Carbocyclic nucleosides as inhibitors of human tumor necrosis factor-α production: Effects of the stereoisomers of (3-hydroxycyclopentyl)adenines

Article abstract of DOI:10.1021/jm950906t

A series of four structurally related carbocyclic nucleosides (6a, 6b, 10a, and 10b) were synthesized and evaluated for their ability to inhibit tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin- 6 (IL-6) production from human primary macrophages. These compounds had little effect on the production of IL-1β and IL-6. It was determined that compound 10a was the most potent inhibitor of TNF-α production (IC₅₀ = 10 μM), having 2-5-fold more activity compared to its enantiomer 10b or its diastereomers 6a and 6b. In addition, these compounds were also tested for their ability to protect mice against lethal challenges of lipopolysaccharide (LPS) and D-galactosamine (D-Gal). Compound 10a showed superior protective effects (100% protection) compared to its enantiomer 10b or its diastereomers 6a and 6b when it was administered to mice which were challenged with 3 times the LD₁₀₀ dose of LPS.

Full text of DOI:10.1021/jm950906t

J . Med. Chem. 1996, 39, 2615-2620
2615
Ca r bocyclic Nu cleosid es a s In h ibitor s of Hu m a n Tu m or Necr osis F a ctor -r
P r od u ction : Effects of th e Ster eoisom er s of (3-Hyd r oxycyclop en tyl)a d en in es
,
†
†
†
†
‡
David R. Borcherding,* Norton P. Peet, H. Randall Munson, Hao Zhang, Paul F. Hoffman,
‡
‡
Terry L. Bowlin, and Carl K. Edwards, III
Discovery Chemistry and Department of Immunology, Hoechst Marion Roussel, Inc., 2110 East Galbraith Road,
P.O. Box 156300, Cincinnati, Ohio 45215-6300
X
Received December 11, 1995
A series of four structurally related carbocyclic nucleosides (6a , 6b, 10a , and 10b) were
synthesized and evaluated for their ability to inhibit tumor necrosis factor-R (TNF-R),
interleukin-1â (IL-1â), and interleukin-6 (IL-6) production from human primary macrophages.
These compounds had little effect on the production of IL-1â and IL-6. It was determined that
compound 10a was the most potent inhibitor of TNF-R production (IC50 ) 10 µM), having 2-5-
fold more activity compared to its enantiomer 10b or its diastereomers 6a and 6b. In addition,
these compounds were also tested for their ability to protect mice against lethal challenges of
lipopolysaccharide (LPS) and D-galactosamine (D-Gal). Compound 10a showed superior
protective effects (100% protection) compared to its enantiomer 10b or its diastereomers 6a
and 6b when it was administered to mice which were challenged with 3 times the LD100 dose
of LPS.
In tr od u ction
Ch em istr y
Under normal conditions the production of cytokines
helps clear viral and bacterial infections and damaged
cells from injured tissues. However, there are many
cases in which the overproduction of proinflammatory
cytokines such as tumor necrosis factor-R (TNF-R),
interleukin-1â (IL-1â), and interleukin-6 (IL-6) can
The cis-configured carbocyclic nucleosides 6a and 6b
Scheme 3) were synthesized from the enantiomerically
(
pure starting materials 2a and 2b (Scheme 1). Com-
pound 2a was prepared from 1 using an enzymatic
1
0
method, and compound 2b was obtained commercially.
Compounds 2a and 2b were converted to the trans-
substituted mesylates 3a and 3b using methanesulfonyl
1
cause multiple-organ diseases and life-threatening shock.
One cytokine that is increasingly recognized as a central
mediator in a wide spectrum of physiological and
chloride (3 equiv) and triethylamine (3 equiv) at 0 °C
1
11
immune functions is macrophage-derived TNF-R. The
in quantitative yield.
Compounds 3a and 3b were
overproduction of TNF-R has been strongly implicated
in septic shock, adult respiratory distress syndrome,
AIDS, inflammatory bowel disease, bacterial meningitis,
used without further purification and were individually
added to stirring solutions containing 3 equiv of sodium
adenide in DMF at 60 °C which afforded the chiral,
protected nucleosides 4a and 4b in 39% and 60% yields,
respectively (Scheme 2). The acetate protecting groups
of 4a and 4b were removed under similar conditions
using potassium carbonate in methanol and purified by
flash chromatography (eluted with 9:1 methylene chlo-
ride/methanol) to give the optically active nucleosides
malaria, multiple sclerosis, atherosclerosis, and rheu-
matoid arthritis.1
-3
Thus, agents which can inhibit the
production of TNF-R have attracted much attention over
the last few years as potential therapies for the treat-
ment of inflammatory diseases.1
It has been reported that adenosine (ADO) and
certain ADO derivatives are potent regulators of mac-
1
1
rophage (Mφ) functions.4
,5
5a and 5b in 67% and 61% yields, respectively. The
individual compounds 5a and 5b were then reduced
with PtO2 in methanol, and the products were purified
Adenosine has been shown
6
to inhibit monocyte/macrophage phagocytosis, chemo-
7
taxis, and the synthesis and secretion of inflammatory
8
,9
11
mediators.
