Design and synthesis of a selective EP4-receptor agonist. Part 4: Practical synthesis and biological evaluation of a novel highly selective EP4-receptor agonist

Article abstract of DOI:10.1016/S0968-0896(02)00085-8

A practical method of synthesizing a highly selective EP4-receptor agonist 1 using Corey lactone 2 as a key intermediate was developed. Selective methanesulfonylation of the primary alcohol of the diol 8 under the newly devised conditions followed by the protection of the remaining secondary alcohol are key reactions in this new method. Further biological evaluation of 1a-b is also reported.

Full text of DOI:10.1016/S0968-0896(02)00085-8

Bioorganic & Medicinal Chemistry 10 (2002) 2103–2110
Design and Synthesis of a Selective EP4-receptor Agonist.
Part 4: Practical Synthesis and Biological Evaluation of a Novel
Highly Selective EP4-Receptor Agonist
Toru Maruyama,* Shin-Itsu Kuwabe, Yasufumi Kawanaka, Tai Shiraishi,
Yoshiyuki Shinagawa, Kiyoto Sakata, Akiteru Seki, Yoko Kishida, Hideyuki Yoshida,
Takayuki Maruyama, Shuichi Ohuchida, Hisao Nakai, Shinsuke Hashimoto,
Masanori Kawamura, Kigen Kondo and Masaaki Toda
Minase Research Institute, Ono Pharmaceutical Co., Ltd., Shimamoto, Mishima, Osaka 618-8585, Japan
Received 21 January 2002; accepted 7 March 2002
Abstract—A practical method of synthesizing a highly selective EP4-receptor agonist 1 using Corey lactone 2 as a key intermediate
was developed. Selective methanesulfonylation of the primary alcohol of the diol 8 under the newly devised conditions followed by
the protection of the remaining secondary alcohol are key reactions in this new method. Further biological evaluation of 1a–b is
also reported. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
further biological evaluation of a newly discovered EP4-
receptor selective agonists 1a–b (Chart 1).
The receptor of PGE2 has been known to be divided into
four subtypes EP1, EP2, EP3 and EP4.1 Among them, the
EP4-receptor, whose cDNA was already cloned, has been
discovered to distribute in thymus, lung, heart, kidney,
bone, womb, liver and other organs.2 Stimulation of the
EP4-receptor by PGE2 leads to an increase of intracel-
lular cAMP. As such, the cytoprotective action of
PGE2, which is thought to mediate EP4-receptor, is
derived from the improvement of blood flow and the
regulation of inflammatory cytokine production.3,4
Therefore, an EP4-receptor agonist would be a useful
drug for the treatment of several deseases, mediated by
inflammatory cytokines, such as shock, rheumatoid
arthritis, hepatitis, osteoporosis and others. However,
the physiological and pathological roles of the EP4-
receptor have not been clarified yet because of a lack of
a highly selective EP4-receptor agonist. In a preceding
paper,5 we reported the discovery of highly selective
EP4-receptor agonists, which will be useful to clarify the
patho-physiological roles of the EP4-receptor subtype.
We report here a practical method for the synthesis and
Chemistry
The synthesis of 5-thiaPGE1 using a three-component
coupling process, was first reported6 in 1985. It was not
a practical method for large-scale synthesis because of
the multi-step reaction process, toxic reagent and poor
reproducibility.
A newly developed method starting with the THP-lac-
tone 2,7 large amounts of which are commercially
available, enabled us to synthesize many analogues of
5-thiaPGE1.
The synthesis of 1a–b is outlined in Scheme 1. Oxidation
of 2 to an aldehyde 3 followed by a Horner–Emmons
reaction of the aldehyde 3 with the phosphonate 4, which
was prepared from (3-methoxymethyl)phenylacetic acid
in two steps, afforded an enone 5. Using ethyl acetate as
a co-solvent, the oxidation reaction of 2 could be car-
ried out at a lower temperature. As a result, the yield of
3 was remarkably improved while 3 was chemically
unstable in the presence of a base.8 Stereoselective
*Corresponding author. Tel.: +81-75-961-1151; fax: +81-75-962-
9314; e-mail: to.maruyama@ono.co.jp
9
reduction of the enone 5 with SBNreagent provided
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00085-8

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T. Maruyama et al. / Bioorg. Med. Chem. 10 (2002) 2103–2110
Scheme 1. Synthesis of 1a–1b. Reagents and conditions: (a) SO3Py, Et3N, EtOAc, DMSO; (b) phosphonate 4, N aH, THF; (c) (S)-BlNOL, LiAlH4, EtOH,
THF; (d) DHP, p-TsOH–H2O, toluene; (e) LiAlH4, THF; (f) MsCl, i-Pr2NEt, MeOH, TMSCl, THF; (g) AcSK, K2CO3, DMF; (h) methyl 4-iodobutyrate,
K2CO3, MeOH; (i) SO3Py, i-Pr2NEt, EtOAc, DMSO; (j) 0.1N HCl, CH3CN, MeOH; (k) PLE, EtOH, phosphate buffer.
Chart 1. Highly selective EP4-receptor agonists.
Scheme 2. Formation of undesired bicyclic ether 14.
15(S)-isomer 6 (84% d.e.) in good yield. Protection of
the newly formed hydroxy group of 6 as a tetra-
hydropyranyl (THP) ether followed by the reduction of
the g-lactone moiety of 7 with lithium aluminum hydride
gave a diol 8 in a nearly quantative yield.
monomethanesulfonylation of 8 by the reported
method10 was carried out as described below.
