SAR studies of acidic dual γ-secretase/PPARγ modulators

Article abstract of DOI:10.1016/j.bmc.2011.08.003

A novel set of dual γ-secretase/PPARγ modulators characterized by a 2-benzyl hexanoic acid scaffold is presented. Synthetic efforts were focused on the variation of the substitution pattern of the central benzene. Finally, we obtained a new class of 2,5-disubstituted 2-benzylidene hexanoic acid derivatives, which act as dual γ-secretase/PPARγ modulators in the low micromolar range. We have explored broad SAR and successfully improved the dual pharmacological activity and the selectivity profile against potential off-targets such as NOTCH and COX. Compound 17 showed an IC₅₀ Aβ42 = 2.4 μM and an EC₅₀ PPARγ = 7.2 μM and could be a valuable tool to further evaluate the concept of dual γ-secretase/ PPARγ modulators in animal models of Alzheimer's disease.

Full text of DOI:10.1016/j.bmc.2011.08.003

Bioorganic & Medicinal Chemistry 19 (2011) 5372–5382
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
SAR studies of acidic dual c-secretase/PPARc modulators
Martina Hieke a,1, Julia Ness b, Ramona Steri a, Christine Greiner c, Oliver Werz d,
a, ,1
⇑
Manfred Schubert-Zsilavecz a, Sascha Weggen b, , Heiko Zettl
⇑
a Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany
b Department of Neuropathology, Heinrich-Heine-University Duesseldorf, Moorenstrasse 5, D-40225 Duesseldorf, Germany
c Department of Pharmaceutical Analytics, Eberhard-Karls-University Tuebingen, Auf der Morgenstelle 8, D-72076 Tuebingen, Germany
d Institute of Pharmacy, Friedrich-Schiller-University Jena, Philosophenweg 14, D-07743 Jena, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel set of dual c-secretase/PPARc modulators characterized by a 2-benzyl hexanoic acid scaffold is
presented. Synthetic efforts were focused on the variation of the substitution pattern of the central ben-
zene. Finally, we obtained a new class of 2,5-disubstituted 2-benzylidene hexanoic acid derivatives, which
Received 25 May 2011
Revised 25 July 2011
Accepted 2 August 2011
Available online 6 August 2011
act as dual
successfully improved the dual pharmacological activity and the selectivity profile against potential off-
targets such as NOTCH and COX. Compound 17 showed an IC50 Ab42 = 2.4 M and an EC50 PPAR = 7.2
-secretase/PPAR modulators in
c-secretase/PPARc modulators in the low micromolar range. We have explored broad SAR and
l
c
lM
Keywords:
SAR
Alzheimer’s disease
and could be a valuable tool to further evaluate the concept of dual
animal models of Alzheimer’s disease.
c
c
Ó 2011 Elsevier Ltd. All rights reserved.
PPARc agonists
c
-Secretase modulators
Carboxylic acids
1. Introduction
proliferation and B-cell differentiation in preclinical studies in
mice.6,7 As an appealing alternative to GSIs, so-called
c-secretase
The accumulation and aggregation of amyloid b-42 peptides
(Ab42) in brain is believed to be a crucial disease causing step in
the pathology of Alzheimer’s disease (AD). Based on the amyloid
hypothesis of AD,1,2 several pharmacological intervention strate-
gies to lower Ab levels in brain have been explored in in vitro and
modulators (GSMs) have been identified.8 GSMs are small mole-
cules that selectively lower generation of the Ab42 peptides while
sparing other substrates of
istically, GSMs appear not to affect the overall enzymatic activity of
-secretase, but shift the ratio of proteolytic APP products by
c-secretase such as NOTCH. Character-
c
in vivo models of AD.3,4 Among those, targeting
c
-secretase appears
to be one of the most promising, potentially disease modifying ap-
proaches for the treatment of AD. -Secretase, an aspartyl-protease,
increasing the generation of shorter and less aggregation-prone
Ab peptides such as Ab38 at the expense of the amyloidogenic
Ab42 species.9
c
is responsible for the last step in the proteolytic release of Ab42
peptides from a large type-I transmembrane protein, the amyloid
precursor protein (APP). Besides APP, numerous other transmem-
brane proteins such as the NOTCH receptor are processed by
The first GSMs were discovered in the class of nonsteroidal anti-
inflammatory drugs (NSAIDs; e.g., ibuprofen and indomethacin;
Fig. 1).8,10 However, these drugs display very low potency against
c-secretase and limited brain permeability. Furthermore, NSAID-
c
-secretase.4,5
Initially,
type GSMs are potent inhibitors of cyclooxygenases (COX) and
chronic COX-inhibition is associated with serious side effects.11
With (R)-flurbiprofen (Fig. 1), a COX-inactive NSAID-type GSM
has been evaluated in clinical trials. Unfortunately, the compound
c-secretase inhibitors (GSIs) have been advanced in
preclinical and clinical development. Unfortunately, the use of GSIs
is limited by severe toxicity resulting from the inhibition of NOTCH
processing. For example, treatment with the GSI LY 411,575 re-
sulted in gastrointestinal bleedings, and inhibition of thymocyte
failed to meet any of the clinical end points in a phase III study.12
A
straightforward interpretation of the trial results and earlier clini-
cal observations exposes the low GSM potency (IC50 = 305 M) as a
l
major weakness of (R)-flurbiprofen, indicating that more potent
GSMs are required to achieve efficacy in the central nervous
system and better clinical outcomes.12,13
Recent developments in the field include highly active acidic
GSMs with some structural similarities to NSAIDs14,15 and structur-
ally different nonacidic GSMs.16,17 Furthermore, some in vivo
⇑
Corresponding authors. Tel.: +49 (0) 211 8104506; fax: +49 (0) 211 8104577
(S.W.); tel.: +1 401 863 6109; fax: +1 401 863 6087 (H.Z.).
E-mail addresses: sweggen@uni-duesseldorf.de (S. Weggen), heiko_zettl@brown.
edu (H. Zettl).
1
Present address: Department of Molecular Pharmacology, Physiology and
Biotechnology, Brown University, 70 Ship Street, Providence, 02903 RI, USA.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.003

M. Hieke et al. / Bioorg. Med. Chem. 19 (2011) 5372–5382
5373
Figure 1. Activity profile of selected NSAIDs and dual active c-secretase/PPARc
modulators.23,46–48
studies of potent GSMs in AD animal models were reported18–22
and a few GSMs appear to have entered clinical trials.17 A detailed
discussion can be found in a recent review of Oehlrich et al.17
We have contributed to recent advances by the characterization
pyrimidine as well as the thioether moiety of 4 by a pure carbon-
based scaffold leading to 2-(3,5-diphenethoxybenzyl)hexanoic acid
7. This new central scaffold enabled the synthetic accessibility of
systematic structural variations of the diphenylethoxy substitution
pattern at the central aromatic. In addition, the phenylethoxy resi-
dues were replaced by previously identified superior substituents,
and SAR of the thioether moiety were explored. The in vitro phar-
macology of the presented compounds includes the determination
of a novel class of GSMs that combines
c-secretase modulation
with another pharmacological activity of therapeutic potential in
AD, that is, modulation of peroxisome proliferator-activated recep-
tor-
plays a central role in lipid and glucose homeostasis.24 Evidence
indicates that activation of PPAR by small molecule agonists
c (PPARc c is a ligand-activated nuclear receptor that
).23 PPAR
of
c
-secretase and PPARc activity as well as effects on other PPAR
c
subtypes (
a
and d) and COX-1 and -2 as potential off-targets. Poten-
might exert beneficial effects in the AD brain by reducing Ab levels,
toxic pro-inflammatory molecules and cerebrovascular impair-
ment.25–29 In addition, the incidence rate of AD appears to be in-
creased around twofold in individuals with type 2 diabetes,30,31
and diabetes and insulin resistance exacerbated amyloid pathology
and cognitive dysfunction in AD animal models.32,33 Therefore,
tial cytotoxic effects were determined for all compounds, and the
influence on NOTCH processing was investigated for the lead
compounds.
