Discovery of novel and potent small-molecule inhibitors of NO and cytokine production as antisepsis agents: Synthesis and biological activity of alkyl 6-(N-substituted sulfamoyl)cyclohex-1-ene-1-carboxylate

Article abstract of DOI:10.1021/jm050623t

To develop a new therapeutic agent for sepsis, screening of the Takeda chemical library was carried out using mouse macrophages stimulated with lipopolysaccharide (LPS) to identify a new class of small-molecule inhibitors of inflammatory mediator production. The lead compound 5a was discovered, from which a series of novel cyclohexene derivatives I bearing a sulfamoyl and ester group were designed, synthesized and tested for their inhibitory activity against nitric oxide (NO) production. Derivatives I were synthesized by the coupling of sulfonyl chlorides and anilines with concomitant double bond migration in the presence of triethylamine, and phenyl ring substitution and modification of the ester and cyclohexene moieties were carried out. Among the compounds synthesized, ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]- cyclohex-1-ene-1-carboxylate [(R)-(+)-5n, TAK-242] was found to exhibit the most potent suppressive activity for the production of not only NO but also inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) induced by LPS-stimulated mouse macrophages with IC ₅₀ values of 1.8, 1.9 and 1.3 nM, respectively. It shows marked beneficial effects in vivo also. Intravenous administration of (R)-(+)-5n at doses of 0.1 mg/kg or more suppressed the production of NO and various cytokines [TNF-α, IL-6 and IL-1β] in the mouse endotoxin shock model. Furthermore, it protected mice from death dose-dependently and all mice survived at a dose of 3 mg/kg. The minimum effective dose to protect mice from lethality in this model was 0.3 mg/kg, which was consistent with those for inhibitory effects on the production of NO and cytokines. Compound CR)-(+)-5n is currently undergoing clinical trials for the treatment of sepsis.

Full text of DOI:10.1021/jm050623t

J. Med. Chem. 2005, 48, 7457-7467
7457
Discovery of Novel and Potent Small-Molecule Inhibitors of NO and Cytokine
Production as Antisepsis Agents: Synthesis and Biological Activity of Alkyl
6-(N-Substituted sulfamoyl)cyclohex-1-ene-1-carboxylate
Masami Yamada,* Takashi Ichikawa, Masayuki Ii, Mie Sunamoto, Katsumi Itoh, Norikazu Tamura, and
Tomoyuki Kitazaki
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 17-85, Jusohonmachi-2-chome,
Yodogawa-ku, Osaka 532-8686, Japan
Received June 30, 2005
To develop a new therapeutic agent for sepsis, screening of the Takeda chemical library was
carried out using mouse macrophages stimulated with lipopolysaccharide (LPS) to identify a
new class of small-molecule inhibitors of inflammatory mediator production. The lead compound
5a was discovered, from which a series of novel cyclohexene derivatives I bearing a sulfamoyl
and ester group were designed, synthesized and tested for their inhibitory activity against
nitric oxide (NO) production. Derivatives I were synthesized by the coupling of sulfonyl chlorides
and anilines with concomitant double bond migration in the presence of triethylamine, and
phenyl ring substitution and modification of the ester and cyclohexene moieties were carried
out. Among the compounds synthesized, ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]-
cyclohex-1-ene-1-carboxylate [(R)-(+)-5n, TAK-242] was found to exhibit the most potent
suppressive activity for the production of not only NO but also inflammatory cytokines, such
as tumor necrosis factor-R (TNF-R) and interleukin-6 (IL-6) induced by LPS-stimulated mouse
macrophages with IC50 values of 1.8, 1.9 and 1.3 nM, respectively. It shows marked beneficial
effects in vivo also. Intravenous administration of (R)-(+)-5n at doses of 0.1 mg/kg or more
suppressed the production of NO and various cytokines [TNF-R, IL-6 and IL-1â] in the mouse
endotoxin shock model. Furthermore, it protected mice from death dose-dependently and all
mice survived at a dose of 3 mg/kg. The minimum effective dose to protect mice from lethality
in this model was 0.3 mg/kg, which was consistent with those for inhibitory effects on the
production of NO and cytokines. Compound (R)-(+)-5n is currently undergoing clinical trials
for the treatment of sepsis.
Introduction
active internal bleeding and recent intracranial surgery.
Therefore, there is still a major unmet medical need for
more effective and safer new antisepsis agents.
The understanding of the pathophysiology of systemic
inflammatory response syndrome (SIRS) has increased
markedly over the past few years.1 Sepsis is generally
defined as SIRS induced by bacterial infection, and
severe sepsis is defined as sepsis associated with acute
organ dysfunction or hypoperfusion abnormalities.2
Septic shock is a subset of severe sepsis and is charac-
terized by hypotension refractory to treatment. In the
United States, more than 500 000 patients per year
develop sepsis. In particular, sepsis and SIRS are in-
creasingly the chief causes of death in intensive care
units, with mortality rates between 30 and 70%.1b,3 De-
spite more than two decades of extensive research, the
heterogeneity of the syndrome has meant that the de-
velopment of an anti-sepsis agent has proved remark-
ably difficult.4 Recently, Drotrecogin-R (Xigris), a recom-
binant human activated protein C, was launched to the
market.5 It lowered the mortality rate in severe sepsis
patients from 31-25%5c and is the only drug to be
approved for the treatment of severe sepsis. However,
the observed mortality difference between Drotrecogin-R
and placebo was limited to patients with high risk of
death. In addition, since Drotrecogin-R increases the
risk of bleeding, it is contraindicated in patients with
It is known that sepsis is caused by some bacterial
components such as lipopolysaccharide (LPS), lipote-
ichoic acid, and peptidoglycan, which prompt host
immune cells to activate and stimulate the production
of various kinds of mediators [cytokines, nitric oxide
(NO), lipid metabolites, etc.].6 Overwhelming inflam-
matory and immune responses are essential features
of sepsis and play a central part in the pathogenesis of
tissue damage, multiple organ failure, and death in-
duced by sepsis.5 A number of inflammatory modifiers
that suppress single inflammatory mediator production,
such as interleukin-1 (IL-1), tumor necrosis factor-R
(TNF-R), NO, or platelet activating factor, have already
entered clinical trials for the treatment of sepsis.7
Unfortunately, most of the candidates which have been
shown to be effective in preclinical studies have not
demonstrated significant efficacy for sepsis patients.4,7
Clinical trials of some of them are still ongoing. Thus,
we considered that a drug suppressing multiple inflam-
matory mediators is necessary for clinical effectiveness.
LPS is an important component of the outer mem-
brane of Gram negative bacteria and has a pivotal role
in inducing sepsis.6,8 LPS triggers the activation of the
immune system through the induction of cytokine
* To whom correspondence should be addressed. Tel: +81-6-6300-
6706. Fax: +81-6-6300-6306. E-mail: Yamada_Masami1@takeda.co.jp.
10.1021/jm050623t CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/14/2005

7458 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Yamada et al.
synthesis, structure-activity relationships, and biologi-
cal properties of this novel series of cyclohexene deriva-
tives.
Chemistry
The synthetic route to compound 5a is shown in
Scheme 1. The thioenolate 2, prepared from com-
mercially available ethyl 2-oxocyclohexanecarboxylate
(1) according to the literature method,10 was oxidized
with sodium trioxoborane tetrahydrate in acetic acid
(AcOH) to give the crude sulfonic acid 3. Subsequent
chlorination was carried out by reaction of 3 with thionyl
chloride (SOCl2), and the resulting sulfonyl chloride 4
was used without purification. Coupling of 4 with
4-chloro-2-fluoroaniline in the presence of triethylamine
(Et3N) gave exclusively the double bond isomerized
product 5a. The structure of 5a was determined by the
1H NMR chemical shifts of the cyclohexene, where the
olefinic and methine protons of 5a were observed at δ
7.12 (1H) and 4.32 (1H) and were assigned to the 2- and
6-position of the cyclohexene ring, respectively. How-
ever, when reaction of 4 with 4-chloro-2-fluoroaniline
was carried out in the absence of Et3N in N,N-dimeth-
ylformamide (DMF), only product 6a (structure deter-
mined by 1H NMR) was obtained in a low yield without
double bond isomerization.
Figure 1. Structure of lead compound 5a and cyclohexene
derivatives I.
release, which leads to damaged blood vessels and a
decrease in vascular resistance and subsequent collapse
of organs and death. Therefore, one promising approach
for treating sepsis is to target LPS itself. Various anti-
LPS agents, such as anti-LPS antibodies, LPS-neutral-
izing proteins, polymyxin B, and LPS antagonists (for
instance, lipid A analogue9) have been evaluated.
To discover new small molecule inhibitors of multiple
inflammatory mediator production from mouse mac-
rophages stimulated with LPS, screening of the Takeda
chemical library was carried out. Thus, a novel cyclo-
hexene derivative 5a bearing a phenylsulfamoyl and
ethyl ester group was identified, which showed suppres-
sive effects on the production of NO, TNF-R, and inter-
leukin-6 (IL-6) with 50% inhibitory concentration (IC50)
values of less than 1 µM in vitro. Furthermore, intra-
peritoneal administration of 5a showed a potent protec-
tive effect at a dose of 10-30 mg/kg in mouse endotoxin
shock model, which is a commonly used sepsis model.
Chemical modification of lead compound 5a, focusing
on the benzene ring substituents, the ester moiety, and
the cyclohexene ring (Figure 1) was carried out to im-
prove the suppressive effects on the production of NO
and various cytokines. In this paper, we describe the
Chemical modification of the substituents on the
phenyl ring was performed according to the synthetic
route shown in Scheme 2. Substituted aniline deriva-
tives 5c,d,f-j,m,q,r were prepared from 3 under the
same conditions as used in the synthesis of 5a (method
A). Thus, the sulfonic acid 3 was converted to sulfonyl
Scheme 1. Synthesis of Compound 5aa
a Reagents: (a) H2S/HCl, EtOH; (b) NaBO3‚4H2O, AcOH; (c) SOCl2; (d) 4-chloro-2-fluoroaniline, Et3N, AcOEt; (e) 4-chloro-2-fluoroaniline,
DMF.
Scheme 2. Synthesis of the N-Arylsulfamoyl Derivativesa
a Reagents: (a) SOCl2; (b) substituted aniline, Et3N, AcOEt; (c) 2-aminothiazole or 2-aminothiadiazole, Et3N, AcOEt; (d) Cl2,
AcOH-H2O.

Inhibitors of NO and Cytokine Production
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7459
Scheme 3. Synthesis of N-(Phenyl- and 4-Fluorophenyl)sulfamoyl Derivativesa
a Reagents: (a) SOCl2; (b) aniline, Et3N, AcOEt; (c) 4-fluoroaniline, Et3N, AcOEt.