Some, but not all, of these effects are
by flash chromatography to give 6a and 6b in 76% and
mediated through classical cell-surface ADO receptors
that are expressed on mononuclear phagocytes. In our
80% yields, respectively (Scheme 3). A hydrogenolysis
product, 9-adenylcyclopentane, was the major side
product (∼20%) in both reductions.
9
attempt to obtain selective inhibitors of TNF-R produc-
tion we have designed and synthesized a carbocyclic
adenosine analogue 6a (Scheme 3) which has been
The trans-configured carbocyclic nucleosides 10a and
10b were prepared from 2a and 2b as shown in Scheme
8
shown to selectively inhibit TNF-R production. In the
4
. The chiral intermediates 2a and 2b were reduced
present study we will describe the synthesis and bio-
logical activity of (cis- and trans-3-hydroxycyclopentyl)-
adenines which were prepared to better understand the
role of the cyclopentanol moiety in the biological activity
of this new class of TNF-R inhibitors.
using Raney nickel and hydrogen to give 7a or 7b in
approximately 55% yield. The chiral cyclopentanols 7a
and 7b were converted to the mesylates 8a and 8b using
the previously described method and then were indi-
vidually treated with sodium adenide to give the pro-
tected trans compounds 9a and 9b in 53% and 46%
yields, respectively. Compound 10a was obtained by
heating a solution of 9a in ethanol at reflux for 48 h
with the pH adjusted to 2.5 using 6 N HCl in 87% yield.
*
To whom correspondence should be addressed at: Discovery
Chemistry, 2110 East Galbraith Rd., P.O. Box 156300, Cincinnati, OH
4
5215-6300. 513-948-6506; 513-948-7585 (Fax).
†
Discovery Chemistry.
Department of Immunology.
‡
X
Abstract published in Advance ACS Abstracts, May 15, 1996.
S0022-2623(95)00906-X CCC: $12.00 © 1996 American Chemical Society

2
616 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Notes
Sch em e 1a
a
Reagents: (a) ref 16; (b) CH3SO2Cl, TEA, CH2Cl2.
Sch em e 2a
a
Reagents: (a) adenine (3 equiv), NaH, DMF; (b) K2CO3, CH3OH.
Sch em e 3a
effects of these compounds on D-Gal sensitized mice
which have received lethal injections of LPS.13
Human Mφ were obtained from healthy volunteers
and were incubated with the compounds (ADO, 6a , 6b,
1
4-17
1
0a , and 10b) and LPS.
the culture supernatants were assayed for TNF-R, IL-
â, and IL-6 using human specific ELISA kits. Shown
in Table 1 are the inhibitory effects that ADO, 6a , 6b,
0a , and 10b have on the production of human TNF-R,
After incubation for 18 h
1
1
IL-1â, and IL-6 elicited from LPS-stimulated Mφ. Only
TNF-R was significantly inhibited by the various car-
bocyclic nucleosides and ADO. ADO and 10a demon-
strated the most potent inhibition of TNF-R production,
having IC50 values of 6 and 9 µM, respectively. ADO
and 10a showed a 2-5-fold separation between their
IC50 values and those obtained for compounds 6a , 6b,
and 10b. Although 10b is the enantiomer of 10a , one
can also view 10b as positional isomer of 10a . The
hydroxyl group on 10a can be viewed as the equivelant
of the 3′-hydroxyl group of adenosine, while the hydroxyl
group on 10b can be considered to reside in the
4′-position. Therefore, compounds 10a and 10b are not
just simply enantiomers, and this may account for the
2-fold separation in the IC50 values for the inhibiton of
TNF-R. Compounds 6a and 6b can also be considered
as positional isomers which may also account for their
a
Reagents: (a) PtO2, H2, MeOH.
Compound 10b was obtained by deprotection of 9b with
potassium carbonate in methanol in 77% yield.
Resu lts a n d Discu ssion
Adenosine and ADO-like compounds have been shown
8
,9,12
to inhibit macrophage-derived TNF-R production.
