toluene, reflux (9: 38%, 10: 50%);(c) p-
TsOH, xylene, reflux, 76%;(d) PCl 5, POCl3, reflux, 57%;(e) KCN, Pd(PPh 3)4, DMF, under Ar, 150 ꢀC, 60%.
Scheme 4. Reagents and conditions: (a) PhMgBr, Pd(PPh3)4, benzene,
60 ꢀC, 36%.
Scheme 5. Reagents and conditions: (a) RXH, K2CO3, DMF. Yields
are shown in Table 2.
pentachloride to give 2-chloropyrimidine 11.17 Reaction
of 11 with potassium cyanide and tetrakis(triphenyl-
phosphine)palladium mainly gave 2-cyanopyrimidine 12.
Results and Discussion
The initial lead compound 1 is a potent IL-1b inhibitor,
but shows many serious symptoms in mice. To avoid
these adverse effects, analogues of compounds 1 and 14
were synthesized and their abilities to inhibit IL-1b
production were evaluated as a preliminary evaluation.
From the results of these tests, two analogues 12 and 14
were found to be non-toxic, but their potencies were
reduced by over one order of magnitude compared to
compound 1 (Table 1). These results supported the
hypothesis that the toxicity of compound 1 is due to the
substituent at the 6-position on the pyridazine nucleus.
This hypothesis prompted us to investigate 3,4-bis(4-
methoxyphenyl)-6-substituted-pyridazines, and conse-
quently we found some potent inhibitors without
adverse effects.
6-Phenylpyridazine was prepared by reaction of 3,4-bis-
(4-methoxyphenyl)-6-chloropyridazine 138 and phenyl-
magnesium bromide in the presence of tetrakis(triphenyl-
phosphine)palladium (Scheme 4).
Pyridazine derivatives modified at the 6-position on the
pyridazine ring were prepared as shown in Scheme 5. 6-
Substituted derivatives 15a–x were synthesized from
compound 13 and the corresponding amines, thiophenols
or phenols in the presence of potassium bicarbonate.18,19
Compounds were evaluated for their abilities to inhibit
IL-1b production in HL-60 cells stimulated with lipo-
polysaccharide (LPS). IC50 values were determined by
comparison of yield with a control to which no test
compound was added.20
The compounds tested are shown in Table 2. In the in
vitro assay, the benzylamino analogue 15d and the
3,4,5-trimethoxyphenylamino analogue 15c were not
potent. N-Alkylation of 2,4-difluorophenylamino ana-
logue 15b decreased the inhibitory potency compared
with non-alkylated analogue 15a. As substituents on the
Acute toxicity of compounds was measured by Irwin’s
method;that is the general observation of behavior in
mice treated at a dose of 30 mg/kg ip.21

T. Matsuda et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2369–2372
Table 1. Biological data for the compounds investigated
2371
Compd
R
X
Y
Z
IL-1b Inhibition IC50 (mM)a
Symptomsb 30 mg/kg ip
1
6
12
CN
CN
CN
Ph
N
N
C
N
C
N
N
C
N
N
C
0.09
6.60
2.88
2.81
0.76
+++ (Death, 6/8c)
+
—
—
—
14
Prednisolone
N
aConcentration (mM) required for 50% inhibition of production of IL-1b.
bGeneral observation of behavior in mice (+++=tremor, dyspnea and difficulty in walking, ++=depression of spontaneous locomotor activity,
+=weak sedation, —=no significant change, NT=not tested).
cNumber of dead mice/total number of mice used.
Table 2. Biological data for the compounds investigated
Compd 15
RX
IL-1b Inhibition IC50 (mM)a
Symptomsb 30 mg/kg ip
Yield (%)
Temperature (ꢀC)
Time (h)
15a
15b
15c
15d
15e
15f
15g
15h
15i
2,4-F2PhNH
2,4-F2PhNPr
3,4,5-(MeO)3PhNH
PhCH2NH
3,4,5-Cl3PhS
2,4-F2PhS
PhS
2,3,4,5,6-F5PhO
PhO
2,3-F2PhO
2-CNPhO
3-CNPhO
2,5-F2PhO
2,3,5,6-F4PhO
2,6-F2PhO
4-MeOPhO
3,4-F2PhO
4-NO2PhO
4-PyO
3,5-F2PhO
2,4-F2PhO
3-NO2PhO
2,4-Cl2PhO
4-CNPhO
1.92
3.16
>100
>100
0.20
1.87
20.5
0.04
0.10
0.12
0.15
0.16
0.18
0.19
0.35
0.45
0.46
0.63
1.24
1.38
2.32
2.36
2.54
>100
—
—
—
NT
—
+
—
++
—
—
—
NT
NT
—
—
NT
—
—
—
—
++
—
—
—
97.0
54.1
71.3
100.0
40.6
37.4
12.9
60.5
70.7
90.0
88.1
75.2
91.5
37.8
39.3
94.7
99.1
74.1
85.2
98.0
52.5
99.9
93.9
57.5
100
170
140
120
100
80
12
10
5
19
72
7
100
150
120
150
150
150
150
150
150
150
150
150
150
150
120
150
150
150
7
24
22
19
24
19
24
12
72
24
14
15
22
6
15j
15k
15l
15m
15n
15o
15p
15q
15r
15s
15t
15u
15v
15w
15x
13
17
15
13
aConcentration (mM) required for 50% inhibition of production of IL-1b.
bGeneral observation of behavior in mice (+++=tremor, dyspnea and difficulty in walking, ++=depression of spontaneous locomotor activity,
+=weak sedation, —=no significant change, NT=not tested).
phenylamino moiety, electron-withdrawing groups
seemed to be favorable (15a vs 15c). As substituents on
the phenylthio moiety, electron-withdrawing groups
were also favorable (15e, 15f vs 15g). The phenoxy
analogue 15i (IC50=0.1 mM) was more effective than the
phenylthio analogue 15g (IC50=20.5 mM). Introduction
of electron-withdrawing groups onto the phenoxy moi-
ety is expected to be useful for development of more

2372
T. Matsuda et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2369–2372
potent inhibitors. Compounds with electron-with-
drawing groups 15j–n were approximately equipotent
with non-substituted phenoxy analogue 15i. However,
other analogues 15p–r and 15t–x showed reduceed
inhibitory potency. Analogue 15s containing a pyridinyl
moiety with electron-withdrawing properties and ana-
logue 15p also showed decreased inhibitory potency.
