Optically active cyclohexene derivative as a new antisepsis agent: An efficient synthesis of ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)-sulfamoyl] cyclohex-1-ene-1-carboxylate (TAK-242)

Article abstract of DOI:10.1248/cpb.54.58

Two new synthetic methods were established for the efficient synthesis of optically active cyclohexene antisepsis agent, ethyl (6R)-6-[N-(2-chloro-4- fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate [(R)-1: TAK-242)]. The first method involved recrystallization from methanol of the diastereomeric mixture (6RS,1′R)-7, obtained by esterification of carboxylic acid 3 with (S)-1-(4-nitrophenyl)ethanol [(S)-5)] to give the desired isomer (6R,1′R)-7 with 99% de in 32% yield. Subsequent catalytic hydrogenolysis and esterification gave (R)-1 with >99% ee. The second method employed enantioselective hydrolysis of acetoxymethyl ester 9a (prepared by alkylation of 3 with bromomethyl acetate) with Lipase PS-D to give the eutomeric enantiomer (R)-9a with excellent enantioselectivity (>99% ee) and high yield (48%). The desired (R)-1 was then obtained by transesterification with ethanol in the presence of concentrated sulfuric acid without loss of ee. Of these, the procedure employing enzymatic kinetic resolution using Lipase PS-D is the more efficient and practical preparation of (R)-1.

Full text of DOI:10.1248/cpb.54.58

58
Chem. Pharm. Bull. 54(1) 58—62 (2006)
Vol. 54, No. 1
Optically Active Cyclohexene Derivative as a New Antisepsis Agent:
An Efficient Synthesis of Ethyl (6R)-6-[N-(2-Chloro-4-fluorophenyl)-
sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242)
Masami YAMADA,* Takashi ICHIKAWA, Toru YAMANO, Fumio KIKUMOTO, Yuji NISHIKIMI,
Norikazu TAMURA, and Tomoyuki KITAZAKI
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited; 2–17–85 Jusohonmachi, Yodogawa-ku,
Osaka 532–8686, Japan. Received July 13, 2005; accepted October 20, 2005
Two new synthetic methods were established for the efficient synthesis of optically active cyclohexene anti-
sepsis agent, ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate [(R)-1: TAK-242)].
The first method involved recrystallization from methanol of the diastereomeric mixture (6RS,1ꢀR)-7, obtained
by esterification of carboxylic acid 3 with (S)-1-(4-nitrophenyl)ethanol [(S)-5)] to give the desired isomer (6R,1ꢀR)-
7 with 99% de in 32% yield. Subsequent catalytic hydrogenolysis and esterification gave (R)-1 with ꢀ99% ee.
The second method employed enantioselective hydrolysis of acetoxymethyl ester 9a (prepared by alkylation of 3
with bromomethyl acetate) with Lipase PS-D to give the eutomeric enantiomer (R)-9a with excellent enantiose-
lectivity (ꢀ99% ee) and high yield (48%). The desired (R)-1 was then obtained by transesterification with
ethanol in the presence of concentrated sulfuric acid without loss of ee. Of these, the procedure employing enzy-
matic kinetic resolution using Lipase PS-D is the more efficient and practical preparation of (R)-1.
Key words TAK-242; optical resolution; lipase; cyclohexene; chiral synthesis
Sepsis and septic shock remain the chief causes of death in ties for further evaluation of this compound. In this paper, we
intensive care units, with mortality rates between 30 and describe the development of two new synthetic methods of
70%.1—3) Although several drug candidates have entered (R)-1 utilizing rac-1 as the synthetic precursor.
clinical trials over the last 20 years, most have not demon-
In order to find an alternative synthesis of optically active
strated significant benefit for patients with sepsis and septic (R)-1 from the rac-1, the following two routes were proposed
shock, and difficulties of the development of an antisepsis (Chart 2): the formation and separation of diastereomeric
agent has been proven. In 2001, Drotrecogin-a (XigrisTM),4) ester derivatives I followed by the conversion to ethyl ester
a recombinant human activated protein C, was launched onto (Route A), and enzymatic hydrolysis of the ester derivative
the market as the first antisepsis agent, but the usefulness of (if necessary, followed by ethyl esterification, Route B).
this drug is limited due to its efficacy and safety.
