Novel cyclohexene derivatives as anti-sepsis agents: Synthetic studies and inhibition of NO and cytokine production

Article abstract of DOI:10.1016/j.bmc.2008.01.030

In order to develop an anti-sepsis agent, a series of cyclohexene derivatives were synthesized and evaluated for their biological activities. Through modification of the sulfonamide spacer moiety depicted by formula II, it was found that the benzylsulfone derivative 10a had potent inhibitory activity against the production of NO. Further modifications of the phenyl ring, ester moiety, and benzyl position of benzylsulfone derivatives III were carried out. Among these compounds, (R)-(+)-10a and (6R, 1S)-(+)-22a showed strong inhibitory activity not only against NO production but also against inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in vitro. Furthermore, (R)-(+)-10a and (6R, 1S)-(+)-22a protected mice from LPS-induced lethality in a dose-dependent manner.

Full text of DOI:10.1016/j.bmc.2008.01.030

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3941–3958
Novel cyclohexene derivatives as anti-sepsis agents:
Synthetic studies and inhibition of NO and cytokine production
Masami Yamada,* Takashi Ichikawa, Masayuki Ii, Katsumi Itoh,ꢀ
Norikazu Tamura and Tomoyuki Kitazaki
Pharmaceutical Research Division, Takeda Pharmaceutical Co., Ltd, 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532-8686, Japan
Received 6 December 2007; revised 16 January 2008; accepted 17 January 2008
Available online 24 January 2008
Abstract—In order to develop an anti-sepsis agent, a series of cyclohexene derivatives were synthesized and evaluated for their bio-
logical activities. Through modification of the sulfonamide spacer moiety depicted by formula II, it was found that the benzylsulfone
derivative 10a had potent inhibitory activity against the production of NO. Further modifications of the phenyl ring, ester moiety,
and benzyl position of benzylsulfone derivatives III were carried out. Among these compounds, (R)-(+)-10a and (6R, 1S)-(+)-22a
showed strong inhibitory activity not only against NO production but also against inflammatory cytokines, such as tumor necrosis
factor-a (TNF-a) and interleukin-6 (IL-6) in vitro. Furthermore, (R)-(+)-10a and (6R, 1S)-(+)-22a protected mice from LPS-induced
lethality in a dose-dependent manner.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
oxide (NO) and cytokine production in vitro.6a Among
these derivatives, ethyl (6R)-6-[N-(2-chloro-4-fluoro-
phenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate [TAK-242,
(R)-1] was found to exhibit potent inhibitory activity for
the production of not only NO but also of various cyto-
kines such as tumor necrosis factor-a (TNF-a) and
interleukin-6 (IL-6) in vitro and in vivo including potent
protective effects in the mouse endotoxin shock model.6
This compound was selected as a clinical candidate for
development as a new class of anti-sepsis agent.
Sepsis and septic shock are the main causes of death in
intensive care units.1 The incidence of sepsis has in-
creased along with the higher occurrence of chronic dis-
ease states, permanent catheters, and resistance to
antimicrobial agents, among other causes.2 The condi-
tion of sepsis is caused by bacterial infection, which
prompts the host immune cells to activate and stimulate
the production of various types of mediators.3 Although
a number of inflammatory modifiers have been devel-
oped for the treatment of sepsis over the last 20 years,
most have not demonstrated significant efficacy in clini-
cal trials.4 In 2001, Drotrecogin-a (XigrisTM),5 a recombi-
nant human activated protein C, was launched onto the
market as the first anti-sepsis agent; however, the useful-
ness of this drug is limited due to its efficacy and safety
issue. Therefore, there is still a major unmet medical
need for more effective and safer new anti-sepsis agents.
We have recently reported the synthesis of novel cyclo-
hexene derivatives and their inhibitory activities of nitric
As an extension of our study on the cyclohexene deriv-
atives depicted by the general formula I in Figure 1,
we next focused on the synthesis of analogs with substi-
tutions of the N-phenylsulfamoyl moiety, such as phen-
ylsulfonylamino, phenyloxysulfonyl, benzylsulfide, and
benzylsulfone derivatives with the general formula II.
Furthermore, we planned the substitution of various
(hetero)aryl groups (Ar), modification of ester moiety
(Z) and introduction of substituents (R1) at the benzyl
position of benzylsulfone derivatives with the general
formula III. Compounds of type III were expected to ex-
hibit potent activity because of their structural similarity
to compounds of type I.
Keywords: Sepsis; Cyclohexene; Benzylsulfone; TAK-242.
*
Corresponding author. Tel.: +81 6 6300 6651; fax: +81 6 6300
6306; e-mail: Yamada_Masami1@takeda.co.jp
ꢀ
We herein describe the synthesis, biological activities,
and structure–activity relationships (SAR) of new cyclo-
hexene derivatives, II and III.
Present address: Kyoto Factory, Riken Vitamin Co. Ltd, 1-2
Kawarajiri-Higashikakiuchi, Kawarabayashicho, Kameoka, Kyoto
621-0007, Japan.
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.01.030

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M. Yamada et al. / Bioorg. Med. Chem. 16 (2008) 3941–3958
Figure 1. Design of cyclohexene derivatives with a sulfone linker.
2. Chemistry
The synthetic route for sulfide and sulfone derivatives is
shown in Scheme 2. Thioacetylation of the acetoxy
group of 7 followed by deacetylation under the acidic
conditions gave thiol 8. Sulfide 9a was prepared by the
coupling reaction of thiol 8 and 2-chloro-4-fluorobenzyl-
bromide. Subsequently, the sulfide was oxidized with m-
chloroperbenzoic acid (mCPBA) to yield the sulfone
10a.
First, in order to examine the effect of linker (Y) on the
inhibition of NO production, we chose functional
groups such as sulfonyl or sulfide in replacing the sul-
fonamide moiety of compound I. The synthesis of phen-
ylsulfonylamino and phenyloxysulfonyl derivatives is
shown in Scheme 1. Bromination of hydroxy cyclohex-
ene 27 by Villieras’ method8 followed by reaction with
sodium azide in N,N-dimethylformamide (DMF) affor-
ded azide 3. Subsequent reduction of 3 with zinc powder
under acidic conditions gave the amine, which was trea-
ted with 2-chloro-4-fluorobenzenesulfonylchloride in the
presence of triethylamine (Et3N) and pyridine in tetra-
hydrofuran (THF) to give phenylsulfonylamido product
4. The phenyloxysulfonyl derivative 6 was prepared by
coupling of sulfonyl chloride 56a with 2-chloro-4-fluoro-
phenol in the presence of 1,4-diazabicyclo[2.2.2]octane
(DABCO).
Substitution on the phenyl ring in compounds of type
III (Z = CO2Et, R1 = H) was performed according to
the synthetic route shown in Scheme 3. The target
benzylsulfone derivatives 10b–o were prepared by oxi-
dation of the corresponding sulfides 9b–o using
mCPBA. Compounds 9b–o were synthesized by the
condensation of thiol 8 and benzylbromides 11b–o in
the presence of an appropriate base [Et3N, 1,8-diazabi-
cyclo [5.4.0]undec-7-ene (DBU) or potassium carbonate
(K2CO3)].
Scheme 1. Reagents: (a) NBS, PPh3, CH2Cl2 (48%); (b) NaN3, DMF (48%); (c) Zn, HCl, acetone; (d) 2-chloro-4-fluorobenzenesulfonylchloride,
pyridine, Et3N, THF (23%, 2 steps); (e) 2-chloro-4-fluorophenol, DABCO, AcOEt (5.3%).
Scheme 2. Reagents: (a) AcSK, Et3N (81%); (b) 4 N HCl/AcOEt, EtOH (94%); (c) 2-chloro-4-fluorobenzylbromide, DBU, DMF (87%); (d) mCPBA,
AcOEt (88%).

M. Yamada et al. / Bioorg. Med. Chem. 16 (2008) 3941–3958
3943
Scheme 3. Reagents: (a) BrCH2–Ar (11b–o), base; (b) mCPBA, AcOEt.
Sulfones containing a heteroaryl ring in place of the phe-
nyl ring were synthesized as shown in Scheme 4. As de-
scribed above, the condensation between thiol 8 and
heteroarylmethylbromides 13b–g9–13 was carried out un-
der the presence of DBU to give sulfides 12b–g. Oxida-
tion with mCPBA yielded the heteroarylmethyl
sulfones 14b–g. The deprotection of 14b was achieved
with trifluoroacetic acid (TFA) to give indole derivative
14a.
pared by transesterification of 10a with the appropriate
alcohol in the presence of a catalytic amount of concen-
trated sulfuric acid (Method A). The other esters were
synthesized from 9a in three steps: ester hydrolysis, fol-
lowed by Mitsunobu reaction with the appropriate
alcohol, and subsequent oxidation of sulfides 16d and
16e to give 15d and 15e, respectively (Method B,
Scheme 5).
Conversion of the ester moiety to hydroxymethyl or ke-
tone is shown in Scheme 6. Reduction of sulfide 9a with
diisobutylaluminum hydride (DIBAL-H) gave the
hydroxymethyl derivative 16f, which was oxidized with
Next, modification of the ester group (Z) in benzylsulf-
one derivatives of type III was carried out as outlined
in Schemes 5 and 6. The esters 15a and 15b were pre-
Scheme 4. Reagents: (a) heteroarylmethylbromides (13b–g); DBU, DMF; (b) mCPBA, AcOEt; (c) TFA (74%).
Scheme 5. Reagents and condition: (a) R3OH, concd H2SO4; (b) 1 N NaOH, EtOH, 40 ꢁC (58%); (c) R3OH, DEAD, PPh3, EtOH-THF; (d) mCPBA,
AcOEt.

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M. Yamada et al. / Bioorg. Med. Chem. 16 (2008) 3941–3958
Scheme 6. Reagents: (a) DIBAL-H, CH2Cl2 (86%); (b) AcCl, Py; (c) TPAP, NMO, CH2Cl2; (d) R4MgBr, THF; (e) mCPBA, AcOEt.
mCPBA to prepare the sulfone derivative 15f. On the
other hand, the reaction of 16f with acetyl chloride fol-
lowed by oxidation gave acetoxy derivative 15g. Further-
more, oxidation of 16f with tetra-n-propylammonium
perruthenate (TPAP) followed by Grignard reaction with
ethyl or n-propylmagnesium bromide gave 16h and 16i as
a diastereomeric mixture, respectively. Each of these com-
pounds was oxidized with TPAP and mCPBA in two steps
to provide ketones 15h and 15i. 1-Hydroxypropyl com-
pound 16h was also converted into sulfone 15j by mCPBA
oxidation.
Compounds possessing an alkyl group in the benzyl po-
sition of compound type III (Z = CO2Et) were synthe-
sized as described in Scheme 7. Condensation of thiol
8 and (1-bromoethyl)benzene (17) according to the con-
ditions shown above in Scheme 2 gave sulfide derivative
18 as a diastereomeric mixture. On the other hand, the
Scheme 7. Reagents: (a) DBU, DMF (61%); (b) mCPBA, AcOEt; (c) Bu3P@CHCN, toluene; (d) Et3N, DBU, DMF (23%); (e) 6 N HCl, MeOH
(89%); (f) DAST, CH2Cl2 (55%).

