Discovery of 13-oxa prostaglandin analogs as antiglaucoma agents: Synthesis and biological activity

Article abstract of DOI:10.1016/j.bmc.2008.11.070

FP-Class prostaglandin analogs have demonstrated utility for the treatment of glaucoma and ocular hypertension. A series of novel FP prostaglandin analogs was designed to optimize topical ocular activity and reduce ocular side-effects by replacing 13-carb

Full text of DOI:10.1016/j.bmc.2008.11.070

Bioorganic & Medicinal Chemistry 17 (2009) 576–584
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of 13-oxa prostaglandin analogs as antiglaucoma agents:
Synthesis and biological activity
a
b
c
b
Zixia Feng a, , Mark R. Hellberg , Najam A. Sharif , Marsha A. McLaughlin , Gary W. Williams ,
*
Daniel Scott c, Tony Wallace c
a Medicinal Chemistry, Ophthalmic Products Research, Alcon Research Ltd., 6201 South Freeway, Mail code: R2-39, Fort Worth, TX 76134, USA
b Molecular Pharmacology, Ophthalmic Products Research, Alcon Research Ltd., 6201 South Freeway, Mail code: R2-39, Fort Worth, TX 76134, USA
c In Vivo Pharmacology, Ophthalmic Products Research, Alcon Research Ltd., 6201 South Freeway, Mail code: R2-39, Fort Worth, Texas 76134, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
FP-Class prostaglandin analogs have demonstrated utility for the treatment of glaucoma and ocular
hypertension. A series of novel FP prostaglandin analogs was designed to optimize topical ocular activity
and reduce ocular side-effects by replacing 13-carbon with oxygen. A facile synthesis was successfully
developed for synthesis of the 13-oxa prostaglandins from the commercially available Corey aldehyde
benzoate. Among the compounds synthesized, AL-16082 was the most potent prostaglandin FP agonist
in vitro. In a prostaglandin FP receptor-linked second-messenger assay, phosphoinositide (PI) turnover,
it exhibited a potency value (EC50) of 1.9 nM (78% max. response relative to fluprostenol). The isopropyl
ester of AL-16082, compound AL-16049, significantly lowered intraocular pressure (IOP) in the ocular
hypertensive monkey eyes by 30%. In the study of acute ocular irritation response in New Zealand albino
Received 25 September 2008
Revised 25 November 2008
Accepted 27 November 2008
Available online 6 December 2008
Keywords:
Glaucoma
13-Oxa prostaglandin
Intraocular pressure (IOP)
rabbits, AL-16049 produced lower incidence of hyperemia, swelling, and discharge than PGF2 (1 lg), and
a
a similar incidence of hyperemia, swelling, and discharge to latanoprost (1.8
l
g). AL-16049 also produced
no signs of ocular irritation or discomfort in the cat at the doses evaluated.
Published by Elsevier Ltd.
1. Introduction
agents devoid of ocular side-effects has been an important advance
in the treatment of glaucoma.3 Agents latanoprost, bimatoprost,
travaprost and bimatoprost4 (Fig. 1), are the active ingredients of
Glaucoma is a progressive disease which leads to optic nerve
damage and loss of vision. The principal risk factor for the disease
is elevated intraocular pressure (IOP). Elevated IOP is caused by a
decrease in the rate aqueous humor exits the anterior chamber
of the eye as opposed to being caused by an increase in the produc-
tion of aqueous humor. Although the mechanisms that lead to re-
duced outflow are poorly understood, it is known that elevated IOP
can be at least partially controlled by administering drugs which
either reduce the production of aqueous humor within the eye,
or increase the outflow of aqueous humor from the eye. FP prosta-
glandin agonists have been shown to effectively reduce IOP by
increasing outflow of aqueous humor through the uveoscleral
pathway.1 Thus, there has been a great deal of interest in develop-
ing synthetic FP prostaglandin agonists with IOP-lowering efficacy.
HO
O
7
HO
1
OH
O
10
15
O
HO
13
OH
HO
OH
Latanoprost
PGF2a
H
HO
HO
N
O
Certain prostaglandins and their pro-drugs, such as PGF2 Fig. 1)
a
O
O
isopropyl ester, reduce IOP in monkeys and in humans, but also
cause conjunctival hyperemia and foreign-body sensation.2 The
development of pro-drugs of potent, selective synthetic prosta-
glandin FP receptor agonists as clinically effective IOP-lowering
HO
HO
O
OH
OH
CF3
Bimatoprost
latanoprost, bimatoprost, travaprost and
Travaprost
* Corresponding author. Tel.: +1 817 551 6983; fax: +1 817 615 3396.
E-mail address: zixia.feng@alconlabs.com (Z. Feng).
Figure 1. Structures of PGF2 ,
a
bimatoprost.
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.11.070

Z. Feng et al. / Bioorg. Med. Chem. 17 (2009) 576–584
577
topically IOP-lowering medications. Their introduction has revolu-
tionized the treatment of ocular hypertension and glaucoma.
As part of our continuing research program targeted at the dis-
covery of prostaglandin agonists that lower IOP with reduced side-
effects, we wish to report the successful discovery of 13-oxa-pros-
taglandins for the treatment of glaucoma and ocular hypertension.
This series was designed to optimize prostaglandin FP agonist top-
ical ocular activity and reduce side effect by replacing 13-carbon
with oxygen. This type of modification usually reduces the hydro-
phobicity of compounds, a property that is believed to be related to
ocular side-effects. A facile synthetic route to 13-oxa prostaglan-
dins was developed.
avoiding a complicated total synthesis. The synthetic routes are de-
scribed in Schemes 1 and 2. The tentative assignment of the stereo-
chemistry at C15 was based on the well-documented difference in
polarity between the 15
a
and 15b epimers.5 Commercially avail-
able Corey aldehyde benzoate 1 was oxidized to carboxylic acid 2
by Jones reagent at 0 °C. Chlorination of 2 afforded acid chloride
3. The crude product 3 was reacted with 3-hydroxy-4-methylthia-
zole-2(3H)-thione to yield the thiohydroxamate ester 4. The nor-
alcohol 5 was obtained in good yield by stirring the thiohydroxa-
mate ester 4 with tris (phenylthio) antimony in dichloromethane
in a flask open to air.6 This synthetic route to intermediate 5 was
an improvement over the previously reported methods.7,8 Alkyl-
ation of alcohol 5 followed by epoxidation using MCPBA provided
the epoxide 7. Regiospecific ring-opening of the epoxide with a
Grignard reagent m-trifluoromethyl-benzylmagnesium bromide
made in situ gave a diastereomer mixture 8.9–11 Protection of the
diol afforded the di-THP-ether 9. Lactone 9 was treated with dii-
2. Chemistry
The primary synthetic target was to develop a facile route to the
13-oxa prostaglandins from commercially available chemicals
O
O
O
O
O
O
O
N
S
O
HO
a
d
b
S
N
S
O
c
COOH
CHO
COCl
O
BzO
O
BzO
BzO
O
S
BzO
2
4
1
3
O
O
O
O
O
O
g
f
e
h
O
CH2
O
O
O
OH
HO
OH
BzO
BzO
BzO
O
5
6
7
8
CF3
O
OH
O
HO
O
i
j
O
O
O
THPO
O
THPO
THPO
THPO
THPO
THPO
CF3
CF3
CF3
9
11
10
HO
HO
O
O
O
k
+
O
O
O
HO
HO
HO
HO
CF3
CF3
AL-16049
12
l
l
HO
HO
OH
OH
O
O
O
O
HO
HO
HO
HO
CF3
CF3
13
AL-16082
Scheme 1. Reagents and conditions: (a) Jones reagent, acetone, 0 °C, 20 min; (b) SOCl2, 65 °C; (c) pyridine, CH2Cl2, rt, 1 h; (d) (PhS)3Sb, CH2Cl2, rt, 16 h, (open flask); (e) allyl
bromide, Ag2O, DMF, rt, 48 h; (f) MCPBA, NaHCO3, CH2Cl2, rt, 24 h; (g) m-trifluromethyl-benzylmagnesium bromide, CuI, ether, À78 °C, 6 h; (h) dihydropyran, p-TsOH, CH2Cl2,
0 °C, 30 min; (i) DIBAL-H, toluene, À78 °C, 1 h; (j) (1) Ph3P+(CH2)4COOHBr+, KOBut, THF; (2) 2-iodopropane, DBU, rt, 16 h; (k) (1) CH3COOH (65%), THF, 550 C, 1 h; (2) chiral
separation by HPLC; (l) LiOH.H2O, MeOH, H2O, rt, 16 h.

