Synthesis and biological evaluation of novel myrtucommulones and structural analogues that target mPGES-1 and 5-lipoxygenase

Article abstract of DOI:10.1016/j.ejmech.2015.06.001

The natural acylphloroglucinol myrtucommulone A (1) inhibits microsomal prostaglandin E₂ synthase (mPGES)-1 and 5-lipoxygenase (5-LO), and induces apoptosis of cancer cells. Starting from 1 as lead, 28 analogues were synthesized following a straightforward modular strategy with high yielding convergent steps. Major structural variations concerned (I) replacement of the syncarpic acid moieties by dimedone or indandione, (II) cyclization of the syncarpic acid with the acylphloroglucinol core, and (III) substitution of the methine bridges and the acyl residue with isopropyl, isobutyl, n-pentyl or phenyl groups, each. The potency for mPGES-1 inhibition was improved by 12.5-fold for 43 (2-(1-(3-hexanoyl-2,4,6-trihydroxy-5-(1-(3-hydroxy-1-oxo-1H-inden-2-yl)-2-methylpropyl)phenyl)-2-methylpropyl)-3-hydroxy-1H-inden-1-one) with IC₅₀ Combining double low line 0.08 μM, and 5-LO inhibition was improved 33-fold by 47 (2-((3-hexanoyl-2,4,6-trihydroxy-5-((3-hydroxy-1-oxo-1H-inden-2-yl) (phenyl)methyl)phenyl) (phenyl)methyl)-3-hydroxy-1H-inden-1-one) with IC₅₀ Combining double low line 0.46 μM. SAR studies revealed divergent structural determinants for induction of cell death and mPGES-1/5-LO inhibition, revealing 43 and 47 as non-cytotoxic mPGES-1 and 5-LO inhibitors that warrant further preclinical assessment as anti-inflammatory drugs.

Full text of DOI:10.1016/j.ejmech.2015.06.001

European Journal of Medicinal Chemistry 101 (2015) 133e149
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and biological evaluation of novel myrtucommulones and
structural analogues that target mPGES-1 and 5-lipoxygenase
,
*
Katja Wiechmann a, Hans Müller b, Volker Huch c, David Hartmann b, Oliver Werz a
,
,
**
Johann Jauch b
a Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, University of Jena, Philosophenweg 14, D-07743 Jena, Germany
b Organic Chemistry II, Saarland University, Campus C 4.2, D-66123 Saarbrücken, Germany
c Institute for Inorganic Chemistry, Saarland University, Campus C 4.1, D-66123 Saarbrücken, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The natural acylphloroglucinol myrtucommulone A (1) inhibits microsomal prostaglandin E2 synthase
(mPGES)-1 and 5-lipoxygenase (5-LO), and induces apoptosis of cancer cells. Starting from 1 as lead, 28
analogues were synthesized following a straightforward modular strategy with high yielding convergent
steps. Major structural variations concerned (I) replacement of the syncarpic acid moieties by dimedone
or indandione, (II) cyclization of the syncarpic acid with the acylphloroglucinol core, and (III) substitution
of the methine bridges and the acyl residue with isopropyl, isobutyl, n-pentyl or phenyl groups, each. The
potency for mPGES-1 inhibition was improved by 12.5-fold for 43 (2-(1-(3-hexanoyl-2,4,6-trihydroxy-5-
(1-(3-hydroxy-1-oxo-1H-inden-2-yl)-2-methylpropyl)phenyl)-2-methylpropyl)-3-hydroxy-1H-inden-1-
Received 30 January 2015
Received in revised form
7 May 2015
Accepted 1 June 2015
Available online 5 June 2015
Keywords:
Myrtucommulone
Acylphloroglucinol
5-Lipoxygenase
mPGES-1
Arachidonic acid
Cyclooxygenase
one) with IC50 ¼ 0.08
trihydroxy-5-((3-hydroxy-1-oxo-1H-inden-2-yl) (phenyl)methyl)phenyl) (phenyl)methyl)-3-hydroxy-
M. SAR studies revealed divergent structural determinants for in-
mM, and 5-LO inhibition was improved 33-fold by 47 (2-((3-hexanoyl-2,4,6-
1H-inden-1-one) with IC50 ¼ 0.46
m
duction of cell death and mPGES-1/5-LO inhibition, revealing 43 and 47 as non-cytotoxic mPGES-1 and 5-
LO inhibitors that warrant further preclinical assessment as anti-inflammatory drugs.
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
pathogenesis and maintenance of inflammation [1]. The starting
point for the biosynthesis of these eicosanoids is the conversion of
Chronic and excessive inflammation is present as a functional
component of many disorders including infections, autoimmune
disorders, atherosclerosis and cancers. Among the pro-
inflammatory lipid mediators the arachidonic acid (AA)-derived
prostaglandins (PG) and leukotrienes (LT) play crucial roles in the
AA to PGH2 catalyzed by cyclooxygenases (COX)-1 and -2 and to
leukotriene LTA4 catalyzed by 5-lipoxygenase (5-LO).
COX-1 is constitutively expressed and responsible for the allo-
cation of basal levels of various PGs (e.g. TXA2, PGE2, PGI2, PGF2a
)
involved in homeostatic functions [1]. During inflammatory re-
actions and certain cancers COX-2 is over-expressed and the levels
of certain PGs are considerably increased [2]. Downstream of COX-
2, the microsomal prostaglandin E2 synthase (mPGES)-1 transforms
PGH2 to PGE2 [3]. High levels of PGE2 mediate pain, fever and other
inflammatory symptoms as well as cancer triggered through
binding to the receptors EP-1 to -4 [4]. The common treatment of
symptoms of inflammation involves the application of non-
steroidal anti-inflammatory drugs (NSAIDs) inhibiting COX-1 and
-2 or selectively COX-2 (i.e., coxibs). However, inhibition of COX-1/2
enzymes interferes severely with the synthesis of homeostatic PGs
causing major side effects like gastrointestinal damages, renal
disturbances or cardiovascular toxicity [5]. Selective inhibition of
mPGES-1 would curb the biosynthesis of pro-inflammatory PGE2
Abbreviations: AA, arachidonic acid; COX, cyclooxygenase; cPLA2, cytosolic
phospholipase A2; FLAP, 5-lipoxygenase-activating protein; 5-HpETE, 5(S)-hydro-
peroxy-6-trans-8,11,14-cis-eicosatetraenoic acid; 5-LO, 5-lipoxygenase; LT, leuko-
triene; MC, myrtucommulones; mPGES-1, microsomal prostaglandin E2 synthase-1;
NSAID, non-steroidal anti-inflammatory drug; PG, prostaglandin; PMNL, poly-
morphonuclear leukocyte; SAR, structureeactivity relationship.
* Corresponding author. Chair of Pharmaceutical/Medicinal Chemistry, Institute
of Pharmacy, Friedrich-Schiller-University Jena, Philosophenweg 14, 07743 Jena,
Germany.
** Corresponding author. Organic Chemistry II, University of Saarland, Campus C
4.2, D-66123 Saarbrücken, Germany.
E-mail addresses: oliver.werz@uni-jena.de (O. Werz), j.jauch@mx.uni-saarland.
de (J. Jauch).
http://dx.doi.org/10.1016/j.ejmech.2015.06.001
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

134
K. Wiechmann et al. / European Journal of Medicinal Chemistry 101 (2015) 133e149
and could thus circumvent unwanted effects caused by COX in-
hibitors [3,6].
efficient method for the synthesis of nonprenylated acylphlor-
oglucinols [21]. The synthesis of 1 combines readily available syn-
carpic acid (15) [22] with isobutyraldehyde (16) and isobutyryl
phloroglucinol (17) in two simple steps (Scheme 1). These are a) a
Mannich reaction of 15 with 16 to yield isobutylidene syncarpic
acid (18) after acid treatment of the intermediate Mannich base,
and b) a double Michael addition of deprotonated 17 to two
equivalents of 18.
The double Michael addition can also be conducted stepwise
with two different Michael acceptors. Thus, the Michael addition of
deprotonated hexanoyl phloroglucinol (19) to one equivalent of 18
leads to compound 20, which can be deprotonated in situ and
added to Michael acceptor 21 to give 9 (Scheme 2). When depro-
tonated 17 is added to Michael acceptor 18, nor-semi-
myrtucommulone (22) is obtained. Compound 22 can be cyclized
to 2 and combined in a second Michael addition with 18 to give 4
(Scheme 2). Synthetic 2 yielded crystals suitable for X-ray analysis
(Fig. 2) [23a], which confirmed the linear structure proposed for 2
[23b].
The general strategy depicted in Scheme 3 offers a high vari-
ability to synthesize different myrtucommulone analogues. All
components of the synthesis can be varied: the central phlor-
oglucinol core, the bridging aldehyde, and the 1,3-dicarbonyl unit.
Additional diversity of the synthesized analogues can be generated
by performing the double Michael addition either in one step
(analogues type A) followed by cyclization (analogues type F) or in
two steps with two different Michael acceptors with cyclization
(analogues type D and E) or without cyclization (analogues type B
and C) after the first Michael addition.
Leukotrienes are synthesized from AA initialized by 5-
lipoxygenase (5-LO) in cooperation with the 5-LO-activating pro-
tein (FLAP) [7]. 5-LO-mediated transformation of AA leads to the
epoxide LTA4 that can be converted to LTB4 via LTA4 hydrolase or to
LTC4 by conjugation with glutathione via LTC4 synthase; cleavage of
LTC4 then leads to LTD4 and LTE4 (collectively named cys-LTs). The
LTs serve as powerful chemoattractants for neutrophils (LTB4) or as
potent bronchoconstrictors in the airways (cys-LTs) at sites of
inflammation and allergic reactions, respectively [8]. Some selec-
tive cys-LT receptor antagonists (montelukast, zafirlukast) are
approved for asthma therapy, however the only approved blocker
of LT synthesis is the 5-LO inhibitor zileuton, which is used as anti-
asthmatic in the US.
Extracts of Myrtus communis L. (myrtle), an evergreen shrub
from the Mediterranean area, are used in traditional medicine as
antibiotic and anti-inflammatory remedies. In 1974, Kashman et al.
isolated myrtucommulone A (MC A,1) and myrtucommulone B (MC
B, 2) from leave extracts of myrtle [9,10]. In 2002, Appendino et al.
found semi-myrtucommulone (3) in myrtle extracts [11]. Shaheen
et al. discovered the myrtucommulones C, D and E (4e6) [12].
Carroll et al. isolated the myrtucommulones F e I (7e10) from
Corymbia scabrida [13], another member of the myrtaceae. The
myrtucommulones J, K and L (11e13) were isolated from Myrtus
communis by Cottiglia et al. [14], and the latest member of the
myrtucommulones, MC M (14), was reported by Choudhary et al.
[15] Recently, Nicoletti et al. discovered that the endophytic fungus
in myrtle Neofusicoccum australe produces 1 and 5 [16]. The
structures of all myrtucommulones are shown in Fig. 1.
Acylphloroglucinols can be synthesized through a straight for-
ward FriedeleCrafts acylation of phloroglucinol (23) with an
appropriate acid chloride (Scheme 4) [24]. The required Michael
acceptors are readily obtained from a,b-unsaturated 1,3-diketones
via a Mannich reaction with an aldehyde in the presence of sec-
ondary amine (piperidine ore pyrrolidine), followed by elimination
We recently identified 1 as the first natural compound active as
a dual inhibitor of mPGES-1 [17] and 5-LO [18]. In a cell-free
mPGES-1 activity assay, 1 inhibited the enzymatic conversion of
PGH2 to PGE2 catalyzed by mPGES-1 in microsomes from stimu-
lated A549 cells with IC50 ¼ 1
and COX-2 in cell-free assays up to concentrations of 30
caused only moderate inhibition of COX-1 activity in platelets with
IC50 ¼ 17 M [17]. Moreover, 1 potently inhibited also 5-LO in
intact cells with IC50 values of 1.8 M as well in cell-free systems
with IC50 in the range of 5e15 M. It is interesting that 1 exhibited
m
M. Notably, 1 did not inhibit COX-1
m
M and
of the amine (Scheme 4) [25e27]. Since these
a,b-unsaturated 1,3-
diketones isomerize easily to -unsaturated 1,3-diketones in the
b,g
m
enol form [25e27], they have to be used immediately in the
Michael addition step.
m
m
With these building blocks, the myrtucommulone analogues
shown in Fig. 3 were synthesized according to Scheme 3 and tested
for inhibition of mPGES-1 and 5-LO as well as for induction of cell
death of leukemic Jurkat cells. Choosing the building blocks was on
the one hand inspired by the respective substituents from isolated
myrtucommulones (Fig. 1), on the other hand the building blocks
represent substituents in the analogues with different steric and
electronic properties. For example, syncarpic acid (15) is more
sterically hindered than dimedone (24), whereas indandione (25) is
flat and also has different electronic properties due to the aromatic
system. This is also valid for the aldehyde building blocks as well as
the acid chloride building blocks. Furthermore, the reported ana-
logues were developed stepwise in an activity-guided way. Thus,
the pharmacological tests revealed that a long unbranched alkyl
group leads to higher activity with respect to mPGES-1- and 5-LO-
inhibition (see below, compare 1 and 7, Table 1). We therefore
synthesized 42 and 43, which proofed this concept also for ana-
logues with indandione as 1,3-diketone building block. This is also
true for analogues based on dimedone (compare 34 and 35),
though in a less distinct way.
only marginal cytotoxicity for non-transformed human monocytes
or fibroblast while it induced apoptosis in cancer cell lines, via the
mitochondrial cytochrome c/Apaf-1/caspase-9 pathway, implying
a potential as anti-tumour agent [19]. Together, 1 represents an
anti-inflammatory compound that targets mPGES-1 and 5-LO,
without interfering with the basal biosynthesis of homeostatic
PGs, and acts as cytotoxic agent against cancer cells. On top of
these in vitro findings, animal experiments confirmed the in vivo
activity of 1, as it reduced the inflammatory response by allevi-
ating the swelling in the carrageenan-induced mouse paw edema
as well as by blocking pleural exudate formation and neutrophil
invasion into damaged tissue in the carrageenan-induced pleurisy
model [20].
Concluding from these promising results we anticipated that a
library of myrtucommulone analogues would offer the possibility
to find compounds with higher activity and greater selectivity than
the natural products and also offer additional insights into
structure-activity-relationships (SAR) for the inhibition of mPGES-1
and 5-LO and structural requirements for induction of cell death.
Structure elucidation of the naturally occurring myrtucommu-
lones and of the analogues from Fig. 3 is difficult when molecules
are not completely rigid. Due to their flexibility, the compounds
exist as mixtures of rotamers and/or keto-enol tautomers which
only interconvert slowly on the NMR time scale [9]. Therefore,
these compounds give rise to very complex 1H-NMR- and 13C-NMR-
2. Results and discussion
2.1. Compound design and chemistry
Our recently published total synthesis of 1, 4 and 7 offered an

K. Wiechmann et al. / European Journal of Medicinal Chemistry 101 (2015) 133e149
135
O
O
O
O
OH HO
OH
O
OHHO
OHHO
O
O
O
OH
O
OH
O
OH
O
OH
O
1
2
3
O
O
O
OH
O
O
O
OH
O
OH
O
O
O
OH
HO
O
O
O
O
O
O
O
OH
O
O
O
4
5
6
O
O
OH
O
O
O
OH
O
OHHO
OHHO
O
O
OHHO
OHHO
O
O
O
O
O
OH
O
O
OH
O
O
7
8
9
O
O
OH
O
O
OH
O
OHHO
O
O
O
H
H
O
O
O
O
O
HO
H
O
O
O
10
11
12
O
O
O
O
O
O
OHHO
O
O
O
O
OH
OH
O
13
14
Fig. 1. Structures of naturally occurring myrtucommulones AeM (1e14).
spectra [9,21]. Additionally, the situation is complicated by the fact
that in constitutionally symmetric natural myrtucommulones and
analogues, the synthesis yields a mixture of the racemate and the
meso form [21]. However, structure elucidation is straight forward
for rigid compounds obtained through cyclization and dehydration
to the respective bis pyrane derivatives [21]. Therefore, every
compound was cyclized in refluxing benzene in the presence of p-
toluene sulfonic acid according to Scheme 5 [21].
Interestingly, all cyclizations led to linear bis pyrane derivatives
except the analogues type A2 and A3, which all gave angularly
cyclized bis pyranes. Compound 55 gave crystals suitable for X-ray
analysis, which unambiguously proofed its structure (Fig. 4) [28].