A highly selective methanesulfonylation reaction took
place in a good yield at the lower temperature using
MsCl/i-Pr2NEt in THF while it was not successful in
dichloromethane or toluene because of the formation of
the cyclized product 14. Et3Nand DMAP were not
acceptable as a base to this reaction because of the pre-
sumed formation of a reactive intermediate different
from that of the successful reaction described above.11
An efficient conversion of 8 to 11 was accomplished by
selective substitution of the primary alcohol group of 8
with a sulfur moiety. The following reaction conditions
were studied. A selective monosubstitution of the pri-
mary alcohol group of 8 with thioacetic acid under
Mitsunobu reaction conditions was attempted to afford
an undesired product 14 (Scheme 2) because of the pre-
sumed formation of an intermediate such as 13. As an
alternative, aryl- or alkyl sulfonylation of the primary
alcohol followed by the introduction of the S-alkyl
moiety was investigated. But a selective monotosylation
of the primary alcohol of 8 by a conventional method
again produced a bicyclic ether 14. Success was achieved
with the application of a selective monomethane-
sulfonylation of a primary alcohol followed by a sub-
stitution reaction with potassium thioacetate. Selective
The addition of methanol to decompose any excess of
the active species followed by the addition of trimethyl-
silyl chloride (TMSCl) enabled us to protect the secondary
alcohol as a trimethylsilyl ether. Decomposition of the
excess reagent prior to the addition of potassium thioa-
cetate was needed to carry out the sequential reactions
in a one-pot manner. The resulting thiol acetate 10 was
converted to 11 by the simultaneous methanolysis of the
thiol acetate and the trimethylsilyl ether followed by S-
alkylation with methyl 4-iodobutanoate in the presence

T. Maruyama et al. / Bioorg. Med. Chem. 10 (2002) 2103–2110
2105
of K2CO3/MeOH. Oxidation of the 9-hydroxy group of
.
11 with SO3 Py complex in DMSO-iPr2NEt afforded
9-keto derivative 12 in good yield. The Swern oxidation
and Corey–Kim oxidation of 11 were not successful
because of a presumed intramolecular interaction of the
9-sulfoxonium moiety with the 5-thia moiety as illu-
strated in Chart 2. This type of intramolecular interaction
.
was estimsted to be avoided by using SO3 Py in DMSO-
Et3Nas an oxidative reagent because of its low reactivity,
while excess reagent and a relatively long reaction time
were needed to complete the reaction. Replacement of
triethylamine with a stronger base (i-Pr2NEt) was again
effective in completing the oxidative reaction at a lower
temperature in a shorter reaction time. Undesired elimina-
tion of the 11-THPOH and a Pummerer type rearrange-
ment were successfully avoided under these improved
oxidation conditions. In the final deprotection reaction
of PGE synthesis, the degradation of PGEs to PGAs
has been a serious problem. To solve this problem, mild
deprotection conditions were investigated. The depro-
tective reaction proceeded without the formation of
PGA on addition of a catalytic amount of diluted HCl
in MeOH-CH3CN. As such, cyclodextrin clathrates of
1a, prepared according to the conventional procedure,
are available as a stable formulation.
Figure 1. Binding affinity of 1b in the receptor subtypes.
hEP4-receptor (KihEP4=1.3 nM). 1b did not show affi-
nity to the other prostanoid receptors such as mFP,
mDP, hTP and hIP at 10 mM.
With regard to the agonist activities of 1b in the EP1,
EP3, FP and TP subtypes, the ability of 1b to increase the
intracellular Ca2+ concentration was evaluated using
fura-2. The agonist activities of 1b in the EP2, EP4, DP
and IP subtypes were evaluated from its ability to
increase the intracellular cAMP level. The subtype selec-
tivity of 1b was also evaluated based on EC50 values. The
EP4-receptor selectivity of 1b was retained also in the
functional assay. Interestingly, the ratio of EC50EP3/
EC50EP4 was increased 250-fold while the ratio of
KiEP3/KiEP4 was increased 80-fold. Compound 1b also
demonstrated potent agonist activity in both mEP4- and
hEP4-receptors (EC50hEP4=1.7 nM) while it did not
show significant EC50 values for the other subtypes
mFP, mDP, hTP and hIP.
Biological Results and Discussion
Binding assay and intracellular signal transduction assay
(Table 1 and Fig. 1)
Results of a binding assay in CHO cells, which express
prostanoid receptors mEP1-EP4, hEP4, mFP, hTP and
hIP, are demonstrated in Table 1. Using the membrane
fractions of the cells, the binding assay of 1b was per-
formed by the reported method12 with minor modifi-
cation. 1b demonstrated potent EP4-receptor affinity
and subtype selectivity although it showed some affinity
also to the other subtypes mEP3 and mEP2. The potent
mEP4-receptor affinity of 1b was reproduced in the
LPS-induced changes of cytokine production in human
whole blood cells (Fig. 2a and b)
As shown in Figure 2a and b, 1b was evaluated to
elucidate whether it regulates LPS-induced cytokine
production in human whole blood cells. The selective
EP4-receptor agonist 1b suppressed TNF-a production
and augmented IL-10 production at 10ꢀ9 M. Based
on the experimental results obtained with this highly
selective agonist, cytokine regulation was strongly
suggested to be one of the EP4-receptor-mediated biolo-
gical activities.13 The compound 1b suppressed TNF-a
production and increased IL-10 production at a lower
concentration than PGE1 while it exhibited the same
potency as PGE1. The EP3-receptor decreased the con-
centration of intracellular cAMP coupled with Gi.12 Thus
the EP3-receptor plays an opposite role in the regulation
of the intracellular cAMP level. Accordingly, PGE1 was
thought to suppress the EP4-receptor-mediated activity
through the EP3-receptor mediated activity. As a result
of the increased EP4-receptor selectivity, 1b exhibited a
regulatory effect on the cytokines at such a low con-
centration. At higher concentrations, the suppression of
Chart 2. Presumed intramolecular interaction.
