Synthesis and biological activity of 1-phenylsulfonyl-4-phenylsulfonylaminopyrrolidine derivatives as thromboxane A2 receptor antagonists

Article abstract of DOI:10.1016/S0968-0896(01)00397-2

The synthesis and biological activity of novel 1-phenylsulfonyl-4- phenylsulfonylaminopyrrolidine analogues are described. All compounds were produced through modification of the substituent formally corresponding to the 1,3-dioxane ring system and the ω-octenol side chain of thromboxane A₂ (TXA₂), in reference to the structure of Daltroban. Several compounds were found to be potent TXA₂ receptor antagonists. Compound 51a was the most effective inhibitor of 9,11-epoxymethano PGH₂ (U-46619)-induced rat aortic strip contraction (IC₅₀ = 0.48 nM).

Full text of DOI:10.1016/S0968-0896(01)00397-2

Bioorganic & Medicinal Chemistry 10 (2002) 1399–1415
Synthesis and Biological Activity of 1-Phenylsulfonyl-4-
Phenylsulfonylaminopyrrolidine Derivatives as
Thromboxane A2 Receptor Antagonists
Hiroshi Marusawa, Hiroyuki Setoi, Akihiko Sawada,* Akio Kuroda, Jiro Seki,
Yukio Motoyama and Hirokazu Tanaka
Exploratory Research Laboratories, Fujisawa Pharmaceutical Co., Ltd. 5-2-3 Tokodai, Tsukuba-shi, Ibaraki 300–2698, Japan
Received 21 September 2001; accepted 12 November 2001
Abstract—The synthesis and biological activity of novel 1-phenylsulfonyl-4- phenylsulfonylaminopyrrolidine analogues are descri-
bed. All compounds were produced through modification of the substituent formally corresponding to the 1,3-dioxane ring system
and the o-octenol side chain of thromboxane A2 (TXA2), in reference to the structure of Daltroban. Several compounds were found
to be potent TXA2 receptor antagonists. Compound 51a was the most effective inhibitor of 9,11-epoxymethano PGH2 (U-46619)-
induced rat aortic stripcontraction (IC 50=0.48 nM). # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
endoperoxides and of antagonizing the effects of various
Thromboxane A2 (TXA2) 11 is recognized as an impor-
tant and potent mediator of acute life threatening dis-
orders. TXA2 displays potent platelet aggregation,2
vasoconstriction3 and bronchoconstriction4 activities,
and is a mediator that can cause problems in various
circulatory disorders5 and in asthmatic conditions.6
Efforts to modulate the actions of TXA2 have focused
on agents that inhibit the biosynthesis of TXA2 or
alternatively block the action of TXA2 at the receptor
level. An example of the former approach is cyclooxy-
genase inhibition. However, cyclooxygenase inhibitors
such as aspirin block not only TXA2 synthesis on plate-
lets, but also the synthesis of vascular prostaglandin I2
(PGI2),7 a powerful antiaggregatory and vasodilatory
substance.8 Several thromboxane synthase inhibitors,
for example Dazoxiben 2,9,10 have been introduced,
however such compounds display possible proag-
gregatory activity of prostaglandin endoperoxides
accumulating after blockage of thromboxane synthase,
which are believed to interact at a common receptor11
for TXA2. Blocking the action of TXA2 at the receptor
level is TXA2 receptor antagonism. TXA2 receptor
antagonists present the advantage12 of inhibiting the
proaggregatory action of both TXA2 and prostaglandin
agonists on smooth muscle cells, thus TXA2 receptor
antagonists are becoming an established method for
treating asthma and various cardiovascular diseases in
the clinic.12À15
Several drugs and development candidates with prosta-
noid structures such as SQ29548 3,16,17 Domitroban
4,18,19 and with non-prostanoid structures, for example
sulfonamide derivatives such as Daltroban 5,20,21 Sera-
trodast 622,23 and Ramatroban 7,24,25 have been found
to be selective TXA2 receptor antagonists.
We recently reported synthesis and biological activity of
4-methyl-3,5-dioxane derivatives as thromboxane A2
receptor antagonists. Among these compounds, sodium
(Z)-7-{(1S,2R,4R) - 2 - [2 - aza - 2 - (((phenyamino)thioxo-
methyl)amino)vinyl]-4-methyl-3,5-dioxanyl}hept-5-eno-
ate was the most potent in vitro antagonist,26 but there
was a need to improve the in vivo duration of action. In
order to achieve this, we next aimed to discover a new
chemical skeleton with a non-prostanoid structure and
focused on the conformational similarities of TXA2 and
Daltroban.
Inspection for the conformational similarities between
their structures with an accurate 3-D molecular model
suggested that the sulfonamides, exemplified by Dal-
troban, might interact with the thromboxane receptor in
*Corresponding author. Fax: +81-3-5256-5370;
e-mail: hiroshi-marusawa@po.fujisawa.co.jp
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00397-2

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H. Marusawa et al. / Bioorg. Med. Chem. 10 (2002) 1399–1415
one of two ways, as shown in Scheme 1. In the first
model, the oxygen atoms of the sulfonamide might
mimic the two oxygen atoms in the 1,3-dioxane ring
system of TXA2. In this conformation, Daltroban could
be viewed as lacking an o-octenol side chain. Alter-
natively, in the second model the sulfonamide might
mimic the allylic alcohol of TXA2. In this case, the
conformation of Daltroban would indicate a structure
without the nuclear interaction of the 1,3-dioxane ring
system. These observations suggested that the sulfona-
mide moiety of Daltroban might tentatively play a role
as mimics of both the nuclear oxygen atoms and the
allylic alcohol. On the basis of this prediction, we
designed a new scaffold with two sulfonamide groups in
the same molecule. After examining various possibilities
for a new ring system and its stereochemistry for the
sulfonamide groups in reference to the conformation
and the distance between the nuclear oxygen atoms and
allylic alcohol in TXA2, amino pyrrolidine compound
18a was finally chosen as a lead. Herein, we report a
series of new pyrrolidine compounds as highly potent
and long-acting TXA2 receptor antagonists.
For comparison of the biological activity, 1-alkylsulfo-
nyl 35 and 1-carbamoyl 36 derivatives were prepared
from 8 as follows. Sulfonylation of 17, prepared by the
method showed in Scheme 2, with n-butylsulfo-
nylchloride and triethylamine in CH2Cl2 followed by
treatment with NaOH in MeOH gave 35. On the other
hand, 36 was obtained by carbamoylation of 15 with
phenylisocyanate and Et3N in CH2Cl2 followed by
treatment with NaOH in MeOH. Various bis-sulfonyl
compounds 37a–g and 50a–55 were prepared in a simi-
lar manner to that for the synthesis of 18a, as described
in Schemes 4 and 5.
Furthermore, in order to investigate the effect of the
side chain at 2-position, different analogues 58a–58d
were synthesized from 42 as shown in Scheme 6. The
aldehyde 42 was prepared from 8 and was subjected to
Wittig reaction with an appropriate ylide. In addition,
the saturated form 55 was also obtained by catalytic
hydrogenation of the mixture of 51a and 51b.
Results and Discussion
In reference to the possible binding conformations
shown in Scheme 1, we first assumed that a molecule
with two sulfonamides moiety might mimic both the
nuclear oxygen atoms and the allylic alcohol, and may
possess more potent TXA2 receptor antagonistic activity
Chemistry
All possible stereoisomers were synthesized, as shown in
Schemes 2 and 3, in order to find the appropriate con-
figurations at the 2- and 4-positions in the pyrrolidine
ring.
Table 1. In vitro activity on platelet aggregation
Commercially available trans-4-hydroxy-l-proline 8 was
esterified with SOCl2 in MeOH, treated with di-tert-
butyldicarbonate, then mesylated at the free hydroxy
groupto afford 9. SN2 substitution of the mesylate
groupof 9 with benzoate in DMSO followed by deben-
zoylation of 10 using K2CO3 in MeOH, gave alcohol 11.
Alcohol 11 was mesylated again to afford 12. The mesyl
groupof 12 was substituted by an azide groupin
DMSO with inversion of configuration at the 4-position
to give 13. Catalytic hydrogenation of the azide group
in 13 in MeOH, followed by acylation with PhSO2Cl
gave sulfonamide 14. Aldehyde 15, obtained from 14 by
DIBAL reduction, was subjected to a Wittig reaction
with the ylide prepared from (4-carboxybutyl)-
triphenylphosphonium bromide and t-BuOK in di-
methylacetamide and afforded the desired acid 16 as the
major product together with approximately 5% of the
trans isomer. The acid 16 was then treated with aqueous
TFA to remove the Boc groupto yield 17. Sulfonylation
of the nitrogen atom in the pyrrolidine ring with
PhSO2Cl and Et3N in CH2Cl2 followed by treatment
with NaOH in MeOH afforded the desired sodium salt
18a. Stereoisomer 18b was obtained in a similar manner
to the methods shown above, starting from azide 19,
which was obtained from 9 by inversion of configura-
tion of the position 4 of the pyrrolidine ring. Each SN2
reaction employed for the preparation of 18a and 18b
occurred stereospecifically, as confirmed by the obser-
vation of no diastereoisomers by 200 MHz NMR. The
other two isomers 18c and 18d were prepared starting
from commercially available cis-4-hydroxy-d-proline 24
using a related strategy, as shown in Scheme 3.
Compound
Inhibition of rabbit platelet aggregation (IC50, mMa)
9,11-azo PGH2
ADP
18a
18b
18c
18d
0.57
100
2.3
>100
>100
>100
>100
100
35
16
2.4
30
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
36
29
37a
37b
37c
37d
37e
37f
37g
0.17
0.26
0.36
0.66
0.34
0.23
0.16
50a
50b
51a
51b
52
53
54
55
0.13
0.68
0.055
0.056
0.14
0.12
0.12
0.23
>100
>100
>100
>100
>100
>100
>100
58a
58b
58c
58d
0.032
0.37
0.31
3.4
>100
>100
>100
>100
Daltroban
0.16
>100
aIC50 value were calculated by regression analysis from three dose
groups, using three different preparations.

H. Marusawa et al. / Bioorg. Med. Chem. 10 (2002) 1399–1415
1401
Scheme 1.
Scheme 2. (a) SOCl2, MeOH; (b) Boc2O, Et3N, CH2Cl2; (c) MsCl, Et3N, CH2Cl2; (d) BzONa, DMSO; (e) K2CO3, MeOH; (f) NaN3, DMSO; (g)
H2-Pd/C, MeOH; (h) PhSO2Cl, Et3N, CH2Cl2; (i) DIBAL, toluene, À78 ꢀC; (j) HOOC(CH2)4P+Ph3BrÀ, t-BuOK, DMAC; (k) 75% TFA; (l) 1N-
NaOH, MeOH; (m) DIBAL, toluene, À25 ꢀC; (n) CrO3, pyridine, CH2Cl2.

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H. Marusawa et al. / Bioorg. Med. Chem. 10 (2002) 1399–1415
Scheme 3. (a) SOCl2, MeOH; (b) PhSO2Cl, Et3N, CH2Cl2; (c) MsCl, Et3N, CH2Cl2; (d) BzONa, DMSO; (e) K2CO3, MeOH; (f) NaN3, DMSO;
(g) H2-Pd/C, MeOH; (h) DIBAL, toluene, À78 ꢀC; (i) HOOC(CH2)4P+Ph3BrÀ, t-BuOK, DMAC; (j) 1N-NaOH, MeOH.
Scheme 4.
than Daltroban. We attempted to design a new com-
pound with a ring system in reference to the conforma-
tion and the distance between the nuclear oxygen
atoms and allylic alcohol in TXA2. As a result, we
designed the 2-alkenyl-4-aminopyrrolidine skeleton
which has two centers of asymmetry at the 2- and 4-
positions of the pyrrolidine ring system. Since we can-
not predict which stereoisomer would be the most

H. Marusawa et al. / Bioorg. Med. Chem. 10 (2002) 1399–1415
1403
Scheme 5. (a) H2-Pd/C, MeOH; (b) p-XPhSO2Cl, Et3N, CH2Cl2; (c) DIBAL, CH2Cl2; (d) HOOC(CH2)4P+Ph3BrÀ, t-BuOK, DMAC; (e) 75%
TFA; (f) p-ClPhSO2Cl, Et3N, CH2Cl2.
Scheme 6. (a) Ph3P+ClÀCH2PhÀ(m- or p-)COOMe, NaH, THF; (b) 90% TFA; (c) p-Cl–PhSO2Cl, Et3N, CH2Cl2; (d) 1N-NaOH, MeOH.
potent TXA2 receptor antagonist, even in reference to
the proposed common spatial pharmacophore27 for
TXA2 receptor antagonists, we first synthesized all four
stereoisomers (18a–18d) as shown in Scheme 2 and 3.
2- and 4-positions of the pyrrolidine ring as 2S and 4R,
respectively, and first synthesized derivatives (16, 35, 36)
with different 1-position substituents on the pyrrolidine
ring. As shown in Table 1, the sulfonamide derivative is
the most potent and demonstrates that our hypothesis
that a molecule with two sulfonamide moieties on a
2-alkenyl-4-aminopyrrolidine skeleton may possess
potent TXA2 receptor antagonistic activity is reasonable.
The target compounds described in Schemes 2 and 3
were evaluated for their ability to inhibit platelet aggre-
gation of rabbit platelet-rich plasma (PRP) induced by
28
9,11-azo PGH2 (5 mL, final concentration 1.0 mM) or
adenosine diphosphate (ADP, 5 mL, final concentration
2.5 mM). The concentrations (IC50) that caused 50%
inhibition of platelet aggregation induced by 9,11-azo
PGH2 are shown in Table 1. Consistent with their
mechanism of action, none of the compounds was
effective in inhibiting ADP induced platelet aggregation.
Since 18a demonstrated more potent TXA2 receptor
antagonistic activity than 35, we investigated the sub-
stituent groupeffect in the para position of the 1-phe-
nylsulfonamide moiety in 18a in reference to Daltroban,
as shown in Table 1. All compounds (37a–37g) showed
TXA2 receptor antagonistic activity, but 37a (p-chloro)
and 37g (p-nitro) were clearly the most potent. Taking
the other biological activities of 37a and 37g into con-
sideration, we fixed the 1-phenylsulfonamide moiety as
p-chloro and investigated the substituent group effect in
the para position of the 4-phenylsulfonamide moiety as
We confirmed that both 18a and 18c displayed TXA2
receptor antagonistic activity and that the 4R form is
essential and 2S form is preferable for antagonistic
activity. Therefore, we fixed the stereochemistry at the