Scheme 4. N-Methylation of Compound 5aa
the appropriate alcohol in the presence of a catalytic
amount of concentrated sulfuric acid. Hydroxyethyl
ester 15 was synthesized via the sodium salt of car-
boxylic acid 14. Thus, 5k was converted to sodium
carboxylate 14 by alkaline hydrolysis with sodium
hydroxide (NaOH) followed by purification using highly
porous polymer resin (CHP-20) and subsequent alky-
lation of 14 with 2-bromoethanol gave 15.
a Reagents: (a) MeI, K2CO3.
The synthesis of cyclopentene and cycloheptene de-
rivatives, 19 and 23, was accomplished as outlined in
Scheme 6. Compound 17 obtained from the commer-
cially available cyclopentene ester 16 using Lawesson’s
reagent was converted in three steps into the corre-
sponding sulfonyl chloride 18 by the same synthetic
route used for 5a. After purification of 18 using silica
gel chromatography, coupling reaction with 2-chloro-4-
fluoroaniline gave 19. The cycloheptene derivative 23
was also prepared from the cycloheptene ester 20,
derived from cycloheptanone according to Mague’s
method,12 using the same conditions employed for the
preparation of 19. The ring-opened derivative 28 was
synthesized from 25, prepared from ethyl 3-oxohex-
anoate,13 via the corresponding sulfonyl chloride 27 by
the same procedure used for 5a. Benzenesulfonyl chlo-
ride 29 was reacted with 2-chloro-4-fluoroaniline to give
30, followed by treatment with sodium methoxide in
ethanol to afford the benzene derivative 31 (Scheme 6).
Among the compounds prepared, compound 5n showed
the most potent suppressive effects on the production
of NO, and therefore separation of the enantiomers of
5n was carried out. Racemic mixture 5n was optically
resolved by preparative high-performance liquid chro-
matography (HPLC) using a chiral column [CHIRAL-
PAK AD,14 hexane/ethanol ) 9:1] to give each enanti-
omers, (+)-5n and (-)-5n, in high enantiomeric purity
(>99%ee). These enantiomers were submitted for X-ray
crystallographic analysis and the absolute configuration
chloride without purification and subsequent treatment
with various anilines in the presence of Et3N to give
5c,d,f-j,m,q,r. With a similar method, thiazole and
thiadiazole derivatives 5s, 5t were synthesized. Alter-
natively, chlorination of 2 with chlorine gas in AcOH-
water followed by chromatography on silica gel gave
purified sulfonyl chloride 4,11 which were reacted with
anilines in the presence of Et3N to give compounds 5k,
l, n-p (method B). In most cases, both methods A and
B exclusively gave the double bond isomerized products
5. However, when unsubstituted aniline was used, 5b
and nonisomerized product 6b were obtained, as shown
in Scheme 3. Additionally, the reaction of 4 with
4-fluoroaniline gave a mixture of 5e, nonisomerized
product 6e and cyclized product 7e, which was formed
by intramolecular cyclization of 6e. On the other hand,
the reaction of 4 with 4-methoxyaniline gave predomi-
nantly the nonisomerized product (data not shown).
From these results, it seems that the formation of the
isomerized cyclohexene derivative depends on the nu-
cleophilicity of the reactant aniline.
Methylation of the sulfonamide nitrogen with methyl
iodide and potassium carbonate proceeded in near-
quantitative yield to give the N-methylsulfonamide 8
(Scheme 4).
Conversion of the ethyl ester was carried out using
the 2,4-difluorophenyl derivative 5k shown in Scheme
5, since 5k showed more potent activity than 5a. The
esters 9-13 were prepared by transesterification with
Scheme 5. Variation of the Ester Group Using 2,4-Difluorophenyl Derivative 5ka
a Reagents: (a) X-OH, c.H2SO4; (b) 1 N NaOH, MeCN; (c) Br(CH2)2OH, DMF.

7460 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Scheme 6. Modification of the Cyclohexene Ringa
Yamada et al.
a Reagents: (a) Lawesson’s reagent; (b) NaBO3‚4H2O, AcOH, then SOCl2; (c) 2-chloro-4-fluoroaniline, Et3N, AcOEt; (d) EtI, NaOEt,
EtOH; (e) H2S, HCl, EtOH; (f) 2-chloro-4-fluoroaniline, pyridine, CH2Cl2; (g) NaOMe, EtOH.
Table 1. Inhibitory Activities of N-Arylsufamoyl Derivatives
5a-t, 6a,b,e, 7e, and 8 on NO Production
compd
R
IC50a (nM) compd
R
IC50a (nM)
Figure 2. X-ray crystal structure of (R)-(+)-5n.
5a
5b
5c
5d
5e
5f
5g
5h
5i
2-F, 4-Cl 160 ( 65
5n 2-Cl, 4-F
3.2 ( 0.89
41 ( 14
H
260 ( 113
75 ( 45
5o
5p
5q
5r
5s
5t
6a
6b
6e
7e
8
2-Cl, 4-Me
2-Cl, 4-CN
of (+)-5n and (-)-5n were confirmed to be (R)- and (S)-
form, respectively. The X-ray crystal structure of (R)-
(+)-5n is shown in Figure 2.
2-F
1600 ( 172
130 ( 16
3-F
4-F
150 ( 14
110 ( 11
12 ( 1.2
66 ( 19
2-Et
2-CO2Me
1100 ( 194
2-Cl
3-Cl
4-Cl
2,3-F2
2,6-F2
2,4-F2
2,4,5-F3
-
-
>10000
>10000
1700 ( 495
>8200
Results and Discussion
400 ( 279
140 ( 49
160 ( 4.9
16 ( 1.4
30 ( 1.8
20 ( 2.0
2-F, 4-Cl
H
The compounds were evaluated for their suppressive
activity against NO production using a murine mac-
rophage cell line, RAW264.7, induced by LPS as the first
in vitro screen. The results are summarized in Tables
1-4 as IC50 values.
5j
5k
5l
4-F
-
1400 ( 363
4100 ( 1742
230 ( 45
-
5m 2,4-Cl2
a The inhibitory activity is shown as an IC50 value, which is
the concentration of test compound required to suppress the
production of NO by 50% of control. Values are the mean ( SD of
two or three experiments.
We initially examined the effect of the substituent on
the phenyl ring of 5a. As shown in Table 1, lead
compound 5a with 4-chloro-2-fluorophenyl showed mod-
erate potency (IC50 ) 160 nM), but removal of the two
halogen atoms on the phenyl ring resulted in a ca. 2-fold
decrease in activity (5b, IC50 ) 260 nM). Introduction
of a fluorine atom at the 2-, 3- or 4-position of the phenyl
ring resulted in high potency (5c-e) compared to
unsubstituted compound 5b, with 2-fluoro derivative 5c
(IC50 ) 75 nM) exhibiting activity over 3-fold greater
than that of 5b. In the case of substitution with a
chlorine atom, 2-chloro derivative 5f (IC50 ) 12 nM)
showed improved activity compared to 3- or 4-chloro
derivatives (5g, h) and was 22-fold more potent than
that of 5b. These results suggested that either a chlorine
or fluorine atom at the 2-position on the phenyl ring
significantly increased the inhibitory activity against
NO production.
Next, the effect of di- or trihalogeno substitution on
the phenyl ring was examined. Introduction of a fluorine
atom at the 3- or 6-position of the 2-fluorophenyl ring
resulted in lower activity, while the 2,4-difluoro deriva-
tive 5k (IC50 ) 16 nM) showed a 5-fold enhancement in
potency compared to 2-fluoro derivative 5c. However,
2,4,5-trifluorophenyl derivative 5l was less potent than
2,4-difluorophenyl 5k. Furthermore, 2-chloro-4-fluo-
rophenyl derivative 5n showed strong activity with IC50

Inhibitors of NO and Cytokine Production
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7461
Table 2. Inhibitory Activities of Cyclohexene Derivatives 5k,
9-13, and 15 on NO Production
Table 3. Inhibitory Activities of Compounds 5n, 19, 23, 28,
and 31 on NO Production
compd
X
IC50a (nM)
compd
n
IC50a (nM)
5k
9
10
11
12
13
15
Et
16 ( 1.4
57 ( 5.4
29 ( 7.2
110 ( 11
140 ( 5.3
49 ( 26
Me
5n
19
23
28
31
1
3.2 ( 0.89
110 ( 16
n-Pr
0
i-Pr
2
240 ( 44
n-Bu
i-Bu
(CH2)2OH
-
-
2600 ( 124
>10000
330 ( 184
a The inhibitory activity is shown as an IC50 value, which is
the concentration of test compound required to suppress the
production of NO by 50% of control. Values are the mean ( SD of
two or three experiments.
a The inhibitory activity is shown as an IC50 value, which is
the concentration of test compound required to suppress the
production of NO by 50% of control. Values are the mean ( SD of
two or three experiments.
bic substituents are favorable for this moiety. Finally,
we examined the effect of changing the size of the
cyclohexene ring. As shown in Table 3, ring contraction
of 5n to the five-membered ring showed over 30-fold
reduction in potency. Ring expansion to seven-mem-
bered ring also exhibited a 75-fold less potency than 5n.
Furthermore, ring-opening of cyclohexene (28) and
replacement of cyclohexene ring with benzene ring (31)
caused a marked decrease of potency.
value of less than 10 nM (IC50 ) 3.2 nM), although 2,4-
dichlorophenyl and 4-chloro-2-fluorophenyl derivatives
(5m and 5a) showed less activity than 5f and 5c,
respectively. From these results, introduction of a
fluorine atom at the 4-position of the phenyl ring
resulted in a 4-fold enhancement in potency, whereas
substitution with a chlorine atom at the 4-position
decreased the inhibitory activity.
When an electron-withdrawing cyano group was
introduced at the 4-position of the phenyl ring in the
2-chloro derivative 5f, a remarkable decrease of activity
was observed. In contrast, introduction of a small,
slightly electron-donating methyl group in 5f retained
activity. In addition, substitution at the 2-position of the
phenyl ring with ethyl (5q) and methoxycarbonyl (5r)
groups showed less potency than for the 2-chlorophenyl
derivative 5f. Replacement of the phenyl group with
heteroaryl groups such as thiazole (5s) and thiadiazole
(5t) resulted in a loss of potency.
Comparison of the substituted phenyl derivatives
indicated that the compounds having 2,4-dihalogeno or
2-halogeno substituents on the phenyl ring were found
to have a strong activity and the order of their potency
was 2-Cl-4-F > 2-Cl; 2,4-F2; 2,4-Cl2; 2-Cl-4-Me; 2-F >2-
F-4-Cl. These results suggest that the potency of inhibi-
tory activity depends significantly on the position and
electronic properties of the phenyl ring substituents.