136
K. Wiechmann et al. / European Journal of Medicinal Chemistry 101 (2015) 133e149
O
O
OH
O
O
O
OHHO
OHHO
O
a
b
O
OH
O
O
O
15
18
1
O
O
HO
OH
16
17
H
OH
Scheme 1. Synthesis of MC A (1). Reagents and conditions: a) i: isobutyraldehyde (16), piperidine, CH2Cl2, r.t., 10 min; ii: 1 N HCl saturated with NH4Cl, 5 min; iii: filter through a
short pad of MgSO4. b) 2 eq. NaH, THF, isobutyryl phloroglucinol (17), r.t., 5 min, then add the solution from a), r.t., 3 h, 96%.
2.2. Biological assays
compounds also inhibit 5-LO inside the cell, we used ionophore
A23187-stimulated human polymorphonuclear leukocytes (PMNL)
Direct inhibition of 5-LO and mPGES-1 activity by the test
compounds was assessed in cell-free assays using partially purified
human recombinant 5-LO and overexpressed mPGES-1 in micro-
in the presence of exogenous AA (20 mM) as 5-LO substrate. Note
that in intact PMNL a given test compound may suppress 5-LO
product synthesis without inhibiting 5-LO directly, for example
by modulating cellular co-factors of 5-LO or by targeting other
enzymes involved in LT synthesis (e.g., FLAP, LTA4H, LTC4S) [29]. The
somal preparations of interleukin-1b-stimulated A549 cells,
respectively. In these assays, reduced enzymatic activity is most
likely due to direct interference of the test compound with the
target enzyme. In order to investigate whether or not the test
5-LO
inhibitor
56
(BWA4C,
(E)-N-hydroxy-N-(3-(3-
phenoxyphenyl)-allyl)acetamide) [30] was used as control.
O
O
O
O
OHHO
OH
O
O
OH
O
O
O
OH
HO
c
d
b
O
OH
O
OH
O
OH
O
22
2
4
O
OH
O
O
a
O
O
15
18
O
O
O
OHHO
OHHO
O
O
OHHO
OH
e
f
O
OH
O
O
OH
20
9
O
HO
OH
19
OH
Scheme 2. Synthesis of MC C (4) and MC H (9). Reagents and conditions: i: isobutyraldehyde (16), piperidine, CH2Cl2, r.t., 10 min; ii: 1 N HCl saturated with NH4Cl, 5 min; iii: filter
through a short pad of MgSO4. b) 1 eq. NaH, THF, isobutyryl phloroglucinol (17), r.t., 5 min, then add the solution from a), r.t., 3 h, 98%. c) pTsOH, benzene, reflux, 30 min, quant. d) 1
eq. NaH, THF, MC B (2), r.t., 5 min, then add the solution from a), r.t., 3 h, 98%. e) 1 eq. NaH, THF, hexanoyl phloroglucinol (19), r.t., 5 min, then add the solution from a), r.t., 3 h, 92%. f)
1 eq. NaH, THF, 20, r.t., 5 min, than add a solution of 21 (prepared according to a) with isovaleraldehyde), r.t. 3 h, 96%.

K. Wiechmann et al. / European Journal of Medicinal Chemistry 101 (2015) 133e149
137
2.3. SARs for 5-LO and mPGES-1
Our synthetic strategy allowed us to approach structural varia-
tions in five directions leading to 28 compounds: (I) preparation of
semi-myrtucommulones with only one methine-bridged syncarpic
acid moiety, (II) cyclization of the syncarpic acid moiety with the
acylphloroglucinol core, (III) replacement of the syncarpic acid
moiety by dimedone or by indandione, (IV) substitution of the
methine bridges with isopropyl, isobutyl or phenyl groups, (V)
varying the acyl substituent at the acylphloroglucinol core applying
methyl, isopropyl, isobutyl, n-pentyl and phenyl groups as well as
replacement by a third syncarpic acid moiety (Fig. 3).
The results of mPGES-1/5-LO dual inhibition by myrtucommu-
lones analogues type A1 (Fig. 3) are presented in Table 1. The
parental compound 1 inhibited mPGES-1 and 5-LO with IC50 ¼ 1.0
and 15.0
mM, respectively [17,18]. Replacing the sterically
demanding isopropyl group as acyl substituent in 1 by the slim n-
pentyl moiety yielding 7, caused a slightly improved potency
regarding mPGES-1 inhibition but not for 5-LO, where it remained
essentially unchanged. When a phenyl was inserted (33), the po-
tency against mPGES-1 was reduced about 5-fold versus 1 but
hardly altered for inhibition of isolated 5-LO, though the potency in
the cell-based assay was reduced.
Replacement of the two isopropyl groups at the methine bridges
between the acylphloroglucinol core and the syncarpic acid by
isobutyl, each, increased the potency against mPGES-1 about 4-fold
Fig. 2. X-ray structure of 2, showing ellipsoids with 50% probability.
Analysis of the inhibition of mPGES-1 activity (i.e., the trans-
formation of PGH2 to PGE2) by a given compound in the cell is not
immediately feasible, since other enzymes than mPGES-1 are
involved in PGE2 formation in stimulated cells (i.e. COX-1/2, cPGES
and mPGES-2), and an appropriate test system is not available. The
mPGES-1 inhibitor 57 (MK-886, 3-(3-(tert-butylthio)-1-(4-
chlorobenzyl)-5-isopropyl-1H-indol-2-yl)-2,2-dimethylpropanoic
acid) [31], was used as reference compound. Analysis of cytotoxic
effects of the test compounds on leukemic Jurkat cells was con-
ducted by MTT assay. Detailed descriptions of these assays are re-
ported in Refs. [19,32], and are summarized in the Experimental
Section.
with IC50 values in the range of 0.22e0.29 mM, regardless of the acyl
substituents (n-pentyl in 34, isopropyl in 35 and isobutyl in 36).
Also for inhibition of 5-LO, isobutyl-substitution of the methine
bridges yielded compounds (i.e., 34, 35, and 36) that exhibited
higher inhibitory activity against the 5-LO enzyme
O
O
R1
R2
n
R3
O
2
1
:
R3
O
OH
R1
R1
OH
HO
OH
R1
HO
OH
MA
HO
:
n
n
n
R3
O
R2
OH
O
R2
Analogue Type A
OH
R2
O
O
1
1
HO
OH
Analogue Type C
OH
AP
R3
O
R1
R1
O
O
n
n
O
O
R2
OH R2
Analogue Type F
R3
O
R3
O
R3
O
R1
OH
HO
HO
R1
OH
R1
R1
OH
O
R1
HO
OH
O
n
n
n
n
n
O
R2
O
R2
O
OH
R4
OH
R4
R2
O
O
OH
Analogue Type E
Analogue Type D
Analogue Type B
Scheme 3. General synthetic possibilities with variation of Michael acceptor (MA, R1 and R2), acylphloroglucinol (AP, R3) and double Michael addition in one step or in two steps.
From analogues type A to analogues type F the flexibility of the molecules decreases.

138
K. Wiechmann et al. / European Journal of Medicinal Chemistry 101 (2015) 133e149
R3
O
OH
O
HO
OH
a
R1
R1
b
HO
OH
n
R2
n
O
O
OH
OH
AP
MA
23
OH
O
OH
O
OH
1,3-diketones
O
O
15
24
25
aldehydes
CHO
CHO
CHO
16
26
27
acid chloride
COCl
COCl
COCl
COCl
nC5H11 COCl
28
29
30
31
32
Scheme 4. Synthesis of the building blocks for the analogues of Scheme 3. Reagents and conditions: a) i: aldehyde, piperidine, CH2Cl2, r.t., 10 min; ii: 1 N HCl saturated with NH4Cl,
5 min; iii: filter through a short pad of MgSO4. b) acid chloride, AlCl3, CH2Cl2/CH3NO2 4:1 (v/v), refl. 1 h.
(IC50 ¼ 2.7e4.8
efficient inhibitors of 5-LO product synthesis in intact cells, with 7-
fold lower IC50 values (0.46 M for 34 versus 3.2 M for 1).
Exchanging only one of the isopropyl groups at the methine bridges
of 34 by isobutyl, yielding 9, only a small loss of potency (against
mPGES-1 and 5-LO) was apparent.
Many mPGES-1 and 5-LO inhibitors are lipophilic acidic mol-
ecules [6] and it appeared possible that the two vinylogous acids
might be structural determinants for bioactivity. Thus, the mono-
cyclized analogues 4 and 52 (type E) of 1 and 7, respectively, with
only one acidic proton as well as the fully cyclized analogue of 1,
namely compound 53 (type F, no acidic proton) were investigated
(Table 2). Whereas the mono-cyclized 4 and 52 with one acidic
m
M). Of interest, these compounds were highly
and 6.4
m
M) whereas inhibition of 5-LO in cell-free and cell-based
M) (Table 2).
assays remained (IC50 in the range of 1.6e6.2
m
m
m
To investigate the necessity of the syncarpic acid moieties in the
myrtucommulones for bioactivity, we replaced both syncarpic acids
by the structurally related dimedone residues. The respective
dimedone analogue of parental 1, that is, compound 37 was about
two-fold less potent against mPGES-1 and 5-LO (cell-free and cell-
based) than 1. Again, the slim n-pentyl as acyl substituent in 39 and
41 and also methyl in 37 governed inhibitory efficiency against
mPGES-1 (IC50 in the range of 0.57e1.1
5-LO (IC50 of 41 ¼ 2.9 M) (Table 3). In contrast, the phenyl-
substituted analogue 40 showed decreased potency against
M), whereas 5-LO inhibitory efficiency was
mM) and partly also against
m
mPGES-1 (IC50 ¼ 3.5
m
proton still inhibited mPGES-1 (IC50 ¼ 0.49 and 0.61
same range as 1 and 7, the non-acidic, fully cyclized 53 was
completely inactive up to 10 M and also the 5-LO inhibitory
m
M) in the
hardly affected in the cell-free assay but potency was lost in intact
cells. Interestingly, also the triple Michael adduct 54, where the acyl
moiety is replaced by a third isopropyl-substituted methine-
bridged dimedone, inhibited mPGES-1 with IC50 ¼ 0.68 equally
well as the related dimedone analogues 37e41, and was even more
m
activity was lost. Thus, at least one vinylogous acid is required to
obtain pronounced activity, but obviously, the altered rigidity and
polarity of 4 compared to 1 also influences the bioactivity. Of
interest, compounds 4 and 52 lost potency against 5-LO in cell-
free assays but gained potency in intact cells, as compared to 1
and 7. The reason for this is unclear and might be due to inter-
ference with 5-LO activation processes such as interaction with
FLAP, 5-LO kinases, Ca2þ mobilization, intracellular redox status
etc. [18,29].
Next, we investigated so-called semi-myrtucommulone de-
rivatives with only one syncarpic acid moiety. Compound 22 with
isopropyl substituent at the acyl moiety and at the methine bridge,
and 50 with isobutyl groups in these positions, as well as 20
with isopropyl at the methine bridge and n-pentyl at the acyl
group were surprisingly still active against mPGES-1
potent against 5-LO (IC50 ¼ 2.8
mM).
Finally, the syncarpic acids were replaced by more extended
and more lipophilic indandione residues, keeping the acidic
property as vinylogous acids, yielding compounds 42e49 (ana-
logues type A3, Fig. 2, Table 4) that structurally vary again in the
acyl residue (isopropyl, isobutyl, n-pentyl, phenyl) or in the
methine bridge substitution (isopropyl, isobutyl or phenyl).
Compared to 1, the potency of the indandione analogue 42 was 3-
fold increased against mPGES-1. The phenyl-substituted acyl de-
rivative 45 was still more potent than 1, but slightly less active than
42, because as in the case for the phenyl-substituted myrtu-
commulone derivative 33, the bulky phenyl is rather detrimental
versus isopropyl. As expected, the n-pentyl-substituted 43 was
(IC50 ¼ 0.39e0.82
m
M) with even slightly improved potency
highly potent against mPGES-1 yielding an IC50 value of 0.08 mM,
being the most potent compound among all synthesized de-
rivatives in this study. In line with these SARs, the isobutyl-
than 1. Again, 20 that was essentially inactive against the 5-LO
enzyme efficiently suppressed 5-LO product formation in intact
cells (IC50 ¼ 1.6
m
M). As expected, the non-acidic cyclized com-
substituted acyl 44 was also quite efficient (IC50 ¼ 0.18
mM). Of
pounds 2 and 51 of the corresponding acidic analogues 22 and 20,
respectively, significantly lost potency against mPGES-1 (IC50 ¼ 7.9
interest, 43, 44 and 45 with extended n-pentyl, isobutyl and phenyl
residues were also more potent against 5-LO in cell-free assays

K. Wiechmann et al. / European Journal of Medicinal Chemistry 101 (2015) 133e149
139
Analogues Type A1
Analogues Type A2
Analogues Type A3
R3
O
R3
O
R3
O
OH
HO
HO
OH
O
OHHO
OHHO
R2
O
OHHO
OHHO
R2
R2
R2
R2
OH
R2
O
OH
O
O
OH
O
O
O
R2
R3
R2
R3
R2
R3
No.
1
No.
No.
37
iPr iPr
42
iPr iPr
iPr Me
iPr iPr
iPr nPent 43
iPr nPent
iPr Ph
7
38
iPr iBu
iPr Ph
Ph iPr
44
45
46
33
iPr nPent 39
iPr Ph 40
iBu nPent 34
iBu iPr
iBu iBu
35
36
iBu nPent 41
Ph nPent 47
Ph iBu
Ph Ph
48
49
Analogues Type B
Analogues Type C
Analogues Type D
R3
O
R3
O
R3
O
O
OHHO
R2
OHHO
R4
O
O
OHHO
OH
O
O
OH
O
OH
O
R2
R2
OH
O
OH
O
R2
R3
R4
R2 R3
iPr iPr
R2
R3
No.
No.
No.
iPr nPent iBu
9
2
iPr iPr
22
iPr nPent 51
iPr nPent 20
iBu iBu 50
Analogues Type E
Triple Michael adduct
Analogues Type F
R3
O
R3
O
O
O
O
OHHO
R2
O
O
O
O
O
OH
R2
OH
O
HO
OH HO
O
R2
OH
R2
O
O
R2
R3
OH
OH
O
R2
iPr iPr
iPr nPent 52
R3
No.
53
No.
O
iPr iPr
4
54
Fig. 3. Analogues and naturally occurring myrtucommulones tested for inhibition of mPGES-1 and 5-LO-inhibition, and for induction of apoptosis.
(IC50 ¼ 1.7, 3.1 and 6.9
substituted 42 (IC50 ¼ 20.0
values were in the range of 1.3e3.2
in the methine bridges were replaced by phenyl then the resulting
analogues with isopropyl (46), isobutyl (48), n-pentyl (47) or
phenyl (49) as acyl substituent efficiently inhibited mPGES-1 with
m
M, respectively) as compared to isopropyl-
M) and also in intact cells the IC50
M. When the isopropyl groups
42e44. Similarly, 5-LO activity was strongly suppressed by 46e49
in the cell free assay (IC50 ¼ 0.46e3.0 M) and in intact cells
(IC50 ¼ 0.69e2.1 M.
Of interest, the n-pentyl derivative 47 was the most potent 5-LO
M, for isolated and cellular 5-LO,
m
m
m
m
inhibitor (IC50 ¼ 0.46 and 0.69
m
respectively). Therefore, substitution of the methine bridges with
the bulky phenyl residues improves 5-LO inhibition versus
IC50 values in the close range of 0.43e0.81 mM but less potent than