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H. Marusawa et al. / Bioorg. Med. Chem. 10 (2002) 1399–1415
Table 2. Potencies of 51a for inhibition of [3H]-U-46619 binding to
washed guinea-pig platelets
Table 4. Inhibition of ex vivo platelet aggregation by oral adminis-
tration of 51a in guinea-pig
Compound
51a
mg/kg
po
1 h (%)a
6 h (%)a
IC50
1.7Â10À9
M
a
U-46619
Collagen
U-46619
Collagen
aIC50 value were calculated by regression analysis from five dose
groups, using three different preparations.
0.032
0.1
0.32
21
97**
100**
10
51*
96**
NTb
NTb
NTb
NTb
100**
95**
a
*p <0.05 and **p<0.01, compared with control group. Data repre-
sents the average percent of inhibition in a group of five animals.
Table 3. In vitro activities of 51a in various species
Species
Agonist
IC50, Ma
5.8Â10À8
Table 5. Effects of 51a in the arachidonic acid-induced pulmonary
infarction mice model
Human PRP
U-46619
Collagen
3.6Â10À8
4.5Â10À8
3.2Â10À9
Compound
51a
Monkey PRP
Dog PRP
U-46619
Collagen
Dose (mg/kg, po)
Survival rate (%)a
0
0
0.32
20
1.0
70**
3.2
80**
Guinea-pig PRP
U-46619
Collagen
1.8Â10À9
a
**p <0.01, compared with control group. Data represent the survi-
val percent in a group of 10 mice.
1.5Â10À9
Rat aortic strips
U-46619
4.8Â10À10
Table 6. Effects of 51a on thrombus formation in extracorporeal
shunt in rat
aIC50 values were calculated by regression analysis from three dose
groups, using four different preparations.
Compound
51a
shown in Table 1. All compounds (50a–55) showed
TXA2 receptor antagonistic activity, but 51a (p-chloro)
was the most potent among them.
Dose (mg/kg, po)
Inhibition (%)a
0.01
19.5
0.032
34.8**
0.1
49.7***
0.32
63.4***
a
**p <0.01 and ***p <0.005, compared with control group. Data
represent the average percent of inhibition in a group of 10 animals.
Finally, we fixed the two phenylsulfonamide moieties as
p-chloro and then modified the nature of the substituent
formally corresponding to the a-heptenoic acid chain.
We first evaluated the potency of cis- and trans- forms
(51a and 51b), respectively, and synthesized the satu-
rated form 55. The activity of 51a was almost equal to
51b, whilst the saturated form 55 was less potent than
the unsaturated forms. Next, we examined benzoic acid
derivatives as a means to fix the conformation of the
carboxylic acid. As shown in Table 1, 58a had the most
potent TXA2 receptor antagonist activity among all
compounds, whilst the other compounds (58b–58d) had
decreased potency. These results demonstrated that
there is a region which recognizes a double bond in the
TXA2 receptor and that the conformational relationship
between the two phenylsulfonamide moieties and the
carboxylic acid in 58a is the most suitable for binding to
the TXA2 receptor.
aortic stripcontraction in various species. As shown in
Table 3, 51a displayed highly specific inhibitory activity,
especially against rat aortic strip contraction
(IC50=4.8Â10À10 M). In addition, we evaluated inhibi-
tory activity against ex vivo platelet aggregation
induced by U-46619 or collagen, following oral admin-
istration of 51a to guinea-pigs, and confirmed the
potent activity, as shown in Table 4. Especially for 0.32
mg/kg p.o. administration, the inhibitory activity after
6 h is the same as that after 1 h and we confirmed the
stability of 51a in vivo. Furthermore, compound 51a
was effective in the arachidonic acid- induced pulmo-
nary infarction mouse model which is well known for
evaluating TXA2 receptor antagonists and improved the
mortality rate apparently (Table 5). In addition, com-
pound 51a also prevents thrombus formation in the
extracorporeal shunt rat model (Table 6). The in vivo
stability of the 1-phenylsulfonyl-4-phenylsulfonylamino-
pyrrolidine skeleton was also demonstrated through
these oral administration experiments.
From these studies, we discovered that 1-phenylsulfo-
nyl-4-phenylsulfonylaminopyrrolidine derivatives based
on the structure of Daltroban possess high TXA2
receptor antagonistic activity and finally selected com-
pound 51a as a potential drug candidate from in vivo
experiments. We examined the biological activities of
51a as shown in Tables 2–6. In a radioligand binding
assay with guinea-pig platelets, we confirmed 51a
inhibited the binding of U-46619,29 which possesses
almost the same agonistic activity30 as 9,11-azo PGH2,
with an IC50 value of 1.7Â10À9 M and demonstrated
high affinity for the TXA2 receptor (Table 2).
Conclusion
We designed a series of1-phenylsulfonyl-4-phenylsulfo-
nylaminopyrrolidines as potent TXA2 receptor antago-
nists, and investigated stereochemistry and various
modifications of the substituent groups on the two phe-
nylsulfonylamide moieties. Through evaluation of
these derivatives, compound 51a was found to be the
most effective inhibitor of 9,11-epoxymethano PGH2
We next evaluated in vitro inhibitory activities against
U-46619- and collagen-induced platelet aggregation and

H. Marusawa et al. / Bioorg. Med. Chem. 10 (2002) 1399–1415
1405
(U-46619)-induced rat aortic stripcontraction
(IC50=4.8Â10À10 M). Further studies on optimizing
this activity will be the subject of subsequent papers.
1-tert-Butyl 2-methyl (2S,4S)-4-((benzoyl)oxy)-1,2-pyr-
rolidinedicarboxylate (10). A mixture of 9 (32.3 g, 100
mmol) and sodium benzoate (28.8 g, 200 mmol) in
dimethylsulfoxide (320 mL) was stirred at 90 ꢀC over-
night and cooled to room temperature. The mixture was
diluted with ethyl acetate (600 mL) and washed succes-
sively with water and brine. The organic phase was
dried over magnesium sulfate and the solvent was eva-
porated in vacuo to give an oil. The oil was crystallized
from n-hexane to give 31.0g (88.7%) of 10 as colorless
Experimental
Proton nuclear magnetic resonance (1H NMR) spectra
were recorded on a Bruker AM-200 Spectrometer using
tetramethylsilane (TMS) or 3-(trimethylsilyl)propionic
acid-d4 sodium salt (TSP-d4) as an internal reference.
The ESI mass spectra (MS) were recorded on a Plate-
form LC-MS Spectrometer (Micromass) and the ESI-
TOF mass spectra were recorded on a Micromass LCT
Mass Spectrometer (Micromass). The optical rotations
([a]2D0) were recorded on a Jasco automatic photoelectric
polarimeter model p-1020. Column chromatography
was carried out on Silica gel 60 (E. Merck, 70–200
mesh).
crystals: mp89–90 ꢀC. H NMR (200 MHz, CDCl3) d
1
1.45 (4.5H, s), 1.48 (4.5H, s), 2.4–2.7 (2H, m), 3.68
(1.5H, s), 3.69 (1.5H, s), 3.69 (1H, m), 3.82 (1H, m), 4.48
(0.5H, dd, J =2, 11 Hz), 4.61 (0.5H, dd, J =4, 11 Hz),
5.53 (1H, m), 7.43 (1H, t, J =7.5 Hz), 7.57 (2H, t, J
=7.5 Hz), 7.98 (2H, d, J =7.5 Hz). ESI-MS m/z 350
(M+H)+. ESITOF-MS m/z 372.1513 (M+Na)+, calcd
for C18H23NO6Na 372.1423.
1-tert-Butyl 2-methyl (2S,4S)-4-hydroxy-1,2-pyrrolidine-
dicarboxylate (11). To a solution of 10 (30 g, 85.9
mmol) in methanol (600 mL) was added potassium car-
bonate (11.9 g, 86.1 mmol) and the mixture was stirred
at room temperature for 1 h. The solution was diluted
with ethyl acetate (1 L) and washed with water. The
organic phase was washed with brine. The aqueous
phase was saturated with sodium chloride, extracted
with chloroform and washed with brine. The combined
organic extracts were dried over magnesium sulfate and
evaporated in vacuo to give an oil. The oil was chro-
matographed on a silica gel (500 g) column with a mix-
ture of n-hexane and ethyl acetate (1:1) as an eluent to
give 20.7g (98.4%) of 11 as colorless crystals: mp59–
1-tert-Butyl 2-methyl (2S,4R)-4-((methylsulfonyl)oxy)-
1,2-pyrrolidinedicarboxylate (9). Methanol (175 mL)
was cooled in a dry ice-acetone bath and to the solution
was added dropwise thionyl chloride (21.4 mL, 293
mmol) over 30 min. After stirring at the same tempera-
ture, to the solution was added portionwise 8 (35.0 g,
267 mmol) and the dry ice–acetone bath was removed.
The reaction mixture warmed to room temperature
then refluxed gently for 6 h. The solvent was evapo-
rated in vacuo and the residue was filtrated and
washed with ether to give 2-methyl (2S,4R)-4-
hydroxy-1,2-pyrrolidinecarboxylate hydrochloride that
was dissolved in dichloromethane (350 mL). The solu-
tion was cooled in an ice bath then triethylamine (75
mL, 538 mmol) and di-tert-butyl dicarbonate (61.4 g,
267 mmol) were added. The reaction mixture was stirred
at the same temperature for 4 h. The mixture was
washed with diluted hydrochloric acid, water, saturated
aqueous sodium hydrogenecarbonate and brine, and
dried over magnesium sulfate. The solvent was evapo-
rated in vacuo and the residue was filtered and washed
with n-hexane to give 53.4 g (81.5% from 8) of 1-tert-
butyl 2-methyl (2S,4R)-4-hydroxy-1,2-pyrrolidinedi-
carboxylate that was dissolved in dichloromethane
(500 mL). The solution was cooled in an ice bath
then triethylamine (36 mL, 258 mmol) and methane-
sulfonyl chloride (20.7 mL, 257 mmol) were added
and the reaction mixture was stirred at the same tem-
perature for 3 h. The solution was washed successively
with diluted hydrochloric acid, saturated aqueous
sodium hydrogenecarbonate and brine, and dried
over magnesium sulfate. The solvent was evaporated
in vacuo and the residue was crystallized from n-
hexane to give 56.2 g (65.0% from 8) of 9 as colorless
62 ꢀC. H NMR (200 MHz, CDCl3) d 1.45 (5.4H, s),
1
1.47 (3.6H, s), 2.10 (1H, m), 2.33 (1H, m), 3.5–3.7 (3H,
m), 3.78 (1.8H, s), 3.80 (1.2H, s), 4.35 (1H, m). ESI-MS
m/z 246 (M+H)+. ESITOF-MS m/z 268.1123
(M+Na)+, calcd for C11H19NO5Na 268.1161.
1-tert-Butyl 2-methyl (2S,4S)-4-((methylsulfonyl)oxy)-
1,2-pyrrolidinedicarboxylate (12). To a solution of 11
(20.0 g, 81.5 mmol) in dichloromethane (500 mL) were
added triethylamine (13.5 mL, 96.9 mmol) and methane-
sulfonyl chloride (7.4 mL, 95.6 mmol) with stirring in an
ice bath and the mixture was stirred at the same tem-
prature for 4 h. The solution was washed successively
with diluted hydrochloric acid, saturated aqueous
sodium hydrogencarbonate and brine, and dried over
magnesium sulfate. The solvent was evaporated in
vacuo to give 26.4 g (100%) of 12 as a pale brown oil:
1H NMR (200 MHz, CDCl3) d 1.43 (5.4H, s), 1.46
(3.6H, s), 2.53 (2H, m), 3.03 (3H, s), 3.76 (3H, s), 3.80
(2H, m), 4.4–4.6 (1H, m), 5.75 (1H, m). ESI-MS m/z 324
(M+H)+.
1-tert-Butyl 2-methyl (2S,4R)-4-azido-1,2-pyrrolidine-
dicarboxylate (13). A mixture of 12 (26.4 g, 81.5 mmol)
and sodium azide (10.6 g, 163 mmol) in dimethyl sulf-
oxide (350 mL) was stirred at 90 ꢀC overnight and the
solution was diluted with ethyl acetate (600 mL). The
solution was washed successively with water and brine.
The solution was dried over magnesium sulfate and the
solvent was evaporated in vacuo to give 20.0 g (90.8%)
crystals: mp73–75 ꢀC. H NMR (200 MHz, CDCl3) d
1
1.43 (6H, s), 1.47 (3H, s), 2.28 (1H, ddd, J =5, 8, 14Hz),
2.63 (1H, m), 3.05 (3H, s), 3.7–3.9 (2H, m), 3.77 (3H, s),
4.41 (0.67H, t, J=8Hz), 4.48 (0.33H, t, J=8Hz), 5.28
(1H, m). ESI-MS m/z 324 (M+H)+. ESITOF-MS m/z
346.0955 (M+Na)+, calcd for C12H21 NO7SNa
346.0936.