M. Yamada et al. / Bioorg. Med. Chem. 16 (2008) 3941–3958
3945
condensation of 8 and 1-(2-chloro-4-fluorophenyl)etha-
nol (20) was successfully carried out under Mitsunobu
conditions with cyanomethylenetributylphosphorane
(CMBP) to give sulfide 21. The reaction of 8 and a-
substituted benzylbromide 23 proceeded under the basic
conditions to give sulfide 24. Furthermore, 24 was trea-
ted with acid followed by diethylaminosulfurtrifluoride
(DAST) to afford a-fluoromethyl sulfide 25. The sulfides
18, 21, and 25 thus prepared were oxidized in the usual
manner with mCPBA to afford the sulfone derivatives
19a, 19b, 22a, 22b, 26a, and 26b, which were isolated
as a single diastereomer through each separation of a
diastereomeric mixture using silica gel column
chromatography.
compound 10b. In the case of 2,3-, 3,4- and 2,4-dichlo-
rinated derivatives (10i, j, k), all compounds showed a
twofold improvement in potency compared to 10b, but
these potencies were less than those of 2- and 3-chloro
derivatives (10e, f). Substitution at the 2-position of
the phenyl ring of 10b with a fluorine atom did not in-
crease potency (10d). Introduction of a fluorine atom
at the 3-position also did not influence activity, as shown
by the IC50 value of 2,3-difluorinated derivative 10o
being nearly equal to that of 2-fluorinated compound
10d. In contrast, the potency of 2,4-difluorophenyl
derivative 10h showed sevenfold stronger potency than
that of 2-fluorinated compound 10d, indicating that
introduction of a fluorine atom at the 4-position was
preferred for activity. 3-Chloro-4-fluoro and 3-chloro-
2-fluoro compounds (10l, 10m) were equipotent to 10a.
On the other hand, introduction of chlorine at the 4-po-
sition of the 2- or 3-halogenophenyl ring reduced poten-
cies (10c, j, k). Comparison of the substituted phenyl
derivatives indicated that the compounds having 2, 4-
dihalogeno or 3-chloro substituents on the phenyl ring
had strong activity. Overall, these results suggest that
inhibitory potency depends significantly on the position
and electronic properties of the phenyl ring substituents-
nearly identical to our findings for the sulfonamide
compounds.6a
3. Results and discussion
The compounds were evaluated for their suppressive
activity against NO production using a murine macro-
phage cell line, RAW264.7 induced by LPS as the pri-
mary in vitro screen. The results are summarized in
Tables 1–6 as IC50 values.
We initially examined the effect of the linker between
cyclohexene ring and 2-chloro-4-fluorophenyl ring. As
shown in Table 1, the aminosulfone (–NHSO2–) deriva-
tive 4 showed a loss of potency. Changing to the sulfo-
nyloxy (–SO2–O–) or sulfanylmethyl (–S–CH2–) linker
(compounds 6 and 9a, respectively) resulted in a remark-
able decrease in activity. In contrast, benzylsulfone com-
pound 10a with the sulfonylmethyl linker (–SO2CH2–)
showed clear activity (IC50 = 10 nM) compared with
that for the sulfonamide linker (–SO2NH–). We there-
fore selected the benzylsulfone type of compound as
our next synthetic target, and investigated the effect of
the substitution on the phenyl ring of 10a.
Subsequently, we investigated the replacement of phenyl
ring moiety with the heteroaryl groups shown in Table
3. All heteroaryl compounds (14a, c–g) exhibited lower
potencies than the benzyl derivative 10a.
Next, modification of the ester group on the cyclohexene
ring of 2-chloro-4-fluorophenyl derivatives was carried
out (Table 4). Replacement of the ethyl ester group with
other alkyl esters resulted in a 10-fold reduction in activ-
ity, although n-propyl ester 15b maintained potency
comparable with 10a. Preparation of reduced com-
pounds 15f, 15g, and 15j led to a substantial loss of
activity (IC50 = >10,000 nM), even though the effect of
reducing the ethyl ester into the hydroxymethyl deriva-
tives on inhibitory activity was of interest to us. On
the other hand, ketones 15h and 15i, though slightly
weaker, exhibited potencies comparable to that of 10a
(15h, IC50 = 64 nM; 15i, IC50 = 11 nM). Thus, it would
As shown in Table 2, removal of the two halogen atoms
on the phenyl ring resulted in a 10-fold decrease in po-
tency (10b, IC50 = 101 nM). Introduction of a chlorine
atom at the 2- or 3-position on the phenyl ring resulted
in a sevenfold enhancement in potency (10e, f) with an
IC50 value of 14 nM, while 4-chloro derivative 10g did
not show increased activity compared to unsubstituted
Table 1. Physicochemical properties and inhibitory activities against NO production of compounds 1, 4, 6, 9a, and 10a
Anal.b
a
IC50 (nM)
Compound
Z
Mp (ꢁC) (solv.)
Formula
1
4
–SO2NH–
–NHSO2–
–SO2O–
4.5
>10,000
904
112–113
C15H17ClFNO4S
C15H17ClFNO4S
C15H16ClFO5S
C16H18ClFO2S
C16H18ClFO4S
CHN
CHN
—
127.0–128.0 (hexane)
Amorphous
Oil
6
9a
10a
–SCH2–
–SO2CH2–
5645
10
—
CH
110.0–111.0 (hexane)
a Inhibitory activities are shown as IC50 values, the concentration of test compound required to suppress the production of NO by 50% of control.
b All compounds gave satisfactory elemental analysis (0.4%) for C, H, and N.

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M. Yamada et al. / Bioorg. Med. Chem. 16 (2008) 3941–3958
Table 2. Physicochemical properties and inhibitory activities against NO production of compounds 10a–o
Anal.b
CH
a
IC50 (nM)
Compound
Ar
Mp (ꢁC) (solv.)
Formula
10a
10
110–111 (hexane)
C16H18ClFO4S
10b
10c
101
104
44.5–45.5 (hexane)
88–89 (hexane)
C16H20O4SÆ1/2H2O
CH
CH
C16H18ClFO4S
l0d
l0e
l0f
97
14
77–78 (i-Pr2O–hexane)
58–59 (AcOEt–hexane)
61–62 (i-Pr2O–hexane)
74.5–76 (hexane)
C16H19FO4S
C16H19ClO4S
C16H19ClO4S
C16H19ClO4S
C16H18F2O4S
CH
CH
CH
CH
CH
14
l0g
l0h
101
14
97.5–98.5 (AcOEt–hexane)
l0i
65
116–117 (hexane)
C16H18Cl2O4S
CH
10j
53
60
89–91 (i-Pr2O–hexane)
C16H18Cl2O4S
C16H18Cl2O4S
CH
CH
10k
104–105 (hexane)
10l
10m
l0n
13
12
Oil
C16H18ClFO4S
C16H18ClFO4S
C16H18ClFO4S
C16H18F2O4S
—
90–91 (i-Pr2O–hexane)
75.5–76.5 (i-Pr2O–hexane)
84–85 (i-Pr2O–hexane)
CH
CH
CH
23
l0o
100
a,b See the corresponding footnotes in Table 1.

M. Yamada et al. / Bioorg. Med. Chem. 16 (2008) 3941–3958
3947
Table 3. Physicochemical properties and inhibitory activities against NO production of compounds 14a and 14c–g
Anal.b
a
IC50 (nM)
Compound
Ar
Mp (ꢁC) (solv.)
Formula
14a
1009
115–117 (dec AcOEt–hexane)
C18H21NO4S
CHN
14c
14d
14e
14f
14g
106
105
123–124 (AcOEt–hexane)
87–88 (hexane)
C18H20O5S
C14H18O4S2
C15H19NO4S
C18H20O4S2
C18H20O5S
CH
—
1412
101
Oil
—
74–75 (i-Pr2O–hexane)
104–106 (i-Pr2O–hexane)
CH
CH
134
a,b See the corresponding footnotes in Table 1.
Table 4. Physicochemical properties and inhibitory activities against NO production of compounds 10a and 15a–j
Anal.b
a
IC50 (nM)
Compound
Z
Mp (ꢁC) (solv.)
Formula
10a
15a
15b
15d
15e
15f
15g
15h
15i
CO2Et
CO2Me
10
99
110.0–111.0 (hexane)
94–95 (i-Pr2O–hexane)
77–78 (i-Pr2O–hexane)
91–93 (i-Pr2O–hexane)
74–76 (i-Pr2O–hexane)
78.5–79.5 (i-Pr2O)
C16H18ClFO4S
C15H16ClFO4S
C17H20ClFO4S
C17H20ClFO4S
C18H22ClFO4S
C14H16ClFO3S
C16H18ClFO4S
C16H18ClFO3S
C17H20ClFO3S
C16H20ClFO3S
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CO2n-Pr
CO2i-Pr
14
100
CO2n-Bu
CH2OH
CH2OAc
C(@O)Et
C(@O)n-Pr
CH(OH)Et
103
>10,000
>10,000
64
70–71 (AcOEt–hexane)
133.5–134.5 (i-Pr2O–hexane)
89–91 (i-Pr2O–hexane)
148–149 (i-Pr2O–hexane)
11
>10,000
15j
a,b See the corresponding footnotes in Table 1.
appear that the carbonyl group at 1-position of the
cyclohexene ring is essential for activity.
ent benzylsulfone case, however, placing the methyl sub-
stituent at the benzylic position of 10b caused relatively
little change in activity. Regarding the diastereomers,
the more polar product 19a exhibited substantially high-
er potency than the less polar product 19b and was al-
most equal to 10b (19a, IC50 = 100 nM; 19b,
IC50 = 1325 nM). Based on these results, we examined
2-chloro-4-fluorophenyl derivatives. Upon introduction
of the methyl group a similar outcome was seen, with
the more polar product 22a showing sevenfold stronger
We examined the effects of substitution at the benzylic
position (R1), as shown in Table 5. Initially, we intro-
duced the methyl group into the benzylic position of
compound 10b to investigate its effect, in particular the
difference in potencies between diastereomers (19a,
19b). Previous studies6a indicated that alkylation of
amide moiety caused a decrease in potency. In the pres-

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M. Yamada et al. / Bioorg. Med. Chem. 16 (2008) 3941–3958
Table 5. Physicochemical properties and inhibitory activities against NO production of compounds 10a, 19, 22, and 26
R1
R2
Mp (ꢁC) (solv.)
Formula
Anal.b
a
IC50 (nM)
Compound
10a
H
Me
2-Cl, 4-F
H
10
100
1325
14
110–111 (hexane)
74–76 (AcOEt–hexane)
95.5–96.5 (hexane)
127.5 (i-Pr2O)
C16H18ClFO4S
C17H22O4S
C17H22O4S
CH
CH
CH
CH
CH
CH
CH
19a more polar
19b less polar
22a more polar
22b less polar
26a more polar
26b less polar
Me
Me
H
2-Cl, 4-F
2-Cl, 4-F
2-Cl, 4-F
2-Cl, 4-F
C17H20ClFO4S
C17H20ClFO4S
C17H19ClF2O4S
C17H19ClF2O4S
Me
CH2F
CH2F
102
435
729
100.4 (i-Pr2O)
92.1 (i-Pr2O)
68.2–68.3 (i-Pr2O)
a,b See the corresponding footnotes in Table 1.
Table 6. Conditions of optical resolution with chiral HPLC and inhibitory activities against NO production of both enantiomers of 10a, 10e, and 22a
Compound
R1
R2
HPLC conditionb
(À)-form
(+)-form
a
IC50 (nM)
a
IC50 (nM)
% ee
% ee
10a
10e
22a
H
2-Cl, 4-F
2-Cl
2-Cl, 4-F
CHIRALPAK AD hexane/EOH = 8:2
CHIRALCEL OJ hexane/EOH = 3:7
CHIRALCEL OJ hexane/EOH = 3:7
10,000
1200
1300
99.8
>99.9
>99
5.7–8.2
11
4.0
>99.9
99.9
>99
H
Me
a See the corresponding footnote in Table 1.
b UV: 230 nm.
potency than that of the less polar product 22b (22a,
IC50 = 14 nM; 22b, IC50 = 102 nM), but had little in-
crease of their potency compared to that of 10a.
Whereas the introduction of a fluoromethyl moiety gave
compounds 26a and 26b, potency was substantially re-
duced for both diastereomers (26a, IC50 = 435 nM;
26b, IC50 = 729 nM).
The (+)-enantiomers showed potent inhibitory activities
of TNF-a and IL-6 production with IC50 values [(+)-
10a, 8.3 and 1.5 nM; (+)-22a, 20 and 1.8 nM, respec-
tively], which were two- to threefold stronger than those
of the racemic compounds (Table 7). Based on these re-
sults, we then selected (+)-10a and (+)-22a as candidates
Table 7. In vitro inhibitory activities against TNF-a and IL-6
production of compounds 10a, (R)-(+)-10a, 22a, and (6R, 1S)-(+)-22a
In accordance with the results of the above SAR study,
it would appear that activity depends on at least two
parts of the molecule (the ester and phenyl moieties)
interacting with the protein, presumably in two different
pockets. This is similar to our findings for the sulfon-
amide derivatives, since it seemed that each moiety
was strictly recognized, as mentioned above.
Finally, we selected three compounds (10a, 10e, 22a),
which exhibited potent suppression of NO production
and potent in vivo efficacy in preliminary study, and sep-
arated each into its two enantiomers using chiral HPLC
in order to evaluate the stereochemical requirements for
activity. The results are summarized in Table 6. As
expected from previous report,6a the (+)-enantiomers
presented 15–300 times more potent activity than the
(À)-enantiomers, as with the sulfonamide derivatives.
Furthermore, the suppressive effects of the (+)-enantio-
mers of 10a and 22a for the production of cytokines
from LPS-stimulated RAW264.7 cells were examined.
a
IC50 (nM)
Compound
TNF-a
IL-6
10a
(R)-(+)-10a
21
4.8
1.5
8.3
22a
(6R, 1S)-(+)-22a
42
20
5.8
1.8
a Inhibitory activities are shown as IC50 values, the concentration of
test compound required to suppress the production of TNF-a or IL-6
by 50% of control. Values are means SD of three or four
experiments.