The synthetic method via the preparation of diastere-
Recently, we reported that ethyl (6R)-6-[N-(2-chloro-4-flu- omeric phenethyl ester derivatives was initially investigated
orophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate [TAK-242: as shown in Chart 3. First, the synthesis of key intermediate
(R)-1] was selected as a new class of clinical candidate for carboxylic acid 3 by hydrolysis of rac-1 was examined using
sepsis.5) TAK-242 exhibited potent inhibitory activity for the standard hydrolytic conditions employing bases such as
production of not only nitric oxide (NO) but also various cy- sodium hydroxide (NaOH), lithium hydroxide, potassium
tokines such as tumor necrosis factor-alfa (TNF-a), inter- carbonate (K2CO3) and cesium carbonate (Cs2CO3) or Lewis
leukin-1-beta (IL-1b) and IL-6 in vitro, as well as potent pro- acids and Brønsted acids. However, in all cases hydrolysis
tective effects in the mouse endotoxin shock model. (R)-1 is was accompanied by significant decomposition resulting in
currently undergoing clinical trails and is expected to be a low yields of 3. In further studies, the use of barium dihy-
promising antisepsis drug.
droxide [Ba(OH)2] was found to give 3 in good yield. Thus,
As shown in Chart 1, (R)-1 was obtained in high enan- the treatment of rac-1 with aqueous Ba(OH)2 solution at
tiomeric purity by the optical resolution of rac-1, prepared 60 °C in acetonitrile (MeCN) gave carboxylic acid 3 in 74%
from commercially available ethyl 2-oxocyclohexane-1-car- isolated yield.
boxylate (2) in three steps, by means of preparative high per-
Next, esterification of 3 was examined using a number of
formance liquid chromatography (HPLC) using a chiral sta- chiral alcohols including (R)- and (S)-1-phenylethanol (4),6)
tionary phase.5) Although separation using preparative HPLC (R)- and (S)-1-(4-nitrophenyl)ethanol (5),6) (R)- and (S)-1-(2-
is a straightforward method for the preparation of small naphtyl)ethanol6) and d- and l-menthol. Ease in separation of
quantities of optically active compounds, applications to diastereomers and appreciable yields made (R)-4 and (S)-5
large scale production are sometimes troublesome. Thus, an the alcohols of choice. Carboxylic acid 3 was allowed to
efficient and practical process for the preparation of optically react with (R)-4 and (S)-5 under usual Mitsunobu reaction
active (R)-1 was required in order to supply sufficient quanti- conditions in tetrahydrofuran (THF) to give the correspond-
Chart
1
∗ To whom correspondence should be addressed. e-mail: Yamada_Masami1@takeda.co.jp
© 2006 Pharmaceutical Society of Japan

January 2006
59
Chart
2
Chart
3
ing diastereomeric esters (6RS,1ꢁS)-6 and (6RS,1ꢁR)-7, re-
spectively. Diastereomeric mixture (6RS,1ꢁS)-6 was sepa-
rated by silica gel column chromatography to preferentially
give the desired isomer (6R,1ꢁS)-67) with 59% diastereomeric
excess (de)8) in 32% yield. The use of a similar procedure af-
forded (6R,1ꢁR)-79) in 32% yield with excellent diastere-
omeric purity (99% de).10) Replacement of the 1-phenethyl
group of (6R,1ꢁS)-6 (59% de) with an ethyl group was subse-
quently carried out by transesterification, followed by crys-
tallization from ethanol (EtOH) to give (R)-1 with ꢀ99%
enantiomeric excess (ee).11) However, the yield of (R)-1 was
low (37%) due to the low diastereomeric purity of (6R,1ꢁS)-6
used. In contrast, (6R,1ꢁR)-7 with 99% de was hydrolyzed to
carboxylic acid (R)-3 in 84% yield by catalytic hydrogenoly-
sis over palladium on carbon (Pd–C) in acetic acid (AcOH),
and subsequent esterification with EtOH in the presence of
concentrated sulfuric acid (conc. H2SO4) gave (R)-1 in 72%
isolated yield with ꢀ99% ee.12) Both products (R)-1 obtained
via the diastereomeric esters, (6R,1ꢁS)-6 and (6R,1ꢁR)-7,
were identical to an authentic sample prepared by HPLC sep-
aration.5)
Chart
4
lution with enzymes has received considerable attention be-
cause of the high enantioselectivities than can be ob-
tained.13—21) Thus, a selection of enzymes was screened in
order to identify those which were capable of catalyzing the
enantioselective hydrolysis of rac-1, however in all cases no
hydrolysis was observed. It is known that enzymatic hydroly-
sis of sterically hindered esters can be problematic,22,23) and
this suggested that the A1,2-strain between the ethoxycar-
bonyl group and sulfamoyl group of rac-1 was preventing
hydrolysis. Achiwa and co-workers introduced acyl-
oxymethyl or carbamoylmethyl groups into the sterically
hindered carboxylic acid, and successfully achieved enantio-
selective enzymatic hydrolysis.22,23) Accordingly, it was
thought that exchange of the ethyl group of rac-1 to acyl-
oxymethyl and carbamoylmethyl group may generate a suit-
able ester substrate for enantioselective enzymatic hydroly-
sis. Esters 9a—c were synthesized by alkylation of 85) as
shown in Chart 4, and were screened for hydrolysis using
various lipases. It was found that several lipases catalyzed the
enantioselective hydrolysis of acetoxymethyl ester 9a to opti-
As described above, a high yielding synthetic route for
(R)-1 in high enantiomeric purity was established via the sep-
aration of diastereomeric ester (6RS,1ꢁR)-7. This procedure,
however, have remained unattractive due to annoying column
chromatography and availability of the optically active alco-
hol (S)-5.