Table 1. Binding affinity (Ki) and agonist activity (EC50) of 1ba
mEP1 mEP2 mEP3 mEP4 hIP mDP mFP hTP
Ki (nM)
EC50 (nM) >104 230
>104 620
56
400
0.70 >104 >104 >104 >104
1.6 >104 >104 >104 >104
aUsing membrane fractions of CHO cells expressing the prostanoid
receptors, Ki values were determined by competitive binding assay,
which was performed according to the method of Kiriyama with some
modifications.12 With regard to the subtype-receptor agonist activity,
EC50 values were determined based on the effects of the test com-
pounds on the increase in the intracellular c-AMP production in EP4,
EP2, IP and DP receptor and in the intracellular Ca2+ production in
EP1, EP3, FP and TP receptor.

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T. Maruyama et al. / Bioorg. Med. Chem. 10 (2002) 2103–2110
Figure 2. (a) Inhibition of LPS-induced TNF-a production in human
whole blood; (b) increase of LPS-induced IL-10 production in human
whole blood.
Figure 3. (a) Inhibition of LPS-induced TNF-a production in rats; (b)
increase of LPS-induced IL-10 production in rats.
TNF-a production by 1b was less potent than that by
PGE1 (Fig. 2). The EP2-receptor agonist activity was
considered to be one of the reasons. Because the EP2
subtype has been known to regulate cytokine produc-
tion by increasing the intracellular cAMP concentration
like the EP4 subtype, the higher concentration of PGE1
is estimated to show maximum activity by EP4- and
EP2-receptor-mediated activity.
the EP4-receptor was considered to mediate various
cytoprotective effects of PGE1 and PGE2 by regulating
cytokine production. Selective stimulation of the
EP4-receptor with 1a was suggested to be a therapeutic
approach to the treatment of several deseases related to
TNF-a and IL-10, such as rheumatoid arthritis,15 hepati-
tis,16 osteoporosis,17 and others. Besides, the therapeutic
dose of 1a in the model, was found to be lower than that
of the non-selective agonist, natural PGE2.
LPS-induced changes of plasma TNF-ꢀ and IL-10 levels
in rats (Fig. 3a and b)
More details about the role of the EP4-receptor are
now being disclosed in our laboratory using the newly
discovered selective EP4-receptor agonists 1a–b.
The effect of 1a, which was metabolically converted to
the free acid form 1b, on LPS-induced changes of TNF-
a and IL-10 levels in the plasma of rats was evaluated.
As demonstrated in Figure 3, increased production of
the plasma TNF- a after the intraveneous administra-
tion of LPS (1 mg/kg) was significantly suppressed by the
intraveneous infusion of 1a (10, 30, 100 and 300 ng/kg/
min) in a dose-dependent manner. The plasma IL-10
level after the administration of LPS was augmented by
the intraveneous infusion of 1a (30, 100 and 300 ng/kg/min)
in a dose-dependent manner.14 Based on these results,
In summary, we have succeeded in the practical synth-
esis of the EP4-receptor agonist 1a starting from Cor-
ey’s lactone 2, which is available in large quantities. The
success was based on complete selectivity in the newly
devised selective mono-methanesulfonylation of the diol
8. As a result, a one-pot reaction to obtain 10 from 8
was accomplished. Further biological evaluations of 1a
and 1b were performed both in vitro and in vivo. Based
on the results of the binding assay and functional assay,

T. Maruyama et al. / Bioorg. Med. Chem. 10 (2002) 2103–2110
2107
the EP4-receptor selectivity and agonist activity of 1a
and 1b are expected to be reproduced in humans.
THF (1.0 L) was added a solution of dimethyl 3-[(3-
methoxymethyl)phenyl]-2-oxopropanephosphonate
4
(42.9 g, 138 mmol) at room temperature under Ar. After
stirring for 1 h, to the resulting suspension was added a
solution of 3 in THF (300 mL). The mixture was stirred
for 30 min before the addition of acetic acid (7.3 mL,
127 mmol). The resulting yellow solution was diluted
with EtOAc (600 mL), washed with water (600 mL) and
brine (300 mL), and dried over MgSO4. The solvent was
removed by evaporation and the residue was purified by
column chromatography on silica gel (Wako gel C-200,
750 g, EtOAc/hexane, 1/1) to give 5 as a pale yellow oil.
(40.3 g, 83.1%).
Experimental
General procedures
Analytical samples were homogeneous as confirmed by
TLC, and afforded spectroscopic results consistent with
the assigned structures. Proton nuclear magnetic reso-
nance spectra (1H NMR) were obtained on a Varian
Gemini-200 or VXR-200s spectrometer using deuterated
chloroform (CDCl3) or deuterated methanol (CD3OD) as
the solvent. Fast atom bombardment mass spectra (FAB-
MS) were obtained on a JEOL JMS-DX303HF spectro-
meter or a JEOL JMS-AX-500 spectrometer. Atmo-
spheric pressure chemical ionization (APCI) was
determined on a Hitachi M1200H spectrometer. Infra-
red spectra (IR) were measured on a Perkin-Elmer FT-
IR 1760X spectrometer or a Jasco Varolar-II spectro-
meter. Optical rotations were measured using a Jasco
DIP-370 polarimeter or a Jasco DIP-1000 polarimeter.
Results of elemental analyses were uncorrected. Column
chromatography was carried out on silica gel [Merck
silica gel 60 (0.063–0.200 mm), Wako gel C-200 or Fuji
Silysia BW235]. Thin layer chromatography was per-
formed on silica gel (Merck TLC or HPTLC plates, silica
gel 60 F254). The following abbreviations for solvents
and reagents are used: ethyl acetate (EtOAc), dimethyl-
sulfoxide (DMSO), tetrahydrofuran (THF), methanol
(MeOH), TBME (t-butylmethylether), hydrochloride
(HCl), sodium bicarbonate (NaHCO3), sulfur trioxide
Rf 0.26 (EtOAc/hexane, 1/1); IR (neat) 2942, 1770,
1695, 1669, 1627, 1444, 1382, 1339, 1185, 1078, 1035,
1
974, 916, 871, 815, 762, 706 cmꢀ1; H NMR (300 MHz,
CDCl3) d 7.37–7.09 (m, 4H), 6.78–6.64 (m, 1H), 6.22 (d,
J=15.6 Hz, 1H), 5.03–4.88 (m, 1H), 4.66–4.53 (m, 1H),
4.44 (s, 2H), 4.22–3.94 (m, 1H), 3.88–3.32 (m, 2H), 3.81
(s, 2H), 3.39 (s, 3H), 2.92–2.05 (m, 5H), 1.90–1.35 (m,
7H); MS (FAB, Pos.) m/z: 415 (M+H)+; HRMS
(MALDI-TOF) calcd for C24H30O6+Na+: 437.1940;
found: 437.1916.
