Synthesis and antirheumatic activity of the metabolites of esonarimod

Article abstract of DOI:10.1016/S0968-0896(02)00084-6

We have developed esonarimod, (±)-2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid, as a new antirheumatic drug. Now we describe herein the preparation of the enantiomers of (±)-deacetylesonarimod, the pharmaceutically active metabolites of esonar

Full text of DOI:10.1016/S0968-0896(02)00084-6

Bioorganic & Medicinal Chemistry 10 (2002) 2713–2721
Synthesis and Antirheumatic Activity of the Metabolites
of Esonarimod
Toshiya Noguchi,* Akira Onodera, Kazuyuki Tomisawa, Miyuki Yamashita,
Kimiyo Takeshita and Sadakazu Yokomori
Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd., Saitama City, Saitama, 330-8530, Japan
Received 21January 2002; accepted 27 February 2002
Abstract—We have developed esonarimod, (ꢀ)-2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid, as a new antirheumatic
drug. Now we describe herein the preparation of the enantiomers of (ꢀ)-deacetylesonarimod, the pharmaceutically active meta-
bolites of esonarimod, and comparison of their antirheumatic activities. No significant difference has been observed between the
two enantiomers. In a pre-clinical study of esonarimod, other metabolites were detected in rat blood or urine. We also synthesized
these compounds as authentic samples to analyze the human metabolites in clinical studies of esonarimod. # 2002 Elsevier Science
Ltd. All rights reserved.
Introduction
enhancement of lymphocyte transformation, an IL-1
antagonistic effect.10
We have developed of esonarimod (1), (ꢀ)-2-acet-
ylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic
acid
In a pre-clinical study of 1, metabolites 2 and 3 were
detected in rat blood, and 4–8 were found in rat urine.
The structures of these metabolites have been studied
and a possible metabolic pathway has been suggested,
as shown in Scheme 1.11,12 To analyze the human
metabolites in clinical studies of 1, it is important to
obtain significant quantities of these compounds as
authentic samples to support the proposed metabolic
pathway for 1. However, preparation of the pharmaco-
logically active metabolite 2 has not been reported.
Although 5 and 7 have been synthesized from 3 by a
biological method,12 chemical syntheses of 5 and 7 have
not been reported.
(Fig. 1), which exhibits beneficial effects in RA patients
in a clinical study.1 Compound 1 inhibits inflammatory
cytokine production by human peripheral blood mono-
nuclear cells,2 interleukin (IL)-6 and matrix metallo-
proteinase (MMP) production by RA synovial
fibroblast-like cells,3,4 and the progression of this dis-
ease in various animal models of arthritis.5ꢁ7 Com-
pound 1 contains a thioacetyl group, which is easily
deacetylated in vivo to form a pharmacologically active
metabolite 2.2,8 The toxicity of esonarimod is sig-
nificantly lower than that of penicillamine in rats. The
low toxicity of esonarimod may be closely related to the
low reactivity of the thiol of 2 with macromolecules in
vivo.9
In this article, we report the formation of pharmacolo-
gically active metabolite 2 and its antirheumatic activ-
ities, as well as the syntheses of other postulated
metabolites 3–8 as authentic samples.13
Compound 1 has been used clinically as a racemate.10
Therefore, we investigated the pharmacological activ-
ities of its optical isomers in comparison with those of
racemic 1 by characteristic tests for 1. Although the
(+)-form of (+)-1, strongly suppressed rat adjuvant
arthritis compared to the (ꢁ)-form, (ꢁ)-1, no difference
was detected between the two isomers with regard to the
Results and Discussion
Preparation of the ammonium salt of 2 in racemic form
(Scheme 2)
Under basic conditions, such as in aqueous ammonia
or potassium hydroxide solutions,
*Corresponding author. Tel.: +81-48-663-1111; e-mail: t.noguchi@
po.rd.taisho.co.jp
1 was easily
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00084-6

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T. Noguchi et al. / Bioorg. Med. Chem. 10 (2002) 2713–2721
1
deacetylated to form thiol 2, which was detected by H
NMR. The thiol 2 seemed to be stable in organic solu-
tion or basic conditions, but when the solvent was
removed to give 2 as a solid, 2 was unstable and readily
converted to dihydrothiophene 9 by cyclization and
subsequent dehydration. By the usual workup (acid-
ification and extraction with organic solvent) under
alkali conditions, facile dehydration of 2 occurred to
give only 9. Therefore, we attempted to isolate 2 as an
ammonium salt. Compound 1 was treated with aqu-
eous ammonia and methanol, and the solvent was
removed under reduced pressure. In this fashion, we
obtained the ammonium salt of 2 as a powder (Scheme
2).
The NMR spectrum of the ammonium salt of 2 in
methanol-d4 showed that 2 existed as an equilibrium
mixture of the keto form 2a and thiolane form 2b (Fig.
2). Compound 1 is racemic, as is the keto form of 2a.
However, the thiolane form 2b has two diastereoi-
somers. Therefore, the ammonium salt of 2 in a solution
consists of a mixture of three compounds, keto form 2a,
and trans and cis thiolane form 2b (Fig. 2). We assigned
each peak in the 1H NMR and 13C NMR (methanol-d4)
spectra, as shown in Table 1.
On the other hand, the IR spectrum (KBr) of the
ammonium salt of 2 exhibited a strong absorption at
1558 cmꢁ1 due to the carboxylate anion. No absorption
peak was observed for the keto group at about 1715
cmꢁ1. Therefore, we presumed that the ammonium salt
of 2 existed almost entirely as the thiolane form in the
solid phase.
Synthesis of optically active 2 as 1,1-dimethyl-3-oxobuty-
lammonium salt (Fig. 3). We next focused on the synth-
esis of optically active 2 from optically active 1.
