Synthesis and bioactivities of novel 5,6-Bis(4-methoxyphenyl)-2H-pyridazin-3-one derivatives: Inhibitors of interleukin-1 beta (IL-1β) production

Article abstract of DOI:10.1016/S0960-894X(01)00433-4

New 5,6-bis(4-methoxyphenyl)-2H-pyridazin-3-one 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)00433-4

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2373–2375
Synthesis and Bioactivities of Novel 5,6-Bis(4-methoxyphenyl)-
2H-pyridazin-3-one Derivatives: Inhibitors of Interleukin-1
Beta (IL-1ꢀ) Production
Takayuki Matsuda,* Taro Aoki, 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 5,6-bis(4-methoxyphenyl)-2H-pyridazin-3-one derivatives were prepared, and their abilities to inhibit IL-1b pro-
duction were evaluated. Some compounds 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
Interleukin-1b (IL-1b) plays a pivotal role in the patho-
genesis of inflammatory joint destruction, including
rheumatoid arthritis (RA).1 Joosten and co-workers
reported that anti-TNF-a monoclonal antibody (mAb)
is effective in the early stages of fully established type-II
collagen-induced arthritis (CIA) in DBA/1J mice,
whereas anti-IL-1b mAbs are most effective in the later
stages of the disease.2,3
Previously, we reported the synthesis and ability to
inhibit IL-1b production of 3,4-bis(4-methoxyphenyl)-6-
Chemistry
phenoxypyridazine 1 (IC50=0.10 mM).4 To find other
pyridazine-related derivatives, we planned to exchange
the pyridazine ring of the compound 1 for a pyr-
idazinone ring, and evaluated the activities of these
compounds. We found a compound effective in CIA.
Here, we describe synthesis and the structure–activity
relationship of the 5,6-bis(4-methoxyphenyl)-2H-pyr-
idazin-3-one analogues.
The majority of the compounds listed in Table 1 were
prepared from the common intermediate 2. This com-
pound was synthesized previously by Nannini et al.,5
but it could be prepared in higher yield under the milder
conditions shown in Scheme 1. Tartaric acid was oxi-
dized with sodium periodate to generate glyoxylic acid
in situ, and the reaction mixture was added to a sus-
pension of desoxyanisoin 3 under basic conditions to
provide aldol adduct 4. Compound 4 was cyclized with
hydrazine, followed by dehydration with p-toluene-
sulfonic acid to give the key intermediate 2.
2-Substituted derivatives 5 were synthesized from the
corresponding halide and intermediate 2 in the presence
of potassium bicarbonate (Scheme 2).6
*Corresponding author. Tel.: +81-42-391-6211;fax: +81-42-395-
0312;e-mail: tamatuda@kowa.co.jp
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00433-4

2374
T. Matsuda et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2373–2375
Scheme 1. Reagents and conditions: (a) (i) tartaric acid, NaIO4;(ii) NaOH, EtOH, 40 ꢀC, 17 h to 70 ꢀC, 1 h, 93%;(b) H 2NNH2 monohydrate,
EtOH, 100 ꢀC, 6 h;(c) p-TsOH, toluene, 120 ꢀC, 3 h, 73%.
Results and Discussion
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 com-
pound was added.7 Results are summarized in Table 1.
Previously, we reported that the pyridazine derivative 1
has inhibitory activity against IL-1b production.4 How-
ever, it was less effective in vivo. We planned to convert
Scheme 2. Reagents and conditions: (a) halide, K2CO3, DMF, 70–
80 ꢀC.
the pyridazine ring of compound 1 to a pyridazinone
ring, with a hydrophobic substituent such as a benzyl
group at the 2-position.
Table 1. IL-1b inhibition assay results for compounds
The activity of the initially designed compound 5a was
more potent than that of unsubstituted compound 2
and similar to that of compound 1. Some halogenated
compounds, 5b–c, showed increased activities, while
others, 5e–i, showed decreased activities. These results
suggested that the bulkiness of chlorine atoms on the N-
substituent might obstruct the interaction between the
N-substituent and a narrow pocket in the drug receptor.
Compd
R
IL-1b Inhibition IC50 (mM)a
On the other hand, among the alkylated compounds,
examined ethyl 5k, cyclopentyl 5p and cyclo-
pentylmethyl 5q were potent inhibitors, while the other
analogues, 5j and 5l–o, showed decreased activity. These
observations suggested that a planar substituent at the
2-position on the pyridazinone ring seems to be favor-
able for activity.
2
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
5y
H
Bn
4-FBn
31.6
0.26
0.15
0.18
0.21
0.53
0.54
1.10
3.16
9.40
1.13
0.61
1.32
>100
7.98
1.80
0.51
0.11
>100
0.43
0.52
1.48
0.62
0.10
0.10
0.17
0.27
0.76
2,4-F2Bn
3,4-Cl2Bn
4-ClBn
3,4-F2Bn
2,4-Cl2Bn
2,6-Cl2Bn
2,4,6-Cl3Bn
Me
Et
iPr
iBu
cPr
cPrCH2
cPn
cPnCH2
CH2¼CHCH2
3-PyCH2
4-PyCH2
2-PyCH2
Cinnamyl
2,4-F2Cinnamyl
4-ClCinnamyl
4-FCinnamyl
2,4-ClCinnamyl
Furthermore, analogues possessing a planar substituent
such as an allyl, pyridinylmethyl or cinnamyl group
were synthesized and their activities were evaluated.
Pyridinylmethyl analogues 5s and 5t, and cinnamyl
derivative 5v showed potent inhibitory activity against
IL-1b production. However, the allyl derivative 5r was
not potent. An aromatic group in the N-substituent
seems to play an important role in the inhibitory effect
against IL-1b production. Moreover, the introduction
of one or two halogen atoms on the phenyl group in the
cinnamyl moiety provided the most potent compounds
in the series of pyridazinones. 4-Chlorocinnamyl and
2,4-difluorocinnamyl analogues showed the same
potency as compound 1.
5z
Prednisolone
In the oral administration study, a series of 5,6-bis(4-
methoxyphenyl)-2H-pyridazin-3-ones were practically
insoluble in water and showed poor oral absorption in
rats. However, an olive oil solution of 4-chlorocinnamyl
aConcentration (mM) required for 50% inhibition of production of IL-
1b.

