Myxochelin-Inspired 5-Lipoxygenase Inhibitors: Synthesis and Biological Evaluation

Article abstract of DOI:10.1002/cmdc.201600536

A total of 48 analogues of the natural product myxochelin A were prepared and evaluated for their inhibitory effects on human 5-lipoxygenase in both cell-free and cell-based assays. Structure–activity relationship analysis revealed that the secondary alcohol function and only chiral center of myxochelin A is not required for biological activity. By expanding the diaminoalkane linker of the two aromatic residues it was possible to generate a myxochelin derivative with superior activity against 5-lipoxygenase in intact cells.

Full text of DOI:10.1002/cmdc.201600536

Accepted Article
Title: Myxochelin-Inspired 5-Lipoxygenase Inhibitors - Synthesis and
Biological Evaluation
Authors: Sebastian Schieferdecker, Stefanie König, Simona Pace,
Oliver Werz, and Markus Nett
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10.1002/cmdc.201600536
ChemMedChem
COMMUNICATION
Myxochelin-Inspired 5-Lipoxygenase Inhibitors – Synthesis and
Biological Evaluation
Sebastian Schieferdecker,[a] Stefanie König,[b] Simona Pace,[b] Oliver Werz,*[b] and Markus Nett*[c]
[a]
Dr. S. Schieferdecker
Department of Biomolecular Chemistry
Leibniz Institute for Natural Product Research and Infection Biology / Hans Knöll Institute
Adolf-Reichwein-Strasse 23, 07745 Jena (Germany)
S. König, Dr. S. Pace, Prof. Dr. O. Werz
[b]
Chair of Pharmaceutical and Medicinal Chemistry
Institute of Pharmacy, Friedrich-Schiller-Universität Jena
Philosophenweg 14, 07743 Jena (Germany)
E-mail: oliver.werz@uni-jena.de
[c]
Prof. Dr. M. Nett
Department of Biochemical and Chemical Engineering
Technische Universität Dortmund
Emil-Figge-Strasse 66, 44227 Dortmund (Germany)
E-mail: markus.nett@bci.tu-dortmund.de
Supporting information for this article is given via a link at the end of the document.
Abstract: A total of 48 analogues of the natural product myxochelin
A were prepared and evaluated for their inhibitory effects on human
5-lipoxygenase in both cell-free and cell-based assays. Structure-
activity relationship analysis revealed that the secondary alcohol
function and only chiral center of myxochelin A is not required for the
biological activity. By expanding the diaminoalkane linker of the two
biosynthesis.[11] In this study, two engineered analogues showed
much less affinity for the coordination of ferric iron than
myxochelin A, but still maintained significant activity against
human 5-LO.[11] To further explore the SAR, we now tested 48
additional derivatives, which were prepared using a recently
developed synthetic strategy.[12] In particular, we set out to
evaluate the effects of different aromatic substitution patterns on
5-LO inhibition. Furthermore, the importance of the lysinol
moiety in myxochelin A was investigated by replacing it with
lysine esters or diaminoalkanes, respectively.
aromatic residues it was possible to generate
a myxochelin
derivative with superior activity against 5-lipoxygenase in intact cells.
The enzyme human 5-lipoxygenase (5-LO) is responsible for the
catalysis of the two initial steps in the biosynthesis of
leukotrienes starting from arachidonic acid. Leukotrienes are
well-known mediators of a variety of inflammatory and allergic
reactions, which include bronchial asthma, rheumatic arthritis,
allergic rhinitis, and cardiovascular diseases.[1,2] The 5-LO
pathway was also associated with cancer including brain, breast,
colon, esophageal mucosa, lung, kidney, mesothelium,
pancreas, prostate and leukemia, and in most of these studies
an increased formation of 5-LO products was observed.[2] This
was confirmed for tissue isolated from patients suffering from
breast and pancreatic cancer.[3,4] The therapeutic value of
inhibitors of the 5-LO pathway is supported by various animal
studies and clinical trials of inflammation and cancer.[4-6] Thus,
human 5-LO is a promising pharmacological target, because its
inhibition blocks the biosynthesis of all leukotrienes and the
bioactive 5-hydroxyeicosatetraenoic acid.
The first set of analogues consisted of 17 compounds
which all contained the lysinol partial structure (Table 1).
