Synthesis and biological evaluation of pyridine-containing lipoxin A4 analogues

Article abstract of DOI:10.1002/cmdc.200900533

A short and efficient synthesis of new pyridine-containing lipoxin A4 analogues was developed. These analogues induce phagocytosis of apoptotic polymorphonuclear leukocytes and display antiinflammatory characteristics by suppressing pro-inflammatory cytokine production by macrophages.

Full text of DOI:10.1002/cmdc.200900533

DOI: 10.1002/cmdc.200900533
Synthesis and Biological Evaluation of Pyridine-Containing Lipoxin A4
Analogues
Colm D. Duffy,[a] Paola Maderna,[b] Ciara McCarthy,[c] Christine E. Loscher,[c] Catherine Godson,[b] and
Patrick J. Guiry*[a]
The natural eicosanoids lipoxin A4 and lipoxin B4 have been
shown to possess multiple biological activities and are funda-
mental in resolving inflammation.[1] A growing interest in this
new class of compounds has emerged over the last 20 years
ever since their role in inflammatory diseases became appar-
ent.[2,3] Discovered and isolated from human leukocytes in the
early 1980s by Serhan and Samuelsson,[4,5] they are a product
of the lipoxygenase pathway produced from arachidonic acid.
These secondary metabolites contain attractive biochemical
lation of differentiated THP-1 cells with all of the compounds
tested caused F-actin rearrangement similar to that described
for native lipoxins. These exciting results, in addition to the po-
tential for an enhanced pharmacological profile, prompted us
to investigate the synthesis of epimeric heteroaromatic LXA4
analogues of type 4 for biological evaluation, as replacing ben-
zene by heteroaromatic bioisosteres is a well-known and suc-
cessful strategy in medicinal chemistry.[12] This would allow a
structure–activity relationship study whereby we could deter-
mine the effect of the decreased electron density of the hetero-
aromatic ring and how the extra heteroatom may alter its abili-
ty to accept hydrogen bonds from the receptor in comparison
with analogue 3.
properties and have the potential to be used as pharmacologi-
cal agents.[6] The first efficient synthetic route to lipoxins (LX)
was described in the 1980s, and soon after their isolation,
many excellent synthetic pathways have emerged.[7,8]
The rapid metabolism of LX in vivo hinders the use of these
compounds as effective pharmaceutical agents.[9,10] LX are rap-
idly metabolized either by oxidation at C15 or reduction at the
C13=C14 double bond. This problem has been recently over-
come by the stereoselective synthesis of aromatic LXA4 of type
3, and LXB4 analogues by our research group.[11] The synthetic
route developed establishes the required stereochemistry by
way of Sharpless epoxidation, palladium-mediated Heck cou-
pling, and diastereoselective reduction reactions. The synthetic
stable LXA4 analogues caused a significant increase in the
phagocytosis of apoptotic human polymorphonuclear neutro-
phils (PMNs) relative to the effect of native LXA4, while the
LXB4 analogue also stimulated phagocytosis. In addition, stimu-
The retrosynthetic analysis of the pyridine-containing LXA4
analogue (Scheme 1) includes an asymmetric reduction of a
ketone, a palladium-catalyzed Heck reaction, a Sharpless asym-
[a] C. D. Duffy, Prof. P. J. Guiry
Centre for Synthesis and Chemical Biology
UCD School of Chemistry and Chemical Biology
University College Dublin, Belfield, Dublin 4 (Ireland)
Fax: (+353)1-7162501
E-mail: patrick.guiry@ucd.ie
Scheme 1. Retrosynthetic analysis of pyridine LXA4 (1S)-4.
[b] Dr. P. Maderna, Prof. C. Godson
UCD Diabetes Research Centre
Conway Institute of Biomolecular and Biomedical Research
UCD School of Medicine and Medical Science
University College Dublin, Belfield, Dublin 4 (Ireland)
metric epoxidation, and a regiospecific pyridine lithiation. The
procedure reported by Gribble and Saulnier[13] allows the for-
mation of 3,4-disubstituted pyridines in excellent yields and
without the formation of unwanted side products. The key to
the success of this reaction is the stability of the lithiated inter-
mediate, which is only stable for 10 min at À788C. The internal
[c] C. McCarthy, Dr. C. E. Loscher
School of Biotechnology
Dublin City University, Dublin 9 (Ireland)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.200900533.