M. Yamada et al. / Bioorg. Med. Chem. 16 (2008) 3941–3958
3949
The in vivo efficacy of compounds (R)-(+)-10a and (6R,
1S)-(+)-22a, which showed high inhibitory potency in
our in vitro assays, was examined using a mouse endo-
toxin shock model, the results of which are summarized
in Table 8. When administrated intravenously 1 h before
lethal LPS challenge, NO level in serum increased signif-
icantly after LPS challenge and both compounds (1–
10 mg/kg) suppressed the production of NO induced
by LPS in a dose-dependent manner. Both (R)-(+)-10a
and (6R, 1S)-(+)-22a demonstrated efficacy against the
LPS-induced cytokines with a high survival rate in mice
in our endotoxin shock model; at a dose of 10 mg/kg all
mice survived.
4. Conclusion
Figure 2. X-ray crystal structure of (R)-(+)-10a.
Based on our previous experience,6a we designed and
synthesized new cyclohexene derivatives with modifica-
tion of sulfonamide group of compound 1 to sulfonylm-
ethyl group. Further investigations of the ester,
substituted phenyl, and sulfonylmethyl moieties were
carried out, and we discovered several compounds with
high activities. As a result of our investigation of the
structure–activity relationship for these compounds,
we succeeded in finding the novel benzylsulfone com-
pounds 10a and 22a, which showed potent inhibitory
activity against NO production from mouse macro-
phages stimulated with LPS. In particular, (R)-(+)-10a
and (6R, 1S)-(+)-22a exhibited highly potent inhibition
of inflammatory cytokines production such as TNF-a,
IL-6, and NO, in addition to showing significant
in vivo efficacy in our mouse endotoxin shock model,
although the activity of TAK-242 is a little superior
compared with those of (R)-(+)-10a and (6R, 1S)-(+)-
22a. These compounds suppressed the increase in serum
levels of NO and protected mice from lethal septic shock
with ED50 values of 1.85–5.6 mg/kg. These results sug-
gest that benzylsulfone inhibitors may be promising
agents for the treatment of sepsis.
Figure 3. X-ray crystal structure of (6R, 1S)-(+)-22a.
for further in vivo evaluation. These enantiomers were
submitted for X-ray crystallographic analysis and the
absolute configuration of (+)-10a and (+)-22a was con-
firmed to be (R)- and (6R, 1S)-form, respectively. The
X-ray crystal structures of both compounds are shown
in Figures 2 and 3.
5. Experimental
5.1. Chemistry
Table 8. Effects of compounds (R)-(+)-10a and (6R, 1S)-(+)-22a on
LPS-induced serum NOx levels and lethality in micea
Melting points were determined on a Yanagimoto micro
melting point apparatus or Buchi, and are uncorrected.
Proton nuclear magnetic resonance (1H NMR) spectra
were recorded on a Varian Gemini-200 (200 MHz) or
Mercury 300 (300 MHz) spectrometer. Chemical shifts
are given in parts per million (ppm) with tetramethylsilane
as an internal standard, and coupling constants (J values)
are given in Hertz (Hz). The following abbreviations are
used: s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet, br = broad. Optical resolutions were re-
corded with Jasco DIP-370 or P-1030 digital polarimeter.
Elemental analyses were carried out by Takeda Analytical
Research Laboratories, Ltd, and results obtained were
within 0.4% of the theoretical values. Reactions were
carried out at room temperature unless otherwise noted
and followed by TLC on silica gel 60 F254 precoated
TLC plates (E. Merck) or by HPLC using an octadecyl
Compound
Dose
(mg/kg) of NO
production
% Inhibition ED50
Survival/
(mg/kg) total
Normal
—
0
—
—
8/8
0/8
Vehicle
(CyD soln)
(R)-(+)-10a
1
3
27.0
54.0
76.0
2/10
6/10
10/10
1.85
5.6
10
(6R, 1S)-(+)-22a
1
3
À10.1
55.7
78.0
0/7
5/7
7/7
10
a Compounds (R)-(+)-10a and (6R, 1S)-(+)-22a (1–10 mg/kg) were
administered intravenously 1 h before LPS (7 mg/kg, ip) challenge.

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M. Yamada et al. / Bioorg. Med. Chem. 16 (2008) 3941–3958
silica (ODS) column (A-303, 4.6 mm i.d. ·250 nm, YMC
Co., Ltd). Standard work-up procedures were as follows.
The reaction mixture was partitioned between the indi-
cated solvent and water. Organic extracts were combined
and washed in the indicated order using the following
aqueous solutions: water, saturated aqueous sodium car-
bonate solution (aqueous NaHCO3), saturated sodium
chloride (NaCl) solution (brine), and 1 N hydrochloric
acid (1 N HCl). Extracts were dried over anhydrous mag-
nesium sulfate (MgSO4), filtered, and evaporated in va-
cuo. Chromatographic separations were carried out on
Silica Gel 60 (0.063–0.200 mm, E. Merck) or ODS
(CPO-273 LR, pre-packed column, 22 mm · 300 mm,
Kusano Kagaku Kikai Co.) using the indicated eluents.
Yields are unoptimized.
2.6 Hz), 7.60 (1H, dd, J = 8.8, 2.6 Hz), 8.05 (1H, dd,
J = 8.8, 8.0 Hz), 8.12 (1H, br s). Anal. Calcd for
C15H17ClFNO4S: C, 49.79; H, 4.74; N, 3.87. Found: C,
49.75; H, 4.51; N, 3.87.
5.4. Ethyl 6-[(2-chloro-4-fluorophenoxy)sulfonyl]cyclo-
hex-1-ene-1-carboxylate (6)
To a mixture of 2-chloro-4-fluorophenol (0.2 mL,
1.89 mmol) and AcOEt (6.0 mL) was added DABCO
(353 mg, 3.15 mmol) and compound 56a (398 mg,
1.58 mmol) dissolved in AcOEt (2.0 mL) at 0 ꢁC, then
the reaction stirred for 4 h at room temperature. The
mixture was diluted with AcOEt and worked up (water,
0.5 N HCl and brine). The residue was purified by flash
silica gel column chromatography (hexane/AcOEt = 8:1,
v/v) and crystallized with hexane to give 6 (30 mg, 5.3%)
5.2. Ethyl 6-azidocyclohex-1-ene-1-carboxylate (3)
1
as a white powder. H NMR (CDCl3) d: 1.26 (3H, t,
(1) A mixture of compound 27 (500 mg, 2.94 mmol)
and CH2Cl2 (4.33 mL) was cooled to 0 ꢁC, PPh3 (1.0 g,
J = 7.0 Hz), 1.71–1.99 (2H, m), 2.13–2.78 (4H, m), 4.23
(2H, q, J = 7.0 Hz), 4.87 (1H, br d, J = 5.4 Hz), 6.96–
7.06 (1H, m), 7.21 (1H, dd, J = 7.8, 3.0 Hz), 7.37 (1H,
t, J = 3.0 Hz), 7.47 (1H, dd, J = 9.2, 5.0 Hz).
3.82 mmol)
and
N-bromosuccinimide
(860 mg,
3.82 mmol) added, then the reaction stirred for 30 min
at 0 ꢁC. The reaction mixture was concentrated under re-
duced pressure, and the residue was purified by flash silica
gel column chromatography (hexane/AcOEt = 20:1, v/v)
to give ethyl 6-bromocyclohex-1-ene-1-carboxylate8
(488 mg, 48%) as a colorless oil.
5.5. Ethyl 6-mercaptocyclohex-1-ene-1-carboxylate (8)
(1) To a solution of 714 (5.22 g, 23.1 mmol) in EtOH
(40 mL) were added potassium thioacetate (3.04 g,
26.6 mmol) and Et3N (3.56 mL, 25.7 mmol), then the
reaction mixture was stirred for 4 days at room temper-
ature and concentrated. The residue was diluted with i-
Pr2O and worked up (water, brine). The residue was
purified by silica gel column chromatography (AcOEt/
hexane = 1:5, v/v) to give the thioacetyl intermediate
(2) To a mixture of above bromide (1.01 g, 4.33 mmol)
and DMF (4.33 mL), sodium azide (1.13 g, 17.3 mmol)
was added and heated at 60 ꢁC for 3 h, then the reaction
stirred during overnight at room temperature. The reac-
tion mixture was diluted with AcOEt and worked up
(water, brine). The residue was purified by silica gel col-
umn chromatography (hexane/AcOEt = 12:1, v/v) to
give 3 (404 mg, 48%) as a colorless oil. 1H NMR
(CDCl3) d: 1.33 (3H, t, J = 7.2 Hz), 1.56–1.76 (3H, m),
1.94–2.10 (1H, m), 2.19–2.41 (2H, m), 4.26 (2H, q,
J = 7.2 Hz), 4.54 (1H, s), 7.23–7.45 (1H, m).
1
(5.11 g, 91%) as a colorless oil. H NMR (CDCl3) d:
1.26 (3H, t, J = 7.0 Hz), 1.65–2.27 (6H, m), 2.31
(3H, s), 4.07–4.21 (2H, m), 4.70 (1H, br s), 7.10–7.14
(1H, m).
(2) To the above product (510 g, 21.1 mmol) in EtOH
(20 mL) was added 4 N HCl–AcOEt soln (25 mL) and
the mixture was stirred for 4 days at room temperature.
The reaction mixture was concentrated and the residue
was purified by silica gel column chromatography
(AcOEt/hexane = 1:20, v/v) to give 8 (3.59 g, 94%) as a
colorless oil. SIMS: 186 (M+). 1H NMR (CDCl3) d:
1.30 (3H, t, J = 7.2 Hz), 1.62–1.82 (2H, m), 1.86–2.10
(2H, m), 2.13–2.22 (2H, m), 3.89 (1H, s), 4.09 (1H, br
s), 4.22 (2H, q, J = 7.2 Hz), 6.89–6.97 (1H, m).
5.3. Ethyl 6-{[(2-chloro-4-fluorophenyl)sulfonyl]amino}
cyclohex-1-ene-1-carboxylate (4)
To a mixture of compound 3 (205 mg, 1.05 mmol) and
acetone (16 mL) were added concd HCl (1.0 mL) and zinc
powder (1.0 g) over 10 min, then the reaction stirred for
15 min at room temperature. The mixture was diluted
with AcOEt and worked up (brine). The residue was dis-
solved with THF (8.0 mL), added 2-chloro-4-fluor-
obenzenesulfonylchloride (241 mg, 1.05 mmol) and
cooled to 0 ꢁC. To the mixture, pyridine (85 lL) was
added and the reaction stirred for 4 h at room tempera-
ture. The mixture was diluted with AcOEt and washed
with 0.5 N HCl and brine, dried over Na2SO4 and concen-
trated under reduced pressure. The residue was purified
by flash silica gel column chromatography (hexane/
AcOEt = 10:1–3:1, v/v) and crystallized with hexane to
give 4 (88 mg, 23%) as a white powder. Mp 127–128 ꢁC.
IR (KBr): 3289, 1713, 1588, 1468, 1429, 1331, 1260,
5.6. Synthesis of sulfide derivatives: typical procedure for
the preparation of compounds 9a–o and 12b–h
5.6.1. Ethyl 6-[(2-chloro-4-fluorobenzyl)sulfanyl]cyclohex-
1-ene-1-carboxylate (9a). To a solution of 8 (40 mg,
0.21 mmol) in DMF (0.9 mL) maintained under a nitro-
gen atmosphere were added 11a (58 mg, 0.26 mmol) and
DBU (0.04 mL, 0.26 mmol). The mixture was then stirred
for 1 h and worked up (AcOEt; water, brine). The residue
was purified by silica gel column chromatography
(AcOEt/hexane = 1:50–1:10, v/v) to give 9a (61 mg,
87%) as a colorless oil. EI-MS: 328 (M+). IR (KBr):
2938, 1715, 1489, 1260, 1242 cmÀ1. 1H NMR (CDCl3) d:
1.26 (3H, t, J = 7.0 Hz), 1.56–2.36 (6H, m), 3.82 (1H,
1
1238, 1215, 1157, 1059 cmÀ1. H NMR (DMSO-d6) d:
1.09 (3H, t, J = 7.2 Hz), 1.33–1.55 (2H, m), 1.71–1.78
(2H, m), 1.96–2.29 (2H, m), 3.71–4.06 (2H, m), 4.09
(1H, br s), 6.96 (1H, t, J = 4.4 Hz), 7.37 (1H, dt, J = 8.0,