Myxochelin S1, in which the two 2,3-dihydroxybenzamide
residues of myxochelin
A
had been replaced by 3,4-
dihydroxybenzamides inhibited 5-LO activity in cell-free assays
at 10 μM by 41.6%, which indicates a significant loss of
bioactivity in comparison to the natural product. While a 2,4-
dihydroxy substitution pattern at the aromatic residues almost
abolished 5-LO inhibition, 2,5-dihydroxy substitution was
surprisingly well tolerated. The corresponding compound,
myxochelin S3, inhibited 5-LO in a concentration-dependent
manner with an IC50 of 2.3 µM. Derivatives featuring two
dimethoxybenzamides were fully inactive (see data for
myxochelins S4-S8 in Table 1), and even single methoxy
substituents at the aromatic rings negatively affected 5-LO
inhibition. Only few compounds of this series, namely
myxochelins S9 and S10, retained some modest 5-LO inhibitory
We recently described the isolation of the catechol
activity. We also tested
a synthetic analogue with 4-
siderophore myxochelin
A from the predatory bacterium
hydroxybenzamide rings. In contrast to related myxochelins with
2- or 3-hydroxybenzamide moieties, which had previously been
shown to sustain 5-LO inhibitory properties,[11] the exclusive
para-hydroxylation pattern in myxochelin S15 turned out to be
disadvantageous for the biological activity.
Since modification of the ring substitution pattern
apparently was not very permissive in terms of 5-LO inhibition,
we then turned our attention to the lysinol motive, which linked
the two aromatic moieties. Here, we explored the importance of
the secondary alcohol by replacing it by a carboxylic acid group
and by ester functions, respectively (Table 2). The most active
derivative hitherto (i.e., myxochelin S3) served as a lead
Pyxidicoccus fallax HKI727.[7] The natural product strongly
suppressed the growth of K-562 leukemia cells and
pharmacological characterization revealed human 5-LO as the
molecular target, which was inhibited with an IC50 of 1.9 ± 0.2
µM in a cell-free assay.[7] The active site of human 5-LO contains
a non-heme iron, which serves as an electron donor (Fe2+) or
acceptor (Fe3+) during catalysis.[8-10] Our original assumption that
the inhibition of human 5-LO was solely due to the complexation
of this active site iron by myxochelin A could be disproven by
structure activity relationship (SAR) studies of myxochelin
derivatives which were generated by precursor-directed
1
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Table 1. Chemical structures of lysinol-derived myxochelin analogues and their 5-LO inhibitory effects in a cell-free assay. The known 5-LO inhibitor zileuton[1]
was used as a control.
R1
R1’
R2
R2’
R3
R3’
R4
R4’
R5
R5’
5-LO activity[a]
[%]
IC50[a]
[µM]
A
OH
H
OH
H
OH
OH
H
OH
OH
H
H
OH
OH
H
H
OH
OH
H
H
H
H
H
H
H
H
H
9.5 ± 3.1
58.4 ± 5.9
87.8 ± 7.0
30.8 ± 6.5
89.5 ± 1.9
85.0 ± 3.0
81.1 ± 7.8
91.5 ± 11.0
91.3 ± 11.1
61.7 ± 6.6
44.7 ± 2.0
84.5 ± 8.1
82.5 ± 3.0
94.9 ± 0.9
106.6 ± 12.6
98.4 ± 9.8
96.4 ± 6.4
78.4 ± 10.0
7.2 ± 2.4
1.9 ± 0.2
n.d.
S1
S2
OH
OH
H
OH
OH
H
H
H
H
H
n.d.
S3
H
H
OH
H
OH
H
H
H
2.3 ± 0.3
n.d.
S4
OMe
H
OMe
H
OMe
H
OMe
H
H
H
S5
OMe
OMe
OMe
OMe
OMe
OMe
OH
OH
H
OMe
OMe
OMe
OMe
OMe
OH
OH
OH
H
OMe
H
OMe
H
H
H
n.d.
S6
OMe
H
OMe
H
H
H
H
H
n.d.
S7
OMe
H
OMe
H
H
H
H
H
n.d.
S8
H
H
H
H
OMe
OH
OH
H
OMe
OH
OH
H
n.d.
S9
H
H
H
H
H
H
n.d.
S10
S11
S12
S13
S14
S15
S16
S17
zileuton
H
H
H
H
H
H
n.d.
H
H
OMe
OMe
OMe
OMe
OH
H
OH
OMe
OMe
OH
OH
H
H
H
n.d.
H
H
H
H
H
H
n.d.
H
H
H
H
H
H
n.d.
H
H
H
H
H
H
H
H
n.d.
H
H
H
H
H
H
H
H
n.d.