140
K. Wiechmann et al. / European Journal of Medicinal Chemistry 101 (2015) 133e149
Table 1
Inhibition of mPGES-1, 5-LO and induction of cell death by natural myrtucommulones and analogues type A1. Data are expressed as means S.E.M. of single determinations
obtained in three to four independent experiments.
Cmpd
mPGES-1 IC50
[
mM]
5-LO
IC50
Cell death induction EC50 [mM]
[mM] (% remaining activity at 10 mM)
Cell-free
Cell-based
1a
7
33
34
35
36
1.0
0.60
5.0 0.9
0.24 0.03
0.29 0.00
0.22 0.02
15.0
7.5
>10 (82.9 7.7)
3.5 0.3
4.8 0.4
2.7 1.2
3.2
3.1
2.4
2.3 0.1
>30
23.5 0.7
12.2 1.3
19.2 1.4
>10 (69.7 7.9)
0.46 0.06
0.97 0.20
0.66 0.24
a
Synthetic 1 and isolated 1 showed in all respects the same pharmacological activities [21].
O
O
a
O
O
OHHO
OHHO
O
O
O
O
OH
O
O
OH
O
O
1
53
O
O
OH
HO
HO
OH
a
O
O
OH
O
O
O
O
42
55
Scheme 5. Cyclization to bis pyrane derivatives for structure elucidation. Only representative examples are shown. Reagents and conditions: a) pTsOH, benzene, reflux, 30 min,
quant.
IC50 ¼ 0.46
mM, exhibiting 12.5-fold and 33-fold improved potency
over parental 1, respectively.
2.4. SAR for induction of cell death of leukemic Jurkat cells
The same 28 analogues from above were investigated for their
ability to induce cell death of leukemic Jurkat cells (Tables 1e4). In
contrast to inhibition of mPGES-1 and 5-LO, the capacity for in-
duction of cell death was generally less pronounced by most of the
analogues as compared to parental 1. Thus, only 7, the n-pentyl
acyl derivative of 1, and 38, the dimedone analogue of 1, exhibited
lower EC50 values (2.3 and 2.0
duction of cell death was evident also for the other dimedone
M. Instead, all
m
M) versus 1 (EC50 ¼ 2.4
mM). In-
analogues 37, 39, 40 and 41 with EC50 ¼ 3.7e13.1
m
indandione analogues 42e49 (type A3) that were highly potent
against mPGES-1 and 5-LO completely failed to induce cell death
Fig. 4. X-ray structure of 55, showing ellipsoids with 50% probability.
up to 30
and 5-LO and for cytotoxicity. Also, the semi-myrtucommulones
20, 22 and 50 (type C) were inactive (EC50 > 30 M), despite
mM, suggesting divergent SARs for inhibition of mPGES-1
m
isopropyl which is actually in agreement with the potent 5-LO
inhibitory effects the myrtucommulones 34e36 carrying more
bulky isobutyl instead of isopropyl moieties at the methine bridges.
Together, the n-pentyl-substituted indandione derivative 43 is the
submicromolar IC50 values for mPGES-1. Interestingly, the corre-
sponding cyclized semi-myrtucommulones 2 and 51, which were
weak inhibitors of mPGES-1, induced cell death with EC50
values ¼ 8.1 and 7.4
myrtucommulones 4 and 52 (type E) were effective inducers of
cell death (EC50 ¼ 3.4 and 2.5 M), while the doubly cyclized
derivative 53 (type F) was less active (EC50 ¼ 17.1 M). We
mM. Along these lines the mono-cyclized
most potent mPGES-1 inhibitor with IC50 ¼ 0.08
mM and the n-
pentyl-substituted indandione derivative 47 with phenyl substi-
m
tution in the methine bridges is most potent against 5-LO with
m

K. Wiechmann et al. / European Journal of Medicinal Chemistry 101 (2015) 133e149
141
Table 2
Inhibition of mPGES-1, 5-LO and induction of cell death by analogues type B, type C, type D, type E, type F and triple Michael adduct 54. Data are expressed as means S.E.M. of
single determinations obtained in three to four independent experiments.
Cmpd
mPGES-1 IC50
[m
M]
5-LO
IC50
Cell death induction EC50 [mM]
[mM] (% remaining activity at 10 mM)
Cell-free
Cell-based
9
0.30 0.08
0.82 0.26
0.39 0.06
0.64 0.04
7.9 0.3
6.4 1.1
0.49 0.07
0.61 0.09
6.6 2.3
1.3 0.2
8.1 0.4
1.6 0.1
1.8 0.3
1.6 0.3
1.6 0.3
0.98 0.39
1.3 0.1
>10 (69.5 4.8)
>10 (83.5 3.5)
n.d.
21.9 0.9
>30
>30
22
20
50
2
51
4
52
53
54
55a
>10 (82.5 4.7)
>10 (72.8 4.9)
4.6 0.8
6.2 0.6
2.5 0.5
>10 (82.3 11.6)
>10 (66.7 6.2)
>10 (95.0 12.9)
2.8 0.6
>30
8.1 0.6
7.4 0.4
3.4 0.5
2.5 0.1
17.1 1.6
6.2 0.1
n.d.
>10 (95.1 8.0)
0.68 0.06
n.d.
n.d.
a
Compound 55 was not sufficiently soluble to be adequately tested in biological assays; n.d., not determined.
Table 3
Inhibition of mPGES-1, 5-LO and induction of cell death by dimedone analogues type A2. Data are expressed as means S.E.M. of single determinations obtained in three to
four independent experiments.
Cmpd
mPGES-1 IC50
[
m
M]
5-LO
IC50
Cell death induction EC50 [mM]
[mM] (% remaining activity at 10 mM)
Cell-free
Cell-based
37
38
39
40
41
1.1 0.3
2.1 0.8
0.57 0.18
3.5 0.5
>10 (69.3 5.1)
25.0
>10 (68.7 7.0)
8.6 0.8
8.1 1.0
13.0
2.7 0.6
>10 (66.1 3.2)
7.3 1.7
7.5 0.9
2.0 0.1
3.7 0.9
4.4 0.9
13.1 2.6
0.81 0.08
2.9 0.2
Table 4
Inhibition of mPGES-1, 5-LO and induction of cell death by indandione analogues type A3. Data are expressed as means S.E.M. of single determinations obtained in three to
four independent experiments.
Cmpd
mPGES-1 IC50
[m
M]
5-LO
IC50
Cell death induction EC50 [mM]
[mM] (% remaining activity at 10 mM)
Cell-free
Cell-based
42
43
44
45
46
47
48
49
0.30 0.03
0.08 0.01
0.18 0.02
0.68 0.04
0.81 0.06
0.43 0.04
0.65 0.17
0.48 0.03
20.0
6.7
>30
>30
>30
>30
>30
>30
>30
>30
1.7 0.3
3.1 0.9
6.9 2.1
0.73 0.03
0.46 0.08
1.2 0.4
3.0 0.6
1.5 0.3
1.3 0.4
3.2 0.6
1.4 0.4
0.69 0.17
2.1 0.3
1.8 0.3
conclude that syncarpic acid (even cyclized) and dimedone moi-
eties are beneficial for cytotoxicity but indandione is not tolerated.
It also appears that isopropyl substituent in the acyl and/or the
methine bridge as well as n-pentyl in acyl governs cytotoxicity
reflected by the low EC50 values of the respective compounds 1, 4,
7, 39, and 52.
conclude that (I) the flat aromatic indandione residues improve the
potency against both mPGES-1 and 5-LO but is not tolerated for
induction of cell death of leukemic cells, (II) the long, unbranched
n-pentyl at the acyl moiety governs cell death and inhibition of 5-
LO and mPGES-1, and is superior over isopropyl, isobutyl and
phenyl groups in this respect, (III) the substituents (isopropyl,
isobutyl or phenyl) at the methine bridge have minor impact on
the bioactivities, nevertheless showing that branching more
distant to the methine bridge leads to higher activity than con-
necting a branched group directly to the methine bridge. Taken
together, the n-pentyl-substituted indandione derivative 43 and
the n-pentyl-substituted indandione derivative 47 with phenyl
substitution in the methine bridges are most potent against
mPGES-1 and 5-LO with 12.5-fold and 33-fold improved potency
over parental 1, respectively. Moreover, these molecules lost the
cytotoxic properties conferring them high potential as anti-
inflammatory drugs.
3. Conclusion
In order to understand the SARs of myrtucommulones as in-
hibitors of mPGES-1 and 5-LO and to identify more potent de-
rivatives we here took advantage of our recently published total
synthesis of myrtucommulones [21] that offers the systematic
variation of the structural components of these acylphlor-
oglucinols. Of interest, semi-myrtucommulones lacking one
methine-bridged syncarpic acid moiety are active against 5-LO and
mPGES-1, even in the cyclized form. From our SARs analysis we

142
K. Wiechmann et al. / European Journal of Medicinal Chemistry 101 (2015) 133e149
4. Experimental section
Michael addition) were added followed by the appropriate 1,3-
dicarbonyl alkylidene (3 eq. for single Michael addition or 6 eq.
for double Michael addition). The mixture was stirred at room
temperature until the starting material was consumed. The solvent
was evaporated and the residue filtered through a 10 cm layer of
silica gel with a mixture of acetone and petroleum ether and
concentrated. The crude product was purified by flash chroma-
tography using mixtures of acetone and petroleum ether.
4.1. Compounds and chemistry
All commercially available reagents were used without further
purification. Solvents were distilled prior to use and where required
were dried by standard laboratory procedures. Flash chromatog-
raphy was performed using Merck silica gel 60 (40e63 mm). NMR
data were recorded on Bruker AV II 400 and AV 500 spectrometers.
Chemical shifts are reported in ppm. In most cases of myrtu-
commulones and analogues no significant 13C NMR spectra could
be acquired, thus none could be reported. Significant 1D and 2D-
NMR data could be obtained after cyclization and allowed the
assignment of protons to carbons and carbon multiplicities. Mul-
tiplicities of protons: b, broad; s, singlet; d, doublet; t, triplet; q,
quartet; quint, quintet; sept, septet; m, multiplet. HRMS spectra
were recorded using a combination of Dionex Ultimate 3000 RSLC
coupled with Advion Nanomate nano-ESI and the mass detector
Thermo Scientific Thermo Fisher Orbitrap. Melting points were
acquired on a Büchi apparatus and are uncorrected. The synthe-
sized myrtucommulones and analogues do not have a sharp
melting point but a melting range, due to their composition of
stereoisomers. For NMR-analysis all myrtucommulones and ana-
logues were cyclized. All compounds were >95% pure.
4.1.5. General procedure E for the cyclization of myrtucommulones
and analogues
The appropriate myrtucommulone or analogue (100 mg) was
suspended in 20 ml of benzene, 3 eq. of pTsOH were added and the
mixture was refluxed for 30 min. The reaction mixture was filtered
through a 10 cm layer of silica gel with a mixture of acetone and
petroleum ether and concentrated. The crude product was either
purified by flash chromatography or by preparative thin layer
chromatography using mixtures of acetone and petroleum ether.
4.1.5.1. Synthesis of 5-hydroxy-2,2,6,6-tetramethyl-4-(2-methyl-1-
(2,4,6-trihydroxy-3-(1-(2-hydroxy-3,3,5,5-tetramethyl-4,6-
dioxocyclohex-1-enyl)-2-methylpropyl)-5-isobutyrylphenyl)propyl)
cyclohex-4-ene-1,3-dione, MC A (1). Following general procedures B
and D, MC A (1) was obtained after flash chromatography (SiO2, PE/
acetone 3:2, Rf ¼ 0.13) as a yellow solid. Yield: 668 mg, (quant.). mp:
140e185 ꢀC. HRMS-ESI, m/z (C38H52O10): calcd. 669.3633 [MþH]þ;
4.1.1. General procedure A for the synthesis of acylphloroglucinols
(AP) [24]
found, 669.3623. 1H NMR (400 MHz, Acetone-d6)
d 15.69 (s, 1H),
A mixture of phloroglucinol (17) (100 mmol) and aluminium
chloride (2 eq.) was suspended in dichloromethane (200 ml) and 2
eq. nitromethane were added dropwise. The mixture was heated to
40e50 ꢀC until no more HCl was formed. 1.1 eq. of the appropriate
acid chloride were added dropwise and the reaction mixture
heated for further 10 min. After cooling the reaction mixture was
hydrolyzed with diluted ice-cold HCl. The organic solvents were
removed in vacuo and the aqueous phase was extracted with
diethyl ether. The organic extract was dried over magnesium sul-
phate, filtered and concentrated. The crude product was purified by
flash chromatography using mixtures of acetone and petroleum
ether (PE) as eluents.
15.29 (s, 1H), 12.99 (m, 2H), 4.23, (m, 2H), 4.09 (m, 1H), 3.16 (m, 2H),
1.36e1.09 (m, 30H), 0.86e0.67 (m, 18H). MC A (1) was cyclized
following procedure E and purified by preparative TLC (PE/acetone)
to obtain compound 53. HRMS-ESI, m/z (C38H48O8): calcd. 633.3422
[MþH]þ; found, 633.3421. 1H NMR (400 MHz, CDCl3)
d 4.53 (d,
J ¼ 3.6 Hz, 2H), 3.18 (sept, J ¼ 6.8 Hz, 1H), 1.96 (m, J1 ¼ 3.6 Hz,
J2 ¼ 6.8 Hz, 2H), 1.55 (s, 6H), 1.45 (s, 6H), 1.43 (s, 6H), 1.42 (s, 6H),
1.26 (s, 6H), 1.24 (s, 6H), 0.78 (t, J ¼ 7.2 Hz, 6H), 0.81 (t, J ¼ 7.2 Hz,
6H). 13C NMR (100 MHz, CDCl3)
d 211.77 (C), 204.27 (C), 198.56 (C),
168.75 (C), 151.93 (C), 108.63 (C), 55.85 (C), 47.53 (C), 42.68 (CH),
35.10 (CH), 32.35 (CH), 25.19 (CH3), 24.80 (CH3), 24.41 (CH3), 19.21
(CH3), 18.65 (CH3), 17.98 (CH3).
4.1.2. General procedure B for the synthesis of Michael acceptors
(MA) from syncarpic acid (15) or dimedone (24) [25e27]
4.1.5.2. Synthesis of 6,8-dihydroxy-5-isobutyryl-9-isopropyl-2,2,4,4-
tetramethyl-2H-xanthene-1,3(4H,9H)-dione,
MC
B
(2).
The 1,3-dicarbonyl compound (6 mmol) was suspended in
dichloromethane (20 ml) and 1.5 eq of the appropriate aldehyde
and 2 eq. piperidine were added. After stirring for 5 min at room
temperature 50 ml of a 1 N HCl, which was saturated with NH4Cl
were added and vigorously stirred for another 5 min. The organic
phase was separated, filtered through a 10 cm layer of silica gel with
a 1:1 mixture of acetone and PE and concentrated. The crude
product was pure enough to be used in the following reaction. The
product was taken up in 10 ml of THF and used immediately.
Compound 22 was cyclized following procedure E to obtain MC B
(2) after flash chromatography (SiO2, PE/acetone 5:1, Rf ¼ 0.25) as a
faint yellow solid. Yield: 680 mg, (82%). mp: 90e95 ꢀC. HRMS-ESI,
m/z (C24H30O6): calcd. 415.2125 [MþH]þ; found, 415.2105. 1H
NMR (400 MHz, CDCl3)
d
6.30 (s, 1H), 4.35 (d, J ¼ 3.6 Hz, 1H), 1.91
(dsept, J1 ¼ 3.6 Hz, J2 ¼ 6.8 Hz, 1H), 0.79 (d, J ¼ 6.8 Hz, 3H), 0.83 (d,
J ¼ 6.8 Hz, 3H), 3.90 (sept, J ¼ 6.8 Hz, 1H), 1.13 (t, J ¼ 6.8 Hz, 3H) 1.26
(t, J ¼ 6.8 Hz, 3H), 1.39 (s, 3H), 1.45 (s, 3H), 1.62 (s, 3H), 1.43 (s, 3H),
13.35 (s, 1H), 6.58 (s, 1H). 13C NMR (100 MHz, CDCl3)
d 211.90 (C),
209.08 (C), 198.65 (C), 168.33 (C), 164.87 (C), 159.61 (C), 153.61 (C),
112.24 (C), 104.11 (C), 103.77 (C), 100.83 (CH), 56.39 (C), 47.46 (C),
39.85 (CH), 34.97 (CH), 31.66 (CH), 25.29 (CH3), 25.11 (CH3), 25.09
(CH3), 24.30 (CH3), 21.00 (CH3), 19.00 (CH3), 18.86 (CH3), 17.84
(CH3).
4.1.3. General procedure C for the synthesis of Michael acceptors
(MA) from 1,3-indandione (25) [33]
A mixture of 1,3-indandione (1 eq.), the appropriate aldehyde
(1.5 eq.), 0.2 eq. of ammonium acetate and 0.5 eq of acetic acid in
chloroform (c ¼ 0.5 mol Lꢁ1) were heated in a reverse DeaneStark
apparatus until the separation of water ceased. The solvent was
evaporated and the crude product was recrystallized from meth-
anol or ethanol/water.
4.1.5.3. Synthesis of (6,8-dihydroxy-7-(1-(2-hydroxy-3,3,5,5-
tetramethyl-4,6-dioxocyclohex-1-enyl)-2-methylpropyl)-5-
isobutyryl-9-isopropyl-2,2,4,4-tetramethyl-2H-xanthene-1,3(4H,9H)-
dione, MC C (4). Following general procedures B and D MC C (4)
was obtained after flash chromatography (SiO2, PE/acetone 3:2,
4.1.4. General procedure D for the alkylation of acylphloroglucinol
with Michael acceptors (MA) from general procedure B or C
The appropriate acylphloroglucinol (1 mmol) was dissolved in
10 ml THF, NaH (1 eq. for single Michael addition or 2 eq. for double
Rf
¼
0.15) as a yellow solid. Yield: 650 mg, (quant.). mp:
180e230 ꢀC. HRMS-ESI, m/z (C38H51O9): calcd. 651.3527 [MþH]þ;
found, 651.3523. 1H NMR (400 MHz, Acetone-d6)
d 13.23e12.10 (m,

