Caulerpenyne and related bis-enol esters are novel-type inhibitors of human 5-lipoxygenase

Article abstract of DOI:10.1002/cmdc.201402065

Caulerpenyne (CYN) is a sesquiterpene from green algae with known inhibitory properties against soybean lipoxygenase. Here we introduce a detailed structure-activity study elucidating the inhibitory effects of CYN and a library of six synthetic CYN analogues on isolated human 5-lipoxygenase (5-LO) and cellular 5-LO in polymorphonuclear leukocytes. Essential structural elements are identified and a structurally simplified inhibitor is introduced. The modes of 5-LO inhibition by CYN and the synthetic inhibitors cannot be assigned to any of the known categories of lipoxygenase inhibitors. These compounds clearly interfere directly with 5-LO and represent rather small and flexible molecules, with unique structures among 5-LO inhibitors identified thus far.

Full text of DOI:10.1002/cmdc.201402065

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DOI: 10.1002/cmdc.201402065
Caulerpenyne and Related Bis-enol Esters Are Novel-Type
Inhibitors of Human 5-Lipoxygenase
Phillipp Richter,[a] Gregor Schubert,[b] Anja Maria Schaible,[b] Levent Cavas,[c] Oliver Werz,*[b]
and Georg Pohnert*[a]
Caulerpenyne (CYN) is a sesquiterpene from green algae with
known inhibitory properties against soybean lipoxygenase.
Here we introduce a detailed structure–activity study elucidat-
ing the inhibitory effects of CYN and a library of six synthetic
CYN analogues on isolated human 5-lipoxygenase (5-LO) and
cellular 5-LO in polymorphonuclear leukocytes. Essential struc-
tural elements are identified and a structurally simplified inhibi-
tor is introduced. The modes of 5-LO inhibition by CYN and
the synthetic inhibitors cannot be assigned to any of the
known categories of lipoxygenase inhibitors. These com-
pounds clearly interfere directly with 5-LO and represent rather
small and flexible molecules, with unique structures among
5-LO inhibitors identified thus far.
due to some functional similarities.[8] However, recent mecha-
nistic and structural investigations as well as comparative in-
hibitor studies with human LOs revealed significant differences
between these LOs.[8a,9] In the present study, we assess the ef-
fects of CYN and a library of synthetic CYN analogues on isolat-
ed human 5-LO and cellular 5-LO in polymorphonuclear leuko-
cytes (PMNL).
For the library design, structural motives of CYN were con-
sidered separately. Initial experiments using CYN derivative 4
revealed that the acetate group at C4 is not essential for activi-
ty. Therefore, the synthetic strategy focused on modification of
the dienyne terminus and of the bis-enolacetate functionality
(dotted and dashed in Figure 1, respectively). Modifications at
Caulerpenyne (CYN (1)) is a sesquiterpene found in several
green algae belonging to the genus Caulerpa. It often repre-
sents the major secondary metabolite in Caulerpales with con-
centrations reaching up to 1.3% of the fresh weight of the
alga.[1] The acetylated sesquiterpene plays a central role in the
rapid wound closure of the siphonous green alga Caulerpa
taxifolia and other members of this genus.[2] During this pro-
cess, CYN (1) is enzymatically deacetylated and thereby trans-
formed to a highly reactive 1,4-bis-aldehyde. Following up re-
ports on the inhibitory activity of CYN (1) on microtubule pro-
tein polymerization, first synthetic CYN analogues in form of
colchicine hybrids were generated, however with limited activi-
ty.[3] CYN (1) itself is also active as inhibitor of telomerase,[4]
amylase,[5] xanthine oxidase[6] and plant lipoxygenases (LOs).[7]
We reported that CYN (1) inhibits soybean LO with an IC50
value of 5.1 mm in an uncompetitive manner.[7b] This plant LO
has long been used as a model enzyme for mammalian LOs
Figure 1. The sesquiterpene caulerpenyne (1), dotted and dashed parts indi-
cate groups modified for SAR studies, the secondary OAc group could be
omitted without significant loss of activity.
the terminus allowed systematically addressing the influence
of the sesquiterpene backbone of the natural product in com-
parison to smaller and less functionalized substituents. Com-
plete truncation of the terminus results in the commercially
available 1,4-diacetoxy-(1E,3E)-butadiene (7). The other test
compounds were synthesized according to a strategy based
on a route to 4 introduced by Richter et al.[10] Starting from
1,4-but-2-ynediol carbomagnesation allows introduction of dif-
ferent alkyl residues or the protected akynyl at position 2.