Introduction of a methyl group at the sulfamoyl
moiety resulted in a slight decrease in potency. Fur-
thermore, the IC50 values of the nonisomerized com-
pounds 6a, 6b, and 6e, and the ring closed compound
7e, were 1700, 8200, 1400, and 4100 nM, respectively,
which were over 10-fold less potent than the corre-
sponding isomers 5a, 5b, and 5e. It is suggested that
the position of the double bond in the cyclohexene ring
is crucial and plays an important role in its inhibitory
effect.
Next, attention was focused on exchange of the ethyl
ester group in 2,4-difluorophenyl derivative 5k (Table
2). Replacement of the ethyl group with a methyl group
resulted in a 4-fold reduction in activity. Substitution
with isopropyl (11), butyl (12) and isobutyl (13) groups
also showed over 3-fold less potency than 5k, while the
propyl ester 10 maintained activity. Additionally, the
2-hydroxyethyl ester derivative (15) showed 20-fold less
potency than ethyl ester 5k, suggesting that hydropho-
From the results of the SAR study, the cyclohexene
derivatives bearing the ester group at 1-position and
substituted phenylsufamoyl group at 6-position had the
inhibitory activity. It was suggested that the activity
was influenced by the shape of molecule from the results
of changing the size of cyclohexene ring, and that there
might be at least two pockets in the binding site on
target molecule (for the ester moiety and the phenyl
moiety), since it seemed that each moiety was strictly
recognized as mentioned above. Among all the com-
pounds prepared, 5n, having the combination of cyclo-
hexene ring, ethyl ester group, and 2-chloro-4-fluorophe-
nylsufamoyl group, exhibited the most potent suppressive
activity for NO production, and the activities of the two
enantiomers, (R)-(+)-5n and (S)-(-)-5n, were evaluated
in order to investigate the stereochemical requirement
for inhibition. As expected, a significant difference was
observed between the enantiomers, and (R)-(+)-5n (IC50
) 1.8 nM) exhibited 350-fold more activity than (S)-
(-)-5n (IC50 ) 640 nM). In addition, the suppressive
effects of 5n and both enantiomers on the production
of cytokines from LPS-stimulated RAW264.7 cells were
examined. (R)-(+)-5n strongly suppressed the produc-
tion of TNF-R and IL-6 with IC50 values of 1.9 and 1.3
nM, respectively (Table 4), which was 120-fold stronger
than those of lead compound 5a.
The in vivo efficacy of (R)-(+)-5n which was the high-
est inhibitor in the in vitro assay was examined using
mouse endotoxin shock model. When administrated
intraveniously 1 h before lethal LPS challenge, (R)-(+)-
5n rescued mice in a dose-dependent manner (Figure
3). At a dose of 3 mg/kg, all mice survived and the
minimum effective dose (ED50) value of (R)-(+)-5n to
protect mice from lethality was 0.3 mg/kg. In this model,
TNF-R, IL-6, IL-1â, and NO levels in serum increased
significantly after LPS challenge, and (R)-(+)-5n (0.1-3
mg/kg) suppressed the production of all cytokines and
NO induced by LPS in a dose-dependent manner. At a

7462 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Yamada et al.
Table 4. In Vitro Inhibitory Activities of NO, TNF-R, and IL-6
Production of 5a,n, (R)-(+)-5n, and (S)-(-)-5n
led to discover of the lead compound 5a. Cyclohexene
derivatives I bearing a 6-(substituted phenyl)sulfamoyl
and 1-(alkyl ester) group were designed and synthesized
by coupling of sulfonyl chlorides with a variety of
anilines in the presence of Et3N with concomitant
migration of the double bond. Chemical modification of
the substituents on the phenyl ring, ethyl ester moiety,
and cyclohexene ring was carried out and an identified
novel series of cyclohexene derivatives exhibited strong
suppressive effects on NO production from mouse mac-
ropharges stimulated with LPS. Among all the com-
pounds prepared, (R)-(+)-5n exhibited the most potent
inhibitory effect on the production of inflammatory
cytokines such as TNF-R and IL-6 as well as NO. These
IC50 values were approximately 100-fold stronger than
those of lead compound 5a. Furthermore, (R)-(+)-5n
showed significant in vivo efficacies in mouse endotoxin
shock model. The ED50 value to protect mice from
lethality was 0.3 mg/kg, and at a dose of 3 mg/kg all
mice survived. In addition, (R)-(+)-5n suppressed the
increase in serum levels of inflammatory mediators such
as TNF-R, IL-6, IL-1â, and NO.
IC50a (nM)
compd
NO
TNF-R
IL-6
5a
160 ( 65
3.2 ( 0.89
1.8 ( 0.30
230 ( 47
3.4 ( 1.3
1.9 ( 0.54
170 ( 20
2.5 ( 0.24
1.3 ( 0.12
5n
(R)-(+)-5n
(TAK-242)
(S)-(-)-5n
640 ( 365
740 ( 242
480 ( 10
a The inhibitory activity is shown as an IC50 value, which is
the concentration of test compound required to suppress the
production of NO, TNF-R, and IL-6 by 50% of control. Values are
the mean ( SD of three or four experiments.
Compound (R)-(+)-5n (TAK-242) was selected as a
candidate for clinical investigation and is a promising
new class of therapeutic agent for the treatment of
sepsis. Although the molecular target of (R)-(+)-5n has
not yet been identified, it is presumed that (R)-(+)-5n
selectively inhibits the Toll-like receptor (TLR) 4-de-
pendent signaling pathway.15
Figure 3. Effect of (R)-(+)-5n on LPS-induced lethality in
mice. (R)-(+)-5n (0.3-3 mg/kg) was administered intra-
venously 1 h before LPS challenge (7 mg/kg, ip). *, p e 0.05
(Tarone’s test with a simple closed step-down method) vs
vehicle. Each group consisted of 10 mice.
Experimental Section
dose of 0.1 mg/kg, (R)-(+)-5n significantly inhibited the
production of all cytokines and NO (Figure 4).
Chemistry. All melting points were determined on a
Yanagimoto micromelting point apparatus and are uncor-
rected. Proton nuclear magnetic resonance (1H NMR) spectra
were recorded on a Varian Gemini-200 (200 MHz) spectrom-
eter and Varian Mercury-300 (300 MHz) in the solvent
indicated. Chemical shifts are given in δ values (ppm) with
Conclusion
In this study, screening for new small-molecule
inhibitors of NO and inflammatory cytokines production
Figure 4. Effects of (R)-(+)-5n on LPS-induced increase in serum NO and proinflammatory cytokine levels in mice. (R)-(+)-5n
(0.1-3 mg/kg) was administered intravenously 1 h before LPS challenge (7 mg/kg, ip). Sera for TNF-R, IL-6, IL-1â, and NO were
collected at 1, 2, and 6 h after LPS challenge, respectively. Data are expressed as means ( SE of 8 mice. *, p e 0.025 (the one-
tailed Shirley-Williams or Williams test) vs vehicle (0 mg/kg).

Inhibitors of NO and Cytokine Production
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7463
tetramethylsilane as an internal standard. The following
abbreviations are used: s ) singlet, d ) doublet, t ) triplet,
q ) quartet, m ) multiplet and br ) broad. Coupling constants
(J values) are given in hertz (Hz). Infrared absorption spectra
(IR) were recorded on a JASCO IR-810 spectrometer. The
secondary ion mass spectra (SI-MS) were recorded with a
Hitachi M-80A mass spectrometer. Optical rotations were
determined with a JASCO DIP-181 or DIP-370 digital pola-
rimeter. Elemental analyses were carried out by Takeda
Analytical Laboratories Ltd. and were within (0.4% of the
theoretical values for the elements indicated unless otherwise
noted.
Reactions were carried out at room temperature unless
otherwise noted and followed by thin-layer chromatography
(TLC) on silica gel 60 F254 precoated TLC plates (E. Merck) or
by HPLC using an octadecyl silica (ODS) column (A-303, 4.6
mm i.d. × 250 mm, YMC Co., Ltd.). Standard workup pro-
cedures were as follows. The reaction mixture was partitioned
between the indicated solvent and water. Organic extracts
were combined and washed in the indicated order using the
following aqueous solutions: water, 5% aqueous sodium
carbonate solution (aqueous NaHCO3), saturated sodium
chloride (NaCl) solution (brine), 1 N aqueous sodium hy-
droxide solution (1 N NaOH) and 1 N hydrochloric acid (1 N
HCl). Extracts were dried over anhydrous magnesium sulfate
(MgSO4), filtered, and evaporated in vacuo. Chromatographic
separations were carried out on Silica gel 60 (0.063-0.200 mm,
E. Merck) or ODS (CPO-273L, prepacked column, 22 mm ×
300 mm, Kusano Kagaku Kikai Co.) using the indicated
eluents. Yields were not maximized.
Ethyl 2-Mercaptocyclohex-1-ene-1-carboxylate (2).10
Hydrogen sulfide (H2S) gas was bubbled through a solution of
ethyl 2-oxocyclohexanecarboxylate (1) (100 g, 538 mmol) in
ethanol (1.0 L) at -50 °C for 2 h, and then hydrogen chloride
(HCl) gas was bubbled into the reaction mixture at -20 °C
for 2 h, followed by the bubbling of H2S gas at -20 °C for 2 h.
After having been allowed to stand for 14 h, the reaction
mixture was poured into an ice-water (600 mL) and worked
up [hexane (2.0 L); water (800 mL × 5)]. The residue was
purified by distillation [bp 136-139 °C/15-16 mmHg (bp 134-
135 °C/15-16 mmHg)10] to give 2 (79.5 g, 73%) as a colorless
oil: IR νmax(KBr), cm-1: 2936, 1728, 1696, 1593, 1366, 1279,
1242, 1181, 1061. 1H NMR (CDCl3): δ 1.30 (3H, t, J ) 7.0
Hz), 1.67 (3H, t, J ) 3.0 Hz), 1.82-1.91 (1H, m), 2.24-2.35
(2H, m), 2.48 (2H, br s), 3.95 (1H, s), 4.22 (2H, q, J ) 7.0 Hz).
2-(Ethoxycarbonyl)cyclohex-1-ene-1-sulfonic Acid (3).