6-{(1E)(3S)-3-Hydroxy-4-[3-(methoxymethyl)phenyl]but-
1-enyl}-7-(2H-3,4,5,6-tetrahydropyran-2-yloxy)(5S,7S,1R,
6R)-2-oxabicyclo[3,3,0]octan-3-one 6. A pellet of lithium
aluminum hydride (8.10 g, 213 mmol) in THF (190 mL)
was stirred vigorously at 60 ꢁC under Ar for 2 h. The
resulting suspension was cooled to room temperature
and a solution of ethanol (9.83 g, 213 mmol) in THF
(100 mL) was added slowly. After stirring for 30 min, a
solution of (S)-(ꢀ)-1,10-bi-2-naphthol (61.1 g, 213 mmol)
in THF (200 mL) was added to the suspension over
20 min. After stirring overnight, the suspension was
cooled with a dry-ice MeOH bath and a solution of 5
(17.7 g, 42.7 mmol) in THF (200 mL) was added slowly
at a temperature below ꢀ60 ꢁC. After stirring for 2 h,
the resulting suspension was quenched with MeOH
(25 mL, 617 mmol) and warmed to 0 ꢁC. The mixture
was poured into aqueous sodium bitartrate (75 g/1.5 L)
and extracted with EtOAc (300 mLꢂ2) repeatedly. The
organic layer was washed with brine (200 mL), and
dried over MgSO4. The solvent was evaporated and the
residual solid was dissolved in 2-propanol (100 mL) and
diisopropylether (100 mL) at 60 ꢁC. To this stirred solu-
tion was added hexane (250 mL) and the mixture was
allowed to cool to room temperature in 30 min. The
precipitates were removed by filtration and the filtrate
was evaporated. The residue was purified by column
chromatography on silica gel (Wako gel C-200, 460 g,
EtOAc, hexane, 2/1) to give 6 as a colorless oil (13.9 g,
78.2%, 15a/15b=92/8).
.
pyridine complex (SO3 Py).
Synthesis of 1
3-(2H-3,4,5,6-Tetrahydropyran-2-yloxy)(1S,3S,2R,5R)-
6-oxa-7-oxobicyclo[3,3,0]octane-2-carbaldehyde 3. To a
stirred solution of 2 (30 g, 117 mmol) and triethylamine
(97.9 mL, 702 mmol) in ethyl acetate (380 mL) was added a
.
solution of SO3 Py (55.9 g, 351 mmol) in EtOAc (100 mL)
and DMSO (189 mL) slowly at a temperature below 10 ꢁC
under Ar. After stirring for an additional 1 h, the mixture
was combined with 1 NHCl (330 mL) slowly. The two
layers were separated and the aqueous layer was extracted
with EtOAc (200 mL) and THF (100 mL) repeatedly.
The combined organic layer was washed with water
(60 mL), saturated aqueous NaHCO3 (60 mL) and brine
(60 mL), and dried over MgSO4. The solvent was removed
by evaporation to give 3 as a pale yellow oil (29.3 g), which
was used in the next reaction without purification.
Rf 0.60 (EtOAc/AcOH, 100/1); IR (neat) 2944, 2731,
1
1768, 1724, 1181 cmꢀ1; H NMR (300 MHz, CDCl3) d
9.73 (m, 1H), 5.11–5.00 (m, 1H), 4.81–4.51 (m, 2H),
3.92–3.79 (m, 1H), 3.59–3.35 (m, 2H), 3.20–3.03 (m,
1H), 2.90 and 2.87 (dd, J=18, 6.3 Hz, 1H), 2.57 and
2.44 (dd, J=18, 3.2 Hz, 1H), 2.40–2.30 (m, 1H), 1.97–
1.42 (m, 7H); MS (FAB, Pos.) m/z: 255 (M+H)+.
Rf 0.42 (EtOAc/benxene, 2/1); IR (neat) 3436, 2942,
1772, 1445, 1381, 1243, 1185, 1078, 1034, 974, 916, 871,
811, 705 cmꢀ1; 1H NMR (300 MHz, CDCl3) d 7.34–7.09
(m, 4H), 5.61 (dd, J=15.6, 6.0 Hz, 1H), 5.52–5.43 (m,
1H), 5.02–4.88 (m, 1H), 4.71–4.66 and 4.66–4.60 (m,
1H), 4.43 (s, 2H), 4.38–4.28 (m, 1H), 4.12–3.89 (m, 1H),
3.89–3.78 (m, 1H), 3.54–3.32 (m, 1H), 3.40 (s, 3H),
2.92–2.24 (m, 5H), 2.18–2.05 (m, 2H), 1.85–1.40 (m,
6-{(1E)-4-[3-(Methoxymethyl)phenyl]-3-oxobut-1-enyl}-
7-(2H-3,4,5,6-tetrahydropyran-2-yloxy)(5S,7S,1R,6R)-2-
oxabicyclo[3,3,0]octan-3-one 5. To a stirred suspension
of sodium hydride (61.1% in oil, 4.98 g, 127 mmol) in

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T. Maruyama et al. / Bioorg. Med. Chem. 10 (2002) 2103–2110
8H); MS (FAB, Pos.) m/z: 417 (M+H)+; HRMS
(MALDI-TOF) calcd for C24H32O6+Na+: 439.2097;
found: 439.2074.