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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reaction vessel temperature must be monitored continuously,
as an increase in temperature above À758C gives rise to lithi-
um/halogen exchange and the formation of 3,4-pyridyne. De-
creasing the temperature to À1008C prevented the formation
of these unwanted by-products and gave the required inter-
mediate 9 in 75% yield (Scheme 2). We had also attempted a
regioselective ortho-lithiation of 3-bromopyridine in order to
Scheme 2. Synthesis of 1-(3-bromopyridin-4-yl)hexan-1-one (5). Reagents
and conditions: a) 1. LDA, À788C, 10 min, 2. 8, À1008C, 2 h, 75%; b) PCC,
AcOH, CH2Cl2, room temperature, 5 h, 70%.
prepare the 2,3-disubstituted pyridine LXA4 analogue. Howev-
er, all reactions carried out at À788C failed to produce the de-
sired product, and instead these conditions led to the forma-
tion of the 3-substituted product obtained from the quenching
of 3-lithiopyridine with hexanal, due to the extremely fast lithi-
um/halogen exchange[14] in this system. The oxidation method
of choice for the preparation of 1-(3-bromopyridin-4-yl)hexan-
1-one (5) was the use of pyridinium chlorochromate (PCC) in
the presence of glacial acetic acid (Scheme 2)[15,16] which, after
5 h at room temperature, gave 5 in 70% yield. Direct prepara-
tion of 1-(3-bromopyridin-4-yl)hexan-1-one (5) was attempted
by quenching the lithiated intermediate of 3-bromopyridine
with the corresponding ester and acid chloride as reported for
related compounds,[17] but the yields obtained for this reaction
were between 5 and 10%.
Scheme 3. Synthesis of pyridine LXA4 (1R)-4 and (1S)-4. Reagents and condi-
tions: a) [h3-(C3H4)Pd(m-Cl)2]2, P(o-tolyl)3, 6, NaOAc, toluene/DMA (3:1), 1158C,
12 h, 82%; b) (À)-DIPCl, Et2O, À258C, 48 h, 69%; c) p-TSA (1.5 equiv), MeOH,
308C, 48 h, 52%; d) (+)-DIPCl, Et2O, À258C, 48 h, 65%; e) p-TSA (1.5 equiv),
MeOH, 308C, 48 h, 62%.
spectively. Removal of the silyl ether protecting groups under
the mild conditions of para-toluenesulfonic acid in methanol
provided pyridine LXA4 (1R)-4 and (1S)-4 in 62 and 52% yields,
respectively (Scheme 3). With these epimeric molecules in
hand, we evaluated their biological activity and compared the
results obtained with the parent aromatic analogues 3.
The palladium-catalyzed Heck reaction is an excellent
method for the formation of trans-alkenes[18] and has been ex-
ploited in many total syntheses because of its high yield and
excellent stereochemical control.[19,20] This reaction has also
been applied to many substrates on an industrial scale in the
synthesis of important pharmaceutical agents.[21] Olefin 6
(Scheme 1) was a substrate for the development of a zirconi-
um-catalyzed one-pot protection/deprotection synthetic meth-
odology[22] and is a key intermediate in the synthesis of aro-
matic lipoxins.[11] However, attempts to apply the conditions
used in the latter synthesis, using palladium acetate and tri-o-
tolylphosphine with tributylamine as the solvent and the base,
only resulted in the isolation of trace amounts of product 10.
Reaction conditions employed by Zhang et al.[23] in the synthe-
sis of nicotinic acetylcholine receptors gave 40% yield, but re-
quired extremely long reaction times (7 days). Robert and co-
workers also reported alternative conditions for the Heck reac-
tion of bromopyridines,[24] and employing these reaction condi-
tions gave product 10 in 82% yield after a relatively short reac-
tion time (Scheme 3). Sodium borohydride was used to reduce
ketone 10 to give a mixture of epimeric alcohols, but unfortu-
nately these compounds were inseparable by column chroma-
tography. Hence, reduction with Brown’s (À)- and (+)-chloro-
diisopinocampheylborane[25] was carried out, and afforded the
desired alcohols (1S)-11 and (1R)-11 in 69 and 65% yields, re-
Differentiated THP-1 cells were exposed to the pyridine LXA4
analogues (1R)-4 and (1S)-4 at concentrations ranging from 0.1
to 10 nm for 15 min at 378C before the addition of apoptotic
human PMNs. The extent of phagocytosis was compared with
that obtained with native LXA4 (1 nm; 15 min at 378C), previ-
ously shown to significantly enhance phagocytosis. Pretreat-
ment of differentiated THP-1 cells with compound (1S)-4 at 1
and 10 nm resulted in a significant increase in phagocytosis of
apoptotic PMNs, similar to the effect of native LXA4 (Figure 1).