Optically active 1 was obtained by a known method,6 as
illustrated in Scheme 3. The absolute configuration of
(+)-1 was determined to be S form.6
However, the ammonium salt of optically active 2 could
not be obtained as a crystalline form. We then con-
verted the ammonium salt to a 1,1-dimethyl-3-oxobuty-
lammonium salt as follows: (+)-1 was dissolved in
aqueous ammonia and acetone, and the solvent was
then removed under reduced pressure. We obtained
(+)-2 as a 1,1-dimethyl-3-oxobutylammonium salt,
which was formed in situ by the reaction of acetone
dimer with ammonia. We then obtained optically active
1,1-dimethyl-3-oxobutylammonium salt of (ꢁ)-2, and
(ꢀ)-2 as a powder by a similar method (Fig. 3).
Scheme 1. A possible metabolic pathway of esonarimod (1) in rat.
Figure 1. Esonarimod (1).
Scheme 2. Synthesis of 2 ammonium salt: (a) KOH or aqueous NH3 (b) usual workup (acidification and extraction with organic solvent); (c) aqu-
eous NH3–MeOH, 0–3 ꢂC, 5 min; (d) solvent removed under reduced pressure, 65%.

T. Noguchi et al. / Bioorg. Med. Chem. 10 (2002) 2713–2721
2715
Figure 2. Keto form (ꢀ)-2a and thiolane form (ꢀ)-2b.
Table 1. Assignment of 2 ammonium salt (1H NMR and 13C NMR in methanol-d4)
Carbon no.
Keto form (ꢀ)-2a
Thiolane form (ꢀ)-2b
cis
trans
dC
dH
dC
dH
dC
dH
1–COO—
2
3
4
5
6
7
8
9
n.d.
–CH—
—
n.d.
3.02
—83.5
52
1 —
3.52
48.3
52.8
or 2.620.41
—
3.38
CHCH2C¼O
413.20, 3.47
201—
27.8
53.4
95.8
2.43 (2H)
—
52.3
98.7
38.6
>C¼O
–CH2S—
Ph–C
2.74, 2.8137
3.29, +3.36
143.2
127.0 or 127.1
129.4
137.8 or 137.9
129.4
127.0 or 127.1
21
3.46,3.52
136
—
7.9
7.29
—
7.29
7.9
2.4
—
7.55
7.1
—
7.1
7.55
2.3
143.2
127.0 or 127.1
129.4
137.8 or 137.9
129.4
—
7.57
7.1
—
7.1
7.57
2.3
Aromatic
Aromatic
Aromatic
Aromatic
Aromatic
Ph–CH3
129.3
130.2
145.1
130.2
129.3
21.6
10
11
12
127.0 or 127.1
21
Figure 3. The structures of (ꢀ), (+), and (ꢁ)-2 1,1-dimethyl-3-oxobutylammonium salt.
Antirheumatic activity of 1,1-dimethyl-3-oxobutylammo-
nium salt of 2 (Fig. 4). All forms of 2 1,1-dimethyl-3-
oxobutylammonium salt [(+) -2, (ꢁ) -2 and (ꢀ) –2]
inhibited IL-1b production,14 as shown in Fig. 4. No
difference in activity was detected between the optical
isomers of 2.
Synthesis of the metabolites 3 and 4 (Scheme 4). We
synthesized 2-methylthiomethyl-4-(4-methylphenyl)-4-
oxobutanoic acid (3) and 4-(4-methylphenyl)-2-methyl-
sulfinylmethyl-4-oxobutanoic acid (4) from 1 (Scheme
4). The acetyl group of 1 was easily hydrolyzed in aqu-
eous sodium hydroxide solution at room temperature to

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T. Noguchi et al. / Bioorg. Med. Chem. 10 (2002) 2713–2721
give the thiol 2. When the reaction mixture was worked
up as usual (acidification and extraction with organic
solvent), 2 was converted to dihydrothiophene 9, indi-
cating that 2 was stable compound under alkaline con-
ditions but unstable under acidic or neutral conditions.
Therefore, the reaction mixture was treated with methyl
iodide immediately after hydrolysis. Since the solvent
was water, the phase transfer catalyst TOMAC (trioc-
tylmethylammonium chloride) was used for methyla-
tion, and 3 was obtained.
Oxidation of sulfide 3 with m-chloroperoxybenzoic acid
(mCPBA) in tetrahydrofuran (THF) gave the sulfoxide 4.
Synthesis of the metabolites 5 and 6 (Scheme 5). We
synthesized 4-(4-hydroxymethylphenyl)-2-methylsulfi-
nylmethyl-4-oxobutanoic acid (5) in eight steps from
terephthalaldehyde monodiethylacetal (12), which was
converted to 4-(4-hydroxymethyl-phenyl)-2-methylsulfi-
nylmethyl-4-oxobutanoic acid (6) by the oxidation of 5
with mCPBA as summarized in Scheme 5.
The key step was indium-promoted coupling of 4-acet-
oxymethylbenzaldehyde (13) and 2-(bromomethyl)
acrylic acid to yield 4-hydroxy-4-(4-acetoxyphenyl)-2-
methylenebutanoic acid (14).15 Benzylalcohol 14 was
oxidized to ketone 15 by Jones reagent.16 Michael
addition of thioacetic acid with 15 gave 16 in high yield.
The sulfide 5 and the sulfoxide 6 were then obtained
Scheme 3. Preparation of optically active esonarimod (1).
Figure 4. Suppressive effect of optical isomers of 2 1,1-dimethyl-3-oxobutylammonium salt on IL-1b 13 production in THP-1 cells. IC50 (95% C.I.).
(ꢀ)-2 1,1-dimethyl-3-oxobutylammonium salt: 29.6 (26.6 -32.9) mg/mL; (ꢀ)-2 1,1-dimethyl-3-oxobutylammonium salt: 30.8 (27.9-33.9) mg/mL; (-)-2
1,1-dimethyl-3-oxobutylammonium salt: 30.4 (26.8-34.3) mg/mL. Each symbol represents the production of IL-1b by THP-1cells with various con-
centrations of optical isomers of 2 (n=4). Results were divided by the mean of the control. The mean and SE of the control were 109.4ꢀ4.4 pg/mL.