M. Yamada et al. / Bioorg. Med. Chem. 16 (2008) 3941–3958
3951
m), 3.94 (2H, s), 4.19 (2H, q, J = 7.0 Hz), 6.96 (1H, dt,
J = 8.6, 2.6 Hz), 7.12 (1H, dd, J = 8.6, 2.6 Hz), 7.46 (1H,
dd, J = 8.6, 6.0 Hz).
5.6.10. Ethyl 6-[(3,4-dichlorobenzyl)sulfanyl]cyclohex-1-
ene-1-carboxylate (9j). Yield 79%. Colorless oil. 1H
NMR (CDCl3) d: 1.27 (3H, t, J = 7.2 Hz), 1.62–1.95
(4H, m), 2.05–2.33 (2H, m), 3.71 (1H, s), 3.79 (2H,
ABq, J = 14Hz), 4.19 (2H, q, J = 7.2 Hz), 6.95–6.97
(1H, m), 7.20–7.23 (1H, m), 7.37 (1H, d, J = 8.1 Hz),
7.46 (1H, d, J = 2.4 Hz).
5.6.2. Ethyl 6-(benzylsulfanyl)cyclohex-1-ene-1-carboxyl-
ate (9b). Yield 67%. Colorless oil. IR (KBr): 2946, 1713,
1
1260, 1242, 1094, 1063 cmÀ1. H NMR (CDCl3) d: 1.27
(3H, t, J = 7.0 Hz), 1.55–2.36 (6H, m), 3.76 (1H, m),
3.86 (2H, s), 4.19 (2H, q, J = 7.0 Hz), 6.95 (1H, t,
J = 4.0 Hz), 7.22–7.39 (5H, m).
5.6.11. Ethyl 6-[(2,4-dichlorobenzyl)sulfanyl]cyclohex-1-
ene-1-carboxylate (9k). Yield 78%. Colorless oil. IR
(KBr): 2937, 1712, 1471, 1259, 1242, 1095, 1062 cmÀ1
.
1H NMR (CDCl3) d: 1.26 (3H, t, J = 7 Hz), 1.64–2.34
(6H, m), 3.81 (1H, m), 3.92 (2H, s), 4.18 (2H, q,
J = 7 Hz), 6.97 (1H, m), 7.19–7.43 (3H, m).
5.6.3. Ethyl 6-[(4-chloro-2-fluorobenzyl)sulfanyl]cyclohex-
1-ene-1-carboxylate (9c). Yield 88%. Colorless oil. IR
(KBr): 2937, 1714, 1489, 1259, 1244, 1093, 1062 cmÀ1
.
1H NMR (CDCl3) d: 1.25 (3H, t, J = 7 Hz), 1.6–2.3 (6H,
m), 3.77 (1H, m), 3.84 (2H, s), 4.17 (2H, q, J = 7 Hz),
6.9–7.5 (4H, m).
5.6.12. Ethyl 6-[(3-chloro-4-fluorobenzyl)sulfanyl]cyclo-
hex-1-ene-1-carboxylate (9l). Yield 75%. Colorless oil.
IR (KBr): 2934, 1713, 1499, 1244, 1094, 1061, 764 cmÀ1
.
1H NMR (CDCl3) d: 1.26 (3H, t, J = 7.2 Hz), 1.64–1.99
(4H, m), 2.05–2.34 (2H, m), 3.82 (1H, s), 3.93 (2H, ABq,
J = 14 Hz), 4.19 (2H, q, J = 7.2 Hz), 6.90 (1H, dt,
J = 8.1, 3.0 Hz), 6.97–6.99 (1H, m), 7.24–7.33 (1H, m).
5.6.4. Ethyl 6-[(2-fluorobenzyl)sulfanyl]cyclohex-1-ene-1-
carboxylate (9d). Yield 92%. Colorless oil. 1H NMR
(CDCl3) d: 1.24 (3H, t, J = 7.2 Hz), 1.63–1.74 (2H, m),
1.83–2.33 (4H, m), 3.80 (1H, br s), 3.88 (2H, ABq,
J = 14 Hz), 4.16 (2H, q, J = 7.2 Hz), 6.94–6.97 (1H,
m), 6.99–7.12 (2H, m), 7.18–7.23 (1H, m), 7.41 (1H,
dd, J = 7.8, 1.5 Hz).
5.6.13. Ethyl 6-[(2-fluoro-3-chlorobenzyl)sulfanyl]cyclo-
hex-1-ene-1-carboxylate (9m). Yield 80%. Colorless oil.
1H NMR (CDCl3) d: 1.24 (3H, t, J = 7.2 Hz), 1.64–1.97
(4H, m), 2.05–2.33 (2H, m), 3.79 (1H, s), 3.88 (2H, ABq,
J = 14 Hz), 4.16 (2H, q, J = 7.2 Hz), 6.95–6.98 (1H, m),
7.04 (1H, td, J = 7.8, 1.2 Hz), 7.25–7.35 (2H, m).
5.6.5. Ethyl 6-[(2-chlorobenzyl)sulfanyl]cyclohex-1-ene-1-
carboxylate (9e). Yield 83%. Colorless oil. 1H NMR
(CDCl3) d: 1.24 (3H, t, J = 7.2 Hz), 1.59–2.32 (6H, m),
3.84 (1H, br s), 3.96, 3.97 (2H, ABq, J = 14 Hz), 4.16
(2H, q, J = 7.2 Hz), 6.95–6.97 (1H, m), 7.16–7.25 (2H,
m), 7.35 (1H, dd, J = 7.5, 2.1 Hz), 7.45 (1H, dd, J = 7.5,
2.1 Hz).
5.6.14. Ethyl 6-[(2-fluoro-5-chlorobenzyl)sulfanyl]cyclo-
hex-1-ene-1-carboxylate (9n). Yield 79%. Colorless oil.
1H NMR (CDCl3) d: 1.24 (3H, t, J = 7.2 Hz), 1.64–
1.72 (2H, m), 1.77–1.96 (2H, m), 2.06–2.34 (2H, m),
3.78 (1H, s), 3.82 (2H, ABq, J = 14 Hz), 4.18 (2H, q,
J = 7.2 Hz), 6.94–7.00 (2H, m), 7.14–7.18 (1H, m), 7.41
(1H, dd, J = 6.6, 2.7 Hz).
5.6.6. Ethyl 6-[(3-chlorobenzyl)sulfanyl]cyclohex-1-ene-1-
carboxylate (9f). Yield 85%. Colorless oil. 1H NMR
(CDCl3) d: 1.27 (3H, t, J = 7.2 Hz), 1.60–1.95 (4H, m),
2.04–2.34 (2H, m), 3.73 (1H, s), 3.81 (2H, ABq,
J = 14 Hz), 4.19 (2H, q, J = 7.2 Hz), 6.94–6.97 (1H,
m), 7.20–7.25 (3H, m), 7.36 (1H, d, J = 1.5 Hz).
5.6.15. Ethyl 6-[(2,3-difluorobenzyl)sulfanyl]cyclohex-1-
ene-1-carboxylate (9o). Yield 74%. Colorless oil. 1H
NMR (CDCl3) d: 1.25 (3H, t, J = 7.2 Hz), 1.62–1.97
(4H, m), 2.05–2.33 (2H, m), 3.79 (1H, br s), 3.84–3.94
(2H, m), 4.17 (2H, q, J = 7.2 Hz), 6.96–7.07 (3H, m),
7.14–7.18 (1H, m).
5.6.7. Ethyl 6-[(4-chlorobenzyl)sulfanyl]cyclohex-1-ene-1-
carboxylate (9g). Yield 93%. Colorless oil. IR (KBr):
2942, 1715, 1644, 1489, 1260, 1242, 1094, 1063 cmÀ1
.
1H NMR (CDCl3) d: 1.27 (3H, t, J = 7.0 Hz), 1.58–
2.32 (6H, m), 3.72 (1H, m), 3.81 (2H, s), 4.18 (2H, q,
J = 7.0 Hz), 6.93–6.96 (1H, m), 7.24–7.32 (4H, m).
5.6.16. tert-Butyl 3({[2-(ethoxycarbonyl)cyclohex-2-en-1-
yl]sulfanyl}methyl)-1H-indole-1-carboxylate (12b). Yield
1
96%. Colorless oil. H NMR (CDCl3) d: 1.20 (3H, t,
J = 7 Hz), 1.60–2.33 (6H, m), 1.67 (9H, s), 3.84 (1H,
br), 3.99 (2H, s), 4.04–4.25 (2H, m), 6.95 (1H, t,
J = 4 Hz), 7.20–7.40 (2H, m), 7.60 (1H, s), 7.68 (1H, d,
J = 8 Hz), 8.12 (1H, d, J = 8 Hz).
5.6.8. Ethyl 6-[(2,4-difluorobenzyl)sulfanyl]cyclohex-1-ene-
1-carboxylate (9h). Yield 66%. Colorless oil. EI-MS:
M+ = 312. IR (KBr): 2938, 1715, 1505, 1260, 1244 cmÀ1
.
1H NMR (DMSO-d6) d: 1.26 (3H, t, J = 7.0 Hz), 1.56–
2.36 (6H, m), 3.78 (1H, m), 3.84 (2H, s), 4.18 (2H, q,
J = 7.0 Hz), 6.76–6.88 (2H, m), 6.97 (1H, t, J = 4.4 Hz),
7.34–7.46 (1H, m).
5.6.17. Ethyl 6-[(1-benzofuran-3-ylmethyl)sulfanyl]cyclo-
hex-1-ene-1-carboxylate (12c). Yield 77%. Colorless oil.
1H NMR (CDCl3) d: 1.19 (3H, t, J = 7 Hz), 1.62–2.30
(6H, m), 3.81 (1H, br), 3.89–4.20 (4H, m), 6.95 (1H, t,
J = 4 Hz), 7.21–7.31 (2H, m), 7.45 (1H, d, J = 7 Hz),
7.64–7.69 (2H, m).
5.6.9. Ethyl 6-[(2,3-dichlorobenzyl)sulfanyl]cyclohex-1-
ene-1-carboxylate (9i). Yield 91%. Colorless oil. IR
(KBr): 2941, 1714, 1504, 1259, 1242, 1097, 1062 cmÀ1
.
1H NMR (CDCl3) d: 1.25 (3H, t, J = 7 Hz), 1.5–2.4
(6H, m), 3.82 (1H, m), 3.99 (2H, s), 4.17 (2H, q,
J = 7 Hz), 6.6–7.5 (4H, m).
5.6.18. Ethyl 6-[(2-thienylmethyl)sulfanyl]cyclohex-1-ene-
1-carboxylate (12d). Yield 98%. Pale-pink oil. 1H NMR

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M. Yamada et al. / Bioorg. Med. Chem. 16 (2008) 3941–3958
1
(CDCl3) d: 1.28 (3H, t, J = 7.2 Hz), 1.63–1.74 (2H, m),
1.81–1.95 (2H, m), 2.05–2.34 (2H, m), 3.81 (1H, s), 4.60,
4.81 (2H, ABq, J = 14 Hz), 4.20 (2H, q, J = 7.2 Hz),
6.90–7.00 (3H, m), 7.19 (1H, dd, J = 4.8, 1.2 Hz).
above for 16d. Yield 98%. H NMR (CDCl3) d: 0.94
(3H, t, J = 7.2 Hz), 1.34–1.45 (2H, m), 1.57–1.75 (4H,
m), 1.83–1.98 (2H, m), 2.05–2.33 (2H, m), 3.82 (1H, s),
3.93 (2H, s), 4.13 (2H, t, J = 6.9 Hz), 6.91–6.98 (2H,
m), 7.10 (1H, dd, J = 8.4, 2.7 Hz), 7.44 (1H, dd,
J = 8.4, 6.0 Hz).
5.6.19. Ethyl 6-[(4-pyridylmethyl)sulfanyl]cyclohex-1-ene-
1-carboxylate (12e). The reaction was carried out by
using of 4-(bromomethyl)pyridine hydrobromide. Yield
93%. Colorless oil. IR (KBr): 2939, 1712, 1599, 1413,
5.8. {6-[(2-Chloro-4-fluorobenzyl)sulfanyl]cyclohex-1-en-
1-yl}methanol (16f)
1259, 1242, 1093, 1062 cmÀ1 1H NMR (CDCl3) d:
.
1.27 (3H, t, J = 7 Hz), 1.5–2.4 (6H, m), 3.73 (1H, m),
3.81 (2H, s), 4.18 (2H, q, J = 7 Hz), 6.9 (1H, m), 7.31
(2H, d, J = 6 Hz), 8.54 (2H, d, J = 6 Hz).
To a solution of 9a (75 mg, 0.23 mmol) in CH2Cl2
(1.5 mL) was added DIBAL-H (0.56 mL, 0.9 M solution
in hexane) at À78 ꢁC. After stirring for 1 h under a
nitrogen atmosphere, Et2O (4.0 mL) and water
(0.56 mL) were added to the reaction mixture. The mix-
ture was stirred for 2 h, then filtered through Celite, and
the filtrate was concentrated under reduced pressure.
The residue was purified by silica gel column chroma-
tography (AcOEt/hexane = 1:4, v/v) to give 16f (56 mg,
5.6.20. Ethyl 6-[(1-benzothiophen-2-ylmethyl)sulfanyl]
cyclohex-1-ene-1-carboxylate (12f). Yield 93%. Colorless
1
oil. H NMR (CDCl3) d: 1.25 (3H, t, J = 7 Hz), 1.60–
2.36 (6H, m), 3.85 (1H, br), 4.05–4.24 (4H, m), 6.97
(1H, t, J = 4 Hz), 7.23–7.35 (3H, m), 7.66–7.79 (2H, m).
1
86%) as a colorless oil. H NMR (CDCl3) d: 1.59–1.68
5.6.21. Ethyl 6-[(1-benzofuran-2-ylmethyl)sulfanyl]cyclo-
hex-1-ene-1-carboxylate (12g). Yield 75%. Colorless oil.
1H NMR (CDCl3) d: 1.21 (3H, t, J = 7 Hz), 1.60–2.35
(6H, m), 3.89 (1H, br), 3.92 (1H, d, J = 15 Hz), 4.03
(1H, d, J = 15 Hz), 4.14 (2H, q, J = 7 Hz), 6.65 (1H,
s), 6.98 (1H, t, J = 4 Hz), 7.16–7.28 (2H, m), 7.43–7.53
(2H, m).
(2H, m), 1.81–2.07 (5H, m), 3.39 (1H, s), 3.85 (2H,
ABq, J = 14 Hz), 3.96–4.17 (2H, m), 5.81–5.83 (1H,
m), 6.95 (1H, td, J = 8.4, 2.7 Hz), 7.12 (1H, dd,
J = 8.4, 2.7 Hz), 7.37 (1H, dd, J = 8.7, 6.0 Hz).
5.8.1. 1-{6-[(2-Chloro-4-fluorobenzyl)sulfanyl]cyclohex-1-
en-1-yl}propan-1-ol (16h). (1) A solution of 16f (497 mg,
˚
1.73 mmol), molecular sieves (4A, 482 mg), 4-methyl-
5.7. 6-[(2-Chloro-4-fluorobenzyl)sulfanyl]cyclohex-1-ene-
1-carboxylic acid (16c)
morpholine N-oxide (482 mg, 4.10 mmol) in CH2Cl2
(10 mL) was added TPAP (31 mg, 86.7 lmol) then the
reaction stirred for 1 h at 0 ꢁC under a nitrogen atmo-
sphere. The reaction mixture was submitted to silica
gel column chromatography (Et2O) to give 6-[(2-
chloro-4-fluorobenzyl)sulfanyl]-1-cyclohexene-1-carbal-
dehyde (332 mg, 67%) as a white amorphous material.
1H NMR (CDCl3) d: 1.61–2.48 (6H, m), 3.76 (1H, s),
3.98 (2H, s), 6.82 (1H, t, J = 3.3 Hz), 6.96 (1H, td,
J = 8.4, 2.7 Hz), 7.11 (1H, dd, J = 8.4, 2.7 Hz), 7.50
(1H, dd, J = 8.7, 6.0 Hz), 9.38 (1H, s).
To a mixture of 9a (780 mg, 2.37 mmol) and EtOH
(24 mL) was added 1 N NaOH (24 mL) and the reaction
mixture was stirred for 5 h at 40 ꢁC. After cooling, 1 N
HCl (24 mL) was added slowly and the mixture was di-
luted with water (200 mL), then the whole was worked
up (AcOEt, brine). The residue was purified by silica
gel column chromatography (hexane/AcOEt = 3:1–1:2,
1
v/v) to give 16c (410 mg, 58%) as a yellow powder. H
NMR (CDCl3) d: 1.59–1.87 (4H, m), 2.07–2.28 (2H,
m), 3.77 (1H, s), 3.90 (2H, m), 6.81 (1H, t, J = 4.0 Hz),
7.15 (1H, td, J = 8.4, 2.6 Hz), 7.36 (1H, dd, J = 8.8,
2.6 Hz), 7.54 (1H, dd, J = 8.4, 6.6 Hz), 12.19 (1H, s).
(2) To a solution of the aldehyde (178 mg, 0.63 mmol)pre-
pared above in THF (3.5 mL), ethylmagnesium bromide
(3.0 M solution in Et2O, 417 lL, 1.25 mmol) was added
dropwise at À20 ꢁC. The mixture was stirred for 1.5 h
and worked up (Et2O; water, brine). The residue was puri-
fied by silica gel column chromatography (AcOEt/hex-
ane = 14:86–26:74, v/v) to afford 16 h (130 mg, 66%) as
a colorless oil. The isolated 16h was a diastereomeric mix-
5.7.1. Isopropyl 6-[(2-chloro-4-fluorobenzyl)sulfanyl]
cyclohex-1-ene-1-carboxylate (16d). To a mixture of
16c (116 mg, 0.39 mmol), triphenylphosphine (202 mg,
0.77 mmol), and 2-PrOH (59 mL, 0.77 mmol) in THF
(3.0 mL) was added diethyl azodicarboxylate (DEAD;
40 wt% toluene solution, 0.35 mL, 0.77 mmol) at 0 ꢁC.
The reaction mixture was then stirred for 23 h under a
nitrogen atmosphere and worked up (AcOEt, water,
brine). The residue was purified by silica gel column
chromatography (AcOEt/hexane = 1:10, v/v) to give
1
ture in the ratio of ca. 3:1. H NMR (CDCl3) d: 0.80
(0.75H, t, J = 7.2 Hz), 0.82 (2.25H, t, J = 7.2 Hz), 1.26–
2.15 (9H, m), 3.12 (0.76H, s), 3.40 (0.24H, s), 3.73–3.93
(2H, m), 3.95 (1H, br s), 5.80 (0.24H, br), 5.89 (0.76H,
br), 6.97 (1H, td, J = 8.4, 2.6 Hz), 7.14 (1H, dd, J = 8.4,
2.6 Hz), 7.38 (1H, dd, J = 8.4, 6.0 Hz).
1
16d (170 mg, 90%) as a colorless oil. H NMR (CDCl3)
d: 1.24 (6H, d, J = 6.2 Hz), 1.61–2.38 (6H, m), 3.83 (1H,
s), 3.93 (2H, s), 5.08 (1H, septet, J = 6.2 Hz), 6.91–7.00
(2H, m), 7.12 (1H, dd, J = 8.4, 2.6 Hz), 7.45 (1H, dd,
J = 8.8, 6.0 Hz).
5.8.2. 1-{6-[(2-Chloro-4-fluorobenzyl)sulfanyl]cyclohex-1-
en-1-yl}butan-1-ol (16i). Compound 16i was prepared
from the above aldehyde derivative by a similar proce-
dure to that described for the synthesis of 16h. The iso-
lated 16i was a diastereomeric mixture in the ratio of ca.
65:35. Yield 54%. Colorless oil. H NMR (CDCl3) d:
0.81–0.90 (3H, m), 1.12–1.45 (4H, m), 1.60–2.08 (7H,
m), 3.11 (0.65H, s), 3.38 (0.35H, s), 3.79, 3.86 (1.3H,
1
5.7.2. Butyl 6-[(2-chloro-4-fluorobenzyl)sulfanyl]cyclo-
hex-1-ene-1-carboxylate (16e). Compound 16e was syn-
thesized from 16c by the same procedure described