2. Results and discussion
2.1. Chemistry
insulin-sensitizing effects of PPAR
cial in AD. Recently, both clinically used PPAR
one (EC50 PPAR = 0.043
M, no brain permeability, phase III)34
c
agonists might also be benefi-
c
agonists rosiglitaz-
c
l
The synthetic procedure leading to compounds 7–19 was previ-
ously described in Hieke et al.39 Of note, most of the presented
compounds also show anti-inflammatory activity by inhibition of
microsomal Prostaglandin E2 synthase-1 (mPGES-1) and/or 5-
lipoxygenase (5-LO). A detailed discussion together with the phar-
macological data can be found elsewhere.39 Preparation of
compounds 7, 8, and 11–22 is presented in Scheme 1 and was car-
ried out in 4–6 steps. Synthesis starts with an Arbuzov reaction
(step i) to yield the generally used phosphonate precursor A. Next,
the two hydroxyl groups of the respective dihydroxybenzaldehy-
des were etherified to intermediates C (step ii). Introduction of
two equal moieties (step iia) was done under Mitsunobu or Wil-
liamson-like conditions using 2 eq of the alcohol or alkylhaloge-
nide as reactants, respectively. The introduction of two different
lipophilic residues (final compounds 19, 21, and 22) was carried
out in two steps via iib and c. The first lipophilic moiety was intro-
duced under Mitsunobu or Williamson–like conditions using 1 eq
alcohol/alkylhalogenide followed by a second Mitsunobu reaction.
All intermediates C were converted to the respective ethyl 2-(ben-
zylidene)hexanoates D in a Wittig-Horner-reaction (step iii) to-
gether with the phosphonate precursor A. The resulting ester
derivatives were hydrolyzed (step v) to the respective disubsti-
tuted 2-(benzylidene)hexanoic acids 8 and 15–19. Alternatively,
and pioglitazone (EC50 PPARc = 0.58 lM, low brain permeability,
phase II)34 failed to meet clinical end points in larger trials.35,36
However, this might be related to the low blood–brain barrier per-
meability of the glitazones, and the application of PPAR
in the treatment of AD warrants further investigation.37 Impor-
tantly, beneficial effects of PPAR agonists and insulin-sensitizing
c agonists
c
drugs might manifest primarily in AD risk reduction in individuals
with type 2 diabetes, and current treatment and prevention trials
for type 2 diabetes include cognition ancillary studies to address
this possibility.38
Our previously reported class of dual
lators is based on 2-[(4,6-diphenethoxypyrimidine-2-yl)thio]hexa-
noic acid (compound 4: IC50 Ab42 = 22.8 M, EC50 PPAR = 8.3 M;
Fig. 1), which was first identified as a GSM through screening of an
in-house library of PPAR
activators.23 Based on compound 4, we
have shown structure–activity relationships (SAR) of broad struc-
c-secretase/PPARc modu-
l
c
l
c
tural variations with a focus on the optimization of the
chain and the terminal phenylalkoxy residues.
a-alkyl
In this Letter, we describe the modification of the central aro-
matic scaffold and of the spacer to the acidic function in order to ex-
plore the SAR of the substitution pattern of the central aromatic and
to further improve the potency. Initially, we replaced the central

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M. Hieke et al. / Bioorg. Med. Chem. 19 (2011) 5372–5382
Scheme 1. Synthesis of compounds 7, 8 and 11–22.39,49 Reagents and conditions: (i) triethylphosphite (1.0 eq),
a-bromo ethylhexanoate (1.0 eq), 120 °C, 12 h. (iia)
Dihydroxybenzaldehyde (1 eq), R1-OH (2.1 eq), TPP (2.5 eq), DEAD (2.5 eq), THF, rt, 1.4–24 h or dihydroxybenzaldehyde (1 eq), R1-bromide (1.2 eq), CsCO3 (1.2 eq), DMF,
50 °C, 5 h. (iib) Dihydroxybenzaldehyde (1 eq), R1-OH (1.0 eq), TPP (1.2 eq), DEAD (1.2 eq), THF, rt, 1.5–3 h or Dihydroxybenzaldehyde (1 eq), R1-bromide (1.2 eq), CsCO3
(1.2 eq), DMF, 50 °C, 5 h. (iic) B (1 eq), R2-OH (1.1 eq), TPP (1.2 eq), DEAD (1.2 eq), THF, rt. (iii) A (1.3 eq), C (1 eq), NaH (1.3 eq), THF, room temperature, 2-22 h. (iv) D, Pd/C
(10%), ethanol, 24 h, rt. (v) D or E (1 eq), LiOH ꢀ H2O (5.0 eq), THF/H2O, 50–60 °C, 6–40 h.
Scheme 2. Synthesis of 2-[(3,5-diphenethoxyphenyl)thio]hexanoic acid 9.39–42 Reagents and conditions: (i) Phloroglucinol (1.0 eq), phenylethyl bromide (2.1 eq), NaH (3 eq),
DMF, room temperature, 20 h. (ii) 9A (1.0 eq), dimethylthiocarbamoyl chloride (1.0 eq), NaH (1.2 eq), DMF, 60 °C, 5 h. (iii) 9B, solvent-free, 240 °C, 5 h. (iv) 9C (1.0 eq), NaOH
(1 mol/l; 10 ml), THF–MeOH, 80 °C, 3 h. (v) 9D (1.0 eq),
H2O, 50 °C, 3 h.
a-bromo ethylhexanoate (1.2 eq), NaH (1.2 eq), DMF, room temperature, 3 h. (vi) 9E (1.0 eq), LiOH ꢀ H2O (5.0 eq), THF/

M. Hieke et al. / Bioorg. Med. Chem. 19 (2011) 5372–5382
5375
Scheme 3. Synthesis of 2-(3,5-diphenethoxyphenoxy)hexanoic acid 10.39,40 Reagents and conditions: (i) Phloroglucinol (1.0 eq), NaH (1.6 eq),
a-bromo ethylhexanoate
(0.8 eq), DMF, room temperature, 8 h. (ii) 10A (1.0 eq), phenylethanol (2.1 eq), DEAD (2.5 eq), TPP (2.5 eq), THF, room temperature, 4 h. (iii) 10B (1.0 eq), LiOH ꢀ H2O (5.0 eq),
THF/H2O, 50 °C, 24 h.
they were first hydrogenated to the corresponding ethyl 2-
(benzyl)hexanoate E (step iv: Pd/C, H2) and subsequently hydro-
lyzed (step v) with LiOH and H2O to yield the substituted benzyli-
dene hexanoic acids 7 and 11–14, 20–22.
description of all experimental procedures has been published
recently.23 In brief, PPAR activity of the final compounds 7–22
was tested in a cellular luciferase-based PPAR transactivation as-
say for all three PPAR subtypes (
compounds, we did not observe significant activity on PPAR
and PPARd (tested concentration: 10 M). Consequently, the fol-
lowing discussion is restricted to PPAR activity. For characteriza-
tion of -secretase activity, the levels of Ab38, Ab40, and Ab42
a, c, and d). Within this set of
2-[(3,5-Diphenethoxyphenyl)thio]hexanoic acid 9 was prepared
in six steps as shown in Scheme 2. First, two of the three hydroxyl
groups of phloroglucinol were etherified (step i).40 The product 9A
was isolated from a mixture of mono-, di-, and trisubstituted
phloroglucinol ethers in low yields. The remaining hydroxyl group
was activated with dimethylthio-carbamoylchloride and sodium
hydride (step ii) to yield 9B. Heating at 240 °C led to a Newman-
Kwart-Rearrangement (step iii),41 which yielded the protected
thiol derivative. Deprotection with NaOH resulted in the free thiol
derivative 9D (step iv),42 which reacted in a nucleophilic substitu-
a
l
c
c
peptides were measured in cell supernatants by ELISA. Inhibition
of COX was tested in a cell-free assay using isolated ovine COX-1
and human recombinant COX-2 enzymes. Potential effects on
proteolytic processing of NOTCH-1 were investigated for lead
compounds (16 and 17) using a previously described reporter
assay.23 Cytotoxicity of all compounds was determined using ala-
marBlue reagent.23
tion with a-bromo ethylhexanoate to 9E (step v). Finally, hydroly-
sis (step vi) yielded compound 9.