A solution of 2 (30.0 g, 161 mmol) in AcOH (50 mL) was added
dropwise to a stirred solution of sodium trioxoboran tetra-
hydrate (74.3 g, 483 mmol) in AcOH (400 mL) for 2 h at 50-
55 °C. The resulting mixture was stirred for 3 h at 50-55 °C
and for 9 h at 80-85 °C, and which was then cooled and
evaporated under reduced pressure to dryness. Acetonitrile
(MeCN) (660 mL) was added to the residue, and the whole
was stirred for 1 h followed by filtration to remove the insoluble
substances. After the filtrate was evaporated under reduced
pressure to dryness, the residue was dissolved in MeCN (500
mL) and then stirred for 2 h. The resulting precipitate was
filtered off, and the filtrate was evaporated under reduced
pressure to dryness. Diisopropyl ether (1.0 L) was added to
the residue to afford 3 (55 g, containing ca. 50% inorganic
substances) as a white powder: 1H NMR (DMSO-d6): δ 1.17
(3H, t, J ) 7.0 Hz), 1.53 (4H, br), 2.08-2.09 (2H, m), 2.22-
2.24 (2H, m), 3.99 (2H, q, J ) 7.0 Hz).
(3.64 g, 24.5 mmol), Et3N (3.41 mL, 24.5 mmol), and ethyl
acetate (54 mL) and then stirred for 18 h. The reaction mixture
was diluted with ethyl acetate (50 mL), and the whole was
then worked up [water (200 mL), brine (100 mL × 3)]. The
residue was crystallized from diisopropyl ether (8.0 mL), and
the precipitate was washed with diisopropyl ether (10 mL) and
ethyl acetate (8.0 mL) to yield 5a (1.60 g, 21.2% from 2) as
colorless crystals. The mother liquor and the washings were
combined and subjected to silica gel column chromatography
(hexane/ethyl acetate, 5:1 to 4:1) followed by crystallization
from ethyl acetate/diisopropyl ether to give second crop of 5a
(1.41 g, 18.7% from 2) as colorless crystals. mp 127-129 °C
(diisopropyl ether); 1H NMR (DMSO-d6): δ 1.10 (3H, t, J )
7.2 Hz), 1.57-1.82 (2H, m), 1.98-2.44 (4H, m), 4.02 (2H, q, J
) 7.2 Hz), 4.32 (1H, d, J ) 4.4 Hz), 7.12 (1H, t, J ) 3.4 Hz),
7.23-7.31 (1H, m), 7.45-7.54 (2H, m), 10.04 (1H, s). Anal.
(C15H17ClFNO4S) C, H, N.
The following compounds 5c, d, f-j, m, q-t were prepared
by a manner similar to that used for 5a.
Ethyl 6-[N-(2-Fluorophenyl)sulfamoyl]cyclohex-1-ene-
1-carboxylate (5c): Yield 43%; mp 110-111 °C (diisopropyl
ether); 1H NMR (DMSO-d6): δ 1.07 (3H, t, J ) 7.2 Hz), 1.58-
1.82 (2H, m), 2.05-2.46 (4H, m), 4.01 (2H, q, J ) 7.2 Hz), 4.32
(1H, d, J ) 4.6 Hz), 7.09-7.32 (4H, m), 7.44-7.54 (1H, m),
9.91 (1H, s). Anal. (C15H18FNO4S) C, H, N.
Ethyl 6-[N-(3-Fluorophenyl)sulfamoyl]cyclohex-1-ene-
1-carboxylate (5d): Yield 29%; mp 113-114 °C (ethyl acetate/
diisopropyl ether); 1H NMR (DMSO-d6): δ 1.16 (3H, t, J ) 7.0
Hz), 1.60-1.80 (2H, m), 2.00-2.33 (4H, m), 3.98-4.15 (2H,
m), 4.37 (1H, d, J ) 4.8 Hz), 6.87 (1H, dt, J ) 8.4, 7.0 Hz),
7.00-7.17 (3H, m), 7.34 (1H, dt, J ) 8.4, 7.0 Hz), 10.33 (1H,
brs). Anal. (C15H18FNO4S) C, H, N.
Ethyl 6-[N-(2-Chlorophenyl)sulfamoyl]yclohex-1-ene-
1-carboxylate (5f): Yield 19%; mp 210-211 °C (ethyl acetate/
hexane); 1H NMR (DMSO-d6): δ 1.05 (3H, t, J ) 7.0 Hz), 1.55-
1.84 (2H, m), 1.99-2.58 (4H, m), 4.00 (2H, q, J ) 7.0 Hz), 4.30
(1H, d, J ) 5.2 Hz), 7.11 (1H, br), 7.19-7.39 (2H, m), 7.48-
7.56 (2H, m), 9.66 (1H, s). Anal. (C15H18ClNO4S) C, H, N.
Ethyl 6-[N-(3-Chlorophenyl)sulfamoyl]cyclohex-1-ene-
1-carboxylate (5g): Yield 10%; mp 113-114 °C (ethyl acetate/
diisopropyl ether); 1H NMR (DMSO-d6): δ 1.16 (3H, t, J ) 7.0
Hz), 1.54-1.81 (2H, m), 1.94-2.38 (4H, m), 4.00-4.15 (2H,
m), 4.36 (1H, d, J ) 4.4 Hz), 7.07 (1H, br), 7.11-7.37 (4H, m),
10.29 (1H, s). Anal. (C15H18ClNO4S) C, H, N.
Ethyl 6-[N-(4-Chlorophenyl)sulfamoyl]cyclohex-1-ene-
1-carboxylate (5h): Yield 16%; mp 145-146 °C (ethyl acetate/
diisopropyl ether); 1H NMR (DMSO-d6): δ 1.15 (3H, t, J ) 7.0
Hz), 1.51-1.78 (2H, m), 1.95-2.20 (4H, m), 3.96-4.13 (2H,
m), 4.32 (1H, d, J ) 4.0 Hz), 7.13 (1H, t, J ) 4.0 Hz), 7.20-
7.24 (2H, m), 7.34-7.39 (2H, m), 10.17 (1H, s). Anal. (C15H18-
ClNO4S) C, H, N.
Ethyl 6-[N-(2,3-Difluorophenyl)sulfamoyl]cyclohex-1-
ene-1-carboxylate (5i): Yield 34%; mp 111-113 °C (ethyl
1
acetate/diisopropyl ether); H NMR (DMSO-d6): δ 1.00 (3H,
t, J ) 7.0 Hz), 1.58-1.83 (2H, m), 1.98-2.43 (4H, m), 4.02 (2H,
q, J ) 7.0 Hz), 4.38 (1H, d, J ) 4.4 Hz), 7.13-7.36 (4H, m),
10.22 (1H, s). Anal. (C15H17F2NO4S) C, H, N.
Ethyl 6-[N-(2,6-Difluorophenyl)sulfamoyl]cyclohex-1-
ene-1-carboxylate (5j): Yield 15%; mp 117-118 °C (ethyl
1
acetate/diisopropyl ether); H NMR (DMSO-d6): δ 1.00 (3H,
t, J ) 7.0 Hz), 1.59-1.88 (2H, m), 2.08-2.56 (4H, m), 3.97 (2H,
q, J ) 7.0 Hz), 4.39 (1H, d, J ) 5.0 Hz), 7.07-7.15 (4H, m),
7.34-7.50 (1H, m), 9.70 (1H, brs). Anal. (C15H17F2NO4S) C,
H, N.
Ethyl 6-[N-(4-Chloro-2-fluorophenyl)sulfamoyl]cyclo-
hex-1-ene-1-carboxylate (5a). Method A. Compound 3 (7.8
g) obtained above was dissolved in thionyl chloride (SOCl2)
(21.0 mL) and heated under reflux for 14 h, and then the
reaction mixture was evaporated under reduced pressure to
dryness. The residue was subjected three times to the proce-
dure involving an addition of hexane (30 mL) followed by
evaporation under reduced pressure to dryness to yield ethyl
2-(chlorosulfonyl)cyclohex-1-ene-1-carboxylate (6.8 g). This (6.2
g) was combined with ethyl acetate (20 mL), and the resultant
mixture was added to a solution of 4-chloro-2-fluoroaniline
Ethyl 6-[N-(2,4-Dichlorophenyl)sulfamoyl]cyclohex-1-
ene-1-carboxylate (5m): Yield 15%; mp 93-94 °C (ethyl
1
acetate/hexane); H NMR (DMSO-d6): δ 1.07 (3H, t, J ) 7.0
Hz), 1.54-1.82 (2H, m), 1.95-2.45 (4H, m), 4.01 (2H, q, J )
7.0 Hz), 4.32 (1H, d, J ) 4.8 Hz), 7.12 (1H, br), 7.40-7.67 (3H,
m), 9.81 (1H, s). Anal. (C15H17Cl2NO4S) C, H, N.
Ethyl 6-[N-(2-Ethylphenyl)sulfamoyl]cyclohex-1-ene-
1-carboxylate (5q): Yield 14%; mp 62-63 °C (ethyl acetate/
hexane); 1H NMR (DMSO-d6): δ 1.06 (3H, t, J ) 7.0 Hz), 1.16

7464 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Yamada et al.
(3H, t, J ) 7.6 Hz), 1.52-1.86 (2H, m), 1.99-2.50 (4H, m),
2.72 (2H, q, J ) 7.6 Hz), 4.01 (2H, q, J ) 7.0 Hz), 4.39 (1H, d,
J ) 4.8 Hz), 7.10 (1H, br), 7.16-7.38 (4H, m), 9.18 (1H, s).
Anal. (C17H23NO4S) C, H, N.
Ethyl 6-[N-(2-Methoxycarbonylphenyl)sulfamoyl]cyclo-
hex-1-ene-1-carboxylate (5r): Yield 18%; mp 91-92 °C
(ethyl acetate/diisopropyl ether); 1H NMR (DMSO-d6): δ 1.21
(3H, t, J ) 7.0 Hz), 1.68-2.36 (6H, m), 3.90 (3H, s), 3.93-4.07
(2H, m), 4.50 (1H, d, J ) 4.4 Hz), 7.15-7.23 (2H, m), 7.61-
7.69 (2H, m), 8.00 (1H, d, J ) 8.8 Hz), 10.39 (1H, s). Anal.
(C17H21NO5S) C, H, N.
mixture was added to a solution of 4-chloro-2-fluoroaniline
(0.55 g, 3.7 mmol) in DMF (5.0 mL). After having been stirred
for 18 h, the reaction mixture was diluted with ethyl acetate
(100 mL), and the whole was then worked up (water, brine).
The residue was purified by silica gel column chromatography
(hexane/ethyl acetate, 4:1) followed by crystallization from
ethyl acetate/diisopropyl ether to give 6a (44 mg, 3.3% from
2) as colorless crystals: mp 105-106 °C (ethyl acetate/
diisopropyl ether); 1H NMR (DMSO-d6): δ 1.06 (3H, t, J ) 7.2
Hz), 1.62 (4H, br), 2.25 (2H, br), 2.39 (2H, br), 3.95 (2H, q, J
) 7.2 Hz), 7.23-7.37 (2H, m), 7.47-7.52 (1H, m), 10.11 (1H,
s). Anal. (C15H17ClFNO4S) C, H, N.