M. Yamada et al. / Bioorg. Med. Chem. 16 (2008) 3941–3958
3953
ABq, J = 13 Hz), 3.83, 3.89 (0.7H, ABq, J = 13 Hz),
3.95–4.08 (1H, m), 5.79–5.90 (1H, m), 6.93–7.01 (1H,
m), 7.11–7.17 (1H, m), 7.33–7.41 (1H, m).
(2) A solution of above product (145 mg, 0.40 mmol) in
CH2Cl2 (10 mL) was cooled to À78 ꢁC and DAST
(107 mL, 0.81 mmol) in CH2Cl2 (10 mL) added drop-
wise. The mixture was stirred for 1 h at À78 ꢁC to
À60 ꢁC, further stirred at À10 ꢁC for 2 h. The whole
was worked up (CH2Cl2; aqueous NaHCO3, water,
brine) and the residue was purified by silica gel column
chromatography (AcOEt/hexane = 1:99–15:5, v/v) to
give 25 (diastereomeric mixture; 80 mg, 55%). 1H
NMR (CDCl3) d: 1.15–1.37 (3H, m), 1.62–2.54 (6H,
m), 2.76–3.22 (1H, m), 3.53–4.32 (4H, m), 4.61–4.88
(1H, m), 6.86–7.22 (3H, m), 7.29–7.74 (1H, m).
5.8.3. Ethyl 6-[(1-phenylethyl)sulfanyl]cyclohex-1-ene-1-
carboxylate (18). Compound 18 was prepared in the same
manner described for compound 9a. The isolated 18 was a
diastereomeric mixture in the ratio of ca. 1:1. Yield 61%.
Colorless oil. 1H NMR (CDCl3) d: 1.22 (1.5H, t,
J = 7.2 Hz), 1.34 (1.5H, t, J = 7.2 Hz), 1.41–1.80 (4H,
m), 1.90–2.32 (2H, m), 3.54 (0.5H, s), 3.65 (0.5H, s),
4.06–4.31 (3H, m), 6.87–6.93 (1H, m), 7.19–7.40 (5H, m).
5.8.4. Ethyl 6-{[1-(2-chloro-4-fluorophenyl)ethyl]sulfa-
nyl}cyclohex-1-ene-1-carboxylate (21). To a solution of
8 (186 mg, 1.0 mmol) in toluene (3.0 mL) maintained un-
der a nitrogen atmosphere were added 20 (175 mg,
1.0 mmol) and CMBP (362 mg, 1.5 mmol), and the reac-
tion stirred for 3 h at 100 ꢁC. The mixture was cooled to
room temperature and concentrated under reduced pres-
sure, then the residue was purified by silica gel column
chromatography (AcOEt/hexane = 1:40–1:20, v/v) to
give 21 (diastereomeric mixture; 123 mg, 36%) as a color-
less oil. Yield 40%. IR (KBr): 2560, 1715, 1489,
5.11. Synthesis of sulfone derivatives: typical procedure
for the preparation of compounds 10a–o (Table 2), 14b–g
(Table 3), 15d, e, f, j (Table 4), and 19a, b, 22a, b, and
26a, b (Table 5)
5.11.1. Ethyl 6-[(2-chloro-4-fluorobenzyl)sulfonyl]cyclohex-
1-ene-1-carboxylate (10a). A mixture of compound 9a
(896 mg, 2.73 mmol) and AcOEt (27 mL) was added
mCPBA (1.48 g, 6.00 mmol) at 0 ꢁC and the reaction stirred
for 2 h. The mixture was diluted with AcOEt and worked
up (aqueous NaHCO3, water, and brine). The residue was
purified by silica gel column chromatography (hexane/
AcOEt = 6:1–4:1, v/v) and crystallized with AcOEt–hexane
to give 10a (869 mg, 88%) as a white powder. IR (KBr):
1242 cmÀ1
.
1H NMR (CDCl3) d: 1.24 (1.73H, t,
J = 7.0 Hz), 1.33 (1.27H, t, J = 7.0 Hz), 1.48 (1.73H, d,
J = 7.0 Hz), 2.05–2.57 (6H, m), 3.53 (0.58H, br s), 3.83
(0.42H, br s), 4.06–4.31 (2H, m), 4.66 (1H, quintet,
J = 7.0 Hz), 6.92–7.06 (3H, m), 7.55 (0.42H, dd,
J = 8.8, 6.2 Hz), 7.73 (0.58H, dd, J = 8.0, 5.8 Hz).
1
2942, 1709, 1605, 1493, 1314, 1250, 1128, 1061 cmÀ1. H
NMR (DMSO-d6) d: 1.32 (3H, t, J = 7.0 Hz), 1.55–2.62
(6H, m), 4.25 (2H, q, J = 7.0 Hz), 4.41 (1H, d,
J = 5.6 Hz), 4.59 (2H, s), 7.03 (1H, dt, J = 8.4, 2.6 Hz),
7.21 (1H, dd, J = 8.4, 2.6 Hz), 7.42 (1H, t, J = 4.0 Hz),
7.62 (1H, dd, J = 8.6, 6.0 Hz). Anal. Calcd for
C16H18ClFO4S: C, 53.26; H, 5.03. Found: C, 53.08; H, 4.95.
5.9. Ethyl 6-({1-(2-chloro-4-fluorophenyl)-2-[(trimethylsilyl)
oxy]ethyl}sulfanyl)-cyclohex-1-ene-1-carboxylate (24)
To a solution of 8 (373 mg, 2.0 mmol) in DMF (5.0 mL)
maintained under a nitrogen atmosphere were added 23
(782 mg, 2.4 mmol), Et3N (0.56 mL, 4.0 mmol), and
DBU (0.3 mL, 2.0 mmol) in DMF (5.0 mL), and the
reaction stirred for 1 h at 0 ꢁC. The mixture was worked
up (AcOEt; water, brine), then the residue was purified
by silica gel column chromatography (AcOEt/hex-
ane = 0.5:99.5–5:95, v/v) to give 24 (diastereomeric mix-
5.11.2. Ethyl 6-(benzylsulfonyl)cyclohex-1-ene-1-carbox-
ylate (10b). Yield 95%. White powder. IR (KBr): 1705,
1
1306, 1250, 1119, 1098, 1061 cmÀ1. H NMR (CDCl3)
d: 1.34 (3H, t, J = 7.2 Hz), 1.41–2.50 (6H, m), 4.28
(2H, q, J = 7.2 Hz), 4.29 (1H, d, J = 13.8 Hz), 4.35
(1H, m), 4.55 (1H, d, J = 14 Hz), 7.37–7.45 (4H, m),
7.50–7.55 (2H, m). Anal. Calcd for C16H20O4SÆ1/
2H2O: C, 60.55; H, 6.67. Found: C, 60.96; H, 6.32.
1
ture; 198 mg, 23%) as a colorless oil. H NMR (CDCl3)
d: À0.06 to À0.02 (9H, m), 1.18 (1.8H, t, J = 7.0 Hz),
1.29 (1.2H, t, J = 7.0 Hz), 1.57–2.23 (6H, m), 3.60–4.25
(5H, m), 4.52–4.68 (0.4H, m), 4.63 (0.6H, t,
J = 4.6 Hz), 6.88–7.22 (3H, m), 7.41 (0.4H, dd, J = 8.8,
6.2 Hz), 7.61 (0.6H, dd, J = 8.8, 6.2 Hz).
5.11.3. Ethyl 6-[(4-chloro-2-fluorobenzyl)sulfonyl]cyclo-
hex-1-ene-1-carboxylate (10c). Yield 50%. IR (KBr):
2941, 1714, 1489, 1313, 1249, 1126, 1097, 1060 cmÀ1
.
1H NMR (CDCl3) d: 1.32 (3H, t, J = 7 Hz), 1.6–2.6
(6H, m), 4.26 (2H, q, J = 7 Hz), 4.36 (1H, m), 4.39,
4.53 (2H, ABq, J = 13 Hz), 7.1–7.2 (2H, m), 7.4–7.6
(2H, m). Anal. Calcd for C16H18ClFO4S: C, 53.26; H,
5.03. Found: C, 53.28; H, 5.10.
5.10. Ethyl 6-{[1-(2-chloro-4-fluorophenyl)-2-fluoroethyl]
sulfanyl}cyclohex-1-ene-1-carboxylate (25)
(1) 6 N HCl (275 lL, 1.65 mmol) was added dropwise to
a solution of 24 (198 mg, 0.46 mmol) in MeOH
(3.0 mL). The mixture was stirred for 0.5 h, then worked
up (AcOEt; water, brine) to give ethyl 6-{[1-(2-chloro-4-
fluorophenyl)-2-hydroxyethyl]sulfanyl}-1-cyclohexene-
1-carboxylate (diastereomeric mixture; 147 mg, 89%) as
a colorless oil. 1H NMR (CDCl3) d: 1.25 (1.8H, t,
J = 7.0 Hz), 1.33 (1.2H, t, J = 7.0 Hz), 1.52–2.36 (6H,
m), 2.90 (0.4H, t, J = 6.4 Hz), 3.81–4.33 (5.6H, m),
4.45–4.55 (1H, m), 6.96–7.18 (3H, m), 7.37 (0.4H, dd,
J = 8.8, 6.2 Hz), 7.66 (0.6H, dd, J = 8.8, 6.2 Hz).
5.11.4. Ethyl 6-[(2-fluorobenzyl)sulfonyl]cyclohex-1-ene-
1-carboxylate (10d). Yield 28%. White powder. 1H
NMR (CDCl3) d: 1.31 (3H, t, J = 7 Hz), 1.56–2.45
(6H, m), 4.26 (2H, q, J = 7 Hz), 4.39–4.43 (1H, m),
4.43 (1H, d, J = 14 Hz), 4.57 (1H, d, J = 14 Hz), 7.09–
7.60 (5H, m). Anal. Calcd for C16H19FO4S: C, 58.88;
H, 5.87. Found: C, 58.99; H, 6.05.
5.11.5. Ethyl 6-[(2-chlorobenzyl)sulfonyl]cyclohex-1-ene-
1-carboxylate (10e). Yield 86%. White powder. 1H