Our next approach to obtain optically active (R)-1 was to
examine the use of enzymatic reaction. Kinetic optical reso-

60
Vol. 54, No. 1
Chart
5
Table 1. Enantioselective Hydrolysis of Acetoxymethyl Ester 9a with Var- vent. Though lipases are more stable in more hydrophobic
ious Lipasesa)
solvents,30) polar or hydrophilic solvents were investigated to
identify a more suitable solvent with improved solubility for
9a. The examination of MeCN and acetone were carried out
under the conditions in Table 2 (runs 4 and 5). Surprisingly,
the reaction rate and E-value using acetone were nearly equal
to those using tert-BME (runs 3 and 5), and thus subsequent
optimization of the reaction conditions was carried out in
acetone.
It was found that the best result was obtained when the
eec) of
(R)-9a
eec) of
(S)-3
concentration of 9a was 50 mg/ml and the ratio of Lipase PS-
D to 9a was 3 to 5. Thus, a mixture of 9a and Lipase PS-D in
acetone containing ca. 10% water was stirred for 20 h at
28 °C to afford (R)-9a with excellent enantiomeric purity
(ꢀ99% ee) and E-value (137) (run 6). To investigate the
practicality of this enzymatic procedure, the synthesis of (R)-
1 was carried out starting from 10 g of 9a. Firstly 9a was hy-
drolyzed under the conditions of run 6 to give (R)-9a in 48%
isolated yield with ꢀ99% ee, without column purification.
The isolation procedure for this synthesis was highly conve-
nient requiring only removal of the enzyme by filtration and
Run
Enzyme
E/Sb)
C
d) (%) Ee)
1
2
3
4
Lipase AK-20
Lipase PS
Lipase F
1/10
1/10
1/10
1/10
86.8
99.2
99.6
96.8
75.3
74.4
53.4
80.6
53.6
20
36
22
38
57.1
65.1
54.5
Lipase QLG
a) The reaction was carried out for 24 h at 35 °C in tert-BME containing ca. 5%
water. Concentration of substrate 9a to solvent was 1 mg/ml. b) E/S, enzyme to sub-
strate ratio (w/w). c) Determined by HPLC analysis. d) C, conversion as deter-
mined by HPLC analysis. e) Calculated according to Chen et al.27)
cally active (R)-9a24) and thus 9a was selected for use as the standard work-up (washing with alkaline water). Avoidance
enzymatic hydrolysis substrate.
of laborious column chromatography rendered this procedure
More practical synthesis of key intermediate 9a was car- quite practical. Transesterification of the resulting (R)-9a was
ried out by esterification of carboxylic acid 3, and optimiza- then carried out with EtOH in the presence of conc. H2SO4 to
tion of the base, solvent and additive,25) indicated that reac- give (R)-1 in 65% isolated yield with ꢀ99% ee (Chart 5).
tion of 3 with bromomethyl acetate in the presence of
Cs2CO3 and tetrabutylammonium iodide (TBAI) at 50 °C in the efficient synthesis of (R)-1 that avoid the use of optical
MeCN gave 9a in 72% isolated yield (Chart 5).
resolution by HPLC. Of these, the procedure employing en-
In conclusion, we have established two new methods for
Next, the hydrolysis of 9a was examined by using fifty li- zymatic kinetic resolution using Lipase PS-D is the more ef-
pases including Lipase AK-20, PS, F and QLG.26) The typi- ficient and practical preparation of (R)-1 (TAK-242).
cal reaction was carried out as follows: a mixture of 9a and
lipase in tert-butylmethyl ether (tert-BME) containing ca.
5% water was stirred for 24 h at 35 °C. As shown in Table 1,
the four lipases gave (R)-9a with high enantiomeric purity
(ꢀ86% ee), although Lipase PS and Lipase F showed the
highest enantioselectivity. In addition, the E-value27,28) using
Lipase PS was considerably superior to that using Lipase F
and thus Lipase PS was selected as the enzyme catalyst for
this hydrolysis.
Taking into account an application to large scale prepara-
tion, it appeared that it would be more appropriate to use Li-
pase PS immobilized on diatomaceous earth (Lipase PS-D)29)
to address issues of ease of operation, stability and cost.
Therefore, further optimization of the reaction conditions
was carried out using Lipase PS-D.