Ethyl 6-[N-(1,3-Thiazol-2-yl)sulfamoyl]cyclohex-1-ene-
Ethyl 2-(Chlorosulfonyl)cyclohex-1-ene-1-carboxylate
(4). Chlorine (Cl2) gas was bubbled through a solution of 2
(1.40 g, 75.2 mmol) in AcOH (18 mL) and water (2.0 mL) below
15 °C for 20 min. After having been stirred for 30 min, nitrogen
gas was bubbled for 10 min. The mixture was concentrated in
vacuo, and the residue was worked up [ethyl acetate (20 mL
× 2); water (20 mL), brine (40 mL)]. The residue was purified
by silica gel chromatography (hexane/ethyl acetate, 9:1) to
afford 4 (1.40 g, 74%) as pale yellow crystals: mp 32-33 °C
(ethyl acetate); IR νmax(KBr), cm-1: 1738, 1375, 1279, 1254,
1-carboxylate (5s): Yield 0.9%; mp 156.5-157.5 °C (ethyl
1
acetate/diisopropyl ether); H NMR (DMSO-d6): δ 1.15 (3H,
t, J ) 7.0 Hz), 1.44-1.78 (2H, m), 2.04-2.36 (4H, m), 4.04 (2H,
m), 4.17 (1H, d, J ) 4.0 Hz), 6.79 (1H, q, J ) 4.6 Hz), 6.97
(1H, t, J ) 4.0 Hz), 7.21 (1H, d, J ) 4.6 Hz), 12.60 (1H, brs).
Anal. (C12H16N2O4S2‚1/4H2O) C, H, N.
Ethyl 6-[N-(1,3,4-Thiadiazol-2-yl)sulfamoyl]cyclohex-
1-ene-1-carboxylate (5t): Yield 4.1%; amorphous powder; 1H
NMR (DMSO-d6): δ 1.16 (3H, t, J ) 7.0 Hz), 1.15-1.77 (2H,
m), 2.00-2.40 (4H, m), 3.98-4.15 (2H, m), 4.20 (1H, d, J )
5.4 Hz), 6.55 (1H, s), 7.01 (1H, t, J ) 5.4 Hz), 8.70 (1H, s).
Ethyl 6-(N-Phenylsulfamoyl)cyclohex-1-ene-1-carbox-
ylate (5b) and Ethyl 2-(N-Phenylsulfamoyl)cyclohex-1-
ene-1-carboxylate (6b). Compound 3 (670 mg) was treated
with SOCl2 (2.0 mL) and aniline (280 mg) according to a
procedure similar to that described for the preparation of 5a.
The obtained residue was purified by silica gel column (hexane/
ethyl acetate, 4:1) and ODS column chromatography (methanol/
water, 7:3) followed by crystallization from methanol/water to
afford 5b (56 mg, 11% from 2) and 6b (37 mg, 7% from 2) as
colorless crystals, respectively.
1
1173, 604, 599. H NMR (CDCl3): δ 1.34 (3H, t, J ) 7.0 Hz),
1.70-1.90 (4H, m), 2.51-2.67 (4H, m), 4.30 (2H, q, J ) 7.0
Hz).
Ethyl 6-[N-(2,4-Difluorophenyl)sulfamoyl]cyclohex-1-
ene-1-carboxylate (5k). Method B. A solution of 4 (19.3 g,
76.4 mmol) in ethyl acetate (100 mL) was added dropwise for
40 min to an ice-cooled solution of 2,4-difluoroaniline (11.6 g,
90.3 mmol), Et3N (21.1 mL, 151 mmol), and ethyl acetate (100
mL) under nitrogen atmosphere. After having been stirred for
14 h, the reaction mixture was diluted with ethyl acetate (250
mL), and the whole was then worked up (water, 0.5 N HCl,
water, brine). The residue was purified by silica gel column
chromatography (hexane/ethyl acetate, 4:1) followed by crys-
tallization from ethyl acetate/diisopropyl ether to give 5k (19.9
g, 75.4%) as colorless crystals: mp 123-124 °C; 1H NMR
(DMSO-d6): δ 1.07 (3H, t, J ) 7.2 Hz), 1.46-1.82 (2H, m),
1.97-2.50 (4H, m), 4.01 (2H q, J ) 7.2 Hz), 4.28 (1H, d, J )
4.8 Hz), 7.04-7.15 (2H, m), 7.29-7.54 (2H, m) 9.86 (1H, brs).
Anal. (C15H17F2NO4S) C, H, N.
5b: mp 110-111 °C (methanol/water); 1H NMR (DMSO-
d6): δ 1.14 (3H, t, J ) 7.2 Hz), 1.55-1.74 (2H, m), 1.98-2.42
(4H, m), 3.97-4.12 (2H, m), 4.32 (1H, d, J ) 4.8 Hz), 7.02-
7.35 (6H, m), 10.03 (1H, brs). Anal. (C15H19NO4S) C, H, N.
6b: mp 112-120 °C (methanol/water); 1H NMR (DMSO-
d6): δ 1.23 (3H, t, J ) 7.0 Hz), 1.54 (4H, br), 2.25 (4H, br),
4.14 (2H, q, J ) 7.0 Hz), 7.02-7.32 (5H, m), 10.13 (1H, brs).
Anal. (C15H19NO4S) C, H, N.
Ethyl 6-[N-(4-Fluorophenyl)sulfamoyl]cyclohex-1-ene-
1-carboxylate (5e), Ethyl 2-[N-(4-Fluorophenyl)sulfa-
moyl]cyclohex-1-ene-1-carboxylate (6e), and 2-(4-Fluo-
rophenyl)-5,6,7,7a-tetrahydro-1,2-benzisothiazol-3(2H)-
one 1,1-Dioxide (7e). Compound 3 (720 mg) was treated with
SOCl2 (2.1 mL) and 4-fluoroaniline (340 mg) according to a
procedure similar to that described for the preparation of 5a.
The obtained residue was purified by silica gel column chro-
matography (hexane/ethyl acetate, 4:1) to give 7e (33 mg, 6%
from 2) as white powdery crystals. The second eluent was
purified with ODS column chromatography (methanol/water,
7:3) to afford 5e (36 mg, 12% from 2) and 6e (25 mg, 5% from
2) as colorless crystals, respectively.
The following compounds 5l, 5n, 5o, and 5p were prepared
by a manner similar to that used for 5k.
Ethyl 6-[N-(2,4,5-trifluorophenyl)sulfamoyl]cyclohex-
1-ene-1-carboxylate (5l): Yield 52%; mp 143-144 °C (ethyl
1
acetate/diisopropyl ether); H NMR (DMSO-d6): δ 1.13 (3H,
t, J ) 7.0 Hz), 1.55-1.85 (2H, m), 1.96-2.48 (4H, m), 4.05 (2H,
q, J ) 7.0 Hz), 4.35 (1H, d, J ) 4.4 Hz), 7.14 (1H, br), 7.47-
7.71 (2H, m), 10.17 (1H, s). Anal. (C15H16F3NO4S) C, H, N.
Ethyl 6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclo-
hex-1-ene-1-carboxylate (5n): Yield 78%; mp 102-103 °C
1
(ethyl acetate/hexane); H NMR (DMSO-d6): δ 1.05 (3H, t, J
) 7.0 Hz), 1.52-1.83 (2H, m), 1.98-2.46 (4H, m), 4.00 (2H, q,
J ) 7.0 Hz), 4.29 (1H, d, J ) 4.8 Hz), 7.10 (1H, br), 7.20-7.30
(1H, m), 7.49-7.58 (2H, m), 9.80 (1H, s). Anal. (C15H17-
ClFNO4S) C, H, N.
5e: mp 125-126 °C (methanol/water); 1H NMR (DMSO-
d6): δ 1.14 (3H, t, J ) 7.2 Hz), 1.55-1.77 (2H, m), 1.98-2.44
(4H, m), 3.97-4.13 (2H, m), 4.28 (1H, d, J ) 4.2 Hz), 7.10-
7.28 (5H, m), 10.03 (1H, brs). Anal. (C15H18FNO4S) C, H, N.
6e: mp 113-118 °C (methanol/water); 1H NMR (DMSO-
d6): δ 1.20 (3H, t, J ) 7.2 Hz), 1.54 (4H, br), 2.25 (4H, br),
4.11 (2H, q, J ) 7.2 Hz), 7.12 (2H, s), 7.16 (2H, s), 10.11 (1H,
brs). Anal. (C15H18FNO4S) C, H, N.
Ethyl 6-[N-(2-Chloro-4-methylphenyl)sulfamoyl]cyclo-
hex-1-ene-1-carboxylate (5o): Yield 46%; mp 76-77 °C
(ethyl acetate/diisopropyl ether); 1H NMR (DMSO-d6): δ 1.06
(3H, t, J ) 7.0 Hz), 1.51-1.83 (2H, m), 1.99-2.46 (4H, m),
2.29 (3H, s), 4.00 (2H, q, J ) 7.0 Hz), 4.29 (1H, d, J ) 5.4 Hz),
7.08 (1H, br), 7.12-7.16 (1H, m), 7.33-7.41 (2H, m), 9.53 (1H,
s). Anal. (C16H20ClNO4S) C, H, N.
Ethyl 6-[N-(2-Chloro-4-cyanophenyl)sulfamoyl]cyclo-
hex-1-ene-1-carboxylate (5p): Yield 28%; mp 95-96 °C
(ethyl acetate/diisopropyl ether); 1H NMR (DMSO-d6): δ 1.12
(3H, t, J ) 7.0 Hz), 1.56-1.84 (2H, m), 1.95-2.42 (4H, m),
4.03 (2H, q, J ) 7.0 Hz), 4.46 (1H, d, J ) 4.8 Hz), 7.20 (1H,
br), 7.70-7.84 (2H, m), 8.07 (1H, br), 10.09 (1H, s). Anal.
(C16H17ClN2O4S) C, H, N.
1
7e: mp 150-153 °C (diisopropyl ether); H NMR (DMSO-
d6): δ 1.75-1.88 (4H, m), 2.42-2.64 (4H, m), 7.40-7.49 (4H,
m). Anal. (C13H12FNO3S) C, H, N.
