A practical synthesis and biological evaluation of 9-halogenated PGF analogues.

Article abstract of DOI:10.1016/S0968-0896(02)00008-1

A series of 9-halo PGF analogues 1-2 and 5-13 were synthesized and biologically evaluated. Among the compounds, 2 was the best EP2-receptor agonist. A practical method of synthesizing 2 via the Julia olefination of an aldehyde 3 with an optically active sulfone 4, which was prepared by Sharpless asymmetric epoxidation of 15, was developed. Other 9-halogenated PGF analogues were synthesized essentially by the same procedure and evaluated. The absolute configuration of 16-OH of 2 was determined as S by the X-ray analysis of a salt consisting of a 1/1 molar ratio of 2 and L-lysine.

Full text of DOI:10.1016/S0968-0896(02)00008-1

Bioorganic & Medicinal Chemistry 10 (2002) 1883–1894
A Practical Synthesis and Biological Evaluation of
9-Halogenated PGF Analogues
Kousuke Tani,a,* Atsushi Naganawa,a Akiharu Ishida,a Hiromu Egashira,a
Yoshihiko Odagaki,a Toru Miyazaki,a Tomoyuki Hasegawa,b Yasufumi Kawanaka,b
Kenji Sagawa,a Hiroyuki Harada,a Mikio Ogawa,a Takayuki Maruyama,a Hisao Nakai,a
Shuichi Ohuchida,a Kigen Kondoa and Masaaki Todaa
aMinase Research Institute, Ono Pharmaceutical Co., Ltd., Shimamoto, Mishima, Osaka 618-8585, Japan
bFukui Research Institute, Ono Pharmaceutical Co., Ltd., Mikuni, Sakai, Fukui 913-8538, Japan
Received 30 October 2001; accepted 20 December 2001
Abstract—A series of 9-halo PGF analogues 1–2 and 5–13 were synthesized and biologically evaluated. Among the compounds, 2
was the best EP2-receptor agonist. A practical method of synthesizing 2 via the Julia olefination of an aldehyde 3 with an optically
active sulfone 4, which was prepared by Sharpless asymmetric epoxidation of 15, was developed. Other 9-halogenated PGF analogues
were synthesized essentially by the same procedure and evaluated. The absolute configuration of 16-OH of 2 was determined as S by
the X-ray analysis of a salt consisting of a 1/1 molar ratio of 2 and l-lysine. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
by this new synthesis (Scheme 1). Additionally, removal
of the Á8,9-olefinic compound, which was formed as a
by-product in the chlorination reaction of the new
method, was found to be easier. We report here full
details on the practical synthesis of 9-halogenated PGF
analogues and their biological evaluations.
In the preceding publications,1 we reported the devel-
opment of the highly selective EP2-receptor agonists 1
and 2, which demonstrate high receptor affinity to the
EP2-receptor (Chart 1). Also in functional studies, 2
showed nearly the same potency as that of PGE2.
Compound 2 suppressed spontaneous uterine motility
in anesthetized rats in a late-term pregnancy, while
1a,1c
Chemistry
PGE2 stimulated uterine motility.
These findings
suggest that 2 is a promising clinical candidate as a
tocolytic with a new mechanism of action. The infor-
mation mentioned above prompted us to develop a
practical synthetic method of the newly discovered EP2-
receptor selective agonist 2. According to the reported
method,1c the undesired 16(R)-isomer of 2 has to be
removed by column chromatography on silica gel, while
this laborious procedure could be successfully avoided
A highly selective EP2-receptor agonist 2 was synthe-
sized by the magnesium-mediated Julia olefination2 of
Chart 1. Structures of EP2-receptor agonists 1 and 2.
*Corresponding author. Tel.: +81-75-961-1151; fax: +81-75-962-
9314; e-mail: k.tani@ono.co.jp
Scheme 1. Retrosynthesis of 2.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00008-1

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K. Tani et al. / Bioorg. Med. Chem. 10 (2002) 1883–1894
an aldehyde 3 with an optically active sulfone 4 as out-
lined in Scheme 1.
converted to 19 (99% ee).5 The hydroxy group of 19
was protected as a tetrahydropyranyl (THP) ether prior
to its use for the Julia olefination.
Preparation of 4
Preparation of 3, 28 and 29
As described in Scheme 2, 4 was prepared from 14,
which was obtained from cyclobutane carboxylic acid in
three steps1b (1) alkylation; (2) reduction; (3) oxidation.
The compound 14 was converted to an allylic alcohol 15
by a Horner–Emmons reaction with diethylethylphos-
phono acetate followed by the reduction of an a,b-
unsaturated ester with diisobutylaluminum hydride.
Enantioselective epoxidation of 15 was conducted by
Sharpless asymmetric epoxidation3 to afford an opti-
cally active epoxy alcohol 16 (94% ee).4 A ring-opening
reaction of the epoxide 16 with Red-Al1 afforded a diol
17. Selective monotosylation of the primary alcohol in
17 followed by a SN2 reaction with a sodium thiophen-
oxide provided 18. Oxidation of 18 with Oxone1 affor-
ded a sulfone 19, which was converted to a benzoate 20
for recrystallization. After purification, 20 was again
The synthesis of aldehydes 3, 28 and 29 is outlined in
Scheme 3. Methanesulfonylation of the 9a-hydroxy
group of 216 followed by the SN2 substitution reaction
with tetrabutylammonium chloride7 afforded 9b-chlo-
ride 23. Selective deprotection of the 1-methyl-1-meth-
oxyethyl group6 of 23 provided 25, which was easily
separated from the by-product (Á8,9-olefinic product)
by column chromatography on silica gel. The ratio of
the desired 9b-chloro product and the Á8,9-olefinic side-
product was approximately 5.4/1.8 The compound 25
was converted to the aldehyde 3 by Swern oxidation.
The corresponding 5,6-dihydro aldehyde 28 was pre-
pared by oxidation of 26, which was obtained by the
catalytic hydrogenation of 25.
The 9b-bromide 29 was obtained from 24, which was pre-
pared by SN2 substitution of 22 with tetrabutylammonium
bromide instead of tetrabutylammonium chloride
according to the procedure described for the prepara-
tion of 28 from 23.
Preparation of 38
The synthesis of 9b-chloro-5(E) aldehyde 38 from the
Corey lactone 309 is outlined in Scheme 4. Conversion
of 30 to 31 was carried out by (1) protection of the pri-
mary alcohol as a t-butyldimethylsilyl ether and (2)
reduction with lithium aluminum hydride. Compound
31 was converted to 32 by (1) selective monoacetyl-
ation of a primary hydroxy group, (2) tosylation of
the a-hydroxy group of 31, (3) a SN2 substitution reac-
tion with tetrabutylammonium chloride, and (4) alka-
line hydrolysis with sodium hydroxide. Oxidation of 32
to the corresponding aldehyde followed by the addition
reaction of a vinyl magnesium bromide provided an
allylic alcohol 33, which was converted to 9b-5(E) chlo-
ride 34 by the rearrangement reaction. One-carbon
homologation of 34 to obtain a nitrile 35 was carried out
in three steps: (1) hydride reduction of 34 with lithium
aluminum hydride, (2) tosylation with p-toluenesulufo-
Scheme 2. Synthesis of an optically active o chain 4. Reagents: (a)
(EtO)2P(O)CH2CO2Et, NaH, THF; (b) i-Bu2AlH, THF, 0 ꢀC; (c) t-
BuOOH, Ti(OiPr)4, D-(ꢁ)-DIPT, CH2Cl2, ꢁ20 ꢀC; (b) Na(Me-
OCH2CH2O)2AlH2, toluene; (c) TsCl, n-Bu4NBr, NaOHaq, toluene,
then PhSH; (d) Oxone1, MeOH, H2O; (e) PhCOCl, DMAP, pyridine;
(f) Recrystallization from EtOH; (g) NaOHaq, MeOH; (h) DHP, p-
TsOH, CH2Cl2.
Scheme 3. Synthesis of 9b-halo aldehyde 3, 28 and 29. Reagents: (a) MsCl, Et3N, CH2Cl2; (b) n-Bu4NCl or n-Bu4NBr, Et3N, toluene, 60 ꢀC; (c)
HClaq, THF; (d) Pd/C, H2, MeOH; (e) SO3–pyridine, Et3N, DMSO.