No effect was observed at a concentration of 0.1 nm (Fig-
ure 1A). Compound (1R)-4 significantly stimulated phagocyto-
sis only at 1 nm, although there is no statistical difference be-
tween the results determined for (1R)-4 and (1S)-4 compared
with those obtained with (1R)-3 and (1S)-3 (Figure 1B).
Given that lipoxins have been reported to affect the produc-
tion of inflammatory cytokines,[26] we assessed the ability of
our pyridine-containing LXA4 analogues (1R)-4 and (1S)-4 to
modulate the production of interleukin-12p40 (IL-12p40), IL-1b,
and monocyte chemoattractant protein-1 (MCP-1) using a J774
murine macrophage cell line. Lipopolysaccharide (LPS;
100 ngmLÀ1) was used to induce cytokine production in the
cells over 24 h. Addition of (1R)-4 at a concentration of 10 mm
1 h before LPS stimulation resulted in the suppression of IL-
12p40 (Figure 2). Exposure of cells to (1S)-4 had a more potent
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ChemMedChem 2010, 5, 517 – 522

Figure 1. LXA4 analogues promote phagocytosis of apoptotic PMNs by differentiated THP-1 cells: A) Differentiated THP-1 cells (5ꢁ105) were treated with vehi-
cle (control), LXA4 (1 nm), or LXA4 analogues at the concentrations indicated for 15 min at 378C prior to co-incubation with apoptotic PMNs (1ꢁ106) for 2 h at
378C. Phagocytosis was detected by staining of PMNs and quantified by light microscopy. Data are expressed as percent phagocytosis and represent mean
values ÆSEM (n=3): *p<0.05 vs. vehicle (control). B) THP-1 cells (5ꢁ105) were treated with vehicle (control), LXA4, benzo analogues or pyridine analogues at
1 nm for 15 min at 378C prior to co-incubation with apoptotic PMNs (1ꢁ106) for 2 h at 378C. Data are expressed as fold induction over basal and represent
mean values ÆSEM (n=3).
effect on IL-12p40 production, with significant suppression of
this cytokine at 10 mm, 1 mm, and 1 nm. Both (1R)-4 and (1S)-4
suppressed LPS-induced production of IL-1b at both 1 mm and
1 nm (Figure 2). There was no effect on MCP-1 production,
demonstrating that the effects of the analogues are specific.
In summary, we report herein a short and efficient synthesis
of two diastereomers of a novel pyridine-containing LXA4 ana-
logue by means of an asymmetric reduction of a ketone, a pal-
ladium-catalyzed Heck reaction, a Sharpless asymmetric epoxi-
dation, and a regiospecific pyridine lithiation. Both compounds
(1R)-4 and (1S)-4 significantly enhanced phagocytosis of PMNs
which is a key requirement for the resolution of inflammation.
Furthermore, they suppressed the production of the pro-in-
flammatory cytokines IL-12p40 and IL-1b, demonstrating their
anti-inflammatory potential. With these preliminary results in
hand for epimeric 3,4-pyridine-containing analogues, future ef-
forts will focus on tuning the pharmacological profile of these
compounds through structural modification, and the results of
these investigations will be reported in due course from these
laboratories.