3954
M. Yamada et al. / Bioorg. Med. Chem. 16 (2008) 3941–3958
NMR (CDCl3) d: 1.30 (3H, t, J = 7.2 Hz), 1.59–1.78
(2H, m), 1.94–2.54 (4H, m), 4.25 (2H, q, J = 7.2 Hz),
4.46 (1H, br d, J = 6 Hz), 4.625, 4.632 (2H, ABq,
J = 14 Hz), 7.29–7.47 (4H, m), 7.59–7.64 (1H, m). Anal.
Calcd for C16H19ClO4S: C, 56.05; H, 5.59. Found: C,
55.88; H, 5.63.
J = 7.2 Hz), 1.49–1.62 (1H, m), 1.72–2.02 (2H, m),
2.16–2.31 (1H, m), 2.38–2.53 (2H, m), 4.19–4.32 (4H,
m), 4.51 (1H, d, J = 14 Hz), 7.15 (1H, t, J = 8.7 Hz),
7.40–7.45 (2H, m), 7.61 (1H, dd, J = 6.9, 2.4 Hz).
5.11.13. Ethyl 6-[(2-fluoro-3-chlorobenzyl)sulfonyl]cyclo-
hex-1-ene-1-carboxylate (10m). Yield 91%. White pow-
der. IR (KBr): 2944, 1705, 1462, 1312, 1250, 1128,
5.11.6. Ethyl 6-[(3-chlorobenzyl)sulfonyl]cyclohex-1-ene-
1-carboxylate (10f). Yield 36%. White powder. 1H NMR
(CDCl3) d: 1.34 (3H, t, J = 7.0 Hz), 1.47–2.55 (6H, m),
4.22–4.34 (4H, m), 4.53 (1H, d, J = 14 Hz), 7.32–7.46
(4H, m), 7.56 (1H, s). Anal. Calcd for C16H19ClO4S:
C, 56.05; H, 5.59. Found: C, 55.82; H, 5.42.
1098, 1061 cmÀ1 1H NMR (CDCl3) d: 1.32 (3H, t,
.
J = 7.2 Hz), 1.59–1.80 (2H, m), 1.92–2.51 (4H, m), 4.27
(2H, q, J = 7.2 Hz), 4.40 (1H, d, J = 5.4 Hz), 4.48, 4.54
(2H, ABq, J = 13.5 Hz), 7.12 (1H, t, J = 7.8 Hz), 7.40–
7.49 (3H, m). Anal. Calcd for C16H18ClFO4S: C,
53.26; H, 5.03. Found: C, 53.02; H, 5.03.
5.11.7. Ethyl 6-[(4-chlorobenzyl)sulfonyl]cyclohex-1-ene-
1-carboxylate (10g). Yield 57%. White powder. IR (KBr):
2980, 2939, 1705, 1493, 1305, 1249, 1120, 1095,
5.11.14. Ethyl 6-[(2-fluoro-5-chlorobenzyl)sulfonyl]cyclo-
hex-1-ene-1-carboxylate (10n). Yield 94%. White pow-
der. IR (KBr): 2946, 1705, 1489, 1312, 1248, 1127,
1
1060 cmÀ1. H NMR (CDCl3) d: 1.34 (3H, t, J = 7 Hz),
1.4–2.6 (6H, m), 4.2–4.4 (3H, m), 4.54 (1H, d,
J = 14 Hz), 7.3–7.6 (5H, m). Anal. Calcd for
C16H19ClO4S: C, 56.05; H, 5.59. Found: C, 56.08; H, 5.41.
1100, 1061 cmÀ1 1H NMR (CDCl3) d: 1.33 (3H, t,
.
J = 7.2 Hz), 1.57–1.80 (2H, m), 1.92–2.09 (1H, m),
2.17–2.33 (1H, m), 2.42–2.53 (2H, m), 4.27 (2H, q,
J = 7.2 Hz), 4.38 (1H, s), 4.43, 4.49 (2H, ABq,
J = 13.8 Hz), 7.07 (1H, t, J = 9.0 Hz), 7.30–7.35 (1H,
m), 7.43 (1H, t, J = 3.6 Hz), 7.55 (1H, dd, J = 6.0,
2.7 Hz). Anal. Calcd for C16H18ClFO4S: C, 53.26; H,
5.03. Found: C, 53.31; H, 5.12.
5.11.8. Ethyl 6-[(2,4-difluorobenzyl)sulfonyl]cyclohex-1-
ene-1-carboxylate (10h). Yield 75%. White powder. H
1
NMR (CDCl3) d: 1.32 (3H, t, J = 7.0 Hz), 1.59–2.50
(6H, m), 4.27 (2H, q, J = 7.0 Hz), 4.35 (1H, d,
J = 14.0 Hz), 4.39 (1H, m), 4.51 (1H, d, J = 14.0 Hz),
6.83–6.96 (2H, m), 7.42 (1H, t, J = 4.0 Hz), 7.49–7.61
(1H, m). Anal. Calcd for C16H18F2O4S: C, 55.80; H,
5.27. Found: C, 55.95; H, 5.40.
5.11.15. Ethyl 6-[(2,3-difluorobenzyl)sulfonyl]cyclohex-1-
ene-1-carboxylate (10o). Yield 88%. White powder. IR
(KBr): 2944, 1709, 1495, 1314, 1250, 1125, 1061 cmÀ1
.
1H NMR (CDCl3) d: 1.33 (3H, t, J = 7.2 Hz), 1.60–
1.79 (2H, m), 1.91–2.11 (2H, m), 2.42–2.52 (2H, m),
4.27 (2H, q, J = 7.2 Hz), 4.40 (1H, br d, J = 4.8 Hz),
4.49, 4.55 (2H, ABq, J = 14 Hz), 7.06–7.33 (3H, m),
7.41–7.44 (1H, m). Anal. Calcd for C16H18F2O4S: C,
55.80; H, 5.27. Found: C, 55.59; H, 5.37.
5.11.9. Ethyl 6-[(2,3-dichlorobenzyl)sulfonyl]cyclohex-1-
ene-1-carboxylate (10i). Yield 50%. White powder. IR
(KBr): 2941, 1699, 1427, 1311, 1249, 1122, 1097,
1060 cmÀ1
.
1H NMR (CDCl3) d: 1.32 (3H, t,
J = 7 Hz), 1.6–2.6 (6H, m), 4.25 (2H, q, J = 7 Hz), 4.44
(1H, m), 4.69 (2H, s), 7.2–7.3 (1H, m), 7.4–7.6 (3H,
m). Anal. Calcd for C16H18Cl2O4S: C, 50.94; H, 4.81.
Found: C, 50.94; H, 4.76.
5.11.16. tert-Butyl 3({[2-(ethoxycarbonyl)cyclohex-2-en-
1-yl]sulfonyl}methyl)-1H-indole-1-carboxylate (14b).
1
Yield 85%. White powder. H NMR (CDCl3) d: 1.34
5.11.10. Ethyl 6-[(3,4-dichlorobenzyl)sulfonyl]cyclohex-1-
ene-1-carboxylate (10j). Yield 79%. White powder. IR
(KBr): 2944, 1707, 1472, 1310, 1250, 1117, 1098,
1061 cmÀ1. 1H NMR (CDCl3) d: 1.35 (3H, t, J = 7.2 Hz),
1.49–1.63 (1H, m), 1.73–2.00 (2H, m), 2.16–2.53 (3H, m),
4.25–4.28 (1H, m), 4.25, 4.49 (2H, ABq, J = 14 Hz), 4.29
(2H, q, J = 7.2 Hz), 7.38 (1H, t, J = 1.8 Hz), 7.42–7.46
(3H, m). Anal. Calcd for C16H18Cl2O4S: C, 50.94; H,
4.81. Found: C, 51.16; H, 4.63.
(3H, t, J = 7.0 Hz), 1.42–2.52 (6H, m), 1.68 (9H, s),
4.29 (2H, q, J = 7.0 Hz), 4.42 (1H, d, J = 14 Hz), 4.44
(1H, br), 4.76 (1H, d, J = 14 Hz), 7.27–7.41 (2H, m),
7.79 (1H, d, J = 7.0 Hz), 7.88 (1H, s), 8.14 (1H, d,
J = 8.8 Hz). Anal. Calcd for C23H29NO6S: C, 61.72; H,
6.53; N, 3.13. Found: C, 61.61; H, 6.32; N, 2.92.
5.11.17. Ethyl 6-[(1-benzofuran-3-ylmethyl)sulfonyl]
cyclohex-1-ene-1-carboxylate (14c). Yield 88%. White
powder. IR (KBr): 1705, 1306, 1250, 1123 cmÀ1 1H
.
5.11.11. Ethyl 6-[(2,4-dichlorobenzyl)sulfonyl]cyclohex-1-
ene-1-carboxylate (10k). Yield 71%. White powder. IR
(KBr): 2943, 1712, 1473, 1311, 1251, 1128, 1097,
NMR (CDCl3) d: 1.32 (3H, t, J = 7.0 Hz), 1.46–2.49
(6H, m), 4.23–4.38 (2H, m), 4.54 (1H, d, J = 5.1 Hz),
4.95 (2H, q, J = 6.9 Hz), 7.41 (1H, t, J = 3.9 Hz), 7.46–
7.59 (3H, m), 7.72 (1H, d, J = 3.9 Hz), 7.85–7.89 (2H,
m), 8.22 (1H, d, J = 9.0 Hz). Anal. Calcd for
C18H20O5S: C, 62.05; H, 5.79. Found: C, 62.12; H, 5.87.
1060 cmÀ1
.
1H NMR (CDCl3) d: 1.32 (3H, t,
J = 7 Hz), 1.5–2.6 (6H, m), 4.26 (2H, q, J = 7 Hz), 4.44
(1H, m), 4.60 (2H, s), 7.2–7.6 (4H, m). Anal. Calcd for
C16H18Cl2O4S: C, 50.94; H, 4.81. Found: C, 50.81; H,
4.98.
5.11.18. Ethyl 6-[(3-thienylmethyl)sulfonyl]cyclohex-1-
ene-1-carboxylate (14d). Yield 69%. White powder. H
1
5.11.12. Ethyl 6-[(3-chloro-4-fluorobenzyl)sulfonyl]cyclo-
hex-1-ene-1-carboxylate (10l). Yield 98%. Colorless oil.
IR (KBr): 2944, 1705, 1501, 1312, 1250, 1125, 1098,
NMR (CDCl3) d: 1.38 (3H, t, J = 7.0 Hz), 1.4–2.5 (6H,
m), 4.28 (2H, q, J = 7.0 Hz), 4.39 (1H, m), 4.42 (1H,
d, J = 17 Hz), 4.86 (1H, d, J = 17 Hz), 7.05 (1H, m),
7.2–7.5 (3H, m).
1063, 781 cmÀ1 1H NMR (CDCl3) d: 1.35 (3H, t,
.