1406
H. Marusawa et al. / Bioorg. Med. Chem. 10 (2002) 1399–1415
of 13 as a pale brown oil: 1H NMR (200 MHz, CDCl3) d
1.41 (6H, s), 1.47 (3H, s), 2.20 (1H, m), 2.32 (1H, m),
3.4–3.7 (3H, m), 3.76 (3H, s), 4.20 (1H, m), 4.36 (1H,
m). ESI-MS m/z 271 (M+H)+.
was added water and the aqueous solution was washed
with ethyl acetate. The aqueous phase was adjusted to
pH 2 with 1 N hydrochloric acid and extracted with
ethyl acetate. The organic phase was washed succes-
sively with water and brine, and dried over magnesium
sulfate. The solvent was evaporated in vacuo and the
residue was chromatographed on a silica gel column
with a mixture of chloroform and methanol (25:1) as
eluent to give 1.70 g (27.6%) of 16 as a pale yellow oil:
1H NMR (200 MHz, CDCl3) d 1.36 (9H, s), 1.6–1.8 (3H,
m), 2.0–2.2 (3H, m), 2.3–2.4 (2H, m), 3.2–3.5 (2H, m),
3.85 (1H, m), 4.57 (1H, m), 5.1–5.5 (2H, m), 5.72 (1H,
broad), 7.3–7.4 (2H, m), 7.4–7.6 (3H, m). ESI-MS m/z
437 (MÀH)À.
1-tert-Butyl 2-methyl (2S,4R)-4-((phenylsulfonyl)amino)-
1,2-pyrrolidinedicarboxylate (14). A solution of 13 (19.5
g, 72.1 mmol) in methanol (150 mL) was shaken under
hydrogen (3 atm) with 10% palladium on carbon (3.0g)
at room temperature for 3 h. After removal of the cata-
lyst by filtration, the solvent was evaporated in vacuo to
give 1-tert-butyl 2-methyl (2S,4R)-4-amino-1,2-pyrroli-
dinedicarboxylate that was dissolved in pyridine (150
mL). The solution was cooled in an ice bath then ben-
zenesulfonyl chloride (11.0 mL, 86.2 mmol) added. The
reaction mixture was stirred at the same temperature for
5 h. The solvent was evaporated in vacuo and the resi-
dure was dissolved in ethyl acetate (300 mL). The solu-
tion was washed successively with water, diluted
hydrochloric acid, saturated aqueous sodium hydro-
gencarbonate and brine, and dried over magnesium
sulfate. The solvent was evaporated in vacuo and the
residue was chromatographed on a silica gel column
with a mixture of n-hexane and ethyl acetate (1:1) as
eluent to give 22.5 g (81.2% from 13) of 14 as colorless
(5E and 5Z)-6-((2S,4R)-4-((Phenylsulfonyl)amino)pyrro-
lidinyl)-5-hexenoic acid trifluoroacetate (17). A solution
of 16 (1.6 g, 3.65 mmol) in 75% aqueous trifluoroacetic
acid (10 mL, 97.4 mmol) was stirred at room tempera-
ture for 1 h and the solvent was evaporated in vacuo. To
the residue was added toluene (10 mL) and the solvent
was evaporated in vacuo to give 1.65 g of 17 (100%) as
a brown oil: 1H NMR (200MHz, D2O) d 1.7–1.9 (4H, m),
2.1–2.3 (2H, m), 2.3–2.4 (2H, m), 3.3–3.6 (2H, m), 4.07
(1H, m), 4.46 (1H, m), 4.80 (1H, m), 5.80 (1H, m), 7.5–7.7
(3H, m), 7.8–7.9 (2H, m). ESI-MS m/z 337 (MÀH)À.
ꢀ
crystals: mp110–111 C. 1H NMR (200 MHz, CDCl3) d
1.40 (9H, s), 2.0–2.3 (2H, m), 3.17 (1H, dd, J =5, 11
Hz), 3.59 (1H, m), 3.98 (1H, m), 4.30 (1H, m), 4.82 (1H,
m), 7.5–7.7 (3H, m), 8.8–8.9 (2H, m). ESI-MS m/z 385
(M+H)+. ESITOF-MS m/z 385.1423 (M+H)+, calcd
for C17H25N2O6S 385.1433.
Sodium (5E and 5Z)-6-((2S,4R)-1-(phenylsulfonyl)-4-
((phenylsulfonyl)amino) - 2 - pyrrolidinyl) - 5 - hexenoate
(18a). To a solution of 17 (322 mg, 0.712 mmol) in di-
chloromethane (5.0 mL) was added triethylamine (0.44
mL, 3.15 mmol) and benzenesulfonyl chloride (126 mg,
0.713 mmol) and the mixture was stirred at room tem-
perature for 2 h. The solution was washed successively
with diluted hydrochloric acid and brine, and dried over
magnesium sulfate. The solvent was evaporated in
vacuo and the residue was chromatographed on a silica
gel column with a mixture of chloroform and methanol
(40:1) as eluent to give 100 mg of (5E and 5Z)-6-
((2S,4R)-1-(phenylsulfonyl)-4-((phenylsulfonyl)amino)-
pyrrolidinyl)-5-hexenoic acid that was dissolved in a
mixture of 1 N sodium hydroxide (0.2 mL, 0.2 mmol)
and water (20 mL). The solution was stirred at room
temperature for 30min and washed with dichloro-
methane. The aqueous phase was applied to a column
of Diaion HP-20 (trademark, sold by Mitsubishi
Chemical Industries Ltd.). The column was washed with
water and the object compound was eluted with a mix-
ture of water and methanol (1:1). The organic solvent
was removed in vacuo and the aqueous residue was
lypholyzed to give 94 mg (26.5% from 17) of 18a as a
1-tert-Butyl (2S,4R)-2-formyl-4-((phenylsulfonyl)amino)-
1,2-pyrrolidinecarboxylate (15). To a solution 14 (10.0
g, 26.0 mmol) in toluene (70 mL) was added dropwise
1.0 M solution of diisobutylaluminum hydride in tolu-
ene (80 mL, 80.0 mmol) at À78 ꢀC. After the resulting
mixture was stirred at the same temperature for 5 h,
saturated aqueous potassium sodium tartrate was added
to the reaction mixture and the mixture was filtered
through Celite. The solid was washed with ethyl acetate
and the combined organic solution was washed with
brine and dried over magnesium sulfate. The solvent
was evaporated in vacuo and the residue was chroma-
tographed on a silica gel column with a mixture of
n-hexane and ethyl acetate (2:1–1:2) as eluent to give
1
8.75 g (95.0%) of 15 as a pale yellow oil: H NMR
(200 MHz, CDCl3) d 1.34 (5.4H, s), 1.37 (3.6H, s), 1.94
(2H, m), 3.17 (1H, m), 3.38 (1H, m), 3.67 (1H, m), 4.15
(1H, m), 7.6–7.7 (3H, m), 7.8–7.9 (2H, m), 8.12 (1H,
broad), 9.36 (1H, broad). ESI-MS m/z 355 (M+H)+.
1
pale yellow powder: H NMR (200 MHz, D2O) d 1.5–
(5E and 5Z)-6-((2S,4R)-1-(tert-Butoxycarbonyl)-4-((phe-
nylsulfonyl)amino)pyrrolidinyl)-5-hexenoic acid (16). To
a solution of (4-carboxybutyl)triphenylphosphonium
bromide (18.8 g, 42.4 mmol) in dimethyl sulfoxide (50
mL) was added sodium methylsulfinylmethide [84.8
mmol, prepared from sodium hydride 3.39 g, 60% in
oil, 84.8 mmol) and dimethyl sulfoxide (50 mL)] and the
solution was stirred at room temperature for 30 min. To
the resulting solution was added 15 (5.0 g, 14.1 mmol) in
dimethyl sulfoxide (50 mL) and the mixture was stirred
at room temperature for 6 h. To the reaction mixture
1.7 (2H, m), 1.7–1.8 (2H, m), 1.9–2.1 (2H, m), 2.15 (2H,
m), 3.33 (1H, m), 3.57 (1H, m), 3.70 (1H, m), 4.38 (1H,
m), 5.3–5.6 (2H, m), 7.5–7.9 (10H, m). ESI-MS m/z 477
(MÀH)À.
1-tert-Butyl 2-methyl (2S,4S)-4-azido-1,2-pyrrolidine-
dicarboxylate (19). By the procedure used for 13, 9
(16.2 g, 50 mmol) and sodium azide (6.5 g, 100 mmol)
1
gave 14.3g (100%) of 19 as a pale brown oil: H NMR
(200 MHz, CDCl3) d 1.43 (5.4H, s), 1.48 (3.6H, s), 2.14
(0.4H, t, J =4 Hz), 2.21 (0.6H, t, J =4 Hz), 2.47 (1H,

H. Marusawa et al. / Bioorg. Med. Chem. 10 (2002) 1399–1415
1407
m), 3.50 (1H, m), 3.73 (1H, m), 3.76 (3H, s), 4.14 (1H,
m), 4.33 (0.6H, dd, J =4, 9 Hz), 4.43 (0.4H, dd, J =4, 9
Hz). ESI-MS m/z 271 (M+H)+.
trifluoroacetate. To a solution of (5E and 5Z)-6-
((2S,4R)-4-((phenylsulfonyl)amino)pyrrolidinyl)-5-hex-
enoic acid trifluoroacetate (300 mg, 0.663 mmol) in di-
chloromethane (10 mL) was added triethylamine (0.5
mL, 3.59 mmol) and benzenesulfonyl chloride (0.1 mL,
0.784 mmol) and the mixture was stirred at room tem-
perature for 6 h. The solution was washed successively
with diluted hydrochloric acid and brine, and dried over
magnesium sulfate. The solvent was evaporated in vacuo
and the residue was chromatographed on a silica gel
column with a mixture of chloroform and methanol
(30:1) as eluent to give 114 mg (35.9% from 22) of 23 as
an oil: 1H NMR (200 MHz, D2O) d 1.5–1.9 (3H, m), 2.0–
2.3 (3H, m), 2.39 (2H, t), 3.1–3.2 (1H, m), 3.4–3.6 (1H,
m), 4.32 (1H, m), 4.89 (1H, m), 5.3–5.5 (2H, m), 7.5–7.7
(6H, m), 7.7–7.9 (4H, m). ESI-MS m/z 477 (MÀH)À.
1-tert-Butyl 2-methyl (2S,4S)-4-((phenylsulfonyl)amino)-
1,2-pyrrolidinedicarboxylate (20). By the procedure
used for 14, 19 (14.0 g, 51.8 mmol), 10% palladium on
carbon (1.4 g) and benzenesulfonyl chloride (6.61 mL,
1
51.8 mmol) gave 10.55 g (53.0%) of 20 as an oil: H
NMR (200 MHz, CDCl3) d 1.37 (9H, s), 1.7–1.9 (1H,
m), 2.81 (1H, m), 3.3–3.5 (2H, m), 3.74 (3H, s), 4.03
(1H, m), 4.20 (1H, m), 5.93 (1H, broad), 7.5–7.6 (3H,
m), 7.8–7.9 (2H, m). ESI-MS m/z 385 (M+H)+.
1-tert-Butyl (2S,4S)-2-hydroxymethyl-4-((phenylsulfonyl)-
amino)-1,2-pyrrolidinecarboxylate (21). To a solution 20
(10.0 g, 26.0 mmol) in toluene (70 mL) was added
dropwise 1.0 M solution of diisobutylaluminum hydride
in toluene (70 mL, 70.0 mmol) at À25 ꢀC and the
resulting mixture was stirred at the same temperature
for 4 h. After saturated aqueous ammonium chloride
was added to the reaction mixture and the mixture was
stirred at room temperature for 1 h. The mixture was
filtered and filtrate was extracted with ethyl acetate. The
organic phase was washed with brine and dried over
magnesium sulfate. The solvent was evaporated in vacuo
Sodium (5E and 5Z)-6-((2S,4S)-1-(phenylsulfonyl)-4-
((phenylsulfonyl)amino) - 2 - pyrrolidinyl) - 5 - hexenoate
(18b). By the procedure used for 18a, 23 (114 mg, 0.238
mmol) and 1 N sodium hydroxide (1 mL, 1.0 mmol)
1
gave 80 mg (67.2%) of 18b as a pale yellow powder: H
NMR (200 MHz, D2O) d 1.4–1.7 (4H, m), 2.0–2.2 (4H,
m), 2.9–3.2 (3H, m), 3.37 (1H, dd, J =6, 12 Hz), 4.28
(1H, m), 5.4–5.5 (2H, m), 7.6–7.8 (10H, m). ESI-MS m/z
477 (MÀH)À.
1
to give 9.44 g (100%) of 21 as a colorless oil: H NMR
(200MHz, CDCl3) d 1.82 (1H, m), 2.23 (1H, m), 3.24 (1H,
dd, J =3, 12 Hz), 3.47 (1H, dd, J =3.5, 12 Hz), 3.53 (1H,
m), 3.8–3.9 (2H, m), 4.03 (1H, dd, J =2.5, 11 Hz), 7.4–7.6
(3H, m), 7.8–7.9 (2H, m). ESI-MS m/z 357 (M+H)+.
2-Methyl (2R,4R)-4-((methylsulfonyl)oxy)-1-(phenylsul-
fonyl)-2-pyrrolidinecarboxylate (25). Methanol (10 mL)
was cooled in a dry ice-acetone bath and to the solution
was added dropwise thionyl chloride (1.22 mL, 16.7
mmol) over 10 min. After stirring at the same tempera-
ture, to the solution was added portionwise 24 (2.0 g,
15.2 mmol) and the dry ice-acetone bath was removed.
The reaction mixture warmed to room temperature then
refluxed gently for 6 h. The solvent was evaporated in
vacuo and the residue was filtrated and washed with
ether to give 2-methyl (2R,4R)-4-hydroxy-1,2-pyrrolidi-
necarboxylate hydrochloride that was dissolved in di-
chloromethane (20 mL). The solution was cooled in an
ice bath then triethylamine (4.22 mL, 30.4 mmol) and
benzenesulfonyl chloride (1.94 mL, 15.2 mmol) were
added. The reaction mixture was stirred at the same
temperature for 3 h. The mixture was washed with
diluted hydrochloric acid, water, saturated aqueous
sodium hydrogenecarbonate and brine, and dried over
magnesium sulfate. The solvent was evaporated in
vacuo and the residue was filtered and washed with
n-hexane to give 1-tert-butyl 2-methyl (2R,4R)-4-
hydroxy-1,2-pyrrolidinedicarboxylate that was dissolved
in dichloromethane (20 mL). The solution was cooled in
an ice bath then triethylamine (2.11 mL, 15.2 mmol)
and methanesulfonyl chloride (1.18 mL, 15.2 mmol)
were added and the reaction mixture was stirred at the
same temperature for 30 min. The solution was washed
successively with diluted hydrochloric acid, saturated
aqueous sodium hydrogenecarbonate and brine, and
dried over magnesium sulfate. The solvent was evapo-
rated in vacuo and the residue was crystallized from n-
hexane to give 5.10gꢀ (94.4% from 24) of 25 as white
crystals: mp114–116 C. 1H NMR (200 MHz, CDCl3) d
2.32 (1H, ddd, J =4.5, 9, 13.5 Hz), 2.58 (1H, m), 2.84
(3H, s), 3.79 (3H, s), 3.7–3.8 (2H, m), 4.46 (1H, t, J =12
(5E and 5Z)-6-((2S,4S)-1-(tert-Butoxycarbonyl)-4-((phe-
nylsulfonyl)amino)pyrrolidinyl)-5-hexenoic acid (22). To
a solution of pyridine (8.5 mL, 106 mmol) in dichloro-
methane (150 mL) was added chromium trioxide (5.55 g,
55.5 mmol) in an ice bath and the mixture was stirred at
room temperature for 1 h. To the solution were added
Celite and a solution of 21 (3.4 g, 9.54 mmol) in dichloro-
methane (20 mL) and the mixture was stirred at room
temperature for 1 h. After being diluted with a mixture of
n-hexane and ethyl acetate (1:1, 150 mL), the solution
was passed through silica gel and the solvent was eva-
porated in vacuo to give 3.38 g (100% from 21) of 1-
tert-butyl (2S,4S)-2-formyl-4-((phenylsulfonyl)amino)-
1,2-pyrrolidinedicarboxylate. By the procedure used for
16, 1-tert-butyl (2S,4S)-2-formyl-4-((phenylsulfonyl)a-
mino)-1,2-pyrrolidinedicarboxylate (3.38 g, 9.54 mmol),
(4-carboxybutyl)triphenylphosphonium bromide (12.7 g,
28.6 mmol), sodium hydride (2.29 g, 60% in oil, 57.3
mmol) and dimethyl sulfoxide (35 mL) gave 2.20 g
1
(52.7% from 21) of 22 as a pale brown oil: H NMR
(200 MHz, CDCl3) d 1.38 (9H, s), 1.5–1.8 (3H, m), 2.0–2.1
(3H, m), 2.3–2.4 (2H, m), 3.08 (1H, dd, J =6.5, 10.5 Hz),
3.6–3.8 (2H, m), 4.44 (1H, m), 5.2–5.5 (2H, m), 7.4–7.6
(3H, m), 7.8–7.9 (2H, m). ESI-MS m/z 437 (MÀH)À.
(5E and 5Z)-6-((2S,4S)-1-(phenylsulfonyl)-4-((phenylsul-
fonyl)amino)-2-pyrrolidinyl)-5-hexenoic acid (23). By the
procedure used for 17, 22 (264 m g, 0.663 mmol) and
75% aqueous trifluoroacetic acid (2.7 mL, 26.3 mmol)
gave 300 mg (100%) of (5E and 5Z)-6-((2S,4R)-4-
((phenylsulfonyl)amino)pyrrolidinyl) - 5 - hexenoic acid