We have previously reported the design and synthesizis
of the carbocyclic adenosine analogue 6a 11 and its ability
2
6
-fold separation in IC50 values. The 3′-epimers 10a and
b and the 4′-epimers 10b and 6b showed a 2-fold
to inhibit TNF-R production from mouse thioglycollate-
elicited peritoneal Mφ and to protect D-galactosamine
D-Gal) sensitized mice from a lethal challenge (LD100)
of lipopolysaccharide (LPS). In this report we describe
the effects of 6a and its stereoisomeric cyclopentanol
analogues (6b, 10a , and 10b) on the production of
TNF-R from primary human Mφ and the protective
differences in their IC50 values with the cis compounds
being the least active.
(
8
These compound were then examined for their protec-
tive effects against lethal injections of D-Gal sensitized,
1
8-20
LPS-challenged (LD100) CF1 mice.
In this septic

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2617
Sch em e 4a
a
Reagents: (a) RaNi, H2, EtOH; (b) MsCl, TEA, CH2Cl2; (c) adenine, NaH, DMF; (d) HCl, EtOH; (e) (i) K2CO3, EtOH; (ii) HCl, H2O.
Ta ble 1. In Vitro and in Vivo Biological Evaluation of
Carbocyclic Nucleosides
potency may be due to a longer duration of action.
Compound 10a was determined not to be a substrate
for purified calf intestinal adenosine deaminase, and it
LPS/D-galactosamine
septic shock model
22
appeared not to be phosphorylated (data not shown).
IC50 (µM)a
IL-1â
% survivalb
We have previously shown that the inhibition of TNF-R
is mediated through the adenosine A3 receptor, and
improved receptor occupancy for compound 10a may
compd
TNF-R
IL-6
LD100
3 × LD100
control
ADO
0
0
0
0
0
0
100
0
1
6
6
40
18
9
16
33
>100
>100
>100
76
>100
>100
>100
>100
>100
>100
>100
>100
also be responsible for its superior potency.
6
6
1
1
a
b
0a
0b
88
50
100
63
66
It has been demonstrated that the carbocyclic adeno-
sine analogues 6a , 6b, 10a , and 10b are effective at
inhibiting TNF-R production from primary human Mφ,
and this activity can be correlated with the protection
seen in the LPS/D-Gal-induced septic shock model. We
have previously shown that the inhibition of TNF-R is
mediated through the adenosine A3 receptor which
PTX
0
a
Control: vehicle (PBS). Compound concentrations (dose re-
sponse): 0.16, 0.8, 4.0, 20.0, and 100.0 µM in PBS. Supernatants
were assayed for TNF-R, IL-1â, and IL-6 using ELISA kits.
b
Control: vehicle (PBS). Compound dosaged: 100 mg/kg IP in
1
2,16
specifically inhibits TNF-R transcription.
TNF-R
PBS.
gene expression is known to be regulated by the NF-kâ
family of transcription factors, and we have also shown
that compound 10a inhibits the activation and trans-
location of the p50/p65 NF-kâ heterodimer complex from
the cytoplasm to the nucleus. Compounds which can
inhibit TNF-R production at the transcriptional level
offer a unique and novel approach to the development
of new therapeutic drugs for the treatment of auto-
shock model, mice were treated with 100 mg/kg ip of
the test compounds (ADO, 6a , 6b, 10a , and 10b) 1 h
prior to challenge with LPS/D-Gal. Pentoxyfylline (PTX)
was used in this experiment as a positive control since
it has been reported to be effective at preventing LPS/
2
1
D-Gal-induced septic shock and death in mice. This
model has been shown to be a TNF-R driven model since
monoclonal anti-TNF-R antibodies can protect D-Gal
1
immune and other inflammatory diseases.
2
0
sensitized mice against the lethal challenge of LPS.
Exp er im en ta l Section
In the first experiment, the D-Gal-sensitized mice were
challenged with an LD100 dose of LPS, and the percent
survival was determined after 8 h. All of the control
animals died, and the PTX positive control showed 66%
protection 8 h postchallenge. ADO showed no protection
at 8 h, presumably due to the rapid metabolism of ADO
to inosine and/or ATP. Compounds 6a and 10a showed
Except where noted otherwise, reagents and starting ma-
terials were obtained from common commercial sources and
used as received. “Dry” reaction solvents refer to solvents in
Aldrich Sure-Seal bottles. The phrase “concentrated in vacuo”
indicates rotary evaporation using a Buchi apparatus at 20-
5
0 °C and 15-30 Torr (KNF Neuberger Model UN 726.12FTP
diaphragm pump). Vacuum drying was done at <10 Torr at
the temperatures noted. Melting points were determined on
a Thomas Hoover Uni-melt capillary melting point apparatus.