M. Yamada et al. / Bioorg. Med. Chem. 16 (2008) 3941–3958
3955
5.11.19. Ethyl 6-[(4-pyridylmethyl)sulfonyl]cyclohex-1-
ene-1-carboxylate (14e). Yield 55%. Pale-yellow oil. IR
(KBr): 2939, 1712, 1599, 1413, 1259, 1242, 1093,
der. IR (KBr): 2942, 1605, 1493, 1316, 1235, 1123,
914 cmÀ1 1H NMR (CDCl3) d: 0.963 (3H, t,
J = 7.2 Hz), 1.34–1.67 (4H, m), 1.75–1.87 (2H, m),
2.04–2.16 (2H, m), 4.28 (1H, d, J = 8.4 Hz), 4.50 (2H,
s), 7.06 (1H, td, J = 8.4, 2.4 Hz), 7.20–7.26 (2H, m),
7.57 (1H, d, J = 8.4, 6.0 Hz).
.
1062 cmÀ1
.
1H NMR (CDCl3) d: 1.27 (3H, t,
J = 7 Hz), 1.5–2.4 (6H, m), 3.73 (1H, m), 3.81 (2H, s),
4.18 (2H, q, J = 7 Hz), 6.9 (1H, m), 7.31 (2H, d,
J = 6 Hz), 8.54 (2H, d, J = 6 Hz).
5.11.26. Ethyl 6-[(1-phenylethyl)sulfonyl]cyclohex-1-ene-
1-carboxylate (19a, 19b). 19a (More polar product):
Yield 26%. 1H NMR (DMSO-d6) d: 1.16 (3H, t,
J = 7.0 Hz), 1.51–1.69 (2H, m), 1.68 (3H, d, J = 7.0 Hz),
1.79–2.40 (4H, m), 4.07 (2H, q, J = 7.0 Hz), 4.26 (1H,
d, J = 4.6 Hz), 4.70 (1H, q, J = 7.0 Hz), 7.18 (1H, t,
J = 3.8 Hz), 7.37–7.45 (5H, m).
5.11.20. Ethyl 6-[(1-benzothiophen-2-ylmethyl)sulfonyl]
cyclohex-1-ene-1-carboxylate (14f). Yield 68%. White
powder. H NMR (CDCl3) d: 1.35 (3H, t, J = 7.0 Hz),
1.27–2.55 (6H, m), 4.30 (2H, q, J = 7.0 Hz), 4.46 (1H,
br), 4.49 (1H, d, J = 14 Hz), 4.98 (1H, d, J = 14 Hz),
7.31–7.44 (3H, m), 7.56 (1H, s), 7.77–7.85 (2H, m). Anal.
Calcd for C18H20O4S2Æ0.2H2O: C, 58.74; H, 5.59.
Found: C, 58.47; H, 5.49.
1
19b (Less polar product): Yield 61%. 1H NMR (CDCl3)
d: 1.17–1.32 (1H, m), 1.35 (3H, t, J = 7.2 Hz), 1.60–1.93
(3H, m), 1.76 (3H, d, J = 7.2 Hz), 2.03–2.43 (2H, m),
4.26–4.33 (1H, m), 4.29 (2H, q, J = 7.2 Hz), 4.64 (1H,
d, J = 4.6 Hz), 7.31–7.33 (1H, m), 7.36–7.42 (3H, m),
7.63–7.66 (2H, m). Anal. Calcd for C17H22O4S: C,
63.33; H, 6.88. Found: C, 62.93; H, 6.89.
5.11.21. Ethyl 6-[(1-benzofuran-2-ylmethyl)sulfonyl]
cyclohex-1-ene-1-carboxylate (14g). Yield 85%. White
powder. IR (KBr): 1705, 1306, 1250, 1123 cmÀ1 1H
.
NMR (CDCl3) d: 1.32 (3H, t, J = 7.0 Hz), 1.46–2.49
(6H, m), 4.23–4.38 (2H, m), 4.54 (1H, d, J = 5.1 Hz),
4.95 (2H, q, J = 6.9 Hz), 7.41 (1H, t, J = 3.9 Hz), 7.46–
7.59 (3H, m), 7.72 (1H, d, J = 3.9 Hz), 7.85–7.89 (2H,
m), 8.22 (1H, d, J = 9.0 Hz). Anal. Calcd for
C18H20O5S: C, 62.05; H, 5.79. Found: C, 62.12; H, 5.87.
5.11.27. Ethyl 6-{[1-(2-chloro-4-fluorophenyl)ethyl]sulfo-
nyl}cyclohex-1-ene-1-carboxylate (22a, 22b). 22a (More
1
polar product): Yield 14%. H NMR (CDCl3) d: 1.29
(3H, t, J = 7.0 Hz), 1.60–1.79 (1H, m), 1.78 (3H, d,
J = 7.0 Hz), 2.05–2.64 (4H, m), 4.09–4.30 (2H, m), 4.35
(1H, d, J = 3.8 Hz), 5.15 (2H, q, J = 7.0 Hz), 7.02–7.14
(1H, m), 7.16 (1H, dd, J = 8.6, 2.6 Hz), 7.38 (1H, t,
J = 4.0 Hz), 7.67 (1H, dd, J = 8.6, 5.8 Hz). Anal. Calcd
for C17H20ClFO4S: C, 54.47; H, 5.38. Found: C, 54.31;
H, 5.54.
5.11.22. Isopropyl 6-[(2-chloro-4-fluorobenzyl)sulfonyl]
cyclohex-1-ene-1-carboxylate (15d). Yield 82%. White
powder. H NMR (CDCl3) d: 1.29 (3H, d, J = 6.3 Hz),
1.31 (3H, d, J = 6.3 Hz), 1.60–1.76 (2H, m), 1.95–2.10
(1H, m), 2.16–2.30 (1H, m), 2.41–2.51 (2H, m), 4.42
(1H, d, J = 4.8 Hz), 4.58 (2H, s), 5.11 (1H, dt,
J = 6.3 Hz), 7.02 (1H, td, J = 8.4, 2.7 Hz), 7.19 (1H,
dd, J = 8.4, 2.7 Hz), 7.38 (1H, t, J = 4.2 Hz), 7.58 (1H,
dd, J = 8.7, 6.0 Hz). Anal. Calcd for C17H20ClFO4S:
C, 54.47; H, 5.38. Found: C, 54.46; H, 5.29.
1
1
22b (Less polar product): Yield 5%. H NMR (CDCl3)
d: 1.34 (3H, t, J = 7.0 Hz), 1.27–2.43 (6H, m), 1.73
(3H, d, J = 7.0 Hz), 4.29 (2H, q, J = 7.0 Hz), 4.36
(1H, br s), 5.11 (1H, q, J = 7.0 Hz), 7.03–7.13 (1H,
m), 7.22 (1H, dd, J = 8.2, 2.6 Hz), 7.30–7.35 (1H, m),
7.79 (1H, dd, J = 8.2, 6.2 Hz). Anal. Calcd for
C17H20ClFO4S: C, 54.47; H, 5.38. Found: C, 54.38;
H, 5.35.
5.11.23. Butyl 6-[(2-chloro-4-fluorobenzyl)sulfonyl]cyclo-
hex-1-ene-1-carboxylate (15e). Yield 73%. White powder.
IR (KBr): 2961, 1707, 1605, 1493, 1314, 1248, 1128, 1098,
1
914 cmÀ1. H NMR (CDCl3) d: 0.96 (3H, t, J = 7.2 Hz),
1.36–1.48 (2H, m), 1.60–1.72 (4H, m), 1.96–2.10 (1H, m),
2.16–2.1 (1H, m), 2.43–2.52 (2H, m), 4.13–4.26 (2H, m),
4.15 (1H, d, J = 4.5 Hz), 4.58 (1H, d, J = 1.5 Hz), 7.02
(1H, td, J = 8.4, 2.4 Hz), 7.19 (1H, dd, J = 8.4, 1.8 Hz),
7.39 (1H, t, J = 3.9 Hz), 7.58 (1H, dd, J = 8.4, 6.0 Hz).
Anal. Calcd for C18H22ClFO4S: C, 55.59; H, 5.70.
Found: C, 55.57; H, 5.74.
5.11.28. Ethyl 6-{[1-(2-chloro-4-fluorophenyl)-2-hydroxy-
ethyl]sulfonyl}cyclohex-1-ene-1-carboxylate (26a, 26b).
26a (More polar product): Yield 46%. IR (KBr): 1713,
1
1495, 1308, 1250, 1132, 1061 cmÀ1. H NMR (CDCl3)
d: 1.31 (3H, t, J = 7.0 Hz), 1.66–2.65 (6H, m), 3.56
(1H, ddd, J = 32.6, 15.0, 2.6 Hz), 3.68–3.87 (1H, m),
4.25 (2H, q, J = 7.0 Hz), 4.44 (1H, d, J = 5.6 Hz), 6.42
(1H, ddd, J = 46.2, 8.8, 2.0 Hz), 7.04–7.19 (2H, m),
7.43 (1H, t, J = 2.8 Hz), 7.53 (1H, dd, J = 8.4, 5.8 Hz).
Anal. Calcd for C17H19ClF2O4S: C, 51.98; H, 4.87.
Found: C, 51.97; H, 5.01.
5.11.24. {6-[(2-Chloro-4-fluorobenzyl)sulfonyl]cyclohex-1-
en-1-yl}methanol (15f). Yield 73%. White powder. IR
(KBr): 3480, 2944, 1605, 1582, 1493, 1308, 1235, 1125,
1044, 914 cmÀ1 1H NMR (CDCl3) d: 1.64–1.76 (1H,
.
m), 1.84–2.42 (5H, m), 2.45 (1H, t, J = 6.8 Hz), 3.96
(1H, br s), 4.12 (1H, dd, J = 13, 6.8 Hz), 4.38–4.48 (1H,
m), 4.48 (2H, s), 6.21 (1H, br), 7.06 (1H, td, J = 8.4,
2.6 Hz), 7.21 (1H, dd, J = 8.4, 2.6 Hz), 7.57 (1H, dd,
J = 8.8, 6.0 Hz). Anal. Calcd for C14H16ClFO3S: C,
52.75; H, 5.06. Found: C, 52.51; H, 4.97.
26b (Less polar product): Yield 31%. IR (KBr): 1705,
1
1495, 1306, 1250, 1128, 1061 cmÀ1. H NMR (CDCl3)
d: 1.31 (3H, t, J = 7.0 Hz), 1.58–2.79 (6H, m), 3.37
(1H, ddd, J = 37.8, 15.4, 1.8 Hz), 4.10–4.29 (3H, m),
4.55 (1H, br s), 6.39 (1H, ddd, J = 44.8, 10.8, 1.8 Hz),
7.05–7.16 (2H, m), 7.43 (1H, t, J = 3.6 Hz), 7.59 (1H,
dd, J = 8.8, 5.8 Hz). Anal. Calcd for C17H19ClF2O4S:
C, 51.98; H, 4.87. Found: C, 51.86; H, 5.01.
5.11.25. 1-{6-[(2-Chloro-4-fluorobenzyl)sulfonyl]cyclo-
hex-1-en-1-yl}propan-1-ol (15j). Yield 36%. White pow-