OMe
OH
-
OMe
OH
-
H
H
F
F
H
H
n.d.
H
H
H
H
F
F
H
H
n.d.
-
-
-
-
-
-
-
-
0.5 ± 0.1
[a] Data are expressed as means ± S.E. of single determinations obtained in three independent experiments; n.d. = not determined
Table 2. Chemical structures of myxochelin analogues with an ester motif and their 5-LO inhibitory effects in a cell-free assay.
R1
R1’
R2
R2’
R3
R3’
R4
R4’
R5
R5’
R
5-LO activity[a]
[%]
IC50[a]
[µM]
S18
S19
S20
S21
S22
S23
S24
S25
S26
S27
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OMe
OH
F
OH
OH
OMe
OH
F
H
H
H
H
CO2Me
CO2Et
CO2Et
CO2H
CO2H
CO2Et
CO2Et
CO2Et
CO2Et
CO2Et
16.4 ± 1.1
12.3 ± 1.0
102.9 ± 5.5
84.4 ± 1.3
77.1 ± 4.6
67.4 ± 3.1
101.2 ± 4.5
62.7 ± 5.5
75.0 ± 8.0
101.8 ± 12.1
0.7 ± 0.1
0.5 ± 0.1
n.d.
OMe
OH
OMe
OH
H
H
H
H
n.d.
OMe
OMe
OMe
OH
OH
H
H
n.d.
OH
F
F
H
H
n.d.
OMe
OH
F
F
H
H
n.d.
H
H
OMe
F
OMe
F
n.d.
OH
OH
H
H
n.d.
OMe
OMe
H
H
F
F
n.d.
[a] Data are expressed as means ± S.E. of single determinations obtained in three independent experiments; n.d. = not determined
compound for this evaluation. The carboxylic acid analogue of
myxochelin S3 was barely active, but the corresponding methyl
and ethyl esters efficiently suppressed 5-LO activity. Myxochelin
S18 and S19 were even more active than their ancestor with
IC50 values of 0.7 µM and 0.5 μM, respectively. Again, this
outcome could be completely reversed through methoxylation of
2
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the aromatic hydroxyl groups (see data for myxochelin S20 in
Table 1). Further attempts to exploit the activity-enhancing effect
of the carboxylic acid ester function for altering the aromatic
substitution pattern were largely unsuccessful, albeit some
derivatives, such as myxochelins S23 and S25, exhibited weak
activities against 5-LO.
To clarify whether the ester function is truly beneficial for 5-LO
inhibition or whether the observed effect could be ascribed to
increasing lipophilicity, a series of myxochelins lacking functional
groups in the linker region was synthesized (Table 3).
the two aromatic residues is not necessary for 5-LO inhibition.
Similar to our previous observations, methoxylation of the
phenolic groups gradually reduced the biological activity. While
the monomethoxylated myxochelin S30 was still capable to
suppress 5-LO-catalyzed product formation (IC50 = 0.8 µM), the
introduction of another methoxy group abolished the activity, as
exemplified by myxochelin S31. Surprisingly, coupling of 2,4-
dihydroxybenzoates with 1,5-diaminopentane led to a derivative
with modest activity, which even tolerated a single methoxylation
(see data for myxochelins S33 and S34 in Table 3). Attempts to
further increase the bioactivity of myxochelin S34 resulted in
myxochelin S41, which contained 2,3,4-trihydroxybenzamide
residues. Structurally, this compound was related to myxochelin
S28 and also showed comparable activity (IC50 = 0.8 µM).
Myxochelin S28, which featured
a cadaverine (i.e., 1,5-
diaminopentane) core, but was otherwise identical to myxochelin
A, turned out as a potent 5-LO inhibitor with an IC50 of 0.6 µM. It
hence became evident that functionalization of the region linking
Table 3. Chemical structures of 1,5-diaminopentane-derived myxochelin analogues and their 5-LO inhibitory effects in a cell-free assay.
R1
R1’
R2
R2’
R3
R3’
R4
R4’
R5
R5’
5-LO activity[a] [%]
IC50[a]
[µM]
S28
S29
S30
S31
S32
S33
S34
S35
S36
S37
S38
S39
S40
S41
S42
OH
OMe
OH
OH
OMe
OH
OH
OH
OMe
H
OH
OMe
OH
OH
OMe
OH
OH
OH
OMe
H
OH
OMe
OMe
OMe
H
OH
OMe
OH
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
F
4.8 ± 0.6
109.4 ± 8.1
11.9 ± 1.4
82.0 ± 13.1
97.7 ± 7.3
66.4 ± 2.0
65.7 ± 3.5
97.3 ± 6.4
102.2 ± 0.4
96.9 ± 7.7
68.8 ± 7.2
90.6 ± 18.7
100.1 ± 6.7
3.8 ± 0.1
0.6 ± 0.1
n.d.