Melting points and boiling points are reported uncorrected.
Thin layer chromatography (TLC) was performed on glass-
backed, silica gel 60F-254 plates (EM) coated to a thickness
of 0.25 mm. The plates were eluted with solvent systems (v/
v) as described and visualized by UV light or a p-anisaldehyde
solution. Flash chromatography was carried out using EM
Science silica gel 60 (40-63 µm) according to the literature
8
8 and 100% protection, respectively, and were more
effective than PTX. Compounds 6b and 10b gave
protection comparable to PTX.
In another experiment, D-Gal sensitized mice were
given 3 times the LD100 dose of LPS to determine if any
of the compounds had superior potency. Only compound
1
0a maintained its ability to protect D-Gal sensitized
mice against the lethal challenge of LPS. The increased

2
618 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13
Notes
procedure using the solvent systems as described. Infrared
IR) spectra were recorded on a Mattson Galaxy Series 5020
infrared spectrophotometer with samples prepared as indi-
cated, and are reported in wave numbers (cm ). H NMR
spectra were recorded on Varian Gemini 300, Unity 300, Unity
methane) 260 (base, (M + 1), 200, 173, 136, 125. Anal.
(
13 5 2
(C12H N O ) C, H, N.
(1S,4R)-1-(9-Ad en yl)-4-h yd r oxycyclop en t -2-en e (5b ).
Compound 4b (5.5 g, 21 mmol) was dissolved in methanol (60
mL), K CO (1.0 g) was added, and the reaction mixture was
2 3
-
1
1
4
00, or Unity 500 spectrometers with chemical shifts (δ)
stirred for 2 h at room temperature. The mixture was filtered
and concentrated in vacuo. The residue was purified using
flash chromatography eluting with 9:1 methylene chloride/
methanol. The fractions containing product were combined
and concentrated to dryness. The product was dissolved in
1
3
reported in ppm relative to tetramethylsilane (0.00 ppm).
C
NMR spectra were recorded on the Varian Gemini instrument
75 MHz) with chemical shifts (δ) reported in ppm relative to
CDCl (77.0 ppm), unless stated otherwise. Mass spectra (MS)
(
3
were obtained on a Finnigan MAT Model TSQ 700 mass
spectrometer system by chemical ionization at 120 eV using
methane (CI, 120 eV) unless otherwise indicated, e.g. with
electron impact, MS (EI, 70 eV). Ultraviolet (UV) spectra were
recorded on a Perkin-Elmer Lambda 4C spectrophotometer
and reported in nanometers (nm). The sample was prepared
in the solvent indicated at a concentration expressed as mg/
mL. Specific rotation [R] was obtained with a J ASCO Model
DEP 360 polarimeter fitted with a 1 dm cell. Measurements
were made at the sodium D line (589 nm) at 20 °C (unless
otherwise specified), and the concentration (g/100 mL) and
solvent are reported. (1R,4S)-1-Acetoxy-4-hydroxycyclopent-
H
2
O, and the pH of the solution was adjusted to 2.5 with 6 N
HCl. The solution was lyophilized to give 2.84 mg (61%) of
1
5b: mp 204-205 °C; [R]
(DMSO-d
(s, 1 H), 6.26 (m, 1 H), 6.03 (m, 1 H), 5.54 (m, 1 H), 4.75 (m, 1
D
) -80° (c ) 0.98, MeOH); H-NMR
6
) δ 9.6 (br s, 1 H), 9.0 (br s, 1 H), 8.57 (s, 1 H), 8.49
H), 2.95 (ddd, 1 H, J ) 16, 8, 8, Hz), 1.79 (ddd, 1 H, J ) 16, 2,
2, Hz); 13C-NMR (DMSO-d
) δ 150.5, 147.8, 144.8, 142.2, 140.3,
6
129.9, 118.0, 73.5, 57.8, 41.3; MS (EI) m/z 217 (M), 188, 173,
135 (base), 108; UV (MeOH) λ ) 261 nm, ꢀ ) 14 500. Anal.
10 11 5
(C H N O‚0.8HCl) C, H, N, Cl.