Synthesis of 2-(3,5-diphenethoxyphenoxy)hexanoic acid 10 is
presented in Scheme 3. The compound was prepared in three steps.
First, one hydroxyl group of phloroglucinol was etherified with
2.3. SAR
Previously, we identified compound 4, a subtype-selective mod-
ethyl
a
-bromohexanoate and sodium hydride to 10A (step i).40 Sec-
ulator of PPAR
NSAID-like GSM with an IC50 Ab42 = 22.8
(Fig. 1), and investigated SAR of the
c
(EC50 = 8.3
l
M, maximal activation 60%) and
M; EC50 Ab38 = 11.3
-alkyl substituent and the
ond, the two remaining hydroxyl groups were etherified with 2-
phenylethanol under Mitsunobu conditions (step ii) to 10B, which
was followed by hydrolysis (step iii) to the carboxylic acid 10.
l
lM
a
phenylalkyl residues.23 In this study, we focused our efforts on
the central part of 4 and replaced the pyrimidine ring and the thi-
oether substructure by a pure carbon-based scaffold (compound 7).
This initial exchange (2-(3,5-diphenethoxybenzyl)hexanoic acid 7)
2.2. Biology
Biological characterization was carried out with established
led to a weaker M) and slightly
c
-secretase (IC50 Ab42 = 38.4
l
cell-based assays for PPAR and
c-secretase activity. A detailed
higher PPAR modulating activity (EC50 = 4.7 lM; see Table 1).
c
Table 1
In vitro pharmacological characterization of compounds 7–10; variation of exocyclic thioether moiety
Compound
X
Y
PPAR
c
activation
Ab42 inhibition (IC50 in
lM)
Ab38 activation (EC50 in lM)
EC50 in
lM
Maximal activation in (%)
7
8
9
10
CH2–
CH@
S
CH
C
CH
CH
4.7 1.8
7.8 2.7
3.4 0.1
4.8 0.9
71 10
95 15
38.4 1.1
37.9 10.9
32.7 0.4
33.7 0.8
28.1 3.3
27.3 10.2
22.2 3.2
26.4 1.4
73
46
1
4
O

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M. Hieke et al. / Bioorg. Med. Chem. 19 (2011) 5372–5382
Table 2
In vitro pharmacological characterization of compounds 11–14; variation of the substitution pattern of the central benzene
Compound Structure
PPAR
c
activation
c-secretase modulation
Activation (EC50 in M) Maximal activation in (%) Ab42 inhibition (IC50 in
l
lM) Ab38 activation (EC50 in lM)
7
4.7 1.8
71 10
38.4 1.1
28.1 3.3
11
3.7 0.8
79
7
>40
>40
12
2.2 0.7
100
9
>40
>40
13
Inactive @ 10
lM
Inverse modulation; IC50 Ab38 ꢁ5 lM
14
10.8 0.8
97
5
19.1 5.2
14.5 5.4
Resulting from a synthetic precursor of 7, we have also prepared
the corresponding 2-(3,5-diphenethoxybenzylidene)hexanoic acid
8 with a double bond connecting the acidic head group with the
pounds 11–14). This subset of compounds yielded clear SAR for
both -secretase and PPAR modulation (see Table 2). Introduction
of the phenylethoxy moieties in 2,3- and 3,4-position (compound
11 and 12) diminished GSM activity (IC50 Ab42 >40 M), but led
to two of the most potent PPAR modulators presented in this
study (EC50 PPAR = 11: 3.7 M, 12: 2.2 M).
c
c
benzene core. This compound showed similar
Ab42 = 37.9 M) and PPAR (EC50 = 7.8 M) modulating activity
as compared to 7.
c
-secretase (IC50
l
l
c
l
c
c
l
l
Next, we have investigated the role of the exocyclic methylene
group of 7 by synthesizing the respective thioether (9) and ether
(10) analogs. Both exocyclic heteroatoms led to a slight improve-
ment in GSM activity with almost identical pharmacological data
The 2,4-diphenethoxy benzene derivative 13 displayed an inter-
esting and for this series unique pharmacological profile: inverse
modulation of
of Ab42 levels). Inverse modulation of
c
-secretase (i.e., reduction of Ab38 and elevation
-secretase was previously
c
for the thioether analog 9 (IC50 Ab42 = 32.7
3.4 M) and the ether analog 10 (IC50 Ab42 = 33.7
PPAR = 4.8 M; Table 1).
Our particular focus in this study was the substitution pattern
of the central benzene of 7. Starting with commercially available
dihydroxybenzaldehydes, we have systematically varied the posi-
tions of the phenylethoxy residues at the benzene ring. Based on
initially introduced 3,5-diphenethoxy-substituted 7, we have pre-
pared the 2,3-, 2,4-, 2,5- and 3,4- disubstituted analogs (com-
l
M, EC50 PPAR
c
observed for some drugs including fenofibrate and celecoxib, and
certain isoprenoids (GGPP, FPP).43 Interestingly, 13 is the first re-
ported inverse GSM containing a carboxylic acid as structural
characteristic.
l
l
M, EC50
c
l
The most favorable pharmacological profile showed the 2,5-
substitution pattern (compound 14).
improved to an Ab42 IC50 of 19.1
PPAR activity (EC50 = 10.8 M), compound 14 was an ideal start-
ing point for further structural optimization.
c
-Secretase modulation was
l
M. With its low micromolar
c
l

M. Hieke et al. / Bioorg. Med. Chem. 19 (2011) 5372–5382
5377
Table 3
In vitro pharmacological characterization of compounds 15–22; structural modifications based on compounds 14 and 12
PPAR
c
activation
c-secretase modulation
Compound
R1
R2
EC50 in
1.8
l
M
Maximal activation in (%)
Ab42 inhibition (IC50 in
lM)
Ab38 activation (EC50 in lM)
Modifications based on compound 14
15
47
M
6
15.4 5.8
3.9 0.9
6.7 2.1
3.1 1.2
16
17
75 8% @ 10
7.2 1.3
l
86 12
2.4 0.4
1.2 0.3
1.6 0.4
1.1 0.1
18
28 8% @ 10
27 5% @ 10
l
l
M
19
M
2.7 0.2
2.5 0.7
Modifications based on compound 12
20
1.1 0.1
69
3
>40
>40
21
2.5 0.4
61
5
>40
>40
22
1.5 0.5
128 18
>40
>40
Next, we synthesized the corresponding 2-(2,5-dipheneth-
oxybenzylidene)hexanoic acid 15. Interestingly, 15 was superior
sized (compound 18), which is the most potent GSM presented
in this study (IC50 Ab42 = 1.2 M). However, PPAR activity was
clearly diminished by this modification (28% activation at
10 M). Finally, a 2-(p-trifluormethyl)phenylmethoxy and a 5-
cyclohexylethoxy moiety were combined in compound 19, which
showed similar potency as 18 (IC50Ab42 = 2.7 M, PPAR 27% acti-
vation at 10 M).
For further investigation of the SAR of PPAR
l
c
to its hydrogenated analog 14 for both GSM and PPAR
c activity
(IC50 Ab42 = 15.4 M, EC50 PPAR = 1.8 M). Therefore, we have
l
c
l
l
used compound 15 as template for further structural optimization
and introduced previously identified privileged substructures for
GSM activity.23 Compounds 5 and 6 bearing cyclohexylethoxy-
and p-(trifluoromethyl)phenylethoxy moieties ( Fig. 1), respec-
l
c
l
c
-selective com-
tively, represented the most potent dual
c
-secretase/PPAR
c
pound 12, three additional compounds with a 3,4-disubstitution
pattern were synthesized (Table 3). In 20, both phenylethoxy res-
idues were replaced by cyclohexylethoxy residues. Furthermore,
the 3,4-disubstitution pattern allowed the stepwise introduction
of two different substituents. This was realized in compounds 21
and 22, where one of each phenylethoxy residues was replaced
by a cyclohexylethoxy in 3- and 4-position, respectively. In sum-
mary, 3,4-dicyclohexylethoxy substitution (20) showed the high-
modulators of our previous study. Consequently, we transferred
these substructures to 2-(2,5-diphenethoxybenzylidene)hexanoic
acid 15.