H
H
0.8 ± 0.3
n.d.
H
H
OMe
OMe
OH
H
OMe
OH
OH
H
n.d.
H
H
n.d.
H
H
n.d.
OMe
H
OMe
H
n.d.
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
n.d.
OMe
H
OMe
H
H
H
n.d.
OH
H
OH
H
H
H
n.d.
OH
OMe
OH
OMe
OH
OH
OH
OMe
H
H
n.d.
H
H
H
H
n.d.
OH
OMe
OH
OMe
OH
OMe
OH
OMe
0.8 ± 0.2
n.d.
85.2 ± 22.3
[a] Data are expressed as means ± S.E. of single determinations obtained in three independent experiments; n.d. = not determined
Next, we evaluated the activities of myxochelins
possessing 1,6-diaminohexane and 1,4-diaminobutane moieties
(Table 4). While the former were slightly more active than the
cadaverine-derived analogues, the latter showed somewhat
impaired 5-LO inhibition. Even though myxochelin S43, which
was prepared from 1,6-diaminohexane, had an excellent IC50
value of 0.1 µM, the 1,4-diaminobutane-derived myxochelin S45
was also a highly potent inhibitor (IC50 = 0.5 µM). Derivatization
of the free phenolic groups in these compounds again led to a
significant activity loss.
The most active myxochelin analogues (i.e., S3, S18, S19,
S28, S30, S41, S43, S45, and S46) were eventually tested in a
cell-based assay using human polymorphonuclear leukocytes
(PMNL) stimulated with Ca2+-ionophore A23187. While the
derivatives S3 and S41 completely failed to inhibit 5-LO product
formation in PMNL, myxochelins S18, S19, S28, S30, S45, and
S46 showed partial inhibition (Table 5). The only potent cellular
5-LO inhibitor was myxochelin S43, which reduced 5-LO product
formation by 88.7% at 10 µM, corresponding to an IC50 value of
1.1 ± 0.3 µM. In comparison, myxochelin A was found to be
much less efficient in the PMNL assay (inhibition of 5-LO
product formation at 10 µM by only 26.5%).
From the obtained data, some general conclusions
concerning the SAR can be drawn. Thus, the secondary alcohol
of myxochelin A is not important for the 5-LO inhibitory activity
and can be omitted. To further improve cellular inhibition of 5-LO,
it might be advisable to extend the length of the carbon chain
linking the two aromatic residues. The latter appear not to be
very permissive for variations of their substitution patterns. Free
phenolic groups in ortho-position to the amide substituent turned
out to be crucial for 5-LO inhibition, which is also consistent with
our previous study.[11] Additional hydroxyl residues in meta-
position further improve the bioactivity, whereas para-oriented
hydroxyl groups are in general detrimental. By applying these
3
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Table 4. Chemical structures of diaminoalkyl-derived myxochelin analogues and their 5-LO inhibitory effects in a cell-free assay.
R1
R1’
R2
R2’
R3
R3’
R4
R4’
R5
R5’
n
5-LO activity[a]
[%]
IC50[a]
[µM]
S43
S44
S45
S46
S47
S48
OH
OMe
OH
OH
OMe
OH
OH
OMe
OH
OH
OMe
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4
4
2
2
2
2
2.3 ± 0.4
102.6 ± 7.1
8.0 ± 0.3
0.1 ± 0.04
n.d.
0.5 ± 0.1
1.8 ± 0.5
n.d.
OH
OH
OMe
OMe
OMe
OH
16.7 ± 0.8
59.2 ± 1.8
107.2 ± 12.5
OH
OH
OMe
OMe
OMe
OMe
n.d.
[a] Data are expressed as means ± S.E. of single determinations obtained in three independent experiments; n.d. = not determined
few guidelines, a new potent 5-LO inhibitor, myxochelin S43,
was designed, which is superior to the natural product
myxochelin A, especially concerning 5-LO inhibition in intact
cells.