578
Z. Feng et al. / Bioorg. Med. Chem. 17 (2009) 576–584
O
O
O
O
a
c
b
7
O
O
O
O
BzO
OH
HO
OH
16: R=H
17: R=Cl
14: R=H
15: R=Cl
R
R
O
OH
O
O
HO
O
d
e
O
O
O
O
O
O
THPO
O
THPO
THPO
THPO
THPO
THPO
R
R
R
18: R=H
19: R=Cl
20: R=H
21: R=Cl
22: R=H
23: R=Cl
HO
HO
O
O
O
f
O
+
O
O
O
O
HO
HO
HO
HO
R
R
26: R=H
27: R=Cl
24: R=H
25: R=Cl
g
g
HO
HO
OH
O
OH
O
+
O
O
O
O
HO
HO
HO
HO
R
R
28: R=H
29: R=Cl
30: R=H
31: R=Cl
Scheme 2. Reagents and conditions: (a) phenol or 3-chlorophenol, K2CO3, CH3CN, 0 °C, 4 h; (b) K2CO3, MeOH, rt, 1.5 h; (c) dihydropyran, p-TsOH, CH2Cl2, 0 °C, 30 min; (d)
DIBAL-H, toluene, À78 °C, 1 h; (e) (1) Ph3P+(CH2)4COOHBr+, KOBut, THF; (2) 2-iodopropane, DBU, rt, 16 h; (f) (1) CH3COOH (65%), THF, 55 °C, 1 h; (2) chiral separation by HPLC;
(g) LiOHÁH2O, MeOH, H2O, rt, 16 h.
sobutylaluminum hydride (DIBAL-H) in toluene at À78 °C to give
the lactol 10. The Wittig reaction with (4-carboxybutyl)triphenyl-
phosphonium bromide and potassium tert-butoxide in THF affor-
ded the acid which was reacted without isolation with isopropyl
iodide and DBU in acetone to give the ester 11.12 Cleavage of the
THP ethers under mildly acidic conditions followed by chiral sepa-
ble 1 were tested for their binding affinity and functional efficacy
to the FP prostaglandin receptor. The acids AL-16082, 13, 28, 29,
30 and 31 were evaluated for their ability to stimulate FP recep-
tor-linked phosphoinositide turnover in Swiss 3T3 mouse fibro-
blast cells (expressing an endogenous FP receptor), and to bind
to FP receptor expressed in bovine corpus luteum, using recently
published procedures.14,15 The results from these studies are sum-
marized in Table 1.
ration on HPLC gave the isopropyl esters AL-16049 (15a) and 12
(15b). Hydrolysis using basic condition provided the corresponding
free acids AL-16082 and 13.13
The 17-carbon-trifluoromethyl substituted phenyl analogs AL-
16082 and 13 had equal affinity and were more potent in the func-
The 16-phenoxy analogues (Scheme 2) were prepared in a sim-
ilar manner except for the ring-opening of the epoxide 7. The epox-
ide was opened by the reaction with phenol or 3-chlorophenol in
the presence of potassium carbonate in acetonitrile to afford the
intermediates 14 and 157 which were hydrolyzed to intermediates
16 and 17.
tional assay than PGF2 and the carboxy acid hydrolysis product of
a
latanoprost (latanoprost acid). The three fold difference in func-
tional potency and the slightly higher affinity of AL-16082 than
13 support the tentative assignment of the 15-OH of AL-16082
as being in the alpha configuration. The glycol analogs 28–31 are
less potent than the standards PGF2 and latanoprost acid and
a
are at least ten fold less potent than the 17-carbon-trifluoromethyl
substituted phenyl analogs in the functional assay. The affinity of
compounds 29–30 for the bovine FP receptor is very similar to each
other and to the standards. Compound 28 has a lower affinity for
3. Results and discussion
The carboxylic acid believed to be the pharmacologically active
form was used in all in vitro studies. The compounds shown in Ta-

Z. Feng et al. / Bioorg. Med. Chem. 17 (2009) 576–584
579
Table 1
Table 3
FP receptor-mediated functional response and fp-receptor binding data
Effect of AL-16049 on pupil diameter in the cat
Dose (
l
g)
AUC1-5
HO
0.1
0.3
1.0
3.12
6.57
10.2
OH
O
O
HO
X
R1
are qualitative, allowing comparison between compounds and be-
tween doses of the same compound. Results are reproducible. No
statistical analysis is performed.
R2
Compd
X
R1
R2
EC50 (nM)
Max.
response (%)
Ki (nM)
AL-16049 produced a dose-dependent reduction in IOP (Fig. 2)
in ocular hypertensive monkeys.18 The lower dose, 1
l
g, was mar-
ginally effective after the first instillation and did not show any in-
crease in IOP reduction on b.i.d. treatment. The higher dose, 3 g,
AL-16082
CH2
CH2
O
O
O
a
b-OH
-OH
CF3
CF3
H
Cl
H
1.9 0.3
5.7 0.3
294 69
52.4 25.4
68.6 13.1
62.2 5.5
24.5 4.4
31.7 4.1
78
82
67
62
67
86
92
85
72 19
130 34
13
28
29
30
31
l
a
a
-OH
-OH
120
9
produced a 22% reduction in IOP after the first instillation which
was increased to 30% after dose 5 b.i.d. There was a sustained
reduction in IOP of 21% evidenced at 16 h after dose 4 b.i.d. Con-
trary to literature reports with related compounds 18, no transient
ocular hypertensive effect was observed. The compound did not
cause miosis in the monkeys and was well tolerated.
680 27
87 37
140 63
b-OH
b-OH
O
Cl
PGF2
130
6
a
Latanoprost acid
98 11
In conclusion, a series of 13-oxa prostaglandin analogues were
synthesized using a facile synthesis we developed. This work dem-
onstrates that these prostaglandins are potential candidates as
ocular hypotensive agents for the treatment of glaucoma. Com-
the bovine receptor despite its potency in the functional assay. The
in vitro results with this narrow series indicate that the substitu-
tion of the 13-carbon with oxygen can result in potent prostaglan-
din analogs.
pound AL-16082 is
a very potent prostaglandin FP agonist
in vitro (EC50 = 1.9 nM) and its ester as a pro-drug, compound
AL-16049, significantly lowers IOP in the ocular hypertensive mon-
key by 30%. In the study of acute ocular irritation response in New
Zealand albino rabbits, it produced less incidence of hyperemia,
Compound AL-16049, an isopropyl ester of the most potent
compound AL-16082, was used as a pro-drug in the in vivo evalu-
ations for corneal penetration and delivery of the hydrolyzed car-
boxylic acid to the aqueous humor. Compound AL-16049 was
evaluated for side effect in the rabbit ocular irritation (ROI) model,
for topical ocular potency in the cat pupil diameter (CPD) constric-
tion model, and for IOP-lowering activity in the lasered ocular
hypertensive monkey model.
swelling, and discharge than PGF2 (1
l
g) and produced less or
no greater incidence of hyperemia, swelling, and discharge than
latanoprost (1.8 g). AL-16049 also produced no signs of ocular
a
l
irritation or discomfort in the cat at the doses evaluated. The fur-
ther study is on the way.