IC50 and 95% confidence interval (CI) were determined by linear regression analysis.
Scheme 4. Synthesis of metabolites 3 and 4: (a) KOH, H20, rt, 30 min; (b) CH3I, TOMAC, rt, 30 min, 80%; (c) mCPBA, THF, rt, 60 min, 37%.

T. Noguchi et al. / Bioorg. Med. Chem. 10 (2002) 2713–2721
2717
Scheme 5. Synthesis of metabolites 5 and 6; (a) NaBH4, EtOH, rt, 60 min; (b) Ac20, pyridine, rt over:night; (c) H2SO4, THF–H2O, rt, 30 in, 96%;
(d) indium metal, THF, 18–39 ꢂC, 35 min, 87%; (e) CrO3, H2SO4, acetone–H2O, ice cooling, 10 min, 83%; (f) AcSH, Et3N, toluene, 60 ꢂC, 2 h, 92%;
(g) NH3, H2O, rt, 60 min; (h) CH3I, TOMAC, Et2O, rt, 30 min, 80%; (i) mCPBA, THF, ice cooling, 60 min, 77%.
ꢂ
0
Scheme 6. Synthesis of metabolites 7 and 8: (a) indium metal, THF, rt, overnight, 62%; (b) (COC1)2 , DMSO, THF, Et3N, ꢁ74 C, 10 min, 42%; (c)
AcSH, Et3N, toluene, 60 ꢂC, 40 min, 86%; (d) NH3, H2O, rt, 60 min; (e) CH3I, TOMAC, rt, 20 min, 61%; (f) mCPBA, THF, ice cooling, 40 min, 80%.
from 16 in the same fashion as for the synthesis of 3
and 4.
benzaldehydes [4-acetoxymethylbenzaldehyde (13) or
terephthalaldehydic acid methyl ester (17)] and 2-(bro-
momethyl)acrylic acid gave the corresponding benzy-
lalcohol derivatives 14 and 18. Using compounds 3–8 as
authentic samples, a possible metabolic route of eso-
narimod 1 in rats was demonstrated.17
Synthesis of metabolites 7 and 8 (Scheme 6). We synthe-
sized 2-methylthiomethyl-4-(4-carboxyphenyl)-4-oxobu-
tanoic acid (7) in five steps from terephthalaldehydic
acid methyl ester 17, and 4-(4-carboxyphenyl)-2-
methylsulfinyl-methyl-4-oxobutanoic acid (8) was
obtained via the oxidation of 7 by the method similar to
that for 3 and 4 (Scheme 6).
Experimental
Melting points were determined by a Buchi 535 melting
point apparatus and were uncorrected. IR spectra were
obtained on a Perkin–Elmer 1760 spectrometer. 1H
NMR and 13C NMR spectra were recorded on a Varian
VXL-200 spectrometer. Chemical shifts are reported in
ppm (d) values, as determined using a JEOL JMS-
SIX102 spectrometer. Elemental analyses were per-
formed on a Perkin–Elmer 2400. TLC was performed
on silica gel pre-coated plates (Merck, Kieselgel
60F254). Column chromatography was performed over
silica gel (Wako, Wako gel C-200). (ꢀ)-Esonarimod (1)
was prepared by the known procedure.10,18
Conclusion
We prepared the pharmacologically active metabolite 2
of esonarimod 1 as an ammonium salt. We confirmed
that the ammonium salt of 2 existed as an equilibrium
of the keto form 2a and thiolane form 2b in basic solu-
tion, while 2 existed as the thiolane form 2b in the solid
phase. We also prepared the optically active 2 1,1-
dimethyl-2-oxopropylammonium salt, and confirmed
that there was no difference in activity between the
optical isomers of 2.
Ammonium salt of (ꢀ)-2-mercaptomethyl-4-(4-methyl-
phenyl)-4-oxobutanoic acid (2). A 42.0 g (150 mmol)
sample of (ꢀ)-1 was dissolved in methanol (150 mL),
We also synthesized other postulated metabolites 3–8 in
racemic form. Indium -promoted coupling between

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T. Noguchi et al. / Bioorg. Med. Chem. 10 (2002) 2713–2721
and 100 mL of 25% ammonia solution was added
dropwise at 0–3 ꢂC over 5 min. The reaction mixture
was stirred at room temperature for 5 min, and then
evaporated in vacuo. The resulting precipitates were
collected by filtration, washed with 250 mL of acetone,
and dried under reduced pressure to give 24.4 g (64%)
of the ammonium salt of (ꢀ)-2 (equilibrated with
ammonium (ꢀ)-5-hydroxy-5-(4-methylphenyl)-3-thio-
lane-carboxylate). Mp: 166–170 ꢂC (decomposed). 1H
NMR and 13C NMR spectra were listed in Table 1. IR
(KBr) cmꢁ1: 3204, 2996, 1558, 1426, 1407, 813, 641.
FAB-Mass m/z: 239 (M+). Anal. calcd for C12H17
NO3S: C, 56.45; H,6.71; N, 5.49; S, 12.56. Found: C,
56.60; H, 6.68; N, 5.48; S, 12.77.
A 25.0 g (89.2 mmol) sample of (+)-1 was dissolved in
acetone (90 mL), and 60 mL of 25% ammonia solution
was added dropwise at 3–10 ꢂC over 10 min. The reac-
tion mixture was stirred at 21–25 ꢂC for 60 min, and
then evaporated in vacuo. The resulting precipitates
were collected by filtration, washed with 20 mL of ace-
tone, and dried under reduced pressure to give 10.0 g
(32%) of 1,1-dimethyl-3-oxobutylammonium salt of
(+)-2 (equilibrated with 1,1-dimethyl-3-oxobutyl-
ammonium (+)-5-hydroxy-5-(4-methylphenyl)-3-thio-
lane-carboxylate). [a]D: +25.3ꢂ (c 10.6, ethanol). Mp
118–119 ꢂC, 1H NMR (methanol-d4) d ppm: 1.35 (s,
6H), 2.18 (s, 3H), 2.30 (s, 3H), 2.37–2.64 (m, 2.2H), 2.87
(s, 2H), 3.35–3.56 (m, 2.8H), 7.10 (d, 1.6H, J=8.1Hz),
7.29 (d, 0.4H, J=8.1Hz), 7.55 (d, 0.8H, J=0.8 Hz),
7.56 (d, 0.8H, J=0.8 Hz), 7.91(d, 0.4H, J=8.1Hz).
keto form/thiolane form=1:4. IR (KBr) cmꢁ1: 2922,
2832, 1720, 1678, 1546, 1396, 1366, 810. Mass m/z: 237
(M+). Anal. calcd for C18H17NO4S: C, 61.16; H, 7.70;
N, 3.96; S, 9.07. Found: C, 61.09; H, 7.74; N, 4.03; S,
9.06.