T. Matsuda et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2373–2375
2375
derivative 5x was absorbed in rats (Fig. 1). The phar-
macokinetic results of 5x are summarized in Table 2.
Among the 5,6-bis(4-methoxyphenyl)-2H-pyridazin-3-
ones evaluated by CIA, strong oral activities were
observed for 5x (Fig. 2).8 Furthermore, 5x was checked
for adverse effects by monitoring the behavior of mice
treated with this compound at a dose of 30 mg/kg ip,
and did not show any undesirable symptoms.
In conclusion, a novel class of 5,6-bis(4-methoxy-
phenyl)-2H-pyridazin-3-ones were synthesized and
shown to be strong inhibitors of IL-1b production.
Among these compounds, 5x showed substantial
plasma concentration profiles after oral administration
in rats and the highest potency in the in vivo test with-
out adverse effects. On the basis of these observations,
we selected compound 5x for further evaluation.
Acknowledgements
Figure 1. Pharmacokinetics of 5x. Plasma concentration of 5x after
oral administration at a dose of 10 mg/kg in olive oil to rats. Data are
the mean and SE of three rats.
The authors thank Ms. Kyoko Yasuoka for providing
synthetic data, and Ms. Natsuyo Kumai, and Ms. Yur-
iko Habata for biological data, and Dr. Hideki Fujino
for pharmacokinetic data. Finally, we are grateful to
Dr. Yoshinori Kyotani for helpful suggestions and
encouragement during the course of this study.
References and Notes
1. Gabay, C. Expert Opin. Invest. Drugs 2000, 9, 113.
2. Joosten, L. A. B.;Helsen, M. M. A.;Saxne, T.;van de Loo,
F. A.;Heinega rd, D.;van den Berg, W. B. J. Immunol. 1999,
163, 5049.
3. Van den Berg, W. B.;Joosten, L. A. B.;Helsen, M.;van de
Loo, F. A. J. Clin. Exp. Immunol. 1994, 95, 237.
4. Matsuda, T.;Aoki, T.;Ohgiya, T.;Koshi, T.;Ohkuchi, M.;
Shigyo, H. Bioorg. Med. Chem. Lett. 2001, 11, 2369.
5. Nannini, G.;Biasoli, G.;Perrone, E.;Forgione, A.;Butti-
noni, A.;Ferrari, M. Eur. J. Med. Chem.-Chim. Ther. 1979,
14, 53.
6. Akita, Y.;Shimazaki, M.;Ohta, A. Synthesis 1981, 974.
7. Rubat, C.;Coudert, P.;Tronche, P.;Bastide, J.;Bastide, P.;
Privat, A. M. Chem. Pharm. Bull. 1989, 37, 2832.
8. 8-week-old DBA/1J mice were used for the arthritis
experiments. Mice were immunized by intradermal injection at
the base of the tail with 100 mg of bovine type-II collagen
emulsified in an equal volume of Freund’s complete adjuvant.
Three weeks later, mice received a booster intradermal injec-
tion at the base of the tail with 100 mg of the same emulsified
collagen. Test compounds were dissolved in olive oil, and
administrated po twice a day from 1 day after first immuniza-
tion to 14 days after collagen booster immunization. The mice
were examined for clinical arthritis and scored by grading each
paw on a scale for 0–3 based on erythema and swelling of the
joint (0=no erythema and swelling;1=erythema and swelling
of one toe;2=erythema and swelling of two or more toes;
3=complete erythema, swelling of the entire paw and
incapacity to bend the ankle). As four legs were scored, the
highest score possible was 12.
Figure 2. Evolution of CIA. Values represent the means of groups of
10 mice. Significantly different from the control: *p<0.05;** p<0.01.
Table 2. Pharmacokinetic parameters of 5x at the Fig. 1 experiments
Compd Tmax (h) Cmax (ng/mL) AUC (mg h/mL)
5x 316.3ꢁ108.0 3.90ꢁ0.98
7.0ꢁ1.0
Data are the meansꢁSE of three rats.
t1/2
4.90ꢁ0.78