As shown in Table 2, when 9a was treated with Lipase PS-
D under the same conditions as used with Lipase PS, a
decrease in E-value was observed (runs 1 vs. 2). This E-value
was significantly improved by increasing the proportion of
both water to solvent and enzyme to substrate (run 3). Unfor-
tunately, the poor solubility of substrate 9a in tert-BME (ca.
Experimental
Melting points were determined using a Yanagimoto melting point appa-
ratus and are uncorrected. IR spectra were obtained on Shimadzu FTIR-
8200PC spectrometer. 1H-NMR spectra were recorded on Varian Gemini
200 (200 MHz) spectrometer or Varian Mercury 300 (300 MHz) spectrome-
ter with tetramethylsilane as an internal standard. The following abbrevia-
tions are used: sꢂsinglet, dꢂdoublet, tꢂtriplet, qꢂquartet, mꢂmultiplet
and brꢂbroad. The optical rotations were recorded with a Jasco DIP-181 or
DIP-370 digital polarimeter. FAB mass spectra were recorded on a JEOL
JMS-HX110. Elemental analyses were carried out by Takeda Analytical
Laboratories Ltd. and were within ꢃ0.4% of the theoretical values for the
elements indicated unless otherwise noted.
Reactions were carried out at room temperature unless otherwise noted
and followed by TLC on silica gel 60 F254 precoated TLC plates (E. Merck)
or by HPLC using an octadecyl silica (ODS) column (YMC-Pack A-303,
4.6 mm i.d.ꢄ250 mm, YMC Co., Ltd.). Standard work-up procedures were
as follows. The reaction mixture was partitioned between the indicated sol-
vent and water. Organic extracts were combined and washed in the indicated
order using the following aqueous solutions: water, 5% aqueous sodium car-
bonate solution (aqueous NaHCO3), saturated sodium chloride (NaCl) solu-
tion (brine), 1 N aqueous sodium hydroxide solution (1 N NaOH) and 1 N hy-
drochloric acid (1 N HCl). Extracts were dried over anhydrous magnesium
sulfate (MgSO4), filtered and evaporated in vacuo. Chromatographic separa-
tions were carried out on Silica gel 60 (0.063—0.200 mm, E. Merck) using
1 mg/ml) would require the use of a large quantity of this sol- the indicated eluents.

January 2006
61
Table 2. Lipase PS-D Catalyzed Enantioselective Hydrolysis of 9aa)
Run
Solv. (Conc., mg/ml)b)
W/Solv.c)
E/Sd)
Time (h)
eee) of (R)-9a
eee) of (S)-3
C
f ) (%)
Eg)
1h)
2
3
4
5
tert-BME (1)
tert-BME (1)
tert-BME (1)
MeCN (20)
Acetone (20)
Acetone (50)
1/20
1/20
1/10
1/10
1/10
1/10
1/7.4
1/7.4
1/5
2/5
2/5
24
30
18
74
20
20
ꢀ99
97.9
ꢀ99
89.6
81.7
ꢀ99
69.5
78.6
74.5
91.4
94.5
84.9
59.0
55.3
57.3
49.5
46.4
54.1
48
34
93
68
91
6
3/5
137
a) The reaction was carried out at 28 °C. b) Conc., concentration of substrate. c) W/Solv., water to solvent ratio (v/v). d) E/S, enzyme to substrate ratio (w/w).
e) Determined by HPLC analysis. f) C, conversion as determined by HPLC analysis. g) Calculated according to Chen et al.27) h) Using Lipase PS.
The de’s of the compounds prepared were determined by HPLC using an
ODS column (YMC-Pack A-303) or silica gel column (YMC-Pack SIL)
under the indicated conditions [column; mobile phase; flow rate; detection].
The ee’s of the compounds prepared were determined by HPLC using a
chiral stationary phase column (CHIRALCEL OC and CHIRALPAK AD,
4.6 mm i.d.ꢄ250 mm, Daicel Chemical Industries, Ltd.) under the indicated
conditions [column; mobile phase; flow rate; detection].
The following lipases were obtained from the Amano Enzyme Inc.: Li-
pase AK-20, Lipase PS and Lipase PS-D. Lipase F was produced by Biocat-
alysts Ltd. Lipase QLG was a product of Meito Sangyo Co., Ltd.