(1S,3R)-1-(9-Aden yl)-3-h ydr oxycyclopen tan e (6b). Com-
pound 5b (100 mg, 0.39 mmol) was dissolved in methanol (50
mL), PtO (30 mg) was added, and the mixture was hydroge-
2
-ene was purchased from Fluka. PTX and lipopolysaccharide
2
nated at 35 psi of hydrogen for 3 h. The catalyst was removed
by filtration through Celite, and the filtrate was concentrated
in vacuo. The residue was purified by flash chromatography
eluted with methylene chloride/methanol (9:1). The fractions
containing product were combined and concentrated and then
dissolved in water which was adjusted to pH 2.5 with 6 N HCl.
(
LPS) from Escherichia coli were purchased from Sigma
Chemical Co. (St. Louis, MO). Human recombinant IL-1â
(
HurIL-1â) was purchased from Upstate Biotechnology Inc.
(Lake Placid, NY), and human recombinant tumor necrosis
factor-R (HurTNF-R) was purchased from R&D Systems (Min-
neapolis, MN).
The solution was lyophilized to give 76 mg (76%) of compound
(
1S,4R)-4-Acetoxy-1-(9-a d en yl)cyclop en t-2-en e (4b). To
1
6
(
)
2
1
b: mp 231 °C dec; [R]
DMSO-d + D
9 Hz), 4.30 (p, 1 H, J ) 2 Hz), 2.49 (m, 1 H), 2.26 (m, 1 H),
) δ 150.5,
48.2, 144.9, 142.5, 120.0, 70.3, 53.9, 41.5, 34.0, 31.0; MS (EI)
O‚
D
) -9.0° (c ) 0.002, MeOH); H-NMR
a 250 mL flask were added (1R,4S)-1-acetoxy-4-hydroxycyclo-
pent-2-ene (2b, 5.0 g, 35.3 mmol), methylene chloride (100 mL),
and methanesulfonyl chloride (12.1 g, 105.9 mmol), and the
contents were stirred and cooled with an ice bath to 0-5 °C.
Neat triethylamine (10.7 g, 105.9 mmol) was added dropwise
over a 10 min period, and after the addition was complete the
ice bath was removed. The reaction mixture was allowed to
come to room temperature and stir for overnight. Progress of
the reaction was monitored by thin layer chromatography
6
2
O) δ 8.62 (s, 1 H), 8.44 (s, 1 H), 5.03 (p, 1 H, J
.13 (m, 1 H), 1.99-1.8 (m, 3 H); 13C-NMR (DMSO-d
6
m/z 219 (M), 202, 162, 135 (base) 108. Anal. (C10
.75H O) C, H, N.
1R,3S)-1-Acetoxy-3-h yd r oxycyclop en ta n e (7a ). Com-
14 l 5
H C N
0
2
(
pound 2a (36 g, 254 mmol) was added to a suspension of
approximately 50 g of Raney nickel in 250 mL of absolute
ethanol. The suspension was stirred under 1-4 bar of
hydrogen at 15-20 °C for approximately 5 h. The mixture
was filtered through a P3 glass filter, and the solid was rinsed
with ethanol. The filtrate was concentrated in vacuo, the
yellow oil was dissolved in ether (100 mL), 1 g of activated
carbon was added, and the mixture was stirred for 5 min and
filtered. The filtrate was evaporated to give 33 g of a colorless
oil which was distilled bulb-to-bulb (80-90 °C at 0.1 mmHg)
(EtOAc/hexane (4:1)), and the reaction was judged to be
complete (R
f
) 0.75). The mixture was extracted with water
(
2 × 25 mL) and brine (1 × 50 mL). The aqueous layers were
combined and extracted with methylene chloride (150mL), and
the combined organic layers were dried (sodium sulfate) and
concentrated in vacuo to give 3b (7.78 g, 100%) as a yellow
oil. This material was used immediately without further
purification: H-NMR (CDCl
1
3
) δ 6.13 (ddd, 1 H, J ) 5, 2, 2
Hz), 6.05 (ddd, 1 H, J ) 5, 2, 1 Hz), 5.85 (m, 1 H), 5.08 (m, 1
to yield 20.0 g (55%) of 7a : 1H-NMR (CDCl
3
) δ 5.14 (m, 1 H),
.33 (m, 1 H), 2.2 (br s, 1 H), 2.09 (m, 1 H), 2.03 (s, 3 H), 2.0-
H), 3.71 (s, 3 H), 2.59 (ddd, 1 H, J ) 16, 8, 4 Hz), 2.38 (ddd, 1
4
1
3
1
3
H, J ) 16, 8, 4 Hz), 2.04 (s, 3 H); C-NMR (CDCl
3
) δ 170.7,
13
.7 (m, 5 H); C-NMR (CDCl
3
) δ 170.8, 75.5, 72.4, 41.7, 33.8,
1
37.5, 133.8, 78.4, 61.3, 41.5, 31.6, 21.0.