Ethyl 2-[N-(4-Chloro-2-fluorophenyl)sulfamoyl]cyclo-
hex-1-ene-1-carboxylate (6a). Compound 3 (2.0 g) was
dissolved in SOCl2 (5.0 mL) and heated under reflux for 14 h,
and then the reaction was evaporated under reduced pressure
to dryness. The residue was subjected three times to the
procedure involving an addition of hexane (10 mL) followed
by evaporation under reduced pressure to dryness to yield
ethyl 2-(chlorosulfonyl)cyclohex-1-ene-1-carboxylate. This was
combined with ethyl acetate (30 mL), and the resultant
Ethyl 6-[N-(4-Chloro-2-fluorophenyl)-N-methylsulfa-
moyl]cyclohex-1-ene-1-carboxylate (8). A mixture of 5a
(250 mg, 0.69 mmol), methyl iodide (118 mg, 1.38 mmol), K2-
CO3 (191 mg, 1.38 mmol) and DMF (2.5 mL) was stirred for 1

Inhibitors of NO and Cytokine Production
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7465
h and worked up (ethyl acetate; water). The residue was
purified by silica gel column chromatography (hexane/ethyl
acetate, 4:1) to give 8 (50 mg, 96%) as a colorless oil: 1H NMR
(DMSO-d6): δ 1.17 (3H, t, J ) 7.2 Hz), 1.56-2.44 (6H, m),
3.19 (3H, s), 4.12 (2H, q, J ) 7.2 Hz), 4.64 (1H, d, J ) 4.4 Hz),
7.16 (1H, t, J ) 3.6 Hz), 7.33-7.39 (1H, m), 7.54-7.62 (2H,
m). Anal. (C16H19ClFNO4S) C, H, N.
Methyl 6-[N-(2,4-Difluorophenyl)sulfamoyl]cyclohex-
1-ene-1-carboxylate (9). A mixture of 5k (300 mg, 0.87
mmol), concentrated H2SO4 (0.4 mL), and methanol (6.0 mL)
was stirred under reflux for 8 days. The reaction mixture was
concentrated in vacuo, and the residue was then worked up
(ethyl acetate; water). The residue was purified by silica gel
column chromatography (hexane/ethyl acetate, 5:1 to 2:1)
followed by crystallization from diisopropyl ether to give 9 (95
mg, 33%) as colorless crystals: mp 117-118 °C; 1H NMR
(DMSO-d6): δ 1.58-1.82 (2H, m), 1.98-2.42 (4H, m), 3.56 (3H,
s), 4.30 (1H, d, J ) 4.6 Hz), 7.05-7.15 (2H, m), 7.28-7.55 (2H,
m), 9.85 (1H, brs). Anal. (C14H15F2NO4S) C, H, N.
Propyl 6-[N-(2,4-Difluorophenyl)sulfamoyl]cyclohex-
1-ene-1-carboxylate (10). Compound 10 was prepared in 19%
yield from 5k and 1-propanol according to a procedure similar
to that described for the preparation of 9, as colorless crys-
tals: mp 110-111 °C (ethyl acetate/diisopropyl ether); 1H NMR
(DMSO-d6): δ 0.79 (3H, t, J ) 7.4 Hz), 1.38-1.82 (4H, m),
2.02-2.45 (4H, m), 3.91 (2H, t, J ) 6.4 Hz), 4.27 (1H, d, J )
4.8 Hz), 7.05-7.12 (2H, m), 7.28-7.53 (2H, m), 9.86 (1H, s).
Anal. (C16H19F2NO4S) C, H, N.
Isopropyl 6-[N-(2,4-Difluorophenyl)sulfamoyl]cyclo-
hex-1-ene-1-carboxylate (11). Compound 11 was prepared
in 10% yield from 5k and 2-propanol according to a procedure
similar to that described for the preparation of 9, as white
powder: mp 134-135 °C (ethyl acetate/diisopropyl ether); 1H
NMR (DMSO-d6): δ 1.04 (3H, d, J ) 6.4 Hz), 1.09 (3H, d, J )
6.4 Hz), 1.58-1.82 (2H, m), 2.02-2.45 (4H, m), 4.25 (1H, d, J
) 4.8 Hz), 4.83 (1H, septet, J ) 6.4 Hz), 7.05-7.15 (2H, m),
7.30-7.54 (2H, m), 9.86 (1H, s). Anal. (C16H19F2NO4S) C, H,
N.
aqueous NaHCO3, and brine). The residue was purified by
silica gel column chromatography (hexane/ethyl acetate, 1:1)
followed by crystallization from diisopropyl ether to give 15
(35 mg, 33%) as white powdery crystals: mp 110-111 °C (ethyl
1
acetate/diisopropyl ether); H NMR (DMSO-d6): δ 1.58-1.81
(2H, m), 2.00-2.42 (4H, m), 3.51 (2H, br), 4.00 (2H, t, J ) 5.0
Hz), 4.34 (1H, d, J ) 4.4 Hz), 4.77 (1H, br), 7.02-7.20 (2H,
m), 7.26-7.37 (1H, m), 7.44-7.56 (1H, m), 9.82 (1H, br). Anal.
(C15H17F2NO5S) C, H, N.
Ethyl 2-(Chlorosulfonyl)cyclopent-1-ene-1-carboxy-
late (18). Compound 1710 (3.90 g, 22.6 mmol), derived from
16 using Lawesson’s reagent, was treated according to a
procedure similar to that described for the preparation of 3 to
afford 2-(ethoxycarbonyl)cyclopent-1-ene-1-sulfonic acid (7.8 g)
as white powder containing inorganic substances. This powder
(1.0 g) was dissolved in SOCl2 (3.0 mL) and then stirred at 80
to 90 °C for 15 h. The mixture was evaporated under reduced
pressure to dryness and then worked up [ethyl acetate (50 mL);
water (50 mL), brine (50 mL)]. The residue was purified by
flash chromatography on silica gel column (hexane/ethyl
acetate, 5:1) to yield 18 (154 mg, 22% from 17) as a yellow oil:
IR νmax(KBr), cm-1: 1738, 1377, 1300, 1265, 1177, 613, 579,
1
550. H NMR (CDCl3): δ 1.35 (3H, t, J ) 7.0 Hz), 2.18 (2H,
quintet, J ) 8.0 Hz), 2.92-3.08 (4H, m), 4.33 (2H, q, J ) 7.0
Hz).
Ethyl 2-(Chlorosufonyl)cyclohept-1-ene-1-carboxylate
(22). Compound 22 was prepared in 34% yield from 21, which
was synthesized from 20, according to a procedure similar to
that described for the preparation of 18, as a brown oil: IR
1
νmax(KBr), cm-1: 2934, 1811, 1736, 1273, 1194, 810. H NMR
(CDCl3): δ 1.34 (3H, t, J ) 7.4 Hz), 1.60-2.00 (6H, m), 2.40-
2.90 (4H, m), 4.29 (2H, q, J ) 7.4 Hz).
Ethyl 5-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclo-
pent-1-ene-1-carboxylate (19). Compound 19 was prepared
in 23% yield from 18 and 2-chloro-4-fluoroaniline according
to a procedure similar to that described for the synthesis of
5k, as pale yellow crystals: mp 116-117 °C (diisopropyl ether/
hexane); 1H NMR (DMSO-d6): δ 1.15 (3H, t, J ) 7.0 Hz), 2.22-
2.74 (4H, m), 4.07 (2H, q, J ) 7.0 Hz), 4.50 (1H, d, J ) 8.0
Hz), 7.10 (1H, s), 7.18-7.28 (1H, m), 7.47-7.56 (2H, m), 9.70
(1H, s). Anal. (C14H15ClFNO4S) C, H, N.
Ethyl 7-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclo-
hept-1-ene-1-carboxylate (23). Compound 23 was prepared
in 3.5% yield from 22 and 2-chloro-4-fluoroaniline according
to a procedure similar to that described for the synthesis of
5k, as pale yellow crystals: mp 71.5-72.5 °C (hexane); 1H
NMR (DMSO-d6): δ 1.23 (3H, t, J ) 7.0 Hz), 1.19-1.38 (1H,
m), 1.67-1.81 (3H, m), 2.02-2.15 (1H, m), 2.15-2.76 (3H, m),
4.05 (2H, q, J ) 7.0 Hz), 4.80 (1H, t, J ) 4.6 Hz), 7.17-7.27
(1H, m), 7.44-7.59 (3H, m), 9.59 (1H, s). Anal. (C16H19-
ClFNO4S) C, H, N.
Butyl 6-[N-(2,4-Difluorophenyl)sulfamoyl]cyclohex-1-
ene-1-carboxylate (12). Compound 12 was prepared in 27%
yield from 5k and 1-butanol according to a procedure similar
to that described for the preparation of 9, as white crystals:
mp 85-86 °C (ethyl acetate/diisopropyl ether); 1H NMR
(DMSO-d6): δ 0.83 (3H, t, J ) 7.0 Hz), 1.18-1.82 (6H, m),
2.00-2.42 (4H, m), 3.95 (2H, br), 4.24 (1H, d, J ) 4.4 Hz), 7.09
(2H, br), 7.30-7.49 (2H, m), 9.86 (1H, brs). Anal. (C17H21F2-
NO4S) C, H, N.
Isobutyl 6-[N-(2,4-Difluorophenyl)sulfamoyl]cyclohex-
1-ene-1-carboxylate (13). Compound 13 was prepared in 16%
yield from 5k and 2-butanol according to a procedure similar
to that described for the preparation of 9, as white crystals:
mp 93-94 °C (ethyl acetate/diisopropyl ether); 1H NMR
(DMSO-d6): δ 0.80 (6H, d, J ) 6.8 Hz), 1.58-1.84 (2H, m),
2.00-2.47 (4H, m), 3.35-3.45 (1H, m), 3.75 (2H, d, J ) 6.8
Hz), 4.27 (1H, d, J ) 4.8 Hz), 7.03-7.13 (2H, m), 7.27-7.53
(2H, m), 9.85 (1H, s). Anal. (C17H21F2NO4S) C, H, N.
Sodium 6-[N-(2,4-Difluorophenyl)sulfamoyl]cyclohex-
1-ene-1-carboxylate (14). Compound 5k (2.50 g, 7.22 mmol)
was dissolved in MeCN (288 mL), and then 1 N NaOH (288
mL) was added to the solution. The mixture was stirred at 55
°C for 12 h and concentrated in vacuo. The residue was
chromatographed on CHP-20P column (water to 50% aqueous
methanol solution). The eluent was concentrated in vacuo, and
the residue was dissolved in water (10 mL) and lyophilized to
yield 14 (0.50 g, 15%) as a white powder: SI-MS m/z: 340 (M
Ethyl (2Z)-3-(Chlorosulfonyl)-2-ethylhex-2-enoate (27).