K. Tani et al. / Bioorg. Med. Chem. 10 (2002) 1883–1894
1885
nyl chloride in pyridine, and (3) substitution reaction
with sodium cyanide in dimethyl sulfoxide. The nitrile 35
was converted to a methyl ester 36 by (1) reduction with
diisobutylaluminum hydride; (2) oxidation with cro-
mium trioxide, and (3) esterification with diazomethane.
Deprotection of 36 followed by oxidation afforded 38.
column chromatography. Alkaline hydrolysis of 43
provided 2. Catalytic hydrogenation of 43 followed by
alkaline hydrolysis afforded 12. Compounds 6, 9 and 10
were synthesized from 29, 38 and 28, respectively,
according to the same procedures used for the prepara-
tion of 2 from 3. The compound 7 was produced as a
by-product in the reductive elimination of 41.
Synthesis of 9ꢀ-halo PG analogues 2, 6, 7, 9, 10 and 12
The absolute configuration of the 16-OH in 2 was
determined by X-ray analysis. X-Ray analysis of a salt
consisting of a 1/1 molar ratio of 2 and l-lysine was
carried out in our laboratory. As shown in Figure 1, 16-
OH was found to be of the S-configuration based on the
absolute configuration of l-lysine in the crystal lattice.
Julia olefination of 3 with 4 using powdered magne-
sium2 was conducted as shown in Scheme 5. Addition
reaction of the carbanion, which was generated from 4,
to the aldehyde 3 afforded 39 as a diastereomeric mix-
ture. Reductive elimination of 39 followed by acidic
deprotection afforded 13(E)-olefin 43, the correspond-
ing 13(Z)-isomer of which was produced as a by-pro-
duct (E/Z=5.0/1)10 and easily removed by silica gel
Inversion of the 9a-hydroxy group of 48 by the Mitsu-
nobu reaction followed by aminolysis with aqueous
Scheme 4. Synthesis of 9b-chloro 5(E)-aldehyde 38. Reagents: (a) TBSCl, imidazole, DMF; (b) LiAlH4, THF; (c) Ac2O, collidine, CH2Cl2; (d) TsCl,
pyridine; (e) n-Bu4NCl, toluene; (f) NaOHaq, MeOH; (g) SO3–pyridine, Et3N, DMSO, CH2Cl2; (h) CH2¼CHMgBr, THF; (i) CH3CH(OEt)3,
EtCO2H, 140 ꢀC; (j) NaCN, DMSO, 100 ꢀC; (k) i-Bu2AlH, CH2Cl2, then HClaq; (l) CrO3, H+, H2O; (m) CH2N2; (n) n-Bu4NF, THF.
Scheme 5. Synthesis of 9b-halo PG analogues 2, 6, 7, 9, 10 and 12. Reagents: (a) 4, n-BuLi, THF, ꢁ78 ꢀC; (b) Mg (powdered, ꢁ50 mesh), TMSCl,
MeOH; (c) p-TsOH, MeOH; (d) NaOHaq, MeOH; (e) Pd/C, H2, MeOH; (f) l-lysine, EtOH, EtOAc.

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K. Tani et al. / Bioorg. Med. Chem. 10 (2002) 1883–1894
ammonia afforded 49, which was converted to 52 by (1)
tosylation of the 9b-hydroxy group of 49, (2) a sub-
stitution reaction with tetrabutylammonium chloride,
and (3) deprotection under acidic conditions.
Schemes 1–6, were biologically evaluated for their affi-
nity to the mouse (m) EP1–4-receptors and their ability
to increase the intracellular cAMP concentration.
Among the compounds tested, 2 demonstrated the best
profile in the EP2-receptor affinity, EP2-receptor selec-
tivity and functional activity (EC50). The EC50 value of
2 was similar to that of PGE2. The compound 2 was
nearly 10-fold more potent in EP2-receptor affinity and
had nearly 3-fold greater EC50 value than the corre-
sponding 9-keto analogue 1.
Conversion of 48 to 51 was carried out by (1) tosylation
of the 9a-hydroxy group of 48 and (2) a SN2 substitu-
tion reaction with tetrabutylammonium fluoride.
The 13,14-dihydro derivative 50 was converted to a 9b-
chloro derivative 53 by the tosylation of 50 followed by
a SN2 substitution reaction with tetrabutylammonium
chloride. Alkaline hydrolysis of 51, 52 and 53 afforded
5 , 8and 11, respectively.
Results of the biological evaluation of the other 9-halo-
16-hydroxy-17,17-trimethylene PG analogues are shown
in Table 1. Replacement of the 9b-chloro group in 2
with a fluorine atom and a bromine atom afforded a 9b-
fluoro analogue 5 and a 9b-bromo analogue 6 which
retained the EP2-receptor affinity and selectivity,
respectively, while losing some of the EP2-receptor
agonistic activity (EC50 value). Removal of the 9-chloro
group from 2 produced 7 with a marked reduction in
agonistic activity although it still showed EP2-receptor
affinity and selectivity. These results clearly indicate that
the 9b-chloro group of 2 plays an important role in both
the receptor affinity and agonistic activity. The corre-
sponding 9a-chloro analogue 8 was also found to be a
selective EP2-receptor agonist while in receptor affinity
and agonistic activity it was less potent than 9b-chloro
analogue 2.
Results and Discussion
A series of 9-halo-15-deoxy-16-hydroxy-17,17-trimethyl-
ene PGF analogues, which were prepared according to
the magnesium-mediated Julia olefination described in
Conversion of the 5(Z)-double bond of 2 to a 5(E)-
double bond and saturated ethylene moiety provided 9
and 10, respectively, also with a reduction in receptor
affinity and agonistic activity. Saturation of the 13(E)-
double bond of 2 afforded 11 with a fair reduction in
activity though its EP2-receptor affinity was nearly 4
times less potent than that of 2. Saturation of both of
the two double bonds of 2 gave 12 with a marked
reduction in agonistic activity. Compounds 10, 11 and
12, the intramolecular double bonds of which were
partially or thoroughly reduced, showed weak affinity
to other receptors such as EP1, EP3 and EP4.
Therefore, both of the intramolecular 5(Z)- and 13(E)-
double bonds of 2 were indispensable for potent recep-
tor affinity, agonistic activity and EP2-receptor selectiv-
ity.
The 16(R)-diastereomer 13 had nearly a 100-fold lower
EC50 value than 2 while its EP2-receptor affinity was
4.8-fold less potent than that of 2. Besides, 13 exhibited
weak affinity to the EP1-receptor (Ki=2200 nM) and
the EP3-receptor (Ki=170 nM).
Fig 1. X-ray crystallographic structure of 2Ly.
Scheme 6. Synthesis of other 9-halo PG analogues 5, 8 and 11. Reagents: (a) diethylazodicarboxylate, PPh3, HCOOH, THF; (b) NH3aq; (c) TsCl,
pyridine; (d) n-Bu4NF or n-Bu4NCl, toluene; (e) HFaq, MeCN; (f) NaOHaq, MeOH.

K. Tani et al. / Bioorg. Med. Chem. 10 (2002) 1883–1894
Table 1. Biological evaluation of 9-halo-16-hydroxy-17,17-trimethylene PG analogues
1887
b
Compds
X, Y
A
B
Binding Ki (nM)a
EC50
mEP1
mEP2
mEP3
mEP4
mEP2
1
2
5
6
7
8
9
10
11
12
X=Y=O
–CH¼CH– (Z)
–CH¼CH– (Z)
–CH¼CH– (Z)
–CH¼CH– (Z)
–CH¼CH– (Z)
–CH¼CH– (Z)
–CH¼CH– (E)
–CH2-CH2–
–CH¼CH– (E)
–CH¼CH– (E)
–CH¼CH– (E)
–CH¼CH– (E)
–CH¼CH– (E)
–CH¼CH– (E)
–CH¼CH– (E)
–CH¼CH– (E)
–CH2–CH2–
>104
>104
>104
>104
5100
>104
>104
5700
>104
3500
2200
18
30
3.3
7.3
2.4
11
13
8.1
8.1
13
17
16
38
>104
>104
>104
>104
>104
>104
>104
>104
2300
>104
170
>104
6100
>104
>104
>104
>104
>104
3100
>104
>104
>104
3.1
11
3.8
10
25
1400
39
31
66
79
590
330
2.0
X=Cl, Y=H
X=F, Y=H
X=Br, Y=H
X=Y=H
X=H, Y=Cl
X=Cl, Y=H
X=Cl, Y=H
X=Cl, Y=H
X=Cl, Y=H
X=Cl, Y=H
–CH¼CH– (Z)
–CH2–CH2–
–CH¼CH– (Z)
–CH2–CH2–
–CH¼CH– (E)
13(16R-OH)
PGE2
5.0
aUsing membrane fractions of Chinese hamster ovary (CHO) cells expressing the mouse prostanoid receptors, Ki values were determined by the
competitive binding assay, which was performed according to the method of Kiriyama et al.13 with some modifications. When the test compound
did not displace binding of radioligands by 50% even at a concentration of 104 nM, the Ki value was not determined (expressed >104).
bWith regard to the subtype-receptor agonistic activity, EC50 values were determined based on the effect of the test compounds on the increase in the
intracellular cAMP production in the mouse EP2 receptor.
Summary
4,4-Trimethylene-2-hexen-1-ol (15). To a stirred suspen-
sion of sodium hydride (306 g, 7.65 mol) in 6 L of THF
was added ethyl diethylphosphonoacetate (1869 g, 8.34
mol) in 2 L of THF at 0 ꢀC and the stirring was con-
tinued for 30 min at room temperature under an argon
atmosphere. To the resulting solution was added the
aldehyde 141b (780 g, 6.95 mol) at 0 ꢀC and the stirring
was continued for 30 min at room temperature. The
reaction mixture was treated with saturated aqueous
ammonium chloride and extracted with EtOAc. The
organic layer was washed with water, then brine, dried
over anhydrous magnesium sulfate and concentrated in
vacuo to afford a crude oil, which was purified by col-
umn chromatography on silica gel to give ethyl 4,4-tri-
methylene-2-hexenoate (1134 g, 81%) as a colorless oil.
TLC Rf=0.53 (n-hexane/EtOAc, 9/1); IR (neat) 2963,
2934, 2855, 1721, 1651, 1463, 1366, 1305, 1267, 1178,
The enantioselective synthesis of the o-chain segment 4
followed by a magnesium-mediated Julia olefination
with 3 provided a practical route to 2. Compounds 6, 7,
9, 10 and 12 were prepared essentially as described
above. Other 9-halo-PGF analogues, 5, 8 and 11, were
also prepared as described in Scheme 6. All of the syn-
thesized 9-halo PGF analogues, 1–2 and 5–13, were
biologically evaluated. Among the compounds, 2 was
the most effective EP2-receptor agonist with regard to
subtype selectivity and agonistic activity.
Experimental
General directions
1
1096, 1041, 986, 861, 721 cmꢁ1; H NMR (200 MHz,
Analytical samples were homogeneous as confirmed by
TLC, and afforded spectroscopic results consistent with
the assigned structures. All 1H NMR spectra were
obtained using a Varian Gemini-200, VXR-200s or
Mercury300 spectrometer. Mass spectra were obtained
on a Hitachi M1200H, JEOL JMS-DX303HF or Per-
Septive Voyager Elite spectrometer. IR spectra were
measured on a Perkin-Elmer FT-IR 1760X or JASCO
FT/IR-430 spectrometer. Elemental analyses for car-
bon, hydrogen, nitrogen and sulfur were carried out on
a Perkin-Elmer PE2400 SeriesII CHNS/O analyzer.
Optical rotations were measured using a JASCO DIP-
1000 polarimeter. Column chromatography was carried
out on silica gel [Merck silica gel 60 (0.063ꢂ0.200 mm)
or Wako Gel C200]. Thin layer chromatography was
performed on silica gel (Merck TLC or HPTLC plates,
silica gel 60 F254).
CDCl3) d 6.98 (d, J=15.8 Hz, 1H), 5.77 (d, J=15.8 Hz,
1H), 4.20 (q, J=7.2 Hz, 2H), 2.10–1.80 (m, 6H), 1.63 (q,
J=7.4 Hz, 2H), 1.31 (t, J=7.2 Hz, 3H), 0.77 (t, J=7.4
Hz, 3H); MS (APCI, Pos, 12V) m/z=183 (M+H)+.
To a stirred solution of ethyl 4,4-trimethylene-2-hex-
enoate (1134 g, 6.22 mol) in 10 L of THF was slowly
added diisobutylaluminum hydride (25% in toluene,
7793 g, 13.7 mol) at 0 ꢀC under an argon atmosphere.
After stirring for 30 min at 0 ꢀC, the reaction mixture
was treated with 1 L of methanol and 24 L of 2 N aqu-
eous hydrochloric acid and extracted with EtOAc. The
organic layer was washed with water, then brine, dried
over anhydrous magnesium sulfate and concentrated in
vacuo to afford a crude oil, which was purified by col-
umn chromatography on silica gel to give 4,4-trimethyl-
ene-2-hexen-1-ol 15 (678 g, 78%) as a colorless oil.