Lipase-mediated acetylation and oxidation using Dess–Martin
periodane provides the central intermediate that can finally be
acetylated under basic conditions using acetic acid anhydride
(Scheme 1, compounds 2, 3 and 4). To evaluate the role of the
bis-enolacetate structural element, the acetates were replaced
with linear carboxylic acids of longer chain length. In these
cases vinyl capronate and Candida rugosa lipase or vinyl stea-
rate and Rhizopus arrhizzus lipase were selected for the enzy-
matic acylation reactions. Acetic acid anhydride was replaced
with anhydrides of the longer chain length fatty acids in the
second acylation (Scheme 1, compounds 5 and 6).
[a] P. Richter, Prof. Dr. G. Pohnert
Friedrich Schiller University Jena
Institute for Inorganic and Analytical Chemistry, Bioorganic Analytics
Lessingstraße 8, 07743 Jena (Germany)
E-mail: georg.pohnert@uni-jena.de
[b] G. Schubert, A. M. Schaible, Prof. Dr. O. Werz
Friedrich Schiller University Jena
Chair of Pharmaceutical/Medicinal Chemistry
Philosophenweg 14, 07743 Jena (Germany)
E-mail: oliver.werz@uni-jena.de
[c] Prof. Dr. L. Cavas
Dokuz Eylꢀl University
Faculty of Sciences, Department of Chemistry
To assess the effects of CYN on 5-LO activity, well-established
cell-free and cell-based assays were applied.[11] The cell-free
assays allow analysis of direct inhibition of 5-LO. They are
˙
Tinaztepe Campus, 35160 Izmir (Turkey)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402065.
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Scheme 1. Synthesis of bis-enol esters with different side chains and acyl
moieties. Reagents and conditions: a) Et2O, reflux; b) vinyl carboxylic ester,
lipase, diisopropyl ether; c) for synthesis of 4, 5 and 6, tetrabutylammonium
fluoride, THF; d) Dess–Martin periodinane, CH2Cl2; e) carboxylic acid anhy-
dride, 4-(dimethylamino)pyridine, Et3N. See the Supporting Information for
detailed procedures.
Figure 2. Inhibition of 5-LO activity by caulerpenyne (CYN (1)). Inhibition of
the formation of 5-LO products (LTB4 and its trans isomers and 5-H(P)ETE) in
A) intact human PMNL and in B) PMNL homogenates. C) Inhibition of the ac-
tivity of isolated human recombinant 5-LO. Data are given as mean +SE,
n=3. *p<0.05; **p<0.01; ***p<0.001 versus vehicle (100%).
based on incubation of isolated human recombinant
5-LO or PMNL homogenates as enzyme source in the
Table 1. IC50 values [mm] of the bisenolacylates and a control compound for 5-LO
presence or absence of inhibitors with arachidonic
acid (AA, 20 mm) as substrate. The cell-based assay
was performed in the presence or absence of exoge-
nous AA as substrate.[11] This assay uses Ca2+-iono-
phore A23187-stimulated human PMNL and can
reveal indirect inhibitory effects on 5-LO product for-
mation (e.g., block of substrate supply, influence on
the 5-lipoxygenase-activating protein (FLAP), and on
5-LO activation by phosphorylation). For both assays,
formation of 5-LO products (i.e., LTB4 and its trans-
isomers and 5-H(P)ETE) derived from AA were deter-
mined by RP-HPLC.[12]
inhibition.
Compd
Purified 5-LO[a] PNMLÀAA[b] PNML+AA[c]
1
4.2
39.1
5.9
18.4
2.1
21.0
n.d.
n.d.
18.8
n.d.