Experimental Section
Chemistry
General experimental conditions: All reactions were carried out
in an inert atmosphere of N2 using oven-dried glassware, and re-
agents were purchased from Sigma–Aldrich. Et2O, THF, and CH2Cl2
were obtained from a PureSolv-300-3-MD dry solvent dispenser
and were used without further purification. N,N-Dimethylacetamide
was purchased from Sigma–Aldrich and was used without further
1
purification; toluene was dried over sodium. H and 13C NMR spec-
tra were recorded on Varian Oxford 500 spectrometer at room tem-
perature using (CH3)4Si as an internal standard. The reference
Figure 2. LXA4 analogues suppress pro-inflammatory cytokine production by
1
values used for CDCl3 were 7.26 and 77.02 ppm for H and 13C NMR
J774 macrophages: J774 macrophages (1ꢁ106) were treated with vehicle
spectra, respectively. Chemical shift (d) values are given in ppm,
(control), (1R)-4, or (1S)-4 at the concentrations indicated for 1 h prior to
stimulation with LPS (100 ngmLÀ1). Cytokine concentrations were deter-
mined by ELISA; data represent mean values ÆSEM (n=4): **p<0.01,
***p<0.001, determined by one-way ANOVA comparing all groups.
and coupling constants are given as absolute values expressed in
Hz. HRMS data were obtained using a Micromass/Waters LCT in-
strument. IR spectra were recorded on a Varian 3100 FTIR Excalibur
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Series spectrometer. Optical rotation values were measured on a
PerkinElmer 241 polarimeter; [a]D values are given in
10À1 degcm2 gÀ1. HPLC analysis was carried out using a Supelco 2-
4304 beta-Dexꢂ 120 (30 mꢁ0.25 mm, 0.25 mm film) and a Chiralcel
OD column (0.46 cm i.d.ꢁ25 cm). Flash chromatography was car-
ried out using Merck Kiesegel 60 F254 (230–400 mesh) silica gel.
Evaporation in vacuo refers to the removal of volatiles on a Bꢃchi
rotary evaporator with an integrated vacuum pump. Thin-layer
chromatography (TLC) was performed on Merck DC-Alufolien
plates precoated with silica gel 60 F254. They were visualized either
by quenching of UV fluorescence or by charring with an acidic va-
nillin solution (vanillin, H2SO4, and AcOH in MeOH).
(neat pentane, then pentane/Et2O 9:1, then 4:1, then 3:2) to afford
10 (486 mg, 82% yield) as a viscous yellow oil. TLC: Rf =0.23 (pen-
tane/Et2O 3:2); [a]D20 =À12.9 10À1 degcm2 gÀ1 (c=0.84, CH2Cl2);
1H NMR (500 MHz, CDCl3): d=8.79 (s, 1H), 8.56 (d, J=5.0 Hz, 1H),
7.27 (d, J=5.0 Hz, 1H), 6.75 (d, J=16.0 Hz, 1H), 6.22 (dd, J=16.0,
6.5 Hz, 1H), 4.16 (dd, J=6.5, 4.8 Hz, 1H), 3.70 (m, 1H), 3.66 (s, 3H),
2.81 (dt, J=7.3, 9.5 Hz, 2H), 2.31 (t, J=7.4, 2H), 1.31–1.79 (m, 10H),
0.91 (s, 9H), 0.87 (s, 12H), 0.05–0.10 ppm (3ꢁs, 12H); 13C NMR
(125 MHz, CDCl3): d=204.1, 173.9, 149.0, 148.4, 144.2, 135.7, 129.9,
125.4, 120.4, 77.0, 76.2, 51.5, 42.4, 34.2, 33.0, 31.3, 26.0, 23.6, 22.5,
20.7, 18.3, 18.2, 13.9, À4.0, À4.2, À4.6, À4.7 ppm; IR (neat): n˜max
=
2995, 2929, 2857, 1740, 1701 cmÀ1; HRMS (EIMS) m/z: found:
592.3870 [M+H]+, C32H57NO5Si2 requires 592.3881.