Compound 2513 (44.2 g, 908 mmol) was treated with H2S and
HCl gases according to a procedure similar to that described
for the preparation of 2 to afford 26 (35.1 g) containing
byproduct. The resulting 26 (10.0 g) was treated with sodium
trioxoboran tetrahydrate (22.8 g), followed by SOCl2 (45.6 mL)
to give 27 (1.76 g, 9.7% from 25): 1H NMR (CDCl3): δ 0.93-
1.44 (9H, m), 1.70-2.82 (6H, m), 4.15-4.37 (2H, m).
Ethyl (2E)-3-[(2-Chloro-4-fluorophenyl)sulfamoyl]-2-
ethylidenhexanoate (28). Compound 28 was prepared in
12% yield from 27 and 2-chloro-4-fluoroaniline according to a
procedure similar to that described for the synthesis of 5k, as
a brown solid: mp 47-48 °C (hexane); IR νmax(KBr), cm-1
:
3260, 2965, 1719, 1495, 1333, 1229, 1159, 1049, 887. 1H NMR
(DMSO-d6): δ 0.86 (3H, t, J ) 7.2 Hz), 1.13 (3H, t, J ) 7.0
Hz), 1.19-2.17 (4H, m), 2.02 (3H, d, J ) 7.2 Hz), 4.08 (2H, q,
J ) 7.0 Hz), 4.28 (1H, dd, J ) 11.6, 3.6 Hz), 6.51 (1H, q, J )
7.2 Hz), 7.19-7.28 (1H, m), 7.46-7.53 (2H, m), 9.59 (1H, s).
Anal. (C16H21ClFNO4S) C, H, N. The obtained compound 28
was determined to be the (E)-isomer by NOE experiment.
Methyl 2-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]ben-
zoate (30). To an ice-cooled solution of methyl 2-(chlorosul-
fonyl)benzoate (29) (1.06 g, 4.52 mmol) in CH2Cl2 (10 mL) were
1
+ H). H NMR (DMSO-d6): δ 1.50-1.65 (2H, m), 1.78-2.41
(4H, m), 4.13 (1H, d, J ) 4.0 Hz), 6.88-6.98 (2H, m), 7.09-
7.20 (1H, m), 7.42 (1H, dt, J ) 9.0, 6.2 Hz). Anal. (C13H12F2-
NO4SNa‚H2O) C, H, N.
2-Hydroxyethyl 6-[N-(2,4-Difluorophenyl)sulfamoyl]-
cyclohex-1-ene-1-carboxylate (15). 2-Bromoethanol (81 mg,
0.65 mmol) was added to a solution of 14 (100 mg, 0.30 mmol)
in DMF (2.0 mL) at 0 °C. The mixture was stirred for 72 h at
room temperature and then worked up (ethyl acetate; water,

7466 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23
Yamada et al.
added 2-chloro-4-fluoroaniline (0.27 mL, 2.26 mmol) and
pyridine (0.77 mL, 9.94 mmol), and then the mixture was
stirred for 3 days. After compound 29 (500 mg, 2.13 mmol)
and pyridine (0.41 mL, 2.34 mmol) were added, the mixture
was stirred for 12 h further and then worked up [ethyl acetate
(50 mL); 1 N HCl (50 mL × 2), brine (50 mL)]. The residue
was purified by silica gel column chromatography (hexane/
ethyl acetate, 20:1 to 5:1) followed by crystallization from
hexane to give 30 (127 mg, 17%) as white powdery crystals:
mp 139.0-139.5 °C; IR νmax(KBr), cm-1: 1725, 1493, 1292,
1173. 1H NMR (CDCl3): δ 4.05 (3H, s), 6.94-7.04 (2H, m),
7.49-7.70 (3H, m), 7.80-7.90 (2H, m), 8.44 (1H, s). Anal.
(C14H11ClFNO4S) C, H, N.
at -80 °C until use. The LPS stock solution was diluted to
appropriate concentrations with saline or culture medium
before use.
Assay for Inhibitory Activity against NO Production.
RAW264.7 cells were plated at 1 × 105 cells/well in 96-well
culture plates (Nunc, Rochester, NY) and incubated overnight.
After removing cell culture supernatants, cells were stimulated
with 10 ng/mL LPS in the presence of various concentrations
of test compounds for 20 h in stimulation medium (phenol red-
free RPMI1640 containing 1% heat-inactivated FBS and 10
µg/mL kanamycin) at 37 °C in a humidified atmosphere of 5%
CO2 in air. The test compounds were dissolved at 10 mmol/L
in DMF, diluted with an RPMI-1640 medium to the appropri-
ate concentrations, and added to the culture.
Ethyl 2-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]ben-
zoate (31). To a solution of compound 30 (70 mg, 0.20 mmol)
in ethanol (5.0 mL) was added 28% sodium methoxide in
methanol (39 mg, 0.20 mmol), and then the mixture was
stirred for 3 days under reflux. The reaction mixture was
concentrated in vacuo and worked up [ethyl acetate (20 mL);
1 N HCl (30 mL), brine (30 mL)]. The residue was purified by
silica gel column chromatography (hexane/ethyl acetate, 10:1
to 5:1) followed by crystallization from hexane to give 31 (46
mg, 64%) as white powdery crystals: mp 138-139 °C; IR
νmax(KBr), cm-1: 1715, 1495, 1281, 1173. 1H NMR (CDCl3): δ
1.47 (3H, t, J ) 7.2 Hz), 4.52 (2H, q, J ) 7.2 Hz), 6.94-7.04
(2H, m), 7.48-7.90 (5H, m), 8.46 (1H, s). Anal. (C15H13-
ClFNO4S) C, H, N.
Production of NO was estimated by measuring the amount
of nitrite, a stable metabolite of NO, according to a fluorometric
method using 2,3-diaminonaphthalene (DAN, Dojindo Labo-
ratories, Kumamoto, Japan). Briefly, 25 µL of 20 µg/mL DAN
was added to 50 µL of the culture supernatant and incubated
at room temperature for 10 min. After adding 25 µL of 0.5 N
NaOH, a fluorescence at 460 nm (excitation wavelength: 355
nm) was measured.
Assay for Inhibitory Activity against Cytokines Pro-
duction. Concentrations of TNF-R, and IL-6 in the culture
supernatants were determined using specific enzyme-linked
immunosorbent assay (ELISA) (Amersham Life Science, Buck-
inghamshire, UK) according to the manufacturer’s instruc-
tions.
Ethyl (6R)-6-[(2-Chloro-4-fluorophenyl)sulfamoyl]cy-
clohex-1-ene-1-carboxylate [(R)-(+)-5n] and Ethyl (6S)-
6-[(2-Chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-
carboxylate [(S)-(-)-5n]. The racemic mixture 5n (2.01 g)
was separated by preparative HPLC using CHIRALPAK AD14
(10.0 mm i.d. × 250 mm) under detection at 254 nm. Elution
with a mixture of hexane/ethanol (9:1) at a flow rate of 8.0
mL/min at room temperature gave two enantiomers, (R)-(+)-
5n (959 mg) and (S)-(-)-5n (979 mg), as pale yellow oils. The
obtained enantiomers were purified by silica gel column
chromatography (hexane/ethyl acetate, 9:2) followed by re-
crystallization from diisopropyl ether/hexane to give (R)-(+)-
5n (634 mg) and (S)-(-)-5n (681 mg) as colorless crystals,
respectively.
Endotoxin Shock Model. Mice were intraperitoneally
injected with LPS at a lethal dose of 7 mg/kg. Survival of mice
was recorded for 7 days following LPS challenge. Compound
(R)-(+)-5n was dissolved in 10% (w/v) Glucuronylglucosyl-â-
cyclodextrin sodium salt (Wako, Osaka, Japan) and adminis-
tered intravenously 1 h before the LPS injection.
Evaluation of Cytokine and NO Production In Vivo.
The blood was collected at the indicated times, and allowed to
clot at room temperature. The serum was separated by
centrifugation and stored at -80 °C until analysis. The serum
levels of TNF-R, IL-6, and IL-1â were determined by specific
ELISA (Amersham Life Science) according to the manufac-
turer’s instructions.
The serum levels of nitrite and nitrate, which are stable end
products of NO metabolism were measured by a fluorometric
method.16 Briefly, 10 µL aliquots of sample were mixed with
20 µL of â-NADPH (0.2 mmol/L, Roche, Mannheim, Germany)
and 20 µL of Aspergillus nitrate reductase (0.25 U/mL, Roche)
and incubated for 20 min at room temperature to convert
nitrate to nitrite. To these samples was added 25 µL of DAN
(25 µg/mL, Dojindo Laboratories) in HCl (2 mol/L) and
incubated for 10 min at room temperature. NaOH (25 µL) (0.5
mol/L) was added, and the fluorescence (excitation: 355 nm,
emission: 460 nm) was measured. The nitrite concentrations
were calculated from a standard curve with sodium nitrite
(Wako).
(R)-(+)-5n (TAK-242): mp 54-55 °C; [R]D20 +111.0° (c 1.0,
methanol). IR νmax(KBr), cm-1: 3245, 2969, 1720, 1649, 1491,
1329, 1256, 1150, 1067, 889. 1H NMR (DMSO-d6): δ 1.05 (3H,
t, J ) 7.0 Hz), 1.56-1.83 (2H, m), 2.01-2.43 (4H, m), 4.00 (2H,
q, J ) 7.0 Hz), 4.30 (1H, d, J ) 5.0 Hz), 7.10 (1H, br), 7.20-
7.30 (1H, m), 7.50-7.58 (2H, m), 9.74 (1H, s). Anal. (C15H17-
ClFNO4S) C, H, N.
(S)-(-)-5n: mp 54-55 °C; [R]D20 -111.0° (c 1.0, methanol).
IR νmax(KBr), cm-1: 2936, 1709, 1605, 1493, 1314, 1250, 1128,
1
1061. H NMR (DMSO-d6): δ 1.05 (3H, t, J ) 7.0 Hz), 1.55-
1.84 (2H, m), 1.96-2.43 (4H, m), 4.00 (2H, q, J ) 7.0 Hz), 4.29
(1H, d, J ) 5.0 Hz), 7.10 (1H, br), 7.20-7.30 (1H, m), 7.50-
7.58 (2H, m), 9.73 (1H, s). Anal. (C15H17ClFNO4S) C, H, N.
Statistical Analysis. For analysis of the production of
cytokine and NO, one-tailed Williams or Shirley-Williams test
was used. The survival rate of mice was evaluated by Tarone’s
test with a simple closed step-down method.