1,1-Dimethyl-3-oxobutylammonium salt of (ꢀ)-2-mer-
captomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (2).
A 42.0 g (150 mmol) sample of (ꢀ)-1 was dissolved in
acetone (150 mL), and 100 mL of 25% ammonia solu-
tion was added dropwise at 6–15 ꢂC over 5 min. The
reaction mixture was stirred at 20–26 ꢂC for 70 min, and
then evaporated in vacuo. The resulting precipitates
were collected by filtration, washed with 50 mL of ace-
tone, and dried under reduced pressure to give 34.7 g
(65%) of 1,1-dimethyl-3-oxobutylammonium salt of
(ꢀ)-2 (equilibrated with 1,1-dimethyl-3-oxobutyl-
ammonium (ꢀ)-5-hydroxy-5-(4-methylphenyl)-3-thio-
1,1-Dimethyl-3-oxobutylammonium salt of (ꢁ)-2-mer-
captomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (2).
The mother liquor, which was given at the preparation
of (+)-1, was washed with concd hydrochloric acid (100
mL)–water (400 mL) and brine (500 mL), dried over
MgSO4, and evaporated in vacuo. The residue was dis-
solved in ethyl acetate (2000 mL) and mixed with R-
(+)-a-phenylethylamine (54.5 g, 0.500 mmol). The pre-
cipitate was recrystallized from ethyl acetate (2000 mL)
to give 177 g of colorless needles. The needles were
treated with ethyl acetate (1000 mL) and concd hydro-
chloric acid (100 mL)–water (400 mL). The organic
layer was washed with concd hydrochloric acid (50
mL)–water (450 mL) and brine (500 mL), dried over
MgSO4, and evaporated in vacuo. The residue was
recrystallized from ether (100 mL) and hexane (200
mL). The crystal was additionally recrystallized from
isopropylether to give 45.5 g (36%) of (ꢁ)-1. [a]D:
ꢁ26.3ꢂ(c 10.5, ethanol). Mp 86–87 ꢂC, 1H NMR
(CDCl3) d 2.38 (s, 3H), 2.40 (s, 3H), 3.16–3.54 (m, 5H),
7.24 (d, 2H, J=8.0 Hz), 7.84 (d, 2H, J=8.0 Hz). IR
(KBr) cmꢁ1: 3040, 1706, 1684, 1244, 1138, 808. MS m/z:
281(MH +). Anal. calcd for C14H16O4S: C, 59.98; H,
5.75; S, 11.44. Found: C, 60.06; H, 5.65; S, 11.73.
lane-carboxylate). Mp: 103–104 ꢂC, H NMR (metha-
1
nol-d4) dppm: 1.35 (s, 6H), 2.17 (s, 3H), 2.31 (s, 3H),
2.36–2.64 (m, 2.2H), 3.35–3.60 (m, 2.8H), 7.10 (d, 1.6H,
J=8.1Hz), 7.24–7.32 (m, 0.4H), 7.54 (d, 0.8H, J=8.1
Hz), 7.58 (d, 0.8H, J=8.1Hz), 7.90 (m, 0.4H); keto
form/thiolane form=1:4. IR (KBr) cmꢁ1 : 3091, 2979,
2931, 1710, 1519, 1397, 614. Mass m/z: 237 (M+). Anal.
calcd for C18H17NO4S: C, 61.16; H, 7.70; N, 3.96; S,
9.07. Found: C, 61.29; H, 7.83; N, 3.93; S, 9.07.
1,1-Dimethyl-3-oxobutylammonium salt of (+)-2-mer-
captomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (2).6
Compound (ꢀ)-1 (250 g, 892 mmol) and (ꢁ)-cinchoni-
dine (158 g, 537 mmol) were dissolved in methanol
(1000 mL), and the solution was concentrated under
reduced pressure. The residue was recrystallized from
ethyl acetate (2000 mL) to give colorless needles [the
mother liquor was used to give (ꢁ)-1], which were
added to fresh ethyl acetate (1000 mL). The mixture was
heated under reflux with stirring for 10 min, then
allowed to cool to obtain 156 g (wet) of purified needles.
The needles were partitioned between ethyl acetate
(1000 mL) and concd hydrochloric acid (100 mL)–water
(400 mL). The organic layer was washed concd hydro-
chloric acid (50 mL)– water (450 mL) and brine (500
mL), dried over MgSO4, and evaporated in vacuo. The
residue was recrystallized from ether (100 mL) and hex-
ane (100 mL) to give 50.4 g (40%) of (+)-1. [a]D
+25.1ꢂ(c 10.9, ethanol). Mp 87–88 ꢂC, 1H NMR
(CDCl3) d 2.36 (s, 3H), 2.42 (s, 3H), 3.18–3.54 (m, 5H),
7.24 (d, 2H, J=8.0 Hz), 7.84 (d, 2H, J=8.0 Hz). IR
(KBr) cmꢁ1: 3056, 2926, 1714, 1697, 1673, 1607, 1573,
1434, 1400, 1364, 1247, 1211, 1188, 1173, 1135, 1097,
1037, 1001, 954, 842, 816, 696, 621, 568. MS m/z: 281
(MH+). Anal. calcd for C14H16O4S: C, 59.98; H, 5.75;
S, 11.44. Found: C, 60.00; H, 5.65; S, 11.62.