1408
H. Marusawa et al. / Bioorg. Med. Chem. 10 (2002) 1399–1415
Hz), 5.23 (1H, m), 7.5–7.7 (3H, m), 7.9–8.0 (2H, m).
ESI-MS m/z 364 (M+H)+. ESITOF-MS m/z 386.0262
(M+Na)+, calcd for C13H17NO7S2Na 386.0344.
solution of diisobutylaluminum hydride in toluene (3 mL,
3.0 mmol), (4-carboxybutyl)triphenylphosphonium bro-
mide (566 mg, 7.08 mmol) and sodium methylsulfinyl-
methide [14.2 mmol, prepared from sodium hydride 3.39
g, 60% in oil, 14.2 mmol) and dimethyl sulfoxide (10
2-Methyl (2R,4S)-4-(benzoyloxy)-1-(phenylsulfonyl)-2-
pyrrolidinecarboxylate (26). By the procedure used for
10, 25 (2.42 g, 6.65 mmol) and sodium benzoate (1.92 g,
1
mL)] gave 207 mg (18.3%) of 31 as an oil. H NMR
(200 MHz, CDCl3) d 1.4–1.8 (3H, m), 2.0–2.2 (3H, m),
2.38 (2H, m), 3.13 (1H, m), 3.50 (2H, m), 4.30 (1H, m),
4.92 (1H, d, J =6.5 Hz), 5.3–5.5 (2H, m), 7.4–7.7 (6H,
m), 7.7–7.9 (4H, m). ESI-MS m/z 477 (MÀH)À.
1
13.3 mmol) gave 1.84 g (71.1%) of 26 as an oil: H
NMR (200 MHz, CDCl3) d 2.35 (1H, ddd, J =4, 9.5, 14
Hz), 2.52 (1H, dt, J =14, 3 Hz), 3.78 (1H, m), 3.80 (3H,
s), 3.89 (1H, dd, J =3.5, 12.5 Hz), 4.42 (1H, dd, J =8,
9.5 Hz), 5.41 (1H, m), 7.3–7.4 (5H, m), 7.5–7.6 (3H, m),
7.8–7.9 (2H, m). ESI-MS m/z 390 (M+H)+.
Sodium (5E and 5Z)-6-((2R,4R)-1-(phenylsulfonyl)-4-
((phenylsulfonyl)amino) - 2 - pyrrolidinyl) - 5 - hexenoate
(18c). By the procedure used for 18a, 31 (200 mg, 0.418
mmol) and 1 N sodium hydroxide (1 mL, 1.0 mmol)
gave 112 mg (54.5%) of 18c as a pale yellow powder: 1H
NMR (200 MHz, D2O) d 1.4–1.7 (4H, m), 2.0–2.2 (4H,
m), 2.9–3.2 (2H, m), 3.37 (1H, m), 4.28 (1H, m), 5.4–5.6
(2H, m), 7.5–7.8 (10H, m). ESI-MS m/z 477 (MÀH)À.
2-Methyl (2R,4S)-4-hydroxy-1-(phenylsulfonyl)-2-pyrro-
lidinecarboxylate (27). By the procedure used for 11, 26
(1.84 g, 4.72 mmol) and potassium carbonate (654 mg,
4.72 mmol) gave 1.11 g (82.5%) of 27 as colorless crys-
tals: mp114–117 ꢀC. 1H NMR (200 MHz, CDCl3) d
2.12 (1H, ddd, J =4.5, 9, 13.5 Hz), 2.24 (1H, m), 3.43
(1H, dt, J =11.5, 2 Hz), 3.62 (1H, dd, J =4, 11.5 Hz),
3.75 (3H, s), 4.45 (1H, t, J =9 Hz), 4.47 (1H, m), 7.5–
7.7 (3H, m), 7.9–8.0 (2H, m). ESI-MS m/z 286
(M+H)+. ESITOF-MS m/z 308.0516 (M+Na)+, calcd
for C12H15NO5SNa 308.0569.
2-Methyl (2R,4S)-4-azido-1-(phenylsulfonyl)-2-pyrrolidine-
carboxylate (32). By the procedure used for 13, 25 (2.42
g, 6.65 mmol) and sodium azide (865 mg, 13.3 mmol)
gave 1.88 g (91.0%) of 32 as pale yellow crystals: mp
97–99 ꢀC. H NMR (200 MHz, CDCl3) d 2.23 (2H, m),
1
3.47 (1H, dd, J =3, 12 Hz), 3.76 (1H, dd, J =5, 12 Hz),
3.78 (3H, s), 4.25 (1H, m), 4.35 (1H, t, J =7 Hz), 7.5–
7.7 (3H, m), 7.9–8.0 (2H, m). ESI-MS m/z 311
(M+H)+. ESITOF-MS m/z 333.0630 (M+Na)+, calcd
for C12H14N4O4SNa 333.0633.
2-Methyl (2R,4S)-4-((methylsulfonyl)oxy)-1-(phenylsul-
fonyl)-2-pyrrolidinecarboxylate (28). By the procedure
used for 12, 27 (1.09 g, 3.82 mmol), triethylamine (0.54
mL, 3.87 mmol) and methanesulfonyl chloride (0.3 mL,
3.88 mmol) gave 1.73 g (100%) of 28 as an oil: 1H NMR
(200 MHz, CDCl3) d 2.30 (1H, ddd, J =4.5, 9, 13.5 Hz),
2.58 (1H, m), 3.82 (3H, s), 3.7–3.9 (2H, m), 3.77 (3H, s),
4.45 (1H, t, J =8 Hz), 5.23 (1H, m), 7.5–7.7 (3H, m), 7.9–
8.0 (2H, m). ESI-MS m/z 364 (M+H)+. ESITOF-MS
m/z 364.0458 (M+H)+, calcd for C13H18NO7S2 364.0524.
2-Methyl (2R,4S)-1-(phenylsulfonyl)-4-((phenylsulfony-
l)amino)-2-pyrrolidinecarboxylate (33). By the proce-
dure used for 14, 32 (1.80 g, 5.80 mmol), 10% palladium
on carbon (500 mg) and benzenesulfonyl chloride (0.74
1
mL, 5.80 mmol) gave 1.02 g (41.4%) of 33 as an oil: H
NMR (200 MHz, CDCl3) d 2.1–2.2 (2H, m), 3.18 (1H,
dd, J =3, 12 Hz), 3.50 (1H, dd, J =5, 12 Hz), 3.77 (3H,
s), 3.99 (1H, m), 4.42 (1H, dd, J =5, 8 Hz), 4.75 (1H, d,
J =7 Hz), 7.5–7.7 (6H, m), 7.8–7.9 (4H, m). ESI-MS m/
z 425 (M+H)+.
2-Methyl (2R,4R)-4-azido-1-(phenylsulfonyl)-2-pyrrolidi-
necarboxylate (29). By the procedure used for 13, 28
(1.39 g, 3.82 mmol) and sodium azide (497 mg, 7.64
mmol) gave 1.15 g (97.0%) of 29 as a pale yellow oil: 1H
NMR (200 MHz, CDCl3) d 2.2–2.4 (2H, m), 3.35 (1H,
dd, J =4, 11 Hz), 3.67 (1H, dd, J =5.5, 11 Hz), 3.73
(3H, s), 4.10 (1H, m), 4.56 (1H, dd, J =4, 8.5 Hz), 7.5–
7.7 (3H, m), 7.9–8.0 (2H, m). ESI-MS m/z 311
(M+H)+. ESITOF-MS m/z 311.0841 (M+H)+, calcd
for C12H15N4O4S 311.0814.
(5E and 5Z)-6-((2R,4S)-1-(phenylsulfonyl)-4-((phenylsul-
fonyl)amino)pyrrolidinyl)-5-hexenoic acid (34). By the
procedure used for 15 and 16, 33 (1.0 g, 2.36 mmol), 1.0
M solution of diisobutylaluminum hydride in toluene
(4 mL, 4.0 mmol), (4-carboxybutyl)triphenylphos-
phonium bromide (3.14 g, 7.08 mmol) and sodium
methylsulfinylmethide [14.2 mmol, prepared from
sodium hydride 566 mg, 60% in oil, 14.2 mmol) and
dimethyl sulfoxide (10 mL)] gave 542 mg (48.0%) of 34
2-Methyl (2R,4R)-1-(phenylsulfonyl)-4-((phenylsulfonyl)-
amino)-2-pyrrolidinecarboxylate (30). By the procedure
used for 14, 29 (1.0 g, 3.22 mmol), 10% palladium on
carbon (200 mg) and benzenesulfonyl chloride (0.41
mL, 3.22 mmol) gave 1.05 g (76.8%) of 30 as white
1
as an oil. H NMR (200 MHz, CDCl3) d 1.5–1.8 (2H,
m), 1.9–2.0 (1H, m), 2.12 (1H, m), 2.2–2.4 (2H, m), 3.4–
3.5 (2H, m), 3.68 (1H, m), 4.45 (1H, m), 5.2–5.5 (2H,
m), 6.27 (1H, d, J =6 Hz), 7.4–7.6 (6H, m), 7.6–7.9 (4H,
m). ESI-MS m/z 477 (MÀH)À.
ꢀ
crystals: mp110–111 C. 1H NMR (200 MHz, CDCl3) d
1.83 (1H, m), 2.18 (1H, ddd, J =6, 10, 15 Hz), 3.2–3.3
(2H, m), 3.77 (3H, s), 4.04 (1H, m), 4.23 (1H, dd, J =2,
6 Hz), 5.97 (1H, d, J =10 Hz), 7.4-7.7 (6H, m), 7.7-7.9
(4H, m). ESI-MS m/z 425 (M+H)+. ESITOF-MS m/z
425.0797 (M+H)+, calcd for C18H21 N2O6S2 425.0763.
Sodium (5E and 5Z)-6-((2R,4S)-1-(phenylsulfonyl)-4-
((phenylsulfonyl)amino) - 2 - pyrrolidinyl) - 5 - hexenoate
(18d). By the procedure used for 18a, 34 (500 mg, 1.04
mmol) and 1 N sodium hydroxide (2 mL, 2.0 mmol)
gave 100 mg (19.1%) of 18d as a pale yellow powder: 1H
NMR (200 MHz, D2O) d 1.5–1.7 (2H, m), 1.81 (1H, m),
(5E and 5Z)-6-((2R,4R)-1-(phenylsulfonyl)-4-((phenylsul-
fonyl)amino)pyrrolidinyl)-5-hexenoic acid (31). By the
procedure used for 15 and 16, 30 (1.0 g, 2.36 mmol), 1.0 M