(6R,1ꢁR)-7 obtained was determined to be 99.5% [column, YMC-pack SIL;
mobile phase, EtOAc–hexane, 1 : 5; flow rate, 0.8 ml/min; detection, UV at
220 nm; retention time, (6R,1ꢁR)-7: 13.2 min, (6S,1ꢁR)-7: 11.2 min]. mp
1
103—104 °C. H-NMR (DMSO-d6) d: 1.31 (3H, d, Jꢂ6.6 Hz), 1.66—2.55
(6H, m), 4.34 (1H, d, Jꢂ5.2 Hz), 5.87 (1H, q, Jꢂ6.6 Hz), 7.14—7.24 (2H,
m), 7.48—7.57 (2H, m), 7.50 (2H, d, Jꢂ8.8 Hz), 8.17 (2H, d, Jꢂ8.8 Hz),
9.82 (1H, s). IR (KBr) cmꢅ1: 3218, 1713, 1520, 1495, 1348, 1329, 1254,
1148, 1067. [a]D20 ꢆ112.2° (cꢂ1.0, MeOH). Anal. Calcd for C21H20-
ClFN2O6S: C, 52.23; H, 4.17; N, 5.80. Found: C, 52.07; H, 4.12; N, 5.92.
(6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-car-
boxylic Acid [(R)-3] A solution of (6R,1ꢁR)-7 (5.0 g, 99.5% de) in AcOH
(300 ml) was hydrogenated over 10% Pd–C (50% wet, 5.0 g) for 2 h under an
atmospheric pressure. The catalyst was removed by filtration, and the filtrate
was concentrated under reduced pressure. The residue was dissolved in
EtOAc (200 ml) and the whole was extracted with 1 N NaOH (200 mlꢄ2).
The aqueous extracts were combined and neutralized with 1 N HCl (500 ml),
then the whole was worked up (EtOAc; brine). The residue was crystallized
from EtOAc–hexane to give (R)-3 (2.92 g, 84%) as white powdery crystals
with ꢀ99% ee [column, CHIRALCEL OC; mobile phase, hexane–EtOH–
trifluoroacetic acid (TFA), 8 : 2 : 0.01; flow rate, 0.8 ml/min; detection, UV at
225 nm; retention time, (R)-3: 10.5 min, (S)-3: 14.4 min]. mp 157.5—158.0 °C.
1H-NMR (DMSO-d6) d: 1.50—2.52 (6H, m), 4.32 (1H, d, Jꢂ4.4 Hz), 7.08
(1H, t, Jꢂ2.8 Hz), 7.17—7.27 (1H, m), 7.47—7.60 (2H, m), 9.60 (1H, br s),
12.35 (1H, br s). IR (KBr) cmꢅ1: 3216, 1709, 1495, 1327, 1223, 1144, 1134.
[a]D20 ꢆ109.7° (cꢂ0.994, MeOH). Anal. Calcd for C13H13ClFNO4S: C,
46.78; H, 3.93; N, 4.20. Found: C, 46.89; H, 4.11; N, 4.27.
(6RS)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-car-
boxylic Acid (3) A hot aqueous Ba(OH)2·8H2O (26.0 g) solution (110 ml)
heated at 70 °C was added to a solution of 15) (10.0 g) in MeCN (150 ml).
The mixture was stirred for 1 h at 60 °C and cooled, then poured into 1 N
HCl (300 ml). The whole was extracted with ethyl acetate (EtOAc, 150 ml)
and the organic layer was extracted with 1 N NaOH (300 ml). The alkaline
aqueous extract was washed with EtOAc (150 ml) and acidified with 10%
citric acid (500 ml). The whole was worked up (EtOAc; brine). The residue
was crystallized from EtOAc–hexane to give 3 (6.82 g, 74%) as white pow-
dery crystals, mp 187—189 °C. 1H-NMR (DMSO-d6) d: 1.55—1.80 (2H,
m), 1.99—2.46 (4H, m), 4.33 (1H, d, Jꢂ4.8 Hz), 7.08 (1H, br s), 7.22 (1H,
dt, Jꢂ8.6, 3.0 Hz), 7.48—7.59 (2H, m), 9.64 (1H, br s), 12.35 (1H, br s).
Anal. Calcd for C13H13ClFNO4S: C, 46.78; H, 3.93; N, 4.20. Found: C,
46.82; H, 4.22; N, 4.03.
(1ꢀS)-1ꢀ-Phenylethyl (6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]-
cyclohex-1-ene-1-carboxylate [(6R,1ꢀS)-6] A solution of diethyl azodi-
carboxylate (DEAD, 40% in toluene, 5.22 g) in THF (60ml) was added drop-
wise to an ice-cooled solution of (R)-4 (7.32 g), triphenylphosphine (PPh3,
15.7 g) and 3 (20.0 g) in THF (200 ml) over a period of 30 min. The mixture
was stirred for 6 h under a nitrogen atmosphere and worked up (EtOAc; 0.1 N
NaOH, water, brine). A mixture of EtOAc–hexane (1 : 2, v/v, 200 ml) was
added to the residue. The resulting insoluble substances were filtered off,
and the filtrate was concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (EtOAc–hexane, 1 : 9→1 : 6,
v/v) to give (6R,1ꢁS)-6 (10.5 g, 32%) as white crystals with 59% de [column,
YMC-pack ODS A-303; mobile phase, methanol (MeOH)–H2O–AcOH,
7 : 3 : 0.02; flow rate, 0.8 ml/min; detection, UV at 254 nm; retention time,
(6R,1ꢁS)-6: 35 min, (6S,1ꢁS)-6: 32 min], which was used for the next step
without purification.