2
3
4
5
>30[d]
11.8
In the cell-based assay, CYN (1) potently and con-
centration-dependently suppressed 5-LO product for-
mation with an IC50 of 5.9 mm (Figure 2A, Table 1). For
the reference 5-LO inhibitor zileuton (approved as
drug for asthma therapy), the IC50 value was deter-
mined at 0.6 mm (not shown). In the cell-free assay,
using PMNL homogenates as source for 5-LO, CYN
(1) inhibited 5-LO activity with an IC50 value of 9.5 mm
(Figure 2B), and for isolated human recombinant
5-LO an IC50 value of 4.2 mm was obtained (Fig-
ure 2C). These data demonstrate that CYN (1) is a
5-LO inhibitor with effectiveness in intact cells.
5.1
48.6
1.0
6
21.0
>100
n.d.
7
8
>100
>100
>100
>100
n.d.
n.d.
The chemical structure of the active CYN (1)
cannot be categorized into known classes of 5-LO in-
hibitors.[13] We thus decided to undertake structure–
activity relationship (SAR) studies in order to dissect
the pharmacophore of CYN (1). Truncation of the
entire terminus results in the bis-enolacetate 1,4-diac-
etoxy-(1E,3E)-butadiene (7), which led to loss of po-
[a] Purified 5-LO incubated with 20 mm AA as substrate. [b] A23187-activated intact
polymorphonuclear leukocytes (PNML) without AA. [c] A23187-activated intact PNML
with 20 mm AA; PMNL were preincubated with test compounds for 15 min and then
activated to induce 5-LO product formation for 10 min. Data are means, n=3–4.
n.d.=not determined. [d] Not soluble at >30 mm.
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tency in PMNL and completely failed to inhibit isolated 5-LO
up to 100 mm. Hence, the alkylation in 2-position of the buta-
diene moiety is essential for bioactivity. Introduction of
a methyl group at this position (2) recovered 5-LO inhibition,
although the potency was almost 10-fold lower as compared
to CYN (1) (Table 1). Similarly, substitution with n-pentyl (3)
that at least partially recovers the hydrophobicity of the dien-
yne terminus of CYN (1) resulted in a moderate inhibitor of
5-LO in the cell-free assay with an IC50 >30 mm. It is surprising
that this compound caused strong suppression of 5-LO activity
in intact cells (IC50 =2.1 mm), which might be related to inter-
ference with 5-LO regulatory processes (e.g., block of substrate
supply, 5-LO phosphorylation or FLAP) or unspecific (acute cy-
totoxic) effects. Interestingly, introduction of a pent-4-inyl resi-
due at 2-position of the butadiene (4) led to comparable po-
tency as found for CYN (1) (Table 1). Unsaturation of the hydro-
phobic terminus thus seems essential for activity. The pent-4-
inyl moiety itself (in hept-6-in-1-ol 8) was completely inactive
in the cell-based and the cell-free assay (Table 1).
Figure 3. Radical scavenging activity. Test compounds were incubated with
5 nmol DPPH for 30 min at RT, and the absorbance was measured at
520 nm. Ascorbic acid and l-cysteine were used as controls. Values are given
as percentage of control (100%) mean +SE, n=3.
structural elements, we suggest that CYN (1) and 4 may
indeed inhibit 5-LO in a non-redox fashion.
The fact that a substantially simplified molecule compared
to CYN (1) exhibited comparable activity opened up new pos-
sibilities for further modifications based on the structure of 4.
The synthetic strategy to 2–4 could be adapted to exchange
the acetyl moieties by longer chain length fatty acids.
To investigate if the compounds inhibit 5-LO in a reversible
manner, wash-out experiments using isolated 5-LO enzyme
were performed. 5-LO activity was inhibited by 1 mm and
10 mm CYN (1) to 25% and 75%, respectively (Figure 4A). A
Capronyloxy (5) and stearyloxy derivatives (6) were
thus accessible for testing. In the cell-based assay
a pronounced influence of the length of the fatty
acid residues on 5-LO inhibition was observed. The
bis-capronyloxy derivative 5 was even more potent
compared to CYN (1) itself and inhibited 5-LO in the
cell-based assay with an IC50 value of 1 mm, while the
bis-enol stearate (6) completely failed to inhibit in
the cell-based assay at 100 mm. Interestingly, 5 was
a poor inhibitor of isolated 5-LO (IC50 =48.6 mm) and
also 6 was less active (IC50 =21 mm) as compared to
Figure 4. Reversibility of 5-LO inhibition and influence of substrate concentration.