A 25.0 g (89.2 mmol) sample of (ꢁ)-1 was dissolved in
acetone (90 mL), and 60 mL of 25% ammonia solution
was added dropwise at 3–9 ꢂC over 10 min. The reaction
mixture was stirred at 19–23 ꢂC for 120 min, and then
evaporated in vacuo. The resulting precipitates were
collected by filtration, washed with 20 mL of acetone,
and dried under reduced pressure to give 13.4 g (42%)
of 1,1-dimethyl-3-oxobutylammonium salt of (ꢁ)-2
(equilibrated with 1,1-dimethyl-3-oxobutyl-ammonium
(ꢁ)-5-hydroxy-5-(4-methylphenyl)-3-thiolane-carbox-
ylate). [a]Dꢁ25.0ꢂ(c 11.1, ethanol). Mp 112–113 ꢂC, H
1
NMR (methanol-d4) d ppm: 1.35 (s, 6H), 2.17 (s, 3H),
2.30 (s, 3H), 2.35–2.64 (m, 2.2H), 3.35–3.60 (m, 2.8H),
7.10 (d, 1.6H, J=8.1Hz), 7.34 (d, 0.4H, J=8.1Hz),
7.59 (d, 0.8H,J=0.8 Hz), 7.60 (d, 0.8H, J=0.8 Hz), 7.94
(d, 0.4H, J=8.1Hz); keto form/thiolane form=1:4. IR

T. Noguchi et al. / Bioorg. Med. Chem. 10 (2002) 2713–2721
2719
(KBr) cmꢁ1 : 2920, 2830, 1720, 1678, 1546, 1396, 1366,
810. Mass m/z: 237 (M+). Anal. calcd for C18H17NO4S:
C, 61.16; H, 7.70; N, 3.96; S, 9.07. Found: C, 61.19; H,
7.74; N, 3.96; S, 9.02.
(s, 1.5H), 2.61 (s, 1.5H), 2.80–3.60 (m, 5H), 7.35 (d, 2H,
J=8.1Hz), 7.88 (d, 2H, J=8.1 Hz), 12.64 (s, 1H). IR
(KBr) cmꢁ1: 3436, 2919, 2507, 1709, 1676, 1232, 1207,
994, 977, 803. Mass (CI) m/z: 269 ((M+H)+). Anal.
calcd for C13H16O4S :C, 58.19;H, 6.01; S, 11.95. Found:
C, 57.98; H, 5.94; S, 12.20.
IL-1ꢀ production in THP-1 cells.14,19,20 THP-1cells
(final concentration; 5ꢃ105 cells/mL, Dainihon Phar-
maceutical. Co. Ltd., Japan), a human monocytic leu-
kemia cell line, were cultured in 96-well microtiterplates
(Corning, USA), with each well containing 1ꢃ106 cells/
mL in 0.1mL of culture medium (RPMI1640 medium
supplemented with 100 U/mL penicillin,100 mg/mL
streptomycin, Life Technologies, Japan), 4000 units/mL
IFN g(final concentration: 1000 units/mL, Genzyme
Corporation, USA) in 0.05 mL of culture medium, and
various concentrations of each drug (final concentra-
tion: 6.25, 12.5, 25, 75, 100, 150 mg/mL in 0.05 mL of
the culture medium or 0.05 mL of culture medium
alone). Culture was performed in 95% air–5% CO2 at
37 ꢂC for 16 h, and 20 mg/mL LPS (final concentration;
1 mg/mL, Difco Laboratories, USA) was added in 0.01
mL of the culture medium. After 24 h of incubation,
culture supernatants were removed and assayed for IL-
1b concentrations using an IL-1b ELISA kit (Amer-
sham Pharmacia Biotech, USA).
(ꢀ)-4-(4-Hydroxymethylphenyl)-2-methylthiomethyl-4-
oxobutanoic acid (5). A 53.5 g (257 mmol) sample of
terephthalaldehyde mono(diethylacetal) (12) was dis-
solved in 500 mL of ethanol and 4.85 g (128 mmol) of
sodium boronhydride under ice cooling. The reaction
mixture was stirred at room temperature for 60 min,
poured into 1500 mL of water, and extracted with 1500
mL of ether. The extract was washed with brine and
water, dried over MgSO4, and evaporated in vacuo to
give 51.7 g of 4-hydroxymethylbenzaldehyde diethyla-
cetal (96%).
A 51.3 g (244 mmol) sample of 4-hydroxymethylbenz-
aldehyde diethylacetal was dissolved in pyridine (200
mL)–acetic anhydride (50 mL), and stirred overnight.
The reaction mixture was evaporated in vacuo, 500 mL
of satd sodium hydrocarbonate water solution was
added, and the mixture was extracted with 1500 mL of
ether. The extract was washed with brine and water,
dried over MgSO4, and evaporated in vacuo to give 61.1
g of 4-acetoxybenzaldehyde diethylacetal (quant).
(ꢀ)-2-Methylthiomethyl-4-(4-methylphenyl)-4-oxobuta-
noic acid (3). A 28.0 g (99.9 mmol) sample of (ꢀ)-1
was dissolved in 85% potassium hydroxide (20.8 g, 315
mmol) water solution (150 mL), and 0.2 g (0.50 mmol)
of trioctylmethylammonium chloride (TOMAC). The
reaction mixture was stirred at room temperature for 30
min. Methyliodide (7.0 mL, 112 mmol) was then added
and the mixture was stirred at room temperature for 30
min and then washed with ether. The aqueous layer was
acidified with 20 mL of concd hydrochloric acid,
extracted with ether. The organic layer was washed with
water, dried over MgSO4, and evaporated in vacuo. The
residue was recrystallized from hexane-ethyl acetate to
A 53.4 g (212 mmol) sample of 4-acetoxybenzaldehyde
diethylacetal was dissolved in THF (267 mL)–water (107
mL). Concd sulfuric acid (2.67 g)–water (27 mL) was
added and the mixture was stirred at room temperature
for 30 min. The reaction mixture was made alkaline with
potassium carbonate, and extracted with 1500 mL of ethyl
acetate. The extract was washed with brine and water,
dried over MgSO4, and evaporated in vacuo to give 37.5 g
(96% from 12) of 4-acetoxymethylbenzaldehyde (13).