A dose response (0.03–100
for AL-16049 (Table 2.). The Max15 (dose at which 15% incidence
of hyperemia occurs) is approximately 1 g. At all doses, AL-
16049 produced a lower incidence of hyperemia, swelling, and dis-
charge than PGF2 (1 g) and produced less or at least no greater
incidence of hyperemia, swelling, and discharge than latanoprost
(1.8
g).16
These data are qualitative, allowing comparison between com-
pounds and between doses of the same compound. Results are
reproducible. No statistical analysis was performed.
Published data suggest that FP prostaglandins produce a dose
related miotic effect in the cat.17 Topical ocular application of
AL-16049 produced a dose-dependent miotic response in the con-
scious cat (Table 3.). Data are Area under the Curve (AUC1-5) for
group average pupil diameter change during one through 5 h after
test compound instillation. AL-16049 produced no signs of ocular
irritation or discomfort in the cat at any dose tested. These data
lg) for hyperemia was established
l
4. Experimental
l
a
Unless otherwise noted, all solvents, chemicals, and reagents
were obtained commercially and used without purification. 1H
NMR and 13C NMR spectra were determined at 200 MHz with a
Varian model VXR-200 spectrometer or at 600 MHz with a Brucker
DRX-600. Low resolution mass spectra were acquired on a Finne-
gan TSQ 46 triple quadrupole mass spectrometer operating in the
positive electrospray mode. High-resolution mass spectra were ac-
quired in the FAB mode by Analytical Instrument Group in Raleigh,
NC. Unless otherwise stated, all reactions without added water
were run under a positive pressure of nitrogen. Concentration re-
fers to removal of solvent in vacuo on a rotary evaporator. Reac-
tions were monitored by TLC on E. Merck silica gel 60 F254
plates, with visualization by UV light, or phosphomolybdic acid.
Column chromatographic purifications were performed under po-
sitive air flow using 230–400 mesh silica gel from E.M. Science.
Chromatography solvents used were HPLC grade from E.M.
Science.
l
Table 2
Dose response effect of AL-16049 on ocular irritation in NZA rabbits*
AL-16049 (lg)
% Incidence
hyperemia
% Incidence
swelling
% Incidence
discharge
4.1. 6-exo-Carboxylicacid-7-endo-benzoyloxy-2-
oxabicyclo[3.3.0]octan-3-one (2)
0.03
0.1
0.3
1
3
10
30
13
13
8
5, 13
25
0, 23
73
23
38
25
3, 30
8
8, 35
45
40, 8, 30
63, 45, 83
5
3
5
3, 5
Corey aldehyde benzoate (15 g, 54.7 mmol) was dissolved in
150 mL of acetone and cooled to 0 °C. Jones reagent was added
dropwise with stirring until the brown color did not change to
green (25 mL). The mixture was stirred at 0 °C for another
15 min. Isopropanol (30 mL) was added to the reaction mixture.
The resulting mixture was filtered through a celite funnel and
the filtrate was concentrated. The residue was dissolved in ethyl
0
15, 8
0
10, 13, 20
68, 55, 93
Latanoprost, 1.8
PGF2 , 1
45, 23, 20
100, 100, 100
a
*
More than one number indicates results from different studies.

580
Z. Feng et al. / Bioorg. Med. Chem. 17 (2009) 576–584
Monkey Intraocular Pressure v
AL-16049, 1.0 ug
20
10
AL-16049, 3.0 ug
PGF2alpha isopropyl ester, 1.0 ug
0
-10
-20
-30
-40
//
//
4
4
0
16
6
2
2
6
Time After Treatment, Hours
=
Treatment
Figure 2. Effect of AL-16049 on intraocular pressure in ocular hypertensive cynomolgus monkey eyes: (a) formulations administered 1 Â 30
1600 h on days 1 and 2; at 0900 h on day 3. N = 9, drug; N = 5, control, (b) OD lasered (hypertensive), and (c) p < 0.001.
lL, OD topical at 0900 and
acetate and washed with water, saturated NaCl, dried over MgSO4,
filtered and concentrated. The residue was crystallized in ethyl
acetate and hexane (5:1). White crystals of 2 (13.4 g) were ob-
tained. MS m/z calcd for C15H15O6 (M+H)+ 291, found 291.
funnel, and washed with ethyl acetate. The filtrate was extracted
with ethyl acetate. The combined organic extracts were washed
with a saturated NaCl solution and dried over anhydrous MgSO4.
Concentration of the combined extracts under reduced pressure,
and chromatography of the residue on silica gel, eluting with 40%
ethyl acetate in hexane, gave 0.36 g of 6 as light yellow oil. 1H
NMR (CDCl3) d 8.01–7.95 (m, 2H), 7.61–7.54 (m, 1H), 7.48–7.41
(m, 2H), 6.00–5.80 (m, 1H), 5.50–5.14 (m, 4H), 4.25–4.15 (m, 1H),
3.12–2.86 (m, 4H), 2.56–2.36 (m, 3H); 13C NMR (CDCl3) d 176.7
(C), 165.7 (C) 133.9 (CH), 133.5 (CH), 129.7 (CH), 129.6 (C), 128.6
(CH), 117.7 (C), 88.7 (CH), 84.8 (CH), 77.9 (CH), 70.7 (CH2), 44.5
(CH), 36.5 (CH2), 33.7 (CH2). MS m/z calcd for C17H19O5Na
(M+Na)+ 325, found 325.
4.2. 7-a-Benzoyloxy-cis-oxabicyclo[3.3.0]octan-3-one-6-
carboxy-4-methylthiazole-2(3H)-thion (4)
Compound 2 (10 g, 34.47 mmol) in 50 mL of thionyl chloride
was gently refluxed for 3 h and then concentrated to chloride 3.
It was dissolved in 30 mL of CH2Cl2 and then added to a solution
of 3-hydroxy-4-methylthiazole-2(3H)-thione (3.56 g, 24.1 mmol)
in 30 mL of CH2Cl2 in dry pyridine (4.2 mL, 51.7 mmol). The result-
ing mixture was stirred at room temperature for 1 h. The solvent
was evaporated and the residue was put in a solution comprised
of 300 ml of ethyl acetate and 100 mL of hexane. The white crystals
formed were washed with ethyl acetate and hexane (3:1) and dried
in vacuum to yield thiohydroxamate ester 4 (10.1 g). MS m/z calcd
for C19H18O6NS2 (M+H)+ 420, found 420.