The obtained compounds 16 (cyclohexyl) and 17 (p-trifluoro-
methyl) showed further increased GSM activity (IC50 Ab42 = 16:
3.9
lation (EC50 <10
shorter p-(trifluoromethyl)phenylmethoxy residues was synthe-
lM, 17: 2.4
lM) combined with low micromolar PPAR
c modu-
lM; see Table 3). Additionally, a derivative with
est activity on PPARc (EC50 = 1.1 lM).

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M. Hieke et al. / Bioorg. Med. Chem. 19 (2011) 5372–5382
2.4. Selectivity profile
Cytotoxicity was determined in the concentration range 10–
100 M using alamarBlue reagent (Fig. 3). None of the presented
compounds displayed detectable cytotoxicity up to a concentration
of 40 M.
l
Most of the acidic NSAID-type GSMs show considerable COX
inhibition with micromolar or even nanomolar activity (Fig. 1).
We screened our set of compounds for their COX inhibitory activity
l
in order to facilitate the design of COX-sparing dual
c
-secretase/
2.5. Discussion
PPAR modulators. COX-1 and -2 inhibition was determined for
c
every presented compound and is summarized in Table 4. Lead
compound 4 is a weak COX inhibitor with an IC50 COX-1 of
In this study, we have investigated the SAR of the central part of
our previously identified dual c-secretase/PPARc
modulator 4.23
16.7
l
M and an IC50 COX-2 >40
l
M. We determined the remaining
By replacement of the central pyrimidine with benzene, we were
able to systematically vary the substitution pattern of the central
aromatic ring. Shifting the ether moieties around the benzene led
to a set of molecules with diverse biological activities: While com-
pound 7 (3,5-substituted) and 14 (2,5-substituted) displayed a
COX-activity at a test concentration of 10
lM (Table 4). None of the
presented compounds reduced the remaining activity lower than
60% for both COX-1 and -2. The most active GSMs 16–19 were even
weaker COX inhibitors with a remaining activity above 75% for
COX-1 and no inhibition of COX-2 at 10
In addition, potential effects on NOTCH processing and signal-
ing were determined for the two most potent dual modulators
l
M (>100%).
micromolar active dual
the 2,3- and 3,4-substituted compounds (11 and 12) were the most
potent PPAR agonists, but inactive on -secretase. Interestingly,
c-secretase/PPARc modulation profile,
c
c
16 and 17 (Fig. 2). NOTCH is a substrate of
bition (e.g., by nonselective -secretase inhibitors) causes severe
side effects.9 Inhibition of NOTCH-1 processing was tested in a
cell-based reporter assay in a concentration range of 0–30
M.44
Importantly, NOTCH-1 reporter activity was not significantly af-
fected by the selected compounds indicating at least an 8- to 12-
fold window of selectivity.
c
-secretase and its inhi-
we obtained the first inverse GSM (13) containing a carboxylic acid
group by introduction of a 2,4-substitution pattern.
c
The most potent GSM 18 with an IC50 of 1.2
by a 2,5-di-p-trifluoromethylbenzyl substitution pattern, whereas
the most potent dual -secretase/PPAR modulators contain a
lM is characterized
l
c
c
2,5-dicyclohexylethoxy-substitution (16) and a 2,5-di-p-trifluoro-
methylphenethoxy-substitution (17).
Table 4
COX inhibition of compounds 7–22 (remaining activity at 10
l
M
SE) as described in Hieke et al.39
Compound
COX-1 inhibition (r.a.@10
l
M)
COX-2 inhibition (r.a.@10
lM)
Compound
COX-1 inhibition (r.a.@10
lM)
COX-2 inhibition (r.a.@10 lM)
7
8
9
10
11
12
13
14
78.7 12.9
81.4 8.5
71.3 2.1
71.6 3.7
66.1 9.8
95.8 21.7
99.1 12.1
63.8 16.6
78.4 11.9
81.4 8.9
109.7 4.9
92.5 2.7
89.6 13.4
86.4 11.8
110.3 5.8
66.5 11.0
15
16
17
18
19
20
21
22
68.0 12.1
76.4 7.3
81.0 11.8
82.6 4.7
92.3 9.2
73.8 15.1
69.1 6.9
82.0 11.7
94.4 10.7
123.4 19.5
102.4 7.3
102.0 5.5
106.1 11.9
79.1 13.2
91.5 5.7
96.6 16.4
Figure 2. Impact on NOTCH-processing.
Figure 3. Cytotoxicity.

M. Hieke et al. / Bioorg. Med. Chem. 19 (2011) 5372–5382
5379
In summary, we have explored broad SAR and obtained selec-
tive GSMs (such as 18) as well as dual -secretase/PPAR modula-
tors (such as 16 and 17), both with low micromolar activity.
Compared to our previously reported compounds, the off-target
selectivity towards COX was clearly improved. With their balanced
25.76 (4C, Cyclohex-C3/5H2), 26.04 (2C, Cyclohex-C4H2), 28.83
(Bu-CH2), 31.27 (Bu-CH2), 32.71 (4C, Cyclohex-C2/6H2), 34.03 (2C,
Cyclohex-CH2), 36.20 (2C, Cyclohex-C1H), 37.44 (Prop-C3), 46.98
(Prop-C2), 59.47 (O–CH2), 66.27 + 66.43 (2C, Ph-O–CH2), 113.85
(Ph-C2), 114.57 (Ph-C5), 120.91 (Ph-C6), 131.86 (Ph-C1), 146.97
(Ph-C4), 148.29 (Ph-C3), 174.78 (COO–). MS (ESI+) = m/e = 487.7
[M+1]+.
Characterization of final product 2-(3,4-bis(2-cyclohexyleth-
oxy)benzyl)hexanoic acid 20: 1H NMR (300.13 MHz, (CD3)2SO):
d = 0.79–0.99 (m, 7H, Bu-CH3 + Bu-CH2), 1.05–1.75 (m, 28H, Bu-
CH2 + Cyclohex-H + –CH2), 2.44–2.75 (m, 3H, Prop-C3H2 + C2H),
3.88–3.98 (q, 4H, J = 6.2 Hz, Ph-O–CH2), 6.61–6.64 (dd, 1H, J = 1.8;
8.1 Hz, Ph-C6), 6.75–6.81 (m, 2H, Ph-C2/5), 12.05 (br s, 1H, COOH).
13C NMR (75.44 MHz, (CD3)2SO): d = 13.78 (Bu-CH3), 22.04 (Bu-
CH2), 25.77 (4C, Cyclohex-C3/5), 26.05 (2C, Cyclohex-C4), 28.92
(Bu-CH2), 31.16 (Bu-CH2), 32.72 (4C, Cyclohex-C2/6), 34.04 (2C,
Cyclohex-CH2), 36.24 (2C, Cyclohex-C1), 37.31 (Prop-C3), 46.90
(Prop-C2), 66.28 + 66.42 (2C, Ph-O–CH2), 113.77 (Ph-C2), 114.63
(Ph-C5), 120.91 (Ph-C6), 146.92 (Ph-C4) 148.27 (Ph-C3), 176.43
(COO–). MS (ESI+) = m/e = 457.6 [M+1]+. Combustion Anal. Calcd
C29H46O4 [458.67]: C, 75.94; H, 10.11. Found: C, 76.07; H,
10.26. Difference: C, 0.13; H, 0.15.
c
c
low micromolar dual
as 16 and 17 could be valuable tools to further investigate the con-
cept of dual -secretase/PPAR modulators in AD animal models.
c-secretase/PPARc activity, compounds such
c
c
3. Experimental
3.1. Compounds and chemistry
The structures of all tested compounds were confirmed by 1H
NMR, 13C NMR and mass spectrometry (ESI). Compounds 7–19
were described previously,39 analytical data of compounds 20–22
(and their intermediates) are presented herein. The purities of
the tested compounds were determined by combustion analysis
and are 98% or higher. Commercial chemicals and solvents were re-
agent grade and used without further purification. 1H and 13C NMR
spectra were measured in DMSO-d6 or CDCl3 on a Bruker ARX 300
or an AV 200 spectrometer. Chemical shifts are reported in parts
per million (ppm) using tetramethylsilane (TMS) as internal stan-
dard. Mass spectra were obtained on a Fissous Instruments VG
Platform 2 spectrometer measuring in the positive- or negative-
ion mode (ESI–MS system). Combustion analysis was performed
by the Microanalytical Laboratory of the Institute of Organic Chem-
istry and Chemical Biology, Goethe-University Frankfurt, on an Ele-
mental Vario Micro Cube. The synthetic procedure for compounds
7–22 is illustrated in Schemes 1–3.