give 21.7 g (80%) of (ꢀ)-3. Mp: 96–97 ꢂC. H NMR
A 5.00 g (28.1mmol) sample of 13 was dissolved in 35
mL of THF, and 3.55 g (30.9 mmol) of indium metal
(ꢁ200 mesh)21 was added. 2-(Bromomethyl)acrylic acid
(5.56 g, 33.7 mmol)–THF (25 mL) was added dropwise
over 25 min under argon atmosphere. The temperature
of the reaction mixture was raised from 18 to 39 ꢂC. The
reaction mixture was stirred for 35 min, 20 mL of 3 mol/
L hydrochloric acid was added, and the mixture was
then extracted with 300 mL of ethyl acetate. The extract
was washed with brine and water, dried over MgSO4,
and evaporated in vacuo. The residue was purified by
column chromatography using ethyl acetate/hex-
ane=1:1–3:1 to give 6.46 g (87%) of 4-(4-acetoxy-
1
(CDCl3, 300 MHz)d ppm: 2.14 (s, 3H), 2.42 (s, 3H), 2.77
(dd, 1H, J=8.2, 13.5 Hz), 2.96 (dd, 1H, J=5.4, 13.5
Hz), 3.28- 3.53 (m, 3H), 7.27 (d, 2H, J=7.9 Hz), 7.88 (d,
2H, J=7.9 Hz). IR (KBr) cmꢁ1: 2916, 1702, 1678, 1607,
1227, 810. Mass (ESI) m/z: 275 ((M+Na)+). Anal.
calcd for C13H16O3S: C, 61.88; H, 6.39; S, 12.71. Found:
C, 61.82; H, 6.37; S, 12.82.
(ꢀ)-4-(4-Methylphenyl)-2-methylsulfinylmethyl-4-oxobu-
tanoic acid (4). An 11.1 g (44.0 mmol) sample of (ꢀ)-3
was dissolved in tetrahydrofuran (THF) and cooled to
4 ꢂC, and 7.97 g (46.2 mmol) of mCPBA was added. The
temperature of the reaction mixture was then raised to
20 ꢂC. The reaction mixture was stirred at 13–20 ꢂC for
60 min, and then evaporated in vacuo. The residue was
washed with 100 mL of THF and then with 250 mL of
ethyl acetate. The product was recrystallized from 220
mL of chloroform and again from 170 mL of chloro-
form to give 4.41g (37%) of ( ꢀ)-4-(4-methylphenyl)-2-
methylsulfinylmethyl-4-oxobutanoic acid (4). The 1H
NMR spectrum of 4 suggested the presence of diaster-
methylphenyl)-4-hydroxy-2-methylenebutanoic
(14).
acid
A 6.19 g (23.4 mmol) sample of 14 was dissolved in 468
mL of acetone, and 6.0 mL of 4 mol/L Jones
reagent16,22 was added under ice cooling. The reaction
mixture was stirred for 5 min, 2.0 mL of Jones reagent
was added, and stirred for 5 min. Jones reagent (2.0 mL;
total 10.0 mL) was added along with 2.0 mL of iso-
propylalcohol. The reaction mixture was stirred for 10
min and extracted with 1000 mL of ether. The extract
ꢂ
1
eomer attached to a sulfur atom. Mp 141–142 C, H
NMR (DMSO-d6, 300 MHz) d ppm: 2.38 (s, 3H), 2.59

2720
T. Noguchi et al. / Bioorg. Med. Chem. 10 (2002) 2713–2721
was washed with water and brine, dried over MgSO4
and evaporated in vacuo. The residue was purified by
column chromatography using ethyl acetate to give 5.80
g (83%) of 4-(4-acetoxymethylphenyl)-2-methylene-4-
oxobutanoic acid (15).
245 mL of THF, and 27.1g (236 mmol) of indium metal
(ꢁ200 mesh) was added. 2-(Bromomethyl)acrylic acid
(43.1g, 261mmol)–THF (105 mL) was then added. The
reaction mixture was heated from 21to 55 ꢂC, and then
stirred at room temperature overnight. Hydrochloric
acid (1mol/L; 700 mL) was added and the mixture was
extracted with 350 mL of ethyl acetate. The extract was
washed with 1mol/L hydrochloric acid and water, dried
over MgSO4, and evaporated in vacuo. The residue was
recrystallized from ethyl acetate to give 33.1g (62%) of
4-hydroxy-4-(4-methoxycarbonylphenyl)-2-methylene-
butanoic acid (18).
A 5.80 g (22.1mmol) sample of 15 was suspended in 32
mL of toluene, and 2.00 g (26.3 mmol) of thioacetic acid
was added. Triethylamine (0.45 g, 4.45 mmol)–toluene
(4.0 mL) was added, and the mixture was stirred at
60 ꢂC for 2 h. The reaction mixture was poured into
cooled aqueous sulfuric acid, and extracted with 200 mL
of ethyl acetate. The extract was washed with brine and
water, dried over MgSO4, and evaporated in vacuo. The
residue was purified by column chromatography using
ethyl acetate/hexane=1:1–3:1 to give 6.88 g (92%) of 4-
(4-acetoxymethylphenyl)-2-acetylthiomethyl-4-oxobuta-
noic acid (16).