A stirred solution of 1-fluoro-3-(methoxymethoxy)benzene (5.3
mmol) in 5 mL of dry THF and 2 mL of dry n-hexane under an
argon atmosphere was cooled to -78 °C. Subsequently, 2 mL of
n-butyl lithium (2.5 M) dissolved in THF were added and the
mixture was stirred for two hours. Carbon dioxide was bubbled
through the solution for 15 min, which induced the formation of a
white precipitate. The solution was allowed to warm up to room
temperature slowly, and then 20 mL of water were added. The
aqueous phase was washed three times with ethyl acetate and
the crude product was purified by reverse phase HPLC.
Table 5. 5-LO activity in PMNL cells after treatment with selected myxochelin
analogues and zileuton, respectively (10 µM).
5-LO activity PMNL [%][a]
S3
92.9 ± 4.9
61.4 ± 6.3
50.3 ± 1.6
36.4 ± 1.4
50.5 ± 3.5
90.3 ± 4.5
11.3 ± 2.6
68.7 ± 11.4
59.3 ± 4.1
10.1 ± 4.1
S18
S19
S28
S30
S41
S43
S45
S46
zileuton
Preparation
benzoic acid
of
6-fluoro-3-methoxy-2-(methoxymethoxy)
A solution of 4-fluoro-1-methoxy-2-(methoxymethoxy)benzene (7
mmol) in 10 mL dry THF was prepared under an argon
atmosphere. To the cooled solution (-78 °C), 5 mL of a 2 M
solution of LDA in dry THF was added and the mixture was
stirred for 1 h. Afterwards, carbon dioxide was bubbled through
the cooled solution for 20 min which resulted in the production of
a yellow precipitate. The solution was then allowed to warm up
to 0 °C and 4 mL of water were added slowly. After 10 more
minutes of stirring at 0 °C, the reaction mixture was warmed to
room temperature. Subsequently, the mixture was diluted by the
addition of 40 mL of water and the THF was distilled off. The
aqueous solution was extracted five times with ethyl acetate (50
mL each), the organic phases were combined, residual water
was removed by the addition of Na2SO4 and afterwards
evaporated to dryness. The crude product was purified by
reverse phase HPLC.
[a] Data are expressed as means ± S.E. of single determinations obtained in
three independent experiments
Experimental Section
Preparation of methoxymethyl acetal protective groups
To a solution of 10 mmol of phenolic compound in 10 mL dry
DMF under an argon atmosphere 20 mmol of N,N-
diisopropylethylamine was added and the mixture was cooled in
an ice water bath. Chloro(methoxy)methane (15 mmol) was
slowly added and the solution was stirred for one hour at 0 °C.
After warming up to room temperature, the solution was stirred
for further three hours. Water (10 mL) was added and the
mixture was extracted three times with dichloromethane. The
organic fractions were combined and the solvent was
evaporated under reduced pressure. Purification of the crude
product was achieved by normal phase flash chromatography
using a mixture of dichloromethane and ethyl acetate (9.5 : 0.5)
or a mixture of ethyl acetate and methanol (9 : 1) as eluent.
Peptide synthesis
To a stirred solution of 1 mmol of aryl carboxylic acid in 5 mL dry
DMF under an argon atmosphere, 1.5 mmol benzotriazol-1-yl-
oxytripyrrolidinophosphonium hexafluorophosphate, 1.5 mmol
N,N-diisopropylethylamine and 0.5 mmol of L-lysine ethyl ester
hydrochloride, hexamethylenediamine, cadaverine or putrescine
were added and the solution was stirred overnight. Afterwards
the mixture was poured into 20 mL water which was extracted
five times with ethyl acetate. If a MOM protective group was
present, 0.5 N HCl was used instead of water and the mixture
Preparation of 2-fluoro-6-(methoxymethoxy)benzoic acid
4
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10.1002/cmdc.201600536
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COMMUNICATION
analyzed by RP-HPLC as described.[14]
was stirred for one hour prior to the extraction with ethyl acetate.
The organic layers were combined, residual water removed by
the addition of Na2SO4 and the solvent removed under reduced
pressure. Crude products were purified by reverse phase HPLC.
Acknowledgements
Dealkylation of methoxy groups
Financial support by the Deutsche Forschungsgemeinschaft
(DFG) within SFB 1127 (Chemical Mediators in Complex
Biosystems) and the Jena School of Microbial Communication
(JSMC) is gratefully acknowledged.
For the dealkylation, 0.5 mmol of educt were dissolved in 5 mL
dry dichloromethane under an argon atmosphere in an ice water
bath. To this solution, 3 mmol of boron tribromide was slowly
added and the solution was stirred for 4 hours at 0 °C.
Afterwards, it was allowed to warm up to room temperature and
the stirring was continued for two hours. The solvent was
removed under reduced pressure and crude products were
purified by reverse phase HPLC.