3.1.1.2. Intermediates of compound 21.
Characterization of
3-hydroxy-4-(2-cyclohexylethoxy)benzaldehyde (21B): 1H NMR
(300.13 MHz, (CD3)2SO): d = 0.92–0.96 (m, 2H, Cyclohex-H), 1.13–
1.20 (m, 3H, Cyclohex-H), 1.25–1.34 (m, 1H, Cyclohex-H), 1.58–
1.77 (m, 8H, Cyclohex-H + –CH2), 4.00–4.11 (m, 4H, Ph-O–CH2),
7.10–7.12 (d, 1H, Ph-C5), 7.23–7.24 (d, 1H, Ph-C2), 7.37–7.38 (dd,
1H, Ph-C6), 9.44 (s, 1H, –OH), 9.85 (s, 1H, CHO). 13C NMR
(75.44 MHz, (CD3)2SO): d = 25.67 (Cyclohex-C3/5), 26.01 (Cyclo-
hex-C4), 32.61 (Cyclohex-C2/6), 33.80 (Cyclohex-C1), 35.84 (Cyclo-
hex-CH2), 66.41 + 66.54 (Ph-O–CH2), 112.44 (Ph-C5), 113.55 (Ph-
C2), 124.29 (Ph-C6), 129.65 (Ph-C1), 147.08 (Ph-C3), 152.73 (Ph-
C4), 191.33 (CHO). MS (ESIꢂ) = m/e = 246.9 [Mꢂ1]ꢂ.
3.1.1. Analytical characterization of compounds 20–22
3.1.1.1. Intermediates of 20.
cyclohexylethoxy)benzaldehyde
Characterization of 3,4-bis(2-
(20C): 1H NMR (300.13 MHz,
(CD3)2SO): d = 0.87–0.96 (m, 4H, Cyclohex-H), 1.13–1.20 (m, 6H,
Cyclohex-H), 1.25–1.34 (m, 2H, Cyclohex-H), 1.58–1.77 (m, 16H,
Cyclohex-H + –CH2), 4.01–4.11 (m, 4H, Ph-O–CH2), 7.13–7.16 (d,
1H, Ph-C5), 7.36–7.37 (d, 1H, Ph-C2), 7.48–7.52 (dd, 1H, Ph-C6),
9.80 (s, 1H, CHO). 13C NMR (75.44 MHz, (CD3)2SO): d = 25.74
(Cyclohex-C3/5), 26.02 (Cyclohex-C4), 32.65 (Cyclohex-C2/6), 34.09
(Cyclohex-CH2), 66.45 + 66.54 (Ph-O–CH2), 111.37 (Ph-C5), 112.40
(Ph-C2), 125.76 (Ph-C6), 129.52 (Ph-C1), 148.90 (Ph-C3), 154.01
(Ph-C4), 191.25 (CHO). MS (ESI+) = m/e = 359.0 [M+1]+.
Characterization of ethyl 2-(3,4-bis(2-cyclohexylethoxy)benzyli-
dene)hexanoate (20D): 1H NMR (300.13 MHz, (CD3)2SO): d = 0.84–
1.70 (m, 38H, Cyclohex-H + –CH2, Bu-CH3, Bu-CH2 + Et-CH3),
2.47–2.50 (m, 2H, Bu-CH2), 3.95–4.02 (m, 4H, Ph-O–CH2), 4.06–
4.20 (m, 4H, O–CH2), 6.96–7.02 (m, 3H, Ph-C2/5/6), 7.49 (s, 1H,
Ph-CH). 13C NMR (75.44 MHz, (CD3)2SO): d = 13.68 (Bu-CH3),
21.46 (Bu-CH2), 25.77 (4C, Cyclohex-C3/5), 26.04 (2C, Cyclohex-
C4), 26.89 (Bu-CH2), 30.77 (Bu-CH2), 32.70 (4C, Cyclohex-C2/6),
34.76 (2C, Cyclohex-CH2), 36.16 (2C, Cyclohex-C1), 60.23 (O–
CH2), 66.36 + 66.49 (2C, Ph-O–CH2), 113.36 (Ph-C2), 114.69 (Ph-
C5), 121.63 (Ph-C6), 127.63 (Ph-C1), 130.72 (C@C–COO), 138.07
(Ph-C@C), 148.22 (Ph-C3), 149.21 (Ph-C4), 167.68 (COO–). MS
(ESI+) = m/e = 485.8 [M+1]+.
Characterization of 4-(2-Cyclohexylethoxy)-3-phenethoxybenzal-
dehyde (21C): 1H NMR (300.13 MHz, (CD3)2SO): d = 0.92–1.75 (m,
13H, Cyclohex-H + –CH2), 3.02–3.06 (t, 2H, J = 6.5 Hz, Ph0-CH2),
4.05–4.09 (t, 2H, J = 6.5 Hz, Ph-C4–O–CH2), 4.19–4.23 (t, 2H,
J = 6.6 Hz, Ph-C3–O–CH2), 7.14–7.38 (m, 7H, Ph-C2/5H + Ph0-H),
7.49–7.52 (dd, 1H, J = 1.8; 8.3 Hz, Ph-C6), 9.79 (s, 1H, CHO). 13C
NMR (75,44 MHz, (CD3)2SO): d = 25.74 (Cyclohex-C3/5), 26.01
(Cyclohex-C4), 32.64 (Cyclohex-C2/6), 33.96 (Cyclohex-C1), 34.92
(Cyclohex-CH2), 35.90 (Ph0-CH2), 66.49 (Ph-C4–O–CH2), 68.94 (Ph-
C3–O–CH2), 111.29 (Ph-C5), 112.37 (Ph-C2), 125.87 (Ph-C6),
126.22 (Ph0-C4), 128.13 (Ph0-C2/6), 129.07 (Ph0-C3/5), 129.43 (Ph-
C1), 138.42 (Ph0-C1), 148.34 (Ph-C3), 153.86 (Ph-C4), 191.31
(CHO). MS (ESI+) = m/e = 353.2 [M+1]+.