The purification of (6R,1ꢁS)-6 was independently carried out and
(6R,1ꢁS)-6 with 97% de was isolated, whose analytical data was shown
below: 1H-NMR (DMSO-d6) d: 1.42 (3H, d, Jꢂ6.6 Hz), 1.50—1.85 (2H,
m), 2.00—2.49 (4H, m), 4.32 (1H, d, Jꢂ4.8 Hz), 5.76 (1H, q, Jꢂ6.6 Hz),
7.14—7.31 (7H, m), 7.48—7.55 (2H, m), 9.72 (1H, br s). Anal. Calcd for
C21H21ClFNO4S: C, 57.60; H, 4.83; N, 3.20. Found: C, 57.64; H, 4.91; N,
3.24.
(1ꢀR)-1ꢀ-(4-Nitrophenyl)ethyl (6R)-6-[N-(2-Chloro-4-fluorophenyl)sul-
famoyl]cyclohex-1-ene-1-carboxylate [(6R,1ꢀR)-7] A solution of DEAD
(40% in toluene, 22.8 ml) in THF (270 ml) was added dropwise to an ice-
cooled solution of (S)-5 (6.7 g), PPh3 (13.2 g) and 3 (16.8 g) in THF (270 ml)
over a period of 1 h. After having been stirred for 7 h under a nitrogen at-
mosphere, the reaction mixture was concentrated under reduced pressure
and toluene (50 ml) was added. The resulting insoluble substances were re-
moved by filtration, and the filtrate was concentrated under reduced pres-
sure. The residue was subjected to silica gel chromatography (EtOAc–
hexane, 1 : 5, v/v) to afford (6RS,1ꢁR)-7 (20.5 g), which was dissolved in
cooled MeOH (15 ml) and the solution was stirred for 30 min at 0 °C. The
crystalline precipitates were collected by filtration and recrystallized from
cooled MeOH to give (6R,1ꢁR)-7 (6.24 g, 32%) as white crystals. The de of
Acetoxymethyl (6RS)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclo-
hex-1-ene-1-carboxylate (9a) A mixture of 3 (10.0 g), bromomethyl ac-
etate (6.41 g), Cs2CO3 (5.86 g), TBAI (5.54 g) and MeCN (200 ml) was
stirred for 2.5 h at 52—55 °C under a nitrogen atmosphere. The mixture was
cooled and worked up (EtOAc; 0.5 N HCl, brine). The residue was crystal-
lized from diisopropyl ether (iso-Pr2O) to give 9a (8.75 g, 72%) as white
powdery crystals, mp 129—130 °C. 1H-NMR (DMSO-d6) d: 1.60—1.80
(2H, m), 2.02 (3H, s), 2.02—2.46 (4H, m), 4.29 (1H, d, Jꢂ5.2 Hz), 5.64
(2H, s), 7.19—7.26 (2H, m), 7.50—7.56 (2H, m), 9.77 (1H, s). IR (KBr)
cmꢅ1: 1761, 1732, 1495, 1208, 1148, 1019. Anal. Calcd for C16H17-
ClFNO4S: C, 47.35; H, 4.22; N, 3.45. Found: C, 47.56; H, 4.16; N, 3.39.
[(2,2-Dimethylpropanoyl)oxy]methyl (6RS)-6-[N-(2-Chloro-4-fluoro-
phenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (9b) Iodomethyl pival-
ate (0.29 g) was added to a solution of 8 (0.36 g) in DMF (3 ml) and the mix-
ture was stirred for 8 h, then worked up (EtOAc; water, brine). The residue
was purified by silica gel column chromatography (EtOAc–hexane, 1 : 3,
v/v) to give 9b (0.28 g, 62%) as a colorless oil: 1H-NMR (DMSO-d6) d: 1.11
(9H, s), 1.6—2.4 (6H, m), 4.30 (1H, br), 5.68 (2H, s), 7.18—7.78 (4H, m),
9.74 (1H, s). FAB-MS m/z: 447 (Mꢆ).
2-Amino-2-oxoethyl (6RS)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]-
cyclohex-1-ene-1-carboxylate (9c) Compound 9c was prepared in 42%
yield by the reaction of 8 with bromoacetamide, as white powdery crystals,
1
mp 146—148 °C. H-NMR (DMSO-d6) d: 1.60—1.85 (2H, m), 2.00—2.42
(4H, m), 4.38 (2H, s), 4.41 (1H, d, Jꢂ5.2 Hz), 7.19—7.29 (4H, m), 7.49—
7.57 (2H, m), 9.75 (1H, s). Anal. Calcd for C15H16ClFN2O5S: C, 46.10; H,
4.13; N, 7.17. Found: C, 46.33; H, 4.24; N, 7.10.