temperature for an additional 30 min and then treated
with MeOH (0.35 mL, 8.63 mmol). After stirring for
30 min, to the reaction mixture was added TMSCl
(4.1 mL, 32.4 mmol) dropwise in 5 min. The mixture was
stirred at a temperature between ꢀ10 and ꢀ5 ꢁC for
another 10 min. Then it was diluted with anhydrous
DMF (165 mL), and potassium carbonate (8.6 g,
63 mmol) and potassium thioacetate (4.9 g, 43.2 mmol)
was added successively. The reaction mixture was stir-
red at 50 ꢁC for 4 h. It was cooled to room temperature
and diluted with TBME (160 mL). The mixture was
washed with water (ꢂ2) and brine, and dried over
MgSO4. Removal of the solvent by rotary evaporator
gave 10 as a brown oil (14.2 g), which was used for the
next reaction without purification.
6-{(1E)(3S)-3-(2H-3,4,5,6-tetrahydropyran-2-yloxy)-4-
[3-(methoxymethyl)phenyl]but -1-enyl}-7-(2H-3,4,5,6-
tetrahydropyran - 2 - yloxy)(5S,7S,1R,6R) - 2 - oxabicyclo
[3,3,0]octan-3-one 7. To a stirred solution of 6 (17.9 g,
43.0 mmol) and 3,4-dihydropyran (4.71 mL, 51.6 mmol)
in toluene (172mL) was added a solution of para-tolu-
enesulfonic acid monohydrate (81.8 mg, 0.430 mmol) in
THF (1mL) at room temperature under Ar. After stirring
for 45min, the solution was treated with triethylamine
(0.090 mL, 0.645 mmol) and then concentrated to give 7
as a colorless oil (21.5 g), which was used for the next
reaction without purification.
Rf 0.74 (EtOAc/hexane, 1/4); IR (neat) 2942, 1693,
1488, 1441, 1383, 1353, 1323, 1251, 1201, 1183, 1134,
1113, 1078, 1021, 975, 915, 885, 870, 842, 815, 749,
Rf 0.79 (EtOAc/hexane, 2/1); IR (neat) 2942, 1778,
1442, 1353, 1243, 1201, 1158, 1117, 1078, 1022, 977,
1
1
917, 870, 815, 705 cmꢀ1; H NMR (300MHz, CDCl3) d
703 cmꢀ1; H NMR (200 MHz, CDCl3) d 7.35–7.05 (m,
7.28–7.05 (m, 4H), 5.60–5.26 (m, 2H), 4.98–4.46 (m, 3H),
4.41 (s, 2H), 4.30–3.72 (m, 2H), 3.54–3.20 (m, 4H), 3.38 (s,
3H), 3.04–2.23 (m, 5H), 2.14–1.90 (m, 2H), 1.90–1.30 (m,
13H); MS (FAB, Pos.) m/z: 501 (M+H)+; HRMS
(MALDI-TOF) calcd for C29H40O7+Na+: 523.2672;
found: 523.2712.
4H), 5.50–5.30 (m, 2H), 4.80–4.40 (m, 2H), 4.34 (s, 2H),
4.35–4.20 (m, 1H), 4.20–4.00 (m, 1H), 3.95–3.60 (m,
3H), 3.50–3.30 (m, 1H), 3.37 (s, 3H), 3.30–3.15 (m, 1H),
3.15–3.00 (m, 1H), 3.00–2.70 (m, 3H), 2.60–2.40 (m,
1H), 2.31 (s, 3H), 1.90–1.10 (m, 17H), 0.10 (s, 9H).
16-(3-Methoxymethyl)phenyl-!-tetranor-5-thiaPGF1
methyl ester 11,15 - bis(tetrahydropyran- 2-yl ether) 11.
To a stirred solution of 10 (14.2 g, 21.6 mmol) and
methyl 4-iodobutyrate (5.91 g, 25.9 mol) in anhydrous
MeOH (88 mL) was added potassium carbonate (7.16 g,
51.8 mol) in one portion at room temperature under Ar.
After stirring for 2 h, the mixture was diluted with
TBME (90 mL) and poured into aqueous NH4Cl. The
organic layer was washed with water (90 mL) and brine
(90 mL). The solution was dried over MgSO4 and deco-
lorized with activated carbon powder (Takeda, Shir-
asagi A, 2.0 g). The mixture was filtered through a Pad
of Celite and the filtrate was concentrated. The residue
was purified by column chromatography on silica gel
(BW235, 220 g, EtOAc/hexane, 2/3ꢃ1/1) to give 11 as a
pale yellow oil (10.5 g, 78.4% in three steps).
11(R),15(S)-Bis(tetrahydro-2-pyranyloxy)-5,9(S)-dihy-
droxy-16-(3-methoxymethyl)phenyl-1,2,3,4,17,18,19,20-
octanorprostanoic acid 8. To a stirred suspension of
LiAlH4 (1.54 g, 40.5 mmol) in anhydrous THF (80 mL),
cooling in an ice-brine bath, was added dropwise a
solution of 7 (21.1 g, 42.2 mmol) over 15 min. After
stirring for 45 min, to the reaction mixture was added
MeOH (4.5 mL) slowly. The mixture was poured into
aqueous sodium bitartrate (14 g/280 mL) and extracted
with EtOAc (100 mL) repeatedly. The combined organic
layer was washed with brine (100 mL), and dried over
MgSO4. The solvent was removed by evaporation to
give 8 as a colorless oil (21.2 g), which was used for the
next reaction without purification.