1-(3-Bromopyridin-4-yl)hexan-1-ol (9). nBuLi (c=2.5m, 2.8 mL,
6.9 mmol) was added to a solution of diisopropylamine (0.88 mL,
6.3 mmol) in THF (20 mL) at À788C under an atmosphere of N2,
and stirring was continued for 15 min. 3-Bromopyridine 7 (0.62 mL,
6.3 mmol) in THF (1 mL) was added over 10 min (maintaining the
internal temperature <À758C). The reaction was brought to
À1008C for 10 min, and hexanal 8 (1.26 g, 12.6 mmol) in THF
(3 mL) was added over 10 min (again maintaining the internal tem-
perature <À758C). The reaction mixture was stirred at À1008C for
1 h and then warmed to À208C over 20 min. The mixture was
quenched with a saturated solution of NH4Cl (3 mL), extracted with
Et2O (3ꢁ25 mL), washed with water (25 mL), brine (25 mL), and
dried over Na2SO4. The solvent was removed in vacuo, and the resi-
due was purified using silica gel chromatography (pentane/EtOAc
9:1, then 4:1) to afford 9 (737 mg, 74% yield) as a viscous yellow
oil. TLC: Rf =0.21 (pentane/EtOAc 4:1); 1H NMR (500 MHz, CDCl3):
d=8.57 (s, 1H), 8.43 (d, J=4.9 Hz, 1H), 7.51 (d, J=4.9 Hz, 1H), 4.98
(m, 1H), 3.22 (brs, 1H, exchanges with D2O), 1.73–1.79 (m, 1H),
1.49–1.65 (m, 1H), 1.40–1.49 (m, 2H), 1.26–1.39 (m, 4H), 0.9 ppm (t,
J=7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3): d=153.3, 151.5, 148.3,
(5S,6R,E)-Methyl-5,6-bis(tert-butyldimethylsilyloxy)-8-{4-[(R)-1-hy-
droxyhexyl]pyridin-3-yl}oct-7-enoate
((1R)-11). Ketone
10
(190 mg, 0.32 mmol) in Et2O (2 mL) was added to a solution of
(+)-chlorodiisopinocampheylborane [(+-DIPCl; 0.41 g, 1.28 mmol)]
in Et2O (2 mL) at À258C, and stirring was continued for 48 h. The
reaction mixture was diluted with pentane and Et2O (2 mL each),
and diethanolamine (64.3 mg, 0.64 mmol) was added. Stirring was
continued for 4 h at room temperature, followed by filtration and
removal of the solvent in vacuo. The residue was purified by silica
gel chromatography (neat pentane, then pentane/Et2O 1:1, then
1:2) to afford (1R)-11 (125 mg, 53% yield) as a viscous yellow oil at
92.3% de as determined by chiral HPLC using an OD column (hex-
anes/iPrOH 99:1) at a flow rate of 1 mLminÀ1: tR =24.0 min for the
R isomer, tR =30.3 min for S. TLC: Rf =0.15 (Et2O/pentane 2:1);
[a]D20 =+29.4 10À1 degcm2 gÀ1 (c=0.36, CHCl3); 1H NMR (500 MHz,
CDCl3): d=8.57 (s, 1H), 8.45 (d, J=5.0 Hz, 1H), 7.40 (d, J=5.0 Hz,
1H), 6.72 (d, J=15.8 Hz, 1H), 6.15 (dd, J=15.8, 6.0 Hz, 1H), 4.94 (t,
J=6.7 Hz, 1H) 4.22 (m, 1H), 3.71 (m, 1H), 3.64 (s, 3H), 2.30 (t, J=
7.5 Hz, 2H), 1.27–1.79 (m, 10H), 0.93 (s, 9H), 0.88 (s, 12H), 0.06–
0.11 ppm (3ꢁs, 12H); 13C NMR (125 MHz, CDCl3): d=174.0, 151.0,
147.9, 147.0, 135.2, 130.7, 124.3, 120.2, 77.0, 76.2, 70.2, 51.5, 37.9,
34.2, 32.8, 31.7, 25.9, 25.4 22.6, 20.9, 18.3, 18.2, 13.9, À4.0, À4.3,
À4.6, À4.7 ppm; IR (neat): n˜max =3365, 2954, 2930, 2857, 1741,
122.2, 120.0, 71.9, 37.1, 31.5, 25.3, 22.5, 14.0 ppm; IR (neat): n˜max
3583, 3296, 2929, 2361, 1588, 1466, 1401, 1343, 1217, 1162, 1084,
756 cmÀ1 cmÀ1 HRMS (EIMS) m/z: found: 258.0486 [M+H]+,
=
;
C11H16BrNO requires 258.0494.