Acetoxymethyl (6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclo-
hex-1-ene-1-carboxylate [(R)-9a] A mixture of 9a (10 g), Lipase PS-D
(6.0 g), water (20 ml) and acetone (200 ml) was stirred for 17.5 h at 28 °C.
The catalyst was removed by filtration. The filtrate was worked up (iso-Pr2O,
EtOAc; saturated aqueous NaHCO3, water, brine) to give (R)-9a (4.82 g,
48%) as a colorless oil. The ee of (R)-9a obtained was determined to be
ꢀ99% ee [column, CHIRALCEL OC; mobile phase, hexane–EtOH–TFA,

62
Vol. 54, No. 1
8 : 2 : 0.01; flow rate, 1.1 ml/min; detection, UV at 225 nm; retention time,
(R)-9a: 16.3 min, (S)-3: 9.97 min]. 1H-NMR (DMSO-d6) d: 1.53—1.89 (2H,
m), 2.02 (3H, s), 2.02—2.43 (4H, m), 4.29 (1H, d, Jꢂ5.2 Hz), 5.64 (2H, s),
7) The stereochemistry of the major isomer was confirmed to be (6R,1ꢁS)
after converting to compound 1.
8) Diastereomeric excesses (de’s) were determined by the HPLC on an
ODS column.
9) The stereochemistry of the major diastereomeric ester was confirmed
to be (6R,1ꢁR) by the same method described in ref. 7.
10) Diastereomeric excesses (de’s) were determined by the HPLC on a sil-
ica gel column.
11) Enantiomeric excesses (ee’s) were determined by the HPLC on a chiral
column (Daicel, CHIRALPAK AD).
12) When direct transesterification of (6R,1ꢁR)-7 to (R)-1 was attempted
using the same method used for (6R,1ꢁS)-6, only decomposition prod-
ucts were obtained.
13) For review see: Santaniello E., Ferraboschi P., Grisenti P., Manzocchi
A., Chem. Rev., 92, 1071—1140 (1992).
14) For review see: Mori K., Synlett, 1995, 1097—1109 (1995).
15) For review see: Robert S. M., J. Chem. Soc., Perkin Trans. 1, 1998,
157—169 (1998).
16) For review see: Robert S. M., J. Chem. Soc., Perkin Trans. 1, 1999,
1—12 (1999).
7.19—7.23 (2H, m), 7.49—7.57 (2H, m), 9.76 (1H, s). IR (KBr) cmꢅ1
:
1761, 1732, 1495, 1208, 1148, 1019. [a]D20 ꢆ84.0° (cꢂ0.205, MeOH).
Ethyl (6R)-6-[N-(2-Chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-
carboxylate [(R)-1] [Method a]: A mixture of (6R,1ꢁS)-6 (10.5 g, 58.8%
de), conc. H2SO4 (5.0 ml) and EtOH (200 ml) was heated under reflux for
15 h. After the addition of conc. H2SO4 (2.0 ml), the whole was refluxed fur-
ther 11 h and cooled. The mixture was concentrated under reduced pressure,
then worked up (EtOAc; ice-water, aqueous NaHCO3, brine). The residue
was purified by silica gel column chromatography (EtOAc–hexane, 1 : 5→
1 : 4, v/v) followed by recrystallization from EtOH to give (R)-1 (2.55 g,
37%) as colorless powdery crystals with ꢀ99% ee [column, CHIRALPAK
AD; mobile phase, EtOH–hexane, 9 : 1; flow rate, 0.6 ml/min; detection, UV
at 254 nm; retention time, (R)-1: 20.0 min, (S)-1: 16.7 min].
[Method b]: A mixture of (R)-3 (2.5 g, ꢀ99% ee), conc. H2SO4 (2.5 ml)
and EtOH (50 ml) was heated under reflux for 19 h. The reaction mixture
was cooled and worked up (EtOAc; water, 0.5 N NaOH, 0.5 N HCl, brine).
The residue was purified by flash silica gel column chromatography (EtOAc–
hexane, 1 : 10→1 : 3, v/v) and crystallized from EtOH–hexane to give (R)-1
(1.94 g, 72%) as colorless prisms with ꢀ99% ee.
17) For review see: Whitesides G. M., Wong C. H., Angew. Chem., Int. Ed.
Engl., 24, 617—718 (1985).