0.5, 21.2; MS (CI, methane) m/z 145 (M + 1), 127, 101; [R]
D
Sodium adenide was prepared by adding adenine (14.40 g,
05.9 mmol) to dry DMF (250 mL) and was added to the slurry
) -5.43° (c ) 1.03, MeOH).
1S,3R)-1-Acetoxy-3-h yd r oxycyclop en ta n e (7b). Com-
pound 7b was prepared in a similar manner as 7a starting
1
(
NaH (60% in mineral oil, 4.24 g, 105.9 mmol), and the reaction
mixture was stirred for 3 h at 55-60 °C. The (1R,4R)-1-
acetoxy-4-[(methylsulfonyl)oxy]cyclopent-2-ene (3b) (7.78 g,
1
with 2b: H-NMR (CDCl ) δ 5.14 (m, 1 H), 4.33 (m, 1 H), 2.2
3
(br s, 1 H), 2.09 (m, 1 H), 2.03 (s, 3 H), 2.0-1.7 (m, 5 H); 13C-
3
5.3 mmol) was added, and the reaction mixture was stirred
NMR (CDCl ) δ 170.8, 75.5, 72.4, 41.7, 33.8, 30.5, 21.2; MS
3
for 48 h at 55-60 °C. The product formation (R ) 0.9) was
f
(CI, methane) m/z 145 (M + 1), 127, 101; [R] ) +5.30° (c )
D
monitored by TLC with methylene chloride/methanol (4:1). The
reaction mixture was cooled to 10 °C (ice bath), and the solid
which formed was collected and washed thoroughly with
methylene chloride. The filtrate was concentrated in vacuo,
and the residue was dissolved in methylene chloride (200 mL)
and filtered to removed undissolved solids. The filtrate was
extracted successively with brine (2 × 50 mL), and the aqueous
layer was extracted with methylene chloride (3 × 100 mL).
The combined organic layers were dried (sodium sulfate) and
concentrated in vacuo. The residue was purified by flash
chromatography eluted with methylene chloride/methanol (19:
1.012, MeOH).
(1R,3S)-1-Acet oxy-3-(m et h ylsu lfon oxy)cyclop en t a n e
(8a ). A solution of (1R,3S)-1-acetoxy-3-hydroxycyclopentane
(7a , 49.0 g, 340 mmol) and methanesulfonyl chloride (46.7g,
408 mmol) in methylene chloride (425 mL) was stirred and
cooled to 0-5 °C (ice bath). To the solution was added
triethylamine (41.3 g, 408 mmol) in 75 mL of methylene
chloride over a 10 min period. After the addition was complete
the ice bath was removed and the reaction mixture was
allowed to come to room temperature and stir for 1 h (TLC;
EtOAc/hexane (4:1); R ) 0.75). The mixture was extracted
f
1
1
6
(
1
1
) to give 5.5 g (60%) of 4b: mp 250 °C dec; [R]
D
) -9.4° (c )
) δ 8.35 (s, 1 H), 7.84 (s, 1 H),
.32 (m, 1 H), 6.18 (m, 1 H), 6.07 (br s, 2 H), 5.7 (m, 2 H), 3.11
with water (1 × 400 mL, 1 × 200 mL) and brine (1 × 200 mL).
The aqueous layers were combined and extracted once with
methylene chloride (200 mL), and the combined organic
solutions were dried (sodium sulfate) and concentrated in
1
.01, MeOH); H-NMR (CDCl
3
ddd, 1 H, J ) 16, 8, 8 Hz), 2.08 (s, 3 H), 1.95 (ddd, 1 H, J )
1
3
vacuo to give 8a as a yellow oil (76.4 g, 98%): 1H-NMR (CDCl
6, 2, 2 Hz); C-NMR (CDCl
3
) δ 170.4, 155.6, 153.1, 149.6,
3
)
38.6, 135.5, 133.7, 119.5, 77.2, 56.7, 38.7, 21.0; MS (CI,
δ 5.14 (m, 2 H), 3.02 (s, 3 H), 2.39 (dt, 1 H), 2.1-1.95 (m, 8 H);

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 13 2619
13C-NMR (CDCl
) δ 170.7, 81.8, 74.0, 39.5, 38.5, 31.7, 30.4,
h and then filtered and concentrated in vacuo. The residue
was purified by flash chromatography eluting with methylene
chloride/metanol (4:1). The fractions containing product were
combined and concentrated, and the residue was dissolved in
water (20 mL). The pH was adjusted to 2.0 with 6 N HCl,
and the solution was lypholyzed to give a white powder (0.9
D
3
2
1.1; IR (neat) 1734, 1352, 1249, 1167, 974, 889; MS (CI,
methane) 223 (M + 1), 163, 127 (base), 67; [R] ) +5.93° (c )
.97, CH Cl ). Anal. (C S) C, H.