Keywords: myxochelin • 5-lipoxygenase • leukotriene •
inflammation • inhibitor
References:
[1]
[2]
J. Z. Haeggström, C. D. Funk, Chem. Rev. 2011, 111, 5866-5898.
O. Rådmark, O. Werz, D. Steinhilber, B. Samuelsson, Biochim. Biophys.
Acta Mol. Cell Biol. Lipids 2015, 1851, 331-339.
Reduction of ethyl ester
For the reduction of the ethyl ester side chains, 0.7 mmol of
ester and 1.4 mmol of LiBH4 were dissolved in 5 mL THF at
room temperature. To this solution, 5 mL of ethanol was added
slowly and the solution was stirred for 16 h. Afterwards 20 mL of
saturated aqueous NH4Cl solution was added. The organic
solvents were removed under reduced pressure and the
aqueous suspension was exhaustively extracted with 20 mL
ethyl acetate. The organic fractions were combined, dried with
Na2SO4 and purified by reverse phase HPLC.
[3]
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Essent. Fatty Acids 2006, 74, 125-134.
R. Hennig, P. Grippo, X.-Z. Ding, S. M. Rao, M. W. Buchler, H. Friess,
M. S. Talamonti, R. H. Bell, T. E. Adiran, Cancer Res. 2005, 65, 6011-
6016.
[5]
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Martínez, J. L. Mulshine, J. Clin. Invest. 1996, 97, 806-813.
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Purification 5-LO from E. coli and cell-free 5-LO activity
assay
[10] J. C. Boyington, B. J. Gaffney, L. M. Amzel, J. Biol. Chem. 1990, 265,
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Human recombinant 5-LO was expressed in E. coli BL21 (DE3)
harboring plasmid pT3-5LO[13] (kindly provided by Dr. Olof
Radmark, Karolinska Institute Stockholm, Sweden). Cells were
lysed in 50 mM triethanolamine/HCl pH 8.0, 5 mM EDTA,
[11] J. Korp, S. König, S. Schieferdecker, H.-M. Dahse, G. M. König, O.
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2003, 17, 949-951.
soybean
trypsin
inhibitor
(60
µg/mL),
1
mM
phenylmethanesulfonyl fluoride, 1 mM dithiothreitol and 1 mg/mL
lysozyme, followed by sonification (3 x 15 s). The homogenate
was centrifuged (10,000 g, 15 min, 4 °C), the supernatant was
centrifuged again (40,000 g, 70 min, 4 °C), and 5-LO in the
resulting supernatant was purified by affinity chromatography on
an ATP-agarose column. Semi-purified 5-LO was diluted in PBS
containing 1 mM EDTA. Samples were pre-incubated with the
test compounds for 10 min at 4 °C. 5-LO product formation was
initiated by addition of CaCl2 (2 mM) and arachidonic acid (20
µM) at 37 °C for 10 min. The reaction was stopped by addition of
1 mL ice-cold methanol and formed 5-LO metabolites were
analyzed by RP-HPLC as previously described.[14]
[14] O. Werz, E. Bürkert, B. Samuelsson, O. Rådmark, D. Steinhilber, Blood
2002, 99, 1044-1052.
Blood cell isolation and activation of 5-LO product
formation in human PMNL
Human PMNL were isolated from leukocyte concentrates of
healthy adult human donors, received from the Institute of
Transfusion Medicine, University Hospital Jena, Germany, by
dextran sedimentation and centrifugation on Nycoprep
cushions.[14] PMNL were washed twice with ice-cold PBS and
resuspended in PBS plus glucose 0.1% containing CaCl2 (1 mM).
Samples were preincubated with the test compounds for 10 min
at 37 °C. 5-LO product formation was triggered by 2.5 µM
A23187 and terminated after 10 min by addition of 1 mL ice-cold
methanol. The production of formed 5-LO metabolites was
5
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10.1002/cmdc.201600536
ChemMedChem
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Entry for the Table of Contents
The enzyme 5-lipoxygenase (5-LO) plays a key role in the biosynthesis of leukotrienes, which are well-known mediators of allergic
and inflammatory reactions. Furthermore, the 5-LO pathway is associated with tumorigenesis, which makes 5-LO a promising
therapeutic target. Recently, the natural product myxochelin A was shown to be a potent 5-LO inhibitor. Here, we report the synthesis
of 48 myxochelin analogues and evaluation of their inhibitory effects on 5-LO in both cell-free and cell-based assays.
6
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