Interestingly, increases in the number of halogen atoms
led to stronger activity, that is the pentafluorophenoxy
analogue 15h was the most effective compound.
References and Notes
1. Dinarello, C. A. Blood 1996, 87, 2095.
2. Dinarello, C. A.;Wolff, S. M. N. Engl. J. Med. 1993, 328, 106.
3. Hannum, C. H.;Wilcox, C. J.;Arend, W. P.;Joslin, F. G.;
Dripps, D. J.;Heimdal, P. L.;Armes, L. G.;Sommer, A.;
Eisenberg, S. P.;Thompson, R. C. Nature 1990, 343, 336.
4. Fischer, E.;Marano, M. A.;Van Zee, K. J.;Rock, C. S.;
Hawes, A. S.;Thompson, W. A.;DeForge, L.;Kenney, J. S.;
Remick, D. G.;Bloedow, D. C.;Thompson, R. C.;Lowry,
S. F.;Moldawer, L. L. J. Clin. Invest. 1992, 89, 1551.
5. Ohlsson, K.;Bjo rk, P.;Bergenfeldet, M.;Hageman, R.;
Thompson, R. C. Nature 1990, 348, 550.
6. Van den Berg, W. B.;Joosten, L. A. B.;Helsen, M.;van de
Loo, F. A. J. Clin. Exp. Immunol. 1994, 95, 237.
7. Gabay, C. Expert Opin. Invest. Drugs 2000, 9, 113.
8. Nannini, G.;Biasoli, G.;Perrone, E.;Forgione, A.;Buttinoni,
A.;Ferrari, M. Eur. J. Med. Chem.-Chim. Ther. 1979, 14, 53.
9. Ogata, M. Chem. Pharm. Bull. 1963, 11, 1522.
10. Karmas, G.;Spoerri, P. E. J. Am. Chem. Soc. 1952, 74, 1580.
11. Akita, Y.;Shimazaki, M.;Ohta, A. Synthesis 1981, 974.
12. Yamanaka, H.;Sakamoto, T.;Nishimura, S.;Sagi, M.
Chem. Pharm. Bull. 1987, 35, 3119.
13. Hardtmann, G. E.;Kathawala, F. G. DE Patent
2,056,407, 1971;US Patent 3,772,288, 1973; Chem. Abstr.
1971, 75, 88635t. This patent may be downloaded from http://
ep.espacenet.com/
14. Wislicenus, W.;Ruthing, A. Liebigs Ann. Chem. 1911,
379, 229.
15. Eiden, F.;Nagar, B. S. Arch. Pharm. 1964, 297, 367.
16. Coppola, G. M.;Fraser, J. D.;Hardtmann, G. E.;Huegi,
B. S.;Kathawala, F. G. J. Heterocycl. Chem. 1979, 16, 545.
17. Coppola, G. M.;Hardtmann, G. E.;Huegi, B. S. J. Het-
erocycl. Chem. 1980, 17, 1479.
Most of the compounds tested were not toxic as deter-
mined by observation of the behavior of mice. It is par-
ticularly noteworthy that compounds 15f, 15h and 15u
possessing fluorine atoms at 2- and 4-positions on the
phenyl rings of phenylthio and phenoxy groups showed
some undesirable effects, but 2,4-difluorophenylamino
analogues 15a and 15b, and 2,4-dichlorophenoxy ana-
logue 15w did not.
In conclusion, a number of analogues with pyridazine,
primidine and pyrazine rings instead of the pyridazine
ring of compound 1 were evaluated for their abilities to
inhibit IL-1b production. Starting from the lead com-
pound 1, we found potent inhibitors without toxicity
among 6-substituted pyridazine compounds, especially
3,4-bis(4-methoxyphenyl)-6-phenoxypyridazine. There-
fore, efforts to expand the structure–activity relation-
ship are currently in progress in our laboratory using
pyridazines as substrates.
18. Coudert, P.;Couquelent, J.;Tronche, P. J. Heterocycl.
Chem. 1988, 25, 799.
19. Shin, M. S.;Kang, Y. J.;Chung, H. A.;Park, J. W.;
Acknowledgements
Kweon, D. H.;Lee, W. S.;Yoon, Y. J.;Kim, S. K.
erocycl. Chem. 1999, 36, 1135.
20. Tanaka, M.;Okita, M.;Akamatsu, H.;Chiba, K.;
J. Het-
The authors thank Ms. Kyoko Yasuoka for providing
synthetic data, and Ms. Natsuyo Kumai, and Ms. Yur-
iko Habata for biological data. Finally, we are grateful
to Dr. Yoshinori Kyotani for helpful suggestions and
encouragement during the course of this study.
Obaishi, H.;Nagakura, N.;Sakurai, H.;Yamatsu, I.
Med. Chem. 1996, 31, 187.
21. Irwin, S. Psychopharmacologia 1968, 13, 222.
Eur. J.