K. Wiechmann et al. / European Journal of Medicinal Chemistry 101 (2015) 133e149
143
2H), 4.36 - 4.29 (m, 1H), 4.01 - 3.92 (m, 1H), 3.56 - 3.44 (m, 1H), 3.37
(CH3), 13.04 (CH3).
- 3.30 (m, 1H), 1.98e1.88 (m, 1H), 1.49 - 1.45 (m, 3H), 1.391 (s, 3H),
1.34e-1.28 (m, 12H), 1.21e1.10 (m, 12H), 0.86 - 0.83 (m, 3H), 0.82 -
0.79 (m, 3H), 0.76 - 0.66 (m, 6H). 13C NMR (100 MHz, Acetone-d6)
4.1.5.6. Synthesis of 4-(1-(3-hexanoyl-2,4,6-trihydroxyphenyl)-2-
methylpropyl)-5-hydroxy-2,2,6,6-tetramethylcyclohex-4-ene-1,3-
dione (20). Following general procedures B and D compound 20
was obtained after flash chromatography (SiO2, PE/acetone 1:1,
Rf ¼ 0.33) as a yellow solid. Yield: 2 g, (87%). mp: 130e160 ꢀC. ESI-
MS, m/z (C26H36O7): calcd. 461.25 [MþH]þ; found, 461.41. 1H NMR
d
207.40, 207.00,198.84,113.18,113.15, 112.93,112.788, 101.89, 57.19,
57.17, 49.12, 49.08, 44.26, 43.67, 43.12, 42.73, 36.83, 36.76, 36.36,
34.31, 34.01, 33.84, 27.45, 27.42, 27.32, 27.24, 27.19, 27.16, 27.10,
27.00, 26.88, 26.81, 26.75, 26.71, 26.64, 26.55, 26.45, 26.39, 26.24,
26.21, 26.17, 26.03, 25.93, 25.88, 23.67, 23.52, 20.65, 20.50, 20.45,
20.27, 20.00, 19.92, 19.87, 19.79, 19.70, 19.34.
(400 MHz, Acetone-d6)
d
14.27e13.66 (m, 2H), 5.70 (d, J ¼ 3.2 Hz,
1H), 3.83 (t, J ¼ 10.4 Hz, 1H), 3.34e3.02 (m, 6H), 1.65 (d p,
J1 ¼ 3.2 Hz, J2 ¼ 7.2 Hz, 2H), 1.35 (m, 3H), 1.16e1.20 (m, 12H), 0.92 -
0.89 (m, 4H), 0.82 (dd, J1 ¼ 2.8 Hz, J1 ¼ 6.4 Hz, 4H), 0.69 (dd, J ¼ 6.4,
4.1.5.4. Synthesis of 4-(1-(3-hexanoyl-2,4,6-trihydroxy-5-(1-(2-
hydroxy-3,3,5,5-tetramethyl-4,6-dioxocyclohex-1-enyl)-2-
methylpropyl)phenyl)-2-methylpropyl)-5-hydroxy-2,2,6,6-
tetramethylcyclohex-4-ene-1,3-dione, MC F (7). Following general
procedures B and D MC F (7) was obtained after flash chromatog-
raphy (SiO2, PE/acetone 3:2, Rf ¼ 0.14) as a yellow solid. Yield:
678 mg, (quant.). mp: 150e190 ꢀC. HRMS-ESI, m/z (C38H52O10):
calcd. 679.3946 [MþH]þ; found, 679.3937. 1H NMR (400 MHz,
11.2 Hz, 4H). 13C NMR (100 MHz, Acetone-d6)
d 208.05, 207.70,
207.49, 207.10, 98.26, 97.31, 53.35, 45.71, 45.57, 43.48, 43.31, 33.63,
27.55, 27.19, 26.84, 26.68, 24.36, 23.78, 23.71, 16.58, 15.31.
4.1.5.7. Synthesis of 5-hydroxy-2,2,6,6-tetramethyl-4-(2-methyl-1-
(2,4,6-trihydroxy-3-isobutyrylphenyl)propyl)cyclohex-4-ene-1,3-
dione (22). Following general procedures B and D compound 22
was obtained after flash chromatography (SiO2, PE/acetone 3:2,
Rf ¼ 0.25) as a yellow solid. Yield: 1.9 g, (88%). mp: 170e205 ꢀC.
HRMS-ESI, m/z (C24H32O7): calcd. 433.2220 [MþH]þ; found,
Acetone-d6) d 15.73 (s, 1H), 15.34 (s, 1H), 14.22e13.68 (m, 1H), 12.99
- 12.91 (m, 2H), 4.23e4.20 (m, 2H), 3.17e3.12 (m, 2H), 3.10 - 3.07
(m, 3H), 1.66e1.65 (m, 2H), 1.36e1.35 (m, 12H), 1.27e1.25 (m, 12H),
1.21 - 1.17 (m, 6H), 0.86 - 0.67 (m, 18H), 0.92e0.88 (m, 3H), 0.86 -
0.84 (m, 6H), 0.81e0.79 (m, 3H), 0.68e0.67 (m, 3H). 13C NMR
433.2216. 1H NMR (400 MHz, Acetone-d6)
d 14.34e14.38 (m, 2H),
5.71 (s, 1H), 4.25e4.11 (m, 1H), 3.87e3.81 (m, 1H), 3.33 - 3.18 (m,
1H), 1.25 (bs, 12H), 1.15e1.08 (m, 6H), 0.84e0.82 (m, 3H), 0.73e0.86
(100 MHz, Acetone-d6)
d 207.36, 206.98, 162.85, 115.95, 115.45,
112.32, 112.22, 67.09, 55.52, 55.39, 51.24, 45.61, 41.37, 33.62, 28.30,
28.10, 26.96, 26.91, 26.88, 26.73, 26.45, 26.26, 26.03, 25.96, 25.86,
24.36, 23.63, 23.38, 23.34, 23.15, 16.59, 15.32. MC F (7) was cyclized
following procedure E and purified by preparative TLC (PE/
acetone). HRMS-ESI, m/z (C40H52O8): calcd. 661.3735 [MþH]þ;
(m, 3H). 13C NMR (100 MHz, Acetone-d6)
d 212.18, 196.18, 113.35,
101.69, 98.48, 97.56, 67.10, 43.27, 40.28, 40.21, 27.60, 26.76, 23.71,
21.39, 21.10, 20.66, 20.36.
4.1.5.8. Synthesis of 4-(1-(3-benzoyl-2,4,6-trihydroxy-5-(1-(2-
hydroxy-3,3,5,5-tetramethyl-4,6-dioxocyclohex-1-enyl)-2-
methylpropyl)phenyl)-2-methylpropyl)-5-hydroxy-2,2,6,6-
tetramethylcyclohex-4-ene-1,3-dione (33). Following general pro-
cedures B and D compound 33 was obtained after flash chroma-
tography (SiO2, PE/acetone 3:2, Rf ¼ 0.19) as a yellow solid. Yield:
703 mg, (quant.). mp: 140e185 ꢀC. HRMS-ESI, m/z (C41H50O10):
calcd. 703.3476 [MþH]þ; found, 703.3475. 1H NMR (400 MHz,
found, 661.3729. 1H NMR (400 MHz, CDCl3)
d 7.56 (s, 1H), 4.53 (d,
J ¼ 3.6 Hz, 2H),1.95 (m, 2H), 0.88 (m, 6H), 0.79 (m, 6H), 2.89 (m,1H),
1.76 (m, 6H), 1.36 (m, 6H), 1.34 (m, 6H), 0.91 (m, 6H), 1.46 (s, 6H),
1.54 (s, 6H) 1.57 (s, 6H) 1.42 (s, 6H). 13C NMR (100 MHz, CDCl3)
d
211.93 (C), 200.80 (C), 198.66 (C), 169.05 (C), 152.21 (C), 147.68 (C),
111.12 (C), 111.04 (C), 108.67 (C), 56.07 (C), 47.67 (C), 45.85 (CH2),
35.21 (CH), 32.49 (CH), 31.57 (CH2), 25.41 (CH3), 24.39 (CH3), 25.09
(CH3), 24.89 (CH3), 24.39 (CH2), 19.29 (CH3), 18.76 (CH3), 14.03
(CH3).
Acetone-d6)
d
11.31e10.47 (m, 2H), 7.72 (s, 1H), 7.64 (d, J ¼ 7.1 Hz,
1H), 7.50e7.41 (m, 1H), 7.40e7.29 (m, 2H), 4.15e3.87 (m, 2H),
3.34e3.17 (m, 1H), 3.16e2.99 (m, 1H), 1.39 (s, 6H), 1.32e1.23 (m,
12H), 1.22e1.16 (m, 6H), 0.90e0.78 (m, 9H), 0.77e0.64 (m, 3H).
Compound 33 was cyclized following procedure E and purified by
preparative TLC (PE/acetone). HRMS-ESI, m/z (C41H46O8): calcd.
667.3265 [MþH]þ; found, 667.3242. 1H NMR (500 MHz, CDCl3)
4.1.5.5. Synthesis of 4-(1-(3-hexanoyl-2,4,6-trihydroxy-5-(1-(2-
hydroxy-3,3,5,5-tetramethyl-4,6-dioxocyclohex-1-enyl)-2-
methylpropyl)phenyl)-3-methylbutyl)-5-hydroxy-2,2,6,6-
tetramethylcyclohex-4-ene-1,3-dione, MC H (9). Following general
procedures B and D MC H (9) was obtained after flash chroma-
tography (SiO2, PE/acetone 3:2, Rf ¼ 0.14) as a yellow solid. Yield:
682 mg, (96%). mp: 170e210 ꢀC. HRMS-ESI, m/z (C41H58O10): calcd.
711.4102 [MþH]þ; found, 711.4105. 1H NMR (400 MHz, Acetone-d6)
d
7.93e7.81 (m, 2H), 7.60 (tdd, J1 ¼ 7.2 Hz, J2 ¼ 6.4 Hz, J3 ¼ 1.3 Hz,
1H), 7.48 (qd, J1 ¼ 8.0 Hz, J2 ¼ 7.5 Hz, J3 ¼ 1.0 Hz, 2H), 4.68e4.53 (m,
2H), 2.06e1.94 (m, 2H), 1.41 (s, 6H), 1.38 (s, 6H), 1.35 (s, 6H), 1.25 (s,
6H), 0.90 (d, J ¼ 6.8 Hz, 6H), 0.86e0.77 (m, 6H). 13C NMR (125 MHz,
d
15.32e13.17 (m, 3H), 4.8 (m, 1H), 4.21 (m, 1H), 3.10 (m, 3H), 2.40
CDCl3) d 211.94 (C), 198.87 (C), 191.80 (C), 169.32 (C), 153.30 (C),
(m, 1H), 1.77e1.42 (m, 3H), 1.36e1.19 (m, 30H), 0.90e0.69 (m, 16H).
MC H (9) was cyclized following procedure E and purified by pre-
parative TLC (PE/acetone). HRMS-ESI, m/z (C41H54O8): calcd.
675.3891 [MþH]þ; found, 675.3907. 1H NMR (500 MHz, CDCl3)
148.77 (C), 138.66 (C), 133.90 (CH), 129.37 (CH), 128.94 (CH), 111.24
(C), 110.94 (C), 108.68 (C), 56.08 (C), 47.60 (C), 35.34 (CH), 32.62
(CH), 25.18 (CH3), 24.94 (CH3), 24.72 (CH3), 24.40 (CH3), 19.37 (CH3),
19.06 (CH3).
d
6.69 (bs, 1H), 4.46 (d, J ¼ 2.7 Hz, 1H), 4.37 (t, J ¼ 6.0 Hz, 1H), 2.88
(m, 2H), 1.93 (td, J1 ¼ 6.9 Hz, J2 ¼ 3.8 Hz, 1H), 1.73 (m, 2H), 1.56 (s,
3H), 1.54 (s, 3H), 1.45 (s, 6H), 1.43 (s, 3H), 1.42 (s, 3H), 1.41 (m, 1H),
1.40 (s, 3H), 1.36 (m, 6H), 0.93 (d, J ¼ 6.5 Hz, 1H), 0.90 (dd,
J1 ¼ 7.5 Hz, J2 ¼ 6.5 Hz, 6H), 0.90 (m, 3H), 0.84 (m, 1H), 0.78 (d,
4.1.5.9. Synthesis of 4-(1-(3-hexanoyl-2,4,6-trihydroxy-5-(1-(2-
hydroxy-3,3,5,5-tetramethyl-4,6-dioxocyclohex-1-enyl)-3-
methylbutyl)phenyl)-3-methylbutyl)-5-hydroxy-2,2,6,6-
tetramethylcyclohex-4-ene-1,3-dione (34). Following general pro-
cedures B and D compound 33 was obtained after flash chroma-
tography (SiO2, PE/acetone 3:2, Rf ¼ 0.14) as a yellow solid. Yield:
725 mg, (quant.). mp: 160e215 ꢀC. HRMS-ESI, m/z (C42H60O10):
calcd. 725.4259 [MþH]þ; found, 725.4252. 1H NMR (400 MHz,
J ¼ 6.9 Hz, 3H). 13C NMR (125 MHz, CDCl3)
d 211.88 (C), 211.79 (C),
200.92 (C), 198.68 (C), 198.07 (C), 169.11 (C), 167.68 (C), 151.75 (C),
147.71 (C), 147.00 (C), 113.64 (C), 111.56 (C), 111.05 (C), 110.10 (C),
108.27 (C), 56.06 (C), 47.67 (C), 47.48 (C), 47.11 (CH2), 45.87 (CH2),
35.59 (CH), 35.24 (CH), 31.74 (CH2), 25.76 (CH), 25.16 (CH), 25.32
(CH3), 25.04 (CH3), 24.98 (CH3), 24.89 (CH3), 24.68 (CH3), 24.54
(CH3), 24.03 (CH2), 23.26 (CH3), 22.67 (CH2), 19.18 (CH3), 18.96
Acetone-d6)
d 13.41 (s, 1H), 12.18e11.27 (m, 1H), 11.05e10.21 (m,
1H), 4.69e4.23 (m, 2H), 3.36e3.05 (m, 1H), 2.37e2.01 (m, 4H),
1.96e1.75 (m, 4H), 1.75e1.61 (m, 24H), 1.58e1.10 (m, 18H),