1888
K. Tani et al. / Bioorg. Med. Chem. 10 (2002) 1883–1894
TLC Rf=0.39 (n-hexane/EtOAc, 4/1); IR (neat) 3322,
2962, 2933, 2875, 2853, 1461, 1377, 1089, 1012, 971
hydroxide, was slowly added p-toluenesulfonyl chloride
(922 g, 4.84 mol) in 1 L of toluene in an ice-bath. After
stirring for 1 h at room temperature, thiophenol (559 g,
5.07 mol) in 1 L of toluene was added to the reaction
mixture. After stirring for additional 1 h, the aqueous
layer was separated. The organic layer was washed with
water three times and concentrated in vacuo to afford a
sulfide 18 (1144 g, 99%) as a colorless oil. TLC Rf=0.52
(n-hexane/EtOAc, 4/1); IR (neat) 3441, 2962, 1584,
1
cmꢁ1; H NMR (200 MHz, CDCl3) d 5.71 (d, J=15.6
Hz, 1H), 5.58 (dt, J=15.6, 5.0 Hz), 4.18 (m, 2H), 2.00–
1.70 (m, 6H), 1.54 (q, J=7.5 Hz, 2H), 1.30 (br, 1H),
0.75 (t, J=7.5 Hz, 3H); MS (APCI, Pos, 12V) m/z=123
(M+H ꢁH2O)+.
(2R,3R)-2,3-Epoxy-4,4-trimethylene-1-hexanol (16). To
a stirred suspension of 480 g of powdered 3A, activated
molecular sieves in 8 L of CH2Cl2 were added sequen-
tially with titanium tetraisopropoxide (275 g, 0.968 mol)
and a solution of d-(ꢁ)-diisopropyltartarate (272 g, 1.16
mol) in 1 L of CH2Cl2 at ꢁ20 ꢀC under an argon atmo-
sphere. After stirring for 1 h at ꢁ20 ꢀC, a solution of the
allyl alcohol 15 (678 g, 4.84 mol) in 1 L of CH2Cl2 was
added and the resulting mixture was stirred for 0.5 h,
whereupon cumyl hydroperoxide (80% in cumene, 1381
g, 7.26 mol) was added. Stirring was maintained for 3 h
at ꢁ20 ꢀC. The resulting reaction mixture was treated
with dimethyl sulfide (1064 mL, 14.5 mol) and stirred
for 3 h at ꢁ20 ꢀC. A 10% aqueous solution of tartaric
acid was then added and the resulting heterogeneous
mixture was filtered to remove molecular sieves. The
organic layer was separated and the aqueous layer was
extracted with EtOAc. The combined organic layers
were washed with aqueous sodium chloride, dried over
anhydrous magnesium sulfate and concentrated in
vacuo to afford a crude epoxy alcohol 16 (4.84 mol,
quant) as a colorless oil. TLC Rf=0.20 (n-hexane/
EtOAc, 4/1); IR (neat) 3402, 2975, 2935, 2877,
1446, 1377, 1256, 1175, 1102, 1074, 1030, 955, 862,
1
1481, 1439, 1310, 1272, 1071, 1026, 737, 690 cmꢁ1; H
NMR (200 MHz, CDCl3) d 7.40–7.10 (m, 5H), 3.80–
3.65 (m, 1H), 3.28–2.94 (m, 2H), 2.00–1.20 (m, 11H),
0.88 (t, J=7.5 Hz, 3H); MS (APCI, Pos, 12V) m/z=233
(M+HꢁH2O)+.
(3R)-1-Benzenesulfonyl-4,4-(trimethylene)hexan-3-ol (19).
To a stirred solution of 18 (1144 g, 4.57 mol) in 19 L of
methanol was added a solution of Oxone1 (4211 g,
13.7 mol as KHSO5) in 19 L of water at 0–10 ꢀC. After
stirring for 3 h at below 20 ꢀC, the reaction mixture was
diluted with 15 L of water and extracted with EtOAc.
The organic layer was washed with water, then brine,
dried over anhydrous magnesium sulfate, and con-
centrated in vacuo to afford a crude sulfone 19 (1291 g,
quant) as an orange oil.
To a stirred mixture of the sulfone 19 (1291 g, 4.57 mol)
and 4-(dimethylamino)pyridine (56 g, 0.457 mol) in 12 L
of pyridine was added benzoyl chloride (964 g, 6.86
mol) at room temperature under an argon atmosphere.
After stirring for 6 h at 50 ꢀC, the reaction mixture was
treated with water and extracted with EtOAc. The
organic layer was washed successively with 2 N aqueous
hydrogen chloride, water, saturated aqueous sodium
bicarbonate, water, and finally brine, dried over anhy-
drous magnesium sulfate, and concentrated in vacuo to
afford a crude crystal, which was purified by recrystalli-
zation from 13 L of EtOH to give an optically pure
(>99%ee) benzoate 20 (1250 g, 71%) as a pale yellow
crystal. TLC Rf=0.58 (n-hexane/EtOAc, 2/1); IR (KBr)
2983, 2962, 2940, 1710, 1320, 1278, 1153, 1113, 1087,
763, 700 cmꢁ1
; d
1H NMR (200 MHz, CDCl3)
4.02–3.90 (m, 1H), 3.72–3.58 (m, 1H), 2.95–2.88 (m,
1H), 2.82 (d, J=2.2 Hz, 1H), 2.10–1.40 (m, 9H),
0.87 (t, J=7.4 Hz, 3H); MS (APCI, Pos, 12V) m/
z=157 (M+H)+.
(3R)-4,4-(Trimethylene)hexane-1,3-diol (17). To a stirred
solution of bis(2-methoxyethoxy)aluminum hydride
(72% in toluene, 6814 g, 24.2 mol) in 13 L of THF was
slowly added the epoxyalcohol 16 (756 g, 4.84 mol) in
THF under an argon atmosphere. After stirring for 4 h,
the reaction mixture was cooled in an ice-bath and
treated with 1400 mL of methanol and 25 L of 2 N
aqueous sodium hydroxide. The mixture was then
extracted with t-butyl methyl ether repeatedly. The
combined organic layers were washed with brine, dried
over anhydrous magnesium sulfate and concentrated in
vacuo to afford a crude oil, which was purified by col-
umn chromatography on silica gel to give the diol 17
(730 g, 95%) as a white solid. TLC Rf=0.27 (n-hexane/
EtOAc, 1/1); IR (neat) 3360, 2962, 2936, 2879, 1463,
715, 598, 531 cmꢁ1 1H NMR (200 MHz, CDCl3) d
;
8.05–7.93 (m, 2H), 7.93–7.82 (m, 2H), 7.70–7.40 (m,
6H), 5.28–5.18 (m, 1H), 3.26–2.98 (m, 2H), 2.20–1.40
(m, 10H), 0.95 (t, J=7.6 Hz, 3H); MS (APCI, Pos, 20V)
m/z=387 (M+H)+, 265, 123.
To a solution of the benzoate 20 (1250 g, 3.23 mol) in
4.5 L of THF and 9 L of methanol was added 4 L of 2 N
aqueous sodium hydroxide at room temperature. After
stirring for 2 h at 50 ꢀC, the reaction mixture was dilu-
ted with 15 L of water and extracted with 10 L of t-butyl
methyl ether twice. The combined organic layers were
washed with water, then brine, dried over anhydrous
magnesium sulfate, and concentrated in vacuo to afford
the sulfone 19 (912 g, quant) as a colorless oil. TLC
Rf=0.32 (n-hexane/EtOAc, 2/1); IR (neat) 3525, 2963,
2936, 2878, 1447, 1304, 1148, 1087, 1072, 748, 689, 599,
1430, 1379, 1055 cmꢁ1
;
1H NMR (200 MHz,
CDCl3) d 4.00–3.76 (m, 3H), 2.52 (br, 1H), 2.28
(br, 1H), 2.05–1.30 (m, 10H), 0.92 (t, J=7.5 Hz,
3H); MS (APCI, Pos, 20V) m/z=159 (M+H)+,
141, 123.
1
533 cmꢁ1; H NMR (200 MHz, CDCl3) d 8.00–7.90 (m,
(3R)-1-Phenylthio-4,4-(trimethylene)hexan-3-ol (18). To
a stirred heterogeneous mixture of diol 17 (730 g, 4.61
mol), tetrabutylammonium bromide (148 g, 0.461 mol)
in 4 L of toluene and 7 L of 2 N aqueous sodium
2H), 7.70–7.50 (m, 3H), 3.66–3.55 (m, 1H), 3.50–3.10
(m, 2H), 2.00–1.30 (m, 11H), 0.88 (t, J=7.5 Hz, 3H);
MS (APCI, Pos, 12V) m/z=283 (M+H)+, 265
(M+HꢁH2O)+.