1S,3R)-1-Acet oxy-3-(m et h ylsu lfon oxy)cyclop en t a n e
8b). Compound 8b was prepared in a similar manner as 8a
starting with 7b (1.44 g, 10.0 mmol) to give 2.22 g (100%) of
D
0
2
2
8 14 5
H O
(
(
g, 77%): mp 239 °C dec; [R] ) -11.6° (c ) 0.1982, MeOH);
1
1
8
b: H-NMR (CDCl
3
) δ 5.14 (m, 2 H), 3.02 (s, 3 H), 2.39 (dt, 1
H-NMR (DMSO-d ) δ 9.6 (br s, 1 H), 9.0 (br s, 1 H), 8.7 (s, 1
6
1
3
H), 2.1-1.95 (m, 8 H); C-NMR (CDCl
3
) 170.7, 81.8, 74.0, 39.5,
8.5, 31.7, 30.4, 21.1; IR (neat) 1734, 1352, 1249, 1167, 974,
89; MS (CI, methane) 223 (M + 1), 163, 127 (base), 67.
1R,3R)-3-Acetoxy-1-(9-a d en yl)cyclop en ta n e (9a ). Ad-
H), 8.6 (s, 1 H), 5.2 (m, 1 H), 4.4 (m, 1 H), 2.4 (m, 1 H), 2.2 (m,
3 H), 2.0 (m, 1 H), 1.7 (m, 1 H); 13C-NMR (DMSO-d ) δ 150.4,
3
8
6
148.3, 144.5, 142.7, 118.2, 70.2, 54.7, 41.5, 33.5, 30.1; MS (CI,
methane) 220 (M, base), 136; UV λmax ) 261.0 nm (95%
ethanol, ꢀ ) 13 800). Anal. (C H ClN O) C, H, N.
(
enine (91.9 g, 680 mmol) was suspended in DMF (700 mL),
and NaH (60% dispersion in mineral oil, 27.2 g, 680 mmol)
was washed with hexane (1 × 200 mL) and was then added to
the stirring adenine suspension. The reaction mixture was
stirred for 3 h at 55 °C, and 8a (76 g, 340 mmol) was added to
the reaction mixture (rinsed with DMF 2 × 100 mL), the
reaction mixture was stirred for 48 h at 55-60 °C (TLC;
1
0
14
5
Cell P r ep a r a tion a n d in Vitr o Dose-Resp on se. Venous
blood was collected from normal volunteers, and peripheral
blood mononuclear cells (PBMC) were isolated by density
gradient centrifugation using LeucoPREP cells separation
tubes (Becton Dickinson, Linclon Park, NJ ). The monocyte/
macrophage populations were obtained by the method de-
methylene chloride/methanol (4:1); R
f
) 0.9). The reaction
17
scribed by Kumagai et al. Briefly, PBMC were allowed to
mixture was then cooled to 10 °C (ice bath), and the solid which
formed was filtered and washed thoroughly with methylene
chloride (1.2 L). The filtrate was concentrated in vacuo, and
the residue was dissolved in methylene chloride (500 mL) and
filtered. The filtrate was extracted with brine (2 × 100 mL),
and the aqueous layer was extracted with methylene chloride
adhere to petri dishes (Falcon) previously coated with heat-
inactivated FCS. Nonadherent cells were rinsed off after a 1
h incubation period, and adherent monocyte/macrophages
populations were removed using RPMI-1640 with 0.2% EDTA
and 5% heat-inactivated FCS. As determined by FACS
analysis, this procedure routinely increased MO-2+/KC56+
(
3 × 100 mL). The combined organic layers were dried (sodium
(
CD14+/CD45+) cells in the population by 3-5-fold over what
sulfate) and concentrated in vacuo. The material was dis-
solved in methylene chloride (300 mL), diluted with hexane
was present in the PBMC population. Compounds were
dissolved in PBS (0.16, 0.8, 4.0, 20.0, and 100 µM) and added
(200 mL), seeded, and kept overnight at room temperature.