Rf 0.35 (EtOAc); IR (neat) 3402, 2941, 1441, 1383, 1352,
1201, 1184, 1134, 1114, 1077, 1021, 976, 913, 869, 813,
Rf 0.42 (EtOAc/hexane, 1/1); IR (neat) 3467, 2942,
1739, 1440, 1368, 1321, 1261, 1201, 1135, 1021, 977,
1
704 cmꢀ1; H NMR (300 MHz, CDCl3) d 7.28–7.07 (m,
1
4H), 5.64–5.24 (m, 2H), 4.78–4.43 (m, 2H), 4.41 (s, 2H),
4.38–4.08 (m, 2H), 4.04–3.26 (m, 7H), 3.39 (s, 3H), 3.10–
1.15 (m, 22H); MS (FAB, Pos.) m/z: 505 (M+H)+;
HRMS (MALDI-TOF) calcd for C29H44O7+Na+:
527.2985; found: 527.3008.
908, 869, 813, 704 cmꢀ1; H NMR (300 MHz, CDCl3) d
7.31–7.07 (m, 4H), 5.65–5.29 (m, 2H), 4.75–4.59 (m,
2H), 4.43 (s, 2H), 4.31–3.19 (m, 7H), 3.67 (s, 3H), 3.37
(s, 3H), 3.00–2.70 (m, 2H), 2.65–2.03 (m, 10H), 2.01–
1.28 (m, 16H); MS (FAB, Pos.) m/z: 621 (M+H)+;
HRMS (MALDI-TOF) calcd for C34H52O8S+Na+:
643.3281; found: 643.3300.
11(R),15(S)-Bis(tetrahydropyran-2-yloxy)-16-(3-methoxy-
methyl)phenyl-1,2,3,4,17,18,19,20-octanor-5-acetylthio-9
(S) - trimethylsilyloxyprostanoic acid 10. To a stirred
solution of MsCl (2.0 mL, 25.9 mmol) in anhydrous
THF (70 mL) was added dropwise diisopropylethyla-
mine (4.5 mL, 25.9 mmol) in 6 min at ꢀ15 ꢁC under Ar
atmosphere. The resulting slightly suspended solution was
stirred at that temperature for 20 min. To the reaction
mixture was added a mixed solution of 8 (10.9 g, 21.6
mmol) and diisopropylethylamine (6.4 mL, 36.7 mmol) in
anhydrous THF (40mL) dropwise over 17 min (ꢀ15 to
ꢀ10 ꢁC). The resulting suspension was stirred at that
16-(3-Methoxymethyl)phenyl-!-tetranor-5-thiaPGE1
methyl ester 11,15 - bis(tetrahydro- 2-pyranyl ether) 12.
To a stirred solution of 11 (10.2 g, 16.4 mmol) and dii-
sopropylethylamine (17.2 mL, 98.6 mmol) in EtOAc
(63 mL) and DMSO (43 mL) was added a solution of
.
SO3 Py (7.85 g, 49.3 mmol) in EtOAc (5 mL) and
DMSO (25 mL) slowly at a temperature below 10 ꢁC
under Ar. After stirring for 50 min, the reaction mixture
was diluted with TBME (165 mL) and quenched with
water (33 mL) slowly. The mixture was washed with

T. Maruyama et al. / Bioorg. Med. Chem. 10 (2002) 2103–2110
2109
ice-cooled 1 NHCl (100 mL) and the aqueous layer was
extracted with TBME (50 mL). The combined organic
layer was washed with water (30 mL), saturated aqueous
NaHCO3 (30 mL) and brine (30 mL), and dried over
MgSO4. The solvent was removed by evaporation and
the residue was purified by column chromatography on
silica gel (BW235, 310 g, EtOAc/hexane, 1/2) to give 12
as a pale yellow oil (9.20 g, 90.7%).
3H), 3.32 (br, 3H), 2.90 (dd, J=14, 5.6 Hz, 1H), 2.83
(dd, J=14, 7.1 Hz, 1H), 2.70 (dd, J=19, 7.8 Hz, 1H),
2.60 (t, J=7.2 Hz, 2H), 2.55–2.16 (m, 7H), 1.94–1.61
(m, 4H); MS (APCI) m/z: 435 (MꢀH)ꢀ; HRMS
(MALDI-TOF) calcd for C23H32O6S+Na+: 459.1817;
found: 459.1850.
Prostanoid receptor binding assay
Rf 0.45 (EtOAc/hexane, 1/1); IR (neat) 2942, 1740,
1440, 1353, 1200, 1133, 1077, 1021, 975, 911, 870, 815,
Membranes from CHO cells expressing prostanoid
receptors were incubated with radioligand (2.5 nM of
[3H]PGE2 for EP1–4, 2.5 nM of [3H]PGF2a for FP,
2.5 nM of [3H]SQ29548 for TP, 5.0 nM of [3H]Iloprost
for IP, or 2.5 nM of [3H]PGD2 for DP) and the test
compounds at various concentrations in assay buffer
[10 mM Kpi (KH2PO4, KOH; pH 6.0), 1 mM EDTA and
0.1 mM NaCl, for EP1-4-receptors; 50mM Tris–HCl (pH
7.5), 1 mM EDTA and 10mM MgCl2 for IP-receptor].
Incubation was carried out at 25ꢁC for 60min except for
EP1 (20 min) and IP (30 min) receptors. 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 for
EP1–4; 10mM Tris–HCl (pH 7.5), 0.1 mM NaCl for IP),
and the radioactivity on the filter was measured in 6 mL of
liquid scintillation (ACSII) mixture with a liquid scintilla-
tion counter. Nonspecific binding was determined by
incubation of unlabeled ligand with assay buffer.
1
704 cmꢀ1; H NMR (200 MHz, CDCl3) d 7.3–7.1 (m,
4H), 5.75–5.5 (m, 2H), 4.8–4.7 and 4.7–4.6 (m, 1H), 4.7–
4.6 and 4.5–4.4 (m, 1H), 4.43 (s, 2H), 4.4–4.25 (m, 1H),
4.2–4.05 and 4.0–3.9 (m, 1H), 3.9–3.7 and 3.55–3.4 (m,
3H), 3.67 (s, 3H), 3.38 (s, 3H), 3.3–3.2 (m, 1H), 3.05–2.9
(m, 1H), 2.9–2.6 (m, 2H), 2.6–2.2 (m, 7H), 2.2–2.0 (m,
1H), 2.0–1.3 (m, 17H); HRMS (MALDI-TOF) calcd for
C34H50O8S+Na+: 641.3124; found: 641.3077.