1252 cmÀ1
;
HRMS (EIMS) m/z: found: 594.4033 [M+H]+,
1-(3-Bromopyridin-4-yl)hexan-1-one (5). Glacial acetic acid
(0.21 mL) was added to a well-stirred solution of PCC (821 mg,
3.81 mmol) in dry CH2Cl2 (20 mL). After 5 min at room temperature,
alcohol 9 (655 mg, 2.54 mmol) in CH2Cl2 (5 mL) was added, and the
mixture was stirred at room temperature for 5 h. Et2O (40 mL) was
added, and the mixture was filtered twice. The solvent was re-
moved in vacuo, and the residue was purified by silica gel chroma-
tography (pentane/EtOAc 4:1) to afford ketone 5 (458 mg, 70%
C32H59NO5Si2 requires 594.4010.
(5S,6R,E)-Methyl-5,6-bis(tert-butyldimethylsilyloxy)-8-{4-[(S)-1-hy-
droxyhexyl]pyridin-3-yl}oct-7-enoate ((1S)-11). Ketone 10 (97 mg,
0.163 mmol) in Et2O (1 mL) was added to a solution of (À)-DIPCl
(210 mg, 0.64 mmol) in Et2O (1 mL) at À258C, and stirring was con-
tinued for 48 h. The reaction mixture was diluted with pentane
and Et2O (1 mL each), and diethanolamine (34 mg, 0.326 mmol)
was added. Stirring was continued for 4 h at room temperature,
followed by filtration and removal of the solvent in vacuo. The resi-
due was purified by silica gel chromatography (neat pentane, then
pentane/Et2O 1:1, then 1:2) to afford (1S)-11 (66 mg, 67% yield) as
1
yield) as an orange oil. TLC: Rf =0.67 (pentane/EtOAc 4:1); H NMR
(500 MHz, CDCl3): d=8.79 (s, 1H), 8.60 (d, J=4.9 Hz, 1H), 7.22 (d,
J=4.9 Hz, 1H), 2.88 (t, J=7.4 Hz, 2H), 1.74–1.69 (m, 2H), 1.37–1.33
(m, 4H), 0.91 ppm (t, J=7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3): d=
202.6, 152.8, 148.5, 148.5, 121.6, 116.0, 42.5, 31.2, 23.2, 22.3,
13.8 ppm; IR (neat):: n˜max =2958, 2931, 1710, 1578, 1466, 1396,
1378, 1276, 1250, 1089, 1022 cmÀ1; HRMS (EIMS) m/z: found:
256.0336 [M+H]+, C11H14BrNO requires 256.0337.
a clear oil at 94.9% de. TLC: Rf =0.15 (Et2O/pentane 2:1); [a]D20
=
À31.9 10À1 degcm2 gÀ1 (c=0.56, CHCl3); 1H NMR (500 MHz, CDCl3):
d=8.60 (s, 1H), 8.45 (d, J=5.0 Hz, 1H), 7.41 (d, J=5.0 Hz, 1H), 6.65
(d, J=15.8 Hz, 1H), 6.18 (dd, J=15.8, 6.5 Hz, 1H), 4.98 (t, J=6.2 Hz,
1H) 4.18 (dd, J=6.5, 4.9 Hz, 1H), 3.70 (m, 1H), 3.65 (s, 3H), 2.31 (t,
J=7.3, 2H), 1.27–1.80 (m, 10H), 0.92 (s, 9H), 0.87 (s, 12H), 0.05–
0.10 ppm (3ꢁs, 12H); 13C NMR (125 MHz, CDCl3): d=174.0, 150.3,
148.7, 147.6, 134.8, 130.5, 124.5, 119.8, 77.2, 76.2, 69.8, 51.5, 38.1,
34.3, 33.0, 31.7, 26.0, 26.0, 25.4, 22.6, 20.7, 18.3, 18.2, 14.0, À4.0,
À4.1, À4.6, À4.6 ppm; IR (neat): n˜max =3350, 2954, 2930, 2857,
1742, 1252 cmÀ1; HRMS (EIMS) m/z: found: 594.3992 [M+H]+,
C32H57NO5Si2 requires 594.4010.