A) Wash-out experiments by 10-fold dilution in incubations of isolated 5-LO and test
compounds. B) Inhibition of isolated 5-LO by test compounds (10 mm, each) at various
AA concentrations. Values, given as percentage of control (vehicle, 100%), are means
+SE, n=4. *p<0.05; **p<0.01; ***p<0.001 versus vehicle (100%).
CYN (1) and 4. This observation would be in accord-
ance with a mechanism where 5 may, in analogy to
3, target 5-LO regulatory events in the cell. The long
C-18 alkyl chains of 6 decrease the solubility and
might hamper cell penetration into the cytosol
where 5-LO resides. In all above assays it has to be
considered that due to the synthetic procedures, all four con-
figurational isomers of 2–6 are present and might differentially
contribute to inhibition of 5-LO, as compared to CYN (1) as ste-
reochemically defined structure.
10-fold dilution of the sample containing 10 mm CYN (1) (final
concentration 1 mm) partially reduced 5-LO inhibition to about
50%. These data suggest an incomplete reversion of 5-LO sup-
pression by CYN (1) after wash-out. In contrast to CYN (1), the
inhibitory effect of compound 4 after 10-fold dilution from
30 mm to 3 mm corresponds to that of an initially administered
3 mm solution (Figure 4A), indicating that the inhibitory effects
are immediately reversible. As expected, Inhibition of 5-LO by
the reference compound zileuton was also reversible in com-
parable wash-out experiments (Figure 4A).[11]
The mode of 5-LO inhibition by the most potent compounds
CYN (1) and the alkynyl derivative 4 was studied in more
detail. Because many direct 5-LO inhibitors act as antioxidants
due to interference with the active site iron by uncoupling its
redox cycle or by radical scavenging during enzymatic cataly-
sis,[13a] we first assessed the radical scavenging activity of CYN
(1) and 4 using the well-established di(phenyl)-(2,4,6-trinitro-
phenyl)iminoazanium (DPPH) assay.[14] Neither compound
caused significant radical scavenging activity at concentrations
up to 200 mm, whereas l-cysteine and ascorbic acid (used as
reference compounds) clearly blocked DPPH radical formation
(Figure 3). Therefore, and supported by the lack of redox-active
Next, we analyzed how 5-LO inhibition by CYN (1) and 4 is
affected by the substrate (AA) concentration. Previous studies
showed that 5-LO activity is maximal at about 10–20 mm AA
but then declines at higher concentrations due to substrate in-
hibition.[13b] As shown in Figure 4B, the magnitude of 5-LO in-
hibition by CYN was enhanced by increasing the AA concentra-
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Experimental Section
tion (from 2 to 40 mm), implying an uncompetitive mode of in-
hibition. Thus, at 2 mm AA almost 60% 5-LO activity remained
in the presence of 10 mm CYN (1), while at 40 mm AA about
less than 10% activity was detectable. In contrast, for 4, in-
creasing the substrate concentration impaired the potency
against 5-LO (Figure 4B). Here, 5-LO activity at 2 mm AA was
suppressed down to 25%, but at 40 mm AA 5-LO inhibition
was moderate and 65% enzyme activity remained. These data
suggest that CYN (1) acts in an uncompetitive manner, while
in sharp contrast 4 inhibits 5-LO in an AA competitive way.
In addition, we analyzed the ability of CYN (1) and 4 to in-
hibit the 5-LO-related human 12-LO and 15-LO-1 that are pres-
ent in PMNL-adherent platelets and eosinophils, respectively.[15]
As shown in Figure 5, CYN (1) concentration-dependently sup-
Details on materials, synthetic procedures and spectroscopic data
can be found in the Supporting Information.
Biological assays
Human PMNL: Human polymorphonuclear leukocytes (PMNL) were
freshly isolated from peripheral blood (Institute for Transfusion
Medicine, University Hospital Jena, Germany) as described.[11] In
brief, venous blood from healthy adult donors was centrifuged
(4000 g/20 min/208C) to prepare leukocyte concentrates. PMNL
were promptly isolated by dextran sedimentation and centrifuga-
tion on Nycoprep cushions. The pellet was resuspended, erythro-
cytes were lysed under hypotonic conditions, and PMNL were re-
covered by centrifugation (purity >96–97%).