K. Tani et al. / Bioorg. Med. Chem. 10 (2002) 1883–1894
1889
(3R)-1-Benzenesulfonyl-3-(tetrahydro-2H-pyran-2-yl)oxy-
4,4-trimethylenehexane (4). To a stirred solution of the
sulfone 19 (1005 g, 3.55 mol) in 7.5 L of CH2Cl2 was
added p-toluene sulfonic acid (10 g, 0.04 mol) under an
argon atmosphere. The mixture was cooled to below
10 ꢀC, and then 3,4-dihydro-2H-pyran (359 g, 4.26
mmol) was slowly added. After the solution had been
stirred for 1 h at 20–30 ꢀC, the reaction was quenched
with saturated aqueous sodium bicarbonate and the
mixture was extracted with EtOAc. The organic layer
was washed with water, then brine, dried over anhy-
drous magnesium sulfate and concentrated in vacuo to
afford a crude oil, which was purified by column chro-
matography on silica gel to give the compound 4 (1244
g, 96%) as a colorless oil. TLC Rf=0.42 and 0.37 (n-
hexane/EtOAc, 4/1); IR (neat) 2939, 2864, 1446, 1305,
1150, 1075, 1031, 987 cmꢁ1; 1H NMR (200 MHz, CDCl3)
d 8.00–7.90 (m, 2H), 7.70–7.50 (m, 3H), 4.40 (m, 1H),
3.94–3.70 (m, 1H), 3.62–3.28 (m, 3H), 3.17–3.04 (m, 1H),
2.24–1.20 (m, 16H), 0.85 (t, J=7.4 Hz, 3H); MS (APCI,
Pos, 20V) m/z=265 (M+HꢁC5H10O2)+, 123.
4.58 (m, 1H), 4.20–3.78 (m, 3H), 3.67 (s, 3H), 3.60–3.20
(m, 3H), 3.20 (s, 3H), 2.38–1.40 (m, 18H), 1.32 (s, 6H).
(5Z)-7-[(1R,2S,3R,5R)-5-Chloro-2-hydroxymethyl-3-(tet-
rahydro-2H-pyran-2-yloxy)cyclopentyl]hept-5-enoic acid
methyl ester (25). To a stirred mixture of 23 (89.4 g,
0.200 mol) in 357 mL of THF was added 1 N aqueous
hydrochloric acid (120 mL, 0.120 mol) below ꢁ5 ꢀC.
After stirring for 30 min at ꢁ5 ꢀC, the reaction mixture
was poured into ice-water and treated with saturated
aqueous sodium bicarbonate. The mixture was then
diluted with 700 mL of EtOAc. The organic layer was
separated and the aqueous layer was extracted with
EtOAc. The combined organic layers were washed with
brine, dried over anhydrous magnesium sulfate, and
concentrated in vacuo to afford a crude oil as a mixture
of 25 and the corresponding Á8,9-olefinic by-product.
The residue was purified by column chromatography on
silica gel to give 25 (45.8 g, 61%) as a colorless viscous
oil. TLC Rf=0.60 (n-hexane/EtOAc, 1/1); MS (FAB,
Pos.) m/z=375 (M+H)+, 291; IR (neat) 3460, 2944,
1740, 1440, 1353, 1202, 1136, 1023, 972, 870, 812 cmꢁ1
;
(5Z)-7-{(1R,2S,3R,5S)-5-Methanesulfonyloxy-2-[(1-meth-
oxy-1-methylethoxy)methyl]-3-(tetrahydro-2H-pyran-2-
yloxy)cyclopentyl}hept-5-enoic acid methyl ester (22).
To a stirred mixture of 21 (90.0 g, 0.210 mol), triethyl-
amine (42.5 g, 0.420 mol) and potassium carbonate
(48.5 g, 0.351 mol) in 420 mL of CH2Cl2, was slowly
added methanesulfonyl chloride (36.1 g, 0.315 mol) at
ꢁ5 ꢀC under an argon atmosphere. After stirring for 30
min, the reaction mixture was quenched with saturated
aqueous sodium bicarbonate and layers were separated.
The aqueous layer was extracted with CH2Cl2 and the
combined organic layers were washed with water, dried
over anhydrous magnesium sulfate, and concentrated in
vacuo to afford a mesylate 22 (111 g, quant) as a pale
yellow oil. TLC Rf=0.60 (CH2Cl2/EtOAc, 4/1); IR
1H NMR (200 MHz, CDCl3) d 5.60–5.33 (m, 2H), 4.70
(m, 0.35H), 4.57 (m, 0.65H), 4.32–3.40 (m, 9H), 2.40–
1.33 (m, 18H). The corresponding bromo-derivative 27
was synthesized from 22 according to the procedure
described above, except that tetrabutylammonium bro-
mide was used instead of tetrabutylammonium chloride.
7-[(1R,2S,3R,5R)-5-Chloro-2-hydroxymethyl-3-(tetrahy-
dro-2H-pyran-2-yloxy)cyclopentyl]heptanoic acid methyl
ester (26). A mixture of 25 (500 mg, 1.33 mmol) and
5% Pd/C in 10 mL of ethanol was stirred for 3 h at
ambient temperature under a hydrogen atmosphere.
The resulting reaction mixture was filtered and con-
centrated in vacuo to afford 26 (500 mg, quant) as an
oil. TLC Rf=0.40 (n-hexane/EtOAc, 1/1); 1H NMR
(200 MHz, CDCl3) ꢀ 4.78–4.54 (m, 1H), 4.35–4.30 (m,
6H), 3.67 (s, 3H), 2.31 (t, J=7.5 Hz, 2H), 2.30–2.10 (m,
2H), 2.00–1.20 (m, 19H).
(neat) 2990, 2942, 2872, 1736, 1455, 1174 cmꢁ1 1H
;
NMR (200 MHz, CDCl3) d 5.55–5.37 (m, 2H), 5.12–
5.02 (m, 1H), 4.71–4.58 (m, 1H), 4.25–4.07 (m, 1H),
3.97–3.79 (m, 1H), 3.67 (s, 3H), 3.67–3.30 (m, 3H), 3.20
(s, 3H), 3.05 and 3.01 (2s, 3H), 2.37–1.40 (m, 18H), 1.33
(s, 6H).
(5Z)-7-[(1R,2S,3R,5R)-5-Chloro-2-formyl-3-(tetrahydro-
2H-pyran-2-yloxy)cyclopentyl]hept-5-enoic acid methyl
ester (3). To a stirred solution of 25 (45.2 g, 0.121 mol)
and triethylamine (73.2 g, 0.727 mol) in 358 mL of
dimethyl sulfoxide was slowly added a solution of sulfur
trioxide-pyridine complex (57.6 g, 0.362 mol) in 215 mL
of dimethylsulfoxide at 20 ꢀC under an argon atmo-
sphere. After stirring for 0.5 h at 20 ꢀC, the reaction
mixture was poured into ice-cold aqueous ammonium
chloride and extracted with EtOAc repeatedly. The
combined organic layers were washed successively with
ice-cold 0.2 N aqueous hydrochloric acid, water, satu-
rated aqueous sodium bicarbonate, water, and finally
brine, before being dried over anhydrous magnesium
sulfate and concentrated in vacuo to afford 3 (46.9 g,
quant) as a dark-brown oil. TLC Rf=0.60 (n-hexane/
EtOAc, 2/1); IR (neat) 2947, 2868, 2736, 1439, 1352,
1323, 1247, 1201, 1154, 1133, 1077, 1034, 1021, 970
7-{(1R,2S,3R,5R)-5-Chloro-2-[(1-methoxy-1-methyl-
ethoxy)methyl]-3-(tetrahydro-2H-pyran-2-yloxy)cyclopen-
tyl}hept-5-enoic acid methyl ester (23). To a stirred
mixture of tetrabutylammonium chloride (104.1 g, 0.374
mol) and triethylamine (105.2 g, 1.04 mol) in 520 mL of
toluene was added 22 (105.4 g, 0.208 mol) under an
argon atmosphere. After stirring for 20 h at 50 ꢀC, the
reaction mixture was cooled to room temperature and
treated with water. The mixture was extracted with
EtOAc and the organic layer was washed with water
repeatedly, and then with brine, dried over anhydrous
magnesium sulfate, and concentrated in vacuo to afford
23 (90.5 g, 97%) as a colorless oil. The ratio of the
desired 9b-chloro product to the Á8,9-olefinic by-pro-
duct was approximately 5.4/1. This by-product was
removed by column chromatography on silica gel in the
next step. TLC Rf=0.86 (CH2Cl2/EtOAc, 4/1); IR
cmꢁ1 1H NMR (200 MHz, CDCl3) d 9.76 and 9.73
;
(2Âd, J=2.0 Hz, 1H), 5.60–5.30 (m, 2H), 4.65–4.50 (m,
2H), 4.15–3.95 (m, 1H), 3.90–3.70 (m, 1H), 3.67 (s, 3H),
3.60–3.40 (m, 1H), 2.80–1.40 (m, 18H). The compounds
(neat) 2943, 2871, 2738, 1740, 1438, 1380, 1367 cmꢁ1
1H NMR (200 MHz, CDCl3) d 5.58–5.28 (m, 2H), 4.70–
;