144
K. Wiechmann et al. / European Journal of Medicinal Chemistry 101 (2015) 133e149
1.04e0.70 (m, 12H). Compound 34 was cyclized following proce-
dure E and purified by preparative TLC (PE/acetone). HRMS-ESI, m/z
(C42H56O8): calcd. 689.4048 [MþH]þ; found, 689.4047. 1H NMR
as a greenish yellow solid. Yield: 425 mg, (76%). mp: 90e130 ꢀC.
HRMS-ESI, m/z (C32H44O8): calcd. 557.3109 [MþH]þ; found,
557.3108. 1H NMR (400 MHz, Acetone-d6)
d 3.90e3.61 (m, 2H), 3.06
(500 MHz, CDCl3)
d
4.34e4.26 (m, 2H), 2.99e2.75 (m, 2H),
(m, 2H), 2.74 (m, 3H), 2.44e2.15 (m, 8H), 1.26e1.15 (m, 4H),
1.15e1.00 (m, 12H), 0.96e0.58 (m, 12H). Compound 37 was cyclized
following procedure E and purified by preparative TLC (PE/
acetone). HRMS-ESI, m/z (C32H40O6): calcd. 521.2898 [MþH]þ;
1.80e1.70 (m, 2H), 1.57e1.32 (m, 10H), 1.52 (s, 6H), 1.42 (s, 6H), 1.41
(s, 6H), 1.39 (s, 6H), 0.93e0.90 (m, 2H), 0.91e0.89 (m, 6H),
0.88e0.85 (m, 6H). 13C NMR (125 MHz, CDCl3)
d 211.76 (C), 200.98
(C), 198.97 (C), 167.77 (C), 151.25 (C), 146.98 (C), 113.43 (C), 112.04
(C), 110.55 (C), 56.06 (C), 47.49 (C), 46.97 (CH2), 45.89 (CH2), 31.72
(CH2), 25.74 (CH), 25.17 (CH), 24.91 (CH3), 24.89 (CH3), 24.55 (CH3),
24.65 (CH3), 23.92 (CH2), 23.37 (CH3), 23.32 (CH3), 22.67 (CH2),
14.04 (CH3).
found, 521.2897. 1H NMR (500 MHz, CDCl3)
d 13.16 (s, 1H), 4.26 (dd,
J1 ¼3.5 Hz, J2 ¼ 1.1 Hz,1H), 4.18 (dd, J1 ¼4.2 Hz, J2 ¼ 0.8 Hz,1H), 2.74
(s, 3H), 2.63e2.51 (m, 2H), 2.48e2.37 (m, 2H), 2.37e2.26 (m, 4H),
1.77 (pd, J1 ¼6.9 Hz, J2 ¼ 4.3 Hz,1H),1.95 (pd, J1 ¼6.9 Hz, J2 ¼ 3.6 Hz,
1H), 1.21 (s, 3H), 1.18 (s, 3H), 1.17 (s, 3H), 1.13 (s, 3H), 0.77e0.74 (m,
9H), 0.70 (d, J ¼ 6.8 Hz, 3H). 13C NMR (125 MHz, CDCl3)
d 202.98 (C),
4.1.5.10. Synthesis of 5-hydroxy-2,2,6,6-tetramethyl-4-(3-methyl-1-
(2,4,6-trihydroxy-3-(1-(2-hydroxy-3,3,5,5-tetramethyl-4,6-
dioxocyclohex-1-enyl)-3-methylbutyl)-5-isobutyrylphenyl)butyl)
cyclohex-4-ene-1,3-dione (35). Following general procedures B and
D compound 34 was obtained after flash chromatography (SiO2, PE/
acetone 3:2, Rf ¼ 0.16) as a yellow solid. Yield: 697 mg, (quant.). mp:
140e180 ꢀC. HRMS-ESI, m/z (C40H56O10): calcd. 697.3946 [MþH]þ;
197.13 (C), 165.90 (C), 165.87 (C), 161.32 (C), 153.87 (C), 151.74 (C),
114.13 (C), 113.52 (C), 110.58 (C), 107.18 (C), 104.12 (C), 51.12 (CH2),
51.06 (CH2), 41.47 (CH2), 41.34 (CH2), 35.38 (CH), 34.72 (CH), 33.56
(CH3), 32.13 (C), 31.47 (CH), 31.22 (CH), 30.20 (CH3), 29.89 (CH3),
27.28 (CH3), 27.23 (CH3), 20.05 (CH3), 19.51 (CH3), 19.48 (CH3), 18.96
(CH3).
found, 697.3938. 1H NMR (400 MHz, Acetone-d6)
d
4.20e4.06 (m,
4.1.5.13. Synthesis of 3-hydroxy-5,5-dimethyl-2-(2-methyl-1-(2,4,6-
trihydroxy-3-(1-(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-enyl)-2-
2H), 2.39e2.19 (m, 4H), 1.73e1.57 (m, 4H), 1.48e1.35 (m, 6H),
1.34e1.29 (m, 6H), 1.29e1.17 (m, 18H), 1.15e1.07 (m, 6H), 0.95 (d,
J ¼ 6.6 Hz, 6H), 0.89e0.75 (m, 12H). Compound 35 was cyclized
following procedure E and purified by preparative TLC (PE/
acetone). HRMS-ESI, m/z (C40H52O8): calcd. 661.3735 [MþH]þ;
methylpropyl)-5-isobutyrylphenyl)propyl)cyclohex-2-enone
(38).
Following general procedures B and D compound 38 was obtained
after flash chromatography (SiO2, PE/acetone 10:1, Rf ¼ 0.16) as a
yellow solid. Yield: 474 mg, (81%). mp: 80e125 ꢀC. HRMS-ESI, m/z
(C34H48O8): calcd. 585.3422 [MþH]þ; found, 585.3413. 1H NMR
found, 661.3727. 1H NMR (500 MHz, CDCl3)
d 6.09 (bs, 1H), 4.33 (t,
J ¼ 5.7 Hz, 2H), 3.17 (sept, J ¼ 6.9 Hz,1H),1.51 (s, 6H),1.48 … 1.38 (m,
(400 MHz, Acetone-d6) d 13.12 (s, 1H), 12.02 (s, 1H), 9.98 (s, 1H),
4H), 1.42 (s, 6H), 1.41 (s, 6H), 1.40 (s, 6H), 1.27 (d, J ¼ 6.9 Hz, 3H), 1.24
4.18e4.05 (m, 1H), 3.96e3.79 (m, 2H), 3.00 (m, 2H), 2.39 (m, 9H),
1.25e1.01 (m, 21H), 0.95e0.85 (m, 6H), 0.80 (m, 3H), 0.72 (m, 3H),
0.63 (m, 3H). Compound 38 was cyclized following procedure E and
purified by preparative TLC (PE/acetone). HRMS-ESI, m/z
(C34H44O6): calcd. 549.3211 [MþH]þ; found, 549.3202. 1H NMR
(d, J ¼ 6.9 Hz, 3H), 0.89 (d, J ¼ 5.8 Hz, 6H), 0.84 (d, J ¼ 5.9 Hz, 6H). 13
C
NMR (125 MHz, CDCl3)
d 211.77 (C), 204.68 (C), 198.11 (C), 167.31
(C), 151.37 (C), 147.00 (C), 113.35 (C), 110.41 (C), 110.21 (C), 56.02 (C),
47.50 (C), 46.74 (CH2), 42.83 (CH), 25.79 (CH), 25.16 (CH), 24.94
(CH3), 24.65 (CH3), 24.58 (CH3), 24.57 (CH3), 23.42 (CH3), 18.19
(CH3), 18.07 (CH3).
(500 MHz, CDCl3)
d
7.64 (s, 1H), 4.33 (t, J ¼ 6.0 Hz, 2H), 3.17 (sept,
J ¼ 6.9 Hz, 1H), 1.48 (m, 6H), 1.44 (s, 6H), 1.41 (m, 4H), 1.38 (m, 4H),
1.27 (d, J ¼ 6.9 Hz, 3H), 1.24 (d, J ¼ 6.9 Hz, 3H), 1.01 (d, J ¼ 6.6 Hz,
4.1.5.11. Synthesis of 5-hydroxy-2,2,6,6-tetramethyl-4-(3-methyl-1-
(2,4,6-trihydroxy-3-(1-(2-hydroxy-3,3,5,5-tetramethyl-4,6-
dioxocyclohex-1-enyl)-3-methylbutyl)-5-(3-methylbutanoyl)phenyl)
butyl)cyclohex-4-ene-1,3-dione (36). Following general procedures
B and D compound 35 was obtained after flash chromatography
(SiO2, PE/acetone 2:1, Rf ¼ 0.12) as a yellow solid. Yield: 711 mg,
(quant.). mp: 150e200 ꢀC. HRMS-ESI, m/z (C41H58O10): calcd.
711.4102 [MþH]þ; found, 711.4108. 1H NMR (400 MHz, Acetone-d6)
6H), 0.87 (d, J ¼ 6.4 Hz, 6H). 13C NMR (125 MHz, CDCl3)
d 211.94 (C),
198.98 (C), 167.73 (C), 164.39 (C), 160.00 (C), 153.18 (C), 114.8 (C),
106.20 (C), 105.64 (C), 100.36 (C), 56.26 (C), 47.43 (C), 47.04 (CH2),
42.83 (CH), 25.18 (CH), 24.63 (CH), 23.57 (CH3), 23.25 (CH3), 22.78
(CH3), 23.04 (CH3), 18.19 (CH3), 18.07 (CH3).
4.1.5.14. Synthesis of 2-(1-(3-hexanoyl-2,4,6-trihydroxy-5-(1-(2-
hydroxy-4,4-dimethyl-6-oxocyclohex-1-enyl)-2-methylpropyl)
phenyl)-2-methylpropyl)-3-hydroxy-5,5-dimethylcyclohex-2-enone
(39). Following general procedures B and D compound 39 was
obtained after flash chromatography (SiO2, PE/acetone 10:1,
Rf ¼ 0.23) as a yellow solid. Yield: 490 mg, (80%). mp: 90e155 ꢀC.
HRMS-ESI, m/z (C36H52O8): calcd. 613.3735 [MþH]þ; found,
d
11.98e11.71 (m, 1H), 10.87e10.45 (m, 2H), 4.58e4.30 (m, 2H),
3.18e3.14 (m, 1H), 3.12e2.96 (m, 1H), 2.34e2.15 (m, 1H), 2.12e1.96
(m, 1H), 1.96e1.78 (m, 1H), 1.56e1.23 (m, 24H), 1.05e0.93 (m, 6H),
0.91e0.71 (m, 12H). Compound 37 was cyclized following proce-
dure E and purified by preparative TLC (PE/acetone). HRMS-ESI, m/z
(C41H54O8): calcd. 675.3891 [MþH]þ; found, 675.3890. 1H NMR
613.3732. 1H NMR (400 MHz, Acetone-d6)
d 3.93e3.61 (m, 1H),
(400 MHz, CDCl3)
d
4.38 (t, J ¼ 8.6 Hz, 2H), 2.93e2.71 (m, 2H), 2.30
3.28e2.93 (m, 2H), 2.45e2.13 (m, 4H), 1.67 (m, 1H), 1.42e1.30 (m,
2H), 1.15e1.00 (m, 7H), 0.96e0.55 (m, 8H). Compound 39 was
cyclized following procedure E and purified by preparative TLC (PE/
acetone). HRMS-ESI, m/z (C36H48O6): calcd. 577.3524 [MþH]þ;
(dsept, J1 ¼13.4 Hz, J2 ¼ 6.8 Hz, 2H), 1.60e1.35 (m, 6H), 1.53 (s, 6H),
1.44 (s, 6H), 1.42 (s, 6H), 1.40 (s, 6H), 0.91e0.86 (m, 6H), 0.86e0.80
(m, 6H), 1.06 (d, J ¼ 5.7 Hz, 3H), 1.04 (d, J ¼ 5.7 Hz, 3H). 13C NMR
(100 MHz, CDCl3)
d
211.74 (C), 200.26 (C), 198.31 (C), 167.94 (C),
found, 577.3522. 1H NMR (500 MHz, CDCl3)
d 13.66 (s, 1H), 4.27 (d,
152.25 (C), 147.00 (C), 113.44 (C), 112.59 (C), 110.59 (C), 56.02 (C),
54.87 (CH2), 47.53 (C), 46.79 (CH2), 25.77 (CH), 25.16 (CH), 24.97
(CH3), 24.87 (CH3), 24.60 (CH3), 23.92 (CH3), 23.45 (CH3), 23.39
(CH3), 23.06 (CH3), 23.00 (CH3), 23.00 (CH3).
J ¼ 3.6 Hz, 1H), 4.19 (d, J ¼ 4.1 Hz, 1H), 3.19e2.98 (m, 2H), 2.65e2.44
(m, 4H), 2.43e2.26 (m, 4H), 1.95 (ddq, J1 ¼ 10.7 Hz, J2 ¼ 7.0 Hz,
J3 ¼ 3.5 Hz, 1H), 1.81e1.75 (m 1H), 1.75e1.70 (m, 2H), 1.44e1.33 (m,
4H), 1.21 (s, 3H), 1.18 (s, 3H), 1.17 (s, 3H), 1.13 (s, 3H), 1.01 (d,
J ¼ 6.6 Hz, 3H), 0.94e0.91 (m, 3H), 0.79e0.73 (m, 6H), 0.70 (d,
4.1.5.12. Synthesis of 2-(1-(3-acetyl-2,4,6-trihydroxy-5-(1-(2-
hydroxy-4,4-dimethyl-6-oxocyclohex-1-enyl)-2-methylpropyl)
phenyl)-2-methylpropyl)-3-hydroxy-5,5-dimethylcyclohex-2-enone
(37). Following general procedures B and D compound 37 was
obtained after flash chromatography (SiO2, PE/CH2Cl2 1:2, Rf ¼ 0.27)
J ¼ 6.9 Hz, 6H). 13C NMR (125 MHz, CDCl3)
d 205.96 (C), 197.10 (C),
197.08 (C), 165.93 (C), 165.80 (C), 161.34 (C), 153.59 (C), 151.54 (C),
114.16 (C), 113.15 (C), 110.73 (C), 107.02 (C), 104.01 (C), 51.14 (CH2),
51.06 (CH2), 45.01 (CH2), 41.46 (CH2), 41.37 (CH2), 31.21 (CH), 35.38
(CH), 34.73 (CH), 32.15 (C), 32.13 (C), 31.82 (CH2), 31.52 (CH), 30.18