(5S,6R,E)-Methyl-5,6-bis(tert-butyldimethylsilyloxy)-8-(4-hexa-
noylpyridin-3-yl)oct-7-enoate (10). [h3-(C3H4)Pd(m-Cl)2]2 (17 mg,
0.048 mmol), P(o-tolyl)3 (34 mg, 0.096 mmol), and NaOAc (234 mg,
2.88 mmol) were dissolved in dry freshly distilled toluene (2 mL) to
which ketone 5 (250 mg, 0.96 mmol) in toluene (1 mL) and olefin 6
(406 mg, 0.96 mmol) in toluene (1 mL) were added. N,N-Dimethyl-
acetamide (DMA; 1.3 mL) was added, and the reaction mixture was
sealed under N2; stirring was continued for 12 h at 1158C followed
by filtration through a pad of Celiteꢂ. The solvent was removed in
vacuo, and the residue was purified by silica gel chromatography
(5S,6R,E)-Methyl-5,6-dihydroxy-8-{4-[(R)-1-hydroxyhexyl]pyridin-
3-yl}oct-7-enoate ((1R)-4). Alcohol (1R)-11 (160 mg, 0.266 mmol)
520
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ChemMedChem 2010, 5, 517 – 522

was dissolved in dry MeOH (1 mL) to which p-toluenesulfonic acid
(p-TSA; 77 mg, 0.4 mmol) was added, and the resultant mixture
was stirred at 308C for 48 h. The solvent was removed in vacuo at
308C to prevent formation of a lactone by-product, and the resi-
due was purified by silica gel chromatography (pentane/EtOAc 1:1,
then neat EtOAc, then EtOAc/MeOH (1%)) to afford (1R)-4
(60.8 mg, 62% yield) as a clear viscous oil. TLC: Rf =0.26 (CH2Cl2/
MeOH 9.5:0.5); [a]D20 =+12.2 10À1 degcm2 gÀ1 (c=2.3, CHCl3);
1H NMR (500 MHz, CDCl3): d=8.39 (s, 1H), 8.35 (appd, J=4.8 Hz
1H) 7.38 (d, J=4.8 Hz, 1H), 6.76 (d, J=15.8 Hz, 1H), 6.15 (dd, J=
15.8, 6.1 Hz, 1H), 4.88 (brs, 1H) 4.22 (brs, 1H), 3.76 (brs, 1H), 3.65
(s, 3H), 3.01 (brs, 3H), 2.35 (t, J=7.27, 2H), 1.26–1.89 (m, 13H),
0.86 ppm (t, J=6.58 Hz, 3H); 13C NMR (125 MHz, CDCl3): d=174.3,
152.2, 147.4, 146.4, 132.9, 130.8, 125.9, 120.7, 75.6, 74.0, 69.3, 51.6,
hyde. For each experiment, the number of THP-1 cells containing
one or more PMN in at least five fields (minimum of 400 cells) was
expressed as a percentage of the total number of THP-1 cells, and
an average between duplicate wells was calculated.
Cytokine production by J774 macrophages
Murine J774 macrophages (European Collection of Cell Cultures,
UK) were maintained in suspension of RPMI 1640 supplemented
with glutamine (2 mmolLÀ1), penicillin (100 IUmLÀ1), streptomycin
(100 mgmLÀ1), and 10% FCS. Cells were seeded at 1ꢁ106 cellsmLÀ1
for experiments and exposed to LPS at a concentration of
100 ngmLÀ1 for 24 h at 378C under 5% CO2. (1R)-4 and (1S)-4
were added to the cells 1 h before addition of LPS, at a concentra-
tions of 1 nm, 1 mm, and 10 mm. After 24 h the supernatants were
collected for cytokine analysis. IL-1b, MCP-1, and IL-12p40 concen-
trations in cell culture supernatants were quantified by commercial
DuoSet ELISA kits (R&D Systems), according to the manufacturer’s
instructions.
37.5, 33.7, 31.9, 31.6, 25.3, 22.5, 21.2, 14.0 ppm; IR (neat): n˜max
=
3389, 2953, 2928, 2856, 1737, 1457, 1235 cmÀ1; HRMS (EIMS) m/z:
found: 366.2278 [M+H]+, C20H31NO5 requires 366.2280.
(5S,6R,E)-Methyl-5,6-dihydroxy-8-{4-[(S)-1-hydroxyhexyl]pyridin-
3-yl}oct-7-enoate ((1S)-4). Alcohol (1S)-11 (50 mg, 0.084 mmol)
was dissolved in dry MeOH (1 mL) to which p-TSA (24.1 mg,
0.126 mmol) and the resultant mixture was stirred at 308C for 48 h.