Characterization of ethyl 2-(4-(2-cyclohexylethoxy)-3-pheneth-
oxybenzylidene)hexanoate (21D): 1H NMR (300.13 MHz, (CD3)2SO):
d = 0.80–0.85 (t, 3H, J = 7.0 Hz, Bu-CH3), 0.91–1.32 (m, 10H, Et-
CH3 + Bu-CH2 + Cyclohex-H), 1.41–1.75 (m, 10H, Cyclohex-H + –
CH2), 2.43–2.50 (m, 1H, Bu-CH2), 3.00–3.04 (t, 2H, J = 6.7 Hz, Ph0-
CH2), 3.97–4.01 (m, 2H, Ph-C4–O–CH2), 4.13–4.20 (m, 4H, O–
CH2 + Ph-C3–O–CH2), 6.96–7.03 (m, 3H, Ph-C2/5/6), 7.17–7.34 (m,
5H, Ph0-H), 7.48 (s, 1H, C@CH). 13C NMR (75.44 MHz, (CD3)2SO):
d = 13.64 (Bu-CH3), 14.14 (Et-CH3), 22.36 (Bu-CH2), 25.73 (2C,
Cyclohex-C3/5), 26.03 (Cyclohex-C4), 26.86 (Bu-CH2), 30.72
(Bu-CH2), 32.67 (2C, Cyclohex-C2/6), 33.99 (Cyclohex-CH2), 34.76
(Ph0-CH2), 36.09 (Cyclohex-C1), 60.22 (O–CH2), 66.26 (Ph-C4–O–
CH2), 69.02 (Ph-C3–O–CH2), 113.25 (Ph-C2), 114.64 (Ph-C5),
122.93 (Ph-C6), 126.19 (Ph-C1), 127.55 (Ph0-C4), 128.13 (2C, Ph0-
Characterization
of
ethyl
2-(3,4-bis(2-cyclohexyleth-
oxy)benzyl)hexanoate (20E): 1H NMR (300.13 MHz, (CD3)2SO):
d = 0.79–0.84 (t, 3H, J = 7.1 Hz, Bu-CH3), 0.89–1.24 (m, 15H, Et-
CH3, Bu-CH2, Cyclohex-H), 1.38–1.77 (m, 20H, Cyclohex-H + –CH2,
Bu-CH2), 2.53–2.73 (m, 2H, Prop-C2H + C3H), 3.39–3.46 (m, 1H,
Prop-C3H), 3.87–4.00 (m, 6H, Ph-O–CH2 + O–CH2), 6.59–6.62 (dd,
1H, J = 1.6; 8.1 Hz, Ph-C6), 6.72–6.73 (d, 1H, J = 1.6 Hz, Ph-C2),
6.79–6.81 (d, 1H, J = 8.1 Hz, Ph-C5). 13C NMR (75.44 MHz,
(CD3)2SO): d = 13.73 (Bu-CH3), 14.04 (Et-CH3), 21.96 (Bu-CH2),
C
2/6), 128.99 (2C, Ph0-C3/5), 130.69 (C@C–COO), 138.02 (Ph-C@C),
138.44 (Ph0-C1), 147.66 (Ph-C3), 149.05 (Ph-C4), 169.01 (COO–).
MS (ESI+) = m/e = 479.5 [M+1]+.

5380
M. Hieke et al. / Bioorg. Med. Chem. 19 (2011) 5372–5382
Characterization of ethyl 2-(4-(2-cyclohexylethoxy)-3-pheneth-
3.04 (t, 2H, J = 6.5 Hz, Ph0-CH2), 3.92–3.97 (t, 2H, J = 6.6 Hz, Ph-C3-
O–CH2), 4.13–4.20 (m, 4H, O–CH2 + Ph-C4-O–CH2), 6.95–7.04 (m,
3H, Ph-C2/5/6), 7.18–7.35 (m, 5H, Ph0-H), 7.49 (s, 1H, C@CH). 13C
NMR (75.44 MHz, (CD3)2SO): d = 13.66 (Bu-CH3), 13.88 (Et-CH3),
21.44 (Bu-CH2), 25.72 (2C, Cyclohex-C3/5), 26.29 (Cyclohex-C4),
26.77 (Bu-CH2), 30.75 (Bu-CH2), 32.67 (2C, Cyclohex-C2/6), 34.05
(Cyclohex-CH2), 34.73 (Ph0-CH2), 36.15 (Cyclohex-C1), 60.21 (O–
CH2), 66.47 (Ph-C3–O–CH2), 68.91 (Ph-C4–O–CH2), 113.36 (Ph-C2),
114.67 (Ph-C5), 122.84 (Ph-C6), 126.18 (Ph-C1), 127.80 (Ph0-C4),
128.11 (2C, Ph0-C2/6), 129.01 (2C, Ph0-C3/5), 130.81 (C@C–COO),
137.99 (Ph-C@C), 138.43 (Ph0-C1), 148.85 (Ph-C4), 148.85 (Ph-C3),
167.64 (COOꢂ). MS (ESI+) = m/e = 479.4 [M+1]+.
oxybenzyl)hexanoate (21E): 1H NMR (300.13 MHz, (CD3)2SO):
d = 0.78–0.83 (t, 3H, J = 7.1 Hz, Bu-CH3), 0.86–0.97 (m, 2H, Bu-
CH2), 1.01–1.06 (t, 3H, J = 7.0 Hz, Et-CH3), 1.15–1.77 (m, 17H, Bu-
CH2 + Cyclohex-H + –CH2), 2.50–2.72 (m, 3H, Prop-C3H2 + –C2H),
2.97–3.01 (t, 2H, J = 6.7 Hz, Ph0-CH2), 3.85–3.98 (m, 4H, Ph-C4–O–
CH2 + OCH2), 4.07–4.12 (t, 2H, J = 6.8 Hz, Ph-C3–O–CH2), 6.60–
6.63 (dd, 1H, J = 1.9; 8.1 Hz, Ph-C6), 6.74 (d, 1H, J = 1.9 Hz, Ph-C2),
6.80–6.82 (d, 1H, J = 8.1 Hz, Ph-C5), 7.17–7.34 (m, 5H, Ph0-H). 13C
NMR (75.44 MHz, (CD3)2SO): d = 13.73 (Bu-CH3), 14.04 (Et-CH3),
21.96 (Bu-CH2), 25.73 (4C, Cyclohex-C3/5), 26.04 (2C, Cyclohex-
C4), 28.82 (Bu-CH2), 31.30 (Bu-CH2), 32.70 (4C, Cyclohex-C2/6),
33.96 (2C, Cyclohex-C1), 35.13 (2C, Cyclohex-CH2), 36.27 (Ph0-
CH2), 37.41 (Prop-C3), 46.97 (Prop-C2), 59.47 (O–CH2), 66.40 (Ph-
C4–O–CH2), 69.04 (Ph-C3-O–CH2), 113.80 (Ph-C2), 114.52 (Ph-C5),
121.09 (Ph-C6), 126.16 (Ph0-C4), 128.13 (2C, Ph0-C2/6), 128.90 (2C,
Ph0-C3/5), 131.86 (Ph-C1) 138.53 (Ph0-C1), 146.88 (Ph-C4), 147.97
(Ph-C3), 174.79 (COO–). MS (ESI+) = m/e = 481.5 [M+1]+.
Characterization of ethyl 2-(3-(2-cyclohexylethoxy)-4-pheneth-
oxybenzyl)hexanoate (22E): 1H NMR (300.13 MHz, (CD3)2SO):
d = 0.79–0.83 (t, 3H, J = 7.1 Hz, Bu-CH3), 0.87–1.73 (m, 23H, Bu-
CH2, Et-CH3, Cyclohex-H + –CH2), 2.52–2.73 (m, 3H, Prop-C3H2 + –
C2H), 2.95–2.99 (t, 2H, J = 6.7 Hz, Ph0-CH2), 3.87–3.99 (m, 4H, Ph-
C3–O–CH2 + OCH2), 4.06–4.11 (t, 2H, J = 6.8 Hz, Ph-C4–O–CH2),
6.59–6.62 (dd, 1H, J = 1.8; 8.2 Hz, Ph-C6), 6.73 (d, 1H, J = 1.8 Hz,
Ph-C2), 6.80–6.83 (d, 1H, J = 8.1 Hz, Ph-C5), 7.17–7.33 (m, 5H, Ph0-
H). 13C NMR (75.44 MHz, (CD3)2SO): d = 13.73 (Bu-CH3), 14.05
(Et-CH3), 21.96 (Bu-CH2), 25.73 (2C, Cyclohex-C3/5), 26.04 (Cyclo-
hex-C4), 28.82 (Bu-CH2), 31.27 (Bu-CH2), 32.69 (2C, Cyclohex-C2/
6), 33.96 (Cyclohex-C1), 35.17 (Cyclohex-CH2), 36.21 (Ph0-CH2),
37.42 (Prop-C3), 46.95 (Prop-C2), 59.48 (O–CH2), 66.25 (Ph-C3–O–
CH2), 69.11 (Ph-C4–O–CH2), 113.77 (Ph-C2), 114.53 (Ph-C5),
120.89 (Ph-C6), 126.13 (Ph0-C4), 128.11 (2C, Ph0-C2/6), 128.99 (2C,
Ph0-C3/5), 132.05 (Ph-C1), 138.58 (Ph0-C1), 146.65 (Ph-C4), 148.20
(Ph-C3), 174.78 (COO–). MS (ESI+) = m/e = 481.5 [M+1]+.