K. Wiechmann et al. / European Journal of Medicinal Chemistry 101 (2015) 133e149
145
(CH3), 29.89 (CH3), 27.30 (CH3), 27.29 (CH3), 24.64 (CH2), 22.80
(CH2), 19.48 (CH3), 18.96 (CH3), 14.14 (CH3).
isobutyrylphenyl)propyl)-1H-inden-1-one (42). Following general
procedure C and D compound 42 was obtained after flash chro-
matography (SiO2, PE/acetone 3:2, Rf ¼ 0.27) as a red solid. Yield:
507 mg, (85%). mp: 100e140 ꢀC. HRMS-ESI, m/z (C36H36O8): calcd.
597.2482 [MþH]þ; found, 597.2474. 1H NMR (400 MHz, Acetone-d6)
4.1.5.15. Synthesis of 2-(1-(3-benzoyl-2,4,6-trihydroxy-5-(1-(2-
hydroxy-4,4-dimethyl-6-oxocyclohex-1-enyl)-2-methylpropyl)
phenyl)-2-methylpropyl)-3-hydroxy-5,5-dimethylcyclohex-2-enone
(40). Following general procedures B and D compound 40 was
obtained after flash chromatography (SiO2, PE/acetone 3:2,
Rf ¼ 0.14) as a yellow solid. Yield: 526 mg, (85%). mp: 150e180 ꢀC.
HRMS-ESI, m/z (C37H46O8): calcd. 619.3265 [MþH]þ; found,
d
13.47 (s,1H), 8.03e7.24 (m,10H), 4.30e4.18 (m,1H), 2.27e2.08 (m,
2H), 1.02 (d, J ¼ 6.6 Hz, 1H), 0.93 (d, J ¼ 6.8 Hz, 3H), 0.87 (d,
J ¼ 6.8 Hz, 3H). Compound 42 was cyclized following procedure E
and purified by preparative TLC (PE/acetone). HRMS-ESI, m/z
(C36H32O6): calcd. 561.2272 [MþH]þ; found, 561.2269. 1H NMR
619.3238. 1H NMR (400 MHz, Acetone-d6)
d
7.72e7.54 (m, 2H),
(500 MHz, CDCl3) d 13.73 (s, 1H), 7.56e7.47 (m, 2H), 7.46e7.41 (m,
7.49e7.25 (m, 3H), 4.02e3.78 (m, 2H), 3.28e3.14 (m, 1H), 3.12e3.04
(m, 1H), 2.25e2.20 (m, 6H),1.07e1.00 (m, 12H), 0.90e0.61 (m, 12H).
Compound 40 was cyclized following procedure E and purified by
preparative TLC (PE/acetone). HRMS-ESI, m/z (C37H42O6): calcd.
583.3054 [MþH]þ; found, 583.3052. 1H NMR (500 MHz, CDCl3)
2H), 7.40e7.32 (m, 2H), 7.28e7.23 (m, 1H), 7.20e7.13 (m, 1H), 4.29
(d, J ¼ 3.0 Hz, 1H), 4.19 (d, J ¼ 3.0 Hz, 1H), 4.06e3.91 (m, 1H), 2.21
(td, J1 ¼ 6.9 Hz, J2 ¼ 3.1 Hz, 1H), 2.06 (td, J1 ¼ 6.9 Hz, J2 ¼ 3.1 Hz, 1H),
1.36 (d, J ¼ 6.8 Hz, 6H),1.14 (d, J ¼ 6.9 Hz, 3H),1.07 (d, J ¼ 7.0 Hz, 3H),
0.65 (d, J ¼ 6.9 Hz, 3H). 13C NMR (125 MHz, CDCl3)
d 210.40 (C),
d
11.52 (s, 1H), 7.62e7.49 (m, 2H), 7.55e7.33 (m, 1H), 7.47e7.37 (m,
192.71 (C), 192.63 (C), 170.23 (C), 170.09 (C), 162.09 (C), 153.89 (C),
151.40 (C), 136.59 (C), 136.61 (C), 132.73 (CH), 132.71 (CH), 132.28
(C), 132.24 (C), 130.57 (CH), 130.38 (CH), 122.43 (CH), 122.22 (CH),
117.82 (CH), 117.68 (CH), 112.19 (C), 110.43 (C), 110.19 (C), 107.25 (C),
41.00 (CH), 34.91 (CH), 33.92 (CH), 33.77 (CH), 33.39 (CH), 22.04
(CH3), 21.33 (CH3), 19.78 (CH3), 19.65 (CH3), 17.66 (CH3).
2H), 4.31 (dd, J1 ¼ 3.7 Hz, J2 ¼ 1.0 Hz, 1H), 4.18 (dd, J1 ¼ 3.7 Hz,
J2 ¼ 0.9 Hz, 1H), 2.63e2.45 (m, 2H), 2.41e2.27 (m, 2H), 2.28e2.17
(m, 2H), 2.00 (ddq, J1 ¼ 10.6 Hz, J2 ¼ 6.9 Hz, J3 ¼ 3.7 Hz, 1H),
1.82e1.74 (m, 1H), 1.77e1.55 (m, 2H), 1.20 (s, 3H), 1.15 (s, 3H), 0.98
(s, 3H), 0.97 (s, 3H), 0.80 (d, J ¼ 6.9 Hz, 3H), 0.76e0.73 (m, 9H). 13
C
NMR (125 MHz, CDCl3) d 199.16 (C), 197.18 (C), 197.12 (C), 166.02 (C),
165.71 (C), 159.25 (C), 153.87 (C), 150.47 (C), 141.64 (C), 131.69 (CH),
128.15 (CH),128.01 (CH),113.47 (C), 113.26 (C),110.72 (C),107.00 (C),
104.61 (C), 51.00 (CH2), 41.37 (CH2), 40.37 (CH2), 35.27 (CH), 34.88
(CH), 31.87 (C), 31.70 (CH), 31.32 (CH), 31.14 (C), 29.92 (CH3), 29.69
(CH3), 27.29 (CH3), 27.20 (CH3), 20.09 (CH3), 19.48 (CH3), 19.00
(CH3).
4.1.5.18. Synthesis of 2-(1-(3-hexanoyl-2,4,6-trihydroxy-5-(1-(3-
hydroxy-1-oxo-1H-inden-2-yl)-2-methylpropyl)phenyl)-2-
methylpropyl)-3-hydroxy-1H-inden-1-one (43). Following general
procedures C and D compound 43 was obtained after flash chro-
matography (SiO2, PE/acetone 3:2, Rf ¼ 0.21) as a red solid. Yield:
487 mg, (78%). mp: 100e145 ꢀC. HRMS-ESI, m/z (C38H40O8): calcd.
625.2796 [MþH]þ; found, 625.2779. 1H NMR (400 MHz, Acetone-
4.1.5.16. Synthesis of 2-(1-(3-hexanoyl-2,4,6-trihydroxy-5-(1-(2-
hydroxy-4,4-dimethyl-6-oxocyclohex-1-enyl)-3-methylbutyl)
phenyl)-3-methylbutyl)-3-hydroxy-5,5-dimethylcyclohex-2-enone
(41). Following general procedures B and D compound 41 was
obtained after flash chromatography (SiO2, PE/CHCl3 10:1, Rf ¼ 0.24)
as a yellow resin. Yield: 520 mg, (81%). HRMS-ESI, m/z (C38H56O8):
calcd. 641.4048 [MþH]þ; found, 641.4039. 1H NMR (400 MHz,
d6)
d
14.00 (s, 1H), 13.88 (s, 1H), 13.76 (s, 1H), 8.22 (d, J ¼ 7.8 Hz, 1H),
8.02 (d, J ¼ 7.7 Hz, 1H), 7.99e7.93 (m, 2H), 7.87 (td, J1 ¼ 7.9 Hz,
J2 ¼ 7.3 Hz, J3 ¼ 1.8 Hz, 2H), 7.83e7.72 (m, 2H), 7.62e7.43 (m, 2H),
7.18e6.99 (m, 2H), 4.04e3.83 (m, 2H), 3.57 (dd, J1 ¼ 9.1 Hz,
J2 ¼ 2.1 Hz, 2H), 3.44e3.29 (m, 2H), 3.20e2.87 (m, 26H), 1.73e1.48
(m, 3H), 1.40e1.24 (m, 3H), 1.16 (dd, J1 ¼ 19.5 Hz, J2 ¼ 12.8 Hz, 3H),
1.02 (d, J ¼ 6.6 Hz, 3H), 0.96e0.83 (m, 3H), 0.80 (d, J ¼ 6.7 Hz, 3H),
0.72 (s, 3H), 0.66 (d, J ¼ 6.7 Hz, 3H), 0.19 (d, J ¼ 6.8 Hz, 3H). Com-
pound 43 was cyclized following procedure E and purified by
preparative TLC (PE/acetone). HRMS-ESI, m/z (C38H36O6): calcd.
589.2585 [MþH]þ; found, 589.2593. 1H NMR (500 MHz, CDCl3)
CDCl3)
d 4.44e4.23 (m, 1H), 3.32 (s, 1H), 3.23e3.09 (m, 1H),
3.06e2.97 (m, 1H), 2.73e2.56 (m, 3H), 2.38e2.24 (m, 4H), 2.16 (s,
8H), 2.08e1.97 (m, 2H), 2.10e1.80 (m, 2H), 1.70e1.63 (m, 2H),
1.44e1.38 (m, 2H), 1.11e0.97 (m, 27H), 0.97e0.76 (m, 12H). 13C NMR
(100 MHz, CDCl3)
d
202.18, 200.73, 197.86, 176.30, 166.19, 117.06,
d 13.93 (s, 1H), 7.47e7.42 (m, 2H), 7.41e7.32 (m, 2H), 7.31e7.28 (m,
98.94, 95.71, 55.17, 54.26, 51.09, 50.82, 49.19, 47.60, 42.68, 40.93,
39.85, 38.62, 35.40, 32.09, 32.05, 31.77, 31.34, 31.26, 31.18, 31.06,
30.39, 29.38, 28.83, 28.31, 28.13, 27.65, 27.62, 27.08, 26.84, 26.72,
26.49, 26.27, 25.89, 25.53, 24.83, 24.77, 24.63, 23.99, 22.74, 22.67,
22.63, 22.41, 21.71, 21.58, 21.14, 20.83, 19.58, 14.14. Compound 41
was cyclized following procedure E and purified by preparative TLC
(PE/acetone). HRMS-ESI, m/z (C34H44O6): calcd. 549.3211 [MþH]þ;
2H), 7.12e7.08 (m, 2H), 4.15 (d, J ¼ 3.0 Hz, 1H), 3.92 (d, J ¼ 2.8 Hz,
1H), 3.46e3.32 (m, 2H), 2.09e2.06 (m, 1H), 1.72e1.74 (m, 2H),
1.44e1.33 (m, 2H), 1.36 (t, J ¼ 7.1 Hz, 2H), 0.76e0.74 (m, 3H),
0.72e0.69 (m, 3H), 0.60 (d, J ¼ 6.8 Hz, 3H), 0.54 (d, J ¼ 6.9 Hz, 1H).
13C NMR (125 MHz, CDCl3)
d 202.33 (C), 192.66 (C), 192.51 (C),
170.30 (C), 170.12 (C), 161.76 (C), 153.72 (C), 150.68 (C), 136.62 (C),
136.40 (C), 132.40 (C), 132.35 (CH), 132.32 (C), 132.21 (CH), 131.89
(CH), 131.02 (CH), 122.71 (CH), 122.66 (CH), 117.47 (CH), 116.95 (CH),
112.28 (C), 111.08 (C), 110.88 (C), 109.99 (C), 107.52 (C), 41.87 (CH2),
35.20 (CH), 34.12 (CH), 33.80 (CH), 33.28 (CH), 32.21 (CH2), 25.03
(CH2), 23.03 (CH2), 22.07 (CH3), 21.52 (CH3), 18.09 (CH3), 17.83
(CH3), 14.17 (CH3).
found, 549.3202. 1H NMR (500 MHz, CDCl3)
d 13.63 (s, 1H), 4.21 (m,
2H), 3.15e3.03 (m, 2H), 1.44e1.33 (m 4H), 2.56e2.42 (m, 4H),
2.39e2.28 (m, 4H), 1.76e1.70 (m, 2H), 1.44e1.33 (m, 4H), 1.30e1.25
(m, 2H), 1.17 (s, 3H), 1.16 (s, 3H), 1.14 (s, 3H), 1.13 (s, 3H), 0.94e0.92
(m, 3H), 0.92 (d, J ¼ 5.1 Hz, 3H), 0.87 (d, J ¼ 6.1 Hz, 3H), 0.80 (d,
J ¼ 6.4 Hz, 3H), 0.78 (d, J ¼ 5.9 Hz, 3H). 13C NMR (125 MHz, CDCl3)
d
205.89 (C), 197.06 (C), 197.01 (C), 165.00 (C), 164.81 (C), 161.23 (C),
4.1.5.19. Synthesis of 3-hydroxy-2-(2-methyl-1-(2,4,6-trihydroxy-3-
(1-(3-hydroxy-1-oxo-1H-inden-2-yl)-2-methylpropyl)-5-(3-
152.63 (C), 150.73 (C), 116.13 (C), 115.90 (C), 112.09 (C), 107.07 (C),
106.42 (C), 51.19 (CH2), 51.13 (CH2), 47.62 (CH2), 46.27 (CH2), 45.00
(CH2), 41.45 (CH2), 41.34 (CH2), 32.32 (C), 32.29 (C), 31.79 (CH2),
29.94 (CH3), 29.63 (CH3), 27.26 (CH3), 27.15 (CH3), 25.30 (CH), 25.25
(CH), 24.75 (CH), 24.52 (CH2), 24.17 (CH), 23.92 (CH3), 23.48 (CH3),
23.35 (CH3), 23.21 (CH3), 22.81 (CH2), 14.15 (CH3).
methylbutanoyl)phenyl)propyl)-1H-inden-1-one
(44). Following
general procedures C and D compound 44 was obtained after flash
chromatography (SiO2, PE/acetone 3:2, Rf ¼ 0.25) as a red solid.
Yield: 470 mg, (77%). mp: 100e145 ꢀC. HRMS-ESI, m/z (C37H38O8):
calcd. 611.2639 [MþH]þ; found, 611.2639. 1H NMR (400 MHz,
Acetone-d6)
d
13.55 (s,1H), 8.05e7.82 (m, 2H), 7.65 (ddd, J1 ¼8.2 Hz,
4.1.5.17. Synthesis of 3-hydroxy-2-(2-methyl-1-(2,4,6-trihydroxy-3-
(1-(3-hydroxy-1-oxo-1H-inden-2-yl)-2-methylpropyl)-5-
J2 ¼ 7.3 Hz, J3 ¼ 1.2 Hz, 2H), 7.57e7.49 (m, 2H), 7.41e7.32 (m, 2H),
7.31e7.23 (m, 2H), 3.49e3.34 (m, 2H), 2.79 (dt, J1 ¼ 17.2 Hz,