H. Marusawa et al. / Bioorg. Med. Chem. 10 (2002) 1399–1415
1409
2.03 (1H, m), 2.1–2.2 (2H, m), 3.40 (1H, m), 3.56 (1H,
dd, J =4.5, 11.5 Hz), 3.70 (1H, m), 4.38 (1H, m), 5.3–
5.6 (2H, m), 7.5–7.9 (10H, m). ESI-MS m/z 477
(MÀH)À.
3.47 (1H, m), 3.76 (3H, s), 4.20 (1H, m), 4.9–5.2 (2H,
m), 6.9–7.0 (2H, m), 7.3–7.4 (2H, m), 7.5–7.6 (4H, m).
ESI-MS m/z 507 (MÀH)À.
Sodium (5E and 5Z)-6-((2S,4R)-1-((4-fluorophenyl)sulfo-
nyl)-4-((phenylsulfonyl)amino)-2-pyrrolidinyl)-5-hexeno-
ate (37d). By the procedure used for 18a, 17 (995 mg,
2.20 mmol), triethylamine (0.62 mL, 4.45 mmol), 4-
fluorobenzenesulfonyl chloride (428 mg, 2.20 mmol)
and 1 N sodium hydroxide (2.42 mL, 2.42 mmol) gave
289 mg (25.4%) of 37d as a white powder: mp 94–98 ꢀC.
1H NMR (200 MHz, D2O) d 1.3–1.6 (4H, m), 1.72 (1H,
m), 1.8–2.1 (3H, m), 2.70 (1H, m), 3.22 (1H, m), 3.47
(1H, m), 4.36 (1H, m), 5.3–5.7 (2H, m), 7.3–7.4 (5H, m),
7.5–7.6 (2H, m), 7.7–7.8 (2H, m). ESI-MS m/z 495
(MÀH)À.
Sodium (5E and 5Z)-6-((2S,4R)-1-(n-butylsulfonyl)-4-
((phenylsulfonyl)amino)-2-pyrrolidinyl)-5-hexenoate (35).
By the procedure used for 18a, 17 (368 mg, 0.813
mmol), triethylamine (0.26 mL, 1.87 mmol), n-butylsul-
fonyl chloride (0.117 mL, 0.902 mmol) and 1 N sodium
hydroxide (0.91 mL, 0.91 mmol) gave 32 mg (8.2%) of
35 as a white powder: mp 96–98 ꢀC. 1H NMR
(200 MHz, D2O) d 0.78 (3H, t, J =7 Hz), 1.2–1.4 (2H,
m), 1.4–1.7 (5H, m), 1.8–2.1 (5H, m), 2.86 (1H, m), 3.03
(2H, m), 3.24 (1H, m), 3.56 (1H, m), 4.50 (1H, m), 5.2–
5.4 (2H, m), 7.4–7.5 (3H, m), 7.6–7.7 (2H, m). ESI-MS
m/z 457 (MÀH)À.
Sodium (5E and 5Z)-6-((2S,4R)-1-((4-bromophenyl)sul-
fonyl) - 4 - ((phenylsulfonyl)amino)-2-pyrrolidinyl)-5-hex-
enoate (37e). By the procedure used for 18a, 17 (990
mg, 2.20 mmol), triethylamine (0.62 mL, 4.45 mmol),
4-bromobenzenesulfonyl chloride (562 mg, 2.20 mmol)
and 1 N sodium hydroxide (2.42 mL, 2.42 mmol) gave
308 mg (24.3%) of 37e as a white powder: mp 108–
Sodium (5E and 5Z)-6-((2S,4R)-1-(anilinocarbonyl)-4-
((phenylsulfonyl)amino)-2-pyrrolidinyl)-5-hexenoate (36).
By the procedure used for 18a, 17 (226 mg, 0.50
mmol), triethylamine (0.153 mL, 1.1 mmol), phenyl iso-
cyanate (0.06 mL, 0.55 mmol) and 1 N sodium hydrox-
ide (0.55 mL, 0.55 mmol) gave 51 mg (21.3%) of 36 as a
pale yellow powder: mp 214–218 ꢀC. 1H NMR
(200 MHz, D2O) d 1.5–1.7 (2H, m), 1.78 (1H, m), 2.0–
2.2 (5H, m), 3.28 (1H, dd, J =5, 11 Hz), 3.57 (1H, dd, J
=6, 11 Hz), 3.88 (1H, m), 4.72 (1H, m), 5.3–5.6 (2H,
m), 7.1–7.3 (3H, m), 7.3–7.4 (2H, m), 7.5–7.6 (3H, m),
7.8–7.9 (2H, m). ESI-MS m/z 456 (MÀH)À.
112 ꢀC. H NMR (200 MHz, D2O) d 1.4–1.7 (3H, m),
1
1.8–2.2 (6H, m), 3.51 (1H, m), 3.64 (1H, m), 4.37 (1H,
m), 5.3–5.6 (2H, m), 7.6–7.9 (5H, m). ESI-MS m/z 556
(MÀH)À.
(5E and 5Z)-6-((2S,4R)-4-((phenylsulfonyl)amino)-1-((4-
(trifluoromethyl)phenyl)sulfonyl) - 2 - pyrrolidinyl)-5-hexe-
noic acid (37f). By the procedure used for 23, 17 (904
mg, 2.00 mmol), triethylamine (0.62 mL, 4.45 mmol),
4-(trifluoromethyl)benzenesulfonyl chloride (489 mg,
2.00 mmol) gave 215 mg (19.7%) of 37f as colorless
crystals: mp108–110 ꢀC. 1H NMR (200 MHz, D2O–
NaOD) d 1.5–1.7 (3H, m), 1.7–1.9 (2H, m), 1.9–2.2 (4H,
m), 3.34 (1H, m), 3.49 (1H, m), 4.43 (1H, m), 5.3–5.6
(2H, m), 7.3–7.5 (3H, m), 7.6–8.0 (6H, m). ESI-MS m/z
545 (MÀH)À.
Sodium (5E and 5Z)-6-((2S,4R)-1-((4-chlorophenyl)sulfo-
nyl)-4-((phenylsulfonyl)amino)-2-pyrrolidinyl)-5-hexeno-
ate (37a). By the procedure used for 18a, 17 (441 mg,
0.975 mmol), triethylamine (0.30 mL, 2.15 mmol), 4-
chlorobenzenesulfonyl chloride (211 mg, 1.00 mmol)
and 1 N sodium hydroxide (1.1 mL, 1.1 mmol) gave 90.5
mg (17.4%) of 37a as a white powder: mp 103–110 ꢀC.
1H NMR (200MHz, D2O) d 1.4–1.6 (2H, m), 1.74 (1H,
m), 1.89 (1H, m), 1.9–2.1 (2H, m), 2.12 (2H, m), 3.2–3.5
(2H, m), 3.62 (1H, m), 4.42 (1H, m), 5.1–5.3 (1H, m), 5.3–
5.6 (1H, m), 7.4–7.7 (9H, m). ESI-MS m/z 511 (MÀH)À.
(5E and 5Z)-6-((2S,4R)-1-((4-nitrophenyl)sulfonyl)-4-
((phenylsulfonyl)amino)-2-pyrrolidinyl)-5-hexenoic acid
(37g). By the procedure used for 23, 17 (998 mg, 2.20
mmol), triethylamine (0.62 mL, 4.45 mmol), 4-nitro-
benzenesulfonyl chloride (488 mg, 2.20 mmol) gave 193
Sodium (5E and 5Z)-6-((2S,4R)-1-((4-methylphenyl)sul-
fonyl)-4 - ((phenylsulfonyl)amino)-2-pyrrolidinyl)-5-hexeno-
ate (37b). By the procedure used for 18a, 17 (606 mg,
1.34 mmol), triethylamine (0.56 mL, 4.02 mmol),
p-toluenesulfonyl chloride (256 mg, 1.34 mmol) and 1 N
sodium hydroxide (1.47 mL, 1.47 mmol) gave 96 mg
ꢀ
mg (16.7%) of 37g as colorless crystals: mp132–134 C.
1H NMR (200 MHz, CDCl3) d 1.6–1.9 (3H, m), 1.9–2.1
(2H, m), 2.1–2.2 (2H, m), 2.3–2.4 (2H, m), 3.42 (1H, m),
3.63 (1H, m), 4.57 (1H, m), 5.3–5.6 (2H, m), 7.4–7.6
(3H, m), 7.7–7.8 (2H, m), 7.9–8.0 (2H, m), 8.3–8.4 (2H,
m). ESI-MS m/z 522 (MÀH)À.
(13.9%) of 37b as a white powder: mp 109–113 ꢀC. H
1
NMR (200 MHz, D2O) d 1.5–1.7 (3H, m), 1.7–1.9 (2H,
m), 1.9–2.1 (2H, m), 2.1–2.2 (2H, m), 2.47 (3H, s), 3.53
(1H, m), 3.68 (1H, m), 4.33 (1H, m), 5.3–5.6 (2H, m),
7.4–7.8 (9H, m). ESI-MS m/z 491 (MÀH)À.
1-tert-Butyl 2-methyl (2S,4R)-4-(((4-chlorophenyl)sulfo-
nyl)amino)-1,2-pyrrolidinedicarboxylate (38). By the
procedure used for 14, 13 (7.27 g, 26.9 mmol), 10%
palladium on carbon (1.12 g) and 4-chlor-
obenzenesulfonyl chloride (6.26 g, 29.7 mmol) gave 9.13
g (81.0%) of 38 as a pale yellow oil: 1H NMR (200 MHz,
CDCl3) d 1.37 (6H, s), 1.40 (3H, s), 2.0–2.4 (2H, m), 3.20
(1H, m), 3.63 (1H, m), 3.72 (3H, s), 3.95 (1H, m), 4.30
(1H, m), 5.0–5.2 (1H, m), 7.52 (2H, d, J =10 Hz), 7.83
(2H, d,J =10 Hz). ESI-MS m/z 419 (M+H)+.
(5E and 5Z)-6-((2S,4R)-1-((4-methoxyphenyl)sulfonyl)-4-
((phenylsulfonyl)amino)-2-pyrrolidinyl)-5-hexenoic acid
(37c). By the procedure used for 23, 17 (665 mg, 1.47
mmol), triethylamine (0.62 mL, 4.45 mmol), 4-methox-
ybenzenesulfonyl chloride (304 mg, 1.47 mmol) gave
333 mg (44.5%) of 37c as colorless crystals: mp130–
131 ꢀC. 1H NMR (200 MHz, D2O–NaOD) d 1.3–1.4
(4H, m), 1.7–2.0 (4H, m), 2.58 (1H, m), 3.19 (1H, m),