1890
K. Tani et al. / Bioorg. Med. Chem. 10 (2002) 1883–1894
28 and 29 was synthesized from 26 and 27, respectively,
according to the procedure described above.
poured into ice-water and extracted with 25% EtOAc–
hexane. The organic layer was washed with water, then
brine, dried over anhydrous magnesium sulfate, and
concentrated in vacuo to afford a crude oil, which was
purified by column chromatography on silica gel to give
a chloro compound (21.0 g, 78%) as a colorless oil.
7-[(1R,2S,3R,5R)-5-Chloro-2-formyl-3-(tetrahydro-2H-
pyran-2-yloxy)cyclopentyl]heptanoic acid methyl ester
(28). Oil. TLC Rf=0.34 (n-hexane/EtOAc, 2/1); 1H
NMR (200 MHz, CDCl3) d 9.77 and 9.74 (2Âd, J=2.0
Hz, 1H), 4.70–4.50 (m, 2H), 4.20–4.00 (m, 1H), 3.90–
3.70 (m, 1H), 3.67 (s, 3H), 3.60–3.40 (m, 1H), 2.80–2.00
(m, 6H), 2.00–1.20 (m, 16H).
To a stirred solution of this compound (21.0 g) in 50 mL
of dimethoxyethane and 150 mL of methanol was added
2 N aqueous sodium hydroxide (50 mL, 100 mmol) at
0 ꢀC. After stirring for 0.5 h at room temperature, the
reaction mixture was poured into ice-water and extrac-
ted with 25% EtOAc–hexane. The organic layer was
washed with water, followed by brine, dried over anhy-
drous sodium sulfate and concentrated in vacuo to
afford a crude oil, which was purified by column chro-
matography on silica gel to give 32 (15.7 g, 83%) as a
colorless oil. TLC Rf=0.40 (n-hexane/EtOAc, 2/1); MS
(1S,2R,3S,4R)-3-(t-Butyldimethylsiloxy)methyl-2-(2-hy-
droxyethyl)-4-(tetrahydro-2H-pyran-2-yloxy)cyclopenta-
nol (31). To a solution of the lactone 30 (21.0 g, 81.9
mmol) and imidazole (10.0 g, 147 mmol) in 50 mL of
dimethyl formamide was added t-butyldimethylsilyl
chloride (14.8 g, 98.3 mmol) at ambient temperature.
After stirring overnight, the reaction mixture was
poured into ice-water and extracted with hexane. The
organic layer was washed with water, then brine, dried
over anhydrous sodium sulfate, and concentrated in
vacuo to afford a TBS ether (30.3 g, quant) as an oil. To
a stirred suspension of lithium aluminum hydride (3.07
g, 81.0 mmol) in 100 mL of THF was slowly added the
TBS ether (30.3 g) in 150 mL of tetrahydrofuran over 2
h at ambient temperature. After the mixture had been
stirred for 1 h at ambient temperature, the reaction was
quenched with a small amount of methanol and satu-
rated aqueous sodium sulfate. The resulting insoluble
substance was removed by filtration and the filtrate was
concentrated in vacuo to afford 31 (30.4 g, 99%) as a
1
(APCI, pos, 20V) m/z=309 (MꢁTHP+H)+, 273; H
NMR (200 MHz, CDCl3) d 4.60 (m, 1H), 4.20–3.94 (m,
2H), 3.95–3.40 (m, 6H), 2.57–1.33 (m, 12H), 0.92 (s,
9H), 0.08 (s, 6H).
(4E)-6-[(1R,2S,3R,5R)-2-(t-Butyldimethylsiloxy)methyl-
5-chloro-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl]-
hex-4-enoic acid ethyl ester (34). To a stirred solution
of 32 (15.6 g, 39.7 mmol) in 15 mL of dimethyl sulfoxide
was added sequentially 30 mL of triethylamine and sul-
fur trioxide–pyridine complex (18.9 g, 119 mmol) at
ambient temperature. After the mixture had been stirred
for 1 h, the reaction was quenched with water. Follow-
ing extraction with 25% EtOAc–hexane, the organic
layer was washed with water, then brine, dried over
anhydrous magnesium sulfate, and concentrated in
vacuo to afford the corresponding aldehyde (15.5 g,
quant) as a pale yellow oil. To a stirred solution of the
aldehyde (12.9 g, 32.0 mmol) in 200 mL of THF was
added a 1.0 M THF solution of vinyl magnesium bro-
mide (64 mL, 64 mmol) at ꢁ78 ꢀC under an argon
atmosphere. After stirring for 1 h at ꢁ78 ꢀC, the reac-
tion mixture was treated with saturated aqueous
ammonium chloride and extracted with 60% ether–
hexane. The organic layer was washed with water, fol-
lowed by brine, dried over anhydrous sodium sulfate,
and concentrated in vacuo to afford 33 (13.4 g) as an oil.
TLC Rf=0.18 (n-hexane/EtOAc, 5/1).
1
colorless oil. H NMR (200 MHz, CDCl3) d 4.69 (m,
1H), 4.30–4.08 (m, 2H), 3.96–3.36 (m, 6H), 3.00–2.20
(br, 2H), 2.15–1.42 (m, 12H), 0.90 (m, 9H), 0.08 (s, 6H).
(1R,2S,3R,5R)-[2-(t-Butyldimethylsiloxy)methyl-5-chloro
- 3 - (tetrahydro - 2H - pyran - 2 - yloxy)cyclopentyl]ethanol
(32). To a stirred solution of 31 (30.4 g, 81.0 mmol)
and 2,4,6-trimethylpyridine (21.4 mL, 162 mmol) in 200
mL of dichloromethane was added acetyl chloride (7.91
mL, 111 mmol) at ꢁ78 ꢀC under an argon atmosphere.
After the mixture had been stirred for 1 h at ꢁ78 ꢀC, the
reaction was quenched with 1.86 mL of methanol. The
mixture was poured into ice-water and extracted with
EtOAc. The organic layer was washed with water, then
brine, dried over anhydrous sodium sulfate, and con-
centrated in vacuo to afford a crude oil, which was
purified by column chromatography on silica gel to give
an acetate (26.3 g, 67.2 mmol) as a colorless oil.
A mixture of 33 (13.4 g, 32 mmol), 80 mL of orthoacetic
acid triethyl ester and catalytic amount of propionic
acid (ca. 250 mg) was stirred for 2 h at 140 ꢀC. After it
had cooled, 0.5 mL of triethylamine was added. The
resulting mixture was concentrated in vacuo to afford a
crude oil, which was purified by column chromato-
graphy on silica gel to give 34 (9.90 g, 63%) as a color-
less oil. TLC Rf=0.44 (n-hexane/EtOAc, 5/1); 1H NMR
(200 MHz, CDCl3) d 5.53–5.42 (m, 2H), 4.59 (m, 1H),
4.22–3.40 (m, 8H), 2.46–1.38 (m, 19H), 0.92 (s, 9H),
0.07 (s, 6H).
To a stirred solution of the acetate (25.9 g, 62.2 mmol)
and 180 mL of pyridine was added p-toluenesulfonyl
chloride (119 g, 622 mmol) at 0 ꢀC and the mixture was
stirred for 16 h at ambient temperature. The reaction
was quenched with 10 mL of water and the mixture was
poured into ice-water, and extracted with EtOAc. The
organic layer was washed first with water, and then with
brine, dried over anhydrous magnesium sulfate, and
concentrated in vacuo to afford a tosylate (35.5 g,
quant). A mixture of this tosylate (35.5 g) and tetra-
butylammonium chloride (34.4 g, 124 mmol), potassium
carbonate (25.7 g, 186 mmol) and 500 mL of toluene
was stirred for 4 h at 50 ꢀC. The reaction mixture was
(5E)-7-[(1R,2S,3R,5R)-2-(t-Butyldimethylsiloxy)methyl-
5-chloro-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl]-
hept-5-enenitrile (35). To a stirred suspension of lithium
aluminum hydride (1.15 g, 30.4 mmol) in 100 mL of