The enantiomeric excess of (R)-(+)-5n and (S)-(-)-5n were
determined by HPLC to be >99%, respectively [column:
CHIRALPAK AD14 (4.6 mm i.d. × 250 mm), room temperature;
eluent: hexane/ethanol (9:1); flow rate: 0.6 mL/min; detec-
tion: UV at 254 nm; tR of (R)-(+)-5n, 20.0 min; tR of (S)-(-)-
5n, 16.6 min].
Biological Methods. Cells, Animals, and Materials.
Murine macrophage cell line RAW 264.7 was purchased from
American Type Culture Collection (Manassas, VA). The cells
were cultured in RPMI1640 (Nikken Bio Medical Laboratories,
Kyoto, Japan) containing 10% heat-inactivated fetal bovine
serum (FBS) and 10 µg/mL kanamycin at 37 °C in a humidified
atmosphere of 5% CO2 in air.
Acknowledgment. We thank Drs. A. Miyake, K.
Okonoigi, Y. Ichimori, Y. Iizawa, and S. Hashiguchi for
their encouragement and helpful advice. We thank Mr.
A. Fujishima and Ms. K. Higashikawa for X-ray crystal-
lographic analysis, Ms. M. Murabayashi for NOE mea-
surement, and Dr. T. Yamano and Mr. M. Tanaka for
optical resolution. We thank Dr. T. Sha, Mr. K. Oda,
Ms. M. Uekata, and Ms. N. Matsunaga for in vitro and
in vivo assays.
Female BALB/c mice (6-week-old) weighing 15-21 g were
obtained from Charles River Japan Inc. (Kanagawa, Japan),
and allowed to acclimate for at least one week before use. LPS
(Escherichia coli O111:B4, Sigma, St. Louis, MO) was dissolved
in saline at a concentration of 10 mg/mL in advance, and kept
Supporting Information Available: X-ray crystallo-
graphic data for (R)-(+)-5n and elemental analyses for the new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.

Inhibitors of NO and Cytokine Production
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 23 7467
sepsis and septic shock. Clin. Microbiol. Rev. 2003, 16, 379-
414. (b) Hack, C. E.; Aarden, L. A.; Thijs, L. G. Role of cytokines
in sepsis. Adv. Immunol. 1997, 66, 101-195. (c) Casey, L. C.;
Balk, R. A.; Bone, R. C. Plasma cytokine and endotoxin levels
correlate with survival in patients with the sepsis syndrome.
Ann. Intern. Med. 1993, 119, 771-778. (d) Riedemann, N. C.;
Guo, R. F.; Ward, P. A. Novel strategies for the treatment of
sepsis. Nature Med. 2003, 9, 517-524.
References
(1) (a) Parrillo, J. E. Pathogenetic mechanism of septic shock. N.
Engl. J. Med. 1993, 328, 1471-1478. (b) Rangel-Frausto, M. S.;
Pittet, D.; Costigan, M.; Hwang, T.; Davis, C. S.; Wenzel, R. P.
The natural history of the systemic inflammatory response
syndrome (SIRS). A prospective study. JAMA 1995, 273, 117-
123. (c) Nystrom, P. O. The systemic inflammatory response
syndrome: definition and aetiology. J. Antimicrob. Chemother.
1998, 41, 1-7. (d) Bone, R. C.; Grodzin, R. J.; Balk, R. A.
Sepsis: a new hypothesis for the pathogenesis of the disease
process. Chest 1997, 112, 235-243. (e) Hotchkiss, R. S.; Karl, I.
E. The pathophysiology and treatment of sepsis. N. Engl. J. Med.
2003, 348, 138-150.
(2) (a) Bone, R. C.; Balk, R. A.; Cerra, F. B.; Dellinger, R. P.; Fein,
A. M.; Knaus, W. A.; Schein, R. M.; Sibbald, W. J.; Definitions
for sepsis and organ failure and guidelines for the use of
innovative therapies in sepsis. The ACCP/SCCM Consensus
Conference Committee. American College of Chest Physicians/
Society of Critical Care medicine. Chest 1992, 101, 1644-1655.
(b) Bone, R. C.; Sprung, C. L.; Sibbald, W. J.; Definitions for
sepsis and organ failure. Crit. Care Med. 1992, 20, 724-726. (c)
Members of the American College of Chest Physicians/Society
of Critical Care Medicine Consensus Conference Committee.
American College of Chest Physicians/Society of Critical Care
Medicine Consensus Conference: Definitions for sepsis and
organ failure and guidelines for the use of innovative therapies
in sepsis. Crit. Care Med. 1992, 20, 864-874. (d) Levy, M. M.;
Fink, M. P.; Marshall, J. C.; Abraham, E.; Angus, D.; Cook, D.;
Cohen, J.; Opal, S. M.; Vincent, J. L.; Ramsay, G. 2001 SCCM/
ESICM/ACCP/ATS/SIS International Sepsis Definitions Confer-
ence. Crit. Care Med. 2003, 31, 1250-1256.
(3) (a) Martin, G. S.; Mannino, D. M.; Eaton, S.; Moss, M. The
epidemiology of sepsis in the United States from 1979 through
2000. N. Engl. J. Med. 2003, 348, 1546-1554. (b) Wheeler, A.
P.; Bernard, G. R. Treating patients with severe sepsis. N. Engl.
J. Med. 1999, 340, 207-214.
(4) (a) Riedemann, N. C.; Ward, P. A. Anti-inflammatory strategies
for the treatment of sepsis. Expert Opin. Biol. Ther. 2003, 3,
339-350. (b) Riedemann, N. C.; Guo, R. F.; Ward, P. A. The
enigma of sepsis. J. Clin. Invest. 2003, 112, 460-467. (c) Arrieta,
O.; Rodriguez-Reyna, T. S.; Sotelo, J. Pharmacological treatment
of septic shock. Exp. Opin. Ther. Patents 2000, 10, 601-622. (d)
Bone, R. C.; Fisher, C. J., Jr.; Clemmer, T. P.; Slotman, G. J.;
Metz, C. A.; Balk, R. A. Sepsis syndrome: a valid clinical entry.
Crit. Care Med. 1989, 17, 389-393. (e) Natanson, C.; Esposito,
C. J.; Banks, S. M. The sirens’ song of confirmatory sepsis
trials: selection bias and sampling error. Crit. Care Med. 1998,
26, 1927-1931.
(7) (a) Beutler, B.; Milsark, I. W.; Cerami, A. C. Passive immuniza-
tion against cachectin/tumor necrosis factor protects mice from
lethal effect of endotoxin. Science 1985, 229, 869-871. (b)
Reinhart, K.; Karzai, W. Anti-tumor necrosis factor therapy in
sepsis: update on clinical trials and lesson learned. Crit. Care
Med. 2001, 29 (Suppl.), S121-S125. (c) Abraham E. Why
immunomodulatory therapies have not worked in sepsis.Inten-
sive Care Med. 1999, 25, 556-566. (d) Calandra, T.; Echtenacher,
B.; Roy, D. L.; Pugin, J.; Metz, C. N.; Hultner, L.; Heumann, D.;
Mannel, D.; Bucala, R.; Glauser, M. P. Protection from septic
shock by neutralization of macrophage migration inhibitory
factor. Nature Med. 2000, 6, 164-170.
(8) (a) Rietschel, E. T.; Brade, H. Bacterial endotoxins. Sci. Am.
1992, 267, 26-33. (b) Rietschel, E. T.; Kirikae, T.; Schade, F.
U.; Mamat, U.; Schmidt, G.; Loppnow, H.; Ulmer, A. J.; Zahr-
inger, U.; Seydel, U.; Padova, F. Di. Bacterial endotoxin:
molecular relationships of structure to activity and function.
FASEB J. 1994, 8, 217-225. (c) Glauser, M. P.; Zanetti, G.;
Baumgatner, J. D.; Cohen, J. Septic shock: pathogenesis. Lancet
1991, 338, 732-736. (d) Karima, R.; Matsumoto, S.; Higashi,
H.; Matsushima, K. The molecular pathogenesis of endotoxic
shock and organ failure. Mol. Med. Today 1999, 5, 123-132.
(9) Mullarkey, M.; Rose, J. R.; Bristol, J.; Kawata, T.; Kimura, A.;
Kobayashi, S.; Przetak, M.; Chow, J.; Gusovsky, F.; Christ, W.
J.; Rossignol, D. P. Inhibition of endotoxin response by E5564,
a nobel Toll-Like Receptor 4-directed endotoxin antagonist. J.
Pharmacol. Exp. Ther. 2003, 304, 1093-1102.
(10) Duus, F. â-Thioxo esters-II: Evidence for ester group rotamerism
and pertubation of the intramolecular hydrogen-bonding in
enethiolized 2-thioxo cycloalkanecarbonylic esters. Tetrahedron
1974, 30, 3753-3763.
(11) Langler, R. F. A facile synthesis of sulfonyl chlorides. Can. J.
Chem. 1976, 54, 498-499.
(12) Li, C. J.; Chen, D. L.; Lu, Y. Q.; Haberman, J. X.; Mague, J. T.
Metal-mediated two-atom carbocycle enlargement in aqueous
medium. Tetrahedron 1998, 54, 2347-2364.
(13) Jpn. Kokai Tokkyo Koho JP 60004182 A2 19850110 Showa, 1985
[Chem. Abstr. 1985, 102, 220891.]
(14) Daicel Chemical Industries, Ltd.
(15) Ii, M.; Sunamoto, M.; Matsunaga, N.; Nakamura, K.; Takashima,
K.; Kitazaki, T.; Sato, J.; Iizawa, Y. TAK-242 suppresses Toll-
like receptor 4-mediated cytokine production and protects mice
from Escherichia coli-induced lethality. Crit. Care Med. 2004,
32(Suppl.), A15.
(16) Misko, T. P.; Schilling R. J.; Salvemini D.; Moore W. M.; Currie
M. G. A fluorometric assay for the measurement of nitrite in
biological samples. Anal. Biochem. 1993, 214, 11-16.
(5) (a) Bernard, G. R.; Vincent, J. L.; Laterre, P. F.; LaRosa, S. P.;
Dhainaut, J. F.; Lopez-Rodriguez, A.; Steingrub, J. S.; Garber,
G. E.; Helterbrand, J. D.; Ely, E. W.; Fisher, C. J. Efficacy and
safety of recombinant human activated protein C for severe
sepsis. N. Engl. J. Med. 2001, 344, 699-709. (b) Warren, H. S.;
Suffredini, A. F.; Eichacker, P. Q.; Munford, R. S. Risks and
benefits on activated protein C treatment for severe sepsis. N.
Engl. J. Med. 2002, 347, 1027-1030.
(6) (a) Van Amersfoort, E. S.; Van Berkel, T. J. C.; Kuiper, J.
Receptors, mediators, and mechanisms involved in bacterial
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