146
K. Wiechmann et al. / European Journal of Medicinal Chemistry 101 (2015) 133e149
J2 ¼ 7.5 Hz, 2H), 2.28e2.11 (m, 2H), 1.17 (d, J ¼ 6.7 Hz, 3H), 1.14e1.06
(m, 3H), 1.00 (d, J ¼ 6.7 Hz, 3H), 0.95e0.85 (m, 6H). Compound 44
was cyclized following procedure E and purified by preparative TLC
(PE/acetone). HRMS-ESI, m/z (C37H34O6): calcd. 575.2428 [MþH]þ;
(125 MHz, CDCl3) d 210.13 (C), 191.61 (C), 191.56 (C), 166.71 (C),
166.17 (C), 163.49 (C), 153.32 (C), 150.76 (C), 143.75 (C), 143.02 (C),
136.36 (C), 136.51 (C), 132.80 (C), 132.55 (C), 132.43 (CH), 132.33
(CH), 130.46 (CH), 130.68 (CH), 130.28 (CH), 130.24 (CH), 128.21
(CH), 127.97 (CH), 122.85 (CH), 121.85 (CH), 118.31 (CH), 118.22 (CH),
118.07 (CH), 117.82 (CH), 111.60 (C), 110.94 (C), 109.33 (C), 107.40 (C),
104.90 (C), 40.86 (CH), 34.16 (CH), 33.58 (CH), 19.64 (CH3), 19.50
(CH3).
found, 575.2422. 1H NMR (500 MHz, CDCl3)
d 13.81 (s, 1H),
7.55e7.49 (m, 1H), 7.50e7.45 (m, 1H), 7.47e7.39 (m, 2H), 7.40e7.30
(m, 2H), 7.32e7.26 (m, 1H), 7.22e7.17 (m, 1H), 4.20 (d, J ¼ 3.0, Hz,
1H), 4.08 (d, J ¼ 3.0 Hz, 1H), 3.21 (ddd, J1 ¼ 70.3 Hz, J2 ¼ 17.2 Hz,
J3 ¼ 6.7 Hz, 2H), 2.39 (sept, J ¼ 6.7 Hz, 1H), 2.24 (pd, J1 ¼ 7.0 Hz,
J2 ¼ 3.0 Hz, 1H), 2.16 (pd, J1 ¼ 6.9 Hz, J2 ¼ 3.0 Hz, 1H), 1.13 (d,
J ¼ 6.9 Hz, 3H), 1.09 (d, J ¼ 6.6 Hz, 3H), 1.08 (d, J ¼ 6.6 Hz, 3H), 1.07
4.1.5.22. Synthesis of 2-((3-hexanoyl-2,4,6-trihydroxy-5-((3-
hydroxy-1-oxo-1H-inden-2-yl) (phenyl)methyl)phenyl) (phenyl)
methyl)-3-hydroxy-1H-inden-1-one (47). Following general pro-
cedures C and D compound 47 was obtained after flash chroma-
tography (SiO2, PE/acetone 1:1, Rf ¼ 0.15) as a red solid. Yield:
623 mg, (90%). mp: 70e125 ꢀC. HRMS-ESI, m/z (C44H36O8): calcd.
693.2483 [MþH]þ; found, 693.2437. 1H NMR (400 MHz, Acetone-
(d, J ¼ 6.9 Hz, 3H), 0.81 (d, J ¼ 6.9 Hz, 3H), 0.68 (d, J ¼ 6.8 Hz, 3H). 13
C
NMR (125 MHz, CDCl3)
d 205.52 (C), 192.77 (C), 192.64 (C), 169.99
(C), 169.94 (C), 161.68 (C), 153.99 (C), 151.40 (C), 136.53 (C), 136.51
(C), 132.61 (CH), 132.24 (CH), 132.24 (C), 132.15 (C), 130.56 (CH),
130.39 (CH), 122.41 (CH), 122.12 (CH), 117.97 (CH), 117.88 (CH),
111.74 (C), 110.43 (C), 110.06 (C), 107.98 (C), 105.46 (C), 54.43 (CH2),
35.43 (CH), 33.83 (CH), 33.74 (CH), 33.23 (CH), 24.78 (CH), 23.07
(CH3), 23.03 (CH3), 21.41 (CH3), 21.13 (CH3), 18.04 (CH3), 17.54 (CH3).
d6)
d 8.64e8.51 (m, 2H), 8.08e7.94 (m, 4H), 7.71e7.53 (m, 6H), 7.17
(dt, J1 ¼ 17.3 Hz, J2 ¼ 6.5 Hz, 4H), 1.70e1.56 (m, 2H), 1.57e1.42 (m,
2H), 1.41e1.16 (m, 4H), 0.99e0.69 (m, 3H). Compound 47 was
cyclized following procedure E and purified by preparative TLC (PE/
acetone). HRMS-ESI, m/z (C44H32O6): calcd. 657.2272 [MþH]þ;
4.1.5.20. Synthesis of 2-(1-(3-benzoyl-2,4,6-trihydroxy-5-(1-(3-
hydroxy-1-oxo-1H-inden-2-yl)-2-methylpropyl)phenyl)-2-
methylpropyl)-3-hydroxy-1H-inden-1-one (45). Following general
procedure C and D compound 45 was obtained after flash chro-
matography (SiO2, PE/acetone 3:2, Rf ¼ 0.15) as a red solid. Yield:
486 mg, (77%). mp: 100e150 ꢀC. HRMS-ESI, m/z (C39H34O8): calcd.
631.2326 [MþH]þ; found, 631.2319. 1H NMR (400 MHz, Acetone-d6)
found, 657.2270. 1H NMR (500 MHz, CDCl3)
d 13.85 (s), 7.48e7.39
(m, 4H), 7.37e7.27 (m, 8H), 7.25e7.19 (m, 2H), 7.18e7.09 (m, 2H),
7.08e6.97 (m, 1H), 5.44 (s, 1H), 5.10 (s, 1H), 3.49e3.22 (m, 2H),
1.90e1.75 (m, 2H), 1.53e1.39 (m, 4H), 1.02e0.93 (m, 3H). 13C NMR
(125 MHz, CDCl3)
d 206.76 (C), 191.59 (C), 191.55 (C), 166.80 (C),
166.21 (C), 163.16 (C), 153.37 (C), 151.25 (C), 143.47 (C), 143.08 (C),
136.55 (C), 136.38 (C), 132.74 (CH), 132.54 (CH), 132.03 (C), 131.88
(C), 130.74 (CH), 130.44 (CH), 130.30 (CH), 130.24 (CH), 128.41 (CH),
128.27 (CH), 127.83 (CH), 127.25 (CH), 122.50 (CH), 122.08 (CH),
118.31 (CH), 118.26 (CH), 111.69 (C), 111.43 (C), 111.33 (C), 108.02 (C),
104.84 (C), 45.69 (CH2), 34.19 (CH), 33.52 (CH), 31.73 (CH2), 24.04
(CH2), 22.81 (CH2), 14.14 (CH3).
d
8.01e7.87 (m, 1H), 7.60e7.48 (m, 2H), 7.47e7.33 (m, 2H),
7.33e7.21 (m, 2H), 7.21e6.97 (m, 2H), 3.50e3.22 (m, 3H), 2.31e2.12
(m, 1H), 1.22e1.06 (m, 6H), 1.01 (d, J ¼ 6.8 Hz, 3H), 0.92 (d,
J ¼ 6.8 Hz, 3H). Compound 45 was cyclized following procedure E
and purified by preparative TLC (PE/acetone). HRMS-ESI, m/z
(C39H30O6): calcd. 595.2115 [MþH]þ; found, 595.2110. 1H NMR
(500 MHz, CDCl3)
d 13.81 (s, 1H), 7.74e7.65 (m, 2H), 7.61e7.54 (m,
1H), 7.53e7.41 (m, 4H), 7.41e7.32 (m, 2H), 7.31 (dt, J1 ¼ 7.0 Hz,
J2 ¼ 0.9 Hz, 1H), 7.21 (ddd, J1 ¼ 8.0 Hz, J2 ¼ 7.1 Hz, J3 ¼ 1.0 Hz, 1H),
7.10 (ddd, J1 ¼8.0 Hz, J2 ¼ 7.1 Hz, J3 ¼ 1.0 Hz,1H), 5.75 (dt, J1 ¼7.1 Hz,
J2 ¼ 0.9 Hz, 1H), 4.21 (d, J ¼ 3.0 Hz, 1H), 4.16 (d, J ¼ 3.1 Hz, 1H), 2.30
(pd, J1 ¼6.9 Hz, J2 ¼ 3.1 Hz,1H), 2.21 (pd, J1 ¼6.9 Hz, J2 ¼ 3.0 Hz,1H),
1.11 (d, J ¼ 6.9 Hz, 6H), 0.82 (d, J ¼ 6.9 Hz, 3H), 0.75 (d, J ¼ 6.8 Hz,
4.1.5.23. Synthesis of 3-hydroxy-2-(phenyl(2,4,6-trihydroxy-3-((3-
hydroxy-1-oxo-1H-inden-2-yl) (phenyl)methyl)-5-(3-
methylbutanoyl)phenyl)methyl)-1H-inden-1-one
(48).
Following general procedures C and D compound 48 was obtained
after flash chromatography (SiO2, PE/acetone 1:1, Rf ¼ 0.24) as a red
solid. Yield: 638 mg, (94%). mp: 70e130 ꢀC. HRMS-ESI, m/z
(C43H34O8): calcd. 679.2326 [MþH]þ; found, 679.2317. 1H NMR
3H). 13C NMR (125 MHz, CDCl3)
d 198.94 (C), 192.74 (C), 192.61 (C),
170.02 (C), 169.57 (C), 159.69 (C), 154.32 (C), 150.34 (C), 141.37 (C),
136.52 (C), 135.89 (C), 132.65 (CH), 132.33 (CH), 132.18 (CH), 131.98
(C), 130.44 (CH), 130.26 (CH), 128.55 (CH), 128.39 (CH), 121.88 (CH),
122.18 (CH), 117.93 (CH), 117.69 (CH), 111.75 (C), 110.34 (C), 109.26
(C), 107.80 (C), 106.20 (C), 35.29 (CH), 33.90 (CH), 33.51 (CH), 21.59
(CH3), 21.04 (CH3), 18.08 (CH3), 17.60 (CH3).
(400 MHz, Acetone-d6) d 8.65e8.42 (m, 1H), 8.14e7.92 (m, 2H), 7.86
(d, J ¼ 13.8 Hz, 1H), 7.72e7.55 (m, 3H), 7.56e7.34 (m, 3H), 7.34e7.25
(m, 1H), 7.24e7.10 (m, 6H), 7.10e7.02 (m, 2H), 7.01e6.91 (m, 1H),
6.91e6.73 (m, 1H), 6.50 (d, J ¼ 46.3 Hz, 1H), 6.26e5.69 (m, 1H),
5.29e4.82 (m, 1H), 3.63e3.20 (m, 1H), 3.19e2.67 (m, 8H), 2.31e2.12
(m, 0H), 1.16e0.68 (m, 7H). Compound 48 was cyclized following
procedure E and purified by preparative TLC (PE/acetone). HRMS-
ESI, m/z (C43H30O6): calcd. 643.2115 [MþH]þ; found, 643.2117. 1H
4.1.5.21. Synthesis of 3-hydroxy-2-(phenyl(2,4,6-trihydroxy-3-((3-
hydroxy-1-oxo-1H-inden-2-yl) (phenyl)methyl)-5-isobutyrylphenyl)
methyl)-1H-inden-1-one (46). Following general procedures C and
D compound 46 was obtained after flash chromatography (SiO2, PE/
acetone 1:1, Rf ¼ 0.19) as a red solid. Yield: 592 mg, (89%). mp:
120e165 ꢀC. HRMS-ESI, m/z (C42H32O8): calcd. 665.2170 [MþH]þ;
NMR (500 MHz, CDCl3)
d
13.86 (s, 1H), 7.46 (dt, J1 ¼ 7.0 Hz,
J2 ¼ 0.8 Hz, 1H), 7.41 (dd, J1 ¼ 2.0 Hz, J2 ¼ 1.6 Hz, 4H), 7.40e7.33 (m,
4H), 7.34e7.28 (m, 2H), 7.28 (d, J ¼ 1.6, 2H), 7.23 (t, J ¼ 1.6 Hz, 1H),
7.21e7.18 (m, 2H), 7.15 (dddd, J1 ¼ 7.5 Hz, J2 ¼ 3.7 Hz, J3 ¼ 2.3 Hz,
J4 ¼ 1.2 Hz, 2H), 7.05 (d, J ¼ 1.8 Hz, 2H), 7.00 (dd, J1 ¼ 7.5 Hz,
J2 ¼ 1.0 Hz, 1H), 5.43 (s, 1H), 5.10 (s, 1H), 3.39e3.09 (m, 2H), 2.40 (dt,
J1 ¼ 13.3 Hz, J2 ¼ 6.7 Hz, 1H), 1.10 (d, J ¼ 6.7 Hz, 3H), 1.06 (d,
found, 665.2167. 1H NMR (400 MHz, Acetone-d6)
d 8.11e7.92 (m,
1H), 7.93e7.74 (m, 1H), 7.73e7.28 (m, 5H), 7.26e7.10 (m, 5H),
7.10e7.02 (m, 1H), 7.01e6.93 (m, 1H), 6.94e6.77 (m, 1H), 5.19e4.97
(m, 1H), 4.21e3.83 (m, 1H), 1.46e1.23 (m, 1H), 1.23e0.96 (m, 4H),
0.96e0.81 (m, 2H). Compound 46 was cyclized following procedure
E and purified by preparative TLC (PE/acetone). HRMS-ESI, m/z
(C42H28O6): calcd. 629.1959 [MþH]þ; found, 629.1953. 1H NMR
J ¼ 6.7 Hz, 3H). 13C NMR (125 MHz, CDCl3)
d 205.22 (C), 191.59 (C),
191.56 (C), 166.78 (C), 166.21 (C), 163.17 (C), 153.38 (C), 151.20 (C),
143.46 (C), 143.08 (C), 136.56 (C), 136.37 (C), 132.77 (CH), 132.54
(CH), 132.04 (C), 131.87 (C), 130.73 (CH), 130.30 (CH), 128.25 (CH),
128.13 (CH), 127.83 (CH), 127.14 (CH), 126.96 (CH), 122.51 (CH),
122.08 (CH), 118.54 (CH), 118.30 (CH), 111.69 (C), 111.42 (C), 111.24
(C), 108.21 (C), 104.52 (C), 54.52 (CH2), 34.19 (CH), 33.52 (CH), 24.69
(CH), 23.16 (CH3), 23.01 (CH3).
(500 MHz, CDCl3)
d 13.78 (s, 1H), 7.53e7.37 (m, 8H), 7.30 (td,
J1 ¼ 7.8 Hz, J2 ¼ 6.0 Hz, 4H), 7.21 (d, J ¼ 7.8 Hz, 2H), 7.20 (d,
J ¼ 17.8 Hz, 2H), 7.08e6.96 (m, 1H), 5.44 (s, 1H), 5.10 (s, 1H),
4.06e3.94 (m, 1H), 1.45e1.36 (m, 3H), 1.35e1.31 (m, 3H). 13C NMR

K. Wiechmann et al. / European Journal of Medicinal Chemistry 101 (2015) 133e149
147
4.1.5.24. Synthesis of 2-((3-benzoyl-2,4,6-trihydroxy-5-((3-hydroxy-
1-oxo-1H-inden-2-yl) (phenyl)methyl)phenyl) (phenyl)methyl)-3-
4.1.5.27. Synthesis of 5-hexanoyl-6,8-dihydroxy-7-(1-(2-hydroxy-
3,3,5,5-tetramethyl-4,6-dioxocyclohex-1-enyl)-2-methylpropyl)-9-
isopropyl-2,2,4,4-tetramethyl-2H-xanthene-1,3(4H,9H)-dione (52).
Following general procedures B and D compound 52 was obtained
after flash chromatography (SiO2, PE/acetone 3:2, Rf ¼ 0.25) as a
yellow solid. Yield: 652 mg, (96%). mp: 120e140 ꢀC. HRMS-ESI, m/z
(C40H54O9): calcd. 679.3840 [MþH]þ; found, 679.3834; 1H NMR
hydroxy-1H-inden-1-one (49). Following general procedures
C
and D compound 49 was obtained after flash chromatography
(SiO2, PE/acetone 1:1, Rf ¼ 0.16) as a red solid. Yield: 643 mg, (92%).
mp: 90e150 ꢀC. HRMS-ESI, m/z (C45H30O8): calcd. 699.2013
[MþH]þ; found, 699.2004. 1H NMR (400 MHz, Acetone-d6)
d
8.62e8.48 (m, 3H), 8.08e7.93 (m, 3H), 7.71e7.54 (m, 6H),
(400 MHz, Acetone-d6) d 4.44e4.29 (m, 1H), 4.05e3.88 (m, 1H),
7.46e7.41 (m, 3H), 7.40e7.32 (m, 3H), 7.31e7.23 (m, 3H), 7.23e6.95
(m, 6H), 5.13e5.06 (m, 2H). Compound 49 was cyclized following
procedure E and purified by preparative TLC (PE/acetone). HRMS-
ESI, m/z (C45H26O6): calcd. 663.1802 [MþH]þ; found, 663.1799. 1H
3.34e3.21 (m, 2H), 3.20e3.06 (m, 2H), 1.96e1.82 (m, 2H), 1.81e1.67
(m, 2H), 1.63 (s, 3H), 1.62 (s, 3H), 1.57 (d, J ¼ 1.6 Hz, 3H), 1.48 (s, 3H),
1.47 (s, 3H), 1.45e1.22 (m, 12H), 1.11 (t, J ¼ 7.0 Hz, 3H), 0.90 (dt,
J1 ¼ 6.1 Hz, J2 ¼ 4.1 Hz, 6H), 0.85e0.69 (m, 6H).
NMR (500 MHz, CDCl3) d 11.77 (s, 1H), 7.77e7.74 (m, 2H), 7.65e7.57
(m, 4H), 7.55e7.47 (m, 4H), 7.38e7.27 (m, 4H), 7.25e7.19 (m, 4H),
7.19e7.13 (m, 2), 7.08 (td, J1 ¼ 7.6 Hz, J2 ¼ 1.1 Hz, 1H), 7.04e6.91 (m,
1H), 5.80e5.73 (m, 1H), 5.35 (s, 1H), 5.15 (s, 1H). 13C NMR (125 MHz,
4.1.5.28. Synthesis of 3-hydroxy-5,5-dimethyl-2-(2-methyl-1-(2,4,6-
trihydroxy-3,5-bis(1-(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-
enyl)-2-methylpropyl)phenyl)propyl)cyclohex-2-enone
(54).
CDCl3)
d
198.92 (C), 192.73 (C), 192.58 (C), 166.29 (C), 165.65 (C),
Following general procedure D phloroglucinol was deprotonated
with 3 eq. of NaH in 20 ml of THF and isobutylidene dimedone (9
eq., prepared according to general procedure B) was added in 5 ml
of THF. After phloroglucinol had been consumed the reaction
mixture was worked up as usual and the product was obtained
after flash chromatography (PE/dichloromethane 1:1 v/v) in 88%
yield as a yellow solid, mp: 110e150 ꢀC. HRMS-ESI, m/z (C42H60O9):
calcd. 709.4310 [MþH]þ; found, 709.4310; 1H NMR (400 MHz,
161.24 (C), 153.51 (C), 149.79 (C), 143.53 (C), 142.84 (C), 141.28 (C),
136.61 (C), 132.60 (CH), 132.42 (CH), 132.22 (C), 131.97 (CH), 131.96
(C), 130.70 (CH), 130.68 (CH), 130.48 (CH), 130.32 (CH), 128.63 (CH),
128.42 (CH), 128.33 (CH), 128.20 (CH), 127.17 (CH), 127.08 (CH),
122.32 (CH), 122.07 (CH), 118.53 (CH), 118.02 (CH), 111.19 (C), 111.30
(C), 110.19 (C), 107.64 (C), 105.58 (C), 34.12 (CH), 33.49 (CH).
CDCl3)
d 12.18e11.60 (m, 3H), 10.81 (s, 3H), 3.87e3.64 (m, 3H),
4.1.5.25. Synthesis of 5-hydroxy-2,2,6,6-tetramethyl-4-(3-methyl-1-
(2,4,6-trihydroxy-3-(3-methylbutanoyl)phenyl)butyl)cyclohex-4-ene-
1,3-dione (50). Following general procedure B and D compound 50
was obtained after flash chromatography (SiO2, PE/acetone 10:1,
Rf ¼ 0.19) as a yellow solid. Yield: 419 mg, (quant.). mp: 150e185 ꢀC.
HRMS-ESI, m/z (C24H34O6): calcd. 419.2428 [MþH]þ; found,
3.17e2.95 (m, 3H), 2.40e2.24 (m, 8H), 2.24-2.10 (m, 3H), 1.13-1.07
(m, 9H), 1.07e0.98 (m, 9H), 0.92e0.88 (m, 4H), 0.88-0.83 (m, 4H),
0.78 (dd, J ¼ 6.4, 1.9 Hz, 2H), 0.75-0.70 (m, 3H), 0.69e0.55 (m, 3H).
13C NMR (100 MHz, CDCl3)
d 151.52; 151.26; 151.18; 151.15; 150.69;
117.54; 116.50; 116.45; 116.37; 116.33; 111.54; 111.49; 111.09;
111.00; 110.72; 110.51; 64.69; 47.92; 45.75; 40.14; 39.72; 31.40;
31.18; 31.07; 31.05; 31.00; 30.98; 30.96; 30.71; 30.68; 30.60; 30.35;
30.26; 27.44; 22.12; 22.01; 21.98; 21.95; 21.81; 21.75; 21.68; 21.53;
15.43. Compound 54 was cyclized following procedure E and pu-
rified by preparative TLC (PE/acetone). mp. >300 ꢀC; HRMS-ESI, m/z
(C42H54O6): calcd. 655.3993 [MþH]þ; found, 655.3992; 1H NMR
419.2403. 1H NMR (400 MHz, Acetone-d6)
d 12.71 (s, 1H), 5.78 (s,
1H), 4.42 (t, J ¼ 8.0 Hz, 1H), 2.99 (d, J ¼ 2.2 Hz, 2H), 2.30e2.21 (m,
2H), 2.16e2.07 (m, 2H), 1.43 (dq, J1 ¼ 13.4 Hz, J2 ¼ 6.6 Hz, 1H),
1.17e1.05 (m, 2H), 1.03 (s, 6H), 0.94 (d, J ¼ 6.6 Hz, 6H), 0.84 (dd,
J1 ¼ 6.6 Hz, J2 ¼ 5.8 Hz, 6H). Compound 50 was cyclized following
procedure E and purified by preparative TLC (PE/acetone). HRMS-
ESI, m/z (C24H32O5): calcd. 401.2323 [MþH]þ; found, 401.2305. 1H
(400 MHz, CDCl3)
d
4.18 (d, J ¼ ꢂ CH); 2.53 (d, J ¼ 3.1 Hz, 6H, 3ꢂ
CH2); 2.30 (d, J ¼ 3.1 Hz, 6H, 3ꢂ CH2); 1.94 (septd, J ¼ 7.0, 3.6 Hz, 3H,
3ꢂ CH); 1.17 (s, 9H, 3 ꢂ CH3); 1.11 (s, 9H, 3ꢂ CH3); 0.87 (d, J ¼ 6.9 Hz,
3ꢂ CH3); 0.76 (d, J ¼ 6.9 Hz, 9H, 3 ꢂ CH3). 13C NMR (100 MHz,
NMR (500 MHz, CDCl3)
d 6.40 (s, 1H), 4.17e4.12 (m, 1H), 3.03e2.87
(m, 2H), 2.48e2.40 (m, 2H), 2.36e2.18 (m, 2H), 1.51e1.34 (m, 3H),
1.41e1.34 (m, 1H), 0.91e0.87 (m, 3H), 1.17e1.11 (m, 3H), 1.17 (s, 3H),
1.11 (s, 3H), 1.06e1.04 (m, 3H), 0.82e0.75 (m, 3H). 13C NMR
CDCl3)
d 197.42 (C); 166.74 (C); 147.72 (C); 113.11 (C); 108.81 (C);
51.23 (CH2); 41.70 (CH2); 35.49 (CH); 32.15 (CH); 32.11 (C); 29.78
(CH3); 27.42 (CH3); 19.90 (CH3); 19.96 (CH3).
(125 MHz, CDCl3)
d 206.59 (C), 198.13 (C), 171.66 (C), 166.74 (C),
152.02 (C), 149.61 (C), 115.14 (C), 106.69 (C), 105.56 (C), 102.44 (CH),
50.99 (CH2), 46.48 (CH2), 43.48 (CH2), 41.60 (CH2), 32.18 (C), 29.69
(CH3), 27.26 (CH3), 25.19 (CH), 24.90 (CH), 23.53 (CH3), 23.42 (CH3),
23.32 (CH3), 22.52 (CH3).
4.2. Biological evaluation and assay systems
4.2.1. Materials
€
PGH2 was from Larodan (Malmo, Sweden). Arachidonic acid,
4.1.5.26. Synthesis of 5-hexanoyl-6,8-dihydroxy-9-isopropyl-2,2,4,4-
tetramethyl-2H-xanthene-1,3(4H,9H)-dione (51). Compound 20
was cyclized following procedure E to obtain compound 51 after
flash chromatography (SiO2, PE/acetone 5:1, Rf ¼ 0.25) as a faint
yellow solid. Yield: 680 mg, (82%). mp: 90e95 ꢀC. HRMS-ESI, m/z
(C24H30O6): calcd. 415.2125 [MþH]þ; found, 415.2105. 1H NMR
Ca2þ ionophore A23187, and all other fine chemicals were from
SigmaeAldrich (Taufkirchen, Germany) unless stated otherwise.
HPLC solvents were from Merck (Darmstadt, Germany).
4.2.2. Cells and cell viability assay
For isolation of human PMNL, venous blood was taken from
healthy adult donors obtained at the University Hospital Jena
(Germany), and leukocyte concentrates were prepared as described
[34]. PMNL were immediately isolated by dextran sedimentation,
centrifugation on LSM 1077 cushions (PAA Laboratories, Linz,
Austria), and hypotonic lysis of erythrocytes. For analysis of 5-LO
product formation, cells were finally resuspended in phosphate-
buffered saline pH 7.4 (PBS) containing 1 mg/ml glucose and
1 mM CaCl2 (PGC buffer).
(500 MHz, CDCl3)
d 13.45 (s, 1H), 6.99 (s, 1H), 6.30 (s, 1H), 4.37 (d,
J ¼ 3.8 Hz, 1H), 3.15 (ddd, J1 ¼ 17.1 Hz, J2 ¼ 8.9 Hz, J3 ¼ 6.0 Hz, 2H),
1.91 (ddq, J1 ¼ 9.9 Hz, J2 ¼ 6.7 Hz, J3 ¼ 3.6 Hz, J4 ¼ 3.1 Hz, 1H), 1.84 …
1.69 (m, 2H),1.66 (s, 3H),1.46 (s, 3H),1.45 (s, 3H),1.44 (s, 3H),1.39 …
1.32 (m, 4H), 0.95 … 0.88 (m, 3H), 0.82 (d, J ¼ 6.8 Hz, 3H) 0.78 (d,
J ¼ 6.8 Hz, 3H). 13C NMR (125 MHz, CDCl3)
d 211.89 (C), 204.64 (C),
199.06 (C), 168.40 (C), 164.53 (C), 153.91 (C), 112.32 (C), 159.73 (C),
105.87 (C), 103.19 (C), 100.64 (CH), 56.26 (C), 47.53 (C), 44.82 (CH2),
35.03 (CH), 31.62 (CH), 31.60 (CH2), 25.49 (CH3), 25.24 (CH3), 25.19
(CH3), 24.29 (CH3), 24.16 (CH2), 22.80 (CH2), 19.04 (CH3), 18.83
(CH3), 14.08 (CH3).
Human leukemic Jurkat T cells were cultured in RPMI 1640
medium supplemented with heat-inactivated fetal calf serum (FCS,
10% v/v), HEPES (10 mM), penicillin (100 U/ml) and streptomycin