K. Tani et al. / Bioorg. Med. Chem. 10 (2002) 1883–1894
1891
THF was slowly added 34 (9.90 g, 20.2 mmol) in 150
mL of THF at ambient temperature. After the mixture
had been stirred for 0.5 h at ambient temperature, the
reaction was quenched with 3.3 mL of methanol and 20
mL of saturated aqueous sodium sulfate. The resulting
insoluble substance was removed by filtration and the
filtrate was concentrated in vacuo to afford an alcohol
(9.03 g, quant) as a colorless viscous oil. A mixture of
this alcohol and p-toluenesulfonyl chloride (9.65 g, 50.6
mmol) in 70 mL of pyridine was stirred for 5 h at
ambient temperature. The reaction was quenched with
0.6 mL of water and the mixture poured into ice-water.
After extraction with ether, the organic layer was
washed with water, then brine, dried over anhydrous
sodium sulfate, and concentrated in vacuo to afford a
tosylate (12.1 g, quant.). A mixture of the crude tosylate
(12.1 g) and sodium cyanide (1.49 g, 30.3 mmol) in 70
mL of dimethyl sulfoxide was stirred for 0.5 h at 100 ꢀC.
The reaction mixture was poured into ice-water and
extracted with ether. The organic layer was washed with
water, then brine, dried over anhydrous sodium sulfate,
and concentrated in vacuo to afford a crude oil, which
was purified by column chromatography on silica gel to
give 35 (6.02 g, 67% in three steps) as a colorless viscous
oil. TLC Rf=0.52 (benzene/EtOAc, 10/1); 1H NMR
(200 MHz, CDCl3) d 5.57–5.47 (m, 1H), 5.43 (dt,
J=15.5, 6.5 Hz, 1H), 4.59 (m, 1H), 4.16 (ddd, J=6.5,
3.5, 3.5 Hz, 0.6H), 4.12 (ddd, J=6.5, 2.5, 2.5 Hz, 0.4H),
4.02 (m, 0.4H), 3.97 (ddd, J=9.0, 9.0, 6.5 Hz, 0.6H),
3.89–3.81 (m, 1H), 3.70 (dd, J=10.0, 5.0 Hz, 0.6H), 3.62
(dd, J=10.0, 5.0 Hz, 0.4H), 3.58 (dd, J=10.0, 5.0 Hz,
0.6H), 3.52–3.45 (m, 1.4H), 2.37–2.15 (m, 6H), 0.92 (s,
9H), 0.07 (s, 6H).
(5E)-7-[(1R,2S,3R,5R)-5-Chloro-2-hydroxymethyl-3-(tet-
rahydro-2H-pyran-2-yloxy)cyclopentyl]hept-5-enoic acid
methyl ester (37). To a stirred solution of 36 (3.02 g,
6.17 mmol) in 50 mL of THF was added 1.0 M tetra-
butylammonium fluoride in THF (8.0 mL, 8.0 mmol).
After stirring for 5 h at ambient temperature, the reac-
tion mixture was poured into ice-water and extracted
with 50% EtOAc–hexane. The organic layer was
washed first with water, and then with brine, dried over
anhydrous sodium sulfate, and concentrated in vacuo to
afford a crude oil, which was purified by column chro-
matography on silica gel to give 37 (1.84 g, 79%) as a
colorless viscous oil. MS (FAB, pos) m/z=375
(M+H)+, 291; IR (neat) 3460, 2944, 1740, 1440, 1353,
1202, 1136, 1023, 972, 870, 812 cmꢁ1
;
1H NMR
(200 MHz, CDCl3) d 5.60–5.33 (m, 2H), 4.70 (m,
0.35H), 4.57 (m, 0.65H), 4.32–3.40 (m, 9H), 2.40–1.33
(m, 18H). The compound 38 was synthesized from 37 by
the same procedure used for the preparation of 3.
(5E)-7-[(1R,2S,3R,5R)-5-Chloro-2-formyl-3-(tetrahydro-
2H-pyran-2-yloxy)cyclopentyl]hept-5-enoic acid methyl
ester (38). Colorless viscous oil. TLC Rf=0.36 (n-hex-
1
ane/EtOAc, 3/1); H NMR (200 MHz, CDCl3) d 9.75
and 9.73 (2Âd, J=0.4 Hz, 1H), 5.60–5.28 (m, 2H), 4.65–
4.50 (m, 2H), 4.17–3.96 (m, 1H), 3.92–3.71 (m, 1H), 3.67
(s, 3H), 3.58–3.39 (m, 1H), 2.74 and 2.58 (2Âm, 1H),
2.52–1.97 (m, 9H), 1.90–1.40 (m, 8H).
(16S)-9-Deoxy-9ꢀ-chloro-15-deoxy-16-hydroxy-17,17-
trimethylene-20-norPGF2 methyl ester (43). To a stirred
solution of 4 (19.8 g, 54.0 mmol) in 125 mL of dry THF
was added n-butyllithium (1.6 M in hexane, 33.8 mL,
54.0 mmol) at ꢁ78 ꢀC under an argon atmosphere. The
reaction mixture was stirred for 1 h at ꢁ78 ꢀC and then
added to a stirred solution of 3 (16.0 g, 41.5 mmol) in
125 mL of dry THF at ꢁ78 ꢀC. After stirring for 1 h at
ꢁ78 ꢀC, the resulting mixture was treated with acetic
anhydride (7.85 mL, 83.0 mmol) and allowed to warm
up to ambient temperature over 1 h. The reaction mix-
ture was treated with saturated aqueous ammonium
chloride and extracted with EtOAc repeatedly. The
combined organic layers were washed with water, then
brine, dried over anhydrous magnesium sulfate, and
concentrated in vacuo to afford a crude 39 (40.1 g) as a
dark-brown viscous oil. TLC Rf=0.41 (n-hexane/
EtOAc, 3/1); IR (neat) 2943, 2863, 1740, 1447, 1371,
(5E)-7-[(1R,2S,3R,5R)-2-(t-Butyldimethylsiloxy)methyl-
5-chloro-3-(tetrahydro-2H-pyran-2-yloxy)cyclopentyl]-
hept-5-enoic acid methyl ester (36). To a stirred solution
of 35 (5.98 g, 13.1 mmol) in 150 mL of CH2Cl2 was
added a 1.01 M toluene solution of diisobutylaluminum
hydride (14.3 mL, 14.4 mmol) at ꢁ70 ꢀC under an argon
atmosphere. The mixture was stirred for 1 h at ꢁ40 ꢀC,
and the mixture was quenched with a small amount of
methanol and 1 N aqueous hydrochloric acid. After
extraction with EtOAc, the organic layer was washed
successively with water, saturated aqueous sodium
bicarbonate, water, and finally brine, dried over anhy-
drous sodium sulfate, and concentrated in vacuo to
afford a crude aldehyde (5.96 g) as an oil. To a stirred
solution of the crude aldehyde (5.96 g, ca. 13 mmol) in
150 mL of acetone was added Jones’ Reagent (7.5 mL)
at ꢁ30 ꢀC. After stirring for 1 h at ꢁ30 ꢀC, the reaction
mixture was treated with a small amount of isopropanol
and stirred for 20 min. After the addition of water, the
mixture was extracted with EtOAc. The organic layer
was washed with water, then brine, dried over anhy-
drous sodium sulfate, and concentrated in vacuo to
afford a carboxylic acid, which was esterified with di-
azomethane to give a crude methyl ester. This crude ester
was purified by column chromatography on silica gel to
afford 36 (3.58 g, 56%) as a colorless viscous oil. TLC
Rf=0.45 (benzene/EtOAc, 5/1); 1H NMR (200 MHz,
CDCl3) d 5.53–5.37 (m, 2H), 4.60 (m, 1H), 4.23–3.40
(m, 9H), 2.40–1.00 (m, 18H), 0.90 (s, 9H), 0.04 (s, 6H).
1307, 1228, 1150, 1131, 1075, 1031 cmꢁ1 1H NMR
;
(200 MHz, CDCl3) ꢀ 8.00–7.45 (m, 5H), 5.70–5.20 (m,
2H), 5.00–3.20 (m, 14H), 2.60–1.20 (m, 37H), 1.10–0.80
(m, 3H); MS (MALDI, Pos) m/z=803 (M+Na)+.
To a stirred solution of the crude 39 (10.6 g, 11.0 mmol)
in 100 mL of methanol was added powdered magnesium
(ꢁ50 mesh, 2.40 g, 100 mmol) under an argon atmo-
sphere. The mixture was again stirred (15 min), and a
catalytic amount of chloro trimethylsilane (0.1 mL) was
added. After stirring for 1 h at ambient temperature, the
reaction mixture was poured into ice-cold aqueous
ammonium chloride and extracted with EtOAc. The
organic layer was washed with water, then brine, dried
over anhydrous magnesium sulfate, and concentrated in
vacuo to afford a crude oil, which was purified by column