16-(3-Methoxymethyl)phenyl-!-tetranor-5-thiaPGE1
methyl ester 1a. To a stirred solution of 12 (10.0 g,
16.2 mmol) in acetonitrile (32 mL) and methanol
(16 mL) was added 0.1 NHCl (16 mL, 1.6 mmol) at
35 ꢁC under Ar. After stirring for 3 h, the solution was
cooled in an ice-bath and diluted with EtOAc (120 mL).
The mixture was washed with aqueous NaHCO3
(2.72 g/90 mL) and the aqueous layer was extracted with
EtOAc (60 mL). The combined organic layer was
washed with brine (50 mL) and dried over MgSO4. The
solvent was removed by evaporation and the residue
was purified by column chromatography on silica gel
(BW235, 300 g, EtOAc–EtOAc/MeOH, 50/1) to give 1a
as a colorless oil (5.40 g, 74.2%).
Measurement of cAMP production
CHO cells expressing EP4-, EP2-, IP- or DP-receptors
were cultured in 24-well plates (1ꢂ105 cells/well). After
2 days, the medium was removed and cells were washed
with 500 mL of Minimum Essential Medium (MEM) and
preincubated for 10min in 450 mL of assay buffer (MEM
containing 1 mM of IBMX, 1% of BSA) at 37ꢁC. Then,
the reaction was started with the addition of the test
compound in 50mL of assay buffer. After incubation for
10min at 37ꢁC, 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).
Rf 0.39 (CHCl3/MeOH, 9/1); [a]2D5 ꢀ41.8 (c 1.0, CHCl3);
IR (neat) 3410, 2923, 1744, 1438, 1366, 1317, 1194,
1
1158, 1090, 1032, 971, 888, 792, 704 cmꢀ1; H N MR
(200 MHz, CDCl3) d 7.30–7.10 (m, 4H), 5.75 (dd, J=6,
15 Hz, 1H), 5.53 (dd, J=8, 15 Hz, 1H), 4.43 (s, 3H),
4.45–4.35 (m, 1H), 3.96 (q, J=9 Hz, 1H), 3.67 (s, 3H),
3.42 (s, 3H), 2.95–2.75 (m, 2H), 2.80–2.00 (m, 10H),
2.00–1.60 (m, 4H); MS (FAB, Pos.) m/z: 451 (M+H)+.
Anal. calcd for C24H34O6S: C, 63.97; H, 7.61; S, 7.12.
Found: C, 63.68; H, 7.90; S, 7.41.
Measurement of intracellular Ca2+ production
16-(3-Methoxymethyl)phenyl-!-tetranor-5-thiaPGE1 1b.
A heterogeneous mixture of 1a (3.03 g, 6.72 mmol) and
porcine liver esterase (PLE, Sigma, 20000U, 3.0 mL) in
EtOH (30 mL) and phosphate buffer (pH 7.4, 300 mL)
was stirred for 1 h at room temperature. The resulting
clear solution was poured into saturated aqueous
(NH4)2SO4 and the mixture was extracted with EtOAc
twice. The organic layer was dried (Na2SO4) and con-
centrated, and the residue was purified by column
chromatography on silica gel (CHCl3 to CHCl3/MeOH,
30/1) to afford 1b as a pale yellow oil (2.67 g, 91%).
The intracellular Ca2+ concentration was measured
using a Jasco CAM220 Spectrofluorometer. Chinese
Hamster Ovary (CHO) cells expressing EP1-, EP3-, TP-
or FP-receptors were cultured for two days. After the
medium was removed, the cells were washed with PBS
and centrifuged at 800 rpm for 3 min. The cells were
incubated at 37 ꢁC for 60 min with fura 2-AM in a con-
ditioned medium consisting of MEM containing 20 mM
indomethacin, 10% FCS and 10 mM HEPES–NaOH
(pH 7.4). The medium containing the cells was cen-
trifuged at 800 rpm for 3 min and the cells were sus-
pended in assay buffer consisting of MEM containing
2 mM indomethacin, 0.1% BSA and 10 mM HEPES-
NaOH (pH 7.4). The test compound was added to the
suspension of the cells with stirring. Intracellular Ca2+
production was calculated from the ratio of the fluores-
cence intensities at 340 and 380 nm.
Rf 0.26 (CHCl3/AcOH/MeOH, 20/2/1); IR (neat) 3392,
2922, 1734, 1447, 1240, 1192, 1159, 1085, 1031, 972,
792, 705 cmꢀ1; 1H NMR (300 MHz, CDCl3) d 7.32–7.11
(m, 4H), 5.76 (dd, J=16, 6.3 Hz, 1H), 5.55 (dd, J=16,
8.4 Hz, 1H), 4.49–4.39 (m, 3H), 3.95 (m, 1H), 3.42 (s,

2110
T. Maruyama et al. / Bioorg. Med. Chem. 10 (2002) 2103–2110
LPS-induced changes of cytokine production in human
whole blood cells
10. Ishihara, K.; Kurihara, H.; Yamamoto, H. J. Org. Chem.
1993, 58, 3791.