148
K. Wiechmann et al. / European Journal of Medicinal Chemistry 101 (2015) 133e149
(100
m
g/ml) at 37 ꢀC and 5% CO2.
under the peaks. All compounds were analyzed in 3e5 indepen-
dent experiments, and data are expressed as means S.E.M.
4.2.3. Determination of product formation by 5-LO in the cell-based
assay
4.2.6. Analysis of cytotoxicity in Jurkat cells
ml per well) were seeded in a 96-well
plate in RPMI 1640 medium supplemented with 10% fetal calf
For analysis of 5-LO product synthesis in intact cells, 5 ꢂ 106
freshly isolated human PMNL were resuspended in 1 ml PGC buffer.
After preincubation with the test compounds (15 min at 37 ꢀC), 5-
Jurkat cells (2 ꢂ 104/100
serum (FCS, SigmaeAldrich, Taufkirchen, Germany), HEPES
LO product formation was initiated by addition of 2.5
ionophore A23187 and 20
M AA. After 10 min at 37 ꢀC, the reaction
was stopped with 1 ml of methanol and 30 l of 1 N HCl, 200 ng
PGB1 and 500 l of PBS were added. Formed 5-LO metabolites were
m -
M Ca2þ
(10 mM), 100
compounds or 0.3% DMSO (as vehicle) were added to each well and
samples were incubated for 48 h. Then, 20 l of thiazolyl blue
mg/ml streptomycin, and 100 units/ml penicillin. Test
m
m
m
m
tetrazolium bromide (MTT, 5 mg/ml PBS) were added and the in-
cubation was continued at 37 ꢀC, 5% CO2 until blue staining of the
vehicle control. Formazan formation was stopped by addition of
extracted and analyzed by HPLC as described [35]. 5-LO product
formation is expressed as ng of 5-LO products per 106 cells, which
includes LTB4 and its all-trans isomers, 5(S),12(S)-di-hydroxy-6,10-
trans-8,14-cis-eicosatetraenoic acid (5(S),12(S)-DiHETE), and 5(S)-
hydro(pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-H(p)
ETE). Cysteinyl LTs C4, D4 and E4 were not detected, and oxidation
products of LTB4 were not determined. All compounds were
analyzed in three to four independent experiments, and
means S.E.M. are given.
100 ml of lysis buffer (SDS, 10%, w/v in 20 mM HCl) and samples
were kept overnight shaking. Absorbance of each well was
measured at 570 nm in a Multiskan™ microplate spectrophotom-
eter (Thermo Scientific, Ulm, Germany).
4.2.7. Statistics
Data are expressed as mean S.E.M. of single determinations
performed in three or five independent experiments at different
days. IC50 values were determined by graphical analysis using
SigmaPlot 12.0 (Systat Software Inc., San Jose, USA).
4.2.4. Expression and purification of human recombinant 5-LO and
determination of 5-LO activity
E.coli BL21 was transformed with pT3-5-LO plasmid, recombi-
nant 5-LO protein was expressed at 37 ꢀC, and 5-LO was purified as
Acknowledgement
described [36]. Purified 5-LO (0.5 mg) was diluted with PBS/EDTA,
1 mM ATP was added, and pre-incubated with test compounds;
We thank Prof. Dr. Rolf Müller and Dr. Thomas Hoffmann,
Institute for Pharmaceutical Biotechnology, University of Saarland,
for the HRMS spectra. The generous support of this project from
Deutsche Forschungsgemeinschaft (JA 616/5-1) is gratefully
acknowledged. Also, we thank University of Saarland, University of
Jena and University of Tübingen for their continuous support of our
projects.
final volume was 1 ml. After 5e10 min at 4 ꢀC, samples were pre-
warmed for 30 s at 37 ꢀC, and 2 mM CaCl2 plus 20
mM AA were
added to initiate 5-LO product formation. The reaction was stopped
after 10 min at 37 ꢀC by addition of ice-cold methanol and the
formed metabolites (all-trans isomers of LTB4 and 5-H(P)ETE) were
analyzed by HPLC as described for intact PMNL. All compounds
were analyzed in three to four independent experiments; data are
given as means S.E.M.
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Pharmacol. 156 (2009) 952e961.
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[18] C. Feisst, L. Franke, G. Appendino, O. Werz, Identification of molecular targets
of the oligomeric nonprenylated acylphloroglucinols from Myrtus communis
and their implication as anti-inflammatory compounds, J. Pharmacol. Exp.
Ther. 315 (2005) 389e396.
[28] X-ray structure determination of compound 55. The data were collected at
153 K on a BrukerAXS X8 Apex CCD diffractometer operating with graphite-
monochromatized Mo
exposed; deriving data in the
Ka
radiation. Frames of 0.5ꢀ oscillation were
q
range of 2e27ꢀ with a completeness of ~99%.
[19] I. Tretiakova, D. Blaesius, L. Maxia, S. Wesselborg, K. Schulze-Osthoff,
J. Cinatl Jr., M. Michaelis, O. Werz, Myrtucommulone from Myrtus communis
induces apoptosis in cancer cells via the mitochondrial pathway involving
caspase-9, Apoptosis 13 (2008) 119e131.
[20] A. Rossi, R. Di Paola, E. Mazzon, T. Genovese, R. Caminiti, P. Bramanti,
C. Pergola, A. Koeberle, O. Werz, L. Sautebin, S. Cuzzocrea, Myrtucommulone
from Myrtus communis exhibits potent anti-inflammatory effectiveness
in vivo, J. Pharmacol. Exp. Ther. 329 (2009) 76e86.
Unit cell: triclinic, P-1, a ¼ 10.7980(8), b ¼ 11.4465(8), c ¼ 14.5669(8)Å,
a
¼ 69.965(2),
b
¼ 77.898(2),
g
¼ 62.190(2)ꢀ, V ¼ 1493.4(2)Å3. Structure
solution and full least-squares refinement with anisotropic thermal parame-
ters of all non-hydrogen atoms and free refinement of the hydrogen were
performed using SHELX [Sheldrick, G. M., Acta Cryst. 2008, A64, 112e22]. The
final refinement result in: R1 ¼ 0.043. Crystallographic data for the structure
have been deposited with the Cambridge Crystallographic Data Centre, CCDC,
12 Union Road, Cambridge CB21EZ, UK. Copies of the data can be obtained
free of charge on quoting the depository number CCDC 1002468. www.ccdc.
cam.ac.uk/data_request/cif.
[21] H. Mueller, M. Paul, D. Hartmann, V. Huch, D. Blaesius, A. Koeberle, O. Werz,
J. Jauch, Total synthesis of myrtucommulone A, Angew. Chem. Int. Ed. 49
(2010) 2045e2049.
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from cellular enzyme regulation, Biochem. Pharmacol. 70 (2005) 327e333.
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J.A. Salmon, L.G. Garland, Selective inhibition of arachidonate 5-lipoxygenase
by novel acetohydroxamic acids: biochemical assessment in vitro and ex vivo,
Br. J. Pharmacol. 94 (1988) 528e539.
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J.A. Mancini, Microsomal prostaglandin E synthase-1 is a major terminal
synthase that is selectively up-regulated during cyclooxygenase-2-dependent
prostaglandin E2 production in the rat adjuvant-induced arthritis model,
J. Immunol. 170 (2003) 4738e4744.
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Pirinixic acid derivatives as novel dual inhibitors of microsomal prostaglandin
E2 synthase-1 and 5-lipoxygenase, J. Med. Chem. 51 (2008) 8068e8076.
[33] The Mannich reaction/elimination sequence gave unsatisfactory yields for
Michael acceptors from 1,3-indandione, These Michael acceptors were pre-
pared by direct condensation of 1,3-indandione with the appropriate alde-
hyde according to Bitter, J.; Leitich, J.; Partale, H.; Polansky, O. E.; Riemer, W.;
Ritter Thomas, U.; Schlamann, B.; Stilkerieg, B, Chem. Ber. 113 (1980)
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blood during pregnancy, Prostagl. Leukot. Essent. Fat. acids 89 (2013)
245e255.
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acid and related beta-oxo delta-enol lactone via selective O-acylation or c-
acylation of preformed enolates, Synth. Commun. 19 (1989) 3241e3247.
[23] a) X-ray structure determination of MC B (2). The data were collected at 213 K
on a Stoe IPDS diffractometer operating with graphite-monochromatized Mo
K
a
radiation. Frames of 1.5ꢀ oscillation were exposed; deriving data in the
q
range of 2e28ꢀ with
a
completeness of ~93%. Unit cell: triclinic, P-1,
a
g
¼
9.196(7),
b
¼
9.640(6), 13.89(2)Å,
a
¼
105.44(9),
b
¼
101.45(9),
¼ 107.81(8)ꢀ, V ¼ 1076.0(6)Å3. Structure solution and full least-squares
refinement with anisotropic thermal parameters of all non-hydrogen atoms
and free refinement of the hydrogen were performed using SHELX [Sheldrick,
G. M., Acta Cryst. 2008, A64, 112e22]. The final refinement result in:
R1 ¼ 0.043. Crystallographic data for the structure have been deposited with
the Cambridge Crystallographic Data Centre, CCDC, 12 Union Road, Cambridge
CB21EZ, UK. Copies of the data can be obtained free of charge on quoting the
depository number CCDC 1002467. www.ccdc.cam.ac.uk/data_request/cif.
b) The structure of isolated myrtucommulone B is not yet finally settled.
Whereas Lit. [9] and [10] assigns structure 2 to MC B, Lit. [12] takes into ac-
count that it could be an isolation artefact and Lit. [13] assigns structure 20 on
the basis of an X-ray analysis.
[35] O. Werz, D. Szellas, M. Henseler, D. Steinhilber, Nonredox 5-lipoxygenase
inhibitors require glutathione peroxidase for efficient inhibition of 5-
lipoxygenase activity, Mol. Pharmacol. 54 (1998) 445e451.
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and stimulus-dependent inhibition of cellular 5-lipoxygenase activity by
nonredox-type inhibitors, FASEB J. 17 (2003) 949e951.
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O. Werz, Licofelone suppresses prostaglandin E2 formation by interference
with the inducible microsomal prostaglandin E2 synthase-1, J. Pharmacol.
Exp. Ther. 326 (2008) 975e982.
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