1410
H. Marusawa et al. / Bioorg. Med. Chem. 10 (2002) 1399–1415
1-tert-Butyl 2-methyl (2S,4R)-4-(((4-methylphenyl)sulfo-
nyl)amino)-1,2-pyrrolidinedicarboxylate (39). By the
procedure used for 14, 13 (5.12 g, 18.9 mmol), 10%
palladium on carbon (787 mg) and p-toluenesulfonyl
chloride (3.98 g, 20.9 mmol) gave 5.38 g (%) of 39
as a pale yellow oil: 1H NMR (200 MHz, CDCl3) d
1.40 (9H, s), 2.1–2.3 (2H, m), 2.45 (3H, s), 3.19
(1H, m), 3.60 (1H, dd, J =6, 11 Hz), 3.71 (3H, s),
3.94 (1H, m), 4.30 (1H, m), 5.20 (1H, m), 7.34 (2H,
d, J =8 Hz), 7.77 (2H, d, J =8 Hz). ESI-MS m/z
399 (M+H)+.
1.88 (1H, m), 2.12 (1H, m), 3.15 (1H, m), 3.7-4.0 (2H,
m), 3.88 (3H, s), 4.93 (1H, m), 5.38 (1H, m), 7.02 (2H, d,
J =8 Hz), 7.25–7.35 (2H, m), 9.44 (1H, broad). ESI-MS
m/z 385 (M+H)+.
1-tert-Butyl (2S,4R)-2-formyl-4-(((4-(trifluoromethyl)-
phenyl)sulfonyl)amino) - 1,2 - pyrrolidinecarboxylate (45).
By the procedure used for 15, 41 (6.00 g, 13.3 mmol)
and 1.0 M solution of diisobutylaluminum hydride in
toluene (30.9 mL, 30.9 mmol) gave 5.20 g (92.8%) of 45
as a pale yellow oil: 1H NMR (200 MHz, CDCl3) d 1.42
(9H, s), 2.1–2.3 (2H, m), 3.3–3.6 (1H, m), 3.83 (1H, m),
4.05 (1H, m), 4.22 (1H, m), 5.47 (1H, m), 7.81 (2H, d, J
=8 Hz), 8.00 (2H, d, J =8 Hz), 9.47 (1H, m). ESI-MS
m/z 423 (M+H)+.
1-tert-Butyl 2-methyl (2S,4R)-4-(((4-methoxyphenyl)sul-
fonyl)amino)-1,2-pyrrolidinedicarboxylate (40). By the
procedure used for 14, 13 (7.00 g, 25.9 mmol), 10%
palladium on carbon (1.5 g) and 4-methoxy-
benzenesulfonyl chloride (6.42 g, 31.1 mmol) gave 8.60g
(5E and 5Z)-6-((2S,4R)-1-(tert-Butoxycarbonyl)-4-(((4-
chlorophenyl)sulfonyl)amino)pyrrolidinyl) - 5 - hexenoic
acid (46). By the procedure used for 16, 42 (5.06 g, 13.0
mmol), (4-carboxybutyl)triphenylphosphonium bro-
mide (17.3 g, 39.0 mmol), sodium hydride (3.12 g, 60%
in oil, 78.0 mmol) and dimethyl sulfoxide (45 mL) gave
6.15 g (100%) of 46 as a pale brown oil: 1H NMR
(200 MHz, CDCl3) d 1.38 (6H, s), 1.39 (3H, s), 1.7–1.8
(2H, m), 2.0–2.2 (4H, m), 2.3–2.5 (2H, m), 3.30 (1H, m),
3.45 (1H, dd, J =5, 12 Hz), 3.86 (1H, m), 4.58 (1H, m),
5.2–5.5 (3H, m), 7.52 (1H, d, J =10 Hz), 7.83 (1H, d, J
=10 Hz). ESI-MS m/z 471 (MÀH)À.
1
(80.1%) of 40 as a pale yellow oil: H NMR (200 MHz,
CDCl3) d 1.48 (9H, s), 2.1–2.3 (2H, m), 3.18 (1H, m),
3.59 (1H, dd, J =6, 11 Hz), 3.70 (3H, s), 3.88 (3H, s),
3.90 (1H, m), 4.28 (1H, m), 5.40 (1H, m), 6.98 (2H, d, J
=8 Hz), 7.80 (2H, d, J =8 Hz). ESI-MS m/z 415
(M+H)+.
1-tert-Butyl 2-methyl (2S,4R)-4-(((4-(trifluoromethyl)-
phenyl)sulfonyl)amino)-1,2-pyrrolidinedicarboxylate (41).
By the procedure used for 14, 13 (6.00 g, 22.2 mmol),
10% palladium on carbon (1.0g) and 4-(tri-
fluoromethyl)benzenesulfonyl chloride (6.51 g, 26.6
mmol) gave 6.40 g (63.7%) of 41 as a pale yellow oil: 1H
NMR (200 MHz, CDCl3) d 1.39 (9H, s), 2.0–2.2 (2H,
m), 2.30 (1H, m), 3.2–3.4 (1H, m), 3.73 (3H, s), 3.98
(1H, m), 4.33 (1H, m), 7.82 (2H, d, J =8 Hz), 8.04 (2H,
d, J =8 Hz). ESI-MS m/z 453 (M+H)+.
(5E and 5Z)-6-((2S,4R)-1-(tert-Butoxycarbonyl)-4-(((4-
methylphenyl)sulfonyl)amino)pyrrolidinyl) - 5 - hexenoic
acid (47). By the procedure used for 16, 43 (3.00 g, 8.17
mmol), (4-carboxybutyl)triphenylphosphonium bro-
mide (10.9 g, 24.5 mmol), sodium hydride (1.96 g, 60%
in oil, 49.0 mmol) and dimethyl sulfoxide (30 mL) gave
1
1-tert-Butyl (2S,4R)-2-formyl-4-(((4-chlorophenyl)sulfo-
nyl)amino)-1,2-pyrrolidinecarboxylate (42). By the pro-
cedure used for 15, 38 (9.01 g, 21.5 mmol) and 1.0M
solution of diisobutylaluminum hydride in toluene (38.7
mL, 38.7 mmol) gave 5.51 g (65.9%) of 42 as pale yel-
low crystals: mp120–122 ꢀC. 1H NMR (200 MHz,
CDCl3) d 1.43 (9H, s), 2.16 (2H, m), 3.33 (1H, m), 3.60
(1H, m), 3.85 (1H, m), 4.26 (1H, m), 4.87 (1H, m), 7.53
(2H, d, J =10 Hz), 7.82 (2H, d, J =10 Hz), 9.4–9.6
(1H, m). ESI-MS m/z 389 (M+H)+.
2.90 g (68.5%) of 47 as a pale brown oil: H NMR
(200 MHz, CDCl3) d 1.41 (9H, s), 1.65–1.85 (3H, m),
2.05–2.25 (2H, m), 2.3–2.45 (3H, m), 2.45 (3H, s), 3.25–
3.5 (1H, m), 3.6–3.9 (1H, m), 4.4–4.65 (1H, m), 5.25–5.5
(2H, m), 7.34 (2H, J =8 Hz), 7.78 (1H, d, J =8 Hz).
ESI-MS m/z 451 (MÀH)À.
(5E and 5Z)-6-((2S,4R)-1-(tert-Butoxycarbonyl)-4-(((4-
methoxyphenyl)sulfonyl)amino)pyrrolidinyl) - 5 - hexenoic
acid (48). By the procedure used for 16, 44 (5.50 g, 14.3
mmol), (4-carboxybutyl)triphenylphosphonium bro-
mide (19.0 g, 42.9 mmol), sodium hydride (3.43 g, 60%
in oil, 85.8 mmol) and dimethyl sulfoxide (50 mL) gave
1-tert-Butyl (2S,4R)-2-formyl-4-(((4-methylphenyl)sulfo-
nyl)amino)-1,2-pyrrolidinecarboxylate (43). By the pro-
cedure used for 15, 39 (5.00 g, 12.6 mmol) and 1.0M
solution of diisobutylaluminum hydride in toluene (25.2
mL, 25.2 mmol) gave 3.42 g (74.0%) of 43 as a pale
1
5.24 g (78.2%) of 48 as a pale brown oil: H NMR
(200 MHz, CDCl3) d 1.39 (9H, s), 1.6–1.8 (3H, m), 2.0–
2.2 (2H, m), 2.25–2.45 (3H, m), 3.35–3.5 (1H, m), 3.75–
3.9 (1H, m), 3.87 (3H, s), 4.55 (1H, m), 5.2–5.5 (2H, m),
6.99 (2H, J =9 Hz), 7.81 (1H, d, J =9 Hz). ESI-MS m/
z 467 (MÀH)À.
1
yellow oil: H NMR (200 MHz, CDCl3) d 1.40 (9H, s),
2.13 (1H, m), 2.47 (3H, s), 3.32 (1H, m), 3.57 (1H, m),
3.82 (1H, m), 4.25 (1H, m), 5.10 (1H, m), 7.34 (2H, d, J
=8 Hz), 7.75 (2H, d, J =8 Hz), 9.50 (1H, broad). ESI-
MS m/z 369 (M+H)+.
(5E and 5Z)-6-((2S,4R)-1-(tert-Butoxycarbonyl)-4-(((4-
(trifluoromethyl)phenyl)sulfonyl)amino)pyrrolidinyl) - 5 -
hexenoic acid (49). By the procedure used for 16, 45
(5.00 g, 11.8 mmol), (4-carboxybutyl)triphenylpho-
sphonium bromide (15.7 g, 35.5 mmol), sodium hydride
(2.84 g, 60% in oil, 71.0 mmol) and dimethyl sulfoxide
(40 mL) gave 5.30 g (88.4%) of 49 as a pale brown oil:
1H NMR (200 MHz, CDCl3) d 1.38 (9H, s), 1.6–1.8 (3H,
1-tert-Butyl (2S,4R)-2-formyl-4-(((4-methoxyphenyl)sul-
fonyl)amino)-1,2-pyrrolidinecarboxylate (44). By the
procedure used for 15, 40 (8.00 g, 22.9 mmol) and 1.0 M
solution of diisobutylaluminum hydride in toluene (45.8
mL, 45.8 mmol) gave 6.00 g (80.9%) of 44 as a pale
1
yellow oil: H NMR (200 MHz, CDCl3) d 1.42 (9H, s),

H. Marusawa et al. / Bioorg. Med. Chem. 10 (2002) 1399–1415
1411
m), 2.0–2.2 (2H, m), 2.25–2.45 (3H, m), 3.3–3.5 (1H, m),
3.86 (1H, m), 4.59 (1H, m), 5.3–5.5 (2H, m), 6.10 (1H,
m), 7.78 (2H, J =8 Hz), 8.02 (1H, d, J =8 Hz). ESI-MS
m/z 505 (MÀH)À.
(5Z)-6-((2S,4R)-1-((4-Chlorophenyl)sulfonyl)-4-(((4-me-
thylphenyl)sulfonyl)amino)pyrrolidinyl) - 5 - hexenoic acid
(52). By the procedure used for 17, 18a and 50a,
47 (3.60 g, 7.72 mmol), 75% aqueous tri-
fluoroacetic acid (25 mL, 243 mmol), triethylamine
(5.38 mL, 38.6 mmol) and 4-chlorobenzenesulfonyl
chloride (1.81 g, 8.58 mmol) gave 630 mg (15.5%)
of 52 as colorless crystals: mp98–101 ꢀC. 1H
NMR (200 MHz, CDCl3) d 1.5–1.8 (5H, m), 1.98
(1H, m), 2.1–2.2 (2H, m), 2.43 (3H, s), 3.4–3.5 (2H,
m), 3.69 (1H, m), 4.52 (1H, q, J =7.5 Hz), 5.2–5.5 (3H,
m), 7.32 (2H, d, J =8 Hz), 7.50 (2H, d, J =8 Hz), 7.68
(2H, d, J =8 Hz), 7.75 (2H, d, J =8 Hz). ESI-MS m/z
525 (MÀH)À.
(5Z)-6-((2S,4R)-1-((4-Chlorophenyl)sulfonyl)-4-((phenyl-
sulfonyl)amino)pyrrolidinyl) - 5 - hexenoic acid (50a). By
the procedure used for 18a, 17 (29.9 g, 66.1 mmol),
triethylamine (40.9 mL, 293 mmol) and 4-chloroben-
zenesulfonyl chloride (14.0 g, 66.3 mmol) gave crude
(5E and 5Z)-6-((2S,4R)-1-((4-chlorophenyl)sulfonyl)-4-
((phenylsulfonyl)amino)pyrrolidinyl) - 5 - hexenoic acid
that was dissolved in chloroform and chromatographed
on a silica gel (Wakogel C300, 700 g) column with
chloroform as an eluent. 50a (10.5 g, 31.0%) was
obtained from the first fractions as crystals: mp121–
123 ꢀC. 1H NMR (200 MHz, CDCl3) d 1.5–1.8 (3H,
m), 1.92 (1H, m), 2.11 (2H, q, J =6.5 Hz), 2.33 (2H,
t, J =6.5 Hz), 3.4–3.6 (2H, m), 3.68 (1H, m), 4.46 (1H,
q, J =8 Hz), 5.37 (1H, dd, J =10.5, 10 Hz), 5.45 (1H,
dt, J =10.5, 7 Hz), 7.5–7.7 (5H, m), 7.7–7.9 (4H, m).
ESI-MS m/z 511 (MÀH)À.
(5Z)-6-((2S,4R)-1-((4-Chlorophenyl)sulfonyl)-4-(((4-
methoxyphenyl)sulfonyl)amino)pyrrolidinyl) - 5 - hexenoic
acid (53). By the procedure used for 17, 18a and 50a,
48 (5.10 g, 10.9 mmol), 75% aqueous trifluoroacetic
acid (32 mL, 312 mmol), triethylamine (6.06 mL,
43.5 mmol) and 4-chlorobenzenesulfonyl chloride
(2.30 g, 10.9 mmol) gave 1.25g (21.1%) of 53 as
colorless crystals: mp90 ꢀC (dec.). 1H NMR
(200 MHz, CDCl3) d 1.5–1.8 (3H, m), 2.02 (1H, m),
2.17 (2H, q, J =7.5 Hz), 2.49 (2H, t, J =6.5 Hz), 3.4–
3.5 (2H, m), 3.66 (1H, m), 3.88 (3H, s), 4.50 (1H, q, J
=7 Hz), 5.2–5.5 (3H, m), 6.98 (2H, d, J =8 Hz), 7.50
(2H, d, J =8 Hz), 7.75 (4H, d, J =8 Hz). ESI-MS m/z
541 (MÀH)À.
(5E)-6-((2S,4R)-1-((4-Chlorophenyl)sulfonyl)-4-((phenyl-
sulfonyl)amino)pyrrolidinyl) - 5 - hexenoic acid (50b). By
the procedure used for 50a, 50b (1.55 g, 4.6%) was
obtained from the second fractions as crystals: mp155–
156 ꢀC. H NMR (200 MHz, CDCl3) d 1.6–1.7 (2H, m),
1
1.7–1.8 (2H, m), 1.9–2.1 (2H, m), 2.28 (2H, t, J =7.5
Hz), 3.18 (1H, dd, J =5.5, 10.5 Hz), 3.47 (1H, m), 3.76
(1H, m), 4.28 (1H, q, J =6.5 Hz), 5.20 (1H, dd, J =8,
15.5 Hz), 5.54 (1H, dt, J =15.5, 6.5 Hz), 7.4–7.6 (5H,
m), 7.6–7.7 (2H, m), 7.75–7.85 (2H, m). ESI-MS m/z
511 (MÀH)À.
(5Z)-6-((2S,4R)-1-((4-Chlorophenyl)sulfonyl)-4-(((4-(tri-
fluoromethyl)phenyl)sulfonyl)amino)pyrrolidinyl)-5-hexe-
noic acid (54). By the procedure used for 17, 18a and
50a, 49 (4.60 g, 9.08 mmol), 75% aqueous tri-
fluoroacetic acid (28 mL, 273 mmol), triethylamine
(6.33 mL, 45.4 mmol) and 4-chlorobenzenesulfonyl
chloride (1.91 g, 9.05 mmol) gave 1.08 g (20.5%) of 54
as colorless crystals: mp140–141 ꢀC. 1H NMR
(200 MHz, CDCl3) d 1.5–1.8 (3H, m), 2.05 (1H, m), 2.18
(2H, q, J =7.5 Hz), 2.40 (2H, t, J =6.5 Hz), 3.41 (1H,
dd, J =4.5, 11 Hz), 3.53 (1H, dd, J =3, 11 Hz), 3.79
(1H, m), 4.65 (1H, q, J =7 Hz), 5.22 (1H, dd, J =11, 10
Hz), 5.44 (1H, dt, J =11, 7.5 Hz), 5.78 (1H, d, J =6.5
Hz), 7.45 (2H, d, J =8 Hz), 7.72 (2H, d, J =8 Hz), 7.89
(2H, d, J =8 Hz), 7.98 (2H, d, J =8 Hz). ESI-MS m/z
581 (M+H)+. ESITOF-MS m/z 603.0549 (M+Na)+,
calcd for C23H24ClF3N2O6S2Na 603.0614.
(5Z)-6-((2S,4R) - 1 - ((4 - Chlorophenyl)sulfonyl) - 4 - (((4 -
chlorophenyl)sulfonyl)amino)pyrrolidinyl) - 5 - hexenoic
acid (51a). By the procedure used for 17, 18a and 50a,
46 (8.14 g, 18.0 mmol), 90% aqueous trifluoroacetic
acid (50 mL, 584 mmol), triethylamine (12.5 mL, 89.7
mmol) and 4-chlorobenzenesulfonyl chloride (3.80 g,
18.0 mmol) gave 2.50 g (25.4%) of 51a as colorless
crystals: mp150.5–151.5 ꢀC. 1H NMR (200 MHz,
CDCl3) d 1.5–1.8 (3H, m), 2.03 (1H, m), 2.1–2.2 (2H,
m), 2.41 (2H, t, J =6.5 Hz), 3.4–3.5 (2H, m), 3.74 (1H,
m), 4.56 (1H, q, J =7 Hz), 5.25 (1H, dd, J =10.5, 9
Hz), 5.48 (1H, dt, J =10.5, 7.5 Hz), 7.4–7.5 (4H, m),
7.7–7.8 (4H, m). ESI-MS m/z 545 (MÀH)À. ESITOF-
MS
m/z
547.0562
(M+H)+,
calcd
for
6-((2S,4R)-1-((4-Chlorophenyl)sulfonyl)-4-(((4-chlorophe-
nyl)sulfonyl)amino)pyrrolidinyl)-5-hexanoic acid (55). A
solution of (5Z and 5E)-6-((2S,4R)-1-((4-chloro-
phenyl)sulfonyl) - 4 - (((4 - chlorophenyl)sulfonyl)amino)-
pyrrolidinyl)-5-hexenoic acid (400 mg, 0.731 mmol) in
methanol (15 mL) was hydrogenated under medium
presure (2 atm) in the presence of 10% palladium car-
bon for 7 h. After removal of the catalyst, the solvent
was evaporated in vacuo and the residue was triturated
with diethyl ether to give 164 mg (41.0%) of 55 as a
white powder: mp 124–125 ꢀC. 1H NMR (200 MHz,
CDCl3) d 1.2–1.4 (4H, m), 1.5–1.7 (3H, m), 1.7–1.9 (3H,
m), 2.35 (2H, t, J =7.5 Hz), 3.09 (1H, m), 3.38 (1H, m),
3.6–3.9 (2H, m), 7.4–7.6 (4H, m), 7.7–7.9 (4H, m). ESI-
MS m/z 547 (MÀH)À.
C22H25Cl2N2O6S2 547.0531. [a]2D0=+7.3ꢀ (0.1 g, 1 mol/
L NaOH, 10 mL, 100 mm).
(5E)-6-((2S,4R) - 1 - ((4 - Chlorophenyl)sulfonyl) - 4 - (((4 -
chlorophenyl)sulfonyl)amino)pyrrolidinyl) - 5 - hexenoic
acid (51b). By the procedure used for 51a, 51b (650 mg,
6.6%) was obtained from the second fractions as crys-
tals: mp111–113 ꢀC. 1H NMR (200 MHz, CDCl3) d
1.6–1.8 (2H, m), 1.8–1.9 (2H, m), 1.9–2.1 (2H, m), 2.31
(2H, t, J =7.5 Hz), 3.22 (1H, dd, J =5, 10 Hz), 3.42
(1H, dd, J =5.5, 10 Hz), 3.83 (1H, m), 4.23 (1H, q, J
=6 Hz), 5.17 (1H, dd, J =7.5, 15.5 Hz), 5.53 (1H, dt, J
=15.5, 6.5 Hz), 7.4–7.5 (4H, m), 7.65–7.8 (4H, m). ESI-
MS m/z 545 (MÀH)À.