Determination of 5-LO activity in intact cells: PMNL (1ꢁ107 mLÀ1
)
were pre-incubated with test compounds for 15 min at 378C.
Then, 5-LO product formation was started by addition of 2.5 mm
Ca2+-ionophore A23187 with or without 20 mm AA. After 10 min at
378C, 5-LO metabolites (LTB4 and its all-trans isomers and
5-H(P)ETE) were extracted and analyzed by RP-HPLC as de-
scribed.[11]
Analysis of LO activities in cell-free assays: Human recombinant
5-LO was expressed in E. coli BL21 transformed with pT3–5LO plas-
mid, and isolated by affinity chromatography on an ATP-agarose
column as described.[11] Alternatively, PMNL were homogenized by
sonication and used as source for 5-LO, 12-LO and 15-LO. Isolated
5-LO or PMNL homogenates were pre-incubated with test com-
pounds for 10 min at 48C and pre-warmed for 30 s at 378C. 5-LO
product formation was initiated by addition of 2 mm CaCl2 and
20 mm AA. After 10 min at 378C, formed 5-LO metabolites (all-trans
isomers of LTB4 and 5-H(P)ETE), 12-H(P)ETE and 15-H(P)ETE were
analyzed by RP-HPLC as described.[11]
Figure 5. Effects of CYN (1) and compound 4 on the activity of 12-LO and
15-LO-1 in homogenates of PMNL. Data are given as means +SE, n=3.
**p<0.01 versus vehicle (100%).
pressed the activity of both 12-LO and 15-LO-1 in PMNL homo-
genates albeit with reduced potency (IC50 =36 and 42 mm, re-
spectively) as compared to 5-LO (IC50 =9.5 mm) under the same
assay conditions. In contrast, compound 4 (up to 100 mm)
failed to significantly repress the activities of these LOs.
Determination of radical scavenging activity: Compounds were dis-
solved in EtOH and combined with an equal volume of 100 mm di-
(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH) in EtOH (final
volume: 200 mL). After incubation for 30 min, absorbance was mea-
sured at 520 nm as described.[11]
Taken together, despite similar potencies against 5-LO, the
mode of 5-LO inhibition by CYN (1) and 4 is substantially dif-
ferent: while 5-LO inhibition by 4 is reversible, impaired by in-
creasing AA concentrations and selective for 5-LO, inhibition of
5-LO by CYN (1) is only partially reversible, markedly improved
at increasing substrate concentrations and unselective as also
12/15-LOs are inhibited. The molecular mechanisms underlying
5-LO inhibition by these compounds are still elusive. 5-LO in-
hibitors have been intensively developed since 1983 in order
to be used as anti-inflammatory or anti-allergic drugs, and
a vast number of compounds had been introduced.[16] These
substances can be categorized as (1) redox-active agents,
(2) iron-ligands, (3) fatty acid competitors that act at the active
5-LO site, or (3) represent agents that interfere with phospho-
lipid binding of 5-LO via the C2-like domain.[13a] Neither CYN
(1) nor compound 4 can be assigned to any of these catego-
ries. Both compounds clearly interfere directly with 5-LO and
represent rather small and flexible molecules, with unique
structures among 5-LO inhibitors identified thus far.
Statistics: Data are expressed as mean +standard error (SE). Statis-
tical evaluation was performed by one-way ANOVAs for independ-
ent or correlated samples followed by Tukey HSD post hoc tests or
by Student’s t test for paired and correlated samples. P values
<0.05 were considered statistically significant. All statistical calcula-
tions were performed using GraphPad InStat 3.10 (GraphPad Soft-
ware Inc., La Jolla, CA, USA). IC50 values were determined by graph-
ical analysis using SigmaPlot 12.0 (Systat Software Inc., San Jose,
CA, USA).
Acknowledgements
We acknowledge the Volkswagen Foundation (Germany) for
funding within the framework of a Lichtenberg Professorship.
Keywords: caulerpenyne · inhibition · lipoxygenase · structure
activity · synthetic analogues
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Received: March 11, 2014
Published online on April 11, 2014
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