The solvent was removed in vacuo at 308C to prevent formation of
a lactone by-product, and the residue was purified by silica gel
chromatography (pentane/EtOAc 1:1, then neat EtOAc, then
EtOAc/MeOH (1%)) to afford (1S)-4 (16.4 mg, 52% yield) as a clear
Acknowledgements
C.D.D. thanks University College Dublin for the award of an
Ad Astra Scholarship. We thank University College Dublin for pro-
viding financial support for this project. We also acknowledge
the facilities provided by the Centre for Synthesis and Chemical
Biology (CSCB), funded by the Higher Education Authority’s Pro-
gramme for Research in Third-Level Institutions (PRTLI). We are
grateful to Dr. Jimmy Muldoon and Dr. Dilip Rai of the CSCB for
NMR and mass spectra, respectively. C.McC. is funded by an
IRCSET PhD scholarship. C.G. and P.M. are funded by Science
Foundation Ireland and by the EU FP6 EICOSANOX programme
LSHM-CT-2004-00503.
viscous oil. TLC: Rf =0.26 (CH2Cl2/MeOH 9.5:0.5); [a]D20
=
À8.8 10À1 degcm2 gÀ1 (c=0.34, CHCl3); 1H NMR (500 MHz, CDCl3):
d=8.36 (appbrs, 2H), 7.36 (d, J=5.0 Hz, 1H), 6.77 (d, J=15.9 Hz,
1H), 6.07 (dd, J=15.9, 6.4 Hz, 1H), 4.85 (dd, J=7.3, 5.2 Hz, 1H) 4.21
(m, 1H), 3.72 (m, 1H), 3.65 (s, 3H), 2.34 (t, J=7.3, 2H), 1.26–1.89
(m, 13H), 0.86 ppm (t, J=6.7 Hz, 3H); 13C NMR (125 MHz, CDCl3):
d=174.3, 151.3, 148.2, 147.2, 132.8, 130.7, 126.1, 120.6, 75.4, 73.9,
70.1, 51.6, 37.6, 33.7, 31.7, 31.6, 25.4, 22.5, 21.1, 14.0 ppm; IR (neat):
n˜max =3383, 2954, 2857, 1733, 1460, 1259 cmÀ1; HRMS (EIMS) m/z:
found: 366.2291 [M+H]+, C20H31NO5 requires 366.2280.
Keywords: cytokines · eicosanoids · inflammation · lipoxins ·
Phagocytosis of apoptotic PMNs by THP-1 cells
synthesis design
The human myelomonocytic cell line THP-1 (European Collection
of Cell Cultures, Salisbury, UK) was maintained as a suspension in
RPMI 1640 supplemented with glutamine (2 mmolLÀ1), penicillin
(100 IUmLÀ1), streptomycin (100 mgmLÀ1), and 10% fetal calf
serum (FCS; Life Technologies Inc., Grand Island, NY, USA). THP-1
cells at 5ꢁ105 mLÀ1 were differentiated to a macrophage-like pheno-
type by treatment with 100 nm phorbol-12-myristate-13-acetate
(PMA) for 48 h at 378C. Human polymorphonuclear neutrophils
(PMNs) were isolated from peripheral venous blood drawn from
healthy volunteers after informed written consent. Briefly, PMNs
were separated by centrifugation on Ficoll-Paque (Pharmacia, Up-
psala, Sweden) followed by dextran sedimentation (Dextran T500,
Pharmacia) and hypotonic lysis of red cells. PMNs were suspended
at 4ꢁ106 cellsmLÀ1, and spontaneous apoptosis was achieved by
culturing PMNs in RPMI 1640 supplemented with 10% autologous
serum, glutamine (2 mmolLÀ1), penicillin (100 IUmLÀ1), and strepto-
mycin (100 mgmLÀ1) for 20 h at 378C under a 5% CO2 atmosphere.
Cells were 25–50% apoptotic on average, with ~3% necrosis as as-
sessed by light microscopy on stained cytocentrifuged prepara-
tions. Differentiated THP-1 cells (5ꢁ105 cells per well) were ex-
posed to the appropriate stimuli as indicated for 15 min at 378C,
before co-incubation with apoptotic PMNs (1ꢁ106 PMNs per well)
at 378C for 2 h. Non-ingested cells were removed by three washes
with cold phosphate-buffered saline. Phagocytosis was assayed by
myeloperoxidase staining of co-cultures fixed with 2.5% glutaralde-
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