4.5. 6b-Glycidoxy-7a-benzoyloxy-cis-2-oxabicyclo[3.3.0]octan-
3-one (7)
The allyl ether 6 (0.36 g, 1.19 mmol) was stirred with m-chloro-
peroxybenzoic acid (0.62 g, 3.56 mmol) and sodium hydrogen car-
bonate (0.36 g, 4.3 mmol) in 10 mL of CH2Cl2 at 0 °C. The mixture
was warmed slowly to room temperature and stirred for 24 h.
The reaction mixture was washed with saturated aqueous sodium
sulphite, saturated sodium hydrogen carbonate, water and brine,
and dried over anhydrous MgSO4. Evaporation of solvent afforded
the crude product, which was chromatographed on a silica gel col-
umn, eluting with 60% ethyl acetate in hexanes to provide the
epoxide 7 as oil 0.12 g. 1H NMR (CDCl3) d 7.99–7.94 (m, 2H),
7.61–7.54 (m, 1H), 7.48–7.40 (m, 2H), 5.48 (s, 1H), 5.27–5.22 (m,
1H), 4.10–3.91 (m, 2H), 3.64–3.46 (1H), 3.17–2.80 (m, 4H), 2.68–
2.42 (m, 4H); 13C NMR (CDCl3) d 176.5 (C), 165.7 (C), 133.5 (CH),
129.7 (CH), 129.6 (C), 128.5 (CH), 89.7 (CH), 84.6 (CH), 77.7 (CH),
71.0 (CH2), 70.2 (CH2), 50.6 (CH), 44.3 (CH), 44.1 (CH2),
36.8(CH2), 33.6 (CH2). MS m/z calcd for C17H19O6 (M+H)+ 340,
found 340.
4.3. 6b-Hydroxy-7a-benzoyloxy-cis-2-oxabicyclo[3.3.0]octan-3-
one (5)
Antimony triphenylsulphide (2 g, 4.45 mmol) was added to a
solution of 4 (0.97 g, 2.23 mmol) in 8 mL of CH2Cl2. The mixture
was stirred in an open flask at room temperature for 16 h. The
white solid was filtered and the solution was evaporated to dry-
ness. Purification of the residue by flash chromatography using
ethyl acetate and hexane (1:1) afforded the desired nor-alcohol 5
(0.4 g). 1H NMR (CDCl3) d 7.99–7.95 (m, 2H), 7.62–7.54 (m, 1H),
7.48–7.27 (m, 2H), 5.32–5.20 (m, 2H), 4.25 (s, 1H), 3.06–2.36 (m,
6H). 13C NMR (CDCl3) d 176.6 (C), 166.5 (C), 113.6 (CH), 129.7
(CH), 128.7 (C), 128.6 (CH), 84.1 (CH), 81.9 (CH), 81.1 (CH), 45.7
(CH), 36.4 (CH2), 33.5 (CH2). MS m/z calcd for C14H15O5 (M+H)+
263, found 263.
4.6. 6b-[2ab-Hydroxy-4-(3-trifluoromethyl)phenyl-butoxy]-7a-
4.4. 6b-Allyloxy-7a-benzoyloxy-cis-2-oxabicyclo[3.3.0]octan-3-
hydroxy-cis-2-oxabicyclo[3.3.0]-octan-3-one (8)
one (6)
Copper(I) iodide (8.3 g, 43.38 mmol) was stirred in dry diethyl
ether (60 mL) at À30 °C under N2. A solution of m-trifluorometh-
ylbenzylmagnesium bromide (86.7 mmol) made in situ from Mg
(2.1 g, 86.7 mmol) and m-trifluoromethylbenzyl bromide
(13.3 mL, 86.7 mmol) in 20 mL of ether was added dropwise and
Compound
5
(0.4 g, 1.53 mmol), allyl bromide (1 mL,
12.2 mmol) and silver(I) oxide (1.1 g, 4.6 mmol) were stirred in
8 mL of dry DMF at room temperature for 48 h. Water (2 mL)
was added and the mixture was filtered through a celite coated

Z. Feng et al. / Bioorg. Med. Chem. 17 (2009) 576–584
581
the mixture was stirred at À30 °C for 15 min. The mixture was
cooled to À78 °C, and the epoxide 7 (2.3 g, 7.23 mmol) in ether
(30 mL) was added dropwise. The resulting mixture was stirred
at À78 °C for 6 h, after which saturated ammonium chloride
(15 mL) was added and the mixture warmed to room temperature
and stirred for 15 min. The two layers were separated and the
aqueous layer was extracted with ethyl acetate (3 Â 30 mL). The
combined organic extracts were washed with a saturated NaCl
solution, dried over anhydrous MgSO4, filtered and concentrated
to a colorless oil, which was chromatographed on a silica gel col-
umn, eluting with ethyl acetate to afford 8 (0.56 g). MS m/z calcd
for C18H21O5F3 (M+NH4)+ 392, found 392.
acetate in hexane, to afford 11 (0.6 g). MS m/z calcd for
36H53O8F3 (M+NH4)+ 688, found 688.
C
4.9. 5Z-(9S,11R,15S)-13-Oxa-17-(3-trifluoromethyl)phenyl-
9,11,15-trihydroxy-18,19,20-trinor-5-prostadienoic acid
isopropyl ester (AL-16049)
Compound 11 (0.6 g, 0.89 mmol) was stirred in 5 mL of
MeOH, 0.5 mL of water and 5 drop of 12 N HCl at 0 °C for
15 min at room temperature for 2 h. Ethyl acetate (10 mL) and
saturated NaHCO3 (10 mL) were added. The two layers were sep-
arated and the aqueous layer was extracted with ethyl acetate
(3 Â 20 mL). The combined organic extracts were washed with
a saturated NaCl solution, dried over anhydrous MgSO4, filtered
and concentrated to an oil residue. The crude product was chro-
matographed on a silica gel column eluting with ethyl acetate
followed by HPLC chiral separation to afford AL-16049 (0.11 g)
and 12 (0.095 g).
4.7. 6b-[2ab-Tetrahydro-2H-pyran-2-yl)oxy-4-(3-trifluoro-
methyl)phenyl-butoxy]-7a-hydroxy-cis-2- oxabicyclo-
[3.3.0]octan-3-one (9)
3,4-Dihydro-2H-pyran (0.54 mL, 5.88 mmol) was added to a
solution of 8 (0.55 g, 1.47 mmol) in CH2Cl2 (25 mL) and the mixture
was cooled to 0 °C. p-Toluenesulfonic acid monohydrate (28 mg,
0.15 mmol) was added and the resulting mixture was stirred at
0 °C for 30 min and then quenched by addition of triethylamine
(0.03 mL). Evaporation of solvent afforded the crude product which
was chromatographed on silica gel (ethyl acetate–hexanes, 1:2) to
provide the title compound 9 as oil 0.75 g. MS m/z calcd for
C28H37O7F3 (M+NH4)+ 560, found 560.
1H NMR (CDCl3) d 7.45–7.27 (m, 4H), 5.48–5.40 (m, 2H), 5.06–
4.93 (m, 1H), 4.21–411 (m, 2H), 3.65–3.62 (m, 1H), 3.57–3.50 (m,
3H), 2.37–1.64 (m, 15H), 1.24–1.21 (d, J = 6 Hz, 6H). 13C NMR
(CDCl3) d 173.59 (C), 142.84 (C), 131.89 (CH), 130.33 (C), 129.94
(CH), 128.87 (CH), 125.14 (CH), 125.07 (CH), 122.77 (CH), 122.70
(CH), 93.04 (CH), 77.53 (CH), 75.03 (CH2), 73.82 (CH), 69.96 (CH),
68.11 (CH), 51.77 (CH), 41.62 (CH2), 34.93 (CH2), 34.33 (CH2),
31.91 (CH2), 26.95 (CH2), 26.16 (CH2), 25.22 (CH2), 22.14 (CH3).