Characterization of 2-(3-(2-cyclohexylethoxy)-4-phenethoxyben-
zyl)hexanoic acid 22: 1H NMR (300.13 MHz, (CD3)2SO): d = 0.79–
0.85 (t, 3H, J = 7.0 Hz, Bu-CH3), 0.91–1.74 (m, 20H, Bu-CH2, Cyclo-
hex-H + –CH2), 2.47–2.71 (m, 3H, Prop-C3H2 + –C2H), 2.96–3.00 (t,
2H, J = 6.6 Hz, Ph0-CH2), 3.87–3.91 (t, 2H, Ph-C3–O–CH2), 4.07–
4.11 (t, 2H, J = 6.7 Hz, Ph-C4–O–CH2), 6.61–6.64 (dd, 1H, J = 1.8;
8.1 Hz, Ph-C6), 6.76 (d, 1H, J = 1.8 Hz, Ph-C2), 6.80–6.83 (d, 1H,
J = 8.2 Hz, Ph-C5), 7.20–7.34 (m, 5H, Ph0-H), 12.06 (br s, 1H, COOH).
13C NMR (75.44 MHz, (CD3)2SO): d = 13.77 (Bu-CH3), 22.04 (Bu-
CH2), 25.74 (2C, Cyclohex-C3/5), 26.06 (Cyclohex-C4), 28.91 (Bu-
CH2), 31.15 (Bu-CH2), 32.72 (2C, Cyclohex-C2/6), 34.00 (Cyclohex-
C1), 35.23 (Cyclohex-CH2), 36.27 (Ph0-CH2), 37.31 (Prop-C3), 46.88
(Prop-C2), 66.36 (Ph-C3–O–CH2), 69.19 (Ph-C4–O–CH2), 113.87
(Ph-C2), 114.73 (Ph-C5), 120.94 (Ph-C6), 126.14 (Ph0-C4), 128.13
(2C, Ph0-C2/6), 128.99 (2C, Ph0-C3/5), 132.53 (Ph-C1) 138.62 (Ph0-
C1), 146.66 (Ph-C4), 148.26 (Ph-C3), 176.40 (COOH). MS (ESI+) =
m/e = 453.5 [M+1]+. Combustion Anal. Calcd C29H40O4 [452.29]: C,
76.95; H, 8.91. Found: C, 76.69; H, 8.87. Difference: C, 0.26; H, 0.04.
Characterization of final product 2-(4-(2-cyclohexylethoxy)-3-phe-
nethoxybenzyl) hexanoic acid 21: 1H NMR (300.13 MHz, (CD3)2SO):
d = 0.79–0.83 (t, 3H, J = 7.0 Hz, Bu-CH3), 0.85–1.30 (m, 9H, Bu-
CH2 + Cyclohex-H), 1.34–1.73 (m, 10H, Cyclohex-H + CH2 + Bu-
CH3), 2.45–2.59 (m, 2H, Prop-C2H + C3H), 2.66–2.74 (dd, 1H,
J = 5.0; 8.4 Hz, Prop-C3H), 2.96–3.01 (t, 2H, J = 6.7 Hz, Ph0-CH2),
3.86–3.90 (t, 2H, J = 6.4 Hz, Ph-C4–O–CH2), 4.08–4.12 (t, 2H,
J = 6.8 Hz, Ph-C3–O–CH2), 6.62–6.66 (dd, 1H, J = 1.8; 8.1 Hz, Ph-
C6), 6.76 (d, 1H, J = 1.8 Hz, Ph-C2), 6.79–6.82 (d, 1H, J = 8.2 Hz, Ph-
C5), 7.18–7.34 (m, 5H, Ph0-H), 12.05 (br s, 1H, COOH). 13C NMR
(75.44 MHz, (CD3)2SO): d = 13.77 (Bu-CH3), 22.03 (Bu-CH2), 25.74
(2C, Cyclohex-C3/5), 26.04 (Cyclohex-C4), 28.92 (Bu-CH2), 31.20
(Bu-CH2), 32.71 (2C, Cyclohex-C2/6), 33.97 (Cyclohex-CH2), 35.16
(Ph0-CH2), 36.31 (Cyclohex-C1), 37.31 (Prop-C3), 46.94 (Prop-C2),
66.39 (Ph-C4–O–CH2), 69.02 (Ph-C3–O–CH2), 113.75 (Ph-C2),
114.58 (Ph-C5), 121.10 (Ph-C6), 126.16 (Ph0-C4), 128.13 (2C, Ph0-
C
2/6), 129.02 (2C, Ph0-C3/5), 132.26 (Ph-C1), 138.54 (Ph0-C1),
146.83 (Ph-C4) 147.93 (Ph-C3), 176.42 (COO–). MS (ESIꢂ) = m/
e = 451.3 [Mꢂ1]ꢂ. Combustion Anal. Calcd C29H40O4 [452.29]: C,
76.95; H, 8.91. Found: C, 77.01; H, 8.95. Difference: C, 0.06; H, 0.04.
3.1.1.3. Intermediates of compound 22.
3-hydroxy-4-phenethoxybenzaldehyde
Characterization of
(22B): NMR
1H
(300.13 MHz, (CD3)2SO): d = 3.05–3.10 (t, 2H, J = 7.0 Hz, Ph0-CH2),
4.24–4.29 (t, 2H, J = 7.0 Hz, Ph-O–CH2), 7.12–7.38 (m, 8H, Ph0-
H + Ph-C2/5/6H), 9.55 (br s, 1H, Ph-OH), 9.79 (s, 1H, CHO). 13C
NMR (75.44 MHz, (CD3)2SO): d = 34.83 (Ph0-CH2), 68.99 (Ph-O–
CH2), 112.58 (Ph-C5), 113.54 (Ph-C2), 124.34 (Ph-C6), 126.31 (Ph0-
C4), 128.27 (Ph0-C2/6), 129.02 (Ph0-C3/5), 129.79 (Ph-C1), 138.31
(Ph0-C1), 147.07 (Ph-C3), 152.45 (Ph-C4), 191.42 (CHO). MS
(ESIꢂ) = m/e = 240.9 [Mꢂ1]ꢂ.
3.2. Determination of c-secretase modulation
Characterization of 3-(2-cyclohexylethoxy)-4-phenethoxybenzal-
dehyde (22C): 1H NMR (300.13 MHz, (CD3)2SO): d = 0.92–1.76 (m,
13H, Cyclohex-H + –CH2), 3.03–3.07 (t, 2H, J = 6.6 Hz, Ph0-CH2),
3.99–4.03 (t, 2H, J = 6.5 Hz, Ph-C3–O–CH2), 4.24–4.29 (t, 2H,
J = 6.6 Hz, Ph-C4–O–CH2), 7.14–7.38 (m, 7H, Ph-C2/5H + Ph0-H),
7.48–7.51 (dd, 1H, J = 1.8, 8.3 Hz, Ph-C6), 9.80 (s, 1H, CHO). 13C
NMR (75.44 MHz, (CD3)2SO): d = 25.72 (Cyclohex-C3/5), 26.02
(Cyclohex-C4), 32.68 (Cyclohex-C2/6), 33.98 (Cyclohex-C1), 34.82
(Ph0-CH2), 36.03 (Cyclohex-C1H), 66.37 (Ph-C3–O–CH2), 69.03 (Ph-
C4–O–CH2), 111.26 (Ph-C5), 112.39 (Ph-C2), 125.75 (Ph-C6),
126.28 (Ph0-C4), 128.15 (Ph0-C2/6), 129.10 (Ph0-C3/5), 129.63 (Ph-
C1), 138.25 (Ph0-C1), 148.60 (Ph-C3), 153.63 (Ph-C4), 191.34
(CHO). MS (ESI+) = m/e = 353.3 [M+1].