3956
M. Yamada et al. / Bioorg. Med. Chem. 16 (2008) 3941–3958
5.12. Ethyl 6-[(1H-indol-3-ylmethyl)sulfonyl]cyclohex-1-
ene-1-carboxylate (14a)
(1H, dd, J = 8.4, 2.7 Hz), 7.45 (1H, dd, J = 8.4,
6.0 Hz). Anal. Calcd for C16H18ClFO2S: C, 58.44; H,
5.52. Found: C, 58.49; H, 5.45.
TFA (2.0 mL) was added dropwise to 14b (224 mg,
0.5 mmol). The mixture was stirred for 2 h. The mixture
was diluted with water and extracted with AcOEt. The or-
ganic layer was washed with aqueous NaHCO3 and brine,
dried (Na2SO4), and concentrated under reduced pres-
sure. The residue was purified by silica gel column chro-
matography (AcOEt/hexane = 1:2, v/v) followed by
crystallization from i-Pr2O to give 14a (17.5 mg, 10%) as
a white powder. 1H NMR (CDCl3) d: 1.33 (3H, t,
J = 7.2 Hz), 1.38–2.47 (6H, m), 4.28 (2H, q, J = 7.2 Hz),
4.42 (1H, br), 4.47 (1H, d, J = 14.4 Hz), 4.80 (1H, d,
J = 14.4 Hz), 7.15–7.24 (2H, m), 7.36–7.38 (2H, m), 7.46
(1H, d, J = 2.1 Hz), 7.83 (1H, d, J = 8.4 Hz), 8.38 (1H,
br). Anal. Calcd for C18H21NO4S: C, 62.23; H, 6.09; N,
4.03. Found: C, 61.95; H, 5.88; N, 3.85.
(2) Compound 15g was prepared from the above prod-
uct by a similar procedure to that described for the syn-
thesis of 10a. Yield 80%. White powder. IR (KBr): 2948,
1
1738, 1605, 1493, 1312, 1235, 1127, 1044, 914 cmÀ1. H
NMR (CDCl3) d: 1.67–1.76 (1H, m), 1.78–1.94 (1H, m),
1.96–2.11 (1H, m), 2.01 (3H, s), 2.14–2.42 (3H, m), 3.81
(1H, br s), 4.39, 4.48 (2H, ABq, J = 14 Hz), 4.66, 4.76
(2H, ABq, J = 14 Hz), 6.25–6.26 (1H, br), 7.02–7.08
(1H, m), 7.20 (1H, dd, J = 8.4, 2.7 Hz), 7.59 (1H, dd,
J = 8.7, 6.0 Hz). Anal. Calcd for C16H18ClFO4S: C,
53.26; H, 5.03. Found: C, 53.14; H, 5.12.
5.13.1. 1-{6-[(2-Chloro-4-fluorobenzyl)sulfonyl]cyclohex-
1-en-1-yl}propan-1-one (15h). (1) To a mixture of 16h
˚
(80 mg, 0.25 mmol), molecular sieves (4A, 71 mg), 4-
5.12.1. Methyl 6-[(2-chloro-4-fluorobenzyl)sulfonyl]cyclo-
hex-1-ene-1-carboxylate (15a). A mixture of 10a (220 mg,
0.61 mmol), concd H2SO4 (0.4 mL), and MeOH (20 mL)
was refluxed for 134 h. The mixture was concentrated in
vacuo, and then worked up (AcOEt, water, brine). The
residue was chromatographed on an ODS column
(MeOH/water = 7:3, v/v) followed by crystallization from
i-Pr2O–hexane to afford 15a (120 mg, 57%) as colorless
methylmorpholine N-oxide (71 mg, 0.61 mmol) in
CH2Cl2 (2.0 mL) was added TPAP (9.0 mg, 0.03 mmol)
and the reaction stirred for 1 h at 0 ꢁC under a nitrogen
atmosphere. The reaction mixture was submitted to sil-
ica gel column chromatography (AcOEt/hexane = 1:4,
v/v) to give 1-{6-[(2-chloro-4-fluorobenzyl)sulfa-
nyl]cyclohex-1-en-1-yl}propan-1-one (43 mg, 54%) as a
colorless oil. 1H NMR (CDCl3) d: 1.09 (3H, t,
J = 7.2 Hz), 1.61–1.72 (2H, m), 1.83–1.96 (2H, m),
2.15–2.41 (2H, m), 2.45–2.81 (2H, m), 3.94 (3H, br s),
6.86 (1H, t, J = 3.2 Hz), 6.96 (1H, td, J = 8.4, 2.6 Hz),
7.11 (1H, dd, J = 8.4, 2.6 Hz), 7.47 (1H, dd, J = 8.4,
6.0 Hz).
1
crystals. H NMR (CDCl3) d: 1.58–2.53 (6H, m), 3.79
(3H, s), 4.39–4.42 (1H, m), 4.59 (2H, s), 7.04 (1H, dt,
J = 8.4, 2.6 Hz), 7.21 (1H, dd, J = 8.4, 2.6 Hz), 7.41 (1H,
t, J = 3.6 Hz), 7.61 (1H, dd, J = 8.4, 6.0 Hz). Anal. Calcd
for C15H16ClFO4S: C, 51.95; H, 4.65. Found: C, 51.96; H,
4.62.
(2) To a solution of the above product (30 mg, 0.10 mmol)
in AcOEt (1.0 mL), mCPBA (52 mg, 0.21 mmol) was
added and the mixture was stirred for 1 h, then worked
up (AcOEt; water, brine). The residue was crystallized
from i-Pr2O–hexane to afford 15h (23 mg, 70%) as white
crystals. IR (KBr): 2940, 1671, 1493, 1310, 1127,
5.12.2. Propyl 6-[(2-chloro-4-fluorobenzyl)sulfonyl]cyclo-
hex-1-ene-1-carboxylate (15b). Compound 15b was pre-
pared from 10a in the same manner as described
above for 15a. Yield 56%. IR (KBr): 2982, 2940, 1705,
1605, 1495, 1314, 1254, 1128, 1111, 912 cmÀ1 1H
.
1
NMR (CDCl3) d: 0.98 (3H, t, J = 7.4 Hz), 1.60–2.53
(8H, m), 4.12–4.20 (2H, m), 4.41 (1H, br), 4.59 (2H,
s), 7.03 (1H, dt, J = 8.8, 3.0 Hz), 7.20 (1H, dd, J = 8.8,
3.0 Hz), 7.42 (1H, t, J = 3.8 Hz), 7.59 (1H, dd, J = 8.8,
6.0 Hz). Anal. Calcd for C17H20ClFO4S: C, 54.47; H,
5.38. Found: C, 54.34; H, 5.54.
912 cmÀ1. H NMR (CDCl3) d: 1.66 (3H, t, J = 7.5 Hz),
1.55–2.56 (4H, m), 2.47–2.58 (2H, m), 2.72–2.86 (2H, m),
4.52, 4.57 (2H, ABq, J = 13.8 Hz), 4.60 (1H, br s), 7.02
(1H, td, J = 8.1, 2.7 Hz), 7.20 (1H, dd, J = 8.1, 2.7 Hz),
7.27–7.30 (1H, m), 7.59 (1H, dd, J = 8.7, 6.0 Hz). Anal.
Calcd for C16H18ClFO3S: C, 55.73; H, 5.26. Found: C,
55.50; H, 5.29.
5.13. {6-[(2-Chloro-4-fluorobenzyl)sulfonyl]cyclohex-1-en-
1-yl}methyl acetate (15g)
5.13.2. 1-{6-[(2-Chloro-4-fluorobenzyl)sulfonyl]cyclohex-
1-en-1-yl}butan-1-one (15i). Compound 15i was prepared
by a similar procedure to that described for the synthesis
of 15h.
(1) To a solution of 16f (200 mg, 0.70 mmol) in pyridine
(4.0 mL) maintained under a nitrogen atmosphere was
added acetylchloride (74 lL, 1.05 mmol) at 0 ꢁC. The
mixture was stirred for 1 h at 0 ꢁC and then room tem-
perature for 17 h, and worked up (AcOEt; 1 N HCl,
water and brine). The residue was purified by silica gel
column chromatography (AcOEt/hexane = 1:7, v/v) fol-
lowed by crystallization from hexane to give {6-[(2-
chloro-4-fluorobenzyl)sulfanyl]cyclohex-1-en-1-yl}methyl
acetate (92 mg, 40%) as white crystals. 1H NMR
(CDCl3) d: 1.60–1.70 (1H, m), 1.76–2.10 (5H, m), 1.97
(3H, s), 3.26 (1H, s), 3.82 (2H, ABq, J = 14 Hz), 4.37
(1H, d, J = 12.3 Hz), 4.68 (1H, dd, J = 12.3, 1.2 Hz),
5.84–5.86 (1H, m), 6.94 (1H, td, J = 8.4, 2.7 Hz), 7.10
(1) 1-{6-[(2-Chloro-4-fluorobenzyl)sulfanyl]cyclohex-1-
en-1-yl}butan-1-one: Yield 65%. Colorless oil. 1H
NMR (CDCl3) d: 0.92 (3H, t, J = 7.2 Hz), 1.59–1.68
(4H, m), 1.85–1.99 (2H, m), 2.10–2.38 (2H, m), 2.46–
2.67 (2H, m), 3.93 (3H, s), 6.85 (1H, t, J = 4.8 Hz),
6.96 (1H, td, J = 8.4, 2.7 Hz), 7.11 (1H, dd, J = 8.4,
2.7 Hz), 7.47 (1H, dd, J = 8.4, 6.0 Hz).
(2) 15i: Yield 29%. IR (KBr): 2963, 1669, 1493, 1312,
1127, 914 cmÀ1 1H NMR (CDCl3) d: 0.98 (3H, t,
.
J = 7.5 Hz), 1.54–1.80 (4H, m), 1.94–2.58 (4H, m), 2.73

M. Yamada et al. / Bioorg. Med. Chem. 16 (2008) 3941–3958
3957
(3H, t, J = 7.5 Hz), 4.52, 4.56 (2H, ABq, J = 13.8 Hz),
4.61 (1H, br s), 6.99–7.06 (1H, m), 7.20 (1H, dd,
J = 8.4, 2.7 Hz), 7.28–7.31 (1H, m), 7.59 (1H, dd,
J = 8.7, 6.0 Hz).
Drs. T. Yamano and T. Ishikawa for synthesis, and
Mr. S. Yamamoto and Mr. M. Tanaka for opitical res-
olution. We thank Ms. K. Higashikawa for X-ray crys-
tallographic analysis. We thank Dr. T. Sha, Mr. K. Oda,
Ms. M. Uekata and Ms. N. Matsunaga for in vitro and
in vivo assays.
5.14. Optical resolution with chiral HPLC (Table 6):
ethyl 6-[(+)- and (À)-][N-(2-chloro-4-fluorobenzyl)sulfo-
nyl]cyclohex-1-ene-1-carboxylate [(+)- and (À)-10a]
References and notes
The racemate 10a was resolved into its enantiomers by
HPLC (column; CHIRALPAK AD,15 20 mm
ID · 250 mm, mobile phase; hexane/EtOH = 8:2, v/v,
flow rate; 8.0 mL/min, temperature; 30 ꢁC, detection;
UV 260 nm), followed by crystallization from hexane
to afford both enantiomers: (+)-10a (49 mg, retention
time: 19.5 min) and (À)-10a (50 mg, retention time:
14.2 min), as white powder, respectively.
1. (a) Parrillo, J. E. N. Eng. J. Med. 1993, 328, 1471; (b)
Rangel-Frausto, M. S.; Pittet, D.; Costigan, M.; Hwang,
T.; Davis, C. S.; Wenzel, R. P. JAMA 1995, 273, 117; (c)
Nystrom, P. O. J. Antimicrob. Chemother. 1998, 41, 1; (d)
Bone, R. C.; Grodzin, R. J.; Balk, R. A. Chest 1997, 112,
235; (e) Hotchkiss, R. S.; Karl, I. E. N. Eng. J. Med. 2003,
348, 138; (f) Sands, K. E.; Bates, D. W.; Lanken, P. N.,
et al. JAMA 1997, 278, 234.
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.
Chest 1992, 101, 1644; (b) Bone, R. C.; Sprung, C. L.;
Sibbald, W. J. Crit. Care Med. 1992, 20, 724.
Compounds 10e and 22a were resolved by chiral HPLC
in the same method as above. The optical purity of each
enantiomer obtained and HPLC conditions are summa-
rized in Table 6.
3. (a) Martin, G. S.; Mannino, D. M.; Eaton, S.; Moss, M.
N. Eng. J. Med. 2003, 348, 1546; (b) Wheeler, A. P.;
Bernard, G. R. N. Eng. J. Med. 1999, 340, 207.
4. (a) Riedemann, N. C.; Ward, P. A. Expert Opin. Biol.
Ther. 2003, 3, 339; (b) Riedemann, N. C.; Guo, R. F.;
Ward, P. A. J. Clin. Invest. 2003, 112, 460; (c) Arrieta,
O.; Rodriguez-Reyna, T. S.; Sotelo, J. Exp. Opin. Ther.
Patents 2000, 10, 601; (d) Bone, R. C.; Fisher, C. J.,
Jr.; Clemmer, T. P.; Slotman, G. J.; Metz, C. A.; Balk,
R. A. Crit. Care Med. 1989, 17, 389; (e) Natanson, C.;
Esposito, C. J.; Banks, S. M. Crit. Care Med. 1998, 26,
1927.
5. (a) Bernard, G. R.; Vincent, J. L.; Laterre, P. F.; LaRosa,
S. P.; Dhainaut, J. F.; Lopez-Rodriguez, A.; Steingrub, J.
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347, 1027.
5.15. Biology
5.15.1. Assay for inhibitory activity against NO produc-
tion. RAW264.7 cells were plated at 1 · 105 cells/well in
96-well culture plates (Nunc, Rochester, NY) and incu-
bated overnight. After removing cell culture superna-
tants, 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 lg/mL kanamycin) at 37 ꢁC in a humidified atmo-
sphere of 5% CO2 in air. The test compounds were pre-
pared as 10 mM solutions in DMF, diluted with an
RPMI-1640 medium to the appropriate concentrations,
and added to the culture.
6. (a) Yamada, M.; Ichikawa, T.; Ii, M.; Sunamoto, M.;
Itoh, K.; Tamura, N.; Kitazaki, T. J. Med. Chem. 2005,
24, 7457; (b) Yamada, M.; Ichikawa, T.; Yamano, T.;
Kikumoto, F.; Nishikimi, Y.; Itoh, K.; Tamura, N.;
Kitazaki, T. Chem. Pharm. Bull. 2006, 54, 58; (c) Ii, M.;
Matsunaga, N.; Hazeki, K.; Nakamura, K.; Takashima,
K.; Seya, T.; Hazeki, O.; Kitazaki, T.; Sato, J.; Iizawa, Y.
Mol. Pharmacol. 2006, 69, 1288; (d) Ii, M.; Sunamoto, M.;
Matsunaga, N.; Nakamura, K.; Takashima, K.; Kitazaki,
T.; Sato, J.; Iizawa, Y. J. Crit. Care Med. 2004, 32(Suppl.),
A15.
Production of NO was estimated by measuring the
amount of nitrite, a stable metabolite of NO, according
to a fluorometric method16 using 2,3-diaminonapthalene
(DAN, Dojindo Laboratories, Kumamoto, Japan).
Briefly, 25 lL of 20 lg/mL DAN was added to 50 lL
of the culture supernatant and incubated at room tem-
perature for 10 min. After adding 25 lL of 0.5 N
NaOH, fluorescence at 460 nm (excitation wavelength:
355 nm) was measured.
7. Villieras, J.; Rambaud, M.; Graff, M. Synth. Commun.
1986, 16, 149.
5.15.2. Endotoxin shock model. Mice were intraperitone-
ally injected with LPS at a lethal dose of 7 mg/kg. Sur-
vival of mice was recorded for 7 days following LPS
challenge. Compounds (R)-(+)-10a and (6R, 1S)-(+)-
22a were dissolved in 10% (w/v) Glucuronylglucosyl-
beta-cyclodextrin sodium salt (Wako, Osaka, Japan)
and administered intravenously 1 h before the LPS
injection.
8. Alami, N. E.; Belaud, C.; Villieras, J. J. Organomet. Chem.
1988, 348, 1.
9. Preparation of 13b: Liu, R.; Zhang, P.; Gan, T.; Cook, J.
M. J. Org. Chem. 1997, 62, 7447.
10. Preparation of 13d: de Dios, A.; Prieto, L.; Martin, J. A.;
Rubio, A.; Ezquerra, J.; Tebbe, M.; de Uralde, B. L.;
´
Martin, J.; Sanchez, A.; LeTourneau, D. L.; McGee, J. E.;
Boylan, C.; Parr, T. R., Jr.; Smith, M. C. J. Med. Chem.
2002, 45, 4559.
11. Preparation of 13c: Henke, B. R.; Aquio, C. J.; Birkemo,
L. S.; Croom, D. K.; Dougherty, R. W., Jr.; Ervin, G. N.;
Grizzle, M. K.; Hirst, G. C.; James, M. K.; Johoson, M.
F.; Queen, K. L.; Sherrill, R. G.; Sugg, E. E.; Suh, E. M.;
Szewczyk, J. W.; Unwalla, R. J.; Yingling, J.; Willson, T.
M. J. Med. Chem. 1997, 40, 2706.
Acknowledgments
We thank Drs. A. Miyake, Y. Iizawa and S. Hashiguchi
for their encouragement and helpful advice. We thank

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M. Yamada et al. / Bioorg. Med. Chem. 16 (2008) 3941–3958
12. Preparation of 13f: Nussbaumer, P.; Gabor, P.; Stuetz, A.
¨
J. Med. Chem. 1991, 34, 65.
13. Preparation of 13g: Martin-Matute, B.; Nevado, C.;
14. Amri, H.; Rambaud, M.; Villieras, J. Tetrahedron 1990,
46, 3535.
15. Daicel Chemical Industries, Ltd.
´
Cardenas, D. J.; Echavarren, A. M. J. Am. Chem. Soc.
2003, 125, 5757.
16. Misko, T. P.; Schilling, R. J.; Salvemini, D.; Moore, W.
M.; Currie, M. G. Anal. Biochem. 1993, 214, 11.