A solution of 8.55 mL (99.7 mmol) of oxalyl chloride in
222 mL of THF was cooled to ꢁ74 ꢂC and dimethyl-
sulfoxide (12.8 mL)–THF (38.5 mL) was added drop-
wise over 5 min. Compound 18 (20.4 g, 81.5 mmol)–
THF (204 mL) was then added dropwise over 11 min,
and 53.0 mL (382 mmol) of triethylamine was added
dropwise over 4 min. After stirring for 10 min, 400 mL
of concd hydrochloric acid was added over 15 min, and
the mixture was extracted with ethyl acetate. The extract
was washed with water and brine, dried over MgSO4,
and evaporated in vacuo. The residue was recrystallized
from ethyl acetate–hexane to give 8.61g (42%) of 2-
methylene-4-(4-methoxycarboxyphenyl)-4-oxobutanoic
acid (19).
A 6.50 g (19.2 mmol) sample 16 was dissolved in 94 mL
of 8% ammonia solution, and the mixture was stirred at
room temperature for 60 min. TOMAC (41mg, 0.10
mmol) and methyliodide (1.5 mL, 24.1 mmol)–ether (7
mL) were added, in this order, and stirred at room
temperature for 30 min. Subsequently, 340 mL of an
aqueous 5% potassium hydroxide solution was added
and the mixture was stirred at room temperature for 20
min. The reaction mixture was cooled and acidified with
3 mol/L hydrochloric acid and then extracted with 300
mL of ethyl acetate. The extract was washed with brine
and water, dried over MgSO4, and evaporated in
vacuo. The residue was purified by column chromato-
graphy using ethyl acetate/hexane=2:1–5:1 to give 4.12
A 6.96 g (28.0 mmol) sample of 19 was suspended in 42
mL of toluene, and 2.57 g (33.8 mmol) of thioacetic acid
was added. Triethylamine (0.57 g, 5.6 mmol)–toluene
(5.25 mL) was added and the mixture was stirred at
60 ꢂC for 40 min. The reaction mixture was cooled, 50
mL of 0.2 mol/L hydrochloric acid was added, and the
mixture was extracted with ethyl acetate. The extract
was washed with brine and water, dried over MgSO4,
and evaporated in vacuo. The residue was recrystallized
from ethyl acetate–hexane to give 5.57 g of 2-acet-
ylthiomethyl-4-(4-methoxycarbonylphenyl)-4-oxobuta-
noic acid (20). Its mother liquor was evaporated. The
residue was purified by column chromatography using
ethyl acetate/hexane=1:1–4:1 to give 2.75 g of 20.
Overall, 8.32 g (86%) of 20 was obtained.
g (80%) of (ꢀ)-5. Mp 98–99 ꢂC, H NMR (DMSO-d6
1
(200 MHz)d 2.08 (s, 3H), 2.71(dd, H1 ,
J=7.5, 13.2
Hz), 2.84 (dd, 1H, J=5.9, 13.2 Hz), 3.01–3.17 (m, 1H),
3.24 (dd, 1H, J=4.6, 18.0 Hz), 3.45 (dd, 1H, J=8.8,
18.0 Hz), 4.59 (s, 2H), 5.38 (br s, 1H), 7.47 (d, 2H,
J=8.3 Hz), 7.95 (d, 2H, J=8.3 Hz), 12.42 (br s, 1H).
IR (KBr) cmꢁ1: 3426, 2915, 2626, 1708, 1677, 1418,
1221, 1028, 816. FAB-MS m/z: 269 (M+H)+). Anal.
calcd for C13H16O4S: C, 58.19; H, 6.01. Found: C,
58.21; H, 5.95.
(ꢀ)-4-(4-Hydroxymethylphenyl)-2-methylsulfinylmethyl-
4-oxobutanoic acid (6). A 1.50 g (5.59 mmol) sample of
(ꢀ)-5 was dissolved in 20 mL of THF and cooled under
an ice bath. MCPBA (1.06 g, 6.14 mmol) was then added.
The reaction mixture was stirred under an ice bath for 60
min, and the crystal was collected by filtration to give
1.23 g (77%) of (ꢀ)-6. The 1H NMR spectrum of 6 sug-
gested the presence of diastereomer attached to a sulfur
atom. Mp 117–118 ꢂC, 1H NMR (DMSO-d6)
(200 MHz)d 2.58 (s, 1.5H), 2.61 (s, 1.5H), 2.80–3.64 (m,
5H), 4.58 (s, 2H), 5.38 (br s, 1H), 7.46 (d, 2H, J=8.0 Hz),
A 3.00 g (9.25 mmol) sample of 20 was dissolved in 43.5
mL of 8% ammonia solution, and stirred at room tem-
perature for 60 min. TOMAC (19 mg, 0.05 mmol) and
methyliodide (0.7 mL, 11.2 mmol)–ether (7 mL) were
added, in this order, and the mixture was stirred at
room temperature for 20 min. Subsequently, 154 mL of
an aqueous 5% potassium hydroxide solution were
added and the mixture was stirred at room temperature
for 30 min. The reaction mixture was cooled, acidified
with 3 mol/L hydrochloric acid and then extracted with
300 mL of ethyl acetate. The extract was washed
with brine and water, dried over MgSO4, and evapo-
rated in vacuo. The residue was purified by column
chromatography using ethyl acetate/THF=4:1–1:1 to
give 1.62 g (61%) of (ꢀ)-7. Compound 7 (0.82 g) was
recrystallized from ethyl acetate to give 0.54 g of purified
7. Mp 169–170 ꢂC, 1H NMR (DMSO-d6)(200MHz)d 2.08
(s, 3H), 2.72 (dd, 1 H,J=7.5, 13.4 Hz), 2.86 (dd, 1H,
J=5.9, 13.4 Hz), 3.04–3.20 (m, 1H), 3.31 (dd, 1H, J=4.6,
7.94 (d, 2H, J=8.0 Hz), 12.63 (br s, 1H). IR (KBr) cmꢁ1
:
3435, 2918, 2513, 1713, 1678, 1415, 1204, 978. FAB-MS
+
.
m/z: 285 (M+H) ). Anal. calcd for C13H16O5S 1/
23THF: C, 55.04; H, 5.73. Found: C, 55.01; H, 5.71.