To characterize the GSM activity of novel analogs, their effects
on the generation of Ab40, Ab42, and Ab38 peptides were deter-
mined in a previously described cell-based ELISA.23,45 In brief,
CHO cells stably overexpressing wild type human amyloid precur-
sor protein and wild type human presenilin-1 were maintained in
DMEM supplemented with 10% FBS, and treated in 96-well plates
for 24 h with increasing concentrations of respective compounds
or DMSO as vehicle. Culture media were collected and analyzed
by ELISA as follows: monoclonal antibody IC16 raised against ami-
no acids 1–15 of the Ab sequence was used as a capture antibody.
To distinguish different Ab species, C-terminal antibodies specific
for Ab40, Ab42, and Ab38 labeled with horseradish peroxidase
(HRP) using the Pierce EZ-Link™ Plus Activated Peroxidase kit
(Thermo Fisher Scientific) were used for detection. 96-well high-
binding microtiter plates were coated overnight at 4 °C with cap-
ture antibody IC16 diluted 1:250 in PBS, pH 7.2. Capture antibody
Characterization of ethyl 2-(3-(2-cyclohexylethoxy)-4-pheneth-
oxybenzylidene)hexanoate (22D): 1H NMR (300.13 MHz, (CD3)2SO):
d = 0.84–0.90 (t, 3H, J = 7.1 Hz, Bu-CH3), 0.94–1.74 (m, 20H, Et-
CH3 + Bu-CH2 + Cyclohex-CH + –CH2), 2.47 (m, 2H, Bu-CH2), 2.99–

M. Hieke et al. / Bioorg. Med. Chem. 19 (2011) 5372–5382
5381
was removed and conditioned media samples (10
l
l for detection
Twenty-four hours after the transfection, cells were treated for
an additional 24 h with increasing concentrations of the com-
of Ab40, 100 l for Ab42, 50 l for Ab38) or standard peptides were
l
l
loaded. HRP-coupled detection antibodies diluted in assay buffer
(PBS containing 0.05% Tween-20, 1% BSA) were added to each well
and incubated overnight at 4 °C. Plates were washed three times
with PBS containing 0.05% Tween-20 and once with PBS. Fifty
microlitre TMB ultra substrate (Thermo Fisher Scientific) was
added, incubated for 1–10 min at RT in the dark, and the reaction
pounds 16 and 17, 5 lM of the c-secretase inhibitor DAPT or DMSO
as vehicle. The cells were then lysed and firefly and renilla lucifer-
ase activities were quantified using the Dual Glo™ Luciferase Assay
System (Promega) and a Paradigm™ microplate reader. Normaliza-
tion for transfection efficacy and cell growth was achieved through
division of the firefly luciferase values by the corresponding renilla
luciferase values. Percent activation of the reporter was then calcu-
lated by normalization of triplicate measurements from each con-
dition to the DMSO control values. Results from three independent
experiments were analyzed by one-way ANOVA with Dunnett’s
posttests using GraphPad Prism. ⁄⁄⁄p <0.001.
was stopped by adding 50 ll 2 M H2SO4. Absorbance at 450 nm
was recorded with a Paradigm™ microplate reader (Beckman–
Coulter). Synthetic Ab40, Ab42 and Ab38 peptides (Bachem AG)
were used to generate standard curves. These Ab peptides were
solubilized in DMSO at a concentration of 1 mg/ml, aliquoted and
stored frozen at ꢂ80 °C. Immediately before use, peptides were di-
luted in assay buffer to 250–3000 pg/ml. Triplicate measurements
from each drug concentration were averaged and normalized to
DMSO control condition. For calculation of IC50 values, cells were
treated with eight increasing concentrations of each compound,
and a nonlinear curve-fit with variable slope model was applied
to the results from 2 to 4 independent experiments. Statistics were
performed using GraphPad Prism Version 5 (GraphPad Software).
3.5. Determination of COX inhibition
Inhibition of the activities of isolated ovine COX-1 and human
recombinant COX-2 was performed as described in Hieke
et al..23,39 Briefly, purified COX-1 (ovine, 50 units) or COX-2 (hu-
man recombinant, 20 units) were diluted in 1 ml reaction mixture
containing 100 mM Tris buffer pH 8, 5 mM glutathione, 5
hemoglobin, and 100 M EDTA at 4 °C and pre-incubated with
the test compounds for 5 min. Samples were pre-warmed for
60 s at 37 °C and arachidonic acid (5 M for COX-1, 2 M for
lM
l
3.3. PPAR transactivation assay
l
l
COS7 cells were grown in DMEM supplemented with FCS, so-
dium pyruvate and penicillin/streptomycine at 37 °C and 5% CO2.
The day before transfection, cells were seeded in 96-well plates
at a density of 30,000 cells per well. Transient transfection was car-
ried out by LipofectamineTM 2000 reagent (Invitrogen) according
COX-2) was added to start the reaction. After 5 min at 37 °C, the
COX product 12-HHT was extracted and then analyzed by HPLC
as described.
3.6. Cytotoxicity assay
´
to the manufacturers protocol with pFR-Luc (Stratagene),
pRL-SV40 (Promega) and the Gal4-fusion receptor plasmids
(pFA-CMV-hPPAR-LBD) of the respective subtype. Five hours after
transfection, medium was changed to DMEM without phenol red
and FCS, containing 0.1% DMSO and the respective concentrations
of the test compounds. Following overnight incubation, cells were
assayed for reporter gene activity using the Dual-GloTM Luciferase
Assay System (Promega) according to the manufacturer’s protocol.
Luminescence was measured with a GENios Pro luminometer
(Tecan Deutschland GmbH, Crailsheim, Germany). Each concentra-
tion of the compounds was tested in triplicate wells and each
experiment was repeated independently at least three times. Nor-
malization for transfection efficacy and cell growth was done
through division of the firefly luciferase data by the corresponding
renilla luciferase values resulting in relative light units. Activation
factors were obtained by dividing through the DMSO control val-
ues. EC50 and standard deviation values were calculated with the
mean values of at least three determinations by SigmaPlot 2001
(Systat Software GmbH) using four parameter logistic regression.
All compounds were evaluated by comparison of the achieved
maximum effect to that of the reference compound (pioglitazone
CHO cells stably overexpressing wild type human amyloid pre-
cursor protein and wild type human presenilin-1 were seeded at
low density in 96-well plates (4000 cells/well) and cultured for
24 h. The cells were then treated in duplicates with increasing con-
centrations (0–100 lM) of the respective compounds or DMSO as
vehicle for an additional 24 h. Cell viability was assessed using
the alamarBlueÒ reagent (Invitrogen). Twenty microlitre alamar-
BlueÒ was added to cells cultured in 200
ll medium and incubated
overnight. Absorbance was measured with a Paradigm™ micro-
plate reader at 570 nm, using 600 nm as the reference wavelength.
Percent viability of vehicle control was calculated from two inde-
pendent experiments.
Acknowledgment
We thank Dr. Jan Näslund (Karoliska Institute, Sweden) for the
NOTCH reporter plasmids, and Drs. Karlheinz Baumann and Man-
fred Brockhaus (F. Hoffmann-La Roche Ltd, Switzerland) for car-
boxyl terminus-specific Ab antibodies. This study was supported
by the Competence Network Degenerative Dementias of the Fed-
eral Ministry of Education (Grant No. 01 GI 1004B, to S.W.) and
the Doktor Robert Pfleger Foundation (Bamberg, Germany).
for PPAR
c
, GW 7647 for PPAR
a
, and L165.041 for PPARd each with
M and
1
l
M). Pioglitazone showed a 6.2 1-fold activation at 1 l
its EC50 was determined as 0.30 0.05 lM.
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