1412
H. Marusawa et al. / Bioorg. Med. Chem. 10 (2002) 1399–1415
1-tert-Butyl (2S,4R)-4-(((4-chlorophenyl)sulfonyl)amino)-
2-((Z)-2-(3-(methoxycarbonyl)phenyl)ethenyl)-1-pyrroli-
dinecarboxylate (56a). To a suspension of triphenyl (4-
methoxycarbonylbenzyl)phosphonium chloride (1.26 g,
2.81 mmol) in tetrahydrofuran (10 mL) was added
sodium hydride (113 mg, 60% in oil, 2.83 mmol) by
portions under an ice bath cooling and the mixture
was stirred in an ice bath for 1 h. To the resultiing
yellow suspension was added dropwise a solution of
42 (995 mg, 2.56 mmol) in tetrahydrofuran (4 mL)
under ice bath cooling and the mixture was stirred
in an ice bath for 1 h. To the mixture were added
saturated aqueous ammonium chloride (1 mL) and
ethyl acetate (30 mL), and the organic layer was washed
successively with water and brine. The organic solution
was dried over magnesium sulfate and the solvent
was evaporated in vacuo to give a crude 1-tert-
1-tert-Butyl (2S,4R)-4-(((4-chlorophenyl)sulfonyl)amino)-
2-((E)-2-(4-(methoxycarbonyl)phenyl)ethenyl)-1-pyrroli-
dinecarboxylate (56d). By the procedure used for 56c,
56d (21.9 g, 23.3%) was obtained from the second frac-
tions as crystals: mp164–165 ꢀC. H NMR (200 MHz,
1
CDCl3) d 1.39 (9H, s), 1.9-2.2 (2H, m), 3.24 (1H, dd, J
=5, 11 Hz), 3.55 (1H, dd, J =6, 11.5 Hz), 3.91 (3H, s),
3.8–4.0 (1H, m), 4.49 (1H, m), 4.91 (1H, m), 6.12 (1H,
dd, J =6.5, 15.5 Hz), 7.37 (2H, d, J =8.5 Hz), 7.50 (2H,
d, J =8.5 Hz), 7.82 (2H, d, J =8.5 Hz), 7.97 (2H, d, J
=8.5 Hz). ESI-MS m/z 521 (M+H)+.
Methyl 3-((Z)-2-((2S,4R)-4-(((4-chlorophenyl)sulfonyl)-
amino) pyrrolidinyl)ethenyl)benzoate (57a). A solution
of 56a (447 mg, 0.858 mmol) in 90% aqueous tri-
fluoroacetic acid (3 mL, 35.1 mmol) was stirred at room
temperature for 30 min and the solvent was evaporated
in vacuo. The residue was suspended in chloroform (10
mL) and the solution was adjusted to pH 8 with satu-
rated aqueous sodium bicarbonate. The organic phase
was separated, washed with brine and dried over mag-
nesium sulfate. The solvent was evaporated in vacuo to
give 330 mg (91.4%) of 57a as an oil: 1H NMR
(200 MHz, CDCl3) d 1.75 (1H, dd, J =7.5, 14 Hz), 1.91
(1H, m), 2.74 (1H, dd, J =5, 12 Hz), 3.26 (1H, dd, J
=6, 12 Hz), 3.90 (1H, m), 3.92 (3H, s), 4.13 (1H, m),
5.62 (1H, dd, J =9.5, 12 Hz), 6.51 (1H, d, J =12 Hz),
7.3–7.5 (5H, m), 7.7–8.0 (3H, m). ESI-MS m/z 421
(M+H)+.
butyl
(2S,4R)-4-(((4-chlorophenyl)sulfonyl)amino)-2-
((E and Z)-2-(3-(methoxycarbonyl)phenyl)ethenyl)-1-
pyrrolidinecarboxylate. The crude product was
separated by using a silica gel column with a mixture of
n-hexane and ethyl acetate (4:1–2:1) as an eluent. 56a
(500 mg, 37.5%) was obtained from the first fractions
(less polar) as crystals: mp 154–156 ꢀC. 1H NMR
(200 MHz, CDCl3) d 1.28 (9H, s), 1.8–2.1 (2H, m), 3.30
(1H, dd, J =5, 12 Hz), 3.56 (1H, dd, J =6, 12 Hz),
3.89 (1H, m), 3.93 (3H, s), 4.79 (1H, m), 4.93 (1H, d, J
=7 Hz), 5.58 (1H, dd, J =9, 12.5 Hz), 6.48 (1H, d, J
=12.5 Hz), 7.3–7.5 (4H, m), 7.83 (2H, d, J =8.5 Hz),
7.92 (2H, m). ESI-MS m/z 521 (M+H)+. ESITOF-MS
m/z 521.1625 (M+H)+, calcd for C25H30ClN2O6S
521.1513.
Methyl 3-((E)-2-((2S,4R)-4-(((4-chlorophenyl)sulfonyl)-
amino)pyrrolidinyl)ethenyl)benzoate (57b). By the pro-
cedure used for 57a, 56b (570 mg, 1.09 mmol) and 90%
aqueous trifluoroacetic acid (10 mL, 117 mmol) gave
1-tert-Butyl (2S,4R)-4-(((4-chlorophenyl)sulfonyl)amino)-
2-((E)-2-(3-(methoxycarbonyl)phenyl)ethenyl)-1-pyrroli-
dinecarboxylate (56b). By the procedure used for 56a,
56b (572 mg, 42.9%) was obtained from the second
fractions (more polar) as crystals: mp 126–128 ꢀC.
1H NMR (200 MHz, CDCl3) d 1.40 (9H, s), 1.8–2.1
(2H, m), 3.24 (1H, dd, J =5.5, 11 Hz), 3.57 (1H,
dd, J =6, 11 Hz), 3.92 (3H, s), 3.97 (1H, m), 4.49
(1H, m), 4.88 (1H, m), 6.09 (1H, dd, J =6.5, 16
Hz), 6.43 (1H, d, J =16 Hz), 7.33 (2H, d, J =8.5
Hz), 7.49 (1H, t, J =7.5 Hz), 7.4–7.6 (3H, m), 7.92 (1H,
d, J =7 Hz), 8.03 (1H, s). ESI-MS m/z 521 (M+H)+.
ESITOF-MS m/z 521.1527 (M+H)+, calcd for
C25H30ClN2O6S 521.1513.
1
450 mg (98.1%) of 57b as an oil: H NMR (200 MHz,
CDCl3) d 1.8--2.0 (2H, m), 2.93 (1H, dd, J =4.5, 11.5
Hz), 3.32 (1H, dd, J =6, 11.5 Hz), 3.90 (1H, m), 3.93
(3H, s), 4.05 (1H, m), 6.28 (1H, dd, J =7.5, 16.5 Hz),
6.52 (1H, d, J =16.5 Hz), 7.36 (1H, t, J =7.5 Hz), 7.4--
7.5 (3H, m), 7.7--7.9 (3H, m), 8.00 (1H, m). ESI-MS m/z
421 (M+H)+.
Methyl 4-((Z)-2-((2S,4R)-4-(((4-chlorophenyl)sulfonyl)-
amino)pyrrolidinyl)ethenyl)benzoate (57c). By the pro-
cedure used for 57a, 56c (15.5 g, 29.8 mmol) and 90%
aqueous trifluoroacetic acid (100 mL, 1.17mol) gave
11.9 g (95.0%) of 57c as a white powder: mp 189–
190 ꢀC. H NMR (200 MHz, CDCl3) d 1.7–2.2 (2H, m),
1
1-tert-Butyl (2S,4R)-4-(((4-chlorophenyl)sulfonyl)amino)-
2-((Z)-2-(4-(methoxycarbonyl)phenyl)ethenyl)-1-pyrroli-
dinecarboxylate (56c). By the procedure used for 56a,
triphenyl (4 - methoxycarbonylbenzyl)phosphonium
chloride (88.5 g, 198 mmol), sodium hydride (7.92 g,
60% in oil, 198 mmol) and 42 (70.0 g, 180 mmol) gave
16.0 g (17.1%) of 56c as colorless crystals: mp178–
179 ꢀC. 1H NMR (200 MHz, CDCl3) d 1.29 (9H, s), 1.8–
2.3 (2H, m), 3.26 (1H, m), 3.51 (1H, dd, J =6, 11 Hz),
3.89 (1H, m), 3.93 (3H, s), 4.78 (1H, m), 5.10 (1H, m),
5.60 (1H, dd, J =9, 11.5 Hz), 6.48 (1H, d, J =11.5 Hz),
7.2–7.4 (2H, m), 7.48 (2H, d, J =8.5 Hz), 7.82 (2H, d, J
=8.5 Hz), 8.02 (2H, d, J =8.5 Hz). ESI-MS m/z 521
(M+H)+. ESITOF-MS m/z 543.1280 (M+Na)+, calcd
for C25H29ClN2O6SNa 543.1333.
2.66 (1H, dd, J =4.5, 11.5 Hz), 3.18 (1H, dd, J =6, 11.5
Hz), 3.88 (1H, m), 3.93 (3H, s), 4.08 (1H, m), 5.61 (1H,
dd, J =9.5, 11.5 Hz), 6.52 (1H, d, J =11.5 Hz), 7.28
(2H, d, J =8.5 Hz), 7.49 (2H, d, J =8.5 Hz), 7.80 (2H,
d, J =8.5 Hz), 8.00 (2H, d, J =8.5 Hz). ESI-MS m/z
421 (M+H)+.
Methyl 4-((E)-2-((2S,4R)-4-(((4-chlorophenyl)sulfonyl)-
amino)pyrrolidinyl)ethenyl)benzoate (57d). By the pro-
cedure used for 57a, 56d (16.0 g, 30.7 mmol) and 90%
aqueous trifluoroacetic acid (100 mL, 1.17 mol) gave
1
12.9 g (100%) of 57d as an oil: H NMR (200 MHz,
CDCl3) d 1.7–2.1 (2H, m), 2.85 (1H, dd, J =4, 11 Hz),
3.27 (1H, dd, J =6, 11 Hz), 3.86 (1H, m), 3.90 (3H, s),
4.09 (1H, m), 6.21 (1H, dd, J =7, 16 Hz), 6.50 (1H, d, J