1892
K. Tani et al. / Bioorg. Med. Chem. 10 (2002) 1883–1894
chromatography on silica gel to give a mixture of 13E-
and 13Z-olefinic compounds (3.0 g, 47% yield from
aldehyde 3) as a pale yellow oil, in which the ratio of
13E to 13Z was approximately 5 to 1. TLC Rf=0.66 (n-
hexane/EtOAc, 3/1); IR (neat) 2942, 2875, 1741, 1440,
(16S)-9-Deoxy-9ꢀ-bromo-15-deoxy-16-hydroxy-17,17-
trimethylene-20-norPGF2 (6). Pale yellow oil. TLC
Rf=0.40 (EtOAc/n-hexane/AcOH, 8/4/0.1); IR (neat)
3369, 2931, 1709, 1434, 1242, 1154, 1069, 968, 735 cmꢁ1
;
1H NMR (200 MHz, CDCl3) d 5.62 (dt, J=15.0, 6.0 Hz,
1H), 5.55–5.30 (m, 3H), 4.22–4.00 (m, 2H), 3.61 (dd,
J=10.0, 2.5 Hz, 1H), 2.37 (t, J=7.0 Hz, 2H), 2.50–1.30
(m, 20H), 0.93 (t, J=7.5 Hz, 3H); MS (APCI, Neg, 20
V) m/z=441, 443 (MꢁH)ꢁ; HRMS calcd for
C22H35BrO4Na [M+Na] +: 465.1616, found 465.1640.
1352, 1200, 1115, 1077, 1032, 977 cmꢁ1 1H NMR
;
(200 MHz, CDCl3) d 5.80–5.25 (m, 4H), 4.60 (m, 2H),
4.15–3.75 (m, 4H), 3.57 (s, 3H), 3.55–3.35 (m, 3H),
2.50–1.40 (m, 34H), 1.00–0.85 (m, 3H); HRMS calcd
for C33H53ClO6Na [M+Na]+: 603.3428, found
603.3446.
(16S)-9-Deoxy-15-deoxy-16-hydroxy-17,17-trimethylene-
20-norPGF2 (7). Pale yellow oil; TLC Rf=0.36 (EtOAc/
n-hexane/AcOH, 8/4/0.1); IR (neat) 3368, 2932, 1709,
To a stirred solution of the olefinic compounds (13.8 g,
23.8 mmol) in 120 mL of methanol was added p-tolu-
enesulfonic acid (226 mg, 1.14 mmol) at 0 ꢀC. After
stirring for 3 h at ambient temperature, the reaction
mixture was treated with saturated aqueous sodium
bicarbonate and extracted with EtOAc repeatedly. The
combined organic layers were washed with water, then
brine, dried over anhydrous magnesium sulfate and
concentrated in vacuo to afford a crude oil as a mixture
of 43 (13E) and the corresponding 13Z isomer. The
residue was purified by column chromatography on
silica gel to give 43 (7.64 g, 78%) as a pale yellow oil.
TLC Rf=0.47 (n-hexane/EtOAc, 1/1); IR (neat) 3369,
2932, 1739, 1437, 1220, 1157, 1070, 968 cmꢁ1; 1H NMR
(200 MHz, CDCl3) d 5.60 (ddd, J=15.0, 8.0, 5.6 Hz,
1H), 5.52–5.35 (m, 3H), 4.18–3.95 (m, 2H), 3.67 (s,
3H), 3.54 (dd, J=10.0, 2.6 Hz, 1H), 2.45–1.30 (m,
22H), 2.33 (t, J=7.6 Hz, 2H), 0.92 (t, J=7.5 Hz, 3H);
MS (APCI, Pos, 20V) m/z=395 (M+HꢁH2O)+, 377
(M+Hꢁ2H2O)+, 341 (M+Hꢁ2H2OꢁHCl)+; HRMS
calcd for C23H37ClO4Na [M+Na]+: 435.2278, found
435.2307.
1
1430, 1261, 1071, 1029, 968, 873, 799 cmꢁ1; H NMR
(200 MHz, CDCl3) d 5.61 (dt, J=15.0, 6.5 Hz, 1H),
5.55–5.25 (m, 3H), 3.88 (q, J=7.5 Hz, 1H), 3.63 (dd,
J=10.0, 2.5 Hz, 1H), 2.34 (t, J=7.0 Hz, 2H), 2.40–1.40
(m, 22H), 0.93 (t, J=7.5 Hz, 3H); MS (APCI, Neg, 20
V) m/z=363 (MꢁH)ꢁ; HRMS calcd for C22H36O4Na
[M+Na]+: 387.2511, found 387.2546.
(16S)-9-Deoxy-9ꢀ-chloro-15-deoxy-16-hydroxy-5,6-
trans-17,17-trimethylene-20-norPGF2 (9). Colorless vis-
cous oil; TLC Rf=0.44 (EtOAc/n-hexane/AcOH, 6/3/
0.1); IR (neat) 3368, 2916, 1715, 1435, 1048, 773 cmꢁ1
;
1H NMR (200 MHz, CDCl3) d 5.65–5.28 (m, 4H), 5.27–
4.40 (br, 3H), 4.19–3.97 (m, 2H), 3.55 (dd, J=10, 1.8
Hz, 1H), 2.48–1.30 (m, 22H), 0.92 (t, J=7.6 Hz, 3H);
MS (APCI, Neg. 20 V) m/z=397 (MꢁH)ꢁ; HRMS
calcd for C22H35ClO4Na [M+Na]+: 421.2122, found
421.2141.
(16S)-9-Deoxy-9ꢀ-chloro-15-deoxy-16-hydroxy-17,17-
trimethylene-20-norPGF1 (10). Colorless oil. TLC
Rf=0.47 (CHCl3/MeOH, 9/1); IR (neat) 3368, 2931,
(16S)-9-Deoxy-9ꢀ-chloro-15-deoxy-16-hydroxy-17,17-
trimethylene-20-norPGF2 (2). To a stirred solution of
43 (7.40 g, 17.9 mmol) in 90 mL of methanol, 18 mL of
2 N sodium hydroxide was added at room temperature.
After stirring for 4 h, the reaction mixture was diluted
with water and the aqueous layer was washed with n-
hexane/ether (1/1). The resulting aqueous layer was
acidified with aqueous hydrochloric acid and extracted
with EtOAc. The organic layer was washed with water,
then brine, dried over magnesium sulfate and con-
centrated in vacuo. The residue was purified by column
chromatography on silica gel to yield 2 (5.79 g, 81%) as
a pale yellow viscous oil. Optical rotation [a]2D5=ꢁ24.4
(c 1.00, EtOH); TLC Rf=0.33 (EtOAc/n-hexane/AcOH,
60/30/1); IR (neat) 3351, 2936, 1709, 1432, 1243, 1069,
1
1713, 1463, 1271, 1069, 969 cmꢁ1; H NMR (300 MHz,
CDCl3) d 5.63–5.53 (m, 1H), 5.42 (dd, J=15, 8 Hz, 1H),
4.16–4.08 (m, 1H), 4.04–3.98 (m, 1H), 3.56 (dd, J=10, 2
Hz, 1H), 2.34 (t, J=7.5 Hz, 2H), 2.30–1.20 (m, 24H),
0.92 (t, J=7.5 Hz, 3H); MS (FAB, Pos) m/z=401
(M+H)+, 365, 329; HRMS calcd for C22H37ClO4Na
[M+Na]+: 423.2278, found 423.2284. The compound 5
was synthesized from 48 according to a procedure
reported previously, except that tetrabutylammonium
fluoride was used instead of tetrabutylammonium chlo-
ride. Compounds 8 and 11 were synthesized from 49
and 50 according to the same procedure.
(16S)-9-Deoxy-9ꢀ-fluoro-15-deoxy-16-hydroxy-17,17-tri-
methylene-20-norPGF2 (5). Colorless oil. TLC Rf=0.38
(EtOAc); IR (neat) 3388, 2934, 2359, 1708, 1436, 1240,
;
1027, 968, 734, 669, 418 cmꢁ1 1H NMR (300 MHz,
CDCl3) d 5.64–5.57 (m, 1H), 5.54–5.36 (m, 3H), 4.90–
4.70 (m, 1H), 4.13–4.05 (m, 1H), 3.61 (dd, J=10, 2 Hz,
1H), 2.40–1.40 (m, 24H), 0.93 (t, J=7.5 Hz, 3H); MS
(APCI, Neg 20 V) m/z=381 (MꢁH)ꢁ; HRMS calcd
for C22H35FO4Na [M+Na]+: 405.2417, found
405.2397.
968, 866 cmꢁ1 1H NMR (200 MHz, CDCl3) d 5.56
;
(ddd, J=15.4, 8.2, 5.2 Hz, 1H), 5.55–5.30 (m, 3H),
5.60–5.00 (br, 3H), 4.20–3.96 (m, 2H), 3.56 (dd, J=10.2,
2.0 Hz, 1H), 2.42–1.30 (m, 20H), 2.35 (t, J=7.0 Hz,
2H), 0.92 (t, J=7.4 Hz, 3H); MS (FAB, Neg) m/z=397
(MꢁH)ꢁ, 361 (MꢁHꢁHCl)ꢁ; HRMS calcd for
C22H35ClO4Na [M+Na]+: 421.2122, found 421.2117.
Compounds 6, 9 and 10 were synthesized from 29, 38
and 28, respectively, by the procedure described above.
The compound 7 was synthesized from 46, which was
obtained as a by-product in the reductive elimination
reaction of 41, according to the procedure outlined
above.
(16S)-11,16-O-Bis(t-butyldimethylsilyl)-15-deoxy-16-
hydroxy-20-norPGF2ꢀ methyl ester (49). To a stirred
solution of 48 (248 mg, 0.399 mmol) in 2.7 mL of THF