6
to the monocyte/macrophage cells (10 /mL) 15 min prior to
The solid that formed was vacuum-dried overnight to give 32.2
g (36%) of 9a . An additional 16.9 g of material was obtained
from the filtrate after silica gel (500 g) chromatography using
methylene chloride/methanol (19:1) to give a total of 49.1 g of
stimulation with LPS (1 µg/mL) in RPMI-1640 supplemented
with 100 units/mL penicillin, 100 µg/mL, 0.18 mM L-glutamine
and 10% nonmitogenic heat-inactivated FCS in 24 well tissue
culture plates. Cultures were incubated at 37 °C in 5% CO
2
9
a . This material was recrystallized from methylene chloride/
for 18 h. Culture supernatants were then harvested by
centrifugation and stored at -70 °C until they were assayed
for cytokines. Culture supernatants were assayed for TNF-
R, IL-1â, and IL-6 proteins using ELISA kits from Cistron,
specific for the protein being assayed. The IC50 data was
generated using a computerized nonlinear regression analysis
as discribed by Baron and Siegel.23
hexane to give 47.3 g (53%) of 9a : mp 148-149 °C; [R]
D
)
1
-
9.03° (c ) 0.997, methanol); H-NMR (CDCl
3
) δ 8.38 (s, 1
H), 7.85 (s, 1 H), 6.75 (br s, 2 H), 5.45 (m, 1 H), 5.10 (p, 1 H),
.45 (m, 3 H), 2.2-1.85 (m, 5 H); 1 C-NMR (CDCl
3
2
1
2
3
) δ 170.5,
55.5, 152.8, 150.1, 138.8, 120.2, 74.6, 54.5, 38.9, 31.1, 30.6,
1.2; UV λmax ) 261 nm (95% ethanol, ꢀ ) 14 500); MS (CI,
methane) 262 (M, base), 218, 202, 136; IR (KBr) 3311, 3144,
1
730, 1666, 1601, 1475, 1419, 1251. Anal. (C12
O2‚0.2H O) C, H, N.
1S,3S)-3-Acetoxy-1-(9-a d en yl)cyclop en ta n e (9b). Com-
H
15
N
5
-
En d otoxin Leth a lity Stu d ies. These studies using CF
1
2
female mice (Charles Rivers) were conducted in a manner
similar to that described by Silverstein.24 After overnight
fasting, mice were pretreated (-1 h) with the test compounds
(
pound 9b was prepared in a similar manner as 9a starting
with 8b (2.2 g, 10.0 mmol), except the crude product was
(100 mg/kg in PBS, ip), followed by an ip challenge of D-GalNH
2
directly chromatographed. For 9b: yield 1.2 g (46%); mp 148-
(20 mg) and S. enteritidis LPS (15-250 ng; DIFCO). The dose
of LPS necessary to cause 100% lethality varied slightly
between different shipments of mice; therefore, an LD100 was
determined prior to each shipment for that group of mice.
Immediately after challenge, the mice were returned to feed
and lethality was recorded 8 h postchallenge.
1
1
49 °C; [R]
D
) +7.8° (c ) 1.007, methanol); H-NMR (CDCl
3
)
δ 8.38 (s, 1 H), 7.85 (s, 1 H), 6.75 (br s, 2 H), 5.45 (m, 1 H),
5
(
.10 (p, 1 H), 2.45 (m, 3 H), 2.2-1.85 (m, 5 H); 13C-NMR
CDCl ) δ 170.5, 155.5, 152.8, 150.1, 138.8, 120.2, 74.6, 54.5,
3
3
1
8.9, 31.1, 30.6, 21.2; UV λmax ) 261 nm (95% ethanol, ꢀ )
4 500); MS (CI, methane) 262 (M + 1, base), 218, 202, 136.
Anal. (C12
15 5 2
H N O ) C, H, N.
Ack n ow led gm en t. The authors thank Prof. Hans
Wynberg, Wolterten Hoeve, and Erik van Echten of
Synlom for providing the chiral cyclopentanols.
(
1R,3R)-1-(9-Ad en yl)cyclop en ta n -3-ol (10a ). Compound
9
a (46.8 g, 177 mmol) was dissolved in ethanol (400 mL), and
the pH was adjusted to 2.5 with 6 N HCl (∼35 mL). The
solution was stirred and heated at reflux for 48 h. The reaction
mixture was cooled to 0-5 °C, and a white solid formed. The
solid was collected, washed with cold ethanol/hexane (1:1) and
hexane, and air-dried overnight to give 29.8 g of 10a . Con-
centration of the filtrate gave an additional 12.97 g (in two
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