MS (ESI) m/z calcd for C26H37O6F3 (M+H)+ 503, found 503, HRMS
calcd for C26H37O6F3 (M+H)+ 503.5778, found 503.2625.
4.8. 5Z-(9S,11R,15RS)-13-Oxa-17-(3-trifluoromethyl)phenyl]-9-
hydroxy-11,15-bis(tetrahydro-2H-pyran-2-yl)oxy-17,18,19,20-
tetranor-5-prostadienoic acid isopropyl ester (11)
4.10. 5Z-(9S,11R,15R)-13-Oxa-17-(3-trifluoromethyl)phenyl-
9,11,15-trihydroxy-18,19,20-trinor-5-prostadienoic acid
isopropyl ester (12)
To a solution of 9 (0.75 g, 1.38 mmol) in 6 mL of toluene at
À78 °C, was added dropwise a 1.5 M solution of DIBAL-H in toluene
(1.2 mL, 1.8 mmol). The resulting mixture was stirred at À78 °C for
1 h. Methanol (3 mL) was added to quench the reaction. Saturated
ammonium chloride (10 mL) and ethyl acetate (10 mL) were added
and the mixture was stirred at room temperature until two layers
were separated. The aqueous layer was separated and was ex-
tracted with ethyl acetate (3 Â 15 mL). The combined organic ex-
tracts were washed with a saturated NaCl solution, dried over
anhydrous MgSO4, filtered and concentrated to a white semi-solid
10 (0.75 g), which was used for next step without further
purification.
Colorless oil. 1H NMR (CDCl3) d 7.45–7.27 (m, 4H), 5.49–5.41 (m,
2H), 5.06–4.94 (m, 1H), 4.23–410 (m, 2H), 3.71–3.56 (m, 3H), 3.42–
4.37 (m, 1H), 2.86–1.65 (m, 15H), 1.24–1.21 (d, J = 6 Hz, 6H). 13C
NMR (CDCl3) d 173.59 (C), 142.84 (C), 131.90 (CH), 129.99 (C),
128.91 (CH), 128.76 (CH), 125.18 (CH), 125.10 (CH), 122.80 (CH),
122.72 (CH), 93.15 (CH), 77.06 (CH), 74.76 (CH2), 73.70 (CH),
69.66 (CH), 67.78 (CH), 51.63 (CH), 41.36 (CH2), 34.47 (CH2),
33.98 (CH2), 31.56 (CH2), 26.61 (CH2), 25.94 (CH2), 24.87 (CH2),
21.82 (CH3). MS (ESI) m/z calcd for C26H37O6F3 (M+H)+ 503, found
503, HRMS calcd for C26H37O6F3 (M+H)+ 503.5778, found 503.2621.
To a suspension of (4-carboxybutyl)triphenylphosphonium bro-
mide (1.53 g, 3.45 mmol) in THF (7 mL) at 0 °C (bath temperature)
was added a 1 M solution of potassium tert-butoxide in THF
(6.9 mL, 6.9 mmol). After stirring 30 min at 0 °C for 10 min at room
4.11. 5Z-(9S,11R,15S)-13-Oxa-17-(3-trifluoromethyl)phenyl-
9,11,15-trihydroxy-18,19,20-trinor-5-prostadienoic acid (AL-
16082)
temperature,
a solution of the crude product 10 (0.75 g,
1.38 mmol) in THF (12 mL) was added dropwise under N2, and
the reaction was stirred at room temperature for 1 h. Saturated
ammonium chloride (15 mL) and ethyl acetate (15 mL) were
added. The aqueous layer was treated with 2 N HCl to adjust the
pH to 5 and was then extracted with ethyl acetate (3 Â 20 mL).
The combined organic layer was washed with a saturated NaCl
solution, dried over anhydrous MgSO4, filtered and concentrated
to a white semi-solid. This was dissolved in acetone (15 mL), the
solution was cooled to 0 °C (bath temperature), and DBU (1.3 mL,
8.28 mmol) was added. After stirring 20 min at room temperature,
isopropyl iodide (0.7 ml, 6.9 mmol) was added and the reaction
was warmed to room temperature and stirred overnight. Saturated
ammonium chloride (15 mL) and ethyl acetate (15 mL) were
added. The aqueous layer was separated and was extracted with
ethyl acetate (3 Â 25 mL). The combined organic extracts were
washed with a saturated NaCl solution, dried over anhydrous
MgSO4, filtered and concentrated to a colorless oil, which was
chromatographed on a silica gel column, eluting with 30% ethyl
A mixture of AL-16049 (25 mg, 0.05 mmol), lithium hydroxide
monohydrate (21 mg, 0.5 mmol), methanol (1 ml) and water
(0.1 mL) was stirred at room temperature overnight. Saturated
KH2PO4 solution was added to adjust the pH to 6 and the mix-
ture was extracted with ethyl acetate (3 Â 3 mL). The combined
organic layer was washed with a saturated NaCl solution, dried
over anhydrous MgSO4, filtered and concentrated to a colorless
oil, which was chromatographed on a short silica gel column,
eluting with 5% methanol in ethyl acetate, to afford AL-16082
(12.5 mg) as a colorless oil. 1H NMR (CD3OD) d 7.51–7.45 (m,
4H), 5.51–5.49 (m, 1H), 5.37–5.35 (m, 1H), 4.09 (s, 1H), 4.0 (s,
1H), 3.66–3.64 (m, 2H), 3.53–3.49 (m, 2H), 2.90–2.87 (m, 1H),
2.73–2.70 (m, 1H), 2.29–2.25 (m, 5H), 2.22–2.11 (m, 2H), 1.80–
1.74 (m, 3H), 1.65–1,62 (m, 3H). 13C NMR (CDCl3) d 177.79 (C),
145.01 (C), 133.36 (CH), 131.76 (C), 130.45 (CH), 130.39 (CH),
130.14 (CH), 126.10 (CH), 124.92.1 (CH), 123.64 (CH), 92.72
(CH), 77.40 (CH), 75.86 (CH2), 71.89 (CH), 70.88 (CH), 51.06
(CH), 47.90 (CH), 42.23 (CH2), 36.58 (CH2), 34.57 (CH2), 32.50

582
Z. Feng et al. / Bioorg. Med. Chem. 17 (2009) 576–584
(CH2), 27.65 (CH2), 26.14 (CH2), 26.07 (CH2). MS (ESI) m/z calcd
for C23H31O6F3 (M+H)+ 461, found 461, HRMS calcd for
C23H31O6F3 (M+H)+ 461.4974, found 461.2150.
4.16. 5Z-(9S,11R,15RS)-13-Oxa-16-(3-chloro)phenoxy-
propoxy]-9-hydroxy-11,15-bis(tetrahydro-2H-pyran-2-yl)oxy-
17,18,19,20-tetranor-5-prostadienoic acid isopropyl ester (23)
To a solution of 19 (0.1 g, 0.2 mmol) in 1 mL of toluene at
À78 °C, was added dropwise a 1.5 M solution of DIBAL-H in toluene
(0.18 mL, 0.26 mmol). The resulting mixture was stirred at À78 °C
for 1 h. Methanol (0.5 mL) was added to quench the reaction. Sat-
urated ammonium chloride (2 mL) and ethyl acetate (3 mL) were
added, the mixture was extracted with ethyl acetate (3 Â 3 mL).