K. Tani et al. / Bioorg. Med. Chem. 10 (2002) 1883–1894
1893
was added sequentially triphenylphosphine (601 mg,
2.29 mmol), diethyl azodicarboxylate (0.375 mL, 2.38
mmol) and formic acid (0.09 mL, 2.39 mmol) at 0 ꢀC.
After stirring for 1 h at 0 ꢀC, the resulting mixture was
treated with water and extracted with EtOAc. The
organic layer was washed with brine, dried over anhy-
drous magnesium sulfate, and concentrated in vacuo to
afford a formate. A mixture of this formate and 0.3 mL
of aqueous ammonia in 3 mL of methanol was stirred
for 14 h at room temperature. The resulting reaction
mixture was extracted with EtOAc. The organic layer was
washed with brine, dried over anhydrous magnesium sul-
fate, and concentrated in vacuo to afford a crude oil,
which was purified by column chromatography on silica
gel to give 49 (152 mg, 0.243 mmol) as a colorless oil. TLC
(CHCl3/CH3OH, 9/1); IR (neat) 3369, 2934, 1708, 1434,
1245, 1070, 1027, 970, 910, 734 cmꢁ1 1H NMR
;
(300 MHz, CDCl3) d 5.65–5.40 (m, 4H), 4.20–4.00 (m,
2H), 3.63 (dd, J=2.1, 10.5 Hz, 1H), 4.00–2.60 (br, 1H),
2.40–1.35 (m, 24H), 0.92 (t, J=7.5 Hz, 3H); MS (FAB,
Pos) m/z=399 (M+H)+; HRMS calcd for
C22H35ClO4Na [M+Na]+: 421.2122, found 421.2128.
(16S)-9-Deoxy-9ꢀ-chloro-15-deoxy-16-hydroxy-17,17-
trimethylene-20-norPGF2 L-lysine salt (2Ly). To a stir-
red solution of 2 (45.9 g, 110 mmol) in 460 mL of etha-
nol was added l-lysine (16.0 g, 110 mmol). To this
suspension was added 1600 mL of ethanol. The mixture
was heated at 80 ꢀC until the precipitates were dissolved,
and then the insoluble substance was removed by filtra-
tion. After cooling, 5 L of EtOAc was added to the fil-
trate. The resulting precipitates were collected by
filtration and dried in vacuo to afford l-lysine salt 2Ly
(54.6 g, 100 mmol, 91% yield) as a colorless crystal. Mp
166–168 ꢀC (dec.); Optical rotation [a]2D5=ꢁ20.4 (c 1.00,
H2O); MS (FAB, Pos) m/z=545 (M+H)+, 421
(MꢁLys+Na)+; IR (KBr) 3424, 2934, 1618, 1542,
1
Rf=0.24 (n-hexane/EtOAc, 5/1); H NMR (200 MHz,
CDCl3) d 5.58–5.35 (m, 3H), 5.25 (m, 1H), 4.04–3.90
(m, 2H), 3.66 (s, 3H), 3.56 (t, J=5.8 HZ, 1H), 2.36–1.40
(m, 22H), 0.94–0.96 (m, 3H), 0.89 (s, 9H), 0.85 (s, 9H),
0.05 (s, 3H), 0.04 (s, 3H), 0.00 (s, 3H), ꢁ0.10 (s, 3H).
(16S)-9-Deoxy-9ꢁ-chloro-15-deoxy-16-hydroxy-17,17-
trimethylene-20-norPGF2 (8). Colorless viscous oil.
TLC Rf=0.40 (EtOAc); IR (neat) 3391, 2932, 1714,
;
1434, 1378, 1262, 1073, 1031, 969 cmꢁ1 1H NMR
(200 MHz, CDCl3) d 5.75–5.31 (m, 3H), 4.41 (m, 1H),
4.02 (m, 1H), 3.64 (m, 1H), 2.68–1.33 (m, 24H), 0.93 (t,
J=7.4 Hz, 3H); MS (FAB, Pos.) m/z=363
(Mꢁ2H2O+H)+, 327 (Mꢁ2H2OꢁHCl+H)+; HRMS
calcd for C22H35ClO4Na [M+Na]+: 421.2122, found
421.2166.
1
1406, 1307, 965 cmꢁ1; H NMR (200 MHz, CD3OD) d
5.70–5.30 (m, 4H), 4.02 (q, J=7.1 Hz, 2H), 3.58–3.45
(m, 2H), 2.92 (t, J=7.3 Hz, 2H), 2.40–1.30 (m, 28H), 0.92
(t, J=7.5 Hz, 3H). Anal. calcd for C28H49ClN2O6; C,
61.69; H, 9.06; N, 5.14; found; C, 61.72; H, 9.21; N, 5.21.
X-ray crystallography of 2Ly
Colorless platelet crystals of 2Ly were obtained from
a methanol/dioxane (1/3) solution. A suitable crystal
of 2Ly having the approximate dimension of
0.10Â0.20Â0.40 mm was mounted on a glass fiber. All
measurements were made on a Rigaku AFC5R dif-
fractometer with graphite monochromated Cu-Ka
radiation and a RU-200 rotating anode X-ray gen-
erator. Of the 9156 reflections which were collected,
4523 were unique. Equivalent reflections were merged.
An empirical absorption correction was applied.
(16S)-9-Deoxy-9ꢀ-chloro-13,14-dihydro-15-deoxy-16-
hydroxy-17,17-trimethylene-20-norPGF2 (11). Colorless
viscous oil. TLC Rf=0.30 (n-hexane/EtOAc/AcOH, 1/
1/0.02); IR (neat) 3369, 2933, 1708, 1459, 1247, 1071
cmꢁ1; 1H NMR (200 MHz, CDCl3) d 5.58–5.38 (m, 2H),
4.50–3.50 (br, 3H), 4.15–4.00 (m, 2H), 3.58 (d, J=8.8
Hz, 1H), 2.40–1.20 (m, 24H), 2.37 (t, J=7.0 Hz, 2H),
0.91 (t, J=7.5 Hz, 3H); MS (FAB, Pos) m/z=401
(M+H)+, 383 (M+HꢁH2O)+, 365 (M+Hꢁ2H2O)+,
329 (M+Hꢁ2H2OꢁHCl)+; HRMS calcd for
C22H37ClO4Na [M+Na]+: 423.2278, found 423.2291.
The program package teXsan11 was used for analysis
and drawing figures. The positions of almost all the
non-H atoms were determined by the program
SHELXS86,12 and carbon atoms of the a chain of 2Ly
were found in the difference Fourier synthesis. Three
methylene atoms of the a chain of 2Ly, C4, C5 and C6,
were disordered over two sites, C40, C50 and C60,
respectively. These atoms were refined isotropically with
50% occupancy. C3 and C7 Carbon atoms were also
refined isotropically, while the other non-H atoms were
refined with anisotropic temperature parameters. The
positions of the H atoms were calculated from the
coordinates of non-H atoms and confirmed by the dif-
ference Fourier synthesis, however the H atoms of the a
chain of 2Ly were not assigned. The assigned H atoms
were included in structure factor calculations but not
refined. Space group C2, cell constant a=32.91 (2) A,
b=5.91(2) A, c=17.64(2) A, b=112.52 (6)ꢀ V=3169 (10)
A3, 1307 reflections with I>3s (I) were used for the final
(16S)-9-Deoxy-9ꢀ-chloro-15-deoxy-16-hydroxy-17,17-
trimethylene-5,6,13,14-tetrahydro-20-norPGF2
(12).
Compound 12 was synthesized from 43, by the same
procedure used for the preparation of compound 26
(catalytic hydrogenation) and compound 2 (alkaline
hydrolysis). Colorless oil. TLC Rf=0.37 (n-hexane/
EtOAc/AcOH, 1/1/0.02); IR (KBr) 3392, 2931, 2857,
1
1709, 1459, 1071 cmꢁ1; H NMR (200 MHz, CDCl3) d
4.50–2.50 (br, 3H), 4.17–3.98 (m, 2H), 3.57 (d, J=9.0
Hz, 1H), 2.35 (t, J=7.0 Hz, 2H), 2.14 (dd, J=7.0, 5.8
Hz, 2H), 2.00–1.20 (m, 26H), 0.92 (t, J=7.5 Hz, 3H);
MS (APCI, Neg., 20V) m/z=401 (MꢁH)ꢁ; HRMS
calcd for C22H39ClO4Na [M+Na] +: 425.2435, found
425.2475.
(16R)-9-Deoxy-9ꢀ-chloro-15-deoxy-16-hydroxy-17,17-
trimethylene-20-norPGF2 (13). Compound 13 was syn-
thesized from the C-16 epimer of compound 48 as
reported previously. White solid. TLC Rf=0.45
structure
factor
calculation,
2y
max=130.1ꢀ,
R=Æ||Fobs|ꢁ|Fcalc||/Æ|Fobs|=0.104, Rw=((Æw(|Fobs|-
|ꢁ|Fcalc|)2/ÆwFobs2)1/2=0.119 where w=1/s2(Fobs).

1894
K. Tani et al. / Bioorg. Med. Chem. 10 (2002) 1883–1894
Prostanoid EP1–4 receptor binding assay
References and Notes
Membranes from CHO cells expressing the mouse
prostanoid receptors were incubated with radioligand
(2.5 nM of [3H]PGE2) and the test compounds at var-
ious concentrations in assay buffer [10 mM Kpi
(KH2PO4, KOH; pH 6.0), 1 mM EDTA and 0.1 mM
NaCl]. Incubation was carried out at 25 ꢀC for 60 min
except for EP1 (20 min). The incubation was terminated
by filtration through Whatman GF/B filters. The filters
were then washed with ice-cold buffer (10 mM Kpi
(KH2PO4, KOH; pH 6.0), 0.1 mM NaCl), and the
radioactivity on the filter was measured in 6 mL of
liquid scintillation (ACSII) mixture with a liquid scin-
tillation counter. Nonspecific binding was determined
by incubation of 10 mM unlabeled PGE2 with assay
buffer.
1. (a) Tani, K.; Naganawa, A.; Ishida, A.; Egashira, H.;
Sagawa, K.; Harada, H.; Ogawa, M.; Maruyama, T.; Ohuchida,
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1
4. The optical purity of epoxide was determined by H NMR
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5. The optical purity of benzoyl ester was determined by
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6. Hayashi, M.; Arai, Y.; Wakatsuka, H.; Kawamura, M.;
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well plates (1Â105 cells/well). After 2 days, the media
were removed and cells were washed with 500 mL of
Minimum Essential Medium (MEM) and preincubated
for 10 min in 450 mL of assay buffer (MEM containing 1
mM of IBMX, 1% of BSA) at 37 ꢀC. Then the reaction
was started with the addition of each test compound in
50 mL of assay buffer. After incubation for 10 min at
37 ꢀC, the reaction was terminated by addition of 500
mL of ice-cold 10% trichloroacetic acid. The cAMP
production was measured by radioimmunoassay using a
cAMP assay kit (Amersham).
8. The ratio of 9b-Cl and Á8,9-olefinic product was deter-
mined by HPLC analysis after deprotection of 13-OH.
9. Corey, E. J.; Schaaf, T. K.; Huber, W.; Koelliker, U.;
Weinshenker, N. M. J. Am. Chem. Soc. 1970, 92, 397.
10. The ratio of 13(E) and 13(Z) was determined by HPLC
anlysis.
11. teXsan: Molecular Structure Corporation: 1993.
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