11. To confirm this hypothesis, these reactions were analyzed
using ReactIR systems. With regard to methanesulfonylation,
two reaction mechanisms are known as shown in Scheme 3a
and b. A reactive intermediate 15 is thought to be produced
using a trialkylamine as a base (Scheme 3a) while 16 is thought
to be produced using pyridine as a base (Scheme 3b).18
Absorption of 15 (1038 cmꢀ1) was observed with decreased
absorption of MsCl (1370, 1177 cmꢀ1) when the reaction was
carried out in dichloromethane using Et3Nor i-Pr2NEt as a
base. Using THF as a solvent instead of dichloromethane, a
completely different result was obtained. In the reaction of
MsCl/Et3Nin THF, no absorption of 15 (1038 cmꢀ1) was
observed with the decrease in absorption of MsCl (1370 and
1177 cmꢀ1). Using MsCl/i-Pr2NEt in THF, the absorption of
MsCl (1370, 1177 cmꢀ1) was retained but the absorption of 15
(1038 cmꢀ1) was not. Based on this information, it was con-
cluded that the above-mentioned reactive intermediates were
not produced under these conditions. The methanesulfonyla-
tion in THF was considered to proceed via unknown reactive
intermediate. i-Pr2NEt was estimated to react with MsCl in
the presence of an alcohol with some interaction, which is dif-
ferent from Scheme 3a.
Heparinized whole blood (from three healthy adult male
volunteers) was diluted 1:10 with Iscove’s modified Dul-
becco’s medium supplemented with 0.1% FBS, penicillin
(100U/mL) and streptomycin (100 mg/mL). Diluted whole
blood was cultured with lipopolysaccharide (LPS) from
WE coli (serotype O55:B5) (100ng/mL) and the test
compound (10ꢀ11ꢃ10ꢀ6 M) at 37ꢁC. After 8 h (TNF-a)
or 20 h (IL-10), supernatants were harvested and the
levels of several cytokines were determined by commer-
cial ELISA kits (Iwaki Glass, Japan).
LPS-induced changes of plasma TNF-ꢀ and IL-10 levels
in rats
LPS and the test compound were dissolved in a sterile
saline solution. Seven-week-old IGS rats (Charles River,
Japan) were injected intravenously with test compounds
(10–300 ng/kg/min), followed by an intravenous adminis-
tration of LPS (10 mg/2 mL/kg) after 30min. 60min after
the LPS injection, blood samples were withdrawn into
heparinized syringes by aorta abdominalis puncture. After
centrifugation at 12,000rpm for 3 min at 4 ꢁC, plasma was
recovered and immediately frozen at ꢀ80ꢁC until
assayed. Plasma TNF-a and IL-10 concentrations were
determined by ELISA kits.
References and Notes
1. Coleman, R. A.; Smith, W. L.; Narumiya, S. Pharmacol.
Rev. 1994, 46, 205.
2. Narumiya, S.; Sugimoto, Y.; Ushikubi, F. Physiol. Rev.
1999, 79, 1193.
Scheme 3. Formation of the presumed reactive intermediates in the
methanesulfonylation.
3. (a) Takahashi, S.; Takeuchi, K.; Okabe, S. Biochem. Phar-
macol. 1999, 58, 1997. (b) Quiroga, J.; Prieto, J. Pharmacol.
Ther. 1993, 58, 67.
4. Shinomiya, S.; Naraba, H.; Ueno, A.; Utsunomiya, I.;
Maruyama, T.; Ohuchida, S.; Ushikubi, F.; Yuki, K.; Nar-
umiya, S.; Sugimoto, Y.; Ichikawa, A.; Oh-ishi, S. Biochem.
Pharmacol. 2001, 61, 1153.
5. Maruyama, T.; Asada, M.; Shiraishi, T.; Sakata, K.; Seki,
A.; Yoshida, H.; Shinagawa, Y.; Maruyama, T.; Ohuchida, S.;
Nakai, H.; Kondo, K.; Toda, M. Bioorg. Med. Chem. Lett.
2001, 11, 2033.
12. Kiriyama, M.; Ushikubi, F.; Kobayashi, T.; Hirata, M.;
Sugimoto, Y.; Narumiya, S. Br. J. Pharmacol. 1997, 122, 217.
13. Shinomiya, S.; Naraba, H.; Ueno, A.; Utsunomiya, I.;
Maruyama, T.; Ohuchida, S.; Ushikubi, F.; Yuki, K.; Nar-
umiya, S.; Sugimoto, Y.; Ichikawa, A.; Oh-ishi, S. Biochem.
Pharmacol. 2001, 61, 1153.
14. Sakata, K.; Maruyama, T.; Seki, A.; Yoshida, H.; Shina-
gawa, Y.; Maruyama, T.; Kondo, K.; Ohuchida, S. Abstract
Book, 11th International Conference on Advances in Pros-
taglandin and Leukotriene Research, Florence, Italy, June 4–
8, 2000.
6. Hazato, A.; Tanaka, T.; Watanabe, K.; Bannai, K.; Toru,
T.; Okamura, N.; Manabe, K.; Ohtsu, A.; Kamimoto, F.;
Kurozumi, S. Chem. Pharm. Bull. 1985, 33, 1815.
7. Corey, E. J.; Shirahama, H.; Yamamoto, H.; Terashima,
S.; Venkateswarlu, A.; Schaaf, T. K. J. Am. Chem. Soc. 1971,
93, 1490.
8. The yield of 3 markedly decreased with an increase in tem-
perature.
9. Noyori, R.; Tomino, I.; Yamada, M.; Nishizawa, M. J.
Am. Chem. Soc. 1984, 106, 6717.
15. Kurihara, Y.; Endo, H.; Akahoshi, T.; Kondo, H. Clin.
Exp. Immunol. 2001, 123, 323.
16. Kasai, K.; Sato, S.; Suzuki, K. Hepatol. Res. 2001, 21, 252.
17. Yoshida, K.; Oida, H.; Kobayashi, T.; Maruyama, T.;
Tanaka, M.; Katayama, T.; Yamaguchi, K.; Segi, E.; Tsu-
boyama, T.; Matsushita, M.; Ito, K.; Ito, Y.; Sugimoto, Y.;
Ushikubi, F.; Ohuchida, S.; Kondo, K.; Nakamura, T.; Nar-
umiya, S. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 4580.
18. O’Donnell, C. J.; Burke, S. D. J. Org. Chem. 1998, 63,
8614 and references cited therein.