The combined organic extracts were washed with a saturated NaCl
solution and dried over anhydrous MgSO4, filtered and concen-
trated to a white semi-solid (0.1 g), which was used for next step
without further purification.
To a suspension of (4-carboxybutyl)triphenylphosphonium bro-
mide (0.22 g, 0.5 mmol) in THF (1 mL) at 0 °C (bath temperature)
was added a 1 M solution of potassium tert-butoxide in THF
(1 mL, 1 mmol). After 40 min, a solution of the crude product above
(0.1 g, 0.2 mmol) in THF (2 mL) was added dropwise under N2, and
the reaction was stirred at room temperature for 1 h. Saturated
ammonium chloride (2 mL) and ethyl acetate (3 mL) were added.
The aqueous layer was treated with 2 N HCl to adjust the pH to 5
and was then extracted with ethyl acetate (3 Â 3 mL). The com-
bined organic layer was washed with a saturated NaCl solution,
dried over anhydrous MgSO4, filtered and concentrated to a white
semi-solid. This was dissolved in acetone (2 ml), the solution was
cooled to 0 °C (bath temperature), and DBU (0.2 mL, 1.2 mmol)
was added. After 20 min isopropyl iodide was added (0.1 mL,
1.0 mmol) and the reaction was warmed to room temperature
and stirred overnight. Saturated ammonium chloride (2 mL) and
ethyl acetate (3 mL) were added, the mixture was extracted with
ethyl acetate (3 Â 3 mL). The combined organic extracts were
washed with a saturated NaCl solution, dried over anhydrous
MgSO4, filtered and concentrated to a colorless oil, which was
chromatographed on a silica gel column, eluting with 30% ethyl
acetate in hexane to afford 23 (0.065 g). MS m/z calcd for
C34H51O9ClNa (M+Na)+, 661, found 661.
4.12. 5Z-(9S,11R,15R)-13-Oxa-17-(3-trifluoromethyl)phenyl-
9,11,15-trihydroxy-18,19,20-trinor-5-prostadienoic acid (13)
Colorless oil. 1H NMR (CD3OD) d 7.51–7.45 (m, 4H), 5.51–
5.49 (m, 1H), 5.37–5.35 (m, 1H), 4.09 (s, 1H), 4.0 (s, 1H),
3.69–3.65 (m, 1H), 3.63–3.61 (m, 1H), 3.55–3.53 (m, 1H),
3.49–3.47 (m, 1H), 2.90–2.87 (m, 1H), 2.73–2.70 (m, 1H),
2.31–2.25 (m, 5H), 2.12–2.09 (m, 2H), 1.85–1.83 (m, 1H),
1.73–1.71 (m, 2H), 1.64–1.60 (m, 3H). 13C NMR (CDCl3)
d
177.80 (C), 145.01 (C), 133.36 (CH), 131.75 (C), 130.50 (CH),
130.36 (CH), 130.15(CH), 126.72 (C), 126.09, 123.64 (CH),
91.21 (CH), 76.00 (CH), 74.15 (CH2), 70.43 (CH), 69.33 (CH),
49.63 (CH), 47.87 (CH), 40.81 (CH2), 35.05 (CH2), 31.21 (CH2),
26.62 (CH2), 24.82 (CH2), 24.63 (CH2). MS (ESI) m/z calcd for
C23H31O6F3 (M+H)+ 461, found 461, HRMS calcd for C23H31O6F3
(M+H)+ 461.4974, found 461.21457.
4.13. 6b-[2ab-Hydroxy-3-(3-chloro)phenoxy-propoxy]-7-a-
benzoyloxy-cis-2-oxabicyclo[3.3.0]octan-3-one (15)
To a solution of 7 (0.1 g, 0.314 mmol) in 2 mL of acetonitrile, 3-
chlorophenol (0.07 ml, 0.63 mmol) and potassium carbonate
(0.13 g, 0.95 mmol) were added and the mixture was refluxed for
4 h. After cooling, the mixture was filtered and the filtrate was con-
centrated. The residue was chromatographed on a silica gel column
eluting with ethyl acetate–hexanes (1:1) to provide the title com-
pound 7 as yellowish oil 0.09 g. MS m/z calcd for C23H23O7ClNa
(M+Na)+ 469, found 469.
4.14. 6b-[2ab-Hydroxy-3-(3-chloro)phenoxy-propoxy]-7a-
hydroxy-cis-2-oxabicyclo[3.3.0]octan-3-one (17)
Potassium carbonate (0.03 g, 0.2 mmol) was added to a solu-
tion of 15 (0.09 g, 0.2 mmol) in 2 mL of methanol. The resulting
mixture was stirred at room temperature for 1.5 h. Ethyl acetate
(2 mL) and 1 N HCl (1 mL) were added. The aqueous layer was
separated and extracted with ethyl acetate (3 Â 3 mL). The com-
bined organic extracts were washed with saturated NaHCO3 and
saturated NaCl solutions and dried over anhydrous MgSO4. Evap-
oration of solvent afforded crude product which was chromato-
graphed on silica gel (ethyl acetate–hexanes, 5:1) to provide
the title compound 17 as yellowish oil 0.075 g. MS m/z calcd
for C16H19O6ClNa (M+Na)+ 365, found 365.
4.17. 5Z-(9S,11R,15R)-13-Oxa-16-phenoxy-propoxy]-9,11,15-
trihydroxy-17,18,19,20-tetranor-5-prostadienoic acid isopropyl
ester (24)
Colorless oil. 1H NMR of 24 (CDCl3) d 7.32–7.24 (m, 2H), 6.99–
6.88 (m, 3H), 5.44–5.41 (m, 2H), 5.02–4.96 (m, 1H), 4.16–3.64
(m, 8H), 2.40–1.64 (m, 11H), 1.24–1.21 (d, J = 6 Hz, 6H). 13C NMR
(CDCl3) d 173.55 (C), 158.51 (C), 130.22 (CH), 129.84 (CH), 129.29
(CH), 121.45 (CH), 114.90 (CH), 93.39 (CH), 77.41 (CH), 73.70
(CH), 71.83 (CH2), 69.84 (CH), 69.14 (CH2), 68.09 (CH), 51.74
(CH), 41.54 (CH2), 34.36 (CH2), 26.96 (CH2), 26.16 (CH2), 25.22
(CH2), 22.17 (CH3). MS (ESI) m/z calcd for C24H36O7 (M+1)+ 437,
found 437.
4.15. 6-[2-Tetrahydro-2H-pyran-2-yl)oxy-3-(3-chloro)phenoxy-
propoxy]-7-tetrahydro-2H-pyran-2-yl)oxy-cis-2-
oxabicyclo[3.3.0]octan-3-one (19)
4.18. 5Z-(9S,11R,15R)-13-Oxa-16-(3-chloro)phenoxy-propoxy]-
9,11,15-trihydroxy-17,18,19,20-tetranor-5-prostadienoic acid
isopropyl ester (25)
3,4-Dihydro-2H-pyran (0.08 ml, 0.85 mmol) was added to a
solution of 17 (0.075 g, 0.211 mmol) in CH2Cl2 (1 mL) and the mix-
ture was cooled to 0 °C. p-Toluenesulfonic acid monohydrate
(2 mg, 0.01 mmol) was added and the resulting mixture was stir-
red at 0 °C for 30 min and then quenched by addition of saturated
NaHCO3 (1 mL). The aqueous layer was separated and extracted
with CH2Cl2 (3 Â 3 mL). The combined organic extracts were
washed with a saturated NaCl solution and dried over anhydrous
MgSO4. Evaporation of solvent afforded the crude product which
was chromatographed on silica gel (ethyl acetate–hexanes, 1:2)
to provide the title compound 19 as oil 0.1 g. MS m/z calcd for
C26H35O8ClNa (M+Na)+ 533, found 533.