(ꢀ)-2-Methylthiomethyl-4-(4-carboxyphenyl)-4-oxobut-
nanoic acid (7). A 35.0 g (213 mmol) sample of ter-
ephthalaldehydic acid methyl ester (17) was dissolved in

T. Noguchi et al. / Bioorg. Med. Chem. 10 (2002) 2713–2721
2721
18.2 Hz), 3.51 (dd, 1H, J=8.4, 18.2 Hz), 8.07 (s, 4H), 12.91
(br s, 2H). IR (KBr) cmꢁ1: 2916, 2676, 1700, 1682, 1432,
1296, 990. FAB-MS m/z: 281((M-H) +). Anal. calcd for
C13H14O5S: C, 55.31; H, 5.00. Found: C, 55.17; H, 4.88.
6. Kameo, K.; Takeshita, K.; Yasuda, Y.; Matsumoto, K.;
Tomisawa, K. Chem. Pharm. Bull. 1996, 44, 602.
7. Hashimoto, M.; Kobayashi, K.; Yamagata, N.; Katsura,
T.; Sugihara, S.; Iwabuchi, H.; Kasama, T.; Kasahara, K.;
Takahashi, T.; Takeshita, K.; Otomo, S. Agents Actions 1992,
37, 99.
(ꢀ)-4-(4-Carboxyphenyl)-2-methylsulfinyl-methyl-4-oxo-
butanoic acid (8). A 450 mg (1.59 mmol) sample of 7
was dissolved in 5.5 mL of THF and cooled under an
ice bath. mCPBA (303 mg, 1.76 mmol) was added. The
reaction mixture was stirred under an ice bath for 40
min, and the crystal was deposited. THF (2.0 mL) and
ethyl acetate (2.0 mL) were added to the reaction mix-
ture, and the resulting precipitates were collected by fil-
8. Yoshida, H.; Kohno, Y.; Endo, H.; Hasegawa, M.; Yamu-
guchi, J.; Tomisawa, K.; Suwa, T. Biol. Pharm. Bull. 1996, 19,
424.
9. Yoshida, H.; Kohno, Y.; Endo, H.; Ohmi, N.; Fukushima,
K.; Suwa, T.; Hayashi, M. Chirality 1997, 9, 22.
10. Kameo, K.; Ogawa, K.; Takeshita, K.; Nakaike, S.;
Tomisawa, K.; Sota, K. Chem. Pharm. Bull. 1988, 36, 2050.
11. Hasegawa, M.; Yoshida, H.; Endo, H.; Kohno, Y.;
Tomisawa, K. Xenobio. Metabol. Dipos 223, 9 (Suppl.) (in
Jananese).
12. Sasaki, J.; Yoshida, H.; Tomisawa, K.; Takeshita, K.;
Adachi, T. Biosci., Biotechnol. Biochem. 1998, 62, 1048.
13. Noguchi, T., Onodera, A., Ota, K., Muto, M., Tomi-
sawa, K., Yokomori, S., Abstract No. 3. The 121st Annual
Meeting of the Pharmaceutical Society of Japan, 2001;
p. 12.
1
tration to give 380 mg (80%) of (ꢀ)-8. The H NMR
spectrum of 8 suggested the presence of diastereomer
ꢂ
1
attached to a sulfur atom. Mp 158–160 C, H NMR
(DMSO-d6)(200 MHz) d 2.60 (s, 1.5H), 2.62 (s, 1.5H),
2.87–3.69 (m, 5H), 8.08 (s, 4H), 13.00 (br s, 2H). IR (KBr)
cmꢁ1: 2924, 1726, 1700, 1683, 1408, 1220, 1014, 764. FAB-
MS m/z: 299 (M+H)+). Anal. calcd for C13H16O6S 1 /
3H2O: C, 53.37; H, 5.19. Found: C, 53.17; H, 5.05.
14. Van den Berg, W. G.; Bresnihan, B. Baillieres Best Pract.
Res. Clin. Rheumatol. 1999, 13 (4), 577.
15. Li, C. J.; Chan, T. H. Tetrahedron Lett. 1991, 32, 7017.
16. Bowden, K.; Heilbron, I. M.; Jones, E. R. H.; Weedon,
B. C. L. J. Chem. Soc. 1946, 39.
References and Notes
1. Abe, T.; Mizushima, Y.; Kashiwazaki, S.; Sugawara, S.;
Nagaya, I.; Tanaka, S.; Nobunaga, M. Proc. XIXth ILAR
Congress Rheum. 1997, 19, 475.
2. Yasuda, Y.; Inoue, T.; Takeshita, K. Drugs Exp. Clin. Res.
1996, 22, 1 .
17. Yamaguchi, J.; Ohmichi, M.; Hasegawa, M.; Yoshida, H.;
Ogawa, N.; Higuchi, S. Drug Metab. Dispos. 2001, 29, 806.
18. Noguchi, T., Onodera, A., Tomisawa, K., Yokomori, S.
Manuscript in preparation.
19. Hayes, M. P.; Wang, J.; Norcross, M. A. Blood 1995,
86 (2), 646.
20. Kubin, M.; Chow, J. M.; Trinchieri, G. Blood 1994, 83 (7),
1847.
21. Soekawa Chemicals, 10–2 Cho-me, Uchikanda, Chiyoda-
Ku, Tokyo, Japan.
3. Takahashi, S.; Inoue, T.; Higaki, M.; Mizushima, Y. Drugs
Exp. Clin. Res. 1998, 24, 67.
4. Sakane, T.; Suzuki, N.; Hirose, Y.; Miura, K.; Wakisaka,
S.; Nagafuchi, H.; Ichino, M.; Tomita, T.; Hashimoto, H.;
Ochi, T.; Mihara, S. J. Rheum. 1997, 24, 2213.
5. Nagai, H.; Takaoka, Y.; Mori, H.; Kawada, K. Pharma-
cology 1996, 53, 190.
22. Meinwald, J.; Crandall, J.; Hymans, W. E. Org. Syn.
1965, 45, 28.