H. Marusawa et al. / Bioorg. Med. Chem. 10 (2002) 1399–1415
1413
=16 Hz), 7.34 (2H, d, J =8.5 Hz), 7.49 (2H, d, J =8.5
Hz), 7.83 (2H, d, J =8.5 Hz), 7.95 (2H, d, J =8.5 Hz).
ESI-MS m/z 421 (M+H)+.
4-((E)-2-((2S,4R)-1-((4-Chlorophenyl)sulfonyl)-4-(((4-
chlorophenyl)sulfonyl)amino)pyrrolidinyl)ethenyl)benzoic
acid (58d). By the procedure used for 18a and 58a, 57d
(12.5 g, 29.7 mmol), triethylamine (4.13 mL, 29.7
mmol), 4-chlorobenzenesulfonyl chloride (6.27 g, 29.7
mmol) and 1 N aqueous sodium hydroxide (83.0 mL,
83.0 mmol) gaveꢀ15.9 g (92.1%) of 58d as a white pow-
der: mp168–171 C (dec.). 1H NMR (200 MHz, CDCl3)
d 1.7–1.9 (2H, m), 3.08 (1H, dd, J =6, 11 Hz), 3.46 (1H,
m), 3.78 (1H, m), 4.32 (1H, m), 6.30 (1H, dd, J =7, 16
Hz), 6.53 (1H, d, J =16 Hz), 7.47 (2H, d, J =8.5 Hz),
7.6–7.7 (4H, m), 7.7–7.8 (4H, m), 7.88 (2H, d, J =8.5
Hz), 8.00 (1H, d, J =6 Hz). ESI-MS m/z 579 (MÀH)À.
ESITOF-MS m/z 581.0468 (M+H)+, calcd for
C25H23Cl2N2O6S2 581.0374. [a]2D0À40.8ꢀ (0.1 g, 1 mol/L
NaOH, 10 mL, 100 mm).
3-((Z)-2-((2S,4R)-1-((4-Chlorophenyl)sulfonyl)-4-(((4-
chlorophenyl)sulfonyl)amino)pyrrolidinyl)ethenyl)benzoic
acid (58a). By the procedure used for 18a, 57a (329 mg,
0.782 mmol), triethylamine (0.11 mL, 0.790 mmol) and
4-chlorobenzenesulfonyl chloride (165 mg, 0.782 mmol)
gave 378 mg (81.2% from 57a) of methyl 3-((Z)-2-
((2S,4R)-1-((4-chlorophenyl)sulfonyl) - 4 - (((4 - chloro-
phenyl)sulfonyl)amino)pyrrolidinyl)ethenyl)benzoate. A
solution of methyl 3-((Z) - 2 - ((2S,4R) - 1 - ((4 - chlor-
ophenyl)sulfonyl)-4-(((4-chlorophenyl)sulfonyl)amino)-
pyrrolidinyl)ethenyl)benzoate (378 mg, 0.635 mmol) in a
mixture of methanol (2.5 mL) and 1 N aqueous sodium
hydroxide (2.5 mL, 2.5 mmol) was stirred at 50 ꢀC for 4
h and the volatile solvent was evaporated in vacuo. The
residual aqueous solution was adjusted to pH 1 with
concentrated hydrochloric acid. The white precipitate
was collected by filtration and washed with water to
give 359 mg (79.0% from 57a) of 58a as a white powder:
Biological methods
Inhibitory effects on 9,11-azo PGH2-induced platelet
aggregation in rabbit. Blood was collected from the car-
otid artery of male rabbits into plastic vessels containing
0.1 volume of 3.8% aqueous sodium citrate. Platelet-
rich plasma (PRP) was then prepared by centrifugation
at 150g for 15 min. Platelet-poor plasma (PPP) was
prepared from the remaining blood after removing
PRP, by centrifugation at 1500g for 10min. PRP was
then adjusted to a concentration of 6.0Â105 cells/mL by
using PPP. Platelet aggregation was investigated using
the turbidometric method with an aggregometer (NKK
Hematracer 1 Model 4A). To 225 mL of PRP, 25 mL of
the test compound solution or vehicle solution (0.1 N
NaOH) was added, and the whole stirred at 1000rpm
for 2 min at 37 ꢀC. Next, 5 mL of 9,11-azo PGH2 (final
concentration 1.0 mM) or ADP (final concentration 2.5
mM, Sigma) was added as an aggregating inducer. The
concentration which caused 50% inhibition (IC50) of
9,11-azo PGH2-induced maximum aggregation was
obtained by regression analysis of the concentration
versus inhibition of aggregation curve.
ꢀ
1
mp127–130 C. H NMR (200 MHz, CDCl3) d 1.8–1.9
(2H, m), 3.23 (1H, dd, J =4.5, 10.5 Hz), 3.50 (1H, dd, J
=5, 10.5 Hz), 3.63 (1H, m), 4.41 (1H, m), 5.67 (1H, dd,
J =9.5, 12 Hz), 6.54 (1H, d, J =12 Hz), 7.4–8.0 (12H,
m). ESI-MS m/z 579 (MÀH)À. ESITOF-MS m/z
581.0354 (M+H)+, calcd for C25H23Cl2N2O6S2
581.0374. [a]2D0À126.6ꢀ (0.05 g, 1 mol/L NaOH, 10 mL,
100 mm).
3-((E)-2-((2S,4R)-1-((4-Chlorophenyl)sulfonyl)-4-(((4-
chlorophenyl)sulfonyl)amino)pyrrolidinyl)ethenyl)benzoic
acid (58b). By the procedure used for 18a and 58a, 57b
(420 mg, 1.00 mmol), triethylamine (0.14 mL, 1.01
mmol), 4-chlorobenzenesulfonyl chloride (211 mg, 1.00
mmol) and 1 N aqueous sodium hydroxide (2.3 mL, 2.3
mmol) gave 312 mg (53.8%) of 58b as a white powder:
ꢀ
1
mp119–121 C. H NMR (200 MHz, CDCl3) d 1.7–1.9
(2H, m), 3.12 (1H, dd, J =5, 9.5 Hz), 3.50 (1H, dd, J
=6.5, 9.5 Hz), 3.79 (1H, m), 4.31 (1H, m), 6.23 (1H, dd, J
=7.5, 16 Hz), 6.57 (1H, d, J =16 Hz), 7.4–8.0 (12H, m).
ESI-MS m/z 579 (MÀH)À. ESITOF-MS m/z 581.0358
(M+H)+, calcd for C25H23Cl2N2O6S2 581.0374.
[a]2D0À55.8ꢀ (0.05 g, 1 mol/L NaOH, 10 mL, 100mm).
Inhibitory effects of 51a on 3H-U46619 binding to
washed guinea-pig platelets. This was carried out
according to the modified method of Kattelman et al.31
PRP was prepared as described above from male Hart-
ley strain guinea-pigs and treated with aspirin (final
concentration 1 mM), then centrifuged at 150g for 15
min to remove residual contaminating red blood cells.
PGI2 (final concentration 40 nM, Funakoshi) was
added to aid in resuspension of the platelets. The PRP
was then spun at 1100g for 15 min to pellet the cells.
The platelet free plasma was discarded, and the platelet
pellets were gently resuspended in buffer [sodium chlo-
ride (138 mM), potassium chloride (5 mM), magnesium
chloride (5 mM), glucose (5.5 mM) and Tris–HCl (25
mM), PH 7.4] to a final cell count of approximately
1.0Â109 cells. Aliquots of platelet suspension (1.0 mL)
were incubated with approximately 5 nM (final con-
centration) [3H]-U46619 ([15-3H(N)]-9,11-epoxymethano
PGH2, 22.4 Ci/mmol, NEN) plus various concentra-
tions of 51a for 5 min at 37 ꢀC with stirring. In order to
block U-46619-induced platelet activation, the cells
were treated with PGI2 (final concentration 250 nM) for
4-((Z)-2-((2S,4R)-1-((4-Chlorophenyl)sulfonyl)-4-(((4-
chlorophenyl)sulfonyl)amino)pyrrolidinyl)ethenyl)benzoic
acid (58c). By the procedure used for 18a and 58a, 57c
(11.5 g, 27.3 mmol), triethylamine (3.80 mL, 27.3
mmol), 4-chlorobenzenesulfonyl chloride (5.77 g, 27.3
mmol) and 1 N aqueous sodium hydroxide (78.5 mL,
78.5 mmol) gave 15.2g (95.7%) of 58c as a white pow-
der: mp206–208 ꢀC (dec). H NMR (200 MHz, CDCl3)
1
d 1.8–2.0 (2H, m), 3.31 (1H, dd, J =3.5, 10.5 Hz), 3.51
(1H, dd, J =5.5, 10.5 Hz), 4.40 (1H, m), 5.68 (1H, dd, J
=9.5, 11.5 Hz), 6.54 (1H, d, J =11.5 Hz), 7.32 (2H, d, J
=8 Hz), 7.40 (2H, d, J =8 Hz), 7.47 (2H, d, J =8 Hz),
7.67 (2H, d, J =8 Hz), 7.78 (2H, d, J =8 Hz), 7.95 (2H,
d, J =8 Hz). ESI-MS m/z 579 (MÀH)À. ESITOF-MS
m/z 581.0358 (M+H)+, calcd for C25H23Cl2N2O6S2
581.0374. [a]2D0À87.5ꢀ (0.1 g, 1 mol/L NaOH, 10 mL,
100 mm).

1414
H. Marusawa et al. / Bioorg. Med. Chem. 10 (2002) 1399–1415
45 s prior to mixing them with drugs. After mixin g, a
0.4 mL aliquot of the binding assay mixture was trans-
ferred to a 1.5 mL Eppendorf tube, and the incubation
was terminated at the appropriate time by centrifuga-
tion at 15,000 rpm for 1 min in a Eppendorf 5414 cen-
trifuge. The supernatant was quickly removed by
aspiration, and the inside of the Eppendorf tubes, as
well as the surface of the pellet was rinsed twice with 0.4
mL of ice-cold resuspension buffer, which was immedi-
ately removed by aspiration. The tip of the tube was cut
off, and placed in a polyethylene vial (Wheaton)
including 0.4 mL of tissue solubilizer (NCS, Amer-
sham). The mixture was incubated for 3–4 h at 50 ꢀC,
then 5 mL of toluene scintilator (Omnifluor 4 g/L tolu-
ene) was added to the mixture. The radioactivity was
measured in a liquid scintillation counter.
after administration of 51a or saline, arachidonic acid
(90 mg/kg, Sigma) was injected intravenously for 20 s.
The effect of 51a was evaluated by measuring the inci-
dence of death within 5 min of injection. Results are
expressed as percent survival. Each group was com-
prised of 10 mice.
Inhibitory effect of 51a on thrombus formation in extra-
corporeal shunt in rat.32 Male Sprague–Dawley rats
weighing 390–530 g were anesthetized with sodium
pentobarbital (50 mg ip). A silk thread (5 cm; Tire No.
50, Fujix Co., Osaka, Japan) was placed in a poly-
ethylene tube (6 cm; Hibiki Size 7, Kunii Co., Tokyo,
Japan). Both ends of this tubing were connected to
other polyethylene tubing (10 cm; Hibiki Size 4, Kunii
Co.). An extracorporeal shunt filled with heparin solu-
tion (50 IU/mL) was introduced between the right
carotid artery and the left jugular vein, and after this,
blood circulation was established. After 20 min, the
blood flow was stopped on the artery side with a pinch-
cock. The middle tubing was then taken away and the
thread coated with thrombus was carefully pulled out of
the tubing. The wet weight of the thrombus was mea-
sured immediately. Another new polyethylene tubing
containing a silk thread and filled with heparin solution
was inserted between the arterial and venous tubing
ends. Blood circulation was re-established by opening
the pinch-cock. After 20 min, the wet weight of the silk
thread was measured as before. The third blood circu-
lation was then performed. Thrombus weight was
determined by subtracting the weight of a silk thread
immersed in heparinized blood from that of the thread
coated with thrombus. The total thrombus weight
obtained from three blood circulation was taken as net
thrombus weight in one experiment. 51a at doses from
0.01 to 0.32 mg/kg or vehicle (water) was given orally 60
min before the first blood circulation. The effect of 51a
were expressed as percent inhibition of thrombus for-
mation compared with vehicle treatment. Each group
was comprised of 10 mice.
Inhibitory effect of 51a on U-46619- or collagen-induced
platelet aggregation in human, monkey, dog or guinea-
pig. These tests were performed by a similar procedure
to the test for platelet aggregation in rabbits. In this
case, the final concentration of U-46619 (Cyman
Chemical) was 1 mM (human), 2 M (monkey) or 0.5 mM
(guinea-pig), and that of collagen (Horm) was 0.5 mg/
mL (human or guinea-pig) or 20 mg/mL (dog). The
concentration of PRP was adjusted to 3.0Â105 cells/ mL
in the case of human, monkey and dog.
Inhibitory effect of 51a on U-46619-induced contractile
response in rat aorta. Male Sprague–Dawley rats (200–
220 g) were killed and their thoracic aorta were placed
in Tyrode solution. Adhering fat and connective tissues
were removed and spiral strips of the aorta were pre-
pared. Each strip (2 mm in width and 15 mm in length)
were mounted in an organ bath containing 25 mL of
Tyrode solution bubbled with 95% O2 and 5% CO2 gas
at 37 ꢀC. After 1 h of incubation period under 0.5 g
resting tension, U-46619 (3.2Â10À8 mol/L) was added
to induce TXA2 receptor-mediated contraction. The
contractile response was isometrically recorded on a
polygraph via a force-displacement transducer. After
obtaining the maximum contractile response by U-46619,
51a in water was then cumulatively added. Finally,
papaverine (1.0Â10À4 mol/L) was added to obtain the
maximum relaxation. The concentration which induce
50% of the maximum relaxation was determined gra-
phically and indicated as an IC50 value.
Acknowledgements
We are grateful to the late Dr. Teruo Oku and Dr.
Masashi Hashimoto for their pertinent advice.
Inhibitory effect of 51a on ex vivo platelet aggregation in
guinea-pig. Male Hartley strain guinea-pigs weighing
about 300 g were used after overnight fasting. Blood
was collected from the abdominal artery at 1 and 6 h
after oral administration of 51a or vehicle (0.1 N
NaOH). PRP was prepared as described before, and
platelet aggregation was induced by adding 5 mL of
U-46619 (final concentration, 0.5 mM) or collagen (final
concentration, 5 mg/mL) to 250 mL of PRP.
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