[Method c]: A mixture of (R)-9a (4.8 g, ꢀ99% ee), conc. H2SO4 (4.8 ml)
and EtOH (96 ml) was heated under reflux for 25 h and cooled. The reaction
mixture was concentrated under reduced pressure, then worked up (EtOAc;
ice-water, saturated aqueous NaHCO3, brine). The residue was purified by
silica gel column chromatography (EtOAc–hexane, 1 : 3, v/v) and crystal-
lized from EtOH–hexane to give (R)-1 (2.76 g, 65%) as colorless prisms
with ꢀ99% ee. mp 68—69 °C.31) 1H-NMR (DMSO-d6) d: 1.05 (3H, t, Jꢂ
7.0 Hz), 1.56—1.83 (2H, m), 2.01—2.43 (4H, m), 4.00 (2H, q, Jꢂ7.0 Hz),
4.30 (1H, d, Jꢂ5.0 Hz), 7.10 (1H, br), 7.20—7.30 (1H, m), 7.50—7.58 (2H,
m), 9.74 (1H, s). IR (KBr) cmꢅ1: 1711, 1649, 1493, 1333, 1150. [a]D20
ꢆ111.0° (cꢂ1.0, MeOH). Anal. Calcd for C15H17ClFNO4S: C, 49.79; H,
4.74; N, 3.87. Found: C, 49.73; H, 4.69; N, 3.81.
18) For review see: Boland W., Frößl C., Lorenz M., Synthesis, 1991,
1049—1072 (1991).
19) Sakamoto K., Tsuzaki K., Shima M., JP 09308497 (1997) [Chem.
Abstr., 128, 74400 (1997)].
20) Yanase H., Arishima T., Kita K., Nagai J., Mizuno S., Takuma Y.,
Endo K., Kato N., Biosci. Biotech. Biochem., 57, 1334—1337 (1993).
21) Brieva R., Crich J. Z., Sih C. J., J. Org. Chem., 58, 1068—1075
(1993).
22) Ebiike H., Terao Y., Achiwa K., Tetrahedron Lett., 32, 5805—5808
(1991).
23) Ebiike H., Matsuyama K., Achiwa K., Chem. Pharm. Bull., 40,
1083—1085 (1992).
These products (R)-1 obtained by the three methods described above, re-
spectively, were identical to TAK-242 prepared by optical resolution by
preparative HPLC5) upon direct comparison with the authentic sample.
24) The stereochemistry of (R)-9a was confirmed after converting to com-
pound 1. Enantiomeric excesses (ee’s) were determined by the HPLC
on a chiral column (Daicel, CHIRALCEL OC).
25) Although dicyclohexylamine, sodium carbonate, lithium carbonate and
K2CO3 were used as another base, the yields of 9a were not superior to
that of Cs2CO3.
26) Lipase AK-20 from Pseudomonas fluorescens, Lipase PS from
Pseudomonas cepacia, Lipase F from Pseudomonas fluorescens and
Lipase QLG from Alcaligenes sp.
Acknowledgements We thank Drs. A. Miyake, K. Itoh and S. Hashiguchi
for helpful discussions throughout this work; Drs. D. Cork and A. Hasuoka
for development of the synthesis of key intermediate 3.
References and Notes
1) Rangel-Frausto M. S., Pittet D., Costigan M., Hwang T., Davis C. S.,
Wenzel R. P., JAMA, 273, 117—123 (1995).
27) Chen C.-S., Fujimoto Y., Girdaukas G., Sih C. J., J. Am. Chem. Soc.,
104, 7294—7299 (1982).
2) Hotchkiss R. S., Karl I. E., N. Engl. J. Med., 348, 138—150 (2003).
3) Martin G. S., Mannino D. M., Eaton S., Moss M., N. Engl. J. Med.,
348, 1546—1555 (2003).
4) 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., N. Engl. J. Med., 344, 699—709 (2001).
5) Yamada M., Ichikawa T., Ii M., Sunamoto M., Ito K., Tamura N., Ki-
tazaki T., J. Med. Chem., 48, 7457—7467 (2005).
28) The enantiomeric ratio (E value) was calculated from Eꢂ
ln[(1ꢅc)(1ꢅees)]/ln[(1ꢅc)(1ꢆees)], where cꢂees/(eesꢆeep).
29) Lipase PS-D is a trade name for Pseudomonas cepacia lipase immobi-
lized on diatomaceous earth.
30) Adlercreutz P., “Enzymatic Reactions in Organic Media,” ed. by Kosk-
inen A. M. P., Klibanov A. M., Wiley-VCH, Weinheim, 1996, p. 9.
31) The melting point of compound (R)-1 obtained above was different
from that of (R)-1 in ref. 5; the crystal form of each compound was
different due to different solvents used for recrystallization.
6) Fujii A., Hashiguchi S., Uematsu N., Ikariya T., Noyori R., J. Am.
Chem. Soc., 118, 2521—2522 (1996).