Compound 23 (65 mg, 0.1 mmol) was stirred in 1 ml of 65% ace-
tic acid and 1 mL of THF at 55 °C for 1.5 h. The solvent was evapo-
rated and the residue was dried under vacuum. The crude product
was chromatographed on a silica gel column eluting with 80% ethyl
acetate in hexane and followed by HPLC chiral separation to afford
25 (20 mg). 1H NMR of 25 (CDCl3) d 7.20–7.12 (m, 1H), 7.08–6.85
(m, 2H), 6.75–6.70 (m, 1H), 5.40–5.31 (m, 2H), 4.95–4.89 (m,
1H), 4.13–3.57 (m, 8H), 2.33–1.57 (m, 11H), 1.17–1.14 (d,
J = 6 Hz, 6H). 13C NMR (CDCl3) d 173.57 (C), 159.27 (C), 134.84
(C), 130.22 (CH), 129.90 (CH), 128.85 (CH), 121.24 (CH), 114.96

Z. Feng et al. / Bioorg. Med. Chem. 17 (2009) 576–584
583
(CH), 113.03 (CH), 93.04 (CH), 77.08 (CH), 73.42 (CH), 71.28 (CH2),
69.29 (CH), 69.12 (CH2), 67.88 (CH), 51.41 (CH), 41.23 (CH2), 34.0
(CH2), 26.61 (CH2), 25.82 (CH2), 24.86 (CH2), 21.82 (CH3). MS
(ESI) m/z calcd for C24H35O7Cl (M+1)+ 471, found 471.
26.10 (CH2). MS (ESI) m/z calcd for C21H29O7Cl (M+1)+ 429, found
429.
4.23. 5Z-(9S,11R,15S)-13-Oxa-16-phenoxy-propoxy]-9,11,15-
trihydroxy-17,18,19,20-tetranor-5-prostadienoic acid (30)
4.19. 5Z-(9S,11R,15S)-13-Oxa-16-phenoxy-propoxy]-9,11,15-
trihydroxy-17,18,19,20-tetranor-5-prostadienoic acid isopropyl
ester (26)
Colorless oil. 1NMR of 28 (CDCl3) d 7.52–7.44 (m, 2H), 7.17–7.09
(m, 3H), 5.75–5.53 (m, 2H), 4.33–3.76 (m, 8H), 2.55–1.85 (m, 11H).
13C NMR (CDCl3) d 173.50 (C), 160.42 (C), 130.39 (CH), 130.26 (CH),
121.78 (CH), 115.53 (CH), 92.78 (CH), 77.32 (CH), 72.29 (CH2),
71.90 (CH), 70.31 (CH), 70.14 (CH2), 51.04 (CH), 42.14 (CH2),
34.47 (CH2), 27.56 (CH2), 26.05 (CH2). MS (APCI) m/z calcd for
C21H30O7 (M+H)+ 395, found 395.
Colorless oil. 1H NMR of 26 (CDCl3) d 7.32–7.24 (m, 2H), 6.99–
6.89 (m, 3H), 5.47–5.30 (m, 2H), 5.06–4.93 (m, 1H), 4.16–3.63
(m, 8H), 2.40–1.64 (m, 11H), 1.24–1.21 (d, J = 6 Hz, 6H). 13C NMR
(CDCl3) d 173.59 (C), 158.56 (C), 130.22 (CH), 129.83 (CH), 129.28
(CH), 121.43 (CH), 114.90 (CH), 93.49 (CH), 77.37 (CH), 73.64
(CH), 71.78 (CH2), 69.77 (CH), 69.18 (CH2), 68.10 (CH), 51.69
(CH), 41.50 (CH2), 34.36 (CH2), 26.96 (CH2), 26.15 (CH2), 25.22
(CH2), 22.17 (CH3). MS (ESI) m/z calcd for C24H36O7 (M+1)+ 437,
found 437.
4.24. 5Z-(9S,11R,15S)-13-Oxa-16-(3-chloro)phenoxy-propoxy]-
9,11,15-trihydroxy-17,18,19,20-tetranor-5-prostadienoic acid
(31)
Colorless oil. 600 mz 1H NMR (CDCl3) d 7.25–7.22 (m, 1H), 6.97
(s, 1H), 6.94–6.92 (d, 1H, J = 12 Hz), 6.89–6.87 (d, 1H, J = 12 Hz),
5.53–5.46 (m, 1H), 5.37–5.30 (m, 1H), 4.09–3.96 (m, 5H), 3.83–
3.80 (m, 1H), 3.69–3.66 (m, 1H), 3.56–3.54 (m, 1H), 2.29–2.24
(m, 5H), 2.11–2.09 (m, 2H), 1.64–1.63 (m, 1H), 1.62–1.61 (m,
3H). 13C NMR (CDCl3) d 178.16(C), 161.30 (C), 135.88 (C), 131.55
(CH), 130.52 (CH), 130.27 (CH), 121.90 (CH), 116.0 (CH), 114.20
(CH), 92.85 (CH), 77.38 (CH), 72.22 (CH2), 71.94 (CH), 70.62
(CH2), 70.19 (CH), 51.09 (CH), 42.22 (CH2), 34.87 (CH2), 27.69
(CH2), 26.52 (CH2), 26.10 (CH2). MS (ESI) m/z calcd for C21H29O7Cl
(M+1)+ 429, found 429.
4.20. 5Z-(9S,11R,15S)-13-Oxa-16-(3-chloro)phenoxy-propoxy]-
9,11,15-trihydroxy-17,18,19,20-tetranor-5-prostadienoic acid
isopropyl ester (27)
Colorless oil. 1H NMR of 27 (CDCl3) d 7.27–7.24 (m, 1H), 7.19–
6.92 (m, 2H), 6.83–6.78 (m, 1H), 5.44–5.30 (m, 2H), 5.03–4.96
(m, 1H), 4.13–3.63 (m, 8H), 2.40–1.64 (m, 11H), 1.24–1.21 (d,
J = 6 Hz, 6H). 13C NMR (CDCl3) d 173.60 (C), 159.30 (C), 134.85
(C), 130.22 (CH), 129.89 (CH), 128.86 (CH), 121.24 (CH), 114.98
(CH), 113.04 (CH), 93.15 (CH), 76.97 (CH), 73.92 (CH), 71.27
(CH2), 69.20 (CH), 69.09 (CH2), 67.73 (CH), 51.27 (CH), 41.11
(CH2), 33.95 (CH2), 26.55 (CH2), 25.74 (CH2), 24.81 (CH2), 21.76
(CH3). MS (ESI), m/z calcd for C24H35O7Cl (M+1)+ 471, found 471.
4.25. FP Receptor binding assay
Washed total particulate bovine corpus luteum membranes
(20 mg wet weight/mL final concentration) were incubated with
4.21. 5Z-(9S,11R,15R)-13-Oxa-16-phenoxy-propoxy]-9,11,15-
trihydroxy-17,18,19,20-tetranor-5-prostadienoic acid (28)
[3H]PGF2 (1–2 nM final) in Krebs buffer (pH 7.4) for 2 h at 23 °C
a
in a total volume of 500
binding was defined with 10 lM unlabeled PGF2 or cloprostenol.
a
l
L as previously described.15 Non-specific
Colorless oil. 1NMR of 28 (CDCl3) d 7.47–7.39 (m, 2H), 7.13–7.05
(m, 3H), 5.70–5.48 (m, 2H), 4.29–3.71 (m, 8H), 2.50–1.76 (m, 11H).
13C NMR (CDCl3) d 173.50 (C), 160.42 (C), 130.87 (CH), 130.74 (CH),
122.27 (CH), 116.02 (CH), 93.31 (CH), 77.79 (CH), 72.88 (CH2),
72.38 (CH), 70.93 (CH2), 70.79 (CH), 51.52 (CH), 42.65 (CH2),
35.00 (CH2), 28.05 (CH2), 26.50 (CH2). MS (APCI) m/z calcd for
C21H30O7 (M+H)+ 395, found 395.
The assays were terminated by rapid vacuum filtration (using
Whatman GF/B glass fiber filter previously soaked in 0.3%
polyethyleneimine) and the filter-bound radioactivity determined
by liquid scintillation spectrometry on a beta-counter. The equilib-
rium concentration of the test prostaglandin required to displace
[3H]PGF2 from the FP-receptor by 50% (Ki) was determined from
a
several experiments. The Ki value is inversely related to the affinity
4.22. 5Z-(9S,11R,15R)-13-Oxa-16-(3-chloro)phenoxy-propoxy]-
9,11,15-trihydroxy-17,18,19,20-tetranor-5-prostadienoic acid
(29)
of the compound for the FP-receptor.
4.26. FP Receptor mediated phosphoinositide (PI) turnover
assays
A mixture of 25 (18 mg, 0.04 mmol), lithium hydroxide mono-
hydrate (17 mg, 0.4 mmol), methanol (1 mL) and water (0.1 mL)
was stirred at room temperature overnight. Saturated KH2PO4
solution was added to adjust the pH to 6 and the mixture was ex-
tracted with ethyl acetate (3 Â 3 mL). The combined organic layer
was washed with a saturated NaCl solution, dried over anhydrous
MgSO4, filtered and concentrated to a colorless oil, which was
chromatographed on a short silica gel column, eluting with 5%
methanol in ethyl acetate, to afford 29 (8 mg). 600 mz 1H NMR
(CDCl3)) d 7.25–7.22 (m, 1H), 6.97 (s, 1H), 6.93–6.92 (d, 1H,
J = 6Hz), 6.89–6.88 (d, 1H, J = 6 Hz), 5.49–5.47 (m, 1H), 5.34–5.33
(m, 1H), 4.09–3.98 (m, 5H), 3.78–3.77 (m, 1H), 3.67–3.65 (m,
1H), 3.57–3.55 (m, 1H), 2.28–2.23 (m, 5H), 2.10–2.09 (m, 2H),
Total [3H]Inositol phosphates ([3H]-IPs) produced by agonist-
mediated activation of phospholipase C were quantified by previ-
ously published procedures using mouse Swiss 3T3 fibroblasts.15
In brief, confluent Swiss 3T3 cells were exposed to 1.0-1.5 lCi of
[3H]-myo-inositol in 0.5 mL DMEM culture medium for 24-30 h
at 37 °C in order to label the cell membranes. Then the cells were
rinsed once with DMEM/F-12 containing 10 mM LiCl, and the ago-
nist stimulation experiment was performed in 0.5 mL of the same
medium to facilitate the accumulation of [3H]-IPs following PI
hydrolysis. Cells were exposed to the agonist or vehicle solvent
for 60 min at 37 °C, followed by aspiration of the medium and
immediate addition of 1 mL of ice-cold 0.1 M formic acid. The cell
lysates (0.9 mL) were loaded on columns containing 1 mL AG 1-X8
anion exchange resin in the formate form. Unincorporated [3H]-
myo-inositol was removed by washing with 10 mL of distilled
water and discarded. The total [3H]-IPs were collected into scintil-
lation vials by washing the columns with 8 mL of 50 mM ammo-
1.63–1.61 (m, 1H), 1.62–1.61 (m, 3H). 13C NMR (CDCl3)
d
178.10(C), 161.33 (C), 135.88 (C), 131.57 (CH), 130.59 (CH),
130.24 (CH), 121.91 (CH), 116.01 (CH), 114.22 (CH), 92.89 (CH),
77.38 (CH), 72.26 (CH2), 71.94 (CH), 70.80 (CH2), 70.31 (CH),
51.09 (CH), 42.25 (CH2), 35.14 (CH2), 27.74 (CH2), 26.536 (CH2),

584
Z. Feng et al. / Bioorg. Med. Chem. 17 (2009) 576–584
nium formate and 4 mL of 1.2 M ammonium formate made in
0.1 M formic acid. The radioactivity associated with the total
[3H]-IPs was determined by scintillation counting on a beta-coun-
ter. Concentration-response curves from several experiments were
used to determine the potency (EC50) and relative intrinsic activity
(efficacy, Emax) for each compound.
were normal and untreated. Intraocular pressure (IOP) was mea-
sured with an Alcon Applanation Pneumatonometer after light cor-
neal anesthesia with 0.1% proparacaine. Following each
measurement, residual anesthetic was washed out with saline.
Test item was administered in one 30 lL aliquot topically to the
lasered (ocular hypertensive) eyes of eight or nine monkeys. Doses
1, 3, and 5 were instilled at approximately 0900 h on three consec-
utive days; doses 2 and 4 at 1630 h on days 1 and 2. Vehicle was
administered on the same schedule to the lasered eyes of five or
six other animals. IOP measurements were taken at baseline, at se-
lected intervals (up to 7 h) after doses 1 and 5 and 16 h after dose
4. The percent change of IOP from baseline was determined for
each animal for every IOP measurement. Group means and stan-
dard error of the mean (SEM) were calculated for each time point.
Student’s t-test and ANOVA were used to analyze data. Significance
4.27. Data analyses
The original data (dpm) from the different ligand binding and
second messenger assay experiments were analyzed using a non-
linear, iterative curve-fitting computer program.14,15
4.28. Formulation for in vivo studies
Prostaglandins were formulated in a trimethamine/boric acid
buffered vehicle containing 4.6% mannitol, HCO-40 (at a ratio of
10–1 with the active ingredient, but not less than 0.1%), 0.01%
EDTA, and 0.01% benzalkonium chloride, pH 7.4.
levels: p < 0.05; a = 0.05. All animal procedures were performed in
accordance with the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research.
Acknowledgments
4.29. Acute ocular irritation response in New Zealand albino
rabbits
We thank Peter Klimko and Paul Zinke for helpful discussions,
Hans Moll and Karen Haggard for HPLC purification, Glen Dillow,
Liang Xue and Olga Papadopoulou for performing and discussing
Mass and NMR spectrometry data.
A method was devised to grossly compare ocular irritation po-
tential of prostaglandin FP agonists using the New Zealand albino
(NZA) rabbit.16,17 The eyes were examined microscopically using
a Topcon SL-6E or SL-7E slit-lamp The eyes scored for conjunctival
hyperemia, conjunctival swelling, and ocular discharge according
to the Hackett and McDonald Scoring System.16 Test article was
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4.30. Acute miotic response in normotensive cats
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line PD measurements, test item was instilled in one 30 lL aliquot
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4.31. Acute intraocular pressure (IOP) response in ocular
hypertensive monkeys
Animals were minimally restrained in specially designed mon-
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